WO2015010210A1 - Compounds for regulating acylated ghrelin - Google Patents

Compounds for regulating acylated ghrelin Download PDF

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Publication number
WO2015010210A1
WO2015010210A1 PCT/CA2014/050700 CA2014050700W WO2015010210A1 WO 2015010210 A1 WO2015010210 A1 WO 2015010210A1 CA 2014050700 W CA2014050700 W CA 2014050700W WO 2015010210 A1 WO2015010210 A1 WO 2015010210A1
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seq
subject
compound according
ghrelin
diabetes
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PCT/CA2014/050700
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French (fr)
Inventor
Alfonso Abizaid BUCIO
Martin WELLMAN
Zachary PATTERSON
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Bucio Alfonso Abizaid
Wellman Martin
Patterson Zachary
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Publication of WO2015010210A1 publication Critical patent/WO2015010210A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/30—Drugs for disorders of the nervous system for treating abuse or dependence
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22

Definitions

  • the present invention relates to the field of ghrelin regulation and, in particular, to peptidic compounds capable of regulating acylated ghrelin levels.
  • Ghrelin was discovered in 1999 (Kojima, M., et al., 1999, Nature, 402(6762):656-660) and quickly identified as a strong obesogenic hormone that, in its acylated form, not only increases food intake, but also increases preference for fatty foods and protects fat reserves within the body (Perello, M., et al., 2010, Biological Psychiatry, 67(9):880-886; Tschop, M., D.L. Smiley, and M . Heiman, 2000, Nature, 407(6806):908-913; Wren, A.M., et al, 2001, J. Clinical Endocrinology & Metabolism, 86(12):5992; and Patterson, Z.R., et ah, 2013, Endocrinology, 154(3):1080-1091).
  • ghrelin seeks to bind to the growth hormone secretagogue receptor- la (GHSR-la), the only known receptor for ghrelin.
  • GHSR-la growth hormone secretagogue receptor- la
  • ghrelin-O- acyl-transferase a member of the membrane bound-O-acyl-transferase (MBOAT) family.
  • MBOAT membrane bound-O-acyl-transferase
  • GOAT is the only known enzyme capable of acylating ghrelin and it is therefore thought to be involved in all processes related to ghrelin.
  • ghrelin Once activated, ghrelin promotes food intake and body weight through interactions with GHSR-la, primarily in the hypothalamus.
  • Circulating acylated ghrelin levels rise prior to a meal when the animal is hungry, and gradually decrease during and after feeding as the animal becomes satiated. Injections of acylated ghrelin promote feeding behaviour and adiposity.
  • acylated ghrelin In addition to its role in obesity, acylated ghrelin has also been identified as an important component of the stress response. During a stressor, acylated ghrelin levels increase and appear to have a protective effect by redirecting the use of appropriate energy resources (Asakawa, A., et ah, 2001, Neuroendocrinolog , 74:143-147). However, depending on the many other variables involved, increases in ghrelin in response to stress may result in changes in appetite and feeding patterns.
  • acylated ghrelin strongly influences glucose metabolism.
  • a reduction in ghrelin levels improves insulin sensitivity and is a promising research target for understanding diabetes as well as treating it (Wiedmer, P., et ah, 2007, Nat Clin Pr act End Met, 3(10):705-712).
  • U.S. Patent Application No. 09/902,556 (US2002/0187938) describes peptides having antagonistic properties to ghrelin.
  • the peptides are based on the ghrelin sequence, comprise between 5 and 10 amino acids and include an octanoyl modification on the third residue.
  • U.S. Patent Application No. 10/522,398 (US2005/0272648) and International Patent Application No. PCT/US2006/038027 (WO 2007/041278) describe peptidyl analogues that possess agonist or antagonist ghrelin activity and therapeutic uses thereof.
  • the peptidyl analogues comprise between 6 and 28 amino acids and include at least one modified amino acid.
  • U.S. Patent Application No. 13/320,408 (US2012/0135918) describes methods of treating obesity, obesity -related disorders, diabetes and metabolic syndrome using a GOAT inhibitor and/or a ghrelin receptor antagonist.
  • the GOAT inhibitor and ghrelin receptor antagonist may each be peptidic compounds comprising an octanoyl or similar modification.
  • the present invention relates generally to compounds for regulating acylated ghrelin.
  • An aspect of the invention relates to a compound of general formula (I)
  • X is absent or a sequence of between 1 and 25 consecutive amino acids of the sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 57, or between 1 and 24 consecutive amino acids of the sequence as set forth in SEQ ID NO: 4 or SEQ ID NO: 77;
  • Y is absent or a peptide linker of between 2 and 10 amino acids in length and comprising amino acids selected from the group of glycine, alanine, valine, lysine and isoleucine;
  • GSA-X-Y is at least 4 amino acids in length
  • B is a cell-penetrating peptide.
  • Another aspect of the invention relates to a pharmaceutical composition comprising a compound of general formula (I), and a pharmaceutically acceptable carrier or diluent.
  • Another aspect of the invention relates to a kit for research use comprising a compound of general formula (I), and instructions for use.
  • Another aspect of the invention relates to a use of a compound of general formula (I) for decreasing levels of acylated ghrelin in a subject.
  • Another aspect of the invention relates to a use of a compound of general formula (I) for treatment or management of obesity in a subject.
  • Another aspect of the invention relates to a use of a compound of general formula (I) for treatment or management of diabetes in a subject.
  • Another aspect of the invention relates to a use of a compound of general formula (I) for treatment or management of pre-diabetes or metabolic syndrome in a subject.
  • Another aspect of the invention relates to a use of a compound of general formula (I) for modifying eating behaviour in a subject.
  • Another aspect of the invention relates to a use of a compound of general formula (I) for treatment or management of post-traumatic stress disorder in a subject.
  • Another aspect of the invention relates to a use of a compound of general formula (I) for the treatment or management of an addictive behaviour in a subject.
  • Another aspect of the invention relates to a method of decreasing acylated ghrelin levels in a subject comprising administering to the subject an effective amount of a compound of general formula (I).
  • Another aspect of the invention relates to a method of treating or managing obesity in a subject comprising administering to the subject an effective amount of a compound of general formula (I).
  • Another aspect of the invention relates to a method of treating or managing diabetes in a subject comprising administering to the subject an effective amount of a compound of general formula (I).
  • Another aspect of the invention relates to a method of treating or managing pre-diabetes or metabolic syndrome in a subject comprising administering to the subject an effective amount of a compound of general formula (I).
  • Another aspect of the invention relates to a method of modifying eating behaviour in a subject comprising administering to the subject an effective amount of a compound of general formula (I).
  • Another aspect of the invention relates to a method of treating or managing post-traumatic stress disorder in a subject comprising administering to the subject an effective amount of a compound of general formula (I).
  • Another aspect of the invention relates to a method of treating or managing an addictive behaviour in a subject comprising administering to the subject an effective amount of a compound of general formula (I).
  • Figure 1 Body weight of animals with choice of high- fat diet or regular chow (ad lib).
  • Figure 2 Caloric intake of animals during baseline (B) and treatment (E). No differences were found between high dose (HD), low dose (LD), vehicle (VEH), and non-injected control (NIC) animals throughout baseline and treatment (A). Similarly, no differences were found when caloric intake was normalized to body mass (B).
  • Figure 3 (A) Average daily regular chow consumption normalized to body mass during baseline and treatment. Animals showed no significant difference in average chow consumption, normalized to weight, during baseline. High dose animals consumed significantly more regular chow on average compared to vehicle animals during treatment (p ⁇ 0.05).
  • FIG. 4 (A) Metabolic efficiency (mass gained in grams per total grams consumed from start to end of baseline or treatment). No differences in metabolic efficiency were found between groups during baseline. Both low dose and high dose animals had significantly reduced metabolic efficiency compared to vehicle and non -injected controls during treatment (p ⁇ 0.01). (B) Fat mass, lean mass, and total water mass of animals following CF801 treatment, as measured by EchoMRI. High-dose animals had significantly lower fat mass compared to vehicle.
  • Figure 5 Open field test for anxiety. Following treatment, no differences were found in time spent around the border or in the center of an open-field apparatus between non-injected controls (NIC), vehicle (VEH), low-dose (LD), and high-dose (HD) animals.
  • NIC non-injected controls
  • VH vehicle
  • LD low-dose
  • HD high-dose
  • FIG. 6 (A) Circulating acylated ghrelin levels after CF801 treatment, measured with enzyme linked immunosorbent assay (ELISA). Plasma acylated ghrelin levels showed a dose-dependent decrease with CF801 treatment. (B) Food consumed following 24 hour fast. High-dose animals consumed significantly less food compared to vehicle controls over a four-hour re-feeding period following reintroduction of food.
  • ELISA enzyme linked immunosorbent assay
  • Figure 7 (A) Chronic social defeat stress leads to an increase in regular chow intake and (B) a decrease in high-fat intake. * p ⁇ 0.05.
  • Figure 8 (A) Stressed animals consumed significantly less high- fat diet when given CF801 treatment, when compared to vehicle and non-injected stressed controls (p ⁇ 0.05). (B) Stressed animals consumed significantly more regular chow compared to vehicle-injected stressed animals (p ⁇ 0.05). (C) CF801 treatment resulted in a significant reduction in average daily caloric intake in stressed animals. This reduction did not reach significance in non-stressed animals, demonstrating an interaction between drug treatment and stress group.
  • Figure 9 (A) CF801 treatment of animals exposed to chronic social defeat stress leads to a reduced body weight. (B) Non-injected and vehicle- injected animals show an increase in plasma acylated ghrelin when exposed to stress. This increase is not observed when animals are treated with daily injections of CF801. [0042] Figure 10: Stressed animals displayed typical responding in the elevated plus maze, with (A) reduced time spent in open arms and (B) no difference in number of entries into the open arms. (C) Stressed animals treated with CF801, however, had fewer entries into the open arms when compared to vehicle-injected stressed controls (p ⁇ 0.05).
  • Figure 12 (A) Glucose tolerance test. Following an overnight fast, animals were given a 2mg/kg bolus of 20% glucose. (B) Circulating levels of corticosterone measured from trunk blood collected at the time of sacrifice. Data represents mean +/- S.E.M. * p ⁇ 0.05 relative to vehicle injected controls.
  • Figure 13 Circulating levels of des-acyl ghrelin (A), acylated ghrelin (B) and total ghrelin (C) in blood plasma samples collected from trunk blood at the time of sacrifice.
  • FIG. 14 Circulating levels of blood glucose (A) and insulin (B) at the time of sacrifice.
  • HomaIR homeostatic model assessment for insulin resistance
  • HomaIR [(Insulin*Glucose) / 22.5]. All values are expressed as mean ⁇ S.E.M. ⁇ ⁇ 0.05 compared to all other groups, ⁇ ⁇ 0.05 compared to vehicle injected controls.
  • Figure 15 mRNA expression of NPY (A), AgRP (B), POMC (C) and SOCS3 (D) in the mediobasal hypothalamus. All values are expressed as mean +/- SEM, relative to GAPDH expression. * p ⁇ 0.05 relative to non-injected controls.
  • Figure 16 Circulating levels of blood glucose (A), insulin (B) and HomaIR (C) at the time of sacrifice. All values are expressed as mean ⁇ S.E.M. * p ⁇ 0.05 compared to vehicle injected control.
  • Figure 17 mR A expression of GOAT (A) and ghrelin (B) in the fundus of the stomach. All values are expressed as mean ⁇ S.E.M., relative to GAPDH and ⁇ - Actin. *** p ⁇ 0.0001; * p ⁇ 0.05 relative to saline injected controls.
  • Figure 18 (A) Enzymatic activity of GOAT with increasing concentrations of CF801. (B) Ghrelin concentrations following application of CF801 to ghrelin secreting SGI cells in vitro.
  • the present invention relates to peptidic compounds of general formula (I), which comprise a ghrelin moiety linked, optionally via a peptide linker, to a cell penetrating peptide moiety.
  • These compounds are linear peptides based on the sequence of ghrelin, which do not include modified amino acids.
  • the compounds surprisingly show sufficient stability and activity to effectively decrease circulating acylated ghrelin levels in vivo.
  • compounds of general formula (I) are capable of reducing circulating acylated ghrelin levels, reducing fat mass and intake of high-fat foods in test animals, blunting rebound feeding in fasting animals and reducing stress- induced calorie intake. Accordingly, in certain embodiments, the compounds of general formula (I) may be useful in the treatment of diseases or disorders associated with obesity, weight gain and/or adiposity. In some embodiments, the compounds of general formula (I) may find use as part of weight management programs or treatment protocols. In some embodiments, the compounds of general formula (I) may find use in stress management programs or treatment protocols. In certain embodiments, the compounds of general formula (I) may find use in the treatment of diabetes, prediabetes or metabolic syndrome.
  • Additional embodiments of the invention relate to the use of compounds of general formula (I) as research tools to assist in the elucidation of the role of acylated ghrelin, for example, in stress, food preferences, body composition and/or weight gain, as well as to study of the effects of changes in the acyl:des-acyl ghrelin ratio and the role of des-acyl ghrelin.
  • Definitions
  • therapy and treatment refer to an intervention performed with the intention of alleviating the symptoms associated with, preventing the development of, or altering the pathology of a disease, disorder or condition.
  • therapy and treatment are used in the broadest sense, and in various embodiments include one or more of the prevention (prophylaxis), moderation, reduction, and/or curing of a disease, disorder or condition at various stages.
  • Subjects in need of therapy/treatment thus may include those already having the disease, disorder or condition as well as those prone to, or at risk of developing, the disease, disorder or condition and those in whom the disease, disorder or condition is to be prevented.
  • subject and patient refer to an animal in need of treatment.
  • the subject or patient may be a human or non-human animal. In certain embodiments, the subject or patient is a human.
  • compositions, use or method excludes the presence of additional elements and/or method steps.
  • a composition, use or method described herein as comprising certain elements and/or steps may also, in certain embodiments consist essentially of those elements and/or steps, and in other embodiments consist of those elements and/or steps, whether or not these embodiments are specifically referred to.
  • the term "about” refers to an approximately +/-10% variation from a given value. It is to be understood that such a variation is always included in any given value provided herein, whether or not it is specifically referred to.
  • compositions and kits of the invention can be used to achieve the disclosed methods and uses.
  • Naturally occurring amino acids are identified throughout by the conventional three- or one-letter abbreviations indicated in Table 1 below, which are as generally accepted in the peptide art and recommended by the IUPAC-IUB commission in biochemical nomenclature.
  • the invention relates to peptidic compounds comprising a ghrelin moiety linked, optionally via a linker, to a cell-penetrating peptide (CPP) moiety.
  • the compounds can be represented by general formula (I): GSA-X-Y-B (I)
  • GSA-X is a ghrelin moiety
  • Y is an optional peptide linker
  • B is a CPP moiety.
  • the combined length of the ghrelin moiety and the optional linker (GSA-X- Y) is at least 4 amino acids.
  • a characteristic of the compounds of general formula (I) is the replacement of the serine residue that naturally occurs at position 3 of the ghrelin peptide with alanine such that the compound cannot be acylated.
  • a shorter GSA-X sequence would be expected to aid in cell penetration and reduce cost.
  • a longer X sequence is expected to better mimic ghrelin and therefore may aid in activity.
  • the ghrelin moiety comprised by the compounds is between 3 and 28 amino acids in length and comprises the sequence Gly- Ser-Ala optionally coupled to a sequence of between 1 and 25 consecutive amino acids of SEQ ID NO: 3 or SEQ ID NO:57, which respectively represent amino acids 4-28 of the naturally occurring human and rodent ghrelin sequence (SEQ ID NO: l and SEQ ID NO: 56), or between 1 and 24 consecutive amino acids of SEQ ID NO: 4 or SEQ ID NO:77, which represent amino acids 4-27 of des Gln-14 ghrelin (SEQ ID NO:2 and SEQ ID NO:76) (Table 2).
  • the ghrelin moiety comprised by the compounds is at least 4 amino acids in length, for example, between 4 and 28 amino acids in length. In some embodiments, the ghrelin moiety is between 5 and 28 amino acids in length, for example, between 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 and 28 amino acids in length, or any range therebetween.
  • the ghrelin moiety comprised by the compounds has a sequence as set forth in any one of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, , SEQ ID NO : 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:
  • the ghrelin moiety comprised by the compounds has a sequence corresponding to a human ghrelin sequence or fragment thereof as set forth in any one of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 31, SEQ ID NO:
  • the ghrelin moiety comprised by the compounds has a sequence corresponding to a mouse or rat ghrelin sequence or fragment thereof as set forth in any one of SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 58, SEQ ID
  • the ghrelin moiety comprised by the compounds includes only the N-terminal region of the ghrelin sequence and thus the ghrelin moiety is between about 4 and 18 amino acids in length, for example, between about 4 and 17 amino acids in length or between about 4 and 16, 15, 14, 13, 12, 11, 10, 9, 8, 7 or 6 amino acids in length, or any range therebetween, and has a sequence corresponding to the N-terminal region of SEQ ID NO:5, 6, 58 or 78.
  • the ghrelin moiety comprised by the compounds has a sequence as set forth in any one of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 73, SEQ ID NO: 74 and SEQ ID NO: 75 (Table 2).
  • the optional peptide linker of compounds of general formula (I) is a sequence of amino acids that may be between 2 and about 10 amino acids in length.
  • the linker when present is between 2 and about 10 amino acids in length and comprises amino acids selected from glycine, alanine, valine, lysine and isoleucine.
  • the linker when present is between 2 and about 10 amino acids in length and comprises amino acids selected from glycine and alanine.
  • the linker when present is a polyglycine or polyalanine peptide linker of between 2 and 10 amino acids in length.
  • the peptide linker functions to spatially separate the ghrelin moiety from the CPP moiety.
  • the compounds may include a longer ghrelin moiety, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more amino acids, and no peptide linker.
  • the CPP moiety comprised by the compounds functions to enhance the cell permeability of the compound.
  • a number of cell penetrating peptides are known in the art and may be included in the compounds as the CPP moiety. Examples include, but are not limited to, the peptide sequences shown in Table 3. Table 3: Cell-Penetrating Peptides
  • Variants of these sequences are known that function as CPPs and thus may be suitable for inclusion in the compounds of general formula (I).
  • examples include variants of the TAT sequence, such as RKKRRQRRR (SEQ ID NO: 52); GRKKRRQRRRPQ (SEQ ID NO:53) and GRKKRRQRRRPPQ (SEQ ID NO:54).
  • the compounds include a CPP moiety that is based on TAT, penetratin or transportin.
  • the compounds may further comprise a modification at the N-terminus, the C-terminus or both. Such modifications may assist for example in purification, stability, bioavailability or the like.
  • N-terminal modifications include, for example, modification with groups such as acetyl, formyl, fatty acid, benzoyl, benzoyloxycarbonyl, bromoacetyl, pyroglutamyl, succinyl and teri-butoxycarbonyl.
  • C- terminal modifications include, for example, modification with groups such as amide, ester and teri-butyl.
  • the compounds of general formula (I) include a modified C-terminus. In some embodiments, the compounds of general formula (I) include a modified C-terminus and a modified N-terminus. In some embodiments, the compounds of general formula (I) include an amidated C-terminus and optionally an acetylated N-terminus. [0079] In certain embodiments, the peptidic compounds have the general formula (I)
  • X is absent or a sequence of between 1 and 25 consecutive amino acids of the sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 57, or between 1 and 24 consecutive amino acids of the sequence as set forth in SEQ ID NO: 4 or
  • Y is absent or a peptide linker of between 2 and 10 amino acids in length and comprising amino acids selected from the group of glycine, alanine, valine, lysine and isoleucine;
  • GSA-X-Y is at least 4 amino acids in length
  • X is a sequence corresponding the sequence as set forth in any one of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO:
  • SEQ ID NO: 24 SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 58, SEQ ID NO: 59, SEQ
  • SEQ ID NO: 60 SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO:
  • SEQ ID NO: 82 SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90 and SEQ ID NO: 91.
  • SEQ ID NO: 5 SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 43.
  • X is a sequence corresponding the sequence as set forth in any one of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 73, SEQ ID NO: 74 and SEQ ID NO: 75.
  • Y is absent or a poly glycine or polyalanine peptide linker of between 2 and 10 amino acids in length.
  • Y is absent.
  • B is a cell-penetrating peptide selected from TAT, penetratin, transportin and variants thereof.
  • B is a cell-penetrating peptide having a sequence as set forth in any one of RKKRRQRRR (SEQ ID NO:52); GRKKRRQRRRPQ (SEQ ID NO:53); YGRKKRRQRRR (SEQ ID NO:44) and GRKKRRQRRRPPQ (SEQ ID NO:54).
  • X is a sequence of between 1 and 14 consecutive amino acids of the sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 57, and Y is absent.
  • X is a sequence of between 1 and 14 consecutive amino acids of the sequence as set forth in SEQ ID NO: 3, and
  • Y is absent.
  • X is a sequence of between 1 and 5 consecutive amino acids of the sequence as set forth in SEQ ID NO: 3, and
  • Y is a peptide linker of between 2 and 10 amino acids in length and comprises amino acids selected from the group of glycine, alanine, valine, lysine and isoleucine.
  • X is a sequence of between 1 and 14 consecutive amino acids of the sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 57;
  • B is a cell-penetrating peptide selected from TAT, penetratin, transportin and variants thereof.
  • compounds of general formula (I) have general formula (II):
  • compounds of general formula (I) have general formula (III):
  • the compounds of general formula (I) as described in any of the above embodiments include a modified C-terminus and optionally a modified N-terminus. In some embodiments, the compounds of general formula (I) as described in any of the above embodiments include an amidated C-terminus and optionally an acetylated N-terminus.
  • the compounds of general formula (I) have general formula (IV): GSA-X-TAT (IV)
  • TAT represents any TAT sequence used for cell penetration, which includes sequences with an amidated C-terminus.
  • the compounds of general formula (I) can be readily prepared by standard chemical synthesis techniques.
  • the principles of solid phase chemical synthesis of polypeptides are well known in the art and may be found in general texts in the area such as Pennington, M.W. and Dunn, B.M., Methods in Molecular Biology, Vol. 35 (Humana Press, 1994); Dugas, H. and Penney, C, Bioorganic Chemistry (1981) SpringerVerlag, New York, pgs. 54-92; Merrifield, J. M, Chern. Soc, 85:2149 (1962), and Stewart and Young, Solid Phase Peptide Synthesis, pp. 24-66, Freeman (San Francisco, 1969).
  • N- and/or C-terminal modifications of the synthesized peptides may also be made by standard techniques. Covalent modifications of the peptide can be introduced, for example, by reacting targeted amino acid residues with an organic derivatizing agent that is capable of reacting with selected terminal residues as is known in the art. Selection of appropriate derivatizing agent(s) can be readily accomplished by a worker skilled in the art. Testing Compounds for Activity
  • toxicity and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals.
  • LD5 0 the dose lethal to 50% of the population
  • ED5 0 the dose therapeutically effective in 50% of the population
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD5 0 /ED5 0 . Large therapeutic indices are preferred for therapeutic uses.
  • compositions that comprise the compound and a pharmaceutically acceptable carrier, diluent, or excipient.
  • the pharmaceutical compositions may be prepared by known procedures using well-known and readily available ingredients.
  • the pharmaceutical compositions may be formulated for administration by a variety of routes, for example, orally (including, for example, buccally or sublingually), topically, parenterally, by inhalation or spray, or rectally.
  • parenteral as used herein may include subcutaneous injections, intradermal, transdermal, intra-articular, intravenous, intramuscular, intravascular, intrasternal, intrathecal injection or infusion techniques.
  • the compounds may optionally be formulated in unit dosage forms containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and/or vehicles.
  • the compounds can be formulated into a form suitable for the selected route of administration, for example, as syrups, elixirs, tablets, troches, lozenges, hard or soft capsules, pills, suppositiories, oily or aqueous suspensions, dispersible powders or granules, emulsions, injectables, solutions, creams, gels, liquids, lotions, sprays or aerosols.
  • a patch typically includes a skin-contacting portion made of any suitable material that is covered or impregnated with a cream or emulsion comprising the compound and which is supported by a backing, one or both of the skin-contacting portion and the backing may have an adhesive segment or other configuration for attaching to the skin surface of a subject.
  • the compounds may be advantageous to complex or associate the compounds with an appropriate delivery vehicle prior to formulation.
  • delivery vehicles may provide beneficial properties to the compounds, for example, improve the stability, solubility, pharmacokinetics, or the like.
  • delivery vehicles include, but are not limited to, polyethylene glycol (PEG), polyacetic acid, polyglycolic acid, hydrogels, dextran, liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts and spheroblasts.
  • compositions intended for oral use may be prepared in either solid or fluid forms.
  • Fluid forms can be prepared according to procedures known in the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavouring agents, colouring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations.
  • An elixir is prepared by using a hydroalcoholic (for example, ethanol) vehicle with suitable sweeteners such as sugar and saccharin, together with an aromatic flavoring agent.
  • Suspensions can be prepared with an aqueous vehicle with the aid of a suspending agent such as acacia, tragacanth, methylcellulose and the like.
  • Solid formulations such as tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets.
  • excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate: granulating and disintegrating agents for example, corn starch, or alginic acid: binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc and other conventional ingredients such as dicalcium phosphate, magnesium aluminum silicate, calcium sulfate, starch, lactose, methylcellulose, and functionally similar materials.
  • inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate
  • granulating and disintegrating agents for example, corn starch, or alginic acid: binding agents, for example starch, ge
  • the tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a time delay material such as glyceryl monostearate or glyceryl distearate may be employed.
  • Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.
  • Soft gelatin capsules are prepared by machine encapsulation of a slurry of the compound with an acceptable vegetable oil, light liquid petrolatum or other inert oil.
  • Aqueous suspensions contain active materials in admixture with excipients suitable for the manufacture of aqueous suspensions.
  • excipients are suspending agents, for example sodium carboxylmethylcellulose, methyl cellulose, hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia: dispersing or wetting agents may be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example hepta-decaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan
  • the aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl- p-hydroxy benzoate, one or more colouring agents, one or more flavouring agents or one or more sweetening agents, such as sucrose or saccharin.
  • preservatives for example ethyl, or n-propyl- p-hydroxy benzoate
  • colouring agents for example ethyl, or n-propyl- p-hydroxy benzoate
  • flavouring agents for example sucrose or saccharin.
  • sweetening agents such as sucrose or saccharin.
  • Oily suspensions may be formulated by suspending the active ingredients in a vegetable oil, for example peanut oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.
  • the oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol.
  • Sweetening agents such as those set forth above, and flavouring agents may be added to provide palatable oral preparations.
  • These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.
  • Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives.
  • a dispersing or wetting agent exemplified by those already mentioned above.
  • Additional excipients for example sweetening, flavouring and colouring agents, may also be present.
  • compositions may also be in the form of oil-in-water emulsions.
  • the oil phase may be a vegetable oil, for example olive oil or peanut oil, or a mineral oil, for example liquid paraffin or mixtures of these.
  • Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate.
  • compositions may also be in the form of a sterile injectable aqueous or oleaginous suspension.
  • This suspension may be formulated according to known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above.
  • the sterile injectable preparation may also be a sterile injectable solution or a suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3-butanediol.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • Adjuvants such as local anaesthetics, preservatives and buffering agents can also be included in the injectable solution or suspension.
  • compositions for rectal administration are typically prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
  • suitable non-irritating excipient include cocoa butter and polyethylene glycols.
  • compositions and methods of preparing pharmaceutical compositions are known in the art and are described, for example, in “Remington: The Science and Practice of Pharmacy ' " (formerly “Remingtons Pharmaceutical Sciences ' “); Gennaro, A., Lippincott, Williams & Wilkins, Philadelphia, PA (2000).
  • Phase I trials are usually an open-label trial (i.e. both the researchers and participants know which treatment is being administered).
  • Phase I trials are used to assess the safety, tolerability, pharmacokinetics, and pharmacodynamics of the compound. They may also be used to determine the best mode of administration (for example, orally or parenterally) if this has not been determined from pre-clinical trials, and/or an appropriate frequency of administration.
  • Phase I studies will include laboratory tests, such as blood or urine tests or biopsies, to evaluate the effects of the compound in the body of the patient.
  • Phase I trials may be conducted in a clinical trial clinic, where the patients can be observed by full-time staff.
  • Clinical trial clinics may be run for example by hospitals or by various contract research organizations (CROs) which conduct the trial on behalf of pharmaceutical companies or other research investigators.
  • CROs contract research organizations
  • Phase I trials usually involve a small number of healthy participants (for example, 10-100) who are treated with a specific dose of the candidate compound. During the trial, the dose is typically increased group by group in order to determine the maximum tolerated dose (MTD) and the dose-limiting toxicities (DLT) associated with the compound. The range of doses to be tested is determined based on the pre-clinical trials and will be a fraction of the dose that was observed to cause toxicity in animal models. Dose escalation studies during Phase I trials determine an appropriate dose to use in a subsequent Phase II trial.
  • MTD maximum tolerated dose
  • DLT dose-limiting toxicities
  • a Phase II trial can be conducted to evaluate the effectiveness of the candidate compound, and to further test the safety of the compound in a larger group of subjects.
  • Phase II trials are usually open-label, but may also be blinded.
  • the compound is administered to a larger group of participants (typically 100-300) who present with the disease for which treatment with the candidate compound is being investigated. The dosage found to be effective in Phase I trials is used.
  • Phase II trials may be designed as case series, to evaluate the safety and activity of the candidate compound in a selected group of subjects, or they may be designed as randomized controlled trials, where some subjects receive the candidate compound and others receive either a placebo or a standard treatment.
  • Phase III trials focus on evaluating the efficacy of the candidate compound, how it compares to the standard, or most widely accepted, treatment, as well as the applicability of the compound to clinical practice.
  • Phase III trials are much larger than Phase II trials, typically with between 500-3,000 participants, and are generally randomized, blinded trials conducted in multiple clinics ("multicentre trials").
  • participants are randomly assigned to one of two or more "arms.” In a trial with two arms, for example, one arm will receive the standard treatment (control group) and the other arm will receive treatment with the candidate compound (investigational group). Other arms may be included, for example, to evaluate against placebo or a different standard treatment.
  • Phase III trials of chronic conditions or diseases often have a short follow-up period for evaluation relative to the period of time the intervention might be used in practice.
  • Phase IV trials are usually conducted as post-marketing surveillance trials and are used to further evaluate the long-term safety and effectiveness of the compound. Phase IV trials are generally designed to detect any rare or long-term adverse effects in a much larger patient population and over a longer time period than was possible during the Phase I-III trials. Phase IV trials may be required by regulatory authorities, or may be undertaken by the sponsoring company for other reasons, such as finding a new market for the drug, testing for interactions with other drugs or in certain sub- populations. USES
  • Exemplary uses for the compounds of general formula (I) include, in various embodiments, use in basic research, use in pre-clinical studies and clinical trials for pharmacological agents to treat a ghrelin associated disorder, and therapeutic uses.
  • certain embodiments contemplate the use of the compounds of general formula (I) in the treatment and/or management of obesity.
  • the compounds may be used for regulating food intake in a subject, for improving compliance of a subject to caloric restriction, and/or for modifying eating behaviour of a subject (for example, by reducing desire for high-calorie foods and/or fats).
  • Some embodiments relate to methods and uses of the compounds of general formula (I) for preventing or reducing weight gain in a subject.
  • the invention relates to methods and uses of the compounds of general formula (I) in the treatment and/or management of diabetes.
  • Diabetes in this context may be insulin-dependent diabetes mellitus (Type I diabetes) or non-insulin-dependent diabetes mellitus (Type II diabetes).
  • the compounds may be administered to a subject having diabetes in order to treat or assist in the management of diabetes, or a diabetes-related condition, such as obesity or weight gain.
  • the compounds may be administered to a subject at risk of developing diabetes in order to prevent the subject from becoming diabetic.
  • Subjects at risk of diabetes include, for example, individuals having pre-diabetes or metabolic syndrome (insulin resistance), as well as overweight, physically inactive individuals.
  • Certain embodiments relate to methods and uses of the compounds of general formula (I) in the treatment and/or management of pre-diabetes. Some embodiments relate to methods and uses of the compounds of general formula (I) in the treatment and/or management of metabolic syndrome (or insulin resistance). In some embodiments, the compounds may be used to modify eating behaviour in individuals having pre-diabetes or metabolic syndrome and thus aid in the treatment and/or management of pre-diabetes or metabolic syndrome.
  • Certain embodiments relate to methods and uses of the compounds of general formula (I) in the treatment and/or management of stress-related eating disorders or other stress-related disorders such as post-traumatic stress disorder (PTSD).
  • PTSD post-traumatic stress disorder
  • Certain embodiments relate to methods and uses of compounds of general formula (I) in the treatment and/or management of addictive behaviours, for example, cravings for activities or substances such as gambling and alcohol.
  • Certain embodiments relate to the use of compounds of general formula (I) as research tools.
  • the compounds may potentially be used in a variety of experimental situations in which acylated and/or des-acylated ghrelin activity are of interest. For example, to study different aspects of diabetes, obesity, and more due to ghrelin's contribution to these diseases.
  • administration of compounds of general formula (I) should lead to improved insulin secretion and sensitivity in response to a glucose load, which aid in glucose tolerance in diabetes.
  • acyl ghrelin and total ghrelin levels can be measured by a variety of techniques, including for example enzyme-linked immunosorbent assays and mass spectrometry.
  • ghrelin has been identified as an important component in the development of post-traumatic stress disorder, with stress-associated increases in ghrelin leading to enhanced fear learning associated with the amygdale (Meyer, R.M., et al., "A Ghrelin-Growth Hormone Axis Drives Stress-Induced Vulnerability To Enhanced Fear,” Mol Psychiatry, advance online publication, October 15, 2013).
  • Compounds of general formula (I) may be used in studies to determine if it is possible to weaken these memories and the fear association during reconsolidation.
  • kits or packs containing a compound of general formula I for research use are typically a collection of biological research products, for example, two or more, that are used together to perform a biological research reaction, procedure, or synthesis, such as an assay, a detection, a separation or a purification, which are shipped together, usually within a common packaging, to an end user.
  • the research kits may optionally include reagents required to conduct a biological procedure, such as buffers, salts, enzymes, enzyme co-factors, substrates, detection reagents, washing reagents, and the like.
  • reagents required to conduct a biological procedure such as buffers, salts, enzymes, enzyme co-factors, substrates, detection reagents, washing reagents, and the like.
  • Other components, such as buffers and solutions for the isolation and/or treatment of a test sample, may also be included in the kit.
  • the kit may additionally include one or more control compounds.
  • one or more of the components of the kit may be lyophilised and the kit may further comprise reagents suitable for the reconstitution of the lyophilised component(s).
  • the container may itself be a suitable vessel for carrying out the biological procedure, for example, a microtitre plate.
  • the kit may also optionally contain reaction vessels, mixing vessels and other components that facilitate the preparation of reagents or a test sample, or the carrying out of the biological procedure.
  • the kit may also include one or more instruments for assisting with obtaining a test sample, such as a syringe, pipette, forceps, or the like.
  • reagents comprised by the kit or their containers may be colour-coded to facilitate their use.
  • reagents are colour-coded, addition of one reagent to another in a particular step may for example result in a change in the colour of the mixture, thus providing an indication that the step was carried out.
  • the kit can optionally include instructions for use, which may be provided in paper form or in computer-readable form, such as a disc, CD, DVD or the like.
  • the kit may also comprise computer readable media comprising software that assists in the interpretation of results obtained from using the kit.
  • kits may be employed in research into, for example, the des-acyl:acyl ghrelin ratio, the role of des-acyl ghrelin, catabolism of fats and carbohydrates, the relationship between ghrelin deletion and metabolic fuel preference, the role of ghrelin in alterations in food preference and cravings for other substances or activities, the role of ghrelin in stress-related eating and/or the role of ghrelin in the development of posttraumatic stress disorder.
  • Certain embodiments of the invention relate to therapeutic kits or packs containing a compound of general formula I.
  • kits Individual components of the kit would typically be packaged in separate containers and, associated with such containers, can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, for use or sale for human or animal administration.
  • the compounds of general formula (I) are provided in the kit in the form of pharmaceutical compositions suitable for administration to a subject.
  • the container may itself be an inhalant, syringe, pipette, eye dropper, or other such like apparatus, from which the composition may be administered to the subject.
  • one or more of the components of the kit can be lyophilized and the kit can additionally contain a suitable solvent for reconstitution of the lyophilized component(s).
  • the kit comprises a transdermal patch comprising a formulation of a compound of general formula (I) which is suitable for application to the skin.
  • the first 10 amino acids of the sequence correspond to the N-terminal sequence of human ghrelin, but with the serine at position 3 substituted with alanine.
  • the remaining amino acids are derived from the TAT sequence.
  • the final CF801 molecule used in the Examples below included an amidated C-terminus, i.e. GSAFLSPEHQRKKRRQRRR-NH 2 (SEQ ID NO:93)
  • CF801 differs in several respects from the Go-CoA-Tat compound described International Patent Application No. PCT/US2009/057512 (WO 2010/039461) with the goal of 1) reducing cost and 2) simplifying synthesis. To achieve these goals, CF801 was designed to be composed solely of amino acids commonly used in peptide synthesis.
  • Go-CoA-Tat contains a Dap3 moiety at the third residue, with a long carbon chain attached to this residue. Additionally, Go-CoA-Tat has an amino-hexanoic acid linker linked to a TAT peptide at the C-terminus.
  • Go-CoA-Tat is more in line with the ternary structure formed during acylation of ghrelin, after the fatty acid is esterified to the ghrelin molecule, as the new species dissociates from the GOAT enzyme.
  • CF801 represents the structure before acylation occurs, that is, it resembles an unacylated ghrelin molecule that potentially could act as a GOAT inhibitor by competing for GOAT but, as it cannot become acylated itself, simply occupying GOAT's active site.
  • CF801 also differs from pentapeptide GSAFL identified by Yang et al. (2008, PNAS, 105(31): 10750-10755) due to the extended sequence that includes additional ghrelin amino acids to mimic more closely a ghrelin molecule, and to provide additional spacing for a TAT sequence with an amidated C-terminus to improve cell penetration.
  • the GSAFL-NH 2 pentapeptide developed by Yang at al. consists of the first five amino acids of unacylated ghrelin with the third residue replaced with an alanine. While the peptide showed inhibitory activity on GOAT it was unlikely that the peptide would effectively cross cell membranes into the intracellular area where GOAT performs its enzymatic reactions.
  • the design of CF801 involved extending and modifying the original GSAFL-NH2 sequence.
  • the first modification was the addition of five more amino acids corresponding to amino acids 6 through 10 of the full-length ghrelin peptide, increasing the similarity to ghrelin as well as providing an extra spacer for the addition of an HIV trans-activator of transcription (Tat) sequence.
  • the Tat sequence utilized consists of the amino acids RKKRRQRRR (SEQ ID NO:52) with an amide group retained on the C-terminus.
  • the addition of the Tat sequence along with the C-terminal amide group are designed to improve cell penetration of the inhibitor. Other cell penetration peptides could be used in place of the Tat sequence to achieve this function.
  • CF801 peptide was synthesized on Rink-mBHA resin (Peptides International, Louisville KY) using Fmoc/tBu with disopropylcarbodiimide/6-CI-HOBT for coupling. Cleavage from the solid support was performed using a TFA-mediated acidolytic procedure for two hours at room temperature. Preparative reverse phase-HPLC using a linear gradient of 0.05% TFA in water vs MeCN was employed to purify the product to >95%. The product was then lyophilized and characterized by analytical RP-HPLC and ES-MS.
  • EXAMPLE 1 CF801 Decreases Plasma Acylated Ghrelin, Body Mass, And Fat Mass, While Increasing Regular Chow And Decreasing High-Fat Diet Intake
  • NIC non-injected control
  • LD low dose CF801
  • HD high dose CF801
  • Post-mortem body composition was analyzed by Echo MRI to determine lean and fat mass composition shortly after decapitation and storage. Measurements of fat mass, lean mass, and total water were obtained.
  • Cohort 2 Rebound Feeding and Plasma Acylated Ghrelin Levels
  • ELISA enzyme-linked immunosorbent assay
  • Body composition using EchoMRI after 13 days of treatment revealed a significantly reduced fat mass in high-dose animals compared to vehicle animals, with no difference in lean mass or total water mass (Figure 4B). This is similar to Go-CoA- Tat treatment, where the difference in body weight was attributed to a reduction in fat mass.
  • EXAMPLE 2 CF801 Effects On Food Intake, Weight, Anxiety, And Acylated Ghrelin Levels During Chronic Social Defeat Stress
  • animals were housed under standard laboratory conditions and received ad libitum access to standard laboratory mouse chow and tap water, with daily 4-hour access to a high- fat diet (60% caloric content from fat; 10 am to 2 pm). Injections of vehicle (0.9% saline) or CF801 (22 ⁇ 1 ⁇ ) were given daily, or no injection for non-injected controls. Injections and stressors began after a two-week baseline.
  • Plasma acylated ghrelin levels as well as corticosterone levels were measured with an enzyme-linked immunosorbent assay (ELISA) kit and an RIA kit, respectively.
  • ELISA enzyme-linked immunosorbent assay
  • CF801 eliminates stress-induced increases in plasma acylated ghrelin
  • CF801 has no effect on stress-induced anxiety, but demonstrates a possible reduction in locomotor activity
  • CF801 treatment during chronic social defeat stress leads to the following results: 1) Reduced high- fat intake, compared to vehicle and non-injected controls.
  • CF801 can be used in the study and/or treatment of stress, in particular chronic social defeat stress, leading to significant reductions in acylated ghrelin levels as well as affecting feeding, caloric preference, and total caloric intake during times of stress. These observed differences can also aid in interpreting acylated ghrelin's role during chronic social defeat stress.
  • mice were fasted overnight on the 8th day of the treatment period. On the morning of the 9 th treatment day, mice were given their daily injection of CF801 followed by a bolus of 20% glucose (2mg/kg). Blood glucose was measure using a glucose meter (Bayer Inc) at the time of injection (time 0), 15, 30 and 60 minutes following injection.
  • mice were sacrificed by rapid decapitation. Circulating levels of blood glucose were measured form trunk blood using a glucose meter (Bayer Inc). Remaining trunk blood was collected and spun down at 3,000Xg for 15 minutes to separate blood plasma from red blood cells. Aliquots of blood plasma were frozen at -80°C until processed. Circulating corticosterone was measured from blood plasma samples using a Radiolmmuno Assay (RIA) kit.
  • RIA Radiolmmuno Assay
  • CF801 reduces respiratory exchange ratio for ⁇ 3hours post-injection.
  • CF801 decreases circulating blood glucose in fasted animals, however does not change tolerance to a glucose bolus
  • CF801 increases circulating levels of corticosterone in non- fasted animals.
  • the reduction in respiratory exchange ratio and heat production indicate that the animals are preferentially metabolizing fat as a fuel source, instead of carbohydrates.
  • EXAMPLE 4 CF801 Reduces Fasting Induced Increases In Circulating Acylated Ghrelin And Reduces Acylated :Des-Acyl Ghrelin Ratio
  • mice 30 C57BL6J male mice were given a 14-day baseline period, wherein they had ad libitum access to regular chow, high-fat diet (60% calories from fat; Harlan Diets) and water.
  • mice were subjected to an overnight fast (excluding animals in the non-fasted group). The following morning, mice were given an injection of vehicle (0.9% saline), low-dose CF801 (11 ⁇ /kg) or high-dose CF801 (22 ⁇ 1 ⁇ ). Exactly 3 hours after receiving an injection, mice were sacrificed by rapid decapitation. Trunk blood was collected for hormonal analysis. Brains were rapidly extracted and the mediobasal hypothalamus was dissected and frozen immediately at - 80°C in 500 ⁇ TRIzol® reagent to preserve RNA integrity. Similarly, the fundus of the stomach was dissected and frozen at -80°C immediately in 500 ⁇ of TRIzol® until processed.
  • trunk blood was collected in EDTA-coated tubes placed on ice and centrifuged at 3000 X g for 15 minutes to separate plasma from red blood cells. Blood plasma was aliquoted separately for each assay to avoid multiple freeze/thaw cycles, and stored at -80°C until processed.
  • acylated ghrelin molecule was treated with of 1.0N HC1 and of lOOmM 4-(hydroxymercuri) benzoic acid before storage. Plasma acylated ghrelin, total ghrelin and insulin were measured using an ELISA kit (Millipore). All samples had a coefficient of variation ⁇ 10%.
  • the HomaIR is a method used to quantify insulin resistance and beta-cell function.
  • Matthews et al. (1985, Diabetologia, 28(7):412-9) first described it under the name HOMA in 1985.
  • the HOMA authors used data from physiological studies to develop a mathematical equations describing glucose regulation as a feedback loop. Therefore, insulin resistance and B-cell function can be estimated from fasting glucose and insulin levels.
  • Mediobasal hypothalamic gene expression was analyzed in a separate cohort of animals who were not fasted, but received no injection (NIC), vehicle injection, low- dose or high-dose CF801. To this end, total RNA from the mediobasal hypothalamus was isolated with TRIzol® and precipitated with ⁇ 3 ⁇ L ⁇ of linear acrylamide. RNA quality and concentrations were determined by absorbance at 280nm and 260nm with a Thermo Scientific Nanodrop 100 spectrophotometer (Thermo Scientific, Rockford, Illinois).
  • 1 oligo(dT) primer (Invitrogen, Carlsbad, California) was added to 9 yL of mRNA and heated to 70°C for 5 minutes.
  • a master mix composed of 4 ⁇ . of 5X first-strand buffer (Invitrogen), 2 ⁇ . of 0.1M dithiothreitol (Invitrogen), l L of RNase inhibitor (Promega Corp, Madison, Wisconsin), l L of lOmM deoxynucleotide triphosphate (Invitrogen), l L of diethylpyrocarbonate water, and of SS2 reverse transcriptase (Invitrogen) were added.
  • Results show that fasting caused a significant increase in total ghrelin (both des-acyl ghrelin and acylated ghrelin; Figure 13) and a significant decrease in circulating blood glucose and insulin (Figure 14).
  • Animals receiving both low-dose and high-dose CF801 showed a significant reduction in circulating acylated ghrelin levels compared to animals receiving vehicle injections.
  • the acylated ghrelin: des-acyl ghrelin ratio tended to be lower in animals receiving both low-dose and high-dose CF801 relative to animals receiving vehicle injections.
  • CF801 reduces circulating acylated ghrelin levels.
  • CF801 does not influence the amount of circulating total ghrelin and des-acyl ghrelin.
  • CF801 reduces circulating blood glucose, even further then vehicle injected fasted animals.
  • CF801 does not change the mRNA expression pattern of NPY, AgRP or SOCS3 in the mediobasal hypothalamus of non-fasted animals. Animals receiving high-dose CF801 showed a slight reduction of POMC in the mediobasal hypothalamus compared to vehicle injected animals.
  • CF801 significantly reduces acylated ghrelin in a fasted animal without influencing total ghrelin levels (acylated and des-acyl ghrelin).
  • EXAMPLE 5 CF801 Reduces GOAT And Ghrelin mRNA Expression In The Fundus Following An Overnight Fast In Wild-Type And GHSR Knock-Out Mice
  • Example 5 Materials and Methods
  • mice received an intraperitoneal injection of either saline or CF801 (22 ⁇ 1 ⁇ ) and were sacrificed exactly three hours later by rapid decapitation. Trunk blood was collected and plasma was separated from red blood cells, aliquoted and stored at -80°C until processed. Samples of the fundus were dissected, placed in TRIzol® reagent and stored at -80°C until processed.
  • trunk blood was collected in EDTA-coated tubes placed on ice and centrifuged at 3000 X g for 15 minutes to separate plasma from red blood cells. Blood plasma was aliquoted separately for each assay to avoid multiple freeze/thaw cycles, and stored at -80°C until processed. Insulin was measured using an ELISA kit (Millipore). All samples had a coefficient of variation ⁇ 10%.
  • RNA from the fundus of the stomach was isolated with 500 ⁇ of TRIzol® and precipitated with ⁇ 3 ⁇ L ⁇ of linear acrylamide. RNA quality and concentrations were determined by absorbance at 280nm and 260nm with a Thermo Scientific Nanodrop 100 spectrophotometer (Thermo Scientific, Rockford, Illinois). To synthesize cDNA, ⁇ , oligo(dT) primer (Invitrogen, Carlsbad, California) was added to of mRNA and heated to 70°C for 5 minutes. To each sample, a master mix composed of 4 ⁇ . of 5X first-strand buffer (Invitrogen), 2 ⁇ .
  • RT-qPCR was conducted on all cDNA samples to determine fold changes using the 2 " ⁇ method using primers detecting the glyceraldehyde 3-phosphate dehydrogenase gene as a control transcript. Briefly 5 ⁇ L ⁇ of each cDNA sample were added to separate wells in a PCR plate. Two microliters of working primer solution, 3 ⁇ L ⁇ of Milli-Q water, and 10 ⁇ L ⁇ of iQ SYBR Green Super Mix with fluorescein (Bio-Rad Laboratories, Hercules, California) were added to each well. Samples were run in duplicate with non-template controls. Primers for GOAT and ghrelin were tested for amplification efficiency using the standard curve method, yielding efficiencies between 95% and 105%. Example 5 Results
  • the homeostatic model of assessment for insulin resistance (HomaIR) was significantly reduced in both WT and GHSR KO animals receiving CF801 compared to their non-stressed controls (Figure 16C).
  • CF801 decreases plasma levels of blood glucose and insulin in both WT and GHSR KO animals. There was a tendency for the reduction in glucose and insulin to be more pronounced in WT animals relative to GHSR KO animals, however this did not attain statistical significance.
  • CF801 potently decreases GOAT expression in the fundus of the stomach in fasted WT and GHSR KO animals.
  • Example 4 As noted above, in this Example, a significant reduction in HomaIR was seen for the CF801 -treated animals, whereas in Example 4 no significant difference was observed between CF801 -treated and control animals. The reason for this difference is most likely due to the limited number of animals included in the HomaIR analysis of Example 4. Specifically, when running the analysis insufficient sample blood remained after total ghrelin and acylated ghrelin measurement to allow a data point for each animal to be obtained. As a result, the number of animals included in the HomaIR scores was reduced in Example 4. With the low number of data points included in the analysis, the effect did not attain statistical significance (p-value 0.140, saline vs. HD CF801) although a visual trend was seen wherein animals receiving the high-dose CF801 in Example 4 seemed to have lower HomaIR scores compared to animals receiving vehicle.
  • GOAT enzymatic activity was measured using the hGOAT assay as previously described by Darling et al (2013, Analytical Biochemistry, 437(l):68-76). Briefly, membrane fractions from Sf9 cells expressing GOAT were thawed on ice and passed through an 18-guage needle 10 times. Assays were initiated by the addition of the acrylodanylated peptide substrate. Assays were incubated at room temperature and stopped by the addition of 50 micro-liters of 20% acetic acid in isopropanol; the addition of the stop solution prior to the acrylodanylated peptide substrate blocked peptide octanoylation.
  • the concentrations required to inhibit the activity of SGI cells is very high, however the SGI cells are known to overexpress both GOAT and ghrelin (Sakata et al, 2013, ibid.) and thus have basal ghrelin levels that are not physiologically relevant.
  • the high concentration of CF801 (i.e. 300 ⁇ ) required to inhibit ghrelin secretion in the SGI cell line is most likely due to the dramatically elevated ghrelin levels seen in this cell line.
  • CF801 decreases ghrelin secretion in the overexpressing SGI cells in vitro at the 300 ⁇ concentration level.
  • CF801 reduces acylated ghrelin levels is not dependent on GOAT activity in vitro.
  • High doses of CF801 i.e. 300 ⁇ significantly reduce secretion of acylated ghrelin for SGI ghrelinoma cells in vitro.
  • CF801 may be used in many different kinds of experiments. These include but are not limited to: 1. Additional experiments examining the role of ghrelin during stress. These include experiments involving different types of stressors, in addition to the chronic social defeat stress paradigm discussed in Example 2. Briefly, a similar protocol to Example 2 is performed with application of various other stressors, including physical and metabolic stressors, such as restraint stress and fox urine. Additional measures are performed, such as a forced-swim task to measure stress-induced depression, along with measurements of the effects CF801 on these measures.
  • Calorimetry experiments such as substrate-utilization experiments with metabolic chambers on mice given CF801. Animals are given doses of CF801 and placed in metabolic chambers capable of measuring respiratory exchange ratio. Changes in RER indicate changes in preferred substrates for metabolism. Time-dependent changes in RER, relative to vehicle injected animals, can also be used to indicate duration since injection to drug effectiveness as well as duration of the drug effect.
  • Glucose tolerance tests to measure CF801's effects on insulin levels and in studies examining diabetes. Briefly, blood glucose is measured using a glucose strip prior to intraperitoneal injection of a glucose solution. Repeated blood glucose measurements are performed post injection for a specific duration of time. Effects of CF801 treatment on blood glucose levels are measured.
  • CF801 Central administration of CF801, such as by ICV to examine the effect on such variables as mass and anxiety to determine if ghrelin may be present and acylated centrally.
  • Current results suggest rapid early effects on feeding when given continuously through osmotic mini -pumps. Treatment by daily injection through the cannula can be performed. 6.

Abstract

Compounds of general formula (I) and their use to reduce circulating acylated ghrelin levels are provided. GSA-X-Y-B (I) in which "GSA-X" is a ghrelin moiety, "Y" is an optional peptide linker and "B" is a CPP moiety. The compounds of general formula (I) may be used as research tools to investigate the roles of ghrelin and acylated ghrelin, as well as in therapeutic contexts to treat diseases or disorders associated with obesity, weight gain and/or adiposity, as well as diabetes, pre-diabetes or metabolic syndrome.

Description

COMPOUNDS FOR REGULATING ACYLATED GHRELIN
FIELD OF THE INVENTION
[0001] The present invention relates to the field of ghrelin regulation and, in particular, to peptidic compounds capable of regulating acylated ghrelin levels.
BACKGROUND OF THE INVENTION
[0002] Ghrelin was discovered in 1999 (Kojima, M., et al., 1999, Nature, 402(6762):656-660) and quickly identified as a strong obesogenic hormone that, in its acylated form, not only increases food intake, but also increases preference for fatty foods and protects fat reserves within the body (Perello, M., et al., 2010, Biological Psychiatry, 67(9):880-886; Tschop, M., D.L. Smiley, and M . Heiman, 2000, Nature, 407(6806):908-913; Wren, A.M., et al, 2001, J. Clinical Endocrinology & Metabolism, 86(12):5992; and Patterson, Z.R., et ah, 2013, Endocrinology, 154(3):1080-1091).
[0003] Once secreted, ghrelin seeks to bind to the growth hormone secretagogue receptor- la (GHSR-la), the only known receptor for ghrelin. The ability of ghrelin to bind to the GHSR-la, and thus carry out its biological activity, is dependent on a post- translation modification of the mature ghrelin protein, wherein an n-octanoic acid is added to the third serine residue of the ghrelin molecule. Modification with an n- octanoyl group induces a conformational change that renders the ghrelin molecule biologically active. The enzyme responsible for the acylation of ghrelin is ghrelin-O- acyl-transferase (GOAT), a member of the membrane bound-O-acyl-transferase (MBOAT) family. To date, GOAT is the only known enzyme capable of acylating ghrelin and it is therefore thought to be involved in all processes related to ghrelin. Once activated, ghrelin promotes food intake and body weight through interactions with GHSR-la, primarily in the hypothalamus. [0004] Circulating acylated ghrelin levels rise prior to a meal when the animal is hungry, and gradually decrease during and after feeding as the animal becomes satiated. Injections of acylated ghrelin promote feeding behaviour and adiposity.
[0005] In addition to its role in obesity, acylated ghrelin has also been identified as an important component of the stress response. During a stressor, acylated ghrelin levels increase and appear to have a protective effect by redirecting the use of appropriate energy resources (Asakawa, A., et ah, 2001, Neuroendocrinolog , 74:143-147). However, depending on the many other variables involved, increases in ghrelin in response to stress may result in changes in appetite and feeding patterns. While this increase in ghrelin during a stressor appears to have a protective effect, chronic elevations may lead to a decrease in the protective effects of ghrelin and potentially to an increase in metabolic disruptions and obesity (Patterson, Z.R., et ah, 2013, ibid.).
[0006] In relation to type II diabetes, acylated ghrelin strongly influences glucose metabolism. A reduction in ghrelin levels improves insulin sensitivity and is a promising research target for understanding diabetes as well as treating it (Wiedmer, P., et ah, 2007, Nat Clin Pr act End Met, 3(10):705-712).
[0007] Analogues of ghrelin for therapeutic uses have been described. For example, International Patent Application No. PCT/US2009/057512 (WO 2010/039461) describes the GOAT inhibitor Go-CoA-Tat and related compounds. Go-CoA-Tat was designed to represent two-thirds of a stable ternary structure formed when GOAT, ghrelin, and a fatty acid combine in the esterification reaction to acylate the ghrelin molecule. This design allows stabilization of the GOAT molecule during binding, competing for the enzyme's active site.
[0008] Yang et al. (2008, PNAS, 105(31):10750-10755) identified several pentapeptides that inhibited GOAT activity in GOAT-transfected homogenized cell extracts. One such pentapeptide was GSAFL. This peptide, however, is unlikely to cross cell membranes, which it must do in order to inhibit GOAT in intact cells.
[0009] U.S. Patent Application No. 09/902,556 (US2002/0187938) describes peptides having antagonistic properties to ghrelin. The peptides are based on the ghrelin sequence, comprise between 5 and 10 amino acids and include an octanoyl modification on the third residue.
[0010] U.S. Patent Application No. 10/522,398 (US2005/0272648) and International Patent Application No. PCT/US2006/038027 (WO 2007/041278) describe peptidyl analogues that possess agonist or antagonist ghrelin activity and therapeutic uses thereof. The peptidyl analogues comprise between 6 and 28 amino acids and include at least one modified amino acid.
[0011] U.S. Patent Application No. 13/320,408 (US2012/0135918) describes methods of treating obesity, obesity -related disorders, diabetes and metabolic syndrome using a GOAT inhibitor and/or a ghrelin receptor antagonist. The GOAT inhibitor and ghrelin receptor antagonist may each be peptidic compounds comprising an octanoyl or similar modification.
[0012] Analogues of des-acyl ghrelin have also been described. Granata et al. 2012, J Med Chem, 55:2585-2596) describe screening various fragments of des-acyl ghrelin to identify fragments with similar activity in promoting survival of pancreatic β-cells and human pancreatic islets to full-length des-acyl ghrelin. The most active fragment identified consisted of residues 6-13 of des-acyl ghrelin. A cyclised version of this sequence was also active. ien et al. (2012, Eur. J. Pharm. Sci., 47:625-637) investigated the in vitro and in vivo stability of the linear and cyclized forms of the amino acid 6-13 fragment and found the cyclized version (AZP531) to be completely protected from degradation. Subsequent studies using AZP531 found that this compound prevents high-fat diet induced dysregulation of glucose homeostasis (Delhanty et al, 2013, FASEB J, 27:1690-1700).
[0013] This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention. SUMMARY OF THE INVENTION
[0014] The present invention relates generally to compounds for regulating acylated ghrelin. An aspect of the invention relates to a compound of general formula (I)
GSA-X-Y-B (I)
[0015] wherein:
X is absent or a sequence of between 1 and 25 consecutive amino acids of the sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 57, or between 1 and 24 consecutive amino acids of the sequence as set forth in SEQ ID NO: 4 or SEQ ID NO: 77;
Y is absent or a peptide linker of between 2 and 10 amino acids in length and comprising amino acids selected from the group of glycine, alanine, valine, lysine and isoleucine;
wherein GSA-X-Y is at least 4 amino acids in length;
and
B is a cell-penetrating peptide. [0016] Another aspect of the invention relates to a pharmaceutical composition comprising a compound of general formula (I), and a pharmaceutically acceptable carrier or diluent.
[0017] Another aspect of the invention relates to a kit for research use comprising a compound of general formula (I), and instructions for use.. [0018] Another aspect of the invention relates to a use of a compound of general formula (I) for decreasing levels of acylated ghrelin in a subject.
[0019] Another aspect of the invention relates to a use of a compound of general formula (I) for treatment or management of obesity in a subject.
[0020] Another aspect of the invention relates to a use of a compound of general formula (I) for treatment or management of diabetes in a subject. [0021] Another aspect of the invention relates to a use of a compound of general formula (I) for treatment or management of pre-diabetes or metabolic syndrome in a subject.
[0022] Another aspect of the invention relates to a use of a compound of general formula (I) for modifying eating behaviour in a subject.
[0023] Another aspect of the invention relates to a use of a compound of general formula (I) for treatment or management of post-traumatic stress disorder in a subject.
[0024] Another aspect of the invention relates to a use of a compound of general formula (I) for the treatment or management of an addictive behaviour in a subject. [0025] Another aspect of the invention relates to a method of decreasing acylated ghrelin levels in a subject comprising administering to the subject an effective amount of a compound of general formula (I).
[0026] Another aspect of the invention relates to a method of treating or managing obesity in a subject comprising administering to the subject an effective amount of a compound of general formula (I).
[0027] Another aspect of the invention relates to a method of treating or managing diabetes in a subject comprising administering to the subject an effective amount of a compound of general formula (I).
[0028] Another aspect of the invention relates to a method of treating or managing pre-diabetes or metabolic syndrome in a subject comprising administering to the subject an effective amount of a compound of general formula (I).
[0029] Another aspect of the invention relates to a method of modifying eating behaviour in a subject comprising administering to the subject an effective amount of a compound of general formula (I). [0030] Another aspect of the invention relates to a method of treating or managing post-traumatic stress disorder in a subject comprising administering to the subject an effective amount of a compound of general formula (I). [0031] Another aspect of the invention relates to a method of treating or managing an addictive behaviour in a subject comprising administering to the subject an effective amount of a compound of general formula (I).
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] These and other features of the invention will become more apparent in the following detailed description in which reference is made to the appended drawings.
[0033] Figure 1: Body weight of animals with choice of high- fat diet or regular chow (ad lib). NIC: Non-injected controls (n = 6), VEH: Vehicle controls (0.9% saline, n =
7) , LD: Low dose CF801 (11 μιηοΐ/kg, n = 8), HD: High dose CF801 (22 μιηοΐ/kg, n =
8) . High dose animals showed significantly reduced body weight compared to vehicle animals after two days of treatment, continuing until end of treatment (p < 0.05).
[0034] Figure 2: Caloric intake of animals during baseline (B) and treatment (E). No differences were found between high dose (HD), low dose (LD), vehicle (VEH), and non-injected control (NIC) animals throughout baseline and treatment (A). Similarly, no differences were found when caloric intake was normalized to body mass (B). [0035] Figure 3: (A) Average daily regular chow consumption normalized to body mass during baseline and treatment. Animals showed no significant difference in average chow consumption, normalized to weight, during baseline. High dose animals consumed significantly more regular chow on average compared to vehicle animals during treatment (p < 0.05). (B) Average daily high-fat diet consumption normalized to body mass during baseline and treatment. High-dose animals (HD) showed a near-significant decrease in high-fat consumption normalized to body weight during treatment compared to vehicle animals (VEH) (p = 0.053).
[0036] Figure 4: (A) Metabolic efficiency (mass gained in grams per total grams consumed from start to end of baseline or treatment). No differences in metabolic efficiency were found between groups during baseline. Both low dose and high dose animals had significantly reduced metabolic efficiency compared to vehicle and non -injected controls during treatment (p < 0.01). (B) Fat mass, lean mass, and total water mass of animals following CF801 treatment, as measured by EchoMRI. High-dose animals had significantly lower fat mass compared to vehicle.
[0037] Figure 5: Open field test for anxiety. Following treatment, no differences were found in time spent around the border or in the center of an open-field apparatus between non-injected controls (NIC), vehicle (VEH), low-dose (LD), and high-dose (HD) animals.
[0038] Figure 6: (A) Circulating acylated ghrelin levels after CF801 treatment, measured with enzyme linked immunosorbent assay (ELISA). Plasma acylated ghrelin levels showed a dose-dependent decrease with CF801 treatment. (B) Food consumed following 24 hour fast. High-dose animals consumed significantly less food compared to vehicle controls over a four-hour re-feeding period following reintroduction of food.
[0039] Figure 7: (A) Chronic social defeat stress leads to an increase in regular chow intake and (B) a decrease in high-fat intake. * p < 0.05.
[0040] Figure 8: (A) Stressed animals consumed significantly less high- fat diet when given CF801 treatment, when compared to vehicle and non-injected stressed controls (p < 0.05). (B) Stressed animals consumed significantly more regular chow compared to vehicle-injected stressed animals (p < 0.05). (C) CF801 treatment resulted in a significant reduction in average daily caloric intake in stressed animals. This reduction did not reach significance in non-stressed animals, demonstrating an interaction between drug treatment and stress group.
[0041] Figure 9: (A) CF801 treatment of animals exposed to chronic social defeat stress leads to a reduced body weight. (B) Non-injected and vehicle- injected animals show an increase in plasma acylated ghrelin when exposed to stress. This increase is not observed when animals are treated with daily injections of CF801. [0042] Figure 10: Stressed animals displayed typical responding in the elevated plus maze, with (A) reduced time spent in open arms and (B) no difference in number of entries into the open arms. (C) Stressed animals treated with CF801, however, had fewer entries into the open arms when compared to vehicle-injected stressed controls (p < 0.05).
[0043] Figure 11: (A) 24 hour respiratory exchange ratio; (B) 24 hour locomotor activity, and (C) 24 hour heat production in animals receiving vehicle injections (n = 5) and CF801 injections (n = 5). Data represents mean +/- S.E.M. * p < 0.05 relative to vehicle injected controls.
[0044] Figure 12: (A) Glucose tolerance test. Following an overnight fast, animals were given a 2mg/kg bolus of 20% glucose. (B) Circulating levels of corticosterone measured from trunk blood collected at the time of sacrifice. Data represents mean +/- S.E.M. * p < 0.05 relative to vehicle injected controls.
[0045] Figure 13: Circulating levels of des-acyl ghrelin (A), acylated ghrelin (B) and total ghrelin (C) in blood plasma samples collected from trunk blood at the time of sacrifice. Acylated ghrelin:des-acyl ghrelin ratio (D). All values are expressed as mean ± S.E.M. p < 0.05 relative to all other groups; * p < 0.05 relative to vehicle injected controls; φρ < 0.10 relative to vehicle injected controls.
[0046] Figure 14: Circulating levels of blood glucose (A) and insulin (B) at the time of sacrifice. Homeostatic model assessment for insulin resistance (HomaIR; C) was calculated as follows: HomaIR = [(Insulin*Glucose) / 22.5]. All values are expressed as mean ± S.E.M. ρ < 0.05 compared to all other groups, δρ < 0.05 compared to vehicle injected controls.
[0047] Figure 15: mRNA expression of NPY (A), AgRP (B), POMC (C) and SOCS3 (D) in the mediobasal hypothalamus. All values are expressed as mean +/- SEM, relative to GAPDH expression. * p < 0.05 relative to non-injected controls.
[0048] Figure 16: Circulating levels of blood glucose (A), insulin (B) and HomaIR (C) at the time of sacrifice. All values are expressed as mean ± S.E.M. * p < 0.05 compared to vehicle injected control. [0049] Figure 17: mR A expression of GOAT (A) and ghrelin (B) in the fundus of the stomach. All values are expressed as mean ± S.E.M., relative to GAPDH and β- Actin. *** p < 0.0001; * p < 0.05 relative to saline injected controls.
[0050] Figure 18: (A) Enzymatic activity of GOAT with increasing concentrations of CF801. (B) Ghrelin concentrations following application of CF801 to ghrelin secreting SGI cells in vitro.
DETAILED DESCRIPTION OF THE INVENTION
[0051] The present invention relates to peptidic compounds of general formula (I), which comprise a ghrelin moiety linked, optionally via a peptide linker, to a cell penetrating peptide moiety. These compounds are linear peptides based on the sequence of ghrelin, which do not include modified amino acids. However, the compounds surprisingly show sufficient stability and activity to effectively decrease circulating acylated ghrelin levels in vivo.
[0052] As demonstrated herein, compounds of general formula (I) are capable of reducing circulating acylated ghrelin levels, reducing fat mass and intake of high-fat foods in test animals, blunting rebound feeding in fasting animals and reducing stress- induced calorie intake. Accordingly, in certain embodiments, the compounds of general formula (I) may be useful in the treatment of diseases or disorders associated with obesity, weight gain and/or adiposity. In some embodiments, the compounds of general formula (I) may find use as part of weight management programs or treatment protocols. In some embodiments, the compounds of general formula (I) may find use in stress management programs or treatment protocols. In certain embodiments, the compounds of general formula (I) may find use in the treatment of diabetes, prediabetes or metabolic syndrome.
[0053] Additional embodiments of the invention relate to the use of compounds of general formula (I) as research tools to assist in the elucidation of the role of acylated ghrelin, for example, in stress, food preferences, body composition and/or weight gain, as well as to study of the effects of changes in the acyl:des-acyl ghrelin ratio and the role of des-acyl ghrelin. Definitions
[0054] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. [0055] The terms "therapy" and "treatment," as used interchangeably herein, refer to an intervention performed with the intention of alleviating the symptoms associated with, preventing the development of, or altering the pathology of a disease, disorder or condition. Thus, the terms therapy and treatment are used in the broadest sense, and in various embodiments include one or more of the prevention (prophylaxis), moderation, reduction, and/or curing of a disease, disorder or condition at various stages. Subjects in need of therapy/treatment thus may include those already having the disease, disorder or condition as well as those prone to, or at risk of developing, the disease, disorder or condition and those in whom the disease, disorder or condition is to be prevented.
[0056] The terms "subject" and "patient" as used herein both refer to an animal in need of treatment. The subject or patient may be a human or non-human animal. In certain embodiments, the subject or patient is a human.
[0057] The use of the word "a" or "an" when used herein in conjunction with the term "comprising" may mean "one," but it is also consistent with the meaning of "one or more," "at least one" and "one or more than one." [0058] As used herein, the terms "comprising," "having," "including" and "containing," and grammatical variations thereof, are inclusive or open-ended and do not exclude additional, unrecited elements and/or method steps. The term "consisting essentially of when used herein in connection with a composition, use or method, denotes that additional elements and/or method steps may be present, but that these additions do not materially affect the manner in which the recited composition, method or use functions. The term "consisting of when used herein in connection with a composition, use or method, excludes the presence of additional elements and/or method steps. A composition, use or method described herein as comprising certain elements and/or steps may also, in certain embodiments consist essentially of those elements and/or steps, and in other embodiments consist of those elements and/or steps, whether or not these embodiments are specifically referred to.
[0059] As used herein, the term "about" refers to an approximately +/-10% variation from a given value. It is to be understood that such a variation is always included in any given value provided herein, whether or not it is specifically referred to.
[0060] It is contemplated that any embodiment discussed herein can be implemented with respect to any disclosed method, use or composition, and vice versa. Furthermore, compositions and kits of the invention can be used to achieve the disclosed methods and uses.
[0061] Naturally occurring amino acids are identified throughout by the conventional three- or one-letter abbreviations indicated in Table 1 below, which are as generally accepted in the peptide art and recommended by the IUPAC-IUB commission in biochemical nomenclature.
Table 1. Amino acid codes
Name 3-Letter 1-Letter Name 3-Letter 1-Letter
Code Code Code Code
Alanine Ala A Leucine Leu L
Arginine Arg R Lysine Lys K
As aragine Asp N Methionine Met M
Aspartic Acid Asp D Phenylalanine Phe F
Cysteine Cys C Proline Pro P
Glutamic Acid Glu E Serine Ser S
Glutamine Gin Q Threonine Thr T
Glycine Gly G Tryptophan Trp w
Histidine His H Tyrosine Tyr Y
Isoleucine lie I Valine Val V
[0062] The peptide sequences set out herein are written according to the generally accepted convention whereby the N-terminal amino acid is on the left and the C- terminal amino acid is on the right. PEPTIDIC COMPOUNDS
[0063] In certain embodiments, the invention relates to peptidic compounds comprising a ghrelin moiety linked, optionally via a linker, to a cell-penetrating peptide (CPP) moiety. The compounds can be represented by general formula (I): GSA-X-Y-B (I)
[0064] in which "GSA-X" is a ghrelin moiety, "Y" is an optional peptide linker and "B" is a CPP moiety. Preferably the combined length of the ghrelin moiety and the optional linker (GSA-X- Y) is at least 4 amino acids.
[0065] A characteristic of the compounds of general formula (I) is the replacement of the serine residue that naturally occurs at position 3 of the ghrelin peptide with alanine such that the compound cannot be acylated. A shorter GSA-X sequence would be expected to aid in cell penetration and reduce cost. A longer X sequence is expected to better mimic ghrelin and therefore may aid in activity.
[0066] Thus, in certain embodiments, the ghrelin moiety comprised by the compounds is between 3 and 28 amino acids in length and comprises the sequence Gly- Ser-Ala optionally coupled to a sequence of between 1 and 25 consecutive amino acids of SEQ ID NO: 3 or SEQ ID NO:57, which respectively represent amino acids 4-28 of the naturally occurring human and rodent ghrelin sequence (SEQ ID NO: l and SEQ ID NO: 56), or between 1 and 24 consecutive amino acids of SEQ ID NO: 4 or SEQ ID NO:77, which represent amino acids 4-27 of des Gln-14 ghrelin (SEQ ID NO:2 and SEQ ID NO:76) (Table 2).
[0067] In certain embodiments, the ghrelin moiety comprised by the compounds is at least 4 amino acids in length, for example, between 4 and 28 amino acids in length. In some embodiments, the ghrelin moiety is between 5 and 28 amino acids in length, for example, between 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 and 28 amino acids in length, or any range therebetween.
[0068] In certain embodiments, the ghrelin moiety comprised by the compounds has a sequence as set forth in any one of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, , SEQ ID NO : 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID
NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID
NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID
NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID
NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID
NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID
NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID
NO: 43, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID
NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID
NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID
NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID
NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID
NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID
NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, ! SEQ ID NO: 90 and SEQ ID NO: 91 (Table 2).
[0069] In certain embodiments, the ghrelin moiety comprised by the compounds has a sequence corresponding to a human ghrelin sequence or fragment thereof as set forth in any one of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 43 (Table 2).
[0070] In certain embodiments, the ghrelin moiety comprised by the compounds has a sequence corresponding to a mouse or rat ghrelin sequence or fragment thereof as set forth in any one of SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29 (Table 2).
[0071] In certain embodiments, the ghrelin moiety comprised by the compounds includes only the N-terminal region of the ghrelin sequence and thus the ghrelin moiety is between about 4 and 18 amino acids in length, for example, between about 4 and 17 amino acids in length or between about 4 and 16, 15, 14, 13, 12, 11, 10, 9, 8, 7 or 6 amino acids in length, or any range therebetween, and has a sequence corresponding to the N-terminal region of SEQ ID NO:5, 6, 58 or 78. In some embodiments, the ghrelin moiety comprised by the compounds has a sequence as set forth in any one of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 73, SEQ ID NO: 74 and SEQ ID NO: 75 (Table 2).
Table 2: Amino Acid Sequences Relating to Ghrelin Moiety GSA-X
Figure imgf000015_0001
Ghrelin Based Peptide Sequences Des Gln-14 Ghrelin Based Peptide
Sequences
SEQ SEQ ID Sequence ID Sequence
NO NO
9 GSAFLSPEHQRVQQRKESKKPPAK 34 GSAFLSPEHQRVQRKESKKPPAK
10 GSAFLSPEHQRVQQRKESKKPPA 35 GSAFLSPEHQRVQRKESKKPPA
11 GSAFLSPEHQRVQQRKESKKPP 36 GSAFLSPEHQRVQRKESKKPP
12 GSAFLSPEHQRVQQRKESKKP 37 GSAFLSPEHQRVQRKESKKP
13 GSAFLSPEHQRVQQRKESKK 38 GSAFLSPEHQRVQRKESKK
14 GSAFLSPEHQRVQQRKESK 39 GSAFLSPEHQRVQRKESK
15 GSAFLSPEHQRVQQRKES 40 GSAFLSPEHQRVQRKES
16 GSAFLSPEHQRVQQRKE 41 GSAFLSPEHQRVQRKE
17 GSAFLSPEHQRVQQRK 42 GSAFLSPEHQRVQRK
18 GSAFLSPEHQRVQQR 43 GSAFLSPEHQRVQR
19 GSAFLSPEHQRVQQ
20 GSAFLSPEHQRVQ
21 GSAFLSPEHQRV
22 GSAFLSPEHQR
23 GSAFLSPEHQ
24 GSAFLSPEH
25 GSAFLSPE
26 GSAFLSP
27 GSAFLS
28 GSAFL
29 GSAF
56 GSSFLSPEHQKAQQRKESKKPPAK 76 GSSFLSPEHQKAQRKESKKPPAKL LQPR QPR
57 LSPEHQKAQQRKESKKPPAKLQPR 77 LSPEHQKAQRKESKKPPAKLQPR
58 GSAFLSPEHQKAQQRKESKKPPAK 78 GSAFLSPEHQKAQRKESKKPPAKL LQPR QPR
59 GSAFLSPEHQKAQQRKESKKPPAK 79 GSAFLSPEHQKAQRKESKKPPAKL LQP QP
60 GSAFLSPEHQKAQQRKESKKPPAK 80 GSAFLSPEHQKAQRKESKKPPAKL LQ Q
61 GSAFLSPEHQKAQQRKESKKPPAK 81 GSAFLSPEHQKAQRKESKKPPAKL L Ghrelin Based Peptide Sequences Des Gln-14 Ghrelin Based Peptide
Sequences
SEQ SEQ ID Sequence ID Sequence
NO NO
62 GSAFLSPEHQKAQQRKESKKPPAK 82 GSAFLSPEHQKAQRKESKKPPAK
63 GSAFLSPEHQKAQQRKESKKPPA 83 GSAFLSPEHQKAQRKESKKPPA
64 GSAFLSPEHQKAQQRKESKKPP 84 GSAFLSPEHQKAQRKESKKPP
65 GSAFLSPEHQKAQQRKESKKP 85 GSAFLSPEHQKAQRKESKKP
66 GSAFLSPEHQKAQQRKESKK 86 GSAFLSPEHQKAQRKESKK
67 GSAFLSPEHQKAQQPvKESK 87 GSAFLSPEHQKAQRKESK
68 GSAFLSPEHQKAQQPvKES 88 GSAFLSPEHQKAQRKES
69 GSAFLSPEHQKAQQRKE 89 GSAFLSPEHQKAQRKE
70 GSAFLSPEHQKAQQRK 90 GSAFLSPEHQKAQRK
71 GSAFLSPEHQKAQQR 91 GSAFLSPEHQKAQR
72 GSAFLSPEHQKAQQ
73 GSAFLSPEHQKAQ
74 GSAFLSPEHQKA
75 GSAFLSPEHQK
[0072] The optional peptide linker of compounds of general formula (I) is a sequence of amino acids that may be between 2 and about 10 amino acids in length. In certain embodiments, the linker when present is between 2 and about 10 amino acids in length and comprises amino acids selected from glycine, alanine, valine, lysine and isoleucine. In certain embodiments, the linker when present is between 2 and about 10 amino acids in length and comprises amino acids selected from glycine and alanine. In some embodiments, the linker when present is a polyglycine or polyalanine peptide linker of between 2 and 10 amino acids in length. [0073] The peptide linker functions to spatially separate the ghrelin moiety from the CPP moiety. This function may also be achieved by the sequence of the ghrelin moiety itself. Thus, in certain embodiments, the compounds may include a longer ghrelin moiety, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more amino acids, and no peptide linker. [0074] The CPP moiety comprised by the compounds functions to enhance the cell permeability of the compound. A number of cell penetrating peptides are known in the art and may be included in the compounds as the CPP moiety. Examples include, but are not limited to, the peptide sequences shown in Table 3. Table 3: Cell-Penetrating Peptides
Figure imgf000018_0001
1 Ho A, et al, 2001, Cancer Res, 61 :474-477; 2 Derossi D, et al, 1994, J Biol Chem, 269: 10444-10450; 3 Elliott E and O'Hare P, 1997, Cell, 88:223-233; 4 Lin YZ, et al, 1995, J Biol Chem, 270: 14255-14258; 5 Lins L, et al, 2008, Biochim Biophys Acta, 1778: 1537-1544; 6 Mitchell DJ, et al, 2000, J Pept Res, 56:318-325; 7 Patel LN, et al, 2007, Pharm Res, 24: 1977-1992; 8 Futaki S, 2002, Int J Pharm, 245: 1-7; 9 Weller K, et al, 2005, Biochemistry, 44: 15799-15811 ; 10 Kim D, et al, 2006, Exp Cell Res, 312: 1277-1288; 11 Elmquist A, 2001, Exp Cell Res, 269:237-244; 12 Morris MC, et al, 1997, Nucleic Acids Res, 25:2730-2736; 13 Delaroche D, et al, 2007, Anal Chem, 79: 1932-1938. [0075] Variants of these sequences are known that function as CPPs and thus may be suitable for inclusion in the compounds of general formula (I). Examples include variants of the TAT sequence, such as RKKRRQRRR (SEQ ID NO: 52); GRKKRRQRRRPQ (SEQ ID NO:53) and GRKKRRQRRRPPQ (SEQ ID NO:54). [0076] In certain embodiments, the compounds include a CPP moiety that is based on TAT, penetratin or transportin.
[0077] In certain embodiments, the compounds may further comprise a modification at the N-terminus, the C-terminus or both. Such modifications may assist for example in purification, stability, bioavailability or the like. Various N- and C-terminal modifications are known in the art. N-terminal modifications include, for example, modification with groups such as acetyl, formyl, fatty acid, benzoyl, benzoyloxycarbonyl, bromoacetyl, pyroglutamyl, succinyl and teri-butoxycarbonyl. C- terminal modifications include, for example, modification with groups such as amide, ester and teri-butyl. [0078] In some embodiments, the compounds of general formula (I) include a modified C-terminus. In some embodiments, the compounds of general formula (I) include a modified C-terminus and a modified N-terminus. In some embodiments, the compounds of general formula (I) include an amidated C-terminus and optionally an acetylated N-terminus. [0079] In certain embodiments, the peptidic compounds have the general formula (I)
GSA-X-Y-B (I) wherein:
X is absent or a sequence of between 1 and 25 consecutive amino acids of the sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 57, or between 1 and 24 consecutive amino acids of the sequence as set forth in SEQ ID NO: 4 or
SEQ ID NO: 77;
Y is absent or a peptide linker of between 2 and 10 amino acids in length and comprising amino acids selected from the group of glycine, alanine, valine, lysine and isoleucine;
wherein GSA-X-Y is at least 4 amino acids in length;
and
B is a cell-penetrating peptide. [0080] In certain embodiments, in general formula (I):
X is a sequence corresponding the sequence as set forth in any one of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO:
23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 58, SEQ ID NO: 59, SEQ
ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO:
82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90 and SEQ ID NO: 91.
[0081] In certain embodiments, in general formula (I):
X is a sequence corresponding the sequence as set forth in any one of SEQ ID
NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 43.
[0082] In certain embodiments, in general formula (I):
X is a sequence corresponding the sequence as set forth in any one of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 73, SEQ ID NO: 74 and SEQ ID NO: 75.
[0083] In certain embodiments, in general formula (I):
Y is absent or a poly glycine or polyalanine peptide linker of between 2 and 10 amino acids in length.
[0084] In certain embodiments, in general formula (I):
Y is absent.
[0085] In certain embodiments, in general formula (I):
B is a cell-penetrating peptide selected from TAT, penetratin, transportin and variants thereof.
[0086] In certain embodiments, in general formula (I):
B is a cell-penetrating peptide having a sequence as set forth in any one of RKKRRQRRR (SEQ ID NO:52); GRKKRRQRRRPQ (SEQ ID NO:53); YGRKKRRQRRR (SEQ ID NO:44) and GRKKRRQRRRPPQ (SEQ ID NO:54).
[0087] In certain embodiments, in general formula (I):
X is a sequence of between 1 and 14 consecutive amino acids of the sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 57, and Y is absent.
[0088] In certain embodiments, in general formula (I):
X is a sequence of between 1 and 14 consecutive amino acids of the sequence as set forth in SEQ ID NO: 3, and
Y is absent.
[0089] In certain embodiments, in general formula (I):
X is a sequence of between 1 and 5 consecutive amino acids of the sequence as set forth in SEQ ID NO: 3, and
Y is a peptide linker of between 2 and 10 amino acids in length and comprises amino acids selected from the group of glycine, alanine, valine, lysine and isoleucine.
[0090] In certain embodiments, in general formula (I):
X is a sequence of between 1 and 14 consecutive amino acids of the sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 57;
Y is absent, and
B is a cell-penetrating peptide selected from TAT, penetratin, transportin and variants thereof.
[0091] In certain embodiments, compounds of general formula (I) have general formula (II):
GSAFL-X-Y-B (II) wherein X, Y and B are as defined above in any embodiment for general formula (I)-
[0092] In certain embodiments, compounds of general formula (I) have general formula (III):
GSAFLSPEHQ-X-Y-B (III) wherein X, Y and B are as defined above in any embodiment for general formula (I)-
[0093] In certain embodiments, the compounds of general formula (I) as described in any of the above embodiments include a modified C-terminus and optionally a modified N-terminus. In some embodiments, the compounds of general formula (I) as described in any of the above embodiments include an amidated C-terminus and optionally an acetylated N-terminus.
[0094] In certain embodiments, the compounds of general formula (I) have general formula (IV): GSA-X-TAT (IV)
[0095] wherein X represents any sequence of amino acids starting at residue 4 up to residue 28 of the ghrelin molecule and TAT represents any TAT sequence used for cell penetration, which includes sequences with an amidated C-terminus.
Preparation of Compounds [0096] The compounds of general formula (I) can be readily prepared by standard chemical synthesis techniques. The principles of solid phase chemical synthesis of polypeptides are well known in the art and may be found in general texts in the area such as Pennington, M.W. and Dunn, B.M., Methods in Molecular Biology, Vol. 35 (Humana Press, 1994); Dugas, H. and Penney, C, Bioorganic Chemistry (1981) SpringerVerlag, New York, pgs. 54-92; Merrifield, J. M, Chern. Soc, 85:2149 (1962), and Stewart and Young, Solid Phase Peptide Synthesis, pp. 24-66, Freeman (San Francisco, 1969).
[0097] In addition, various commercial peptide synthesis companies can synthesize such a molecule at a relatively low cost (for example, Peptides International Inc., GenScript USA Inc. and Phoenix Pharmaceuticals Inc.).
[0098] Optional N- and/or C-terminal modifications of the synthesized peptides may also be made by standard techniques. Covalent modifications of the peptide can be introduced, for example, by reacting targeted amino acid residues with an organic derivatizing agent that is capable of reacting with selected terminal residues as is known in the art. Selection of appropriate derivatizing agent(s) can be readily accomplished by a worker skilled in the art. Testing Compounds for Activity
[0099] The ability of the compounds of general formula (I) to increase circulating acylated ghrelin levels may be tested in animal models using standard techniques, such as those described in the Examples herein.
[00100] Other properties of the compounds, such as those described in the Examples, may also be tested if desired.
[00101] When the compounds are intended for therapeutic use, toxicity and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. For example, LD50 (the dose lethal to 50% of the population) and/or ED50 (the dose therapeutically effective in 50% of the population) values maybe determined. The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Large therapeutic indices are preferred for therapeutic uses.
PHARMACEUTICAL COMPOSITIONS
[00102] Certain embodiments of the invention relate to the therapeutic use of the compounds of general formula (I). For therapeutic use, the compounds are typically formulated as pharmaceutical compositions that comprise the compound and a pharmaceutically acceptable carrier, diluent, or excipient. The pharmaceutical compositions may be prepared by known procedures using well-known and readily available ingredients. [00103] The pharmaceutical compositions may be formulated for administration by a variety of routes, for example, orally (including, for example, buccally or sublingually), topically, parenterally, by inhalation or spray, or rectally. The term parenteral as used herein may include subcutaneous injections, intradermal, transdermal, intra-articular, intravenous, intramuscular, intravascular, intrasternal, intrathecal injection or infusion techniques. The compounds may optionally be formulated in unit dosage forms containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and/or vehicles. [00104] The compounds can be formulated into a form suitable for the selected route of administration, for example, as syrups, elixirs, tablets, troches, lozenges, hard or soft capsules, pills, suppositiories, oily or aqueous suspensions, dispersible powders or granules, emulsions, injectables, solutions, creams, gels, liquids, lotions, sprays or aerosols. In some embodiments, it may be useful to formulate the compounds for transdermal delivery, for example by applying or impregnating a bandage or a patch with the compound for application to the skin of a subject. A patch typically includes a skin-contacting portion made of any suitable material that is covered or impregnated with a cream or emulsion comprising the compound and which is supported by a backing, one or both of the skin-contacting portion and the backing may have an adhesive segment or other configuration for attaching to the skin surface of a subject.
[00105] In certain embodiments, it may be advantageous to complex or associate the compounds with an appropriate delivery vehicle prior to formulation. Such vehicles may provide beneficial properties to the compounds, for example, improve the stability, solubility, pharmacokinetics, or the like. Examples of delivery vehicles include, but are not limited to, polyethylene glycol (PEG), polyacetic acid, polyglycolic acid, hydrogels, dextran, liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts and spheroblasts.
[00106] Compositions intended for oral use may be prepared in either solid or fluid forms. Fluid forms can be prepared according to procedures known in the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavouring agents, colouring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. An elixir is prepared by using a hydroalcoholic (for example, ethanol) vehicle with suitable sweeteners such as sugar and saccharin, together with an aromatic flavoring agent. Suspensions can be prepared with an aqueous vehicle with the aid of a suspending agent such as acacia, tragacanth, methylcellulose and the like.
[00107] Solid formulations such as tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate: granulating and disintegrating agents for example, corn starch, or alginic acid: binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc and other conventional ingredients such as dicalcium phosphate, magnesium aluminum silicate, calcium sulfate, starch, lactose, methylcellulose, and functionally similar materials. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed.
[00108] Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil. Soft gelatin capsules are prepared by machine encapsulation of a slurry of the compound with an acceptable vegetable oil, light liquid petrolatum or other inert oil.
[00109] Aqueous suspensions contain active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxylmethylcellulose, methyl cellulose, hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia: dispersing or wetting agents may be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example hepta-decaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl- p-hydroxy benzoate, one or more colouring agents, one or more flavouring agents or one or more sweetening agents, such as sucrose or saccharin.
[00110] Oily suspensions may be formulated by suspending the active ingredients in a vegetable oil, for example peanut oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavouring agents may be added to provide palatable oral preparations. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid. [00111] Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavouring and colouring agents, may also be present.
[00112] Pharmaceutical compositions may also be in the form of oil-in-water emulsions. The oil phase may be a vegetable oil, for example olive oil or peanut oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavoring agents. [00113] Pharmaceutical compositions may also be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or a suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. Adjuvants such as local anaesthetics, preservatives and buffering agents can also be included in the injectable solution or suspension.
[00114] Pharmaceutical compositions for rectal administration are typically prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter and polyethylene glycols.
[00115] Other pharmaceutical compositions and methods of preparing pharmaceutical compositions are known in the art and are described, for example, in "Remington: The Science and Practice of Pharmacy'" (formerly "Remingtons Pharmaceutical Sciences'"); Gennaro, A., Lippincott, Williams & Wilkins, Philadelphia, PA (2000).
CLINICAL TRIALS
[00116] When the compounds are intended for therapeutic use and following the demonstrated effectiveness of a compound in vitro and in animal models, the compound will need to be submitted to standard GLP animal toxicology and pharmacokinetic studies (pre-clinical trials) and then be entered into Clinical Trials in order to further evaluate its efficacy in the treatment of the target disease and to obtain regulatory approval for therapeutic use. As is known in the art Clinical Trials involving new drugs progress through phases of testing, which are identified as Phases I, II, III, and rv. Each stage or phase of pre-clinical study and clinical testing represents a significant development, regulatory and commercialization milestone towards providing the public with therapeutic options for diseases and health management.
[00117] Initially, the candidate compound will be evaluated in a Phase I trial, which is usually an open-label trial (i.e. both the researchers and participants know which treatment is being administered). Typically Phase I trials are used to assess the safety, tolerability, pharmacokinetics, and pharmacodynamics of the compound. They may also be used to determine the best mode of administration (for example, orally or parenterally) if this has not been determined from pre-clinical trials, and/or an appropriate frequency of administration. Phase I studies will include laboratory tests, such as blood or urine tests or biopsies, to evaluate the effects of the compound in the body of the patient. Phase I trials may be conducted in a clinical trial clinic, where the patients can be observed by full-time staff. Clinical trial clinics may be run for example by hospitals or by various contract research organizations (CROs) which conduct the trial on behalf of pharmaceutical companies or other research investigators.
[00118] Phase I trials usually involve a small number of healthy participants (for example, 10-100) who are treated with a specific dose of the candidate compound. During the trial, the dose is typically increased group by group in order to determine the maximum tolerated dose (MTD) and the dose-limiting toxicities (DLT) associated with the compound. The range of doses to be tested is determined based on the pre-clinical trials and will be a fraction of the dose that was observed to cause toxicity in animal models. Dose escalation studies during Phase I trials determine an appropriate dose to use in a subsequent Phase II trial.
[00119] A Phase II trial can be conducted to evaluate the effectiveness of the candidate compound, and to further test the safety of the compound in a larger group of subjects. Phase II trials are usually open-label, but may also be blinded. In Phase II trials, the compound is administered to a larger group of participants (typically 100-300) who present with the disease for which treatment with the candidate compound is being investigated. The dosage found to be effective in Phase I trials is used. Phase II trials may be designed as case series, to evaluate the safety and activity of the candidate compound in a selected group of subjects, or they may be designed as randomized controlled trials, where some subjects receive the candidate compound and others receive either a placebo or a standard treatment.
[00120] Phase III trials focus on evaluating the efficacy of the candidate compound, how it compares to the standard, or most widely accepted, treatment, as well as the applicability of the compound to clinical practice. Phase III trials are much larger than Phase II trials, typically with between 500-3,000 participants, and are generally randomized, blinded trials conducted in multiple clinics ("multicentre trials"). In Phase III trials, participants are randomly assigned to one of two or more "arms." In a trial with two arms, for example, one arm will receive the standard treatment (control group) and the other arm will receive treatment with the candidate compound (investigational group). Other arms may be included, for example, to evaluate against placebo or a different standard treatment. For practical reasons, Phase III trials of chronic conditions or diseases often have a short follow-up period for evaluation relative to the period of time the intervention might be used in practice.
[00121] Once a candidate compound has proved satisfactory in Phase III trials, the trial results are used as a basis for the regulatory submission to the appropriate drug approval agency (for example, U.S. Food and Drug Administration (FDA), Health Canada or European Medicines Agency (EMEA)), which will also include the methods and results of human and animal studies, manufacturing procedures, formulation details, and shelf life.
[00122] Phase IV trials are usually conducted as post-marketing surveillance trials and are used to further evaluate the long-term safety and effectiveness of the compound. Phase IV trials are generally designed to detect any rare or long-term adverse effects in a much larger patient population and over a longer time period than was possible during the Phase I-III trials. Phase IV trials may be required by regulatory authorities, or may be undertaken by the sponsoring company for other reasons, such as finding a new market for the drug, testing for interactions with other drugs or in certain sub- populations. USES
[00123] Exemplary uses for the compounds of general formula (I) include, in various embodiments, use in basic research, use in pre-clinical studies and clinical trials for pharmacological agents to treat a ghrelin associated disorder, and therapeutic uses. [00124] The result of the use of the compounds of general formula (I) in vivo, whereby circulating levels of acylated ghrelin are reduced, indicates that these compounds have potential therapeutic use in treatment and/or management of various conditions associated with elevated levels of acylated ghrelin.
[00125] For example, certain embodiments contemplate the use of the compounds of general formula (I) in the treatment and/or management of obesity. In this context, the compounds may be used for regulating food intake in a subject, for improving compliance of a subject to caloric restriction, and/or for modifying eating behaviour of a subject (for example, by reducing desire for high-calorie foods and/or fats). Some embodiments relate to methods and uses of the compounds of general formula (I) for preventing or reducing weight gain in a subject.
[00126] In certain embodiments, the invention relates to methods and uses of the compounds of general formula (I) in the treatment and/or management of diabetes. Diabetes in this context may be insulin-dependent diabetes mellitus (Type I diabetes) or non-insulin-dependent diabetes mellitus (Type II diabetes). The compounds may be administered to a subject having diabetes in order to treat or assist in the management of diabetes, or a diabetes-related condition, such as obesity or weight gain. Alternatively, the compounds may be administered to a subject at risk of developing diabetes in order to prevent the subject from becoming diabetic. Subjects at risk of diabetes include, for example, individuals having pre-diabetes or metabolic syndrome (insulin resistance), as well as overweight, physically inactive individuals.
[00127] Certain embodiments relate to methods and uses of the compounds of general formula (I) in the treatment and/or management of pre-diabetes. Some embodiments relate to methods and uses of the compounds of general formula (I) in the treatment and/or management of metabolic syndrome (or insulin resistance). In some embodiments, the compounds may be used to modify eating behaviour in individuals having pre-diabetes or metabolic syndrome and thus aid in the treatment and/or management of pre-diabetes or metabolic syndrome.
[00128] Certain embodiments relate to methods and uses of the compounds of general formula (I) in the treatment and/or management of stress-related eating disorders or other stress-related disorders such as post-traumatic stress disorder (PTSD).
[00129] Certain embodiments relate to methods and uses of compounds of general formula (I) in the treatment and/or management of addictive behaviours, for example, cravings for activities or substances such as gambling and alcohol. [00130] Certain embodiments relate to the use of compounds of general formula (I) as research tools. The compounds may potentially be used in a variety of experimental situations in which acylated and/or des-acylated ghrelin activity are of interest. For example, to study different aspects of diabetes, obesity, and more due to ghrelin's contribution to these diseases. As an example, administration of compounds of general formula (I) should lead to improved insulin secretion and sensitivity in response to a glucose load, which aid in glucose tolerance in diabetes.
[00131] The use of compounds of general formula (I) to inhibit ghrelin acylation will lead to increased levels of des-acyl ghrelin and an increased des-acyl:acyl ghrelin ratio, allowing the compounds to be used in studies examining this ratio as well as des-acyl ghrelin itself, of which relatively little is currently known. Furthermore, one can determine if inhibition of acylation can lead to changes in total ghrelin levels, which may indicate feedback mechanisms induced by changes in acylated or des-acylated ghrelin levels.
[00132] Due to ghrelin's role in metabolism and substrate utilization, catabolism of fats and carbohydrates may be studied using the compounds of general formula (I). The compounds would be expected to shift catabolism to adipose tissue. Studies previously examining the relationship between ghrelin deletion and metabolic fuel preference can be replicated using the compounds in wild-type animals, such as those conducted by Wortley et al. (2004, PNAS, 101(21):8227-8232). Along with metabolic changes, alterations in food preference can be induced by inhibition of ghrelin acylation, resulting in a reduction in cravings for highly palatable foods. It is expected that treatment with compounds of general formula (I) can reduce this ghrelin-associated preference for palatable foods. Studies similar to that of Disse et al. (2010, Physiology & Behavior, 101(2):277-281), as well as Chuang et al. (2011, J. Clinical Investigation, 121(7):2684-2692) (in the case of food preference in response to stress) can be conducted to confirm this. Additionally, cravings for other activities may potentially be reduced, such as gambling and alcohol. In all of these examples, acyl ghrelin and total ghrelin levels can be measured by a variety of techniques, including for example enzyme-linked immunosorbent assays and mass spectrometry.
[00133] Recently, ghrelin has been identified as an important component in the development of post-traumatic stress disorder, with stress-associated increases in ghrelin leading to enhanced fear learning associated with the amygdale (Meyer, R.M., et al., "A Ghrelin-Growth Hormone Axis Drives Stress-Induced Vulnerability To Enhanced Fear," Mol Psychiatry, advance online publication, October 15, 2013). Compounds of general formula (I) may be used in studies to determine if it is possible to weaken these memories and the fear association during reconsolidation.
KITS
Research Kits [00134] Certain embodiments of the invention relate to kits or packs containing a compound of general formula I for research use. A research kit is typically a collection of biological research products, for example, two or more, that are used together to perform a biological research reaction, procedure, or synthesis, such as an assay, a detection, a separation or a purification, which are shipped together, usually within a common packaging, to an end user.
[00135] In addition to the compound of general formula (I), the research kits may optionally include reagents required to conduct a biological procedure, such as buffers, salts, enzymes, enzyme co-factors, substrates, detection reagents, washing reagents, and the like. Other components, such as buffers and solutions for the isolation and/or treatment of a test sample, may also be included in the kit. The kit may additionally include one or more control compounds.
[00136] In some embodiments, one or more of the components of the kit may be lyophilised and the kit may further comprise reagents suitable for the reconstitution of the lyophilised component(s).
[00137] The various components of the kit are provided in suitable containers. In some embodiments, the container may itself be a suitable vessel for carrying out the biological procedure, for example, a microtitre plate. Where appropriate, the kit may also optionally contain reaction vessels, mixing vessels and other components that facilitate the preparation of reagents or a test sample, or the carrying out of the biological procedure. The kit may also include one or more instruments for assisting with obtaining a test sample, such as a syringe, pipette, forceps, or the like.
[00138] In some embodiments, reagents comprised by the kit or their containers may be colour-coded to facilitate their use. When reagents are colour-coded, addition of one reagent to another in a particular step may for example result in a change in the colour of the mixture, thus providing an indication that the step was carried out.
[00139] The kit can optionally include instructions for use, which may be provided in paper form or in computer-readable form, such as a disc, CD, DVD or the like. The kit may also comprise computer readable media comprising software that assists in the interpretation of results obtained from using the kit.
[00140] The kits may be employed in research into, for example, the des-acyl:acyl ghrelin ratio, the role of des-acyl ghrelin, catabolism of fats and carbohydrates, the relationship between ghrelin deletion and metabolic fuel preference, the role of ghrelin in alterations in food preference and cravings for other substances or activities, the role of ghrelin in stress-related eating and/or the role of ghrelin in the development of posttraumatic stress disorder.
Therapeutic Kits
[00141] Certain embodiments of the invention relate to therapeutic kits or packs containing a compound of general formula I.
[00142] Individual components of the kit would typically be packaged in separate containers and, associated with such containers, can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, for use or sale for human or animal administration.
[00143] In certain embodiments, the compounds of general formula (I) are provided in the kit in the form of pharmaceutical compositions suitable for administration to a subject. In this case, if desired, the container may itself be an inhalant, syringe, pipette, eye dropper, or other such like apparatus, from which the composition may be administered to the subject. In some embodiments, one or more of the components of the kit can be lyophilized and the kit can additionally contain a suitable solvent for reconstitution of the lyophilized component(s). In certain embodiments, the kit comprises a transdermal patch comprising a formulation of a compound of general formula (I) which is suitable for application to the skin.
[00144] To gain a better understanding of the invention described herein, the following examples are set forth. It will be understood that these examples are intended to describe illustrative embodiments of the invention and are not intended to limit the scope of the invention in any way.
EXAMPLES [00145] The following examples utilise a peptide-based compound, CF801, that was initially developed as a potential GOAT inhibitor. The amino acid sequence of CF801 is:
GSAFLSPEHQRKKRRQRRR (SEQ ID NO: 92)
[00146] The first 10 amino acids of the sequence correspond to the N-terminal sequence of human ghrelin, but with the serine at position 3 substituted with alanine. The remaining amino acids are derived from the TAT sequence. The amino acid at position 11 (arginine) of CF801, while belonging to the TAT sequence, also corresponds to amino acid 11 of the human ghrelin sequence.
[00147] The final CF801 molecule used in the Examples below included an amidated C-terminus, i.e. GSAFLSPEHQRKKRRQRRR-NH2 (SEQ ID NO:93)
[00148] CF801 differs in several respects from the Go-CoA-Tat compound described International Patent Application No. PCT/US2009/057512 (WO 2010/039461) with the goal of 1) reducing cost and 2) simplifying synthesis. To achieve these goals, CF801 was designed to be composed solely of amino acids commonly used in peptide synthesis. Go-CoA-Tat contains a Dap3 moiety at the third residue, with a long carbon chain attached to this residue. Additionally, Go-CoA-Tat has an amino-hexanoic acid linker linked to a TAT peptide at the C-terminus. The structure of Go-CoA-Tat is more in line with the ternary structure formed during acylation of ghrelin, after the fatty acid is esterified to the ghrelin molecule, as the new species dissociates from the GOAT enzyme. CF801 represents the structure before acylation occurs, that is, it resembles an unacylated ghrelin molecule that potentially could act as a GOAT inhibitor by competing for GOAT but, as it cannot become acylated itself, simply occupying GOAT's active site. Although the results obtained from the following Examples suggest that CF801 is likely not a GOAT inhibitor, the compound's use and efficacy in reducing, preventing increases in, or otherwise ameliorating the effects of circulating levels of acylated ghrelin was clearly demonstrated.
[00149] CF801 also differs from pentapeptide GSAFL identified by Yang et al. (2008, PNAS, 105(31): 10750-10755) due to the extended sequence that includes additional ghrelin amino acids to mimic more closely a ghrelin molecule, and to provide additional spacing for a TAT sequence with an amidated C-terminus to improve cell penetration.
Design and use of CF801
[00150] The GSAFL-NH2 pentapeptide developed by Yang at al. (ibid) consists of the first five amino acids of unacylated ghrelin with the third residue replaced with an alanine. While the peptide showed inhibitory activity on GOAT it was unlikely that the peptide would effectively cross cell membranes into the intracellular area where GOAT performs its enzymatic reactions. The design of CF801 involved extending and modifying the original GSAFL-NH2 sequence. The first modification was the addition of five more amino acids corresponding to amino acids 6 through 10 of the full-length ghrelin peptide, increasing the similarity to ghrelin as well as providing an extra spacer for the addition of an HIV trans-activator of transcription (Tat) sequence. The Tat sequence utilized consists of the amino acids RKKRRQRRR (SEQ ID NO:52) with an amide group retained on the C-terminus. The addition of the Tat sequence along with the C-terminal amide group are designed to improve cell penetration of the inhibitor. Other cell penetration peptides could be used in place of the Tat sequence to achieve this function.
Preparation of CF801
[00151] CF801 peptide was synthesized on Rink-mBHA resin (Peptides International, Louisville KY) using Fmoc/tBu with disopropylcarbodiimide/6-CI-HOBT for coupling. Cleavage from the solid support was performed using a TFA-mediated acidolytic procedure for two hours at room temperature. Preparative reverse phase-HPLC using a linear gradient of 0.05% TFA in water vs MeCN was employed to purify the product to >95%. The product was then lyophilized and characterized by analytical RP-HPLC and ES-MS.
EXAMPLE 1: CF801 Decreases Plasma Acylated Ghrelin, Body Mass, And Fat Mass, While Increasing Regular Chow And Decreasing High-Fat Diet Intake
Example 1 Materials & Methods
Cohort 1: Treatment
[00152] 29 Male C57/BL6J mice were given a 10-day baseline with ad libitum access to regular chow, high-fat diet (60% calories from fat; Harlan) and water. Mice were randomly assigned to non-injected control (NIC, n=6), vehicle control (0.9% saline, n=7), low dose CF801 (LD; 11 umol/kg of CF801, n=8), and high dose CF801 (HD; 22 umol/kg of CF801, n=8) groups. [00153] Following baseline, each morning for 13 consecutive days, animals were given by intraperitoneal injection either 0.9% saline, 11 umol/kg CF801 (low-dose), 22 μιηοΐ/kg CF801 (high-dose), or no injection (NIC). During treatment animals were maintained with ad lib access to both regular chow and a high-fat diet. Quantities consumed of regular chow and high- fat diet, as well as body mass were measured and recorded daily.
Open-Field Test
[00154] Animals were subjected to an open-field test following the two-week treatment period to measure anxiety levels. Standard procedures were followed for measurements of total time spent in the center and around the borders.
Recovery Following Treatment
[00155] Following the 13 day treatment period, half the animals were rapidly decapitated and carcasses were stored at -80°C until further processed. The other half were given an 8-day recovery period over which no injections were given, followed by rapid decapitation and storage at -80°C.
Fat Mass, Lean Mass, and Total Water with EchoMRI
[00156] Post-mortem body composition was analyzed by Echo MRI to determine lean and fat mass composition shortly after decapitation and storage. Measurements of fat mass, lean mass, and total water were obtained. Cohort 2: Rebound Feeding and Plasma Acylated Ghrelin Levels
[00157] A second cohort of animals were obtained and randomly assigned to vehicle (n=5), low dose (n=5), and high dose (n=6) groups, using the same concentrations of CF801 as cohort 1. Following a five-day baseline, treatment began with ad lib access to regular chow (in the absence of high- fat diet). On day four of treatment, animals were fasted for 24 hours, after which chow was reintroduced to measure rebound feeding. Treatment continued until day 7 when animals were sacrificed and trunk-blood collected for acylated ghrelin analysis using enzyme-linked immunosorbent assay (ELISA). Example 1 Results
[00158] No differences in mass were found in animals throughout baseline. After two days of treatment high dose animals were significantly lighter than vehicle animals (p < 0.05). This difference persisted for the remainder of the experiment (Figure 1). This is relatively similar to results obtained for Go-CoA-Tat, where significant differences appeared by day 10 of treatment. While these differences in mass were found between groups, there were no differences in caloric intake (Figure 2). An analysis of type of food consumed (high-fat vs. regular chow) showed that animals in the high-dose group showed a reduced preference for high fat food, shifting a considerable amount of caloric intake towards regular chow (Figure 3 A and B). Metabolic efficiency (weight gained per calorie consumed) was significantly reduced in both low and high-dose animals (p < 0.01; Figure 4A).
[00159] Body composition using EchoMRI after 13 days of treatment revealed a significantly reduced fat mass in high-dose animals compared to vehicle animals, with no difference in lean mass or total water mass (Figure 4B). This is similar to Go-CoA- Tat treatment, where the difference in body weight was attributed to a reduction in fat mass.
[00160] As expected in the second cohort, circulating acylated ghrelin levels, as measured by enzyme-linked immunosorbent assay, showed a dose-dependent decrease with CF801 after seven days of treatment (Figure 6A). Again, these data are in agreement to results obtained with Go-CoA-Tat, although the timing of ghrelin measurements for Go-CoA-Tat was much more acute, with measurements made at 6, 12, and 24 hours with significance beginning at 12 hours.
[00161] Following a 24-hour fast after three days of treatment, high-dose animals consumed significantly less food over a four-hour recovery period (Figure 6B).
Example 1 Summary
[00162] The following effects of CF801 were observed when given by IP injection: 1) Reduction in circulating acylated ghrelin levels. 2) Reduction in body mass with significance after two days of treatment.
3) After 13 days of treatment animals on CF801 had significantly less fat mass compared to vehicle controls, with no difference in lean mass or total water.
4) Increase in regular chow intake and a decrease in high-fat diet consumption when given ad lib access to both. There were no differences in total caloric intake.
5) Following a 24-hour fast, animals given CF801 showed reduced caloric intake over a four-hour period once food was reintroduced.
6) No difference in anxiety levels as measured with the open field task (Figure 5).
[00163] The findings under points 2-6 are in-line with the observed reduction in acylated ghrelin levels (point 1).
EXAMPLE 2: CF801 Effects On Food Intake, Weight, Anxiety, And Acylated Ghrelin Levels During Chronic Social Defeat Stress
Example 2 Materials & Methods
[00164] 52 Male C57/BL6J mice were weight-matched and assigned to one of six groups: non-injected controls non-stressed (NIC NS; n=10), non-injected controls stressed (NIC S; n=10), vehicle non-stress (VEH NS; n=8), vehicle stress (VEH S; n=8), CF801 non-stressed (CF801 NS; n=8), and CF801 stressed (CF801 S; n=8). Throughout the experiment, animals were housed under standard laboratory conditions and received ad libitum access to standard laboratory mouse chow and tap water, with daily 4-hour access to a high- fat diet (60% caloric content from fat; 10 am to 2 pm). Injections of vehicle (0.9% saline) or CF801 (22μιηο1^) were given daily, or no injection for non-injected controls. Injections and stressors began after a two-week baseline.
Chronic Social Defeat Stress Paradigm [00165] Each animal in the stress groups were housed for 21 consecutive days in a cage shared with a much larger sexually experienced male CD-I mouse, with a wire- mesh divider separating the two mice. Each day the animals were permitted to interact until the experimental mouse was subdued or until 15 minutes had passed. Elevated Plus Maze
[00166] All animals were subjected to a standard elevated plus maze to measure anxiety levels. During a 5 minute period, duration spent in open and closed arms were measured, along with the number of entries into the open arms.
Hormone Analysis
[00167] Animals were rapidly decapitated and trunk blood collected. Plasma acylated ghrelin levels as well as corticosterone levels were measured with an enzyme-linked immunosorbent assay (ELISA) kit and an RIA kit, respectively.
Example 2 Results
CF801 in stressed animals leads to an amplified disruption of stress-induced feeding changes
[00168] As expected, chronic social defeat stress led to an increase in regular chow intake and a decrease in high-fat diet intake (p < 0.05; Figure 7). However, between the three stressed groups, stressed animals treated with CF801 consumed significantly less high-fat diet (p < 0.0001; Figure 8A) and, compared to vehicle-injected controls, significantly more regular chow (p < 0.05; Figure 8B). Furthermore, CF801 treatment in stressed animals led to an overall reduction in caloric intake during the stressor period, compared to vehicle (p < 0.05) and non-injected controls (p < 0.05) also receiving stress, with this overall reduction not being significant when comparing non- stressed groups (Figure 8C). This interaction was significant (p < 0.05), suggesting a possible stress-induced amplification in the disruption of metabolic processes as a result of the reduction in acylated ghrelin levels. [00169] As with the results in non-stressed animals (Example 1), stressed animals treated with CF801 also showed a reduction in body mass when compared to vehicle and non-injected controls (p < 0.05; Figure 9A).
CF801 eliminates stress-induced increases in plasma acylated ghrelin
[00170] In response to chronic social defeat stress, increases in plasma acylated ghrelin levels were observed in both vehicle and non-injected controls (Figure 9B). This increase, however, was not observed in CF801 animals, supporting the role of CF801 as regulator of acylated ghrelin levels (Figure 9B).
CF801 has no effect on stress-induced anxiety, but demonstrates a possible reduction in locomotor activity
[00171] As expected, stressed animals spent significantly less time in the open arms of the elevated plus maze (p < 0.005) while no differences were found in number of entries into the open arms (p > 0.05) (Figure 10A & B). When stress and no-stress groups are collapsed together, CF801 treated animals made significantly fewer entries into the open arm when compared to vehicle treated animals (p < 0.05), but not when compared to non-injected controls (Figure IOC).
Example 2 Summary
[00172] CF801 treatment during chronic social defeat stress leads to the following results: 1) Reduced high- fat intake, compared to vehicle and non-injected controls.
2) Overall reduced caloric intake.
3) Reduced body mass.
4) Lack of a stress-induced increase in acylated ghrelin levels.
[00173] Combined, these data suggest that CF801 can be used in the study and/or treatment of stress, in particular chronic social defeat stress, leading to significant reductions in acylated ghrelin levels as well as affecting feeding, caloric preference, and total caloric intake during times of stress. These observed differences can also aid in interpreting acylated ghrelin's role during chronic social defeat stress.
EXAMPLE 3: CF801 Decreases Respiratory Exchange Ratio And Heat Production in C57BL6J Male Mice Example 3 Materials and Methods
Treatment
[00174] 10 Male C57/BL6J mice were given a 14-day baseline period wherein they had ad libitum access to regular chow, high-fat diet (60% calories from fat; Harlan Diets) and water. Mice were randomly assigned to one of two groups: vehicle injected controls (VEH; 0.9% saline, n = 5) or high dose CF801 (HD; 22nmol/kg of CF801, n = 5). Following the baseline period, each animal received an intraperitoneal injection of either 0.9% saline (VEH) or 22μιηο1/] ¾ CF801 (HD) each morning, for 17 consecutive days. During the treatment period, animals were maintained with ad libitum access to both regular chow and the high-fat diet. Body weight and the amount of each diet consumed were recorded each day for the entire experiment.
Respiratory Exchange Ratio, Heat Production, Locomotor Activity
[00175] During the baseline period, all animals were placed in metabolic chambers (TSE Systems; LabMaster/Phenomaster) for 48 hours. The metabolic chambers use indirect calorimetry to measure respiratory exchange ratio and heat production, as well as infrared beams to measure locomotor activity. Data collected from the first 24 hours of being housed in the metabolic chambers was discarded as the animals habituate to the environment. Data from the second 24 hours in the metabolic chambers was used as a baseline measure. Mice were returned back to their home cage following the 48-hour exposure to metabolic chambers during the baseline period. On treatment day 12, mice were returned to the metabolic chambers for 24 hours. Respiratory exchange ratio, heat production and locomotor activity were measured for each animal every 30 minutes.
Glucose Tolerance [00176] Mice were fasted overnight on the 8th day of the treatment period. On the morning of the 9th treatment day, mice were given their daily injection of CF801 followed by a bolus of 20% glucose (2mg/kg). Blood glucose was measure using a glucose meter (Bayer Inc) at the time of injection (time 0), 15, 30 and 60 minutes following injection.
Blood Glucose & Corticosterone Analysis
[00177] Following the last injection of CF801 on treatment day 17, mice were sacrificed by rapid decapitation. Circulating levels of blood glucose were measured form trunk blood using a glucose meter (Bayer Inc). Remaining trunk blood was collected and spun down at 3,000Xg for 15 minutes to separate blood plasma from red blood cells. Aliquots of blood plasma were frozen at -80°C until processed. Circulating corticosterone was measured from blood plasma samples using a Radiolmmuno Assay (RIA) kit.
Example 3 Results
[00178] There were no differences in any measures taken from the metabolic chambers between any animals during the baseline period. On the 12th day of treatment, animals receiving HD CF801 showed a significant reduction in respiratory exchange ratio, as well as a significant reduction in heat production for 3 consecutive hours post-injection (Figure 11A,C). RER and Heat production in animals receiving CF801 returned to control levels 3 hours post-injection and remained equivalent to control levels for the remainder of time spent in the metabolic chambers. There were no differences in locomotor activity between any animals at any time point during the treatment period (Figure 11B).
[00179] On the 9th day of CF801 treatment, all animals were subjected to a glucose tolerance test. Results show that following an overnight fast, animals receiving CF801 injections have significantly lower levels of blood glucose compared to animals receiving vehicle injections (Figure 12A). Following a bolus of glucose, both animals receiving vehicle injections and animals receiving CF801 injections demonstrated equivalent glucose clearing capabilities. [00180] Following sacrifice, trunk blood was collected for hormonal analysis. Results demonstrate that animals receiving CF801 injections had significantly higher levels of circulating corticosterone compared to animals receiving vehicle injections (Figure 12B). Example 3 Summary
[00181] The following effects were observed:
1) CF801 reduces respiratory exchange ratio for ~3hours post-injection.
2) CF801 reduces heat production for ~3hours post-injection.
3) There was no change in locomotor activity between animals receiving vehicle or CF801 injections.
4) CF801 decreases circulating blood glucose in fasted animals, however does not change tolerance to a glucose bolus
5) CF801 increases circulating levels of corticosterone in non- fasted animals. [00182] The reduction in respiratory exchange ratio and heat production (points 1 & 2) indicate that the animals are preferentially metabolizing fat as a fuel source, instead of carbohydrates. These data demonstrate how animals may lose body fat mass without any reductions in caloric intake (as seen in Example 1).
EXAMPLE 4: CF801 Reduces Fasting Induced Increases In Circulating Acylated Ghrelin And Reduces Acylated :Des-Acyl Ghrelin Ratio
Example 4 Materials and Methods
Treatment
[00183] 30 C57BL6J male mice were given a 14-day baseline period, wherein they had ad libitum access to regular chow, high-fat diet (60% calories from fat; Harlan Diets) and water. Mice were randomly assigned to 1 of 4 groups: non-fasted, non injected controls (non-fasted, NIC; n = 7), fasted vehicle injected controls (0.9% saline - VEH; n = 7), fasted low-dose CF801 (Ι ΐμιηοΐ/kg - Fasted, LD; n = 8) or fasted high dose CF801 (22μηιο1/1 ¾ - Fasted HD; n = 8). There were no differences in chow intake, high-fat diet intake or body weight between any groups at the end of the baseline period. On the last day of the baseline period, mice were subjected to an overnight fast (excluding animals in the non-fasted group). The following morning, mice were given an injection of vehicle (0.9% saline), low-dose CF801 (11 μιηοΐ/kg) or high-dose CF801 (22μιηο1^). Exactly 3 hours after receiving an injection, mice were sacrificed by rapid decapitation. Trunk blood was collected for hormonal analysis. Brains were rapidly extracted and the mediobasal hypothalamus was dissected and frozen immediately at - 80°C in 500μί TRIzol® reagent to preserve RNA integrity. Similarly, the fundus of the stomach was dissected and frozen at -80°C immediately in 500μί of TRIzol® until processed.
Hormonal Analysis
[00184] Immediately following rapid decapitation, trunk blood was collected in EDTA-coated tubes placed on ice and centrifuged at 3000 X g for 15 minutes to separate plasma from red blood cells. Blood plasma was aliquoted separately for each assay to avoid multiple freeze/thaw cycles, and stored at -80°C until processed. To protect the acylated ghrelin molecule, a
Figure imgf000046_0001
aliquot of blood plasma was treated with
Figure imgf000046_0002
of 1.0N HC1 and of lOOmM 4-(hydroxymercuri) benzoic acid before storage. Plasma acylated ghrelin, total ghrelin and insulin were measured using an ELISA kit (Millipore). All samples had a coefficient of variation < 10%.
Homeostatic Model of Assessment for Insulin Resistance (HomaIR)
[00185] The HomaIR is a method used to quantify insulin resistance and beta-cell function. Matthews et al. (1985, Diabetologia, 28(7):412-9) first described it under the name HOMA in 1985. The HOMA authors used data from physiological studies to develop a mathematical equations describing glucose regulation as a feedback loop. Therefore, insulin resistance and B-cell function can be estimated from fasting glucose and insulin levels.
Mediobasal Hypothalamic mRNA Expression [00186] Mediobasal hypothalamic gene expression was analyzed in a separate cohort of animals who were not fasted, but received no injection (NIC), vehicle injection, low- dose or high-dose CF801. To this end, total RNA from the mediobasal hypothalamus was isolated with TRIzol® and precipitated with \3μL· of linear acrylamide. RNA quality and concentrations were determined by absorbance at 280nm and 260nm with a Thermo Scientific Nanodrop 100 spectrophotometer (Thermo Scientific, Rockford, Illinois). To synthesize cDNA, 1 oligo(dT) primer (Invitrogen, Carlsbad, California) was added to 9 yL of mRNA and heated to 70°C for 5 minutes. To each sample, a master mix composed of 4μΙ. of 5X first-strand buffer (Invitrogen), 2μΙ. of 0.1M dithiothreitol (Invitrogen), l L of RNase inhibitor (Promega Corp, Madison, Wisconsin), l L of lOmM deoxynucleotide triphosphate (Invitrogen), l L of diethylpyrocarbonate water, and
Figure imgf000047_0001
of SS2 reverse transcriptase (Invitrogen) were added. Samples where then run on a PTC-200 Thermal Cycler (MJ Research, Watertown, Massachusetts) at 42°C for 1.5 hours followed by 90°C for 10 minutes. Samples were stored at -20°C. RT-qPCR was conducted on all cDNA samples to determine fold changes using the 2"ΔΔα method using primers detecting the glyceraldehyde 3 -phosphate dehydrogenase gene as a control transcript. Briefly
Figure imgf000047_0002
of each cDNA sample were added to separate wells in a PCR plate. Two microliters of working primer solution, 3μΙ. of Milli-Q water, and ΙΟμΙ. of iQ SYBR Green Super Mix with fluorescein (Bio-Rad Laboratories, Hercules, California) were added to each well. Samples were run in duplicate with non-template controls. Primers for NPY, AgRP, POMC and SOCS3 were tested for amplification efficiency using the standard curve method, yielding efficiencies between 95% and 105%.
Example 4 Results
[00187] Results show that fasting caused a significant increase in total ghrelin (both des-acyl ghrelin and acylated ghrelin; Figure 13) and a significant decrease in circulating blood glucose and insulin (Figure 14). Animals receiving both low-dose and high-dose CF801 showed a significant reduction in circulating acylated ghrelin levels compared to animals receiving vehicle injections. The acylated ghrelin: des-acyl ghrelin ratio tended to be lower in animals receiving both low-dose and high-dose CF801 relative to animals receiving vehicle injections. [00188] As previously seen, animals receiving the high-dose CF801 tended to have lower levels of circulating blood glucose relative to animals receiving vehicle injections (Figure 14A). There were no differences in blood glucose between animals receiving low-dose or high-dose CF801. As an index of insulin resistance, the homeostatic model assessment was calculated for each group (HomaIR). Results demonstrate that an overnight fast significantly decreased the HomaIR, however there were no differences between animals that received vehicle injections, low-dose or high-dose CF801 injections (Figure 14C).
Example 4 Summary
[00189] The following effects were observed:
1) Following an overnight fast, CF801 reduces circulating acylated ghrelin levels.
2) Following an overnight fast, CF801 does not influence the amount of circulating total ghrelin and des-acyl ghrelin.
3) Following an overnight fast, CF801 significantly decreases the acy des- acyl ghrelin ratio.
4) Following an overnight fast, CF801 reduces circulating blood glucose, even further then vehicle injected fasted animals.
5) CF801 does not change the mRNA expression pattern of NPY, AgRP or SOCS3 in the mediobasal hypothalamus of non-fasted animals. Animals receiving high-dose CF801 showed a slight reduction of POMC in the mediobasal hypothalamus compared to vehicle injected animals.
[00190] Animals in a fasted state have extremely high levels of circulating acylated ghrelin. CF801 significantly reduces acylated ghrelin in a fasted animal without influencing total ghrelin levels (acylated and des-acyl ghrelin).
EXAMPLE 5: CF801 Reduces GOAT And Ghrelin mRNA Expression In The Fundus Following An Overnight Fast In Wild-Type And GHSR Knock-Out Mice Example 5 Materials and Methods
Treatment
[00191] 11 wild-type (WT) and 14 growth hormone secretagogue receptor (GHSR) knock-our (KO) animals were given a 14-day baseline period wherein they received ad libitum access to standard laboratory chow, high fat diet (60% calories from fat, Harlan Diets) and water. Mice were randomly assigned to one of four treatment groups: WT Saline (n = 5), GHSR KO Saline (n = 5), WT CF801 (22μιηο1¾; n = 6), GHSR KO CF801 (22μιηο1^; n = 8). There were no differences in chow intake, high- fat diet intake or body weight between any groups at the end of the baseline period. On the last day of baseline, animals were fasted overnight. The following morning, mice received an intraperitoneal injection of either saline or CF801 (22μιηο1^) and were sacrificed exactly three hours later by rapid decapitation. Trunk blood was collected and plasma was separated from red blood cells, aliquoted and stored at -80°C until processed. Samples of the fundus were dissected, placed in TRIzol® reagent and stored at -80°C until processed.
Hormonal Analysis
[00192] Immediately following rapid decapitation, trunk blood was collected in EDTA-coated tubes placed on ice and centrifuged at 3000 X g for 15 minutes to separate plasma from red blood cells. Blood plasma was aliquoted separately for each assay to avoid multiple freeze/thaw cycles, and stored at -80°C until processed. Insulin was measured using an ELISA kit (Millipore). All samples had a coefficient of variation < 10%.
Ghrelin and GOAT mRNA Expression
[00193] Total RNA from the fundus of the stomach was isolated with 500μί of TRIzol® and precipitated with \3μL· of linear acrylamide. RNA quality and concentrations were determined by absorbance at 280nm and 260nm with a Thermo Scientific Nanodrop 100 spectrophotometer (Thermo Scientific, Rockford, Illinois). To synthesize cDNA, ΙμΙ, oligo(dT) primer (Invitrogen, Carlsbad, California) was added to of mRNA and heated to 70°C for 5 minutes. To each sample, a master mix composed of 4μΙ. of 5X first-strand buffer (Invitrogen), 2μΙ. of 0.1M dithiothreitol (Invitrogen), Ι μΙ, of RNase inhibitor (Promega Corp, Madison, Wisconsin), Ι μΙ, of 1 OmM deoxynucleotide triphosphate (Invitrogen), 1 of diethylpyrocarbonate water, and Ι μΙ^ of SS2 reverse transcriptase (Invitrogen) were added. Samples where then run on a PTC-200 Thermal Cycler (MJ Research, Watertown, Massachusetts) at 42°C for 1.5 hours followed by 90°C for 10 minutes. Samples were stored at -20°C. RT-qPCR was conducted on all cDNA samples to determine fold changes using the 2"ΔΔα method using primers detecting the glyceraldehyde 3-phosphate dehydrogenase gene as a control transcript. Briefly 5μL· of each cDNA sample were added to separate wells in a PCR plate. Two microliters of working primer solution, 3μL· of Milli-Q water, and 10μL· of iQ SYBR Green Super Mix with fluorescein (Bio-Rad Laboratories, Hercules, California) were added to each well. Samples were run in duplicate with non-template controls. Primers for GOAT and ghrelin were tested for amplification efficiency using the standard curve method, yielding efficiencies between 95% and 105%. Example 5 Results
[00194] CF801 significantly reduced circulating levels of blood glucose and insulin in WT animals. Interestingly, CF801 tended to reduce blood glucose (p = 0.081) and insulin levels (p = 0.098) in GHSR KO animals (Figure 16A,B). The homeostatic model of assessment for insulin resistance (HomaIR) was significantly reduced in both WT and GHSR KO animals receiving CF801 compared to their non-stressed controls (Figure 16C).
[00195] Results from RT-qPCR analysis on the fundus of the stomach revealed that CF801 potently reduces GOAT mRNA expression in both WT and GHSR KO animals, relative to their vehicle injected controls (Figure 17A). Ghrelin mRNA expression in the fundus, on the other hand, was significantly increased in WT animals receiving CF801 relative to their vehicle injected controls. In contrast, there was no change in ghrelin mRNA expression in the fundus between GHSR KO animals receiving vehicle or CF801 , suggesting the effects of CF801 on ghrelin mRNA expression are dependent on GHSR signaling. Example 5 Summary [00196] The following effects were observed:
1) CF801 decreases plasma levels of blood glucose and insulin in both WT and GHSR KO animals. There was a tendency for the reduction in glucose and insulin to be more pronounced in WT animals relative to GHSR KO animals, however this did not attain statistical significance.
2) CF801 decreased the HomaIR score in fasted WT and GHSR KO animals.
3) CF801 potently decreases GOAT expression in the fundus of the stomach in fasted WT and GHSR KO animals.
4) There was a significant increases in ghrelin mR A expression in the fundus of the stomach in fasted WT animals, but not fasted GHSR KO animals.
[00197] As noted above, in this Example, a significant reduction in HomaIR was seen for the CF801 -treated animals, whereas in Example 4 no significant difference was observed between CF801 -treated and control animals. The reason for this difference is most likely due to the limited number of animals included in the HomaIR analysis of Example 4. Specifically, when running the analysis insufficient sample blood remained after total ghrelin and acylated ghrelin measurement to allow a data point for each animal to be obtained. As a result, the number of animals included in the HomaIR scores was reduced in Example 4. With the low number of data points included in the analysis, the effect did not attain statistical significance (p-value 0.140, saline vs. HD CF801) although a visual trend was seen wherein animals receiving the high-dose CF801 in Example 4 seemed to have lower HomaIR scores compared to animals receiving vehicle.
[00198] In this Example, the HomaIR analysis was repeated using a larger number of animals resulting in more data points and a stronger statistical power for the HomaIR analysis. As can be seen above, the effect in this case reached statistical significance.
EXAMPLE 6: Effect of CF801 On Enzymatic Activity Of GOAT And Activity Of Ghrelin Secreting Cells in vitro Example 6 Materials and Methods
GOAT Activity Assay
[00199] GOAT enzymatic activity was measured using the hGOAT assay as previously described by Darling et al (2013, Analytical Biochemistry, 437(l):68-76). Briefly, membrane fractions from Sf9 cells expressing GOAT were thawed on ice and passed through an 18-guage needle 10 times. Assays were initiated by the addition of the acrylodanylated peptide substrate. Assays were incubated at room temperature and stopped by the addition of 50 micro-liters of 20% acetic acid in isopropanol; the addition of the stop solution prior to the acrylodanylated peptide substrate blocked peptide octanoylation. Assays were then analyzed by reverse phase HPLC using a gradient from 30% acetonitrile in water containing 0.05% TFA to 63% acetonitrile in water containing 0.05% TFA flowing at lml/min over 14 min, followed by 100% acetonitrile for 5 min. Peptide substrates typically eluted with a retention time of 5-7 minutes, with octanoylated peptide product eluting at approximately 12 minutes. Chromotogram analysis and peak integration were performed using Chemstation for LC (Agilent Technologies).
In vitro Ghrelin Secretion Studies
[00200] In vitro secretion studies were performed as previously described by Sakata et al (2013, PLoS ONE, 8(6):e64882). Briefly, cells from the immortalized ghrelin cell line SG-1 were used for these studies. SG-1 cells were plated in DMEM/R-12 50:50 medium containing 10% (vol/vol) fetal bovine serum and supplemented with 50 microliters octanoate-BSA, 100 U/mL penicillin and 100 microgram/mL streptomycin sulfate. The cells were placed into the wells of poly-L-lysine-coated culture dishes and incubated in humidified 95% air and 5% C02 at 37°C. Cells were incubated with CF801 overnight in humidified 95% air and 5% C02 at 37.5°C, and then the media was collected and centrifuged at 3,000 X g for 5 minutes. The supernatant was stored at - 80°C until analyzed. Acyl-ghrelin concentrations were determined using a mouse/rat acylated ghrelin enzyme immunoassay kit (#A05117, Bertin pharma, Montingy le Bretonneau, France) according to the manufacturer's guidelines. Example 6 Results [00201] Results demonstrate that CF801 does not inhibit GOAT activity at the micoM concentration levels (Figure 18 A). When the concentrations of CF801 was increased to l.OmM, there was a sharp decline in GOAT activity, however these results are not physiologically relevant and a precipitate formed at these high concentrations. [00202] Overnight incubation of SGI ghrelin secreting cells in a CF801 bath, demonstrated that CF801 does not influence ghrelin secretion activity of SGI cells at low concentrations (i.e. 10, 20 and 1 OOmicroM), but significantly reduces ghrelin secretion at the 300μΜ concentration (Figure 18B). The concentrations required to inhibit the activity of SGI cells is very high, however the SGI cells are known to overexpress both GOAT and ghrelin (Sakata et al, 2013, ibid.) and thus have basal ghrelin levels that are not physiologically relevant. The high concentration of CF801 (i.e. 300μΜ) required to inhibit ghrelin secretion in the SGI cell line is most likely due to the dramatically elevated ghrelin levels seen in this cell line.
Example 6 Summary
[00203] The following effects were observed:
1) CF801 does not inhibit GOAT activity.
2) CF801 decreases ghrelin secretion in the overexpressing SGI cells in vitro at the 300μΜ concentration level.
[00204] The mechanism by which CF801 reduces acylated ghrelin levels is not dependent on GOAT activity in vitro. High doses of CF801 (i.e. 300μΜ) significantly reduce secretion of acylated ghrelin for SGI ghrelinoma cells in vitro.
EXAMPLE 7: Other Experiments
[00205] Due to ghrelin's wide range of roles, CF801 may be used in many different kinds of experiments. These include but are not limited to: 1. Additional experiments examining the role of ghrelin during stress. These include experiments involving different types of stressors, in addition to the chronic social defeat stress paradigm discussed in Example 2. Briefly, a similar protocol to Example 2 is performed with application of various other stressors, including physical and metabolic stressors, such as restraint stress and fox urine. Additional measures are performed, such as a forced-swim task to measure stress-induced depression, along with measurements of the effects CF801 on these measures.
Establishing recommended doses by constructing dose-response curves. Current findings suggest that 22 μιηοΐ/kg daily given intraperitoneally to male C57/BL6J mice is sufficient to observe significant differences in various measures including body weight and fat mass. Briefly, responses to various doses centered around 22 μιηοΐ/kg are measured to determine effective doses for each measure. Measures can include body mass, fat mass, as well as measures on anxiety.
Calorimetry experiments, such as substrate-utilization experiments with metabolic chambers on mice given CF801. Animals are given doses of CF801 and placed in metabolic chambers capable of measuring respiratory exchange ratio. Changes in RER indicate changes in preferred substrates for metabolism. Time-dependent changes in RER, relative to vehicle injected animals, can also be used to indicate duration since injection to drug effectiveness as well as duration of the drug effect.
Glucose tolerance tests to measure CF801's effects on insulin levels, and in studies examining diabetes. Briefly, blood glucose is measured using a glucose strip prior to intraperitoneal injection of a glucose solution. Repeated blood glucose measurements are performed post injection for a specific duration of time. Effects of CF801 treatment on blood glucose levels are measured.
Central administration of CF801, such as by ICV to examine the effect on such variables as mass and anxiety to determine if ghrelin may be present and acylated centrally. Current results suggest rapid early effects on feeding when given continuously through osmotic mini -pumps. Treatment by daily injection through the cannula can be performed. 6. CF801 's effect on reward given data that suggests ghrelin is highly involved in reward and salience (Egecioglu, E., et al, 2010, Addiction Biology, 15(3):304- 311 ; Abizaid, A., et al, 2006, J Clin Invest, 116(12):3229-3239; Naleid, A.M., et al, 2005, Peptides, 26(l l):2274-2279; Jerlhag, E., et al, 2007, Addiction Biology, 12(1):6-16), with these effects being blunted in ghrelin knockout (Bahi, A., et al, 2013, Peptides, 43(0):48-55; Jerlhag, E., et al, 2009, PNAS, 106(27): 11318-11323) and GOAT knockout animals (Davis, J.F., et al, 2012, Hormones and Behavior, 62(5):598-604).
[00206] The disclosures of all patents, patent applications, publications and database entries referenced in this specification are hereby specifically incorporated by reference in their entirety to the same extent as if each such individual patent, patent application, publication and database entry were specifically and individually indicated to be incorporated by reference.
[00207] Although the invention has been described with reference to certain specific embodiments, various modifications thereof will be apparent to those skilled in the art without departing from the spirit and scope of the invention. All such modifications as would be apparent to one skilled in the art are intended to be included within the scope of the following claims.

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A compound of general formula (I)
GSA-X-Y-B (I)
wherein:
X is absent or a sequence of between 1 and 25 consecutive amino acids of the sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 57, or between 1 and 24 consecutive amino acids of the sequence as set forth in SEQ ID NO: 4 or SEQ ID NO: 77;
Y is absent or a peptide linker of between 2 and 10 amino acids in length and comprising amino acids selected from the group of glycine, alanine, valine, lysine and isoleucine;
wherein GSA-X-Y is at least 4 amino acids in length;
and
B is a cell-penetrating peptide.
The compound according to claim 1 , wherein X is a sequence as set forth in any of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,
SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14,
SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19,
SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24,
SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29,
SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34,
SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39,
SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 58,
SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63,
SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68,
SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73,
SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78,
SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90 and SEQ ID NO: 91.
3. The compound according to claim 1, wherein X is a sequence as set forth in any one of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 73, SEQ ID NO: 74 and SEQ ID NO: 75.
4. The compound according to claim 1, wherein X is a sequence of between 1 and 14 consecutive amino acids of the sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 57, and Y is absent.
5. The compound according to claim 1, wherein X is a sequence of between 1 and 5 consecutive amino acids of the sequence as set forth in SEQ ID NO: 3.
6. The compound according to claim 1, wherein the compound has general formula (II):
GSAFL-X-Y-B (II)
7. The compound according to claim 1, wherein the compound has general formula (III):
GSAFLSPEHQ-X-Y-B (III)
8. The compound according to any one of claims 1 to 3 and 5 to 7, wherein Y is absent or a poly glycine or polyalanine peptide linker of between 2 and 10 amino acids in length.
9. The compound according to any one of claims 1 to 7, wherein Y is absent.
10. The compound according to any one of claims 1 to 9, wherein B is a cell- penetrating peptide selected from TAT, penetratin, transportin and variants thereof.
11. The compound according to claim 10, wherein B is a cell-penetrating peptide having a sequence as set forth in any one of RKKRRQRRR (SEQ ID NO: 52); GRKKRRQRRRPQ (SEQ ID NO:53); YGRKKRRQRRR (SEQ ID NO:44) or GRKKRRQRRRPPQ (SEQ ID NO:54).
12. The compound according to claim 1, wherein the compound has a sequence as set forth in SEQ ID NO: 92.
13. The compound according to any one of claims 1 to 12, further comprising a modified C-terminus, a modified N-terminus, or both.
14. The compound according to any one of claims 1 to 12, further comprising a modified C-terminus.
15. The compound according to claim 13 or 14, wherein the modified C-terminus is an amidated C-terminus.
16. A pharmaceutical composition comprising the compound according to any one of claims 1 to 15, and a pharmaceutically acceptable carrier or diluent.
17. A kit for research use comprising a compound according to any one of claims 1 to 15, and instructions for use.
18. The kit according to claim 17, further comprising one or more reagents, reaction vessels or mixing vessels.
19. The kit according to claim 18, wherein the one or more reagents are selected from buffers, salts, enzymes, enzyme co-factors, substrates, detection reagents and washing reagents.
20. The kit according to any one of claims 17 to 19, wherein the research use comprises an investigation into one or more of the des-acyl:acyl ghrelin ratio, the role of des-acyl ghrelin, catabolism of fats and carbohydrates, the relationship between ghrelin deletion and metabolic fuel preference, the role of ghrelin in alterations in food preference, the role of ghrelin in cravings for substances or activities, the role of ghrelin in stress-related eating and/or the role of ghrelin in the development of post-traumatic stress disorder.
21. A compound according to any one of claims 1 to 15 for use in decreasing acylated ghrelin levels in a subject.
22. Use of a compound according to any one of claims 1 to 15 in the manufacture of a medicament for decreasing levels of acylated ghrelin in a subject.
23. The compound according to claim 21, or the use according to claim 22, wherein the subject is obese, is overweight, has diabetes, has pre-diabetes, has metabolic syndrome, is at risk of developing diabetes, has a stress-related eating disorder, or has post-traumatic stress disorder.
24. A compound according to any one of claims 1 to 15 for use in the treatment or management of obesity in a subject.
25. Use of a compound according to any one of claims 1 to 15 in the manufacture of a medicament for treatment or management of obesity in a subject.
26. A compound according to any one of claims 1 to 15 for use in the treatment or management of diabetes in a subject.
27. Use of a compound according to any one of claims 1 to 15 in the manufacture of a medicament for treatment or management of diabetes in a subject.
28. A compound according to any one of claims 1 to 15 for use in the treatment or management of pre-diabetes or metabolic syndrome in a subject.
29. Use of a compound according to any one of claims 1 to 15 in the manufacture of a medicament for treatment or management of pre-diabetes or metabolic syndrome in a subject.
30. The compound according to claim 28, or the use according to claim 29, wherein the treatment or management reduces the risk of the subject developing diabetes.
31. The compound according to any one of claims 21, 23, 24, 26, 28 or 30, or the use according to any one of claims 22, 23, 25, 27, 29 or 30, wherein the compound modifies the eating behaviour of the subject.
32. A compound according to any one of claims 1 to 15 for use to modify eating behaviour of a subject.
33. Use of a compound according to any one of claims 1 to 15 in the manufacture of a medicament for modifying eating behaviour in a subject.
34. The compound according to claim 32, or the use according to claim 33, wherein the subject is obese, is overweight, has diabetes, has pre-diabetes, has metabolic syndrome, is at risk of developing diabetes, or has a stress-related eating disorder.
35. The compound according to any one of claims 31, 32 or 34, or the use according to any one of claims 31, 33 or 34, wherein the modified eating behaviour comprises a reduction in fat intake.
36. A compound according to any one of claims 1 to 15 for use to treat or manage post-traumatic stress disorder in a subject.
37. Use of a compound according to any one of claims 1 to 15 in the manufacture of a medicament for treatment or management of post-traumatic stress disorder in a subject.
38. A compound according to any one of claims 1 to 15 for use in the treatment and/or management of an addictive behaviour in a subject.
39. Use of a compound according to any one of claims 1 to 15 in the manufacture of a medicament for treatment or management of an addictive behaviour in a subject.
40. A method of decreasing acylated ghrelin levels in a subject comprising administering to the subject an effective amount of a compound according to any one of claims 1 to 15.
41. The method according to claim 40, wherein the subject is obese, is overweight, has diabetes, has pre-diabetes, has metabolic syndrome, is at risk of developing diabetes, has a stress-related eating disorder, or has post-traumatic stress disorder.
42. A method of treating or managing obesity in a subject comprising administering to the subject an effective amount of a compound according to any one of claims 1 to 15.
43. A method of treating or managing diabetes in a subject comprising administering to the subject an effective amount of a compound according to any one of claims 1 to 15.
44. A method of treating or managing pre-diabetes or metabolic syndrome in a subject comprising administering to the subject an effective amount of a compound according to any one of claims 1 to 15.
45. The method according to claim 44, wherein the treatment or management reduces the risk of the subject developing diabetes.
46. The method according to any one of claims 40 to 45, wherein the compound modifies the eating behaviour of the subject.
47. The method according to claim 46, wherein the modified eating behaviour comprises a reduction in fat intake.
48. A method of modifying eating behaviour in a subject comprising administering to the subject an effective amount of a compound according to any one of claims 1 to 15.
49. The method according to claim 48, wherein the subject is obese, is overweight, has diabetes, has pre-diabetes, has metabolic syndrome, is at risk of developing diabetes, or has a stress-related eating disorder.
50. The method according to claim 48 or 49, wherein the modified eating behaviour comprises a reduction in fat intake.
51. A method of treating or managing post-traumatic stress disorder in a subject comprising administering to the subject an effective amount of a compound according to any one of claims 1 to 15.
52. A method of treating or managing an addictive behaviour in a subject comprising administering to the subject an effective amount of a compound according to any one of claims 1 to 15.
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WO2010039461A2 (en) * 2008-10-03 2010-04-08 The Johns Hopkins University Methods for synthesis and uses of inhibitors of ghrelin o-acyltransferase as potential therapeutic agents for obesity and diabetes

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