Exploration Stage

During the exploration stage, high throughput screening (HTS) reveals compounds ("hits") that are active in modifying the action of the target protein. Pharmaceutical profiling contributes by confirmation of HTS hits using an integrity and purity assay. Often, the majority of compounds in a HTS screening library were synthesized many years earlier, came from an extramural source, or were members of a combinatorial screening library that were not completely characterized. The integrity and purity assay start the discovery team off with known structures on which to base synthetic series. If the HTS hit is impure, due to degradation or reaction by-products, the integrity and purity assay will serve as the first stage of structure elucidation, if the team wants to identify the active component in the sample.

The integrity and purity assay also serves to qualify compounds for nonspecific assays of biological activity or pharmaceutical properties. For example, if an assay uses a 96-well plate UV spectrophotometer, the presence of an impurity can significantly change the assay results, especially if the impurity's properties or activity differ from the parent compound.

Also at this stage, pharmaceutical profiling assays, such as solubility, permeability, and metabolic stability provide a multivariate property profile of HTS hits that contribute to the team's holistic selection of leads for synthetic optimization. The pharmaceutical-property profile is considered in relation to the ensemble of activity properties for making informed decisions. Conversely, an alert to poor properties can save the team effort and time on problematic structures, or forewarn of issues that must later be solved via synthetic modification, salt form, or formulation.

In many organizations, advancement from the exploratory stage requires in vivo model. Some of the profiling assays can provide evidence of in vivo exposure to the blood and penetration into the target tissue. Thus, the assays can provide evidence for meeting advancement criteria.

Early-Discovery Stage

In early discovery, pharmaceutical-profiling assays can assist with monitoring properties of series analogs as they are synthesized. This can provide feedback about how synthetic modifications that are intended to explore SAR also change the properties of the analogs. Comparisons are made to the historical baseline of the HTS hits and leads. It is very common for synthetic modifications to add molecular mass and lipophilicity to the molecule, often resulting in higher lipophilicity that leads to lower solubility and permeability. Monitoring these property changes as series compounds are synthesized keeps the team informed of movement of the compounds away from optimum properties. The effective activity of the drug is a function of both its intrinsic activity with the target protein and the concentration of the drug that is delivered to the target.

As discovery research moves forward, great value is derived from dosing experiments with living animals. In vitro profiling assays are used to predict the in vivo ADME performance, in order to select compounds for animal studies.

Simple in vivo assays of blood or tissue provide the first measurement of the ADME properties of the compound. In early discovery, studies of mixtures of compounds [23-25], which are co-administered to a single animal, can efficiently provide this exposure and tissue-penetration information. Early in vivo exposure studies help to establish dosing levels for animal models. Blood samples can also be obtained from the animal activity model following dosing to correlate in vivo pharmacology with plasma levels. Lower than expected in vivo activity can often be correlated with low plasma or tissue levels.

When inadequate in vivo exposure or PK properties are observed, pharmaceutical profiles can be used to troubleshoot the cause of poor in vivo exposure or PK [26]. Assays for solubility, permeability, and stability (metabolic, plasma, acid) can help to track down the inadequate properties responsible for poor in vivo performance. Property optimization synthesis can then be initiated. Subsequent series analogs can be assayed to rank order compounds by properties for subsequent in vivo tests, in order to give the highest likelihood of success. Often animal studies are expensive and time-consuming, especially if they are performed using the animal activity model. Simple in vitro profiling assays can provide information for improved decisions and efficiency.

Early discovery also makes extensive use of in vitro and cell-culture activity models. Activity measurements can be inconsistent when the compound has low aqueous solubility. Thus, the solubility assay, can assist with the interpretation of biological activity tests. Another cause of poor bioactivity in vitro is poor stability in the assay medium. Also, there can be discrepancies with activity when compounds are moved from biological models in 96-384-well format to living-cell models. Differences in activity can often be traced to permeability of the compound through the cellular lipid membrane and correlate with results of the permeability assay.

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