Solubility Assays

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... 

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. 

Measuring compound remaining in solution has the advantage that the endpoint is quantitative and similar to that in a thermodynamic solubility assay. Within reasonable experimental parameters so that Beer s law is followed a UV absorbance is linearly related to concentration in solution. Measuring compound concentration in solution is very well established technology with a wealth of available instrumentation. 

Visual inspechon frequently cannot differenhate between an amorphous or crystalline material, e.g. at Pfizer medicinal chemists were required to submit only crystalline and not amorphous compounds to an automated thermodynamic solubility assay. In prachce half the white powders that they produced for the assay and that they thought were crystalline were actually amorphous. Prior to 2000 the vast majority of these medicinal chemistry labs had no melting point equipment and it was only in 2000 that the pharmaceuhcal sciences department started a workshop to teach medicinal chemists the importance of solid state properhes, how to crystallize compounds and the importance of salt forms. 

Compound solubility is important because it affects the bioavailability of compounds in vivo, the behavior of compounds in in vitro assays, and the ease with which preclinical in vivo studies can be run. Solubility assays that measure either... 

At the hit triage stage, it is most common to be able to characterize sets of compounds in a kinetic solubility assay. In the assessment and utilization of these data, the potential disconnects between kinetic and thermodynamic solubility must be considered. Low kinetic solubility for a series of compounds should lead a project team to be concerned about the behavior of compounds in biological assays and buffers, as well as the potential for optimizing drug-like properties in that series. Conversely, while high kinetic solubility is a desirable property, chemists should still remain cognizant of the need to assess thermodynamic solubility as compounds are further optimized. 

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. 

The results of a pH 4-9.5 solubility assay of chlorpromazine are shown in Fig. 6.10. The horizontal line represents the upper limit of measurable solubility (e.g., 125 pg/mL), which can be set by the instrument according to the requirements of the assay. When the measured concentration reaches the line, the sample is completely dissolved, and solubility cannot be determined. This is automatically determined by the instrument, based on the calculated value of R. When measured points fall below the line, the concentration corresponds to the apparent solubility Sapp. 

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-... 

Solubility and permeability were measured by a high throughput solubility assay and parallel artificial membrane permeation assay (PAMPA), respectively [56], The assays categorized 14 out of 18 drugs based on the BCS consistent with their known solubility and permeability characteristics [56],... 

Obata K, Sugano K, Machida M, Aso Y (2004) Biopharmaceutics classification by high throughput solubility assay and PAMPA. Drug Dev Ind Pharm 30 181-185. 

FIGURE I Schematic diagram of high-throughput LC/UV/MS analysis for equilibrium solubility assay. ... 

Solubility screens using LC/MS detection do not require an ultra-pure sample of the test compound due to the selective detection of the mass spectrometer. Mass spectrometric detection offers high selectivity and low detection limits, which eliminates the need to develop complex chromatographic methods. The LC/MS-based solubility screen surpasses the traditional HPLC/UV-based equilibrium solubility assay with increased throughput, minimal manual intervention, and high sensitivity and selectivity. 

All compounds are tested as purified active ingredients. Most compounds are obtained from commercial sources, some are purified at Cerep from a formulated product, and some have been synthesized at Cerep. The purity (chromatographic purity) is measured by the aqueous solubility assay, which uses LC/MS with UV or evaporative light scattering detection to analyze the sample. More than 99% of BioPrint compoimds are >95% pure. The remaining compounds are >80% pure and are primarily compounds purified from natural extracts. 

A solubility assay with a detection range from 10 to 2 x 10 is performed as a part of the BioPrint profile and shows that about one third of the BioPrint compounds are insoluble at 10 " M. Thus, aqueous solubility limits the high-end concentration for IC50 determination to 10 " M. The final DMSO concentration tolerated in the assay reaction medium must not exceed 1%. This also limits the high-end concentration to 10 M. 

We distinguish between two families of solubility assays based on their use for different purposes ... 

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. 

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