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  • 1. Abu-Bakar, A'edah
    et al.
    Lämsä, Virpi
    Arpiainen, Satu
    Moore, Michael R.
    Lang, Matti A.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Pharmaceutical Biochemistry.
    Hakkola, Jukka
    Regulation of CYP2A5 gene by the transcription factor nuclear factor (erythroid-derived 2)-like 22007In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 35, no 5, p. 787-794Article in journal (Refereed)
    Abstract [en]

    We have previously shown that cadmium, a metal that alters cellular redox status, induces CYP2A5 expression in nuclear factor (erythroid-derived 2)-like 2 wild-type (Nrf2(-/-)) mice but not in the knockout (Nrf2(-/-)) mice. In the present studies, the potential role of Nrf2 in cadmium-mediated regulation of Cyp2a5 gene was investigated in mouse primary hepatocytes. Cadmium chloride (CdCl2) caused a time-dependent induction of the CYP2A5 at mRNA, protein, and activity levels, with a substantial increase observed within 3 h of exposure. Immunoblotting showed cadmium-dependent nuclear accumulation of Nrf2 within 1 h of exposure. Cotransfection of mouse primary hepatocytes with Cyp2a5 promoter-luciferase reporter plasmids and Nrf2 expression plasmid resulted in a 3-fold activation of Cyp2a5 promoter-mediated transcription relative to the control. Deletion analysis of the promoter localized the Nrf2 responsive region to an area from -2656 to -2339 base pair. Computer-based sequence analysis identified two putative stress response elements (StRE) within the region at positions -2514 to -2505 and -2386 to -2377. Chromatin immunoprecipitation and electrophoretic mobility shift assays showed that interaction of the more proximal StRE with Nrf2 was stimulated by CdCl2. Finally, site-directed mutagenesis of the proximal StRE in Cyp2a5 promoter-luciferase reporter plasmids abolished Nrf2 mediated induction. Collectively, the results indicate that Nrf2 activates Cyp2a5 transcription by directly binding to the StRE in the 5'-flanking region of the gene. This acknowledges Cyp2a5 as the first phase I xenobiotic-metabolizing gene identified under the control of the StRE-Nrf2 pathway with a potential role in adaptive response to cellular stress.

  • 2.
    Ahlin, Gustav
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Hilgendorf, Constanze
    AstraZeneca R&D, Mölndal.
    Karlsson, Johan
    AstraZeneca R&D, Mölndal, Sweden.
    Al-Khalili Szigyarto, Cristina
    Department of Proteomics, The Royal Institute of Technology, Stockholm, Sweden.
    Uhlén, Mathias
    Department of Proteomics, The Royal Institute of Technology, Stockholm, Sweden.
    Artursson, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Endogenous Gene and Protein Expression of Drug Transporting Proteins in Cell Lines Routinely used in Drug Discovery Programs2009In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 37, no 12, p. 2275-2283Article in journal (Refereed)
    Abstract [en]

    The aim of this study was to investigate the gene and protein expression profiles of important drug transporting proteins in human cell lines commonly used for studies of drug transport mechanisms. Human cell lines used to transiently or stably express single transporters (HeLa, HEK293) and leukaemia cell lines used to study drug resistance by ABC-transporters (HL-60, K562) were investigated, and compared with organotypic cell lines (HepG2, Saos-2, Caco-2 and Caco-2 TC7). For gene expression studies, real-time PCR was used, while monospecific polyclonal antibodies were generated and used to investigate protein expression by immunohistochemistry. Thirty-six transporters were studied for gene expression and nine for protein expression. The antibodies were validated using expression patterns in human tissues. Finally, the function of one ubiquitously expressed transporter, MCT1; SLC16A1 was investigated using 14C-lactic acid as a substrate. In general, the adherent cell lines (HeLa, HEK293) displayed low transporter expression and the expression patterns were barely affected by transfection. The leukaemia cell lines (K562, HL-60) and Saos-2 also had low endogenous transporter expression, while the organotypic cell lines (HepG2 and Caco-2) showed higher expression of some transporters. Comparison of gene and protein expression profiles gave poor correlations, but better agreement was obtained for antibodies with a good validation score, indicating that antibody quality was a significant variable. Importantly, the monocarboxylic acid transporting protein MCT1 was significantly expressed in all, and functional in most of the cell lines, indicating that MCT1 may be a confounding factor when the transport of small anionic drugs is investigated.

  • 3.
    Andersson, Helén
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Helmestam, Malin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Women's and Children's Health, Obstetrics and Gynaecology.
    Zebrowska, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Olovsson, Matts
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Women's and Children's Health, Obstetrics and Gynaecology.
    Brittebo, Eva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Tamoxifen-Induced Adduct Formation and Cell Stress in Human Endometrial Glands2010In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 38, no 1, p. 200-207Article in journal (Refereed)
    Abstract [en]

    The beneficial effects of tamoxifen in the prevention and treatment of breast cancer are compromised by an increased risk of endometrial polyps, hyperplasia, and cancer. Tamoxifen is metabolized to an array of metabolites with estrogenic effects but also to reactive intermediates that may form protein and DNA adducts. The aim of this study was to investigate cellular [(3)H]tamoxifen adduct formation by light microscopic autoradiography and cell stress by immunohistochemical analysis of glucose-regulating protein 78 (GRP78), nuclear factor kappaB (NF-kappaB), and caspase 3 in human endometrial explants after short-term incubation with tamoxifen. The cellular expression of tamoxifen-metabolizing enzymes in human endometrial biopsy samples was also determined by immunohistochemistry. The results showed selective [(3)H]tamoxifen adduct formation in glandular and surface epithelia after incubation with a nontoxic concentration of [(3)H]tamoxifen (6 nM). There was also a selective expression of the endoplasmic reticulum stress chaperone GRP78 and activated caspase 3 at these sites after incubation with cytotoxic concentrations of tamoxifen (10-100 microM). The cell stress was preferentially observed in samples from women in the proliferative menstrual phase. No treatment-related expression of NF-kappaB was observed. Constitutive expression of the tamoxifen-metabolizing enzymes CYP1B1, CYP2A6, CYP2B6, CYP2C8/9/19, CYP2D6, and SULT2A1 in glandular and surface epithelia was shown, but there was a large interindividual variation. The colocalization of [(3)H]tamoxifen adducts, expression of GRP78, caspase 3, and tamoxifen-metabolizing enzymes in human glandular and surface epithelia suggest a local bioactivation of tamoxifen at these sites and that epithelial cells are early target sites for tamoxifen-induced cell stress.

  • 4.
    Bergman, Ebba
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Lundahl, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Fridblom, Patrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences.
    Hedeland, Mikael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Bondesson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Knutson, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences.
    Lennernäs, Hans
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    The Enterohepatic Disposition of Rosuvastatin in Pigs and The Impact of Concomitant Dosing with Cyclosporine and Gemfibrozil2009In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 37, no 12, p. 2349-2358Article in journal (Refereed)
    Abstract [en]

    The hepatobiliary transport and local disposition of rosuvastatin in pig was investigated, along with the impact of concomitant dosing with two known multiple transport inhibitors; cyclosporine and gemfibrozil. 80 mg rosuvastatin was administered as an intrajejunal bolus dose in Treatments I, II and III (TI, TII, and TIII, respectively; n=6 per treatment). Cyclosporine (300 mg) and gemfibrozil (600 mg) were administered in addition to the rosuvastatin dose in TII and TIII, respectively. Cyclosporine was administered as a two hour intravenous infusion and gemfibrozil as an intrajejunal bolus dose. In Treatment IV (TIV, n=4) was 5.9 mg rosuvastatin administered as an intravenous bolus dose. The study was conducted using a pig model, which enabled plasma sampling from the portal (VP), hepatic (VH) and femoral veins and bile from the common hepatic duct. The biliary recovery of the administered rosuvastatin dose was 9.0±3.5% and 35.7±15.6% in TI and TIV, respectively. Rosuvastatin was highly transported into bile as the median AUCbile/AUCVH ratio was 1770 (1640-11300) in TI. Gemfibrozil did not have an effect on the plasma pharmacokinetics of rosuvastatin, most likely because the unbound inhibitor concentrations did not exceed the reported IC50-values. However, cyclosporine significantly reduced the hepatic extraction of rosuvastatin (TI; 0.89±0.06, TII; 0.46±0.13) and increased the AUCVP and AUCVH by 1.6 and 9.1-fold, respectively. In addition, the biliary exposure and fe, bile were reduced by ≈50%. The strong effect of cyclosporine was in accordance with inhibition of sinusoidal uptake transporters, such as members of the OATP-family, rather than canalicular transporters.

  • 5.
    Boström, Emma
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Division of Pharmacokinetics and Drug Therapy.
    Simonsson, Ulrika S. H.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Division of Pharmacokinetics and Drug Therapy.
    Hammarlund-Udenaes, Margareta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Division of Pharmacokinetics and Drug Therapy.
    In Vivo Blood-Brain Barrier Transport of Oxycodone in the Rat: Indications for Active Influx and Implications for Pharmacokinetics/Pharmacodynamics2006In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 34, no 9, p. 1624-1631Article in journal (Refereed)
    Abstract [en]

    The blood-brain barrier (BBB) transport of oxycodone was studied in rats. Microdialysis probes were inserted into the striatum and vena jugularis. Ten animals were given a bolus dose followed by a 120-min constant rate infusion to study the steady-state concepts of oxycodone BBB equilibration. Another 10 animals were given a 60-min constant rate infusion to study the rate of equilibration across the BBB. Oxycodone-D3 was used as a calibrator for the microdialysis experiments. The samples were analyzed with a liquid chromatography-tandem mass spectrometry method and a population pharmacokinetic model was used to simultaneously fit all the data using NONMEM. A two-compartment model which allowed for a delay between the venous and arterial compartments best described the pharmacokinetics for oxycodone in blood and plasma, whereas a one-compartment model was sufficient to describe the pharmacokinetics in the brain. The BBB transport of oxycodone was parameterized as CL(in) and K(p,uu). CL(in) describes the clearance of oxycodone across the BBB into the brain, whereas K(p,uu) describes the extent of drug equilibration across the BBB. CL(in) across the BBB was estimated to 1910 microl/min x g brain. K(p,uu) was estimated to 3.0, meaning that the unbound concentration of oxycodone in brain was 3 times higher than in blood, which is an indication of active influx of oxycodone at the BBB. This is the first evidence of an opioid having an unbound steady-state concentration in brain that is higher than unity, which can explain potency discrepancies between oxycodone and other opioids.

  • 6.
    Bylund, Johan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Annas, Anita
    Hellgren, Dennis
    Bjurström, Sivert
    Andersson, Håkan
    Svanhagen, Alexander
    Amide hydrolysis of a novel chemical series of microsomal prostaglandin e synthase-1 inhibitors induces kidney toxicity in the rat2013In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 41, no 3, p. 634-641Article in journal (Refereed)
    Abstract [en]

    A novel microsomal prostaglandin E synthase 1 (mPGES-1) inhibitor induced kidney injury at exposures representing less than 4 times the anticipated efficacious exposure in man during a 7-day toxicity study in rats. The findings consisted mainly of tubular lesions and the presence of crystalline material and increases in plasma urea and creatinine. In vitro and in vivo metabolic profiling generated a working hypothesis that a bis-sulfonamide metabolite (determined M1) formed by amide hydrolysis caused this toxicity. To test this hypothesis, rats were subjected to a 7-day study and were administered the suspected metabolite and two low-potency mPGES-1 inhibitor analogs, where amide hydrolysis was undetectable in rat hepatocyte experiments. The results suggested that compounds with a reduced propensity to undergo amide hydrolysis, thus having less ability to form M1, reduced the risk of inducing kidney toxicity. Rats treated with M1 alone showed no histopathologic change in the kidney, which was likely related to underexposure to M1. To circumvent rat kidney toxicity, we identified a potent mPGES-1 inhibitor with a low propensity for amide hydrolysis and superior rat pharmacokinetic properties. A subsequent 14-day rat toxicity study showed that this compound was associated with kidney toxicity at 42, but not 21, times the anticipated efficacious exposure in humans. In conclusion, by including metabolic profiling and exploratory rat toxicity studies, a new and active mPGES-1 inhibitor with improved margins to chemically induced kidney toxicity in rats has been identified.

  • 7.
    Bylund, Johan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Macsari, Istvan
    Besidski, Yevgeni
    Olofsson, Susanne
    Petersson, Carl
    Arvidsson, Per I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Bueters, Tjerk
    Novel bioactivation mechanism of reactive metabolite formation from phenyl methyl-isoxazoles2012In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 40, no 11, p. 2185-2191Article in journal (Refereed)
    Abstract [en]

    Recently, we described a series of phenyl methyl-isoxazole derivatives as novel, potent, and selective inhibitors of the voltage-gated sodium channel type 1.7 (Bioorg Med Chem Lett 21:3871-3876, 2011). The lead compound, 2-chloro-6-fluorobenzyl [3-(2,6-dichlorophenyl)-5-methylisoxazol-4-yl]carbamate, showed unprecedented GSH and cysteine reactivity associated with NADPH-dependent metabolism in trapping studies using human liver microsomes. Additional trapping experiments with close analogs and mass spectra and NMR analyses suggested that the conjugates were attached directly to the 5′-methyl on the isoxazole moiety. We propose a mechanism of bioactivation via an initial oxidation of the 5′-methyl generating a stabilized enimine intermediate and a subsequent GSH attack on the 5′-methylene. Efforts to ameliorate reactive metabolite generation were undertaken to minimize the potential risk of toxicity. Formation of reactive metabolites could be significantly reduced or prevented by removing the 5′-methyl, by N-methylation of the carbamate; by replacing the nitrogen with a carbon or removing the nitrogen to obtain a carboxylate; or by inserting an isomeric 5′-methyl isoxazole. The effectiveness of these various chemical modifications in reducing GSH adduct formation is in line with the proposed mechanism. In conclusion, we have identified a novel mechanism of bioactivation of phenyl 5-methyl-isoxazol-4-yl-amines. The reactivity was attenuated by several modifications aimed to prevent the emergence of an enimine intermediate. Whether 5′-methyl isoxazoles should be considered a structural alert for potential formation of reactive metabolites is dependent on their context, i.e., 4′-nitrogen.

  • 8.
    Edwards, David
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Radiology.
    Battle, Mark
    Lear, Rochelle
    Farrar, Gill
    Barnett, D. Jon
    Godden, Vanessa
    Coombes, Catherine
    Oliveira, Alexandra
    Ahlström, Håkan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Radiology.
    The in vivo and in vitro metabolic profile of 99mTc-NC100668, a new tracer for imaging venous thromboembolism: identification and biodistribution of the principal radiolabeled metabolite2006In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 34, no 7, p. 1128-1135Article in journal (Refereed)
    Abstract [en]

    (99m)Tc-NC100668 [Acetyl-Asn-Gln-Glu-Gln-Val-Ser-Pro-Tyr(3-iodo)-Thr-Leu-Leu-Lys-Gly-NC100194] is a radiopharmaceutical imaging agent being developed to aid the diagnosis of thromboembolism. The stability profile of (99m)Tc-NC100668 was investigated by high-performance liquid chromatography (HPLC) after in vitro exposure to blood and plasma obtained from rat and human, as well as to urine and bile obtained from rat. The metabolic profile of (99m)Tc-NC100668 exposed to human and rat hepatic S9 (a liver homogenate-rich cytochrome P450) was also studied. The profile of (99m)Tc-labeled species in plasma, urine, and bile was investigated following i.v. administration of (99m)Tc-NC100668 to rat. The major species observed in vitro and in vivo consisted of the (99m)Tc-chelator (NC100194) [N,N-Bis(N-(1,1-dimethyl-2-(hydroxylimino-)propyl)aminoethyl)aminoethylamine] attached to the C-terminal amino acid residue and referred to as (99m)Tc-complex of Gly-NC100194. The identity of the major metabolite was confirmed by cochromatography with an authentic standard and the genuine metabolite using a second HPLC method. The minor metabolites were sodium pertechnetate ((99m)Tc) and (99m)Tc-NC100194. In addition, a small number of other species were transiently observed in vitro; they were not investigated further. The biodistribution of the major metabolite was studied in male Wistar rats. The affinity of the major metabolite toward plasma clot was established using a plasma clot-forming assay. A minor uptake of (99m)Tc-complex of Gly-NC100194 in the plasma clot and a rapid removal from the body were noted. In conclusion, the metabolites of (99m)Tc-NC100668 are not anticipated to have a negative impact on the ability of the test substance to image blood clots.

  • 9. Englund, Gunilla
    et al.
    Lundquist, Patrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Skogastierna, Cristine
    Johansson, Jenny
    Hoogstraate, Janet
    Afzelius, Lovisa
    Andersson, Tommy B.
    Projean, Denis
    Cytochrome P450 Inhibitory Properties of Common Efflux Transporter Inhibitors2014In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 42, no 3, p. 441-447Article in journal (Refereed)
    Abstract [en]

    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.

  • 10. Fransson, Martin N.
    et al.
    Green, Henrik
    Litton, Jan-Eric
    Friberg, Lena E.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Influence of Cremophor EL and Genetic Polymorphisms on the Pharmacokinetics of Paclitaxel and Its Metabolites Using a Mechanism-Based Model2011In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 39, no 2, p. 247-255Article in journal (Refereed)
    Abstract [en]

    The formulation vehicle Cremophor EL has previously been shown to affect paclitaxel kinetics, but it is not known whether it also affects the kinetics of paclitaxel metabolites. This information may be important for understanding paclitaxel metabolism in vivo and in the investigation of the role of genetic polymorphisms in the metabolizing enzymes CYP2C8 and CYP3A4/CYP3A5 and the ABCB1 transporter. In this study we used the population pharmacokinetic approach to explore the influence of predicted Cremophor EL concentrations on paclitaxel (Taxol) metabolites. In addition, correlations between genetic polymorphisms and enzyme activity with clearance of paclitaxel, its two primary metabolites, 6 alpha-hydroxypaclitaxel and p-3'-hydroxypaclitaxel, and its secondary metabolite, 6 alpha-p-3'-dihydroxypaclitaxel were investigated. Model building was based on 1156 samples from a study with 33 women undergoing paclitaxel treatment for gynecological cancer. Total concentrations of paclitaxel were fitted to a model described previously. One-compartment models characterized unbound metabolite concentrations. Total concentrations of 6 alpha-hydroxypaclitaxel and p-3'-hydroxypaclitaxel were strongly dependent on predicted Cremophor EL concentrations, but this association was not found for 6 alpha-p-3'-dihydroxypaclitaxel. Clearance of 6 alpha-hydroxypaclitaxel (fraction metabolized) was significantly correlated (p < 0.05) to the ABCB1 allele G2677T/A. Individuals carrying the polymorphisms G/A (n = 3) or G/G (n = 5) showed a 30% increase, whereas individuals with polymorphism T/T (n = 8) showed a 27% decrease relative to those with the polymorphism G/T (n = 17). The correlation of G2677T/A with 6 alpha-hydroxypaclitaxel has not been described previously but supports other findings of the ABCB1 transporter playing a part in paclitaxel metabolism.

  • 11.
    Franzén, Anna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Carlsson, Carina
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology, Environmental Toxicology.
    Hermansson, Veronica
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology, Environmental Toxicology.
    Lang, Matti
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Brittebo, Eva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    CYP2A5-mediated activation and early ultrastructural changes in the olfactory mucosa: studies on 2,6-dichlorophenyl methylsulfone2006In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 34, no 1, p. 61-68Article in journal (Refereed)
    Abstract [en]

    2,6-Dichlorophenyl methylsulfone (2,6-diClPh-MeSO2) is a potent olfactory toxicant reported to induce endoplasmic reticulum (ER) stress, caspase activation, and extensive cell death in mice. The aim of the present study was to examine cytochrome P450 (P450)-dependent bioactivation, nonprotein sulfhydryl (NP-SH) levels, and early ultrastructural changes in mouse olfactory mucosa following an i.p. injection of 2,6-diClPh-MeSO2 (32 mg/kg). A high covalent binding of 2,6-diClPh-14C-MeSO2 in olfactory mucosa S9 fraction was observed, and the CYP2A5/CYP2G1 substrates coumarin and dichlobenil significantly decreased the binding, whereas the CYP2E1 substrate chlorzoxazone had no effects. An increased bioactivation was detected in liver microsomes of mice pretreated with pyrazole, known to induce CYP2A4, 2A5, 2E1, and 2J, and addition of chlorzoxazone reduced this binding. 2,6-DiClPh-14C-MeSO2 showed a marked covalent binding to microsomes of recombinant yeast cells expressing mouse CYP2A5 or human CYP2A6 compared with wild type. One and 4 h after a single injection of 2,6-diClPh-MeSO2, the NP-SH levels in the olfactory mucosa were significantly reduced compared with control, whereas there was no change in the liver. Ultrastructural studies revealed that ER, mitochondria, and secretory granules in nonneuronal cells were early targets 1 h after injection. We propose that lesions induced by 2,6-diClPh-MeSO2 in the mouse olfactory mucosa were initiated by a P450-mediated bioactivation in the Bowman's glands and depletion of NP-SH levels, leading to disruption of ion homeostasis, organelle swelling, and cell death. The high expression of CYP2A5 in the olfactory mucosa is suggested to play a key role for the tissue-specific toxicity induced by 2,6-diClPh-MeSO2.

  • 12.
    Fridén, Markus
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Bergström, Fredrik
    Wan, Hong
    Rehngren, Mikael
    Ahlin, Gustav
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Hammarlund-Udenaes, Margareta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Bredberg, Ulf
    Measurement of Unbound Drug Exposure in Brain: Modelling of pH Partitioning Explains Diverging Results between the Brain Slice and Brain Homogenate Methods2011In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 39, no 3, p. 353-362Article in journal (Refereed)
    Abstract [en]

    Currently used methodology for determining unbound drug exposure in brain combines measurement of the total drug concentration in the whole brain in vivo with estimation of brain tissue binding from one of two available in vitro methods: equilibrium dialysis of brain homogenate and the brain slice uptake method. This study of 56 compounds compares the fraction of unbound drug in brain (f(u,brain)), determined using the brain homogenate method, with the unbound volume of distribution in brain (V(u,brain)), determined using the brain slice method. Discrepancies were frequent and primarily related to drug pH partitioning, due to the preservation of cellular structures in the slice that are absent in the homogenate. A mathematical model for pH partitioning into acidic intracellular compartments was derived to predict the slice V(u,brain) from measurements of f(u,brain) and drug pKa. This model allowed prediction of V(u,brain) from f(u,brain) within a 2.2-fold error range for 95% of the drugs, as compared to a 4.5-fold error range using the brain homogenate f(u,brain) method alone. The greatest discrepancies between the methods occurred with compounds that are actively transported into brain cells, including gabapentin, metformin and prototypic organic cation transporter substrates. It is concluded that intra-brain drug distribution is governed by several diverse mechanisms in addition to non-specific binding and that the slice method is therefore more reliable than the homogenate method. Alternatively, predictions of V(u,brain) can be made from homogenate f(u,brain) using the presented pH partition model, although this model does not take into consideration possible active brain cell uptake.

  • 13.
    Fridén, Markus
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Ducrozet, Frederic
    Middleton, Brian
    Antonsson, Madeleine
    Bredberg, Ulf
    Hammarlund-Udenaes, Margareta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Development of a High-Throughput Brain Slice Method for Studying Drug Distribution in the Central Nervous System2009In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 37, no 6, p. 1226-1233Article in journal (Refereed)
    Abstract [en]

    New, more efficient methods of estimating unbound drug concentrations in the CNS combine the amount of drug in whole brain tissue samples measured by conventional methods with in vitro estimates of the unbound brain volume of distribution (Vu,brain). While the brain slice method is the most reliable in vitro method for measuring Vu,brain, it has not previously been adapted for the needs of drug discovery research. The aim of this study was to increase the throughput and optimize the experimental conditions of this method. Equilibrium of drug between the buffer and the brain slice within the 4-5 hours of incubation is a fundamental requirement. However, it is difficult to meet this requirement for many of the extensively binding, lipophilic compounds in drug discovery programmes. In this study, the dimensions of the incubation vessel and mode of stirring influenced the equilibration time, as did the amount of brain tissue per unit buffer volume. The use of casette experiments for investigating Vu,brain in a linear drug concentration range increased the throughput of the method. The Vu,brain for the model compounds ranged from mL*g brain(-1); the sources of variability are discussed. The optimized set-up of the brain slice method allows precise, robust estimation of Vu,brain for drugs with diverse properties, including highly lipophilic compounds. This is a critical step forward for the implementation of relevant measurements of CNS exposure in the drug discovery setting.

  • 14.
    Fridén, Markus
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Gupta, Anubha
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Antonsson, Madeleine
    Bredberg, Ulf
    Hammarlund-Udenaes, Margareta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    In vitro methods for estimating unbound drug concentrations in the brain interstitial and intracellular fluids2007In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 35, no 9, p. 1711-1719Article in journal (Refereed)
    Abstract [en]

    Concentrations of unbound drug in the interstitial fluid of the brain are not rapidly measured in vivo. Therefore, measurement of total drug levels, i.e., the amount of drug per gram of brain, has been a common but unhelpful practice in drug discovery programs relating to central drug effects. This study was designed to evaluate in vitro techniques for faster estimation of unbound drug concentrations. The parameter that relates the total drug level and the unbound interstitial fluid concentration is the unbound volume of distribution in the brain (V(u,brain)). It was measured in vitro for 15 drugs using brain slice uptake and brain homogenate binding methods. The results were validated in vivo by comparison with V(u,brain) microdialysis results. The slice method results were within a 3-fold range of the in vivo results for all but one compound, suggesting that this method could be used in combination with total drug levels to estimate unbound interstitial fluid concentrations within reasonable limits. Although successful in 10 of 15 cases, the brain homogenate binding method failed to estimate the V(u,brain) of drugs that reside predominantly in the interstitial space or compounds that are accumulated intracellularly. Use of the simple methods described in this article will 1) allow quantification of active transport at the blood-brain barrier in vivo, 2) facilitate the establishment of a relationship between in vitro potency and in vivo activity for compounds acting on central nervous system targets, and 3) provide information on intracellular concentrations of unbound drug.

  • 15.
    Garcia, Luna Prieto
    et al.
    AstraZeneca, IMED Biotech Unit, Drug Metab & Pharmacokinet, Cardiovasc & Metab Dis, Gothenburg, Sweden.
    Janzen, David
    AstraZeneca, IMED Biotech Unit, Drug Metab & Pharmacokinet, Cardiovasc & Metab Dis, Gothenburg, Sweden.
    Kanebratt, Kajsa P.
    AstraZeneca, IMED Biotech Unit, Drug Metab & Pharmacokinet, Cardiovasc & Metab Dis, Gothenburg, Sweden.
    Ericsson, Hans
    AstraZeneca, IMED Biotech Unit, Quantitat Clin Pharmacol, Early Clin Dev, Gothenburg, Sweden.
    Lennernäs, Hans
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Lundahl, Anna
    AstraZeneca, IMED Biotech Unit, Drug Metab & Pharmacokinet, Cardiovasc & Metab Dis, Gothenburg, Sweden.
    Physiologically Based Pharmacokinetic Model of Itraconazole and Two of Its Metabolites to Improve the Predictions and the Mechanistic Understanding of CYP3A4 Drug-Drug Interactions2018In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 46, no 10, p. 1420-1433Article in journal (Refereed)
    Abstract [en]

    Physiologically based pharmacokinetic (PBPK) modeling for itraconazole using a bottom-up approach is challenging, not only due to complex saturable pharmacokinetics (PK) and the presence of three metabolites exhibiting CYP3A4 inhibition, but also because of discrepancies in reported in vitro data. The overall objective of this study is to provide a comprehensive mechanistic PBPK model for itraconazole in order to increase the confidence in its drug-drug interaction (DDI) predictions. To achieve this, key in vitro and in vivo data for itraconazole and its major metabolites were generated. These data were crucial to developing a novel bottom-up PBPK model in Simcyp (Simcyp Ltd., Certara, Sheffield, United Kingdom) for itraconazole and two of its major metabolites: hydroxy-itraconazole (OH-ITZ) and keto-itraconazole (keto-ITZ). Performance of the model was validated using prespecified acceptance criteria against different dosing regimens, formulations for 29 PK, and DDI studies with midazolam and other CYP3A4 substrates. The main outcome is an accurate PBPK model that simultaneously predicts the PK profiles of itraconazole, OH-ITZ, and keto-ITZ. In addition, itraconazole DDIs with midazolam and other CYP3A4 substrates were successfully predicted within a 2-fold error. Prediction precision and bias of DDI expressed as geometric mean fold error were for the area under the concentration-time curve and peak concentration, 1.06 and 0.96, respectively. To conclude, in this paper a comprehensive data set for itraconazole and its metabolites is provided that enables bottom-up mechanism-based PBPK modeling. The presented model is applicable for studying the contribution from the metabolites and allows improved assessments of itraconazole DDI.

  • 16.
    Granberg, Lizette
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology, Environmental Toxicology.
    Östergren, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Brandt, Ingvar
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology, Environmental Toxicology.
    Brittebo, Eva B.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    CYP1A1 and CYP1B1 in blood-brain interfaces: CYP1A1-dependent bioactivation of 7,12-dimethylbenz(a)anthracene in endothelial cells.2003In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 31, no 3, p. 259-265Article in journal (Refereed)
    Abstract [en]

    Immunohistochemistry and autoradiography were used to identify sites of the cytochrome P450 enzymes (P450) 1A1 and 1B1 expression and activation of 7,12-dimethylbenz(a)anthracene (DMBA), in the brain of rodents pretreated with the aryl hydrocarbon receptor (AhR) agonists beta-naphthoflavone (BNF), 3,3',4,4',5-pentachlorobiphenyl or vehicle. Immunohistochemistry revealed that CYP1A1 was preferentially induced in endothelial cells (EC) in the choroid plexus, in veins in the leptomeninges, and in cerebral veins of AhR agonist-pretreated mice. No induction occurred in cerebral capillary EC. In vehicle-treated mice no localization of CYP1A1 in EC was observed. CYP1B1 was expressed in smooth muscle cells of arteries in the leptomeninges, in cerebral arteries/arterioles and to a low extent in ependymal cells of AhR agonist- and vehicle-treated mice. No CYP1B1 was detected in capillary loops of the choroid plexus or in cerebral capillaries. Following administration of [(3)H]DMBA to BNF-pretreated mice, a marked irreversible binding in EC of the choroid plexus and of veins in the leptomeninges was observed but not in cerebral capillaries. In vehicle-treated mice, there was no [(3)H]DMBA-binding at these sites. Furthermore, a high level of irreversibly bound [(3)H]DMBA occurred in EC at these sites in precision-cut mouse/rat brain slices and in excised blood-brain interfaces incubated with [(3)H]DMBA. Since [(3)H]DMBA binding sites corresponded with the sites of CYP1A1 induction, we conclude that rodents express a constitutively low but highly inducible and functional CYP1A1 in EC of some of the blood-brain interfaces. The role of CYP1A1/1B1 and environmental pollutants in the etiology of cerebrovascular disease needs further consideration.

  • 17.
    Granberg, Lizette
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolutionary Biology, Environmental Toxicology.
    Östergren, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Brandt, Ingvar
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolutionary Biology, Environmental Toxicology.
    Brittebo, Eva B
    CYP1A1 and CYP1B1 in blood-brain interfaces:: CYP1A1-dependent bioactivation of 7,12-dimethylbenz[a]anthracene in endothelial cells2003In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 31, no 3, p. 259-265Article in journal (Refereed)
    Abstract [en]

    Immunohistochemistry and autoradiography were used to identify sites of the cytochrome P450 enzymes (P450) 1A1 and 1B1 expression and activation of 7,12-dimethylbenz(a)anthracene (DMBA), in the brain of rodents pretreated with the aryl hydrocarbon receptor (AhR) agonists beta-naphthoflavone (BNF), 3,3',4,4',5-pentachlorobiphenyl or vehicle. Immunohistochemistry revealed that CYP1A1 was preferentially induced in endothelial cells (EC) in the choroid plexus, in veins in the leptomeninges, and in cerebral veins of AhR agonist-pretreated mice. No induction occurred in cerebral capillary EC. In vehicle-treated mice no localization of CYP1A1 in EC was observed. CYP1B1 was expressed in smooth muscle cells of arteries in the leptomeninges, in cerebral arteries/arterioles and to a low extent in ependymal cells of AhR agonist- and vehicle-treated mice. No CYP1B1 was detected in capillary loops of the choroid plexus or in cerebral capillaries. Following administration of [(3)H]DMBA to BNF-pretreated mice, a marked irreversible binding in EC of the choroid plexus and of veins in the leptomeninges was observed but not in cerebral capillaries. In vehicle-treated mice, there was no [(3)H]DMBA-binding at these sites. Furthermore, a high level of irreversibly bound [(3)H]DMBA occurred in EC at these sites in precision-cut mouse/rat brain slices and in excised blood-brain interfaces incubated with [(3)H]DMBA. Since [(3)H]DMBA binding sites corresponded with the sites of CYP1A1 induction, we conclude that rodents express a constitutively low but highly inducible and functional CYP1A1 in EC of some of the blood-brain interfaces. The role of CYP1A1/1B1 and environmental pollutants in the etiology of cerebrovascular disease needs further consideration.

  • 18.
    Gupta, Anubha
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Chatelain, Pierre
    Massingham, Roy
    Jonsson, E Niclas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Hammarlund-Udenaes, Margareta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Brain distribution of cetirizine enantiomers: comparison of three different tissue-to-plasma partition coefficients : K(p), K(p,u), and K(p,uu)2006In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 34, no 2, p. 318-323Article in journal (Refereed)
    Abstract [en]

    The objective of this study was to compare the blood-brain barrier (BBB) transport and brain distribution of levo- (R-CZE) and dextrocetirizine (S-CZE). Microdialysis probes, calibrated using retrodialysis by drug, were placed into the frontal cortex and right jugular vein of eight guinea pigs. Racemic CZE (2.7 mg/kg) was administered as a 60-min i.v. infusion. Unbound and total concentrations of the enantiomers were measured in blood and brain with liquid chromatography-tandem mass spectrometry. The brain distribution of the CZE enantiomers were compared using the parameters K(p,) K(p,u,) K(p,uu), and V(u,br). K(p) compares total brain concentration to total plasma concentration, K(p,u) compensates for binding in plasma, whereas K(p,uu) also compensates for binding within the brain tissue and directly quantifies the transport across the BBB. V(u,br) describes binding within the brain. The stereoselective brain distribution indicated by the K(p) of 0.22 and 0.04 for S- and R-CZE, respectively, was caused by different binding to plasma proteins. The transport of the CZE enantiomers across the BBB was not stereoselective, since the K(p,uu) was 0.17 and 0.14 (N.S.) for S- and R-CZE, respectively. The K(p,uu) values show that the enantiomers are effluxed to a large extent across the BBB. The V(u,br) of approximately 2.5 ml/g brain was also similar for both the enantiomers, and the value indicates high binding to brain tissue. Thus, when determining stereoselectivity in brain distribution, it is important to study all factors governing this distribution, binding in blood and brain, and the BBB equilibrium.

  • 19. Hilgendorf, Constanze
    et al.
    Ahlin, Gustav
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Seithel, Annick
    Artursson, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Ungell, Anna-Lena
    Karlsson, Johan
    Expression of thirty-six drug transporter genes in human intestine, liver, kidney, and organotypic cell lines2007In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 35, no 8, p. 1333-1340Article in journal (Refereed)
    Abstract [en]

    This study was designed to quantitatively assess the mRNA expression of 36 important drug transporters in human jejunum, colon, liver, and kidney. Expression of these transporters in human organs was compared with expression in commonly used cell lines (Caco-2, HepG2, and Caki-1) originating from these organs to assess their value as in vitro transporter system models, and was also compared with data obtained from the literature on expression in rat tissues to assess species differences. Transporters that were highly expressed in the intestine included HPT1, PEPT1, BCRP, MRP2, and MDR1, whereas, in the liver, OCT1, MRP2, OATP-C, NTCP and BSEP were the main transporters. In the kidney, OAT1 was expressed at the highest levels, followed by OAT3, OAT4, MCT5, MDR1, MRP2, OCT2, and OCTN2. The best agreement between human tissue and the representative cell line was observed for human jejunum and Caco-2 cells. Expression in liver and kidney ortholog cell lines was not correlated with that in the associated tissue. Comparisons with rat transporter gene expression revealed significant species differences. Our results allowed a comprehensive quantitative comparison of drug transporter expression in human intestine, liver, and kidney. We suggest that it would be beneficial for predictive pharmacokinetic research to focus on the most highly expressed transporters. We hope that our comparison of rat and human tissue will help to explain the observed species differences in in vivo models, increase understanding of the impact of active transport processes on pharmacokinetics and distribution, and improve the quality of predictions from animal studies to humans.

  • 20.
    Karlgren, Maria
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. Uppsala Univ, Dept Pharm, Box 580, SE-75123 Uppsala, Sweden.
    Simoff, Ivailo
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Keiser, M.
    Univ Med Greifswald, Ctr Drug Absorpt & Transport, Dept Clin Pharmacol, Greifswald, Germany.
    Oswald, S.
    Univ Med Greifswald, Ctr Drug Absorpt & Transport, Dept Clin Pharmacol, Greifswald, Germany.
    Artursson, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. Uppsala University, Science for Life Laboratory, SciLifeLab.
    CRISPR-Cas9: A New Addition to the Drug Metabolism and Disposition Tool Box2018In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 46, no 11, p. 1776-1786Article, review/survey (Refereed)
    Abstract [en]

    Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9), i.e., CRISPR-Cas9, has been extensively used as a gene-editing technology during recent years. Unlike earlier technologies for gene editing or gene knockdown, such as zinc finger nucleases and RNA interference, CRISPR-Cas9 is comparably easy to use, affordable, and versatile. Recently, CRISPR-Cas9 has been applied in studies of drug absorption, distribution, metabolism, and excretion (ADME) and for ADME model generation. To date, about 50 papers have been published describing in vitro or in vivo CRISPR-Cas9 gene editing of ADME and ADME-related genes. Twenty of these papers describe gene editing of clinically relevant genes, such as ATP-binding cassette drug transporters and cytochrome P450 drug-metabolizing enzymes. With CRISPR-Cas9, the ADME tool box has been substantially expanded. This new technology allows us to develop better and more predictive in vitro and in vivo ADME models and map previously underexplored ADME genes and gene families. In this mini-review, we give an overview of the CRISPR-Cas9 technology and summarize recent applications of CRISPR-Cas9 within the ADME field. We also speculate about future applications of CRISPR-Cas9 in ADME research.

  • 21. Koeck, Kathleen
    et al.
    Ferslew, Brian C.
    Netterberg, Ida
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Yang, Kyunghee
    Urban, Thomas J.
    Swaan, Peter W.
    Stewart, Paul W.
    Brouwer, Kim L. R.
    Risk Factors for Development of Cholestatic Drug-Induced Liver Injury: Inhibition of Hepatic Basolateral Bile Acid Transporters Multidrug Resistance-Associated Proteins 3 and 42014In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 42, no 4, p. 665-674Article in journal (Refereed)
    Abstract [en]

    Impaired hepatic bile acid export may contribute to development of cholestatic drug-induced liver injury (DILI). The multidrug resistance-associated proteins (MRP) 3 and 4 are postulated to be compensatory hepatic basolateral bile acid efflux transporters when biliary excretion by the bile salt export pump (BSEP) is impaired. BSEP inhibition is a risk factor for cholestatic DILI. This study aimed to characterize the relationship between MRP3, MRP4, and BSEP inhibition and cholestatic potential of drugs. The inhibitory effect of 88 drugs (100 mu M) on MRP3- and MRP4-mediated substrate transport was measured in membrane vesicles. Drugs selected for investigation included 50 BSEP non-inhibitors (24 non-cholestatic; 26 cholestatic) and 38 BSEP inhibitors (16 non-cholestatic; 22 cholestatic). MRP4 inhibition was associated with an increased risk of cholestatic potential among BSEP non-inhibitors. In this group, for each 1% increase in MRP4 inhibition, the odds of the drug being cholestatic increased by 3.1%. Using an inhibition cutoff of 21%, which predicted a 50% chance of cholestasis, 62% of cholestatic drugs inhibited MRP4 (P < 0.05); in contrast, only 17% of non-cholestatic drugs were MRP4 inhibitors. Among BSEP inhibitors, MRP4 inhibition did not provide additional predictive value of cholestatic potential; almost all BSEP inhibitors were also MRP4 inhibitors. Inclusion of pharmacokinetic predictor variables (e. g., maximal unbound concentration in plasma) in addition to percent MRP4 inhibition in logistic regression models did not improve cholestasis prediction. Association of cholestasis with percent MRP3 inhibition was not statistically significant, regardless of BSEP-inhibition status. Inhibition of MRP4, in addition to BSEP, may be a risk factor for the development of cholestatic DILI.

  • 22.
    Krogstad, Veronica
    et al.
    Univ Oslo, Sect Pharmacol & Pharmaceut Biosci, Dept Pharm, POB 1068, N-0316 Oslo, Norway;Oslo Univ Hosp, Dept Transplantat Med, Rikshosp, Oslo, Norway.
    Peric, Alexandra
    AstraZeneca Gothenburg, BioPharmaceut R&D, Res & Early Dev, Cardiovasc Renal & Metab, Gothenburg, Sweden.
    Robertsen, Ida
    Univ Oslo, Sect Pharmacol & Pharmaceut Biosci, Dept Pharm, POB 1068, N-0316 Oslo, Norway.
    Kringen, Marianne K.
    Diakonhjemmet Hosp, Ctr Psychopharmacol, Oslo, Norway;OsloMet Oslo Metropolitan Univ, Dept Hlth Sci, Oslo, Norway.
    Wegler, Christine
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. AstraZeneca Gothenburg, BioPharmaceut R&D, Res & Early Dev, Cardiovasc Renal & Metab, Gothenburg, Sweden.
    Angeles, Philip Carlo
    Vestfold Hosp Trust, Morbid Obes Ctr, Tonsberg, Norway;Vestfold Hosp Trust, Dept Surg, Tonsberg, Norway.
    Hjelmesaeth, Jöran
    Vestfold Hosp Trust, Morbid Obes Ctr, Tonsberg, Norway;Univ Oslo, Inst Clin Med, Dept Endocrinol Morbid Obes & Prevent Med, Oslo, Norway.
    Karlsson, Cecilia
    AstraZeneca Gothenburg, BioPharmaceut R&D, Late Stage Dev Metab, Cardiovasc Renal & Metab, Gothenburg, Sweden;Univ Gothenburg, Sahlgrenska Acad, Inst Med, Dept Mol & Clin Med, Gothenburg, Sweden.
    Andersson, Shalini
    AstraZeneca Gothenburg, BioPharmaceut R&D, Res & Early Dev, Cardiovasc Renal & Metab, Gothenburg, Sweden.
    Artursson, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Åsberg, Anders
    Univ Oslo, Sect Pharmacol & Pharmaceut Biosci, Dept Pharm, POB 1068, N-0316 Oslo, Norway;Oslo Univ Hosp, Dept Transplantat Med, Rikshosp, Oslo, Norway.
    Andersson, Tommy B.
    AstraZeneca Gothenburg, BioPharmaceut R&D, Res & Early Dev, Cardiovasc Renal & Metab, Gothenburg, Sweden;Karolinska Inst, Sect Pharmacogenet, Dept Physiol & Pharmacol, Stockholm, Sweden.
    Christensen, Hege
    Univ Oslo, Sect Pharmacol & Pharmaceut Biosci, Dept Pharm, POB 1068, N-0316 Oslo, Norway.
    A Comparative Analysis of Cytochrome P450 Activities in Paired Liver and Small Intestinal Samples from Patients with Obesity2020In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 48, no 1, p. 8-17Article in journal (Refereed)
    Abstract [en]

    The liver and small intestine restrict oral bioavailability of drugs constitute the main sites of pharmacokinetic drug-drug interactions. Hence, detailed data on hepatic and intestinal activities of drug metabolizing enzymes is important for modeling drug disposition and optimizing pharmacotherapy in different patient populations. The aim of this study was to determine the activities of seven cytochrome P450 (P450) enzymes in paired liver and small intestinal samples from patients with obesity. Biopsies were obtained from 20 patients who underwent Roux-en-Y gastric bypass surgery following a 3-week low-energy diet. Individual hepatic and intestinal microsomes were prepared and specific probe substrates in combined incubations were used for determination of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A activities. The activities of CYP2C8, CYP2C9, CYP2D6, and CYP3A were quantified in both human liver microsomes (HLM) and human intestinal microsomes (HIM), while the activities of CYP1A2, CYP2B6, and CYP2C19 were only quantifiable in HLM. Considerable interindividual variability was present in both HLM (9- to 23-fold) and HIM (5- to 55-fold). The median metabolic HLM/HIM ratios varied from 1.5 for CYP3A to 252 for CYP2C8. The activities of CYP2C9 in paired HLM and HIM were positively correlated (r = 0.74, P < 0.001), while no interorgan correlations were found for activities of CYP2C8, CYP2D6, and CYP3A (P > 0.05). Small intestinal CYP3A activities were higher in females compared with males (P < 0.05). Hepatic CYP2B6 activity correlated negatively with body mass index (r = -0.72, P < 0.001). These data may be useful for further in vitro-in vivo predictions of drug disposition in patients with obesity. SIGNIFICANCE STATEMENT Hepatic and intestinal drug metabolism is the key determinant of oral drug bioavailability. In this study, paired liver and jejunum samples were obtained from 20 patients with obesity undergoing gastric bypass surgery following a 3-week low-energy diet. We determined the hepatic and small intestinal activities of clinically important P450 enzymes and provide detailed enzyme kinetic data relevant for predicting in vivo disposition of P450 substrates in this patient population.

  • 23.
    Lundahl, Anna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Hedeland, Mikael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Bondesson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Lennernäs, Hans
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    In Vivo Investigation in Pigs of Intestinal Absorption, Hepatobiliary Disposition, and Metabolism of the 5 alpha-Reductase Inhibitor Finasteride and the Effects of Coadministered Ketoconazole2011In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 39, no 5, p. 847-857Article in journal (Refereed)
    Abstract [en]

    The overall aim of this detailed investigation of finasteride's pharmacokinetics (PK) and metabolism in pigs was to improve the understanding of the in vivo PK for this drug and its metabolites. Specific aims were to examine the effects of ketoconazole co-administration on the PK in three plasma compartments (the portal, hepatic and femoral veins), bile and urine and to utilize these data to in detail study the intestinal absorption, the liver extraction ratio and apply a semi-physiological based PK model to the data. The pigs received an intrajejunal dose of finasteride (0.8 mg/kg) either alone (n=5) or together with ketoconazole (10 mg/kg) (n=5), or an intravenous dose (0.2 mg/kg) (n=3). Plasma, bile and urine (collected from 0-6 hours) were analyzed with ultra performance liquid chromatography tandem mass spectrometry. Ketoconazole increased the bioavailability of finasteride from 0.36±0.23 to 0.91±0.1 (p<0.05) and the terminal half-life from 1.6±0.4 to 4.0±1.1 hours (p<0.05). From deconvolution it was found that the absorption rate from the intestine to the portal vein was rapid and the product of the fraction absorbed and the fraction that escaped gut wall metabolism was high (fa*FG≈1). Interestingly, the apparent absorption rate constant (k(a)) to the femoral vein was lower compared to the portal vein, probably because of binding and distribution within the liver. The liver extraction ratio was time-dependent and varied with the two routes of administration. After intrajejunal administration, from 1 6 hours the liver extraction ratio was significantly (p<0.05) reduced by the ketoconazole treatment from intermediate (0.41±0.21) to low (0.21±0.10).

  • 24.
    Lundahl, Anna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Lennernäs, Hans
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Knutson, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences.
    Bondesson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Hedeland, Mikael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Identification of Finasteride Metabolites in Human Bile and Urine by High-Performance Liquid Chromatography/Tandem Mass Spectrometry2009In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 37, no 10, p. 2008-2017Article in journal (Refereed)
    Abstract [en]

    The objective of this study was to further investigate the metabolism of the 5α-reductase inhibitor, finasteride, and to identify previously unknown phase I and phase II metabolites in vitro and in vivo in human bile and urine. Healthy volunteers were given 5 mg of finasteride, directly to the intestine, and bile and urine were collected for 3 and 24 h, respectively. A single-pass perfusion technique, Loc-I-Gut, was used for drug administration and bile collection from the proximal jejunum, distal to papilla of Vater. Incubations with human liver microsomes/S9 fractions and different cofactors were performed with finasteride and the previously known metabolites, ω-hydroxy finasteride (M1) and finasteride-ω-oic acid (M3). Liquid chromatography coupled to triple quadrupole mass spectrometry (MS) with positive/negative electrospray ionization and ion trap with MSn measurements were used for structural investigations and identification of metabolites. Two hydroxy metabolites of finasteride, other than M1, and one intact hydroxy finasteride glucuronide were identified in vitro and in bile and urine. The glucuronide and at least one of the hydroxy metabolites were previously unidentified. M1 and M3 were glucuronidated in vitro by specific human UDP-glucuronosyltransferases, UGT1A4 and UGT1A3, respectively. M1 glucuronide was not identified in vivo, and M3 glucuronide, an acyl glucuronide, was present in low amounts in bile from a few individuals. In conclusion, previously undescribed metabolites were identified, in vitro and in human urine and bile. Bile collection using the Loc-I-Gut technique followed by sensitive mass spectrometry analysis led to the discovery of novel, both phase I and phase II, finasteride metabolites in human bile.

  • 25.
    Lundquist, Patrik
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Englund, Gunilla
    Skogastierna, Cristine
    Loof, Johan
    Johansson, Jenny
    Hoogstraate, Janet
    Afzelius, Lovisa
    Andersson, Tommy B.
    Functional ATP-Binding Cassette Drug Efflux Transporters in Isolated Human and Rat Hepatocytes Significantly Affect Assessment of Drug Disposition2014In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 42, no 3, p. 448-458Article in journal (Refereed)
    Abstract [en]

    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.

  • 26.
    Lundquist, Patrik
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Loof, Johan
    Fagerholm, Urban
    Sjogren, Ingemo
    Johansson, Jenny
    Briem, Sveinn
    Hoogstraate, Janet
    Afzelius, Lovisa
    Andersson, Tommy B.
    Prediction of In Vivo Rat Biliary Drug Clearance from an In Vitro Hepatocyte Efflux Model2014In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 42, no 3, p. 459-468Article in journal (Refereed)
    Abstract [en]

    Well-established techniques are available to predict in vivo hepatic uptake and metabolism from in vitro data, but predictive models for biliary clearance remain elusive. Several studies have verified the expression and activity of ATP-binding cassette (ABC) efflux transporters central to biliary clearance in freshly isolated rat hepatocytes, raising the possibility of predicting biliary clearance from in vitro efflux measurements. In the present study, short-term plated rat hepatocytes were evaluated as a model to predict biliary clearance from in vitro efflux measurements before major changes in transporter expression known to take place in long-term hepatocyte cultures. The short-term cultures were carefully characterized for their uptake and metabolic properties using a set of model compounds. In vitro efflux was studied using digoxin, fexofenadine, napsagatran, and rosuvastatin, representing compounds with over 100-fold differences in efflux rates in vitro and 60-fold difference in measured in vivo biliary clearance. The predicted biliary clearances from short-term plated rat hepatocytes were within 2-fold of measured in vivo values. As in vitro efflux includes both basolateral and canalicular effluxes, pronounced basolateral efflux may introduce errors in predictions for some compounds. In addition, in vitro rat hepatocyte uptake rates corrected for simultaneous efflux predicted rat in vivo hepatic clearance of the biliary cleared compounds with less than 2-fold error. Short-term plated hepatocytes could thus be used to quantify hepatocyte uptake, metabolism, and efflux of compounds and considerably improve the prediction of hepatic clearance, especially for compounds with a large biliary clearance component.

  • 27.
    Lundquist, Patrik
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Loof, Johan
    Sohlenius-Sternbeck, Anna-Karin
    Floby, Eva
    Johansson, Jenny
    Bylund, Johan
    Hoogstraate, Janet
    Afzelius, Lovisa
    Andersson, Tommy B.
    The Impact of Solute Carrier (SLC) Drug Uptake Transporter Loss in Human and Rat Cryopreserved Hepatocytes on Clearance Predictions2014In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 42, no 3, p. 469-480Article in journal (Refereed)
    Abstract [en]

    Cryopreserved hepatocytes are often used as a convenient tool in studies of hepatic drug metabolism and disposition. In this study, the expression and activity of drug transporters in human and rat fresh and cryopreserved hepatocytes was investigated. In human cryopreserved hepatocytes, Western blot analysis indicated that protein expression of the drug uptake transporters [human Na+-taurocholate cotransporting polypeptide (NTCP), human organic anion transporting polypeptides (OATPs), human organic anion transporters, and human organic cation transporters (OCTs)] was considerably reduced compared with liver tissue. In rat cryopreserved cells, the same trend was observed but to a lesser extent. Several rat transporters were reduced as a result of both isolation and cryopreservation procedures. Immunofluorescence showed that a large portion of remaining human OATP1B1 and OATP1B3 transporters were internalized in human cryopreserved hepatocytes. Measuring uptake activity using known substrates of OATPs, OCTs, and NTCP showed decreased activity in cryopreserved as compared with fresh hepatocytes in both species. The reduced uptake in cryopreserved hepatocytes limited the in vitro metabolism of several AstraZeneca compounds. A retrospective analysis of clearance predictions of AstraZeneca compounds suggested systematic lower clearance predicted using metabolic stability data from human cryopreserved hepatocytes compared with human liver microsomes. This observation is consistent with a loss of drug uptake transporters in cryopreserved hepatocytes. In contrast, the predicted metabolic clearance from fresh rat hepatocytes was consistently higher than those predicted from liver microsomes, consistent with retention of uptake transporters. The uptake transporters, which are decreased in cryopreserved hepatocytes, may be rate-limiting for the metabolism of the compounds and thus be one explanation for underpredictions of in vivo metabolic clearance from cryopreserved hepatocytes.

  • 28.
    Matsson, Elin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Palm, Johan E.
    Eriksson, Ulf
    Bottner, Pernilla
    Lundahl, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Knutson, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences.
    Lennernäs, Hans
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Effects of ketoconazole on the in vivo biotransformation and hepatobiliary transport of the thrombin inhibitor AZD0837 in pigs2011In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 39, no 2, p. 239-246Article in journal (Refereed)
    Abstract [en]

    Ketoconazole has been shown in clinical trials to increase the plasma exposure of the oral anticoagulant prodrug AZD0837 [(2S)-N-{4- [(Z)-amino(methoxyimino)methyl]benzyl}-1-{(2R)-2-[3-chloro-5-(difluoromethoxy)phenyl]-2-hydroxyethanoyl}-azetidine-2-carboxamide] and its active metabolite, AR-H067637 [(2S)-N-{4-[amino(imino)methyl]benzyl}-1-{(2R)-2-[3-chloro-5-(difluoromethoxy)phenyl]-2-hydroxyethanoyl}-azetidine-2-carboxamide]. To investigate the biotransformation of AZD0837 and the effect of ketoconazole on this process, we used an experimental model in pigs that allows repeated sampling from three blood vessels, the bile duct, and a perfused intestinal segment. The pigs received AZD0837 (500 mg) given enterally either alone (n = 5) or together with single-dose ketoconazole (600 mg) (n = 6). The prodrug (n = 2) and its active metabolite (n = 2) were also administered intravenously to provide reference doses. The plasma data revealed considerable interindividual variation in the exposure of the prodrug, intermediate metabolite, and active metabolite. However, AR-H067637 was detected at very high concentrations in the bile with low variability (Ae(bile) = 53 ± 6% of the enteral dose), showing that the compound had indeed been formed in all of the animals and efficiently transported into the bile canaliculi. Concomitant dosing with ketoconazole increased the area under the plasma concentration-time curve for AZD0837 (by 99%) and for AR-H067637 (by 51%). The effect on the prodrug most likely arose from inhibited CYP3A-mediated metabolism. Reduced metabolism also seemed to explain the increased plasma exposure of the active compound because ketoconazole prolonged the terminal half-life with no apparent effect on the extensive biliary excretion and biliary clearance. These in vivo results were supported by in vitro depletion experiments for AR-H067637 in pig liver microsomes with and without the addition of ketoconazole.

  • 29. Okura, Takashi
    et al.
    Hattori, Asami
    Takano, Yusuke
    Sato, Takenori
    Hammarlund-Udenaes, Margareta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Terasaki, Tetsuya
    Deguchi, Yoshiharu
    Involvement of the pyrilamine transporter, a putative organic cation transporter, in blood-brain barrier transport of oxycodone.2008In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 36, no 10, p. 2005-2013Article in journal (Refereed)
    Abstract [en]

    The purpose of this study was to characterize blood-brain barrier (BBB) transport of oxycodone, a cationic opioid agonist, via the pyrilamine transporter, a putative organic cation transporter, using conditionally immortalized rat brain capillary endothelial cells (TR-BBB13). Oxycodone and [3H]pyrilamine were both transported into TR-BBB13 cells in a temperature- and concentration-dependent manner with Km values of 89 and 28 microM, respectively. The initial uptake of oxycodone was significantly enhanced by preloading with pyrilamine and vice versa. Furthermore, mutual uptake inhibition by oxycodone and pyrilamine suggests that a common mechanism is involved in their transport. Transport of both substrates was inhibited by type II cations (quinidine, verapamil, and amantadine), but not by classic organic cation transporter (OCT) substrates and/or inhibitors (tetraethylammonium, 1-methyl-4-phenylpyridinium, and corticosterone), substrates of OCTN1 (ergothioneine) and OCTN2 (L-carnitine), or organic anions. The transport was inhibited by metabolic inhibitors (rotenone and sodium azide) but was insensitive to extracellular sodium and membrane potential for both substrates. Furthermore, the transport of both substrates was increased at alkaline extracellular pH and decreased in the presence of a protonophore (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone). Intracellular acidification induced with ammonium chloride enhanced the uptakes, suggesting that the transport is driven by an oppositely directed proton gradient. The brain uptake of oxycodone measured by in situ rat brain perfusion was increased in alkaline perfusate and was significantly inhibited by pyrilamine. These results suggest that blood-brain barrier transport of oxycodone is at least partly mediated by a common transporter with pyrilamine, and this transporter is an energy-dependent, proton-coupled antiporter.

  • 30.
    Sadiq, Muhammad Waqas
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Salehpour, Mehran
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Ion Physics.
    Forsgård, Niklas
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Ion Physics.
    Possnert, Göran
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Ion Physics.
    Hammarlund-Udenaes, Margareta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Morphine brain pharmacokinetics at very low concentrations studied with Accelerator Mass Spectrometry and Liquid Chromatography-tandem Mass Spectrometry2011In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 39, no 2, p. 174-179Article in journal (Refereed)
    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.

  • 31.
    Sjödin, Elin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Fritsch, Holger
    Eriksson, Ulf G
    Logren, Ulrika
    Nordgren, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences.
    Forsell, Patrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences.
    Knutson, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences.
    Lennernäs, Hans
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Intestinal and hepatobiliary transport of ximelagatran and its metabolites in pigs2008In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 36, no 8, p. 1519-1528Article in journal (Refereed)
    Abstract [en]

    The direct thrombin inhibitor melagatran is formed from ximelagatran via two intermediate metabolites, OH-melagatran and ethylmelagatran. The biotransformation of ximelagatran does not involve cytochrome P450 isoenzymes, and it has been suggested that a reported interaction with erythromycin may instead be mediated by transport proteins. A pig model that simultaneously enables bile collection, sampling from three blood vessels and perfusion of a jejunal segment, was used to investigate the biotransformation of ximelagatran and the effect of erythromycin on the intestinal and hepatobiliary transport of ximelagatran and its metabolites. The pigs received enteral ximelagatran (n = 6), enteral ximelagatran together with erythromycin (n = 6), i.v. ximelagatran (n = 4), or i.v. melagatran (n = 4). The plasma exposure of the intermediates was found to depend on the route of ximelagatran administration. Erythromycin increased the area under the plasma concentration-time curve (AUC) of melagatran by 45% and reduced its biliary clearance from 3.0 +/- 1.3 to 1.5 +/- 1.1 ml/min/kg. Extensive biliary exposure of melagatran and ethylmelagatran, mediated by active transport, was evident from the 100- and 1000-fold greater AUC, respectively, in bile than in plasma. Intestinal efflux transporters seemed to be of minor importance for the disposition of ximelagatran and its metabolites considering the high estimated f(abs) of ximelagatran (80 +/- 20%) and the negligible amount of the compounds excreted in the perfused intestinal segment. These findings suggest that transporters located at the sinusoidal and/or canalicular membranes of hepatocytes determine the hepatic disposition of ximelagatran and its metabolites, and are likely to mediate the ximelagatran-erythromycin pharmacokinetic interaction.

  • 32.
    Sjögren, Erik
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Lennernäs, Hans
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Andersson, Tommy B
    Gråsjö, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Bredberg, Ulf
    The multiple depletion curves method provides accurate estimates of intrinsic clearance (CLint), maximum velocity of the metabolic reaction (Vmax), and Michaelis constant (Km): accuracy and robustness evaluated through experimental data and Monte Carlo simulations2009In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 37, no 1, p. 47-58Article in journal (Refereed)
    Abstract [en]

    The use of multiple depletion curves for the estimation of maximum velocity of the metabolic reaction (V(max)), the Michaelis constant (K(m)), and intrinsic clearance (CL(int)) was thoroughly evaluated by means of experimental data and through a series of Monte Carlo simulations. The enzyme kinetics of seven compounds were determined using the multiple depletion curves method (MDCM), the traditional initial formation rate of metabolite method (IFRMM), and the "in vitro t(1/2)" method, and the parameter estimates that were derived from the three methods were compared. The impact of a change in enzyme activity during the incubation period on the parameter estimates and the possibility to correct for this were also investigated. The MDCM was in good overall agreement with the IFRMM. Correction for a change in enzyme activity was possible and resulted in a better concordance in CL(int) estimates. The robustness of the method in coping with different rates of substrate turnover and variable starting concentrations were also demonstrated through Monte Carlo simulations. Furthermore, the limitations imposed by assumptions inherent in the in vitro t(1/2) method were demonstrated both experimentally and by simulations. This study demonstrates that the MDCM is a robust and efficient method for estimating enzyme kinetic variables with high accuracy and precision. The method may potentially be used in a wide range of applications, from pure enzyme kinetics to in vitro-based predictions of the pharmacokinetics of compounds with multiple and/or unknown metabolic pathways.

  • 33.
    Sjögren, Erik
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Nyberg, Joakim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Magnusson, Mats O.
    Lennernäs, Hans
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Hooker, Andrew C.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Bredberg, Ulf
    Optimal experimental design for assessment of enzyme kinetics in a drug discovery screening environment2011In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 39, no 5, p. 858-863Article in journal (Refereed)
    Abstract [en]

    A penalized ED-optimal design with a discrete parameter distribution was used to find an optimal experimental design for assessment of enzyme kinetics in a screening environment. A data set for enzyme kinetic data (Vmax and Km) was collected from previously reported studies and every Vmax/Km pair (n=76) was taken to represent a unique drug compound. The design was restricted to 15 samples, an incubation time of up to 40 min and starting concentrations (C0) for the incubation between 0.01 and 100 µM. The optimization was performed by finding the sample times and C0 returning the lowest uncertainty (SE) of the model parameter estimates. Individual optimal designs (I-OD), one general optimal design (G-OD) and one for laboratory practice pragmatically modified design (OD) were obtained. In addition, a standard design (STD-D), representing a commonly applied approach for metabolic stability investigations, was constructed. Simulations were performed for OD and STD-D using the Michaelis-Menten (MM) equation and enzyme kinetic parameters were estimated both with MM and a mono exponential (EXP) decay. OD generated a better result (RSE) for 99% of the compounds and an equal or better result (RMSE) for 78% of the compounds. Furthermore, high-quality estimates (RMSE <30%) of both Vmax and Km could be obtained for a considerable number (26%) of the investigated compounds. The results presented in this study demonstrate that the output could generally be improved when compared to that obtained from the standard approaches used today.

  • 34.
    Sjögren, Erik
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Svanberg, Petter
    Kanebratt, Kajsa P.
    Optimized Experimental Design for the Estimation of Enzyme Kinetic Parameters: An Experimental Evaluation2012In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 40, no 12, p. 2273-2279Article in journal (Refereed)
    Abstract [en]

    A set of compounds (n = 30), including traditional cytochrome P450 substrates and compounds from AstraZeneca's compound library, was used in an experimental evaluation of an optimal design approach (ODA) for the estimation of enzyme kinetic parameters (CLint, V-max, and K-m). A depletion method previously shown to provide reliable results, the multiple depletion curves method (MDCM), was used as reference. Experiments were conducted with human liver microsomes, and samples were analyzed using liquid chromatography-tandem mass spectrometry. CLint estimated with the ODA were in >90% of the cases within a 2-fold difference compared with MDCM estimates. In addition, good agreement was generally seen for V-max and K-m estimates between the two methods as >80% of the estimates were within or almost within a 2-fold difference. The variability in V-max and K-m estimates were generally higher than for CLint estimates. In addition, decreased substrate turnover considerably increased the variability in V-max and Km estimates, whereas only a modest increase was observed for CLint estimates. The experimental design of using multiple starting concentrations for the estimation of enzyme kinetics was shown to be appropriate even when there was a limitation to the number of samples. The method allowed for good estimates of CLint and also for V-max and K-m in many cases. Hence, this approach is a good alternative for the estimation of enzyme kinetic parameters, especially if enzyme saturation and an assessment of a potential risk for nonlinear metabolism are of interest.

  • 35.
    Svensson, Ulrika S H
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Sandstrom, R
    Carlborg, Örjan
    Swedish University of Agricultural Sciences, Uppsala, Sweden.
    Lennernas, H
    Ashton, M
    High in situ rat intestinal permeability of artemisinin unaffected by multiple dosing and with no evidence of P-glycoprotein involvement1999In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 27, no 2, p. 227-232Article in journal (Refereed)
    Abstract [en]

    The objective of this study was to investigate whether the decrease in artemisinin bioavailability after repeated oral dosing in humans can be a result of increased efflux of artemisinin by P-glycoprotein or decreased membrane transport at the intestinal barrier, The effective jejunal permeability (P-eff) of artemisinin was investigated using an in situ rat perfusion model. Fifty-four rats were randomized to one of three treatment arms: no pretreatment, pretreatment with artemisinin emulsion for 5 days (60 mg/kg/day, p.o.), or pretreatment with emulsion vehicle for 5 days, The rats within each treatment arm were randomized further to be jejunally perfused with either low (500 ng/ml) or high (5000 ng/ml) artemisinin concentration or low artemisinin concentration plus the P-glycoprotein inhibitor R,S-verapamil (400 mu g/ml). Perfusate samples were assayed for content of artemisinin, R,S-verapamil, and perfusion viability markers. Artemisinin P-eff was 1,44 +/- 0.38, 1.17 +/- 0.32, and 1.71 +/- 0.29 (.10(-4), cm/s) in rats receiving no pretreatment and perfused with low, high, or low artemisinin concentration plus verapamil, respectively. Multiple oral dosing of artemisinin did not affect the jejunal permeability of artemisinin, R,S-verapamil P-eff was similar in artemisinin-pretreated rats (1.09 +/- 0.54.10(-4), cm/s) and rats pretreated with only vehicle (1.07 +/- 0.37.10(-4), cm/s), The decrease in artemisinin bioavailability after multiple oral dosing in human is probably not a result of changes in P-glycoprotein expression or general intestinal transport. It seems more likely attributed to increased hepatocellular activity. Furthermore, artemisinin exhibits high jejunal permeability and is neither a substrate nor inducer of P-glycoprotein.

  • 36.
    Svensson, Ulrika S H
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Sandstrom, R
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Carlborg, Örjan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Lennernas, H
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Ashton, M
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    High in situ rat intestinal permeability of artemisinin unaffected bymultiple dosing and with no evidence of P-glycoprotein involvement1999In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 27, no 2, p. 227-232Article in journal (Refereed)
  • 37.
    Syvänen, Stina
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Lindhe, Örjan
    Palner, Mikael
    Kornum, Birgitte R
    Rahman, Obaidur
    Långström, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Knudsen, Gitte M
    Hammarlund-Udenaes, Margareta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Species differences in blood-brain barrier transport of three positron emission tomography radioligands with emphasis on P-glycoprotein transport2009In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 37, no 3, p. 635-643Article in journal (Refereed)
    Abstract [en]

    Species differences occur in the brain concentrations of drugs, but the reasons for these differences are not yet apparent. This study was designed to compare brain uptake of three radiolabeled P-glycoprotein (P-gp) substrates across species using positron emission tomography. Brain concentrations and brain-to-plasma ratios were compared; [11C]verapamil in rats, guinea pigs, and monkeys; [11C](S)-(2-methoxy-5-(5-trifluoromethyltetrazol-1-yl)-phenylmethylamino)-2(S)-phenylpiperidine (GR205171) in rats, guinea pigs, monkeys, and humans; and [18F]altanserin in rats, minipigs, and humans. The fraction of the unbound radioligand in plasma was studied along with its metabolism. The effect of P-gp inhibition was investigated by administering cyclosporin A (CsA). Pronounced species differences were found in the brain and brain-to-plasma concentrations of [11C]verapamil, [11C]GR205171, and [18F]altanserin with higher brain distribution in humans, monkeys, and minipigs than in rats and guinea pigs. For example, the brain-to-plasma ratio of [11C]GR205171 was almost 9-fold higher in humans compared with rats. The species differences were still present after P-gp inhibition, although the increase in brain concentrations after P-gp inhibition was somewhat greater in rats than in the other species. Differences in plasma protein binding and metabolism did not explain the species-related differences. The findings are important for interpretation of brain drug delivery when extrapolating preclinical data to humans. Compounds found to be P-gp substrates in rodents are likely to also be substrates in higher species, but sufficient blood-brain barrier permeability may be retained in humans to allow the compound to act at intracerebral targets.

  • 38. Tarning, J.
    et al.
    Bergqvist, Yngve
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Day, N.P.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry.
    Arvidsson, Björn
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry.
    White, N.J.
    Ashton, M.
    Lindegårdh, Niklas
    Characterization of Human Urinary Metabolites of the Antimalarial Piperaquine2006In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 34, no 12, p. 2011-2019Article in journal (Refereed)
    Abstract [en]

    Five metabolites of the antimalarial piperaquine (PQ) (1,3-bis-[4-(7chloroquinolyl-4)-piperazinyl-1]-propane) have been identified and their molecular structures characterized. After a p.o. dose of dihydroartemisinin-piperaquine, urine collected over 16 h from two healthy subjects was analyzed using liquid chromatography (LC)/UV, LC/tandem mass spectrometry (MS/MS), Fourier transform ion cyclotron resonance (FTICR)/MS, and H NMR. Five different peaks were recognized as possible metabolites [M1, 320 m/z; M2, M3, and M4, 551 m/z (PQ + 16 m/z); and M5, 567 m/z (PQ + 32 m/z)] using LC/MS/MS with gradient elution. The proposed carboxylic M1 has a theoretical monoisotopic molecular mass of 320.1166 m/z, which is in accordance with the FTICR/MS (320.1168 m/z) findings. The LC/MS/MS results also showed a 551 m/z metabolite (M2) with a distinct difference both in polarity and fragmentation pattern compared with PQ, 7-hydroxypiperaquine, and the other 551 m/z metabolites. We suggest that this is caused by N-oxidation of PQ. The results showed two metabolites (M3 and M4) with a molecular ion at 551 m/z and similar fragmentation pattern as both PQ and 7-hydroxypiperaquine; therefore, they are likely to be hydroxylated PQ metabolites. The molecular structures of M1 and M2 were also confirmed using H NMR. Urinary excretion rate in one subject suggested a terminal elimination half-life of about 53 days for M1. Assuming formation rate-limiting kinetics, this would support recent findings that the terminal elimination half-life of PQ has been underestimated previously.

  • 39.
    Thörn, Helena Anna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Hedeland, Mikael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Bondesson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Knutson, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences.
    Yasin, Mohammed
    Dickinson, Paul
    Lennernäs, Hans
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Different effects of ketoconazole on the stereoselective first-pass metabolism of R/S-verapamil in the intestine and the liver: important for the mechanistic understanding of first-pass drug-drug interactions2009In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 37, no 11, p. 2186-2196Article in journal (Refereed)
    Abstract [en]

    In this acute study a pig jejunal intestinal perfusion model with multiple plasma sampling sites and three different administration routes was used to investigate the quantitative contribution of the intestine versus liver to the first-pass extraction of each enantiomer of verapamil (VER). A subclinical dose of ketoconazole (8 mg) was coadministered in the perfusion solution to selectively inhibit gut wall CYP3A. Both enantiomers of VER and its main metabolite norverapamil (NOR) as well as the inhibitor ketoconazole were quantified in all plasma compartments by liquid chromatography-tandem mass spectrometry. The overall first-pass metabolic extraction of VER and the metabolite NOR was shown to be stereoselective with the S-isomer being more extensively extracted. For VER the ratio of R- enantiomer to S-enantiomer was greater in the hepatic vein than in the portal vein (approximately 2.2 versus 1.4), indicating that the stereoselective metabolism of VER in pigs mainly occurs on the first pass through the liver and not in the intestine. Ketoconazole increased the area under the curve from time 0 to 6 h and C(max) of R- and S-VER at least 3-fold in the portal vein, most likely explained by inhibition of gut wall metabolism. Conversely, hepatic extraction was increased because the effect of gut wall metabolism was not observed at the peripheral sampling sites. In conclusion, this study provided novel and more direct information on the contribution of the intestine and the liver, respectively, to the overall first-pass extraction of racemic VER.

  • 40.
    Vildhede, Anna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Karlgren, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Svedberg, Elin K.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Wisniewski, Jacek R.
    Lai, Yurong
    Norén, Agneta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Upper Abdominal Surgery.
    Artursson, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Hepatic Uptake of Atorvastatin: Influence of Variability in Transporter Expression on Uptake Clearance and Drug-Drug Interactions2014In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 42, no 7, p. 1210-1218Article in journal (Refereed)
    Abstract [en]

    Differences in the expression and function of the organic anion transporting polypeptide (OATP) transporters contribute to interindividual variability in atorvastatin clearance. However, the importance of the bile acid transporter sodium taurocholate cotransporting polypeptide (NTCP, SLC10A1) in atorvastatin uptake clearance (CLupt) is not yet clarified. To elucidate this issue, we investigated the relative contribution of NTCP, OATP1B1, OATP1B3, and OATP2B1 to atorvastatin CLupt in 12 human liver samples. The impact of inhibition on atorvastatin CLupt was also studied, using inhibitors of different isoform specificities. Expression levels of the four transport proteins were quantified by liquid chromatography tandem mass spectrometry. These data, together with atorvastatin in vitro kinetics, were used to predict the maximal transport activity (MTA) and interindividual differences in CLupt of each transporter in vivo. Subsequently, hepatic uptake impairment on coadministration of five clinically interacting drugs was predicted using in vitro inhibitory potencies. NTCP and OATP protein expression varied 3.7- to 32-fold among the 12 sample donors. The rank order in expression was OATP1B1 > OATP1B3 approximate to NTCP approximate to OATP2B1. NTCP was found to be of minor importance in atorvastatin disposition. Instead, OATP1B1 and OATP1B3 were confirmed as the major atorvastatin uptake transporters. The average contribution to atorvastatin uptake was OATP1B1 > OATP1B3 >> OATP2B1 > NTCP, although this rank order varied among individuals. The interindividual differences in transporter expression and CLupt resulted in marked differences in drug-drug interactions due to isoform-specific inhibition. We conclude that this variation should be considered in in vitro to in vivo extrapolations.

  • 41.
    Vildhede, Anna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Mateus, André
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Khan, Elin K.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Lai, Yurong
    Bristol Myers Squibb Co, Princeton, NJ USA.
    Karlgren, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Artursson, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Kjellsson, Maria C.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Mechanistic Modeling of Pitavastatin Disposition in Sandwich-Cultured Human Hepatocytes: A Proteomics-Informed Bottom-Up Approach2016In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 44, no 4, p. 505-516Article in journal (Refereed)
    Abstract [en]

    Isolated human hepatocytes are commonly used to predict transporter-mediated clearance in vivo. Such predictions, however, do not provide estimations of transporter contributions and consequently do not allow predictions of the outcome resulting from a change in the activity of a certain transporter, e.g., by inhibition or a genetic variant with reduced function. Pitavastatin is a drug that is heavily dependent on hepatic transporters for its elimination and it is mainly excreted as unchanged drug in the bile. For this reason, pitavastatin was used as a model drug to demonstrate the applicability of a bottom-up approach to predict transporter-mediated disposition in sandwich-cultured human hepatocytes (SCHH), allowing for the estimation of transporter contributions. Transport experiments in transfected HEK293 cells and inverted membrane vesicles overexpressing each of the relevant transport proteins were used to generate parameter estimates for the mechanistic model. By adjusting for differences in transporter abundance between the in vitro systems and individual SCHH batches, the model successfully predicted time profiles of medium and intracellular accumulation. Our predictions of pitavastatin bile accumulation could, however, not be confirmed due to a very low biliary excretion of pitavastatin in relation to the hepatic uptake in our SCHH. This study is, to our knowledge, the first to successfully simulate transporter-mediated processes in a complex system such as SCHH at the level of individual transport proteins using a bottom-up approach.

  • 42.
    Weiss, Frederik
    et al.
    Univ Tubingen, NMI Nat & Med Sci Inst, Markwiesenstr 55, D-72770 Reutlingen, Germany;SIGNATOPE GmbH, Reutlingen, Germany.
    Hammer, Helen S.
    Univ Tubingen, NMI Nat & Med Sci Inst, Markwiesenstr 55, D-72770 Reutlingen, Germany;SIGNATOPE GmbH, Reutlingen, Germany.
    Klein, Kathrin
    Dr Margarete Fischer Bosch Inst Clin Pharmacol, Stuttgart, Germany;Univ Tubingen, Dept Clin Pharmacol, Tubingen, Germany.
    Planatscher, Hannes
    Univ Tubingen, NMI Nat & Med Sci Inst, Markwiesenstr 55, D-72770 Reutlingen, Germany;SIGNATOPE GmbH, Reutlingen, Germany.
    Zanger, Ulrich M.
    Dr Margarete Fischer Bosch Inst Clin Pharmacol, Stuttgart, Germany;Univ Tubingen, Dept Clin Pharmacol, Tubingen, Germany.
    Norén, Agneta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Upper Abdominal Surgery.
    Wegler, Christine
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. Innovat Med & Early Dev Biotech Unit, Cardiovasc & Metab Dis, Molndal, Sweden.
    Artursson, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Joos, Thomas O.
    Univ Tubingen, NMI Nat & Med Sci Inst, Markwiesenstr 55, D-72770 Reutlingen, Germany;SIGNATOPE GmbH, Reutlingen, Germany.
    Poetz, Oliver
    Univ Tubingen, NMI Nat & Med Sci Inst, Markwiesenstr 55, D-72770 Reutlingen, Germany;SIGNATOPE GmbH, Reutlingen, Germany.
    Direct Quantification of Cytochromes P450 and Drug Transporters-A Rapid, Targeted Mass Spectrometry-Based Immunoassay Panel for Tissues and Cell Culture Lysates2018In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 46, no 4, p. 387-396Article in journal (Refereed)
    Abstract [en]

    The quantification of drug metabolizing enzymes and transporters has recently been revolutionized on the basis of targeted proteomic approaches. Isotope-labeled peptides are used as standards for the quantification of the corresponding proteins in enzymatically fragmented samples. However, hurdles in these approaches are low throughput and tedious sample prefractionation steps prior to mass spectrometry (MS) readout. We have developed an assay platform using sensitive and selective immunoprecipitation coupled with mass spectrometric readout allowing the quantification of proteins directly from whole cell lysates using less than 20,000 cells per analysis. Peptide group-specific antibodies (triple X proteomics antibodies) enable the enrichment of proteotypic peptides sharing a common terminus. These antibodies were employed to establish a MS-based immunoassay panel for the quantification of 14 cytochrome P450 (P450) enzymes and nine transporters. We analyzed the P450 enzyme and transporter levels in genotyped liver tissue homogenates and microsomes, and in samples from a time course induction experiment in human hepatocytes addressing different induction pathways. For the analysis of P450 enzymes and transporters only a minute amount of sample is required and no prefractionation is necessary, thus the assay platform bears the potential to bridge cell culture model experiments and results from whole organ tissue studies.

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