Related Experiment Video
Updated: Jan 12, 2026
10:44
Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures
Published on: March 28, 2017
10.2K
Human cytochrome P4502C8 metabolizes repaglinide to 4'-hydroxyrepaglinide, not 3'-hydroxyrepaglinide.
Raman Sharma1, Amanda Balesano1, Angela C Doran1
1Pfizer Inc., Groton, Connecticut.
Drug Metabolism and Disposition: the Biological Fate of Chemicals
|October 30, 2025
Summary
Previous studies incorrectly identified the main metabolite of repaglinide. This research corrects the structure, showing hydroxylation occurs at the 4-position, not the 3-position, impacting CYP2C8 activity studies.
Area of Science:
- Drug Metabolism and Pharmacokinetics
- Enzyme Kinetics
- Organic Chemistry
Background:
- Cytochrome P450 2C8 (CYP2C8) is crucial for metabolizing drugs like repaglinide.
- The 3'-hydroxylation of repaglinide has been used as a biomarker for CYP2C8 activity.
- Previous reports misidentified the site of repaglinide hydroxylation.
Purpose of the Study:
- To unequivocally determine the correct site of repaglinide hydroxylation catalyzed by CYP2C8.
- To correct the structural assignment of the major hydroxyrepaglinide metabolite.
- To ensure accurate interpretation of CYP2C8 activity in drug metabolism studies.
Main Methods:
- Biosynthesis of the repaglinide metabolite using recombinant CYP2C8.
- Isolation and structural elucidation using high-resolution mass spectrometry and 1D/2D NMR spectroscopy.
- Comparative analysis with synthetic standards and low-temperature 2D NMR for definitive structural assignment.
Main Results:
- Demonstrated that CYP2C8 hydroxylates repaglinide at the 4-position, not the 3-position.
- Determined Michaelis constants for 4'-hydroxylation in human liver microsomes (10.2 μM) and recombinant CYP2C8 (5.4 μM).
- Confirmed the structure of the 4'-hydroxymetabolite through advanced spectroscopic techniques.
Conclusions:
- The primary hydroxy metabolite of repaglinide generated by CYP2C8 is 4'-hydroxymetabolite, not 3'-hydroxyrepaglinide.
- Accurate identification of this metabolite is critical for reliable assessment of CYP2C8 activity.
- This finding necessitates a re-evaluation of previous in vitro and in vivo studies relying on this metabolic marker.
Related Concept Videos
01:06Oral Hypoglycemic Agents: Glinides
585
Repaglinide (Prandin) and Nateglinide (Starlix), known as glinides, are oral insulin secretagogues that stimulate insulin release from pancreatic β cells by closing the ATP-sensitive potassium channels (KATP channel). Repaglinide controls insulin release from pancreatic β cells by managing potassium efflux. It shares two binding sites with sulfonylureas and also has a unique site, indicating overlapping mechanisms of action. With a rapid onset and a 4-7 hour duration, it effectively...
585
01:17Drug Metabolism: Phase I Reactions
4.6K
A phase I reaction is a biochemical process that introduces a functionally reactive polar group to a substance. This transformation predominantly occurs in the liver, facilitated by the cytochrome P450 system of hemoproteins situated in the lipophilic endoplasmic reticulum of cells. The metabolite generated through this process can have varying polarities. If it is sufficiently polar, it can be easily excreted in the urine due to its water compatibility. However, if the metabolite is nonpolar,...
4.6K
01:14Drug Metabolism: Phase II Reactions
4.8K
Phase II reactions are essential for the detoxification and elimination of drugs from the body. These reactions involve the conjugation of parent drugs or their phase I metabolites with endogenous molecules, resulting in more hydrophilic drug conjugates. The primary conjugation reactions in this phase are sulfation and glucuronidation. Both sulfation and glucuronidation typically produce biologically inactive metabolites. However, in some cases involving prodrugs, active metabolites may be...
4.8K
01:23Dipeptidyl Peptidase 4 Inhibitors
574
Dipeptidyl peptidase 4 (DPP-4) is a serine protease widely distributed in the body. It's involved in the inactivation of GLP-1 and GIP hormones, which are crucial for insulin regulation. DPP-4 inhibitors, such as sitagliptin (Januvia), saxagliptin (Onglyza), linagliptin (Tradjenta), alogliptin (Nesina), and vildagliptin (Galvus), help increase the proportion of active GLP-1, enhancing insulin secretion. These inhibitors work by competitively binding to DPP-4. This binding causes a...
574
01:20Pharmacokinetics in Obese Patients: Drug Metabolism and Excretion
164
Drug metabolism, a critical process in the liver, involves two primary phases: Phase I reactions and Phase II conjugation. Obesity introduces significant alterations in this metabolic process, primarily due to fatty infiltration of the liver, leading to conditions such as nonalcoholic fatty liver disease (NAFLD). This condition can modify the activities of both Phase I and II enzymes, impacting how drugs are metabolized in obese patients.Phase I metabolism sees variable effects across...
164
01:24Phase II Reactions: Glucuronidation
1.6K
Glucuronidation, a pivotal phase II biotransformation process, involves the coupling of glucuronic acid to a drug or xenobiotic. Given its widespread occurrence and critical role in drug metabolism, it's considered the most crucial phase II reaction. It enhances the water solubility of substances, aiding their expulsion from the body. The driving force behind these reactions is a group of enzymes known as UDP-glucuronosyltransferases (UGTs). UGTs facilitate the transfer of a glucuronic acid...
1.6K