Application of Cytochrome p450 (CYP450) and UDP-glucuronosyltransferase (UGT) Enzymes in Drug-drug Interaction Studies Regarding Metabolic Phenotype and Enzyme Inhibition

Key words: drug-drug interaction (DDI), cytochrome p450 (CYP450 enzyme), udp-glucuronosyltransferase (UGT), Enzyme inhibition, CYP450 enzyme metabolic phenotyping study, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4/5, CYP2A6, CYP2E1, UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B7, UGT2B15, UGT2B17.

• IPHASE Produce

Note: CYP Enzymes are usually used with NADPH Regeneration System/ NADPH and PBS
          UGT Enzymes are usually used with UGT Incubation System and PBS.

Metabolism plays a crucial role in the fate of drugs, affecting their disposal throughout the body and thus impacting target site exposure and impact. This mainly occurs in the liver, but it may also occur in extrahepatic organs. Recent studies have revealed the existence and functional significance of drug metabolizing enzymes (DME) in the brain. Cytochrome P450 enzyme (CYP) and UDP-glucuronosyltransferase (UGT) are also key participants in drug biotransformation within the central nervous system (CNS).

• Cytochrome p450 (CYP)

Cytochrome p450 dominates phase I metabolism (oxidation, reduction, hydrolysis), accounting for over 75% of drug metabolism. Key subtypes include CYP3A4 (50% drug metabolism) and CYP2D6 (20% drug metabolism). CYP converts lipophilic drugs into polar metabolites, promoting excretion.

Primary hepatocytes are hepatocytes isolated directly from human or animal liver, and have become the preferred model for drug metabolism research due to their preservation of intact CYP enzyme activity and physiological relevance. The primary hepatocytes model is the most widely accepted evaluation model for CYP enzyme induction in industry, academia, and regulatory agencies. As a cellular system, human hepatocyte are composed of nuclear receptors, co activators and inhibitors, target genes and promoters, as well as drug metabolizing enzyme capable of those of the liver and can effectively simulate the induction of candidate drugs and their metabolites.

• UDP-glucuronosyltransferase (UGT)

UDP-glucuronosyltransferases is a primary phase Ⅱ enzyme that uses glucuronic acid as a sugar donor to catalyze the binding of glucuronic acid with exogenous substances and polar groups, promoting its clearance. Human UGT is widely distributed and expressed in tissues such as the liver, small intestine, kidney, stomach, and lungs. The liver is the main organ in the human body that undergoes glucuronic acid binding reactions, and most UGT subtypes are expressed in the liver. UGT1A7, UGT1A8, UGT1A10, and UGT2A1 are distributed in extrahepatic tissues, and the glucuronic acid binding reaction occurring in extrahepatic tissues is mainly related to the absorption and excretion of drugs.

• Key Applications in Drug Development

In vitro screening model: CYP or UGT enzymes such as liver microsomes/primary hepatocytes are used for liver microsome/ hepatocytes incubation experiments to evaluate the metabolic rate and half-life of candidate drugs, and then evaluate the metabolic stability of candidate drugs (such as CYP3A4 inhibition experiments). Using gene edited cells to predict clinical metabolic differences.

Drug-drug interaction (DDI) study: Conduct CYP inhibition experiments to detect whether candidate drugs inhibit key CYP enzymes (such as CYP3A4, CYP2C9) and predict clinical DDI risk. Conduct UGT inhibition experiments to evaluate the effect of drugs on UGT activity. Detecting the induction effect of drugs on CYP/UGT through primary hepatocytes analysis.

Development of biological analysis methods: The study of CYP and UGT enzymes plays a critical role in bioanalytical method development and validation for drug metabolism and pharmacokinetic (DMPK) studies. Use biological matrices containing CYP/UGT metabolites (such as bile and plasma) for matrix effect evaluation, optimize LC-MS/MS methods, and avoid ion inhibition/enhancement effects.

Genetic polymorphism research: UGT1A1 and other genes that control UGT enzyme expression are important genes involved in human metabolic cycles. With the development of pharmacogenomics, it has been found that their genetic polymorphism is related to certain drug metabolism levels, which in turn affects the occurrence, development, and treatment of diseases and many other aspects.

Study on species differences: Compare the differences in CYP enzyme activity of primary hepatocytes such as humans, rats, and dogs, and optimize the transition strategy from preclinical to clinical use.

• Enzyme Inhibition

CYP enzyme mediated enzyme inhibition refers to the phenomenon where certain compounds can inhibit the activity of certain CYP450 metabolic enzymes, resulting in slowed metabolism, reduced clearance rates, and increased exposure of certain drugs when used in combination, thereby posing a safety hazard. According to the different mechanisms of inhibition, the inhibitory effect of drugs on CYP450 enzymes can be divided into reversible inhibition and time-dependent inhibition (TDI). Time dependent inhibition, also known as irreversible inhibition, generally refers to the formation of a complex between a candidate drug and a CYP enzyme through covalent bonds, resulting in irreversible enzyme inactivation. The inhibitory effect of the inhibitor on the enzyme does not immediately disappear after the inhibitor is removed, but exhibits time-dependent characteristics.

• CYP450 Enzyme Metabolic Phenotyping Study

At present, there are three main methods for identifying the enzyme metabolic phenotype of CYP450: selective inhibition method, recombinant human CYP450 isoenzyme method, and correlation analysis method. Selective inhibition method can be divided into chemical inhibition method and antibody inhibition method. It involves measuring the metabolic activity of human liver microsomes on drugs with and without the addition of a series of CYP450 enzyme subtype selective chemical inhibitors or antibodies, in order to investigate whether the metabolism of drugs is affected by the selective inhibition of CYP450 enzyme subtypes in human liver microsomes, calculate the relative inhibition percentage, and infer the CYP450 enzyme metabolic phenotype. Among them, chemical inhibition method has been widely used due to its simple operation and low cost.

• Conclusion

CYP enzymes and UGT enzymes, as the core enzyme systems of drug metabolism, respectively dominate phase I (oxidation, reduction) and phase II (glucuronidation) reactions, profoundly affecting the efficacy, toxicity, and personalized use of drugs. Subtypes such as CYP3A4 and CYP2D6, along with enzymes such as UGT1A1 and UGT2B7, form a complex metabolic network. Their activity differences (such as gene polymorphism and species specificity) can be accurately analyzed through primary hepatocytes models and LC-MS/MS technology, providing key data support for drug development.

Reference

Zhang, M., Rottschäfer, V., & CM de Lange, E. (2024). The potential impact of CYP and UGT drug-metabolizing enzymes on brain target site drug exposure. Drug Metabolism Reviews, 56(1), 1-30.

Ghosal, A., Ramanathan, R., Kishnani, N. S., Chowdhury, S. K., & Alton, K. B. (2005). Cytochrome P450 (CYP) and UDP-Glucuronosyltransferase (UGT) enzymes: role in drug metabolism, polymorphism, and identification of their involvement in drug metabolism. In Progress in Pharmaceutical and Biomedical Analysis (Vol. 6, pp. 295-336). Elsevier.