0022-3565/97/2801-0402$03.

00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 280, No. 1
Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A.
JPET 280:402–409, 1997

Blood-Brain Barrier Permeability and Bioavailability of a Highly

Potent and m-Selective Opioid Receptor Antagonist, CTAP:
Comparison with Morphine1

THOMAS J. ABBRUSCATO, SARAH A. THOMAS, VICTOR J. HRUBY and THOMAS P. DAVIS

Departments of Pharmacology (T.J.A., S.A.W., T.P.D.) and Chemistry (V.J.H.), University of Arizona, College of Medicine, Tucson, Arizona
Accepted for publication September 3, 1996

Downloaded from jpet.aspetjournals.org at Univ Of Calif San Francisco on March 20, 2012
ABSTRACT
D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) is a cyclic, of this peptide offers enzymatic resistance. Additionally,
penicillamine-containing octapeptide that is structurally similar [3H]CTAP was found to be extensively protein-bound to albu-
to somatostatin and displays greater antagonist potency and min in the perfusion medium (68.2%) and to proteins in rat
selectivity for m-opioid receptors, compared with the classical serum (84.2%). Entry into the brain and CSF was not inhibited
m-selective antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr- by the addition of unlabeled CTAP to the perfusion medium,
NH2. The aim of this study was to determine whether CTAP can suggesting that passage into the CNS is most likely through
enter the central nervous system (CNS) by crossing either the diffusion across the membranes that comprise the blood-brain
blood-brain barrier or the blood-cerebrospinal fluid barrier barrier, rather than by saturable transport. Also, greater
(CSF) and to characterize the mechanism of CNS entry. CNS amounts of [3H]morphine entered both the brain and CSF after
entry of [3H]CTAP was compared with that of the vascular a 20-min brain perfusion, compared with [3H]CTAP. The in-
space marker [14C]inulin and the m-agonist [3H]morphine. By creased CNS penetration observed for [3H]morphine, com-
using an in situ brain perfusion technique coupled to high- pared with [3H]CTAP, is likely due to the increased lipophilicity
performance liquid chromatographic analysis, greater amounts of morphine, as shown by its higher octanol/saline partition
of radioactivity were detected in the brain or CSF at most time coefficient. Based on the pharmacokinetic profile, CTAP may
points for [3H]CTAP, compared with [14C]inulin. [3H]CTAP was be a promising m-selective antagonist that can be used as a
found to remain predominantly intact in the brain after a 20-min treatment for opiate overdose or addiction and also as a phar-
rat brain perfusion (62.8%). CTAP was also stable in the blood macological tool to further understand opioid neurobiology.
and serum of rats (T1/2 . 500 min), showing that the structure

Since the discovery of multiple types of opioid receptors et al., 1991, 1992, 1993; Brownson et al., 1994; Williams et al.,
(Martin et al., 1976; Lord et al., 1977), attempts have been 1996) has been shown to be both enzymatically stable and
made to elucidate the physiological function of these recep- able to enter the CNS through a saturable mechanism at the
tors (m, k and d). The development of potent, specific antag- BBB. Opioid antagonists that are commercially available
onists and agonists is essential for clarification of the multi- have historically been modeled from alkaloid opioid agonists,
ple biological effects thought to be mediated by each receptor. i.e., naloxone and naltrexone, both of which are not receptor
Several receptor agonists have been developed that are se- selective. This paper describes the blood-to-CNS pharmaco-
lective for the various receptor subtypes, for example, kinetics of a cyclic peptidergic analog of somatostatin, CTAP,
[D-Pen2,D-Pen5]-enkephalin (d) (Mosberg et al., 1983), which has been shown to be extremely potent and selective
[D-Ala2,N-MePhe4,Gly-ol5]-enkephalin (m) (Handa et al.,
for m-opioid receptors (Kramer et al., 1989).
1981) and U50,488H (k) (VonVoigtlander et al., 1983). These
Somatostatin is a 28-amino acid, regulatory peptide hor-
agonists have been extensively characterized pharmacologi-
mone that has numerous effects within the CNS and periph-
cally, and a few have been evaluated for their ability to enter
eral nervous system, such as controlling growth hormone,
the brain. For example, [D-Pen2,D-Pen5]-enkephalin (Weber
insulin and glucagon release. It is also postulated that, after
neurosecretion of somatostatin, there is a metabolic interac-
Received for publication June 24, 1996.
1
This work was supported by National Institute on Drug Abuse Grant
tion that occurs with brain capillary endothelial cells
DA06284. (Pardridge et al., 1985).

ABBREVIATIONS: BBB, blood-brain barrier; CNS, central nervous system; CSF, cerebrospinal fluid; CTAP, d-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-
Thr-NH2; CTOP, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2; CTP, D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2; HPLC, high-performance liquid
chromatography; TFA, trifluoroacetic acid.

402
1997 Blood/CNS Pharmacokinetics of CTAP 403
Several analogs of somatostatin have been developed that 1992). This is advantageous, because naloxone has been
may provide clinical intervention for the treatment of endo- shown to elicit agonist-like effects at high doses (Crain and
crine disturbances such as acromegaly, diabetes mellitus Shen, 1992; Nestler, 1993). Based on the pharmacological
(Karashima and Schally, 1988) and peptic ulcer disease (Las- profile, CTAP may be a promising and selective antagonist
zlo et al., 1989). Cancer treatment has been shown to be that can be used for both opiate overdose and addiction and
another important application for the use of somatostatin as a pharmacological tool.
analogs (Schally et al., 1986). A recently developed soma- CNS penetration and biological stability are deciding fac-
tostatin analog that shows promise for treating abnormal tors for the clinical efficacy of CTAP. The aim of this study
hormone secretion by cancerous tumors is Sandostatin, D- was to characterize the blood-to-CNS pharmacokinetics and
Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-OH. (Lamberts, 1986, biological stability of CTAP, because only central routes, i.e.,
1987). Another analog, RC-121 (D-Phe-Cys-Tyr-D-Trp-Lys- i.c.v., have been examined for the related analog CTP (Shook
Val-Cys-Thr-NH2), has been shown to be approximately 100 et al., 1987). In the present study CNS entry of [3H]CTAP
times more potent than somatostatin-1-14 in the inhibition of was compared with that of [3H]morphine, the classical m-re-
growth hormone release but ,5 times more potent in the ceptor agonist, and the vascular space marker [14C]inulin.
inhibition of gastric acid release (Cai et al., 1986, 1987). CNS uptake and stability studies were also performed using
Several years ago, somatostatin-1-14 was shown to display a well-characterized in situ brain perfusion technique cou-

Downloaded from jpet.aspetjournals.org at Univ Of Calif San Francisco on March 20, 2012
affinity for opioid receptors, despite the apparent lack of pled to HPLC analysis (Takasato et al., 1984; Abbruscato et
structural similarity to endogenous opioid peptides or opiate al., 1996; Williams et al., 1996). Comparisons were made
alkaloids (Terenius, 1976). Thus, interest within our re- between the brain and CSF uptake of [3H]CTAP, [14C]inulin
search group focused on the development of opioid receptor- and [3H]morphine after a 20-min perfusion. The existence of
selective and enzymatically stable somatostatin analogs that saturable uptake mechanisms controlling the CNS entry of
could be used to characterize opioid receptors. Additionally, [3H]CTAP was also investigated.
m-selective antagonists that can reverse the unwanted side If CTAP is able to cross the BBB and/or blood-CSF barrier,
effects of m-receptor-activated analgesia often seen with mor- then it may provide a useful means to clinically treat narcotic
phine and heroin, such as respiratory depression, convul- drug overdose and addiction without the unwanted precipi-
sions, nausea, vomiting, decreased gastrointestinal motility, tated withdrawal symptoms seen with the use of naloxone.
changes in mood, alterations in endocrine and autonomic CTAP could also be used as a pharmacological tool, with
nervous systems, tolerance and physical dependence, are systemic administration, for further understanding of opioid
needed (Pasternak, 1993). The m-receptor has often been neurobiology.
cited as playing a vital role in the expression of central opiate
dependence, and the d- and k-receptors appear to play minor
roles (Maldonado et al., 1992). Methods
CTAP, CTOP and CTP are a series of conformationally
constrained, penicillamine-containing octapeptides synthe- Supplies and chemicals. CTAP, [3H]CTAP (22.5 Ci/mmol) and
sized by Pelton et al. (1985, 1986). CTAP, CTOP and CTP are [3H]morphine (50 mCi/mmol) were generous gifts from the National
Institute on Drug Abuse. [14C]Inulin (2.7 mCi/g) was purchased from
conformationally constrained peptides because they contain
DuPont-New England Nuclear (Boston, MA).
a disulfide linkage between the cysteine and the penicilla-
In situ brain perfusion studies. The protocol described below
mine, which provides a useful approach to improving selec- was approved by the Institutional Animal Care and Use Committee
tivity of flexible peptides (Kazmierski et al., 1988). This syn- at the University of Arizona. Adult Sprague-Dawley rats (250–300 g)
thesis approach eliminates the low-energy conformations of were anesthetized with sodium pentobarbital (64.8 mg/kg) and hep-
the peptide and provides insight into the topological features arinized (10,000 U/kg). The jugular veins were located and the com-
that are required for high-affinity binding to a specific opioid mon carotid arteries were cannulated using fine silicone tubing
receptor subtype. Another advantage exists, in that there is connected to a perfusion system, as previously described (Abbruscato
an elimination of activity at the natural receptor for the et al., 1996).
peptide, i.e., somatostatin receptor. CTAP was shown to dis- Perfusion was performed with a thoroughly oxygenated (pO2 5
642–727 mm Hg) mammalian Ringer solution (37°C). After the de-
play greater antagonist potency and selectivity for m-opioid
sired perfusion pressure and rate were achieved (approximately 100
receptors, compared with the classical m-selective antagonist
mm Hg and 3.1 ml/min, respectively), the right jugular vein was cut
CTOP (Kramer, et al., 1989). CTAP is 1200-fold more selec- and allowed to drain. The contralateral carotid was cannulated and
tive for the m- vs. d-receptor binding sites and .4000-fold perfused in a manner similar to that described above. [3H]CTAP
selective for m-opioid receptor binding vs. somatostatin bind- (molecular weight, 1107) in the presence or absence of 100 mM CTAP,
ing in rat brain (Pelton et al., 1986). CTAP has also been [3H]morphine (molecular weight, 758.8) or [14C]inulin (molecular
shown to reduce the morphine-tolerant state (antinocicep- weight, 5000–5500) was infused, using a slow-drive syringe pump,
tion) in mice and block the m-receptor without causing severe into the inflowing mammalian Ringer solution. After the set perfu-
withdrawal, as measured by withdrawal jumping in mor- sion time (2.5, 10, 15 or 20 min), a cisterna magna CSF sample was
phine-dependent mice (Wang et al., 1994). Furthermore, taken with a glass cannula. The animal was then decapitated and
the brain was removed. The choroid plexuses were excised and a
CTAP is a neutral antagonist, showing low intrinsic activity,
portion of the brain was homogenized in 26% dextran and capillary
and has considerable potential for the clinical treatment of
depletion buffer. The perfusion outflow was collected from the ca-
narcotic overdose, particularly in addicts, where naloxone rotid cannulae at the end of the time, to serve as a reference. The
precipitates immediate withdrawal (Wang et al., 1994). In a brain and CSF samples were then weighed and prepared for liquid
model of acute morphine tolerance in mice, CTAP has been scintillation counting in a model LS 5000 TD b-counter (43% effi-
shown to block the effects of both morphine and naloxone, ciency for 3H and 93% efficiency for 14C; Beckman Instruments,
without any effect on the m-receptor alone (Maldonado et al., Fullerton CA).
404 Abbruscato et al. Vol. 280

Capillary depletion. Measurement of the vascular contribution tube to achieve a final concentration of 100 mM and was incubated
to total brain uptake was performed using a capillary depletion step, for 0, 30, 60, 120, 240 or 360 min. At the end of the set incubation
as previously described (Triguero et al., 1990). Briefly, the brain was period, enzyme activity was terminated by the addition of 200 ml of
removed and the choroid plexuses were excised. The brain tissue acetonitrile with 0.5% acetic acid, and the tubes were placed on ice.
(500 mg) was homogenized (Polytron homogenizer; Brinkmann In- Each tube was then centrifuged at 3000 3 g, and 300 ml of the
struments, Westbury, NY) in 1.5 ml of physiological buffer [10 mM supernatant was transferred to a clean 1.5-ml conical tube. An equal
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 141 mM NaCl, 4 volume of water was added to reduce the final acetonitrile concen-
mM KCl, 2.8 mM CaCl2, 1 mM MgSO4, 1 mM NaH2PO4, 10 mM tration to 25%, and the sample was stored for HPLC analysis.
D-glucose, pH 7.4] kept on ice. Two milliliters of ice-cold 26% dextran HPLC analysis. Brain extractions of [3H]CTAP were analyzed
(molecular weight, 60,000) were then added and homogenization was using a Series 410 HPLC gradient system (Perkin-Elmer, Norwalk,
performed again. Two aliquots of homogenate were taken and cen- CT). Samples were eluted from an Inertsil ODS-2 column (4.6 3 150
trifuged at 5400 3 g for 15 min in a microfuge (Beckman Instru- mm; Metachem Technologies Inc., Torrance, CA) with a curvilinear
ments Inc.). The capillary-depleted supernatant was then separated gradient of 0.1% TFA in acetonitrile (20–50%) vs. 0.1% aqueous TFA
from the vascular pellet. All of the homogenization procedures de- over 30 min, at 1.5 ml/min; the column temperature was maintained
scribed above were performed within 2 min. The homogenate, super- at 37°C. After separation on the HPLC column, the outflow was
natant and pellet were then aliquoted for radioactive counting (Beck- routed to an on-line A200 Flo-One radioactivity detector equipped
man 5500 beta counter). with a 2.5-ml flow cell (Packard Radiomatic Instruments and Chem-

Downloaded from jpet.aspetjournals.org at Univ Of Calif San Francisco on March 20, 2012
Expression of results. The amount of radioactivity in the whole icals, Tampa Bay, FL).
brain, CSF, homogenate, supernatant and pellet was expressed as Peptide stability studies in rat brain homogenate and serum were
the percentage ratio of the tissue concentration (CTissue, in dpm per analyzed using a reverse-phase Perkin Elmer 250 HPLC gradient
gram or dpm per milliliter) to the concentration in the perfusion fluid system, a model 710B WISP autoinjector (Waters Associates), a
(CPerf, in dpm per milliliter), expressed as RTissue (in milliliters per Perkin Elmer LC-15 UV absorbance detector, a Hewlett-Packard
gram or milliliters per millilter). model 3396A integrator and a Vydac 218TP54 column (4.6 3 250
mm). Samples were eluted using a curvilinear gradient of acetoni-
RTissue~%! 5 CTissue/CPerf 3 100 trile (20–50%) vs. 0.1% NaH2PO4, pH 7.4, over 30 min. The flow rate
was 1.5 ml/min, and the column temperature was maintained at
The unidirectional transfer constant (Kin) and the initial volume of 37°C.
distribution (Vi) were graphically determined from the multiple-time Protein binding studies. The amount of [3H]CTAP binding to
uptake data (2.5–20 min) using the following equation (Zlokovic et either bovine albumin in the perfusion medium or proteins in rat
al., 1986): serum was determined by ultrafiltration centrifugal dialysis (Pau-
lus, 1969). Rat serum was obtained by harvesting blood from
CTissue~T!/CPerf~T! 5 ~Kin!~T! 1 Vi
Sprague-Dawley rats and allowing the blood to clot for 30 min on ice
where CTissue(T) and CPlasma(T) are radioactivities per unit weight of and 30 min at room temperature. The whole blood was then centri-
tissue and perfusion fluid, respectively, at time T. The above equa- fuged (Sorvall RC2-B centrifuge; DuPont Medical Products, Wil-
tion describes a straight line, where the slope is Kin (in milliliters per mington, DE) at 20,000 3 g for 20 min, to produce a serum super-
minute per gram) and the y-intercept is Vi (in milliliters per gram). natant. [3H]CTAP was dissolved in either perfusion medium or rat
Any brain-to-blood movement of the test compound can be observed serum warmed to 37°C and was ultrafiltered using a Centrifree 228
as a departure from linearity of the experimental points. To deter- micropartition device (Amicon, Beverly, MA). The total concentra-
mine blood-to-CSF transfer constants, a two compartment/single- tion (T) of [3H]CTAP introduced into the system and found in the
time uptake analysis was used (Abbruscato et al., 1996; Williams et ultrafiltrate (F) was determined by liquid scintillation counting
al., 1996). This can be performed by using the following equation: (Beckman 5500). The percentage of [3H]CTAP bound to either albu-
min in the perfusion medium or proteins in the rat serum was
Kin 5 RCSF/T expressed as [(T 2 F)/T] 3 100. To verify that bovine albumin was
not found in the ultrafiltrate, the protein concentration was deter-
Blood-to-brain unidirectional transfer constants were also deter- mined by the method of Lowry et al. (1951).
mined by single-time uptake analysis. The vascular space was cor- Data analysis. All experiments were expressed as means 6
rected for by subtracting [14C]inulin (RBrain) values from the test S.E.M. Analysis of variance was used to compare the slopes, deter-
drug values at the same time point. mined by least-squares linear regression analysis of the multiple-
Extraction of radiolabeled peptide. Brain extractions were time uptake data. Student’s t test was used for comparison of the two
performed using a modified method of Erchegyi et al. (1991). Briefly, means, and statistical significance was taken as P , .01 or P , .05.
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 Results
[3H]CTAP from the cerebral vasculature. The animal was decapi-
tated, and the brain was removed and immediately placed in 7.5 ml In situ brain perfusion experiments. Multiple-time
of ice-cold 10% TFA. Each sample was then homogenized (Polytron analysis was performed for both [3H]CTAP and [14C]inulin in
homogenizer) and centrifuged at 20,000 3 g for 20 min. The super- the brain and CSF. Uptake was expressed as RTissue, which is
natants were collected and an equal volume of ether was added. The the percentage ratio of tissue to plasma radioactivities (mil-
ether phase was discarded and the remaining samples were lyophi- liliters per gram or milliliters per milliliter). As shown in
lized to dryness. The samples were then diluted to 500 ml with 10% figure 1, the uptake of [3H]CTAP and [14C]inulin into the
acetonitrile and stored for HPLC analysis. brain and CSF was linear with time. These results indicate
In vitro brain stability studies. Mouse brain homogenates were
that the brain uptake of [3H]CTAP was statistically greater
prepared by a modified method of Davis and Culling-Berglund
(1985). The protein concentration was determined to be 6.8 mg/ml by
than that of the vascular space marker [14C]inulin (P , .05).
the method of Lowry et al. (1951). Aliquots (180 ml) of resuspended, After consideration of the vascular space, the brain and CSF
twice-washed, 15% rat brain homogenate were placed into 1.5-ml uptake values of [3H]CTAP were not statistically different.
centrifuge tubes and, together with a buffer control, warmed to 37°C Table 1 shows that the unidirectional transfer constant for
in a rolling water-bath incubator. At time 0, CTAP was added to each [3H]CTAP into the brain and CSF was 5.96- and 2.43-fold
1997 Blood/CNS Pharmacokinetics of CTAP 405
TABLE 1
Calculated unidirectional transfer constants (K) and initial
volumes of distribution (V) for [H]CTAP and [C]inulin.
Kin and Vi values were determined as the slope and ordinate intercept, respec-
tively, of the computed regression lines.
Brain
Compound CSF, Kin
Vi Kin
ml/100 g ml/min/g ml/min/g
[14C]Inulin 1.18 6 0.45 0.27 6 0.03a 0.88 6 0.33b
[3H]CTAP 1.91 6 0.09 1.61 6 0.07a 2.14 6 0.82b
a
Values (mean 6 S.E.M.) were determined from the computer-generated lines
of regression (brain value is corrected for vascular space).
b
Values (mean 6 S.E.M.) were determined from a single-time uptake analysis
at a perfusion time of 20 min.

Inhibition experiments with 100 mM CTAP. Entry into

Downloaded from jpet.aspetjournals.org at Univ Of Calif San Francisco on March 20, 2012
the brain and CSF was not statistically different after a
20-min brain perfusion with [3H]CTAP in the presence and
absence of 100 mM CTAP (fig. 4). Thus, the entry into the
brain of [3H]CTAP was not inhibited by the addition of un-
labeled CTAP to the perfusion medium.
Protein binding studies with [3H]CTAP. [3H]CTAP
was found to be bound to protein in both the perfusion me-
dium (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).
RTissue and octanol/saline partition coefficients de-
termined 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]mor-
phine entered the CSF, compared with [14C]inulin (P , .05).
Octanol/saline partition coefficients for [3H]CTAP and
Fig. 1. Multiple-time uptake plots of [3H]CTAP (f) and [14C]inulin (å) [3H]morphine were higher and statistically different, com-
uptake into brain (A) and CSF (B) of in situ perfused rats. Uptake is
pared with that for [14C]inulin (P , .01). Furthermore, the
expressed as the percentage ratio of tissue to plasma radioactivities
(milliliters per gram or milliliters per milliliter). Each point represents the RTissue values correlated well (r 5 0.946) with the octanol/
mean 6 S.E.M. (n 5 3–7 animals for each point). The brain uptake of saline partition coefficients for [3H]CTAP, [3H]morphine and
[3H]CTAP was statistically greater than that of the vascular space [14C]inulin.
marker [14C]inulin (P , .05). However, after consideration of vascular
space, the brain and CSF uptake values for [3H]CTAP were not statis-
tically different. Discussion
14
higher than that calculated for [ C]inulin. Also, the initial The present studies have led to two major findings. First,
volume of distribution into the brain for [3H]CTAP was 1.62- [3H]CTAP can enter the brain by crossing the BBB; second,
fold higher than that determined for [14C]inulin. [3H]CTAP is stable in the brain and serum of rats but re-
Extraction of [3H]CTAP. After a 20-min vascular brain mains extensively bound to albumin in the perfusion me-
perfusion, the majority (62.8%) of the [3H]CTAP coeluted dium.
with the radioactive standard (fig. 2). Five metabolites that [14C]Inulin was used as an extravascular space marker.
comprised 37.2% of the total area counts were also observed. Our data demonstrate that very little [14C]inulin actually
In vitro brain and serum stability studies with enters the brain and CSF. The unidirectional transfer con-
CTAP. The percent recovery of intact CTAP incubated for stants for [14C]inulin transfer into the brain and CSF were
240 min in 15% twice-washed brain membranes or 100% 0.27 6 0.03 and 0.88 6 0.33 ml/min/g, respectively. These
plasma was determined using HPLC analysis. The T1/2 of values are quite low and compare well with previously pub-
CTAP was .500 min in both the brain and serum, as deter- lished values for [14C]sucrose (0.32 6 0.02 and 0.07 6 0.02
mined by HPLC analysis (fig. 3). ml/min/g into the brain and CSF, respectively) (Abbruscato et
406 Abbruscato et al. Vol. 280

Downloaded from jpet.aspetjournals.org at Univ Of Calif San Francisco on March 20, 2012
Fig. 2. HPLC Flo-One radioactive detector chromato-
gram 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.

al., 1996). These data confirm that the overall physiology of a greater amount of radioactivity detected in the brain and/or
the BBB remains intact during the in situ brain perfusion CSF at all time points for [3H]CTAP, in comparison with
experiments, because these high-molecular weight com- [14C]inulin, and that [3H]CTAP enters the CNS predomi-
pounds ([14C]inulin molecular weight, 5000 –5500; [14C]su- nantly through the BBB, whereas the blood-CSF barrier
crose molecular weight, 342) were not detected at high levels plays a minor role. This can be explained by the fact that the
in the CNS. CSF is more likely to act as a “sink” to the brain than the
The data presented show that [3H]CTAP can enter the brain is to act as a sink to the CSF (Davson et al., 1961) and
CNS. The unidirectional transfer constant of [3H]CTAP into that the surface area of the choroid plexus is approximately
the brain and CSF was 5.96- and 2.43-fold higher than that 5000 times smaller than the surface area of the cerebral
calculated for [14C]inulin. These data also show that there is capillary endothelium (Bradbury, 1979). The small amount of
1997 Blood/CNS Pharmacokinetics of CTAP 407

Downloaded from jpet.aspetjournals.org at Univ Of Calif San Francisco on March 20, 2012
Fig. 5. RBrain percentage, representing the ratio of homogenate, su-
pernatant or pellet to plasma radioactivities. Supernatant represents
brain homogenate depleted of the cerebral capillary endothelium. Per-
Fig. 3. Percent recovery of intact CTAP in rat brain and serum over a fusion time was 20 min. Values are the mean 6 S.E.M. of three or four
240-min time course. A half-time of disappearance of .500 min in both experiments each. * Pellet contained significantly smaller counts than
brain and serum was determined by HPLC analysis. homogenate for both [3H]CTAP and [3H]morphine (P , .05).

20-min in situ brain perfusion ensured that we were measur-

ing 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 pepti-
dases 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 m-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 iden-
tified, 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
Fig. 4. Uptake expressed as a percentage ratio of tissue to perfusate potent antagonist. This peptide may actually enter into the
radioactivities (RTissue, in milliliters per gram or milliliters per milliliter). brain via diffusion and then become trapped in the brain
Perfusion time was 20 min and values are the mean 6 S.E.M. for three compartment.
animals. The uptake of [3H]CTAP, in the absence and presence of 100
mM unlabeled CTAP, into the brain and CSF was found not to be Other important experiments were performed to ensure
statistically different. the biological stability of this drug. In vitro stability studies
were conducted in serum and brain homogenate of rats.
TABLE 2 [3H]CTAP was shown to be stable in the blood and serum of
Percent of [H]CTAP bound to protein in the perfusion medium or rats (T1/2 . 500 min), showing that the structure of this
rat serum
peptide offers enzymatic resistance to blood-borne pepti-
The levels of radioactivity found in the total sample and ultrafiltrate were deter-
mined by ultrafiltrational centrifugal dialysis using Amicon Centrifree microparti- dases. The biological stability of CTAP is probably due to the
tion devices. penicillamine-cysteine disulfide linkage, which allows the
[3H]CTAP compound to become conformationally constrained and bio-
logically active. The metabolic half-lives were quite long,
Perfusion medium Rat serum compared with that of another octapeptide analog of soma-
dpm/0.1 ml tostatin, Sandostatin. Sandostatin has numerous clinical
Total sample 271,127 1,196,935 uses in the treatment of endocrine disturbances, especially
Ultrafiltrate 85,841 189,294 those resulting from inappropriate hormone secretion by tu-
Protein-bound (%) 68.2 84.2 mors. The pharmacokinetic half-life of Sandostatin was de-
termined to be 113 min after s.c. administration (Lamberts,
[3H]CTAP detected in the CSF is most likely due to the 1986, 1987). These experiments confirm that CTAP can over-
diffusion of drug from the stagnant brain extracellular fluid come a problem that impedes the clinical use of naturally
to the rapidly flowing CSF environment. occurring peptides, i.e., a short metabolic half-life.
The measurement of intact [3H]CTAP in the brain after a Another component of CNS biodistribution that needs to be
408 Abbruscato et al. Vol. 280

TABLE 3
R (%) and octanol/saline partition coefficient for [H]CTAP, [H]morphine and [C]inulin
R (%) represents the ratio of tissue to plasma radioactivities 3 100. Data are mean 6 S.E.M. Perfusion time was 20 min (n 5 3–5 animals/compound). Octanol/saline
partition coefficients were calculated as the ratio of labeled substance in the octanol phase to that in the aqueous phase. For each compound, triplicate determinations
were made.
[3H]CTAP [3H]Morphine [14C]Inulin

RBrain (%) 5.10 6 0.66** 8.33 6 1.21** 1.46 6 0.36

RCSF (%) 4.27 6 1.06 6.97 6 0.76* 1.75 6 0.66
Octanol/saline 0.0380 6 0.0047** 0.0455 6 0.0036** 0.0008 6 0.0001
* P , .05, Student’s t test.
** P , .01, Student’s t test.

measured when evaluating the blood-to-CNS pharmacoki- walls, to achieve enzymatic degradation. High levels of pep-
netics is the ability of a given test solute to bind to serum tidases are known to be expressed at the membranes of brain
proteins. [3H]CTAP was found to be extensively protein microvessel endothelial cells (Brownson et al., 1994).
bound to albumin in the perfusion medium (68.2%) and to rat A greater amount of [3H]morphine entered both the brain
serum proteins (84.2%) (table 2). A protein-binding compo- and CSF after a 20-min brain perfusion, compared with

Downloaded from jpet.aspetjournals.org at Univ Of Calif San Francisco on March 20, 2012
nent has also been observed with other analogs of somatosta- [14C]inulin (P , .01 and P , .05, respectively) (table 3). The
tin (Banks et al., 1990). This suggests that actual CNS up- increased CNS penetration by [3H]morphine, compared with
take values may be higher without this protein binding [3H]CTAP, is likely due to increased lipophilicity, as shown
component being taken into consideration. Extensive binding by the high octanol/saline partition coefficient. In addition,
of [3H]CTAP to albumin in the perfusion medium and rat the RTissue values correlate well with octanol/saline partition
serum proteins may actually be protecting CTAP from enzy- coefficients for [3H]CTAP, [3H]morphine and [14C]inulin (r 5
matic degradation by systemic peptidases. 0.946 for brain and r 5 0.926 for CSF). Thus, lipophilicity
It is apparent that [3H]CTAP can enter into the CNS, may be a determining factor for CNS entry of these drugs.
based on in situ brain perfusion experiments coupled to This also confirms the reliability of using our in situ brain
HPLC analysis. The next step was to determine whether the perfusion technique to mimic or predict in vivo situations,
mechanism of entry was by means of passive diffusion or such as compounds attempting to traverse the BBB and/or
saturable transport. In situ brain perfusion experiments blood-CSF barrier.
were performed with [3H]CTAP in the presence of 100 mM This work supports the hypothesis that the m-selective
CTAP. Entry into the brain and CSF was not inhibited by the somatostatin analog CTAP can cross the BBB at therapeutic
addition of unlabeled CTAP (100 mM) to the perfusion me- levels. The actual amount of CTAP that crosses both the BBB
dium. This suggests that passage into the CNS was most and blood-CSF barrier is quantitatively comparable to that of
likely directed through diffusion across the membranes that the efficacious, m-selective agonist morphine. It is surprising
comprise the BBB, rather than by saturable transport. These that a compound with the clinical efficacy of morphine does
results concur with the findings of Banks et al. (1990), show- not enter the brain at high levels. The absolute percentage of
ing that somatostatin analogs can cross the murine BBB by injected dose of morphine that enters the brain has been
diffusion. This does not rule out the possibility of a saturable calculated at 0.02%/g of brain tissue (Banks and Kastin,
transport mechanism that may facilitate CTAP transport 1994). The present study shows that CTAP may play an
from the brain back into the blood, which has been described important clinical role in treating narcotic addiction, depen-
previously as PTS-5 and which is involved in the brain-to- dence or overdose. Because CTAP has excellent biological
blood transport of somatostatin and certain other analogs stability and blood-CNS penetration, it may be an improve-
(Banks and Kastin, 1992). This brain-to-blood transport ment over the classical opioid antagonist naloxone for treat-
seems less likely to occur with CTAP, because there were ing opioid crisis. Naloxone has a relatively short duration of
considerable amounts of intact [3H]CTAP detected in the action and must be administered repeatedly or by infusion.
brain after a 20-min vascular brain perfusion. Also, one must be precise in titrating the dose, for fear of
Comparisons were made between CTAP and the classical, precipitating severe withdrawal (Goodman and Gilman,
clinically efficacious, opioid agonist morphine, in reference to 1996). CTAP may therefore provide improved antagonism at
the amount of intact compound that crossed either the BBB the m-receptor, without intrinsic activity, and a longer dura-
or blood-CSF barrier and the contribution of binding to the tion of action, with less severe withdrawal.
endothelial space. The vascular component contributes sig-
nificantly to the uptake of both [3H]CTAP and [3H]morphine Acknowledgments
(44% and 32%, respectively). [3H]CTAP and [3H]morphine The authors thank Steve Waters for insightful comments about
may be sequestered in the endothelial cell component due to the manuscript.
either high lipophilicity and/or binding to brain microvessels.
References
Previously it has been shown that brain microvessels rapidly
ABBRUSCATO, T. J., WILLIAMS, S. A., HRUBY, V. J. AND DAVIS, T. P.: Blood-to-CNS
sequester and degrade somatostatin analogs (Pardridge et entry and stability of biphalin, a unique double-enkephalin analog, and its
al., 1985). This may represent one mechanism for the rapid halogenated derivatives. J. Pharmacol. Exp. Ther. 276: 1049–1057, 1996.
BANKS, W. A. AND KASTIN, A. J.: Bi-directional passage of peptides across the
inactivation of brain-derived neuropeptides after neurosecre- blood-brain barrier. Prog. Brain Res. 91: 139–148, 1992.
tion. A potential reason for the high concentration of BANKS, W. A. AND KASTIN, A. J.: Opposite direction of transport across the
[3H]CTAP detected in the microvasculature pellet may in- blood-brain barrier for Tyr-MIF-1 and MIF-1: Comparison with morphine.
Peptides 15: 23–29, 1994.
volve the binding of [3H]CTAP to a receptor on the cell BANKS, W. A., SCHALLY, A. V., BARRERA, C. M., FASOLD, M. B., DURHAM, D. A.,
membrane of the endothelial cells that comprise the vessel CSERNUS, V. J., GROOT, K. AND KASTIN, A. J.: Permeability of the murine
1997 Blood/CNS Pharmacokinetics of CTAP 409
blood-brain barrier to some octapeptide analogs of somatostatin. Proc. Natl. NESTLER, E. J.: Cellular responses to chronic treatment with drugs of abuse.
Acad. Sci. U.S.A. 87: 6762–6766, 1990. Crit. Rev. Neurobiol. 7: 23–29, 1993.
BRADBURY, M. W. B.: The Concept of the Blood-Brain Barrier, John Wiley and PARDRIDGE, W. M., EISENBERG, J. AND YAMADA, T.: Rapid sequestration and
Sons, New York, 1979. degradation of somatostatin analogues by isolated brain microvessels.
BROWNSON, E. A., ABBRUSCATO, T. J., GILLESPIE, T. J., HRUBY, V. J. AND DAVIS, T. J. Neurochem. 44: 1178–1184, 1985.
P.: Effect of peptidases at the blood-brain barrier on the permeability of PASTERNAK, G. W.: Pharmacological mechanisms of opioid analgesics. Clin.
enkephalin. J. Pharmacol. Exp. Ther. 270: 675–680, 1994. Neuropharmacol. 16: 1–18, 1993.
CAI, R.-Z., KARASHIMA, T., GUOTH, J., SZOKE, B., OLSEN, D. AND SCHALLY, A. V.: PAULUS, H.: A rapid and sensitive method for measuring the binding of radio-
Superactive octapeptide somatostatin analogs containing tryptophan at po- active ligands to proteins. Anal. Biochem. 32: 91–100, 1969.
sition 1. Proc. Natl. Acad. Sci. U.S.A. 84: 2502–2506, 1987.
PELTON, J. T., GULYA, K., HRUBY, V. J., DUCKLES, S. P. AND YAMAMURA, H. I.:
CAI, R.-Z., SZOKE, B., LU, R., FU, D., REDDING, T. W. AND SCHALLY, A. V.:
Conformationally restricted analogs of somatostatin with high mu-opiate
Synthesis and biological activity of highly potent octapeptide analogs of
receptor specificity. Proc. Natl. Acad. Sci. U.S.A. 82: 236–238, 1985.
somatostatin. Proc. Natl. Acad. Sci. U.S.A. 83: 1896–1900, 1986.
CRAIN, S. M. AND SHEN, K.-F.: After GM1 ganglioside treatment of sensory PELTON, J. T., KAZMIERSKI, W., GULYA, K., YAMAMURA, H. I. AND HRUBY, V. J.:
neurons naloxone paradoxically prolongs the action potential but still an- Design and synthesis of somatostatin analogs with high potency and speci-
tagonizes opioid inhibition. J. Pharmacol. Exp. Ther. 260: 182–186, 1992. ficity for mu opioid receptors. J. Med. Chem. 29: 2370–2374, 1986.
DAVIS, T. P. AND CULLING-BERGLUND, A.: High-performance liquid chromato- SCHALLY, A. V., CAI, R.-Z., TORRES-ALEMAN, I., REDDING, T. W., SZOKE, B., FU, D.,
graphic analysis of in vitro central neuropeptide processing. J. Chromatogr. HIEROWSKI, M. T., COLALUCA, J. AND KONTUREK, S.: In Neural and Endocrine
327: 279–292, 1985. Peptides and Receptors, ed. by T. W. Moody, pp. 73–88, Plenum, New York,
DAVSON, H., KLEEMAN, C. R. AND LEVIN, E.: Blood-brain barrier and extracellular 1986.
space. J. Physiol. (Lond.) 159: 1961. SHOOK, J. E., PELTON, J. T., LEMCKE, P. K., PORRECA, F., HRUBY, V. J. AND BURKS,

Downloaded from jpet.aspetjournals.org at Univ Of Calif San Francisco on March 20, 2012
ERCHEGYI, J., KASTIN, A. J., ZADINA, J. E. AND QUI, X.-D.: Isolation of a heptapep- T. F.: Mu opioid antagonist properties of a cyclic somatostatin octapeptide in
tide Val-Val-Tyr-Pro-Trp-Thr-Gln (valorphin) with some opiate activity. Int. vivo: Identification of mu receptor-related functions. J. Pharmacol. Exp.
J. Peptide Protein Res. 39: 477–488, 1991. Ther. 242: 1–7, 1987.
GOODMAN, L. S. AND GILMAN, A. G.: The Pharmacological Basis of Therapeutics, TAKASATO, Y., RAPOPORT, S. I. AND SMITH, Q. R.: An in situ brain perfusion
9th ed., 550–551, 1996. technique to study cerebrovascular transport in the rat. Am. J. Physiol. 247:
HANDA, B. K., LANE, A. C., LORD, J. A. H., MORGAN, B. A., RANCE, M. J. AND SMITH, H484–H493, 1984.
C. F. C.: Analogues of B-LPH61–64 possessing selective agonist activity at TERENIUS, L.: Somatostatin and ACTH are peptides with partial antagonist-
mu-opiate receptors. Eur. J. Pharmacol. 70: 531–540, 1981. like selectivity for opiate receptors. Eur. J. Pharmacol. 38: 211–215, 1976.
KARASHIMA, T. AND SCHALLY, A. V.: Superactive somatostatin analog decreases TRIGUERO, D., BUCIAK, J. B., YANG, J. AND PARDRIDGE, W. M.: Capillary depletion
plasma glucose and glucagon levels in diabetic rats. Peptides 9: 561–565,
method for quantification of blood-brain barrier transport of circulating
1988.
peptides and plasma proteins. J. Neurochem. 54: 1882–1888, 1990.
KAZMIERSKI, W., WIRE, W. S., LUI, G. K., KNAPP, R. J., SHOOK, J. E., BURKS, T. F.,
VONVOIGTLANDER, P. F., LAHTI, R. A. AND LUDENS, J. H.: U-50,488: A selective
YAMAMURA, H. I. AND HRUBY, V. J.: Design and synthesis of somatostatin
analogues with topographical properties that lead to highly potent and and structurally novel non-mu (kappa) opioid agonist. J. Pharmacol. Exp.
specific mu opioid receptor antagonists with greatly reduced binding at Ther. 224: 7–12, 1983.
somatostatin receptors. J. Med. Chem. 31: 2170–2177, 1988. WANG, Z., BILSKY, E. J., PORRECA, F. AND SADEE, W.: Constitutive mu opioid
KRAMER, T. H., SHOOK, J. E., KAZMIERSKI, W., AYRES, E. A., WIRE, W. S., HRUBY, receptor activation as a regulatory mechanism underlying narcotic tolerance
V. J. AND BURKS, T. F.: Novel peptidic mu opioid antagonists: Pharmacologic and dependence. Life Sci. 54: PL339–PL350, 1994.
characterization in vitro and in vivo. J. Pharmacol. Exp. Ther. 249: 544–549, WEBER, S. J., ABBRUSCATO, T. J., BROWNSON, E. A., LIPKOWSKI, A. W., POLT, R.,
1989. MISICKA, A., HAASETH, R. C., BARTSOV, H., HRUBY, V. J. AND DAVIS, T. P.:
LAMBERTS, S. W. J.: Non-pituitary action of somatostatin: A review on the Assessment of an in vitro blood-brain barrier model using several
therapeutic role of SMS 201–995 (Sandostatin). Acta Endocrinol. 112: suppl. [Met5]enkephalin opioid analogs. J. Pharmacol. Exp. Ther. 266: 1649–1655,
276, 41–55, 1986. 1993.
LAMBERTS, S. W. J.: A guide to the clinical use of the somatostatin analog WEBER, S. J., GREENE, D. L., HRUBY, V. J., YAMAMURA, H. I., PORRECA, F. AND
SMS201–995h (Sandostatin). Acta Endocrinol. 116: suppl. 286, 54–66, 1987. DAVIS, T. P.: Whole body and brain distribution of [3H]cyclic [D-Pen2,d-
LASZLO, F., PAVO, I., PENKE, B. AND BALINT, G. A.: Protective effect of an orally Pen5]enkephalin after intraperitioneal, intravenous, oral and subcutaneous
administered, highly potent somatostatin analog (RC-121) against absolute administration. J. Pharmacol. Exp. Ther. 263: 1308–1316, 1991.
ethanol-induced hemorrhagic erosions of rat gastric mucosa. Life Sci. 44: WEBER, S. J., GREENE, D. L., SHARMA, S. D., YAMAMURA, H. I., KRAMER, T. H.,
1573–1578, 1989. BURKS, T. F., HRUBY, V. J., HERSH, L. B. AND DAVIS, T. P.: Distribution and
LORD, J. A. H., WATERFIELD, A. A., HUGHES, J. AND KOSTERLITZ, H. W.: Endoge- analgesia of [3H][d-Pen2,d-Pen5]enkephalin and two halogenated analogs
nous opioid peptides: Multiple agonists and receptors. Nature (Lond.) 267: after intravenous administration. J. Pharmacol. Exp. Ther. 259: 1109–1117,
495–499, 1977.
1992.
LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L. AND RANDAL, R. J.: Protein
WILLIAMS, S. A., ABBRUSCATO, T. J., HRUBY, V. J. AND DAVIS, T. P.: The passage
measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265–275,
1951. of a delta-opioid receptor selective enkephalin, DPDPE, across the blood-
MALDONADO, R., NEGUS, S. AND KOOB, G. F.: Precipitation of morphine with- brain and/or the blood-cerebrospinal fluid barriers. J. Neurochem. 66: 1289–
drawal syndrome in rats by administration of mu-, delta- and kappa- 1299, 1996.
selective opioid antagonists. Neuropharmacology 31: 1231–1241, 1992. ZLOKOVIC, B. V., BEGLEY, D. J., DJURICIC, B. M. AND MITROVICK, D. M.: Measure-
MARTIN, W. R., EADES, C. G., THOMPSON, J. A., HUPPLER, R. E. AND GILBERT, P. E.: ment of solute transport across the blood-brain barrier in the perfused
The effects of morphine- and nalorphine-like drugs in the nondependent and guinea-pig brain: Method and application to N-methyl-a-aminoisobutyric
morphine-dependent chronic spinal dog. J. Pharmacol. Exp. Ther. 197: 517– acid. J. Neurochem. 46: 1444–1451, 1986.
532, 1976.
MOSBERG, H. I., HURST, R., HRUBY, V. J., GEE, K., YAMAMURA, H. I., GALLIGAN, J. Send reprint requests to: Thomas P. Davis, Ph.D., Department of Pharma-
J. AND BURKS, T. F.: Bis-penicillamine enkephalins possess highly improved cology, University of Arizona College of Medicine, 1609 N. Warren St., Tucson,
specificity toward delta-opioid receptors. Proc. Natl. Acad. Sci. U.S.A. 80: AZ 85724.
5871–5874, 1983.