In vitro and in vivo investigations on fluoroquinolones; effects of the P-glycoprotein efflux transporter on brain distribution of sparfloxacin

Abstract

The role of mdr1a-encoded P-glycoprotein on transport of several fluoroquinolones across the blood–brain barrier was investigated. In vitro, P-glycoprotein substrates were selected by using a confluent monolayer of MDR1-LLC-PK1 cells. The inhibition of fluoroquinolones (100 μM) on transport of rhodamine-123 (1 μM) was compared with P-glycoprotein inhibitors verapamil (20 μM) and SDZ PSC 833 (2 μM). Subsequently, transport polarity of fluoroquinolones was studied. Sparfloxacin showed the strongest inhibition (26%) and a large polarity in transport, by P-glycoprotein activity. In vivo, using mdr1a (−/−) and wild-type mice, brain distribution of pefloxacin, norfloxacin, ciprofloxacin, fleroxacin and sparfloxacin was determined at 2, 4, and 6 h following intra-arterial infusion (50 nmol/min). Brain distribution of sparfloxacin was clearly higher in mdr1a (−/−) mice compared with wild-type mice. Sparfloxacin was infused (50 nmol/min) for 1, 2, 3 and 4 h in which intracerebral microdialysis was performed. At 4 h, in vivo recovery (dynamic-no-net-flux method) was 6.5±2.2 and 1.5±0.5%; brain_ECF concentrations were 5.1±0.2 and 26±21 μM; and total brain concentrations were 7.2±0.3 and 23±0.3 μM in wild-type and mdr1a (−/−) mice, respectively. Plasma concentrations were similar (18.4±0.7 and 17.9±0.5 μM, respectively). In conclusion, sparfloxacin enters the brain poorly mainly because of P-glycoprotein activity at the blood–brain barrier.

Introduction

Fluoroquinolones are used in the chemotherapy of various infectious diseases because of their broad and strong antibacterial activity, especially against Gram-negative bacteria (Moellering, 1996). Fluoroquinolones tend to distribute rapidly into peripheral tissues and fluids, and reach concentrations often higher than found in serum or plasma (Sörgel et al., 1989). However, distribution of the unbound drugs into the cerebrospinal fluid (CSF) and brain extracellular fluid (brain_ECF) was shown to be poor (Kitzes-Cohen, 1989, Ooie et al., 1996a, Ooie et al., 1997a). On the other hand, severe CNS side effects like hallucinations, anxiety, agitation, depression, and convulsions have been reported following administration of fluoroquinolones (Ooie et al., 1997a), probably related to their penetration into the brain since seizures have been shown to be related to CSF concentrations in laboratory animals Delon et al., 1997, Delon et al., 1998

For optimal therapy, knowledge of factors involved in the transport of fluoroquinolones into the brain should be known. Transport into the brain is to a large extent determined by transport across the blood–brain barrier. Blood–brain barrier transport of fluoroquinolones has been studied in vitro (Jaehde et al., 1993), and in vivo (Ooie et al., 1996a, Ooie et al., 1997a, Ooie et al., 1997b) where mostly transport into the CSF was investigated (Jaehde et al., 1992, Ooie et al., 1996b, Ooie et al., 1996c, Delon et al., 1999). These studies indicated that distribution of fluoroquinolones into the brain was restricted, especially for the most hydrophilic compounds (Delon et al., 1999).

The P-glycoprotein is a transmembrane transport protein that actively extrudes its substrates from cells. It is found in many cancers, where it is associated with multidrug resistance (MDR). In humans, the drug transporting P-glycoprotein is encoded by the MDR1 gene and in rodents by the mdr1a and mdr1b genes. P-glycoprotein is also present in normal cellular systems such as the endothelial cells of the blood–brain barrier Cordon-Cardo et al., 1989, Thiebaut et al., 1989, testes, gastro-intestinal tract, and kidney. At the blood–brain barrier it effluxes a variety of substrates from the endothelial compartment into blood. The observation that brain penetration of several compounds was much smaller than predicted on the basis of their lipophilicity (Levin, 1980) could be explained by the fact that a number of these drugs are P-glycoprotein substrates Schinkel et al., 1994, Schinkel et al., 1995.

The aim of this study was to investigate whether transport of fluoroquinolones across the blood–brain barrier was affected by P-glycoprotein mediated transport. In vitro studies were performed using MDR1-LLC-PK1 pig kidney epithelial cells Schinkel et al., 1995, Ito et al., 1997, in order to select P-glycoprotein substrates among the fluoroquinolones. In these cells P-glycoprotein is expressed at the apical side. Confluent monolayers of these cells were used to determine the transport of compounds from apical-to-basolateral and basolateral-to-apical side. Polarity in drug transport is an indication for the compound being a P-glycoprotein substrate. Firstly, the effect of the fluoroquinolones ciprofloxacin, cinoxacin, fleroxacin, lomefloxacin, norfloxacin, pefloxacin, and sparfloxacin on transport of the P-glycoprotein substrate rhodamine-123 (Kessel, 1989) was estimated in vitro. These effects were compared with the known P-glycoprotein inhibitors verapamil and SDZ PSC 833. Secondly, for those fluoroquinolones showing a significant inhibition of P-glycoprotein mediated efflux of rhodamine-123, the polarity in transport was determined.

For in vivo studies the mdr1a (−/−) mice model was used. These mice lack the expression of P-glycoprotein at the blood–brain barrier, which allows in vivo investigations on the role of P-glycoprotein on drug transport across the blood–brain barrier (Schinkel et al., 1994). Fluoroquinolones that showed to be P-glycoprotein substrates in the in vitro studies (ciprofloxacin, fleroxacin, norfloxacin, pefloxacin, and sparfloxacin) were administered intravenously by constant infusion. At various times total brain and plasma concentrations were estimated and the ratio of total brain over plasma was used as a measure for brain uptake.

Brain disposition of sparfloxacin was studied in more detail following intra-arterial infusion and performing intracerebral microdialysis in mdr1a (−/−) and wild-type mice. This technique allows the monitoring of free drug concentrations in brain_ECF and is therefore well suited for pharmacokinetic investigations in rats (De Lange et al., 1994, De Lange et al., 1995a, De Lange et al., 1995b, De Lange et al., 1995c) and in mice (De Lange et al., 1998). The dynamic-no-net-flux (Olson and Justice, 1993) method was applied to determine in vivo recovery and concentration-time profiles of sparfloxacin in brain_ECF.