In Vitro Applications

As shown in Fig. 13.1, part of the DMPK lead optimization process would include various in vitro assays used to screen out compounds before they would be considered for the (in vivo) PK studies [14, 40-42]. Kassel has provided an overview of strategies that can be used for increasing the throughput of these in vitro assays [16]. Typical in vitro screens would include microsomal stability, Caco-2 assay and p450 enzyme inhibition studies [14, 15, 18, 20, 22, 43-48]. Microsomal stability is used to give an indication of a compound's metabolic stability (this can also be done by using hepatocytes) [18, 22, 44, 49]. An early example of a higher throughput microsomal stability assay was described by Korfmacher et al. [50] in 1999; in this assay, the HPLC-MS analysis step was automated and the system was able to handle 75 compounds per week. A more recent example is described by Di et al. [51] who used robotic separation and a rapid HPLC-MS/MS system. A very high throughput highly automated microsomal stability assay has been reported by Xu et al. [52]; this assay had the capacity to handle up to 176 compounds per day. This system used a high throughput eight-channel parallel HPLC system along with intelligent software to process the data.

The Caco-2 cell monolayer permeability assay is one of the standard in vitro assays that are used to predict human absorption of NCEs [16, 22, 40, 43, 5355]. The Caco-2 cell assay measures the permeability potential for a compound and can be used to help sort through large numbers of compounds. While various robotic procedures have been developed to perform the Caco-2 assay, it is now common to use HPLC-MS/MS to assay the samples that are produced in the procedure [53]. There have been several reports in the literature on various ways to use HPLC-MS/MS for Caco-2 assays [56-60]. For example, Fung et al. [57] described a procedure for higher throughput Caco-2 sample analysis that made use of the multiplexed electrospray interface (MUX) that can handle four parallel HPLC systems that are then monitored by a single MS/MS system. By using generic fast gradient HPLC conditions and special software for data processing, the one LC-MS/MS system could be used to assay the samples from 100 NCEs per week. In another example, Hakala et al. [58] discussed the possibility of performing Caco-2 assays on a mixture of compounds instead of the normal single compound studies. In addition, Hakala et al. [58] compared the utility of using electrospray ionization (ESI) to atmospheric pressure photoionization (APPI) for this application; the authors concluded that APPI provided a bigger linear quantitative range than was obtained from ESI (3-4 orders of magnitude vs 2-3 orders of magnitude, respectively).

Another higher throughput in vitro assay is the human cytochrome P450 enzyme inhibition assay. This assay is used to make sure that a compound does not have the potential for producing drug-drug interactions in a clinical setting due to the inhibition of one or more human P450 isozymes [61-66]. Often, these P450 assays are carried out in a higher throughput manner using 96-well plates for sample preparation and combining multiple isozyme measurements into one-sample analysis. For example, Chu et al. [67] described an analysis based on HPLC-MS/MS that measured CYP2D6 and 3A4 using human liver microsomes. Bu et al. [68] reported that they were able to evaluate the inhibition potential for five P450 isozymes (CYP2D6, 3A4, 2A6, 2C9, 2E1) using a single assay based on human liver microsomes and five probe substrates. In another example, Testino and Patonay [69] validated an analysis for the high throughput inhibition screening of the five major P450 isozymes (CYP1A2, 2C9, 2C19, 2D6, 3A4) using human liver microsomes and HPLC-MS/MS for the assay [64]. Peng et al. [70] reported on the use of monolithic HPLC columns as part of an HPLC-MS/MS system that was used for high throughput screening of the same five major human cytochrome p450 isozymes; their HPLC-MS/MS assay was able to be completed in just 24 s, due to the higher flowrate capabilities of the monolithic column. Recently, Kim et al. [71] described a high throughput P450 enzyme inhibition assay based on HPLC-MS/MS that measures a compound's inhibition potential for nine p450 enzymes.

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