Arteaga-Henríquez et al. Trials (2020) 21:161 https://doi.org/10.1186/s13063-019-4040-x STUDY PROTOCOL Open Access Treating impulsivity with probiotics in adults (PROBIA): study protocol of a multicenter, double-blind, randomized, placebo-controlled trial Gara Arteaga-Henríquez1 , Silvia Karina Rosales-Ortiz1, Alejandro Arias-Vásquez2,3, Istvan Bitter4, Ylva Ginsberg5, Pol Ibañez-Jimenez1, Tünde Kilencz4, Catharina Lavebratt6,7, Silke Matura8, Andreas Reif8, Janos Rethelyi4, Vanesa Richarte1,9, Nanda Rommelse3,10, Anne Siegl8 and J. Antoni Ramos-Quiroga1,9,11,12* Abstract Background: Impulsivity and compulsivity are related to emotional and social maladjustment and often underlie psychiatric disorders. Recently, alterations in microbiota composition have been shown to have implications for brain development and social behavior via the microbiota–gut–brain axis. However, the exact mechanisms are not fully identified. Recent evidence suggests the modulatory effect of synbiotics on gut microbiota and the contribution of these agents in ameliorating symptoms of many psychiatric diseases. To date, no randomized controlled trial has been performed to establish the feasibility and efficacy of this intervention targeting the reduction of impulsivity and compulsivity. We hypothesize that supplementation with synbiotics may be an effective treatment in adults with high levels of impulsivity and/or compulsivity. Methods/design: This is a prospective, multicenter, double-blind, randomized controlled trial with two arms: treatment with a synbiotic formula versus placebo treatment. The primary outcome is the response rate at the end of the placebo- controlled phase (response defined as a Clinical Global Impression–Improvement Scale score of 1 or 2 = very much improved or much improved, plus a reduction in the Affective Reactivity Index total score of at least 30% compared with baseline). A total of 180 participants with highly impulsive behavior and a diagnosis of attention deficit/hyperactivity disorder (ADHD) and/or borderline personality disorder, aged 18–65 years old, will be screened at three study centers. Secondary outcome measures, including changes in general psychopathology, ADHD symptoms, neurocognitive function, somatic parameters, physical activity, nutritional intake, and health-related quality of life, will be explored at assessments before, during, and at the end of the intervention. The effect of the intervention on genetics, microbiota, and several blood biomarkers will also be assessed. Gastrointestinal symptoms and somatic complaints will additionally be explored at 1-week follow-up. Discussion: This is the first randomized controlled trial to determine the effects of supplementation with synbiotics on reducing impulsive and compulsive behavior. This clinical trial can contribute to explaining the mechanisms involved in the crosstalk between the intestinal microbiome and the brain. If effects can be established by reducing impulsive and compulsive behavior, new cost-effective treatments might become available to these patients. (Continued on next page) * Correspondence: jaramos@vhebron.net 1 Psychiatric Genetics Unit, Vall d’Hebron Research Institute (VHIR), Barcelona, Catalonia, Spain 9 Department of Psychiatry, Hospital Universitari Vall d’Hebron, Barcelona, Catalonia, Spain Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Arteaga-Henríquez et al. Trials (2020) 21:161 Page 2 of 12 (Continued from previous page) Trial registration: ClinicalTrials.gov, NCT03495375. Registered on 26 February 2018. Keywords: Impulsivity, Compulsivity, Aggression, Microbiome, Probiotics, Synbiotics, ADHD, Borderline personality disorder, Nutrition Background Importantly, the type of food intake can influence Impulsivity may be defined as “a predisposition towards brain development and function in all age groups, with a rapid, unplanned reactions to internal or external stimuli, recent study showing that prenatal exposure to an un- with diminished regard to the negative consequences that healthy diet was associated with ADHD symptoms, fur- such reactions may have for the impulsive individual or ther linked to altered epigenetic modification of blood- others” [1–4]. In contrast, compulsivity represents “the per- derived DNA [17, 18]. However, these findings have not formance of repetitive and functionally impairing overt or been consistently replicated in large-sample randomized covert behavior without an adaptive function, performed in controlled trials (RCTs), and the underlying mechanisms a habitual or stereotyped fashion, either according to rigid remain unknown [19–23]. rules or as a means of avoiding perceived negative conse- quences” [2, 5]. Both traits share neuronal mechanisms in- volving a dysfunctional inhibition of thoughts and behavior Gut microbiome, microbiota, and behavior [1, 6], and, rather than unitary phenomena, they are consid- Although microbiota refers to the specific microorganisms ered as multidimensional constructs that involve disruption that are found within a specific environment, microbiome within a range of neural processes, including attention, refers to the collection of genomes from all the microor- perception, and coordination of motor and/or cognitive ganisms found in this particular environment [24]. It is hy- processes. Impulsive and compulsive symptoms are over- pothesized that an imbalance in the gut microbiota represented in individuals with several psychiatric disorders, (dysbiosis) may have a negative effect on neurodevelop- such as attention-deficit/hyperactivity disorder (ADHD), ment, behavior, and cognition [25–31]. Related to this, borderline personality disorder (BPD), and/or obsessive- changes in human microbiome and/or microbiota compos- compulsive disorder [7]. Importantly, maladaptive impulsiv- ition have been consistently found in individuals with aut- ity and compulsivity can lead to serious consequences not ism spectrum disorder (ASD) [32–36], and a pilot study on only for affected individuals and their families but also for the microbiome has demonstrated, for the first time, a dif- society. They predispose individuals to aggressive or addict- ference in several bacterial taxa between subjects with ive behaviors, increasing the risk for mortality [8]. Despite ADHD and healthy control subjects [37]. Specifically, lower this, data about modifiable risk and protective factors are Firmicutes genus and higher Bifidobacterium genus were largely lacking. found in subjects with ADHD than in healthy individuals, with the increase in the Bifidobacterium genus relating to decreased ventral striatal functional magnetic resonance Nutrition, brain, and behavior imagining responses during reward anticipation [37]. Not only heritability, sex, and socioeconomic status (SES) The relationship between gut microbiota and the brain but also diet may play a pivotal role in impulsive and com- seems to be bidirectional. The gut microbiota modulates pulsive symptomatology [9, 10]. In line with this, a num- brain function and development, and the brain can alter ber of studies have shown that different nutrient the gut microbiota, allowing colonization by pathogenic combinations may have interactive effects on cognitive bacteria [38–40]. Illustrating this idea, some studies have function and behavior, including antioxidants; omega-3 shown an overrepresentation of gastrointestinal symp- polyunsaturated fatty acids (PUFAs); monounsaturated toms in patients with both neurodevelopmental and fatty acids; polyphenols; potassium; calcium; zinc; fiber; neuropsychiatric disorders [41–48], and a study in folate; and/or vitamins A, B12, C, D, or E [11–13]. More healthy students found that during exams, psychological specifically, recent reports have linked aggressive behavior stress increased at the same time that numbers of lacto- with low blood omega-3 PUFA levels and/or with low sea- bacilli in stool samples decreased [49]. food consumption [14], and several animal studies have But how does this bidirectional communication—the found that the long-term consumption of a low-calorie gut–brain axis—work? The “enteric nervous system” is diet enhances autophagy and protects neurons effectively complex and, regarding neurotransmitters and signaling against aging, maintaining learning and memory capacity, molecules, similar to the brain [50]. As an example, 95% whereas long-term consumption of a high-calorie diet fa- of all circulating serotonin, dopamine, or noradrenaline cilitates neuronal loss in the hippocampus, aggravating precursors are produced by our gut microbiota [50]. age-related cognitive impairments [15, 16]. This system is connected to the central nervous system Arteaga-Henríquez et al. Trials (2020) 21:161 Page 3 of 12 through hormonal or innate neuronal pathways [51] that improve their daily life function. Moreover, we will are critical for its development and vice versa [52]. Fur- evaluate the composition of gut microbiota in this popu- thermore, studies have shown that the gut flora is critic- lation and link it to inflammatory/immunological abnor- ally involved in immunoregulation [53–55], whereas malities that can underlie core symptoms of these some reports have shown the immune system as an im- disorders. portant regulator of neurodevelopment and synaptic function in the brain [56]. In line with this, immune dys- Methods/design function and/or autoimmunity have been speculated to This protocol is presented in accordance with the 2013 be associated with many neuropsychiatric and neurode- SPIRIT (Standard Protocol Items: Recommendations for velopmental disorders, such as ADHD [57, 58]. Support- Interventional Trials) Statement (See Additional file 1 ing this idea, an increased incidence of immune- for the populated SPIRIT Checklist (83). mediated disorders (e.g., asthma, allergic rhinitis, atopic dermatitis, allergic conjunctivitis, psoriasis, thyrotoxi- Trial design and study setting cosis, or type 1 diabetes) has been found among patients The Treatment of impulsivity in adults with probiotics with ADHD [59–62]. Moreover, elevated inflammatory (PROBIA) trial is designed as a 10-week multicenter, pro- markers (especially interleukin [IL]-6) [63–65] or auto- spective, randomized, double-blind, placebo-controlled, antibody levels (e.g., anti–basal ganglia antibodies, anti- parallel design study. It will be performed in close cooper- bodies against the dopamine transporter) [66, 67] have ation between three European clinical centers: Goethe been found both in serum/plasma and in cerebrospinal University Hospital Frankfurt (Department of Psychiatry, fluid of these patients [63, 68, 69]. Psychosomatic Medicine and Psychotherapy), Frankfurt, Germany; Vall d’Hebron Research Institute (Psychiatry, Probiotic/synbiotic interventions Mental Health and Addictions Group), Barcelona, Spain; Probiotic bacteria, or “probiotics,” are live, nonpatho- and Semmelweis University (Department of Psychiatry genic microorganisms that normally inhabit the intes- and Psychotherapy), Budapest, Hungary. These centers tines and contribute to the health of the host by are all affiliated with or part of university hospitals and, improving microbial balance [70]. On the other hand, with around 300 new inpatients and outpatients visiting prebiotics are nondigestible ingredients that selectively each center every year, are considered as reference centers stimulate the growth and activity of these probiotic mi- for the treatment of ADHD, BPD, and other disorders croorganisms [71]. The synergic combination of probio- characterized by high impulsivity levels. tics and prebiotics is referred to as synbiotics. Recent findings suggest that probiotics and/or synbiotics can Study population and recruitment not only revert dysbiosis but also modulate brain activity Participants will be eligible for participation in this study and improve cognition, mood, and behavior [72–78]. if they meet all the inclusion criteria and none of the ex- Importantly, a recent study has shown that oral adminis- clusion criteria listed in Table 1. With the aim of gender tration with Lactobacillus during the first 6 months of balancing the study, at least 30% female participants will life reduced the prevalence of ADHD or ASD at the age be included. of 13 [79]. However, the exact mechanisms by which Recruitment began on 22 February 2019 and will have probiotics and/or synbiotics exert their action remain a duration of 2 years. A total of 180 participants (60 unknown. Recent findings suggest immunomodulatory subjects per site) will be recruited by trained psychia- and anti-inflammatory properties of these agents [80], trists and/or psychologists at the different sites (see Fig. possibly by selectively targeting T-helper (Th) type 1 1). Strategies aimed at optimizing the recruitment [81] and Th17 cell lineages [82]. process will include the distribution of handouts or The aim of the present multicenter, prospective, flyers to colleagues, physicians, and families, as well as double-blind, placebo-controlled, parallel-group study is an offer of reimbursement of study-related travel costs to investigate the effect of a synbiotic formula (Synbiotic to participants. Furthermore, and depending on local 2000 Forte 400; Synbiotic AB, Höganäs, Sweden) on re- regulations, information via presentations, websites, ducing impulsive, compulsive, and aggressive behaviors and social media campaigns will be provided. Regular in a sample of highly impulsive adults with a diagnosis calls will be made to patients to avoid participant with- of attention-deficit/hyperactivity disorder (ADHD) and/ drawal from the study. or borderline personality disorder (BPD). In this study, we will test the idea that supplementation with probio- Randomization and study intervention tics will, by modifying the gut microbiota structure and After eligibility checks have been conducted and writ- metabolism, reduce impulsive, compulsive, and aggres- ten consent has been obtained by the different psychi- sive behaviors in this specific population and thereby atrists and/or psychologists of the trial team, eligible Arteaga-Henríquez et al. Trials (2020) 21:161 Page 4 of 12 Table 1 Inclusion and exclusion criteria of the PROBIA trial Inclusion criteria Both males and females aged 18–65 years A high level of multidimensional impulsivity based on both a Clinical Global Impression–Severity Scale (CGI-S) score ≥ 4 and an Affective Reactivity Index (ARI) ≥ 5 DSM-5 criteria for attention deficit/hyperactivity disorder (ADHD) and/or borderline personality disorder (BPD) confirmed by a structured diagnostic interview (ADHD: Diagnostic Interview for Adult ADHD [DIVA 2.0]; BPD: Structured Clinical Interview for DSM-IV [SCID-II]) Deemed reliable and compliant with the protocol by the investigator Ability to speak and comprehend the native language of the country in which the assessments take place Informed consent signed Exclusion criteria Antibiotherapy within the last 6 weeks prior to study Currently taking probiotics Presence of a major psychiatric disorder with psychotic symptoms or other major psychiatric conditions requiring hospitalization (e.g., significant mood disorders) Neurological disorders involving central functions (e.g., epilepsy, multiple sclerosis, narcolepsy) Intelligence quotient (IQ) < 70 (measured by WAIS, if available) Major physical illnesses of the cardiovascular, endocrine, pulmonal, immune, or gastrointestinal system or undergoing immunosuppression History of/present clinically relevant somatic acute or chronic disorders that, in the opinion of the investigator, might confound the results of tolerability/safety assessments or prohibit the patient from completing the study or would not be in the best interest of the patient Documented allergy, hypersensitivity, or intolerance to any of the ingredients of the intervention Subject has taken another investigational product or taken part in a clinical study within 30 days prior to entering the study. Abbreviations: DSM Diagnostic and Statistical Manual of Mental Disorders, PROBIA Treatment of impulsivity in adults with probiotics, WAIS Wechsler Adult Intelligence Scale participants will be randomized to the experimental However, due to the large sample size, a balanced ra- (EG) or the control (CG) group in a 1:1 allocation ra- tio of baseline characteristics between the EG and CG tio using an independent web-based computerized is expected. service (www.randomization.com). Randomization will Individual participant treatment kits will contain all be center-stratified and have a fixed length per center, required daily synbiotic or placebo envelopes for the 10- being independent of participant characteristics. week RCT and will be prepared in advance by sequentially Fig. 1 Timeline of the PROBIA study Arteaga-Henríquez et al. Trials (2020) 21:161 Page 5 of 12 numbering and labeling them as “A” or “B.” Because this separate scores for inattentive, hyperactive, and impulsive is a double-blind study, neither the participants nor the subscales, providing a better characterization of patients. clinicians involved in the trial will have access to the Changes in general psychopathology, major stressors, randomization list. neurocognitive function, sleep disturbances, body-related Synbiotic 2000 Forte 400 (SF) is a synbiotic formula cur- measures (e.g., pulse rate, blood pressure, body mass rently produced by Synbiotic AB in Höganäs, Sweden. Each index, waist–hip circumference ratio), medications taken, dose (powder-containing sachet) of SF contains 100 billion physical activity (via mobile health application and move- of each of Pediococcus pentosaceus 5-33:3, Lactobacillus ment sensors), health-related quality of life, microbiome paracasei subsp. paracasei 19, Lactobacillus plantarum composition, and blood markers will also be evaluated. 2362, and Leuconostoc mesenteroides 77:1, in combination Blood markers will include genetic parameters, hormones, with four bioactive fermentable fibers (2.5 g each of β- neurotransmitters, nutrients, and inflammatory/anti-in- glucan, inulin, pectin, and resistant starch). SF is catego- flammatory molecules (see Table 2). rized as a generally regarded as safe agent, being tested on Blood samples (34.5 ml) will be fractioned into serum several hundreds of critically ill patients (e.g., pancreatitis, and plasma and stored at − 80 °C. Then, they will be liver transplant) with no side effects or specific and/or rele- shipped to the Department of Human Genetics of Rad- vant risks conferred to the trial participants [83]. The pla- boudumc, Nijmegen, The Netherlands, for storage. There- cebo (also provided by Synbiotic AB) is composed of the after, blood markers will be measured at the Department polysaccharide maltodextrin and has a texture and flavor of Clinical Biochemistry at Radboudumc in Nijmegen ac- similar to SF. Participants will be asked to continue with cording to validated protocols. Participants willing to take their previous medications and/or usual treatments and part in the assessment of their microbiome will receive a add the synbiotic or placebo once daily on top of cold foods specially designed container for feces collection, allowing such as yogurt, muesli, or salad. To secure bacterial viabil- participants to send the samples by ordinary post to the ity, participants will be asked to store both SF and placebo individual centers, where they will be processed and stored at + 4–6 °C. at − 80 °C for later shipping to the Department of Human Genetics of Radboudumc for sequencing and analysis of bacterial DNA. Crucially, before and during the interven- Outcome measures tion, the participants will be asked to send back their fecal Primary outcomes samples within 24 h after the test sessions. The primary outcome measure will be response to treat- Another secondary outcome measure will be the ment, defined by a reduction in the Affective Reactivity change in nutritional intake. Participants will be asked to Index–Self-Report (ARI-S) scale total score of at least complete at least three 24-h dietary recalls (24HDRs): at 30% compared with baseline plus a Clinical Global Im- baseline, during the intervention, and directly afterward, pression–Improvement Scale total score of 1 or 2 (very including two weekdays and one weekend day on non- much improved or much improved) at the end of the consecutive days. They will need to describe the type placebo-controlled phase. and amount (in grams) of all drinks and foods consumed during the previous days, starting with the first intake Secondary outcomes after waking up in the morning. Moreover, they will The secondary outcome measures are selected to assess have to specify the time for every meal occasion during intervention effects on symptoms of impulsivity, compul- the day (breakfast, lunch, dinner, or snack). sivity, and aggression via a series of selected scales and Participant compliance with the intervention (SF or questionnaires (e.g., the Strengths and Difficulties Ques- placebo) will be monitored with the Probabilistic Medi- tionnaire, Yale-Brown Obsessive Compulsive Scale, Diffi- cation Adherence Scale throughout the 10 weeks of the culties in Emotion Regulation Scale, and the Urgency, intervention. Moreover, participants will be asked to re- Premeditation [lack of], Perseverance [lack of], Sensation turn unused envelopes from the previous visit. This will Seeking, Positive Urgency, Impulsive Behavior Scale enable us to secure bacterial viability and at the same [UPPS-P]). Interestingly, the UPPS-P consists of a 59-item time estimate adherence to treatment. Finally, the pres- self-report assessing five subscales (negative and positive ence of somatic complaints/side effects or gastrointes- urgency, premeditation, perseverance, and sensation seek- tinal symptoms (via the Bristol Stool Scale) will also be ing), with a mean value being calculated separately for evaluated. each scale, allowing the estimation of distinct dimensions of impulsive behavior [84]. Another secondary outcome Participant timeline measure will be the change in ADHD symptoms (via the The trial timeline is shown in Table 3. Time points per ADHD Rating Scale [ADHD-RS]). Like the UPPS-P, the intervention will be selected on the basis of known dur- ADHD-RS provides not only a total ADHD score but also ation to exert effect. Arteaga-Henríquez et al. Trials (2020) 21:161 Page 6 of 12 Table 2 Measurement of blood markers Type of marker Marker name Link with: Tissue Inflammation TNF-α Inflammation/exercise*/diet Serum/plasma IL-1β Inflammation/exercise*/diet Serum/plasma IL-6 Inflammation/exercise*/diet Serum/plasma CRP Inflammation/diet Serum/plasma Bacterial lipoprotein Inflammation/diet Serum/plasma Vitamin B12 Diet Serum/plasma Folic acid Diet Serum/plasma Vitamins and minerals Vitamin D Diet Serum/plasma Iron Diet Serum/plasma Cu Diet Serum/plasma Nutrients Cholesterol Diet Serum/plasma Glucose Diet Serum/plasma Homocysteine Diet Serum/plasma BDNF Diet Serum/plasma 5-HT Diet Serum/plasma Other markers SCFA Diet Feces GLP-1 Hormone-diet Serum/plasma Insulin Hormone-diet Serum/plasma Paraoxonase Diet Serum/plasma Abbreviations: 5-HT serotonin, BDNF brain-derived neurotrophic factor, CRP C-reactive protein, GLP-1 glucagon-like peptide 1, IL interleukin, SCFA short-chain fatty acid, TNF-α tumor necrosis factor-α At the screening visit (TS), the rate of impulsivity will three 24HDRs on a web-based tool or paper on two be established by performing structured interviews, the nonconsecutive weekdays and one weekend day (dur- ARI-S, and the Clinical Global Impression–Severity ation, 15 min each). Individuals will also be asked to Scale. After having established the presence of high im- collect feces on one day during the 1-week period at pulsivity, the diagnosis of ADHD and/or BPD will be home and to bring it along at T0. assessed via the Diagnostic Interview for Adult ADHD, In addition to responding to the questionnaires and Second Edition, and the Structured Clinical Interview scales listed in Table 1, participants will be instructed on for DSM-IV Axis II Disorders section for BPD, respect- how to store and eat the study product (SF or placebo). ively. Thereafter, screening for eligibility based on clin- The intervention will begin on the day after T0, and par- ical history, demographics (including SES), tests, and ticipants will be asked to continue with their usual medi- questionnaires (e.g., Mini-International Neuropsychiatric cations and/or treatments. Participants will have to Interview [M.I.N.I.-Plus], IQ tests [Wechsler Adult complete another three 24HDRs, collect their feces be- Intelligence Scale, Fourth Edition]) will be performed, fore the next visit, and bring it along at T1, following and informed consent will be obtained. Once this the same guidelines as during the screening assessments. process is finished, a mobile health system as well as a T1 (midintervention assessment; duration, approxi- food record will be introduced to participants (duration, mately 3 h) will take place 5 weeks after T0. This midin- approximately 1 h). The mobile health system will enable tervention assessment aims at obtaining the primary and patients to share (via a mobile application) data of inter- secondary outcome measures during the ongoing trial. est regarding their physical activity (acceleration, num- Participants will be asked once again to wear movement ber of steps). sensors, complete another three 24 HDRs, and collect Trial-specific assessments will be done at T0 (dur- their feces 1 week before T2 and bring it along at T2. At ation, approximately 3 h), scheduled within 1 week 10 weeks after initiating the probiotic or placebo interven- after TS. During the week between TS and the base- tion (T2), primary and some secondary outcome measures line visit (T0), the mobile health system will be used will be assessed again (duration, approximately 3 h). at home. Here and during 24 h, participants will wear Within 1 week after T2 and with the aim of measuring movement sensors on two working days and on Sat- safety aspects of the intervention (i.e., somatic com- urday and Sunday, and they will be asked to complete plaints/side effects as well as gastrointestinal symptoms), Arteaga-Henríquez et al. Trials (2020) 21:161 Page 7 of 12 Table 3 Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) schematic schedule of enrollment, interventions, and assessments Study procedures Screening Open label Follow-up Visit TS T0 T1 T2 T3 Week −1 1 5 10 11 Day −7 to −1 1 35 ± 2 70 ± 2 77 ± 2 Informed consent form X Inclusion/exclusion criteria X Demographics (including SES) X IQ (IQ score based on WAIS, if available) X M.I.N.I.-Plus assessment X Confirmation of diagnosis/-es X Confirmation of ADHD DIVA 2.0 X Confirmation of BPD SCID-II X Primary outcome Clinical Global Impression–Improvement Scale CGI-I X X X Affective Reactivity Index ARI-S X X X X X Secondary outcomes Clinical Global Impression–Severity Scale CGI-S X X X X Clinician rating of ADHD symptoms ADHD-RS X X X Self-rating of multidimensional impulsivity UPPS-P X X X Questionnaire on well-being X X Functioning Assessment Short Test FAST X X X Self/other rating of strength and difficulties SDQ X X X Clinician rating of compulsivity Y-BOCS X X X Sleep problems 5-item questionnaire X X X Self-rating of emotion regulation difficulties DERS-16 X X X Somatic complaints/side effects Body system questions X X X Gastrointestinal symptoms Bristol Stool Scale X X X X Self-rating of perceived stress PSS X X X Major stressors X X X Blood pressure, pulse rate, height, weight, Assessed by clinician X X X waist, hip, medical history, medication evaluation Physical activity Actigraphy; mobile health application X X Blood biomarkers Blood sample X X X Microbiome Stool sample X X X DNA and RNA Blood sample X X X Neurocognitive measures CPT-II X X X Nutritional intake Food diary X X X Treatment adherence/satisfaction/beliefs Probabilistic Medication Adherence Scale ProMAS X X X Abbreviations: ADHD attention-deficit/hyperactivity disorder, ADHD-RS Attention-Deficit/Hyperactivity Disorder Rating Scale, ARI-S Affective Reactivity Index–Self- Report, BPD borderline personality disorder, CGI-I Clinical Global Impression–Improvement Scale, CGI-S Clinical Global Impression–Severity Scale, CPT-II Continuous Performance Test, DERS-16 Difficulties in Emotion Regulation Scale, DIVA 2.0 Diagnostic Interview for Adult ADHD, Second Edition, FAST Functioning Assessment Short Test, IQ intelligence quotient, M.I.N.I.-Plus Mini-International Neuropsychiatric Interview, PROBIA Treatment of impulsivity in adults with probiotics, ProMAS Probabilistic Medication Adherence Scale, PSS Perceived Stress Scale, SCID-II Structured Clinical Interview for DSM-IV Axis II Disorders, SDQ Strengths and Difficulties Questionnaire, SES socioeconomic status, UPPS-P Urgency, Premeditation (lack of), Perseverance (lack of), Sensation Seeking, Positive Urgency, Impulsive Behavior Scale, WAIS Wechsler Adult Intelligence Scale, Y-BOCS Yale-Brown Obsessive Compulsive Scale Arteaga-Henríquez et al. Trials (2020) 21:161 Page 8 of 12 the follow-up (T3) assessment will take place (duration, Data management approximately 20 min). Participants and clinical staff will be able to fill out all requested questionnaires online via the Castor database Criteria for discontinuing allocated interventions system (http://castoredc.com/). Then, data will be dir- Any undesirable experience (of either a physical or psy- ectly uploaded to a central database using a macro soft- chological nature) occurring to a participant during the ware. Both the Castor database system and macro study, whether or not considered related to undergoing software are fully secure and in accordance with current treatment with SF or placebo, will be considered an ad- standards (i.e., Good Clinical Practice [GCP], 21 CFR verse event (AE). Participants will be asked for AEs at Part 11, EU Data Protection Directive, ISO27001 and each visit and then will be discussed with the study ISO9001 hosting). Both systems are also approved by physician at the site. Thus, if an AE occurs in response the Clinical Research Centre Nijmegen, Nijmegen, The to SF or placebo, this will immediately be recorded by Netherlands, because it will be responsible for tracking the principal investigator (PI) or the PI’s staff, and the any changes made manually to raw data. Any other data participant may be discontinued from the study. will be uploaded (after site-specific quality control) by Serious adverse events (SAEs), serious adverse reac- the research team to the central database using tions (SARs) or unexpected serious adverse reactions are encrypted mass storage devices. All information col- defined, respectively, as any adverse event, reaction, or lected in the study will be coded with a unique pseudo- unexpected adverse reaction that results in death; is life- code identifier (PSC) in such a way that participants threatening and/or requires hospitalization or prolonga- cannot be identified from the corresponding data (dir- tion of existing hospitalization; results in persistent or ective 95/46/EC) and ensuring that the information col- significant disability or incapacity; or consists of a con- lected for the study remains strictly confidential. genital anomaly or birth defect. Any of them will be Likewise, collected samples of biomaterial (i.e., blood communicated immediately to the lead coordinator of and microbiota) will be labeled with the corresponding the trial at Vall d’Hebron Research Institute (VHIR) PSC. All patient-related data will be stored at the clinical and to the coordinating PI at the clinical site, who will sites in a database with password protection. A separate be responsible for reporting the event to the ethics single database on the central server of VHIR will link committee that approved the protocol. Suspected unex- the PSC to the participant’s personal data (including pected serious adverse reactions (SUSARs) that are fatal name, address, telephone details, and date of birth). Da- or life-threatening must also be reported not later than tabases will be accessible only to the immediate research 7 days after the sponsor is first made aware of the reac- team at each site. tion. Any additional relevant information must be re- ported within 8 days. SUSARs that are not fatal or life- Study monitoring threatening must be reported within 15 days of the Proper conduct of data collection in the trial will be moni- sponsor first becoming aware of the reaction. All on- tored via on-site visits of a monitoring staff member going AEs will be followed up until no more signs and throughout the study; quality of data collected will further symptoms are verifiable or until stability has been be monitored regularly by a statistical supervision team. reached. Depending on the event, additional tests or After the first five participants per site are included, the medical procedures, as well as referrals to a general quality of data (on item/trial level) per participant will be physician or a medical specialist, will be indicated dur- checked, aiming at detecting any error that may occur in ing this follow-up phase. the beginning of the project and to prevent these errors In the case that a participant’s psychological state dete- from recurring. After including participant numbers 5 to riorates to a clinically significant degree during the trial, 10, the completeness and accuracy of the data on sum- the investigators will either discuss with the participant mary/scale level will be checked for all participants. the possibility of withdrawing from the study or decide Thereafter, data on a summary/scale level will be checked that the participant should withdraw. If a participant, for randomly for 1 in 5 participants. Outcomes of this data any reason, requires treatment with certain therapeutic check will be written in an overview and reported to all re- agents (i.e., antibiotics), the agent taken and time of search personnel. If adjustments to the database or proce- administration will be noted. If any other protocol exclu- dures need to be made, these will be made as soon as sion violation has occurred, the participant’s involve- possible. Moreover, the investigators will permit quality ment will be discontinued. Participants may at any time data checks, audits, and inspections by providing the request to be withdrawn from the study or revoke their sponsor direct access to source data and other documents consent to participate. If a patient is discontinued from (e.g., medical files) by request. the trial, a follow-up visit will be carried out to ensure In each participating center, the PI will be responsible the well-being of the participant. for the local ethics application, on-site training of Arteaga-Henríquez et al. Trials (2020) 21:161 Page 9 of 12 clinicians, identification and recruitment of participants Ethics and dissemination including randomization, data collection according to Before the first subject is enrolled in the trial, all ethical the study protocol, completion of the case report forms and legal requirements will be met. The study protocol, including answering queries, maintaining and updating participant information, and the respective consent form the investigator site file, participation in monitoring, and will be approved according to the respective local and reporting SAEs to the study PI in Barcelona. Each site national regulations at each of the participating centers will have regular meetings with their clinical team in before the start of the trial, and participants will be made order to ensure progress of the trial. As the leading co- aware of the investigational nature and the core aspects ordinator center and with the aim of monitoring the of the study as well. This study will be conducted in ac- study progress, the VHIR team will arrange telephone cordance with the principles of the Declaration of meetings with the local sites throughout the project on a Helsinki (2008) and the Medical Research Involving Hu- regular basis. man Subjects Act (WMO). It will follow the principles Due to the need for research groups to share their data of the Guideline for Good Clinical Practice (ICH GCP to maximize the value of each research contribution, to guideline E6), EU Directive 2001/20/EC, and applicable pool them to address research questions that require lar- regulatory requirements and guidelines in the participat- ger numbers, or to carry out meta-analyses, participants ing countries/regions. The handling of personal data will and, if applicable, their legal guardians will be asked be done in accordance with the new General Data Pro- whether they consent that their data (or portions of their tection Regulation that started in May 2018 in the Euro- data) will be shared in anonymized fashion with other pean Union. research projects. Trial findings will be reported to public and private healthcare providers, specific stakeholders such as policy makers, the medical community, or academic and com- Statistical analysis mercial parties interested in therapy development, as Primary outcome measures will be analyzed by applying well as the general public via publications, conferences, logistic regression analyses with type of treatment, diag- press releases, public talks, and the internet- (e.g., You- nostic group (e.g., ADHD, BPD, or both), gender, and Tube, Facebook, Twitter). All trial results will be re- center as independent variables. A sample size of 180 ported in accordance with the Consolidated Standards of participants will allow for detection of an odds ratio of Reporting Trials (CONSORT) guidelines (www.consort- 2.55 with 90% power, assuming a response rate of 20% statement.org). for the CG at a significance level of 0.05. For simplifica- tion purposes, calculations will assume no effects of the Discussion covariates on the response rate. Secondary analyses will We have presented a design and protocol for an RCT of be performed on the secondary outcome measures a nonpharmacological intervention—synbiotic treat- assessed at the different time points. General linear ment—for the reduction of impulsive and compulsive models will be applied to examine effects of treatment symptomatology in a sample of adult patients with group on continuous outcome variables while control- ADHD and/or BPD. The PROBIA study is the first ling for baseline assessment, gender, diagnostic group, multinational RCT evaluating the effects of probiotics and center. Categorical outcome measures will be inves- on cognitive function, impulsivity, and compulsivity in a tigated using generalized linear models. Treatment ef- large ADHD and/or BPD sample. Existing studies primar- fects and their 95% confidence intervals will be reported. ily addressing the effects of supplementation with probio- To investigate potential long-term effects of probiotics, tics in psychiatric disorders mostly come from animal data of the EG for 10 weeks will be analyzed. General studies or have been done in smaller samples. Moreover, linear models for repeated measurements will be applied to date, no RCT has evaluated the composition of gut to study changes from baseline to follow-up visit for this microbiota in ADHD and/or BPD populations with a large group, and missing data will be handled by the last ob- sample size. Thus, whether the microbial community in servation carried forward method. Statistical significance these individuals is different from that in mentally healthy will be defined at the 0.05 level. humans remains unknown. Once the primary and secondary outcome measures re- Our study will also allow us to identify links between the sults are obtained, all responsible investigators at all study microbiome and hallmark characteristics of ADHD and/or sites will get access to the data to be able to reanalyze the BPD patients (e.g., impulsivity, compulsivity, or aggressive data regarding specific additional research questions. behavior) and also to investigate whether dietary patterns In case of an external request for replication, the re- and probiotics can induce alterations in the gut microbiota, spective statistical analysis will be provided by the trial which may mediate/moderate effects on these behaviors. statistician. Linking these data with blood biomarkers as well as genetic Arteaga-Henríquez et al. Trials (2020) 21:161 Page 10 of 12 and epigenetic parameters will provide integrated mechan- Funding istic knowledge on diet/lifestyle–gut–brain–behavior rela- The project is sponsored by the European Commission and funded by the European Union’s Horizon 2020 research and innovation program under tionships relevant to impulsivity and compulsivity. In order grant agreement no. 728018. The sponsors and funders played no part in to create a study sample population that is highly represen- study design; collection, management, analysis, and interpretation of the tative of impulsivity, broad inclusion criteria will be ap- data; writing of the report; or the decision to submit the report for publication. plied, with most psychiatric comorbidities being accepted, allowing us to provide a better illustration of possible ef- Availability of data and materials fects that could be expected if the intervention were intro- Not applicable. duced in a clinical setting. Understanding the microbiota would be important both for better comprehension of the Ethics approval and consent to participate Written consent will be obtained from every study participant. The Ethical biological bases that underlie the studied disorders and for Committee of the Department of Psychiatry, Psychosomatic Medicine and the future development of new evidenced-based medica- Psychotherapy at the University Hospital Frankfurt-Goethe University in tions for these conditions. Frankfurt, Germany, approved the study protocol on 14 November 2018 with protocol number 269/18. The ethical committee approval for the study protocol requested by Istvan Bitter, Department of Psychiatry and Psycho- Trial status therapy, Semmelweis University, Budapest, Hungary, was issued by the Minis- try of Human Capacities, dated 3 September 2019. The Ethical Committee of This trial is registered with ClinicalTrials.gov the University Hospital Vall d’Hebron in Barcelona, Spain, approved the study (NCT03495375) and was first posted on 12 April 2018. protocol on 11 January 2019 with protocol number 311/2018. An addendum The first participant gave consent on 22 February 2019 was approved on 12 April 2019 with protocol number 311/2018. and was randomized on 27 February 2019. Recruitment Consent for publication is expected to be completed 1 April 2021. The most re- Not applicable. cent version of the protocol (V2.0) was approved by the Ethical Committee of the University Hospital Vall Competing interests d’Hebron, Barcelona, Spain, on 12 April 2019 [PR IB has served as an advisor/consultant in the last 5 years for Angelini, Eli Lilly, Gedeon Richter, and Pierre Fabre and has lectured for Eli Lilly, Janssen, (AG)311-2018]. Lundbeck, Gedeon Richter, and Servier. JARQ was on the speaker's bureau and/or acted as consultant for Eli Lilly, Janssen-Cilag, Novartis, Shire, Takeda, Bial, Shionogui, Lundbeck, Almirall, Braingaze, Sincrolab, Medice and Rubio in Supplementary information the last 5 years. He has also received travel awards (air-tickets + hotel accom- Supplementary information accompanies this paper at https://doi.org/10. odation) for taking part in psychiatric meetings organized by Janssen-Cilag, 1186/s13063-019-4040-x. Rubio, Shire, Medice and Eli Lilly. The Department of Psychiatry, chaired by him, has received unrestricted educational and research support from Eli Lilly, Additional file 1. Standard Protocol Items: Recommendations for Lundbeck, Janssen-Cilag, Actelion, Shire, Ferrer, Oryzon, Roche, Psious and Interventional Trials (SPIRIT) 2013 checklist: recommended items to Rubio in the last 5 years. YG has received speaker fees, has received reim- address in a clinical trial protocol and related documents. bursement for travel costs, and/or has served as a consultat for Novartis, HB Pharma, Shire, Eli Lilly, Hogrefe, Broadman, Clarke Partners, Medscape, Medi- bas, and Natur & Kultur. All other authors declare that they have no compet- Abbreviations ing interests. 24HDR: 24-h dietary recall; ADHD: Attention-deficit/hyperactivity disorder; ADHD-RS: Attention-Deficit/Hyperactivity Disorder Rating Scale; AE: Adverse Author details event; ARI-S: Affective Reactivity Index–Self-Report; ASD: Autism spectrum 1 Psychiatric Genetics Unit, Vall d’Hebron Research Institute (VHIR), Barcelona, disorder; BPD: Borderline personality disorder; CGI-I: Clinical Global Catalonia, Spain. 2Department of Human Genetics, Donders Institute for Impression–Improvement Scale; CGI-S: Clinical Global Impression–Severity Brain, Cognition and Behavior, Radboud University Medical Centre, Nijmegen, Scale,; CPT-II: Continuous Performance Test; DERS-16: Difficulties in Emotion The Netherlands. 3Department of Psychiatry, Donders Institute for Brain, Regulation Scale; DIVA 2.0: Diagnostic Interview for Adult ADHD, Second Cognition and Behavior, Radboud University Medical Centre, Nijmegen, The Edition; EG: Experimental group (probiotic); GCP: Good clinical practice; Netherlands. 4Department of Psychiatry and Psychotherapy, Semmelweis IL: Interleukin; IQ: Intelligence quotient; LAB: Lactic acid bacteria; M.I.N.I.- University, Budapest, Hungary. 5Department of Clinical Neuroscience, Centre Plus: Mini-International Neuropsychiatric Interview; PI: Principal investigator; for Psychiatry Research, Karolinska Institute, Stockholm, Sweden. 6Department ProMAS: Probabilistic Medication Adherence Scale; PSC: Pseudocode of Molecular Medicine and Surgery, Karolinska Institute, Stockholm, Sweden. identifier; PUFA: Polyunsaturated fatty acid; RCT: Randomized controlled trial; 7 Center for Molecular Medicine (CMM), Karolinska University Hospital, SAE: Serious adverse event; SCID-II: Structured Clinical Interview for DSM-IV Stockholm, Sweden. 8Department of Psychiatry, Psychosomatic Medicine and Axis II Disorders; SDQ: Strengths and Difficulties Questionnaire; Psychotherapy, University Hospital, Goethe University, Frankfurt, Germany. SES: Socioeconomic status; SF: Synbiotic 2000 Forte; SUSAR: Suspected 9 Department of Psychiatry, Hospital Universitari Vall d’Hebron, Barcelona, unexpected serious adverse reaction; UPPS-P: Urgency, Premeditation (lack Catalonia, Spain. 10Karakter Child and Adolescent Psychiatry University of), Perseverance (lack of), Sensation Seeking, Positive Urgency, Impulsive Centre, Nijmegen, The Netherlands. 11Biomedical Network Research Centre Behavior Scale; VHIR: Vall d’Hebron Research Institute; WAIS: Wechsler Adult on Mental Health (CIBERSAM), Barcelona, Catalonia, Spain. 12Department of Intelligence Scale; Y-BOCS: Yale-Brown Obsessive Compulsive Scale Psychiatry and Legal Medicine, Universitat Autònoma de Barcelona, Barcelona, Catalonia, Spain. Acknowledgements Not applicable. Received: 13 May 2019 Accepted: 30 December 2019 Authors’ contributions GAH and SKRO drafted the manuscript. JR,IB,YG,CL,AR,AS, and JARQ critically References reviewed the manuscript. 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