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NEUROPHARMACOLOGY

Characterization of the Antinociceptive Actions of Bicifadine in Models of Acute, Persistent, and Chronic Pain

DOV Pharmaceutical, Inc., Somerset, New Jersey (A.S.B., P.A.K., A.S.L., P.S., E.K.); Veterans Affairs Medical Center and Departments of Psychiatry and Behavioral Neuroscience, Oregon Health and Science University, Portland, Oregon (A.J.); Department of Drug Development and Behavioral Neuroscience (P.P., A.N., M.K.), Department of Neurobiology (G.N.), and Department of Pharmacology (K.G., M.K.), Institute of Pharmacology Polish Academy of Sciences, Smetna Krakow, Poland; Department of Physiology and Pharmacology, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Israel (E.T.); Department of Anatomy and Neurobiology, Morehouse School of Medicine, Atlanta Georgia (M.B.); and Department of Cytobiology and Histochemistry, Collegium Medicum, Jagiellonian University, Krakow, Poland (G.N.)

Received for publication October 31, 2006
Accepted February 21, 2007.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Bicifadine (1-p-tolyl-3-azabicyclo[3.1.0]hexane) inhibits monoamine neurotransmitter uptake by recombinant human transporters in vitro with a relative potency of norepinephrine > serotonin > dopamine (1:2:17). This in vitro profile is supported by microdialysis studies in freely moving rats, where bicifadine (20 mg/kg i.p.) increased extrasynaptic norepinephrine and serotonin levels in the prefrontal cortex, norepinephrine levels in the locus coeruleus, and dopamine levels in the striatum. Orally administered bicifadine is an effective antinociceptive in several models of acute, persistent, and chronic pain. Bicifadine potently suppressed pain responses in both the Randall-Selitto and kaolin models of acute inflammatory pain and in the phenyl-p-quinone-induced and colonic distension models of persistent visceral pain. Unlike many transport inhibitors, bicifadine was potent and completely efficacious in both phases of the formalin test in both rats and mice. Bicifadine also normalized the nociceptive threshold in the complete Freund's adjuvant model of persistent inflammatory pain and suppressed mechanical and thermal hyperalgesia and mechanical allodynia in the spinal nerve ligation model of chronic neuropathic pain. Mechanical hyperalgesia was also reduced by bicifadine in the streptozotocin model of neuropathic pain. Administration of the D₂ receptor antagonist (-)-sulpiride reduced the effects of bicifadine in the mechanical hyperalgesia assessment in rats with spinal nerve ligations. These results indicate that bicifadine is a functional triple reuptake inhibitor with antinociceptive and antiallodynic activity in acute, persistent, and chronic pain models, with activation of dopaminergic pathways contributing to its antihyperalgesic actions.

Activation of the noradrenergic and serotonergic receptors subserving pain-modulating pathways can be achieved through blockade of the NE (NET) and 5-HT (SERT) transporters. Dual NET and SERT inhibitors, such as the tricyclic antidepressants (TCAs) amitriptyline and desipramine (Owens et al., 1997), possess greater analgesic efficacy, particularly in painful neuropathic states such as herpes neuralgia, diabetic neuropathy, and nerve crush syndromes (Sindrup et al., 2005), than either selective NET or SERT inhibitors (Fishbain et al., 2000). Likewise, the contribution of dopaminergic pathways to analgesic processes is supported by observations that dopamine transport (DAT) inhibitors (Pedersen et al., 2005) and D₂ agonists (Magnusson and Fisher, 2000) are antinociceptive in models of acute and chronic pain elicited by a number of modalities. Clinical evidence of a role for DA in modulating nociceptive inputs is provided by the hyperalgesia associated with dopaminergic hypofunction, such as in Parkinson's disease (Drake et al., 2005), whereas the DAT inhibitor bupropion (Semenchuk and Davis, 2000) and the DA precursor levodopa (Ertas et al., 1998) are analgesic in neuropathic pain syndromes (Hagelberg et al., 2004).

Although the analgesic efficacy of TCAs has been established, they exhibit a significant and dose-limiting side effect profile, including xerostomia, nausea, and intractable arrythmias, leading to problems of compliance (Sindrup et al., 2005). Dual uptake inhibitors, such as venlafaxine and duloxetine, which are clinically effective in reducing neuropathic pain (Lang et al., 1996; Iyengar et al., 2004), have mitigated many of these limiting side effects. However, enhancement of dopaminergic neurotransmission may further expand the favorable analgesic profile of dual NET and SERT inhibitors (Pedersen et al., 2005). To this end, we have characterized the antinociceptive properties of bicifadine (1-p-tolyl-3-azabicyclo[3.1.0]hexane), an inhibitor of biogenic amine transporters, in animal models of acute and chronic pain.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Animals. Male Sprague-Dawley rats [175–260 g; Elevage Janvier, Le Genest St Isle, France (Randall-Selitto, kaolin tests); Harlan, Indianapolis, IN (CFA, SNL models); Prokocim, Krakow, Poland (open field; rotarod)], male Wistar rats [150–160 g; Elevage Janvier (colonic distension); Institute of Pharmacology, Krakow, Poland (microdialysis)], and male Swiss CD-1 mice [18–30 g: Charles River, Lyon, France (PPQ); Institute of Pharmacology (tail-flick test), Bio-LASCO, Taipei, Taiwan (formalin test)] were used in this study. The investigators adhered to experimental procedures and housing conditions in accordance with the Animal Protection Act of August 21, 1997 (Poland's Government Regulations and Laws Gazette), the French Ministry for Agriculture and Fisheries, the International Associations for the Study of Pain guidelines, and the University of Minnesota Animal Care and Use Committee.

Upon receipt, the rats and mice were allowed at least 5 days to acclimate to their surroundings before testing. These animals were housed and fed in accredited facilities maintained on a standard 12-h light/dark cycle (lights on, 6:00 AM; lights off, 6:00 PM) at a room temperature of 19.5–24.5°C and relative humidity of 45 to 65%. Free access to food and water was maintained throughout the study period. Animals were assigned randomly to treatment groups.

Biochemistry and Physiology
Recombinant Human Monoamine Transporters: Binding and Uptake Studies. [¹²⁵I]RTI-55 [3-(4-iodophenyl)tropan-2-carboxylic acid methyl ester] was used in competition binding assays characterizing bicifadine affinity for the three monoamine neurotransmitter transporters using previously described techniques (Eshleman et al., 1999). In brief, bicifadine, [¹²⁵I]RTI-55 (40–80 pM final concentration, PerkinElmer Life and Analytical Sciences, Boston, MA), and Krebs-HEPES assay buffer were added to an aliquot of recombinant human transporter preparation (12–30-µg protein) to yield a final volume of 250 µl. Specific binding was defined using either 5 µM mazindol (hDAT, hNET), or 5 µM imipramine (hSERT). The reaction was incubated for 90 min at room temperature in the dark and was terminated by vacuum filtration.

The potency of bicifadine in suppressing monoamine neurotransmitter uptake was determined using suspensions of cell lines recombinantly expressing human transporters. These suspensions were prepared by removing the medium from cells grown on 150-mm-diameter tissue culture dishes and then washing the plates twice with Ca²⁺,Mg²⁺-free phosphate-buffered saline. Fresh Ca²⁺,Mg²⁺-free phosphate-buffered saline solution (2.5 ml) was then added to each plate, and the plates placed into a 25°C water bath for 5 min. The cells were gently scraped from the plates, and cell clusters were separated by trituration with a pipette for 5 to 10 aspiration/ejection cycles (Eshleman et al., 1999). Bicifadine and Krebs-HEPES assay buffer were added to these suspensions, and after a 10-min preincubation of the isolated cells at 25°C, either [³H]DA, [³H]5-HT, or [³H]NE (56, 26.9, or 60 Ci/mmol, respectively, 20 nM final concentration) was added. The assay was incubated for an additional 10 min, and the radiolabeled neurotransmitter uptake was terminated by vacuum filtration. Specific uptake was defined as the difference in uptake observed in the absence and presence of 5 µM mazindol (hDAT and hNET) or 5 µM imipramine (hSERT).

Characterization of Bicifadine Interactions with Other Receptor Systems. The affinity of bicifadine for a number of receptor systems was assessed using validated radioligand competition binding assays under conditions defined by the contractor [MDS Pharma Services, King of Prussia, PA (http://www.discovery.mdsps.com/Catalog/Assays); or Cerep, Rueil-Malmaison, France (http://www.cerep.fr/Cerep/Users/pages/catalog/assay)] (Table 1). For those receptors where the K_i for bicifadine was <10 µM, additional investigations were performed to identify the pharmacological nature of the interaction (i.e., agonist or antagonist) using previously described biochemical and physiological assays performed under conditions validated by the contractor (MDS Pharma Services or Cerep).

View this table: [in this window] [in a new window]   TABLE 1 Bicifadine interactions with neurotransmitter transporters, receptors, and ion channels: radioligand-receptor binding screen The receptors tested, their sources, the radioligands used to define the binding sites, and the relative affinity of bicifadine for these receptors are indicated. Additional information on the assay conditions can be obtained from the contractor's website. Bicifadine showed significant affinity (Ki < 10 µM) for the 1-, 2-, and 1-adrenergic; 5-HT1A- and 5-HT1B-serotonergic; and 1-receptors. Studies were performed at: 1Oregon Health Sciences University/VA Medical Center; 2Cerep; 3MDS Pharma Services; and 4Polish Academy of Sciences.

N-Methyl-D-aspartate Receptor Electrophysiology. The effect of bicifadine was investigated on N-methyl-D-aspartate (NMDA)-gated currents in primary cultures of rat hippocampal pyramidal neurons (Nahum-Levy et al., 2002). Whole-cell patch-clamp experiments were conducted at room temperature between 1 and 2 weeks after plating the neurons, with a holding potential for the voltage-clamp experiments of -60 mV.

Microdialysis. Rats were anesthetized with ketamine (75 mg/kg i.m.) and xylazine (10 mg/kg i.m.) and placed into a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA). The skulls were exposed, and small holes were drilled for insertion of the microdialysis probes using the following coordinates:1.8 mm anterior from the bregma; 2.7 mm lateral from the sagittal suture; -7.0 mm ventral from the dural surface (striatum); 2.9 mm anterior from the bregma; 0.8 mm lateral from sagittal suture; -4.5 mm ventral from the dural surface (prefrontal cortex); -9.8 mm posterior from the bregma; 1.3 mm lateral from the sagittal suture; and -5.7 mm ventral from the dural surface (locus coeruleus). Sampling from the striatum and prefrontal cortex was conducted using microdialysis probes constructed by inserting two fused silica tubes (30 and 35 mm long, 150 µm o.d.; Polymicro Technologies Inc., Phoenix, AZ) into a microdialysis fiber (220 µm o.d., AN69; Hospal, Bologna, Italy). The tube assembly was placed into a stainless steel cannula (22G, 10 mm) forming the shaft of the probe. Portions of the inlet and outlet tubes were individually placed inside polyethylene PE-10 tubing and glued. The free end of the dialysis fiber was sealed, and 4 or 3 mm of the exposed length was used for dialysis in the striatum or prefrontal cortex, respectively. For the locus coeruleus, CMA/11 microdialysis probes (Carnegie Medicin, Stockholm, Sweden) were used. All probes were connected to a syringe pump (BAS Instruments, West Lafayette, IN), which delivered an artificial cerebrospinal fluid composed of 145 mM NaCl, 2.7 mM KCl, 1.0 mM MgCl₂, 1.2 mM CaCl₂, pH 7.4 at a flow rate of 1.5 µl/min. Baseline samples were collected every 20 min after the washout period to obtain a stable extracellular neurotransmitter level. Bicifadine was then administered, and dialysate fractions were collected for 240 min. At the end of the experiment, the rats were sacrificed and their brains were histologically examined to validate probe placement.

Neurotransmitter Analysis. DA, 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA), as well as 5-HT and its metabolite 5-hydroxyindoleacetic acid (5-HIAA), were analyzed by HPLC with electrochemical detection. Chromatography was performed using an LC-10 AD pump (Shimadzu Europa GmbH, Warsaw, Poland), an LC-4B amperometric detector with a cross-flow detector cell (BAS), and a BDS-Hypersil C₁₈ analytical column (3 x 100 mm). The mobile phase was composed of 0.1 M monochloroacetic acid adjusted to pH 3.7 with 3 M sodium hydroxide, 0.5 mM EDTA, 25 mg/l 1-octanesulfonic acid sodium salt, 5.7% methanol, and 2.5% acetonitrile. The flow rate was 0.5 ml/min, and the applied potential at the 3-mm glassy carbon electrode was +600 mV, with a sensitivity of 2 nA/V. NE was measured using an HPLC system equipped with a P580 pump (Dionex Corp, Sunnyvale, CA) connected to an injection valve with a 10-µl loop and a BDS-Hypersil analytical column (2.0 x 100 mm). The mobile phase was composed of 0.05 M KH₂PO₄ (adjusted to pH 3.7 with ortho-phosphoric acid), 0.5 mM EDTA, 150 mg/l 1-octanesulfonic acid sodium salt, 10 mM NaCl, and 1.2% acetonitrile. The flow rate was 180 µl/min. NE was detected in dialysates with a radial flow detector cell coupled to a LC-4B amperometric detector (BAS). The applied potential at the 3-mm glassy carbon electrode was +600 mV with a sensitivity of 2 nA/V. The chromatographic data were processed by Chromax 2001 (Pol-Lab, Warsaw, Poland) software run on a PC computer. The values were not corrected for in vitro probe recovery, which was approximately 15%.

Pharmacokinetic Analysis. Male rats were orally administered 6, 20, or 60 mg/kg (n = 3/dose) bicifadine, and blood samples (approximately 1 ml) were taken 1, 2, 4, 8, and 24 h postdosing. Plasma levels of bicifadine were determined using a validated liquid chromatography-mass spectrometry/mass spectrometry assay (WIL Research Laboratories, Ashland, OH). In brief, plasma aliquots (0.2 ml) were transferred to tubes containing 50 µl of 0.5 M KOH, 25 µl of internal standard [8 µg/ml (1R,5S)-(+)-1-(3,4-dichlorophenyl)-3-azabicyclo[3.1.0]hexane], and 300 µl of blank rat plasma, mixed, and allowed to stand for 15 min. Diethyl ether (8 ml) was then added to each tube, which was shaken for 15 min, and then centrifuged at 3500 rpm for 5 min. The organic fraction was transferred to clean tubes, and the samples were evaporated to dryness under nitrogen. This step was repeated, and the samples finally reconstituted in 200 µl of methanol. Aliquots of the fully reconstituted samples were then transferred to autosampler tubes.

The extracted samples (40 µl) were then injected onto an HPLC (2695; Waters, Milford, MA) with a Hypersil C18-BDS column (50 x 4.6 mm, 3 µm particle size) and a C18 guard column (Phenomenex Security Guard). The sample was isocratically eluted with a mobile phase consisting of 30% acetonitrile, 35% methanol, 0.5% formic acid, and 34.5% ammonium acetate (5 mM) at a flow rate of 0.4 ml/min. The retention time for bicifadine was approximately 2.3 min, with a retention time of 3.0 min for the internal standard. Total run time was approximately 6 min.

Bicifadine was detected with a tandem mass spectrometer (Micromass Quattro Micro) equipped with an atmospheric pressure chemical ionization interface in positive ionization mode. Detection settings used were: corona, 20 µA; cone (bicifadine), 35 V; extractor, 2 V; RF lens, 0.2 V; source temperature, 130°C; desolvation temperature, 400°C; cone gas flow, 100 l/h nitrogen; and desolvation gas flow, 700 l/h nitrogen. Bicifadine was monitored using an m/z = 174/133 (parent/product ion), using argon as the collision gas at a pressure of 2.5 x 10^-3 mbar and an energy of 18 eV. Data acquisition and analysis were performed using MassLynx software, version 3.5.

In addition to the plasma samples taken from the dosed rats, each set of analyses consisted of calibration samples (8–9 concentrations) in duplicate: one solvent blank; one blank rat plasma sample; one blank rat plasma sample doped with internal standard; and quality control samples (three concentrations in triplicate). Valid sample runs required that two thirds of quality control samples not deviate more than ±15% from target concentration values. The lower limit of detection of this assay was 8.26 ng/ml bicifadine, and the upper limit was 1652 ng/ml.

Models of Acute Inflammatory Pain
Randall-Selitto Test. These studies were performed by Calvert Laboratories, Inc. (Olyphant, PA). Inflammation was induced by the subplantar injection of 0.1 ml of 20% Brewer's yeast suspension into the plantar surface of the rat right hind paw (Le Bars et al., 2001). Test agents or vehicle were orally administered 1 h after the yeast injection. Two hours after the yeast injection, the mechanical nociceptive threshold was measured using an analgesiometer (Ugo Basile, Milan, Italy). The nociceptive threshold was defined as the force at which the rat vocalized or withdrew its paw. The upper limit for pressure administration was 250g.

Kaolin-Induced Arthritis Model. Studies using this model were performed by Cerep. Arthritis was induced by injecting 100 µl of 10% (w/v) kaolin suspension in saline into the knee joint of the rat right hind paw (Hertz et al., 1980). The vehicle and test substances were orally administered 30 min after kaolin injection. Behavioral assessments of gait behavior were conducted every hour from 1 to 5 h after drug dosing using the following indices: 0 = normal gait; 1 = mild disability; 2 = intermittent raising of paw; 3 = elevated paw.

Models of Acute Nociceptive Pain
Tail-Flick Test. The tail-flick latency is the time taken by a mouse to withdraw its tail from a radiant heat source as measured using a semi-automated device (H. Sachs Elektronik #812, Hugstetten, Germany) (Le Bars et al., 2001). The latency data were converted to the maximal possible effect using the equation: MPE = 100 x ([Latency_Drug - Latency_Baseline]/[Latency_cutoff - Latency_Baseline]. The heat source was adjusted for testing subjects under high intensity (12 s cut-off latency) or low intensity (25 s cut-off latency) stimuli. Vehicle-treated mice responded to the high and low intensity stimuli, with an average latency of 6.3 ± 0.18 and 16.8 ± 0.41 s, respectively. Subjects were tested before and 60 min after p.o. or s.c. administration of test compounds under blinded conditions.

Hot Plate Test. Mice were placed on a heated surface maintained at either 55 or 59 ± 0.5°C. The time between placement on the hot plate and the occurrence of licking of the hind paws, shaking or jumping off the surface, was recorded as the response latency (Le Bars et al., 2001). Animals with baseline latencies of less than 12 s or more than 18 s were excluded from the study. A maximal cutoff time of 30 s was used to prevent tissue damage, with a control rat response time of approximately 6 to 8 s.

Models of Persistent Pain
Phenyl-p-quinone-Induced Abdominal Contractions. Studies using this model were performed by Cerep. Test compounds were administered 30 to 60 min before phenyl-p-quinone (PPQ) administration. Abdominal contractions were induced by i.p. injection of PPQ (1 mg/kg) (Le Bars et al., 2001). Mice were placed individually into observation boxes 15 min after PPQ injection, and a number of writhes (i.e., stretching, twisting a hind leg inward, or contracting the abdomen) were recorded over a 3-min period. Control (vehicle-treated) mice produce approximately 30 ± 5 (mean ± S.D.) incidences of these behaviors during this period. ED₅₀ values were calculated as the dose required to reduce the number of writhes to <18 in 50% of the treated mice.

Colonic Distension Model. Rats received an intracolonic application of 1.5 ml of 1% acetic acid, followed 2.5 h later by catheter-induced colonic distension lasting for 10 min. Pain was scored by visual counting of the abdominal contractions over a 10-min period. Vehicle and test substances were administered 60 min before initiation of colonic distension. This investigation was performed by Porsolt and Partners (Le Genest Saint Isle).

Formalin Test. Vehicle or test substances were orally administered 60 min before formalin injection. Sprague-Dawley rats were injected with 50 µl of 5% formalin in 0.9% NaCl into the dorsal surface of the right hind paw (Le Bars et al., 2001). Likewise, CD-1 mice were injected with 20 µl of 5% formalin solution. Hind paw licking time was recorded between 0 and 10 min (Phase 1) and between 15 and 40 min (Phase 2) after formalin injection in the rats. Licking time was recorded over 0 to 5 (Phase 1) and 20 to 30 (Phase 2) min in mice. Results are expressed as the mean ± S.E.M. of the time spent licking paws during each phase of the study. These investigations were performed by MDS.

The Complete Freund's Adjuvant Model of Persistent Inflammatory Pain. Rats were anesthetized with 2 to 3% isoflurane, and 50 µlofa Mycobacterium tuberculosis (1 mg/ml) suspended in CFA were injected subcutaneously into the plantar surface of the left paw (Stein et al., 1988). One week later, the baseline and postdrug treatment paw-withdrawal thresholds to mechanical stimulus were measured as outlined below. These investigations were performed by Algos Therapeutics, Inc. (St. Paul, MN).

Models of Chronic Pain
The Spinal Nerve Ligation Model. The SNL model (Kim and Chung, 1992) was used to induce chronic neuropathic pain. Rats were anesthetized with isoflurane; the left L₅ and L₆ spinal nerves were exposed by removing the paravertebral muscle and a part of the left spinous process of the L₅ vertebrae. The L₅ and L₆ spinal nerves were isolated and tightly ligated with 6-0 silk suture (NC-silk black, USP 5/0, metric 1). The muscle and adjacent fascia were sutured, and the skin incision was closed externally with wound clips. The clips were removed 10 days after surgery, and the animals were allowed to recover for at least 2 weeks before testing. These investigations were performed by Porsolt and Partners and Algos Therapeutics.

Streptozotocin-Induced Diabetic Neuropathy. This model was utilized in studies performed by Cerep. Diabetes was induced by administration of a single dose of streptozotocin (STZ) (75 mg/kg i.p.; Courteix et al., 1993), prepared as a solution in citrate buffer (pH 4.2). Twenty-three days after injection, the presence of diabetes was confirmed by measuring hyperglycemia using a glucometer with blood glucose strips (Glucotrend type 1895729 and Accu-Check strips; Roche Diagnostics, Indianapolis, IN). Animals with glucose levels lower than 250 mg/dl were not used for further studies.

Behavioral Testing Models of Chronic Pain
Mechanical Hyperalgesia. Baseline and post-treatment values for mechanical nociceptive sensitivity in all models of chronic pain were evaluated using a Paw Pressure Analgesymeter (7200; Ugo Basile, Comerio, Italy), which generates a linearly increasing mechanical force. For the assessment of mechanical hyperalgesia, the force was applied to the plantar surface of the injured rat hind paw via a cone-shaped stylus with a rounded plastic tip (2 mm²) placed between the third and fourth metatarsus. The nociceptive threshold was defined as the force at which the first pain behavior (paw withdrawal, struggle, and/or vocalization) was expressed. The maximal force for testing was set at 390g. The pain threshold was measured in both hind paws.

Thermal Hyperalgesia. Rats were placed into an acrylic box (17 x 11 x 13 cm; Ugo Basile 7371) on an elevated glass floor and left to habituate for 10 min. A mobile radiant heat source (96 ± 10 mW/cm²) was focused under the nonlesioned and lesioned hindpaws, and the paw-withdrawal latency was automatically recorded. The cut-off latency was set for 45 s of exposure.

Mechanical Allodynia. Rats were placed on a metal mesh and covered with a plastic dome. They were allowed to habituate until the exploratory behavior diminished (15 min). Mechanical sensitivity (paw-withdrawal threshold) was evaluated at baseline, postinjury (day 14 postsurgery), and post-treatment (day 14 postsurgery) using eight Semmes-Weinstein filaments (Stoelting Co., Wood Dale, IL) with varying stiffness (0.4, 0.7, 1.2, 2.0, 3.6, 5.5, 8.5, and 15g) according to the up-down method (Chaplan et al., 1994). Because this stimulus is normally not considered painful, significantly larger responses after surgery are interpreted as a measure of mechanical allodynia.

Side Effect Profile
Motor Function Assessments. The sedative, ataxic, and myorelaxant effects of bicifadine and desipramine were determined using the open field, rotarod, and grip strength meter (Popik et al., 2006). In the open-field test, rats were placed in the corner of a dimly lit (40 lux) open field of black plywood (66 x 56 x 30 cm) beginning 60 min after drug or vehicle administration, and the distance traveled over a 21-min period was measured using the Any-maze tracking system (Stoelting Co.). The presence of ataxia was assessed using a rotarod apparatus (ENV-577; MED Associates, St. Albans, VT) rotating at 6 rpm. Those animals that did not fall off of the apparatus within 2 min were considered to have normal balance and coordination. The degree of myorelaxation was determined next using a grip strength meter (Columbus Instruments, Columbus, OH). The forepaws of a rat were placed on the metal mesh attached to the meter, and the body was gently pulled until the rat released the grid. Three measures of grip strength were taken sequentially for each rat, averaged, and corrected for body weight.

Drug Administration. Bicifadine (5–100 mg/kg) was dissolved in distilled water (5 ml/kg, Randall-Selitto, tail-flick, colonic distension, CFA, SNL models), 0.9% saline (10 ml/kg, formalin), or 1% carboxymethylcellulose (10 ml/kg, kaolin, STZ models) administered orally to mice or rats 60 min before measures of hyperalgesia or allodynia commenced. For the microdialysis studies, bicifadine (20 mg/kg i.p.) was administered in 0.9% saline solution (5 ml/kg) to freely moving animals during the procedure. In the local administration studies, all test substances were dissolved in distilled water and injected subcutaneously in a volume of 50 µl. All solutions were prepared immediately before administration.

Gabapentin (GBP, 300 mg/kg p.o.), morphine sulfate (128 mg/kg p.o.), or the -opioid receptor agonist U-50,488 (3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide, 10 mg/kg p.o.) was dissolved in distilled water and administered in a volume of 5 ml/kg either 60 or 30 min before testing as reference agents. The D₂ antagonist (-)-sulpiride (15 mg/kg i.p., 5 ml/kg distilled water) was administered 60 min before testing. Indomethacin was administered orally in 1% methylcellulose vehicle (10 ml/kg). Animals were acclimated to the test room for at least 1 h before testing. Drug solutions were coded by a separate experimenter uninvolved in conducting the behavioral testing. The blind was not broken until the end of the study. Test substances and vehicles were administered in random order by a blinded investigator.

Bicifadine was synthesized by EaglePicher Technologies (Lenexa, KS). Gabapentin (no. 006569) was obtained from Hawkins Pharmaceutical Group (Minneapolis, MN). Indomethacin was purchased from either Sigma-Aldrich (St. Louis, MO) or Fluka (Buchs, Switzerland). Acetaminophen (A7085), codeine (C5901), complete Freund's adjuvant (F5881), desipramine (D3900), ibuprofen (I4883), morphine (M8777), streptozotocin (S0130), (-)-sulpiride (S7771), and U-50,488 (D8040) were obtained from Sigma-Aldrich.

View larger version (36K): [in this window] [in a new window]   Fig. 1. Bicifadine-induced changes in the extracellular levels of biogenic amine neurotransmitters and their metabolites as determined using microdialysis in normal rats. 5-HT (F(11,1,11) = 83 treatment; 7.7 time; 7.5 interaction, all P < 0.01) and NE (F(11,1,11) = 299 treatment; 25.4 time; 24.8 interaction, all P < 0.01) levels were measured in the prefrontal cortex (PFCtx) 40 min before and up to 180 min after administration of bicifadine to freely moving rats (A). NE levels were measured in the locus coeruleus (LC) (F(8,1,8) = 28.8 treatment; 2.6 time; 2.5 interaction, all P < 0.01) after bicifadine administration (B). In the striatum (Str), levels of 5-HT (F(11,1,11) = 95.9 treatment; 8.5 time; 9.1 interaction, all P < 0.01) (C), DA (F(11,1,11) = 127 treatment; 7.8 time; 8.3 interaction, all P < 0.01) (E) and their metabolites 5-HIAA (F(11,1,11) = 54.1 treatment; 4.3 time; 4.5 interaction, all P < 0.01) (D), DOPAC (F(11,1,11) = 285 treatment; 6.2 time; 12.6 interaction, all P < 0.01) (F), and HVA (F(11,1,11) = 87.5 treatment; 5.7 time; 5.4 interaction, all P < 0.01) (F) were measured after bicifadine administration. The dose of bicifadine administered was 20 mg/kg i.p. in all cases. Each sample of microdialysate was accumulated over a 20 min period, with each point representing the mean percentage change (±S.E.M.) from baseline levels for bicifadine (solid symbols, n = 4) (B) or n = 8 (A, C-F) and vehicle-treated (open symbols, n = 4, all panels) rats. Absolute baseline levels (in picograms/10 µl) were: 2.8 ± 0.2 (5-HT, PFCtx) and 3.4 ± 0.2 (NE, PFCtx) (A); 0.6 ± 0.15 (NE, LC) (B); 2.9 ± 0.1 (5-HT, Str) (C); 615 ± 29 (5-HIAA, Str) (D); 10.2 ± 0.8 (DA, Str) (E); 2418 ± 159 (DOPAC, Str) and 1829 ± 120 (HVA, Str) (F). *, individual points or point ranges falling within the brackets were significantly different from the mean baseline level; P < 0.05, 2-way ANOVA followed by Bonferroni's post hoc comparison matrix.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Biochemical Pharmacology. The ability of bicifadine to inhibit radioligand binding to a variety of receptors, including those implicated in nociception, was investigated in a radioligand receptor binding screen (Table 1). Bicifadine inhibited [¹²⁵I]RTI-55 binding to sites on recombinant human 5-HT, NE, and DA transporters with moderate (micromolar) but equivalent affinities (1:2:2, respectively; Table 1). Subsequent functional tests (Table 2) indicated that bicifadine potently inhibited the uptake of [³H]NE and [³H]5-HT by cell lines expressing recombinant human monoamine transporters (IC₅₀ = 55 and 117 nM, respectively), whereas its potency in blocking [³H]DA uptake was approximately one order of magnitude lower (IC₅₀ = 910 nM) (1:2:17, respectively).

View larger version (17K): [in this window] [in a new window]   Fig. 2. Plasma concentrations of bicifadine in male rats. Bicifadine was administered as a single oral dose of 6 (), 20 (), or 60 () mg/kg, and blood drawn at the indicated times. Plasma levels of bicifadine were determined using a validated liquid chromatography-mass spectrometry/mass spectrometry method. Data represent the mean ± S.D. of observations from three animals.

In addition to its interactions with biogenic amine transporters, bicifadine exhibits moderate affinity for ₁-, ₂-, and ₁-adrenergic receptors; 5-HT_1A- and 5-HT_1B-serotonergic receptors; and ₁ receptors, as indicated by K_i values 10 µM for these sites (Table 1). Bicifadine inhibits radioligand binding to ₁ receptors (K_i = 773 nM) and acts as an antagonist of these receptors (IC₅₀ = 18.6 µM), as indicated by the blockade of phenylephrine-stimulated contractions of rat vas deferens (Table 2). [³H]Rauwolscine binding to ₂ receptors was inhibited by bicifadine with a K_i = 1.01 µM, but it exhibited lower affinities for recombinantly expressed human ₂ receptor subtypes (IC₅₀: _2A = 6.0 µM, _2B = 4.2 µM; _2C = 23.9 µM, [³H]RX 821002 [0.7 nM], [³H]RX 821002 [2.5 nM], and [³H]MK 912 [0.2 nM], respectively). Bicifadine acted as an ₂-receptor agonist, reducing the neurogenic twitch response of the rat vas deferens with an EC₅₀ = 6 µM. Radioligand binding to ₁ adrenoceptors was inhibited by bicifadine (Table 1), which acted as a relatively low potency antagonist at this site (Table 2).

View larger version (20K): [in this window] [in a new window]   Fig. 3. The antinociceptive effects of bicifadine in a model of acute inflammatory pain (Randall-Selitto test). A, dose-response curve for bicifadine in the Randall-Selitto test. Rats received a subplantar injection of Brewer's yeast suspension, followed 1 h later by orally administered test substances or saline vehicle. Testing began 1 h after administration of the test substances. Bicifadine () dose-dependently increased the threshold pressure for paw withdrawal with an ED50 = 11.3, (3.38, 37.7) mg/kg (mean, 95% CI), with 20 mg/kg bicifadine increasing the withdrawal threshold to 206 ± 22g. In comparison, indomethacin (, 20 mg/kg p.o.) increased the nociceptive threshold to 142 ± 20.8g. Response to vehicle (dashed line across bottom of graph) was 91 ± 13.5g. Each point represents the mean ± S.E.M. of data from n = 10 animals. The sigmoidal curve was fitted to bicifadine data, and the ED50 determined using nonlinear regression. B, duration of action of bicifadine. Bicifadine was administered orally at t = 0 at a dose of 50 mg/kg. Data represents the mean (n = 8) pressure threshold for withdrawal of inflamed paw 2 h after subplantar injection of yeast suspension. C, bicifadine exhibits antinociceptive activity after local administration. Sixty minutes after injecting the yeast suspension into the plantar surface of the hind paw, the test substances (bicifadine, ibuprofen [Ib], morphine [Mor], 50 µl) were injected into the dorsal surface of the inflamed paw, and withdrawal thresholds were measured in the inflamed paws 60 min later. In one cohort (dotted bars), bicifadine (8 µg) was injected into the normal paw, and withdrawal thresholds were measured in the inflamed (BI) and normal (BN) paws of the bicifadine-treated rats 60 min after administration 60 min later. Bicifadine (1, 4, 8, and 12 µg/paw, solid bars) reduced the paw-withdrawal threshold when injected into the inflamed paw, as did ibuprofen (80 µg/paw, cross-hatched bar) and morphine (50 µg/paw, hatched bar). Dashed line, mean antinociceptive activity achieved after administration of 20 mg/kg bicifadine p.o.. Data represent mean ± S.E.M. of 10 to 20 observations. **, significantly different from vehicle-treated, inflamed paw, P < 0.01, respectively, one-way ANOVA followed by Dunnett's post hoc comparison test (F(4,55) = 7.50, P < 0.01; F(2,37) = 17.9, P < 0.01). ##, significantly different from vehicle-treated, inflamed paw (ibuprofen t28 = 5.29; morphine t28 = 5.39; all P < 0.01). aa, significantly different from vehicle-treated, uninflamed paw (t38 = 7.58, P < 0.01). Veh, vehicle-injected normal paw; Veh Infl, vehicle-injected inflamed paw.

Additional studies indicated that bicifadine did not significantly inhibit (<50% inhibition at the highest concentration tested) radioligand binding to: A₁ and A_2A adenosine; AMPA; _2,3-adrenergic; angiotensin₁; benzodiazepine; bradykinin_1,2; calcitonin-gene related peptide; cannabinoid_1,2; cholecystokinin_A; corticotrophin-releasing factor₁; D_1,2S,3,4 dopamine; endothelin_A; epidermal growth factor; GABA_A; glucocorticoid; glutamate binding site on NMDA receptor; melanocortin₄; neurokinin₁; neuropeptide Y₁; ₇-nicotinic cholinergic; M₁,₂,_3,4 cholinergic; µ, , and opioids; orphanin₁;DPand EP_2,4 prostanoid; P₂X purine; 5-HT₃ serotonin; vanilloid₁; and vasopressin_1A receptors (Table 1). Bicifadine had no measurable affinity for the glutamate binding site on NMDA receptors and exhibited low potency (IC₅₀ = 23 ± 7.9 µM) in displacing [³H]MK-801 from its binding site in the NMDA receptor ionophore. There was no evidence that bicifadine had significant affinity for the ₂ Ca²⁺, N-type Ca²⁺,K⁺_ATP; saxitoxin-sensitive K⁺_V; and human ether-a-go-go-related gene K⁺_V channels.

The two principal metabolites of bicifadine, the lactam [5-p-tolyl-3-aza-bicyclo[3.1.0]hexan-2-one] and the lactam acid [4-(4-oxo-3-aza-bicyclo[3.1.0]hexan-1-yl)benzoic acid], were also screened. Neither of these showed any affinity (<50% inhibition) for any of the receptors or transporters investigated above at concentrations up to 30 µM (data not shown).

Pharmacokinetics. The plasma levels of bicifadine were determined 1, 2, 4, 8, and 24 h after oral administration of single doses of 6, 20, or 60 mg/kg to male rats (Fig. 2). The t_max was approximately 1 h for all the doses tested, and the C_max values were 483, 1517, and 3361 ng/ml (approximately 2.3, 7.2, and 16 µM) following the administration of 6, 20, and 60 mg/kg, respectively.

Bicifadine Activity in Models of Acute Pain. The antinociceptive activity of bicifadine was examined in models of acute inflammatory, visceral, and nociceptive pain. In the yeast-inflamed hindpaw model of acute inflammatory pain (Randall-Selitto test; Table 3), bicifadine increased the threshold for paw withdrawal (Fig. 3A). Compared with reference opiate and nonsteroidal anti-inflammatory (NSAIDs) analgesics, bicifadine was approximately 15 times more potent (on a milligram/kilogram basis, 25-fold on a micromolar/kilogram basis) as an oral analgesic than acetaminophen and four times more potent than codeine (3.3-fold on a micromolar/kilogram basis; Table 3). Based on its potency in the Randall-Selitto test (ED₅₀ = 9.2 mg/kg p.o.; Table 3) and an LD₅₀ = 370 mg/kg, the therapeutic index (LD₅₀/ED₅₀) for orally administered bicifadine in the rat is approximately 40. Bicifadine was approximately equipotent in increasing the withdrawal threshold in the Randall-Selitto test following either subcutaneous (ED₅₀ = 10.1 mg/kg) or oral administration, with a potency comparable to that of subcutaneously administered pentazocine, d-propoxyphene, and codeine (data not shown). The analgesic action of a maximally effective dose of bicifadine (50 mg/kg p.o.) in the Randall-Selitto test was observed by 1 h after administration but was no longer apparent by 4 h after administration (Fig. 3B). Bicifadine was also an effective analgesic when administered locally in the Randall-Selitto test (Fig. 3C). Bicifadine (4, 8, and 12 µg/paw) significantly increased the paw-withdrawal latency when administered into the plantar surface of the inflamed paw. Paw-withdrawal thresholds for both the inflamed and uninflamed paws were not altered when bicifadine was administered into the uninflamed paw (8 µg/paw; Fig. 3C). Although locally administered bicifadine had no effect on inflamed paw volume (1.518 ± 0.045 and 1.534 ± 0.077 ml, Veh and bicifadine, respectively), orally administered bicifadine (40 mg/kg) significantly reduced the inflamed paw volume 21% (1.37 ± 0.04 and 1.08 ± 0.06 ml, Veh and bicifadine, respectively, P < 0.05, t test). The antinociceptive effects of morphine and ibuprofen were comparable to bicifadine when locally administered at doses of 50 and 80 µg/paw, respectively.

View larger version (24K): [in this window] [in a new window]   Fig. 4. Bicifadine is an effective antinociceptive agent in the kaolin-induced arthritis model. Arthritis was induced by the intra-articular injection of kaolin suspension. One hour after kaolin administration, the test substances or saline vehicle were orally administered. Gait scores were recorded hourly, starting 1 h after kaolin administration (A) and hourly for up to 5 h (B–E). Bicifadine (12.5 and 25 mg/kg, closed bars) and indomethacin (3 mg/kg p.o., hatched bars) significantly improved the gait scores of kaolin-treated rats only at 4 (D) and 5 (E) h after administration. **, significantly different from vehicle-treated control animals, P < 0.01, respectively, Kruskal-Wallis test followed by Dunn's post hoc comparison matrix. ##, significantly different from vehicle-treated control animals, P < 0.01, Mann-Whitney U test.

Bicifadine was also examined in models of acute nociception. Orally administered bicifadine did not show consistent efficacy in the tail-flick test at high stimulus intensity (tail-withdrawal latency of 6.14 ± 0.24 s in vehicle-treated mice) (Fig. 5). However, bicifadine dose-dependently increased tail-flick latencies (ED₅₀ = 38.8, 30.7–49.0 mg/kg p.o., mean, 95% CI, nonlinear regression analysis, 185 µmol/kg) when a lower intensity radiant heat stimulus was used (tail-withdrawal latency of 15.3 ± 0.35 s in vehicle treated mice) or following subcutaneous administration (data not shown). Likewise, ibuprofen (150 mg/kg i.p.) was more effective in increasing tail-flick latency under low intensity stimuli (49.7 ± 8.0%MPE) than under high intensity (9.0 ± 3.5%MPE). Morphine was equieffective at both stimulus intensities (ED₅₀ = 2.69, 2.51–2.88 mg/kg S.C., 4.0 µmol/kg, low intensity, 2.84, 2.73–2.98 mg/kg S.C., 4.2 µmol/kg, high intensity). Bicifadine (s.c. or p.o.) was ineffective in mice placed on a 59°C hot plate. However, when bicifadine (25 mg/kg s.c., 30 min postadministration) was administered to mice placed on a hot plate maintained at 55°C, it significantly increased the response time by 43% above vehicle levels. By comparison, morphine (10 mg/kg s.c., 15 µmol/kg 30 min postadministration) increased the response latency by 88%.

View larger version (11K): [in this window] [in a new window]   Fig. 5. Bicifadine activity in the tail-flick test. The antinociceptive actions of orally administered bicifadine (25, 50, 75, and 100 mg/kg) were investigated in the tail-flick test at high and low stimulus intensities in albino Swiss mice. The heat source was adjusted for testing subjects under high intensity (12 s cut-off latency, open symbols) or low intensity (25 s cut-off latency, closed symbols) stimuli. Vehicle-treated mice responded to the high- and low-intensity stimuli with an average latency of 6.3 ± 0.18 and 16.8 ± 0.41 s, respectively. The tail-flick latencies at low intensity of all doses of bicfadine-treated animals (squares) are significantly different from vehicle-treated control, P < 0.01, one-way ANOVA and Dunnett's post hoc comparison test (Bicifadine, F(4,44) = 13.48, P < 0.01). Responses of the ibuprofen (150 mg/kg i.p.)-treated animals (circles) were significantly different from vehicle-treated controls under low-intensity stimuli, P < 0.01 (t17 = 5.286).

Bicifadine Activity in Models of Persistent Pain. Bicifadine was an effective analgesic in two models of tonic visceral pain, the PPQ-induced abdominal contraction test (Table 3) and the colonic distension test (Fig. 6). Bicifadine suppressed the abdominal contractions induced by PPQ (ED₅₀ = 13, 6–29 mg/kg p.o., 4.8 mg/kg s.c.), with a potency equivalent to codeine (on a milligram/kilogram basis, 0.5-fold less on a micromolar/kilogram basis), and greater than acetaminophen [2.8-fold (milligram/kilogram), 3.9-fold (micromolar/kilogram); Table 3]. In the colonic distension model, the number of abdominal contractions observed in response to acetic acid infusion followed by colonic distension was significantly reduced by bicifadine, with a minimal effective oral dose of 5 mg/kg, (23 µmol/kg) and a maximal effect comparable to that of the reference antinociceptive agent used in this study, the -opioid agonist U-50,488 (10 mg/kg, 24.6 µmol/kg, p.o.; Fig. 6).

View larger version (18K): [in this window] [in a new window]   Fig. 6. Bicifadine reduces the number of abdominal contractions in the rat colonic distension model. Anesthetized rats received colonic irrigation with an irritant (acetic acid) solution, followed by oral administration of bicifadine. Sixty minutes later, a balloon catheter was inserted into the colon and inflated, and the number of abdominal contractions was counted. Bicifadine (solid bars) significantly reduced the number of abdominal contractions at all of the doses tested, with a maximal efficacy comparable with that of the reference agent U-50,488 (10 mg/kg p.o., hatched bar). **, significantly different from vehicle-treated control animals, P < 0.01, one-way ANOVA and Dunnett's post hoc comparison test (F(4,45) = 15.6, P < 0.01). ##, significantly different from vehicle-treated control animals, unpaired two-tailed t test, t18 = 8.17, P < 0.01.

View larger version (28K): [in this window] [in a new window]   Fig. 7. Bicifadine is an effective antinociceptive agent in both phases of the formalin test in rats and mice. One hour after administration of bicifadine, rats and mice received a midplantar injection of 5% formalin solution. For rats, hindpaw licking time was recorded between 0 and 10 min (Phase 1, A) and between 15 and 40 min (Phase 2, B) after formalin injection; whereas for mice, the licking time was recorded over 0 to 5 (Phase 1, C) and 20 to 30 (Phase 2, D) min. Bicifadine (solid bars, 10, 20, and 30 mg/kg p.o. rats or 5, 10, 20, 40, and 60 mg/kg p.o. mice) dose-dependently decreased the amount of time spent licking in both phases of the test, and in both species of rodent, with maximal efficacies that were comparable to or greater than those of the reference agent morphine (Mor, hatched bars, 80 mg/kg p.o. rats, 60 mg/kg p.o. mice). *, **, significantly different from vehicle-treated control, P < 0.05, 0.01, respectively, one-way ANOVA and Dunnett's post hoc comparison test (A, F(6,63) = 17.0, P < 0.01; B, F(6,63) = 19.1, P < 0.01; C, F(5,54) = 6.0, P < 0.01; D, F(5,54) = 10.7, P < 0.01). ##, significantly different from vehicle-treated control, P < 0.01, two-tailed, unpaired t test (A, t(18) = 3.0; B, t(18) = 3.8; C, t(18) = 13.1; D, t(18) = 6.2).

As in animal models of acute inflammatory pain, bicifadine was found to be active in a model of persistent inflammation. One week after the subplantar administration of CFA, bicifadine (40 and 60 mg/kg, 190 and 290 µmol/kg p.o.) normalized the nociceptive thresholds at 1 and 3 h after administration (Fig. 8, A and B), an effect comparable with that of indomethacin (30 mg/kg, 84 µmol/kg p.o.). This antinociceptive effect was absent by 24 h after administration of either drug (Fig. 8C).

View larger version (18K): [in this window] [in a new window]   Fig. 8. Duration of the antinociceptive actions of bicifadine in the CFA model of persistent inflammatory pain. Animals received an intraplantar injection of CFA (1 mg/ml) under anesthesia 1 week before administration of test substances (bicifadine: 5, 10, 20, 40, and 60 mg/kg p.o., solid bars; indomethacin: 30 mg/kg p.o., hatched bars). Mechanical hyperalgesia (paw-withdrawal latency) was assessed at 1 (A), 3 (B), and 24 (C) h after administration of the test substances. Both bicifadine (20 and 40 mg/kg) and indomethacin (Indo) significantly elevated the nociceptive threshold of the lesioned paw at 1 and 3 h after administration. *,**, significantly different from vehicle-treated control, P < 0.05, 0.01, one-way ANOVA and Dunnett's post hoc comparison test (A, F(5,54) = 5.9, P < 0.01; B, F(5,54) = 4.48, P < 0.01). #, ##, significantly different from vehicle-treated control, P < 0.05, 0.01, two-tailed, unpaired t test (A, t(18) = 2.8; B, t(18) = 2.1).

View larger version (31K): [in this window] [in a new window]   Fig. 9. The antinociceptive effects of bicifadine in the SNL rat model of neuropathic pain: mechanical (A) and thermal (B) hyperalgesia and mechanical allodynia (C). Bicifadine (20, 40, and 60 mg/kg p.o., closed bars) and gabapentin (300 mg/kg p.o., hatched bar) significantly increased the withdrawal threshold of lesioned paws above those observed in vehicle-treated animals (Veh, open bar) in the mechanical hyperalgesia assessment (A). In a separate group of animals, bicifadine (12.5, 25, and 100 mg/kg p.o.) and morphine (Mor, 128 mg/kg p.o., hatched bar) significantly increased the withdrawal latency for lesioned paws above those for vehicle-treated paws (Veh, open bar) (B). Bicifadine (40 mg/kg p.o.) and gabapentin (300 mg/kg p.o.) also increased the response threshold to Von Frey hairs in a test of mechanical allodynia (C). Evidence for the involvement of dopamine in the suppression of mechanical hyperalgesia by bicifadine is indicated in D. The antihyperalgesic action of bicifadine (40 mg/kg, closed bar) was inhibited by pretreatment with the D2 antagonist (-)-sulpiride (15 mg/kg, cross-hatched bar), a dose that did not alter responsiveness in this assay (gray bar). Values represent the mean ± S.E.M. of results from 9 to 10 rats per group. *,**, responses are significantly different from those for lesioned paws from vehicle-treated rats, P < 0.05, 0.01, respectively, one-way ANOVA followed by Dunnet's post hoc comparison test. (A, F(5,51) = 6.9, P < 0.01; B, F(4,45) = 4.7, P < 0.01; C, F(5,53) = 6.3, P < 0.01). **, responses are significantly different from those for lesioned paws from vehicle-treated rats, P < 0.01, and from (-)-sulpiride and Bic/Sulpiride groups, P < 0.01, respectively, one-way ANOVA followed by Tukey's post hoc comparison matrix. (D, F(3,32) = 18.0, P < 0.01). #, ##, significantly different from vehicle-treated control, P < 0.05, 0.01, respectively, two-tailed, unpaired t test (A, t(18) = 2.9; B, t(18) = 4.1; C, t(18) = 3.1; D, t(15) = 2.4).

The effect of the D₂ dopamine receptor antagonist (-)-sulpiride on the antinociceptive actions of bicifadine was investigated in the mechanical hyperalgesia assessment using SNL rats (Fig. 9D). Although bicifadine (40 mg/kg p.o.) effectively raised the lesioned paw-withdrawal threshold to levels comparable with unlesioned paws, (-)-sulpiride (15 mg/kg s.c., a nonsedating dose) alone had no significant effect. Combining (-)-sulpiride with bicifadine significantly attenuated the bicifadine-induced elevation of the withdrawal threshold by 38%. The antiallodynic actions of bicifadine (40 mg/kg) on the Von Frey thresholds (8.6 ± 2.1g) were not significantly altered by (-)-sulpiride (15 mg/kg, 6.6 ± 1.5g).

The temporal profile of the antinociceptive actions of bicifadine differed in the mechanical and thermal hyperalgesia assessments (Fig. 10). Bicifadine (25 mg/kg p.o.) was maximally effective in increasing the force required to induce lesioned paw withdrawal at 60 min, with a significant antinociceptive effect observed up to 120 min after administration (Fig. 10A). In contrast, bicifadine (25 mg/kg, 120 µmol/kg p.o.) was effective in the thermal hyperalgesia assessment as early as 30 min after administration, with significant activity at 90 and 240 min (Fig. 10B). Comparable results in both assessments were observed following the administration of morphine (128 mg/kg p.o.).

View larger version (32K): [in this window] [in a new window]   Fig. 10. The duration of action of bicifadine in assessments of mechanical (A) and thermal (B) hyperalgesia in the SNL rat model of neuropathic pain. The force inducing lesioned paw withdrawal was significantly increased above vehicle-treated control levels at 60 and 120 min after administration of bicifadine (25 mg/kg p.o., A). The latency to withdraw the lesioned paw was significantly increased in rats 30, 90, and 240 min after administration of bicifadine (25 mg/kg p.o., B). In contrast, morphine (128 mg/kg p.o.) significantly increased the force inducing paw withdrawal at 60 min and the latency to paw withdrawal at 15, 60, 120, and 240 min after administration. Values represent the mean ± S.E.M. of results from eight rats per group. *, **, significantly different from the contemporaneous, vehicle-treated lesioned paw response, P < 0.05, 0.01, two-way ANOVA followed by Bonferroni's post hoc comparison matrix. A, bicifadine, F(6,1,6) = 18.7 treatment, P < 0.01; morphine, F(3,1,3) = 36 treatment, 11.7 time, 11.1 interaction, all P < 0.01; B, bicifadine, F(1,6,6) = 47.2 treatment, 2.1 time, 2.7 interaction, all P < 0.01; morphine, F(1,3,3) = 68.9 treatment, P < 0.01).

View larger version (46K): [in this window] [in a new window]   Fig. 11. The antinociceptive properties of bicifadine are maintained after subchronic administration. The force inducing lesioned paw withdrawal in SNL rats (mechanical hyperalgesia assessment) was significantly increased above vehicle control levels following either a single dose of bicifadine (Bic, 50 mg/kg p.o., dotted bar), or after subchronic administration (50 mg/kg/day, 5 days, p.o., black bar). Acute, but not subchronic morphine (Mor, 128 mg/kg p.o., hatched bar) and acute gabapentin (Gbp, 300 mg/kg p.o., vertical stripes) also significantly increased the paw-withdrawal force above vehicle control. Values represent the mean ± S.E.M. of results from 10 rats per group. *, **, significantly different from vehicle-treated lesioned paw response, P < 0.05, 0.01, one-way ANOVA followed by Dunnet's test, F(4,44) = 6.9, P < 0.01. ##, significantly different from vehicle-treated control, P < 0.01, two-tailed, unpaired t test, t(18) = 5.0.

View larger version (17K): [in this window] [in a new window]   Fig. 12. The antinociceptive actions of bicifadine in the STZ-treated rat model of diabetic neuropathy. Bicifadine (12.5 and 25 mg/kg p.o.) significantly increased the paw-withdrawal force in rats with STZ-induced mechanical hyperalgesia above vehicle-treated, diabetic controls. Values represent the mean ± S.E.M. of results from 10 rats per group. **, response thresholds are significantly different from those from vehicle-treated rats, P < 0.01, one-way ANOVA followed by Dunnet's post hoc comparison test, F(4,45) = 8.3, P < 0.01.

View larger version (33K): [in this window] [in a new window]   Fig. 13. Effects of bicifadine and desipramine on motor activity, rotarod performance, and grip strength. Bicifadine (12.5–100 mg/kg p.o.) had no significant effect on the distance traveled by rats in the open field (A), whereas desipramine (25, 50 mg/kg i.p., B) significantly reduced the distance traveled in the open field relative to vehicle-treated rats. Bicifadine (C) did not cause a decrement in rotarod performance, whereas rats treated with desipramine (D) showed a trend toward decreased rotarod performance, which did not reach significance (E). Bicifadine (F) did not alter grip strength, whereas rats treated with desipramine (50 mg/kg) showed a significant decrement in grip strength (G). *, brackets, Significantly different from vehicle control, P < 0.05, two-way ANOVA and Bonferroni's corrected post hoc comparison test, F(2,6,12) = 654; treatment, 328 time, 150 interaction, all P < 0.01. *, significantly different from vehicle control, P < 0.05, one-way ANOVA and Dunnett's post hoc comparison test, F(2,19) = 6.2, P < 0.01.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Bicifadine is a functional inhibitor of biogenic amine transporters, inhibiting radioligand binding to the DAT, NET, and SERT with moderate affinity and suppressing [³H]NE and [³H]5-HT uptake with approximately an order of magnitude greater potency than [³H]DA uptake. Such discrepancies between radioligand binding and neurotransmitter uptake assays are not unusual (Owens et al., 1997, Eshleman et al., 1999), and may reflect differences in the nature of the assays (an equilibrium binding assay versus a nonequilibrium functional test), substrate affinity (high affinity binding site probes versus lower affinity neurotransmitters), chemical structure of the radioligand, and the lack of contiguity between binding sites. Nonetheless, the ability of bicifadine to inhibit DA, NE, and 5-HT uptake is supported by in vivo microdialysis studies, where bicifadine significantly increased extrasynaptic levels of these neurotransmitters in a number of brain regions. Whereas the kinetics of the neurotransmitter changes differed slightly from region to region, the overall increases in neurotransmitter levels (as indicated by the AUC_0–180) were not significantly different in the prefrontal cortex (NE and 5-HT). This "balanced" inhibition of NET and SERT may yield an antinociceptive synergy resulting from the simultaneous activation of noradrenergic and serotonergic systems (Danzebrink and Gebhart, 1991; Iyengar et al., 2004, Leventhal et al., 2007), suggesting that dual uptake inhibitors may have an enhanced antinociceptive profile relative to uptake inhibitors more selective for the SERT or NET.

However, additional aspects of the molecular pharmacology of bicfadine may contribute to a novel antinociceptive profile. In addition to its actions as a functional DAT inhibitor, bicifadine is an ₂-adrenoceptor agonist at pharmacologically relevant concentrations (K_i = 1 µM; EC₅₀, neurogenic twitch = 6 µM). In addition, bicifadine is a full agonist at the 5-HT_1A receptor. These actions, combined with the blockade of biogenic amine neurotransmitter uptake, may broaden the antinociceptive profile of bicifadine or enhance its efficacy, particularly at doses where ₂ adrenoceptors and 5-HT_1A receptors are activated (Stone et al., 1997, Boyd, 2001).

Given a molecular pharmacology profile consistent with antinociceptive activity, bicifadine was tested in vivo in models of acute, persistent, and chronic nociception. Bicifadine is readily absorbed following oral administration, producing maximal plasma concentrations of approximately 16 µMat 60 mg/kg, with an elimination half-life of approximately 3.5 h. Bicifadine was found to be potent and fully efficacious in two models of acute (yeast-inflamed hindpaw and intra-articular kaolin) and persistent (CFA) inflammatory pain. Although the efficacy of bicifadine was lost at higher doses (50 mg/kg) in the kaolin model, this is not likely to reflect a disruption of motor function that affects gait (see below) but may reflect an inhibition of NE release by activation of presynaptic ₂ adrenoceptors. In addition to its systemic activity, bicifadine was active following local administration in the hindpaw inflammation model, consistent with its actions as an indirect -adrenoceptor agonist inhibiting the secretion of pro-inflammatory agents by activated immunocytes (Elenkov et al., 2000). Nonetheless, bicifadine is not a classic anti-inflammatory, as indicated by its lack of effect on COX-1 and COX-2 activity. Contrasting with its efficacy in models of acute inflammatory pain, bicifadine was less effective in the tail-flick and hot plate models of acute nociception and then only after reducing the stimulus intensity or administering bicifadine subcutaneously. These assays are particularly sensitive to opiates, and the relative lack of antinociceptive activity of bicifadine in these models is shared with a number of other analgesic classes, including other NET/SERT blockers, gabapentin, and NSAIDs (Le Bars et al., 2001).

The central actions of bicifadine are also important in its antinociceptive profile, as indicated by its high potency and efficacy in three models of persistent pain (PPQ, colonic distension, and formalin tests). Activating central adrenergic pathways increase the threshold for the visceromotor response in visceral pain models (Danzebrink and Gebhart, 1991), an effect synergistically enhanced by stimulation of serotonergic systems (Danzebrink and Gebhart, 1991). Thus, the inhibition of NET and SERT and the direct activation of 5-HT_1A receptors may contribute to the efficacy and potency of bicifadine in these models (Rouzade et al., 1998). However, it is the antinociceptive profile of bicifadine in the formalin test that seems unique for a biogenic amine uptake inhibitor. Thus, bicifadine potently reduced both early and late-phase paw licking after formalin injection. This effect on both phases of the response to formalin contrasts with other uptake blockers (e.g., duloxetine, amitriptyline) (Iyengar et al., 2004, Bomholt et al., 2005), NSAIDs (Hunskaar and Hole, 1987), and gabapentin (Field et al., 1997). The ability of bicifadine to completely suppress paw licking in Phase 1 is consistent with its ability to inhibit acute pain by mechanisms independent of 5-HT or NE uptake blockade. Although opiates are effective analgesics in both phases of the formalin test, the mechanism of action of bicifadine does not involve direct activation of opioid pathways as indicated by its lack of affinity for opioid receptor subtypes and the inability of naloxone to inhibit its antinociceptive actions (data not shown). Alternatively, bicifadine may suppress Phase 1 licking by direct activation of serotonergic and adrenergic pathways (Kanui et al., 1993; Buritova et al., 2005).

Antinociceptive efficacy in the second phase of the formalin test is predictive of antihyperalgesic activity in neuropathic pain models (Fishbain et al., 2000; Le Bars et al., 2001). Bicifadine effectively suppressed mechanical hyperalgesia in both the SNL and STZ-induced models of neuropathic pain at doses comparable with those effective in treating acute and persistent pain syndromes. In these models, bicifadine exhibited greater oral potency and equivalent antinociceptive efficacy to morphine and gabapentin. Unlike morphine, the antinociceptive activity of bicifadine in the mechanical hyperalgesia assessment did not significantly decline following subchronic administration. While suppressing mechanical allodynia in the SNL model, further examination of the actions of bicifadine indicated that it was effective in suppressing thermal hyperalgesia, despite the insensitivity of this endpoint in nerve-ligation models of neuropathic pain (Wang and Wang, 2003; Dowdall et al., 2005). In the SNL model, the antinociceptive profile of bicifadine resembles that of dual uptake blockers. However, what seems to be unique is that a nonsedating dose of (-)-sulpiride significantly inhibited the effects of bicifadine on mechanical hyperalgesia in SNL rats without altering its actions on mechanical allodynia. Although consistent with the involvement of bicifadine-induced elevations in striatal DA in its antinociceptive actions, these observations contrast with previous reports of the involvement of dopaminergic mechanisms in mechanical allodynia in this model (Pedersen et al., 2005). Investigating the effect of D₂ antagonists on the antiallodynic effects of bicifadine using a full dose-response curve may be a more sensitive way to establish the role of dopaminergic pathways in bicifadine-induced antinociception.

Bicifadine shares many antinociceptive attributes with other uptake inhibitors, but it does not share their side-effect profile. Desipramine significantly reduced motor activity in the open field, reduced grip strength, and showed a trend toward inducing ataxia, all at antinociceptive doses (Sawynok and Reid, 2001; Bomholt et al., 2005). In contrast, bicifadine had no significant effects on motor activity. Although striatal DA levels are increased by antinociceptive doses of bicifadine, there is no evidence of bicifadine-induced hyperlocomotion or stereotypy, even at doses 10-fold greater than its antinociceptive ED₅₀ in the Randall-Selitto test. The ₁-antagonist properties of bicifadine may serve to suppress any motor hyperactivity or stereotypy resulting from dopaminergic activation, given the role of ₁ adrenoceptors in mediating these activities (Drouin et al., 2002). In contrast, the lack of sedation, myorelaxation, or ataxia associated with bicifadine administration may result from its low affinity for muscarinic cholinergic and H₁ histamine receptors. In addition to these preclinical observations, clinical studies administering analgesic doses of bicifadine (600 mg p.o.) reported no significant changes in basic cardiovascular function relative to placebo (4 mm Hg increase in systolic blood pressure, 3 mm Hg increase in diastolic blood pressure) and observed no effect on pulse, respiration rate, or body temperature (Stern et al., 2006). Finally, whereas many psychostimulants with abuse liability (e.g., amphetamine, cocaine) inhibit dopamine uptake, bicifadine does not induce dependence in rodents or primates after as much as 48 days of administration (unpublished observation), nor does it substitute for cocaine in discrimination studies with cocaine-trained rats at doses that do not impair bar-pressing performance (R. Balster, personal communication).

Dual NET/SERT inhibitors have demonstrated clinical utility as analgesics, but recent data suggest that the inhibition of DAT may have a further salutary effect on the treatment of peripheral neuropathic pain, as evidenced by the NNT score (number of patients needed to be treated in order to obtain a positive response [50% reduction in pain intensity]). The number needed to treat values for a serotonin-selective reuptake inhibitor as an analgesic is as high as 6.8. These values decline to 5.5 with a SERT/NET inhibitor such as venlafaxine, further decreasing to 2.2 with TCAs and finally setting at 1.6 for the DAT inhibitor bupropion (Sindrup et al., 2005). The results of this trend suggest that the transporter inhibition profile of bicifadine, a functional inhibitor of DAT, NET, and SERT with antinociceptive activity, may provide greater clinical efficacy in treating neuropathic pain states than currently available SNRIs and TCAs, but without the side-effect profile associated with TCAs, a hypothesis that awaits further study in the clinic.

	Footnotes

 
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.116483.

ABBREVIATIONS: NE, norepinephrine; ANOVA, analysis of variance; AUC, area under the curve; CFA, complete Freund's adjuvant; CI, confidence interval; C_max, maximal plasma concentration; [¹²⁵I]RTI-55, 3-(4-iodophenyl)tropan-2-carboxylic acid methyl ester; COX, cyclooxygenase; DA, dopamine; DAT, dopamine transporter; U-50,488, 3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide; DOPAC, 3,4-dihydroxyphenylacetic acid; ED₅₀, dose yielding a 50% of maximal response; 5-HT, serotonin; GABA, -aminobutyric acid; h, human; 5-HIAA, 5-hydroxyindoleacetic acid; HPLC, high pressure liquid chromatography; HVA, homovanillic acid; LC, locus coeruleus; LD₅₀, dose lethal to 50% of subjects; %MPE, percentage of maximum possible effect; NET, norepinephrine transporter, NMDA, N-methyl-D-aspartate; NSAID, nonsteroidal anti-inflammatory; pfCTX, prefrontal cortex; PPQ, phenylparaquinone; SERT, serotonin transporter; SNL, spinal nerve ligation; STZ, streptozotocin, Str, striatum; TCA, tricyclic antidepressant; Veh, vehicle; RX 821002, 2-(2,3-dihydro-2-methoxy-1,4-benzodioxin-2-yl)-4,5-dihydro-1H-imidazole hydrochloride; SKF-10047, [2S-(2,6,11R^*]-1,2,3,4,5,6-hexahydro-6,11-dimethyl-3-(2-propenyl)-2,6-methano-3-benzazocin-8-ol hydrochloride; MK 912, (2S-trans)-1,3,4,5',6,6',7,12b-octahydro-1',3',-dimethyl-spiro[2H-benzofuro[2,3-a]quinolizine-2,4'(1'H)-pyrimidin]-2'-(3'H)-one hydrochloride hydrate; MK-801, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate.

Address correspondence to: Dr. Anthony S. Basile, DOV Pharmaceutical, Inc., 150 Pierce St., Somerset, NJ 08873-4185. E-mail: abasile{at}dovpharm.com

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