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Serotonin1A Receptor Activation by Flesinoxan in Humans Body Temperature and Neuroendocrine Responses B. Seletti, M.D., C. Benke/fat, M.D., P. Blier, M.D., Ph.D., L. Annable, B.Sc., Dip. Stat., F. Gilbert, M.D., and C. de Montigny, M.D., Ph.D.
The effects of tlze selectir 1e 5-HT,A re(eptor agonist lwnnone response to flesinoxan was blocked by pindolol flesinoxan on neuroendouine funct/011, temperature, a11d but not by methysergide, whereas the prolactin response belzavior were assessed i11 male healt/1y volunteers usi11g a ,uas blocked by methysergide but not by pindolol. The double-blind, placebo-controlled crossouer design. ACTH and cortisol responses to flesinoxan were Flexinoxan (7 and 14 µg/kg), administered intravenously potentiated by methysergide. The flesinoxan-induced in 11 healthy voluntl'ers, elicited a dose-related deuease !1ypothermia was attenuated by both methysergide and in b,)dy temperature and increases i11 growth lzormone, pindolol, although the latter effects did not reach adrenocorticotropic hormone (ACTH), cortisol, a11d statistical significance. The present results suggest that prolactin plasma levels. In a second independent study, the growth hormone response and the hypothermic 12 healthy rnlunteers were pretreated sequentially, at response to the intravenous infusion of flesinoxan may one-week intervals, ,uith either the S-HT1" antago11ist serz,e as a ualid index of S-HT1A receptor function in pindolol (30 mg, PO), t/ie nonselectil·e S-HT1 c /1w11ans. [Neuropsychopharmacology 13:93-104, antagonist methysergide (4 mg, PO!, or placebo. prior to 1995/ being administered flesinoxa11 (1 mg, IV). The growtli
KEY WORDS: Flesinoxan; Hypothermia; Growth probes such as buspirone (Meltzer and Maes 1994; hormone; Pindolol; Methysergide; 5-HT1A receptor Moeller et al. 1994), gepirone (Anderson et al. 1990), tandospirone (Miller et al. 1990), and ipsapirone (Lesch Over the past decade, the development of nontoxic, et al. 1989). Although buspirone has a high affinity for selective serotonin (5-HT) receptor agonists has made 5-HT 1A receptors, its complex pharmacological effects it possible to probe 5-HT receptor function in humans on dopaminergic neurotransmission make us question in vivo by means of pharmacological challenges. More its validity as an index of S-HT1A function, particularly specifically, studies exploring the role of brain S-HT1A with respect to measurements of buspirone-induced receptor function in major affective and anxiety dis prolactin release (Meltzer et al. 1983). Moreover, many orders have now been made possible using receptor of these agents share a common metabolite, 1-(2-pyri midinyl) piperazine (1-PP), which displays a2-adreno From the Neurobiological Psychiatry Unit, Department of Psy receptor antagonist properties (Gobbi et al. 1990; Blier chiatry, McGill University, Montreal. Canada H3A lA 1. and the et al. 1991 ), which may constitute a confounding factor Department of Endocrinology, Maisonneuvc-Rosemont Hospital, Montreal, Canada HlT 2M4. in the interpretation of these studies. Address correspondence to C. Benkelfat, M.D., l\eurobiological Flesinoxan is a newly developed substituted ben Psychiatry Unit, Department of Psychiatrv, McGill University, 1033 zamide with high affinity and selectivity for the 5-HT1A Pine Ave. West, Montreal, Canada H3A lAI. Received August 31, 1994; revised Januarv 10, 1995; accepted Janu receptor subtype (2 nM); (Olivier et al. 1991). In autora ary 27, 1995 . diographic studies, flesinoxan showed a regional dis-
.'Jl:UROl'~Y(HOPlfAR\1,\LOltlCI ;c1ci, \~\I ,';, \.1.) . ..:' © 19 tribution cumparable t,1 that ot 8-hvdroxy-2-(di-n-pfll 29 years (SD = 4.8; range = 21 to 37 years; weight = pylamino)tetralin (8-01-1-DPAT; Schipper et al. 1991) 73.2 ± 10.5 kg; and height = 1.76 ± 0.15 m) each re It decreased body temperature and reduced aggressivitv ceived two intravenous infusions of flesinoxan (7 µg/kg scores in animal models (Schipper et al. 1991). Flesi and 14 µg/kg) and one of saline, over a period of 10 noxan displays high putency at both somatodendritic minutes, on three separate occasions, a minimum of and postsynaptic 5-HT I.\ receptors 1Hadrava et al. 1 week apart, between August and November 1992. 1995). For instance, in microiontophoretic studies, it was Neuroendocrine, physiological, and psychological mea found 17 times more potent than gepirone in suppress sures were collected over a 2-hour period during each ing the firing rate of dorsal hippocampus pvramid,,1 session. neurons (de Montignv et al. 1991), These characteris tics, together with the fact that. contrary to the azap1- Antagonist Study. In a randomized, double-blind, 3 x rones, it does not generate I-PP, make it a potentiallv 3 Latin-square design, 12 men (age = 28.3 ± 4.7 years; valuable probe for assessing =;-HT 1\ rL'Ceptor function range= 20 to 40 years; weight 71.3 ± 5.6 kg; height = in humans. 1.78 ± 0.15 m) received orally, on three separate occa The present studv determrnl'd tht· effects of flesinli sions each, a placebo, 30 mg of racemic pindolol, or xan on body temperature, and neuruendocrine and bl' 4 mg of methysergide, 90 minutes prior to the infusion havioral measures in vnung male healthv volunteer-. of l mg flesinoxan over 10 minutes. Each subject was The selectivity uf these respunst·s for the 5-HT svstem. tested on three separate occasions, a minimum of 1 and more specifJCally for the ''i-HT \ receptor, was fur 1 week apart, between November 1992 and February ther investigated in volunteers pretredted with the mixed 1993. Five subjects participated in both the dose-re 5-HT1 ::i antagonist nwthvsl'l)~ilk. tlw 5-HT1 \ antagt•• sponse and the antagonist studies. Neuroendocrine, nist pindolnl, and pl,Kebu phvsiulogical, and psychological measures were col lected over a 3.5-hour period during each session. MATERIALS AND METHODS Subjects Twenty-eight healthv niluntl'er-. were recruited through Procedure local nev,·spaper advertisements. All potential research Subjects arrived at the Clinical Investigation Unit of the subjects underwent physical and psychiatric examina Royal Victoria Hospital in Montreal at 8:30 A.M., hav tions (using the Structured Climcal Interview for DSM ing fasted since midnight. They wore normal indoor IE-R, nonpatient version. '::ipit1er et al. 1992). had an attire and, after being weighed, reclined on a bed in electrocardiogram and routine laboratorv tests, includ a comfortable position with the head elevated. They ing serum sequential multiple analysis by computer were not allowed to eat, sleep, or watch television un complete bluod count. thvroid !unction tests, HIV test til the procedure was completed. Two indwelling ve u ~ine analysis. and a urine tuxil·ulug\'-screen for drugs nuus catheters were inserted into the antecubital vein of abuse. Unlv ph\'SJC all\' heal th v su biects \\ ithou t .i and kept opened with a slow infusion of 5% dextrose personal histtin of psHhidtric illness Pr a family h1-- in a normal saline solution. After insertion of the intra tory of mood disprdn-. ur ciln,hPlism in first-degree rela venous catheters, baseline blood samples were obtained tives were in\'ited tu partiCipate Subjects scoring 2 m folluwmg a 45-min rest period and again, 15 minutes mnre on at least three svmptom-, ( m the Hopkins Svmp· later. to determine baseline hormone concentrations. tom Checklist (HSCI -90l and mun· than 3 on the B,'rk Immediately following the collection of the last base lnventur\· flH Dq1r1c'-s1on 11. en· c'\dudt·d. All subjt•ct.; line sample, around 10:00 A.M., subjects received an were mediLatlun free· tor .i minimum ot 2 weeks priur rntravenous infusion of either flesinoxan or saline de to testing, smuked iv-,-, than H' Cigarettes per dav, <1nd l!\·ered over a 10-minute period. Additional blood sam ingested nu more th,w the l'(jUI\ alent of five beer-, pe1· ples were collected at 15, 30, 45, 60, and 120 minutes ,,.-eek and three cup.., Pt ( L'ftt·t' ~w1· dav. fh1s studv \\ d.., attl'r the start of the infusion. ln the antagonist study, approved by thl' Re-.t•,m h Uh1c-, Board nf t\w Dep,irt the first blood sample was obtained 45 minutes after rwnt ut Pc-\ ch1,1tn , it \ k( ,ill L nivers;t\ . \,\ ntten in insertion of the intravenous catheters. Then, subjects f..irmed consent\\ ,b obt.rnwd trum all ·,ubject-, lwkn· hen· administered orally either a placebo, pindolol, or enrullment 111 tlw ,tu,h methysergide 90 minutes prior to the flesinoxan infu sHin Blood samples were collected at -15 minutes, im Experimental Design rnediatelv prior to the flesinoxan infusion, and at 15, Dose-Respo11se!,f11d11, l11 ,, 1c1nd,,m1,1ed, ,iuuble-hl111d ;u, 45, hO, and 120 minutes following the flexinoxan in :, x 3 Latin-squ,Ht' de-,1.~n 11 nwn 1,itli a nwan c1g,· ,1t tus1un NEUROPSYCHOl'HARMACOLOL) ['!Cle; \ lll.. I,, I\:()_ 2 Flesinoxan and Serotonin1A Receptors 95 Neuroendocrine, Behavioral, and tiunal-Deficit. The somatic symptom checklist is a Physiological Measurements compendium of the most frequent somatic complaints cummonly encountered during treatment with seroto Neuroendocrine Measurements. Blood samples for nergic agents, in particular flesinoxan (flesinoxan in measuring hormone plasma levels were collected in ice wstigator brochure, Solvay-Duphar, Weesp, Holland); cold plastic tubes containing 10 µI/ml of 0.5 M Na2 it consists of 29 physical symptoms rated by subjects EDT A. They were centrifuged (20 minutes at 2,500 x on a five-point scale (none, slight, moderate, much, se at 4cq within one hour of sampling. Plasma was ali vere). In the dose-response study, the psychological quuted into plastic tubes and stored at -- 70' C until time questionnaires were administered 15 minutes prior to, of assay. Plasma prulactin (PRL), growth hormone and 30 and 120 minutes follmving the flesinoxan infu (GH), and adrenocorticotropic hormone (ACTH) lev sion; in the antagonist study, the psychological ques els were determined by standard immunoradiometric tionnaires were administered 90 and 15 minutes prior assays using commercially available kits (Immunocorp, t(1, and 30 and 120 minutes following, the flesinoxan Montreal, QC; ICN Biomedicals, Montreal, QC; Nichols infusion. Institute Diagnostics, San Juan Capistrano, CA). Plasma cortisol was determined bv radiuimmunoassay using a CPmmerciallv available kit (Kallestad, Austin, TX). The Drugs inter- and intra-assay coefficients of variation were as Flcsinoxan was provided by Solvay-Duphar in lyophi follows: PRL, 3.9°/4, and 2.4%, CH. 12. 1% and 12.3%; li1:ed powder contained in a closed sterile container. ACTH, 7.8% and 3.2%; ct1rtisul, 7.6% and 8.4%. At the Flesinoxan and saline infusion vials were prepared in end of each study, all measurenwnts were carried out identical forms, by the pharmacy department of the in one assay by a techninan blind to the order and na Roval Victoria Hospital. Flesinoxan was dissolved in ture of drug treatments. -;,iline 0.9':lo, following Solvay-Duphar instructions, and Assessment of Hypothennic and Cardiovascular Re storl'd in individual vials of l3 ml each (0.05 mg/ml; 7 sponses. Sublingual bodv temperature \Vas measured pg kg; 0.1 mgiml: 14 µg/kg). The concentration, iden using a thermistor probe (Electro-therm, model TM99A) titv. pH, purity, lack of pyrogenicity, and stability of and digital readings were obtained at the end of a flesinoxan were verified by Societe d' Analyses Biophar 2-minute recording period. Blood pressure and pulse rnaceutiques (Laval, QC). Flesinoxan was diluted in 20 were measured and recorded using an automated mon ml Llf saline 0.9% and administered via an antecubital itor (Criticare 508, CritiL:,m' '-i\stems, Inc., Waukeshc1. , cin over 10 minutes. The use of flesinoxan as an intra WI:,. Sublingual bodv temperatun'. blood pressure, and ' l'llous infusion was approved by the Health Protec tiun Branch nf Health and Welfare, Canada. heart rate \\ere ren1rded l'\Tf\ I; minut!:'s thrnughuut the pron_,dun' l'indolol I Visken(!{) and methysergide (Sansert®) are l0 tith commercially available in Canada. Placebo (lac Assessment of Mood and Other Psychological Respon tll'>l'L pindolol, and methysergide pills were provided ses. Three questionnaires \Vere used tu assess the psv L,,- the pharmacy department of the Royal Victoria Hos chological and somatic changes associated \Vith fles1- p1tc1l _ l'indolol and methysergide were administered noxan infusions: the Bipolar Profile of Mood States l,lmdl\ in random order 90 rninut!:'s prior to flesinoxan (McNair et al. 1988), the NlMH Self-Rating Scale (Mur 111tusiun c1t c1 dose of 30 mg and 4 mg, respectively. phy et al. 1989), and a somatic symptom checklist. The Bipolar Profile is highly sensitive to subclinical changes Statistical Analysis in mood states and consists of 72 adjectives commonh used to describe transitory mood states. Subjects an' Bec,iuse some samples were accidentally destroyed dur required to rate each adjective un a four-point scall' ing storage and centrifugation, neuroendocrine results ranging from "not at all" (0) tP "extremely" (3). Adjec Me reported for IO subjects only, except for levels of tivE·s are grouped together in six dusters representing ACTH. which are reported for 9 subjects in the dose specific mood states (Agreeable-Hostile, Compused rt>sponse studv and 7 subjects in the antagonist study. An> A 30 same subjects, the hypothesis of no treatment x time ... interaction was tested by multivariate analysis, by com :::l' 25 ... puting Wilks's lambda statistic transformed to an F var 'iii T T iate (Morrison 1976). When the treatment x time inter z. 20 9----0--·-oT .... I ·, .. action was significant (p < .05), indicating differences ! I. '· ,,, ... 15 . ,, ... between the treatments with respect to the profile of .!! I •, Q response over time, the equality of treatment effects was .. JO / -..... ,.-6 =- I tested at each time point, by separate univariate analy T.' "E 5 ses of covariance. In the absence of a significant treat "' _/_,, 0 ment x time interaction, the treatments were compared s:::" ...... <:] 0 with respect to the average response over time by a univariate analyses of covariance. The homogeneity -S of the regression slopes of the response variable on 0 15 30 45 60 75 90 105 120 the covariate (baseline value) for each treatment group Time (min) (parallelism of regression lines) was tested by the treat B ment x covariate interaction. No significant departures 20 ... from homogeneity occurred (p > .05). Thus, the mag T T nitude of the differences between treatments can be as 15 p-·-0. ' sumed to be independent of the baseline values of the ; '' covariate. Because of missing data for the ACTH re 10 I , .. , sponse to flesinoxan, in the dose-response study, only / , ...... maximum change (independent of time) relative to base p-r;' ' 5 ...... , .. line(~ peak) could be analyzed. The hypothesis of no ,' 0 difference between the treatments with respect to the , T 0 incidence of side effects was tested by the chi-square ·······································() test for contingency tables. All statistical analyses were -5 1 performed with the Systat software package (MacIntosh <:] version, 1989). All data are expressed as means ± SD, -10 ~.....-----r----r-----ir----,-----,-----,--.....----....--- unless specified othenvise. 0 15 30 45 60 75 90 105 120 Time (min) RESULTS C 400 Dose-Response Study ... Comparison of Baseline Measurements 300 - T Prior to placebo or flesinoxan administration (whether ... T .-.-----·-0 7 or 14 µg/kg), baseline neuroendocrine, temperature, 200 i.--o------·- -, blood pressure, and pulse were within normal limits T / p for all subjects: plasma PRL (µg/L), 5.8 ± 3.2, 6.7 ± 2.9, 100 I and 6.1 ± 2.5; plasma GH (µg/L), 3.5 ± 4.1, 6.0 ± 11.6, I / and 2.1 ± 0.9; plasma cortisol (nmol/L), 248.1 ± 59.8, 0 I 279.8 ± 112.1, and 280.1 ± 77.9; plasma ACTH ·--o-· T T ··,, ········<>········<>········-<>·············· ··················· T (pmol/L), 4. 7 ± 2.1, 5.1 ± 2. 7, and 4. 7 ± 2.2; tempera ture ('C), 36.5 ± 0.2, 36.5 ± 0.3, and 36.6 ± 0.2. For these measurements, subjects showed negligible vari 0 15 30 45 60 75 90 105 120 ations between each baseline session, with the excep Time (min) tion of one subject, who showed a high baseline plasma GH on one occasion. Figure 1. Time-dependent effect of flesinuxan (7 µgikg, di amonds; 14 µg/kg, circles) or placebo (squares) on plasma Neuroendocrine Measurements prolactin (A), growth hormone (B), and cortisol (C) concen trations. Each point represents mean ± SEM obtained from PROLACTIN. The 7 µg/kg dose of flesinoxan produced 10 healthy volunteers. **I'< .OJ; *** p < .001, flesinoxan 14 no alteration of PRL levels (Figure 1A). However, at µglkg vs. placebo, ANCOVA. the dosage 14 µg/kg, flesinoxan significantly increased PRL levels over baseline (placebo: Li peak = - 2%; flesi noxan [7 µg/kg]: ~peak= 15%; flesinoxan [14 µg/kg]: NEUROPSYCHOPHARMACOLOG'! l99'i- VOL. 11, NO. 2 Flesinoxan and Serotonin1A Receptors 97 ** 0.25 l 40 0 .1 .1 30 I t .l. "- ..., <~-i-····· .....: .. ;= 20 .,,_ Q, -0.25 \~-, "- .~········1·······1· ··~1·········--1 .,E +t t ·,. • • • * ••• I- 'o----o • • • 10 < .1 .1 ·-·-0._ +tt ttt .1 --o-----0--. -0.5 ttt 1 1 --o ...., ttt ttt 1 o L...c:,,:=,=:, ::::, ,=:, ::::, -::,::::- ,2,1::==~=- ttt Placebo FLS 7 µglkg FLS 14 µg/kg -0.75--'---~-~~-~-~-~---~ Figure 2. Effect of flesinoxan or placebo on plasma ACTH 0 15 30 45 60 75 90 105 120 levels (maximum response). Data obtained from 9 healthy Time (min) volunteers; mean ± SEM. ** p < .005, flesinoxan 14 µgikg vs. placebo, ANCOV A. Figure 3. Time-dependent effect of flesinoxan (7 µg/kg, di amonds; 14 µg/kg, circles) or placebo (squares) on oral tem ll peak = 366%). Multivariate analysis indicates a sig perature. Each point represents mean ± SEM (obtained nificant interaction [treatment x time] for PRL (Wilks's from 11 healthy volunteers). Flesinoxan 7 µg/kg vs. placebo, F8,24 = 6.93; p < .001). The maximal PRL response oc ** p < .01, *** p < .001; flesinoxan 14 µg/kg vs. placebo, ttt p < .001, ANCOV A. curred 45 minutes after administration of the high dose of flesinoxan (14 µgikg) (F1 1, = 44.80; p < .001) and plasma PRL levels were still significantly increased at 120 minutes (Figure 1A). · (Figure 2). The ANCOVA showed a significant treat GROWTH HORMONE. Flesinoxan, at the dosage 14 ment effect (F2,13 = 8.18; p < .005) due to a greater µg/kg, increased significantly plasma GH levels over ACTH response associated with administration of the baseline (placebo: ll peak= - 7%; flesinoxan [7 µg/kgj: high dose of flesinoxan. ll peak= 5% flesinoxan [14 µg,kgj: ll peak = 743%). Body Temperature, Blood Pressure, Multivariate analysis revealed a significant interaction and Heart Rate [treatment x time) for GH (Wilks's FK24 = 5.44; p < .001). With the high dose of flesinoxan the maximal GH Flesinoxan decreased oral temperature in a dose response occurred at 60 minutes (Fi.1, = 54.12; p < dependent manner (Figure 3). The maximum effect was .001), then declined rapidly over the next hour in all recorded at 120 minutes after administration of either subjects (Figure 18). Plasma GH levels remained signi dose of flesinoxan (placebo: ll peak = 0°C; flesinoxan ficantly increased over baseline at 120 minutes. [7 µg/kg]: ll peak = -0.25°C; flesinoxan (14 µg/kg]: CORTISOL. Significant long-lasting increases in ll peak = -0.50°C) (Figure 3). Multivariate analysis re plasma cortisol were produced by flesinoxan at the high vealed a significant interaction [treatment x time] for dosage (placebo: ll peak = - 25%; flesinoxan (7 µg/kgj: oral temperature (Wilks's fi4,22; p < .001). At the time ll peak= -19%; flesinoxan 114 µg/kg]: ll peak= 92%). of maximum response, there was a greater hypother Multivariate analysis revealed a significant interaction mic response to flesinoxan (7 µg/kg) (F1,1s = 18.55; p < [treatment x time) for cortisol (Wilks's fH,2 4 = 7.00; p < .001) and to flesinoxan (14 µg/kg) (F1,1s = 74.93; p < .001). The maximal cortisol response occurred 120 min .001) than to placebo. There was no significant effect utes after administration of the high dose of flesinoxan of flesinoxan at either dosage level or placebo on both (F1,15 = 75.34; p < 0.001) (Figure lC). Consistent with blood pressure and heart rate. circadian rhythm, a decrease uver time was noted for Psychological Effects plasma cortisol levels in the placebo group. The peak PRL and cortisol responses elicited by the high dose of PROFILE OF Mooo STATES. The high dose of flesinoxan flesinoxan correlated significantly (rH = .68, p < .05). was associated with a moderate increase in feelings of ACTH. Flesinoxan at the dosage 14 µgikg in tiredness and fatigue. Multivariate analysis revealed no creased significantly plasma ACTH levels over base significant interaction [treatment x time], but a line (placebo: ll peak = - 21<¼i; flesinoxan (7 µg/kg]: significant treatment effect for the factors energetic/tired ll peak= -6%; flesinoxan (14 µg/kg]: ll peak= 585%) (h 17 = 11.05; p < .001), confident/unsure (F2,17 = 98 R. Seletti et al. NEUROPSYCHOPHARMACOLOGY 1995-VOL. 13, NO. 2 \ B :!S 20 15 - !() ;J oi, :I 2, .. 10 15 C 0 -,,'-' E .. 5 0 :c 10 T Figure 4. Effect of pindulol (30 " i 0 mg, diamonds), methysergide (4 -~ 0.. mg, circles), and placebo (squares) 5 [,!l .. • • - -5 administered orally, 90 minutes E"' ttt Cl> prior to flesinoxan intravenous 0 T ·-- ---o 5:::"' -10 infusion (1 mg) on plasma concen· <1 trations of prolactin (A), growth w5 -15 hormone (B), cortisol (C). and () 15 30 45 60 75 90 105 120 0 15 30 45 60 75 90 105 120 ACTH (D). Each point represents the mean ± SEM obtained frnm Time lmin) Time (min) 10 healthy volunteers, with the D JOO 30 exception of ACTH response~. where each point represenb mean ± SEM from 7 subjech * p <. .05; ** p < .01; ***I'< .lXll. zoo 20 [pindolol + tlesinoxanj vs. [pl,i 0 E ceb,i + flesinoxan ]; and t /' < _();; Q, ttp < .01; tttp < .001, [methv~cr gide + flesinoxan] vs. [plciceb(, 100 + flesinoxanj, ANCOVA. () 0 15 JO 45 60 75 90 105 120 0 15 30 45 60 75 90 105 120 Time (min) Time (min) 12.14; p < .001), and clearheaded;confused (F2 17 = 6.8; effects for the following side effects: drowsiness, light p < .01), Maximal responses occurred at 30 minutes: headedness, nausea, numbness, headache, faintness, energetiC!tired (f117 = 21.57; p < .001); confident un asthenia, dizziness, and sweating. Two subjects com sure (F111 = 17.64; p < .001); and clearheadediconfused plained of headache and difficulty concentrating after (F1 17 = 14.10; p < .002). placebo. Ratings of nausea (ry = .63, p < .05) and light NIMH SELF-RATI:--JC SCALI. Subjects noticed headedness (r9 = .71, p < .05) were correlated signi significant but moderate feelings uf fatigue and lack llf ficantly with the peak flesinoxan-induced cortisol re energy after the high dose of flesinoxan but not after sponse. the low dose. Multivariate analysis revealed a significant interaction [treatment x time] 11(F217 = 4.76, p < .ll:'i) for altered self-awareness and a significant treatment Antagonist Study effect (h 17 = 4.49, p < .0:i) for functional deficit. Max Comparison of Baseline Measurements. All baseline imum responses occurred at 30 minutes, and altered measurements collected prior to the administration of self-awareness (F1 1- = 4 3'i, p < .05) and functional placebo, pindolol, and methysergide were within nor deficit (F1 17 = 9.21, p < (Jl). The effect decreased mal limits: PRL (µg/L), 8.9 ± 7.2, 7.1 ± 5.7, and 7.2 ± slightly over the followmg hour 6.3; CH (µg/L), 5.1 ± 8.1, 4.3 ± 4.9, and 11.9 ± 12.9; Side Effects. Six subiecb reported transient side effects cortisol (nmol/L), 246.9 ± 71.8, 246.8 ± 98.7, and356.1 ± following the l0vv dose of flesinoxan, and 10 reported l:i6.4; ACTH (pmol!L), 4.0 ± 1.3, 4.6 ± 1.9, and 9.7 ± them following tht' high dose uf flesinoxan. Contin 9.8; temperature CC), 36.4 ± 0.2, 36.3 ± 0.2, and gencv table analyses -.h11wl·d c;ignificant treatment 3b.4 ± 0.2. NEUROPSYCHOPHARMACOLOC't i'IY>- \ OL. I\, NO. Flesinoxan and Serotonin1A Receptors 99 Effects of Pindolol and Methysergide. Administration min. F1.1Y = 9.19; p < .05) (placebo~ peak= -0.32°C; of either pindolol or methysergide did not significantly pindolol '1 peak = -0.21 °C; methysergide peak = (p > .05) alter baseline measurements of hormone lev -0.17°C) (Figure 5). els, body temperature, systolic blood pressure, pulse, Effects of Pindolol and Methysergide on Cardiovascu and psychological subscores prior to flesinoxan infu lar Parameters. Multivariate analysis revealed no sion. However, methysergide showed a trend toward significant interaction [treatment x time] for either increasing GH (interaction [treatment x time]: h 1s = blood pressure or pulse values following flesinoxan in 2.96; p = .083) and cortisol (treatment effect: F2_1c; = fusion. 3.52; p = .056). Moreover, methysergide increased ACTH levels slightly but not significantly. Diastolic Effects of Pindolol and Methysergide on Flesinoxan blood pressure was significantly affected by both pin lnduced Psychological Effects. As in the dose-re dolol and methysergide (treatment effect; F21'; = sponse study, flesinoxan infusion produced a mild to 25.78; p < .001): the former drug decreased it by 4 mm moderate increase in the subjects' feelings of tiredness of Hg (h 1g = 12.81; p < .01), whereas the latter in and fatigue, although of a lesser magnitude. This effect creased it by 2 mm of Hg (F1 JY = 5.59; p < 0.05). was potentiated by methysergide but was unaffected by pindolol (interaction [treatment x time]; ener Effects of Pindolol and Methysergide on getic/tired: F2.19 = 7.86; p < .01; confident/unsure: Flesinoxan-Induced Neuroendocrine Responses h IY = 5.72; p < .05). In addition, some subjects reported a subclinical increase in the subjective percep PROLACTJN. Methysergide, but not pindolol, signifi tion of altered self-awareness, dysphoria, and func cantly blocked the PRL response to flesinoxan (interac tional deficit as measured by the NIMH Self-Rating tion [treatment x time]; Wilks's F,_24 = 3.78; p < .005) Scale. None of these responses were affected by pre (placebo, peak = 194%; pindolol, peak = 203%; treatment with either methysergide or pindolol. methysergide, peak = 40%) (Figure 4A). GROWTH HORMONE. PindoloL but not methyser Side Effects. Overall, this procedure was well toler gide, completely abolished the GH response to ated, with subjects reporting even fewer or less intense flesinoxan (interaction [treatment x time]; Wilks's side effects after flesinoxan than they reported in the Fs.24 = 3.52; p < .008) (placebo, '1 peak = 224%; pin dolol, peak = 6%; methysergide, '1 peak = 79%) (Figure 4B). CORTISOL. Both methysergide and pindolol failed to block the cortisol response to flesinoxan. It was even potentiated by methysergide (interaction [treatment x time]; Wilks's Fs.24 = 3.59; p < .05) (placebo, '1 peak 0 51 %; pindolol, peak = 63(¾_); methysergide, '1 peak = 59%) (Figure 4C). ;..; ACTH. As for the cortisol response, methysergide *** I.."' showed a trend toward potentiating the ACTH re ::, ';, ** I.. sponse to flesinoxan (treatment effect; hY = 3.04; p = '11 Q. hand, pretreatment with pindolol E .098). On the other '11 resulted in a slight attenuation of peak ACTH levels f- -0.2 < (placebo, peak = 148%; pindolol, '1 peak = 113%; methysergide, peak = 206%) (Figure 4D). Effects of Pindolol and Methysergide on Flesinoxan Induced Hypothermia. Both methysergide and pin dolol showed a trend toward attenuating the maximal -0.4 ..L..-.---r---.---.--r--.--,---,,---,- hypothermic response to flesinoxan (interaction [treat 0 15 30 45 60 75 90 105 120 = = ment x time]; Wilks's Fi-t.26 1.83; p .09). Strik Time (min) ingly, the hypothermic effect of flesinoxan was pres Figure 5. Effect of pindolol (30 mg, diamonds), methyser ent in the first hour irrespective of the nature of the gide (4 mg, circles), and placebo (squares) administered pretreatment administered (Figure 5). When tempera orally, 90 minutes prior to flesinoxan intravenous infusion were analyzed separately for each time point, ture data (1 mg) on oral body temperature. Each point represents the statistically significant treatment differences emerged mean ± SEM obtained from 12 healthy volunteers. * p < .05; indicating an at:t'nuation bv methysergide of the * * p < . 01; * * *p < . 005, [methysergide + flesinoxan] vs. flesinoxan-induced '"~:-.,therm1<1 ,it 90, 105, and 120 I placebo + flesinoxan], ANCOVA. There is no significant minutes following flesinll,an admirnstration 1at ](le; effect of pindolol pretreatment. 100 B. Seletti et al. NEUROPSYCIIOPHARMACOLOGY 1995-VOL. 13, NO. 2 dose-response study. Drowsiness, lightheadedness, 5-HTIA agonist buspirone on GH release is unaffected and difficulty concentrating were reported by the same in humans with experimentally lowered serotonergic proportion of subjects. Pindolol showed a trend toward neurotransmission, induced by a dietary depletion of attenuating and methysergide a trend toward poten tryptophan (Blier et al. 19946). tiating the side effects induced by flesinoxan. However, In the present study, flesinoxan infusion resulted the chi-square test demonstrated no significant (p > .05) in a time-dependent increase in plasma GH levels, con differences between the two treatment conditions. Two sistent with results obtained with other selective 5-HT1A subjects dropped out of the study following the ad agonists in humans (Meltzer et al. 1983; Lesch et al. ministration of methysergide administered in combi 1989; Anderson et al. 1990; Miller et al. 1990). Blockade nation with flesinoxan (vomiting, 11 = 1; vertigo, n = 1). of the GH response to flesinoxan by pindolol but not by methysergide is consistent with a S-HT1A-mediated phenomenon. This finding concurs with previous Test-Retest reports of pindolol blocking the GH response to ipsapi Five subjects participated in both studies, 40 days apart, rone (Lesch 1991) and buspirone (Anderson and Co and hence were suitable for a test-retest comparison of wen 1992). The failure of the S-HT112 receptor an their responsiveness to flesinoxan. The subjects' PRL tagonist methysergide to antagonize the GH response and GH responses in both studies were of comparable to flesinoxan was not unexpected, given the limited magnitude (plasma PRL [µg/L]: peak = 16.4 ± 11.1 effectiveness of methysergide and metergoline (another and 11.8 ± 11.7; plasma GH [µg/Lj: ~peak= 12.8 ± similar S-HT112 antagonist) in attenuating S-HT1A 12.2 and 7.4 ± 16.3), whereas their ACTH and cortisol receptor-mediated functional responses (Koenig et al. responses to flesinoxan were lower during retest 1987; McCance et al. 1987; Neuhauser et al. 1988; Sharp (plasma ACTH [pmol/Lj: max = 16.5 ± 13.4 and et al. 1989; Fuller and Snoddy, 1990; Gartside et al. 8.8 ± 11.6; plasma cortisol [nmoliLI: ,~peak= 83.2 :<:- 1992). This might be related to the weak capacity of 162.9 and 26.0 ± 80.2). There was a significant correla methysergide to displace [1251] LSD from 5-HT1A bind tion in the peak GH (ri = 0.881, p < .05) but not in thl' ing sites (Lovenberg et al. 1993). peak PRL or cortisol responsiveness ac,.oss the t\\'l 1 studies. Flesinoxan-Induced Hypothermia The hypothermic respunse tu tlesinoxan was of thl' same intensity in both studies(~ peak= --0.5 ± 0.2 There is now good evidence that 5-HTiA agonists pro and -0.4 ± 0.2 C). duce hypothermia in rodents via postsynaptic 5-HT1A receptors (for review see Millan et al. 1993). Recent ex perimental evidence, using the hypothermic response DISCUSSION to buspirone as an index of 5-HT IA receptor function in healthy volunteers undergoing a tryptophan deple Flesinoxan administered intravenously to male healthy tion paradigm, suggests that humans respond similarly volunteers elicited a dose- and time-dependent hypo (Blier et al. 1994a). The S-HT1A agonists ipsapirone, thermic effect as well as increases in plasma GH, ACTH; gepirone, and buspirone induce hypothermia in both cortisol, and PRL levels. The rise in plasma levels of rats (Koenig et al. 1988) and humans (Anderson et al. GH was blocked by pindolol but was not affected bv 1990; Lesch et al. 1990a; Anderson and Cowen, 1992), methysergide. Conversely, the PRL response to flesi which is blocked by pindolol (Lesch et al. 1990a; An noxan was blocked by methysergide but not by pin derson and Cowen, 1992). Consistent with 5-HT mech dolol. The hypothermic response to flesinoxan was at anisms, methysergide significantly attenuated the tenuated by both pindolol and methysergide but univ hypothermic response to flesinoxan. A similar effect significantly so by methysergide. Methysergide poten was obst>rved with pindolol but did not reach statisti tiated the ACTH and rnrtisol responses to flesinoxan cal significance. Although these data could indicate that tlt>sinoxan-induced hypothermia is a S-HT2 recep tor-mediated event, such an interpretation is unlikely Flesinoxan-Induced Growth Hormone Release for the following reasons: (1) the affinity of flesinoxan In general, studies with selective 5-HT agonists or an for 5-HT 2 receptors in binding studies is very low tagonists tend to support the stimulatory action of 5-HT (>4,500 nM) (Olivier et al. 1992); (2) in rodents and hu on GH secretion (Tuomisto and Mannish) 1985). This mans, 5-HT 2 agonists cause hyper- rather than 5-HT effect seems due to its postsynaptic action in the hypothermia (Strassman and Qualls 1994); (3) in data rodent hypothalamus, since the 5-HT synthesis inhibi recently obtained from our laboratory, the hypother tor parachlorophenylalanine was reported to decrease mic response elicited in rats by flesinoxan was pre basal GH levels (Tuomisto and Mannistti 1985). In sup vented by pindolol (Hadravci et al. 1995). pl1rt of this contention, tht> stimulatory effect uf tht' A more conservative interpretation of this relative NELROPSYCHOPHARMACOLOCY 194;-voL. 1,, NO. 2 Flesinoxan and Serotonin1A Receptors 101 failure of pindolol to significantly block the flesinoxan that 8-OH-DPAT elicits PRL release in rats (Simonovic induced hypothermia may be that pharmacokinetic et al. 1984; Willoughby et al. 1988; Aulakh et al. 1988; (dose, duration of treatment, brain penetration) or, per Di Sciullo et al. 1990; Kellar et al. 1992; Flores et al. 1992), haps to a lesser extent, pharmacodynamic factors (rel which is blocked by methysergide (Flores et al. 1992) ative selectivitylaffmity of pindolol for 5-HT1A versus or metergoline (Willoughby et al. 1988; Aulakh et al. other 5-HT receptors and/or adrenoceptors in humans) 1988; Kellar et al. 1992). In humans, increased PRL may have prevented the achievement of sufficient secretion was reported with buspirone (Meltzer et al. blockade of S-HT1A receptors. In fact, it is noteworthy 1983; Coccaro et al. 1990; Anderson and Cowen, 1992) that, for both 5-HT antagonists, the rate at which and gepirone (Anderson et al. 1990), but not with ip hypothermia manifested itself was unaffected in the first sapirone (Lesch et al. 1989) or tandospirone (Miller et hour following flesinoxan injection and that an an al. 1990). Differences in the magnitude of the PRL re tagonism was apparent only in the second hour. sponse to various S-HT1A receptor probes in humans might be explained by differences in their respective Flesinoxan-Induced Release of ACTH and Cortisol pharmacological profiles, in particular with respect to their relative affinity for 5-HTIA versus 02 receptors In this study, the high but not the low dose of flesinoxan (Olivier et al. 1992; Nash and Meltzer 1989). Interpre produced significant increases in plasma ACTH and tation of the PRL data is further complicated by the re cortisol levels. Hypothalamic-pituitary-adrenal activa port that nonselective S-HT1-2 antagonists, such as tion was not likely due to anxiety since all subjects metergoline, block the PRL release produced by halo reported feeling free of anxiety throughout the study; peridol, thus suggesting dopamine-mediated mecha nor was it due to a reflexive cardiovascular effect, as nisms (Ellis et al. 1991). The pattern observed in the neither blood pressure nor heart rate was affected by present study-that the PRL response is blocked by the procedure. The finding of a positive correlation be methysergide but not by pindolol-suggests that the tween the nausea and lightheadedness induced by flesinoxan-induced rise in PRL might not be 5-HT 1A flesinoxan and cortisol plasma levels suggests, how receptor-mediated. Thus the blockade by methysergide ever, that part of the flesinoxan-induced release of cor of the PRL response to flesinoxan could indicate either tisol might be related to somatic side effects. a 5-HT? or 02 receptor-mediated event (Krulich et al. In contrast to results obtained with selective 5-HT tA 1981; Hoyer 1991). Given the high affinity of methyser agonists in rats (Koenig et al. 1987; Gilbert et al. 1988; gide for S-HT7 receptors (32 nM; Lovenberg et al., Haleem et al. 1989; Fuller 1990; Fuller and Snoddy 1990; 1993), the lack of affinity of pindolol for this recently Di Sciullo et al. 1990; Gartside et al. 1992; Pan and Gil cloned receptor, and the moderate affinity of flesinoxan bert 1992, 1993) and in humans (Lesch et al. 1990b), the for 02 receptors (140 nM; Olivier et al. 1992), it is pos effect of flesinoxan on ACTH or cortisol release was not sible that the PRL response to flesinoxan may involve prevented by pindolol (Figure 4C,D). The failure of pin both 5-HT and dopaminergic activation. However, dolol to block the flesinoxan-induced rise in ACTH and presently it is not known whether flesinoxan, like cortisol plasma levels suggests that this effect was not 8-OH-DPAT, has significant affinity for S-HT7 recep 5-HT 1A receptor-mediated. This conclusion is consis tors, which are present in high density in the hypothala tent with a recent report that 8-OH-DPAT, administered mus (Lovenberg et al. 1993). centrally in rodents, exerts a biphasic effect on the hypo The differential effectiveness of pindolol to an thalamic-pituitary-adrenal axis, with the stimulatory tagonize 5-HT1A responses in the present study might effect being non-5-HT IA receptor-mediated (Welch et be due to the involvement of different populations of al. 1993). However, given the breadth of the literature 5-HT IA receptors with distinct pharmacological prop suggesting otherwise (Gilbert et al. 1988; Pan and Gil erties. Indeed, in rats, a low dose of racemic pindolol bert 1992, 1993), these conclusions are tentative and (4 mg/kg) blocks the 8-OH-DPAT-induced hypother await replication. An alternative interpretation may be mia but leaves unaffected the PRL increase produced that, as in the case of flesinoxan-induced hypothermia, by this S-HT1A receptor agonist (Aulakh et al. 1988). other pharmacological factors may have limited the Yet, ( - ) pindolol, which is the enantiomer with the ability of pindolol to block potently 5-HTtA receptor 5-HT1A affinity, at a dose of 15 mg/kg, blocks the PRL mediated responses. The potentiation of the cortisol re response to 8-OH-DPAT (Blier et al. 1994b), thus indi sponse by methysergide is most likely due to its partial cating that the PRL-response in rats to 8-OH-DPAT is 5-HT agonistic properties (Apperley et al. 1980; Dumuis et al. 1988; Hoyer 1988; Schoeffter and Hoyer 1988). mediated by a 5-HT IA receptor. Interestingly, the sup pressant effect of microiontophoretically applied 8-0H Flesinoxan-Induced Release of Prolactin DPA Ton the firing activity of rat dorsal hippocampus pyramidal neurons is not blocked by ( - ) pindolol. Sev The demonstration of enhanced PRL secretion in re eral other lines of evidence accumulated in recent years sponse to flesinoxan is consistent with earlier reports also indicate that pre- and postsynaptic 5-HTIA recep- 102 B. Seletti l't al NEUROPSYCHOPHARMACOLOGY 1995-VOL. 13, NO. 2 tors have different pharmacological properties. For in Blier!', Lista A, de Montigny C (1993): Differential proper stance, the buspirone analogue BMY 7378 readily blocks ties of pre- and postsynaptic 5-hydroxytryptamine1A re ceptors in the dorsal raphe and hippocampus, I: Effect 5-HT A receptors in the rat dorsal hippocampus while 1 of spiperone. J Pharmacol Exp Ther 265:7-15 ineffectively blocking S-HT1A autoreceptors in the dor sal raphe (Chaput and de Montigny 1988). In contrast, Blier P, Seletti B, Bouchard C, Artigas F, de Montigny C ( 1994a): Functional evidence for the differential respon acute spiperone injection blocks the effect of microion siveness of pre and postsynaptic 5-HTJA receptors in rat tophoretic applications of 5-HT onto 5-HT neurons but brain. Soc Neurosci Abstr 20:632.9 not onto hippocampus neurons (Blier et al. 1993). Con Blier P, Seletti B, Young SN, Benkelfat C, de Montigny C seguently, the failure of pindolol in the present studv (1994b): SerotoninIA receptor activation and hypother to block the effect of a rather selective 5-HT 1A receptor mia: Evidence for a postsynaptic mechanism in humans. agonist, such as flesinoxan, by no means rules out the l\'europsychopharmacology 10(suppL 35, part 2):92S possibility that the observed physiological response is Chaput Y, de Montigny C (1988): Effects of the 5-hydroxy not mediated by a 5-HT v. receptor. tryptamine1 receptor antagonist, BMY 7378, on 5-hy droxytryptamine neurotransmission: Electrophysiolog ical studies in the rat central nervous system. J Pharmacol Exp Ther 246:359-370 CONCLUSION l occaro EF, Gabriel S, Siever LJ (1990): Buspirone challenge: ['reliminary evidence for a role for central 5-HT1A recep The present results provide novel evidence that the in tor function in impulsive aggressive behavior in humans. travenous infusion of flesinoxan is a safe and valid probe Psychopharmacol Bull 26:393-405 of ~erotonergic function. More specifically, the attenu de Montigny C, Ortemann C, Blier P (1991): Effect of 5-HT1A aticm or blockade by pindolol of the flesinoxan-induced agonist flesinoxan on the 5-HT system: Electrophysio hypothermic and GH responses suggests that these logical studies in the rat CNS. Soc NeurosciAbstr 17:193 measures qualify as reliable indices of 5-HT 1A receptor Di Sciullo A, Bluet-Pajot MT, Mounier F, Oliver C, Schmidt tunction in man. B, Kordon C (1990): Changes in anterior pituitary hor mone levels after serotonin JA receptor stimulation. 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