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Results
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Bayesian evidence supported a small beneficial effect of creatine. The creatine effect bordered significance for BDS (
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Conclusions
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Our study, in combination with the literature, implies that creatine might have a small beneficial effect. Larger studies are needed to confirm or rule out this effect. Given the safety and broad availability of creatine, this is well worth investigating; a small effect could have large benefits when scaled over time and over many people.
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Trial registration
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The trial was prospectively registered (drks.de identifier: DRKS00017250,
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Supplementary Information
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The online version contains supplementary material available at 10.1186/s12916-023-03146-5.
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PMC10647179
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Keywords
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Open Access funding enabled and organized by Projekt DEAL.
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Background
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Given the important role cognition plays in daily life, enhancing cognition safely and cheaply is highly desirable. Creatine is safe, well-tolerated, and cheap [While the safety and athletic benefits of creatine are well established, its potential cognitive benefits are still unclear. A systematic review tentatively suggests that creatine supplementation may improve “short-term memory”/working memory and “intelligence/reasoning” in healthy individuals [Supplementing creatine may benefit cognition as muscle and brain cells use creatine to access more energy when demand is high. They store creatine as phosphocreatine, which acts to regenerate the energy-providing adenosine triphosphate (ATP) [Dietary creatine is primarily contained in meat, fish, and a small amount in some dairy products [Creatine intake increases the level of creatine in the blood serum [It is unclear if creatine supplementation has similar effects on omnivores and vegetarians. Rae et al. [To test these hypotheses, we approximately replicated the study design and treatment (5 g per day of creatine for 6 weeks) used by Rae et al. [
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Methods
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PMC10647179
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Trial design
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We conducted a randomised, placebo-controlled, double-blind, cross-over study. The primary endpoints are the scores in the cognitive tasks after 6 weeks of each supplementation. Six weeks is the duration used by Rae et al. [Participant flow through the study
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Participants
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Participants were 18 years or older (see
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Interventions and similarity of treatment groups
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SETTLE
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Participants took the supplements daily for 6 weeks, including the day of the testing. The creatine supplement consisted of creatine monohydrate powder “CreaPure PG” produced by the company Alzchem (Trostberg, Germany). The placebo supplement consisted of maltodextrin powder “Maltodextrin 6” produced by the company Nutricia (Frankfurt am Main, Germany).The cans looked exactly the same except for clear markings of which one was the first and which one the second supplement. The two powders looked exactly the same and were flavourless. The solubility was somewhat different: While the placebo powder was completely soluble in water and did not settle, the creatine powder slowly settled. We were initially not aware of this difference in solubility. After we noticed it (after the first 40 participants), we asked participants to stir the powder into yoghurt or food with a similar consistency, as we had found no perceptible difference then. To check to what extent blinding was achieved, directly after the last testing, participants were asked to guess what their first supplement had been.
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Outcomes
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We had two primary outcomes:A standardised 10-min subtest of Raven Advanced Progressive Matrices (RAPM) [The Wechsler auditory Backward Digit Span (BDS) [RAPM is a test of abstract reasoning. Each item in the test consists of a 3 × 3 matrix with pictures of geometric forms. One of the pictures is missing and the task consists of choosing the right picture to fill this gap out of eight alternatives. The full RAPM consists of 80 items and has a time limit of 40 min. We used the same standardised 10-min subtests of the RAPM as Rae et al. [The Backward Digit Span is a test of working memory. The tester reads increasingly longer series of digits to the participant whose task it is to remember and repeat them in reverse order. The task starts with two digits. Each length has two series of digits. The test ends after wrong answers to two series of the same length. The BDS score consists of the sum of correct responses.We had eight further exploratory outcomes:The D2 Test of Attention [The Trail-Making-Test A (TMT-A), a test of visual attention [The Trail-Making-Test B (TMT-B), a test of task switching [The Block-Tapping-Test, a test of visuospatial working memory [The Auditory Verbal Learning Test (AVLT, in German: VLMT), a word-learning test including immediate recall, delayed recall, and recognition [The Brief-Visuospatial-Memory Test—Revised (BVMT-R), a test of visuospatial memory [The Stroop test (in German: Farb-Wort-Interferenz Test, a test of inhibitory control [Regensburger Wortflüssigkeitstest, a test of verbal fluency [Participants reported side effects experienced during the supplementation period in a free text form on the day of testing. How side effects would be grouped for the report was determined after evaluating all entries. At baseline testing, participants performed a test of crystallised intelligence called “Mehrfachwahl-Wortschatztest (MWT-B)” [
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Sample size
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The sample size of 123 was powered (with power = 0.8, alpha = 0.05, calculated with GPower [Block-tapping was originally performed with physical blocks and later on the website Psytoolkit [
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Randomisation and blinding
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The order of the two supplements was randomised with Excel by the pharmacy of the University Hospital Heidelberg. They labelled each of the cans of supplements with the participant code and “A” or “B”, corresponding to the first and second supplement. The staff members who tested participants also provided the participants with the supplement cans. Allocation concealment was performed using sequentially numbered, opaque sealed envelopes (SNOSE). Participants and all staff who interacted with them were kept blinded to the allocation (also see intervention section).
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Statistical methods
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For each cognitive test, we conducted a mixed ANOVA with test score after supplementation as the dependent variable, supplement (creatine vs placebo) as the within-subjects factor and supplement order (creatine-first vs placebo-first) as the between-subjects factorWe conducted a frequentist analysis, because it is widely understood and it offers the insight of how likely the data is under the null hypothesis. However, we also wanted to know whether the data was more likely under the null or under the alternative hypotheses, i.e. in which direction to update our credence, and to what extent. We conducted a Bayesian analysis, because it answers this question. We were interested in comparing the null hypothesis to different alternative hypotheses—postulating a large effect like in the study we replicated [
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Confirmatory analyses
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As preregistered, our two confirmatory cognitive tasks are the Backward Digit Span and Raven’s Advanced Progressive Matrices. All other cognitive tasks are analysed in an exploratory fashion. There is one deviation from our preregistered analyses. We had preregistered
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Robustness checks
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We checked the robustness of our normality-assuming ANOVAs by performing: an ANOVA on 20%-trimmed data, an ANOVA on 5%- and 20%-winsorised data, and a robust ANOVA which uses trimming and bootstrapping (performed with the sppb functions in the WRS2 R package). The latter ANOVA provides the most robust estimate of these methods [
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Bayes factors
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For the calculation of the Bayes factors, we used the estimated marginal means (EMMs) of the creatine and placebo score. The EMMs are the means weighed for the order groups (creatine-first and placebo-first), so that imbalances in the sizes of the order groups do not affect the means. So, we only had two groups for the Bayes factor calculation (creatine and placebo), simplifying the analysis. The mean difference and standard error of the mean difference were used to describe the data. Using the Bayesplay package [
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Exploratory analyses
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In addition to the confirmatory analyses of BDS and RAPM, we analysed the other cognitive tasks in the same way in an exploratory fashion.We also looked in an exploratory fashion at the first supplementation and the second supplementation separately and at participants with a low and high baseline performance separately (see
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Results
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PMC10647179
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Participant flow
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See participant flow in Fig. Drop-outs were due to supplements failing to arrive (
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Recruitment
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Participants were recruited through flyers and social media between 05/2019 and 05/2022 and tested between 05/2019 and 08/2022.
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Baseline data
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MINOR
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We analysed all available participant data apart from two minor exceptions (see Participant baseline characteristicsData is given as mean (standard deviation) or as percentage. The MWT-B (Mehrfach-Wahl-Wortschatztest) is a test of crystallised intelligence [
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Blinding, adherence, and side effects
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After their final testing session, the last 73 participants were asked to guess the order of their supplements (as the idea did not occur to us before). Forty-three (59%) guessed correctly and 30 (41%) guessed incorrectly. A binomial test reveals that the probability of 43 or more correct guesses out of 73 by pure chance is A Adherence and negative side effectsAdherence (self-reported) was high (Table
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Interaction with diet
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There was no significant interaction between diet and supplement nor between diet, supplement, and supplement order for neither BDS (
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Bayes factors
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To facilitate the interpretation of the results of the confirmatory analysis, we provide Bayes factors. A Bayes factor (BFWe compare several alternative hypotheses postulating small beneficial effects of creatine to the null hypothesis. For RAPM, the data was very insensitive, very weakly favouring the alternative hypotheses. For BDS, the data was more sensitive, providing weak to moderate support in favour of the alternative hypotheses. Two different approaches to calculating these Bayes factors were used (see statistical analysis) and the results were similar (Table Results of Bayesian analysisBayes factors (BFThere was strong evidence in favour of the null hypothesis compared to the alternative hypothesis postulating the effect size found by Rae et al. [The results were similar whether using normal or Cauchy distributions. For more details on this and the aforementioned calculations, see the In summary, this study provides weak to moderate evidence for a small cognitive benefit of creatine and strong evidence against the effect size by Rae et al. [
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Robustness checks
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We checked the robustness of our confirmatory analysis (the normal ANOVA) by performing an ANOVA on 20%-trimmed data, an ANOVA on 5%- and 20%-winsorised data, and an ANOVA which uses bootstrapping and 20% trimming.For RAPM, all of these methods gave overall similar results to that of the normal ANOVA (Table Results of robustness checksCreatine effect For BDS, whose skewness statistic was slightly further from 0 than that of RAPM, these methods gave results that differ from each other and from the normal ANOVA to a relevant extent (Table Thus, the result for RAPM was robust and for BDS much less so.
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Exploratory cognitive tasks
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There was no indication that creatine improved the performance of our exploratory cognitive tasks. The distribution of Results for exploratory cognitive tasksCreatine effect
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Discussion
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PMC10647179
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Summary of results
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This is the largest study on the cognitive effects of creatine to date. As part of our study, we aimed to replicate Rae et al. [We found Bayesian evidence for a small beneficial effect of creatine on cognition for both tasks. Cohen’s
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Effect of diet
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Dietary creatine is primarily contained in meat, fish, and a small amount in some dairy products [Rae et al. [Observational data on the role of dietary creatine in cognition is conflicting. Ostojic et al. [Overall, observational evidence is mixed. Evidence from RCTs and brain creatine studies does not support the idea that dietary creatine affects brain health.
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BDS and other short-term memory tasks
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While the creatine effect for BDS in our study bordered significance, it was smaller (One reason for this could be that there might have been more noise in our study, maybe due to the COVID pandemic starting in the midst of the study. This reason might apply to all of our tests. However, the standard deviation for BDS in this study was almost exactly the same as in the study we aimed to replicateThree further studies tested the effect of creatine supplementation on BDS in a different population than healthy young adults. In two of these studies, participants were healthy and elderly [In a review by Avgerinos et al. [Five of our exploratory tasks, the forward digit span, forwards and backwards block-tapping task (spatial), the BVMT-R, and the immediate recall part of the VLMT also tested short-term memory and there was no indication of an effect for these tasks. A review by Dolan et al. [Overall, the effect size for BDS in the present study lies in between that of other studies, some of which found very large effects and some of which found no effect. The reason why we found an effect for BDS, which is a short-term memory task, but not for our exploratory short-term memory tasks, might be that BDS is probably more difficult and thus requires a higher ATP turnover, which is where creatine helps. Short-term memory is critical for language comprehension, learning, planning, reasoning, and general fluid intelligence [
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RAPM and other abstract reasoning tasks
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In our study, the creatine effect for Raven’s Advanced Progressive Matrices (RAPM) was much smaller (Rae et al. [In a review by Avgerinos et al. [In sum, two studies found much larger creatine effects for RAPM than that in the present study, while a third study did not find a significant effect. Possibly the real effect lies in the middle like our study. RAPM, while not a pure measure of g [
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Exploratory cognitive tasks other than short-term memory
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We did not find a creatine effect for our exploratory tasks. Our negative finding for the verbal fluency task is in line with the only other study using the same task [In sum, for the exploratory tasks, overall the evidence does not support a creatine effect. However, as the evidence for the Stroop task shows, this might be only when the tasks are made too easy for participants, so that creatine has no chance to help.
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Effect of age
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It has been claimed that creatine supplementation is more likely to benefit older adults more than younger ones [One theory behind an effect of age is that brain creatine levels might decrease with age. There is evidence that this happens with muscle creatine levels [Experimental evidence suggests an effect of age. A meta-analysis by Prokopidis et al. [
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Effect of gender
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In their review, Smith-Ryan et al. [There have only been three RCTs on the effect of creatine on cognition in healthy women and three in healthy men. One of the studies with women found a creatine effect and two did not find an effect (one of these with only elderly participants). Two of the studies with men (one of these with sleep-deprived participants) found a creatine effect and one did not find an effect. Apart from the present study, which found no effect of sex, studies who included both men and women did not report the effect of sex. In their meta-analysis on the effect of creatine on memory performance, Prokopidis et al. [
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Limitations
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There are a number of limitations to this study. Despite the large sample size compared to other studies, a larger sample size would be needed to be powered for effects that are smaller but still relevant. Some of the data (4%) could not be analysed because it was not labelled with the participant and timepoint. The COVID-19 pandemic started in the middle of the study, might have added noise to the data, and meant that we had to switch from in-person cognitive testing to testing via video call. However, we do not see this potential source of noise reflected in the standard deviations compared to pre-pandemic studies. We assessed baseline days per week of meat consumption (median = 3.5, mean = 3.7, SD = 2.0), but not grammes per day of consumption. However, creatine intake through meat is usually substantially lower than the supplemented dose [
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Conclusions
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Supplementing creatine is safe, easy, and very cheap. The real effect of creatine on cognition is likely smaller than that reported in Rae et al. [
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Acknowledgements
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We thank the doctors of the University Clinic Bonn who collected blood samples. We thank Prof. Dr. Rand R. Wilcox, Dr. Lincoln Colling, Dr. Christian Stark, Jan Speller, Maximilian Meier, and David Reinstein for their feedback on statistical questions. We thank Thomas Szpejewski and Tom Lieberum for their help with verifying data quality. We thank all data entry helpers.
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Authors’ contributions
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JFS and JB conceived of the study. JFS, XK, SL, AP, HM, and JB designed the study. AP and HM managed staff members as the director and vice director of the hospital. JFS and JB wrote the grant proposal. XK wrote the proposal for the ethics committee. JFS preregistered the study. XK prepared the logistics of the study and supervised EKK. JFS, AF, and EKK tested participants. JFS managed data entry and analysed the data. JFS wrote and revised the manuscript with help from XK, GA, SL, UE, and JB. UE contributed to the supervision of JFS. JB was the main supervisor and senior author of the study. All authors read and approved the final manuscript.
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Authors’ Twitter handles
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Twitter handles: @FabienneSand (Julia Fabienne Sandkühler)Twitter handles: @JanMBrauner (Jan Brauner)
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Funding
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Open Access funding enabled and organized by Projekt DEAL. Funding was provided by the non-profit organisation Effective Ventures Foundation, 2443 Fillmore St., #380–16662, San Francisco, CA 94115, the non-profit organisation Effective Altruism Foundation, Efringerstrasse 25, CH-4057 Basel, Switzerland, and private donor Paul Christiano. The trial funders had no role in the design of the study; the collection, analysis, or interpretation of data; the writing of the report; or the decision to submit the article for publication.
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Availability of data and materials
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The appendix, protocol, data, code, and output of this study are openly available at the Open Science Framework,
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Declarations
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Ethics approval and consent to participate
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Ethical approval was obtained from the ethics committee of the University of Bonn (060/19). Participants gave informed consent to participate before being enrolled.
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Consent for publication
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This manuscript contains only anonymised data. Participants gave consent for the publication of this data.
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Competing interests
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The authors declare that they have no competing interests.
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References
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Introduction
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CHF
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CHF, CHRONIC HEART FAILURE
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Sodium-glucose co-transporter 2 (SGLT2) inhibitors have cardiovascular protective properties in addition to the metabolic effects and represent a cornerstone of treating patients with chronic heart failure (CHF). We hypothesised that empagliflozin reduces tissue sodium content in patients with CHF.
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PMC9849317
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Methods
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CHF
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CHF
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In a double-blind, randomised (2:1), placebo-controlled, parallel-group, clinical trial, 74 patients with NYHA class II–III CHF and an ejection fraction of 49% or less received empagliflozin 10 mg once daily or placebo for 3 months. In each patient, tissue sodium content of the lower leg was assessed non-invasively by sodium-MRI (
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PMC9849317
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Results
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After 1 and 3 months treatment with empagliflozin (
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Conclusion
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This trial showed a significant decrease in skin sodium content after 1 and 3 months of treatment with empagliflozin. The decrease in skin sodium content may reflect a decrease in subclinical micro-oedema or/and in non-osmotic bound tissue sodium, both reported to impair left ventricular function.
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PMC9849317
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Trial registration number
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NCT03128528 (
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PMC9849317
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Trial registration date
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25th April 2017.
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PMC9849317
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Supplementary Information
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The online version contains supplementary material available at 10.1007/s00392-022-02119-7.
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PMC9849317
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Keywords
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Open Access funding enabled and organized by Projekt DEAL.
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PMC9849317
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Introduction
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hyperglycaemia, T2D, type 2 diabetes, CHF
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CHRONIC HEART FAILURE, EXCESS SODIUM, CHF, HYPERGLYCAEMIA, TYPE 2 DIABETES
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Sodium glucose co-transporter 2 (SGLT2) inhibitors were originally developed to treat hyperglycaemia in patients with type 2 diabetes (T2D) and became further a cornerstone of treating patients with chronic heart failure (CHF) [For years, excess of intravascular sodium (e.g. due to excessive sodium intake) has been assumed to be always accompanied by water retention, leading to increased plasma volume and higher cardiac output. However, knowledge of sodium metabolism has progressed and the 2-compartment model has been replaced by the 3-compartment model, in which excess sodium is bound non-osmotically to negative charged glycosaminoglycans present in the extracellular space, mainly in the skin, endothelial surface layer, muscle and bone [Recent developments in sodium-MRI (In the current trial, we focused on patients with chronic heart failure (CHF) and mid-range (HFmrEF) or reduced (HFrEF) ejection fraction in stable euvolemic condition. Our aim was to analyse the changes of tissue sodium content in these patients after 1 and 3 months of treatment with empagliflozin from baseline and compared to placebo.
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PMC9849317
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Material and methods
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PMC9849317
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Trial design
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HEART
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The trial was a prospective, investigator initiated, double-blind, randomised, placebo controlled, parallel-group clinical trial, performed at the Clinical Research Centre of the University Hospital Erlangen, Germany. We recruited patients with HFrEF or HFmrEF, according the 2016 ESC guidelines definition, from the University outpatient clinic as well as through newspaper advertisements. All patients had to be in a stable condition with New York Heart Association class (NYHA) II–III.Each patient entered, after being screened for eligibility, a 2-weeks run-in phase where, if necessary, change of medication to meet the patient’s clinical condition or additional diagnostics (e.g. echocardiogram) took place. After the 2-weeks run-in phase, being naive to or off any SGLT2 inhibitor for at least 10 weeks, patients underwent the first Trial design: summary of study visits and tests performed in each visitAfter
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PMC9849317
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Trial population
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diabetes, T2D, CHF
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CHF, DIABETES
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Patients between 18 and 85 years of age and who had a CHF with an ejection fraction below 40% (HFrEF) or an ejection fraction between 40 and 49% as well as a N-terminal pro-brain natriuretic peptide (NTproBNP) level > 125 pg/ml and at least one structural abnormality of the left atrium or ventricle (HFmrEF), in stable conditions, were included. Individuals were excluded, if they had any other form of diabetes than T2D, were treated with insulin or more than one oral anti-diabetic drug or any SGLT2 inhibitor within the last 10 weeks prior to the screening visit or were treated with loop diuretics (above furosemide > 80 mg/day, torasemide > 40 mg/day or piretanide > 6 mg/day), had an uncontrolled diabetes (glycated haemoglobin (HbA1c) ≥ 10% or fasting-plasma glucose level > 240 mg/dl) or uncontrolled blood pressure (BP ≥ 180/100 mmHg), had an estimated glomerular filtration rate (eGFR) < 30 ml/min/1.73 m
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PMC9849317
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Endpoints
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The primary endpoint of the trial was to analyse the effect of 3 months treatment with empagliflozin on change in tissue (skin, muscle, bone marrow) sodium content compared to placebo. Secondary endpoints were the effect of 1 month treatment with empagliflozin on change in tissue sodium content compared to placebo as well as the effect of 1 and 3 months treatment with empagliflozin compared to baseline.
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PMC9849317
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Clinical parameters
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At the first visit, all demographic data were recorded. At the randomisation visit, a fasting blood sample was taken in order to determine the NTproBNP level, serum sodium level, HbA1c and fasting plasma glucose as well as other biochemical safety parameters (e.g. serum creatinine, eGFR). Twenty-four-hour urine was collected to assess the urine volume, sodium as well as glucose excretion over 24 h. The 24-h urine collections were primarily frozen in order to keep the double-blind aspect of the trial maintained, the analyses were performed after the end of the trial.Office BP and heart rate was assessed after 5 min of rest according to guidelines [
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PMC9849317
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Body composition measurements
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overhydration
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OVERHYDRATION
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Fluid status of the patients was assessed by a whole-body bioimpedance spectroscopy (Body Composition Monitor (BCM), Fresenius Medical Care, Bad Homburg, Germany) at baseline, after 1 and 3 months. From the impedance data and additional clinical parameters, extracellular water, intracellular water and total water was calculated by the equations proposed by Moissl [Fluid status is represented by overhydration (OH in litre), based on a three-compartment model developed by Chamney [
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PMC9849317
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Statistical analysis
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T2D
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In our trial tissue sodium, muscle water and muscle fat content in the lower leg were specially measured with a 3.0 T clinical MR system (Magnetom Skyra, Siemens Healthineers, Erlangen, Germany) using a custom-made transit/receive sodium RF birdcage knee coil (32.6 MHz, Stark Contrast, Erlangen, Germany). Reliability as well as accuracy have been shown previously [In brief, patients were in supine position with the thickest part of their left or right calf muscle at the centre of the sodium RF birdcage knee coil with calibration tubes placed in a phantom holder underneath the calf and included in the field of view. Partial volume effects and short relaxation times of sodium ions result in a more than threefold underestimation of measured sodium concentrations and in order to reflect this, the signal intensities are reported in arbitrary units (AU). Proton and sodium images were then acquired (Fig. Sample of Interrater variability calculations showed according to Bland Altman [Statistical analysis was performed using SPSS Statistics 28. The sample size was calculated for the primary endpoint using data acquired during a previous trial, the SD of change in skin sodium content was 4.17 AU in patients with T2D [All analyses of this trial were performed in patients presenting at least a
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Results
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PMC9849317
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Adherence
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Adherence to trial medication (pill counting) as well as overall adherence was given with 99 ± 3%. All patients in the empagliflozin group presented significant urinary glucose excretion at 1 and 3 months follow-up visits. None of the patients in the placebo group showed urinary glucose excretion (no drop-ins).
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Correlations
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After 1 month treatment with empagliflozin, we observed a significant correlation between the change in skin sodium content and the change in muscle sodium content (Relationship between change in skin sodium content and change in muscle sodium content No relationship was found between the change in haematocrit, 24-h urine volume excretion, 24-h urine sodium or urine glucose excretion, body weight and BMI and either the change in skin sodium content nor the change in muscle sodium content after 1 and 3 months treatment with empagliflozin (data not given).
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Discussion
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hypertrophic, T2D, myocardial interstitial oedema, renal failure, cardiac dysfunction, CHF, heart failure, glucosuria, muscle inversion
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CHF, RENAL FAILURE, HEART FAILURE, HYPERTROPHIC
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Randomised clinical trials conducted with empagliflozin or dapagliflozin in patients with CHF have demonstrated protective effects of SGLT2 inhibitors concerning development of heart failure, incident heart failure hospitalisations, all-cause mortality and renal failure [These results are in accordance with the results of a small exploratory analysis showing a significant reduction in skin sodium content with no reduction in muscle sodium and muscle water content in patients with T2D after 6 weeks treatment with dapagliflozin [With the current MRI technique, we are not able to accurately differentiate between intracellular and extracellular interstitial sodium ion signals and thus to separate whether the observed decrease in skin sodium content is indicative of reduced micro-oedema or/and reduced water-independent bound sodium or both (and this is most likely the case in our opinion). The muscle inversion recovery Indirect signs of our trial, supporting the fact that the measured change in skin sodium content is related to the non-osmotic sodium, are the absence of a correlation between change in skin sodium content and change in muscle water content as well as in 24-h urine volume excretion, 24-h urine sodium excretion and 24-h urine glucose excretion. However, a close relationship between change in skin sodium content and change in muscle sodium content was observed after 1 and 3 months treatment with empagliflozin, indicating a parallel line in the changes. Possible explanations for the lack of significant difference in change in muscle sodium content are either smaller and more heterogenous changes in the muscle than in the skin or the short follow-up. Further, there is a relation between change in muscle sodium content and muscle water content that persisted after 3 months. These observations may reflect the glycoaminoglycan bound sodium in the tissue. Others have shown that in patients on haemodialysis, sodium content in muscle (Increased tissue sodium content may lead, in addition to micro-oedema, to cardiac as well as vascular effects, which ultimately lead to CHF. In experimental and in vivo studies, increased tissue sodium content (high sodium concentration in the extracellular fluid) lead to increased hypertrophic signalling, e.g. induced directly hypertrophic responses in myocardial myoblasts and vascular smooth muscle cells of neonatal rats [It has been demonstrated that patients with CHF have myocardial interstitial oedema, which causes systolic and diastolic cardiac dysfunction and increased stiffness of the left ventricle [We showed reduced office BP and improved metabolic control, findings well described in our previous studies [The broad known mode of action of SGLT2 inhibitors is enhanced glucosuria and natriuresis. Initially at 1 month, a significant increase in 24 h urinary sodium excretion was observed, as already by others, which was although no longer evident at 3 months and may explain in part the rapid onset of reduced incidence of heart failure hospitalisation observed in the prospective endpoint trial [We think that the changes in bone marrow sodium content mirror the changes observed in the skin and constitute a finding of unclear significance. Furthermore, the measurement of bone marrow sodium content lacks validation against a gold standard method as shown for skin and muscle sodium content [Several limitations should be pointed out. This was a single-center trial with an overall small sample size. However, this trial was not designed to be a clinical outcome trial, but a mechanistic trial. The chosen sample size was based on power calculation according to the data of our previous trial [
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PMC9849317
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Conclusion
|
vascular dysfunction, CHF
|
CHF
|
We found a significant reduction in tissue sodium content after 1 and 3 months treatment with the SGLT2 inhibitor empagliflozin. Our data support the concept that increased skin sodium content in patients with CHF has clinically relevant pathogenic effects, leading to cardiac as well as vascular dysfunction and consequently to CHF. The reduction of extravascular sodium content may reflect a decrease in subclinical micro-oedema or/and in non-osmotic bound sodium, both reported to impair left ventricular function.
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PMC9849317
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Supplementary Information
|
Below is the link to the electronic supplementary material.Supplementary file1 (DOCX 192 KB)
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PMC9849317
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Abbreviations
|
HEART, TYPE 2 DIABETES MELLITUS
|
Sodium-MRIArbitrary unitsBody composition monitorBlood pressureChronic heart failureEstimated glomerular filtration rateFlow mediated vasodilatationGlycated haemoglobinHeart failure with mid-range ejection fractionHeart failure with reduced ejection fractionN-terminal pro-brain natriuretic peptideNew York Heart AssociationOverhydrationRadiofrequencySodium-glucose co-transporter 2Type 2 diabetes mellitus
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PMC9849317
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Acknowledgements
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HYPERTENSION
|
We gratefully acknowledge the expert technical assistance of Dorothea Bader-Schmieder, Ingrid Fleischmann, Kerstin Fröhlich-Endress, Ulrike Heinritz, Wiebke Maurer and Simone Pejkovic (Clinical Research Centre, Department of Nephrology and Hypertension, University Hospital Erlangen, Friedrich-Alexander University Erlangen-Nuremberg, Germany). The present work was performed in fulfilment of the requirements for obtaining the degree Dr. med. for L.N.
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PMC9849317
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Author contributions
|
JK and RES designed the study. Material preparation, data collection and analysis were performed by Julie Kolwelter and the data were reviewed by RES. The first draft of the manuscript was written by JK and all authors contributed to the discussion as well as commented on previous versions of the manuscript. All authors read and approved the final manuscript.
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PMC9849317
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Funding
|
Open Access funding enabled and organized by Projekt DEAL. This investigator-initiated clinical trial was supported by a grant to the University Hospital Erlangen, Germany, provided by Boehringer Ingelheim.
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PMC9849317
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Declarations
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PMC9849317
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Conflict of interest
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RES received speaker fees and advisory board fees and PB has received research funding (related and unrelated to the present work) from Boehringer Ingelheim Pharma GmbH & Co. KG during the conduct of the trial. All other authors declare that there is no conflict of interest.
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PMC9849317
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Ethical standards
|
The trial was approved by the Ethics Committee of the University of Erlangen. The trial was performed in accordance with the principles of the Declaration of Helsinki. All patients provided written informed consent prior to inclusion in the trial. The trial was registered at
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PMC9849317
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References
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PMC9849317
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BACKGROUND
|
IgE-mediated anaphylaxis, B cell malignancies
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PEANUT ALLERGY, ALLERGIC REACTIONS, SYSTEMIC ALLERGIC REACTION
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IgE-mediated anaphylaxis is a potentially fatal systemic allergic reaction for which there are no currently FDA-approved preventative therapies. Bruton’s tyrosine kinase (BTK) is an essential enzyme for IgE-mediated signaling pathways and is an ideal pharmacologic target to prevent allergic reactions. In this open-label trial, we evaluated the safety and efficacy of acalabrutinib, a BTK inhibitor that is FDA approved to treat some B cell malignancies, in preventing clinical reactivity to peanut in adults with peanut allergy.
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PMC10425211
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METHODS
|
’
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After undergoing graded oral peanut challenge to establish their baseline level of clinical reactivity, 10 patients had a 6-week rest period, then received 4 standard doses of 100 mg acalabrutinib twice daily and underwent repeat food challenge. The primary endpoint was the change in patients’ threshold dose of peanut protein to elicit an objective clinical reaction.
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PMC10425211
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RESULTS
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ADVERSE EVENTS, EVENTS
|
At baseline, patients tolerated a median of 29 mg of peanut protein before objective clinical reaction. During subsequent food challenge on acalabrutinib, patients’ median tolerated dose significantly increased to 4,044 mg (range 444–4,044 mg). 7 patients tolerated the maximum protocol amount (4,044 mg) of peanut protein with no clinical reaction, and the other 3 patients’ peanut tolerance increased between 32- and 217-fold. 3 patients experienced a total of 4 adverse events that were considered to be possibly related to acalabrutinib; all events were transient and nonserious.
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PMC10425211
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CONCLUSION
|
Acalabrutinib pretreatment achieved clinically relevant increases in patients’ tolerance to their food allergen, thereby supporting the need for larger, placebo-controlled trials.
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PMC10425211
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TRIAL REGISTRATION
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ClinicalTrials.gov NCT05038904
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PMC10425211
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FUNDING
|
LUDWIG
|
AstraZeneca Pharmaceuticals, the Johns Hopkins Institute for Clinical and Translational Research, the Ludwig Family Foundation, and NIH grants AI143965 and AI106043.
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PMC10425211
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|
Introduction
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stinging insect venom, systemic allergic reaction, allergic, Anaphylaxis
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ANAPHYLAXIS, SYSTEMIC ALLERGIC REACTION
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Anaphylaxis is an acute, potentially life-threatening systemic allergic reaction that may be caused by foods, medications, or stinging insect venom in allergic individuals (Acalabrutinib (Calquence; Acerta Pharma and AstraZeneca) is a second-generation oral, covalent inhibitor of Bruton’s tyrosine kinase (BTK), an essential enzyme for high-affinity IgE receptor (FcεRI) signaling in human mast cells and basophils (
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PMC10425211
|
Results
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PMC10425211
|
|||
Trial design and patient characteristics.
|
IgE-mediated peanut allergy
|
This prospective, open-label trial enrolled adult patients with IgE-mediated peanut allergy (A total of 28 patients were screened, of whom 14 met eligibility criteria (
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PMC10425211
|
|
Primary endpoint.
|
’
|
The predetermined primary endpoint was the change in patients’ threshold dose of ingested peanut protein to elicit an objective clinical reaction during OFC after acalabrutinib pretreatment compared with patients’ baseline. At baseline, patients tolerated a median of 29 mg of peanut protein (range, 1–444) before experiencing an objective clinical reaction during OFC. If needed, all patients were treated with intramuscular epinephrine for their reaction per standard of care in addition to adjunct therapies (e.g., antihistamines or albuterol) at the discretion of the investigator; a summary of rescue medications that were administered to patients during their baseline OFC is listed in
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PMC10425211
|
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Secondary endpoints.
|
SKIN, SECONDARY
|
A key secondary endpoint included the change in the severity of clinical reactions during OFC, as assessed by using a modified PRACTALL scale to score symptoms (Skin puncture testing to peanut extract was included as a secondary endpoint and surrogate marker of mast cell reactivity in vivo. All patients had a positive skin puncture test to undiluted peanut extract at baseline, with a median wheal area of 126 mmAn additional secondary endpoint was the percentage of basophils activated ex vivo by peanut extract. All patients had a positive basophil activation test at baseline to at least 1 dilution of peanut extract (mean peak response to peanut, 31.7%; range, 3.5–70.7;
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PMC10425211
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Exploratory endpoints.
|
allergy
|
ALLERGY
|
Because BTK plays an important role in B cell receptor signaling and therefore affects plasma B cell survival, exploratory endpoints included markers of humoral immunity function and allergy. Based on earlier studies (
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PMC10425211
|
Safety.
|
deaths, wheezing, concussions
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ADVERSE EVENTS
|
Safety endpoints included electrocardiography and laboratory blood testing, including complete blood counts and differentials, serum chemistries, and liver function tests. A total of 15 adverse events occurred in 5 of 10 patients (50%; Among adverse events not attributed to acalabrutinib, 1 patient experienced recurrent wheezing after her baseline OFC despite treatment with epinephrine and was sent to the emergency department for further management. Two sports-related concussions occurred in 2 patients during the trial that were considered to be unrelated to study drug or procedures; 1 occurred before the patient received acalabrutinib, and 1 occurred 18 days after the patient’s last dose of acalabrutinib. One of these same patients also experienced a separate mechanical fall due to tripping over an object, which occurred before acalabrutinib therapy. No deaths or treatment-related serious adverse events occurred during the trial. No electrocardiographic changes were observed during acalabrutinib treatment.
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PMC10425211
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Discussion
|
allergic, increases peanut-allergic, first-ever, ’, IgE-mediated anaphylaxis, systemic allergic reactions
|
FOOD-INDUCED ANAPHYLAXIS
|
This trial has demonstrated the first-ever treatment to achieve rapid-onset prevention of IgE-induced food reactivity. Results showed that a short course of premedication with standard dosing of the BTK inhibitor acalabrutinib can achieve marked reduction or even complete elimination of clinical reactivity to ingestion of food allergens in allergic patients. Because tolerance to at least 300 mg of peanut protein is considered to be protective against reaction from an accidental exposure (In parallel with their clinical tolerance, patients’ highest negative skin test dilution of peanut also increased several logs. However, acalabrutinib treatment did not completely suppress skin tests as was observed for basophil activation, which was abolished by acalabrutinib treatment. This is in line with prior studies showing that BTK inhibitor doses that can suppress ex vivo basophil activation responses do not completely inhibit skin tests (It is unknown why some patients did not achieve the same magnitude of clinical protection from food-induced anaphylaxis with acalabrutinib treatment as others. Interestingly, these results illustrate the role of mast cells in food-induced anaphylaxis, given that some patients still reacted to peanut ingestion despite complete inhibition of their basophils by acalabrutinib. No correlation was observed between patients’ clinical response and their baseline skin test size or percentage reduction; baseline basophil activation; specific IgE to peanut or components; total IgE or specific-to-total IgE ratios; baseline tryptase level (data not shown); body weight or body mass index, though our study was not powered to detect such correlations. Further trials are necessary to determine the minimum dose of BTK inhibitors required to attain reliable protection against systemic allergic reactions for all patients.Given their remarkably rapid onset of action (within 2 days), BTK inhibitors may be a superior choice as adjunct prophylactic therapy for procedures such as high-risk immunotherapy or desensitizations compared with the anti-IgE monoclonal antibody omalizumab, which requires at least 4–8 weeks to achieve an effect and which does not reliably increase tolerance in every patient (Our results, in addition to prior trials utilizing the first-in-class BTK inhibitor ibrutinib to suppress allergen skin tests (Limitations of our study include a small patient population, the lack of fully blinded OFCs, and the lack of a placebo treatment arm. We attempted to mitigate any potential placebo effects or bias by using a modified PRACTALL scale (In conclusion, we have shown that pretreatment with the oral BTK inhibitor acalabrutinib for just 2 days significantly increases peanut-allergic patients’ tolerance to peanut during oral exposure. These results support the need for larger, placebo-controlled trials to further evaluate the safety and efficacy of these drugs in preventing IgE-mediated anaphylaxis. In particular, dose-finding trials will be necessary to determine the minimal effective dose of BTK inhibitors needed to prevent morbidity and/or mortality from either therapeutic or accidental allergen exposures before these medications can be used in clinical practice.
|
PMC10425211
|
Methods
|
PMC10425211
|
|||
Study design.
|
The full trial protocol is available in Supplementary information. While the trial was partially supported by a research agreement from AstraZeneca, the design and conduct of the study were performed entirely by the investigators. All study procedures were conducted at a single site — Johns Hopkins University School of Medicine — in accordance with international ethics guidelines and local ethical and legal requirements, including the Declaration of Helsinki. Study visits were completed in the Clinical Research Unit at the Johns Hopkins University Bayview Campus in Baltimore, Maryland after written informed consent was obtained. All study visits occurred between December 2021 and October 2022. Patients stopped all antihistamines and medications with antihistamine properties at least 1 week before study visits in preparation for skin puncture testing and OFC. At Visit 1, medical history, vital signs, height, and weight were collected, and an electrocardiogram was performed (
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PMC10425211
|
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Patient recruitment, screening, and eligibility.
|
infection, IgE-mediated allergy, hepatitis
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ALLERGY, CARDIOVASCULAR DISEASE, CEREBROVASCULAR ACCIDENT, BLEEDING DISORDER, INFECTION, HEPATITIS
|
Eligible patients were 18 years of age or older at screening with a history of an IgE-mediated allergy to peanut. Patients were required to have a positive skin puncture test to peanut extract and an objective clinical reaction to cumulative dose of 1,044 mg of peanut protein or less during baseline OFC. Key exclusion criteria included: cardiovascular disease or prior cerebrovascular accident; active infection; history of bleeding disorder or receiving anticoagulants; any immunomodulatory therapies or oral corticosteroids within 1 month before study participation; active infection or latent hepatitis; use of strong CYP3A4 inducers or inhibitors; and pregnancy or nursing. Patients were also excluded if they had ever received peanut immunotherapy or omalizumab. Complete eligibility criteria are listed in Patients were recruited from the Johns Hopkins University Allergy and Clinical Immunology outpatient clinic and through IRB-approved advertising on social media. Patients who responded to advertisements were initially screened by telephone to determine eligibility. If determined eligible, patients were consented by teleconference before visit 1 in compliance with FDA 21 CFR Part 11.
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PMC10425211
|
Medical history and demographics.
|
atopic disorders
|
FOOD ALLERGIES, ATOPIC DISORDERS
|
Age and information about medical comorbidities including food allergies and other atopic disorders were collected at intake. Patients were asked to report their biologic sex (options included male and female) and gender identity (options included male, female, unspecified, and prefer not to answer). Patients were also asked to report their race (options included American Indian or Alaskan Native, Asian, Black or African American, Native Hawaiian or other Pacific Islander, White, unknown, or not reported) and ethnicity (options included Hispanic or Latino, not Hispanic or Latino, unknown, and not reported).
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PMC10425211
|
Endpoints.
|
’
|
SECONDARY
|
The predetermined primary endpoint was the change in patients’ threshold dose of ingested peanut protein to elicit an objective clinical reaction during OFC after acalabrutinib pretreatment compared with the patients’ baseline. A key secondary endpoint included the change in the severity of clinical reactions during OFC. Other secondary endpoints included size of the skin test wheal to peanut extract and the percent of basophils activated ex vivo by peanut extract while receiving acalabrutinib compared with baseline. Safety endpoints included electrocardiography and laboratory blood testing, including complete blood counts and differentials, serum chemistries, and liver function tests. Exploratory endpoints included changes in circulating quantitative immunoglobulins and serum-specific IgE to peanut and peanut components.
|
PMC10425211
|
Skin puncture testing.
|
SKIN
|
End-point titration skin puncture testing was performed using whole peanut extract (Greer), undiluted and in 9 serial 1:10 dilutions (original units given by manufacturer, weight/volume). Histamine (1 mg/mL; ALK) and saline (Greer) were used as positive and negative controls, respectively. Lincoln Diagnostics Multi-Test II devices and testing trays were used for skin testing application for all extract dilutions and controls. Skin tests were read 15 minutes after application. The wheal and flare were each circled with a ballpoint pen and transferred to a clear adhesive sheet, from which the largest diameter and its shortest perpendicular diameter for each wheal was measured in millimeters. Skin test wheal area was calculated as π × (average radius)
|
PMC10425211
|
|
Oral food challenge to peanut.
|
ADVERSE EFFECT
|
All patients underwent a patient-blinded, placebo-controlled, graded OFC to peanut at visit 1 to establish their baseline level of clinical reactivity. The food challenge protocol was designed to detect the “no observed adverse effect level”, or the highest dose observed not to produce any adverse effect, for each patient (
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PMC10425211
|
|
Symptom score assessment.
|
Adapted from the PRACTALL scale (
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PMC10425211
|
||
Basophil activation testing.
|
MP
|
Whole blood samples drawn into 4 mL lithium heparin phlebotomy tubes (BD Biosciences) before food challenge at each visit were utilized for basophil activation testing. Whole blood was incubated with mouse IgM anti-human-IgE monoclonal antibody (clone 6061P, Hybridoma Labs), the indicated dilutions of peanut extract (Greer), 1 μM fMLP (Sigma), or vehicle (Greer incipient control solution) in PAGCM buffer (piperazine-N,N′-bis[2-ethanesulfonic acid] + bovine serum albumin [MP Biomedicals] + glucose [Sigma-Aldrich] + 1.7 mM calcium + 1.7 mM magnesium) for 30 minutes at 37°C. Cells were then fixed using Phosflow Fix Buffer (BD Biosciences), centrifuged at 400
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PMC10425211
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