Cytochrome P450 Reaction Phenotyping for 4 Additional CYPs: Why and How?

Metabolism is the major clearance mechanism listed for approximately three-quarters of the top 200 prescribed drugs in the United States in 2002¹. Successful identification of the major enzyme(s) of a New Chemical Entity (NCE) is critical for the pharmaceutical industry. Cytochrome P450 (CYP) enzymes are listed as contributors to clearance for 48% of drugs cleared by metabolism, with CYP3A4 being the major isoform among all CYPs. Besides, some literature suggests that additional enzymes, such as CYP2A6, CYP2J2, CYP4F2, and CYP2E1, also play a major role in drug metabolism^2-4. Therefore, the objective of this study is to establish additional human CYP reaction phenotyping assays using recombinant human CYPs, which successfully identified the enzymes involved in drug metabolism. This article will illustrate the meaning, methods, and results of the additional Cytochrome P450 reaction phenotyping assays, and provide case studies on its applications.

Why do we need to establish additional human CYP reaction phenotyping assays?

If the drug is metabolized by a relatively singular enzyme and metabolism is the major route of elimination of the drug from the body, the risk of metabolism-based drug-drug interaction (DDI) with co-administered drugs may be much higher. Reaction phenotyping is a study to identify and characterize the major enzymes involved in the metabolism of a drug. Accurate identifications help provide an early risk assessment of their potential to exhibit significant metabolism-based DDI.

(1) The requirements of CYP enzyme research scope in FDA Guidance

In the Draft FDA Guidance (2006), CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A were listed as enzymes which should be evaluated in in vitro phenotyping experiments routinely to determine which enzymes metabolized the investigated drug. By 2012, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A are still recommended as routine ones, but additional enzymes are first mentioned. If a drug is not metabolized by the major CYPs (listed above), the drug’s likelihood of being a substrate for other CYP enzymes (e.g., CYP2A6, CYP2J2, CYP4F2, CYP2E1) or non-CYP Phase I enzymes should be considered. In the Final FDA Guidance (2020), CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A were listed as enzymes which should be evaluated in in vitro phenotyping experiments routinely to determine which enzymes metabolize the investigational drug. If the investigational drug is not found to undergo significant in vitro metabolism by these major CYP enzymes, the sponsor should then determine what additional enzymes contribute to the metabolism of the investigational drug. These additional enzymes include but are not limited to:

• CYP enzymes including CYP2A6, CYP2J2, CYP4F2, and CYP2E1
• Other Phase I enzymes
• Phase II enzymes

(2) Additional enzymes play a major role in the metabolism of a drug

According to the Final FDA Guidance (2020), the contribution of a specific metabolizing enzyme to an investigational drug’s clearance is considered significant if the enzyme is responsible for > 25% of the drug’s elimination based on the in vitro phenotyping studies and human PK data. Under these circumstances, the sponsor should conduct clinical DDI studies using strong index inhibitors and/or inducers of the enzyme. Thus, reaction phenotyping has become standard practice in identifying and characterizing the major enzymes in drug discovery and development.

With the number of new molecular entities (NME) increasing, additional enzymes (e.g., CYP2A6, CYP2J2, CYP4F2, CYP2E1) have played a major role in the metabolism of the investigated drug. For example, CYP4F2 is the major enzyme in the metabolism of Fingolimod Hydrochloride (trade name: Gilenya) and the prodrug (Compound 3) for Gemcitabine, respectively.

Considering (1) the requirements of FDA Guidance, (2) the research and development trend of investigating drugs, (3) the abundance of individual CYPs in native human liver and (4) the proportion of CYPs involved in the metabolism of drugs, the scope of investigation for additional CYP enzymes in this section will be defined as follows: CYP2A6, CYP2J2, CYP4F2, CYP2E1 using recombinant human CYPs as the experimental system.

When should we conduct additional human CYP reaction phenotyping assays?

There are three methods used for identifying the individual CYP enzymes responsible for a drug's metabolism: (1) chemicals, drugs, or antibodies as specific enzyme inhibitors in human liver microsomes; (2) individual human recombinant CYP enzymes; (3) correlation analysis which involves measuring the reaction velocity of the biotransformation pathway of interest in several individual human liver samples and correlating the reaction rates with the activity of the individual CYP enzymes in the same bank of microsome samples. The two approaches, including CYP isozyme-selective chemical inhibition and metabolism study using recombinant human CYP enzymes, are widely used in reaction phenotyping.

1. CYP enzyme-selective chemical inhibition method: the investigated drug will be incubated with human liver microsome in the presence and absence of a selective chemical inhibitor to each CYP enzyme. The percentage inhibition of metabolism of the investigated drug by the CYP-selective inhibitor will be calculated. The CYP enzyme, which showed significant inhibition in the presence of its selective chemical inhibitor, is assigned as the metabolic enzyme of the investigated drug.
2. Metabolism study using individual human recombinant CYP enzymes: the investigated drug will be incubated with each human recombinant CYP enzyme to measure the reaction velocity. Scaled reaction velocity in microsomes will be calculated by multiplying the reaction velocity by the specific CYP with its expression abundance in native human liver microsomes. %Contribution by individual CYP in human liver microsomes will be calculated.

Generally, both the chemical inhibition study and recombinant CYP metabolism study consistently confirmed that CYPx is the major enzyme for the metabolism of an investigated drug. However, in some cases, the results of the chemical inhibition study and recombinant CYP metabolism study are inconclusive. Thus, the investigational drug is not found to undergo significant metabolism by these major CYP enzymes. Under such circumstances, additional human cytochrome P450 (CYP450) reaction phenotyping assays should be conducted.

How do we establish additional Cytochrome P450 reaction phenotyping assays?

Unlike the common CYP isozymes, the role of additional human CYP could not be confirmed in both the selective chemical inhibition method and metabolism study using individual human recombinant CYP enzymes. Thus, the objective of this study is to obtain consistent values of the enzyme kinetic parameters (e.g., K_m and V_max) or intrinsic clearance (CL_int) using substrates for individual CYPs, as reported in the literature, based on the characteristics of drug metabolic enzymes. Taking CYP2J2 as examples, the methodology is introduced below.

Example 1: CYP2J2

CYP2J2 is the only member of the human CYP2J subfamily known for metabolizing arachidonic acid to physiologically important epoxides. The mRNA level of CYP2J2 is highest in the small intestine and heart, while it is relatively lower in the liver.

The active site cavities for CYP2J2 and CYP3A4 are depicted individually in Fig. 1, A and B, respectively, and Fig. 1C depicts an overlay of the two active sites with the I helix included for orientation. The homology model suggests that the active site volume for CYP2J2 is similar to that of CYP3A4. Moreover, Ketoconazole, a CYP3A inhibitor commonly applied in the chemical inhibition study in human liver microsomes, can inhibit the activity of CYP2J2 under certain conditions. Therefore, an apparent metabolism of a compound of interest by CYP3A in a chemical inhibitor approach could actually be mediated by CYP2J2. Since no selective chemical inhibitors are available for CYP2J2, CYP2J2 phenotyping couldn’t be identified using CYP enzyme-selective chemical inhibition method in human liver microsome. Therefore, a metabolism study using recombinant CYP2J2 will be established by measuring intrinsic clearance (CLint) of Astemizole (CYP2J2 probe substrate).

Figure 1. Comparison between the active site cavity volumes for human CYP2J2

(Green, based on homology model) (A), CYP3A4 (red, Protein Data Bank 1tqn)

active sites (B), and overlay of CYP2J2 and CYP3A4 (C).

Results of additional human CYP reaction phenotyping assays

The enzyme kinetic parameters (e.g., K_m and V_max) of metabolites from probe substrates for CYP2A6, CYP4F2, and CYP2E1 were determined. The results are shown below.

Under the optimized linear conditions, the enzyme kinetic parameters (e.g., K_m and V_max) or intrinsic clearance (CL_int) using probe substrates for individual CYPs (CYP2A6, CYP4F2, CYP2E1, and CYP2J2) were determined and showed in Table 1 and Table 2. The values for enzyme kinetic parameters (e.g., K_m and V_max) or intrinsic clearance (CL_int) are consistent with the values from literature (within 2-fold). Therefore, additional human CYP reaction phenotyping assays (including CYP2A6, CYP4F2, CYP2E1, and CYP2J2) were established successfully.

Table 1. K_m Values in WuXi AppTec vs. Publication

Note: WuXi AppTec (blue), Publication (red)

Table 2. CL_int Values in WuXi AppTec VS Publication

Note: WuXi AppTec (blue), Publication (red)

The CYP2J2 metabolism study was performed according to the method of Caroline A Lee (2010). The results indicated that Astemizole was highly metabolized in recombinant human CYP2J2 at 100 pmol/mL. And the %remaining of Astemizole was less than 10 at 5-minute timepoint, suggesting the value of CL_int might be underestimated at 100 pmol/mL. The CL_int value for CYP2J2 from WuXi AppTec was higher than the value from the publication using the optimized recombinant enzyme concentration (5 pmol/mL or 10 pmol/mL).

Application of additional human CYP reaction phenotyping assays

Case 1: For initial metabolite M1 from Compound A, the results of chemical inhibition study and recombinant CYP metabolism study were contradictory. The chemical inhibition study showed that CYP3A was the major metabolic enzyme catalyzing the formation of M1. However, the recombinant CYP metabolism study showed that CYP2C9 was the major metabolic enzyme (Fig. 2). The additional metabolism study using recombinant human CYP isozyme showed that CYP2J2 was the major metabolic enzyme (Fig. 3). In conclusion, the results above showed that CYP2J2 was the major metabolic enzyme for the formation of M1.

Figure 2. %Contribution of Individual CYP (7CYPs) to the Metabolite M1 from Compound A in Human Liver Microsomes

Figure 3. The relative formation rate of M1 in recombinant CYP2J2 and CYP2C9

The cases above results indicate that additional recombinant CYP assays provided more important and reliable information to researchers.

A final word

The in vitro studies on identifying important hepatic enzymes responsible for the metabolism of the drugs provide insights into the pharmacokinetic properties of these drugs. From these findings above, some additional enzymes, such as CYP2J2, have been identified to play a major role in drug metabolism. Therefore, establishing a reliable system to identify the role of additional human CYPs involved in drug metabolism is essential. WuXi AppTec has established a comprehensive and reliable system for testing additional CYP enzymes. Including additional CYPs (including CYP2A6, CYP2J2, CYP4F2, and CYP2E1) is critical to accurately identify the metabolic enzymes in the drug discovery and development stage. 

Talk to a WuXi AppTec expert today to get the support you need to achieve your drug development goals.

References:

1. Donglu Zhang et al. ADME-enabling technologies in drug design and development. 2012, 550-559.

2. Raccor BS and Kaspera R. Extra-hepatic isozymes from the CYP1 and CYP2 families as potential chemotherapeutic targets. Curr Top Med Chem. 2013, 13:1441–1453.

3. Caroline A. Lee, J. P. Jones III, Jonathan Katayama. Identifying a Selective Substrate and Inhibitor Pair for the Evaluation of CYP2J2 Activity. Drug Metab Dispos. 2012, 40:943–951.

4. Hege Christensen, Anette L. Hestad. CYP3A5-mediated metabolism of midazolam in recombinant systems is highly sensitive to NADPH cytochrome P450 reductase activity. Xenobiotica, 2011, 41(1): 1–5.

5. Yoshiya Yamamura, Noriyuki Koyama & Ken Umehara. Comprehensive kinetic analysis and influence of reaction components for chlorzoxazone 6-hydroxylation in human liver microsomes with CYP antibodies. Xenobiotica, 2015, 45(4): 353–360.

6. Michael Zhuo Wang, Janelle Y. Saulter, Etsuko Usuki. CYP4F Enzymes Are the Major Enzymes in Human Liver Microsomes That Catalyze the O-Demethylation of the Antiparasitic Prodrug DB289 [2,5-Bis(4-amidinophenyl) furan-bis-O-methylamidoxime]. Drug Metab Dispos. 2006, December; 34(12): 1985–1994.

7. Robert L. Walsky and R. Scott Obach. VALIDATED ASSAYS FOR HUMAN CYTOCHROME P450 ACTIVITIES. Drug Metab Dispos. 2004, February; 32: 647–660.

8. L. B. VON Weymarn and S. E. Murphy* CYP2A13-catalysed coumarin metabolism: comparison with CYP2A5 and CYP2A6. Xenobiotica. 2003, vol. 33, no. 1, 73–81

9. Xiaoliang Zhuo, Jun Gu, Qing-Yu Zhang. Biotransformation of Coumarin by Rodent and Human Cytochromes P-450: Metabolic Basis of Tissue-Selective Toxicity in Olfactory Mucosa of Rats and Mice. The Journal of Pharmacology and Experimental Therapeutics.1999, 288 (2) 463-471.

10. Caroline A. Lee, David Neul, Andrea Clouser-Roche, Deepak Dalvie. Identification of Novel Substrates for Human Cytochrome P450 2J2. Drug Metab Dispos. 2010, 38:347–356.

Author: Huan Liu, Lifang Jiang, Genfu Chen

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