# Patent

## Title
Ghrelin analogues

## Abstract

The present invention relates, inter alia, to ghrelin analogs and their medical use, for example in the treatment of cachexia, chronic obstructive pulmonary disease, gastrointestinal disorders (e.g., gastroparesis and/or inflammatory disorders such as colitis, gut barrier dysfunction, and ischemia reperfusion injury), loss of body weight, and decreased appetite.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is the U.S. National Stage of the International Patent Application No. PCT/EP2013/052107, filed Feb. 1, 2013, which claims the benefit of U.S. Provisional Application No. 61/704,393, filed Sep. 21, 2012, and U.S. Provisional Application No. 61/594,970, filed Feb. 3, 2012, each of which is incorporated herein by reference.


FIELD OF THE INVENTION
The present invention relates, inter alia, to ghrelin analogues and their medical use, for example in the treatment of a condition caused or characterized by loss of body weight or decreased appetite, e.g. the treatment and/or prevention of cachexia, effects of gastrectomy or vagectomy, anorexia or bulimia, chronic obstructive pulmonary disease, and gastrointestinal disorders (e.g., gastroparesis and/or inflammatory disorders such as colitis, gut barrier dysfunction, and ischemia reperfusion injury), in an individual in need thereof.
BACKGROUND OF THE INVENTION
Cachexia is a wasting syndrome characterized by physical wasting associated with loss of weight and muscle mass. These symptoms leave individuals affected by the syndrome extremely weak and unable to perform everyday tasks. Cachexia often is associated with diseases such as cancer, AIDS, and cardiovascular disease. According to the National Cancer Institute, almost one-third of cancer deaths may be caused by cachexia. Despite this remarkable statistic, there currently appear to be no effective treatments for cachexia.
A number of hormones that influence satiety, gastrointestinal motility, and blood glucose levels are secreted in the gastrointestinal tract. One of these hormones is ghrelin.
The peptide ghrelin was identified as the ligand of the growth-hormone secretagogue receptor type 1a (GHSR1a), and plays an important role in a number of biological systems, such as the release of growth hormone, the release of insulin, the stimulation of appetite, and gastric motility. Ghrelin is a 28 amino acid peptide hormone with its third residue (Ser3) modified with an octanoyl acid moiety. This latter modification appears to be important for ghrelin's interaction with GHSR1a, and, thus, its activity. Ghrelin is formed by the cleavage of preproghrelin, which produces at least two distinct peptides, ghrelin and obestatin.
SUMMARY OF THE INVENTION
The invention provides a compound of the formula I:

R1—Z—R2  (I)

wherein

R1 is hydrogen, C1-4 alkyl, formyl, acetyl, trifluoroacetyl or benzoyl;

R2 is OH or NH2; and

Z is an amino acid sequence having the formula Ia:

X1-X2-X3-Phe-Leu-Ser-Pro-X8-His-X10-X11-X12-X13-X14-X15-X16-Glu-Ser-X19-X20-Pro-Pro-Ala-X24-Leu-X26-Pro-X28  (Ia) (SEQ ID NO: 2)

or is a truncated form thereof lacking from 1 to 23 amino acids from the C-terminus;

wherein

X1 is an amino acid residue selected from Ala, Inp, Pro, DPro, Sar, Aib, Phe and Ser;

X2 is Ser; or

X1 and X2 together constitute a residue of a non-naturally occurring amino acid, wherein the shortest bond separation between the amino nitrogen atom and the carbon atom of the carbonyl group in the non-naturally occurring amino acid is 3 to 7 bonds;

when X1 is an amino acid residue selected from Pro, DPro, Phe and Ser, or when X1 and X2 together constitute a residue of the non-naturally occurring amino acid, then X3 is selected among:

—NH—CH[(CH2)n—O—R3]—C(O)—;  (i)

—NH—CH(R4)—C(O)—;  (ii)

—NH—CH[(CH2)m—O—R5]—C(O)—; and  (iii)

—NH—CH[(CH2)p—NH—R6]—C(O)—;  (iv)

and when X1 is an amino acid residue selected from Ala, Sar, Inp and Aib, then X3 is selected among:

—NH—CH[(CH2)n—O—R3]—C(O)—; and  (i)

—NH—CH(R4)—C(O)—;  (ii)

wherein

R3 and R4 are, independently, C1-35-alkyl or -alkenyl groups;

R5 and R6 are, independently, C1-35-acyl groups; and

n, m and p are integers 1, 2, 3, 4 or 5;

when present

X8 is selected from Glu, Lys and Tyr;

X10 is selected from Gin, Glu and Ser;

X11 is selected from Arg, Lys and Ser;

X12 is selected from Val, Ala, Pro, Lys, Thr and Leu;

X13 is selected from Gin and Ser;

X14 is selected from Gin, Lys, Tyr and Ser, or is absent;

X15 is selected from Arg and Ser;

X16 is selected from Lys, Ser and Pro;

X19 is selected from Lys and Ser;

X20 is selected from Lys, Glu and Ser;

X24 is selected from Lys, Tyr and Ser;

X26 is selected from Gin and Ser; and

X28 is selected from Arg and Ser;

and, optionally, one or more of said amino acid residues X8, X10, X11, X13, X14, X16, X19, X20, X24, X26 and X28, independently, is replaced with a modified lysine residue comprising a lipophilic substituent having the formula Z1Z2—, wherein Z2 is a spacer moiety or is absent, and Z1 is a lipophilic moiety conjugated, either via Z2 or directly, to the side-chain of said lysine residue;

or a pharmaceutically acceptable salt or solvate of any of the above.

In some embodiments, the present invention provides a compound comprising the formula I:

R1—Z—R2  (I)

wherein

R1 is hydrogen, C1-4 alkyl, formyl, acetyl, trifluoroacetyl or benzoyl;

R2 is OH or NH2; and

Z is an amino acid sequence having the formula Ia:

X1-X2-X3-Phe-Leu-Ser-Pro-Glu-His-X10-X11-X12-X13-X14-Arg-X16-Glu-Ser-X19-X20-Pro-Pro-Ala-X24-Leu-X26-Pro-X28  (Ib) (SEQ ID NO: 3)

or is a truncated form thereof lacking from 1 to 23 amino acids from the C-terminus;

wherein

X1 is an amino acid residue selected from Ala, Inp, Pro, DPro, Sar, Aib, Phe and Ser;

X2 is Ser; or

X1 and X2 together constitute a residue of a non-naturally occurring amino acid, wherein the shortest bond separation between the amino nitrogen atom and the carbon atom of the carbonyl group in the non-naturally occurring amino acid is 3 to 7 bonds;

when X1 is an amino acid residue selected from Pro, DPro, Phe and Ser, or when X1 and X2 together constitute a residue of the non-naturally occurring amino acid, then X3 is selected among:

—NH—CH[(CH2)n—O—R3]—C(O)—;  (i)

—NH—CH(R4)—C(O)—;  (ii)

—NH—CH[(CH2)m—O—R5]—C(O)—; and  (iii)

—NH—CH[(CH2)p—NH—R6]—C(O)—;  (iv)

and when X1 is an amino acid residue selected from Ala, Sar, Inp and Aib, then X3 is selected among:

—NH—CH[(CH2)n—O—R3]—C(O)—; and  (i)

—NH—CH(R4)—C(O)—;  (ii)

wherein

R3 and R4 are, independently, C1-35-alkyl or -alkenyl groups;

R5 and R6 are, independently, C1-35-acyl groups; and

n, m and p are integers 1, 2, 3, 4 or 5;

when present

X10 is selected from Gin and Ser;

X11 is selected from Arg and Lys;

X12 is selected from Val, Ala, Thr and Leu;

X13 is selected from Gin and Ser;

X14 is selected from Gin and Ser, or is absent;

X16 is Lys;

X19 is Lys;

X20 is Lys;

X24 is Lys; and

X26 is selected from Gin and Ser;

X28 is Arg;

and, optionally, one or more of said amino acid residues X10, X11, X13, X14, X16, X19, X20, X24, X26 and X28, independently, is replaced with a modified lysine residue comprising a lipophilic substituent having the formula Z1Z2—, wherein Z2 is a spacer moiety or is absent, and Z1 is a lipophilic moiety conjugated, either via Z2 or directly, to the side-chain of said lysine residue;

or a pharmaceutically acceptable salt or solvate of any of the above.

In some embodiments, the present invention provides a compound comprising the formula I:

R1—Z—R2  (I)

wherein

R1 is hydrogen, C1-4 alkyl, formyl, acetyl, trifluoroacetyl or benzoyl;

R2 is OH or NH2; and

Z is an amino acid sequence having the formula Ia:

X1-X2-X3-Phe-Leu-Ser-Pro-Glu-His-X10-X11-X12-X13-X14-Arg-X16-Glu-Ser-X19-X20-Pro-Pro-Ala-X24-Leu-X26-Pro-X28  (Ib) (SEQ ID NO: 3)

or is a truncated form thereof lacking from 1 to 23 amino acids from the C-terminus;

wherein

X1 is an amino acid residue selected from Ala, Inp, Pro, DPro, Sar, Aib, Phe and Ser;

X2 is Ser; or

X1 and X2 together constitute a residue of a non-naturally occurring amino acid, wherein the shortest bond separation between the amino nitrogen atom and the carbon atom of the carbonyl group in the non-naturally occurring amino acid is 3 to 7 bonds;

when X1 is an amino acid residue selected from Pro, DPro, Phe and Ser, or when X1 and X2 together constitute a residue of the non-naturally occurring amino acid, then X3 is selected from among:

—NH—CH[(CH2)n—O—R3]—C(O)—;  (i)

—NH—CH(R4)—C(O)—;  (ii)

—NH—CH[(CH2)m—O—R5]—C(O)—; and  (iii)

—NH—CH[(CH2)p—NH—R6]—C(O)—;  (iv)

and when X1 is an amino acid residue selected from Ala, Sar, Inp and Aib, then X3 is selected from among:

—NH—CH[(CH2)n—O—R3]—C(O)—; and  (i)

—NH—CH(R4)—C(O)—;  (ii)

wherein

R3 and R4 are, independently, C1-35-alkyl or -alkenyl groups;

R5 and R6 are, independently, C1-35-acyl groups; and

n, m and p are integers 1, 2, 3, 4 or 5;

when present

X10 is Gin;

X11 is selected from Arg and Lys;

X12 is selected from Val, Ala, Thr and Leu;

X13 is Gin;

X14 is Gin or is absent;

X16 is Lys;

X19 is Lys;

X20 is Lys;

X24 is Lys; and

X26 is Gin;

X28 is Arg;

and optionally, wherein one or more of said amino acid residues X10, X11, X13, X14, X16, X19, X20, X24, X26 and X28 independently, is replaced with a modified lysine residue comprising a lipophilic substituent having the formula Z1Z2—, wherein Z2 is a spacer moiety or is absent, and Z1 is a lipophilic moiety conjugated, either via Z2 or directly, to the side-chain of said lysine residue;

or a pharmaceutically acceptable salt or solvate of any of the above.

In some embodiments X28 is not one of the residues which may be replaced by the modified lysine residue.
In any of the above formulae, it may be desirable that X1 is Pro and X3 is selected from

—NH—CH[(CH2)n—O—R3]—C(O)—; and  (i)

—NH—CH[(CH2)p—NH—R6]—C(O)—.  (iv)

Thus the invention further provides a compound comprising the formula I:

R1—Z—R2  (I)

wherein

R1 is hydrogen, C1-4 alkyl, formyl, acetyl, trifluoroacetyl or benzoyl;

R2 is OH or NH2; and

Z is an amino acid sequence having the formula Ic:

Pro-Ser-X3-Phe-Leu-Ser-Pro-Glu-His-X10-X11-X12-X13-X14-Arg-X16-Glu-Ser-X19-X20-Pro-Pro-Ala-X24-Leu-X26-Pro-X28  (Ic) (SEQ ID NO: 4)

or is a truncated form thereof lacking from 1 to 23 amino acids from the C-terminus;

wherein

X3 is selected from among:

—NH—CH[(CH2)n—O—R3]—C(O)—; and  (i)

—NH—CH[(CH2)p—NH—R6]—C(O)—;  (iv)

wherein

R3 is a C1-35-alkyl or -alkenyl group;

R6 is a C1-35-acyl group; and

n and p are integers 1, 2, 3, 4 or 5;

when present

X10 is selected from Gin and Ser;

X11 is selected from Arg and Lys;

X12 is selected from Val, Ala, Thr and Leu;

X13 is selected from Gin and Ser;

X14 is selected from Gin and Ser, or is absent;

X16 is Lys;

X19 is Lys;

X20 is Lys;

X24 is Lys; and

X26 is selected from Gin and Ser;

X28 is Arg;

and optionally, wherein one or more of said amino acid residues X10, X11, X13, X14, X16, X19, X20, X24, X26 and X28 independently, is replaced with a modified lysine residue comprising a lipophilic substituent having the formula Z1Z2—, wherein Z2 is a spacer moiety or is absent, and Z1 is a lipophilic moiety conjugated, either via Z2 or directly, to the side-chain of said lysine residue;

or a pharmaceutically acceptable salt or solvate of any of the above

In certain embodiments, X3 is —NH—CH[(CH2)n—O—R3]—C(O)— (where n and R3 are as defined above), X12 is Val and X20 is Ser.
Thus the compound may have the formula I:

R1—Z—R2  (I)

wherein

R1 is hydrogen, C1-4 alkyl, formyl, acetyl, trifluoroacetyl or benzoyl;

R2 is OH or NH2; and

Z is an amino acid sequence having the formula Id:

Pro-Ser-X3-Phe-Leu-Ser-Pro-Glu-His-X10-X11-Val-X13-X14-Arg-X16-Glu-Ser-X19-Ser-Pro-Pro-Ala-X24-Leu-X26-Pro-X28  (Id) (SEQ ID NO: 5)

or is a truncated form thereof lacking from 1 to 23 amino acids from the C-terminus;

wherein

X3 is —NH—CH[(CH2)n—O—R3]—C(O)—

wherein

R3 is a C1-35-alkyl or -alkenyl group and n is an integer 1, 2, 3, 4 or 5;

when present

X10 is selected from Gin and Ser;

X11 is selected from Arg and Lys;

X13 is selected from Gin and Ser;

X14 is selected from Gin and Ser, or is absent;

X16 is Lys;

X19 is Lys;

X24 is Lys; and

X26 is selected from Gin and Ser;

X28 is Arg;

and optionally, wherein one or more of said amino acid residues X10, X11, X13, X14, X16, X19, X24, X26 and X28 independently, is replaced with a modified lysine residue comprising a lipophilic substituent having the formula Z1Z2—, wherein Z2 is a spacer moiety or is absent, and Z1 is a lipophilic moiety conjugated, either via Z2 or directly, to the side-chain of said lysine residue;

or a pharmaceutically acceptable salt or solvate of any of the above.

In alternative embodiments, X3 is —NH—CH[(CH2)p—NH—R6]—C(O)—, X12 is Val and X20 is Ser.
Thus the compound may have the formula I:

R1—Z—R2  (I)

wherein

R1 is hydrogen, C1-4 alkyl, formyl, acetyl, trifluoroacetyl or benzoyl;

R2 is OH or NH2; and

Z is an amino acid sequence having the formula Id:

Pro-Ser-X3-Phe-Leu-Ser-Pro-Glu-His-X10-X11-Val-X13-X14-Arg-X16-Glu-Ser-X19-Ser-Pro-Pro-Ala-X24-Leu-X26-Pro-X28  (Id) (SEQ ID NO: 5)

or is a truncated form thereof lacking from 1 to 23 amino acids from the C-terminus;

wherein

X3—NH—CH[(CH2)p—NH—R6]—C(O)—

wherein

R6 is a C1-35-acyl group and p is an integer 1, 2, 3, 4 or 5;

when present

X10 is selected from Gin, Glu and Ser;

X11 is selected from Arg and Lys;

X13 is selected from Gin and Ser;

X14 is selected from Gin and Ser, or is absent;

X16 is Lys;

X19 is Lys;

X24 is Lys; and

X26 is selected from Gin and Ser;

X28 is Arg;

and optionally, wherein one or more of said amino acid residues X10, X11, X13, X14, X16, X19, X24, X26 and X28 independently, is replaced with a modified lysine residue comprising a lipophilic substituent having the formula Z1Z2—, wherein Z2 is a spacer moiety or is absent, and Z1 is a lipophilic moiety conjugated, either via Z2 or directly, to the side-chain of said lysine residue;

or a pharmaceutically acceptable salt or solvate of any of the above.

In some embodiments X28 is not one of the residues which may be replaced by the modified lysine residue.
Further aspects of the present invention include, but are not limited to, a compound, or a pharmaceutically acceptable salt or solvate thereof, of the invention for use in therapy (e.g., in treatment of any of the diseases, disorders, conditions, or syndromes described herein).
Further aspects of the present invention include, but are not limited to, a pharmaceutical composition comprising a compound, or a pharmaceutically acceptable salt or solvate thereof, of the invention and a pharmaceutically acceptable carrier.
Further aspects of the present invention include, but are not limited to, a method of treating any of the diseases, disorders, conditions, or syndromes described herein in a subject in need thereof, the method comprising administering a therapeutically effective amount of a compound, or a pharmaceutically acceptable salt or solvate thereof, of the invention, or a pharmaceutical composition of the invention to the subject.
Further aspects of the present invention include, but are not limited to, use of a compound, or a pharmaceutically acceptable salt or solvate thereof, of the invention, or a pharmaceutical composition of the invention, in the manufacture of a medicament for use in therapy (e.g. in treatment of any of the diseases, disorders, conditions, or syndromes described herein).
Further aspects of the present invention include, but are not limited to, a nucleic acid molecule (which may comprise DNA, RNA, or a mixture thereof) encoding a compound (peptide) of the invention.
Further aspects of the present invention include, but are not limited to, an expression vector comprising such a nucleic acid molecule of the invention.
Further aspects of the present invention include, but are not limited to, a host cell containing such a nucleic acid construct or expression vector of the invention.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1: Effects of ghrelin or vehicle administration early in the light phase on food intake in normal mice. (A) Food intake when animals had free access to food directly after administration; (B) food intake when animals had access to food 2 hours after administration.
FIG. 2: Effects of administration of test compounds of the invention or vehicle early in the light phase on food intake in normal mice. Animals had access to food 2 hours after compound administration. (A) 0-6 hour accumulated food intake (B) 0-24 hour accumulated food intake.



DETAILED DESCRIPTION OF THE INVENTION
Throughout the present specification, the conventional one-letter and three-letter codes for residues of naturally occurring amino acids are used. Unless otherwise indicated, reference is made to the L-isomeric forms of the amino acids in question. Where appropriate, the D-isomeric form of an amino acid is indicated in the conventional manner by the prefix “D” before the conventional three-letter code (e.g. DPro). Frequently employed three- or four-letter codes are employed for residues of a number of less common or non-naturally occurring amino acids, including Orn (ornithine, i.e. 2,5-diaminopentanoic acid), Dbu (2,4-diaminobutyric acid), Gaba (gamma-aminobutyric acid), Sar (sarcosine, i.e. N-methylglycine), Aib (α-aminoisobutyric acid), Bal (β-alanine, i.e. 3-aminopropanoic acid, also referred to as β-ala), Dap (2,3-diaminopropanoic acid, also referred to as Dpr), Inp (isonipecotic acid, i.e. 4-carboxypiperidine), IPro (i.e. pyrrolidine-3-carboxylic acid), cACC (cis-4-aminocyclohexane carboxylic acid), tACC (trans-4-aminocyclohexane carboxylic acid), Mamb (3-aminomethyl-benzoic acid) and Cmp (4-carboxymethyl-piperidine).
Unless otherwise indicated, the term “ghrelin” refers to native human ghrelin having the sequence H-Gly-Ser-Ser(O-octanoyl)-Phe-Leu-Ser-Pro-Glu-His-Gln-Arg-Val-Gln-Gln-Arg-Lys-Glu-Ser-Lys-Lys-Pro-Pro-Ala-Lys-Leu-Gln-Pro-Arg-OH (SEQ ID NO: 1).
In the latter sequence, the abbreviation Ser(O-octanoyl) for the residue in position 3 designates a modified (acylated) serine residue in which the hydrogen atom of the side-chain hydroxy group in serine is replaced by an n-octanoyl group.
Among sequences disclosed herein are sequences (including SEQ ID NO: 1, above) incorporating an “H—” moiety at the amino terminus (N-terminus) of the sequence, and either an “—OH” moiety or an “—NH2” moiety at the carboxy terminus (C-terminus) of the sequence. In such cases, and unless otherwise indicated, an “H—” moiety at the N-terminus of the sequence in question indicates a hydrogen atom [i.e. R1═H in formula I; corresponding to the presence of a free primary or secondary amino group at the N-terminus], while an “—OH” (as in SEQ ID NO: 1, above) or an “—NH2” moiety at the C-terminus of the sequence indicates a hydroxy group [i.e. R2═OH; corresponding to the presence of a carboxy (COOH) group at the C-terminus] or an amino group [i.e. R2═NH2; corresponding to the presence of an amido (CONH2) group at the C-terminus], respectively. In each sequence of the invention, a C-terminal “—OH” moiety may be substituted for a C-terminal “—NH2” moiety, and vice-versa.
Compounds (ghrelin analogues) of the invention exhibit agonist activity at the human ghrelin receptor, i.e. the growth hormone secretagogue receptor type 1a (GHSR1a) having the sequence encoded by the sequence with accession number AY429112.1 (GI:38016896) (SEQ ID NO: 6). Activity may be determined by measuring Ca2+ signalling in cells expressing the ghrelin receptor. The cells are preferably mammalian cells. They may be human cells, such as HEK293 cells, or non-human mammalian cells e.g. rodent cells. In certain embodiments the cells do not express an endogenous ghrelin receptor and have been transfected with nucleic acid encoding the human ghrelin receptor such that they express the human ghrelin receptor. The agonist activity of a given compound may be quantified by determining an EC50 value for the compound. A suitable assay may be performed at a concentration of 20000 cells per 100 μl medium, e.g. in standard 96 well tissue culture plates, or more specifically may be performed under the conditions described in Example 3 below.
The compound of the invention may have greater agonist activity at the ghrelin receptor than human ghrelin (including the octanoyl moiety at Ser3, i.e. compound 1 described in the examples). For example, the compound may have a lower EC50 than human ghrelin when determined under identical conditions, e.g. the conditions mentioned above. Data for in vitro activation of the human GHSR1a receptor [expressed as an EC50 value, i.e. the concentration at which the test compound in question exhibits 50% of its maximal activity, as determined by a ghrelin receptor functional assay as described herein (vide infra)] by more than 60 widely representative compounds of the invention are shown in Table 1 (vide infra).
Under the conditions described in Example 3, compounds of the invention will then typically exhibit EC50 values of ≦40 nM, such as ≦30 nM, for example ≦20 nM, e.g. ≦10 nM, and numerous compounds of the invention exhibit EC50 values of ≦5 nM. Some compounds of the invention exhibit EC50 values of, ≦1 nM, such as ≦0.5 nM, or ≦0.3 nM.
The compounds of the invention may be capable of modulating (e.g. increasing) food intake in mammals, such as: primates including humans, great apes (e.g. gorillas, chimpanzees, orangutans), old world monkeys, new world monkeys; rodents (e.g. mice, rats, guinea pigs, hamsters); cats; dogs; lagomorphs (including rabbits); cows; sheep; goats; horses; pigs; or any other livestock, agricultural, laboratory or domestic mammals. Preferably they have activity in humans.
For example, the compounds may increase aggregate food intake in a subject as compared to an equivalent untreated subject. They may increase aggregate food intake in a subject as compared to an equivalent subject equivalently treated with human ghrelin (including the octanoyl moiety at Ser3, i.e. compound 1 described in the examples), e.g. a subject treated with the same dose (w/w) of human ghrelin in the same dosing pattern.
In either instance, aggregate food may be increased over a period of 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 10 hours, 12 hours or 24 hours after administration.
Aggregate food intake compared to untreated subjects, or compared to subjects treated with human ghrelin, may be increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or even more over the respective period.
The compounds may be capable of exerting their effect on food intake when the subjects are provided with access to food at the same time as administration of the compound. Additionally or alternatively, the compounds may be capable of exerting their effect on food intake when the subjects are provided with access to food only after administration of the compound, e.g. 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, or even longer, after administration of the compound. In certain embodiments the compounds are capable of increasing food intake when the subjects are provided with access to food only 2 hours after administration of the compound.
The effect of the compounds may be measured in any suitable mammalian test subject, such as a rodent. An example is the C57BL/6J mouse as used in the examples. A suitable individual test dosage may be 300 nmol/kg body weight, although the skilled person will be capable of determining suitable dosages.
As discussed above, one aspect of the present invention relates to a compound having the formula I:

R1—Z—R2  (I)

wherein

R1 is hydrogen, C1-4 alkyl, formyl, acetyl, trifluoroacetyl or benzoyl;

R2 is OH or NH2; and

Z is an amino acid sequence having the formula Ia:

X1-X2-X3-Phe-Leu-Ser-Pro-X8-His-X10-X11-X12-X13-X14-X15-X16-Glu-Ser-X19-X20-Pro-Pro-Ala-X24-Leu-X26-Pro-X28  (Ia) (SEQ ID NO: 2)

or is a truncated form thereof lacking from 1 to 23 amino acids from the C-terminus;

wherein

X1 is an amino acid residue selected from Ala, Inp, Pro, DPro, Sar, Aib, Phe and Ser;

X2 is Ser; or

X1 and X2 together constitute a residue of a non-naturally occurring amino acid, wherein the shortest bond separation between the amino nitrogen atom and the carbon atom of the carbonyl group in the non-naturally occurring amino acid is 3 to 7 bonds;

when X1 is an amino acid residue selected from Pro, DPro, Phe and Ser, or when X1 and X2 together constitute a residue of the non-naturally occurring amino acid, then X3 is selected among:

—NH—CH[(CH2)n—O—R3]—C(O)—;  (i)

—NH—CH(R4)—C(O)—;  (ii)

—NH—CH[(CH2)m—O—R5]—C(O)—; and  (iii)

—NH—CH[(CH2)p—NH—R6]—C(O)—;  (iv)

and when X1 is an amino acid residue selected from Ala, Sar, Inp and Aib, then X3 is selected among:

—NH—CH[(CH2)n—O—R3]—C(O)—; and  (i)

—NH—CH(R4)—C(O)—;  (ii)

wherein

R3 and R4 are, independently, C1-35-alkyl or -alkenyl groups;

R5 and R6 are, independently, C1-35-acyl groups; and

n, m and p are integers 1, 2, 3, 4 or 5;

when present

X8 is selected from Glu, Lys and Tyr;

X10 is selected from Gin, Glu and Ser;

X11 is selected from Arg, Lys and Ser;

X12 is selected from Val, Ala, Pro, Lys, Thr and Leu;

X13 is selected from Gin and Ser;

X14 is selected from Gin, Lys, Tyr and Ser, or is absent;

X15 is selected from Arg and Ser;

X16 is selected from Lys, Ser and Pro;

X19 is selected from Lys and Ser;

X20 is selected from Lys, Glu and Ser;

X24 is selected from Lys, Tyr and Ser;

X26 is selected from Gin and Ser; and

X28 is selected from Arg and Ser;

and, optionally, one or more of said amino acid residues X8, X10, X11, X13, X14, X16, X19, X20, X24, X26 and X28 independently, is replaced with a modified lysine residue comprising a lipophilic substituent having the formula Z1Z2—, wherein Z2 is a spacer moiety or is absent, and Z1 is a lipophilic moiety conjugated, either via Z2 or directly, to the side-chain of said lysine residue;

or a pharmaceutically acceptable salt or solvate of any of the above.

In certain embodiments, the invention provides a compound of formula I

R1—Z—R2  (I)

wherein

R1 is hydrogen, C1-4 alkyl, formyl, acetyl, trifluoroacetyl or benzoyl;

R2 is —OH or —NH2; and

Z is an amino acid sequence having the formula Ib:

X1-X2-X3-Phe-Leu-Ser-Pro-Glu-His-X10-X11-X12-X13-X14-Arg-X16-Glu-Ser-X19-X20-Pro-Pro-Ala-X24-Leu-X26-Pro-X28  (Ib) (SEQ ID NO: 3)

or is a truncated form thereof lacking from 1 to 23 amino acids from the C-terminus;

wherein

X1 is an amino acid residue selected from Ala, Inp, Pro, DPro, Sar, Aib, Phe and Ser;

X2 is Ser; or

X1 and X2 together constitute a residue of a non-naturally occurring amino acid, wherein the shortest bond separation between the amino nitrogen atom and the carbon atom of the carbonyl group in the non-naturally occurring amino acid is 3 to 7 bonds;

when X1 is an amino acid residue selected from Pro, DPro, Phe and Ser, or when X1 and X2 together constitute a residue of the non-naturally occurring amino acid, then X3 is selected among:

—NH—CH[(CH2)n—O—R3]—C(O)—;  (i)

—NH—CH(R4)—C(O)—;  (ii)

—NH—CH[(CH2)m—O—R5]—C(O)—; and  (iii)

—NH—CH[(CH2)p—NH—R6]—C(O)—;  (iv)

and when X1 is an amino acid residue selected from Ala, Sar, Inp and Aib, then X3 is selected among:

—NH—CH[(CH2)n—O—R3]—C(O)—; and  (i)

—NH—CH(R4)—C(O)—;  (ii)

wherein

R3 and R4 are, independently, C1-35-alkyl or -alkenyl groups;

R5 and R6 are, independently, C1-35-acyl groups; and

n, m and p are integers 1, 2, 3, 4 or 5;

when present

X10 is selected from Gin and Ser;

X11 is selected from Arg and Lys;

X12 is selected from Val, Ala, Thr and Leu;

X13 is selected from Gin and Ser;

X14 is selected from Gin and Ser, or is absent;

X16 is Lys;

X19 is Lys;

X20 is Lys;

X24 is Lys; and

X26 is selected from Gin and Ser;

X28 is Arg;

and, optionally, one or more of said amino acid residues X10, X11, X13, X14, X16, X19, X20, X24, X26 and X28 independently, is replaced with a modified lysine residue comprising a lipophilic substituent having the formula Z1Z2—, wherein Z2 is a spacer moiety or is absent, and Z1 is a lipophilic moiety conjugated, either via Z2 or directly, to the side-chain of said lysine residue;

or a pharmaceutically acceptable salt or solvate of any of the above.

In some embodiments of compounds of the invention the compound is a compound of formula (I):

R1—Z—R2  (I)

wherein

R1 is hydrogen, C1-4 alkyl, formyl, acetyl, trifluoroacetyl or benzoyl;

R2 is —OH or —NH2; and

Z is an amino acid sequence having the formula Ib:

X1-X2-X3-Phe-Leu-Ser-Pro-Glu-His-X10-X11-X12-X13-X14-Arg-X16-Glu-Ser-X19-X20-Pro-Pro-Ala-X24-Leu-X26-Pro-X28  (Ib) (SEQ ID NO: 3)

or is a truncated form thereof lacking from 1 to 23 amino acids from the C-terminus;

wherein

X1 is an amino acid residue selected from Ala, Inp, Pro, DPro, Sar, Aib, Phe and Ser;

X2 is Ser; or

X1 and X2 together constitute a residue of a non-naturally occurring amino acid, wherein the shortest bond separation between the amino nitrogen atom and the carbon atom of the carbonyl group in the non-naturally occurring amino acid is 3 to 7 bonds;

when X1 is an amino acid residue selected from Pro, DPro, Phe and Ser, or when X1 and X2 together constitute a residue of the non-naturally occurring amino acid selected from cACC, tACC, Mamb and Cmp, then X3 is selected from among:

—NH—CH[(CH2)n—O—R3]—C(O)—;  (i)

—NH—CH(R4)—C(O)—;  (ii)

—NH—CH[(CH2)m—O—R5]—C(O)—; and  (iii)

—NH—CH[(CH2)p—NH—R6]—C(O)—;  (iv)

and when X1 is an amino acid residue selected from Ala, Sar, Inp and Aib, then X3 is selected from among:

—NH—CH[(CH2)n—O—R3]—C(O)—; and  (i)

—NH—CH(R4)—C(O)—;  (ii)

wherein

R3 and R4 are, independently, C1-35-alkyl or -alkenyl groups;

R5 and R6 are, independently, C1-35-acyl groups; and

n, m and p are integers 1, 2, 3, 4 or 5;

when present

X10 is Gin;

X11 is selected from Arg and Lys;

X12 is selected from Val, Ala, Thr and Leu;

X13 is Gin;

X14 is Gin or is absent;

X16 is Lys;

X19 is Lys;

X20 is Lys;

X24 is Lys; and

X26 is Gin;

X28 is Arg;

and optionally, wherein one or more of said amino acid residues X10, X11, X13, X14, X16, X19, X20, X24, X26 and X28 independently, is replaced with a modified lysine residue comprising a lipophilic substituent having the formula Z1Z2—, wherein Z2 is a spacer moiety or is absent, and Z1 is a lipophilic moiety conjugated, either via Z2 or directly, to the side-chain of said lysine residue;

or a pharmaceutically acceptable salt or solvate of any of the above.

In any of the above formulae, it may be desirable that X1 is Pro and X3 is selected from

—NH—CH[(CH2)n—O—R3]—C(O)—; and  (i)

—NH—CH[(CH2)p—NH—R6]—C(O)—.  (iv)

Thus the invention further provides a compound comprising the formula I:

R1—Z—R2  (I)

wherein

R1 is hydrogen, C1-4 alkyl, formyl, acetyl, trifluoroacetyl or benzoyl;

R2 is OH or NH2; and

Z is an amino acid sequence having the formula Ic:

Pro-Ser-X3-Phe-Leu-Ser-Pro-Glu-His-X10-X11-X12-X13-X14-Arg-X16-Glu-Ser-X19-X20-Pro-Pro-Ala-X24-Leu-X26-Pro-X28  (Ic) (SEQ ID NO: 4)

or is a truncated form thereof lacking from 1 to 23 amino acids from the C-terminus;

wherein

X3 is selected from among:

—NH—CH[(CH2)n—O—R3]—C(O)—; and  (i)

—NH—CH[(CH2)p—NH—R6]—C(O)—;  (iv)

wherein

R3 is a C1-35-alkyl or -alkenyl group;

R6 is a C1-35-acyl group; and

n and p are integers 1, 2, 3, 4 or 5;

when present

X10 is selected from Gin and Ser; X11 is selected from Arg and Lys;

X12 is selected from Val, Ala, Thr and Leu;

X13 is selected from Gin and Ser;

X14 is selected from Gin and Ser, or is absent;

X16 is Lys;

X19 is Lys;

X20 is Lys;

X24 is Lys; and

X26 is selected from Gin and Ser;

X28 is Arg;

and optionally, wherein one or more of said amino acid residues X10, X11, X13, X14, X16, X19, X20, X24, X26 and X28 independently, is replaced with a modified lysine residue comprising a lipophilic substituent having the formula Z1Z2—, wherein Z2 is a spacer moiety or is absent, and Z1 is a lipophilic moiety conjugated, either via Z2 or directly, to the side-chain of said lysine residue;

or a pharmaceutically acceptable salt or solvate of any of the above

In certain embodiments, X3 is —NH—CH[(CH2)n—O—R3]—C(O)— (where n and R3 are as defined above), X12 is Val and X20 is Ser.
Thus the compound may have the formula I:

R1—Z—R2  (I)

wherein

R1 is hydrogen, C1-4 alkyl, formyl, acetyl, trifluoroacetyl or benzoyl;

R2 is OH or NH2; and

Z is an amino acid sequence having the formula Id:

Pro-Ser-X3-Phe-Leu-Ser-Pro-Glu-His-X10-X11-Val-X13-X14-Arg-X16-Glu-Ser-X19-Ser-Pro-Pro-Ala-X24-Leu-X26-Pro-X28  (Id) (SEQ ID NO: 5)

or is a truncated form thereof lacking from 1 to 23 amino acids from the C-terminus;

wherein

X3 is —NH—CH[(CH2)n—O—R3]—C(O)—

wherein

R3 is a C1-35-alkyl or -alkenyl group and n is an integer 1, 2, 3, 4 or 5;

when present

X10 is selected from Gin and Ser;

X11 is selected from Arg and Lys;

X13 is selected from Gin and Ser;

X14 is selected from Gin and Ser, or is absent;

X16 is Lys;

X19 is Lys;

X24 is Lys; and

X26 is selected from Gin and Ser;

X28 is Arg;

and optionally, wherein one or more of said amino acid residues X10, X11, X13, X14, X16, X19, X24, X26 and X28 independently, is replaced with a modified lysine residue comprising a lipophilic substituent having the formula Z1Z2—, wherein Z2 is a spacer moiety or is absent, and Z1 is a lipophilic moiety conjugated, either via Z2 or directly, to the side-chain of said lysine residue;

or a pharmaceutically acceptable salt or solvate of any of the above.

In alternative embodiments, X3 is —NH—CH[(CH2)p—NH—R6]—C(O)—, X12 is Val and X20 is Ser.
Thus the compound may have the formula I:

R1—Z—R2  (I)

wherein

R1 is hydrogen, C1-4 alkyl, formyl, acetyl, trifluoroacetyl or benzoyl;

R2 is OH or NH2; and

Z is an amino acid sequence having the formula Id:

Pro-Ser-X3-Phe-Leu-Ser-Pro-Glu-His-X10-X11-Val-X13-X14-Arg-X16-Glu-Ser-X19-Ser-Pro-Pro-Ala-X24-Leu-X26-Pro-X28  (Id) (SEQ ID NO: 5)

or is a truncated form thereof lacking from 1 to 23 amino acids from the C-terminus;

wherein

X3—NH—CH[(CH2)p—NH—R6]—C(O)—

wherein

R6 is a C1-35-acyl group and p is an integer 1, 2, 3, 4 or 5;

when present

X10 is selected from Gin, Glu and Ser;

X11 is selected from Arg and Lys;

X13 is selected from Gin and Ser;

X14 is selected from Gin and Ser, or is absent;

X16 is Lys;

X19 is Lys;

X24 is Lys; and

X26 is selected from Gin and Ser;

X28 is Arg;

and optionally, wherein one or more of said amino acid residues X10, X11, X13, X14, X16, X19, X24, X26 and X28 independently, is replaced with a modified lysine residue comprising a lipophilic substituent having the formula Z1Z2—, wherein Z2 is a spacer moiety or is absent, and Z1 is a lipophilic moiety conjugated, either via Z2 or directly, to the side-chain of said lysine residue;

or a pharmaceutically acceptable salt or solvate of any of the above.

In some embodiments X28 is not one of the residues which may be replaced by the modified lysine residue.
In some embodiments of a compound (or a pharmaceutically acceptable salt or solvate thereof, said salts or solvates to be understood as being included in any embodiment of the invention described herein unless specifically excluded), of the invention, X1 is an amino acid residue selected from Ala, Inp, Pro, DPro, Sar, Aib, Phe and Ser; and X3 is selected from among: (i) —NH—CH[(CH2)n—O—R3]—C(O)—; and (ii) —NH—CH(R4)—C(O)—.
In further embodiments of a compound, or a pharmaceutically acceptable salt or solvate thereof, of the invention, X1 is an amino acid residue selected from Pro, DPro, Phe and Ser; and X3 is selected from among: (i) —NH—CH[(CH2)n—O—R3]—C(O)—; (ii) —NH—CH(R4)—C(O)—; (iii) —NH—CH[(CH2)m—O—R5]—C(O)—; and (iv) —NH—CH[(CH2)p—NH—R6]—C(O)—.
In certain embodiments of a compound, or a pharmaceutically acceptable salt or solvate thereof, of the invention, X1 is an amino acid residue selected from Pro, DPro and Aib, such as an amino acid residue selected from Pro and DPro.
More generally, X1 and X2 together may constitute one of a wide variety of amino acid residues in which the spatial relationship of at least some atoms or functional groups is similar to that of the Gly1-Ser2 fragment of ghrelin, such as the spatial relationship between the carbonyl carbon of Ser and the basic α-amino nitrogen of Gly. Spatial relationship may, for example, be similar in terms of three-dimensional orientation of, or distances between, atoms or functional groups, but spatial relationship of atoms or functional groups may also be with reference to simpler features, such as the number of bonds between atoms or functional groups.
In the present invention, the spatial relationship of atoms or functional groups relates to the shortest bond separation (3 to 7) between the amino nitrogen atom and the carbon atom of the carbonyl group in the non-naturally occurring amino acid.
For a Gly-Ser fragment the number of bonds between the carbonyl carbon of Ser and the α-amino nitrogen of Gly is 5. In residues of the specific non-naturally occurring amino acids cACC, tACC, Cmp, and Mamb that are present in certain embodiments of compounds of the present invention, a separation of 5 bonds is likewise present between the carbonyl carbon and the amino nitrogen. A separation of from 3 to 7 bonds (such as 5 bonds) provides spatial similarity to the corresponding distance in a Gly-Ser fragment. Such residues may or may not contain cyclic moieties.
Thus, in some embodiments, X1 and X2 together constitute a residue of a non-naturally occurring amino acid, wherein the shortest bond separation between the amino nitrogen atom and the carbon atom of the carbonyl group in the non-naturally occurring amino acid is 3 to 7 bonds.
In some embodiments of a compound, or a pharmaceutically acceptable salt or solvate thereof, of the invention, X1 and X2 together constitute a residue of a non-naturally occurring amino acid selected from cACC, tACC, Mamb and Cmp.
Examples of other non-naturally occurring amino acid residues that may be of relevance in this connection include, but are not limited to, the following:




Further non-naturally occurring amino acid residues that may likewise be of relevance in this connection include ornithine (Orn) and α-N-acetylornithine(AcOrn).
For the avoidance of doubt, “non-naturally occurring amino acid residue” and equivalent terms are used to signify amino acid residues other than the group of 20 amino acids normally incorporated into proteins by mammalian transcription and translation systems before any post-translational modification (i.e. amino acids other than Glycine, Alanine, Valine, Leucine, Isoleucine, Serine, Cysteine, Threonine, Methionine, Proline, Phenylalanine, Tyrosine, Tryptophan, Histidine, Lysine, Arginine, Aspartate, Glutamate, Asparagine and Glutamine). They should not be taken to require that the amino acid itself does not occur in nature (although such amino acids may certainly be used).
In certain embodiments of a compound, or a pharmaceutically acceptable salt or solvate thereof, of the invention, X3 is selected among: (i) —NH—CH[(CH2)n—O—R3]—C(O)—; and (ii) —NH—CH(R4)—C(O)—. In certain embodiments, X3 is selected from among: (iii) —NH—CH[(CH2)m—O—R5]—C(O)—; and (iv) —NH—CH[(CH2)p—NH—R6]—C(O)—.
In some embodiments of a compound, or a pharmaceutically acceptable salt or solvate thereof, of the invention, R3, when present, is a C5-16-alkyl or -alkenyl group. In some embodiments of a compound, or a pharmaceutically acceptable salt or solvate thereof, of the invention, R3, when present, is a C7-10-alkyl or -alkenyl group. In certain embodiments of a compound, or a pharmaceutically acceptable salt or solvate thereof, of the invention, R3, when present, is a C8-alkyl or -alkenyl group
In some embodiments of a compound, or a pharmaceutically acceptable salt or solvate thereof, of the invention, R4, when present, is a C7-18-alkyl or -alkenyl group. In some embodiments of a compound, or a pharmaceutically acceptable salt or solvate thereof, of the invention, R4, when present, is a C7-12-alkyl or -alkenyl group, such as a C7-10-alkyl or -alkenyl group. In certain embodiments of a compound, or a pharmaceutically acceptable salt or solvate thereof, of the invention, R4, when present, is a C10-alkyl or -alkenyl group
In certain embodiments of compound, or a pharmaceutically acceptable salt or solvate thereof, of the invention, R5, when present, is a C5-16-acyl group. In some embodiments of compound, or a pharmaceutically acceptable salt or solvate thereof, of the invention, R5, when present, is a C7-10-acyl group. In certain embodiments of compound, or a pharmaceutically acceptable salt or solvate thereof, of the invention, R5, when present, is a C8-acyl group.
In certain embodiments of a compound, or a pharmaceutically acceptable salt or solvate thereof, of the invention, R6, when present, is a C5-16-acyl group. In some embodiments of compound, or a pharmaceutically acceptable salt or solvate thereof, of the invention, R6, when present, is a C7-10-acyl group. In certain embodiments of compound, or a pharmaceutically acceptable salt or solvate thereof, of the invention, R6, when present, is a C8-acyl group.
In the compound, or a pharmaceutically acceptable salt or solvate thereof, of the invention, the alkyl or alkenyl groups R3 and R4, and the alkyl or alkenyl groups present in the acyl groups R5 and R6 may independently be linear or branched.
Examples of linear saturated C5-16-alkyl groups and C7-18-alkyl groups include, but are not limited to, —(CH2)nCH3 where n is an integer from 4 to 17, —CH2CH2CH2CH—2CH2CH2CH3 (heptyl), —CH2CH2CH2CH2CH2CH2CH2CH3 (octyl) and —CH2CH2CH2CH2CH2CH2CH2CH2CH3 (nonyl).
Examples of branched saturated C5-16-alkyl groups and C7-18-alkyl groups include, but are not limited to, —CH(CH3)CH2CH2CH2CH2CH2CH3, —CH2CH(CH3)CH2CH2CH2CH2CH3, —CH2CH2CH(CH3)CH2CH2CH2CH3, —CH2CH2CH2CH(CH3)CH2CH2CH3, —CH2CH2CH2CH2CH(CH3)CH2CH3, —CH2CH2CH2CH2CH2CH(CH3)2, —CH(CH2CH3)CH2CH2CH2CH2CH2—, —CH2CH(CH2CH3)CH2CH2CH2CH3, —CH2CH2CH(CH2CH3)CH2CH2CH3, —CH2CH2CH2CH(CH2CH3)CH2CH3, —CH2CH2CH2CH2CH2CH(CH2CH3)CH3.
Examples of linear partially unsaturated C5-16-alkenyl groups and C7-16-alkenyl groups include, but are not limited to, —CH═CH—CH2—CH2—CH2—CH2—CH3, —CH2—CH═CH2—CH2—CH2—CH2—CH3, —CH2—CH2—CH═CH2—CH2—CH2—CH3, —CH2—CH2—CH2—CH═CH2—CH2—CH3, —CH2—CH2—CH2—CH2—CH═CH2—CH3, —CH2—CH2—CH2—CH2—CH2—CH═CH2, —CH═CH—CH═CH—CH2—CH2—CH3, —CH═CH—CH═CH—CH2—CH═CH2, —CH═CH—CH2—CH2—CH═CH—CH3, —CH═CH—CH═CH—CH2—CH═CH2, —CH═CH—CH═CH—CH═CH—CH═CH2.
Examples of branched partially unsaturated C5-16-alkenyl groups and C7-16-alkenyl groups include, but are not limited to, —C(CH3)═CH—CH2—CH2—CH2—CH3, —C(CH3)═CH—CH2—CH2—CH2—CH2—CH3, —CH═CH—CH2—CH2—CH2—CH(CH3)2, —CH2—CH2—CH═CH—CH2—CH(CH3)2.
C5-16-acyl groups may be represented as —C(═O)RA, wherein RA is a linear or branched C4-15-alkyl group (in which case such an acyl group may also be referred to as a C5-16-alkanoyl group) or a linear or branched C4-15-alkenyl group (in which case such an acyl group may also be referred to as a C5-16-alkenoyl group). Examples of acyl groups include, but are not limited to, —C(═O)CH2CH2CH2CH2CH2CH3 (heptanoyl), —C(═O)CH2CH2CH2CH2CH2CH2CH3 (octanoyl) and —C(═O)CH2CH2CH2CH2CH2CH2CH2CH3 (nonanoyl).
In some embodiments of a compound, or a pharmaceutically acceptable salt or solvate thereof, of the invention, the integer n, m, or p is 1.
The compounds of the invention may carry one or more intramolecular bridge within the peptide sequence Z. Each such bridge is formed between the side chains of two amino acid residues of Z. The two residues may be separated by one, two, three or more amino acids in the linear sequence of Z. Thus the bridge may be formed between a pair of residues at positions XA and XA+2, positions XA and XA+3, positions XA and XA+4, or any other suitable pair of residues.
For example, a Lys residue at position X8 may form an intramolecular bridge with any Glu residue in the molecule, e.g. a Glu residue at position X10 or X20.
A Lys residue at position X11 may form an intramolecular bridge with any Glu residue in the molecule, e.g. a Glu residue at position X8 or X20.
A Lys residue at position X12 may form an intramolecular bridge with any Glu residue in the molecule, e.g. a Glu residue at position X8, X10 or X20.
A Lys residue at position X16 may form an intramolecular bridge with any Glu residue in the molecule, e.g. a Glu residue at position X8, X10 or X20.
A Lys residue at position X19 may form an intramolecular bridge with any Glu residue in the molecule, e.g. a Glu residue at position X8 or X10.
A Lys residue at position X20 may form an intramolecular bridge with any Glu residue in the molecule, e.g. a Glu residue at position X8 or X10.
A Lys residue at position X24 may form an intramolecular bridge with any Glu residue in the molecule, e.g. a Glu residue at position X8, X10 or X20.
A Glu residue at position X8 may form an intramolecular bridge with any Lys residue in the molecule, e.g. a Lys residue at position X11, X12, X14, X16, X19, X20 or X24.
A Glu residue at position X10 may form an intramolecular bridge with any Lys residue in the molecule, e.g. a Lys residue at position X8, X12, X14, X16, X19, X20 or X24.
A Glu residue at position X20 may form an intramolecular bridge with any Lys residue in the molecule, e.g. a Lys residue at position X8, X11, X12, X14, X16 or X24.
By way of example, the bridge may be formed between the side chains of residue pairs X8 and X12, X10 and X14, or X16 and X20. The two side chains can be linked to one another through ionic interactions, or by covalent bonds, typically to form a lactam ring.
Typically, the two amino acids which form the intramolecular bridge are Lys and Glu.
Thus, when a Glu is present at position X8, a Lys may be present at position X12.
When a Glu is present at position X10, a Lys may be present at position X14.
When a Lys is present at position X16, a Glu may be present at position X20.
In some embodiments of a compound, or a pharmaceutically acceptable salt or solvate thereof, of the invention, Z1 is a long-chain fatty acid acyl moiety, such as a fatty acid moiety selected from dodecanoyl, 2-butyloctanoyl, tetradecanoyl, hexadecanoyl, heptadecanoyl, octadecanoyl, and eicosanoyl.
In some embodiments of a compound, or a pharmaceutically acceptable salt or solvate thereof, of the invention, Z2 is a spacer moiety comprising one or more amino acid residues. Z2 may, for example, be an amino acid residue selected from Glu, γGlu (derived from γ-glutamic acid) and Bal, or a residue of an amino acid selected from 3-aminopropanoic acid, 4-aminobutanoic acid, 8-aminooctanoic acid and 8-amino-3,6-dioxaoctanoic acid.
Specific embodiments of compounds of the invention include compounds selected from the group consisting of:
H-PS-S(O-octanoyl)-FLSPEHQRVQQRKESKKPPAKLQPR-OH (SEQ ID NO: 7);
H-[DPro]-S-S(O-octanoyl)-FLSPEHQRVQQRKESKKPPAKLQPR-OH (SEQ ID NO: 8);
H-FS-S(O-octanoyl)-FLSPEHQRVQQRKESKKPPAKLQPR-OH (SEQ ID NO: 9);
H-SS-S(O-octanoyl)-FLSPEHQRVQQRKESKKPPAKLQPR-OH (SEQ ID NO: 10);
H-Cmp-S(O-octanoyl)-FLSPEHQRVQQRKESKKPPAKLQPR-OH (SEQ ID NO: 11);
H-Mamb-S(O-octanoyl)-FLSPEHQRVQQRKESKKPPAKLQPR-OH (SEQ ID NO: 12);
H-[cACC]-S(O-octanoyl)-FLSPEHQRVQQRKESKKPPAKLQPR-OH (SEQ ID NO: 13);
H-[tACC]-S(O-octanoyl)-FLSPEHQRVQQRKESKKPPAKLQPR-OH (SEQ ID NO: 14);
H-Inp-S-S(O-octyl)-FLSPEHQRV-K(hexadecanoyl)-QRKESKKPPAKLQPR-OH (SEQ ID NO: 15);
H-Inp-S-S(O-octyl)-FLSPEHQRVQQRKES-K(hexadecanoyl)-KPPAKLQPR-OH (SEQ ID NO: 16);
H-Inp-S-S(O-octyl)-FLSPEHQRVQQRKESKKPPAKL-K(hexadecanoyl)-PR-OH (SEQ ID NO: 17);
H-PS-S(O-octyl)-FLSPEHQRVQQRKES-K(hexadecanoyl)-KPPAKLQPR-OH (SEQ ID NO: 18);
H-[DPro]-S-S(O-octyl)-FLSPEHQRVQQRKES-K(hexadecanoyl)-KPPAKLQPR-OH (SEQ ID NO: 19);
H-Aib-S-S(O-octyl)-FLSPEHQRVQQRKES-K(hexadecanoyl)-KPPAKLQPR-OH (SEQ ID NO: 20);
H-PS-S(O-octyl)-FLSPEHQRVQQRKESKKPPAKLQPR-OH (SEQ ID NO: 21);
H-[DPro]-S-S(O-octyl)-FLSPEHQRVQQRKESKKPPAKLQPR-OH (SEQ ID NO: 22); and
H-Aib-S-S(O-octyl)-FLSPEHQRVQQRKESKKPPAKLQPR-OH (SEQ ID NO: 23).
Pharmaceutically acceptable salts or solvates of any of the above compounds are likewise within the scope of the invention.
Further specific embodiments of compounds of the invention include compounds selected from the group consisting of:
H-AS-S(O-octyl)-FLSPEHQRVQQRKESKKPPAKLQPR-OH (SEQ ID NO: 24);
H-[cACC]-S(O-octyl)-FLSPEHQRVQQRKESKKPPAKLQPR-OH (SEQ ID NO: 25);
H-Cmp-S(O-octyl)-FLSPEHQRV-K(hexadecanoyl)-QRKESKKPPAKLQPR-OH (SEQ ID NO: 26);
H-FS-S(O-octyl)-FLSPEHQRVQQR-K(hexadecanoyl)-ESKKPPAKLQPR-OH (SEQ ID NO: 27);
H-[DPro]-S-S(O-octyl)-FLSPEHQRVQQRKES-K(hexadecanoyl)-KPPAKLQPR-OH (SEQ ID NO: 28);
H-Aib-S-S(O-octyl)-FLSPEHQRVQQRKESK-K(hexadecanoyl)-PPAKLQPR-OH (SEQ ID NO: 29);
H-Sar-S-S(O-octyl)-FLSPEHQRVQQRKESKKPPAKL-K(hexadecanoyl)-PR-OH (SEQ ID NO: 30);
H-AS-S(O-octyl)-FLSPEHQRVQQRKESKKPPAKLQPR-NH2 (SEQ ID NO: 31);
H-Inp-S-S(O-octyl)-FLSPEHQRVQQRKESKKPPAKLQPR-NH2 (SEQ ID NO: 32);
H-PS-S(O-octyl)-FLSPEHQRVQQRKESKKPPAKLQPR-NH2 (SEQ ID NO: 33);
H-[DPro]-S-S(O-octyl)-FLSPEHQRVQQRKESKKPPAKLQPR-NH2 (SEQ ID NO: 34);
H-PS-S(O-octyl)-FLSPEHQRV-K(hexadecanoyl)-QRKESKKPPAKLQPR-NH2 (SEQ ID NO: 35);
H-Mamb-S(O-octyl)-FLSPEHQRV-K(hexadecanoyl)-QRKESKKPPAKLQPR-NH2 (SEQ ID NO: 36);
H-SS-S(O-octyl)-FLSPEHQRVQQRKES-K(hexadecanoyl)-KPPAKLQPR-NH2 (SEQ ID NO: 37);
H-AS-S(O-octyl)-FLSPEHQRVQQRKESKKPPAKL-K(hexadecanoyl)-PR-NH2 (SEQ ID NO: 38);
H-Aib-S-S(O-octyl)-FLSPEHQRVQQRKESKKPPAKL-K(hexadecanoyl)-PR-NH2 (SEQ ID NO: 39);
H-PS-[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQQRKESKKPPAKLQPR-OH (SEQ ID NO: 40);
H-[DPro]-S—[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQQRKESKKPPAKLQPR-OH (SEQ ID NO: 41);
H-Cmp-[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQQRKESKKPPAKLQPR-OH (SEQ ID NO: 42);
H-[DPro]-S—[NH—CH(n-C10H21)—C(O)]-FLSPEHQRV-K(hexadecanoyl)-QRKESKKPPAKLQPR-OH (SEQ ID NO: 43);
H-Sar-S—[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQQR-K(hexadecanoyl)-ESKKPPAKLQPR-OH (SEQ ID NO: 44);
H-Aib-S—[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQQR-K(hexadecanoyl)-ESKKPPAKLQPR-OH (SEQ ID NO: 45);
H-[cACC]-[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQQRKES-K(hexadecanoyl)-KPPAKLQPR-OH (SEQ ID NO: 46);
H-[DPro]-S—[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQQRKESK-K(hexadecanoyl)-PPAKLQPR-OH (SEQ ID NO: 47);
H-AS-[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQQRKESKKPPAKL-K(hexadecanoyl)-PR-OH (SEQ ID NO: 48);
H-Mamb-[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQQRKESKKPPAKLK(hexadecanoyl)-PR-OH (SEQ ID NO: 49);
H-AS-[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQQRKESKKPPAKLQPR-NH2 (SEQ ID NO: 50);
H-Inp-S—[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQQRKESKKPPAKLQPR-NH2 (SEQ ID NO: 51);
H-PS-[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQQRKESKKPPAKLQPR-NH2 (SEQ ID NO: 52);
H-[DPro]-S—[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQQRKESKKPPAKLQPR-NH2 (SEQ ID NO: 53);
H-SS-[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQQRKESKKPPAKLQPR-NH2 (SEQ ID NO: 54);
H-Cmp-[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQQRKESKKPPAKLQPR-NH2 (SEQ ID NO: 55);
H-[cACC]-[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQQRKESKKPPAKLQPR-NH2 (SEQ ID NO: 56);
H-Sar-S—[NH—CH(n-C10H21)—C(O)]-FLSPEHQRV-K(hexadecanoyl)-QRKESKKPPAKLQPR-NH2 (SEQ ID NO: 57);
H-Aib-S—[NH—CH(n-C10H21)—C(O)]-FLSPEHQRV-K(hexadecanoyl)-QRKESKKPPAKLQPR-NH2 (SEQ ID NO: 58);
H-Cmp-[NH—CH(n-C10H21)—C(O)]-FLSPEHQRV-K(hexadecanoyl)-QRKESKKPPAKLQPR-NH2 (SEQ ID NO: 59);
H-Inp-S—[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQQR-K(hexadecanoyl)-ESKKPPAKLQPR-NH2 (SEQ ID NO: 60);
H-[DPro]-S—[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQQRKES-K(hexadecanoyl)-KPPAKLQPR-NH2 (SEQ ID NO: 61);
H-SS-[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQQRKES-K(hexadecanoyl)-KPPAKLQPR-NH2 (SEQ ID NO: 62);
H-Sar-S—[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQQRKESK-K(hexadecanoyl)-PPAKLQPR-NH2 (SEQ ID NO: 63);
H-AS-[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQQRKESKKPPAKL-K(hexadecanoyl)-PR-NH2 (SEQ ID NO: 64); and
H-PS-[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQQRKESKKPPAKL-K(hexadecanoyl)-PR-NH2 (SEQ ID NO: 65);
H-PS-S(O-octanoyl)-FLSPEHQRV-K(hexadecanoyl)-QRKESKKPPAKLQPR-NH2 (SEQ ID NO: 66);
H-[DPro]-S-S(O-octanoyl)-FLSPEHQRV-K(hexadecanoyl)-QRKESKKPPAKLQPR-NH2 (SEQ ID NO: 67);
H-SS-S(O-octanoyl)-FLSPEHQRV-K(hexadecanoyl)-QRKESKKPPAKLQPR-NH2 (SEQ ID NO: 68);
H-Cmp-S(O-octanoyl)-FLSPEHQRV-K(hexadecanoyl)-QRKESKKPPAKLQPR-NH2 (SEQ ID NO: 69);
H-Cmp-S(O-octanoyl)-FLSPEHQRVQQR-K(hexadecanoyl)-ESKKPPAKLQPR-NH2 (SEQ ID NO: 70);
H-[cACC]-S(O-octanoyl)-FLSPEHQRVQQR-K(hexadecanoyl)-ESKKPPAKLQPR-NH2 (SEQ ID NO: 71);
H-[tACC]-S(O-octanoyl)-FLSPEHQRVQQRKES-K(hexadecanoyl)-KPPAKLQPR-NH2 (SEQ ID NO: 72);
H-SS-S(O-octanoyl)-FLSPEHQRVQQRKESK-K(hexadecanoyl)-PPAKLQPR-NH2 (SEQ ID NO: 73);
H-PS-Dap(N-octanoyl)-FLSPEHQRVQQRKESKKPPAKLQPR-OH (SEQ ID NO: 74);
H-[DPro]-S-Dap(N-octanoyl)-FLSPEHQRVQQRKESKKPPAKLQPR-OH (SEQ ID NO: 75);
H-SS-Dap(N-octanoyl)-FLSPEHQRVQQRKESKKPPAKLQPR-OH (SEQ ID NO: 76);
H-PS-Dap(N-octanoyl)-FLSPEHQRVQQR-K(hexadecanoyl)-ESKKPPAKLQPR-OH (SEQ ID NO: 77);
H-Mamb-Dap(N-octanoyl)-FLSPEHQRVQQRKES-K(hexadecanoyl)-KPPAKLQPROH (SEQ ID NO: 78);
H-PS-Dap(N-octanoyl)-FLSPEHQRVQQRKESKKPPAKLQPR-NH2 (SEQ ID NO: 79);
H-[DPro]-S-Dap(N-octanoyl)-FLSPEHQRVQQRKESKKPPAKLQPR-NH2 (SEQ ID NO: 80);
H-SS-Dap(N-octanoyl)-FLSPEHQRVQQRKESKKPPAKLQPR-NH2 (SEQ ID NO: 81);
H-PS-Dap(N-octanoyl)-FLSPEHSRVSSRKESKKPPAKLSPR-NH2 (SEQ ID NO: 82);
H-Cmp-Dap(N-octanoyl)-FLSPEHQRVQQRKESKKPPAKLQPR-NH2 (SEQ ID NO: 83);
H-[cACC]-Dap(N-octanoyl)-FLSPEHQRVQQRKESKKPPAKLQPR-NH2 (SEQ ID NO: 84);
H-PS-Dap(N-octanoyl)-FLSPEHQRV-K(hexadecanoyl)-QRKESKKPPAKLQPR-NH2 (SEQ ID NO: 85);
H-[DPro]-S-Dap(N-octanoyl)-FLSPEHQRV-K(hexadecanoyl)-QRKESKKPPAKLQPRNH2 (SEQ ID NO: 86);
H-FS-Dap(N-octanoyl)-FLSPEHQRV-K(hexadecanoyl)-QRKESKKPPAKLQPR-NH2 (SEQ ID NO: 87);
H-SS-Dap(N-octanoyl)-FLSPEHQRV-K(hexadecanoyl)-QRKESKKPPAKLQPR-NH2 (SEQ ID NO: 88);
H-[DPro]-S-Dap(N-octanoyl)-FLSPEHQRVQQR-K(hexadecanoyl)-ESKKPPAKLQPR-NH2 (SEQ ID NO: 89);
H-Mamb-Dap(N-octanoyl)-FLSPEHQRVQQR-K(hexadecanoyl)-ESKKPPAKLQPR-NH2 (SEQ ID NO: 90);
H-PS-Dap(N-octanoyl)-FLSPEHQRVQQRKESK-K(hexadecanoyl)-PPAKLQPR-NH2 (SEQ ID NO: 91);
H-SS-Dap(N-octanoyl)-FLSPEHQRVQQRKESK-K(hexadecanoyl)-PPAKLQPR-NH2 (SEQ ID NO: 92);
and H-Cmp-Dap(N-octanoyl)-FLSPEHQRVQQRKESKKPPAKL-K(hexadecanoyl)-PR-NH2 (SEQ ID NO: 93).
Further specific embodiments of compounds of the invention include compounds selected from the group consisting of:
H-GS-S(octanoyl)-FLSPE(HQRK(QQRKESKKPPAKLQPR-OH (SEQ ID NO: 94);
H-GS-S(octanoyl)-FLSPEHE(RVQK(RKESKKPPAKLQPR-OH (SEQ ID NO: 95);
H-GS-S(octanoyl)-FLSPEHQRVQQRK(ESKE(PPAKLQPR-OH (SEQ ID NO: 96);
H-PS-S(O-octyl)-FLSPEOH( )QRKQQRKESKKPPAKLQPR-NH2 (SEQ ID NO: 97);
H-PS-S(O-octyl)-FLSPEHE(RVQK(RKESKKPPAKLQPR-NH2 (SEQ ID NO: 98);
H-Orn-S(O-octyl)-FLSPEHQRVQQRKESKKPPAKLQPR-NH2 (SEQ ID NO: 99);
Ac-Orn-S(O-octyl)-FLSPEHQRVQQRKESKKPPAKLQPR-NH2 (SEQ ID NO: 99);
H-PS-Dap(N-octanoyl)-FLSPEHQRVQRKESKKPPAKLQPR-NH2 (SEQ ID NO: 100);
H-PS-S(O-octyl)-FLSPEHQRVQRKESKKPPAKLQPR-NH2 (SEQ ID NO: 101);
H-PS-Dap(N-octanoyl)-FLSPK-NH2 (SEQ ID NO: 102);
H-PS-S(O-octyl)-FLSPK-NH2 (SEQ ID NO: 103);
H-PS-[NH—CH(n-C10H21)—C(O)]-FLSPK-NH2 (SEQ ID NO: 104);
H-PS-Dap(N-octanoyl)-FLSPY-NH2 (SEQ ID NO: 105);
H-PS-S(O-octyl)-FLSPEHQRVQQRKES-K(hexadecanoyl-isoGlu)-KPPAKLQPR-NH2 (SEQ ID NO: 106);
H-PS-Dap(N-octanoyl)-FLSPEHSRVSSRKES-K(hexadecanoyl-isoGlu)-KPPA-NH2 (SEQ ID NO: 107);
H-PS-S(O-octyl)-FLSPEHSRVSSRKESSKPPAKLSPR-NH2 (SEQ ID NO: 108);
H-PS-S(O-octyl)-FLSPEHSRVSSRKESKSPPAKLSPR-NH2 (SEQ ID NO: 109);
H-PS-S(O-octyl)-FLSPEHSRVSSRKESSSPPAKLSPR-NH2 (SEQ ID NO: 110);
H-PS-S(O-octyl)-FLSPEHSRPSSR-NH2 (SEQ ID NO: 111);
H-PS-S(O-octyl)-FLSPEHSRVSSRPESSSPPAKLSPR-NH2 (SEQ ID NO: 112);
H-PS-S(O-octyl)-FLSPEHSRPSSRPESSSPPAKLSPR-NH2 (SEQ ID NO: 113);
H-PS-S(octanoyl)-FLSPEHQSVQQRKESKKPPAKLQPR-NH2 (SEQ ID NO: 114);
H-PS-S(octanoyl)-FLSPEHQRVQQSKESKKPPAKLQPR-NH2 (SEQ ID NO: 115);
H-PS-S(octanoyl)-FLSPEHQRVQQRSESKKPPAKLQPR-NH2 (SEQ ID NO: 116);
H-PS-S(octanoyl)-FLSPEHQRVQQRKESSKPPAKLQPR-NH2 (SEQ ID NO: 117);
H-PS-S(octanoyl)-FLSPEHQRVQQRKESKSPPAKLQPR-NH2 (SEQ ID NO: 118);
H-PS-S(octanoyl)-FLSPEHQRVQQRKESKKPPASLQPR-NH2 (SEQ ID NO: 119);
H-PS-S(octanoyl)-FLSPEHQRVQQRKESKKPPAKLQPS-NH2 (SEQ ID NO: 120);
H-PS-S(octanoyl)-FLSPYHQRVQQRKESKKPPAKLQPR-NH2 (SEQ ID NO: 121);
H-PS-S(octanoyl)-FLSPEHQRVQYRKESKKPPAKLQPR-NH2 (SEQ ID NO: 122);
H-PS-S(octanoyl)-FLSPEHQRVQQRKESKKPPAYLQPR-NH2 (SEQ ID NO: 123);
H-PS-S(octanoyl)-FLSPYHQRVQYRKESKKPPAYLQPR-NH2 (SEQ ID NO: 124);
H-PS-S(O-octyl)-FLSPEHQRVQQRKESKKPPAKLQPR-NH2 (SEQ ID NO: 125);
H-PS-S(O-octyl)-FLSPEHQRVQRKESKKPPAKLQPR-NH2 (SEQ ID NO: 126);
H-PS-S(O-octyl)-FLSPEHQRVQQRKESKKPPA-NH2 (SEQ ID NO: 127);
H-PS-S(O-octyl)-FLSPEHQRVQQRKES-NH2 (SEQ ID NO: 128);
H-PS-[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQQRKESKKPPAKLQPR-NH2 (SEQ ID NO: 129);
H-PS-[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQRKESKKPPAKLQPR-NH2 (SEQ ID NO: 130);
H-[cACC]-S(O-octyl)-FLSPEHQRVQQRKESKKPPAKLQPR-NH2 (SEQ ID NO: 131);
H-PS-S(O-octyl)-FLSPEHSR-NH2 (SEQ ID NO: 132);
H-PS-S(O-octyl)-FLSPEHSRVSSR-NH2 (SEQ ID NO: 133); and
H-PS-S(O-octyl)-FLSPEHSRVSSRKESKKPPAKLSPR-NH2 (SEQ ID NO: 134).
In the present description and claims, double parentheses “0” after the one-letter abbreviations for two amino acid residues in an amino acid sequence, e.g. “E( )” and “K( )”, signify the presence of an intramolecular bond formed between the side-chains of the amino acid residues in question. In the case of “E( )” and “K( )”, the intramolecular bond will typically be a lactam bridge.
Additional specific embodiments of compounds of the invention include compounds selected from the group consisting of:
H-PS-Dap(N-octanoyl)-FLSPEHQRVQQRKESKKPPAKLQP-NH2 (SEQ ID NO: 135);
H-PS-Dap(N-octanoyl)-FLSPEHQRVQQRKESKKPPAKLQ-NH2 (SEQ ID NO: 136);
H-PS-Dap(N-octanoyl)-FLSPEHQRVQQRKESKKPPAKL-NH2 (SEQ ID NO: 137);
H-PS-Dap(N-octanoyl)-FLSPEHQRVQQRKESKKPPAK-NH2 (SEQ ID NO: 138);
H-PS-Dap(N-octanoyl)-FLSPEHQRVQQRKESKKPPA-NH2 (SEQ ID NO: 139);
H-PS-Dap(N-octanoyl)-FLSPEHQRVQQRKESKKPP-NH2 (SEQ ID NO: 140);
H-PS-Dap(N-octanoyl)-FLSPEHQRVQQRKESKKP-NH2 (SEQ ID NO: 141);
H-PS-Dap(N-octanoyl)-FLSPEHQRVQQRKESKK-NH2 (SEQ ID NO: 142);
H-PS-Dap(N-octanoyl)-FLSPEHQRVQQRKESK-NH2 (SEQ ID NO: 143);
H-PS-Dap(N-octanoyl)-FLSPEHQRVQQRKES-NH2 (SEQ ID NO: 144);
H-PS-Dap(N-octanoyl)-FLSPEHQRVQQRKE-NH2 (SEQ ID NO: 145);
H-PS-Dap(N-octanoyl)-FLSPEHQRVQQRK-NH2 (SEQ ID NO: 146);
H-PS-Dap(N-octanoyl)-FLSPEHQRVQQR-NH2 (SEQ ID NO: 147);
H-PS-Dap(N-octanoyl)-FLSPEHQRVQQ-NH2 (SEQ ID NO: 148);
H-PS-Dap(N-octanoyl)-FLSPEHQRVQ-NH2 (SEQ ID NO: 149);
H-PS-Dap(N-octanoyl)-FLSPEHQRV-NH2 (SEQ ID NO: 150);
H-PS-Dap(N-octanoyl)-FLSPEHQR-NH2 (SEQ ID NO: 151);
H-PS-Dap(N-octanoyl)-FLSPEHQ-NH2 (SEQ ID NO: 152);
H-PS-Dap(N-octanoyl)-FLSPEH-NH2 (SEQ ID NO: 153);
H-PS-Dap(N-octanoyl)-FLSPE-NH2 (SEQ ID NO: 154);
H-PS-Dap(N-octanoyl)-FLSP-NH2 (SEQ ID NO: 155);
H-PS-Dap(N-octanoyl)-FLS-NH2 (SEQ ID NO: 156);
H-PS-Dap(N-octanoyl)-FL-NH2 (SEQ ID NO: 157);
H-PS-Dap(N-octanoyl)-FLSPEHSRVSSRKESKKPPAKLSP-NH2 (SEQ ID NO: 158);
H-PS-Dap(N-octanoyl)-FLSPEHSRVSSRKESKKPPAKLS-NH2 (SEQ ID NO: 159);
H-PS-Dap(N-octanoyl)-FLSPEHSRVSSRKESKKPPAKL-NH2(SEQ ID NO: 160);
H-PS-Dap(N-octanoyl)-FLSPEHSRVSSRKESKKPPAK-NH2 (SEQ ID NO: 161);
H-PS-Dap(N-octanoyl)-FLSPEHSRVSSRKESKKPPA-NH2 (SEQ ID NO: 162);
H-PS-Dap(N-octanoyl)-FLSPEHSRVSSRKESKKPP-NH2 (SEQ ID NO: 163);
H-PS-Dap(N-octanoyl)-FLSPEHSRVSSRKESKKP-NH2 (SEQ ID NO: 164);
H-PS-Dap(N-octanoyl)-FLSPEHSRVSSRKESKK-NH2 (SEQ ID NO: 165);
H-PS-Dap(N-octanoyl)-FLSPEHSRVSSRKESK-NH2 (SEQ ID NO: 166);
H-PS-Dap(N-octanoyl)-FLSPEHSRVSSRKES-NH2 (SEQ ID NO: 167);
H-PS-Dap(N-octanoyl)-FLSPEHSRVSSRKE-NH2 (SEQ ID NO: 168);
H-PS-Dap(N-octanoyl)-FLSPEHSRVSSRK-NH2 (SEQ ID NO: 169);
H-PS-Dap(N-octanoyl)-FLSPEHSRVSSR-NH2 (SEQ ID NO: 170);
H-PS-Dap(N-octanoyl)-FLSPEHSRVSS-NH2 (SEQ ID NO: 171);
H-PS-Dap(N-octanoyl)-FLSPEHSRVS-NH2 (SEQ ID NO: 172);
H-PS-Dap(N-octanoyl)-FLSPEHSRV-NH2 (SEQ ID NO: 173);
H-PS-Dap(N-octanoyl)-FLSPEHSR-NH2 (SEQ ID NO: 174);
H-PS-Dap(N-octanoyl)-FLSPEHS-NH2 (SEQ ID NO: 175);
H-PS-[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQQRKESKKPPA-NH2 (SEQ ID NO: 176);
H-PS-[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQQRKESKKPP-NH2 (SEQ ID NO: 177);
H-PS-[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQQRKESKKP-NH2 (SEQ ID NO: 178);
H-PS-[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQQRKESKK-NH2 (SEQ ID NO: 179);
H-PS-[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQQRKESK-NH2 (SEQ ID NO: 180);
H-PS-[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQQRKES-NH2 (SEQ ID NO: 181);
H-PS-[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQQRKE-NH2 (SEQ ID NO: 182);
H-PS-[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQQRK-NH2 (SEQ ID NO: 183);
H-PS-[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQQR-NH2 (SEQ ID NO: 184);
H-PS-[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQQ-NH2 (SEQ ID NO: 185);
H-PS-[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQ-NH2 (SEQ ID NO: 186);
H-PS-[NH—CH(n-C10H21)—C(O)]-FLSPEHQRV-NH2 (SEQ ID NO: 187);
H-PS-[NH—CH(n-C10H21)—C(O)]-FLSPEHQR-NH2 (SEQ ID NO: 188);
H-PS-[NH—CH(n-C10H21)—C(O)]-FLSPEHQ-NH2 (SEQ ID NO: 189);
H-PS-[NH—CH(n-C10H21)—C(O)]-FLSPEH-NH2 (SEQ ID NO: 190);
H-PS-[NH—CH(n-C10H21)—C(O)]-FLSPE-NH2 (SEQ ID NO: 191);
H-PS-[NH—CH(n-C10H21)—C(O)]-FLSP-NH2 (SEQ ID NO: 192);
H-PS-[NH—CH(n-C10H21)—C(O)]-FLS-NH2 (SEQ ID NO: 193); and
H-PS-[NH—CH(n-C10H21)—C(O)]-FL-NH2. (SEQ ID NO: 194).
Further specific embodiments of compounds of the invention include compounds selected from the group consisting of:
H-PS-Dap(N-octanoyl)-FLSPEHQRVQQRKESKKPPAKLQP-OH (SEQ ID NO: 135);
H-PS-Dap(N-octanoyl)-FLSPEHQRVQQRKESKKPPAKLQ-OH (SEQ ID NO: 136);
H-PS-Dap(N-octanoyl)-FLSPEHQRVQQRKESKKPPAKL-OH (SEQ ID NO: 137);
H-PS-Dap(N-octanoyl)-FLSPEHQRVQQRKESKKPPAK-OH (SEQ ID NO: 138);
H-PS-Dap(N-octanoyl)-FLSPEHQRVQQRKESKKPPA-OH (SEQ ID NO: 139);
H-PS-Dap(N-octanoyl)-FLSPEHQRVQQRKESKKPP-OH (SEQ ID NO: 140);
H-PS-Dap(N-octanoyl)-FLSPEHQRVQQRKESKKP-OH (SEQ ID NO: 141);
H-PS-Dap(N-octanoyl)-FLSPEHQRVQQRKESKK-OH (SEQ ID NO: 142);
H-PS-Dap(N-octanoyl)-FLSPEHQRVQQRKESK-OH (SEQ ID NO: 143);
H-PS-Dap(N-octanoyl)-FLSPEHQRVQQRKES-OH (SEQ ID NO: 144);
H-PS-Dap(N-octanoyl)-FLSPEHQRVQQRKE-OH (SEQ ID NO: 145);
H-PS-Dap(N-octanoyl)-FLSPEHQRVQQRK-OH (SEQ ID NO: 146);
H-PS-Dap(N-octanoyl)-FLSPEHQRVQQR-OH (SEQ ID NO: 147);
H-PS-Dap(N-octanoyl)-FLSPEHQRVQQ-OH (SEQ ID NO: 148);
H-PS-Dap(N-octanoyl)-FLSPEHQRVQ-OH (SEQ ID NO: 149);
H-PS-Dap(N-octanoyl)-FLSPEHQRV-OH (SEQ ID NO: 150);
H-PS-Dap(N-octanoyl)-FLSPEHQR-OH (SEQ ID NO: 151);
H-PS-Dap(N-octanoyl)-FLSPEHQ-OH (SEQ ID NO: 152);
H-PS-Dap(N-octanoyl)-FLSPEH-OH (SEQ ID NO: 153);
H-PS-Dap(N-octanoyl)-FLSPE-OH (SEQ ID NO: 154);
H-PS-Dap(N-octanoyl)-FLSP-OH (SEQ ID NO: 155);
H-PS-Dap(N-octanoyl)-FLS-OH (SEQ ID NO: 156);
H-PS-Dap(N-octanoyl)-FL-OH (SEQ ID NO: 157);
H-PS-Dap(N-octanoyl)-FLSPEHSRVSSRKESKKPPAKLSP-OH (SEQ ID NO: 158);
H-PS-Dap(N-octanoyl)-FLSPEHSRVSSRKESKKPPAKLS-OH (SEQ ID NO: 159);
H-PS-Dap(N-octanoyl)-FLSPEHSRVSSRKESKKPPAKL-OH (SEQ ID NO: 160);
H-PS-Dap(N-octanoyl)-FLSPEHSRVSSRKESKKPPAK-OH (SEQ ID NO: 161);
H-PS-Dap(N-octanoyl)-FLSPEHSRVSSRKESKKPPA-OH (SEQ ID NO: 162);
H-PS-Dap(N-octanoyl)-FLSPEHSRVSSRKESKKPP-OH (SEQ ID NO: 163);
H-PS-Dap(N-octanoyl)-FLSPEHSRVSSRKESKKP-OH (SEQ ID NO: 164);
H-PS-Dap(N-octanoyl)-FLSPEHSRVSSRKESKK-OH (SEQ ID NO: 165);
H-PS-Dap(N-octanoyl)-FLSPEHSRVSSRKESK-OH (SEQ ID NO: 166);
H-PS-Dap(N-octanoyl)-FLSPEHSRVSSRKES-OH (SEQ ID NO: 167);
H-PS-Dap(N-octanoyl)-FLSPEHSRVSSRKE-OH (SEQ ID NO: 168);
H-PS-Dap(N-octanoyl)-FLSPEHSRVSSRK-OH (SEQ ID NO: 169);
H-PS-Dap(N-octanoyl)-FLSPEHSRVSSR-OH (SEQ ID NO: 170);
H-PS-Dap(N-octanoyl)-FLSPEHSRVSS-OH (SEQ ID NO: 171);
H-PS-Dap(N-octanoyl)-FLSPEHSRVS-OH (SEQ ID NO: 172);
H-PS-Dap(N-octanoyl)-FLSPEHSRV-OH (SEQ ID NO: 173);
H-PS-Dap(N-octanoyl)-FLSPEHSR-OH (SEQ ID NO: 174);
H-PS-Dap(N-octanoyl)-FLSPEHS-OH (SEQ ID NO: 175);
H-PS-[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQQRKESKKPPA-OH (SEQ ID NO: 176);
H-PS-[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQQRKESKKPP-OH (SEQ ID NO: 177);
H-PS-[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQQRKESKKP-OH (SEQ ID NO: 178);
H-PS-[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQQRKESKK-OH (SEQ ID NO: 179);
H-PS-[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQQRKESK-OH (SEQ ID NO: 180);
H-PS-[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQQRKES-OH (SEQ ID NO: 181);
H-PS-[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQQRKE-OH (SEQ ID NO: 182);
H-PS-[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQQRK-OH (SEQ ID NO: 183);
H-PS-[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQQR-OH (SEQ ID NO: 184);
H-PS-[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQQ-OH (SEQ ID NO: 185);
H-PS-[NH—CH(n-C10H21—C(O)]-FLSPEHQRVQ-OH (SEQ ID NO: 186);
H-PS-[NH—CH(n-C10H21)—C(O)]-FLSPEHQRV-OH (SEQ ID NO: 187);
H-PS-[NH—CH(n-C10H21)—C(O)]-FLSPEHQR-OH (SEQ ID NO: 188);
H-PS-[NH—CH(n-C10H21)—C(O)]-FLSPEHQ-OH (SEQ ID NO: 189);
H-PS-[NH—CH(n-C10H21)—C(O)]-FLSPEH-OH (SEQ ID NO: 190);
H-PS-[NH—CH(n-C10H21)—C(O)]-FLSPE-OH (SEQ ID NO: 191);
H-PS-[NH—CH(n-C10H21)—C(O)]-FLSP-OH (SEQ ID NO: 192);
H-PS-[NH—CH(n-C10H21)—C(O)]-FLS-OH (SEQ ID NO: 193); and
H-PS-[NH—CH(n-C10H21)—C(O)]-FL-OH (SEQ ID NO: 194).
Pharmaceutically acceptable salts or solvates of any of the compounds of the invention, e.g., compounds within the latter two groups, are likewise within the scope of the present invention.
In each of the sequences described above, and in each sequence described herein, a C-terminal “—OH” moiety may be substituted for a C-terminal “—NH2” moiety, and vice-versa.
Each of the specific compounds of the invention mentioned above, taken alone, constitutes an individual embodiment of a compound of the invention.
In the above, the abbreviations Ser(O-octanoyl) and Ser(O-octyl) for the residue in position 3 (as they occur, e.g., in the compound of the invention:
H-PS-S(O-octanoyl)-FLSPEHQRVQQRKESKKPPAKLQPR-OH (SEQ ID NO: 7); in the compound of the invention:
H-[DPro]-S-S(O-octanoyl)-FLSPEHQRVQQRKESKKPPAKLQPR-OH (SEQ ID NO: 8); in the compound of the invention:
H-PS-S(O-octanoyl)-FLSPEHQRV-K(hexadecanoyl)-QRKESKKPPAKLQPR-NH2 (SEQ ID NO: 66); in the compound of the invention:
H-[DPro]-S-S(O-octanoyl)-FLSPEHQRV-K(hexadecanoyl)-QRKESKKPPAKLQPR-NH2 (SEQ ID NO: 67); in the compound of the invention:
H-SS-S(O-octanoyl)-FLSPEHQRVQQRKESK-K(hexadecanoyl)-PPAKLQPR-NH2 (SEQ ID NO: 73); in the compound of the invention:
H-[DPro]-S-S(O-octyl)-FLSPEHQRVQQRKESKKPPAKLQPR-OH (SEQ ID NO: 22); and in the compound of the invention:
H-Aib-S-S(O-octyl)-FLSPEHQRVQQRKESKKPPAKLQPR-OH) (SEQ ID NO: 23) refer to a modified serine residue in which the hydrogen atom of the side-chain hydroxy group in serine is replaced by an n-octanoyl group or n-octyl group, respectively. The abbreviation Dap(N-octanoyl) for the residue in position 3 (as it occurs, by way of example, in the compound of the invention:

H-PS-Dap(N-octanoyl)-FLSPEHQRVQQRKESKKPPAKLQPR-OH (SEQ ID NO: 74); in the compound of the invention:

H-[DPro]-S-Dap(N-octanoyl)-FLSPEHQRVQQRKESKKPPAKLQPR-OH (SEQ ID NO: 75); in the compound of the invention:

H-Mamb-Dap(N-octanoyl)-FLSPEHQRVQQRKES-K(hexadecanoyl)-KPPAKLQPROH (SEQ ID NO: 78); in the compound of the invention:

H-PS-Dap(N-octanoyl)-FLSPEHQRVQQRKESKKPPAKLQPR-NH2 (SEQ ID NO: 79); in the compound of the invention:

H-[DPro]-S-Dap(N-octanoyl)-FLSPEHQRVQQRKESKKPPAKLQPR-NH2 (SEQ ID NO: 80); in the compound of the invention:

H-PS-Dap(N-octanoyl)-FLSPEHSRVSSRKESKKPPAKLSPR-NH2 (SEQ ID NO: 82); and in the compound of the invention:

H-SS-Dap(N-octanoyl)-FLSPEHQRVQQRKESKKPPAKLQPR-NH2) (SEQ ID NO: 81); refers to a modified residue of the non-naturally occurring amino acid 2,3-diaminopropanoic acid in which the side-chain amino group (3-amino group) is N-acylated with an n-octanoyl group.

In further aspects of the invention, in addition to those residues specified above in relation to position X1, additional amino acid residues which may be of relevance as X1 residues in compounds of the invention include, but are not limited to, Bal, βPro and Lys.
In still further aspects of the invention, in addition to those residues specified above in relation to the positions X10, X13, X14 and X26, the amino acid residue at one or more of those four positions may, independently, be selected from the group consisting of Glu, Leu, Lys, Ser and Arg.
The peptide sequence Z in formula I may be considered to consist of up to 28 positions, designated X1 to X28. The amino acid residues at positions X4 to X7, X9, X17, X18, X21 to X23, X25 and X27, when present, are fixed as set out in formula Ia. The amino acid residues at positions X1 to X3, X8, X10 to X16, X19, X20, X24, X26 and X28, when present, may vary within the parameters described in this specification.
In certain embodiments described by formula Ib, positions X8, X15 and X28 are also fixed.
In some aspects, Z is full-length. That is to say, at least positions X1 to X13 and X15 to X28 are present. Position X14 may independently be present or absent.
In yet further aspects, the invention also provides compounds having an amino acid sequence (Z in formula I above) that is truncated (shortened), from the C-terminus, relative to the full length of the sequence Z in compounds of the invention of formula I as disclosed above. In some embodiments, the truncated compounds are truncated by up to 23, e.g., up to 22, residues counting from the C-terminus of a compound of the invention of formula I as disclosed above (which in its longest form as disclosed above has an Arg residue at the C-terminus). Thus, such compounds may be truncated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 amino acid residues, counting from the C-terminus of a compound of the invention of formula I as disclosed above.
Thus, the peptide sequence Z will always comprise residue positions X1 to X5, and may comprise X1 to X6, X1 to X7, X1 to X8, X1 to X9, X1 to X10, X1 to X11, X1 to X12, X1 to X13, X1 to X14, X1 to X15, X1 to X16, X1 to X17, X1 to X18, X1 to X19, X1 to X20, X1 to X21, X1 to X22, X1 to X23, X1 to X24, X1 to X25, X1 to X26, X1 to X27, or X1 to X28. In any of these, position X14 may be independently absent.
Without wishing to be bound by theory, the more highly truncated certain compounds of the invention are, the more advantageous they may be with regard to ghrelin receptor agonist activity to have an —NH2 group rather than an —OH group at the C-terminus thereof (R2 in compounds of the invention of formula I as already disclosed above).
In some embodiments, the compounds of the invention may have at least 40%, e.g., at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, 99.5%, or 99.9% amino acid sequence identity to native human ghrelin. The percentage identity is calculated only over the length of overlap between the relevant compound and the native human ghrelin sequence. Thus, for example, a compound consisting solely of positions X1 to X14 of native human ghrelin would considered to have 100% identity to ghrelin, not 50%. (Needless to say, such a compound is not a compound of the invention.) For the avoidance of doubt, a modified residue (such as a Lys residue carrying a lipophilic substituent) is considered to be different to the unmodified form for the purposes of determining sequence identity. If position 14 is absent, that should not be taken into account in the assessment of sequence identity.
As an alternative to percentage sequence identity, it may be convenient to consider the number of residues in a given compound which are different from the corresponding residues in native human ghrelin.
The compounds of the invention differ from native human ghrelin at least at positions 1 and 3. They may have a maximum of 3 differences, a maximum of 4 differences, a maximum of 5 differences, a maximum of 6 differences, a maximum of 7 differences, a maximum of 8 differences, a maximum of 9 differences, a maximum of 10 differences, a maximum of 11 differences, a maximum of 12 differences, a maximum of 13 differences, a maximum of 14 differences, a maximum of 15 differences or a maximum of 16 differences compared to native human ghrelin, depending on the length of the compound itself, including any absence of position 14. It may be desirable that one of these differences is a Lys modified by acylation as described herein.
Thus, a compound consisting of positions X1 to X5, X1 to X6, or X1 to X7 has a maximum of 3 residues which are different to the corresponding residues in human ghrelin.
A compound consisting of positions X1 to X8 or X1 to X9 may have a maximum of 3 or a maximum of 4 residues which are different to the corresponding residues in human ghrelin.
A compound consisting of positions X1 to X10 may have a maximum of 3, a maximum of 4 or a maximum of 5 residues which are different to the corresponding residues in human ghrelin.
A compound consisting of positions X1 to X1 may have a maximum of 3, a maximum of 4, a maximum of 5, or a maximum of 6 residues which are different to the corresponding residues in human ghrelin.
A compound consisting of positions X1 to X12 may have a maximum of 3, a maximum of 4, a maximum of 5, a maximum of 6, or a maximum of 7 residues which are different to the corresponding residues in human ghrelin.
A compound consisting of positions X1 to X13 may have a maximum of 3, a maximum of 4, a maximum of 5, a maximum of 6, a maximum of 7, or a maximum of 8 residues which are different to the corresponding residues in human ghrelin.
A compound consisting of positions X1 to X14 may have a maximum of 3, a maximum of 4, a maximum of 5, a maximum of 6, a maximum of 7, a maximum of 8, or a maximum of 9 residues which are different to the corresponding residues in human ghrelin.
A compound consisting of positions X1 to X15 may have a maximum of 3, a maximum of 4, a maximum of 5, a maximum of 6, a maximum of 7, a maximum of 8, a maximum of 9, or a maximum of 10 residues which are different to the corresponding residues in human ghrelin.
A compound consisting of positions X1 to X16, X1 to X17, or X1 to X18 may have a maximum of 3, a maximum of 4, a maximum of 5, a maximum of 6, a maximum of 7, a maximum of 8, a maximum of 9, a maximum of 10, or a maximum of 11 residues which are different to the corresponding residues in human ghrelin.
A compound consisting of positions X1 to X19 may have a maximum of 3, a maximum of 4, a maximum of 5, a maximum of 6, a maximum of 7, a maximum of 8, a maximum of 9, a maximum of 10, a maximum of 11, or a maximum of 12 residues which are different to the corresponding residues in human ghrelin.
A compound consisting of positions X1 to X20, X1 to X21, or X1 to X22 or X1 to X23, may have a maximum of 3, a maximum of 4, a maximum of 5, a maximum of 6, a maximum of 7, a maximum of 8, a maximum of 9, a maximum of 10, a maximum of 11, a maximum of 12, or a maximum of 13 residues which are different to the corresponding residues in human ghrelin.
A compound consisting of positions X1 to X24 or X1 to X25 may have a maximum of 3, a maximum of 4, a maximum of 5, a maximum of 6, a maximum of 7, a maximum of 8, a maximum of 9, a maximum of 10, a maximum of 11, a maximum of 12, a maximum of 13, or a maximum of 14 residues which are different to the corresponding residues in human ghrelin.
A compound consisting of positions X1 to X26 or X1 to X27 may have a maximum of 3, a maximum of 4, a maximum of 5, a maximum of 6, a maximum of 7, a maximum of 8, a maximum of 9, a maximum of 10, a maximum of 11, a maximum of 12, a maximum of 13, a maximum of 14, or a maximum of 15 residues which are different to the corresponding residues in human ghrelin.
A compound consisting of positions X1 to X28 may have a maximum of 3, a maximum of 4, a maximum of 5, a maximum of 6, a maximum of 7, a maximum of 8, a maximum of 9, a maximum of 10, a maximum of 11, a maximum of 12, a maximum of 13, a maximum of 14, a maximum of 15, or a maximum of 16 residues which are different to the corresponding residues in human ghrelin.
For the purposes of this assessment, absence of position X14 is considered a difference relative to human ghrelin.
In some embodiments, the polypeptides of the invention may comprise the amino acid sequence of any one of Compound Nos: 5-23 (see Table 1, below), or a functional variant thereof that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% sequence identity to ghrelin. A functional variant of a polypeptide of the invention may have at least, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 100% of at least one biological activity of a native ghrelin polypeptide. For example, in some embodiments, a polypeptide of the invention may comprise one or more amino acid substitutions, e.g., conservative amino acid substitutions, and bind to and/or activate the ghrelin receptor by at least 50% compared to the binding and/or activation by native human ghrelin.
The term “conservative substitution” as used herein denotes that one or more amino acids are replaced by another, biologically similar residue. Examples include substitution of amino acid residues with similar characteristics, e.g. small amino acids, acidic amino acids, polar amino acids, basic amino acids, hydrophobic amino acids and aromatic amino acids. See, for example, the table below. An example of a conservative substitution with a residue normally not found in endogenous, mammalian peptides and proteins is the conservative substitution of Arg or Lys with, for example, ornithine, canavanine, aminoethylcysteine or another basic amino acid. For further information concerning phenotypically silent substitutions in peptides and proteins, see, e.g., Bowie et al., Science 247, 1306-1310, 1990. In the scheme below are conservative substitutions of amino acids grouped by physicochemical properties. I: neutral, hydrophilic, II: acids and amides, III: basic, IV: hydrophobic, V: aromatic, bulky amino acids.














I
II
III
IV
V







A
N
H
M
F


S
D
R
L
Y


T
E
K
I
W


P
Q 

V



G


C









In some embodiments, the polypeptide of the invention may comprise functional fragments or variants thereof that have, at most, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions compared to a wild-type or native human ghrelin sequence. The polypeptide of the invention may further be with or without a signal sequence.
In some embodiments, a polypeptide of the invention shares at least 95% amino acid sequence identity to any one of Compound Nos: 5-21.
As used herein, the term “pharmaceutically acceptable salt” refers to a salt that is not harmful to a patient or subject to which the salt in question is administered. It may be a salt chosen, e.g., among acid addition salts and basic salts. Examples of acid addition salts include chloride salts, citrate salts and acetate salts. Examples of basic salts include salts wherein the cation is selected from alkali metal cations, such as sodium or potassium ions, alkaline earth metal cations, such as calcium or magnesium ions, as well as substituted ammonium ions, such as ions of the type N(R1)(R2)(R3)(R4)+, wherein R1, R2, R3 and R4 independently will typically designate hydrogen, optionally substituted C1-6-alkyl groups or optionally substituted C2-6-alkenyl groups. Examples of relevant C1-6-alkyl groups include methyl, ethyl, 1-propyl and 2-propyl groups. Examples of C2-6-alkenyl groups of possible relevance include ethenyl, 1-propenyl and 2-propenyl. Other examples of pharmaceutically acceptable salts are described in “Remington's Pharmaceutical Sciences”, 17th edition, Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, Pa., USA, 1985 (and more recent editions thereof), in the “Encyclopaedia of Pharmaceutical Technology”, 3rd edition, James Swarbrick (Ed.), Informa Healthcare USA (Inc.), NY, USA, 2007, and in J. Pharm. Sci. 66: 2 (1977).
The term “solvate” in the context of the present invention refers to a complex of defined stoichiometry formed between a solute (in casu, a peptide compound or pharmaceutically acceptable salt thereof according to the invention) and a solvent. The solvent in this connection may, for example, be water, ethanol or another pharmaceutically acceptable, typically small-molecular organic species, such as, but not limited to, acetic acid or lactic acid. When the solvent in question is water, such a solvate is normally referred to as a hydrate.
Some embodiments of the invention relate to a peptide compound or pharmaceutically acceptable salt or solvate thereof according to the invention for use as or for the manufacture or preparation of a pharmaceutical composition, or for the use of a peptide compound or pharmaceutically acceptable salts or solvates thereof in methods of treating or preventing a variety of conditions, diseases, disorders, and syndromes, for example, cachexia, low body weight, loss of body weight, low or decreased appetite, low muscle mass, effects of gastrectomy or vagectomy, anorexia, bulimia, chronic obstructive pulmonary disease, growth hormone deficiency, cardiovascular indications (e.g., low blood flow, low cardiac output, and high blood pressure), osteoporosis, arthritis pain, inflammatory disorders (e.g., arthritis and systemic lupus erythematosus) and gastrointestinal disorders including but not limited to gastroparesis, post-operative ileus, inflammatory disorders such as colitis (e.g., ulcerative colitis), gut barrier dysfunction, inflammatory bowel disease (IBD), Crohn's disease, and ischemia reperfusion injury.
In some embodiments, a peptide compound of the invention is a peptide compound for use in treating a subject in need thereof.
In further embodiments, the peptide compound of the invention is a peptide compound for use in increasing, in a subject in need thereof, food intake and/or body weight.
In further embodiments, the peptide compound of the invention is a peptide compound for inducing, in a subject in need thereof, the production of growth hormone.
In further embodiments, the peptide compound of the invention is a peptide compound for increasing, in a subject in need thereof, blood flow (e.g., colonic blood flow) and/or cardiac output.
In further embodiments, the peptide compound of the invention is a peptide compound for decreasing, in a subject in need thereof, blood pressure.
In further embodiments, the peptide compound of the invention is a peptide compound for inducing, in a subject in need thereof, increased motility of the gastrointestinal tract.
In further embodiments, the peptide compound of the invention is a peptide compound for decreasing, in a subject in need thereof, inflammation.
In some embodiments, a pharmaceutical composition of the invention is a pharmaceutical composition for use in treating a subject in need thereof.
In further embodiments, the pharmaceutical composition of the invention is a pharmaceutical composition for use in treating cachexia and/or chronic obstructive pulmonary disease.
In further embodiments, the pharmaceutical composition of the invention is a pharmaceutical composition for use in increasing, in a subject in need thereof, food intake and/or body weight.
In further embodiments, the pharmaceutical composition of the invention is a pharmaceutical composition for inducing, in a subject in need thereof, the production of growth hormone.
In further embodiments, the pharmaceutical composition of the invention is a pharmaceutical composition for increasing, in a subject in need thereof, blood flow (e.g., colonic blood flow) and/or cardiac output.
In further embodiments, the pharmaceutical composition of the invention is a pharmaceutical composition for decreasing, in a subject in need thereof, blood pressure.
In further embodiments, the pharmaceutical composition of the invention is a pharmaceutical composition for inducing, in a subject in need thereof, increased motility of the gastrointestinal tract.
In further embodiments, the pharmaceutical composition of the invention is a pharmaceutical composition for decreasing, in a subject in need thereof, inflammation.
Further embodiments of the invention relate to the use of a peptide compound of the invention in the manufacture or preparation of a pharmaceutical composition for the treatment, in a subject in need thereof, of one or more of the conditions, diseases, disorders, and syndromes disclosed herein.
Among related, additional embodiments of the invention are corresponding methods of treatment of conditions, diseases or disorders among those disclosed herein. Thus, exemplary embodiments of the invention relate to a method for treatment, in a subject in need thereof, of one or more of the conditions, diseases, disorders, and syndromes disclosed herein, the method comprising administering to the subject a therapeutically effective amount of a peptide compound or pharmaceutically acceptable salt or solvate thereof according to the invention.
Other embodiments of the invention relate to a method for increasing food intake and/or body weight in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a peptide compound or pharmaceutically acceptable salt or solvate thereof according to the invention.
Other embodiments of the invention relate to a method for inducing the production of growth hormone in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a peptide compound or pharmaceutically acceptable salt or solvate thereof according to the invention.
Other embodiments of the invention relate to a method for increasing blood flow (e.g., colonic blood flow) and/or cardiac output in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a peptide compound or pharmaceutically acceptable salt or solvate thereof according to the invention.
Other embodiments of the invention relate to a method for decreasing blood pressure in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a peptide compound or pharmaceutically acceptable salt or solvate thereof according to the invention.
Other embodiments of the invention relate to a method for increasing motility of the gastrointestinal tract in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a peptide compound or pharmaceutically acceptable salt or solvate thereof according to the invention.
Other embodiments of the invention relate to a method for decreasing inflammation in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a peptide compound or pharmaceutically acceptable salt or solvate thereof according to the invention.
In some embodiments, a pharmaceutical composition of the invention is a pharmaceutical composition for use in preventing a disease, condition, or syndrome in a subject in need thereof.
In some embodiments, a pharmaceutical composition of the invention is a pharmaceutical composition for use in preventing, in a subject in need thereof, cachexia, and/or chronic obstructive pulmonary disease.
In further embodiments, the pharmaceutical composition of the invention is a pharmaceutical composition for use in preventing, in a subject in need thereof, decreased food intake and/or loss of body weight.
In further embodiments, the pharmaceutical composition of the invention is a pharmaceutical composition for preventing, in a subject in need thereof, a deficiency in the production of growth hormone.
In further embodiments, the pharmaceutical composition of the invention is a pharmaceutical composition for preventing, in a subject in need thereof, low blood flow (e.g., low colonic blood flow) and/or low cardiac output.
In further embodiments, the pharmaceutical composition of the invention is a pharmaceutical composition for preventing, in a subject in need thereof, elevated blood pressure.
In further embodiments, the pharmaceutical composition of the invention is a pharmaceutical composition for preventing, in a subject in need thereof, reduced motility of the gastrointestinal tract.
In further embodiments, the pharmaceutical composition of the invention is a pharmaceutical composition for preventing, in a subject in need thereof, inflammation.
Further embodiments of the invention relate to the use of a peptide compound of the invention in the manufacture or preparation of a pharmaceutical composition for the prevention, in a subject in need thereof, of one or more of the conditions, diseases, disorders, and syndromes disclosed herein.
Among related, additional embodiments of the invention are corresponding methods of preventing conditions, diseases, disorders, and syndromes among those disclosed herein. Thus, exemplary embodiments of the invention relate to a method for prevention, in a subject in need thereof, of one or more of the conditions, diseases, disorders, and syndromes disclosed herein, the method comprising administering to the subject a therapeutically effective amount of a peptide compound or pharmaceutically acceptable salt or solvate thereof according to the invention.
Other embodiments of the invention relate to a method for preventing decreased food intake and/or loss of body weight in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a peptide compound or pharmaceutically acceptable salt or solvate thereof according to the invention.
Other embodiments of the invention relate to a method for preventing reduced production of growth hormone in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a peptide compound or pharmaceutically acceptable salt or solvate thereof according to the invention.
Other embodiments of the invention relate to a method for preventing decreased blood flow (e.g., colonic blood flow) and/or low cardiac output in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a peptide compound or pharmaceutically acceptable salt or solvate thereof according to the invention.
Other embodiments of the invention relate to a method for preventing high blood pressure in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a peptide compound or pharmaceutically acceptable salt or solvate thereof according to the invention.
Other embodiments of the invention relate to a method for preventing decreased motility of the gastrointestinal tract in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a peptide compound or pharmaceutically acceptable salt or solvate thereof according to the invention.
Other embodiments of the invention relate to a method for preventing inflammation in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a peptide compound or pharmaceutically acceptable salt or solvate thereof according to the invention.
Other embodiments of the invention relate to veterinarian use (e.g., use in a non-human animal) of a peptide compound or pharmaceutically acceptable salt or solvate thereof according to the invention.
The term “therapeutically effective amount” as used herein in the context of the above-described methods of treatment, prevention or other therapeutic interventions according to the invention refers to an amount that is sufficient to cure, ameliorate, alleviate or partially arrest the clinical manifestations of the particular disease, disorder or condition that is the object of the treatment, prevention or other therapeutic intervention in question e.g. as measured by established clinical endpoints or other biomarkers (established or experimental). A therapeutically relevant amount may be determined empirically by one skilled in the art based on the indication being treated or prevented and the subject to whom the therapeutically relevant amount is being administered. For example, the skilled worker may measure one or more of the clinically relevant indicators of bioactivity described herein, e.g., growth hormone levels, nutrient levels, and inflammatory cytokine levels. The skilled worker may determine a clinically relevant amount through in vitro or in vivo measurements. Other exemplary measures include weight loss, food intake, and appetite.
An amount adequate to accomplish any one or more of these effects is defined as a therapeutically effective amount. The administered amount and the method of administration can be tailored to achieve optimal efficacy. An amount effective for a given purpose will depend, inter alia, on the severity of the disease, disorder or condition that is the object of the particular treatment, prevention or other therapeutic intervention, on the body weight and general condition of the subject in question, on diet, on possible concurrent medication, and on other factors well known to those skilled in the medical arts. Determination of an appropriate dosage size and dosing regimen most appropriate for administration of a peptide compound or pharmaceutically acceptable salt or solvate thereof according to the invention to a human may be guided by the results obtained by the present invention, and may be confirmed in properly designed clinical trials. An effective dosage and treatment protocol may be determined by conventional means, starting with a low dose in laboratory animals and then increasing the dosage while monitoring the effects, and systematically varying the dosage regimen as well. Numerous factors may be taken into consideration by a clinician when determining an optimal dosage for a given subject. Such considerations are well known to the skilled person.
The terms “treatment” and grammatical variants thereof (e.g. “treated”, “treating”, “treat”) as employed in the present context refer to an approach for obtaining beneficial or desired clinical results. For the purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilization (i.e., not worsening) of state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival relative to expected survival time if not receiving treatment. A subject (e.g., a human) in need of treatment may thus be a subject already afflicted with the disease or disorder in question. The term “treatment” includes inhibition or reduction of an increase in severity of a pathological state or symptoms (e.g., weight loss) relative to the absence of treatment, and is not necessarily meant to imply complete cessation of the relevant disease, disorder or condition.
The terms “preventing” and grammatical variants thereof (e.g., “prevented”, “preventing”, “prevent”) as employed in the present context refer to an approach for preventing the development of, or altering the pathology of, a condition, disease or disorder. Accordingly, “prevention” may refer to prophylactic or preventive measures. For the purposes of this invention, beneficial or desired clinical results include, but are not limited to, prevention or slowing of symptoms, progression or development of a disease, whether detectable or undetectable. A subject (e.g., a human) in need of prevention may thus be a subject not yet afflicted with the disease or disorder in question. The term “prevention” thus includes slowing the onset of disease relative to the absence of treatment, and is not necessarily meant to imply permanent prevention of the relevant disease, disorder or condition.
In some embodiments of the invention, a subject in need of the particular treatment or other therapeutic intervention referred to in connection with the various aspects of the invention described above is a mammal.
In some embodiments, a mammal is a livestock animal such as, but not limited to, a cow, a sheep, a pig, a goat, or a horse. In some embodiments, a mammal is a pet such as, but not limited to, a cat or a dog. In some embodiments, a mammal is, e.g., a mouse, a rat, a rabbit, a monkey, or a chimpanzee. In some embodiments, a mammal is a non-human mammal. In some embodiments, a mammal is a non-human primate.
In further embodiments, the mammal is a human.
Additional embodiments of the invention relate to pharmaceutical compositions comprising a peptide compound, or a pharmaceutically acceptable salt or solvate thereof, according to the invention, together with a pharmaceutically acceptable carrier, excipient or vehicle.
Synthesis of Peptide Compounds
The peptide compounds of the invention may be manufactured by standard peptide synthetic methods, by use of recombinant expression systems, or by any other suitable method. Thus, the compounds may be synthesized in a number of ways, including, e.g., methods comprising:
(a) synthesizing the peptide compound by standard solid-phase or liquid-phase methodology, either stepwise or by fragment assembly, and isolating and purifying the final peptide compound product;
(b) expressing a nucleic acid construct that encodes the peptide compound or a fragment or precursor thereof in a host cell and recovering the expression product from the host cell culture; or
(c) effecting cell-free in vitro expression of a nucleic acid construct encoding the peptide compound or a fragment or precursor thereof, and recovering the expression product; or by any combination of the methods of (a), (b) or (c) to obtain fragments of the peptide compound, subsequently joining (e.g., ligating) the fragments to obtain the peptide compound, and recovering the peptide compound.
The method of synthesis may comprise the step of chemically modifying one of more amino acid side chains in a precursor peptide to yield a compound of the invention. Such modification may, for example, introduce a non-naturally occurring amino acid, convert one or more amino acids into non-naturally occurring amino acids, introduce an intramolecular bridge between two amino acid side chains, e.g. by forming a lactam ring between a Glu and a Lys residue, or introduce a lipophilic substituent at a lysine residue. It may be preferable to synthesize the peptide compounds of the invention by means of solid-phase or liquid-phase peptide synthesis. In this context, reference may be made to WO 98/11125 or, inter alia, Fields, G. B. et al., “Principles and Practice of Solid-Phase Peptide Synthesis”; in: Synthetic Peptides, Gregory A. Grant (ed.), Oxford University Press (2nd edition, 2002) and the synthesis examples herein.
Accordingly, the present invention also provides methods for producing a polypeptide of the invention according to above recited methods; a nucleic acid molecule encoding part or all of a polypeptide of the invention or a precursor thereof, a vector comprising at least one nucleic acid of the invention, expression vectors comprising at least one nucleic acid of the invention capable of producing a polypeptide of the invention when introduced into a host cell, and a host cell comprising a nucleic acid molecule, vector or expression vector of the invention.
It will be understood that a nucleic acid will typically only be capable of encoding a polypeptide of the invention when the polypeptide sequence Z consists entirely of the 20 naturally occurring amino acids.
Lipophilic Substituents
As explained above, one or more positions in the formulae Ia, Ib and Ic may independently be replaced by a Lys residue conjugated (i.e. covalently attached) to a lipophilic substituent.
Without wishing to be bound by any particular theory, it is thought that the lipophilic substituent binds albumin in the blood stream, thus shielding the compounds of the invention from enzymatic degradation and thereby enhancing the half-life of the compounds. It may also modulate the potency of the compound, e.g. with respect to the glucagon receptor and/or the GLP-1 receptor.
The positions in question may be any one or more of X8, X10, X11, X13, X14, X16, X19, X20, X24, X26 and (in some formulae) X28. In certain embodiments, only one such position comprises a Lys conjugated to a lipophilic substituent. In other embodiments, two amino acid side chains are each conjugated to a lipophilic substituent. In yet further embodiments, three or even more amino acid side chains are each conjugated to a lipophilic substituent. When a compound contains two or more lipophilic substituents, they may be the same or different. Typically, there are no more than two, or no more than three such positions in any given molecule.
The lipophilic substituent comprises or consists of a lipophilic moiety Z1 which may be covalently bonded directly to an atom in the amino acid side chain, or alternatively may be conjugated to the amino acid side chain by a spacer Z2.
The term “conjugated” is used here to describe the physical attachment of one identifiable chemical moiety to another, and the structural relationship between such moieties. It should not be taken to imply any particular method of synthesis.
The lipophilic moiety may be attached to the amino acid side chain or to the spacer via an ester, a sulphonyl ester, a thioester, an amide, a carbamate, a urea or a sulphonamide. Accordingly it will be understood that preferably the lipophilic substituent includes an acyl group, a sulphonyl group, an N atom, an O atom or an S atom which forms part of the ester, sulphonyl ester, thioester, amide or sulphonamide. Preferably, an acyl group in the lipophilic substituent forms part of an amide or ester with the amino acid side chain or the spacer.
The lipophilic moiety may include a hydrocarbon chain having 4 to 30 C atoms. Preferably it has at least 8 or 12 C atoms, and preferably it has 24 C atoms or fewer, or 20 C atoms or fewer. The hydrocarbon chain may be linear or branched and may be saturated or unsaturated. It will be understood that the hydrocarbon chain is preferably substituted with a moiety which forms part of the attachment to the amino acid side chain or the spacer, for example an acyl group, a sulphonyl group, an N atom, an O atom or an S atom. Most preferably the hydrocarbon chain is substituted with acyl, and accordingly the hydrocarbon chain may be part of an alkanoyl group, for example palmitoyl, caproyl, lauroyl, myristoyl or stearoyl.
Accordingly, the lipophilic moiety may have the formula shown below:




A may be, for example, an acyl group, a sulphonyl group, NH, N-alkyl, an O atom or an S atom, preferably acyl. n is an integer from 3 to 29, preferably from 7 to 25, more preferred 11 to 21, even more preferred 15 to 19.
The hydrocarbon chain may be further substituted. For example, it may be further substituted with up to three substituents selected from NH2, OH and COOH, especially at the free end of the molecule distal from the spacer or peptide. For example, it may comprise a free carboxylic acid group.
If the hydrocarbon chain is further substituted, preferably it is further substituted with only one substituent. Alternatively or additionally, the hydrocarbon chain may include a cycloalkane or heterocycloalkane, for example as shown below:




Preferably the cycloalkane or heterocycloalkane is a six-membered ring. Most preferably, it is piperidine.
Alternatively, the lipophilic moiety may be based on a cyclopentanophenanthrene skeleton, which may be partially or fully unsaturated, or saturated. The carbon atoms in the skeleton each may be substituted with Me or OH. For example, the lipophilic substituent may be cholyl, deoxycholyl or lithocholyl.
As mentioned above, the lipophilic moiety may be conjugated to the amino acid side chain by a spacer. When present, the spacer is attached to the lipophilic moiety and to the amino acid side chain. The spacer may be attached to the lipophilic moiety and to the amino acid side chain independently by an ester, a sulphonyl ester, a thioester, an amide, a carbamate, a urea or a sulphonamide. Accordingly, it may include two moieties independently selected from acyl, sulphonyl, an N atom, an O atom or an S atom. The spacer may have the formula:




wherein B and D are each independently selected from acyl, sulphonyl, NH, N-alkyl, an O atom and an S atom, preferably from acyl and NH. Preferably, n is an integer from 1 to 10, preferably from 1 to 5. The spacer may be further substituted with one or more substituents selected from C0-6 alkyl, C0-6 alkyl amine, C0-6 alkyl hydroxy and C0-6 alkyl carboxy.

Alternatively, the spacer may have two or more repeat units of the formula above. B, D and n are each selected independently for each repeat unit. Adjacent repeat units may be covalently attached to each other via their respective B and D moieties. For example, the B and D moieties of the adjacent repeat units may together form an ester, a sulphonyl ester, a thioester, an amide or a sulphonamide. The free B and D units at each end of the spacer are attached to the amino acid side chain and the lipophilic moiety as described above.
Preferably the spacer has five or fewer, four or fewer or three or fewer repeat units. Most preferably the spacer has two repeat units, or is a single unit.
The spacer (or one or more of the repeat units of the spacer, if it has repeat units) may be, for example, a natural or unnatural amino acid. It will be understood that for amino acids having functionalised side chains, B and/or D may be a moiety within the side chain of the amino acid. The spacer may be any naturally occurring or unnatural amino acid. For example, the spacer (or one or more of the repeat units of the spacer, if it has repeat units) may be Gly, Pro, Ala, Val, Leu, Ile, Met, Cys, Phe, Tyr, Trp, His, Lys, Arg, Gin, Asn, -Glu, -Glu, Asp, Ser Thr, Gaba (gamma-aminobutyric acid), Aib, Bal (also referred to as -Ala), 5-aminopentanoyl, 6-aminohexanoyl, 7-aminoheptanoyl, 8-aminooctanoyl, 9-aminononanoyl or 10-aminodecanoyl.
For example, the spacer may be a single amino acid selected from -Glu, Gaba, Bal and -Glu.
A lipophilic substituent may be conjugated to any amino acid side chain in a compound of the invention. Preferably, the amino acid side chain includes a carboxy, hydroxyl, thiol, amide or amine group, for forming an ester, a sulphonyl ester, a thioester, an amide or a sulphonamide with the spacer or lipophilic substituent. For example, the lipophilic substituent may be conjugated to Asn, Asp, Glu, Gin, His, Lys, Arg, Ser, Thr, Tyr, Trp, Cys or Dbu, Dap or Orn. Preferably, the lipophilic substituent is conjugated to Lys. An amino acid shown as Lys in the formulae provided herein may be replaced by, e.g., Dbu, Dap or Orn where a lipophilic substituent is added.
An example of a lipophilic substituent comprising lipophilic moiety and spacer is shown in the formula below:




Here, a Lys residue in the compound of the present invention is covalently attached to -Glu (the spacer) via an amide moiety. Palmitoyl (i.e. hexadecanoyl) is covalently attached to the -Glu spacer via an amide moiety, thus creating a hexadecanoyl-isoGlu group.
Nucleic Acids and Expression Methods
For recombinant expression, nucleic acids encoding entire peptides conjugates, precursors, or fragments thereof will normally be inserted in suitable vectors to form cloning or expression vectors; such novel vectors are also part of the invention. The vectors can, depending on purpose and type of application, be in the form of plasmids, phages, cosmids, mini-chromosomes, or virus, but also naked DNA which is only expressed transiently in certain cells is an important vector. Preferred cloning and expression vectors (plasmid vectors) of the invention are capable of autonomous replication, thereby enabling high copy-numbers for the purposes of high-level expression or high-level replication for subsequent cloning.
In general outline, an expression vector comprises the following features in the 5′→3′ direction and in operable linkage: a promoter for driving expression of the nucleic acid fragment of the invention, optionally a nucleic acid sequence encoding a leader peptide enabling secretion (to the extracellular phase or, where applicable, into the periplasma), the nucleic acid fragment encoding the peptide of the invention, and optionally a nucleic acid sequence encoding a terminator. They may comprise additional features such as selectable markers and origins of replication. When operating with expression vectors in producer strains or cell lines it may be preferred that the vector is capable of integrating into the host cell genome. The skilled person is very familiar with suitable vectors and is able to design one according to their specific requirements.
The vectors are used to transform host cells to produce the required compound. Such transformed cells can be cultured cells or cell lines used for propagation of the nucleic acid fragments and vectors, or used for recombinant production of the peptides.
Preferred host cells are micro-organisms such as bacteria [such as the species Escherichia (e.g. E. coli), Bacillus (e.g. Bacillus subtilis), Salmonella, or Mycobacterium (preferably non-pathogenic, e.g. M. bovis BCG), yeasts (e.g., Saccharomyces cerevisiae and Pichia pastoris), and protozoans. Alternatively, the transformed cells may be derived from a multicellular organism, i.e. it may be fungal cell, an insect cell, an algal cell, a plant cell, or an animal cell such as a mammalian cell. For the purposes of cloning and/or optimised expression it is preferred that the transformed cell is capable of replicating the encoding nucleic acid. Cells expressing the nucleic fragment are useful embodiments of the invention; they can be used for small-scale or large-scale preparation of the peptides of the invention.
When producing peptides of the invention or fragments or precursors thereof by means of transformed cells, it is convenient, although far from essential, that the expression product is secreted into the culture medium.
Therapeutic Uses
The peptide compounds of the invention have the surprising benefit of a significantly longer half-life as compared to a native human ghrelin polypeptide, and exhibit reduced degradation as compared to a native human ghrelin polypeptide. Thus, the peptide compounds of the invention are well suited for use in the treatment of a variety of conditions, diseases, disorders, and syndromes.
Uses of the peptide compounds of the invention also encompass uses of pharmaceutically acceptable salts or solvates thereof.
The compounds of the invention may provide an attractive treatment option for, inter alia, diseases characterized, in whole or in part, by loss of body weight.
Cachexia is characterized by loss of body weight, loss of appetite, and wasting in an individual. Individuals affected by cachexia also experience fatigue, muscle atrophy, and weakness. Cachexia often is associated with diseases and conditions such as cancer, AIDS, physical trauma, chronic kidney disease and heart failure. Although cachexia represents a serious health problem, its underlying causes are not well-known. However, ghrelin is thought to play a role in the development of cachexia.
The compounds of the present invention may therefore be used as pharmaceutical agents for treating or preventing cachexia, promoting weight gain, reducing weight loss (e.g. by increasing appetite, food intake, and calorie intake), as well as associated diseases and health conditions. For example, the compounds of the invention may be used to treat low or loss of body weight, low or loss of body fat, low or loss of muscle mass, loss of appetite, decreased food intake, anorexia, bulimia, or the inability to gain weight or increase body mass in an individual that has been gastrectomized and/or vagectomized. The compounds of the invention may also be used for treatment of chronic obstructive pulmonary disease, growth hormone deficiency, osteoporosis, cardiovascular indications (e.g., low blood flow, low cardiac output, and high blood pressure), arthritis pain, inflammatory disorders (e.g., arthritis and systemic lupus erythematosus), and gastrointestinal disorders including but not limited to gastroparesis, post-operative ileus, inflammatory disorders such as colitis (e.g., ulcerative colitis), gut barrier dysfunction, inflammatory bowel disease (IBD), Crohn's disease, and ischemia reperfusion injury.
Thus, the invention provides use of a compound of the invention in the treatment of a condition as described above, in an individual in need thereof.
The invention also provides a compound of the invention for use in a method of medical treatment, particularly for use in a method of treatment of a condition as described above.
In preferred embodiments, the compounds described may be used in preventing weight loss or promoting weight gain.
In specific embodiments, the present invention comprises use of a compound of the invention for preventing weight loss or promoting weight gain in an individual in need thereof.
In specific embodiments, the present invention comprises use of a compound of the invention in a method of treatment of a condition caused or characterized by loss of body weight, e.g. the treatment and/or prevention of cachexia or wasting (for example in patients with burns, AIDS, kidney failure or sepsis), or treatment and/or prevention of the effects of gastrectomy, vagectomy, anorexia (e.g. anorexia nervosa) or bulimia, in an individual in need thereof.
In some embodiments, the present invention comprises use of a compound of the invention for treating chronic obstructive pulmonary disease.
In some embodiments, a compound of the invention may be used in promoting the production of growth hormone.
In specific embodiments, the present invention comprises use of a compound of the invention for promoting the production of growth hormone in an individual in need thereof.
In specific embodiments, the present invention comprises use of a compound of the invention in a method of treatment of a condition caused or characterized by low production of growth hormone.
In some embodiments, a compound of the invention may be used in promoting an increase in blood flow or cardiac output.
In specific embodiments, the present invention comprises use of a compound of the invention for promoting an increase in blood flow or cardiac output in an individual in need thereof.
In specific embodiments, the present invention comprises use of a compound of the invention in a method of treatment of a condition caused or characterized by low blood flow or cardiac output, e.g., low cardiac output syndrome or poor circulation.
In some embodiments, a compound of the invention may be used in treating post-operative ileus.
In some embodiments, a compound of the invention may be used in increasing gastrointestinal motility.
In specific embodiments, the present invention comprises use of a compound of the invention for increasing gastrointestinal motility in an individual in need thereof.
In specific embodiments, the present invention comprises use of a compound of the invention in a method of treatment of a condition caused or characterized by low gastrointestinal motility, e.g., gastroparesis.
In some embodiments, a compound of the invention may be used in reducing inflammation.
In specific embodiments, the present invention comprises use of a compound of the invention for reducing inflammation in an individual in need thereof.
In specific embodiments, the present invention comprises use of a compound of the invention in a method of treatment of a condition caused or characterized by inflammation, e.g., arthritis, systemic lupus erythematosus, colitis (e.g., ulcerative colitis), gut barrier dysfunction, inflammatory bowel disease (IBS), Crohn's disease, and ischemia reperfusion injury.
Exemplary effects of treatment using the compounds of the invention include preventing weight loss, promoting weight gain, treating obesity (e.g. by increasing appetite, food intake, and calorie intake), increasing production of growth hormone, increasing blood flow, increasing cardiac output, decreasing blood pressure, increasing gastrointestinal motility, and decreasing inflammation.
In some embodiments, the present invention comprises a pharmaceutical composition or method for suppressing or treating spinal nerve degeneration, damage, or cell death.
In some embodiments, the present invention comprises a pharmaceutical composition or method for promoting spinal nerve regeneration, proliferation or repair.
In some embodiments, the present invention comprises a pharmaceutical composition or method for suppressing or treating hyperglycemia.
In some embodiments, the present invention comprises a pharmaceutical composition or method for promoting insulin production in pancreatic beta cells.
In some embodiments, the present invention comprises a pharmaceutical composition or method for suppressing degeneration or disease of pancreatic beta cells.
In some embodiments, the present invention comprises a pharmaceutical composition or method for promoting health, longevity, neogenesis, or regeneration of pancreatic beta cells.
Pharmaceutical Compositions
The inclusion of one or more of a peptide compound of the invention in a pharmaceutical composition also encompasses inclusion of a pharmaceutically acceptable salt or solvate of a peptide compound of the invention.
The peptide compounds of the present invention may be formulated as pharmaceutical compositions which are suited for administration with or without storage, and which typically comprise a therapeutically effective amount of at least one peptide compound of the invention, together with a pharmaceutically acceptable carrier, excipient or vehicle.
The term “pharmaceutically acceptable carrier” includes any of the standard pharmaceutical carriers. Pharmaceutically acceptable carriers for therapeutic use are well known in the pharmaceutical art and are described, for example, in “Remington's Pharmaceutical Sciences”, 17th edition, Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, Pa., USA, 1985. For example, sterile saline and phosphate-buffered saline at slightly acidic or physiological pH may be used. Suitable pH-buffering agents may, e.g., be phosphate, citrate, acetate, tris(hydroxymethyl)aminomethane (TRIS), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), ammonium bicarbonate, diethanolamine, histidine, arginine, lysine or acetate (e.g. as sodium acetate), or mixtures thereof. The term further encompasses any carrier agents listed in the US Pharmacopeia for use in animals, including humans.
A pharmaceutical composition of the invention may be in unit dosage form. In such form, the composition is divided into unit doses containing appropriate quantities of the active component or components. The unit dosage form may be presented as a packaged preparation, the package containing discrete quantities of the preparation, for example, packaged tablets, capsules or powders in vials or ampoules. The unit dosage form may also be, e.g., a capsule, cachet or tablet in itself, or it may be an appropriate number of any of these packaged forms. A unit dosage form may also be provided in single-dose injectable form, for example in the form of a pen device containing a liquid-phase (typically aqueous) composition. Compositions may be formulated for any suitable route and means of administration. Pharmaceutically acceptable carriers or diluents include those used in formulations suitable for e.g. oral, intraviteral, rectal, vaginal, nasal, topical, enteral or parenteral (including subcutaneous (SC), intramuscular (IM), intravenous (IV), intradermal and transdermal) administration or administration by inhalation. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmaceutical formulation.
Further embodiments of the invention relate to devices, dosage forms and packages used to deliver the pharmaceutical formulations of the present invention. Thus, at least one peptide compound or specified portion or variant in either the stable or preserved formulations or solutions described herein, can be administered to a patient in accordance with the present invention via a variety of delivery methods including SC or IM injection; transdermal, pulmonary, transmucosal, implant, osmotic pump, cartridge, micro pump, or other means appreciated by the skilled artisan, as well-known in the art.
Still further embodiments of the invention relate to oral formulations and administration. Formulations for oral administration may rely on the co-administration of adjuvants (e.g., resorcinols and nonionic surfactants such as polyoxyethylene oleyl ether and n-hexadecylpolyethylene ether) to artificially in-crease the permeability of the intestinal walls, as well as the co-administration of enzymatic inhibitors (e.g., pancreatic trypsin inhibitors, diisopropylfluorophosphate (DFF) and trasylol) to inhibit enzymatic degradation. The active constituent compound of the solid-type dosage form for oral administration can be mixed with at least one additive, including sucrose, lactose, cellulose, mannitol, trehalose, raffinose, maltitol, dextran, starches, agar, arginates, chitins, chitosans, pectins, gum tragacanth, gum arabic, gelatin, collagen, casein, albumin, synthetic or semisynthetic polymer, and glyceride. These dosage forms can also contain other type(s) of additives, e.g., inactive diluting agent, lubricant such as magnesium stearate, paraben, preserving agent such as sorbic acid, ascorbic acid, alpha-tocopherol, antioxidants such as cysteine, disintegrators, binders, thickeners, buffering agents, pH adjusting agents, sweetening agents, flavoring agents, and perfuming agents.
Dosages
A typical dosage of a peptide compound of the invention as employed in the context of the present invention may be in the range from about 0.001-1,000 mg/subject per day, such as about 0.001 mg/subject, 0.01 mg/subject, 0.1 mg/subject, 1 mg/subject, 10 mg/subject, 100 mg/subject, and 1,000 mg/subject per day, administered in one or more doses, such as from one to three doses. As already indicated to some extent above, the exact dosage employed will depend, inter alia, on: the nature and severity of the disease or disorder to be treated; the sex, age, body weight and general condition of the subject to be treated; possible other, concomitant disease or disorder that is undergoing or is to undergo treatment; as well as other factors that will be known to a medical practitioner of skill in the art.
A peptide compound of the invention may be administered continuously (e.g., by intravenous administration or another continuous drug administration method), or may be administered to a subject in intervals, typically in regular time intervals, depending on the desired dosage and the pharmaceutical composition selected by the skilled practitioner for the particular subject. Regular administration dosing intervals include, e.g., once daily, twice daily, once every two, three, four, five or six days, once or twice weekly, once or twice monthly, and the like. Such regular peptide compound administration regimens of the invention may, in certain circumstances such as, e.g., during chronic long term administration, be advantageously interrupted for an interval of time so that the medicated subject reduces the level of or stops taking the medication, often referred to as taking a “drug holiday.” Drug holidays are useful for, e.g., maintaining or regaining sensitivity to a drug especially during long term chronic treatment, or, to reduce unwanted side-effects of long term chronic treatment of the subject with the drug. The timing of a drug holiday depends on the timing of the regular dosing regimen and the purpose for taking the drug holiday (e.g., to regain drug sensitivity and/or to reduce unwanted side effects of continuous, long term administration). In some embodiments, the drug holiday may be a reduction in the drug (e.g., below the therapeutically effective amount for a certain interval of time). In other embodiments, administration of the drug is stopped for a certain interval of time before administration is started again, at the same or at a different dosing regimen (e.g., at a lower or higher dose and/or frequency of administration). A drug holiday of the invention may thus be selected from a wide range of times and dosage regimens. An exemplary drug holiday is two or more days, one or more weeks, one or more months, up to about 24 months of drug holiday. So, for example, a regular daily dosing regimen with a peptide compound of the invention may be interrupted by a drug holiday of a week, two weeks, or four weeks, after which time a daily or a weekly dosing schedule is resumed. A variety of other drug holiday regimens are envisioned to be useful for administering the peptide compounds of the invention.
Combination Therapy
As noted above, it will be understood that reference in the following to a peptide compound of the invention also extends to a pharmaceutically acceptable salt or solvate thereof as well as to a composition comprising more than one different peptide compound of the invention.
A peptide compound of the invention may be administered as part of a combination therapy together with another active agent for the treatment of the disease or disorder in question, e.g., cachexia, and in such cases, the two active agents may be given together or separately, e.g. as constituents in the same pharmaceutical composition or formulation, or as separate formulations.
A peptide compound of the invention may further be used in combination with an anti-hypertension agent of a known type, including, but not limited to, an angiotensin-converting enzyme inhibitor, an angiotensin II receptor blocker, a diuretic, a beta-blocker and a calcium channel blocker.
A peptide compound of the invention may still further be used in combination with an anti-dyslipidemia agent of known type, including, but not limited to, a statin, a fibrate, a niacin and/or a cholesterol absorption inhibitor.
A peptide compound of the invention may also be used in combination with a proton pump inhibitor (i.e., a pharmaceutical agent possessing pharmacological activity as an inhibitor H+/K+-ATPase) of known type, including, but not limited to, an agent of the benzimidazole derivative type or of the imidazopyridine derivative type, such as OmeprazoleTM, LansoprazoleTM, DexlansoprazoleTM, EsomeprazoleTM, PantoprazoleTM, RabeprazoleTM, ZolpidemTM, AlpidemTM, SaripidemTM or NecopidemTM.
A peptide compound of the invention may also be used in combination with a pharmaceutical used to treat impaired gastrointestinal motility of known type, including, but not limited to, Leul3-motilin, prostaglandin F2 alpha, Cisapride®, Propulsid®, metoclopramide, domperidone, ondansetron, tropisetron, mosapride and itopride.
A peptide compound of the invention may also be used in combination with, for example, adenosine-antagonizing pyrazolopyridine compounds, pituitary adenylate cyclase activating peptide (PACAP) receptor antagonist, fedotozine; neuropeptides, and proteinase-activated receptor-2 antagonists (U.S. Pat. No. 5,929,035).
A peptide compound of the invention may, moreover, be used in combination with an anti-inflammatory agent of known type, including, but not limited to: steroids and corticosteroids, such as beclomethasone, methylprednisolone, betamethasone, prednisone, dexamethasone, and hydrocortisone; non-steroidal antiinflammatory agents (NSAIDs), such as propionic acid derivatives (e.g. alminoprofen, benoxaprofen, bucloxic acid, carprofen, fenbufen, fenoprofen, fluprofen, flurbiprofen, ibuprofen, indoprofen, ketoprofen, miroprofen, naproxen, oxaprozin, pirprofen, pranoprofen, suprofen, tiaprofenic acid and tioxaprofen); acetic acid derivatives (e.g. indomethacin, acemetacin, alclofenac, clidanac, diclofenac, fenclofenac, fenclozic acid, fentiazac, furofenac, ibufenac, isoxepac, oxpinac, sulindac, tiopinac, tolmetin, zidometacin and zomepirac); fenamic acid derivatives (e.g. flufenamic acid, meclofenamic acid, mefenamic acid, niflumic acid and tolfenamic acid); biphenylcarboxylic acid derivatives (e.g. diflunisal and flufenisal); oxicams (e.g. isoxicam, piroxicam, sudoxicam and tenoxicam); salicylates (e.g. acetylsalicylic acid and sulfasalazine); and pyrazolones (e.g. apazone, bezpiperylon, feprazone, mofebutazone, oxyphenbutazone and phenylbutazone); COX II inhibitors, such as rofecoxib and celecoxib; preparations of interferon beta (e.g. interferon beta-1a or interferon beta-1b); and certain other compounds, such as 5-aminosalicylic acid and prodrugs and pharmaceutically acceptable salts thereof.
Each of the embodiments of the invention may be combined individually or in combination with one or more other embodiments of the invention.
Each of the compounds of the invention comprises, consists essentially of, or consists of a sequence as described herein.
EXAMPLES
Abbreviations employed in the following are as follows:

    BSA: bovine serum albumin
    COMU: 1-[(1-(cyano-2-ethoxy-2-oxoethylideneaminooxy)-dimethylamino-morpholinomethylene)]methanaminium hexafluorophosphate
    DCM: dichloromethane
    DMEM: Dulbecco's Modified Eagle's Medium
    DMF: N,N-dimethylformamide
    DMSO: dimethylsulfoxide
    DIPEA: diisopropylethylamine
    EtOH: ethanol
    Et2O: diethyl ether
    Fmoc: 9-fluorenylmethoxycarbonyl
    HATU: N-[(dimethylamino)-1H-1,2,3-triazol[4,5-b]pyridine-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide
    HEPES: 4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid
    HPLC: high-performance liquid chromatography
    MeCN: acetonitrile
    MS: mass spectroscopy
    NEP: N-ethylpyrrolidone
    NMP: N-methylpyrrolidone
    PBS: phosphate-buffered saline
    PCR: polymerase chain reaction
    RP: reverse phase
    TFA: trifluoroacetic acid
    TIS: triisopropylsilane

General Synthesis Procedure for Ghrelin Analogues



Solid-phase peptide synthesis (SPPS) was performed on a microwave assisted synthesizer using standard Fmoc strategy in NMP or NEP on a polystyrene resin [such as TentaGel S PHB-Arg(Pbf)]. HATU or COMU was used as coupling reagent together with DIPEA as base. Piperidine (20% in NEP or DMF) was used for deprotection. Pseudoproline: Fmoc-Glu(OtBu)-Ser(Psi. Me, Me Pro)-OH (purchased from NovaBiochem) was used where applicable. Human ghrelin and rat ghrelin were likewise synthesized and purified using synthesis methodology and purification procedures as described herein.
Cleavage:
The crude peptide was cleaved from the resin by treatment with 95/2.5/2.5% (v/v) TFA/TIS/water at room temperature for 2 h. Most of the TFA was removed at reduced pressure, and the crude peptide was precipitated and washed with diethyl ether and allowed to dry at ambient temperature.
Peptide Purification
The crude peptides were purified by standard RP HPLC with a gradient of buffer A (0.1% aqueous TFA) and buffer B (aqueous solution containing 0.1% TFA and 90% MeCN). Fractions were analysed by analytical HPLC and MS, and relevant fractions were pooled and lyophilised.
The peptides were further purified by preparative RP HPLC using a gradient of buffer A′ (0.1% aqueous TFA) and buffer B′ (aqueous solution containing 0.1% TFA and 90% MeCN). The final product was characterised by analytical HPLC and MS.
Analytical HPLC Method
The peptides were analyzed by an analytical HPLC method using a gradient of buffer A′ (see above) and buffer B′ (see above).
Example 1
Synthesis of H-PS-S(O-octyl)-FLSPEHQRVQQRKESKKPPAKLQPR-OH (Compound No. 19) (SEQ ID NO: 21)
TentaGel S PHB Fmoc-Arg(Pbf) resin (1.30 g; 0.326 mmol; Rapp Polymere) was treated with 20% piperidine in NEP for initial deprotection. A temperature increase to approx. 40° C. within 30 seconds was observed. The reaction vessel was drained and a second portion of 20% piperidine in NEP was added. This round of deprotection took place for 3 minutes, keeping the temperature between 70 and 75° C. The resin was then subjected to repeated introduction of Fmoc-amino acid by COMU/DIPEA, and elimination of Fmoc by 20% piperidine in NEP.
Coupling Step:
the mixture was heated to 75° C. and then allowed to cool down. This heating and cooling cycle was repeated over a period of 5 minutes. During the coupling, nitrogen was bubbled through the reaction vessel.
Fmoc Deprotection Step:
20% piperidine in NEP was added for initial deprotection. A temperature increase to approx. 40° C. within 30 seconds was observed. The reaction vessel was drained and a second portion of 20% piperidine in NEP was added. This round of deprotection took place for 3 minutes, keeping the temperature between 70 and 75° C.
In this sequential manner, the sidechain-protected peptide was assembled on the resin. The O-octyl-derivatised serine residue (at position 3) was introduced using the building block Fmoc-L-Ser(O-octyl)-OH (obtained from WuXi AppTec, China). Pseudoproline used: Fmoc-Glu(OtBu)-Ser(Psi. Me, Me Pro)-OH.
The dried peptide-containing resin (694 mg) was subjected to cleavage using 20 ml of the following mixture: TFA:TIS:water (95:2.5:2.5). After keeping the resin in contact with the cleavage mixture for 2 hours it was removed by filtration. The filtrate was concentrated in vacuo, and diethyl ether was added. The resulting precipitate was recovered by centrifugation and allowed to dry at room temperature, giving the crude peptide (450 mg).
The crude peptide was then dissolved in 10 ml buffer A and applied to a Gemini NX AXIA column (C18; 50 mm×250 mm; 110 Å, 10 micron). The column was eluted with a linear gradient of 95-85% buffer A for the first 3 minutes, and then with 85-60% buffer A for 47 minutes. Flow rate: 35 ml/min. The desired fractions were collected and lyophilized to give approx. 91 mg of the desired compound. Molecular mass determined by MS: 3394.93 Da (theoretical: 3394.93 Da).
Example 2
Synthesis of H-PS-S(O-octanoyl)-FLSPEHQRVQQRKESKKPPAKLQPR-OH (Compound No. 5) (SEQ ID NO: 7)
TentaGel S PHB Fmoc-Arg(Pbf) resin (3.759 g, divided into three portions which were run in parallel; 0.940 mmol; Rapp Polymere) was treated with 20% piperidine in NEP for initial deprotection. A temperature increase to approx. 40° C. within 30 seconds was observed. The reaction vessel was drained and a second portion of 20% piperidine in NEP was added. This round of deprotection took place for 3 minutes, keeping the temperature between 70 and 75° C. The resin was then subjected to repeated introduction of Fmoc-amino acid by COMU/DIPEA, and elimination of Fmoc by 20% piperidine in NEP.
Coupling Step:
the mixture was heated to 75° C. and then allowed to cool down. This heating and cooling cycle was repeated over a period of 5 minutes. During the coupling, nitrogen was bubbled through the reaction vessel.
Fmoc Deprotection Step:
20% piperidine in NEP was added for initial deprotection. A temperature increase to approx. 40° C. within 30 seconds was observed. The reaction vessel was drained and a second portion of 20% piperidine in NEP was added. This round of deprotection took place for 3 minutes, keeping the temperature between 70 and 75° C. In this sequential manner, the 25 first residues of the sidechain-protected peptide were assembled on the resin. The synthesis was then continued using approx. one-sixth of the total amount of this peptide resin. The O-octanoyl-derivatised serine residue (at position 3) was introduced using the building block Fmoc-L-Ser(O-octanoyl)-OH (obtained from WuXi AppTec, China).
The dried peptide-containing resin (1419 mg) was subjected to cleavage using 40 ml of TFA:TIS:water (95:2.5:2.5). After keeping the resin in contact with the cleavage mixture for 2 hours it was removed by filtration. The filtrate was concentrated in vacuo, and diethyl ether was added. The resulting precipitate was recovered by centrifugation and allowed to dry at room temperature, giving the crude peptide (600 mg).
The crude peptide was then dissolved in 10 ml buffer A and applied to a Gemini NX AXIA column (C18; 50 mm×250 mm; 110 Å, 10 micron). The column was eluted with a linear gradient of 95-85% buffer A for the first 3 minutes, and then with 85-60% buffer A for 47 minutes. Flow rate: 35 ml/min. The desired fractions were collected and lyophilized to give approx. 61 mg of the desired compound. Molecular mass determined by MS: 3408.91 Da (theoretical: 3408.90 Da)
Example 3
Ghrelin Receptor Functional Assay
To test activation of the ghrelin receptor, stable cell lines expressing the human ghrelin receptor were produced. In brief, HEK293 cells were used for transfection of the cDNA for human ghrelin receptor cloned into the transfection plasmid pIRESneo2dNGFR. More specifically, the cDNA encoding the human growth hormone secretagogue receptor type 1a (GHSR1a; ghrelin receptor) was cloned from the cDNA clone AY429112 (accession no. AY429112.1; GI:38016896) (SEQ ID NO: 6). The DNA encoding the ghrelin receptor was amplified by PCR using primers also encoding terminal restriction sites for subcloning. The ghrelin receptor was subcloned into a mammalian expression vector containing a neomycin (G418) resistance marker. The fidelity of the DNA encoding the receptor was confirmed by DNA sequencing. HEK293 cells were used for transfection of the ghrelin receptor expression plasmid. The cells were grown according to standard protocol in growth medium and transfected with the plasmids using Lipofectamin (Invitrogen) according to the manufacturer's protocol. The cells stably expressing ghrelin receptor were selected using G418 in the growth medium (in that only cells that have taken up and incorporated the cDNA expression plasmid survive). The polyclonal cell line was propagated and stocks of cells were frozen for later use.
In vitro effects of compounds of the invention were examined by measuring Ca2+ induction (using Fluo-4 DirectTM Calcium Assay Kit, Invitrogen F10471) in the HEK293 cell line stably expressing the human ghrelin receptor. The cells were seeded in Pre-coated Poly-L-lysine 96-well tissue culture plates with 20000 cells per well in 100 μl of standard growth medium (DMEM with Glutamax-I containing (Invitrogen 61965), 10% v/v FBS, Penicillin (100 IU/ml), Streptomycin (100 μg/ml), 1 mM sodium pyruvate, 1× non-essential amino acids (Invitrogen 11140) and 1% G418 (Gibco 10131-027). The cells were placed in an incubator (37° C., 5% CO2) until the next day.
Fluo-4 Mix was prepared by mixing 10 ml of phenol-free DMEM with 10 ml 2× Fluo-4 DirectTM Calcium Reagent Loading Solution. Medium was removed from the cells and replaced with 100 μl of pre-warmed (37° C.) Fluo-4 Mix, and the cells were then placed in the incubator for 30 minutes. Peptides were dissolved to 1 mM in DMSO and diluted to 2× final concentration in modified peptide buffer (0.2% w/v BSA and 2 mM HEPES in PBS, pH 7.4.) in a 96-well plate. Positive control wells contained 200 nM human ghrelin (100 nM final) and negative control wells contained buffer with no peptide.
The plate with test compounds was pre-warmed before starting the measurements. The cell plate and compound plate were placed in a FLIPR instrument (Molecular Devices), and the program was initiated. The program measures basal level of fluorescence of Fluo-4 for 10 seconds, adds 100 μl of peptide solution to the cells and measures the change in fluorescence as a consequence of the changes in Ca2+ concentration in the cells. The output used for calculation of activity was selected to be the maximal response (expressed as a ratio to the basal level. EC50 values) and maximal response values were determined based on these values.







TABLE 1







EC50 results for test compounds









Compound

EC50


No.
Sequence
(nM)












 1*
H-GS-S(O-octanoyl)-FLSPEHQRVQQRKESKKPPAKLQPR-OH (SEQ
0.307



ID NO: 1)



 


 2*
H-GSSFLSPEHQRVQQRKESKKPPAKLQPR-OH (SEQ ID NO: 1)
>1,000


 


 3*
H-GS-S(O-octanoyl)-FLSPEHQKAQQRKESKKPPAKLQPR-OH
0.204



(SEQ ID NO: 195)



 


 4*
H-GSSFLSPEHQKAQQRKESKKPPAKLQPR-OH (SEQ ID NO: 195)
>1,000


 


 5
H-PS-S(O-octanoyl)-FLSPEHQRVQQRKESKKPPAKLQPR-OH
0.298



 (SEQ ID NO: 7)



 


 6
H-[DPro]-S-S(O-octanoyl)-FLSPEHQRVQQRKESKKPPAKLQPR-OH
0.786



(SEQ ID NO: 8)



 


 7
H-FS-S(O-octanoyl)-FLSPEHQRVQQRKESKKPPAKLQPR-OH
0.714



(SEQ ID NO: 9)



 


 8
H-SS-S(O-octanoyl)-FLSPEHQRVQQRKESKKPPAKLQPR-OH
0.385



(SEQ ID NO: 10)



 


 9
H-Cmp-S(O-octanoyl)-FLSPEHQRVQQRKESKKPPAKLQPR-OH
0.558



(SEQ ID NO: 11)



 


10
H-Mamb-S(O-octanoyl)-FLSPEHQRVQQRKESKKPPAKLQPR-OH
1.56



(SEQ ID NO: 12)



 


11
H-[cACC]-S(O-octanoyl)-FLSPEHQRVQQRKESKKPPAKLQPR-OH
0.379



(SEQ ID NO: 13)



 


12
H-[tACC]-S(O-octanoyl)-FLSPEHQRVQQRKESKKPPAKLQPR-OH
1.89



(SEQ ID NO: 14)



 


13
H-Inp-S-S(O-octyl)-FLSPEHQRV-K(hexadecanoyl)-
2.55



QRKESKKPPAKLQPR-OH (SEQ ID NO: 15)



 


14
H-Inp-S-S(O-octyl)-FLSPEHQRVQQRKES-K(hexadecanoyl)-
2.52



KPPAKLQPR-OH (SEQ ID NO: 16)



 


15
H-Inp-S-S(O-octyl)-FLSPEHQRVQQRKESKKPPAKL-K
1.18



(hexadecanoyl)-PR-OH (SEQ ID NO: 17)



 


16
H-PS-S(O-octyl)-FLSPEHQRVQQRKES-K(hexadecanoyl)-
2.88



KPPAKLQPR-OH (SEQ ID NO: 18)



 


17
H-[DPro]-S-S(O-octyl)-FLSPEHQRVQQRKES-K(hexadecanoyl)-
4.63



KPPAKLQPR-OH (SEQ ID NO: 19)



 


18
H-Aib-S-S(O-octyl)-FLSPEHQRVQQRKES-K(hexadecanoyl)-
2.9



KPPAKLQPR-OH (SEQ ID NO: 20)



 


19
H-PS-S(O-octyl)-FLSPEHQRVQQRKESKKPPAKLQPR-OH
0.579



(SEQ ID NO: 21)



 


20
H-[DPro]-S-S(O-octyl)-FLSPEHQRVQQRKESKKPPAKLQPR-OH
0.77



(SEQ ID NO: 22)



 


21
H-Aib-S-S(O-octyl)-FLSPEHQRVQQRKESKKPPAKLQPR-OH
0.46



(SEQ ID NO: 23)



 


22
H-PS-Dap(N-octanoyl)-FLSPEHQRVQQRKESKKPPAKLQPR-NH2
0.193



(SEQ ID NO: 74)



 


23
H-PS-Dap(N-octanoyl)-FLSPEHSRVSSRKESKKPPAKLSPR-NH2
0.224



(SEQ ID NO: 82)



 


24
H-PS-Dap(N-octanoyl)-FLSPEHQRVQQRKESKKPPA-NH2
0.136



(SEQ ID NO: 139)



 


25
H-PS-Dap(N-octanoyl)-FLSP-NH2 (SEQ ID NO: 155)
0.26


 


26
H-PS-Dap(N-octanoyl)-FLS-NH2 (SEQ ID NO: 156)
1.2


 


27
H-PS-Dap(N-octanoyl)-FL-NH2 (SEQ ID NO: 157)
1.69


 


28
H-PS-[NH-CH(n-C10H21)-C(O)]-FLSPEHQRVQQRKES-NH2
0.164



(SEQ ID NO: 181)



 


29
H-PS-[NH-CH(n-C10H21)-C(O)]-FLSPEHQRVQQRKESKKPPA-NH2
0.255



(SEQ ID NO: 176)



 


30
H-GS-S(octanoyl)-FLSPEOHQRKOQQRKESKKPPAKLQPR-OH
0.534



(SEQ ID NO: 94)



 


31
H-GS-S(octanoyl)-FLSPEHEURVQKORKESKKPPAKLQPR-OH
0.597



(SEQ ID NO: 95)



 


32
H-GS-S(octanoyl)-FLSPEHQRVQQRKOESKEOPPAKLQPR-OH
1.29



(SEQ ID NO: 96)



 


33
H-PS-S(O-octyl)-FLSPEOHQRKOQQRKESKKPPAKLQPR-NH2
0.365



(SEQ ID NO: 97)



 


34
H-PS-S(O-octyl)-FLSPEHEURVQKORKESKKPPAKLQPR-NH2
0.155



(SEQ ID NO: 98)



 


35
H-Orn-S(O-octyl)-FLSPEHQRVQQRKESKKPPAKLQPR-NH2
0.527



(SEQ ID NO: 99)



 


36
Ac-Orn-S(O-octyl)-FLSPEHQRVQQRKESKKPPAKLQPR-NH2
1.4



(SEQ ID NO: 99)



 


37
H-PS-Dap(N-octanoyl)-FLSPEHQRVQRKESKKPPAKLQPR-NH2
0.253



(SEQ ID NO: 100)



 


38
H-PS-S(O-octyl)-FLSPEHQRVQRKESKKPPAKLQPR-NH2
0.33



(SEQ ID NO: 101)



 


39
H-PS-Dap(N-octanoyl)-FLSPK-NH2 (SEQ ID NO: 102)
0.364


 


40
H-PS-S(O-octyl)-FLSPK-NH2 (SEQ ID NO: 103)
0.388


 


41
H-PS-[NH-CH(n-C10H21)-C(O)]-R-SPK-NH2 (SEQ ID NO: 104)
0.432


 


42
H-PS-Dap(N-octanoyl)-FLSPY-NH2 (SEQ ID NO: 105)
0.195


 


43
H-PS-S(O-octyl)-FLSPEHQRVQQRKES-K(hexadecanoyl-
0.496



isoGlu)-KPPAKLQPR-NH2 (SEQ ID NO: 106)



 


44
H-PS-Dap(N-octanoyl)-FLSPEHSRVSSRKES-K(hexadecanoyl-
0.566



isoGlu)-KPPA-NH2 (SEQ ID NO: 107)



 


45
H-PS-S(O-octyl)-FLSPEHSRVSSRKESSKPPAKLSPR-NH2
0.359



(SEQ ID NO: 108)



 


46
H-PS-S(O-octyl)-FLSPEHSRVSSRKESKSPPAKLSPR-NH2
0.296



(SEQ ID NO: 109)



 


47
H-PS-S(O-octyl)-FLSPEHSRVSSRKESSSPPAKLSPR-NH2
0.454



(SEQ ID NO: 110)



 


48
H-PS-S(O-octyl)-FLSPEHSRPSSR-NH2 (SEQ ID NO: 111)
0.341


 


49
H-PS-S(O-octyl)-FLSPEHSRVSSRPESSSPPAKLSPR-NH2
0.525



(SEQ ID NO: 112)



 


50
H-PS-S(O-octyl)-FLSPEHSRPSSRPESSSPPAKLSPR-NH2
0.642



(SEQ ID NO: 113)



 


51
H-PS-S(octanoyl)-FLSPEHQSVQQRKESKKPPAKLQPR-NH2
0.149



(SEQ ID NO: 114)



 


52
H-PS-S(octanoyl)-FLSPEHQRVQQSKESKKPPAKLQPR-NH2
0.168



(SEQ ID NO: 115)



 


53
H-PS-S(octanoyl)-FLSPEHQRVQQRSESKKPPAKLQPR-NH2
0.182



(SEQ ID NO: 116)



 


54
H-PS-S(octanoyl)-FLSPEHQRVQQRKESSKPPAKLQPR-NH2
0.095



(SEQ ID NO: 117)



 


55
H-PS-S(octanoyl)-FLSPEHQRVQQRKESKSPPAKLQPR-NH2
0.086



(SEQ ID NO: 118)



 


56
H-PS-S(octanoyl)-FLSPEHQRVQQRKESKKPPASLQPR-NH2
0.183



(SEQ ID NO: 119)



 


57
H-PS-S(octanoyl)-FLSPEHQRVQQRKESKKPPAKLQPS-NH2
0.039



(SEQ ID NO: 120)



 


58
H-PS-S(octanoyl)-FLSPYHQRVQQRKESKKPPAKLQPR-NH2
0.719



(SEQ ID NO: 121)



 


59
H-PS-S(octanoyl)-FLSPEHQRVQYRKESKKPPAKLQPR-NH2
0.198



(SEQ ID NO: 122)



 


60
H-PS-S(octanoyl)-FLSPEHQRVQQRKESKKPPAYLQPR-NH2
0.252



(SEQ ID NO: 123)



 


61
H-PS-S(octanoyl)-FLSPYHQRVQYRKESKKPPAYLQPR-NH2
0.594



(SEQ ID NO: 124)



 


62
H-PS-S(O-octyl)-FLSPEHQRVQQRKESKKPPAKLQPR-NH2
0.396



(SEQ ID NO: 125)



 


63
H-PS-S(O-octyl)-FLSPEHQRVQRKESKKPPAKLQPR-NH2
0.33



(SEQ ID NO: 126)



 


64
H-PS-S(O-octyl)-FLSPEHQRVQQRKESKKPPA-NH2
0.231



(SEQ ID NO: 127)



 


65
H-PS-S(O-octyl)-FLSPEHQRVQQRKES-NH2 (SEQ ID NO: 128)
0.221


 


66
H-PS-[NH-CH(n-C10H21)-C(O)]-FLSPEHQRVQQRKESKKPPAKLQPR-
0.333



NH2 (SEQ ID NO: 129)



 


67
H-PS-[NH-CH(n-C10H21)-C(O)]-FLSPEHQRVQRKESKKPPAKLQPR-
0.23



NH2 (SEQ ID NO: 130)



 


68
H[cACC]-S(O-octyl)-FLSPEHQRVQQRKESKKPPAKLQPR-NH2
0.271



(SEQ ID NO: 131)



 


69
H-PS-S(O-octyl)-FLSPEHSR-NH2 (SEQ ID NO: 132)
0.44


 


70
H-PS-S(O-octyl)-FLSPEHSRVSSR-NH2 (SEQ ID NO: 133)
0.467


 


71
H-PS-S(O-octyl)-FLSPEHSRVSSRKESKKPPAKLSPR-NH2
0.48



(SEQ ID NO: 134)





*Compound No. 1: human ghrelin with an octanoyl moiety at position 3


*Compound No. 2: human ghrelin without octanoyl moiety at position 3


*Compound No. 3: rat ghrelin with octanoyl moiety at position 3


*Compound No. 4: rat ghrelin without octanoyl moiety at position 3






Example 4
Effect of Compounds of the Invention on Food Intake in Normal Mice
Normal chow-fed C57BL/6J male mice were used. The mice were kept in an automated food- and water-intake monitoring system (HM-2) which allows automated on-line measurements of accumulated food intake (FI) and other parameters. Food intake was monitored by the HM-2 system up to 24 h after dosing. The mice were kept 6 per cage, and each dosing group consisted of 6 animals. The animals were dosed once subcutaneously with vehicle (PBS) or with test compound [human ghrelin (Compound 1 in Table 1) or Compound 16, 22, 23 or 43 of the invention] at a dose of 300 nmol/kg body weight, just after the start of the light period in which the mice are inactive and normally do not eat.
In the initial experimental set-up with ghrelin as test compound, animals were allowed free access to food directly after treatment, and the resulting data for food intake are shown in FIG. 1A herein (vide infra).
In order to further investigate the “duration of action” of ghrelin and the other test compounds a new protocol was defined. In the modified protocol the gate to the food chambers was closed prior to dosing with vehicle or test compound and was re-opened 2 hours thereafter. The resulting data for food intake following dosing with ghrelin or vehicle are summarized in FIG. 1B (vide infra), while the corresponding data for food intake following dosing with each of the of the other four test compounds. (Compound 16, 22, 23 or 43 of the invention) or with vehicle are summarized in FIG. 2A (data for monitoring up to 6 hours after dosing) and FIG. 2B (data for monitoring up to 24 hours after dosing).
Results:
When animals had free access to food immediately after dosing, the administration of ghrelin resulted in increased FI when compared to PBS treated controls (FIG. 1A). In contrast, when access to food was delayed for 2 h after ghrelin administration, the effect of ghrelin was essentially lost (FIG. 1B).
Since ghrelin had little effect on FI in the light phase of the modified protocol, this protocol was used for screening compounds (ghrelin analogues) of the invention having a longer duration of action than ghrelin per se. Examples of FI responses (0-6 h) and (0-24 h) for four compounds of the invention are presented in FIG. 2, while data for accumulated food intake (FI) for a total of 11 compounds of the invention (determined using the modified protocol) are presented in Table 2 (below). Interestingly, Compound 23 of the invention produced moderate effects in the 0-6 h light period (FIG. 2A), but had significant effects on FI during the dark period (FIG. 2B), while Compound 22 had significant effects on FI in both the light period and the dark period. Treatment with vehicle (PBS) had little effect on FI during the light period (FIG. 2A), and animals ate normally during the dark period (FIG. 2B).







TABLE 2







Effect of administration of test compounds (compounds of the invention) 


or vehicle (PBS) on FI in the light phase (0-6 h) using the modified 


protocol (vide supra). Data are expressed as accumulated FI (g) over 6 h. 









Accumulated FI at 6h 


Compound
(g)





PBS
0.26 ± 0.04


16
0.65 ± 0.1 


22
1.87 ± 0.23


43
0.37 ± 0.14


23
0.72 ± 0.3 


PBS
 0.03 ± 0.002


29
1.36 ± 0.2 


34
0.53 ± 0.08


23
0.32 ± 0.23


PBS
0.08 ± 0.04


42
 0.2 ± 0.08


PBS
 0.6 ± 0.19


45
0.38 ± 0.13


46
1.68 ± 0.2 


47
1.55 ± 0.16


PBS
0.12 ± 0.05


48
0 ± 0


49
 0.6 ± 0.09


50
0.19 ± 0.05

## Claims

1. A compound having the formula I:


R1—Z—R2  (I)

wherein
R1 is hydrogen, C1-4 alkyl, formyl, acetyl, trifluoroacetyl or benzoyl;
R2 is OH or NH2; and
Z is an amino acid sequence having the formula Ia:


X1-X2-X3-Phe-Leu-Ser-Pro-X8-His-X10-X11-X12-X13-X14-X15-X16-Glu-Ser-X19-X20-Pro-Pro-Ala-X24-Leu-X26-Pro X28  (Ia) (SEQ ID NO: 2)


or is a truncated form thereof lacking from 1 to 23 amino acids from the C-terminus;
wherein
X1 is Pro;
X2 is Ser; and
X3 is selected from the group consisting of:


—NH—CH[(CH2)n—O—R3]—C(O)—;  (i)



—NH—CH(R4)—C(O)—;  (ii)



—NH—CH[(CH2)m—O—R5]—C(O)—; and  (iii)



—NH—CH[(CH2)p—NH—R6]—C(O)—;  (iv)


wherein
R3 and R4 are, independently, C1-35-alkyl or C1-35-alkenyl groups;
R5 and R6 are, independently, C1-35-acyl groups; and
n, m and p are integers 1, 2, 3, 4 or 5; and
when present
X8 is selected from Glu, Lys and Tyr;
X10 is selected from Gin, Glu and Ser;
X11 is selected from Arg, Lys and Ser;
X12 is selected from Val, Ala, Pro, Lys, Thr and Leu;
X13 is selected from Gin and Ser;
X14 is selected from Gin, Lys, Tyr and Ser, or is absent;
X15 is selected from Arg and Ser;
X16 is selected from Lys, Ser and Pro;
X19 is selected from Lys and Ser;
X20 is selected from Lys, Glu and Ser;
X24 is selected from Lys, Tyr and Ser;
X26 is selected from Gin and Ser; and
X28 is selected from Arg and Ser;
or a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1, having the formula I:


R1—Z—R2  (I)

wherein
R1 is hydrogen, C1-4 alkyl, formyl, acetyl, trifluoroacetyl or benzoyl;
R2 is —OH or —NH2; and
Z is an amino acid sequence having the formula Ib:


X1-X2-X3-Phe-Leu-Ser-Pro-Glu-His-X10-X11-X12-X13-X14-Arg-X16-Glu-Ser-X19-X20-Pro-Pro-Ala-X24-Leu-X26-Pro-X28  (Ib) (SEQ ID NO: 3)


or is a truncated form thereof lacking from 1 to 23 amino acids from the C-terminus;
wherein
X1 is Pro;
X2 is Ser; and
X3 is selected from the group consisting of:


—NH—CH[(CH2)n—O—R3]—C(O)—;  (i)



—NH—CH(R4)—C(O)—;  (ii)



—NH—CH[(CH2)m—O—R5]—C(O)—; and  (iii)



—NH—CH[(CH2)p—NH—R6]—C(O)—;  (iv)


wherein
R3 and R4 are, independently, C1-35-alkyl or C1-35-alkenyl groups;
R5 and R6 are, independently, C1-35-acyl groups; and
n, m and p are integers 1, 2, 3, 4 or 5; and
when present
X10 is selected from Gin and Ser;
X11 is selected from Arg and Lys;
X12 is selected from Val, Ala, Thr and Leu;
X13 is selected from Gin and Ser;
X14 is selected from Gin and Ser, or is absent;
X16 is Lys;
X19 is Lys;
X20 is Lys;
X24 is Lys;
X26 is selected from Gin and Ser; and
X28 is Arg;
or a pharmaceutically acceptable salt thereof.

3. The compound according to claim 2, having the formula I:


R1—Z—R2  (I)

wherein
R1 is hydrogen, C1-4 alkyl, formyl, acetyl, trifluoroacetyl or benzoyl;
R2 is —OH or —NH2; and
Z is an amino acid sequence having the formula Ia:


X1-X2-X3-Phe-Leu-Ser-Pro-Glu-His-X10-X11-X12-X13-X14-Arg-X16-Glu-Ser-X19-X20-Pro-Pro-Ala-X24-Leu-X26-Pro X28  (Ib) (SEQ ID NO: 3)


or is a truncated form thereof lacking from 1 to 23 amino acids from the C-terminus;
wherein
X1 is Pro;
X2 is Ser; and
X3 is selected from the group consisting of:


—NH—CH[(CH2)n—O—R3]—C(O)—;  (i)



—NH—CH(R4)—C(O)—;  (ii)



—NH—CH[(CH2)m—O—R5]—C(O)—; and  (iii)



—NH—CH[(CH2)p—NH—R6]—C(O)—;  (iv)


wherein
R3 and R4 are C1-35-alkyl or C1-35-alkenyl groups;
R5 and R6 are C1-35-acyl groups; and
n, m and p are integers 1, 2, 3, 4 or 5; and
when present
X10 is Gin;
X11 is selected from Arg and Lys;
X12 is selected from Val, Ala, Thr and Leu;
X13 is Gin;
X14 is Gin or is absent;
X16 is Lys;
X19 is Lys;
X20 is Lys;
X24 is Lys;
X26 is Gin; and
X28 is Arg;
or a pharmaceutically acceptable salt thereof.

4. The compound according to claim 1, wherein
Z is an amino acid sequence having the formula Ic:


Pro-Ser-X3-Phe-Leu-Ser-Pro-Glu-His-X10-X11-X12-X13-X14-Arg-X16-Glu-Ser-X19-X20-Pro-Pro-Ala-X24-Leu-X26-Pro-X28  (Ic) (SEQ ID NO: 4)


or is a truncated form thereof lacking from 1 to 23 amino acids from the C-terminus;
wherein
X3 is selected from the group consisting of:


—NH—CH[(CH2)n—O—R3]—C(O)—; and  (i)



—NH—CH[(CH2)p—NH—R6]—C(O)—;  (ii)


wherein
R3 is a C1-35-alkyl or C1-35-alkenyl group;
R6 is a C1-35-acyl group; and
n and p are integers 1, 2, 3, 4 or 5; and
when present
X10 is selected from Gln and Ser;
X11 is selected from Arg and Lys;
X12 is selected from Val, Ala, Thr and Leu;
X13 is selected from Gin and Ser;
X14 is selected from Gin and Ser, or is absent;
X16 is Lys;
X19 is Lys;
X20 is Lys;
X24 is Lys;
X26 is selected from Gin and Ser; and
X28 is Arg;
or a pharmaceutically acceptable salt thereof.

5. The compound according to claim 1, wherein
Z is an amino acid sequence having the formula Id:


Pro-Ser-X3-Phe-Leu-Ser-Pro-Glu-His-X10-X11-Val-X13-X14-Arg-X16-Glu-Ser-X19-Ser-Pro-Pro-Ala-X24-Leu-X26-Pro-X28  (Id) (SEQ ID NO: 5)


or is a truncated form thereof lacking from 1 to 23 amino acids from the C-terminus;
wherein
X3 is —NH—CH[(CH2)n—O—R3]—C(O)—
wherein
R3 is a C1-35-alkyl or C1-35-alkenyl group and n is an integer 1, 2, 3, 4 or 5; and
when present
X10 is selected from Gin and Ser;
X11 is selected from Arg and Lys;
X13 is selected from Gin and Ser;
X14 is selected from Gin and Ser, or is absent;
X16 is Lys;
X19 is Lys;
X24 is Lys;
X26 is selected from Gin and Ser; and
X28 is Arg;
or a pharmaceutically acceptable salt thereof.

6. The compound according to claim 1, wherein
Z is an amino acid sequence having the formula Id:


Pro-Ser-X3-Phe-Leu-Ser-Pro-Glu-His-X10-X11-Val-X13-X14-Arg-X16-Glu-Ser-X19-Ser-Pro-Pro-Ala-X24-Leu-X26-Pro-X28  (Id) (SEQ ID NO: 5)


or is a truncated form thereof lacking from 1 to 23 amino acids from the C-terminus;
wherein
X3—NH—CH[(CH2)p—NH—R6]—C(O)—
wherein
R6 is a C1-35-acyl group and p is an integer 1, 2, 3, 4 or 5; and
when present
X10 is selected from Gin, Glu and Ser;
X11 is selected from Arg and Lys;
X13 is selected from Gin and Ser;
X14 is selected from Gin and Ser, or is absent;
X16 is Lys;
X19 is Lys;
X24 is Lys;
X26 is selected from Gin and Ser; and
X28 is Arg;
or a pharmaceutically acceptable salt thereof.

7. The compound according to claim 1, wherein R3 is a C5-16-alkyl or C5-16-alkenyl group, or a pharmaceutically acceptable salt thereof.

8. The compound according to claim 1, wherein R4 is a C7-18-alkyl or C7-18-alkenyl group, or a pharmaceutically acceptable salt thereof.

9. The compound according to claim 1, wherein R5 is a C5-16-acyl group, or a pharmaceutically acceptable salt thereof.

10. The compound according to claim 1, wherein R6 is a C5-16-acyl group, or a pharmaceutically acceptable salt thereof.

11. The compound according to claim 1, wherein n, m or p is 1, or a pharmaceutically acceptable salt thereof.

12. The compound according to claim 1, wherein at least positions X1 to X13 and X15 to X28 of Z are present, or a pharmaceutically acceptable salt thereof.

13. The compound according to claim 1, selected from the group consisting of:
H-PS-S(O-octanoyl)-FLSPEHQRVQQRKESKKPPAKLQPR-OH (SEQ ID NO: 7);
H-PS-S(O-octyl)-FLSPEHQRVQQRKESKKPPAKLQPR-OH (SEQ ID NO: 21);
H-PS-Dap(N-octanoyl)-FLSPEHQRVQQRKESKKPPAKLQPR-NH2 (SEQ ID NO: 74);
H-PS-Dap(N-octanoyl)-FLSPEHSRVSSRKESKKPPAKLSPR-NH2 (SEQ ID NO: 82);
H-PS-Dap(N-octanoyl)-FLSPEHQRVQQRKESKKPPA-NH2 (SEQ ID NO: 139);
H-PS-Dap(N-octanoyl)-FLSP-NH2 (SEQ ID NO: 155);
H-PS-Dap(N-octanoyl)-FLS-NH2 (SEQ ID NO: 156);
H-PS-Dap(N-octanoyl)-FL-NH2 (SEQ ID NO: 157);
H-PS-[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQQRKES-NH2 (SEQ ID NO: 181);
H-PS-[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQQRKESKKPPA-NH2 (SEQ ID NO: 176);
H-PS-S(O-octyl)-FLSPE(HQRK( )QQRKESKKPPAKLQPR-NH2 (SEQ ID NO:97);
H-PS-S(O-octyl)-FLSPEHE( )RVQK( )RKESKKPPAKLQPR-NH2 (SEQ ID NO: 98);
H-PS-Dap(N-octanoyl)-FLSPEHQRVQRKESKKPPAKLQPR-NH2 (SEQ ID NO: 100);
H-PS-S(O-octyl)-FLSPEHQRVQRKESKKPPAKLQPR-NH2 (SEQ ID NO: 101);
H-PS-Dap(N-octanoyl)-FLSPK-NH2 (SEQ ID NO: 102);
H-PS-S(O-octyl)-FLSPK-NH2 (SEQ ID NO: 103);
H-PS-[NH—CH(n-C10H21)—C(O)]-FLSPK-NH2 (SEQ ID NO: 104);
H-PS-Dap(N-octanoyl)-FLSPY-NH2 (SEQ ID NO: 105);
H-PS-S(O-octyl)-FLSPEHSRVSSRKESSKPPAKLSPR-NH2 (SEQ ID NO: 108);
H-PS-S(O-octyl)-FLSPEHSRVSSRKESKSPPAKLSPR-NH2 (SEQ ID NO: 109);
H-PS-S(O-octyl)-FLSPEHSRVSSRKESSSPPAKLSPR-NH2 (SEQ ID NO: 110);
H-PS-S(O-octyl)-FLSPEHSRPSSR-NH2 (SEQ ID NO: 111);
H-PS-S(O-octyl)-FLSPEHSRVSSRPESSSPPAKLSPR-NH2 (SEQ ID NO: 112);
H-PS-S(O-octyl)-FLSPEHSRPSSRPESSSPPAKLSPR-NH2 (SEQ ID NO: 113);
H-PS-S(octanoyl)-FLSPEHQSVQQRKESKKPPAKLQPR-NH2 (SEQ ID NO: 114);
H-PS-S(octanoyl)-FLSPEHQRVQQSKESKKPPAKLQPR-NH2 (SEQ ID NO: 115);
H-PS-S(octanoyl)-FLSPEHQRVQQRSESKKPPAKLQPR-NH2 (SEQ ID NO: 116);
H-PS-S(octanoyl)-FLSPEHQRVQQRKESSKPPAKLQPR-NH2 (SEQ ID NO: 117);
H-PS-S(octanoyl)-FLSPEHQRVQQRKESKSPPAKLQPR-NH2 (SEQ ID NO: 118);
H-PS-S(octanoyl)-FLSPEHQRVQQRKESKKPPASLQPR-NH2 (SEQ ID NO: 119);
H-PS-S(octanoyl)-FLSPEHQRVQQRKESKKPPAKLQPS-NH2 (SEQ ID NO: 120);
H-PS-S(octanoyl)-FLSPYHQRVQQRKESKKPPAKLQPR-NH2 (SEQ ID NO: 121);
H-PS-S(octanoyl)-FLSPEHQRVQYRKESKKPPAKLQPR-NH2 (SEQ ID NO: 122);
H-PS-S(octanoyl)-FLSPEHQRVQQRKESKKPPAYLQPR-NH2 (SEQ ID NO: 123);
H-PS-S(octanoyl)-FLSPYHQRVQYRKESKKPPAYLQPR-NH2 (SEQ ID NO: 124);
H-PS-S(O-octyl)-FLSPEHQRVQQRKESKKPPAKLQPR-NH2 (SEQ ID NO: 125);
H-PS-S(O-octyl)-FLSPEHQRVQRKESKKPPAKLQPR-NH2 (SEQ ID NO: 126);
H-PS-S(O-octyl)-FLSPEHQRVQQRKESKKPPA-NH2 (SEQ ID NO: 127);
H-PS-S(O-octyl)-FLSPEHQRVQQRKES-NH2 (SEQ ID NO: 128);
H-PS-[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQQRKESKKPPAKLQPR-NH2 (SEQ ID NO: 129);
H-PS-[NH—CH(n-C10H21)—C(O)]-FLSPEHQRVQRKESKKPPAKLQPR-NH2 (SEQ ID NO: 130);
H-PS-S(O-octyl)-FLSPEHSR-NH2 (SEQ ID NO: 132);
H-PS-S(O-octyl)-FLSPEHSRVSSR-NH2 (SEQ ID NO: 133); and
H-PS-S(O-octyl)-FLSPEHSRVSSRKESKKPPAKLSPR-NH2 (SEQ ID NO: 134);
or a pharmaceutically acceptable salt thereof;
wherein parentheses indicate an intramolecular bridge between a Lys and Glu residue.

14. The compound according to claim 1, wherein the compound has at least one biological activity of native human ghrelin.

15. A pharmaceutical composition comprising a compound of claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

16. A method for:
(i) treating cachexia, low body weight, loss of body weight, low or decreased appetite, inability to gain weight due to gastrectomy or vagectomy, gut barrier dysfunction, inflammatory bowel disease (IBD), Crohn's disease, colitis, ulcerative colitis, or ischemia reperfusion injury;
(ii) increasing food intake and/or body weight;
(iii) inducing the production of growth hormone; or
(iv) inducing increased motility of the gastrointestinal tract;
the method comprising the step of administering to a subject in need thereof a therapeutically effective amount of a compound of claim 1, or a pharmaceutically acceptable salt thereof.