Info

Recievet

Lo calibrator (G.6 (M in solvent)

Hi calibrator (3.G (M in solvent)

Donor fG(dilute-1GX-3 (M)

Donor t6hr(dilute-10X

Receiver

FIGURE 13.12 Sample generation for PAMPA assay.

Lo calibrator (G.6 (M in solvent)

Hi calibrator (3.G (M in solvent)

Donor fG(dilute-1GX-3 (M)

S (L of 3mM soln in DMSO

Donor t6hr(dilute-10X

Receiver

Total: 415 samples

FIGURE 13.12 Sample generation for PAMPA assay.

well suited to providing the required selectivity, sensitivity, and throughput, and would afford the opportunity to incorporate controls to ensure the quality of the data. Rather than simply measuring the MS responses for donor solution before incubation and the receiver solution after incubation for determining permeability, the determination of actual concentrations from a two-point calibration curve made by dilution into organic solvent was more reliable. The permeability measurement is calculated from the concentrations of donor and receiver solutions at the incubation endpoint (6 hours). A donor at time zero solution is also measured and compared to the high standard to assess adequate solubility in the assay buffer and to monitor mass balance. The work flow for preparing the sample for analysis is shown in Figure 13.12. The incubation is performed in duplicate to provide an indication of well-to-well variability. The entire process, including preparation of the lipid bilayer, has been automated on a CyBio liquid handler. Each 96-well plate of compounds to be analyzed results in a 96-well plate containing all the low standards for the compounds, a 96-well plate containing all the high standards for the compounds, and 96-well plates for donor at time zero, donor at time 6 hours, and receiver at time 6 hours. From an automation perspective, it would appear to be simplest to run one full plate at a time. However, this procedure would separate in time the various measurements for individual compounds that will be used to calculate the permeability readout. The software prepares the instrument run lists to acquire all related analytical measurements sequentially. A Leap HTS Pal autosampler equipped with multiple trays is used to select the samples from the various plates without impacting the injection-to-injection time. The software then automatically integrates the analytical runs and provides the data in an Excel summary (Figure 13.10) as previously described. This assay has a capacity of 1000 compounds/month, with a turnaround time of two days.

Metabolic Stability

The use of liver microsomal assays prepared from various species for the early prediction of stability of compounds to first pass phase-one metabolism is well documented [49]. A number of approaches that use LC-MS for the analytical portion of the assay have been reported [12-18]. Automated LC-MS/MS methods appear to be well suited to provide the necessary selectivity, sensitivity, and throughput and may incorporate controls to ensure the quality of the data [50]. Automated methods are created for each compound as described earlier. The percent of parent drug remaining and rate of clearance are calculated. Comparison of the parent-drug concentration in the incubation at time-zero solution to the high standard allows for the calculation of recovery. The work flow for preparing the samples for analysis is shown (Figure 13.13). Incubations with rat, mouse, and human microsomes are routinely performed; other species are run by request. The entire process of preparation of standards in organic solvent, serial dilutions, and incubations in duplicate is carried out on a Tecan liquid handler. As described previously for the PAMPA assay, each plate contains all compounds to be analyzed, with each plate consisting of a particular species and time point, duplicate, or analytical standard. The software prepares the instrument run lists to acquire all related analytical measurements, then automatically integrates the analytical runs and provides the data in an Excel summary (Figure 13.14) that reports percent recovery, percent remaining, and rate for each individual compound. Red flags indicate data that are outside the range of values set by the operator, alerting to manual review. The data are then uploaded into the corporate database, as described for the PAMPA assay. This assay is currently configured with a capacity of 720 compounds/month with a turnaround of 4 days.

1 x standards

45 compounds + 3 controls

2 x t10-rat

2 x t10-human

2 x t0-human

2 x t0-rat

2 x t0-mouse

Total: 144 assays 624 samples

2 x t10-mouse

FIGURE 13.13 Sample generation for metabolic-stability assay.

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