Anna-Karin Sohlenius-Sternbeck

AstraZeneca, Tukholma, Stockholm, Sweden

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Publications (9)25.86 Total impact

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    ABSTRACT: This review details how drug metabolism and pharmacokinetics (DMPK) and physicochemical deliveries played an important role in data interpretation and compound optimization at AstraZeneca R&D in Södertälje, Sweden. A selection of assays central in the evaluation of the DMPK properties of new chemical entities is presented, with guidance and consideration on assay outcome interpretation. Early in projects, solubility, LogD, permeability and metabolic stability were measured to support effective optimization of DMPK properties. Changes made to facilitate high throughput, efficient bioanalysis and the handling of large amounts of samples are described. In order for a drug to have a desired pharmacological effect it has to have the right properties to be able to reach the target site in sufficient concentration. The disposition of a drug is dependent on interactions between the body and the drug, its molecular properties and the physical and biological barriers presented in the body. The optimization of chemical series is a complex process that is dependent on a range of assessments and considerations. Already early in drug discovery, we used an integrated approach for the prediction of the fate of drugs in human (early dose to man) based on data obtained from in vitro experiments. The early dose to man was refined with project progression, which triggered more intricate assays and experiments. At later stages, preclinical in vivo pharmacokinetic (PK) data was integrated with pharmacodynamics (PD) to allow predictions of required dose, dose intervals and exposure profile to achieve the desired effect in man.
    No preview · Article · Dec 2015 · Current Drug Metabolism
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    ABSTRACT: 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 (hNTCP, hOATPs, hOATs and hOCTs) was considerably reduced compared to 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 hOATP1B1 and hOATP1B3 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 to 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 to 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 under-predictions of in vivo metabolic clearance from cryopreserved hepatocytes.
    Preview · Article · Jan 2014 · Drug metabolism and disposition: the biological fate of chemicals
  • Sanja Juric · Patrik Lundquist · Yin Hu · Anna Juréus · Anna-Karin Sohlenius-Sternbeck
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    ABSTRACT: Abstract 1. Human hepatocytes that had been cold-preserved in SureTran(TM) matrix (Abcellute Ltd, Cardiff, UK) were used for studies on cell viability, cytochrome P450 (CYP) 3A4, 2B6 and 1A2 induction and hepatic drug transporters. It has recently been shown that basal CYP activities are maintained in cold-preserved hepatocytes (Palmgren et al., 2012). 2. After 5 d of cold preservation, the viability was still more than 70%, and after 8 d it was around 60%. In hepatocytes that had been cold-preserved for 3 d, the activity of CYP3A4 was induced around 15-fold upon treatment with 8 µM rifampicin for 72 h. For CYP2B6, the activity was induced 4- to 16-fold in hepatocytes that had been cold-preserved for 3 d and thereafter treated with 1 mM phenobarbital for 72 h. The activity of CYP1A2 was low and close to the limit of detection in non-treated cells that had been cold-preserved for up to 3 d, while the activity increased in cells treated with 0.3-25 µM β-naphthoflavone for 72 h. CYP3A4, 2B6 and 1A2 mRNA levels were only determined with hepatocytes from one donor and increased upon treatment with the inducers. 3. Hepatic uptakes of estrone-3-sulfate, taurocholate, ipratropium and rosuvastatin were stable in human hepatocytes that had been cold-preserved for up to 2 d. 4. In summary, cold-preserved human hepatocytes demonstrate retained viability and can advantageously be used for in vitro induction studies and for studies of hepatic uptake transporters.
    No preview · Article · Apr 2013 · Xenobiotica
  • Anna-Karin Sohlenius-Sternbeck · Urban Fagerholm · Johan Bylund
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    ABSTRACT: Abstract 1. We applied the regression offset approach to predict rat in vivo intrinsic clearance (CL(int)) for 54 new chemical acid entities with high plasma protein binding values and low renal clearance (CL). The prediction success was correlated to volume of distribution (V(d)), molecular weight (Mw) and CL. 2. A correlation between poor in vitro-in vivo extrapolation (IVIVE) and V(d) values distinct from the V(d) of albumin (0.1-0.2 L/kg) was revealed. For compounds with a V(d) value above 0.5 L/kg, 0% of the predictions of in vivo CL(int) was within twofold of the observed value, compared to 69% for compounds with a V(d) value below 0.5 L/kg. 3.  Compounds with a Mw below 450 g/mol demonstrated more accurate in vivo CL(int) predictions than compounds with a Mw above 450 g/mol, i.e. 63% compared to 21% within twofold. For compounds with in vivo CL below 30% of the liver blood flow (LBF), 53% of the predictions was within twofold of the observed value, compared to 0% for compounds with CL above 30% of the LBF. 4. We show that accurate IVIVE for acidic compounds with high plasma protein binding and low renal CL can be associated with a low V(d) (i.e. around the V(d) of albumin) and with a low in vivo CL, and that Mw is an important optimization parameter for pharmacokinetic. This study also further demonstrates the advantages of the application of the regression method for identifying cases when metabolic CL is not the single major elimination pathway.
    No preview · Article · Jan 2013 · Xenobiotica
  • Tjerk Bueters · Sanja Juric · Anna-Karin Sohlenius-Sternbeck · Yin Hu · Johan Bylund
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    ABSTRACT: Abstract 1. Intestinal loss, 1 - (F(obs)/f(H)), is the missing fraction of the dose that is unexplained by systemic clearance. Here, we investigated whether intestinal loss in rat is predictive for human, and whether intestinal metabolism explained observed differences between rat and human. 2. For 81 marketed drugs, human and rat intestinal loss values were calculated from the literature and in-house sources. To examine the contribution of intestinal cytochrome P450-mediated metabolism to the high observed intestinal loss in the rat, metabolism was determined in rat and human intestinal microsomes for 15 compounds. 3. Oral bioavailability poorly correlated between rat and human. Twenty-two compounds in the human and 47 compounds in the rat showed an intestinal loss of more than 20%. The intestinal availability for many compounds was higher in human than in rat. Selected compounds, however, were more stable in rat than in human intestinal microsomes. 4. The rat poorly predicts the risk for intestinal loss in human; many compounds in rat had lower bioavailability than anticipated based on the hepatic clearance, but demonstrated little intestinal loss in human. This discrepancy appeared not to be caused by a higher cytochrome P450-mediated intestinal metabolism in the rat.
    No preview · Article · Jan 2013 · Xenobiotica
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    ABSTRACT: Systematic under-prediction of clearance is frequently associated with in vitro kinetic data when extrapolated using physiological scaling factors, appropriate binding parameters and the well-stirred model. The present study describes a method of removing this systematic bias through application of empirical correction factors derived from regression analyses applied to the in vitro and in vivo data for a defined set of reference compounds. Linear regression lines were established with in vivo intrinsic clearance (CLint), derived from in vivo clearance data and scaled in vitro intrinsic clearance from isolated hepatocyte incubations. The scaled CLint was empirically corrected to a predicted in vivo CLint using the slope and intercept from a uniform weighted linear regression applied to the in vitro to in vivo extrapolation. Cross validation of human data demonstrated that 66% of the reference compounds had a predicted in vivo CLint within two-fold of the observed value. The average absolute fold error (AAFE) for the in vivo CLint predictions was 1.90. For rat, 54% of the compounds had a predicted value within two-fold of the observed and the AAFE was 1.98. Three AstraZeneca projects are used to exemplify how a two-sided prediction interval, applied to the rat regression corrected reference data, can form the basis for assessing the likelihood that, for a given chemical series, the in vitro kinetic data is predictive of in vivo clearance and is therefore appropriate to guide optimisation of compound metabolic stability.
    No preview · Article · Apr 2012 · Xenobiotica
  • Anna-Karin Sohlenius-Sternbeck
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    ABSTRACT: Biologically based scaling factors have to be used to predict in vivo metabolic clearance of xenobiotics from data obtained in vitro. Although standard values for the hepatocellularity numbers for different species are used in the literature, detailed information on the determination of these values has only been presented for humans and rats, and somewhat different results have been obtained in different studies. The present work was undertaken in order to determine the number of hepatocytes per gram of liver for human, dog, rabbit, rat and mouse livers. Hepatocellularity numbers were calculated from the ratio between the liver protein concentration and the protein concentration in the corresponding hepatocyte suspension. For human, rabbit, rat and mouse livers, the hepatocellular values were in the same range, more precisely 139+/-25, 114+/-20, 117+/-30 and 135+/-10 million cells per gram of liver, respectively. However, for the dog liver, the corresponding value was as high as 215+/-45 million cells per gram. These values should be of importance during the scaling process of intrinsic clearance for xenobiotics in hepatocytes to in vivo hepatic clearance.
    No preview · Article · Dec 2007 · Toxicology in Vitro
  • Jose Castro-Perez · John Shockcor · Peter Jackson · Sveinn Briem · Anna-Karin Sohlenius-Sternbeck · Eva Floby
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    ABSTRACT: Over the years we have seen many developments on the hardware and software for the mass spectrometers that has allowed the users to benefit from lower levels of detection and ease-of use. One area in which development pace has been slower is in HPLC. Improving the chromatographic resolution will allow us to obtain superior separations with high peak capacity, thus reducing co-elution of metabolites and also enhance the sensitivity in the MS system but reducing ion suppression. Having better chromatographic separations will not only help to detect more metabolites which were co-eluting before but also to reduce ion suppression'. To investigate this we have employed an Ultra Performance Liquid Chromatography system (UPLCTM). By the use of this technology, it will allow us to run faster and separations with excellent peak capacities for very complicated biological matrices. This instrument has been coupled to a hybrid quadrupole-TOF mass spectrometer (QTof Premier TM). In order to show the potential of this particular development, we will show the results from the analysis of a number of marker compounds Midazolam, Phenacetin, Diclofenac, Diazepam and all drugs mentioned incubated as a cocktail. The substrates were incubated at 2uM using rat hepatocytes. The full scan TOF-MS sensitivity allowed very good levels of detection for all samples analyzed with exact mass and excellent semi-quantitative properties. The run times for each sample were 5 minutes in comparison with a typical run time of 20 minutes when the same experiments were carried out using HPLC. No difference in the CLint of parent compounds and their metabolites formed was observed when incubated individually or as a cocktail. Moreover, with a single injection both rate of disappearance of the parent compound and metabolites formed were reported simultaneously by the use of an in-house software algorithm which data mined the raw data. This software algorithm used an exact mass data filter which allowed the removal of false positives.
    No preview · Conference Paper · Jan 2006
  • Anna-Karin Sohlenius-Sternbeck · Achim Orzechowski
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    ABSTRACT: Metabolism of testosterone to various products (catalyzed by several different CYP isozymes) and the activities of phenol sulfotransferase (pST) and glutathione transferase (GST) in S9 fractions prepared from the mucosa of the duodenum, jejunum, ileum, caecum and upper and lower colon of male Sprague-Dawley rats were determined and compared to the corresponding hepatic and renal activities. Incubation of the S9 fraction prepared from the jejunum with testosterone and NADPH resulted in the formation of 2alpha-, 6alpha-, 6beta- and 16alpha-hydroxytestosterone and androstenedione at rates that were 1.6, 24, 1.3, 0.6 and 1.3%, respectively, of the corresponding hepatic values. The production of 2alpha-hydroxytestosterone was catalyzed only by the preparations from the duodenum and jejunum; whereas 6alpha-, 6beta- and 16alpha-hydroxytestosterone and androstenedione were produced in all regions of the intestine. In the case of the rat kidney, the rates of formation of the different testosterone metabolites were between 0.6 and 35% of the corresponding liver activity. The activity of glutathione transferase was approximately 12-26% of the corresponding hepatic activity throughout the intestine. The highest activity of phenol sulfotransferase was observed in the lower colon (almost 6% of the liver activity) and the lowest activity in the duodenum (1%). The renal activities of GST and pST were 70 and 1%, respectively, of the corresponding liver values. In summary, the metabolism of testosterone and the activities of GST and pST in rat intestine are generally low to very low in comparison to the corresponding activities in rat liver. In most cases, these activities are present throughout the entire intestine and not restricted to a particular portion(s) of this organ.
    No preview · Article · Jul 2004 · Chemico-Biological Interactions