Metabolism Studies Help to Complete Picture of Experimental Drug Activity

Determining the therapeutic effects and relative toxicity of an experimental drug completes only a part of the pre-clinical picture. Before a drug can be tested on humans, scientists first must understand the biotransformation that occurs when it is metabolized.

Because of the significant role that drug metabolism plays in determining in vivo drug action, many drug companies are including examination of drug metabolism properties as part of the screening process in their drug development efforts.

Identification of Metabolites

Part of the drug-development process involves identifying and quantitating the metabolites formed as the drug is metabolized. If a drug produces numerous metabolites, it introduces increased possibility of problems due to polymorphism, the variability in the way that different hosts will metabolize it, said Dr. Michael J. Cwik, Analytical Chemistry supervisor for IITRI's Life Sciences Group.

"The regulatory agencies want to see the metabolism of a compound to understand whether the metabolites are potentially more toxic than the drug itself," said Dr. Cwik, who holds a Ph.D. in medicinal chemistry and has more than 20 years of experience in the development of analytical methods for drug analysis. If the drug is metabolized extremely slowly, it can accumulate in the body to toxic levels, whereas if it is metabolized too quickly, its efficacy is diminished.

Analytical Methods

IITRI scientists use a combination of in vivo and in vitro studies to elucidate the metabolism of specific drugs. During in vitro studies, the drug is incubated with biological metabolizing agents (such as human liver or intestinal microsomes, S9 fraction or hepatocytes), and the outcome is then analyzed. With in vivo studies, the blood or urine of the laboratory animals treated with the drug is analyzed.

	"In a drug metabolism study, LC/MS/MS will first identify the intact metabolites produced from reaction, then target components of interest for further cleavage to get detailed information of their structure," Dr. Lina Long, Research Biologist Toxicology and Carcinogenesis

Liquid chromatography-mass spectrometry (LC/MS/MS) instrumentation provides a clear view of the biotransformation that occurs in the metabolic process. LC separates the parent compound and its individual metabolites, and MS/MS determines the structure of the metabolites, explained Dr. Lina Long, research chemist for the Toxicology and Carcinogenesis Division of IITRI's Life Sciences Group. Dr. Long is responsible for the development and validation of LC/MS/MS methods to identify drug metabolites, as well as for the design and conduct of in vitro studies of drug metabolism and drug-drug interaction at IITRI.

"In a drug metabolism study, LC/MS/MS will first identify the intact metabolites produced from reaction, then target components of interest for further cleavage to get detailed information of their structure," said Dr. Long, who holds a Ph.D. in medicinal chemistry and has more than 10 years of experience with in vitro and in vivo studies of the metabolism of cancer drugs.

Identification of Drug-Metabolizing Enzymes

Another important component of any drug metabolism study is to determine which enzyme or enzymes are responsible for metabolizing the drug. Scientists can identify the metabolizing enzymes by isolating one enzyme at a time and testing for a reaction. The results are useful in explaining or predicting pharmacokinetic variability, which may occur when a drug is metabolized by an enzyme that is expressed differently in different hosts. "It also lets you know if there is the possibility of a drug-drug interaction, if two drugs administered concurrently compete for the same enzyme," Dr. Long explained.

Determination of Drug-Drug Interactions

The co-administration of two or more drugs can alter the clearance of one of the agents. Drugs that are metabolized by the same enzyme will compete with each other for a binding site on the enzyme, thereby decreasing the rate of metabolism of the lower affinity drug. If the affected pathway is the major route of elimination for the drug, then increased plasma levels of the drug and prolonged or exaggerated pharmacological effects are possible.

Drug-drug interactions also can occur when one drug induces the metabolism of a second drug. In this case, the clearance of the drug would be increased, and the pharmacological effect diminished.

Scientists can reveal these possibilities by incubating two or more drugs together in vitro and testing for an altered reaction. This information is crucial to obtain before the drugs are administered to humans. If drug levels become too low or too high in patients with serious chronic illnesses such as heart disease or epilepsy who are taking multiple drugs, the effects can be life-threatening.

Outcomes

By elucidating the biotransformation that occurs when a drug is metabolized, these studies aid in the design of clinical trials to evaluate the drug's efficacy in humans. Results are combined with data on the efficacy and toxicity of the drug to support the selection of optimal patient populations (disease targets) for clinical studies. This data also helps to identify possible confounders of drug activity, and to investigate the pharmacologic mechanisms responsible for drug efficacy and toxicity, thereby creating a more complete picture of the drug's potential worth.