Predicting CYP induction in stable cell lines

Assessing gene transcription as an endpoint for determining induction of drug metabolizing enzymes and transporters has become the “method of choice” in the FDA’s latest draft Guidance for Industry drug interaction studies. Increased gene transcription due to NCE exposure can be determined by two distinct methods, nuclear receptor activation and changes in mRNA levels in primary hepatocyte cultures.

Human hepatocytes are generally considered the “preferred system” for predicting clinical inducers. Alternatives for assessing CYP3A4 induction by potential drugs, are cell-based transactivation assays, which employ HepG2 cells transfected either transiently or stably with a pregnane X receptor (PXR) expression vector, plus a luciferase reporter gene containing CYP3A4 response elements (proximal promoter and distal xenobiotic-responsive enhancer module). DPX2 cells (developed by Puracyp), which are stable hPXR transfectants, show CYP3A enzyme induction (measured as increased luciferase activity) by therapeutics which is comparable to that reported with human hepatocytes (Krueger et al., 2002; Trubetskoy et al., 2005; Raucy and Lasker, 2010). Despite lacking certain phenotypic properties inherent to human hepatocytes, such as constitutive expression of P450 and nuclear receptor proteins, DPX2 cells nevertheless respond to typical CYP3A inducers [e.g., rifampicin (RIF)] with a marked increase in CYP3A metabolic activity (Trubetskoy et al., 2005), eliminate screening dependence on an uninterrupted supply of human hepatocytes, and exhibit much lower interexperimental variability. The use of hepatocytes in high-throughput screening and structure-activity relationship (SAR) studies during early discovery is limited, whereas DPX2 cells can readily surmount such issues (Shukla et al., 2011).

DXP2 to assess PXR inducers – Fee-for-services

Receptor activation screening services using proprietary cell lines by Puracyp enable researchers to identify NCEs that have the potential to activate human, rat, mouse, dog or monkey SXR/PXR, AhR and CAR. These services are offered in Europe through tebu-bio, at their own labs based near Paris.

Rifampicin dose response

A typical dose-response curve for rifampicin generated in human PXR, DPX2, cells. Results are expressed as fold induction above the negative control (DMSO) and were calculated by determining luminescence and normalizing to the viable cell number. Commitment is to provide high quality results in a rapid throughput manner, study turnaround time is 7 business days from receipt of compounds.

The test system is high-throughput consisting of a stably transformed tumor cell line (DPX2) plated on 96-well microtiter plates. The enhancers (PXRE) and promoters linked to a reporter (luciferase) are stably integrated into the tumor cells. Activation is assessed by monitoring reporter gene activity and by comparing results to analogous cells treated with solvent. Positive controls will consist of cells treated with 6 concentrations of rifampicin. In this manner, compounds activating PXR can be easily and expediently identified. Because these are stably integrated cell lines, three to 30 fold PXR activation may be observed.

To  aid  in  interpreting  the  results  produced  in  Puracyp’s  cell  lines,  controls  producing  various  levels  of  receptor  activation  (fold  induction)  are  included  with  your compounds. Controls  included  in  the  assay  represent  potent  levels  of  receptor  activation.    Results  from  screening  of  your  compounds  are  expressed  as  fold  induction  and  as  a  %  of  each  control.  In  this  manner,  compounds  that  exhibit  >75%  of  a  control  at  10  μM  can  be  considered  as  a  similar  activator.  An example of  two  unknowns,  UK1  and  UK2,  are  shown  below  in  the  table.

Table

RIF  =  rifampicin;  UK  =  unknown a Activation  (induction)  potency  is  defined  as  negative,  weak,  moderate  and  strong.    Negative,  weak,  moderate  and  strong  activators  are  compounds  giving  <15%,  <40%,  <69%  and  >70%,  respectively,  of  the  response  obtained  with  10  μM  RIF  *  at  equimolar  concentrations.

 

UK1  is  considered  to  be  a  moderate  PXR  activator  based  on  the  response  produced  in  DPX2  cells  and  expressed  as  %  of  10  μM  rifampicin.  Compounds  that  are  weak  or  moderate and   potent   inducers   in Puracyp’s   cell   lines  may   or   may   not  produce   drug-drug   interaction  in  vivo (1). Conversely,  compounds  classified  as  potent  nuclear  receptor  activators,  i.  e.  >75%  of  the  potent  positive  control   at   equimolar   concentrations,   will  most   likely   produce   drug-­‐drug interactions (1). When using in vitro data  to  predict  in  vivo,  caution  needs  to be  exercised. In  particular,  plasma  concentrations  and  dose  administered  must  be  considered  in  the interpretation  of  results.

It  is  important  to  emphasize  that  the  patterns  or  trends  in  receptor  activation  will  be  the  same  within  a  specific  cell-‐based  assay.  For  this  reason,  other  positive  controls will always be included at  concentrations  that  give  potent  activation.    Test  compound  response  is  then  compared  to  the  response  given  by  the  positive  control  at  an  equimolar dose  in  each  cell  line (2-­4)

DPX2 Conclusion

Puracyp servicesStudies are designed to evaluate the effect of one or multiple new chemical entities (NCEs) on the activation of the human PXR nuclear receptor. Cell lines stably transfected with the hPXR and the CYP3A4 response elements will be seeded in a 96-well plate. Twenty-four hours after seeding the cells will be treated with various concentrations (at least six) of compounds in triplicate wells and cells returned to the incubator for an additional 24 h. At the end of this incubation period, the number of viable cells per well will be determined using Promega’s Cell Titer Fluor Cytotoxicity Assay. Following this assay, ONE-Glo will be added to the same wells and reporter gene activity assessed.

tebu-bio has been a Puracyp service provider for more than 10 years now. If you’re interested in benefiting from their expertise in fee-for-services based on Puracyp’s technology, make a note of their easy address to start designing your project: adme@tebu-bio.com.

What about doing it yourself?

sidebox2You might be interested in these cell-based kits, which contain everything you’ll need for multiplex-type assays, including division arrested cell lines, Promega’s One-Glo™ for assessing receptor activation (via luciferase), P450-Glo™ substrates for assessing CYP3A or CYP1A mediated metabolism, and CellTiter-Fluor™ for measuring cell viability. All you’ll have to supply are the test compounds!

Results obtained with Puracyp’s Receptor Activation Assay kits are highly reproducible and lack the variability inherent to other methodology. Little effort is required to perform the tests and the entire experiment is completed in 2 days with a total of 3-6 hours research time. Several different kit formats and cell lines are available, including  human and rodent PXR and AhR (human).

Start by taking a look at the assays here! 

 

References

1)  Fahmi,  O.A.,  Raucy,  J.  L.,  Ponce,  E.,  Hassanali,  S.  and  Lasker,  J.  M.  Drug  Metab.  Disp.  40:  2201-­‐2211,  2012.
2)  Bjornsson  et  al.,  Drug  Metab.  Dispos.  31:815-­‐832  2003.
3)  2012  FDA  Draft  Guideline  (www.fda.gov/cder)
4)  Sinz  et  al., Curr  Drug  Metab.  7:375-­‐388  2006
4)  Chu  et  al.,  Drug  Metab  Dispos 37:1339-­‐1354  20

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