Metabolite ID




CYP450 inhibition

Fluorimetric probes


a ACD is Advanced Chemistry Development Inc.

a ACD is Advanced Chemistry Development Inc.

standardization of protocols and biological reagents is most common within an organization to ensure comparability of data. Variations in the biological reagents and protocols between organizations make the data less comparable between organizations. Many analytical instrument vendors have developed common methods on their instruments and provide them as "solutions."

It is anticipated that integrated workstations will be developed that incorporate all of the steps of a method in one system, thus reducing the burden on the laboratory scientist to integrate the system and placing the emphasis on generating results, rather than developing methods.

Pharmaceutical research organizations select assays that meet their own expertise and goals in assessing the performance of their compounds against pharmaceutical hurdles. While it is not easy, from the literature, to assemble a list of what each organization is actively using, Table 15.3 and Figure 15.3 provide an example from the Wyeth Research organization.

Compound quality is addressed with a rapid LC/UV/MS assay [12]. Many groups use compounds that were synthesized several years before or obtained from an outside source. Structure assignment inaccuracies, impurities, and degradants can cause SAR and SPR confusion for the drug-discovery team.

Traditional medicinal chemistry quantitative structure-activity relationship (QSAR) studies often involve octanol-water distribution coefficients (Log D). These can be miniaturized and automated for profiling. The Log D experiment

FIGURE 15.3 Example of a multivariate pharmaceutical profiling program.

may seem simple, but many variables (e.g., pH, ionic strength, counterions, co-solvents) have a major effect on the results. Log D is often measured by chromatographic techniques for higher throughput [13-16], or it may be calculated for efficiency.

Three common properties that affect intestinal absorption of drugs after oral administration are solubility, permeability, and pKa. Traditional solubility experiments measure solubility of solids placed into aqueous phases (thermodynamic solubility), but these methods are too slow or they consume too much material for drug discovery. Higher throughput methods must be used. The direct ultraviolet (UV) method [17] adds compound dissolved in dimethyl sulfoxide (DMSO) to an aqueous buffer and measures the UV absorption of the aqueous phase using a 96-well plate reader after equilibration and filtration (kinetic solubility). Lipinski has discussed the pitfalls that inadequate solubility information can have for a drug-discovery organization [18].

Many organizations use colon adenocarcinoma (Caco-2) for detailed study of permeability; however, this method can be resource intensive. Parallel artificial-membrane permeability (PAMPA) [19] has proven to be a reliable predictor of passive transcellular permeability for intestinal absorption prediction. It is also useful to interpret results of cell-based discovery assays, in which cell-membrane permeability is limiting. Finally, pKa provides insight into the pH dependence of solubility and permeability. It can be measured [20] or calculated to get an understanding of the regions of the intestine in which the compound will be best absorbed, as well as to anticipate the effect of pH on solubility and permeability. Permeability at the blood-brain barrier (BBB) also can be rapidly profiled [21].

FIGURE 15.3 Example of a multivariate pharmaceutical profiling program.

Metabolism is also important to assess, and most organizations have implemented microsomal, hepatocyte, or other in vitro assays to assess relative physiological stability. Other destabilizing factors are plasma, acid (stomach), base (large intestine), light (laboratory and factory), and heat (storage) [22].

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