Effect of Peptidase Inhibitors on Dynorphin A ( 1-17 ) or ( 1-13 )-Induced Antinociception and Toxicity at Spinal Level

Our group has earlier demonstrated that three enzymes sensitive to peptidase inhibitors (PIs), amastatin (A)-, captopril (C)-, and phosphoramidon (P), played an important role in inactivation of enkephalins at the spinal level. Dynorphin-converting enzyme (DCE) hydrolyzes dynorphin (Dyn) A (1-17) or Dyn A (1-13) mainly at the Arg-Arg bond. Dynorphin A and its derived peptides interact with opioid and glutamate receptors at their Nand C-terminals, respectively. The purpose of the present study was to evaluate the antinociceptive potency and toxicity of intrathecal administered Dyn A (1-17), Dyn A (1-13), or Dyn A (1-6) under pretreatment with ACP and/or the DCE inhibitor p-hydroxymercuribenzoate (PHMB). The effect of these PIs on Dyn A (1-17)-induced inhibition of electrically-evoked contractions in mouse vas deferens was also investigated. The inhibitory potency of Dyn A (1-17) on electrically-evoked contractions in mouse vas deferens under pretreatment with ACP was higher than that with AC, AP, or CP. Pretreatment with ACP augmented Dyn A (1-17) or (1-13)-induced antinociception by approximately 50or 30-fold with no sign of allodynia when administered intrathecally at low doses. Pretreatment with ACP and PHMB induced neuropathy. These findings showed that intrathecal administration of low-dose Dyn A (1-17) or DynA (1-13) increased antinociception under pretreatment with ACP, but without signs of allodynia in rat.

Three peptidases, an aminopeptidase N (APN), a dipeptidylcarboxypeptidase, and neutral endopeptidase-24.11(NEP), play an important role in degradation of opioid peptides.High-performance liquid chromatography revealed that [Leu 5 ]enkephalin (LE) [11] or Dyn A (1-8) [12] remained intact in the presence of a mixture of peptidase inhibitors (PIs) when incubated with membrane preparation, but was completely hydrolyzed after incubation in their absence.
The purpose of the present study was to evaluate antinociceptive potency and toxicity with i.t.administration of Dyn A (1-17), Dyn A (1-13), or Dyn A (1-6) under pretreatment with ACP and/or DCE inhibitor p-hydroxymercuribenzoate (PHMB) in rat spinal cord under physiological conditions.In an attempt to further characterize the pharmacological action of PIs, the effect of ACP on Dyn A (1-17)-induced inhibition of electrically-evoked contractions in MVD was also evaluated.

Materials and Methods
The present animal experiments were performed in strict accordance with the guidelines (http://www.u-tokai.ac.jp/about/concept/guidance.html) of Tokai Uni-versity and with the approval of the Animal Investigation Committee of this institute.

Intrathecal Administration
Intrathecal catheters were implanted in Male Wistar rats (180 -220 g each; Nihon Clea, Tokyo, Japan) under inhalation anesthesia with nitrous oxide, oxygen, and isoflurane (2%) as described previously [17] [25].After surgery, all rats were housed individually in a temperature-and light-controlled environment with free access to food and water.Only rats with normal motor function and behavior were used for the experiments 7 days later.Drugs were injected at a volume of 10 μl followed by 10 μl saline over 1 min.

Tail-Flick Test
The investigators were blind to all drug treatments carried out in these experiments.Induction of antinociception by Dyn A (1-17), Dyn A (1-13), or Dyn A (1-6) was measured by the tail immersion assay, with 55˚C as the nociceptive stimulus [26] [27].The latency to flick the tail was measured as described previously [17].A cut-off time of 5 sec was used to prevent any injury to the tail.The % of maximal possible effect (MPE) for each animal at each time was calculated using the following formula: %MPE = [(test latency − baseline latency)/(5 − baseline latency)] × 100.The area under the curve (AUC) value for the antinociceptive action of each drug was also calculated in some of the experiments.

Von Frey Test
The threshold for tactile allodynia was measured with a series of von Frey filaments (von Frey Filaments; Bioseb, Vitrolles, France), ranging from 2.44 to 5.88 (0.03 -60.0 g), according to the methods of Park et al. [28] and Zhu et al. [29].
The rats were placed in individual transparent plastic boxes with a wire mesh floor at least 15 min before testing began to allow acclimatization to the environment.The filaments were pushed against the plantar surface of the right hind paw.Tactile thresholds were measured at intervals before and after administration of drugs.Results were reported as the mean value of 4 readings from the right hind paw in each rat.

Statistical Analyses
The results are given as the mean and standard error of the mean (S.E.M.) of the data.The statistical analysis was conducted using computer software (Prism, version 6.0 c, Graph Pad Software, San Diego, CA, USA) for a comparison across experimental conditions.When a significant difference among the %MPE data after drug administration was obtained in a two-way (drugs and time) repeated measures analysis of variance (ANOVA), Dunn's multiple comparison test was applied to determine the significance at each time point.When a significant difference was observed in the AUC data among the groups in a two-way (drugs and dose) repeated ANOVA, Dunn's multiple comparison test was applied to determine the significance at each dose.When a significant difference within groups was obtained in the Kruskal-Wallis test, Dunn's comparison test was applied to determine significance.

Enhanced Effect of PIs in Paired Combinations or all Together on Dyn A (1-17)-Induced Inhibition in Isolated Preparation
The results showed that Dyn A (1-17) significantly inhibited electrically-evoked contractions in MVD.The inhibitory potency of Dyn A (1-17) was dose-dependently augmented by ACP (Table 1).Administration of paired combinations (2 µM each of AP, AC, or CP) or all three PIs together (2 µM ACP) revealed that any two combination or ACP increased Dyn A (1-17)-induced inhibition of electrically-evoked contractions in MVD.This effect was significantly stronger with ACP than with CP; it was also stronger than with administration of AP or AC, but not significantly so (Table 2).

Effect of PHMB on Dyn A (1-17)-Induced Antinociception
The antinociceptive potency of 0.3 nmol Dyn A (1-17) under pretreatment with ACP was significantly higher than that of 0.3 nmol Dyn A (1-17) alone or with PHMB (10 nmol) (Figure 7).The antinociceptive potency of 0.  of the tail flick and von Frey tests in all of four of these animals are reported here.The two remaining rats, however, showed prolonged and severe paralysis, making both the tail flick and von Frey test unviable.Thus, 4 out of 6 rats showed some signs of neuropathy following i.t.administration of 0.3 nmol Dyn A (1-17) under pretreatment with both PHMB and ACP.

ACP Attenuates Allodynia Induced by i.t. Administration of Dyn A (1-17)
No significant differences were observed in the baseline threshold stimulus be-

Discussion
The present results revealed that the antinociceptive potency of Dyn A (1-17), Dyn A (1-13), or Dyn A (1-6) depended on the dose and length of the peptide when administered i.t. in the absence of, or under pretreatment with, ACP.This is in good agreement with the results of earlier studies showing that Dyn A (1-17)-induced antinociception at the supraspinal level was greater than that of Dyn A (1-6) or Dyn A (1-13) at the same dose [17], and that Dyn A (1-17) had a greater analgesic effect than Dyn A (1-13) in the absence of ACP [30].Pretreatment with ACP increased antinociception with low-dose Dyn A (1-17), with neither hyperalgesia nor paralysis.The present findings then provide further support for the view that C-terminal peptide fragments of Dyn A (1-17), such as Dyn A (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17), are catabolized into shorter products that are extremely weak toxicity under pretreatment with ACP [17], thus reducing neuropathic symptoms.Intrathecal administration of Dyn A (1-17) and LE [31] under pretreatment with ACP augmented antinociception by at least 50-and 100-fold, respectively.These findings coincide well with the results of earlier histological studies showing that the distribution of NEP and APN was highly concentrated in the substantia gelatinosa of the spinal cord, a region closely associated with µ-opioid receptors and enkephalins [32] [33] [34] [35].Taken together, this suggests that this co-localization of peptidases and opioid peptides plays a critical role in nociception through the latter's inactivation in the spinal cord.
Pretreatment of paired combinations (2 µM each of AP, AC, or CP) or all three PIs together (2 µM ACP) revealed that AP or ACP significantly increased Dyn A (1-17)-induced inhibition of electrically-evoked contractions in MVD.This effect was significantly stronger with ACP than with AC or CP; it was also stronger than with administration of AP, but not significantly so.These results demonstrate that ACP is required to inhibit degradation of intact Dyn A (1-17), and that any residual pair of peptidases inactivates substantial amounts of Dyn A (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17).This is in good agreement with the results of an earlier study by this group demonstrating that the antinociceptive potency of Dyn A (1-17) was higher under pretreatment with ACP than with any paired combination of these PIs at the supraspinal level [17].
Neuropathy was observed in a total of 4 out of 6 rats after i.t.administration of 0.  [10].Further support for this hypothesis comes from evidence that high doses of Dyn A enhance intracellular levels of Ca 2+ via simultaneous activation of NMDA and κ-opioid receptors, whereas low doses activate only κ-opioid receptors [7].

Conclusion
In conclusion, the present results showed that inactivation of A-, C-, or P-sensitive enzymes leads to an increase in low-dose Dyn A (1-17)-induced antinociception without signs of allodynia at the spinal level.The antinociceptive potency and induction of allodynia by Dyn A (1-17), Dyn A (1-13), or their peptide fragments depended on their dose and length.The present findings suggest that PIs and other inhibitors of opioid peptide-degrading enzymes may have potential as novel therapeutic compounds for treatment of pain.
Change over time in Dyn A (1-17)-induced antinociception with i.t.administration of saline and ACP is shown in Figure 1(a) and Figure 1(b), respectively.

Figure 1 .
Figure 1.Dose-dependent antinociception by i.t.administration of Dyn A (1-17) under pretreatment with saline or ACP.Upper (a) and middle panels (b) indicate time course of %MPE of Dyn A (1-17) (0.03 -1 nmol) under pretreatment with saline and ACP, respectively.Significantly different from saline-saline or ACP-saline treated control by Dunn's post-hoc test following two-way repeated measures ANOVA; *P < 0.05, **P < 0.01, and ***P < 0.001.Lower panel (c) shows AUC 0-120min for value of %MPE indicated in upper (a) and middle panels (b).Where asterisks have been placed above AUC 0-120min values for Dyn A (1-17) under pretreatment with ACP, this indicates significant differences in comparison with for saline alone or saline under pretreatment with ACP according to Dunn's post-hoc test following the Kruskal-Wallis test; *P < 0.05, **P < 0.01, and ***P < 0.001.Where sharp symbols have been placed above AUC 0-120min values for Dyn A (1-17) under pretreatment with ACP, this indicates significant differences in comparison with those for Dyn A (1-17) under pretreatment with saline according to Dunn's post-hoc test following two-way repeated measures ANOVA; ## P < 0.01 and ### P < 0.001.

Figure 2 .
Figure 2. Dose-dependent antinociception by i.t.administration of Dyn A (1-13) under pretreatment with saline or ACP.Upper (a) and middle panels (b) indicate time course of %MPE of Dyn A (1-13) (0.1 -3 nmol) under pretreatment with saline and ACP, respectively.Significantly different from saline-saline treated control in Dunn's post-hoc test following two-way repeated measures ANOVA; *P < 0.05, **P < 0.01, and ***P < 0.001.Lower panel (c) shows AUC 0-120min for value of %MPE indicated in upper (a) and middle panels (b).Where asterisks have been placed above AUC 0-120min values for Dyn A (1-13) under pretreatment with ACP, this indicates significant differences in comparison with for saline alone or saline under pretreatment with ACP according to Dunn's post-hoc test following the Kruskal-Wallis test; *P < 0.05 and **P < 0.01.Where sharp symbols have been placed above AUC 0-120min values for Dyn A (1-13) under pretreatment with ACP, this indicates significant differences in comparison with those for Dyn A (1-13) under pretreatment with saline according to Dunn's post-hoc test following two-way repeated measures ANOVA; ## P < 0.01 and ### P < 0.001.

Figure 4 (Figure 3 .
Figure 4(a) shows change over time in Dyn A (1-6)-induced antinociception after i.t.administration of saline, while Figure 4(b) shows that with ACP.A

Figure 4 .
Figure 4. Dose-dependent antinociception by i.t.administration of Dyn A (1-6) under pretreatment with saline or ACP.Upper (a) and middle panels (b) indicate time course of %MPE of Dyn A (1-6) (0.3 -3 nmol) under pretreatment with saline and ACP, respectively.Significantly different from saline-saline treated control in Dunn's post-hoc test following two-way repeated measures ANOVA; *P < 0.05, **P < 0.01, and ***P < 0.001.Lower panel (c) shows AUC 0-60min for value of %MPE indicated in upper (a) and middle panels (b).Where asterisks have been place above AUC 0-60min values for Dyn A (1-6) under pretreatment with ACP, this indicates significant differences in comparison with saline under pretreatment with ACP according to Dunn's post-hoc test following the Kruskal-Wallis test; **P < 0.01.Where sharp symbols have been place above AUC 0-60min values for Dyn A (1-6) under pretreatment with ACP, this indicates significant differences in comparison with that for Dyn A (1-6) under pretreatment with saline according to Dunn's post-hoc test after two-way repeated measures ANOVA; ### P < 0.001.

Figure 7 .
Figure 7. Antinociception by i.t.administration of Dyn A (1-17) under pretreatment with saline or ACP together with/without PHMB.Upper panel (a) indicates time course of %MPE of Dyn A (1-17) (0.3 nmol) under pretreatment with saline and ACP with and without PHMB, respectively.Significantly different from saline-ACP treated control in Dunn's post-hoc test following two-way repeated measures ANOVA; *P < 0.05, **P < 0.01, and ***P < 0.001.Lower panel (b) shows AUC 0-120min for value of %MPE indicated in upper panel (a).Significantly different from saline-ACP treated control according to Dunn's post-hoc test following Kruskal-Wallis test; *P < 0.05 and **P < 0.01.

Table 1 .
IC 50 values and ratio of potency of Dyn A (1-17) under pretreatment of ACP.
Each value represents mean ± S.E.M. of data obtained from four MVD.The ratio of potency was significantly increased in 2 or 5 µM ACP as compared to these in 0 µM ACP; *P < 0.05 by Dunn's post-hoc test following Kruskal-Wallis test.

Table 2 .
IC 50 values and ratio of potency of Dyn A (1-17) under pretreatment of combination of PIs.