CPSA Digest 2001

Proceedings -Thursday, October 11, 2001

ThOA3

Predictive Models for Tissue Metabolism - Screening Using Organ Perfusion Methods

Background
Various in vitro systems have been relied upon quite heavily in drug discovery optimization to guide the selection of optimal drug candidates. However, many questions are asked of these screens‹Which in vitro system should be used (e.g., cells, liver slices, microsomes), What if the liver is not a primary site of metabolism (e.g., gut wall actually detoxifies phenol), Which cells should be used as representative?

While in vivo experiments may provide good evidence that a certain substance has an effect on an organ, only the testing in isolated organs allows for an assessment of its immediate impact excluding extraorgan influences. The concentration of the substance applied to isolated organs can be well controlled. Samples of the perfusate (venous effluate) or the organ itself may be obtained easily and frequently.

Unlike experiments in intact animals, isolated organs allow the investigator to retain control over internal or external parameters such as type and compositions of the perfusate (nutrition), or a certain treatment before and during the experiment. Perfused organs allow for continuous monitoring of many aspects of physiology and pathophysiology at the same time and under the same genetic or experimental conditions, which is often not possible in laboratory animals for ethical or technical reasons. In addition, isolated organs offer the opportunity of investigating the administration of multiple agents by different routes.

The figure below presents the position of isolated perfused organs compared with cell cultures, either human- or animal-derived.

Premise
The major organ(s) that are responsible for metabolism in vivo can determined when individual organs are perfused with whole blood at flow rates comparable to those seen in vivo. Addition of drugs to the perfusate allows for the time-dependent generation of metabolites, and measurement of drug concentrations at each time point provides essential pharmacokinetic information. It cannot be assumed that the liver is the primary organ of metabolism, and instead the lung, gut wall and kidney should also be examined for their influences on metabolism.

When the primary tissue or organ of metabolism in an animal model (e.g., rat) is identified, it is sensible to use the same tissue or organ in another species for cross-species comparisons. Note that, however, in humans there are only in vitro systems.

How should it be decided which in vitro system to use? Which system will provide real data on the relative rates of metabolism in different species? Should it be tissue slices, isolated cells, homogenates or sub cellular fractions? The presenter¹s approach to these questions is to use the rate of metabolism per gram of tissue (or mg protein) derived from the perfused organ studies in rat as the yardstick by which to validate the rate of metabolism in the in vitro systems. Oftentimes, the rate of metabolism in slices, isolated cells, homogenates or sub cellular fractions using an arbitrary incubation medium is well below what is expected. However, by judicious manipulation of the incubation medium it should be possible to improve the nature and rates of metabolism in vitro so that they may approach that of the whole perfused organ. If the improved rates are 80% or more than that in the whole organ, then cross-species comparisons using the same conditions should provide reliable data that can be used for go/no-go decisions.

LC-API-MS is the method of choice for the analysis of drugs in biological fluids and the generation of pharmacokinetic parameters. This technique is ideal, also, for use in these organ perfusion studies. In order to increase the throughput of pharmacokinetic screening, simultaneous dosing of numerous compounds followed by multiple component analysis using LC-MS/MS (the N-in-1) approach has been developed and proved to be effective.

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