Application Note. ADME and cytotoxicity assays Use of the CyBi -Well for reagent addition and washing procedures

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1 ADME and cytotoxicity assays Use of the CyBi -Well for reagent addition and washing procedures Heidi Prüfer, Michael Busch; CyBio Screening GmbH, Jena Cells have several strategies which they can use to react following exposure to various substances. For the purposes of drug development it is important to know how a potential drug may be treated by such cellular defence mechanisms. Thus, in the course of the drug discovery process the aim of ADME/Tox studies is to investigate the reaction of cells to a specific compound. In this application note the following examples of ADME/Tox assays are presented: CyP 45 Assays Neutral Red Assay Alamar Blue Assay MTT Assay The focus was set on the application of the CyBi -Well simultaneous pipettor as an instrument for the automation of the above tests. CyP 45 Assays In addition to transport mechanisms (active pumping of a drug out of individual cells) and storing strategies (encapsulation of drugs), there are several metabolising mechanisms which convert a substance into species that the organism can then handle in different ways. The main enzymes involved in this metabolising strategy fall into two classes, the phase I and phase II biotransforming enzymes. The phase I enzymes (e.g. the family of cytochrome P45 isoforms; CYP) prepare xenobiotic substances for further treatment: e.g. by hydrolysis or redox processes. The drugs handled by these phase I enzymes are then derivatised by the phase II enzymes (mostly transferases like the glutatione-s-transfersases). Since the interaction of a compound with CYPs has consequences for drug metabolism or drug-drug interaction, assays targeting these enzymes are used increasingly in the ADME/Tox. Two isoforms of this class of drug metabolising enzyme were tested: the 2D6 and the 3A4 isoforms. Commercial kits (BD Biosciences) were employed to compare a series of known inhibitors for CYP45 3A4 and 2D6. In principle, both of the assays work in the same way. The cytochromes metabolise a substrate which is converted into a fluorescent species. productinfo@cybio-ag.com Tel:

2 The CYP-assays were performed in 96 well plates according to kit manufacturers protocols. The CyBi - Well was used for all liquid handling steps. The liquid handling procedure and time taken for each step are listed below. Addition of 1µL cofaktor mix duration 2 s. Tip washing duration 5 s. Addition of 6µL compound dilutions duration 9 s. Incubation at 37 for 1 minutes duration 1 min Addition 1 µl enzyme/substrate solution and mixing duration 63 s Tip washing duration 5 s. Incubation at 37 for 3 minutes duration 3 min Addition of 75µL of stop solution duration 67 s Readout duration 2 min The resulting Z values for the 2D6 isoform were around.6, those for the 3A4 were around.7, which is sufficient in both cases. In general the assay with the 3A4 isoform showed a better performance. This difference may be due to the use of two different fluorescent dyes for these two isoforms. Readout / FFU Tamoxiphen: Cyp3A4 and 2D6 (96) In this example Tamoxiphen showed a similar inhibition to both of the isoforms (see Fig.1). The variation of the data is similar for both isoforms. The raise of the signal for tamixophen at higher concentrations is a typical result of auto fluorescence effects, which are not uncommon for certain classes of compounds Inhibitor / µm Fig1: IC5s of tamoxiphen in CyP45 3A4 (upper curve) and 2D6 (lower curve) assay in the 96 well format productinfo@cybio-ag.com Tel:

3 Neutral Red Assay Cells can store xenobiotic substances in lyosomes. This is the basis of this type of cytotoxicity assay. The neutral red assay works via the encapsulation of neutral red (NR) into lyosomes of living cells. Cells containing NR encapsulated in their lyosomes are first washed then cell lysed. The amount of the optical absorption caused by liberated NR is a measure of the number of living cells. This assay proceeds over several days including several long incubation steps. The steps for a 96-well plate-based assay are summarised below. All liquid handling steps were performed on a CyBi -Well: Addition of 1 µl/well of cell suspension (A549 cells, 1e5 cells/ml). Replacement of the medium by 1µL/well of compound solution. Washing 3 times with 2 µl/well 37 C buffer, using a CyBi -Well. Addition of 1µL neutral red solution. Incubation 3 h. Washing with 2 µl/well 37 C buffer, using a CyBi -Well Addition of 1 µl/well of lysis solution. Measurement of the absorption at 54 nm. Sterility and even washing were the critical factors for the success of this assay. The use of a CyBi -Well with sterilised (autoclaved) tips together with sterilised tip-washing water were the key to success in this assay. It is important to note, that for all cellular assays described here a CyBi -Well, and not a special washer, was applied for washing as well as for reagent addition. This demonstrates the versatility of this highly precise liquid handling instrument. Absorption Controls: Neutral Red Assay (384) The Z -values, which were achieved with the Neutral Red assay were between.6 and.7 in both 96- and 384-well microplates. An example of control wells in the 384-well format is given in Fig. 2. Fig.2: Controls in a neutral red assay plate (384-well format). productinfo@cybio-ag.com Tel:

4 cell activity / % Neutral Red Assay (96) concentration p-nitrophenol / mm In Fig.3 a dose response of p-nitro phenol is given. The IC5 found (.2 mm) was comparable to literature data. The variation of the data demonstrates the accuracy of all washing and reagents addition steps. Fig.3: A p-nitro phenol dose-response in a neutral red assay plate (96). Alamar Blue Assay In contrast to the MTT and the NR assay the Alamar Blue assay is a cytotoxicity assay with fluorescence readout. It is based upon the reduction of resazurin by living cells, which results in a fluorescent reaction product (resorufin). Unlike the MTT and Neutral Red assay, no cell lysis is necessary for the Alamar Blue assay. The basic assay steps in a 96-well plate are as follows: Addition of 1 µl per well of cell suspension (A549 cells, 1e5 cells/ml). Incubation of the MTP for 48 h. Replacement of the medium by 1µl/well fresh medium Addition of 1 µl/well compound solution. Incubation of the MTP for h. Addition of 1µL/well resazurin solution Incubation for 5 h. Measurement of the fluorescence at 53/59 nm. productinfo@cybio-ag.com Tel:

5 Use of the CyBi -Well resulted in a highly reproducible assay (Fig. 4). Three replicates were tested for each compound with excellent reproducibility. Naldixic Acid: 3 Plates Trichlorphon: 3 Plates inhibition / % inhibition / % concentration / mm concentration / mm Propranolol: 3 Plates 12 inhibition / % Fig.4: A series of 3 dose response experiments for three different substances determined with the Alamar Blue assay. For each compound three micro plates (96-well format) were analysed concentration / mm The IC5s determined were very close to literature data [NIH Publication No: 1-45]. Substance nalidixic acid propranolol*hcl trichlorfon IC5 found literature The Z -values were >.7 (nalixic acid), >.5 (propranolol) and >.6 (trichlorphon) which shows that this assay can be screened with significant results. productinfo@cybio-ag.com Tel:

6 MTT Assay This is a typical cytotoxicity assay. Here the viability of cells is reflected in the capability of living cells to convert a colourless substrate (MTT, a methyl-thiazol-tetrazolium salt) into a coloured reaction product (a formazan). This reaction takes place in the mitochondria of intact cells catalysed by the mitochondrial succinate dehydrogenases. The reaction product is located as needles at the cellular surface. Here all the assay steps are summarised (96-well plates): Addition of 1 µl/well of cell suspension (A549 cells, 1e5 cells/ml). Replacement of the medium by 1µL/well compound solution. Washing 3 times with 37 C PBS, using a CyBi -Well Addition of 1µL MTT solution. Incubation 4 h. Replacement of the MTT solution by 1 µl/well of lysis solution (carefully). Shaking for 15 min for the dilution of the formazan crystals. Measurement of the absorption at 56nm. One critical factor in this assay is extremely careful washing in order to avoid washing away the fragile formazan needles from the cell surfaces. As the data below demonstrate, using a CyBi -Well even for washing guarantees excellent data quality. The MTT assay worked similarly in both, 96- and 384-well plate formats. The results were slightly better in the 96 plate format. This may confirm the assumption, that washing has an impact on assay performance, since cell layers are more likely to remain intact when a larger well volume is involved. During the MTT assay the Z -values found were between.7 and.8. The main reason for these good values in comparison to the neutral red assay is the relative high dynamic range of the readout of this assay. absorbance Controls of MTT Assay (384) well Fig. 5: Raw data of controls of the MTT assay in a 384-well plate. In Fig. 5 an example of control wells in the 384-well plate format is given, which show the good reproducibility of this assay using a CyBi -Well. productinfo@cybio-ag.com Tel:

7 p-nitrophenol: MTT Assay (96) absorbance R7 R4 R1 As in the case of the neutral red assay, some spikes were observed. An explanation for these spikes was found: In wells which showed spikes at the readout a contamination of micro organisms had taken place. When a high degree of sterility was maintained during the compound addition (sterile tips, compounds diluted in freshly prepared medium, sterile wash water), these spikes did not occur. Fig. 6: Raw data of a p-nitro phenol dose response of the MTT assay in a 96-well plate. Fig. 6 shows the raw data of a dose response in a 96-well plate. The slightly higher values in rows 1 and 8 indicate that evaporation had a different effect depending on incubation time, but in this case these effects were tolerable. Conclusion The examples above provide a brief overview of the broad range of ADME/Tox related applications, where a CyBi -Well can be applied as the central liquid handling device. The reliability and excellent reproducibility of this instrument makes it a versatile liquid handler for this important class of assays. The results of the cellular assays demonstrate that a CyBi -Well shows excellent performance even when applied for critical assay steps such as washing. During these washing steps, parameters for liquid handling (location of the tips in the wells during aspiration and dispensing, speed of pistons) could be optimised in order to avoid disruption of the sensitive cell layers at the bottom of the individual wells, thereby helping to guarantee excellent results. productinfo@cybio-ag.com Tel: