The Supplementation Therapy in Autism and Response to Treatment Study

1. RATIONALE FOR CURRENT STUDY

The main purpose of the present study is to address a major unmet clinical need for Level 1 autistic individuals with comorbid anxiety and/or depressive symptoms, with respect to a condition of psychic vulnerability not responding to conventional psychopharmacological approaches. We will thus perform an investigator-initiated proof-of-concept study (Phase-2 Pilot Study), with the purpose to examine:

(i) Palmitoylethanolamide (PEA) ability to alleviate symptoms of psychic distress (i.e., anxiety and/or depression) in Level 1 autistic individuals; (ii) PEA safety and tolerability; (iii) The biological basis of PEA effect. 2. TRIAL OBJECTIVES

To evaluate:

(i) The viability of identifying and consenting Level 1 autistic volunteers into a trial with PEA; (ii) The efficacy of PEA in providing relief to anxiety and/or depressive symptoms in Level 1 autistic individuals; (iii) Whether sustained PEA treatment is well tolerated by Level 1 autistic individuals over a period of at least 12 weeks; (iv) The biological mechanisms underpinning PEA beneficial effects in Level 1 autistic individuals (e.g., modulation of the endocannabinoid (eCB) system, immunological response, metabolic fingerprinting, gut microbiome composition).

2.1. Objective (i) Feasibility questions

Over the first 12 months from first patient recruited we will assess whether:

(i) A minimum of 20 eligible volunteers have consented to be enrolled into the study; (ii) At least 80% of recruited patients have completed the 12-week follow-up; Feasibility endpoints (i) Number of subjects giving consent; (ii) Proportion of participants completing the 12-week follow-up.

2.2. Objective (ii) Research Questions

Our primary clinical research question is whether PEA added to treatment as usual (TAU) in Level 1 autistic individuals:

(i) Improves psychic distress (i.e., anxiety and depressive symptoms) to the extent of impacting on levels of self-sufficiency;

Our secondary clinical research questions are whether PEA added to TAU in Level 1 autistic individuals:

(ii) Reduces the intensity of anxiety and/or depressive symptoms, with particular regards to the subdomains of somatisation, anxiety and depression; (iii) Improves interpersonal and neurocognitive functioning. Primary endpoints Assessing the global improvement in symptom intensity and psychological distress associated with autism through the Global Score Index (GSI) of the SCL-90-R.

Secondary endpoints (i) Impact on levels of personal autonomy as measured through the WHODAS 2.0 total score.

(ii) Severity of anxiety symptoms as assessed using the Hospital Anxiety and Depression Scale (HADS) and the somatisation, anxiety and depression subscales of the SCL-90-R; (iii) Severity of neurocognitive and interpersonal functioning as assessed using the subdomains 1 'cognitive activities' and 4 'interpersonal relationships' of the WHODAS 2.0.

All the study endpoints for the 12-week clinical trial will be assessed by comparing follow-up (FUP) visits and baseline. For those participants continuing in the 24-week extension phase, change (FUP visit minus baseline) in symptom intensity and psychological distress associated with autism as measured through the GSI of the SCL-90-R and the impact on levels of personal autonomy measured through the WHODAS 2.0 total score will be compared with the group of those willing to discontinue PEA and continue with TAU.

2.3. Objective (iii) Safety questions We will evaluate if sustained PEA treatment is well tolerated, with minimal side-effects.

Safety endpoints Incidence of adverse effects during the study period, as measured using the UKU side-effect rating scale. 3. TRIAL DESIGN We will evaluate the study viability through an internal pilot that will assess the ability of the study to identify, consent, and follow up Level 1 autistic volunteers experiencing anxiety and/or depressive symptoms in the study. We propose a 12-week, open-label, investigator-initiated proof of concept study (Phase-2 Pilot Study) of PEA (600 mg/day) for the treatment of psychic distress (i.e., sub-threshold anxiety and/or depressive symptoms) in Level 1 autistic individuals. We plan to enrol 20 young adults diagnosed with Autism Spectrum Disorder (ASD). Those completing the 12-week initial phase, will be proposed to enter a 24-week extension phase to evaluate the clinical stability of treatment, with the possibility of PEA titration to 1200 mg/day based on clinical improvement obtained so far. 4. PARTICIPANTS 4.1. Selection of participants The proposed single-centre study will involve a university clinical research facility in Italy. Participants will be enrolled into the internal pilot, that will progress to the open-label trial. Volunteers who express an interest in the study and are identified as having Level 1 autistic individuals with comorbid anxiety and/or depressive symptoms by their clinical teams will be approached by study researchers and given a patient information sheet. Those who agree to take part in the study will be invited for a screening visit.

4.2. Heterogeneity in autism spectrum disorder ASD comprises three subgroups of patients based on the severity of symptoms whose classification is based on impairment of social communication and restricted, repetitive behaviour patterns: (i) Level 1 'Support is needed', (ii) Level 2 "Significant support is needed", (iii) Level 3 "Very significant support is needed very significant support'. As the present study was designed to assess the effect of PEA on mentally fragile conditions with interference in the global functioning of autistic individuals, we will focus on subjects with severity level 1, more affected by depressive and anxious symptoms than autistic subjects with higher levels of impairment, requiring a prioritisation of these manifestations compared to core symptoms. Symptoms of psychological distress in ASD individuals, below the threshold of clinical criteria for the diagnosis of a full-blown psychiatric disorder and not requiring the introduction of conventional psychotropic medications, are nevertheless often deserving of an intervention to support their well-being. 5. INTERVENTION 5.1. Trial Medication Oral Palmitoylethanolamide (PEA; 600 mg per day) in tablet form. PEA will be obtained by a pharmaceutical company operating under good manufacturing practice conditions with appropriate certification. The information presented on the labels for PEA will comply with applicable national and local regulations.

5.2. Dosing Regimen PEA is to be taken orally once a day (600 mg per day) around mealtime during the 12-week initial phase of the study. During the 24-week extension phase of the study, the trial medication is to be taken from once a day up to twice a day (600-1200 mg per day), based on clinical judgment of the improvement obtained so far, around mealtime. Each participant will undergo PEA treatment for a maximum of 36 weeks. The information presented on the labels for the PEA will comply with applicable national and local regulations.

5.3. Medication Risks Being a food supplement/nutraceutical, PEA can be purchased at pharmacies without a medical prescription. While unknown risks cannot be excluded, serious adverse events including overdose have not been documented.

5.4. Drug Accountability Study specific prescriptions can be used for dispensing the study product. The study medication can only be prescribed by qualified physicians clearly given this role on the study delegation log. Only people designated by the Principal Investigator (PI) can collect medication. Only PEA supplied for this study can be dispensed against the study specific prescription. Full accountability records will be completed including recording the batch, expiry date, people dispensing/checking the prescription, quantity and date of drug returns, empty packaging. Nothing is destroyed without the authorization from the PI.

5.5. Storage of study medication PEA will be stored at room temperature (\< 25 °C) and not kept in a refrigerator, in compliance with local regulations. It will be stored in a secure area away from other treatments and clearly marked for this study.

Removal of study medication outside of expiry date/at trial conclusion and destruction 5.6. The expiry date will always be reported on the study medication. Study medication outside of the expiry date will not be dispensed and will be destroyed, subject to authorization, on an ongoing basis.

5.7. Withdrawal of Subjects According to Declaration of Helsinki, all participants will have the right to withdraw from the study at any time without giving any reason, without any prejudice to their future medical care, and will be informed as such before consent. A participant's withdrawal will be discussed in terms of only discontinuing the study treatment and continuing follow-up visits. Should the patient request to completely withdraw from the study, the decision will be respected. All reasonable efforts will be made to ascertain the reason for discontinuation, even though participants will be not obliged to give any explanations. Already collected data will be kept and included in the final analysis. The investigator themselves may also withdraw participants for various reasons, including but not limited to the following: protocol violations, inter-current illness, adverse events, serious adverse events, suspected unexpected serious adverse reactions, administrative reasons, participation in the trial affecting their ongoing care, symptomatic worsening. In the latter case, participants will be followed up with the same schedule of research assessments as those who continue in the study, till they complete the 12-week follow-up period or till they progress to frank psychiatric disturbance (whichever is earlier). In case of autistic individuals experiencing progression to a full-blown psychiatric disorder, they will exit from the study intervention, be deemed as treatment failure, and will only be assessed for safety outcomes until they complete the 12-week follow-up period.

5.8. Subject Compliance Pill-counts will be performed at FUP visits 1, 2, 3, and 4, in order to assess compliance with PEA treatment. Participants will be defined as complying in the presence of a pill count greater than 50% the expected number taken. Participants who are defined as non-complying with the medication will be coded as protocol deviators.

5.9. Concomitant Medication Based on participants' clinical history, concomitant requirement of psychotropic medication is an exclusion criterion for the study, with the exception of patients undergoing Selective Serotonin Reuptake Inhibitor (SSRI) stable monotherapy (at least 8 months). Patients requiring continued treatment with other classes of psychotropic medications during the treatment phase may be withdrawn from the study by the PI. Very short-term treatment with rescue medications that have a well-established sedative or calming effect (e.g., Benzodiazepines) during the study may be allowed. Throughout the study, any other concomitant medications or treatments deemed necessary to provide adequate supportive care may be prescribed by investigators. A record will be kept to list all concomitant medications received during the treatment phase. 6. VISIT ASSESSMENTS

The following visit assessments will be performed:

6.1. First Screening Visit This will take place approximately one week prior to the Second Screening Visit and two to three weeks prior to the Baseline Visit. First, informed consent will be obtained from those who wish to take part to the study. Consenting patients will then be screened against the study inclusion and exclusion criteria and collecting information on their medical history. Individuals satisfying the criteria will be recruited by employed or delegated investigators. Also, consent w

• World Health Organization Disability Assessment Schedule 2.0 [Time frame: 12 weeks for the initial phase, further 24 weeks for the extension phase]
• Hospital Anxiety and Depression Scale [Time frame: 12 weeks for the initial phase, further 24 weeks for the extension phase]
• Symptom-Checklist-90-R [Time frame: 12 weeks for the initial phase, further 24 weeks for the extension phase]
• World Health Organization Disability Assessment Schedule 2.0 [Time frame: 12 weeks for the initial phase, further 24 weeks for the extension phase]