Influence of probenecid on the delivery of morphine-6-glucuronide to the brain
Introduction
Morphine-6-glucuronide (M6G) is an active metabolite of morphine. Rat studies have shown that this metabolite is equipotent to morphine after systemic administration, and 6–650 times more potent than morphine after intracerebroventricular administration (Abbott and Palmour, 1988, Frances et al., 1992, Paul et al., 1989). Therefore, it is of clinical interest to investigate the blood–brain barrier (BBB) transport of this metabolite.
In the central nervous system (CNS), several active transporters such as P-glycoprotein (P-gp), multidrug resistance associated proteins (MRPs), organic anion transporters (Oats) and organic anion transporting polypeptides (Oatps) are expressed at the BBB and at the blood–cerebrospinal fluid (CSF) barrier. These transporters may hinder influx of drugs from the blood into the brain, and/or enhance drug efflux from the brain back to the blood. Therefore, discrepancies from equal unbound concentrations in the brain and the blood at steady state could be explained by active processes at the level of the BBB and/or the blood–CSF barrier (Hammarlund-Udenaes et al., 1997). Microdialysis measures unbound drug concentrations simultaneously in various tissues, and it has been used to assess the involvement of efflux mechanisms at the BBB (Tunblad et al., 2003, Wong et al., 1993; Xie et al., 2000). The insertion of a microdialysis probe into the brain tissue makes it possible to study the local pharmacokinetics of a drug, and drug–drug interactions at the BBB.
Active efflux mediated by P-gp has been shown to restrict the penetration of various drugs from the blood to the brain using P-gp knock-out mice (de Lange et al., 1998, Megard et al., 2002, Schinkel et al., 1994; Xie et al., 1999) and P-gp inhibitors (Desrayaud et al., 1997, Letrent et al., 1999, Potschka and Loscher, 2001). In addition, probenecid, a non-specific inhibitor of organic anion transporters, has been used to influence the distribution of drugs to the brain (Tunblad et al., 2003, Wong et al., 1993; Xie et al., 2000). The ratio between the unbound concentration of M6G in the brain to that in blood at steady state was shown to be below unity, indicating the involvement of efflux transport mechanisms at the BBB (Bouw et al., 2001). Recently, it was demonstrated that P-gp is not involved in the brain efflux of M6G (Bourasset et al., 2003). These results were inconsistent with a previous report using an in vitro system of porcine brain capillary endothelial cells (Huwyler et al., 1996). In addition, Lötsch and co-workers have demonstrated that the ratio of unbound concentrations of M6G in the spinal cord to the total concentrations in plasma increased from 0.08 in rats receiving only M6G to 0.17 in rats that were pretreated with PSC833 (Lötsch et al., 2002a), which mainly modulates P-gp, but also Oatp1 and Oatp2 (Cvetkovic et al., 1999). Despite co-administration of PSC833 the spinal cord/plasma ratio was below unity, indicating that the transporters inhibited by PSC833 are not the only transporters involved in the brain efflux of M6G. The same group reported that inhibition of the probenecid-sensitive transporters did not affect the spinal cord/plasma ratio of M6G in rats, although the pharmacologic effect of M6G was altered (Lötsch et al., 2002b). Since the BBB and the blood–CSF barrier have different properties both regarding the structure of the barrier and the expression of specific transporters, transport across these barriers may well differ for a drug.
The objective of the present study was to investigate if the transport of M6G across the BBB is influenced by the probenecid-sensitive transporters. For this microdialysis was used to measure the unbound concentrations of M6G in the brain extracellular fluid (ECF) and in the blood after administration of M6G alone, and after M6G and probenecid co-administration. Nonlinear mixed effects modelling was used to obtain estimates of the influx clearance (CLin), i.e., the clearance from the blood to the brain, the efflux clearance (CLout), i.e., the clearance from the brain to the blood, and the ratios between these clearances (CLin/CLout). In addition, the random effects, including the residual error, were estimated by the mixed modelling approach. This study shows the first application of the integrated model for microdialysis data, which was recently presented (Tunblad et al., 2004).
Section snippets
Animals
Male Sprague–Dawley rats (n = 10) (Møllegaard, Denmark) weighing 262–300 g were used. The rats were acclimatised for at least seven days at 22 °C controlled humidity prior to the experiment. During this period the animals had free access to food and water. The Animal Ethics Committee of Uppsala University approved the protocol (C 144/99).
Probes and chemicals
Microdialysis probes for measurements in the brain (CMA/12 (3 mm)) and in venous blood (CMA/20 (10 mm)) were purchased from CMA, Stockholm, Sweden. The membranes of
Results
Using the integrated model it was possible to fit a model to all data simultaneously, i.e., the total arterial concentrations, the brain and blood dialysate concentrations and the recovery measurements from the blood and brain probes.
The typical value of the blood probe recovery was estimated as 54.1%, with an interprobe variability of 17% (Table 1). The brain probe recovery was best described by a typical value which varied randomly between experimental days (inter occasion variability). The
Discussion
The main finding of this study is that probenecid has no effect on the brain concentrations of M6G. This was concluded from the CLin/CLout ratio, which remained unchanged upon probenecid co-administration. In contrast, probenecid affects the pharmacokinetics in the blood by decreasing the systemic elimination.
The CLin/CLout ratio was estimated as 0.29, indicating that bulk flow and/or active processes act on M6G at the BBB. Similarly, the CLin/CLout ratio and the steady state ratio for unbound
Acknowledgements
The authors would like to thank Jessica Strömgren for excellent assistance with the animal surgery and the experiments. This work was supported by the Swedish Foundation for Strategic Research, Stockholm, Sweden and by the Swedish Research Council no. 11558.
References (33)
- et al.
Morphine-6-glucuronide: analgesic effects and receptor binding profile in rats
Life Sci.
(1988) - et al.
Steady-state theory for quantitative microdialysis of solutes and water in vivo and in vitro
Life Sci.
(1990) - et al.
Effect of the P-glycoprotein inhibitor, SDZ PSC 833, on the blood and brain pharmacokinetics of colchicine
Life Sci.
(1997) - et al.
Delayed antinociceptive effect following morphine-6-glucuronide administration in the rat—pharmacokinetic/pharmacodynamic modelling
Pain
(1998) - et al.
An improved method for the simultaneous determination of morphine and its principal glucuronide metabolites
J. Chromatogr.
(1988) - et al.
Regional response of cerebral blood volume to graded hypoxic hypoxia in rat brain
Br. J. Anaesth.
(2002) - et al.
The influence of inhibition of probenecid sensitive transporters on the central nervous system (CNS) uptake and the antinociceptive activity of morphine-6-glucuronide in rats
Neurosci. Lett.
(2002) - et al.
A co-culture-based model of human blood–brain barrier: application to active transport of indinavir and in vivo–in vitro correlation.
Brain Res.
(2002) - et al.
Disruption of the mouse mdr1a P-glycoprotein gene leads to a deficiency in the blood–brain barrier and to increased sensitivity to drugs
Cell
(1994) - et al.
NONMEM user's guide, NONMEM Project Group
(1994)