Introduction

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Since the discovery of multiple types of opioid receptors (Martin et al., 1976; Lord et al., 1977), attempts have been made to elucidate the physiological function of these receptors (µ,  and ). The development of potent, specific antagonists and agonists is essential for clarification of the multiple biological effects thought to be mediated by each receptor. Several receptor agonists have been developed that are selective for the various receptor subtypes, for example, [D-Pen²,D-Pen⁵]-enkephalin () (Mosberg et al., 1983), [D-Ala²,N-MePhe⁴,Gly-ol⁵]-enkephalin (µ) (Handa et al., 1981) and U50,488H () (VonVoigtlander et al., 1983). These agonists have been extensively characterized pharmacologically, and a few have been evaluated for their ability to enter the brain. For example, [D-Pen²,D-Pen⁵]-enkephalin (Weber et al., 1991, 1992, 1993; Brownson et al., 1994; Williams et al., 1996) has been shown to be both enzymatically stable and able to enter the CNS through a saturable mechanism at the BBB. Opioid antagonists that are commercially available have historically been modeled from alkaloid opioid agonists, i.e., naloxone and naltrexone, both of which are not receptor selective. This paper describes the blood-to-CNS pharmacokinetics of a cyclic peptidergic analog of somatostatin, CTAP, which has been shown to be extremely potent and selective for µ-opioid receptors (Kramer et al., 1989).

Somatostatin is a 28-amino acid, regulatory peptide hormone that has numerous effects within the CNS and peripheral nervous system, such as controlling growth hormone, insulin and glucagon release. It is also postulated that, after neurosecretion of somatostatin, there is a metabolic interaction that occurs with brain capillary endothelial cells (Pardridge et al., 1985).

Several analogs of somatostatin have been developed that may provide clinical intervention for the treatment of endocrine disturbances such as acromegaly, diabetes mellitus (Karashima and Schally, 1988) and peptic ulcer disease (Laszlo et al., 1989). Cancer treatment has been shown to be another important application for the use of somatostatin analogs (Schally et al., 1986). A recently developed somatostatin analog that shows promise for treating abnormal hormone secretion by cancerous tumors is Sandostatin, D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-OH. (Lamberts, 1986, 1987). Another analog, RC-121 (D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH₂), has been shown to be approximately 100 times more potent than somatostatin-1-14 in the inhibition of growth hormone release but <5 times more potent in the inhibition of gastric acid release (Cai et al., 1986, 1987).

Several years ago, somatostatin-1-14 was shown to display affinity for opioid receptors, despite the apparent lack of structural similarity to endogenous opioid peptides or opiate alkaloids (Terenius, 1976). Thus, interest within our research group focused on the development of opioid receptor-selective and enzymatically stable somatostatin analogs that could be used to characterize opioid receptors. Additionally, µ-selective antagonists that can reverse the unwanted side effects of µ-receptor-activated analgesia often seen with morphine and heroin, such as respiratory depression, convulsions, nausea, vomiting, decreased gastrointestinal motility, changes in mood, alterations in endocrine and autonomic nervous systems, tolerance and physical dependence, are needed (Pasternak, 1993). The µ-receptor has often been cited as playing a vital role in the expression of central opiate dependence, and the - and -receptors appear to play minor roles (Maldonado et al., 1992).

CTAP, CTOP and CTP are a series of conformationally constrained, penicillamine-containing octapeptides synthesized by Pelton et al. (1985, 1986). CTAP, CTOP and CTP are conformationally constrained peptides because they contain a disulfide linkage between the cysteine and the penicillamine, which provides a useful approach to improving selectivity of flexible peptides (Kazmierski et al., 1988). This synthesis approach eliminates the low-energy conformations of the peptide and provides insight into the topological features that are required for high-affinity binding to a specific opioid receptor subtype. Another advantage exists, in that there is an elimination of activity at the natural receptor for the peptide, i.e., somatostatin receptor. CTAP was shown to display greater antagonist potency and selectivity for µ-opioid receptors, compared with the classical µ-selective antagonist CTOP (Kramer, et al., 1989). CTAP is 1200-fold more selective for the µ- vs. -receptor binding sites and >4000-fold selective for µ-opioid receptor binding vs. somatostatin binding in rat brain (Pelton et al., 1986). CTAP has also been shown to reduce the morphine-tolerant state (antinociception) in mice and block the µ-receptor without causing severe withdrawal, as measured by withdrawal jumping in morphine-dependent mice (Wang et al., 1994). Furthermore, CTAP is a neutral antagonist, showing low intrinsic activity, and has considerable potential for the clinical treatment of narcotic overdose, particularly in addicts, where naloxone precipitates immediate withdrawal (Wang et al., 1994). In a model of acute morphine tolerance in mice, CTAP has been shown to block the effects of both morphine and naloxone, without any effect on the µ-receptor alone (Maldonado et al., 1992). This is advantageous, because naloxone has been shown to elicit agonist-like effects at high doses (Crain and Shen, 1992; Nestler, 1993). Based on the pharmacological profile, CTAP may be a promising and selective antagonist that can be used for both opiate overdose and addiction and as a pharmacological tool.

CNS penetration and biological stability are deciding factors for the clinical efficacy of CTAP. The aim of this study was to characterize the blood-to-CNS pharmacokinetics and biological stability of CTAP, because only central routes, i.e., i.c.v., have been examined for the related analog CTP (Shook et al., 1987). In the present study CNS entry of [³H]CTAP was compared with that of [³H]morphine, the classical µ-receptor agonist, and the vascular space marker [¹⁴C]inulin. CNS uptake and stability studies were also performed using a well-characterized in situ brain perfusion technique coupled to HPLC analysis (Takasato et al., 1984; Abbruscato et al., 1996; Williams et al., 1996). Comparisons were made between the brain and CSF uptake of [³H]CTAP, [¹⁴C]inulin and [³H]morphine after a 20-min perfusion. The existence of saturable uptake mechanisms controlling the CNS entry of [³H]CTAP was also investigated.

If CTAP is able to cross the BBB and/or blood-CSF barrier, then it may provide a useful means to clinically treat narcotic drug overdose and addiction without the unwanted precipitated withdrawal symptoms seen with the use of naloxone. CTAP could also be used as a pharmacological tool, with systemic administration, for further understanding of opioid neurobiology.

	Methods

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Supplies and chemicals. CTAP, [³H]CTAP (22.5 Ci/mmol) and [³H]morphine (50 mCi/mmol) were generous gifts from the National Institute on Drug Abuse. [¹⁴C]Inulin (2.7 mCi/g) was purchased from DuPont-New England Nuclear (Boston, MA).

In situ brain perfusion studies. The protocol described below was approved by the Institutional Animal Care and Use Committee at the University of Arizona. Adult Sprague-Dawley rats (250-300 g) were anesthetized with sodium pentobarbital (64.8 mg/kg) and heparinized (10,000 U/kg). The jugular veins were located and the common carotid arteries were cannulated using fine silicone tubing connected to a perfusion system, as previously described (Abbruscato et al., 1996).

Capillary depletion. Measurement of the vascular contribution to total brain uptake was performed using a capillary depletion step, as previously described (Triguero et al., 1990). Briefly, the brain was removed and the choroid plexuses were excised. The brain tissue (500 mg) was homogenized (Polytron homogenizer; Brinkmann Instruments, Westbury, NY) in 1.5 ml of physiological buffer [10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 141 mM NaCl, 4 mM KCl, 2.8 mM CaCl₂, 1 mM MgSO₄, 1 mM NaH₂PO₄, 10 mM D-glucose, pH 7.4] kept on ice. Two milliliters of ice-cold 26% dextran (molecular weight, 60,000) were then added and homogenization was performed again. Two aliquots of homogenate were taken and centrifuged at 5400 × g for 15 min in a microfuge (Beckman Instruments Inc.). The capillary-depleted supernatant was then separated from the vascular pellet. All of the homogenization procedures described above were performed within 2 min. The homogenate, supernatant and pellet were then aliquoted for radioactive counting (Beckman 5500 beta counter).

Expression of results. The amount of radioactivity in the whole brain, CSF, homogenate, supernatant and pellet was expressed as the percentage ratio of the tissue concentration (C_Tissue, in dpm per gram or dpm per milliliter) to the concentration in the perfusion fluid (C_Perf, in dpm per milliliter), expressed as R_Tissue (in milliliters per gram or milliliters per millilter). The unidirectional transfer constant (K_in) and the initial volume of distribution (V_i) were graphically determined from the multiple-time uptake data (2.5-20 min) using the following equation (Zlokovic et al., 1986): where C_Tissue(T) and C_Plasma(T) are radioactivities per unit weight of tissue and perfusion fluid, respectively, at time T. The above equation describes a straight line, where the slope is K_in (in milliliters per minute per gram) and the y-intercept is V_i (in milliliters per gram). Any brain-to-blood movement of the test compound can be observed as a departure from linearity of the experimental points. To determine blood-to-CSF transfer constants, a two compartment/single-time uptake analysis was used (Abbruscato et al., 1996; Williams et al., 1996). This can be performed by using the following equation: Blood-to-brain unidirectional transfer constants were also determined by single-time uptake analysis. The vascular space was corrected for by subtracting [¹⁴C]inulin (R_Brain) values from the test drug values at the same time point.

Extraction of radiolabeled peptide. Brain extractions were performed using a modified method of Erchegyi et al. (1991). Briefly, rats were perfused with [³H]CTAP as described previously. At the end of a 20-min perfusion period, the animal was perfused with mammalian Ringer solution for 2 min to remove any remaining [³H]CTAP from the cerebral vasculature. The animal was decapitated, and the brain was removed and immediately placed in 7.5 ml of ice-cold 10% TFA. Each sample was then homogenized (Polytron homogenizer) and centrifuged at 20,000 × g for 20 min. The supernatants were collected and an equal volume of ether was added. The ether phase was discarded and the remaining samples were lyophilized to dryness. The samples were then diluted to 500 µl with 10% acetonitrile and stored for HPLC analysis.

In vitro brain stability studies. Mouse brain homogenates were prepared by a modified method of Davis and Culling-Berglund (1985). The protein concentration was determined to be 6.8 mg/ml by the method of Lowry et al. (1951). Aliquots (180 µl) of resuspended, twice-washed, 15% rat brain homogenate were placed into 1.5-ml centrifuge tubes and, together with a buffer control, warmed to 37°C in a rolling water-bath incubator. At time 0, CTAP was added to each tube to achieve a final concentration of 100 µM and was incubated for 0, 30, 60, 120, 240 or 360 min. At the end of the set incubation period, enzyme activity was terminated by the addition of 200 µl of acetonitrile with 0.5% acetic acid, and the tubes were placed on ice. Each tube was then centrifuged at 3000 × g, and 300 µl of the supernatant was transferred to a clean 1.5-ml conical tube. An equal volume of water was added to reduce the final acetonitrile concentration to 25%, and the sample was stored for HPLC analysis.

HPLC analysis. Brain extractions of [³H]CTAP were analyzed using a Series 410 HPLC gradient system (Perkin-Elmer, Norwalk, CT). Samples were eluted from an Inertsil ODS-2 column (4.6 × 150 mm; Metachem Technologies Inc., Torrance, CA) with a curvilinear gradient of 0.1% TFA in acetonitrile (20-50%) vs. 0.1% aqueous TFA over 30 min, at 1.5 ml/min; the column temperature was maintained at 37°C. After separation on the HPLC column, the outflow was routed to an on-line A200 Flo-One radioactivity detector equipped with a 2.5-ml flow cell (Packard Radiomatic Instruments and Chemicals, Tampa Bay, FL).

Protein binding studies. The amount of [³H]CTAP binding to either bovine albumin in the perfusion medium or proteins in rat serum was determined by ultrafiltration centrifugal dialysis (Paulus, 1969). Rat serum was obtained by harvesting blood from Sprague-Dawley rats and allowing the blood to clot for 30 min on ice and 30 min at room temperature. The whole blood was then centrifuged (Sorvall RC2-B centrifuge; DuPont Medical Products, Wilmington, DE) at 20,000 × g for 20 min, to produce a serum supernatant. [³H]CTAP was dissolved in either perfusion medium or rat serum warmed to 37°C and was ultrafiltered using a Centrifree 228 micropartition device (Amicon, Beverly, MA). The total concentration (T) of [³H]CTAP introduced into the system and found in the ultrafiltrate (F) was determined by liquid scintillation counting (Beckman 5500). The percentage of [³H]CTAP bound to either albumin in the perfusion medium or proteins in the rat serum was expressed as [(T  F)/T] × 100. To verify that bovine albumin was not found in the ultrafiltrate, the protein concentration was determined by the method of Lowry et al. (1951).

Data analysis. All experiments were expressed as means ± S.E.M. Analysis of variance was used to compare the slopes, determined by least-squares linear regression analysis of the multiple-time uptake data. Student's t test was used for comparison of the two means, and statistical significance was taken as P < .01 or P < .05.

	Results

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In situ brain perfusion experiments. Multiple-time analysis was performed for both [³H]CTAP and [¹⁴C]inulin in the brain and CSF. Uptake was expressed as R_Tissue, which is the percentage ratio of tissue to plasma radioactivities (milliliters per gram or milliliters per milliliter). As shown in figure 1, the uptake of [³H]CTAP and [¹⁴C]inulin into the brain and CSF was linear with time. These results indicate that the brain uptake of [³H]CTAP was statistically greater than that of the vascular space marker [¹⁴C]inulin (P < .05). After consideration of the vascular space, the brain and CSF uptake values of [³H]CTAP were not statistically different.

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Multiple-time uptake plots of [3H]CTAP () and [14C]inulin () uptake into brain (A) and CSF (B) of in situ perfused rats. Uptake is expressed as the percentage ratio of tissue to plasma radioactivities (milliliters per gram or milliliters per milliliter). Each point represents the mean ± S.E.M. (n = 3-7 animals for each point). The brain uptake of [3H]CTAP was statistically greater than that of the vascular space marker [14C]inulin (P < .05). However, after consideration of vascular space, the brain and CSF uptake values for [3H]CTAP were not statistically different.

Extraction of [³H]CTAP. After a 20-min vascular brain perfusion, the majority (62.8%) of the [³H]CTAP coeluted with the radioactive standard (fig. 2). Five metabolites that comprised 37.2% of the total area counts were also observed.

View larger version (10K): [in this window] [in a new window]   Fig. 2.   HPLC Flo-One radioactive detector chromatogram of a TFA extract of [3H]CTAP from the brain after a 20-min vascular perfusion. The majority of the sample coeluted with purified radioactive standard.

In vitro brain and serum stability studies with CTAP. The percent recovery of intact CTAP incubated for 240 min in 15% twice-washed brain membranes or 100% plasma was determined using HPLC analysis. The T_1/2 of CTAP was >500 min in both the brain and serum, as determined by HPLC analysis (fig. 3).

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Percent recovery of intact CTAP in rat brain and serum over a 240-min time course. A half-time of disappearance of >500 min in both brain and serum was determined by HPLC analysis.

Inhibition experiments with 100 µM CTAP. Entry into the brain and CSF was not statistically different after a 20-min brain perfusion with [³H]CTAP in the presence and absence of 100 µM CTAP (fig. 4). Thus, the entry into the brain of [³H]CTAP was not inhibited by the addition of unlabeled CTAP to the perfusion medium.

View larger version (29K): [in this window] [in a new window]   Fig. 4.   Uptake expressed as a percentage ratio of tissue to perfusate radioactivities (RTissue, in milliliters per gram or milliliters per milliliter). Perfusion time was 20 min and values are the mean ± S.E.M. for three animals. The uptake of [3H]CTAP, in the absence and presence of 100 µM unlabeled CTAP, into the brain and CSF was found not to be statistically different.

Protein binding studies with [³H]CTAP. [³H]CTAP was found to be bound to protein in both the perfusion medium (68.2%) and rat serum (84.2%) (table 2). No protein was detected in the ultrafiltrate with the Lowry protein assay.

Capillary depletion analysis. The vascular component of the brain uptake of [³H]CTAP and [³H]morphine (44% and 32%, respectively) contributed extensively to overall brain uptake (fig. 5). The homogenate and the supernatant were not statistically different, in both cases. The counts detected in the pellet were found to be significantly smaller than counts detected in the homogenate for both [³H]CTAP and [³H]morphine (P < .05).

View larger version (26K): [in this window] [in a new window]   Fig. 5.   RBrain percentage, representing the ratio of homogenate, supernatant or pellet to plasma radioactivities. Supernatant represents brain homogenate depleted of the cerebral capillary endothelium. Perfusion time was 20 min. Values are the mean ± S.E.M. of three or four experiments each. * Pellet contained significantly smaller counts than homogenate for both [3H]CTAP and [3H]morphine (P < .05).

R_Tissue and octanol/saline partition coefficients determined for [³H]CTAP, [³H]morphine and [¹⁴C]inulin. Table 3 shows that a significantly greater amount of [³H]CTAP and [³H]morphine entered the brain, compared with [¹⁴C]inulin, after a 20-min vascular brain perfusion (P < .01). In addition, a significantly greater amount of [³H]morphine entered the CSF, compared with [¹⁴C]inulin (P < .05). Octanol/saline partition coefficients for [³H]CTAP and [³H]morphine were higher and statistically different, compared with that for [¹⁴C]inulin (P < .01). Furthermore, the R_Tissue values correlated well (r = 0.946) with the octanol/saline partition coefficients for [³H]CTAP, [³H]morphine and [¹⁴C]inulin.

	Discussion

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The present studies have led to two major findings. First, [³H]CTAP can enter the brain by crossing the BBB; second, [³H]CTAP is stable in the brain and serum of rats but remains extensively bound to albumin in the perfusion medium.

[¹⁴C]Inulin was used as an extravascular space marker. Our data demonstrate that very little [¹⁴C]inulin actually enters the brain and CSF. The unidirectional transfer constants for [¹⁴C]inulin transfer into the brain and CSF were 0.27 ± 0.03 and 0.88 ± 0.33 µl/min/g, respectively. These values are quite low and compare well with previously published values for [¹⁴C]sucrose (0.32 ± 0.02 and 0.07 ± 0.02 µl/min/g into the brain and CSF, respectively) (Abbruscato et al., 1996). These data confirm that the overall physiology of the BBB remains intact during the in situ brain perfusion experiments, because these high-molecular weight compounds ([¹⁴C]inulin molecular weight, 5000-5500; [¹⁴C]sucrose molecular weight, 342) were not detected at high levels in the CNS.

The data presented show that [³H]CTAP can enter the CNS. The unidirectional transfer constant of [³H]CTAP into the brain and CSF was 5.96- and 2.43-fold higher than that calculated for [¹⁴C]inulin. These data also show that there is a greater amount of radioactivity detected in the brain and/or CSF at all time points for [³H]CTAP, in comparison with [¹⁴C]inulin, and that [³H]CTAP enters the CNS predominantly through the BBB, whereas the blood-CSF barrier plays a minor role. This can be explained by the fact that the CSF is more likely to act as a "sink" to the brain than the brain is to act as a sink to the CSF (Davson et al., 1961) and that the surface area of the choroid plexus is approximately 5000 times smaller than the surface area of the cerebral capillary endothelium (Bradbury, 1979). The small amount of [³H]CTAP detected in the CSF is most likely due to the diffusion of drug from the stagnant brain extracellular fluid to the rapidly flowing CSF environment.

The measurement of intact [³H]CTAP in the brain after a 20-min in situ brain perfusion ensured that we were measuring intact [³H]CTAP in the brain and not just free tritium due to water exchange. HPLC verification also allowed for the monitoring of potential peptide metabolism due to peptidases that may be expressed in the brain or at the blood-brain interface (Brownson et al., 1994). [³H]CTAP remained predominantly intact (62.8%) in the brain after a 20-min rat brain perfusion. The HPLC verification of detectable amounts of [³H]CTAP measured in the brain ensures that this µ-selective antagonist can enter into the brain intact and be available to elicit a pharmacological response. Although other metabolites produced by brain perfusion were not identified, they may represent enzymatic metabolism either at the blood-brain interface or in the CNS after passage. The large amounts of intact [³H]CTAP detected in the brain after a 20-min brain perfusion may explain why CTAP is such a potent antagonist. This peptide may actually enter into the brain via diffusion and then become trapped in the brain compartment.

Other important experiments were performed to ensure the biological stability of this drug. In vitro stability studies were conducted in serum and brain homogenate of rats. [³H]CTAP was shown to be stable in the blood and serum of rats (T_1/2 > 500 min), showing that the structure of this peptide offers enzymatic resistance to blood-borne peptidases. The biological stability of CTAP is probably due to the penicillamine-cysteine disulfide linkage, which allows the compound to become conformationally constrained and biologically active. The metabolic half-lives were quite long, compared with that of another octapeptide analog of somatostatin, Sandostatin. Sandostatin has numerous clinical uses in the treatment of endocrine disturbances, especially those resulting from inappropriate hormone secretion by tumors. The pharmacokinetic half-life of Sandostatin was determined to be 113 min after s.c. administration (Lamberts, 1986, 1987). These experiments confirm that CTAP can overcome a problem that impedes the clinical use of naturally occurring peptides, i.e., a short metabolic half-life.

Another component of CNS biodistribution that needs to be measured when evaluating the blood-to-CNS pharmacokinetics is the ability of a given test solute to bind to serum proteins. [³H]CTAP was found to be extensively protein bound to albumin in the perfusion medium (68.2%) and to rat serum proteins (84.2%) (table 2). A protein-binding component has also been observed with other analogs of somatostatin (Banks et al., 1990). This suggests that actual CNS uptake values may be higher without this protein binding component being taken into consideration. Extensive binding of [³H]CTAP to albumin in the perfusion medium and rat serum proteins may actually be protecting CTAP from enzymatic degradation by systemic peptidases.

It is apparent that [³H]CTAP can enter into the CNS, based on in situ brain perfusion experiments coupled to HPLC analysis. The next step was to determine whether the mechanism of entry was by means of passive diffusion or saturable transport. In situ brain perfusion experiments were performed with [³H]CTAP in the presence of 100 µM CTAP. Entry into the brain and CSF was not inhibited by the addition of unlabeled CTAP (100 µM) to the perfusion medium. This suggests that passage into the CNS was most likely directed through diffusion across the membranes that comprise the BBB, rather than by saturable transport. These results concur with the findings of Banks et al. (1990), showing that somatostatin analogs can cross the murine BBB by diffusion. This does not rule out the possibility of a saturable transport mechanism that may facilitate CTAP transport from the brain back into the blood, which has been described previously as PTS-5 and which is involved in the brain-to-blood transport of somatostatin and certain other analogs (Banks and Kastin, 1992). This brain-to-blood transport seems less likely to occur with CTAP, because there were considerable amounts of intact [³H]CTAP detected in the brain after a 20-min vascular brain perfusion.

Comparisons were made between CTAP and the classical, clinically efficacious, opioid agonist morphine, in reference to the amount of intact compound that crossed either the BBB or blood-CSF barrier and the contribution of binding to the endothelial space. The vascular component contributes significantly to the uptake of both [³H]CTAP and [³H]morphine (44% and 32%, respectively). [³H]CTAP and [³H]morphine may be sequestered in the endothelial cell component due to either high lipophilicity and/or binding to brain microvessels. Previously it has been shown that brain microvessels rapidly sequester and degrade somatostatin analogs (Pardridge et al., 1985). This may represent one mechanism for the rapid inactivation of brain-derived neuropeptides after neurosecretion. A potential reason for the high concentration of [³H]CTAP detected in the microvasculature pellet may involve the binding of [³H]CTAP to a receptor on the cell membrane of the endothelial cells that comprise the vessel walls, to achieve enzymatic degradation. High levels of peptidases are known to be expressed at the membranes of brain microvessel endothelial cells (Brownson et al., 1994).

A greater amount of [³H]morphine entered both the brain and CSF after a 20-min brain perfusion, compared with [¹⁴C]inulin (P < .01 and P < .05, respectively) (table 3). The increased CNS penetration by [³H]morphine, compared with [³H]CTAP, is likely due to increased lipophilicity, as shown by the high octanol/saline partition coefficient. In addition, the R_Tissue values correlate well with octanol/saline partition coefficients for [³H]CTAP, [³H]morphine and [¹⁴C]inulin (r = 0.946 for brain and r = 0.926 for CSF). Thus, lipophilicity may be a determining factor for CNS entry of these drugs. This also confirms the reliability of using our in situ brain perfusion technique to mimic or predict in vivo situations, such as compounds attempting to traverse the BBB and/or blood-CSF barrier.

This work supports the hypothesis that the µ-selective somatostatin analog CTAP can cross the BBB at therapeutic levels. The actual amount of CTAP that crosses both the BBB and blood-CSF barrier is quantitatively comparable to that of the efficacious, µ-selective agonist morphine. It is surprising that a compound with the clinical efficacy of morphine does not enter the brain at high levels. The absolute percentage of injected dose of morphine that enters the brain has been calculated at 0.02%/g of brain tissue (Banks and Kastin, 1994). The present study shows that CTAP may play an important clinical role in treating narcotic addiction, dependence or overdose. Because CTAP has excellent biological stability and blood-CNS penetration, it may be an improvement over the classical opioid antagonist naloxone for treating opioid crisis. Naloxone has a relatively short duration of action and must be administered repeatedly or by infusion. Also, one must be precise in titrating the dose, for fear of precipitating severe withdrawal (Goodman and Gilman, 1996). CTAP may therefore provide improved antagonism at the µ-receptor, without intrinsic activity, and a longer duration of action, with less severe withdrawal.