Specific modulation of sigma binding sites by the anxiolytic drug opipramol

DOI 10.1007/s00702-003-0019-5

J Neural Transm (2003) 110: 1169–1179

Specific modulation of sigma binding sites by the anxiolytic

drug opipramol

G. Holoubek and W. E. Muller

Department of Pharmacology, Biocenter University Frankfurt,Frankfurt, Germany

Received February 7, 2003; accepted March 24, 2003Published online July 30, 2003; # Springer-Verlag 2003

Summary. The atypical anxiolytic and antidepressive drug opipramol bindswith high affinity to s1 and somewhat lower affinity to s2 sites. After subchron-ic treatment, opipramol significantly down-regulated s2 but not s1 sites. Thiseffect was not seen for imipramine, citalopram, and reboxetine under similarconditions. On the other hand, only imipramine reduced the number of s1 sites.It is suggested that effects at s2 sites are involved in the anxiolytic properties ofopipramol.

Keywords: Opipramol, sigma binding sites, subchronic treatment, anxiolytics,antidepressants, forced swimming test, rats.

Introduction

Opipramol (Fig. 1) is structurally related to the tricyclic antidepressant imi-pramine and the anticonvulsant and mood stabilizing drug carbamazepine.Originally developed in the 1960s as an antidepressant (Theobald et al.,1964; Rajotte et al., 1966) its even more pronounced anxiolytic properties weresoon recognized (Arfwidsson et al., 1971; Grosser and Ryan, 1965). They haverecently been confirmed in a well-designed clinical trial in patients sufferingfrom generalized anxiety disorders. In this study opipramol demonstratedsuperiority over placebo and had similar efficacy as alprazolam, however, witha more favorable side effect profile (M€ooller et al., 2001). Opipramol is clinicallyused in Germany and a few other European countries with increasing tendencyas a substitute of benzodiazepines for long-term application.

Although structurally related to imipramine, even the initial pharmaco-logical studies with opipramol indicated major differences to the tricyclics,especially since opipramol is not an inhibitor of the neuronal reuptake ofnorepinephrine and serotonin (Theobald et al., 1964; M€uuller and Siebert,1998; Ferris et al., 1991). It is active in animal models of antidepressive activ-ity, but its anxiolytic properties are already seen at much lower concentrations

(M€uuller and Siebert, 1998), which is also different to imipramine. The differentmechanisms of action for opipramol and imipramine remain unclear. Ferris et al.(1991) reported a potent interaction of opipramol with sigma binding sites as apossible pharmacological mechanism. With the emerging role of sigma recep-tors in psychopharmacology (Leonard, 1997) and the development of specificsigma ligands as antidepressants and anxiolytics (Sanchez et al., 1997; Akunneet al., 2001; Heading, 2001) the potential role of sigma binding site interactionscame into the focus of discussion, recently (M€uuller and Siebert, 1999).

The link of sigma sites with the pharmacology of antidepressants and anxio-lytics is based upon the observation that imipramine and some other antide-pressants might alter sigma binding site properties after subchronic treatment,which in some cases might be even independent of a direct interaction withthese sites (Leonard, 1997; Shirayama et al., 1997; Faherty et al., 1998).Accordingly, we investigated the possible effects of opipramol on the propertiesof sigma binding sites in rat brain after subchronic treatment in comparisonwith typical antidepressants like imipramine, reboxetine and citalopram, whichexhibit weak interactions with sigma binding sites (Narita et al., 1996). Sincethe total population of sigma sites (stot) consists at least of s1 and s2 sites withdifferent properties and different receptor proteins (McCann et al., 1994), bothsubsites were included in our study. Furthermore, well confirmed pharmacolo-gical effects of antidepressants like down-regulation of b adrenoceptor and5HT2 receptor densities, and reduction of immobility time in the forced swim-ming test were investigated for opipramol. The aim of our investigation was thesearch for adaptive neurochemical changes following opipramol treatment pos-sibly related to its potent interaction with sigma receptors. The drugs used forcomparison were antidepressants and anxiolytics possessing reuptake inhibitingproperties with (imipramine) or without (citalopram, reboxetine) receptor pro-file, but each without relevant affinity to sigma sites.

Fig. 1. Structures and receptor profiles of opipramol and imipramine (data taken from M€uullerand Siebert, 1998, 1999)

1170 G. Holoubek and W. E. M€uuller

Material and methods

Animals

Male Sprague Dawley rats (220–250 g) were obtained from Harlan Winkelmann, Borchen(Germany). Animals were housed in plastic cages with water and food ad libitum and weremaintained on a 12 hour light=dark cycle. All experiments were performed in accordance withthe German animal right regulations.

Drug treatment

Opipramol in comparison with imipramine: Solutions of opipramol and imipramine were pre-pared freshly every day at a concentration of 6 mg=ml in 0.2% agarose gel. Rats were weighedimmediately before treatment and obtained 20 mg drug=kg body weight or vehicle by stomachtube. Treatment was performed over 14 days and rats were sacrificed by guillotine 24 h after thelast administration. The dose of 20 mg=kg has previously been shown to give maximum effects inex vivo binding as well as behavioral experiments (M€uuller and Siebert, 1998, 1999).

Opipramol in comparison with reboxetine and citalopram: Because of the short eliminationhalf-lives of reboxetine and citalopram (Dostert et al., 1997; Fredricson et al., 1982) the upperapplication scheme was modified according to a standardized protocol of the forced swimmingtest. Rats were orally treated twice daily with opipramol, reboxetine, citalopram (10 mg=kg) orvehicle over 9 days. The last drug application took place 1 h before the forced swimming test.Rats were sacrificed by guillotine immediately after the test.

Forced swimming test

Rats were placed into a glass cylinder (50 cm height� 25 cm diameter) filled with water (25�C)up to 20 cm. Under these conditions rats are unable to touch the bottom. 48 h before the mainsession (test¼ 5 min) the pretest was performed over 15 min. During the test rats were observedand immobility times stopped.

Preparation of rat brain membranes

After removal of the brains, distinct brain areas typically used to investigate adaptive changes ofthe individual receptor systems were dissected on ice (frontal cortices, hippocampi, cerebelli andmid=hindbrain), immediately frozen in liquid nitrogen, and stored until analysis at � 80�C.

For preparation of rat brain membranes the required brain area was weighed, homogenized in30 ml ice-cold Tris-HCl buffer (50 mM; pH 7.7, 20–25�C) and centrifuged at 48,000 g for 20 min(4�C). Pellets were resuspended in 30 ml ice-cold Tris-HCl buffer and centrifuged as describedabove. The washing procedure was repeated and the pellet resuspended in the required volumeTris-HCl buffer (20 mg=ml for s receptor binding, 6 mg=ml for b and 5HT2 receptor binding).200ml of the membrane homogenates were immediately used in receptor binding experiments(final assay volume: 250ml).

Receptor binding experiments

Saturation experiments were performed using 8 different concentrations of the specific radioli-gand (see Table 1). Reaction mixture was incubated for 30–60 min at 37�C. Reaction was stoppedby a rapid vacuum filtration (24-sample Brandel Cell Harvester) through Whatman GF=C filters.Filters were washed 3 to 7 times with 1 ml icecold buffer and were eluted with 3 ml Lumasafeplus (Packard, Dreieich, Germany). Radioactivity was determined using a TR 1900 scintillationcounter (Canberra-Packard, Dreieich, Germany). Specific binding was calculated subtractingblank from total binding (each measured as triplicate) using unlabelled inhibitors. For detailssee Table 1.

Modulation of sigma binding sites by opipramol 1171

Analysis of receptor binding assays and statistics

Bmax and KD values were calculated using nonlinear regression (Graph Pad+ Prism 3). Effectswere evaluated using two-way and one-way ANOVA with Newman–Keuls multiple comparisonpost test and Student’s t-test (unpaired two-tailed), depending on the groups analyzed. AllANOVAs were significant with a p<0.05 except those comparing KD values of stot. Differenceswere deemed significant when �p<0.05; ��p<0.01; ��p<0.001 in comparison with thevehicle treated control group and are depicted in the graphs and tables.

Protein determination

The protein concentration was determined according to the method of Lowry et al. (1951) usingBioRad+ Protein Assay solutions and bovine serum albumine as standard.

Chemicals

The following drugs were kindly donated: Opipramol from Novartis (Nuremberg, Germany),reboxetine from Pharmacia & Upjohn (Kalamazoo, USA), citalopram from H. Lundbeck A=S(Copenhagen, Denmark). Imipramine was purchased from Sigma Aldrich (Munich, Germany).

3H-DTG (3-Di-O-tolylguanidine, 31 Ci=mmol), 3H(þ)-Pentazocine (28 Ci=mmol), 3H(�)-CGP-12177 (36 Ci=mmol) and 3H-Ketanserin hydrochloride (63 Ci=mmol) were purchased fromPerkinElmer Life Sciences (Cologne, Germany).

Unlabeled haloperidol, DL-propranolol hydrochloride, mianserin hydrochloride were pur-chased from Research Biochemicals Incorporated (Natick, MA, USA), Sigma Aldrich (Munich,Germany) and ICN (Eschwege, Germany), respectively.

Results

Effects of opipramol and imipramine treatmenton s receptor densities

14 days of p.o. treatment with opipramol and imipramine (20 mg=kg body-weight) changed s receptor densities in the mid=hindbrain structure (Rivaand Creese, 1990) compared to the vehicle treated control group, however, witha quite different pattern (Fig. 2). Opipramol treatment caused a reduction of stot

densities by 26% and of s2 receptor densities by 37%. s1 receptor densities

Table 1. Details of the receptor binding assays

Assay Radioligand Blank Membrane Incubation Filter pre-treatmentsuspension time [37�C]

stot3H-DTG, 10 mM mid=hindbrain, 60 min 0.3%0.3–40 nM haloperidol 16 mg ww=ml buffer polyethyleneimine

s13H(þ)-Pentazocine 10 mM mid=hindbrain, 60 min 0.3%0.2–40 nM haloperidol 16 mg ww=ml buffer polyethyleneimine

s2 Bmax were calculatedby subtraction of s1

from stot

b 3H(�)-CGP-12177, 10 mM (DL)- frontal cortex, 4.8 30 min –0.02–2.0 nM propranolol HCl mg ww=ml buffer

5HT23H-Ketanserin HCl, 10 mM mianserin frontal cortex, 4.8 30 min 0.03%0.2–4.0 nM HCl mg ww=ml buffer brij 35+


were not significantly altered. In contrast, imipramine treatment caused noreduction of s2 receptor densities, but a weak reduction of s1 receptor densitiesby 16% compared to control.

As shown in Table 2, only opipramol treatment resulted in an elevation of theKD values for s1 sites, probably indicating remaining opipramol bound to thesesites, confirming the high in vitro affinity of opipramol for s1 sites (Fig. 1).

Fig. 2. Effects of opipramol and imipramine on s receptor densities. s receptor densitieswere determined in mid=hindbrains of rats orally treated with opipramol (20 mg=kg),imipramine (20 mg=kg) and vehicle (0.9% saline) over 14 continuous days. Maximum numberof s binding sites (Bmax values [fmol=mg protein]) were obtained by 8 point saturation experi-ments using the specific radioligands 3H-DTG (stot) and 3H-(þ)-Pentazocine (s1). Bmax valuesof s2 receptors were calculated by subtracting s1 from stot. Data are means� S.E.M. from

9–11 rats

Table 2. Effects of opipramol and imipramine on KD values

KD [nmol=l]

Treatment stot receptors

Control 34.33� 8.54Opipramol 32.31� 10.49Imipramine 38.06� 14.32

s1 receptors

Control 19.65� 10.89Opipramol 45.73� 28.69��Imipramine 18.58� 4.19

KD values were determined with saturation experiments using3H-DTG (stot) and 3H(þ)-Pentazocine (s1) and were calculatedby Graph Pad+ Prism 3 using nonlinear regression. Data aremeans� S.D. of 9–11 rats


Effects of opipramol and imipramine treatment on band 5HT2 receptor densities

As demonstrated in Fig. 3 (left), b adrenoceptor densities, determined with theselective b ligand 3H-CGP 12177, were not affected by treatment with opi-pramol over 14 days, neither in the frontal cortex nor in hippocampus comparedto control. Imipramine showed the well known b adrenoceptor down-regulation,both in the frontal cortex and hippocampus (39% and 36%, respectively).

The same pattern was found for 5HT2 receptor densities in frontal cortices.Opipramol treatment had no effect, whereas imipramine treatment caused areduction of 5HT2 receptor densities of 30%. KD values were slightly elevatedby imipramine treatment in frontal cortices but not by opipramol (data notshown).

Comparison of opipramol with reboxetine and citalopram

In order to investigate whether anxiolytic and antidepressant properties per seare connected with modulation of sigma sites, two drugs with low sigma affin-ities were investigated for comparison (IC50 values in nmol=l, citalopram: s1:264, s2: 2700; reboxetine: s1: >10000, s2: 6390, own data). Because of theshort half lifes of reboxetine and citalopram the effect of both selective reuptakeinhibitors were investigated in a slightly different treatment regime (10 mg=kgbodyweight b.i.d. over 9 days, see material and methods). However, neitherreboxetine nor citalopram had any effect on s receptor densities in the mid=hindbrain region (Fig. 4), whereas the effect of opipramol on stot and s2

receptor densities was confirmed: Opipramol treatment lead to a significant

Fig. 3. Effects of opipramol and imipramine treatment on b adrenoceptor densities (A) and5HT2 receptor densities (B). Drugs were orally given over 14 continuous days (20 mg=kg).Data are means� S.E.M. of 9–11 rats. A b adrenoceptor densities were determined in frontalcortices (FC) and hippocampi (Hip) of treated rats using the specific radioligand 3H-CGP-12177 (Bmax values [fmol=mg protein]). The outcome of two-way ANOVA was a highlysignificant (p<0.001) drug and brain region difference. B 5HT2 receptor densities were de-termined in frontal cortices (FC) of the same rats using 3H-Ketanserin HCl (Bmax values

[fmol=mg protein])


reduction (28%) of stot receptor densities and a 44% reduction of s2 receptordensities. s1 receptor density was again not changed. Silmilar to the firstexperiment, opipramol treatment showed a highly significant increased KD

value of s1 receptors (Table 3). All other KD values were unchanged.Immobility time was decreased by all three tested substances (Fig. 5),

though to a different extent (reboxetine>citalopram>opipramol).

Fig. 4. Comparison with reboxetine and citalopram. s receptor densities were determined in mid/hindbrains of rats orally treated with opipramol, reboxetine and citalopram (10 mg=kg, b.i.d.) over9 days. Bmax values [fmol=mg protein] were obtained by 8 point saturation experiments using thespecific radioligands 3H-DTG (stot) and 3H-(þ)-Pentazocine (s1). Bmax values of s2 receptors

were calculated by subtracting s1 from stot. Data are means� S.E.M. from 9–10 rats

Table 3. Effects of opipramol, reboxetine and citalopram on KD values

KD [nmol=l]

Treatment stot receptors

Control 38.32� 17.59Opipramol 29.23� 9.41Reboxetine 39.70� 12.87Citalopram 34.75� 8.87

s1 receptors

Control 14.62� 5.09Opipramol 30.02� 10.31��Reboxetine 19.48� 6.77Citalopram 18.70� 6.04

KD values were determined with saturation experiments using3H-DTG (stot) and 3H(þ)-Pentazocine (s1) and were calculated byGraph Pad+ Prism 3 using nonlinear regression. Data are means� S.D.of 9–10 rats


Discussion

Our data clearly indicate that opipramol interacts with sigma sites in vivo, thus,confirming previous findings in the mouse (M€uuller and Siebert, 1999). Opi-pramol exhibits higher affinity fors1 sites in vitro and in vivo (M€uuller and Siebert,1999; see also Fig. 1), but the properties of these sites are not directly altered bysubchronic treatment with opipramol. The small increase of KD-values is prob-ably not reflecting changes of s1 site properties, but an indication for receptorbound opipramol. Very interestingly, opipramol down-regulated s2 sites in ourstudy. This was not seen for any of the other antidepressants and seems torepresent a specific feature of this drug only. Opipramol did not alter b adre-noceptor and 5HT2 receptor densities, which is in agreement with its biochem-ical profile (Fig. 1), e.g. no inhibition of norepinephrine uptake and a weaker5HT2 binding.

Several observations have demonstrated that s1 sites can be down-regulatedby subchronic treatment with the s1 antagonist haloperidol, probably indepen-dent of alterations in s1 receptor gene transcription (Ericson and Ross, 1992;Inoue et al., 2000; Riva and Creese, 1990; Klein et al., 1994; Zamanillo et al.,2000). This effect of haloperidol was pronounced in the mid=hindbrain regionof the rat (Riva and Creese, 1990), but was also present in whole brain mem-branes of guinea pigs and rats (Klein et al., 1994; Ericson and Ross, 1992), aswell as in many different regions of the rat or the guinea pig brain (Inoue et al.,2000). Treatment with the s1 agonist pentazocine is usually without effect ons1 sites (Kizu et al., 1991; Weissman and De Souza, 1991). Therefore weconclude that opipramol behaves as an s1 agonist in our experiments.The assumption is further supported by results of the forced swimming test.Since treatment with opipramol resulted in a significant reduction of immobilitytime it fits to reports that only s1 agonists but not antagonists are effective inthis model (Matsuno et al., 1996; Urani et al., 2001).

Fig. 5. Effects of opipramol, reboxetine and citalopram in the forced swimming test. Immo-bility times (sec) were stopped during 5 minutes after the last drug application (10 mg=kg, p.o.,1 h before the test). Rats were pre-treated over 9 days with the drugs (10 mg=kg b.i.d., p.o.).

Data are means� S.E.M. of 9–10 rats


Even if opipramol’s in vitro affinity for s2 sites is less than for s1 sites, itspecifically down-regulates s2 density. Although much less is known about s2

than s1 sites, s2 sites have recently moved into the focus of interest because ofthe impressive anxiolytic profile of the specific s2 ligand siramesine (Sanchezet al., 1997; Heading, 2001). Very little is known about adaptive changes of s2

sites following drug treatment, except that haloperidol might have no effectunder treatment conditions where s1 sites are down-regulated (Itzhak and Stein,1991). Our consistent findings about a specific down-regulation of s2 sites byopipramol are novel and probably suggest a specific role of this effect in therather unusual preclinical and clinical profile of this anxiolytic drug. However,it is not yet clear whether this observation represents a direct effect followings2 site occupation by opipramol or must be explained by other properties of thedrug.

Down-regulation of sigma sites by antidepressant drugs has been suggestedas one possible aspect of their pharmacological properties (Leonard, 1997) butseems to be restricted to s1 sites and to some antidepressants only (Shirayamaet al., 1993). Our positive data with imipramine and our negative findings withcitalopram and reboxetine are basically in line with these observations. Themechanism by which imipramine down-regulates s1 sites is not known, butis probably not associated with direct in vivo binding to these sites due to itslow affinity (Fig. 1). This is supported by the lack of any effect of citalopramwhen given under similar conditions, since citalopram binds with similarlyweak affinity as imipramine to s1 sites (Narita et al., 1996; Sanchez and Meier,1997). Takebayashi et al. (2002) found an increased s1 density in PC12 cellsusing Western blotting after a short-term treatment of 2 days with imipramineand fluvoxamine. However, this condition is not comparable with our treatmentregime and brain tissue.

In conclusion, opipramol is the first drug shown to specifically down-reg-ulate s2 sites under pharmacological relevant doses.

Acknowledgements

This research was supported by a research grant from Novartis, Nuremberg, Germany.

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Authors’ address: W. E. M€uuller, Department of Pharmacology, Biocenter N 260, 1. OG,Marie-Curie-Strasse 9, D-60439 Frankfurt, Germany, e-mail: [email protected]


Specific modulation of sigma binding sites by the anxiolytic drug opipramol

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