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Vol. 280, Issue 3, 1147-1151, 1997
The Laboratory of the Biology of Addictive Diseases (J.Y., M.J.K.), and the Laboratory of Mass Spectrometry and Gaseous Ion Chemistry (B.T.C.), the Rockefeller University, New York, New York and the Department of Pharmacology (E.R.B., J.H.W.), the University of Michigan, Ann Arbor, Michigan 48109
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Abstract |
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 Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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E-2078 is a dynorphin A (1-8) analog, [N-methyl-Tyr1, N-methyl-Arg7-D-Leu8] Dyn A (1-8) ethylamide. Its biochemical stability against enzymatic degradation in vitro in human and rhesus monkey blood, and in vivo in rhesus monkey blood was studied using matrix-assisted laser desorption/ionization mass spectrometry. In vitro studies were carried out in freshly drawn human and rhesus monkey blood, incubated at 37°C for various time periods. In vivo studies were conducted by E-2078 i.v. injection to rhesus monkeys, and blood samples were collected at various time points after the injection. It was found that E-2078 was stable against enzymatic degradation in vitro in freshly drawn human and rhesus monkey blood. Minor biotransformation products from E-2078, such as E (1-4), E (1-5) and E (3-6), were detected in vitro in some human and rhesus monkey blood, but they made up less than 5% of the total starting E-2078 peptide. No biotransformation products were detected in the blood samples from in vivo studies. The apparent half-life of elimination of E-2078 in vivo from the rhesus monkey blood was determined to be 44.0 min.
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Introduction |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Dyn A (1-17) is an endogenous
neuropeptide. Studies have indicated that this peptide and its
congeners may attenuate the behavioral symptoms of the opioid
withdrawal syndrome in morphine-dependent rodents and may be useful as
a therapeutic agent for the treatment of opioid dependency in man
(Takemori et al., 1992
, 1993). It has also been shown that
Dyn A peptides have analgesic properties and may have potential
application in the management of pain (Hooke and Lee, 1995; Smith and
Lee, 1988). However, natural Dyn A peptides are susceptible to
enzymatic degradation in vitro and in vivo in a
biological system (Goldstein et al., 1979; Young et
al., 1987). Dyn A peptides undergo proteolytic cleavages in a
biological matrix to form a variety of biotransformation products
(Butelman et al., 1996; Chou et al., 1993, 1994a,
1994b; Yu et al., 1995, 1996a, 1996b).
To stabilize Dyn A peptides against enzymatic degradation, a group of
researchers synthesized a variety of Dyn A analogs (Tachibana et
al., 1988; Yoshino et al., 1990). These analogs were
evaluated and studied for their stability, receptor-binding properties, and biological activities. They finally arrived at
[N-methyl-Tyr1,
N-methyl-Arg7-D-Leu8] Dyn A (1-8)
ethylamide, designated as E-2078.
Studies showed that E-2078 was stable against enzymatic degradation
(Nakazawa et al., 1990). E-2078 bound to 
-opioid receptor just like Dyn A (1-17) (Yoshino et al., 1990), and
exhibited a 2-fold more potent analgesic effect than morphine. This
enhanced analgesic action was attributed to its stability against
enzymatic degradation (Nakazawa et al., 1990; Yoshino
et al., 1990). E-2078 exhibited analgesic properties when
used in patients with severe pain after lower abdominal surgery in
clinical studies (Fujimoto and Momose, 1995; Tachibana, 1996).
We report studies of E-2078 biotransformation in vitro in freshly drawn human and rhesus monkey blood and also in vivo in rhesus monkey blood. These studies were conducted using our recently developed techniques of matrix-assisted laser desorption/ionization mass spectrometry, which permits sensitive and specific simultaneous detection of both parent peptide compounds and multiple biotransformation products in a biological matrix.
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Materials and Methods |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Subjects. Three healthy human male subjects, average age 28.7 yr (range 24-31), served as donors for the blood used in the in vitro studies. Five adult rhesus monkeys, Macaca mulatta, two females and three males, average age 12 yr (range 8-18), mean weight, 8.7 kg (range 6.5-12 kg) were used for the in vitro and in vivo studies. They were housed singly with free access to water, and were fed approximately 30 Purina (St. Louis, MO) monkey chow biscuits daily and fresh fruit twice per week. The monkeys were housed on a 12:12 light:dark cycle (light on at 7:00). Experiments were carried out between 10:00 and 15:00.
Chemicals. [N-methyl-Tyr1, N-methyl-Arg7-D-Leu8] Dyn A (1-8) ethylamide (E-2078) was synthesized and kindly supplied by Eisai Co. Ltd. (Ibaraki, Japan). Dyn A (1-8) was purchased from Peninsula Laboratories, Inc. (Belmont, CA). Angiotensin III and diphenhydramine HCl were purchased from Sigma Chemical Co. (St. Louis, MO). Ketamine HCl was obtained from Fort Dodge (Fort Dodge, IA). Heparin was purchased from Elkins-Sinn (Cherry Hill, NJ). Saline (0.9% NaCl) and dextrose were from Abbott Laboratories (North Chicago, IL). 4HCCA was obtained from Aldrich Chemical Company, Inc. (Milwaukee, WI). High-performance liquid chromatography grade acetonitrile was purchased from Burdick & Jackson (Muskegon, MI) and TFA from Fisher Scientific (Fair Lawn, NJ).
Dyn A (1-8) and its analog, E-2078, in vitro biotransformation in human and rhesus monkey blood. Blood was drawn from subjects into vacutainer tubes preconditioned with EDTA (Becton Dickinson, Rutherford, NJ). In the E-2078 processing experiments, 14.0 ml of freshly drawn blood was placed in a Falcon polypropylene tube, 50 ml (Becton Dickinson, Lincoln Park, NJ). After removal of 1.0 ml blood as control, E-2078 in amount of 7.67 mg, which was dissolved in 500 µl of saline, was immediately added to the blood (i.e., 0.59 mg/ml). This E-2078-containing blood was incubated in a water bath at 37°C (Bench Scale Equipment Co., Inc., Dayton, OH). Blood samples were taken from the incubating tube at the following time-points after the addition of E-2078: 0, 15, 30, 60, 90, 120, 180, 240, 300, 360, 420, 480 min and 24 hr. One ml of blood was used for each time point. In the similar Dyn A (1-8) processing experiments, 8 and 11 ml of rhesus monkey and human blood, respectively, were used. Dyn A (1-8), 4.13 mg for monkey blood and 5.90 mg for human blood, was used (0.59 mg/ml). Blood samples, 1 ml, were taken at the following time-points: rhesus monkey blood, 0, 1, 5, 10, 15, 30 min; human blood, 0, 1, 5, 10, 15, 30, 45, 60 and 90 min.
Immediately after removal of the blood from the incubating tube, plasma was separated from the blood by centrifugation in a Sorvall RC-5B refrigerated (0-5°C) superspeed centrifuge (Sorvall Instruments, Du Pont Company, Newtown, CT), at 3400 × g for 5 min. A total of 200 µl of plasma containing E-2078 and proteolytic products was added to 1.8 ml of 1.0% TFA aqueous solution. The samples were frozen at
70°C until analysis.
Before analysis, the samples were thawed at room temperature. To 2.0 ml
of sample plasma solution was added 100 µl of 630 µM (0.029 mg/ml)
Dyn A (1-8) in 0.05% aqueous TFA as the internal standard for the
E-2078 processing studies. For the Dyn A (1-8) processing studies, 100 µl of 500 µM (0.022 mg/ml) angiotensin III in 0.05% aqueous TFA as
the internal standard was added to 2.0 ml of sample plasma solution.
The solution containing plasma, the peptide, the internal standard and
TFA, was centrifuge-filtered with Centricon-SR3 concentrators (Amicon
Inc., Beverly, MA), with molecular weight cut-off of 3000 Da. A total
of 20 µl of the filtrate was mixed with 10 µl of acetonitrile for
analysis by mass spectrometry.
E-2078 in vivo studies in rhesus monkeys. Monkeys received injections with diphenhydramine (1.2 mg/kg, intramuscularly) as a pretreatment to limit the possible consequences of histamine release after administration of relatively large amounts of E-2078. Thirty min later, they were anesthetized with ketamine (10 mg/kg, i.m.). The back of the lower leg area was carefully shaved to avoid any adventitious bleeding. An indwelling catheter (Angiocath, 22 gauge, 1 inch long, Becton Dickinson, Sandy, UT) was acutely placed in a superficial vein of each leg, secured and flushed with heparinized saline (20 U/ml), and connected to a multisample injection port. One milliliter of blood sample was obtained as control and placed in a 2-ml vacutainer preconditioned with EDTA on ice. All blood sampling was followed by port and catheter flushing with heparinized saline. The animal was then placed on a heating pad (37°C) on a surgery table.
During the sampling period, the animal received a dextrose/saline i.v. infusion (approximately 30 ml/kg/hr) from the catheter that had previously been used for the E-2078 injection. Supplemental ketamine (approximately 5 mg/kg) was administered at hourly intervals to maintain the animal in an anesthetized state throughout the sampling period. The required amount of E-2078 was dissolved in saline (10 mg/kg in 20 mg/ml solution), and injected in one of the leg catheters (injection time was approximately 15 sec). At the end of injection, a timer was started, and the injection catheter was flushed with heparinized saline. Blood sampling was performed at the following time points from the contralateral catheter: 0, 5, 15, 30, 60, 90, 120 and 180 min. Samples were centrifuged (3400 × g at 0-5°C for 5 min). A 0.2-ml aliquot of plasma was placed in a cryovial containing 1.8 ml of 1% TFA and kept at40°C until the time of analysis.
In vivo blood samples were treated following the same
procedures described earlier for in vitro blood samples with
the exception that 40 µl of 80 µM (1.54 µg/ml) of Dyn A (1-8)
was used as the internal standard for 2.0 ml of plasma sample solution.
Mass spectrometry.
The samples were analyzed with a custom
matrix-assisted laser desorption/ionization time-of-flight mass
spectrometer (MALDI-MS) constructed at the Rockefeller University
(Beavis and Chait, 1989, 1990). The instrument incorporates an HY-400
Nd-YAG laser (Lumonics Inc., Kanata, Ontario, Canada) operated at a
wavelength of 355 nm, a pulse duration of ca. 10 ns, and a pulse energy
density of ca. 15 mJ/cm2; a 30 KV ion acceleration
potential; and a 2 m long linear time-of-flight mass analyzer. Ion
detection and signal amplification are accomplished with a hybrid
microchannel plate detector-discrete dynode electron multiplier
assembly (detector potential being 3.6 KV).
-cyano-4-hydroxycinnamic acid in acetonitrile and 0.1% aqueous TFA
[1:2 (v/v)]. Plasma containing E-2078 biotransformation products was
mixed with matrix solution in a ratio of 1:2 (v/v). A 0.5-µl aliquot
of the resulting solution was applied to the mass spectrometer sample
probe tip, and allowed to evaporate to dryness in the air. The sample
probe was inserted into the mass spectrometer vacuum system where
solvents in the sample were completely removed. After 5 min, a working
pressure of approximately 10
7 torr was achieved. To
obtain adequate statistics, i.e., to achieve a
signal-to-noise ratio of the peak of interest of more than 10:1, the
results from 100 laser shots were added to produce each mass spectrum.
In this study, one spectrum was obtained for each sample from each
subject.
A standard curve was constructed using E-2078 standard solutions. The
standard solutions were made by serial dilution of E-2078 in 0.05% TFA
aqueous solution. A constant amount of internal standard, Dyn A (1-8)
(1 µl of 136 µg/ml, or 0.136 µg), was added to 100 µl of
standard solutions. The E-2078 standard solutions containing Dyn A
(1-8) were each mixed with acetonitrile (2:1, v/v), and the resulting
solutions were separately mixed 4HCCA matrix solution (1:2, v/v).
Half-microliter aliquots of the resulting solutions were applied to the
mass spectrometer sample probe tip for MALDI MS analysis. Mass
spectrometric measurements were carried out in triplicate for each
aliquot of the standard solutions. The ratio of the signal intensity
from E-2078 to that from the internal standard, and the S.E. of the
triplicate measurements were calculated.
In the Dyn A (1-8) in vitro biotransformation studies, the
use of angiotensin III as the internal standard was to show the appearance and disappearance of Dyn A (1-8) and the processing products.
In vivo pharmacokinetic analysis. The pharmacokinetics of the disappearance of E-2078 in vivo in rhesus monkey blood was analyzed with SAAM II (version 1.0.2, SAAM Institute, University of Washington, Seattle, WA), a program for kinetic analysis. The kinetic curves were plotted with both the experimental data and calculated data obtained with SAAM II using KaleidaGraph (Version 3.0.5, Abelbeck Software, Reading, PA) with nonlinear curve-fitting routine. The half-life of E-2078 was calculated by plotting the logarithm of the relative signal intensity vs. the time after injection.
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Results |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Dyn A (1-8), on in vitro incubation at 37°C in
freshly drawn human and rhesus monkey blood, was rapidly processed to
form two biotransformation products: Dyn A (2-8) and Dyn A (1-6)
(fig. 1). Dyn A (1-8) and the biotransformation
products were cleared from human blood at 90 min incubation, and
cleared from the rhesus monkey blood at 30 min incubation. E-2078 was
also incubated in freshly drawn human and rhesus monkey blood at 37°C
for up to 24 hr. In stark contrast to the results observed with Dyn A
(1-8), no major biotransformation products were detected. As shown in figure 2, at 24 hr incubation time point, intact E-2078
was still the major species present in the blood. Minor
biotransformation products such as E (1-4), E (1-5) and E (3-6) were
detected in blood of some human and rhesus monkey subjects at certain
incubation time points (data not shown). Mass spectrometric analysis of
the control blood for in vitro Dyn A (1-8) and E-2078
processing studies in human and rhesus monkey blood recorded a flat
baseline, besides a few very small background peaks.
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In vivo studies of E-2078 after i.v. injection to rhesus monkey indicated that E-2078 was stable against enzymatic cleavage in blood. No biotransformation products were detected in any of the samples collected at the various time points up to 180 min after the injection.
With the use of an internal standard, Dyn A (1-8), quantitative
analysis of the peptide in the samples could be carried out by using
the relative signal intensity of the signal from E-2078 against that
from the internal standard. As shown in the calibration curve (fig.
3) within the concentration range between 22.22 pg and
555.55 pg of E-2078 applied to the sample probe tip, the relative signal intensity is linearly correlated to the amount of peptide applied.
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The pharmacokinetics of disappearance of E-2078 in vivo in
rhesus monkey blood was analyzed by using Dyn A (1-8) as the internal standard. The pharmacokinetic curves were plotted (fig.
4). The plots constructed based on experimental data and
data obtained from kinetic calculation coincided with each other (fig.
4a). Assessment of the goodness-of-fit (Foster, 1995) indicated that the fit for the monoexponential model
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). The first linear section, from 0 to 10 min,
represented a rapid distribution of the peptide out of the vascular
system into various tissues after injection. The second linear section, from 15 to 180 min, represented the elimination of E-2078 from both the
blood and the tissues, once the distribution had been completed and the
plasma and tissue concentration were in equilibrium. The calculated
apparent half-life was t1/2(
) = 44.0 min; thus it would take approximately 4.4 hr (around six elimination half-lives) for E-2078 to be eliminated from the rhesus monkey (Wartak,
1983).
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Discussion |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Matrix-assisted laser desorption/ionization mass spectrometry is
an analytical technique developed in recent years. It permits sensitive, simultaneous as well as specific detection of the presence of multiple components in a sample. This technique can tolerate complex
sample conditions such as biological matrices, requiring minimal sample
pretreatment. In our laboratories, this technique has been successfully
applied to several studies of biotransformation of neuropeptides
including Dyn A peptides in a variety of biological matrices, such as
blood, brain tissues and cerebrospinal fluid (Butelman et
al., 1996; Chou et al., 1993, 1994a, 1994b; Yu et al., 1995, 1996a, 1996b, 1996c).
In this study, biotransformation of Dyn A (1-8) in human and rhesus
monkey blood was found to exhibit the same patterns as that of the
endogenous opioid peptide Dyn A (1-17) (Yu et al., 1995,
1996a, 1996b). Two major biotransformation pathways could be
identified. Cleavage of N-terminal, tyrosine, to form Dyn A (2-8), and
the cleavage between Arg(6) and Arg(7) linkage to form Dyn A (1-6). No
cleavage from the C-terminal was detected. Biotransformation of Dyn A
(1-8) in rhesus monkey blood occurred at a faster rate than in human
blood, as seen in figure 1. This result corroborates our previous
findings in studies of Dyn A (1-17) (Yu et al., 1995, 1996a, 1996b).
The modified Dyn A (1-8), E-2078, did not exhibit significant biotransformation either in vitro in freshly drawn human and rhesus monkey blood, or in vivo in rhesus monkey blood. Modification with a methyl group at Tyr1, N-methyl-Tyr1, effectively protected the cleavage at the N-terminal Tyr1 position. No biotransformation products from this processing were detected. Peptide linkage of Arg(6)-Arg(7) is usually the other site of cleavage of Dyn A peptides in a biological matrix. Modification with a methyl group at the N-Arg7 position effectively blocked this biotransformation.
Minor biotransformation products of E-2078, E(1-4), E(1-5) and E(3-6), were detected in vitro in human and rhesus monkey blood. Based on the signal abundance of all the peptides, these biotransformation products made up less than 5% of the total starting E-2078 peptide. These biotransformation products were not constantly detectable at all time points. No biotransformation products were detected in vivo in rhesus monkey blood, possibly because the formation of processing products were in very small amount that they were absorbed onto the tissues or blood cells or rapidly eliminated from the system.
Peptide biotransformation may be tissue specific. Reported stability
studies on E-2078 in comparison with Dyn A (1-17) have shown that no
E-2078 degradation product was detected after incubation with mouse
serum (10%) for 2 hr, whereas Dyn A (1-17) was degraded completely in
that time period (Nakazawa et al., 1990). However, E-2078
was biotransformed in mice brain homogenate (1%) with a half-life
about 4 hr; Dyn A (1-17) biotransformation half-life was about 0.5 hr
(Nakazawa et al., 1990). The cleavage sites of E-2078 in
mouse brain homogenates were identified to be Gly(3)-Phe(4), Phe(4)-Leu(5), and Leu(5)-Arg(6) by high-performance liquid
chromatography (Tachibana, 1996). In this study, no biotransformation
products from Gly(3)-Phe(4) cleavage were detected.
In summary, Dyn A (1-8) analog, E-2078, was very stable in
vitro in freshly drawn human and rhesus monkey blood and in
vivo in rhesus monkey blood. Minor processing products from
E-2078, such as E(1-4), E(1-5) and E(3-6), were detected in
vitro in some human and rhesus monkey blood. The apparent
half-life of elimination of E-2078 in vivo from the rhesus
monkey blood was about 44.0 min. Based on these studies, E-2078 is a
suitable candidate for further neurobiological and pharmacological
studies, also for possible development as a therapeutic agent for the
management of pain, opioid addiction and cocaine addiction (Kreek,
1996; Spangler, 1993, 1996).
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Acknowledgment |
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The authors thank the Eisai Co. Ltd. (Ibaraki, Japan), especially Dr. Y. Yamanishi, for their generous gift of E-2078 peptide. The authors thank Dr. Ann Ho for her valuable suggestions in the preparation of this manuscript.
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Footnotes |
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Accepted for publication November 19, 1996.
Received for publication August 22, 1996.
1 This work was supported in part by NIH-NIDA Research Center Grant DA P50-05130 (M.J.K.); NIH-NIDA Research Scientist Award DA 00049 (M.J.K.); NIH-CRR General Clinical Research Center Grant M01-RR00102 (M.J.K.); NIH P41 RR 00862 (B.T.C.); and NIH-NIDA Grant DA 00254 (J.H.W.).
Send reprint requests to: Dr. Jim Yu, The Rockefeller University, Box 171, 1230 York Avenue, New York, NY 10021.
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Abbreviations |
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Dyn A, dynorphin A;
4HCCA, -cyano-4-hydroxycinnamic acid;
TFA, trifluoroacetic acid;
Arg, arginine;
Tyr, tyrosine;
Leu, leucine;
Phe, phenylalanine;
Gly, glycine;
MALDI-MS, matrix-assisted laser desorption/ionization mass
spectrometry.
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References |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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This article has been cited by other articles:
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  J. Yu, E. R. Butelman, J. H. Woods, B. T. Chait, and M. J. Kreek Dynorphin A (1-8) Analog, E-2078, Crosses the Blood-Brain Barrier in Rhesus Monkeys J. Pharmacol. Exp. Ther., August 1, 1997; 282(2): 633 - 638. [Abstract] [Full Text]
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