This article deals with an important topic in the pharmaceutical industry, that of screening and eliminating potential drug candidates that manifest toxic properties in vivo. Absorption, distribution, metabolism, excretion, and toxicity (ADME/Tox) is generally applied to the ensemble of those tests that are used to characterize a compound's properties with respect to absorption by the intestine, distribution to the organism, metabolism by the liver, excretion by the kidney, and toxicity profiles. ADME/Tox screening in the drug discovery and development setting is taking center stage given the large fraction of lead compound and drug failures associated with toxicity properties. See Figure 1, which illustrates this point. Thirty percent of the total new drug attrition in the developmental pipeline is attributed to toxicity profiles and side effects. Hence, this is an area that the pharmaceutical companies and other drug discovery and development entities are paying close attention to.

Figure 1

Traditional deployment of ADME/Tox approaches in drug development has been in the latter stages of drug development—essentially late in the process subsequent to the initial phases of discovery of "hit compounds." Such a set-up was feasible when the number of drug discovery targets was few and the numbers of high-throughput screening assay points were relatively low across the pharmaceutical enterprise.

With the changing paradigm in the drug discovery space, the number of drug targets is expanding and so is the volume of assay points performed in high-throughput screens. Therefore, it is imperative for the industry to quickly and efficiently triage "potential hits," which have significant ADME and toxicity profiles (inconsistent with their potential as drug candidates). In this manner, ADME/Tox screening needs to be performed earlier in the process of drug discovery and development. By earlier, it is meant upstream and close to the primary (high-throughput screening) and secondary screening sectors, as illustrated in the Figure 2.

Figure 2

Note in Figure 2 how ADME and toxicity screening overlaps into the secondary screening space, this overlap of the sectors is meant to illustrate the utilization of ADME and toxicity screening assays earlier in the drug discovery ensemble.

In terms of the quantitative market opportunity in the ADME and toxicity screening space, almost $ 3 billion was spent on various ADME and toxicity studies in 2001. Note that this figure includes the hugely-expensive animal studies that take place late in the process of drug development, which are not affected by the upstream migration of ADME and toxicity screening, and overlapping into the screening (primary and secondary) space, as a result of growth in drug targets and hence their interrogation. Figure 3 presents a breakout of how this total (quantitative) market opportunity segregates into its individual components.

Figure 3

Note that the in vitro toxicology portion of the pie (valued at $200 million) is the current estimated market size for screening out the toxic hits out of the total pool of hits generated in the primary-secondary screening programs. Therefore, it appears that the in vitro screening marketplace (for screening out toxic compounds) is a significant market opportunity, and significant growth is predicted in this market segment.

Here we discuss the various approaches and technologies that have gained traction and are currently deployed for ADME and toxicity screening. Perhaps the best-known indicators (or proxies) for toxicity screening (of compounds) in cells are the ensemble of cytochrome P450 enzymes whose role it is in vivo to "detoxify" drugs. Therefore, systems that enable the induction of the various biologically relevant cytochrome P450 enzymes by various drugs offer value to the pharmaceutical industry as they enable the latter to set up assays to "screen out" these toxic molecular entities. Table 1 presents the key biological assay systems for the evaluation of the various cytochrome P450 enzyme isoforms.

Table 1 provides a snapshot of the various biological assay systems for ADME and toxicity screening. Given the central and essential role of the liver in processing out toxic compounds from the system, the pharmaceutical industry is interested in having proxies of the liver for the purpose of screening compounds against in their quest to "screen out" hit compounds with problematic ADME and toxicity profiles. For this reason, hepatocytes have attracted a significant share of the marketplace for ADME and toxicity screening, and Table 2 presents different hepatocyte assay systems and their respective value drivers (in this manner, we explore the landscape of approaches and technologies that the industry is deploying to address the fundamental ADME/Tox properties of compounds).

In the above table, we have highlighted (shaded) those model systems and approaches that are predictive of human toxicity. This is perhaps the most crucial element of an ADME and toxicity screen—how strongly predictive is the assay approach? Generally, human hepatocytes are highly predictive in this setting, as they are the "closest proxy" to the in vivo situation. However, their availability and reproducibility of the conditions of the experiment are critical bottlenecks in the process, and hence, the industry continues to search for technologies that are predictive, robust (reproducible), and cost-effective.

The above discussion focused exclusively on cytochrome P450 enzymes and hepatocytes as proxies and model systems, respectively, for toxicity screening in the pharmaceutical industry. In addition to the above, there are a number of other markers for ADME and toxicity profiling that are deployed by the industry, in the drug development engine, and these are discussed below.

In addition to assays using hepatocytes as model systems for the induction of the various cytochrome P450 enzyme isoforms, there are other types of assays that are performed in the ADME and toxicity space, e.g., use of the CaCo-2 cell line (which is an intestinal epithelial cell line) for the measurement of absorption (transport) of compounds across cells. Inhibition of P-glycoprotein (Pgp-1) is another proxy in the ADME/Tox paradigm for profiling drug-like compounds.

Figure 4 presents a list of the various common products on the market for ADME/Tox screening in the drug discovery and development space.

Note from this chart how the ADME and toxicity segments of this market are segmented into subsectors, as follows:

ADME Marketplace

• Cytochrome P450 inhibition

• Cytochrome P450 induction

• Pgp-1 inhibition assay

• Caco-2 cells for absorption assay

Also note how many of the assays that are performed have applicability in the cytochrome P450 enzyme space. This is because this enzyme system (the various isoforms being represented in, and having value for, different individuals in the population) has gained the validation of the community as bona fide metabolism markers.

An emerging area in the homogeneous potentially high-throughput screening for toxicity profiles is the use of fluorescent probes as oxygen sensors. These compounds can be added to cells in culture, and as cells respire, they consume oxygen. The probes then fluoresce upon oxygen utilization, and in this manner, oxygen utilization and respiration (and viability) of a culture (with or without drugs added) can be studied. In this manner, oxygen sensors can be used in a mix and measure format to interrogate drug candidates for various ADME and toxicity properties.

It is becoming abundantly clear that the pharmaceutical and biotechnology drug discovery landscape is moving towards cell-based assays as a means to study and screen targets in vivo in physiologically relevant formats. As a consequence, it is becoming necessary to perform ADME and toxicity triage screening, also in cell-based formats, to enable physiologically relevant ADME and toxicity profiles to be generated. As mentioned earlier in this article, primary human hepatocytes are a gold standard for performing cell-based ADME and toxicity assays (in contrast to in vitro assays performed with purified cytochrome P450 enzymes in vitro). However, primary hepatocytes are difficult and unpredictable to obtain and are expensive, as they are obtained from primary human explants. A number of cell lines are being developed that seek to recapitulate in vivo ADME and toxicity profiles of compounds. These cell lines have the advantage of being capable of indefinite propagation ex vivo and are cost-effective, plus, because of their clonal nature, provide highly reproducible data consistently.

An emerging trend in this space is the generation of engineered cell lines that contain transfected elements that can be viewed as biological reporter systems for ADME and toxicity screening in vivo. An example here is the hERG (human ether-a-go-go gene) potassium ion channel that has been implicated as a cardio-toxicity marker. Therefore, hERG channel screening for ADME and toxicity properties of potential hit compounds is exceedingly important and is gaining acceptance in the pharmaceutical industry. In this manner, many vendors are providing hERG-transfected stable cell lines for screening in drug discovery and development. Going forward, as more markers (proxies) for ADME and toxicity are discovered, it is expected that menus of cell lines will be generated that are hardwired with the appropriate signaling apparatus (and reporter systems) to enable cell-based ADME and toxicity screening to be coupled to the primary and secondary screening paradigms.

This article was featured in the July 2003 issue of Drug & Market Development. This article was written by Enal Razvi, Ph.D., Vice President of Business Development at DiscoveRx Corporation, Fremont, CA, USA. He can be contacted at erazvi@earthlink.net

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