N-terminal truncations of substance P1–7 amide affect its action on spinal cord injury-induced mechanical allodynia in rats

European Journal of Pharmacology 738 (2014) 319–325

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Neuropharmacology and analgesia

N-terminal truncations of substance P1–7 amide affect its action on spinal cord injury-induced mechanical allodynia in rats Anna Carlsson-Jonsson a,n, Tianle Gao b, Jing-Xia Hao b, Rebecca Fransson c, Anja Sandström c, Fred Nyberg a, Zsuzsanna Wiesenfeld-Hallin b, Xiao-Jun Xu b a

Department of Pharmaceutical Biosciences, Uppsala University, P.O. Box 591, SE-751 24 Uppsala, Sweden Department of Physiology and Pharmacology, Karolinska Institutet, Nanna Svartz väg 2, SE-171 77 Stockholm, Sweden c Department of Medicinal Chemistry, Uppsala University, P.O. Box 574, SE-751 24 Uppsala, Sweden b

art ic l e i nf o

a b s t r a c t

Article history: Received 16 February 2014 Received in revised form 30 April 2014 Accepted 23 May 2014 Available online 13 June 2014

Central neuropathic pain can arise from injury of the spinal cord and can become chronic. Treatment is difficult and, because complete pain relief is currently very hard to achieve, there is a need for new, more effective treatment options. In this study we used an animal model of spinal cord injury to evaluate the potency of a bioactive fragment of substance P (SP), i.e. SP1–7, in alleviating signs of allodynia and acute pain. SP1–7 is known from earlier studies to possess antinociceptive properties. We also studied the effects of intraperitoneal injection of an amidated analog of this heptapeptide and of its truncated analogs, all of which had high affinity to the SP1–7 binding site, to evaluate the importance of the removed amino acids for the biodistribution and stability of the peptides. Most of the examined compounds alleviated mechanical allodynia without any signs of sedation or motor impairment in the rats. In contrast, the response threshold to acute nociceptive stimulation was not affected by any of the compounds tested. Most of the amino acids in the heptapeptide structure were essential for retaining the biological effect after peripheral injection. These observations suggest that the heptapeptide and its N-terminal truncated hexa- and pentapeptide analogs could be of interest for further development of analgesics in the management of mechanical allodynia. & 2014 Elsevier B.V. All rights reserved.

Keywords: Chronic pain Neuropathic pain Spinal cord injury Substance P fragment SP1–7 Anti-allodynia Substance P-cleaving enzymes Chemical compounds studied in this article: Substance P (PubChem CID: 36511) Substance P(1–7) (PubChem CID: 115322)

1. Introduction Central pain can occur from a lesion or dysfunction in the central nervous system (CNS). It can arise from many different kinds of lesions in the brain or spinal cord, such as multiple sclerosis or spinal cord injury (SCI), and the pain causes suffering because of its constant and irritating character (Boivie, 2006). Chronic neuropathic pain due to SCI, which occurs in 26–96% of the patients (Dijkers et al., 2009) is difficult to treat. Results from studies of the anti-epileptic drugs gabapentin and pregabalin and the tricyclic antidepressant amitriptyline in patients with SCIrelated pain are conflicting (Rekand et al., 2012). It is uncertain whether treatment with opioid analgesics is favorable for the patient and complete pain relief is hard to achieve (Boivie, 2006). It is evident that new approaches to the medical treatment of neuropathic pain are needed. A major metabolite of the neuropeptide substance P (SP), known as SP1–7 ( Fig. 1), exhibits many effects opposing those of the parent compound, including analgesia in animal models of

n

Corresponding author. Tel.: þ 46 18 4715053. E-mail address: [email protected] (A. Carlsson-Jonsson).

http://dx.doi.org/10.1016/j.ejphar.2014.05.060 0014-2999/& 2014 Elsevier B.V. All rights reserved.

both acute and chronic pain (Carlsson et al., 2010; Stewart et al., 1982). Specific binding sites for SP1–7 that are distinct from the tachykinin receptors have been identified in rodent brain and spinal cord (Botros et al., 2006, 2008; Igwe et al., 1990). However, while the endogenous opioid tetrapeptide endomorphin-2 and the selective m-opioid receptor agonist DAMGO have equal affinity for this binding site, other opioids and tachykinins have very low, if any, affinity for it (Botros et al., 2006). Thus, SP1–7 and its binding site may offer a novel approach to the problem of chronic pain. We have previously synthesized pseudopeptides based on SP1–7 and endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) (Fransson et al., 2008; Fransson et al., 2010). Chemical modifications to endomorphin-2 and SP1–7 have generated interesting findings. For example, the amino acid Phe7 is essential for binding, C-terminal amidation of SP1–7 induces a five-fold increase in binding affinity, and N-terminal truncation of the heptapeptides to tripeptides does not affect the binding affinity (Fransson et al., 2008). Truncation of endomorphin-2 resulted in a dipeptide, Phe-Phe amide, with high affinity and potent effects after intrathecal administration (Fransson et al., 2010; Ohsawa et al., 2011) but the efficacy was lost after peripheral administration (unpublished data). We thus turned our focus to SP1–7 and its analogs with a C-terminal amide. Native SP1–7 induced antinociception when

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ACE SPE

DPP-IV

H-Arg1-Pro2-Lys3-Pro4-Gln5-Gln6-Phe7-Phe8-Gly9-Leu10-Met11-NH2

PPCE

NEP

Fig. 1. Possible sites for enzymatic cleavage of substance P. DPP-IV ¼ dipeptidyl peptidase IV; PPCE ¼post-proline cleaving enzyme; ACE¼ angiotensin-converting enzyme; SPE ¼substance P endopeptidase; NEP ¼ neutral endopeptidase.

administered peripherally to mice (Stewart et al., 1982) and dosedependently attenuated the vasodilation response when administered locally in an animal model of inflammation (Wiktelius et al., 2006). However, the effects on neuropathic pain of peripherally administered SP1–7 and truncated forms of SP1–7 thereof have not yet been evaluated in vivo. Thus, the aim of this study was to investigate the effect of peripherally administered SP1–7 and its amidated analog on SCI-induced pain. In addition, we administered N-terminal truncated fragments of the amidated heptapeptide in order to investigate the role of the N-terminal sequence in the peptide in terms of the overall biological activity.

2. Material and methods

Rat behavior was tested before and 15, 30 and 60 min after each peptide injection. 2.3.1. Mechanical allodynia Sensitivity to mechanical stimulation was tested by application of calibrated von Frey filaments (Stoelting, USA) onto the skin. The animal was gently restrained and the filaments were applied with increasing pressure until they bent. Each filament was applied 5– 10 times at a frequency of 1/s and the intensity of stimulation which induced vocalization at a 475% response rate was considered the pain threshold. 2.3.2. Tail-flick test The reaction of the animals to an acute nociceptive stimulus was tested with the tail-flick test. The animal was gently restrained and placed on a glass surface with a heated light source (Ugo Basile, Italy) underneath, focused at the tip of the tail. The intensity of the light was set so that the animal responded within 10 s. The time from the onset of the heat source to the withdrawal of the tail was registered. 2.3.3. Motor tests A combined motor test of walking in an open field and a righting reflex to detect the potential motor and sedative effect of SP and its fragments in spinally injured rats has been used, as described by Gao et al. (2013). The following scores were used in

2.1. Animals Female Sprague-Dawley rats (Möllegård, Denmark), weighing 250 g at the start of the experiment, were housed four per cage in an animal house with a 12-h light/dark cycle and a constant temperature of 22 71 1C. Food and water was available ad libitum. The experiments were carried out according to the ethical guidelines of the International Association for the Study of Pain and were approved by the local animal research ethics committee. 2.2. Photochemically induced spinal cord ischemic injury (SCI) An ischemic SCI was induced photochemically as described previously (Hao et al., 2004, 1996; Xu et al., 1992). Briefly, animals were anaesthetized with 0.5 mg/kg intraperitoneal (i.p.) medetomidine (Domitor, LÄÄkefarmos, Finland) and 60 mg/kg i.p. ketamine (Ketalar, Parke-Davis, Sweden) and one jugular vein was cannulated. A midline incision was made on the skin overlying the vertebral segments T12–L1. The animals were positioned with the vertebral segment T12 or T13 (spinal segments L3–5) beneath the beam of a tunable argon laser (Innova model 70, Coherent Laser Product Division, Palo Alto, CA; operating at 514 nm with an average power output of 160 mW) and irradiated for 10 min. Immediately prior to and 5 min after the start of the irradiation, erythrosine B (Red N13, Aldrich-Chemie, Steinheim, Germany) dissolved in 0.9% saline was injected intravenously at a dose of 32.5 mg/kg. The rat temperature was maintained at 37–38 1C during the irradiation. The animals were allowed to recover from surgery for one week before any behavioral testing. 2.3. Behavioral tests The behavior of the rats was tested weekly to establish the development and maintenance of allodynia. The peptides were evaluated approximately 8 weeks after induction of the SCI for their effects on mechanical allodynia (Section 2.3.1), acute antinociception (Section 2.3.2) and motor impairment (Section 2.3.3).

Fig. 2. Effects of spinal cord injury (SCI) on mechanical and nociceptive stimulation in rats. (A) Hypersensitivity to mechanical stimulation of the flanks with von Frey hairs developed within one week after SCI (n¼ 8). Data were plotted as medians 7MAD for vocalization threshold; nPo 0.05 vs baseline (Wilcoxon Signed Rank test). (B) There are no significant changes in tail-flick latency after SCI (n¼ 8; paired t test). Data are plotted as means 7S.E.M.

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the tests. Walking: 0 ¼normal walking; 5 ¼walks with only mild deficit; 15 ¼hindlimb can support weight; 25¼ frequent movement of hindlimb, no weight bearing; 40¼minor movement in hindlimb, no weight bearing; 45¼ no movement of hindlimb, no weight bearing;Righting: 0 ¼normal righting counter to direction of the roll; 5 ¼weakened attempt to righting; 10 ¼delayed attempt to righting; 15 ¼no attempt to righting. The results are illustrated in Fig. 6.

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(50  4.6 mm) and ACE 5 Phenyl (50  4.6 mm) columns with an H2O/MeCN gradient and 0.1% trifluoroacetic acid and ultraviolet detection at 220 nm. All the tested peptides were 498% pure (Fransson et al., 2008). The peptides were dissolved and diluted in acidified saline (0.9% saline with 0.01 M acetic acid) and acidified saline was used as control. Cumulative doses of the peptides (1.85, 18.5 and 185 nmol/kg) were administered i.p. at 60 min intervals; the doses were adapted from Stewart et al. and Zhou et al. (Stewart et al., 1982; Zhou et al., 2011).

2.4. Chemicals 2.5. Statistics SP was purchased from Bachem (Bubendorf, Switzerland), SP1–7 was purchased from Polypeptide Group, Strasbourg, France, SP1–7 amide, SP2–7 amide, SP3–7 amide and SP4–7 amide were prepared as described previously (Fransson et al., 2008) using solid-phase peptide synthesis, with Fmoc protection. The crude peptides were purified by reverse-phase high-performance liquid chromatography (RP-HPLC), pooled and lyophilized. The purity of each peptide was determined by RP-HPLC using ACE 5 C18

The data are expressed as medians 7the median absolute deviation (MAD) or means 7the standard error of the mean (S.E.M.). ANOVA with repeated measurements was followed by Fisher's PLSD test to identify any differences between the treatment groups. Post-drug time point results and baselines were compared using the Wilcoxon Signed Rank test (mechanical stimulation threshold) or Dunnett's test (thermo-latency), and

Fig. 3. Effects of (A) substance P (SP) fragment SP1–7, (B) SP1–7 amide, (C) SP2–7 amide, (D) SP3–7 amide, (E) SP, and (F) saline, on vocalization thresholds in spinal cord-injured (SCI) rats with mechanical stimulation hypersensitivity. The data are plotted as means7 S.E.M; n¼ 8 in all except B, where n¼ 6. Fisher's PLSD test following ANOVA with repeated measurements indicates that there is a significant difference (po 0.05) in vocalization threshold between group B (receiving SP1–7 amide) and the control group F (receiving saline). nPo 0.05 versus baseline (time 0; Wilcoxon Signed Rank test). #P o 0.05 versus the vocalization threshold for saline-treated SCI rats at the corresponding time point (Mann–Whitney U-test). SP has no significant effects on vocalization thresholds (E).

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the thresholds of the drug-treated groups and the saline-treated vehicle group at each time point were compared using the Mann– Whitney U test (mechanical stimulation threshold) or the unpaired t test (thermo-latency).

3. Results As can be seen in Fig. 2A, animals that underwent SCI surgery developed hypersensitivity to mechanical stimulation of the flanks with von Frey hairs within one week; the allodynia remained stable during the 8-week observation period. In contrast, tail-flick latencies were not altered by surgery and remained unaltered throughout the study period (Fig. 2B). The pharmacological study was performed 8 weeks after surgery. I.p. administration of SP1–7 (Fig. 3A) and the SP1–7 amide (Fig. 3B) reduced mechanical allodynia. A significant anti-allodynic effect versus baseline (P o0.05) was detected 15 and 30 min after administration of the highest dose of SP1–7 (185 nmol/kg). In contrast, a significant anti-allodynic effect was detected with only 18.5 nmol/kg of SP1–7 amide indicating higher potency than SP1–7. When the truncated peptides were tested, it was found that certain amino acids could be removed without any changes to the pharmacological effect. For example, removal of the N-terminal Arg1 yielded a peptide fragment (SP2–7 amide) with an anti-allodynic effect in the same range as that of the native heptapeptide, with a significant effect 30 min after administration of the highest dose (Fig. 3C). Similarly, removal of an additional amino acid (Pro2) at the N-terminal end (SP3–7 amide) did not alter the anti-allodynic effect (Fig. 3D). Thus, both the N-terminal truncated pentapeptide and the hexapeptide retained the antiallodynic activity of the heptapeptide entity. However, further truncation by removing the Lys3 (SP4–7 amide; Fig. 4A) and then the Pro4 (SP5–7 amide; Fig. 4B) resulted in complete loss of this activity.

Fig. 4. Effects of (A) SP4–7 amide and (B) SP5–7 amide on mechanical stimulation hypersensitivity in spinal cord-injured rats. The data are plotted as means 7S.E.M; n¼ 8 for both A and B. No significant effects are observed with the Wilcoxon Signed Rank test or Mann–Whitney U test.

To investigate whether SP mimics the effects of the SP fragments in terms of modulating the allodynic response in the SCI animals, SP was injected and evaluated in the same manner as the peptide fragments. No change in vocalization threshold was detected at any time point (Fig. 3E). In the tail-flick assay, neither SP nor any of the peptide fragments included in this study had an analgesic effect, at any dose tested (Fig. 5). The pharmacological effects on mechanical allodynia are summarized in Table 1. I.p. injection of saline had no effect. No signs of motor impairment or sedation were noted after administration of any peptide, at any dose tested (Fig. 6).

4. Discussion This study clearly demonstrates that a peripheral injection of the endogenous SP fragment SP1–7 can alleviate signs of mechanical allodynia in rats with SCI. The amidated analog was even more potent, as demonstrated by an equivalent significant anti-allodynic effect at a 10-fold lower dose than that of the endogenous compound. Truncation of the heptapeptides generated smaller molecules with sustained affinity to the binding site (Fransson et al., 2008). Removal of one or two amino acids resulted in hexaand pentapeptides with retained anti-allodynic effect. However, further truncation led to complete loss of the pharmacological effect. Importantly, the tested peptides did not generate any signs of sedation in the animals, even at the highest dose tested. Molecules with anti-allodynic effects and without a sedative component have great potential for development of specific effective drugs for the treatment of allodynia. The enzymes responsible for the metabolism of SP (Fig. 1) might also act on fragments of SP, thus potentially explaining the lack of effect of the tri- and tetrapeptides in the study. Post-proline cleaving enzyme degrades SP at the Pro4–Gln5 position (Yoshimoto et al., 1981) and dipeptidyl peptidase IV cleaves the bonds between Pro2–Lys3 and Pro4–Gln5 (Ahmad et al., 1992). In addition, angiotensin-converting enzyme could act as a dipeptidyl peptidase on SP1–7, yielding dipeptide fragments (Yokosawa et al., 1983). Thus, it is possible that these enzymes could degrade the tri-and tetrapeptides before they reach their target (and faster than the degradation of SP1–7) resulting in loss of efficacy. Another explanation for the discrepancy between the binding data and the pharmacological effects could be altered cell permeability to the shorter peptides. Thus, the loss of two amino acids with positive charges, Arg1 and Lys3, from the original heptapeptide to form the SP4–7 and SP5–7 fragments might not necessarily affect the binding affinity, but could negatively affect the permeation of the molecules through the relevant tissues, making it more difficult for the fragments reach their targets. The size and presence of positively charged residues in peptides are known to be relevant factors in determining the permeability of cells and the blood–brain barrier to the molecules, e.g. in adsorptive endocytosis (Brasnjevic et al., 2009). These factors have, in fact, demonstrably affected the passage of SP through the blood–brain barrier in vitro (Freed et al., 2002). It thus appears that most of the N-terminal sequence of SP1–7 must be retained in order to keep the analgesic effect in vivo. Consequently, if analgesic compounds are to be developed from the heptapeptide, the N-terminal sequence should be considered in addition to the Phe7 at the C-terminal, which is already known to be crucial for binding (Fransson et al., 2008) and thus for the pharmacological effect. An interest in peptides as potential drug candidates has been resurrected recently, as a result of the decreasing number of approved drugs. In fact, the use of synthetic strategies for limiting metabolism and the exploration of alternative routes of administration have enabled a large number of peptide-based drugs to reach the market (Vlieghe et al., 2010). This

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Fig. 5. Effects of (A) substance P fragment SP1–7, (B) SP1–7 amide, (C) SP2–7 amide, (D) SP3–7 amide, (E) SP, and (F) saline, on tail-flick latency in spinal cord-injured rats; n¼ 8 in all except B, where n¼ 6. The data are plotted as means 7 S.E.M. Dunnett's test or the unpaired t test indicate no effects of any of the compounds on tail-flick latency.

clearly demonstrates that it is not necessary to remove the peptide character of potential drug molecules completely and, in fact, small pseudopeptides can be used as drugs. A peptide-based product with the ability to alleviate chronic neuropathic pain, which could have great potential from a clinical perspective, is thus not out of the question. Chronic pain is a common health problem (the prevalence ranges from 12% to 30% in the general population) that often has unsatisfactory treatment results and is possibly undertreated (Breivik et al., 2006; Harker et al., 2012; Moulin et al., 2002; Schopflocher et al., 2011). The animal SCI model (Hao et al., 2004) is believed to represent chronic central pain in humans well; like humans, the animals display long-lasting symptoms such as spontaneous pain and allodynia. Although gabapentin is effective in relieving allodynia (Wu et al., 2004), the SCI animal model has indicated high treatment resistance: baclofen, clonidine, pentobarbital and carbamazepine did not alleviate allodynia, or did so only slightly (Xu et al., 1992). Allodynia is also relatively insensitive to morphine, which was analgesic only in sedative doses in the model (Hao et al., 2004). Thus, the SCI animal model reflects the problems encountered in treating chronic central pain in humans.

In this study, SP1–7 and its amide reduced mechanical hypersensitivity in SCI rats, but did not alter tail flick latency. We have previously shown that the hypersensitivity to mechanical stimulation in these rats is probably mediated by input through myelinated A-fibers, since it was not affected by the ultrapotent capsaicin analog resiniferatoxin, while the tail flick latency, which is mediated by unmyelinated C-fibers, was markedly prolonged (Hao et al., 1996). An important issue in this context is the mechanism of action of the SP fragment and its analogs. Although the peptides investigated were fragments of SP, their mechanism of action differs from that of the native undecapeptide. The affinity of SP1–7 for its specific binding sites in rat and mouse brain and spinal cord greatly exceeds that of SP and the SP C-terminal fragments. SP1–8 exhibits some affinity for the SP1–7 binding sites, while C-terminal truncated forms of the SP1–7 sequence have no or negligible affinity, and none of the typically used selective ligands for tachykinin receptors have significant affinity (Botros et al., 2006). All the SP fragments tested in this study have high affinity for the SP1–7 binding sites, while the affinity of SP itself is 100-fold lower (Table 1). In line with this, only the compounds with a high affinity

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for the SP1–7 binding sites had an anti-allodynic effect in this model, and the mechanical stimulation threshold did not change after SP administration. The fact that SP1–7 does not have any affinity for the neurokinin-1 (NK1) receptor (Vigna, 2001) further strengthens the theory that the SP1–7 binding site is unique, and distinct from the NK1 receptor (Botros et al., 2006; Igwe et al., 1990).

Since SP has been shown to possess both pronociceptive and antinociceptive properties (Stewart et al., 1976), the question may arise as to why it had no pharmacological effect in this study. In previous studies, the antinociceptive effect of SP occurred about 30 min after it was injected into the brain, which indicates that it might need to be enzymatically cleaved, possibly to the bioactive heptapeptide fragment, before producing its antinociceptive effect

Table 1 Amino acid sequences and Ki values for SP1–7 radioligand binding to rat spinal cord membrane (Ki data by Fransson et al., 2008, SP data from Botros et al., 2006), and effects of the respective peptides on mechanical allodynia. Peptide

Amino acid sequence

Ki 7 S.E.M. (nM)

Effect

SP SP1–7 SP1–7 SP2–7 SP3–7 SP4–7 SP5–7

H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-OH H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-NH2 H-Pro-Lys-Pro-Gln-Gln-Phe-NH2 H-Lys-Pro-Gln-Gln-Phe-NH2 H-Pro-Gln-Gln-Phe-NH2 H-Gln-Gln-Phe-NH2

159 712 1.6 70.06 0.3 70.02 2.8 70.25 4.4 70.1 4.5 70.3 1.9 70.05

— þ þþ þ þ — —

amide amide amide amide amide

(þ moderate effect; þ þ good effect; –no effect).

Fig. 6. Effects of (A) substance P fragment SP1–7, (B) SP1–7 amide, (C) SP2–7 amide, (D) SP3–7 amide, (E) SP, and (F) saline, on motor deficits in spinal cord-injured rats; n ¼8 in all except B, where n ¼6. The data are plotted as means 7 S.E.M. No significant effects are observed with the Wilcoxon Signed Rank test or Mann–Whitney U test.

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(Stewart et al., 1976, 1982). However, the conversion rate of SP into bioactive fragments appears to be quite slow in the rat brain, as the ratio between SP and SP1–7 is quite high in several brain regions (Hallberg et al., 2000). This slow rate could mean that SP is only partly metabolized, not enough to be able to generate any behavioral change in the animals. The lack of a pronociceptive effect in the assay might be due to the reduced mechanical stimulation threshold caused by the SCI. However, the exact anti-allodynic mechanism of action of these peptides at a molecular level has not yet been fully clarified. Although the specific binding sites for SP1–7 in the CNS indicate unique sites distinct from the known tachykinin or opioid receptors, SP1–7 may also affect peripheral receptors, since it has antivasodilatory properties (Wiktelius et al., 2006). It is unclear exactly where the relevant binding sites are located. As with many other peptides, it thus appears that SP1–7 may have both peripheral and central effects. In accordance with this, it appears that the SP1–7 peptide fragment can be produced both centrally and peripherally, since enzymes capable of generating it are available in both the CNS and the periphery (Persson et al., 1995) as well as at the blood–brain barrier (Freed et al., 2002). in vitro studies have shown that SP and its C- and N-metabolites can pass this barrier (Freed et al., 2002). The alleviating effect of peripherally administered SP1–7 on the development of opioid tolerance (Zhou et al., 2011), which is centrally mediated, indicates that it is able to reach the CNS in vivo. Behavioral symptoms such as allodynia in SCI rats appear to be the result of neuronal changes in the dorsal horn of the spinal cord, around the injury (Hao et al., 2004), and abnormal input by the primary afferents (Wiesenfeld-Hallin et al., 1997). Thus, the ability of the studied compounds to ameliorate this type of pain-like behavior could indicate that they are acting on spinal or supraspinal sites. Additional studies are needed to confirm this hypothesis. Nonetheless, the results presented here suggest that the examined peptides interact with pain-processing pathways that reach the CNS.

5. Conclusions We have shown that peripheral injection of the endogenous bioactive peptide fragment SP1–7 into animals with SCI can reduce signs of mechanical allodynia without any signs of sedation or motor impairment. Moreover, amidation of the heptapeptide results in an even more potent response. The analgesic effects were preserved on truncation of the SP1–7 amide to generate hexa- and pentapeptides, but further truncation led to complete loss of the biological activity. This change in efficacy is thought to be associated with enzymatic inactivation of the molecules or altered cell permeability. These results are of potential interest for the development of SP1–7 pseudopeptides and increase the hope of finding effective strategies for the treatment of human neuropathic pain.

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