Padsevonil

High Affinity Binding Profile to SV2A, SV2B, and SV2C Isoforms

Padsevonil demonstrates high affinity binding to all three isoforms of synaptic vesicle protein 2: SV2A, SV2B, and SV2C. researchgate.netoup.comucb.comnih.govresearchgate.netpatsnap.comaesnet.org This pan-SV2 binding profile distinguishes it from levetiracetam and brivaracetam, which are selective SV2A ligands. nih.govnih.govresearchgate.netpatsnap.comaesnet.orgtiho-hannover.de

Quantitative Binding Kinetics and Comparative Analysis (e.g., vs. Levetiracetam, Brivaracetam)

Quantitative binding studies have characterized the affinity of this compound for the SV2 isoforms. This compound exhibits nanomolar affinity for SV2A, SV2B, and SV2C. researchgate.netoup.comnih.govresearchgate.netlarvol.com Specifically, its affinity for SV2A (pKi 8.5) is notably higher than that of levetiracetam (pKi 5.2) and brivaracetam (pKi 6.6). nih.govnih.govresearchgate.netpatsnap.com This indicates that this compound binds more potently to SV2A than these established SV2A ligands. nih.govwikipedia.org

Beyond affinity, this compound also displays distinct binding kinetics compared to levetiracetam and brivaracetam. nih.govresearchgate.netpatsnap.comaesnet.org It exhibits slower dissociation kinetics from human recombinant SV2A at 37°C, with a dissociation half-life (t½) of 30 minutes, in contrast to the rapid dissociation of levetiracetam and brivaracetam (t½ <0.5 minutes). nih.govresearchgate.netpatsnap.com This suggests a longer target occupancy for this compound at SV2A. nih.gov

The following table summarizes the binding affinities:

Note: Data primarily from references nih.govresearchgate.netpatsnap.com. Levetiracetam and Brivaracetam are selective for SV2A.

Characterization of Molecular Interactions and Putative Binding Sites on SV2 Proteins

Anticonvulsants like levetiracetam and brivaracetam are known to bind to a highly conserved central site within SV2 proteins. researchgate.netrcsb.org Cryo-electron microscopy structures of SV2A and SV2B complexed with anticonvulsant ligands suggest that these compounds bind to a central site in a luminal-occluded conformation, resembling an inhibited transporter. rcsb.org

This compound's interaction with SV2A differs from that of levetiracetam and brivaracetam. nih.govresearchgate.netpatsnap.comaesnet.org Notably, the binding of this compound to SV2A is not potentiated by the allosteric modulator UCB1244283, unlike the binding of brivaracetam and levetiracetam. nih.govresearchgate.netpatsnap.comaesnet.org This suggests that this compound may interact with a different binding site or have a unique mode of interaction within the SV2A protein compared to levetiracetam and brivaracetam. nih.govpatsnap.comaesnet.org While levetiracetam is thought to be sandwiched between conserved tryptophan residues in SV2A, the specific molecular interactions for this compound across all three SV2 isoforms require further detailed characterization. researchgate.net

Elucidation of Functional Modulation on Synaptic Vesicle Dynamics

While the precise mechanism by which SV2 ligands exert their antiseizure effects is not fully elucidated, it is widely believed to involve the modulation of neurotransmitter release via their interaction with synaptic vesicles. oup.compatsnap.comontosight.ai SV2 proteins play a critical role in the synaptic vesicle cycle, including exocytosis and endocytosis. nih.gov Their interaction with proteins like synaptotagmin, which is involved in calcium-dependent neurotransmitter release, highlights their importance in synaptic function. nih.govresearchgate.net

This compound's high-affinity binding to all three SV2 isoforms suggests it may influence synaptic vesicle dynamics more broadly than selective SV2A ligands. researchgate.netoup.comucb.comaesnet.org By binding to SV2A, SV2B, and SV2C, this compound could potentially modulate neurotransmitter release in various neuronal populations where these isoforms are expressed. SV2A is found in nearly all neurons, while SV2B is primarily in glutamatergic neurons, and SV2C is largely restricted to specific brain regions like the striatum and midbrain, being the main SV2 paralog in dopaminergic neurons. nih.gov The functional consequences of this compound's pan-SV2 binding profile on synaptic vesicle dynamics and neurotransmitter release across different neuronal types are an area of ongoing research.

Binding to the Benzodiazepine Recognition Site

This compound binds to the benzodiazepine recognition site on GABA-A receptors. researchgate.netnih.govoup.comucb.comnih.govresearchgate.netpatsnap.comdovepress.comfrontiersin.org This site is located at the interface of the alpha and gamma subunits of the pentameric GABA-A receptor complex. frontiersin.org Binding to this site allosterically modulates the receptor, enhancing the effects of GABA. frontiersin.org

This compound exhibits low-to-moderate affinity for the benzodiazepine site on recombinant and native GABA-A receptors. researchgate.netnih.govnih.govresearchgate.netpatsnap.comresearchgate.net In electrophysiological studies, this compound acts as a positive allosteric modulator of GABA-A receptors with a partial agonist profile at the benzodiazepine site. researchgate.netnih.govnih.govresearchgate.netfrontiersin.orgresearchgate.netaesnet.org Its relative efficacy compared to a full agonist like zolpidem has been reported to be around 40-44%. nih.govresearchgate.netaesnet.org

Studies have shown that this compound can potentiate GABA-mediated chloride currents in cultured neurons. researchgate.netresearchgate.net It shows activity across a range of benzodiazepine-sensitive GABA-A receptor subtypes containing α1, α2, α3, and α5 subunits in combination with β2 and γ2 subunits. nih.govfrontiersin.orgresearchgate.netaesnet.org this compound demonstrated higher potency at receptors containing α1 and α5 subunits compared to those with α2 and α3 subunits. nih.govfrontiersin.orgresearchgate.net Importantly, it does not show activity at benzodiazepine-insensitive receptors containing the α4 subunit or receptors lacking the γ2 subunit, further confirming its interaction specifically with the benzodiazepine binding site. nih.govresearchgate.netaesnet.org

In vivo receptor occupancy studies in mice have corroborated the in vitro findings, demonstrating SV2A occupancy at lower doses and benzodiazepine site occupancy at higher doses. nih.govresearchgate.netpatsnap.comaesnet.orgtiho-hannover.delarvol.com

Identification of Receptor Subunit Interactions (e.g., α1 and γ2 subunits)

This compound interacts with GABAA receptors at the benzodiazepine binding site, which is typically located at the interface between an alpha (α) and a gamma (γ) subunit. researchgate.net Specifically, benzodiazepine-sensitive GABAA receptors commonly contain α1, α2, α3, or α5 subunits in combination with a γ2 subunit. nih.govfrontiersin.org

Electrophysiological studies using recombinant human GABAA receptors expressed in Xenopus oocytes have provided insights into this compound's selectivity for different receptor subtypes. nih.govaesnet.orgresearchgate.net this compound demonstrated potentiation of GABA-induced currents in receptors containing α1, α2, α3, and α5 subunits in combination with β2 and γ2 subunits. nih.govaesnet.orgresearchgate.net Higher potency was observed at α1- and α5-containing receptors compared to α2- and α3-containing receptors. nih.govresearchgate.net Importantly, this compound showed no activity at benzodiazepine-insensitive GABAA receptors containing the α4 subunit (α4β2γ2), further confirming its interaction with the benzodiazepine site. nih.govnih.govaesnet.orgresearchgate.net Studies also indicated inactivity on receptors lacking the γ2 subunit (α1β2 receptors), reinforcing its dependence on the α/γ interface for binding. aesnet.org

Here is a summary of this compound's potency at different recombinant GABAA receptor subtypes:

Functional Characterization as a Partial Agonist

Functional studies have characterized this compound as a positive allosteric modulator with a partial agonist profile at the benzodiazepine site of GABAA receptors. nih.govfrontiersin.orgnih.govresearchgate.net

Electrophysiological Analysis of Allosteric Modulation Properties

Patch-clamp experiments on recombinant human α1β2γ2 GABAA receptors have shown that this compound does not elicit chloride currents in the absence of the endogenous agonist GABA, indicating a lack of intrinsic agonist activity on its own. nih.govresearchgate.netnih.govresearchgate.net However, when co-applied with GABA at a low effective concentration (EC20), this compound potentiated the GABA response. nih.govresearchgate.netnih.govaesnet.orgresearchgate.net This potentiation effect confirms its action as a positive allosteric modulator, enhancing the activity of the receptor in the presence of GABA. nih.govresearchgate.netnih.govresearchgate.net The potentiation reached 167% at the EC20 of GABA, with an EC50 for this compound of 138 nmol/L in recombinant α1β2γ2 receptors. nih.govresearchgate.netnih.govresearchgate.net Similar GABA-potentiating activity was observed in native GABAA receptors in cultured rat cortical neurons, with an EC50 of 208 nmol/L. nih.govaesnet.orgresearchgate.net

Assessment of Intrinsic Agonist Activity in Recombinant Systems

As noted in electrophysiological analyses, studies using recombinant GABAA receptors have demonstrated that this compound does not induce chloride currents when applied alone, even at concentrations up to 30 µmol/L. nih.govresearchgate.netnih.govresearchgate.net This lack of effect in the absence of GABA confirms that this compound acts allosterically and does not possess significant intrinsic agonist activity at these receptors. nih.govresearchgate.net

The partial agonist nature of this compound at the benzodiazepine site has been further characterized by comparing its maximal potentiation effect to that of full agonists like zolpidem or chlordiazepoxide. researchgate.netnih.govaesnet.orgresearchgate.net In recombinant α1β2γ2 receptors, this compound showed a relative efficacy of approximately 40-44% compared to zolpidem. researchgate.netnih.govaesnet.orgresearchgate.net When compared to the nonselective full agonist chlordiazepoxide across different receptor subtypes, this compound's relative efficacy varied: 60% for α1β2γ2, 26% for α2β2γ2, 56% for α3β2γ2, and 41% for α5β2γ2. nih.govaesnet.orgresearchgate.net This profile of lower maximal efficacy compared to full agonists is consistent with its classification as a partial agonist. nih.govaesnet.orgresearchgate.net

Here is a summary of this compound's relative efficacy compared to full agonists:

Theoretical Frameworks for Combined Presynaptic and Postsynaptic Modulation

The rationale behind developing a drug with both presynaptic and postsynaptic mechanisms stems from the observation that combining agents acting on SV2A and GABAA receptors can lead to enhanced protection against seizures in nonclinical models. nih.gov This suggests that simultaneously modulating neurotransmitter release at the presynaptic terminal via SV2 proteins and enhancing inhibitory neurotransmission at the postsynaptic membrane via GABAA receptors could offer a more comprehensive approach to regulating neuronal excitability. nih.gov

The theoretical advantage of a single compound with dual action, as opposed to co-administering two separate drugs, lies in the potential for coordinated pharmacokinetic and pharmacodynamic properties, potentially leading to a more predictable and optimized therapeutic effect. Preclinical studies with this compound have demonstrated robust efficacy across various seizure and epilepsy models, including those considered to be drug-resistant. researchgate.netfrontiersin.org In some models, this compound has shown greater protection than the combination of a selective SV2A ligand (levetiracetam or brivaracetam) with a benzodiazepine (diazepam), suggesting that its unique dual mechanism may confer superior antiseizure properties. researchgate.netfrontiersin.org This observation supports the theoretical framework that simultaneous modulation of both targets by a single molecule can lead to synergistic effects that are not simply additive. researchgate.netfrontiersin.org The partial agonist activity at the GABAA receptor benzodiazepine site is hypothesized to contribute to a better tolerability profile and potentially a lower risk of tolerance development compared to full agonists. nih.govnih.govaesnet.orgresearchgate.netresearchgate.netresearchgate.net

Hypothesized Contributions to Enhanced Antiseizure Efficacy

This compound is a novel antiepileptic drug (AED) candidate designed with a unique dual mechanism of action, targeting both presynaptic and postsynaptic sites nih.govucb.comnih.govresearchgate.netnih.gov. This dual targeting is hypothesized to contribute to its enhanced antiseizure efficacy, particularly in drug-resistant epilepsy nih.govresearchgate.netnih.govonderzoekmetmensen.nlfrontiersin.org.

The rationale behind developing a single molecular entity that interacts with both synaptic vesicle protein 2 (SV2) and γ-aminobutyric acid type A (GABAA) receptors stems from observations in nonclinical models. These studies indicated that co-administration of levetiracetam (an SV2A ligand) with agents acting on GABAA receptors led to enhanced seizure protection without increasing adverse effects nih.gov. Consequently, it was hypothesized that a single drug simultaneously targeting these pathways could translate to improved clinical outcomes, especially in patients whose seizures are not controlled by existing AEDs nih.govonderzoekmetmensen.nl.

This compound exhibits high affinity binding to all three isoforms of synaptic vesicle protein 2: SV2A, SV2B, and SV2C ucb.comnih.govatriumhealth.org. Its affinity for SV2A is notably higher than that of earlier SV2A ligands like levetiracetam and brivaracetam nih.govwikipedia.org. Specifically, this compound has a nanomolar affinity (pKi 8.5) for SV2A, which is approximately 100-fold greater than brivaracetam and 2000-fold greater than levetiracetam nih.govnih.gov. It also binds with high affinity to SV2B (pKi 7.9) and SV2C (pKi 8.5) nih.gov. The interaction with SV2 proteins is thought to modulate neurotransmitter release, a key factor in seizure generation frontiersin.orgmdpi.com.

In addition to its presynaptic effects, this compound interacts with the benzodiazepine (BZD) binding site on the postsynaptic GABAA receptor nih.govucb.comnih.govresearchgate.netatriumhealth.org. Electrophysiological studies using patch-clamp experiments on recombinant GABAARs have shown that this compound acts as a positive allosteric modulator nih.gov. While it has no intrinsic activity in the absence of GABA, it potentiates the GABA response nih.gov. Research suggests this compound acts as a nonselective partial agonist at the BZD site of GABAARs, demonstrating potentiation across various alpha subunits (α1, α2, α3, and α5) nih.gov. The low-to-moderate affinity binding and partial agonist profile at the GABAA receptor are hypothesized to limit the level of receptor occupancy and activation, potentially reducing the risk of side effects and tolerance compared to classic benzodiazepines nih.gov.

The combination of high-affinity binding to SV2 isoforms and moderate-affinity partial agonism at the GABAA receptor's BZD site is the cornerstone of this compound's hypothesized enhanced antiseizure efficacy nih.govucb.comnih.govresearchgate.net. This dual action is believed to provide a more comprehensive approach to modulating neuronal excitability than targeting either pathway alone frontiersin.orgatriumhealth.org. Preclinical studies have investigated this compound's efficacy in various seizure and epilepsy models, including those considered resistant to existing AEDs researchgate.netonderzoekmetmensen.nlresearchgate.net.

In the amygdala kindling model, which is considered predictive of efficacy against drug-resistant focal epilepsy, this compound demonstrated significant protection and was found to be more potent than several other AEDs with different mechanisms of action researchgate.netonderzoekmetmensen.nlfrontiersin.orgresearchgate.net. Furthermore, in the 6 Hz mouse model of focal seizures, this compound provided greater protection than combinations of diazepam (a benzodiazepine) with either levetiracetam or brivaracetam, suggesting that its unique dual mechanism confers superior antiseizure properties compared to the combined effects of single-target drugs frontiersin.orgresearchgate.net.

Translational studies using PET imaging in healthy volunteers aimed to identify optimal target occupancy levels for both SV2A and GABAA receptors that correlated with effective antiseizure activity observed in rodent models nih.govresearchgate.net. These studies suggested that high, sustained SV2A occupancy (>90%) and transient, lower GABAA receptor occupancy (10-15%) were associated with preclinical efficacy nih.govresearchgate.net. This understanding informed the rational dose selection for clinical trials nih.govresearchgate.net.

While preclinical data supported the potential for enhanced efficacy due to this dual mechanism researchgate.netonderzoekmetmensen.nlresearchgate.net, clinical trial results have been mixed. A Phase IIa proof-of-concept trial in patients with treatment-resistant focal epilepsy showed a reduction in median weekly seizure frequency with this compound compared to placebo, and a higher percentage of patients achieving a ≥75% reduction in seizure frequency, although the latter did not reach statistical significance nih.govresearchgate.net. However, a subsequent Phase IIb study did not reach statistical significance for its primary efficacy endpoints ucb.comnih.gov. Despite the outcome of the Phase IIb study, the rational design targeting both presynaptic SV2 and postsynaptic GABAA receptors represented a novel approach in the search for more effective treatments for drug-resistant epilepsy nih.govfrontiersin.orgmdpi.com.

Preclinical Efficacy Findings in Select Models

This compound Target Binding Affinities

Acute Seizure Models

In acute seizure models, this compound has shown potent, dose-dependent protection against seizures induced by various methods. researchgate.netnih.govresearchgate.net

This compound provided potent, dose-dependent protection against seizures induced by administration of pilocarpine or 11-deoxycortisol. researchgate.netnih.govresearchgate.net It was found to be less potent in the pentylenetetrazol (PTZ) and bicuculline models. researchgate.netnih.govresearchgate.net

11-Deoxycortisol is known to induce seizures that can be refractory to certain first-generation AEDs, suggesting its utility as a model for drug-resistant epilepsy. nih.govresearchgate.net Studies have shown that 11-deoxycortisol impedes GABAergic neurotransmission, which may contribute to its seizure-inducing properties. nih.gov

This compound demonstrated potent, dose-dependent protection against seizures induced by 6 Hz stimulation and acoustically-induced seizures. researchgate.netnih.govresearchgate.net It was less potent in the maximal electroshock seizure (MES) model. researchgate.netnih.govresearchgate.net

In the 6 Hz (44 mA) model, this compound provided significantly greater protection than the combination of diazepam with either levetiracetam or brivaracetam, suggesting its unique mechanism of action confers superior antiseizure properties in this model. researchgate.netnih.gov The 6 Hz model is considered a useful tool for identifying compounds with potential efficacy against pharmacoresistant seizures. acs.org

This compound has shown protective effects in the mouse model of acute epilepsy induced by 6 Hz stimulation, acoustic stimulation, and pilocarpine, with reported ED50 values of 0.16, 0.17, and 0.19 mg/kg, respectively. medchemexpress.com

Protection Against Chemoconvulsant-Induced Seizures (e.g., Pilocarpine, 11-Deoxycortisol, Pentylenetetrazol, Bicuculline)

Chronic Epilepsy Models

This compound has also displayed dose-dependent protective effects in chronic epilepsy models, including those that represent human epilepsy syndromes and drug resistance. researchgate.netnih.gov

In the amygdala kindling model, which is considered predictive of efficacy against focal to bilateral tonic-clonic seizures in the clinical setting, this compound provided significant protection in kindled rodents. researchgate.netnih.govnih.gov In mice, this compound was reported to be the most potent AED compared with nine others with different mechanisms of action in this model. researchgate.netnih.govnih.gov Its therapeutic index was also noted as being the highest, potentially indicating a favorable efficacy profile. researchgate.netnih.gov

The amygdala kindling model is one of the few animal models that has shown clinical validation, as its outcomes have been predictive of antiseizure efficacy in patients with difficult-to-treat focal seizures. mdpi.com Full seizure protection was observed in the amygdala kindling model at concentrations corresponding to clinical doses. aesnet.org

In the murine amygdala kindling model, the ED50 for protection against focal-to-bilateral seizures was 1.2 mg/kg. nih.gov At this dose, estimated occupancy was greater than 90% at SV2A and approximately 10% at GABAARs. nih.gov

This compound displayed dose-dependent protective effects in the intrahippocampal kainate and Genetic Absence Epilepsy Rats from Strasbourg (GAERS) models. researchgate.netnih.govresearchgate.net These models represent human mesial temporal lobe and absence epilepsy, respectively. researchgate.netnih.gov

The intrahippocampal kainate model in mice is a model of mesial temporal lobe epilepsy (mTLE) where spontaneous recurrent seizures (SRSs) develop after the induction of status epilepticus. frontiersin.org this compound demonstrated activity in the murine intrahippocampal kainate model, showing a significant decrease in the number and cumulated duration of hippocampal paroxysmal discharges in a dose-dependent manner. researchgate.netmedchemexpress.com

The GAERS model is a recognized animal model of absence epilepsy, characterized by spontaneous spike-and-wave discharges (SWDs). wikipedia.orgplos.org This model exhibits a pharmacological profile similar to human absence seizures. plos.org

Preclinical Antiseizure Efficacy of this compound in Rodent Models

In Vitro Drug Transporter Interactions

In vitro studies have been conducted to assess the interaction of this compound and its metabolites with a range of human ATP-binding cassette (ABC) efflux transporters and solute carrier (SLC) uptake transporters. These studies aim to understand the potential for transporter-mediated drug-drug interactions and the influence of transporters on this compound's disposition in the body. Two in vitro studies specifically provided data on the interaction of this compound (PSL), its desmethyl metabolite, and its carboxylic acid metabolite with human ABC transporters P-glycoprotein (P-gp), BCRP, and BSEP, as well as human uptake transporters OATP1B1, OATP1B3, OAT1, OAT3, OCT1, OCT2, MATE1, and MATE2-K. tandfonline.comtandfonline.com

Analysis of Interactions with Efflux Transporters (e.g., P-glycoprotein, BCRP, BSEP)

This compound demonstrated weak inhibition of P-gp. tandfonline.comtandfonline.com Specifically, this compound showed a dose-dependent reduction in P-gp-mediated digoxin transport, with an IC50 value of 60.46 µM. tandfonline.comtandfonline.com In contrast to this compound, its desmethyl metabolite appears to be a P-gp substrate based on in vitro studies. aesnet.org Other transporters, including BCRP and BSEP, were not significantly inhibited by this compound and its metabolites in these studies. tandfonline.comtandfonline.com P-gp, BCRP, and BSEP are important efflux transporters located in various tissues, including the intestine, liver, kidney, and blood-brain barrier, influencing drug absorption, distribution, and excretion. mdpi.comnih.govnih.govsigmaaldrich.com

Evaluation of Interactions with Uptake Transporters (e.g., OCT1, OCT2, MATE1, MATE2-K, OATP1B1, OATP1B3, OAT1, OAT3)

Among the evaluated uptake transporters, this compound and its desmethyl metabolite demonstrated potent inhibition of the OCT2 transporter. tandfonline.comtandfonline.com The IC50 values for OCT2 inhibition were 1.1 µM for this compound and 2.2 µM for the desmethyl metabolite. tandfonline.comtandfonline.com Inhibition of the human transporter MATE2-K by this compound and its desmethyl metabolite was found to be moderate, with IC50 values of 69.55 µM and 160.5 µM, respectively. tandfonline.comtandfonline.com this compound was also identified as a weak inhibitor of OCT1 and MATE2-K. tandfonline.comtandfonline.com Other uptake transporters, including OATP1B1, OATP1B3, OAT1, and OAT3, were not significantly inhibited by this compound and its metabolites. tandfonline.comtandfonline.com OCT1, OCT2, OAT1, OAT3, MATE1, and MATE2-K are involved in the uptake and excretion of various compounds, particularly in the kidneys and liver. d-nb.infoabdominalkey.com

Here is a summary of this compound's in vitro transporter interactions:

Note: IC50 values for weak or not significantly inhibited transporters were not consistently provided in the source material.

Assessment of Metabolic Stability in Preclinical In Vitro Systems (e.g., Human Liver Microsomes)

Preclinical in vitro studies, including those utilizing human liver microsomes (HLMs), have assessed the metabolic stability of this compound. Metabolic stability in liver microsomes is a key indicator of how readily a compound is metabolized by hepatic enzymes, primarily cytochrome P450 (CYP) enzymes. nuvisan.comevotec.comcreative-diagnostics.com In preclinical studies, CYP3A4 was identified as the main P450 isoform involved in this compound metabolism, with a potential minor contribution from CYP2C19. tandfonline.comtandfonline.comresearchgate.netnih.gov this compound was shown to be a time-dependent inhibitor of CYP2C19 and a weak inducer of CYP3A4. tandfonline.comtandfonline.comresearchgate.netnih.gov High metabolic stability in human liver microsomes has been reported for a lead molecule, (R)-32, which is related to this compound. researchgate.net

Antinociceptive Efficacy in Rodent Pain Models

Preclinical studies in rodent models have investigated the potential antinociceptive efficacy of this compound or related compounds. researchgate.netacs.orgnih.gov A lead molecule, (R)-32, identified as a phenyl-glycinamide derivative, demonstrated potent antinociceptive efficacy in various pain models in mice following intraperitoneal administration. researchgate.netacs.org

Investigation in Inflammatory Pain Models (e.g., Formalin-Induced Pain)

The formalin test in rodents is a widely used model to assess both acute and persistent pain, including inflammatory pain. creative-bioarray.comnih.govcreative-biolabs.comnuchemsciences.com The test typically involves injecting formalin into the paw, which elicits a biphasic pain response: an early phase (neurogenic pain) and a late phase (inflammatory pain). nih.govcreative-biolabs.comnuchemsciences.com The related compound (R)-32 demonstrated potent antinociceptive efficacy in formalin-induced pain in mice. researchgate.netacs.org This indicates a potential effect of this class of compounds on inflammatory pain pathways.

Evaluation of Neurodevelopmental Effects in Neonatal Rodent Models

Studies in neonatal rodent models, specifically Sprague-Dawley rat pups at postnatal day (P)7, P14, and P21, have investigated the effects of this compound. researchgate.netnih.govresearchgate.netnus.edu.sgresearchgate.net These studies assessed various outcomes, including seizure activity and the induction of cell death. This compound suppressed pentylenetetrazole (PTZ)-evoked seizures across multiple age groups in a dose-dependent manner. researchgate.netnih.govresearchgate.netnus.edu.sgresearchgate.net

Comparative Studies on Induction of Cell Death (e.g., vs. Phenobarbital)

A key aspect of the developmental neurotoxicity research involved comparing this compound's effect on cell death induction with that of other ASMs, notably phenobarbital. researchgate.netnih.govresearchgate.netnus.edu.sgresearchgate.net Phenobarbital has been extensively reported to induce cell death in neonatal rodents when administered at P7. researchgate.netnih.govresearchgate.netnus.edu.sgresearchgate.netnih.gov In comparative studies, a separate cohort of P7 rat pups was treated with neonatal ASMs, including this compound and phenobarbital, and evaluated for cell death 24 hours later using PathoGreen staining. researchgate.netnih.govnus.edu.sgresearchgate.net

The research findings indicate that this compound did not produce increased cell death compared to the vehicle control group. researchgate.netnih.govnus.edu.sgresearchgate.net This is in contrast to the effects observed with phenobarbital, which is known to induce neuronal apoptosis in the immature brain. researchgate.netresearchgate.net The data suggest that while many ASMs, including phenobarbital, induce cell death in neonatal rats, this compound does not. researchgate.netnih.govnus.edu.sgresearchgate.net

The following table summarizes the comparative findings on cell death induction in neonatal rats:

These findings suggest that this compound may offer a favorable safety profile concerning developmental neurotoxicity compared to some established ASMs like phenobarbital. researchgate.netnih.gov

Optimization of Receptor Binding Assays (e.g., Radioligand Displacement Methodologies)

Radioligand displacement assays have been utilized to determine this compound's affinity for SV2 protein isoforms and the benzodiazepine site of GABAA receptors. These studies have shown that this compound binds with nanomolar affinity to all three SV2 isoforms (SV2A, SV2B, and SV2C). nih.govresearchgate.netpatsnap.com Its affinity for SV2A is notably higher than that of levetiracetam and brivaracetam, two other SV2A ligands. nih.govresearchgate.netpatsnap.comnih.gov Specifically, this compound's affinity for SV2A (pKi 8.5) is approximately 100-fold greater than brivaracetam (pKi 6.6) and 2000-fold greater than levetiracetam (pKi 5.2). nih.govresearchgate.netpatsnap.comnih.gov Unlike levetiracetam and brivaracetam, which are selective for SV2A, this compound also exhibits high affinity for SV2B (pKi 7.9) and SV2C (pKi 8.5). nih.govresearchgate.netpatsnap.com

Furthermore, these assays revealed that this compound's interaction with SV2A involves slower binding kinetics compared to levetiracetam and brivaracetam, with a dissociation half-life of 30 minutes from the human protein at 37°C, in contrast to less than 0.5 minutes for levetiracetam and brivaracetam. nih.govresearchgate.netpatsnap.com The binding of this compound to SV2A was not potentiated by the allosteric modulator UCB1244283, suggesting a different binding site on the SV2A protein compared to levetiracetam and brivaracetam. nih.govresearchgate.netnih.gov

For the benzodiazepine site on GABAA receptors, radioligand binding assays demonstrated that this compound has low-to-moderate affinity (pIC50 ≤ 6.1, pKi 6.4). nih.govresearchgate.netpatsnap.comresearchgate.netresearchgate.net

Table 1: In Vitro Binding Affinities of this compound

Application of Functional Electrophysiological Assays (e.g., Patch-Clamp Techniques on Recombinant Receptors)

Electrophysiological studies, including patch-clamp techniques on recombinant GABAA receptors, have been used to characterize the functional effects of this compound. These studies showed that this compound acts as a positive allosteric modulator of GABAA receptors. researchgate.net It potentiated GABA responses in recombinant human GABAA receptors and native GABAA receptors in cultured rat cortical neurons. nih.govresearchgate.net this compound did not evoke chloride currents in the absence of GABA, indicating it is not a direct agonist. researchgate.net Its relative efficacy compared to zolpidem, a full agonist reference drug, was approximately 40%, classifying it as a partial agonist at the benzodiazepine site. nih.govresearchgate.netpatsnap.comresearchgate.netresearchgate.net this compound demonstrated higher potency at α1- and α5-containing GABAA receptors compared to α2- and α3-containing receptors in Xenopus oocytes expressing recombinant human GABAA receptors. nih.govresearchgate.net

Positron Emission Tomography (PET) for Target Occupancy in Animal Models

PET imaging allows for the quantitative assessment of receptor occupancy by a drug in vivo. This is particularly valuable in drug development to understand the relationship between drug exposure and target engagement.

Development and Validation of Radiotracers (e.g., [11C]UCB-J for SV2A)

The development and validation of specific radiotracers are critical for PET imaging of drug targets. [11C]UCB-J is a widely used and well-characterized radiotracer for imaging SV2A in vivo. turkupetcentre.netsnmjournals.org It binds to SV2A, which is expressed ubiquitously in synapses throughout the brain. turkupetcentre.net The specificity of [11C]UCB-J for SV2A has been demonstrated through displacement studies using known SV2A ligands like levetiracetam and this compound. turkupetcentre.net [11C]UCB-J has shown favorable characteristics as a PET tracer, including high specific and reversible binding. turkupetcentre.netbiorxiv.org

Quantitative Assessment of SV2A and GABA-A Receptor Occupancy in Rodents

In vivo receptor occupancy studies in mice using radioligand binding assays have supported the in vitro findings regarding this compound's target profile. These studies showed that this compound occupies SV2A at lower doses than those required for significant occupancy of the benzodiazepine site on GABAA receptors. nih.govresearchgate.netpatsnap.comresearchgate.netacs.orgkuleuven.be

Specifically, the median effective dose (ED50) for SV2A occupancy in mice was reported as 0.2 mg/kg, while the ED50 for benzodiazepine site occupancy was considerably higher at 36 mg/kg. nih.govresearchgate.netpatsnap.comresearchgate.netacs.orgkuleuven.be This indicates a greater affinity of this compound for SV2A in vivo, consistent with the in vitro binding data. kuleuven.be

Translational studies have shown that the differential proportionality of target engagement observed in rodents is retained in humans. nih.gov In the murine amygdala kindling model, an ED50 of 1.2 mg/kg for protection against focal-to-bilateral seizures corresponded to over 90% SV2A occupancy and approximately 10% GABAA receptor occupancy. nih.gov Human PET studies with this compound at a dose of 400 mg twice daily showed similar target occupancy, with sustained high-level SV2A occupancy (>90%) and low, transient GABAA receptor occupancy (around 13.4%). nih.govresearchgate.netoup.com

Table 2: In Vivo Receptor Occupancy of this compound in Mice

Pharmacokinetic/Pharmacodynamic (PK/PD) Modeling in Animal Studies

PK/PD modeling in animal studies is a powerful tool used to establish relationships between drug exposure (pharmacokinetics) and pharmacological effect (pharmacodynamics). accp1.orgcatapult.org.uk This approach helps in understanding the time course of drug action and predicting optimal dosing schedules. accp1.orgcatapult.org.uk

For this compound, animal models predictive of human epilepsy were carefully selected to assess its antiseizure efficacy and inform translational studies. nih.govresearchgate.netresearchgate.net A population PK (popPK) model was developed based on data from clinical trials, including rich PK sampling. nih.govresearchgate.net This model was utilized to simulate plasma concentration-time profiles for various doses, taking into account inter-patient variability. nih.gov

Studies in mice, particularly in the amygdala kindling model, provided data on the relationship between this compound dose, target occupancy, and efficacy. nih.gov At a dose of 1.2 mg/kg, which provided protection against focal-to-bilateral seizures in 50% of mice (ED50), estimated occupancy was reported to be >90% at SV2A and approximately 10% at GABAARs. nih.gov This differential target engagement observed in animal models was found to be translatable to humans in subsequent PET studies. nih.govresearchgate.net

In Silico Approaches for Predicting Drug-Drug Interactions

In silico approaches are valuable for predicting potential drug-drug interactions (DDIs) early in the development process, helping to identify risks and guide further investigation. tandfonline.comtandfonline.comresearchgate.netnih.govplos.org These methods often involve analyzing the interaction of a drug candidate with drug-metabolizing enzymes and transporters. tandfonline.comtandfonline.comnih.govresearchgate.netnih.gov

Preclinical in vitro studies identified cytochrome P450 (CYP) 3A4 as the primary isoform involved in this compound metabolism, with a potential minor contribution from CYP2C19. tandfonline.comtandfonline.comnih.govresearchgate.net In silico and in vitro DDI studies characterized this compound's potential as a perpetrator of interactions. tandfonline.comtandfonline.comnih.govresearchgate.net this compound was shown to be a time-dependent inhibitor of CYP2C19 and a weak inducer of CYP3A4. tandfonline.comtandfonline.comnih.govresearchgate.net It also demonstrated weak inhibition of P-gp, OCT1, and MATE2-K, and potent inhibition of OCT2. tandfonline.comtandfonline.comnih.govresearchgate.net These preclinical findings informed quantitative predictions using mechanistic static modeling to assess DDI risk, guiding the design of Phase I clinical studies. tandfonline.comtandfonline.comresearchgate.net

Molecular Modeling and Docking Studies for Receptor-Ligand Interactions

Molecular modeling and docking studies are computational techniques used to predict how a small molecule like this compound might interact with its target receptors at an atomic level. mdpi.comopenaccessjournals.comnih.gov These studies help to understand binding affinities, preferred binding orientations, and the nature of interactions within the binding site. openaccessjournals.com

For this compound, which targets SV2 proteins and the benzodiazepine site of GABAA receptors, molecular modeling and docking studies would be instrumental in understanding the molecular basis of its dual mechanism of action. ucb.comresearchgate.netnih.gov While specific detailed results of this compound docking studies were not extensively detailed in the provided search results, the general application of these techniques involves simulating the binding of the ligand (this compound) to the receptor structures (SV2 proteins and GABAA receptor) to predict the most stable binding pose and the types of interactions involved (e.g., hydrogen bonding, van der Waals forces, electrostatic interactions). openaccessjournals.com Studies on related compounds or general approaches to receptor-ligand interactions highlight the use of techniques like induced fit docking and molecular dynamics simulations to evaluate binding mechanisms and molecular flexibility. mdpi.com Functional investigations on recombinant and native neuronal GABAARs have shown that this compound acts as a positive allosteric modulator with a partial agonist profile at the benzodiazepine site, providing experimental validation for the insights gained from molecular modeling approaches. nih.gov

Application of Theoretical Calculations for Elucidating Stereoisomer Properties

Theoretical calculations, often based on quantum chemistry methods like Density Functional Theory (DFT), can be applied to study the properties of different stereoisomers of a compound. scielo.org.za Stereoisomers have the same molecular formula and connectivity but differ in the spatial arrangement of their atoms, which can lead to significant differences in their pharmacological properties, including pharmacodynamic, pharmacokinetic, and toxicological profiles. researchgate.netlibretexts.orgnih.gov

This compound has a specific stereochemistry, indicated by the (4R) designation in its chemical name: (4R)-4-(2-chloro-2,2-difluoroethyl)-1-[[2-(methoxymethyl)-6-(trifluoromethyl)imidazo[2,1-b] atriumhealth.orgucb.comresearchgate.netthiadiazol-5-yl]methyl]pyrrolidin-2-one. wikipedia.orguni.lu While direct theoretical calculations specifically on this compound stereoisomers were not detailed in the search results, the importance of such calculations is highlighted in the context of other compounds. For instance, theoretical calculations have been performed on transition states leading to different stereoisomers of other molecules to understand the origin of stereoselectivity in synthesis. researchgate.net These calculations can provide insights into the relative stability, reactivity, and other physical properties of different stereoisomers, which is crucial for developing stereoselective synthesis methods and understanding potential differences in biological activity. scielo.org.zaresearchgate.netresearchgate.net The regulatory requirement to assess stereoisomers separately for their biological activities underscores the importance of theoretical and experimental approaches in this area. researchgate.net