Influence of probenecid on the delivery of morphine-6-glucuronide to the brain

https://doi.org/10.1016/j.ejps.2004.09.009Get rights and content

Abstract

The objective was to evaluate the influence of probenecid on the blood–brain barrier (BBB) transport of morphine-6-glucuronide (M6G).

Microdialysis probes were placed in the striatum and into the jugular vein of Sprague–Dawley rats. Each probe was calibrated in vivo using retrodialysis by drug. M6G was administered as a 4-h exponential i.v. infusion, and the experiment was repeated the following day with the addition of probenecid. The data were analysed using NONMEM.

An integrated model including the total arterial concentrations, the dialysate concentrations in brain and blood, and the recovery measurements, was developed. The extent of BBB transport, expressed as the ratio between clearance into the brain and clearance out of the brain (CLin/CLout), was estimated as 0.29 on both days, indicating that efflux transporters act on M6G at the BBB. However, the probenecid-sensitive transporters are not involved in the brain efflux, as the ratio was unaltered although probenecid was co-administered. In contrast, the systemic elimination of M6G decreased by 22% (p < 0.05) upon probenecid co-administration. The half-life of M6G was longer in the brain than in blood on both experimental days (p < 0.05).

In conclusion, probenecid decreased the systemic elimination of M6G, but had no effect on the BBB transport of M6G.

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)

  • U. Bickel et al.

    Poor permeability of morphine 3-glucuronide and morphine 6-glucuronide through the blood–brain barrier in the rat

    J. Pharmacol. Exp. Ther.

    (1996)
  • F. Bourasset et al.

    Evidence for an active transport of morphine-6-beta-d-glucuronide but not P-glycoprotein-mediated at the blood–brain barrier

    J. Neurochem.

    (2003)
  • M.R. Bouw et al.

    Pharmacokinetic-pharmacodynamic modelling of morphine transport across the blood–brain barrier as a cause of the antinociceptive effect delay in rats—a microdialysis study

    Pharm. Res.

    (2000)
  • M.R. Bouw et al.

    Methodological aspects of the use of a calibrator in in vivo microdialysis—further development of the retrodialysis method

    Pharm. Res.

    (1998)
  • M.R. Bouw et al.

    Blood–brain barrier transport and brain distribution of morphine-6-glucuronide in relation to the antinociceptive effect in rats—pharmacokinetic/pharmacodynamic modelling

    Br. J. Pharmacol.

    (2001)
  • M. Cvetkovic et al.

    OATP and P-glycoprotein transporters mediate the cellular uptake and excretion of fexofenadine

    Drug Metab. Dispos.

    (1999)
  • Cited by (0)

    View full text