Auditory N1 event-related potential amplitude is predictive of serum concentration of BPN14770 in fragile x syndrome

Auditory N1 event-related potential amplitude is predictive of serum concentration of BPN14770 in fragile x syndrome | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Auditory N1 event-related potential amplitude is predictive of serum concentration of BPN14770 in fragile x syndrome Jordan E. Norris, Elizabeth M. Berry-Kravis, Mark D. Harnett, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4474353/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Nov, 2024 Read the published version in Molecular Autism → Version 1 posted 9 You are reading this latest preprint version Abstract Fragile X syndrome (FXS) is a rare neurodevelopmental disorder caused by a CGG repeat expansion ≥ 200 repeats in 5’ untranslated region of the FMR1 gene, leading to intellectual disability and cognitive difficulties, including in the domain of communication. A recent phase 2a clinical trial testing BPN14770, a phosphodiesterase 4D inhibitor, showed improved cognition in 30 adult males with FXS on drug relative to placebo. The initial study found significant improvements in clinical measures assessing cognition, language, and daily functioning in addition to marginal improvements in electroencephalography (EEG) results for the amplitude of the N1 event-related potential (ERP) component. EEG results suggest BPN14770 improved neural hyperexcitability in FXS. The current study investigated the relationship between BPN14770 pharmacokinetics (PK) and the amplitude of the N1 ERP component from the initial data. Consistent with the original group-level finding in period 1 of the study, participants who received BPN14770 in the period 1 showed a significant correlation between N1 amplitude and serum concentration of BPN14770. These findings strengthen the validity of the original result, indicating that BPN14770 improves cognitive performance by modulating neural hyperexcitability. This study represents the first report of significant correlation between a reliably abnormal EEG marker and serum concentration of a novel pharmaceutical in FXS. biomarker fragile x syndrome zatolmilast EEG pharmacokinetics Figures Figure 1 Figure 2 Background Fragile X syndrome (FXS) is a rare neurodevelopmental disorder caused by a repeat expansion of > 200 CGG repeats in the 5’ untranslated region of the FMR1 gene located on the X chromosome (Santoro et al., 2012; Straub et al., 2023). Clinical features of FXS include high rates of intellectual disability, anxiety, and difficulties with executive function. Cognitive features of FXS, including communication difficulties, are often cited as among the most distressing for individuals with FXS and their families with no treatment currently existing for ameliorating cognitive symptoms (Weber et al., 2019). A recent phase 2 clinical trial assessing BPN14770 (now Zatolmilast), a first-in-class phosphodiesterase 4D (PDE4D) inhibitor, in adult males with FXS demonstrated cognitive improvements on the performance-based NIH Toolbox Cognitive Battery and in caregiver reports of language and daily functioning (Berry-Kravis et al., 2021). BPN14770 works to increase cyclic AMP (cAMP) levels by reducing phosphodiesterase activity (Gurney et al., 2019). The a priori secondary physiological measures included electroencephalography (EEG) which measured brain activity during an auditory habituation task. Specifically, the amplitude of the N1 event related potential (ERP) component was assessed and demonstrated marginal reductions suggesting improvements in neural hyperexcitability (Berry-Kravis et al., 2021). Generally, individuals with FXS exhibit increased neural responses to auditory stimuli compared to typically developed controls with the consensus that heighted neural responses to auditory stimulation reflects overall neural hyperexcitability as well as increased sensory sensitivity (Contractor et al., 2015; Ethridge et al., 2019). While BPN14770 does not target mechanisms related directly to neural hyperexcitability, a PDE4D inhibitor may support neural network organization by improving long-term potentiation through improved cyclic AMP signaling (Mierau et al., 2017). Given the validity of the EEG findings, the N1 amplitude outcomes provide a physiological bridge between known molecular mechanisms of BPN14770 and improvements in clinical outcomes. However, reductions in usable data for ERP results reduced power to detect effects, raising the question of whether the marginal efficacy findings for the N1 ERP reflect true, but statistically underpowered, reductions in neural hyperexcitability. To confirm validity of the finding, N1 amplitude reductions were assessed against plasma BPN14770 levels from pharmacokinetic assessment. Methods Participants were 30 males (age 18-41 years, M = 31.63, SD = 7.32; IQ 24.63-66.19, M = 42.78, SD = 11.6) with FXS participating in a single-center, phase 2 clinical trial assessing the efficacy and safety of BPN14770 (ClinicalTrials.gov identifier: NCT03569631, registration date: June 26, 2018). All participants or their legal guardians signed informed consent which included consent for EEG data collection. All study procedures were approved by the Institutional Review Board at Rush University Medical Center; EEG analysis and data management procedures were additionally approved by the IRB at University of Oklahoma. Procedure Habituation Task The auditory habituation task consisted of 150 paired 50-ms white-noise bursts with a 500 ms interstimulus-onset interval. Each stimulus train was separated by a 4,000 ms intertrial interval for a total participation time of 11.5 minutes. Event related potential (ERP) values were measured at baseline, crossover, and end of trial. The N1 amplitude was calculated for both stimuli in the habituation pair and defined as the most negative peak between 50 and 200 ms post-stimulus. EEG Recording and Preprocessing EEG data were continuously recorded and digitized at 512Hz using a 32-channel BioSemi ActiveTwo system (BioSemi) with a 5th order Bessel anti-aliasing filter at 200 Hz. Sensors were all active referenced to a driven right leg feedback loop between Cz and a central posterior ground electrode during recording. Data were inspected offline, resampled to 500 Hz, and preprocessed to remove artifacts prior to analysis using MATLAB 2018. In order, 1.) data were digitally filtered offline from 0.5 to 100 Hz with a 57-63 Hz notch, 2.) bad channels were visually inspected and interpolated with no more than ~ 5% of sensors interpolated (max of 2 channels), 3.) segments with high artifact contamination (i.e., large movement-related artifacts) were manually rejected, 4.) data were then submitted to independent component analysis (ICA) for further artifact correction, and 5.) re-referenced to the average of all channels (Delorme & Makeig, 2004). Pharmacokinetics Pharmacokinetic (PK) samples were collected at baseline and at the end of each crossover arm to confirm the study drug was present when expected (Berry-Kravis et al., 2021). The original study did not have a sufficient wash-out period and therefore the main analyses were conducted on data from period 1 between subjects. As a result, the period 1 PK value variable has 0 ng ml -1 BPN14770 if the participant received placebo during period 1. Statistical Analysis A Spearman rank correlation analysis was conducted to determine whether PK values correspond to a reduction in the N1 amplitude to the first stimulus in the habituation pair which demonstrated marginal significance in the original analysis of the EEG N1 ERP component. Correlations were run separately 1) including both placebo and treatment groups, and 2) including just the treatment group, to account for inherent baseline variability in N1 amplitude across participants that may skew correlation values when all individuals in the placebo group have a PK value of 0 ng ml -1 . Additional exploratory correlations were run to assess PK relationships with the second stimulus in the habituation pair. Results Including both placebo and treatment groups in the analysis, there was a marginally significant positive correlation between the period 1 PK values and the N1 amplitude to the first stimulus (rho = .396, p = .055; N = 24 valid) but not for the second repeated stimulus (rho = .296, p = .160; N = 24 valid). The follow up correlation run on only those in period 1 who received BPN14770 confirmed the positive relationship between PK values and the N1 amplitude for stimulus 1 (rho = .608, p = .036; N = 12 valid; Fig. 1 ). Interestingly, the correlation between the N1 amplitude of the second peak and BPN14770 PK values was marginally significant when evaluating only those who received BPN14770 during period 1 (r = .524, p = .08; N = 12 valid; Fig. 2 ). It is important to note that the N1 ERP amplitude is a negative value, so positive correlations indicate a reduction in N1 amplitude with increased serum concentration of BPN14770. Discussion Individuals with FXS tend to exhibit increased neural responses reflected by increased ERP amplitudes to auditory stimuli compared to typically developed individuals, likely due to neural hyperexcitability (Ethridge et al., 2016 ; 2019 ; Tempio et al., 2023 ). The original results from the phase 2a clinical trial suggested BPN14770 reduced the ERP amplitude of the first stimulus in a habituation pair. Given the N1 is a negative-going ERP component, the positive correlation presented in the current study indicates a decrease in negative amplitude with BPN14770, which is an improvement that corresponds to a decreased neural response to the stimulus. Figure 1 demonstrates the expanded negative range of ERP values for the placebo group, highlighting the group-level shift in ERP amplitudes toward smaller values, as well as the relationship between BPN14770 serum concentration and decreases in N1 negative amplitude. The individual with the largest plasma concentration of BPN14770 shows a nearly zero-amplitude value for N1, suggesting a potential upper limit to effective dosing for this individual for maintaining the N1 amplitude within typical levels. The current results raise confidence in the validity of the original, marginal ERP amplitude group-level result in favor of BPN14770 improvements. Confirmation of prior results lends support to the conclusion that BPN14770 reduces neural hyperexcitability during stimulus processing. Variations in the auditory N1 ERP have been associated with language and communication as well as sensory outcomes in FXS (Ethridge et al., 2016 ; 2019 ; Schmitt et al., 2020 ) the results have implications for language processing and may underlie the noted improvements in clinical outcomes from the original study (Berry-Kravis et al., 2021 ). Limitations Limitations included the small sample size consistent with Phase 2 studies, expected data loss in EEG data due to movement which further reduced statistical power, and carry-over effects in placebo measures from period 2. While the correlation supports the original findings, and suggests that the effect was present but underpowered, the period 1 analyses within group reflect only 12 individuals. Nevertheless, this study represents the first report of significant correlation between a reliably abnormal EEG marker and serum concentration of a novel pharmaceutical in FXS. The N1 amplitude will be assessed as part of the ongoing phase 3 trial testing BPN14770 in a larger sample which will provide a sufficiently powered statistical model for assessing N1 amplitude improvement with BPN14770 treatment. Abbreviations FXS: fragile x syndrome FMR1: fragile x messenger ribonucleoprotein 1 EEG: electroencephalography ERP: event-related potential PK: pharmacokinetics NIH: National Institutes of Health PDE4D: phosphodiesterase 4D Declarations Consent to Participate Declaration: All participants or their legal guardians signed informed consent which included consent for EEG data collection. Human Ethics Approval Declaration: All study procedures were approved by the Institutional Review Board at Rush University Medical Center; EEG analysis and data management procedures were additionally approved by the Institutional Review Board at University of Oklahoma, following the guidelines of the Declaration of Helsinki. Consent for publication: N/A Availability of data and materials: All data generated or analyzed during this study are included in this published article. All data points reported in the analyses are presented in Figures 1 and 2. Competing Interests: J.E.N, E.M.B.-K., L.E.E., M.A.R., A.O., C.M. and J.F. declare no competing interests. M.D.H. and S.D.R. are paid consultants to Tetra Therapeutics. M.E.G. is an employee of Tetra Therapeutics, which is a wholly owned subsidiary of Shionogi & Co., Ltd that has a financial interest in BPN14770. Funding: Direct clinical costs were funded by the FRAXA Research Foundation. Access to and training on the NIH-TCB was obtained in association with work on HD076189 (E.M.B.K.). Tetra Therapeutics provided drug product and funded trial administration and independent data analysis. Authors contributions: J.E.N. conducted all analyses and drafted the manuscript E.M.B.-K. designed and conducted the clinical trial M.D.H. led the biostatistical analysis for the original manuscript S.A.R. designed the clinical trial and served as medical monitor M.A.R. preprocessed and organized the EEG dataset A.H.O., C.M. and J.F. conducted the clinical trial M.E.G. contributed to the clinical protocol L.E.E. supervised all aspects of EEG experimental design, data analysis, and manuscript preparation. All authors contributed significantly to manuscript preparation. Acknowledgements: We thank the patients and their families for participating in the clinical trial. References Berry-Kravis, EM, Harnett, MD, Reines, SA, Reese, MA, Ethridge, LE, Outterson, AH, Michalak, C, Furman, J, & Gurney, ME. Inhibition of phosphodiesterase-4D in adults with fragile X syndrome: a randomized, placebo-controlled, phase 2 clinical trial. Nat Med, 2021; 27(5), 862-870. https://doi.org/10.1038/s41591-021-01321-w Contractor, A, Klyachko, VA, & Portera-Cailliau, C. Altered Neuronal and Circuit Excitability in Fragile X Syndrome. Neuron, 2015; 87(4), 699–715. https://doi.org/10.1016/j.neuron.2015.06.017 Delorme, A, & Makeig, S. EEGLAB: an open source toolbox for analysis of single-trial EEG dynamics including independent component analysis. J Neurosci Methods, 2004; 134(1), 9-21. https://doi.org/10.1016/j.jneumeth.2003.10.009 Ethridge LE, De Stefano LA, Schmitt LM, Woodruff NE, Brown KL, Tran M, Wang J, Pedapati EV, Erickson CA and Sweeney JA. Auditory EEG Biomarkers in Fragile X Syndrome: Clinical Relevance. Front. Integr. Neurosci. 2019; 13:60. doi: 10.3389/fnint.2019.00060 Ethridge, LE, White, SP, Mosconi, MW, Wang, J, Byerly, MJ, & Sweeney, JA. Reduced habituation of auditory evoked potentials indicate cortical hyper-excitability in Fragile X Syndrome. Translational psychiatry, 2016; 6(4), e787. https://doi.org/10.1038/tp.2016.48 Gurney ME, Nugent, RA, Mo, X, Sindac, JA, Hagen, TJ, Fox, D, O’Donnell, JM, Zhang, C, Xu, Y, Zhang, H-T, Groppi, VE, Bailie, M, White, RE, Romero, DE, Vellekoop, AS, Walker, JR, Surman, MD, Zhu, L, & Campbell RF. Journal of Medicinal Chemistry, 2019; 62 (10), 4884-4901 http://doi.org/10.1021/acs.jmedchem.9b00193 Mierau, A, Klimesch, W, & Lefebvre, J. State-dependent alpha peak frequency shifts: Experimental evidence, potential mechanisms and functional implications. Neuroscience, 2017; 360, 146-154. https://doi.org/10.1016/j.neuroscience.2017.07.037 Santoro, MR, Bray, SM, & Warren, ST. Molecular mechanisms of fragile X syndrome: a twenty-year perspective. Annu Rev Pathol, 2012; 7, 219-245. https://doi.org/10.1146/annurev-pathol-011811-132457 Schmitt, LM, Wang, J, Pedapati, EV, Thurman, AJ, Abbeduto, L, Erickson, CA, Sweeney, JA. A neurophysiological model of speech production deficits in fragile X syndrome. Brain Communications, 2020; 2 (1), 2020, fcz042, https://doi.org/10.1093/braincomms/fcz042 Straub, D, Schmitt, LM, Boggs, AE, Horn, PS, Dominick, KC, Gross, C, & Erickson, CA. A sensitive and reproducible qRT-PCR assay detects physiological relevant trace levels of FMR1 mRNA in individuals with Fragile X syndrome. Sci Rep, 2023; 13(1), 3808. https://doi.org/10.1038/s41598-023-29786-4 Tempio A, Boulksibat A, Bardoni B and Delhaye S. Fragile X Syndrome as an interneuronopathy: a lesson for future studies and treatments. Front. Neurosci. 2023; 17:1171895. doi: 10.3389/fnins.2023.1171895 Weber, JD, Smith, E, Berry-Kravis, E, Cadavid, D, Hessl, D, & Erickson, C. Voice of People with Fragile X Syndrome and Their Families: Reports from a Survey on Treatment Priorities. Brain Sci, 9(2). https://doi.org/10.3390/brainsci9020018 Additional Declarations Competing interest reported. J.E.N, E.M.B.-K., L.E.E., M.A.R., A.O., C.M. and J.F. declare no competing interests. M.D.H. and S.D.R. are paid consultants to Tetra Therapeutics. M.E.G. is an employee of Tetra Therapeutics, which is a wholly owned subsidiary of Shionogi & Co., Ltd that has a financial interest in BPN14770. Cite Share Download PDF Status: Published Journal Publication published 02 Nov, 2024 Read the published version in Molecular Autism → Version 1 posted Editorial decision: Revision requested 02 Aug, 2024 Reviews received at journal 24 Jun, 2024 Reviews received at journal 21 Jun, 2024 Reviewers agreed at journal 05 Jun, 2024 Reviewers agreed at journal 05 Jun, 2024 Reviewers invited by journal 05 Jun, 2024 Editor assigned by journal 01 Jun, 2024 Submission checks completed at journal 31 May, 2024 First submitted to journal 24 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4474353","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":312629867,"identity":"8fc8dbac-398f-44b3-bdb5-df233975f999","order_by":0,"name":"Jordan E. 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All PK values at 0 are individuals who received placebo during period 1.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4474353/v1/c89efe3e83b9238ec76aa554.jpeg"},{"id":58385306,"identity":"ea01e3aa-16ec-4965-80e0-845c12258e46","added_by":"auto","created_at":"2024-06-14 18:39:32","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":61381,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ePeriod 1 PK – N1 Correlation for the Second ERP Peak. \u003c/em\u003eCorrelation plot for period 1 PK values and N1 amplitude. All PK values at 0 are individuals who received placebo during period 1.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4474353/v1/a763ac4e5806d0374d80d7d4.jpeg"},{"id":68207245,"identity":"54262d59-bdc4-4b4e-bb5e-dedcebf4d32a","added_by":"auto","created_at":"2024-11-04 16:36:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":363763,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4474353/v1/1f07b75e-0024-4d38-87e8-d3904023647d.pdf"}],"financialInterests":"Competing interest reported. J.E.N, E.M.B.-K., L.E.E., M.A.R., A.O., C.M. and J.F. declare no competing interests. M.D.H.\nand S.D.R. are paid consultants to Tetra Therapeutics. M.E.G. is an employee of Tetra\nTherapeutics, which is a wholly owned subsidiary of Shionogi \u0026 Co., Ltd that has a\nfinancial interest in BPN14770.","formattedTitle":"Auditory N1 event-related potential amplitude is predictive of serum concentration of BPN14770 in fragile x syndrome","fulltext":[{"header":"Background","content":"\u003cp\u003eFragile X syndrome (FXS) is a rare neurodevelopmental disorder caused by a repeat expansion of \u0026gt; 200 CGG repeats in the 5\u0026rsquo; untranslated region of the \u003cem\u003eFMR1\u003c/em\u003e gene located on the X chromosome (Santoro et al., 2012; Straub et al., 2023). Clinical features of FXS include high rates of intellectual disability, anxiety, and difficulties with executive function. Cognitive features of FXS, including communication difficulties, are often cited as among the most distressing for individuals with FXS and their families with no treatment currently existing for ameliorating cognitive symptoms (Weber et al., 2019).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA recent phase 2 clinical trial assessing BPN14770 (now Zatolmilast), a first-in-class phosphodiesterase 4D (PDE4D) inhibitor, in adult males with FXS demonstrated cognitive improvements on the performance-based NIH Toolbox Cognitive Battery and in caregiver reports of language and daily functioning (Berry-Kravis et al., 2021). BPN14770 works to increase cyclic AMP (cAMP) levels by reducing phosphodiesterase activity (Gurney et al., 2019). The \u003cem\u003ea priori\u003c/em\u003e secondary physiological measures included electroencephalography (EEG) which measured brain activity during an auditory habituation task. Specifically, the amplitude of the N1 event related potential (ERP) component was assessed and demonstrated marginal reductions suggesting improvements in neural hyperexcitability (Berry-Kravis et al., 2021). Generally, individuals with FXS exhibit increased neural responses to auditory stimuli compared to typically developed controls with the consensus that heighted neural responses to auditory stimulation reflects overall neural hyperexcitability as well as increased sensory sensitivity (Contractor et al., 2015; Ethridge et al., 2019). While BPN14770 does not target mechanisms related directly to neural hyperexcitability, a PDE4D inhibitor may support neural network organization by improving long-term potentiation through improved cyclic AMP signaling (Mierau et al., 2017).\u003c/p\u003e\n\u003cp\u003eGiven the validity of the EEG findings, the N1 amplitude outcomes provide a physiological bridge between known molecular mechanisms of BPN14770 and improvements in clinical outcomes. However, reductions in usable data for ERP results reduced power to detect effects, raising the question of whether the marginal efficacy findings for the N1 ERP reflect true, but statistically underpowered, reductions in neural hyperexcitability. To confirm validity of the finding, N1 amplitude reductions were assessed against plasma BPN14770 levels from pharmacokinetic assessment.\u0026nbsp;\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eParticipants were 30 males (age 18-41 years, \u003cem\u003eM\u003c/em\u003e = 31.63, \u003cem\u003eSD\u003c/em\u003e = 7.32; IQ 24.63-66.19, \u003cem\u003eM\u003c/em\u003e = 42.78, \u003cem\u003eSD\u0026nbsp;\u003c/em\u003e= 11.6) with FXS participating in a single-center, phase 2 clinical trial assessing the efficacy and safety of BPN14770 (ClinicalTrials.gov identifier: NCT03569631, registration date: June 26, 2018). All participants or their legal guardians signed informed consent which included consent for EEG data collection. All study procedures were approved by the Institutional Review Board at Rush University Medical Center; EEG analysis and data management procedures were additionally approved by the IRB at University of Oklahoma.\u003c/p\u003e\n\u003cp\u003eProcedure\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eHabituation Task\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe auditory habituation task consisted of\u0026nbsp;150 paired 50-ms white-noise bursts with a 500\u0026thinsp;ms interstimulus-onset interval. Each stimulus train was separated by a 4,000\u0026thinsp;ms intertrial interval for a total participation time of 11.5 minutes.\u0026nbsp;Event related potential (ERP) values were measured at baseline, crossover, and end of trial. The N1 amplitude was calculated for both stimuli in the habituation pair and defined as the most negative peak between 50 and 200 ms post-stimulus.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEEG Recording and Preprocessing\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eEEG data were continuously recorded and digitized at 512Hz using a 32-channel BioSemi ActiveTwo system (BioSemi) with a 5th order Bessel anti-aliasing filter at 200 Hz. Sensors were all active referenced to a driven right leg feedback loop between Cz and a central posterior ground electrode during recording. Data were inspected offline, resampled to 500 Hz, and preprocessed to remove artifacts prior to analysis using MATLAB 2018. In order, 1.) data were digitally filtered offline from 0.5 to 100 Hz with a 57-63 Hz notch, 2.) bad channels were visually inspected and interpolated with no more than ~ 5% of sensors interpolated (max of 2 channels), 3.) segments with high artifact contamination (i.e., large movement-related artifacts) were manually rejected, 4.) data were then submitted to independent component analysis (ICA) for further artifact correction, and 5.) re-referenced to the average of all channels (Delorme \u0026amp; Makeig, 2004).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePharmacokinetics\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePharmacokinetic (PK) samples were collected at baseline and at the end of each crossover arm to confirm the study drug was present when expected (Berry-Kravis et al., 2021). The original study did not have a sufficient wash-out period and therefore the main analyses were conducted on data from period 1 between subjects. As a result, the period 1 PK value variable has 0 ng ml\u003csup\u003e-1\u003c/sup\u003e BPN14770 if the participant received placebo during period 1.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eStatistical Analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA Spearman rank correlation analysis was conducted to determine whether PK values correspond to a reduction in the N1 amplitude to the first stimulus in the habituation pair which demonstrated marginal significance in the original analysis of the EEG N1 ERP component. Correlations were run separately 1) including both placebo and treatment groups, and 2) including just the treatment group, to account for inherent baseline variability in N1 amplitude across participants that may skew correlation values when all individuals in the placebo group have a PK value of 0 ng ml\u003csup\u003e-1\u003c/sup\u003e. Additional exploratory correlations were run to assess PK relationships with the second stimulus in the habituation pair.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eIncluding both placebo and treatment groups in the analysis, there was a marginally significant positive correlation between the period 1 PK values and the N1 amplitude to the first stimulus (rho\u0026thinsp;=\u0026thinsp;.396, p\u0026thinsp;=\u0026thinsp;.055; N\u0026thinsp;=\u0026thinsp;24 valid) but not for the second repeated stimulus (rho\u0026thinsp;=\u0026thinsp;.296, p\u0026thinsp;=\u0026thinsp;.160; N\u0026thinsp;=\u0026thinsp;24 valid). The follow up correlation run on only those in period 1 who received BPN14770 confirmed the positive relationship between PK values and the N1 amplitude for stimulus 1 (rho\u0026thinsp;=\u0026thinsp;.608, p\u0026thinsp;=\u0026thinsp;.036; N\u0026thinsp;=\u0026thinsp;12 valid; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eInterestingly, the correlation between the N1 amplitude of the second peak and BPN14770 PK values was marginally significant when evaluating only those who received BPN14770 during period 1 (r\u0026thinsp;=\u0026thinsp;.524, p\u0026thinsp;=\u0026thinsp;.08; N\u0026thinsp;=\u0026thinsp;12 valid; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). It is important to note that the N1 ERP amplitude is a negative value, so positive correlations indicate a \u003cem\u003ereduction\u003c/em\u003e in N1 amplitude with increased serum concentration of BPN14770.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIndividuals with FXS tend to exhibit increased neural responses reflected by increased ERP amplitudes to auditory stimuli compared to typically developed individuals, likely due to neural hyperexcitability (Ethridge et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Tempio et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The original results from the phase 2a clinical trial suggested BPN14770 reduced the ERP amplitude of the first stimulus in a habituation pair. Given the N1 is a negative-going ERP component, the positive correlation presented in the current study indicates a decrease in negative amplitude with BPN14770, which is an improvement that corresponds to a decreased neural response to the stimulus. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e demonstrates the expanded negative range of ERP values for the placebo group, highlighting the group-level shift in ERP amplitudes toward smaller values, as well as the relationship between BPN14770 serum concentration and decreases in N1 negative amplitude. The individual with the largest plasma concentration of BPN14770 shows a nearly zero-amplitude value for N1, suggesting a potential upper limit to effective dosing for this individual for maintaining the N1 amplitude within typical levels.\u003c/p\u003e \u003cp\u003eThe current results raise confidence in the validity of the original, marginal ERP amplitude group-level result in favor of BPN14770 improvements. Confirmation of prior results lends support to the conclusion that BPN14770 reduces neural hyperexcitability during stimulus processing. Variations in the auditory N1 ERP have been associated with language and communication as well as sensory outcomes in FXS (Ethridge et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Schmitt et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) the results have implications for language processing and may underlie the noted improvements in clinical outcomes from the original study (Berry-Kravis et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eLimitations\u003c/h2\u003e \u003cp\u003eLimitations included the small sample size consistent with Phase 2 studies, expected data loss in EEG data due to movement which further reduced statistical power, and carry-over effects in placebo measures from period 2. While the correlation supports the original findings, and suggests that the effect was present but underpowered, the period 1 analyses within group reflect only 12 individuals. Nevertheless, this study represents the first report of significant correlation between a reliably abnormal EEG marker and serum concentration of a novel pharmaceutical in FXS. The N1 amplitude will be assessed as part of the ongoing phase 3 trial testing BPN14770 in a larger sample which will provide a sufficiently powered statistical model for assessing N1 amplitude improvement with BPN14770 treatment.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eFXS: fragile x syndrome\u003c/p\u003e\n\u003cp\u003eFMR1: fragile x messenger ribonucleoprotein 1\u003c/p\u003e\n\u003cp\u003eEEG: electroencephalography\u003c/p\u003e\n\u003cp\u003eERP: event-related potential\u003c/p\u003e\n\u003cp\u003ePK: pharmacokinetics\u003c/p\u003e\n\u003cp\u003eNIH: National Institutes of Health\u003c/p\u003e\n\u003cp\u003ePDE4D: phosphodiesterase 4D\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eConsent to Participate Declaration: All participants or their legal guardians signed informed consent which included consent for EEG data collection.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHuman Ethics Approval Declaration: All study procedures were approved by the Institutional Review Board at Rush University Medical Center; EEG analysis and data management procedures were additionally approved by the Institutional Review Board at University of Oklahoma, following the guidelines of the Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003eConsent for publication: N/A\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials: All data generated or analyzed during this study are included in this published article. All data points reported in the analyses are presented in Figures 1 and 2.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCompeting Interests: J.E.N, E.M.B.-K., L.E.E., M.A.R., A.O., C.M. and J.F. declare no competing interests. M.D.H. and S.D.R. are paid consultants to Tetra Therapeutics. M.E.G. is an employee of Tetra Therapeutics, which is a wholly owned subsidiary of Shionogi \u0026amp; Co., Ltd that has a financial interest in BPN14770.\u003c/p\u003e\n\n\u003cp\u003eFunding: Direct clinical costs were funded by the FRAXA Research Foundation. Access to and training on the NIH-TCB was obtained in association with work on HD076189 (E.M.B.K.). Tetra Therapeutics provided drug product and funded trial administration and independent data analysis.\u003c/p\u003e\n\u003cp\u003eAuthors contributions: J.E.N. conducted all analyses and drafted the manuscript\u003c/p\u003e\n\u003cp\u003eE.M.B.-K. designed and conducted the clinical trial\u003c/p\u003e\n\u003cp\u003eM.D.H. led the biostatistical analysis for the original manuscript\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eS.A.R. designed the clinical trial and served as medical monitor\u003c/p\u003e\n\u003cp\u003eM.A.R. preprocessed and organized the EEG dataset\u003c/p\u003e\n\u003cp\u003eA.H.O., C.M. and J.F. conducted the clinical trial\u003c/p\u003e\n\u003cp\u003eM.E.G. contributed to the clinical protocol\u003c/p\u003e\n\u003cp\u003eL.E.E. supervised all aspects of EEG experimental design, data analysis, and manuscript preparation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll authors contributed significantly to manuscript preparation.\u003c/p\u003e\n\n\u003cp\u003eAcknowledgements:\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eWe thank the patients and their families for participating in the clinical trial.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBerry-Kravis, EM, Harnett, MD, Reines, SA, Reese, MA, Ethridge, LE, Outterson, AH, Michalak, C, Furman, J, \u0026amp; Gurney, ME. Inhibition of phosphodiesterase-4D in adults with fragile X syndrome: a randomized, placebo-controlled, phase 2 clinical trial. Nat Med, 2021; 27(5), 862-870. https://doi.org/10.1038/s41591-021-01321-w \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/li\u003e\n \u003cli\u003eContractor, A, Klyachko, VA, \u0026amp; Portera-Cailliau, C. Altered Neuronal and Circuit Excitability in Fragile X Syndrome.\u0026nbsp;Neuron,\u0026nbsp;2015; 87(4), 699\u0026ndash;715. https://doi.org/10.1016/j.neuron.2015.06.017\u003c/li\u003e\n \u003cli\u003eDelorme, A, \u0026amp; Makeig, S. EEGLAB: an open source toolbox for analysis of single-trial EEG dynamics including independent component analysis. J Neurosci Methods, 2004; 134(1), 9-21. https://doi.org/10.1016/j.jneumeth.2003.10.009 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/li\u003e\n \u003cli\u003eEthridge LE, De Stefano LA, Schmitt LM, Woodruff NE, Brown KL, Tran M, Wang J, Pedapati EV, Erickson CA and Sweeney JA. Auditory EEG Biomarkers in Fragile X Syndrome: Clinical Relevance. Front. Integr. Neurosci. 2019; 13:60. doi: 10.3389/fnint.2019.00060\u003c/li\u003e\n \u003cli\u003eEthridge, LE, White, SP, Mosconi, MW, Wang, J, Byerly, MJ, \u0026amp; Sweeney, JA. Reduced habituation of auditory evoked potentials indicate cortical hyper-excitability in Fragile X Syndrome.\u0026nbsp;Translational psychiatry,\u0026nbsp;2016; 6(4), e787. https://doi.org/10.1038/tp.2016.48\u003c/li\u003e\n \u003cli\u003eGurney ME, Nugent, RA, Mo, X, Sindac, JA, Hagen, TJ, Fox, D, O\u0026rsquo;Donnell, JM, Zhang, C, Xu, Y, Zhang, H-T, Groppi, VE, Bailie, M, White, RE, Romero, DE, Vellekoop, AS, Walker, JR, Surman, MD, Zhu, L, \u0026amp; Campbell RF. Journal of Medicinal Chemistry, 2019; 62 (10), 4884-4901 http://doi.org/10.1021/acs.jmedchem.9b00193\u003c/li\u003e\n \u003cli\u003eMierau, A, Klimesch, W, \u0026amp; Lefebvre, J. State-dependent alpha peak frequency shifts: Experimental evidence, potential mechanisms and functional implications. Neuroscience, 2017; 360, 146-154. https://doi.org/10.1016/j.neuroscience.2017.07.037\u003c/li\u003e\n \u003cli\u003eSantoro, MR, Bray, SM, \u0026amp; Warren, ST. Molecular mechanisms of fragile X syndrome: a twenty-year perspective. Annu Rev Pathol, 2012; 7, 219-245. https://doi.org/10.1146/annurev-pathol-011811-132457 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/li\u003e\n \u003cli\u003eSchmitt, LM, Wang, J, Pedapati, EV, Thurman, AJ, Abbeduto, L, \u0026nbsp;Erickson, CA, Sweeney, JA. A neurophysiological model of speech production deficits in fragile X syndrome. Brain Communications, 2020; 2 (1), 2020, fcz042, https://doi.org/10.1093/braincomms/fcz042\u003c/li\u003e\n \u003cli\u003eStraub, D, Schmitt, LM, Boggs, AE, Horn, PS, Dominick, KC, Gross, C, \u0026amp; Erickson, CA. A sensitive and reproducible qRT-PCR assay detects physiological relevant trace levels of FMR1 mRNA in individuals with Fragile X syndrome. Sci Rep, 2023; 13(1), 3808. https://doi.org/10.1038/s41598-023-29786-4\u003c/li\u003e\n \u003cli\u003eTempio A, Boulksibat A, Bardoni B and Delhaye S. Fragile X Syndrome as an interneuronopathy: a lesson for future studies and treatments. Front. Neurosci. 2023; 17:1171895. doi: 10.3389/fnins.2023.1171895\u003c/li\u003e\n \u003cli\u003eWeber, JD, Smith, E, Berry-Kravis, E, Cadavid, D, Hessl, D, \u0026amp; Erickson, C. Voice of People with Fragile X Syndrome and Their Families: Reports from a Survey on Treatment Priorities. Brain Sci, 9(2). https://doi.org/10.3390/brainsci9020018 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-autism","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mola","sideBox":"Learn more about [Molecular Autism](http://molecularautism.biomedcentral.com/)","snPcode":"13229","submissionUrl":"https://submission.nature.com/new-submission/13229/3","title":"Molecular Autism","twitterHandle":"@MolecularAutism","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"biomarker, fragile x syndrome, zatolmilast, EEG, pharmacokinetics","lastPublishedDoi":"10.21203/rs.3.rs-4474353/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4474353/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFragile X syndrome (FXS) is a rare neurodevelopmental disorder caused by a CGG repeat expansion\u0026thinsp;\u0026ge;\u0026thinsp;200 repeats in 5\u0026rsquo; untranslated region of the FMR1 gene, leading to intellectual disability and cognitive difficulties, including in the domain of communication. A recent phase 2a clinical trial testing BPN14770, a phosphodiesterase 4D inhibitor, showed improved cognition in 30 adult males with FXS on drug relative to placebo. The initial study found significant improvements in clinical measures assessing cognition, language, and daily functioning in addition to marginal improvements in electroencephalography (EEG) results for the amplitude of the N1 event-related potential (ERP) component. EEG results suggest BPN14770 improved neural hyperexcitability in FXS. The current study investigated the relationship between BPN14770 pharmacokinetics (PK) and the amplitude of the N1 ERP component from the initial data. Consistent with the original group-level finding in period 1 of the study, participants who received BPN14770 in the period 1 showed a significant correlation between N1 amplitude and serum concentration of BPN14770. These findings strengthen the validity of the original result, indicating that BPN14770 improves cognitive performance by modulating neural hyperexcitability. This study represents the first report of significant correlation between a reliably abnormal EEG marker and serum concentration of a novel pharmaceutical in FXS.\u003c/p\u003e","manuscriptTitle":"Auditory N1 event-related potential amplitude is predictive of serum concentration of BPN14770 in fragile x syndrome","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-14 18:39:27","doi":"10.21203/rs.3.rs-4474353/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-08-02T13:59:08+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-24T09:02:31+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-21T12:41:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"25886653835236623922839276101852927469","date":"2024-06-05T11:49:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"169104786504654037732064357774594081255","date":"2024-06-05T09:22:16+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-05T09:17:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-01T09:32:39+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-31T08:54:13+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Autism","date":"2024-05-24T20:45:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-autism","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mola","sideBox":"Learn more about [Molecular Autism](http://molecularautism.biomedcentral.com/)","snPcode":"13229","submissionUrl":"https://submission.nature.com/new-submission/13229/3","title":"Molecular Autism","twitterHandle":"@MolecularAutism","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"768a2c2c-ffff-4c2d-a112-c751a34b55d0","owner":[],"postedDate":"June 14th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-11-04T16:27:37+00:00","versionOfRecord":{"articleIdentity":"rs-4474353","link":"https://doi.org/10.1186/s13229-024-00626-0","journal":{"identity":"molecular-autism","isVorOnly":false,"title":"Molecular Autism"},"publishedOn":"2024-11-02 16:20:20","publishedOnDateReadable":"November 2nd, 2024"},"versionCreatedAt":"2024-06-14 18:39:27","video":"","vorDoi":"10.1186/s13229-024-00626-0","vorDoiUrl":"https://doi.org/10.1186/s13229-024-00626-0","workflowStages":[]},"version":"v1","identity":"rs-4474353","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4474353","identity":"rs-4474353","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}