In Vitro Metabolism Assay Kits

In vitro drug metabolism is the use of an ex vivo system, such as cells, tissue homogenates, subcellular fractions, or purified enzymes, to mimic the biotransformation of drugs in vivo. In vitro metabolism study is an important step in drug development. In the early stage of drug development, candidates that are metabolically unstable or may produce toxic metabolites can be rapidly screened through in vitro drug metabolism tests, thereby reducing the risk and cost for later clinical trials. The in vitro data can be used to predict the metabolic pathways, metabolic rates, and main metabolites of candidates in vivo, which provides an important reference for in vivo pharmacokinetic (PK) studies. In addition, whether a drug acts as a substrate, inhibitor, or inducer of metabolic enzymes can be evaluated, and potential drug-drug interactions (DDI) can be predicted by in vitro studies, which is essential for ensuring drug safety.

Amerigo Scientific offers a range of kits specifically designed for in vitro drug metabolism studies. The utilization of these kits can eliminate the labor-intensive process of preparing liver microsomes/recombinases and reagents, significantly reducing the experimental timeframe.

Metabolic Stability Assay

Drug metabolism in vivo generally converts nonpolar, lipophilic pharmacologically active drug molecules into polar, inactive, or nontoxic metabolites that are readily eliminated by the kidneys or other organs. Generally, drug metabolism pathways are classified into phase I and phase II. Phase I metabolism involves functionalization of drug substrates to yield more polar derivatives such as alcohols, phenols, or carboxylic acids, and phase II metabolism involves bimolecular conjugation with endogenous hydrophilic moieties to yield products such as glucuronides and sulfates that are readily excreted by the kidneys.

The metabolic stability of a drug directly influences its clearance rate in the body. Drugs with low metabolic stability are rapidly metabolized and cleared, potentially resulting in insufficient drug exposure and difficulty in achieving effective therapeutic concentrations. For orally administered drugs, hepatic first-pass metabolism is a critical factor affecting bioavailability. Evaluation of metabolic stability in the early stage of drug development allows for timely identification and elimination of compounds with poor metabolic properties.

Amerigo Scientific provides easy-to-use kits for the assessment of phase l and phase ll metabolic stability. The kits contain all components to be directly used for metabolic stability assay, such as NADPH regeneration system and/or other incubation system, liver microsomes, buffer, and positive substrate.

Phase I Metabolism Kits

Liver microsomes contain most of the phase I enzymes, the most important of which is cytochrome P450s (CYPs). When NADPH enzyme cofactors are added to the liver microsomes, the phase I metabolic system can be reestablished. Amerigo Scientific provides phase I metabolism kits to study the phase I metabolic stability of drug candidates.

Phase II Metabolism Kits

Phase II metabolism, also known as conjugation reactions, involves the covalent attachment of endogenous hydrophilic groups to phase I metabolites or parent drugs to yield polar metabolites, resulting in reduced toxicity, activity, or polarity of the prodrug. In phase II metabolism, the most common reaction is glucuronidation, during which uridine-5′-diphospho-α-d-glucuronic acid (UDPGA) is bound to a drug molecule under the catalysis of UDP-glucuronosyltransferases (UGTs) in microsomes. Amerigo Scientific provides phase II metabolism kits that contain liver microsomes and UGTs for evaluating the phase II metabolic stability of drug candidates in vitro.

Features of Phase I/II Metabolism Kits

• Convenience: Kits contain all reagents required for in vitro metabolic studies, making them easy to use and saving preparation time.
• Accuracy: Each component in the kit undergoes rigorous quality testing to ensure that the test results are accurate, reliable, and highly reproducible.
• Stability: The high stability of the kits makes them easy to transport and store.

Cytochrome P450 Phenotyping

Drug-metabolizing enzyme phenotyping, also known as reaction phenotyping, is an important step of metabolism research in drug development to identify drug metabolizing enzymes that are mainly involved in the metabolism of drugs in vivo. By identifying the major enzymes responsible for drug metabolism, the major clearance pathways of drugs in vivo can be predicted.

CYPs is a superfamily of hemoproteins that catalyze the phase I biotransformation of many endogenous substrates and exogenous substances. Multiple forms of CYP enzymes are present in all eukaryotes, animals, plants, and fungi. Human drug metabolism CYPs are concentrated in the smooth endoplasmic reticulum of the Zone 3 hepatocytes in the liver. The major human drug metabolism CYPs belong to families 1, 2, and 3, and the specific isoforms are 1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5, and 3A7.

CYP Metabolic Phenotyping Kits

Amerigo Scientific provides a range of kits that can be directly used for the identification of the metabolic phenotype of drugs. The kits contain all the reagents required for metabolic phenotyping, such as NADPH regeneration system, liver microsome/recombinase, buffer, positive substrate, and inhibitor.

Cytochrome P450 Inhibition (IC50) Assay

Combination therapy involves using multiple treatment modalities to obtain increased therapeutic effects. However, the combined efficacy may be accompanied by adverse events caused by drug-drug interactions (DDIs). If two drugs are metabolized by the same enzyme, or a drug is an inhibitor or inducer of a metabolic enzyme, DDIs may occur when the two drugs are used at the same time. DDIs may change the blood concentration of drugs, resulting in altered efficacy or increased toxic side effects.

DDIs are a leading cause of serious adverse reactions in clinical practice. CYP-related DDIs primarily involve enzyme inhibition and enzyme induction. Enzyme inhibition occurs when a compound suppresses the activity of drug-metabolizing enzymes, thereby slowing the metabolism of co-administered drugs. This results in increased blood concentrations of the affected drug, potentially leading to toxicity. Enzyme induction refers to a compound enhancing the activity of drug-metabolizing enzymes, which accelerates the metabolism of co-administered drugs. This leads to decreased blood concentrations of the affected drug, causing reduced therapeutic efficacy.

Figure 1. CYP-related Drug- Drug Interaction

CYP Inhibition Assay Kits

Amerigo Scientific provides easy-to-use kits that can be directly used for the rapid assessment of CYP enzyme inhibition by drug candidates.