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In Vivo Active Drug Uptake and Efflux at the Blood-Brain Barrier: With Focus on Drug Transport Interactions
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. (Translational PKPD Group)
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The blood-brain barrier (BBB) controls the movement of substances into and out of the brain. The tight junctions between endothelial cells and energy dependent transporters in the BBB influence rate and extent of drug distribution to the brain.

The aim of this thesis was to study different methodological and pharmacokinetic aspects of drug transport at the BBB by characterizing possible active uptake and drug-drug interactions. Therefore, advanced tools for data acquisition and analysis were applied. The role of BBB transport in early drug development, with particular emphasis on in vitro-in vivo comparisons and species differences, was also investigated.

Microdialysis in rats was used to study the BBB pharmacokinetics of oxymorphone, diphenhydramine (DPHM), oxycodone and morphine. Oxymorphone, DPHM and verapamil were all found to be actively taken up at the BBB, with brain to blood unbound drug ratios of 2, 5 and 2, respectively. The effect profile for oxycodone was successfully described using the modified M3 method for censored observations. In vitro experiments indicated a competitive interaction between DPHM and oxycodone on active uptake transport to the brain. No such interaction was observed in vivo due to much lower unbound concentrations achieved, compared with the in vitro Ki values. Active uptake of morphine at the BBB was not demonstrated even at very low concentrations as it was not possible to separate the active uptake transport process from active efflux by decreasing the morphine concentration. Mice carrying the human P-gp gene (hMDR1) were used to evaluate possible species differences in P-gp function. Differences were evident between the hMDR1 and normal mice in BBB penetration of various P-gp substrates and in the effect of blockers on P-gp function. Quantitative measurements of P-gp expression levels at the BBB and a comparison with human data are crucial for the future use of the hMDR1 model.

In conclusion, this thesis reports active uptake of oxymorphone, DPHM and verapamil at the BBB. In vivo interaction of DPHM and oxycodone at the BBB was found not to be significant at therapeutic drug concentrations. Furthermore species differences were found between human and mouse P-gp function at the BBB.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2012. , 50 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 165
Keyword [en]
Blood-brain barrier, Active uptake transport, Microdialysis, P-glycoprotein, Drug interactions, Species differences, Pharmacokinetics, Pharmacodynamics, NONMEM
National Category
Pharmaceutical Sciences
Research subject
Pharmaceutical Science
Identifiers
URN: urn:nbn:se:uu:diva-180824ISBN: 978-91-554-8472-9 (print)OAI: oai:DiVA.org:uu-180824DiVA: diva2:551315
Public defence
2012-10-26, B:42, BMC, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2012-10-04 Created: 2012-09-10 Last updated: 2013-01-23Bibliographically approved
List of papers
1. Oxymorphone active uptake at the blood-brain barrier and population modeling of its pharmacokinetic-pharmacodynamic relationship
Open this publication in new window or tab >>Oxymorphone active uptake at the blood-brain barrier and population modeling of its pharmacokinetic-pharmacodynamic relationship
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2013 (English)In: Journal of Pharmaceutical Sciences, ISSN 0022-3549, E-ISSN 1520-6017, Vol. 102, no 9, 3320-3331 p.Article in journal (Refereed) Published
Abstract [en]

The aim of this study was to characterize the blood–brain barrier (BBB) transport and pharmacokinetics–pharmacodynamics (PKPD) relationship of oxymorphone and to further elucidate its possible contribution to oxycodone analgesia. The BBB transport of oxymorphone was studied using microdialysis in male Sprague–Dawley rats. Samples from microdialysis blood and brain probes, brain tissue, and plasma were analyzed by liquid chromatography with tandem mass spectrometry. The effect was measured as tail-flick latency. The study consisted of a PKPD experiment with combined microdialysis and antinociceptive measurements (n = 8), and another antinociceptive effect experiment (n = 9) using a 10 times lower dose. The combined data were analyzed with an integrated PKPD model in nonlinear mixed effect modeling utilizing a specific method (M3) for handling missing PD observations. The concentration of unbound oxymorphone was higher in brain than in blood, with a ratio of 1.9 (RSE, 9.7%), indicating active uptake at the BBB. The integrated PKPD model described the oxymorphone BBB transport and PKPD relationship successfully, with an EC50 in the brain of 63 ng/mL, and the M3 method was able to address the issue of censored observations. Oxymorphone has active uptake transport at the BBB in rats, with moderate uptake clearance to the brain. Its contribution to analgesia after oxycodone administration is not significant.

Place, publisher, year, edition, pages
Wiley: , 2013
National Category
Pharmaceutical Sciences
Research subject
Pharmaceutical Science
Identifiers
urn:nbn:se:uu:diva-180822 (URN)10.1002/jps.23492 (DOI)000330240900038 ()
Available from: 2012-09-10 Created: 2012-09-10 Last updated: 2017-12-07Bibliographically approved
2. Diphenhydramine Active Uptake at the Blood-Brain Barrier and Its Interaction with Oxycodone in Vitro and in Vivo
Open this publication in new window or tab >>Diphenhydramine Active Uptake at the Blood-Brain Barrier and Its Interaction with Oxycodone in Vitro and in Vivo
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2011 (English)In: Journal of Pharmaceutical Sciences, ISSN 0022-3549, E-ISSN 1520-6017, Vol. 100, no 9, 3912-3923 p.Article in journal (Refereed) Published
Abstract [en]

Diphenhydramine (DPHM) and oxycodone are weak bases that are able to form cations. Both drugs show active uptake at the blood-brain barrier (BBB). There is thus a possibility for a pharmacokinetic interaction between them by competition for the same uptake transport system. The experiments of the present study were designed to study the transport of DPHM across the BBB and its interaction with oxycodone in vitro and in vivo. In vitro, the interaction between the drugs was studied using conditionally immortalized rat brain capillary endothelial cells (TR-BBB13 cells). The in vivo relevance of the in vitro findings was studied in rats using brain and blood microdialysis. DPHM was actively transported across the BBB in vitro (TR-BBB13 cells). Oxycodone competitively inhibited DPHM uptake with a K(i) value of 106 mu M. DPHM also competitively inhibited oxycodone uptake with a K(i) value of 34.7 mu M. In rats, DPHM showed fivefold higher unbound concentration in brain interstitial fluid (ISF) than in blood, confirming a net active uptake. There was no significant interaction between DPHM and oxycodone in vivo. This accords with the results of the in vitro experiments because the unbound plasma concentrations that could be attained in vivo, without causing adverse effects, were far below the Ki values.

Keyword
blood-brain barrier, drug interactions, organic cation transporters (OCTs), pharmacokinetics, active transport, HPLC (high-performance/pressure liquid chromatography), mass spectrometry
National Category
Medical and Health Sciences Pharmaceutical Sciences
Research subject
Pharmacokinetics and Drug Therapy
Identifiers
urn:nbn:se:uu:diva-159250 (URN)10.1002/jps.22567 (DOI)000294666600030 ()
Available from: 2011-09-26 Created: 2011-09-26 Last updated: 2017-12-08Bibliographically approved
3. Morphine brain pharmacokinetics at very low concentrations studied with Accelerator Mass Spectrometry and Liquid Chromatography-tandem Mass Spectrometry
Open this publication in new window or tab >>Morphine brain pharmacokinetics at very low concentrations studied with Accelerator Mass Spectrometry and Liquid Chromatography-tandem Mass Spectrometry
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2011 (English)In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 39, no 2, 174-179 p.Article in journal (Refereed) Published
Abstract [en]

Morphine has been predicted to show nonlinear blood-brain barrier (BBB) transport at lower concentrations. Present study investigated the possibility of separating active influx of morphine from its efflux by using very low morphine concentrations, and to compare AMS with LC-MS/MS as method for analysing microdialysis samples. A 10-min bolus infusion of morphine, followed by a constant-rate infusion, was given to male rats (n=6) to achieve high (250 ng.ml(-1)), medium (50 ng.ml(-1)) and low (10 ng.ml(-1)) steady-state plasma concentrations (C(ss)). An additional rat received infusions to achieve low (10 ng.ml(-1)), very low (2 ng.ml(-1)) and ultra low (0.4 ng.ml(-1)) concentrations. Unbound morphine concentrations from brain extracellular fluid and blood were sampled with microdialysis and analysed by LC-MS/MS and AMS. The average K(p,uu) for the low and medium steady-state levels were 0.22±0.08 and 0.21±0.05, when measured with AMS (NS; p=0.5). For the medium and high steady-state levels, K(p,uu) values were 0.24±0.05 and 0.26±0.05, measured with LC-MS/MS (NS; p=0.2). For the low, very low and ultra low levels, K(p,uu) values were 0.16±0.01, 0.16±0.02 and 0.18±0.03, respectively, measured with AMS. The medium-concentration K(p,uu) values were, on average, 16% lower with AMS than with LC-MS/MS. There were no significant changes in K(p,uu) over a 625-fold concentration range (0.4-250 ng.ml(-1)). It was not possible to separate active uptake transport from active efflux using these low concentrations. The two analytical methods provided indistinguishable results for blood plasma concentrations, but differed by up to 38% for microdialysis samples; however, this did not affect our conclusions.

National Category
Pharmaceutical Sciences
Research subject
Pharmacokinetics and Drug Therapy
Identifiers
urn:nbn:se:uu:diva-141822 (URN)10.1124/dmd.110.036434 (DOI)000286317900004 ()21059857 (PubMedID)
Available from: 2011-01-12 Created: 2011-01-12 Last updated: 2017-12-11Bibliographically approved
4. Comparison of  blood-brain barrier transport of P-glycoprotein substrates in P-glycoprotein humanized, knock-out and wild type mice
Open this publication in new window or tab >>Comparison of  blood-brain barrier transport of P-glycoprotein substrates in P-glycoprotein humanized, knock-out and wild type mice
(English)Manuscript (preprint) (Other academic)
National Category
Medical and Health Sciences
Research subject
Pharmaceutical Science
Identifiers
urn:nbn:se:uu:diva-180823 (URN)
Available from: 2012-09-10 Created: 2012-09-10 Last updated: 2013-01-23

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