Repurposing the Dihydropyridine Calcium Channel Inhibitor Nicardipine as a Na_v1.8 inhibitor in vivo for Pitt Hopkins Syndrome

Abstract

Purpose:

Individuals with the rare genetic disorder Pitt Hopkins Syndrome (PTHS) do not have sufficient expression of the transcription factor 4 (TCF4) which is located on chromosome 18. TCF4 is a basic helix-loop-helix E protein that is critical for the normal development of the nervous system and the brain in humans. PTHS patients lacking sufficient TCF4 frequently display gastrointestinal issues, intellectual disability and breathing problems. PTHS patients also commonly do not speak and display distinctive facial features and seizures. Recent research has proposed that decreased TCF4 expression can lead to the increased translation of the sodium channel Na_v1.8. This in turn results in increased after-hyperpolarization as well as altered firing properties. We have recently identified an FDA approved dihydropyridine calcium antagonist nicardipine used to treat angina, which inhibited Na_v1.8 through a drug repurposing screen.

Methods:

We have now performed behavioral testing in groups of 10 male Tcf4(+/−) PTHS mice dosing by oral gavage at 3 mg/kg once a day for 3 weeks using standard methods to assess sociability, nesting, fear conditioning, self-grooming, open field and test of force.

Results:

Nicardipine returned this spectrum of behavioral deficits in the Tcf4(+/−) PTHS mouse model to WT levels and resulted in statistically significant results.

Conclusions:

These in vivo results in the well characterized Tcf4(+/−) PTHS mice may suggest the potential to test this already approved drug further in a clinical study with PTHS patients or suggest the potential for use off label under compassionate use with their physician.

Introduction

It is suggested that there are well over 7000 rare diseases, while only a fraction have current treatments approved there is increasing interest in developing treatments for many more and there are several government provided incentives for doing this (1). One approach to potentially bring treatments to rare disease patients faster is by repurposing or repositioning existing FDA approved drugs for new applications and there are several ways to do this experimentally or computationally (2–6). Individuals with the rare genetic disorder Pitt Hopkins Syndrome (PTHS) do not have sufficient expression of the transcription factor 4 (TCF4) which is located on chromosome 18. While PTHS was initially described by D. Pitt and I. Hopkins (7), the linkage to TCF4 was only recently established in 2007. Further the protein product of TCF4 is known to be a basic helix-loop-helix E protein that has a fundamental role in the development of the human nervous system and brain (8–10) such that over 140 unique aberrations have been reported in the literature associated with TCF4 (11). For example, the alteration of the TCF4 regulatory networks is known to result in abnormal cortical development in the brain, while pre-natal and post-natal development are also known to be sensitive to the altered expression of TCF4 (12). Additionally TCF4 has been shown to regulate dendritic spine density as well as morphology hence loss of this would likely contribute to the neurological symptoms observed with PTHS (13). The clinical phenotype for PTHS is characterized by frequent display of gastrointestinal mobility issues, intellectual disability, developmental delay and breathing problems. PTHS patients also commonly do not speak and display distinctive facial features and seizures (11, 14). Other clinical features include recurrent seizures/epilepsy, lack of speech, delay in walking and behavioral problems (11, 14). The developmental spectrum of PTHS can also encompass anxiety, sensory disorders and attention deficit hyperactivity disorder (ADHD). The recent high throughput sequencing of the human genome coding regions has resulted in an improved diagnosis of PTHS (15). In summary, the decreased expression of TCF4 has a dramatic effect on child development and is frequently described as a cause of intellectual disability (15). There is a wide spectrum of physical abilities for PTHS patients and some patients can be identified that that have physical abilities beyond those classically noted in the literature (16). Close to 1000 PTHS patients have now been identified worldwide (Pitt Hopkins Research Foundation, personal communication) while many are likely undiagnosed or misdiagnosed with other syndromes like Angelman or Rett. A recent consensus statement provided a clinically accepted definition of PTHS, as well as a clear molecular diagnostic pathway and suggestions for managing related health problems (11). This study also provided information to enable a differential diagnosis with Angelman and Rett syndromes. A recent large patient case series also indicated that whole exome sequencing or commercial panels were needed to provide a clear molecular diagnosis for PTHS (14). There are also many comorbidities that when managed can increase the quality of life for both PTHS patients and families (14).

While there is no FDA approved treatment for PTHS, there have been preclinical studies with histone deacetylase inhibitors (17) and NMDA receptor antagonists (18) which have also been suggested for further study. Maher and colleagues have shown that in a Pitt Hopkins mouse model (19): 1. Loss of TCF4 function leads to altered intrinsic excitability of the prefrontal neurons; 2. The loss of TCF4-dependent excitability Is reversed by both sodium channel (SCN10a) and potassium channel (KCNQ1) antagonists; and 3. that in the central neurons of wild type animals TCF4 represses the expression of both SCN10a and KCNQ1 genes. This work clearly suggested that these ion channels represented targets in the central nervous system (CNS) that could be modulated with drugs. This may then in turn ameliorate the previously described cognitive deficits that are frequently observed in PTHS. The potential for these ion channels to represent validated targets for PTHS (20) builds on decades of knowledge accumulated for the many well-known voltage-gated potassium and sodium channels (21) and more recent work using subtype-selective modulators of these ion channels.

One of these ion channels of perhaps highest clinical relevance is Na_v1.8 which is a sodium ion channel subunit (voltage-gated channel subtype) that is encoded by the SCN10A gene in humans (22–24). Na_v1.8 is known to be expressed in the peripheral sensory neurons. Additionally, in the dorsal root ganglion (DRG), Na_v1.8 is expressed in small-diameter sensory neurons that are unmyelinated. These are also called C-fibers and are involved in nociception (25, 26). Na_v1.8 is therefore an important therapeutic target and is frequently targeted with new analgesics (27) and other potential treatments for chronic pain (28). Our previous in vitro study performed a drug repurposing high-throughput screen against Na_v1.8 and identified a number of FDA-approved dihydropyridine calcium channel antagonists (2). One of these, nicardipine was shown to inhibit Na_v1.8 with a sub micromolar IC₅₀ (2) and we now demonstrate that it may rescue the TCF4-dependent excitability deficit as demonstrated using behavioral testing in Tcf4(+/−) PTHS mice using standard methods as described in earlier studies (29).

Materials and Methods

Compounds

Nicardipine was purchased from Sigma Aldrich (St. Louis, MO).

Ethical approval

All experimental procedures were performed in strict compliance with the 1986 UK Home Office regulation for the Care and Use of Laboratory Animals and approved by the IEB Ethical Committee.

Animals

In this study male B6;129-Tcf4tm1Zhu/J mice were bred with female B6129SF1/J mice to produce Tcf4(+/−) and WT littermates (provided by Dr. Andrew J. Kennedy, University of Alabama at Birmingham). All experiments described as follows were subsequently performed on male mice at 2 months of age. 10 animals were used in each group.

Genotyping

We used TransnetXY Automated Genotyping (TransnetYX, Inc. Cordova, TN) to genotype the mice.

Animal housing

The mice were housed in 35 × 30 × 12 cm plastic cages, 5 mice were housed in each and they were habituated to the animal facilities for minimum of a week before testing. The room temperature was kept at a constant 21 ± 2°C with 55 ± 5% relative humidity and a 12-h light–dark cycle (where lights were on from 7 a.m.–7 p.m.). In addition the air exchange in the facility was automatically controlled. The mice had free access to commercial food pellets and water throughout. Testing was consistently performed during the light phase of the day.

Animal’s humane end points

Humane endpoints commonly include one or more of the following observations. 1. Impaired ambulation of the animals which prevents them from reaching their food or water. 2. An excessive weight loss or extreme emaciation of the animals 3. A loss of physical or mental alertness of the animals. 4. Animals demonstrate difficult or labored breathing. 5. Animals demonstrate a prolonged inability to remain upright. In addition these endpoints can be further described where Weight loss is the gradual loss of weight over an extended period of time that then leads to rapid weight loss of 15–20 per cent within a few days or emaciation. Breathing: The animal clearly demonstrates an increased respiratory rate and/or effort when breathing. Such labored respiration is very often then accompanied by a strong noticeable abdominal component to the breathing. Dehydration: During dehydration the skin loses its elasticity such that when the skin is pinched over the back it should then return to its normal position after release. However, in a dehydrated animal, this does not happen as the skin will remain tented. Body Condition: can be identified by scoring, poor coat condition, inability of ambulation, unresponsive to manual stimulation, isolation from cage mates, not interested in burrowing. Following the exposure to the test compound the animals were observed for signs of toxicity, at a minimum twice-daily observations took place, more frequent observations were undertaken immediately after dosing the mice.

Randomization and Blinding

All experiments were conducted with the experimenter blind to the genotype as well as the drug treatment. We ensured that separate investigators 1. prepared and coded dosing solutions, 2. allocated the mice to the different study treatment groups, 3. dosed the animals, and 4. collected the behavioral data. 5. analyzed the data.

Dosing

Groups of 10 male Tcf4(+/−) PTHS mice were dosed by oral gavage at 3 mg/kg once a day for 3 weeks.

Behavioural Testing

The behavioural testing methods described below have been recently described in detail (29, 30) as well as by others. For all experiments undertaken, the mice were tested once in the same piece of apparatus. Prior to this testing, mice that were not used in the study were placed in the same apparatus for several minutes before the experiment. The apparatus was then thoroughly cleaned with moist and then with dry tissues prior to testing each mouse in the same equipment. The aim of this effort was to add a low but constant background mouse odor that was available for all experimental subjects in the study.

Open Field

The open field apparatus was used to test the mice for anxiety/hyperactivity behaviour and habituation to a novel environment. This is one of the most fundamental forms of learning, in which the decreased exploration as a function of repeated exposure to the same environment is taken as direct measure of the animal’s memory. This behaviour was studied in two sessions of exposure to the open field, occurring at 60 min and 24 h post treatment. The open field assay was performed during the light cycle between 8 a.m. and 4 p.m. using an automated system which included a VersaMax activity monitor chamber with VersaDat software (AccuScan Instruments, Columbus, OH). The study mice were brought to the experimental room 20 min before testing in the apparatus. The study involved placing a mouse into a comer square facing the corner and then observed for 60 min. The distance which the mouse travelled (as measured by the number of squares entered with whole body or locomotor activity) and rears (both the front paws off the ground, but not as part of grooming) was then recorded.

Sociability

The measurement of sociability has been described in detail by (30). Briefly, in the three-chambered sociability task, a subject mouse was evaluated for its exploration of a novel social stimulus (namely a novel mouse). The approach using the three-chambered social approach task is that it monitors the direct social approach behaviours when a subject mouse is presented with an option of spending time with either a novel or a familiar mouse. The preference for social novelty is therefore observed when a subject mouse spends more time with the novel mouse. The apparatus for this test is a rectangular three-chamber box (where each chamber is 20 cm (length) × 40.5 cm (width) × 22 cm (height)). Dividing walls in the chamber are made from clear perplex, with openings (10 cm width × 5 cm height) which permit mouse entry into each chamber. The apparatus is lit from below (10 lux). The study mice were brought to the experimental room 20 min before testing for sociability.

Fear conditioning

Fear conditioning to a context is a type of associative learning used in many species. The dependent measure used in contextual (delay) fear conditioning is a freezing response. This occurs following pairing of an unconditioned stimulus (US), for example a foot shock with a conditioned stimulus (CS), which is a particular context. Contextual fear conditioning is the most basic of the conditioning procedures and involves taking an animal, placing it in a novel environment, then providing an aversive stimulus, before removing it. When the animal is next returned to the identical environment, it will demonstrate a ‘freezing response’ if it can remember and associate that same environment with the prior aversive stimulus. Such “freezing” is a species-specific response to fear. We therefore programmed a 120-sec habituation period before the first of two identical trials began. This allows the mouse to explore briefly and to take in the aspects of the chamber used in the study. An auditory tone (80 dB) for 15–30 sec as a cue is then presented. A mild foot shock (0.17–0.8 mA) for 1–2 sec is also administered during the last 2 sec of the tone presentation and co-terminated with the tone. After the shock presentation to the mouse, an intertrial interval (60–210 sec) precedes a second identical trial. Following the final shock presentation to the mouse, the house light remained on for an additional 60 sec, to enable removing the mouse in a 30–60 sec time period after the last trial. Three shocks were separated by 30 seconds. Memory was tested 24 h after training for 5 minutes. Nicardipine was given before the training to improve memory acquisition.

Nesting

The loss of the ability to perform ones “Activities of daily living” (ADL) is sign of neurological diseases such as Alzheimer’s disease. Therefore, preclinical behavioural screening of possible treatments for AD currently focuses on cognitive testing, however, the human episodic memory (the most severely affected aspect of memory in AD) is different to rodent memory, which is largely non-episodic. Preclinical screening therefore needs to characterise the ADL of mice. Nesting is a sensitive and widely characterised test of this behaviour. Both male and female mice make nests, for thermoregulation as well as for reproduction. It has been demonstrated that for C57BL/6 mice, nest scores for males and females are in the same range, (score of 4–5 on nest construction), but when the animal’s hippocampus is lesioned the median score decreases (score of 1–2) while a score of 3 is unlikely to be exceeded in this case (31). This nesting test used the same individual cages as described earlier. Normal bedding covered the floor of the cage to a depth of 0.5 cm. Each cage was also supplied with a ‘Nestlet’, which is a 5 cm square of pressed cotton batting (Ancare). Mice were then placed individually into the nesting cages one hour before the dark phase, and the nesting results were visually assessed the following morning. The nests were assessed on a standardized 5-point scale described below and the amount of untorn nestlet was weighed. The nest building was therefore scored on a 5 point scale as follows: Score 1: The Nestlet was largely untouched by the animal (>90% intact); Score 2: The Nestlet was partially torn up (50–90% remaining intact); Score 3: The Nestlet was mostly shredded but often there was no identifiable nest site: <50% of the Nestlet remained intact but <90% was within a quarter of the cage floor area, i.e. the cotton was not gathered into a nest but spread around the cage; Score 4: An identifiable, but flat nest: >90% of the Nestlet was torn up, the material was gathered into a nest within a quarter of the cage floor area, but the nest was flat, with walls higher than mouse body height (curled up on its side) on less than 50% of its circumference; Score 5: A (near) perfect nest: >90% of the Nestlet was torn up, the nest was a crater, with walls higher than mouse body height on more than 50% of its circumference.

Self-grooming: stereotypy behaviour

During the open field each mouse was scored for cumulative time spent grooming all body regions. Autism Spectrum Disorder phenotypes include irritability, self-grooming, self-injury, aggression, and tantrums. Self-grooming is known to be a complex innate behaviour with a well-studied evolutionary conserved sequencing pattern. It is also one of the most commonly tested behavioural activities in rodents. In addition, self-grooming may be a useful approach to test repetitive behaviour in the PTHS mouse model, and it hence has some use to current translational pharmacology. The assessment of self-grooming in mice is also a useful tool for understanding the neural circuits that are involved in complex sequential patterns of action such as those tested in this model.

Test of force or grip strength

This measure tests the neuromuscular function of the hindlimb grip strength. The hindlimbs of mice were tested with a standard grip strength meter. This involved taking mice that were scuffed by the back of the neck, then held by the tail and lifted into vertical upright position. The mice were then allowed to grasp the metal grid with the hindlimbs and then the mice were pulled backward in the horizontal plane. The force applied to the metal grid at the moment the grasp was subsequently recorded in Newtons.

Statistical Analysis of Behavioral Data

All behavioral data were analyzed by two-way analysis of variance (ANOVA) which was then followed by post-test comparisons where appropriate using Tukey’s Multiple Comparison Test. The data are also represented in figures as the mean and standard deviation (SD). All of these statistical analyses were performed in Prism 8.2.1. (GraphPad, SanDiego, CA).

Results

The transcription factor TCF4 is master regulator of schizophrenia (32) and is associated with PTHS (10), which results in severe language impairment and development delay. Tcf4(+/−) mice have therefore been very well characterized (17, 18), demonstrating deficits in habituation. These mice are also hyperactive in the open field as well as demonstrating learning and memory deficits as observed in the Morris water maze (18). While there are certainly subtle differences identified between mouse models, these mice possess a large array of behavioural deficits that allow them to be used for preclinical studies to obtain a reliable readout for efficacy. Therefore, in this study a behavioural battery was undertaken with Tcf4(+/−) mice to determine whether orally dosed nicardipine (3 mg/kg dosed once a day for 3 weeks) was sufficient to rescue hindlimb force control, learning and memory deficits, hyperactivity, sociability, nesting and stereotypy associated with autism spectrum disorder. Treatment with nicardipine did not result in any visible side effects. Nicardipine treatment had an effect in the sociability test. In comparison with WT mice, PTHS Tcf4(+/−) mice had levels of sociability with familiar and novel mice comparable to the WT mice when treated with nicardipine (Figure 1). This is not because Tcf4(+/−) animals recognize both types of mice as familiar, but rather they seem to treat both as novel individuals. As Tcf4(+/−) mice do explore target mice, they do not appear to have sociability deficits, but rather a social recognition impairment, which prevents them from identifying the familiar mice as familiar. Tcf4(+/−) mice treated with a dose of 3 mg/kg per day for 3 weeks were virtually undistinguishable from WT mice (Figure 1). Tcf4(+/−) showed poorer nesting behaviour compared with WT mice (Figure 2). Nicardipine at a dose of 3 mg/kg per day for 3 weeks improved this behaviour in Tcf4(+/−) mice (Figure 2). Tcf4(+/−) mice also showed a significant deficit in contextual fear conditioning memory to an aversive stimulus compared with WT mice (Figure 3). In Tcf4(+/−) mice, treatment with Nicardipine before acquisition improved long-term memory retention 24 h after training. There was memory improvement to WT levels in Tcf4(+/−) mice treated with nicardipine (3 mg/kg per day for 3 weeks) (Figure 3). Tcf4(+/−) mice also showed increased self-grooming compared with WT (Figure 4). Nicardipine decreased self-grooming in Tcf4(+/−) mice when treated with nicardipine (Figure 4). Nicardipine also decreased the distance travelled in Tcf4(+/−) mice in the open field test (Figure 5) and improved the grip strength to wild type levels in Tcf4(+/−) mice (Figure 6).

Figure 1.

Nicardipine (3 mg/Kg per day (3 weeks) by oral gavage) improved social recognition of the familiar mice, which was impaired in PTHS Tcf4(+/−) mouse model. Data represent mean ± SD. ****p<0.0001.

Figure 2.

Nicardipine (3 mg/Kg per day (3 weeks) by oral gavage) improved nesting in the PTHS Tcf4(+/−) mouse model. Data represent mean ± SD. ****p<0.0001.

Figure 3.

Nicardipine (3 mg/Kg per day (3 weeks) by oral gavage) improved fear conditioning in the PTHS Tcf4(+/−) mouse model. Data represent mean ± SD. ****p<0.0001.

Figure 4.

Nicardipine (3 mg/Kg per day (3 weeks) by oral gavage) improved self-grooming in the PTHS Tcf4(+/−) mouse model. Data represent mean ± SD. ****p<0.0001.

Figure 5.

Nicardipine (3 mg/Kg per day (3 weeks) by oral gavage) improved open field in the PTHS Tcf4(+/−) mouse model. Data represent mean ± SD. ****p<0.0001.

Figure 6.

Nicardipine (3 mg/Kg per day (3 weeks) by oral gavage) improved test of force in the PTHS Tcf4(+/−) mouse model. Data represent mean ± SD. ****p<0.0001.

Discussion

The dihydropyridine calcium channel antagonist nicardipine was identified previously for the first time as a relatively potent inhibitor of Na_v1.8 (IC₅₀ 0.6μM) (2). Na_v1.8 is a well characterized sodium ion channel subunit that is encoded by the SCN10A gene (22–24) and it has been demonstrated that TCF4-dependent excitability deficits are rescued in the PTHS mouse by sodium channel antagonists (19). We therefore evaluated nicardipine dosed orally to PTHS mouse in different behavioural tests. We found the PTHS mouse had levels of sociability comparable to the WT mice when treated with nicardipine (Figure 1). There are two possible explanations for the effect of the treatment. Tcf4(+/−) mice might have explored both the familiar and novel mice at equally high rates because of their hyperactivity. By decreasing locomotor activity, Nicardipine might have unveiled the social novelty preference. A second possibility is that Nicardipine enhanced retrieval of the recognition memory for the familiar mouse. The second explanation seems more likely as the total time spent with novel and familiar mice is similar for WT and Tcf4(+/−). Nesting behaviour was also returned to WT levels when treated with nicardipine (Figure 2). Although nesting behaviour is simple and innate, there can be many causes for the observed deficits. Nesting behaviour may also represent a measure of animal health and general wellbeing. Deficits in nesting have been observed in models of psychiatric disease (33), suggesting that anxiety-like processes could be affecting these mice and could explain the phenotype. Memory was also improved in the PHTS mouse to WT levels with nicardipine (Figure 3). Nicardipine could have improved memory retention by enhancing memory acquisition, memory consolidation or both processes. Importantly, freezing behaviour is sensitive to the anxiety state of the animals. However, if nicardipine affected freezing behaviour by decreasing anxiety, it would have decreased, and not increased performance in this test. This indicates that the effect of the treatment can be explained by its improvement of memory and not decreasing anxiety. Self-grooming also improved in the PHTS mice (Figure 4). One possible interpretation is that Tcf4(+/−) animals are more prone to repetitive action patterns. Abnormal grooming has been seen in animal models of Autism Spectrum Disorder and thus it is of interest in translational neuropharmacology.

Together, these results (Figures 1–6) point to a possible dysfunction of the hippocampus and perhaps the amygdala in PTHS using this mouse model. Social recognition memory and contextual fear conditioning memory are both dependent on the integrity of the hippocampus. In addition, malfunction of this region is also associated with increased anxiety-like behaviour. The hippocampus has also been implicated in the nesting task, in which Tcf4(+/−) mice perform poorly. Thus, the impairments observed in Tcf4(+/−) mice are likely consistent with hippocampal dysfunction. It is possible that one target region for nicardipine is indeed the hippocampus. The amygdala has also been implicated in both fear memory and anxiety-like behaviour. Hyperactivity of this structure could be implicated in increased anxiety, while deficits in fear learning are usually linked to amygdala hypofunction. The abnormal self-grooming in Tcf4(+/−) mice suggest that there might also be a dysregulation of the basal ganglia, as the striatum and substantia nigra are both part of the neural network involved in this behaviour (34, 35). Since Tcf4(+/−) mice are impaired in many behavioural tasks, there are many neurotransmitter systems that could be targeted by nicardipine. Based on our in vitro data this would appear to be via Na_v1.8. although the compound is well known as a calcium channel antagonist and it is possible this molecule is inhibiting other ion channels or additional targets. In addition, several other structurally related dihydropyridine calcium channel inhibitors also inhibit Na_v1.8 to differing extents (2). There would appear to be a structure activity relationship for this channel in the same way as there is for the calcium channel. Maher et al., (19) have previously reported TCF4-dependent excitability deficits can be are rescued by either SCN10a and KCNQ1 antagonists. In addition, they reported that intact Tcf4 can repress the expression of SCN10a and KCNQ1 genes. This group did not show a role for the calcium channel. It is likely therefore that dihydropyridine compounds like nicardipine are promiscuous and show overlap for different ion channels. There has been some discussion of compounds inhibiting both sodium and calcium channels (36) likely based on the evolution of these ion channels. Also it should be noted that there is a long history of similar dihydropyridines inhibiting drug transporters such a P-gp (37) as well as TGFβ/Smad signaling (37), suggesting that such compounds can have activities at very different proteins with diverse functions. In the current study we provide extensive data to show for the first time that nicardipine improved a spectrum of behavioral deficits in the Tcf4(+/−) PTHS mouse model comparable to WT levels (Figure 1–6).

Certainly, while the current study only uses one of the several PTHS mouse models available, it is unknown whether the observations for nicardipine may transfer to other mouse models. Further studies in different mouse models may therefore be warranted. In addition, we only considered a single dose and dosing interval. While others have dosed nicardipine in mice at 5 and 10mg/kg in drug interaction studies (38), it may be important to consider several lower doses which have been used in other animal (rabbits and dogs) models of ischemia which showed protection as reviewed elsewhere (39). The high lipophilicity and pKa of nicardipine may enable it to accumulate in tissues which then allows lower doses to be used as demonstrated in rat cerebrocortical synaptosomes previously (40). Nicardipine is widely used as an FDA approved therapy available under prescription to predominantly treat angina and hypertension which are chronic diseases. PTHS is also chronic syndrome so a drug would need to be safe for long term use over the patient’s life. More recently it has also been evaluated as a clinical treatment for neuroprotection of microglia (41) as well as increasing the clearance of beta amyloid (42). The accessibility of this drug (20mg and 30mg doses (43)), and the fact that it has been used in millions of patients would suggest its viability for clinical assessment though it has not obtained approval for use in pediatric populations to our knowledge. The potential for nicardipine to be a practical treatment for PTHS is demonstrated as the effective dose in mouse is 3 mg/kg, which can be scaled to a human dose based on body surface areas by dividing by 12.3 (44), which equates to 0.24 mg/kg in human. Taking an average human of 60 kg, this represents a dose of 14.6 mg. This predicted dose could be even lower in children (based on weight) which would suggest the lowest dose of nicardipine (20 mg given every 8 hours) would be an ideal starting point. This would suggest that half a tablet would still be within the effective dose level, potentially lowering the likelihood of any cardiac side effects. These results would indicate the need for a potential clinical trial to test the safety in PTHS patients. Further preclinical studies could assess the combination of this drug with other compounds such as histone deacetylase inhibitors (17) or NMDA receptor antagonists (18) which have also been suggested as potential treatments for PTHS. This work therefore demonstrates for the first time that an approved drug identified as a potent inhibitor of Na_v1.8 is efficacious in multiple behavioral tests in the well characterized and widely used Tcf4(+/−) mouse model of PHTS. It also paves the way for testing the additional inhibitors of different drug classes which we identified previously (such as cinnarizine, pimozide, carvedilol, etc.)(2) either as monotherapies or combination therapies for PTHS.