CYP identification, inhibition and induction

 

Our experience in CYP identification, inhibition and induction

We provide our customers with a comprehensive set of CYP enzyme – drug interaction studies. These include the studies describe below.

Identification of CYP enzymes

The methods we use for the studies of CYP enzymes responsible for oxidative metabolism include

  • using recombinant CYP enzymes
  • using specific inhibitors
  • using antibodies

reCYPid

reCYP study uses recombinant CYP proteins to pinpoint metabolizing CYP enzymesUsed for lead molecule selection and drug candidate characterisation.

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CYPid Pilot

This study pinpoints involved CYP enzymes in an indirect way utilizing the CYP selective inhibitors (small molecule inhibitors or antibodies). The used small molecule inhibitors can include: fluvoxamine (CYP1A2), tranylcypromine (CYP2A6), ticlodipine (CYP2B6), montelukast (CYP2C8), Sulphphenazole (CYP 2C9), fluconazole (CYP2C19), quinidine (CYP2D6), pyridine (CYP2E1), and ketoconazole (CYP3A4).

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CYPid Pilot and reCYPid are complementary: both studies should point to the same direction to be able to reliably state that a certain CYP enzyme produces a certain metabolite. Metabolite profile of NCE should be known before the studies can be performed.

MetaboEnzyme

This study identifies the involvement of many other than CYP enzymes activities on the compound. This is based on using recombinant enzymes from UGT, SULT, AldO, GE, GST, FMA and MAO families.

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CYP inhibition

InhibCYP

Cost-effective study to unravel the inhibition potential of NCE towards CYP enzymes. Utilizes cocktail incubation of CYP specific probe compounds. Evaluation of potential drug-drug interactions. Used for lead molecule selection and drug candidate characterization.
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cyp_inhib_1
IC50 values and parameters to evaluate time-dependent CYP3A4 inhibition, for Ketoconazole, Gestodene and three study compounds (CMPD1-3)

Mechanism based inhibition

Mechanism based inhibition analysis the potential inhibition caused by the compound on the known activity of CYPs on a cocktail of known small molecules.
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cyp_inhib_2
The effect of study compound (CMPD2) on the CYP3A4 model activities after 0 (circles) and 30 (triangles) min preincubation with (closed symbols, solid line) and without (open symbols, dashed line) NADPH.

CYP induction

Direct CYP inhibition

Plated fresh human hepatocytes are exposed for 72 h to 3 different concentrations of NCE in duplicate. Controls include vehicle, rifampicin (CYP3A4), omeprazole (CYP1A2) and Phenobarbital (CYP2B6). The medium is replaced with fresh medium containing specific substrates to the prototypically induced genes CYP1A2, CYP2B6 and CYP3A4. Samples are withdrawn at 4 h. Possible direct cell toxicity is followed visually. If required, also induction of five other main drug metabolizing CYP isoforms can be studied simultaneously by a N-in-one cocktail multiplexing approach.
Alternatively mRNA level induction is measured after 24 h exposure. Measured genes include CYP1A2, CYP2B6 and CYP3A4.
FDA recommends using at least three different donors to detect CYP induction.
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Indirect CYP inhibition

Plated cells of human hepatoma cell line transfected with nuclear receptors (PXR and CAR form several species and other receptors available) and corresponding luciferase reporter plasmid. Cells are exposed to studied drug compound and relevant CYP specific inducers.
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Background

Proteins of the cytochrome P450 (CYP) superfamily form a large group of enzymes. CYP enzymes are present throughout the biological taxonomy, including animals, plants, bacteria and fungi. CYP enzyme activity is to catalyze the oxidation and reduction reactions of various substrates, including both endogenous organic small molecules and their metabolites as well as xenobiotic molecules, like drug substances. Therefore CYPs are able to modify often lipophilic drug molecules to more polar metabolites and also becoming substrates of the enzymes involved in the phase II conjugation metabolism.. CYP enzyme family is in focus of major part of metabolism and activity for drug compounds and most drugs undergo CYP mediated metabolism (so called Phase I metabolism, which is mainly carried out by CYP enzymes). CYP enzymes are primarily cell membrane associated proteins, located in the mitochondria and endoplasmic reticulum (ER).

As the same CYP enzymes are typically involved in enzymatic reactions for multiple molecules, both endogenous and xenobiotics, regulation of the CYP activity by the xenobiotic drugs via inhibition or induction of the CYP enzymes, may severe consequences for the metabolism, pharmacokinetics and consequently pharmacodynamics. In addition, many drug-drug interaction reactions take place via the CYP enzyme system and therefore one drugs’ ability to alter the CYP activity can increase (via CYP induction) or decrease (via CYP inhibition) the activity of the co-dosed other drug(s). Especially the altered metabolism and clearance affected by co-dosed drugs, is of central concern for those drugs with a narrow therapeutic window. In addition, some normal dietary substances are capable to alter CYP activity, grape fruit juice caused CYP3A4 inhibition being a classical example.

Different human populations as well as individuals within a given population segment can have differences in the activity levels of various CYP enzymes. There are large number of single nucleotide polymorphisms (SNPs) and haplotypes found in the CYP enzymes, both in their coding regions as well as on various gene regulation regions (like 5′ region). These are either directly affecting the activity of the given CYP enzymes or altering their induction potential. The most concerned CYP in regards of the genetic differences in various populations is CYP2D6. CYP2D6 is known to be involved in the metabolism of over 50 drugs (including antipsychotics, antiarrhythmics, antidepressants, selective serotonin reuptake inhibitors and beta-adrenoceptor blockers). Some 8% of Caucasians are classified as poor metabolizers due to lowered CYP2D6 activity. ON the other hand, approximately 1% of Caucasians carry more than one copies of active CYP2D6 genes, making them ultrarapid metabolizers. Similar kind of pharmacogenomic differences are found at least in CYP2C19, CYP2C9.

Identification of metabolizing CYP enzyme

Identification of the metabolizing CYP enzyme(s), or so called reaction phenotyping, is of great importance in drug discovery. Often the developed new drugs are intended or likely to be co-administered with other drugs. Identification of the metabolizing CYP enzymes gives data for potential drug interactions with the other existing drugs with known CYP profiles as occupancy of the same metabolic route is considered a potential risk factor. Other main reason for identification of metabolizing CYP enzyme(s) is the above mentioned pharmacogenomics observations, if the CYPs with known population polymorphisms are involved in the metabolism this would need to be taken into consideration.

There are different ways to identify the CYP enzymes involved in metabolism of the drug compounds.

  • Using recombinant CYP enzymes
  • Using CYP specific small molecule inhibitors
  • Using CYP specific inhibiting antibodies

Using recombinant CYP enzymes

Recombinant CYP enzymes are used in an incubation with the tested drug compound and co-factors (NADPH) and the possible formation of a CYP specific metabolite is detected by LC-MS. The results from activity of different CYP isoforms are interpreted accordingly to the known amounts of each CYP isoform in the liver. In addition to various CYPs, also other enzymes known to be involved in xenobiotic metabolism, are available as recombinant proteins from commercial vendors and can be studied in a similar settings.

Using CYP specific inhibitors

The specific CYP activity present in microsomes towards the studied drug compound can be detected by using CYP specific inhibitors (small molecules or antibodies). A metabolic profile generated in the same settings without any CYP inhibitors is compared to those profiles, where each major CYP enzyme is individually inhibited and the difference of metabolites in the presence of a given specific inhibitor is taken as a proof of the involvement of the inhibited CYP isoform. Further, by using different concentrations of the specific inhibitors, IC50 values can be calculated.

CYP inhibition

CYP inhibition is studied typically in either microsomal fractions and co-factors for CYP activity (NADPH) or in fresh or cryopreserved hepatocytes. The study setup involves know, CYP specific substrates with known metabolites. The potential effect by the tested drug compound on the quantity of the generated CYP specific known metabolite is measured, i.e. if the tested compound is able to inhibit a specific CYP enzyme, there is less amount of the metabolite generated from the used CYP specific reference compound. Similarly, by using different concentrations, IC50 values can be calculated in a more detailed study.

A multiplexed way to study CYP inhibition, so called N-in-one cocktail approaches, have also been developed. In those assays, specific substrates for more than one CYP enzyme are added to the same incubation reaction with the studied drug compound and the effect of the studied compound on the different CYPs as the quantity of the produced metabolites is measured at the same time.

The CYP inhibition can also be covalent inhibition, i.e. that the drug compound is able to irreversibly inhibit the CYP enzyme, until the enzyme is recycled by new biosysnthesis. This is called mechanism based inhibition (MBI; or time based/dependent inhibition or suicide inhibition). The prediction of MBI is critical in drug development, since MBI can result in long-lasting CYP inhibition and even associated toxicity. The study setup for detecting MBI involves a microsome system with NADH and a pre-incubation (typically 30 min) with the studied new drug compound. After the pre-incubation, the reaction is diluted and a set of known CYP specific substrates are added. The secondary incubation is stopped after some time (like 20 min) and the amounts of generated known metabolites from the CYP specific reference compounds are quantitated by LC-MS. These results are compared to an arrangement, where the tested drug compound and the CYP specific inhibitors are added at the same time (ie without a preincubation with the tested drug compound alone). If more inhibition is observed with the preincubated reaction, MBI is assumed.

CYP induction

CYP induction is typically measured in a couple of ways. A direct measurement uses fresh/cryopreserved primary human hepatocytes. The cells are plated and after forming as a monolayer (1-2 days), the studied drug compound is added on the culture medium as well as known CYP inducers on separate plates as control. After the induction period (typically 24-72h), allowing the synthesis of increased amounts of the particular CYP enzyme, CYP specific substrates are added. The rate of metabolism is compared betwen non-induced, specifically induced with the particular known CYP inducer and the tested drug compound – increased quantity of metabolites indicated CYP induction. Alternatively the mRNA levels of specific CYP transcripts can be quantitated by qPCR after the incubation with the drug compound and controls.

An alternative way to measure CYP induction is to detect the activation of nuclear receptors transcription factors. Nuclear receptors, like aryl hydrocarbon receptor (AhR), pregnane X receptor (PXR), constitutive androstane receptor (CAR), peroxisome proliferator-activated receptor (PPAR), farnesoid X receptor (FXR), liver X receptor (LXR), hepatic nuclear factor (HNF) family members, glucocorticoid receptor (GR) and CCAAT/enhancer-binding proteins (C/EBPs) can be activated by the xenobiotic and in turn are activating the transcription of the controlled CYP enzymes. The various CYP isofomrs are differentially regulated by these transpiration factors.

The activity of these nuclear receptors is typically done as a luciferase reporter plasmid activity measurements in human hepatoma cell line (HepG2). The cells are co-transfected with the measured nuclear receptor cDNA and the relevant luciferase reporter gene fusion, containing promoter region the co-expressed nuclear receptor is binding to and thus activating the luciferase transcription. The studied drug compounds are added in different concentrations to the culture medium and the luciferase activity is measured (typically after 24 h). The study setup includes known inducers of the nuclear receptors as controls. Compared to the direct induced CYP activity measurements, the indirect reporter gene fusion setup is less expensive since the primary hepatocytes are not needed.