CPSA Digest 2002

Proceedings -Wednesday, October 9, 2002

WeOD2

The Rat:
A Model for Higher Throughput Evaluation of Oral Absorption

Background
An important challenge in drug discovery bioanalytical support studies is to develop an in vivo pharmacokinetic screen for the high throughput evaluation of orally dosed compounds. Potential pharmacokinetic liabilities can thus be identified very early in the discovery process. The rat is often used as the screening model for these studies and several strategies have been devised within the pharmaceutical industry in an effort to improve throughput and reduce cost.

One strategy is n-in-1 dosing in which multiple compounds are dosed in one animal and the selectivity of the mass spectrometer is used to individually quantitate their concentrations from the mixture. This approach is also called simultaneous multiple compound dosing or cassette dosing. The data generated by this approach yield meaningful pharmacokinetic data in a much shorter time frame, and fewer animals and fewer numbers of samples are used than in traditional methods. However, disadvantages of this technique include potential drug-drug interactions on pharmaco-kinetics and metabolic conversion of one compound to another compound already included in the series.

Another strategy is to use pooled plasma from multiple animals dosed with single unique compounds. Using this approach, 10 animals may be dosed with one compound each and then all of the 1 hr time point samples are pooled and subjected to sample preparation, and so on for each time point. Sample pooling in this manner eliminates the concern from cassette dosing of potential drug-drug interactions on pharmacokinetics and metabolic conversion of one compound to another compound already included in the series. However, disadvantages of this technique include reduced sensitivity and increased complexity.

An alternative strategy has been developed at Schering-Plough called the Cassette Accelerated Rapid Rat Screen (CARRS). Simply, this approach can be described as one in which drug candidates are dosed individually (n=2 rats per compound) in batches of six compounds per set, and then samples are pooled across time points to provide a small number of test samples for analysis.

Premise
The Cassette Accelerated Rapid Rat Screen comprises "cassette planning:"

• Six compounds per set (from the same therapeutic program)
• Each set of 6 is dosed on the same day
• Each set of 6 is assayed on the same night
• The study is designed to fit within the 96-well microplate format
• The pre-set conditions are fairly rigid

The general procedure follows:

• Each new chemical entity (NCE) is dosed orally to 2 rats
• Blood is collected over a 6 hr period (0.5, 1, 2, 3, 4, 6 hr)
• Equal volumes of plasma from each time point are pooled across rats (6 samples/NCE)
• LC-MS/MS analysis uses only a mini standard curve that brackets the expected plasma concentrations
• An Area Under the Curve (AUC) from 0-6 hr can be calculated from the plasma concentrations
• A large number of NCEs can be ranked by AUC to prioritize compounds for further investigation

Sample preparation prior to analysis involves semi-automated protein precipitation in the microplate format (50 mL plasma: 150 mL acetonitrile) followed by centrifugation and isolation of the resulting supernatant. A particular arrangement of prepared samples along with standards and blanks within the microplate format is shown below.

An example of data obtained using this technique is shown below. The estimated AUC from 0-6 hr is obtained for each NCE and expressed in both ng/mL and nM concentrations. Data from multiple NCE are then compared and ranked for prioritization.

Value of the Technology
Dosing in cassettes of six compounds per therapeutic project and sample pooling across the two rats dosed with one compound result in a total of 36 study samples to be analyzed per cassette. A useful time-concentration curve is obtained. The method development required for LC-MS/MS analysis is streamlined by using a fast LC gradient for the six compounds; since the six are from the same therapeutic series, this approach has been shown to be appropriate. Since compounds are dosed individually into the rats and samples are not pooled across the six compounds, it is possible to perform metabolite screening, in which additional SRM transitions are included to detect potential metabolites. Overall, 36 NCEs can be dosed per week and throughput is increased over
traditional methods such as the "rapid rat oral PK screen."

References and/or Links
W.A. Korfmacher, K.A. Cox, K.J. Ng, J. Veals, Y. Hsieh, S. Wainhaus, L. Broske, D. Prelusky, A. Nomeir and R.E White, "Cassette-Accelerated Rapid Rat Screen: A Systematic Procedure for the Dosing and Liquid Chromatography/Atmospheric Pressure Ionization Tandem Mass Spectrometric Analysis of New Chemical Entities as Part
of New Drug Discovery", Rapid Commun. Mass Spectrom. 15(5) (2001)
335-340.

K.A. Cox, K. Dunn-Meynell, W.A. Korfmacher, L. Broske, A.A. Nomeir, C-C Lin, M.N. Cayen and W.H. Barr, "Novel in vivo procedure for rapid pharmacokinetic screening of discovery compounds in rats", Drug Discov. Today 4(5) (1999) 232-237.

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