Prospects of pharmacotranscriptomics in understanding the effects of antiepileptic drugs and searching for new classes of antiepileptic drugs

Introduction

Epilepsy is a genetically and clinically heterogeneous, prevalent, and socially significant disorder affecting all age groups, with approximately 1-2% of the global population diagnosed with the condition [1]. It is characterized by recurrent unprovoked seizures caused by an imbalance between excitation and inhibition in neuronal circuits. This disease requires long-term, and in some cases lifelong, administration of antiepileptic drugs (AEDs) [1]. Chronic AED use, high AED doses, and AED polytherapy are associated with a high risk of adverse drug reactions (ADRs), including teratogenicity [2], neurotoxicity [3], cardiotoxicity [4], metabolic syndrome [5, 6], among others, as well as the development of therapeutic resistance with inadequate seizure control in a significant proportion of patients. Pharmacometabolomics (therapeutic drug monitoring (TDM) of AEDs, gas-liquid chromatography-mass spectrometry (GLC-MS) of active AED metabolites) and pharmacogenomics (pharmacogenetic testing of non-functional polymorphisms in genes encoding key AED metabolism and transport enzymes) are actively developing fields and represent priority areas in personalized neurology [7]. Despite growing researcher interest in the relationship between epigenetic biomarkers and the efficacy or safety of epilepsy pharmacotherapy, which influence the risk of AED-induced ADRs, approaches based on pharmacotranscriptomics [8] remain in their infancy and are not yet applied in routine clinical epileptology practice. The success of the multiomics approach (pharmacometabolomics, pharmacogenomics, and pharmacotranscriptomics) to epilepsy pharmacotherapy will largely depend on the criteria used for selecting methods to analyze AEDs and their active metabolites in biological fluids (plasma, serum, saliva, urine, hair), pharmacogenetic panels for identifying polymorphisms (variants) in candidate genes encoding AED metabolism and transport pathways, and epigenetic biomarkers (primarily microRNAs influencing changes in patient gene expression (transcriptome) in response to AED exposure (dose, duration, etc.), especially during long-term use. Identifying promising epigenetic biomarkers will increase the chances of success in microRNA-based association studies and may ultimately facilitate the development of a new class of drugs for patients with therapeutically resistant epilepsy.

Pharmacotranscriptomics and Epigenetic Biomarkers of Antiepileptic Drugs

Pharmacotranscriptomics is an emerging research field that has only recently begun to develop and holds promise for identifying targets, defining epigenetic biomarkers, and assessing AED efficacy [9] beyond the scope of pharmacogenomics and pharmacometabolomics [10]. Key research directions in the field of AED pharmacotranscriptomics include:

1. Elucidating AED mechanisms of action:
   a) RNA sequencing (RNA-seq) to analyze the entire transcriptome (all RNA molecules) in cells or tissues exposed to AEDs, enabling the determination of which genes are activated or suppressed by AEDs;
   b) Investigation of hub genes and their pathways to identify genes that play a central role in AED-induced changes in gene expression and the pathways in which they are involved;

2. Discovering new AEDs:
   a) Drug repurposing, where transcriptomic data identify drugs that modify the transcriptomic signature of epilepsy, including achieving seizure freedom; pharmacotranscriptomics-based drug repurposing helps identify new classes of drugs with potential antiepileptic effects;

3. Personalized neurology/epileptology:
   a) Predicting response to AEDs (efficacy and safety), where analyzing transcriptome data from epilepsy patients can aid in developing predictive models to determine which AEDs are most likely to be effective for a specific individual and which might lead to clinically significant ADRs (teratogenesis, neurotoxicity, cardiotoxicity, metabolic syndrome);

4. Understanding epilepsy pathophysiology, where transcriptomic studies can reveal molecular pathways involved in the pathogenesis, progression of epilepsy, and development of therapeutic resistance to AEDs, potentially leading to the identification of novel drug targets [11].

The mechanism of action of AEDs is linked to their effects on various molecular targets that selectively reduce neuronal excitability and provide adequate seizure control. First-generation and newer AEDs possess diverse mechanisms of action, which can be broadly categorized into two groups based on their regulatory functions concerning voltage-gated ion channels and synaptic excitability [12]. However, recent studies convincingly demonstrate that AEDs and their active metabolites can exert regulatory effects on gene expression as epigenetic modifiers [13].

Epigenetic modifications and regulators represent potential molecular elements that control relevant physiological and pathological processes, thereby influencing the natural course of epilepsy and the response to AEDs in a given individual. These epigenetic modulators can be used as biomarkers of AED efficacy and safety because they offer several advantages and provide information about gene function, thus explaining differences between endophenotypes of individual patients with epilepsy. Pharmacotranscriptomic technologies employed for analyzing epigenetic biomarkers are being developed and refined, becoming simpler and more accessible for use [9, 11, 13].

In 2017, García-Giménez et al. [8] proposed a modified definition of an epigenetic biomarker as any epigenetic mark or altered epigenetic mechanism that: 1) is stable and reproducible upon sample processing and can be measured in biological fluids or primary tissue specimen types (fresh, frozen, and formalin-fixed paraffin-embedded); 2) predicts the future risk of disease development (risk); 3) identifies disease (diagnosis); 4) reveals information about the natural history of the disease; 5) predicts disease outcome (prognosis); 6) responds to therapy (prediction); 6) monitors response to therapy or drugs (therapy monitoring); 7) allows simultaneous diagnosis and targeted therapy (theragnosis).

The advantages of pharmacotranscriptomics over pharmacometabolomics and pharmacogenomics in epileptology are explained by the fact that epigenetic biomarkers: 1) can provide crucial information about gene function in individual cell types, filling clinical gaps and showing the extent to which specific genetic programs are controlled; 2) can incorporate environmental information and may include data on the lifestyle of the patient with epilepsy, thereby explaining, for example, how nutrition and metabolic factors affect patient health and disease course; 3) can provide information about the natural history of epilepsy, acting as true bioarchives; 4) a wide range of epigenetic biomarkers (particularly microRNAs and post-translational histone modifications) are extremely stable in fluids (e.g., plasma, serum, urine, saliva, etc.) and most are also highly stable in major types of tissue specimens (e.g., fresh and frozen tissues, dried blood spots (Guthrie cards), paraffin-embedded tissue samples, etc.); 5) microRNAs are very stable molecules even in low-quality samples; 6) can provide valuable information for disease diagnosis, prognostication, and treatment monitoring; 7) can enable simultaneous diagnosis and targeted therapy, thus facilitating theranosis [8].

Significant epigenetic biomarkers include DNA methylation, histone protein modifications, and the functions of non-coding RNAs.

Cell-free circulating DNA (cfDNA) has been proposed as an epigenetic biomarker in various pathological conditions [8, 9] and could potentially be used in epileptology. The amount of cfDNA in healthy individuals is typically very low (less than 5 ng/mL in plasma) and can increase 8-10 fold in individuals with some forms of epilepsy. Limitations for the clinical use of cfDNA include challenges in its isolation from biological fluids and quantification due to the small amount and fragmented nature of cfDNA in available biospecimens. Furthermore, the extraction and purification step is critical for developing reproducible, standardized cfDNA isolation methods, including quality control to measure extraction efficiency, fragment size bias, and yield [14].

Histone proteins. The use of histone proteins as disease epigenetic biomarkers is based on analyzing post-translational histone modifications and their variations in the disease context and investigating histones in the extracellular environment (in blood). In the latter case, analyzing post-translational modifications of histone proteins is a valuable tool for diagnosing and/or predicting disease progression [8, 9]. Most kits are designed for rapid isolation of core histone proteins through simple procedures, providing acceptable yield, although they do not preclude the simultaneous isolation of other nuclear proteins. Their primary application is functional analysis performed via western blotting. However, using histone proteins in epigenetic regulation research is far from their application as epigenetic biomarkers of clinical significance, for instance, in assessing AED efficacy and safety. Limitations also include challenges underlying histone protein isolation methods with contamination by other nuclear proteins and components. Most kits and purification methods require high cell densities, which can be obtained through tissue homogenization or isolation of blood cells. There are currently few available methods for purifying histone proteins from biological fluids [11, 13].

It is known that histone modifications influence transcription and other functions of DNA as a template [13]. This process is regulated by specific enzymatic mechanisms in which metabolites act as co-substrates or activators/inhibitors. One of the most common ways to modify histone proteins is acetylation, which neutralizes the positively charged lysine residues abundant in histones, thereby "opening" chromatin and making DNA more accessible to other protein factors [15]. Histone acetylation status is regulated by the balance between histone acetyltransferase and histone deacetylase (HDAC) activity. HDAC inhibition causes an accumulation of acetylated histone forms, thus regulating gene expression, cell proliferation, and cell death. Some AEDs may act as HDAC inhibitors and play a crucial role in multiple mechanisms of gene expression. For example, valproic acid (VPA) was the first AED known to non-selectively inhibit HDAC [16, 17]. Subsequently, carbamazepine (CBZ), topiramate (TPM), and lacosamide (LCM) have also been shown to be HDAC inhibitors [18, 19]. Levetiracetam (LEV) cannot directly affect HDAC activity, but 2-pyrrolidinone-n-butyric acid (the major metabolite of LEV) promotes histone deacetylation in HeLa cells [34].

Circulating microRNAs. MicroRNAs can also be detected in biological fluids or via liquid biopsy, and since some show altered levels in patients with various clinical forms of epilepsy [20], the number of studies demonstrating their promise as epigenetic biomarkers of therapeutic resistance to AEDs and the development of ADRs (e.g., AED-induced metabolic syndrome [6]) has increased in recent years. Depending on the laboratory diagnostic protocol used, one can distinguish: free circulating microRNAs; protein-bound microRNAs; microvesicle-associated microRNAs; all microRNAs present in a blood sample. However, a limitation of using circulating microRNAs as epigenetic biomarkers is the lower efficiency and yield from blood plasma and serum compared to microRNA isolation from cells and tissues [21].

Abnormal microRNA expression can lead to aberrant protein expression, and these unintended responses may be induced by AEDs. For instance, prenatal exposure to VPA leads to overexpression of miR-132 in the mouse embryonic brain, subsequently reducing the levels of its molecular targets — methyl-CpG-binding protein 2 (MECP2) and Rho-GTPase-activating protein (p250GAP) — which may result in autistic-like behavior and pathological changes in the mouse cerebral cortex [22]. Phenobarbital (PB) can cause changes in the expression levels of the gene encoding delta-like homolog 1 and the gene encoding type 3 deiodinase (Dlk1-Dio3), which can express microRNA clusters, resulting in hepatocyte hypertrophy and reprogramming, increasing the risk of PB-induced liver cancer in rodents [23]. CBZ-induced dermatotoxicity (specifically Stevens-Johnson syndrome) is associated with microRNA dysregulation in an experimental analysis of immune cells [24].

Discussion

Recent studies have shown that AEDs can: alter DNA methylation; influence histone protein modification by affecting enzymes such as DNA methyltransferases, histone deacetylases, and methyl-binding proteins; change the expression levels of microRNAs [25]. In this context, cfDNA, modified histone proteins, and circulating microRNAs can be considered promising epigenetic biomarkers of AED efficacy and safety, influencing the expression of target genes of AED action. This explains the predictive, preventive, diagnostic, and therapeutic role of pharmacotranscriptomics based on the aforementioned biomarkers in epilepsy, therapeutic resistance to AEDs, and AED-induced ADRs [26, 27], alongside pharmacogenomics [7, 28, 29] and pharmacometabolomics [10, 17].

Thus, it has been shown that VPA-induced hepatotoxicity with the development of non-alcoholic fatty liver disease is associated with DNA methylation and dysregulation of the PPARγ, PPARα, AHR, and CD36 genes [30], while VPA-induced disruption of folate metabolism is associated with DNA methylation and dysregulation of the MTHFR gene [2, 31]. In animal models (rodents), VPA-induced overexpression of miR-132 and miR-134-5p has been associated with the development of autism spectrum disorders [22, 32]. PB-induced disruption of histone deacetylase H3 acetylation and methylation affects the metabolism of this drug [33], and overexpression of miR-200b and miR-221 is associated with PB-induced carcinogenesis [34]. CBZ-induced disruption of histone acetylation alters the regulation of the CYP3A4 gene, slowing the metabolism of this drug [35], and CBZ-induced overexpression of miR-155, miR-18a, and miR-21 is associated with dermatotoxicity [24]. AED-induced neurotoxicity, particularly concerning the fetal brain in pregnant women with epilepsy, is explained by multiple pathogenetic mechanisms, including disruption of folate metabolism and altered expression of placental transporter proteins [2, 36]. In the future, pharmacotranscriptomics may help develop new strategies for predicting, preventing, and correcting these ADRs [37, 38].

Pharmacotranscriptomics helps reshape our understanding of AED mechanisms of action. For example, VPA increases methylation of the -39C locus in the SCN3A gene promoter and may increase levels of fat mass and obesity-associated protein (FTO), which in turn inhibits the expression of MBD2 and NaV1.3 genes, providing a new explanation for the antiseizure effect of this drug [39]. The antiseizure effect of ethosuximide is associated with overexpression of DNMT gene mRNA in the cerebral cortex in an animal model of epilepsy (rats) [40].

Furthermore, results from pharmacotranscriptomics research are leading to AED repurposing. For example, the molecular effects of VPA include DNA methylation, histone acetylation, and modification of histones H3 and H4 by histone deacetylases [41, 42], and altered expression of microRNAs (hsa-miR-124, hsa-miR-125a, hsa-miR-125b, hsa-miR-133b, hsa-miR-145-5p, hsa-miR-205) [43, 44, 45]. As a result of VPA-induced DNA methylation, the regulation of various genes and their pathways is altered (e.g., BRD1, CD133, NANOG, NGN1, OCT4, SCN3A, SOX2, etc.), enabling the repurposing of VPA from an antiseizure and mood-stabilizing drug to an antineoplastic and immunomodulatory agent [46].

The molecular effects of CBZ and LEV include DNA methylation, histone acetylation, and modification of histone deacetylase H3 [13, 33, 37, 47]. CBZ-induced DNA methylation has been shown to alter the regulation of the BRD1 gene [13]. Administration of VPA and CBZ can induce transcription activation of the BRD1 gene, associated with schizophrenia predisposition, through demethylation of its promoter, making BRD1 a new target for these drugs in treating schizophrenia spectrum disorders [48].

Molecular effects of lacosamide include histone acetylation and altered microRNA expression [49]. Lacosamide and brivaracetam decrease hsa-miR-107 expression and increase hsa-miR-195-5p expression, explaining their antineoplastic effect [50]. Oxcarbazepine-induced DNA methylation leads to altered regulation of the GABRB2 gene, through which its psychotropic effect is achieved [51]. Lamotrigine affects histone acetylation [33], and ethosuximide affects DNA methylation, altering the regulation of DNMT1 and DNMT3 genes [38]. Experiments using animal models have shown that cannabidiol influences DNA methylation, altering the regulation of the CB1 gene and mitochondrial ferritin, mediating its psychotropic and neuroprotective effects [52, 53].

In recent years, the pharmaceutical industry has faced declining productivity in research and new development in epileptology, resulting in fewer AEDs reaching the market despite increased investment. This is because some AED candidates fail during later stages of development due to safety concerns and/or previously undetected ADRs. The QSTAR project demonstrated that pharmacotranscriptomics, through gene expression profiling, can identify compound ADRs and is a valuable tool for decision-making in the early stages of new AED development [54]. Single-cell RNA sequencing (scRNA-Seq) combined with parallel CRISPR-based systems, also known as Perturb-seq, CRISP-Seq, and CROP-seq, could aid in developing new AEDs [55]. This approach allows screening of genes involved in therapeutic resistance to AEDs or specific cellular targets, combining the resolution of massively parallel scRNA-Seq with the scale of genome editing in pooled CRISPR screens, providing functional information about the impact of a specific genetic perturbation on the measurable epilepsy phenotype in an individual patient [56, 57].

A study by Lin WH et al. [58] demonstrated that RNA interference screening could help identify novel targets for next-generation AEDs based on increased expression of the homeostatic regulator pumilio (Pum). Pum activity is known to be regulated by depolarization of brain neurons. Enhanced synaptic excitation increases Pum expression and enhances translational repression of voltage-gated sodium channel (Nav) transcripts, which is sufficient to inhibit Na+ ion current (INa) in neurons and ultimately reduce the frequency of action potential generation and epileptic seizures.

Wang L et al. [59] showed that miR-139-5p increases sensitivity to AEDs in therapeutically resistant epilepsy by inhibiting multidrug resistance-associated protein 1 (MRP1). Furthermore, miR-139-5 expression levels influence cerebral cortex development, with miR-139-5p overexpression potentially attenuating cerebral cortex damage through regulatory effects on cortical migration by targeting Lis1 [60]. miR-139-5p agonists (ago-miR-139-5p) attenuate damage in epilepsy patients by inhibiting human transforming growth factor [59]. Thus, a new class of AEDs that increases miR-139-5p expression could prevent further epilepsy development and reduce the risk of developing therapeutic resistance to AEDs.

Conclusion

Knowledge of transcriptome variants and their influence in the context of molecular changes causing epigenetic modification of epilepsy course and individual response to AEDs in patients with epilepsy, using non-integrated technologies, may help reduce the risk of developing therapeutic resistance to AEDs and serious ADRs. The multifaceted nature of epilepsy as a genetically and clinically heterogeneous disease and its subcellular heterogeneity play a crucial role in AED efficacy and safety, therapeutic resistance, and toxicity. Pharmacotranscriptomics is a powerful tool for understanding the molecular mechanisms of AED action, discovering new microRNA-based classes of AEDs, and advancing personalized medicine to achieve an optimal balance between the efficacy and safety of epilepsy pharmacotherapy.