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A dataset consisting of measured values for LogD, solubility, metabolic stability in human liver microsomes (HLM) and Caco-2 permeability was used to evaluate the prediction models for lipophilicity (S+LogD), water solubility (S+Sw_pH), metabolic stability in HLM (CYP_HLM_Clint), intestinal permeability (S+Peff) and P-gp substrate identification (P-gp substrate) in the software ADMET PredictorTM (AP) from Simulations Plus. The dataset consisted of a total of 4794 compounds, with at least data from metabolic stability determinations in HLM, from multiple discovery projects at Medivir. Our evaluation shows that the global AP models can be used for categorization of high and low values based on predicted results for metabolic stability in HLM and intestinal permeability, and to give good predictions of LogD (R2=0.79), guiding the synthesis of new compounds and for prioritzing in vitro ADME experiments. The model seems to overpredict solubility for the Medivir compounds, however. We also used the in-house datasets to build local models for LogD, solubility, metabolic stability and permeability by using artificial neural network (ANN) models in the optional ModelerTM module of AP. Predictions of the test sets were performed with both the global and the local models and the R2 value for linear regression for predicted versus measured HLM CLint based on logarithmic data was 0.72 for the in-house model and 0.53 for the AP model. The improved predictions with the local models are likely explained both by the specific chemical space of the Medivir dataset and lab specific assay conditions for parameters which require biological assay systems. Significance Statement AP is useful early in projects for predicting and categorizing LogD, metabolic stability and permeability, to guide the synthesis of new compounds and for prioritizing in vitro ADME experiments. The building of local in-house prediction models with the optional AP Modeler Module can give improved prediction success since these models are built on data from the same experimental setup and can also be based on compounds with similar structures.

1. A set of reference compounds for time-dependent inhibition (TDI) of cytochrome P450 with available literature data for kinact and KI was used to predict clinical implications using the GastroPlusTM software. Comparisons were made to in vivo literature interaction data. 2. The predicted AUC ratios (AUC+inhibitor/AUCcontrol) could be compared with the observed ratios from literature for all compounds with detailed information about in vivo administration, pharmacokinetics and in vivo interactions (N = 21). For this dataset, the difference between predicted and observed AUC ratios for interactions with midazolam was within twofold for all compounds except one (telaprevir, for which non-CYP-mediated metabolism likely plays a role after multiple dosing). 3. The sensitivity, specificity and accuracy of the GastroPlusTM predictions using a binary classification as no-to-weak interaction versus moderate-to-strong interaction for all compounds with available in vivo interaction data, were 80%, 82% and 81%, respectively (N = 31). 4. As a result of our evaluations of the DDI module in GastroPlusTM, we have implemented an early TDI risk assessment decision tree for our drug discovery projects involving in vitro screening and early GastroPlusTM predictions. Shifted IC50 values are determined and kinact/KI estimated (by using a regression line established with in house-shifted IC50 values and literature kinact/KI ratios), followed by GastroPlusTM predictions.

Background: 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. 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. 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 Sodertalje, Sweden. Methods: 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. 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. Results and Conclusions: A well-defined work flow of DMPK activities from early lead identification up to the selection of a candidate drug was developed. This resulted in a cost effective and efficient optimization of chemical series, and facilitated informed decision making throughout project progress.

1. A set of reference compounds for time-dependent inhibition (TDI) of cytochrome P450 with available literature data for k

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.

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). 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. 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. 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.

1. We compared the intrinsic clearance (CL(int)) of a number of substrates in suspensions of fresh and cryopreserved human hepatocytes from seven donors. 2. CL(int) values for a cocktail incubation of phenacetin, diclofenac, diazepam, bufuralol, midazolam, and hydroxycoumarin were 4.9 +/- 3.4, 18 +/- 7.2, 5.1 +/- 4.9, 6.3 +/- 3.3, 9.8 +/- 5.8 and 22 +/- 14 microl min(-1)/10(6) cells, respectively, and they correlated well with corresponding CL(int) values using cryopreserved hepatocytes from 25 different donors. 3. CL(int) values of each cocktail substrate and 20 AstraZeneca new chemical entities were compared in fresh and cryopreserved hepatocytes from the same three donors. There was a statistically significant correlation between CL(int) in fresh and cryopreserved hepatocytes for each of the three livers (p0.002) and the geometric mean of the ratio of fresh to cryopreserved CL(int) values was 1.03. 4. In conclusion, the results add further support to the use of cryopreserved human hepatocytes as a screening model for the intrinsic clearance of new chemical entities.

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.

The activity of glutathione transferase was measured in sonicates of fresh rat hepatocytes and of cryopreserved rat, human and dog hepatocytes in the presence of added glutathione and by using 1-chloro-2,4-dinitrobenzene (CDNB) as non-selective substrate. The glutathione-conjugating capacity was also investigated in the presence of CDNB alone (without glutathione) with intact fresh rat hepatocytes and cryopreserved rat and human hepatocytes. Finally, the intracellular level of glutathione was measured in these hepatocytes. The specific activity of glutathione transferase in sonicates of fresh rat hepatocytes (in the presence of added GSH and CDNB) was about 415 nmol/min/10(6) cells. The corresponding activities in cryopreserved rat, human and dog hepatocytes were approximately 320, 440 and 540 nmol/min/10(6) cells, respectively. In contrast, glutathione conjugation by the intact cryopreserved human and rat hepatocytes in the presence of CDNB alone was less than 10% of the corresponding conjugation by fresh rat hepatocytes, indicating that glutathione was depleted in these cryopreserved hepatocytes. Glutathione depletion was confirmed after analytical measurement of the glutathione levels in fresh and cryopreserved hepatocytes. In fresh rat hepatocytes the level of glutathione was 44 nmol/10(6) cells, whereas it was 2.5 and 4.4 nmol/10(6) cells in cryopreserved rat and human hepatocytes, respectively. In summary, glutathione transferase was active in these cryopreserved hepatocytes but the cryopreservation procedure likely causes depletion in the intracellular level of glutathione, resulting in an overall reduced glutathione conjugating capacity.

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.

A cocktail of the following probe substrates for human drug-metabolizing enzymes was used to characterize hepatocyte preparations: phenacetin (for CYP1A2), diclofenac (CYP2C9), diazepam (CYP2C19), bufuralol (CYP2D6), midazolam (CYP3A4/5) and 7-hydroxycoumarin (for glucuronidation and sulphation). The cocktail was incubated with cryopreserved human, dog or minipig hepatocytes or with freshly prepared rat hepatocytes. Sample analysis was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in an Open Access environment that allowed less experienced MS operators to login, submit and analyse sample sets using predefined settings without the immediate attendance of an experienced analyst. Intrinsic clearances (CLint) were calculated from the disappearance of the compounds from the incubations. Initially, the cocktail used for human, rat and dog hepatocyte incubations contained 7-ethoxycoumarin instead of 7-hydroxycoumarin. However, 7-ethoxycoumarin had an inhibitory effect on the metabolism of phenacetin. The highest CLint estimated with human and dog hepatocytes was observed for 7-hydroxycoumarin. For rat and minipig hepatocytes, the highest CLint was observed for bufuralol. In incubations with dog and minipig hepatocytes, the lowest CLint was seen with diclofenac, whereas for human and rat hepatocytes, the lowest value was observed with diazepam and phenacetin, respectively. When the cocktail was incubated together with human hepatocytes and 1 microM ketoconazole, the CLint of midazolam was decreased to about 7.5% of the control value, whereas the metabolism of the other cocktail compounds was virtually unaffected by this CYP3A inhibitor. It is suggested that a cocktail of specific human probe substrates for drug-metabolizing enzymes can be used routinely for the determination of the metabolic capacity of hepatocyte preparations in order to ensure the quality and reproducibility of experiments. Moreover, a cocktail of specific probe substrates can also be a useful tool for studies on enzyme inhibition.

1. Slices of human and rat liver were cryopreserved in 18% dimethyl sulphoxide (DMSO) and subsequently stored in liquid nitrogen for periods up to as long as 6 months. After thawing, the metabolism of testosterone to hydroxylated products and conjugation of 7-hydroxycoumarin were investigated. 2. Rat liver slices stored in liquid nitrogen for 6 months exhibited rates of formation of 7alpha-, 6beta- 16alpha- and 2alpha-hydroxytestosterone, and of androstenedione that did not differ significantly from those observed with fresh slices. 3. No formation of 2alpha-hydroxytestosterone was detected with slices of human liver. However, in contrast with the rat, human slices produced 2beta-hydroxytestosterone. The rates of formation of 7alpha-, 6beta-, 16alpha- and 2beta-hydroxytestosterone and of androstenedione by human liver slices after 6 months of storage in liquid nitrogen were 82, 71, 236, 66 and 92%, respectively, of the corresponding rates by fresh slices. 4. The rates of sulphation and glucuronidation of 7-hydroxycoumarin by slices from rat liver were 97 and 119%, respectively, of the corresponding fresh values after 6 months of storage in liquid nitrogen. 5. 7-Hydroxycoumarin glucuronidation by human liver slices was 53% of the corresponding fresh values after 6 months of storage. However, human slices showed little or no capacity to conjugate 7-hydroxycoumarin with sulphate. 6. It was demonstrated that slices of both human and rat liver can be cryopreserved and stored in liquid nitrogen for at least 6 months without major changes in their rates of metabolism of testosterone to its hydroxylated products and of 7-hydroxycoumarin conjugation. These findings further emphasize that cryopreservation of liver slices can be an effective tool in the use of biological material of limited availability.

1. We applied the regression offset approach to predict rat in vivo intrinsic clearance (CLint) 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 (Vd), molecular weight (Mw) and CL. 2. A correlation between poor in vitro-in vivo extrapolation (IVIVE) and Vd values distinct from the Vd of albumin (0.1-0.2 L/kg) was revealed. For compounds with a Vd value above 0.5 L/kg, 0% of the predictions of in vivo CLint was within twofold of the observed value, compared to 69% for compounds with a Vd value below 0.5 L/kg. 3. Compounds with a Mw below 450 g/mol demonstrated more accurate in vivo CLint 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 Vd (i.e. around the Vd 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.

1. Intestinal loss, 1 - (Fobs/fH), 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.

Background: The aim of this study was to develop potent, selective and cell-permeable inhibitors of MALT1 protease activity. MALT1 is an Arg-specific protease that cleaves multiple substrates, acting as a key component in T- and B-cell receptor signaling in lymphocytes. MALT1 protease activity is reported to mediate normal and disease-associated lymphocyte proliferation and survival as part of the CARMA-1/BCL-10/MALT1 (CBM) signaling complex, making MALT1 an attractive therapeutic target. Constitutive MALT1 activity is a hallmark of Diffuse Large B Cell Lymphoma of the activated B cell type (ABC-DLBCL). This aberrant activation is observed as a consequence of mutations in CARMA-1/CARD11, and in multiple regulatory proteins upstream of the CBM complex. Methods: MALT1 enzyme assay with Ac-Leu-Arg-Ser-Arg-AMC as substrate was used to measure Ki. Selectivity towards other Arg-specific proteases was measured by inhibition of Trypsin and Thrombin. For determination of cellular potency the ABC-DLBCL cell line OCI-LY3, carrying the L251P mutation of CARMA-1/CARD11, was used. A MALT1 cell assay to measure A20 substrate cleavage was adapted to capillary electrophoresis and quantification. MALT1 down-stream signaling was measured by inhibition of IL-6 expression. Results: Published inhibitors of MALT1 were profiled for potency, selectivity, DMPK properties and activity in cell-based assays, and showed several drawbacks such as low MALT1 activity, low cell permeability and/or selectivity. We developed several novel reversible small molecule inhibitors of MALT1 with Ki 100μM, Thrombin Ki>100μM, and Caco-2 Papp = 1×10−6cm/s shows intracellular A20 cleavage IC50 = 1100nM, whereas compound B with MALT1 Ki = 47nM and Caco-2 Papp = 5.6×10−6cm/s, displays intracellular A20 cleavage IC50 = 600nM. We observe a clear dissociation between intracellular inhibition of MALT1 and inhibition of cell proliferation of DLBCL cell lines of ABC-type with constitutive MALT1 activity, e.g. compound C (MALT1 Ki = 35nM, Trypsin Ki>100μM, Thrombin Ki>100μM and intracellular A20 cleavage IC50 = 260nM) inhibits the proliferation of OCI-LY3 cells and TMD8 cells with CC50 values of 73μM and 96μM respectively, after 5 days exposure. Conclusion: Inhibition of MALT1 protease activity did not translate into significant anti-proliferative effects in DLBCL cell lines. These new MALT1 inhibitors provide the opportunity to test other therapeutic hypotheses involving the targeting of MALT1 protease activity. Citation Format: Fredrik G. Oberg, Sofia Karlstrom, Ian Henderson, Ellen Hewitt, Anders Kallin, Susanne Sedig, Jimmy Lindberg, Anna-Karin Sohlenius-Sternbeck, Richard Bethell, Mark Albertella. Development of selective small-molecule inhibitors of cellular MALT1 protease activity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3011.

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.

We compare three different approaches to scale clearance (CL) from human hepatocyte and microsome CL(int) (intrinsic CL) for 52 drug compounds. By using the well-stirred model with protein binding included only 11% and 30% of the compounds were predicted within 2-fold and the average absolute fold errors (AAFE) for the predictions were 5.9 and 4.1 for hepatocytes and microsomes, respectively. When predictions were performed without protein binding, 59% of the compounds were predicted within 2-fold using either hepatocytes or microsomes and the AAFE was 2.2 and 2.3, respectively. For hepatocytes and microsomes there were significant correlations (P = 8.7 x 10(-13), R(2) = 0.72; P = 2.8 x 10(-9), R(2) = 0.61) between predicted CL(int in vivo) (obtained from in vitro CL(int)) and measured CL(int in vivo) (obtained using the well-stirred model). When CL was calculated from the regression, 76% and 70% of the compounds were predicted within 2-fold and the AAFE was 1.6 and 1.8 for hepatocytes and microsomes, respectively. We demonstrate that microsomes and hepatocytes are in many cases comparable when scaling of CL is performed from regression. By using the hepatocyte regression, CL for 82% of the compounds in an independent test set (n = 11) were predicted within 2-fold (AAFE 1.4). We suggest that a regression line that adjusts for systematic under-predictions should be the first-hand choice for scaling of CL.

Male C57Bl/6 mice were treated for 5 days with 0.05% perfluorooctanoic acid (PFOA) in their diet. This treatment resulted in a potent induction of peroxisomal fatty acid beta-oxidation in the liver. In order to investigate recovery from treatment with PFOA, mice were given normal laboratory chow for up to 20 days after termination of PFOA administration. It was established that the activities of peroxisomal lauoryl-CoA oxidase and palmitoyl-CoA oxidation were still elevated 2-3 weeks after termination of treatment. The catalase activity recovered in the cytosolic fraction was also still significantly elevated after 20 days with normal laboratory chow. Furthermore, the protein content of the mitochondrial fraction was increased by PFOA and had not returned to control level at the end of the recovery period. Perfluorooctanoic acid also caused a persistent effect in omega hydroxylation of lauric acid (cytochrome P-452). The activities of cytosolic DT-diaphorase and glutathione transferase were also enhanced by PFOA. However, these two enzymes recovered relatively rapidly from the treatment (2-20 days). This study reveals two different patterns of recovery from PFOA treatment, one involving parameters that recovered completely, or almost completely, from PFOA treatment after 20 days and another involving parameters that were still elevated at the end of the recovery period.

In the present study we have investigated the disappearance of chlorzoxazone, dextromethorphan, 7-ethoxycoumarin, imipramine, quinidine, testosterone and verapamil from the medium in which fresh and cryopreserved rat liver slices were incubated. These compounds are all substrates of major isoforms of cytochrome P450 expressed in the liver. The metabolism of five of these compounds in microsomes from rat liver was also examined. Determinations of the concentrations of the compounds were performed employing LC/MS. Intrinsic clearance values (CL(ints)) were calculated on the basis of the concentration-vs.-time curves. No significant differences in the CL(int) values obtained with fresh and cryopreserved rat liver slices were observed for any of the compounds. The highest CL(int) value estimated with liver slices was observed for testosterone and the lowest values were with chlorzoxazone and 7-ethoxycoumarin. The total CL(int) values for 7-ethoxycoumarin and imipramine, calculated using scaling factors, were similar for liver slices and microsomes. In the case of testosterone, this total CL(int) was approximately 3.7-fold lower, whereas for dextromethorphan and quinidine it was 2.5- and 8.5-fold higher, respectively, with liver slices than with microromes. In conclusion, the rate of metabolism of the seven compounds tested with rat liver slices was not affected by cryopreservation. This finding adds further support to the general conclusion that the major activities involved in drug metabolism are not affected by cryopreservation of rat liver slices.

1. Xenobiotic-metabolizing enzymes, including both cytochrome P450 and phase II-conjugating systems, have been characterized in rat liver slices cryopreserved in 12 or 18% dimethylsulphoxide (DMSO). 2. Several cytochrome P450 isoforms in rat liver slices metabolized testosterone to a variety of hydroxylated products. The rates of formation of these same products were well maintained during cryopreservation of the slices in both 12 or 18% DMSO. 3. After cryopreservation of rat liver slices in 18% DMSO, the rates of metabolism of ropivacaine to 3-hydroxyropivacaine, 4-hydroxyropivacaine and PPX (all catalysed by different cytochrome P450 isoforms) were approximately 94, 79 and 82% respectively of the corresponding rates observed with fresh slices. 4. The rates of conjugation of 7-hydroxycoumarin and 1-naphthol by rat liver slices were significantly decreased after cryopreservation in 12% DMSO, but they were maintained when the concentration of this cryopreservant was increased to 18% 5. After cryopreservation in 12% DMSO, the mitochondrial reduction of the tetrazolium salt MTT by rat liver slices was significantly lowered. In contrast, slices cryopreserved in 18% DMSO demonstrated no significant decrease in their capacity to reduce MTT. 6. Thus, in agreement with previous studies, it was found that cytochrome P450-dependent activities are retained after cryopreservation of liver slices. Although phase II-conjugating enzyme activities are more sensitive to cryopreservation, it was shown that increasing the concentration of DMSO present during cryopreservation could circumvent the problem. This modification improves the usefulness of cryopreserved rat liver slices as a tool in drug metabolism studies.

Male and female C57B1/6 mice were rendered vitamin A-deficient, and the effects of this deficiency on certain xenobiotic-metabolizing enzymes and defenses against oxidative stress were examined. Vitamin A deficiency significantly increased the levels of DT-diaphorase, glutathione transferase, and catalase in the hepatic cytosolic fraction from male mice (5.2-, 1.6-, and 3.5-fold, respectively), as well as from female mice (4.8-, 3.3-, and 2.4-fold, respectively). In the hepatic mitochondrial fraction (containing peroxisomes) from male animals, the activities of urate oxidase and catalase were increased 3.4- and 1.7-fold, respectively. The activity of catalase in the mitochondrial fraction from female mice was not affected by vitamin A deficiency, whereas the activity of peroxisomal urate oxidase was increased 2.9-fold. The hepatic level of ubiquinone was increased somewhat. The significance of the increases observed here is presently unclear, but it may be speculated that vitamin A and/or its metabolites are somehow involved in the down-regulation of these proteins. Another possibility is that these enzymes are increased as a result of hepatic oxidative stress caused by vitamin A deficiency. However, vitamin A deficiency had no effect on the activity of superoxide dismutase in this study, whereas the activity of glutathione peroxidase was slightly decreased (27%) in the hepatic cytosolic fraction from male mice. In addition, the hepatic level of alpha-tocopherol was decreased dramatically in the vitamin A-deficient animals.

Vitamin A-adequate and vitamin A-deficient C57B1/6 mice were treated for ten days with 0.02% (w/w) perfluorooctanoic acid (PFOA) in their diet. Treated vitamin A-adequate and -deficient mice demonstrated approximately the same increases in liver somatic index (g liver/g body weight) (somewhat more than 2-fold) and mitochondrial protein content (5-fold). PFOA treatment resulted in a 26-fold increase in hepatic peroxisomal lauroyl-CoA oxidase activity in vitamin A-adequate mice, whereas the same activity was unchanged in vitamin A-deficient mice. Vitamin A deficiency itself caused a 3- to 4-fold increase in cytosolic catalase activity and a smaller increase in the activity of microsomal cytochrome P-450 IVA (lauric acid ω- and ω-1 hydroxylase) in this same organ. The induction of the activities of these enzymes was less prominent in vitamin A-deficient mice compared with the effect caused by PFOA in vitamin A-adequate mice, resulting in approximately the same maximal values for these parameters in both groups (i.e. approx. 21 mmol/g liver · min and 350 nmol/g liver · min, respectively). A 70 kDa protein, presumably the multifunctional protein, was shown by Commassie blue staining of SDS-polyacrylamide gels and by immunoblotting (with antibodies towards the multifunctional protein) to be induced to approximately the same degree in vitamin A-adequate and -deficient mice. A morphometric study revealed that PFOA causes the same extent of hepatic peroxisome proliferation in vitamin A-deficient as in vitamin A-adequate mice. The possibility that PFOA exerts its effect in vivo through at least two different mechanisms is discussed.

Vitamin A-deficient male mice were treated for 10 days with 0.02% perfluorooctanoic acid (PFOA) in their diet. The effects of this highly potent peroxisome proliferator on peroxisomal palmitoyl-CoA oxidation and lauroyl-CoA oxidase activities were reduced by 3.1–7.5 times in comparision to the values obtained with mice receiving a vitamin A-adequate diet. The activity of peroxisomal catalase was virtually unaffected by vitamin A deficiency and treatment with PFOA had no significant effect on this activity in vitamin A-adequate or vitamin A-deficient mice. However, vitamin A deficiency itself caused a more than 800% increase in cytosolic catalase activity. Thus, the precentage increase caused by PFOA on cytosolic catalase activity was reduced 12.6 times in vitamin A-deficent mice compared to the increase in vitamin A-adequate mice, although the total absolute activities were similar. These findings suggest that the peroxisome proliferation caused by this peroxisome proliferator is highly depedent on the vitamin A status of the mouse.

Morris hepatoma 7800C1 cells (a Wistar rat cell line) were exposed to 100 microM arachidonic acid in the medium for seven days. This treatment resulted in 150% and 60% increases (above control activities) in acyl-CoA oxidase (which catalyzes the first step in peroxisomal beta-oxidation) and catalase activities, respectively. Arachidonic acid (C20:4) can be metabolized to 20- and 19-hydroxy-arachidonic acid by cytochrome P-450IVA and it was shown that our cells are capable of forming 20-hydroxyarachidonic acid. However, 20-hydroxyarachidonic acid (0.1-0.8 microM, 4 days) had no effects on lauroyl-CoA oxidase and catalase activities in Morris hepatoma cells. Treatment of 7800C1 cells with 100 microM all-trans-retinoic acid resulted in inductions of catalase (160% above the control activity) and carnitine acetyltransferase (140% above the control activity) activities. The activity of lauroyl-CoA oxidase was often, but not always, slightly induced by treatment with all-trans-retinoic acid. When all-trans-retinoic acid was administered together with arachidonic acid, these two compounds had a synergistic effect on the induction of acyl-CoA oxidase activity (almost 700% above the control activity). However, treatment of Morris hepatoma cells with the man-made peroxisome proliferator, perfluorooctanoic acid, together with all-trans-retinoic acid did not result in any synergistic effect on this same enzyme activity. In summary, this study (1) corroborates findings from transfection experiments indicating that the heterodimer PPAR-RXR alpha activates transcription of the acyl-CoA oxidase gene using the Morris hepatoma cell line; (2) shows that arachidonic acid induces the activity of lauroyl-CoA oxidase; (3) suggests that transcription of the catalase gene is not regulated by a PPAR-RXR alpha heterodimer in this system; and (4) demonstrates that peroxisome proliferation in Morris hepatoma cells by perfluorooctanoic acid is not as dependent on the level of retinoic acid as is the same process caused by arachidonic acid.

In this study, Morris hepatoma 7800C1 cells (from rat) were exposed to 500 microM perfluorooctanoic acid (PFOA) in the culture medium for 7 days. This treatment resulted in inductions of catalase, lauroyl-CoA oxidase (which catalyzes the first step in peroxisomal beta-oxidation) and of cytochrome P-450IVA (specialized for omega- and omega-1 hydroxylation of fatty acids). Northern blot analysis revealed that the level of mRNA for peroxisomal fatty acyl-CoA oxidase was enhanced in cells treated with PFOA. Inductions of the enzymes mentioned above are generally connected with peroxisome proliferation in vivo. This work also includes a comparison between the activities of catalase, lauroyl-CoA oxidase, DT-diaphorase and glutathione transferase in rat liver homogenate and 7800C1 cells in order to investigate to what extent this cell line differs from the situation in vivo. The findings suggest that the cells selectively lost most of their peroxisomes during transformation into a cell line and subsequent propagation. The control activities of catalase and lauroyl-CoA oxidase (marker enzymes for peroxisomes) were only about 2% of the corresponding enzyme activities in rat liver. In addition, a morphological study revealed that the frequency of peroxisomes in 7800C1 cells is very low. The control activity of glutathione transferase in 7800C1 cells was 11% of the corresponding activity in rat liver homogenate, whereas the level of DT-diaphorase was virtually the same in 7800C1 cells as in rat liver. Electron microscopic investigation of the control cultures revealed all signs of viable cells, with well-developed cell organelles. Treatment of 7800C1 cells with 500 microM PFOA has little effect on cellular morphology.

Male C57 BL/6 mice were exposed to 1.0% (w/w) acetylsalicylic acid (ASA) in their diet for 10 days and effects related to peroxisome proliferation were subsequently examined. A 2.2-fold increase in mitochondrial protein content was obtained. The activities of the peroxisomal enzymes, lauroyl-CoA oxidase, palmitoyl-CoA oxidation and catalase, were enhanced 4.5-, 4.0- and 2.1-fold, respectively. There was a dramatic increase (9.1-fold) in microsomal cytochrome P450 IVA-catalysed activity, a 1.6-fold induction of total microsomal P450 content and a 2-fold induction of microsomal cytochrome P450 reductase activity (measured as NADPH-cytochrome c reductase). Catalase activity in the cytosol was induced 5.2-fold and DT-diaphorase activity was increased 3.5- and 3.2-fold in the cytosol and mitochondria, respectively. There was a significant increase in the susceptibility of microsomes to lipid peroxidation. Smaller increases in superoxide dismutase, glutathione transferase and glutathione peroxidase activities were also observed. The possible relevance of these effects to the pharmacology of ASA is discussed.

Perfluorooctane sulfonic acid is almost as potent as perfluorooctanoic acid in causing increases in peroxisomal fatty acid beta-oxidation, peroxisomal catalase activity, omega-hydroxylation of lauric acid, cytosolic epoxide hydrolase activity and cytosolic DT-diaphorase activity. Octane sulfonic acid was ineffective at doses used for perfluorooctane sulfonic acid and perfluorooctanoic acid. The results support the theory of co-regulation of these parameters and peroxisome proliferation. The fact that perfluorooctane sulfonic acid causes peroxisome proliferation challenges the hypothesis that the first step in this process is formation of a thioester between the proliferator (the carboxylic group) and coenzyme A.

Male and female C57Bl/6 mice were administered perfluor-octanoic acid PFOA; 0.02-0.05% w/w; 5-10 days) in their diet. This treatment resulted in a several-fold induction of hepatic peroxisomal fatty acid beta-oxidation (monitored as increases in cyanide-insensitive palmitoyl-CoA oxidation, lauroyl-CoA oxidase and catalase activity) in all animals. The protein content of the hepatic mitochondrial fraction was also increased in all mice exposed to PFOA. Furthermore, studies on xenobiotic-metabolizing enzymes revealed no sex-related difference in the response to PFOA. All mice demonstrated a dramatic increase in omega-hydroxylation of lauric acid. Cytosolic epoxide hydrolase, glutathione transferase and DT-diaphorase activities were increased about 2-5-fold. These results with mice differ dramatically from previous studies and our own experiments here with Wistar rats, in which exposure to PFOA causes hepatic peroxisome proliferation in male animals, whereas females are unaffected.