Discovery solubility assay

Discovery solubility assays can be subdivided depending on how the solubility point is determined. One type of assay measures compound coming out of solu-hon. The other type of assay measures compound in solution. Both methods have advantages and limitahons. 

These three areas will be discussed as they relate to aqueous solubility. This will be followed by a general discussion of experimental approaches to measuring aqueous solubility in a discovery setting. Detailed descriptions of varied discovery solubility assays can be found in the excellent review by Kerns [1],... 

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-... 

The quantitation of solubility in the current discovery setting differs markedly from that of a traditional aqueous solubility assay. In the traditional solubility assay the crystalline well-characterized solid is equilibrated in aqueous solution for sufficient time to reach equilibrium (generally 24-48 hours). The solution composition is important in the comparison with a discovery solubility assay. The traditional thermodynamic solubility experiment is performed in the absence of any organic co-solvent. By comparison the discovery solubility assay is frequently performed in the presence of a co-solvent, usually DMSO. The physical state of the starting solid in the development thermodynamic assay is relevant to the comparison with discovery solubility assays. As was previously mentioned, the solids in discovery today are frequently noncrystalline and therefore invariably more aqueous soluble. 

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. 

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... 

When compounds are selected for preliminary PK studies, the identification of an appropriate dosing vehicle for iv studies requires solubility studies in various vehicles. Also, the study of thermodynamic solubility is useful as this more closely reflects the environment experienced by compounds on oral dosing. Higher throughput thermodynamic solubility assays have been introduced recently [23] so it will be possible to introduce this type of assay earlier in the discovery process. 

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. 

PURPOSE AND RATIONALE Solubility assays are gaining growing attention in drug discovery, because many pharmaceutically active compounds can be adjusted to in vivo testing merely with co-solvents. Furthermore, in vitro assays may also lead to false results, simply for precipitation of a compound in the assay media. Solubility assays vary in one main point they are either performed from solids or stock solutions. A nomenclature has been established in the literature which tries to distinguish between these methods. Determinations from stock solutions are often called kinetic solubility whereas thermodynamic solubility stands for solubility of solids (Kerns). Thermodynamic solubility takes the crystal lattice forces into account. Batch to batch variations, polymorphism... 

PURPOSE AND RATIONALE In vitro testing of drug candidates and combinatorial chemistry lead to an increase of lipophilic compounds with poor solubility (Lipinsky). Poor solubility itself may lead to poor oral availability of a potential drug. The growing demand for solubility data in lead phase of drug discovery is answered by a variety of simple solubility assays, which allow the classification of a compound without real quantification (see previous section). One of the easiest way to detect saturation in a solvent is the turbidity of the solution if precipitation occurs. The turbidity caused by precipitation of a poorly soluble compound can be detected by a couple of detection methods (Van de Hulst, Hongve). Lipinsky describes the first methodology, which use UV as detection method and is able to screen hundreds of compounds a day with one instrument. 

In the lead identification and lead optimization phases of discovery, there is greater focus on thermodynamic solubility measurements. Thermodynamic solubility assays are designed to determine the solubility of the stable crystalline form of the compound, since this is the physical form that will be sought in the development phase for orally administered drugs. As such, thermodynamic solubilities provide discovery projects with a better risk assessment of likely formulation issues in development. Thermodynamic solubilities, unlike kinetic solubilities, are less dependent on the initial physical form of the compound and being less time critical also tend to be more reproducible. This is particularly important from a molecular design perspective where chemists are seeking to modify molecular structure to improve solubility. 

More recently in discovery there has been a trend toward developing high-throughput thermodynamic solubility assays, which incorporate a solid-state assessment at the end of the period of agitation. This assessment aids interpretation of the solubility data and is an important consideration when relating the solubility data to molecular structure. Solid-state characterization methods include the use of PLM [16], microscopic analysis [34], PXRD [32, 33], and Raman microscopy [22]. With all these... 

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. 

Kerns EH, Di L, Carter GT (2008) In vitro solubility assays in drug discovery. Curr Drug Metab 9 879-885... 

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... 

Palladium and platinum are the longest known and most studied of the six platinum metals [1-11], a reflection of their abundance and consequent availability. Platinum occurs naturally as the element, generally with small amounts of the other platinum metals. It was used as a silver substitute by Colombian Indians and first observed there by Ulloa (1736), who called it platina del Pinto ( little silver of the Pinto river ) but the first sample was actually brought to Europe in 1741 by Charles Wood, Assay Master of Jamaica. Palladium was isolated in 1803 by W.H. Wollaston, who was studying the aqua regia-soluble portion of platinum ores (he announced his discovery by an anonymous leaflet advertising its sale through a shop in Soho) and named it after the newly discovered asteroid Pallas [12],... 

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