In vitro metabolic stability is an important early ADME test for predicting in vivo half-life and clearance of the test compounds. Although the metabolism of drugs generally occurs in a variety of tissues such as the intestinal wall, kidneys, lung, skin and blood, the liver is considered to be the major site of drug metabolism. Hepatocytes or liver microsomes are, therefore, the fast and cost-effective way to determine the intrinsic clearance (CLint) of a drug candidate. These CLint assays, along with other parameters including plasma protein binding and blood-to-plasma ratio, often allow a good prediction of in vivo hepatic clearance.
The Advantages of Enhancing Metabolic Stability
- Increasing bioavailability and half-life, less frequent dosing
- Better consistency between dose and plasma concentration, reducing need for therapeutic monitoring
- Reduction in turnover rates from different preclinical species, may improve extrapolations from animal data to humans
- Lower patient to patient variability in drug levels, as patient variability is largely due to differences in drug metabolism capacity
- Reducing the need for further studies of metabolites in animals and humans
Whether the drug is designed to be administered orally, intravenously, or topically will affect the type of in vitro testing required. Creative Bioarray offers a wide range of biological systems to determine in vitro metabolic stability including primary hepatocytes, tissue homogenates and tissue fractions (e.g. microsomes, mitochondrial preparations, S9 fractions and intestinal fluid) from a variety of preclinical species.
Liver microsomes are a recognized source of drug metabolizing enzymes. They contain cytochrome P450, FMO carboxylesterases, epoxide hydrolase and UGT. Liver microsomes are widely used to determine in vitro intrinsic clearance (CLint), which can then be scaled to predict in vivo hepatic clearance.
S9 fractions can be used to evaluate phase I metabolism and some phase II (conjugation) reactions, including sulfation and glucuronidation, depending on the co-factors added. It is highly enriched in cytoplasm and microsomes.
Intact hepatocytes are often referred to as the "gold standard" for in vitro evaluation of metabolism, as they allow for measurement of phase I and II metabolism under conditions where compound movement is restricted by membranes as it is in vivo and in the presence of similar co-factor concentration.
Intestinal Fluid Metabolic Stability
The intestinal fluid are comprised of bile salts, phospholipids, cholesterol and bicarbonate as well as a series of digestive enzymes such as proteases, peptidases, lipases, carbohydrases and nucleases. However, their value to assess drug stability is limited, as they do not contain digestive enzymes.
Metabolic Stability in Small Intestinal Mucosal Homogenate
The small intestinal mucosa consists mainly of mucus and enterocytes. These cells are rich in phase I and phase II enzymes and can significantly lower oral bioavailability. Small intestinal mucosal homogenate is a powerful and cost-effective tool for assessing drug metabolic stability in the presence of relevant co-factors.
Key Features of Our Metabolic Stability Services
- LC/MS method development for analysis of the test articles
- Screening are performed at four optional time points (short-, medium-, long-time course options)
- Incubating in a suitable test system (S9, microsome, hepatocyte, recombinant CYP enzyme) with appropriate cofactors and assessing at multiple time-points in triplicate, with additional replicates for critical samples
- Comparisons of time and cofactor blanks
- Incubation of marker substrates as positive controls to check for metabolic competency of the test system
- Data analysis
Creative Bioarray has a leading portfolio of compound stability services designed to help you understand the metabolic profile of your compounds. All our assays can be ordered individually or in combination to provide cost savings.
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