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Table 3. Pharmacological selectivity of A-803467 (CEREP)

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The response to acute thermal stimulation was determined using a commercially available paw thermal stimulator (UARDG; University of California, San Diego, CA). Rats were placed individually in Plexiglas cubicles mounted on a glass surface maintained at 30°C, and allowed a 30 min habituation period. A thermal stimulus, in the form of radiant heat emitted from a focused projection bulb, was then applied to the plantar surface of each hind paw. In each test session, each rat was tested in three sequential trials at »5-min intervals. Paw withdrawal latencies (PWL) were calculated as the mean of the two shortest latencies. An assay cut off was set at 20.5 s.

The response to noxious mechanical stimulation was determined by measuring withdrawal threshold to paw pressure by using the Ugo Basile analgesymeter (Comerio, Italy). The animals were gently restrained, and steadily increasing pressure was applied to the dorsal surface of a hind paw via a dome-shaped plastic tip (diameter = 1 mm). The pressure required to elicit paw withdrawal was determined twice and values averaged.

After a 30-min acclimation period to individual observation cages, 50 ml of a 5% formalin solution was injected (s.c.) into the dorsal aspect of the right hind paw and the rats were then returned to the clear observation cages. Rats were observed for periods of time corresponding to phase 1 (0-10 min) and phase 2 (30-50 min) of the formalin test. Nociceptive behaviors were recorded from animals during the session by observing each animal for one 60-sec observation period during each 5-min interval. Nociceptive behaviors recorded included flinching, licking or biting the injected paw.

Unilateral inflammation was induced by injecting 100 ml of a 1% solution of l-carrageenan or 150 ml of a 50% solution of CFA (Sigma Chemical Co., St. Louis, MO) in physiological saline into the plantar surface of the right hind paw of the rat. The hyperalgesia to thermal stimulation was determined 2 h after carrageenan by using the same apparatus as described above for the noxious acute thermal assay. A-803467 was injected 90 min after carrageenan injection (i.e., 30 min before testing in inflamed rats). In the CFA experiments, CFA was injected 2 or 4 days before behavioral testing for thermal hyperalgesia as described above. Animals were also tested for mechanical allodynia by using calibrated von Frey filaments (Stoelting, Wood Dale, IL). Briefly, rats were placed into individual Plexiglas containers and allowed to acclimate for 15-20 min before testing. Paw withdrawal threshold was determined by increasing and decreasing stimulus intensity and estimated by using a Dixon nonparametric test. Only rats with threshold scores £4.5 g were considered allodynic and used in compound testing experiments.

. As previously described in detail by Kim and Chung (6), a 1.5-cm incision was made dorsal to the lumbosacral plexus. In anesthetized rats, the paraspinal muscles (left side) were separated from the spinous processes, and the L5 and L6 spinal nerves isolated and tightly ligated with 3-0 silk threads. After hemostasis, the wound was sutured and coated with antibiotic ointment. The rats were allowed to recover and then placed in a cage with soft bedding for 14 days before behavioral testing for mechanical allodynia.

As described in detail by Bennett and Xie (1), in anesthetized rats, a 1.5-cm incision was made 0.5 cm below the pelvis, and the biceps femoris and the gluteous superficialis (right side) were separated. The sciatic nerve was exposed and isolated, and four loose ligatures (5-0 chromic catgut) with 1-mm spacing were placed around it. The rats were allowed to recover and then placed in a cage with soft bedding for 14 days before behavioral testing for mechanical allodynia as described above. In addition, animals were also tested for cold allodynia by dipping their hind paw in a cold-water bath (4.5°C) and determining the paw withdrawal latency.

As described in detail by Lynch et al. (8), chemotherapy-induced neuropathic pain was induced by a continuous i.v. infusion of vincristine. In anesthetized rats, the right external jugular vein was catheterized (PE60 tubing) with a vincristine-filled osmotic pump (0.5 ml/h, 14 days; Alzet Model 2002; Durect Corporation, Cupertino, CA) that had been primed overnight to deliver 30 mg/kg.day vincristine sulfate (Sigma-Aldrich, St Louis, MO). The rats were allowed to recover and were then placed in a cage with soft bedding for 14 days before behavioral testing for mechanical allodynia as described above.

Three hours after intraplantar injection of capsaicin (10 mg/10 ml of 10% ethanol and 2-hydroxypropyl cyclodextrin), rats were tested for mechanical allodynia, away from the site of injection. A-803467 was injected 30 min before behavioral testing (150 min after capsaicin injection).

. As described by Brennan et al. (3), a 1-cm longitudinal incision was made through the skin and fascia of the plantar aspect of the foot, starting 0.5 cm from the proximal edge of the heel and extending toward the toes. The plantaries muscle was elevated and incised longitudinally with origin and insertion of the muscle remaining intact. The skin was then closed with two mattress sutures of 5-0 nylon. After surgery, the animals were allowed to recover and were housed individually with soft bedding. Animals were tested for mechanical allodynia by using von Frey hairs as described above 2 and 24 h after surgery.

The test used was a modification of the abdominal constriction test described by Collier et al. (4). Each animal received an i.p. injection of 0.3 ml of 0.6% acetic acid in normal saline to evoke writhing. Abdominal constriction was defined as a mild constriction and elongation passing caudally along the abdominal wall, accompanied by a slight twisting of the trunk and followed by bilateral extension of the hind limbs. The total number of abdominal constrictions was recorded from 5 to 20 min after acetic acid injection.

Rats were allowed to acclimate to the study room 1 h before treatment in an individual clear, 20 ´20 ´ 30-cm Plexiglass cage. Three behavioral parameters (breathing rate, closing/opening of the eyes, and posture) were used as pain indices and scored in a manner similar to that previously described [Lanteri-Minet et al. (7); Boucher et al. (2)]. Animals received either saline or cyclophosphamide (CP; Sigma/Aldrich, Milwaukee, WI) 100 mg/kg, i.p. and were evaluated for behavioral changes 70 min later. Only rats that showed visceral pain with decrease in respiratory rate and increase in nociceptive behaviors were included in the study. The difference between the rats receiving saline and the rats receiving CP for respiratory rate and nociceptive behaviors determined the "window" of visceral pain. At that time, animals received either vehicle or A-803467 and were tested again for behavioral changes 30 min later.

CRD methodology was essentially as described by Jones and Gebhart (5). The visceromotor response to CRD was determined by recording electromyographic (EMG) activity of the external oblique musculature. Bipolar EMG electrodes (Teflon-insulated multistrand 40-guage stainless steel wire) were sutured into the external oblique musculature just above the inguinal ligament of rats anesthetized with ketamine/pentobarbital [Youth et al. (9)]. The electrodes were passed s.c. to the nape of the neck and externalized for future access. The animals were allowed to recover for a minimum of 4 days, with food and water ad libitum.

All studies were performed in conscious rats. Rats were placed in an ECU Experimental Conditioning Unit (Braintree Scientific, Braintree MA). A 7-cm flexible latex balloon was inserted transanally, and kept in place by taping to the base of the tail. A series of four baseline distensions (60 mmHg, 20-s duration) were administered at 4-min intervals. A-803467 was then administered by i.p. injection (2 ml/kg), and, beginning 30 min after drug injection, a series of additional 60-mmHg distensions were administered over the next 60 min. A-803467 was tested at doses of 10, 30, and 100 mg/kg. Vehicle was 5% DMSO in PEG 400. EMG signals were amplified (×10 k), bandpass-filtered (low cutoff: 300 Hz, high: 5kHz), and digitized at 1,000 Hz with a Power1401 data acquisition interface (Cambridge Electronic Design, England) connected to a PC running Spike2 software (v.5.01, CED). The EMG waveforms were rectified and integrated with Spike2. Each rat's postdrug EMG responses to CRD were normalized to the mean EMG value of the four baseline distensions. The area under the curve (AUC) of each rat's EMG time course was calculated (SigmaPlot 7.1) and averaged for each dose group. One-way ANOVA, followed by Dunnett's multiple comparison procedure (Minitab release 11.13) was used to detect significant decreases in AUC (analgesic efficacy).

. Locomotor activity was measured in an open field by using photobeam activity monitors (AccuScan Instruments, Columbus, OH), and rotorod performance was measured by using an accelerating rotorod apparatus (Omnitech Electronics, Inc., Columbus, OH). For the rotorod assay, rats were allowed a 30-min acclimation period in the testing room and then placed on a 9-cm-diameter rod, which increased in speed from 0 to 20 rpm over a 60-s period. The time required for the rat to fall from the rod was recorded, with a maximum score of 60 s. Each rat was given three training sessions. To test for balance, rats were also assessed for their ability to remain on top of 0.5-cm-thick edges of a 40 ´40 ´ 36-cm Plexiglas box. The rats had to pull themselves up on to the edge and avoid falling with a cutoff time of 2 min. The average (latency to fall) of two trials was recorded.

1. Bennett GJ, Xie YK (1988) Pain 33:87-107.

2. Boucher M, Meen M, Codron JP, Coudore F, Kemeny JL, Eschalier A (2000) J Urol 164: 203-208.

3. Brennan TJ, Vandermeulen EP, Gebhart GF (1996) Pain 64:493-501.

4. Collier HOJ, Dinneen LC, Johnson CA, Schneider C (1968) Br J Pharmacol Chemother 32:295-310.

5. Jones RC, III, Gebhart GF (2004) Models of Visceral Pain: Colorectal Distention (CRD) in Current Protocols in Pharmacology (Wiley, New York), pp 5.36.1-5.36.17.

6. Kim SH, Chung JM (1992) Pain 50:355-363.

7. Lanteri-Minet M, Bon K, Pommery J, Michiels JF, Menetrey D (1995) Exp Br Res 105:220-232.

8. Lynch JJ, III, Wade CL, Zhong CM, Mikusa JP, Honore P (2004) Pain 110:56-63.

9. Youth RA, Simmerman SJ, Newell R, King RA (1973) Physiol Behav 10:633-6.