Early discovery solubility assay

As previously discussed, compound form differs markedly between early discovery and the late discovery/development interface. The early discovery compound is poorly characterized as to its crystalline form - it may be nonsolid, amorphous, or possibly crystalline but uncharacterized as to polymorphic form. The late discovery/development interface compound is crystalline as defined by phase-contract microscopy or powder X-ray diffraction, and its polymorphic and salt form is frequently characterized. This difference has profound implications for the design of a discovery solubility assay. The key question is this Is it better to design an early discovery solubility assay as a separate type of experiment, or is it better to try to automate a traditional thermodynamic solubility assay to handle the very large number of compounds likely to be encountered in early discovery Another way to state this question is this Does it make sense to run a thermodynamic solubility assay on poorly crystalline early discovery compounds This is the type of question about which reasonable people could disagree. However, this author does have a distinct opinion. It is much better to set up a distinctively different solubility assay in early discovery and to maintain a clear distinction between the assay type appropriate in early discovery and the assay type appropriate at the late discovery/ development interface. Two issues are relevant to this opinion One relates to the need for a solubility assay to reflect/predict early discovery stage oral absorption and the other relates to people/chemistry issues. 

Adding compounds solubilized in DMSO to aqueous medium as part of a discovery solubility assay can lead to two types of solubility assay with different uses. At one extreme, the quantity of DMSO is kept very low (<1%). At this low level of DMSO, the solubility is only slightly affected by the DMSO content. For example, data from a poster by Ricerca Ltd. [11] suggest that a DMSO content of 1% should not elevate apparent solubility by more than about 65%. At 5% DMSO, this group reported an average solubility increase of 145% due to the DMSO content. Solubility in an early discovery assay containing one percent DMSO can however exceed thermodynamic solubility by much more than 65%. However, this is very likely due to the time scale. Studies by the Avdeef (plon Inc.) group show a close approximation of early discovery solubility (quantitated by UV) to literature ther-... 

As previously discussed, the compound s form differs markedly from early discovery to the late discovery and development interface. The early discovery compound is poorly characterized as to crystalline form. It may be non-solid, amorphous, or even crystalline but uncharacterized as to polymorphic form. The late discovery/development interface compound is crystalline as defined by phase contract microscopy or powder X-ray diffraction and its polymorphic and salt form is frequently characterized. This difference has profound implications for the design of a discovery solubility assay. 

Solubility in an early discovery assay containing 1% DMSO can however exceed thermodynamic solubility by a lot more than 65%. However, this is very likely due to the time scale. Studies by the Avdeef (plon Inc) group show a close approximation of early discovery solubility (quantitated by UV) to literature thermodynamic solubility if the early discovery assay is allowed to approach equilibrium, for example by sitting overnight. The... 

The generalization as to binned solubility ranges is based on experimental aqueous solubility assays that are primarily intended for early discovery use. Most often these assays employ a drug in DMSO stock solution rather than a powder... 

Many factors may confound the assessment of the D DI potential of early discovery compounds [93], Limited or no solubility data exist to understand the likelihood that the compound will precipitate out of an in vitro incubation. The compounds have generally not been analyzed from a spectroscopic perspective their characteristics may interfere with a fluorogenic DDI assay. Metabolism data are typically not available. The binding of a compound to plasma proteins or microsomal incubation constituents is not well understood, which may lead to underprediction of its inhibitory potential. The compounds are typically delivered in DMSO, which may cause solvent-related inhibition of the enzymatic assay. Also, since little is known about in vivo concentrations or projected dose, framing the consequences of an early DDI in vitro experiment may be difficult. With these factors in mind, general experimental paradigms have been developed to help minimize their potential impact. 

Zhou, L., Yang, L., Tilton, S. and Wang, J. (2007) Development of a high throughput equilibrium solubility assay using miniaturized shake-flask method in early drug discovery. Journal of Pharmaceutical Sciences, 96, 3052-3071. 

The equilibrium shake flask method is the gold standard for thermodynamic solubility determination (Glomme et al., 2005). The assay is performed by adding a solvent to solid material, and mixing for 24-48 hrs. The solution is filtered and the supernatant is diluted and assayed using LC-UV-MS. This method is low throughput compared to the needs of HTS and early discovery. 

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. 

Bioavailability can be assessed early in the discovery process by combining data from a number of independent in vitro assays run as part of a pharmaceutical properties profile. These assays generally include some measurement of aqueous solubility, lipophilicity, cell or membrane permeability, and metabolic stability. In most cases, proper interpretation of... 

Solubility is usually expressed as log S, where S is the saturated compound concentration in mol/1 in equilibrium with its most stable crystalline form under certain defined conditions (e.g., physiological pH at room temperature over an extended period oftime, typically 24—48 h). This is also known as the thermodynamic solubility. The typical log S values for discovery compounds vary from —3(1 mM) down to —7 (0.1 pM). In contrast, kinetic solubility refers to the solubility value determined within a defined period of time, which is usually much shorter than 24 h. Since equilibrium conditions are not often achieved in this time frame, the compound is typically not in its most stable crystalline form. Therefore, the kinetic solubility value is normally higher than that obtained from the thermodynamic approach. Despite these caveats, kinetic solubility measurement can be set up in a high-throughput assay format and has been used by some pharmaceutical companies to identify poorly soluble compounds in the very early stage of drug discovery. 

As with turbidimetric assays, many of the direct UV absorbance assays are set up to determine kinetic solubility. However, the UV absorbance method also lends itself well to thermodynamic solubility determination by extending the period of sample agitation prior to filtration to 24 h or more. This offers a number of advantages. The solubility data generated are less dependent on the physical form of the initial material precipitated from DMSO and are much closer to thermodynamic solubility values determined later in discovery and in early development. As such, it gives more consistent solubility data through the discovery phase and enables a better quality early assessment to be made of the likely difficulties or otherwise of progressing a lead series into development. 

Solubility issues are universal for activity and property screening, both HTS and traditional bench-top assays. It significantly impairs the quality of biological assays, ADME/TOX screens and in vivo activity measurements. This chapter focuses on solubility issues and potential solutions for screening assays in early drug discovery. The effects of solubility on absorption and oral bio availability will be addressed elsewhere in this book. 

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