Carrier-mediated processes at several rat brain interfaces determine the neuropharmacokinetics of morphine and morphine-6-β-d-glucuronide
Introduction
Morphine and its active metabolite, morphine-6-β-d-glucuronide (M6G), both have analgesic activity. Although M6G has been shown to be equally or slightly more potent than morphine after systemic administration, M6G has a higher potency when administered by the intracerebroventricular route (Shimomura et al., 1971, Abbott and Palmour, 1988, Paul et al., 1989, Hanna et al., 2005). In addition, it has been shown that M6G produced a longer antinociceptive effect than morphine (Frances et al., 1992, Stain et al., 1995). However, the affinity of the two opioids for the μ-type receptors has been shown to be almost the same or slightly higher for morphine (Christensen and Jorgensen, 1987, Pasternak et al., 1987, Abbott and Palmour, 1988, Paul et al., 1989). Thus it is not evident that the difference in analgesic potency between morphine and M6G depends mainly on differences in types of opioid receptors and signal transduction processes (Osborne et al., 2000, Romberg et al., 2003, Kilpatrick and Smith, 2005). In any case, the higher analgesic potency of M6G compared to morphine could be related to differences in their neuropharmacokinetic behavior. Indeed, it has been shown that after a subcutaneous or an intracerebroventricular injection of morphine or M6G in the rat, morphine partitions more into brain intracellular fluid (bICF) than into brain extracellular fluid (bECF), whereas M6G distributes almost exclusively into bECF (Stain-Texier et al., 1999, Okura et al., 2003). We hypothesize that these two opposite patterns for morphine and M6G distribution into specific brain compartments could depend on two factors. Firstly, on their distinct logP values (− 0.2 and − 2.4, respectively (Murphey and Olsen, 1994)) which favor the bECF distribution for the more polar M6G, and secondly on the involvement of transporters at several brain interfaces including the blood–brain barrier (BBB), the bICF/bECF and bECF/CSF (cerebrospinal fluid) interfaces—which correspond to the neuronal/glial and ependymal cell membranes, respectively. The involvement of transporters at the BBB has been studied both for morphine and M6G, whereas such involvement at other brain interfaces has not yet been studied. At the BBB level, morphine is carried from the brain to the blood by at least two efflux proteins, one being probenecid-sensitive (Tunblad et al., 2003), and the other being the P-glycoprotein (P-gp) (Xie et al., 1999, Cisternino et al., 2001), an ATP-binding cassette (ABC) protein expressed at the luminal membrane of the brain microvessel endothelial cells forming the BBB. The BBB transport of M6G has been shown not to be P-gp-mediated (Bourasset et al., 2003, Skarke et al., 2004), but to be mediated by a glucose-sensitive transporter (which may be GLUT-1) and a digoxin-PSC833 co-sensitive transporter (which could be oatp2) (Bourasset et al., 2003).
In order to check whether any transporters were also involved at the bICF/bECF barrier and/or bECF-CSF barrier, and were potentially responsible for the heterogeneity of morphine and M6G brain distributions, we carried out two studies. Firstly, we performed transcortical microdialysis in the rat, perfusing morphine or M6G through the dialysis probe, with and without probenecid, a known overall inhibitor of several organic anion transporters. Thus, we measured the in vitro and in vivo probe recoveries of morphine and M6G by retrodialysis, with and without probenecid. This allowed us to calculate the apparent bECF permeability factor for morphine and M6G, which depends on both probe characteristics and morphine and M6G transport processes from bECF to adjacent fluids, i.e., to blood, bICF, and CSF (Bungay et al., 1990).
Secondly, we used experimental morphine and M6G pharmacokinetic data previously obtained in rat plasma, bECF, bICF, and CSF (Stain-Texier et al., 1999), to establish several brain pharmacokinetic models, which assumed the presence or absence of capacity-limited transport systems at the BBB, bICF/bECF, and bECF/CSF interfaces. The objective was to find the most appropriate model fitting these experimental data. Taken together, these two approaches allowed us to evaluate at which brain interface capacity-limited transport processes might be involved in M6G and morphine neuropharmacokinetics.
Section snippets
Drugs
Morphine and M6G were obtained from Francopia-Sanofi (Paris, France) and were dissolved in brain perfusion fluid (CMA, Phymep, France) to obtain solutions containing 10 μM of morphine or M6G. Probenecid and morphine-d3 were purchased from SIGMA (St Quentin, France). Probenecid was dissolved in 8.4% NaHCO3 and was added to the perfusion fluid to obtain a probenecid concentration of 30 mM. Chemicals were of HPLC grade and purchased from Fluka and Riedel-de-Haën (SIGMA, St Quentin, France).
Animals
Male
Transcortical microdialysis
In the absence of probenecid, the in vitro RD of M6G and morphine were significantly 1.4-fold (p = 0.015) decreased and 1.5-fold (p = 0.005) enhanced compared with their in vivo RD, respectively (Fig. 1). This difference of RD between in vitro and in vivo experiments can be attributed to the permeability of the tissue surrounding the probe, i.e., the bECF, and then to the capabilities of morphine and M6G to distribute from bECF to plasma and other brain fluids. The calculation of the apparent bECF
Discussion
Based upon the previous data of Sun et al. (2001) who showed by using transcortical microdialysis that the probe recovery can be changed by adding a nonspecific organic anion carrier modulator such as probenecid, and that these recovery variations can be attributed to changes in the distribution of studied drugs, from bECF to adjacent fluids, we hypothesized that a probenecid-mediated increase or decrease in the probe recovery under steady-state conditions can result from a distribution of the
Conclusion
We conclude that the neuropharmacokinetics of morphine and M6G are governed by several brain transporters. M6G is carried from plasma to bECF by two influx transporters and then trapped in bECF by the simultaneous action of another transporter located at the ependymal barrier, which limits the extrusion of M6G into CSF. Inversely, the neuropharmacokinetics of morphine is governed by at least one probenecid-sensitive transporter located at neuronal and/or glial cell membranes, influxing morphine
Acknowledgement
We thank Pharsight Corporation for allowing our university to benefit from the Academic Licensing Program (Pharsight Corporation, 800 West El Camino Real, Mountain View, California 94040, USA) and we thank Alan Strickland for editing the English text.
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