In Vivo Applications

While there are several high throughput in vitro screens as described above, there is still a significant need for in vivo assays as shown in Fig. 13.1. Several recent reports have discussed using HPLC-MS/MS for the bioanalytical step in discovery PK studies [5, 6, 8, 23, 72-74]. In the following sections, various aspects of the strategies that can be utilized in these in vivo studies are discussed. While the focus of this section will be on PK studies, another aspect of the lead optimization process is the understanding of the PK/pharmacodynamic (PD) relationships. While the topic of PK/PD is outside the scope of this chapter, the interested reader is directed to articles by Rohatigi or Chaikin et al. [75-77] for more information on this important topic. Finally, a discussion of metabolite identification methods and new technologies will also be included.

One of the issues that is still debated is whether or not to use cassette dosing as a way to increase the throughput for discovery PK studies. As shown in Fig. 13.4, cassette dosing (also called N-in-one dosing) is the practice of dosing multiple NCEs into one laboratory animal and then collecting blood samples from the animal and using HPLC-MS/MS for analysis of the samples [78]. It is the ability of HPLC-MS/MS to assay multiple compounds in one sample that has allowed this technique to be utilized. As discussed recently by Manitpisitkul and White [78], although cassette dosing is still used by about half of the major pharmaceutical companies, it does have problems that should be considered before implementing it as part of a drug discovery strategy. For example, drug-drug interactions between the multiple compounds dosed into one animal can lead to erroneous PK conclusions for one or more of the dosed compounds. Indeed, the possibility of drug-drug interactions from cassette dosing was proposed previously by White

Cassette Dosing
Fig. 13.4 Conventional dosing vs cassette dosing. In conventional dosing, only one compound is dosed in each rat; in cassette dosing, multiple compounds are dosed in each rat. Adapted from [78], with permission from Elsevier.

and Manitpisitkul [79], who predicted that cassette dosing would lead to both false positive as well as false negative values for certain PK parameters. This prediction was confirmed recently by Smith et al. [80], who reported on problems with using cassette dosing to assess PK parameters for a group of compounds in mice. There are also practical issues with the cassette dosing approach. For example, it is important to make sure that the dosed compounds are not isomers that would likely be difficult to assay in a mixture. Some researchers also try to make sure that compounds are not within 14-16 Da from another dosed compound, so that +16 Da or —14 Da metabolites cannot interfere with the assay of a co-dosed compound [81]. There can also be formulation issues - often single compounds produce poor oral suspensions such that a mixture of these poorly soluble compounds might result in a formulation that is not suitable for dosing.

In spite of these concerns, there are still groups that find cassette dosing to be a useful strategy. For example, Ohkawa et al. [82] described using cassette dosing for 200 compounds in 50 cassettes. Zhang et al. [83] have reported the use of cassette dosing for measuring brain and plasma levels as part of new drug discovery. In their study, three or four compounds were grouped in a cassette and were dosed as a mixture to rats. Tamvakopoulos et al. [81] reported on the use of cassette dosing in rats in order to measure brain/plasma (B/P) ratios as part of a screening process; in their study, they found that the B/P ratios were largely un affected when using the cassette dosing method. More recently, Zhang et al. [83] discussed the use of cassette dosing in rats in order to get B/P ratios for a series of discovery compounds; in this work, they also described the use of a fast chromatographic separation as part of the HPLC-MS/MS assay. Another recent example of cassette dosing was provided by Sadagopan et al. [84]; in their work, rats were dosed with a mixture of compounds either by intravenous (IV) or oral (PO) routes.

An alternative to cassette dosing, Korfmacher et al. [85] described the use of the cassette accelerated rapid rat screen (CARRS). CARRS uses cassettes of six compounds as the basis for a systematic higher throughput rat oral PK screen. The six compounds in a given cassette are selected by the medicinal chemists from one drug discovery team. This is not cassette dosing; instead, each compound is dosed into two rats that are sampled at six timepoints (0.5, 1, 2, 3, 4, 6 h) by the serial-bleeding procedure. The plasma samples from the two rats are pooled at each timepoint, so that each dosed compound results in six plasma samples to be assayed by HPLC-MS/MS. As described by Korfmacher et al. [85], all the samples and standards that are needed for the assay for the six dosed compounds in one cassette can be placed in a single 96-well plate. With current fast HPLC-MS/ MS methods, the methods can be developed and the 96-well plate can be assayed in no more than a few days. The CARRS system was set up to accept multiple (originally five and recently up to eight) cassettes of six compounds on a weekly basis [78]. Therefore, this system allows multiple (currently up to eight) drug discovery teams to be able to select six NCE's for testing in this rapid PK screen each week [78]. Each week, all of the selected compounds are dosed by the drug discovery dosing group and the resulting plasma samples are delivered to the discovery bioanalytical group for analysis during the following week. Within two weeks of the compound selection request, electronic PK summary reports are issued to the discovery teams that requested the compounds to be dosed. The PK report shows the individual plasma concentrations for the six timepoints as well as the calculated area under the curve (AUC) for each dosed compound in an Excel format. Because it is a discrete dosing procedure, it avoids the potential problems of cassette dosing (vide supra) while still providing a systematic process for testing multiple NCEs in an in vivo PK model. This higher throughput assay has continued to be very useful as a rat oral PK screen; in the first four years that it was in place, CARRS was used to screen over 7000 compounds [78].

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