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JPET 295:1070–1076, 2000
Vol. 295, No. 3
2901/866636
Printed in U.S.A.
Opioid Receptor Imaging with Positron Emission Tomography
and [18F]Cyclofoxy in Long-Term, Methadone-Treated Former
Heroin Addicts1
MITCHEL A. KLING, RICHARD E. CARSON, LISA BORG, ALAN ZAMETKIN, JOHN A. MATOCHIK, JAMES SCHLUGER,
PETER HERSCOVITCH, KENNER C. RICE, ANN HO, WILLIAM C. ECKELMAN, and MARY JEANNE KREEK
Laboratory of the Biology of Addictive Diseases, The Rockefeller University, New York, New York (L.B., J.S., A.H., M.J.K.); Clinical Center, PET
Department (R.E.C., P.H., W.C.E.), National Institute of Mental Health (M.A.K., A.Z.), and National Institute of Diabetes and Digestive and
Kidney Diseases (K.C.R.), National Institutes of Health, Bethesda, Maryland; and National Institute on Drug Abuse, National Institutes of Health,
Baltimore, Maryland (J.A.M.)
Accepted for publication August 25, 2000
This paper is available online at http://www.jpet.org
We have found that well stabilized, methadone-maintained, former heroin addicts (MTPs) treated with effective
dosages, with no ongoing polydrug or alcohol abuse, have
markedly reduced or eliminated drug craving and normalized neuroendocrine function including both stress-responsive hypothalamic-pituitary-adrenal axis function and also
reproductive, biology-related hypothalamic-pituitary-gonadal axis function (Kreek, 1973a, 1978, 1987, 1992, 1996a,b;
Kreek et al., 1981, 1984; Kreek and Koob, 1998). In contrast,
profound alterations in both the stress-responsive and the
reproductive axes have been documented in active heroin
addicts during cycles of short-acting opiate abuse, primarily
of heroin (which has a half-life of only 3 min in humans) and
its major metabolite, morphine (which has a half-life of 4 – 6
Received for publication May 15, 2000.
1
This work was supported in part by NIH National Institute on Drug Abuse
Grants DA-P50-05130 and DA-KO5-00049 and NIH Center for Research Resources Grant M01-RR00102.
and kappa opioid receptors. Imaging was performed in the
morning, 22 h after the last dose of methadone in patients, and
concurrent plasma levels of methadone were determined. Five
brain regions of specific interest for addiction and pain research
(thalamus, amygdala, caudate, anterior cingulate cortex, and
putamen) were among the six regions of highest [18F]cyclofoxy
binding. Specific binding of [18F]cyclofoxy was lower by 19 to
32% in these regions in MTPs compared with those in normal
volunteers. The degree to which specific binding was lower in
caudate and putamen correlated with methadone plasma levels
(P ⬍ .01 and P ⬍ .05, respectively), suggesting that these lower
levels of binding may be related to receptor occupancy with
methadone and that significant numbers of opioid receptors
may be available to function in their normal physiological roles.
h) (Kreek, 1973a, 1978, 1987, 1996a,b; Kreek et al., 1981,
1984; Kreek and Koob, 1998). Many studies have shown that
both these functional systems are modulated by the mu opioid receptor-directed endogenous opioid ligands in normal,
healthy humans (Kreek, 1987, 1996b; Kreek and Koob, 1998;
Schluger et al., 1998). Immune function, which may also be
modulated in part by the endogenous opioid system and is
abnormal during cycles of heroin addiction, becomes normal
during long-term methadone treatment (Novick et al., 1989).
The subjective responses to acute and chronic pain are similar in methadone-maintained patients and non-opioid-dependent persons; acute and chronic pain in maintenance
patients similarly responds to treatment with short-acting,
primarily mu opioid receptor-directed analgesic agents such
as morphine, hydromorphone, and fentanyl (Ho and Dole,
1979).
We have hypothesized that although a proportion of mu
opioid receptors would be occupied by methadone at all times
during stabilized, long-term methadone maintenance with
ABBREVIATIONS: MTP, methadone-maintained former heroin addicts; PET, positron emission tomography; ROI, region of interest; RUH,
Rockefeller University Hospital; NIH, National Institutes of Health.
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ABSTRACT
Stabilized methadone-maintained former heroin addicts (MTPs)
treated with effective doses of methadone have markedly reduced drug craving; reduction or elimination of heroin use;
normalized stress-responsive hypothalamic-pituitary-adrenal,
reproductive, and gastrointestinal function; and marked improvement in immune function and normal responses to pain,
all of which are physiological indices modulated in part by
endogenous and exogenous opioids directed at the mu and, in
some cases, the kappa-opioid systems. This study was performed to explore opioid receptor binding in MTPs. Fourteen
normal, healthy volunteers and 14 long-term MTPs in treatment
for 2 to 27 years and receiving 30 to 90 mg/day of methadone
were studied with positron emission tomography using tracer
amounts of [18F]cyclofoxy, an opioid antagonist that labels mu
2000
Human Opioid Receptor Imaging by PET
Materials and Methods
Twenty-eight subjects were studied: 14 normal, healthy volunteers (7 males) with a mean age of 38 years (23– 65 years) and 14
long-term, methadone-treated patients (8 males), all former heroin
addicts, with a mean age of 37 years (20 – 60 years). Each subject in
this second group met the requirements of the federal regulations for
entry into long-acting opioid agonist treatment with methadone, or
l-␣-acetylmethadol, i.e., at least 1 year of multiple, daily self-administrations of a short-acting opiate, primarily heroin, with the devel-
opment of tolerance and physical dependence. These subjects were
maintained on methadone administered orally each day at doses
ranging from 30 to 90 mg/day, with a mean dose of 62 mg/day. Their
duration of methadone-maintenance treatment ranged from 2 to 27
years, with a mean of 7.6 years.
Subjects were initially evaluated in the Rockefeller University
Hospital (RUH), a National Institutes of Health (NIH) General Clinical Research Center. Each subject signed a written informed consent at the RUH General Clinical Research Center. Later, each
subject signed a separate informed consent at the NIH Clinical
Center. Study protocol and informed consents were approved by the
appropriate institutional review boards and radiation safety committees at both institutions.
Potential study subjects were first seen in the outpatient clinic on
two or more occasions for evaluation and explanations of the entire
study procedure. Subjects were admitted to the RUH General Clinical Research Center for at least 1 day for baseline neuroendocrine
re-evaluation immediately before going to the NIH Clinical Center.
They were then accompanied to Bethesda, MD, the location of the
NIH Clinical Center, by a Rockefeller University physician or nurse
for 1) an initial outpatient visit, 2) a magnetic resonance imaging
scan to rule out any gross structural abnormality of the brain, and 3)
PET imaging using [18F]cyclofoxy, which was performed in the morning for all subjects 22 ⫾ 1 h after the last dose of methadone in the
methadone-maintained patients. All studies were performed within
2 days on an outpatient basis at the NIH Clinical Center.
Cyclofoxy, an opioid antagonist originally synthesized and developed by the laboratory group of Dr. Kenner Rice (National Institute
for Diabetes, Digestive and Kidney Diseases, NIH), made imaging of
opioid receptors possible (Pert et al., 1984; Burke et al., 1985; Channing et al., 1985). On each study day, [18F]cyclofoxy was synthesized
in the NIH-CC PET Department. A Scanditronix scanner (model
PC2048-15B; Scanditronix, Uppsala, Sweden), which provides 15
simultaneous slices with 6- to 7-mm axial and transverse resolution,
was used for PET imaging. Subjects were positioned so that transverse slices were acquired along the canthomeatal line.
Before the study began, a physician placed an intra-arterial line in
the radial artery of one arm and an intravenous line in the antecubital fossa of the other arm of the subject. A molded thermoplastic
face mask was made for each subject to maintain head position
throughout the PET study. [18F]Cyclofoxy (approximately 4 mCi i.v.)
was injected over 1 min. [18F]Cyclofoxy was prepared with a sufficiently high specific activity to contain less than 8 ␮g of total cyclofoxy to prevent precipitating any opioid withdrawal symptoms in the
methadone-maintained patients and to prevent demonstrable
changes in receptor occupancy in any study subject. Twenty-three
scan frames were obtained over 90 min at predetermined time intervals (Cohen et al., 1997). Thus, cyclofoxy, which is administered
by bolus, approaches equilibrium over the time of the 90-min scans.
Arterial blood samples were taken at predetermined time points to
measure the plasma input function. Metabolite analysis was performed at 13 time points by extraction of plasma with ethyl acetate
and radiolabel counting (Carson et al., 1993). Protein binding of
[18F]cyclofoxy measured by ultrafiltration was found to be similar in
the control and patient groups (unbound fractions for controls,
63.9 ⫾ 3.2%; MTP, 63.4 ⫾ 4.1%). Blood samples were also taken to
measure plasma levels of methadone before the beginning of the
study and at 30, 60, and 90 min. The daily dose of methadone was
administered to maintenance patients after the study was completed
approximately 24 h after the last dose.
The total binding of [18F]cyclofoxy was determined in 14 brain
regions of interest (ROIs), which were identified on the reconstructed
PET images (Table 1). The procedure for data analysis for this study
is based on previous [18F]cyclofoxy studies in baboons and in humans
(Theodore et al., 1992; Carson et al., 1993; Cohen et al., 1997). The
time series of cyclofoxy images were first corrected for subject motion
(Woods et al., 1992). Each image obtained 10 min after injection was
matched to an image obtained by summing images from 0 to 10 min.
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moderate to high doses of methadone, significant numbers of
mu receptors remain unoccupied and thus available for action by the endogenous opioid ligands in critical brain regions. These unoccupied receptors are also available for action by exogenous opioid ligands commonly used for the relief
of pain. The half-life of methadone in the racemic (d,l or S,R)
form commonly used in treatment is 24 h in humans, and
additional studies using stable isotope technology have
shown that the half-life of the active (l,R) enantiomer of
methadone in humans is 36 to 48 h (Inturrisi and Verebely,
1972; Kreek, 1973b; Rubenstein et al., 1978; Kreek et al.,
1979). We would predict that a significant proportion of mu
opioid receptors would be occupied by this ligand in steady
state during treatment. We also hypothesized that if in longterm, stabilized, methadone maintenance patients, the occupancy of mu opioid receptors by methadone uses only a portion of available receptors, then this would allow the
documented gradual normalization of those aspects of physiology that are under modulation by mu opioid receptordirected endogenous ligands. Furthermore, these unoccupied
receptors may bind endogenous opioids to allow normal responsivity to pain and stressors as well as to exogenous
opioids used in analgesia.
In an extensive series of double blind studies, it was shown
that adequate doses of methadone fully protected against any
perception of superimposed short-acting opiates, including
objective and subjective potentially priming effects and any
adverse reactions (Dole et al., 1966; Kreek, 1992). However,
we have also shown that if very large amounts of heroin are
superimposed, narcotic-like, including euphorogenic, effects
can be produced by exceeding the degree of tolerance and
cross-tolerance to other opioids achieved during steady-state,
moderate- to high-dose (60 –150 mg/day) methadone treatment (Dole et al., 1966; Kreek, 1992).
Therefore, we conducted a study using positron emission
tomography (PET) with [18F]cyclofoxy as the radioligand, an
opioid antagonist with mu and kappa opioid receptor-directed binding (Pert et al., 1984; Burke et al., 1985; Channing
et al., 1985; Rothman and McLean, 1988; Theodore et al.,
1992; Carson et al., 1993; Cohen et al., 1997). The purposes of
this study were 1) to extend earlier PET studies in young and
middle-aged healthy volunteers with no history of substance
abuse by mapping the extent of opioid receptor binding
throughout the human brain, including five regions of special
interest for research in addictive diseases that are also related to pain and analgesia (caudate, putamen, amygdala,
anterior cingulate cortex, and thalamus) and 2) to conduct
PET studies in long-term, stabilized, methadone-maintained
former heroin addicts to determine the possible changes in
the binding of [18F]cyclofoxy assumed to be attributable to
occupancy by methadone and to determine whether there are
any regional variations in changes of opioid receptor binding.
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Kling et al.
Vol. 295
TABLE 1
Total and specific [18F]cyclofoxy binding in different brain regions
Total [18F]Cyclofoxy Bindinga
Normal Volunteers
Methadone-Treated
Patients
Specific [18F]Cyclofoxy Bindinga
Normal Volunteers
ml plasma/ml tissue
Thalamus
Amygdala
Caudate
Insula
Anterior cingulate cortex
Putamen
Middle temporal cortex
Middle frontal cortex
Parietal cortex
Cerebellum
Inferior temporal cortex
Hippocampus
White matter
Occiptal cortex
a
ml plasma/ml tissue
15.7 ⫾ 2.0
14.6 ⫾ 3.5
14.1 ⫾ 2.4
12.9 ⫾ 2.5
13.0 ⫾ 2.2
13.9 ⫾ 2.5
11.3 ⫾ 1.7
11.0 ⫾ 1.7
10.4 ⫾ 1.7
9.7 ⫾ 1.1
9.6 ⫾ 1.3
9.1 ⫾ 1.5
6.4 ⫾ 1.0b
6.1 ⫾ 0.7
14.1 ⫾ 2.8
11.2 ⫾ 3.6
10.5 ⫾ 2.5
9.7 ⫾ 2.0
9.7 ⫾ 1.9
9.6 ⫾ 2.3
7.7 ⫾ 1.4
7.1 ⫾ 1.7
6.0 ⫾ 1.5
4.9 ⫾ 1.5
4.9 ⫾ 1.7
4.3 ⫾ 1.7
0.7 ⫾ 0.9
9.6 ⫾ 2.1
8.5 ⫾ 3.4
8.0 ⫾ 2.0
6.7 ⫾ 2.2
6.9 ⫾ 1.9
7.7 ⫾ 2.0
5.1 ⫾ 1.2
4.9 ⫾ 1.4
4.3 ⫾ 1.4
3.6 ⫾ 0.9
3.5 ⫾ 1.0
2.9 ⫾ 1.8
0.3 ⫾ 1.0b
Values are mean ⫾ S.D.
n ⫽ 13.
Circular ROIs (37 pixels, 2 mm/pixel) were placed on the summed
images (Cohen et al., 1997). A sample ROI using the methods of this
study has been published previously (Cohen et al., 1997). Timeactivity curves were obtained from the motion-corrected images.
Then data were fitted to a model with one tissue compartment and
three parameters, K1, k2, and Vb, to define tissue uptake (K1), clearance (k2), and vascular volume (volume of blood, Vb), respectively.
Although this model is simplified, it is adequate to describe the
kinetics of cyclofoxy in humans (Theodore et al., 1992).
The primary parameter of interest in this analysis is the total
tissue volume of distribution, Vt, which is calculated from the fitted
parameters. We define Vt as the ratio at true equilibrium of tissue
concentration to metabolite-corrected plasma radioactivity. For this
model, Vt is calculated as K1/k2, i.e., the one-tissue-compartment
model estimate of the total tracer volume of distribution. Vt measures total tracer binding, i.e., free tracer, nonspecifically bound
tracer, and specifically bound tracer. In a region with little or no
specific binding such as the occipital cortex, Vt measures only free
and nonspecifically bound tracer. Published data of opioid receptor
density in human brain post mortem have shown modest kappa
opioid receptor density but little to no mu opioid receptor density in
specific subregions of the occipital cortex (Hiller and Fan, 1996).
Assuming that nonspecific binding is uniform throughout the
brain, we define the specific volume of distribution, Vs, of an ROI as
Vs (ROI) ⫽ Vt (ROI) ⫺ Vt (occipital). Vs is thus the ratio of specifically
bound tracer to free plasma concentration at equilibrium. It is linearly proportional to the ratio of the unoccupied receptor (Bmax) to
the dissociation equilibrium constant (KD), called the binding potential (Mintun et al., 1984). Changes in this measure reflect changes in
total receptor concentration, receptor occupancy, or affinity. As cyclofoxy binds to both mu and kappa receptors, Vs will be linearly
proportional to the sum of its binding potential at each receptor
subtype [Bmax(␮)/KD(␮) ⫹ Bmax(␬)/KD(␬)]. Rothman and McLean
(1988) reported that cyclofoxy labels both mu and kappa opioid
receptors, but their estimates of KD in rat were 0.34 nM for ␮ and 1.5
nM for ␬, suggesting a 5-fold affinity difference.
Statistical Analysis. The ROI with the least total binding is the
occipital area; the mean total binding of right and left occipital
regions of each subject, considered nonspecific binding, was used to
calculate specific binding in all other ROIs for that subject as described above. The validity of this approach was shown by a t test
confirming that in the occipital area, there was no difference in total
cyclofoxy binding between control subjects (6.6 ⫾ 0.6; mean ⫾ S.D.)
and MTPs (6.1 ⫾ 0.7) (t ⫽ 1.80, df ⫽ 24, P ⬍ .10).
The values from each bilateral region of interest were used to
calculate a mean value of that ROI for each subject. The group
mean ⫾ S.E.M. of total binding of cyclofoxy for each of the 14 ROIs
was determined and ranked in descending order of magnitude of the
means in the control group.
ANOVA, group ⫻ ROI, with repeated measures on the last factor
was used to evaluate the significance of differences of specific cyclofoxy binding between the subject groups. Because the error term for
post hoc tests in the between- and within-subject interaction is not
clear, an individual one-way ANOVA was used to evaluate the differences between groups for each ROI. The level of significance was
.05, with Bonferroni correction for multiple comparisons.
To investigate the relationship between the circulating plasma
levels of methadone and the degree of lowering in comparison with
normal volunteers of specific cyclofoxy binding in the six ROIs with
greatest specific cyclofoxy binding, regression analysis with correlation was used. For each ROI of the 12 MTPs for whom mean plasma
methadone levels across the 90-min period of scanning were available, the mean value of the specific binding of the control group
minus each individual’s value was plotted against plasma levels of
methadone.
Results
The total binding for normal, healthy subjects and longterm, stabilized MTP subjects is presented (Table 1). The
specific binding in healthy volunteers in all brain regions
studied is compared with that in the long-term MTPs (Fig. 1
and Table 1).
ANOVA across the 13 brain regions, group ⫻ ROI with
repeated measures on the last factor, showed that there was
a significantly lower level of cyclofoxy binding in the MTP
group than in the healthy volunteers (F(1,26) ⫽ 17.83; P ⬍
.0005). As expected, there was a significant difference among
the ROIs (F(12,312) ⫽ 123.31; P ⬍ .000001). There was also
a significant group ⫻ ROI interaction (F(12,312) ⫽ 4.13; P ⬍
.00001). The mean ⫾ S.E.M. values for specific cyclofoxy
binding for each group in each ROI are shown in Fig. 1, with
an asterisk indicating significance of difference in each region after Bonferroni correction for multiple comparisons.
The five regions of special interest for research related to
addictive diseases and pain management, caudate, putamen,
anterior cingulate, amygdala, and thalamus, were found to
be among the six regions with the highest specific opioid
receptor binding (Fig. 1). The mean ⫾ S.D. differences in
cyclofoxy binding in these brain regions between the long-
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b
20.7 ⫾ 2.5
17.8 ⫾ 3.6
17.1 ⫾ 2.7
16.4 ⫾ 2.1
16.3 ⫾ 1.9
16.2 ⫾ 2.5
14.4 ⫾ 1.7
13.7 ⫾ 1.6
12.6 ⫾ 1.5
11.5 ⫾ 1.4
11.5 ⫾ 1.8
10.9 ⫾ 1.7
7.3 ⫾ 0.7
6.6 ⫾ 0.6
Methadone-Treated
Patients
2000
Human Opioid Receptor Imaging by PET
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term, stabilized, methadone-maintained patients and the
normal, healthy volunteers are shown in Table 2.
Plasma levels of methadone were at a plateau, as expected
from previous studies, and within the expected therapeutic
range (Fig. 2). The degree to which specific binding of [18F]cyclofoxy was lower in MTPs in comparison with normal volunteers, although small, was significantly correlated with
the mean plasma levels of methadone (0 –90 min) in caudate
(P ⬍ .01) and putamen (P ⬍ .05) and was near significant
levels in anterior cingulate cortex (P ⬍ .06) and insula (P ⬍
.06) (Fig. 3).
Discussion
To further our understanding of opioidergic function in
opiate addiction and its effective treatment, specific binding
of [18F]cyclofoxy was determined by PET imaging in 14 stabilized, long-term, methadone-maintained former heroin addicts (mean age, 38 years) and compared with binding in
normal, healthy volunteers (mean age, 37 years). The five
brain regions, which were of especial interest to us from the
start of the study because of their involvement in addictive
diseases and pain, were found to be five of the six areas with
the most cyclofoxy binding in human brain: thalamus, amygdala, caudate, anterior cingulate cortex, and putamen. In a
previously reported study of patients with Alzheimer’s disease and similarly older, age-matched healthy subjects from
51 to 75 years of age, similar subcortical (caudate, putamen,
and thalamus) and limbic and paralimbic (anterior cingulate
cortex, amygdala, and insula) brain regions were found to
have the highest values for cyclofoxy-specific binding (Cohen
et al., 1997). Specific binding of [18F]cyclofoxy in these five
regions of special interest in methadone-maintained former
heroin addicts studied 22 h after the last oral dose of methadone was determined to be 19 to 32% lower than that
observed in normal volunteers. Furthermore, the lower specific binding in MTPs compared with normal volunteers was
correlated with their plasma methadone levels.
Since the cloning of first the rodent and then the human
mu opioid receptor, it has been possible to study the binding
of mu, kappa, and delta opioid ligands. In all these studies, as
TABLE 2
Percentage lower binding in five ROIs in MTPs compared with normal volunteersa
Thalamus
Amygdala
Caudate
Anterior cingulate cortex
Putamen
a
Total [18F]Cyclofoxy Binding
Specific [18F]Cyclofoxy Binding
24 ⫾ 10
18 ⫾ 20
18 ⫾ 14
21 ⫾ 13
15 ⫾ 15
32 ⫾ 15
24 ⫾ 30
24 ⫾ 19
29 ⫾ 20
19 ⫾ 21
Percentage lower binding ⫽ (mean of normal volunteers ⫺ individual MTP’s values). Values are mean ⫾ S.D.
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Fig. 1. Specific binding of [18F]cyclofoxy (mean ⫹ S.E.M.) in 13 brain regions of normal volunteers and long-term, methadone-treated former heroin
addicts. Regions of interest (full region names are presented in Table 1) are ordered from most to least-specific binding in normal volunteers ( n ⫽ 14
for each brain region in each group, except n ⫽ 13 for white matter in methadone-treated patients). Group ⫻ ROI ANOVA with repeated measures
on the last factor of [18F]cyclofoxy binding showed a significantly lower level between groups and a significant group ⫻ ROI interaction (see Results).
*, each region in which individual ANOVA for each ROI with Bonferroni correction for multiple comparisons yielded a significantly lower level of
binding in MTPs compared with normal volunteers. Thl, thalamus; Amy, amygdala; Caud, caudate; Ins, insula; ACg, anterior cingulate cortex; Put,
putamen; MT, middle temporal cortex; MFr, middle frontal cortex; Par, parietal cortex; Crb, cerebellum; IT, interior temporal cortex; Hip, hippocampus; WMt, white matter.
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Kling et al.
well as in recent signal transduction studies, methadone has
been demonstrated to be a pure and full agonist directed
specifically at the mu opioid receptor. However, the only
available published data for cyclofoxy have been generated in
in vitro experiments using rodent tissue and indicate that
cyclofoxy binds to both mu and kappa receptors (Rothman
and McLean, 1988). To date, there is only indirect evidence
suggesting that cyclofoxy binds preferentially at mu receptors, such as the finding in this study of low binding in the
occipital cortex, an area with little mu but significant kappa
opioid receptor density in humans as well as similar in vivo
findings from previous PET studies using this ligand (Hiller
and Fan, 1996; Cohen et al., 1997). The methodology of our
study did not permit the determination of whether these
findings are consistent with an overall decrease, increase, or
lack of change in opioid receptor number (with occupancy
alone accounting for the lower binding), affinity, or function
in MTPs compared with normal volunteers. Interestingly,
there is one recent report that includes the assessment of mu
opioid receptor density in human heroin addicts post mortem
in comparison with healthy controls, which showed no
change in opioid receptor density (Gabilondo et al., 1994).
Potentially relevant preclinical studies have not yielded a
consensus on the effects of chronic exposure to short-acting
mu opioid receptor agonists on receptor density or binding
capacity. There are no studies delivering methadone by
pump, which is necessary in rodents to model the long-acting
pharmacokinetic properties of methadone in humans (Zhou
et al., 1996). We hypothesize that the lower binding in MTPs
in comparison with normal volunteers reflects steady-state
methadone occupancy of mu opioid receptors. The half-life of
methadone in the racemic (d,l or S,R) form, usually used in
treatment of heroin addiction or chronic pain, is 24 h in
humans, and the half-life of the active (l,R) enantiomer in
humans is 36 to 48 h (Inturrisi and Verebely, 1972; Kreek,
1973b; Rubenstein et al., 1978; Kreek et al., 1979). The peak
plasma levels of methadone occur 2 to 4 h after oral dosing,
and the plasma levels return to plateau levels within 4 to 8 h
after each oral dose (Inturrisi and Verebely, 1972; Kreek,
1973b; Kreek et al., 1979). In addition, we have found that
the cerebrospinal fluid levels of methadone vary in relationship to plasma levels of methadone, with peak levels occurring at approximately the same time and with levels of methadone in cerebrospinal fluid approximately 10 to 20% of those
in plasma at most time points studied (Rubenstein et al.,
1978). Therefore, these PET studies were performed at a time
that, in other studies, we have found plasma levels of methadone to be sustained at a plateau, usually approximately
Fig. 3. The amount of each methadone-treated patient’s [18F]cyclofoxy
binding less than the mean of that region for the normal volunteers is plotted against the mean plasma methadone level across the PET scan
session. The scatterplot with regression line and 95% confidence interval
are shown for four of the six regions
with the highest levels of specific binding. The amount by which specific
binding is lower than the mean of that
for the normal volunteers is significantly correlated in caudate (A) and in
putamen (B), whereas the relationship
fails to reach significance in the anterior cingulate cortex (C) and in insula
(D) in the 12 patients for whom plasma
methadone levels were available.
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Fig. 2. Plasma levels of methadone in long-term, methadone-treatment
patients sampled across the 90-min PET scan session (starting approximately 22 h after the last dose of methadone). Values for the 12 patients
for whom the plasma was available are expressed as mean ⫾ S.E.M.
Vol. 295
2000
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cycles of internalization or to actual increased production of
opioid receptors, with an even higher percentage of occupancy by methadone, which in turn could account for the
apparent modest lowered specific [18F]cyclofoxy binding observed (19 –32%) during steady-state occupancy by methadone. The correlation of lower cyclofoxy binding levels with
plasma methadone levels, along with all previous studies of
mu opioid agonist effects on opioid receptor density in adult
animals, and the recent study of mu receptor density in
heroin addicts post mortem suggest that receptor occupancy
with methadone represents a significant component of the
lower [18F]cyclofoxy binding observed in MTPs.
Studying additional specific patient populations could help
elucidate whether either relative down-regulation or up-regulation occurs in living humans as well as the time course of
potential changes in reference to exposure to short-acting
illicitly used opiates and long-acting treatment agents. Such
populations might include active heroin addicts before entry
into treatment as well as long-term illicit drug- and medication-free, well stabilized former addicts without any continuing drug or alcohol abuse in comparison with both normal
controls and stabilized long-term MTPs. Studying such subjects presents particular challenges, however, because active
heroin addicts before entry into treatment may have difficulty cooperating with the lengthy study protocol and given
the approximately 80 to 90% relapse rate of heroin addicts
not receiving long-acting opioid pharmacotherapy, significant numbers of truly illicit drug- and medication-free former
heroin addicts are very difficult to identify for study. Studying MTPs after a medication “washout” period would also be
of theoretical benefit. However, the protracted period required to ethically reduce the dose and discontinue methadone, thus avoiding the discomfort of opiate withdrawal as
well as the additional time required to reach a new homeostasis in a medication-free, illicit drug-free status and the very
high documented rate of relapse would outweigh the potential value of attempting such a study.
Whether either relative down-regulation or up-regulation
of opioid receptors ever occurs in living adult humans could
be potentially determined by studying additional specific
populations. A large proportion of heroin addicts (70 –90%) in
many regions of the United States are also cocaine dependent, and a substantial percentage (approximately 30%) of
methadone-maintained former heroin addicts remain dependent on cocaine (Borg et al., 1999). Several years ago, our
laboratory showed that chronic “binge” pattern cocaine administration causes an increase in mu opioid receptor density
in the rat (Unterwald et al., 1992, 1994). Subsequently, these
studies were extended in humans by Zubieta et al. (1996),
who found an increase of mu opioid receptor density in several specific brain regions in recently abstinent cocaine addicts using PET with [11C]carfentanil as the radiolabeled mu
opioid receptor-directed ligand. Recently, Yuferov et al.
(1999) have shown that acute binge pattern cocaine administration in a rat model significantly increases the mRNA
levels of the mu opioid receptor, which may contribute to the
significantly increased density of the mu opioid receptors
seen after 7 and 14 days of binge pattern cocaine administration. This is the one example in which a drug of abuse or
treatment agent has been shown unequivocally to increase or
decrease opioid receptor density in humans as well as in
rodent models. Chronic naltrexone administration has been
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150 to 400 ng/ml. In this study, the plasma levels at 22 h after
the last oral dose of methadone (dose range, 30 –90 mg; mean
dose, 62 mg) ranged from 127 to 673 ng/ml, with a mean
approximately 350 ng/ml.
Many studies exploring the mechanism of action of methadone and its effectiveness have shown the disruption by chronic
use of short-acting opiates such as heroin of multiple physiological processes including modulation of normal stress responsivity, actions at hypothalamic-anterior pituitary sites of release of
corticotropin-releasing factor and pro-opiomelanacortin peptides (adrenocorticotropin and ␤-endorphin), control of reproductive biology, specifically by impairing luteinizing hormone
release, alterations of many indices of immune and gastrointestinal function, and a variety of other physiological systems
(Kreek, 1973, 1978, 1987, 1992, 1996a,b; Ho and Dole, 1979;
Kreek et al., 1981, 1984; Novick et al., 1989; Kreek and Koob,
1998). The early findings of effects of short-acting opioids, in
contrast to the gradual normalization during steady-dose,
steady-state treatment with methadone, led to the discovery of
the role of endogenous opioids in a variety of physiological
processes (Kreek, 1973a, 1978, 1992, 1996b; Kreek et al., 1981,
1984). It has been demonstrated and reported that very large
amounts of heroin, greater than those usually self-administered, are necessary to overcome the “blockade” provided by
moderate to high (60 –150 mg) doses of methadone. This is
compatible with the concept that methadone is occupying some,
but not all, of the available opioid receptors. However, this is not
necessarily the full explanation for the blockade or for the
gradual normalizations of functions under mu opioid receptor
modulation during long-term treatment. The mechanisms that
allow the long-acting mu opioid agonist methadone, at doses
used in maintenance treatment, to block the effects of the usual
self-administered doses of heroin, but that allow normalization
of other physiological functions modulated by opioids as well as
adequate response to opioid analgesics, have not been fully
elucidated. The findings of the present study are not inconsistent with a substantial fraction of opioid receptors being available for binding both to endogenous ligands (which would presumably be related to normalized function of physiological
systems known to be modulated in part by endogenous opioids)
as well as to additional exogenous opioids (potentially related to
the adequate response to opioid analgesia seen in MTPs).
In addition, or alternatively, the lower binding in MTPs
may have been attributable to an overall modest reduction of
mu (and/or kappa) opioid receptors on cell surfaces during
steady-dose methadone maintenance treatment. Such lower
binding could be attributable either to occupancy alone
and/or to methadone-induced internalization or to other
mechanisms yielding actual reduction in receptor density
possibly induced by chronic exposure to this long-acting, mu
opioid ligand. Alternatively, lower binding could have been
present on an a priori basis, such as the presence of an allelic
variant of the receptor, with altered binding (Bond et al.,
1998). Recent studies have suggested that methadone acts
like the natural endogenous opioid peptides with respect to
effecting prompt internalization of opioid receptors after cell
surface receptor binding in contrast to most exogenous opioids including morphine, the major metabolite of heroin,
which does not effect rapid internalization (Keith et al.,
1998). The findings of this also would be compatible with a
modest increase in opioid receptor density, attributable either to increased receptor presentation on cell surfaces after
Human Opioid Receptor Imaging by PET
1076
Kling et al.
repeatedly shown to increase mu opioid receptor density in
living adult, whole animal studies; however, no studies of its
effects in humans have yet been reported. Additional PET
studies in unstabilized methadone-maintained patients who
continue to abuse cocaine, as contrasted with well stabilized
patients receiving methadone treatment and also with “pure”
cocaine abusers, would be of interest, along with studies,
when possible, in long-term, drug-free former heroin addicts
not receiving pharmacotherapy.
Acknowledgments
We thank Drs. R. Maslansky, E. Khuri, and A. Wells for the
clinical characterizations of each subject in methadone maintenance
treatment as well as for the general patient and staff education
concerning PET studies. We also acknowledge Susan Lampert, R.N.,
research nurse, Rockefeller University Hospital, who made this
study possible and Jason Kreuter and Lori Lefter for generous technical assistance. We also acknowledge Michael Channing and the
staff of the NIH PET Department.
Bond C, Laforge KS, Tian M, Melia D, Zhang, S, Borg L, Gong J, Schluger J, Strong
JA, Leal SM, Tischfield JA, Kreek MJ and Yu L (1998) Variation in receptor
function from a single nucleotide polymorphism in the human ␮-opioid receptor
gene: Possible implications for opioid addiction. Proc Natl Acad Sci USA 95:9608 –
9613.
Borg L, Broe DM, Ho A and Kreek MJ (1999) Cocaine abuse sharply reduced in an
effective methadone maintenance program. J Addict Dis 18:63–75.
Burke TR, Rice KC and Pert CB (1985) Probes for narcotic receptor mediated
phenomena. II. Synthesis of 17-methyl and 17-cyclopropylmethyl-3,14-dihydroxy4,5 alpha-epoxy-6-beta-fluoromorphinans (foxy and cyclofoxy) as models of opioid
ligands suitable for positron emission transaxial tomography. Heterocycles 23:69 –
99.
Carson RE, Channing MA, Blasberg RG, Dunn BB, Cohen RM, Rice KC and Herscovitch P (1993) Comparison of bolus and infusion methods for receptor quantitation: Application to [18F]cyclofoxy and positron emission tomography. J Cereb
Blood Flow Metab 13:24 – 42.
Channing MA, Eckelman WC, Bennett JM, Burke TR and Rice KC (1985) Radiosynthesis of [18F]3-acetylcyclofoxy: A high affinity opiate antagonist. Int J Appl
Radiat Isot 36:429 – 433.
Cohen RM, Andreason PJ, Doudet DJ, Carson RE and Sunderland T (1997) Opiate
receptor avidity and cerebral blood flow in Alzheimer’s disease. J Neurol Sci
148:171–180.
Dole VP, Nyswander ME and Kreek MJ (1966) Narcotic blockade. Arch Intern Med
118:304 –309.
Hiller JM and Fan L (1996) Laminar distribution of the multiple opioid receptors in
the human cerebral cortex. Neurochem Res 21:1333–1345.
Ho A and Dole VP (1979) Pain perception in drug-free and in methadone-maintained
human ex-addicts. Proc Soc Exp Biol Med 162:392–395.
Gabilondo AM, Meana JJ, Barturen F, Sastre M and Garcia-Sevilla JA (1994)
␮-Opioid receptor and ␣-2-adrenoreceptor agonist binding sites in the postmortem
brain of heroin addicts. Psychopharmacology 115:135–140.
Inturrisi CE and Verebely K (1972) The levels of methadone in the plasma in
methadone maintenance. Clin Pharmacol Ther 18:633– 637.
Keith DE, Anton B, Murray SR, Zaki PA, Chu PC, Lissin DV, Monteillet-Agius G,
Stewart PL, Evans CJ and Von Zastrow M (1998) ␮-Opioid receptor internalization: Opiate drugs have different effects on a conserved endocytic mechanism in
vitro and in the mammalian brain. Mol Pharmacol 53(3):377–384.
Kreek MJ (1973a) Medical safety and side effects of methadone in tolerant individuals. J Am Med Assoc 223:665– 668.
Kreek MJ (1973b) Plasma and urine levels of methadone. NY State J Med 73:2773–
2777.
Kreek MJ (1978) Medical complications in methadone patients. Ann NY Acad Sci
311:110 –134.
Kreek MJ (1987) Multiple drug abuse patterns and medical consequences, in Psychopharmacology: The Third Generation of Progress (Meltzer HY ed) pp 1597–
1604, Raven Press, New York.
Kreek MJ (1992) Rationale for maintenance pharmacotherapy of opiate dependence,
in Addictive States (O’Brien CP and Jaffe JH eds) pp 205–230, Raven Press, New
York.
Kreek MJ (1996a) Opiates, opioids and addiction. Mol Psychiatry 1:232–254.
Kreek MJ (1996b) Opioid receptors: Some perspectives from early studies of their
role in normal physiology, stress responsivity and in specific addictive diseases.
Neurochem Res 21:1469 –1488.
Kreek MJ, Hachey DL and Klein PD (1979) Stereoselective disposition of methadone
in man. Life Sci 24:925–932.
Kreek MJ and Koob GF (1998) Drug dependence: Stress and dysregulation of brain
reward pathways. Drug Alcohol Depend 51:23– 47.
Kreek MJ, Raghunath J, Plevy S, Hamer D, Schneider B and Hartman N (1984)
ACTH, cortisol and beta-endorphin response to metyrapone testing during chronic
methadone maintenance treatment in humans. Neuropeptides 5:277–278.
Kreek MJ, Wardlaw SL, Friedman J, Schneider B and Frantz AG (1981) Effects of
chronic exogenous opioid administration on levels of one endogenous opioid (betaendorphin) in man, in Advances in Endogenous and Exogenous Opioids (Simon E
and Takagi H eds) pp 364 –366, Kodansha, Tokyo.
Mintun MA, Raichle ME, Kilbourn MR, Wooton GF and Welch MJ (1984) A quantitative model for the in vivo assessment of drug binding sites with positron
emission tomography. Ann Neurol 15:217–227.
Novick DM, Ochshorn M, Ghali V, Croxson TS, Mercer WD, Chiorazzi N and Kreek
MJ (1989) Natural killer cell activity and lymphocyte subsets in parenteral heroin
abusers and long-term methadone maintenance patients. J Pharmacol Exp Ther
250:606 – 610.
Pert CB, Danks JA, Channing MA, Eckelman WC, Larson SM, Bennett JM, Burke
TR and Rice KC (1984) 3-[18F]Acetylcyclofoxy: A useful probe for the visualization
of opiate receptors in living animals. FASEB Lett 177:281–286.
Rothman RB and McLean SA (1988) An examination of the opiate receptor subtypes
labeled by [3H]cyclofoxy: An opiate antagonist suitable for positron emission tomography. Biol Psychiatry 22:423– 458.
Rubenstein RB, Kreek MJ, Mbawa N, Wolff WI, Korn R and Gutjahr CL (1978)
Human spinal fluid methadone levels. Drug Alcohol Depend 3:l03–106.
Schluger JH, Ho A, Borg L, Porter M, Maniar S, Gunduz M, Perret G, King A and
Kreek MJ (1998) Nalmefene causes greater hypothalamic-pituitary-adrenal axis
activation than naloxone in normal volunteers: Implications for the treatment of
alcoholism. Alcohol Clin Exp Res 22:1430 –1436.
Theodore WH, Carson RE, Andreason P, Zametkin A, Blasberg R, Leiderman DB,
Rice K, Newman A, Channing M, Dunn B, Simpson N and Herscovitch P (1992)
PET imaging of opiate receptor binding in human epilepsy using [18F]cyclofoxy.
Epilepsy Res 13:129 –139.
Unterwald EM, Horne-King J and Kreek MJ (1992) Chronic cocaine alters brain
␮-opioid receptors. Brain Res 584:314 –318.
Unterwald EM, Rubenfeld JM and Kreek MJ (1994) Repeated cocaine administration upregulates ␬- and ␮-, but not ␦-, opioid receptors. Neuroreport 5:1613–1616.
Woods RP, Cherry SR and Mazziotta JC (1992) Rapid automated algorithm for
aligning and reslicing PET images. J Comput Assisted Tomogr 16:620 – 633.
Yuferov V, Zhou Y, Spangler R, Maggos CE, Ho A and Kreek MJ (1999) Acute “binge”
cocaine increases ␮-opioid receptor mRNA levels in areas of the rat mesolimbic
mesocortical dopamine system. Brain Res Bull 48:109 –112.
Zhou Y, Spangler R, Maggos CE, LaForge KS, Ho A and Kreek MJ (1996) Steadystate methadone in rats does not change mRNA levels of corticotropin-releasing
factor, its pituitary receptor or proopiomelanocortin. Eur J Pharmacol 315:31–35.
Zubieta J-K, Gorelick DA, Stauffer R, Ravert HT, Dannals RF and Frost JJ (1996)
Increased ␮-opioid receptor binding detected by PET in cocaine-dependent men is
associated with cocaine craving. Nat Med 2:1225–1229.
Send reprint requests to: Mary Jeanne Kreek, M.D., Laboratory of the
Biology of Addictive Diseases, The Rockefeller University, 1230 York Ave., Box
171, New York, NY 10021. E-mail: [email protected]
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References
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