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Recombinant peptide derived from the venom the Phoneutria nigriventer spider relieves nociception by nerve deafferentation
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Recombinant peptide derived from the venom the Phoneutria nigriventer spider relieves nociception by nerve deafferentation Flavia Tasmin Techera Antunesa, Stephanie Gonçalves Angelob, Eliane Dallegravec, Jaqueline Nascimento Picadad, Norma Possa Marronie, Elizangela Schemitte, ⁎ Alice Gomes Ferrazb, Marcus Vinicius Gomezf, Alessandra Hubner de Souzaa, a
Program of Postgraduation in Cellular and Molecular Biology Applied to Health (PPGBioSaúde), Lutheran University of Brazil (ULBRA), Canoas, RS, Brazil Laboratory of Pharmacology, Lutheran University of Brazil (ULBRA), Canoas, RS, Brazil c Department of Pharmacoscience, University Federal of Science of Health of Porto Alegre (UFCSPA), Rio Grande do Sul, Brazil d Laboratory of Genetic Toxicology, PPGBioSaúde and PPGGTA, Lutheran University of Brazil (ULBRA), Canoas, RS, Brazil e Laboratory of Oxidative Stress and Antioxidants, PPGBioSaúde and PPGGTA, Lutheran University of Brazil (ULBRA), Canoas, RS, Brazil f Nucleus of Postgraduation, Institute of Teaching and Research of Santa Casa de Belo Horizonte, Belo Horizonte, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: Neuropeptide Antinociception Neuropathic pain Voltage-gated calcium channel
The avulsion of nerve roots of the brachial plexus that is commonly seen in motorcycle accidents is a type of neuropathy due to deafferentation. This type of pain is clinically challenging since therapeutical protocols fail or have severe side effects. Thus, it is proposed to evaluate the antinociceptive activity of the recombinant CTK 01512-2 peptide that is derived from the venom of the Phoneutria nigriventer spider, as a future new therapeutical option. The neuropathic pain was surgically induced by avulsion of the upper brachial plexus trunk in groups of male Wistar rats and after 17 days, they were treated intrathecally with morphine, ziconotide, and CTK 01512–2. Behavioral tests were performed to evaluate mechanical and thermal hyperalgesia, cold allodynia, the functional activity of the front paw, and exploratory locomotion after the treatments. The peripheral blood samples were collected 6 h after the treatments and a comet assay was performed. The spinal cord was removed for the lipoperoxidation dosing of the membranes. The cerebrospinal fluid was analyzed for the dosage of glutamate. The recombinant peptide showed an antinociceptive effect when compared to the other drugs, without affecting the locomotor activity of the animals. Mechanical and thermal hyperalgesia, as well as cold allodynia, were reduced in the first hours of treatment. The levels of glutamate and the damage by membrane lipoperoxidation were shown to be improved, and genotoxicity was not demonstrated. In a scenario of therapeutical failures in the treatment of this type of pain, CTK 01512–2 was shown as a new effective alternative protocol. However, further testing is required to determine pharmacokinetics.
1. Introduction Neuropathic pain rises as a direct consequence of an injury or a disease that affects the somatosensory system (Treede et al., 2008). These lesions cause symptoms that are associated with the triggering or the amplification of the spontaneous neuronal signaling, leading to the development of central sensitization, which is an increased pain perception (hyperalgesia) and a threshold decrease (allodynia) that extends beyond the injured area (Teixeira et al., 2015). The brachial plexus is formed by the union of the ventral branches
of the spinal cord, C5-C8, and T1, and this provides motor and sensory innervation for the entire upper limb (Johnson et al., 2006). Brachial plexus injuries (BPI) are more common in young patients that are involved in motorcycle accidents due to the traction mechanism, and approximately 70% of these develop neuropathic pain (Flores, 2006; Schnick et al., 2018). Multiple BPI-patterns may occur, but half of these patients present partial lesions, as well as supraclavicular (91%), together with those involving the upper plexus (C5 to C8) (94%) (Bertelli et al., 2017). The deafferentation pain that characterizes BPI is severe, disabling, and a challenge for pharmacological protocols (Hussein et al.,
Abbreviations: BPI, Brachial plexus injuries; Cav2.2, N-type voltage-sensitive calcium channels; TBARS, thiobarbituric acid reactive substances; MDA, malondialdehyde; ROS, reactive oxygen species; TRPA1, transient receptor potential ankyrin 1 ⁎ Corresponding author at: Lutheran University of Brazil, Av. Farroupilha, 8001, District São José, Canoas, Rio Grande do Sul 92425900, Brazil. E-mail address: [email protected] (A.H. de Souza). https://doi.org/10.1016/j.npep.2019.101980 Received 18 June 2019; Received in revised form 10 October 2019; Accepted 13 October 2019 0143-4179/ © 2019 Published by Elsevier Ltd.
Please cite this article as: Flavia Tasmin Techera Antunes, et al., Neuropeptides, https://doi.org/10.1016/j.npep.2019.101980
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2018). It is clinically recommended to administer a first-line treatment of tricyclic antidepressants, serotonin reuptake inhibitors (duloxetine and venlafaxine), and calcium channel blockers (gabapentin and pregabalin) (Finnerup et al., 2015). Even opioids are being considered by the guidelines as a second-line treatment and they are still commonly prescribed for the treatment of neuropathic pain (McNicol et al., 2017). The N-type voltage-sensitive calcium channels (Cav2.2) are responsible for the release of neurotransmitters into the nociceptive pathway, such as glutamate. For this reason, drug development targets, such as ziconotide, are a synthetic version of the marine cone toxin (ωconotoxin MVIIA) (Patel et al., 2018; Stevens and Stephens, 2018). The drug is approved for the treatment of chronic pain, however, an intrathecal administration, a low therapeutical index, and severe neurological side effects (such as suicidal and homicidal ideations) restrict its use (Safavi-Hemami et al., 2018; Goga, 2018). CTK 01512-2 is a recombinant peptide of the Phtx3-6 (Phα1β) fraction of the toxin that is derived from the venom of the Phoneutria nigriventer spider. With 55 amino acids and 6 disulfide bonds (Wormwood et al., 2018), it produced a prolonged effect of prevention and a reduction of nociception, without causing adverse effects in rodents (Peigneur et al., 2018). The peptide has demonstrated reversibly, and not specifically a blockade of Cav 2.2 that affects the intracellular Ca2+ influx and the glutamate release. With a similar efficacy to ziconotide, it has displayed a higher therapeutical index (Souza et al., 2008). The peptide has proved to have a therapeutical potential in preclinical studies of pain models, such as inflammation (Silva et al., 2015), viscera (Diniz et al., 2014), neuropathic (Souza et al., 2008), cancer (Rigo et al., 2013), and fibromyalgia (Souza et al., 2014). It has been evaluated for its antinociceptive effects following an intrathecal administration of the CTK 01512-2 peptide in a neuropathic pain model, by avulsion of the upper trunk of the brachial plexus in rats. Besides, its genotoxic potential and its neuroprotective role against reactive oxygen species have been evaluated. Given the need to develop a drug that contemplates an enduring analgesia reaction, without causing any dependence or severe side effects, the peptide shows promise.
minutes before use.
2. Methods
2.4. Treatments
2.1. Materials
In order to evaluate the antinociceptive effects of the CTK 01512–2 peptide in the deafferentation neuropathic pain models, the animals that developed nociception were treated intrathecally with a vehicle, (10 μl PBS), ziconotide (100 pmol / site, positive control), morphine (1000 pmol / site, positive control), and CTK 01512–2 (200 pmol / site). After 6 h of treatment, the animals were euthanized (data, in brief, Fig. 1).
2.2. Animals 60 male Wistar rats aged between 6 and 8 weeks, with a mean weight of 340 g, were purchased from the Central Biotério de Pelotas (Rio Grande do Sul - Brazil). They were randomized into 6 groups. The researchers estimated a group size of seven rats for each experimental group. The present study was conducted by following the ARRIVE guidelines (Kilkenny et al., 2012) and the study was held at the Lutheran University of Brazil (ULBRA) in the city of Canoas, in accordance with the guidelines of the National Council for Control of Animal Experimentation (2013). It received approval from the Ethics Committee on the Use of Animals of the said University.
2.3. Surgery All of the surgical procedures were performed on the right side of the rats after anesthesia that was induced intraperitoneally by ketamine (50 mg/kg) and xylazine (10 mg / kg). The upper trunk of the brachial plexus was pinched with a forceps and extracted from the spinal cord by traction, with sufficient mechanical force so that the upper trunk was pulled together with the dorsal root ganglion, according to the technique of Liu et al. (2017). In the sham group, the brachial plexus was exposed and dissected without any nerve damage, and the wound was closed. The animals received subcutaneous tramadol (10 mg / kg every 24 h for 4 days) for the pain relief post-surgery. The treatments were commenced when the nociceptive responses decreased by 50% relative to control (seen 17 days after surgery). The PBS group represented the untreated animals with lower nociceptive thresholds for comparison with the other groups and for the validation of the nociceptive model. The sham group was used to differentiate itself from the neuropathic group, by presenting only tissue damage and by having different responses in the behavioral nociception.
The drugs used were ziconotide (ω-conotoxin MVIIA), which was purchased from Latoxan (Valence, France); morphine sulfate (Cristália, São Paulo, Brazil); and the recombinant form of the Phα1β (CTK 015122) fraction from Giotto Biotech S.R.I. (Florence, Italy). The solutions were prepared in PBS (pH 7.4) and diluted to the desired concentrations
Fig. 1. Antiallodynic mechanical effects by morphine, CTK 01512-2, and ziconotide, when intrathecally administered after the treatments at 1, 3, and 5 h, in comparison to the basal values (B) and the neuropathic animals (N), after 17 days of surgery, as verified by the Von Frey test. Each point and each vertical line represents the mean and SEM values that were obtained from 7 animals. The statistical analysis was performed by using a two-way analysis of variance (ANOVA), followed by Bonferroni's post hoc test. (*p < .05 and **p < .01 when compared to PBS).
2
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(crossing) and the vertical investigation of the number of suspensions of the previous paws (rearing) were measured (Stanford, 2007).
2.5. Grasping test The tail was gently raised in the rats and they were allowed to grab a grid that was attached to a standard electronic balance. While holding the animal, it continued to be raised by the tail with an increased strength until it lost its control. At that moment, the value shown by the balance was recorded (Bertelli and Mira, 1993). This procedure was repeated 3 times in order to register the mean. The test was performed 17 days after surgery, to ensure that the animal had an impairment in the functional activity of the front paws.
2.10. Measurement of glutamate in the cerebrospinal fluid After euthanasia by an isoflurane overdose, the cerebrospinal fluid was extracted by the technique of a puncture of the cisterna magna. The analyses of glutamate were performed enzymatically after increasing the fluorescence, due to the production of NADPH in the presence of glutamate dehydrogenase and NADP+ (Nicholls et al., 1987).
2.6. Mechanical allodynia
2.11. Comet assay
The 50% paw threshold (50% PWT) of the rats was evaluated by using an up-and-down motion (Chaplan et al., 1994). The animals were placed on a metal mesh floor that was covered by a transparent plastic box, which was erected 30 cm above the ground. The plantar surface of the right hind paw was perpendicularly stimulated with a series of Von Frey monofilaments (4 g, 6 g, 8 g, 10 g, 15 g, 26 g, and 60 g) and the stress response responses were analyzed. The sessions started with an application of 10 g filaments. If the voltage-response was harmful, a filament with a smaller value in (g) was used. However, if the response voltage was innocuous, the filament with the higher subsequent value in (g) was attempted from the last response. The monofilaments were applied for six sessions, with calculations as already described (Dixon, 1980). The mechanical allodynia was considered as a threshold decrease when compared to the baseline. The test was performed after the administration of the treatment protocols at 1 h, 3 h, and 5 h.
Immediately after euthanasia, the peripheral blood was collected in order to perform the comet assay as described by Tice (2000). 10 mL aliquots of the samples were mixed with a thin layer of 0.75% low melting agarose (95 mL) and placed on pre-coated slides with standard 1.5% agarose. These slides were dipped in a lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH 10 with the addition of 1% Triton X100, and 10% DMSO at the time of use) for at least 1 h up to 72 h at 4 °C, for the disruption of the cell membranes. The cell lysis allowed for the migration of the DNA fragments, which was performed after the incubation of the slides in the alkaline buffer (300 mM NaOH and 1 mM EDTA, pH 13) for 20 min - and the subsequent application of 300 mA and 25 V (0.90 V / cm) for 15 min to the lysed cells on the slides for microscopy of the DNA electrophoresis cells. The slides were neutralized shortly after electrophoresis with 0.4 M Tris buffer, pH 7.5, and stained with a silver solution. The results were expressed as damage index (DI) and the frequency of damage (FD). The DI was obtained by a visual evaluation of the damage classes (from 0 to 4), by extracting an index that expressed the general damage suffered by a population of cells (50 cells per slide, in duplicate).
2.7. Cold allodynia The animals remained in the acrylic boxes on a raised wire mesh platform, in order to allow for access to the plantar surface of the right hind paw, where 250 μl of acetone were applied (Choi et al., 1994). The retreat response was evaluated by using a scale of 0 to 3 points after a single application of acetone: (a) no response, 0 points, characterized by the absence of paw movement; (b) light response, 1 point, where the rat expressed a response in which the paw had little or no weight on it; (c) moderate response, 2 points, characterized by a raised leg that did not come into contact with the surface; (d) sharp response, 3 points, a response in which the rat licked, bit, or kicked (Shimizu et al., 2000). The test was performed to verify the installation of the pain model and the treatment times.
2.12. Lipid peroxidation assessment In order to evaluate the neuroprotective effects of the treatments through the membrane lipoperoxidation index, the spinal cord was homogenized with a phosphate buffer for two minutes at 0–2 °C. This homogenate was centrifuged in a refrigerated centrifuge (SORVALL RC5B Refrigerated Superspeed Centrifuge) for 10 min at 4000 rpm (Luesuy et al., 1985). The precipitate was discarded and the supernatant was used. The protein concentrations in the homogenate of the spinal cord were determined using the Bradford method (Bradford, 1976). The lipoperoxidation through the thiobarbituric acid reactive substances (TBARS) method was conducted by heating the homogenized material in the presence of thiobarbituric acid. The TBARS test quantified malondialdehyde (MDA), one of the products of the decomposition of hydroperoxides of polyunsaturated fatty acids that were formed during the oxidative process. The concentrations obtained were expressed as nmol/mg protein (Buege and Aust, 1978).
2.8. Thermal hyperalgesia The rats were acclimatized for 24 h before the test and then for 3 min inside the appliance when it was off (cold). During the test, the plate temperature was maintained at 52 °C ± 0.5° (Ugo Basile Hot / Cold Plate 35,100®). The animals were placed one by one in the transparent polypropylene cylinder on the heated surface. The time between the placement of the animals and the beginning of the leg withdrawal or “tapping” was recorded as a response latency in seconds, with a single measurement being in each period (Spindola et al., 2010). The cut-off time was 30 s, to avoid tissue damage. The test was performed in order to verify the installation of the pain model and the treatment times.
2.13. Data analysis The differences between 3 or more groups at one time were analyzed by one-way ANOVA, followed by Dunnett's post hoc test. The differences between 3 or more groups in the time course curves were analyzed by two-way ANOVA, followed by the Bonferroni post hoc test. The results are expressed as ± S.E.M. Values of p < .05 were considered significant. The statistical analysis was performed using GraphPad 5.0.
2.9. Open field In order to evaluate whether the drugs interfered with the locomotor or exploratory activity of the animals, the Open Field test was performed after the first hour of treatment. The animals were subjected to an open field in white sand measuring 100 × 60 × 40, where the ground was divided into 9 units by black lines. So as evaluate the horizontal exploration, the number of crossings with all of the paws
3. Results 3.1. Grasping test The animals that were submitted to surgery (334.2 ± 18.5 g) 3
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Fig. 2. Cold antiallodynic effects of CTK 01512-2 after treatments at 2 and 5 h, in comparison to basal (B) and the neuropathic animals (N), after 17 days of surgery, as verified by the Acetone Test. Each point and each vertical line represents the mean and SEM values that were obtained from 7 animals. The statistical analysis was performed when using a two-way analysis of variance (ANOVA), followed by Bonferroni's post hoc test. (*p < .05 when compared to PBS).
Fig. 3. Antihyperalgesic thermal effects of morphine, CTK 01512-2, and morphine after treatments at 1:30, 3:30, and 5:30 h, in comparison to the values of the neuropathic animals (N), 17 days after surgery, as verified by the Hot Plate test. Each point and each vertical line represents the mean and SEM values that were obtained from 7 animals. The statistical analysis was performed by using a two-way analysis of variance (ANOVA), followed by Bonferroni's post hoc test. (*p < .05 and **p < .001 when compared to the sham group).
3.3. Effects on cold allodynia
showed a decrease in seizure force at day 17, when compared to the baseline values (436.4 ± 17.97 g), as well as when compared to the control group (431.89 ± 35.63 g on day 0 and 600.2 ± 43.73 g on day 17) and sham (489.24 ± 25.38 g on day 0 and 537.92 ± 21.13 g on day 17) (data in brief, Fig. 2). The two-way ANOVA revealed all of this as statistically significant.
When comparing the 17-day nociception scores after the brachial plexus avulsion surgery (PBS: 0.75 ± 0.25, morphine: 1.0 ± 0.24, CTK 01512–2: 1.11 ± 0.20, ziconotide: 0.86 ± 0.14) with the values following the dose administrations, it was verified that CTK 01512-2 produced an antiallodynic effect within 2 h after administration (0.44 ± 0.18), which did not occur with the PBS: 1.40 ± 0.40, morphine: 1.11 ± 0.20, or ziconotide: 1.14 ± 0.26 (Fig. 2).
3.2. Effects on mechanical allodynia The animals departed from the close baseline values (PBS: 31.12 ± 6.96 g, morphine: 37.53 ± 5.21 g, CTK 01512–2: 36.30 ± 4.35 g, ziconotide: 38.01 ± 5.54 g) and after 17 days of surgery, they presented a decrease in the mechanical threshold (this being an indication of neuropathy), with values similar to each other (PBS: 12.87 ± 0.46 g; morphine: 11.42 ± 0.76 g; CTK 01512–2: 11.38 ± 1.11 g; ziconotide: 11.46 ± 1.60 g). After the administration of the treatments, the neuropathic animals received the vehicle (PBS) and continued with a low nociception threshold (15.90 ± 0.71 g), demonstrating that the PBS treatment did not decrease the nociception. Thus, it was possible to verify that the animals that received morphine had a higher threshold, totally reversing the neuropathic pain after 1 h of administration (43.15 ± 6.50 g). This antinociceptive effect lasted for up to 3 h after the treatment (36.35 ± 6.80 g), when compared to the PBS group (15.97 ± 3.22 g). Likewise, ziconotide and CTK 015012-2 exhibited an antiallodynic effect within the third hour after administration (CTK 01512–2: 37.24 ± 4.70 g; ziconotide: 42.13 ± 6.64 g) and this tended to remain with this antiallodynic activity for up to 5 h after its administration (CTK 01512-2: 26.40 ± 4.02 g; ziconotide: 22.70 ± 7.87 g), with a threshold greater than PBS (14.32 ± 1.47 g) (Fig. 1).
3.4. Effects on thermal hyperalgesia This antinociceptive effect was observed at 1:30 h after the administration of CTK 01512–2 (19.0 ± 3.64 s), which was the maximum effect observed in this test when compared to the sham group (6.65 ± 1.17 s). Ziconotide (100 pmol/site) produced an antinociceptive effect at 3:30 h after application (17.0 ± 3.16 s), lasting up to 5:30 h (13.0 ± 3.16 s). Morphine (1000 pmol/site), in a dose 5 times higher than CTK 01512-2, caused an antinociceptive effect that started at 1:30 h (25.33 ± 4.71 s) after its administration (this being its maximum effect), and remained for 5:30 h (19.0 ± 2.23 s) (Fig. 3). 3.5. Assessment of locomotor activity in the open field The number of invaded squares (crossovers), measured within 5 min after 2 h of the treatments, were not different between the control (54.55 ± 7.22), sham (59.12 ± 5.20), PBS (64.66 ± 5.30), CTK 01512–2 (59.5 ± 8.53), ziconotide (61.66 ± 11.09), and morphine (48 ± 8.86) groups. Likewise, the exploratory activities were not different in the control (19.88 ± 2.44), sham (21.63 ± 1.67), PBS 4
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(28 ± 2.65), CTK 01512–2 (25.75 ± 3.19), (20.44 ± 2.88), and morphine (25.83 ± 3.21) groups.
that were used for the analysis. The expression of the vesicular glutamate transporters was shown to be enhanced in the TRPA1 positive neurons (Kim et al., 2018), and cytosolic Ca2+ was an important regulator for their activation (Zygmunt and Högestätt, 2014). The activation of TRPA1 not only triggered the Ca2+ influx across the plasmatic membranes but also induced the Ca2+ release from the endolysosomes in the rat DRG neurons. This played a significant role in nociception and in the pain hypersensitivity (Shang et al., 2016). CTK 01512–2 increased the cold allodynia threshold, however, ziconotide and morphine did not show this result. This effect was due to the mechanism of the selective antagonism of the transient receptor potential ankyrin 1 (TRPA1) and the Cav2.2 blockade may represent a potential advantage in pain conditions involving TRPA1 (Tonello et al., 2017). In agreement with this mechanism, CTK 01512–2 did not affect the itching behavior that was generated by the intradermal application of the oxidizing agent H2O2 (which activates TRPA1) (Maciel et al., 2014). It is known that increased intracellular calcium activates the reactive oxygen species (ROS) production pathways, so that the Cav2.2 blockade reduces the influx of calcium into the cells, having the potential to reduce the production of free radicals, and also to attenuate the lipid peroxidation reactions (Ates et al., 2007). TRPA1 is an oxidative stress-sensitive Ca2+ permeable channel and as the neuronal antioxidant defenses are weak, increased levels of ROS lead to an exacerbation of nitro-oxidative stress, mitochondrial dysfunction, glial activation, and the inflammatory response (Carrasco et al., 2018). It has been proven that the native toxin Phα1β reduces the ROS levels in pain models (Diniz et al., 2012), but Coelho (2016, unpublished data) tested the recombinant form CTK 01512-2, demonstrating the generation of an MDA lower index in the evaluation of peroxidation in the medullary tissues. We suggest that, CTK 01512-2, probabily for being a TRPA1 antagonist (Tonello et al., 2017), was shown to decrease the damage to the lipid membranes after the deafferentation of the nerve branches; consequently, reducing the oxidative damage to the DNA, as reflected in the Comet Assay. Calcium can increase the production of ROS. On the other hand, ROS can significantly affect the calcium influx into the cellular and intracellular calcium stocks (Görlach et al., 2015). In addition, the oxidative stress-generated activation of TRPA1 in the Schwann cells maintains the macrophage infiltration into the injured nerves and sends paracrine signals to activate the nociceptor TRPA1 (de Logu et al., 2017). Silva et al. (2015) proved that CTK 01512-2 significantly improved the neuroinflammatory response in a multiple sclerosis model. Knowing that the TRPA1 antagonism may also decrease the ROS levels and the neuropathic pain (Inceoglu et al., 2015), this toxin shows a high pharmacological potential when compared to ziconotide. Endoplasmic reticulum stress (ERS) leads to an unfolded protein response (UPR) as a mechanism for cell survival. This can be activated by high levels of ROS and neuroinflammation, among others (Liu et al., 2019). The spinal nerve ligation depletes Ca2+ from the endoplasmic reticulum, thus, it may trigger UPR (Mekahli et al., 2011). The ERS pathways play an important role in regulating the excitability of the nociceptive system. The UPR promotes axonal regeneration after a peripheral nerve injury (Oñate et al., 2016), but continued and persistent activation can make the spinal neurons vulnerable to neuropathic injury pain stimuli (Khangura et al., 2019). Since the toxin decreases neuroinflammation and oxidative stress, an indirect action in this pathway is suggested. However, further studies are needed to confirm this hypothesis. Adverse effects were not noticed throughout the behavioral testing, ensuring the safety of the administration of CTK 01512-2. However, 40% of the ziconotide treated animals showed snakelike motions, shortly after the injection and up to the fifth hour, as were also seen by Wang et al. (2015); Jayamanne et al. (2013); and Dallegrave et al. (2018). This demonstrates the adverse effects related to ziconotide and it reinforces that CTK 01512–2 produces maximum analgesia at doses in
ziconotide
3.6. Measurements of glutamate in the cerebrospinal fluid Both CTK 01512–2 (0.95 ± 0.23) and ziconotide (0.97 ± 0.21) demonstred lower glutamate levels in the cerebrospinal fluid when compared to the group receiving the vehicle (PBS) (1.45 ± 0.19). Morphine also showed a decrease in the glutamate levels (0.83 ± 0.13), when compared to sham (1.28 ± 0.28) and PBS (1.45 ± 0.19), however no significant differences were showed (p = .28, one-way ANOVA) (data, in brief, Fig. 3). 3.7. Assessment of genotoxicity by the comet assay Before the behavior tests, the peripheral blood was collected. The DNA damage in the comet assay was measured using the parameters of the Damage Index and this ranged from 0 (completely undamaged, 100 cells × 0) to 400 (with a maximum damage of 100 × 4). The damage frequency was calculated based on the number of cells with tails versus those with no tail). No significant differences were found between the groups (Table 1). 3.8. Assessment role of neuroprotection on lipid peroxidation The levels of TBARS (nmol / mg protein) in the spinal cord of the rats were similar between the groups – sham (0.94 ± 0.08), CTK 01512–2 (0.81 ± 0.27), ziconotide (0.97 ± 0.15), and morphine (0.73 ± 0.16). These levels, however, were significantly higher in the PBS group (1.41 ± 0.01) when compared to the previous groups (Fig. 4). 4. Discussion The antiallodynic mechanical and antihyperalgesic thermal effects of CTK 01512–2 were due to Cav2.2 blocking (Souza et al., 2008). This has been proven in chronic neuropathic pain models by chronic constriction injuries (Rosa et al., 2014), or in peripheral mononeuropathy by partial ligation of the sciatic nerve (Souza et al., 2008). The current study's data shows that the dose of CTK 01512-2, which was administered in these experiments, was sufficient to increase the mechanical allodynia threshold at the third hour, lasting up to the fifth hour, like ziconotide. Morphine had its effect from the first hour of application, lasting up to the third hour, in this acute treatment (Cosgrave et al., 2017). The blocking of Cav2.2 led to decreased glutamate in the synaptic cleft; consequently, there was a modulation of the nociceptive pathway (Kuraishi et al., 1985). Thus, the current researchers' data confirms the mechanism actions of CTK 01512-2, by a decrease of glutamate in the cerebrospinal fluid of the treated rats. However, the difference was not significant between the groups, due to the reduced number of animals Table 1 Damage Index and Damage Frequency (Comet Assay) in the peripheral blood cells of the treated animals before the deafferentation of the upper trunk brachial plexus.
Control Sham PBS Morphine CTK 01512–2 Ziconotide
Damage index Mean ± SD
Damage frequency Mean ± SD
8.00 8.75 7.60 6.50 5.20 5.20
7.00 8.50 7.60 6.75 3.20 5.00
± ± ± ± ± ±
5.91 1.25 4.93 1.0 2.38 2.38
± ± ± ± ± ±
4.95 1.29 4.93 1.70 0.83 2.23
(n = 4 or 5 animals, statistical analysis by ANOVA, and Tukey's test). 5
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Fig. 4. Levels of TBARS in the homogenates of the spinal cord of the rats that were treated with PBS, morphine, CTK 01512–2, and ziconotide, when in comparison to the sham group. Each point and each vertical line represents the mean and SE values that were obtained from 7 animals. The statistical analysis was performed by using oneway analysis of variance (ANOVA), followed by the Student-NewmanKeuls post hoc test. (**p < .01 when compared to PBS/CTK 01512–2, PBS/Ziconotide, and PBS/Sham. ***p < .001 when compared to PBS/ Morphine).
Fig. 5. Graphical presentation of the main conclusion of study.
Antunes, Alessandra Hubner de Souza. Data collection: Flavia Tasmim Techera Antunes, Stephanie Gonçalves Angelo, Alice Gomes Ferraz. Data analysis and interpretation: Flavia Tasmim Techera Antunes, Stephanie Gonçalves Angelo, Elizangela Schemitt, Alessandra Hubner de Souza. Drafting the article: Flavia Tasmim Techera Antunes, Alessandra Hubner de Souza. Critical revision of the article and final approval of the version to be published: Alessandra Hubner de Souza, Jaqueline Nascimento Picada, Norma Possa Marroni, Marcus Vinicius Gomez, Eliane Dallegrave. Specimen collection: Alice Gomes Ferraz, Elizangela Schemitt, Eliane Dallegrave.
which it does not induce adverse effects. The assessments of genotoxicity in the peripheral blood showed no evidence of damage to the genetic material, as did the study by Souza et al. (2018), with a recombinant toxin in healthy animals. The current researchers have shown that rats with chronic peripheral deafferentation neuropathy have an impaired paw functionality, in addition to developing a central sensitization. An acute intrathecal administration of CTK 01512-2 has been effective in reducing mechanical allodynia, as well as cold and thermal hyperalgesia, without altering the locomotor and exploratory activities of the animals. These effects were mediated by the decrease of glutamate in the cerebrospinal fluid and by the reduction of lipoperoxidation of the membranes at the level of the spinal cord, without demonstrating genotoxic effects. 5. Conclusion
Declaration of Competing Interest
In the clinic, patients with an avulsion brachial plexus injury develop central sensitization and the available treatments are sparse and refractory, in addition to manifesting severe adverse effects or dose tolerances. Given that this present study has suggested that CTK 015122 has an analgesic potential in an animal model of pain by deafferentation (Fig. 5), it can provide a viable alternative treatment. However, repeated dose toxicity testing is necessary since the needs of patients with chronic pain usually use their medication for an extended period.
All of the authors declare no competing interests. Acknowledgment This research group was supported by FAPEMIG (Fundação de Amparo a Pesquisa do Estado de Minas Gerais), Belo Horizonte, State of Minas Gerais, Brazil. Appendix A. Supplementary data
Funding
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.npep.2019.101980.
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
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Contents lists available at ScienceDirect
Neuropeptides journal homepage: www.elsevier.com/locate/npep
Recombinant peptide derived from the venom the Phoneutria nigriventer spider relieves nociception by nerve deafferentation Flavia Tasmin Techera Antunesa, Stephanie Gonçalves Angelob, Eliane Dallegravec, Jaqueline Nascimento Picadad, Norma Possa Marronie, Elizangela Schemitte, ⁎ Alice Gomes Ferrazb, Marcus Vinicius Gomezf, Alessandra Hubner de Souzaa, a
Program of Postgraduation in Cellular and Molecular Biology Applied to Health (PPGBioSaúde), Lutheran University of Brazil (ULBRA), Canoas, RS, Brazil Laboratory of Pharmacology, Lutheran University of Brazil (ULBRA), Canoas, RS, Brazil c Department of Pharmacoscience, University Federal of Science of Health of Porto Alegre (UFCSPA), Rio Grande do Sul, Brazil d Laboratory of Genetic Toxicology, PPGBioSaúde and PPGGTA, Lutheran University of Brazil (ULBRA), Canoas, RS, Brazil e Laboratory of Oxidative Stress and Antioxidants, PPGBioSaúde and PPGGTA, Lutheran University of Brazil (ULBRA), Canoas, RS, Brazil f Nucleus of Postgraduation, Institute of Teaching and Research of Santa Casa de Belo Horizonte, Belo Horizonte, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: Neuropeptide Antinociception Neuropathic pain Voltage-gated calcium channel
The avulsion of nerve roots of the brachial plexus that is commonly seen in motorcycle accidents is a type of neuropathy due to deafferentation. This type of pain is clinically challenging since therapeutical protocols fail or have severe side effects. Thus, it is proposed to evaluate the antinociceptive activity of the recombinant CTK 01512-2 peptide that is derived from the venom of the Phoneutria nigriventer spider, as a future new therapeutical option. The neuropathic pain was surgically induced by avulsion of the upper brachial plexus trunk in groups of male Wistar rats and after 17 days, they were treated intrathecally with morphine, ziconotide, and CTK 01512–2. Behavioral tests were performed to evaluate mechanical and thermal hyperalgesia, cold allodynia, the functional activity of the front paw, and exploratory locomotion after the treatments. The peripheral blood samples were collected 6 h after the treatments and a comet assay was performed. The spinal cord was removed for the lipoperoxidation dosing of the membranes. The cerebrospinal fluid was analyzed for the dosage of glutamate. The recombinant peptide showed an antinociceptive effect when compared to the other drugs, without affecting the locomotor activity of the animals. Mechanical and thermal hyperalgesia, as well as cold allodynia, were reduced in the first hours of treatment. The levels of glutamate and the damage by membrane lipoperoxidation were shown to be improved, and genotoxicity was not demonstrated. In a scenario of therapeutical failures in the treatment of this type of pain, CTK 01512–2 was shown as a new effective alternative protocol. However, further testing is required to determine pharmacokinetics.
1. Introduction Neuropathic pain rises as a direct consequence of an injury or a disease that affects the somatosensory system (Treede et al., 2008). These lesions cause symptoms that are associated with the triggering or the amplification of the spontaneous neuronal signaling, leading to the development of central sensitization, which is an increased pain perception (hyperalgesia) and a threshold decrease (allodynia) that extends beyond the injured area (Teixeira et al., 2015). The brachial plexus is formed by the union of the ventral branches
of the spinal cord, C5-C8, and T1, and this provides motor and sensory innervation for the entire upper limb (Johnson et al., 2006). Brachial plexus injuries (BPI) are more common in young patients that are involved in motorcycle accidents due to the traction mechanism, and approximately 70% of these develop neuropathic pain (Flores, 2006; Schnick et al., 2018). Multiple BPI-patterns may occur, but half of these patients present partial lesions, as well as supraclavicular (91%), together with those involving the upper plexus (C5 to C8) (94%) (Bertelli et al., 2017). The deafferentation pain that characterizes BPI is severe, disabling, and a challenge for pharmacological protocols (Hussein et al.,
Abbreviations: BPI, Brachial plexus injuries; Cav2.2, N-type voltage-sensitive calcium channels; TBARS, thiobarbituric acid reactive substances; MDA, malondialdehyde; ROS, reactive oxygen species; TRPA1, transient receptor potential ankyrin 1 ⁎ Corresponding author at: Lutheran University of Brazil, Av. Farroupilha, 8001, District São José, Canoas, Rio Grande do Sul 92425900, Brazil. E-mail address: [email protected] (A.H. de Souza). https://doi.org/10.1016/j.npep.2019.101980 Received 18 June 2019; Received in revised form 10 October 2019; Accepted 13 October 2019 0143-4179/ © 2019 Published by Elsevier Ltd.
Please cite this article as: Flavia Tasmin Techera Antunes, et al., Neuropeptides, https://doi.org/10.1016/j.npep.2019.101980
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2018). It is clinically recommended to administer a first-line treatment of tricyclic antidepressants, serotonin reuptake inhibitors (duloxetine and venlafaxine), and calcium channel blockers (gabapentin and pregabalin) (Finnerup et al., 2015). Even opioids are being considered by the guidelines as a second-line treatment and they are still commonly prescribed for the treatment of neuropathic pain (McNicol et al., 2017). The N-type voltage-sensitive calcium channels (Cav2.2) are responsible for the release of neurotransmitters into the nociceptive pathway, such as glutamate. For this reason, drug development targets, such as ziconotide, are a synthetic version of the marine cone toxin (ωconotoxin MVIIA) (Patel et al., 2018; Stevens and Stephens, 2018). The drug is approved for the treatment of chronic pain, however, an intrathecal administration, a low therapeutical index, and severe neurological side effects (such as suicidal and homicidal ideations) restrict its use (Safavi-Hemami et al., 2018; Goga, 2018). CTK 01512-2 is a recombinant peptide of the Phtx3-6 (Phα1β) fraction of the toxin that is derived from the venom of the Phoneutria nigriventer spider. With 55 amino acids and 6 disulfide bonds (Wormwood et al., 2018), it produced a prolonged effect of prevention and a reduction of nociception, without causing adverse effects in rodents (Peigneur et al., 2018). The peptide has demonstrated reversibly, and not specifically a blockade of Cav 2.2 that affects the intracellular Ca2+ influx and the glutamate release. With a similar efficacy to ziconotide, it has displayed a higher therapeutical index (Souza et al., 2008). The peptide has proved to have a therapeutical potential in preclinical studies of pain models, such as inflammation (Silva et al., 2015), viscera (Diniz et al., 2014), neuropathic (Souza et al., 2008), cancer (Rigo et al., 2013), and fibromyalgia (Souza et al., 2014). It has been evaluated for its antinociceptive effects following an intrathecal administration of the CTK 01512-2 peptide in a neuropathic pain model, by avulsion of the upper trunk of the brachial plexus in rats. Besides, its genotoxic potential and its neuroprotective role against reactive oxygen species have been evaluated. Given the need to develop a drug that contemplates an enduring analgesia reaction, without causing any dependence or severe side effects, the peptide shows promise.
minutes before use.
2. Methods
2.4. Treatments
2.1. Materials
In order to evaluate the antinociceptive effects of the CTK 01512–2 peptide in the deafferentation neuropathic pain models, the animals that developed nociception were treated intrathecally with a vehicle, (10 μl PBS), ziconotide (100 pmol / site, positive control), morphine (1000 pmol / site, positive control), and CTK 01512–2 (200 pmol / site). After 6 h of treatment, the animals were euthanized (data, in brief, Fig. 1).
2.2. Animals 60 male Wistar rats aged between 6 and 8 weeks, with a mean weight of 340 g, were purchased from the Central Biotério de Pelotas (Rio Grande do Sul - Brazil). They were randomized into 6 groups. The researchers estimated a group size of seven rats for each experimental group. The present study was conducted by following the ARRIVE guidelines (Kilkenny et al., 2012) and the study was held at the Lutheran University of Brazil (ULBRA) in the city of Canoas, in accordance with the guidelines of the National Council for Control of Animal Experimentation (2013). It received approval from the Ethics Committee on the Use of Animals of the said University.
2.3. Surgery All of the surgical procedures were performed on the right side of the rats after anesthesia that was induced intraperitoneally by ketamine (50 mg/kg) and xylazine (10 mg / kg). The upper trunk of the brachial plexus was pinched with a forceps and extracted from the spinal cord by traction, with sufficient mechanical force so that the upper trunk was pulled together with the dorsal root ganglion, according to the technique of Liu et al. (2017). In the sham group, the brachial plexus was exposed and dissected without any nerve damage, and the wound was closed. The animals received subcutaneous tramadol (10 mg / kg every 24 h for 4 days) for the pain relief post-surgery. The treatments were commenced when the nociceptive responses decreased by 50% relative to control (seen 17 days after surgery). The PBS group represented the untreated animals with lower nociceptive thresholds for comparison with the other groups and for the validation of the nociceptive model. The sham group was used to differentiate itself from the neuropathic group, by presenting only tissue damage and by having different responses in the behavioral nociception.
The drugs used were ziconotide (ω-conotoxin MVIIA), which was purchased from Latoxan (Valence, France); morphine sulfate (Cristália, São Paulo, Brazil); and the recombinant form of the Phα1β (CTK 015122) fraction from Giotto Biotech S.R.I. (Florence, Italy). The solutions were prepared in PBS (pH 7.4) and diluted to the desired concentrations
Fig. 1. Antiallodynic mechanical effects by morphine, CTK 01512-2, and ziconotide, when intrathecally administered after the treatments at 1, 3, and 5 h, in comparison to the basal values (B) and the neuropathic animals (N), after 17 days of surgery, as verified by the Von Frey test. Each point and each vertical line represents the mean and SEM values that were obtained from 7 animals. The statistical analysis was performed by using a two-way analysis of variance (ANOVA), followed by Bonferroni's post hoc test. (*p < .05 and **p < .01 when compared to PBS).
2
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(crossing) and the vertical investigation of the number of suspensions of the previous paws (rearing) were measured (Stanford, 2007).
2.5. Grasping test The tail was gently raised in the rats and they were allowed to grab a grid that was attached to a standard electronic balance. While holding the animal, it continued to be raised by the tail with an increased strength until it lost its control. At that moment, the value shown by the balance was recorded (Bertelli and Mira, 1993). This procedure was repeated 3 times in order to register the mean. The test was performed 17 days after surgery, to ensure that the animal had an impairment in the functional activity of the front paws.
2.10. Measurement of glutamate in the cerebrospinal fluid After euthanasia by an isoflurane overdose, the cerebrospinal fluid was extracted by the technique of a puncture of the cisterna magna. The analyses of glutamate were performed enzymatically after increasing the fluorescence, due to the production of NADPH in the presence of glutamate dehydrogenase and NADP+ (Nicholls et al., 1987).
2.6. Mechanical allodynia
2.11. Comet assay
The 50% paw threshold (50% PWT) of the rats was evaluated by using an up-and-down motion (Chaplan et al., 1994). The animals were placed on a metal mesh floor that was covered by a transparent plastic box, which was erected 30 cm above the ground. The plantar surface of the right hind paw was perpendicularly stimulated with a series of Von Frey monofilaments (4 g, 6 g, 8 g, 10 g, 15 g, 26 g, and 60 g) and the stress response responses were analyzed. The sessions started with an application of 10 g filaments. If the voltage-response was harmful, a filament with a smaller value in (g) was used. However, if the response voltage was innocuous, the filament with the higher subsequent value in (g) was attempted from the last response. The monofilaments were applied for six sessions, with calculations as already described (Dixon, 1980). The mechanical allodynia was considered as a threshold decrease when compared to the baseline. The test was performed after the administration of the treatment protocols at 1 h, 3 h, and 5 h.
Immediately after euthanasia, the peripheral blood was collected in order to perform the comet assay as described by Tice (2000). 10 mL aliquots of the samples were mixed with a thin layer of 0.75% low melting agarose (95 mL) and placed on pre-coated slides with standard 1.5% agarose. These slides were dipped in a lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH 10 with the addition of 1% Triton X100, and 10% DMSO at the time of use) for at least 1 h up to 72 h at 4 °C, for the disruption of the cell membranes. The cell lysis allowed for the migration of the DNA fragments, which was performed after the incubation of the slides in the alkaline buffer (300 mM NaOH and 1 mM EDTA, pH 13) for 20 min - and the subsequent application of 300 mA and 25 V (0.90 V / cm) for 15 min to the lysed cells on the slides for microscopy of the DNA electrophoresis cells. The slides were neutralized shortly after electrophoresis with 0.4 M Tris buffer, pH 7.5, and stained with a silver solution. The results were expressed as damage index (DI) and the frequency of damage (FD). The DI was obtained by a visual evaluation of the damage classes (from 0 to 4), by extracting an index that expressed the general damage suffered by a population of cells (50 cells per slide, in duplicate).
2.7. Cold allodynia The animals remained in the acrylic boxes on a raised wire mesh platform, in order to allow for access to the plantar surface of the right hind paw, where 250 μl of acetone were applied (Choi et al., 1994). The retreat response was evaluated by using a scale of 0 to 3 points after a single application of acetone: (a) no response, 0 points, characterized by the absence of paw movement; (b) light response, 1 point, where the rat expressed a response in which the paw had little or no weight on it; (c) moderate response, 2 points, characterized by a raised leg that did not come into contact with the surface; (d) sharp response, 3 points, a response in which the rat licked, bit, or kicked (Shimizu et al., 2000). The test was performed to verify the installation of the pain model and the treatment times.
2.12. Lipid peroxidation assessment In order to evaluate the neuroprotective effects of the treatments through the membrane lipoperoxidation index, the spinal cord was homogenized with a phosphate buffer for two minutes at 0–2 °C. This homogenate was centrifuged in a refrigerated centrifuge (SORVALL RC5B Refrigerated Superspeed Centrifuge) for 10 min at 4000 rpm (Luesuy et al., 1985). The precipitate was discarded and the supernatant was used. The protein concentrations in the homogenate of the spinal cord were determined using the Bradford method (Bradford, 1976). The lipoperoxidation through the thiobarbituric acid reactive substances (TBARS) method was conducted by heating the homogenized material in the presence of thiobarbituric acid. The TBARS test quantified malondialdehyde (MDA), one of the products of the decomposition of hydroperoxides of polyunsaturated fatty acids that were formed during the oxidative process. The concentrations obtained were expressed as nmol/mg protein (Buege and Aust, 1978).
2.8. Thermal hyperalgesia The rats were acclimatized for 24 h before the test and then for 3 min inside the appliance when it was off (cold). During the test, the plate temperature was maintained at 52 °C ± 0.5° (Ugo Basile Hot / Cold Plate 35,100®). The animals were placed one by one in the transparent polypropylene cylinder on the heated surface. The time between the placement of the animals and the beginning of the leg withdrawal or “tapping” was recorded as a response latency in seconds, with a single measurement being in each period (Spindola et al., 2010). The cut-off time was 30 s, to avoid tissue damage. The test was performed in order to verify the installation of the pain model and the treatment times.
2.13. Data analysis The differences between 3 or more groups at one time were analyzed by one-way ANOVA, followed by Dunnett's post hoc test. The differences between 3 or more groups in the time course curves were analyzed by two-way ANOVA, followed by the Bonferroni post hoc test. The results are expressed as ± S.E.M. Values of p < .05 were considered significant. The statistical analysis was performed using GraphPad 5.0.
2.9. Open field In order to evaluate whether the drugs interfered with the locomotor or exploratory activity of the animals, the Open Field test was performed after the first hour of treatment. The animals were subjected to an open field in white sand measuring 100 × 60 × 40, where the ground was divided into 9 units by black lines. So as evaluate the horizontal exploration, the number of crossings with all of the paws
3. Results 3.1. Grasping test The animals that were submitted to surgery (334.2 ± 18.5 g) 3
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Fig. 2. Cold antiallodynic effects of CTK 01512-2 after treatments at 2 and 5 h, in comparison to basal (B) and the neuropathic animals (N), after 17 days of surgery, as verified by the Acetone Test. Each point and each vertical line represents the mean and SEM values that were obtained from 7 animals. The statistical analysis was performed when using a two-way analysis of variance (ANOVA), followed by Bonferroni's post hoc test. (*p < .05 when compared to PBS).
Fig. 3. Antihyperalgesic thermal effects of morphine, CTK 01512-2, and morphine after treatments at 1:30, 3:30, and 5:30 h, in comparison to the values of the neuropathic animals (N), 17 days after surgery, as verified by the Hot Plate test. Each point and each vertical line represents the mean and SEM values that were obtained from 7 animals. The statistical analysis was performed by using a two-way analysis of variance (ANOVA), followed by Bonferroni's post hoc test. (*p < .05 and **p < .001 when compared to the sham group).
3.3. Effects on cold allodynia
showed a decrease in seizure force at day 17, when compared to the baseline values (436.4 ± 17.97 g), as well as when compared to the control group (431.89 ± 35.63 g on day 0 and 600.2 ± 43.73 g on day 17) and sham (489.24 ± 25.38 g on day 0 and 537.92 ± 21.13 g on day 17) (data in brief, Fig. 2). The two-way ANOVA revealed all of this as statistically significant.
When comparing the 17-day nociception scores after the brachial plexus avulsion surgery (PBS: 0.75 ± 0.25, morphine: 1.0 ± 0.24, CTK 01512–2: 1.11 ± 0.20, ziconotide: 0.86 ± 0.14) with the values following the dose administrations, it was verified that CTK 01512-2 produced an antiallodynic effect within 2 h after administration (0.44 ± 0.18), which did not occur with the PBS: 1.40 ± 0.40, morphine: 1.11 ± 0.20, or ziconotide: 1.14 ± 0.26 (Fig. 2).
3.2. Effects on mechanical allodynia The animals departed from the close baseline values (PBS: 31.12 ± 6.96 g, morphine: 37.53 ± 5.21 g, CTK 01512–2: 36.30 ± 4.35 g, ziconotide: 38.01 ± 5.54 g) and after 17 days of surgery, they presented a decrease in the mechanical threshold (this being an indication of neuropathy), with values similar to each other (PBS: 12.87 ± 0.46 g; morphine: 11.42 ± 0.76 g; CTK 01512–2: 11.38 ± 1.11 g; ziconotide: 11.46 ± 1.60 g). After the administration of the treatments, the neuropathic animals received the vehicle (PBS) and continued with a low nociception threshold (15.90 ± 0.71 g), demonstrating that the PBS treatment did not decrease the nociception. Thus, it was possible to verify that the animals that received morphine had a higher threshold, totally reversing the neuropathic pain after 1 h of administration (43.15 ± 6.50 g). This antinociceptive effect lasted for up to 3 h after the treatment (36.35 ± 6.80 g), when compared to the PBS group (15.97 ± 3.22 g). Likewise, ziconotide and CTK 015012-2 exhibited an antiallodynic effect within the third hour after administration (CTK 01512–2: 37.24 ± 4.70 g; ziconotide: 42.13 ± 6.64 g) and this tended to remain with this antiallodynic activity for up to 5 h after its administration (CTK 01512-2: 26.40 ± 4.02 g; ziconotide: 22.70 ± 7.87 g), with a threshold greater than PBS (14.32 ± 1.47 g) (Fig. 1).
3.4. Effects on thermal hyperalgesia This antinociceptive effect was observed at 1:30 h after the administration of CTK 01512–2 (19.0 ± 3.64 s), which was the maximum effect observed in this test when compared to the sham group (6.65 ± 1.17 s). Ziconotide (100 pmol/site) produced an antinociceptive effect at 3:30 h after application (17.0 ± 3.16 s), lasting up to 5:30 h (13.0 ± 3.16 s). Morphine (1000 pmol/site), in a dose 5 times higher than CTK 01512-2, caused an antinociceptive effect that started at 1:30 h (25.33 ± 4.71 s) after its administration (this being its maximum effect), and remained for 5:30 h (19.0 ± 2.23 s) (Fig. 3). 3.5. Assessment of locomotor activity in the open field The number of invaded squares (crossovers), measured within 5 min after 2 h of the treatments, were not different between the control (54.55 ± 7.22), sham (59.12 ± 5.20), PBS (64.66 ± 5.30), CTK 01512–2 (59.5 ± 8.53), ziconotide (61.66 ± 11.09), and morphine (48 ± 8.86) groups. Likewise, the exploratory activities were not different in the control (19.88 ± 2.44), sham (21.63 ± 1.67), PBS 4
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(28 ± 2.65), CTK 01512–2 (25.75 ± 3.19), (20.44 ± 2.88), and morphine (25.83 ± 3.21) groups.
that were used for the analysis. The expression of the vesicular glutamate transporters was shown to be enhanced in the TRPA1 positive neurons (Kim et al., 2018), and cytosolic Ca2+ was an important regulator for their activation (Zygmunt and Högestätt, 2014). The activation of TRPA1 not only triggered the Ca2+ influx across the plasmatic membranes but also induced the Ca2+ release from the endolysosomes in the rat DRG neurons. This played a significant role in nociception and in the pain hypersensitivity (Shang et al., 2016). CTK 01512–2 increased the cold allodynia threshold, however, ziconotide and morphine did not show this result. This effect was due to the mechanism of the selective antagonism of the transient receptor potential ankyrin 1 (TRPA1) and the Cav2.2 blockade may represent a potential advantage in pain conditions involving TRPA1 (Tonello et al., 2017). In agreement with this mechanism, CTK 01512–2 did not affect the itching behavior that was generated by the intradermal application of the oxidizing agent H2O2 (which activates TRPA1) (Maciel et al., 2014). It is known that increased intracellular calcium activates the reactive oxygen species (ROS) production pathways, so that the Cav2.2 blockade reduces the influx of calcium into the cells, having the potential to reduce the production of free radicals, and also to attenuate the lipid peroxidation reactions (Ates et al., 2007). TRPA1 is an oxidative stress-sensitive Ca2+ permeable channel and as the neuronal antioxidant defenses are weak, increased levels of ROS lead to an exacerbation of nitro-oxidative stress, mitochondrial dysfunction, glial activation, and the inflammatory response (Carrasco et al., 2018). It has been proven that the native toxin Phα1β reduces the ROS levels in pain models (Diniz et al., 2012), but Coelho (2016, unpublished data) tested the recombinant form CTK 01512-2, demonstrating the generation of an MDA lower index in the evaluation of peroxidation in the medullary tissues. We suggest that, CTK 01512-2, probabily for being a TRPA1 antagonist (Tonello et al., 2017), was shown to decrease the damage to the lipid membranes after the deafferentation of the nerve branches; consequently, reducing the oxidative damage to the DNA, as reflected in the Comet Assay. Calcium can increase the production of ROS. On the other hand, ROS can significantly affect the calcium influx into the cellular and intracellular calcium stocks (Görlach et al., 2015). In addition, the oxidative stress-generated activation of TRPA1 in the Schwann cells maintains the macrophage infiltration into the injured nerves and sends paracrine signals to activate the nociceptor TRPA1 (de Logu et al., 2017). Silva et al. (2015) proved that CTK 01512-2 significantly improved the neuroinflammatory response in a multiple sclerosis model. Knowing that the TRPA1 antagonism may also decrease the ROS levels and the neuropathic pain (Inceoglu et al., 2015), this toxin shows a high pharmacological potential when compared to ziconotide. Endoplasmic reticulum stress (ERS) leads to an unfolded protein response (UPR) as a mechanism for cell survival. This can be activated by high levels of ROS and neuroinflammation, among others (Liu et al., 2019). The spinal nerve ligation depletes Ca2+ from the endoplasmic reticulum, thus, it may trigger UPR (Mekahli et al., 2011). The ERS pathways play an important role in regulating the excitability of the nociceptive system. The UPR promotes axonal regeneration after a peripheral nerve injury (Oñate et al., 2016), but continued and persistent activation can make the spinal neurons vulnerable to neuropathic injury pain stimuli (Khangura et al., 2019). Since the toxin decreases neuroinflammation and oxidative stress, an indirect action in this pathway is suggested. However, further studies are needed to confirm this hypothesis. Adverse effects were not noticed throughout the behavioral testing, ensuring the safety of the administration of CTK 01512-2. However, 40% of the ziconotide treated animals showed snakelike motions, shortly after the injection and up to the fifth hour, as were also seen by Wang et al. (2015); Jayamanne et al. (2013); and Dallegrave et al. (2018). This demonstrates the adverse effects related to ziconotide and it reinforces that CTK 01512–2 produces maximum analgesia at doses in
ziconotide
3.6. Measurements of glutamate in the cerebrospinal fluid Both CTK 01512–2 (0.95 ± 0.23) and ziconotide (0.97 ± 0.21) demonstred lower glutamate levels in the cerebrospinal fluid when compared to the group receiving the vehicle (PBS) (1.45 ± 0.19). Morphine also showed a decrease in the glutamate levels (0.83 ± 0.13), when compared to sham (1.28 ± 0.28) and PBS (1.45 ± 0.19), however no significant differences were showed (p = .28, one-way ANOVA) (data, in brief, Fig. 3). 3.7. Assessment of genotoxicity by the comet assay Before the behavior tests, the peripheral blood was collected. The DNA damage in the comet assay was measured using the parameters of the Damage Index and this ranged from 0 (completely undamaged, 100 cells × 0) to 400 (with a maximum damage of 100 × 4). The damage frequency was calculated based on the number of cells with tails versus those with no tail). No significant differences were found between the groups (Table 1). 3.8. Assessment role of neuroprotection on lipid peroxidation The levels of TBARS (nmol / mg protein) in the spinal cord of the rats were similar between the groups – sham (0.94 ± 0.08), CTK 01512–2 (0.81 ± 0.27), ziconotide (0.97 ± 0.15), and morphine (0.73 ± 0.16). These levels, however, were significantly higher in the PBS group (1.41 ± 0.01) when compared to the previous groups (Fig. 4). 4. Discussion The antiallodynic mechanical and antihyperalgesic thermal effects of CTK 01512–2 were due to Cav2.2 blocking (Souza et al., 2008). This has been proven in chronic neuropathic pain models by chronic constriction injuries (Rosa et al., 2014), or in peripheral mononeuropathy by partial ligation of the sciatic nerve (Souza et al., 2008). The current study's data shows that the dose of CTK 01512-2, which was administered in these experiments, was sufficient to increase the mechanical allodynia threshold at the third hour, lasting up to the fifth hour, like ziconotide. Morphine had its effect from the first hour of application, lasting up to the third hour, in this acute treatment (Cosgrave et al., 2017). The blocking of Cav2.2 led to decreased glutamate in the synaptic cleft; consequently, there was a modulation of the nociceptive pathway (Kuraishi et al., 1985). Thus, the current researchers' data confirms the mechanism actions of CTK 01512-2, by a decrease of glutamate in the cerebrospinal fluid of the treated rats. However, the difference was not significant between the groups, due to the reduced number of animals Table 1 Damage Index and Damage Frequency (Comet Assay) in the peripheral blood cells of the treated animals before the deafferentation of the upper trunk brachial plexus.
Control Sham PBS Morphine CTK 01512–2 Ziconotide
Damage index Mean ± SD
Damage frequency Mean ± SD
8.00 8.75 7.60 6.50 5.20 5.20
7.00 8.50 7.60 6.75 3.20 5.00
± ± ± ± ± ±
5.91 1.25 4.93 1.0 2.38 2.38
± ± ± ± ± ±
4.95 1.29 4.93 1.70 0.83 2.23
(n = 4 or 5 animals, statistical analysis by ANOVA, and Tukey's test). 5
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Fig. 4. Levels of TBARS in the homogenates of the spinal cord of the rats that were treated with PBS, morphine, CTK 01512–2, and ziconotide, when in comparison to the sham group. Each point and each vertical line represents the mean and SE values that were obtained from 7 animals. The statistical analysis was performed by using oneway analysis of variance (ANOVA), followed by the Student-NewmanKeuls post hoc test. (**p < .01 when compared to PBS/CTK 01512–2, PBS/Ziconotide, and PBS/Sham. ***p < .001 when compared to PBS/ Morphine).
Fig. 5. Graphical presentation of the main conclusion of study.
Antunes, Alessandra Hubner de Souza. Data collection: Flavia Tasmim Techera Antunes, Stephanie Gonçalves Angelo, Alice Gomes Ferraz. Data analysis and interpretation: Flavia Tasmim Techera Antunes, Stephanie Gonçalves Angelo, Elizangela Schemitt, Alessandra Hubner de Souza. Drafting the article: Flavia Tasmim Techera Antunes, Alessandra Hubner de Souza. Critical revision of the article and final approval of the version to be published: Alessandra Hubner de Souza, Jaqueline Nascimento Picada, Norma Possa Marroni, Marcus Vinicius Gomez, Eliane Dallegrave. Specimen collection: Alice Gomes Ferraz, Elizangela Schemitt, Eliane Dallegrave.
which it does not induce adverse effects. The assessments of genotoxicity in the peripheral blood showed no evidence of damage to the genetic material, as did the study by Souza et al. (2018), with a recombinant toxin in healthy animals. The current researchers have shown that rats with chronic peripheral deafferentation neuropathy have an impaired paw functionality, in addition to developing a central sensitization. An acute intrathecal administration of CTK 01512-2 has been effective in reducing mechanical allodynia, as well as cold and thermal hyperalgesia, without altering the locomotor and exploratory activities of the animals. These effects were mediated by the decrease of glutamate in the cerebrospinal fluid and by the reduction of lipoperoxidation of the membranes at the level of the spinal cord, without demonstrating genotoxic effects. 5. Conclusion
Declaration of Competing Interest
In the clinic, patients with an avulsion brachial plexus injury develop central sensitization and the available treatments are sparse and refractory, in addition to manifesting severe adverse effects or dose tolerances. Given that this present study has suggested that CTK 015122 has an analgesic potential in an animal model of pain by deafferentation (Fig. 5), it can provide a viable alternative treatment. However, repeated dose toxicity testing is necessary since the needs of patients with chronic pain usually use their medication for an extended period.
All of the authors declare no competing interests. Acknowledgment This research group was supported by FAPEMIG (Fundação de Amparo a Pesquisa do Estado de Minas Gerais), Belo Horizonte, State of Minas Gerais, Brazil. Appendix A. Supplementary data
Funding
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.npep.2019.101980.
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
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