ES2550536T3 - Reasons for the family of pancreatic polypeptides, polypeptides and methods that comprise them - Google Patents

Abstract

A PPF polypeptide, wherein said polypeptide comprises: the amino acid sequence of SEQ ID NO: 438.

Description

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Figure 45 depicts the effects of administration of an illustrative PPF polypeptide in gastric emptying, compared to PYY (3-36), in rats.

Figure 46 depicts the effects of the administration of an illustrative PPF polypeptide on heart rate and mean arterial pressure (MAP) in rats.

Figure 47 depicts the effects of the administration of an illustrative PPF polypeptide on heart rate and mean arterial pressure (MAP) in rats.

Figure 48 depicts the effects of an illustrative PPF polypeptide, compared to PYY (3-36) with and without co-administration of amylin on body weight in OID-prone rats

Figure 49 depicts the effects of two illustrative PPF polypeptides with and without the joint administration of amylin on body weight in OID-prone rats.

Figure 50 depicts the effects of an illustrative PPF polypeptide with and without the joint administration of amylin on body composition in OID-prone rats.

Figure 51 depicts the effects of an illustrative PPF polypeptide with and without the joint administration of amylin on body composition in OID-prone rats.

Figure 52 depicts the effects of PYY (3-36) or an illustrative PPF polypeptide with and without the joint administration of amylin in fasting insulin levels in rats.

Figure 53 depicts the effects of an illustrative PPF polypeptide with and without the co-administration of amylin in RQ and GE in rats.

Figure 54 compares the calculated degradation rates of several PPF polypeptides with respect to those of PYY (3-36).

Detailed description

The present disclosure relates, in general, to polypeptides of the family of pancreatic polypeptides ("PPF") having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at minus 92%, at least 94% or at least 97% sequence identity with PYY (3-36) along the entire length of PYY (3-36). PPF polypeptides may comprise no more than 10, no more than 5, no more than 3, no more than 2 or no more than 1 amino acid substitutions. PPF polypeptides also comprise at least two PPF motifs that include at least the N-terminal polyfoline PPF motif and the C-terminal tail PPF motif. As used herein, "motif" refers to an amino acid sequence that is characteristic of a specific biochemical function or defines an independently folded domain. The additional PPF motives of the disclosure may correspond to a motif of any of the polypeptides of the PP family, including PP, PYY and NPY, for example, the motive of the II type II spin region of PYY or the motive -helical of the C end of PYY. In certain disclosures, the PPF polypeptides of the disclosure may not include any artificial amino acids.

The present invention according to the claims also relates to PPF polypeptides useful in the treatment and prevention of metabolic disorders and disorders. In some embodiments, the PPF polypeptides of the invention may have comparable or superior potency in the treatment and / or in the prevention of metabolic conditions and disorders, as compared to native human PP, PYY, PYY (3-36) or NPY. In some embodiments, the PPF polypeptides of the invention may have lower potency, but may have other desirable characteristics such as greater ease of fabrication, stability and / or ease of formulation, compared to PP, PYY, PYY (3-36) or NPY.

In some embodiments, and without intending to be limited by theory, it is believed that peripheral administration of the new PPF polypeptides of the invention to a subject reduces the availability of nutrients and, therefore, is useful in treatment and prevention. of obesity, and related metabolic disorders or disorders. As such, the present invention provides PPF polypeptide compositions and their use in reducing the availability of nutrients in a subject in need thereof for use in the treatment and in the prevention of metabolic conditions or disorders that may benefit from a reduction in Nutrient availability These PPF polypeptides may be useful in the treatment of, for example, obesity, diabetes, including, but not limited to, type 2 or non-insulin-dependent diabetes, eating disorders, insulin resistance syndrome, disease. cardiovascular or a combination of these conditions.

It has now been discovered that a PYY polypeptide, PYY agonist or PPF polypeptide can have metabolic effects in the body, and can preferably be used to reduce or maintain unwanted body fat.

or increasing lean body mass.

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The present invention is directed, in part, to PPF polypeptides according to the claims for use in modifying body composition by reducing body weight, maintaining body weight.

or reduction of body weight gain, while selectively reducing body fat, or reducing or increasing body fat, while maintaining or increasing lean body mass. However, in certain situations, for example, in bodybuilding, the increase in body weight may be desired, for example, through the selective intake of nutrients (for example, increasing fat or calorie content), while the percentage of body fat is reduced or maintained.

The uses of the present invention contemplate the administration of an effective amount of a PYY polypeptide, PYY agonist or PPF to a subject to affect the desired results as described in the present application.

The PYY polypeptide, PYY agonist or PPF administered may be in the form of a peptide, a prodrug or as pharmaceutical salts thereof. The term "prodrug" refers to a compound that is a pharmacological precursor that, after administration, releases the drug in vivo by some chemical or physiological process, for example, proteolytic cleavage, or upon reaching an environment with a certain pH .

PPF polypeptides according to the invention can be used in any individual in need of such methods and in individuals for whom the practice of the methods is desired. These individuals can be any mammal including, but not limited to, humans, dogs, horses, cows, pigs, chickens, turkeys and other commercial or companion value animals.

In this document, the titles of the sections are used only for organizational purposes, and should not be interpreted in any way as limitations of the subject matter described.

PPF polypeptides of the invention and PPF motifs

As described above, the present invention relates, at least in part, to new PPF polypeptides according to the claims. The disclosure relates to PPF polypeptides comprising at least two PPF motifs, in which the at least two PPF motifs include at least the N-terminal polyproline PPF motif and the C-terminal tail PPF motif. The PPF polypeptides of the PPF disclosure will also have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 94% or at least 97% of sequence identity with a native PYY (3-36) along the entire length of the PYY (3-36). In some disclosures, the polypeptides of the present disclosure will retain, at least in part, a biological activity of native human PP, PYY or NPY, for example, the polypeptides of the present disclosure will, in general, be agonists or antagonists of PP, PYY and / or NPY. In some embodiments, the polypeptides of the present invention will exhibit biological activity in the treatment and in the prevention of metabolic disorders or disorders. In addition, the PPF polypeptides of the invention may include internal linker compounds, may include chemical modifications to internal amino acid residues, or may be chemically modified in the N-terminal or C-terminal moiety. In some disclosures, the polypeptides of the disclosure include only natural L amino acid residues and / or modified natural L amino acid residues. In some disclosures, the polypeptides of the disclosure do not include artificial amino acid residues.

The PPF motives of the disclosure may correspond to any motive of any of the native polypeptides of the PP family, including PP, PYY and NPY. A "PPF motive" is, in general, a structural component, primary, secondary or tertiary, of a native polypeptide of the PP family that is essential for biological activity, that is, biological activity is substantially reduced in the absence. or because of the disturbance of the motive. PPF motifs may include any of those known in the art, including, but not limited to, the type II motif of N-terminal polyproline of a native polypeptide of the PP family, the type II spin motif of a native polypeptide of the PP family, the C-terminal -helical motif of a native polypeptide of the PP family and the C-terminal tail motif of a native polypeptide of the PP family. More particularly, in the N-terminal polyproline PPF motif, the amino acids corresponding to residues 5 and 8 of a native polypeptide of the PP family are generally conserved in the form of proline. The type II rotation motif will generally include the amino acids corresponding to residues 12-14 of a native polypeptide of the PP family. The -helical motif can be extended, in general, from the amino acids corresponding to approximately the remainder 14 of a native polypeptide of the PP family to any point and including the C-terminal end, provided that the -helical motif includes a sufficient number of amino acid residues that allow the formation of a -helical turn in solution. The -helical motif may also include substitutions, insertions and deletions of amino acids with respect to the native sequence of the PP family, provided that the -helical turn can still be formed in solution. The C-terminal tail PPF motif, in general, includes amino acids that correspond approximately to the last 10 residues of a native polypeptide of the PP family. In some disclosures, the C-terminal tail motif includes the last 7, 6 or 5 residues of a polypeptide native to the PP family. In some disclosures, the C-terminal tail motif includes amino acid residues 32-35.

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In one disclosure, the PPF polypeptides of the disclosure do not include any artificial amino acid residues, and also with the proviso that the PPF polypeptides of the invention do not include any native PPF polypeptide (e.g., PP, NPY (1-36), NPY (3-36), PYY (1-36), PYY (3-36), NPY (2-36), NPY (4-36), PYY (2-36), PYY (4-36), PP (236), PP (3-36) or PP (4-36)). In some disclosures, the PPF disclosure polypeptides do not include: Tyr1hPP, Lys4hPP, Asn7hPP, Arg19hPP, Tyr21hPP, Glu21hPP, Ala23hPP, Gln23hPP, Gln34hPP, Phe6Arg19hPP, Phe6Tyr21hPP, Phe6Glu21hPP, Phe6Ala23hPP, Phe6Gln23hPP, Pro13Ala14hPP, Ile31Gln34PP, Arg19Tyr20Tyr21Ser22Ala23hPP, Lys4Arg19Tyr20Tyr21Ser22Ala23hPP, Lys4Arg19Tyr20Tyr21Ser22Ala23hPP (2-36), Ala1NPY, Tyr1NPY, Ala2NPY, Leu2NPY, Phe2NPY, His2NPY, Ala3NPY, Ala4NPY, Ala6NPY, Tyr7pNPY, Ala7NPY, Ala9NPY, Ala10NPY, Ala11NPY, Gly12, Gly12, Ply12, Gly12 Lys19NPY, Ala20NPY, Ala21NPY, Ala22NPY, Gly23NPY, Ala24NPY, Trpz4pNPY, Ala25NPY, Lys25NPY, Ala26NPY, Ala27NPY, Phe27NPY, Ala28NPY, Ala29NPY, Glnz9NPY, Ala30NPY, Phe30NPY, Ala31NPY, Trp31pNPY, Ala32NPY, Trp32NPY, Ala33NPY, Lys33NPY, Ala34NPY, Pro34NPY, Leu34NPY, Ala35NPY, Lys35NPY, Ala36NPY, Phe36NPY, His36NPY, Glu4Pro34pNPY, Arg6Pro34pNPY, Phe6Pro34pNPY, Cys6Pro34pNPY, Asn6Pro34pNPY, Phe7Pro34pNPYPro 7Pro34 pNPY, Asp7Pro34 pNPY, Phe8Pro34 pNPY, Arg8Pro34 pNPY, Cys8Pro34 pNPY, Asp8Pro34 pNPY, Asn8Pro34pNPY, Pro11Pro34pNPY, Ser13Pro14pNPY, Trp24,31pNPY, Ala31Pro32pNPY, Cys31Pro34pNPY, Leu31Pro34NPY, Phe32Pro34pNPY, Ala21,25Pro34pNPY, Pro11Tyr13Pro14Pro34pNPY, Ahx (9-22) pNPY, Ahx (9-17) pNPY, des-AA (10-20) Cys7,21Pro34-pNPY, des-AA (10-17) -pNPY, des-AA (10-17) -Cys2,27-pNPY, des- AA (10-17) -Ala7,21-pNPY, des-AA (10-17) -Cys7,21-pNPY, des-AA (10-17) -Glu7Lys21-pNPY, des-AA (10-17) Cys7 , 21Pro34pNPY, des-AA (10-17) Glu7Lys21Pro34pNPY, des-AA (10-17) Cys7,21Leu31Pro34pNPY, des-AA (11-17) Cys7,21Pro34pNPY, Pro34PYY, His34PYY, Lys25hPYY (5-36), Arg4h (5-36), Arg4h (5-36), Arg4h (5-36), Arg4h 4-36), Gln4hPYY (4-36), Asn4hPYY (4-36), Lys25hPYY (4-36), Leu3hPYY (3-36), Val3hPYY (3-36), Lys25hPYY (3-36), Tyr1,36pPYY , Pro13Ala14hPYY, Leu31Pro34PYY, FMS-PYY, FMS-PYY (3-36), Fmoc-PYY, Fmoc-PYY (3-36), FMS2-PYY, FMS2-PYY (3-36), Fmoc2-PYY, Fmoc2- PYY (3-36), hPP (1-7) -pNPY, hPP (1-17) pNPY, hPP (19-23) -pNPY, hPP (19-23) -Pro34pNPY, hPP (19-23) -His34pNPY , rPP (19-23) -pNPY, rPP ( 19-23) -Pro34pNPY, rPP (19-23) -His34pNPY, hPP (1-7) -pNPY, hPP (1-17) -pNPY, hPP (1-17) -His34pNPY, pNPY (1-7) - hPP, pNPY (1-7, 19-23) -hPP, cPP (1-7) -pNPY (19-23) -hPP, cPP (1-7) -NPY (19-23) -His34hPP, hPP (1 -17) -His34pNPY, hPP (19-23) -pNPY, hPP (19-23) -Pro34pNPY, hPP (19-23) -His34pNPY, rPP (19-23) -pNPY, rPP (19-23) -Pro34pNPY , rPP (19-23) -His34pNPY, pNPY (1-7) -hPP, pNPY (19-23) -hPP, pNPY (19-23) -Gln34hPP, pNPY (19-23) -His34hPP, pNPY (19- 23) -Phe6Gln34hPP, pNPY (19-23) -Phe6His34hPP, pNPY (1-7,19-23) -hPP, pNPY (1-7,19-23) -Gln34hPP, cPP (20-23) -Pro34pNPY, cPP (21-23) -Pro34-pNPY, cPP (22-23) -Pro34-pNPY, cPP (1-7) -Pro34-pNPY, cPP (20-23) -Pro34-pNPY, cPP (1-7.20 -23) -Pro34-pNPY, cPP (1-7) -pNPY (19-23) -hPP, cPP (1-7) -pNPY (19-23) -His34hPP or cPP (1-7) -gPP (19 -23) -hPP.

In another disclosure, said PPF polypeptides of the disclosure also do not include: Thr27hPYY (3-36), Ile30hPYY (3-36), Ser32hPYY (3-36), Lys33hPYY (3-36), Asn34hPYY (3-36), Lys35hPYY ( 3-36), Thr36hPYY (3-36), Lys25 Th27hPYY (3-36), Lys25Ile30hPYY (3-36), Lys25Ser32hPYY (3-36), Lys25Lys33hPYY (3-36), Lys25Asn24hPYY (3-36), Lys25Yys (HP) 3-36), Lys25Thr36hPYY (3-36), Thr27Ile28hPYY (3-36), Thr27Val28hPYY (3-36), Thr27Gln29hPYY (3-36), Thr27Ile30hPYY (3-36), Thr27Val30hPYY (3-36), Thr27 (3-36), Thr27 -36), Thr27Leu31hPYY (3-36), Thr27Ser32hPYY (3-36), Thr27Lys33hPYY (3-36), Thr27Asn34hPYY (3-36), Thr27Lys35hPYV (3-36), Thr27Thr36hPYY (3-36), Thr27P (3-36), Thr27P (3-36) 36), Phe27Ile30hPYY (3-36), Phe27Ser32hPYY (3-36), Phe27Lys33hPYY (3-36), Phe27Asn34hPYY (3-36), Phe27Lys35hPYY (3-36), Phe27Thr36hPYY (336), Gln29Yle 3-7h (3-36) Gln29Ser32hPYY (3-36), Gln29Leu33hPYY (3-36), Gln29Asn34hPYY (3-36), Gln29Leu35hPYY (336), Gln29Thr36hPYY (3-36), Ile30Ile31hPYY (3-36), Ile30Ye30h (3-30h) -36), Ile30Lys33hPYY (3-36), Ile30As n34hPYY (3-36), Ile30Lys35hPYY (3-36), Ile30Thr36hPYY (3-36), Ile30Phe36hPYY (3-36), Val30Ser32hPYY (3-36), Val30Lys33hPYY (3-36), Val30Asn34hPYY (3-36), Val35 (3-36), Val30Thr36hPYY (3-36), Ile31Ser32hPYY (3-36), Ile31Lys33hPYY (3-36), Ile31Asn34hPYY (3-36), Ile31Lys35hPYY (3-36), Ile31Thr36hPYY (3-36), Ile31Phe (h36) 3-36), Leu31Ser32hPYY (3-36), Leu31Lys33hPYY (3-36), Leu31Asn34hPYY (3-36), Leu31Lys35hPYY (3-36), Leu31Thr36hPYY (336), Ser32Lys33hPYY (3-36), Ser32Asn34h 3-3P ), Ser32Lys35hPYY (3-36), Ser32Thr36hPYY (3-36), Ser32Phe36hPYY (3-36), Lys33Asn34hPYY (3-36), Lys33Lys35hPYY (3-36), Lys33Thr36hPYY (3-36), Lys33PY36hPY36hPY36hPY36hPY36hPY36hPY36hPY36hPY36hPY36 (3-36), Asn34Phe36hPYY (3-36), Lys35Thr36hPYY (3-36), Lys35Phe36hPYY (3-36), Thr27hPYY (436), Phe27hPYY (4-36), Ile28hPYY (4-36), Val28hPYY (4- 36), Gln29hPYY (4-36), Ile30hPYY (4-36), Val30hPYY (4-36), Ile31hPYY (4-36), Leu31hPYY (4-36), Ser32hPYY (4-36), Lys33hPYY (4-36) ), Asn34hPYY (4-36), Lys35hPYY (4-36), Thr36hPYY (4-36), Phe36hPYY (4-36), Lys25Thr27hPYY (4-36), Lys25Ph e27hPYY (4-36), Lys25Ile28hPYY (4-36), Lys25Val28hPYY (4-36), Lys25Gln29hPYY (4-36), Lys25Ile30hPYY (4-36), Lys25Val30hPYY (4-36), Lys25Ile31ePyY (YY) (4-36), Lys25Ser32hPYY (4-36), Lys25Lys33hPYY (4-36), Lys25Asn24hPYY (4-36), Lys25Lys35hPYY (4-36), Lys25Thr36hPYY (4-36), Lys25Phe36hPYY (4-36), Thr27I (4-36), Thr27 4-36), Thr27Val28hPYY (4-36), Thr27Gln29hPYY (4-36), Thr27Ile30hPYY (4-36), Thr27Val30hPYY (4-36), Thr27Ile31hPYY (4-36), Thr27Leu31hPYY (4-36), Thr27 -36), Thr27Lys33hPYY (4-36), Thr27Asn34hPYY (4-36), Thr27Lys35hPYY (4-36), Thr27Thr36hPYY (4-36), Thr27Phe36hPYY (4-36), Phe27Ile28hPYY (4-36), Phe27 (4-36) 36), Phe27Gln29hPYY (4-36), Phe27Ile30hPYY (4-36), Phe27Val30hPYY (4-36), Phe27Ile31hPYY (4-36), Phe27Leu31hPYY (4-36), Phe27Se32hPYY (4-36), Phe27 (4-36), Phe27 (4-36), Phe27 (4-36), Phe27 (4-36) ), Phe27Asn34hPYY (436), Phe27Lys35hPYY (4-36), Phe27Thr36hPYY (4-36), Phe27Phe36hPYY (4-36), Gln29Ile30hPYY (4-36), Gln29Val30hPYY (4-36), Gln29Yle (4-36) (4-36), Gln29Ser32, hPYY (4-36) Gln29Leu33hPYY (4-36 ), Gln29Asn34hPYY (4-36), Gln29Leu35hPYY (4-36), Gln29Thr36hPYY (4-36), Gln29Phe36hPYY (4-36), Ile30Ile31hPYY (4-36), Ile30Leu31hPYY (4-36), Ile30 (4-36) , Ile30Lys33hPYY (4-36), Ile30Asn34hPYY (4-36), Ile30Lys35hPYY (4-36), Ile30Thr36hPYY (4-36), Ile30Phe36hPYY (4-36), Val30Ile31hPYY (4-36), Val30Yeh (4-36), Val30Yeh (4-36) Val30Ser32hPYY (4-36), Val30Lys33hPYY (4-36), Val30Asn34hPYY (4-36), Val30Lys35hPYY (4-36), Val30Thr36hPYY (4-36), Val30Phe36hPYY (4-36), Ile31Ser32hPYY (4-36) (4-36), Ile31Asn34hPYY (4-36), Ile31Lys35hPYY (4-36), Ile31Thr36hPYY (4-36), Leu31Phe36hPYY (4-36), Leu31Phe36hPYY (4-36), Leu31Ser32hPYY (4-33), Val31L (4-36), Val31L 4-36), Leu31Asn34hPYY (436), Leu31Lys35hPYY (4-36), Leu31Thr36hPYY (4-36), Leu31Phe36hPYY (4-36), Ser32Lys33hPYY (4-36),

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Thr27Val30hPYY (5-36), Thr27Ile31hPYY (5-36), Thr27Leu31hPYY (5-36), Thr27Thr36hPYY (5-36), Thr27Phe36hPYY (5-36), Phe27Ile28hPYY (5-36), Phe27Y27H (5-27) (5-36), Phe27Ile30hPYY (5-36), Phe27Val30hPYY (5-36), Phe27Ile31hPYY (5-36), Phe27Leu31hPYY (5-36), Phe27Thr36hPYY (5-36), Phe27Phe36hPYY (5-36), Gly29 (5-36), Gly29 (5-36) 5-36), Gln29Val30hPYY (5-36), Gln29Ile31hPYY (5-36), Gln29Leu31hPYY (5-36), Gln29Thr36hPYY (5-36), Gln29Phe36hPYY (5-36), Ile30Ile31hPYY (5-36e), Ile30Y (5-36e), Ile30 (5) -36), Ile30Thr36hPYY (5-36), Ile30Phe36hPYY (5-36), Val30Ile31hPYY (5-36), Val30Leu31hPYY (5-36), Val30Thr36hPYY (5-36), Val30Phe36hPYY (5-36), Ile31Thr36 (5-31) 36), Leu31Phe36hPYY (5-36), Leu31Phe36hPYY (5-36), Leu31Thr36hPYY (5-36), Leu31Phe36hPYY (5-36).

In yet another disclosure, the PPF polypeptides of the disclosure do not include those PPF related polypeptides disclosed in WO 03/026591 and WO 03/057235.

In another disclosure, the polypeptides of the disclosure are at least 34 amino acids in length. In other disclosures, PPF polypeptides may be at least 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33 amino acids in length. In addition, in one disclosure, the polypeptides of the disclosure include only natural L amino acid residues and / or modified natural L amino acid residues. Alternatively, in another disclosure, the polypeptides of the disclosure do not include artificial amino acid residues.

In yet another disclosure, the PPF polypeptides of the disclosure may have at least 60%, 65%, 70%, 80%

or 90% sequence identity with respect to a native PYY (3-36) along the entire length of PYY (3-36). Such PPF polypeptides of the disclosure may also have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 94% or at least 97% identity of sequence with respect to a native PP. In yet another disclosure, said PPF polypeptides of the disclosure may have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 94% or at least 97% sequence identity with respect to a native NPY.

More specifically, in one aspect, the present disclosure relates to new PPF polypeptides comprising at least two PPF motifs, in which the at least two PPF motifs include at least the N-terminal polyproline PPF motif and the motif. of C-terminal tail PPF, and the PPF polypeptide does not include any artificial amino acid residues. Such PPF polypeptides of the disclosure will have at least a 50% sequence identity with respect to a native PYY (3-36) along the entire length of the PYY (3-36). In some disclosures, said PPF polypeptides have at least 34 amino acid residues. In some disclosures, said PPF polypeptides of the disclosure may have at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 94% or at least 97% sequence identity with respect to a native PYY (3-36) along the entire length of the PYY (3-36). Such PPF polypeptides of the disclosure may also have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 94% or at least 97% identity of sequence with respect to a native PP. In some disclosures, PPF polypeptides may comprise no more than 10, no more than 5, no more than 3, no more than 2 or no more than 1 amino acid substitutions. In yet another disclosure, said PPF polypeptides of the disclosure may have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 94% or at least 97% sequence identity with respect to a native NPY.

In another aspect, the PPF polypeptides of the disclosure include PYY analog polypeptides. In yet another aspect of the disclosure, the PPF polypeptides of the disclosure include chimeric PPF polypeptides comprising a fragment of a PP, PYY or NPY polypeptide covalently linked to at least one additional fragment of a PP, PYY or NPY polypeptide, in which Each fragment of PP, PYY or NPY includes a PPF motif. Such PPF analog polypeptides and PPF chimeric polypeptides of the disclosure will have at least 50% sequence identity with respect to a native PYY (3-36) along the entire length of the PYY (3-36). In some disclosures, said PPF polypeptides of the disclosure may have at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 94% or at least 97% sequence identity with respect to a native PYY (3-36) along the entire length of the PYY (3-36). The PPF polypeptides of the disclosure may also have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 94% or at least 97% identity of sequence with respect to a native PP. In yet another disclosure, the PPF polypeptides of the disclosure may have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 94% or at least 97% sequence identity with respect to a native NPY. In certain disclosures, desirable PPF polypeptides may not include N PP terminals in combination with C-terminal NPY fragments.

PPF polypeptides useful in the disclosure may have a PPF activity higher or lower than that of the native compounds for a particular activity. Thus, for example, PYY agonists may have 3, 5, 10, 50, 100, 500, 1,000 times or more activity than the native PYY. On the other hand, while it is desirable to use a PYY agonist that has activity similar or superior to that of the native PYY, the person skilled in the art would understand that agonists who had less activity than native PYY would also be useful in the present disclosure. Such agonists, for example, may have any value selected from 2, 5, 10, 15 or 20 times less activity than native PYY, but possess other desirable characteristics, for example, solubility, bioavailability, ease of manufacture or formulation, or less effects. secondary. In some disclosures, a PPF polypeptide useful in the disclosure may be an PYY antagonist.

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In U.S. Pat. No. 5,574,010, WO04 / 089279, WO 04/066966, WO 03/057235, WO 03/026591, WO 98/20885, WO 94/22467, examples of PYY agonists, more particularly agonists or analogues of agonists of PYY analogues (analogues and derivatives of PYY). Other agonists of PYY analogs are described in Balasubramaniam et al., Pept Res 1 (1): 32-5, 1998, Balasubramaniam et al., Peptides 14: 1011-1016, 1993, 5 Boublik et al., J. Med Chem 32: 597-601, 1989, Liu et al., J. Gastrointest Surg 5 (2): 147-52, 2001, Gehlert et al., Proc Soc Exp Biol Med, 218: 7-22, 1998, Sheikh et al., Am J Physiol. 261: G701-15, 1991, Potter et al., Eur J Pharmacol 267 (3): 253-362, 1994, Lebon et al., J. Med. Chem 38: 1150-57, 1995, Fournier et al., Mol Pharmacol 45 (1): 93-101, 1994, Kirby et al., J. Med Chem 38: 4579-86, 1995, Beck et al., FEBS Letters 244 (1): 119-122, 1989, Rist et al., Eur J Biochemistry 247: 1019-1028, 1997, Soll et al., Eur J Biochem 268 (10): 2828-37, 2001, Cabrele et al., J Pept Sci

10 6 (3): 97-122, 2000, Balasubramaniam et al., J Med Chem 43: 3420-3427, 2000, Kirby et al., J Med Chem 36: 380208, 1993, Grundemar et al., Regulatory Peptides 62 : 131-136, 1996, Feinstein et al. J Med Chem 35: 2836-2843, 1992, Cox et al. Regulatory Peptides 75-76: 3-8, 1998, Cabrele et al., Peptides 22: 365-378, 2001, Keire et al. Biochemistry 39: 9935-9942, 2000, Keire et al. Am. J. Physiol Gastrointest Liver Liver Physiol 279: G126-G131, 2000.

15 PYY, NPY, PP constitute a family of C-terminally amidated peptides that participate in the regulation of gastrointestinal function, blood pressure and feeding behavior. Without intending to be limited by theory, the ability of these peptides to selectively bind and activate receptor subtypes and is believed to depend largely on a stable structure in solution, including the so-called "PP fold." Table 1 (below) shows the potencies of the PP family ligands in the receptors

20 known and the rank order of the potencies of various ligands.

Table 1 Summary of receptor pharmacology for the PP receptor family

RECEIVERS

PHARMACOLOGY REFERENCE

Inhibition of food intake (peripheral)

PYY (3-36)  PYY >> NPY, NPY (3-36), PP, Ac-PYY (2236)

Gastric emptying

PYY (3-36)  PYY >> NPY, NPY (3-36), PP, Ac-PYY (2236)

Stimulation of food intake (central)

PYY  PYY (3-36) = NPY = NPY (3-36)> PP Iyengar et al., J. Pharmacol. Exp. Ther. 289: 1031-40, 1999

Y1

NPY = PYY> NPY (3-36) = PYY (3-36) = PP Iyengar et al., J. Pharmacol. Exp. Ther. 289: 1031-40, 1999; Gehlert, Proc. Soc. Exp. Biol. Med. 218: 7-22, 1998; Michel et al., Pharmacol. Rev. 50: 143-50, 1998; US 5,968,819

Y2

NPY = PYY = PYY (3-36) = NPY (3-36) >> PP Iyengar et al., J. Pharmacol. Exp. Ther. 289: 1031-40, 1999; Gehlert, Proc. Soc. Exp. Biol. Med. 218: 7-22, 1998; Michel et al., Pharmacol. Rev. 50: 143-50, 1998; US 5,968,819

Y3

NPY> PP> PYY Gehlert, Proc. Soc. Exp. Biol. Med. 218: 7-22, 1998; Michel et al., Pharmacol. Rev. 50: 143-50, 1998.

Y4

PP> PYY> NPY> PYY (336) = NPY (3-36) Iyengar et al., J. Pharmacol. Exp. Ther. 289: 1031-40, 1999; Gehlert, Proc. Soc. Exp. Biol. Med. 218: 7-22, 1998; Michel et al., Pharmacol. Rev. 50: 143-50, 1998; US 5,968,819

Y5

NPY = PYY ≥ PP ≥ PYY (336) = NPY (3-36) Iyengar et al., J. Pharmacol. Exp. Ther. 289: 1031-40, 1999; Gehlert, Proc. Soc. Exp. Biol. Med. 218: 7-22, 1998; Michel et al., Pharmacol. Rev. 50: 143-50, 1998; document 5,968,819

Y6

NPY = PYY ≥ NPY (3-36)> PP Iyengar et al., J. Pharmacol. Exp. Ther. 289: 1031-40, 1999; Gehlert, Proc. Soc. Exp. Biol. Med. 218: 7-22, 1998; Michel et al., Pharmacol. Rev. 50: 143-50, 1998; US 5,968,819

(Y7)

PYY> NPY >> PYY (3-36) = PP Yang et al., Br. J. Pharmacol. 123: 1549-54,1998

(Y7)

PYY (3-36) ≥ PYY> NPY >> PP Haynes et al., Br. J. Pharmacol. 122: 1530-6, 1997

(Y7)

PYY >> NPY = PYY (3-36) = PP Kawakubo et al., Brain Res. 854: 30-4, 2000

Research has suggested that differences in binding affinities of Y receptors correlate with

25 secondary and tertiary structural differences. See, for example, Keire et al., Biochemistry 2000, 39, 99359942. The native swine PYY has been characterized as one that includes two C-terminal helical segments of residues 17 to 22 and 25 to 33 separated by a curve in the remains 23, 24 and 25, a turn around remains 12

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Phe27Leu31hPYY (5-36), Phe27Ser32hPYY (5-36), Phe27Lys33hPYY (5-36), Phe27Asn34hPYY (5-36), Phe27Lys35hPYY (536), Phe27Thr36hPYY (5-36), Phe27PY36h (5-7) -36), Gln29Val30hPYY (5-36), Gln29Ile31hPYY (536), Gln29Leu31hPYY (5-36), GlnZ9Ser32, hPYY (5-36), Gln29Leu33hPYY (5-36), Gln29Asn34hPYY (5-36), GlY29 (5-36), 5-L) 36), Gln29Thr36hPYY (5-36), Gln29Phe36hPYY (5-36), Ile30Ile31hPYY (5-36), Ile30Leu31hPYY (5-36), Ile30Ser32hPYY (5-36), Ile30Lys33hPYY (5-36), IY30Y (5-36) ), Ile30Lys35hPYY (5-36), Ile30Thr36hPYY (5-36), Ile30Phe36hPYY (5-36), Val30Ile31hPYY (5-36), Val30Leu31hPYY (5-36), Val30Ser32hPYY (S-36), Val30Y (5-36) , Val30Asn34hPYY (5-36), Val30Lys35hPYY (5-36), Val30Thr36hPYY (5-36), Val30Phe36hPYY (5-36), Ile31Ser32hPYY (5-36), Ile31Lys33hPYY (5-36), Ile31Asn34hPYY (5-36) Ile31Lys35hPYY (5-36), Ile31Thr36hPYY (5-36), Leu31Phe36hPYY (5-36), Leu31Phe36hPYY (5-36), Leu31Ser32hPYY (5-36), Val31Lys33hPYY (5-36), Leu31Asn34hPY35YPY35YPY31 (536), Leu31Thr36hPYY (5-36), Leu31Phe36hP YY (5-36), Ser32Lys33hPYY (5-36), Ser32Asn34hPYY (5-36), Ser32Lys35hPYY (5-36), Ser32Thr36hPYY (5-36), Ser32Phe36hPYY (5-36), Lys33Asn34hPYY (5-36), Lys33Yys (5-36), Lys33Thr36hPYY (S-36), Lys33Phe36hPYY (S-36), Asn34Lys35hPYY (5-36), Asn34Phe36hPYY (5-36), Lys35Thr36hPYY (536) or Lys35Phe36hPYY (5-36).

In some disclosures, the PYY analog polypeptides of the disclosure do not include any artificial amino acid residues, and comprise a C-terminal tail motif of hPYY. The C-terminal motif may comprise amino acid residues 32-35 of hPYY, for example, Thr, Arg, Gln, Arg (SEQ ID NO: 351). In said disclosure, the PYY analog polypeptides of the disclosure do not include any native PYY polypeptide or 1-4 Final deletions thereof (eg, PYY (1-36), PYY (2-36), PYY (3-36) ) and, PYY (4-36)). In some disclosures, such PYY analogs do not include: Lys25hPYY (5-36), Arg4hPYY (4-36), Glr4hPYY (4-36), Asn4hPYY (4-36), Lys25hPYY (4-36), Leu3hPYY (3- 36), Val3hPYY (3-36), Lys25hPYY (3-36), Tyr1,36pPYY, Pro13Ala14hPYY, FMS-PYY, FMS-PYY (3-36), Fmoc-PYY, Fmoc-PYY (3-36), FMS2 -PYY, FMS2-PYY (3-36), Fmoc2-PYY or Fmoc2-PYY (3-36).

In another aspect, said PYY analog polypeptides of the disclosure comprising a hPYY Cterminal tail motif also do not include: Thr27hPYY (3-36), Ile30hPYY (3-36), Thr36hPYY (3-36), Lys25Thr27hPYY (3-36 ), Lys25Ile30hPYY (3-36), Lys25Asn24hPYY (3-36), Lys25Thr36hPYY (3-36), Thr27Ile28hPYY (3-36), Thr27Val28hPYY (3-36), Thr27Gln29hPYY (3-3630), Thr27Ile (3-36), Thr27I (3-36), ThrY , Thr27Val30hPYY (3-36), Thr27Ile31hPYY (3-36), Thr27Leu31hPYY (3-36), Thr27Thr36hPYY (3-36), Thr27Phe36hPYY (3-36), Phe27Ile30hPYY (3-36), Phe27Thr (3-36), Phe27Thr (3-36), Phe27Thr (3-36) Gln29Ile30hPYY (3-36), Gln29Thr36hPYY (3-36), Ile30Ile31hPYY (3-36), Ile30Leu31hPYY (3-36), Ile30Thr36hPYY (3-36), Ile30Phe36hPYY (3-36), Val30TH36h (3-30) (3-36), Ile31Phe36hPYY (3-36), Leu31Thr36hPYY (3-36), Thr27hPYY (4-36), Phe27hPYY (4-36), Ile28hPYY (4-36), Val28hPYY (4-36), Gln29hPYY ( 4-36), Ile30hPYY (4-36), Val30hPYY (4-36), Ile31hPYY (4-36), Leu31hPYY (4-36), Thr36hPYY (4-36), Phe36hPYY (4-36), Lys25Thr27hPYY (4 -36), Lys25Phe27hPYY (436), Lys25Ile28hPYY (4-36), Lys2 5Val28hPYY (4-36), Lys25Gln29hPYY (4-36), Lys25Ile30hPYY (4-36), Lys25Val30hPYY (436), Lys25Ile31hPYY (4-36), Lys25Leu31hPYY (4-36), Lys25YPY36 (4-36) -36), Thr27Ile28hPYY (436), Thr27Val28hPYY (4-36), Thr27Gln29hPYY (4-36), Thr27Ile30hPYY (4-36), Thr27Val30hPYY (4-36), Thr27Ile31hPYY (436e) Thr27 (Y6), Thr27 (4-36), Thr27Phe36hPYY (4-36), Phe27Ile28hPYY (4-36), Phe27Val28hPYY (436), Phe27Gln29hPYY (4-36), Phe27Ile30hPYY (4-36), Phe27Val30hPYY (4-36), Phe27 (4-36), Phe27 (4-36) 36), Phe27Leu31hPYY (4-36), Phe27Thr36hPYY (4-36), Phe27Phe36hPYY (4-36), Gln29Ile30hPYY (4-36), Gln29Val30hPYY (4-36), Gln29Ile31hPYY (4-36), Gln29Y 4-16 (Gl36) ), Gln29Thr36hPYY (4-36), Gln29Phe36hPYY (4-36), Ile30Ile31hPYY (4-36), Ile30Leu31hPYY (4-36), Ile30Th36hPYY (4-36), Ile30Phe36hPYY (4-36), Val30Yle (4-36), Val30Yle (4-36) , Val30Leu31hPYY (4-36), Val30Thr36hPYY (4-36), Val30Phe36hPYY (4-36), Ile31Thr36hPYY (4-36), Leu31Phe36hPYY (4-36), Leu31Phe36hPYY (4-36), Leu31Thr36hPY36 (4-36) Leu31Phe36hPYY (4-36), Thr27hPYY (5-36), Ph e27hPYY (5-36), Ile28hPYY (5-36), Val28hPYY (536), Gln29hPYY (5-36), Ile30hPYY (5-36), Val30hPYY (5-36), Ile31hPYY (5-36), Leu31hPYY (5 -36), Thr36hPYY (5-36), Phe36hPYY (5-36), Lys25Thr27hPYY (5-36), Lys25Phe27hPYY (5-36), Lys25Ile28hPYY (5-36), Lys25Val28hPYY (5-36), Lys25Yln (5-29) 36), Lys25Ile30hPYY (5-36), Lys25Val30hPYY (5-36), Lys25Ile31hPYY (5-36), Lys25Leu31hPYY (5-36), Lys25Thr36hPYY (5-36), Lys25Phe36hPYY (5-36), Thr27I 5-36), Thr27 ), Thr27Val28hPYY (5-36), Thr27Gln29hPYY (5-36), Thr27Ile30hPYY (5-36), Thr27Val30hPYY (5-36), Thr27Ile31hPYY (5-36), Thr27Leu31hPYY (5-36), Thr27Thr-36) , Thr27Phe36hPYY (5-36), Phe27Ile28hPYY (5-36), Phe27Val28hPYY (5-36), Phe27Gln29hPYY (5-36), Phe27Ile30hPYY (5-36), Phe27Val30hPYY (5-36), Phe27Ile 5-6) Phe27Leu31hPYY (5-36), Phe27Thr36hPYY (5-36), Phe27Phe36hPYY (536), Gln29Ile30hPYY (5-36), Gln29Val30hPYY (5-36), Gln29Ile31hPYY (5-36), Gln29Y29Y29 (533) ), Gln29Phe36hPYY (5-36), Ile30Ile31hPYY (5-36), Ile30Leu31hPYY (5-36), Ile30Thr36hPYY (5-36), Ile30Phe36hPYY (5-36), Val30Ile31hPYY (5-36), Val30Leu31hPYY (5-36), Val30Thr36hPYY (5-36), Val30Phe36hPYY (5-36), Ile31Thr36hPYY (5-36), Leu31Phe36hPY36YPY36YPY36YPY36YPY36YPY36YPY36YPY36 (5-36), Leu31Thr36hPYY (5-36) or Leu31Phe36hPYY (5-36).

In some disclosures, PYY analog polypeptides of the disclosure are at least 34 amino acids in length. In some disclosures, PYY analog polypeptides of the disclosure include only natural L amino acid residues and / or modified natural L amino acid residues. In some disclosures, PYY analog polypeptides of the disclosure do not include artificial amino acid residues.

More particularly, in one aspect, the present disclosure relates to PYY analog polypeptides that include one or more amino acid modifications of the sequence. Such modifications include substitutions, insertions and / or deletions, alone or in combination. In some disclosures, PYY analog polypeptides of the disclosure include one or more modifications of a "non-essential" amino acid residue. In the context of the disclosure, a "non-essential" amino acid residue is a residue that can be modified, that is, deleted or substituted, in the amino acid sequence of native human PYY without substantially inhibiting or reducing the agonist activity of PYY of the PYY analog polypeptide. In some embodiments, the PYY analog polypeptides of the disclosure

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they retain at least about 25%, or from about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98% or about 99% of the biological activity of native human PYY with respect to the reduction of nutrient availability. In another disclosure, the PYY analog polypeptides of the disclosure exhibit a better PYY agonist activity. In some disclosures, PYY analog polypeptides of the disclosure have at least about 110%, about 125%, about 130%, about 140%, about 150%, about 200%, or more of the Biological activity of native human PYY in terms of reducing nutrient availability.

PYY analog polypeptides are those that have a potency in one of the assays described herein (including food intake, gastric emptying, pancreatic secretion, body composition or weight reduction) tests that are equal to or greater than NPY, PYY or PYY power (3-36) in that same test. In some disclosures, PYY analog polypeptides of the disclosure may show greater ease of manufacture, stability and / or ease of formulation, compared to PP, NPY, PYY or PYY (3-36).

to. Substitutions

In one disclosure, the PYY analog polypeptides of the disclosure may have one or more substitutions in the amino acid sequence of native human PYY (SEQ ID NO: 2), alone or in combination with one or more insertions

or eliminations. In some disclosures, the substitution does not substantially inhibit or reduce the PYY agonist activity of the PYY analog polypeptide. In one aspect, the present disclosure relates to PYY analog polypeptides that have a single substitution, or the consecutive or non-consecutive substitution of more than one amino acid residue in the amino acid sequence of native human PYY (SEQ ID NO: 2). In some disclosures, PYY analog polypeptides of the disclosure include one, two, three, four, five, six, seven, eight, nine or ten amino acid substitutions.

In some disclosures, the amino acid residues of native human PYY (SEQ ID NO: 2) of the helical C-terminal region of PYY (eg, residues 20, 24, 25, 27 and 29), the end-of- tail (32-36) and / or the N-terminal prolines of position 5 and 8 are not substituted. In some disclosures, amino acid residues are not substituted at positions 32 to 36 of native human PYY (SEQ ID NO: 2). In another disclosure, amino acid residues of native human PYY (SEQ ID NO. 2) are not substituted at one or more amino acid sequence positions selected from: 5, 7, 8, 20, 24, 25, 27, 29, 32, 33, 34, 35, 36, and any combination thereof.

Substitutions may include conserved amino acid substitutions. A "conservative amino acid substitution" is one in which the amino acid residue is replaced by an amino acid residue that has a similar, or physicochemical, side chain (eg, electrostatic characteristics, hydrogen bonds, isosteric, hydrophobic) . Families of amino acid residues that have similar side chains are known in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, Threonine, tyrosine, methionine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan), -branched side chains (e.g. threonine, valine, isoleucine) and side chains aromatic (for example, tyrosine, phenylalanine, tryptophan, histidine).

In another disclosure, PYY analog polypeptides of the disclosure may include substitutions of one or more artificial amino acids and / or non-amino acids, for example, amino acid mimetics, in the PYY sequence (SEQ ID NO: 2). In some disclosures, the non-amino acids inserted in the PYY sequence (SEQ ID NO: 2) may be spin mimetics  or linker molecules, such as -NH-X-CO-, in which X = (CH2) n (where n can be 2 -20) or -NH-CH2CH2 (-O-CH2CH2-O-) m-CH2-CO- (where m = 1-5). The linker molecules may include aminocaproyl ("Aca"), -alanyl and 8-amino-3,6-dioxaoctanoyl. Mim Turn mimetics are commercially available (BioQuadrant Inc, Québec, Canada) and have been described in the literature (Hanessian et al., Tetrahedron 12789-854 (1997); Gu et al., Tetrahedron Letters 44: 5863- 6 (2003); Bourguet et al., Bioorganic & Medicinal Chemistry Letters 13: 1561-4 (2003); Grieco et al., Tetrahedron Letters 43: 6297-9 (2002); Souers et al., Tetrahedron

57: 7431-48 (2001); Tsai et al., Bioorganic & Medicinal Chemistry 7: 29-38 (1999); Virgil et al., Tetrahedron 53: 6635-44 (1997)). The spin mimetics  may include the mimetic A and the mimetic B illustrated below.

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Modify with a Bolton-Hunter group. In some disclosures, there may be multiple Bolton-Hunter modification sites along the PYY analog polypeptide. Bolton-Hunter reagents used for the modification of polypeptides are commercially available, and may include, but are not limited to, water-soluble Bolton-Huntet reagent, sulfosuccinimidyl-3- [4-hydroxyphenyl] propionate (Pierce Biotechnology, Inc ., Rockford, IL) and reagent 2 of Bolton-Hunter, N-succinimidyl-3- (4-hydroxy-3-iodophenyl) propionate (Wako Pure Chemical Industries, Ltd., Japan, catalog No. 199-09341). Next, an illustrative Bolton-Hunter group conjugated through an amide bond to a PYY analog polypeptide is illustrated, in which the broken line passes through the amide bond:

PYY analog polypeptides can be iodized (such as radiolabeled with 125I) before or after Bolton-Hunter modification. PYY or PYY analogues modified with Bolton-Hunter and marked with 125I can also be purchased from Amersham Corporation (Arlington Heights, IL). Bolton-Hunter derivatives are abbreviated as "modified with BH" in Table 4 (SEQ ID No. 475-480).

and. Analogues and derivatives

In some disclosure, PYY analog polypeptides include combinations of the modifications described above, that is, removal, insertion and substitution.

By way of example, PYY analog polypeptides may include N-terminal deletions in combination with one or more amino acid substitutions. For example, PYY analog polypeptides include PYY (3-36) with one or more of the following amino acid substitutions: Ala3, Leu3, Pro3, Ala4, Gly4, d-Ala4, homoLys4, Glu4, Ala5, Ala6, Val6, d-Ala7, Tyr7, His7, Ala8, Ala9, Ala10, Ala11, d-Ala12, Ala13, homoSer13, Ala14, Ala15, Gln15, Ala16, Ala17, Met17, Ala18, Ser18, nor-Val18, Ala19, N-Me- Ala19, Lys19, homoArg19, Ala20, Ala21, d-Ala22, Ala23, Ala24, Ala25, Lys25, homoArg25, Ala26, Ala27, Ala28, Ala29, Ala30, Ala31, Ala32, Ala33, Lys33, Ala34, Ala35, Ala36, His36, Trp36, N-Me-Tyr36 and Phe36. In some disclosures, the PYY analog polypeptide includes one, two or three amino acid substitutions. Certain PYY analog polypeptides comprise deletions in combination with amino acid insertions (see, for example, SEQ ID No. 89-174)

PYY analog polypeptides include polypeptides of Formula (V) (SEQ ID No. 350):

Xaa3 Xaa4 Pro Xaa6 Xaa7 Pro Xaa9 Xaa10 Xaa11 Xaa12

Xaa13 Xaa14 Xaa15 Xaa16 Xaa17 Xaa18 Xaa19 Tyr Xaa21 Xaa22

Xaa23 Leu Arg Xaa26 Tyr Xaa28 Asn Xaa30 Xaa31 Thr

Arg Gln Arg Xaa36

in which:

Xaa3 is Ile, Ala, Pro, Ser, Thr or NorVal;

Xaa4 is Lys, Ala, Gly, Glu, Asp, d-Ala, homoLys or homoArg;

Xaa6 is Glu, Ala, Val, Asp, Asn or Gln;

Xaa7 is Ala, Asn, His, Ser or Tyr;

Xaa9 is Gly, Ala, Ser, sarcosina, Pro or Aib;

Xaa10 is Glu, Ala, Asp, Asn, Gln, Pro, Aib or Gly;

Xaa11 is Asp, Ala, Glu, Asn, Gln, Pro, Aib or Gly;

Xaa12 is Ala or d-Ala;

Xaa13 is Ser, Ala, Thr or homoSer;

Xaa14 is Pro, Ala, homoPro, hydroxyPro, Aib or Gly;

Xaa15 is Glu, Ala, Asp, Asn, Gln, Pro, Aib or Gly;

Xaa16 is Glu, Ala, Asp, Asn or Gln;

Xaa17 is Leu, Ala, Met, Trp, Ile, Val or NorVal;

Xaa18 is Asn, Asp, Ala, Glu, Gln, Ser or Thr;

Xaa19 is Arg, Tyr, Lys, Ala, Gln or N (Me) Ala;

Xaa21 is Tyr, Ala, Met, Phe or Leu;

Xaa22 is Ala, Ser, Thr or d-Ala;

Xaa23 is Ser, Ala, Thr or homoSer;

Xaa26 is His or Ala;

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presence of fatty acid and the activating agent (HBTU / HOBt) in DMF. The resulting derivatives are purified by reverse phase HPLC and purity is confirmed by LC / MS.

Modification with PEG can be carried out in solution in a lysine-free amino group or an amino group of a purified peptide using commercially available activated PEG esters. The resulting PEGylated derivatives are purified to homogeneity by reverse phase HPLC and purity is confirmed by LC / MS and MALDI-MS.

The formation of intramolecular disulfide bonds can be performed in free cysteines using iodine / acetic acid as an oxidizing agent.

The PPF polypeptides of the disclosure can be assayed in a variety of in vitro biological assays, including receptor binding assays Y using binding assay methodologies generally known to those skilled in the art, or in vivo, using ingestion assays of food, body weight and body composition, using methodologies generally known to those skilled in the art. Illustrative essays include those described below.

Signaling assays by Gi-G0-coupled receptors: Without intending to be limited by theory, the signaling of G-protein-coupled receptors (GPCR) mediated by heterotrimeric G-proteins can be classified into signaling classes based on a subunit composition  The Gs, Gq and Gi / G0 proteins mediate intracellular signaling through the activation of signaling pathways that lead to different physiological assessment criteria. Activation of Gs and Gi / G0 coupled receptors leads to stimulation or inhibition of adenylate cyclase, respectively, while activation of Gq coupled receptors results in stimulation of phospholipase C (PLC) and an increase in the concentration of intracellular calcium The measurement of a reduction in cAMP produced as a result of the activation of the receptors coupled to Gi / G0 can be technically difficult, while the measurement of an increase coupled to Gq in the intracellular calcium is relatively easy. Therefore, assays have been developed to evaluate the activity of Gi / G0 coupled receptors using cotransfected cells with promiscuous  subunits of G to redirect Gi / G0 signaling through PLC, and the use of indicator or fluorophores genes Calcium sensitive is known in the art. These assays can be used to evaluate the ability of PPF polypeptides to act as agonists or antagonists in Y receptors. See, for example, Stables, et al., (1997) Anal. Biochem, 252 (1): 115-26.

NPY Y1 receptor binding assay: confluent culture membranes of SK-N-MC cells that endogenously express Y1 neuropeptide receptors are prepared. The membranes are incubated with human YY peptide labeled with [125I] 60 pM (2,200 Ci / mmol, Perkin Elmer Life Sciences), and with unlabeled PPF polypeptide for 60 minutes at room temperature in a 96-well polystyrene plate. Next, the contents of the wells of a 96-well fiberglass plate are collected using a Perkin Elmer plate collector. The dry fiberglass plates are combined with scintillation and counting is performed in a Perkin Elmer scintillation counter.

NPY Y2 receptor binding assay: confluent culture membranes of SK-N-BE cells are prepared that endogenously express Y2 neuropeptide receptors. The membranes are incubated with human YY peptide labeled with [125I] 30 pM (2200 Ci / mmol, PerkinElmer Life Sciences), and with unlabeled PPF polypeptide for 60 minutes at room temperature in a 96-well polystyrene plate. Next, the contents of the wells of a 96-well fiberglass plate are collected using a Perkin Elmer plate collector. The dry fiberglass plates are combined with scintillation and counting is performed in a Perkin Elmer scintillation counter.

NPY Y4 receptor binding assay: CHO-K1 cells are transfected transiently with cDNA encoding the Y4 neuropeptide gene, and 48 hours later, confluent cell culture membranes are prepared. The membranes are incubated with human pancreatic polypeptide labeled with [125I] 18 pM (2,200 Ci / mmol, PerkinElmer Life Sciences), and with unlabeled PPF polypeptide for 60 minutes at room temperature in a 96-well polystyrene plate. Next, the contents of the wells of a 96-well fiberglass plate are collected using a Perkin Elmer plate collector. The dry fiberglass plates are combined with scintillation and counting is performed in a Perkin Elmer scintillation counter.

NPY Y5 receptor binding assay: CHO-K1 cells are transfected transiently with cDNA encoding the Y5 neuropeptide gene, and 48 hours later, confluent cell culture membranes are prepared. The membranes are incubated with human YY peptide labeled with [125I] 44 pM (2,200 Ci / mmol, PerkinElmer Life Sciences), and with unlabeled PPF polypeptide for 60 minutes at room temperature in a 96-well polystyrene plate. Next, the contents of the wells of a 96-well fiberglass plate are collected using a Perkin Elmer plate collector. The dry fiberglass plates are combined with scintillation and counting is performed in a Perkin Elmer scintillation counter.

46

image37

E05854234

10-20-2015

Table 2:

SEQ ID NO.

Y1RBA (nM) Y2RBA (nM) Y4RBA (nM) Y5RBA (nM)

one

10 1000 0.034 1.6

2

0.2 0.058 4,5 0.31

3

6.2 0.041 54 0.85

4

0.48 0.24 39 0.43

5

> 1000 229 > 1000 0.59

6

0.42 0.19 0.84 0.19

7

1000 twenty-one 1000 1000

8

1000 12 1000 1000

9

0.61 0.085 51 0.47

10

1.3 0.023 107 0.49

eleven

2.6 0.059 96 0.41

12

1.7 0.14 16 0.31

13

3.2 0.42 169 0.54

14

1000 1.6 1000 6.8

fifteen

1.6 0.026 52 0.33

16

4.1 0.048 29 0.15

17

eleven 0.037 104 0.36

18

0.32 0.031 19 0.32

19

5.4 0.036 117 0.73

twenty

2.9 0.04 93 0.42

twenty-one

24 0.31 182 3.3

22

12 0.1 75 7.4

2. 3

13 0.2 54 3.2

26

4.4 0.04 120 0.42

27

7 0.18 104 1.3

28

0.55 0.032 9.2 0.23

29

14 0.46 178 0.95

fifty

0.86 0.15 14 0.6

51

0.68 0.14 7.7 0.56

52

2.7 0.19 twenty-one 0.93

53

2.2 0.084 7.4 0.64

89

4.7 0.11 38 0.99

90

fifteen 0.46 fifty 7.3

91

9.2 0.35 99 1.9

92

9.8 0.36 107 5

93

8.6 0.28 99 5.6

94

1.8 0.048 27 0.54

95

8.2 0.67 101 7.3

96

7.4 0.29 56 6.6

97

8.6 0.19 54 2.9

98

4.4 0.099 49 2.1

99

3.5 0.065 43 0.99

100

5.9 0.28 70 4

101

8.6 0.18 65 3.4

102

7.8 0.09 58 1.8

103

1.8 0.038 22 0.66

104

4.6 0.053 27 0.89

105

4.4 0.3 68 3.3

106

5.4 0.081 37 0.92

107

eleven 0.27 70 5.1

108

8.8 0.12 51 2.1

109

9.5 0.73 74 3. 4

110

twenty 0.81 97 8.7

111

17 0.41 71 10

112

5.6 0.33 76 6.3

113

6.8 0.1 37 1.2

114

71 0.25 119 14

115

3. 4 6.2 193 55

116

8.9 0.23 40 10

117

7.3 0.21 74 5.8

118

88 0.97 180 31

48

E05854234

10-20-2015

SEQ ID NO.

Y1RBA (nM) Y2RBA (nM) Y4RBA (nM) Y5RBA (nM)

119

158 1.1 92 47

120

17 1.5 44 27

121

14 0.19 51 14

122

36 0.4 68 2.4

123

Four. Five 9.2 66 1.7

124

> 1000 86 > 1000 56

125

28 9.1 129 8.4

126

24 3. 4 88 2.4

127

> 1000 > 1000 > 1000 > 1000

128

> 1000 113 > 1000 > 1000

130

4,5 0.25 46 5.2

131

6.8 0.28 80 2.9

132

17 0.56 113 7

133

1000 8.3 1000 138

135

293 43 1000 1000

136

88 0.081 863 1.8

138

7.9 0.43 165 5

139

301 twenty 1000 354

140

1000 380 1000 1000

142

6.2 0.12 61 1.2

143

3.8 0.19 56 2.3

144

4,5 0.39 52 4.6

145

5.4 0.12 47.5 1.5

146

8.7 0.19 73 2.3

147

5.1 0.092 48 1.7

148

5 0.1 fifty 1.8

150

276 eleven 1000 118

151

7.6 0.25 115 2.1

152

3.7 0.24 3.9 0.82

153

8.4 0.28 135 2.9

155

7.6 0.24 108 2.3

156

7.3 0.35 147 3.3

157

5.8 0.11 63 1.6

158

6.1 0.11 66 2.1

160

6.3 0.56 71 2.9

162

eleven 0.47 86 2.8

165

4.8 0.072 59 1.3

171

33 0.53 97 10

172

22 3.3 59 9.1

173

14 0.99 52 7.8

174

eleven 0.35 64 80

175

twenty 0.72 > 1000 > 1000

176

7.6 0.84 120 8.5

177

5.8 0.34 46 eleven

178

7.7 0.29 38 17

179

30 5.4 33 208

180

4.3 0.11 49 3.9

181

6.3 0.41 46 2.4

182

4.4 0.21 65 5.8

183

4.7 0.071 60 9.2

184

26 0.14 54 42

185

3 0.13 38 3.8

186

0.85 0.11 29 2.8

187

1000 62 1000 128

188

1000 102 1000 968

189

1000 57 1000 202

190

1000 24 1000 578

193

308 78 331 180

194

32 1.5 89 fifteen

195

fifteen 1.7 146 5.7

196

1000 612 1000 1000

197

1000 46 611 1000

49

E05854234

10-20-2015

SEQ ID NO.

Y1RBA (nM) Y2RBA (nM) Y4RBA (nM) Y5RBA (nM)

198

10 0.7 88 9.9

199

38 4.1 143 58

200

106 7 426 74

201

27 2.2 99 29

202

36 148 2. 3 80

203

33 4.4 108 78

204

47 1.1 223 37

205

44 1.5 172 18

206

66 fifteen 204 Four. Five

207

180 0.69 1000 114

208

228 93 407 568

211

3.7 0.24 fifty 5.4

212

2.9 0.046 59 0.8

225

6.7 0.15 79 1.8

226

3 0.059 35 0.57

227

one 0.032 38 0.11

228

4.1 0.1 61 1.1

229

8.2 0.23 57 2.7

230

3.4 0.1 Four. Five 1.2

231

5.6 0.37 55 9.4

235

8.7 0.65 77 12

236

6.5 0.24 62 4.6

237

2.1 0.11 35 2.8

239

0.18 0.092 18 0.27

240

2.4 0.059 89 0.58

241

4 0.15 61 0.88

242

2.7 0.13 71 one

243

18 0.74 124 7.2

244

eleven 1.5 88 7.5

245

0.19 0.077 16 0.35

246

3.9 0.11 119 0.7

247

0.38 0.12 25 0.76

248

0.48 0.12 24 0.44

249

0.36 0.11 twenty-one 0.34

250

2.2 0.075 73 0.51

251

0.42 0.12 28 0.52

252

2.1 0.074 52 0.64

253

1.3 0.041 3. 4 0.29

254

2.3 0.051 85 0.56

255

5.7 0.26 208 2

256

1.7 0.039 395 0.48

258

0.39 0.12 22 0.89

260

0.42 0.16 22 0.74

261

2.9 0.11 71 one

262

1.7 0.087 61 0.91

263

3.2 0.1 141 1.2

264

1.8 0.22 98 0.48

265

7.3 1.1 272 eleven

266

2 0.13 193 1.7

267

0.25 0.1 9.5 0.32

268

0.31 0.14 twenty-one 0.57

269

3.8 0.084 77 0.74

270

3.3 0.13 97 1.4

271

0.51 0.094 4.2 0.25

272

0.26 0.1 12 0.27

273

0.32 0.18 twenty-one 0.89

274

4.9 0.42 181 1.5

275

0.59 0.099 81 1.5

276

0.68 0.3 8.3 1.3

277

3.4 0.16 150 2.5

278

3.6 0.078 138 1.4

279

6.4 1.2 200 12

fifty

E05854234

10-20-2015

SEQ ID NO.

Y1RBA (nM) Y2RBA (nM) Y4RBA (nM) Y5RBA (nM)

280

2.1 0.38 108 1.6

281

2.8 0.1 117 0.67

282

0.55 0.04 18 0.15

283

30 3.4 87 10.6

284

1.1 0.071 47 0.56

285

0.67 0.18 16 0.54

286

0.65 0.11 0.75 0.3

287

5.2 0.16 10 1.2

288

1.8 0.35 eleven 1.1

289

48 0.83

290

187 0.51

291

186 201 9.5 0.71

292

1.4 0.17 0.77 0.32

293

0.82 0.18 0.87 0.48

294

0.94 0.17 0.98 0.51

295

one 0.18 one 0.63

296

2.7 0.76 2.9 2.1

297

3.6 0.32 4 1.8

298

5.5 1.2 3.4 3.9

299

eleven 3.2 16 7.5

300

83 16 311 78

301

26 3.7 70 28

302

5.1 0.68 93 2.9

303

6 0.5 7.1 3.3

304

0.51 0.14 0.48 0.28

306

0.6 0.16 1.2 0.27

307

0.53 0.13 0.73 0.47

308

one 0.56 2.1 1.4

309

3.3 78 5.6 1.5

310

29 454 27 5.1

311

16 0.49 51 1.8

312

70 0.42 91 3.4

313

9.2 0.57 151 2.6

314

8.2 0.67 202 2.5

315

9.2 2.1 467 5.6

316

7.1 0.63 52 1.1

317

4.3 0.097 16 0.69

318

100 1.3 84 1.9

319

35 1.04 77 1.2

320

77 3.1 243 13

321

12 3.7 57 5.6

332

13 0.54 38 one

333

4.8 0.54 37 0.87

334

twenty-one 0.45 101 2.4

335

3. 4 0.72 109 3.6

338

8.1 0.68 46 1.1

341

1.8 0.15 eleven 0.3

342

fifteen 0.62 84 1.4

343

12 0.38 69 1.3

347

35 18 740 51

436

4 0.07 36 1.4

437

5.1 0.45 371 2.1

438

1.5 0.079 167 1.2

439

0.93 0.05 176 0.47

440

1.6 0.1 100 1.2

441

4.8 0.65 224 7

442

1.6 0.11 214 1.3

443

474 113 914 592

444

6.6 0.36 97 3.8

445

9.1 0.56 269 6.3

446

13 one 141 6.6

447

8.3 0.5 206 25

51

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Claims (1)

image 1 image2