Crotalphine induces potent antinociception in neuropathic pain by acting at peripheral opioid receptors

European Journal of Pharmacology 594 (2008) 84–92

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European Journal of Pharmacology

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Crotalphine induces potent antinociception in neuropathic pain by acting atperipheral opioid receptors

Vanessa Pacciari Gutierrez a, Katsuhiro Konno b,1, Marucia Chacur c, Sandra Coccuzzo Sampaio a,Gisele Picolo a, Patricia Brigatte a,2, Vanessa Olzon Zambelli a, Yara Cury a,⁎a Laboratory of Pathophysiology, Butantan Institute, Av. Vital Brazil, 1500, 05503-900 Sao Paulo-SP, Brazilb Center for Applied Toxinology, Av. Vital Brazil, 1500, 05503-900 Sao Paulo-SP, Brazilc Department of Physiology and Biophysics, Biomedical Science Institute, University of Sao Paulo, Av. Prof. Lineu Prestes, 1524, 05508-900 Sao Paulo-SP, Brazil

⁎ Corresponding author. Laboratório de FisiopatologiVital Brazil, 1500, 05503-900, São Paulo, SP, Brazil. Tel.:3726 1505.

E-mail address: [email protected] (Y. Cury).1 Present address: Institute of Natural Medicine, Unive

Toyama-shi, Toyama 930-0194, Japan.2 Present address: Department of Physiology and B

Institute, University of Sao Paulo, Av. Prof. Lineu Prestes,Brazil.

0014-2999/$ – see front matter © 2008 Elsevier B.V. Aldoi:10.1016/j.ejphar.2008.07.053

a b s t r a c t

Article history:

Keywords:CrotalphineAntinociceptionNeuropathic painOpioid receptorsMorphineGabapentin

rtant clinical problem and it is usually resistant to the current therapy. We haverecently characterized a novel analgesic peptide, crotalphine, from the venom of the South Americanrattlesnake Crotalus durissus terrificus. In the present work, the antinociceptive effect of crotalphine wasevaluated in an experimental model of neuropathic pain induced in rats by chronic constriction of sciaticnerve. The effect of the peptide was compared to that induced by the crude venom, which confirmed thatcrotalphine is responsible for the antinociceptive effect of the crotalid venom on neuropathic pain. Forcharacterization of neuropathic pain, the presence of hyperalgesia, allodynia and spontaneous pain wasassessed at different times after nerve constriction. These phenomena were detected 24 h after surgery andpersisted at least for 14 days. The pharmacological treatments were performed on day 14 after surgery.Crotalphine (0.2–5 µg/kg) and the crude venom (400–1600 µg/kg) administered p.o. inhibited hyperalgesia,allodynia and spontaneous pain induced by nerve constriction. The antinociceptive effect of the peptide andcrude venom was long lasting, since it was detected up to 3 days after treatment. Intraplantar injection ofnaloxone (1 µg/paw) blocked the antinociceptive effect, indicating the involvement of opioid receptors in thisphenomenon. Gabapentin (200 mg/kg, p.o.), and morphine (5 mg/kg, s.c.), used as positive controls, blockedhyperalgesia and partially inhibited allodynia induced by nerve constriction. These data indicate thatcrotalphine induces a potent and long lasting opioid antinociceptive effect in neuropathic pain that surpassesthat observed with standard analgesic drugs.

© 2008 Elsevier B.V. All rights reserved.

1. Introduction

Chronic neuropathic pain is the result of disease or damage to theperipheral or central nervous system (Taylor, 2006). The major clinicalsymptoms of neuropathic pain are hyperalgesia, allodynia andspontaneous ongoing pain (Dworkin et al., 2003). Treatment of thiscondition involves many therapeutic approaches, including the use ofanalgesic drugs such as opioids, mainly morphine, gabapentin andanti-inflammatory drugs (Caviedes and Herranz, 2002; Dworkin et al.,2003; Romero-Sandoval et al., 2007). However, the therapeutic

a, Instituto Butantan, Avenida+55 11 3726 7222; fax: +55 11

rsity of Toyama, 2630 Sugitani,

iophysics, Biomedical Science1524, 05508-900 Sao Paulo-SP ,

l rights reserved.

approaches are not completely effective in relieving neuropathicpain, leading to an impairment of patients' life quality (Meyer-Rosberget al., 2001). The mechanisms involved in neuropathic pain are stillunclear, although the understanding of neuropathic pain has beenaccelerated by the use of animal models, such as the chronicconstriction injury, the spinal nerve ligation, the partial sciaticligation, the tibial and sural transection, and the complete sciatictransection models, which reproduce some of the symptoms ofhuman neuropathic pain (Dowdall et al., 2005).

Several lines of evidence have indicated that the venom of the SouthAmerican rattlesnake Crotalus durissus terrificus induces a long-lastingantinociceptive effect mediated by activation of κ- and δ-opioidreceptors (Brigatte et al., 2001; Giorgi et al., 1993; Picolo and Cury,2004; Picolo et al., 2000). We have recently isolated and characterized,from the crude crotalid venom, a novel peptide, named crotalphine thatdisplays antinociceptive activity. Crotalphine, administered in lowdosesby oral, i.v. or intraplantar routes, induces antinociceptive effect in theprostaglandin E2- and carrageenin-induced mechanical hyperalgesiamodels in rats and in the hot-plate test in mice. In the mechanicalhyperalgesia models, crotalphine shows a long-lasting (5 days)

Fig. 1. Hyperalgesia, allodynia and spontaneous pain induced by chronic constrictioninjury of sciatic nerve. For induction of chronic constriction injury, the rat commonsciatic nerve was exposed and 4 ligatures were tied loosely around it. In sham-operated(Sham) rats the sciatic nerve was exposed but left unaffected. Pain threshold wasestimated in the rat paw pressure test (A) and low-threshold mechanical allodynia wasmeasured using the von Frey test (B). Spontaneous pain was assessed by cumulativeduration (in seconds, sec) of licking (C) and lifting (D) of the left (Operated) and right(control) hind paws, during 10 min. The tests were applied before and on days 1, 7 and14 after surgery in the left sciatic nerve or right paw (control). Data represent meanvalues±S.E.M. for 6 rats. ⁎Significantly different from mean values obtained before(Time 0) surgery (Pb0.05).

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antinociceptive effect mediated by activation of peripheral κ-opioidreceptors (Konno et al., 2008). These results indicate that this peptidemay contribute to the antinociceptive effect of the crotalid venom.

Despite the demonstration that crotalphine presents high biolo-gical stability, being orally active in low doses and inducing a longlasting antinociceptive effect, which emphasize its therapeuticpotential, the effect of this peptide in models of chronic pain has notbeen determined yet. The present work was undertaken to character-ize the antinociceptive effect of crotalphine in a rat model ofneuropathic pain induced by chronic constriction of the sciatic nerve.

2. Materials and methods

2.1. Animals

Male Wistar rats (170–190 g) were used throughout this study.Animals were housed in a temperature-controlled (21±2 °C) andlight-controlled (12/12 h light/dark cycle) room. All behavioural testswere performed between 9:00 am and 4:00 pm. Standard food andwater were available ad libitum until 2 h before crotalphineadministration. After this period, only water was available. Theanimals were kept deprived of food for a period of no longer than5 h. All procedures were in accordance with the guidelines for theethical use of conscious animals in pain research published by theInternational Association for the Study of Pain (Zimmermann, 1983)and were approved by the Institutional Animal Care Committee of theButantan Institute (CEUAIB, protocol number 098/2002).

2.2. Peptide synthesis

The peptide crotalphine (bE-F-S-P-E-N-C-Q-G-E-S-Q-P-C, wherebE is pyroglutamic acid and the two cysteine residues forming adisulfide bond) was manually synthesized using H-Cys(Trt)-2-ClTrtresin (Novabiochem) as a carrier with Fmoc chemistry in the solidphase, as previously described (Konno et al., 2008). The synthesizedcrotalphine was stored at −20 °C until use.

2.3. Crotalus durissus terrificus venom

Lyophilized venom of C. durissus terrificus was obtained from theLaboratory of Herpetology, Instituto Butantan, São Paulo, Brazil, andstored at −20 °C.

2.4. Chronic constriction injury

For induction of neuropathic pain, chronic constriction of the sciaticnerve was performed as described by Bennett and Xie (1988). Ratswere anesthetized with halothane. The common sciatic nerve wasexposed at the level of the middle of the thigh by blunt dissectionthrough biceps femoris. Proximal to the sciatic's trifurcation (about7 mm), the nerve was freed of adhering tissue and 4 ligatures (4.0chromic gut) were tied loosely around it with about 1 mm spacing.Great care was taken to tie the ligatures, such that the diameter of thenerve was seen to be just barely constricted. The incisionwas closed inlayers. In sham-operated rats the sciatic nerve was exposed but leftunaffected. For characterization of neuropathic pain, the developmentof hyperalgesia, allodynia and spontaneous pain (gross behaviour)were evaluated on days 1, 7, and 14 after surgery.

2.5. Evaluation of mechanical hyperalgesia

The rat paw pressure test (Randall and Selitto, 1957) was used fordetermination of hyperalgesia. An Ugo-Basile pressure apparatus wasused to assess pressure pain thresholds prior to nerve ligation andagain at different periods of time later on. Testing was blind in regardto group designation. Briefly, increasing force (in g, 16 g/s) was applied

to the hind paw. The force needed to induce paw withdrawal wasrecorded as the pain threshold. To reduce stress, the rats werehabituated to the testing procedure the day before the experiment.

2.6. Evaluation of low threshold mechanical allodynia

The von Frey test (Chaplan et al., 1994) was used to assess low-thresholdmechanical pain thresholds prior to nerve ligation and againat different periods of time later on. Testing was blind in regard to

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group designation. This test was performed as previously described indetail, using the modified up-down method (Milligan et al., 2000).Briefly, a logarithmic series of 10 calibrated Semmes-Weinsteinmonofilaments (von Frey hairs, Stoelting, Wood Dale, IL, USA) wasapplied to the right hind paw to determine the stimulus intensitythreshold stiffness required to elicit a paw withdrawal response. Logstiffness of the hairs is determined by log10 (mg×10) and ranged from3.61 (407 mg) to 5.18 (15,136 mg). Basal line assessment was initiatedwith the 2041mg hair. In the event of a pawwithdrawal, the same hairwas again presented 30–60 s later. If the response was again elicited,the 407 mg monofilament was presented. In the absence of a pawwithdrawal response to the 407 mg stimulus, the next strongermonofilament was presented (692 mg). The monofilament thatelicited a clear response was recorded, and was presented onceagain 30–60 s later. If the animal withdrew his paw on twoconsecutive trials with the same stiffness value, no further von Freyhairs were tested. However, in the absence of a response to the initial2041 mg monofilament, presentation of monofilaments continued inascending order until two consecutive responses were elicited fromthe same monofilament. All single responses were recorded, butassessment was complete only after two consecutive responses wereelicited from the same monofilament. In instances when rats failed torespond to the strongest stimulus (15,136 mg), it was considered thecut-off value. Responses that occurred to the weakest stimulus(407 mg) were assigned the lower cut-off value for that time point.To reduce stress, rats were habituated to the experimental environ-ment each of four days before experiments. Behavioural responseswere used to calculate the 50% paw withdrawal threshold (absolutethreshold), by fitting a Gaussian integral psychometric function usinga maximum-likelihood fitting method (Harvey, 1986; Milligan et al.,2000, 2001, 2003; Treutwein and Strasburger, 1999). This fittingmethod allows parametric analyses (Harvey, 1986; Milligan et al.,2000, 2001, 2003; Treutwein and Strasburger, 1999).

2.7. Evaluation of gross behaviour

Ratswere observed for signs of spontaneous pain before and on days1, 7 and 14 after nerve constriction. For observation of signs ofspontaneous pain, ratswere placed on a glass floor at room temperature(21±1 °C). After an acclimatization period of 30 min, animals wereobserved for 10 min and the cumulative duration of the lifting andlickingof thehind limbswasmeasured. The liftingand lickingof thepawas a part of grooming behaviour was not taken into account.

2.8. Evaluation of general motor activity

Possible changes in general activity induced by crotalphine wereinvestigated in an open-field arena (Broadhurst,1960). Hand-operatedcounters were used to score ambulation (locomotion) frequency(number of floor units entered) and rearing frequency (number oftimes the animal stood on hind legs). Each animal was individuallyplaced in the centre of the open field and behavioural parameterswere recorded for 3 min. The open field was washed with water–alcohol (5%) before the animals were placed in it to avoid possiblebiasing effects of odour clues left by previous subjects.

Fig. 2. Crotalphine inhibits hyperalgesia, allodynia and spontaneous pain induced bychronic constriction injury of the sciatic nerve. For induction of chronic constriction injury,the rat common sciatic nervewas exposed and 4 ligatureswere tied loosely around it. Painthreshold was estimated in the rat paw pressure test (A) and low-threshold mechanicalallodynia was measured using the von Frey test (B). Spontaneous pain was assessed bycumulative duration (in seconds, sec) of licking (C) and lifting (D) of thepaw, during10min.The tests were applied 14 days after surgery, before and at different times after oraladministration of crotalphine (C, 0.0016–5 µg/kg) or saline (control group). Dash linerepresents the mean values observed before surgery. Data represent mean values ±S.E.M.for 6 rats. ⁎Significantly different from mean values of control group; §Significantlydifferent from mean values obtained before treatments (Time 0); # Significantly differentfrom mean values obtained before surgery (dash line) (Pb0.05).

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2.9. Pharmacological treatments

In order to exclude the role of prostanoids in hyperalgesia andallodynia induced by nerve constriction, the cyclooxygenase inhibitorindomethacin and the cyclooxygenase-2 inhibitor celecoxib wereused. Both drugs were administered on day 14 after surgery.Indomethacin (Sigma Chem. Co., USA, 4 mg/kg, i.v.) or Tris (control)was administered directly into the tail vein of the rats 60 min beforenociceptive evaluation. Celecoxib (Sigma Chem. Co., USA, 10 mg/kg) orCMC 1% (Carboxi-methyl-cellulose, control) was administered by oralroute 120 min before nociceptive evaluation.

For evaluation of the antinociceptive activity, crotalphine wasdissolved in sterile saline and administered by oral (gastral cannula,2 ml) route on day 14 after surgery. The antinociceptive activity wasevaluated at different times after treatment and compared to thatinduced by the crude venom. Sterile saline was used as control.

In addition, standard analgesic drugs were also tested on day 14after surgery. Morphine (morphine sulfate, União Química, Brazil,5 mg/kg, s.c. or 6 µg/paw, intraplantar route), and gabapentin (SigmaChem. Co., USA, 200 mg/kg, p.o.) were injected 60 min beforenociceptive evaluation. Gabapentin and morphine were dissolved insterile saline. In these assays, sterile saline, administered by s.c., oral orintraplantar routes, was used as control. The doses of the drugs werebased on previous work of our group or on data from the literature(Chacur et al., 2003; Joshi et al., 2006; Nagakura et al., 2008).

To characterize the involvement of opioid receptors in theantinociceptive effect of crotalphine, naloxone, a non-specific opioidreceptor antagonist, were injected by intraplantar route (1 µg/paw)immediately after oral administration of crotalphine.

In all assays, testing was blind in regard to group designation.

2.10. Statistical analysis

Results are presented as mean±S.E.M. Statistical evaluation of datawas carried out by analysis of variance (ANOVA) and sequentialdifferences among means were compared according to Tukey contrastanalysis at Pb0.05 (Gad and Weil, 1989). Data from the von Frey testwere analyzed as the interpolated 50% threshold (absolute threshold)in log base 10 of stimulus intensity (monofilament stiffness inmilligrams×10).

3. Results

3.1. Evaluation of nociceptive behaviour induced by chronic constrictionof the sciatic nerve

The chronic constriction injury caused a significant decrease inpain threshold (Fig. 1A), as measured by the Randall and Sellito test,and lowered withdrawal thresholds as measured by the von Frey test(Fig. 1B). Mechanical hyperalgesia and low-threshold mechanicalallodynia were detected on day 1 after surgery and persisted at least,for 14 days. The contralateral intact paw did not show alterations inpain threshold (data not shown). Sham-operated rats did not presentalterations in threshold measurements as compared to basal values(Fig. 1A and B).

Fig. 3.^C. durissus terrificus venom inhibits hyperalgesia, allodynia and spontaneous pain

induced by chronic constriction injury of the sciatic nerve. For induction of chronicconstriction injury, the rat common sciatic nerve was exposed and 4 ligatures were tiedloosely around it. Pain threshold was estimated in the rat paw pressure test (A) and low-thresholdmechanical allodyniawasmeasured using thevon Frey test (B). Spontaneous painwas assessedbycumulativeduration (inseconds, sec) of licking (C) and lifting (D) of thepaw,during 10 min. The tests were applied 14 days after surgery, before and at different timesafter oral administration of venom (V, 100–1,600 µg/kg) or saline (control group). Dash linerepresents themeanvalues observed before surgery. Data representmeanvalues±S.E.M. for6 rats. ⁎Significantly different from mean values of control group; §Significantly differentfrommean values obtained before treatments (Time 0); # Significantly different frommeanvalues obtained before surgery (dash line) (Pb0.05).

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Sciatic nerve constriction also induced a significant increase inlicking (Fig. 1C) and lifting (Fig. 1D) of the ipsilateral paw, on days 1, 7,and 14 after surgery, as compared to the values observed in thecontralateral paw.

3.2. Effect of oral administration of crotalphine on hyperalgesia,allodynia and spontaneous pain induced by chronic constriction injury

Hyperalgesia (Fig. 2A) and allodynia (Fig. 2B) induced by nerveconstriction was inhibited by crotalphine (0.2 a 5 µg/kg) administeredp.o. on day 14 after surgery. The peptide caused graded inhibition ofboth phenomena at low doses (0.2 a 1 µg/kg), as well as an increase inmechanical threshold in the Randall and Selitto test, to values abovethose recorded at baseline (i.e. antinociception) at the higher dose(Fig. 2A). The results also demonstrated that crotalphine (5 µg/kg)blocked the increase in licking (Fig. 2C) and lifting (Fig. 2D) of thepaws. The antinociceptive effect of the peptide was detected for up to72 h after treatment (Fig. 2). The effect of crotalphinewas compared tothe effect induced by the crude venom. Oral administration of thecrotalid venom (200–1600 µg/kg) blocked mechanical hyperalgesia(Fig. 3A) and low-threshold mechanical allodynia (Fig. 3B) induced bychronic constriction injury and also induced (400 to 1600 µg/kg) anincrease in mechanical threshold to values above those recorded atbaseline. The results also demonstrated that the crotalid venom(800 µg/kg) blocked the increase in licking (Fig. 3C) and lifting(Fig. 3D) of the paws. The antinociceptive effect of the venomwas alsodetected for up to 72 h after treatment (Fig. 3).

Fig. 4. Effect of morphine, gabapentin, indomethacin and celecoxib on hyperalgesia andallodynia induced by chronic constriction injury of the sciatic nerve. For induction ofchronic constriction injury, the rat common sciatic nerve was exposed and 4 ligatureswere tied loosely around it. Pain threshold was estimated in the rat paw pressure test(A) and low-threshold mechanical allodynia was measured using the von Frey test (B).The tests were applied on day 14 after surgery. Morphine (5 mg/kg, s.c.), Gabapentin(200 mg/kg, p.o.), and Indomethacin (4 mg/kg, i.v.) were administered 60 min beforenociceptive evaluation, whereas Celecoxib (10 mg/kg, p.o.) was administered 120 minbefore the tests. Saline and Crotalphine (5 µg/kg, p.o., 60 min before nociceptiveevaluation) were used as controls. Dash line represents the mean values observedbefore surgery. Data represent mean values±S.E.M. for 6 rats. ⁎Significantly differentfrom mean values of saline group; # Significantly different from mean values obtainedbefore surgery (dash line) (Pb0.05).

Fig. 5. Peripheral opioid receptors mediate the antinociceptive effect of crotalphine onchronic constriction injury of the sciatic nerve. For induction of chronic constriction injury,the rat common sciatic nervewas exposed and 4 ligatureswere tied loosely around it. Painthreshold was estimated in the rat paw pressure test (A) and low-threshold mechanicalallodynia wasmeasured using the von Frey test (B). The tests were applied on day 14 aftersurgery. Crotalphine (5 µg/kg, p.o.) or saline (p.o.) was administered 60 min beforenociceptive evaluation. C. durissus terrificus venom (800 µg/k, p.o.) was used as positivecontrol. Naloxone (1 µg/paw) or saline (100 µl/paw) was injected by intraplantar routeimmediately before crotalphine or venom administration. Dash line represents the meanvalues observed before surgery. Data represent mean values±S.E.M. for 6 rats.⁎Significantly different from mean values of saline group; # Significantly different frommean values obtained before surgery (dash line) (Pb0.05).

In the present work, the effects of standard analgesic drugs werealso evaluated. First, to evaluate the involvement of inflammatorymediators, such as prostanoids, in the mechanical hyperalgesia andlow-threshold mechanical allodynia induced by nerve constriction,indomethacin (4 mg/kg, i.v.) and celecoxib (10 mg/kg, p.o.) wereadministered to the animals 14 days after surgery. Both antiinflam-matory drugs did not interfere with the nociceptive phenomena(Fig. 4A and B), indicating that alterations in pain threshold observedin this period of time after surgery were not due to the presence ofinflammation induced by the surgery. In addition, the effect ofgapapentin (200 mg/kg, p.o.), an anticonvulsant used for thetreatment of neuropathic pain, and morphine (5 mg/kg, s.c.), thestandard opioid drug, was also evaluated. Both drugs, administered onday 14 after surgery, blocked mechanical hyperalgesia (Fig. 4A) andpartially inhibited low-threshold mechanical allodynia (Fig. 4B)induced by nerve constriction, in contrast to crotalphine that blockedboth phenomena and also increased the pain threshold of the animalsin comparison to basal values (Fig. 4A and B).

3.3. Effect of oral administration of crotalphine on spontaneous motoractivity

As motor activity inhibition could interfere with the behaviour ofthe rats in the nociceptive test, the possible influence of crotalphine onspontaneousmotor activity was investigated in the Open Field, 60min

Fig. 6. Crotalphine induces peripheral (local) antinociceptive effect on chronicconstriction injury of the sciatic nerve. For induction of chronic constriction injury, therat common sciatic nerve was exposed and 4 ligatures were tied loosely around it. Painthreshold was estimated in the rat paw pressure test (A) and low-threshold mechanicalallodynia was measured using the von Frey test (B). The tests were applied on day 14after surgery. Crotalphine (0.0006 µg/paw) or saline (100 µl/paw, control) wasadministered by intraplantar route 60 min before nociceptive evaluation. Morphine(6 µg/paw) used as positive control, was injected 60 min before the assays. Dash linerepresents themeanvalues observed before surgery. Data representmeanvalues±S.E.M.for 6 rats. ⁎Significantly different from mean values of saline group; # Significantlydifferent from mean values obtained before surgery (dash line) (Pb0.05).

Fig. 7. Effect of prolonged treatment with crotalphine on its antinociceptive effect onchronic constriction injury of the sciatic nerve. For induction of chronic constrictioninjury, the rat common sciatic nerve was exposed and 4 ligatures were tied looselyaround it. Pain threshold was estimated in the rat paw pressure test (A) and low-threshold mechanical allodynia was measured using the von Frey test (B). Spontaneouspain was assessed by cumulative duration (in seconds, sec) of licking (C) and lifting (D)of the paw, during 10 min. Crotalphine (5 µg/kg, p.o.) or saline (control) wasadministered each 3 days, during 75 days. The administration of the peptide or salinestarted on day 14 after surgery. The results were compared to those obtained with crudevenom administered to the animals according to same protocol (800 µg/kg).Nociception was estimated on day 14, immediately before (Time 0) beginningtreatments and each 3 days, during 75 days, 60 min after crotalphine, venom or salineadministration. Data represent mean values±S.E.M. for 6 rats. ⁎Significantly differentfrom mean values of control group and from mean values obtained before treatments(Time 0).

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after treatment. Oral administration of the peptide (5 µg/kg) had noeffect on frequencies of locomotion (number of floor units entered bythe animals) (crotalphine=80±0.98 and saline=80±1.2) and rearing(number of times the animal stood on hind legs) (crotalphine=21±0.92 and saline=22±1).

The effect of morphine and gabapentin on the locomotor activity ofthe animals was also evaluated. Administration of morphine (5mg/kg)or gabapentin (200 mg/kg) had no effect on frequencies of locomotion(morphine=80±0.98; gabapentin=75±1.22)) and rearing (mor-phine=21±0.92; gabapentin=25±0.83).

3.4. Opioid receptors are involved in the antinociceptive effect ofcrotalphine on neuropathic pain

To evaluate the possible mechanisms involved in the antinocicep-tive effect of crotalphine, rats were injected, by intraplantar route,with naloxone, a non-specific opioid receptor antagonist (1 µg/paw).The antinociceptive activity of crotalphine (5 µg/kg, p.o.) and also ofthe crude venom (800 µg/kg, p.o.) was abolished by the opioidantagonist, indicating the involvement of opioid receptors in thiseffect (Fig. 5A and B).

The results described above, showing that intraplantar injection ofnaloxone abolished the antinociceptive effect of crotalphine, suggeststhat peripheral opioid receptors mediate the action of the peptide inthis model of neuropathic pain. In order to confirm that crotalphinedisplays peripheral (local) activity, the peptide (0.0006 µg/paw) wasinjected 14 days after surgery, by intraplantar route. The effect of thepeptide was compared to the effect of morphine (6 µg/paw). Previouswork has demonstrated that this dose of the peptide (Konno et al.,

2008) and of morphine (Cury, Y. and Pereira, L.M., unpublished data)did not induce a systemic antinociceptive effect. Crotalphine andmorphine, administered by intraplantar route, inhibited mechanical

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hyperalgesia (Fig. 6A) and low-threshold mechanical allodynia(Fig. 6B) induced by nerve constriction.

3.5. Effect of prolonged treatment with crotalphine on hyperalgesia,allodynia and spontaneous pain induced by chronic constriction injury

The demonstration that crotalphine displays opioid activityprompted us to investigate the possible development of tolerance toits antinociceptive effect after prolonged treatment. For induction oftolerance, crotalphine (5 µg/kg, p.o.) was administered every 3 days,during a period of 75 days. The administration of the peptide startedon day 14 after surgery. The results were compared to that obtainedwith crude venom administered to the animals according to sameprotocol described above. The results presented in Fig. 7 showed thatprolonged administration of crotalphine or crude venom did notinduce the development of tolerance to their antinociceptive effect.

4. Discussion

Data presented herein demonstrate that crotalphine, a 14-amino-acid peptide obtained from the venom of the South Americanrattlesnake C. durissus terrificus, induces a long-lasting and opioidreceptor-mediated antinociceptive effect in a ratmodel of neuropathicpain induced by chronic constriction of sciatic nerve.

The chronic constriction of rat sciatic nerve is a model of peripheralmononeuropathy produced in adult rats by placing loosely constric-tive ligatures around the common sciatic nerve. This model repro-duces the neural mechanisms and manifestation of nociceptivesymptoms (hyperalgesia, allodynia and spontaneous pain) of neuro-pathic pain disorders observed in humans (Bennett and Xie, 1988). Inthe present work, mechanical hyperalgesia, low-thresholdmechanicalallodynia and spontaneous pain were observed within 1 day andlasted for at least 14 days after surgery, a result consistent with otherreports (Attal et al., 1990; Bennett and Xie, 1988; Dowdall et al., 2005;Moller et al., 1998; Tal and Bennett, 1994). In addition, the resultsdemonstrated that these nociceptive phenomena are not due to thepresence of inflammatory mediators, such as prostanoids, since thenon-steroidal antiinflammatory drugs indomethacin and celecoxib,administered 14 days after surgery, did not modify hyperalgesia andallodynia induced by nerve constriction. Taken together, the observa-tion of a long lasting nociceptive phenomena and the lack ofcontribution of an inflammatory process caused by nerve constrictionto these phenomena indicate that nerve constriction resulted inneuropathic pain. It is important to stress that administration ofindomethacin and celecoxib on days 1 and 7 after surgery waseffective in inhibiting the nociceptive phenomena (data not shown),suggesting that nociception observed in the early periods of time afternerve constriction results, at least partially, from an inflammatoryresponse.

The antinociceptive profile of crotalphine in attenuating thenociceptive phenomena induced by nerve constriction was evaluatedafter oral administration of the peptide. Recent work of our group hasdemonstrated that crotalphine, administered in low doses by oralroute, induces a long lasting (5 days) antinociceptive effect inprostaglandin E2- and carrageenin-induced hyperalgesia (Konnoet al., 2008). In the present work, the peptide, administered 14 daysafter nerve constriction, inhibited mechanical hyperalgesia, low-threshold mechanical allodynia and spontaneous pain. The higheffectiveness of crotalphine is evidenced by the results observed in thedose-response curve study and also by the observation of the longlasting (3 days) antinociceptive effect in this model of neuropathicpain.

As pointed out in our previous work (Konno et al., 2008), it isintriguing that crotalphine, a 14-amino-acid peptide, displays long-lasting antinociceptive effect after oral administration. The mechan-isms by which oral administration of the peptide produces such effect

have not been determined yet, but various peptides do displaybioactivity, including antinociception, when administered by oralroute (Heimann et al., 2007; Karaki et al., 1990, 1993; Seppo et al.,2003; Sipola et al., 2001).

Konno et al. (Konno et al., 2008) observed that in the prostaglandinE2- and carrageenin-induced hyperalgesia, the dose of 1 µg/kg ofcrotalphine blocked hyperalgesia and also induced an increase in thepain threshold of the animals. In the present work, we observed thatthe dose of 5 µg/kg of the peptide induced a similar effect, i.e., itblocked mechanical hyperalgesia induced by nerve constriction andalso caused an increase in the pain threshold of the animals, ascompared to basal values obtained before surgery. Therefore, the doseof 5 µg/kg was chosen for characterization of the antinociceptiveproperties of crotalphine in the chronic constriction injury model.

We have previously shown that the amino acid sequence ofcrotalphine is identical to the γ-chain of crotapotin (Konno et al.,2008), a non-toxic and non-enzymatic component of crotoxin, themain neurotoxin of crude venom (Aird et al., 1985; Bon et al., 1989;Faure et al., 1991; Vital-Brazil, 1982). In order to evaluate the possibleinfluence of crotalphine on the locomotor activity of the animals,which could alter their performance in the nociceptive tests presentlyused, animals treated with the peptide were evaluated in the OpenField. The results indicated that the antinociceptive effect ofcrotalphine is not due to alterations in the general activity of theanimals since the frequency of locomotion and rearing was not alteredby the peptide.

The antinociceptive activity of standard analgesic drugs was alsohere evaluated. In contrast to the effect detected for crotalphine,morphine, a standard opioid drug and gabapentin, an anticonvulsantclinically employed in the management of neuropathic pain, in thedoses used, only partially inhibited the nociceptive phenomenainduced by nerve constriction, showing limited efficacy in thismodel of neuropathic pain. The doses of both drugs were based ondata from the literature and are within the range of the doses thatdisplay efficacy and potency in experimental models of neuropathicpain, including the chronic constriction injury model (Chapman et al.,1998; Hama and Borsook, 2005; Hunter et al., 1997; Joshi et al., 2006;Lee et al., 1995; Pelissier et al., 2003). However, data from theliterature have indicated that the effectiveness of morphine andgabapentin can vary depending on the experimental model ofneuropathic pain tested and the type (mechanical or thermal) ofnoxious stimulus applied for the investigation of hyperalgesia andallodynia (Erichsen and Blackburn-Munro, 2002; Idanpaan-Heikkilaand Guilbaud, 1999; Walczak et al., 2006). It is important to stress thatthe doses of morphine and gabapentin here used did not causealterations in the general activity of the animals, as evaluated in theOpen Field test or as evidenced by data from the literature (Hunteret al., 1997; Lee et al., 1995). In the present work, doses of morphinehigher than 5 mg/kg were not used since previous data of our grouphave demonstrated that they alter the parameters analyzed in theOpen Field test and also interfered with the respiratory frequency ofthe animals (Gutierrez, V. and Cury, Y., unpublished data).

Konno et al. (Konno et al., 2008) showed that the antinociceptiveeffect of crotalphine observed in the prostaglandin E2- and carragee-nin-induced hyperalgesia model, is mediated by activation of opioidreceptors. The involvement of opioid receptors in the effect of thepeptide in the chronic constriction injury model was here evidencedby the demonstration that naloxone, injected by intraplantar route,blocked the antinociceptive effect of crotalphine. The dose of naloxone(1 µg/paw) used in this assay is devoid of systemic effect, as previouslydemonstrated (Picolo et al., 2000). Therefore, these results indicatethat peripheral opioid receptors mediate the effect of crotalphine andalso that the peptide induces a peripheral antinociceptive effect. Theperipheral activity was confirmed by the results demonstrating thatcrotalphine injected in low doses into the plantar surface of the rats,reversed the nociceptive behaviour induced by nerve constriction. The

91V.P. Gutierrez et al. / European Journal of Pharmacology 594 (2008) 84–92

dose of 0.0006 µg/paw of the peptide was based in our previous workshowing that this dose induces a local long-lasting antinociceptiveeffect in the prostaglandin E2-induced hyperalgesia model, withoutcausing systemic effects (Konno et al., 2008).

Activation of opioid receptors may result from a direct action ofcrotalphine on opioid receptors and/or from the release of endogen-ous opioid peptides. As pointed out in our recent work (Konno et al.,2008), preliminary results indicate that crotalphine does not directlyactivate opioid receptors, since the peptide did not displace binding of[3H] naloxone to opioid receptors in rat brainmembrane homogenates(Correa, F., unpublished data). The contribution of endogenous opioidsto the antinociceptive effect of crotalphine is now under investigation.

The type of opioid receptor involved in the antinociceptive effect ofcrotalphine was not determined. Konno et al. (Konno et al., 2008)showed that κ opioid receptors are involved in the antinociceptiveeffect of the peptide in the models of hyperalgesia induced byprostaglandin E2 and carrageenin. In addition, in an experimentalmodel of cancer pain induced in rats by intraplantar injection ofWalker 256 carcinoma cells (Brigatte et al., 2007) the antinociceptiveeffect of crotalphinewas demonstrated to bemediated by activation ofperipheral κ and δ opioid receptors (Cury, y. and Brigatte, P.,unpublished data). The involvement of distinct types of opioidreceptors was also observed for the crude crotalid venom, adminis-tered by oral route. Picolo et al. (Picolo et al., 2003, 2000)demonstrated that the effect of the venom in models of hyperalgesiais mediated by activation of peripheral κ- and/or δ-opioid receptors,being the type of opioid receptor dependent on the type and durationof the nociceptive stimulus. In addition, Picolo et al. (Picolo et al., 2003;Picolo and Cury, 2004) evidenced that activation of the L-arginine–nitricoxide–cGMO pathway, with subsequent opening of peripheral ATP-sensitive K+ channels is responsible, at least partially, for the molecularmechanisms of the venom effect. Based on the results observed for thecrude venom, studies on the characterization of the type of opioidreceptor activated by crotalphine, in the chronic constriction injurymodel and the subsequent molecular mechanisms involved in itsantinociceptive effect are now in progress.

In the present work we also investigated the possible developmentof tolerance to the antinociceptive effect of the peptide. Several linesof evidence have indicated that repeated administration of drugsendowed with opioid activity caused the development of tolerance totheir analgesic effect (Andoh et al., 2008; Koob and Bloom, 1988).Results described herein indicate that prolonged treatment withcrotalphine did not cause the development of tolerance to theantinociceptive activity of the peptide in the chronic constrictioninjury model. For these studies, crotalphine was administered by oralroute every 3 days, during a period of 75 days, as it was observed in thetime–response curve studies that the effect of the peptide is detectedfor up to 3 days. In addition, we also evaluated the possibledevelopment of tolerance for crotalphine administered daily, during14 days. The daily treatment with the peptide also did not induce thedevelopment of tolerance to its antinociceptive effect (data notshown). These results are in contrast with data from the literatureshowing that prolonged treatment with opioid drugs, such asmorphine, leads to the development of tolerance to their antinoci-ceptive activity. The peripheral activity of crotalphine might possiblycontribute to the lack of development of tolerance to its antinocicep-tive effect. Experimental data, despite still controversial, haveindicated that peripheral analgesia induced by opioid drugs is resistantto the development of tolerance evenwhen its central effect is reducedafter prolonged treatment (Stein et al., 2001; Tokuyama et al., 1998;Zollner et al., 2008).

In the present work, the antinociceptive effect of the peptide wasalso compared to that observed with the crude venom of the crotalidsnake, since we have previously shown that the venom induces, inexperimental models of acute pain, a long-lasting antinociceptiveeffect mediated by activation of opioid receptors (Brigatte et al., 2001;

Giorgi et al., 1993; Picolo and Cury, 2004; Picolo et al., 2000). Theresults showed that the crotalid venom, administered by oral route,also induces a long-lasting antinociceptive effect in the chronicconstriction injury model, mediated by activation of opioid receptors.These results confirms our previous observation that crotalphine isresponsible for the opioid-like antinociceptive effect of the crotalidvenom administered by oral route (Konno et al., 2008).

In conclusion, data presented herein demonstrate that crotalphine,a 14-amino acid peptide isolated from the venom of the SouthAmerican rattlesnake C. durissus terrificus, induces marked antinoci-ceptive effect in an experimental model of neuropathic pain inducedby chronic constriction of rat sciatic nerve. The antinociceptive effectis long lasting and observed for the peptide administered in low dosesby oral route. Despite being mediated by opioid receptors, prolongedtreatment with the peptide did not cause the development oftolerance to its antinociceptive activity.

Chronic neuropathic pain usually responds somewhat poorly topharmacotherapy and the available drugs either fail to provide painrelief or are often associated with side-effects. Therefore, advances inanalgesic research and new medications development are necessaryto provide effective treatment of chronic pain. Our results, showingthe potent and long lasting antinociceptive effect of crotalphine,points to its therapeutic potential for the control of chronic pain.

Acknowledgements

This work was supported by Center for Applied Toxinology (CAT/CEPID/FAPESP), COINFAR Pesquisa e Desenvolvimento Farmacêuticoand Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP,grant numbers 02/04918-8, 07/00135-2 and 07/03404-4).

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