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Paroxetine binding to the rat norepinephrine transporter in vivo
BRIEF REPORT Paroxetine Binding to the Rat Norepinephrine Transporter In Vivo Michael J. Owens, David L. Knight, and Charles B. Nemeroff Background: The norepinephrine transporter (NET)/uptake site is an antidepressant-sensitive transporter located on plasma membranes of noradrenergic neurons and other specialized cells that remove norepinephrine (NE) from the synapse to terminate the actions of NE. The antidepressant paroxetine is believed to produce its therapeutic effects primarily by acting as a highly selective antagonist of the serotonin transporter (SERT). However, in vitro data indicates that paroxetine inhibits the NET. The present study was designed to determine whether paroxetine inhibits in NET in vivo. Methods: Rats were administered paroxetine (6.5, 10.0, or 15.0 mg/kg/day) via osmotic minipumps for 1 week. Following attainment of steady state serum concentrations, cortical NET function was assessed by both [3H]nisoxetine binding and [3H]-norepinephrine uptake assays conducted ex vivo. Results: In unwashed brain homogenates, serum paroxetine concentrations greater than 100 ng/mL were positively correlated with the observed Kd for [3H]-nisoxetine. At [3H]-nisoxetine concentrations associated with 50% transporter occupancy in vehicle treated rats, [3H]-nisoxetine binding was decreased 21% and 34% in rats exhibiting serum paroxetine concentrations ⬎ 100 ng/mL and ⬎ 500 ng/mL, respectively. Conclusions: Although paroxetine is a very potent inhibitor of the SERT, paroxetine also inhibits the NET at serum concentrations ⬎ 100 ng/mL. This novel finding may underlie the broad therapeutic utility of paroxetine in mood and anxiety disorders. Biol Psychiatry 2000;47: 842– 845 © 2000 Society of Biological Psychiatry Key Words: Paroxetine, norepinephrine transporter, norepinephrine uptake, selectivity, [3H]-nisoxetine binding, drug concentrations
Introduction
T
he serotonin transporter (SERT) and norepinephrine transporter (NET)/uptake sites are antidepressant-sensitive transporters located on plasma membranes of seroFrom the Laboratory of Neuropsychopharmacology, Department of Psychiatry and Behavioral Sciences, Emory University School of Medicine, Atlanta, Georgia. Address reprint requests to Charles B. Nemeroff, M.D., Ph.D., Emory University School of Medicine, Department of Psychiatry and Behavioral Sciences, 1639 Pierce Drive, Suite 4000, Atlanta GA 30322. Received March 29, 1999; revised October 25, 1999; accepted December 2, 1999.
© 2000 Society of Biological Psychiatry
tonergic and noradrenergic neurons and other specialized cells that remove these monoamines from the extracellular milieu near the synapse to terminate the actions of these neurotransmitters. The antidepressant paroxetine is a potent and highly selective SERT antagonist in vitro (Barker and Blakely 1995; Bolden-Watson and Richelson 1993); however, in a previous study paroxetine, compared to other so-called selective serotonin reuptake inhibitors (SSRIs), possesses moderate affinity for the rat and human NET in vitro (Owens et al 1997). Thus, the Ki values for paroxetine at the rat and human NET are 59 and 85 nmol/L, respectively. For comparison with other SSRIs, fluoxetine is the next most potent antagonist of the NET with Ki values of 473 and 777 nmol/L at the same target proteins. Although there is no demonstrable therapeutic window for paroxetine, many patients responding to this SSRI for treatment of depression, panic disorder, or obsessive-compulsive disorder (OCD) exhibit serum concentrations of 40 – 80 ng/mL (range 30 –120 ng/mL) or 122–244 nmol/L (range 91–364 nmol/L). Although much of the paroxetine in serum is protein bound and free concentrations of paroxetine in cerebrospinal fluid or extracellular brain fluid are not well characterized, we hypothesized that the moderate affinity of paroxetine for the NET in vitro also occurs in vivo.
Methods and Materials Drug Treatment Groups of adult, male Sprague-Dawley rats (200 –225 g, Harlan Sprague Dawley, Raleigh, NC) were implanted with subcutaneous Alzet 2ML2 osmotic minipumps containing vehicle (50% PEG 400) or paroxetine (6.5–15 mg/kg/day). Our group has previously observed that the terminal elimination half-life of paroxetine in adult male rats is approximately 8.0 hours (unpublished observations). Therefore steady-state concentrations are reached in less than 2 days. After 1 week of treatment, rats were killed by decapitation, and serum and brains collected. The prefrontal cortex was immediately dissected and used for [3H]norepinephrine uptake studies. The remaining brain was frozen on dry ice and stored at ⫺70°C until utilized for later [3H]nisoxetine binding studies.
Paroxetine Concentrations Serum paroxetine concentrations were determined via highperformance liquid chromatography (HPLC) with ultraviolet 0006-3223/00/$20.00 PII S0006-3223(99)00314-5
Paroxetine Binding to the NET
(UV) detection. Briefly, citalopram was added as an internal standard to all serum samples. Serum samples were then subjected to solid phase extraction (100 mg Extrasep C18 cartridges, Nalge Nunc International, Rochester, NY). Following extraction, quantification was accomplished via an isocratic HPLC separation using a 100 ⫻ 2 mm MOS-2 Hypersil (C8) 3 m reverse phase column (Keystone Scientific, Bellefonte, PA) followed by UV detection at 225 nm. Analyses were performed with a model 1100 Hewlett Packard HPLC chemstation equipped with a diode array detector. The mobile phase consisted of 20 mmol/L potassium phosphate monobasic, 110 L N,N-dimethyloctylamine/L, 28% acetonitrile, pH 6.83. Flow rate was set at 0.6 mL/minute. Calibration curves were constructed from drug free rat serum by addition of varying amounts of paroxetine (0 –500 ng). A five-point standard curve and two quality control specimens were included in each assay. The limit of detection was 2.0 ng/mL serum. Average correlation coefficients of variation are 3% intra-assay and 8% inter-assay, at a concentration of 100 ng/mL. Absolute recovery is approximately 85%.
NET Binding and Transport Assays [3H]-Norepinephrine transport was determined as described in Owens et al (1995) with modifications for use of [3H]-norepinephrine (Owens et al 1997). Briefly, brains were rapidly removed and placed in cold 0.32 mol/L sucrose containing 11 mmol/L glucose. Approximately 150 mg (wet weight) of prefrontal and frontal cortex was homogenized in 4 mL of the sucrose solution using a small Teflon on glass homogenizer using 8 up and down strokes at 30 rpm. The homogenate was centrifuged at 600 g for 5 min. The supernatant was immediately decanted and centrifuged at 24,000 g for 5 min and the resulting pellet resuspended in an assay buffer at 12.5 mg original wet weight/mL using 8 strokes of the Teflon on glass homgenizer as before. Of this suspension, 200 L was used in each assay tube. The assay buffer consists of: 20 mmol/L HEPES, 10 mmol/L glucose, 145 mmol/L NaCl, 4.5 mmol/L KCl, 1.2 mmol/L MgCl2, 1.5 mmol/L CaCl2 (added last). Just before use, pargyline (0.2 mmol/L) and 10 mg/ml L-ascorbate were added to the assay buffer and the pH adjusted to 7.45 at 37°C. For the assay, 600 L of assay buffer, 100 L of buffer with or without 100 mol/L norepinephrine (final concentration), 100 L of [3H]norepinephrine (25–2000 nmol/L final concentration), and 200 L of tissue suspension (150 –175 g protein). Samples were incubated for exactly 10 min at 37°C. The reaction was terminated by the addition of 5 mL cold 0.15 mol/L NaCl followed by vacuum filtration over GF/B glass fiber filters. Filters were washed four times with 5 mL of cold NaCl solution and counted on a liquid scintillation counter at 50% efficiency. [3H]-Nisoxetine binding was determined as previously described (Owens et al 1997).
Data Analysis Binding and transport saturation data was analyzed with the computer program PRISM (GraphPad, San Diego). Linear regression, Pearson correlations, and analyses of variance (ANO-
BIOL PSYCHIATRY 2000;47:842– 845
843
Figure 1. Serum paroxetine concentrations are positively correlated with the apparent Kd of [3H]-nisoxetine for the norepinephrine transporter. Rats were treated with varying doses of paroxetine for 1 week to achieve steady state concentrations. Linear regression (solid line; Kd ⫽ 1.44 ⫹ 0.00216 * [serum paroxetine]) and 95% confidence intervals (dotted lines) are shown. Data analyzed by Pearson correlation.
VAs) were analyzed with the program SigmaStat (SPSS, Chicago).
Results The presence of paroxetine bound to the NET would increase the apparent Kd of [3H]-nisoxetine. As shown in Figure 1, serum concentrations of paroxetine were positively correlated with the Kd values for [3H]-nisoxetine binding (r ⫽ 0.655; p ⬍ .001). Paroxetine-treated rats were divided into subgroups based on serum concentration ranges. At a concentration of [3H]-nisoxetine equivalent to the Kd in vehicle-treated rats (1.46 nmol/L), rats with paroxetine concentrations between 100 and 500 ng/mL or 500 and 1000 ng/mL exhibited decreased [3H]-nisoxetine binding of 21% and 34%, respectively (Figure 2). As expected, paroxetine treatment did not alter the Bmax or Vmax of [3H]-nisoxetine binding or [3H]-NE transport, respectively (data not shown). In contrast to our observations with [3H]-nisoxetine, serum paroxetine concentrations were not correlated with the Km for [3H]-NE transport (data not shown).
Discussion Numerous studies have shown that paroxetine is a potent antagonist of the SERT in vitro and in vivo (Barker and Blakely 1995). Moreover, in vitro studies have shown that paroxetine is highly selective for the SERT versus the NET. Depending on the methodology and tissue source, we have observed that paroxetine
844
BIOL PSYCHIATRY 2000;47:842– 845
Figure 2. Rats were subdivided into groups based on range of serum paroxetine concentrations. BOUND represents amount of bound [3H]-nisoxetine at the concentration of [3H]-nisoxetine calculated to be the mean Kd (1.46 nmol/L) in vehicle control rats. The amount bound for each rat at 1.46 nmol/L [3H]nisoxetine was obtained from the individual saturation binding curves for each rat. Data expressed as mean ⫾ SE. Data analyzed by one-way analysis of variance followed by Dunnett’s test. *p ⬍ .05.
possess anywhere from 395- to 1308-fold greater affinity for the SERT (Owens et al 1997). Nevertheless, compared to other SSRIs and the dual SERT and NET antagonist, venlafaxine, paroxetine has a greater affinity for the NET in vitro. Very similar findings have been reported by Richelson and colleagues (Tatsumi et al 1997). Based on reported serum concentrations of paroxetine in humans during treatment for depression, panic, or OCD, and this affinity of paroxetine for the NET, we hypothesized that, at certain doses/serum concentrations, paroxetine would antagonize the NET in addition to its strong antagonism of the SERT. [3H]-Nisoxetine is a selective radioligand for labeling NETs in vitro and ex vivo (Tejani-Butt 1992). Our ex vivo binding experiment revealed that serum paroxetine concentrations are positively correlated with an increased apparent Kd for [3H]-nisoxetine (Figure 1). This is consistent with receptor binding theory, in which drugs occupying or competing for the same site increase the apparent Kd, without altering Bmax. In an attempt to quantify the magnitude of the paroxetine antagonism of the NET, we examined the decrease in [3H]-nisoxetine binding at a fixed concentration of [3H]-nisoxetine that occupied 50% of all available NETs in vehicle controls. When paroxetine-treated rats were subgrouped by serum concentration ranges, modest but statistically significant decreases in the ability of [3H]-nisoxetine to bind to the NET at serum paroxetine concentrations greater than 100 ng/mL were observed (Figure 2).
M.J. Owens et al
Although there was a significant overall correlation between serum paroxetine and increased apparent Kd values, we are unable to explain why four samples with concentrations ⬎ 500 ng/mL did not show an increased Kd. Similarly, we did not observe evidence for paroxetine antagonism of [3H]-NE transport in our ex vivo experiments (data not shown). The logistics of measuring binding or active transport in these samples may have artificially decreased the true magnitude of NET antagonism. Prior to harvesting the tissue, extracellular concentrations and NET occupancy by paroxetine in the CNS are at some semistable level; however, the sample must be prepared prior to the assay, and incubated in vitro for a given time during the assay. Both tissue preparation and assay incubation functionally dilute the concentration of paroxetine initially in the sample, thereby decreasing the observable magnitude of NET antagonism. Indeed, analogous studies examining chronic SSRI treatment in rats carried out in this lab found ⬎95% inhibition of [3H]-citalopram binding to the SERT but minimal inhibition of [3H]-5-HT uptake measured ex vivo (unpublished observations). Future studies will be carried out with modifications that attempt to decrease both the preparation and assay volume (minimize dilution of paroxetine concentrations) and assay length (minimize time in which paroxetine can dissociate from the NET). Similar modifications of autoradiographic protocols for determining receptor occupancy will be utilized. Finally, in vivo microdialysis studies are planned which have previously been able to show increases in norepinephrine in dialysate following administration of drugs that inhibit the NET (eg., desipramine). The clinical importance of these observations is currently obscure. Unlike certain antidepressants that exhibit observable effects of NET antagonism under certain conditions (e.g., dose-dependent increases in blood pressure; Effexor [Wyeth-Ayerst Laboratories, Philadelphia] package insert), we are unaware of any reports of indirect evidence suggesting NET antagonism by paroxetine. The development of NET ligands for positron emission tomography or single photon emission computed tomography would be able to unequivocally determine whether paroxetine binds to the NET in vivo. Finally, it is not known whether a small inhibition of the NET as seen here would produce functionally important changes in noradrenergic transmission, and/or whether these are of any measurable therapeutic benefit.
Supported by National Institutes of Health, grant number MH-51761.
Paroxetine Binding to the NET
References Barker EL, Blakely RD (1995): Norepinephrine and serotonin transporters: Molecular targets of antidepressant drugs. In: Bloom FE, Kupfer DJ, editors. Psychopharmacology: The Fourth Generation of Progress. New York: Raven, 321–333. Bolden-Watson C, Richelson E (1993): Blockade by newly developed antidepressants of biogenic amine uptake into rat brain synaptosomes. Life Sci 52:1023–1029. Owens MJ, Ieni JR, Knight DL, Winders K, Nemeroff CB (1995): The serotonergic antidepressant nefazodone inhibits the serotonin transporter: In vivo and ex vivo studies. Life Sci 57:PL373–PL380.
BIOL PSYCHIATRY 2000;47:842– 845
845
Owens MJ, Morgan WN, Plott SJ, Nemeroff CB (1997): Neurotransmitter receptor and transporter binding profile of antidepressants and their metabolites. J Pharmacol Exp Ther 283:1305–1322. Tatsumi M, Groshan K, Blakely RD, Richelson E (1997): Pharmacological profile of antidepressants and related compounds at human monoamine transporters. Eur J Pharmacol 340:249 –258. Tejani-Butt SM (1992): [3H]-Nisoxetine: A radioligand for quantitation of norepinephrine uptake sites by autoradiography or by homogenate binding. J Pharmacol Exp Ther 260:427– 436.
Introduction
T
he serotonin transporter (SERT) and norepinephrine transporter (NET)/uptake sites are antidepressant-sensitive transporters located on plasma membranes of seroFrom the Laboratory of Neuropsychopharmacology, Department of Psychiatry and Behavioral Sciences, Emory University School of Medicine, Atlanta, Georgia. Address reprint requests to Charles B. Nemeroff, M.D., Ph.D., Emory University School of Medicine, Department of Psychiatry and Behavioral Sciences, 1639 Pierce Drive, Suite 4000, Atlanta GA 30322. Received March 29, 1999; revised October 25, 1999; accepted December 2, 1999.
© 2000 Society of Biological Psychiatry
tonergic and noradrenergic neurons and other specialized cells that remove these monoamines from the extracellular milieu near the synapse to terminate the actions of these neurotransmitters. The antidepressant paroxetine is a potent and highly selective SERT antagonist in vitro (Barker and Blakely 1995; Bolden-Watson and Richelson 1993); however, in a previous study paroxetine, compared to other so-called selective serotonin reuptake inhibitors (SSRIs), possesses moderate affinity for the rat and human NET in vitro (Owens et al 1997). Thus, the Ki values for paroxetine at the rat and human NET are 59 and 85 nmol/L, respectively. For comparison with other SSRIs, fluoxetine is the next most potent antagonist of the NET with Ki values of 473 and 777 nmol/L at the same target proteins. Although there is no demonstrable therapeutic window for paroxetine, many patients responding to this SSRI for treatment of depression, panic disorder, or obsessive-compulsive disorder (OCD) exhibit serum concentrations of 40 – 80 ng/mL (range 30 –120 ng/mL) or 122–244 nmol/L (range 91–364 nmol/L). Although much of the paroxetine in serum is protein bound and free concentrations of paroxetine in cerebrospinal fluid or extracellular brain fluid are not well characterized, we hypothesized that the moderate affinity of paroxetine for the NET in vitro also occurs in vivo.
Methods and Materials Drug Treatment Groups of adult, male Sprague-Dawley rats (200 –225 g, Harlan Sprague Dawley, Raleigh, NC) were implanted with subcutaneous Alzet 2ML2 osmotic minipumps containing vehicle (50% PEG 400) or paroxetine (6.5–15 mg/kg/day). Our group has previously observed that the terminal elimination half-life of paroxetine in adult male rats is approximately 8.0 hours (unpublished observations). Therefore steady-state concentrations are reached in less than 2 days. After 1 week of treatment, rats were killed by decapitation, and serum and brains collected. The prefrontal cortex was immediately dissected and used for [3H]norepinephrine uptake studies. The remaining brain was frozen on dry ice and stored at ⫺70°C until utilized for later [3H]nisoxetine binding studies.
Paroxetine Concentrations Serum paroxetine concentrations were determined via highperformance liquid chromatography (HPLC) with ultraviolet 0006-3223/00/$20.00 PII S0006-3223(99)00314-5
Paroxetine Binding to the NET
(UV) detection. Briefly, citalopram was added as an internal standard to all serum samples. Serum samples were then subjected to solid phase extraction (100 mg Extrasep C18 cartridges, Nalge Nunc International, Rochester, NY). Following extraction, quantification was accomplished via an isocratic HPLC separation using a 100 ⫻ 2 mm MOS-2 Hypersil (C8) 3 m reverse phase column (Keystone Scientific, Bellefonte, PA) followed by UV detection at 225 nm. Analyses were performed with a model 1100 Hewlett Packard HPLC chemstation equipped with a diode array detector. The mobile phase consisted of 20 mmol/L potassium phosphate monobasic, 110 L N,N-dimethyloctylamine/L, 28% acetonitrile, pH 6.83. Flow rate was set at 0.6 mL/minute. Calibration curves were constructed from drug free rat serum by addition of varying amounts of paroxetine (0 –500 ng). A five-point standard curve and two quality control specimens were included in each assay. The limit of detection was 2.0 ng/mL serum. Average correlation coefficients of variation are 3% intra-assay and 8% inter-assay, at a concentration of 100 ng/mL. Absolute recovery is approximately 85%.
NET Binding and Transport Assays [3H]-Norepinephrine transport was determined as described in Owens et al (1995) with modifications for use of [3H]-norepinephrine (Owens et al 1997). Briefly, brains were rapidly removed and placed in cold 0.32 mol/L sucrose containing 11 mmol/L glucose. Approximately 150 mg (wet weight) of prefrontal and frontal cortex was homogenized in 4 mL of the sucrose solution using a small Teflon on glass homogenizer using 8 up and down strokes at 30 rpm. The homogenate was centrifuged at 600 g for 5 min. The supernatant was immediately decanted and centrifuged at 24,000 g for 5 min and the resulting pellet resuspended in an assay buffer at 12.5 mg original wet weight/mL using 8 strokes of the Teflon on glass homgenizer as before. Of this suspension, 200 L was used in each assay tube. The assay buffer consists of: 20 mmol/L HEPES, 10 mmol/L glucose, 145 mmol/L NaCl, 4.5 mmol/L KCl, 1.2 mmol/L MgCl2, 1.5 mmol/L CaCl2 (added last). Just before use, pargyline (0.2 mmol/L) and 10 mg/ml L-ascorbate were added to the assay buffer and the pH adjusted to 7.45 at 37°C. For the assay, 600 L of assay buffer, 100 L of buffer with or without 100 mol/L norepinephrine (final concentration), 100 L of [3H]norepinephrine (25–2000 nmol/L final concentration), and 200 L of tissue suspension (150 –175 g protein). Samples were incubated for exactly 10 min at 37°C. The reaction was terminated by the addition of 5 mL cold 0.15 mol/L NaCl followed by vacuum filtration over GF/B glass fiber filters. Filters were washed four times with 5 mL of cold NaCl solution and counted on a liquid scintillation counter at 50% efficiency. [3H]-Nisoxetine binding was determined as previously described (Owens et al 1997).
Data Analysis Binding and transport saturation data was analyzed with the computer program PRISM (GraphPad, San Diego). Linear regression, Pearson correlations, and analyses of variance (ANO-
BIOL PSYCHIATRY 2000;47:842– 845
843
Figure 1. Serum paroxetine concentrations are positively correlated with the apparent Kd of [3H]-nisoxetine for the norepinephrine transporter. Rats were treated with varying doses of paroxetine for 1 week to achieve steady state concentrations. Linear regression (solid line; Kd ⫽ 1.44 ⫹ 0.00216 * [serum paroxetine]) and 95% confidence intervals (dotted lines) are shown. Data analyzed by Pearson correlation.
VAs) were analyzed with the program SigmaStat (SPSS, Chicago).
Results The presence of paroxetine bound to the NET would increase the apparent Kd of [3H]-nisoxetine. As shown in Figure 1, serum concentrations of paroxetine were positively correlated with the Kd values for [3H]-nisoxetine binding (r ⫽ 0.655; p ⬍ .001). Paroxetine-treated rats were divided into subgroups based on serum concentration ranges. At a concentration of [3H]-nisoxetine equivalent to the Kd in vehicle-treated rats (1.46 nmol/L), rats with paroxetine concentrations between 100 and 500 ng/mL or 500 and 1000 ng/mL exhibited decreased [3H]-nisoxetine binding of 21% and 34%, respectively (Figure 2). As expected, paroxetine treatment did not alter the Bmax or Vmax of [3H]-nisoxetine binding or [3H]-NE transport, respectively (data not shown). In contrast to our observations with [3H]-nisoxetine, serum paroxetine concentrations were not correlated with the Km for [3H]-NE transport (data not shown).
Discussion Numerous studies have shown that paroxetine is a potent antagonist of the SERT in vitro and in vivo (Barker and Blakely 1995). Moreover, in vitro studies have shown that paroxetine is highly selective for the SERT versus the NET. Depending on the methodology and tissue source, we have observed that paroxetine
844
BIOL PSYCHIATRY 2000;47:842– 845
Figure 2. Rats were subdivided into groups based on range of serum paroxetine concentrations. BOUND represents amount of bound [3H]-nisoxetine at the concentration of [3H]-nisoxetine calculated to be the mean Kd (1.46 nmol/L) in vehicle control rats. The amount bound for each rat at 1.46 nmol/L [3H]nisoxetine was obtained from the individual saturation binding curves for each rat. Data expressed as mean ⫾ SE. Data analyzed by one-way analysis of variance followed by Dunnett’s test. *p ⬍ .05.
possess anywhere from 395- to 1308-fold greater affinity for the SERT (Owens et al 1997). Nevertheless, compared to other SSRIs and the dual SERT and NET antagonist, venlafaxine, paroxetine has a greater affinity for the NET in vitro. Very similar findings have been reported by Richelson and colleagues (Tatsumi et al 1997). Based on reported serum concentrations of paroxetine in humans during treatment for depression, panic, or OCD, and this affinity of paroxetine for the NET, we hypothesized that, at certain doses/serum concentrations, paroxetine would antagonize the NET in addition to its strong antagonism of the SERT. [3H]-Nisoxetine is a selective radioligand for labeling NETs in vitro and ex vivo (Tejani-Butt 1992). Our ex vivo binding experiment revealed that serum paroxetine concentrations are positively correlated with an increased apparent Kd for [3H]-nisoxetine (Figure 1). This is consistent with receptor binding theory, in which drugs occupying or competing for the same site increase the apparent Kd, without altering Bmax. In an attempt to quantify the magnitude of the paroxetine antagonism of the NET, we examined the decrease in [3H]-nisoxetine binding at a fixed concentration of [3H]-nisoxetine that occupied 50% of all available NETs in vehicle controls. When paroxetine-treated rats were subgrouped by serum concentration ranges, modest but statistically significant decreases in the ability of [3H]-nisoxetine to bind to the NET at serum paroxetine concentrations greater than 100 ng/mL were observed (Figure 2).
M.J. Owens et al
Although there was a significant overall correlation between serum paroxetine and increased apparent Kd values, we are unable to explain why four samples with concentrations ⬎ 500 ng/mL did not show an increased Kd. Similarly, we did not observe evidence for paroxetine antagonism of [3H]-NE transport in our ex vivo experiments (data not shown). The logistics of measuring binding or active transport in these samples may have artificially decreased the true magnitude of NET antagonism. Prior to harvesting the tissue, extracellular concentrations and NET occupancy by paroxetine in the CNS are at some semistable level; however, the sample must be prepared prior to the assay, and incubated in vitro for a given time during the assay. Both tissue preparation and assay incubation functionally dilute the concentration of paroxetine initially in the sample, thereby decreasing the observable magnitude of NET antagonism. Indeed, analogous studies examining chronic SSRI treatment in rats carried out in this lab found ⬎95% inhibition of [3H]-citalopram binding to the SERT but minimal inhibition of [3H]-5-HT uptake measured ex vivo (unpublished observations). Future studies will be carried out with modifications that attempt to decrease both the preparation and assay volume (minimize dilution of paroxetine concentrations) and assay length (minimize time in which paroxetine can dissociate from the NET). Similar modifications of autoradiographic protocols for determining receptor occupancy will be utilized. Finally, in vivo microdialysis studies are planned which have previously been able to show increases in norepinephrine in dialysate following administration of drugs that inhibit the NET (eg., desipramine). The clinical importance of these observations is currently obscure. Unlike certain antidepressants that exhibit observable effects of NET antagonism under certain conditions (e.g., dose-dependent increases in blood pressure; Effexor [Wyeth-Ayerst Laboratories, Philadelphia] package insert), we are unaware of any reports of indirect evidence suggesting NET antagonism by paroxetine. The development of NET ligands for positron emission tomography or single photon emission computed tomography would be able to unequivocally determine whether paroxetine binds to the NET in vivo. Finally, it is not known whether a small inhibition of the NET as seen here would produce functionally important changes in noradrenergic transmission, and/or whether these are of any measurable therapeutic benefit.
Supported by National Institutes of Health, grant number MH-51761.
Paroxetine Binding to the NET
References Barker EL, Blakely RD (1995): Norepinephrine and serotonin transporters: Molecular targets of antidepressant drugs. In: Bloom FE, Kupfer DJ, editors. Psychopharmacology: The Fourth Generation of Progress. New York: Raven, 321–333. Bolden-Watson C, Richelson E (1993): Blockade by newly developed antidepressants of biogenic amine uptake into rat brain synaptosomes. Life Sci 52:1023–1029. Owens MJ, Ieni JR, Knight DL, Winders K, Nemeroff CB (1995): The serotonergic antidepressant nefazodone inhibits the serotonin transporter: In vivo and ex vivo studies. Life Sci 57:PL373–PL380.
BIOL PSYCHIATRY 2000;47:842– 845
845
Owens MJ, Morgan WN, Plott SJ, Nemeroff CB (1997): Neurotransmitter receptor and transporter binding profile of antidepressants and their metabolites. J Pharmacol Exp Ther 283:1305–1322. Tatsumi M, Groshan K, Blakely RD, Richelson E (1997): Pharmacological profile of antidepressants and related compounds at human monoamine transporters. Eur J Pharmacol 340:249 –258. Tejani-Butt SM (1992): [3H]-Nisoxetine: A radioligand for quantitation of norepinephrine uptake sites by autoradiography or by homogenate binding. J Pharmacol Exp Ther 260:427– 436.