Structure-Based Optimization of Multifunctional Agonists for Opioid

Oct 31, 2016 - *For R.W.: phone, +86-9318912567; fax, 86-931-891-1255; E-mail, [email protected]., *For Q.F.: phone, +86-9318915322; E-mail, ...
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Structure-Based Optimization of Multifunctional Agonists for Opioid and Neuropeptide FF Receptors with Potent Nontolerance Forming Analgesic Activities Zi-Long Wang, Jia-Xin Pan, Jing-Jing Song, Hong-Hai Tang, Hong-Ping Yu, Xu-Hui Li, Ning Li, Ting Zhang, Run Zhang, Meng-Na Zhang, Biao Xu, Quan Fang,* and Rui Wang* Key Laboratory of Preclinical Study for New Drugs of Gansu Province, and Institute of Physiology, School of Basic Medical Sciences, Lanzhou University, 199 Donggang West Road, Lanzhou, 730000, PR China S Supporting Information *

ABSTRACT: The opioid and neuropeptide FF pharmacophore-containing chimeric peptide 0 (BN-9) was recently developed and produced potent nontolerance forming analgesia. In this study, 11 analogues of 0 were designed and synthesized. An in vitro cAMP assay demonstrated that these analogues behaved as multifunctional agonists at both opioid and NPFF receptors. In mouse tail-flick test, most of the analogues produced potent nontolerance forming antinociception. Notably, 11 (DN-9) was 33-fold more potent than 0 at analgesic effects, which was mediated by μ- and κ-opioid receptors. In addition, 11 also produced powerful analgesic effects in the formalin pain and CFA-induced chronic inflammatory pain models. Strikingly, following its repeated administration for 6 days, 11 did not produce antinociceptive tolerance in the tailflick test and CFA-induced pain model. The present work indicates that it is reasonable to design multifunctional peptide ligands for opioid and NPFF receptors in a single molecule producing effective nontolerance forming antinociception.



INTRODUCTION Pain is an unpleasant sensory and emotional experience and serves a protective role to warn us of potential injuries or diseases. However, chronic pain can significantly influence the individual’s quality of life, and millions of people are suffering from various pains worldwide.1,2 The treatment of pain, especially chronic pain, is a major challenge in the clinic. Although opioid analgesics play important roles in the treatment of severe acute pain and chronic pain,3,4 currently available opioid analgesics have a number of unwanted side effects that hamper their clinical use, particularly in chronic pain management.3,4 Therefore, the need to develop novel analgesics with enhanced efficacy and limited side effects is becoming more and more urgent. The generation of pain is very complex, and numerous neurotransmitters and inflammatory mediators participate in the pain pathway.5−7 The opioid system is one of the main systems engaged in pain perception.3 The activation of opioid receptors can strongly inhibit pain transmission but is also the source of the undesirable effects of opioid analgesics.3 Previous studies have revealed that some neurotransmitters could modulate opioid activities.8,9 Therefore, to reduce or eliminate opioid-related side effects, developing compounds that target opioid receptors and some other receptors with modulatory effects on opioid activities might be a promising strategy.10−13 The successful examples of this strategy include bifunctional opioid compounds that target opioid receptors and the nociceptin receptor, neurokinin-1 receptor (NK-1), cannabi© 2016 American Chemical Society

noid receptor 1 (CB1), cholecystokinin type 2 receptor (CCK2), or metabotropic glutamate receptor 5 (mGluR5).14−21 Nociceptin is one of the major compounds for developing bifunctional opioid analgesics, and a number of bifunctional compounds have been developed.20,22−24 Recently, Guillemyn et al. reported that a hybrid peptide behaved as an agonist at opioid receptors and an antagonist at the nociceptin receptor. This hybrid peptide permeates the BBB and produces potent antinociception after intravenous administration with limited respiratory depression.20 Hruby and colleagues have developed a series of bifunctional peptides with both the opioid agonist and NK-1 antagonist pharmacophores, including TY005, TY027, NP30, and their analogues.15,25,26 These compounds exhibit potent analgesia in both acute and chronic pain models with limited tolerance. PK20 and [Ile9]PK20, two chimeric peptides with both opioid and neurotensin pharmacophores, produced potent antinociceptive effects and had limited motor coordination impairments.27,28 Taken together, these bifunctional opioid compounds induce powerful analgesic effects with limited side effects such as a lack of antinociceptive tolerance, respiratory depression, and motor coordination impairments. Neuropeptide FF (NPFF) was identified as an important endogenous opioid-modulating peptide and causes complicated opioid-modulating actions.8,29−31 NPFF blocks opioid-induced antinociception at the supraspinal level32,33 but induces an Received: August 9, 2016 Published: October 31, 2016 10198

DOI: 10.1021/acs.jmedchem.6b01181 J. Med. Chem. 2016, 59, 10198−10208

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Table 1. Structures of Multifunctional Peptide Analogues Synthesized in the Present Study

a

compd

abbreviated names

sequences

0 1 2 3 4 5 6 7 8 9 10 11

BN-9a [NMe.Tyr1]-BN-9 [D.Ser2]-BN-9 [D.Met2]-BN-9 [NMe.Phe4]-BN-9 [Gly5]-BN-9 [Leu5]-BN-9 [Met5]-BN-9 [D.Met5]-BN-9 [D.Leu5]-BN-9 [D.Ala5]-BN-9 DN-9

Tyr-D.Ala-Gly-Phe-Gln-Pro-Gln-Arg-Phe-NH2 NMe.Tyr-D.Ala-Gly-Phe-Gln-Pro-Gln-Arg-Phe-NH2 Tyr-D.Ser-Gly-Phe-Gln-Pro-Gln-Arg-Phe-NH2 Tyr-D.Met-Gly-Phe-Gln-Pro-Gln-Arg-Phe-NH2 Tyr-D.Ala-Gly-NMe.Phe-Gln-Pro-Gln-Arg-Phe-NH2 Tyr-D.Ala-Gly-Phe-Gly-Pro-Gln-Arg-Phe-NH2 Tyr-D.Ala-Gly-Phe-Leu-Pro-Gln-Arg-Phe-NH2 Tyr-D.Ala-Gly-Phe-Met-Pro-Gln-Arg-Phe-NH2 Tyr-D.Ala-Gly-Phe-D.Met-Pro-Gln-Arg-Phe-NH2 Tyr-D.Ala-Gly-Phe-D.Leu-Pro-Gln-Arg-Phe-NH2 Tyr-D.Ala-Gly-Phe-D.Ala-Pro-Gln-Arg-Phe-NH2 Tyr-D.Ala-Gly-NMe.Phe-Gly-Pro-Gln-Arg-Phe-NH2

Data cited from our previous report.40

activation of each opioid and NPFF receptors subtypes may be a key factor in developing multifunctional opioid−NPFF agonists with more potent analgesic effects and reduced side effects. In the current work, we synthesized a series of analogues of 0 with the following modifications: (1) selective replacement of Try1, D.Ala2, and Phe4 by the corresponding amino acids to selectively decrease δ-opioid potency or improve μ-opioid potency (analogues 1−4) based on the reported data obtained from the structure−activity relationships of opioid peptides.41 Previous studies suggested that [NMe.Tyr1]-Leuenkephalin maintained μ-opioid activity and was 5-fold less active at the δ-opioid receptor compared with its parent peptide;44 therefore, analogue 1 ([NMe.Tyr1]-BN-9) was designed to selectively decrease activity at the δ-opioid receptor. Furthermore, analogues 2−4 were designed based on the successful modifications of enkephalins. Substitution of hydrophilic D-amino acids at the Gly2 position of enkephalin could increase the potency and selectivity for μ-opioid, and D.Ser and D.Met were used successfully in previous studies.41,45,46 The Gly3 position in enkephalin is very intolerant to substitutions, and the substitution of Phe4 with NMe.Phe was often used in the powerful and stable ligands for the μ-opioid receptor;41,47,48 (2) substitution of Gln5 with Gly, Leu, Met, D.Leu, D.Met, and D.Ala (analogues 5−10). Gln5 of the parent peptide 0 is an overlapping position of the opioid and NPFF pharmacophores of the parent peptide and changes at the 5-position of Leu- or Met-enkephalin yield active, selective μ-opioid receptor ligands.41 In addition, the Gln4 position of NPFF can be replaced with Gly, Glu, or Asn without significantly decreasing the potency at NPFF receptors.49 Therefore, Gly, Leu, Met, D.Leu, D.Met, and D.Ala were used to replace the 5-position of analogue 0; (3) analogue 11 was designed by combining successful chemical modifications to enhance μ-opioid potency via in vitro functional assay. The structures of the synthesized analogues of 0 are presented in Table 1. All of these analogues were synthesized by manual solidphase synthesis using standard N-fluorenylmethoxycarbonyl (Fmoc) chemistry. Synthesis of the analogues was conducted on Rink amide 4-methybenzhydrylamine (MBHA) resin. Hexafluorophosphate (HBTU), 1-hydroxybenzotriazole (HOBt), and N,N′-diisopropylethylamine (DIEA) were used as coupling agents. The established peptides were desalted by gel filtration, purified using semipreparative reversed-phase high pressure liquid chromatography (RP-HPLC), and characterized by RP-HPLC, magnetic deflection time-of-flight mass spec-

opioid-like analgesia or potentiated morphine analgesia at the spinal level.34,35 In addition, previous studies demonstrated that the NPFF system was involved in opioid antinociceptive tolerance and dependence.29,36−39 In view of these findings, we recently synthesized and developed a multifunctional agonist toward opioid and NPFF receptors, 0 (BN-9, Tyr-D.Ala-GlyPhe-Gln-Pro-Gln-Arg-Phe-NH2).40 0 is a chimeric peptide containing the half structure of biphalin (Tyr-D.Ala-Gly-PheNH−) and the C-terminal of NPFF. Our data showed that 0 has potent nontolerance forming antinociception in mice. Thus, 0 may be used as a promising lead compound to develop analgesics with reduced or eliminated tolerance. To further identify the structural and conformational requirements of 0 for opioid and NPFF receptors interaction, we synthesized 11 analogues of 0 in an effort to balance opioid and NPFF receptors activation and improve antinociceptive potencies in this study. In addition, the in vitro functional activities, antinociceptive activities, and tolerance developments of these analogues were evaluated and compared with 0. Most strikingly, analogue 11 (DN-9) was identified as a mixed agonist for μ, κ, δ-opioid, NPFF1, and NPFF2 receptors and exerted a potent analgesic effect without tolerance (ED50 = 0.0163 nmol), which is approximately 33-fold more potent than 0 in the tail-flick assay. 11 also produced a dramatic pain reduction in the mouse models of formalin pain and complete Freund’s adjuvant (CFA)-induced chronic inflammatory pain. Moreover, the substitution modification from 0 to 11 improved the metabolic stability in the mouse brain homogenate.



RESULTS AND DISCUSSION Chemistry. The analogues in this work were based on 0, which functions as a mixed agonist of opioid and NPFF receptors. Our design of peptides that simultaneously interact with opioid and NPFF receptors was based on reasonable overlapping pharmacophores of opioid and NPFF receptors ligands in a single molecule. Previous studies for the structure− activity relationships of opioid and NPFF peptides indicated that the N-terminus of opioid peptides was an opioid pharmacophore, whereas the C-terminus RFamide of NPFF and related peptides was a NPFF pharmacophore.41−43 As shown in the chemical structure of 0, the N-terminal amino acids unique to biphalin and the C-terminal part of NPFF were fused in a single molecule.40 As a multifunctional ligand, the agonism and selectivity for each receptor may affect its in vivo pharmacological activities. Therefore, the identification of a proper balance between the 10199

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Table 2. Analytical Data for Multifunctional Peptide Analogues RP-HPLC 10−80% in 30 min a

b

compd

tR (min)

purity (%)

1 2 3 4 5 6 7 8 9 10 11

13.945 13.485 15.004 13.499 14.808 15.590 16.085 17.241 18.535 15.638 14.850

99.50 98.27 98.92 95.65 96.06 99.15 99.33 99.18 99.79 98.78 97.49

RP-HPLC 10−100% in 30 min

MD-TOF-MS [M + H]+

tRa

purity (%)

calcd

identified

mp (°C)

99.76 98.05 99.13 95.39 96.07 99.02 99.24 99.50 99.91 98.76 97.43

1126.54 1128.54 1172.54 1126.54 1041.50 1097.56 1115.52 1115.52 1197.56 1055.52 1055.50

1126.57 1128.58 1172.59 1126.58 1041.53 1097.59 1115.55 1115.55 1197.59 1055.54 1055.54

147−149 146−148 145−147 146−148 145−146 144−146 144−145 144−146 146−148 147−149 147−149

b

(min)

12.842 12.435 13.655 14.739 13.542 14.086 14.512 15.530 16.447 14.203 13.613

tR with XBridge BEH 130 Prep C18 (4.6 mm × 250 mm, 5 μm). The gradients 10−80% and 10−100% indicate solvent gradients of Me3CN in water from 10 to 80% in 30 min and from 10 to 100% in 30 min, respectively. bPurity determination based on analytical RP-HPLC. a

Table 3. Functional Activities of Multifunctional Peptide Analogues at Opioid Receptors μ-opioid receptor compd 0 1 2 3 4 5 6 7 8 9 10 11

a

EC50 (nM)

0.15b 0.23 0.25 0.19 0.04 0.42 0.23 0.11 0.10 0.09 0.08 0.04

308.2b 196.2 562.7 423.6 20.1 28.4 250.2 126.9 409.0 583.3 248.6 38.6

log EC50 −6.51 −6.71 −6.25 −6.37 −7.70 −7.55 −6.60 −6.90 −6.39 −6.23 −6.61 −7.41

± ± ± ± ± ± ± ± ± ± ± ±

κ-opioid receptor Emax (%) 92.4 86.7 97.1 89.4 91.6 80.4 98.9 89.2 89.7 86.3 85.6 81.6

± ± ± ± ± ± ± ± ± ± ± ±

9.7b 16.2 16.7 13.3 1.9 8.6 13.2 5.4 5.6 5.5 4.3 2.3

a

EC50 (nM)

0.25b 0.04 0.06 0.08 0.03 0.06 0.08 0.16 0.34

563.6b 2586 1466 1263 31.0 1631 120.3 239.8 2457 c c 1488

log EC50 −6.25 −5.59 −5.83 −5.90 −7.51 −5.79 −6.92 −6.62 −5.61

± ± ± ± ± ± ± ± ±

−5.83 ± 0.21

δ-opioid receptor a

Emax (%)

log EC50

± ± ± ± ± ± ± ± ± ± ± ±

−6.23 ± 0.06b

105.6 73.6 96.9 91.5 95.6 84.2 96.8 69.6 60.8 59.1 46.6 95.7

20.7b 3.0 4.7 5.1 1.3 3.5 4.4 6.0 13.9 5.0d 6.6d 13.7

−6.93 −6.94 −6.81 −6.05 −7.31 −7.41 −6.92 −7.25 −6.09 −6.24

± ± ± ± ± ± ± ± ± ±

0.16 0.12 0.16 0.09 0.14 0.07 0.05 0.12 0.19 0.09

EC50 (nM) 586.7b c 117.7 113.8 156.7 898.3 49.3 38.8 119.9 56.9 810.2 574.7

Emax (%) 82.2 70.0 84.9 84.7 84.9 75.7 79.1 81.2 78.4 86.4 85.3 85.4

± ± ± ± ± ± ± ± ± ± ± ±

3.7b 4.5d 5.6 4.8 6.7 3.9 3.3 1.3 1.7 2.8 9.4 4.3

The log EC50 ± standard error is expressed as logarithmic values determined from the nonlinear regression analysis of data collected from at least three independent experiments performed in duplicate. bValues were cited from our previous report.40 cThe top of the agonist concentration response curve was not obtained even at the highest test concentration of the peptide; an EC50 value cannot be determined. dIn these instances, the percentage of agonist response is provided for the response at 10 μM peptide. a

potency in both the guinea pig ileum (GPI) and mouse vas deferens (MVD) assays.41,45,46 In this study, the substitution of position 2 with D.Ser or D.Met (analogues 2 and 3) exhibited a very similar in vitro profile toward μ-opioid receptor. However, the κ-opioid activities were decreased approximately 2-fold, and the δ-opioid activities increased approximately 4-fold. In addition, our data showed that analogue 4 from the Nmethylation of the Phe in position 4 exhibited increased the potency for both μ- and κ-opioid receptors. This result is consistent with the previous report that NMe.Phe4-dermophin significant improved μ and δ potency.47 DAMGO (Tyr-D.AlaGly-NMe.Phe-Gly-ol), which has an NMe.Phe at position 4, is one of most potent μ-opioid receptor agonists.48 Moreover, modification at the Gln5 position of 0 was performed in an effort to alter opioid receptor selectivity and balance the activities between opioid and NPFF receptors subtypes. Interestingly, the replacement of Gln in position 5 with Gly (analogue 5) significantly increased μ-opioid potency. The substitution of position 5 with other amino acids (analogues 7−10) in this study did not alter agonistic activities at the μ-opioid receptor; however, the substitution of position 5 with both L- and D-types of Leu or Met (analogues 6−10) significantly enhanced δ-opioid activity. In addition, the

trometry (MD-TOF MS), and melting point (mp). The peptides with the purities that exceeded 95% as characterized by analytical RP-HPLC were used in the following studies. The detailed analytical properties of these analogues are provided in Table 2. Biological Results. In Vitro cAMP Functional Activities. To investigate the agonistic activities of the new analogues of 0 toward opioid and NPFF receptors in vitro, the inhibition of forskolin-stimulated cyclic adenosine monophosphate (cAMP) accumulation was evaluated in HEK-293A cells stably expressing opioid or NPFF receptors. The results of the functional activities at opioid receptors are summarized in Table 3. Most of the analogues exhibited concentrationdependent inhibition in forskolin-stimulated cAMP accumulation in the cell expressing μ-, κ-, and δ-opioid receptors. N-Methylation of the Tyr in position 1 (analogue 1) results in considerably reduced potency at the δ-opioid receptor, indicating the Tyr1 plays an important role in activating the δopioid receptor. Our results are consistent with the previous structure−activities studies of opioid peptides.44 The previous studies reported that the alkylation of the Nα of Tyr1 of enkephalins resulted in a loss of potency at the δ-opioid receptor.44 However, the substitution of D.Ala, D.Ser, or D.Met at the position 2 of enkephalins could increase stability and 10200

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Table 4. Functional Activities of Multifunctional Peptide Analogues at NPFF Receptors NPFFR1 compd 0 1 2 3 4 5 6 7 8 9 10 11

log EC50a −6.23 −6.03 −5.50 −6.18 −6.54 −6.06 −7.27 −6.52

± ± ± ± ± ± ± ±

0.18 0.23 0.42 0.39 0.31 0.38 0.36 0.30

−6.01 ± 0.30

EC50 (nM) b

b

591.0 927.7 3195 661.3 289.1 880.7 54.4 301.3 c c c 973.4

NPFFR2 log EC50a

Emax (%) 59.9 72.4 82.3 73.5 67.7 66.8 68.4 69.2 26.6 30.7 15.1 69.8

± ± ± ± ± ± ± ± ± ± ± ±

−6.73 −6.88 −6.61 −7.38 −6.57 −6.73 −6.59 −7.06 −6.50 −6.19 −5.30 −6.51

b

9.3 13.8 17.3 17.9 7.2 11.0 9.4 10.3 4.6d 4.6d 8.4d 8.6

± ± ± ± ± ± ± ± ± ± ± ±

EC50(nM)

0.36 0.28 0.25 0.47 0.19 0.45 0.31 0.38 0.32 0.24 0.34 0.14

184.6 132.2 254.0 41.3 271.7 184.8 258.7 87.0 318.2 651.4 5012 310.8

Emax (%) 58.4 49.5 63.0 57.9 54.5 56.2 57.3 58.4 70.18 37.2 45.9 73.3

± ± ± ± ± ± ± ± ± ± ± ±

15.3 9.3 8.6 16.0 6.4 16.6 10.9 11.5 12.0 6.3 18.3 7.4

The log EC50 ± standard error is expressed as logarithmic values determined from the nonlinear regression analysis of data collected from at least three independent experiments performed in duplicate. bValues were cited from our previous report.40 cThe top of the agonist concentration response curve was not obtained even at the highest test concentration of the peptide; an EC50 value cannot be determined. dIn these instances, the percentage of agonist response is provided for the response at 10 μM of peptide. a

replacement of Gln5 with a D-type amino acid (analogues 8− 10) led to reduced κ-opioid potencies. Furthermore, on the basis of the successful chemical modifications at the 4 and 5 positions to enhance μ-opioid potency in the in vitro functional assay, analogue 11 with substitutions at Phe4 and Gln5 residues improved its potency at the μ-opioid receptor but a slight loss of κ-opioid activity was observed. The data of the functional activities of the analogues at NPFF receptors are summarized in Table 4. For the NPFF1 receptor, only analogue 6 had a significantly improved activity, with approximately 11-fold potency compared with 0. Analogue 2 had a slight decrease in NPFF1 receptor potency, and analogues 8−10 exhibited a dramatic decrease in NPFF1 receptor activation. Analogues 8−10 are D-type amino acid substitutions at the 5 position, indicating the configuration of amino acid at position 5 may be critical for the NPFF1 receptor potency of these analogues. For the NPFF2 receptor, analogue 4 had an increase, whereas analogue 11 exhibited a significant decrease in agonistic properties. The remaining analogues have the similar activities for the NPFF2 receptor compared with 0. Our results indicate the contribution of the N-terminal amino acids of NPFF and related peptides to bind and activate NPFF receptors, which is consistent with previous studies.49,50 Antinociception and Tolerance Studies in the Tail-Flick Test. The antinociception and tolerance for the acute pain of these analogues were determined using the mouse tail-flick test. All analogues were evaluated for antinociception after icv administration. To evaluate antinociceptive tolerance, mice were icv injected 3 × ED50 doses of the related analogues once daily for 6 days. The difference between the antinociceptive responses to analogues at the same dose on days 1 and 6 was compared to determine whether tolerance was developed. These data are summarized in Table 5. In the present study, 0 produced potent nontolerance forming antinociception with an ED50 value of 0.533 nmol after icv administration, which is similar to our recent data that 0 produced nontolerance antinociception with an ED50 value of 0.39 nmol.40 All of the new analogues produced potent doseand time-dependent antinociception with ED50 values ranging from 0.0163 to 1.298 nmol (Figure 1, Table 5, and Supporting Information, Figure S1). Among these analogues, analogues 3

Table 5. Antinociception and Tolerance Evaluation after the icv Administration of Multifunctional Peptide Analogues via Mouse Tail-Flick Test compd 0 1 2 3 4 5 6 7 8 9 10 11 a

ED50a nmol/mouse (95% CI) 0.5330 1.2980 0.6062 0.0314 0.3862 0.4172 0.1865 0.2967 0.3986 0.4367 0.3361 0.0163

(0.5007, (1.2000, (0.5255, (0.0289, (0.3590, (0.3798, (0.1509, (0.2637, (0.3716, (0.3865, (0.2864, (0.0145,

0.5675) 1.4050) 0.6993) 0.0341) 0.4154) 0.4583) 0.2304) 0.3338) 0.4275) 0.4934) 0.3944) 0.0182)

relative potency to 0

6 days tolerance

1.00 0.41 0.88 16.97 1.38 1.28 2.86 1.80 1.34 1.22 1.59 32.78

no tolerance no tolerance tolerance tolerance no tolerance no tolerance no tolerance no tolerance no tolerance no tolerance no tolerance no tolerance

ED50 was determined at the time of peak effect.

and 11 exhibited significant improved antinociceptive potencies that were approximately 17- and 33-fold more potent compared with the parent peptide 0, respectively. The high antinociceptive potency of 11 might result from its high potency at the μ-opioid receptor in vitro. However, it is difficult to explain the high antinociceptive potency of 3. Compared with 0, 3 displayed increased potency at δ-opioid and NPFF2 receptors, but the functional interaction between NPFF and δ-opioid systems in pain modulation are completely unknown to date. It appears that the antinociceptive potency of the multifunctional peptides depends on their potencies and selectivities at opioid and NPFF receptors, the details of which need further elaboration. In cAMP assays, both analogues 2 and 3 activate both opioid and NPFF receptors. To our surprise, only analogues 2 and 3 developed antinociceptive tolerance after 6 days of repeated icv injections (Table 5 and Supporting Information, Figure S2). It is difficult to explain these exceptional cases. However, their functional selectivity at the μ-opioid receptor might result in the different pharmacological effects on antinociceptive tolerance. Previous studies have suggested that β-arrestin pathway signaling downstream of the μ-opioid receptor may 10201

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ligands for opioid and NPFF receptors in a single molecule, producing effective nontolerance forming antinociception. Antinociceptive Profiles of 11 in the Tail-Flick Test. The dose- and time-related antinociceptive effects and tolerance evaluation of 11 are presented in Figure 1. 11 exhibited a strong and sustained antinociceptive effect for longer than 60 min that peaked at 20 min (F3, 31 = 531.7, P < 0.001). The antinociception of 11 in the tail-flick test was approximately 33-fold more potent compared with 0 with calculated ED50 values of 0.0163 nmol for 11 and 0.533 nmol for 0 (Table 5). According to our previous data, the ED50 value for morphine was 1.02 nmol after icv administration in the tail-flick test,40 indicating 11 has approximately 62-fold more potent antinociception compared with morphine in the tail-flick test. Combined with the cAMP assay data described above, the enhanced μ-opioid agonistic activity might contribute to the potent antinociception of 11. In addition, another potential explanation for the enhanced antinociception might due to its high stability in resisting the enzymatic hydrolysis. Our data in the metabolic stability test revealed that 11 displayed a half-life of 86.29 ± 4.00 min in the brain membrane homogenate, which is approximately 2-fold longer compared with 0 based on our previous report.40 Furthermore, 11 exhibited similar potency of antinociception on days 1 and 6 after 6 days of repeated treatments (P > 0.05) (Figure 1C). Taken together, our results clearly indicate that 11 produces the most potent analgesic effect among these new analogues and has no antinociceptive tolerance after repeated administration in acute pain model. Furthermore, to investigate the roles of opioid and NPFF receptors in the central antinociception of 11, the antagonists of opioid and NPFF receptors were used in the present study. As shown in Figure 2A, pretreatment with the nonselective opioid receptor antagonist naloxone (5 nmol, icv) 10 min prior to 11 completely inhibited its supraspinal antinociception, suggesting the involvement of opioid receptors in the central antinociception of 11 (F3,28 = 270.3, P < 0.001). To further identify the role of individual opioid receptor subtypes in this process, β-funaltrexamine (β-FNA), nor-binaltorphimine (norBNI), or naltrindole (NTI), the selective antagonists for μ-, κ-, and δ- receptors, respectively, were icv injected 4 h, 30 min, or 20 min, respectively, prior to treatment with 11. Our data showed that the central antinociception of 11 was completely inhibited by pretreatment with the μ- and κ-opioid receptor antagonists β-FNA (10 nmol) and nor-BNI (10 nmol) (F7,59 = 194.1, P < 0.001; P < 0.001) but not by the δ-opioid receptor antagonist NTI (10 nmol, P > 0.05). These data strongly suggest that 11 produces supraspinal antinociception via μ- and κ-opioid receptors. Next, the possible role of the NPFF system in the antinociception of 11 was also investigated. Our results illustrated that the antinociception of 11 was significantly augmented by pretreatment with the nonselective NPFF receptors antagonist RF9 (10 nmol, icv) (Figure 2C; F3,30 = 100.5, P < 0.001), which is similar to the property of its parent peptide 0.40 Previous studies have indicated that NPFF caused antiopioid activities at the supraspinal level.32,33 Given that 11 could activate both NPFF and opioid receptors, its NPFF moiety might exert antiopioid activities and partially reduce its opioid moiety-mediated antinociception after supraspinal administration. In the present study, pretreatment with RF9 prevented the antiopioid activities of the NPFF moiety and endogenous NPFF systems and might further potentiate the μopioid antinociception of 11 after icv administration. This

Figure 1. Antinociceptive effect of 11 in the mouse tail-flick test after central administration. (A) Antinociceptive dose− and time−response curve for 11 after icv administration. (B) AUC date calculated from 0 to 60 min in the dose− and time−response curves. (C) Antinociceptive tolerance evaluation of 11 after 6 days of repeated icv administration. Each value represents the mean ± SEM. Group size is indicated in the figures. ***P < 0.001 indicates significant differences compared with the saline group according to one-way ANOVA followed by Dunnett’s post-hoc test.

contribute to the desensitization of the μ-opioid receptor and the development of opioid antinociceptive tolerance.51,52 In the future, the functional selectivities of these multifunctional peptides should be further investigated. Overall, these results indicate that it is reasonable to design multifunctional peptide 10202

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result was consistent with above deduction and supports an antiopioid character of the NPFF system in the brain. The antinociceptive effects of 11 were further investigated after ip administration in the tail-flick test. Our results demonstrated that 11 produced dose-dependent antinociceptive effects after ip administration (Figure 3; F3,30 = 34.328, P