Some energy-sensing molecules and metabolism regulators (including the mammalian target of rapamycin, mTOR) have been recently indicated as possible downstream targets of KGD and may be involved in neuroprotective effects associated to the diet (84). Defects in the mTOR pathway have been linked to ASD (85– 87). Failure to inhibit mTOR pathway could lead to MD due to decreased mitophagy (88) resulting in an accumulation of dysfunctional mitochondria as observed in a mouse model of ASD with phosphatase and tensin homolog on chromosome ten (*Pten*) gene haploinsuffciency (89). Indeed, inhibition of mTOR has been linked to a delay in the occurrence of the epileptic episodes (90) and KGD-fed rats showed inhibition of the activation of the mTOR pathway in brain (21), thus representing an appropriate treatment to control seizures while enhancing the clearance of defective/damaged mitochondria.

#### **ANTIOXIDANT AND NEUROPROTECTIVE ROLE OF THE KGD**

Ketone bodies (without glucose and at concentrations 10-times higher than physiological ones) inhibit mitochondrial reactive oxygen species (ROS) production in rat neurocortical neurons by increasing NADH oxidation following glutamate (Glu) excitotoxicity (32). It has been suggested that the production of NADPH via oxidation of succinate semialdehyde (SSA) into succinate in the Glu decarboxylase (GAD)/γ-aminobutyric acid (GABA) pathway may buffer the redox changes likely to occur in stressful conditions (91–93). However, other mitochondrial NADPH sources are quantitatively more important than SSADH and fatty acid oxidation produces more mitochondrial ROS than pyruvate oxidation (94).

Thus, the use of KGD could be beneficial in ASD given that higher rates of mitochondrial ROS production and compromised cellular antioxidant status (69, 95, 96) have been reported in peripheral cells from children with ASD (44, 68, 69).

#### **EFFECT OF THE KGD ON GABAergic AND CHOLINERGIC SYSTEMS DISTURBANCES**

The GABA shunt bypasses two steps of the tricarboxylic acid cycle – the α-ketoglutarate (KG) dehydrogenase complex and the succinylCoA synthase – for the conversion of KG into succinate (**Figure 1**). It involves three enzymes: a GAD, catalyzing the Glu decarboxylation to GABA, a GABA transaminase, converting GABA to SSA, and an SSADH, catalyzing the oxidation of SSA to succinate (97). This metabolic route (the GAD/GABA pathway) is conserved from bacteria, through yeast and plants, to vertebrates. In higher eukaryotes, SSA can be reduced to γ-hydroxybutyric acid (GHB) by an alternative reaction catalyzed by a GHB dehydrogenase (98–100). It has been proposed that KGD may limit the availability of oxaloacetate to aspartate aminotransferase, an enzyme involved in brain Glu metabolism, resulting in increased Glu or Gln availability to produce GABA (101). The increased conversion of Glu to GABA would be potentially beneficial in ASD (102–105) (**Figure 1**).

Changes in GABA neurotransmission by KGD might explain the decrease in seizure frequencies and improved behavior observed in Rett syndrome (106). Studies in patients with ASD strongly suggest a dysfunction in the GABAergic system (107– 109). However, changes in other components (including Gly, taurine, and GABA) cannot be excluded (34). In the case of SSADH deficiency (SSADH), the KGD may work through restitution of GABAergic neurotransmission (36), although the use of KGD in SSADHD has been strongly argued until more research is performed to test its potential detrimental effects in humans (110). Conversely, ketotic rodents fed on KGD showed no changes

in whole brain (GABA) [between brackets = concentrations; (33, 111)]; however, regional (GABA) changes cannot be ruled out (112), in addition to species-specific differences in the expression of GABA receptors subtypes (113, 114). Considering that cerebrospinal fluid from children treated with KGD showed higher (GABA) (34), it would be of interest to evaluate GABA and amino acid concentrations in different brain areas in animal models of ASD fed KGD.

Dysfunction in the cholinergic system has been observed when PDH deficits are present (115) because a block in this enzyme decreases (citrate), the precursor of acetylcholine via citrate lyase (116). Studies in humans and animal models of ASD suggested that dysfunction of the cholinergic system underlies ASD-related behavioral symptoms (117–119). Trials conducted on ASD individuals have shown beneficial effects of galantamine (an acetylcholinesterase inhibitor) in the management of aberrant behaviors in children and adolescents with ASD (120–122). Treatment of BTBR mice with the acetylcholinesterase inhibitor donepezil hydrochloride improved social preference, social interaction and decreased cognitive rigidity (123). Thus, a KGD has the potential to exhibit beneficial effects in individuals with both ASD and PDH deficiency because the metabolism of KB overcomes the decrease in (citrate) (124) and that of (acetylcholine).

#### **POTENTIAL SIDE EFFECTS OF KGD IN ASD**

Several side effects of KGD have been reported, among them: (a) limitation in protein, carbohydrate, and other nutrients intake can result in a lack of weight gain and growth inhibition (42), which could be detrimental in ASD because of a predisposition for being underweight (125) and the presence of eating disorders (126). Thiamine, lipoic acid, and l-carnitine supplementation have been helpful in selected cases (25). (b) Dyslipidemia from KGD (127, 128) would need to be supervised in ASD patients with β-oxidation deficits, including carnitine deficiency (64, 129) and, for older patients, the additional increased risk in heart disease and atherosclerosis (130).  (c) KGD has an increased risk of systemic ketosis, which may result in lower affinity of hemoglobin for oxygen, resulting in severe outcomes (e.g., coma and death) especially in anemic ASD patients (133). (d) Adverse events experienced by patients with RC complex deficits and epilepsy, which could be extrapolated to those with ASD, included symptomatic persistent hypoglycemia, persistent metabolic acidosis, aspiration pneumonia, and pneumonia followed by respiratory failure (27). (e) Initial fasting and prolonged caloric restriction can cause acute metabolic decompensation in ASD patients with metabolic disorders (134). To reduce the adverse effects of fasting, some studies have omitted the initial fasting period and substituted it with a gradual increase in calories (135). (g) Other side effects include constipation, slower growth, kidney stones, and gastroesophageal reflux (136), although most of them are treatable and/or preventable.

#### **CONCLUDING REMARKS**

More research is necessary to understand the potential therapeutic use of KGD in ASD as discussed at length for SSADHD (110). More specifically, how this diet may improve mitochondrial function in ASD and how this putative improvement derived from a better energy and/or neurotransmitter management may influence behavioral symptoms. There are concerns about utilizing KGD in patients with metabolic encephalopathies, with specific contraindications in pyruvate carboxylase deficiency, fatty acid oxidation disorders, and Krebs cycle disorders. Thus, given that the mechanism of action of KGD has not been yet fully understood, even in cases of improved behavioral symptoms, KGD in ASD might need to be prescribed on a case-by-case basis, upon careful biochemical characterization and metabolic profiling.

#### **AUTHOR CONTRIBUTIONS**

All authors contributed to the design of the work and interpretation of the literature, drafted the work, and gave final approval of the version to be published. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

#### **ACKNOWLEDGMENTS**

This work was supported by Simons Foundation (SFARI #271406 to Cecilia Giulivi), R01-ES011269, R01-ES015359, and R01- ES020392.

#### **REFERENCES**

refractory epilepsy. *Epilepsy Res* (2005) **64**:115–25. doi:10.1016/j.eplepsyres. 2005.03.008

ketogenic and standard diets. *Epilepsy Behav* (2009) **15**:133–41. doi:10.1016/j. yebeh.2009.02.038

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 30 May 2014; accepted: 17 June 2014; published online: 30 June 2014. Citation: Napoli E, Dueñas N and Giulivi C (2014) Potential therapeutic use of the ketogenic diet in autism spectrum disorders. Front. Pediatr. 2:69. doi: 10.3389/fped.2014.00069*

*This article was submitted to Child and Neurodevelopmental Psychiatry, a section of the journal Frontiers in Pediatrics.*

*Copyright © 2014 Napoli, Dueñas and Giulivi. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

### Repeated insulin-like growth factor 1 treatment in a patient with Rett syndrome: a single case study

#### **Giorgio Pini 1\*, M. Flora Scusa1 , Alberto Benincasa1 , Ilaria Bottiglioni 1 , Laura Congiu1 , Cyrus Vadhatpour 2 , Anna Maria Romanelli 1 , Ilaria Gemo1 , Chetti Puccetti 1 , Rachel McNamara2 , Seán O'Leary 2 , Aiden Corvin3 , Michael Gill 3 and Daniela Tropea1,3\***

1 Tuscany Rett Center, Versilia Hospital, Viareggio, Italy

2 Medical School, Trinity College Dublin, Dublin, Ireland

3 Department of Psychiatry, Trinity College Dublin, Dublin, Ireland

#### **Edited by:**

Roberto Canitano, University Hospital of Siena, Italy

#### **Reviewed by:**

Carlo Sala, CNR Institute of Neuroscience, Italy Magdalena Romanowicz, Stanford University, USA Iliyan Ivanov, Mount Sinai School of Medicine, USA Arianna Maffei, State University of New York at Stony Brook, USA

#### **\*Correspondence:**

Giorgio Pini, Tuscany Rett Center, Versilia Hospital, Via Aurelia 335, Lido di Camaiore, ASL 12 di Viareggio, Italy e-mail<EMAIL_ADDRESS>Daniela Tropea, Department of Psychiatry, Trinity College Dublin, College Green, Dublin 2, Ireland e-mail<EMAIL_ADDRESS>
Rett syndrome (RTT) is a devastating neurodevelopmental disorder that has no cure. Patients show regression of acquired skills, motor, and speech impairment, cardiorespiratory distress, microcephaly, and stereotyped hand movements.The majority of RTT patients display mutations in the gene that codes for the Methyl-CpG binding protein 2 (MeCP2), which is involved in the development of the central nervous system, especially synaptic and circuit maturation.Thus, agents that promote brain development and synaptic function are good candidates for ameliorating the symptoms of RTT. In particular, insulinlike growth factor 1 (IGF1) and its active peptide (1–3) IGF1 cross the Blood Brain Barrier, and therefore are ideal treatments for RTT Indeed, both (1–3) IGF1 and IGF1 treatment significantly ameliorates RTT symptoms in a mouse model of the disease In a previous study, we established that IGF1 is safe and well tolerated on Rett patients. In this open label clinical case study, we assess the safety and tolerability of IGF1 administration in two cycles of the treatment. Before and after each cycle, we monitored the clinical and blood parameters, autonomic function, and social and cognitive abilities, and we found that IGF1 was well tolerated each time and did not induce any side effect, nor it interfered with the other treatments that the patient was undergoing. We noticed a moderate improvement in the cognitive, social, and autonomic abilities of the patient after each cycle but the benefits were not retained between the two cycles, consistent with the pre-clinical observation that treatments for RTT should be administered through life. We find that repeated IGF1 treatment is safe and well tolerated in Rett patients but observed effects are not retained between cycles.These results have applications to other pathologies considering that IGF1 has been shown to be effective in other disorders of the autism spectrum.

**Keywords: Rett syndrome, insulin-like growth factor 1, social cognition, seizures, autonomic functions**

#### **INTRODUCTION**

The clinical case study performed in this experiment depicts the efficacy and safety of insulin-like growth factor 1 (IGF1) exposure upon two cycles of treatment. The patient is a young girl with a clinical and genetic diagnosis of Rett syndrome (RTT). The study started when the patient was 5 years old. The patient received first round of treatment on the 25th of May 2010 and discontinued on the 11th of November 2010. The second round of treatment started on the 20th of November 2012 and discontinued on the 28th of February 2013.

#### **BACKGROUND**

Rett syndrome is taxonomized as a pervasive neurodevelopmental disorder affecting mainly female children (1:10000) due to its X-linked method of inheritance (1). Methy-CpG binding protein 2 (MeCP2) dysfunction is associated with several neurodevelopmental disorders (1), and can cause a wide variety of phenotypes in females. Most males do not live past the first year due to neonatal encephalopathy and death in the classic RTT karyotype (2). The effects of MECP2 mutations lead to a loss of function or overexpression of the MeCP2 protein. If it results in a loss of function state, some examples of effects yield: classic RTT, atypical RTT, autism, and mild mental retardation (2). The majority of RTT patients (85%) have mutations in the MECP2 gene that lead to loss of MECP2 protein function. At this point of time, there is no cure to RTT syndrome, however treatment strategies are starting to emerge.

Rett syndrome is a progressive neurodevelopmental disorder that initially presents in females just past 18 months. Patients with RTT appear to have apparent normal development and milestone achievements up to the first 18 months of age (3). Early indication of neurological disease progression includes: deceleration of head growth, general growth retardation, weight loss, and weak posture via muscle hypotonia (2). Progression of RTT leads to the loss of purposeful use of their hands and the development of stereotypic hand wringing or washing movements: other hand movements are clapping, flapping, and mouthing (2). Withdrawal of social behaviors are noticeable where patients have shown symptoms such as irritability, self-abusive mannerisms, blank faces, audio hypersensitivity, reduction of eye contact, unresponsiveness to social cues, and indifference to variable environments (2). Eventual losses in motor coordination, ataxia, and gait apraxia are symptoms of motor pathway deterioration (2). Most girls eventually require wheel chair assistance during teenage years. Autonomic perturbations include hyperventilation (and other breathing abnormalities), seizures with variable severity, cardiac abnormalities, constipation, parkinsonian features, and oropharyngeal dysfunction (2). Patients may have a normal appetite but still experience weight loss and osteopenia. Structural abnormalities include scoliosis and rigidity. Behavioral complications involve teeth grinding, temper tantrums, depression, and anxiety. The condition eventually plateaus and the patient may live up to their seventh decade (2).

MeCP2 is an example of a mammalian protein that binds to methylated CpGs. For its ability to bind DNA and influence gene expression, MeCP2 is mostly believed to be a transcription factor (1), however localization of MeCP2 outside the nucleus and in the synaptic compartments (4) suggest that MeCP2 protein may have also functions directly related to synaptic activity. Inheritance of the MECP2 gene follows an X-linked dominant pattern and sporadic mutations are prevalent in 85% of classic RTT cases (2). Mutations that have been noted on the spectrum include missense, nonsense, and frame shift mutations for this protein. Mutations impacting on the nuclear localization sequence of MeCP2 or early truncating mutations have more severe phenotypes in comparison to missense mutations; while C-terminal deletions are thought to cause milder phenotypes. There is an association between the severity of the mutation and the severity of the phenotype (5). The phenotype of the female is highly variable depending on the expression levels of the unaffected copy of MECP2 on the other X chromosome.

The identification of the gene associated with Rett made possible the generation of mutant mice defective in MeCP2 functions. The mice have signs that resemble patients' symptoms and therefore constitute a valid model for the study of neurobiology of Rett and for testing candidate therapeutics. A significant discovery has emerged from two independent groups concluding that in the mouse model, the reactivation of the natural Mecp2 gene in the adult mutant mice partially reestablished normal phenotype (6, 7). This finding implies that when the normal brain has impairment by a dysfunctional MeCP2, control conditions can be restored in the central nervous system. However, Zoghbi's lab has shown that drug treatment for RTT has to be administered during the whole life of the patients (8).

Neurobiological analysis shows that MeCP2 mutations produce defects in central nervous system development, specifically in the maturation of circuits and synapses (4, 9). Therefore, medical treatments focused on brain development and synaptic functioning is promising for the regression of RTT. Indeed, a research study noted that overexpression of brain-derived neurotrophic growth factor (BDNF), which is a neurotrophin that has involvement in brain development and plasticity, resulted in the neutralization of the MeCP2 deficiency (10). This allowed for the consideration of growth factors, which are centralized around their involvement of neuronal and synaptic growth, toward potential treatment options for RTT syndrome. BDNF is incapable of crossing the blood brain barrier, so it is of no use for immediate pharmacological therapy consideration. However, IGF1 and its active peptide (1–3) IGF1, are capable of crossing the blood brain barrier. This quality makes them an ideal drug for treatment in neurodevelopmental disorders like RTT. Research has shown that IGF1 peptide treatment partially restored spine density and synaptic amplitude in the mouse model (9). The treatment was started at 2 weeks of age and was noted to stabilize cortical plasticity to the wild-type level. One of the major effects noted in this study was after the treatment of IGF1; MeCP2 KO with treatment mice improved from an average of 60-day lifespan to an average of 90-day lifespan in comparison with KO without treatment mice. Not only did the KO mice with treatment have increased life span, but also improved respiratory, cardiac, and locomotor function. In the study from Tropea et al. (9) (1–3) IGF1, has already shown to ameliorate the symptoms of RTT in the mouse model of the disease. IGF1 has also been shown to be an effective therapy in MeCP2 KO mice (11). Other studies show that IGF1 is a suitable treatment for human RTT patients and that there are no known risks associated with IGF1 administration (12, 13). In our previous study, six girls with classic RTT, between 4 and 11 years of age, were administered IGF1 subcutaneously twice per day for 6 months. They were evaluated based on the effectiveness of the treatment and it was reported that IGF1 partially restored a significant amount of the symptoms of RTT. From this study, all families and caretakers noticed an improvement in cognitive abilities of the girls and their interactions with the surrounding environment. Considered McGraw's study, lifelong therapy is necessary to combat RTT syndrome, like IGF1 for MeCP2 dysfunction (8).

The aim of this paper is to evaluate if repeated cycles of IGF1 is a suitable method, in respect to safety and efficacy, for a life-long treatment option in the human model. The clinical case study in this report depicts a patient receiving two cycles of IGF1 treatment and clinical parameter assessments. Also, we provide insight on how the patient responds after the treatment with two clinical evaluations after the treatment cycles. The study is an open label study, and the child displays classic RTT syndrome, where the phenotypic onset of the disease took place between the eighth and ninth month. The child started the first IGF1 cycle in May 2010, when the child was in the fifth year of age. IGF1 was administered twice daily (0.1 mg/kg) for 6 months. Two years later, another cycle of treatment was started. The dosage and routes of administration were the same, but the second cycle lasted 4 months. In between each cycle, blood parameters were monitored and autonomic responses evaluated. During both cycles, IGF1 was well tolerated in each cycle and no direct side effect was observed. IGF1 is also shown to cause no interference with the anti-epileptic medications that the patient was undergoing. Based on the conclusion of this experiment, the treatment is believed to promise a strong impact on RTT syndrome.

#### **SUBJECTS AND METHODS**

#### **CLINICAL ANALYSIS**

We evaluated the RTT patient's overall physical health, RTTassociated phenotypic expressions of the patient, and growth developments. We took notes, recorded in the Section "Results," during various stages of the treatment process during two cycles

of IGF1 exposure. The parents kept a diary with daily annotations of glycemia levels and alterations in behavior (sleep, eating, etc.)

#### **Patient screening**

Patient was chosen based on the presentation of classic RTT syndrome and confirmed mutation in the MECP2 gene. The family gave written consent for partaking in this study before any treatment took place. The selection criteria are described in Pini et al. (12).

#### **Treatment administration**

Mecasermin–Increlex (IGF1) was subcutaneously administered twice daily (0.1 mg/kg) in two cycles: the first cycle lasted nearly 6 months, and the second cycle lasted 4 months, with a 2 years gap between the two cycles. Possible side effects include hypoglycemia (headache, dizziness, nausea, and perspiration), tonsil hypertrophia, and local irritation at the injection site. The family was thoroughly informed about the treatment process; the caretakers of the patient were trained to perform daily injections, and were provided with a kit to measure glycemic levels. Patients were observed for side effects during every visit and critical events were recorded and are in summarized in the Section "Results."

#### **Post treatment examination**

The patient was followed up twice after each cycle to evaluate general health after IGF1 exposure. Patient was physically examined based on cardio-respiratory factors, body growth parameters, and motor abilities. Critical events were recorded along with the corresponding epileptic treatment at that time. Behavioral examination along the parameters of social interaction and mood were also evaluated.

#### **VIDEO ANALYSIS OF COGNITIVE AND SOCIAL ABILITIES**

Video analysis utilized approximately 20 h of video recordings of RTT patients and was filmed in Versilia Hospital (Italy). The RTT patient observed in this paper was an analyzed participant in this study. The investigators, analyzing the videos, were not aware that the patient had received IGF1 treatment. Observed preliminary footage from additional patients yielded the design of the scoring criteria. It was decided to score the girls on 20 features,10 negatives, and 10 positives. The patient was scored in each feature on a scale from 1 to 5, with 1 indicating extreme RTT, 2 indicating severe RTT, 3 indicating moderate RTT,4 indicating mild RTT,and 5 indicating absent RTT. In this way, a lower score was related to a more prevalent RTT-like behavior. The 10 negative features chosen were: hand wringing, biting, rocking, hitting, indiscriminate moaning, tongue chewing, vacant staring, bruxism, breath-holding/apnea, and Valsalva maneuver. The 10 positive features chosen were: pointing, manipulating, reaching for something, ability to mimic/imitate, indicating yes/no with head gesture, reactivity to a call, reactivity to an object,smiling in response to a stimulus,deliberate vocalization, and attention.

#### **RESULTS**

The patient was born from the mother's first pregnancy. The mother suffers from epilepsy and was treated with anti-epileptic drugs (AEDs) since adolescence. The patient was born at week 38 of gestation by cesarean section due to an emergency response. At birth, the patient had the following dimensions: 2600 g in weight, 45.5 cm in length, and 33 cm in head circumference. The newborn received artificial feeding due to maternal agalactia.  The patient's motor development showed independent seat positioning acquired at 8 months with kyphosis of the bust: unimanual walking with support at 18 months with mild postural tremor. At 18 months, the patient's use of hands was discrete and she could grab objects and press buttons. The patient's language development showed production of vocalization and infrequent simple syllabic doublings (bi-bi-, ma-ma, ba-ba). The patient showed bruxism with variable power. The patient had tendency to be constipated and sphincter control was not acquired. The sleep–wake rhythm was characterized by frequent nocturnal awakening and was prescribed Niaprazine (Nopron). On the 2nd of August 2006, genetic testing for RTT indicated a mutation c. 397C > T (R133C) of exon 4 of the MECP2 gene.

The clinical case study performed in this experiment depicts the efficacy and safety of IGF1 exposure upon two cycles of treatment. Careful consideration was executed in monitoring to the effects of IGF1 in both autonomic function and blood parameters. The patient received first round of treatment on the 25th of May 2010 and discontinued on the 11th of November 2010. The second round of treatment started on the 20th of November 2012 and discontinued on the 28th of February 2013. The dosage for IGF1 was the same in both cycles: 0.1 mg/kg maintenance dosages and 0.05 mg/kg first and last weeks were adjustment dosages. Our findings are noted below during each visit with the patient during their cycles of IGF1 treatment.

#### **FIRST CYCLE OF IGF1 TREATMENT**

The first experimentation with IGF1 started on 25th of May 2010 (first cycle), when the patients were 5 years and 8 months. We clinically assessed the patient and results were noted as follows: patient has slight ataxic ambulation, which required minimum support; slight hypotonia was noticeable and babinsky positive ROT hyper-excitability; wide-spread bouts of tremor often associated with tachypnea, and possible grasping of objects upon request. Patient demonstrated expressive language, characterized by varied and modulated vocalizations. The patient had a calm demeanor with discreet interest in external environment and surrounding people.

Second visit was on the 31st of August 2010. The patient showed good clinical health and similar ambulation as the first visit. The patient showed coastal navigation with the family. The hypotonia and wide-spread tremor was the same as first visit. The patient showed improvement in grip of objects. The patient continued to demonstrate expressive language, characterized by varied and modulated vocalizations. Communicative intentions were observed and actions were occasionally preceded by anticipatory behaviors. The demeanor remained calm.

Third visit took place on the 23th of November 2010. The patient maintained good general clinical condition and the parents reported a good clinical outcome at home. The patient was able to ambulate with unimanual support with tendency to externally rotate the march of the lower limbs, and launch of the same. The use of hands to engage in patterns of action with objects have been a new observation, such as pressing a button or lever to get a visual or sound effect. The patient demonstrates good conduct and interaction with unfamiliar people. The food and sleep–wake rhythm is regular, while obstinate constipation persists.

The patient was discontinued from IGF1 on the 27th of November 2010 and clinical assessments were continued.

The fourth visit was on the 15th of February 2011. The clinical observations were almost unchanged from the previous visit. The gait seemed to have improved, even when the child fatigued easily. There was a submission of critical events that took place. On the 8th of February 2011, at 10:40 a.m. the first epileptic crisis lasting 2–3 min was characterized by tremor, hypotonia, eyes wide and staring into space. This was followed by sleep. Secondary crisis showed facial and perioral cyanosis, tremor, and blank stares, followed by sleep. A third crisis at 6:10 p.m. noted guttural verses, staring, stiffening, and blank stares followed by sleep. A fourth crisis occurred at 7:30 p.m. where stiffening, staring, perioral cyanosis, pallor of the face, and strange vocalizations followed by sleep. Vomiting, facial and perioral cyanosis, and guttural sounds characterized the fifth crisis at 9:30 p.m. On the 9th of February 2011, the patient had a fever of 37.3°C. At 12:35 p.m., the first crisis lasted a minute and was characterized by salivation, perioral and facial cyanosis, absent eyes, and potential hypotonia causing the patient to fold on itself.

The second crisis (on February 9th) occurred at 1:38 p.m. and lasted 3 min; characterized by perioral and facial cyanosis and mouth tremors followed by sleep. The third crisis occurred at 5:27 p.m. and lasted for 2 min, characterized by an absent gaze. On the 11th of February 2011 at 10:16 a.m., the patient was noticed to have staring eyes and making guttural sob like sounds. On the 12th of February 2011 at 10:00 a.m., the patient woke up with a frightened look, began trembling with tears, and guttural vocalizations. A second crisis occurred at 3:40 p.m. characterized by neck muscle failure and appearance of sleep while sitting on the toilet, with guttural vocalizations. On the 13th of February 2011, the patient presented with a fever of 38.3°C, pharyngitis, and began Zithromax treatment.

The fifth visit took place on the 24th of May 2011. The clinical examination showed good general condition and parents reported a stable clinical experience. The child had unimanual-supported ambulation with tendency to externally rotate and launch of the lower limbs. The clinician noted a decreased resistance during walking. The patient was observed to have a discreet use of hands when she grabbed objects and pressed buttons to get a visual or sound effect. Social interaction showed observation of the environment and the maintenance of eye contact for a longer duration of time. The patient maintained a serene mood. The food and sleep–wake rhythm were regular and constipation still persisted. Critical incidents occurred almost daily and were characterized by staring, gaze deviation to the left, and clonus of the limbs. On the 13th of April 2011, the child presented a crisis with the semiotics for a 10-min period, which required Micronoan administration. The patient's anti-epileptic therapy dosage was increased: valproate 200 + 200 mg/day.

The sixth visit took place on the 21st of October 2011. The clinical examination reported a good general clinical condition. The parents informed that the child was attentive, observational of the environment, interacting with people, and mood was serene. The critical events were reported to occur on awakening and were characterized by wide-eyed, clonus of the limbs, apnea, and cyanosis of the face. The patient then took Valproate: dosage of 250 + 200 mg/day.

Upon collection of the clinical data set for the first cycle, blood test showed no morbidity related to the treatment and a noticeable improvement in bone density. Glycemic values taken during each visit all remained well within reference value (60– 110 mg/dl), where the patients' average was 80 mg/dl and the range was between 75 and 86 mg/dl. The starting bone density measurement was taken on the 26th of May 2010 and a second one was taken at the end of first cycle on the 15th of February 2011. This measurement showed a BMD of 0.333 and 0.451, respectively.

Analysis of cognitive and social abilities show mixed results in positive and negative RTT features, in terms of improvement and disease progression (**Figures 1** and **2**). **Figures 1** and **2**, representing video analysis, note that the negative features (9 out of 10) increased more than positive features (5 out of 10). Hitting and breath-holding/apnea were the most improved negative features. Smiling in response to a stimulation, deliberate vocalization, and yes/no with head gesture were the most improved positive features. The patient shows an improvement in height percentile during the course of IGF1 treatment in almost immediate results, however, it returned to previous percentile among the discontinuation of the IGF1 treatment. No other significant correlations to the IGF1 treatment can be made from **Table 1**. In the ISS evaluation, we noted a starting score of 4 in autonomic function, at the first dose of the cycle; and upon the last dose, the patient reduced to a score of 3 in autonomic function. This implies that the autonomic function restored as a result of IGF1 treatment. Another improvement noted by the ISS score was in growth and development from an ISS score of 6, at first treatment, to a score of 5, at last dosage. This also implies that IGF1 treatment improved the patient's growth and development. ISS scores are shown in **Figure 3**.

#### **SUMMARY OF IGF1 EFFECTS AFTER THE FIRST CYCLE OF TREATMENT**

We found that IGF1 was well tolerated during the first cycle of treatment and did not show any side affect. The treatment produced an increase in height and BMI of the patient mostly during the first 3 months of treatment, and returned to baseline after the discontinuation of the drug (**Tables 1** and **4**). Significant benefits were noted as reduction of negative stereotypes, while the social and cognitive abilities did not show any significant improvement (**Figures 1** and **2**; **Table 5**). IGF1 did not show any significant effect on ISS, with the exception of the autonomic function, that appear improved also after 6 months from the last IGF1 administration (**Figure 3**; **Table 6**).

#### **SECOND CYCLE OF IGF1 TREATMENT**

The next cycle of IGF1 treatment started on the 20th of November 2012, when the patient was 8 years and 2 months. Since the last visit, the patient is continuing the anti-epileptic therapy: Valproate

**FIGURE 1 | IGF1 treatment affects different features in different cycles**. Comparison of the scores of individual features at T0 and T1 for negative **(A)** and positive **(B)** features in cycle 1 (left) and cycle 2 (right). Paired comparison with Wilcoxon test shows that IGF1 treatment improved

significantly negative features in cycle 1 (Wilcoxon test, p-value = 0.014), but not in cycle 2 (Wilcoxon test, p-value = 0.07). Viceversa, for positive features the effect was significant in cycle 2 (Wilcoxon test, p-value = 0.016), but not in cycle 1 (0.3).

(250 + 200 mg/day) and Levetiracetam (300 + 300 mg/day). The clinical examination shows that health is fair and reported: patient has slight ataxic ambulation, which required minimum support; slight hypotonia was noticeable and Babinski positive ROT hyperexcitability; wide-spread bouts of tremor often associated with tachypnea; and possible grasping of objects upon request, but released after a few seconds. The patient has expressive language that is characterized by production of varied and modulated vocalizations. The mood of the patient is serene and there is a discreet interest in the environment and in surrounding people.

The second visit of the second cycle took place on the 29th of January 2013. The patient continues to have seizures three to four times a week characterized by eyes turned in backwards. The patient has an increased in dosage of Levetiracetam (350 + 350 mg/day) and maintained Valproate (250 + 200 mg/day). The parents reported an increase in bruxism with a decrease in stereotypic mannerisms. The patient showed longer attention span, improved eye contact, and smiling. The dietary intake was good and constipation improved. The clinical examination reported: patient has slight ataxic ambulation, which required minimum support; slight hypotonia was noticeable and Babinski positive ROT hyper-excitability; wide-spread bouts of tremor often associated with tachypnea; and possible grasping of objects upon request, but released after a few seconds. The patient had expressive language characterized by production of varied and modulated vocalizations. The mood of the patient was serene and there was a discreet interest in the environment and in surrounding people. We took extra note in the slight improvement of attention: specifically indicating that the patient holds her gaze for the duration of the smile and she seems more interested in the environment. Motor stereotypes decreased slightly while bruxism increased.

The third visit took place on the 3rd of December 2013. The grandfather informs about an improvement in attention span to external stimuli throughout the visual field laterally. The communication capability is improved and characterized by the improved ability to stare at an object that the patient wants to play with or a place that the patient wants to go. He also noticed an improved grip on objects. The mother reports that the patient eats less and tremors and bruxism have increased. She also states that sleep patterns are altered by presence of numerous episodes of pavor during sleep and wake states. The clinical examination reported:

**Table 1 | Growth parameters during first cycle of treatment**.

This table compares the growth parameters of the RTT patient in the first cycle with the average child of same age.The patient remains in low percentiles, despite improvements in height during IGF1 application.

patient has slight ataxic ambulation, which required minimum support; slight hypotonia was noticeable and Babinski positive ROT hyper-excitability; wide-spread bouts of tremor often associated with tachypnea; and possible grasping of objects upon request, but released after a few seconds. The patient has expressive language that is characterized by production of varied and modulated vocalizations. The mood of the patient is serene and there is a discreet interest in the environment and in surrounding people. The clinician took another extra note in the slight improvement of attention: specifically indicating, again, that the patient holds her gaze for the duration of the smile and she seems more interested in the environment. Motor stereotypes decreased slightly while bruxism increased.

Suspension of the second cycle of IGF1 occurred on the 28th of February 2013.

The following clinical observation was recorded on the 21st of May 2013. An anamnestic incident was recorded with a slightly increased frequency of seizures and every crisis has a longer duration of action. There is a constant intense tremor present. Parents observe the patient suffering from pharyngitis and a decrease in appetite. The clinical observation includes the patient being pale and sleepy, while being poorly responsive to environmental stimulation. They informed about two critical episodes in the morning with semiology and in few seconds durations. The gait was possible with minimal support and appeared ataxic. Slight hypotonia was noticeable and Babinski positive ROT hyper-excitability. Widespread bouts of tremor were noted in association with tachypnea. There was grasping of objects on presentation, but released after a few seconds. The stereotypes were unchanged.

Clinical data sets provided no insight into the second cycle of IGF1 treatment in blood work or growth parameters (**Table 2**). Glycemic values taken during each visit all remained well within reference value (60–110 mg/dl), where the patients' average was 78.2 mg/dl and the range was between 73 and 84 mg/dl. The starting bone density measurement was taken on the 20th of November 2012 and a second one was taken at the end of the second cycle on the 21st of May 2013. This measurement showed a BMD of 0.426 and 0.465, respectively.

This table shows the growth parameters during the second cycle of IGF1 treatment. There are no significant contributions to these characteristics given by the treatment. The patient maintains in low percentiles during the second IGF1 treatment cycle.

The patient shows mixed results for positive and negative phenotypes based on the video analysis (**Figures 1** and **2**), however no adverse effects are noted in association with the second cycle of IGF1 treatment. **Figures 1** and **2**, representing video analysis, note that the negative features (4 out of 10) increased less than positive features (6 out of 10). Hand wringing and biting showed most improvement in negative features. Reactivity to an object and deliberate vocalization showed the most improvement in positive features. In the ISS evaluation: we noted a starting score of 6 in autonomic function, at the first dose of the cycle; and upon the last dose, the patient reduced to a score of 5 in autonomic function. This implies that the autonomic function restored, again, as a result of IGF1 treatment. Another consistent improvement noted by the ISS score was in growth and development from an ISS score of 6, at first treatment, to a score of 5, at last dosage. This also implies that IGF1 treatment improved the patient's growth and development. ISS scores are shown in **Figure 3**.

#### **SUMMARY OF IGF1 EFFECTS AFTER THE SECOND CYCLE OF TREATMENT**

In the second cycle of treatment, there were no significant effects of IGF1 on the growth parameters of the patient (**Tables 1** and **4**). The scoring of the cognitive and social abilities, as well as the negative features of Rett, shows a general worsening of the conditions between the end of the first cycle and the starting of the second cycle of treatment. The second treatment with IGF1 produces a significant improvement in the scoring for the social and cognitive abilities, but no effects are visible on the negative symptoms of RTT (**Figures 1** and **2**; **Table 6**). The ISS components show a mixed effect of IGF1 on different features, with overall a long term improvement of movement and autonomic functions (**Figure 3**; **Table 5**).

#### **FOLLOW-UP EXAMINATIONS POST CYCLES**

On September 24, 2013, the patient was 9 years old with a body mass index of 10.13 (normal values between 19 and 25 kg/m2 ). The occipital–frontal cortex circumference was 48.2 cm and her weight was 14.6 kg. The patient's results in this report came from the evaluations of the autonomic nervous system in quiet conditions. **Table 3** depicts the cardiovascular examination that took place on this date.

The results showed normal breathing patterns for 21.2–35.9% of mixed tachypnea breath, 12.2% of deep breaths, 7.4% of

#### **Table 3 | Cardiovascular reflexes range during cycles**.

This table depicts that the patient falls with in the normal reference range for three out of the four cardiovascular reflexes that we observed. Cardiac vagal tone is just slightly under the reference value.

breath-holding episodes, 8% of hyperpnea, and 3.8% of apnea episodes. The average heart rate recorded is about 90 beats per minute, while the mean values of CVT in relaxation amounted in about 5.6; where the registered CVT peaks results are of average and significant.

The phenotype of the cardiopulmonary patient was classified as forced (40.2%). The general autonomic framework was that of a basal level vagal tone, which is slightly lower than normal. There were multiple types of respiratory rhythm abnormalities that were indicative of instability within the respiratory centers of the brainstem.

On 11 March 2014, a second follow-up examination was recorded. The parents noted some changes in seizure related events. Since November of 2013, the patient took Carbamazepine (25 mg in the morning and 50 mg in the evening), which was suspended while being treated with Levetiracetam. The latest crisis occurred a week before this examination where the patient had a cluster of six episodes on the same day. The patient grew agitated and had difficulty falling asleep, so Valium was given by the parents.

At this time, the patient was currently attending third grade class with support and rehabilitative physiotherapy continues 2 days a week (partially at ASL and partially privately) with swimming integrated. She was on the following AEDs: depakote (250 mg in morning and 250 mg in the evening), Carbamazepine (5 mg in morning and 50 mg in the evening), and Pepidax.

The general health conditions were satisfactory. The patient was able to autonomously maintain the sitting position with ataxia of the trunk and ambulation requires support. Tremors were noticed and the detection of bruxism. Scoliosis was absent, while there was reduced muscle tropism. The patient made little facial expression, with eye contact being only possible if the child is evoked several times; language was absent. During the visit, there were no manual stereotypes; however, the parents report it occurred when the patient was tired. The patient showed fluctuation in vigilance, and during the visit the child was alert and responsive. During neurological examinations, the child had suddenly fallen asleep for few minutes at a couple of occasions. The physician recommended that it was advisable for the patient to continue the rehabilitation treatment that was already in progress.

#### **COMPARISON OF IGF1 EFFECTS DURING AND AFTER THE TWO CYCLES**

In order to compare the effects of IGF1 between the two cycles, we considered the relative values of each parameter (growth parameters, ISS scores, cognitive, and social abilities scores) during and after the termination of the drug administration, grouping

together the effects during IGF1 administration and after the suspension of the drug (**Figure 4**). In particular, for the effects during the two cycles, was considered the differences between the last visit during treatment and the visit before treatment (**Figure 4A**). For the preservation of the effects after the cycles, we considered the difference between the values at the last visit post treatment and the values of the last visit during treatment (**Figure 4B**). We observed that the effects of the drug were comparable between the two cycles, and even with a slight positive effect during the first cycle, there were no significant differences (two tailed *t*-test, *p*-value = 0.28).

#### **EPILEPTIC HISTORY DURING OBSERVATION**

The patient was enlisted into the study in March 2010, but because of an onset of epilepsy during this month the start treatment was postponed to May. There were two critical incidents between the months of March to May and on average the crisis occurred at a frequency of four/month. The patient then commenced the first cycle of IGF1 treatment on the 25th of May 2010. The epileptic treatment during this time was Valproate. From the time of the previous visit to the next visit on the 31st of August 2010, there were five critical episodes reported while on the Valproate therapy, on average the crisis during IGF1 administration have the same intensity and frequency than before the treatment. More frequent critical incidents occurred between the second and third visit, where the patient was on Valproate. Following this visit, there was the suspension of IGF1 on the 27th of November in 2010. On the fourth visit,15th of February 2011,after the suspension of IGF1 administration, the crisis appeared in clusters on several occasions. At this point, the epileptic therapy was adjusted to Valproate plus Frisium (for when the patient has many crises). By the fifth visit on the 24th of May 2011, critical episodes became more frequents and occurred on almost a daily basis. The therapy was Valproate, while Frisium was suspended. Visit six, on the 21st of October 2011, indicated critical episodes occurring upon awakening. Still the patient remained on Valproate.

The second trial start date occurred on the 20th of November of 2012. Critical episodes are daily and the epileptic therapy was Valproate and Levatiracetam. The second visit showed similar critical episodes on a daily basis. Suspension of IGF1 occurred on the

**FIGURE 4 |The effects of IGF1 treatment for each cycle are comparable**. The plots represent the differential scores of growth parameters, ISS scores and Social and cognitive scores, before and after IGF1 Administration [EFFECTS **(A)**], and in follow-up studies [PRESERVE **(B)**]. Although we observe a slightly major efficacy of IGF1 during the first cycle of treatment, the differences are limited and they are not significant.

28th of February 2013. The third visit took place on the 12th of March 2013, where the critical episodes remain daily. The crisis did not change with the administration of IGF1 treatment and the epileptic therapy remained on Valproate and Levatiracetam. On the 24th of September 2013, the critical episodes were reported the same on a daily basis. During this fourth visit, the patient was on the same epileptic treatment. On the 28th of October 2013, the epileptic therapy was changed: Levitiracetam was removed and Carbamazepine was added. The sixth visit, on the 3rd of November 2014, reported that Carbamazepine caused a thinning out of critical episodes, but they were clustered. The epileptic therapy was carbamazepine and valproic acid.

#### **DISCUSSION**

The use of IGF1 represents an appealing therapy for RTT. Due to IGF1 affecting multiple symptoms of RTT in animal models, its noticeable benefits from pediatric use, and its physical properties allowing it to cross the blood brain barrier; pilot trials (12, 13), indicated that IGF1 is safe and tolerable for treatment of RTT patients.

Considering the finding that any treatment for RTT should be administered for life (8), our research addresses the issue of multiple IGF1 treatments and tolerability in children with RTT syndrome. During our first study (12), we observed the major improvements during the first weeks of IGF1 treatment, and for this reason, we decided to reduce the time of the second treatment to 20 weeks. These findings are in line with the study by Khwaja et al. (13), where the amount of IGF1 in the serum decreases during the administration period, and the authors suggest that an adjustment of the dosage and administration time could be more beneficial to the Rett patients.

Although our study is mostly oriented in confirming the safety and tolerability of IGF1 through multiple cycles, we observed an overall improvement of autonomic function during IGF1 treatment, and then a lessening of improvement upon suspension of IGF1 (**Figure 3**; **Table 5**). We recorded no hypoglycemia, which indicates that IGF1 shows good toleration in patients. This is important because it is one of the main risks associated with IGF1 treatment (13).

This table summarizes the differential scores for each component of the growth (listed on the left), considering the difference at the end versus the beginning of the treatment (effect) and the difference after 6 months from the last administration versus the scores on the day of the last administration (preserve).The original scores are those reported in**Tables 1** and **2**.The improvements are highlighted in green, and the worsenings are highlighted in red.

In this study, similarly to what reported in the past and by other researchers (12, 13), we did not observed any specific influence of IGF1 treatment on epileptic episodes: the appearance of the epileptic crisis preceded the first cycle of IGF1 administration, and the intensity and frequency of the episodes remained on average the same before and after the treatment. The intense episodes reported between the second and third week during the first cycle of treatment, can be due to an endogeneous evolution of the crisis, as suggested also by the increased frequency after the interruption of the treatment (which required an adjustment of the therapy). Before starting the second IGF1 administration, the crisis had a daily frequency and they remained unaltered during the whole cycle of treatment. These observations are in accordance to the literature in pediatric syndromes, where IGF1 is not associated to an increase in seizures, but rather epileptic phenotype is associated to decreased IGF1 levels in the serum (14, 15). On the contrary, it is possible that the seizure medication hindered the positive effects of IGF1. It would be interesting to compare the effects of IGF1 administration in patients treated with valproate versus patients treated with alternative AEDs.

Insulin-like growth factor 1 has also been considered to be more effective when given in acute intermittent pulses, versus chronic dosing. One noted side effect from IGF1, was that long term IGF1 administration has linkage with potential puberty effects; which is already accelerated in RTT (13). Being that IGF1 has a strong feedback system, patient tolerance over cycles is worth exploring into for future studies. This implies the evaluation of lower doses in life-long treatment. An assessment of total effects on single analyzed relative parameters (ISS scores, cognitive and social abilities, and growth parameters) showed that the effects observed with the first cycle of treatment were not significantly different from those in the second cycle of treatment (**Figure 4**, two tailed *t*-test *p*-value = 0.28).

While there were some phenotypic benefits shown by the assessment of social and cognitive abilities, nine of the improvements remained after the first cycle was over, and no phenotypic improvement remained in the second trial as different phenotypes improved aside from attention. However, we observed motor control as another form of improvement. However, our research insight into motor control improvement may be a result of subjectivity further research is suggested to elucidate the validity of this potential benefit. Our analysis confirms the results of Zoghbi's team, that whatever treatment is decided for RTT syndrome patients, it must be for life (8).

The two studies of IGF1 safety and tolerability in RTT patients (12, 13), showed similar results: IGF1 did not produce hypoglycemia and it is not associated to seizures, and overall is well tolerated by patients. In addition, we observed an improvement in autonomic function, and Khwaja and colleagues concluded that motor ability showed no measurable improvement, but also found

#### **Table 6 | Differential scores of ISS scores during cycles**.

This table summarizes the differential scores for each component of the ISS (listed on the left), considering the difference at the end versus the beginning of the treatment (effect) and the difference after 6 months from the last administration versus the scores on the day of the last administration (preserve). The improvements are highlighted in green, and the worsenings are highlighted in red. Note that the decrease in the scoring are improvements for the ISS; on the contrary, increases in the scoring represent worsening.

#### **Table 5 | Differential scores of social and cognitive abilities during cycles**.

This table summarizes the differential scores for negative and positive features of the social and cognitive abilities assessment. Each number represents the difference of the score for the specific ability at the end of the treatment (T1) versus the beginning of the treatment (T0). Improvements are therefore positive numbers and they are highlighted in green, while worsening are negative numbers and are highlighted in red.

that respiratory complications associated with RTT syndrome showed improvement (13).

One factor to consider in our findings is the environmental surrounding that the patient experienced during and after the IGF1 treatment. The patient lived with the mother during the time of treatment. The mother suffers from anxiety and seizures, which may have limited the benefits of IGF1 treatment due to high stress levels being perceived by the patient. The consideration is due to a follow-up communication, post IGF1 second cycle treatment, where the grandparents noted an immediate amelioration of the patient's phenotypic mannerisms. The stressful environment could have caused misperception of the severity level to phenotypic hindrances, which are RTT associated.

The personal environmental surrounding, together with the onset and progression of the epileptic crisis are two of the limits of this case study for assessing the benefits of IGF1 therapy. Appropriate double blind and placebo controlled studies are necessary to define the efficacy of IGF1 treatment. However, considering the rarity of RTT (1:10,000), and the recent discovery of IGF1 as possible Rett treatment, data coming from a single case study are nonetheless useful to further develop the treatment.

Our research show that repeated treatments with IGF1 is safe and tolerable for the patient. This finding along with other findings on IGF1 treatment with RTT, should encourage further research in IGF1 treatment for additional neurodevelopmental disorders.

#### **CONCLUDING REMARKS**

Our research shows that repeated treatments with IGF1 are safe and tolerable for the patient. We observed improved autonomic function and social and cognitive abilities in each cycle but the benefits were lost between cycles. The results in this clinical case study suggest that IGF1 is beneficial for our patient, but a larger cohort of patients and proper clinical trials are requested to establish the overall efficacy in RTT.

#### **ACKNOWLEDGMENTS**

We thank our patient and her family for their collaboration. We thank the funding agencies: Region Tuscany, Fondazione Cassa di Risparmio di Lucca, ASL12 Viareggio, TIAMO foundation, and Marie Curie IRG (248284). We are also thankful to: Enrico Rossi, Ledo Gori, Prof. Michele Zappella, Anna Rita Bertellotti, all the doctors and operators in Versilia hospital, Fabrizio Bianchi Istituto di Fiologia Clinica, Centro Nazionale delle Ricerche Pisa.

#### **REFERENCES**

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 12 April 2014; accepted: 20 May 2014; published online: 03 June 2014. Citation: Pini G, Scusa MF, Benincasa A, Bottiglioni I, Congiu L, Vadhatpour C, Romanelli AM, Gemo I, Puccetti C, McNamara R, O'Leary S, Corvin A, Gill M and Tropea D (2014) Repeated insulin-like growth factor 1 treatment in a patient with Rett syndrome: a single case study. Front. Pediatr. 2:52. doi: 10.3389/fped.2014.00052 This article was submitted to Child and Neurodevelopmental Psychiatry, a section of*

*the journal Frontiers in Pediatrics.*

*Copyright © 2014 Pini, Scusa, Benincasa, Bottiglioni, Congiu,Vadhatpour, Romanelli, Gemo, Puccetti, McNamara, O'Leary, Corvin, Gill and Tropea. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# New experimental treatments for core social domain in autism spectrum disorders

#### **Roberto Canitano\***

Division of Child Neuropsychiatry, University Hospital of Siena, Siena, Italy

#### **Edited by:**

Yuri Bozzi, University of Trento, Italy

#### **Reviewed by:**

Maria Luisa Scattoni, Istituto Superiore di Sanità, Italy Latha Soorya, Rush University Medical Center, USA

#### **\*Correspondence:**

Roberto Canitano, Division of Child Neuropsychiatry, University Hospital of Siena, Viale Bracci 14, Siena 53100, Italy

e-mail<EMAIL_ADDRESS>
Current therapeutics in autism spectrum disorders (ASD) only treat the associated symptoms, without addressing core social dysfunctions. A paradigm shift in research of the pathogenesis of ASD, its synaptic abnormalities and altered signaling in multiple dynamic systems, have led to new experimental treatments for treating the core social abnormalities of ASD. NMDA antagonists, especially memantine, have been introduced in clinical trials addressing glutamatergic transmission in children and adolescents with ASD. GABAergic signaling has been targeted in trials using the GABAB receptor agonist arbaclofen for ASD patients with promising results. Oxytocin has been recognized as implicated in social development and affiliative behaviors. Preliminary findings from clinical trials using oxytocin in children with ASD show encouraging improvements in social cognition, but larger studies are needed. In two of the single gene disorders associated with ASD, Insulin Growth Factor (IGF-1) is a new treatment that has been tested in Rett syndrome and Phelan-McDermid syndrome (Chromosome 22 deletion syndrome). IGF-1 has been demonstrated to reverse the reduction in the number of excitatory synapses and the density of neurons that characterize these conditions in animal studies and it is being introduced as an experimental treatment. As a novel approach to verify treatment efficacy, neural processing modifications were recently evaluated by fMRI after a pivotal response training intervention. Another study of neural changes in response to treatment examined variations in EEG signaling in patients after an Early Start Denver Model (ESDM) intervention.

**Keywords: autism spectrum disorders, experimental treatments, preclinical models, clinical trials, childhood and adolescence**

#### **INTRODUCTION**

Autism spectrum disorders (ASD) are early-onset neurodevelopmental disorders characterized by major difficulties in social interaction, communication, and repetitive or restricted interests and behaviors. Autism is defined as a spectrum disorder due to the heterogeneity of clinical presentation, the degree of social impairment,intellectual ability,associated symptoms,and possible etiology. ASD are included in the diagnostic category of neurodevelopmental disorders in the Diagnostic and Statistical Manual of Mental Disorders V (1). The diagnosis of ASD is based on two major symptoms: social-communication deficits, restricted and repetitive interests, and behaviors. By definition, these symptoms must occur during the early childhood of individuals with ASD.

The currently accepted prevalence of ASD, based on consistent reports from multiple sources in different populations, is about 1% worldwide. ASDs are therefore among the most common pervasive developmental disorders and there is great concern regarding its growing incidence (2–4).

To date, the only FDA-approved treatments for ASD are the atypical antipsychotics risperidone and aripiprazole, which are mainly directed at treating the associated symptoms and not the core social dysfunctions that characterize this heterogeneous group of disorders. Treatments with these two medications have been demonstrated to reduce and attenuate irritability (e.g., tantrums, aggression, hyperactivity, and self-injurious behaviors)

in children and adolescents with ASD (5–8). Improvements in social interaction and reciprocity have been observed as well, but this is probably a secondary effect of an overall reduction in maladaptive behaviors and not a primary therapeutic effect of these medications.

Targeted treatments for ASD are developed through the understanding of molecular and cellular abnormalities that guide specific interventions, hypothesizing that the wide variety of genetic variants in ASD converge in a core set of molecular pathways that mediate phenotypic expression in some identifiable core symptoms (9). Most research for new therapeutics currently uses preclinical models, such as "knockout" mice displaying specific molecular abnormalities. Genetic studies of ASD and related neurodevelopmental disorders have provided classes of potentially useful compounds. Proof-of-principle assays with agents that reversed phenotypes in mouse models have paved the way for clinical trials (10).

#### **EXCITATORY/INHIBITORY IMBALANCE IN ASD AND NEW TREATMENTS**

An imbalance of excitatory (glutamate) to inhibitory (GABA) neurotransmission (E/I imbalance) is thought to be implicated in the pathogenesis of ASD (11–13). Excessive excitatory glutamatergic neurotransmission with a loss of inhibitory GABA transmission, as well as abnormalities in synaptic plasticity due to dysfunctions

in the NMDA, AMPA, and/or GABA receptor systems, have been detected in mouse models and support this conceptualization of ASD pathogenesis (10). Pharmacological evidence emerged from mouse models with deletions in synaptic genes Fmr1, Mecp2, and Shank2, and the BTBR inbred strain, which have demonstrated favorable outcomes from treatments with glutamatergic agents, including memantine (14). After these preclinical investigations, clinical trials were carried out to test the potential role of glutamatergic and GABAergic agents in reversing core social dysfunction in ASD.

#### **GLUTAMATERGIC TARGETING IN ASD**

Altered glutamatergic excitatory transmission involves different receptors, including down-regulation of AMPA receptors, abnormalities of NMDA receptor-mediated plasticity, and altered metabotropic glutamate receptor subtype 5(mGluR5) signal transduction. The mGluR5 antagonists have been tested in preclinical models of autism with variable effects on social and stereotypic behaviors (15). MPEP, an antagonist of the mGluR5 receptor, reduced repetitive self-grooming in BTBR mice. BTBR is an autism mouse model with behavioral abnormalities in social interaction and communication, as well as repetitive movements and behaviors. Risperidone also reduced repetitive self-grooming in BTBR, but only at doses that induced sedation (16). GRN-259, a selective negative allosteric modulator of the mGluR5 receptor, was tested in BTBR mice. GRN-529 reduced repetitive behaviors in three cohorts of BTBR mice and it attenuated social withdrawal with an associated increase in communication, providing overall improvement in behavioral conditions (17). Treatment with the two AMPAKINE compounds CX1837 and CX1739, agents active on AMPA receptors that enhance excitatory glutamatergic action, succeeded in recovering social impairment but it did not attenuate the high levels of repetitive self-grooming in BTBR (18). In 4 week-old Balb/c mice, the NMDA receptor agonist d-cycloserine was found to improve both sociability and repetitive behaviors, emphasizing the potential role of NMDA agents in modulating the core social dysfunctions of ASD (19). Studies of Shank3 heterozygous mice detected a reduction in basal neurotransmission, reflecting reduced AMPA receptor-mediated transmission, which was targeted with insulin growth factor (IGF-1) treatment (20, 21).

Clinical trials followed the preclinical phase of investigation with glutamatergic agents. Memantine, an NMDA receptor antagonist, was tested in children and adolescents with ASD. Overall, the memantine trials conducted so far have yielded inconsistent findings due to methodological flaws, thus they did not allow for firm conclusions. No recommendations on the use of memantine or of other glutamatergic agents in ASD treatment are warranted as yet (22).

#### **GABAergic TARGETING IN ASD**

GABAergic signaling has been demonstrated to be implicated in E/I imbalance in ASD (23). Engrailed2 (En2) knockout mice have been proposed as a model for ASD. In this mutant, the cerebellum and hippocampus have been shown to have abnormal synaptic transmission, altered developmental processes and defective GABA transmission convergent with ASD pathways. A link between altered function of En2, deficits of GABAergic forebrain neurons, and the pathogenesis of ASD has been postulated (24, 25). In addition, social and cognitive abnormalities have been detected in En2 knockout mice, further highlighting its role in ASD. Deficits in reciprocal social interactions growing over time were found in En2 null mutants (26). Interestingly, mice lacking Mecp2 from GABA-releasing neurons demonstrated Rett syndrome and autistic features, including repetitive behaviors. In these mice, MeCP2-deficient GABAergic neurons showed a presynaptic reduction in glutamic acid decarboxylase 1 (Gad1) and glutamic acid decarboxylase 2 (Gad2) levels, and GABA immunoreactivity (27).

In clinical trials, GABA signaling has been probed with specific compounds. Arbaclofen, a GABAB receptor agonist that acts upstream of mGluR5 receptor signaling and is thought to augment inhibitory neurotransmission, was investigated. Initially, a preclinical study (28) followed by a clinical trial with arbaclofen was carried out in Fragile X syndrome, the most common cause of intellectual disability and ASD (29), and favorable findings were reported regarding social domains (30). A recent open-label trial with STX209, a form of Arbaclofen, was conducted in ASD patients (31). Safety, tolerability, and efficacy were tested in ASD individuals in an 8-week open-label trial enrolling 32 children and adolescents with a score of ≥17 on the aberrant behavior checklist (ABC)-irritability subscale. STX209 was generally well tolerated. Improvements were reported on several outcome measures, including the ABC-Irritability (the primary endpoint) and the Lethargy/Social Withdrawal subscales, and the Social Responsiveness Scale. Randomized controlled trials are needed to confirm these preliminary positive findings.

#### **mTOR TARGETING IN ASD**

The mammalian target of rapamycin (mTOR) pathway is central to synaptic protein synthesis and it integrates inputs from different sources, including NMDA and metabotropic glutamate receptors. In addition, it is directly involved in the maintenance of the physiological synaptic E/I ratio. Abnormalities in mTOR signaling have been found in ASD (32). Activation of mTORC1 promotes the formation of the eIF4F initiation complex. Mutations in genes upstream of mTOR, as detected in tuberous sclerosis complex (TSC1/2), neurofibromatosis 1(NF1), phosphatase and tensin homolog (PTEN), or the Fragile X syndrome gene (FMR1) cause hyperactivity of the mTORC1–eIF4E pathway and lead to syndromic forms of ASDs (33, 34). Accordingly, mTOR inhibitors are first line candidates for the treatment of ASD in TSC (35). Everolimus, an mTOR inhibitor, is currently being studied in a double-blind controlled trial in children and adolescents (6–21 years old) with TSC, ASD and seizures (NCT01289912).1 The primary aim of this trial is improvement in cognition, while ASD symptoms attenuation and seizure frequencies are secondary objectives. Rapamycin, an mTORC1 inhibitor and immune-suppressor, was investigated in a recent preclinical study as an agent to ameliorate social behaviors and attenuate stereotypies in BTBR mice. Rapamycin was found to improve many components of sociability in the BTBR mouse,

1http://clinicaltrials.gov

pointing to mTOR overactivation as a therapeutic target in ASD. However, no effects of rapamycin on stereotypic behaviors were detected (36).

An important regulatory action was recently described in the other component of this pathway, the downstream effectors of mTOR. Signaling molecules in the downstream of the mTOR pathway have been demonstrated to play crucial roles in ASD pathogenesis, further highlighting its role (37, 38). Furthermore, 4E-BP2 inhibits protein translation by competing with eIF4G for eIF4E binding. The deletion of the gene encoding 4E-BP2 (EIF4ebp2) leads to autistic-like behaviors in mice. It was demonstrated that the removal of 4E-BP2, or overexpression of eIF4E, enhances protein translation; increased translation of neuroligins (NLGNs) is the next step that in turn causes an increased synaptic E/I ratio, which may eventually lead to ASD phenotypes. Pharmacological inhibition of eIF4E reversed social behavior deficits in EIF4ebp2 knockout mice and restored the E/I balance (37). These findings thus established a strong link between eIF4E-dependent translational control of NLGNs, E/I balance, and the development of ASD-like behaviors. Pharmacological targeting of downstream effectors of mTOR may represent promising experimental therapeutics in ASDs.

#### **SINGLE GENE DISORDERS WITH ASD AND EXPERIMENTAL TREATMENTS**

Single gene disorders associated with high rates of comorbid ASD, such as Rett syndrome and Phelan-McDermid syndrome (PMS) have provided significant models of targeted interventions with novel experimental treatments.

#### **IGF-1 IN PHELAN-McDERMID SYNDROME (CHROMOSOME 22q13 DELETION SYNDROME)**

The loss of a functional copy of the SHANK3 gene leads to 22q13 deletion syndrome, a complex clinical condition known as PMS. Haploinsufficiency of SHANK3 accounts for about 0.5% of cases of ASD and/or developmental delay (39). There is mounting evidence for a wider role of SHANK3 and glutamate signaling abnormalities in ASD and related conditions. IGF-1 regulates synapse formation, neurotransmitter release, and neuronal excitability via posttranslational modification of NMDA and AMPA receptors (40).

Therapeutic approaches with IGF-1 aimed at reversing deficits in SHANK3-haploinsufficiency have been carried out in mice. IGF-1 has been demonstrated to reverse the reduction in excitatory synapse numbers and density of neurons. Administration of daily intraperitoneal injections of human IGF-1 over a 2-week period was found to reverse deficits in hippocampal AMPA signaling, long-term potentiation (LTP), and motor performance in Shank3-deficient mice (41). These findings led to a current trial of IGF-1 as a treatment in PMS, which is still underway and expected to provide important clues on its role in the treatment of this ASD subtype (see text footnote 1 NCT0152590).

In a recent study, induced pluripotent stem cells (iPSCs) from individuals with PMS and autism were used to produce functional neurons. It was demonstrated that PMS neurons have reduced SHANK3 expression and major defects in excitatory, but not inhibitory, synaptic transmission. IGF-1 treatment promoted the formation of mature excitatory synapses that lack SHANK3 but contain PSD95 and NMDA receptors with fast deactivation kinetics. These findings provided evidence for a disruption in the ratio of E/I in PMS neurons, and they point to a molecular pathway that can be recruited to restore it (42).

#### **IGF-1 IN RETT SYNDROME**

Studies in mouse and human neuronal models of Rett syndrome also demonstrated benefits with IGF-1 administration. In 90% of cases, Rett syndrome is caused by abnormalities on the MECP2 gene, by either deletions or mutations. IGF-1, highly expressed during brain development and very important for spine maturation, has been tested to overcome MECP2 deficit. Synaptic abnormalities have been observed in Rett syndrome related to genetic defects on the MECP2 gene (43). Treatment with IGF-1 in a mouse model of Rett syndrome has been shown to increase synaptic growth and to rescue phenotype defects (44). A phase II randomized placebo-controlled trial is underway to evaluate the safety and efficacy of IGF-1 in treating ASD symptoms and respiratory/autonomic dysfunction in patients with Rett syndrome (see text footnote 1 NCT01777542).

#### **INTRANASAL OXYTOCIN IN ASD**

There has been growing interest in oxytocin as it is recognized to be implicated in social development and affiliative behaviors. Intranasal oxytocin has been shown to increase social initiative and motivation (45), as well as social cognition (46), and to reduce repetitive and restricted behaviors in ASD. Previous studies with oxytocin in ASD were conducted by single-dose administration to adults, therefore, the long-term effect of nasal oxytocin and its effect on children must still be clarified (47).

There have been only a few trials with intranasal oxytocin in children with ASD, and most were pilot studies on small numbers of patients. In one of the first double-blind studies, oxytocin nasal spray was found to improve emotion recognition in 16 male youth aged 12–19 with ASD (48). In a recent pilot study, 15 children and adolescents with ASD with verbal IQs of ≥70 participated in a trial on the safety and tolerability of intranasal oxytocin. Repeated measures of regression analysis controlled for week, dose, age, and sex were employed. The highest dose evaluated, 0.4 IU/kg, was found to be well tolerated. Over 3 months of treatment, social cognition, repetitive behaviors, and anxiety were improved with the maintenance of effect for 3 months after discontinuation of treatment (49). In an open-label study, oxytocin was administered intranasaly over a long term of 7 months to eight male youths with ASD (10–14 years of age; IQ: 20–101). Six of the eight participants had improvement in the communication and social interaction domains at ADOS-G evaluation and at *T*-scores of the CBCL; however, no statistically significant improvement was found in the ABC (50). In a double-blind study with a controlled design, a group of 38 male youths (7– 16 years old) with ASD were administered 24 or 12 IU, depending on weight, of intranasal placebo or oxytocin once daily over four consecutive days. The oxytocin or placebo was administered in a dynamic social condition, i.e., during parent–child interaction training sessions. In this study, intranasal oxytocin did not significantly improve social interaction skills, however it must be

mentioned that it was an untested behavioral intervention, limiting the interpretation of results (51). Collectively, these studies demonstrated mixed results as to the outcome of core social impairment, with a tendency toward an improvement in social domains. Large-scale, double-blind placebo-controlled studies are needed to confirm the role of intranasal oxytocin in core social ASD dysfunction.

A randomized, double-blind cross over fMRI trial with intranasal oxytocin was conducted in a group of 17 ASD children and adolescents (age 8–16.5 years) to evaluate neural circuit modifications hypothesized after oxytocin administration (52). The Reading the Mind in the Eyes Test (RMET), a social cognition test (53) was used to investigate the brain areas under examination, and a remarkable variation was observed 45 min after oxytocin administration. Structures and circuits within the so-called social brain, such as the dorsal and ventral striatum, premotor cortex, posterior cingulate, and posterior–superior temporal sulcus, demonstrated enhanced activity after oxytocin administration. Moreover, changes in salivary oxytocin concentrations from baseline to 30 min post administration were positively associated with increased activity in the right amygdala and orbito-frontal cortex during social vs. non-social judgments. Furthermore, it was highlighted that oxytocin enhanced the salience of social stimuli, thus positively interfering with social cognition. These changes in the relevant brain areas of interest were observed following a single oxytocin administration and further research to test the persistence of these changes over time in long-term treatments is needed.

#### **NEW APPROACHES TO VERIFY TREATMENT EFFICACY**

Neural circuit modification following treatment for ASD is a novel field of research that could shed light on the mechanisms involved in neural changes and in the search for the biomarkers of treatment response (54). Functional MRI is the main instrument for a dynamic investigation of the neural substrate. A case study in two children with ASD was carried out with functional MRI performed before and after an intensive intervention of 4 months of pivotal response training (PRT). PRT is an empirically validated behavioral treatment that has widespread positive effects on communication, behavior, and social skills in young children with ASD. Neural processing modifications during a biological motion task, a form of social information, were evaluated by fMRI. Outcome measures included the evaluation of dysfunctional areas identified in children with ASD, compared to typically developing children (right amygdala, bilateral fusiform gyri, left ventrolateral prefrontal, and right posterior superotemporal sulcus). After treatment, ASD children showed increased activation in the above listed areas, indicating that neural modifications had occurred and they overlapped with the brain area activations of their typically developing peers (55).

Another study, investigating neural changes in response to treatment, examined variations in EEG signaling after an intervention with the Early Start Denver Model (ESDM) in a group of 48 children with ASD, aged 18–30 months. Children with ASD were randomized into two groups; one received an ESDM intervention for 2 years and the other received only their previous treatment in the community. A typically developing group of children served as controls. Event related potentials (ERP) and EEG activity (spectral power) were detected during the presentation of faces versus objects. The ESDM group demonstrated a better outcome in ASD symptoms, IQ, language, and adaptive behavior than the others. Increased cortical activation, i.e., a reduction in alfa power and increased theta band, was detected in this group of children as well as in typically developing children when viewing faces, in contrast to the community intervention group that showed the opposite pattern with greater cortical activation when viewing objects. The limitation of this research was that it lacked a baseline evaluation (56).

#### **CONCLUSION AND FUTURE DIRECTIONS**

Currently available medications mostly act upon selective symptoms of ASD, possibly offering a chance to further explore the specific mechanisms and circuits involved in etiological factors. Synaptic or circuit mechanisms associated with circumscribed aspects of ASD, e.g., social and communication domains, need to be thoroughly evaluated at the molecular and synaptic levels and possibly separated from the other symptoms/domains. Core mechanisms that cover distinct fractions of ASD would parallel the concept of core symptoms in ASD, based on the hypothesis that a group of protein factors converges on common pathways to be targeted (13). The next generation of animal models carrying human mutations will be of the utmost importance in uncovering the neural and molecular bases of ASD and to pave the way for clinical trials of treatments for core social domains. A homogeneous group of ASD individuals with phenotype and synaptic defects should be enrolled in pharmacological trials to test the efficacy and the safety of specific compounds. These trials would measure outcomes in the core social domain of ASD and also identify specific biomarkers for a wider perspective of treatment outcomes. Investigations of dynamic brain changes in ASD patients, using fMRI after treatments, would be extremely valuable in order to shed light on the neural underpinnings of core social impairment in these individuals.

#### **REFERENCES**

selective activation of GABAB receptors with arbaclofen. *Sci Transl Med* (2012) **4**(152):ra128. doi:10.1126/scitranslmed.3004218

autism. *J Am Acad Child Adolesc Psychiatry* (2012) **51**:1150–9. doi:10.1016/j.jaac. 2012.08.018

**Conflict of Interest Statement:** The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 24 April 2014; accepted: 29 May 2014; published online: 20 June 2014. Citation: Canitano R (2014) New experimental treatments for core social domain in autism spectrum disorders. Front. Pediatr. 2:61. doi: 10.3389/fped.2014.00061*

*This article was submitted to Child and Neurodevelopmental Psychiatry, a section of the journal Frontiers in Pediatrics.*

*Copyright © 2014 Canitano. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Autism as a disorder of biological and behavioral rhythms: toward new therapeutic perspectives

**Sylvie Tordjman1,2\*, Katherine S. Davlantis 3 , Nicolas Georgieff 4 , Marie-Maude Geoffray 4 , Mario Speranza5 , George M. Anderson6 , Jean Xavier 7 , Michel Botbol 1,8, Cécile Oriol 2 , Eric Bellissant 9,10 , Julie Vernay-Leconte11, Claire Fougerou9,10, Anne Hespel 9,10, Aude Tavenard9,10, David Cohen7 , Solenn Kermarrec 1,2, Nathalie Coulon1 , Olivier Bonnot 1 and Geraldine Dawson3**

#### **Edited by:**

Roberto Canitano, University Hospital of Siena, Italy

#### **Reviewed by:**

Rajshekhar Bipeta, Gandhi Medical College and Hospital, India Felipe Ortuño, Clínica Universidad de Navarra, Spain Roberto Canitano, University Hospital of Siena, Italy

#### **\*Correspondence:**

Sylvie Tordjman, Hospitalo-Universitaire de Psychiatrie de l'Enfant et de l'Adolescent, 154 rue de Châtillon, Rennes 35200, France e-mail<EMAIL_ADDRESS>
There is a growing interest in the role of biological and behavioral rhythms in typical and atypical development. Recent studies in cognitive and developmental psychology have highlighted the importance of rhythmicity and synchrony of motor, emotional, and interpersonal rhythms in early development of social communication. The synchronization of rhythms allows tuning and adaptation to the external environment.The role of melatonin in the ontogenetic establishment of circadian rhythms and the synchronization of the circadian clocks network suggests that this hormone might be also involved in the synchrony of motor, emotional, and interpersonal rhythms. Autism provides a challenging model of physiological and behavioral rhythm disturbances and their possible effects on the development of social communication impairments and repetitive behaviors and interests. This article situates autism as a disorder of biological and behavioral rhythms and reviews the recent literature on the role of rhythmicity and synchrony of rhythms in child development. Finally, the hypothesis is developed that an integrated approach focusing on biological, motor, emotional, and interpersonal rhythms may open interesting therapeutic perspectives for children with autism. More specifically, promising avenues are discussed for potential therapeutic benefits in autism spectrum disorder of melatonin combined with developmental behavioral interventions that emphasize synchrony, such as the Early Start Denver Model.

**Keywords: autism spectrum disorder, biological rhythms, motor, emotional and relational rhythms, synchronization of rhythms, melatonin, Early Start Denver Model, therapeutics**

#### **INTRODUCTION**

Endogenous physiological variations involved in biological rhythms reflect adaptation to the environment. Thus, the sleep– wake rhythm associated with biological circadian rhythms can be viewed as an adaptation to the day–night cycle. Circadian rhythms allow temporal organization of biological functions in relation to environmental changes (1). The periodicity of activities applies to all biological, physiological, and psychological functions; recently, the science of biological rhythms, chronobiology, has emerged with its own theory, science, and education (2).

Furthermore, recent studies in the field of cognitive and developmental psychology have highlighted the importance of rhythmicity and synchrony of motor, emotional, and relational rhythms in early development of social communication. Given the major role of the sleep hormone melatonin in the ontogenetic establishment of diurnal rhythms, the synchronization of peripheral oscillators (also termed clocks) and the regulation of human circadian rhythms (1), melatonin might be involved in the synchrony of motor, emotional, and relational rhythms.

Indeed, relationships might exist, based on the hypothesis of ergodicity (3), between cellular communication networks involving a cellular synchrony (synchronization of cellular oscillations by melatonin) and early social communication development involving a synchrony of motor, emotional, and interpersonal rhythms. Autism spectrum disorder (ASD) – a developmental disorder characterized by social communication impairments associated with repetitive interests and behaviors – provides an interesting and challenging model of abnormal melatonin production in early developmental disorders and its possible relationship with autistic behavioral impairments.

This article proposes a central role of rhythmicity and synchrony of rhythms in typical child development and offers a new integrative approach, which considers autism as a disorder of biological and behavioral rhythms. In this perspective, promising avenues will be discussed in this article for potential therapeutic benefits in ASD of melatonin and developmental behavioral interventions that emphasize rhythms and synchronization, such as the Early Start Denver Model (ESDM).

#### **PHYSIOLOGICAL AND BEHAVIORAL RHYTHM DISTURBANCES IN AUTISM**

#### **AUTISM AS A DISORDER OF BIOLOGICAL RHYTHMS**

Alterations in circadian sleep–wake rhythm have frequently been reported in autism (4, 5). More specifically, reduced total sleep and longer sleep latency as well as nocturnal and early morning awakenings are often observed in individuals with ASD (6– 12). Furthermore, prior studies on melatonin in autism have all reported abnormalities in melatonin secretion (see **Table 1**). In addition, abnormalities in cortisol circadian rhythm have also been reported in autism [for a review, see Tordjman et al. (13)]. In particular, significantly higher frequency of absence of circadian variation in melatonin and cortisol levels was observed in individuals with autism compared to typically developing controls. Golombek et al. (14) described the effects of circadian desynchronization that can enhance susceptibility to certain disorders

#### (metabolic, immune, cognitive, and somatic disorders including cancer). It is noteworthy that congenitally blind children with consequently abnormal melatonin secretion and synchronization [the production of pineal-derived melatonin depends on the light acting through the retinohypothalamic tract (15)] very frequently display autism [up to 42% (16)], whereas hearing impaired children, including hearing loss, show autism less frequently [up to 10% (17)]. More specifically, abnormally low daytime and nighttime melatonin secretion was associated with an absence of melatonin circadian variation in some individuals with autism (18, 19), which in turn, given the role of synchronizer of melatonin, also has consequences on the circadian rhythms network, including the cortisol circadian rhythm (13, 20).

This blunted circadian rhythmicity with no or little variability might be related to the difficulties in adapting to changes typically observed in individuals with autism. Thus, children with autism who are confronted with physiological continuity due to absent circadian rhythms may have difficulties adapting to changes in either their external or their internal environment (26). Indeed, as previously underlined, the circadian clocks network, synchronized by melatonin and involving an internal system of continuity/discontinuity, allows adaptation to environmental changes.

Similarly, blunted circadian rhythmicity may explain the difficulty observed in many children with autism in adapting to

#### **Table 1 | Studies of melatonin levels in individuals with autism**.

changes in rhythms of their external and internal environment. Thus, rapid rhythms of sensory stimuli in their external environment (e.g., repeated visual stimuli provided by a stroboscopic light) can provoke epileptic seizure in some individuals with autism [approximately one-third of children with autism have epilepsy (27)]. Interestingly, EEG changes tended to be correlated with an abnormal rhythm of melatonin in young adults with autism (22). Furthermore, some parents have reported that their daughters with autism experience epileptic seizures toward the 14th day of their menstrual cycle, which is when luteinizing hormone (LH) levels peak (Tordjman, personal communication), suggesting quite tentatively that individuals with autism may have difficulties adapting to periodic hormonal changes in their internal environment. We can state the following hypothesis: a change in rhythm associated with excessive environmental stimuli might strongly increase arousal and lead to physiological stress which, for some individuals with autism, can disturb the rhythmic activity of a particular brain area, leading it to fall out of sync with the rest of the brain and causing its population of neurons to fire (depolarization and occurrence of an epileptic seizure). This underlines the importance of stable physiological rhythms.

Finally, significant relationships have been found between lower nocturnal melatonin excretion and increased severity of autistic social communication impairments, especially for verbal communication and social imitative play (18, 23). These findings are in agreement with studies suggesting an association between reduced melatonin production and language impairment (22, 28). Along the same line, the systemic administration in the animal model of Zebra Finch of a melatonin-1B receptor antagonist at the beginning of the night shortens the song and motif length and affects the song syllable lengths produced the next day (29). Reduced melatonin activity might create timing problems in biological clocks with physiological and psychological effects that might be, according to Boucher's model of autism (30) and Wimpory's theory (31), involved in autistic impairments, notably in autistic social communication impairments. It is noteworthy that deficiency in oxytocin [oxytocin is considered a bonding hormone (32, 33)] has also been reported in autism (34, 35) and bonding is involved in the development of very early social interaction in infants. Interestingly, the release of oxytocin by the posterior pituitary gland follows a robust circadian rhythm in mammals. Further studies are needed to better understand the underlying mechanisms of oxytocin anomalies in autism and to explore, in particular, possible oxytocin rhythm disturbances in ASD. The importance of the synchrony of rhythms in the development of social interaction and communication is detailed in the next section.

#### **IMPORTANCE OF SYNCHRONY OF RHYTHMS FOR SOCIAL COMMUNICATION IN TYPICAL DEVELOPMENT AND AUTISM SPECTRUM DISORDER**

Several studies, based on animal models and human perinatal development, suggest that stable patterns of repeated stimuli in the form of maternal physiological rhythms, involving cross-modal perception such as regular cardiac rhythm, which provides the fetus with auditory and vibratory stimuli, allow the fetus to integrate sensory information facilitating prenatal perceptual learning and develop a coherent representation of his or her internal and external environment (36–38). Fluctuations in the physiological rhythms (variants), such as variations in the maternal cardiac rhythm and also variations in hormone levels involved in the circadian rhythms that are already present during the fetus life (the fetus' circadian rhythms are the mother's ones), occurring in a background of regular repetition of identical sequences (invariants), may help the fetus to develop the ability to adapt to change in an environment characterized by high regularity. As previously emphasized (38), very early mother–infant relations provide a secure environment based on the repetition of invariants, while at the same time promoting adaptation to change through the presence of variants. It is through the regular repetition of identical sequences of discontinuity, such as circadian rhythms, that a continuum is constructed associated with the development of adaptation to changes.

The development of the very earliest form of communication relies on the sharing of emotions between mother and infant, when, for example, the infant is suckling in his or her mother's arms, through emotional synchrony that enhances the integration of sensory inputs (39, 40). Concerning memory processes, emotions enable to "fix" events, just as a photographic fixing agent sets images (38). Cortisol (a stress and arousal neurohormone) crosses the placental barrier. The cortisol circadian rhythm, as well as the melatonin circadian rhythm in the fetus and infant after birth, are those of maternal cortisol and melatonin. Indeed, the infant's circadian cortisol and melatonin rhythms are only established between 2 and 3 months of age in typical development, at the same time that infants begin to have more regular sleep–wake cycles associated with nighttime sleep lasting 6–8 h (41, 42). Interestingly, this period coincides in typical development with the emergence of social smiling by the second month of life (43), the advent of mirror self-recognition at around 3 months of age (44), and increased brain activation to speech occurring between 3 and 4 months of age (45).

At birth, the human immaturity of the cerebral cortex allows initial learning to influence the neural architecture through perceptual-action mapping (46, 47). The infant's social skills, especially including imitation, shared attention, and empathic understanding, also contribute to the development of learning (46, 48). Social synchrony can be defined as the dynamic and reciprocal adaptation of the temporal structure of behaviors between interactive partners (49). In typically developing children, the quality of social interaction depends on an active dialog between the parent and the infant (50, 51). Numerous studies have been emphasizing the importance of parent–infant synchrony and the construction of shared timing in social communication development (52).

Also, biological markers were associated with relational synchrony. First, oxytocin administration to parent enhances infant physiological and behavioral readiness for social engagement and parallels an oxytocin increase in infants (32). Second, neural correlates were found using hyper-scanning recordings of EEG brain activity and measures of neural synchronization between distant brain regions of interacting individuals through a free imitation task (53). Dumas and colleagues' study (53) was the first to record dual EEG activity in dyads of subjects during spontaneous non-verbal interaction. Eleven same-sex pairs were scanned. They found that interpersonal rhythmic oscillations were correlated with the emergence of synchronization in the brain's alpha–mu band between the right centro-parietal regions (an area involved in social interaction) of both participants. Developmental psychologists now study interaction not only as the addition of two behaviors but also as a global phenomenon in which synchrony is considered as social *per se*. To describe the dialog between two partners engaged in behavioral and affective exchange, developmental psychologists more and more take into consideration rhythm and temporal course of both behavior and affect, regarded as key expressions of adaptation during interaction (49, 52).

Only a few studies have addressed the importance of infant– caregiver synchrony/reciprocity in the development of social communication involving infants who subsequently are diagnosed with autism. It appears appropriate in the field of autism to consider the combined domain of social communication, as the most recent version of the ADOS scale does, as well as the recently released DSM-5 American classification. Methods to investigate this issue include studies using early home videos (54), parental interviews focusing on early abnormalities (55), and prospective assessment of children at risk of ASD (e.g., siblings) (56, 57). Studies have revealed a pervasive developmental course in infants who were later diagnosed with ASD. Thus, the first signs were abnormalities in eye contact, imitation, disengagement, joint attention, orienting to name, and body language. These behaviors are parts of the affective tuning disturbances; the term affective tuning, defined by Stern (58) as the "execution of behaviors expressing the emotional ownership of a shared affective state," refers to infant– caregiver emotional communication based on rhythmic similarity from the second semester of life onward. Also, these behaviors are important precursors of later-developing symptoms. However, whether these first signs impair early infant–parent interactions and whether they reflect already autistic behavioral impairments in the infant remain to be explored. In two related studies based on home movies of children later diagnosed with autism, Saint-Georges et al. (54) and Cohen et al. (59) showed that motor and emotional asynchrony was present between infants and parents before 12 months of age, and parents perceived weaker initiatives from their children. In addition, parents experienced weaker interactive responsiveness from their children and increasingly tried to compensate this perceived deficit by soliciting behaviors through touching the infant. This was particularly observed after 12 months of age for the fathers of infants who were later diagnosed with autism.

Many authors have studied imitation in children with autism (imitation of other people's faces, gestures, or vocal signals) in order to better understand the development of autistic social communication impairments. This specific type of imitation is referred to as "spatial" imitation to highlight the capacity to produce an instantaneous copy of the form of the signal. However, another way to communicate with others is to perform a "temporal" imitation of their behavior (60). This is what humans do through rhythmic finger or foot taping, dancing, singing, and drumming in synchrony with others (61). Xavier et al. (62) highlighted the importance of rhythmicity and synchrony in the development of children's imitative exchanges with peers. From birth, a child has a predisposition to engage, intersubjectively, with the rhytmic actions and awareness of other persons, and to move in synchrony with them (44, 63). Synchronic imitation is an important preverbal way to communicate among peers (64, 65). This reciprocal experience concerns two children able and motivated to coordinate their behavior with the non-ritualized behavior of the other, in both form and timing and to alternate turns between model and imitator (66). The impression of fluidity in the coordination of movements between partners is underlined by mutual attention, engagement, continuous adaptation, and turn taking (67). This rhythmic process made of ludic spontaneous imitation reveals moments of discontinuity occurring in a background of continuity. Neural bases of this coupling activity are constituted by the neuron mirrors system (68), with the same evidence showing that the neuronal structures involved when a mental state is experienced, are also recruited during the observation of others. Guionnet et al. (69) designed a free imitation paradigm in an fMRI study to examine some neural correlates of social interaction. Their results agree with those previously evidenced (70, 71) concerning the core circuit of imitation, but they found different activations between the situation of imitating and that of being imitated.

A special quality of temporal imitation is the ability to use different motor movements in order to communicate. Thus, simple finger tapping can be synchronized with another's head nodding or trunk movements, whether entrained by one of the movements or in response to external synchronizing stimuli such as music. Although animals and humans can perceive rhythms and produce rhythmic motor patterns, only humans can adapt their rhythmic movements to external rhythms (72) [with the exception of the cockatoo (73)]. The ability to be rhythmically synchronized with the environment appears important for infant development in the emotional, cognitive, social, and sensorimotor realms (44, 74). It has been demonstrated that the human fetus and newborn already have the capacity to perceive and produce rhythms (75). The ability to produce temporally adapted motor patterns comes later and depends on the specific motor system involved and the relationship between the beat presented and the spontaneously occurring motor tempo of the infant (76, 77). It should be fruitful to longitudinally examine children with autism in terms of ability to adapt their own rhythm to external rhythms. Interestingly, clinical observations suggest that some children with autism are able to respond to an external rhythmic vocalization by a similar rhythmic motor pattern such as hand flapping (Tordjman, personal communication). However, previous studies reported disorganized rhythms, stereotypies, and poor synchrony in most of these children (78), which might be related to the low melatonin levels reported to be associated in autism with the severity of verbal communication and social imitative play impairments (18, 23). Melatonin, as a regulator of physiological rhythms and oscillations, might enhance the capacity of children with ASD to synchronize their movements with movements of others (this synchronization of movements is needed for imitative play) and with external rhythmic auditory stimuli (such as music and/or human voice enhancing their verbal skills). Interestingly, in a study of social smiling in infants (79), there was no difference in frequency of smiling between 2- and 5-month-old infants with and without ASD during infant–caregiver face-to-face interactions. However, whereas typically developing infants showed a significant increase

in smiling rate when caregivers were smiling, smiling in infants later diagnosed with ASD was not synchronized with smiling in caregivers and was not contingent upon caregiver behavior (caregiver facial expressions and vocalizations). Furthermore, it is noteworthy that, as previously indicated in this section, the infant's melatonin circadian rhythm is established between 2 and 3 months of age, and a study (80) reported that eye contact was normal in 2-month-old infants later diagnosed with autism but declined between 2 and 6 months of age, suggesting an additional argument in favor of a possible relationship between the wellreplicated nocturnal melatonin deficit in autism (see **Table 1**) and the development of autistic social communication impairments.

#### **REPETITIVE BEHAVIORS AND INTERESTS**

Repetitive behaviors and interests are defined, according to DSM-5 criteria (81), as a repetition of identical sequences of behaviors for motor stereotypies (motor stereotypies involve repetitive maladaptive movements) or thoughts for restrictive patterns of interests (restrictive and repetitive interests involve fixated interests, adherence to routines, or ritualized and rigid thinking patterns). Thus,repetitive behaviors and interests can be viewed as behavioral responses to the need to create discontinuity that is repeated at regular intervals, which could have been fundamentally lacking in the physiological development of children with autism due to the melatonin deficit reported in autism. Our finding (18) observed in a sample of 43 adolescents and young adults with autism (nocturnal excretion of 6-SM was significantly negatively correlated with repetitive use of objects), taken together with improvement of stereotyped behaviors following administration of melatonin in 24 children and adolescents with ASD (82), supports this hypothesis.

The autistic deficit in melatonin secretion might lead to physiological rhythm disturbances in autism impairing biological circadian rhythms and even, in certain cases, to an "endless" physiological continuity provoked by the absence of variation in melatonin levels. From this perspective, stereotyped behaviors and interests can be seen as offering to children with autism rhythmic forms providing rhythmicity and discontinuity through the creation of repeated identical patterns. Albert Goldbeter (83), director of the Chronobiology Unit at the Brussels Sciences University, underlines that life is rhythm. It is noteworthy that the word pineal comes from pine cone that is a Mesopotamian symbol representing the source of life and the power of regeneration. In Asia, the pineal gland is considered as the 7th chakra and represents the location of the soul and the highest level of consciousness.

Donald Winnicott (84) emphasized that "the main problem for a typically developing child is to be able to create a continuum out of discontinuity." According to him, the first optimal container ("holding environment") for the newborn is the progressive internalization of the rhythmic structures of feeding (rhythmic ebb and flow corresponding to the kinesthetic experience of suckling), providing a sense of continuing existence. We can nevertheless hypothesize, as we did in the previous section and in a prior article (26), that this internalization process starts far earlier, in the womb, through maternal physiological rhythms; conversely, based on clinical observations in autism, children with autism create discontinuity out of continuity. We can also state the hypothesis that children with autism, confronted with an"endless" physiological continuity (or at least with an absence of physiological discontinuities repeated at regular intervals), would develop anxiety and stress due to the lack of secure stable rhythms, and would therefore try to create by themselves rhythmicity in order to control them through stereotyped behaviors. However, the relationships between stereotyped behaviors and stress responses or anxiety need to be clarified given discrepant results reported in some studies (85, 86).

Finally, it should be noted that several studies have also recently opened new behavioral, neurobiological, and pharmacological perspectives on autistic repetitive behaviors, especially on selfinjurious behaviors [for a review, see Adler et al. (87), Minshawi et al. (88), and Stigler (89)]. Thus, research on potential therapeutic effects for repetitive behaviors and interests should investigate benefits of melatonin in ASD (see next section) but should not be limited to the administration of melatonin.

#### **THERAPEUTIC BENEFITS OF MELATONIN IN AUTISM SPECTRUM DISORDER**

Published melatonin treatment studies in autism are presented in **Table 2**. Many have been most concerned with effects of melatonin on sleep. Reviews, commentaries, and meta analyses of melatonin treatment studies are presented in **Table 3**. These two tables come from a prior article of our team (26).

As previously discussed (26), the treatment studies have a number of limitations. In some studies individuals covering a wide age range were included with pre-pubertal, pubertal, and post-pubertal individuals being studied (102, 103, 105). Reported differences in pineal melatonin secretion according to age and pubertal stage, coupled with the clearly developmental nature of autism, suggests that therapeutic effects of melatonin might vary considerably with age and pubertal status (23). Studies have also been limited by small sample sizes (90–93, 95, 96, 98, 102, 104). In larger studies, heterogeneous groups are often examined and have included blind individuals and individuals with various neurological disabilities with concomitant intellectual disability. In such studies, often results seen for the autism subgroup are not separately presented (97, 100, 103–107). The specificity and interpretation of the results with respect to autism are often unclear. Additional research is needed to determine whether and which findings might be specific to autism or whether melatonin's effect might be similar across groups. It can be hypothesized that melatonin's effects are due to actions on certain behavioral dimensions and that they can be observed across disorders. However, it should be pointed out that in our studies (18, 23), lower melatonin excretion was significantly associated with social communication impairments rather than with sleep problems. Future studies of melatonin in ASD should simultaneously examine melatonin levels, sleep problems, autistic behavioral impairments, and level of functioning so that a more complete picture can emerge.

It is worth noting that just a few of the therapeutic trials of melatonin have assessed effects on autistic behavioral impairments. These include reports of improved communication (105), reduced social withdrawal (82, 99), decreased stereotyped behaviors and rigidity (82, 102), and reduced anxiety (99, 103). Furthermore, at time the improvements noted were not sufficiently detailed. For example, Wright et al. (105) reported significant

**Table 2 | Studies on potential**

 **therapeutic**

 **benefits of melatonin**

 **in autism**.

**Table 2 | Continued**

similar between the 2 groups

**Table 2 | Continued**

communication improvement without reporting separate verbal or non-verbal scores. In other instances, investigators did not use validated instruments; thus, Garstang and Wallis (102) reported improvements in rigidity based solely on parents' and teachers' comments, while Wasdell et al. (103) reported reductions in anxiety based on caregivers' comments. All of the published clinical trials of melatonin in autism/ASD have used sleep measures as their main outcome variable and none have studied the dose– response relationship for melatonin. To reiterate, there is a real need for further studies on the therapeutic effects of melatonin that are conducted on large, relatively homogeneous samples and that employ validated behavioral assessments.

#### **SYNCHRONIZATION OF MOTOR, EMOTIONAL, AND RELATIONAL RHYTHMS: THERAPEUTIC BENEFITS OF THE EARLY START DENVER MODEL IN AUTISM SPECTRUM DISORDER**

Although there is a growing interest in the role of motor, emotional, and interpersonal rhythms in autism, there have been only a few attempts to focus on behavioral synchronization of social communication during therapeutic and educational intervention in autism. The ESDM, developed by Sally Rogers and Geraldine Dawson (119, 120), is a comprehensive behavioral and developmental early intervention approach designed for delivery to 12- to 60-month-old children. The model uses the knowledge of how typical infants, toddlers, and pre-schoolers develop, in addition to knowledge of the ways in which autism affects early development, in order to facilitate an appropriate developmental trajectory in young children with autism beginning at the earliest ages. The goals of the ESDM are to reduce the severity of disabling autism symptoms in very young children, and to accelerate children's developmental rates in a multitude of domains, including cognitive, social–emotional, and language domains. In order to do so, there is a strong focus on interpersonal engagement marked by synchrony and reciprocity during teaching, as research demonstrates that these are closely tied to successful developmental outcomes (121–124). Within the ESDM, the therapist and child work together to develop and sustain coordinated, synchronous activity routines into which teaching opportunities are embedded. The ESDM reflects an understanding of the importance of synchrony of motor, emotional, and relational rhythms for the early development of young children with autism.

Thus, the ESDM allows one to initiate interaction with the child by synchronizing the therapist's motor, emotional, and relational rhythms with the child's rhythms (providing invariants/continuity). For example, if the child uses an object with a rhythmic motor pattern, the therapist will reproduce this behavior with exactly the same rhythmic pattern. The evolution of the child is then facilitated by introducing new and different rhythms (providing variants/discontinuity). In addition, the adjustment of the therapist's behaviors, movements, level of arousal, and posture (for example, standing up, lying down, or sitting) to those of the child contributes to foundational invariants necessary during the initial phase. Furthermore, the ESDM requires the program to be implemented in different environments (such as home, school, and caregiver settings), which also contributes to the introduction of variants.

#### **Table 3 | Review, meta-analysis, and discussion of therapeutic uses of melatonin in autism**.

(Continued)

#### **Table 3 | Continued**

The ESDM is both a curriculum and a set of teaching practices. A specific developmental curriculum, administered every 3 months in typical practice, defines the skills to be taught at any given time. In addition, a manual of teaching practices outlines the ways in which these skills are to be taught. Embedded within both the curriculum and the set of teaching practices is a focus on rhythms and synchrony. In other words, when therapists are working within the model, a focus on rhythms permeates both what they teach and how they teach it.

The ESDM curriculum outlines skills to be taught in multiple areas, including cognitive, social–emotional, and language domains. The curriculum focuses heavily upon the teaching of skills that promote engagement with other people in a synchronous, rhythmic way. Many young children with autism enter an ESDM program with weaknesses in these skills, such as imitation, joint attention, orienting to name, and eye contact, and these have broad influences upon the manner in which these children can engage with others. It is well recognized that learning occurs within a social context and that social skills, such as imitation and shared attention, provide a foundation for many aspects of learning, including language, cognitive, and social–emotional abilities. Thus, the ESDM focuses heavily on the development of social engagement and interactional synchrony early on in order to further support learning of a wide range of skills.

One area of focus within the ESDM, which is closely tied to the concept of rhythmicity, is imitation. Young children with autism often exhibit deficits in their imitation skills, including those of imitating others' facial expressions and movements, gestures, body actions, and actions on objects. From the beginning of a young child's ESDM program, teaching of imitation is stressed, from basic imitation of actions on objects to more nuanced imitation of sound effects produced in play. Spontaneous and appropriate imitation of others is typically rhythmic and marked by mutual attention, continuous adaptation, and turn taking. The beginning stages of facilitating imitation and social engagement often start with imitating the child, thereby entering into a rhythmic interaction with the child. By imitating the child's movements and establishing a synchronous interaction, eye contact and mutual engagement in the interaction are promoted (125).

Rhythmicity is also involved in the way teaching occurs within the ESDM upon multiple levels, from the basic structure of each interactive routine to the ways in which adults engage with children during interaction. Social communication is about sharing moments of synchrony with others, and the ESDM supports the emergence of interactional synchrony. The therapist facilitates the occurrence of synchronous moments and strings them together into routines into which the therapist can naturally embed teaching opportunities. The primary vehicle through which all teaching is accomplished in this model is the joint activity routine, which is permeated by moments of rhythmic, synchronous interaction, and enriched with positive affect. Adult and child are attuned to one another, both taking the lead, both following the other's lead, taking turns, and creating a positive and motivating activity in conjunction with one another.

The joint activity routine, although flexible and naturalistic, adheres to a four-part structure: (1) opening, (2) theme, (3) elaboration, and (4) closing. This structure provides a set of invariants, against which multiple variants can occur at differing levels. In order to begin engagement within a joint activity routine, the adult works hard to *find the child's smile*. This can often be accomplished by getting into the child's own rhythm (imitating a child's actions is a very powerful tool to facilitate motivated response and interaction) or by finding a rhythm that the child likes. For example, perhaps a child finds a toy drum and begins banging on it with his hand. In the ESDM, the therapist would join the child and would likely take her/his own drum and imitate the child's actions, joining his rhythm. This would be considered the opening phase of the joint activity routine.

Next, the child and adult would develop a theme. This can be considered a continuation of the rhythm introduced during the opening phase – in our example, banging a drum slowly with hands. The adult and child would take turns doing so, each leading and each following. The skillful adult would embed teaching opportunities into these turns – perhaps a focus on eye contact during dyadic engagement, or on imitation of actions on objects, or on giving and taking objects with eye contact. Both partners then play within this rhythm for a while, until one introduces a variant, or elaboration. In the ESDM, elaboration occurs by introducing a change into the joint activity routine, a change in the rhythm which often allows different teaching targets to be practiced. In this example, the child and adult might start to play peek-a-boo behind the drums. A new rhythm would need to be established, and both partners would work together in order to do so. An elaboration of this sort is a rhythmic fluctuation occurring against a background of invariants (e.g., the same interactive partner, the same material, the same setting), which allows the child to learn to adapt to change and teaches the child how to engage with people and materials differently. Finally, this rhythm would likely begin to fade, the teaching value of the activity would start to diminish, and/or the child would begin to lose motivation. The fourth stage of the joint activity routine – the closing – is the last part of the joint activity routine. Adult and child might help one another clean up the materials, and then both partners would begin an entirely new joint activity routine. This could often involve a change in location, activity level, or teaching domain, and an entirely new set of coordinated interactions would be developed.

Within the joint activity routine, synchrony pervades within several different levels and in several different areas. Adult and child are attuned to one another and responsive to one another in terms of sensory input and output, motor actions, and emotions. Coordinated dyadic engagement, well-balanced in terms of leader and follower, pervades a high-quality joint activity routine. It is quite natural to view this ever-present synchrony in terms of a focus on shared nuanced rhythms.

There is also a focus on rhythmicity within some of the broader teaching practices of the ESDM; for example, in the way in which a teaching session is constructed. As mentioned above, teaching is delivered within joint activity routines. For a therapist-delivered session, these routines are strung together within a session to occupy a full 1- or 2-h time period. Within that time period, therapist and child move around to occupy several spaces. In a well-coordinated way, they engage together in a joint activity routine at the table, and then may move onto the floor for the next. They may sit together, then they may stand or walk or run. The child's level of arousal is carefully monitored, and the session is marked by alternations of quiet thoughtfulness and active play, all coordinated artfully by a skilled adult. All of these changes occur against a stable background, as the session is marked by stability and continuity, with the introduction of variants when appropriate. The ESDM, a comprehensive, behavioral, and developmental early intervention designed for infants and toddlers with autism, is rooted within a sense of and appreciation for rhythmicity and synchrony at multiple levels, ranging from the specific skills that are taught to how those skills are taught, woven together and supported by a broader structure focused on maintaining a well-coordinated, synchronous set of dyadic interactions.

#### **CONCLUSION: TOWARD AN INTEGRATIVE APPROACH COMBINING THE USE OF MELATONIN WITH THE ESDM**

Taken all together, ASD could be seen as a disorder of rhythmicity with, more specifically, impairments in the synchrony of rhythms. Alternatively, such asynchrony might play an important role in a possibly large subgroup of individuals that forms part of the heterogeneous ASD category. In this article, we proposed an integrative approach to study desynchronization in biological and psychological rhythms in ASD and develop an etiopathogenic hypothesis as well as therapeutic perspectives for ASD based on this integrative approach. Indeed, this integrated physiological and psychological approach opens important therapeutic perspectives for ASD based on regulation of physiological rhythms (in particular, through the use of chronobiotics such as melatonin, and also through light exposure, use of regularly scheduled bedtime, wake up, meals, or activities) combined with synchronization of motor, emotional, and relational rhythms through developmental behavioral intervention such as the ESDM. Further studies are required to better ascertain the underlying mechanisms of physiological alterations induced by temporal desynchronization and to better understand the role of biological rhythms and rhythmicity in the development of social communication, repetitive behaviors, and interests or adaptation to changes, and therefore in the development of autism involving impairments in these domains.

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