Enhanced brain delivery with lower hepatic exposure of lazaroid loaded nanostructured lipid carriers developed using a design of experiment approach.

The current study was designed to develop and optimize lazaroid loaded nano-structured lipid carriers (LAZ-NLCs) using design of experiment approach for enhancing lazaroid brain exposure. Response surface plots were used to determine the effects of independent variables (amount of PEGylating agent and liquid lipid) on dependent variables (particle size, zeta potential and encapsulation efficiency), while numerical optimization was used for optimizing LAZ-NLCs composition. The optimal LAZ-NLCs were spherical in shape with measured size of 172.3 ±â€¯3.54 nm, surface charge of -4.54 ±â€¯0.87 mV and encapsulation efficiency of 85.01 ±â€¯2.60%. The optimal LAZ-NLCs were also evaluated for hemolytic potential, storage stability and solid-state properties. The plasma pharmacokinetics along with brain and hepatic distributions of control lazaroid citrate solution and optimal LAZ-NLCs formulation were evaluated in Sprague-Dawley rats after the single bolus intravenous administration. The optimized LAZ-NLCs and the control lazaroid citrate solution had similar plasma pharmacokinetic profiles; however, differential organ bio-distributions were observed. The lazaroid exposure in brain was enhanced by two times with a decreased liver exposure by half for the NLCs group compared to the solution group.

UPLC-MS/MS assay of 21-aminosteroid (lazaroid U74389G) for application in pharmacokinetic study.

Lazaroids are potent inhibitors of lipid peroxidation, both in vitro and in vivo. Additionally, a member of the lazaroid family, U-74389G (LAZ) has been shown to have specific radio-protective and anti-proliferative effects. However, there is no quantitative analytical method developed for measuring the therapeutic levels of LAZ for the aforementioned effects. This article highlights the development and validation of a sensitive UPLC-MS/MS method for the quantification of LAZ, and its subsequent application in pharmacokinetic studies in rats with the lower limit of quantification (LLOQ) of 1.95 ng/mL. LAZ and internal standard diadzein (IS) were separated using ACQUITY UPLC(®) BEH C18 column. Gradient elution was used at a flow rate of 0.45 mL/min with mobile phases consisting of 0.1% formic acid in water and 0.1% formic in acetonitrile. LAZ (m/z 612→260) and IS (m/z 255→199) were detected by electrospray ionization (ESI) using multiple reaction monitoring (MRM) in a positive mode on QTRAP(®) 5500 System. The UPLC-MS/MS method was validated as per the US FDA Guidelines for Bio-analytical Validation. LAZ was extracted from rat plasma (100 µL) using protein precipitation by acetonitrile with mean recovery and matrix factor in range of 47.7-56.1%, and 85.6-89.4%, respectively. The calibration curve for LAZ was linear in the range of 1.95-250 ng/mL. The inter-day and intra-day accuracy and precision values for LLOQ, low, medium, high and very high concentration QC samples were within ±15%. LAZ was tested under different storage conditions, for short-term bench-top stability (1h and 3h at 25°C), long-term stability (1 month at -80°C), freeze-thaw cycle stability (1 cycle and 3 cycles) and stability of processed samples in auto-sampler (24h at 10°C) with stability values within ±15% range of nominal concentrations. The validated UPLC-MS/MS method was further applied to a pharmacokinetic study in rats after a single intravenous dose of LAZ at 5 mg/kg.

Rationale, design and critical end points for the Riluzole in Acute Spinal Cord Injury Study (RISCIS): a randomized, double-blinded, placebo-controlled parallel multi-center trial.

BACKGROUND: Riluzole is a sodium channel-blocking agent used in treating amyotrophic lateral sclerosis. It has been approved by the U.S. Food and Drug Administration, Canadian and Australian authorities, and in many other countries. A phase I trial of riluzole for acute spinal cord injury (SCI) provided safety and pharmacokinetic data and suggested neuroprotective benefits. A phase IIB/III double-blinded randomized controlled trial (RCT) started in January 2014 (https://clinicaltrials.gov, NCT01597518). This article describes the pathophysiological rationale, preclinical experience and design of the phase IIB/III RCT of Riluzole in Acute Spinal Cord Injury Study (RISCIS). OBJECTIVES: The primary objective of the trial is to evaluate the superiority of riluzole, at a dose of 100 mg BID in the first 24 h followed by 50 mg BID for the following 13 days post injury, compared with placebo in improving neurological motor outcomes in patients with C4-C8 level, International Standards for Neurological Classification of Spinal Cord Injury Examination (ISNCSCI) grade A, B or C acute (within 12 h post injury) SCI. SETTING: Acute trauma centers worldwideMethods:A double-blind, multi-center, placebo-controlled RCT will enroll 351 participants randomized 1:1 to riluzole and placebo. The primary end point is the change between 180 days and baseline in ISNCSCI Motor Score. This study has 90% power to detect a change of nine points in ISNCSCI Motor Score at one-sided α=0.025. RESULTS: Currently enrolling in 11 centers. CONCLUSION: This study will provide class I evidence regarding the safety and neuroprotective efficacy of riluzole in patients with acute cervical SCI.

High performance liquid chromatography-tandem mass spectrometric assay of dexmedetomidine in plasma, urine and amniotic fluid samples for pregnant ewe model.

Dexmedetomidine (DEX; Precedex(®)), approved by the Food and Drug Administration (FDA) in 1999 as a sedative for use in the intensive care unit, is a potent and highly selective α2-adrenoceptor agonist with significant sedative, analgesic and anxiolytic effects. However, the research of DEX use during pregnancy is limited and the impact of DEX on the fetal development is unclear. This article describes a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay suitable for various biomatrices of plasma, urine and amniotic fluid, as a prerequisite for pharmacokinetic characterization of DEX in the pregnant ewe model. DEX and testosterone (internal standard; IS) were extracted from 200µL of plasma, urine or amniotic fluid with ethyl acetate. The HPLC resolution was achieved on an Agilent ZORBAX SB-CN column with a gradient elution at a flow rate of 0.5mL/min using a mobile phase of 5-100% of acetonitrile with 0.5% formic acid (mobile phase B) in water (mobile phase A). The detection was performed by a triple quadrupole tandem mass spectrometer with positive electrospray ionization. The precursor/product transitions (m/z) in the positive ion mode [M+H](+) were m/z 201.5→95.4 for DEX and m/z 289.2→109.1 for IS. The method was validated in the concentration range of 25 (lower limit of quantification; LLOQ)-5000pg/mL for both maternal and fetal plasma, and of 50 (LLOQ)-5000pg/mL for urine and amniotic fluid, respectively. The intra- and inter-day precision and accuracy were within ±9%. The overall recoveries of DEX were 82.9-87.2%, 85.7-88.4%, 86.2-89.7% and 83.7-88.1% for maternal plasma, urine, fetal plasma and amniotic fluid, respectively. The percentage matrix factors in different biomatrices were less than 120%. Stability studies demonstrated that DEX was stable after three freeze/thaw cycles, in the autosampler tray at 20°C for 24h and during the 3h sample preparation at room temperature. The validated HPLC-MS/MS method has been successfully employed for pharmacokinetic evaluation of DEX in pregnant ewes and fetuses.

CYP3A-dependent drug metabolism is reduced in bacterial inflammation in mice.

BACKGROUND AND PURPOSE: Gene expression of Cyp3a11 is reduced by activation of Toll-like receptors (TLRs) by Gram-negative or Gram-positive bacterial components, LPS or lipoteichoic acid (LTA) respectively. The primary adaptor protein in the TLR signalling pathway, TIRAP, plays differential roles in LPS- and LTA-mediated down-regulations of Cyp3a11 mRNA. Here, we have determined the functional relevance of these findings by pharmacokinetic/pharmacodynamic (PK/PD) analysis of the Cyp3a substrate midazolam in mice. Midazolam is also metabolized by Cyp2c in mice. EXPERIMENTAL APPROACH: Adult male C57BL/6, TIRAP+/+ and TIRAP-/- mice were pretreated with saline, LPS (2 mg·kg⁻¹) or LTA (6 mg·kg⁻¹). Cyp3a11 protein expression, activity and PK/PD studies using midazolam were performed. KEY RESULTS: Cyp3a11 protein expression in LPS- or LTA-treated mice was reduced by 95% and 60% compared with saline-treated mice. Cyp3a11 activity was reduced by 70% in LPS- or LTA-treated mice. Plasma AUC of midazolam was increased two- to threefold in LPS- and LTA-treated mice. Plasma levels of 1'-OHMDZ decreased significantly only in LTA-treated mice. Both LPS and LTA decreased AUC of 1'-OHMDZ-glucuronide. In the PD study, sleep time was increased by ∼2-fold in LPS- and LTA-treated mice. LTA-mediated decrease in Cyp3a11 protein expression and activity was dependent on TIRAP. In PK/PD correlation, AUC of midazolam was increased only in LPS-treated mice compared with saline-treated mice. CONCLUSIONS AND IMPLICATIONS: LPS or LTA altered PK/PD of midazolam. This is the first study to demonstrate mechanistic differences in regulation of metabolite formation of a clinically relevant drug by Gram-negative or Gram-positive bacterial endotoxins.