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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics
JPET 283:1305–1322, 1997
Vol. 283, No. 3
Printed in U.S.A.
Neurotransmitter Receptor and Transporter Binding Profile of
Antidepressants and Their Metabolites1
MICHAEL J. OWENS, W. NEAL MORGAN, SUSAN J. PLOTT and CHARLES B. NEMEROFF
Laboratory of Neuropsychopharmacology, Department of Psychiatry and Behavioral Sciences, Emory University School of Medicine,
Atlanta, Georgia
Accepted for publication August 12, 1997
Nefazodone and venlafaxine are two of several newer antidepressants that have been introduced in the United States
in the past several years. These drugs and their metabolites,
like the TCAs and SSRIs, are both antagonists of monoamine
transporters and receptors in the CNS. The potency of transporter antagonism and receptor binding can theoretically
predict both clinical efficacy and side effect profile. With
radioligand binding assays, the potency of a given drug for a
specific receptor or transporter can be calculated by obtaining the equilibrium inhibition constant (Ki). This constant,
unlike IC50 calculations which have been performed in numerous receptor binding studies, is independent of the specific radioligand used or the concentration of radioligand in
the assay. This allows for comparison of Ki values across
laboratories.
Nefazodone has a chemical structure (fig. 1) seemingly
unrelated to SSRIs, TCAs, tetracyclics, bupropion or monoamine oxidase inhibitors. Nefazodone is effective in the treatReceived for publication February 14, 1997.
1
Supported by a grant from Bristol Myers-Squibb, the Stanley Foundation
Scholars Program, and NIMH MH-40524.
paroxetine, generally thought of as a selective SERT antagonist, possesses moderately high affinity for the NET and that
venlafaxine, which has been described as a “dual uptake inhibitor”, possesses weak affinity for the NET. We observed significant correlations in SERT (r 5 0.965) or NET (r 5 0.983) affinity
between rat and human transporters. Significant correlations
were also observed between muscarinic cholinergic and NET
affinity. There are several significant correlations between affinities for the 5-HT1A, 5-HT2A, histamine1, alpha-1 and alpha-2
receptors. These novel findings, not widely described previously, suggest that many of the individual drugs studied in
these experiments possess some structural characteristic that
determines affinity for several G protein-coupled, but not muscarinic, receptors.
ment of depression, and it has a more favorable side effect
profile than the structurally similar antidepressant trazodone (Fontaine et al., 1994; Rickels et al., 1994; Taylor et al.,
1995; Robinson et al., 1996). Nefazodone has three major
active metabolites (Mayol et al., 1994): hydroxynefazodone,
mCPP and a triazoledione tautomer of desethylhydroxynefazodone hereafter termed triazoledione. Venlafaxine, another
effective antidepressant, is marketed as a dual serotonin and
norepinephrine uptake inhibitor. Its major metabolite is Odesmethylvenlafaxine.
In the present study, we have determined the Ki for 19
commonly used antidepressants or their metabolites for the
rat and human SERT and NET. Eleven of these compounds
were tested further to determine their affinity for the rat
5-HT1A, 5-HT2A, and muscarinic cholinergic receptors as well
as for the guinea pig histamine1 (H1) receptor and the human
alpha-1 and alpha-2 receptors. These studies build upon the
seminal studies of Richelson and colleagues (Richelson and
Nelson, 1984; Bolden-Watson, 1993; Cusack et al., 1994) by
examining the most up-to-date series of antidepressants and
their metabolites that target the monoamine transporters.
ABBREVIATIONS: SERT, serotonin transporter; NET, norepinephrine transporter; 5-HT, 5-hydroxytryptamine; mCPP, meta-chlorophenylpiperazine; AUC, area under the curve; HPLC, high-pressure liquid chromatography; SSRI, serotonin selective reuptake inhibitor; CNS, central nervous
system; TCA, tricyclic antidepressant.
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ABSTRACT
Several new antidepressants that inhibit the serotonin (SERT)
and norepinephrine transporters (NET) have been introduced
into clinical practice the past several years. This report focuses
on the further pharmacologic characterization of nefazodone
and its metabolites within the serotonergic and noradrenergic
systems, in comparison with other antidepressants. By use of
radioligand binding assays, we measured the affinity (Ki) of 13
antidepressants and 6 metabolites for the rat and human SERT
and NET. The Ki values for eight of the antidepressants and
three metabolites were also determined for the rat 5-HT1A,
5-HT2A and muscarinic cholinergic receptors, the guinea pig
histamine1 receptor and the human alpha-1 and alpha-2 receptors. These data are useful for predicting side effect profiles and
the potential for pharmacodynamic drug-drug interactions of
antidepressants. Of particular interest were the findings that
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Owens et al.
Vol. 283
Moreover, their affinity at both the rat and human variants
of the SERT and NET are compared.
Methods
Tissue sources. These studies were conducted in accordance with
the Declaration of Helsinki and/or with the Guide for the Care and
Use of Laboratory Animals as adopted and promulgated by the
National Institutes of Health. Male Sprague-Dawley rats or guinea
pigs were housed with food and water available ad libitum in an
environmentally controlled animal facility. Animals were sacrificed
by guillotine decapitation without anesthesia as approved by the
Emory University Animal Use and Care Committee.
For the SERT, NET, 5-HT2A and muscarinic cholinergic binding
studies, pooled rat frontal cortex (anterior to the hippocampus) was
collected and stored at 280°C until needed. Similarly stored rat
hippocampus was used for the 5-HT1A receptor assays. Pooled whole
guinea pig brain was used for the H1 receptor assay. Human frontal
and parietal cortex was pooled from six normal control brains obtained from the brain bank of the Emory University Alzheimer’s
Disease Research Center for use in the alpha-1 and alpha-2 receptor
assays. Postmortem delay in these samples ranged from 6 to 11
hours, and none of the patients were treated with any medications at
the time of death that are known to interact with alpha adrenergic
receptors.
Samples were homogenized with a Polytron PT 3000 (Brinkmann;
20,000 rpm 3 12 seconds) in 30 volumes of their individual assay
buffers (table 1) at 4°C, and centrifuged at 43,000 3 g for 10 min. The
supernatants were decanted and resuspended in 30 volumes of
buffer, homogenized, separated into several individual aliquots and
centrifuged. For membrane pellets that were used in the 5-HT2A,
5-HT1A, alpha-1 and alpha-2 binding assays, the pellets following
the second centrifugation were resuspended in 30 volumes of buffer
and the suspensions were preincubated in an oscillating water bath
at 37°C for 10 min. After preincubation, these suspensions were
recentrifuged at 43,000 3 g for 10 min, the supernatants decanted
and resuspended in 30 volumes of cold buffer, homogenized, separated into several individual aliquots and centrifuged. The resulting
pellets were stored at 270°C until assayed.
Stable transfection of human SERT (Qian et al., 1997) or human
NET cDNA (Galli et al., 1995) into HEK-293 (human embryonic
kidney) cells has resulted in cell lines exhibiting high-affinity, Na1dependent transport of serotonin or norepinephrine with pharmacological properties identical with those of native membranes. These
have been provided to us by Randy Blakely, Ph.D. (Vanderbilt University). Both cell lines were grown in ten tray Nunc cell factories
(6,320 cm2) to confluence in Dulbecco’s modified Eagle’s medium
containing 10% fetal bovine serum supplemented with L-glutamine
(2 mmol/l final concentration), penicillin (100 mg/ml) and 100
units/ml streptomycin in a humidified incubator at 37°C containing
5% CO2. The selecting antibiotic geneticin sulfate (250 mg/ml) was
used during all growth phases. Membranes were harvested using
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Fig. 1. Chemical structures of the antidepressants and metabolites used in the studies. Nortriptyline, desipramine, norfluoxetine and desmethylsertraline are the N-desmethyl metabolites of amitriptyline, imipramine, fluoxetine and sertraline, respectively. The active metabolite of
venlafaxine is the O-desmethyl derivative.
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Antidepressant Receptor Binding
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TABLE 1
Assay conditions for binding and transport assays
Receptor/
Transporter
Ligand
Membrane Source
(mg protein/tube)
SERT
[3H]Citalopram
Rat cortex (150 mg)
hSERT
[3H]Citalopram
HEK-293/hSERT cDNA (35 mg)
hSERT
[3H]Serotonin
HEK-293/hSERT cDNA
NET
[3H]Nisoxetine
Rat cortex (175 mg)
[ H]Nisoxetine
HEK-293/hNET cDNA (75 mg)
hNET
[3H]Norepinephrine
HEK-293/hNET cDNA
5-HT1A
[3H]8-OH-DPAT
Rat hippocampus (175 mg)
5-HT2A
[3H]Ketanserin
Rat cortex (175 mg)
alpha-1
alpha-2
Histamine (H1)
[3H]Prazosin
[O-methyl-3H]Rauwolscine
[3H]Pyrilamine
Human cortex (125 mg)
Human cortex (200 mg)
Guinea pig whole brain (400 mg)
Muscarine
[3H]-N-methylscopolamine
Rat cortex (25 mg)
Final
Volumeb
52.6 mM Tris-HCl, 126.4 mM NaCl, 5.26 mM
KCl, pH 7.9, 22°C, 1 hr
52.6 mM Tris-HCl, 126.4 mM NaCl, 5.26 mM
KCl, pH 7.9, 22°C, 1 hr
Krebs-Ringer Henseleit bufferc
105 mM ascorbate, 105 mM pargyline, pH 7.4,
37°C, 10 min
52.6 mM Tris-HCl, 316 mM NaCl, 5.26 mM KCl,
pH 7.4, 4°C, 4 hr
52.6 mM Tris-HCl, 316 mM NaCl, 5.26 mM KCl,
pH 7.4, 4°C, 4 hr
Krebs-Ringer Henseleit bufferc
105 mM ascorbate, 105 mM pargyline, pH 7.4,
37°C, 10 min
52.6 mM Tris-HCl, 1.05 mM MnCl2, pH 7.7,
22°C, 1 hr
52.6 mM Tris-HCl, 4.2 mM CaCl2, 30 nM prazosin, pH 7.4, 37°C, 1 hr
52.6 mM Tris-HCl, pH 7.5, 22°C, 1 hr
52.6 mM Tris-HCl, pH 7.4, 22°C, 1.5 hr
10.5 mM K2HPO4, 52.6 mM NaCl, pH 7.5, 22°C,
1 hr
77.4 mM Na2HPO4, 22.6 mM NaH2PO4, 5.27
mM MgCl2, pH 7.4, 22°C, 1 hr
2 ml
2 ml
1 ml
1 ml
1 ml
1 ml
2 ml
1 ml
5 ml
2 ml
5 ml
5 ml
a
Assay buffer composition, temperature and length of incubation.
All competing drugs added as 1/20th the final volume in 5 mM HCl.
126.3 mM NaCl, 4.95 mM KCl, 1.26 mM KH2PO4, 1.26 mM MgSO4, 10.53 mM N-2-hydroxyethylpiperazine-N1-2-ethanesulfonic acid (HEPES), 2.32 mM CaCl2,
5.52 mM glucose.
b
c
37°C phosphate-buffered saline containing 0.53 mmol/l ethylenediaminetetraacetic acid, separated into aliquots, and centrifuged at
2000 3 g for 10 min. The supernatants were decanted and the pellets
homogenized as described above.
General radioligand binding assay methods. For all data
shown in the manuscript, serial dilution of radioligands or competing
drugs was carried out in borosilicate glass tubes silanized with Prosil
28 (PCR Inc., Gainesville FL). Fresh competing drug was weighed
out for each individual competition curve. All competing drugs were
initially dissolved in 50% ethanol containing 5 mmol/l HCl at a drug
concentration of 1 mg/ml. Subsequent serial dilutions were performed in silanized glass tubes in 5 mmol/l HCl and added as 1/20th
the final total volume of the assay tubes. This did not alter the pH of
any of the buffer systems. We compared the use of silanized glass
tubes with polystyrene and polypropylene tubes and found that
silanized glass tubes were preferable for preparing serial dilutions
(M. J. Owens and W. N. Morgan, unpublished observations). The
total incubation volumes and membrane protein concentrations of all
assays were adjusted such that the free ligand concentration was at
least 95% of the total ligand concentration (see table 1). For all
membrane binding assays, with the exception of those using human
brain tissue which we were not able to study, we observed that the Kd
values of freshly prepared tissue pellets and previously frozen tissue
pellets are identical, although a 0 to 8% decrease in Bmax was observed among the various assays (M. J. Owens and W. N. Morgan,
unpublished observations). Competition assays used 19 to 20 concentrations of competing ligand in triplicate over a maximum concentration range of 10213 to 1024.6 mol/l. The chosen concentrations
of competing ligand were adjusted for each assay to provide at least
10 points on the curve between 10% and 90% displacement. The only
exceptions were the transport and radioligand binding assays with
the hSERT and hNET cell lines which used 12 concentrations of
competing ligand. All competition binding assays used a single concentration of 3H-labeled radioligand equal to the calculated Kd of
that ligand for its receptor (table 2). Competitive transport assays
used a single concentration of either [3H]serotonin (final concentration 20 nmol/l; 5 nmol/l [3H]serotonin, 15 nmol/l serotonin) or
[3H]norepinephrine (final concentration 20 nmol/l; 5 nmol/l [3H]norepinephrine, 15 nmol/l (2)-norepinephrine).
Assay conditions for each receptor or transporter are shown in
table 1. Total and nonspecific binding was determined in triplicate at
each concentration of ligand. All incubations were terminated by the
addition of an excess of ice-cold assay buffer, vacuum filtration over
Whatman GF/B filters (presoaked in buffer containing 0.3% polyethyleneimine) and washed four times with 5 ml of ice-cold buffer.
Filters were dried and suspended in 10 ml of Aquasol (DuPont/New
England Nuclear, Boston, MA) scintillation fluid and equilibrated for
a minimum of 60 min before counting in a liquid scintillation counter
at 50% efficiency.
For transport assays, HEK-293 cells stably transfected with either
the hSERT cDNA or hNET cDNA were grown to confluence in
Dulbecco’s modified Eagle’s medium as described above and plated
out at a density of approximately 100,000 cells/well into poly-L-lysine
(0.5 mg/ml)-coated 24-well culture plates. Cells were allowed to adhere to plates for 24 hr before use in the assay (Galli et al., 1995;
Qian et al., 1997). Wells were preincubated for 10 min with competing ligand before addition of radioligand. Incubations were terminated by the addition of 1 ml of buffer (pH 7.4, 22°C), quickly
aspirated and washed once with 1 ml of 37°C buffer. Cells were
removed from plates by the addition of 500 ml of 1% sodium dodecyl
sulfate.
For each different receptor assay, the results of six separate saturation studies were simultaneously analyzed by use of the computer
program LIGAND (Munson and Rodbard, 1980). In competition assays, the results of at least three separate competition assays were
analyzed by use of the computer program PRISM 2.0 (GraphPad
Software, Inc., San Diego, CA). In all instances, LIGAND or PRISM
analysis revealed that the data were best fit by a one-site model
rather than a two-site model. All Ki data are expressed as geometric
mean 6 S.E. in nanomoles per liter. Geometric means and S.E. were
calculated by the method described by De Lean et al. (1982). pKi
correlations were conducted by Pearson correlations with SAS software (Cary, NC).
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hNET
3
Incubation Conditionsa
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Owens et al.
Vol. 283
TABLE 2
Affinity of the radioligands and their Bmax/Vmax in the tissues used in these studies
Ligand
Receptor
Concentration
Range
Specific Bindinga
nmol/l
[3H]Citalopram
[3H]Citalopram
[3H]Serotonin
[3H]Nisoxetine
[3H]Nisoxetine
[3H]Norepinephrine
[3H]8-OH-DPAT
[3H]Ketanserin
[3H]-N-methylscopolamine
[3H]Pyrilamine
[3H]Prazosin
[O-methyl-3H]Rauwolscine
Rat SERT
hSERT
hSERT
Rat NET
hNET
hNET
Rat 5-HT1A
Rat 5-HT2A
Rat muscarinic
Guinea pig H1
Human alpha-1
Human alpha-2
0.05–2.50
0.02–2.50
40–6000
0.07–5.50
0.05–5.0
40–6000
0.025–2.0
0.015–10.0
0.005–0.6
0.075–3.0
0.004–2.50
0.15–5.0
89%;
85%;
95%;
84%;
87%;
95%;
94%;
80%;
98%;
97%;
89%;
75%;
1 mmol/l chloroimipramine
1 mmol/l chloroimipramine
500 nmol/l paroxetine
1 mmol/l mazindol
1 mmol/l mazindol
1 mmol/l mazindol
10 mmol/l 5-HT
1 mmol/l cinanserin
250 nmol/l atropine
1 mmol/l chlorpheniramine
10 mmol/l phentolamine
1 mmol/l yohimbine
Kd/Km
Bmax/Vmax
nmol/l
fmol/mg protein
0.56
1.06
533
0.73
1.24
1608
0.13
0.77
0.036
0.15
0.037
0.86
357
1575
4.10 3 10217 mol/cell/min
194
2531
216
1.73 3 10
mol/cell/min
187
233
994
137
88
184
a
Data presented as % specific binding as a function of total binding in competition curves (fig. 2,A to L); final concentration of drug used to defined specific
binding/transport.
Results
Affinity of the radioligands and their Bmax/Vmax in
the tissues used in these studies. Table 2 shows the
results of the saturation analyses for the radioligands used in
the present studies, which served to determine radioligand
and tissue concentrations necessary for the competition studies. Calculation of Ki values required determination of radioligand Kd values in this laboratory as values reported in the
literature under similar assay conditions are not always
identical. The specific assay methods were based on those we
have previously used successfully in this laboratory or were
based on those reported in the literature. Specifically, [3H]8OH-DPAT binding was performed as described and characterized by Hall et al. (1985, 1986); [3H]ketanserin binding
was performed as described in McKenna et al. (1989) and
Owens et al. (1991); and [3H]N-methylscopolamine binding
was modified from the binding described by Dörje et al.
(1991). [3H]Prazosin binding used human cortical tissue because prazosin showed affinity for both alpha-1 and alpha-2
receptors in rat cortex, unlike human cortex in which prazosin is selective for the alpha-1 receptor (Cheung et al., 1982).
[O-methyl-3H]rauwolscine binding was also performed in human brain because yohimbine and rauwolscine, two alpha-2
antagonists, have significantly different affinities in rat versus human cortex (Summers et al., 1983; Cheung et al., 1982;
Ruffolo et al., 1991). [3H]Pyrilamine binding to H1-histamine
receptors was performed in guinea pig brain tissue because
the pharmacological profile of pyrilamine in this species more
closely resembles that observed with the human H1 receptor
than does binding in rat brain tissue (Chang et al., 1979; Hill
and Young, 1980; Hill, 1990).
Affinities of antidepressants and their metabolites.
Table 3 lists the affinities (Ki) of the various antidepressants
and their metabolites for the transporters and receptors examined in the present studies. The averaged competition
curves for each transporter/receptor system are shown in
figure 2, A to L. The data for the SERT and NET in table 3
were used to determine relative selectivity among the various drugs for the two transporters (fig. 3). Dividing the Ki for
the NET by the Ki for the SERT yields a unitless number
where 1 equals no selectivity (i.e., equal affinity for both
transporters). Values .1 represent greater SERT selectivity.
Values ,1 represent greater NET selectivity.
Correlation of affinities between receptors and species. Table 4 lists the significant correlations observed comparing affinity at one transporter/receptor and affinity at
another. Figure 4, A to D, compares the affinities with use of
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 12, 2017
Drugs. [3H]Citalopram (3012 GBq/mmol), [3H]8-OH-2-(dipropylamino)tetralin (4914 GBq/mmol), [3H]ketanserin (2290 GBq/
mmol), [3H]prazosin (2886 GBq/mmol) and [3H]pyrilamine (866 GBq/
mmol) were obtained from New England Nuclear (Boston MA).
[3H]Serotonin (4084 GBq/mmol), [3H]nisoxetine (3105 GBq/mmol),
[3H]norepinephrine (1441 GBq/mmol), [O-methyl-3H]rauwolscine
(3111 GBq/mmol) and [3H]N-methylscopolamine (855 GBq/mmol)
were obtained from Amersham Inc. (Buckinghamshire UK). Nefazodone, hydroxynefazodone, mCPP, trazodone and triazoledione were
gifts from Bristol Myers-Squibb (Wallingford, CT). Fluoxetine, norfluoxetine, and nortriptyline were gifts from the Eli Lilly and Co.
(Indianapolis, IN). Venlafaxine and O-desmethylvenlafaxine were
gifts from Wyeth-Ayerst Pharmaceuticals (Princeton, NJ). Paroxetine was a gift from SmithKline Beecham Pharmaceuticals (West
Sussex, England). Fluvoxamine was a gift from Solvay Pharmaceuticals (Marietta, GA). Citalopram was a gift from H. Lundbeck A/S
(Copenhagen-Valby, Denmark). Sertraline and desmethysertraline
were gifts from Pfizer Pharmaceuticals (Groton, CT). Imipramine
and desipramine were purchased from Sigma (St. Louis, MO). Amitriptyline, atropine, chloroimipramine, chlorpheniramine, cinanserin, mazindol, phentolamine, serotonin HCl and yohimbine were
purchased from Research Biochemicals Inc. (Natick, MA)
Measurement of nefazodone concentrations in serial dilutions from competition assays. To determine why serial dilution
of certain competing drugs in assay buffer resulted in steep competition curves, nefazodone concentrations were directly measured in
individual serial dilutions prepared in silanized glass tubes. Nefazodone concentrations in serial dilutions prepared in 5 mmol/l HCl or
assay buffer from the [3H]citalopram binding experiments were measured by HPLC with a modification of the method of Franc et al.
(1991). Of the various individual dilutions, 20 to 100 ml were injected
onto a 150 3 4.6 mm BDS-Hypersil-Phenyl 5-mm column with guard
(Keystone Scientific, Inc., Bellefonte, PA) with a mobile phase consisting of 20 mmol/l NH4C2H3O2 buffer (pH 5 3.0), methanol and
acetonitrile at a ratio of 53:15:32 at a flow rate of 1.0 ml/min. Peaks
were identified by an LDC (Riviera Beach, FL) ultraviolet detector
set at 250 nm. Sensitivity of the assay was 2.5 ng.
Serial dilutions were prepared fresh every 60 min to ensure that
samples sitting on the bench were not degraded/altered. As in the
binding experiments, peak heights of serial dilutions in 5 mmol/l HCl
represented 100% recovery. Samples from identical serial concentrations from both diluent conditions were injected before measurement
of the next concentration. The order of samples (i.e., 5 mmol/l HCL
vs. assay buffer) were randomized for each concentration.
1997
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Antidepressant Receptor Binding
TABLE 3
Inhibition constants (Ki, nmol/l) of antidepressants for various monoamine transporters and receptorsa
Rat SERT [3H]Citalopram
Rat Cortex
Human SERT [3H]Citalopram
Transfected Cells
Human SERT [3H]5-HT
Transfected Cells
Nefazodone
Hydroxynefazodone
Triazoledione
mCPP
Trazodone
Amitriptyline
Desipramine
Paroxetine
Sertraline
Citalopram
Fluoxetine
Fluvoxamine
Venlafaxine
O-Desmethylvenlafaxine
Chloroimipramine
Nortriptyline
Imipramine
Norfluoxetine
Desmethylsertraline
220 6 4
612 6 11
26,471 6 3,019
218 6 7
237 6 16
16 6 0.8
129 6 7
0.05 6 0.0003
0.29 6 0.01
0.75 6 0.002
2.0 6 0.1
1.5 6 0.01
19 6 1.7
20 6 0.1
0.47
60 6 3.3
8.7 6 0.04
1.9 6 0.04
4.6 6 0.05
459 6 28
1,015 6 27
34,527 6 6,773
202 6 9
252 6 10
2.8 6 0.1
22 6 1
0.065 6 0.006
0.15 6 0.01
1.5 6 0.03
0.90 6 0.06
1.6 6 0.1
7.5 6 0.4
7.7 6 0.3
0.05 6 0.001
15 6 0.1
1.3 6 0.04
2.3 6 0.10
3.7 6 0.26
549 6 26
1203 6 23
70122 6 4514
432 6 24
690 6 47
36 6 1
163 6 5
0.83 6 0.06
3.3 6 0.4
8.9 6 0.7
20 6 2
14 6 1
102 6 9
115 6 6
ND
279 6 20
20 6 2
54 6 4
187 6 15
Receptor assay Radioligand Tissue
source
rat NET [3H]Nisoxetine
Rat Cortex
Human NET [3H]Nisoxetine
Transfected Cells
Human NET [3H]NE
Transfected Cells
Nefazodone
Hydroxynefazodone
Triazoledione
mCPP
Trazodone
Amitriptyline
Desipramine
Paroxetine
Sertraline
Citalopram
Fluoxetine
Fluvoxamine
Venlafaxine
O-Desmethylvenlafaxine
Mazindol
Nortriptyline
Imipramine
Norfluoxetine
Desmethylsertraline
555 6 8
728 6 32
.100,000
699 6 5
20,887 6 703
8.6 6 0.04
0.31 6 0.01
59 6 0.7
1,597 6 44
3,042 6 58
473 6 11
940 6 27
1,067 6 29
938 6 86
0.47
0.99 6 0.03
11 6 0.74
4,997 6 206
3,411 6 127
618 6 12
664 6 24
.100,000
1,940 6 152
37,419 6 1,433
19 6 1
0.63 6 0.03
85 6 5
817 6 80
7,865 6 304
777 6 37
2,950 6 103
2,269 6 84
2,753 6 114
0.46 6 0.004
1.8 6 0.07
20 6 0.54
3,947 6 222
811 6 17
713 6 27
1,053 6 49
.100,000
4,360 6 263
30,751 6 2,110
102 6 9
3.5 6 0.6
328 6 25
1,716 6 151
30,285 6 1,600
2,186 6 142
4,743 6 639
1,644 6 84
3,152 6 103
1.93
21 6 0.77
142 6 8
3,050 6 146
2,365 6 12
Receptor Assay
Radioligand
Tissue Source
5-HT2A
[3H]Ketanserin
Rat Cortex
5-HT1A
[3H]8-OH-DPAT
Rat Hippocampus
H1
[3H]Pyrilamine
Guinea Pig Brain
Alpha-1
[3H]Prazosin
Human Cortex
Alpha-2
[3H]Rauwolscine
Human Cortex
Muscarinic
[3H]NMSb
Rat Cortex
Nefazodone
Hydroxynefazodone
Triazoledione
mCPP
Trazodone
Amitriptyline
Desipramine
Paroxetine
Sertraline
Fluoxetine
Venlafaxine
Cinanserin
Serotonin
Chlorpheniramine
Phentolamine
Yohimbine
Atropine
7.1 6 0.4
7.2 6 0.4
159 6 16
110 6 3
20 6 1
5.3 6 0.2
115 6 13
6,320 6 608
2,207 6 60
141 6 9
.100,000
3.5
52 6 1
56 6 1
1,371 6 42
16 6 0.2
42 6 1
129 6 2
2,272 6 10
21,168 6 2401
3,663 6 82
8,313 6 62
.100,000
30 6 1
28 6 0.2
11 6 0.9
449 6 10
29 6 1
0.17 6 0.01
31 6 1
13,746 6 404
5,042 6 165
933 6 23
12,909 6 1,075
5.5 6 0.3
8.0 6 0.4
173 6 4
97 6 3
12 6 0.2
4.4 6 0.2
23 6 1
995 6 35
36 6 2
1,353 6 17
39,921 6 810
84 6 4
63 6 2
1,915 6 90
112 6 6
106 6 2
114 6 2
1,379 6 39
3,915 6 114
477 6 17
3,090 6 121
.100,000
4,569 6 42
11,357 6 313
.100,000
4,702 6 59
12,188 6 185
2.6 6 0.1
37 6 1
42 6 2
232 6 11
512 6 12
29,966 6 1,464
0.12
0.44
3.4
0.71
0.080
Geometric mean 6 S.E. from three to six separate experiments (see “Methods”).
b 3
[ H]-N-methylscopolamine.
c
ND, not determined.
a
the same ligand ([3H]citalopram or [3H]nisoxetine) to bind to
the rat and human SERT and NET, respectively, or the
affinities calculated with the radioligands above versus active transport of [3H]5-HT or [3H]NE. As expected, highly
significant correlations were found between the affinity of
the antidepressants for the transporters measured by
[3H]citalopram or [3H]nisoxetine and active monoamine
transport in cultured cells (fig. 4, B and D). As shown in
figure 4, A and C, competition assays with either [3H]citalopram or [3H]nisoxetine showed highly significant correla-
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Receptor Assay Radioligand Tissue
Source
1310
Owens et al.
Vol. 283
tions (P , .0001) comparing the rat and human versions of
the respective transporters. Indeed, linear regression yielded
almost a perfect one-to-one correlation for both the SERT and
NET. However, in the SERT, the TCAs amitriptyline, nortriptyline, imipramine, desipramine and chloroimipramine
were 4.5 to 10 times more potent (table 3) at the human
SERT. This increased potency is shown by the TCAs being
below the regression line in figure 4A.
As shown in table 4, several correlations were observed in
the affinities between different receptor/transporter systems.
The potency of the various antidepressants for either the
SERT or NET was positively correlated with the affinity at
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Fig. 2. Averaged competition curves for
each of the transporters and receptors examined. The amount bound for each individual curve was converted to % total
binding and the means of three to six separate experiments are plotted as the averaged curve for each drug. All curves were
best fit by PRISM to a one-site competition model. Πrepresents the compound
used to define nonspecific binding in each
of the assays: A and C, chloroimipramine;
D to F, mazindol; G, serotonin; H, cinanserin; I, atropine; J, phentolamine; K, yohimbine; L, chlorpheniramine.
1997
Antidepressant Receptor Binding
1311
rat muscarinic receptors. Positive correlations between the
affinities for the 5-HT2A, 5-HT1A, H1, alpha-1 and alpha-2
receptors were also observed. The strongest correlations were
observed between alpha-1 and alpha-2 receptors (r 5 0.920)
and 5-HT1A and alpha-2 receptors (r 5 0.933). The results
suggest that the antidepressants tested here possess certain
individual structural features that render a certain degree of
potency to a variety of G protein-coupled receptors, the exception being muscarinic receptors. Finally, if not for triazoledione which possesses negligible SERT activity and venlafaxine which is inactive at the 5-HT2A and 5-HT1A
receptors, a negative correlation would be observed between
SERT affinity and 5-HT2A or 5-HT1A affinity (P , .01, data
not shown).
As shown in table 5, highly significant correlations between the affinity of the antidepressants for rodent and human versions of the 5-HT1A, 5-HT2A, H1 and muscarinic
receptors were observed. Although the affinities were highly
correlated between species, we observed that the absolute
potencies were higher in our studies with rat or guinea pig
brain. However, a similar higher affinity for all drugs was
observed in our studies with the alpha-1 and alpha-2 receptors which used human cortex. We believe this could be the
result of our using 5 mmol/l HCl to prepare our serial dilu-
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Fig. 2cd.
1312
Owens et al.
Vol. 283
tions of competing ligand, which does not allow loss of competing drug as observed when serial dilutions are prepared in
assay buffer (see below). Additionally, differences in assay
conditions could contribute to the differences, but this should
be minimal when Ki or Kd values are calculated rather than
IC50 values. Perhaps more likely are species differences that
result in different absolute affinities, but do not change rank
orders of potency. With a limited amount of human tissue
available, we did note that the phenylpiperazine antidepressants (nefazodone, its metabolites and trazodone), but not
the other antidepressants examined, were 5- to 10-fold less
potent at the human H1 than the guinea pig H1 receptor
(data not shown). However, these values were still substantially more potent for nefazodone than those reported by
Cusack et al. (1994).
HPLC analysis of nefazodone concentrations in serial dilutions. Serial dilutions of competing drugs are routinely prepared in assay buffer after dissolution in the appropriate solvent. We observed that after complete
dissolution of the various drugs in an acid/ethanol mixture
and further serial dilution in assay buffer, several drugs
produced competition curves with very high Hill coefficients
(nH . 1.8) and could not be accurately curve fitted (fig. 5)
(Morgan et al., 1995). These very steep curves were observed
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Fig. 2ef.
1997
Antidepressant Receptor Binding
1313
in rat SERT, 5-HT2A and 5-HT1A receptor assays (they were
not examined in the other assays) and were most pronounced
for nefazodone, hydroxynefazodone, trazodone, sertraline
and amitriptyline (data not shown). They were more modestly observed for triazoledione and paroxetine, and not observed at all for mCPP, venlafaxine, fluoxetine and desipramine. In addition to the very steep competition curves
produced by certain drugs, the drugs also appeared substantially less potent as shown by IC50 values. (Ki could not be
calculated in the steep curves because of the poor curve fit.)
To compare the absolute amount of nefazodone in several
types of serial dilutions, we prepared fresh serial dilutions of
nefazodone in: 5 mmol/l HCl as was used in all data pre-
sented in table 3 and figure 2, A to L, and in buffer from the
[3H]citalopram assay. Samples were immediately subjected
to HPLC analysis and compared for nefazodone concentrations (table 6). Serial dilution of nefazodone prepared in 5
mmol/l HCl was equal to that prepared in mobile phase (M.
Owens, unpublished observations) and was taken to equal
100% recovery. As shown in table 6, dilutions prepared in
assay buffer were significantly lower. Moreover, recovery
decreased with increasing dilution. The addition of H1 (25 ml
of 1 mol/l HCl) to the assay buffer increased recovery to a
limited extent (data not shown). Using the actual nefazodone
concentrations as determined by HPLC (table 6), we compared the [3H]citalopram competition curves in rat cortex
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Fig. 2gh.
1314
Owens et al.
Vol. 283
produced by nefazodone in 5 mmol/l HCl assay buffer, and
assay buffer corrected for actual nefazodone concentrations
(fig. 6). As shown in figure 6, the corrected nefazodone curve
was now similar to the nefazodone in HCl curve in terms of
the observed potency of nefazodone. Moreover, the curve
could now be fit to an idealized one-site competition model
and had a Hill coefficient near 1.0.
Discussion
Nefazodone, venlafaxine, fluvoxamine and mirtazapine
have all recently been introduced in the United States for
clinical use. These compounds are inhibitors of monoamine
transporters, with the exception of mirtazapine, which appears to be primarily an alpha-2 antagonist at auto- and
heteroreceptors as well as a potent 5-HT2A and 5-HT3 antagonist (de Boer and Ruigt, 1995). Unlike venlafaxine and
fluvoxamine, which only have high affinity for monoamine
transporters, nefazodone possesses potent 5-HT2A receptor
antagonism as well as monoamine transporter antagonist
properties (Taylor et al., 1995; Owens et al., 1995). Several
years have passed since the comprehensive studies of Richelson and colleagues (Bolden-Watson and Richelson, 1993;
Cusack et al., 1994) appeared in which the receptor binding
profile of several antidepressants and metabolites were examined. Many earlier studies either focused on a single re-
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Fig. 2ij.
1997
Antidepressant Receptor Binding
1315
ceptor/transporter or did not include active metabolites for
many of the compounds. Moreover, many studies reported
IC50 inhibition values which highly depended on both the
radioligand used and assay conditions, and were difficult to
compare across laboratories. Thus, we examined in detail the
binding profile of several antidepressants and their metabolites that are monoamine transporter antagonists with particular attention paid to nefazodone (fig. 1).
Antagonism/inhibition of the SERT was established by
competition for [3H]citalopram from either rat frontal cortical membranes or from a HEK-293 cell line stably transfected with the human SERT or antagonism of [3H]5-HT
transport into intact HEK-293 cells expressing the human
SERT. [3H]Citalopram possesses several advantages compared with other ligands for labeling the SERT including the
greatest selectivity, higher specific activity and an affinity
which, unlike [3H]paroxetine, provides sufficient signal without depleting free ligand concentration at typical assay volumes and protein amounts (D’Amato et al., 1987; Owens et
al., 1996). Although it is not known for certain, citalopram
and paroxetine probably label the site near or at the site
which serotonin itself occupies for transport (Barker et al.,
1994; Barker and Blakely, 1996). Nevertheless, actual inhibition of [3H]monoamine transport may represent the best
measure of transporter antagonism.
As shown in table 3 and figure 2, A to C, only the nefaz-
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Fig. 2kl.
1316
Owens et al.
Vol. 283
odone metabolite, triazoledione, did not possess any affinity
for the SERT. Only moderate affinity for the SERT was
observed for nefazodone and its metabolites. Previously, both
nefazodone and hydroxynefazodone have demonstrated Ki
values between 137 and 181 nmol/l for inhibition of rat [3H]5HT transport (Bolden-Watson and Richelson, 1993; Taylor et
al., 1995). Moreover, serum levels observed in rats which
significantly, but not completely, inhibit 5-HT transport are
consistent with those obtained with oral nefazodone dosages
of 300 to 500 mg/day in humans which demonstrated clinical
antidepressant efficacy (Owens et al., 1995; Robinson et al.,
1996).
The potency of the various antidepressants at the rat
SERT agrees very well with those of other studies which
typically included only a few compounds (D’Amato et al.,
1987; Plenge and Mellerup, 1991; Cheetham et al., 1993).
Unlike, sertraline, amitriptyline and imipramine, the desmethyl metabolites of fluoxetine and venlafaxine had potency
similar to their parent compounds. Desmethylsertraline still
retains high potency and accumulates to 1.6- to 2.1-fold
higher levels than sertraline in plasma, but in vivo data
suggest that it may not contribute significantly to inhibition
of 5-HT transport clinically (Sprouse et al., 1996). This conclusion may be born out in the finding of significantly reduced potency versus sertraline in 5-HT transport via the
human SERT.
The affinity of the various compounds for displacing
[3H]citalopram from the rat and human versions of the SERT
were very similar and highly correlated (tables 3 and 4, fig.
4A). The exceptions are the clearly higher potencies of the
TCAs amitriptyline, nortriptyline, imipramine, desipramine
and chloroimipramine for the human SERT. The increased
potency of the tricyclics at the human SERT compared with
the rat SERT agrees very well with the findings of Barker
and colleagues (Barker et al., 1994; Barker and Blakely,
1996) who used chimeric rat and human SERTs. This property of the tricyclics appears to be attributed to a region near
putative transmembrane domain 12 of the human SERT
which imparts some species (human) preference for TCAs.
Inhibition of [3H]5-HT transport was also highly correlated
with the ability of individual compounds to displace [3H]citalopram (tables 3 and 4, fig. 4B). These results agree very well
with those of Bolden-Watson and Richelson (1993) and
Cheetham et al. (1993).
[3H]Nisoxetine was used to label rat and human NETs.
Nisoxetine is 400- and 1000-fold more potent in binding to
the NET than the DAT and SERT, respectively. The binding
is saturable and Na1-dependent to a single class of binding
sites (Tejani-Butt, 1992). As shown in table 3 and figure 2, D
and E, other than the TCAs, paroxetine was the only other
compound possessing moderately high affinity. Even in the
face of its high relative SERT selectivity (fig. 3), preliminary
studies with serum concentrations of paroxetine similar to
those used to treat panic disorder antagonize the NET in rats
(M. J. Owens, D. L. Knight and C. B. Nemeroff, unpublished
observations). Although nefazodone and trazodone possess
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Fig. 3. Relative selectivities for the antidepressants and metabolites for the SERT and NET. The Ki in nanomoles per liter for the NET was divided
by the Ki for the SERT and resulted in a unitless value in which 1 equals equipotency for both transporters. Values ,1 represent relatively greater
NET selectivity and values .1 represent relatively greater SERT selectivity. There is no unit for the y-axis. Individual drugs are spread out along
the y-axis to aid in viewing the data only. HUMAN 3H-AMINE represents [3H]NE and [3H]5-HT transport in the cell lines expressing the hNET or
hSERT. RAT 3H-NIS/3H-CIT represents [3H]nisoxetine and [3H]citalopram binding in rat frontal cortex. HUMAN 3H-NIS/3H-CIT represents
[3H]nisoxetine and [3H]citalopram binding in membranes prepared from the cell lines expressing the hNET or hSERT. DMI, desipramine; NOR,
nortriptyline; OH-NEF, hydroxynefazodone; TRIAZ, triazoledione; NEF, nefazodone; AMI, amitriptyline; IMI, imipramine; DMS, desmethysertraline;
VEN, venlafaxine; O-DMVEN, O-desmethylvenlafaxine; TRAZ, trazodone; NORFLUOX, norfluoxetine; FLUOX, fluoxetine; FLUVOX, fluvoxamine;
PAROX, paroxetine; SER, sertraline; CIT, citalopram.
1997
Antidepressant Receptor Binding
TABLE 4
Pearson correlations between binding affinity at the various
transporters and receptorsa
a
P value
n
0.964
0.962
0.961
0.980
0.982
0.957
0.618
0.704
0.701
0.806
0.820
0.751
0.847
0.842
0.865
0.868
0.847
0.639
0.803
0.933
0.842
0.639
0.739
0.613
0.865
0.803
0.739
0.920
0.868
0.933
0.613
0.920
,.0001
,.0011
,.0001
,.0001
,.0001
,.0001
.043
.016
.016
.003
.002
.008
.001
.001
,.001
,.001
.001
.034
.003
,.001
.001
.034
.009
.046
,.001
.003
.009
,.001
,.001
,.001
.046
,.001
19
18
18
19
19
19
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
Only those correlations that were significant are shown.
similar SERT affinity, trazodone is inactive at the NET suggesting that important structural requirements for NET activity are found in either the phenoxyethyl substituent or the
5-substituent of the triazole moiety, or both (fig. 1).
Our findings for the inhibition of [3H]NE transport in cells
expressing the human NET are very similar to those observed for inhibition of [3H]5-HT transport by the human
SERT in that the rank order of potency was similar to that
observed for radioligand binding (table 3, figs. 2, D and E,
and 4D). Of the same drugs tested, our calculated Ki values
were identical with those reported by Pacholczyk et al. (1991)
for the transfected human NET.
As mentioned above, absolute affinity was highly correlated among the compounds for the rat and human NET and
between the use of [3H]nisoxetine and [3H]NE (table 3, fig. 4,
C and D). Indeed, this correlation was even stronger than
that observed for the SERTs because of the similar potencies
of the TCAs for rat and human NETs. These data suggest
that rat tissue does provide useful data regarding the potency of various compounds for the human NET and that this
can be reflected in radioligand binding studies which are
easier to perform.
The relative selectivities for the SERT and NET are shown
in figure 3. The rank order of selectivity varies slightly depending on the assay method. Desipramine and nortriptyline
are clearly the most NET selective of the compounds tested,
and sertraline and citalopram are the most SERT selective.
Although relatively weak antagonists of the SERT and NET,
nefazodone and hydroxynefazodone are the closest to being
called “dual uptake inhibitors.” Our data show that venlafaxine and O-desmethylvenlafaxine have, at a minimum, a .15fold selectivity for the SERT. These selectivities are relative,
and with escalating dosages and increased free drug concentrations in serum, even relatively selective drugs can begin
to, or fully, bind to other transporters/receptors.
Amitriptyline, nefazodone and hydroxynefazodone were
potent at displacing [3H]ketanserin binding from the rat
cortical 5-HT2A receptor (table 3). Trazodone also displayed
significant potency (Ki 5 20 nmol/l). These data agree closely
with data reported previously (Wander et al., 1986; Seeman,
1993; Cusack et al., 1994). Our results from rat tissue are
highly correlated with those of Cusack et al. (1994) who used
human cortex (table 5), although our antidepressants displayed somewhat higher affinity in all instances. Once again,
we believe this could be related to the method of serial dilution and/or species differences in absolute affinity.
The phenylpiperazine derivatives possessed moderate affinity for the rat hippocampal 5-HT1A receptor (table 3).
mCPP is thought to act as an agonist, whereas it is not
known whether the parent drugs are antagonists or agonists,
although antagonism is more likely. It has been suggested
that combined with the potent 5-HT2A antagonism, these
compounds do augment 5-HT1A-mediated function (Taylor et
al., 1995). These findings agree with those of Richelson
and colleagues (Wander et al., 1986; Cusack et al., 1994)
and those reported in Seeman (1993). Our data in rat
hippocampus was highly correlated with that in human
tissue (table 5).
As previously reported by the manufacturer (Paxil, package insert), paroxetine possesses moderately high affinity for
muscarinic receptors similar to that observed for the TCA
desipramine (table 3). Once again, our data in rats are highly
correlated with data observed in humans (Stanton et al.,
1993; Cusack et al., 1994) and in Seeman (1993).
As noted under “Methods,” H1 receptors in guinea pig brain
display closer pharmacology to the human H1 receptor than
do rat H1 receptors. Amitriptyline was the most potent drug
tested. However, trazodone, nefazodone, hydroxynefazodone
and triazoledione all displayed moderately high potency (Ki
values , 30 nmol/l). These findings are in sharp contrast to
those reported by Cusack et al. (1994) who reported Ki values
for nefazodone and trazodone as 24,000 and 1,100 nmol/l,
respectively. This discrepancy may be explained by our findings of loss of drug accompanying serial dilution of the drugs
(see below) and species differences for the binding affinity of
these two drugs (see “Results”). Even with these discrepancies, our data in guinea pig brain was highly correlated with
the data reported by Cusack et al. (1994) in human cortex
(table 5).
Previous work has provided substantial evidence that rat
cortical alpha adrenoceptors have pharmacological characteristics that are different from their human counterparts
(Ruffolo et al., 1991). Therefore, we used human cortical
tissue for our studies of antidepressant alpha-1 and alpha-2
affinities. For each drug tested, we observed that they possessed a higher affinity for alpha-1 receptors than for alpha-2
receptors (table 3). The high potency displayed by nefazodone
and hydroxynefazodone is surprising because the former is
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rat SERT 3 hSERT [ H]citalopram
hSERT [3H]5-HT 3 hSERT [3H]citalopram
hSERT [3H]5-HT 3 rat SERT
Rat NET 3 hNET [3H]nisoxetine
hNET [3H]NE 3 hNET [3H]nisoxetine
hNET [3H]NE 3 rat NET
Muscarinic 3 rat SERT
hSERT [3H]citalopram
hSERT [3H]5-HT
rat NET
hNET [3H]nisoxetine
hNET [3H]NE
5-HT2A 3 5-HT1A
H1
alpha-1
alpha-2
5-HT1A 3 5-HT2A
H1
alpha-1
alpha-2
H1 3 5-HT2A
5-HT1A
alpha-1
alpha-2
Alpha-1 3 5-HT2A
5-HT1A
H1
alpha-2
Alpha-2 3 5-HT2A
5-HT1A
H1
alpha-1
3
Correlation
Coefficient (r)
1317
1318
Owens et al.
Vol. 283
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Fig. 4. Scatter plots, Pearson correlations, linear regressions and 95% confidence intervals comparing the same radioligand across species (A
and C) and radioligand binding versus active transport in cells expressing the human SERT (B) or human NET (D). DMI, desipramine; NOR,
nortriptyline; OH-NEF, hydroxynefazodone; TRIAZ, triazoledione; NEF, nefazodone; AMI, amitriptyline; IMI, imipramine; DMS, desmethysertraline;
VEN, venlafaxine; O-DMVEN, O-desmethylvenlafaxine; TRAZ, trazodone; NORFLUOX, norfluoxetine; FLUOX, fluoxetine; FLUVOX, fluvoxamine;
PAROX, paroxetine; SER, sertraline; CIT, citalopram; CHLORO, chloroimipramine; MAZ, mazindol.
reportedly associated with significantly less orthostasis
when compared with amitriptyline. Nefazodone and hydroxynefazodone were the only drugs tested that had moderate affinities (,100 nmol/l) for the alpha-2 receptor. As
expected, because of the use of the same species tissues, both
our alpha-1 and alpha-2 data were highly correlated with the
data reported by Cusack et al. (1994). However, they were not
any greater than that observed with the 5-HT2A, 5-HT1A or
muscarinic receptors in which we used rat tissue (table 5).
Although we do not know of any studies of structure-
activity relationships between the compounds studied here,
the strong positive correlations observed between the affinity
of individual compounds for the 5-HT1A, 5-HT2A, H1, alpha-1
and alpha-2 receptors suggests that these individual drugs
possess some structural characteristics that determine affinity for several G protein-coupled receptors, the only exception
being the muscarinic receptors. Standard hydropathy analyses of the primary sequences for these receptors found that
the N-terminal region of the rat m1 receptor, which contributes most of the muscarinic binding in rat cortex, possesses
1997
1319
Antidepressant Receptor Binding
TABLE 5
Correlations between rodent and human brain for the affinities of various antidepressantsa
Human
Human
Human
Human
Human
Human
Correlation
Coefficient (r)
P value
n
0.974
0.945
0.854
0.992
0.985
0.933
,.0001
.0004
.0065
,.0001
,.0001
.0007
8
8
8
8
8
8
5-HT2A 3 rat 5-HT2A
5-HT1A 3 rat 5-HT1A
H1 3 guinea pig H1
muscarinic 3 rat muscarinic
alpha-1
alpha-2
Linear regressionb
y 5 mx 1 b
y
y
y
y
y
y
5
5
5
5
5
5
0.88x
0.89x
0.80x
0.94x
0.82x
0.67x
2
2
2
2
2
2
0.49
0.25
0.41
0.05
0.68
1.41
a
pKi values were compared for the following antidepressants using data from the present study and the study of Cusack et al. (1994), which used human tissue
exclusively: amitriptyline, desipramine, nefazodone, fluoxetine, paroxetine, sertraline, trazodone and venlafaxine. Human tissue was used in both studies of alpha-1 and
alpha-2 receptor affinities.
b
y represents pKi values obtained in human tissue by Cusack et al. (1994); x represents pKi values obtained in the present manuscript. A regression formula of y 5
1.0x 1 0 would represent an absolute one-to-one correspondence between the two studies. Slopes ,1 represent that a relatively higher affinity for a given compound
was observed in the present study compared with that of Cusack et al. (1994).
TABLE 6
Nefazodone concentrations (mmol/l) in serial dilution tubes
determined by HPLCa
5 mmol/l HCl
Assay Bufferb
502.4
317.0
200.0
126.2
79.6
50.2
31.7
20.0
183.8 6 35.2
72.0 6 6.0
33.6 6 2.6
17.5 6 3.7
8.4 6 0.9
3.9 6 0.3
1.7 6 0.4
0.82 6 0.04
a
Serial dilutions performed in 5 mmol/l HCl were identical with those made in
mobile phase. Only a single injection was tested for each concentration for each
series of dilutions. n 5 5 for each concentration.
b
Assay buffer is that used for [3H]citalopram binding (table 1).
structural features significantly different from the other receptors (Arnold J. Mandell, personal communication). This
portion of the m1 receptor does not appear to be important in
binding of “muscarinic” ligands (Brann et al., 1993).
Nefazodone is an effective antidepressant that is marketed
as a serotonergic modulating agent that possesses a favorable side effect profile compared with the structurally related
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Fig. 5. Nefazodone competition curves for the 5-HT2A receptor in rat
cortex. NEFAZODONE in HCl is the final averaged curve taken from
figure 2H (Ki 5 7.1 nmol/l). NEFAZODONE in buffer is a representative
of several curves obtained during the use of assay buffer to perform
serial dilutions. A one-site competition curve was attempted to fit the
data by PRISM but could not be accurately accomplished, thus a Ki
could not be calculated. However, the IC50 was 4753 nmol/l whereas
that of nefazodone in HCl was 14.2 nmol/l. The calculated Hill coefficient for NEFAZODONE in buffer was 3.05.
antidepressant, trazodone, and compared with the TCAs
(Taylor et al., 1995). Unlike the SERT selective antagonists,
nefazodone neither produces sexual dysfunction nor alters
sleep architecture. Pharmacokinetic analyses find that, at
steady-state concentrations, the molar hydroxynefazodone
AUC is approximately 35% that of nefazodone. The molar
AUC of mCPP is approximately 13% of that of nefazodone
and that of the triazoledione tautomer is 160% that of nefazodone (Kaul et al., 1995; Mayol et al., 1994). Although nefazodone is not particularly potent in vitro at antagonizing the
SERT and NET, because of the high circulating plasma concentrations of drug, it can produce some transporter inhibition in vivo (Hemrick-Leucke et al., 1994; Owens et al., 1995).
Based on metabolic patterns and affinity, the three major
metabolites of nefazodone are unlikely to contribute to any
transporter inhibition in vivo. Nefazodone and hydroxynefazodone have similar affinity for all the other receptors tested
and include potent affinity for the 5-HT2A site which is
thought to represent an important, although not well understood, aspect of its clinical effectiveness (Taylor et al., 1995).
These two drugs are also quite potent at the alpha-1 receptor,
which is not consistent with the lack of orthostatic side effects and is one instance in which in vitro binding affinity
may not accurately predict potential side effects. The relatively potent affinity of nefazodone, hydroxynefazodone and
triazoledione at the H1 receptor are consistent with the sedative properties of nefazodone. Nefazodone and hydroxynefazodone possess moderate affinity for the alpha-2 receptor.
One could speculate that this may allow nefazodone to act, in
part, in a manner similar to the new antidepressant mirtazapine on alpha-2 auto- and heteroreceptors. Additionally, the
moderately high affinity for 5-HT1A receptors suggests that
nefazodone may have some inherent properties that mimic
the strategy being utilized by the use of pindolol augmentation as a means to block 5-HT1A somatodendritic autoreceptors and hasten clinical response and/or convert antidepressant nonresponders to responders. Although trazodone
shows a striking similarity to nefazodone in vitro, with the
exception of lack of NET antagonism, trazodone rarely produces priapism, whereas nefazodone does not. The mechanism of this difference is not known or discernible from the
present binding studies.
The TCAs exhibited a binding profile very similar to that
reported previously. Thus, amitriptyline, imipramine, nortriptyline and desipramine showed high affinity for the
SERT, particularly the human version, and for the NET in
which the secondary amines were more potent. In agreement
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Owens et al.
Vol. 283
Fig. 6. Nefazodone competition curves
for the rat SERT. NEFAZODONE in HCl
and NEFAZODONE in buffer are single
representative curves observed in the
experiments. Corrected NEFAZODONE
in buffer is the NEFAZODONE in buffer
curve corrected for the actual nefazodone concentrations observed by
HPLC analysis in table 6.
SERT antagonism are considerably smaller than those
needed for alpha-1 blockade.
The curves shown in figure 5 represent the curves we
initially observed for several drugs in many different assays.
Steep slopes are generally a sign of positive cooperativity
which is observed with enzyme kinetics, but not typically in
radioligand assays. We considered whether this was a solubility issue; however, an incomplete dissolution of drug would
indeed shift the curve to the right but would not change the
shape of the curve as observed here. Because these drugs are
all weak bases, even at the physiological pH of the buffers,
most of the drug would be in an ionized form and likely more
soluble in the aqueous buffer than as the free base. Thus, the
high degree of ionization produced by the 5 mmol/l HCl
dilutions was probably not important for drug dissolution. All
the compounds, with the exception of desmethylsertraline
and mazindol, were easily solubilized in the 50% ethanol:50%
5 mmol/l HCl solution at 1 mg/ml. Even when using assay
buffer alone to perform serial dilutions, the dilution from 1
mg/ml to the most concentrated dilution for the assay was
200 to 250 mg drug/ml assay buffer and resulted in apparently complete solubility. We further confirmed the fact that
the drug was fully dissolved and not a particulate suspension
by measuring drug levels directly from the serial dilution
tubes where we observed a significant loss of drug (table 6
and fig. 6). We further observed that addition to the assay
buffer serial dilutions of 25 ml of 1 mol/l HCl directly before
HPLC analysis resulted in a significant, but not complete,
recovery of drug that was observed to be “missing” in the
dilutions of assay buffer alone (data not shown).
The apparent loss of potency and the steep competition
curves could plausibly be explained by the presence of a
saturable nonspecific binding site not affected by the silanization process on the walls of the glass tubes. Although
this is only speculative, this would mean that at lower drug
concentrations all the drug is bound to this unidentified site,
which results in no competition for radioligand. At the point
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with previous data, the TCAs had high affinity for the H1,
alpha-1 and muscarinic receptors, which correlates well with
their known side effect pattern of sedation, orthostatic hypotension, dry mouth, constipation and tachycardia. One could
also speculate that amitriptyline (table 3) and nortriptyline,
but not imipramine or desipramine (Cusack et al., 1994), may
also act therapeutically via 5-HT2A antagonism.
Venlafaxine, paroxetine, sertraline, citalopram, fluoxetine,
fluvoxamine and their various metabolites are all potent
antagonists of the SERT. Although marketed as a “dual
uptake inhibitor” (Effexor, package insert; Muth et al., 1986),
venlafaxine and O-desmethylvenalfaxine are not potent NET
antagonists in vitro, although they do show activity in vivo.
This may be explained by the fact that free drug concentrations in vivo may be relatively high because venlafaxine
shows considerably less plasma protein binding (;20 –25%
bound) than any of the other compounds and, therefore,
likely antagonizes the NET as well as the SERT in vivo. Of
the other non-TCAs tested, only paroxetine showed moderately high affinity for the NET, although it was still 2 to 3
orders of magnitude more potent at the SERT. However, as
noted earlier, we have preliminary evidence that paroxetine
administration in the high therapeutic range may affect the
NET in vivo.
In general, the SERT selective antagonists were devoid of
any meaningful potency at the various other receptors we
examined with two exceptions. Paroxetine has moderately
high potency for both muscarinic receptors (Ki 5 41 nmol/l)
and the NET as noted above. This former finding is undoubtedly responsible for the dry mouth and occasional blurry
vision observed with paroxetine use but it may reduce the
frequency of diarrhea and loose stools that accompany the
use of other SERT antagonists. The second exception is the
moderately high potency of sertraline for alpha-1 receptors
(Ki 5 36 nmol/l). Although one might predict significant
orthostasis as a result, this has not been observed clinically,
perhaps because concentrations necessary for adequate
1997
where this site becomes saturated (i.e., significantly higher
concentrations of serial drug dilution), there is now a large
amount of drug available and considerable competition occurs. Because the data are plotted based on assumed concentrations, a very sharp drop in binding (i.e., steep curve)
occurs. Whatever the mechanism, we believe that this may
represent one explanation why in almost all instances in the
present set of studies, the drugs we tested had higher affinity
than those previously reported by others for the same compounds. We also believe that, in the future, unless every
compound to be tested is examined comparing assay buffer
versus dilute acid serial dilution, that serial dilutions for all
drugs that are weak bases should be performed in dilute acid
rather than assay buffer.
Acknowledgments
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The authors thank David L. Knight of the Department of Psychiatry & Behavioral Sciences, and Joseph Daley and Laura Wurthheimer of the Emory University School of Medicine for their technical
assistance. We are grateful to our departmental colleagues Arnold J.
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University Alzheimer’s Disease Research Center for generously providing the human brain tissue.
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Send reprint requests to: Michael J. Owens, Ph.D., Laboratory of Neuropsychopharmacology, Department of Psychiatry & Behavioral Sciences, 1639
Pierce Drive, Suite 4000, Emory University School of Medicine, Atlanta, GA
30322.
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