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ANALGESICS AND METHODS OF USE THEREOF

20210355166 · 2021-11-18

    Inventors

    Cpc classification

    International classification

    Abstract

    The present invention relates to peptides with alternating stereochemistry. In particular, the invention relates to peptides comprising alternating stereochemistry of (LDLD) in the first four amino acid residues. The invention further contemplates the use of peptides with alternating stereochemistry in treating pain.

    Claims

    1. An isolated peptide comprising Formula I ##STR00071## wherein, counting from the N-terminus, the first and third amino acid residues are L-amino acid residues and the second and fourth amino acid residues are D-amino acid residues; and wherein R.sup.1 is hydrogen, C.sub.1-C.sub.3 alkyl, or a bio-reversible moiety optionally comprising a sugar moiety; R.sup.2 is hydrogen, C.sub.1-C.sub.3 alkyl, or a bio-reversible moiety optionally comprising a sugar moiety; wherein R.sup.1 and R.sup.2 may together form one bio-reversible moiety; R.sup.3 and R.sup.4 are independently selected from hydrogen or C.sub.1-C.sub.3 alkyl, preferably —CH.sub.3; R.sup.5 is hydrogen, —OH, or a bio-reversible moiety optionally comprising a sugar moiety; R.sup.6 is a side chain of an amino acid or C.sub.1-C.sub.6 alkyl; R.sup.7 is a side chain of an amino acid or C.sub.1-C.sub.6 alkyl; R.sup.8 is —OH, —NH.sub.2, —O(C.sub.1-C.sub.3 alkyl), ##STR00072## or 1 to about 30 L-amino acid residues; Y.sub.1 is —OH, —NH.sub.2, or 1 to about 30 L-amino acid residues; Y.sub.2 is hydrogen or a sugar moiety, preferably a disaccharide moiety; and wherein when R.sup.8 is 1 to about 30 L-amino acid residues (1) the L-amino acid residues are optionally residues that may be optionally glycosylated with a sugar moiety, preferably a disaccharide moiety, and (2) the C-terminus is optionally amidated.

    2. The peptide according to claim 1, wherein R.sup.6 is C.sub.1-C.sub.6 alkyl and R.sup.7 is C.sub.1-C.sub.6 alkyl; and/or wherein R.sup.6 and R.sup.7 are independently selected from the side chain of alanine, valine, norvaline, leucine, norleucine, or isoleucine; and/or wherein R.sup.6 and R.sup.7 are each a valine side chain (—CH(CH.sub.3).sub.2); and/or wherein R.sup.6 and R.sup.7 are each a threonine side chain; and/or wherein R.sup.3 and R.sup.4 are —CH.sub.3; and R.sup.5 is —OH.

    3.-8. (canceled)

    9. An isolated peptide comprising Formula I ##STR00073## wherein, counting from the N-terminus, the first and third amino acid residues are L-amino acid residues and the second and fourth amino acid residues are D-amino acid residues; and wherein R.sup.1 is hydrogen, single bond, or a —C.sub.1-C.sub.3 alkyl; R.sup.2 is hydrogen, single bond, or a —C.sub.1-C.sub.3 alkyl; R.sup.3 and R.sup.4 are independently selected from hydrogen or C.sub.1-C.sub.3 alkyl, preferably —CH.sub.3; R.sup.5 is hydrogen, —OH, or —O(C.sub.1-C.sub.3)alkyl; R.sup.6 is a side chain of an amino acid or C.sub.1-C.sub.6 alkyl; R.sup.7 is a side chain of an amino acid or C.sub.1-C.sub.6 alkyl; R.sup.8 is —OH, —NH.sub.2, —O(C.sub.1-C.sub.3 alkyl), ##STR00074## or 1 to about 30 L-amino acid residues; Y.sub.1 is —OH, —NH.sub.2, or 1 to about 30 L-amino acid residues; Y.sub.2 is hydrogen or a sugar moiety, preferably a disaccharide moiety; and wherein when R.sup.8 is 1 to about 30 L-amino acid residues (1) the L-amino acid residues are optionally residues that may be optionally glycosylated with a sugar moiety, preferably a disaccharide moiety, and (2) the C-terminus is optionally amidated; wherein when R.sup.8 is a linker, the linker comprises a sugar moiety, preferably a disaccharide moiety such as lactose, and wherein when one of R.sup.1 or R.sup.2 is a single bond, one of R.sup.1 and R.sup.2 is hydrogen and the single bond is a peptide bond to an L-amino acid residue that may optionally be N-terminally alkylated, preferably singly methylated.

    10. The peptide according to claim 9, wherein one of R.sup.1 and R.sup.2 is hydrogen and one of R.sup.1 and R.sup.2 is —CH.sub.3; and/or wherein R.sup.5 is —O(C.sub.1-C.sub.3)alkyl, preferably —OCH.sub.3; preferably wherein R.sup.3 and R.sup.4 are —CH.sub.3; or wherein R.sup.3 and R.sup.4 are —CH.sub.3 and R.sup.5 is —OH; and/or wherein R.sup.6 and R.sup.7 are each a valine side chain (—CH(CH.sub.3).sub.2); or wherein R.sup.6 and R.sup.7 are each a threonine side chain; and/or wherein one of R.sup.1 or R.sup.2 is a single bond, one of R.sup.1 and R.sup.2 is a hydrogen, and the single bond is a peptide bond to an L-amino acid residue; optionally wherein the L-amino acid residue has at least one N-terminal methylation; and/or optionally wherein the L-amino acid residue is an L-alanine residue.

    11.-21. (canceled)

    22. The peptide according to claim 1, wherein R.sup.8 is —OH, —NH.sub.2, —O(C.sub.1-C.sub.3 alkyl), ##STR00075## Y.sub.1 is —OH, —NH.sub.2, or 1 to about 30 L-amino acid residues; and Y.sub.2 is hydrogen or a sugar moiety, preferably a disaccharide moiety; and wherein R.sup.8 is ##STR00076## Y.sub.1 is —OH, —NH.sub.2, or 1 to about 30 L-amino acid residues; and Y.sub.2 is hydrogen or a sugar moiety, preferably a disaccharide moiety; or wherein Y.sub.1—NH.sub.2; or wherein Y.sub.1 is 1 to about 30 L-amino acid residues; or wherein Y.sub.1 is 1 to about 25 L-amino acid residues, 1 to about 20 L-amino acid residues, 1 to about 15 L-amino acid residues, 1 to about 11 L-amino acid residues, or 1 to about 5 L-amino acid residues; optionally wherein Y.sup.1 is 1 to about 11 L-amino acid residues.

    23.-29. (canceled)

    30. The peptide according to claim 1, wherein R.sup.8 is 1 to about 11 L-amino acid residues, wherein the 1 to about 11 L-amino acid residues comprise at least one glycosylated L-amino acid residue, preferably comprising at least one O-glycosylated L-serine residue.

    31. (canceled)

    32. The peptide according to claim 1, wherein R.sup.1 and R.sup.2 are hydrogen; R.sup.3, R.sup.4, and R.sup.5 are hydrogen; R.sup.6 and R.sup.7 are each —CH(CH.sub.3).sub.2; and R.sup.8 is —OH, —NH.sub.2, —O(C.sub.1-C.sub.3 alkyl), ##STR00077## Y.sub.1 is —OH, —NH.sub.2, or 1 to about 30 L-amino acid residues; and Y.sub.2 is hydrogen or a sugar moiety, preferably a disaccharide moiety; or wherein R.sup.1 and R.sup.2 are hydrogen; R.sup.3 and R.sup.4 are both hydrogen or both —CH.sub.3; R.sup.5 is —OH; R.sup.6 and R.sup.7 are each —CH(CH.sub.3).sub.2; and R.sup.8 is —OH, —NH.sub.2, —O(C.sub.1-C.sub.3 alkyl), ##STR00078## Y.sub.1 is —OH, —NH.sub.2, or 1 to about 30 L-amino acid residues; Y.sub.2 is hydrogen or a sugar moiety, preferably a disaccharide moiety; optionally wherein R.sup.8 is —NH.sub.2, ##STR00079## Y.sub.1 is —OH, —NH.sub.2, or 1 to about 30 L-amino acid residues; and Y.sub.2 is hydrogen or a sugar moiety, preferably a disaccharide moiety; optionally wherein R.sup.8 is ##STR00080## Y.sub.1 is —OH or —NH.sub.2; and Y.sub.2 is hydrogen or a sugar moiety, preferably a disaccharide moiety; or optionally wherein Y.sub.1 is 1 to about 25 L-amino acid residues, 1 to about 20 L-amino acid residues, 1 to about 15 L-amino acid residues, 1 to about 11 L-amino acid residues, or 1 to about 5 L-amino acid residues; preferably wherein Y.sup.1 is 1 to about 11 L-amino acid residues.

    33.-37. (canceled)

    38. The peptide according to claim 1, wherein Y.sub.2 is a sugar moiety, preferably a disaccharide moiety; optionally wherein Y.sub.2 the disaccharide moiety is a lactose moiety or melibiose moiety; optionally wherein Y.sub.2 the disaccharide moiety is a lactose moiety; optionally wherein the disaccharide moiety is attached through a beta linkage.

    39.-41. (canceled)

    42. The peptide according to claim 1, wherein R.sup.8 is 1 to about 25 L-amino acid residues, 1 to about 20 L-amino acid residues, 1 to about 15 L-amino acid residues, 1 to about 11 L-amino acid residues, or 1 to about 5 L-amino acid residues; wherein said L-amino acid residues comprises at least one amino acid residue that is N-glycosylated, O-glycosylated, C-glycosylated, S-glycosylated, or Se-glycosylated; optionally wherein said L-amino acid residues comprise at least one L-amino acid residue that is O-glycosylated; optionally wherein said amino acid residue that is O-glycosylated is an L-serine residue.

    43.-46. (canceled)

    47. The peptide according to claim 1, wherein R.sup.1 and R.sup.2 together form a bio-reversible moiety; optionally wherein said bio-reversible moiety is ##STR00081## or ═N═N (azido moiety) or the peptide according to claim 1 wherein one of R.sup.1 or R.sup.2 is hydrogen and one of R.sup.1 or R.sup.2 is —C(═O)OCH.sub.2CH.sub.3 or —C(═O)OCH.sub.2OC(═O)CH.sub.3; or wherein R.sup.5 is a bio-reversible moiety optionally wherein the bio-reversible moiety is —C(═O)CH.sub.3.

    48.-51. (canceled)

    52. The peptide according to claim 1, selected from the group consisting of: L-Phe-D-Val-L-Val-D-Phe (peptide 1a, Bilaid A); L-Phe-D-Val-L-Val-D-Phe-NH.sub.2 (peptide 1e); L-Tyr-D-Val-L-Val-D-Phe (peptide 3a, Bilaid C); L-Tyr-D-Val-L-Val-D-Phe-NH.sub.2 (peptide 3b); 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-NH.sub.2 (peptide 3c; Bilorphin); 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-L-Ser(β-Lac)-NH.sub.2 (peptide 3g; Bilactorphin); 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-OCH.sub.2CH.sub.3 (peptide 4); 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-L-Pro-L-Asn-L-Leu-L-Ala-L-Glu-L-Lys-L-Ala-L-Leu-L-Lys-L-Ser-L-Leu-NH.sub.2 (peptide 11); 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-NH.sub.2 wherein the N-terminus is substituted with the bio-reversible moiety —C(═O)OCH.sub.2OC(═O)CH.sub.3 (peptide 10); 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-NH.sub.2 wherein the N-terminus is substituted with the bio-reversible moiety ##STR00082## (peptide 8); 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-NH.sub.2 wherein the hydroxy group on 2,6-dimethyl-L-tyrosine is substituted with the bio-reversible moiety —C(═O)CH.sub.3 (peptide 5); 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-OCH.sub.2CH.sub.3 wherein the hydroxy group on 2,6-dimethyl-L-tyrosine is substituted with the bio-reversible moiety —C(═O)CH.sub.3 (peptide 6); 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-NH.sub.2 wherein the N-terminus is substituted with the bio-reversible moiety —C(═O)OCH.sub.2CH.sub.3 (peptide 7); and 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-NH.sub.2 wherein the N-terminus is substituted with the bio-reversible moiety, ═N═N, to form an N-terminal azido group (peptide 9).

    53. A peptide comprising: L-AA-L-Tyr-D-Val-L-Val-D-Phe-linker-sugar moiety; L-AA-L-Tyr-D-Thr-L-Thr-D-Phe-linker-sugar moiety; L-AA-L-Dmt-D-Val-L-Val-D-Phe-linker-sugar moiety; L-AA-L-Dmt-D-Thr-L-Thr-D-Phe-linker-sugar moiety; wherein L-AA is any L-amino acid residue optionally with at least one N-terminal —CH.sub.3; wherein the hydroxyl group of L-Tyr or L-Dmt is optionally alkylated; and wherein the linker is preferably L-Ser or L-Thr.

    54. A peptide comprising Formula II ##STR00083## wherein, counting from the N-terminus, the first amino acid residue is an L-amino acid residue and the second and fourth amino acid residues are D-amino acid residues; wherein R.sup.9 is hydrogen or a bio-reversible moiety optionally comprising a sugar moiety; R.sup.10 is hydrogen or a bio-reversible moiety optionally comprising a sugar moiety; wherein when one of R.sup.9 or R.sup.10 is a hydrogen and one of R.sup.9 or R.sup.10 is a bio-reversible moiety, the bio-reversible moiety is preferably —C(═O)OCH.sub.2CH.sub.3 or —C(═O)OCH.sub.2OC(═O)CH.sub.3; wherein R.sup.9 and R.sup.10 may together form one bio-reversible moiety, wherein preferably the bio-reversible moiety is ##STR00084## or ═N═N (azido moiety); R.sup.11 and R.sup.12 are independently selected from hydrogen or C.sub.1-C.sub.3 alkyl, preferably —CH.sub.3; R.sup.13 is hydrogen, —OH, or a bio-reversible moiety optionally comprising a sugar moiety; R.sup.14 is a side chain of an amino acid or C.sub.1-C.sub.6 alkyl, preferably C.sub.1-C.sub.4 alkyl, more preferably —CH(CH.sub.3).sub.2; R.sup.15 is hydrogen, —OH, or a bio-reversible moiety; and R.sup.16 is —OH, —O(C.sub.1-C.sub.3 alkyl), —NH.sub.2, ##STR00085## or 1 to about 30 L-amino acid residues; Y.sub.3 is —OH, —NH.sub.2, or 1 to about 30 L-amino acid residues; Y.sub.4 is hydrogen or a sugar moiety, preferably a disaccharide moiety; and wherein when R.sup.16 is 1 to about 30 L-amino acid residues (1) the L-amino acid residues are optionally residues that may be optionally glycosylated with a sugar moiety, preferably a disaccharide moiety, and (2) the C-terminus is optionally amidated.

    55. (canceled)

    56. (canceled)

    57. The peptide according to claim 54, wherein R.sup.14 is a valine side chain (—CH(CH.sub.3).sub.2); or wherein R.sup.14 is a threonine side chain; wherein optionally R.sup.11 and R.sup.12 are —CH.sub.3; and R.sup.13 is —OH; wherein optionally R.sup.9, R.sup.10, R.sup.11, R.sup.12, and R.sup.13 are each hydrogen; wherein optionally R.sup.9, R.sup.10, R.sup.11, R.sup.12, and R.sup.13 are hydrogen; R.sup.14 is C.sub.1-C.sub.4 alkyl; R.sup.15 is —OH.

    58.-62. (canceled)

    63. A peptide comprising Formula II ##STR00086## wherein, counting from the N-terminus, the first amino acid residue is an L-amino acid residue and the second and fourth amino acid residues are D-amino acid residues; wherein R.sup.9 is hydrogen, a single bond, or —C.sub.1-C.sub.3 alkyl, preferably —CH.sub.3; R.sup.10 is hydrogen, a single bond, or —C.sub.1-C.sub.3 alkyl, preferably —CH.sub.3; R.sup.11 and R.sup.12 are independently selected from hydrogen or C.sub.1-C.sub.3 alkyl, preferably —CH.sub.3; R.sup.13 is hydrogen, —OH, or —O(C.sub.1-C.sub.3)alkyl; R.sup.14 is a side chain of an amino acid or C.sub.1-C.sub.6 alkyl, preferably C.sub.1-C.sub.4 alkyl, more preferably —CH(CH.sub.3).sub.2; R.sup.15 is hydrogen, —OH, or a bio-reversible moiety; and R.sup.16 is —OH, —O(C.sub.1-C.sub.3 alkyl), —NH.sub.2, ##STR00087## 1 to about 30 L-amino acid residues, or a linker; Y.sub.3 is —OH, —NH.sub.2, or 1 to about 30 L-amino acid residues; Y.sub.4 is hydrogen or a sugar moiety, preferably a disaccharide moiety; wherein when R.sup.16 is 1 to about 30 L-amino acid residues (1) the L-amino acid residues are optionally residues that may be optionally glycosylated with a sugar moiety, preferably a disaccharide moiety, and (2) the C-terminus is optionally amidated; wherein when R.sup.16 is a linker, the linker comprises a sugar moiety, preferably a disaccharide moiety such as lactose, and wherein when one of R.sup.9 or R.sup.10 is a single bond, one of R.sup.9 or R.sup.10 is hydrogen and the single bond is a peptide bond to an L-amino acid residue optionally N-terminally alkylated, preferably singly methylated.

    64. The peptide according to claim 63, wherein one of R.sup.9 and R.sup.10 is hydrogen and one of R.sup.9 and R.sup.10 is —CH.sub.3; or wherein R.sup.13 is —O(C.sub.1-C.sub.3)alkyl, preferably —OCH.sub.3, optionally wherein R.sup.11 and R.sup.12 are —CH.sub.3; or wherein R.sup.11 and R.sup.12 are —CH.sub.3, and R.sup.13 is —OH; or wherein one of R.sup.9 or R.sup.10 is a single bond, one of R.sup.9 or R.sup.10 is hydrogen, and the single bond is a peptide bond to an L-amino acid residue; optionally wherein the L-amino acid residue has at least one N-terminal methylation; and/or optionally wherein the L-amino acid residue is an L-alanine residue; or wherein R.sup.14 is a valine side chain (—CH(CH.sub.3).sub.2); or wherein R.sup.14 is threonine side chain.

    65.-73. (canceled)

    74. The peptide according to claim 54, wherein R.sup.16 is —NH.sub.2.

    75. The peptide according to claim 54, wherein R.sup.16 is 1 to about 30 L-amino acid residues; Y.sub.3 is —OH, —NH.sub.2, or 1 to about 30 L-amino acid residues; Y.sub.4 is hydrogen or a sugar moiety, preferably a disaccharide moiety; and wherein when R.sup.16 is 1 to about 30 L-amino acid residues (1) the L-amino acid residues are optionally residues that may be optionally glycosylated with a sugar moiety, preferably a disaccharide moiety, and (2) the C-terminus is optionally amidated; wherein said L-amino acid residues comprises at least one amino acid residue that is N-glycosylated, O-glycosylated, C-glycosylated, S-glycosylated, or Se-glycosylated; optionally wherein said L-amino acid residues comprise at least one amino acid residue that is O-glycosylated; optionally wherein said amino acid residue that is O-glycosylated is an L-serine residue.

    76.-83. (canceled)

    84. The peptide according to claim 54, wherein R.sup.16 is ##STR00088## Y.sub.3 is —OH, —NH.sub.2, or 1 to about 30 L-amino acid residues; and Y.sub.4 is hydrogen or a sugar moiety, preferably a disaccharide moiety; or wherein R.sup.9, R.sup.10, R.sup.11, R.sup.12, and R.sup.13 are hydrogen; R.sup.14 is —CH(CH.sub.3).sub.2; R.sup.15 is —OH; and R.sup.16 is ##STR00089## Y.sub.3 is —OH or —NH.sub.2; and Y.sub.4 is hydrogen or a sugar moiety, preferably a disaccharide moiety; or wherein the peptide is L-Phe-D-Val-Gly-D-Tyr-NH.sub.2.

    85. The peptide according to claim 54, wherein R.sup.16 is ##STR00090## Y.sub.3 is —OH or —NH.sub.2; and Y.sub.4 is hydrogen or a sugar moiety, preferably a disaccharide moiety; or the peptide according to claim 54, wherein said sugar moiety is a disaccharide moiety, preferably wherein the disaccharide moiety is attached through a beta linkage; optionally wherein said disaccharide moiety is a lactose moiety or a melibiose moiety, preferably wherein the disaccharide moiety is attached through a beta linkage; optionally wherein said disaccharide moiety is a lactose moiety, preferably wherein the lactose moiety is attached through a beta linkage; or the peptide according to claim 54, wherein R.sup.9 and R.sup.10 together form a bio-reversible moiety; optionally wherein said bio-reversible moiety is ##STR00091## or ═N═N (azido moiety); or wherein one of R.sup.9 or R.sup.10 is hydrogen and one of R.sup.9 or R.sup.10 is —C(═O)OCH.sub.2CH.sub.3 or —C(═O)OCH.sub.2OC(═O)CH.sub.3; or wherein R.sup.13 is a bio-reversible moiety; optionally wherein the bio-reversible moiety is —C(═O)CH.sub.3.

    86.-95. (canceled)

    96. An isolated peptide comprising Formula III
    X.sup.1—X.sup.2—X.sup.3—X.sup.4  (III) wherein: X.sup.1 is the N-terminal amino acid residue comprising an N-terminal moiety —NR.sup.17R.sup.18; X.sup.4 comprises a C-terminal moiety selected from —C(═O)OH, —C(═O)O(C.sub.1-C.sub.3 alkyl) —C(═O)NH.sub.2, ##STR00092## wherein Y.sub.5 is —OH or —NH.sub.2, and Y.sub.6 is hydrogen or a sugar moiety, preferably a disaccharide moiety; X.sup.1 is an L-amino acid residue selected from L-tyrosine, 2,6-dimethyl-L-tyrosine, or L-phenylalanine, wherein when X.sup.1 is L-tyrosine or 2,6-dimethyl-L-tyrosine, the residue is optionally O-substituted at the 4-position with a bio-reversible moiety optionally comprising a sugar moiety; X.sub.2 is a D-amino acid residue, preferably D-alanine, D-valine, D-leucine, or D-isoleucine, more preferably, D-valine; X.sub.3 is glycine or an L-amino acid residue, wherein when X.sub.3 is an L-amino acid residue, X.sub.3 is preferably L-alanine, L-valine, L-leucine, or L-isoleucine, more preferably L-valine; X.sub.4 is a D-amino acid residue selected from D-tyrosine or D-phenylalanine, wherein when X.sub.4 is D-tyrosine, the residue is optionally O-substituted with a bio-reversible moiety; R.sup.17 and R.sup.18 are independently selected from hydrogen or a bio-reversible moiety optionally comprising a sugar moiety, or R.sup.17 and R.sup.18 together form a bio-reversible moiety optionally comprising a sugar moiety; and wherein the peptide is a MOPr agonist.

    97. The peptide according to claim 96, wherein the C-terminal moiety is —C(═O)OH, ##STR00093## and Y.sub.5 is —OH and Y.sub.6 is hydrogen or a sugar moiety; the peptide further comprises about 5, 8, 11, 12, 20 or 26 additional L-amino acid residues on the C-terminus; and/or wherein R.sup.17 and R.sup.18 are each hydrogen, X.sup.2 is a D-valine residue, X.sup.3 is glycine or an L-valine residue, X.sup.4 comprises a C-terminal moiety selected from —C(═O)OH, —C(═O)NH.sub.2, ##STR00094## wherein Y.sub.5 is —OH or —NH.sub.2, and Y.sub.6 is hydrogen or a sugar moiety, preferably a disaccharide moiety.

    98. (canceled)

    99. An isolated peptide comprising Formula III
    X.sup.1—X.sup.2—X.sup.3—X.sup.4  (III) wherein: X.sup.1 is the N-terminal amino acid residue comprising an N-terminal moiety —NR.sup.17R.sup.18; X.sup.4 comprises a C-terminal moiety selected from —C(═O)OH, —C(═O)O(C.sub.1-C.sub.3 alkyl)-C(═O)NH.sub.2, ##STR00095## or a linker, wherein Y.sub.5 is —OH or —NH.sub.2, and Y.sub.6 is hydrogen or a sugar moiety, preferably a disaccharide moiety; X.sup.1 is an L-amino acid residue selected from L-tyrosine, 2,6-dimethyl-L-tyrosine, or L-phenylalanine, wherein when X.sup.1 is L-tyrosine or 2,6-dimethyl-L-tyrosine, the residue is optionally O-substituted at the 4-position with C.sub.1-C.sub.3 alkyl; X.sup.2 is a D-amino acid residue, preferably D-threonine, D-alanine, D-valine, D-leucine, or D-isoleucine, more preferably, D-threonine or D-valine; X.sup.3 is glycine or an L-amino acid residue, wherein when X.sup.3 is an L-amino acid residue, X.sup.3 is preferably L-threonine, L-alanine, L-valine, L-leucine, or L-isoleucine, more preferably L-threonine or L-valine; X.sup.4 is a D-amino acid residue selected from D-tyrosine or D-phenylalanine, wherein when X.sup.4 is D-tyrosine, the residue is optionally O-substituted with a bio-reversible moiety; R.sup.17 and R.sup.18 are independently selected from hydrogen, a single bond, or a —C.sub.1-C.sub.3 alkyl, preferably —CH.sub.3; and wherein when X.sup.4 comprises a linker, the linker comprises a sugar moiety, preferably a disaccharide moiety such as lactose, wherein when one of R.sup.17 or R.sup.18 is a single bond, one of R.sup.17 or R.sup.18 is hydrogen and the single bond is a peptide bond to an L-amino acid residue that may optionally be N-terminally alkylated, preferably singly methylated; and wherein the peptide is a MOPr agonist.

    100. The peptide according to claim 99, wherein the C-terminal moiety is —C(═O)OH, ##STR00096## and Y.sub.5 is —OH and Y.sub.6 is hydrogen or a sugar moiety; the peptide further comprises about 5, 8, 11, 12, 20 or 26 additional L-amino acid residues on the C-terminus; and/or wherein X.sup.2 is a D-valine residue, X.sup.3 is glycine or an L-valine residue, X.sup.4 comprises a C-terminal moiety selected from —C(═O)OH, —C(═O)NH.sub.2, ##STR00097## wherein Y.sub.5 is —OH or —NH.sub.2, and Y.sub.6 is hydrogen or a sugar moiety, preferably a disaccharide moiety; and/or wherein X.sup.2 is a D-threonine residue, X.sup.3 is glycine or an L-threonine residue, X.sup.4 comprises a C-terminal moiety selected from —C(═O)OH, —C(═O)NH.sub.2, ##STR00098## wherein Y.sub.5 is —OH or —NH.sub.2, and Y.sub.6 is hydrogen or a sugar moiety, preferably a disaccharide moiety; and/or wherein one of R.sup.17 and R.sup.18 is hydrogen and one of R.sup.17 and R.sup.18 is —CH.sub.3; or wherein one of R.sup.17 or R.sup.18 is a single bond, one of R.sup.17 or R.sup.18 is a hydrogen, and the single bond is a peptide bond to an L-amino acid residue; optionally wherein the L-amino acid residue has at least one N-terminal methylation; and/or wherein the L-amino acid residue is an L-alanine residue; or wherein X.sup.4 comprises a linker; optionally wherein the linker comprises an amino acid based linker, peptide based linker, an amino acid comprising linker, and/or maleimide based linker, and/or a combination thereof; and/or wherein X.sup.1 is L-tyrosine or 2,6-dimethyl-L-tyrosine and wherein the L-tyrosine or 2,6-dimethyl-L-tyrosine is O-substituted at the 4-position with C.sub.1-C.sub.3 alkyl; optionally wherein X.sup.1 is 2,6-dimethyl-L-tyrosine and wherein 2,6-dimethyl-L-tyrosine is O-substituted at the 4-position with C.sub.1-C.sub.3 alkyl.

    101.-110. (canceled)

    111. The peptide according to claim 96, wherein X.sup.4 comprises a C-terminal moiety selected from, ##STR00099## wherein Y.sub.5 is —OH or —NH.sub.2, and Y.sub.6 is a disaccharide moiety, preferably wherein the disaccharide moiety is attached through a beta linkage; optionally wherein the disaccharide moiety is a lactose moiety, preferably wherein the lactose moiety is attached through a beta linkage; or wherein said additional L-amino acids comprise at least one amino acid residue that is N-glycosylated, O-glycosylated, C-glycosylated, S-glycosylated, or Se-glycosylated; or wherein R.sup.17 and R.sup.18 together form a bio-reversible moiety; optionally wherein said bio-reversible moiety is ##STR00100## or ═N═N (azido moiety); or wherein one of R.sup.17 or R.sup.18 is hydrogen and one of R.sup.17 or R.sup.18 is —C(═O)OCH.sub.2CH.sub.3 or —C(═O)OCH.sub.2OC(═O)CH.sub.3.

    112.-116. (canceled)

    117. The peptide according to claim 96 selected from the group consisting of L-Phe-D-Val-L-Val-D-Phe (peptide 1a, Bilaid A); L-Phe-D-Val-L-Val-D-Phe-NH.sub.2 (peptide 1e); L-Tyr-D-Val-L-Val-D-Phe (peptide 3a, Bilaid C); L-Tyr-D-Val-L-Val-D-Phe-NH.sub.2 (peptide 3b); 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-NH.sub.2 (peptide 3c; Bilorphin); 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-L-Ser(β-Lac)-NH.sub.2 (peptide 3g; Bilactorphin); L-Phe-D-Val-Gly-D-Tyr-NH.sub.2 (peptide 2d); 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-OCH.sub.2CH.sub.3 (peptide 4); 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-L-Pro-L-Asn-L-Leu-L-Ala-L-Glu-L-Lys-L-Ala-L-Leu-L-Lys-L-Ser-L-Leu-NH.sub.2 (peptide 11); 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-NH.sub.2 wherein the N-terminus is substituted with the bio-reversible moiety —C(═O)OCH.sub.2OC(═O)CH.sub.3 (peptide 10); 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-NH.sub.2 wherein the N-terminus is substituted with the bio-reversible moiety ##STR00101## (peptide 8); 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-NH.sub.2 wherein the hydroxy group on 2,6-dimethyl-L-tyrosine is substituted with the bio-reversible moiety —C(═O)CH.sub.3 (peptide 5); 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-OCH.sub.2CH.sub.3 wherein the hydroxy group on 2,6-dimethyl-L-tyrosine is substituted with the bio-reversible moiety —C(═O)CH.sub.3 (peptide 6); 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-NH.sub.2 wherein the N-terminus is substituted with the bio-reversible moiety —C(═O)OCH.sub.2CH.sub.3 (peptide 7); and 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-NH.sub.2 wherein the N-terminus is substituted with the bio-reversible moiety, ═N═N, to form an N-terminal azido group (peptide 9).

    118.-123. (canceled)

    124. A pharmaceutical composition comprising a peptide according to claim 1 and at least one pharmaceutical excipient; wherein optionally (i) said composition is formulated for oral administration; or (ii) said peptide is not glycosylated and said composition is formulated for nasal administration or intrathecal administration.

    125. (canceled)

    126. The pharmaceutical composition according to claim 124, wherein said peptide is glycosylated and said composition is formulated for oral administration, administration by injection, or intrathecal administration.

    127.-134. (canceled)

    135. A method of: (i) treating pain, preferably wherein the pain is post-operative pain, pain associated with nerve damage, pain associated with bone fracture, pain associated with a burn, or pain associated with a wound; (ii) delivering analgesia; or (iii) treating pain or delivering analgesia with reduced adverse side effect(s), preferably reduced in comparison to morphine and preferably wherein said adverse side effect(s) is gastrointestinal (GI) inhibition and/or respiratory depression, in a subject in need thereof, comprising administering to the subject the peptide according to claim 1.

    136.-138. (canceled)

    Description

    BRIEF DESCRIPTION OF THE FIGURES

    [0252] Embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings as follows.

    [0253] FIG. 1: Competitive Binding Assay. Peptides [YvVf-OH (3a), YvVf-NH.sub.2 (3b), [Dmt]-vVf-NH.sub.2 (3c)] tested for competitive binding to hMOPr against the MOPr agonist [.sup.3H]DMAGO. [Dmt]-vVf-NH.sub.2 (3c) was tested for competitive binding to hDOPr against the DOPr agonist [.sup.3H]DADLE and for competitive binding to hKOPr against the KOPr agonist [.sup.3H]U69593. Symbols in figure: hMOPr: YvVf-OH (3a) x in circle; YvVf-NH.sub.2 (3b) circle in circle, [Dmt]-vVf-NH.sub.2 (3c) large circle; DOPr: [Dmt]-vVf-NH.sub.2 (3c) * in circle; KOPr: [Dmt]-vVf-NH.sub.2 (3c)+in circle.

    [0254] FIG. 2: FIG. 2A: Example of GIRK current recorded from rat LC neuron in response to met-enkephalin (1 μM), [Dmt]-vVf-NH.sub.2 (3c, Bilorphin) (1 μM), and its reversal by co-application of the MOPr selective antagonist, CTAP ((D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH.sub.2) (SEQ ID NO: 31) (1 μM). Scale bars: 50 pA, 5 min. FIG. 2B: Partial antagonist effect of [Dmt]-vVf-NH.sub.2 (3c, Bilorphin) (1 μM) on the GIRK current evoked by supramaximal desensitizing concentration of met-enkephalin (10 μM, 10 min; same scale as FIG. 2A).

    [0255] FIG. 3: Agonist concentration-response relationships of exemplar opioids and [Dmt]-vVf-NH.sub.2 (3c, Bilorphin) for activation of GIRK current in LC neurons normalised to 1 μM met-enkephalin applied as a probe in each cell (N=4-13 cells per data point). [Dmt]-vVf-NH.sub.2 (3c, Bilorphin, * in circle) in comparison to met-enkephalin (X in circle), morphine (+ in circle), and endomorphin-2 (circle in circle).

    [0256] FIG. 4: Exemplary record of G.sub.GIRK in mMOPr expressing AtT20 cell in response to somatostatin (SST) and the concentrations of opioids shown and duration of bars, after alkylation of a fraction of receptors by the irreversible MOPr antagonist β-chlornaltrexamine (β-CNA). Scale bar 0.2 ns, 1 min.

    [0257] FIG. 5: Concentration-response curves of G.sub.GIRK induced by opioids in AtT20 cells after reducing the receptor reserve by β-CNA pretreatment to produce a maximum response to met-enkephalin to 80% of that produced by somatostatin (SST). [Dmt]-vVf-NH.sub.2 (3c, Bilorphin, * in circle (purple)) in comparison to met-enkephalin (X in circle), morphine (+ in circle), endomorphin-2 (circle in circle) and oliceridine (large circle). Patch-clamp recordings in AtT20 cells stably expressing FLAG tagged mouse MOPr (mMOPr).

    [0258] FIG. 6: C-terminal phosphorylation induction by [Dmt]-vVf-NH.sub.2 (3c, Bilorphin) in comparison to met-enkephalin, morphine, and endomorphin-2 using a phosphosite specific antibody. Representative images of Serine 375 phosphorylation in AtT20 cells induced by a saturating concentration (30 μM) of met-enkephalin, endomorphin 2, morphine and bilorphin after 5 min incubation. Colours enhanced uniformly for presentation purposes.

    [0259] FIG. 7: β-Arrestin recruitment induced by [Dmt]-vVf-NH.sub.2 (3c, Bilorphin) in comparison to met-enkephalin, morphine, and endomorphin-2 as determined by MOPr-luciferease and β-arrestin2-YFP constructs. Time course of ligand-induced BRET signal (light emission of 535 nm/475 nm) indicating β-arrestin 2 recruitment after agonist exposure (shown by the arrow). The band represents the standard error of experiments repeated independently 6 times (each experiment had triplicates).

    [0260] FIG. 8: MOPr internalisation. Example images of MOPr internalisation 30 min after treatment with 30 μM of agonists. Dual staining was employed for quantification (membrane receptor in green (appearing light grey) and internalized receptor in red (appearing darker grey), colours enhanced uniformly for presentation purposes).

    [0261] FIG. 9: Maximal efficacy values of endomorphin 2, morphine and bilorphin relative to met-enkephalin for GIRK channel activation, Serine 375 phosphorylation, β-arrestin 2 recruitment and normalization internalisation. [Dmt]-vVf-NH.sub.2 (3c, Bilorphin) in comparison to morphine, and endomorphin-2, and met-enkephalin, presented in order from foreground to background).

    [0262] FIG. 10: MOPr internalisation in cells expressing GRK2-YFP. Examples of enhanced internalization (green (appearing light grey) and red (appearing darker grey) as in panel C) produced by oliceridine, bilorphin and morphine in cells overexpressing both GRK2 (yellow (appearing grey)) and β-arrestin 2.

    [0263] FIG. 11: FIG. 11A: Internalization for each agonist in cells (ratio of fluorescence in green/[green+red] channels) transiently transfected with both GRK2 and β-arrestin 2 (n=40 cells from 2 experiments). FIG. 11B: Bias ratios calculated from GGIRK maxima normalized to met-enkephalin (from FIG. 5) internalization (from FIG. 11A) normalized to Met-enkephalin for bilorphin indicates greater G-protein bias than both olicerideine and morphine. FIG. 11C: Internalization for each agonist in cells (ratio of fluorescence in green/[green+red] channels) transiently transfected with both GRK2 and β-arrestin 2 (n=5 experiments, with greater than 10 cells in each). FIG. 11D: Bias ratios calculated from GGIRK maxima normalized to met-enkephalin (from FIG. 5) and internalization (from FIG. 11C) normalized to Met-enkephalin for bilorphin indicates greater G-protein bias than both olicerideine and morphine.

    [0264] FIG. 12: In vivo analgesia assay of analogues of [Dmt]-vVf-NH.sub.2 (3c, Bilorphin). [Dmt]-vVf-L-Ser(β-Lac)-NH.sub.2 (3g, Bilactorphin) produced dose-dependent analgesia in mice on the 54° C. hotplate, after sub-cutaneous administration and was antagonised by naltrexone. Doses in μmol/kg are indicated in parentheses (n=7-12 per data point except naltrexone [n=4]). Vehicle (circle with =), morphine (circle with +), [Dmt]-vVf-L-Ser(β-Lac)-NH.sub.2 (3g, Bilactorphin (14 μmol/kg, circle with 1 star (*); 28 μmol/kg, circle with 2 stars (**); 56 μmol/kg, circle with 3 stars (***); 112 μmol/kg, circle with 4 stars (****)); [Dmt]-vVf-L-Ser(β-Lac)-NH.sub.2 (3g, Bilactorphin) with naltrexone (circle with x).

    [0265] FIG. 13: Peripherally administered [Dmt]-vVf-L-Ser(β-Lac)-NH.sub.2 (3g, Bilactorphin) was equipotent with morphine on the hotplate test (n=5-12). [Dmt]-vVf-L-Ser(β-Lac)-NH.sub.2 (3g, Bilactorphin, large circle (dark grey)) and morphine (x in circle (light grey)).

    [0266] FIG. 14: Representative trace indicating time course of GIRK current in MOPr expressing AtT20 cell in response to [Dmt]-vVf-NH.sub.2 (3c, Bilorphin) and [Dmt]-vVf-L-Ser(β-Lac)-NH.sub.2 (3g, Bilactorphin) and morphine relative to a probe of 1 μM somatostatin. The scale bars represent 0.2 nS and 1 min.

    [0267] FIG. 15: Concentration response curves of potassium conductance induced by [Dmt]-vVf-NH.sub.2 (3c, Bilorphin, x in circle) and [Dmt]-vVf-L-Ser(β-Lac)-NH.sub.2 (3g, Bilactorphin, large circle) normalised to 1 μM somatostatin applied as a probe in individual cells.

    [0268] FIG. 16: Example images of MOPr internalization induced by 30 μM [Dmt]-vVf-NH.sub.2 (3c, Bilorphin) and [Dmt]-vVf-L-Ser(β-Lac)-NH.sub.2 (3g, Bilactorphin) after GRK2 overexpression (membrane and normalised MOPr in green (appearing light grey) and red (appearing darker grey) respectively and GRK2 in Yellow).

    [0269] FIG. 17: Maximal efficacy values of morphine (green), [Dmt]-vVf-NH.sub.2 (3c, Bilorphin, purple) and [Dmt]-vVf-L-Ser(β-Lac)-NH.sub.2 (3g, Bilactorphin, dark green) relative to met-enkephalin (30 μM exposure of the agonists) to produce receptor internalisation. For each set, morphine on the left, bilprophin is in the middle, and bilactorphin is on the right.

    [0270] FIG. 18: Predicted binding pose of [Dmt]-vVf-NH.sub.2 (3c, Bilorphin) (A and B) and endomorphin-2 (C and D) from MD simulations. FIGS. 18A and 18C: Predicted binding poses of bilorphin (dark grey) (18A) and endomorphin-2 (light grey) (18C), and the positions of the surrounding binding pocket residues (lightest grey) obtained after molecular docking and 1 μs of MD simulations. The salt bridge between protonated amine of the ligands and Asp147.sup.3.32 is marked as a dashed black line. TM7 has been removed for clarity. FIGS. 18B and 18D: Alternative viewpoint from (18A/18C) of the predicted binding poses of bilorphin (dark grey) (18B), and endomorphin-2 (light grey) (18D), and the positions of the surrounding binding pocket residues (lightest grey) obtained after molecular docking and 1 μs of MD simulations. The salt bridge between protonated amine of the ligands and Asp147.sup.3.32 is marked as a dashed black line. This time TM4 has been removed for clarity.

    [0271] FIG. 19: RMSD plot of [Dmt]-vVf-NH.sub.2 (3c, Bilorphin) (A) and endomorphin-2 (B). FIG. 19A: RMSD calculations performed on the heavy atoms of bilorphin, compared to the initial docked pose (darker grey), and the alpha carbons of the receptor transmembrane domains, compared to the first frame of the MD simulation (lighter grey). FIG. 19B: RMSD calculations performed on the heavy atoms of endomorphin-2, compared to the initial docked pose (lighter grey), and the alpha carbons of the receptor transmembrane domains, compared to the first frame of the MD simulation (grey). Inset: fluctuations of Phe.sup.4 in endomorphin-2 during the MD simulation showing 3 different positions of Phe.sup.4.

    [0272] FIG. 20: FIG. 20A: Ligand-residue interaction fingerprints for the bilorphin-MOPr complex (dark grey) and endomorphin-2-MOPr complex (light grey). Data is expressed as the percentage of simulation time each residue is within 4.5 Å of the ligand, with points radiating outwards from 0% to 100% in 20% increments. FIG. 20B: Principal component analysis was performed on the alpha carbons of the receptor transmembrane domains, before projecting the receptor conformations at each simulation time point onto PC1 and PC2. The bilorphin-MOPr complex is in purple, the endomorphin-2-MOPr complex in orange, and the black point indicates the conformation of the inactive MOPr model to which the peptides were docked.

    [0273] FIG. 21: Extracted structures representing the extremes of PC1 demonstrate the conformational differences between the bilorphin-MOPr complex (dark grey) and the endomorphin-2-MOPr complex (light grey). Loops have been removed from the image to depict only the part of the receptor the PCA was performed on. White arrows indicate conformational changes in the helices moving from bilorphin—bound to endomorphin-2-bound MOPr.

    [0274] FIG. 22: Calculation of the volume of the orthosteric binding site using CASTp showed the binding pocket was larger for the bilorphin-MOPr complex (dark grey) compared to the endomorphin2-MOPr complex (light grey). CASTp calculations were performed on structures averaged over the final 100 ns of each simulation.

    [0275] FIG. 23: Maximal effect of agonists in each signalling and calculation of bias for G-protein activation versus other pathways: Non-normalized maximal efficacy (±S.E.M.) for activation of A, GIRK, B, Ser.sup.371 phosphorylation, C β-arrestin 2 recruitment and D internalization that was used to calculate ratios presented in FIG. 9, and for calculation of Δ Normalized E.sub.Max in E, or included in the operational model in F. Data represented in E and F are mean and 95% confidence intervals. Met-enkephalin is shown in lightest grey, endomorphin 2 in dark grey, morphine in lighter grey and bilorphin (peptide 3c, [Dmt]-vVf-NH.sub.2) is shown in darkest grey.

    [0276] FIG. 24: Antinociceptive action of oral bilactorphin and morphine: FIG. 24A: Time-response (mean±SEM) for oral gavage of [Dmt]-vVf-L-Ser(β-Lac)-NH.sub.2 (3g, Bilactorphin) and morphine on hot-plate latency. Vehicle (large circle); Morphine (circle with +); [Dmt]-vVf-L-Ser(p-Lac)-NH.sub.2 (3g, Bilactorphin) (100 μmol/kg, 6, circle with 1 star (*); 300 μmol/kg, 6, circle with 2 stars (**); 1000 μmol/kg, 6, circle with 3 stars (***)). FIG. 24B: Area under the curve (AUC) of the full time-response data for each animal shown in FIG. 24A for 300 min after gavage. Ordinary one-way ANOVA of AUC data revealed statistically significant differences between all doses of bilactorphin above 100 μmol/kg and morphine 90 μmol/kg.

    [0277] FIG. 25: Structures of bilaids, bilorphin, and bilactorphin, including [0278] L-Phe-D-Val-L-Val-D-Phe (peptide 1a, Bilaid A); [0279] L-Phe-D-Val-L-Val-D-Phe-NH.sub.2 (peptide 1e); [0280] L-Tyr-D-Val-L-Val-D-Phe (peptide 3a, Bilaid C); [0281] L-Tyr-D-Val-L-Val-D-Phe-NH.sub.2 (peptide 3b); [0282] 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-NH.sub.2 (peptide 3c; Bilorphin); and [0283] 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-L-Ser(β-Lac)-NH.sub.2 (peptide 3g; Bilactorphin).

    [0284] FIG. 26: Analogues of Bilaid C. Including the following peptides: [0285] 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-OCH.sub.2CH.sub.3 (peptide 4); [0286] 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-NH.sub.2 wherein the hydroxy group on 2,6-dimethyl-L-tyrosine is substituted with the bio-reversible moiety —C(═O)CH.sub.3 (peptide 5); [0287] 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-OCH.sub.2CH.sub.3 wherein the hydroxy group on 2,6-dimethyl-L-tyrosine is substituted with the bio-reversible moiety —C(═O)CH.sub.3 (peptide 6); [0288] 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-NH.sub.2 wherein the N-terminus is substituted with the bio-reversible moiety —C(═O)OCH.sub.2CH.sub.3 (peptide 7); [0289] 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-NH.sub.2 wherein the N-terminus is substituted with the bio-reversible moiety

    ##STR00038##

    (peptide 8); [0290] 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-NH.sub.2 wherein the N-terminus is substituted with the bio-reversible moiety, ═N═N, to form an N-terminal azido group (peptide 9); [0291] 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-NH.sub.2 wherein the N-terminus is substituted with the bio-reversible moiety —C(═O)OCH.sub.2OC(═O)CH.sub.3 (peptide 10); [0292] 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-L-Pro-L-Asn-L-Leu-L-Ala-L-Glu-L-Lys-L-Ala-L-Leu-L-Lys-L-Ser-L-Leu-NH.sub.2 (peptide 11); [0293] 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-L-Ser(β-Lac)-NH.sub.2 (peptide 3g; Bilactorphin); and [0294] 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-L-Ser(β-D-Glc)-NH.sub.2 (peptide 3h).

    [0295] FIG. 27: Antinociceptive response of the peptides as labelled presented as integrated Area Under the Curve over one hour (AUC response in seconds×time in minutes) for hotplate responses measured, 5, 10, 20, 30 and 60 minutes after subcutaneous injection of each peptide or saline. Asterisks show significantly different AUC response from saline (One way ANOVA with Fisher's LSD post-hoc tests).

    [0296] FIG. 28: Cryo-EM structure and Molecular Dynamics simulations with DAMGO: A. The binding pose of DAMGO in the cryo-EM structure of the MOPr-Gi complex (Koehl Nature (2018) 558: 547-552). DAMGO is shown in dark grey, with surrounding binding pocket residues and the receptor helices in light grey. B. Predicted binding pose of DAMGO after docking with BUDE and 1 μs MD simulation starting from the inactive MOPr structure (Manglik Nature (2012) 485: 321-326.) DAMGO is shown in middle grey and surrounding residues and helices in lighter grey. Koehl Nature (2018) 558: 547-552 reported poor resolution of the C-terminal portion of DAMGO and high flexibility of this region in an MD simulation. With this flexible C-terminal ethanolamine omitted, the RMSD between all heavy atoms of DAMGO in the cryo-EM structure and in our final pose after 1 μs MD was 2.83 Å. Thus the DAMGO-MOPr interactions in the cryo-EM structure and in the model were virtually identical.

    DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

    [0297] The following are embodiments of the invention.

    [0298] Embodiment 1: An isolated peptide comprising Formula I

    ##STR00039##

    wherein, counting from the N-terminus, the first and third amino acid residues are L-amino acid residues and the second and fourth amino acid residues are D-amino acid residues; wherein
    R.sup.1 is hydrogen or a bio-reversible moiety optionally comprising a sugar moiety;
    R.sup.2 is hydrogen or a bio-reversible moiety optionally comprising a sugar moiety; [0299] wherein R.sup.1 and R.sup.2 may together form one bio-reversible moiety;
    R.sup.3 and R.sup.4 are independently selected from hydrogen or C.sub.1-C.sub.3 alkyl, preferably —CH.sub.3;
    R.sup.5 is hydrogen, —OH, or a bio-reversible moiety optionally comprising a sugar moiety;
    R.sup.6 is a side chain of an amino acid or C.sub.1-C.sub.6 alkyl;
    R.sup.7 is a side chain of an amino acid or C.sub.1-C.sub.6 alkyl;
    R.sup.8 is —OH, —NH.sub.2, —O(C.sub.1-C.sub.3 alkyl),

    ##STR00040##

    or 1 to about 30 L-amino acid residues; [0300] Y.sub.1 is —OH, —NH.sub.2, or 1 to about 30 L-amino acid residues; [0301] Y.sub.2 is hydrogen or a sugar moiety, preferably a disaccharide moiety; and [0302] wherein when R.sup.8 is 1 to about 30 L-amino acid residues (1) the L-amino acid residues are optionally residues that may be optionally glycosylated with a sugar moiety, preferably a disaccharide moiety, and (2) the C-terminus is optionally amidated.

    [0303] Embodiment 2: The peptide according to Embodiment 1, wherein R.sup.6 is C.sub.1-C.sub.6 alkyl and R.sup.7 is C.sub.1-C.sub.6 alkyl.

    [0304] Embodiment 3: The peptide according to Embodiment 1 or Embodiment 2, wherein R.sup.6 and R.sup.7 are independently selected from the side chain of alanine, valine, norvaline, leucine, norleucine, or isoleucine.

    [0305] Embodiment 4: The peptide according to any one of Embodiments 1 to 3, wherein R.sup.6 and R.sup.7 are each a valine side chain (—CH(CH.sub.3).sub.2).

    [0306] Embodiment 5: The peptide according to Embodiment 1, wherein R.sup.6 and R.sup.7 are each a threonine side chain.

    [0307] Embodiment 6: The peptide according to any one of Embodiments 1 to 5, wherein R.sup.3 and R.sup.4 are —CH.sub.3; and R.sup.5 is —OH.

    [0308] Embodiment 7: The peptide according to any one of Embodiments 1 to 6, wherein R.sup.1 and R.sup.2 are each hydrogen.

    [0309] Embodiment 8: The peptide according to any one of Embodiments 1 to 5, wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, and R.sup.5 are each hydrogen.

    [0310] Embodiment 9: An isolated peptide comprising Formula I

    ##STR00041##

    wherein, counting from the N-terminus, the first and third amino acid residues are L-amino acid residues and the second and fourth amino acid residues are D-amino acid residues; wherein
    R.sup.1 is hydrogen, single bond, or a —C.sub.1-C.sub.3 alkyl;
    R.sup.2 is hydrogen, single bond, or a —C.sub.1-C.sub.3 alkyl;
    R.sup.3 and R.sup.4 are independently selected from hydrogen or C.sub.1-C.sub.3 alkyl, preferably —CH.sub.3;
    R.sup.5 is hydrogen, —OH, or —O(C.sub.1-C.sub.3)alkyl;
    R.sup.6 is a side chain of an amino acid or C.sub.1-C.sub.6 alkyl;
    R.sup.7 is a side chain of an amino acid or C.sub.1-C.sub.6 alkyl;
    R.sup.8 is —OH, —NH.sub.2, —O(C.sub.1-C.sub.3 alkyl),

    ##STR00042##

    or 1 to about 30 L-amino acid residues; [0311] Y.sub.1 is —OH, —NH.sub.2, or 1 to about 30 L-amino acid residues; [0312] Y.sub.2 is hydrogen or a sugar moiety, preferably a disaccharide moiety;
    wherein when R.sup.8 is 1 to about 30 L-amino acid residues (1) the L-amino acid residues are optionally residues that may be optionally glycosylated with a sugar moiety, preferably a disaccharide moiety, and (2) the C-terminus is optionally amidated;
    wherein when R.sup.8 is a linker, the linker comprises a sugar moiety, preferably a disaccharide moiety such as lactose, and
    wherein when one of R.sup.1 or R.sup.2 is a single bond, one of R.sup.1 and R.sup.2 is hydrogen and the single bond is a peptide bond to an L-amino acid residue that may optionally be N-terminally alkylated, preferably singly methylated.

    [0313] Embodiment 10: The peptide according to Embodiment 9, wherein one of R.sup.1 and R.sup.2 is hydrogen and one of R.sup.1 and R.sup.2 is —CH.sub.3.

    [0314] Embodiment 11: The peptide according to Embodiment 9 or Embodiment 10, wherein R.sup.5 is —O(C.sub.1-C.sub.3)alkyl, preferably —OCH.sub.3.

    [0315] Embodiment 12: The peptide according to Embodiment 11, wherein R.sup.3 and R.sup.4 are —CH.sub.3.

    [0316] Embodiment 13: The peptide according to Embodiment 11, wherein R.sup.3 and R.sup.4 are hydrogen.

    [0317] Embodiment 14: The peptide according to Embodiment 9 or Embodiment 10, wherein R.sup.3 and R.sup.4 are —CH.sub.3 and R.sup.5 is —OH.

    [0318] Embodiment 15: The peptide according to any one of Embodiments 9 to 14, wherein R.sup.6 and R.sup.7 are each a valine side chain (—CH(CH.sub.3).sub.2).

    [0319] Embodiment 16: The peptide according to any one of Embodiments 9 to 14, wherein R.sup.6 and R.sup.7 are each a threonine side chain.

    [0320] Embodiment 17: The peptide according to any one of Embodiments 9 to 16, wherein one of R.sup.1 or R.sup.2 is a single bond, one of R.sup.1 and R.sup.2 is a hydrogen, and the single bond is a peptide bond to an L-amino acid residue.

    [0321] Embodiment 18: The peptide according to Embodiment 17, wherein the L-amino acid residue has at least one N-terminal methylation.

    [0322] Embodiment 19: The peptide according to Embodiment 17 or Embodiment 18, wherein the L-amino acid residue is an L-alanine residue.

    [0323] Embodiment 20: The peptide according to any one of Embodiments 9 to 19, wherein R.sup.8 is a linker.

    [0324] Embodiment 21: The peptide according to Embodiment 20, wherein the linker comprises an amino acid based linker, peptide based linker, an amino acid comprising linker, and/or maleimide based linker, and/or a combination thereof.

    [0325] Embodiment 22: The peptide according to any one of Embodiments 1 to 19, wherein R.sup.8 is —OH, —NH.sub.2, —O(C.sub.1-C.sub.3 alkyl),

    ##STR00043## [0326] Y.sub.1 is —OH, —NH.sub.2, or 1 to about 30 L-amino acid residues; and [0327] Y.sub.2 is hydrogen or a sugar moiety, preferably a disaccharide moiety.

    [0328] Embodiment 23: The peptide according to Embodiment 22, wherein R.sup.8 is

    ##STR00044## [0329] Y.sub.1 is OH NH.sub.2, or 1 to about 30 L-amino acid residues and [0330] Y.sub.2 is hydrogen or a sugar moiety, preferably a disaccharide moiety.

    [0331] Embodiment 24: The peptide according to Embodiment 22 or Embodiment 23, wherein Y.sub.1—NH.sub.2.

    [0332] Embodiment 25: The peptide according to Embodiment 22 or Embodiment 23, wherein Y.sub.1 is 1 to about 30 L-amino acid residues.

    [0333] Embodiment 26: The peptide according to Embodiment 25, wherein Y.sub.1 is 1 to about 25 L-amino acid residues, 1 to about 20 L-amino acid residues, 1 to about 15 L-amino acid residues, 1 to about 11 L-amino acid residues, or 1 to about 5 L-amino acid residues.

    [0334] Embodiment 27: The peptide according to Embodiment 26, wherein Y.sup.1 is 1 to about 11 L-amino acid residues.

    [0335] Embodiment 28: The peptide according to any one of Embodiments 1 to 19, wherein R.sup.8 is 1 to about 30 L-amino acid residues.

    [0336] Embodiment 29: The peptide according to Embodiment 28, wherein R.sup.8 is 1 to about 25 L-amino acid residues, 1 to about 20 L-amino acid residues, 1 to about 15 L-amino acid residues, 1 to about 11 L-amino acid residues, or 1 to about 5 L-amino acid residues.

    [0337] Embodiment 30: The peptide according to Embodiment 29, wherein R.sup.8 is 1 to about 11 L-amino acid residues.

    [0338] Embodiment 31: The peptide according to Embodiment 30, wherein the 1 to about 11 L-amino acid residues comprise at least one glycosylated L-amino acid residue, preferably comprising at least one O-glycosylated L-serine residue.

    [0339] Embodiment 32: The peptide according to Embodiment 1, wherein R.sup.1 and R.sup.2 are hydrogen; R.sup.3, R.sup.4, and R.sup.5 are hydrogen; R.sup.6 and R.sup.7 are each —CH(CH.sub.3).sub.2; and R.sup.8 is —OH, —NH.sub.2, —O(C.sub.1-C.sub.3 alkyl),

    ##STR00045## [0340] Y.sub.1 is —OH, —NH.sub.2, or 1 to about 30 L-amino acid residues; and [0341] Y.sub.2 is hydrogen or a sugar moiety, preferably a disaccharide moiety.

    [0342] Embodiment 33: The peptide according to Embodiment 1, wherein R.sup.1 and R.sup.2 are hydrogen; R.sup.3 and R.sup.4 are both hydrogen or both —CH.sub.3; R.sup.5 is —OH; R.sup.6 and R.sup.7 are each —CH(CH.sub.3).sub.2; and R.sup.8 is —OH, —NH.sub.2, —O(C.sub.1-C.sub.3 alkyl),

    ##STR00046## [0343] Y.sub.1 is —OH, —NH.sub.2, or 1 to about 30 L-amino acid residues; [0344] Y.sub.2 is hydrogen or a sugar moiety, preferably a disaccharide moiety.

    [0345] Embodiment 34: The peptide according to Embodiment 32 or Embodiment 33, wherein R.sup.8 is —NH.sub.2,

    ##STR00047## [0346] Y.sub.1 is —OH, —NH.sub.2, or 1 to about 30 L-amino acid residues; and [0347] Y.sub.2 is hydrogen or a sugar moiety, preferably a disaccharide moiety.

    [0348] Embodiment 35: The peptide according to any one of Embodiments 32 to 34, wherein R.sup.8 is

    ##STR00048## [0349] Y.sub.1 is —OH or —NH.sub.2; and [0350] Y.sub.2 is hydrogen or a sugar moiety, preferably a disaccharide moiety.

    [0351] Embodiment 36: The peptide according to any one of Embodiments 32 to 34, wherein Y.sub.1 is 1 to about 25 L-amino acid residues, 1 to about 20 L-amino acid residues, 1 to about 15 L-amino acid residues, 1 to about 11 L-amino acid residues, or 1 to about 5 L-amino acid residues.

    [0352] Embodiment 37: The peptide according to Embodiment 36, wherein Y.sup.1 is 1 to about 11 L-amino acid residues.

    [0353] Embodiment 38: The peptide according to any one of Embodiments 1 to 19, 22, 23, and 32 to 35, wherein Y.sub.2 is a sugar moiety, preferably a disaccharide moiety.

    [0354] Embodiment 39: The peptide according to Embodiment 38, wherein Y.sub.2 the disaccharide moiety is a lactose moiety or melibiose moiety.

    [0355] Embodiment 40: The peptide according to Embodiment 38, wherein Y.sub.2 the disaccharide moiety is a lactose moiety.

    [0356] Embodiment 41: The peptide according to Embodiment 39 or Embodiment 40, wherein the disaccharide moiety is attached through a beta linkage.

    [0357] Embodiment 42: The peptide according to Embodiment 1, wherein R.sup.8 is 1 to about 25 L-amino acid residues, 1 to about 20 L-amino acid residues, 1 to about 15 L-amino acid residues, 1 to about 11 L-amino acid residues, or 1 to about 5 L-amino acid residues.

    [0358] Embodiment 43: The peptide according to Embodiment 42, wherein R.sup.8 is 1 to about 11 L-amino acid residues.

    [0359] Embodiment 44: The peptide according to Embodiment 42 or Embodiment 43, wherein said L-amino acid residues comprises at least one amino acid residue that is N-glycosylated, O-glycosylated, C-glycosylated, S-glycosylated, or Se-glycosylated.

    [0360] Embodiment 45: The peptide according to Embodiment 44, wherein said L-amino acid residues comprise at least one L-amino acid residue that is O-glycosylated.

    [0361] Embodiment 46. The peptide according to Embodiment 45, wherein said amino acid residue that is O-glycosylated is an L-serine residue.

    [0362] Embodiment 47: The peptide according to any one of Embodiments 1 to 6, 22 to 31, and 38 to 46, wherein R.sup.1 and R.sup.2 together form a bio-reversible moiety.

    [0363] Embodiment 48: The peptide according to Embodiment 47, wherein said bio-reversible moiety is

    ##STR00049##

    or ═N═N (azido moiety).

    [0364] Embodiment 49: The peptide according to any one of Embodiments 1 to 6, 22 to 31, and 38 to 46, wherein one of R.sup.1 or R.sup.2 is hydrogen and one of R.sup.1 or R.sup.2 is —C(═O)OCH.sub.2CH.sub.3 or —C(═O)OCH.sub.2OC(═O)CH.sub.3.

    [0365] Embodiment 50: The peptide according to any one of Embodiments 1 to 5, 7, 22 to 31, and 38 to 46, wherein R.sup.5 is a bio-reversible moiety.

    [0366] Embodiment 51: The peptide according to Embodiment 50, wherein the bio-reversible moiety is —C(═O)CH.sub.3.

    [0367] Embodiment 52: The peptide according to Embodiment 1, selected from the group consisting of: [0368] L-Phe-D-Val-L-Val-D-Phe (peptide 1a, Bilaid A); [0369] L-Phe-D-Val-L-Val-D-Phe-NH.sub.2 (peptide 1e); [0370] L-Tyr-D-Val-L-Val-D-Phe (peptide 3a, Bilaid C); [0371] L-Tyr-D-Val-L-Val-D-Phe-NH.sub.2 (peptide 3b); [0372] 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-NH.sub.2 (peptide 3c; Bilorphin); [0373] 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-L-Ser(β-Lac)-NH.sub.2 (peptide 3g; Bilactorphin); [0374] 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-OCH.sub.2CH.sub.3 (peptide 4); [0375] 2,6-dimethyl-L-tyrosine-D-Va-L-Val-D-Phe-L-Pro-L-Asn-L-Leu-L-Aa-L-Glu-L-Lys-L-AIa-L-Leu-L-Lys-L-Ser-L-Leu-NH.sub.2 (peptide 11); [0376] 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-NH.sub.2 wherein the N-terminus is substituted with the bio-reversible moiety —C(═O)OCH.sub.2OC(═O)CH.sub.3 (peptide 10); [0377] 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-NH.sub.2 wherein the N-terminus is substituted with the bio-reversible moiety

    ##STR00050##

    (peptide 8); [0378] 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-NH.sub.2 wherein the hydroxy group on 2,6-dimethyl-L-tyrosine is substituted with the bio-reversible moiety —C(═O)CH.sub.3 (peptide 5); [0379] 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-OCH.sub.2CH.sub.3 wherein the hydroxy group on 2,6-dimethyl-L-tyrosine is substituted with the bio-reversible moiety —C(═O)CH.sub.3 (peptide 6); [0380] 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-NH.sub.2 wherein the N-terminus is substituted with the bio-reversible moiety —C(═O)OCH.sub.2CH.sub.3 (peptide 7); and [0381] 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-NH.sub.2 wherein the N-terminus is substituted with the bio-reversible moiety, ═N═N, to form an N-terminal azido group (peptide 9).

    [0382] Embodiment 53: A peptide comprising [0383] L-AA-L-Tyr-D-Val-L-Val-D-Phe-linker-sugar moiety; [0384] L-AA-L-Tyr-D-Thr-L-Thr-D-Phe-linker-sugar moiety; [0385] L-AA-L-Dmt-D-Val-L-Val-D-Phe-linker-sugar moiety; [0386] L-AA-L-Dmt-D-Thr-L-Thr-D-Phe-linker-sugar moiety; [0387] wherein L-AA is any L-amino acid residue optionally with at least one N-terminal —CH.sub.3; [0388] wherein the hydroxyl group of L-Tyr or L-Dmt is optionally alkylated; and [0389] wherein the linker is preferably L-Ser or L-Thr.

    [0390] Embodiment 54: The peptide according to any one of Embodiments 1 to 53, wherein said peptide, in comparison to morphine: [0391] (a) exhibits a lower ratio of induction of C-terminal phosphorylation of MOPr versus G-protein activation; and/or [0392] (b) exhibits a lower ratio of induction of β-arrestin recruitment versus G-protein activation; and/or [0393] (c) exhibits a lower ratio of induction of MOPr internalisation versus G-protein activation.

    [0394] Embodiment 55: The peptide according to any one of Embodiments 1 to 53, wherein said peptide, in comparison to morphine, exhibits a lower ratio of induction of β-arrestin recruitment versus G-protein activation.

    [0395] Embodiment 56: The peptide according to any one of Embodiments 1 to 55, wherein said peptide exhibits an increase in inhibition of cAMP formation in comparison to vehicle at a concentration of about 10 μM in an assay using hMOPr.

    [0396] Embodiment 57: The peptide according to any one of Embodiments 1 to 56, wherein said peptide in a competitive binding assay using [.sup.3H]DAMGO exhibits a K.sub.i of less than about 5 μM, less than about 3.5 μM, less than about 1 μM, less than about 0.8 μM, less than about 0.5 μM, or less than about 0.3 μM.

    [0397] Embodiment 58: The peptide according to Embodiment 57, wherein said peptide exhibits a K.sub.i of less than about 0.5 μM or less than about 0.3 μM.

    [0398] Embodiment 59: The peptide of any one of Embodiments 1 to 58, wherein said peptide crosses the blood brain barrier.

    [0399] Embodiment 60: A pharmaceutical composition comprising a peptide according to any one of Embodiments 1 to 59 and at least one pharmaceutical excipient.

    [0400] Embodiment 61: The pharmaceutical composition according to Embodiment 60, wherein said composition is formulated for oral administration.

    [0401] Embodiment 62: The pharmaceutical composition according to Embodiment 60, wherein said peptide is glycosylated and said composition is formulated for oral administration, administration by injection, or intrathecal administration.

    [0402] Embodiment 63: The pharmaceutical composition according to Embodiment 60, wherein said peptide is not glycosylated and said composition is formulated for nasal administration or intrathecal administration.

    [0403] Embodiment 64: A method of treating pain comprising administering to a subject a peptide according to any one of Embodiments 1 to 59 or the pharmaceutical composition according to any one of Embodiments 60 to 63.

    [0404] Embodiment 65: The method of Embodiment 64, wherein the pain the pain is post-operative pain, pain associated with nerve damage, pain associated with bone fracture, pain associated with a burn, or pain associated with a wound.

    [0405] Embodiment 66: Use of a peptide according to any one of Embodiments 1 to 59 or the pharmaceutical composition according to any one of Embodiments 60 to 63 in the manufacture of a medicament for treating pain.

    [0406] Embodiment 67: The use of Embodiment 66, wherein the pain the pain is post-operative pain, pain associated with nerve damage, pain associated with bone fracture, pain associated with a burn, or pain associated with a wound.

    [0407] Embodiment 68: A peptide according to any one of Embodiments 1 to 59 for use in a method of treating pain.

    [0408] Embodiment 69: A pharmaceutical composition according to any one of Embodiments 60 to 63 for use in a method of treating pain.

    [0409] Embodiment 70: The peptide for use in the method of Embodiment 68 or the pharmaceutical composition for use in the method of Embodiment 69 wherein the pain is post-operative pain, pain associated with nerve damage, pain associated with bone fracture, pain associated with a burn, or pain associated with a wound.

    [0410] Embodiment 71: A method of delivering analgesia comprising administering to a subject a peptide according to any one of Embodiments 1 to 59 or the pharmaceutical composition according to any one of Embodiments 60 to 63.

    [0411] Embodiment 72: Use of peptide according to any one of Embodiments 1 to 59 or the pharmaceutical composition according to any one of Embodiments 60 to 63 for the manufacture of a medicament for delivering analgesia.

    [0412] Embodiment 73: A peptide according to any one of Embodiments 1 to 59 or the pharmaceutical composition according to any one of Embodiments 60 to 63 for use in a method of delivering analgesia.

    [0413] Embodiment 74: A method of treating pain or delivering analgesia with reduced adverse side effect(s), preferably reduced in comparison to morphine, comprising administering to a subject a peptide according to any one of Embodiments 1 to 59 or the pharmaceutical composition according to any one of Embodiments 60 to 63.

    [0414] Embodiment 75: The method according to Embodiment 74, wherein said adverse side effect(s) is gastrointestinal (GI) inhibition and/or respiratory depression.

    [0415] Embodiment 76: Use of peptide according to any one of Embodiments 1 to 59 or the pharmaceutical composition according to any one of Embodiments 60 to 63 for the manufacture of a medicament for a method of treating pain or delivering analgesia with reduced adverse side effect(s), preferably reduced in comparison to morphine.

    [0416] Embodiment 77: The use according to Embodiment 76, wherein said adverse side effect(s) is gastrointestinal (GI) inhibition and/or respiratory depression.

    [0417] Embodiment 78: A peptide according to any one of Embodiments 1 to 59 or the pharmaceutical composition according to any one of Embodiments 60 to 63 for use in a method of treating pain or delivering analgesia with reduced adverse side effect(s), preferably reduced in comparison to morphine.

    [0418] Embodiment 79: The peptide for use in the method of Embodiment 78 or the pharmaceutical composition for use in the method of Embodiment 78, wherein said adverse side effect(s) is gastrointestinal (GI) inhibition and/or respiratory depression.

    [0419] Embodiment 80: A peptide comprising Formula II

    ##STR00051##

    wherein, counting from the N-terminus, the first amino acid residue is an L-amino acid residue and the second and fourth amino acid residues are D-amino acid residues; wherein
    R.sup.9 is hydrogen or a bio-reversible moiety optionally comprising a sugar moiety;
    R.sup.10 is hydrogen or a bio-reversible moiety optionally comprising a sugar moiety; [0420] wherein when one of R.sup.9 or R.sup.10 is a hydrogen and one of R.sup.9 or R.sup.10 is a bio-reversible moiety, the bio-reversible moiety is preferably —C(═O)OCH.sub.2CH.sub.3 or —C(═O)OCH.sub.2OC(═O)CH.sub.3; [0421] wherein R.sup.9 and R.sup.10 may together form one bio-reversible moiety, wherein preferably the bio-reversible moiety is

    ##STR00052##  or ═N═N (azido moiety);
    R.sup.11 and R.sup.12 are independently selected from hydrogen or C.sub.1-C.sub.3 alkyl, preferably —CH.sub.3;
    R.sup.13 is hydrogen, —OH, or a bio-reversible moiety optionally comprising a sugar moiety;
    R.sup.14 is a side chain of an amino acid or C.sub.1-C.sub.6 alkyl, preferably C.sub.1-C.sub.4 alkyl, more preferably —CH(CH.sub.3).sub.2;
    R.sup.15 is hydrogen, —OH, or a bio-reversible moiety; and
    R.sup.16 is —OH, —O(C.sub.1-C.sub.3 alkyl), —NH.sub.2,

    ##STR00053##

    or 1 to about 30 L-amino acid residues; [0422] Y.sub.3 is —OH, —NH.sub.2, or 1 to about 30 L-amino acid residues; [0423] Y.sub.4 is hydrogen or a sugar moiety, preferably a disaccharide moiety; and [0424] wherein when R.sup.16 is 1 to about 30 L-amino acid residues (1) the L-amino acid residues are optionally residues that may be optionally glycosylated with a sugar moiety, preferably a disaccharide moiety, and (2) the C-terminus is optionally amidated.

    [0425] Embodiment 81: The peptide according to Embodiment 80, wherein R.sup.14 is C.sub.1-C.sub.6 alkyl.

    [0426] Embodiment 82: The peptide according to Embodiment 80 or Embodiment 81, wherein R.sup.14 is selected from the side chain of alanine, valine, norvaline, leucine, norleucine, or isoleucine.

    [0427] Embodiment 83: The peptide according to any one of Embodiments 80 to 82, wherein R.sup.14 is a valine side chain (—CH(CH.sub.3).sub.2).

    [0428] Embodiment 84: The peptide according to Embodiment 80, wherein R.sup.14 is a threonine side chain.

    [0429] Embodiment 85: The peptide according to any one of Embodiments 80 to 84, wherein R.sup.11 and R.sup.12 are —CH.sub.3; and R.sup.13 is —OH.

    [0430] Embodiment 86: The peptide according to any one of Embodiments 80 to 84, wherein R.sup.9 and R.sup.10 are each hydrogen.

    [0431] Embodiment 87: The peptide according to any one of Embodiments 80 to 84, wherein R.sup.9, R.sup.10, R.sup.11, R.sup.12, and R.sup.13 are each hydrogen.

    [0432] Embodiment 88: The peptide according to any one of Embodiments 80 to 84, wherein R.sup.9, R.sup.10, R.sup.11, R.sup.12, and R.sup.13 are hydrogen; R.sup.14 is C.sub.1-C.sub.4 alkyl; R.sup.15 is —OH.

    [0433] Embodiment 89: A peptide comprising Formula II

    ##STR00054##

    wherein, counting from the N-terminus, the first amino acid residue is an L-amino acid residue and the second and fourth amino acid residues are D-amino acid residues; wherein
    R.sup.9 is hydrogen, a single bond, or —C.sub.1-C.sub.3 alkyl, preferably —CH.sub.3;
    R.sup.10 is hydrogen, a single bond, or —C.sub.1-C.sub.3 alkyl, preferably —CH.sub.3;
    R.sup.11 and R.sup.12 are independently selected from hydrogen or C.sub.1-C.sub.3 alkyl, preferably —CH.sub.3;
    R.sup.13 is hydrogen, —OH, or —O(C.sub.1-C.sub.3)alkyl;
    R.sup.14 is a side chain of an amino acid or C.sub.1-C.sub.6 alkyl, preferably C.sub.1-C.sub.4 alkyl, more preferably —CH(CH.sub.3).sub.2;
    R.sup.15 is hydrogen, —OH, or a bio-reversible moiety; and
    R.sup.16 is —OH, —O(C.sub.1-C.sub.3 alkyl), —NH.sub.2,

    ##STR00055##

    1 to about 30 L-amino acid residues, or a linker; [0434] Y.sub.3 is —OH, —NH.sub.2, or 1 to about 30 L-amino acid residues; [0435] Y.sub.4 is hydrogen or a sugar moiety, preferably a disaccharide moiety;
    wherein when R.sup.16 is 1 to about 30 L-amino acid residues (1) the L-amino acid residues are optionally residues that may be optionally glycosylated with a sugar moiety, preferably a disaccharide moiety, and (2) the C-terminus is optionally amidated;
    wherein when R.sup.16 is a linker, the linker comprises a sugar moiety, preferably a disaccharide moiety such as lactose, and
    wherein when one of R.sup.9 or R.sup.10 is a single bond, one of R.sup.9 or R.sup.10 is hydrogen and the single bond is a peptide bond to an L-amino acid residue optionally N-terminally alkylated, preferably singly methylated.

    [0436] Embodiment 90: The peptide according to Embodiment 89, wherein one of R.sup.9 and R.sup.1 is hydrogen and one of R.sup.9 and R.sup.10 is —OH.sub.3.

    [0437] Embodiment 91: The peptide according to Embodiment 89 or Embodiment 90, wherein R.sup.13 is —O(C.sub.1-C.sub.3)alkyl, preferably —OCH.sub.3.

    [0438] Embodiment 92: The peptide according to Embodiment 91, wherein R.sup.11 and R.sup.12 are —OH.sub.3.

    [0439] Embodiment 93: The peptide according to Embodiment 91, wherein R.sup.11 and R.sup.12 are hydrogen.

    [0440] Embodiment 94: The peptide according to Embodiment 89 or Embodiment 90, wherein R.sup.11 and R.sup.12 are —CH.sub.3; and R.sup.13 is —OH.

    [0441] Embodiment 95: The peptide according to Embodiment 89, wherein one of R.sup.9 or R.sup.10 is a single bond, one of R.sup.9 or R.sup.10 is hydrogen, and the single bond is a peptide bond to an L-amino acid residue.

    [0442] Embodiment 96: The peptide according to Embodiment 95, wherein the L-amino acid residue has at least one N-terminal methylation.

    [0443] Embodiment 97: The peptide according to Embodiment 95 or Embodiment 96, wherein the L-amino acid residue is an L-alanine residue.

    [0444] Embodiment 98: The peptide according to any one of Embodiments 89 to 97, wherein R.sup.14 is a valine side chain (—CH(CH.sub.3).sub.2).

    [0445] Embodiment 99: The peptide according to any one of Embodiments 89 to 97, wherein R.sup.14 is threonine side chain.

    [0446] Embodiment 100: The peptide according to any one of Embodiments 80 to 99, wherein R.sup.16 is —NH.sub.2.

    [0447] Embodiment 101: The peptide according to any one of Embodiments 80 to 99, wherein

    [0448] R.sup.16 is

    ##STR00056##

    or 1 to about 30 L-amino acid residues; [0449] Y.sub.3 is —OH, —NH.sub.2, or 1 to about 30 L-amino acid residues; [0450] Y.sub.4 is hydrogen or a sugar moiety, preferably a disaccharide moiety; and [0451] wherein when R.sup.8 is 1 to about 30 L-amino acid residues (1) the L-amino acid residues are optionally residues that may be optionally glycosylated with a sugar moiety, preferably a disaccharide moiety, and (2) the C-terminus is optionally amidated.

    [0452] Embodiment 102: The peptide according to Embodiment 101, wherein R.sup.16 is 1 to about 30 L-amino acids.

    [0453] Embodiment 103: The peptide of Embodiment 102, wherein R.sup.16 is 1 to about 25 L-amino acid residues, 1 to about 20 L-amino acid residues, 1 to about 15 L-amino acid residues, 1 to about 11 L-amino acid residues, or 1 to about 5 L-amino acid residues.

    [0454] Embodiment 104: The peptide according to Embodiment 103, wherein R.sup.16 is 1 to about 11 L-amino acid residues.

    [0455] Embodiment 105: The peptide according to any one of Embodiments 102 to 104, wherein said L-amino acid residues comprises at least one amino acid residue that is N-glycosylated, O-glycosylated, C-glycosylated, S-glycosylated, or Se-glycosylated.

    [0456] Embodiment 106: The peptide according to Embodiment 105, wherein said L-amino acid residues comprise at least one amino acid residue that is O-glycosylated.

    [0457] Embodiment 107: The peptide according to Embodiment 106, wherein said amino acid residue that is O-glycosylated is an L-serine residue.

    [0458] Embodiment 108: The peptide according to any one of Embodiments 89 to 99, wherein R.sup.16 is a linker.

    [0459] Embodiment 109: The peptide according to Embodiment 108, wherein the linker comprises an amino acid based linker, peptide based linker, an amino acid comprising linker, and/or maleimide based linker, and/or a combination thereof.

    [0460] Embodiment 110: The peptide according to Embodiment 101, wherein

    R.SUP.16 .is

    [0461] ##STR00057## [0462] Y.sub.3 is —OH, —NH.sub.2, or 1 to about 30 L-amino acid residues; and [0463] Y.sub.4 is hydrogen or a sugar moiety, preferably a disaccharide moiety.

    [0464] Embodiment 111: The peptide according to any one of Embodiments 80 to 99, wherein

    R.SUP.16 .is

    [0465] ##STR00058##

    Y.sub.3 is —OH or —NH.sub.2; and Y.sub.4 is hydrogen or a sugar moiety, preferably a disaccharide moiety.

    [0466] Embodiment 112: The peptide according Embodiment 80, wherein R.sup.9, R.sup.10, R.sup.11, R.sup.12, and R.sup.13 are hydrogen; R.sup.14 is —CH(CH.sub.3).sub.2; R.sup.15 is —OH; and [0467] R.sup.16 is

    ##STR00059## [0468] Y.sub.3 is —OH or —NH.sub.2; and Y.sub.4 is hydrogen or a sugar moiety, preferably a disaccharide moiety.

    [0469] Embodiment 113: The peptide according to any one of Embodiments 80 to 99, 101, 110, 111, and 112, wherein said sugar moiety is a disaccharide moiety, preferably wherein the disaccharide moiety is attached through a beta linkage.

    [0470] Embodiment 114: The peptide according to Embodiment 113, wherein said disaccharide moiety is a lactose moiety or a melibiose moiety, preferably wherein the disaccharide moiety is attached through a beta linkage.

    [0471] Embodiment 115: The peptide according to Embodiment 113 or Embodiment 114, wherein said disaccharide moiety is a lactose moiety, preferably wherein the lactose moiety is attached through a beta linkage.

    [0472] Embodiment 116: The peptide according to any one of Embodiments 80 to 85, wherein R.sup.9 and R.sup.10 together form a bio-reversible moiety.

    [0473] Embodiment 117: The peptide according to Embodiment 116, wherein said bio-reversible moiety is

    ##STR00060##

    or ═N═N (azido moiety).

    [0474] Embodiment 118: The peptide according to any one of Embodiments 80 to 85, wherein one of R.sup.9 or R.sup.10 is hydrogen and one of R.sup.9 or R.sup.10 is —C(═O)OCH.sub.2CH.sub.3 or —C(═O)OCH.sub.2OC(═O)CH.sub.3.

    [0475] Embodiment 119: The peptide according to any one of Embodiments 80 to 85, wherein R.sup.13 is a bio-reversible moiety.

    [0476] Embodiment 120: The peptide according to Embodiment 119, wherein the bio-reversible moiety is —C(═O)CH.sub.3.

    [0477] Embodiment 121: The peptide according to Embodiment 80 which is L-Phe-D-Val-Gly-D-Tyr-NH.sub.2.

    [0478] Embodiment 122: The peptide of any one of Embodiments 80 to 121 wherein said peptide in comparison to morphine: [0479] (a) exhibits a lower ratio of induction of C-terminal phosphorylation of MOPr versus G-protein activation; and/or [0480] (b) exhibits a lower ratio of induction of β-arrestin recruitment versus G-protein activation; and/or [0481] (c) exhibits a lower ratio of induction of MOPr internalisation versus G-protein activation.

    [0482] Embodiment 123: The peptide of any one of Embodiments 80 to 121, wherein said peptide, in comparison to morphine, exhibits a lower ratio of induction of β-arrestin recruitment versus G-protein activation.

    [0483] Embodiment 124: The peptide of any one of Embodiments 80 to 123, wherein said peptide exhibits an increase in inhibition of cAMP formation in comparison to vehicle at a concentration of about 10 μM in an assay using hMOPr.

    [0484] Embodiment 125: The peptide of any one of Embodiments 80 to 124, wherein said peptide in a competitive binding assay using [.sup.3H]DAMGO exhibits a K.sub.i of less than about 5 μM, less than about 3.5 μM, less than about 1 μM, less than about 0.8 μM, less than about 0.5 μM, or less than about 0.3 μM.

    [0485] Embodiment 126: The peptide of Embodiment 125, wherein said peptide exhibits a K.sub.i of less than about 0.5 μM or less than about 0.3 μM.

    [0486] Embodiment 127: The peptide of any one of Embodiments 80 to 126, wherein said peptide crosses the blood brain barrier.

    [0487] Embodiment 128: A pharmaceutical composition comprising a peptide according to any one of Embodiments 80 to 127 and at least one pharmaceutical excipient.

    [0488] Embodiment 129: The pharmaceutical composition according to Embodiment 128, wherein said composition is formulated for oral administration.

    [0489] Embodiment 130: The pharmaceutical composition according to Embodiment 128, wherein said peptide is glycosylated and said composition is formulated for oral administration, administration by injection, or intrathecal administration.

    [0490] Embodiment 131: The pharmaceutical composition according to Embodiment 128, wherein said peptide is not glycosylated and said composition is formulated for nasal administration or intrathecal administration.

    [0491] Embodiment 132: A method of treating pain comprising administering to a subject a peptide according to any one of Embodiments 80 to 127 or the pharmaceutical composition according to any one of Embodiments 128 to 131.

    [0492] Embodiment 133: The method of Embodiment 132, wherein the pain is post-operative pain, pain associated with nerve damage, pain associated with bone fracture, pain associated with a burn, or pain associated with a wound.

    [0493] Embodiment 134: Use of a peptide according to any one of Embodiments 80 to 127 or the pharmaceutical composition according to any one of Embodiments 128 to 131 in the manufacture of a medicament for treating pain.

    [0494] Embodiment 135: The use of Embodiment 134, wherein the pain is post-operative pain, pain associated with nerve damage, pain associated with bone fracture, pain associated with a burn, or pain associated with a wound.

    [0495] Embodiment 136: A peptide according to any one of Embodiments 80 to 127 for use in a method of treating pain.

    [0496] Embodiment 137: A pharmaceutical composition according to any one of Embodiments 128 to 131 for use in a method of treating pain.

    [0497] Embodiment 138: The peptide for use in the method of Embodiment 136 or the pharmaceutical composition for use in the method of Embodiment 137 wherein the pain is post-operative pain, pain associated with nerve damage, pain associated with bone fracture, pain associated with a burn, or pain associated with a wound.

    [0498] Embodiment 139: A method of delivering analgesia comprising administering to a subject a peptide according to any one of Embodiments 80 to 127 or the pharmaceutical composition according to any one of Embodiments 128 to 131.

    [0499] Embodiment 140: Use of peptide according to any one of Embodiments 80 to 127 or the pharmaceutical composition according to any one of Embodiments 128 to 131 for the manufacture of a medicament for delivering analgesia.

    [0500] Embodiment 141: A peptide according to any one of Embodiments 80 to 127 or the pharmaceutical composition according to any one of Embodiments 128 to 131 for use in a method of delivering analgesia.

    [0501] Embodiment 142: A method of treating pain or delivering analgesia with reduced adverse side effect(s), preferably reduced in comparison to morphine, comprising administering to a subject a peptide according to any one of Embodiments 80 to 127 or the pharmaceutical composition according to any one of Embodiments 128 to 131.

    [0502] Embodiment 143: The method according to Embodiment 142, wherein said adverse side effect(s) is gastrointestinal (GI) inhibition and/or respiratory depression.

    [0503] Embodiment 144: Use of peptide according to any one of Embodiments 80 to 127 or the pharmaceutical composition according to any one of Embodiments 128 to 131 for the manufacture of a medicament for treating pain or delivering analgesia with reduced adverse side effect(s), preferably reduced in comparison to morphine.

    [0504] Embodiment 145: The use according to Embodiment 144, wherein said adverse side effect(s) is gastrointestinal (GI) inhibition and/or respiratory depression.

    [0505] Embodiment 146: A peptide according to any one of Embodiments 80 to 127 or the pharmaceutical composition according to any one of Embodiments 128 to 131 for use in a method of treating pain or delivering analgesia with reduced adverse side effect(s), preferably reduced in comparison to morphine.

    [0506] Embodiment 147: The peptide for use in the method of Embodiment 146 or the pharmaceutical composition for use in the method of Embodiment 146, wherein said adverse side effect(s) is gastrointestinal (GI) inhibition and/or respiratory depression.

    [0507] Embodiment 148: An isolated peptide comprising Formula III


    X.sup.1—X.sup.2—X.sup.3—X.sup.4  (III)

    wherein:
    X.sup.1 is the N-terminal amino acid residue comprising an N-terminal moiety —NR.sup.17R.sup.18;
    X.sup.4 comprises a C-terminal moiety selected from —C(═O)OH, —C(═O)O(C.sub.1-C.sub.3 alkyl), —C(═O)NH.sub.2,

    ##STR00061##

    wherein Y.sub.5 is —OH or —NH.sub.2, and Y.sub.6 is hydrogen or a sugar moiety, preferably a disaccharide moiety;
    X.sup.1 is an L-amino acid residue selected from L-tyrosine, 2,6-dimethyl-L-tyrosine, or L-phenylalanine, wherein when X.sup.1 is L-tyrosine or 2,6-dimethyl-L-tyrosine, the residue is optionally O-substituted at the 4-position with a bio-reversible moiety optionally comprising a sugar moiety;
    X.sup.2 is a D-amino acid residue, preferably D-alanine, D-valine, D-leucine, or D-isoleucine, more preferably, D-valine;
    X.sup.3 is glycine or an L-amino acid residue, wherein when X.sup.3 is an L-amino acid residue, X.sup.3 is preferably L-alanine, L-valine, L-leucine, or L-isoleucine, more preferably L-valine;
    X.sup.4 is a D-amino acid residue selected from D-tyrosine or D-phenylalanine, wherein when X.sup.4 is D-tyrosine, the residue is optionally O-substituted with a bio-reversible moiety;
    R.sup.17 and R.sup.18 are independently selected from hydrogen or a bio-reversible moiety optionally comprising a sugar moiety, or R.sup.17 and R.sup.18 together form a bio-reversible moiety optionally comprising a sugar moiety; and
    wherein the peptide is a MOPr agonist.

    [0508] Embodiment 149: The peptide according to Embodiment 148, wherein the C-terminal moiety is —C(═O)OH,

    ##STR00062##

    and Y.sub.5 is —OH and Y.sub.6 is hydrogen or a sugar moiety; the peptide further comprises about 5, 8, 11, 12, 20 or 26 additional L-amino acid residues on the C-terminus.

    [0509] Embodiment 150: The peptide according to Embodiment 148 or Embodiment 149, wherein R.sup.17 and R.sup.18 are each hydrogen, X.sup.2 is a D-valine residue, X.sup.3 is glycine or an L-valine residue, X.sup.4 comprises a C-terminal moiety selected from —C(═O)OH, —C(═O)NH.sub.2,

    ##STR00063##

    wherein Y.sub.5 is —OH or —NH.sub.2, and Y.sub.6 is hydrogen or a sugar moiety, preferably a disaccharide moiety.

    [0510] Embodiment 151: An isolated peptide comprising Formula III


    X.sup.1—X.sup.2—X.sup.3—X.sup.4  (III)

    wherein:
    X.sup.1 is the N-terminal amino acid residue comprising an N-terminal moiety —NR.sup.17R.sup.18;
    X.sup.4 comprises a C-terminal moiety selected from —C(═O)OH, —C(═O)O(C.sub.1-C.sub.3 alkyl), —C(═O)NH.sub.2,

    ##STR00064##

    or a linker, wherein
    Y.sub.5 is —OH or —NH.sub.2, and Y.sub.6 is hydrogen or a sugar moiety, preferably a disaccharide moiety;
    X.sup.1 is an L-amino acid residue selected from L-tyrosine, 2,6-dimethyl-L-tyrosine, or L-phenylalanine, wherein when X.sup.1 is L-tyrosine or 2,6-dimethyl-L-tyrosine, the residue is optionally O-substituted at the 4-position with C.sub.1-C.sub.3 alkyl;
    X.sup.2 is a D-amino acid residue, preferably D-threonine, D-alanine, D-valine, D-leucine, or D-isoleucine, more preferably, D-threonine or D-valine;
    X.sup.3 is glycine or an L-amino acid residue, wherein when X.sup.3 is an L-amino acid residue, X.sup.3 is preferably L-threonine, L-alanine, L-valine, L-leucine, or L-isoleucine, more preferably L-threonine or L-valine;
    X.sup.4 is a D-amino acid residue selected from D-tyrosine or D-phenylalanine, wherein when X.sup.4 is D-tyrosine, the residue is optionally O-substituted with a bio-reversible moiety;
    R.sup.17 and R.sup.18 are independently selected from hydrogen, a single bond, or a —C.sub.1-C.sub.3 alkyl, preferably —CH.sub.3; and
    wherein when X.sup.4 comprises a linker, the linker comprises a sugar moiety, preferably a disaccharide moiety such as lactose,
    wherein when one of R.sup.17 or R.sup.18 is a single bond, one of R.sup.17 or R.sup.18 is hydrogen and the single bond is a peptide bond to an L-amino acid residue that may optionally be N-terminally alkylated, preferably singly methylated; and
    wherein the peptide is a MOPr agonist.

    [0511] Embodiment 152: The peptide according to Embodiment 151, wherein the C-terminal moiety is —C(═O)OH,

    ##STR00065##

    and Y.sub.5 is —OH and Y.sub.6 is hydrogen or a sugar moiety; the peptide further comprises a out 5, 8, 11, 12, 20 or 26 additional L-amino acid residues on the C-terminus.

    [0512] Embodiment 153: The peptide according to Embodiment 151 or Embodiment 152, wherein X.sup.2 is a D-valine residue, X.sup.3 is glycine or an L-valine residue, X.sup.4 comprises a C-terminal moiety selected from —C(═O)OH, —C(═O)NH.sub.2,

    ##STR00066##

    wherein Y.sub.5 is —OH or —NH.sub.2, and Y.sub.6 is hydrogen or a sugar moiety, preferably a disaccharide moiety.

    [0513] Embodiment 154: The peptide according to Embodiment 151 or Embodiment 152, wherein X.sup.2 is a D-threonine residue, X.sup.3 is glycine or an L-threonine residue. X.sup.4 comprises a C-terminal moiety selected from —C(═O)OH, —C(═O)NH.sub.2,

    ##STR00067##

    wherein Y.sub.5 is —OH or —NH.sub.2, and Y.sub.6 is hydrogen or a sugar moiety, preferably a disaccharide moiety.

    [0514] Embodiment 155: The peptide according to any one of Embodiments 151 to 154, wherein one of R.sup.17 and R.sup.18 is hydrogen and one of R.sup.17 and R.sup.18 is —CH.sub.3.

    [0515] Embodiment 156: The peptide according to any one of Embodiments 151 to 154, wherein one of R.sup.17 or R.sup.18 is a single bond, one of R.sup.17 or R.sup.18 is a hydrogen, and the single bond is a peptide bond to an L-amino acid residue.

    [0516] Embodiment 157: The peptide according to Embodiment 156, wherein the L-amino acid residue has at least one N-terminal methylation.

    [0517] Embodiment 158: The peptide according to Embodiment 156 or Embodiment 157, wherein the L-amino acid residue is an L-alanine residue.

    [0518] Embodiment 159: The peptide according to Embodiment 151, wherein X.sup.4 comprises a linker.

    [0519] Embodiment 160: The peptide according to Embodiment 154, wherein the linker comprises an amino acid based linker, peptide based linker, an amino acid comprising linker, and/or maleimide based linker, and/or a combination thereof.

    [0520] Embodiment 161: The peptide according to any one of Embodiments 151 to 160, wherein X.sup.1 is L-tyrosine or 2,6-dimethyl-L-tyrosine and wherein the L-tyrosine or 2,6-dimethyl-L-tyrosine is O-substituted at the 4-position with C.sub.1-C.sub.3 alkyl.

    [0521] Embodiment 162: The peptide according to Embodiment 161, wherein X.sup.1 is 2,6-dimethyl-L-tyrosine and wherein 2,6-dimethyl-L-tyrosine is O-substituted at the 4-position with C.sub.1-C.sub.3 alkyl

    [0522] Embodiment 163: The peptide according to any one of Embodiments 148 to 162, wherein X.sup.4 comprises a C-terminal moiety selected from,

    ##STR00068##

    wherein Y.sub.5 is —OH or —NH.sub.2, and Y.sub.6 is a disaccharide moiety, preferably wherein the disaccharide moiety is attached through a beta linkage.

    [0523] Embodiment 164: The peptide according to Embodiment 162, wherein the disaccharide moiety is a lactose moiety, preferably wherein the lactose moiety is attached through a beta linkage.

    [0524] Embodiment 165: The peptide according to Embodiment 149, wherein said additional L-amino acids comprise at least one amino acid residue that is N-glycosylated, O-glycosylated, C-glycosylated, S-glycosylated, or Se-glycosylated.

    [0525] Embodiment 166: The peptide according to any one of Embodiments 148, 149, and 163 to 165, wherein R.sup.17 and R.sup.18 together form a bio-reversible moiety.

    [0526] Embodiment 167: The peptide according to Embodiment 166, wherein said bio-reversible moiety is

    ##STR00069##

    or ═N═N (azido moiety).

    [0527] Embodiment 168: The peptide according to any one of Embodiments 148, 149 and 163 to 165, wherein one of R.sup.17 or R.sup.18 is hydrogen and one of R.sup.17 or R.sup.18 is —C(═O)OCH.sub.2CH.sub.3 or —C(═O)OCH.sub.2OC(═O)CH.sub.3.

    [0528] Embodiment 169: The peptide according to Embodiment 148 selected from the group consisting of [0529] L-Phe-D-Val-L-Val-D-Phe (peptide 1a, Bilaid A); [0530] L-Phe-D-Val-L-Val-D-Phe-NH.sub.2 (peptide 1e); [0531] L-Tyr-D-Val-L-Val-D-Phe (peptide 3a, Bilaid C); [0532] L-Tyr-D-Val-L-Val-D-Phe-NH.sub.2 (peptide 3b); [0533] 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-NH.sub.2 (peptide 3c; Bilorphin); [0534] 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-L-Ser(β-Lac)-NH.sub.2 (peptide 3g; Bilactorphin); [0535] L-Phe-D-Val-Gly-D-Tyr-NH.sub.2 (peptide 2d); [0536] 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-OCH.sub.2CH.sub.3 (peptide 4); [0537] 2,6-dimethyl-L-tyrosine-D-Va-L-Val-D-Phe-L-Pro-L-Asn-L-Leu-L-Aa-L-Glu-L-Lys-L-AIa-L-Leu-L-Lys-L-Ser-L-Leu-NH.sub.2 (peptide 11); [0538] 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-NH.sub.2 wherein the N-terminus is substituted with the bio-reversible moiety —C(═O)OCH.sub.2OC(═O)CH.sub.3 (peptide 10); [0539] 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-NH.sub.2 wherein the N-terminus is substituted with the bio-reversible moiety

    ##STR00070##

    (peptide 8); [0540] 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-NH.sub.2 wherein the hydroxy group on 2,6-dimethyl-L-tyrosine is substituted with the bio-reversible moiety —C(═O)CH.sub.3 (peptide 5); [0541] 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-OCH.sub.2CH.sub.3 wherein the hydroxy group on 2,6-dimethyl-L-tyrosine is substituted with the bio-reversible moiety —C(═O)CH.sub.3 (peptide 6); [0542] 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-NH.sub.2 wherein the N-terminus is substituted with the bio-reversible moiety —C(═O)OCH.sub.2CH.sub.3 (peptide 7); and [0543] 2,6-dimethyl-L-tyrosine-D-Val-L-Val-D-Phe-NH.sub.2 wherein the N-terminus is substituted with the bio-reversible moiety, ═N═N, to form an N-terminal azido group (peptide 9).

    [0544] Embodiment 170: The peptide of any one of Embodiments 148 to 169, wherein said peptide in comparison to morphine: [0545] (a) exhibits a lower ratio of induction of C-terminal phosphorylation of MOPr versus G-protein activation; and/or [0546] (b) exhibits a lower ratio of induction of β-arrestin recruitment versus G-protein activation; and/or [0547] (c) exhibits a lower ratio of induction of MOPr internalisation versus G-protein activation.

    [0548] Embodiment 171: The peptide of any one of Embodiments 148 to 169 wherein said peptide, in comparison to morphine, exhibits a lower ratio of induction of β-arrestin recruitment versus G-protein activation.

    [0549] Embodiment 172: The peptide of any one of Embodiments 148 to 171, wherein said peptide exhibits an increase in inhibition of cAMP formation in comparison to vehicle at a concentration of about 10 μM in an assay using hMOPr.

    [0550] Embodiment 173: The peptide of any one of Embodiments 148 to 172, wherein said peptide in a competitive binding assay using [.sup.3H]DAMGO exhibits a K.sub.i of less than about 5 μM, less than about 3.5 μM, less than about 1 μM, less than about 0.8 μM, less than about 0.5 μM, or less than about 0.3 μM.

    [0551] Embodiment 174: The peptide of Embodiment 173, wherein said peptide exhibits a K.sub.i of less than about 0.5 μM or less than about 0.3 μM.

    [0552] Embodiment 175: The peptide of any one of Embodiments 148 to 174, wherein said peptide crosses the blood brain barrier.

    [0553] Embodiment 176: A pharmaceutical composition comprising a peptide according to any one of Embodiments 148 to 175 and at least one pharmaceutical excipient.

    [0554] Embodiment 177: The pharmaceutical composition according to Embodiment 176, wherein said composition is formulated for oral administration.

    [0555] Embodiment 178: The pharmaceutical composition according to Embodiment 176, wherein said peptide is glycosylated and said composition is formulated for oral administration, administration by injection, or intrathecal administration.

    [0556] Embodiment 179: The pharmaceutical composition according to Embodiment 176, wherein said peptide is not glycosylated and said composition is formulated for nasal administration or intrathecal administration.

    [0557] Embodiment 180: A method of treating pain comprising administering to a subject a peptide according to any one of Embodiments 148 to 175 or the pharmaceutical composition according to any one of Embodiments 176 to 179.

    [0558] Embodiment 181: The method of Embodiment 180, wherein the pain is post-operative pain, pain associated with nerve damage, pain associated with bone fracture, pain associated with a burn, or pain associated with a wound.

    [0559] Embodiment 182: Use of a peptide according to any one of Embodiments 148 to 175 or the pharmaceutical composition according to any one of Embodiments 176 to 179 in the manufacture of a medicament for treating pain.

    [0560] Embodiment 183: The use of Embodiment 182, wherein the pain is post-operative pain, pain associated with nerve damage, pain associated with bone fracture, pain associated with a burn, or pain associated with a wound.

    [0561] Embodiment 184: A peptide according to any one of Embodiments 148 to 175 for use in a method of treating pain.

    [0562] Embodiment 185: A pharmaceutical composition according to any one of Embodiments 176 to 179 for use in a method of treating pain.

    [0563] Embodiment 186: The peptide for use in the method of Embodiment 184 or the pharmaceutical composition for use in the method of Embodiment 185, wherein the pain is post-operative pain, pain associated with nerve damage, pain associated with bone fracture, pain associated with a burn, or pain associated with a wound.

    [0564] Embodiment 187: A method of delivering analgesia comprising administering to a subject a peptide according to any one of Embodiments 148 to 175 or the pharmaceutical composition according to any one of Embodiments 176 to 179.

    [0565] Embodiment 188: Use of peptide according to any one of Embodiments 148 to 175 or the pharmaceutical composition according to any one of Embodiments 176 to 179 for the manufacture of a medicament for delivering analgesia.

    [0566] Embodiment 189: A peptide according to any one of Embodiments 148 to 175 or the pharmaceutical composition according to any one of Embodiments 176 to 179 for use in a method of delivering analgesia.

    [0567] Embodiment 190: A method of treating pain or delivering analgesia with reduced adverse side effect(s), preferably reduced in comparison to morphine, comprising administering to a subject a peptide according to any one of Embodiments 148 to 175 or the pharmaceutical composition according to any one of Embodiments 176 to 179.

    [0568] Embodiment 191: The method according to Embodiment 190, wherein said adverse side effect(s) is gastrointestinal (GI) inhibition and/or respiratory depression.

    [0569] Embodiment 192: Use of peptide according to any one of Embodiments 148 to 175 or the pharmaceutical composition according to any one of Embodiments 176 to 179 for the manufacture of a medicament for a method of treating pain or delivering analgesia with reduced adverse side effect(s), preferably reduced in comparison to morphine.

    [0570] Embodiment 193: The use according to Embodiment 192, wherein said adverse side effect(s) is gastrointestinal (GI) inhibition and/or respiratory depression.

    [0571] Embodiment 194: A peptide according to any one of Embodiments 148 to 175 or the pharmaceutical composition according to any one of Embodiments 176 to 179 for use in a method of treating pain or delivering analgesia with reduced adverse side effect(s), preferably reduced in comparison to morphine.

    [0572] Embodiment 195: The peptide for use in the method of Embodiment 194 or the pharmaceutical composition for use in the method of Embodiment 194, wherein said adverse side effect(s) is gastrointestinal (GI) inhibition and/or respiratory depression.

    [0573] Embodiment 196: A peptide according to any one of Embodiments 1 to 59, 80 to 127, and 148 to 175 for use in medicine.

    [0574] Embodiments of the invention will now be described, by way of example, with reference to the accompanying drawings.

    TABLE-US-00001 TABLE 1 Sequence Name # (SEQ ID NO) N- term P1 P2 P3 P4 bilaid A 1a H-FvVf-OH H L-Phe D-Val L-Val D-Phe (SEQ ID NO: 1) 1b H-fVvF-OH — D-Phe L-Val D-Val L-Phe (SEQ ID NO: 4) 1c H-FVvf-OH — — L-Val D-Val — (SEQ ID NO: 5) 1d H-fvVF-OH — D-Phe — — L-Phe (SEQ ID NO: 6) 1e H-FvVf-NHtext missing or illegible when filed — — — — — (SEQ ID NO: 7) 1f H-fVvF-NHtext missing or illegible when filed — D-Phe L-Val D-Val L-Phe (SEQ ID NO: 8) 1g H-FVvf-NHtext missing or illegible when filed — — L-Val D-Val — (SEQ ID NO: 9) 1h H-FVVF-NHtext missing or illegible when filed — — L-Val — L-Phe (SEQ ID NO: 10) bilaid B 2a H-FvVy-OH H L-Phe D-Val L-Val D-Tyr (SEQ ID NO: 2) 2b H-FvVy-NHtext missing or illegible when filed — — — — — (SEQ ID NO: 11) 2c H-FVVY-NHtext missing or illegible when filed — — L-Val — L-Tyr (SEQ ID NO: 12) 2d H-FvGy-NHtext missing or illegible when filed — — — Gly — (SEQ ID NO: 13) 2e H-FGVy-NHtext missing or illegible when filed — — Gly — — (SEQ ID NO: 14) 2f H-FGGy-NHtext missing or illegible when filed — — Gly Gly — (SEQ ID NO: 15) bilaid C 3a H-YvVf-OH H L-Tvr D-Val L-Val D-Phe (SEQ ID NO: 3) 3b H-YvVf-NHtext missing or illegible when filed — — — — — (SEQ ID NO: 16) bilorphin 3c H-[Dmt]-vVf-NHtext missing or illegible when filed — Dmt — — — (SEQ ID NO: 17) 3d Aε-YvVf-NHtext missing or illegible when filed CHtext missing or illegible when filed CO — — — — (SEQ ID NO: 18) 3e H-YVVF-OH — — L-Val — L-Phe (SEQ ID NO: 19) 3f H-YVVF-NHtext missing or illegible when filed — — L-Val — L-Phe (SEQ ID NO: 20) Bilactorphin 3g H-[Dmt]-vVfS(β- Lac)-NHtext missing or illegible when filed — Dmt — — — (SEQ ID NO: 21) 3h H-[Dmt]-vVfS(β-D-Glc)-NHtext missing or illegible when filed — Dmt — — — (SEQ ID NO: 22) TetraQ, initial screen % inhibition of forskolin- Agonist stimulate cAMP potency formation in Binding in LC hMOPr HEK cells screen neurons (10 um CEREP Ki (nM) EC50 Name C- term DAMGO = 94%) MOPr DOPr KOPr (nM) bilaid A OH 21% 3,100 — 0 — 0 — 0 NHtext missing or illegible when filed 47% 750 NHtext missing or illegible when filed 0 NHtext missing or illegible when filed 0 NHtext missing or illegible when filed 19% >10,000 bilaid B OH 0 NHtext missing or illegible when filed 0 NHtext missing or illegible when filed 0 NHtext missing or illegible when filed 31% 1600 NHtext missing or illegible when filed 0 NHtext missing or illegible when filed 0 bilaid C OH 77% 210 4,200 NHtext missing or illegible when filed 93 bilorphin NHtext missing or illegible when filed 1.1 190 770 130 NHtext missing or illegible when filed >10,000 — >10,000 NHtext missing or illegible when filed >10,000 Bilactorphin L-Ser(β- Lac)-NHtext missing or illegible when filed L-Ser(β- D-Glc)-NHtext missing or illegible when filed Dmt = 2,6-dimethyl-L-tyrosine Parent (native) peptide of each set highlighted “—” indicates no change from parent peptide where values are missing they were not determined text missing or illegible when filed indicates data missing or illegible when filed

    Example 1: Peptide Synthesis

    [0575] The peptides in Table 1 were synthesized using Fmoc chemistry.

    [0576] Novel Peptide Synthesis:

    [0577] General synthetic details: Initial HPLC was performed on a system consisting of two Shimadzu LC-8A Preparative Liquid Chromatographs with static mixer, Shimadzu SPD-M10AVP Diode Array Detector and Shimadzu SCL-10AVP System Controller. Further HPLC was performed using an Agilent 1100 Series separations module equipped with Agilent 1100 Series diode array and/or multiple wavelength detectors, Polymer Laboratories PL-ELS1000 ELSD and Agilent 1100 Series fraction collector and running ChemStation (Revisions 9.03A or 10.0A). NMR spectra were acquired in DMSO-d.sub.6 on a Bruker Avance 500 or a Bruker Avance 600 spectrometer under XWIN-NMR or Topspin control, referenced to residual .sup.1H signals. Electrospray ionisation mass spectra (ESIMS) were acquired using an Agilent 1100 series separations module equipped with an Agilent 1100 series LC/MSD mass detector and Agilent 1100 series diode array detector. High-resolution (HR) ESIMS measurements were obtained on a Finnigan MAT 900 XL-Trap instrument with a Finnigan API III source. Unless otherwise specified, a constant level of 0.1% TFA was used in all HPLC separations. Chiroptical measurements ([α].sub.D) were obtained on a Jasco P-1010 Intelligent Remote Module type polarimeter in a 100×2 mm cell.

    [0578] Fmoc-L- and D-amino acids were obtained from Novabiochem (Laufelfingen, Switzerland) or Peptide Institute (Osaka, Japan). 2-Chlorotrityl chloride and Rinkamide resins were purchased from Novabiochem (Laufelfingen, Switzerland). 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) was obtained from Richelieu Biotechnologies (Quebec, Canada). Trifluoroacetic acid (TFA), N,N-diisopropylethylamine (DIEA) and N,N-dimethylformamide (DMF), all peptide synthesis grade, were purchased from Auspep (Melbourne, Australia).

    [0579] Characterisation of Bilaids A-C:

    [0580] bilaid A (FvVf-OH) (1a). light brown oil; [α].sub.D+9 (c 0.2, 0.1% TFA/MeOH); HRESI(+)MS m/z 533.2745 [(M+Na), C.sub.28H.sub.38N.sub.4O.sub.5Na requires 533.2740]; For .sup.1H, .sup.13C and 2D NMR (600 MHz, DMSO-d.sub.6) see FIG. 25 and Table 1.

    [0581] bilaid B (FvVy-OH) (2a). light brown oil; HRESI(+)MS m/z 549.2692 [(M+Na).sup.+, C.sub.28H.sub.38N.sub.4O.sub.6Na requires 549.2689]; For .sup.1H NMR (600 MHz, DMSO-d.sub.6) see FIG. 25 and Table 1.

    [0582] bilaid C (YvVf-OH) (3a). light brown oil; HRESI(+)MS m/z 527.2879 [(M+H).sup.+, C.sub.28H.sub.39N.sub.4O.sub.6 requires 527.2870]; For .sup.1H NMR (600 MHz, DMSO-d.sub.6) see FIG. 25 and Table 1.

    [0583] General peptide synthesis procedure: All peptides were assembled manually by stepwise solid-phase peptide synthesis. 2-Chlorotrityl chloride resin (0.176 g, 0.25 mmol; for peptide acids) or Rink amide resin (0.176 g, 0.25 mmol; for peptide amides) was swollen in DMF for 2 h and drained. The first Fmoc protected amino acid (1 mmol) was dissolved in DMF (2 mL) and DIEA (174 μL, 1 mmol) was added. After complete dissolution of the amino acid, the mixture was added to the reaction vessel and shaken for 2 h. The resin was flow washed with DMF for 1 min. The Fmoc protecting group was removed by shaking the resin with 5% piperidine/DMF mixture (2×10 mL, each cycle for 1 min). After deprotection the resin was again flow washed for 1 min. The next amino acid (1 mmol) was activated with 0.5 M HBTU solution (2 mL) and DIEA (174 μL, 1 mmol) and was added to the reaction vessel. The mixture was shaken for 10 min and a ninhydrin test was performed to calculate the coupling yield. This test was repeated after each coupling. After completion of the assembly, the terminal Fmoc group was removed as described above, the resin washed with DMF followed by DCM and dried under nitrogen. The peptide was cleaved from the resin by shaking with 10 mL of cleavage mixture (TFA:water: 95:5) for 2 h. TFA was evaporated under N.sub.2 gas.

    [0584] Purification of synthetic peptide acids from solid phase synthesis: Reaction products for the synthetic peptides, were purified by preparative HPLC (Zorbax SB-C.sub.18 column, 250×21.2 mm, 7 μm, isocratic 45% H.sub.2O(0.1% TFA):MeOH for bilaid A (FvVf-OH, 35.3 mg) (1a) and fVvF-OH, 41.0 mg (1b); 30% H.sub.2O(0.1% TFA):MeOH for FVvf-OH, 25.0 mg (1c) and fvVF-OH, 30.8 mg (1d); 40% H.sub.2O(0.1% TFA); bilaid B (FvVy-OH, 44.8 mg) (2a) and bilaid C (YvVf-OH, 45.9 mg) (3a).

    [0585] Solution conversion of tetrapeptide acids to amides: To a solution of di-tert-butyl-dicarbonate in 1,4-dioxane (29.3 mg/mL) was added pyridine (10.5 μL/mL) and ammonium bicarbonate (10.5 mg/mL). To 1a, 1b, 1c and 1d was added 300 μL of this solution and to 2a and 3a was added 600 μL, equating to 4 eq. After stirring for 3 d at 50° C., the reaction products were purified by preparative HPLC (Zorbax SB-C.sub.8 150×21.2 mm column; gradient of 90% H.sub.2O(0.1% TFA):MeCN to MeCN over 15 min), to yield pure samples.

    [0586] LC/MS analyses on synthetic peptides: Performed on Zorbax SB-C.sub.8 150×4.6 mm, 5 μm column (flow 1 mL/min; gradient 10-100% MeCN/H.sub.2O (+isocratic 0.05% HCO.sub.2H) over 15 min.

    [0587] Chiral HPLC analysis on peptides: Performed on Astec Chirobiotic T column, 150×4.6 mm, 5 μm, 0.5 mL/min, isocratic MeOH (0.1% triethylamine, 0.2% AcOH, pH 6.23).

    N.SUP.α.-(9-fIuorenylmethoxycarbonyl)-3-O-[2,3,6-tri-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-β-D-glucopyranosyl]-L-serine (Fmoc-L-Ser[β-Lac(Ac.SUB.7.)]—OH)

    [0588] β-lactose peracetate was prepared according to the procedure outlined by Xu et al Journal of Carbohyrate Chemistry (2012) 31(9): 711-720. Briefly, α-lactose monohydrate (20.0 g) was added in portions to a stirring suspension of sodium acetate (5.0 g) in acetic anhydride (200 mL) with the temperature maintained at 135° C. After 1 h the solution was poured into ice-water (1 L) and stirred overnight. The resulting precipitate was collected by filtration, redissolved in CH.sub.2Cl.sub.2, washed with satd. NaHCO.sub.3 and dried over MgSO.sub.4. Following removal of solvent under reduced pressure it was crystallised from CH.sub.2Cl.sub.2/MeOH (16.1 g, 42%). ESI-MS (m/z): calc. 619.2 [M-OAc].sup.+ found 619.3.

    [0589] Fmoc-L-Ser-OH was O-β-lactosylated based on the procedure described by Salvador et al Tetrahedron (1995) 51(19):5643-5656. To a mixture of β-lactose peracetate (5.0 g) and Fmoc-L-Ser-OH (2.9 g) in anhydrous CH.sub.2Cl.sub.2 (100 mL) was added BF.sub.3-Et.sub.2O (2.8 mL) and stirred under nitrogen for 20 h. The solution was washed with 1 M HCl then water and dried over MgSO.sub.4. Purified by silica gel chromatography (1% AcOH/2% MeOH in CH.sub.2Cl.sub.2) followed by RP-HPLC (50% B isocratic) (1.9 g, 27% from β-lactose peracetate). ESI-MS (m/z): calc. 946.3 [M+H].sup.+ found 946.3.

    H-[2,6-dimethyl-L-tyrosyl]-D-Val-L-Val-D-Phe-L-Ser(β-Lac)-NH.SUB.2 .(Bilactorphin, 3g)

    [0590] Peptide assembly was performed using Fmoc chemistry on 0.5 mmol scale on Fmoc-Rink-amide polystyrene resin (substitution value 0.67 mmol/g). Fmoc deprotections were accomplished by treatments with 50% piperidine/DMF (2×1 min). Couplings were performed using three equivalents of Fmoc amino acid/HBTU/DIEA (1:1:1) relative to resin loading (30 min). N.sup.α-Fmoc-O-β-lactosyl-L-serine was incorporated as the hepta-O-acetate (prepared as described above); N.sup.α-Boc-2,6-dimethyl-L-tyrosine was used without side-chain protection. Cleavage from the resin and removal of side-chain protecting groups was achieved by treatment with 95% TFA/2.5% TIPS/2.5% H.sub.2O for 2 h at room temperature. TFA was removed under a stream of nitrogen, and the product was precipitated using cold diethyl ether/n-hexane (1:1), washed with Et.sub.2O, redissolved in 50% acetonitrile/0.1% TFA/H.sub.2O and lyophilised. ESI-MS (m/z): calc. 1259.5 [M+H]+, found 1259.7. The crude product was deacetylated by treatment with a solution of 5% hydrazine/30% acetonitrile/H.sub.2O for 5 h then purified by RP-HPLC (10 to 50% B over 40 min). (190 mg, 39% from initial resin loading). ESI-MS (m/z): calc. 965.5 [M+H]+, found 965.4.

    [0591] General Materials and Methods

    [0592] RP-HPLC solvent A was 0.05% TFA/H.sub.2O and solvent B was 0.043% TFA/90% acetonitrile/H.sub.2O. Analytical HPLC was performed on a Shimadzu LC20AT system using a Thermo Hypersil GOLD C18 2.1×100 mm column at flow rate of 0.3 mL/min. Absorbance was recorded at 214 nm. Preparative HPLC was performed on a Waters DeltaPrep 3000 system using a Vydac 208TP 50×250 mm column at a flow rate of 80 mL/min. Mass spectra were recorded in positive ionisation mode on an API 2000 triple quadrupole mass spectrometer (AB SCIEX, Framingham, Mass., USA). Fmoc amino acids and O-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) were from Iris Biotech (Marktredwitz, Germany), dimethylformamide (DMF) and diisopropylethylamine (DIEA) were from Auspep (Melbourne, Australia). Boc-2,6-dimethyl-L-tyrosine was purchased from AstaTech Inc (Bristol Pa., USA). All other reagents were obtained from Sigma Aldrich.

    Example 2: Inhibition of Forskolin Induced cAMP Formation

    [0593] Procedure for measuring the level of intracellular cAMP produced upon modulation of adenylate cyclase activity by G-protein-coupled receptors (GPCRs). To test the functional interaction between opioids, and opioid-like compounds, and specific opioid GPCRs by measuring changes in intracellular cAMP levels relative to basal levels. The assay can measure agonist and antagonist activity on GPCRs by stimulating cells to either increase or decrease intracellular cAMP levels.

    [0594] Materials

    [0595] Reagents supplied in cAMP assay kit: Anti-cAMP acceptor beads, Streptavidin-coated donor beads, Biotinylated cAMP, cAMP standard, 3% Tween-20 solution. Additional reagents: 1 M HEPES (Muticel), 10% Tween-20 (Pierce), BSA (Sigma), 1×PBS (Gibco BRL), Sterile distilled water (Gibco BRL), Forskolin (Sigma), 10×HBSS (Hepes Buffered Salt Solution, Gibco BRL), IBMX solution (3-Isobutyl-1-Methylxanthine, Sigma), DMSO (Sigma), 95% Ethanol (Sigma), Complete growth media, Versene (Gibco). Equipment: Envision multilabel plate reader (Perkin Elmer), Optiplate-384 well plates (Perkin Elmer), TopSeal adhesive sealing film (Perkin Elmer), 96-well plates (Axigen, Polypropylene V bottom), Silicon 96-well plate sealing mats (Axigen), Single channel pipettors, Multichannel pipettor, Centrifuge, Vortex, 75 cm2 vented tissue culture flask, 50 ml conical tubes, 15 ml conical tubes, Electronic pipette aid, Disposable sterile transfer pipettes, haemoytometer, Microscope.

    [0596] Method

    [0597] The assay is performed in a 384-well plate and each data point is performed in triplicate. The test compounds should be analyzed on the same plate as controls and cAMP standards. The number of compounds to be screened for activity will determine the volume of reagents and cells required for each experiment. The test compounds, controls and cAMP standards can be added to the plate in advance or while the cells are incubating with stimulation buffer. The plate should be sealed with TopSeal adhesive sealing film to avoid evaporation.

    [0598] Assay background: Detection of cAMP is based on the competition between intracellular cAMP and biotinylated cAMP linked streptavidin-coated donor beads for anti-cAMP conjugated acceptor beads. When the donor and acceptor beads are in close proximity a signal emitted at 520-620 nm is detected using the Envision multilabel plate reader.

    [0599] Preparation of Reagents

    Stocks:

    [0600] 500 mM IBMX solution: Dissolve 100 mg IBMX in 900 μl DMSO to give a 500 mM stock solution. Aliquot and store at 20° C.
    50 mM forskolin solution: Dissolve 5 mg forskolin in 244 μl of 95% ethanol to give a 50 mM stock solution. Store at 20° C. and use as required.
    Fresh reagents-Prepare the following reagents fresh in 50 ml conical tubes:
    Stimulation buffer (1×HBSS, 0.1% BSA, 1 mM IBMX): For 50 ml add 5 ml 10×HBSS to a 50 ml tube then make up to 50 ml with water. Add 50 mg BSA, place at 37° C. until BSA is dissolved then add 100 μl IBMX while the buffer is at 37° C. to ensure that the IBMX does not precipitate.
    Lysis buffer (0.3% Tween-20, 5 mM HEPES, 0.1% BSA): For 40 ml add 1.2 ml 10% Tween-20 and 200 μl 1 M HEPES to a 50 ml tube then make up to 40 ml with water. Add 50 mg BSA, place at 37° C. until BSA is dissolved.
    Stimulation buffer with forskolin (*200 μM in stimulation buffer): Add forskolin to a dilution of 1:250 from the 50 mM stock to the required amount of stimulation buffer. It should be noted that the final concentration in the assay plate will be halved. *The optimal concentration of forskolin in the assay is cell line specific and should be optimized.

    [0601] Preparation of test peptides: Test peptides are typically tested at a final concentration of 10 μM. As most test peptides are dissolved in 100% DMSO it is recommended that the DMSO concentration is limited to 2% v/v during cell stimulation to ensure maximum cell viability and responsiveness. Prepare 1 mM and 100 μM stock solutions of test peptides in an appropriate diluent and store at 4° C. For a library of peptides this can be performed in 96 well plates sealed with silicon sealing mats. Test peptides are diluted fresh from stock solutions to a working concentration in stimulation buffer with forskolin. It should be noted that as peptides are diluted 1:1 with cells in the assay the working concentration should be twice the final required concentration. It may be necessary to perform assays on compounds freshly diluted from DMSO stocks compounds to avoid experimental variability.

    [0602] Control compounds: Compounds with known activity are used as controls. Controls are typically used at 1 μM and are diluted fresh in stimulation buffer with forskolin from stocks stored at −20° C. For the assay 5 μl of prepared control compounds are added per well in triplicate.

    [0603] Preparation of cells: For best results cells should be low passage at 70-90% confluence. To prepare cells for the assay remove growth medium, add Versene and then incubate at 37° C. for approximately 5 minutes to allow cells to detach from the tissue culture plastic. Collect cells and centrifuge for 2 minutes at 275×g. Decant the supernatant and resuspend the cell pellet in 1×PBS. Determine the cell concentration using a haemocytometer. Re-centrifuge cells for 2 minutes at 275×g and decant supernatant. Resuspend cells in stimulation buffer to a final concentration of 1-4×10.sup.4 cells per ml. Note that the cells number will influence the cAMP levels prevailing before (basal) and after adenylate cyclase activation. A cell titration should be performed to optimize the difference between basal and stimulation levels of cAMP. Cells are incubated in stimulation buffer for 20 to 30 minutes at 37° C. prior to adding 5 μl to wells containing test and control compounds. Note that cells are not added to the cAMP standards.

    [0604] Preparation of cAMP standard curve: Prepare a standard cAMP dilution series from the kit supplied 50 μM cAMP solution. Vortex at maximum intensity for 5 seconds before use. Serially dilute to provide a final concentration range from 5 μM to 0.5 nM cAMP (for example: 5 μM, 0.5 μM, 50 nM, 15 nM, 5 nM, 1.5 nM, and 0.5 nM cAMP). A positive control (no cAMP) should also be included. For the assay 10 μl of standard dilutions are added per well in triplicate.

    [0605] Preparation of acceptor and donor bead solutions: The anti-cAMP conjugated acceptor beads and streptavidin-coated donor beads are light sensitive and should be handled under subdued lighting or under lights fitted with green filters. Once the beads have been added to the assay plate it should be wrapped in foil so that incubations are performed in the dark. Prepare acceptor and donor bead solutions in 15 ml conical tubes while the cells are incubating and keep in the dark until required. For the acceptor bead solution gently mix 10 μl bead suspension per ml of lysis buffer. For the donor bead solution use 10 μl bead suspension per ml of lysis buffer and 0.75 μl/ml of cAMP-biotin and mix gently.

    [0606] cAMP Assay Procedure

    1. Add standards (10 μl/well), control compounds (5 μl/well) and test peptides (5 μl/well) into 384-well plates and seal with Top Seal adhesive sealing film. Leave at room temperature until the cell incubation is complete.
    2. Add 5 μl cells incubated in stimulation buffer to the wells containing test peptides and control compounds, but not to the standards. Incubate cells and compounds for 30 min at 37° C.
    3. Add 10 μl lysis buffer per well.
    4. Under subdued lighting add 5 μl acceptor bead solution to each well. Wrap plate in foil and incubate at room temperature with gentle mixing on an orbital shaker for 60 min.
    5. Also under subdued lighting add 5 μl donor bead solution to each well, wrap the plate in foil, and then incubate overnight at room temperature with gentle mixing on an orbital shaker.
    6. Measure cAMP levels on Envision multilabel plate reader.

    [0607] Results analysis: Analyse results using PRISM software to calculate the intracellular levels of cAMP for each triplicate data point and the standard deviation of these data points.

    [0608] In accordance with the method described above, the peptides were screened for inhibition of forskolin induced cAMP formation in HEK cells expressing the human MOPr (hMOPr).

    [0609] Neither Bilaid B (2a) nor its C-terminal carboxamide analogue (2b) showed activity at 10 μM, indicating the phenylalanine at the fourth amino acid position is more favourable.

    [0610] As shown in Table 1, bilaid A (peptide 1a, H-FvVf-OH) and bilaid C (peptide 3a, H-YvVf-OH) showed activity at 10 μM. In contrast, analogues of bilaid A having DLDL (1b) stereochemistry or LLDD (1c) stereochemistry were inactive at 10 μM and an analogue of bilaid A having LLLL stereochemistry (1h) was less active at 10 μM, highlighting the importance of the LDLD motif for maintaining MOPr activity.

    Example 3: Competitive Binding Assay (MOPr)

    [0611] Peptides were tested for competitive binding to hMOPr against the MOPr agonist [.sup.3H]DAMGO. Bilaid A (1a) showed modest affinity (K.sub.i=3.1 μM) that was improved 4-fold through C-terminal amidation (1e, K.sub.i=0.75 μM). The N-terminal tyrosine containing Bilaid C (3a) displayed sub-micromolar binding (K.sub.i=210 nM) at the hMOPr. C-terminal amidation (3b) doubled MOPr affinity (K.sub.i=93 nM), consistent with the increased binding of 1e over 1a. Dimethylation of the N-terminal tyrosine ([Dmt]-vVf-NH.sub.2 (3c)) (Dmt=2,6-dimethyl-L-tyrosine) see Zhao et al J Pharmacol Exp Ther (2003) 307(3): 947-54) resulted in a further increase to yield a K.sub.i of 1.1 nM. Peptide 3c (designated bilorphin), bound with nearly 200-fold selectivity for hMOPr over hDOPr (K.sub.i=190 nM) and 700-fold selectivity over hKOPr (K.sub.i=770 nM). See FIG. 1 and Table 1.

    [0612] Competitive MOPr binding was determined using a filtration separation followed by liquid scintillation counting procedure after incubation of membranes prepared from human recombinant MOPr expressed in HEK-293 cells (Human Embryonic Kidney cell line) with [.sup.3H]DAMGO (0.5 nM) plus various concentrations of unlabelled peptides for 120 minutes at 22° C. The specific ligand binding to the receptors is defined as the difference between the total binding and the nonspecific binding determined in the presence of an excess of an unlabelled opioid ligand (naloxone, 10 μM). The results are expressed as a percent of control specific binding ((measured specific binding/control specific binding)×100) obtained in the presence of unlabelled peptides of interest. The IC.sub.50 values (concentration causing a half-maximal inhibition of control specific binding) and Hill coefficients (nH) were determined by non-linear regression analysis of the competition curves generated with mean replicate values using Hill equation curve fitting (Y=D+[(A−D)/(1+(C/C.sub.50nH)], where Y=specific binding, D=minimum specific binding, A=maximum specific binding, C=compound concentration, C.sub.50=IC.sub.50, and nH=slope factor).

    Example 4: Competitive Binding Assay (DOPr)

    [0613] Peptides were tested for competitive binding to hDOPr against the DOPr agonist [.sup.3H]DADLE. See FIG. 1 and Table 1.

    [0614] Inhibition of binding to human recombinant DOPr (hDOPr) expressed in CHO (Chinese Hamster Ovary) cell line was performed as described for MOPr binding (Example 3) except that incubation was in [3H]DADLE (0.5 nM) for 120 min at 22° C.

    Example 5: Competitive Binding Assay (KOPr)

    [0615] Peptides were tested for competitive binding to hKOPr against the KOPr agonist [.sup.3H]U69593. See FIG. 1 and Table 1.

    [0616] Inhibition of binding to human recombinant KOPr (hKOPr) expressed in CHO (Chinese Hamster Ovary) cell line was performed as described for MOPr binding (Example 3) except that incubation was in [.sup.3H]U69593 (2 nM) for 60 min at 22° C.

    Example 6: Patch Clamp Recordings of Activated G-Protein

    [0617] To assess functional activity of bilorphin, patch-clamp recordings of G-protein activated, inwardly rectifying potassium channel (GIRK) currents were made in rat locus coeruleus (LC) neurons, which natively express MOPr but not DOPr or KOPr (North et al Proc Natl Acad Sci USA. (1987) 84(15):5487-91). Bilorphin acted as an agonist with potency greater than morphine. Its actions were completely reversed by the highly MOPr selective antagonist CTAP (n=9, FIG. 2A; Table 1) (Pelton et al J Med Chem (1986) 29(11):2370-5) establishing that bilorphin does not act on closely related receptors expressed by LC neurons such as NOPr or somatostatin receptors (Connor et al Br J Pharmacol (1996) 119(8): 1614-8). Bilaid C analogues with an acetylated N-terminal (3d), or all L-stereoisomers (3e and 3f), were inactive in LC neurons at 10-30 μM, indicating the importance of a free N-terminal and the native LDLD motif for maintaining MOPr activity (Table 1). The submaximal, partial agonist-like action of bilorphin in LC neurons was confirmed by its partial antagonism of the full agonist actions of met-enkephalin. This was tested after MOPr was desensitized for 10 min to produce a stable activated response after acute desensitization, as well as to reduce functional receptor reserve (n=12, FIG. 2B).

    [0618] Brain slice electrophysiology: Brain slices containing locus coeruleus LC neurons were prepared from male Sprague Dawley rats (4-6 weeks) as described previously (Sadeghi M et al Br J Pharmacol (2015) 172(2):460-8, incorporated by reference). Briefly, rats were anesthetized with isoflurane and decapitated. The brain was dissected and mounted in a vibratome chamber (Leica biosystem, VT100, Wetzlar, Germany) in order to prepare horizontal brain slices (280 μm). Slices were cut and stored in warm (34° C.) artificial cerebrospinal fluid (ACSF) containing the following (in mM): 125 NaCl, 2.5 KCl, 2 CaCl.sub.2, 1 MgCl.sub.2, 1.25 NaH.sub.2PO.sub.4, 25 NaHCO.sub.3 and 11 glucose supplemented with 0.01 (+) MK801 (95% O.sub.2-5% CO.sub.2). Slices were incubated in warm oxygenated ACSF for at least 30 min before recording. Slices were transferred to the recording chamber while warm ACSF (34° C.) was superfused at a rate of ˜2 mL/min. Whole-cell voltage-clamp recordings were acquired from LC neurons with Multiclamp 700B amplifier (Molecular Devices, CA, USA) at holding potential of −60 mV. Recording pipettes (2-4 MΩ) were filled with internal solution containing (in mM): 135 potassium gluconate, 8 NaCl, 10 HEPES, 0.5 EGTA, 2 Mg-ATP and 0.3 Na-GTP; pH 7.3, 280-285 mOsM. Continuous current recordings were collected in chart mode at 500 Hz and filtered at 20-50 Hz using Axograph X (Axograph Scientific, Sydney, Australia). Series resistance monitored throughout the experiments and remained <15 MΩ; otherwise the data were discarded. Outward current was measured as the difference between baseline current and peak current of drug application.

    Example 7: Assessment of Relative Potency and Intrinsic Efficacy

    [0619] The relative potency and intrinsic efficacy (maximum response) of bilorphin also in other signalling pathways was examined in AtT20 cells stably expressing FLAG tagged mouse MOPr (mMOPr) to enable analysis of bias (FIG. 5). To reliably determine the relative intrinsic efficacy of bilorphin to activate G-proteins (G.sub.GIRK), it was ensured that no MOPr agonist reached a ceiling effect. β-chlornaltrexamine was used to irreversibly inactivate a sufficient fraction of receptors to reduce the maximum response of met-enkephalin to 80% of that produced by a supramaximal concentration of somatostatin acting on native SST receptors in the same cells (FIGS. 4, 5). Maximal activation of SST receptors normally produces a G.sub.GIRK increase equivalent to a maximal activation of MOPr (Yousuf et al Mol Pharmacol (2015) 88(4): 825-35). Under these conditions, bilorphin, morphine and endomorphin-2 all displayed similar maximal responses indicating they have similar intrinsic efficacies. With the exception of met-enkephalin all agonists displayed similar potencies in brain slices and the cell line. The reduced potency of Met-enkephalin in brain slices is known to result from its degradation by enkephalinases and other peptidases (Williams, et al. J. Pharmac. Exp. Ther. (1987) 243: 397-401.) As expected from brain slice experiments all three opioids were moderately efficacious but less so than Met-enkephalin (FIGS. 5, 23A). In contrast, the G-protein biased, small molecule agonist oliceridine, activated G.sub.GIRK significantly less efficaciously than either morphine or bilorphin (FIGS. 5, 23A).

    [0620] Expression of MOPr in AtT20 Cells:

    [0621] Wild type mMOPr was cloned in pcDNA3.1 plasmids with FLAG-tag and expressed stably in AtT20 cells at a deliberately low level of expression (8 μmol/mg protein; 2×10.sup.5 receptors/cell estimated from cytometry), as previously described Borgland et al J Biol Chem (2003) 278(21): 18775-84. For patch clamp experiments AtT20 cells were seeded on 35-mm polystyrene culture dishes (Beckton, Dickinson Biosciences) in Dulbecco modified Eagle medium (Gibco, Life Technologies, Australia) containing 4.5 g/L glucose, penicillin-streptomycin (100 μl/ml), G418 (50 mg/ml.) (Gibco, Invitrogen) and 10% FBS. Cell cultures were kept in humidified 5% CO.sub.2 atmosphere at 37° C. Cells were ready for recording after 24 hours incubation.

    [0622] Cultured cell electrophysiology: Perforated patch clamp recordings were performed as previously described (Yousuf et al. Mol Pharmacol. (2015) 88(4):825-35). Pipettes were pulled from borosilicate glass (AM Systems, Everett, Wash., USA) yielding input resistances between 3.5-4.5 MΩ and were filled with internal solution containing 135 mM potassium gluconate, 3 mM MgCl.sub.2, 10 mM HEPES (adjusted to pH 7.4 with KOH). The recording electrodes were first front filled with this internal solution and then backfilled with the same solution containing 200 μg/ml amphotericin B (in 0.8% DMSO). For measuring I.sub.GIRK the KCl concentration in the bath was increased to 20 mM (substituted for NaCl) before the start of the measurements and was maintained throughout the experiments as previously described in Yousuf et al. (2015). Liquid junction potential was calculated to be +16 mV and was adjusted before the start of each recording. Currents were recorded at 37° C. in a fully enclosed, temperature-controlled recording chamber using an Axopatch 200B amplifier and pCLAMP 9.2 software, and digitized using Digidata 1320 (Axon Instruments, Molecular Devices, Sunnyvale, Calif., USA). Currents were sampled at 100 Hz, low pass filtered at 50 Hz and recorded on hard disk for later analyses. I.sub.GIRK was recorded using a 200 ms voltage step to −120 mV from a holding potential of −60 mV delivered every 2 s. Drugs were perfused directly onto cells using a ValveLink 8.2 pressurized pinch valve perfusion system (AutoMate Scientific, USA).

    [0623] Data Analyses. All data are shown as the mean±SEM and analysed using GraphPad Prism v7. All data points are plotted as chord GIRK conductance (G.sub.GIRK, nS) using the following calculation: [I.sub.GIRK (−60 mV)−I.sub.GIRK (−120 mV)]pA/60 mV.

    [0624] Assessment of Inducing C-Terminal Phosphorylation

    [0625] MOPr C-terminal phosphorylation, β-arrestin recruitment and internalisation are thought to contribute to on-target opioid analgesic side effects so that G-protein biased opioids that avoid arrestin signalling should show an improved side effect profile (Manglik et al Nature (2016) 537(7619):185-190; Schmid et al Cell (2017) 171(5):1165-1175; DeWire et al J Pharmacol Exp Ther (2013) 344(3):708-17). Agonist-induced phosphorylation of residue serine 375 (Ser.sup.375) drives β-arrestin recruitment and internalisation (El Kouhen et al J Biol Chem (2001) 276(16): 12774-80). The activity of bilorphin for inducing C-terminal phosphorylation, β-arrestin recruitment and MOPr internalisation was assayed in the same AtT20 cell line used to determine G-protein activation. The activity of bilorphin-induced Ser.sup.375 phosphorylation was determined using a phosphosite specific antibody (FIGS. 6, 9; Doll et al Br J Pharmacol (2011) 164(2): 298-307). Surprisingly, and unlike other opioid peptides (Thompson et al Biochem Pharmacol (2016) 113:70-87), bilorphin produced very low levels of pSer375 immunoreactivity at saturating concentrations (30 μM, FIGS. 6, 9, 23B). Maximal phosphorylation by bilorphin was similar to and displayed a trend to be less than that produced by morphine, which is known to only weakly phosphorylate MOPr at Ser.sup.375 (FIG. 23) (McPherson et al (2010) Mol Pharmacol 78: 756-766).

    [0626] Ser.sup.375 Phosphorylation assay: AtT20 cells stably expressing MOPr were grown on coverslip to ˜50% confluence. Cells were serum starved for at least 30 min and then incubated in the absence or presence of the indicated ligand for 5-10 min at 37° C. Phosphorylation was terminated by fixing the cells with −30° C. methanol followed by 10 min incubation on ice. Cells were washed three times with phosphate buffered saline (PBS) and then heated in sodium citrate buffer (10 mM, 0.05% Tween 20, pH.6) for 20 min at 95° C. Cells were incubated with anti-phospho Ser.sup.375 antibody (1:200, Cell Signalling) overnight at room temperature. Next day, labeled receptors were stained with Alexa-fluor 488 antibody (1 μg/ml, 1 h at room temperature, Thermo Fisher Scientific). Imaging was performed as detailed below.

    [0627] Imaging: Images of receptor phosphorylation and internalization (Example 10) were acquired using Zeiss 510 Meta laser scanning confocal microscope at a resolution of 1024×1024 pixels using a 60× oil emulsion objective. Imaging parameters including laser intensity, photomultiplier tube (PMT) voltage and offset remained constant for each experiment. Mean fluorescence intensity was measured using ImageJ software to calculate mean gray value of an area defined outside a single cell. Each experiment was normalized to the mean of untreated cells as 0% and the mean of cells treated with saturating concentrations of Met-enk (30 μM) as 100%.

    Assessment of β-Arrestin 2 Recruitment

    [0628] Using MOPr-luciferease and β-arrestin2-YFP constructs, a BRET assay was performed to determine β-arrestin 2 recruitment to the receptor (FIGS. 7, 9) (Thompson et al Biochem Pharmacol (2016) 113:70-87). Similar to Ser.sup.375 phosphorylation, saturating concentrations of bilorphin induced very low levels of BRET efficiency relative to known agonists, and was also significantly less than morphine (up to 30 μM) (FIG. 7).

    [0629] Arrestin recruitment: Agonist-induced recruitment of β-arrestin2 to MOPr was examined using a BRET-based approach. AtT20 cells were plated in 10-cm dishes and co-transfected with MOPr C-terminally tagged with Rluc8 (MOPr-RLuc8), β-arrestin2-YFP and GRK2 (1 μg, 4 μg and 2 μg, respectively). 24 h after transfection wells were replated into white opaque 96-well plates (CulturPlate, PerkinElmer) and allowed to adhere overnight. Cells were washed with Hank's Balanced Salt Solution (HBSS) and equilibrated in HBSS for 30 min at 37° C. prior to the experiment. Coelenterazine h was added to a final concentration of 5 μM 10 min before dual fluorescence/luminescence measurement in a LUMIstar Omega plate reader (BMG LabTech). Baseline BRET was measured for 30 sec prior to addition of the indicated ligand. The BRET signal was calculated as the ratio of light emitted at 530 nm by YFP over the light emitted at 430 nm by Renilla luciferase 8 (RLuc8).

    [0630] Assessment of MOPr Internalization

    [0631] MOPr internalisation was assessed immunocytochemically after 30 minutes of agonist treatment (FIGS. 8, 9). Bilorphin produced almost no detectable internalisation of MOPr, compared to low level internalisation induced by morphine and robust internalisation driven by both endomorphin-2 and met-enkephalin (FIG. 9). Furthermore, co-incubation of bilorphin (10 μM) with an efficaciously internalizing agonist reduced internalisation (3 independent experiments using 3 μM DAMGO as agonist, data not shown). In summary, when normalised to the maximum response to met-enkephalin in each pathway, bilorphin displayed similar maximal G-protein efficacy to morphine with progressive reduction in relative efficacy across pathways from Ser.sup.371 phosphorylation, β-arrestin recruitment to internalisation (FIG. 9), suggesting that bilorphin is a G-protein biased opioid.

    [0632] Endocytosis assay: Receptor internalization was quantified using a ratiometric staining of membrane and internalized receptors. Briefly, AtT20 cells expressing FLAG-tagged MOPr were incubated with 1 μg/ml Alexa594-conjugated M1 monoclonal anti-FLAG (prepared from Alexa-fluor 594 with a succinimidyl ester moiety, Molecular Probes) for 30 min to label membrane receptors. Cells were then incubated for an additional 30 min with indicated agonist at 37° C. To unbind the M1 anti-FLAG antibody from the surface receptors, cells were quickly washed three times with ice-cold PBS lacking Mg.sup.2+ and Ca.sup.2+ and supplemented with 0.04% EDTA (pH 7.4). Cells were fixed with 4% paraformaldehyde in PBS for 20 min under non-permeabilized condition and then were incubated with anti-FLAG polyclonal antibody (1 μg/ml, 2 h at room temperature, Sigma Aldrich) followed by Alexa-fluor 488 goat anti-rabbit antibody (1 μg/ml, 1 h at room temperature, Thermo Fisher Scientific). Therefore, surface receptors were labeled with Alexa-fluor 488, while internalized receptors were labeled with Alexa-fluor 594. Percentage of internalized receptors was calculated as a ratio of mean 594 nm fluorescence intensity to total mean fluorescence intensity at 594 nm and 488 nm.

    [0633] In experiments where GRK2-YFP and R-arrestin 2-HA were expressed, Alexa-fluor 405 goat anti-rabbit (2 μg/mL, 1 h at room temperature, Abcam) was used as a secondary antibody in place of Alexa-fluor 488 goat anti-rabbit to avoid fluorescence spectral overlap with YFP. 405 nm fluorescence was false-colored to green for representative images. Only YFP positive cells were analyzed for internalization.

    Example 8: A E.SUB.MAX .and Δ log τ Determination

    [0634] Operational analysis, the de facto standard for quantifying biased signaling (Kenakin Curr Protoc Pharmacol. (2016) 74:2.15.1-2.15.15; Kelly Br J Pharmacol (2013) 169(7): 1430-46), suggests that bilorphin is G-protein biased relative to morphine (FIG. 23) and relative bias values for other agonists, including the arrestin-biased endomorphin-2 were similar to those previously reported (Rivero et al Mol Pharmacol (2012) 82(2): 178-88). However, operational analysis requires accurate determination of EC.sub.50, which was impractical for bilorphin due to very low internalisation efficacy, yielding poor functional affinity estimates with large error terms (FIG. 23) (Kelly Br J Pharmacol (2013) 169(7): 1430-46). Calculation of ΔE.sub.Max ratios from maximum response in each pathway and, Δ log τ provide complementary estimate of bias in signalling assays where no ceiling exists, i.e. all agonists are partial agonists (Burgueño et al Sci Rep (2017) 7(1):15389; Kelly Br J Pharmacol (2013) 169(7): 1430-46). The current assay conditions, under which the maximum possible G.sub.GIRK response for the high intrinsic efficacy agonist, met-enkephalin, was reduced to approximately 80% of the maximum possible response satisfies this criterion. This approach and accompanying data (FIGS. 18-22; Table 2) substantiated the G-protein bias of bilorphin relative to morphine, and to the strongly internalising peptides met-enkephalin (Thompson et al Mol Pharmacol (2015) 88(2): 335-346; McPherson et al (2010) Mol Pharmacol 78: 756-766) and endomorpin-2 (FIG. 23).

    [0635] Bias calculation and statistics: Agonist concentration response curves were fitted to a three-parameter concentration response curve, a logistic function with constrained slope of 1, in GraphPad Prism 7 producing estimates of curve location (EC.sub.50) and asymptote (E.sub.max). As basal activity was subtracted in all pathways the bottom of the curve was constrained to 0.

    [0636] The de facto standard for quantifying agonist affinity and efficacy to accurately determine biased signalling is the operational model of agonism (Black and Leff Proc R Soc Lond B Biol Sci (1983) 220(1219):141-62, Kenakin Mol Pharmacol (2015) 88(6):1055-61). Agonist concentration response data for each pathway was fitted to the operational model. Maximal effect in the system was defined by the reference full-agonist met-enkephalin and the slope of the transducer curve constrained to one. Efficacy (T) and affinity (KA) estimates were produced from the curve fit for test agonists endomorphin 2, bilorphin and morphine. log(τ/KA) values for each agonist were normalized by subtraction of the reference agonist met-enkephalin log(τ/KA) value within each pathway to produce Δ log(τ/KA). Subtraction across pathways produced ΔΔ log(τ/KA), a normalized estimated of each agonist's signaling bias. Previous papers on the operational model have advocated application of pooled variance in order to increase the power of these comparisons (Kenakin s al ACS Chem Neurosci (2012) 3(3):193-203). This approach is not suitable here, or in any situation with variable curve fit quality, due to the very low signaling efficacy of the biased agonists producing much larger error for the calculated parameters and invalidating the assumptions of pooled variance (Table 2). Standard error of the linear combinations of parameters was therefore propagated exactly under standard rules (Farrance and Frenkel Clin Biochem Rev (2012) 33(2):49-75, ISO/IEC Guide to Uncertainty in Measurement). Poor curve fit due to low signaling efficacy reduced the power of the ΔΔ log(τ/KA) approach generally and prevented confident interpretation of bias estimates from this approach (FIG. 23).

    [0637] In cases where all test agonists are partial compared to the reference agonist efficacy alone has been used to quantify bias (Burgueño et al Sci Rep (2017) 7(1):15389). In systems with low receptor reserve the asymptote of the logistic function, E.sub.max, is a robust, assumption free and affinity independent estimate of efficacy that approaches the value of operational efficacy. In systems with a linear relationship between agonist occupancy and effect such as the β-arrestin pathways studied here where there is no signal amplification, E.sub.max approximates operational efficacy, ‘τ’.

    [0638] E.sub.max was normalized to the reference agonist within each pathway and subtracted across comparison pathways to produce Δ normalized E.sub.max, an efficacy measure of bias. Observation of concentration-response curve position, which approaches operational affinity for all partial agonists presented here, across pathways measured shows bias in this instance does not appear affinity driven due to conservation of rank potency.

    [0639] Degrees of freedom were calculated by first conservatively taking the lower of the two sample sizes when the normalizing to met-enkephalin. In the case of Δ normalized E.sub.max, variance across the pathways could be assumed to be equal allowing degrees of freedom to be summed. In the case of ΔΔ log(τ/KA), low efficacy in the β-arrestin pathways caused heterogeneity of variance and degrees of freedom was approximated using the Welch-Satterwaite equation (ISO/IEC Guide to Uncertainty in Measurement).

    [0640] Bias of each agonist, in both A normalized E.sub.max and ΔΔ log(τ/KA) calculations, was tested by a one-way t-test to 0, the value of the reference agonist. The bias of bilorphin was then compared to morphine using a two-sample t-test, equal or unequal variance as appropriate. All comparisons were multiplicity adjusted using the Holm-Sidak ranking method (GraphPad Prism 7) within each pair of pathways examined.

    TABLE-US-00002 TABLE 2 Pathway log(tau/ Agonist E.sub.max log(EC.sub.50) K.sub.A K.sub.A) GIRK (80% knockdown) met-enkephalin  0.84 ± 0.01 −7.47 ± 0.03  *** 7.4 ± 0.03 endomorphin-2  0.64 ± 0.01 −6.9 ± 0.05 −6.5 ± 0.06 7.5 ± 0.04 morphine  0.52 ± 0.02 −7.6 ± 0.04 −6.9 ± 0.07 6.7 ± 0.05 bilorphin  0.51 ± 0.01 −7.2 ± 0.05 −6.8 ± 0.06 7.0 ± 0.05 oliceridine  0.33 ± 0.01 −7.6 ± 0.06 −7.3 ± 0.09 7.2 ± 0.08 Phosphorylation met-enkephalin 100* −7.1 ± 0.06 *** 7.1 ± 0.05 endomorphin-2 98 ± 2 −7.2 ± 0.07 *** 7.2 ± 0.05 morphine 52 ± 3 −6.4 ± 0.10 −5.8 ± 0.12 5.9 ± 0.09 bilorphin 39 ± 4 −6.1 ± 0.11 −6.4 ± 0.12 6.2 ± 0.10 oliceridine Not tested β-arrestin 2 recruitment met-enkephalin 102 ± 4  −6.3 ± 0.07 *** 6.2 ± 0.06 endomorphin-2 100 ± 3  −7.0 ± 0.06 *** 7.0 ± 0.06 morphine 41 ± 4 −6.1 ± 0.20 −5.9 ± 0.24 5.7 ± 0.19 bilorphin 18 ± 4 −6.1 ± 0.34 −6.0 ± 0.39 5.3 ± 0.32 oliceridine Not tested Internalisation met-enkephalin 100* −6.1 ± 0.05 *** 6.1 ± 0.03 endomorphin-2 94 ± 3 −6.4 ± 0.07 *** 6.2 ± 0.05 morphine 23 ± 2 −5.4 ± 0.12 −5.4 ± 0.21 4.8 ± 0.08 bilorphin  3 ± 1 −5.1 ± 0.40 −6.6 ± 2.4  4.9 ± 0.47 oliceridine No activity *Defined as 100% ***Full agonist, constrained K.sub.A

    Example 9: MOPr Internalisation in Cells Overexpressing GRK-YFP

    [0641] To compare the bias of bilorphin with the established, small non-peptidic, G-protein biased agonist, oliceridine, both of which produce very little internalisation, MOPr internalisation in cells overexpressing GRK2-YFP and B-arrestin 2-HA was examined, which was undertaken to enhance internalisation of morphine (Zhang et al Proc Natl Acad Sci USA (1998) 95(12):7157-62). In GRK2 positive cells, morphine, oliceridine and bilorphin all produced clear internalisation signals (FIG. 10). Quantification shows that even under these amplified conditions, bilorphin induces similar MOPr internalisation to oliceridine but less than morphine at saturating concentrations (FIGS. 10, 11A, 11B). Calculation of bias relative to morphine with the ΔE.sub.max method suggests that bilorphin exhibits similar or greater G-protein bias than oliceridine, establishing it is a novel, and also potentially safer G-protein biased opioid (FIG. 11B). See also FIGS. 11C and 11D.

    Example 10: Molecular Docking and Molecular Dynamics

    [0642] To investigate whether there is a conformational basis for bias, molecular docking and molecular dynamics (MD) simulations (Sutcliffe et al J Mol Biol (2017) 429(12):1840-1851) were performed with bilorphin and endomorphin-2 at mMOPr. Conformations (10,000) were taken from one microsecond simulations of each peptide in water. These ensembles were docked to the orthosteric binding pocket of the inactive MOPr crystal structure (Manglik et al Nature (2016) 537(7619): 185-190) using the Bristol University Docking Engine (BUDE) (McIntosh-Smith et al Int J High Perform Comput Appl (2015) 29(2):119-134). The lowest energy structures were inspected visually and prioritised according to the distance between the protonated amine of the ligand and Asp147.sup.332 (superscript numbers follow the Ballesteros-Weinstein numbering system for GPCR residues [Ballesteros and Weinstein Methods in Neurosciences (1995) 25:366-428] of less than 3 Å. Selected peptide-MOPr complexes were then embedded in a lipid and cholesterol bilayer and used in all-atom MD simulations to evaluate binding poses, residue interactions and receptor conformational changes. 8×125 ns simulations were performed, with different initial velocities, to give a total of 1 μs of trajectory data for each peptide. Endomorphin-2 was modelled as the cis isomer, as this conformation had the most stable and lowest energy binding pose after docking with BUDE and 125 ns of MD simulation.

    [0643] The predicted binding pose of bilorphin determined from the MD simulations is shown in FIGS. 18A and 18B. Bilorphin was predicted to bind within the orthosteric binding site, with the Dmt-Tyr1 orientated towards the intracellular side of MOPr. The rest of the tetrapeptide chain extended out towards the extracellular side of MOPr, making contacts with residues at the top of TM2 and TM7. Endomorphin-2 was also predicted to bind in the orthosteric site (FIGS. 18C, 18D), with the phenol group of Tyr1 interacting with His297.sup.6.52, and the rest of the peptide chain extending towards ECL1 and ECL2 and the top of TM2. The RMSD plot in FIG. 19A shows that the binding pose of bilorphin was relatively stable over the 1 μs simulation time after an initial deviation from the docked pose of ˜1.5 Å. The backbone of endomorphin-2 was stable in its bound pose, with some fluctuation in the RMSD plot introduced by the flexibility of the Phe4 aromatic ring which switched between 3 main poses (FIG. 19B).

    [0644] Both peptides maintained an ionic interaction with the essential opioid binding residue Asp147.sup.3.32 of the MOPr complex, and interacted with the conserved rotamer toggle switch Trp293.sup.6.48, for the entire simulation time (FIG. 20A). Both peptides also interacted with His297.sup.6.52; endomorphin-2 directly, and bilorphin switching between a direct interaction and hydrogen bonding via a bridging water molecule (inset in FIG. 18A). However, there were differences in how these peptides interact with the MOPr binding pocket. For instance, bilorphin, but not endomorphin-2, interacted with Tyr75.sup.1.39 in TM1. On the other hand, endomorphin-2 interacted with the extracellular loops, contacting W133.sup.ECL1 in ECL1 for the entire simulation time, and transiently interacting with Cys217.sup.ECL2, Thr218.sup.ECL2 and Leu219.sup.ECL2 of ECL2, but these interactions were absent for bilorphin. Moreover, endomorphin-2 made a greater number of contacts with TM3 and TM5 than bilorphin.

    [0645] Principal component analysis (PCA) was employed to examine the conformational changes in the receptor transmembrane helices. After fitting to remove global rotation and translation of the system, the covariance matrix was generated from just the alpha carbons of the MOPr transmembrane domains, to avoid including the highly dynamic loops in the analysis. The receptor conformations at each time point were projected onto principal components (PC) 1 and 2, and plotted in FIG. 20B. PC1 and PC2 accounted for 28.9% and 10.9% of the variance, respectively. Both peptide-MOPr complexes sampled conformations across PC2, but clustered differently based on PC1. By generating a pseudo-trajectory of PC1, and extracting structures from the actual simulations that represent extremes of PC1, we were able to visualise the helix movements contributing to the principal component.

    [0646] As depicted in FIG. 21, PC1 primarily described alternative conformations in the extracellular region of the receptor close to the orthosteric binding site, and to a lesser extent differences in the intracellular portions of the helices. With bilorphin bound, there was a bulging of the middle portion of TM1 and a shift outwards from the helix bundle, relative to the endomorphin-2-bound receptor. There were also substantial movements of the extracellular ends of TMs 2, 6 and 7, and a kink formed in TM4 allowed the extracellular part of this helix to move towards TM3 with endomorphin-2 bound. A smaller movement of TM3 towards TM2 in the endomorphin-2-bound receptor, whereby Met151.sup.3.36 shifted ˜1.7 Å from its initial position, is in agreement with the active conformation of TM3 observed in the agonist-bound crystal structure (Huang et al Nature (2015) 524(7565): 315-21). These alternative conformations of the helices around the orthosteric binding site were also reflected in the volume of the binding pocket, as calculation of the volume of the orthosteric binding site using CASTp revealed the bilorphin binding pocket volume to be on average 1.6 times greater than with endomorphin-2 bound (Dundas et al Nucleic Acids Res (2006) 34(Web Server issue):W116-8) (FIG. 22).

    [0647] On the intracellular side of MOPr, PC1 described inward movements of TMs 5, 6 and 7 with endomorphin-2 bound, compared to the bilorphin-bound MOPr (FIG. 21).

    [0648] The cryo-electron microscopy structure of the MOPr-Gi complex bound to DAMGO was recently resolved (Koehl Nature (2018) 558: 547-552). Prior to the release of this structure, the inventors performed MD simulations with DAMGO at the MOPr (FIG. 28) using the methods described here for bilorphin and endomorphin-2. The position of DAMGO as well as the ligand residue interactions in the cryo-EM structure and in the model (FIG. 28) were nearly identical, providing confidence that our docking and MD strategy for bilorphin and endomorphin-2 is likely to be relevant to the ligand-MOPr interactions occurring in vivo.

    [0649] Analysis of the MD data therefore suggests that the different ligand-residue interactions for these oppositely biased peptides may lead to the alternative receptor conformations described by the PCA, and hence the opposing bias profiles of bilorphin and endomorphin-2.

    [0650] Molecular dynamics: Generation of peptide conformations: Generation of peptide conformations: The 3D conformers of bilorphin and endomorphin-2 (EM2) were built in Chimera (Petterson et al J Comput Chem (2004) 25(13): 1605-12). Two endomorphin-2 conformers were used, with the Tyr1-Pro2 peptide bond modelled as either the cis or trans isomer, and treated as separate ligands for MD simulation and docking. Peptides were protonated at the N-terminal tyrosine and parameterised with Antechamber and the general Amber force field (Wang et al J Mol Graph Model (2006) 25(2): 247-60, Wang et al J Comput Chem (2004) 25(9): 1157-74). Conformer generation was performed by running 1 μs MD simulations of each peptide in explicit solvent (0.15 M NaCl and TIP3P water) under the Amber ff14SB force field. These trajectory data were analysed with cpptraj (Roe et al J Chem Theory Comput (2013) 9(7): 3084-95) to extract 10000 conformations for each peptide to use in molecular docking.

    [0651] Docking of peptides to MOPr: Molecular docking was performed with the Bristol University Docking Engine (BUDE) (McIntosh-Smith et al Int J High Perform Comput Appl. (2015) 29(2): 119-134.). Peptides were docked to an inactive MOPr model obtained from the x-ray crystal structure of the antagonist-bound MOPr (PDB: 4DKL) (Manglik et al Nature (2012) 485(7398): 321-6). The protein was prepared in Insight II (Accelrys) as follows; ligands and the T4 lysozyme were removed, and a loop search performed to find a homologous loop to model in the missing intracellular loop 3. A loop was selected by visual inspection and the residues changed to the correct mouse MOPr sequence. Molecular docking to this MOPr structure was performed with each of the three peptides, bilorphin, cis-endomorphin-2 and trans-endomorphin-2, independently. The following describes the docking procedure for one peptide. Multi-conformer docking was run such that the 10000 conformations of the peptide were treated as independent molecules. A box of size 15, 15, 15 Å centred on the orthosteric binding site was designated as the search space. BUDE's genetic algorithm was used to search the available pose space for the best energy poses. A total of 105,000 poses were sampled for each of the 10,000 peptide conformers. The total possible number of poses was 1.57×10.sup.8 for each conformer, corresponding to x,y,z translation within the box and 360° rotation in all axes in 10° increments. The 50 lowest energy binding poses were inspected visually and subjected to a distance constraint between the protonated amine of the ligand and Asp147.sup.3.32 of less than 3 Å. The selected peptide-MOPr complexes were used in short (125 ns) MD simulations to assess the stability of the binding pose, before a full 1 μs of trajectory data was collected, as described below. Based on the docking data and the initial 125 ns MD simulations, the cis-endomorphin-2 conformer was chosen for further simulation.

    [0652] MD simulations: Each peptide-MOPr complex was embedded in a POPC:POPE:cholesterol lipid bilayer at a 5:5:1 ratio using the replacement method, and the simulation box (initial dimensions: 90, 110, 90 Å) solvated with TIP3P water and NaCl (150 mM), using the CHARMM-GUI software (Jo et al J Comput Chem (2008) 29(11): 1859-65). Amber parameter topology and coordinate files were prepared in LEaP. Structures were minimised over 10000 steps, then the system was heated under constant volume and pressure with lipids restrained, from 0 K to 100 K over 5 μs, and then from 100 K to 310 K over 100 μs. 10 rounds of 500 μs equilibration was performed under constant pressure to equilibrate the periodic box dimensions. Simulations were run in 8×125 ns parallel steps under the Amber ff14SB and Lipid14 force fields (Maier et al J Chem Theory Comput (2015) 11(8): 3696-713; Dickson et al J Chem Theory Comput (2014) 10(2): 865-79), producing 1 μs of simulation data for each peptide-MOPr complex. Temperature and pressure were controlled using the Langevin thermostat and the anisotropic Berendsen barostat, with a 2 fs time step and trajectories written every 100 μs. Trajectories were visualised in VMD (Humphrey et al J Mol Graph (1996) 14(1): 33-8, 27-8), analysis was performed using cpptraj (Roe et al J Chem Theory Comput (2013) 9(7): 3084-95), and images were prepared in Chimera (Petterson et al J Comput Chem (2004) 25(13): 1605-12).

    [0653] Principal Component Analysis: Trajectories were aligned to a set of “core residues” showing the least fluctuation during the simulation time to remove general translation and rotation of the protein in analysis. Principal component analysis was performed on the 3D Cartesian coordinates of the alpha carbons of the transmembrane domains of all trajectories, yielding 567 eigenvalues. Receptor conformations at each simulation time point were projected onto the first 2 PCs, accounting for ˜40% of the variance.

    Example 11: In Vivo Assessment

    [0654] The actions of bilorphin were assessed in vivo. Bilorphin failed to inhibit nociception in the hotplate test when administered subcutaneously (100 mg/kg, n=4) or intravenously (50 mg/kg n=4) versus vehicle. By contrast, bilorphin was antinociceptive after intrathecal injection (5 nmol/mouse, peak effect 41±9% MPE n=4, versus 0±1.5% for vehicle, n=4), suggesting the lack of systemic activity is due to poor penetration of the blood brain barrier (BBB). Several bilorphin analogues were developed with substitutions thought to enhance BBB permeability, including gycosylation near the C-terminal. The di-glycosylated analogue, bilactorphin (3g), was a potent analgesic after systemic administration (s.c; ED.sub.50 of 34 μmol/kg, 95% CI=28-40 μmol/kg; FIGS. 12, 13) and was nearly equipotent with morphine (ED.sub.50 of 27 μmol/kg, 95% CI=24-30 μmol/kg; FIG. 13) and was antagonised by co-administration of the opiod antagonist, naltrexone (FIG. 12). Bilactorphin was also active after i.v (peak effect of 88.9±11.8 versus 14.4±1.8% MPE for vehicle, n=3-4) or oral administration (FIGS. 24A, 24B) In contrast the mono-glycosylated analogue (3h) was systemically inactive, consistent with the greater analgesic effects elicited by systemic administration of disaccharide vs. monosaccharide modified opioid peptide. (Li et al Future Med Chem (2012) 4(2): 205-26). These findings establish that the LDLD opioid peptide backbone is a viable framework for further development of G-protein biased opioid analgesics. Like bilorphin, bilactorphin, was a potent partial opioid agonist in AtT20 cells (without fractional inactivation of MOPr) but exhibited a small loss of potency compared with bilorphin (FIGS. 14, 15). Bilactorphin did, however, display modest internalization and β-arrestin recruitment compared to bilorphin suggesting the potential superiority of other substitutions that do not disrupt G-protein bias of the parent bilorphin (FIGS. 16, 17).

    [0655] Nociception (Analgesia) testing: All experiments involving animals were approved by the University of Sydney Animal Ethics Committee (AEC. Protocol number K00/12-2011/3/5650). Experiments were performed under the guidelines of the Australian code of practice for the care and use of animals for scientific purposes (National Health and Medical Research Council, Australia, 7th Edition). Great care was taken to minimise animal suffering during these experiments and to reduce the number of animals used. Adult male C57BL/6 mice (20-25 g) were housed 5-6 per cage in individually ventilated cages under controlled light (12:12 h, lights on at 6 am) and climate (18-23° C., 40-60% humidity) conditions. Food and water was available ad libitum. Mice were given at least 7 days to habituate to housing facilities prior to handling, and handled by the experimenter for 4 days prior to testing. Experiments were conducted between 8 am and 6 μm in a quiet, temperature-controlled room (21±1° C.). The experimenter was blind to all drugs tested. Animals were tested on a 54° C. hotplate, with a maximum cut-off time of 20 seconds to prevent tissue damage. Endpoints were hindpaw lick, hindpaw flutter or jump. Baseline latency was recorded immediately before subcutaneous injection with morphine, bilactorphin or vehicle (20% PEG400/saline v/v) in a total volume of 200 μL. Mice were tested 30, 60, 90, 150, 210, 330 and 450 min following injection. The percentage of maximal possible effects (% MPE) were calculated as follows: % MPE=(test latency−baseline latency)/(cutoff latency−baseline latency)×100%. The cut-off latency was 20 seconds. Significant differences were assessed with a two-way ANOVA and Tukey's post hoc multiple comparisons test. The dose-response curves for bilactorphin and morphine were calculated using the maximal responses for each dose between 30-90 min. Doses were transformed to the logarithm of μmol/kg. A two-way ANOVA was used to compare data at equimolar doses.

    Example 12: In Vivo Assessment

    [0656] The peptides of FIG. 27 were tested exactly as described above in Example 11. Experiments were performed under the guidelines of the Australian code of practice for the care and use of animals for scientific purposes (National Health and Medical Research Council, Australia, 7th Edition). Great care was taken to minimise animal suffering during these experiments and to reduce the number of animals used. Adult male C57BL/6 mice (20-25 g) were housed 5-6 per cage in individually ventilated cages under controlled light (12:12 h, lights on at 6 am) and climate (18-23° C., 40-60% humidity) conditions. Food and water was available ad libitum. Mice were given at least 7 days to habituate to housing facilities prior to handling, and handled by the experimenter for 4 days prior to testing. Experiments were conducted between 8 am and 6 μm in a quiet, temperature-controlled room (21±1° C.). The experimenter was blind to all drugs tested. Animals were tested on a 54° C. hotplate, with a maximum cut-off time of 20 seconds to prevent tissue damage. Endpoints were hindpaw lick, hindpaw flutter or jump. Baseline latency was recorded immediately before subcutaneous injection with morphine, peptide, or vehicle (20% PEG400/saline v/v) in a total volume of 200 μL. Mice were tested 30, 60, 90, 150, 210, 330 and 450 min following injection. The percentage of maximal possible effects (% MPE) were calculated as follows: % MPE=(test latency−baseline latency)/(cutoff latency−baseline latency)×100%. The cut-off latency was 20 seconds. Integrated Area Under the Curve over one hour (AUC: response in seconds×time in minutes) for hotplate responses measured, 5, 10, 20, 30 and 60 minutes after subcutaneous injection of each drug or saline were calculated for each drug by measuring triangulated areas of response (seconds) multiplied by time of testing (minutes) for 60 minutes at the times specified. Differences were analysed with a one way ANOVA with Fisher's LSD post-hoc tests.

    Discussion

    [0657] The invention relates to the identification of a novel peptidic backbone useful for the development of a new peptidic class of G-protein biased opioids. The results generated indicate that his peptidic backbone can be used to develop an orally active opioid agonist with G-protein biased pharmacology. This novel LDLD structure has not previously been isolated from a eukaryote. The parent natural product, bilaid C (3a in Table 1) from which bilorphin was derived, is a relatively weak opioid and the potential natural function of the opioid agonist activity for this estuarine yeast is uncertain. The unexpected biostability of the LDLD structure and its novel opioid pharmacology, can be used to develop safer opioids.

    [0658] G-protein biased opioid agonists have been proposed as a route to improving therapeutic profile. Among known peptidergic opioid agonists, which have little bias or bias toward β-arrestin signalling, bilorphin's pharmacological profile is most unusual as it is atypically G-protein biased compared with other natural opioid peptides, although a synthetic opioid cyclopeptide with G-protein bias was recently reported. Bilorphin is comparably biased to the Phase III drug candidate oliceridine. Glycosylation produced an analogue active in vivo via subcutaneous and oral administration, validating the bilorphin tetrapeptide backbone as a platform for further development of biased opioid agonists. Pre-clinical development of such G-protein biased agonists show a strikingly favourable profile with reduced respiratory depression and constipation. The first such compound to reach clinical trials, oliceridine (TRV130), was reported to have an increased window between antinociceptive and respiratory depressive activity and appears to be safer in humans than morphine for equi-analgsesic doses. Similarly, a series of substituted fentanyl analogues was observed to produce increased therapeutic window for respiratory depression correlating with increased G-protein versus β-arrestin 2 recruitment. PZM21, developed via in silico screening with novel receptor interactions, appears to be a G-protein biased agonist when compared to morphine. It was reported to produce no respiratory depression but this has not been reproduced by others (Hill et al Br J Pharmacol (26 Mar. 2018) epub PMID: 29582414).

    [0659] To investigate whether bias could be explained by the differential interaction of bilorphin and endomorphin-2 with MOPr, or by distinct receptor conformational changes initiated by each, Molecular Dynamics simulations with bilorphin or endomorphin-2 bound to MOPr were undertaken. Both peptides were docked to the orthosteric binding site of MOPr and displayed differences in ligand-residue interactions, which may translate to their differing bias profiles. Notably, endomorphin-2 transiently interacted with residues in ECL2, including the conserved residue Leu219, proposed to be important for arrestin-bias and ligand residence time at the 5HT2A and 5HT2B receptors and other aminergic GPCRs. In contrast, bilorphin did not contact the extracellular loops. The interactions between the peptides and the MOPr binding pocket appear to translate to the divergent conformational changes observed by the PCA. Specifically, with endomorphin-2 bound the extracellular portions of the TMDs moved inwards so that the orthosteric binding pocket contracted relative to the bilorphin-bound MOPr. On the intracellular side of the receptor TM5, 6 and 7 adopted distinct positions depending on the bound peptide, mainly an inward shift of these helices in the presence of endormorphin-2. As has been previously suggested, interaction with a G protein or arrestin may be required for MOPr to achieve a fully active state, and therefore it is unsurprising that in these MD simulations of the receptor and agonist alone, the intracellular portion of the complex did not sample the fully active conformation captured in the agonist and nanobody-bound crystal structure.

    [0660] Whilst it remains challenging at present to associate ligand-induced GPCR conformations with differential coupling to G proteins or arrestins, the subtle differences in ligand-residue interactions and conformations of the MOPr helices that we have modelled here may represent the initial changes induced by the oppositely biased peptides, bilorphin and endomorphin-2, which lead to their different signalling profiles and potentially adverse effect liabilities.

    [0661] It remains uncertain, however, whether G-protein bias per se is the sole property contributing to improved safety of drugs such as oliceridine (TRV130) (Singla et al J Pain Res (2017) 10: 2413-2424). Using receptor knockdown, it is shown here that TRV130 has very low G-protein efficacy compared with morphine. Similar results have been reported for another opioid, PZM21, claimed to be safer than morphine (Hill et al Br J Pharmacol (26 Mar. 2018) epub PMID: 29582414) and it is difficult to evaluate G-protein efficacy of novel biased agonists in other studies because assays were insensitive to the relatively low G-protein efficacy of morphine (Schmid et al Cell (2017) 171(5): 1165-1175; DeWire et al J Pharmacol Exp Ther (2013) 344(3): 708-17). Very low G-protein efficacy may indeed be a confounding factor in the pre-clinical and clinical studies of side effect profile, given that agonists with very low G-protein efficacy such as buprenorphine are not strongly biased but are well characterised to produce less respiratory depression and overdose death than highly efficacious agonists such as morphine and methadone. Because bilorphin is strongly G-protein biased and has nearly equivalent G-protein efficacy to morphine, its analogs will facilitate the direct test of the influence of bias without being confounded by differing G-protein efficacy.