{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,5]],"date-time":"2026-09-05T05:54:16Z","timestamp":1788587656022,"version":"build-2803163510"},"update-to":[{"DOI":"10.12688\/f1000research.52607.1","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2021,7,26]],"date-time":"2021-07-26T00:00:00Z","timestamp":1627257600000}}],"reference-count":50,"publisher":"F1000 Research Ltd","license":[{"start":{"date-parts":[[2021,7,26]],"date-time":"2021-07-26T00:00:00Z","timestamp":1627257600000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["f1000research.com"],"crossmark-restriction":false},"short-container-title":["F1000Res"],"abstract":"<ns3:p><ns3:bold>Background:\u00a0<\/ns3:bold>Women with epilepsy face difficult choices whether to continue antiepileptic drug treatment during pregnancy, as uncontrolled seizures carry great risk to mother and fetus but continuing treatment may have adverse effects on baby\u2019s development. This study aimed at evaluating antiepileptic drug entry into developing brain.<\/ns3:p><ns3:p><ns3:bold>Methods:\u00a0<\/ns3:bold>Anaesthetised pregnant, non-pregnant adult females, postnatal and fetal rats were injected intraperitoneally with different doses, single or in combinations, of valproate and lamotrigine,\u00a0within clinical range. Injectate included\u00a0<ns3:sup>3<\/ns3:sup>H-labelled drug. After 30min, CSF, blood and brain samples were obtained; radioactivity\u00a0measured using liquid scintillation counting. Some animals were also exposed to valproate in feed throughout pregnancy and into neonatal period.\u00a0Drug levels\u00a0measured by liquid chromatography coupled to mass spectrometry (LC-MS). Results\u00a0given as CSF or tissue\/plasma% as index of drug entry.<\/ns3:p><ns3:p><ns3:bold>Results:\u00a0<\/ns3:bold>Entry of valproate into brain and CSF was higher at E19 and P4 compared to adult and was dose-dependent except at E19; placental transfer increased significantly at highest dose of 100mg\/kg. Lamotrigine entry into the brain was dose dependent only at E19. Chronic valproate treatment, or combination of valproate and lamotrigine had little effect on either drug entry, except for reduced valproate brain entry in adult brain with chronic treatment. Placental transfer decreased significantly after chronic valproate treatment. LC-MS measurement of valproate in adults confirmed that rat plasma values were within the clinical range and CSF\/plasma and brain\/plasma ratios for LC-MS and\u00a0<ns3:sup>3<\/ns3:sup>H-valproate were similar.<\/ns3:p><ns3:p><ns3:bold>Conclusion:\u00a0<\/ns3:bold>Results suggest that entry of valproate may be higher in developing brain, the capacity of barrier mechanism is mostly unaffected by doses within the clinical range, with or without addition of lamotrigine. Chronic valproate exposure may result in upregulation in cellular mechanisms restricting its entry into the brain. Entry of lamotrigine was little different at different ages and was not dose dependent.<\/ns3:p>","DOI":"10.12688\/f1000research.52607.2","type":"journal-article","created":{"date-parts":[[2021,7,26]],"date-time":"2021-07-26T11:30:06Z","timestamp":1627299006000},"page":"384","update-policy":"https:\/\/doi.org\/10.12688\/f1000research.crossmark-policy","source":"Crossref","is-referenced-by-count":11,"title":["Entry of antiepileptic drugs (valproate and lamotrigine) into the developing rat brain"],"prefix":"10.12688","volume":"10","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0323-9589","authenticated-orcid":false,"given":"Samuel 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status","group":{"name":"current-referee-status","label":"Current Referee Status"}},{"value":"10.5256\/f1000research.55908.r85603, Alan Leviton, Department of Neurology, Boston Children's Hospital's Hospital, Harvard Medical School, Boston, MA, USA, 07 Jun 2021, version 1, indexed","URL":"https:\/\/f1000research.com\/articles\/10-384\/v1#referee-response-85603","order":0,"name":"referee-response-85603","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"<b>Norman Saunders<\/b>; <i>Posted: 28 Jun 2021<\/i>; We should like to thank Dr Leviton for his review of our paper. We appreciate his comment that the study was a thorough evaluation and likely to be of value to clinicians in advising their patients who are required to take antiepileptic drugs during pregnancy.","URL":"https:\/\/f1000research.com\/articles\/10-384\/v2#referee-comment-6843","order":1,"name":"referee-comment-6843","label":"Referee Comment","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.5256\/f1000research.55908.r86533, Neil Dani, Department of Pathology, Boston Children's Hospital, Boston, MA, USA, 15 Jun 2021, version 1, indexed","URL":"https:\/\/f1000research.com\/articles\/10-384\/v1#referee-response-86533","order":2,"name":"referee-response-86533","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"<b>Norman Saunders<\/b>; <i>Posted: 28 Jun 2021<\/i>; We should like to thank Dr Dani for his review of our paper and his comment that our study will provide guidelines for future experiments. In fact, we are already embarking on these.","URL":"https:\/\/f1000research.com\/articles\/10-384\/v2#referee-comment-6844","order":3,"name":"referee-comment-6844","label":"Referee Comment","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.5256\/f1000research.55908.r86365, Stefan Liebner, Elif Fidan, Institute of Neurology (Edinger-Institute), Interdisciplinary Centre for Neuroscience Frankfurt, Frankfurt, Germany, 24 Jun 2021, version 1, indexed","URL":"https:\/\/f1000research.com\/articles\/10-384\/v1#referee-response-86365","order":4,"name":"referee-response-86365","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"<b>Norman Saunders<\/b>; <i>Posted: 18 Jul 2021<\/i>; We thank Drs Liebner and Fidan for their detailed appraisal of our paper. We list our responses below. Where appropriate changes in the ms have been tracked. <b>Major points:<\/b> The major points of critique on the manuscript by Toll&nbsp; <i>et al.<\/i>&nbsp;are listed below:&nbsp; Figure 3E shows that there is a significant difference in the entry of 30 and 100 mg\/kg valproate into cortex at P4. However, the authors claim on Page 13, in Paragraph 2: &quot;For a dose of 30 mg\/kg (within the clinical range, Johannessen &amp; Johannessen, 2003) and 100 mg\/kg above the usual clinical range there was no difference in entry at any of the three ages studied.&quot;&nbsp;The authors should comment on this discrepancy. <i>We thank the reviewers for drawing out attention to this discrepancy which has now been corrected. \u201cFor a dose of 30 mg\/kg (within the clinical range, Johannessen &amp; Johannessen, 2003) and 100 mg\/kg (above the usual clinical range) there was no difference in entry into CSF at any of the three ages studied; in cortex and brainstem the entry of valproate was higher at P4 with the larger clinical dose, but not at the other ages (Figure 3).\u201d<\/i> &nbsp; Figure 1 shows the residual vascular space in the organs, cortex, brainstem, and thymus. The authors report that each point represents the result from a single animal. Assuming that one animal was used to collect these three organs, why is the number of animals different for the different organs? For example the figure shows in A that n=3 animals were used to analyze the residual vascular space in the adult cortex, while the number of animals for the analysis of the brainstem is only n=2. <i>We initially only took cortex but later on decided to investigate brainstem as well so the first adult used for dextran blood space didn\u2019t have a brainstem value. <\/i> &nbsp; There is a high variation in the n numbers between different groups and\/or different time points. Moreover, in several cases, the n number is not high enough to perform statistical analysis. For instance, in Figure 4E, one of the three animals might be outlier, suggesting that more replicates are needed. The authors should comment on this. <i>We do comment on this in \u201cLimitations of the Methods\u201d and we agree, that more experiments would have been desirable.<\/i> &nbsp; The authors have evaluated the entry of&nbsp; <sup>3<\/sup>H-valproate into the cortex of E19 fetuses following chronic feeding of the mother. Considering the idea of combining valproate with lamotrigine, additional studies in which the entry of the drugs when combined in a chronic treatment paradigm would be complementary. <i>We agree with this point and these experiments are planned. &nbsp;It takes considerable time to prepare feeds, check if animals consume enough to obtain a clinical range of the drug in plasma and make sure they are willing to mate when on this treatment.<\/i> &nbsp; It is not very clear in the methods part whether the animals in the chronic group received 100 mg\/kg i.p. valproate injection or not. If they did, isn\u2019t it a combination of chronic+acute instead of just a chronic phase? If they did not, isn\u2019t this time frame following the i.p. injection too short to evaluate a chronic phase outcome? <i>All animals with chronic feeding received a final injection with radiolabelled valproate (described on page 6). The final acute injection is used to measure permeability to find out whether chronic exposure alters this aspect of the barrier mechanisms and therefore drug entry (see Koehn et al, 2019).<\/i> As it is written in the figure legend and on Page 12, Paragraph 6, Figures 9A and 9C show results from only one animal for each time point which does not seem to be sufficient to draw a conclusion. Moreover, Figures 9B and 9C show aggregated data along the time frame following the injection. A setup in which enough number of replicates were used for each time point and in which the aggregated data is from those replicates for each time point would be better to understand the dose and time-dependent entry of the drug into the fetus. <i>Each point is one fetus but the whole line graph shows data for that litter. Fetuses are taken one after the next therefore there will always be a time lag between littermates.<\/i> &nbsp; The authors write in the thymus permeability section: &quot;At P4 for doses of 30 and 100 mg\/kg the entry was substantially less than at E19 or in the adult but increased with a dose of 200 mg\/kg valproate.&quot;&nbsp;However, Figures 10B and C show that valproate entry to the thymus at 100 mg\/kg dose is higher in P4 compared to the adult. The authors should clarify this. <i>We thank the reviewers for drawing our attention to this error which has been corrected. \u201cAt P4 for the dose of 30 mg\/kg the entry was substantially less than at E19 or in the adult but increased with a dose of 200 mg\/kg valproate. At 100 mg\/kg the entry declined with age.\u201d<\/i> &nbsp; Considering the increase in the entry of lamotrigine to CSF and brain with age, it is suggested in the manuscript that mechanisms protecting the brain against entry of lamotrigine are well established at E19 but appear less effective at older ages. This sounds like an assertive explanation for the observed phenomena with statistically not satisfying numbers of replicates. Given that lamotrigine potentially is a substrate of P-glycoprotein, the authors should comment on the regulation of the ABC transporter between E19 and postnatal stages up to adulthood. <i>As the reviewers comment lamotrigine is at best a potential substrate for P-glycoprotein. We have modified the statement in the text. \u201cOne possible explanation for the observed age-related differences in the entry of LTG and VPA into the brain could be the time frame of when responsible transporters become functional (see Discussion in Koehn et al, 2019).\u201d&nbsp;<\/i> &nbsp; The &quot;Limitations of the study&quot;&nbsp;part does not add anything to the scientific quality of the article. These limitations might be valid for any research group during the pandemic. The reviewer can only judge if the presented data are valid and if experimental and statistical rigidity were applied. Why and how it came about, and their ability to present solid data, cannot be judged by the reviewer. <i>We disagree and note that two other Reviewers have not commented upon this aspect of the paper. We have nevertheless restricted the comment on the impact of the pandemic to a single sentence. &nbsp;We feel strongly that all papers should carry a section detailing limitations. If more authors were willing to acknowledge the experimental limitations of their studies it might lead to an improvement in standards of research and help readers assess validity of results (e.g. Begley &amp; Ioannidis, 2015, Circ Res. 2015;116:116-126. DOI: 10.1161\/CIRCRESAHA.114.303819.). <\/i> <b>Minor points:<\/b> Page 1, Results section: The sentence &quot;Entry of valproate into brain and CSF was higher at E19 and P4 compared to adult but was not dose-dependent;..&quot;&nbsp;could be written in a different way to make it clear that valproate entry is dose dependent but only at E19 this is not the case. <i>Revised to read \u201cEntry of valproate into brain and CSF was higher at E19 and P4 compared to adult and was dose-dependent, except at E19.&quot;&nbsp;<\/i> &nbsp; Page 3, line 69: Check the format of the reference &quot;(rat, Koehn&nbsp; <i>et al<\/i>., 2019; Koehn&nbsp; <i>et al<\/i>., 2020)&quot;. Was the word &quot;rat&quot;&nbsp;supposed to be in the reference? Can remove? <i>This was to indicate that the animal model was rat. Text modified to indicate this.<\/i> &nbsp; Page 7: &quot;The ratio of 3H-dextran into cortex and brainstem compared with plasma, illustrated in Figure 1A and B, showed no significant difference at any of the ages studied and was around 2\u20134%.&quot;&nbsp;Was it supposed to be &quot;entry into&quot;&nbsp;instead of just &quot;into&quot;? <i>Detectable dextran does not enter the brain in 5 minutes. The wording has been changed to \u201cin brain tissue\u201d to clarify this.<\/i> &nbsp; Figure 9C: Why the 20mg\/kg case lasts only until app. 95 min? It is written on Page&nbsp; 12 &quot;Figures 9B and 9D show the aggregated data up to 110 min.&quot; However, the 20mg\/kg does not reach that time point. <i>Usually fetuses are collected at 10 minutes intervals, the particular dam used for the 20mg\/kg experiment had 5 pups. We limit the time to present results from all experiments to 110 min to allow longer experiments (more fetuses) to be comparable with shorter experiments.<\/i> &nbsp; Figure 10: Why is &quot;Thymus&quot;&nbsp;written on top of some graphs while on top of others not? This &quot;subtitle&quot;&nbsp;is not needed anyway, since there is already main title of the figure. <i>Thank you for drawing our attention to this anomaly. We have asked for \u201cThymus\u201d to be removed from Figure 10 B and C and Figure 12 B.<\/i>","URL":"https:\/\/f1000research.com\/articles\/10-384\/v2#referee-comment-6930","order":5,"name":"referee-comment-6930","label":"Referee Comment","group":{"name":"article-reports","label":"Article Reports"}},{"value":"The author(s) declared that no grants were involved in supporting this work","order":6,"name":"grant-information","label":"Grant Information"},{"value":"This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.","order":0,"name":"copyright-info","label":"Copyright"}]}}