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Vol. 283, Issue 3, 1305-1322, 1997
Laboratory of Neuropsychopharmacology, Department of Psychiatry and Behavioral Sciences, Emory University School of Medicine, Atlanta, Georgia
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Abstract |
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Abstract
Introduction
Methods
Results
Discussion
References
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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 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 = 0.965) or NET (r = 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.
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Introduction |
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Abstract
Introduction
Methods
Results
Discussion
References
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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 treatment 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
O-desmethylvenlafaxine.
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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. Moreover, their affinity at both the rat and human
variants of the SERT and NET are compared.
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Methods |
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Abstract
Introduction
Methods
Results
Discussion
References
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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
80°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 × 12 seconds) in 30 volumes of their individual assay buffers
(table 1) at 4°C, and centrifuged at
43,000 × 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 × 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 70°C until assayed.
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) or
human NET cDNA (Galli et al., 1995) into HEK-293 (human
embryonic kidney) cells has resulted in cell lines exhibiting
high-affinity, Na+-dependent 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 µg/ml) and 100 units/ml streptomycin in a humidified incubator at 37°C containing 5%
CO2. The selecting antibiotic geneticin sulfate
(250 µg/ml) was used during all growth phases. Membranes were
harvested using 37°C phosphate-buffered saline containing 0.53 mmol/l
ethylenediaminetetraacetic acid, separated into aliquots, and
centrifuged at 2000 × 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
10
13 to 104.6
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
()-norepinephrine).
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; 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 µl 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 ± 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).
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 µl
were injected onto a 150 × 4.6 mm BDS-Hypersil-Phenyl 5-µm
column with guard (Keystone Scientific, Inc., Bellefonte, PA) with a
mobile phase consisting of 20 mmol/l NH4C2H3O2
buffer (pH = 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.
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Results |
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Abstract
Introduction
Methods
Results
Discussion
References
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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]8-OH-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.
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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 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 correlations (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.
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.
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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 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.)
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Discussion |
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Abstract
Introduction
Methods
Results
Discussion
References
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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 receptor/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 nefazodone
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]5-HT 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
Na+-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 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 >15-fold 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 = 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
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 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 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 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 = 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 = 36 nmol/l). Although one might predict significant orthostasis as a result, this has not been observed clinically, perhaps because concentrations necessary for adequate 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 µg 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 µl 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 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.
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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. Mandell, M.D., and Karen Selz, Ph.D., of the Laboratory of Biological Dynamics and Theoretical Neuroscience for assistance with hydropathy analyses. We also thank Dr. Suzanne Mirra of the Emory University Alzheimer's Disease Research Center for generously providing the human brain tissue.
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Footnotes |
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Accepted for publication August 12, 1997.
Received for publication February 14, 1997.
1 Supported by a grant from Bristol Myers-Squibb, the Stanley Foundation Scholars Program, and NIMH MH-40524.
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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Abbreviations |
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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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References |
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Abstract
Introduction
Methods
Results
Discussion
References
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represents 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.
