Testing the nonclinical Comprehensive In Vitro Proarrhythmia Assay (CiPA) paradigm with an established anti-seizure medication: Levetiracetam case study.

Levetiracetam (LEV), a well-established anti-seizure medication (ASM), was launched before the original ICH S7B nonclinical guidance assessing QT prolongation potential and the introduction of the Comprehensive In Vitro Proarrhythmia Assay (CiPA) paradigm. No information was available on its effects on cardiac channels. The goal of this work was to "pressure test" the CiPA approach with LEV and check the concordance of nonclinical core and follow-up S7B assays with clinical and post-marketing data. The following experiments were conducted with LEV (0.25-7.5 mM): patch clamp assays on hERG (acute or trafficking effects), NaV 1.5, CaV 1.2, Kir 2.1, KV 7.1/mink, KV 1.5, KV 4.3, and HCN4; in silico electrophysiology modeling (Virtual Assay® software) in control, large-variability, and high-risk human ventricular cell populations; electrophysiology measurements in human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and dog Purkinje fibers; ECG measurements in conscious telemetered dogs after single oral administration (150, 300, and 600 mg/kg). Except a slight inhibition (<10%) of hERG and KV 7.1/mink at 7.5 mM, that is, 30-fold the free therapeutic plasma concentration (FTPC) at 1500 mg, LEV did not affect any other cardiac channels or hERG trafficking. In both virtual and real human cardiomyocytes, and in dog Purkinje fibers, LEV induced no relevant changes in electrophysiological parameters or arrhythmia. No QTc prolongation was noted up to 2.7 mM unbound plasma levels in conscious dogs, corresponding to 10-fold the FTPC. Nonclinical assessment integrating CiPA assays shows the absence of QT prolongation and proarrhythmic risk of LEV up to at least 10-fold the FTPC and the good concordance with clinical and postmarketing data, although this does not exclude very rare occurrence of QT prolongation cases in patients with underlying risk factors.

Use of a physiologically based pharmacokinetic-pharmacodynamic model for initial dose prediction and escalation during a paediatric clinical trial.

AIMS: To build and verify a physiologically based pharmacokinetic (PBPK) model for radiprodil in adults and link this to a pharmacodynamic (PD) receptor occupancy (RO) model derived from in vitro data. Adapt this model to the paediatric population and predict starting and escalating doses in infants based on RO. Use the model to guide individualized dosing in a clinical trial in 2- to 14-month-old children with infantile spasms. METHODS: A PBPK model for radiprodil was developed to investigate the systemic exposure of the drug after oral administration in fasted and fed adults; this was then linked to RO via a PD model. The model was then expanded to include developmental physiology and ontogeny to predict escalating doses in infants that would result in a specific RO of 20, 40 and 60% based on average unbound concentration following a twice daily (b.i.d.) dosing regimen. Dose progression in the clinical trial was based on observed concentration-time data against PBPK predictions. RESULTS: For paediatric predictions, the elimination of radiprodil, based on experimental evidence, had no ontogeny. Predicted b.i.d. doses ranged from 0.04 mg/kg for 20% RO, 0.1 mg/kg for 40% RO to 0.21 mg/kg for 60% RO. For all infants recruited in the study, observed concentration-time data following the 0.04 mg/kg and subsequent doses were within the PBPK model predicted 5th and 95th percentiles. CONCLUSION: To our knowledge, this is the first time a PBPK model linked to RO has been used to guide dose selection and escalation in the live phase of a paediatric clinical trial.

Advantageous safety profile of a dual selective alpha(2C) agonist/alpha(2A) antagonist antinociceptive agent.

A selective α2C -adrenoceptor (AR) agonist was developed for the treatment of neuropathic pain. The objective was to dissociate analgesic activity from cardiovascular and sedative side effects commonly observed with nonselective agents. A 2-amino-oxazoline derivative (compound A), identified as a dual α2C -AR agonist/α2A -AR antagonist in in vitro-binding assays, exhibited in vivo efficacy in rodent pain models. Its safety profile was compared with that of clonidine in six different in vivo models. Contrary to clonidine, compound A did not induce hypotension in pentobarbital-anesthetized rats, in conscious spontaneous hypertensive rats, or in telemetered dogs. Both agents induced similar dose-dependent decreases in heart rate in dogs and rats. In anesthetized pithed rats, clonidine showed dose-dependent hypertension and inhibited electrical nerve stimulation-induced tachycardia at doses close to its efficacious doses in the mouse formalin test, while compound A had much weaker vasoconstrictive and antichronotropic effects. Finally, in a mouse Irwin test, no sedation was observed with compound A at 30-fold its ED50 in the mouse formalin test, while sedative effects of clonidine started from three-fold its ED50 . These data confirm the advantageous safety profile of the new dual α2C -AR agonist/α2A -AR antagonist agent vs. the nonselective agonist clonidine.

Seletracetam (UCB 44212).

Better pharmacotherapies for epilepsy are needed for patients who are refractory to or have tolerability difficulties with current treatments. Seletracetam, a new drug in epilepsy development, is a pyrrolidone derivative structurally related to levetiracetam (trade name Keppra). It was discovered because of its high binding affinity to the synaptic vesicle 2A (SV2A) protein, which is now known to be the binding site for this family of compounds. Seletracetam shows very potent seizure suppression in models of acquired or genetic epilepsy, as well as high CNS tolerability in various animal models. Pharmacokinetic studies in animals suggest that seletracetam is rapidly and highly absorbed, with linear and time-independent pharmacokinetics. Seletracetam appears neither to inhibit nor to induce the major human drug metabolizing enzymes, and it demonstrates low plasma protein binding (<10%), which suggests a low potential for drug-drug interactions. Initial studies in humans demonstrated first-order monocompartmental kinetics with a half-life of 8 h and an oral bioavailability of >90%. Studies in healthy volunteers showed that the treatment emergent adverse events were of mild to moderate severity, were mostly of CNS origin and were resolved within 24 h. Altogether, these results suggest that seletracetam represents a promising new antiepileptic drug candidate, one that demonstrates a potent, broad spectrum of seizure protection and a high CNS tolerability in animal models, with initial clinical findings suggestive of straightforward pharmacokinetics and good tolerability.

Determination of phospholipidosis potential based on gene expression analysis in HepG2 cells.

Phospholipidosis (PLD) is characterized by an intracellular accumulation of phospholipids in lysosomes and the concurrent development of concentric lamellar bodies. Recently, H. Sawada et al. (2005, Toxicol. Sci. 83, 282-292) identified 17 genes as potential biomarkers of PLD in HepG2 cells. The present study was undertaken to determine if this set of genes measured by quantitative PCR could be validated in the same cell line. The objective was also to investigate the dose-response relationship to further validate the assay and to select the concentrations to use for screening activities. In a first experiment (one concentration tested), out of the 17 genes, the best gene biomarkers of PLD (i.e., 11 genes) were selected for practical screening reasons. Based on these genes, 91.6% (i.e., 11 of 12) of the compounds known to induce PLD were identified as positive and all the negative compounds (i.e., five of five) were also confirmed. When the data obtained in the first experiment were compared to the data by Sawada et al., (2005) the coefficient of correlation calculated was slightly higher than 75%. In the second experiment (26 compounds [all 17 compounds from the first experiment plus 9 other compounds] tested at a minimum of three concentrations), 93.3% (14/15) of the compounds known to induce PLD were identified as such and all the negative controls (six compounds) were also confirmed. Three compounds likely to induce PLD were identified as positive in our assay. Finally, two compounds for which no data are available were also tested. When both experiments 1 and 2 were compared, the coefficient of correlation for 16 compounds tested at the same concentrations reached 87.7%. In conclusion, the present study further confirms the utility of gene expression in HepG2 cells to identify a potential to induce PLD. Finally, based on the data presented, researchers are encouraged to use a range of minimum three concentrations (e.g., 12.5, 25, and 50 microM) to screen for PLD in the human HepG2 cell line.