J Pharm Sci. 2013 Feb;102(2):750-60. doi: 10.1002/jps.23395.

An alternative approach for quantitative bioanalysis using diluted blood to profile oral exposure of small molecule anticancer drugs in mice.

Liederer BM, Berezhkovskiy LM, Ubhayakar SS, Deng Y.

Genentech, Inc., Drug Metabolism and Pharmacokinetics, South San Francisco, California 94080, USA. liederer.bianca@gene.com

Abstract

A quantitative bioanalytical method for pharmacokinetic studies using diluted whole blood from serially bled mice was developed. Oral exposure profiles in mice for five model anticancer compounds dacarbazine, gefitinib, gemcitabine, imatinib, and topotecan were determined following discrete and cassette (five-in-one) dosing. Six micro blood samples per animal were collected and added to a fixed amount of water. This dilution served several purposes: the red blood cells were lysed; an anticoagulant was unnecessary and the fluid volume of diluted sample was sufficient for bioanalytical assays. AUC values obtained from blood concentrations were within twofold for discrete and cassette dosing except for imatinib (2.1-fold difference) and in agreement with those obtained from plasma concentrations after discrete dosing. All compounds were stable in plasma and diluted blood samples for at least 2 weeks at approximately -80°C. Matrix and intermatrix effects were evaluated to ensure robustness and integrity of the bioanalytical assays. This method provides significant process improvement by enhancing efficiency for sample collection and processing and reducing resources (e.g., reduced compound, cost, and animal requirement) compared with conventional methods. Our study demonstrates the applicability of using diluted micro blood samples for small molecule quantitative bioanalysis to support mouse studies in drug discovery. Copyright © 2012 Wiley Periodicals, Inc.

PMID: 23225118

 

SUPPLEMENT:

Recent advances in synthetic procedures, molecular biology and robotics have resulted in a large increase in the number of compounds requiring evaluation for their drug-like properties and pharmacokinetic (PK)/pharmacodynamics (PD) profiles during drug discovery.  The increasing demand requires more animal studies, thus increasing animal usage.  To obtain PK parameters in these animal studies multiple plasma samples are taken throughout a time course study.  However, in smaller rodents, such as mice, collection of plasma is more complicated, labor-intensive and time-consuming compared to larger animals.  Each mouse can be bled for only a limited amount of time points as the blood collection volume should not exceed approximately 10% of the blood volume over a 24 hour period.  Therefore, a large number of mice per study are needed, increasing animal handling as well as compound requirements and costs.  In order to reduce the animal usage, we proposed two changes to the drug discovery screening process for mouse PK studies: 1) to micro bled (10 or 15 µL) mice serially instead of taking composite samples allowing more samples to be taken from one animal without exceeding “animal welfare regulations” and 2) to use cassette dosing instead of discrete dosing which involves the simultaneous administration of several compounds to a single animal instead of only one compound.

To evaluate our proposal, five small molecule anticancer model compounds: dacarbazine, gemcitabine, gefitinib, imatinib and topotecan were used for PK studies.  Traditional plasma samples were collected from discrete studies.  In this study, two plasma samples were collected from each mouse, one via retro-orbital bleed (approximately 0.1 mL) and one via terminal cardiac puncture (approximately 0.2 mL) and a total of three mice were needed for a 6-timepoint PK study to evaluate one model compound.  To evaluate 1) blood samples were collected from a parallel discrete dosing PK study.  In this study, six micro blood samples were collected via tail nick bleeding and only one mouse was needed for the 6-timepoint PK study.  The concentrations of each compound in the plasma and blood samples were analyzed by LC-MS/MS assays.    For all compounds, the blood concentration-time curves and PK parameters were similar to the plasma concentration-time curves and PK parameters (Table 1 and Figure 1).  The statistical t-test showed that there was no difference between PK parameters (AUClast) within the 2-fold range at 95% confidence level.  The results demonstrated that utilizing blood PK over plasma PK with the described study design reduces number of animals and the amount of material by at least 50%.

Further evaluation was carried out to validate 2) by using these five model compounds for a 5-in-1 cassette dosing PK study.  The blood samples were collected as described above via tail nick and the concentration of each compound in the blood samples were analyzed by LC-MS/MS assays.  The AUC values obtained from blood concentrations were within 2-fold for discrete and cassette dosing except, for imatinib (2.1-fold difference), and in agreement with those obtained from plasma concentration after discrete dosing (Table 1 and Figure 1).  The results demonstrated that using cassette dosing for PK studies as a screening tool can further significantly reduce animal usage and increase efficiency for sample collection and processing and reduce resources (e.g. five times reduction in animals used in the case).

In summary, our proposal for using cassette dosing PK study with serially micro bled mice as a screening tool provides a remarkable reduction in animal usage and (e.g. 15-times in this specific example if 1) and 2) are utilized) significant process improvements by enhancing efficiency for sample collection and processing and reducing resources compared to conventional methods.

Bianca Liederer-tab1

Table 1. Mean ± standard deviation (SD) pharmacokinetic parameters of dacarbazine, gefitinib, gemcitabine, imatinib and topotecan in female CD-1 mice following oral administration of a single 10 mg/kg dose.

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