Effects of different doses of venlafaxine on serotonin and norepinephrine reuptake in healthy volunteers

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1 International Journal of Neuropsychopharmacology (2007), 10, Copyright f 2006 CINP doi: /s Effects of different doses of venlafaxine on serotonin and norepinephrine reuptake in healthy volunteers ARTICLE CINP Pierre Blier 1,3,Élise Saint-André 1, Chantal Hébert 1,3, Claude de Montigny 1, Normand Lavoie 2 and Guy Debonnel 1 1 Department of Psychiatry, McGill University, Montréal, QC, Canada 2 Department of Urology, McGill University, Montréal, QC, Canada 3 University of Ottawa Institute of Mental Health Research, Ottawa, ON, Canada Abstract Venlafaxine is generally considered to be a dual 5-HT and NE reuptake inhibitor when it is used at doses above 75 mg/d in humans. While its 5-HT reuptake-inhibiting property has been demonstrated, some controversy still exists regarding the doses of venlafaxine required to inhibit NE reuptake. Healthy male volunteers received, on a double-blind basis, paroxetine (20 mg/d), desipramine (100 mg/d), nefazodone (300 mg/d), or venlafaxine (150 or 300 mg/d) in the last 5 d of a 7-d period of administration. Inhibition of 5-HT reuptake was estimated by determining the degree of depletion of whole-blood 5-HT, while that of NE was assessed by measuring the attenuation of the systolic blood pressure increases produced by intravenous injections of tyramine. Paroxetine, both regimens of venlafaxine, and to a lesser extent desipramine significantly decreased whole-blood 5-HT content. Nefazodone failed to produce any significant change. Desipramine abolished the tyramine pressor response, whereas all other drug regimens left this parameter unaltered. Venlafaxine and paroxetine acted as potent 5-HT reuptake inhibitors in the present study. In contrast, neither the moderate nor the high dose of venlafaxine displayed any significant inhibiting activity in this model assessing NE reuptake in peripheral NE terminals. The validity of the model was confirmed by the potent inhibitory action of desipramine on NE reuptake. While the reasons for this unexpected lack of action remain unclear, venlafaxine appeared to be an effective NE reuptake agent in depressed patients using the same approach. Received 13 September 2005; Reviewed 30 October 2005; Revised 14 November 2005; Accepted 22 November 2005; First published online 11 May 2006 Key words: Antidepressant, nefazodone, paroxetine, reuptake inhibition, tyramine. Introduction Almost all tricyclic antidepressant drugs are potent norepinephrine (NE) reuptake inhibitors. In contrast, their potency to inhibit the reuptake of serotonin (5-hydroxytryptamine; 5-HT) is more variable, ranging from extremely potent for clomipramine to ineffective for trimipramine (Hyttel, 1982). In contrast, it is acknowledged that the non-tricyclic agent venlafaxine is an effective 5-HT reuptake inhibitor, but there remains uncertainty regarding the doses necessary to inhibit the NE transporter in humans. In synaptosomal preparations of rodent brains, venlafaxine displays only moderate affinity for 5-HT Address for correspondence : Professor P. Blier, University of Ottawa Institute of Mental Health Research, Royal Ottawa Hospital, Lady Grey Building, Suite 2043, Ottawa, Ontario, Canada K1Z 7K4. Tel. : (ext. 6908) Fax : pblier@rohcg.on.ca reuptake sites, and at NE reuptake sites, its affinity has generally been reported to be in the micromolar range, which may be considered of questionable physiological significance (Béïque et al., 1998). Specifically, its activity on NE reuptake is about 10 times weaker than that of sertraline, which is considered a selective 5-HT reuptake inhibitor (SSRI; Bolden-Watson and Richelson, 1993). Similar in-vitro data have been reported in human cell lines (Owens et al., 1997; Tatsumi et al., 1997). These observations using different preparations and assays show considerable variability in the potency and selectivity of venlafaxine, thereby raising concerns about the mechanism of action of venlafaxine in the treatment of mood and anxiety disorders. The in-vivo assessment of the potency of venlafaxine to inhibit 5-HT and NE reuptake in the rat brain have, however, yielded very different results. For instance, the dose of venlafaxine necessary to

2 42 P. Blier et al. inhibit the firing rate of 5-HT neurons in anaesthetized rats is the same as for the SSRI paroxetine (Béïque et al., 1999). Such a suppression of firing activity is due to the increased activation of cell body 5-HT 1A autoreceptors by enhanced amounts of synaptic 5-HT, resulting from 5-HT reuptake inhibition. This dose equivalency between these two drugs is, therefore, quite different from the 100-fold greater in-vitro potency of paroxetine when compared to that of venlafaxine (Béïque et al., 1998; Tatsumi et al., 1997). In a similar model indirectly assessing NE reuptake activity, venlafaxine was only three times less active than the potent tricyclic NE reuptake inhibitor desipramine in suppressing the firing rate of NE neurons in the locus coeruleus (Béïque et al., 1999). Among the possible explanations for this discrepancy between in-vitro and in-vivo results is the low plasma protein binding of venlafaxine when compared to those of paroxetine and desipramine, being y30% for venlafaxine vs. 95% for the latter two drugs (Ereshefsky and Dugan, 2000; Puozzo and Leonard, 1996). Presumably, the greater free fraction of venlafaxine may allow greater brain penetration. Despite the fact that the 5-HT and NE systems in the rodent and the human brain are similar, the effectiveness of various antidepressant drugs to inhibit 5-HT and NE reuptake needs to be evaluated in healthy volunteers and in patient populations. Studies examining such actions of venlafaxine in both groups of subjects are still lacking. In the present study, the capacity of venlafaxine and of other reference antidepressant agents to inhibit 5-HT reuptake was evaluated from their ability to deplete whole-blood content of 5-HT. Since blood platelets can only store 5-HT through a 5-HT reuptake transporter that is nearly identical to that present on 5-HT neurons in the brain, and that y90% of the total blood 5-HT is in platelets, the sustained inhibition of 5-HT reuptake depletes whole-blood 5-HT (Artigas et al., 1985; Demet et al., 1978). The activity of the NE transporter was evaluated by measuring the increase in the systolic blood pressure (BP) produced by the intravenous injection of tyramine. Tyramine penetrates peripheral NE terminals through their NE transporters and then releases NE which transiently increases BP (Hoffman and Lefkowitz, 1990). NE reuptake inhibition therefore attenuates this response (Ghose, 1984). Subjects and methods Study subjects Volunteers were recruited among students, outside the faculty of medicine, on the McGill University campus. Prior to their inclusion in the study, 40 volunteers underwent a structured clinical interview (SCID for non-patients), a physical examination, an electrocardiogram and laboratory tests consisting of a complete blood count, electrolytes, liver enzymes, urea and creatinine, hepatitis B and HIV testing, and urine drug screening. Participants met the following inclusion criteria: (1) male, between the ages of 18 and 40 years old; females were not included to avoid having to control for menstrual cycle or possible metabolic interference with oral contraceptives; (2) body weight in the normal range as indicated by body mass index; (3) no significant medical illness, including migraines, high blood pressure (BP>140/90), hepatitis B or HIV; (4) no evidence suggestive of a past or present history of mental disorders. Subjects were excluded from the study for any of the following reasons: (1) use of an investigational drug within 30 d of study entry; (2) orthostatic hypotension (a fall in systolic BP>10 mmhg); (3) use of any psychotropic medication or any illicit drug; (4) regular use of any medication other than acetaminophen or vitamins and mineral supplements; (5) history of difficult phlebotomy. None of the subjects smoked tobacco. Any significant abnormality upon physical examination or laboratory tests was also a reason for exclusion. A total of 37 healthy male volunteers entered the study. There was one dropout in the high-dose venlafaxine group due to side-effects. This subject was replaced to have six persons in each group. Drug regimens Volunteers were randomly assigned to one of six groups: placebo, paroxetine (Paxil 1 ) 20 mg/d, nefazodone (Serzone 1 ) 300 mg/d, desipramine 100 mg/d (Norpramine 1 ), venlafaxine immediate release (IR) 150 mg/d (Effexor IR 1, intermediate dose), or venlafaxine IR 300 mg/d (Effexor IR 1, high dose). The regimens were chosen on the basis of their clinical efficacy in depression. The dose of paroxetine was the minimal effective dose for depression and was not meant to test its potential noradrenergic action reported to occur with high doses (Gilmor et al., 2002). The two venlafaxine regimens on the basis of their antidepressant action were superior to SSRIs, at least in some studies (Kelsey, 1996; Thase et al., 2001). Following randomization, investigators and subjects remained blind to the drug administration groups. The capsules containing placebo or one of the five different medications were identical. For the first 2 d, all subjects took one capsule in the morning and one

3 5-HT/NE reuptake inhibition in healthy volunteers 43 capsule at bedtime. They were started at bedtime on the day the baseline data were collected. The paroxetine group received 10 mg in the morning, the nefazodone group received 75 mg b.i.d., the desipramine group 25 mg b.i.d., the low-dose venlafaxine group 37.5 mg b.i.d., and the high-dose venlafaxine group 75 mg b.i.d. For the next 5 d, all subjects took one capsule in the morning and two capsules at bedtime. The paroxetine group received 10 mg b.i.d., the nefazodone group received 150 mg b.i.d., the desipramine group 50 mg b.i.d., the low-dose venlafaxine group 75 mg b.i.d., and the high-dose venlafaxine group 150 mg b.i.d.. The assessment of NE and 5-HT reuptake processes was performed at baseline (day 0) and on day 7 with the last dose being taken about 2 3 h before testing. Taking into account these gradual titrations, the half-life of the drugs and that of their metabolites, it was deemed that 5 d at the same dose would be sufficient to achieve steady-state plasma levels (Devane, 1998). The plasma levels of venlafaxine and desmethyl venlafaxine were carried out by Dr Gilles Caillé at the University of Montreal using a method approved by Wyeth and summarized in Debonnel et al. (2006). Assessment of 5-HT reuptake Platelet 5-HT content has become widely used for studying the activity of the 5-HT transporter on serotonergic neurons (Arora et al., 1984). Blood platelets can take up and store 5-HT in a fashion very similar to that of 5-HT neurons, but lack the enzymatic system necessary to synthesize 5-HT and most of the 5-HT receptors subtypes (Demet et al., 1978; Pletscher, 1986). Thus, any 5-HT present in the platelets has been taken up by the transport system, and concentration constitutes an index of the efficacy of 5-HT uptake sites on 5-HT neurons. Following blockade of the 5-HT reuptake process, platelet 5-HT content gradually decreases as older platelets that have previously taken up 5-HT are removed from the circulation. They are replaced by new ones that are rendered incapable of effective 5-HT uptake by the reuptake- inhibiting drugs. Moreover, as a very large proportion (90 95%) of blood 5-HT is sequestrated in the platelets, wholeblood 5-HT content largely reflects platelet 5-HT content (Artigas et al., 1985; Demet et al., 1978; Pletscher, 1986). Blood 5-HT content measured on day 7 was compared to the baseline level and its reduction was used as an index of the degree of 5-HT reuptake inhibition. An adapted HPLC procedure using an electrochemical detector for assessing 5-HT levels in samples from total blood, from approaches previously described was utilized (Kilts et al., 1981; Koch and Kissinger, 1980; Korpi, 1984; Sasa et al., 1978). The procedure involves deproteination of blood samples by addition of perchloric acid (4 M) in presence of antioxidants and application of the supernatant on an amberlite CG-50 cation exchange resin column from which 5-HT is eluted with an acetate buffer (ph 5.1). We used an internal standard (2 nmol/ml N-methyl- 5-hydroxytryptamine oxalate salt) and its per cent recovery was used to correct for methodological losses during the extraction and chromatographic procedures and ensure correction for losses during quantification by HPLC. One-millilitre whole-blood aliquots were thawed and 0.5 ml of the internal standard working solution was added as well as acetate buffer to make up the volume to 2 ml. A total of 0.2 ml perchloric acid was used to precipitate serum proteins, the mix was vortexed for 1 min and centrifuged at 4000 rpm for 10 min with a Brinkmann Eppendorf centrifuge. The supernatant was transferred to glass tubes containing 2 ml of sodium acetate buffer. The ph was adjusted to with 6 mol/l ammonium hydroxide. The HPLC equipment consisted of a Waters HPLC pump (510 model; Oregon City, OR, USA), a standard analytical Hypersil column (Chromatographic Sciences Company, Montreal, Quebec), an electrochemical detector (model 400, Princeton Applied Research, Oakridge, TN, USA) with an Ag/AgCl reference electrode set at +0.5 V potential to eliminate interference caused by catecholamines, metanephrines and other metabolites of catecholamines, and a gold working electrode. An autosampler (Hitachi model 7200 L, Chromabec, Montreal, Quebec) was used for automatic injection of samples and a Waters 740 data module integrator was used to record chromatograms, calculate the area under the curve and obtain ratios of 5-HT and internal standard. The mobile phase containing 100 mg disodium EDTA, 25.3 ml concentrated ammonium hydroxide, 32.3 ml glacial acetic acid, and ml methanol with a ph adjusted to 5.1 was filtered through 0.45 picometres membrane filters and degassed for 15 min before use. Assessment of NE reuptake Tyramine acts indirectly as a sympathomimetic agent, i.e. it is taken up in NE neurons by the NE transporter and then it produces a release of NE from its intraneuronal stores (Hoffman and Lefkowitz, 1990). NE reuptake inhibition therefore attenuates this response (Ghose, 1984). In turn, NE acts on the

4 44 P. Blier et al. vascular post-synaptic a 1 -adrenoceptors, which causes a vasoconstriction and a secondary elevation of systolic BP (Ghose, 1984). Pretreatment with drugs that block the high-affinity NE reuptake site, prevent the increase in BP that usually follows tyramine administration by preventing tyramine entry into NE neurons (Hoffman and Lefkowitz, 1990). The tyramine test consists of measuring the increase in systolic BP of patients receiving a fixed dose of tyramine, or measuring the dose of tyramine necessary to cause a predetermined increase in systolic BP (Ghose, 1984; Ghose and Turner, 1975). Either a decrease in the pressor response to the same doses of tyramine, or an increase in the dose of tyramine necessary to cause a given increase in systolic BP, is considered to be a reliable index of NE reuptake blockade. In the present study, the effects of two doses of intravenous tyramine (4 mg and 6 mg) on systolic BP were assessed. The following procedure was followed at each visit: the subject was connected to a Criticare 508 apparatus, which automatically registered the heart rate (HR) and BP, and a catheter was installed in an antecubital vein in one arm. Following a resting period of 30 min in the supine position, during which HR and BP were recorded every 10 min, blood was drawn for 5-HT level measurements. A test dose of 0.5 mg tyramine was then administered intravenously in order to rule out the presence of a pheochromocytoma, which would not have been detected by the physical examination and laboratory tests. After 5 min, and if there was no significant elevation in BP after the test dose, a dose of 4 mg tyramine was administered. The BP and HR were then measured every 2 min for 10 min. This time period allowed us to detect the peak increase in BP, which usually occurs 2 min after a bolus injection of tyramine, as well as sufficient time to let BP return to its baseline level. If the increase in BP did not reach 30 mmhg after the 4-mg tyramine dose, a second dose of 6 mg was given, and the BP and HR were measured as previously described. The difference between the systolic BP measured just prior to the tyramine load and that measured 2 min after was considered the index for the action of tyramine. Blockade of NE reuptake manifests itself by a decrease in the elevation of BP shown prior to beginning the drug regimen (effect of tyramine measured on day 1). Evaluation and statistical analysis Serotonin reuptake data are expressed in whole-blood 5-HT concentrations in pmol/l compared to baseline levels. These results, as well as the systolic, diastolic BPs and the pulses were analysed using the two-tailed paired Student s t test. The systolic BP response to tyramine was considered to be the difference between the systolic BP measured just prior to the tyramine load and that measured 2 min post-administration. ANOVA for repeated measures for doses of tyramine and treatments were conducted on the data to assess the effects of the different drug regimens on the pressor response to loads of 4 mg and 6 mg tyramine. Post-hoc analyses were conducted where appropriate with Bonferroni s method. All values are expressed as means S.E.M. The level of significance was set at p<0.05. Ethics This study was carried out in accordance with the Declaration of Helsinki and was approved by the Research Ethics Board of the Royal Victoria Hospital, McGill University. All subjects gave informed written consent. Subjects were given $150 (CDN) as compensation for lost time related to their participation. Results Plasma levels of the antidepressant drugs In the six subjects who received 150 mg/d venlafaxine, the mean level of the parent compound was 67 ng/ml (range 43 97) and that of its metabolite, desmethyl venlafaxine, was 338 ng/ml (range ). The total level of venlafaxine plus its metabolite was ng/ml (range ). In the six subjects who received 300 mg/d venlafaxine, the mean level of the parent compound was 170 ng/ml (range ) and that of the metabolite was 540 ng/ml (range ). The total level of venlafaxine plus its metabolite in this second group was ng/ml (range ). These levels are consistent with those obtained in depressed patients (Debonnel et al., 2006). The mean ratio of the metabolite/parent compound was 4.7 (range ). Despite this apparent wide range of metabolic activity, it is important to mention that the total concentration of venlafaxine active moieties varied by less than 2-fold in both groups of subjects. Furthermore, the two subjects who had the lowest and the highest metabolite/parent compound ratio still had a total level of active agents within 15% of the mean of their respective group. The mean level of paroxetine was ng/ml (range , n=6), values that are fully consistent with those reported using 20 mg/d (Gilmor et al.,

5 5-HT/NE reuptake inhibition in healthy volunteers ). The mean desipramine level was ng/ ml (range , n=6). The levels of nefazodone in the six subjects varied from 66 to 2990 ng/ml (mean S.E.M ), those of the hydroxymetabolite varied from 35 to 499 ng/ml (mean S.E.M ), and mcpp levels were low as expected (mean S.E.M ng/ml, range <3 79 ng/ml). The mean concentration of des-ethyl-hydroxynefazodone, a metabolite with an uncharacterized biological activity, was of ng/ml (range ). Modifications of cardiovascular parameters by the antidepressants Despite the small number of subjects tested in each group, and the numerous factors that can alter such parameters, it was decided to assess BP and pulse at baseline and after the 1-wk duration of the study, when the subjects were lying down immediately before starting the tyramine infusions. In the placebo group, there was a mean significant drop of 13 beats/ min (range +2 to x27; d.f.=5, t=2.92, p=0.03), with five of the six subjects presenting a decrease at day 7. In the desipramine group, there was a mean significant increase of 14 beats/min (range x4 to +32; d.f.=5, t=2.63, p=0.046), with five of the six subjects presenting an increase during the second session. The other medications did not produce any significant change in pulse (p>0.5, data not shown). With regards to systolic and diastolic BPs, the only significant difference noted among all the treatment groups, was the increased mean diastolic pressure by 5 mmhg in the paroxetine group (range +2 to +9; d.f.=5, t=3.51, p=0.017). Depletion of whole-blood 5-HT levels by the antidepressant drugs The mean concentration of 5-HT was pmol/l at baseline in the placebo group and d later, with five of the six subjects having a f10% variation within that 1-wk period. This mean difference was not statistically significant (d.f.=5, t=1.05, p=0.34; see Figure 1). Similarly in the nefazodone group, there was a non-significant mean 10 pmol/l decrease after 7 d administration (d.f.=5, t=0.15, p=0.884), suggesting a lack of sustained effect of nefazodone on 5-HT reuptake (Figure 1). In the desipramine group, however, there was a small but significant mean 19% decrease in whole-blood 5-HT (d.f.=5, t=3.19, p=0.02), all six subjects showing a decrease in this parameter after 7 d administration (range 8 33% decreases, Figure 1). In the paroxetine % change in whole-blood 5-HT Desipramine 100 mg/d Placebo * * * * Paroxetine 20 mg/d Nefazodone 300 mg/d Venlafaxine 150 mg/d 300 mg/d Figure 1. Percentage changes of whole-blood 5-HT levels after 7 d administration of placebo or the various antidepressant agents. There were six subjects in each group. They received half of the indicated doses for the first 2 d of administration and the full doses for the next 5 d on a twice-daily regimen. The last dose was given 2 3 h prior to drawing blood. The baseline levels, expressed in pmol/l, were in the placebo group, in the desipramine group, in the paroxetine group, in the nefazodone group, in the 150 mg/d venlafaxine group and in the 300 mg/d venlafaxine group. * Indicates p<0.05 using the paired two-tailed Student s t test. group, all subjects had a robust decrease in wholeblood 5-HT (range 45 96%; d.f.=5, t=3.02, p=0.03; Figure 1). In the 150 mg/d venlafaxine group, all subjects also had a marked decrease in their whole-blood 5-HT level (range 52 85%; d.f.=5, t=4.45, p=0.007; Figure 1). Finally, in the high-dose venlafaxine group, the decrease was profound and more homogeneous than in the last two groups (range 73 82%; d.f.=5, t=8.02, p<0.001; Figure 1). Effects of the antidepressant medications on the tyramine pressor response The modifications in systolic BP were compared using a two-way ANOVA, after ensuring that the normality and equal variance tests were passed (p=0.23 and 0.70 respectively). It revealed a significant source of variance of dose of tyramine (F=61.43, d.f.=1, p<0.001), of treatment (F=6.54, d.f.=11, p<0.001), but not of treatmentrdose (F=1.44, d.f.=11, p=0.16). All the subsequent pairwise comparisons were carried out using Bonferroni s method considering a p value smaller than 0.05 as significant. The 6-mg dose of tyramine produced a numerically greater increase in systolic BP than the 4-mg dose in all the groups at baseline, although it reached statistical significance only in the nefazodone group (t=4.37). The mean

6 46 P. Blier et al. 30 Placebo Desipramine 100 mg/d Paroxetine 20 mg/d Nefazodone 300 mg/d Increase in systolic blood pressure by tyramine (mmhg ± S.E.M.) increase produced by the 4-mg dose was 12 mmhg and a statistically significant further increase of 9 mmhg was produced by the 6-mg dose. Placebo administration did not significantly alter the tyramine response (t=1.99), thus indicating test retest reliability of this paradigm over a 1-wk period. The only medication that significantly altered the tyramine pressor response was desipramine (t=5.96). This drug nearly abolished the pressor response to both doses of tyramine (t values of 5.06 and 5.45 at 4 mg and 6 mg respectively). The paroxetine, nefazodone and the low- and high-dose venlafaxine regimens yielded non-significant t values of 0.15, 1.90, 1.28 and 0.64 respectively (Figures 2, 3). Discussion * * mg 6 mg 4 mg 6 mg 4 mg 6 mg 4 mg 6 mg Figure 2. Alterations of systolic blood pressures within 2 min of the intravenous injection of tyramine in a bolus manner. The number of subjects studied in each group is indicated at the bottom of the histograms. They received half of the indicated doses for the first 2 d of administration and the full doses for the next 5 d on a twice-daily regimen. The last dose was given 2 3 hours prior to the tyramine challenge. * Indicates p<0.05 using the Bonferroni method as post-hoc tests following an ANOVA for repeated measures. The difference between the effects of the 4-mg and 6-mg doses of tyramine at baseline was significant only in the nefazodone group. %, Prior to administration; &, after 7 d administration. The results of the present study indicate that paroxetine and venlafaxine are potent inhibitors of 5-HT reuptake. Desipramine exerted a small, albeit significant, inhibition of 5-HT reuptake, whereas nefazodone was devoid of sustained inhibitory action on this process (Figure 1). Among these four antidepressant drugs, only desipramine exerted a significant attenuation of the tyramine pressor response (Figures 2 and 3). These data indicate that venlafaxine, even at a dose that is four times higher than its minimal effective dose in depression (i.e. 75 mg/d), did Increase in systolic pressure produced by tyramine (mmhg ± S.E.M.) Venlafaxine 150 mg/d 300 mg/d mg 6 mg 4 mg 6 mg Figure 3. Alterations of systolic blood pressures within 2 min of the intravenous injection of tyramine in a bolus manner. The number of subjects studied in each group is indicated at the bottom of the histograms. They received half of the indicated doses for the first 2 d of administration and the full doses for the next 5 d on a twice-daily regimen. The last dose was given 2 3 h prior to the tyramine challenge. The treatments did not produce significant alterations of the tyramine pressor response, using the Bonferroni method as post-hoc tests following an ANOVA for repeated measures. %, Prior to administration; &, after 7 d administration.

7 5-HT/NE reuptake inhibition in healthy volunteers 47 not appear to inhibit the NE reuptake process in the present study in healthy volunteers. The assessment of whole-blood 5-HT diminutions by reuptake inhibitors yields an underestimation of the actual degree of reuptake inhibition. This is because, even if a reuptake inhibitor would rapidly and completely inhibit the 5-HT reuptake pump of platelets, their content needs an extended delay to be depleted as a result of 5-HT being released and not being refilled through reuptake. Indeed, when the treatment with a dose of 75 mg/d venlafaxine is extended from 1 to 4 wk, the degree of depletion increases over time despite venlafaxine achieving its steady-state level within a few days (Debonnel et al., 2006). Nevertheless, the advantage of this approach is that it readily permits detection if various substances exert a sustained action on the 5-HT reuptake transport system. In the case of nefazodone, for instance, it was reported that 200 mg nefazodone administered twice daily inhibited by 34% the ex-vivo platelet 5-HT reuptake in healthy male volunteers 2 4 h after its oral administration, but not 12 h after the last dose. In the same study, nefazodone was devoid of effects on the whole-blood levels of 5-HT. In contrast, a daily dose of 20 mg fluoxetine produced a sustained inhibition of platelet 5-HT reuptake and a whole-blood 5-HT depletion of up to 80% (Salazar et al., 1994). It may appear surprising that desipramine at a relatively low dose actually produced a significant reduction of whole-blood 5-HT. Nevertheless, prior reports have documented the lack of absolute specificity of this drug for the NE reuptake process. For instance, Javors et al. (2000) estimated that after a prolonged administration of desipramine in patients, the IC 50 required to inhibit 5-HT platelet depletion was 195 ng/ml. Moreover, Gilmor et al. (2002) showed that the reuptake of 5-HT was also significantly inhibited (by 18%) when using the plasma of the patients containing 100 ng/ml desipramine in a human cell line in vitro. The present results are thus strikingly similar to those previously published. Although the three sets of data indicate that desipramine produces a consistent inhibition of 5-HT reuptake transporters in humans, they do not imply that it would necessarily alter 5-HT levels in the brain. This is presumably because there is a reserve in the number of 5-HT transporters (5-HTT) in order to maintain the effectiveness of the process. In humans, it is estimated that y80% of the 5-HTT have to be occupied to produce an antidepressant response using drugs that inhibit 5-HT reuptake (Meyer et al., 2004). Indeed, that study has clearly shown that the minimal effective doses of various 5-HT reuptake inhibitors, including venlafaxine, produced an 80% occupancy of 5-HTT sites in the human brain, as assessed using a labelled 5-HTT ligand and positron emission tomography. Perhaps the strongest clinical evidence that tricyclic antidepressants do not exert a potent inhibitory action on the 5-HT reuptake process, with the exception of clomipramine, is that they are not effective in the treatment of obsessive compulsive disorder, unlike the SSRIs. In contrast, clomipramine is a potent 5-HT reuptake inhibitor and it is the only tricyclic drug with an anti-obsessional action (Goodman et al., 1997). The robust inhibitory action of desipramine on the tyramine pressor response was expected based on prior reports from ours, and several other laboratories. Indeed, amitriptyline and imipramine (which are metabolized to nortriptyline and desipramine respectively) potently inhibit this response, as does nortriptyline itself, maproptiline, clomipramine (which is metabolized to the potent NE reuptake inhibitor desmethyl-clomipramine), and the nontricyclic NE reuptake inhibitors tomoxetine (now denoted atomoxetine) and reboxetine (Gobbi et al., 2003; Harvey et al., 2000; Hassan et al., 1985, 1989; Seppala et al., 1981; Slater et al., 2000; Turcotte et al., 2001; Zerbe et al., 1985). In contrast, the SSRI paroxetine at its minimal effective dose in depression of 20 mg/d was devoid of effect in this test (Hassan et al., 1985, 1989; Figure 2). This is consistent with the results of Gilmor et al. (2002) showing a 27% inhibition of NE reuptake ex vivo using the plasma of patients containing 100 ng/ml paroxetine. It remains to be determined, however, if higher regimens of paroxetine (50 mg/d or more), consistently producing levels of y200 ng/ml paroxetine, would attenuate the tyramine pressor response, given that the ex-vivo inhibitory action of such plasma levels of NE reuptake is 40% (Gilmor et al., 2002). Nefazodone did not alter the tyramine pressor response consistently with its relative absence of effect at the human NE transporter (K i =618 nmol/l; Owens et al, 1997). However, it is conceivable, although improbable, that its high affinity for a 1 -adrenoceptors may have prevented a rise in systolic BP. Indeed, nefazodone did not even inhibit 5-HT reuptake despite having a higher affinity for 5-HT than for NE reuptake sites (K i =459 nmol/l; Owens et al., 1997). The lack of inhibitory action of venlafaxine on the tyramine pressor response was unexpected, especially at the 300 mg/d regimen. It is noteworthy that Harvey et al. (2000) reported an attenuation of this parameter using a 375 mg/d regimen of venlafaxine. The attenuation was, however, only of y20% with respect to the baseline value with the standard error

8 48 P. Blier et al. for that mean value nearly reaching the 1.0 ratio level (i.e. no change prior to and following treatment). It should be noted that this value was not significantly different from those of the low-dose venlafaxine regimen and of the SSRI sertraline due to the relatively high standard deviations in all three conditions. The authors proposed that 5-HT reuptake inhibition could somehow enhance the tyramine pressor response, but prior evidence and the present report indicate that this not the case (Hassan et al., 1985, 1989; Figure 2). Their conclusion was solely based on the observation that the 375 mg/d venlafaxine regimen failed to separate from the maproptiline group. Consequently, it appears that these experiments did not produce any evidence for a significant attenuation of the tyramine pressor response using 375 mg/d, as did the present ones using 300 mg/d. In contrast, in a companion paper, a significant attenuation was demonstrated at regimens of 225 and 375 mg/d venlafaxine in depressed patients (Debonnel et al., 2006). Possible bases for this healthy volunteer/depressed patient difference are discussed in that paper. Nevertheless, it is important to emphasize here that this healthy volunteer/depressed patient difference is not present with amitriptyline, with the drug being effective in both groups of subjects (Ghose and Coppen, 1977; Hassan et al., 1989; Wilkins et al., 1985). Aside from the tyramine pressor test on BP, other peripheral human models have been utilized to demonstrate a NE reuptake action of venlafaxine. For instance, tyramine-induced pupillary response, reduced salivary flow, BP and HR alterations, and cardiac NE reuptake have all reported significant effects for 75 mg/d venlafaxine (Bitsios et al., 1999; Melichar et al., 2001; Watkins et al., 2003). These approaches, although they demonstrate a significant degree of NE reuptake inhibition in the periphery, probably did not correspond to a threshold of NE reuptake inhibition sufficient to alter the central NE reuptake process necessary to produce an antidepressant response potentially superior to that of SSRIs (Kelsey, 2000; Thase et al., 2001). It is interesting to note, however, that in the NE-induced constriction of the dorsal vein of the hand, 75 mg/d venlafaxine and 20 mg/d paroxetine were ineffective, but 100 mg/d desipramine as well as 150 mg/d venlafaxine produced significant inhibitory effects (Abdelmawla et al., 1999). The latter model would thus correspond to a clinical action of venlafaxine superior to that of SSRIs at doses greater than 75 mg/d, presumably being mediated by an additional NE reuptake inhibition. All these physiological models represent attempts at estimating NE reuptake inhibition in the brain. Upon the availability of a specific ligand of NE reuptake sites for use in positron emission tomography studies, the degree of occupancy of NE transporters by various regimens of venlafaxine will be directly measurable. Nevertheless, such results will have to be correlated with clinical data, as done by Meyer et al. (2004) for the 5-HT reuptake process, in order to determine at what degree of occupancy of NE transporters is the NE reuptake process significantly inhibited. In summary, 5-HT reuptake inhibition was clearly documented with 20 mg/d paroxetine and with 150 and 300 mg/d venlafaxine. The adequacy of the tyramine pressor response to document the presence of NE reuptake inhibition was indicated by the inhibitory action of the NE reuptake inhibitor desipramine and the lack of effect of paroxetine and nefazodone. In contrast, the two regimens of venlafaxine did not result in NE reuptake inhibition in healthy volunteers. Acknowledgements This work was supported by Wyeth Canada. We would like to thank Dr Gilles Caillé and his team for carrying out, under contract, the venlafaxine plasma level determinations. Details of this procedure are provided in Debonnel et al. (2006). We would like to thank GlaxoSmithKline and Bristol Myers Squibb for the plasma level assays of paroxetine and nefazodone respectively. Desipramine levels were determined in the main laboratory of the Royal Victoria Hospital in Montreal. P. Blier is an Adjunct Professor in the Department of Psychiatry at McGill University. Statement of Interest P. Blier, G. Debonnel and C. de Montigny have been paid consultants, remunerated speakers and have received other research grants from Wyeth. P. Blier has been contracted to produce an animated programme for educational purposes for Wyeth. References Abdelmawla A, Langley R, Szabadi E, Bradshaw C (1999). Comparison of the effects of venlafaxine, desipramine, and paroxetine on noradrenaline- and methoxamine-evoked constriction of the dorsal hand vein. 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