Riluzole in Spinal Cord Injury Study (RISCIS)-Pharmacokinetic (PK) Sub-Study: An Analysis of Pharmacokinetics, Pharmacodynamics, and Impact on Axonal Degradation of Riluzole in Patients With Traumatic Cervical Spinal Cord Injury Enrolled in the RISCIS Phase III Randomized Controlled Trial.

To date, no drug therapy has shown significant efficacy in improving functional outcomes in patients with acute spinal cord injury (SCI). Riluzole is an approved benzothiazole sodium channel blocker to attenuate neurodegeneration in amyotrophic lateral sclerosis (ALS) and is of interest for neuroprotection in SCI. In a Phase I clinical trial (ClinicalTrials.gov Identifier: NCT00876889), riluzole was well tolerated with a 2-week treatment at the dose level approved for ALS and exhibited potential efficacy in patients with SCI. The acute and progressive nature of traumatic SCI and the complexity of secondary injury processes alter the pharmacokinetics (PK) of therapeutics. In the PK sub-study of the multi-center, randomized, placebo-controlled, double-blinded Riluzole in Spinal Cord Injury Study (RISCIS) Phase II/III trial (ClinicalTrials.gov Identifier: NCT01597518), a total of 32 SCI patients were enrolled, and most of our patients were middle-age Caucasian males with head and neck injuries. We studied the PK and pharmacodynamics (PD) of riluzole on motor recovery, measured by International Standards for Neurological Classification of SCI (ISNCSCI) Motor Score at injury and at 3-month and 6-month follow-ups, along with levels of the axonal injury biomarker phosphorylated neurofilament heavy chain (pNF-H), during the 2-week treatment. PK modeling, PK/PD correlations were developed to identify the potential effective exposure of riluzole for intended PD outcomes. The longitudinal impacts of SCI on the PK of riluzole are characterized. A time-varying population PK model of riluzole is established, incorporating time-varying clearance and volume of distribution from combined data of Phase I and Phase II/III trials. With the developed model, a rational, optimal dosing scheme can be designed with time-dependent modification to preserve the required therapeutic exposure of riluzole. The PD of riluzole and the relationship between PK and neurological outcomes of the treatment were established. The time course of efficacy in total motor score improvement (ΔTMS) and pNF-H were monitored. A three-dimensional (3D) PK/PD correlation was established for ΔTMS at 6 months with overall riluzole exposure area under the curve for Day 0-Day14 (AUCD0-D14) and baseline TMS for individual patients. Patients with baseline TMS between 1 and 36 benefited from the optimal exposure range of 16-48 mg*h/mL. The PD models of pNF-H revealed the riluzole efficacy, as treated subjects exhibited a diminished increase in progression of pNF-H, indicative of reduced axonal breakdown. The independent parameter of area between effective curves (ABEC) between the time profiles of pNF-H in placebo and treatment groups was statistically identified as a significant predictor for the treatment effect on the biomarker. A mechanistic clinical outcomes (CO)/PD (pNF-H) model was established, and the proposed structure demonstrated the feasibility of PK/PD/CO correlation model. No appreciable hepatic toxicity was observed with the current riluzole treatment regimen. The development of effective treatment for SCI is challenging. However, the future model-informed and PK-guided drug development and regimen modification can be rationally executed with the optimal dosing regimen design based on the developed 3D PK/PD model. The PK/PD/CO model can serve as a rational guide for future drug development, PKPD model refinement, and extension to other studies in SCI settings.

Low-Luminance Blue Light-Enhanced Phototoxicity in A2E-Laden RPE Cell Cultures and Rats.

N-retinylidene-N-retinylethanolamine (A2E) and other bisretinoids are components of lipofuscin and accumulate in retinal pigment epithelial (RPE) cells-these adducts are recognized in the pathogenesis of retinal degeneration. Further, blue light-emitting diode (LED) light (BLL)-induced retinal toxicity plays an important role in retinal degeneration. Here, we demonstrate that low-luminance BLL enhances phototoxicity in A2E-laden RPE cells and rats. RPE cells were subjected to synthetic A2E, and the effects of BLL on activation of apoptotic biomarkers were examined by measuring the levels of cleaved caspase-3. BLL modulates the protein expression of zonula-occludens 1 (ZO-1) and paracellular permeability in A2E-laden RPE cells. Early inflammatory and angiogenic genes were also screened after short-term BLL exposure. In this study, we developed a rat model for A2E treatment with or without BLL exposure for 21 days. BLL exposure caused fundus damage, decreased total retinal thickness, and caused neuron transduction injury in the retina, which were consistent with the in vitro data. We suggest that the synergistic effects of BLL and A2E accumulation in the retina increase the risk of retinal degeneration. These outcomes help elucidate the associations between BLL/A2E and angiogenic/apoptotic mechanisms, as well as furthering therapeutic strategies.

Ultrapressure liquid chromatography-tandem mass spectrometry assay using atmospheric pressure photoionization (UPLC-APPI-MS/MS) for quantification of 4-methoxydiphenylmethane in pharmacokinetic evaluation.

4-Methoxydiphenylmethane (4-MDM), a selective augmenter of Leukotriene A4 Hydrolase (LTA4H), is a new anti-inflammatory compound for potential treatment of chronic obstructive pulmonary disease (COPD). Currently, there is no liquid chromatography tandem mass spectrometric (LC-MS/MS) method for the quantification of 4-MDM. A major barrier for developing the LC-MS/MS method is the inability of electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) to ionize 4-MDM due to its hydrophobicity and lack of any functional group for ionization. With the advent of atmospheric pressure photoionization (APPI) technique, many hydrophobic compounds have been demonstrated to ionize by charge transfer reactions. In this study, a highly sensitive ultrapressure liquid chromatography tandem mass spectrometry assay using atmospheric pressure photoionization (UPLC-APPI-MS/MS) for the quantifications of 4-MDM in rat plasma has been developed and validated. 4-MDM was extracted from the plasma by solid phase extraction (SPE) and separated chromatographically using a reverse phase C8 column. The photoionization (PI) was achieved by introducing anisole as a dopant to promote the reaction of charge transfer. The assay with a linear range of 5 (LLOQ)-400ngmL(-1) met the regulatory requirements for accuracy, precision and stability. The validated assay was employed to quantify the plasma concentrations of 4-MDM after an oral dosing in Sprague Dawley (SD) rats.

The Utility of the Swine Model to Assess Biological Rhythms and Their Characteristics during Different Stages of Residence in a Simulated Intensive Care Unit: A Pilot Study.

The purpose of this pilot study was to explore the utility of the mammalian swine model under simulated intensive care unit (sICU) conditions and mechanical ventilation (MV) for assessment of the trajectory of circadian rhythms of sedation requirement, core body temperature (CBT), pulmonary mechanics (PM) and gas exchange (GE). Data were collected prospectively with an observational time-series design to describe and compare circadian rhythms of selected study variables in four swine mechanically ventilated for up to seven consecutive days. We derived the circadian (total variance explained by rhythms of τ between 20 and 28 h)/ultradian (total variance explained by rhythms of τ between 1 and <20 h) bandpower ratio to assess the robustness of circadian rhythms, and compare findings between the early (first 3 days) and late (subsequent days) sICU stay. All pigs exhibited statistically significant circadian rhythms (τ between 20 and 28 h) in CBT, respiratory rate and peripheral oxygen saturation, but circadian rhythms were detected less frequently for sedation requirement, spontaneous minute volume, arterial oxygen tension, arterial carbon dioxide tension and arterial pH. Sedation did not appear to mask the circadian rhythms of CBT, PM and GE. Individual subject observations were more informative than group data, and provided preliminary evidence that (a) circadian rhythms of multiple variables are lost or desynchronized in mechanically ventilated subjects, (b) robustness of circadian rhythm varies with subject morbidity and (c) healthier pigs develop more robust circadian rhythm profiles over time in the sICU. Comparison of biological rhythm profiles among sICU subjects with similar severity of illness is needed to determine if the results of this pilot study are reproducible. Identification of consistent patterns may provide insight into subject morbidity and timing of such therapeutic interventions as weaning from MV.

Evaluation of the sodium-glutamate blocker riluzole in a preclinical model of ervical spinal cord injury.

STUDY TYPE: Basic research Introduction: Because sodium and glutamate play integrated roles in the pathology of spinal cord injury (SCI), there is intense interest in the potential role of the sodium-glutamate blocker riluzole as a neuroprotective agent for spinal cord injury. A phase I safety clinical trial of riluzole is about to commence in the USA and Canada. OBJECTIVE: The key challenges in translating riluzole to the clinic relate to uncertainty regarding the time window for post-injury administration of riluzole and the optimal dose. Hence, the current study was undertaken to study these issues in a preclinical model of cervical SCI in rodents. METHODS: Adult female rats received moderate cervical spinal cord clip compression injury (35 g × 1 min at C7-T1) and were randomly assigned to one of the following three groups (n = 12/group): a) 8 mg/kg of riluzole intraperitoneally at 1 hour (P1 group) ; b) 3 hours (P3 group) after injury or c) a control group (vehicle). The P1 and P3 groups then received 6 mg/kg of riluzole intraperitoneally every 12 hours for 7 days. A smaller group of rats (n = 10) received a higher dose (8 mg/kg IP Q12 h for 7 days) of riluzole following the initial dosing. Functional recovery was tested by open field locomotion (BBB score, BBB subscore) and grid walk. Sensory function (tactile allodynia) was assessed by quantitative von Frey filament testing. Somatosensory evoked potentials (SSEP) were performed to quantitatively assess central axonal conduction. Western blotting for neurofilament 200 (NF200) was used to assess axonal integrity. Quantitative image analysis of lesion histology (HE/LFB stained sections) was undertaken to assess tissue sparing at the injury site. High performance liquid chromatography (HPLC) was used to test pharmacokinetics of riluzole. ANOVA followed by the Tukey's post-hoc testing was used to compare the results. RESULTS: A total of 92.3% of injured rats that received repeated dose of 6 mg/kg riluzole survived during the term of 6 weeks, showing tolerance to this dosage. In contrast, higher doses of riluzole (8mg/kg IP q12h post-injury) were associated with significant respiratory depression and high mortality (7/10 rats). Riluzole treatment (P1 group) decreased the functional deficit significantly (P < 0.05) at 6 weeks after injury by showing 11.4 in BBB, 3.8 in BBB subscore and 2.8 in grid walk test, compared with 10.25 in BBB, 1.1 in BBB subscore, and 8.0 in grid walk test of control group (Figure 1). Quantitative analysis of SSEPs confirmed the neuroprotective effects of riluzole (P1 group) by showing significantly larger amplitude (control = 20.67 µV, P1 = 42.67 µV and P3 = 37.00 µV, Figure 2) and reduced response latency (control = 4.05 ms and P1 = 3.9 ms). Western blotting of NF200 indicated greater axonal preservation with riluzole treatment (P1 group) at 1 week after injury. The P3 group showed trends to improved neurobehavioral recovery and axonal preservation that did not attain significance. HPLC showed riluzole penetrated into the spinal cord as fast as 15 minutes, accumulated in the spinal cord at a concentration six times higher than in plasma, and two times higher than in brain. The drug stayed in spinal cord with a high concentration before the next injection. Figure 1 Riluzole treatment improved functional recovery. The most robust effects were seen when the drug was injected at one hour after SCIFigure 2 Riluzole treatment increased amplitude of somatosensory evoked potentials Conclusions: The sodium-glutamate blocker riluzole confers neurobehavioral and anatomical neuroprotection when administered one hour after moderately severe cervical contusive/compressive injury.