| 1. |
- Aomori, Tohru, et al.
(författare)
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Rapid Single-Nucleotide Polymorphism Detection of Cytochrome P450 (CYP2C9) and Vitamin K Epoxide Reductase (VKORC1) Genes for the Warfarin Dose Adjustment by the SMart-Amplification Process Version 2
- 2009
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Ingår i: Clinical Chemistry. - : Oxford University Press (OUP). - 0009-9147 .- 1530-8561. ; 55:4, s. 804-812
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Polymorphisms of the CYP2C9 (cytochrome P450, family 2, subfamily C, polypeptide 9) gene (CYP2C9*2, CYP2C9*3) and the VKORC1 (vitamin K epoxide reductase complex, subunit 1) gene (-1639G>A) greatly impact the maintenance dose for the drug warfarin. Prescreening patients for their genotypes before prescribing the drug facilitates a faster individualized determination of the proper maintenance dose, minimizing the risk for adverse reaction and reoccurrence of thromboembolic episodes. With current methodologies, therapy can be delayed by several hours to 1 day if genotyping is to determine the loading dose. A simpler and more rapid genotyping method is required. METHODS: We developed a single-nucleotide polymorphism (SNP)-detection assay based on the SMart Amplification Process version 2 (SMAP 2) to analyze CYP2C9*2, CYP2C9*3, and VKORC1 -1639G>A polymorphisms. Blood from consenting participants was used directly in a closed-tube real-time assay without DNA purification to obtain results within 1 h of blood collection. RESULTS: We analyzed 125 blood samples by both SMAP 2 and PCR-RFLP methods. The results showed perfect concordance. CONCLUSIONS: The results validate the accuracy of the SMAP 2 for determination of SNPs critical to personalized warfarin therapy. SMAP 2 offers speed, simplicity of sample preparation, the convenience of isothermal amplification, and assay-design flexibility, which are significant advantages over conventional genotyping technologies. In this example and other clinical scenarios in which genetic testing is required for immediate and better-informed therapeutic decisions, SMAP 2-based diagnostics have key advantages.
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| 2. |
- Englund, Gunilla, et al.
(författare)
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Cytochrome P450 Inhibitory Properties of Common Efflux Transporter Inhibitors
- 2014
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Ingår i: Drug Metabolism And Disposition. - : American Society for Pharmacology & Experimental Therapeutics (ASPET). - 0090-9556 .- 1521-009X. ; 42:3, s. 441-447
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Tidskriftsartikel (refereegranskat)abstract
- Drug transporter inhibitors are important tools to elucidate the contribution of transporters to drug disposition both in vitro and in vivo. These inhibitors are often unselective and affect several transporters as well as drug metabolizing enzymes, which can make experimental results difficult to interpret with confidence. We therefore tested 14 commonly used P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug-resistance associated protein (MRP) inhibitors as inhibitors of cytochrome P450 (P450) enzyme activities using recombinant enzymes. A subset of P-gp and/or CYP3A inhibitors were selected (cyclosporin A, elacridar, ketoconazole, quinidine, reserpine, and tacrolimus) for a comparison of P450 inhibition in human microsomes and hepatocytes. Most P-gp inhibitors showed CYP3A4 inhibition, with potencies often in a similar range as their P-gp inhibition, as well as less potent CYP2C19 inhibition. Other P450 enzymes were not strongly inhibited except a few cases of CYP2D6 inhibition. MRP and BCRP inhibitors showed limited P450 inhibition. Some inhibitors showed less P450 inhibition in human hepatocytes than human liver microsomes, for example, elacridar, probably due to differences in binding, permeability limitations, or active, P-gp mediated efflux of the inhibitor from the hepatocytes. Quinidine was a potent P450 inhibitor in hepatocytes but only showed weak inhibition in microsomes. Quinidine shows an extensive cellular uptake, which may potentiate intracellular P450 inhibition. Elacridar, described as a potent and selective P-gp inhibitor, displayed modest P450 inhibition in this study and is thus a useful model inhibitor to define the role of P-gp in drug disposition without interference with other processes.
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| 3. |
- Lundquist, Patrik, et al.
(författare)
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Functional ATP-Binding Cassette Drug Efflux Transporters in Isolated Human and Rat Hepatocytes Significantly Affect Assessment of Drug Disposition
- 2014
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Ingår i: Drug Metabolism And Disposition. - : American Society for Pharmacology & Experimental Therapeutics (ASPET). - 0090-9556 .- 1521-009X. ; 42:3, s. 448-458
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Tidskriftsartikel (refereegranskat)abstract
- Freshly isolated hepatocytes are considered the gold standard for in vitro studies of hepatic drug disposition. To ensure a reliable supply of cells, cryopreserved human hepatocytes are often used. ABC-superfamily drug efflux transporters are key elements in hepatic drug disposition. These transporters are often considered lost after isolation of hepatocytes. In the present study, the expression and activity of ABC transporters BCRP, BSEP, P-gp, MRP2, MRP3, and MRP4 in human and rat cryopreserved hepatocytes were investigated. In commercially available human cryopreserved hepatocytes, all drug efflux transporters except human BCRP (hBCRP) exhibited similar expression levels as in fresh liver biopsies. Expression levels of hBCRP were 60% lower in cryopreserved human hepatocytes than in liver tissue, which could lead to, at most, a 2.5-fold reduction in hBCRP-mediated efflux. Fresh rat hepatocytes showed significantly lower levels of rat BCRP compared with liver expression levels; expression levels of other ABC transporters were unchanged. ABC transporters in human cryopreserved cells were localized to the plasma membrane. Functional studies could demonstrate P-gp and BCRP activity in both human cryopreserved and fresh rat hepatocytes. Inhibiting P-gp-mediated efflux by elacridar in in vitro experiments significantly decreased fexofenadine efflux from hepatocytes, resulting in an increase in apparent fexofenadine uptake. The results from the present study clearly indicate that ABC transporter-mediated efflux in freshly isolated as well as cryopreserved rat and human hepatocytes should be taken into account in in vitro experiments used for modeling of drug metabolism and disposition.
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| 4. |
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| 5. |
- Skogastierna, Cristine
(författare)
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Pleiotropic effects of HMG-CoA reductase inhibitors : studies in vitro and in vivo
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cardiovascular disease (CVD) is the prime cause of death in industrialized countries, resulting from the progression of atherosclerotic lesions. An increased level of plasma cholesterol, particularly within low-density lipoproteins, is a principle risk factor, but also elevated triglycerides and low levels of high-density lipoprotein cholesterol. Recognition of dyslipidemia as a risk factor for CVD has led to an international consensus that anti-hyperlipidemic drug therapy is essential for the primary and secondary prevention in risk patients. HMG-CoA reductase inhibitors, i.e. statins, are the most common drugs used for this purpose, inhibiting the rate-limiting step in cholesterol biosynthesis. The medical significance of HMG-CoA reductase inhibitors have in recent years been expanded to include cholesterol-independent or "pleiotropic" vascular effects, including improvement of vascular endothelial function, inhibition of vascular smooth muscle cell proliferation and migration, anti-inflammatory actions, anti-oxidative effects or stabilization of vulnerable plaques. These effects have potential in the treatment of coronary artery disease in various settings, such as prevention of its onset as well as its progression. The major aim of this thesis was to contribute to the general knowledge about the pleiotropic effects of statins, particularly in relation to the vascular endothelium and to carcinogenesis. We found that fluvastatin at clinically relevant doses up-regulates transcription and translation of prostacyclin synthase and endothelial nitric oxide synthase in human vascular endothelial cells, associated with an increase in cellular production of prostacyclin (PGI2) and nitric oxide (NO), and induces rapid dilatation in isolated human arteries via contribution of endothelium derived factors like NO and PGI2. These results suggest beneficial effects of fluvastatin on endothelial maintenance in vivo and a protective role on the cardiovascular system, particularly at the level of vascular endothelium. We also show that simvastatin induces a potentially negative alteration of vascular endothelial cells both in vivo and in vitro. Exposure of clinically comparable concentrations of simvastatin led to decreased production and release of PGI2 in endothelial cells and to significantly increased urinary thromboxane A2 (TXA2) levels in healthy volunteers, possibly resulting in a shift of the balance between PGI2 and TXA2, favoring the thromboxane pathway. Fluvastatin and simvastatin were also shown to have different impact on vascular endothelial oxidative stress status, both in cultivated endothelial cells and in healthy volunteers. Compared to simvastatin, fluvastatin seems beneficial both regarding expression of anti-oxidative stress enzymes and reactive oxygen species activity in vitro and anti-oxidative capacity in vivo. The suggested anti-carcinogenic properties of statins in relation to expression of the redox- active enzymes thioredoxin reductases (TrxR) were investigated in paper IV. We found that statin treatment decreases the hepatic expression of TrxR1 in humans and rats. In addition, the decreased TrxR1-levels were correlated with inhibited carcinogenesis in rat liver. The effect on TrxR1 levels might explain some of the anti-carcinogenic effects of statins. Overall, our results add to the conjecture that the impact on production of vascular active substances may be significantly different among the statins, and thus the consequences of statin exposure may not be drug class related but rather compound specific, highlighting the importance of more comparative studies where several statins are included. These findings add novel insight in the field of pleiotropic effects of statins and provide some insight for functional differences among statins.
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