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Thursday, November 18, 2010

TECHNIQUES AND APPROACHES FOR STUDIES IN VITRO OF DRUG METABOLISM AND

TECHNIQUES AND APPROACHES FOR STUDIES IN VITRO OF DRUG METABOLISM AND DRUG INTERACTIONS

A. Cytochrome P-450, Microsomes, and Related Tools
B. Other Hepatic Enzymes
C. Gastrointestinal Drug Metabolism
D. Interspecies Metabolic Comparisons and Other Uses of Animal Data

TECHNIQUES AND APPROACHES FOR STUDIES IN VITRO OF DRUG METABOLISM AND DRUG INTERACTIONS

The goals in evaluating in vitro drug metabolism are: (1) to identify all of the major metabolic pathways that affect the test drug and its metabolites, including the specific enzymes responsible for elimination and the intermediates formed; and (2) to explore and anticipate the effects of the test drug on the metabolism of other drugs and the effects of other drugs on its metabolism. Pharmacologic effects of the test drug and its major metabolites also should be studied, if feasible. Knowledge that a particular drug is not a substrate for certain metabolic pathways is helpful. For example, if it is learned early in drug development that a molecule is not a substrate for CYP450 3A4 or that this pathway represents only a minor contribution to overall metabolism, then concern is lessened or eliminated for possible inhibition of 3A4 metabolism by drugs such as ketoconazole and erythromycin or possible induction of metabolism by drugs such as rifampin and anticonvulsants. Studies in vitro also could indicate whether a drug itself is or is not an inhibitor of common metabolic pathways. The potential for a drug inhibiting the metabolism of other drugs is almost always present for drugs metabolized by the same pathway, but can also be present for entirely separate pathways, including the principal metabolic route for a compound. This potential was first appreciated for quinidine, which is a substrate for metabolism by CYP450 3A4 and is also a very potent inhibitor of CYP450 2D6.

A. Cytochrome P-450, Microsomes, and Related Tools

1. Assessing the metabolism of a test drug The most mature technology for the study in vitro of drug metabolism (enzymes involved, metabolites formed, and potential inhibitors) is associated with the set of enzymes contained in the cytochrome CYP450 superfamily. These enzymes are responsible for the metabolism of the majority of drugs given to humans.

Metabolism usually occurs in the liver, but the enzymes (especially CYP450
3A4) also are important in gut metabolism. Human liver microsomes provide
the most convenient way to study CYP450 metabolism. Microsomes are a
subcellular fraction of tissue obtained by differential high-speed centrifugation.

All of the CYP450 enzymes are collected in the microsomal fraction. The
CYP450 enzymes retain their activity for many years in microsomes or whole
liver stored at low temperature (e.g., -70o C). Cofactor requirements for
CYP450-mediated reactions are well characterized, consisting primarily of a
redox sustaining system such as NADPH. Hepatic microsomes can be obtained
commercially, with or without prior phenotyping, for most important drug metabolizing enzymes.

During studies to identify metabolic routes of elimination for an investigational new drug, microsomes from several donors should be used, either individually or pooled, to avoid reliance on microsomes that are deficient in one or more metabolic pathways, unless this is a specific objective of the study. With the use of selective chemical inhibitors for each major pathway, the metabolic pathways for a new drug can be readily demonstrated or ruled out. Careful consideration of incubating concentrations of both inhibitor and substrate is essential to maintain a selective approach. For example, quinidine and ketoconazole are relatively selective inhibitors of 2D6 and 3A4, respectively, at concentrations below 1 micromolar, but both will also inhibit other CYP450 enzymes at higher concentrations, an inhibition that is not known to be clinically ertinent. Antibodies to specific CYP450 enzymes also can be used to attempt elective inhibition of metabolic pathways, but at present this approach is limited by lack of wide commercial availability of the antibodies, incomplete haracterization of their selectivity, and high laboratory-to-laboratory variation for antibody inhibition results in comparison to chemical inhibitors. The cDNAs for the common CYP450s have been cloned, and the recombinant human enzymatic proteins have been expressed in a variety of cells. After the apparent metabolic pathway has been determined using microsomes, use of these recombinant enzymes provides an excellent way to confirm results identified in microsomes.

The most complete picture for hepatic metabolism can be obtained with intact liver systems, in which the cofactors are self-sufficient and the natural orientation for linked enzymes is preserved. Isolated hepatocytes and precisioncut slices have these desirable features. Radiolabeled drugs are very helpful at this stage. A major logistic problem with these preparations, however, is that enzymatic activities are not stable for much longer than 24 hours. Overcoming that limitation will be valuable for investigating induction of enzyme activity.

Studies in vitro can identify critical metabolic pathways for a new drug and metabolites that are formed by these pathways. The clinical significance of this information should generally be confirmed via studies in the clinic. Absence of a finding that certain metabolic pathways are important via in vitro studies may obviate the need for further clinical investigations or at least help focus the design of these studies.

2. Assessing effects on other drugs
Human microsomes are also the most useful tool for screening for the effects of a new drug on common CYP450 pathways and for providing rapid initial information on potential drug-drug interactions. A general assessment of effects on major metabolic pathways can be obtained by simultaneous incubation of the investigational new drug with standard probe compounds, which are available for many CYP450 pathways. The experiments are exceptionally rapid and straightforward, requiring no special equipment. In general, if appropriate concentrations of the test drug are used with established probes, a negative result in vitro (no interaction identified) is reassuring and can generally eliminate the need for further clinical evaluation. Positive results suggest the need for further clinical evaluation.

B. Other Hepatic Enzymes
Although the CYP450 superfamily is the dominant group of metabolizing enzymes, other classes of important enzymes for drug metabolism are present in humans, including enzymes responsible for acetylation, methylation, glucuronidation, sulfation,and de-esterification (esterases). Approaches in vitro are not as widely applied for these enzymes as to the CYP450s, but considerable progress has been made, and further important efforts are underway.

In addition to the CYP450 enzymes, microsomes contain other enzymes, including a variety of transferases. For conjugating reaction pathways, supplementation of microsomal preparations with conjugating moieties as added cofactors has been successful. Cytosolic (soluble) enzymes are not contained in the microsomal fraction,but may be readily investigated using other subcellular fractions (e.g., S9).

C. Gastrointestinal Drug Metabolism
Much emphasis in metabolic research and development has focused on the liver, because this organ has always been regarded as the principal site of drug metabolism.
For particular drugs, however, other tissues may predominate (e.g., the kidney or gastrointestinal mucosa). Because most drugs are given orally, interest has been increasing in the effect of gastrointestinal mucosal enzymes on drug entry to the systemic circulation. Drugs susceptible to metabolism via CYP450 3A4 may exhibit low and/or variable bioavailability. Thus, determining the susceptibility of a drug to metabolism by CYP450 3A4 may be important not only in identifying routes of elimination but also in predicting the likelihood of significant first-pass metabolism first-pass metabolism
.

D. Interspecies Metabolic Comparisons and Other Uses of Animal Data
Animal toxicology studies are an important component of assessing safety for subsequent human exposure. Although comparative metabolism has long been of interest, this emphasis has grown in recent years, and many drug development programs now produce extensive characterization of metabolites in animals. This work has not regularly been linked to parallel findings in humans, but the availability of tools for the study of human metabolism in vitro provides an opportunity to refocus and enhance the goals of pharmacokinetic and metabolic studies in animals.
Animal studies provide the means to determine whether new chemical species generated by human metabolic studies in vitro are active pharmacologically (toxicologically) and how they compare to the parent compound, often a critical determinant of the effect of drug-drug interactionsor genetic diversity. Early identification of human metabolic routes of elimination and metabolites by studies in vitro can provide clear direction for
preclinical studies in animals.

An especially valid application of in vitro and appropriate clinical follow-up studies is to compare drug and metabolite exposure in humans and animals. Reasonably similar exposure supports the relevance of a particular animal species to the assessment of a potential human risk, and knowledge of differences (e.g., a toxic metabolite in animals, but not in humans) could aid in interpretation of clinical data. The earlier this is done, the easier it will be to use the information in planning and interpreting clinical studies.
Although the use of in vitro techniques to determine the most metabolically relevant species for nonclinical testing may enhance the value of these studies, selecting appropriate species or strains is not a simple matter. The need for historical control data and prior experience in toxicology studies for a particular species and strain could limit the ability to select species and strains based on similarities of metabolic pathways to humans. Nonetheless, major metabolic dissimilarities between the test species and humans reduce the confidence in these studies as predictors of safety in humans.

Also see :

DRUG METABOLISM/DRUG INTERACTION STUDIES IN THE DRUG DEVELOPMENT PROCESS: STUDIES IN VITRO

CORRELATION BETWEEN STUDIES IN VITRO AND IN VIVO


TIMING OF METABOLISM STUDIES, LABELING, RELATED APPLICATIONS AND CONSIDERATIONS

TECHNIQUES AND APPROACHES FOR STUDIES IN VITRO OF DRUG METABOLISM AND DRUG INTERACTIONS

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