Philippe Coassolo

Prediction of Hepatic Metabolic Clearance Based on Interspecies Allometric Scaling Techniques and In Vitro-In Vivo Correlations

This article reviews the methods available for predicting hepatic metabolic clearance in humans, and discusses their application to the processes of drug discovery and development. The application of these techniques has increased markedly during the past few years because of the improved availability of human liver samples, which has increased the opportunities to use in vitro studies to predict human clearance. The techniques available involve both empirical and physiologically based approaches. Allometric scaling using in vitro data from animals and humans combines certain aspects of both approaches.An evaluation of data retrieved from the literature indicates that, together with in vitro human data, allometric scaling based on a combination of in vitro and in vivo preclinical data can accurately predict clearance in humans. With this approach, 80% of the predictions were within a 2-fold factor of actual human clearance values, with an overall accuracy of 1.6-fold.The uncertainties and inaccuracies in predicting human clearance are related to: (i) the specific method that is used to make the prediction; (ii) the experimental design and the model used to determine the in vitro clearance; (iii) protein binding within the in vitro test system; and (iv) various in vivo factors such as the involvement of extrahepatic metabolism and active transport processes, interindividual variability and nonlinearity in pharmacokinetics.In contrast to purely empirical approaches, the physiological approach to predicting clearance gives an opportunity to integrate some of these complexities and, therefore, should provide more confidence in the prediction of clearance in humans.

Evaluation of the effect of culture configuration on morphology, survival time, antioxidant status and metabolic capacities of cultured rat hepatocytes.

We evaluated the antioxidant status, namely cellular lipid peroxidation, by measuring thiobarbituric acid reactive substances (TBARS), cellular reduced glutathione (GSH) content, glutathione reductase (GSSG-R), glutathione transferase (GST), glutathione peroxidase (GSH-Px) and catalase activities in rat liver, hepatocytes immediately after isolation and in two-dimensional (2D) culture (on non-coated or collagen-coated dishes, as collagen-collagen or collagen-Matrigel sandwich cultures) or three-dimensional (3D) culture on Matrigel-coated dishes. Microsomal cytochrome P450 (CYP)- and UDP-glucuronosyl transferase (UGT)- dependent activities were also assessed in rat livers and hepatocyte cultures. The overall antioxidant status of rat hepatocytes immediately after isolation was not significantly different from that of rat livers. During culture, GSH was increased in 2D but not in 3D cultures in accordance with morphological observations; that is that matrix-cell interactions involving GSH, important in 2D, are minimal in 3D cultures. While UGT- and GST-dependent activities were equivalent in cultured hepatocytes and in rat livers, both catalase and GSH-Px activities decreased with time in all culture configurations. Constitutive CYP-dependent activities were drastically decreased in hepatocytes after isolation and attachment and did not recover in any culture configuration tested. Our results highlight that, although 2D sandwich cultures and 3D cultures on Matrigel allow longevity of rat hepatocyte cultures and optimal induction of CYPs, an imbalance in phase I/phase II detoxication processes in cultured rat hepatocytes occurs, whatever the culture configuration.

Prediction of hepatic metabolic clearance: comparison and assessment of prediction models.

ObjectiveTo perform a comparative quantitative evaluation of the prediction accuracy for human hepatic metabolic clearance of 5 different mathematical models: allometric scaling (multiple species and rat only), physiologically based direct scaling, empirical in vitro-in vivo correlation, and supervised artificial neural networks.MethodsThe mathematical prediction models were implemented with a publicly available dataset of 22 extensively metabolised compounds and compared for their prediction accuracy using 3 quality indicators: prediction error sum of squares (PRESS), r2 and the fold-error.ResultsApproaches such as physiologically based direct scaling, empirical in vitro-in vivo correlation and artificial neural networks, which are based on in vitro data only, yielded an average fold-error ranging from 1.64 to 2.03 and r2 values greater than 0.77, as opposed to r2 values smaller than 0.44 when using allometric scaling combining in vivo and in vitro preclinical data. The percentage of successful predictions (less than 2-fold error) ranged from 55% (rat allometric scaling) to between 64 and 68% with the other approaches.ConclusionsOn the basis of a diverse set of 22 metabolised drug molecules, these studies showed that the most cost-effective and accurate approaches, such as physiologically based direct scaling and empirical in vitro-in vivo correlation, are based on in vitro data alone. Inclusion of in vivo preclinical data did not significantly improve prediction accuracy; the prediction accuracy of the allometric approaches was at the lower end of all methods compared.

Prediction of hepatic clearance using cryopreserved human hepatocytes: a comparison of serum and serum-free incubations

Cryopreserved human hepatocytes have been used to predict hepatic in‐vivo clearance. Physiologically‐based direct scaling methods generally underestimate human in‐vivo hepatic clearance. Cryopreserved human hepatocytes were incubated in 100% serum and in serum‐free medium to predict the in‐vivo hepatic clearance of six compounds (phenazone (antipyrine), bosentan, mibefradil, midazolam, naloxone and oxazepam). Monte Carlo simulations were performed in an attempt to incorporate the variability and uncertainty in the measured parameters to the prediction of hepatic clearance. The intrinsic clearance (CLint) and the associated variability of the six compounds decreased in the presence of serum and the values were reproducible across donors. The predicted CLhep, in‐vivo obtained with hepatocytes from donors incubated in serum was more accurate than the prediction obtained in the absence of serum. For example, the CLhep, in‐vivo of mibefradil in donor GNG was 4.27 mL min−1 kg−1 in the presence of serum and 0.46 mL min−1 kg−1 in the absence of serum (4.88 mL min−1 kg−1 observed in‐vivo). Using the results obtained in this study together with an extended data set (26 compounds), the clearance of 77% of the compounds was predicted within a 2‐fold error in the absence of serum. In the presence of serum, 85% of the compounds were successfully predicted within a 2‐fold error. In conclusion, cryopreserved human hepatocyte suspensions represented a convenient and predictive model to assess human drug clearance.

Interspecies Scaling of Bosentan, A New Endothelin Receptor Antagonist and Integration of in Vitro Data into Allometric Scaling

AbstractPurpose. The goal of this study was to find a rational and reliable method of using animal data to predict the clearance of metabolised drugs in humans. Methods. One such approach is to use in vitro liver models (e.g. hepatocytes and microsomes) to determine the relative capacities of the various animal species and humans to metabolise the test compound. These data can then be combined with the in vivo clearances in animals, to calculate the in vivo clearance in humans using allometric scaling techniques. In this study, this approach was evaluated with a new endothelin receptor antagonist, bosentan, which is eliminated mainly through metabolism and is characterized by very large interspecies differences in clearance. Therefore, this compound provided a stringent test of our new extrapolation method for allometric scaling. Results. The results obtained with bosentan showed that adjusting the in vivo clearance in the different animal species for the relative rates of metabolism in vitro gave a far better prediction of human clearance than an empirical correcting factor (brain weight). Conclusions. This approach provided a more rational basis for predicting the clearance of metabolised compounds in humans.

Animal Pharmacokinetics and Interspecies Scaling from Animals to Man of Lamifiban, a New Platelet Aggregation Inhibitor

Relating pharmacokinetic information obtained in animal species to man (interspecies scaling) can play an important role in enabling understanding of the differences and similarities between species, and helping to predict the kinetic profile of a new compound in man.

Strain difference (WKY, SPRD) in the hepatic antioxidant status in rat and effect of hypertension (SHR, DOCA). Ex vivo and in vitro data

We assessed the hepatic antioxidant status of spontaneously (SHR) and desoxicorticosterone acetate (DOCA)-induced hypertensive rats and that of respective normotensive Wistar Kyoto (WKY) and Sprague-Dawley (SPRD) rats. For this we evaluated, ex vivo in liver cytosols, reduced glutathione (GSH) content, glutathione-related enzyme (peroxidase, reductase and transferase) activities as well as the rate of lipid peroxidation in 9–11 week-old rats. The antioxidant status and the cytotoxicity of acetaminophen, a radical- and hydrogen peroxide-mediated hepatotoxic compound, were also assessed in vitro in cultured hepatocytes isolated from hypertensive (SHR, DOCA) and normotensive control (WKY, SPRD) rats. Our results suggest that a difference exists in the hepatic antioxidant status between rat strains, with GSH levels being lower (–15%) and lipid peroxidation rate higher (+30%) in WKY compared to SPRD rats. In hepatocyte cultures from WKY rats, both GSH content and catalase activity were lower (–30 and –70% respectively) compared to hepatocyte cultures from SPRD rats. This was associated with a 35% higher cytotoxicity of acetaminophen in cultured hepatocytes from WKY rats compared to that in hepatocytes from SPRD rats. Hypertension in DOCA rats (mmHg: 221 ± 9 vs. 138 ± 5 in control SPRD rats) was associated with decreases (about 30%) in both glutathione peroxidase (GSH-Px) and catalase activities, ex vivo in livers and in vitro in hepatocyte cultures. Hypertension in SHR (mmHg: 189 ± 7 vs. 130 ± 5 in control WKY rats) was also associated with decreases (about 50%) in GSH-Px activity, ex vivo in livers and in vitro in hepatocyte cultures but catalase activity was not modified. The IC50 of acetaminophen was also lower in hepatocytes from hypertensive rats compared to respective controls, which could be related to the weakened antioxidant status in hepatocytes from hypertensive rats. Our data thus suggest that hepatocyte cultures are appropriated tools in which to assess hepatotoxicity and hepatoprotection in hypertension.

Follow-up to the pre-validation of a harmonised protocol for assessment of CYP induction responses in freshly isolated and cryopreserved human hepatocytes with respect to culture format, treatment, positive reference inducers and incubation conditions

We have compared induction responses of human hepatocytes to known inducers of CYP1A2, CYP2B6, CYP2C and CYP3A4/5 to determine whether the culture format, treatment regimen and/or substrate incubation conditions affected the outcome. CYP induction responses to prototypical inducers were equivalent regardless of pre-culture time (24h or 48h), plate format (60mm or 24-well plates) used or whether CYP activities were measured in microsomes or whole cell monolayers. Fold-induction of CYP3A4/5 by 1000muM PB and 10microM RIF were equivalent. In contrast, the fold-induction of CYP2B6 by PB was 3-fold higher that by 10microM RIF. In addition to inducing CYP1A2, 50microM OME also induced CYP3A4/5 in 50% of the donors tested. CYP2B6 was induced in 14 out of 21 donors by BNF; however CYP3A4/5 was unaffected by BNF in these donors. In order to confirm that donor-to-donor variation was not due to inter-laboratory differences, the induction responses of 5 different batches of cryopreserved human hepatocytes were compared in two different laboratories. The induction of CYP1A2, CYP2B6 and CYP3A4 measured in our laboratory were equivalent to those obtained by the commercial companies, proving good between-laboratory reproducibility. In conclusion, there is some flexibility in the treatment and incubation protocols for classical CYP induction assays on human hepatocytes. Both RIF and PB are suitable positive control inducers of CYP3A4/5 but PB may be more appropriate for CYP2B6 induction. BNF may be more appropriate for CYP1A2 induction than OME since, in contrast to the latter, it does not induce CYP3A4. Induction responses using hepatocytes from the same donor but in different labs can be expected to be similar. The good reproducibility of induction responses between laboratories using cryopreserved hepatocytes underlines the usefulness of these cells for these types of studies.

Estimation of flavin-containing monooxygenase activity in intact hepatocyte monolayers of rat, hamster, rabbit, dog and human by using N-oxidation of benzydamine

The flavin-containing monooxygenase (FMO)-dependent N-oxidation of benzydamine has been assessed as a method for monitoring the activity of FMOs in monolayer cultures of hepatocytes from rat, dog, rabbit, hamster and human. The advantage of this substrate is that benzydamine N-oxide formation can be measured directly in extracts of cellular incubations without an intensive work-up procedure. Benzydamine and its N-oxide are readily separated by HPLC with fluorometric detection. This assay proved sensitive enough to monitor FMOs activity in intact monolayer of cultured hepatocytes. The formation of benzydamine N-oxide was inhibited when hepatocytes were coincubated with methimazole (another FMO substrate) in a dose-dependent manner, whereas N-octylamine (an inhibitor of cytochrome P450) had no inhibitory effect. In contrast to cytochrome P450, FMO activity assessed by benzydamine N-oxidation was relatively stable for all species studied during 72-h cultures.

Comparison of the stability of some major cytochrome P450 and conjugation reactions in rat, dog and human hepatocyte monolayers

SummaryThe stability of four major cytochrome P450 isoenzymes (CYP1A, CYP2B, CYP2E1 and CYP3A) and of two phase II conjugation enzymes (glucuronyl- and sulfotransferases) was investigated in primary cultures of rat, dog and human hepatocytes in the same conditions. 7-ethoxyresorufin deethylation (EROD), 7-methoxycoumarin demethylation (MCOD), chlorzoxazone (CLOX) 6-hydroxylation, 1′- and 4-hydroxylation of midazolam (MDZ), and p-nitrophenol glucuronidation and sulfation, were used respectively. The EROD activity was stable over 72 hours in rat and dog and only 48 hours in human hepatocytes. The MCOD activity was also stable in rat but decreased in dog by 30% within 72 hours the CLOX hydroxylase activity was most stable in human whereas in rat and dog it fell down to 30% within 72 and 24 hours, respectively. The MDZ hydroxylase activity showed the same unstability profile in the three species investigated. Both conjugation reactions were either stable or showed an increase by up to 60–70% in all three species over 72 hours. The enzymes tested showed different stabilities in rat, dog and human hepatocytes over 72 hours, thus demonstrating the limitations of hepatocyte monolayers as models for metabolic investigations and emphasising the need for validation/characterization studies before routine use.