Discussion

The kinetic analysis of [³H]DPDPE uptake into the CNS was determined by using six concentrations of unlabeled DPDPE in the range 1 to 100 μM (fig. 1). The results of this study indicated that the brain uptake of [³H]DPDPE consisted of both saturable and nonsaturable components, which could be described by Michaelis-Menten type kinetics with K _m = 45.5 ± 27.6 μM, V _max = 51.1 ± 13.2 pmol · min⁻¹ · g⁻¹ andK _d = 0.6 ± 0.3 μl · min⁻¹ · g⁻¹ (Table 1). These kinetic constants reflect the presence of a saturable uptake system for [³H]DPDPE with a relatively large affinity and a low capacity. Interestingly, although leucine-enkephalin failed to inhibit significantly the CNS uptake of [³H]DPDPE (table 2;Williams et al., 1996), leucine-enkephalin has previously been found to enter the brain by a saturable uptake system with a relatively large K _m value, 34 to 41 μM, and a low capacity, 0.14 to 0.16 nmol · min⁻¹ · g⁻¹ (Zlokovic et al., 1989). In contrast to brain uptake, the CSF entry of [³H]DPDPE was found to be purely nonsaturable with a diffusion constant of 0.9 ± 0.1 μl · min⁻¹ · g⁻¹ (fig. 1). Several peptides have been shown to cross the BBB and/or the blood-CSF barriervia passive diffusion, e.g.,thyrotropin-releasing hormone (Zlokovic et al., 1985; 1988).

DPDPE is currently undergoing Phase 1 clinical trials to determine its therapeutic plasma concentration range. However, it is known that DPDPE, at a concentration of 60 mg/kg, can elicit significant analgesia within 5 min of being administered i.v. in the mouse (Weber et al., 1991). It is also known that approximately 4% of [³H]DPDPE administered i.v. can be found in the blood after 5 min. If we suppose that 4% of a 60-mg/kg dose of DPDPE remains in the blood, then this will result in a total blood concentration in a 30-g mouse of 0.072 mg. A mouse has approximately 2 ml of blood,i.e., 0.036 mg/ml or 56 μM. The K _mvalue determined in the present study was 46 μM, so if 0.036 mg/ml was a therapeutic blood concentration, then both the saturable and the nonsaturable uptake systems characterized in the present study for [³H]DPDPE would be important for the brain entry of this analgesic.

The antitumor amino acid analog, acivicin, has been shown to be transported across the BBB by the large neutral amino acid carrier (Williams et al., 1990; Takada et al., 1991;Chikhale et al., 1995). Although DPDPE (MW 645.8) is considerably larger than acivicin (MW 178.6), it is possible that the N-terminal tyrosine of DPDPE enables it to use this large neutral amino acid carrier at the BBB. However, BCH, a nonmetabolizable analog that is known to be specific for this transporter, failed to inhibit the uptake of [³H]DPDPE into either the brain or the CSF (table 2). This indicates that [³H]DPDPE does not utilize the large neutral amino acid transporter to enter the CNS and confirms our previous work, which indicates that [³H]DPDPE crossed the BBB intact and was not metabolized to produce [³H]tyrosine (Williams et al., 1996). It is unclear why there was a significant increase in the uptake of [³H]DPDPE into the brain in the presence of BCH, but one could hypothesize that the transport mechanism for [³H]DPDPE was stimulated by the saturation of the large neutral amino acid transport mechanism.

Peptides have been found to enter the CNS by several saturable mechanisms, including absorptive-mediated endocytosis (Kumagai et al., 1987), receptor-mediated transport (Pardridge et al., 1985) and carrier-mediated transport (Banks and Kastin, 1987). In an attempt to clarify further the saturable mechanism by which [³H]DPDPE enters the CNS, we examined uptake in the presence of a variety of substances (table 2; fig. 3).

It is known that the luminal and abluminal endothelial cell membranes of the BBB have anionic sites (Vorbrodt, 1989), and it has been suggested that these sites restrain vesicle and channel formation for transport into or through the endothelial cell (Hardebo and Kåhrström, 1985). In addition, the negative surface charge prevents the adhesion of anionized substances on the intima (Danon and Skutelsky, 1976), so cationized macromolecules are more capable of nonspecific adsorption onto the cell membrane (Griffin and Giffels, 1982). The isoelectric point (pI) of a compound therefore plays an important role in its uptake by brain capillaries (Nagy et al., 1983; Vorbrodt et al., 1995) and in its passage across the choroid plexus (Griffin and Giffels, 1982). For example, positively charged peptides, such as cationized albumin, have been shown to use absorptive-mediated endocytosis to cross the BBB (Kumagaiet al., 1987). Furthermore, in contrast to cationized ferritin, native (anionic) ferritin has been shown to have a limited passage across the choroid plexus epithelium (Van Deurs et al., 1981). DPDPE (pI ∼ 6.5) is slightly negatively charged at physiological pH, so it would seem unlikely to use absorptive-mediated endocytosis to enter the CNS (Baldwin and Chien, 1984; Shen et al., 1992).

Poly-l-lysine (3–300 μM) has been shown to inhibit the binding of E-2078, ebiratide and cationized albumin, to isolated brain capillaries (Kumagai et al., 1987; Terasaki et al., 1989; Shimura et al., 1991). However, in vivo intracarotid artery infusions of 5 μM poly-l-lysine have been shown to produce significant Evans blue/albumin leakage, a result that suggests loss of BBB integrity (Westergren and Johansson, 1993). In the present study, poly-l-lysine, at concentrations in the perfusion medium ranging from 5 to 300 μM, also caused visible extravasation of Evans blue, as well as an increase in [¹⁴C]sucrose/vascular space, compared with control groups (fig. 2). In contrast, 0.3 μM poly-l-lysine in the perfusion medium did not significantly affect the visible extravasation of Evans blue or the [¹⁴C]sucrose/vascular space. The polycation protamine sulphate has also been shown to affect the extravasation of a vascular space marker in a dose-dependent manner (Nagy et al., 1983). Furthermore, poly-l-lysine at picomolar concentrations has been shown not to affect the passage of albumin or horseradish peroxidase across mouse fibroblast membranesin vitro (Shen and Ryser, 1978). Interestingly, in the presence of 0.3 μM poly-l-lysine, the uptake of [³H]DPDPE into the brain did increase approximately 3-fold (table 2). This would suggest that 0.3 μM poly-l-lysine significantly affects the cellular uptake of [³H]DPDPE, not its passage through interendothelial junctions. Previous studies have suggested that the focal points of this increase in barrier permeability are the endothelial tight junctions (Nagy et al., 1983), and in the present study, this may be so at poly-l-lysine concentrations of ≥5 to 300 μM. Poly-l-lysine has also been shown to result in glomerular epithelial cell swelling (Seiler et al., 1975), and it may be that endothelial/epithelial cell death is responsible for the loss of BBB integrity (Nagy et al., 1983). Although it is known that polycation infusions result in neutralization of the negative surface charge (Nagy et al., 1983), how 0.3 μM poly-l-lysine increases the blood-brain passage of [³H]DPDPE has not been established. It may be due to the opening up of hydrophilic channels in the endothelial cell membrane (Nagy et al., 1983) or to stimulation of transendothelial vesicular transport (Westergaard, 1980). However, the results of the present study do suggest that poly-l-lysine is not in competition with [³H]DPDPE. It is likely that if absorptive-mediated endocytosis is responsible for the poly-l-lysine-mediated increased passage of [³H]DPDPE, it is not the saturable uptake system identified in the absence of poly-l-lysine (fig. 1).

Receptors have been shown to mediate the endocytosis of insulin, insulin-like growth factors and transferrin across the BBB (Pardridgeet al., 1985; Duffy et al., 1988; Fishmanet al., 1987), and insulin receptors are known to be expressed at the blood-CSF barrier (Baskin et al., 1986). Figure 3 illustrates the absence of an effect by the deltaopioid receptor antagonist ICI 174,864 at a concentration above that of the K _m value for saturable DPDPE transport. This would suggest that delta opioid receptors do not mediate the saturable uptake of this delta opioid receptor-selective peptide, [³H]DPDPE, through the BBB (Terasakiet al., 1989; Zlokovic et al., 1989). In addition, the uptake of [³H]DPDPE into the brain and CSF was not significantly decreased in the presence of insulin, even at a concentration of 10 μM, which can occupy the receptors (Shimuraet al., 1991). This suggests that the receptor-mediated endocytosis system responsible for the passage of insulin into the CNS is not involved in the saturable uptake of [³H]DPDPE into the brain. Furthermore, it has been hypothesized that different saturable mechanisms of peptide transport could be manifested in the different rate and saturation constants for individual peptides and proteins (Zlokovic et al., 1990; Shimura et al.,1991; Poduslo et al., 1994). The kinetic constant (K _m) determined for the brain flux of [³H]DPDPE was in the low micromolar range (table 1), which would also appear to reflect carrier-mediated, rather than receptor-mediated, [³H]DPDPE uptake (Zlokovic et al., 1990).

It is of interest that insulin was found to increase significantly the uptake of [³H]DPDPE into the brain and CSF (fig. 3), while not affecting [¹⁴C]sucrose/vascular space. It is known that insulin affects the transmembrane transport of different substances, particularly glucose, into numerous different kinds of cells (Ayre, 1989). Previous reports have provided evidence for insulin potentiation of drug uptake by cells; for example, insulin has been shown to increase the cytotoxic effect of methotrexate, an anticancer agent, in human breast cancer cells in vitro (Alabasteret al., 1981). Although the authors suggested that the increased cytotoxicity was related to an increase in the sensitivity of the cells to methotrexate that resulted from a metabolic modification by insulin, a later proposal suggested that insulin had induced changes in cellular lipid synthesis and membrane lipid profile, resulting in changes in membrane fluidity and enhanced methotrexate transport (Schilsky et al., 1981). The enhanced cytotoxicity was suggested to be related to an increased capacity of the cells to accumulate free intracellular methotrexate. Another study showed that insulin increased the passage across the BBB of the deoxyribonucleoside analog azidodeoxythymidine (Ayre et al., 1989). It has been proposed that “through an unidentified interaction with its specific receptors on brain capillary endothelial cells, insulin facilitates the passage of drug molecules across the BBB into the brain” (Ayre, 1989). The results of the present study indicate that insulin can also alter the brain uptake of the analgesic [³H]DPDPE. Thus this study provides further evidence for the insulin-potentiation theory (fig. 3). The significance of insulin as a therapeutic modality in the alleviation of pain could prove invaluable if we could safely control its known side effects.

In conclusion, the present study has demonstrated that an enzymatically stable enkephalin analog, DPDPE, can enter the brain viaboth saturable and nonsaturable uptake mechanisms, which can be described by Michaelis-Menten type kinetics with aK _m value of 45.5 ± 27.6 μM, aV _max value of 51.1 ± 13.2 pmol · min⁻¹ · g⁻¹ and aK _d value of 0.6 ± 0.3 μl · min⁻¹ · g⁻¹. In addition, the CSF uptake of [³H]DPDPE could not be self-inhibited (K _d = 0.9 ± 0.1 μl · min⁻¹ · g⁻¹). Further experiments revealed that [³H]DPDPE was not entering the CNSvia the large neutral amino acid transporter, by binding to opioid receptors or by the leucine-enkephalin uptake system. It would also appear that although [³H]DPDPE is not in competition with either poly-l-lysine or insulin to enter the CNS, both substances do have significant effects on the uptake of [³H]DPDPE, and this may have valuable clinical implications. It is still unclear which saturable uptake mechanism is responsible for the entry of [³H]DPDPE into the CNS, but overall the results suggest a carrier-mediated transport system, which may be valuable for other peptide and nonpeptide drugs.