A sensitive LC-MS/MS method for quantification of phenytoin and its major metabolite with application to in vivo investigations of intravenous and intranasal phenytoin delivery.

Phenytoin is a powerful antiseizure drug with complex pharmacokinetic properties, making it an interesting model drug to use in preclinical in vivo investigations, especially with regards to formulations aiming to improve drug delivery to the brain. Moreover, it has a major metabolite, 5-(4-hydroxyphenyl)-5-phenylhydantoin, which can be simultaneously studied to achieve a better assessment of its behaviour in the body. Here, we describe the development and validation of a sensitive LCMS/MS method for quantification of phenytoin and 5-(4-hydroxyphenyl)-5-phenylhydantoin in rat plasma and brain which can be used in such preclinical studies. Calibration curves produced covered a range of 7.81 to 250 ng/mL (plasma) and 23.4 to 750 ng/g (brain tissue) for both analytes. The method was validated for specificity, sensitivity, accuracy, and precision and found to be within the acceptable limits of ±15% over this range in both tissue types. The method when applied in two in vivo investigations: validation of a seizure model and to study the behaviour of a solution of intranasally administered phenytoin as a foundation for future studies into direct nose-to-brain delivery of phenytoin using specifically developed particulate systems, was highly sensitive for detecting phenytoin and 5-(4-hydroxyphenyl)-5-phenylhydantoin in rat plasma and brain.

A sensitive LC-MS/MS method for the study of exogenously administered 13 C-oleoylethanolamide in rat plasma and brain tissue.

Oleoylethanolamide is an endogenous molecule with neuroprotective effects. It has been reported that exogenous oleoylethanolamide can be administered therapeutically, but the confounding presence of the endogenous molecule has led to conflicting reports regarding the mechanisms of the effects and highlights a need for an adequate methodology to differentiate them. We have developed a liquid chromatography-tandem mass spectrometry method to study oleoylethanolamide in rat plasma and brain using a 13 C-labeled isotope, 13 C-oleoylethanolamide. 13 C-oleoylethanolamide was extracted using a liquid-liquid extraction employing acetonitrile and tert-butyl methyl ether (1:4). Analysis was performed using a gradient with a total run time of 12 min. 13 C-oleoylethanolamide, d4 -oleoylethanolamide (internal standard), and 12 C-oleoylethanolamide (endogenous background) eluted simultaneously at 1.64 min. The method was validated for specificity, sensitivity, accuracy, and precision and found to be capable of quantification within acceptable limits of ±15% over the calibration range of 0.39-25 ng/mL for the plasma and 1.17-75 ng/g for the brain. It was then applied to quantify 13 C-oleoylethanolamide over 90 min after intravenous administration of a solution (1 mg/kg) in rats. Results suggest that 13 C-oleoylethanolamide does not reach therapeutic concentrations in the brain, despite a relatively prolonged plasma circulation, suggesting that rapid degradation in the brain remains an obstacle to its clinical application to neurological disease.

Incorporation of an Endogenous Neuromodulatory Lipid, Oleoylethanolamide, into Cubosomes: Nanostructural Characterization.

Oleoylethanolamide (OEA) is an endogenous lipid with neuroprotective properties and the fortification of its concentration in the brain can be beneficial in the treatment of many neurodegenerative disorders. However, OEA is rapidly eliminated by hydrolysis in vivo, limiting its therapeutic potential. We hypothesize that packing OEA within a nanoparticulate system such as cubosomes, which can be used to target the blood-brain barrier (BBB), will protect it against hydrolysis and enable therapeutic concentrations to reach the brain. Cubosomes are lipid-based nanoparticles with a unique bicontinuous cubic phase internal structure. In the present study, the incorporation and chemical stability of OEA in cubosomes was investigated. Cubosomes containing OEA had a mean particle size of less than 200 nm with low polydispersity (polydispersity index <0.25). Infrared spectroscopy and high-performance liquid chromatography showed chemical stability and the encapsulation of OEA within cubosomes. Cryo-TEM and SAXS measurements were used to probe the influence of the addition of OEA on the internal structure of the cubosomes. Up to 30% w/w OEA (relative to phytantriol) could be incorporated into phytantriol cubosomes without any significant disruption of the nanostructure of the cubosomes. Combined, the results indicate that OEA-loaded cubosomes have the potential for application as a colloidal carrier for OEA, potentially preventing hydrolysis in vivo.

Translational Considerations in the Development of Intranasal Treatments for Epilepsy.

Epilepsy is a common and serious neurological disorder, to which a high proportion of patients continue to be considered "drug-resistant", despite the availability of a host of anti-seizure drugs. Investigation into new treatment strategies is therefore of great importance. One such strategy is the use of the nose to deliver drugs directly to the brain with the help of pharmaceutical formulation to overcome the physical challenges presented by this route. The following review explores intranasal delivery of anti-seizure drugs, covering the link between the nose and seizures, pathways from the nose to the brain, current formulations in clinical use, animal seizure models and their proposed application in studying intranasal treatments, and a critical discussion of relevant pre-clinical studies in the literature.

Comparison of cubosomes and hexosomes for the delivery of phenytoin to the brain.

The ability to formulate cubosomes and hexosomes with a single lipid by changing only the colloidal stabiliser presents a unique opportunity to directly compare the biological performance of these uniquely structured nanoparticles. This was explored here via the encapsulation and brain delivery of a model anti-seizure drug, phenytoin, in selachyl alcohol cubosomes and hexosomes. Nanoparticles were prepared with Pluronic® F127 or Tween 80® as the stabiliser and characterised. The internal nanostructure of nanoparticles shifted from hexosomes when using Pluronic® F127 as the stabiliser to cubosomes when using Tween 80® and was conserved following loading of phenytoin, with high encapsulation efficiencies (>97%) in both particle type. Cytotoxicity towards brain endothelial cells using the hCMEC/D3 line was comparable regardless of stabiliser type. Finally, in vivo brain delivery of phenytoin encapsulated in cubosomes and hexosomes after intravenous administration to rats was studied over a period of 60 min, showing cubosomes to be superior to hexosomes, both in terms of brain concentrations and brain to plasma ratio. While the role of stabiliser and/or internal nanostructure remains to be conclusively determined, this study is the first in vivo comparison of cubosomes and hexosomes for the delivery of a therapeutic drug molecule across the BBB and into the brain.

Deuterated phytantriol - A versatile compound for probing material distribution in liquid crystalline lipid phases using neutron scattering.

Phytantriol is an interfacially-active lipid that is chemically robust, non-digestible and forms particles with internal bicontinuous cubic phase structures (cubosomes) when dispersed with non-ionic surfactants at ambient and physiological temperatures. The liquid crystalline internal structure of phytantriol-based cubosomes can be changed to alter the interfacial contact area/topology with the aqueous dispersant to trigger bioactive payload release or to alter the local membrane curvature around bound or embedded proteins. To enable the study of payload distribution, structure and transformation kinetics within phytantriol particles by neutron scattering techniques it is desirable to have access to a deuterated version of this molecule but to date a synthetic route has not been available. The first successful synthesis of phytantriol-d39 is presented here alongside a preliminary physical characterisation of related particle structures when phytantriol-d39 is dispersed using two non-ionic surfactants, Tween® 80 and Pluronic® F127. Synchrotron small angle X-ray scattering (SAXS) was used to confirm that phytantriol-d39-based nanoparticles in D2O form similar liquid crystalline structures to those of their natural isotopic abundance (phytantriol/H2O) counterparts as a function of temperature. Finally, small angle neutron scattering (SANS) with solvent contrast to match out the phytantriol-d39 structuring was used to show that the spatial correlations between the Tween® and Pluronic® non-ionic surfactant molecules are different within dispersed phytantriol-d39 particles with different liquid crystalline structures in D2O. The surfactant molecules in phytantriol-d39/Tween® 80 particles with Im3m cubic structures were found to follow a self-avoiding walk, whereas in phytantriol-d39/Pluronic® F127 particles with Pn3m cubic structures they were found to follow a more rod-like packing arrangement.

Pharmacy students' use of and beliefs about traditional healthcare.

Health professional students come from many different cultural backgrounds, and may be users of traditional healthcare (also known as ethnomedicine or folk medicine). This study aimed to explore New Zealand pharmacy students' knowledge and beliefs about traditional healthcare, and to examine whether these changed during the course. A questionnaire was administered to students in 2011 and again in 2013. Students were from a wide range of ethnic groups. Their reported use of traditional healthcare increased (from 48% in 2011 to 61% in 2013) and was usually for minor illness or prevention. Non New Zealand European students were more likely to use traditional healthcare. Use of traditional healthcare was relatively common, and after exposure to a biomedical curriculum students seemed to be more, rather than less likely to report using traditional healthcare. Education about traditional healthcare should not be based on the assumption that all healthcare students are unfamiliar with, or non-users of, traditional healthcare.