Although Attention Deficit Hyperactivity Disorder (ADHD) is associated with an increased risk of substance use disorder (SUD), existing literature on how SUD interacts with ADHD outcomes is limited. This study investigates whether SUD among individuals with ADHD is associated with worse ADHD outcomes and prognosis, and the association between overall functioning and SUD. In addition, we seek to understand whether heavy cannabis use is a better predictor of poorer outcomes compared to SUD status alone.We conducted a retrospective analysis on 50 ADHD patient charts, which were allocated based on SUD status. Subgroup analysis was performed on the total sample population, with allocation based on heavy cannabis use. Mann-Whitney and Chi-Square tests were used for both the primary and subgroup analyses.p = < 0.0001) on objective cognitive testing ) than ADHD patients without SUD. The correlation with poorer ADHD outcomes was more pronounced when groups were allocated based on heavy cannabis use status; in addition to significantly lower IVA/CPT scores (p = 0.0011), heavy cannabis use was associated with more severe fine motor hyperactivity and self-reported hyperactivity/impulsivity scores .SUD status highly correlated with more ADHD-related cognitive impairments and poorer functional outcomes at the time of diagnosis. ADHD patients with comorbid ADHD-SUD scored significantly lower (Future research is needed to determine how substance abuse can be a barrier to improved ADHD outcomes, and the effect cannabis and other substances have on cognitive function and pharmacotherapy of ADHD. Attention Deficit Hyperactivity Disorder (ADHD) is a neurodevelopmental disorder characterized as a persistent pattern of inattention, hyperactivity, and/or impulsivity . The undThe acute effects of cannabis on cognition has been investigated, and has been shown to impair the following; verbal memory and learning, working memory, attention (task and dose dependent), inhibition, and psychomotor function . A studyThese findings are fairly consistent across reviews and meta analyses, all of which identify specific subgroups of cannabis user that are at risk of more severe neurocognitive deficits, such as early (adolescent) use onset , 21–23, Despite the overwhelming amount of studies that support the harms of cannabis use on ADHD outcomes, there are still studies that investigate the therapeutic use of cannabinoids in ADHD patients  and showThe Integrated Auditory and Visual Continuous Performance test (IVA/CPT) was the objective measure used in our study, which is a standardized test that has been validated for diagnostic accuracy in comparison to clinician diagnosed ADHD, with EEG correspondence. The IVA/CPT computes a sustained auditory and visual attention quotient , response quotient, and fine motor hyperactivity quotient. The attention quotient consists of a summary of separate audio and visual measures of vigilance (a measure of inattention based on omission of errors), focus , and speed (average reaction time for correct responses). The response control quotient is comprised of separate auditory and visual scores for prudence (a measure of impulsivity and response inhibition based on commission errors), consistency (a measure of one’s ability to stay on task by variability in response times), and stamina (a measure of sustained effort over time by comparing mean reaction times of the first 200 correct responses vs last 200 correct responses) .This study is unique as it is one of the few studies that specifically focuses on both objective cognitive measures and psychosocial functioning. Our explorative study seeks to understand: 1) How do the cognitive profiles of patients who have ADHD and SUD differ from patients with ADHD without SUD, 2) Does SUD among adults in a naturalistic outpatient community setting result in more functional impairment and worse ADHD outcomes, and 3) how do the cognitive profiles of ADHD patients differ based varying cannabis use.The study is a retrospective analysis and chart review of the first 50 patients with a new ADHD diagnosis, between the dates of January 1st, 2017 and June 1st, 2019 – the first 25 ADHD patients without SUD, and the first 25 ADHD patients with SUD.Every patient had been assessed and diagnosed by a psychiatrist at an outpatient psychiatric clinic that specializes in ADHD, after a full psychiatric evaluation. In addition, all participants completed a set of questionnaires to gain more information on their personal and psychiatric history, and level of functioning. All patients were tested by the IVA/CPT.The independent variables in the study are ADHD patients with substance use disorder (SUD group), and ADHD patients without substance use disorder (Non SUD group).The dependent variables are as follows; demographics , suicide risk level, medical comorbidities, alcohol and substance use history, psychiatric comorbid disorders, IVA/CPT score, number of non-valid IVA/CPT scores, parental breakups, family history of substance use disorder, childhood adversity events such as sexual and physical abuse, highest attained education level, interactions with the law, and employment status. The dependent variable data was collected from diagnosed patients through the many questionnaires’ given to all patients at the clinic. These dependent variables function as markers for ADHD response and function. They served as a way to gain more background information on each patient, and to better identify potential confounding variables, which facilitated proper analysis and interpretation of the results. Subgroup analysis was performed within the total sample population, and compared heavy cannabis users (CU group) with non-heavy cannabis users (Non CU group).Participants were allocated to one of two groups; 1) participants with ADHD who have DSM-5 defined SUD, and 2) participants with ADHD who do not have DSM-5 defined SUD.Inclusion criteria: newly diagnosed patients with ADHD who are under the care of a psychiatrist specializing in ADHD, between the dates of January 1st, 2017 and June 1st, 2019.Exclusion criteria: Patients who got diagnosed with ADHD; 1) without an Integrated Auditory and Visual Continuous Performance test score, 2) Whose reported substance use is too ambiguous to determine substance use status (in accordance with the DSM-5 Substance use disorder and intoxication criteria), or have skipped section(s) in the substance use questionnaire. The number of excluded charts are small, and would not affect statistical analysis and results. Additionally, CPT results were not known for participants who were excluded due to incomplete substance use questionnaire. Therefore, CPT results did not influence the decision to exclude participants due to incomplete questionnaires.In order to obtain our target of 50 patient charts in total, the supervising investigator reviewed patient charts sequentially (by decreasing date), starting on June 1st, 2019 until 25 charts in each group are identified. The maximum date range for participant recruitment was between the dates of January 1st, 2017 to June 1st, 2019. All substances in the DSM-5 SUD criteria were used to allocate patient charts based on substance use status except for nicotine and caffeine, as these are substances that can enhance ADHD-related cognition [When patient charts satisfied inclusion and exclusion criteria, and met DSM-5 criteria for SUD, they were allocated to the SUD group. When patient charts satisfied inclusion and exclusion criteria, and did not meet DMS-5 criteria for SUD, they were allocated to the Non SUD group. The maximum number of charts that could be reviewed, in order to attain our goal of 25 charts per group, was set at 500 charts. This selection process was chosen as a way of randomization, and to control for selection & allocation bias, where participant selection was dependent on when they got diagnosed, not on the choice of the researchers.After the primary analysis, Subgroup analyses was performed. The subgroup analysis compared heavy cannabis users (CU group) with those who either did not use cannabis, or light users of cannabis below the heavy cannabis use threshold (Non CU group). We chose a more conservative threshold as to whether one’s cannabis use met heavy cannabis use status, as the substance was recently legalized in the country. Heavy cannabis use in this study was defined as use 3 times a week or greater, or last use within 72 h at the time of diagnosis among weekly users. We chose this method to capture the cannabis users that are at higher risk (daily or near daily use) of neurocognitive deficits, based on numerous meta-analyses , 22, 24.Measure of ADHD outcomes were classified and calculated based on objective cognitive testing or subjective patient report. Objective cognitive testing measures were calculated based on participant’s completion of the Integrated Visual and Auditory Continuous Performance task. The following objective measures are computed by the IVA/CPT; Sustained Auditory & Visual Attention Quotients , and Fine motor Hyperactivity. Fine Motor Hyperactivity measures off-task impulsive fine motor activity with the mouse, and is computed as a severity score . Severity score on ADHD symptoms were calculated based on self-reported symptoms consistent with the DSM-5 diagnostic criteria for Inattention and Hyperactivity/Impulsivity. Both hyperactivity/impulsivity and inattention measures were ranked out of 9 – if participants meet any of the 9 symptoms in each checklist, then they were given a point for the respective symptom. For both Inattention and Hyperactivity/Impulsivity checklists, if adult participants had 5 points out of 9 or less, then their symptom severity is mild (given a score of 1). If 6 or 7 points out of 9 then symptom severity is moderate (given a score of 2), and if 8 or 9 points out 9, then symptom severity is severe (given a score of 3).All patients were assessed by a psychiatrist, whom used DSM-5 Criteria to diagnose the following; ADHD, SUD, major depressive disorder, generalized anxiety disorder, borderline personality disorder. Participants were given questionnaires for all of the above disorder, which were designed based off of DSM-5 symptom criteria. The completed questionnaires were then used in conjunction with the psychiatrists history and mental status exam, in order to confirm these diagnoses, and to ensure all DSM-5 criteria were met.Highest attained education level was ranked as follows: 1 = less then grade 10, 2 = grades 10–12, 3 = completion of grade 12, 4 = Community college, 5 = Bachelor’s degree, 6 = completion of Master’s degree, 7 = Doctorate degree.Was graded as either mild, moderate or severe, and was given the numerical values 1, 2 and 3 respectively. Participants were ranked based on the rules of The Nova Scotia Tool for Suicide Risk assessment. Patients were ranked as severe if they had an active plan or intent of suicide with ongoing suicidal ideation. Participants were moderate if they had suicidal ideation, multiple risk factors but not current intent nor plan. Participants were low risk if they had no history of cutting or suicide attempts.Personality disorder and traits were computed based on responses to the personality disorder questionnaire. Each question represents a DSM-5 symptom criteria for a given personality disorder where participants circle either ‘yes’, ‘maybe’, or ‘no’. Participants would meet DSM-5 symptom criteria if they circled ‘yes’, and would not meet the DSM-5 symptom criteria if they circled ‘maybe’ or ‘no’. If the questionnaire contained all DSM-5 symptom criteria for a given personality disorder, and a participant checked off all the symptoms required for a diagnosis of a given personality disorder, then they were said to meet DSM-5 diagnostic criteria for that personality disorder. If the questionnaire contained most DSM-5 symptom criteria for a given personality disorder – but not enough to be diagnostic - then they were said to have personality traits for that personality disorder. Outcome measures for personality traits were analyzed as a ratio of total symptoms checked off for a given personality disorder, divided by total symptoms for that personality disorder.The substance use questionnaire asks about the daily amount, frequency, and date of last use for the following substances; alcohol, cannabis, hallucinogens, stimulants, cocaine, pain medication not prescribed, and barbiturates. Nicotine use was inquired by checking a yes or no box depending on whether one currently uses it, with a space for the participant to elaborate if they feel necessary. Caffeine use wasn’t collected. These questionnaires were used in conjunction with the patient history to establish a DSM-5 diagnosis of SUD.T tests and Chi squared tests were used to determine statistical significance between SUD status and measures of ADHD outcome and function. Odds ratios were calculated for each comparison. The same tests were used for the subgroup analysis.Table Table Cannabis was the most commonly used drug for the SUD group, whereas alcohol was the most common substance used in the Non SUD group – although alcohol use was still higher for the SUD group compared to the Non SUD group. Cocaine and Hallucinogen use was the least used substance in both groups in terms of frequency.In order to test our hypothesis that Substance use among the ADHD population results in worse ADHD outcomes, statistical analysis (Mann-Whitney Non Parametric T test) was done between SUD groups and Non SUD groups. Table Table P value of 0.462. One participant in the Non SUD group was excluded in the analysis of borderline personality disorder diagnosis, as they were in late adolescence and therefore did not receive the DSM-5 personality disorder questionnaire.Table In order to test our hypothesis that comorbid ADHD and SUD results in poorer functional impairment within society, data pertaining to violence, interactions with law, academic and occupational achievement and trauma history was analyzed Table . Chi-squSignificance was found for measures relating to violence, charges, physical abuse, and poor relationship with parents. In terms of academic and occupational achievement, significance was found for not completing high school, and the highest attained education level. The SUD group had a median education level at grade 12, whereas those in the non SUD group had a median college degree education level. History of failing grade, academic difficulty, and dropping out of college/university were approaching significance at an alpha level of 0.05, and met significance with an alpha level of 0.1.P values, are as follows; Drinking/Drugging Leads to Violence (P = 0.014), Previous Charges (P = 0.054), Previous Arrests (=0.247), Previous Jail time (P > 0.99), Dropped out of College or University (P = 0.209), and Current Employment (P > 0.99).As the event rate for some variables were low, the Yates continuity chi-square was used if any cell in the chi-square had an expected count less than 5. These variables, along with their corrected Although subgroup analyses can be difficult to interpret, we elected to compare heavy cannabis users (CU group) with non-heavy cannabis users (Non CU group). Table The CU group differed significantly from the Non CU group by having more impairment in objective (IVA/CPT) and subjective (self-reported ADHD symptoms) cognitive functioning. Fine motor activity and Hyperactivity/impulsivity symptoms were significantly higher in the CU group – both of which were not seen when comparing groups by SUD status. No significant differences were found when comparing inattention symptoms. Like the comparison of SUD groups, the CU subgroups had significantly poorer performance on IVA/CPT, higher suicide risk, poorer education attainment, more borderline and antisocial personality traits, and higher major depression disorder severity.We aimed to investigate how substance abuse among the ADHD population may interact with different outcomes measures, and how the profiles of ADHD patients differ according to SUD status. Our results show that according to IVA/CPT results between groups Fig. , Table 3No significant difference was found between groups for reported number of ADHD symptoms Fig. , Table 3Although no significant differences were found for fine motor hyperactivity Fig. , Table 3Subgroup analyses investigating heavy cannabis abuse shows even more impairment in objective and subjective ADHD outcomes Table . SignifiThese results suggest that SUD status at diagnosis predicts poorer ADHD outcomes and prognosis. These results bring up the question of how ADHD and SUD interact with one another, and the other factors  that interplay to produce a more severe ADHD phenotype. Are individuals with severe ADHD symptoms  more likely to abuse substances , and/or Studies investigating the effect cannabis has on brain structure and function shows that cannabis use is associated with altered brain structure and function , 45, 46.Table The results presented in Table Our study has several limitations; this study is a retrospective analysis with no blinding of participants or investigators. Identified patients are allocated into independent variable groups depending on their reported substance use on questionnaires. Therefore, whether a participant fits into the “ADHD with SUD” group or “ADHD without SUD” group is dependent on the integrity of their self-reported substance use patterns on questionnaires. Due to the study’s retrospective nature, not all confounding variables were controlled for, which may include the chronicity of substance use (i.e. whether they used cannabis regularly during adolescence), age of ADHD diagnosis, and other comorbidities and social circumstances not asked about in the questionnaires.Our study served as a pilot study and is exploratory in nature to gain more background information on SUD in the ADHD population, and how patients may differ in many psychosocial variables based on SUD status. Our results suggest that more research is needed to determine the effect that certain substances may have on neuromaturation and cognition, and how substances can be a barrier to improved ADHD outcomes. Considering how the use of cannabis recently became legalized in Canada, this study has a special importance, as it further draws attention to the complexity of heavy cannabis use, and accentuates the need to further understand how cannabis use disorder interacts with cognitive functioning.


Attention-Deficit/Hyperactivity Disorder (ADHD) in children is associated with several adverse family characteristics, such as higher parenting stress, more conflicted parent–child relationships, lower parental competence, and higher levels of parental psychopathology. Hence, children with ADHD more often grow up under suboptimal circumstances, which may impact the development of their attachment representations. Here, we investigated whether children with ADHD have more insecure and disorganized attachment representations than their typically developing peers, and which factors could explain this association. We included 104 children between 4 and 11 years old, 74 with ADHD (without Conduct Disorder) and 30 typically developing control children. Children completed a state-of-the-art story stem task to assess their attachment representation, and we measured parents’ expressed emotion , parents’ perceived sense of competence, parental education levels, and parent-rated ODD symptoms of the child. We found that, after controlling for multiple comparisons, children with ADHD had less secure and more ambivalent and disorganized attachment representations relative to their typically developing peers. These group differences were independent of comorbid ODD and parental education levels. There were no group differences on avoidant attachment representations. Explorative analyses within the ADHD group showed that attachment representations were not related to parent–child relationship quality, perceived parenting competence, parental education levels, and comorbid ODD symptoms. We conclude that children with ADHD disproportionately often have attachment problems. Although this conclusion is important, treatment implications of this co-occurrence are yet unclear as research on ADHD and attachment is still in its infancy. Attention-Deficit/Hyperactivity Disorder (ADHD) is a neurodevelopmental disorder characterized by age-inappropriate levels of inattention, hyperactivity, and/or impulsivity, causing impairment in several domains of life . In chilGenerally, three different attachment representations are distinguished. Sensitive and supportive parenting in response to stress will help the child to develop a secure base script, ultimately leading to a secure attachment representation . On the Despite its potential relevance, empirical research on the link between attachment and ADHD is scarce. A recent meta-analysis including 62 studies found a link between children’s attention problems and attachment quality: children with more insecure and children with more disorganized attachment representations had more attention problems than children with secure or organized attachment representations, although the magnitude of the effects was modest . A systeFirst, most studies investigated the link between attachment and ADHD symptoms in samples of typically developing children, or in children at risk for attachment problems . Only a Second, many studies investigating the link between ADHD and attachment did not control for comorbid disruptive behavior problems. Abundant literature shows a significant association between disruptive behavior and insecure attachment ,10. The behavior has become too subtle [representations are derived from the completion of a story by the child [A third limitation of the literature on ADHD and attachment relates to the measurement of attachment. From school-age, observational methods such as the Strange Situation Test  are lesso subtle . In addio subtle ,25. Manyo subtle ; (iii) ao subtle . A solidhe child ,28.In the current study, we take these limitations of previous studies into account, by investigating attachment representations in a carefully diagnosed sample of school-age children with and without ADHD, matched by age, sex, and intelligence, controlling for comorbid disruptive behavioral symptoms and using state-of-the-art story stem procedures to measure attachment representations. We hypothesize that children with ADHD have more insecure and more disorganized attachment representations, relative to typically developing children, and—based on the available literature on ADHD and insecure attachment—that this cannot be entirely explained by comorbid disruptive behavior. Then, we zoom into the group of children with ADHD to explore whether insecure and/or disorganized attachment representations in these children could be accounted for by parents’ expressed emotion , parents’ perceived sense of competence, parental education levels, and children’s comorbid ODD symptoms. Ultimately, this may guide interventions for children with ADHD and their families.N = 44, inattentive presentation: N = 21, hyperactive/impulsive presentation: N = 9) and 30 Typically Developing (TD) children participated. Children with ADHD were recruited for a microtrial on behavioral parent training in six child mental healthcare outpatient clinics in the Netherlands [A total of 74 children with ADHD  [The Disruptive Behavior Disorders module of the Dutch version of the structured Diagnostic Interview Schedule for Children (DISC-IV)  was admictively) . The DISTwo subtests (Block Design and Vocabulary) of the Dutch Wechsler Intelligence Scale for Children (WISC-III) ,32 or, wParent’s highest level of education  was obtained. We could not obtain this information for two participants.We used a story stem task to assess attachment representation. In this task, the researcher starts telling four stories to the child while using two dolls  and a standardized set of attributes. Each story has an attachment-related theme, and the child is asked to finish all stories . The theκ = 0.82 − 0.96). Raters followed a training in the assessments and scoring of the tapes. All disagreements were solved after discussion. Ultimately, this resulted in a score between 1 (no signs) and 5  on each of the four attachment representations .The session was recorded, and all tapes were scored independently afterwards by two raters  and intraclass correlation coefficients for the frequency of positive/critical comments  were excellent.Five Minute Speech Samples (FMSS)  were colThe Parenting Sense of Competence scale (PSOC)  was usedEligible parents were provided with an information letter. When interested, procedures were explained and parents provided informed consent. Parents received an online questionnaire with demographic questions and the PSOC. The remaining measures were administered during a home visit or a visit to the clinic, in which the parents and the child were assessed simultaneously for about two hours. Parents received compensation for participating (EUR 10).As preliminary analyses, we used independent t-tests, Mann–Whitney tests , and chi-square analyses to assess between-group differences on age, sex, intelligence, parental education levels, and ADHD and ODD symptoms.As primary analyses, to test whether children with and without ADHD differ in attachment representations, we planned to use an ANCOVA with group (ADHD vs. TD) as independent variable and the continuous score on the attachment representation as dependent variable. However, as Levene’s and Bartlett’s test both indicated unequal variances , we instead performed multilevel regression analyses with group as fixed effect in which we freely estimated group variances in attachment representations. As we had four measures of attachment representations, this analysis was performed four times, once for each attachment representation.We included age as covariate as the literature shows attachment and age are correlated . HoweverInstead, to understand whether associations between ADHD and attachment were independent of ODD symptoms or parental education levels, we performed separate analyses with ODD symptoms and parental education level as independent variables instead of group .within the group of children with ADHD. Participants were excluded from specific secondary analyses when data were missing for any of the relevant measures (note: we had missing data on the PSOC for 7 participants and on the FMSS for 6 participants).As secondary analyses, to elucidate which children with ADHD are particularly at risk for insecure and disorganized attachment representations, we used bootstrapped (as assumptions were violated) hierarchical linear regression analyses to assess the influence of age and parental education levels (block 1), comorbid ODD symptoms (added in block 2), expressed emotions, and parenting sense of competence (both added in block 3) In both sets of analyses, we applied a Benjamini–Hochberg correction  for multGroups did not differ in age, sex distribution, and intelligence . ParentaMultilevel regression analyses with age as covariate revealed that children with ADHD differed significantly from TD children on three of the four attachment representations. Children with ADHD had lower scores on secure attachment representations and higher scores on ambivalent and disorganized attachment representations relative to TD children. Groups did not differ in avoidant attachment representations. Statistics on group differences are depicted in Importantly, we found no significant effects on any attachment representation when ODD symptoms or parental education levels were analyzed as independent variables instead of group. This suggested that the observed associations between group and secure/ambivalent/disorganized attachment were independent of ODD symptoms or parental education levels , and parents’ perceived sense of competence were not associated with any of the attachment representations. For detailed results, see None of the bootstrapped linear regression analyses The aim of this study was to investigate whether children with ADHD had more insecure and/or disorganized attachment representations relative to their peers without ADHD. Using story-stem procedures, we found that—in line with our hypothesis—children with ADHD had less secure attachment representations relative to typically developing children. Additionally, children with ADHD had more ambivalent and disorganized attachment representations. No between-group differences were found on avoidant attachment representations. Crucially, we found no effects of ODD symptoms and parental education on any attachment representation, which indicated that the link between insecure and disorganized attachment and ADHD was not explained by these factors.The link between insecure and disorganized attachment and ADHD could be explained by several mechanisms. First, it may be more challenging to form a secure attachment relationship with children with ADHD relative to typically developing children, because of the inattentive, hyperactive, and impulsive behavior of these children. This may increase parenting stress, interfere with sensitive and responsive parenting behavior, and challenge the parent–child interaction, together challenging parents’ ability to provide a secure base for their child ,23,49.Second, early life stress in general, and more specifically the insensitive/unresponsive caretaking behavior that leads to insecure attachment representations, may contribute to the development of ADHD. High levels of early life stress may have direct impact on the maturation of the brain ,51, poteAn explanation for the association between insecure attachment and ADHD could be that children with insecure attachment relationships with their parents constantly devote part of their attention to their safety and their caregiver , which To better understand which characteristics of children with ADHD were particularly associated with insecure or disorganized attachment representations, we investigated four potentially relevant mechanisms within the ADHD group. Based on earlier work, we reasoned that parent–child relationship quality , parentsFirst, a recent model describing the development of attachment from a learning-theory perspective proposes that secure attachment is learned by reductions in cortisol after caregivers’ support, which is a reinforcer in the safety conditioning of the child, thereby leading to increased trust in the caregiver . More spDRD4 7-repeat allele, a polymorphism that was also linked to reward sensitivity and ADHD [Second, a related mechanism to explain attachment problems in children with ADHD could come from the differential susceptibility theory, which posits that some children are more susceptible and others are more resilient concerning environmental influences, regardless whether these influences are negative or positive ,72,73. Eand ADHD . This teand ADHD .The confidence in our findings is increased by several strengths of the study. Contrary to most studies on ADHD and attachment, we adopted a case-control design with a homogeneous ADHD group in which all children were medication-naive, and used a state-of-the-art story-stem task to measure attachment representations . MoreoveAlthough all participating families of children with ADHD actively pursued behavioral treatment, a limitation of our study was that we potentially included a sample of relatively high-functioning families of children with ADHD, as established by average mean intelligence levels in the ADHD group, the low number of high scores on expressed emotions, the fact that none of the children were taking medication, and the fact that we excluded children with comorbid CD. However, it is most likely that between-group differences in attachment representations would rather be larger than smaller if our ADHD group would have been more severely impaired.Based on our findings, we suggest considering both ADHD and attachment when assessing children referred with either ADHD behaviors and/or attachment-related problems. The co-occurrence of ADHD and insecure/disorganized attachment representations is likely explained by several mutually interactive mechanisms  and VIPWe demonstrated a link between ADHD and insecure/disorganized attachment representations in school-age children. This study adds to a small but important literature, and there is an urgent need for more studies on the clinical meaning and implications of the association between ADHD and attachment problems.


Bartonella is a rapidly expanding group of ubiquitous bacteria that occur mainly in different animal species, but some can also be transmitted to humans. Three species, B. henselae, B. bacilliformis, and B. quintana, are responsible for the majority of human cases. The severity of the clinical symptoms often depends on the immune status of the patient, but others factors such as the species of the pathogen, virulence factors, and bacterial load also can play an important role. As the information on the occurrence of bartonellosis in the human population in Slovakia is absent, the aim of our pilot study was to determine the seroprevalence against B. henselae and B. quintana in the population of people living in Eastern Slovakia, and to identify the impact of related risk factors. Of 536 people included in the study, 126 (23.5%) showed positivity for anti-B. henselae antibodies and 133 (24.8%) against B. quintana. A statistically higher prevalence was confirmed only in the case of B. quintana in women regardless of the risk group. In analyzing the risk factors, we found significant differences between B. henselae seropositive and seronegative groups only in uric acid levels and serum creatinine, both, however, clinically irrelevant. Significant, but clinically irrelevant differences were observed also in alanine aminotransferase (ALT) levels and creatinine in people seropositive to B. quintana. The genus  Bartonella infections, belonging to a group of emerging diseases, are caused by a Gram-negative, aerobic, pleomorphic, intracellular pathogens of the order Rhizobiales, genus Bartonella.Bartonella were known. Since the last reclassification in 1993, the number of Bartonella species has increased to 45 [Bartonellosis was first described in 1909, and by 1990 only two species of Bartonella species, there are at least 16 species that have been associated with the human disease—Bartonella (B.) alsatica, B. ancashensis, B. bacilliformis, B. bovis, B. clarridgeiae, B. doshiae, B. elizabethae, B. grahamii, B. henselae, B. koehlerae, B. mayotimonensis, B. quintana, B. rochalimae, B. tamiae, B. vinsonii subsp. arupensis and B. vinsonii subsp. berkhoffi [B. bacilliformis, which has a human reservoir, other species are zoonotic. Multiple reservoirs for the development of human Bartonella infections are both domestic and wild animals. In Europe, these are mainly cats, dogs, and rodents [Among all the erkhoffi ,3. With  rodents ,5.B. henselae, B. bacilliformis, and B. quintana—are mainly responsible for most human Bartonella diseases.Three species—B. henselae is the etiological agent of one of the most well-known zoonotic human infections caused by Bartonella—cat scratch disease (CSD) [B. bacilliformis [B. quintana is the causative agent of Trench fever [se (CSD) . Carrionliformis , and B. ch fever . Bartonella infections  and some are suspected to be involved in transmission , depending on the bacteria species and their vertebrate reservoirs [Bartonella became evident only in the early 1990s, when B. quintana and B. henselae were first described in HIV-infected patients and subsequently in immunocompetent persons [Diseases caused by these microorganisms are mainly transmitted by the bite or scratch of infected animals or by different insect vectors, while some arthropods were proved as a vector of servoirs ,12,13,14 persons . Infecti persons . The sevBartonella spp. varies among different geographic regions, groups of examined people, and the diagnostic method used [The prevalence of hod used . Most ofhod used . As there is no law in Slovakia that imposes systematic screening for this infection, information on the occurrence of this disease in humans are absent. Thus far, this infection has been confirmed in Slovakia only in small mammals, with positivity ranging between 9% and 73.8% ,19. Bartonella infections include direct contact with infected animals . A variety of insects, including fleas, body lice, and ticks, act as vectors and play an important role in transmission not only between animals but also between animals and humans [B. henselae and B. quintana in the population of people living in Eastern Slovakia and to identify the impact of related risk factors.Risk factors associated with d humans ,10. Theid humans . For theAnalysis of baseline information of the people living in segregated Roma settlements (risk group) and people from the general population showed that this two populations are completely different in terms of demographics, socioeconomic status, and biochemical parameters (except transaminases), .B. henselae and B. quintana, an apple-green fluorescence was detected. Samples that showed fluorescence at titers of 1:64 and above were considered positive.In the case of a positive immunological response to the presence IgG antibodies against B. henselae. In the case of B. quintana, IgG antibodies were confirmed in 133 people and overall prevalence was 24.8% (95% CI 21.3–28.7). Basic differentiation of seroprevalence based on gender and risk group is presented in B. quintana IgG positivity and no difference was observed in B. henselae IgG seropositivity in regards to gender or risk group.Of the 536 respondents included in the study, 126  showed positivity for IgG antibodies against Because of no significant difference in any antibody prevalence between Roma and non-Roma, differences in potential risk factors were analyzed in the whole cohort .B. henselae seropositivity, but with low OR. Analogous analysis with B. quintana showed strong association with female gender (OR 2.1) and weaker association with basic education. All other analyzed risk factors were insignificant.Roma ethnicity was entered as a confounding factor, along with gender in the multivariate regression model . After aBartonella have been characterized since the beginning of the 1990s. As a result, the spectrum of natural reservoirs, vectors, and subsequent human diseases caused by these species has expanded considerably [Several new species and subspecies of the genus iderably .B. henselae and anti-B. quintana antibodies in people living in Eastern Slovakia and identification of risk factors that may be involved in the development of the disease.In our study, we focused on the investigation of the prevalence of anti-B. henselae, the most prevalent zoonotic Bartonella species occurring endemically worldwide, cats are the primary reservoir, but DNA from this pathogen has also been found in dogs, cows, horses, marine mammals, and sea turtles [Ctenocephalides felis) also [B. henselae and can play an important role in their transmission to humans and animals [In the case of  turtles ,21,22,23is) also . Other v animals .B. quintana has a worldwide distribution often associated with war zones and poor hygiene, which is related to the predisposition to contamination of the human body louse (Pediculus humanus humanus) [B. quintana has been isolated from feline fleas and ticks, and this bacteria has been confirmed in dogs, in addition to feral cats [humanus) ,27. Thishumanus) . In addihumanus) . DNA of ral cats ,31,32.Bartonella infection in humans can be difficult to diagnose. Culture, molecular, and serological tests can be used, but since the causative agent cannot be easily cultured, the diagnosis usually relies on epidemiological, clinical, and serological proof.Generally, Bartonella species is often associated with poor hygiene, unfavorable social conditions, and a low standard of living. In Slovakia, these conditions mainly concern people living in segregated Roma settlements. There is generally a higher prevalence of infectious diseases, poisoning, and injuries in this group [In Slovakia, to date, bartonellosis in humans has not been officially reported. As is known from foreign studies, the occurrence of many is group . These pBartonella infections would be higher in the Roma population due to poor hygienic standards and increased contact with stray animals as a risk factor, our study did not confirm this. A statistically higher prevalence was confirmed only in the case of B. quintana in women regardless of the risk group. The explanation might be that having high hygienic standards in an area where other community could not afford it and where stray animals  are increasingly present without proper management of risk does not prevent Bartonella infections efficiently.Although our study confirmed that Roma live in socially weaker conditions compared to the majority , and it B. henselae seropositive and seronegative groups only in uric acid levels and serum creatinine, both, however, clinically irrelevant. Significant, but clinically irrelevant differences were observed also in ALT levels and creatinine in people seropositive to B. quintana.In analyzing the risk factors, we found significant differences between B. henselae and 24.8% for B. quintana) we found is comparable to the results of other studies. Generally, the prevalence of Bartonella infections in humans varies from 0.2% to 30% on average, depending on the investigated group and the diagnostic method used. For example, in a study conducted in Germany, the seropositivity rates among the healthy adults are 30%, in Greece 19.8%, in Poland 25.85%, in Austria 26.5%, in Sweden 16%, and 8.7% in Spain [Bartonella infections in the population and to study associations of the occurrence of Bartonella antibodies in humans and animals.The overall seropositivity  kit. This test uses Vero cells that have been infected with either B. henselae or B. quintana as the individual substrates permitting the qualitative detection and semi-quantitation of human serum IgG antibodies to Bartonella. This test uses only a small blood sample for reliable diagnosis; however, cross-reactive between species may occur and, therefore, the results should be interpreted with caution.Another limitation may be the diagnostic test used. In our study, detection of IgG antibodies to Samples and data from the cross-sectional population-based Hepa-Meta study conducted in Eastern Slovakia were used. The study focused on the prevalence of viral hepatitis diseases, metabolic syndrome, and selected bacterial and parasitic infectious diseases in the population living in segregated settlements. The inhabitants from the general population from the same region were used as a control group. The methodology of this study was previously described in detail by Madarasová Gecková et al. .B. henselae and B. quintana was comprised of 536 people aged 18–55, randomly selected from the overall HepaMeta database (n = 855). The study cohort consisted of 283 people living in 10 different segregated Roma settlements in Eastern Slovakia  and 253 people from the general population from the same region  who were used as a control group.The final sample that examined the presence of IgG antibodies against vena mediana cubiti under standard conditions. After centrifugation of the blood, serum samples were collected and stored at −80 °C until their use in the immunofluorescence test. Hyperlipemic, hemolyzed, and contaminated sera were excluded from testing.Data were collected via analyzed blood and a questionnaire. Venous blood was collected from The questionnaire gathered information about the sociodemographic background , living conditions , and poverty .Bartonella infections was done using a commercial indirect immunofluorescent assay (IFA) kit .Detection of IgG antibodies to B. henselae or B. quintana for detecting IgG antibodies was used according to the manufacturer’s instructions.A kit that uses Vero cells infected with either B. henselae and B. quintana antigens. After incubation for 60 min at room temperature and then washing, the fluorescently labelled anti-IgG antibodies were added to the wells. Afterwards, the slide was washed, dried and mounted and was examined using fluorescence microscopy at 400× magnification.Antibody determination was performed in two phases. In the first step, the blood sera were diluted at a 1:64 screening dilution and then added to appropriate slide wells containing Biochemical parameters—aspartate aminotransferase (AST), alanine aminotransferase (ALT), uric acid, albumin, creatinine, and C-reactive protein (CRP) were determined by routine biochemical methods on the analyzer ADVIA 2400. Reference values for men were for AST 0.17–0.85 ukal/L, ALT 0.3–0.8 ukat/L, uric acid 220–420 ukat/L, albumin 32–48 g/L, creatinine 66–106 umol/L, and CRP 0–5 mg/L. For women the reference values were for AST 0.17–0.6 ukal/L, ALT 0.2-0.7 ukat/L, uric acid 140–340 ukat/L, albumin 32–48 g/L, creatinine 44–84 umol/L, and CRP 0–5 mg/L.p value < 0.05 was considered statistically significant.Categorical variables are reported as absolute and relative frequency (n (%)). Interval variables are reported as mean ± standard error of mean. Baseline comparisons of the prevalence between groups based on various demographic, socio-economic, and laboratory variables were performed by Chi-squared test in case of categorical variables or Mann–Whitney test in case of interval variables. Adjusted odds ratios of variables that significantly differed between positive and negative groups were obtained by multivariate logistic regression adjusted for confounders. Two-sided


Small intestinal content recovery of repotrectinib was decreased 4.9-fold in −/−Abcb1a/1b and 13.6-fold in −/−Abcb1a/1b;Abcg2 mice. Intriguingly, −/−Abcb1a/1b;Abcg2 mice displayed transient, mild, likely CNS-localized toxicity. Oatp1a/1b deficiency caused a 2.3-fold increased oral availability and corresponding decrease in liver distribution of repotrectinib. In −/−Cyp3a mice, repotrectinib plasma AUC0–h was 2.3-fold increased, and subsequently reduced 2.0-fold in humanized CYP3A4 transgenic mice. Collectively, Abcb1 and Abcg2 restrict repotrectinib brain accumulation and possibly toxicity, and control its intestinal disposition. Abcg2 also limits repotrectinib oral availability. Oatp1a/1b mediates repotrectinib liver uptake, thus reducing its systemic exposure. Systemic exposure of repotrectinib is also substantially limited by CYP3A activity. These insights may be useful to optimize the therapeutic application of repotrectinib.Repotrectinib shows high activity against ROS1/TRK/ALK fusion-positive cancers in preclinical studies. We explored the roles of multidrug efflux transporters ABCB1 and ABCG2, the OATP1A/1B uptake transporter(s), and the CYP3A complex in pharmacokinetics and tissue distribution of repotrectinib in genetically modified mouse models. In vitro, human ABCB1 and ABCG2, and mouse Abcg2 efficiently transported repotrectinib with efflux transport ratios of 13.5, 5.6, and 40, respectively. Oral repotrectinib (10 mg/kg) showed higher plasma exposures in Abcg2-deficient mouse strains. Brain-to-plasma ratios were increased in  Chromosomal rearrangements involving ROS proto-oncogene 1 (ROS1) and the anaplastic lymphoma kinase (ALK) gene have been associated with many types of malignancies, including non-small cell lung cancer (NSCLC) . There iRepotrectinib  transporters and solute carrier (SLC) transporters . The ABCDrug transporters often modulate drug absorption and availability, distribution, and elimination together with drug-metabolizing enzymes. Cytochrome P450 (CYP) is a major metabolizing enzyme superfamily, among which the CYP3A enzymes account for the metabolism of around 50% of currently used drugs . The plaHere, we studied the roles of the multidrug transporters ABCB1, ABCG2, and OATP1A/1B in repotrectinib plasma exposure and tissue accumulation, using wild-type and genetically modified knockout and transgenic mouse models. Moreover, we evaluated to what extent CYP3A affects the oral availability of repotrectinib.Repotrectinib  was purchased from TargetMol . Ko143 was supplied by Tocris Bioscience . Zosuquidar was obtained from Sequoia Research Products . Bovine serum albumin (BSA) fraction V was supplied by Roche Diagnostics . Heparin (5000 IU/mL) and isoflurane were obtained from Leo Pharma  and Pharmachemie , respectively. All other chemicals and reagents were purchased from Sigma-Aldrich . Chemicals that were used in the bioanalytical repotrectinib method were previously reported .Polarized Madin-Darby Canine Kidney (MDCK-II) cells (ECACC 00062107) stably transduced with either human (h) ABCB1, hABCG2 or mouse (m) Abcg2 cDNA were generated in our institute between 1995–2005. The proper identity and functionality, such as highly characteristic growth and drug transporter properties, including inhibitor sensitivity, were regularly checked in these epithelial cells. Mycoplasma routinely tested negative in these cells. The passage number was 10–15 when used in the transport experiments.5 cells in each well and maintained and grown in DMEM with 10% fetal bovine serum , 100 U/mL penicillin, and 100 µg/mL streptomycin, at 37 °C in 5% CO2 and 95% humidified air, for three days to form an intact monolayer. Prior to and after the transport phase, the integrity of monolayer membrane was checked and confirmed by the measurement of the transepithelial electrical resistance (TEER).Transepithelial transport experiments were performed as previously described with 12-well plates  containing microporous polycarbonate membrane filters . The parental MDCK-II cells and their subclones were seeded on the membrane at a density of 2.5 × 102 and 95% humidified environment during the experiment. At 1-, 2-, 4-, and 8-h time points, 50 µL aliquots were taken from the acceptor compartment and stored at −30 °C until LC-MS/MS measurement of repotrectinib. The active transport ratio r, calculated by dividing the amount of apically directed drug transport by basolaterally directed drug translocation after 8 h, was used to assess active transport of repotrectinib.After pre-incubation with 5 µM zosuquidar (ABCB1 inhibitor) and/or 5 µM Ko143 (ABCG2/Abcg2 inhibitor) in both compartments (when appropriate) for 1 h, the transport phase was initiated (t = 0) by replacing the medium in apical and basolateral compartments with fresh DMEM with 10% FBS and the appropriate inhibitor(s), and repotrectinib at 5 µM in the donor compartment. The cells were kept at 37 °C, pH~7.4, in a 5% COStandard animal housing and care were according to the institutional guidelines complying with Dutch and EU legislations. All experimental animal protocols, including power calculations, designed under the nationally approved DEC/CCD project AVD301002016595, were evaluated and approved by the Institutional Animal Care and Use Committee. Based on the principles of 3Rs , experimental procedures were optimized. Animal experiments are reported also in compliance with the ARRIVE 2.0 guidelines. In view of the intended clinical application of repotrectinib, either male or female mice were used in different parts of our studies, to minimize the unnecessary culling of mice, and in order to get experimental data for both genders and thus avoid systematic gender bias in our study.−/−Abcb1a/1b, −/−Abcg2, −/−Abcb1a/1b;Abcg2, −/−Oatp1a/1b, −/−Cyp3a and Cyp3aXAV mice [Wild-type, XAV mice ,25,26, ov/v), followed by further dilution with a 10 mM hydrochloric acid solution, yielding a dosing solution of 1 mg/mL, freshly prepared on the day of experiment. Final concentrations of the solvents in the administration solution were: 2%, 2%, 2%, and 94%  for DMSO, ethanol, polysorbate 80, and 10 mM hydrochloric acid solution, respectively.Repotrectinib was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 50 mg/mL. This stock solution was first diluted 3-fold with ethanol/polysorbate 80 , respectively. Plasma fraction was isolated from blood by centrifugation at 9000× g for 6 min at 4 °C. All samples were stored at −30 °C until analysis.To minimize variation in absorption upon oral administration, mice were fasted for 2–3 h before repotrectinib (10 mg/kg body weight) was administered by gavage into the stomach at 10 µL/g body weight, with a blunt-ended needle. Tail vein serial blood samples were collected at 0.125, 0.25, 0.5, 1, 2 and 4 h using heparinized capillary tubes  for the 8-h experiments. For the 4- and 2-h experiments, blood samplings were performed from the tail vein with the same schedule, but terminated at 2 and 1 h, respectively. Eight, four, or two hours after oral administration, mice were deeply anesthetized with 2–3% isoflurane, followed by cardiac puncture to collect blood in Eppendorf tubes containing heparin as an anticoagulant. Brain, liver, spleen, kidney, lung, small intestine, and testis were rapidly collected after sacrificing the mice by cervical dislocation. The small intestinal contents (SIC) were separated from small intestinal tissue (SI), which was flushed and rinsed with cold saline to remove any residual feces. Brain, liver, spleen, kidney, lung, SI, SIC, and testis were homogenized with 1, 3, 1, 2, 1, 3, 2, and 1 mL of 2% BSA  was applied when multiple groups were compared, and the Bonferroni post hoc correction was used to accommodate multiple testing. Differences were considered statistically significant when P < 0.05.A non-compartmental model using the PKSolver add-in program for Microsoft Excel was adopted to calculate the pharmacokinetic parameters of repotrectinib . Oral avr = 2.5, r = 1.0, r = 13.5, r = 1.1, Transepithelial drug transport was evaluated using polarized monolayers of MDCK-II parental cells and its subclones stably overexpressing hABCB1, hABCG2 or mAbcg2. Repotrectinib at 5 µM was modestly transported in the apical direction  relative to wild-type mice.To study the possible roles of ABCB1 and ABCG2 in controlling plasma exposure and tissue accumulation of repotrectinib, an 8-h pilot experiment was performed in female wild-type and −/−Abcb1a/1b;Abcg2 mice compared to wild-type mice  and brain-to-plasma ratios (9.5-fold) of repotrectinib in ype mice . Interesype mice . This suype mice . For the−/− mice .−/−Abcb1a/1b;Abcg2 mice, including hypoactivity and continuously closed eyes, at 1–2 h after oral administration. These signs of toxicity disappeared around 4 h. In contrast, no noticeable toxicity symptoms were found in wild-type mice.Of note, during the experiment we observed mild toxicity signs in −/−Abcb1a/1b, −/−Abcg2, and −/−Abcb1a/1b;Abcg2 mice were tested. This experiment was terminated at 4 h to achieve relatively high plasma levels of repotrectinib, which are more relevant for assessing the overall impact of ABC transporters on repotrectinib exposure. As shown in −/−Abcg2 and −/−Abcb1a/1b;Abcg2 mice compared to wild-type mice, with significantly higher peak plasma concentrations, but not in single −/−Abcb1a/1b mice. Moreover, there were no significant differences in plasma AUC0–4h of repotrectinib between −/−Abcg2 and −/−Abcb1a/1b;Abcg2 mice. These data together indicate that mAbcg2 could limit repotrectinib plasma exposure, but mAbcb1a/1b could not, at least not to a significant extent.To further investigate the separate or combined impact of ABCB1 and ABCG2 on plasma pharmacokinetics and tissue distribution of oral repotrectinib at 10 mg/kg, male wild-type, −/−Abcb1a/1b mice (4.1-fold) and especially −/−Abcb1a/1b;Abcg2 mice (29.1-fold) than in wild-type mice, but not in single −/−Abcg2 mice. Wild-type mice showed a relatively low brain-to-plasma ratio (0.15), which was substantially increased by 4.1-fold in −/−Abcb1a/1b mice and 14.2-fold in combination −/−Abcb1a/1b;Abcg2 mice (both P < 0.001). These results suggest that mAbcb1 could strongly limit the brain distribution of repotrectinib, whereas mAbcg2 also contributes to this process, but the latter effect can only be clearly observed when mAbcb1 is absent. Interestingly, similar signs of transient toxicity as before were observed in −/−Abcb1a/1b;Abcg2 mice, but not in any of the other strains. This suggests a possible role of the dramatically increased brain concentration of repotrectinib in the −/−Abcb1a/1b;Abcg2 strain. Qualitatively similar tissue distribution results were observed for the testis, where Sertoli cells form a similar physical and biochemical protective barrier as the brain, the blood-testis barrier (BTB). The testis-to-plasma ratios of repotrectinib were increased by 5.6-fold when both ABC transporter systems were absent, suggesting that the relative impact of mAbcb1a/1b and mAbcg2 was somewhat lower than that in the brain  and −/−Abcb1a/1b;Abcg2 mice (0.4%) compared to the other strains (−/−Abcg2 mice (4.8%), the recovery of repotrectinib was significantly lower in −/−Abcb1a/1b;Abcg2 mice. These data suggest that Abcg2 may affect primarily biliary excretion, and Abcb1a/1b primarily intestinal efflux of repotrectinib. A similar profile of repotrectinib distribution was seen in the SI tissue of these strains, whereas in spleen and kidney no significant differences were observed when assessing tissue-to-plasma ratios  was substantially increased by 1.5-fold (P < 0.05) in −/−Oatp1a/1b mice compared to wild-type mice. However, liver-to-plasma ratios were not significantly different between wild-type and −/−Oatp1a/1b mice at 8 h, when the plasma levels of repotrectinib in both mouse strains were much reduced compared to the peak concentration.OATP-mediated uptake can impact the oral availability and tissue distribution of drugs. The possible interactions of repotrectinib with OATP/SLCO uptake transporters are largely unknown. To study this aspect, a pilot experiment administering repotrectinib (10 mg/kg) orally to female wild-type and −/−Oatp1a/1b mice was terminated at 4 h after oral administration of 10 mg/kg repotrectinib. In −/−Oatp1a/1b mice, the plasma AUC0–4h of repotrectinib was significantly increased by 2-fold compared to that in wild-type mice  combined was collected given the similarity in results between these two compartments obtained in previous experiments. As shown in P < 0.001), respectively, compared to these parameters in wild-type mice. Importantly, while a similar amount of repotrectinib (% of dose) was recovered in the SIWC, after correction for plasma concentration or plasma exposure (AUC0–2h), the relative intestinal (SIWC) recovery of repotrectinib was significantly decreased by 2.8-fold and 1.9-fold, respectively, in −/−Oatp1a/1b mice compared to that in wild-type mice. Overall, these data demonstrate that mouse Oatp1a/1b transporters could mediate substantial uptake of repotrectinib into the liver, and thus markedly control the oral availability, liver distribution, and enterohepatic circulation of repotrectinib.To further study the relevance of these phenomena at a higher plasma concentration, a 2-h experiment was performed in male wild-type and −/−Cyp3a (Cyp3a knockout) and Cyp3aXAV mice, which are specifically overexpressing human CYP3A4 in the liver and intestine of −/−Cyp3a mice. Serial blood samples at different time points and organs at 8 h were collected and processed as described above. The plasma exposure of repotrectinib was significantly increased by 2.3-fold in −/−Cyp3a mice relative to wild-type mice. In addition, the repotrectinib plasma AUC0–8h in Cyp3aXAV mice was markedly decreased by 2.0-fold compared to −/−Cyp3a mice  and −/−Abcb1a/1b;Abcg2 (14-fold) mice. These transporters in the BBB thus actively keep repotrectinib out of the brain, with ABCB1 playing a dominant role, and may also restrict its CNS toxicity. A similar impact was observed for repotrectinib distribution in the testis. While somewhat indirect, liver concentration and SIC data suggested that Abcg2 plays a substantial role in biliary excretion/concentration, and Abcb1a/1b in direct intestinal efflux of repotrectinib. Markedly increased systemic exposure and decreased liver distribution of repotrectinib in −/−Oatp1a/1b compared to wild-type mice indicated that mouse Oatp1a/1b transporters can mediate the hepatic uptake of repotrectinib. Repotrectinib oral availability, but not relative tissue distribution, was further restricted by mouse Cyp3a and human CYP3A4. This shows that CYP3A is an important factor in determining metabolic clearance of repotrectinib in both species.Repotrectinib was found to be efficiently transported in vitro by human ABCB1, human ABCG2, and mouse Abcg2, and modestly by canine ABCB1. The oral availability of repotrectinib was significantly increased in max around 30 min, followed by gradual elimination. Importantly, the systemic exposure in wild-type mice dosed with repotrectinib at 10 mg/kg reached clinically relevant exposure levels, similar to those in patients taking repotrectinib at 160 mg once daily [After oral administration of repotrectinib, the absorption among all tested mouse strains was relatively fast, with a T−/−Abcg2 and −/−Abcb1a/1b;Abcg2 mice, but not in single −/−Abcb1a/1b mice, compared to wild-type mice. These data may reflect a partially reduced Abcg2-mediated excretion from hepatocytes into bile, thus leading to increased plasma exposure. The relatively reduced liver-to-plasma ratio of repotrectinib in Abcg2 knockout mice may reflect strongly decreased intrahepatic bile accumulation of repotrectinib due to the Abcg2 deficiency. The markedly reduced recovery of repotrectinib in the small intestinal content in the absence of especially Abcb1a/1b and, to a lesser extent, Abcg2  of repotrectinib in Abcg2-deficient strains. Moreover, the liver-to-plasma ratios of repotrectinib were significantly reduced by 30–35% in t, Abcg2  is likelrectinib .Considering the therapeutic importance of readily reaching the brain tumors and brain metastases commonly occurring in NSCLC, the interactions between repotrectinib and the ABC transporters in the BBB should be thoroughly investigated. Similar to brigatinib and many other TKIs ,28, repoCNS metastases represent a major cause of morbidity and mortality in patients with ROS1- and ALK- driven NSCLC. Given the limited brain exposure of repotrectinib found in wild-type mice because of Abcb1a/1b and Abcg2 functions, and their potential clinical relevance for limiting therapeutic efficacy against brain malignancies as well as tumor cells that themselves express ABC transporters, we could consider to use this insight to enhance the CNS (and tumor) exposure of repotrectinib by the application of potent pharmacological ABCB1 and ABCG2 inhibitors such as elacridar.The mild toxicity of repotrectinib observed in mice in which both Abcb1a/1b and Abcg2 were absent was probably related to on-target adverse events due to TRK inhibition, most likely in the brain (see below). The transient toxicity symptoms were very similar to those recently reported in an on-going phase I clinical trial NCT03063116), in which dizziness is the most common adverse effects of repotrectinib. This side effect could tentatively have resulted from decreased proprioception and cerebellar dysfunction 16, in wh.−/−Cyp3a and −/−Oatp1a/1b mice were at least as high as those in −/−Abcb1a/1b;Abcg2 mice, and yet no noticeable signs of toxicity were observed in either of these strains. The pronounced difference in susceptibility suggests that the markedly increased CNS distribution of repotrectinib is most likely behind this toxicity. Given this apparently mild and manageable toxicity together with the potential benefits for treatment of brain metastases, attempts to improve the CNS distribution of repotrectinib in patients by using an efficacious ABCB1/ABCG2 inhibitor may still be worth considering, but should obviously be carefully monitored for possibly increased toxicity symptoms.The toxicity of repotrectinib in mice appears not to be directly related to the plasma concentration and exposure of repotrectinib, as the systemic exposures in Among ROS1, TRK, and ALK inhibitors approved by the FDA or currently under clinical investigation, there are two other novel macrocyclic TKIs, lorlatinib (ALK/ROS1 inhibitor) and selitrectinib .−/−Cyp3a and Cyp3aXAV mice. This suggests that coadministration of a CYP3A inhibitor, such as ritonavir, may present an approach to boost the systemic exposure of repotrectinib in patients without invoking the risk of altering relative tissue distribution [Based on publicly available information, little is known about the interaction of repotrectinib with CYP3A. Our experiments clearly demonstrate that both mouse and human CYP3A can markedly reduce the plasma levels and thus overall systemic exposure of repotrectinib. This suggests that variable activity of CYP3A in patients, due to either drug–drug interactions or genetic polymorphisms, would likely have a noticeable impact on metabolic clearance and oral availability of repotrectinib. This probably should be taken into consideration in clinical dosing of repotrectinib for its future broader clinical use. It is worth mentioning that the relative tissue distribution of repotrectinib was similar among wild-type, ribution . In addiThis is the first study documenting that repotrectinib is transported by ABCB1 and ABCG2 in vitro, that its oral availability is limited by Abcg2, and that Abcb1a/1b and Abcg2 together substantially restrict repotrectinib brain accumulation in mice. Additionally, the absence of Abcb1 and Abcg2 markedly affects the enterohepatic cycling of repotrectinib through the diminished intestinal and/or hepatobiliary excretion of repotrectinib into the lumen of small intestine. Moreover, we demonstrated that, unexpectedly, OATP1A/1B could restrict the plasma exposure of repotrectinib by mediating its hepatic uptake and thus facilitating hepatobiliary excretion. Finally, human CYP3A4 and mouse Cyp3a can markedly reduce the oral availability of repotrectinib, without affecting its relative tissue distribution. These insights may be used to further enhance the therapeutic application, efficacy, and safety of repotrectinib.


In research about attention-deficit/hyperactivity disorder (ADHD) there is growing interest in evaluating cortical activation and using neurofeedback in interventions. This paper presents a case study using monopolar electroencephalogram recording (brain mapping known as MiniQ) for subsequent use in an intervention with neurofeedback for a 10-year-old girl presenting predominantly inattentive ADHD. A total of 75 training sessions were performed, and brain wave activity was assessed before and after the intervention. The results indicated post-treatment benefits in the beta wave (related to a higher level of concentration) and in the theta/beta ratio, but not in the theta wave (related to higher levels of drowsiness and distraction). These instruments may be beneficial in the evaluation and treatment of ADHD. Attention-deficit/hyperactivity disorder (ADHD) is one of the most common childhood disorders, affecting between 5.9% and 7.2% of the infant and adolescent population. The fifth edition of the Diagnostic and Statistical Manual of Mental Disorders  describeThe determination of ADHD symptoms, along with the underlying neuropsychology, as outlined by the theories above, have led in recent years to the incorporation of evaluation and intervention techniques that do not solely focus on the behavioral aspects of the disorder. More specifically, techniques such as electroencephalography in ADHD evaluation and neurofeedback in interventions may provide greater benefits in detection and treatment.The present study analyzes a specific case of ADHD with predominantly inattentive presentation, covering monopolar electroencephalogram recording  and intervention via neurofeedback.The study was approved by the relevant Ethics Committee of the Principality of Asturias , and all procedures complied with relevant laws and institutional guidelines.The current diagnostic criteria for ADHD can be found in the DSM-5  and in tOne alternative to qEEG is monopolar EEG recording , called MiniQ . The MiniQ is an instrument for evaluating brain waves from 12 cortical locations  . This tyThe distribution of electrical brain activity must be analyzed considering each site and the expected frequency. A regulated subject is characterized by more rapid activity in the frontal regions (predominantly beta) which decreases toward the posterior  regions, where slower waves (theta and delta) are expected ,11. SlowIn contrast, the electrical activity in a subject with predominantly inattentive ADHD is characterized by a predominance of theta waves (compared to beta) in the frontal regions, particularly on the left (F3). During tasks , a subject with predominantly inattentive ADHD will exhibit increased slower (theta) waves, and there will be a slowdown in the frontal regions that hinders attentional quality, as suggested by researchers such as Clarke et al.  and moreMany studies have examined the efficacy of the various treatments and interventions aimed at improving symptoms associated with ADHD , such as medication, behavioral treatments, and neurofeedback . NeurofThe use of neurofeedback in interventions for ADHD began in 1973, although the first study with positive results was published in 1976 . Since tNeurofeedback training is normally done two or three times a week, and around 40 sessions are needed to see changes in symptomatology . AlthougThe intervention protocol must be tailored to each individual case based on prior assessment, especially when using results from tests such as the MiniQ. In this context, the objective of the current study is to present the process of analyzing brainwaves in a case with ADHD (predominantly inattentive presentation) via the MiniQ test, the protocol for intervention using neurofeedback, and its efficacy. Although the alteration of brainwaves in specific areas in subjects with ADHD is well documented, and the efficacy of neurofeedback has been observed in various studies, the present study aims to provide a specific procedure for assessment and intervention. Researchers and professionals need specific protocols and procedures that allow them to determine what is effective for each individual case.This was a case study using monopolar electroencephalogram recording (brain mapping known as MiniQ) for subsequent use in an intervention with neurofeedback for a 10-year-old girl presenting predominantly inattentive ADHD.The subject was a 10-year-old girl in the fourth year of primary education. Her academic performance was poor, with the worst results in language, social sciences, and science. She found it difficult to go to school and was shy and reserved. She was the younger of two sisters, the older being an outstanding pupil. Her mother characterized her as a quiet girl who needed a lot of time to do any kind of task. In addition, during the study and academic tasks, she would often gaze into space, as if she were in her own world. Both her father and her mother evidenced concern for her school results, but also for her social relationships, as her self-absorption appeared in all contexts, making it hard for her to have conversations, pay attention to others, or follow the rules in games.The consultation was for poor academic performance, slowness doing tasks, and wandering attention from when she had started school, although that had increased in the previous year. Initially, the subject did not demonstrate any great willingness to attend the consultations, but over time, she demonstrated a participative attitude with good involvement in doing the tasks she was set.The subject’s school history was one of failure in the main school subjects. She had not had to repeat a school year, but her form tutors repeatedly raised this possibility with her parents. At the time of the study, there had been no clinical or educational psychology assessments. Previous diagnosis of ADHD was by her neuropediatrician one month before the assessment in the Psychology clinic consultation. From that point, guidelines were given for pharmacological treatment, which had not begun.An assessment was performed using a MiniQ . Assessment using the MiniQ is a two-step process  which is simple, relatively fast, and inexpensive.The first step is to make the recording from the 12 cortical sites, which can be done with eyes closed or open, and either with or without tasks (reading or arithmetic). This gives information about the values of the different brainwaves at each site. To begin, electrodes are placed on the earlobes and two active electrodes in each of the sites indicated by the program. Before beginning the assessment for each site, the impedance level—the quality of the connection—for each of the electrodes must be checked, both on the ears and on the scalp, to avoid artefacts. When the impedance level is below 4, the recording process can begin. The subject is instructed to remain still and to look at the computer screen where there is an image of a landscape. They must keep their eyes open and keep silent. The program guides the application, which is based on the placement of electrodes in groups of two following the sequence: Cz–Fz, Cz–Pz, F3–F4, C3–C4, P3–P4, O1–O2, and T3–T4. For sites F3–F4, subjects are asked to read a story quietly and to do some simple arithmetic . Once recordings have been made at all of the sites, the program filters the data to remove artefacts. Finally, the recorded data is interpreted, and the values are analyzed, allowing the state of the subjects’ brainwaves to be determined. Applying the test takes approximately 60 min.The second step is to analyze the collected data considering the site and the frequency ranges at each. The sites are labelled based on the four quadrants of the cortex: anterior, posterior, left hemisphere (odd numbers), and right hemisphere (even numbers). The instrument gives the results in two formats, an Excel spreadsheet and a PowerPoint. In addition to the measurements or wave values  at the sites noted above, the spreadsheet also includes the values for the ratios of theta/alpha, theta/beta, SMR/theta, and peak alpha. The PowerPoint presentation gives the same information, although over a background image of a brain, which allows scores to be seen at the relevant site see . With thThe values of the theta/beta ratios are interpreted based on Monastra et al. (1999) , bearinghttps://thoughttechnology.com/, accessed on 23 December 2021). Two protocols were used in the intervention process, an SMR protocol and a theta/beta protocol. The protocol and specific sites selected were based on the prior evaluation.The intervention was carried out using the Biograph Infiniti biofeedback software  and a blue square . The subject is told that the game involves trying to make the pink square as small as possible and the blue square as large as possible. The computer automatically generates the ranges over which the waves are worked, although they can be changed manually by the therapist. The desired working theta/beta ratio can also be set manually. The protocol begins with high ratios, close to three, such that the task is simple and the subject achieves reinforcement on many occasions. The ratio is progressively reduced according to the subject’s progress.The intervention lasted for a year and consisted of 75 neurofeedback sessions. There were two phases to the training. The first phase, “the regulation phase”, covered the first 15 sessions, during which the SMR protocol was followed at Cz. The aim of this first phase was to strengthen SMR and inhibit theta and beta3 in the central region. These sessions were around 45 min each. To avoid tiredness, different presentations of neurofeedback were used (videogame or film) during the sessions, with five-minute breaks between each presentation.The second phase ran from session 16 to session 75. In these sessions, the SMR protocol at Cz was applied for 20 min, followed by a five-minute break before the theta/beta protocol at Fz was applied for another 20 min. For the first six months of the intervention, sessions were 45 min, twice weekly. During the remaining six months, the sessions were weekly and remained 45 min long.Based on the information obtained over the evaluation of the case, and considering the prior diagnosis from her pediatric neurologist, the subject presented ADHD with predominantly inattentive presentation. As Given the brainwave profile, the aim of the intervention was to reduce theta and increase beta in the frontal zones. That indicated using the SMR and theta/beta protocols .Once the neurofeedback intervention was completed, brainwave activity was assessed again using the MiniQ. Theta activity fell following the intervention, both at Fz (by 0.77) and at Cz (by 1.56). To put it another way, there was a reduction in the slow wave at both sites . This is in line with expected values of theta at the cortical level, as they should be higher in posterior areas and lower in frontal areas.There was also an increase in beta at the two sites, with a 3.60-point increase at Fz and a 4.2-point increase at Cz. In this case, the intervention produced considerable increases in the rapid wave values at both sites, although the value was slightly higher in the central area than in the frontal. Values for beta waves are expected to be higher in frontal areas than central areas, and although that was not the case here, the values were very close. The SMR wave also increased notably, by 2.57 points at Fz and 2.89 points at Cz. In short, the intervention led to a slight reduction in the slow wave, with lower values at post-treatment (less distraction), and increases in fast waves, beta, and SMR, with higher values after the intervention (better ability to concentrate). The theta/beta ratio also decreased at post-treatment , both at Fz (by 0.69) and Cz (by 0.96), from values close to those for ADHD to scores more indicative of a subject without ADHD.In addition, as initially proposed, the assessment with the MiniQ also considered the subject’s activation levels during reading and arithmetic tasks. Measurement of these values was at sites F3 and F4. The subject did three types of task for two minutes each: Paying attention to the screen on which a landscape appeared, reading a story, and doing simple arithmetic (addition and subtraction). As In the first task (pay attention to the screen), the values for theta, beta, and beta3 at F3 and F4 all rose. In the second and third tasks (reading and arithmetic), there were variations in all of the waves, both slow and fast. These results indicate that there was no improvement during tasks following the intervention, because although the fast waves (beta and beta3) increased, the slow wave (theta) did not diminish. Following the intervention, the expectation was to have increased levels of beta and beta3 , while reducing levels of theta. However, as The aim of this study was to present the process for detecting a case of ADHD (predominantly inattentive presentation) using the MiniQ test, along with the neurofeedback intervention protocol and its efficacy. In terms of detection, the MiniQ showed the subjects’ brain activity, which together with behavioral symptoms, provided details of their characteristic profile and allowed tailored treatment. Various studies in the literature have concluded that children with ADHD exhibit higher levels of theta waves and lower levels of beta waves, particularly in frontal areas ,11. In aIn the current case study, the MiniQ was relatively simple to apply, and it provided large amounts of information related to brainwave values at the 12 different sites. More specifically, the EEG record of the 10-year-old subject showed lower levels of beta activity in the frontal regions and a higher level of theta activity in the frontal and central regions. However, the slow wave (theta) should be higher in posterior regions and fall in the central area, whereas the fast waves (beta and beta3) should be higher in the anterior regions and lower in the posterior. The subject’s theta/beta ratio was high (Cz: 2.05) and close to values seen in subjects with ADHD according to Monastra et al. . and JarThe intervention produced a variety of results. Firstly, there was a small reduction in theta activity and an increase in SMR, which would indicate better levels of attention. In addition, the theta/beta ratio fell to levels which were closer to those in subjects without ADHD. However, this improvement in the theta/beta ratio was due to increased beta rather than by the reduction of theta. Janssen et al. found similar results in 38 children with ADHD by analyzing the learning curve during 29 neurofeedback training sessions [On similar lines, during tasks after the intervention (reading and arithmetic), there was no reduction in theta but there was an increase in beta and beta3, again in line with the results from Janssen et al. . For reaAs Enriquez-Geppert et al.  and DuriThese results point toward the hypothesis that the low baseline cortical activation seen in subjects with ADHD would be found to be the basis of the disorder. While neurofeedback training may produce a positive progression, difficulties would persist, particularly during specific tasks in which subjects with ADHD are unable to achieve an ideal profile of brainwave activity for optimum performance. This is a reflection of the fact that the disorder persists throughout life, and hence, despite improvements in the cortical activation profile and the subject learning to strengthen their beta wave activity to concentrate, there will continue to be high levels of theta.In this context, various studies such as Doppelmayr and Weber  and VernIt is also worth noting that, while previous studies employed similar protocols , the numbers of sessions and the session durations varied between studies. These variations may be related to the differences in the results and indicate the need to establish intervention protocols not only about what to work with (brain waves) but also how to do it . At the same time, the present study underscores the need to tailor protocols to subjects’ profiles, along the same lines as previous studies, for instance Cueli et al. , who notIn the future, it would be advisable to assess subjects’ levels of activation every 10 to 15 sessions of neurofeedback training in order to tailor the protocols to their progress and to study the theta/beta ratio learning curve as mentioned above. One limitation it is important to note is that multidomain assessments before, during, and after treatment (and adequate follow-up) should include blinding and sham inertness Another limitation of the present study is the lack of a behavioral assessment that would allow for an in-depth analysis of the subject’s progress in line with the protocol from Holtmann et al. . At the Finally, despite the limitations described above, it would also be useful to consider the possibility of incorporating this type of training in more cases of subjects with ADHD, because neurofeedback intervention may offer long-term benefits in terms of improving the attentional abilities of subjects with ADHD, especially if one considers that approximately a third of ADHD patients do not respond to, or sufficiently tolerate, pharmacological treatment . In this