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NEUROPHARMACOLOGY

Dual, Hyperalgesic, and Analgesic Effects of the High-Efficacy 5-Hydroxytryptamine 1A (5-HT_1A) Agonist F 13640 [(3-Chloro-4-fluoro-phenyl)-[4-fluoro-4-{[(5-methyl-pyridin-2-ylmethyl)-amino]-methyl}piperidin-1-yl]methanone, Fumaric Acid Salt]: Relationship with 5-HT_1A Receptor Occupancy and Kinetic Parameters

Centre de Recherche Pierre Fabre (L.B., M-.B.A., M.P., A.N.-T., J.-P.R., F.C.C.) and Preclinical DMPK Departement (I.R.-U., F.S.), Castres, France; and Alcohol and Drug Addiction Division, Department of Psychiatry, University of Texas Health Science Center, San Antonio, Texas (W.K.)

Received for publication September 13, 2004
Accepted November 3, 2004.

	Abstract

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The aim of the present study was to establish the relationship between the plasma and brain concentration-time profiles of F 13640 [(3-chloro-4-fluoro-phenyl)-[4-fluoro-4-{[(5-methyl-pyridin-2-ylmethyl)-amino]-methyl}piperidin-1-yl]methanone, fumaric acid salt] after acute administration and both its hyper- and hypoanalgesic effects in rats. The maximal plasma concentration (C_max) of F 13640 after i.p. administration of 0.63 mg/kg was obtained at 15 min and decreased to half its maximal value after about 1 h. The amount of F 13640 collected by means of in vivo microdialysis in hippocampal dialysates could be measured reliably after 0.63 and 2.5 mg/kg, reached its maximum at about 1 h, and fell to half of its maximal value at about 3 h. 5-Hydroxytryptamine 1A (5-HT_1A) receptor occupancy was estimated by ex vivo binding in rat brain sections. F 13640 inhibited [³H]8-hydroxy-2-[di-n-propylamino] tetralin binding ex vivo in rat hippocampus, entorhinal cortex, and frontal cortex (ED₅₀, 0.34 mg/kg i.p.). Maximal inhibition was reached at approximately 30 min after 0.63 mg/kg F 13640 and fell to half of its value after about 4 to 8 h. After injection (15 min) in the paw pressure test, F 13640 (0.63 mg/kg i.p.) induced an initial hyperalgesia that was followed 4 h later by a paradoxical analgesia that lasted until 8 h. In contrast, in the formalin test, F 13640 inhibited pain behaviors until 4 h after drug administration. F 13640 also produced elements of the 5-HT syndrome that lasted up to 4 h after administration. These results demonstrate that F 13640 induces hyperalgesia and/or analgesia with a time course that parallels the occupancy of 5-HT_1A receptors and the presence of the compound in blood and brain.

We have recently reported on the discovery of F 13640, a uniquely selective and high-efficacy 5-HT_1A receptor agonist that induces powerful, broad-spectrum analgesia by two unprecedented neuroadaptive mechanisms (Colpaert et al., 2002). Inverse tolerance develops to F 13640-induced analgesia so that, with chronic administration, its analgesic effects grow rather than decay, as is the case with opioids (Colpaert et al., 2002; Bruins Slot et al., 2003). Also, F 13640 cooperates with ongoing nociceptive stimulation; the magnitude of its analgesic effect increases with the intensity and duration of nociception. For instance, whereas 0.63 mg/kg F 13640 produced hyperalgesia in normal rats 15 min after its i.p. injection, the compound produced profound analgesia in rats that were exposed to the severe, tonic nociception induced by the s.c. injection of 50 µl of formaldehyde (2.5%) into the plantar surface of the hindpaw (Bardin et al., 2003). Also, the cooperation should grow more effective, because the time during which the subject is coexposed to 5-HT_1A receptor activation and nociception is longer (Colpaert, 1996). As a result of these remarkable neuroadaptive actions, continuous infusion of F 13640 by means of osmotic pumps produces analgesia in rat models of chronic nociceptive pain and neuropathic allodynia that surpasses that, if any, of morphine and other agents that exemplify further molecular mechanisms of central analgesia [i.e., the serotonin (5-HT) and noradrenaline reuptake inhibitor imipramine, the N-methyl-D-aspartate antagonist ketamine, and the anticonvulsant gabapentin) (Colpaert et al., 2002; Deseure et al., 2003; Wu et al., 2003). Thus, very high-efficacy 5-HT_1A receptor stimulation constitutes a novel molecular mechanism of central analgesia that may be particularly relevant to the treatment of chronic pain states (Colpaert et al., 2002; Xu et al., 2003; Colpaert et al., 2004).

The studies reported herein used ex vivo and in vivo experiments to characterize the plasma and brain concentration-time profiles of F 13640 after its acute administration and establish the relationship between these profiles and both the hyper- and hypoanalgesic effects of F 13640 in rats. In the first series of experiments, we determined both the dose response and time course of plasma F 13640 concentration. The presence of F 13640 in rat brain after systemic injection using in vivo microdialysis coupled with liquid chromatography/tandem mass spectrometry (LC/MS/MS) was also measured. The occupancy of 5-HT_1A receptors by F 13640 was estimated ex vivo by measuring the ability of F 13640 to inhibit [³H]8-hydroxy-2-[di-n-propylamino] tetralin (8-OH-DPAT) binding in rat brain sections. In a second series of experiments, the time course of the paradoxical effects that the i.p. injection of F 13640 (0.63 mg/kg) produced on nociception (Colpaert et al., 2002) were measured in the paw pressure test and formalin model of tonic pain. Vocalization in response to mechanical stimulation is a highly integrated response (Le Bars et al., 2001) that is particularly susceptible to the hyperalgesic effects of 5-HT_1A receptor agonists as well as opioids (Colpaert et al., 2002); these same 5-HT_1A receptor agonists also demonstrate specific analgesia in the formalin test (Bardin et al., 2003). In addition, and since 5-HT_1A agonists induce a so-called 5-HT syndrome in rats (Berendsen et al., 1989), several behavioral signs of the 5-HT syndrome were also monitored. The F 13640 dose used (0.63 mg/kg i.p.) corresponds with that at which the drug produces both hyperalgesic and analgesic effects (Colpaert et al., 2002).

	Materials and Methods

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Animals
Male Sprague-Dawley rats [Ico: OFA SD (specific pathogen-free); Iffa Credo, L'Arbresele, France] were used in all experiments. Upon arrival, the animals were housed in groups in an environmentally controlled room (temperature, 21 ± 1°C; relative humidity, 55 ± 5%) on a 12-h light/dark cycle (lights on at 7:00 AM). Food (standard rat chow, A04; SAFE, Epinay sur Orge, France) and filtered (0.22 µm) water were continuously available. A 5-day acclimatization period was allowed before animals were used in experiments. Animals were handled and cared for in accordance with the Guide for the Care and Use of Laboratory Animals and the Guidelines of the Ethics Committee of the International Association for the Study of Pain, and all procedures were approved by the institutional Ethical Review Committee.

Detection of F 13640 in Plasma
Dose Response. Rats (160–180 g) received an i.p. injection of F 13640 (0.01–2.5 mg/kg) or saline, and 15 min afterward, blood samples from the abdominal aorta were collected under isoflurane anesthesia into plastic tubes containing heparinate. The blood samples were centrifuged as soon as possible, and the plasma was kept frozen at –20°C.

Time Response. Rats (160–180 g) received an injection of F 13640 (0.63 mg/kg), and under isoflurane anesthesia, blood samples from the abdominal aorta were collected into plastic tubes containing heparinate before and 15, 30, 45, 60, and 90 min and 2, 2.5, 3, 3.5, 4, 8, and 24 h after the i.p. injection of F 13640. The blood samples were centrifuged as soon as possible, and the plasma was kept frozen at –20°C.

Quantification of F 13640. Twenty-five microliters of internal standard solution (final, 10 ng/ml) and 0.5 ml of acetonitrile for protein precipitation were added to each centrifuged rat plasma sample. After mixing and centrifugation, the supernatant was transferred onto a 1-ml ChemElut cartridge (Varian, Inc., Palo Alto, CA) and allowed to wait for 10 min. Elution was performed using 2 x 3 ml of ethyl acetate. The eluate was evaporated under a slight stream of nitrogen. The dry residue was dissolved in 150 µl of mobile phase, and 30 µl was injected into the chromatographic system.

The LC/MS/MS system consisted of a liquid chromatography Alliance 2690 system (Waters, Milford, MA) coupled with a triple quadrupole mass spectrometer LCZ Quattro (Waters). Chromatographic separation was performed on an RP18 Symmetry 50 x 2.1-mm i.d., 3.5-µm particle size column (Waters). Eluent was performed at 0.2 ml/min with 15 mM ammonium acetate buffer, pH 3/acetonitrile [76/24 (v/v)]. A positive electrospray ionization was applied, and the mass spectrometry analysis in MS/MS mode was realized using the Multiple Reaction Monitoring mode. The transitions were 394.3 (M + H)⁺ 157.1 amu and 408.3 (M + H)⁺ 157.2 amu for F 13640 and internal standard, respectively.

The calibration curve was generated at 0.1, 0.5, 2, 10, 50, and 100 ng/ml in duplicate by spiking control rat plasma with 25 µl of appropriate F 13640 standard solutions. Quality control (QC) samples were prepared from separate weighing at 0.3, 5, and 75 ng/ml in duplicate. The QC samples were stored at –20°C until analysis under the same conditions as pharmacokinetic samples and considered as unknown samples. F 13640 was quantified in processed plasma samples by using Masslynx 3.4 software (Waters). The calibration curve was obtained by least square regression of F 13640 and internal standard peak area ratio versus theoretical concentration using a 1/C weighting model.

Accuracy and precision of the bioanalytical method were 3.75 and 10.24%, respectively, at the limit of quantification (0.1 ng/ml). The maximal inaccuracy of QC samples was 14.58% (at 0.3 ng/ml). Imprecision was lower than 4.69% (at 5 ng/ml).

Detection of F 13640 in Microdialysis Samples
Microdialysis. Rats weighing 240 to 260 g were anesthetized with chloral hydrate (400–500 mg/kg i.p. and supplementary doses to maintain anesthesia). A microdialysis probe (3 mm in length, 0.5 mm diameter; CMA/Microdialysis AB, Solna, Sweden) was stereotaxically implanted into the ventral hippocampus; the stereotaxic coordinates were –4.8 mm rostral, 4.6 mm lateral, and –7.5 mm ventral from bregma and dura surface, according to Paxinos and Watson (1986). The probe was continuously perfused (0.5 µl/min) with artificial cerebrospinal fluid containing 1 µM citalopram. Starting approximately 2 h after probe implantation, a 30-min sample was collected as vehicle baseline, and F 13640 (0.63 mg/kg) was injected. After 15 min, eight consecutive 30-min samples were collected. At the end of the experiment, the animal was killed by decapitation, and the brain was removed, frozen, and cut in a cryomicrotome (Jung Frigocut 2800; Leica Instruments GmbH, Nubloch, Germany) to verify the placement of the probe. The samples were frozen (–70°C) until analyzed by LC/MS/MS.

Measurement of F 13640. A method based on LC/MS/MS was developed to quantify F 13640 in microdialysis samples. The inherent selectivity offered by tandem mass spectrometry eliminated chemical noise, thereby improving the detectability of F 13640. LC/MS/MS was performed on a Finnigan Mat TSQ 7000 mass spectrometer (Thermo Finnigan, San Jose, CA) equipped with an atmospheric pressure chemical ionization interface. A Thermo Separation Products LC system consisting of a 4100MS quaternary pump and an AS3000 autosampler was used to introduce samples into the mass spectrometer via a Valco electric six-port injector valve (Valco Instruments Co. Inc., Houston, TX). This diverter valve was installed to allow for automated control of the flow to the mass spectrometer (flow between 5 and 8 min). F 13640 was chromatographed on a Waters Symmetry C8 column (5 µm, 4.6 x 250 mm) eluted with acetonitrile/water/ammonium acetate/acetic acid (350 ml/650 ml/3.87 g/50 ml) with a resulting pH of about 4 and at a flow rate of 1 ml/min. The nebulizer probe and heated capillary were maintained at a temperature of 500 and 175°C, respectively. The corona discharge needle was set at 5 µA. Nitrogen was the sheath and auxiliary gas at a pressure of 80 psi and graduation of 10 psi on the flowmeter, respectively.

Quantitation was conducted using selected reaction monitoring detection. The mass spectrometer was programmed to transmit the protonated molecules of the analyte [M + H]⁺ through the first quadrupole (Q1) at m/z 394. Collision-induced dissociation caused fragmentation within the second quadrupole collision cell (Q2). Argon was the collision gas in Q2 at a pressure of 1 mTorr with a collision energy of –25eV. The product ion monitored by the third quadrupole (Q3) was m/z 374 [MH⁺–HF]. Calibration curves of F 13640 were constructed after analyzing 10 µl of each standard at concentrations of 0.4, 1, 4, 10, and 40 pg/µl. Linear regression analysis of the reconstructed ion chromatogram peak area versus analyte concentration was performed [coefficients of determination (r²) were higher than or equal to 0.9996] before assaying samples of each rat. Quality control samples (40 pg/µl) were injected after three analyzed microdialysis samples (10 µl injected into the mass spectrometer without sample preparation).

Data Analysis. The biphasic appearance of the time-response data could be described adequately by an equation as described for 5-HT_1A receptor binding ex vivo, except that the maximum amount was in picograms per microliter of F 13640. Results are mean ± S.E.M. of four animals. Student's t test was used to compare the parameter values derived from the results obtained at 0.63 and 2.5 mg/kg F 13640.

5-HT_1A Receptor Binding ex Vivo
Treatment. Rats weighing 170 to 200 g were treated with different doses (0.04–10 mg/kg) of F 13640 and sacrificed 30 min later or treated with 0.63 mg/kg F 13640 and sacrificed 30 min or 1, 2, 4, or 8 h after administration of the compound.

Autoradiography. [³H]8-OH-DPAT binding in brain sections and autoradiography were carried out essentially as described previously (Gozlan et al., 1995). Brains were frozen in isopentane at –30°C and kept at –70°C. The brain was then cut in a cryomicrotome and kept at –70°C until use. Horizontal sections (20 µm) were thaw-mounted on gelatin-coated glass slides and stored at –70°C until use. Slide-mounted sections were thawed during 30 min at room temperature and then incubated for 90 min at room temperature with 1 nM [³H]8-OH-DPAT in the presence or absence of 10 µM of unlabeled 5-HT to estimate nonspecific binding. The assay buffer was Tris and 50 mM HCl, pH 7.4, at 25°C. Slides were then rinsed 2 x 5 min in ice-cold buffer, quickly dipped 2 x 5 s in ice-cold deionized water, and air-dried. Tritium-sensitive film ([³H]Hyperfilm; Amersham Biosciences Inc., Orsay, France) was apposed to the incubated slides and stored for 3 weeks in a light-proof Kodak X-ray exposure holder. Films were developed manually. A computerized image analysis system (Imagena 2000; Biocom, Les Ulis, France) was used to estimate receptor densities in different brain regions (hippocampus, entorhinal cortex, and frontal cortex). Optical density was converted to femtomoles per milligram of tissue using a standard curve produced from presliced ³H standards (microscales; Amersham Biosciences Inc.) containing known amounts of radioactivity.

Data Analysis. For the dose-response experiments, three brain sections were used to determine total binding in each rat, and three sections were used to determine nonspecific binding. For each animal and brain region, two values (right and left hemisphere) are the mean of total minus nonspecific binding, expressed as the percentage of the matching control animal. For the three brain regions examined, logistic dose-response curves were simultaneously fitted to the percentage of binding inhibition by means of the program ALLFIT (Windows version 2.12; C. and A. De Léan, University of Montréal, Montréal, QC, Canada) assuming a minimum of 0, a maximum of 100, a common slope, and a common ED₅₀ value.

For the kinetic experiments, three brain sections were used to determine total binding in each rat, and one section was used to determine nonspecific binding. For each animal and brain region, two values (right and left hemisphere) are the mean of three sections minus nonspecific binding, expressed as the percentage of the matching control animal. Results are means of three experiments. The biphasic appearance of the time-response data could be described adequately by an equation consisting of the sum of two exponentials [i.e., response = (A₁ *e^(–k1*time)) + (A₂ *e^(–k2*time)) + C, where A₁ = –A₂ and C = 0 (asymptote)], which was fitted to the data by the solver function of Microsoft Excel. From this equation, the following measures were calculated: maximum binding inhibition (max), time (in minutes) at maximum (t_max), time (in minutes) at which half-maximum was reached (t_{1/2 max}), and time (in minutes) at which half-asymptote was reached (t_{1/2 asym}). Results are mean ± S.E.M. of three experiments. One-way repeated measures (RM) analysis of variance (ANOVA) was used to compare the parameter values in the different brain regions.

Time Course of F 13640 Effects on the Paw Pressure Test
Nociceptive thresholds were determined using the Randall-Selitto method (Randall and Selitto, 1957) and measured with a Ugo Basile analgesimeter (Apelex; Ugo Basile, Comerio, Italy; tip diameter of the probe, 1 mm; weight, 30 g) by applying increasing pressure to the left hind paw until a squeak (vocalization threshold) was obtained. Results are expressed in grams. A 750-g cut-off value was used to prevent tissue damage. Rats weighing 180 to 200 g received an i.p. injection of either saline or F 13640 (0.63 mg/kg), and the vocalization threshold was determined before as well as 15 and 30 min and 1, 2, 4, and 8 h after the injection.

Data Analysis. Data represent the mean ± S.E.M. vocalization threshold of nine animals per group. Time-response data were analyzed by two-way RM ANOVA followed, where appropriate, by post hoc Student-Newman-Keuls test. p < 0.05 was considered to be significant.

Time Course of F 13640 Analgesic Effects on the Formalin Test
The formalin test was carried out as described previously (Bardin et al., 2003). Rats weighing 180 to 200 g were individually placed in a clear plastic chamber (with a mirror placed under the floor at a 45° angle to allow an unobstructed view of the paws) for a 30-min habituation period. Thereafter, rats received a 50-µl s.c. injection of diluted (2.5%) formaldehyde into the plantar surface of the right hind paw. Following this injection, the rats were returned to the chamber, and behaviors were observed during the early (0–5 min) and late (22.5–27.5 min) phases, during which the effects of formalin were typically apparent (Abbott et al., 1999). During each of these two 5-min periods, observations were made in the following way. Every 30 s, rats were observed for the presence or absence of spontaneous pain behaviors; i.e., 1) the injected paw was elevated and not in contact with any surface, and 2) the injected paw was licked. This observation cycle was repeated 10 times during the 5-min period; thus, the incidence of a particular behavior could vary from 0 to 10 for each of the two observation periods. To determine the time course of F 13640-mediated analgesia, separate groups of animals received F 13640 (0.63 mg/kg) or saline either 15 or 30 min or 1, 2, 4, or 8 h prior to the formalin injection.

The presence or absence of flat body posture (FBP), forepaw treading (FPT), and lower lip retraction (LLR) was also recorded before the injection of formalin. The results obtained at each time were expressed as the percentage of rats showing FPT, FBP, or LLR.

Data Analysis. Data were expressed as the mean ± S.E.M. score of seven animals, and each group was analyzed, per time, using Student's t test. p < 0.05 was considered to be significant.

Drugs
[³H]8-OH-DPAT (TRK.850; 160–240 Ci/mmol) was purchased from Amersham Biosciences Inc., and 5-HT creatinine sulfate was purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France). Chloral hydrate was purchased from Fluka (Saint-Quentin Fallavier, France). F 13640 is the property of the company sponsoring the present work and was synthesized in house. F 13640 was dissolved in distilled water and administered i.p. using a volume of 10 ml/kg; doses are expressed as the weight of the free base.

	Results

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Dose Response and Time Course of Plasma F 13640 Concentration Analysis. Fifteen minutes after i.p. administration of F 13640 (0.01–2.5 mg/kg), the concentration of F 13640 measured in plasma samples was dose-dependent [F(5,22) = 14.1; p < 0.001] (Fig. 1A). The time-course concentrations of F 13640 in plasma after a dose of 0.63 mg/kg are reported in Fig. 1B. The mean C_max was obtained at the first sampling time (i.e., 15 min) and was 293.43 ng/ml. The concentration of F 13640 decreased slowly over the 8 h but remained quantifiable until 24 h (0.19 ng/ml; not shown). The mean area under the curve over the 24 h was 422 ng/ml/h.

View larger version (12K): [in this window] [in a new window]   Fig. 1. A, dose response of plasma concentration of F 13640 (0.01–2.5 mg/kg) collected 15 min after i.p. administration of the compound and measured by LC/ESI-MS/MS. Results are means ± S.E.M. of five animals. B, plasma concentration of F 13640 collected at various times after i.p. administration of the compound at the dose of 0.63 mg/kg and measured by LC/ESI-MS/MS. Results are means ± S.E.M. of five animals.

Detection of F 13640 in Microdialysis Samples. After the administration of 0.63 and 2.5 mg/kg, F 13640 was detected in the dialysis samples. The limit of detection was reached at 0.16 mg/kg. The maximal amount detected and time to reach this maximum are reported in Table 1. The amounts of F 13640 measured in the hippocampus of a representative animal at each dose are shown in Fig. 2.

View larger version (20K): [in this window] [in a new window]   Fig. 2. Amounts of F 13640 collected in microdialysis samples in rat hippocampus and measured by LC/MS/MS after i.p. administration of F 13640 (0.16, 0.63, and 2.5 mg/kg). Shown are the values for a representative rat at each dose.

The concentration of F 13640 in the dialysis samples increased dose-dependently to a maximum of 13.5 pg/µl64 min after the administration of 2.5 mg/kg F 13640. The concentration obtained after 0.63 mg/kg F 13640 was 3.8-fold lower than that observed after a 4-fold higher dose but reached its maximum at about the same time (i.e., 51 min). The concentration fell to half of its maximal value at about 3 h after the administration of 0.63 or 2.5 mg/kg F 13640.

5-HT_1A Receptor Binding ex Vivo. F 13640 (0.04–10 mg/kg) administered 30 min before decapitation dose-dependently inhibited [³H]8-OH-DPAT binding in rat hippocampus, entorhinal cortex, and frontal cortex with a common ED₅₀ value: 0.34 mg/kg (see Fig. 3 for representative autoradiograms; Fig. 4, left). [³H]8-OH-DPAT binding was inhibited in a time-dependent manner by 0.63 mg/kg F 13640 (see Fig. 4, right, for a representative experiment). The parameters of this inhibition are reported in Table 2. The effects of F 13640 were not significantly different in the three brain regions examined and reached a maximum within about 15 to 35 min after injection; the maximum fell to half of its value within 3.5 to 8.5 h.

View larger version (41K): [in this window] [in a new window]   Fig. 3. Autoradiograms of horizontal brain sections labeled by [3H]8-OH-DPAT. Brain sections are from rats treated with different doses of F 13640 administered i.p. (Hip, hippocampus; Ent, entorhinal cortex; Fr, frontal cortex).

View larger version (16K): [in this window] [in a new window]   Fig. 4. [3H]8-OH-DPAT binding to rat brain sections of rats treated with F 13640. Left, effects of different doses (0.04–10 mg/kg) administered 30 min before decapitation (n = 3 per dose). Right, effects of 0.63 mg/kg in rats sacrificed at various times after administration (shown are the results from a representative experiment; n = 3 per time interval).

Time Course of F 13640 Effects on the Paw Pressure Test. The time course of the nociceptive threshold following the injection of F 13640 at the dose of 0.63 mg/kg is shown in Fig. 5. Two-way RM ANOVA indicated a significant effect of treatment [F(1,80) = 31.4; p < 0.001], time [F(5,80) = 72.4; p < 0.001], and the treatment x time interaction [F(5,80) = 75.1; p < 0.001]. The injection of F 13640 in rats displayed a biphasic time-dependent response compared with the saline control group; the earlier response was associated with a marked decrease in nociceptive threshold, and the later response was associated with a rise of the nociceptive threshold. The decrease in pain threshold (hyperalgesia) reached a maximum early upon injection (15 min; p < 0.001) and was statistically significant at 1 and 2 h after injection (p < 0.05). Thereafter, the nociceptive threshold increases (analgesia) significantly at 4 and 8 h after injection (p < 0.05). At the 15-min interval, vocalization required no experimental mechanical stimulation and seemed to be provoked by the men handling the animal; at later intervals (e.g., 1 and 2 h), however, the mechanical stimulation was definitely required for vocalization to occur.

View larger version (18K): [in this window] [in a new window]   Fig. 5. Time effects of acute administration of F 13640 (0.63 mg/kg i.p.) or saline on the threshold for mechanical, nociceptive stimulation to produce vocalization in rats. Measures were taken immediately before and at stated times after the administration of saline or F 13640. Values represent the means ± S.E.M. for nine animals. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 compared with saline (post hoc; Student-Newman-Keuls test).

Time Course of F 13640 Analgesic Effects on the Formalin Test. The potential antinociceptive effects of F 13640 (0.63 mg/kg) on the formalin test was assessed during the 8 h following i.p. administration (Fig. 6). The analgesic effects of F 13640 were long-lived, with a significant inhibition (p < 0.05) of paw elevation and paw licking on both phases lasting 2 and 4 h after drug administration, respectively.

View larger version (18K): [in this window] [in a new window]   Fig. 6. Time effects of acute administration of F 13640 (0.63 mg/kg i.p.) or saline on the paw elevation and paw licking that occur during the early (i.e., 0–5 min; solid symbols) and late phase (i.e., 22.5–27.5 min; open symbols) after intraplantar injection of 50 µl of formalin (2.5%) in rats. Values represent the mean ± S.E.M. score (maximal score = 10) of seven animals. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 compared with saline (post hoc; Student's t test).

Time Course of Behavioral 5-HT Syndrome Induced by F 13640. After the injection of 0.63 mg/kg in rats, F 13640 induced flat body posture, forepaw treading, and lower lip retraction. The incidence of all three elements of the 5-HT syndrome was maximal (100%) 15 and 30 min after administration of the compound and completely gone at 4 h, except for the lower lip retraction, which disappeared at 8 h (Fig. 7).

View larger version (11K): [in this window] [in a new window]   Fig. 7. Induction of elements of the behavioral 5-HT syndrome by F 13640 expressed as the percentage of animals showing the recorded behavior: FBP, FPT, and LLR.

	Discussion

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The studies presented herein examined the concentrations of F 13640, a novel central analgesic (Colpaert et al., 2002), after i.p. administration in brain and plasma, as well as the ability of F 13640 to bind 5-HT_1A receptors in rat brain sections. The time course of the paradoxical, hyperalgesic, and analgesic effects that F 13640 produced on nociception (Colpaert et al., 2002; Bruins Slot et al., 2003) was also measured to compare the time dependence of the analgesic actions of F 13640. Indeed, in normal rats, F 13640 causes an initial hyperalgesia followed by analgesia. Thus, in mimicking the effects of nociceptive stimulation (Colpaert et al., 2002; Buritova et al., 2003), F 13640 sets off two distinct actions. First, repeated or chronic F 13640 causes an analgesia that grows rather than decays. Second, F 13640 cooperates with nociceptive stimulation in paradoxically causing analgesia (Colpaert et al., 2002; Bruins Slot et al., 2003; Deseure et al., 2003).

F 13640 appeared in plasma and brain within 15 and 30 min, respectively, after i.p. injection of the drug. The peak concentration in plasma was reached rapidly at 15 min, and then it decreased slowly over the 8 h. Prior to this study, it had not been demonstrated that F 13640 crosses the blood-brain barrier upon systemic administration, although pharmacological data strongly suggest central effects. The present data indicate that F 13640 is detectable in central tissues following i.p. administration. The maximal concentration in brain was reached within 1 h, whatever the doses used, and decreased to half-maximum at about 3 h after injection. A concentration of F 13640, sufficient to be detected by LC/MS/MS, reached the hippocampus after administration of 0.63 and 2.5 mg/kg of the compound. Very few data are available in the literature concerning the measurement of drug concentrations in the brain, using microdialysis, after systemic administration. Estimations of morphine concentrations measured in microdialysis samples have been reported previously (Barjavel et al., 1995). In this case, the concentrations of morphine were measured by radioimmunoassay. Other authors measured 5-HT_1B/1D agonists after i.v. injection using electrochemical detection (Johnson et al., 2001). Another group measured a 5-HT_2A antagonist and its metabolite using microdialysis coupled to LC/MS/MS (Scott and Heath, 1998). Although present data preclude estimation of the actual concentration of F 13640 at the hippocampal level, they demonstrate that the amounts of the compound measured were proportional to the injected dose.

Concerning the occupancy of central 5-HT_1A receptors, a previous study was described using a lower efficacy 5-HT_1A partial agonist, buspirone (Sethy and Francis, 1988); however, that study employed membrane homogenates. In contrast, better resolution of receptor occupancy can be obtained by autoradiographic measurements. Indeed, occupancy of monoamine receptors by autoradiography at relevant pharmacological doses has been reported for antipsychotics (Schotte et al., 1993). Herein, consistent with good penetration into the brain, ex vivo autoradiographic binding studies indicate that, in agreement with its high in vitro affinity (pK_i, 9.07; Colpaert et al., 2002), F 13640 also has high affinity for 5-HT_1A receptors in vivo in hippocampus, entorhinal cortex, and frontal cortex. The inhibition of [³H]8-OH-DPAT binding by F 13640 was not significantly different in the three brain regions examined with an ED₅₀ value of 0.34 mg/kg. At a dose producing submaximal inhibition of [³H]8-OH-DPAT binding 30 min after its administration (i.e., 0.63 mg/kg), F 13640 reached its maximal effect within 15 to 35 min after injection. The receptor occupancy slowly decreased over the 8-h time period studied.

The pharmacodynamic part of this study was performed in the parallel groups of rats of the same strain and sex as in the pharmacokinetic part. The same dose as used in the pharmacokinetic experiments was used for studying the time-response relationships for several effects induced by F 13640. Thus, in the paw pressure test, the i.p. administration of F 13640 (0.63 mg/kg; Fig. 5) produced a maximal decrease in the vocalization threshold to paw pressure 15 min after its injection. This initial hyperalgesia was followed 4 h later by an analgesia that lasted until 8 h. Together, these findings indicate that the first-order effect (i.e., hyperalgesia) induced by F 13640 appeared with higher brain tissue and plasma concentrations, and the activation of the 5-HT_1A receptors and second-order effect (i.e., analgesia) occurred when the drug concentrations had almost disappeared. Thus, the findings reported herein are in accordance with a theory of signal transduction (Colpaert, 1996; Colpaert and Fregnac, 2001) that predicts that the first-order effect results directly from receptor activation, whereas the paradoxical second-order effect occurs after the implementation and subsequent discontinuation of receptor activation. A reciprocal phenomenon was found with morphine, where the hyperalgesia that was observed upon a single exposure probably coincided with the elimination of the agents (Bruins Slot and Colpaert, 1999; Bruins Slot et al., 2003).

Paradoxically, in the formalin pain test, F 13640 administered at the same dose (also 15 min after i.p. injection) induced analgesia in both the early and late phases, as measured by the elevation or the licking of the formalin-injected paw. These results confirm our previous data (Bardin et al., 2001, 2003) and earlier reports that 5-HT_1A receptor agonists produce analgesic effects in the formalin model (Millan et al., 1996; Oyama et al., 1996; Shannon and Lutz, 2000). This analgesic property of F 13640 was long-lived, with a significant inhibition of paw elevation and paw licking, on both phases, lasting 2 and 4 h after drug administration, respectively. It is noteworthy that over the same time period, F 13640 produced hyperalgesia in the paw pressure but not the formalin test. However, that the same i.p. treatment with F 13640 can produce both hyper- and hypoalgesic actions is also in agreement with a theory (Colpaert, 1996) and data (Colpaert et al., 2002; Deseure et al., 2002; Bardin et al., 2003) that suggest that, by mimicking the central effects of nociceptive stimulation, 5-HT_1A receptor activation cooperates with nociception in both an intensity- and duration-dependent manner to paradoxically produce analgesia in the formalin test. Thus, the absence of F 13640-induced hyperalgesia in the formalin test is probably due to the nociceptive stimulation that is produced by formalin injection being more intense and/or longer-lasting than the definitely shorter-lasting and possibly lower-intensity stimulation produced in the paw pressure test.

In the course of these experiments, we also monitored the 5-HT syndrome that F 13640 (Bruins Slot et al., 2003), like other 5-HT_1A receptor agonists (De Vry, 1995), is expected to produce (i.e., flat body posture, forepaw treading, and lower lip retraction). The incidence of all three elements of the 5-HT syndrome was maximal (100%) 15 and 30 min after administration of F 13640 (0.63 mg/kg) and lasted approximately 4 h, except for the lower lip retraction, which persisted until 8 h. This is in accordance with the time-concentration and occupancy of 5-HT_1A receptors' profile of F 13640 in plasma and brain (Fig. 2, 3, 4) and the other pharmacodynamic results cited above. The 5-HT syndrome does not, however, seem to explain analgesic effects that were observed. Thus, earlier data indicate that tachyphylaxis develops to these signs and that prazosin blocks them (Bardin et al., 2001; Bruins Slot et al., 2003), but the ability of 5-HT_1A agonists to inhibit formalin-induced paw elevation and paw licking remains unaltered after tachyphylaxis has developed; it also can not be blocked by prazosin (Bardin et al., 2001). Therefore, as in previous studies (Bardin et al., 2001, 2003; Colpaert et al., 2002), the analgesic effects of 5-HT_1A receptor activation reported herein were behaviorally specific. The behavioral specificity of 5-HT_1A receptor activation-induced hyperalgesic effects, however, remains to be examined. In the present studies, the hyperalgesia and behavioral signs of the 5-HT_1A syndrome displayed a similar time course, and the continuous infusion of F 13640 in earlier studies caused tachyphylaxis to both effects (Colpaert et al., 2002; Bruins Slot et al., 2003).

The data presented herein demonstrate that the hyper- and hypoalgesic effects of F 13640 that occur after i.p. injection were related to the peak concentration of the compound in brain tissue. The large distribution of 5-HT_1A receptors in the central nervous system makes it possible for F 13640's actions to involve various supraspinal and spinal systems that modulate pain processes (Hamon and Bourgoin, 1999). Indeed, i.c.v. injection of the 5-HT_1A receptor agonist 8-OH-DPAT inhibited the early phase of formalin-induced licking in mice (Fasmer et al., 1986). Selective depletion of cerebral 5-HT also induced antinociception in the formalin test, suggesting that the antinociceptive action of 5-HT_1A ligands may reflect a reduction in serotonergic transmission by stimulation of 5-HT_1A autoreceptors (Hole et al., 1990). Nevertheless, in vivo electrophysiological and behavioral studies have also demonstrated that spinal 5-HT_1A receptors mediate the inhibitory effect of the descending 5-HT pathway from the nucleus raphe magnus on the transmission of nociceptive information in the spinal cord (Liu et al., 2002). In addition, when injected intrathecally in rats, 5-HT_1A receptor agonists are generally found to exert marked antinociceptive effects (Lin et al., 1996; Nadeson and Goodchild, 2002; Bardin and Colpaert, 2004). We have recently reported that F 13640 induces c-Fos expression in deep dorsal horn laminae (Buritova et al., 2003), where the expression of 5-HT_1A receptor mRNA is particularly dense (Zhang et al., 2002).

Although F 13640's hyper- and hypoalgesic effects can be described in terms of the signal transduction concept discussed above, it remains to be determined whether this concept fully accounts for the complex role of 5-HT_1A receptors in pain processing. 5-HT_1A receptors are present in different areas of the brain and spinal cord as well as in peripheral tissues (Barnes and Sharp, 1999), mediating different and often paradoxical functional effects (Millan, 1995) that may vary depending on the extent of concurrent nociceptive input (Hains et al., 2002; Liu et al., 2002). In addition, pain processing demonstrates a remarkable time-dependent neuroplasticity (e.g., Woolf and Salter, 2000) that involves serotonergic systems (Porreca et al., 2002).

In conclusion, F 13640 induces hyperalgesia and/or analgesia, with a time course that parallels the occupancy of 5-HT_1A receptors and the presence of the compound in brain and blood. However, although these effects seem to be related to the presence of F 13640 at the supraspinal level, the neuroanatomical site(s) at which the action of F 13640 is produced remains to be identified. Further research is also required to elucidate the cellular features and neurophysiological pathways that 5-HT_1A ligands share in producing these effects.

	Acknowledgements

 
We thank Nathalie Danty, Nathalie Consul-Denjean, Valérie Rigal, Véronique Ravailhe, and Nathalie Malfètes for expert technical assistance.

	Footnotes

 
Part of this work was presented at the 10th International Conference on in Vivo Methods: Assié M-B, Pélissou M, Ribet J-P, Koek W, Newman-Tancredi A, and Colpaert FC (2003) Detection of the 5-HT_1A agonist analgesic, F 13640, in rat hippocampus: microdialysis coupled to LC/MS/MS, and ex vivo occupancy of 5-HT_1A receptors, in Monitoring Molecules in Neuroscience: Proceedings of the 10th Conference on in Vivo Methods (Kehr J ed); 2003 Jun 24–27; Stockholm, Sweden. 400 p., Karolinska Institutet.

doi:10.1124/jpet.104.077669.

ABBREVIATIONS: 5-HT, 5-hydroxytryptamine; F 13640, (3-chloro-4-fluoro-phenyl)-[4-fluoro-4-{[(5-methyl-pyridin-2-ylmethyl)-amino]-methyl}piperidin-1-yl]methanone, fumaric acid salt; LC, liquid chromatography; MS/MS, tandem mass spectometry; [³H]8-OH-DPAT, 8-hydroxy-2-[di-n-propylamino] tetralin; amu, atomic mass units; QC, quality control; RM, repeated measures; ANOVA, analysis of variance; FBP, flat body posture; FPT, forepaw treading; LLR, lower lip retraction; ESI, electrospray ionization.

Address correspondence to: Dr. Laurent Bardin, Department of General Pharmacology, Centre de Recherche Pierre Fabre, 17 avenue Jean Moulin, 81106 Castres Cedex. E-mail: laurent.bardin{at}pierre-fabre.com

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