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Quantitative analysis of the opioid peptide DAMGO in rat plasma and microdialysis samples using liquid chromatography-tandem mass spectrometry
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. (Translational PKPD)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. (Translational PKPD)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. (Translational PKPD)
2012 (English)In: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376X, Vol. 900, 11-17 p.Article in journal (Refereed) Published
Abstract [en]

A liquid chromatography–electrospray ionization-tandem mass spectrometry (LC–ESI-MS/MS) method for the quantification of the opioid peptide DAMGO in rat plasma, as well as DAMGO and the microdialysis recovery calibrator [13C2,15N]-DAMGO in microdialysis samples, is described. The microdialysis samples consisted of 15 μL Ringer solution containing 0.5% bovine serum albumin. Pretreatment of the samples involved protein precipitation with acetonitrile followed by dilution with 0.01% formic acid. The lower limits of quantification were 0.52 ng/mL and 0.24 ng/mL for DAMGO and [13C2,15N]-DAMGO respectively and the response was linear up to 5000 fold higher concentrations. The plasma samples (50 μL) were precipitated with acetonitrile containing the isotope labeled analog [13C2,15N]-DAMGO as internal standard. The method was linear in the range of 11–110,000 ng/mL. The separations were conducted on a HyPurity C18 column, 50 × 4.6 mm, 3 μm particle size, with a mobile phase consisting of acetonitrile, water and formic acid to the proportions of 17.5:82.5:0.01. Low energy collision dissociation tandem mass spectrometric (CID-MS/MS) analysis was carried out in the positive ion mode using multiple reaction monitoring (MRM) of the following mass transitions: m/z 514.2 → 453.2 for DAMGO and m/z 517.2 → 456.2 for [13C2,15N]-DAMGO. The intra-day precision and accuracy did not exceed 5.2% and 93–104% for both compounds and sample types described. The inter-day precision an accuracy were <6.8% and 95–105% respectively. The method described is simple, reproducible and suitable for the analysis of small sample volumes at low concentrations.

Place, publisher, year, edition, pages
2012. Vol. 900, 11-17 p.
Keyword [en]
Opioid peptide, DAMGO, LC-MS/MS, Microdialysis
National Category
Pharmaceutical Sciences
Research subject
Pharmacokinetics and Drug Therapy
Identifiers
URN: urn:nbn:se:uu:diva-181430DOI: 10.1016/j.jchromb.2012.05.014ISI: 000306881000002OAI: oai:DiVA.org:uu-181430DiVA: diva2:557346
Available from: 2012-09-27 Created: 2012-09-24 Last updated: 2017-12-07Bibliographically approved
In thesis
1. Quantitative Aspects of Nanodelivery Across the Blood-Brain Barrier: Exemplified with the Opioid Peptide DAMGO
Open this publication in new window or tab >>Quantitative Aspects of Nanodelivery Across the Blood-Brain Barrier: Exemplified with the Opioid Peptide DAMGO
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The use of nanocarriers is an intriguing approach in the development of efficacious treatment for brain disorders. The aim of the conducted research was to evaluate and quantify the impact of a liposomal nanocarrier formulation on the brain drug delivery. A novel approach for investigating the blood-brain barrier transport of liposomal DAMGO is presented, including in vivo microdialysis in rat, a high quality LC-MS/MS bioanalytical method and pharmacokinetic model analysis of the data. Factors limiting the brain distribution of the free peptide DAMGO were also investigated. Microdialysis, in combination with plasma sampling, made it possible to separate the released drug from the encapsulated and to quantify the active substance in both blood and brain interstitial fluid over time.

The opioid peptide DAMGO entered the brain to a limited extent, with a clearance out of the brain 13 times higher than the clearance into the brain. The brain to blood ratio of unbound drug was not affected when the efflux transporter inhibitors cyclosporine A and elacridar were co-administered with DAMGO. Nor was the transport affected in the in vitro Caco-2 assay using the same inhibitors. This indicates that DAMGO is not transported by P-glycoprotein (Pgp) or breast cancer resistant protein (Bcrp). The blood-brain barrier transport was significantly increased for DAMGO when formulated in liposomes, resulting in 2-3 fold higher brain to blood ratio of unbound DAMGO. The increased brain delivery was seen both for glutathione tagged PEGylated liposomes, as well as for PEGyalted liposomes without specific brain targeting. The improvement in brain delivery was observed only when DAMGO was encapsulated into the liposomes, thus excluding any effect of the liposomes themselves on the integrity of the blood-brain barrier. Modeling of the data provided additional mechanistic understanding of the brain uptake, showing that endocytosis or transcytosis of intact liposomes across the endothelial cell membranes were unlikely. A model describing fusion of the liposomes with the luminal membrane described the experimental data the best.

In conclusion, the studies presented in this thesis all contribute to an increased understanding of how to evaluate and improve brain delivery of CNS active drugs and contribute with important insights to the nanocarrier field.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. 70 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 208
Keyword
blood-brain barrier, liposomes, nanocarriers, brain delivery, pharmacokinetics, modeling and simulation, microdialysis, opioid peptide, DAMGO, LC-MS/MS
National Category
Pharmaceutical Sciences
Research subject
Pharmaceutical Science
Identifiers
urn:nbn:se:uu:diva-267599 (URN)978-91-554-9428-5 (ISBN)
Public defence
2016-01-15, B42, Biomedicinskt centrum (BMC), Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2015-12-21 Created: 2015-11-24 Last updated: 2016-01-13

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Lindqvist, AnnikaJansson, BrittHammarlund-Udenaes, Margareta

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