Circulating microRNAs and isomiRs as biomarkers for the initial insult and epileptogenesis in four experimental epilepsy models: The EPITARGET study.

OBJECTIVE: Structural epilepsies can manifest months or years after the occurrence of an initial epileptogenic insult, making them amenable for secondary prevention. However, development of preventive treatments has been challenged by a lack of biomarkers for identifying the subset of individuals with the highest risk of epilepsy after the epileptogenic insult. METHODS: Four different rat models of epileptogenesis were investigated to identify differentially expressed circulating microRNA (miRNA) and isomiR profiles as biomarkers for epileptogenesis. Plasma samples were collected on day 2 and day 9 during the latency period from animals that did or did not develop epilepsy during long-term video-electroencephalographic monitoring. miRNAs and isomiRs were identified and measured in an unsupervised manner, using a genome-wide small RNA sequencing platform. Receiver operating characteristic analysis was performed to determine the performance of putative biomarkers. RESULTS: Two days after an epileptogenic insult, alterations in the levels of several plasma miRNAs and isomiRs predicted epileptogenesis in a model-specific manner. One miRNA, miR-3085, showed good sensitivity (but low specificity) as a prognostic biomarker for epileptogenesis in all four models (area under the curve = .729, sensitivity = 83%, specificity = 64%, p < .05). SIGNIFICANCE: Identified plasma miRNAs and isomiRs are mostly etiology-specific rather than common prognostic biomarkers of epileptogenesis. These data imply that in preclinical and clinical studies, it may be necessary to identify specific biomarkers for different epilepsy etiologies. Importantly, circulating miRNAs like miR-3085 with high negative predictive value for epileptogenesis in different etiologies could be useful candidates for initial screening purposes of epileptogenesis risk.

Emerging Molecular Targets for Anti-Epileptogenic and Epilepsy Modifying Drugs.

The pharmacological treatment of epilepsy is purely symptomatic. Despite many decades of intensive research, causal treatment of this common neurologic disorder is still unavailable. Nevertheless, it is expected that advances in modern neuroscience and molecular biology tools, as well as improved animal models may accelerate designing antiepileptogenic and epilepsy-modifying drugs. Epileptogenesis triggers a vast array of genomic, epigenomic and transcriptomic changes, which ultimately lead to morphological and functional transformation of specific neuronal circuits resulting in the occurrence of spontaneous convulsive or nonconvulsive seizures. Recent decades unraveled molecular processes and biochemical signaling pathways involved in the proepileptic transformation of brain circuits including oxidative stress, apoptosis, neuroinflammatory and neurotrophic factors. The "omics" data derived from both human and animal epileptic tissues, as well as electrophysiological, imaging and neurochemical analysis identified a plethora of possible molecular targets for drugs, which could interfere with various stages of epileptogenetic cascade, including inflammatory processes and neuroplastic changes. In this narrative review, we briefly present contemporary views on the neurobiological background of epileptogenesis and discuss the advantages and disadvantages of some more promising molecular targets for antiepileptogenic pharmacotherapy.

Cloning and Functional Analysis of Rat Tweety-Homolog 1 Gene Promoter.

Tweety-homolog 1 protein (Ttyh1) is abundantly expressed in neurons in the healthy brain, and its expression is induced under pathological conditions. In hippocampal neurons in vitro, Ttyh1 was implicated in the regulation of primary neuron morphology. However, the mechanisms that underlie transcriptional regulation of the Ttyh1 gene in neurons remain elusive. The present study sought to identify the promoter of the Ttyh1 gene and functionally characterize cis-regulatory elements that are potentially involved in the transcriptional regulation of Ttyh1 expression in rat dissociated hippocampal neurons in vitro. We cloned a 592 bp rat Ttyh1 promoter sequence and designed deletion constructs of the transcription factors specificity protein 1 (Sp1), E2F transcription factor 3 (E2f3), and achaete-scute homolog 1 (Ascl1) that were fused upstream of a luciferase reporter gene in pGL4.10[luc2]. The luciferase reporter gene assay showed the possible involvement of Ascl1, Sp1, and responsive cis-regulatory elements in Ttyh1 expression. These findings provide novel information about Ttyh1 gene regulation in neurons.

Lipopolysaccharide Upregulates Palmitoylated Enzymes of the Phosphatidylinositol Cycle: An Insight from Proteomic Studies.

Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria that induces strong proinflammatory reactions of mammals. These processes are triggered upon sequential binding of LPS to CD14, a GPI-linked plasma membrane raft protein, and to the TLR4/MD2 receptor complex. We have found earlier that upon LPS binding, CD14 triggers generation of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], a lipid controlling subsequent proinflammatory cytokine production. Here we show that stimulation of RAW264 macrophage-like cells with LPS induces global changes of the level of fatty-acylated, most likely palmitoylated, proteins. Among the acylated proteins that were up-regulated in those conditions were several enzymes of the phosphatidylinositol cycle. Global profiling of acylated proteins was performed by metabolic labeling of RAW264 cells with 17ODYA, an analogue of palmitic acid functionalized with an alkyne group, followed by detection and enrichment of labeled proteins using biotin-azide/streptavidin and their identification with mass spectrometry. This proteomic approach revealed that 154 fatty-acylated proteins were up-regulated, 186 downregulated, and 306 not affected in cells stimulated with 100 ng/ml LPS for 60 min. The acylated proteins affected by LPS were involved in diverse biological functions, as found by Ingenuity Pathway Analysis. Detailed studies of 17ODYA-labeled and immunoprecipitated proteins revealed that LPS induces S-palmitoylation, hence activation, of type II phosphatidylinositol 4-kinase (PI4KII) ß, which phosphorylates phosphatidylinositol to phosphatidylinositol 4-monophosphate, a PI(4,5)P2 precursor. Silencing of PI4KIIß and PI4KIIα inhibited LPS-induced expression and production of proinflammatory cytokines, especially in the TRIF-dependent signaling pathway of TLR4. Reciprocally, this LPS-induced signaling pathway was significantly enhanced after overexpression of PI4KIIß or PI4KIIα; this was dependent on palmitoylation of the kinases. However, the S-palmitoylation of PI4KIIα, hence its activity, was constitutive in RAW264 cells. Taken together the data indicate that LPS triggers S-palmitoylation and activation of PI4KIIß, which generates PI(4)P involved in signaling pathways controlling production of proinflammatory cytokines.

miR-124-3p is a chronic regulator of gene expression after brain injury.

Traumatic brain injury (TBI) initiates molecular and cellular pathologies that underlie post-injury morbidities, including hippocampus-related memory decline and epileptogenesis. Non-coding small RNAs are master regulators of gene expression with the potential to affect multiple molecular pathways. To evaluate whether hippocampal gene expression networks are chronically regulated by microRNAs after TBI, we sampled the dentate gyrus of rats with severe TBI induced by lateral fluid-percussion injury 3 months earlier. Ingenuity pathway analysis revealed 30 upregulated miR-124-3p targets, suggesting that miR-124-3p is downregulated post-TBI (z-score = - 5.146, p < 0.05). Droplet digital polymerase chain reaction (ddPCR) and in situ hybridization confirmed the chronic downregulation of miR-124-3p (p < 0.05). Quantitative PCR analysis of two targets, Plp2 and Stat3, indicated that their upregulation correlated with the miR-124-3p downregulation (r = - 0.647, p < 0.05; r = - 0.629, p < 0.05, respectively). Immunohistochemical staining of STAT3 confirmed the increased protein expression. STRING analysis showed that 9 of the 30 miR-124-3p targets belonged to a STAT3 network. Reactome analysis and data mining connected the targets especially to inflammation and signal transduction. L1000CDS2 software revealed drugs (e.g., importazole, trichostatin A, and IKK-16) that could reverse the observed molecular changes. The translational value of our data was emphasized by in situ hybridization showing chronic post-traumatic downregulation of miR-124-3p in the dentate gyrus of TBI patients. Analysis of another brain injury model, status epilepticus, highlighted the fact that chronic downregulation of miR-124 is a common phenomenon after brain injury. Together, our findings indicate that miR-124-3p is a chronic modulator of molecular networks relevant to post-injury hippocampal pathologies in experimental models and in humans.

Standardization procedure for plasma biomarker analysis in rat models of epileptogenesis: Focus on circulating microRNAs.

The World Health Organization estimates that globally 2.4 million people are diagnosed with epilepsy each year. In nearly 30% of these cases, epilepsy cannot be properly controlled by antiepileptic drugs. More importantly, treatments to prevent or modify epileptogenesis do not exist. Therefore, novel therapies are urgently needed. In this respect, it is important to identify which patients will develop epilepsy and which individually tailored treatment is needed. However, currently, we have no tools to identify the patients at risk, and diagnosis of epileptogenesis remains as a major unmet medical need, which relates to lack of diagnostic biomarkers for epileptogenesis. As the epileptogenic process in humans is typically slow, the use of animal models is justified to speed up biomarker discovery. We aim to summarize recommendations for molecular biomarker research and propose a standardized procedure for biomarker discovery in rat models of epileptogenesis. The potential of many phylogenetically conserved circulating noncoding small RNAs, including microRNAs (miRNAs), as biomarkers has been explored in various brain diseases, including epilepsy. Recent studies show the feasibility of detecting miRNAs in blood in both experimental models and human epilepsy. However, the analysis of circulating miRNAs in rodent models is challenging, which relates both to the lack of standardized sampling protocols and to analysis of miRNAs. We will discuss the issues critical for preclinical plasma biomarker discovery, such as documentation, blood and brain tissue sampling and collection, plasma separation and storage, RNA extraction, quality control, and RNA detection. We propose a protocol for standardization of procedures for discovery of circulating miRNA biomarkers in rat models of epileptogenesis. Ultimately, we hope that the preclinical standardization will facilitate clinical biomarker discovery for epileptogenesis in man.

Ttyh1 protein is expressed in glia in vitro and shows elevated expression in activated astrocytes following status epilepticus.

In a previous study, we showed that Ttyh1 protein is expressed in neurons in vitro and in vivo in the form of punctuate structures, which are localized to neuropil and neuronal somata. Herein, we provide the first description of Ttyh1 protein expression in astrocytes, oligodendrocytes and microglia in vitro. Moreover, using double immunofluorescence, we show Ttyh1 protein expression in activated astrocytes in the hippocampus following amygdala stimulation-induced status epilepticus. We demonstrate that in migrating astrocytes in in vitro wound model Ttyh1 concentrates at the edges of extending processes. These data suggest that Ttyh1 not only participates in shaping neuronal morphology, as previously described, but may also play a role in the function of activated glia in brain pathology. To localize Ttyh1 expression in the cellular compartments of neurons and astrocytes, we performed in vitro double immunofluorescent staining using markers for the following subcellular structures: endoplasmic reticulum (GRP78), Golgi apparatus (GM130), clathrin-coated vehicles (clathrin), early endosomes (Rab5 and APPL2), recycling endosomes (Rab11), trans-Golgi network (TGN46), endoplasmic reticulum membrane (calnexin), late endosomes and lysosomes (LAMP1) and synaptic vesicles (synaptoporin and synaptotagmin 1). We found that Ttyh1 is present in the endoplasmic reticulum, Golgi apparatus and clathrin-coated vesicles (clathrin) in both neurons and astrocytes and also in late endosomes or lysosomes in astrocytes. The presence of Ttyh1 was negligible in early endosomes, recycling endosomes, trans-Golgi network, endoplasmic reticulum membrane and synaptic vesicles.

Are alterations in transmitter receptor and ion channel expression responsible for epilepsies?

Neuronal voltage-gated ion channels and ligand-gated synaptic receptors play a critical role in maintaining the delicate balance between neuronal excitation and inhibition within neuronal networks in the brain. Changes in expression of voltage-gated ion channels, in particular sodium, hyperpolarization-activated cyclic nucleotide-gated (HCN) and calcium channels, and ligand-gated synaptic receptors, in particular GABA and glutamate receptors, have been reported in many types of both genetic and acquired epilepsies, in animal models and in humans. In this chapter we review these and discuss the potential pathogenic role they may play in the epilepsies.

Circulating microRNAs from plasma as preclinical biomarkers of epileptogenesis and epilepsy.

Epilepsy frequently develops as a result of brain insult; however, there are no tools allowing to predict which patients suffering from trauma will eventually develop epilepsy. microRNAs are interesting candidates for biomarkers, as several of them have been described to change their levels in the brains, and in the plasma of epileptic subjects. This study was conducted to evaluate the usefulness of plasma miRNAs as epileptogenesis/epilepsy biomarkers. In our studies, we used a rat model of temporal lobe epilepsy. An epileptogenic insult was status epilepticus evoked by stimulation of the left lateral nucleus of the amygdala. Next, animals were continuously video and EEG monitored for 3 months. Blood was collected at 14, 30, 60, and 90 days after stimulation. Blood plasma was separated and miRNA levels were analyzed. We compared miRNA levels between sham-operated and stimulated animals, and between animals with high and low numbers of seizures. We propose three miRNAs that could be biomarkers of epilepsy: miR-671, miR-9a-3p and miR-7a-5p. According to us, miR-206-5p is a potential biomarker of epileptogenesis, and miR-221-3p is a potential biomarker of epilepsy severity. We think that these five miRNAs can be considered in the future as potential treatment targets.

Status epilepticus induces long lasting increase in S100A6 expression in astrocytes.

In the present work we examined expression and localization of the S100A6 protein in rat brain in a model of epilepsy induced by Status Epilepticus evoked by amygdala stimulation. We demonstrate, through the use of the reverse transcriptase-polymerase chain reaction technique, that mRNA level of S100A6 was increased in cortex while, as found by immunoblotting, the level of the S100A6 protein was significantly higher in the cortex and in the CA1 area of the hippocampus at day 14 after stimulation. Immunohistochemical studies performed on rat brain slices indicated that S100A6 immunoreactivity was elevated in GFAP-positive astrocytes in the hippocampus and cortex starting from day 1, and further increased at day 4 and 14 after stimulation. Interestingly, in a subpopulation of astrocytes, up-regulation of S100A6 was associated with an increased level of ß-catenin, a protein involved in regulation of S100A6 expression. Altogether, our data show a widespread and prolonged up-regulation of S100A6 in the epileptic brain and indicate that an increase in S100A6 immunoreactivity is related to astrogliosis.

New insight on the mechanisms of epileptogenesis in the developing brain.

The incidence of epilepsy is at its highest in childhood and seizures can persist for a lifetime. As brain tissue from pediatric patients with epilepsy is rarely available, the analysis of molecular and cellular changes during epileptogenesis, which could serve as targets for treatment approaches, has to rely largely on the analysis of tissue from animal models. However, these data have to be analyzed in the context of the developmental stage when the insult occurs. Here we review the current status of the available animal models, the molecular analysis done in these models, as well as treatment attempts to prevent epileptogenesis in the immature brain. Considering that epilepsy is one of the major childhood neurological diseases, it is remarkable how little is known on epileptogenesis in the immature brain at a molecular level. It is a true challenge for the future to expand the armamentarium of clinically relevant animal models, and systematic analysis of molecular and cellular data to enhance the probability of developing syndrome specific antiepileptogenic treatments and biomarkers for acquired pediatric epileptogenesis.

Behavioral characteristics as potential biomarkers of the development and phenotype of epilepsy in a rat model of temporal lobe epilepsy.

The present study performed a detailed analysis of behavior in a rat model of epilepsy using both established and novel methodologies to identify behavioral impairments that may differentiate between animals with a short versus long latency to spontaneous seizures and animals with a low versus high number of seizures. Temporal lobe epilepsy was induced by electrical stimulation of the amygdala. Rats were stimulated for 25 min with 100-ms trains of 1-ms biphasic square-wave pluses that were delivered every 0.5 s. Electroencephalographic recordings were performed to classify rats into groups with a short latency (< 20 days, n = 7) and long latency (> 20 days, n = 8) to the first spontaneous seizure and into groups with a low number of seizures (62 ± 64.5, n = 8) and high number of seizures (456 ± 185, n = 7). To examine behavioral impairments, we applied the following behavioral tests during early and late stages of epilepsy: behavioral hyperexcitability, open field, novel object exploration, elevated plus maze, and Morris water maze. No differences in stress levels (e.g., touch response in the behavioral hyperexcitability test), activity (e.g., number of entries into the open arms of the elevated plus maze), or learning (e.g., latency to find the platform in the Morris water maze test during training days) were observed between animals with a short versus long latency to develop spontaneous seizures or between animals with a low versus high number of seizures. However, we found a higher motor activity measured by higher number of entries into the closed arms of the elevated plus maze at week 26 post-stimulation in animals with a high number of seizures compared with animals with a low number of seizures. The analysis of the Morris water maze data categorized the strategies that the animals used to locate the platform showing that the intensity of epilepsy and duration of epileptogenesis influenced swimming strategies. These findings indicate that behavioral impairments were relatively mild in the present model, but some learning strategies may be useful biomarkers in preclinical studies.

Expression of Ttyh1, a member of the Tweety family in neurons in vitro and in vivo and its potential role in brain pathology.

We have previously shown that Ttyh1 mRNA is expressed in neurons and its expression is up-regulated in the brain during epileptogenesis and epilepsy. In this study, we aimed to elucidate the role of Ttyh1 in neurons. We found widespread expression of Ttyh1 protein in neurons in vivo and in vitro. Ttyh1 immunoreactivity in vitro was frequently found in invaginations of dendritic spines; however, Ttyh1, seldom co-localized with synaptic markers in vivo. Silencing Ttyh1 expression with siRNA in hippocampal cultures resulted in alterations of MAP2 distribution along neurites causing it to appear in the form of chains of beads. Over-expression of Ttyh1 caused intense neuritogenesis and the formation of numerous filopodia-like protrusions. Similar protrusions were also produced in SH-SY5Y neuroblastoma cells over-expressing Ttyh1. Using a biotin-streptavidin pull-down assay and mass spectrometry, we identified proteins that can form complexes with Ttyh1 in the brain. Ttyh1 binding proteins are often expressed in the endoplasmic reticulum or the Golgi apparatus or are localized at synapses. Finally, we found increased expression of Ttyh1 in the inner molecular layer of the dentate gyrus in an animal model of epilepsy. On the basis of our findings, we propose Ttyh1 involvement in brain pathology.

Molecular and cellular basis of epileptogenesis in symptomatic epilepsy.

Epileptogenesis refers to a process in which an initial brain-damaging insult triggers a cascade of molecular and cellular changes that eventually lead to the occurrence of spontaneous seizures. Cellular alterations include neurodegeneration, neurogenesis, axonal sprouting, axonal injury, dendritic remodeling, gliosis, invasion of inflammatory cells, angiogenesis, alterations in extracellular matrix, and acquired channelopathies. Large-scale molecular profiling of epileptogenic tissue has provided information about the molecular pathways that can initiate and maintain cellular alterations. Currently we are learning how these pathways contribute to postinjury epileptogenesis and recovery process and whether they could be used as treatment targets.

Lack of migration and neurological benefits after infusion of umbilical cord blood cells in ischemic brain injury.

The finding of stem/progenitor cells in postnatal bone marrow and umbilical cord blood, opens up a possibility of using stem cells to treat neurologic diseases. There is a controversy, whether intravenously administered human umbilical cord blood cells (HUCBC) migrate to the brain, differentiate and improve recovery after ischemia. In this study, 1-3 x10;6 cells from non-cultured (non-committed) mononuclear HUCBC fraction were intravenously infused 1, 2, 3 or 7 days after a transient middle cerebral artery occlusion (MCAo) in adult rats. We found few human cells only in the ischemic area, localized mostly around blood vessels with few positive cells in the brain parenchyma. Timing of HUCBC delivery after ischemia or injection of Cyclosporin A at the time of delivery, had no effect on the number of human cells detected in the ischemic brain. Infusion of HUCBC did not reduce infarct volume and did not improve neurologic deficits after MCAo, suggesting that HUCBC failed to migrate/survive in the ischemic brain and did not provide significant neurological benefits.

SorCS2 Controls Functional Expression of Amino Acid Transporter EAAT3 and Protects Neurons from Oxidative Stress and Epilepsy-Induced Pathology.

VPS10P domain receptors emerge as central regulators of intracellular protein sorting in neurons with relevance for various brain pathologies. Here, we identified a role for the family member SorCS2 in protection of neurons from oxidative stress and epilepsy-induced cell death. We show that SorCS2 acts as sorting receptor that sustains cell surface expression of the neuronal amino acid transporter EAAT3 to facilitate import of cysteine, required for synthesis of the reactive oxygen species scavenger glutathione. Lack of SorCS2 causes depletion of EAAT3 from the plasma membrane and impairs neuronal cysteine uptake. As a consequence, SorCS2-deficient mice exhibit oxidative brain damage that coincides with enhanced neuronal cell death and increased mortality during epilepsy. Our findings highlight a protective role for SorCS2 in neuronal stress response and provide a possible explanation for upregulation of this receptor seen in surviving neurons of the human epileptic brain.

A generalised framework for detailed classification of swimming paths inside the Morris Water Maze.

The Morris Water Maze is commonly used in behavioural neuroscience for the study of spatial learning with rodents. Over the years, various methods of analysing rodent data collected during this task have been proposed. These methods span from classical performance measurements to more sophisticated categorisation techniques which classify the animal swimming path into behavioural classes known as exploration strategies. Classification techniques provide additional insight into the different types of animal behaviours but still only a limited number of studies utilise them. This is primarily because they depend highly on machine learning knowledge. We have previously demonstrated that the animals implement various strategies and that classifying entire trajectories can lead to the loss of important information. In this work, we have developed a generalised and robust classification methodology to boost classification performance and nullify the need for manual tuning. We have also made available an open-source software based on this methodology.

A companion to the preclinical common data elements for physiologic data in rodent epilepsy models. A report of the TASK3 Physiology Working Group of the ILAE/AES Joint Translational Task Force.

The International League Against Epilepsy/American Epilepsy Society (ILAE/AES) Joint Translational Task Force created the TASK3 working groups to create common data elements (CDEs) for various aspects of preclinical epilepsy research studies, which could help improve standardization of experimental designs. This article concerns the parameters that can be measured to assess the physiologic condition of the animals that are used to study rodent models of epilepsy. Here we discuss CDEs for physiologic parameters measured in adult rats and mice such as general health status, temperature, cardiac and respiratory function, and blood constituents. We provide detailed CDE tables and case report forms (CRFs), and with this companion manuscript we discuss the monitoring of different aspects of physiology of the animals. The CDEs, CRFs, and companion paper are available to all researchers, and their use will benefit the harmonization and comparability of translational preclinical epilepsy research. The ultimate hope is to facilitate the development of biomarkers and new treatments for epilepsy.

Chronically dysregulated NOTCH1 interactome in the dentate gyrus after traumatic brain injury.

Traumatic brain injury (TBI) can result in several dentate gyrus-regulated disabilities. Almost nothing is known about the chronic molecular changes after TBI, and their potential as treatment targets. We hypothesized that chronic transcriptional alterations after TBI are under microRNA (miRNA) control. Expression of miRNAs and their targets in the dentate gyrus was analyzed using microarrays at 3 months after experimental TBI. Of 305 miRNAs present on the miRNA-array, 12 were downregulated (p<0.05). In parallel, 75 of their target genes were upregulated (p<0.05). A bioinformatics analysis of miRNA targets highlighted the dysregulation of the transcription factor NOTCH1 and 39 of its target genes (NOTCH1 interactome). Validation assays confirmed downregulation of miR-139-5p, upregulation of Notch1 and its activated protein, and positive enrichment of NOTCH1 target gene expression. These findings demonstrate that miRNA-based transcriptional regulation can be present at chronic time points after TBI, and highlight the NOTCH1 interactome as one of the mechanisms behind the dentate gyrus pathology-related morbidities.

Meta-Analysis of MicroRNAs Dysregulated in the Hippocampal Dentate Gyrus of Animal Models of Epilepsy.

The identification of mechanisms transforming normal to seizure-generating tissue after brain injury is key to developing new antiepileptogenic treatments. MicroRNAs (miRNAs) may act as regulators and potential treatment targets for epileptogenesis. Here, we undertook a meta-analysis of changes in miRNA expression in the hippocampal dentate gyrus (DG) following an epileptogenic insult in three epilepsy models. We identified 26 miRNAs significantly differentially expressed during epileptogenesis, and five differentially expressed in chronic epilepsy. Of these, 13 were not identified in any of the individual studies. To assess the role of these miRNAs, we predicted their mRNA targets and then filtered the list to include only target genes expressed in DG and negatively correlated with miRNA expression. Functional enrichment analysis of mRNA targets of miRNAs dysregulated during epileptogenesis suggested a role for molecular processes related to inflammation and synaptic function. Our results identify new miRNAs associated with epileptogenesis from existing data, highlighting the utility of meta-analysis in maximizing value from preclinical data.