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Developmental Aspects of Drug Transport Across the Blood-Brain Barrier
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. (Margareta Hammarlund-Udenaes)
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The developmental aspect of drug transport across the blood-brain barrier (BBB) was investigated. Microdialysis was used to study unbound morphine BBB transport at different ages in sheep. An in vitro study was performed to find differentially expressed genes in brain capillary-rich fractions of the brain in rats of different ages. Microdialysis and brain-to-plasma ratios were used to study the contribution of breast cancer resistance protein (Bcrp) to the transport of nitrofurantoin (NTF) across the BBB of rats during development as well as in adult rats and mice.

A method of analysing morphine and its metabolites in plasma and microdialysis samples was developed and validated. The in vivo recovery of deuterated morphine, used as a calibrator in microdialysis experiments, was not affected by the presence of morphine in the tissue. A net influx of morphine was observed in premature lambs and adult sheep, in contrast to the efflux seen in other species. This influx decreased with age, indicating that the morphine transport across the BBB changes with age. In contrast, the transport of the morphine metabolite morphine-3-glucuronide (M3G) did not change with age. Microarray data indicated that several active transporters are differentially expressed with age. Moreover, the mRNA expression levels of Abcg2 (Bcrp) and Slc22a8 (organic anion transporter 3) changed with age when quantified using real-time polymerase chain reaction. In contrast, the expression of Abcb1 (P-glycoprotein) and occludin (a tight junction protein) did not change with age. In rats, the brain distribution of NTF decreased with age due to increased protein binding in plasma. The concentration ratio of unbound NTF across the BBB was low in the adult rat, due to intra-brain metabolism and/or efflux by other transporters. Bcrp did not appear to have a significant contribution in the developing rat or in knock-out mice compared to wild-type controls with regard to NTF BBB transport.

In conclusion, in vitro studies showed that the expression levels of some genes changed with age, presumably affecting subsequent drug distribution to the brain. Further, in vivo studies showed that distribution across the BBB changed with age for morphine but not for M3G or NTF.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2009. , p. 60
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 110
Keywords [en]
Blood-brain barrier, development, active transport, tight junction proteins, microdialysis, recovery, morphine, nitrofurantoin, Bcrp, microarray, real-time PCR, in vitro, in vivo, LC-MS/MS
National Category
Pharmaceutical Sciences
Research subject
Pharmacokinetics and Drug Therapy
Identifiers
URN: urn:nbn:se:uu:diva-108374ISBN: 978-91-554-7627-4 (print)OAI: oai:DiVA.org:uu-108374DiVA, id: diva2:272147
Public defence
2009-11-26, B42, Biomedicinskt Centrum, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2009-11-04 Created: 2009-09-17 Last updated: 2018-01-12Bibliographically approved
List of papers
1. On-line desalting and determination of morphine, morphine-3-glucuronide and morphine-6-glucuronide in microdialysis and plasma samples using column switching and liquid chromatography/tandem mass spectrometry
Open this publication in new window or tab >>On-line desalting and determination of morphine, morphine-3-glucuronide and morphine-6-glucuronide in microdialysis and plasma samples using column switching and liquid chromatography/tandem mass spectrometry
2005 (English)In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 19, no 15, p. 2116-2122Article in journal (Refereed) Published
Abstract [en]

A sensitive and reproducible method for the determination of morphine and the metabolites morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) was developed. The method was validated for perfusion fluid used in microdialysis as well as for sheep and human plasma. A C18 guard column was used to desalt the samples before analytical separation on a ZIC HILIC (hydrophilic interaction chromatography) column and detection with tandem mass spectrometry (MS/MS). The mobile phases were 0.05% trifluoroacetic acid (TFA) for desalting and acetonitrile/5 mM ammonium acetate (70:30) for separation. Microdialysis samples (5 microL) were directly injected onto the system. The lower limits of quantification (LLOQ) for morphine, M3G and M6G were 0.50, 0.22 and 0.55 ng/mL, respectively, and the method was linear from LLOQ to 200 ng/mL. For plasma, a volume of 100 microL was precipitated with acetonitrile containing internal standards (deuterated morphine and metabolites). The supernatant was evaporated and reconstituted in 0.05% TFA before the desalting process. The LLOQs for sheep plasma were 2.0 and 3.1 ng/mL and the ranges were 2.0-2000 and 3.1-3100 ng/mL for morphine and M3G, respectively. For human plasma, the LLOQs were 0.78, 1.49 and 0.53 ng/mL and the ranges were 0.78-500, 1.49-1000 and 0.53-500 ng/mL for morphine, M3G and M6G, respectively.

Place, publisher, year, edition, pages
John Wiley & Sons, 2005
National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-103231 (URN)10.1002/rcm.2035 (DOI)15988726 (PubMedID)
Available from: 2009-05-15 Created: 2009-05-15 Last updated: 2018-01-13Bibliographically approved
2. The use of a deuterated calibrator for in vivo recovery estimations in microdialysis studies
Open this publication in new window or tab >>The use of a deuterated calibrator for in vivo recovery estimations in microdialysis studies
2008 (English)In: Journal of Pharmaceutical Sciences, ISSN 0022-3549, E-ISSN 1520-6017, Vol. 97, no 8, p. 3433-3441Article in journal (Refereed) Published
Abstract [en]

One of the crucial issues in quantitative microdialysis is the reliability of recovery estimates to correctly estimate unbound drug tissue concentrations. If a deuterated calibrator is used for retrodialysis, the calibrator has the same properties as the study drug. However, recovery of the calibrator may be affected by the presence of the drug in the tissues. The aim of this study was to investigate the recovery of deuterated morphine with time in the absence and presence of morphine in rat tissues. Microdialysis probes were placed in the brain and blood of eight rats. Ringer's solution containing D3-morphine was perfused throughout the study and recovery was estimated. After a stabilization period of 3 h, an exponential infusion of morphine was administered over 4 h. The presence of morphine did not affect the recovery of D3-morphine from brain or blood. The average recovery values (SD) were 0.145 (0.039) and 0.131 (0.048) during the stabilization and infusion periods, respectively, for the brain probe and 0.792 (0.055) and 0.790 (0.084), respectively, for the blood probe. The recovery of deuterated morphine was stable over time in the brain and in blood, and was not affected by the presence of pharmacologically concentrations of morphine.

Keywords
microdialysis, active transport, blood-brain barrier, efflux pumps, ABC transporters
National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-13336 (URN)10.1002/jps.21217 (DOI)000258081100042 ()17990306 (PubMedID)
Available from: 2008-01-22 Created: 2008-01-22 Last updated: 2018-01-12Bibliographically approved
3. The influence of age on the distribution of morphine and morphine-3-glucuronide across the blood-brain barrier in sheep
Open this publication in new window or tab >>The influence of age on the distribution of morphine and morphine-3-glucuronide across the blood-brain barrier in sheep
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2009 (English)In: British Journal of Pharmacology, ISSN 0007-1188, E-ISSN 1476-5381, Vol. 157, no 6, p. 1085-1096Article in journal (Refereed) Published
Abstract [en]

Background and purpose

 The effect of age on the distribution of morphine and morphine-3-glucuronide (M3G) across the blood-brain barrier (BBB) was studied in a sheep model utilizing intracerebral microdialysis. The effect of neonatal asphyxia on brain drug distribution was also studied.

Experimental approach

 Microdialysis probes were inserted into the cortex, striatum and blood of 11 lambs (127 gestation days) and six ewes. Morphine, 1 mg.kg(-1), was intravenously administered as a 10 min constant infusion. Microdialysis and blood samples were collected for up to 360 min and analysed using liquid chromatography-tandem mass spectrometry. The half-life, clearance, volume of distribution, unbound drug brain : blood distribution ratio (K(p,uu)) and unbound drug volume of distribution in brain (V(u,brain)) were estimated.

 Key results

 Morphine K(p,uu) was 1.19 and 1.89 for the sheep and premature lambs, respectively, indicating that active influx into the brain decreases with age. Induced asphyxia did not affect transport of morphine or M3G across the BBB. Morphine V(u,brain) measurements were higher in sheep than in premature lambs. The M3G K(p,uu) values were 0.27 and 0.17 in sheep and premature lambs, indicating a net efflux from the brain in both groups.

Conclusions and implications

 The morphine K(p,uu) was above unity, indicating active transport into the brain; influx was significantly higher in premature lambs than in adult sheep. These results in sheep differ from those in humans, rats, mice and pigs where a net efflux of morphine from the brain is observed.

Keywords
microdialysis, morphine, active efflux, active influx, blood-brain barrier
National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-103204 (URN)10.1111/j.1476-5381.2009.00242.x (DOI)000268260800023 ()19438510 (PubMedID)
Available from: 2009-05-15 Created: 2009-05-15 Last updated: 2018-01-13Bibliographically approved
4. Differences in gene expression in the developing rat brain using cDNA microarray and real-time PCR
Open this publication in new window or tab >>Differences in gene expression in the developing rat brain using cDNA microarray and real-time PCR
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

The blood-brain barrier (BBB) is formed by endothelial cells, connected by tight junction proteins restricting paracellular transport. Transport of substances into the brain may also be limited due to active efflux transporters. Not much is known about the development of tight junction proteins and active transporters in the BBB, and how this affects drug distribution to the brain at different ages. The aim of this study was to analyze possible differences in expression of tight junction proteins and active transporters in the BBB at different postnatal ages in the rat. Brain capillary-rich fractions (BCRF) were collected and gene expression levels were compared at three postnatal ages using microarray analysis. BCRF was also analyzed for the mRNA expression levels of P-gp Abcb1 (P-gp), Abcg2 (Bcrp), Slc22a8 (Oat3) and occludin using real-time PCR at 12 different postnatal ages between postnatal Day 1 and Adult. In the array analysis, 28 genes of interest were found to be differentially expressed. The mRNA levels of Abcg2 decreased to one seventh from postnatal Day 1 to Day 15. The levels of Slc22a8 increased from birth and reached a plateau at Day 3 - 12 followed by a decrease to Day 15. These findings show that active transporters are differentially expressed in the developing BBB, possibly affecting drug distribution to the brain. No change in the mRNA levels for Abcb1 or occludin was observed.

Keywords
Blood-brain barrier, development, active transport, tight junctions, microarray, real-time PCR
National Category
Pharmaceutical Sciences
Research subject
Pharmacokinetics and Drug Therapy
Identifiers
urn:nbn:se:uu:diva-109367 (URN)
Available from: 2009-10-14 Created: 2009-10-14 Last updated: 2018-01-12
5. Bcrp does not influence transport of nitrofurantoin across the blood-brain barrier at different ages
Open this publication in new window or tab >>Bcrp does not influence transport of nitrofurantoin across the blood-brain barrier at different ages
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

In the blood-brain barrier (BBB), tight junction proteins together with active efflux transporters efficiently restrict access of many compounds to the brain. The contribution of breast cancer resistance protein (Bcrp, coded by Abcg2) for drug efflux in the BBB is not clear. The aim of this study was to investigate the contribution of Bcrp in the rat BBB and how development affects distribution of a Bcrp substrate with age. Nitrofurantoin (NTF) is a good substrate for Bcrp and was used as model substance. Brain-to-plasma concentration ratio (Kp) of NTF was measured at postnatal Day 1, Day 4, Day 11 and in adult rats. Microdialysis was used to measure concentration ratio of unbound NTF across the BBB (Kp,uu) with or without blockers for active transport (PSC833 and probenecid). To investigate the in vivo contribution of Bcrp, Kp was also measured in Bcrp-/- and wild-type control mice with or without the selective Bcrp blocker Ko143. The Kp decreased with age, but due to an increase in the protein binding. The Kp,uu was on average 0.047 and not affected by the presence of any blocker. Possible explanations for the low Kp,uu is intra-brain metabolism and/or efflux due to other transporter(s). No difference was observed in the Kp of NTF for Bcrp-/- compared to wild-type mice, independent of co-administration with Ko143. Thus, no in vivo contribution of Bcrp to the BBB brain transport of NTF was detected.

Keywords
Blood-brain barrier, development, active transport, Bcrp, nitrofurantoin, intra-brain metabolism, LC-MS/MS
National Category
Pharmaceutical Sciences
Research subject
Pharmacokinetics and Drug Therapy
Identifiers
urn:nbn:se:uu:diva-109160 (URN)
Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2018-01-13

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