Entry of antiepileptic drugs (valproate and lamotrigine) into the developing rat brain

Abstract

Background: 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’s development. This study aimed at evaluating antiepileptic drug entry into developing brain. Methods: Anaesthetised pregnant, non-pregnant adult females, postnatal and fetal rats were injected intraperitoneally with different doses, single or in combinations, of valproate and lamotrigine, all within clinical range. Injectate included 3H-labelled drug. After 30min, CSF, blood and brain samples were obtained; radioactivity was measured using liquid scintillation counting. Some animals were also exposed to valproate in feed throughout pregnancy and into neonatal period. Drug levels were measured by liquid chromatography coupled to mass spectrometry (LC-MS). Results are given as CSF or tissue/plasma% as index of drug entry. Results: Entry of valproate into brain and CSF was higher at E19 and P4 compared to adult but was not dose-dependent; 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 3H-valproate were similar. Conclusion: 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.

Developmental changes in the extent of drug binding to rat plasma proteins

“…Two hundred μl of plasma was spiked with 10 μl of test marker or drug at clinically relevant concentration and traced with 0.1 μCi of their respective radiolabelled form (Table 1 ). Samples were incubated for 30 min at 37 °C with gentle agitation, intended to be consistent with conditions of in vivo permeability experiments 35 – 37 , before undergoing ultrafiltration using 30 kDa molecular weight cut-off centrifugal filters (Centrifree®, Merck Millipore) for 3 min to obtain samples of the protein-free fraction. Filtrate volumes were kept to a minimum of about 15% (< 30 μl) of the total sample volume.…”

“… Concentrations of drugs used were based on previous in vivo studies (digoxin and paracetamol 35 ; ivacaftor 36 ; valproate and lamotrigine 37 ; olanzapine, Huang et al 2023 in preparation). …”

“…To validate that the in vitro ultrafiltration method for determination of extent of protein binding reflects protein binding in vivo, two drugs, valproate and lamotrigine, were also tested in vivo in P4 pups. Briefly, animals received an injection of one of the drugs and blood was collected after 30 min reflecting the protocol of the in vitro method and as detailed previously 35 – 37 . Immediately following sampling of blood, plasma was separated and used for ultrafiltration as described above.…”

“…Immediately following sampling of blood, plasma was separated and used for ultrafiltration as described above. Quantitation of valproate and lamotrigine in whole plasma and the protein-free fractions was performed using liquid chromatography coupled to mass spectrometry (LC–MS/MS) methods established previously 37 , detailed methods are described in Supplementary Information . Data from in vivo and in vitro measurements are summarised and compared in Table 2 .…”

“…The validity of the method used was confirmed using samples from in vivo experiments for two of the drugs (lamotrigine and valproate) that were measured using liquid chromatography coupled to mass spectrometry. In the Discussion the measurements of the drugs’ protein binding have been related to their in vivo brain and CSF levels following systemic administration using previously published data 35 – 37 .…”