Antinociceptive effect of Brazilian armed spider venom toxin Tx3-3 in animal models of neuropathic pain

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www.elsevier.com/locate/pain

Antinociceptive effect of Brazilian armed spider venom toxin Tx3-3 in animal models of neuropathic pain Gerusa Duarte Dalmolin a, Cássia Regina Silva b, Flávia Karine Rigo a, Guilherme Monteiro Gomes b, Marta do Nascimento Cordeiro c, Michael Richardson c, Marco Aurélio Romano Silva d, Marco Antonio Máximo Prado a,e, Marcus Vinicius Gomez a,d,f, Juliano Ferreira a,b,⇑ a

Programa de Pós-graduação em Farmacologia Bioquímica e Molecular, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil Programa de Pós-graduação em Ciências Biológicas: Bioquímica Toxicológica, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil Fundação Ezequiel Dias, Belo Horizonte, MG, Brazil d Laboratório de Neurociência, Departamento de Saúde Mental, Faculdade de Medicina, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil e Robarts Research Institute, University of Western Ontario, London, Ontario, Canada f Núcleo de Pós-graduação, Santa Casa de Belo Horizonte, Brazil b c

Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.

a r t i c l e

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Article history: Received 16 June 2010 Received in revised form 4 February 2011 Accepted 8 April 2011

Keywords: Neuropathic pain P/Q-type calcium channel R-type calcium channel Calcium channel blocker Peptide toxin Antinociception

a b s t r a c t Venoms peptides have produced exceptional sources for drug development to treat pain. In this study we examined the antinociceptive and side effects of Tx3-3, a peptide toxin isolated from Phoneutria nigriventer venom, which inhibits high-voltage-dependent calcium channels (VDCC), preferentially P/Q and R-type VDCC. We tested the effects of Tx3-3 in animal models of nociceptive (tail-flick test), neuropathic (partial sciatic nerve ligation and streptozotocin-induced diabetic neuropathy), and inflammatory (intraplantar complete Freund’s adjuvant) pain. In the tail-flick test, both intrathecal (i.t.) and intracerebroventricular (i.c.v.) injection of Tx3-3 in mice caused a short-lasting effect (ED50 and 95% confidence intervals of 8.8 [4.1–18.8] and 3.7 [1.6–8.4] pmol/site for i.t. and i.c.v. injection, respectively), without impairing motor functions, at least at doses 10–30 times higher than the effective dose. By comparison, x-conotoxin MVIIC, a P/Q and N-type VDCC blocker derived from Conus magus venom, caused significant motor impairment at doses close to efficacious dose in tail flick test. Tx3-3 showed a long-lasting antinociceptive effect in neuropathic pain models. Intrathecal injection of Tx3-3 (30 pmol/site) decreased both mechanical allodynia produced by sciatic nerve injury in mice and streptozotocin-induced allodynia in mice and rats. On the other hand, i.t. injection of Tx3-3 did not alter inflammatory pain. Taken together, our data show that Tx3-3 shows prevalent antinociceptive effects in the neuropathic pain models and does not cause adverse motor effects at antinociceptive efficacious doses, suggesting that this peptide toxin holds promise as a novel therapeutic agent for the control of neuropathic pain. Ó 2011 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

1. Introduction Venoms from spiders, cone snails, snakes, and scorpions contain a pharmacopoeia of toxins that block receptor or channel activation as a means of producing shock, paralysis, or death in their prey. Alternatively, these venoms have produced exceptional leads for drug development [19,37]. For example, ziconotide, the synthetic version of the peptide x-conotoxin MVIIA found in the venom of the marine cone snail Conus magus, was approved

⇑ Corresponding author at: Department of Chemistry, Universidade Federal de Santa Maria, Avenida Roraima n° 1000 Bairro Camobi, Santa Maria, RS, Brazil. Tel.: +55 55 32208053; fax: +55 55 32208031. E-mail address: [email protected] (J. Ferreira).

(commercial name Prialt; Azur Pharma International, Philadelphia, PA, USA) for the treatment of pain in patients who require intrathecal analgesia and are refractory to opioid therapy. Ziconotide was demonstrated to be efficacious in the management of severe and chronic pain by blocking spinal N-type voltage-dependent calcium channels [46]. Voltage-dependent calcium channels (VDCC) are a family of ion channels classified by both their electrophysiological and pharmacological properties. They have been generally divided into low-threshold (T-type) and high-threshold (L-, N-, P/Q-, and R-types) [34]. In addition, the contribution of different VDCCs to nociception processes has gained considerable interest in recent years [5,64,71]. In fact, their presence in pain-modulation areas, such as the spinal cord, dorsal root ganglia, and brainstem, indicate

0304-3959/$36.00 Ó 2011 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.pain.2011.04.015

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the essential role of these types of VDCCs in the processing of painrelated information in the central nervous system [24,31,51,75]. Furthermore, VDCCs seem to be implicated in the central pain sensitization that occurs following nerve injury and during inflammatory states [44,45,63]. The management of chronic inflammatory and, mainly, neuropathic pain, is still a major challenge to clinicians because of its unresponsiveness to most of the currently used analgesic drugs [18,76]. The discovery of an efficacious analgesic substance from cone-snail venom led to an interest in animal venom toxins with the potential to treat chronic pain. In this sense, the venom of the Brazilian armed spider Phoneutria nigriventer possesses a cocktail of peptide toxins that modulate ion channels [22]. The purified fraction 3 of Phoneutria nigriventer venom (PhTx3) contains 6 toxin isoforms (Tx3-1 to -6) [12] that target VDCCs with different affinity patterns. Recently, one toxin, Tx3-6, isolated from PhTx3, which we have patented, named Pha1b, demonstrated that it preferentially blocks the N-type calcium current [72]. Pha1b has been demonstrated to be as potent as x-conotoxin MVIIA to produce antinociception, but presented higher therapeutic index than x-conotoxin MVIIA in preclinical experiments [65]. Here, we focus on another PhTx3-purified toxin, the polypeptide Tx3-3, which blocks VDCCs with a preference against P/Q- and R-type calcium currents [36]. Thus, the present study aimed to investigate the analgesic potential of Tx3-3 in different animal models of pain, and to compare its effects with those elicited by another P/Q-type VDCC blocker, the x-conotoxin MVIIC, extracted from the marine cone snail Conus magus [27].

2. Methods 2.1. Animals Male and female adult Swiss mice weighing 25–35 g, and male adult Wistar rats weighing 250–300 g were maintained in home cages under a 12:12-hour light-dark cycle (lights on 6:00 am) and at a constant room temperature (22 ± 2 °C), with standard laboratory chow and water ad libitum. The animals were acclimatized to the experimental room for at least 1 hour before testing (environmental habituation period). The experiments were approved by the ethics committee of the Universidade Federal de Santa Maria (process number: 23081.005024/2010-88), and were carried out in accordance with the current guidelines for the care of laboratory animals and the ethical guidelines for investigations of experiments in conscious animals [81]. The number of animals and the intensity of the noxious stimuli were the minimum necessary to demonstrate consistent effects of drug treatments. 2.2. Drugs The toxin Tx3-3 was purified from the venom of the spider Phoneutria nigriventer by a combination of chromatographic steps according to the method of Cordeiro et al. [12]. The criteria for the purity and/or identity of the Tx3-3 are based on: (1) the shape/morphology of the toxin peak in the final RP-HPLC; (2) the results of ES-Q-TO mass spectroscopy; and (3) the determination of the N-terminal sequence of 20–20 amino acids of every sample. All of the samples that we have used presented a purity of better than 95%, as determined by N-terminal sequencing. Tx3-3 has a molecular weight of 510,000 Da, and its amino acid sequence is GKCADAWESCDNYPCCVVNGYSRTCMCSANRCNCDDTKTLREHFG [8]. The x-conotoxin-MVIIC was purchased from Latoxan (Valence, France). Complete Freund’s adjuvant (CFA), streptozotocin (STZ), and morphine sulfate were obtained from Sigma (St. Louis, MO, USA).

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2.3. Drug treatments Tx3-3 (1–300 pmol/site), MVIIC (0.3–300 pmol/site), and morphine (35 and 70 nmol/site) were administered by intrathecal (i.t.) or intracerebroventricular (i.c.v.) routes, according to the techniques described by Hylden and Wilcox [29] and Laursen and Belknap [35], respectively. The i.t. and i.c.v. injections were delivered in a volume of 5 lL/site to mice and 10 lL/site to rats. STZ was administered by intraperitoneal route in a dose of 200 and 50 mg/kg to mice and rats, respectively. Tx3-3, MVIIC, and morphine were dissolved in phosphate-buffered saline (pH 7.4), and STZ was dissolved in citrate buffer (pH 4.5). Behavioral testing was performed by an experienced observer blind with respect to drug administration. 2.4. Behavioral procedures 2.4.1. Tail-flick test Measurement of nociceptive pain was carried out as described by D’Amour and Smith [13], with some modifications [68]. Briefly, after the environmental habituation period, the mice were gently handled and had two-thirds of their tail dipped into a bath containing water kept at 48 ± 1 °C. This low-intensity stimulus yields baseline latencies (9–12 seconds) that are long enough to observe hyperalgesia or analgesia. The latency to withdraw the tail from the hot bath was recorded with a stopwatch. Each mouse was tested twice before the administration of drugs to obtain the baseline withdrawal latency, and several times after drug treatments. If no response occurred within 24 seconds the test was interrupted to avoid tissue damage. The results were expressed in % baseline latency, calculated as follows: % baseline latency = (latency postdrug latency predrug)/(latency predrug)  100. Male and female mice were used in this test because no difference was observed in baseline withdrawal latency between male and female mice in earlier pilot tests (mean latency of 10.95 ± 1.73 and 10.10 ± 1.28 seconds for males and females, respectively). 2.4.2. von Frey test The measurement of the mechanical paw withdrawal threshold was carried out using the up-down paradigm, as described previously by Chaplan et al. [10], with minor modifications [65]. Briefly, mice or rats were first acclimatized (1–2 hours) in individual clear Plexiglass boxes on an elevated wire mesh platform to allow access to the plantar surface of the hind paws. von Frey filaments of increasing stiffness (0.02–10 g and 4–100 g for mice and rats, respectively) were applied to the hind paw plantar surface of the animals with a pressure high enough to bend the filament. The absence of a paw lifting after 5 seconds led to the use of the next filament with increasing weight, whereas paw lifting indicated a positive response and led to the use of the next weaker filament. This paradigm continued for a total of 6 measurements, including the one before the first paw-lifting response had been made, or until 4 consecutive positive (assigned a score of 0.030 or 6.59 g for mice or rats, respectively) or 4 consecutive negative (assigned a score of 6.76 or 83.22 g for mice or rats, respectively) responses occurred. The 50% mechanical paw withdraw threshold (PWT) response was then calculated from the resulting scores as described previously by Dixon [14]. The PWT was expressed in grams (g) and was evaluated before and several times after i.t. injection of Tx3-3. A significant decrease in PWT compared to baseline values was considered as mechanical allodynia. 2.4.3. Assessment of side-effects The locomotor performance was tested in the rotarod apparatus as described previously [17], with some adaptations [47]. Five

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minutes after i.t. or i.c.v. administration of different doses of Tx3-3 or MVIIC, the animals were placed on a rotarod (3.7 cm in diameter, 8 rpm), and during a 2-minute period we counted the number of falls and recorded the first fall latency. High doses of morphine (70 nmol/ site, i.t. or 35 nmol/site, i.c.v.), which are known to disrupt motor activity in mice [38], were used as a positive control to the test. To evaluate muscular weakness, the animals were submitted to a righting reflex test, according to Luvisetto et al. [39], which consisted of placing the mouse horizontally on its back and recording with a stopwatch the time it takes to regain the upright position. Intended to identify any other abnormal behavior usually elicited by the central administration of toxins that block VDCCs [41], we qualitatively attempted to assess gross behaviors of treated animals, such as whole-body shaking, coordination problems, muscle weakness, paralysis, and serpentine-like movements of the tail. 2.5. Induction of inflammatory pain model The CFA inflammatory pain model was performed according to Ferreira et al. [21]. Briefly, male mice received a 20-lL intradermal injection of CFA (1 mg/mL of heat inactivated Mycobacterium tuberculosis in paraffin oil [85%] and mannide monoleate [15%]) into the right hind paw. The Tx3-3 (30 pmol/site, i.t.) was administered concomitantly or 48 hours after the CFA injection. The CFA-induced mechanical allodynia was evaluated through von Frey filaments before and several times after Tx3-3 administration. 2.6. Induction of traumatic neuropathy For the induction of peripheral traumatic mononeuropathy, male mice were first anesthetized (90 mg/kg of ketamine plus 3 mg/kg of xylazine hydrochloride, intraperitoneally), and then a partial ligation of the right sciatic nerve was made by tying onethird to one-half of the dorsal portion of the sciatic nerve, using a previously described procedure [40]. In sham-operated mice, the control group, the nerve was only exposed without any ligation. Seven days after the surgical procedure the mechanical sensitivity was measured with von Frey filaments to verify the development of allodynia. 2.7. Induction of diabetic neuropathy For the induction of diabetic polyneuropathy, male mice and male rats received 200 and 50 mg/kg of STZ, respectively, according to the methods described by Ohsawa and Kamei [54] and Calcutt and Chaplan [7]. Hyperglycemia was confirmed through a strip-operated reflectance meter (Accu-Check; Roche Diagnostics, São Paulo, Brazil) using blood taken by a tail prick. Further, paw mechanical sensitivity was evaluated using von Frey filaments to verify the development of neuropathic allodynia. Glucose values and mechanical allodynia were verified after 15 or 21 days of diabetic induction in mice and rats, respectively. Only animals with a blood glucose concentration >300 mg/dL and established mechanical allodynia were included in the diabetic group. 2.8. Statistical analysis Statistical analysis was carried out by Student’s t-test, v2 test, or a 1-way or 2-way analysis of variance followed by StudentNewman–Keuls’ or Bonferroni posttests when appropriate. P values <0.05 were considered significant. When possible, the ED50 values were calculated by nonlinear regression using a dose-response equation adjusted to provide the best description of the values of the individual experiments, using GraphPad Software 4.0 (GraphPad, La Jolla, CA, USA); these were reported as geometric means accompanied by their respective 95% confidence limits.

3. Results 3.1. Effects produced by intrathecal and intracerebroventricular injection of Tx3-3 on nociceptive pain and motor behavior in mice Intrathecal administration of Tx3-3 (1–100 pmol/site) produced a short-lasting effect (about 15 minutes) in the tail-flick (Fig. 1B), with ED50 value (and the 95% confidence limits) of 8.8 (4.1– 18.8) pmol/site, and maximum effect (Emax) of 51 ± 14% estimated for Tx3-3 peaked effect (at 5 minutes) (Fig. 1A). There was no difference in duration or magnitude of the Tx3-3 effect between male and female mice (the % baseline latency 5 minutes after Tx3-3 i.t. injection was 46 ± 9 and 56 ± 17 for males and females, respectively; data not shown). Importantly, Tx3-3 did not produce side effects usually attributed to VDCC blockade [41], at least following a spinal injection of 300 pmol/site (Table 1). Furthermore, no locomotor impairment was observed at any dose of Tx3-3 tested in the rotarod test (Table 1) or in the righting reflex test (data not shown). As positive control, morphine (70 nmol/site, i.t.) impaired the performance of mice in the rotarod test (Table 1). We then compared the effects of Tx3-3 with that produced by x-conotoxin MVIIC. Intrathecal administration of 300 pmol/site of x-conotoxin MVIIC produced a marked increase in the tail-flick latency during the first 15 minutes after its administration (Fig. 1D). However, the development of motor side effects prevented the following tail-flick measurements. The motor side effects manifested initially as hind limb flaccidity evolved to a hind limb paralysis after around 30 minutes of injection. This motor disability was quantified using the righting reflex test. Intrathecal x-conotoxin MVIIC delayed the righting reflex test (2.78 ± 0.67 seconds compared to 0.12 ± 0.03 seconds for i.t. phosphate-buffered saline; P < 0.001, Student’s t-test). The flaccid paralysis, though not systematically examined, was found to be reversed within 24 hours. A minor dose of MVIIC (100 pmol/site) did not change the tail-flick withdrawal response (Fig. 1C) but, albeit with less frequency, produced hind limb flaccid paralysis (Table 1). Intracerebroventricular injection of Tx3-3 (1-30 pmol/site) also produced a short-lasting antinociceptive effect (Fig. 2B), with ED50 value (95% confidence limits) of 3.7 (1.6–8.4) pmol/site and maximum effect (Emax) of 84 ± 16% (Fig. 2A) estimated for Tx3-3 peaked effect (at 5 minutes) (Fig. 2A). Side effects were not observed at any dose up to 300 pmol/site (Table 1). In comparison, i.c.v. injection of x-conotoxin MVIIC (0.3– 3.0 pmol/site) produced antinociceptive effect (Emax = 62 ± 13% of baseline latency; Fig. 2C) and time-course similar to Tx3-3 (Fig. 2C and D), but with major potency (ED50 = 0.6 (0.1– 2.8) pmol/site). However, at higher doses (10–300 pmol/site), the x-conotoxin MVIIC induced some motor behavioral effects consisting of intense whole-body shaking, coordination problems, circling behavior, and muscle weakness beginning within a few minutes after injection (Table 1). When locomotor performance was examined in the rotarod test, injection of 10–30 pmol/site of x-conotoxin MVIIC impaired mice performance (Table 1), and higher doses (100 and 300 pmol/site) caused severe motor disability, which prevented testing mice in the rotarod test. Both i.t. and i.c.v. injection of Tx3-3 produced similar effect in the nociceptive pain test but, as the former route is often used to deliver analgesic drugs directly into the central nervous system, we used i.t. route in the clinically relevant models of pain. 3.2. Effects of intrathecal injection of Tx3-3 in chronic pain models Mice injected with CFA developed mechanical allodynia, characterized by a significant reduction in the PWT when von Frey filaments were applied in injected paw (PWT diminished from

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Fig. 1. Effects produced by intrathecal (i.t.) injection of peptide toxins in the tail-flick test in mice. Dose–response curve (A and C) and time-course effect (B and D) caused by i.t. injection of Tx3-3 (1–100 pmol/site) or MVIIC (100–300 pmol/site) in mice. The effects are expressed as % baseline latency. For dose–response curves, the % baseline latency was calculated at the antinociceptive peak (5 minutes). Each column or point represents the mean of 6–9 animals, and vertical lines show the SEM. Asterisks denote the significance levels in comparison with control (phosphate-buffered-saline [PBS]-treated group) values. Statistical analysis was performed using a 1-way analysis of variance (ANOVA) followed by Student-Newman–Keuls’ posttest (A and C) or a 2-way ANOVA followed by Bonferroni’s posttest (B and D); ⁄P < 0.05, ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001.

Table 1 Side-effects assessment of central administration of Tx3-3 or MVIIC on mice behavior. Drug/route

PBS/i.t Morphine/i.t. Tx3-3/i.t.

MVIIC/i.t. PBS/i.c.v. Morphine/i.c.v. Tx3-3/i.c.v.

MVIIC/i.c.v.

Dose (pmol/site)

70  103 1 3 10 30 48 100 300 100 300 35  103 1 3 10 30 100 300 0.3 1 3 10 30 100 300

Muscle

Rotarod test

Shaking

Weakness

Paralysis

Number of falls

First fall latency

0/7 n.d. 0/5 0/4 0/4 0/4 0/4 0/4 0/4 0/6 0/8 0/6 n.d. 0/6 0/6 0/5 0/5 0/6 0/6 0/5 0/5 0/6 3/4** 6/8** 3/3** 2/2**

0/7 n.d. 0/5 0/4 0/4 0/4 0/4 0/4 0/4 2/6* 6/8** 0/6 n.d. 0/6 0/6 0/5 0/5 0/6 0/6 0/5 0/5 0/6 3/4** 6/8** 3/3** 2/2**

0/7 0/5 0/5 0/4 0/4 0/4 0/4 0/4 0/4 2/6* 6/8** 0/6 0/4 0/6 0/6 0/5 0/5 0/6 0/6 0/5 0/5 0/6 0/4 0/8 0/3 0/2

0.9 ± 0.4 3.0 ± 0.9*** 0.4 ± 0.2 0.3 ± 0.3 0.3 ± 0.3 0.5 ± 0.3 0.8 ± 0.6 0.8 ± 0.5 0.2 ± 0.5 n.d. n.d. 0.8 ± 0.7 7.8 ± 1.8*** 0.2 ± 0.2 0.3 ± 0.2 0.8 ± 0.6 0.8 ± 0.4 0.7 ± 0.4 1.3 ± 0.5 0.8 ± 0.4 1.6 ± 0.6 0.6 ± 0.3 7.3 ± 2.5** 4.8 ± 2.3* n.d. n.d.

84.3 ± 14.3 31.6 ± 6.6* 99.8 ± 12.5 108.3 ± 11.8 94.8 ± 25.3 115.0 ± 5.0 85.0 ± 22.1 96.6 ± 15.7 106.4 ± 13.6 n.d. n.d. 93.5 ± 16.8 25.3 ± 13.7* 112.5 ± 7.5 91.2 ± 18.9 91.0 ± 18.7 86.6 ± 14.4 98.8 ± 13.8 74.6 ± 20.5 92.2 ± 14.2 60.8 ± 17.2 92.0 ± 38.9 40.3 ± 26.7 7.3 ± 3.0** n.d. n.d.

PBS, phosphate-buffered saline; i.t., intrathecal; i.c.v., intracerebroventricular; n.d., not determined. Data are mean ± SEM of 2–8 animals. Statistical analysis was performed using a 1-way analysis of variance followed by Student-Newman–Keuls’ test, for rotarod test or v2 test for muscle dysfunctions. * P < 0.05 denote the significance levels in comparison with control (PBS-treated) groups. ** P < 0.01 denote the significance levels in comparison with control (PBS-treated) groups. *** P < 0.001 denote the significance levels in comparison with control (PBS-treated) groups.

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Fig. 2. Effects produced by intracerebroventricular (i.c.v.) injection of peptide toxins in the tail-flick test in mice. Dose–response curve (A and C) and time-course effect (B and D) caused by i.c.v. injection of Tx3-3 (1–30 pmol/site) or MVIIC (0.3–3 pmol/site) in mice. The effects are expressed as % baseline latency. For dose-response curves, the % baseline latency was calculated at the antinociceptive peak (5 minutes). Each column or point represents the mean of 8–11 animals, and vertical lines show the SEM. Asterisks denote the significance levels in comparison with control (phosphate-buffered-saline [PBS]-treated group) values. Statistical analysis was performed using a 1-way analysis of variance (ANOVA) followed by Student-Newman–Keuls’ posttest (A and C) or a 2-way ANOVA followed by Bonferroni’s posttest (B and D); ⁄P < 0.05, ⁄⁄P < 0.01.

2.16 ± 0.44 g in baseline to 0.21 ± 0.07 g 48 hours after CFA administration; P < 0.001, Student’s t-test). Tx3-3 neither prevented (when administered immediately before CFA injection) nor reversed (when injected 48 hours after the CFA) the development of CFA-mechanical allodynia (data not shown). The partial sciatic nerve ligation caused mechanical allodynia in mice (PWT diminished from 1.46 ± 0.25 to 0.19 ± 0.05 g 7 days after nerve ligation; P < 0.001, Student’s t-test), that was readily reduced by i.t. administration of 30 pmol/site of Tx3-3. As show in Fig. 3A, the effect of Tx3-3 was detected from 30 minutes of administration (46 ± 13% of inhibition), remained unaltered at 60 minutes (53 ± 22% of inhibition), ending approximately 120 min postinjection. No change in mechanical sensitivity was observed in the sham-operated group (PWT values before and after the surgical procedure were 1.85 ± 0.33 and 2.21 ± 0.29 g, respectively; data not shown). Besides increasing blood glucose values (455.0 ± 17.5 and 125.5 ± 4.8 mg/dL of glycemia in STZ-treated and control mice, respectively), the treatment with STZ produced mechanical allodynia, which was measured 15 days after STZ injection (PWT of diabetic mice was 2.68 ± 0.36 g, whereas in the control group it was 0.73 ± 0.23 g; P < 0.001, Student’s t-test). Intrathecal injection of Tx3-3 (30 pmol/site) substantially reduced mechanical allodynia of diabetic mice (maximum effect of 83 ± 15% at 60 minutes) from 30 minutes (69 ± 9% of inhibition), lasting for at least 120 minutes (57 ± 11% of inhibition) (Fig. 3B). We confirm the antinociceptive effect of Tx3-3, testing its effects in another rodent species. Similar to mice, rats treated with STZ showed increased blood glucose values (488.5 ± 41.4 mg/dL

and 120.4 ± 12.1 mg/dL of glycemia in STZ-treated and control rats, respectively; P < 0.001, Student’s t-test) and mechanical allodynia (values of PWT of control and STZ-treated rats were 27.5 ± 4.8 and 8.1 ± 1.3 g, respectively; P < 0.001, Student’s t-test). Intrathecal administration of Tx3-3 (30 pmol/site) was also effective in inhibiting the allodynia induced by diabetic neuropathy in rats with time-course and efficacy similar to that observed in mice, although the effect appeared earlier in rats (70.7 ± 19.3% of inhibition at 10 minutes) (Fig. 3C). Finally, although effective in reducing the mechanical allodynia of neuropathic mice and rats, Tx3-3 did not alter their normal mechanical sensitivity per se (Fig. 4A and B). 4. Discussion The venom of the Brazilian ‘‘armed’’ spider Phoneutria nigriventer is a rich source of biologically active peptides, including the toxin Tx3-3, a high-threshold VDCC blocker. Our results show that Tx3-3 caused a short-lasting antinociceptive effect in the nociceptive pain test and a long-lasting antinociceptive effect in neuropathic pain models, without producing detectable side effects. However, Tx3-3 did not change the inflammatory pain. We first demonstrated that spinal and supraspinal administration of Tx3-3 inhibited nociceptive pain. The involvement of glutamate release in acute pain transmission is well known [16,43]. In previous studies, Tx3-3 was shown to inhibit calcium influx [49] and glutamate release in rat brain cortical synaptosomes through a mechanism coupled to calcium-dependent exocytosis [23,58]. These actions were next related to the Tx3-3 blockade of P/Q- and

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Fig. 4. Effect of intrathecal (i.t.) injection of Tx3-3 on mechanical threshold of mice and rats in the physiological state. Effect of Tx3-3 (30 pmol/site) on mechanical sensitivity in mice (A) and rats (B). The effects of Tx3-3 are expressed as 50% paw withdraw thresholds (PWT). Each point represents the mean of 5–8 animals, and vertical lines show the SEM. Statistical analysis was performed using a 2-way analysis of variance. PBS, phosphate-buffered saline.

Fig. 3. Effect of intrathecal (i.t.) administration of Tx3-3 on neuropathic pain models. (A) Effect of Tx3-3 (30 pmol/site) on allodynia induced by partial sciatic ligation in mice. (B) Effect Tx3-3 (30 pmol/site) on streptozotocin (STZ)-induced neuropathic allodynia in mice. (C) Effect of Tx3-3 (30 pmol/site) on STZ-induced neuropathic allodynia in rats. The effects of Tx3-3 are expressed as 50% paw withdraw threshold (PWT). PWT was calculated 7 (A), 15 (B), or 21 (C) days after neuropathy induction. Each point represents the mean of 5–11 animals, and vertical lines show the SEM. Asterisks denote the significance levels in comparison with phosphate-buffered saline (PBS)-treated neuropathic group values. Statistical analysis was performed using a 2-way analysis of variance followed by Bonferroni’s posttest; ⁄P < 0.05, ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001.

R-type VDCCs [36]. VDCCs regulate calcium influx, initiating the release of various neurotransmitters at the spinal and supraspinal levels [48,56]. Thus, the inhibition of calcium-dependent glutamate release by Tx3-3 could explain, at least in part, the fast antinociceptive effect produced by both spinal and supraspinal injection of this toxin in the tail-flick test. Importantly, tail withdrawal is not only a simple spinal reflex, but it is under control of supraspinal structures such as the rostral ventromedial medulla and periaqueductal gray, involved in the descending painmodulating pathway [2]. The VDCCs expressed in such supraspinal structures contribute to the neurotransmitter-mediated activation of descending facilitatory systems [26,57,60,70]. Therefore, we believe that i.c.v.-injected Tx3-3 produced its effect by blocking supraspinal VDCCs implicated in descending facilitatory pain pathway. Because it has been suggested that the generalizability of the findings from basic studies of pain could be improved by the

inclusion of both sexes in the study [50], we tested the antinociceptive effect of Tx3-3 in male and female mice and did not detect any difference between the sexes. Thus, our results indicate that the antinociceptive effect of Tx3-3 is not gender-dependent. A known peptide toxin extracted from Conus magus, x-conotoxin MVIIC, has been shown to mimic some actions produced by Tx33, both on glutamate release and on intracellular calcium changes [58], besides blocking preferentially P/Q-type VDCCs [27]. For these reasons, we compared the effects of x-conotoxin MVIIC and Tx3-3 in the nociceptive pain test. The i.t. and i.c.v. injection of x-conotoxin MVIIC caused not only an increase in tail-flick response latency but also significant motor dysfunctions. Some of them were earlier described by Olivera and colleagues [55], who classified the x-conotoxin MVIIC as a ‘‘shaker peptide,’’ as well as by Malmberg and Yaksh [41], who reported the establishment of flaccid paralysis of rat hind limbs about 30 minutes after MVIIC injection. It is tempting to speculate that the better safety profile of Tx3-3 in relation to x-conotoxin MVIIC is due to the additional affinity of Tx3-3 for R-type calcium channels [36], which play a considerable role in nociception [44,60,61] but seem not to be involved in motor functions, generally attributed to P/Q-type calcium channels [30,39]. Moreover, the x-conotoxin MVIIC had a higher degree of effect in the tail flick test than Tx3-3 when given via i.t. injection, but Tx3-3 was much more potent than x-conotoxin MVIIC at spinal site. On the other hand, x-conotoxin MVIIC showed higher potency than Tx3-3 when injected via i.c.v. route. This fact could be attributed to the high density of P/Q-type VDCCs expressed in nociceptive pathways at the supraspinal site [26] that gives rise to a powerful effect of x-conotoxin MVIIC injected by i.c.v. route. At the spinal site, P/Q-type VDCCs are mainly involved in motor function [30,75]. Although of high magnitude, the effect of x-conotoxin MVIIC in the tail-flick test was detected only at a dose that caused motor impairment. Thus, we cannot be sure that the effect observed in the tail-flick test, which settled down after 5 minutes of administration and remained unchanged over time, was a true antinociceptive effect. Anyway, Tx3-3 inhibited nociceptive

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pain at doses at least 10 times lower than the maximum dose tested without producing detectable side effects in any case. Most importantly, the antinociceptive effect of Tx3-3 was observed only in situations that involved the activation of pain pathways, such as thermal nociception and mechanical allodynia. On the other hand, Tx3-3 did not change mechanical sensitivity except when alterations of nociceptive processing secondary to persistent injury were present, as in mechanical allodynia produced by nerve injury. Noxious thermal and mechanical stimuli are detected by different types of sensory fibers [9,15,62]. VDCCs are found both in unmyelinated and myelinated sensory neurons. N-type and Rtype VDCCs are preferentially detected in heat-activated peptidergic unmyelinated neurons [20,75]. In contrast, P/Q-type VDCCs predominate in nonpeptidergic C fibers and in the Ad fibers, which are high-threshold mechanical nociceptors [62,75]. The lack of effect of Tx3-3 on normal mechanical sensitivity may be due to the low-threshold mechanical stimulation produced by von Frey filaments, whose main purpose was to detect allodynia, or due to a minor role of P/Q-type VDCC on the mechanical sensitivity in the physiological state. Accordingly, previous studies using the agatoxin IVA, a selective P/Q-type VDCC blocker extracted from the venom of spider Agenelopsis aperta, support the thesis that spinal P/Q-type VDCCs are engaged in responses to noxious stimuli once, and only if, the central sensitization is established [52,53,63]. In fact, the nerve injury may establish a reorganization in the dorsal horn of the spinal cord, with the switching of neuronal phenotype by changing the pattern of expression or activity of some ion channels such as P/Q- and R-type VDCCs [1,28,32,42,69,77,79], producing central sensitization. These adaptations may explain the outcome of mechanical allodynia in neuropathic states and could account for the long-lasting action of Tx3-3 in mechanical allodynia induced by neuropathy. Alternatively, the higher therapeutic safety profile of Tx3-3 compared with the x-conotoxin MVIIC and the longer Tx3-3 action in cases of neuropathy could be explained by the modulated receptor hypothesis, as described for local anesthetics on sodium channels and for dihydropyridines on L-type VDCCs [3,25]. This hypothesis predicts that the interaction of a drug with its receptor site in the channel complex changes as a function of the channel state, that is, a state-dependent blockade. Based on this, the kinetics of the onset and removal of Tx3-3 block would be strongly affected by the gating conformation of the channel. VDCCs may exist in some conformation states, including the inactivated state (a state intermediate between the resting and activated state) [80]. The access to the inactivated channel is greater during highfrequency neuronal firing conditions, as found in neuropathy [67]. Like some x-conotoxins that preferentially blocked VDCC in the inactivated state [4,67] the Tx3-3 could be interacting more strongly to the inactivated state of VDCC, giving rise to a slower dissociation from the channel and, therefore, a long-lasting effect on neuropathic pain. Moreover, the modulated receptor hypothesis explains shifts in channel inactivation by drugs via a drug-induced stabilization of the inactivated state [25]. Interestingly, Tx3-3 alters the kinetics of P/Q and R-type VDCC by increasing the degree of channel inactivation [36]. Furthermore, a state-dependent blockade could account for the better safety profile of Tx3-3 in relation to x-conotoxin MVIIC. However, further studies are required to elucidate the ability of Tx3-3 to discriminate between different states of VDCCs. Although numerous analgesic agents are available, neuropathic pain is still difficult to treat. A significant functional role of VDCCs in neuropathic pain mechanisms has been substantiated by several lines of evidence [11,28,33,78]. In recent years, a diversity of toxins isolated from animal venoms targeting VDCCs has been tested to treat neuropathic pain [6,66,73,74]. Here, we showed that Tx3-3 has a promising profile to treat neuropathic pain. Our data are in

accordance with studies demonstrating that P/Q- and R-type VDCCs are linked to adaptive changes involved in sensitization following nerve ligation [39,44,45,60,61]. Tx3-3 was also effective in a diabetic neuropathic pain model. There are reports of increased levels of P/Q-type in dorsal root ganglia, especially in large myelinated afferent fibers, in mice and rats treated with STZ [69,79]. Such alteration in fibers originally responsive to innocuous stimuli justifies the appearance of Tx3-3 effect on the mechanical sensitivity of diabetic mice and rats. Importantly, we observed a similar pattern of action of Tx3-3 in mice and rats submitted to the diabetic neuropathy model. In fact, the degree of similarity of VDCCs is significant across species, including mice, rats, and humans [64], which is consistent with the conservation of Tx3-3 antiallodynic property between mice and rats. Conversely, Tx3-3 did not prevent or reverse CFA-induced mechanical allodynia. Although relevant in central sensitization, the role of spinal VDCCs in inflammatory pain remains poorly understood. Nevertheless, a study showed that CFA-induced inflammation produces adaptive changes in N-type currents at primary afferent synaptic transmission without, however, changes to the P/Q- or R-type currents [59]. Taken together, our data show that the armed spider toxin Tx33 effectively alleviates neuropathic pain, without promoting the adverse effects usually developed by VDCC blockers. In line with this view, our results hold a significant promise that Tx3-3 can represent a novel therapeutic agent to manage neuropathic pain. Conflict of interest statement The authors declare no conflict of interest. Acknowledgments This study was supported by the Instituto do Milenio MCT/ CNPq, Instituto Nacional de Ciência e Tecnologia em Medicina Molecular MCT/CNPq, CAPES, PRONEX and FAPEMIG. The fellowships from CNPq, CAPES and FAPEMIG are also acknowledged. References [1] Abe M, Kurihara T, Han W, Shinomiya K, Tanabe T. Changes in expression of voltage-dependent ion channel subunits in dorsal root ganglia of rats with radicular injury and pain. Spine (Phila Pa 1976) 2002;27:1517–24. [2] Basbaum AI, Fields HL. Endogenous pain control systems: brainstem spinal pathways and endorphin circuitry. Ann Rev Neurosci 1984;7:309–38. [3] Bean BP. Nitrendipine block of cardiac calcium channels: high-affinity binding to the inactivated state. Proc Natl Acad Sci USA 1984;81:6388–92. [4] Berecki G, Motin L, Haythornthwaite A, Vink S, Bansal P, Drinkwater R, Wang CI, Moretta M, Lewis RJ, Alewood PF, Christie MJ, Adams DJ. Analgesic (omega)conotoxins CVIE and CVIF selectively and voltage-dependently block recombinant and native N-type calcium channels. Mol Pharmacol 2010;77:139–48. [5] Bourinet E, Zamponi GW. Voltage gated calcium channels as targets for analgesics. Curr Top Med Chem 2005;5:539–46. [6] Brose WG, Gutlove DP, Luther RR, Bowersox SS, McGuire D. Use of intrathecal SNX-111, a novel, N-type, voltage-sensitive, calcium channel blocker, in the management of intractable brachial plexus avulsion pain. Clin J Pain 1997;13:256–9. [7] Calcutt NA, Chaplan SR. Spinal pharmacology of tactile allodynia in diabetic rats. Br J Pharmacol 1997;122:1478–82. [8] Cardoso FC, Pacífico LG, Carvalho DC, Victória JM, Neves AL, Chávez-Olórtegui C, Gomez MV, Kalapothakis E. Molecular cloning and characterization of Phoneutria nigriventer toxins active on calcium channels. Toxicon 2003;41:755–63. [9] Cavanaugh DJ, Lee H, Lo L, Shields SD, Zylka MJ, Basbaum AI, Anderson DJ. Distinct subsets of unmyelinated primary sensory fibers mediate behavioral responses to noxious thermal and mechanical stimuli. Proc Natl Acad Sci USA 2009;106:9075–80. [10] Chaplan SR, Bach FW, Pogrel JW, Chung JM, Yaksh TL. Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Methods 1994;53:55–63.

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