Development of a P-Glycoprotein Knockout Model in Rodents to Define Species Differences in its Functional Effect at the Blood–brain Barrier

doi:10.1002/jps.20658

Journal of Pharmaceutical Sciences

Volume 95, Issue 9, September 2006, Pages 1944-1953

https://doi.org/10.1002/jps.20658 Get rights and content

Abstract

The objective of this study was to establish the optimal blood concentrations of the potent P-glycoprotein (P-gp) inhibitor GF120918 (Elacridar) required to achieve maximal knockout of this efflux transporter in the blood–brain barrier (BBB) of mice, rats, and guinea pigs. Genetic mdr1a/b(−/−) knockout mice and “chemical” P-gp knockout mice, rats, and guinea pigs, generated by 24 h continuous infusion of GF120918, were used to investigate the effects of P-gp modulation on the brain penetration of SB-487946. Genetic mdr1a/b(−/−) knockout mice demonstrated a >70-fold increase in brain:blood ratio of SB-487946 compared to mdr1a/b(+/+) wild-type mice. There was a similar increase in SB-487946 brain:blood ratio in GF120918-treated mice (ca. >67-fold) and rats (ca. 95-fold) but a significantly smaller increase (ca. 10-fold) in guinea pigs treated with GF120918. An appreciable difference was found in the BBB functional effect of P-gp efflux in rodents. GF120918 blood EC90 in mice and rats were similar however, the EC90 in guinea pigs was ca. 10-fold higher, suggesting a species difference in the activity of P-gp at the BBB in some rodents. This study establishes the optimal blood concentrations of GF120918 in relation to SB-487946, to achieve chemically induced P-gp knockout in rodents.

Section snippets

INTRODUCTION

The blood–brain barrier (BBB) consists of endothelial cells lining the brain capillaries connected by tight junctions.¹ Xenobiotics cross this endothelial cell monolayer via the transcellular pathway² to enter into the brain and exert pharmacological activity in the central nervous system (CNS). Physiochemical properties, for example, lipophilicity, molecular weight, and polar surface area (PSA), are known to be the major influencing factors that limit the extent to which xenobiotics cross the

Chemicals and Materials

SB-487946 and GF120918 were obtained from GlaxoSmithKline Compound Registry. Saline aq. 0.9% (w/v) and glucose aq. 5% (w/v) were obtained from Fresenlus Kabi Limited (Lancs, UK). 1-Methyl 2-Pyrrolidinone and 1,2 propanediol were obtained from Sigma Chemical Company (St. Louis, MO). Hydroxypropyl-beta-cyclodextrin (KleptoseHPB™) was obtained from Roquette UK Limited (Northants, UK). All other reagents and chemicals were of an analytical grade or higher.

SB-487946 was dissolved in 5% (w/v) glucose

Genetic Knockout and Control Animals

Blood and brain concentrations of SB-487946 following continuous infusion of SB-487946 to mdr1a/b(−/−) knockout and mdr1a/b(+/+) wild-type mice are shown in Table 1. Mean brain:blood ratio of SB-487946 is shown in Figure 1. The levels of SB-487946 in the mdr1a/b(+/+) wild-type mice brain could not be determined as the levels were below the lower limit of quantification (LLQ = 0.010 µM). A greater than 70-fold increase in the brain concentration was seen in the mdr1a/b(−/−) knockout mice

DISCUSSION

In these studies, we evaluated P-gp-mediated transport of SB-487946, a probe compound identified as a substrate for P-gp, into the mouse, rat, and guinea pig brain and the inhibition of P-gp by GF120918 at escalating target dose levels to produce a “chemical” P-gp knockout rodent. A common tool for in vivo study of the effects of P-gp efflux on brain penetration has traditionally been the mdr1a/b(−/−) knockout mouse model¹² which is deficient in the gene and hence P-gp protein expression.

Acknowledgements

The authors thank the following people for their contributions, Alison Gilbert, Leanne Westgate, Evangelia Grigorakou, Scott Summerfield, Michael Briggs, Ishrut Hussain, and David Begley.

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Research ArticlesDevelopment of a P-Glycoprotein Knockout Model in Rodents to Define Species Differences in its Functional Effect at the Blood–brain Barrier

Abstract

Section snippets

INTRODUCTION

Chemicals and Materials

Genetic Knockout and Control Animals

DISCUSSION

Acknowledgements

Adv Drug Deliv Rev

Life Sci

Eur J Cancer

J Pharmeut Sci

J Pharmeut Sci

Cell

Adv Drug Deliv Rev

Crit Rev Oncol Hematol

Adv Drug Deliv Rev

Molec Imag Biol

Adv Drug Deliv Rev

Trends Pharmacol Sci

The role of drug transporters at the blood–brain barrier

Annu Rev Pharmacol Toxicol

P-glycoprotein and multidrug resistance-associated proteins limit the brain uptake of saquinavir

J Pharmacol Exp Ther

A general pattern for substrate recognition by P-glycoprotein

Eur J Biochem

Utility of mdr1-gene deficient mice in assessing the impact of P-glycoprotein on the pharmacokinetics and pharmacodynamics in drug discovery and development

Curr Drug Metab

Cellular localization of the multidrug-resistance gene product P-glycoprotein in normal human tissues

Proc Natl Acad Sci USA

Detection of P-glycoprotein isoforms by gene-specific monoclonal antibodies

Proc Natl Acad Sci USA

Role of P-glycoprotein in pharmacokinetics. Clinical implications

Clin Pharmacokinet

The impact of P-glycoprotein on the disposition of drugs targeted for indications of the central nervous system: Evaluation using the mdr1a/b knockout mouse model

Drug Metab Dispos

The drug transporter P-glycoprotein limits oral absorption and brain entry of HIV-1 protease inhibitors

J Clin Invest

Pharmacological inhibition of P-glycoprotein transport enhances the distribution of HIV-1 protease inhibitors into brain and testes

Drug Metab Dispos

Increased drug delivery to the brain by P-glycoprotein inhibition

Clin Pharmacol Ther

Tissue distribution and chemical induction of multiple drug resistance genes in rats

Drug Met Dispos

The MDR1 (ABCB1) gene polymorphism and its clinical implications

Clin Pharmacokinet

The three mouse multidrug resistant (mdr) genes are expressed in a tissue-specific manner in normal mouse tissue

Mol Cell Biol

The gene encoding multidrug resistance is induced and expressed at high levels during pregnancy in the secretory epithelium of the uterus

Proc Natl Acad Sci USA

Research Articles
Development of a P-Glycoprotein Knockout Model in Rodents to Define Species Differences in its Functional Effect at the Blood–brain Barrier