Glycosylated cyclic endomorphin analogs

Abstract

For example, the formula (I): A, which is useful in the treatment of pain 1 -Cyclo[A 2 -A 3 -A 4 -A 5 -A 6 -O-Carb, the formula (II): A 1 -Cyclo[A 5 -A 3 -A 4 -A 2 -A 6 -O-Carb glycosylated cyclic peptides, and pharmaceutically acceptable salts thereof, are described herein. In some embodiments, A 1 is L-Tyr; A 2 is D-Lys, D-Orn, D-Dab, or D-Dpr; A 3 is L-Trp; A 4 is L-Phe; A 5 is an amino acid residue selected from the group consisting of Asp, Glu, iso-Asp, and iso-Glu; A 6 is (a) a hydroxy-substituted amino acid residue (HO-AA), or (b) an oligopeptide containing 2-5 amino acid residues including HO-AA; Carb is a carbohydrate group bonded to the side-chain oxygen of HO-AA by a β-D-glycoside bond, and optionally, the C-terminus of A 6 is amidated, for example, as a primary amide.

Description

Technical Field

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 347,182, filed May 31, 2022, which is hereby incorporated by reference in its entirety.

[0002] This invention was made with government support under I01BX003776 awarded by the Department of Veterans Affairs of the United States of America and DA052539 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] The biological sequence information of this application is included in an XML file named "TU-685 PCT.xml" with a file size of 29,122 bytes and created on May 24, 2023, which is hereby incorporated by reference in its entirety.

[0004] The present invention relates, among other things, to novel opioid receptor agonists for use in methods of treating pain and opioid use disorder. More specifically, the present invention relates to methods of treatment using glycosylated cyclic endomorphin-1 (EM1) and endomorphin-2 (EM2) analogs and analogs.

Background Art

[0005] Opioids that act on the μ-opioid receptor (MOR) are the gold standard for moderate to severe pain relief, but their use is limited by serious side effects, specifically a tendency to abuse. Balancing the risk of addiction with concerns about inadequately treated pain has led to fluctuations in the increasing restrictions following the increase in opioid prescriptions. However, the spread of opioid overdose has steadily increased in approximately three waves, namely, a steady increase since the 1990s due to the increase in opioid prescriptions, a sharp increase starting in 2010 due to heroin overdose, and a recent increase due to fentanyl overdose (CDC1).

[0006] According to the US Centers for Disease Control and Prevention (CDC), opioids were involved in 49,860 overdose deaths in 2019, or 137 deaths per day, accounting for 70.6% of all drug overdose deaths (CDC2), exceeding the reports of motor vehicle accidents by the US National Highway Traffic Safety Administration (NHTSA) (93 per day; NHTSA). 38 of these deaths per day (28%) are directly related to prescription drugs, but recent studies have shown that 75 percent of humans who began misusing opioids in the 2000s reported that their first opioid was a prescription drug (Cicero et al. 2014). Limitations on drugs available for pain treatment play an important role in the epidemic, as shown by the CDC proposal (CDC1): "To cover this epidemic, we need to improve how we treat pain. We must prevent misuse, regular use, and overdose before they start." Therefore, compounds that provide effective pain relief without toxicity can help prevent the onset of addiction and contribute to its treatment.

[0007] Currently available treatments for OUD include various forms of methadone, buprenorphine, naltrexone, and buprenorphine + naloxone. Methadone and buprenorphine play beneficial roles in the treatment of OUD. They produce an effective opioid substitution effect through their relatively long-lasting action, which can reduce the need for the next dose. However, they retain rewarding properties. They have a propensity for abuse as indicated by strong increases in intravenous self-administration (SA), locomotor sensitization, and conditioned place preference (CPP) behavior in rats, and are thus strictly regulated (Martin et al., 2007; Steinpreis, Rutell, and Parrett, 1996; Tzschentke, 2004; Wade et al., 2015). In humans, buprenorphine and methadone have clinical utility in reducing the positive subjective effects of opioids, but both compounds are self-administered and produce positive reinforcing effects (Comer, Sullivan, and Walker, 2005; Jones, Madera, and Comer, 2014). Buprenorphine is combined with the antagonist naloxone in several formulations such as Suboxone, Bunavail, and Zubsolv, and all of them are vulnerable to influencing the induction of withdrawal effects. Naltrexone, a complete opioid antagonist, can block opioid craving but precipitates withdrawal symptoms and cannot be used prior to medically managed opioid withdrawal. Thus, currently available treatments are successful but have significant limitations, and new treatments are urgently needed. Therefore, new treatments with reduced rewarding properties could increase the options for the treatment and management of OUD.

[0008] Drugs that can effectively treat pain without a rewarding effect could play an important role in reducing OUD. Such compounds could also contribute as a treatment for OUD. The μ-opioid receptor is the target of the most effective current treatments for OUD and includes the μ-agonist methadone and the partial agonist buprenorphine. These substances are effective but are themselves abused, produce positive reinforcing effects, and result in withdrawal symptoms. Alternative treatments with low or no propensity for abuse could change the treatment of OUD.

[0009] In recent years, MOR has been designated as a (clinic) protein by the Target Development Level (TDL) classification of the Genomic Analysis (IDC) Knowledge Management Center for Drugable Therapies. clin (Oprea et al., 2018). This name reflects a target that is related to at least one approved drug / active pharmaceutical ingredient by mechanism of action (MoA). MOR is one of the most widely studied drug targets, providing a knowledge base that enables many approaches to separate desirable effects (analgesics) from undesirable effects mediated by receptors. Multiple mechanisms of action are currently known to be mediated by MOR, including G protein and β-arrestin activation, ion channel modulation, and many intracellular signaling functions (Al-Hasani and Bruchas, 2011). Bohn and colleagues showed that β-arrestin knockout mice exhibited enhanced morphine-induced analgesia, reduced respiratory depression, and GI dysfunction (Bohn et al., 1999; Raehal, Walker, and Bohn, 2005). These findings raised the possibility that optimal criteria for analgesics brought about by MOR activation could be achieved with fewer side effects based on differential activation of the various mechanisms mediated by MOR.

[0010] Many programs are thought to develop biased agonists - compounds that selectively activate one signaling pathway over another - in which case it was the G protein rather than β-arrestin signaling. Indeed, this approach has been successful in generating compounds that increase analgesia but decrease respiratory depression and GI dysfunction (e.g., Schmid et al. 2017). However, findings particularly relevant to OUD are that β-arrestin knockout mice and G protein-biased agonists also show increased sensitivity to the rewarding effects of morphine, including increased CPP and striatal dopamine release (Bohn et al. 2003) and changes in intracranial self-stimulation (Altarifi et al. 2017) compared to wild-type mice. Furthermore, G protein-biased agonists show increased hyperalgesia (Araldi, Ferrari, and Levine 2018), and β-arrestin knockout mice show increased allodynia (Chen et al. 2016). These studies have shown that G protein / β-arrestin-biased agonists can worsen, rather than improve, abuse liability and pain duration. Thus, biased agonists reflect the potential to ultimately separate desirable from undesirable effects, but many studies remain to address this potential with respect to OUD.

[0011] Endomorphin (EM) is a potent and selective natural short-chain peptide agonist of the μ-opioid receptor (MOR), which is the major analgesic target of currently used opioids such as morphine. Soon after their discovery (Zadina et al., 1997), EM showed a promising profile of a potent analgesic with reduced some side effects, including a reduction in the reward (Wilson et al., 2000) and respiratory depression (Czapla et al., 2000) of EM1. Since natural peptides are unstable in plasma, EM-based pharmaceuticals require chemical modification of the structure (EM analogs). Cyclic, D-amino acid-containing EM analogs were described by Zadina et al. and were evaluated for (1) metabolic stability of favorable drug properties, (2) highly effective pain suppression, and (3) a marked reduction in harmful side effects. After extensive screening of many analogs for these properties, four that showed considerable promise were thoroughly characterized (Zadina et al., 2016; and U.S. Pat. Nos. 8,716,436 and 10,919,939). These substances, specifically the EM1 analog designated ZH853, showed several useful properties but still had room for improvement, for example, improving blood-brain barrier and blood-intestinal barrier penetration.

[0012] The pain suppression test showed that ZH853 can penetrate important barriers including the blood-brain barrier (BBB) that shows central activation and the gut-blood barrier that results in oral efficacy. However, there are significant limitations that clearly indicate that these analogs need to be formulated or modified to improve central penetration and be reliably effective as oral formulations. This is particularly important for the treatment of potential drug abuse disorders since the injection route can provide a trigger for relapse.

[0013] For example, in a pain test comparing ZH853 to morphine (Feehan et al., 2017), the ED 50 for pain relief was similar for morphine and ZH853 after peripheral (i.v.) administration. The mean ED 50On a weight basis (mg / kg), ZH853 is 1.4 times higher than morphine and 1.7 times lower on a molar basis, showing an approximate morphine equivalence of about 1. On the other hand, intrathecal administration of ZH853 in 3 models was on average 62 times more potent than morphine. These data indicate that less ZH853 than morphine penetrates into the central tissue, but the analog is considerably more potent in activating these targets, resulting in an intravenous (i.v.) effect of approximately equal efficacy. Thus, substances with improved blood-brain barrier penetration were able to substantially reduce the required dose and limit peripherally mediated side effects. Furthermore, the increased oral efficacy provided a preferred route of administration for pain and OUD treatment.

[0014] Furthermore, compounds that selectively bind to the δ and κ opioid receptors (DOR and KOR, respectively) have also been reported to be useful in the treatment of pain and, to some extent, other symptoms such as drug dependence, with some success.

[0015] Given the problems with current opioid compounds and methods for treating pain (e.g., chronic pain, neuropathic pain, inflammatory pain, etc.), drug dependence, and OUD, there is a continuing need for new opioid compounds. The compounds and methods described herein address these needs.

Prior Art Documents

Patent Documents

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Patent Document 1

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Non-Patent Documents

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Summary of the Invention

[0018] (Abstract) The compounds described herein are cyclic peptide analogs of EM1 and EM2, having a cyclic peptide pharmacophore with a tyrosine or tyrosine derivative adjacent to the N-terminus and a glycosylated amino acid or glycosylated short-chain peptide adjacent to the C-terminus. In some embodiments, the EM analogs are of formula (I): A 1 -cyclo[A 2 -A 3 -A 4 -A 5 -A 6 -O-Carb, or of formula (II): A 1 -cyclo[A 5 -A 3 -A 4 -A 2 -A 6 -O-Carb peptides; and pharmaceutically acceptable salts thereof. These compounds can be used in methods for treating pain (e.g., chronic pain, neuropathic pain, inflammatory pain, etc.), as well as in methods for treating drug dependence and OUD. The innovative approach to the development of analgesics described herein departs from strategies for modifying mostly opium-derived compounds, including century-old compounds such as oxycodone and hydrocodone. The latter in combination with acetaminophen (e.g., Vicodin) was the most widely prescribed of all drugs in the United States in 2013 (Imformatics 2104). In contrast, the pharmacophore utilized herein is a glycosylated cyclic analog of EM1 similar to some of the substances described by Zadina et al. (Zadina et al., 2016, which is hereby incorporated by reference in its entirety; and U.S. Patent Nos. 8,716,436 and 10,919,939).

[0019] As used herein, the partial peptide formulas indicated by "cyclo[... ]" and "c[... ]" refer to cyclic peptides in which the peptide ring is formed by a non-peptide crosslinked amide bond between the first and last amino acid residues within the square brackets.

[0020] For reference, the abbreviations of the amino acids described herein are alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), valine (Val), ornithine (Orn), 1-naphthylalanine (Nal), 2,3-diaminopropionic acid (Dpr), 2,4-diaminobutyric acid (Dab), allothreonine (allo-Thr), cis-4-hydroxyproline (cis-4-hydroxy-Pro), trans-4-hydroxyproline (trans-4-hydroxy-Pro), cis-3-hydroxyproline (cis-3-hydroxy-Pro), trans-3-hydroxyproline (trans-3-hydroxy-Pro), homoglutamic acid (hGlu; also called 2-amino-1,6-hexanedioic acid), homolysine (hLys; also called 2,7-diaminoheptanoic acid), bis-homoglutamic acid (bhGlu; also called 2-amino-1,7-heptanedioic acid), and bis-homolysine (bhLys; also called 2,8-diaminooctanoic acid).

[0021] Many examples of natural and unnatural amino acids are described in Chapter 14 “Amino Acid Properties and Consequences of Substitutions” by Matthew J. Betts and Robert B. Russell in Bioinformatics for Geneticists, Matthew R. Barns and Ian C. Cray, Eds., John Wiley & Sons, Ltd., pp. 298 - 316 (2003), which is hereby incorporated by reference in its entirety and hereinafter referred to as Betts and Russell.

[0022] As used herein, “isoAsp,” “isoGlu,” “iso-hGlu,” and “iso-bhGlu” refer to aspartic acid, glutamic acid, homoglutamic acid, or bis-homoglutamic acid residues of the peptide chain that are each bonded to an adjacent succeeding amino acid residue (extending in the N-terminal to C-terminal direction) in the chain by an amide bond between the side chain carboxyl of aspartic acid, glutamic acid, homoglutamic acid, or bis-homoglutamic acid and the α-amino group of the succeeding amino acid residue. In other words, the α-carboxyl group of an isoAsp, isoGlu, or iso-hGlu residue does not form part of the peptide backbone.

[0023] As used herein, the term “hydroxy-substituted amino acid” (“HO-AA”) refers to an amino acid having a hydroxyl substitution on its side chain. Non-limiting examples of hydroxy-substituted amino acids include Ser, Thr, allo-Thr, homoser, cis-4-hydroxy-Pro, trans-4-hydroxy-Pro, cis-3-hydroxy-Pro, and trans-3-hydroxy-Pro.

[0024] In Formulas (I) and (II): A 1 is an amino acid residue selected from the group consisting of Tyr, N-methyl-Tyr, 2,6-dimethyl-Tyr, and N-methyl-2,6-dimethyl-Tyr; A 2is an amino acid residue selected from the group consisting of Lys, hLys, bhLys, Orn, Dab, and Dpr; A 3 is an amino acid residue selected from the group consisting of Trp, N-methyl-Trp, Phe, N-methyl-Phe, p-X-Phe, and N-methyl-p-X-Phe; A 4 is an amino acid residue selected from the group consisting of Phe, N-methyl-Phe, p-X-Phe, and N-methyl-p-X-Phe; wherein X is F, Cl, Br, or NO2 at the 4-position of the phenyl group of the Phe side chain; A 5 is an amino acid residue selected from the group consisting of Asp, Glu, hGlu, bhGlu, iso-Asp, iso-Glu, iso-hGlu, and iso-bhGlu; A 6 is (a) a hydroxy-substituted amino acid residue (HO-AA) (e.g., an HO-AA selected from the group consisting of Ser, Thr, homo-Ser, cis-4-hydroxy-Pro, trans-4-hydroxy-Pro, cis-3-hydroxy-Pro, and trans-3-hydroxy-Pro); or (b) an oligopeptide containing 2 to 5 amino acid residues (e.g., a dipeptide, tripeptide, tetrapeptide, or pentapeptide) containing at least one HO-AA; Carb is a carbohydrate group such as glucose (D-Glc), lactose (D-Lact, or D-Glc-β-D-Gal), etc., which is bonded to the side chain oxygen of the HO-AA of A 6 by a β-D-glycosidic bond; The notations cyclo[A 2 -A 3 -A 4 -A 5 and cyclo[A 5 -A 3 -A 4 -A 2 mean that the side chain amino group of A 2 is bonded to A 5 by an amide bond, thereby forming a ring, and A 5When A is isoAsp, isoGlu, iso-hGlu, or iso-bhGlu, the side-chain amino group of A 2 is bonded to the α-carboxyl group of A 5 by an amide bond, but when A 5 is Asp, Glu, hGlu, or bhGlu, the side-chain amino group of A 2 is bonded to the side-chain carboxyl group of A 5 by an amide bond, representing a cyclic peptide ring; Optionally, the C-terminus of A 6 is amidated (e.g., as a primary amide).

[0025] In some embodiments, the N-terminal residue of the oligopeptide is HO-AA. In some other embodiments, the C-terminal residue of the oligopeptide is HO-AA. In still other embodiments, the oligopeptide contains two or more HO-AA residues. In some preferred embodiments, the C-terminus of A 6 is amidated as a primary amide.

[0026] In addition to HO-AA, the oligopeptide can contain any combination of amino acid residues, e.g., D-amino acid residues, L-amino acid residues, and Gly.

[0027] As used herein, the term EM1 analog refers to a peptide in which A 3 = Trp or N-methyl-Trp, while the term EM2 analog refers to a peptide in which A 3 = Phe, N-methyl-Phe, p-X-Phe, or N-methyl-p-X-Phe.

[0028] In some preferred embodiments of formula (I), A 2 is a D-amino acid residue.

[0029] In some preferred embodiments of formula (II), particularly when A 5 is Asp, Glu, hGlu, or bhGlu, A 5 is a D-amino acid residue.

[0030] The compounds of formula (I) and formula (II) are useful for the treatment of pain, drug dependence, and opioid use disorder.

[0031] In some preferred embodiments, the peptide is a cyclic peptide of formula (I).

[0032] In some embodiments, the pharmaceutical composition comprises a glycosylated peptide of formula (I) and / or formula (II), or a pharmaceutically acceptable salt thereof, in a pharmaceutically acceptable carrier.

[0033] In some embodiments, a method of treating pain comprises administering to a patient in need of treatment for pain (i.e., a patient suffering from pain including, but not limited to, chronic pain, neuropathic pain, and inflammatory pain) a glycosylated peptide of formula (I) and / or formula (II), or a pharmaceutically acceptable salt thereof, in a pharmaceutically acceptable carrier.

[0034] In some embodiments, a method of treating drug dependence comprises administering to a patient in need of treatment for drug dependence (i.e., a patient dependent on or addicted to drugs such as opioid drugs) a glycosylated peptide of formula (I) and / or formula (II), or a pharmaceutically acceptable salt thereof, in a pharmaceutically acceptable carrier.

[0035] In another embodiment, a method of treating opioid use disorder comprises administering to a subject in need of treatment for opioid use disorder a pharmaceutical composition comprising a glycosylated peptide of formula (I) and / or formula (II), or a pharmaceutically acceptable salt thereof, in a pharmaceutically acceptable carrier. For example, the peptide can be administered in place of, and as an alternative to, a μ-opioid agonist to which the subject is addicted. In some embodiments, the subject is addicted to one or more opioids, such as morphine, oxycodone, hydrocodone, codeine, heroin, etc., and blocks withdrawal symptoms induced, for example, by interrupting chronic exposure to the opioid to which the subject is addicted. The subject has often been previously treated for OUD using drugs such as methadone, buprenorphine, naltrexone, etc.

[0036] In some embodiments for treating pain, drug dependence, or OUD, the subject is treated intravenously with a glycosylated peptide of formula (I) and / or formula (II), or a pharmaceutically acceptable salt thereof. In other embodiments, the subject is treated orally with the glycosylated peptide. In the treatment of OUD, the initial dose of the cyclic peptide can be a low dose, such as a dose less than the ED50 of the peptide for analgesia. In some embodiments, treatment starts at a low dose and is increased over time to a higher maintenance level during the course of treatment.

[0037] Without wishing to be bound by theory, it is believed that glycosylation at or near the C-terminus of the cyclic endomorphin analog modulates the membrane affinity of the pharmacophore, enables "hopping" from membrane to membrane, and allows the drug to reach the brain and other μ-receptors by passing through the cellular gate (i.e., the blood-brain barrier). Together with cyclization, glycosylation results in more effective analgesia mediated by the μ-receptors of the brain.

[0038] Non-limiting embodiments are described below, illustrating certain features and aspects of the compositions and methods described herein.

[0039] Embodiment 1 is formula (I): A 1 -Cyclo[A2 -A 3 -A 4 -A 5 -A 6 -O-Carb, formula (II): A 1 -Cyclo[A 5 -A 3 -A 4 -A 2 -A 6 -O-Carb glycosylated cyclic peptides, and pharmaceutically acceptable salts thereof; wherein: A 1 is an amino acid residue selected from the group consisting of Tyr, N-methyl-Tyr, 2,6-dimethyl-Tyr, and N-methyl-2,6-dimethyl-Tyr; A 2 is an amino acid residue selected from the group consisting of Lys, hLys, bhLys, Orn, Dab, and Dpr; A 3 is an amino acid residue selected from the group consisting of Trp, N-methyl-Trp, Phe, N-methyl-Phe, p-X-Phe, and N-methyl-p-X-Phe; A 4 is an amino acid residue selected from the group consisting of Phe, N-methyl-Phe, p-X-Phe, and N-methyl-p-X-Phe; X is F, Cl, Br, or NO2 at the 4-position of the phenyl group of the Phe side chain; A 5 is an amino acid residue selected from the group consisting of Asp, Glu, homo-Glu, bis-homo-Glu, iso-Asp, iso-Glu, iso-homo-Glu, and iso-bis-homo-Glu; A 6 is an amino acid residue or an oligopeptide containing 2 to 5 amino acid residues, A 6 contains at least one hydroxy-substituted amino acid residue; Carb is a carbohydrate group linked to the side chain oxygen of HO-AA by a β-D-glycosidic bond; Cyclo[A 2 -A 3 -A 4 -A 5 and Cyclo[A2 -A 3 -A 4 -A 5 each represents a cyclic peptide ring in which A 2 is A 5 is linked to form a cyclic peptide ring; A 5 When A is isoAsp, isoGlu, isohomoGlu, or isobishomoGlu, the side-chain amino group of A 2 is linked to the α-carboxyl group of A 5 by an amide bond; however, when A 5 is Asp, Glu, homoglu, or bishomoglu, the side-chain amino group of A 2 is linked to the side-chain carboxyl group of A 5 by an amide bond; Optionally, the C-terminal carboxyl group of A 6 is amidated.

[0040] Embodiment 2 is the peptide of Embodiment 1, wherein A 1 is Tyr.

[0041] Embodiment 3 is the peptide of Embodiment 1, wherein A 1 is N-methyl-Tyr.

[0042] Embodiment 4 is the peptide of any one of Embodiments 1 to 3, wherein A 3 is Trp.

[0043] Embodiment 5 is the peptide of any one of Embodiments 1 to 3, wherein A 3 is N-methyl-Trp.

[0044] Embodiment 6 is the peptide of any one of Embodiments 1 to 3, wherein A 3 is Phe.

[0045] Embodiment 7 is the peptide of any one of Embodiments 1 to 6, wherein A 4 is Phe.

[0046] Embodiment 8 is the peptide of Embodiment 1, wherein A 4is the peptide of any one of Embodiments 1 to 6, where A is N-methyl-Phe.

[0047] Embodiment 9 is a peptide of any one of Embodiments 1 to 6, where A is p-X-Phe and X is F, Cl, Br, or NO2 at the 4-position of the phenyl group in the Phe side chain. 4 is the peptide of any one of Embodiments 1 to 6, where A is p-X-Phe and X is F, Cl, Br, or NO2 at the 4-position of the phenyl group in the Phe side chain.

[0048] Embodiment 10 is a peptide of any one of Embodiments 1 to 6, where A is N-methyl-p-X-Phe and X is F, Cl, Br, or NO2 at the 4-position of the phenyl group in the Phe side chain. 4 is the peptide of any one of Embodiments 1 to 6, where A is N-methyl-p-X-Phe and X is F, Cl, Br, or NO2 at the 4-position of the phenyl group in the Phe side chain.

[0049] Embodiment 11 is the peptide of Embodiment 1, where A is Tyr, A is Trp, and A is Phe. 1 where A is Tyr, 3 where A is Trp, 4 where A is Phe.

[0050] Embodiment 12 is a peptide of any one of Embodiments 1 to 11, where A is L-Asp. 5 is the peptide of any one of Embodiments 1 to 11, where A is L-Asp.

[0051] Embodiment 13 is a peptide of any one of Embodiments 1 to 11, where A is L-Glu. 5 is the peptide of any one of Embodiments 1 to 11, where A is L-Glu.

[0052] Embodiment 14 is a peptide of any one of Embodiments 1 to 11, where A is D-Asp. 5 is the peptide of any one of Embodiments 1 to 11, where A is D-Asp.

[0053] Embodiment 15 is a peptide of any one of Embodiments 1 to 11, where A is D-Glu. 5 is the peptide of any one of Embodiments 1 to 11, where A is D-Glu.

[0054] Embodiment 16 is a peptide of any one of Embodiments 1 to 11, where A is L-isoAsp. 5 is the peptide of any one of Embodiments 1 to 11, where A is L-isoAsp.

[0055] Embodiment 17 is, where A is 5It is a peptide of any one of Embodiments 1 to 11, where A is L-IsoGlu.

[0056] Embodiment 18 is such that A 5 It is a peptide of any one of Embodiments 1 to 11, where A is D-IsoAsp.

[0057] Embodiment 19 is such that A 5 It is a peptide of any one of Embodiments 1 to 11, where A is D-IsoGlu.

[0058] Embodiment 20 is such that A 6 It is a peptide of any one of Embodiments 1 to 19, where A is L-Ser.

[0059] Embodiment 21 is such that A 6 It is a peptide of any one of Embodiments 1 to 19, where A is L-Ser-NH2.

[0060] Embodiment 22 is such that A 6 It is a peptide of any one of Embodiments 1 to 19, where A is L-Thr.

[0061] Embodiment 23 is such that A 6 It is a peptide of any one of Embodiments 1 to 19, where A is L-Thr-NH2.

[0062] Embodiment 24 is a peptide of any one of Embodiments 1 to 23, where Carb is β-D-glucose.

[0063] Embodiment 25 is a peptide of any one of Embodiments 1 to 23, where Carb is β-D-lactose.

[0064] Embodiment 26 is a peptide of any one of Embodiments 1 to 25, where the peptide is a cyclic peptide of formula (I).

[0065] Embodiment 27 is a peptide of any one of Embodiments 1 to 25, where the peptide is a cyclic peptide of formula (II).

[0066] Embodiment 28 is the peptide of Embodiment 1 or Embodiment 26, which is Tyr-c[D-Lys-Trp-Phe-Glu]-Ser(β-Lact)-NH2 (SEQ ID NO: 1).

[0067] Embodiment 29 is the peptide of Embodiment 1 or Embodiment 26, which is Tyr-c[D-Lys-Trp-Phe-Glu]-Ser(β-Glc)-NH2 (SEQ ID NO: 5).

[0068] Embodiment 30 is the peptide of Embodiment 1 or Embodiment 26, which is Tyr-c[D-Lys-Trp-Phe-Glu]-Ser(β-Glc)-Ser(β-Glc)-NH2 (SEQ ID NO: 10).

[0069] Embodiment 31 is a pharmaceutical composition comprising any one of the peptides of Embodiments 1 to 30, or a pharmaceutically acceptable salt thereof, in a pharmaceutically acceptable carrier.

[0070] Embodiment 32 is a method for treating pain, comprising the step of administering the pharmaceutical composition of Embodiment 31 to a subject in need of treatment for pain.

[0071] Embodiment 33 is the method of Embodiment 32, wherein the pain is chronic pain.

[0072] Embodiment 34 is the method of Embodiment 32 or 33, wherein the pain is neuropathic pain.

[0073] Embodiment 35 is any one of the methods of Embodiments 32 to 34, wherein the pain is inflammatory pain.

[0074] Embodiment 36 is a method for treating drug dependence, comprising the step of administering a therapeutically effective amount of the pharmaceutical composition of Embodiment 31 to a subject in need of treatment for drug dependence.

[0075] Embodiment 37 is a method for treating opioid use disorder, comprising the step of administering to a subject in need of treatment for opioid use disorder a therapeutically effective amount of the pharmaceutical composition of Embodiment 31.

[0076] Embodiment 38 is the method of Embodiment 37, wherein the composition is administered instead of, and as an alternative to, a μ-opioid receptor agonist to which the subject is addicted.

[0077] Embodiment 39 is any one of the peptides of Embodiments 1 to 30 for treating pain.

[0078] Embodiment 40 is the peptide of Embodiment 39, wherein the pain is chronic pain.

[0079] Embodiment 41 is the peptide of Embodiment 39 or 40, wherein the pain is neuropathic pain.

[0080] Embodiment 42 is any one of the peptides of Embodiments 39 to 41, wherein the pain is inflammatory pain.

[0081] Embodiment 43 is any one of the peptides of Embodiments 1 to 30 for treating drug dependence.

[0082] Embodiment 44 is any one of the peptides of Embodiments 1 to 30 for treating opioid use disorder.

Brief Description of the Drawings

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Figure 11

DETAILED DESCRIPTION OF THE INVENTION

[0084] Formula (I): A 1 -Cyclo[A2 -A 3 -A 4 -A 5 -A 6 -O-Carb, formula (II): A 1 -Cyclo[A 5 -A 3 -A 4 -A 2 -A 6 -Glycosylation of O-Carb, cyclic EM analogs, and pharmaceutically acceptable salts thereof are described herein. In formulas (I) and (II), A 1 is an L-amino acid residue selected from the group consisting of Tyr, N-methyl-Tyr, 2,6-dimethyl-Tyr, and N-methyl-2,6-dimethyl-Tyr; A 2 is a D- or L-amino acid residue selected from the group consisting of Lys, hLys, bhLys, Orn, Dab, and Dpr; A 3 is an amino acid residue selected from the group consisting of Trp and N-methyl-Trp; A 4 is a residue selected from the group consisting of Phe, N-methyl-Phe, p-X-Phe, and N-methyl-p-X-Phe, wherein X is F, Cl, Br, or NO2 at the 4-position of the phenyl group of the Phe side chain; A 5 is an amino acid residue selected from the group consisting of Asp, Glu, hGlu, bhGlu, isoAsp, isoGlu, isohGlu, and isobhGlu. In some embodiments of formula (I), A 2 is a D-amino acid, and A 5 is either a D- or L-amino acid residue. In some embodiments of formula (II), specifically when A 5 is Asp, Glu, hGlu, or bhGlu, A 5 is a D-amino acid residue. A 3 and A 4 can be D- or L-amino acid residues.

[0085] In both formula (I) and formula (II), A 6is a hydroxy-substituted amino acid residue (HO-AA) which can be a D- or L-amino acid; (b) the C-terminal amide of the first HO-AA; (c) an oligopeptide containing 2 to 5 amino acid residues in which at least one of those residues is HO-AA; or (d) the C-terminal amide of the oligopeptide; the other residues of the oligopeptide include one or more residues selected from D-amino acid residues, L-amino acid residues, Gly, and a glycosylated second HO-AA; Carb is a carbohydrate group linked to the side-chain oxygen of HO-AA by a β-glycosidic bond; A 5 When A 2 is Asp, Glu, hGlu, or bhGlu, the side-chain amino group of A 5 is linked to the side-chain carboxyl group of A 5 When A 2 is isoAsp, isoGlu, iso hGlu, or iso bhGlu, the side-chain amino group of A 5 is linked to the α-carboxyl group of A 6 Unless otherwise specified, the glycoside sugar is a D-sugar. Optionally, the C-terminus of A

[0086] In some embodiments of the compounds of formulas (I) and (II), A 1 is L-Tyr.

[0087] In some embodiments of the compounds of formulas (I) and (II), A 3 is L-Trp, in which case the peptide is an EM1 analog, but in other embodiments, A 3 is L-Phe, in which case the peptide is an EM2 analog.

[0088] In some embodiments of the compounds of formulas (I) and (II), A 4 is L-Phe.

[0089] In some embodiments of the compounds of formulas (I) and (II), A 1 is L-Tyr, A 3 is L-Trp, A 4is L-Phe, but in other embodiments, A 1 is L-Tyr, and A 3 is L-Phe, and A 4 is L-Phe.

[0090] In some embodiments of the compound of formula (II), A 5 is Asp or Glu, preferably D-Asp or D-Glu.

[0091] Non-limiting examples of HO-AA residues include, for example, HOAA such as Ser, Thr, homoser, allo-Thr, cis-4-hydroxy-Pro, trans-4-hydroxy-Pro, cis-3-hydroxy-Pro, trans-3-hydroxy-Pro, etc., and C-terminal amides of any of the aforementioned HO-AA residues. HO-AA can have an L or D amino acid structure.

[0092] As used herein, the notations “-NH2”, “-NH2”, and “CONH2” at the C-terminus of a peptide are interchangeably used to refer to the amidation of the C-terminal carboxyl group as a primary amide (i.e., -(C=O)NH2).

[0093] A 6The side-chain oxygen of HO-AA is glycosylated by a Carb group via a β-glycosidic bond between the carbohydrate and the hydroxyl group. Non-limiting examples of Carb groups include monosaccharides such as D-glucose (D-Glc), and disaccharides such as D-lactose (D-Glc-β-D-Gal), β-maltose, β-lactose, β-melibiose, trisaccharides such as β-maltotriose, both linear and branched, and more complex carbohydrate moieties. Other examples include sucrose, trehalose, saccharose, maltose, cellobiose, gentiobiose, isomaltose, and primeveose. Other glycosyl groups include galactose, xylose, mannose, manosaminic acid, fucose, GalNAc, GlcNAc, idose, iduronic acid, glucuronic acid, and sialic acid. Unless otherwise specified, the carbohydrates of formulas (I) and (II) are D-carbohydrates.

[0094] A of the oligopeptide 6 group may include any amino acid residue of the oligopeptide chain in addition to HO-AA. A 6 In some embodiments where A is an oligopeptide, the oligopeptide includes amino acids classified as "small amino acids" such as Gly, Ala, Cys, and Pro (see Betts and Russell p. 299), avoiding steric interference at the opioid receptor active site. The oligopeptide A having small amino acid residues 6Non-limiting examples of the group include, for example, Gly-Ser, Gly-Ser-NH2, Gly-Thr, Gly-Thr-NH2, Ala-Ser, Ala-Ser-NH2, Ala-Thr, Ala-Thr-NH2, Pro-Ser, Pro-Ser-NH2, Pro-Thr, Pro-Thr-NH2, Gly-Gly-Ser-NH2, Gly-Gly-Thr-NH2, Gly-Gly-Pro-NH2, Xaa-Xaa-Xaa-Ser-NH2, Xaa-Xaa-Xaa-hydroxy-Pro-NH2, Xaa-Xaa-Xaa-Xaa-Ser-NH2, Xaa-Xaa-Xaa-Xaa-hydroxy-Pro-NH2, etc., where each Xaa can independently be, for example, Gly, Ala, Cys or Pro. Further, the oligopeptide A 6 group contains more than one HO-AA and optionally each HO-AA can be glycosylated.

[0095] As described herein, Formula (III) provides an alternative structural representation of the compounds encompassed by Formula (I) to emphasize the size of the ring portion and the relative size of the side chain, where A 2 is a D-amino acid residue; A 3 is L-Trp or L-N-Me-Trp; A 4 is L-Phe, L-N-Me-Phe, L-p-X-Phe, and L-N-Me-p-X-Phe, where X is F, Cl, Br, or NO2:

[0096]

Chemical formula

[0097] In some preferred embodiments of formula (III), the C-terminus of A 6 is a primary amide.

[0098] In some preferred embodiments of formula (III), A 6 is Ser, and Carb is D-glucose or D-lactose.

[0099] In some preferred embodiments of formula (III), A 6 is Ser-Ser, and the side-chain oxygen of each Ser is bonded to Carb (preferably D-glucose or D-lactose).

[0100] A 3 is L-Trp or L-N-Me-Trp, and A 4 is L-Phe, L-N-Me-Phe, L-p-X-Phe, and L-N-Me-p-X-Phe, where X is F, Cl, Br, or NO2. The compound of formula (II) is structurally represented as formula (IV) and shows ring-size variations:

[0101]

Chemical formula

[0102] In some preferred embodiments of formula (IV), the C-terminus of A 6 is a primary amide.

[0103] In some preferred embodiments of formula (IV), A 6 is Ser and Carb is D-glucose or D-lactose.

[0104] In some preferred embodiments of formula (IV), A 6 is Ser-Ser and the side-chain oxygen of each Ser is bonded to Carb (preferably D-glucose or D-lactose).

[0105] Table 1 shows the variations in the ring size for various combinations of A of formula (I) 2 and A 5 Table 2 shows the variations in the ring size for various combinations of A of formula (II). The ability to select the relative positions of A 2 and A 5 (comparing formula (I) with formula (II)), together with the ability to match the ring size of the peptide, can be advantageous for optimizing or matching different characteristics of the glycosylated peptide, such as, for example, vehicle solubility, blood stability, gastric acid stability, small intestine stability, storage stability, opioid receptor affinity, opioid receptor selectivity, etc. 2 and A 5 The ring size of the cyclic peptide moiety of the compounds of formula (I) can vary from 13 atoms to 22 atoms. This is evident from the examination of the chemical structure of formula (III): R

[0106] When R 5 is a covalent bond and x is 1 (i.e., when A 2 of formula (I) is Dpr and A 5 is isoAsp, isoGlu, iso hGlu, or iso bhGlu), there are 13 atoms forming the ring, but when R 5 is -(CH2) y - and x is 6 and y is 4 (i.e., when A 2 of formula (I) is bhLys and A 5 is bhGlu), there are 22 atoms in the ring. Similar to the case of formula (I), the ring size of formula (II) also varies depending on which A 2 and A 5 residues are included in the substance, but the variations are due to different patterns of amino acid selection. The smallest ring size of the compounds of formula (II), 14 atoms, is obtained by A 2 which is Dpr and A 5 which is Asp or isoAsp. The largest ring size (22 atoms) of the compounds of formula (II) is obtained by A 2 which is bhLys and A 5 which is bhGlu or iso bhGlu. As is evident in Tables 1 and 2, the same ring size can be obtained with A 2 and A 5It can be obtained by many different selections, giving considerable flexibility to the ability to tailor the structure of the compound in order to optimize and / or alter the physical, chemical, and biological properties of the compound.

[0107] Furthermore, A 2 and A 5 Along with the differences in ring size that can be obtained by the selection of residues, the relative positions of A 2 and A 5 can have an effect on receptor binding affinity, binding strength, binding selectivity, and / or physical properties such as crystallization, solubility, and solution stability. This provides many options for tailoring and optimizing activity as well as physical properties.

[0108] [Table 1]

[0109] [Table 2]

[0110] As shown in formula (III), when R 5 is a covalent bond, R 6 is a methylene chain in the range from -CH2- to -CH2-CH2-CH2-CH2, and the residue A 6 is separated from the cyclic peptide ring by 1 to 4 carbon atoms compared to the case where R 6 is a covalent bond. When A 6 is an oligopeptide, the spacing of the cyclic peptide ring from HO-AA of A 6 is increased by 3 atoms for each additional residue of the oligopeptide chain. Thus, the distance between the peptide ring and HO-AA is significantly different and can be advantageous for optimizing various characteristics of the glycosylated peptide, such as solubility in vehicles, blood stability, gastric acid stability, intestinal stability, storage stability, opioid receptor affinity, opioid receptor selectivity, etc.

[0111] Figure 1 shows the chemical structures of two glycosylated cyclic EM1 analogs of formula (I) having different peptide ring sizes. Panel A shows Tyr-cyclo[D-Lys-Trp-Phe-Glu]-Ser-NH2 (SEQ ID NO: 1) glycosylated by analog A2: β-D-Glc-β-D-Gal (also known as β-D-Lact) and having an 18-membered cyclic peptide ring, and panel B shows Tyr-cyclo[D-Dpr-Trp-Phe-isoGlu]-(β-D-Glc) cis-4-hydroxy-L-Pro-NH2 (SEQ ID NO: 2) having a 13-membered cyclic peptide ring.

[0112] Synthesis of Glycosylated Cyclic EM1 Analogs The glycosylated peptides of formula (I) and (II) can be prepared by conventional liquid or solid-phase methods using appropriate protecting groups and coupling agents. Such methods generally utilize various protecting groups for the various amino acid residues of the peptide. Suitable deprotection methods are used to remove specific (or all) protecting groups and, when solid-phase synthesis is applied, include separation of the resin. Apostol et al. (Peptides, 131, 2020, 170369) reviewed peptide glycosylation. The peptides can be synthesized, for example, as described below.

[0113] The peptides of formula (I) and formula (II) are synthesized by Fmoc-based solid-phase peptide synthesis (SPPS) starting with a glycylated A residue directly attached to MBHA-Rink resin 6 4-methylbenzhydrylamine hydrochloride, on a polymer-bound Rink (MBHA-Rink) resin, and then the remaining residues are added in the order of A 5 、A 4 、A 3 、A 2 、and A 1 、for compounds of formula (I) (see, for example, Figure 2, panel A), and in the order of A 2 、A 4 、A 3 、A 5 、and A 1 for compounds of formula (II). The t-butyl ether group is A 1Included for side-chain protection, Boc (t-butyloxycarbonyl) is A 1 used for amino protection. Peptides are assembled on MBHA-Rink resin by repetitive removal of Fmoc protecting groups and coupling of protected amino acids. For example, HBTU (O-benzotriazol-N,N,N’,N’-tetramethyluronium hexafluorophosphate; CAS#94790-37-1) and HOBT (N-hydroxybenzotriazole; CAS#2592-95-2) are used as coupling reagents in N,N-dimethylformamide (DMF), and diisopropylethylamine (DIPEA) is used as a base. Finally coupled, A that forms the peptide ring structure 2 and A 5 Amino and carboxyl groups are protected orthogonally as follows (Isidro-Llobet, Alvarez, and Albericio 2009): A 2 The amino side chain of (i.e., D-lysine, D-Orn, D-Dab or D-Dpr) is protected by an aryloxycarbonyl (Alloc) group, while A 5 the side-chain carboxyl of is protected as an aryl (All) ester when A 5 is Asp, Glu, hGlu, or bhGlu; or the α-carboxyl group of A 5 is protected as an aryl ester when A 5 is isoAsp, isoGlu, iso hGlu, or iso bhGlu (see, for example, Figure 2, panel B). These two protecting groups are removed simultaneously by hydrogen atom transfer (e.g., by Pd 0 and phenylsilane, PhSiH3), and cyclization is achieved by a peptide condensation reagent such as 6-chloro-benzotriazol-1-yloxy-tris-pyrrolidinophosphonium hexafluorophosphate (PyClock), which can result in a cyclic peptide ring. The resulting peptide is deprotected by hydrazine hydrate, the acetate group from the sugar, then trifluoroacetic acid (TFA and triethylsilane, Et3SiH) is removed, the remaining side-chain protection is removed, and the completed glycopeptide is cleaved from the resin.

[0114] The crude peptide is precipitated with diethyl ether and recovered by filtration. The peptide can be purified by high performance liquid chromatography or other methods of peptide purification known in the peptide synthesis field. For example, the solid obtained above can be dissolved in DMF or a similar polar aprotic solvent. The mixture is stirred at room temperature for about 1 hour. The solvent is removed in vacuo. The residual oil is dissolved in 10% aqueous acetonitrile (MeCN / H2O) and lyophilized to obtain the peptide in solid form. The purification of the crude lyophilized peptide is carried out by reverse phase high performance liquid chromatography (RP-HPLC) using techniques well known in the peptide and protein fields.

[0115] Pharmaceutical Also described herein are pharmaceutical products containing a pharmaceutically effective amount of a glycosylated peptide in a pharmaceutically acceptable carrier (e.g., diluent, complexing agent, additive, excipient, adjuvant, etc.). The glycosylated peptide can exist, for example, in salt form, microcrystalline form, nanocrystalline form, cocrystalline form, nanoparticle form, microparticle form, or amphiphilic form. The salt form can be, for example, salts of inorganic acids such as hydrochloride, phosphate, sulfate, bisulfate, hemisulfate, etc.; or salts of organic acids such as acetate, aspartate, citrate, fumarate, maleate, malate, lactate, hippurate, tartrate, gluconate, succinate, etc.

[0116] The carrier can be an organic or inorganic carrier, or a combination thereof, which is suitable for topical, enteral or parenteral application. The glycosylated peptide can be formulated with a conventional non-toxic pharmaceutically acceptable carrier, for example, for tablets, pellets, capsules, liposomes, suppositories, nasal sprays, solutions, emulsions, suspensions, aerosols, targeted chemical delivery systems, and any other form suitable for use. Non-limiting examples of carriers that can be used include water, glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, urea, and other carriers suitable for use in the manufacture of formulations in solid, semi-solid, liquid or aerosol form. In addition, adjuvants, stabilizers, thickeners, coloring agents, and fragrances can be used in the composition.

[0117] In another aspect, pharmaceutical compositions useful for treating pain and related symptoms that utilize the glycosylated peptide compounds of formula (I) and formula (II) are described herein. The pharmaceutical composition comprises at least one glycosylated peptide of formula (I) and / or formula (II) in combination with a pharmaceutically acceptable carrier, vehicle, or diluent, such as an aqueous buffer at a physiologically acceptable pH (e.g., pH 7-8.5), a polymer-based nanoparticle vehicle, liposomes, etc. The pharmaceutical composition can be delivered in any suitable dosage form, such as a liquid, gel, solid, cream, or paste dosage form. In one embodiment, the composition can be adapted to provide sustained release of the peptide.

[0118] In some embodiments, the pharmaceutical composition includes, but is not limited to, forms suitable for oral, rectal, intranasal, inhalation, topical (including buccal and sublingual), transdermal, vaginal, parenteral (including intramuscular, subcutaneous, and intravenous), spinal (epidural, intrathecal), and central (intraventricular) administration. The composition can be conveniently provided in separate dosage units where appropriate. The pharmaceutical composition can be prepared by any method well known in the pharmaceutical art. Some preferred modes of administration include intravenous (iv), topical, subcutaneous, oral, and spinal.

[0119] Pharmaceutical compositions suitable for oral administration include capsules, cachets, or tablets, each containing a predetermined amount of one or more peptides as a powder or granule. In another embodiment, the oral composition is a solution, suspension, or emulsion. Alternatively, the peptide can be provided as a bolus, lozenge, or paste. Tablets and capsules for oral administration can contain conventional excipients such as binders, fillers, lubricants, disintegrants, colorants, flavorants, preservatives, or wetting agents. Tablets can be coated, if desired, by methods well known in the art. Oral liquid preparations include, for example, aqueous or oily suspensions, solutions, emulsions, syrups, or elixirs. Alternatively, the composition can be provided as a dry product for constitution with water or another suitable vehicle before use. Such liquid preparations can contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which can include edible oils), preservatives, and the like. Additives, excipients, and the like are typically included in the composition for oral administration within a concentration range suitable for their intended use or function in the composition and are well known in the pharmaceutical formulation art. The glycosylated peptides of formula (I) and / or formula (II) are included in the composition within a therapeutically useful and effective concentration range as determined by conventional methods well known in the medical and pharmaceutical arts. For example, a typical composition can contain one or more peptides at a concentration in the range of at least about 0.01 nanomolar to about 1 molar, preferably at least about 1 nanomolar to about 100 millimolar.

[0120] Pharmaceutical compositions for parenteral, spinal, or central administration (e.g., by bolus injection or continuous infusion) or injection into amniotic fluid are provided in unit dosage forms in ampoules, prefilled syringes, small volume infusions, or multi-dose containers and may preferably contain additional preservatives. The compositions for parenteral administration may be suspensions, solutions, or emulsions and may contain excipients such as suspending agents, stabilizers, and dispersing agents. Alternatively, the glycosylated peptides of formula (I) and / or formula (II) may be provided in powder form obtained by sterile isolation of a sterile solid or by lyophilization from a solution for constitution prior to use with a suitable vehicle such as sterile, pyrogen-free water. Additives, excipients, etc. are typically included in the compositions for parenteral administration within a concentration range suitable for the intended use or function in the composition and are well known in the pharmaceutical formulation art. The glycosylated peptides of formula (I) and / or formula (II) are included in the composition within a concentration range useful and effective for treatment as determined by conventional methods well known in the medical and pharmaceutical arts. For example, typical compositions may contain one or more peptides at a concentration in the range of at least about 0.01 nanomolar to about 100 millimolar, preferably at least about 1 nanomolar to about 10 millimolar.

[0121] Pharmaceutical compositions for topical administration of the glycosylated peptides of formula (I) and / or formula (II) to the epithelium (mucosa or skin surface) can be formulated as ointments, creams, lotions, gels, or as transdermal patches. Such transdermal patches may contain a penetration enhancer, such as linalool, carvacrol, thymol, citral, menthol, t-anethole, etc. Ointments and creams may contain, for example, an aqueous or oily base to which suitable thickeners, gelling agents, coloring agents, etc. are added. Lotions and creams contain an aqueous or oily base and typically may also contain one or more emulsifiers, stabilizers, dispersants, suspending agents, thickeners, coloring agents, etc. Gels preferably contain an aqueous carrier base and include a gelling agent, such as a cross-linked polyacrylic acid polymer, a derivatized polysaccharide (e.g., carboxymethyl cellulose), etc. Additives, excipients, etc. are typically included in the composition for topical administration to the epithelium within a concentration range suitable for the intended use or function in the composition and are well known in the pharmaceutical formulation art. The glycosylated peptides of formula (I) and / or formula (II) are included in the composition within a concentration range useful and effective for treatment, as determined by conventional methods well known in the medical and pharmaceutical arts. For example, a typical composition may contain one or more peptides at a concentration in the range of at least about 0.01 nanomolar to about 1 molar, preferably at least about 1 nanomolar to about 100 millimolar.

[0122] Pharmaceutical compositions suitable for local administration in the mouth (e.g., oral or sublingual administration) include tablets containing a peptide in a flavor base such as sucrose, acacia, or tragacanth; troches containing a peptide in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes containing the active ingredient in a suitable liquid carrier. The pharmaceutical compositions for local administration in the mouth may contain, if desired, a penetration enhancer. Additives, excipients, etc. are typically included in the compositions for local oral administration within a concentration range suitable for the intended use or function in the composition and are well-known in the pharmaceutical formulation art. The peptides of the present invention are included in the composition within a therapeutically useful and effective concentration range as determined by conventional methods well-known in the medical and pharmaceutical arts. For example, typical compositions may contain one or more peptides at a concentration in the range of at least about 0.01 nanomolar to about 1 molar, preferably at least about 1 nanomolar to about 100 millimolar.

[0123] Pharmaceutical compositions suitable for rectal administration include the glycosylated peptides of formula (I) and / or formula (II) in combination with a solid or semi-solid (e.g., cream or paste) carrier or vehicle. For example, such rectal compositions may be provided as unit dose suppositories. Suitable carriers or vehicles include cocoa butter and other substances commonly used in the art. Additives, excipients, etc. are typically included in the compositions for rectal administration within a concentration range suitable for the intended use or function in the composition and are well-known in the pharmaceutical formulation art. The glycosylated peptides of formula (I) and / or formula (II) are included in the composition within a therapeutically useful and effective concentration range as determined by conventional methods well-known in the medical and pharmaceutical arts. For example, typical compositions may contain one or more peptides at a concentration in the range of at least about 0.01 nanomolar to about 1 molar, preferably at least about 1 nanomolar to about 100 millimolar.

[0124] According to one embodiment, a pharmaceutical composition suitable for vaginal administration is provided as a pessary, tampon, cream, gel, paste, foam, or spray containing the peptide of the present invention in combination with a carrier known in the art. Alternatively, a composition suitable for vaginal administration can be delivered in a liquid or solid dosage form. Additives, excipients, etc. are typically included in the composition for vaginal administration within a concentration range suitable for the intended use or function in the composition and are well-known in the pharmaceutical formulation art. The glycosylated peptides of formula (I) and / or formula (II) are included in the composition within a therapeutically useful and effective concentration range as determined by conventional methods well-known in the medical and pharmaceutical arts. For example, a typical composition can contain one or more peptides at a concentration in the range of at least about 0.01 nanomolar to about 1 molar, preferably at least about 1 nanomolar to about 100 millimolar.

[0125] Pharmaceutical compositions suitable for intranasal administration are also described herein. Such intranasal compositions include a vehicle for delivering a liquid spray, dispersible powder, or instillation drug and a glycosylated peptide of formula (I) and / or formula (II) in a suitable administration device. The instillation drug can be formulated with an aqueous or non-aqueous base that also includes one or more dispersants, solubilizers, or suspending agents. The liquid spray is conveniently delivered from a pressurized pack, inhaler, nebulizer, or other convenient means for delivering an aerosol containing the peptide. The pressurized pack contains a suitable high-pressure gas, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gases well known in the art. The aerosol dosage form can be controlled by providing a valve that delivers a measured amount of the peptide. Alternatively, the pharmaceutical composition for administration by inhalation or insufflation can be provided in the form of a dry powder composition, such as a powder mixture of the peptide and a suitable powder base, such as lactose or starch. Such powder compositions can be provided in unit dosage forms in, for example, capsules, cartridges, gelatin packs, or blister packs from which the powder can be administered with the aid of inhalation or insufflation. Additives, excipients, etc. are typically included in the intranasal administration composition within a concentration range suitable for the intended use or function in the composition and are well known in the pharmaceutical formulation art. The glycosylated peptide of formula (I) and / or formula (II) is included in the composition within a therapeutically useful and effective concentration range as determined by conventional methods well known in the medical and pharmaceutical arts. For example, a typical composition can contain one or more peptides at a concentration in the range of at least about 0.01 nanomolar to about 1 molar, preferably at least about 1 nanomolar to about 100 millimolar.

[0126] Optionally, the pharmaceutical composition may include one or more other therapeutic agents, for example, as a combination therapy. The additional therapeutic agent is included in the composition within a concentration range that is useful and effective for treatment, as determined by conventional methods well known in the medical and pharmaceutical arts. The concentration of any particular additional therapeutic agent may be in the same range as is typical for the use of that agent as monotherapy, or the concentration may be less than that typical for monotherapy if there is a synergistic effect when combined with the peptide of the present invention.

[0127] In another aspect, the present invention provides the use of a glycosylated peptide of formula (I) and / or formula (II) for the treatment of pain, the treatment of discomfort associated with gastrointestinal disorders, the treatment of drug dependence, and for OUD. A method of providing analgesia (reduction or decrease in pain), relief from gastrointestinal disorders such as diarrhea, and the treatment of drug dependence and OUD in a patient such as a mammal including a human, comprises administering to a patient suffering from one of the aforementioned conditions an effective amount of a glycosylated peptide of formula (I). Diarrhea can be caused by the effects or side effects of various agents or treatments, including many causes, such as infections, cholera, or those used for cancer treatment. Preferably, the peptide is administered parenterally or enterally. The dosage of the effective amount of the peptide can vary depending on the age and condition of each individual patient being treated. However, a suitable unit dosage form is typically in the range of about 0.01 to about 100 mg. For example, the unit dosage can be in the range of about 0.2 mg to about 50 mg. Such unit dosages can be administered more than once a day, for example, 2 or 3 times a day.

[0128] All embodiments of the glycosylated peptides of formula (I) and formula (II) can be in an "isolated" state. For example, an "isolated" peptide is one that is either fully or partially purified. In some examples, an isolated compound is part of a larger composition, buffer system, or reagent mixture. Optionally, the isolated peptide can be in the form of a pharmaceutically acceptable salt (e.g., hydrochloride, trifluoroacetate, trifluoromethanesulfonate, etc.). In other situations, the isolated peptide can be homogeneously purified. The composition can also contain the peptide or compound at a level of at least about 50, 80, 90, or 95% (mole or weight basis) of all other species present therein. A mixture of the glycosylated peptides of formula (I) and / or formula (II) can be used in the practice of the methods described herein.

[0129] For example, methods of using the peptides of formula (I) and formula (II) disclosed herein in a pharmaceutical formulation or therapeutic agent are also described herein. These methods can include the use of a single peptide, or a combination of multiple peptides (i.e., a mixture). Thus, certain embodiments are included in pharmaceuticals comprising the glycosylated peptides of formula (I) and / or formula (II), and methods of manufacturing such pharmaceuticals.

[0130] As used herein, the terms "reducing," "inhibiting," "blocking," "preventing," "alleviating," or "relieving," when referring to a compound (e.g., a peptide), mean that the compound reduces the occurrence, severity, size, volume, or related symptoms of a condition, event, or activity by at least about 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, 27.5%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, or 100% as compared to the degree of the condition, event, or activity that would normally occur without application of the compound or composition comprising the compound. The terms "increasing," "elevating," "enhancing," "upregulating," "improving," or "activating," when referring to a compound, mean that the compound increases the occurrence or activity of a condition, event, or activity by at least about 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, 27.5%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 400%, 500%, 750%, or 1000% as compared to the degree of the condition, event, or activity that would normally occur without application of the compound or composition comprising the compound.

[0131] The following examples are included to illustrate certain aspects of the materials and methods described herein. It should be understood by those skilled in the art that the techniques disclosed in the examples, which present techniques known to function well for the production or use of the described glycosylated peptides, may constitute preferred materials and methods. However, in light of the present disclosure, those skilled in the art will understand that many modifications can be made to the specific disclosed embodiments and that similar or analogous results can be obtained without departing from the spirit and scope of the invention as set forth in the appended claims. The examples are provided for illustrative purposes only and are not intended to be limiting.

Example

[0132] [Example 1] Peptide Synthesis Chemical / Peptide Synthesis: Morphine was generously supplied by the NIDA Drug Supply Program. ZH853 was custom synthesized by Anaspec (Fremont, CA) and guaranteed to be >95% pure. Peptides A1 - A8 were synthesized as described above by the laboratory of Robin Plot (see Figures 2, 3, and Table 3).

[0133]

Table 3

[0134] Figure 2 schematically shows the mode of synthesis of analog A2: Tyr-cyclo[D-Lys-Trp-Phe-Glu]-(β-Lact)Ser-NH2 (SEQ ID NO: 1). Panel A shows the order in which the functional moiety of the MBHA-Rink resin and the residues of the peptide are added to the resin-bound peptide of formula (I). Panel B shows the ring closure of the cyclic peptide A2, as well as the deprotection and cleavage of the peptide from the resin.

[0135] The non-glycosylated cyclic peptide ZH853 was custom synthesized as the acetate by Anaspec (Fremont, CA) and guaranteed to be >95% pure. The glycopeptides A1 - A7 and the non-glycosylated control peptide A8 were synthesized by HPLC to an estimated >95% purity as described below.

[0136] O-linked glycopeptides were synthesized using an automated PRELUDE peptide synthesizer (Gyros-PTI) or manually by simple frit syringe using a well-established solid-phase method based on Fmoc protection (Isidro-Llobet et al., 2009).

[0137] Either of the first amino acids on the resin, e.g., Fmoc-Ser[O-β-D-Glc(OAc)4]-OH or Fmoc-Ser[O-β-Lact(OAc)7]-OH, was manually coupled to MBHA-Rink amide resin (200 mesh, original loading capacity of 0.83 mmol / g), and capping with 0.8 equivalents of Fmoc-amino acid and Ac2O provided a final resin loading of 0.5 - 0.7 mmol / g of Fmoc-amino acid.

[0138] For each gram of MBHA-Rink amide resin (200 mesh, 0.83 mmol / gram), 0.8 mmol of amino acid was used. A fritted syringe was used as the reaction vessel, and the resin was swollen in DMF for 10 minutes and then washed twice with dimethylformamide (DMF) for 2 minutes each. Fmoc removal was achieved using 2% DBU + 2% piperidine in DMF. After treatment, the resin was washed six times with DMF for 2 minutes each. For Fmoc sugar amino acid activation, chloro-hydroxybenzotriazole (Cl-HOBt) and diisopropylcarbodiimide (DIC) were used. Subsequent loading of Fmoc-amino acid (3 equivalents / resin mmol) was carried out using N,N,N’,N’-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate, O-(benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HBTU, (3 equivalents / resin mmol), and N-methylmorpholine (12 equivalents / resin mmol) using a PRELUDE peptide synthesizer.

[0139] At the cyclization site, Fmoc-Glu(All) and Fmoc Lys(Alloc) were used to provide selective deprotection (Thieret et al., 1997). After synthesizing the full-length peptide on the resin, the All and Alloc protecting groups were removed by using 0.1 equivalent / resin mmol of Pd(PPh3)4 in the presence of 20 equivalents / resin mmol of PhSiH3 in dichloromethane (DCM), then washed six times with DCM for 2 minutes each, and then four times with DMF for 2 minutes each.

[0140] The lactam ring was formed using 4 equivalents / resin mmol of PyClock and 12 equivalents of diisopropylethylamine (DIEA) in NMP. The resin was then washed four times with DMF for 2 minutes, twice with 0.5% sodium diethyldithiocarbamate (DEDTC) in DMF for 4 minutes, then six times with DMF for 2 minutes each, and six times with DCM for 2 minutes each. The resin was dried in vacuo.

[0141] The acetyl group was removed from the sugar moiety in NMP (H2NNH2·H2O / NMP) for 1 hour with gentle mixing using 50% hydrazine hydrate. The solution was drained, the resin was refilled with the H2NNH2·H2O / NMP solution, gently mixed for an additional 3 hours, washed 6 times with DMF for 2 minutes and 6 times with DCM for 2 minutes, and dried in vacuo.

[0142] A cleavage cocktail containing trifluoroacetic acid (TFA), DCM, H2O, the scavenger triethylsilane (Et3SiH), and anisole (9:1:0.2:0.3:0.05) was used to simultaneously remove the amino acid side chains and the N-terminal protecting groups (t-butyl ether and Boc protecting groups, respectively) and cleave the cyclic glycopeptide from the resin. The resin was treated with 1 mL of the cleavage cocktail per 100 mg of resin (based on the initial mass) and rotated for 1 hour. The cleavage cocktail solution was collected and the volume was reduced to approximately 1 / 3 of the original volume under argon. Addition of diethyl ether (Et2O) at 0 °C caused precipitation of the crude glycopeptide, which was then centrifuged, the Et2O was decanted to obtain the crude peptide, which was then dried in vacuo. The resulting grayish solid was dissolved in H2O, frozen, and lyophilized prior to purification by reverse-phase high-performance liquid chromatography (RP-HPLC) using a semi-preparative (250×25 mm) or preparative (250×55 mm) C-18 column with an H2O:CH3CN gradient containing 0.1% trifluoroacetic acid (TFA). The purity of the product (typically 9 - 10% yield) was confirmed using analytical RP-HPLC (250×4.0 mm) and MS / MS by Collision Induced Decomposition (CID), as well as high-field (600 or 800 MHz) NMR.

[0143] [Example 2] Peptide Bond Test Receptor binding profile and Ki: Receptor binding and Ki determinations were generously provided by the National Institute of Mental Health's Psychiatric Drug Screening Program (PDSP) under the direction of Bryan L. Roth at the University of North Carolina at Chapel Hill and Jamie Driscoll, the project officer at NIMH in Bethesda, MD, USA.

[0144] Figure 4 illustrates receptor binding affinities to mu, delta and kappa (μ, δ, κ, MOR, DOR, KOR) opioid receptors. Table 4 provides binding (Ki) values (in nM; high values indicate low binding affinity) and log Ki, as well as the ratios of DOR to MOR (delta / mu) and KOR to MOR binding, for the glycosylated analogs A1, A2, A3, A4, A5, A6 and A7 compared to the non-glycosylated A8 and ZH853, for the mu opioid receptor (MOR), delta opioid receptor (DOR) and kappa opioid receptor (KOR).

[0145] The reference compound ZH853, which has a Ki of less than nanomolar, showed the highest MOR affinity, followed by the non-glycosylated Ser analog A8 (shown as 8 in the figure). Glycosylation reduced the MOR affinity by approximately 5-fold in analogs A1, A2, A6, and A7 (shown as 1, 2, 6, and 7, respectively, in Figure 4). Substitution of Trp at the 3-position with D-Nal (analogs A4 and A5, shown as 4 and 5, respectively, in Figure 4) further reduced MOR binding by 11-fold and eliminated delta and kappa binding. However, L-Nal substitution (A6, shown as 6 in Figure 4) did not reduce MOR affinity but increased KOR affinity, resulting in the least selective compound. In contrast, the double glycoside (A7, shown as 7 in Figure 4) did not change MOR affinity, but DOR and KOR affinities decreased below detectable levels, resulting in the most selective MOR binding. Unexpectedly, insertion of a Gly spacer in front of Ser + glucoside (A3, shown as 3 in Figure 4) reduced MOR affinity and increased KOR affinity, resulting in the only analog with the lowest MOR affinity and a KOR affinity higher than the DOR affinity. Binding assays also showed that the analogs did not significantly bind (>10 μM) to 28 major off-target sites: 8 adrenergic receptor sites, 7 serotonin receptor sites, 4 dopamine receptor sites, 3 histamine receptor sites, 2 muscarinic receptor sites, 2 benzodiazepine sites, the GABA receptor, and the sigma-2 receptor, as well as the dopamine transporter (DAT) receptor.

[0146] [Table 4]

[0147] [Example 3] In Vivo Evaluation of Peptides Animals: Male and female CD-1 and DBA mice (21 - 29 g body weight at the time of the test) were obtained from Charles River (Wilmington, MA) and housed on a 12-hour light / dark cycle. All experiments were approved by the Tulane Institute's Animal Care and Use Committee and were conducted in accordance with the NIH guidelines for the care and use of laboratory animals. All efforts were made to minimize the suffering of the animals and to reduce the number of animals used.

[0148] Antinociception: Antinociception was determined in the standard tail-flick (TF) and hot plate (HP) tests described below.

[0149] Tail-flick (TF): CD1 mice were used for this test. The latency to withdraw the tail from a heat source was automatically measured (IITC, Woodland Hills, CA). The baseline latency was 3 - 4 seconds, and the cut-off time was set at 9 seconds to prevent tissue damage. The percent maximum possible effect (%MPE) was determined as * [(latency - baseline latency) / 9 - (baseline latency)] 50 × 100. The peak response was slower for the glycopeptides (40 - 60 minutes) compared to the reference compounds ZH853 (30 - 40 minutes) and morphine (20 - 30 minutes). Therefore, the mean MPE at each of those times was calculated and used as the peak response for determining ED 50In the calculation (Figure 6), all animals were used. The duration of pain suppression was evaluated using both the area under the curve (AUC) and the duration of pain suppression, and calculated as the time from the first to the last time point that exceeded or was equal to 50% MPE. In these two measurements, the majority of animals in the top two doses of A2 and the top one dose of A1 were higher than 50% MPE at 3 hours, underestimating the actual AUC and duration. Therefore, only the 5-hour subgroup in these groups was used for the calculation of AUC and duration. As shown in Figures 5 and 6, this method was further supported as being conservative because the average score remained above 50% MPE at the 5-hour time point even for the top dose of A2.

[0150] Hot plate (HP) test: The HP test (Chapman et al, 1985), which reflects a systematic complex response above the spinal cord, was used to evaluate the CNS activation of pain suppression by compounds administered peripherally (sc). In preliminary experiments, DBA mice were found to show HP responses to all tested compounds containing lower doses of morphine than CD1 mice. Therefore, they were used in this test to reduce the test compound requirement. The HP apparatus (HP, IITC, Woodland Hills, CA) was set at 55 °C, the temperature that elicits a response after 7 - 9 seconds. Three baseline HP latencies of quickly lifting the hind paw, licking, or shaking were examined before drug injection. Mice were removed from the HP after a maximum of 30 seconds. Mice were injected with various analogs (0 - 5.6 mg / kg s.c.) and tested 30 - 300 minutes after injection. The data were converted to the maximum possible effect (%MPE) as described for TF. The peak response was delayed (45 - 60 minutes) with the glycopeptide compared to the reference compounds ZH853 and morphine (30 - 45 minutes). Therefore, the average MPE at each of these times was calculated and used as the peak response for determining ED 50 and used to calculate the area under the curve (AUC). The duration of pain suppression higher than 50% MPE was calculated as for TF.

[0151] Conditioned place preference (CPP): Male CD-1 mice were tested for CPP in a modified two-chamber apparatus (Noldus, Leesburg, VA). The 20×20 cm chambers had different visual and tactile cues: 1) vertical striped walls with round holes in a metal floor or 2) gray walls with clover-shaped holes. Absence of bias was indicated by baseline times over all tests, which were 51.5% and 48.5% respectively. The eight regions balanced the cue patterns of the left and right chambers. The test was conducted in four phases: acclimation (1 - 2 days, 30’), baseline (4 - 5 days, 20’), conditioning (6 - 8 days, 40’), and testing (9th day, 20’). Two sessions per day were conducted in each phase (9:30 am and 10:30 am), and the animals were placed in the left chamber in session 1 and in the right chamber in session 2. For baseline and testing, both chambers were freely movable. For conditioning, the animals received vehicle in the first session and were confined to one chamber, and in the second session, they were administered drug (vehicle, morphine, or A2) in the other chamber. These protocols balanced exposure to the chambers and minimized drug carry-over effects for conditioning. CPP was determined as the change in time spent on the drug-associated side minus the time spent on the same side during the baseline test.

[0152] Statistics / Data analysis: Data were analyzed by GRAPHPAD PRISM software (GraphPad Software, San Diego CA). Analysis of variance (ANOVA) and Newman-Kuels or Bonferroni tests after mixed model analysis were used to evaluate group differences when appropriate. Dose-response curves were analyzed by non-linear regression models. Outliers were evaluated by recognized statistical methods, e.g., by Walfish in 2006. Differences were considered statistically significant at p < 0.05. Test compounds were coded during in vivo experiments and tested by blinded observers.

[0153] Results and discussion Three glycosylated EM1 analogs (A1, A2, and A7) were selected for the TF test in male and female CD-1 mice and compared to two reference compounds, ZH853 and morphine. In pilot studies, analogs A3 - A6 showed lower TF and hot plate responses than the reference compounds and were not further tested at this point.

[0154] Figure 5 shows the time - course of analgesia in the tail flick test and Figure 6 shows the area under the curve (AUC) and duration (%MPE>50%) of analgesia. Three - way analysis of the AUC for each pair of compounds (drug×dose×gender) showed a predicted significant effect of dose for all drugs (p<0.0001), reflecting a significant dose - dependence. A significant effect of drug was also observed (p<0.0001), reflecting the overall rank order of efficacy as A2>A7>A1>morphine>ZH853. A main effect of gender (p<0.05) was observed in the comparison of A1 vs A2, reflecting the overall lower values in females for these two compounds. The comparison of A1 vs A7 revealed an interaction of dose×drug÷gender (p<0.05), reflecting a sharp increase in the score of females for A7, including higher scores than males at the highest dose. However, two - way analysis of dose×gender for each compound did not show a significant main effect of gender for the glycopeptides. A7 showed a dose×gender effect (p<0.01), confirming the above - mentioned enhanced responsiveness by females. A significant effect of gender (p<0.05) was seen in the three - way comparison of morphine and ZH853, and then two - way analysis of drug×dose showed significant effects (p<0.05) and a statistical trend (p<0.1) for gender differences for morphine and ZH853, respectively. Similar statistical effects were observed for the duration of >50% MPE.

[0155] In summary, the overall significant differences among the compounds showed the rank order of efficacy as analog A2 (Lac) > A7 (Glc) > A1 (Glc) > morphine > ZH853. The efficacy and duration of pain suppression for male and female of A2, and female of A7 were conservative estimates because some animals in each group had > 50% %MPE score at the maximum test time of 5 hours. To more accurately determine the relative efficacy, another group of mice (n = 6) was tested with 10 mg / kg of morphine and compared with a dose of 1.8 mg / kg of A2 that produced complete (100% MPE) pain suppression and a duration of ≥ 3 hours. Panel B of Figure 6 shows that these doses provided time-course data that were well-matched. Panel F provides AUC data for equal pain suppression doses versus morphine. The data showed that analog A2 produced an equivalent total pain suppression effect as morphine at doses 5.55-fold to 11-fold lower on a mg / kg basis and up to 21.8-fold lower on a molar basis.

[0156] The effect of gender generally reflected lower scores in females. This occurred across the doses of A1 and ZH853, while for A2, A7, and morphine, female scores were lower at low doses but closer to males at high doses. For analog A7, female scores showed a sharp increase and exceeded males at 5.6 mg / kg, reflecting a significant drug × dose × gender interaction in the comparison of analogs A1 and A7.

[0157] The dose-response curves and relative potency (ED 50) is shown in Fig. 7. The rank order of potency was the same as that of the above-mentioned efficacy / total analgesia in mg / kg calculation (A2 > A7 > A1 > morphine > ZH853), but in terms of molar basis (μmol / kg), the rank order of morphine and ZH853 was reversed. Potency was lower in females than in males for all compounds, and significant effects were shown for analogs A2 and ZH853. However, there was no ratio exceeding 2-fold. In both males and females, analog A2 was significantly more potent than all other compounds except for male A2 vs A7 which showed a statistical trend (p = 0.0517) (p < 0.01 - 0.0001). The potency of analog A2 compared to the control was 2 - 3-fold in mg / kg and 4 - 8-fold on a molar basis (μmol / kg).

[0158] Fig. 8 shows the time-dependent analgesia in HP, and Fig. 9 shows the AUC (A and B) and duration (C and D). Similar to the tail flick test, analogs A2 and A7 produced potent analgesia in this test, which is consistent with the concept that these compounds activate pain regulated by the central mechanism. However, in contrast to the TF test, the responses of animals given analog A1 were not significantly greater than those of the reference compounds ZH853 and morphine, and no significant sex differences were observed.

[0159] Three-way analysis (drug × dose × sex) of the hot plate (HP) curve under area (AUC) data for each pair of compounds confirmed dose-dependence due to a significant effect of dose (p < 0.0001 in all cases). The effect of the drug was not significant between analogs A2 and A7, or between analogs A1, ZH853, and morphine, showing two layers of efficacy by analogs A2 and A7 that produced a higher pain suppression effect than the other three compounds. The significant effect of dose × drug for analog A2 or A7 compared to the other compounds showed an increase at a greater rate in response to increasing dose. Sex differences (and interactions) were not significant, showing similar effects in both sexes for all compounds in this study. Univariate analysis at each dose across groups showed that analogs A2 and A7 produced significantly larger AUCs than analogs A1, morphine, and ZH853 at three of the four doses tested: 1.8, 3.2, and 5.6 mg / kg. As shown in Figure 9, the AUC range for 1.8 mg / kg of analogs A2 and A7 was similar to that of 5.6 mg / kg of analogs A1, ZH853, and morphine, showing approximately three-fold higher efficacy for analogs A2 and A7. Figure 9, panel E, directly shows this comparison by stating that 1.8 mg / kg of analog A2 and 5.6 mg / kg of morphine produced similar peak effects, time courses, and areas under the curve, supporting the estimate of three-fold higher efficacy for analog A2. The duration of >50% MPE (C and D) showed similar statistical effects.

[0160] The dose-response curves and relative potency (ED 50 ) of the compounds are shown in Figure 10. The rank order of potency was the same as the above efficacy / total pain suppression in TF, except that ZH853 was more potent than morphine at both mg / kg and μmol / kg values (A2 > A7 > A1 > ZH853 > morphine). Potency was lower in females than males in all compounds, showing a significant effect for A1, A2, and ZH853. However, the ratio did not exceed two-fold. Males and females given analogs A2 and A7 showed significantly higher potency than the ZH853 and morphine controls, but not with analog A1.

[0161] Figure 11 shows the conditioned place preference (CPP) results with three equal analgesic doses of morphine and A2, providing graphs of both the time-course (left) and area under the curve (AUC; right inset) results. The absence of CPP is an indicator that the drug may be useful for the treatment of OUD. After two habituation and two baseline sessions, conditioning was performed for 3 days. Vehicle, morphine, or A2 was administered s.c., and the mice were confined to one side of the apparatus as described in the method. On a later day, a test session was performed, and CPP was determined as the change in time spent on the drug-associated side minus the time spent on the same side during the baseline test. The inset shows the three doses of each compound that produce equal analgesic effects (dotted line) in the tail-flick test. Reward tests often show biphasic effects, and higher doses do not produce higher effects. Here, the lowest dose of morphine tested produced the highest CPP. Morphine, but not A2, showed 1) significant drug (p<0.01) and drug×dose (p<0.05) effects, and a significant difference between two low doses ( *** , p<0.001) in a two-way mixed-effects analysis of variance, 2) a one-way analysis of variance showing morphine at 3.2 mg / kg higher than vehicle (++, p<0.01), and 3) all three morphine doses, but not the analog doses, produced CPP as confirmed by a t-test showing that all were significantly higher than zero (#, p<0.5), with n = 12.

[0162] Reference

[0163]

Table 5

[0164] All references, including publications, patent applications, and patents, cited in this specification are hereby incorporated by reference in their entirety to the same extent as if each reference had been individually and specifically indicated to be incorporated by reference and were set forth in full herein.

[0165] The use of the terms "a", "an", and "the" and of similar referents in the context of descriptions of materials and methods (in particular, in the context of the following claims) is considered to include both the singular and the plural, unless specifically stated otherwise herein or clearly negated by the context. The terms "comprising", "having", "including", and "containing" are considered to be open-ended terms (i.e., meaning "including, but not limited to") unless otherwise specified. The terms "consisting of" and "consist of" are considered to be restrictive terms that limit any composition or method to the specific components or steps recited in a given claim or portion of the specification. Further, by its open-ended nature, the term "comprising" broadly encompasses compositions and methods that "consist essentially of" or "consist of" the specific components or steps recited in a given claim or portion of the specification, in addition to compositions and methods that include other components or steps beyond those recited. The recitation of ranges of values herein is merely intended to serve as a convenient method of referring individually to each separate value falling within the range, and each separate value is incorporated into the specification as if it were individually recited herein. All numerical values obtained by measurement (e.g., weight, concentration, physical dimensions, removal rate, flow rate, etc.) are considered not to be exact numbers and should be considered to include values within the known limits of the measurement techniques commonly used in the art, whether or not the term "about" is explicitly recited. All methods described herein can be performed in any suitable order unless otherwise specified herein or clearly negated by the context. The use of any and all examples or exemplary language (e.g., "such as") provided herein is merely intended to more clearly illustrate a particular aspect of the materials or methods described herein and is not intended to limit the scope of the claims unless otherwise specified.No language in this specification should be construed as indicating any non-claim element as essential to the practice of the claims.

[0166] Preferred embodiments are described herein and include the best mode known to the inventors for carrying out the claimed invention. Variations of those preferred embodiments may become apparent to those skilled in the art upon reading the foregoing description. The inventors expect those skilled in the art to employ such variations as appropriate, and the inventors intend the claimed invention to be practiced otherwise than as specifically described herein. Accordingly, the claimed invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Further, any combination of the above-described elements in all possible variations thereof is included in the claimed invention unless otherwise specified herein or clearly precluded by context.

Claims

1. Formula (I): A １ -Cyclo[A ２ -A ３ -A ４ -A ５ ]-A ６ -O-Carb, Formula (II): A １ -Cyclo[A ５ -A ３ -A ４ -A ２ ]-A ６ -O-Carb's glycosylated cyclic peptides, and pharmaceutically acceptable salts thereof; Wherein: A １ is an amino acid residue selected from the group consisting of Tyr, N-methyl-Tyr, 2,6-dimethyl-Tyr, and N-methyl-2,6-dimethyl-Tyr; A ２ is an amino acid residue selected from the group consisting of Lys, hLys, bhLys, Orn, Dab, and Dpr; A ３ is an amino acid residue selected from the group consisting of Trp, N-methyl-Trp, Phe, N-methyl-Phe, p-X-Phe, and N-methyl-p-X-Phe; A ４ is an amino acid residue selected from the group consisting of Phe, N-methyl-Phe, p-X-Phe, and N-methyl-p-X-Phe; X is F, Cl, Br, or NO at the 4-position of the phenyl group of the Phe side chain ２ ; A ５ is an amino acid residue selected from the group consisting of Asp, Glu, homoGlu, bis-homoGlu, isoAsp, isoGlu, isohomoGlu, and isobis-homoGlu; A ６ is an amino acid residue or an oligopeptide containing 2 to 5 amino acid residues, A ６ contains at least one hydroxy-substituted amino acid residue; Carb is a carbohydrate group bonded to the side chain oxygen of HO-AA by a β-D-glycoside bond; Cyclo[A ２ -A ３ -A ４ -A ５ and Cyclo[A ２ -A ３ -A ４ -A ５ each represent a cyclic peptide ring in which A ２ is A ５ bonded to A; when A ５ is isoAsp, isoGlu, isohomoGlu, or isobishomoGlu, the side-chain amino group of A ２ is bonded to the α-carboxyl group of A ５ by an amide bond, but when A ５ is Asp, Glu, homoglu, or bis-homoglu, the side-chain amino group of A ２ is bonded to the side-chain carboxyl group of A ５ by an amide bond; Optionally, the C-terminal carboxyl group of A ６ is amidated, a peptide.

2. The peptide according to claim 1, wherein A １ is Tyr.

3. The peptide according to claim 1, wherein A １ is N-methyl-Tyr.

4. The peptide according to any one of claims 1 to 3, wherein A ３ is Trp.

5. The peptide according to any one of claims 1 to 3, wherein A ３ is N-methyl-Trp.

6. The peptide according to any one of claims 1 to 3, wherein A ３ is Phe.

7. The peptide according to any one of claims 1 to 6, wherein A ４ is Phe.

8. The peptide according to any one of claims 1 to 3, wherein A ４The peptide according to any one of claims 1 to 6, wherein A is N-methyl-Phe. **Claim 9** A ４ is p-X-Phe, and X is F, Cl, Br, or NO at the 4-position of the phenyl group of the Phe side chain ２ The peptide according to any one of claims 1 to 6. **Claim 10** A ４ is N-methyl-p-X-Phe, and X is F, Cl, Br, or NO at the 4-position of the phenyl group of the Phe side chain ２ The peptide according to any one of claims 1 to 6. **Claim 11** A １ is Tyr, A ３ is Trp, A ４ is Phe, the peptide according to claim 1. **Claim 12** A ５ is L-Asp, the peptide according to any one of claims 1 to 11. **Claim 13** A ５ is L-Glu, the peptide according to any one of claims 1 to 11. **Claim 14** A ５ is D-Asp, the peptide according to any one of claims 1 to 11. **Claim 15** A ５ is D-Glu, the peptide according to any one of claims 1 to 11. **Claim 16** A ５ is L-isoAsp, the peptide according to any one of claims 1 to 11. **Claim 17** A ５ is L-isoGlu, the peptide according to any one of claims 1 to 11. **Claim 18** A ５ is D-isoAsp, the peptide according to any one of claims 1 to 11. Claim 19 A ５ is D - isoGlu, the peptide according to any one of claims 1 to 11. Claim 20 A ６ is L - Ser, the peptide according to any one of claims 1 to 19. Claim 21 A ６ is L - Ser - NH ２ is the peptide according to any one of claims 1 to 19. Claim 22 A ６ is L - Thr, the peptide according to any one of claims 1 to 19. Claim 23 A ６ is L - Thr - NH ２ is the peptide according to claim 1. Claim 24 Carb is β - D - glucose, the peptide according to any one of claims 1 to 23. Claim 25 Carb is β - D - lactose, the peptide according to any one of claims 1 to 23. Claim 26 The peptide is a cyclic peptide of formula (I), the peptide according to any one of claims 1 to 25. Claim 27 The peptide is a cyclic peptide of formula (II), the peptide according to any one of claims 1 to 25. Claim 28 Tyr - c[D - Lys - Trp - Phe - Glu] - Ser(β - Lact) - NH ２ (SEQ ID NO: 1), the peptide according to claim 1 or claim 26. Claim 29 Tyr - c[D - Lys - Trp - Phe - Glu] - Ser(β - Glc) - NH ２ (SEQ ID NO: 5), the peptide according to claim 1 or claim 26. Claim 30 Tyr-c[D-Lys-Trp-Phe-Glu]-Ser(β-Glc)-Ser(β-Glc)-NH ２ (SEQ ID NO: 10), the peptide according to claim 1 or claim 26.

31. A pharmaceutical composition comprising the peptide according to any one of claims 1 to 30, or a pharmaceutically acceptable salt thereof, in a pharmaceutically acceptable carrier.

32. A method for treating pain, comprising the step of administering the pharmaceutical composition according to claim 31 to a subject in need of treatment for pain.

33. The method according to claim 32, wherein the pain is chronic pain.

34. The method according to claim 32 or claim 33, wherein the pain is neuropathic pain.

35. The method according to any one of claims 32 to 34, wherein the pain is inflammatory pain.

36. A method for treating drug dependence, comprising the step of administering a therapeutically effective amount of the pharmaceutical composition according to claim 31 to a subject in need of treatment for drug dependence.

37. A method for treating opioid use disorder, comprising the step of administering a therapeutically effective amount of the pharmaceutical composition according to claim 31 to a subject in need of treatment for opioid use disorder.

38. The method according to claim 37, wherein the composition is administered in place of and as an alternative to a μ-opioid receptor agonist that causes the subject to become addicted.

39. The peptide according to any one of claims 1 to 30 for treating pain.

40. The peptide according to claim 39, wherein the pain is chronic pain.

41. The peptide according to claim 39 or 40, wherein the pain is neuropathic pain.

42. The peptide according to any one of claims 39 to 41, wherein the pain is inflammatory pain.

43. The peptide according to any one of claims 1 to 30 for treating drug dependence.

44. The peptide according to any one of claims 1 to 30 for treating opioid use disorder.

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