Dermorphin tetrapeptide analogs as potent and long-lasting analgesics with pharmacological profiles distinct from morphine

Peptides 32 (2011) 421–427

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Review

Dermorphin tetrapeptide analogs as potent and long-lasting analgesics with pharmacological profiles distinct from morphine Hirokazu Mizoguchi a , Giacinto Bagetta b , Tsukasa Sakurada c , Shinobu Sakurada a,∗ a b c

Department of Physiology and Anatomy, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558, Japan Department of Pharmacobiology, University of Calabria, Arcavacata di Rende, Cosenza 87036, Italy First Department of Pharmacology, Daiichi College of Pharmaceutical Sciences, 22-1 Tamagawa-cho, Minami-ku, Fukuoka 815-8511, Japan

a r t i c l e

i n f o

Article history: Received 4 May 2010 Received in revised form 21 November 2010 Accepted 21 November 2010 Available online 30 November 2010 Keywords: Dermorphin analog Analgesics ␮-Opioid receptor ␬-Opioid receptor Dynorphins Dependence

a b s t r a c t Dermorphin (Tyr-d-Ala-Phe-Gly-Tyr-Pro-Ser-NH2 ) is a heptapeptide isolated from amphibian skin. With a very high affinity and selectivity for ␮-opioid receptors, dermorphin shows an extremely potent antinociceptive effect. The structure–activity relationship studies of dermorphin analogs clearly suggest that the N-terminal tetrapeptide is the minimal sequence for agonistic activity at ␮-opioid receptors, and that the replacement of the d-Ala2 residue with d-Arg2 makes the tetrapeptides resistant to enzymatic metabolism. At present, only a handful of dermorphin N-terminal tetrapeptide analogs containing d-Arg2 have been developed. The analogs show potent antinociceptive activity that is greater than that of morphine with various injection routes, and retain high affinity and selectivity for ␮-opioid receptors. Interestingly, some analogs show pharmacological profiles that are distinct from the traditional ␮-opioid receptor agonists morphine and [d-Ala2 ,NMePhe4 ,Gly-ol5 ]enkephalin (DAMGO). These analogs stimulate the release of dynorphins through the activation of ␮-opioid receptors. The activation of ␬-opioid receptors by dynorphins is suggested to reduce the side effects of ␮-opioid receptor agonists, e.g., dependence or antinociceptive tolerance. The dermorphin N-terminal tetrapeptide analogs containing d-Arg2 may provide a new target molecule for developing novel analgesics that have fewer side effects. © 2010 Elsevier Inc. All rights reserved.

Contents 1. 2. 3. 4. 5. 6.

7.

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dermorphin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Endogenous dermorphins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Synthetic dermorphin analogs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Distinct antinociceptive profiles of dermorphin tetrapeptide analogs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adverse effects of dermorphin tetrapeptide analogs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Respiratory depression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Antinociceptive tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Dependence liability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Preface Morphine, one of the oldest analgesics in the history of human beings, is still the best analgesic in the clinic, especially for cancer pain and post-operative pain. Its analgesic effect is mediated through ␮-opioid receptors, which are mainly involved in the

∗ Corresponding author. Tel.: +81 22 727 0124; fax: +81 22 727 0125. E-mail address: [email protected] (S. Sakurada). 0196-9781/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2010.11.013

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endogenous pain control systems among the three major subtypes of opioid receptors. Beside its potent analgesic effect, morphine also has several side effects, including constipation, itchiness, respiratory depression, antinociceptive tolerance, physical dependence, and psychological dependence. These side effects of morphine seriously affect its effectiveness in the clinic. Therefore, new analgesics that are more potent and have fewer side effects than morphine have been explored. At present, several ␮-opioid receptor agonists along with morphine, e.g., fentanyl, oxycodone, and methadone, are available as analgesics in the clinic. However, the pharmacological

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Table 1 Endogenous opioid peptides. Endogenous ␮-opioid peptides Endomorphin-1 Endomorphin-2 ␤-Endorphin

Dermorphin Endogenous ␦-opioid peptides [Met5 ]Enkephalin [Leu5 ]Enkephalin Endogenous ␬-opioid peptides Dynorphin A

Dynorphin B ␣-Neo-endorphin ␤-Neo-endorphin

Tyr1 -Pro-Trp-Phe4 -NH2 Tyr1 -Pro-Phe-Phe4 -NH2 Tyr1 -Gly-Gly-Phe-Met5 -Thr-Ser-GluLys-Ser10 -Gln-Thr-Pro-Leu-Val15 -ThrLeu-Phe-Lys-Asn20 -Ala-Ile-Ile-LysAsn25 -Ala-Tyr-Lys-Lys-Gly30 -Gln31 -OH Tyr1 -d-Ala-Phe-Gly-Tyr5 -Pro-Ser7 NH2 Tyr1 -Gly-Gly-Phe-Met5 -OH Tyr1 -Gly-Gly-Phe-Leu5 -OH Tyr1 -Gly-Gly-Phe-Leu5 -Arg-Arg-IleArg-Pro10 -Lys-Leu-Lys-Trp-Asp15 -AsnGln17 -OH Tyr1 -Gly-Gly-Phe-Leu5 -Arg-Arg-GlnPhe-Lys10 -Val-Val-Thr13 -OH Tyr1 -Gly-Gly-Phe-Leu5 -Arg-Lys-TyrPro-Lys10 -OH Tyr1 -Gly-Gly-Phe-Leu5 -Arg-Lys-TyrPro9 -OH

characteristics of these analgesics are quite similar to morphine, and their intrinsic activities are similar to or lower than morphine [24]. Unfortunately, most of the side effects of morphine, as well as its analgesic effects, are mediated through ␮-opioid receptors [39]. Therefore, more valuable ␮-opioid receptor agonists than morphine as analgesics are still not available in the clinic. During the past two decades, selective ␦-opioid receptor agonists (TAN-67, SNC80, etc.) or selective ␬-opioid receptor agonists (U-50, 488H, TRK-820, etc.) have been developed as new analgesics that have fewer side effects than morphine [2,27,57,58]. However, none of these compounds are used in the clinic as analgesics because their analgesic effects are much weaker than morphine. The low intrinsic activity of ligands mediated through ␦- or ␬-opioid receptors and the low density of ␦- or ␬-opioid receptors in the site involved in pain transmission limit their analgesic effect [14,19]. Theoretically, there is no possibility that selective ␦-opioid receptor agonists or selective ␬-opioid receptor agonists can be better analgesics than morphine in the clinic. Like [d-Ala2 ,NMePhe4 ,Gly-ol5 ]enkephalin (DAMGO), ␤endorphin or dermorphin, several ␮-opioid receptor agonists that have higher intrinsic activity than morphine are available [23,24]. However, all of those compounds are peptides; therefore, none of those compounds were originally expected to be effective after peripheral injection. Interestingly, dermorphin and its analogs were unexpectedly effective after peripheral injection [40]. In the present article, the antinociceptive profiles of dermorphin analogs are reviewed. Dermorphin and its analogs may provide a new target molecule for developing novel analgesics that have higher intrinsic activity and fewer side effects than morphine.

Fig. 1. The increase in [35 S]GTP␥S binding by DAMGO, dermorphin or morphine in spinal cord membranes obtained from ICR mice. Membranes were incubated with 50 pM [35 S]GTP␥S and 30 ␮M GDP in the absence/presence of various concentrations (1 nM–10 ␮M) of DAMGO, dermorphin or morphine for 2 h at 25 ◦ C. Non-specific binding was measured in the presence of 10 ␮M unlabeled GTP␥S. The data are expressed as the percentage of basal [35 S]GTP␥S binding measured in the presence of GDP and in the absence of agonist, and represent the mean ± S.E.M. for at least three independent experiments. The concentration-effect curves were calculated with GraphPad Prism, a computer-assisted curve-fitting program. The maximum stimulation with DAMGO, dermorphin or morphine is 93.77%, 94.90% or 49.55%, respectively.

2. Dermorphin Dermorphin (Tyr-d-Ala-Phe-Gly-Tyr-Pro-Ser-NH2 ) is a heptapeptide isolated from amphibian skin [26]. As shown in Table 1, the amino acid sequence of dermorphin is quite different from the other endogenous opioid peptides. Dermorphin does not contain the common N-terminal sequence for the traditional endogenous opioid peptides (Tyr-Gly-Gly-Phe), and its sequence is completely different from that of the endomorphins, which were identified as endogenous ␮-opioid peptides that also do not contain the common sequence. Interestingly, dermorphin contains d-Ala (d-isomer amino acid) in its sequence. In the opioid receptor binding assay using the mouse brain, dermorphin shows a very high affinity for ␮-opioid receptors with a low affinity for ␦-opioid receptors (Table 2) [12]. Without any significant affinity for ␬-opioid receptors, dermorphin shows a higher selectivity for ␮-opioid receptors than morphine. The intrinsic activity of dermorphin for G-protein activation is similar to that of DAMGO and higher than that of morphine (Fig. 1), suggesting that dermorphin, as well as DAMGO, is a full agonist for ␮-opioid receptors, whereas morphine is a partial agonist for that receptor. With a high affinity and intrinsic activity for ␮-opioid receptors, dermorphin shows a strong and prolonged antinociceptive effect with various injection routes. After i.c.v. injection, dermorphin is approximately 200 times more potent than morphine for antinociception [11]. Unlike other pep-

Table 2 Affinities of ligands for ␮-, ␦- and ␬-opioid receptors in mouse brain. Ligands

Morphine Dermorphin Endomorphin-1 [d-Ala2 ]Deltorphin II U-50,488H

IC50 values with their 95% confidence intervals (nM) [3 H]DAMGO (␮)

[3 H]DPDPE (␦)

[3 H]U-69,593 (␬)

2.84 (2.57–3.14) 0.76 (0.69–0.83) 2.86 (2.49–3.28) n.d. n.d.

133 (100–176) 73.5 (59.1–91.4) n.d. 2.29 (1.98–2.66) n.d.

141 (114–174) >1000 n.d. n.d. 0.76 (0.60–0.95)

Membranes obtained from ICR mouse whole brain without the cerebellum were incubated at 25 ◦ C for 2 h with 1 nM of [3 H]DAMGO, [3 H]DPDPE or [3 H]U-69,593 in the absence/presence of various concentrations (0.03–1000 nM) of the agonists. Non-specific binding was measured in the presence of 10 ␮M naloxone, DPDPE or U-69,593 for the [3 H]DAMGO, [3 H]DPDPE or [3 H]U-69,593 binding assays, respectively. The IC50 values with their 95% confidence intervals for the agonists in each binding assay were calculated with GraphPad Prism, a computer-assisted curve-fitting program. n.d., not determined.

H. Mizoguchi et al. / Peptides 32 (2011) 421–427 Table 3 Dermorphin and its endogenous analogs. Dermorphin [Hyp6 ]Dermorphin [Lys7 ]Dermorphin [Lys7 ]Dermorphin-OH [Trp4 ,Ans7 ]Dermorphin [Trp4 ,Ans7 ]Dermorphin-OH [Trp4 ,Ans5 ]Dermorphin(1-5)-OH

Tyr1 -d-Ala-Phe-Gly-Tyr5 -Pro-Ser7 -NH2 Tyr1 -d-Ala-Phe-Gly-Tyr5 -Hyp6 -Ser7 -NH2 Tyr1 -d-Ala-Phe-Gly-Tyr5 -Pro-Lys7 -NH2 Tyr1 -d-Ala-Phe-Gly-Tyr5 -Pro-Lys7 -OH Tyr1 -d-Ala-Phe-Trp4 -Tyr5 -Pro-Ans7 -NH2 Tyr1 -d-Ala-Phe-Trp4 -Tyr5 -Pro-Ans7 -OH Tyr1 -d-Ala-Phe-Trp4 -Ans5 -OH

tidic analgesics, dermorphin shows potent antinociceptive effects with peripheral injection routes, such as s.c. and i.v. injections [40]. Although the blood–brain barrier (BBB) permeability of dermorphin is markedly lower than for morphine, dermorphin still has a four-fold more potent antinociceptive effect than morphine after s.c. injection [31]. The peripherally-administered dermorphin is mainly degraded to the deamidated N-terminal tetrapeptide (dermorphin[1-4]-OH) by cleaving enzymes in the blood, lever, brain, or spinal cord [30]. However, its rate of degradation is more than 30 times slower than for other endogenous opioid peptides [50]. Moreover, dermorphin[1-4]-OH, the main metabolite of dermorphin, does not have remarkable agonistic activity for ␮-opioid receptors [17]. The potency of dermorphin[1-4]-OH for inhibiting the guinea pig ileum contraction is only 1/525 of dermorphin. Therefore, the potent and prolonged antinociceptive effect of dermorphin may be due to its slow degradation, but not to its active metabolites. 3. Endogenous dermorphins At present, seven endogenous analogs of dermorphin have been identified (Table 3). Unlike dermorphin, three of the endogenous analogs do not have a C-terminus that is protected by amidation. Without the C-terminal amidation, the affinity of these peptides for ␮-opioid receptors is dramatically reduced by 5–63 fold [29]. Dermorphin is degraded by enzymes that cleave its C-terminal amino acid [30]. Since C-terminal amidation prevents this degradation by cleaving enzymes, endogenous dermorphin analogs that contain C-terminal amidation may have more potent agonistic activity for ␮-opioid receptors than the analogs that lack C-terminal amidation. Among the endogenous dermorphin analog identified, [Lys7 ]dermorphin has been suggested to have high BBB permeability [31]. The antinociceptive effect of s.c.-administered [Lys7 ]dermorphin is 6.6 times stronger than dermorphin, whereas the effect of i.c.v.-administered [Lys7 ]dermorphin is almost identical to dermorphin, suggesting that BBB permeability of [Lys7 ]dermorphin is higher than dermorphin. Moreover, the duration of the antinociceptive effect of [Lys7 ]dermorphin is 2 times longer than dermorphin. Since [Lys7 ]dermorphin is more resistant than dermorphin to the cleaving enzymes in the brain [29], the slow degradation of [Lys7 ]dermorphin may be responsible for its prolonged antinociceptive effect [31]. It is note worthy that [Lys7 ]dermorphin has two binding sites on ␮-opioid receptors: one is a [Lys7 ]dermorphin high-affinity site and the other is a [Lys7 ]dermorphin low-affinity site [29]. Interestingly, [Trp4 ,Asn7 ]dermorphin, one of the endogenous dermorphin analogs, has a very high affinity for the [Lys7 ]dermorphin low-affinity ␮-opioid receptors. [Trp4 ,Asn7 ]dermorphin administered i.c.v. is 7 times more potent than [Lys7 ]dermorphin as an inducer of catalepsy, whereas the antinociceptive effect of i.c.v.-administered [Trp4 ,Asn7 ]dermorphin is only 1/80 of [Lys7 ]dermorphin. Moreover, [Trp4 ,Asn7 ]dermorphin is 2 times more potent than [Lys7 ]dermorphin as a ␮-opioid receptor agonist in the mouse vas deference or guinea pig ileum. This evidence clearly suggests that the pharmacological profile of the [Lys7 ]dermorphin high-affinity ␮-opioid receptors is

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quite different with that of the [Lys7 ]dermorphin low-affinity ([Trp4 ,Asn7 ]dermorphin high-affinity) ␮-opioid receptors. In fact, the antinociceptive effect of i.c.v.-administered [Lys7 ]dermorphin is potently suppressed by pretreatment with the ␮1 -opioid receptor antagonist naloxonazine, whereas the cataleptic response of i.c.v.-administered [Lys7 ]dermorphin is not affected by naloxonazine [31]. The [Lys7 ]dermorphin high-affinity ␮-opioid receptors may be ␮1 -opioid receptors that are mainly involved in central antinociception, whereas the [Lys7 ]dermorphin low-affinity ([Trp4 ,Asn7 ]dermorphin high-affinity) ␮-opioid receptors may be ␮2 -opioid receptors that are involved in the cataleptic response and smooth muscle contraction.

4. Synthetic dermorphin analogs Since the discovery of dermorphin, more than 100 dermorphin analogs have been synthesized, and their structure–activity relationships have been well established. The remarkable characteristic of the structure of dermorphin is the presence of a d-isomer amino acid (d-Ala) located between two aromatic amino acids (Tyr1 and Phe3 residues) in its sequence. Interestingly, the d-Ala2 residue is essential for the agonistic activity of dermorphin for ␮-opioid receptors, since an analog with the d-Ala2 residue replaced by l-Ala2 ([l-Ala2 ]dermorphin) is almost inactive [17]. The affinity of [l-Ala2 ]dermorphin for ␮-opioid receptors is more than 4000 times lower than dermorphin. Ordinarily, the N-terminal fragments of dermorphin have reduced opioid activity compared to the native heptapeptide (dermorphin) because of the lost C-terminal amino acids [16]. The N-terminal hexapeptide (dermorphin[1–6]) and pentapeptide (dermorphin[1–5]) of dermorphin retain approximately half of the affinity for ␮-opioid receptors compared to dermorphin. The N-terminal tetrapeptide of dermorphin (dermorphin[1-4]) still has a moderate affinity for ␮opioid receptors (approximately 1/20 of dermorphin), whereas the N-terminal tripeptide of dermorphin (dermorphin[1–3]) does not have any significant affinity for ␮-opioid receptors. The N-terminal tetrapeptide of dermorphin may be the minimal sequence required for agonistic activity at ␮-opioid receptors. The most fundamental finding in structure–activity relationship studies for dermorphin analogs is that replacement of the d-Ala2 residue with d-Arg2 may increase the selectivity of dermorphin analogs for ␮-opioid receptors involved in central antinociception, but not in their peripheral effects. In fact, the antinociceptive effect of i.c.v.-administered [d-Arg2 ]dermorphin is only 4 times weaker than that of dermorphin, although [d-Arg2 ]dermorphin is 26 times weaker than dermorphin as an inhibitor of the guinea pig ileum contraction [11]. Unlike i.c.v.-administered [dArg2 ]dermorphin, which shows a weaker antinociceptive effect than dermorphin, s.c.-administered [d-Arg2 ]dermorphin shows an equipotent antinociceptive effect with dermorphin [46]. The evidence clearly suggests that the substitution with the d-Arg2 residue in dermorphin increases its BBB permeability. More interestingly, the N-terminal tetrapeptide of [d-Arg2 ]dermorphin ([d-Arg2 ]dermorphin[1-4]) shows a 2.2 time more potent antinociceptive effect than [d-Arg2 ]dermorphin at supraspinal sites [11], whereas the antinociceptive effect of dermorphin[1-4] is 1/4 of dermorphin [3]. Deamidated [d-Arg2 ]dermorphin[14], [d-Arg2 ]dermorphin[1-4]-OH, also shows a very potent and long-lasting antinociceptive effect [46,48]. [d-Arg2 ]dermorphin[14]-OH is the main and most potent active metabolite of [d-Arg2 ]dermorphin [45]. Since [d-Arg2 ]dermorphin[1-4] and [dArg2 ]dermorphin[1-4]-OH are enzymatically very stable [5,45], their potent and long-lasting antinociceptive effect may be due to their high resistance to enzymatic degradation.

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Table 4 Synthetic dermorphin analogs containing d-Arg2 . [d-Arg2 ]Dermorphin [d-Arg2 ]Dermorphin[1-4] [d-Arg2 ]Dermorphin[1-4]-OH [d-Arg2 ,␤-Ala4 ]Dermorphin[1-4] (TAPA-NH2 ) [d-Arg2 ,␤-Ala4 ]Dermorphin[1-4]-OH (TAPA) N␣ -Amidino-[d-Arg2 ,␤Ala4 ]Dermorphin[1-4]-OH (Amidino-TAPA) N␣ -Amidino-[d-Arg2 ,Me␤Ala4 ]Dermorphin[1-4]-OH (ADAMB) [d-Arg2 ,Sar4 ]Dermorphin[1-4]-OH (TAPS) [d-Arg2 ,Lys4 ]Dermorphin (DALDA) [Dmt1 ,d-Arg2 ,Lys4 ]Dermorphin ([Dmt1 ]DALDA)

Tyr1 -d-Arg2 -Phe-Gly-Tyr5 -ProSer7 -NH2 Tyr1 -d-Arg2 -Phe-Gly4 -NH2 Tyr1 -d-Arg2 -Phe-Gly4 -OH Tyr1 -d-Arg2 -Phe-␤-Ala4 -NH2 Tyr1 -d-Arg2 -Phe-␤-Ala4 -OH

H2 NC(=NH)-Tyr1 -d-Arg2 -Phe-␤Ala4 -OH

H2 NC(=NH)-Tyr1 -d-Arg2 -PheMe␤-Ala4 -OH Tyr1 -d-Arg2 -Phe-Sar4 -OH Tyr1 -d-Arg2 -Phe-Lys4 -NH2 (CH3 )2 Tyr1 -d-Arg2 -Phe-Lys4 -NH2

analgesic for obstetrical use [55]. [Dmt1 ,d-Arg2 ,Lys4 ]Dermorphin ((CH3 )2 Tyr-d-Arg-Phe-Lys-NH2 : [Dmt1 ]DALDA), a DALDA derivative containing 2 ,6 -dimethyl-Tyr, is a most potent derivative of DALDA as a ␮-opioid receptor agonist [51]. [Dmt1 ]DALDA shows an extremely potent antinociception, especially in the spinal cord. The potencies of [Dmt1 ]DALDA for antinociception are 218 times, 119 times and 5148 times higher than those of morphine after s.c., i.c.v. and i.t. injections, respectively [32]. Interestingly, these [d-Arg2 ]dermorphin[1-4] derivatives developed as new analgesics, contain the common sequence Tyr-d-Arg-Phe. This common sequence may be essential for the production of an extremely potent and prolonged antinociceptive effect. [dArg2 ,Sar4 ]dermorphin[1-4]-OH (Tyr-d-Arg-Phe-Sar-OH: TAPS) is another new analgesic that contains this common sequence [47]. With a high affinity and selectivity for ␮-opioid receptors [59], TAPS produces an extremely potent antinociceptive effect with various injection routes. The antinociceptive effect produced by i.c.v., i.t., s.c. and p.o. administrations of TAPS was 600 times, 393 times, 21 times and 1.5 times more potent than that produced by morphine, respectively [18,33,34,47–49].

Dmt, dimethyl-tyrosine; Me␤-Ala, methyl-␤-Ala.

With the above information, many N-terminal tetrapeptide analogs of dermorphin containing d-Arg2 have been developed as new analgesics (Table 4). The most remarkable molecule for a new analgesic is [d-Arg2 ,␤-Ala4 ]dermorphin[1-4]-OH (Tyr-dArg-Phe-␤-Ala-OH: TAPA) [4]. TAPA shows an extremely potent and long-lasting antinociception, which is 2000 times, 1100 times and 9.1 times greater than that of morphine after i.c.v., i.t. and s.c. injections, respectively [4,43,49]. TAPA has a very high affinity and selectivity for ␮-opioid receptors [43] and its antinociceptive effect is selectively mediated by ␮-opioid receptors [4,43]. However, the bioavailability of TAPA was unexpectedly low, and the magnitude of the antinociception induced by TAPA administered p.o. was reduced to a level comparable to morphine [7]. Sasaki et al. developed the C-terminal amidated form of TAPA, [d-Arg2 ,␤-Ala4 ]dermorphin[1-4]-NH2 (Tyr-d-Arg-Phe-␤-Ala-NH2 : TAPA-NH2 ) [44]. TAPA-NH2 has a high affinity for ␮-opioid receptors, which is comparable to TAPA, and shows a greater selectivity for ␮-opioid receptors than TAPA. TAPA-NH2 also shows a more potent antinociceptive effect than morphine, which is mediated by ␮-opioid receptors; however, its potency is still weaker than that of TAPA [25]. The structure–activity relationships study for dermorphin analogs showed that N␣ -amidination strengthens the antinociceptive activities of dermorphin analogs [15]. Based on this evidence, N␣ -amidinated TAPA N␣ -amidino-[d-Arg2 ,␤-Ala4 ]dermorphin[14]-OH (H2 NC( NH)-Tyr-d-Arg-Phe-␤-Ala-OH: amidino-TAPA) was developed [33]. Amidino-TAPA retained high affinity and selectivity for ␮-opioid receptors [22], and produced a more potent antinociception than TAPA after both s.c. and p.o. injections [7,33]. N␣ -Amidino-[d-Arg2 ,Me␤-Ala4 ]dermorphin[1-4]-OH (H2 NC( NH)-Tyr-d-Arg-Phe-Me␤-Ala-OH: ADAMB) is a more potent derivative of TAPA for antinociception [33]. The potencies of ADAMB for antinociception are 37 times and 3.8 times higher than those of morphine after s.c. and p.o. injections, respectively. Another remarkable candidate molecule for a new analgesic is [d-Arg2 ,Lys4 ]dermorphin (Tyr-d-Arg-Phe-Lys-NH2 : DALDA) [52]. DALDA has two positively charged amino acids in its sequence, and shows an extremely high selectivity for ␮-opioid receptors. Unfortunately, its antinociceptive effect in the spinal cord is only 14 times higher than morphine, and relatively very low compared to TAPA or its derivatives [54]. However, unlike other ␮-opioid receptor agonists, DALDA is highly restricted in distribution to the placenta, and may be a promising opioid

5. Distinct antinociceptive profiles of dermorphin tetrapeptide analogs Interestingly, some of the dermorphin tetrapeptide analogs containing d-Arg2 have antinociceptive profiles that are distinct from traditional ␮-opioid receptor agonists. The antinociceptive effects of i.t.-administered [Dmt1 ]DALDA, TAPS, TAPA, TAPANH2 , and amidino-TAPA are potently suppressed by the ␬-opioid receptor antagonist nor-binaltorphimine, although those peptides are very selective for ␮-opioid receptors and do not have any significant affinity for ␬-opioid receptors [18,22,51,56,59, unpublished observations]. This phenomenon is also observed with the endogenous ␮-opioid peptide endomorphin-2, which causes the release of dynorphin A via activation of ␮-opioid receptors [41]. Like endomorphin-2, the above peptides also evoke the spinal release of dynorphins through ␮-opioid receptors, and the released dynorphins subsequently stimulate ␬-opioid receptors. Intriguingly, the released dynorphins are variable with these peptides. TAPS and TAPA-NH2 cause the release of dynorphin B [18,unpublished observation], whereas TAPA causes the release of ␣-neo-endorphin [unpublished observation]. On the other hand, similarly to endomorphin-2, [Dmt1 ]DALDA causes the release of dynorphin A [56]. Amidino-TAPA causes the release of all three endogenous ␬-opioid peptides: dynorphin A, dynorphin B and ␣neo-endorphin [22]. In addition, [Dmt1 ]DALDA and amidino-TAPA evoke the spinal release of [Met5 ]enkephalin and [Leu5 ]enkephalin, respectively [22,56]. In fact, their spinal antinociceptive effects are also attenuated by the ␦-opioid receptor antagonists naltriben and naltrindole. The traditional ␮-opioid receptor agonists, such as DAMGO and morphine, do not have these effects. Therefore, the distinct antinociceptive profiles of these opioid peptides, which include the release of the endogenous opioid peptides, may be mediated by the activation of distinct ␮-opioid receptors that are insensitive to traditional ␮-opioid receptor agonists. Since the evidence supporting the distinct antinociceptive profiles of these opioid peptides is only from behavioral pharmacological experiments, more direct evidence from neurochemical or molecular biological experiments is needed to prove the distinctness of their antinociceptive profiles. The ␮-opioid receptor gene MOR-1 was first cloned in 1993 [8,60], and since then 33 splice variants have been identified in mouse MOR-1 mRNA [9,10,13,37,38]. Although the distributions of most of the splice variants in the rodent central nervous system have been described [9,10,35–38], the selectivity and intrinsic

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activity of ␮-opioid receptor agonists for each splice variant and the physiological roles of each splice variant are still unknown. We have recently found that the spinal antinociceptive effect of amidino-TAPA, which has a distinct antinociceptive profile (see above), is mediated through the activation of the MOR1J, MOR-1K and MOR-1L, which are amidino-TAPA-sensitive and DAMGO-insensitive splice variants of MOR-1 [20]. Interestingly, the release of the endogenous ␬-opioid peptides dynorphin A, dynorphin B and ␣-neo-endorphin and the endogenous ␦-opioid peptide [Leu5 ]enkephalin in the spinal cord by amidino-TAPA was eliminated under conditions that cause the desensitization of the MOR-1J, MOR-1K and MOR-1L, suggesting that the distinct antinociceptive profile of amidino-TAPA is mediated through the activation of amidino-TAPA-sensitive and DAMGO-insensitive MOR-1 splice variants [unpublished observations]. The distinct antinociceptive profiles of the other d-Arg2 -containing dermorphin tetrapeptide analogs may also be mediated by the activation of specific splice variants of MOR-1 that are insensitive to DAMGO or morphine. 6. Adverse effects of dermorphin tetrapeptide analogs Beside its potent antinociceptive effect, morphine shows many side effects in the clinic, e.g., constipation (inhibition of gastrointestinal transit), itchiness, catalepsy, respiratory depression, antinociceptive tolerance, physical dependence, and psychological dependence. Those side effects, as well as its analgesic effect, are mediated through the activation of ␮-opioid receptors. Therefore, it has long been considered that it is theoretically impossible to develop potent analgesics without severe side effects. However, as mentioned above, several new analgesics, which have antinociceptive profiles distinct from morphine, have been developed. As described below, some of these analogs also show different profiles for their side effects than morphine. 6.1. Respiratory depression Morphine shows significant respiratory depression in the clinic at doses 10 times higher than the analgesic dose (AD). In rodents, the morphine-induced respiratory depression is observed at doses of 10 × AD50 , 30 × AD50 , and 5 × AD80 after s.c., i.t. and i.c.v. administration, respectively [34,53,54]. Unlike morphine, TAPS does not induce respiratory depression even at a more than 30 times higher dose than the AD80 after i.c.v. administration [34]. Interestingly, relatively low doses of TAPS (up to 10 × AD80 ) injected i.c.v. cause respiratory stimulation. The lack of respiratory depression with TAPS may be a clinical benefit as an analgesic. In contrast, DALDA injected i.t. causes respiratory depression at a dose of 3 × AD50 [54]. [Dmt1 ]DALDA at a dose of 32 × AD50 after i.t. injection, does not show respiratory depression; however, s.c.-administered [Dmt1 ]DALDA, in contrast, causes respiratory depression at 10 × AD50 [53,54].

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cross-tolerance in mice that have developed antinociceptive tolerance to morphine [6]. Interestingly, [Dmt1 ]DALDA shows the same characteristics as TAPA for the antinociceptive cross-tolerance to morphine. Morphine injected s.c. shows antinociceptive crosstolerance in mice that have developed antinociceptive tolerance to [Dmt1 ]DALDA, whereas [Dmt1 ]DALDA injected s.c. does not show marked antinociceptive cross-tolerance in mice that have developed antinociceptive tolerance to morphine [1,61]. The evidence that TAPA and [Dmt1 ]DALDA, but not TAPS, are still effective for producing antinociception under the condition of morphine antinociceptive tolerance, suggests a clinical benefit for TAPA and [Dmt1 ]DALDA as analgesics. 6.3. Dependence liability Morphine is well known to develop the both physical dependence and psychological dependence. The physical dependence liability of the dermorphin tetrapeptide analogs containing dArg2 has only been evaluated for TAPA and TAPS [4,28]. Like morphine, chronic s.c. treatment with TAPA or TAPS causes physical dependence, and withdrawal symptoms are observed after their abrupt withdrawal. However, the withdrawal symptoms for TAPA and TAPS are relatively milder than those for morphine, suggesting that the physical dependence liability of TAPA and TAPS is lower than that of morphine. Regarding the psychological dependence liability, no research has been conducted on the dermorphin tetrapeptide analogs containing d-Arg2 . However, we have recently found that unlike morphine, amidino-TAPA lacks the psychological dependence liability [21]. As described above, amidino-TAPA causes the release of dynorphins via activation of ␮-opioid receptors. The activation of ␬-opioid receptors by the released dynorphins may block the psychological dependence from amidino-TAPA, since amidino-TAPA shows a remarkable rewarding effect in prodynorphin-knockout mice [21]. The other dermorphin tetrapeptide analogs mentioned above also cause the release of dynorphins via activation of ␮-opioid receptors. Although it has not been determined at present, those analogs may also lack the psychological dependence liability. 7. Conclusion The dermorphin N-terminal tetrapeptide analogs containing d-Arg2 show antinociceptive activity that is greater than that of morphine with various injection routes, even after peripheral injections. Some analogs have distinct antinociceptive profiles from the traditional ␮-opioid receptor agonist morphine. They stimulate the release of dynorphins through the activation of distinct ␮-opioid receptors. The activation of ␬-opioid receptors by the released dynorphins is suggested to reduce the side effects of the ␮opioid receptor agonists. The dermorphin N-terminal tetrapeptide analogs containing d-Arg2 may provide a new target molecule for developing novel analgesics that have fewer side effects and more therapeutic benefits than morphine.

6.2. Antinociceptive tolerance Acknowledgements Like morphine, most of the dermorphin tetrapeptide analogs containing d-Arg2 cause antinociceptive tolerance. However, the characteristics of their antinociceptive cross-tolerance to morphine are variable. TAPS injected s.c. shows antinociceptive crosstolerance in rats that have developed antinociceptive tolerance to morphine, whereas morphine injected s.c. does not show antinociceptive cross-tolerance in rats that have developed antinociceptive tolerance to TAPS [42]. In contrast, morphine injected s.c. and i.c.v., but not i.t., shows antinociceptive cross-tolerance in mice that have developed antinociceptive tolerance to TAPA, whereas TAPA injected s.c., i.c.v. and i.t. does not show antinociceptive

This work was supported by a Grant-in-Aid for Scientific Research (C) [KAKENHI 21600013 and 22600009] from the Japan Society for the Promotion of Science, and a Matching Fund Subsidy for Private Universities from the Ministry of Education, Culture, Sports, Science, and Technology Japan (2010–2014). References [1] Ben Y, Smith AP, Schiller PW, Lee NM. Tolerance develops in spinal cord, but not in brain with chronic [Dmt1 ]DALDA treatment. Br J Pharmacol 2004;143:987–93.

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