A Brief Survey on Recent Endomorphin-1 and Endomorphin-2 Analogues
Rossella De Marco*
Department of Chemistry “G. Ciamician”, University of Bologna, via Selmi 2, 40126 Bologna, Italy
*Corresponding author: Rossella De Marco, Department of Chemistry “G. Ciamician”, University of Bologna, via Selmi 2, 40126 Bologna, Italy, Tel: +39 0512099570; Fax: +39 0512099456; E-mail: firstname.lastname@example.org
Citation: De Marco R (2017) A Brief Survey on Recent Endomorphin-1 and Endomorphin-2 Analogues. J Chem Allied Res 2017: 14-23. doi:https://doi.org/10.29199/JCAR-101012
Received: 09 August, 2017; Accepted: 10 October, 2017; Published: 24 October, 2017
The neurophysiological distribution of the Endomorphins (EMs), i.e., Endomorphin-1 (EM1) and Endomorphin-2 (EM2), reflects their potential role in many major biological processes. These include perception of pain, responses related to stress, and complex functions such as reward, arousal, and vigilance, as well as autonomic, cognitive, neuroendocrine, and limbic homeostasis. In particular, EM1 and EM2 have demonstrated potent antinociception without some of the undesired side effects of opiate drugs. Unfortunately, their clinical applications as painkillers remain unrealistic due to their poor metabolic stability, inability to cross the Blood-Brain Barrier (BBB), and efficient efflux. In this review is reported a brief collection of recently published modified endomorphins, for which the potentiality as painkiller was clearly demonstrated in vitro and in vivo. Selected structures of bioactive EM analogues highlighting the modified residues, with references, are reported in the table of the review.
Keywords: Analgesic Activity; Bioavailability; Blood Brain Barrier; Endomorphins; Enzymatic Stability; Opioid; Peptidomimetic
The neuropeptides Endomorphin-1, H-Tyr-Pro-Trp-PheNH2 (EM1) (Table 1, 1) and Endomorphin-2, H-Tyr-Pro-Phe-PheNH2 (EM2) (Table 1, 2), have been discovered by James Zadina in 1997 . These tetrapeptides showed a noteworthy potency and selectivity for m-Opioid Receptor (MOR), and are currently regarded as the endogenous MOR agonists in the mammalian Central Nervous System (CNS), with a strong antinociceptive effect against neuropathic and acute pain. The neurophysiological distribution of EMs and the MORs in the CNS  reflects their role in many important biological processes, including pain perception, responses to stress, and complex functions such as reward, arousal, vigilance, and autonomic, cognitive, neuroendocrine functions, and limbic homeostasis [3,4]. EMs also involved in the feeding regulation .
Table 1: Selected examples of bioactive EM analogues highlighting the modified residue(s), with references.
The endomorphins have been deeply studied to developing drugs for the treatment of pain in humans [6,41]. The increasing interest encourages the development of modern green chemistry protocols for the production of large quantities of EMs [42-44].
At present, their clinical use as painkillers is hampered, due to their low metabolic stability [45,46], inability to cross the Blood-Brain Barrier (BBB), and efficient efflux [47,48]. As a consequence, in the recent years, many structural modifications have been introduced into EMs to improve their pharmacological properties: incorporation of unnatural amino acids [49-51] introduction of structural constraints  nitrogen quaternization  alteration of lipophilicity [33,53] replacement of the natural L-amino acids by their D-enantiomers  N-alkylation [54,55] introduction of α-substituted α-amino acids  β-substituted α-amino acids  proline analogues [56-58] γ-and β-amino acids [59-63] substituted β-amino acids . These issues have been extensively reviewed elsewhere [3,41,50,51,64]. In this review, we focus on modified sequences of EMs to improve bioavailability, stability, and in vivo efficacy.
EM Analogues Containing Modified Tyrosine
Tyrosine is regarded as the essential residue for EM binding and activation of MOR, and hence it is also referred to as the “message” of the peptide, therefore a few modifications at this residue have been tolerated . Very often, the derivatization or deletion of the protonable amino group of Tyr resulted in inactive compounds, or turned the agonist behavior into antagonism.
Among these, the replacement of the Tyr1 by 2′,6′-Dimethyltyrosine (Dmt) resulted in greatly increased bioactivity  however, the transit of such modified analogs into the CNS after peripheral administration in some cases remained insufficient.
In 2005, Li et al.,  introduced at the N-terminus of EM2 some Tyr analogs containing either single or multiple alkyl groups at positions 2′,3′, and 6′ of the aryl ring. The EM2 analogues containing 2′′,6′′-Diethyltyrosine (Det), 2′′-Ethyl-6′′-Methyltyrosine (Emt), and 2′′,3′′,6′′Trimethyltyrosine (Tmt) showed high MOR affinity and a potent functional agonism, as determined by the Guinea Pig Ileum (GPI) and Mouse Vas Deferens (MVD) assays, while large substituents gave inferior results. The analgesic effect of 2′,6′-dimethyltyrosine [Dmt1]EM2 (Table 1, 3) and [Det1]EM2 was determined in mice by the tail-flick test and the hot-plate test. Intracerebroventricular (i.c.v.) administration of the compounds elicited a dose-dependent antinociception response. In particular, the duration of the analgesic response [Dmt1]EM2 lasted 60 min compared with 20 min for EM2.
Jinsmaa et al.,  observed that [Dmt1]EM1 (Table 1, 4) had a weak MOR/DOR selectivity, and acted as a MOR agonist and as a potent DOR antagonist, in contrast to [Dmt1]EM2, showing that Dmt substitution affected both receptor selectivity and agonist/antagonist properties. In vivo, [Dmt1]EM1 was slightly more potent than [Dmt1]EM2 in tail-flick and hot-plate tests .
In 2012, Varamini et al.,  modified the N-terminus of EM1 by acylation of the Tyr amino group with succinamic acid, esterified in turn on the C-terminus with lactose (Table 1, 5). This modification led to a reduction of receptor binding affinity and agonist activity at MOR, but in increased stability in human plasma, and hence to significant pain easing effect in rats, after both Intravenous (i.v.) and oral administration . Indeed, 5 exhibited a dose-dependent antinociceptive activity, following i.v. administration in a Chronic Constriction Injury (CCI) rat model of neuropathic pain, with an ED50 of 8.3 μmol/kg (for morphine, 2.6 μmol/kg), and produced dose dependent pain relief after oral administration in CCI rats, ED50 19.6 μmol/kg, that was equivalent with that of morphine (20.7 μmol/kg). Interestingly, antinociception was not accompanied by constipation, a major side effect of morphine.
Wang et al., designed the analog of EM1 N α-amidino-Tyr(Me)-D-Pro-Gly-Trp-p-Cl-Phe-NH2 (Table 1, 6) that displayed a potent and prolonged antinociceptive activity upon s.c. administration through a central mechanism . The compound 6 showed a significant increase in brain and serum stability compared to EM1, with half-life (t1/2) in brain exceeding 3 h. The t1/2 of the halogenated analog was shorter than that of non-halogenated forms in the brain homogenates, whereas in serum, the former was found to be more stable .
Hau et al.,  introduced a guanidinium group on Tyr1 in EM2. This insertion decreased the MOR affinity but moderately increased metabolic stability and the analgesia after i.c.v. and i.v. administration in the tail-flick test.
Jinsmaa and co-workers  utilized unbranched alkyl chains containing 2 to 8 methylene groups, with Dmt on both ends. The best compound, 1,4-bis(Dmt-NH)butane, had a higher MOR affinity and functional efficacy, with central and systemic antinociceptive activity in vivo. A further improvement was achieved by coupling two Dmt residues to a pyrazinone ring scaffold by means of alkyl chains to yield the class of 3,6-bis[Dmt-NH-(CH2)n]-2(1H)-pyrazinones. These new compounds showed high MOR affinity, selectivity and functional agonism. In particular, 3,6-bis[Dmt-NH(CH2)3]-5-methyl-2(1H)-pyrazinone produced analgesia in mice in a naloxone reversible manner after i.c.v. and also peripheral (s.b. and oral) administration. These findings indicate that endomorphin mimetics may be useful candidates in the search for novel painkillers that pass through the gut and the BBB to target brain receptors.
EM Analogues Containing Modified Proline
The proline residue at position 2 of the EMs was found to act as a stereochemical spacer responsible for the proper orientation of the pharmacophores of the EMs . Therefore, modifications aimed at increasing peptide stability and bioavailability can be tolerated only when the peptide mimetics can still adopt the required bioactive conformation.
Shi et al., introduced in place of proline a cyclic asparagine derivative, 6-oxo-2-phenyl hexahydropyrimidine-4-carboxylic acid (cycloAsn), into the structure of EM2 . [cycloAsn]2EM2 (Table 1, 7) was more effective than EM2 upon peripheral administration in the visceral pain model, since it reduced abdominal constrictions elicited by AcOH (77% versus 62% at the same dose, respectively). The highest inhibition (97%) was observed 30-35 min. after Intraperitoneal (i.p.) injection of AcOH . However, the analgesia of 7 happened later than that of aspirin in AcOH-induced mice writhing test after i.p. injection.
Peptides containing β-amino acids generally display better pharmacokinetic properties . Gentilucci et al.,  replaced the Pro2 with (R)-pyrrolidine-3-carboxilic acid, (R)-β2-Pro, giving the tetrapeptide 8 (Table 1), which showed high MOR affinity, with Ki = 0.16 nM and IC50 = 0.5 nM, comparable to that of EM1. The stereoisomers 9 and 10 (Table 1) showed Ki = 3.8 nM, IC50 = 180 nM and Ki = 10.4 nM, IC50 = 72.0 nM, respectively . These analogues of EM1 showed increased metabolic stability compared to the native peptide. Indeed, EM1 was degraded in 3 h by α-chymotrypsin giving a mixture of Tyr-Pro-Trp and Phe-NH2; the enzymatic digestion of EM1 by aminopeptidase-M gave a mixture of Tyr-Pro and Trp-Phe-NH2, and the latter dipeptide was degraded to Trp and PheNH2; carboxypeptidase-Y hydrolyze the C-terminal amide of Phe-NH2 giving the tetrapeptide Tyr-Pro-Trp-Phe, which was degraded to Tyr-Pro-Trp, and after 24 hours only 23% of the initial amount of EM1 was detected. In contrast, under the same conditions carboxypeptidase-Y, the analogue 9 remained almost completely intact, >90%, while 10 remained intact about 80%, and 8 was degraded till 20% of the initial amount. The compounds 8 and 10 were completely degraded to 18-25% of the initial amount by carboxypeptidase-Y, while peptide 9 was still present, about 90%. Under the action of the aminopeptidase-M, the peptides 8-10 were scarcely degraded being still present in 85-90%.
Gentilucci et al.,  replaced all residues in turn in EM1 with the corresponding β3 homologues. The compound H-Tyr-β3-Pro-Trp-PheNH2 (Table 1, 11) showed a low nanomolar affinity and selectivity for MOR (Ki = 2.1 nM and IC50 = 4.0 nM) in a competitive binding assay. However, the analogue 11 was less potent compared to the parent peptide 1 . The peptide 12 (Table 1) with D-β-homo-Pro showed lower MOR affinity, Ki = 67 nM and IC50 = 79 nM. A significant loss of affinity was observed with the substitution of all amino acids of EM1. The peptides 11 and 12 showed MOR agonism, since their inhibited the accumulation of cAMP induced by forkoline in the cAMP test, with IC50 values of 6.5 and 45.0 nM, respectively (for DAMGO, 1.1 nM). The presence of homo-Pro conferred higher enzymatic stability . The analogue 12 was found more stable compared the stereoisomer 11 in the presence of carboxypeptidase-Y, only 50% of 12 being degraded after 24 h, and 23% degraded in 3 h by α-chymotrypsin. On the other hand, after 3 h of incubation with aminopeptides-M, both 11 and 12 showed excellent resistance, being intact in 89% and 96%, respectively.
The analgesic effect of 11 in vivo was evaluated by the tail flick and visceral pain tests. The peripheral administration was more effective in visceral models of pain than the tail-flick test, suggesting a predominant peripheral mode of action; indeed, peptide 11 showed antinociceptive efficacy in the tail flick test with a ED50 = 9.2 mg/kg, while the acetic acid-induced abdominal constriction test showed a ED50 = 1.2 mg/kg, after Subcutaneous (s.c.) injection in mice. The gastrointestinal propulsion was decreases after s.c. of 11 (ED50 10.0 mg/kg) .
The antinociceptive activity of systematically administered 11 was partially blocked by both MOR antagonists, namely naloxone given i.c.v., and naloxone methiodide given i.p. The KOR antagonist nor-binaltorphimine and the DOR antagonist naltrindole resulted ineffective. Apparently, the compound 11 does not readily cross the BBB, but at higher doses was observed that this cyclopeptide can produce antinociception through both peripheral and central opioid receptors in mouse models , thus implicating both central and peripheral mechanisms of action. The peptide acts preferentially through central and peripheral MOR to produce antinociception and to inhibit gastrointestinal transit. This EM1 mimetic is the first analogue of EM1 showing antinociceptive activity after systemic administration.
Yu et al., in 2015 combined the introduction of Tic, i.e., 1,2,3,4-tetrahydroisoquinoline carboxylic acid, in place of Pro2, and of α-hydroxy-β-amino acids instead of Tryptophan (Trp) and Phenylalanine (Phe) at positions 3 and 4, respectively . The resulting analogues H-Dmt-TicTrp-(2S,3S)AHPBA-NH2 (Table 1, 13) and H-Dmt-Tic-(2R,3S)AHPBA-Phe-NH2 (Table 1, 14), showed a great increase in enzymatic stability (t1/2>2 h). The antinociceptive activity of 14 was equal to that of morphine in the mouse warm-water (55°C) tail-withdrawal test. In conclusion, the replacement of Tyr1-Pro2 by Dmt1-Tic2 and the introduction of α-hydroxy-β-amino acid led to a dramatic increase in enzymatic stability of the EM analogues .
Methylation of amide bonds is an important modification that can tune biological functions. This simple method increased the lipophilicity of peptides, reduced conformational flexibility, which is usually an undesired property, and inhibits the action of proteolytic enzymes, finally increasing the BBB transport of small peptides . Kruszynski et al.,  described analogues of EM2 containing N-methylated residues. [Sar2]EM2 (Sar = N-methylglycine) (Table 1, 15) showed the same potency compared to EM2 in triggering antinociception in mice after central administration, but displayed also a significant pain-relieving effect when given peripherally .
Varamini et al., proposed the replacement of Pro2 by 2-Aminocyclopentanecarboxylic Acid or 2-Aminocyclohexanecarboxylic Acid residues (Acpc and Achc) to improve the lipophilicity of EM2 . These analogues [(1S,2R)Acpc2]EM2 (Table 1, 16) and [(1S,2R)Achc2]EM2 (Table 1, 17) penetrated across the rat brain cells (co-culture of primary cerebral ECs and glial cells) via passive diffusion and the permeability coefficient of the analogs was significantly higher than that of parent peptide, suggesting increased BBB permeation.
EM Analogues Containing Modified Tryptophan or Phenylalanine at Position 3
The residues tryptophan or phenylalanine at position 3, belong to the address portion of EM1 or EM2 . However, some atypical peptides deprived of some fundamental pharmacophores have been shown to interact with the opioid receptors mainly through interactions involving the Trp as the message portion. The prototypic compound in this series is the cyclic pentapeptide c[Tyr-D-Pro-D-Trp-Phe-Gly] (Table 1, 18). This compound emerged from a library of cyclic analogues of EM1 by Cardillo et al., . The analog 18 showed nanomolar affinity and selectivity for MOR, albeit lacking the fundamental protonable amino group of Tyr. This analogue was more lipophilic and resistant to enzymatic degradation than EM1, and produced preemptive antinociception in a mouse visceral pain model when given i.p. or s.c. . The replacement of DPro by glycine gave the analogue c[Tyr-Gly-D-Trp-Phe-Gly] (Table 1, 19), for which the affinity towards MOR was almost 10-fold better than 18 . These cyclic analogues 18 and 19 can still interact and activate the opioid receptor as agonists by alternative mechanisms , and produce antinociceptive effect via the peripheral opioid receptors .
De Marco et al., in 2014 determined the minimal bioactive sequence of 18 and 19, the tripeptide Ac-D-Trp-Phe-GlyNH2 (Table 1, 20) . This short peptide 20 showed nanomolar affinity and selectivity toward MOR with Ki = 5.6 nM, determined by displacement binding assays. The tripeptide 20 acted as a partial agonist, since it inhibited forskolin induced cAMP accumulation with moderate maximal effect, similarly to EM1 and EM2 [71,72] (the full agonism of the EMs has been observed in few experiments ).
The introduction of the nitro group, halogens, alkyl groups, and combinations of different groups into the indole ring of Trp in 20 [74,75], gave the 5-NO2-tripeptide (Table 1, 21) and 7-Br-2Me-tripeptide (Table 1, 22) with interesting behavior in vitro and in vivo . The compound 21 showed a significant MOR affinity in vitro, with Ki = 51.9 nM. The 7-Br-2-Me-tripeptide was an excellent MOR ligand with Ki = 4.03 nM, despite the absence of any cationic group for a strong ionic interaction with the receptor, and the presence of two sterically demanding groups. The modification at indole ring of Trp increased the enzymatic stability, indeed after 3 h the nitrotripeptide 21 was degraded <10%, and the 7-Br-2-Me-22 about ~20%, in mouse serum. The antinociceptive tests in vivo gave unexpected results. The antinociceptive response was expressed as a percentage of Maximal Possible Effect (% MPE); the tripeptide 21 displayed the highest analgesic effect in vivo, 46% MPE at 30 min as determined by the tail flick test, while the 7-Br-2Me-tripeptide 22, under the same conditions, gave only 32% MPE at 15 min. For comparison, the correlated cyclopeptide 19 and tripeptide 20, showed a mild analgesic effect, which peaked at 30 min of exposure with 32% MPE and MPE 37% at 30 min, respectively (Figure1) .
To rationalize these data, the physicochemical indicators were calculated and analyzed. Lipophilicity is a chemical-physical parameter implicated in penetration across biological barriers . The limitation to lipophilicity as a strategy to improve bioavailability is the solubility of analogs in aqueous media, and their partition into the brain’s interstitial fluid to exert an effect. Compounds too lipid-soluble cannot traverse the Brain-Blood-Barrier (BBB) because they are in effect trapped in the membrane [77,78]. The partition coefficient (logP) is often utilized to describe the lipophilicity of a compound and its ability to cross the BBB . When the logP > 2, the peptides can reach the brain, but for logP > 3.5 the solubility in water is poor and bioavailability is reduced [80,81].
Another important parameter is the topological Polar Surface Area (tPSA) that correlates with drug transport properties. In particular, it represents the quantity of surface donation of polar atoms in a molecule .
The logP and tPSA values for the peptides 19-22 were calculated and analyzed; the ranking for clogP was 22 >> 19 > 20 > 21, and for tPSA was 22 ~ 20 < 19 < 21. It is generally accepted that low clogP and/or higher tPSA correspond to poor BBB permeation, therefore these calculated values do no account for the experimental efficacy in the mouse tail flick assay. The compound 21 showed significantly higher antinociception compared to the other compounds despite its clearly less favorable clogP (too low) and tPSA (too large), and even despite of the reduced MOR affinity (10-8 M) compared to 22.
In contrast, compounds’ efficacy in the mouse tail flick assay nicely correlated to the Lipophilic Efficiency Indices (LLE) and the Ligand Efficiency Dependent Lipophilicity Index (LELP) (Figure 1). LLE can be used to identify low potency target compounds with small size and low lipophilicity. LELP has been recently proposed to combine lipophilicity, molecular size and potency into a single parameter, thus overcoming the size limitation of LLE. Large LLE and low LELP values have been shown to correlate well with in vivo efficacy. The best LLE scores are ~5-7 or greater based on an average oral drug clogP ~2.5, and potency in the range of ~1-10 nM. The lower limit of LLE is 0.3 and the lipophilicity range is -3< log P <3 which defines a range of best LELP scores between -10 and +10 .
Figure 1: The antinociceptive efficacy in vivo correlated to the LLE, LELP, tPSA, clogP and MPE% at 30 min.
The calculated LLE values were: 20 ~21 > 19 > 22. For LELP: 21 << 20 < 19 < 22. The large LLE (>7) and the very low LELP of 21, perfectly agreed with the higher efficacy observed in vivo. The major contributor to the lipophilicity index in 22 is the halogen, and the values of clogP and tPSA explained why 22 probably crossed the BBB very rapidly .
EM Analogues Containing Modified Phenylalanine at Position 4
Together with Phe3/Trp3, PheNH24 belongs to the address portion of the EMs, and exerts a certain influence on the binding properties of the ligands .
Wang et al., studied analogues of EMs, where the last amino acid was replaced by D-Ala and ended with a benzyl  group displayed higher MOR affinity, Ki = 4.56 and 8.67 nM, respectively. The substitution of Phe4 with D-Val gave 23 (Table 1) that exhibited a MOR affinity about 2-fold higher (Ki = 2.32 nM), and a DOR affinity 1.6-fold higher, in binding assay. In contrast, the selectivity decreased about 3-fold compared to parent peptide EM2. The antinociceptive response after i.c.v. administration and the inhibition effect reached the peak 5 min after injection. Compound 23 still displayed about 50% MPE, 60 min after i.c.v. administration at dose 6.7 nmol/kg. Moreover, the duration of the hot plate response inhibition, induced by all the parents and analogs, appeared to be shorter than morphine .
Fujita et al.,  prepared analogues of EM2 by introduction of aromatic amines at the Cterminal amide. The compounds with 1-naphthyl, 5-quinolyl, cyclohexyl, and 2-adamantyl showed MOR affinity with Ki = 2.41-6.59 nM. As discussed in the first section, the replacement of Tyr1 by Dmt exerted profound effects. In vivo, H-Dmt-Pro-Phe-NH-5-Isq (5-isoquinolyl) (Table 1, 24) after Intracisternal (i.c.) administration in the tail pressure test in mice produced a dose-dependent antinociceptive effect that was antagonized completely by naltrexone. The maximal effect of 24 (30 μg/mouse) was about 1/10 of that obtained for morphine .
Introduction of methyl, ethyl, and tert-butyl esters at the C-terminus of EM2 gave analogues which were tested in vivo for contractions of the longitudinal muscle of distal colon . The ED50 values of the analogs were about 1.5-fold higher, 2- and 8-fold lower than EM2, respectively. This approach appeared promising to reduce undesirable side effects. Antinociception was tested in vivo by the warm water (50°C) tail flick assay after i.c.v. injection (0.67-20 nmol/kg). All compounds induced a dose-related antinociceptive effect, with peak at 5 min after injection. At the dose of 20 nmol/kg, the MPE values of the analogs were 61%, 73% and 61%, respectively, similar to EM2 (58%).
The conversion of the C-terminal amide into a hydrazide in EMs did not markedly change their MOR binding affinities. Nonetheless, EM2-NHNH2 (Table 1, 25) showed decreased GPI and MVD potencies (10- and 5-fold compared to EM2, respectively) . It is notable that EM1-NHNH2 (Table 1, 26) exhibited the highest antinociception effect after i.c.v. injection, being 1.5-fold more potent than EM1, but with moderate colonic contractile and expulsive effects, comparable with the parent peptide . Further, 25 showed a slightly lower analgesia than EM2, at higher doses (i.c.v., 1.5 and 5 nmol/mouse). The inhibitory effects of colonic propulsion were drastically attenuated, that would be helpful for the development of suitable MOR drugs, without some undesirable side effects.
Wang et al., introduced α-methylene-β-amino acids (Map) at diverse positions of EM1 . Compared to normal classic β-amino acid, these unnatural residues are highly constrained due to the double bond at the Cα. The compounds 27 and 28 (Table 1), in which Phe4 was substituted with a furyl ring, displayed high activity and selectivity for MOR (EC50 = 0.0334 and 0.0342 nM, respectively). The enzymatic stability of EM1, 27, and 28, was assessed in the mouse brain homogenate; EM1 was degraded very rapidly, with a t1/2 of 16.9 min, while t1/2 for [Map4]EM1 was ranging from 62 to 90 min. Compounds 27 and 28 showed strong antinociceptive effect in vivo in the mouse tail-flick test (10.9- and 9.8-fold more potent than EM1, respectively).
The same authors further modified the sequence of EM1, by combining diverse unnatural residues: Dmt1, (R)-β-Pro2, and (Ph)-Map/(2-furyl)Map4. The resulting analogue H-Dmt-(R)-β-ProTrp-(2-furyl)Map-NH2 (Table 1, 29) showed picomolar affinity and subpicomolar potency for MOR (EC50 = 0.0420 pM, Ki = 3.72 pM) . In mouse brain homogenate, 29 showed high stability, t1/2 > 600 min. The agonist behavior was assessed by the forskolin-induced cAMP accumulation test, that displayed an extremely high potency, with EC50 = 0.0421 pM, and Emax 99.5%. This compound induced a strong and consistent antinociceptive effect in vivo in the tail-flick test, ED50=0.532nmol/kg .
Tóth et al.,  introduced another combination of substitutions into the sequence of EM1: 2-Aminocyclohexanecarboxylic Acid (Achc) and Dmt1, pF-Phe4, or βMe-Phe4. The analogue 30 with Dmt1, cis-(1S,2R)Achc2, and pF-Phe4 (Table 1) preserved MOR activity and selectivity at [35S]GTPγS binding assays. The replacement of Phe4 by (2R,3R)βMe-Phe4 gave 31 (Table 1) that reduced selectivity and affinity toward MOR, while the introduction of (2S,3S)βMe-Phe4 (Table 1, 32) showed increased affinity and potency toward MOR. The metabolic stability of 30, 31 and 32 (Table 1) was significantly increased (t1/2 > 20 h) compared to the EM1 (t1/2 = 5-7 min) .
Hybrid Analogues of EM1 and EM2
In this section, hybrid structures of EMs and other neuropeptides are reported. In the examples proposed, the conjugation of sequences belonging to different peptides was shown to improve stability, and/or enhanced BBB permeability, while high MOR potency was maintained.
Mollica et al.,  fused the structures of two highly selective MOR ligands, namely EM2 and DAMGO, H-Tyr-D-Ala-Gly-(N-Me)Phe-Gly-ol. DAMGO was known to be more than 100-fold potent than morphine in mouse writhing nociception assay, and displayed a higher metabolic stability compared to other opioid peptides. The hybrid 33 (Table 1) showed in vivo an AD50 = 3.77 and 2.65 nmol in the hot plate test and tail flick assay, respectively, (for EM2 in the same tests AD50 = 2.29 and 1.49 nmol). With a t1/2 of about 40 min., the metabolic stability of 33 was much higher than EM2, showing that multi-N-methylation and C-terminal modification with ethanolamide moiety gave increase plasma stability and could be a useful tool to develop stable candidates as opioid drugs .
Koda et al.,  introduced Lipoamino Acids (Laas) at both ends of EM1, obtaining an increase in lipophilicity and stability of the endogenus ligand. The pentapeptide analogue [C8LaaDmt1]EM1 (Table 1, 34) showed a t1/2 of 43.5 min in a mixture of digestive enzymes, 8-fold greater than that of EM1 . This compound 34 exhibited potent in vitro MOR agonist affinity (Ki = 0.08 nM, >140-fold better that of the parent peptide) and a subnanomolar inhibition of cAMP production .
Varamini et al.,  synthesized the analogue of EM1 35 (Table 1), in which Tyr1 was substituted by 2-aminodecanoic acid gave. This analogue showed promising potential for the treatment of neuropathic pain, exhibiting strong analgesic activity after systemic administration, without producing constipation, a major side-effect of morphine.
The same authors proposed an analogue of EM1 with a C10-Laa at the N-terminus by attaching various Substance P (SP) fragments at the C-terminus (Table 1, 36) . SP is the undecapeptide neurotransmitter associated with liaising the hyperalgesic response. Nevertheless, low doses of SP were shown to produce a modest pain-relieving effect  and were capable of intensifying opioid-mediated analgesia . In 2007, Kream and co-workers  demonstrated that C-terminal SP fragments conjugated with opioid peptides, produced strong antinociceptive effect with little or no development of opioid tolerance or dependence in rats. The hybrid peptides proposed by Varamini, showed an important amelioration in permeability across Caco-2 monolayers, enzymatic stability, high MOR affinity and agonist activity with nanomolar inhibition of forskolin stimulated cAMP production .
Janecka et al., designed the compound Tyr-c[D-Lys-Phe-Phe-Asp]NH2 (Table 1, 37), in which the aspartate was used as a β-amino acid, and the α-carboxylate was connected to the amino-side chain of Lys, giving a EM2 analogue which conserved the free N-terminus of Tyr1 . Compound 37 showed MOR affinity comparable to EM2, higher enzymatic stability in rat brain homogenate, and a longer-lasting antonociceptive activity after i.c.v. administration . Instead, the dimer of 37 (Tyr-c[D-Lys-Phe-Phe-Asp]NH2)2 (Table 1, 38) displayed a potent peripheral antinociceptive activity in the mouse model of visceral inflammatory pain , possibly associated to the larger molecular size which prevented the access into the CNS.
The replacement in the sequence of 37 of Phe at positions 3 and 4 by D-1-naphthyl-3alanine (D-1-Nal) or D-2-naphthyl-3-alanine (D-2-Nal), and replacement of Tyr1 by Dmt , gave 39 and 40 (Table 1) . The introduction of residues of opposite stereochemistry tended to stabilize alternative secondary structures. Both compounds were excellent ligands for MOR with Ki = 0.25 and 0.44 nM, respectively, and good ligand for KOR (Ki = 1.78 and 1.02 nM). The analogues 39 and 40 displayed high antinociceptive activity in mice when delivered by i.c.v. but also i.p. injection, but slightly lower activity after systemic (i.p.) administration .
Gentilucci et al.,  mixed the structure of EM1 with the structure of another opioid ligand of natural origins, the KOR/MOR cyclotetrapeptide ligand CJ-15,208 c[Phe-D-Pro-Phe-Trp], isolated from the fermentation broth of the fungus Ctenomyces serratus ATCC15502 . The introduction of a β- and a γ-amino acid gave the compounds 41, c[β-Ala-D-Pro-Phe-Trp], and 42, c[GABA-D-Pro-Phe-Trp], (Table 1). The analogue 41 displayed selectivity towards MOR and revealed full agonist activity in vitro, while 42 showed certain selectivity for DOR over MOR, acting as a strong DOR antagonist and a weak agonist at MOR. Interestingly, 41 revealed a strong, MOR dependent antinociceptive effect in vivo upon systemic administration, consistent with the cyclic nature. It is well acknowledged that peripheral and central MOR may contribute to modulate visceral pain.
Nowadays, in the academic community there is increasing interest towards the use of the endogenous opioid peptides EM1 and EM2 as potent analgesics for the treatment of forms of pain resistant to morphine and the other alkaloids, such as neuropathic pain. Indeed, there is clear evidence that the antinociception mediated by EM is dissociated from the typical, severe side effects of the opiate drugs currently utilized in clinical practice. In this review, the recent modifications adopted to increase stability and/or bioavailability, and in vivo analgesic activity, of the native peptides are presented and discussed. Several of the EM analogues proved to be highly active in vivo in animal models, therefore they represent suitable candidates for clinical investigations. Nonetheless, it appears that renewed effort has to be dedicated to raise the interest of the pharmaceutical companies to develop and exploit these peptidomimetics as painkillers.
Conflict of Interest
The author confirm that this article content has no conflict of interest.
We thank Fondazione Umberto Veronesi for financial support.