BCTC (N-(4-tertiarybutylphenyl)-4-(3-cholorphyridin-2...

JPET #46268 1

BCTC (N-(4-tertiarybutylphenyl)-4-(3-cholorphyridin-2-yl)tetrahydropryazine-

1(2H)-carbox-amide), a novel, orally-effective vanilloid receptor 1 antagonist with

analgesic properties: II. In vivo characterization in rat models of inflammatory and

neuropathic pain.

James D. Pomonis, James E. Harrison, Lilly Mark, David R. Bristol, Kenneth J.

Valenzano and Katharine Walker

Purdue Pharma Discovery Research, 6 Cedar Brook Drive, Cranbury, NJ 08512

Copyright 2003 by the American Society for Pharmacology and Experimental Therapeutics.

JPET Fast Forward. Published on April 29, 2003 as DOI:10.1124/jpet.102.046268This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on April 29, 2003 as DOI: 10.1124/jpet.102.046268

at ASPE

T Journals on July 8, 2020

jpet.aspetjournals.orgD

ownloaded from

http://jpet.aspetjournals.org/

JPET #46268 2

Running Title: Selective blockade of vanilloid receptor 1 in rat models of pain

Corresponding author: Kenneth J. Valenzano, 6 Cedarbrook Drive, Cranbury, NJ 08512, Tel

(609) 409-5770; Fax (609) 409-6922; E-mail: [email protected]

Number of Pages: 28

Number of Tables: 0

Number of Figures: 6

Number of References: 40

Number of words in Abstract: 217

Number of words in Introduction: 569

Number of words in Discussion: 1295

Abbreviations: BCTC, N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-

yl)tetrahydropyrazine-1(2H)-carbox-amide; DRG: dorsal root ganglion, FCA: Freund’s

complete adjuvant, FW50: Median fiber weight, NSAID: nonsteroidal anti-inflammatory

drug, PWL: paw withdrawal latency, PWT: paw withdrawal threshold, VR1: vanilloid

receptor 1

Recommended section assignment: Neuropharmacology

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on April 29, 2003 as DOI: 10.1124/jpet.102.046268

at ASPE



ownloaded from


JPET #46268 3

ABSTRACT

The vanilloid receptor 1 (VR1) is a cation channel expressed predominantly by

nociceptive sensory neurons and is activated by a wide array of pain-producing stimuli

including capsaicin, noxious heat and low pH. Although the behavioral effects of

injected capsaicin and the VR1 antagonist capsazepine have indicated a potential role

for VR1 in the generation and maintenance of persistent pain states, species differences

in the molecular pharmacology of VR1 and a limited number of selective ligands have

made VR1 difficult to study in vivo. BCTC is a recently described inhibitor of capsaicin-

and acid-mediated currents at rat VR1 (Valenzano et al., accompanying manuscript).

Here we report the effects of BCTC on acute, inflammatory, and neuropathic pain in rats.

Administration of BCTC (30 mg/kg, p.o.) significantly reduced both mechanical and

thermal hyperalgesia induced by intraplantar injection of 30 µg capsaicin. In rats with

Freund’s complete adjuvant-induced inflammation, BCTC significantly reduced the

accompanying thermal and mechanical hyperalgesia (3 mg/kg and 10 mg/kg, p.o.,

respectively). BCTC also reduced mechanical hyperalgesia and tactile allodynia 2

weeks following partial sciatic nerve injury (10 and 30 mg/kg, p.o.). BCTC did not affect

motor performance on the rotarod following administration of doses up to 50 mg/kg p.o.

These data suggest a role for VR1 in persistent and chronic pain arising from

inflammation or nerve injury.


at ASPE



ownloaded from


JPET #46268 4

The vanilloid receptor type 1 (VR1) is a pivotal molecular integrator of noxious stimuli

that is expressed on somatic and autonomic primary afferent neurons. VR1 has been

confirmed as a ligand-gated ion channel following its cloning from rat and human

tissues, and has been shown to be highly expressed in small diameter primary afferent

neurons (Caterina et al., 1997; Hayes et al., 2000; McIntyre et al., 2001). In vitro studies

have shown that, like the native vanilloid receptor, recombinant VR1 can be activated by

a variety of chemical as well as physical stimuli. In vitro, VR1 responds to plant-derived

compounds including capsaicin, a pungent component of chilli peppers, lipid mediators

such as anandamide (Smart et al., 2000), the lipoxygenase product 12-(S) HPETE

(Hwang et al., 2000), as well as noxious heat (Caterina et al., 1997), and low pH

(Tominaga et al., 1998).

A potential role for VR1 in nociception has been evident for some time as injection of the

VR1 agonist capsaicin induces nocifensive and hyperalgesic behaviors in rodents and

pain in humans (Szolcsanyi, 1977; Carpenter and Lynn, 1981; Simone et al., 1987;

Simone et al., 1989; Gilchrist et al., 1996). Further support for VR1 as a therapeutic

target arose from experiments involving capsazepine. Capsazepine is a VR1 antagonist

that has been shown to competitively inhibit capsaicin-mediated responses in isolated

dorsal root ganglion (DRG) neurons (Bevan et al., 1992a) and tissues from rat (Bevan et

al., 1992b; Cholewinski et al., 1993; Maggi et al., 1993; Santicioli et al., 1993; Jerman et

al., 2000), mouse (Urban and Dray, 1991) and guinea-pig (Ellis and Undem, 1994; Fox

et al., 1995; Auberson et al., 2000). In vivo, capsazepine has been shown to inhibit

nocifensive and hyperalgesic responses to capsaicin in mice, rats and guinea pigs

(Santos and Calixto, 1997; Walker et al., 2002). However, both in vitro and in vivo

studies have indicated that capsazepine also has species-dependent activity. In vitro,

capsazepine has been shown to block low pH-mediated activation of human or guinea


at ASPE



ownloaded from


JPET #46268 5

pig, but not rat VR1 (Lou and Lundberg, 1992; Satoh et al., 1993; Fox et al., 1995;

McIntyre et al., 2001; Savidge et al., 2002). These in vitro results correlate with the

finding that capsazepine reverses inflammatory and neuropathic hyperalgesia in the

guinea pig, but not in the rat (Walker et al., 2002).

Together, these data suggest that blockade of low pH-induced VR1 activation may be

predictive of antihyperalgesic efficacy in vivo. If this hypothesis is valid, then molecules

that inhibit pH-induced activation of rat VR1, in vitro, would also produce a reduction in

hyperalgesia in rat models of chronic pain. In the accompanying report, Valenzano and

colleagues describe N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-

1(2H)-carbox-amide (BCTC) as a potent, selective and orally bioavailable antagonist of

rat VR1 (Valenzano et al., accompanying manuscript). In contrast to capsazepine, BCTC

not only blocks the activation of rat VR1 by capsaicin, but also by low pH at the native rat

VR1 in a skin-nerve preparation. Thus, BCTC has provided us with an opportunity to test

our hypothesis that the inhibition of low pH-induced activation of VR1 confers in vivo

efficacy in models of chronic pain. This report describes the effects of BCTC in models

of, inflammatory,neuropathic, and capsaicin-induced pain in the rat. The efficacy and

side effect profile of BCTC in these models were compared to those of non-steroidal

anti-inflammatory drugs (NSAIDs) and anti-epileptic drugs currently used for the clinical

therapy of inflammatory and neuropathic pain, respectively.


at ASPE



ownloaded from


JPET #46268 6

MATERIALS AND METHODS

Compounds and administration procedures. BCTC was synthesized according to known

methods and was used in all experiments as its free base (molecular weight = 372.89).

Indomethacin (Sigma, St. Louis, MO) and BCTC were administered orally in 2% beta-

cyclodextrin (Sigma, St. Louis, MO) by gastric gavage in a dose volume of 10 ml/kg body

weight. Gabapentin (Kemprotec, Middlesborough, UK) was dissolved in saline, and

administered via i.p. injection in a dose volume of 2 ml/kg. Capsaicin (Sigma, St. Louis,

MO) was used in all experiments as its free base. The procedure of Gilchrist et al (1996)

was used to dissolve the capsaicin. Briefly, 6 mg of capsaicin was first dissolved in 0.14

mL (20) sorbitan monooleate (Tween 80) by gently heating the solution to approximately

70oC. The solution was then diluted with 1.86 mL 0.9% sodium chloride using an

ultrasonic bath, and passed through a 0.20 µm filter. The final concentration of the

capsaicin solution was 3 µg/µL. Intraplantar injections of the capsaicin solution were

given in a 10 µL volume using a 100 µL Hamilton syringe fitted with a 27-gauge needle.

The Tween 80/saline vehicle was used for control injections.

Animals. The Purdue Institutional Animal Care and Use Committee (IACUC) approved

all animal procedures according to the guidelines of the Office of Laboratory Animal

Welfare. Male Sprague-Dawley rats (Taconic Farms, Germantown, NY), weighing 180-

200g at the start of acute and inflammatory experiments, or 90-110g at the start of nerve

ligation experiments were used. Animals were group-housed and had free access to

food and water at all times, except prior to oral administration of drugs when food was

removed for 16 h before dosing. For comparison to compound-treated groups, animals

treated with the appropriate drug vehicle were included in each experiment. The volume


at ASPE



ownloaded from


JPET #46268 7

of administration was identical for vehicle- and compound-treated rats and rats were

identical with respect to all other experimental procedures.

Capsaicin-induced hyperalgesia. Intraplantar injection of capsaicin (30 µg) was used to

induce mechanical and thermal hyperalgesia, as previously described (Gilchrist et al.,

1996). The paw pressure assay was used to assess capsaicin-induced mechanical

hyperalgesia. For this assay, hind paw withdrawal thresholds (PWT) to a noxious

mechanical stimulus were determined using an analgesymeter (7200, Ugo Basile,

Varese, Italy) (Walker et al., 2001). Cut-off was set at 250 g and the end point was taken

as complete paw withdrawal. PWT were determined once for each rat at each time

point. All rats were tested for baseline PWT and one hour later, animals received a

single dose of 1, 3, 10, or 30 mg/kg BCTC or vehicle (p.o., volume = 10 mL/kg, n =

10/group). Thirty minutes later, under isofluorane/oxygen anesthesia, rats received a

single intraplantar injection of 30 µg capsaicin or vehicle in a 10 µL volume. Ninety

minutes after the intraplantar injection of capsaicin, PWT were determined again.

The plantar test was used to assess capsaicin-induced thermal hyperalgesia (n =

8/group). For this test, hind paw withdrawal latencies (PWL) to a noxious thermal

stimulus were determined using the technique described by Hargreaves et al (1988)

using a plantar test apparatus (Model # 7370-371, Ugo Basile, Varese, Italy). Cut-off

was set at 32 seconds, and any directed paw withdrawal from the heat source was taken

as the end point. To assess the effects of BCTC on the development of capsaicin-

induced thermal hyperalgesia rats were treated as described above for capsaicin-

induced mechanical hyperalgesia, but PWL were measured 30 min after capsaicin

injection.


at ASPE



ownloaded from


JPET #46268 8

Inflammatory hyperalgesia. The efficacy of BCTC against hyperalgesia associated with

inflammation was investigated using the Freund’s Complete Adjuvant (FCA) model

(Walker et al., 2001). Mechanical and thermal hyperalgesia were measured in separate

groups of rats (n = 8-20/group) according to the procedure described above. Baseline

PWT or PWL were determined, and rats were then anaesthetized with

isofluorane/oxygen and received an intraplantar injection of 100% FCA (50 µL). Twenty-

four hours following FCA treatment, pre-drug PWT or PWL measurements were taken

and then rats received a single dose of 1, 3, 10, or 30 mg/kg BCTC, 30 mg/kg

indomethacin, or vehicle (p.o., volume = 10 mL/kg). PWT or PWL were determined again

2, 4, 6, and 24 hours post-drug administration.

Hyperalgesia and allodynia following nerve-injury. The partial sciatic nerve ligation

model was used as a model of nerve-injury-related pain in rats, as previously described

by Seltzer and colleagues (Seltzer et al., 1990). Partial ligation of the left sciatic nerve

was performed under isoflurane/O2 inhalation anesthesia. Following induction of

anesthesia, the left thigh was shaved and cleaned. The sciatic nerve was exposed at

high thigh level through a small incision and was carefully cleared of surrounding

connective tissues at a site near the trocanther just distal to the point at which the

posterior biceps semitendinosus nerve branches off the common sciatic nerve. A 7-0 silk

suture was inserted into the nerve with a 3/8 curved, reversed-cutting mini-needle, and

tightly ligated so that the dorsal 1/3 to 1/2 of the nerve thickness was held within the

ligature and the wound was closed in two layers. Sham-operated control rats underwent

an identical dissection on the left hind limb, but the sciatic nerve was not dissected away

from the surrounding muscle nor ligated. Following surgery animals were weighed and

placed on a warm pad until they recovered from anesthesia.


at ASPE



ownloaded from


JPET #46268 9

The effects of BCTC on mechanical hyperalgesia and tactile allodynia were measured

14-17 days post-surgery (n = 8-10/group). Mechanical hyperalgesia was assessed

using the paw pressure technique prior to, 2, 4, 6 and 24 hours after drug administration.

To assess tactile allodynia, rats (N = 10/group) were placed in clear, plexiglass

compartments with a wire mesh floor and were allowed to habituate for a period of at

least 15 minutes. After habituation, a series of von Frey monofilaments was presented

to the plantar surface of the left (operated) foot of each rat. The series consisted of six

monofilaments of increasing diameter, with the smallest diameter fiber presented first.

Five trials were conducted with each filament, with each trial separated by approximately

2 minutes. Each presentation lasted for a period of 4-8 seconds, or until a nocifensive

withdrawal behavior was observed. Flinching, paw withdrawal, or licking of the paw

were all considered nocifensive behavioral responses. Tactile allodynia was assessed

prior to and 2 hours post drug administration.

Ataxia/motor coordination. To examine the potential effects of BCTC on motor

performance, rats were tested using the rotarod assay. The rotarod speed was

maintained at 30 rpm, with the maximum time spent on the rotarod set at 300 sec. Rats

(N = 8-10/group) received two training trials on the first day, were fasted overnight, and

then received a single dose of either 10, 30, or 50 mg/kg BCTC, or 100 mg/kg

gabapentin (i.p.) as a positive control. Rats were tested on the rotarod 2 and 4 hours

after drug administration.

Statistical analysis. For studies involving mechanical or thermal hyperalgesia, tail flick,

or rotarod latencies, untransformed data were analyzed using a one-way analysis of

variance. In instances where a significant main effect was detected, planned


at ASPE



ownloaded from


JPET #46268 10

comparisons were made using Fisher’s PLSD test. The level of significance was set at p

<0.05. For studies involving tactile allodynia, analyses were performed using an

analysis of covariance model for each time, with log value of the median fiber weight

(FW50) required to evoke a nocifensive response at Hour 0 (baseline) as the covariate. A

t-test for each pair wise comparison based on this model was performed, using PROC

GLM of SAS (Version 6.12).


at ASPE



ownloaded from


JPET #46268 11

RESULTS

Capsaicin-induced hyperalgesia. Injection of 30 µg of capsaicin into the plantar surface

of the left hindpaw induced a profound, but short-lasting decrease in PWL to thermal

stimulation. Initial experiments revealed that this thermal hyperalgesia was present from

15-45 min after injection, and was most robust 30 min post-injection (data not shown).

We therefore tested whether pretreatment of rats with BCTC would prevent the

development of capsaicin-mediated thermal hyperalgesia. Injection of 30 µg capsaicin

resulted in a significant decrease in PWL (27.1 + 1.0 sec versus 9.5 + 2.25 sec for

vehicle- or capsaicin-treated rats, respectively). Pre-treatment with BCTC (30 mg/kg,

p.o.) prior to capsaicin injection significantly inhibited the development of capsaicin-

induced thermal hyperalgesia (18.3 + 2.34 sec, p < 0.01), as compared to rats treated

with capsaicin alone.

As reported previously (Gilchrist et al., 1996) intraplantar injection of capsaicin (30 µg)

also induced mechanical hyperalgesia that lasted longer than the coincident thermal

hyperalgesia. Our preliminary experiments indicated that maximal decreases in PWT

occurred 90 min post-capsaicin injection (data not shown). There was a significant main

effect of treatment on PWT, indicating a dose-dependent reversal of capsaicin-induced

mechanical hyperalgesia by BCTC (F(5,41) = 4.008, p < 0.01). Pre-treatment with BCTC

(30 mg/kg) 30 min prior to capsaicin injection significantly inhibited capsaicin-induced

mechanical hyperalgesia relative to rats treated with capsaicin alone (p < 0.05, Fig. 1).

BCTC reduces thermal and mechanical hyperalgesia associated with inflammation.

Intraplantar injection of 50 µL FCA resulted in the development of thermal hyperalgesia

as indicated by decreased PWL to a noxious thermal stimulus (Fig. 2). Oral


at ASPE



ownloaded from


JPET #46268 12

administration of BCTC produced a dose-dependent reduction in thermal hyperalgesia 2

(F(5,42) = 8.892, p < 0.001) and 4 hours post-administration (F(5,42) = 6.306, p < 0.001.

BCTC (3 and 10 mg/kg, p.o.) produced significant antihyperalgesia 2 hours following

administration. (p < 0.05). These effects were comparable both in terms of efficacy and

duration of action to the NSAID, indomethacin (30 mg/kg, p.o; p < 0.05, Fig. 2). The

effects of BCTC were restricted to the hyperalgesia associated with inflammation or

injury and do not reflect an acute analgesic activity as PWT from the non-injured hind

paw were not affected by systemic BCTC administration (data not shown).

Intraplantar injection of 50 µL FCA also resulted in the development of mechanical

hyperalgesia, determined using the paw pressure assay (Fig. 3). BCTC (1-30 mg/kg,

p.o.) produced a dose-dependent reversal of FCA-induced mechanical hyperalgesia at

2, 4 and 6 hours post-administration (p < 0.001 at each time point). BCTC significantly

reduced FCA-induced mechanical hyperalgesia following oral administration at doses as

low as 3 mg/kg (4 h) and at higher doses (10 and 30 mg/kg, p.o.) were effective for up to

6 h (Fig. 3). The effects of BCTC were similar to indomethacin (30 mg/kg, p.o.) in terms

of efficacy, but indomethacin was only effective at 2 and 4 h following administration.

BCTC reduces mechanical hyperalgesia associated with nerve injury. Partial ligation of

the sciatic nerve resulted in the development of mechanical hyperalgesia within two

weeks of surgery. Administration of BCTC to rats 14-18 days after partial ligation of the

sciatic nerve resulted in a significant and dose-dependent reversal of this mechanical

hyperalgesia (F(6,89) = 10.675, p < 0.001, Fig. 4). Oral administration of BCTC (1-30

mg/kg) produced a significant reversal of mechanical hyperalgesia in the partially

denervated rat hind paw for at least 6 h following administration (p < 0.01), The effects of

administration of BCTC (10 mg/kg, p.o.) were similar to those seen following


at ASPE



ownloaded from


JPET #46268 13

administration of gabapentin (100 mg/kg, i.p,), but with a longer duration of action (6 vs.

4 h, Fig. 4).

BCTC reduces tactile allodynia associated with nerve injury. Partial ligation of the sciatic

nerve also resulted in the development of tactile allodynia that was evident within 14

days of surgery (Fig. 5A). Administration of 10 mg/kg BCTC to rats 14-18 days after

partial ligation of the sciatic nerve resulted in the development of tactile allodynia as

determined by a significant decrease in the median fiber weight, or the fiber weight

required to evoke a hindpaw withdrawal with a 50% frequency (FW50) relative to vehicle-

treated controls. Administration of 10 mg/kg BCTC significantly increased the log FW50

at 2 hours post-administration (p <0.01, Fig. 5B). Administration of 100 mg/kg

gabapentin (i.p.) also significantly increased the log FW50 in nerve-injured rats 2 hours

post-administration (p < 0.01 Fig. 5B).

Side effect profiling: ataxia. A common side effect of compounds used to treat

neuropathic pain is ataxia, which can confound the interpretation of behavioral assays.

A role for VR1 in motor function has not been described, however we tested rats for

motor function using the rotarod assay. BCTC did not affect rotarod performance at 2 or

4 h following administration at doses up to 50 mg/kg, whereas gabapentin (100 mg/kg,

i.p.) produced in a significant decrease in rotarod performance 2 hours post-

administration (p < 0.05, Fig. 6).


at ASPE



ownloaded from


JPET #46268 14

DISCUSSION

VR1 has been extensively investigated as an intriguing target for the pharmacotherapy

of pain. Its expression is predominantly restricted to nociceptive neurons, and it is

activated by pathological rather than physiological stimuli, including well-known

nociceptive stimuli such as acid, temperatures above 48o C, and the pungent spice

capsaicin. Here we report that systemic administration of the novel VR1 antagonist,

BCTC, significantly reversed mechanical and thermal hyperalgesia associated with

injection of capsaicin, with FCA-induced inflammation, or following nerve injury. These

effects were seen in the absence of ataxia that is commonly associated with anti-

epileptic drugs used to treat neuropathic pain.

The VR1 antagonist capsazepine has also been used in many studies to investigate the

role of VR1 in acute and chronic nociception. However, recent studies have revealed

species-specific pharmacological actions of capsazepine that may limit its applicability in

some animal models (McIntyre et al., 2001; Savidge et al., 2002; Walker et al., 2002).

Although capsazepine can inhibit activation of rat VR1 by capsaicin, Valenzano et al.,

(accompanying manuscript) found it to be ineffective at blocking acid-mediated VR1

currents, and is similarly ineffective at reversing pain behaviors associated with

inflammation in rats (Walker et al., 2002). However, the pharmacological profile of

capsazepine is markedly different in guinea pigs compared with rats. Capsazepine is

effective at inhibiting both the acid- and capsaicin-induced VR1 currents at guinea pig

VR1 (Savidge et al., 2002), and has been shown to reduce inflammatory and nerve-

injury related pain in guinea pigs (Walker et al., 2002). Thus, the species-dependent

activity of capsazepine to inhibit low pH-induced currents at VR1 correlates with its

ability to reduce hyperalgesia in models of chronic pain.


at ASPE



ownloaded from


JPET #46268 15

In contrast to capsazepine, BCTC inhibits the activation of rat VR1 by either capsaicin or

low pH (Valenzano et al., accompanying manuscript). The unique pharmacological

profile for BCTC at the rat VR1 provided us with an opportunity to test the hypothesis

that the effects of VR1 blockers in chronic pain models may be predicted by their ability

to inhibit low pH-induced activation of VR1. We found that pretreatment with BCTC

effectively inhibits the VR1 agonist effects of capsaicin in the rat in vivo (Fig. 1). These

results were consistent with the findings of Valenzano, et al., (accompanying

manuscript) who demonstrated using an ex-vivo rat skin-nerve preparation that BCTC

completely inhibited the activation of nociceptors by capsaicin.

However the most striking findings of this study were those indicating that BCTC is

effective in reversing established thermal and mechanical hyperalgesia associated with

inflammation (Figs. 2 and 3) or nerve injury (Figs. 4 and 5). The effects of BCTC were

restricted to the hyperalgesia associated with inflammation or injury and do not reflect an

acute analgesic activity as PWT from the non-injured hind paw were not affected by

systemic BCTC administration (data not shown). Likewise, BCTC did not produce any

impairment of locomotor activity over and above the dose range that produced anti-

hyperalgesic effects (Fig. 6). These findings are interesting in light of the recent study by

Walker et al. (2002) indicating that systemic administration of the VR1 antagonist

capsazepine effectively reverses mechanical hyperalgesia associated with inflammation

or nerve injury in the guinea pig but was ineffective in rat models of chronic pain. These

differences are reflected by the different molecular pharmacological profiles of

capsazepine vs. BCTC at rat VR1, in vitro. Valenzano et al., (accompanying manuscript)

have demonstrated that unlike capsazepine, BCTC potently antagonizes low pH-induced

activation of recombinant or native rat VR1. This suggests that the in vivo efficacy of

VR1 antagonists in models of chronic pain may be predicted by their ability to inhibit pH-


at ASPE



ownloaded from


JPET #46268 16

induced activation of VR1. Since both capsazepine and BCTC antagonize capsaicin-

induced activation of VR1, in vitro and in vivo, this action appears to be less predictive of

a VR1 antagonist’s efficacy in chronic pain states.

We have found that the VR1 antagonist, BCTC (3-30 mg/kg, p.o.), effectively reverses

both thermal and mechanical hyperalgesia associated with inflammation in the rat (Figs.

2 and 3). However, whereas Walker et al., (2002) reported that capsazepine was weakly

active against thermal hyperalgesia associated with inflammation in the guinea pig, we

found that VR1 blockade via BCTC provided effective reversal of inflammatory

hyperalgesia in the rat. Although this may indicate further differences in the molecular

pharmacology of BCTC vs. capsazepine at VR1, we cannot rule out the possibility that

different behavioral phenotypes make the measurement of thermal hyperalgesia more

robust in the rat as opposed to the guinea pig. Regardless the explanation, we know that

the hyperalgesic effects of heat cannot be explained by the action of VR1 alone, since

mice lacking VR1 retain the sensation of noxious heat (Davis et al., 2000; Caterina et al.,

2000) and two VR1 homologues, VRL-1 (TRPV2) and TRPV3 have been reported to be

insensitive to capsaicin or protons but respond to either high or low threshold heat

stimulation (Caterina et al., 1999; Peier et al., 2002).

We have found that BCTC effectively reversed hypersensitivity to mechanical stimuli

following inflammation or nerve injury. Stimuli that activate VR1 have been shown to

induce mechanical hypersensitivity. To this end, intraplantar injection of either capsaicin

(Simone et al., 1987; Simone et al., 1989; Gilchrist et al., 1996) or hyaluronic acid

(Hamamoto et al., 1998) results in the development of mechanical hyperalgesia. In fact,

the mechanical hyperalgesia observed after capsaicin injection is more robust than the

thermal hyperalgesia both in terms of duration of action and the size of the area of


at ASPE



ownloaded from


JPET #46268 17

secondary hyperalgesia (Gilchrist et al., 1996). There is also evidence that the

mechanisms underlying the thermal and mechanical hyperalgesia following capsaicin

injection differ. Thermal hyperalgesia is thought to be due to sensitization of nociceptors

because it is restricted to the immediate area of injection, whereas central mechanisms

may underlie the mechanical hyperalgesia as the sensitivity is seen in a larger area

surrounding the site of injection (Simone et al., 1987; Simone et al., 1989; LaMotte et al.,

1991). It has been shown that intradermal capsaicin injection facilitates the responses of

dorsal horn neurons due to input of low threshold mechanoreceptors and nociceptors

(LaMotte et al., 1991; Simone et al., 1991). In inflamed tissue, local pH decreases may

ultimately activate VR1, sensitizing nociceptors and low threshold mechanoreceptors,

decreasing the threshold at which mechanical stimuli result in the detection of noxious

stimuli.

The ability of BCTC to reduce tactile allodynia and mechanical hyperalgesia following

partial ligation of the sciatic nerve may be attributed, in part, to altered expression of

VR1 in this nerve injury model. Hudson and colleagues (2001) recently demonstrated

that partial ligation of the sciatic nerve results in decreased VR1 immunoreactivity in

damaged (ligated) neurons, but increased VR1 immunoreactivity in undamaged

neurons. In another rat model of neuropathy, tight ligation of the L5 spinal nerve

resulted in a significant decrease of VR1-immunoreactivity in the injured L5 dorsal root

ganglion (DRG) neurons, with a concomitant increase in VR1-immunoreactivity in the

uninjured L4 DRG neurons. Interestingly, the increased VR1-immunoreactivity was

present not only in C fibers, but also in myelinated A fibers, as well, which could also

explain the ability of BCTC to block mechanical sensitivity following nerve injury (Hudson

et al., 2002). The increased levels of VR1 may then also prime the sensory neurons to

respond to other physiological consequences of nerve damage that occur post injury,


at ASPE



ownloaded from


JPET #46268 18

such as the release of inflammatory mediators from macrophages during Wallerian

degeneration (Tracey and Walker, 1995).

The findings reported here provide strong support for a role for VR1 in the pathology of

chronic pain, both of inflammatory and neuropathic origin. Moreover, the in vitro

characterization of BCTC (Valenzano et al., accompanying manuscript) indicates that

the ability of VR1 antagonists to block low pH-induced channel opening may be a key

component in predicting the in vivo efficacy of VR1 antagonists in models of chronic

pain.


at ASPE



ownloaded from


JPET #46268 19

ACKNOWLEDGEMENTS

The authors would like to thank Dan Waligora for his assistance with small

animal surgery.


at ASPE



ownloaded from


JPET #46268 20

REFERENCES

Auberson S, Lacroix JS and Lundberg JM (2000) Modulation of capsaicin-sensitive

nerve activation by low pH solutions in guinea-pig lung. Pharmacol & Toxicol. 86:16-23.

Bevan S, Hothi S, Hughes G, James IF, Rang HP, Shah K, Walpole CS and Yeats JC

(1992a) Capsazepine: a competitive antagonist of the sensory neurone excitant

capsaicin. Br J Pharmacol. 107:544-552.

Bevan S, Rang H and Shah K (1992b) Capsazepine does not block the proton-induced

activation of rat sensory neurones. Br J Pharmacol. 107:235P.

Carpenter SE and Lynn B (1981) Vascular and sensory responses of human skin to mild

injury after topical treatment with capsaicin. Br J Pharmacol. 73:755-758.

Caterina MJ, Leffler A, Malmberg AB, Martin WJ, Trafton J, Petersen-Zeitz KR,

Koltzenburg M, Basbaum AI and Julius D (2000) Impaired nociception and pain

sensation in mice lacking the capsaicin receptor. Science. 288: 306-313.

Caterina MJ, Rosen TA, Tominaga M, Brake AJ and Julius D (1999) A capsaicin-

receptor homologue with a high threshold for noxious heat. Nature. 398:436-441.

Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD and Julius D (1997)

The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature.

389:816-824.


at ASPE



ownloaded from


JPET #46268 21

Cholewinski A, Burgess GM and Bevan S (1993) The role of calcium in capsaicin-

induced desensitization in rat cultured dorsal root ganglion neurons. Neuroscience.

55:1015-1023.

Davis JB, Gray J, Gunthorpe MJ, Hatcher JP, Davey PT, Overend P, Harries MH,

Latcham J, Clapham C, Atkinson K, Hughes SA, Rance K, Grau E, Harper AJ, Pugh PL,

Rogers DC, Bingham S, Randall A and Sheardown SA (2000) Vanilloid receptor 1 is

essential for inflammatory thermal hyperalgesia. Nature. 405:183-187.

Ellis JL and Undem BJ (1994) Inhibition by capsazepine of resiniferatoxin- and

capsaicin-induced contractions of guinea pig trachea. J Pharmacol Exp Ther. 268:85-89.

Fox AJ, Urban L, Barnes PJ and Dray A (1995) Effects of capsazepine against

capsaicin- and proton-evoked excitation of single airway C-fibres and vagus nerve from

the guinea-pig. Neuroscience. 67:741-752.

Gilchrist HD, Allard BL and Simone DA (1996) Enhanced withdrawal responses to heat

and mechanical stimuli following intraplantar injection of capsaicin in rats. Pain 67:179-

188.

Hamamoto DT, Ortiz-Gonzalez XR, Honda JM and Kajander KC (1998) Intraplantar

injection of hyaluronic acid at low pH into the rat hindpaw produces tissue acidosis and

enhances withdrawal responses to mechanical stimuli. Pain. 74:225-234.


at ASPE



ownloaded from


JPET #46268 22

Hargreaves K, Dubner R, Brown F, Flores C and Joris J (1988) A new and sensitive

method for measuring thermal nociception in cutaneous hyperalgesia. Pain. 32:77-88.

Hayes P, Meadows HJ, Gunthorpe MJ, Harries MH, Duckworth DM, Cairns W, Harrison

DC, Clarke CE, Ellington K, Prinjha RK, Barton AJ, Medhurst AD, Smith GD, Topp S,

Murdock P, Sanger GJ, Terrett J, Jenkins O, Benham CD, Randall AD, Gloger IS and

Davis JB (2000) Cloning and functional expression of a human orthologue of rat vanilloid

receptor-1. Pain. 88:205-215.

Hudson LJ, Bevan S, McNair K, Gentry C, Fox A, Kuhn R and Winter J (2002)

Metabotropic glutamate receptor 5 upregulation in A-fibers after spinal nerve injury: 2-

methyl-6-(phenylethynyl)-pyridine (MPEP) reverses the induced thermal hyperalgesia. J

Neurosci. 22:2660-2668.

Hudson LJ, Bevan S, Wotherspoon G, Gentry C, Fox A and Winter J (2001) VR1 protein

expression increases in undamaged DRG neurons after partial nerve injury. Eur J

Neurosci. 13:2105-2114.

Hwang SW, Cho H, Kwak J, Lee SY, Kang CJ, Jung J, Cho S, Min KH, Suh YG, Kim D

and Oh U (2000) Direct activation of capsaicin receptors by products of lipoxygenases:

endogenous capsaicin-like substances. Proc Natl Acad Sci USA. 97:6155-6160.

Jerman JC, Brough SJ, Prinjha R, Harries MH, Davis JB and Smart D (2000)

Characterization using FLIPR of rat vanilloid receptor (rVR1) pharmacology. Br J

Pharmacol. 130:916-922.


at ASPE



ownloaded from


JPET #46268 23

LaMotte RH, Shain CN, Simone DA and Tsai EF (1991) Neurogenic hyperalgesia:

psychophysical studies of underlying mechanisms. J Neurophys. 66:190-211.

Lou YP and Lundberg JM (1992) Inhibition of low pH evoked activation of airway

sensory nerves by capsazepine, a novel capsaicin-receptor antagonist. Biochem

Biophys Res Comm. 189:537-544.

Maggi CA, Bevan S, Walpole CS, Rang HP and Giuliani S (1993) A comparison of

capsazepine and ruthenium red as capsaicin antagonists in the rat isolated urinary

bladder and vas deferens. Br J Pharmacol. 108:801-805.

McIntyre P, McLatchie LM, Chambers A, Phillips E, Clarke M, Savidge J, Toms C,

Peacock M, Shah K, Winter J, Weerasakera N, Webb M, Rang HP, Bevan S and James

IF (2001) Pharmacological differences between the human and rat vanilloid receptor 1

(VR1). Br J Pharmacol. 132:1084-1094.

Peier AM, Reeve AJ, Andersson DA, Moqrich A, Earley TJ, Hergarden AC, Story GM,

Colley S, Hogenesch JB, McIntyre P, Bevan S and Patapoutian A (2002) A heat-

sensitive TRP channel expressed in karatinocytes. Science. 296:2046-2049.

Santicioli P, Del Bianco E, Figini M, Bevan S and Maggi CA (1993) Effect of

capsazepine on the release of calcitonin gene-related peptide-like immunoreactivity

(CGRP-LI) induced by low pH, capsaicin and potassium in rat soleus muscle. Br J

Pharmacol. 110:609-612.


at ASPE



ownloaded from


JPET #46268 24

Santos AR and Calixto JB (1997) Ruthenium red and capsazepine antinociceptive effect

in formalin and capsaicin models of pain in mice. Neurosci Lett. 235:73-76.

Satoh H, Lou YP and Lundberg JM (1993) Inhibitory effects of capsazepine and SR

48968 on citric acid-induced bronchoconstriction in guinea-pigs. Eur J Pharmacol.

236:367-372.

Savidge J, Davis C, Shah K, Colley S, Phillips E, Ranasinghe S, Winter J, Kotsonis P,

Rang H and McIntyre P (2002) Cloning and functional characterization of the guinea-pig

vanilloid reeptor 1. Neuropharmacology 43:450-456.

Seltzer Z, Dubner R and Shir Y (1990) A novel behavioral model of neuropathic pain

disorders produced in rats by partial sciatic nerve injury. Pain. 43:205-218.

Simone DA, Baumann TK and LaMotte RH (1989) Dose-dependent pain and

mechanical hyperalgesia in humans after intradermal injection of capsaicin. Pain. 38:99-

107.

Simone DA, Ngeow JY, Putterman GJ and LaMotte RH (1987) Hyperalgesia to heat

after intradermal injection of capsaicin. Brain Res. 418:201-203.

Simone DA, Sorkin LS, Oh U, Chung JM, Owens C, LaMotte RH and Willis WD (1991)

Neurogenic hyperalgesia: central neural correlates in responses of spinothalamic tract

neurons. J Neurophys. 66:228-246.


at ASPE



ownloaded from


JPET #46268 25

Smart D, Gunthorpe MJ, Jerman JC, Nasir S, Gray J, Muir AI, Chambers JK, Randall AD

and Davis JB (2000) The endogenous lipid anandamide is a full agonist at the human

vanilloid receptor (hVR1). Br J Pharmacol. 129:227-230.

Szolcsanyi J (1977) A pharmacological approach to elucidation of the role of different

nerve fibres and receptor endings in mediation of pain. Journal de Physiologie. 73:251-

259.

Tominaga M, Caterina MJ, Malmberg AB, Rosen TA, Gilbert H, Skinner K, Raumann

BE, Basbaum AI and Julius D (1998) The cloned capsaicin receptor integrates multiple

pain-producing stimuli. Neuron. 21:531-543.

Tracey DJ and Walker JS (1995) Pain due to nerve damage: are inflammatory mediators

involved? Inflamm Res. 44:407-411.

Urban L and Dray A (1991) Capsazepine, a novel capsaicin antagonist, selectively

antagonises the effects of capsaicin in the mouse spinal cord in vitro. Neurosci Lett.

134:9-11.

Valenzano KJ, Grant E, Hachicha M, Schmid L, Limberis J, Tafesse L, Q S, Rotshteyn

Y, Whittemore E and Hodges D (accompanying manuscript) BCTC (N-(4-

tertiarybutylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carbox-amide), a

novel, orally-effective vanilloid receptor 1 antagonist with analgesic properties: 1. In vitro

characterization and pharmacokinetic properties. Submitted J Pharmacol Exp Ther

#045674.


at ASPE



ownloaded from


JPET #46268 26

Walker K, Bowes M, Panesar M, Davis A, Gentry C, Kesingland A, Gasparini F, Spooren

W, Stoehr N, Pagano A, Flor PJ, Vranesic I, Lingenhoehl K, Johnson EC, Varney M,

Urban L and Kuhn R (2001) Metabotropic glutamate receptor subtype 5 (mGlu5) and

nociceptive function. I. Selective blockade of mGlu5 receptors in models of acute,

persistent and chronic pain. Neuropharmacology. 40:1-9.

Walker K, Urban L, Medhurst SJ, Patel S, Panesar M, Fox AJ and McIntyre P (2003)

The VR1 antagonist, capsazepine, reverses mechanical hyperalgesia in models of

inflammatory and neuropathic pain. J Pharmacol Exp Ther. 304: 56-62.


at ASPE



ownloaded from


JPET #46268 27

LEGENDS FOR FIGURES

Figure 1. Pretreatment with BCTC dose-dependently inhibits capsaicin-induced

mechanical hyperalgesia. Rats received oral administration of beta-cyclodextrin vehicle

(V) or BCTC 30 minutes prior to an intraplantar injection of 30 µg capsaicin or

saline/Tween vehicle (V). Administration of 30 mg/kg BCTC significantly reduced

capsaicin-induced mechanical hyperalgesia 90 min post-capsaicin injection. Asterisks

denote significance (P < 0.05) from capsaicin (CAP)/V group according to Fisher’s PLSD

test, error bars represent s.e.m (n = 9-10 rats/group).

Figure 2. BCTC dose-dependently inhibits inflammatory thermal hyperalgesia. Rats

received an intraplantar injection of 50 µL saline (open squares) or 100% FCA (all other

groups), followed 24 hours later by oral administration of BCTC, indomethacin or vehicle.

Asterisks denote significance (P < 0.05) from FCA/vehicle group according to Fisher’s

PLSD test, error bars represent s.e.m (n = 8 rats/group).

Figure 3. BCTC dose-dependently reverses inflammatory mechanical hyperalgesia.

Rats received an intraplantar injection of 50 µL saline (open squares) or 100% FCA (all

other groups), followed 24 hours later by oral administration of BCTC, indomethacin, or

vehicle. Asterisks denote significance (P < 0.05) from FCA/vehicle group according to

Fisher’s PLSD test, error bars represent s.e.m (n = 10-20 rats/group).

Figure 4. BCTC dose-dependently reverses mechanical hyperalgesia associated with

nerve injury. Rats underwent surgery involving partial ligation of the sciatic nerve. 14-17


at ASPE



ownloaded from


JPET #46268 28

days post-surgery, rats received oral administration of vehicle or BCTC, or

intraperitoneal administration of gabapentin (100 mg/kg, open circles). Asterisks denote

significance (P < 0.05) from vehicle-treated control group according to Fisher’s PLSD

test, error bars represent s.e.m (n = 10-20 rats/group.

Figure 5. BCTC significantly reduces tactile allodynia associated with nerve injury.

Rats underwent surgery involving partial ligation of the sciatic nerve. Rats were tested

for tactile allodynia 14-17 days post-surgery by application of von Frey monofilaments

prior to (A), and 2 hours (B) post-administration of vehicle, BCTC, or intraperitoneal

administration of gabapentin (100 mg/kg, open circles). N = 10 rats/group.

Figure 6. BCTC has no effect on motor performance as measured using the rotarod

assay. Rats were placed on a rotarod set at a fixed speed of 30 rpm and the latency to

fall off the rotarod was recorded 2 and 4 hours post-administration of vehicle, BCTC

(p.o.), or 100 mg/kg gabapentin (i.p.). Asterisks denote significance (P < 0.05) from

vehicle-treated group, error bars represent s.e.m. N = 8 rats/group.


at ASPE



ownloaded from


Figure 1

V/V CAP/V 1 3 10 300

100

200

Paw

Wit

hdr

awal

Thr

esho

ld (

g)

BCTC (mg/kg)

*

This article has not been copyedited and form

atted. The final version m

ay differ from this version.

JPET

Fast Forward. Published on A

pril 29, 2003 as DO

I: 10.1124/jpet.102.046268 at ASPET Journals on July 8, 2020 jpet.aspetjournals.org Downloaded from


Figure 2

10

20

5

ShamVehicle1 mg/kg BCTC3 mg/kg BCTC10 mg/kg BCTC30 mg/kg Indomethacin

*** *

*

Pre-FCA 2 4

Time Post-Administration (hours)

Paw

Wit

hd

raw

alL

aten

cy (

seco

nds)




JPET


pril 29, 2003 as DO



Figure 3

100

150

ShamVehicle1 mg/kg BCTC3 mg/kg BCTC10 mg/kg BCTC30 mg/kg BCTC30 mg/kg Indomethacin

0 2 4 6 24

**

*

****

*

*


Paw

Wit

hdr

awal

Thr

esho

ld (

gram

s)




JPET


pril 29, 2003 as DO



Figure 4

100

150ShamVehicle1 mg/kg BCTC3 mg/kg BCTC10 mg/kg BCTC30 mg/kg BCTC100 mg/kg Gabapentin

0 2 4 6 24

** *

*

*

**


Paw

Wit

hdr

awal

Thre

shol

d (g

ram

s)




JPET


pril 29, 2003 as DO



Figure 5

0 1 10 1000

20

40

60

80

100APre-dose

Fiber Weight (grams)

Res

po

nse

Fre

qu

ency

(%)

0 1 10 1000

20

40

60

80

100Vehicle3 mg/kg BCTC10 mg/kg BCTC30 mg/kg BCTC100 mg/kg GabapentinSham

B2 hrs post-dose

Fiber Weight (grams)

Res

pon

se F

requ

ency

(%)




JPET


pril 29, 2003 as DO



Figure 6

0 2 40

50

100

150Vehicle3 mg/kg BCTC10 mg/kg BCTC50 mg/kg BCTC100 mg/kg Gabapentin

*

Time Post-Administration (hrs)

Lat

ency

(se

con

ds)




JPET


pril 29, 2003 as DO



BCTC (N-(4-tertiarybutylphenyl)-4-(3-cholorphyridin-2...

Documents

Transcript of BCTC (N-(4-tertiarybutylphenyl)-4-(3-cholorphyridin-2...