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Use of loperamide as a phenotypic probe of mdr1a status in CF-1 mice
J Cory Kalvass1, Candace L Graff, Gary M Pollack
1Division of Drug Delivery and Disposition, School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
Purpose:
A subpopulation of the CF-1 mouse strain (approximately 25%) lacks P-gp expression, and consequently, increased brain penetration of many substrates is observed in these animals. Mice lacking the mdr1a gene represent an important research tool to study the potential effects of P-gp on CNS substrate disposition.
Methods:
Adult CF-1 mice were used in all experiments. Loperamide-induced antinociception was determined by the hotplate latency test at 0.25, 2, and 4 h post-dose. At the conclusion of the pharmacodynamic experiment(s), trunk blood and brain tissue were collected and analyzed by high-performance liquid chromatography-mass spectrometry (LC-MS/MS). Mice were also genotyped for their mdrla status via RT-PCR.
Results:
All mice with three consecutive effects of maximum hotplate latency (60 s) showed considerable opioid-like behavior in addition to antinociception. Mice without three consecutive effects of maximum hotplate latency (< or = 30 s) showed no opioid-like behavior. The loperamide brain-to-serum ratio in mice identified as P-gp-deficient was 65-fold higher compared to the P-gp-competent animals (10.1 +/- 1.0 vs. 0.155 +/- 0.018). All animals identified as phenotypically P-gp-competent based on the hotplate assay evidenced the mdrla(+/+) genotype.
Conclusion:
This assay appears to offer a rapid and unambiguous measure via a relatively non-invasive, simple technique to identify P-gp status in the CF-1 subpopulation of mice.
Insights
A novel assay effectively identifies P-glycoprotein (P-gp) deficient CF-1 mice, crucial for studying P-gp's role in central nervous system (CNS) drug disposition. This method offers a rapid, non-invasive way to determine P-gp status in research animals.
Area of Science:
- Pharmacology
- Neuroscience
- Genetics
Background:
- A subpopulation of CF-1 mice (~25%) lacks P-glycoprotein (P-gp) expression.
- This P-gp deficiency leads to increased brain penetration of various substrates.
- Mice lacking the mdr1a gene are valuable tools for investigating P-gp effects on CNS substrate disposition.
Purpose of the Study:
- To develop and validate a rapid, non-invasive assay for identifying P-gp deficient CF-1 mice.
- To correlate P-gp status with loperamide brain penetration and antinociception.
Main Methods:
- Adult CF-1 mice were subjected to loperamide administration.
- Antinociception was measured using the hotplate latency test.
- Brain and blood samples were analyzed for loperamide concentration using LC-MS/MS.
- Mice were genotyped for mdr1a status via RT-PCR.
Main Results:
- A 65-fold higher loperamide brain-to-serum ratio was observed in P-gp deficient mice compared to P-gp competent mice.
- Phenotypically P-gp competent mice exhibited the mdr1a(+/+) genotype.
- Hotplate latency correlated with P-gp status and opioid-like behavior.
Conclusions:
- The hotplate assay provides a rapid and unambiguous method for identifying P-gp status in CF-1 mice.
- This assay is a simple, relatively non-invasive technique suitable for research settings.
- The findings facilitate further research into P-gp's role in CNS drug disposition.
