The in vitro biokinetics of chlorpromazine and diazepam in aggregating rat brain cell cultures after repeated exposure.

Neurotoxic effects of compounds can be tested in vitro using cell systems. One example is aggregating rat brain cell cultures. For the extrapolation of in vitro data to the in vivo situation, it is important to take the biokinetics of the test compound into account. In addition, the exposure in vivo is often for a longer period of time; therefore, it is crucial to incorporate this into in vitro assays as well. In this study, aggregating rat brain cell cultures were exposed to chlorpromazine (CPZ) and diazepam (DZP) for 12-days with repeated exposure. Samples were taken from the stocks, test media, cell culture media and cells at specific time points on the first and last exposure day. These samples were analysed by HPLC-UV. The amount of CPZ in the medium decreased over time, whereas the amount in the cells showed an increase. Accumulation of CPZ in the cells was seen over the 12-day repeated exposure. The amount of DZP in the medium remained stable over time and only up to 2% of DZP added was found in the cells. Different biokinetic behaviour was found for CPZ and DZP. Possible explanations are differences in uptake into the cells or efflux out of the cells. The decrease of CPZ in the medium versus the stable amount of DZP results in differences in exposure concentrations over time, which should be taken into account when interpreting in vitro effect data.

Biokinetics of chlorpromazine in primary rat and human hepatocytes and human HepaRG cells after repeated exposure.

Since drug induced liver injury is difficult to predict in animal models, more representative tests are needed to better evaluate these effects in humans. Existing in vitro systems hold great potential to detect hepatotoxicity of pharmaceuticals. In this study, the in vitro biokinetics of the model hepatotoxicant chlorpromazine (CPZ) were evaluated in three different liver cell systems after repeated exposure in order to incorporate repeated-dose testing into an in vitro assay. Primary rat and human hepatocytes, cultured in sandwich configuration and the human HepaRG cell line were treated daily with CPZ for 14 days. Samples were taken from medium, cells and well plastic at specific time points after the first and last exposure. The samples were analysed by HPLC-UV to determine the amount of CPZ in these samples. Based on cytotoxicity assays, the three models were tested at 1-2 µM CPZ, while the primary rat hepatocytes and the HepaRG cell line were in addition exposed to a higher concentration of 15-20 µM. Overall, the mass balance of CPZ decreased in the course of 24 h, indicating the metabolism of the compound within the cells. The largest decrease in parent compound was seen in the primary cultures; in the HepaRG cell cultures the mass balance only decreased to 50%. CPZ accumulated in the cells during the 14-day repeated exposure. Possible explanations for the accumulation of CPZ are a decrease in metabolism over time, inhibition of efflux transporters or binding to phospholipids. The biokinetics of CPZ differed between the three liver cell models and were influenced by specific cell properties as well as culture conditions. These results support the conclusion that in vitro biokinetics data are necessary to better interpret chemical-induced cytotoxicity data.

In vitro biokinetics of chlorpromazine and the influence of different dose metrics on effect concentrations for cytotoxicity in Balb/c 3T3, Caco-2 and HepaRG cell cultures.

The extrapolation of in vitro to in vivo toxicity data is a challenge. Differences in sensitivity between cell systems may be due to intrinsic properties of the cell but also because of differences in exposure. In this study, the cytotoxicity and biokinetics of the antipsychotic chlorpromazine (CPZ) were studied in in vitro assays using different cell types and exposure conditions. Different dose metrics were assessed to express the sensitivity to CPZ. The biokinetics of CPZ were measured in cell cultures of Balb/c 3T3, Caco-2 and HepaRG cells. Cytotoxicity was measured by Alamar Blue and expressed using different dose metrics, including the nominal, measured total and measured free CPZ medium concentrations. CPZ was taken up by the cells; the highest amounts in the cell compartments were found in the Caco-2 and HepaRG cells. CPZ was highly protein-bound in the Caco-2 cell medium containing 10% fetal bovine serum, resulting in lower bioavailable exposure concentrations. Moreover, also uptake into the cells strongly influenced the concentration in the medium. The Balb/c 3T3 cells were the most sensitive to the toxic effect of CPZ. The use of different dose metrics influenced the cytotoxicity results found in the three cell types. The data show that in comparing the sensitivity of the tested cell systems, the freely dissolved concentration is a more appropriate dose metric than total concentration in the medium. The ranking in sensitivity of the three cell types for CPZ was dependent on the dose metric used.

Transport of chlorpromazine in the Caco-2 cell permeability assay: a kinetic study.

The intestinal transport of compounds can be measured in vitro with Caco-2 cell monolayers. We took a closer look at the exposure and fate of a chemical in the Caco-2 cell assay, including the effect of protein binding. Transport of chlorpromazine (CPZ) was measured in the absorptive and secretory direction, with and without albumin basolaterally. Samples were taken from medium, cells, and well plastic. For the secretory transport experiments with albumin, the free CPZ concentration at the start of the experiment was measured by negligible depletion-solid phase microextraction (nd-SPME). Recovery of CPZ from the medium was low, especially in the absorptive transport direction. CPZ was found in the cells (≤20%) and bound to the well plastic (≤25%), and 94% of CPZ was bound to albumin. An initial lag phase was observed, which was likely caused by partitioning of CPZ between the donor concentration and the Caco-2 cells; after 20 min, transport of CPZ to the receiver compartment was linear. The low recovery and the test compound found both inside the Caco-2 cells and bound to the well plastic complicate the calculation of the fraction transported and render reliable estimates of permeability constants impossible. For a chemical like chlorpromazine, which is hydrophobic in its neutral form, but in general also for more lipophilic compounds, the Caco-2 cell assay might not be straightforward, and a more detailed study into the fate and exposure of the test compound might be needed to arrive at meaningful data for transport and permeability.

Development of a negligible depletion-solid phase microextraction method to determine the free concentration of chlorpromazine in aqueous samples containing albumin.

The addition of proteins to in vitro systems influences the free concentration of the test compound in the medium. The objective of this study was to set up a negligible depletion-solid phase microextraction method, coupled to high-performance liquid chromatography (nd-SPME-HPLC) to measure the free concentration of chlorpromazine (CPZ) in medium containing albumin. The nd-SPME method was optimized for coating thickness (polyacrylate coating) and exposure time, and potential effects from the addition of bovine serum albumin (BSA) were studied. It was shown that the addition of albumin did not cause fouling or influenced the uptake kinetics of CPZ into the fiber. At a realistic in vivo albumin concentration of 40 g/L of albumin, 94% of CPZ was protein bound. This is in line with findings in vivo, reporting a protein binding for CPZ of 92-99%. The nd-SPME-HPLC method described in this study can be used to measure the free concentration of chlorpromazine in medium containing proteins. These findings can be used to correct in vitro data for protein binding of chlorpromazine and this information is essential for the extrapolation to in vivo data.