OPIOID PEPTIDE

Abstract

The present invention relates to an opioid peptide represented by general formula TyrGlyGly-X1-X2-X3-X4, wherein: X1 is represented by Thr or Glu; X1 is represented by Gly or Thr or Ala; X3 is represented by Ala or Val or Gly or Glu; and X4 is represented by His or Gln or Thr or SEQ ID NO:1, or HisTyr or GlnTyr or ThrTyr.

The invention also relates to pharmaceutical and food compositions comprising the peptide and to the use of the same for analgesic purposes, and/or for providing a feeling of satiety to a subject.

Claims

1. Opioid peptide or salt thereof having naloxone-like binding activity characterized by being represented by formula:
TyrGlyGly-X1-X2-X3-X4 where: X1 is represented by Thr or Glu; X1 is represented by Gly or Thr or Ala; X3 is represented by Ala or Val or Gly or Glu; and X4 is represented by His or Gln or Thr or SEQ ID NO:1, or HisTyr or GlnTyr or ThrTyr.

2. A peptide according to claim 1 characterized by being SEQ D NO: 2.

3. A peptide according to claim 1 characterized by being SEQ D NO: 3.

4. A peptide according to claim 1 characterized by being SEQ D NO: 4.

5. A peptide according to claim 1 characterized by being SEQ D NO: 5.

6. A peptide according to claim 1 characterized by being SEQ D NO: 6.

7. A pharmaceutical composition characterized by comprising at least one opioid peptide having naloxone-like binding activity defined in claim 1 or salt thereof and at least one pharmaceutically acceptable carrier.

8. A pharmaceutical composition according to claim 7, characterized by having a concentration of peptide or salt thereof in the composition ranging from 0.001% (w/w) 99.999% (w/w).

9. A pharmaceutical composition according to claim 7 characterized by comprising an analgesic compound.

10. A pharmaceutical composition according to claim 9 characterized by comprising morphine as the analgesic compound.

11. A pharmaceutical composition according to claim 7 characterized in that it is in the pharmaceutical form selected from a tablet, capsule, elixir, solution, suspension, emulsion, lotion, cream, ointment, granulate or powder.

12. A pharmaceutical composition characterized by comprising at least one pharmaceutically acceptable carrier and a promoter-forming agent of at least one peptide defined in claim 1.

13. A pharmaceutical composition according to claim 12 characterized by having a genetically modified microorganism as promoter agent.

14. A pharmaceutical composition according to claim 12 characterized in that it is in the pharmaceutical form selected from a tablet, capsule, elixir, solution, suspension, lotion, cream, ointment, granulate or powder.

15. A food composition characterized in that it comprises at least one food grade substance and at least one peptide defined in claim 1.

16. A food composition according to claim 15 characterized in that it comprises an amount of peptide or salt thereof ranging from 0.001% (w/w) to 99.999% (w/w).

17. A food composition characterized by comprising at least one food-grade substance and one promoter-forming agent of at least one peptide defined in claim 1.

18. A food composition according to claim 17 characterized in that it comprises a genetically modified micro-organism as promoter agent.

19. A nutraceutical composition characterized in that it comprises at least one food grade substance and at least one peptide defined in claim 1.

20. A nutraceutical composition according to claim 19 characterized in that it comprises an amount of peptide or salt thereof ranging from 0.001% (w/w) to 99.999% (w/w).

21. A nutraceutical composition characterized in that it comprises at least one food-grade substance and one promoter-forming agent of at least one peptide defined in claim 1.

22. A nutraceutical composition according to claim 21 characterized in that it comprises a genetically modified micro-organism as promoter agent.

23. The use of the peptide defined in claim 1 characterized in that it is for providing analgesia in a subject.

24. The use of the peptide defined in claim 1 characterized in that it is for providing a feeling of satiety to a subject.

25. The use of the pharmaceutical composition defined in claim 7, characterized in that it is for providing analgesia in a subject.

26. The use of the pharmaceutical composition defined in claim 7 characterized in that it is for providing a feeling of satiety to a subject.

27. The use of the food composition defined in claim 15 characterized in that it is for providing a feeling of satiety to a subject.

28. The use of the nutraceutical composition defined in claim 19 characterized in that it is for providing a feeling of satiety to a subject.

29. A method of activating the opioid receptor characterized by comprising administering to a subject an effective amount of at least one peptide defined in claim 1.

30. A method of activating the opioid receptor characterized by comprising administering to a subject a pharmaceutical composition as defined in claim 7.

31. A method of activating the opioid receptor characterized by comprising administering to a subject a pharmaceutical composition as defined in claim 15.

32. A method of activating the opioid receptor characterized by comprising administering to a subject a nutraceutical composition as defined in claim 19.

33. A method of providing analgesia characterized by comprising administering to a subject an effective amount of at least one peptide defined in claim 1.

34. A method of providing analgesia characterized by comprising administering to a subject a pharmaceutical composition as defined in claim 7.

35. A method of providing satiety to a subject characterized by comprising administering to a subject an effective amount of at least one peptide defined in claim 1.

36. A method of providing satiety to a subject characterized by comprising administering to a subject a pharmaceutical composition as defined in claim 7.

37. A method of providing satiety to a subject characterized by comprising administering to a subject a food composition as defined in claim 15.

38. A method of providing satiety to a subject characterized by comprising administering to a subject a nutraceutical composition as defined in claim 19.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0053] FIG. 1—Latency index acquired at different times after application (i.p.) in mice using the Hot Plate test for 120 minutes.

[0054] FIG. 2—Latency index acquired at different times after application (i.p.) in mice using the Hot Plate test for 150 minutes.

[0055] FIG. 3—Latency index acquired at different times after application (i.p.) in mice using the Hot Plate test for 240 minutes. At 150 minutes the application of naloxone was suspended in the animals treated with the peptide sequence SEQ ID NO: 6.

[0056] FIG. 4—Latency index acquired at different times after application (i.p.) in mice using the Tail Flick test for 240 minutes.

[0057] FIG. 5—Latency index acquired at different times after application (i.p.) in mice using the Hot Plate test for 240 minutes.

[0058] FIG. 6—Latency index acquired at different times after application (i.p.) in mice using the Tail Flick test for 240 minutes.

[0059] FIG. 7—Latency index acquired at different times after application (i.p.) in mice using the Hot Plate test for 240 minutes.

[0060] FIG. 8—Latency index acquired at different times after application (i.p.) in mice using the Tail Flick test for 240 minutes.

[0061] FIG. 9—Latency index acquired at different times after application (i.p.) in mice using the Hot Plate test for 240 minutes.

[0062] FIG. 10—Latency index acquired at different times after application (i.p.) in mice using the Tail Flick test for 240 minutes.

[0063] FIG. 11—Representation of the fragments obtained at the different incubation times with the enzyme trypsin. Analysis performed by mass spectrometry, MALDI. The first spectrum, marked with the letter A, refers to the time of 15 minutes; the second spectrum marked with the letter B indicates the time of 30 minutes; C indicates the time of 2 hour2; D indicates the time of 4 hours; and, finally, E is the control peptide (SEQ ID NO: 6).

[0064] FIG. 12—Representation of the fragments obtained at the different incubation times with the enzyme trypsin. Analysis performed by mass spectrometry, MALDI. The first spectrum, marked with the letter A, refers to the time of 30 minutes; the second spectrum marked with the letter B indicates the time of 1 hour; C indicates the time of 2 hours; D indicates the time of 4 hours; and, finally, E is the control peptide (Leu-enkephalin).

DETAILED DESCRIPTION OF THE INVENTION

[0065] The term “patient” used in this invention includes humans and also other mammals such as intensive and extensive breeding animals, zoo animals, companion animals such as cat, dog and horse.

[0066] The term “pharmaceutically acceptable carrier” used in the present invention refers to a diluent or adjuvant or excipient or carrier with which or in which the active compound is administered.

[0067] The term “therapeutically effective amount” used in the invention refers to the amount of active compound which, when administered to a patient to prevent or treat a condition (such as pain, hunger, etc.) is sufficient to effect such treatment. The therapeutically effective amount varies according to the active compound, the patient's condition, the severity of the condition, the age, weight and other characteristics of the patient.

[0068] The term “nutraceutical” used in the invention refers to a food or part of a food that provides medical and health benefits, including prevention and/or treatment of a condition.

[0069] The present invention relates to an opioid peptide or salt thereof having naloxone-like binding activity represented by the sequence:

TyrGlyGly-X1-X2-X3-X4 where:

[0070] X1 is represented by Thr or Glu;

[0071] X1 is represented by Gly or Thr or Ala;

[0072] X3 is represented by Ala or Val or Gly or Glu; and

[0073] X4 is represented by His or Gln or Thr or SEQ ID NO:1, or HisTyr or GlnTyr or ThrTyr.

[0074] Preferably, the present invention relates to peptides or salts thereof having naloxone-like binding activity and sequences represented by SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

[0075] The present invention further relates to a pharmaceutical composition comprising at least one opioid peptide or salt thereof having naloxone-like binding activity and at least one pharmaceutically acceptable carrier. The said peptide of the pharmaceutical composition is represented by the sequence:

TyrGlyGly-X1-X2-X3-X4 where:

[0076] X1 is represented by Thr or Glu;

[0077] X1 is represented by Gly or Thr or Ala;

[0078] X3 is represented by Ala or Val or Gly or Glu; and

[0079] X4 is represented by His or Gln or Thr or SEQ ID NO:1, or HisTyr or GlnTyr or ThrTyr.

[0080] Preferably, the peptides of the pharmaceutical composition of the present invention are represented by SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

[0081] In the pharmaceutical composition of the invention the concentration of peptide or its salt having naloxone-like binding activity ranges preferably from 0.001% (w/w) to 99.999% (w/w).

[0082] In an alternative embodiment of the pharmaceutical composition of the invention, said composition further comprises an analgesic compound. Preferably, said analgesic compound is morphine.

[0083] The pharmaceutical composition of the invention may take various pharmaceutical forms. Among them, we can mention: tablet, capsule, elixir, solution, suspension, emulsion, lotion, cream, ointment, granulate, powder or lyophilized powder.

[0084] The present invention also relates to a pharmaceutical composition comprising at least one pharmaceutically acceptable carrier and a promoter-forming agent of at least one peptide having naloxone-like binding activity. The said peptide is represented by the sequence:

TyrGlyGly-X1-X2-X3-X4 where:

[0085] X1 is represented by Thr or Glu;

[0086] X1 is represented by Gly or Thr or Ala;

[0087] X3 is represented by Ala or Val or Gly or Glu; and

[0088] X4 is represented by His or Gln or Thr or SEQ ID NO:1, or HisTyr or GlnTyr or ThrTyr.

[0089] Preferably, the peptides of the pharmaceutical composition of the present invention are represented by SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

[0090] In a preferred embodiment of said pharmaceutical composition, the promoter is a genetically modified microorganism.

[0091] There are several pharmaceutical forms in which the pharmaceutical compositions of this invention may be present. Among these forms are the tablet, capsule, elixir, solution, suspension, lotion, cream, ointment, granulate, powder or lyophilized powder.

[0092] The present invention further relates to a food composition comprising at least one food grade substance and one opioid peptide or salt thereof having naloxone-like binding activity. The said peptide is represented by the sequence:

TyrGlyGly-X1-X2-X3-X4 where:

[0093] X1 is represented by Thr or Glu;

[0094] X1 is represented by Gly or Thr or Ala;

[0095] X3 is represented by Ala or Val or Gly or Glu; and

[0096] X4 is represented by His or Gln or Thr or SEQ ID NO:1, or HisTyr or GlnTyr or ThrTyr.

[0097] Preferably, the peptides of the pharmaceutical composition of the present invention are represented by SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

[0098] In the food composition of the present invention the concentration of peptide or its salt having naloxone-like binding activity ranges preferably from 0.001% (w/w) to 99.999% (w/w).

[0099] The present invention also relates to a food composition comprising at least one food grade substance and a promoter-forming agent of at least one opioid peptide or salt thereof having naloxone-like binding activity. The said peptide is represented by the sequence:

TyrGlyGly-X1-X2-X3-X4 where:

[0100] X1 is represented by Thr or Glu;

[0101] X1 is represented by Gly or Thr or Ala;

[0102] X3 is represented by Ala or Val or Gly or Glu; and

[0103] X4 is represented by His or Gln or Thr or SEQ ID NO:1, or HisTyr or GlnTyr or ThrTyr.

[0104] Preferably, the peptides of the pharmaceutical composition of the present invention are represented by SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. In a preferred embodiment of said food composition of the invention, the promoter-forming agent is a genetically modified microorganism.

[0105] The present invention further relates to a nutraceutical composition comprising at least one food grade substance and one opioid peptide or salt thereof having naloxone-like binding activity. The said peptide is represented by the sequence:

TyrGlyGly-X1-X2-X3-X4 where:

[0106] X1 is represented by Thr or Glu;

[0107] X1 is represented by Gly or Thr or Ala;

[0108] X3 is represented by Ala or Val or Gly or Glu; and

[0109] X4 is represented by His or Gln or Thr or SEQ ID NO:1, or HisTyr or GlnTyr or ThrTyr.

[0110] Preferably, the peptides of the nutraceutical composition of the present invention are represented by SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

[0111] In the nutraceitucal composition of the present invention the concentration of peptide or its salt having naloxone-like binding activity ranges preferably from 0.001% (w/w) to 99.999% (w/w).

[0112] The present invention also relates to a nutraceitucal composition comprising at least one food grade substance and a promoter-forming agent of at least one opioid peptide or salt thereof having naloxone-like binding activity. The said peptide is represented by the sequence:

TyrGlyGly-X1-X2-X3-X4 where:

[0113] X1 is represented by Thr or Glu;

[0114] X1 is represented by Gly or Thr or Ala;

[0115] X3 is represented by Ala or Val or Gly or Glu; and

[0116] X4 is represented by His or Gln or Thr or SEQ ID NO:1, or HisTyr or GlnTyr or ThrTyr.

[0117] Preferably, the peptides of the nutraceutical composition of the present invention are represented by SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. In a preferred embodiment of said nutraceitucal composition of the invention, the promoter-forming agent is a genetically modified microorganism.

[0118] The present invention further relates to the use of the peptide defined in the invention in order to provide analgesia in an individual. The said peptide is an opioid peptide or salt thereof having naloxone-like binding activity represented by the sequence:

TyrGlyGly-X1-X2-X3-X4 where:

[0119] X1 is represented by Thr or Glu;

[0120] X1 is represented by Gly or Thr or Ala;

[0121] X3 is represented by Ala or Val or Gly or Glu; and

[0122] X4 is represented by His or Gln or Thr or SEQ ID NO:1, or HisTyr or GlnTyr or ThrTyr.

[0123] Preferably, the invention relates to the use of at least one peptide selected from those having sequence represented by SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 for the purpose of providing analgesia in a subject.

[0124] The present invention further relates to the use of the peptide defined in the invention in order to provide a sense of satiety to a subject. The said peptide is an opioid peptide or salt thereof having naloxone-like binding activity represented by the sequence:

TyrGlyGly-X1-X2-X3-X4 where:

[0125] X1 is represented by Thr or Glu;

[0126] X1 is represented by Gly or Thr or Ala;

[0127] X3 is represented by Ala or Val or Gly or Glu; and

[0128] X4 is represented by His or Gln or Thr or SEQ ID NO:1, or HisTyr or GlnTyr or ThrTyr.

[0129] Preferably, the invention relates to the use of at least one peptide selected from those having sequence represented by SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 for the purpose of providing a sense of satiety to a subject.

[0130] The invention further relates to the use of the pharmaceutical compositions described in the invention for providing analgesia in a subject. Said analgesia occurs via the opioid receptor.

[0131] The invention further relates to the use of the pharmaceutical compositions described in the invention for providing a sense of satiety to a subject. Said sense of satiety occurs via the opioid receptor.

[0132] The invention further relates to the use of the food compositions described in the invention for providing a sense of satiety to a subject. Said sense of satiety occurs via the opioid receptor.

[0133] The invention further relates to the use of the nutraceutical compositions described in the invention for providing a sense of satiety to a subject. Said sense of satiety occurs via the opioid receptor.

[0134] Another object of the invention is a method of activating the opioid receptor, such method comprising administering to a subject an effective amount of at least one opioid peptide or salt thereof having naloxone-like binding activity.

[0135] The said peptide is represented by the sequence:

TyrGlyGly-X1-X2-X3-X4 where:

[0136] X1 is represented by Thr or Glu;

[0137] X1 is represented by Gly or Thr or Ala;

[0138] X3 is represented by Ala or Val or Gly or Glu; and

[0139] X4 is represented by His or Gln or Thr or SEQ ID NO:1, or HisTyr or GlnTyr or ThrTyr.

[0140] Preferably, the peptides administered are represented by SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

[0141] Said administration of the opioid peptide of the invention may occur through external, enteral or parenteral application.

[0142] It is also an object of the invention a method of activating the opioid receptor, such method comprising administering to a subject a pharmaceutical composition comprising at least one opioid peptide or salt thereof having naloxone-like binding activity and at least one pharmaceutically acceptable carrier. The said peptide of the pharmaceutical composition is represented by the sequence:

TyrGlyGly-X1-X2-X3-X4 where:

[0143] X1 is represented by Thr or Glu;

[0144] X1 is represented by Gly or Thr or Ala;

[0145] X3 is represented by Ala or Val or Gly or Glu; and

[0146] X4 is represented by His or Gln or Thr or SEQ ID NO:1, or HisTyr or GlnTyr or ThrTyr.

[0147] Preferably, the peptides of the pharmaceutical composition are represented by SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

[0148] Said administration of the pharmaceutical composition of the invention may be effected through external, enteral or parenteral application.

[0149] Another object of the invention is a method of activating the opioid receptor, such method comprising administering to a subject a pharmaceutical composition comprising at least one pharmaceutically acceptable carrier and one promoter-forming agent of at least one peptide having naloxone-like binding activity. The said peptide is represented by the sequence:

TyrGlyGly-X1-X2-X3-X4 where:

[0150] X1 is represented by Thr or Glu;

[0151] X1 is represented by Gly or Thr or Ala;

[0152] X3 is represented by Ala or Val or Gly or Glu; and

[0153] X4 is represented by His or Gln or Thr or SEQ ID NO:1, or HisTyr or GlnTyr or ThrTyr.

[0154] Preferably, the peptides of the pharmaceutical composition are represented by SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. Also preferably, the promoter of the pharmaceutical composition is a genetically modified microorganism.

[0155] Said administration of the pharmaceutical composition of the invention may be effected through external, enteral or parenteral application.

[0156] Another object of the invention is a method of activating the opioid receptor, such method comprising administering to a subject a food composition comprising at least one food grade substance and an opioid peptide or salt thereof having naloxone-like binding activity. The said peptide is represented by the sequence:

TyrGlyGly-X1-X2-X3-X4 where:

[0157] X1 is represented by Thr or Glu;

[0158] X1 is represented by Gly or Thr or Ala;

[0159] X3 is represented by Ala or Val or Gly or Glu; and

[0160] X4 is represented by His or Gln or Thr or SEQ ID NO:1, or HisTyr or GlnTyr or ThrTyr.

[0161] Preferably, the peptides of the food composition are represented by SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

[0162] Another object of the invention is a method of activating the opioid receptor, such method comprising administering to a subject a food composition comprising at least one food grade substance and a promoter-forming agent of at least one opioid peptide or salt thereof having naloxone-like binding activity. The said peptide is represented by the sequence:

TyrGlyGly-X1-X2-X3-X4 where:

[0163] X1 is represented by Thr or Glu;

[0164] X1 is represented by Gly or Thr or Ala;

[0165] X3 is represented by Ala or Val or Gly or Glu; and

[0166] X4 is represented by His or Gln or Thr or SEQ ID NO:1, or HisTyr or GlnTyr or ThrTyr.

[0167] Preferably, the peptides are represented by SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. In a preferred embodiment, the peptide promoter-forming agent is a genetically modified microorganism. Another object of the invention is a method of activating the opioid receptor, such method comprising administering to a subject a nutraceutical composition comprising at least one food grade substance and an opioid peptide or salt thereof having naloxone-like binding activity. The said peptide is represented by the sequence:

TyrGlyGly-X1-X2-X3-X4 where:

[0168] X1 is represented by Thr or Glu;

[0169] X1 is represented by Gly or Thr or Ala;

[0170] X3 is represented by Ala or Val or Gly or Glu; and

[0171] X4 is represented by His or Gln or Thr or SEQ ID NO:1, or HisTyr or GlnTyr or ThrTyr.

[0172] Preferably, the peptides of the nutraceutical composition are represented by SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. Another object of the invention is a method of activating the opioid receptor, such method comprising administering to a subject a nutraceutical composition comprising at least one food grade substance and a promoter-forming agent of at least one opioid peptide or salt thereof having naloxone-like binding activity. The said peptide is represented by the sequence:

TyrGlyGly-X1-X2-X3-X4 where:

[0173] X1 is represented by Thr or Glu;

[0174] X1 is represented by Gly or Thr or Ala;

[0175] X3 is represented by Ala or Val or Gly or Glu; and

[0176] X4 is represented by His or Gln or Thr or SEQ ID NO:1, or HisTyr or GlnTyr or ThrTyr.

[0177] Preferably, the peptides are represented by SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. In a preferred embodiment, the peptide promoter-forming agent is a genetically modified microorganism. Another object of the invention is a method for providing analgesia, such method comprising administering to a subject an effective amount of at least one opioid peptide or salt thereof having naloxone-like binding activity. The said peptide is represented by the sequence:

TyrGlyGly-X1-X2-X3-X4 where:

[0178] X1 is represented by Thr or Glu;

[0179] X1 is represented by Gly or Thr or Ala;

[0180] X3 is represented by Ala or Val or Gly or Glu; and

[0181] X4 is represented by His or Gln or Thr or SEQ ID NO:1, or HisTyr or GlnTyr or ThrTyr.

[0182] Preferably, the peptides administered are represented by SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

[0183] Said administration of the opioid peptide of the invention may be effected through external, enteral or parenteral application.

[0184] It is also an object of the invention a method of providing analgesia, such method comprising administering to a subject a pharmaceutical composition comprising at least one opioid peptide or salt thereof having naloxone-like binding activity and at least one pharmaceutically acceptable carrier. The said peptide of the pharmaceutical composition is represented by the sequence:

TyrGlyGly-X1-X2-X3-X4 where:

[0185] X1 is represented by Thr or Glu;

[0186] X1 is represented by Gly or Thr or Ala;

[0187] X3 is represented by Ala or Val or Gly or Glu; and

[0188] X4 is represented by His or Gln or Thr or SEQ ID NO:1, or HisTyr or GlnTyr or ThrTyr.

[0189] Preferably, the peptides of the pharmaceutical composition are represented by SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

[0190] Said administration of the pharmaceutical composition of the invention may be effected through external, enteral or parenteral application.

[0191] Another object of the present invention is a method of providing analgesia, such method comprising administering to a subject a pharmaceutical composition comprising at least one pharmaceutically acceptable carrier and one promoter-forming agent of at least one peptide having naloxone-like binding activity. The said peptide is represented by the sequence:

TyrGlyGly-X1-X2-X3-X4 where:

[0192] X1 is represented by Thr or Glu;

[0193] X1 is represented by Gly or Thr or Ala;

[0194] X3 is represented by Ala or Val or Gly or Glu; and

[0195] X4 is represented by His or Gln or Thr or SEQ ID NO:1, or HisTyr or GlnTyr or ThrTyr.

[0196] Preferably, the peptides of the pharmaceutical composition are represented by SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. Also preferably, the promoter of the pharmaceutical composition is a genetically modified microorganism.

[0197] Said administration of the pharmaceutical composition of the invention may be effected through external, enteral or parenteral application.

[0198] It is also an object of the invention a method of providing satiety to a subject, such method comprising administering to a subject an effective amount of at least one opioid peptide or salt thereof having naloxone-like binding activity. The said peptide is represented by the sequence:

TyrGlyGly-X1-X2-X3-X4 where:

[0199] X1 is represented by Thr or Glu;

[0200] X1 is represented by Gly or Thr or Ala;

[0201] X3 is represented by Ala or Val or Gly or Glu; and

[0202] X4 is represented by His or Gln or Thr or SEQ ID NO:1, or HisTyr or GlnTyr or ThrTyr.

[0203] Preferably, the peptides administered are represented by SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

[0204] Another object of the invention is a method of providing satiety to a subject, such method comprising administering to a subject a pharmaceutical composition comprising at least one opioid peptide or salt thereof having naloxone-like binding activity and at least one pharmaceutically acceptable carrier. The said peptide of the pharmaceutical composition is represented by the sequence:

TyrGlyGly-X1-X2-X3-X4 where:

[0205] X1 is represented by Thr or Glu;

[0206] X1 is represented by Gly or Thr or Ala;

[0207] X3 is represented by Ala or Val or Gly or Glu; and

[0208] X4 is represented by His or Gln or Thr or SEQ ID NO:1, or HisTyr or GlnTyr or ThrTyr.

[0209] Preferably, the peptides of the pharmaceutical composition are represented by SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

[0210] It is also an object of the present invention a method of providing satiety to a subject, such method comprising administering to a subject a pharmaceutical composition comprising at least one pharmaceutically acceptable carrier and one promoter-forming agent of at least one peptide having naloxone-like binding activity. The said peptide is represented by the sequence:

TyrGlyGly-X1-X2-X3-X4 where:

[0211] X1 is represented by Thr or Glu;

[0212] X1 is represented by Gly or Thr or Ala;

[0213] X3 is represented by Ala or Val or Gly or Glu; and

[0214] X4 is represented by His or Gln or Thr or SEQ ID NO:1, or HisTyr or GlnTyr or ThrTyr.

[0215] Preferably, the peptides of the pharmaceutical composition are represented by SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. Also preferably, the promoter of the pharmaceutical composition is a genetically modified microorganism.

[0216] Another object of the invention is a method of providing satiety to a subject, such method comprising administering to a subject a food composition comprising at least one food grade substance and an opioid peptide or salt thereof having naloxone-like binding activity. The said peptide is represented by the sequence:

TyrGlyGly-X1-X2-X3-X4 where:

[0217] X1 is represented by Thr or Glu;

[0218] X1 is represented by Gly or Thr or Ala;

[0219] X3 is represented by Ala or Val or Gly or Glu; and

[0220] X4 is represented by His or Gln or Thr or SEQ ID NO:1, or HisTyr or GlnTyr or ThrTyr.

[0221] Preferably, the peptides of the food composition are represented by SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

[0222] Another object of the invention is a method of providing satiety to a subject, such method comprising administering to a subject a food composition comprising at least one food grade substance and a promoter-forming agent of at least one opioid peptide or salt thereof having naloxone-like binding activity. The said peptide is represented by the sequence:

TyrGlyGly-X1-X2-X3-X4 where:

[0223] X1 is represented by Thr or Glu;

[0224] X1 is represented by Gly or Thr or Ala;

[0225] X3 is represented by Ala or Val or Gly or Glu; and

[0226] X4 is represented by His or Gln or Thr or SEQ ID NO:1, or HisTyr or GlnTyr or ThrTyr.

[0227] Preferably, the peptides are represented by SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. In a preferred embodiment, the peptide promoter-forming agent is a genetically modified microorganism. Another object of the invention is a method of providing satiety to a subject, such method comprising administering to a subject a nutraceutical composition comprising at least one food grade substance and an opioid peptide or salt thereof having naloxone-like binding activity. The said peptide is represented by the sequence:

TyrGlyGly-X1-X2-X3-X4 where:

[0228] X1 is represented by Thr or Glu;

[0229] X1 is represented by Gly or Thr or Ala;

[0230] X3 is represented by Ala or Val or Gly or Glu; and

[0231] X4 is represented by His or Gln or Thr or SEQ ID NO:1, or HisTyr or GlnTyr or ThrTyr.

[0232] Preferably, the peptides of the nutraceutical composition are represented by SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. Another object of the invention is a method of providing satiety to a subject, such method comprising administering to a subject a nutraceutical composition comprising at least one food grade substance and a promoter-forming agent of at least one opioid peptide or salt thereof having naloxone-like binding activity. The said peptide is represented by the sequence:

TyrGlyGly-X1-X2-X3-X4 where:

[0233] X1 is represented by Thr or Glu;

[0234] X1 is represented by Gly or Thr or Ala;

[0235] X3 is represented by Ala or Val or Gly or Glu; and

[0236] X4 is represented by His or Gln or Thr or SEQ ID NO:1, or HisTyr or GlnTyr or ThrTyr.

[0237] Preferably, the peptides are represented by SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. In a preferred embodiment, the peptide promoter-forming agent is a genetically modified microorganism.

[0238] Experiments and results obtained:

[0239] Peptide synthesis: The synthesis of the peptides of the present invention was conducted using the Fmoc/t-butyl (9-fluorenylmethoxycarbonyl) strategy of manual synthesis in solid support (Chan & White, 2000) followed by purification by means of high performance liquid chromatography (HPLC) using a C18 reverse-phase semipreparative column (Vydac).

[0240] In vivo analyzes: Tests were carried out on Swiss mice of the Mus musculus species, in which nociception tests (Hot Plate and Tail Flick) were used according to Le Bars et al. (2001). Injections were administered via intraperitoneal (IP). Doses were based upon dose and molaridade of the morphine (10 mg/kg animal). As a positive control, Leu-enkephalin (19.3 mg/kg animal) was used in addition to morphine. Sodium chloride saline was used as the negative control and, in order to suspend the antinociceptive effect of Leu-enkephalin and the peptide, serial doses (every hour) of Naloxone (4 mg/kg animal) were injected.

[0241] Digestion of the immobilized pepsin precursor: the peptide sequence SEQ ID NO: 6 was digested using immobilized pepsin for a period of 4 hours at 37° C. under agitation of 1400 RPM evaluating the fragmentation index at the times of 15 minutes, 30 minutes, 2 hours and 4 hours.

[0242] To initiate the nociceptive tests and verify the efficiency of the same, a pilot experiment was carried out using the peptide sequence SEQ ID NO: 2 and evaluating the behavior of the animals in the course of two hours. At the end of the Hot Plate experiment (FIG. 1), it was evaluated that the test should be performed longer because the peptide showed delayed and prolonged activity.

[0243] With the prolonged experiment time, the nociceptive tests demonstrated that the peptide sequence SEQ ID NO: 6 showed opioid activity. As can be seen in FIG. 2, the peptide sequence SEQ ID NO: 6 and enkephalin (positive control) initiated antinociceptive activity at 60 minutes after application up to 150 minutes. The saline solution (negative control) showed no effect throughout the experiment. Peptide sequence SEQ ID NO: 6 and enkephalin had their effect cancelled when the substance naloxone was applied (at 0.60 and 120 min.).

[0244] This result shows that the peptide binding activity is similar to naloxone because, in the same way that the main morphine antagonist is naloxone, which interacts with opioid receptors (mu, kappa and delta) (Goldeberg, 2010; Kane et al., 2006), it is also an antagonist of the peptide. For this reason naloxone was used in the synthetic peptide assays aiming at antagonism to confirm the route of action, which was via opioid receptors.

[0245] The antinociceptive activity of Leu-enkephalin and the peptide sequence SEQ ID No: 6 resulted in a prolonged effect in animals, as shown in FIG. 2, in which 4 hours of hot plate experiment were evaluated.

[0246] In the period in which naloxone was applied (zero to 150 minutes), the effect of the peptide sequence SED ID NO: 6 was antagonized. After 150 minutes naloxone was no longer injected and the activity of peptide sequence SEQ ID: 6 was noted. (FIG. 3).

[0247] In the other nociception test, the Tail Flick test, the activity of Leu-enkephalin and the peptide sequence SEQ ID NO: 6 also showed a prolonged effect, as can be seen in FIG. 4 where the onset of antinociception occurred between 60 and 90 minutes after the injection, the experiment lasting 4 hours. The application of naloxone caused the inhibition of the effect of the peptide sequence SEQ ID NO: 6 and enkephalin.

[0248] Experiments with the sequence peptide SEQ ID NO: 3 were also carried out using the same parameters of the nociceptive tests. In FIGS. 5 and 6 it is possible to visualize the activity of said peptide.

[0249] The antinociception activity of the sequence peptide SEQ ID NO: 4 can be seen in the graphs obtained from the Hot Plate and Tail Flick tests shown in FIGS. 7 and 8, respectively. In this experiment, the Naloxone test was not performed.

[0250] Experiments were also carried out with the peptide sequence SEQ ID NO: 5 as can be seen in FIGS. 9 and 10, which were also antinociceptive.

[0251] The peptide sequence SEQ ID NO: 6 was digested using immobilized pepsin for a period of 4 hours, evaluating the fragmentation index at the times of 15 minutes, 30 minutes, 2 hours and 4 hours. It was possible to verify (FIG. 11) an increase in the intensity of the lower mass ions over time and the decrease of the precursor ion.

[0252] It shows that during incubation with trypsin enzyme (enzymes present in the gastrointestinal tract) the intensity of the major peak of SEQ ID NO: 6 is reducing. By means of this experiment it was possible to observe the in vitro peptide digestion by the enzyme present in the organism.

[0253] Enkephalin (Leu) was digested using immobilized pepsin for a period of 4 hours, evaluating the fragmentation index at the times of 30 minutes, 1 hour, 2 hours and 4 hours. It has been observed in FIG. 12 the decrease of the precursor ion over time.

[0254] It shows that during incubation with trypsin enzyme (enzymes present in the gastrointestinal tract) the intensity of the major peak of enkephalin (Leu) is reducing. By means of this experiment it was possible to try to observe the in vitro peptide digestion by the enzyme present in the organism.