Overview of preclinical and clinical studies investigating pharmacokinetics and drug-drug interactions of padsevonil.

BACKGROUND: Padsevonil is an antiseizure medication candidate intended to benefit patients with drug-resistant epilepsy. Our investigations aimed at characterizing pharmacokinetics and drug-drug interaction (DDI) profile of padsevonil. RESEARCH DESIGN AND METHODS: An overview of preclinical and clinical pharmacology studies conducted during padsevonil development is provided. RESULTS: In preclinical studies, cytochrome (CYP) 3A4 was identified as the main P450 isoform involved in padsevonil metabolism, with potential minor contribution from CYP2C19. Padsevonil was shown to be a time-dependent CYP2C19-inhibitor, weak CYP3A4-inducer, weak inhibitor of P-gp/OCT1/MATE2-K, and potent OCT2-inhibitor. Initial clinical pharmacology studies in healthy participants showed that padsevonil had (i) good absorption, (ii) clearance mediated mainly by metabolism, and (iii) time-dependent kinetics. A study in genotyped participants confirmed the role of CYP2C19 in clearance and time-dependent kinetics; the major contribution of CYP3A4 was confirmed in DDI studies with CYP3A4-inducers (carbamazepine, oxcarbazepine) and -inhibitor (erythromycin). Padsevonil did not affect pharmacokinetics of valproate/lamotrigine/levetiracetam/oxcarbazepine or oral contraceptives. In a cocktail clinical study, padsevonil showed moderate CYP2C19 inhibition (omeprazole) and weak CYP3A4 induction (oral midazolam). No specific effects on CYP1A2 (caffeine), CYP2C9 (S-warfarin), and CYP2D6 (dextromethorphan) were observed. CONCLUSIONS: The studies presented helped in understanding padsevonil disposition and risks of DDIs, which would inform dosing and prescribing. CLINICAL TRIAL REGISTRATION: https://www.clinicaltrials.gov identifiers are NCT04131517, NCT03480243, NCT03695094, NCT04075409.

Pharmacokinetics of Staccato® alprazolam in healthy adult participants in two phase 1 studies: An open-label smoker study and a randomized, placebo-controlled ethnobridging study.

OBJECTIVE: Staccato® alprazolam is a single-use, drug-device combination delivering alprazolam to the deep lung that is being evaluated as treatment for rapid and early seizure termination. This article reports pharmacokinetic (PK) data from two phase 1 studies of Staccato alprazolam in healthy adult participants. METHODS: The smoker study (EPK-002/NCT03516305) was an open-label, nonrandomized, single-dose, PK study in smokers and nonsmokers aged 21-50 years, administered a single inhaled dose of 1 mg Staccato alprazolam. The ethnobridging study (UP0101/NCT04782388) was a double-blind, placebo-controlled study in Japanese, Chinese, and Caucasian participants aged 18-55 years randomized 4:1 to a single inhaled dose of Staccato alprazolam 2 mg or Staccato placebo. RESULTS: In the smoker study, 36 participants (18 smokers, 18 nonsmokers) were enrolled and received Staccato alprazolam. Following Staccato administration, alprazolam was rapidly absorbed, with a median time to peak drug plasma concentration (Tmax) of 2 min in both smokers (range = 2-30 min) and nonsmokers (range = 2-60 min). Staccato alprazolam was rapidly absorbed to a similar extent in both smokers and nonsmokers. The most commonly reported treatment-emergent adverse events (TEAEs) were somnolence and dizziness. In the ethnobridging study, 10 participants each of Japanese, Chinese, and Caucasian ethnicities were randomized 4:1 to Staccato alprazolam or Staccato placebo. Following Staccato administration, alprazolam was rapidly absorbed and distributed, with a median Tmax of 1.5-2 min in Japanese (range = 1-2 min), Chinese (range = 1-34 min), and Caucasian (range = 1-120 min) participants. Somnolence and sedation were the most commonly reported TEAEs. In both studies, there were no deaths, and no participants reported serious or severe TEAEs, or discontinued due to TEAEs. SIGNIFICANCE: Alprazolam was rapidly absorbed, and therapeutic drug levels were achieved within 2 min postdose when administered to the lung with the Staccato device. Staccato alprazolam was generally well tolerated and displayed a safety profile consistent with that known from other alprazolam applications. No new safety signals were identified.

Clinical Bridging Studies and Modeling Approach for Implementation of a Patient Centric Sampling Technique in Padsevonil Clinical Development.

Volumetric absorptive microsampling (VAMS) techniques have gained popularity these last years as innovative tool for collection of blood pharmacokinetic (PK) samples in clinical trials as they offer many advantages over dried blood spot and conventional venous blood sampling. The use of Mitra®, a blood collection device based on volumetric absorptive microsampling (VAMS) technology, was implemented during clinical development of padsevonil (PSL), an anti-seizure medication (ASM) candidate. The present study describes the approach used to bridge plasma (obtained from conventional venous blood sampling) and blood exposures (obtained with Mitra®) to support the use of Mitra as sole blood PK sampling method in clinical trials. Paired blood (using Mitra®) and plasma samples (using conventional venous blood sampling) were collected in healthy volunteers as well as in patients with epilepsy. PSL concentration in plasma and blood were analyzed using different approaches which included evaluation of blood-to-plasma ratios (B/P) over time, linear regression, Bland-Altman analysis as well as development of a linear-mixed effect model based on clinical pharmacology studies. Results showed that the observed in vivo B/P and the measured bias between the 2 collection methods were consistent with the measured in vitro B/P. Graphical analysis demonstrated a clear time effect on the B/P which was confirmed in the linear mixed effect model with sampling time identified as significant covariate. Finally, the built-in model was validated using independent datasets and was shown to adequately predict plasma concentration based on blood concentration with a mean bias of less than 9% (predicted versus observed plasma concentration).

Target occupancy biomarker assay development using a conformation-selective antibody against small-molecule-bound TNF.

Background: An antibody specific to small-molecule inhibitor-bound TNF has enabled the development of target occupancy biomarker assays to support the development of novel treatments for autoimmune disorders. Materials & methods: ELISAs were developed for inhibitor-bound and total TNF to determine the percentage of TNF occupancy in samples from stimulated blood. Inhibitor-saturated samples allowed measurement of total and inhibitor-bound TNF in a single electrochemiluminescence immunoassay. Results: TNF occupancy was proportional to inhibitor concentration in plasma samples. An electrochemiluminescence method for inhibitor-bound TNF was validated for use as a potential clinical occupancy biomarker assay. Conclusion: Development of these assays has allowed measurement of a target occupancy biomarker, which has supported progression of the first small-molecule inhibitors of TNF.

Effectively utilizing the Sponsor Contract Research Organization interaction for successful implementation of critical flow cytometry in the clinic.

As the number of therapeutic modalities expand, and the field of scientific research evolves toward finding treatment solutions for complex and rare disease, an ability to demonstrate efficacy through biomarker end points in clinical development studies is becoming increasingly important. Implementing flow cytometry in a clinical setting is challenging and many sponsor organizations take a hybrid approach, developing complex analytical methods internally before identifying and forming partnerships with contract research organizations to conduct the formal analytical method validation and sample bioanalysis. Ensuring that these interactions are effective is critical to the delivery of high-quality, impactful clinical data. This paper provides a review of the recommendations, challenges and solutions for the implementation of decision-making flow cytometry end points effectively utilizing the Sponsor Contract Research Organization interaction.

Unraveling the mechanism and the risk behind seizure liability of lead compounds in a neuroscience project.

INTRODUCTION: Several compounds from a neuroscience project induced convulsions in animals, at low exposure levels via a hypothetical off-target mechanism. A set of in vitro and in vivo experiments were conducted in order to 1) identify the mechanism behind convulsions; 2) characterize the convulsions, 3) detect premonitory signs that could be monitored clinically, and 4) assess the development of tolerance after repeat dosing. METHODS: Patch clamp assays were conducted on 12 different ion channels (e.g. sodium, potassium, calcium, AMPA, NMDA, GABAA and purinergic receptors) known to be associated with seizures, to identify the off-target culprit. A multiphase study was conducted with UCB-A and UCB-B in Beagle dogs telemetered for video EEG/EMG monitoring to further characterize the convulsive pattern. First, both compounds were administered by intravenous constant infusion (dose: 5 mg/kg/h) over 2 h. Thereafter, the same dogs received a daily oral administration of UCB-A (8 mg/kg/day) for 7 days. RESULTS: Compounds inducing convulsions showed strong inhibitory activity on GABAA channels (IC50 values <10 µM), whereas compounds with partial or no inhibitory effect on these channels did not induce seizures. In EEG experiments, convulsions were preceded by premonitory clinical signs (e.g. tremors, myoclonic jerks) and morphological EEG abnormalities (e.g. sharp waves, spike and wave patterns), confirming their CNS origin. No attenuation of the seizurogenic effects was observed over the 7-day treatment period. DISCUSSION: A well-designed set of experiments including electrophysiological assays on seizure-related ion channels and EEG/EMG assessment in telemetered dogs allowed a proper seizure liability risk assessment, leading to a rapid no go decision for the two most advanced leads.

Cross-species pharmacological characterization of the allylglycine seizure model in mice and larval zebrafish.

Treatment-resistant seizures affect about a third of patients suffering from epilepsy. To fulfill the need for new medications targeting treatment-resistant seizures, a number of rodent models offer the opportunity to assess a variety of potential treatment approaches. The use of such models, however, has proven to be time-consuming and labor-intensive. In this study, we performed pharmacological characterization of the allylglycine (AG) seizure model, a simple in vivo model for which we demonstrated a high level of treatment resistance. (d,l)-Allylglycine inhibits glutamic acid decarboxylase (GAD) - the key enzyme in γ-aminobutyric acid (GABA) biosynthesis - leading to GABA depletion, seizures, and neuronal damage. We performed a side-by-side comparison of mouse and zebrafish acute AG treatments including biochemical, electrographic, and behavioral assessments. Interestingly, seizure progression rate and GABA depletion kinetics were comparable in both species. Five mechanistically diverse antiepileptic drugs (AEDs) were used. Three out of the five AEDs (levetiracetam, phenytoin, and topiramate) showed only a limited protective effect (mainly mortality delay) at doses close to the TD50 (dose inducing motor impairment in 50% of animals) in mice. The two remaining AEDs (diazepam and sodium valproate) displayed protective activity against AG-induced seizures. Experiments performed in zebrafish larvae revealed behavioral AED activity profiles highly analogous to those obtained in mice. Having demonstrated cross-species similarities and limited efficacy of tested AEDs, we propose the use of AG in zebrafish as a convenient and high-throughput model of treatment-resistant seizures.

Rapid epileptogenesis in the mouse pilocarpine model: video-EEG, pharmacokinetic and histopathological characterization.

Temporal lobe epilepsy (TLE) is the most common form of drug-resistant epilepsy and several rodent models allow studying the pathophysiology of this disorder. One of the best characterized models of TLE is the pilocarpine model. The model has been widely used in rats, but relatively few studies report data obtained with mice. This triggered the present study to perform a comprehensive characterization of the mouse pilocarpine model. We used male NMRI mice (28-32 g) and first established the dose-response relationship for pilocarpine (250-400 mg/kg; ip) to induce status epilepticus (SE). This enabled to define the optimal dose (300 mg/kg) producing the highest SE response (50%) associated with the best survival rate of the animals (90%). The impact of different durations of SE (0.5-3.0 h) on the time to the onset of the first spontaneous recurrent seizures (SRS) was recorded during 5-day continuous video monitoring following the SE. Virtually no "latent" period was observed as the seizures appeared already within 24-48 h after the pilocarpine-induced SE and 0.5 h duration of the SE was sufficient to trigger SRS. Pharmacokinetics assessment showed that these seizures were not associated with residual pilocarpine exposure as it was cleared from the blood and brain already within 24 h post-injection. Consistent with previous reports from the rat pilocarpine model we observed that the extent of hippocampal reorganization and neuronal loss correlates with the duration of SE. However, the shorter durations of SE (0.5-2.0 h) appeared to produce cell loss restricted mainly to the hilus of the dentate gyrus. Interestingly, we also observed that the number of seizures occurring within 5 days after SE appeared to correlate with the degree of hippocampal damage. Continuous 7-week video-EEG monitoring after the SE revealed that SRS were expressed in a particular pattern of clusters. Taken together, the current study provides an in-depth characterization of the mouse pilocarpine model and confirms several features of the epileptogenesis process previously reported from the rat pilocarpine model. However, the mouse pilocarpine model differs by the rapid onset of seizures and an apparent correlation between their numbers and the degree of histopathological changes. Our findings highlight that the pilocarpine model of TLE in mice is associated with brain pathology akin to different stages of human disease and may provide a valuable tool for the discovery of future antiepileptic drugs with disease-modifying properties.