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- Bäckström, Gunilla, et al.
(författare)
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Genetic variation in the ATP-binding cassette transporter gene ABCG2(BCRP) in a Swedish population
- 2003
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Ingår i: European Journal of Pharmaceutical Sciences. - 0928-0987 .- 1879-0720. ; 18:5, s. 359-364
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Tidskriftsartikel (refereegranskat)abstract
- The ATP-binding cassette transporter ABCG2 (also named breast cancer resistance protein, BCRP) functions as a drug efflux transporter and is expressed at high levels in the human small intestine. The aim of this study was to screen the human ABCG2 gene for genetic variation. The regions of the gene most likely to affect function, namely the coding parts, exon/intron boundaries, 5' untranslated region and 3' untranslated region and the proposed promoter region, were included in the screening. DNA was obtained from 60 Swedish individuals. The screening was performed using a polymerase chain reaction-denaturing high-performance liquid chromatography approach followed by sequence analysis. Eight sites of genetic variation were identified. The sequence variations considered to be most likely to affect transcription level or transport function were a CTCA deletion in the 5' flanking region, a single nucleotide polymorphism (SNP) in a 5' flanking CpG island, two non-synonymous SNPs, changing valine at amino acid position 12 to methionine and glutamine at position 141 to lysine, respectively. Genotyping of these sequence variations revealed linkage between the CTCA deletion and the SNP changing glutamine 141 for lysine. This information forms the basis for future association studies to investigate the genetic basis of differences of drug disposition due to sequence variation in the ABCG2 gene.
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| 4. |
- Haglund, Sofie, 1975-, et al.
(författare)
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IMPDH activity in thiopurine-treated patients with inflammatory bowel disease - Relation to TPMT activity and metabolite concentrations
- 2008
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Ingår i: British Journal of Clinical Pharmacology. - : Wiley. - 0306-5251 .- 1365-2125. ; 65:1, s. 69-77
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Tidskriftsartikel (refereegranskat)abstract
- AIMS: Azathioprine and 6-mercaptopurine are steroid-sparing drugs used in inflammatory bowel disease (IBD). The polymorphic enzyme thiopurine S-methyltransferase (TPMT) is of importance for thiopurine metabolism and occurrence of adverse events. The role of other thiopurine-metabolizing enzymes is less well known. This study investigated the role of inosine-5′- monophosphate dehydrogenase (IMPDH), which is a key enzyme in the de novo synthesis of guanine nucleotides and also strategically positioned in the metabolic pathway of thiopurines. METHODS: IMPDH was measured in 100 healthy blood donors. IMPDH, TPMT and metabolite concentrations were studied in 50 patients with IBD on stable thiopurine therapy. IMPDH activity was measured in peripheral blood mononuclear cells. TPMT activity, 6-methylthioinosine 5′-monophosphate (meTIMP) and 6-thioguanine nucleotide (6-TGN) concentrations were measured in red blod cells, which is the current practice in clinical monitoring of thiopurines. Enzyme activities were related to metabolite concentrations and clinical characteristics. RESULTS: A wide range of IMPDH activity was observed both in healthy blood donors (median 13.1, range 4.7-24.2 nmol mg-1 protein h-1) and IBD patients (median 14.0, range 7.0-21.7). There was a negative correlation between IMPDH activity and dose-normalized meTIMP concentrations (rs = -0.31, P = 0.03), but no evident correlation to 6-TGN concentration or the meTIMP/6-TGN ratio. There were no significant correlations between TPMT activity and metabolite concentrations. CONCLUSION: Even though the meTIMP concentrations correlated inversely to the IMPDH activity, the role of IMPDH in balancing the formation of methylated and phosphorylated metabolites was not evident. Taken together, the results give cause to question established opinions about thiopurine metabolism. © 2007 The Authors.
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| 5. |
- Lindqvist Appell, Malin, 1976-, et al.
(författare)
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Identification of two novel sequence variants affecting thiopurine methyltransferase enzyme activity
- 2004
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Ingår i: Pharmacogenetics. - : Ovid Technologies (Wolters Kluwer Health). - 0960-314X .- 1473-561X. ; 14:4, s. 261-265
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Tidskriftsartikel (refereegranskat)abstract
- The polymorphic enzyme thiopurine methyltransferase (TPMT) is involved in the methylation of thiopurines. On comparing the phenotype with the genotype in Swedish patients with inflammatory bowel disease and healthy individuals, we found two discordant cases with low TPMT enzyme activity (0.3 and 0.4 U/ml packed red blood cells (pRBC). Genotyping by pyrosequencing revealed that they carried the nucleotide substitutions 460G>A and 719A>G, giving two possible genotypes (TPMT*1/*3A or TPMT*3B/ *3C). DNA sequencing of exon III to X was performed in the patients and their parents. We identified an A>G transition in the start codon (exon III, 1A>G, Met>Val, TPMT*14) in one of the patients and her father (6.3 U/ml pRBC). The mother in this family carried the 460G>A and 719A>G nucleotide substitutions (TPMT*3A, 5.0 U/ml pRBC). In the second family, sequencing revealed a G>A transition in the acceptor splice site in intron VII/exon VIII (IVS7 - 1G>A, TPMT*15) in the patient and his mother (6.9 U/ml pRBC). His father was genotyped as TPMT*1/*3A (6.0 U/ml pRBC). Hence, we report the identification of two novel sequence variants, present in highly conserved nucleotide positions of the human TPMT gene, resulting in a loss of enzyme activity.
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| 6. |
- Taipalensuu, Jan, et al.
(författare)
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Correlation of gene expression of ten drug efflux proteins of the ATP-binding cassette transporter family in normal human jejunum and inhuman intestinal epithelial Caco-2 cell monolayers
- 2001
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Ingår i: Journal of Pharmacology and Experimental Therapeutics. - 0022-3565 .- 1521-0103. ; 299:1, s. 164-170
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Tidskriftsartikel (refereegranskat)abstract
- This investigation describes the expression and interindividual variability in transcript levels of multiple drug efflux systems in the human jejunum and compares the expression profiles in these cells with that of the commonly used Caco-2 cell drug absorption model. Transcript levels of ten-drug efflux proteins of the ATP-binding cassette (ABC) transporter family [MDR1, MDR3, ABCB5, MRP1-6, and breast cancer resistance protein (BCRP)], lung resistance-related protein (LRP), and CYP3A4 were determined using quantitative polymerase chain reaction in jejunal biopsies from 13 healthy human subjects and in Caco-2 cells. All genes except ABCB5 were expressed, and transcript levels varied between individuals only by a factor of 2 to 3. Surprisingly, BCRP and MRP2 transcripts were more abundant in jejunum than MDR1 transcripts. Jejunal transcript levels of the different ABC transporters spanned a range of three log units with the rank order: BCRP approximately MRP2 > MDR1 approximately MRP3 approximately MRP6 approximately MRP5 approximately MRP1 > MRP4 > MDR3. Furthermore, transcript levels of 9 of 10 ABC transporters correlated well between jejunum and Caco-2 cells (r2 = 0.90). However, BCRP exhibited a 100-fold lower transcript level in Caco-2 cells compared with jejunum. Thus, the expression of a number of efflux protein transcripts in jejunum are equal to, or even higher than, that of MDR1, suggesting that the roles of these proteins (in particular BCRP and MRP2) in intestinal drug efflux have been underestimated. Also, we tentatively conclude that the Caco-2 cell line is a useful model of jejunal drug efflux, if the low expression of BCRP is taken into account.
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| 7. |
- Tavelin, Staffan, et al.
(författare)
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An improved cell culture model based on 2/4/A1 cell monolayers for studies of intestinal drug transport : characterization of transport routes
- 2003
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Ingår i: Pharmaceutical research. - 0724-8741 .- 1573-904X. ; 20:3, s. 373-381
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Tidskriftsartikel (refereegranskat)abstract
- Purpose. To improve the viability of the 2/4/A1 cell culture model and to investigate different routes of drug transport in this cell line. Methods. Two approaches were taken to decrease apoptosis. First, rat intestinal 2/4/A1 cells were transfected to overexpress the antiapoptotic protein Bcl-2. Second, normal 2/4/A1 cells were cultivated under conditions that stimulate differentiation and limit apoptosis. The monolayer integrity was investigated by transepithelial electrical resistance, permeability, and microscopy. The expression of drug transporters was investigated by RT-PCR, and transport function was assessed using specific markers. Results. Normal 2/4/A1 cells died by apoptosis at 39°C. Bcl-2-expressing 2/4/A1 cells were viable but adopted a morphology of less-differentiated epithelial cells. Optimization of the culture conditions for 2/4/A1 cells inhibited cell death. The integrity was comparable to that of the human jejunum (50 Ω × cm2), making this approach preferable to Bcl-2 overexpression. Transcriptional analysis showed that some (e.g., MDR1), but not all (e.g., PepT1), transporters were found in 2/4/A1 cells. Studies using substrates for PepT1, P-gp, MRP2, and BCRP showed that none of the transporters were functional in 2/4/A1. Conclusions. The improved culture procedure will facilitate the use of 2/4/A1 cells. 2/4/A1 lack several transporters, which makes them a promising alternative to Caco-2 cells and artificial membranes in studies of passive drug transport.
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