Classifications

• • 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
  • C07K14/60—Growth hormone-releasing factor [GH-RF], i.e. somatoliberin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
  • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
• • 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
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/12—Antidiuretics, e.g. drugs for diabetes insipidus
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the invention relates to inhibitors of ghrelin O-acyltransferase, and methods of making and using them.
• acyl-ghrelin involves an unusual post-translational octanoylation of the serine at the 3 position of the ghrelin peptide. This octanoylation is necessary for its bioactivity, which occurs via interaction with the growth hormone secretagogue receptor (GHSR).
• GHSR growth hormone secretagogue receptor
• the present inventors have found that an inhibitor of GOAT can induce satiety and weight reduction in the obese patient. Furthermore, blocking acylated ghrelin can improve the glucose responsiveness of islets, and thus a GOAT inhibitor is also expected to be an effective therapeutic for use in the Type II diabetic patient population.
• a bisubstrate analog ghrelin O- acyltransferase (GOAT) inhibitor such as, for example, GO-CoA-Tat
• GOAT bisubstrate analog ghrelin O- acyltransferase
• GO-CoA-Tat ghrelin O- acyltransferase
• the invention provides compounds that can inhibit membrane- bound O-acyltransferase (mBOAT) membrane proteins, such as ghrelin O-acyltransferase.
• mBOAT membrane- bound O-acyltransferase
• the compounds are of the formula
• R is absent, or is an organic moiety selected from the group consisting of a substituted or unsubstituted alkyl group, a substituted or unsubstituted peptide chain, a substituted or unsubstituted polysaccharide, or a peptoid residue such as, for example, N-methyl or other N-alkyl substituted glycine.
• R is a peptide chain, it can be, for example, a chain of 1- 20 amino acid residues.
• R is a peptide chain corresponding to ghrelin residues 4-28, or 4-15, or 4-10, or 4-5.
• amino acid includes both naturally occurring and synthetic amino acids. These include, inter alia, alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, 5 -hydroxy lysine, 4-hydroxyproline, thyroxine, 3-methylhistidine, ⁇ -N-methyllysine, ⁇ -N,N,N-trimethyllysine, aminoadipic acid, ⁇ - carboxyglutamic acid, phosphoserine, phosphothreonine, phosphotyrosine, N-methylarginine, and N-acetyllysine.
• amino acid residue may be the residue of an alpha, beta, gamma, or higher order amino acid, preferably one corresponding to a naturally occurring amino acid in other respects.
• amino acid residue encompasses, for example, any amino acid listed above when incorporated into, e.g., an amino acid sequence or other chain structure, whether such chain is linear or branched.
• compositions for example, a composition can comprise a compound of the invention and a pharmaceutically acceptable excipient or carrier.
• the invention provides methods for preparing the compounds of the invention.
• the method can comprise the steps of providing a ghrelin peptide sequence comprising from 3 to 15 N-terminal amino acid residues, wherein the serine residue at the 3 position is replaced with alloc-protected 1 ,2-diaminopropionic acid and wherein D4-Tat is bound to one end of the peptide sequence; combining the ghrelin peptide sequence with Pd(PPh 3 ) 4 palladium reagent, n-bromo octanoic anhydride and Reagent K in one or more reaction vessels to obtain a bromo-octanoylated intermediate; and combining the bromo- octanoylated intermediate with coenzyme A.
• the invention provides methods for treating a disease, disorder or condition using the compounds and compositions of the invention.
• the methods are used to treat obesity. These methods can comprise administering to a subject a composition comprising a therapeutically effective amount of one or more of the compounds of the invention.
• the methods can, for example, bring about a greater loss of fat mass than lean mass in the subject, or they can bring about an increase in the ratio of ghrelin to acyl-ghrelin in the subject.
• the methods can be used to treat diabetes, for example type II diabetes. These methods can comprise administering to a subject a composition comprising a therapeutically effective amount of one or more of the compounds of the invention.
• these methods can, for example, bring about an increased production of insulin in the subject, or an increased responsiveness to a glucose challenge in the subject, or a reduction in uncoupling- protein 2 (UCP-2) mRNA levels in the subject.
• the methods can be used to treat irritable bowel syndrome.
• These methods can comprise administering to a subject a composition comprising a therapeutically effective amount of one or more of the compounds of the invention.
• the invention provides methods for treating mBOAT-associated diseases.
• these methods can comprise administering to a subject a composition comprising a therapeutically effective amount of one or more compounds of the invention.
• kits can comprise a composition comprising at least one dose of a therapeutically effective amount of the compounds and compositions disclosed herein.
• the kits also include at least one dose of an additional weight loss treatment.
• FIG. 1 Figures IA-I G: GO-CoA-Tat is a bisubstrate inhibitor that inhibits GOAT, lowering acyl ghrelin levels.
• A Bisubstrate analog inhibitors covalently combine two substrates, thereby increasing potency and specificity.
• B Structure of D4-Tat and synthetic scheme for bisubstrate inhibitors, which consist of three components or their substantial equivalents: coenzyme A; an octanoylated moiety such as, for example, a substituted or unsubstituted ghrelin peptide; and a Tat peptide.
• C Structures of compounds according to the present invention.
• FIG. 1C Acyl-ghrelin levels (pg/ml) in HEL cell lysate (2.5 x 10 6 cells) after 24 hr incubation with one of the seven compounds listed in Figure 1C.
• Figures 2A-2D GO-Co A-Tat lowers circulating acyl- but not desacyl- ghrelin levels in mice.
• FIGS 4A-4D GO-Co A-Tat increases insulin, decreases glucose levels, and down-regulates UCP2 mRNA.
• IPGTT intraperitoneal glucose tolerance test
• C QRT-PCR of islets and
• Figure 5A-5E GO-Co A-Tat lowers acyl- but not desacyl-ghrelin levels in AGS and Kato III cells.
• A Dose-response inhibition of acyl-ghrelin (pg/ml) in AGS cell lysate (2.5 x 10 6 cells) after 24 hr incubation with various concentrations of GO-Co A-Tat.
• B Temporal inhibition of 6 ⁇ M GO-CoA-Tat in AGS cell lysate (2.5 x 10 6 cells).
• C Dose-response of des- acyl ghrelin (pg/ml) in HEL cell lysate (2.5 x 10 6 cells) after 24 hr incubation with various concentrations of GO-CoA-Tat and D4-Tat.
• D Dose-response inhibition after 24 hr incubation and
• E temporal inhibition of 6 ⁇ M GO-CoA-Tat in Kato III cells.
• FIGS 6A-6B Establishment of an in vitro assay for GOAT.
• GOAT was present in microsomes isolated from HEK293T GnTI(-) cells.
• HEK293T GnTI(-) cells were transfected with GOAT-3xFlag or empty vector (Control) and microsomes were prepared as described in the Examples.
• A Immunoblot analysis with 10 ⁇ g/mL monoclonal anti-FLAG antibody of microsomes (50 ⁇ g of protein) from transfected and untransfected cells.
• B GOAT activity is present in microsomes from transfected but not untransfected cells and is completely inhibited by GO-CoA-Tat at 10OnM.
• Figures 7A-7C Assay for non-specific inhibition and toxicity of GOCoA-Tat.
• A Percent activity in the presence of 10 ⁇ M of GO-CoA-Tat in p300, PCAF and AANAT.
• B Viability of HepG2 cells and
• C HEL cells after 24 hr incubation with varying concentrations of GO-CoA-Tat.
• Figures 8A-8D Effects of GO-Co A-Tat on intake and excretion.
• B water consumption.
• C The cumulative feces and (D) urine production of these mice also were not different to a statistically significant degree.
• Figure 9 Blood panel from mice treated with D4-Tat and GOCoA-Tat.
• Figures lOA-lOC Effect of GO-CoA-Tat on insulin and glucose in islets and mice.
• C a statistically significant decrease in blood glucose.
• Figures 1 IA-11C Weight gain in wild type or ghrelin knockout mice on a medium chain triglyceride diet, with or without GOAT antagonist. The GOAT inhibitor GO-
• CoA-Tat affects the weight of wild type but not genetically altered (ghrelin knockout) mice, confirming the targeting of the ghrelin pathway
• Figures 12A-12B Glucose tolerance test in wild type and ghrelin knockout mice given GOAT antagonist.
• the GOAT inhibitor GO-CoA-Tat affects the glucose levels of wild type but not genetically altered (ghrelin knockout) mice, confirming the targeting of the ghrelin pathway.
• FIGS 13A-13C Photoactivatable GO-CoA-Tat analogs can cross-link solubilized GOAT. Two photoactivatable versions of GO-CoA-Tat can directly bind and chemically crosslink to GOAT.
• Figures 14A-14B GOAT inhibitor suppresses blood IGFl levels in male and female mice. This is consistent with an effect on acyl-ghrelin.
• Ghrelin a powerful appetite-stimulating hormone secreted by the stomach, has been implicated as a key peptide hormone that stimulates weight gain in animals and people.
• ghrelin requires acylation, an unusual post-translational octanoylation of the serine residue at the 3 position.
• the octanoylated form of ghrelin is commonly referred to as acyl-ghrelin.
• the octanoylation of ghrelin is mediated by ghrelin O-acyltransferase (GOAT).
• GOAT is a member of the membrane-bound O-acyltransferase (mBOAT) family of membrane proteins. Disclosed herein is a bisubstrate analog GOAT inhibitor, GO-CoA-Tat, which is potent, selective, and effective in vitro, in cell culture, and in mice.
• mBOAT membrane-bound O-acyltransferase
• the invention provides compounds for use in treating, for example, obesity and/or diabetes.
• the compounds can have the generic formula (I):
• R may be absent, or R can be any organic moiety consistent with the proper functioning of the compound, as disclosed herein.
• R can be, for example, an alkyl group, as well as combinations of peptoid residues (e.g., N-alkyl GIy), beta amino acids, aminobutyrate, and phenylGly.
• R is a peptide chain made up of amino acid residues, such as, for example, those comprising a fragment of the ghrelin peptide. The fragment of the ghrelin peptide can begin, for example, at residue 4.
• R can be made up, for example, of residues 4-5, or 4-10, or 4-15, or 4-28 of the ghrelin peptide. Indeed, the invention contemplates that R can be any number of residues that is capable of functioning according to the invention as disclosed herein.
• the compound of formula I is also referred to as "GO-CoA-Tat.”
• specific examples of compounds that fall within the genus defined by Formula I can also be referred to as "GO-CoA-Tat.”
• GO-CoA-Tat is an inhibitor of GOAT, which mediates the octanoylation of ghrelin to produce acyl-ghrelin.
• GS is made up of the amino acid residues corresponding to glycine and serine, respectively, according to their conventional one-letter designations.
• the glycine and serine can be substituted with any organic moiety, including a peptide chain of any length, that can function according to the invention as disclosed herein.
• suitable substitutes for the serine residue include, for example, D-amino acids such as D-AIa or D-Ser, which can also be truncated to an acetyl, or propionyl group; or a hydroxyacetyl substituent.
• Suitable substitutes for the glycine residue include those listed for serine as well as a peptoid residue (e.g., N-alkyl GIy), a beta amino acid, aminobutyrate, and phenylGly.
• peptoid residue e.g., N-alkyl GIy
• beta amino acid aminobutyrate
• phenylGly e.g., phenylGly.
• peptide chain encompasses moieties that include peptoid residues and other variants or derivatives of amino acids; it also encompasses moieties made of combinations of naturally occurring amino acids, peptoid residues and other amino acid variants or derivatives, or any subset thereof.
• GS-Dap-R refers to 1 ,2-diaminopropionic acid.
• Dap replaces the serine normally present at the 3 position. Dap is a serine isostere with a nitrogen in place of oxygen in the side chain, leading to an amide linkage.
• Ahx refers to an amino-hexanoyl linker, which is used to link R with the Tat sequence. Ahx can be, for example, aminohexanoic acid, glycine, amino propionic acid, amino butyric acid, aminopentanoic acid, and ethyleneglycol-based substituents.
• Tat which is shorthand for "trans-acting activator of transcription,” refers to a peptide sequence commonly found in the HIV virus.
• Tat can be an 11 -residue peptide comprising the amino acids YGRKKRRQRRR.
• Tat proteins can mediate the transport of molecules, such as proteins or other moieties, across cell membranes. Such mediation appears to be independent of molecule size and does not appear to involve any disruption of the plasma membrane.
• the Tat moiety in GO-CoA-Tat aids in the delivery of the compound into the interior of the cell. Accordingly, any moiety that would also aid in such delivery can be substituted for Tat in the compounds of the present invention.
• Suitable substituents for Tat include, for example, oligoArg, oligoalkyl guanidiniums, and either d- or 1- stereoisomers for Tat.
• D4-Tat comprises a peptide of four aspartate residues bound to a Tat sequence. D4-Tat were prepared using the Fmoc strategy or a modification thereof.
• GO-CoA-Tat is based on the theory that, if GOAT uses a ternary complex mechanism which templates octanoyl-CoA and ghrelin peptide, linking the two substrates with a non-cleavable bridge could combine the binding energies of the individual ligands without the entropic loss associated with forming the ternary complex (Fig IA).
• a related successful strategy has been used for other peptide-modifying enzymes, including histone acetyltransferases (HAT) and protein kinases.
• ghrelin peptide is used as the R group in Formula I, we were uncertain about the ghrelin peptide length needed for recognition by GOAT, we selected amino acids 1-10 for coupling to octanoyl-CoA, to maximize inclusion of highly conserved ghrelin residues.
• An Tat-derived peptide sequence for example an 11 -mer HIV Tat, can also be attached to the C- terminus via an amino-hexanoyl linker to enhance cell penetration.
• GO-CoA-Tat appears to be a selective antagonist for GOAT because, at 10 ⁇ M, it showed less than 15% inhibition of three other acetyl-CoA utilizing enzymes in vitro, including p300 HAT, PCAF HAT, and serotonin N-acetyltransferase (Fig. 7). Moreover, 10 ⁇ M GO-CoA- Tat appears non-toxic to HEL cell and HEPG2 cell viability (Fig. 7). Furthermore, GO-CoA-Tat is likely to be acting as a bona fide bisubstrate analog in antagonizing GOAT activity (Fig. IG). The fact that inclusion of the Tat sequence, or other moiety that aids in cell entry, increases inhibitory activity suggests that cell penetration is involved, and the compound is not acting on a cell surface receptor.
• GO-Co A-Tat is administered, for example, intraperitoneally, to enhance insulin response to a glucose load and lead to a relative weight reduction in a mammal, e.g., in mice fed a high fat diet.
• Quantitative MR spectroscopy has established that GO-CoA-Tat treatment leads to a selective reduction in fat versus lean mass.
• GOAT can be targeted in treatment of obesity and glucose intolerance, and GO-CoA-Tat provides an effective GOAT inhibitor.
• the invention provides compositions comprising a compound of formula I and a pharmaceutically acceptable excipient.
• pharmaceutically acceptable encompasses those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem complications commensurate with a reasonable benefit/risk ratio.
• pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopoeia or other generally recognized international pharmacopoeia for use in animals, and more particularly in humans.
• excipient refers to a substance that is used in the formulation of pharmaceutical compositions, and, by itself, may be without therapeutic value.
• excipients can be used in the invention, including those described in Remington: The Science and Practice of Pharmacy, 21 st Ed. (2006), which is incorporated by reference. Excipients include, but are not limited to, antioxidants, anti-bacterial agents that prevent the decay of the formulation itself (as opposed to those exhibiting a therapeutic effect), preservatives, chelating agents, buffering agents, agents for adjusting toxicity, colorings, flavorings and diluting agents, emulsifying and suspending agents, and other substances with pharmaceutical applications.
• compositions of the invention can be administered in conjunction with other forms of obesity treatment, such as diet and exercise or bariatric surgery.
• other forms of obesity treatment such as diet and exercise or bariatric surgery.
• the compositions of the invention can be administered before, simultaneously with, or after the other form of obesity treatment.
• compositions for use in accordance with the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
• Suitable routes of administration may, include, for example, oral, rectal, transmucosal, especially transnasal, topical, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
• the pharmaceutical composition may be administered locally or systemically.
• the composition can be administered locally via injection of the preparation directly into a specific region of a patient's body.
• the active ingredients of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
• penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
• the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
• Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient.
• Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores.
• Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP).
• disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
• Dragee cores are provided with suitable coatings.
• suitable coatings may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
• Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
• compositions that can be used orally include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
• the push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers.
• the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
• stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
• compositions may take the form of tablets or lozenges formulated in conventional manner.
• the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
• a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
• the dosage unit may be determined by providing a valve to deliver a metered amount.
• Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
• compositions described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion.
• Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative.
• the compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
• compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
• the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
• a suitable vehicle e.g., sterile, pyrogen-free water based solution
• the active ingredient may also be subjected to PEGylation according to methods known in the art.
• the preparations of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
• compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount is an amount of one or more active ingredients effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated.
• Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals.
• the data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
• the dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.
• dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
• compositions to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
• compositions including the preparation of the present invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
• compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
• Methods of Preparing are well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
• the invention provides methods of preparing the compounds of
• the methods comprise the steps of providing a ghrelin peptide sequence comprising from 3 to 15 N-terminal amino acid residues, wherein the serine residue at the 3 position is replaced with alloc-protected 1 ,2-diaminopropionic acid and wherein a Tat sequence is bound to one end of the peptide sequence; combining the ghrelin peptide sequence with Pd(PPh 3 ) 4 palladium reagent, n-bromo octanoic anhydride and Reagent K in one or more reaction vessels to obtain a bromo-octanoylated intermediate; and combining the bromo- octanoylated intermediate with coenzyme A.
• the Tat can be, for example, bound to one end of the R sequence of formula 1.
• Tat can be attached to the C-terminal end of the peptide sequence.
• Tat is attached through a linking moiety, such as, for example, an amino-hexanoyl linker, though any linking moiety that does not interfere with the proper functioning of the GO-Co A-Tat is within the scope of the present invention.
• Reagent K refers to a standard mixture of trifluoroacetic acid, water, thioanisole, phenol and ethanedithiol. It is used to deblock protected amino acids and cleave them from resin.
• Synthesis of GO-CoA-Tat (e.g., Compound 1 as listed in Figure 1C), can be performed using, for example, a solid phase strategy (Fig. IB).
• a set of related compounds (Compounds 2-7, Fig. 1C) with different peptide lengths and individual deletion of CoA, octyl, and Tat, respectively, can also be synthesized (Fig. 1C) and used according to the present ivnention.
• the invention provides methods of preventing and/or treating various diseases, disorders or conditions using a composition comprising therapeutically effective amount of a compound of Formula I.
• Treatment refers to prophylactic or preventative measures in a subject who has not developed signs or symptoms of the disorder.
• Treatment encompasses, for example, alleviation of symptoms; diminishment of the extent of the condition, disorder or disease; stabilization (i.e., not worsening) of the state of the condition, disorder or disease; slowing of the condition, disorder or disease progression; amelioration of the condition, disorder or disease state; remission, whether partial or total; or enhancement or improvement of the condition, disorder or disease.
• Those in need of treatment include those already with the disorder.
• Those in need of prevention or prophylaxis include those in which the disorder can be prevented.
• the patient to be administered the compounds disclosed herein may have been diagnosed as having the disorder or may be predisposed or susceptible to the disorder.
• Treatment includes eliciting a measurable response, preferably a clinically significant response, without excessive levels of side effects.
• Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.
• the subject according to the present invention is a mammal, such as a human, that is diagnosed with one of the diseases, disorders or conditions described herein, or alternatively is predisposed to at least one type of the diseases, disorders or conditions described herein.
• the compositions of the present invention can be administered to any mammal in need of the composition that can experience the beneficial effects of the compounds of the invention. Any such mammal is considered a "subject.”
• Such subjects include humans and non-humans, such as humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc.
• the subject can be a man or a woman.
• the amount of active agent required to constitute a therapeutically effective amount will vary based on a number of factors, including the severity of the disorder to be treated; the identity, age, body weight, general health, gender, diet and chemical make-up of the patient; the type and degree of the cellular response to be achieved; the specific agents or composition employed, and its activity; the time of administration, route of administration, and rate of excretion of the agent; the duration of the treatment; drugs used in combination or coincidental with the specific agent; and similar factors well known in the medical arts.
• a therapeutically effective amount is an amount that produces a measurable alleviation of one or more symptoms associated with a disease or disorder.
• a therapeutically effective amount for treating or preventing obesity can be up to or at least about 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 5 mg/kg, 7.5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg or more of the active agent per day.
• a therapeutically effective dose can be up to or at least about 50 mg, 60 mg,
• the invention provides methods of treating or preventing obesity.
• the methods can comprise administering to a subject a composition comprising a therapeutically effective amount of the compound of Formula I.
• administering the composition can bring about, for example, a greater loss of fat mass than lean mass in the subject. It can also bring about, for example, an increase in the ratio of ghrelin to acyl-ghrelin in the subject.
• the invention provides methods of treating or preventing diabetes, for example type II diabetes.
• the methods can comprise administering to a subject a composition comprising a therapeutically effective amount of the compound of Formula I.
• Administering the composition can bring about, for example, an increased production of insulin in the subject. It can also bring about, for example, an increased responsiveness to a glucose challenge in the subject, and/or a reduction in uncoupling-protein 2 (UCP-2) mRNA levels in the subject.
• UCP-2 uncoupling-protein 2
• the therapeutically effective amount of the compounds of Formula I for treating or preventing diabetes can be determined by one of ordinary skill in the art, though it can be, for example, similar to that described above for obesity.
• a therapeutically effective amount for treating or preventing diabetes can be up to or at least about 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 5 mg/kg, 7.5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg or more of the active agent per day.
• a therapeutically effective dose can be up to or at least about 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 120 mg, 140 mg, 160 mg, 180 mg, 200 mg, 210 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg, 2000 mg or more of the active agent per day.
• GOAT inhibition appears to treat diabetes by decreasing levels of uncoupling protein-2 (UCP-2) in pancreatic islet cells. That GOAT inhibition modulates UCP2 levels so dramatically further substantiates the connection of acyl-ghrelin to obesity and type 2 diabetes.
• Acyl ghrelin induces feeding behavior by causing an increase in UCP2 levels in arcuate nucleus neurons that co-express neuropeptide Y and agouti-related protein.
• UCP2 can be markedly upregulated in islets of ob/ob mice, a model of obesity-induced diabetes.
• ob/ob mice lacking UCP2 exhibit restored first-phase insulin secretion, increased serum insulin levels, and greatly decreased levels of glycemia.
• a human UCP2 polymorphism, A55V is associated with obesity and markedly increased insulin and leptin concentrations.
• type 2 diabetic patients homozygous for the -866A UCP2 promoter polymorphism have elevated UCP2 expression and require a higher frequency of insulin treatment. These patients also have significantly lower glucose-induced insulin secretion (GIIS).
• GIIS glucose-induced insulin secretion
• the islets isolated from -866 A/A donors have significantly lower GIIS than those isolated from -866 G/A heterozygous and G/G homozygous individuals.
• the invention provides methods of treating mBOAT- associated diseases.
• the methods can comprise administering to a subject a composition comprising a therapeutically effective amount of the compound of Formula I, thereby treating the mBOAT-associated disease.
• mBOAT associated disease is a disease characterized by dysregulation of a pathway in which an mBOAT peptide is involved.
• mBOAT genes There are at least sixteen mBOAT genes.
• a gene product known as "porcupine” which has structural similarities to mBOAT proteins, is integral to Wnt signaling, a process associated with cancer. Wnt requires acyl modification by the porcupine acyltransferase enzyme. Wnt signaling may contribute to many cancers and could be blocked by porcupine inhibitors.
• mBOAT associated diseases include various forms of lipid metabolism dysregulation. Altered lipid metabolism can contribute to cancer, and blocking mBOATs can provide cancer treatments.
• the invention provides methods of treating or preventing disorders associated with gastric motility dysregulation, such as, for example, irritable bowel syndrome.
• Ghrelin administration increases gastric emptying and is associated with decreased gastroparesis. Therefore, administration of GO-CoA-TAT can be used to inhibit gastric motility via its inhibition of GOAT and the concomitant decrease in acyl-ghrelin levels, exerting a palliative effect in patients with irritable bowel syndrome and other disorders associated with gastric motility dysregulation, as would be understood by a person of ordinary skill in the art.
• kits comprising kits.
• the kits comprise a composition comprising at least one dose of a therapeutically effective amount of the compound of Formula I.
• kits can further comprise at least one dose of an additional weight loss treatment.
• additional treatments include, for example, antibodies against acyl- ghrelin as well as antagonists to GHSR.
• acyl-ghrelin hormone offers several advantages over receptor antagonists.
• these enzyme inhibitors do not need to cross the blood-brain barrier, in contrast to acyl-ghrelin receptor blockers, for which many of the key sites of action are in the brain.
• the effect of an enzyme inhibitor is likely to be more global in scope. For example, there may be classes of acyl-ghrelin receptors other than GHSR that have not yet been identified, and which would presumably not be affected by receptor antagonists targeting known receptors. However, the activities of these as-yet unknown receptors would of course be impacted by decreased hormone levels.
• receptor blockers have the disadvantage that animals may have feedback mechanisms that would drive higher acyl-ghrelin formation in response to receptor blockade, which might overwhelm receptor blockers.
• a feedback mechanism would be effectively counteracted by an enzyme inhibitor, which targets the biosynthetic pathway and thus prevents the feedback mechanism from increasing acyl-ghrelin levels.
• the GOAT enzyme acts catalytically, a given dose of GOAT inhibitor is likely to be proportionately more effective than a similar dose of an acyl-ghrelin receptor blocker, which is only capable of more or less one-to-one effectiveness.
• this invention sets forth a novel approach for the pharmacologic management of weight and glucose control.
• Example 1 Effect of GO-CoA-Tat on Acyl-Ghrelin Production in Three Cell
• GO-Co A-Tat appears to be a selective GOAT antagonist since at 10 ⁇ M, it showed less than 15% inhibition of three acetyl-CoA utilizing enzymes in vitro including p300 HAT, PCAF HAT, and serotonin N-acetyltransferase (Fig. 7). Moreover, 10 ⁇ M GO-CoA-Tat appears non-toxic to HEL cell and HEPG2 cell (ATCC, Manassas, VA) viability (Fig. 7).
• Example 2 Effect of GO-CoA-Tat Administration On Acyl-Ghrelin
• Example 3 Effect of GO-CoA-Tat Administration on Weight Gain in Mice
• mice were treated every 24 h with GO-CoA-Tat (29 mg/kg IP) and studied in metabolic cages allowing daily monitoring of intake and output as well as body mass.
• the mice were subjected every 3 d to quantitative magnetic resonance (QMR) spectroscopy to evaluate the animals' fat and lean mass (15).
• QMR quantitative magnetic resonance
• Example 4 Effect of GO-CoA-Tat On Insulin Response to Glucose
• QRT-PCR of islets isolated from mice treated with GO-CoA-Tat demonstrated a 20-fold reduction in UCP2 mRNA levels as compared to D4-tat treated mice (Fig. 4C) but no change in levels of insulin, ghrelin, or GHSR.
• QRT-PCR showed non-statistically significant effects on UCP2 in the gastric fundus (Fig. 4D).
• ob/ob mice lacking UCP2 exhibit restored first-phase insulin secretion, increased serum insulin levels, and greatly decreased levels of glycemia (19).
• a human UCP2 polymorphism, A55V is associated with obesity and markedly increased insulin and leptin concentrations (22).
• Type 2 diabetic patients homozygous for the -866A UCP2 promoter polymorphism have elevated UCP2 expression and require a higher frequency of insulin treatment. These patients also have significantly lower glucose-induced insulin secretion (GIIS) (23).
• GIIS glucose-induced insulin secretion
• the islets isolated from -866 A/A donors have significantly lower GIIS than those isolated from -866 G/A heterozygous and G/G homozygous individuals (24).
• Synthesis of GO-CoA-Tat and related derivatives was analogous to previously described methods (33, 34) beginning with ghrelin and using the Fmoc strategy. Variable lengths of ghrelin peptide (3 to 15 C-terminal sequences) were employed and Ser3 in ghrelin was replaced with Alloc (allyloxycarbonyl) protected- 1 ,2-diaminopropionic acid.
• the collected fractions were concentrated under reduced pressure and lyophilized to give the final products as white solids.
• Their matrix-assisted laser desorption / ionization (MALDI) and electrospray (ES)-mass spectrometry data were consistent with the calculated values and the final concentrations of the compounds in aqueous solution for assay were determined by amino acid analysis.
• Example 6 Cell culture, cell Iy sate preparation and EIA measurements
• HEL cells and AGS cells were grown in RPMI
• Cells (7.5x10 6 cells) were centrifuged at 1000 rpm in a table-top centrifuge for 5 minutes. The cell pellet was then washed and to this 0.25 ml of ice-cold modified RIPA buffer containing was added and vigorously pipetted and vortexed and then allowed to sit for 10 minutes at 4° C to thoroughly Iy se cells. The lysate was then spun down at 2,000 rpm in a precooled centrifuge for 15 minutes. Immediately after centrifugation, the supernatant was stored at -80°C.
• GOAT was cloned by a two-stage nested RT-PCR scheme from mouse stomach.
• a band at ⁇ 1 .3kB was excised from the gel and ligated into pCR ® 2.1 -TOPO ® (Invitrogen).
• GOAT was amplified from this vector using primers designed for 5' blunt ligation and 3' EcoRI cleavage, then cloned into the p ⁇ H vector, derived from pHLsec (Aricescu et al. Acta Cryst. (2006). D62, 1243-1250) using Kpnl (blunted) and EcoRI.
• Cells were lysed using a dounce homogenizer (type B pestle, 30 strokes). Lysates were centrifuged at 2,135 x g at 4°C for 10 min, and the supernatant was transferred to a new tube and centrifuged again under the same conditions.
• the assay protocol was modified from Yang et al. (36).
• the assay mixture contained 50 mM HEPES, pH 7.0, 1 ⁇ M octanoyl-CoA (33 dpm/fMol, American Radiolabeled Chemicals), 10 ⁇ M Ghrelin27-Biotin, 50 ⁇ g microsome protein, and 50 ⁇ M palmitoyl-CoA (Avanti Polar Lipids). Reactions were initiated by the addition of membrane protein and incubated at 37°C for 5 min. Where indicated, GO-Co A-Tat was pre-incubated for 5 min with the membrane protein.
• HEL cell line and the human immortalized hepatocyte cell line HepG2 (ATCC, Manassas, VA) treated with the varying concentrations of GO-CoA-Tat was determined using a LIVE/DEAD viability assay kit (Molecular Probes).
• Rat bone marrow stromal cells (RBMSCs) were incubated with two probes, 2 ⁇ M calcein-AM (green color) for 30 min and 4 ⁇ M ethidium homodimer-1 (EtdD-1, bright red color) for 10 min, for intracellular esterase activity and plasma membrane integrity, respectively.
• islets were incubated in serum-free RPMI media with 5 ⁇ M GO-CoA-Tat for 24 hrs prior to a static incubation assay for thirty minutes in glucose-free RPMI media with 50, 150 and 300 mg/dl glucose added and the insulin secreted into the medium was assessed with ELISA (Alpco Diagnostics).
• All reactions contain 200 ⁇ M thiamine pyrophosphate, 5 mM MgCl 2 , 1 mM DTT, 50 ⁇ g/mL BSA, 200 ⁇ M NAD, 2.4 mM ⁇ - ketoglutarate, 10 ⁇ M GO-Co A-Tat, and 50 ⁇ M acetyl-CoA.
• Reactions with p300 were performed in 100 mM HEPES, pH 7.9, and contain 0.1 units ⁇ -KGDH and 200 ⁇ M H4-15, a 15- mer peptide substrate based on the sequence of the histone H4 tail.
• p300 reactions are incubated at 30 0 C prior to initiation with addition of 100 nM p300 and take place at 30 0 C.
• Reactions with PCAF are performed in 100 mM HEPES, pH 7.9, and contain 0.037 units ⁇ -KGDH and 100 ⁇ M H3-20, a 20-mer peptide substrate based on the sequence of the histone H3 tail.
• PCAF reactions are incubated at 30 0 C for 10 minutes prior to initiation with addition of 100 nM PCAF and take place at 30 0 C.
• Reactions with AANAT are performed in 100 mM NH 4 OAc, pH 6.8, and contain 0.1 units ⁇ -KGDH and 200 ⁇ M tryptamine.
• AANAT reactions are incubated at 25°C for 10 minutes prior to initiation with addition of 10.83 nM AANAT and take place at 25°C. All reactions are followed over the linear portion of the progress curve, which provides the initial velocity via linear regression.
• Example 13 Murine Experiments
• Serum levels of desacyl- and acyl-ghrelin were determined by ELISA (Alpco Diagnostics). For diabetic outcomes, the above protocol was followed with the exception that an oral glucose challenge (lg/kg) was administered thirty minutes prior to blood sampling. For diabetic outcomes compound was administered to non-fasted mice 6 hours prior to an 18 hour fast, at which time a 2.5g/kg intraperitoneal glucose tolerance test (IP-GTT) was performed on conscious mice. Blood was sampled from the tail vein at 0, 15, 30, 60 and 120 min post IP-GTT. Glucose was measured with a glucometer (LifeScan OneTouch) and insulin values were assessed by ELISA (Alpco Diagnostics).
• IP-GTT intraperitoneal glucose tolerance test
• mice body profile of mice was assessed with QNMR every three days (EchoMRI). For each body measurement, the average of three separate scans was obtained.
• Example 14 QRT-PCR
• Islets were isolated as described in Song et al. (41) after collagenase and
• RT-PCR was performed using the one-tube RT-PCR Sybr green mix (BioRad) according to standard protocols. Fold changes in expression levels were calculated using the DDCT method. Duplicate results were analyzed using Student's t-Test.
• Primers used for RT-PCR Insulin Fw: CGAGGCTTCTTCTACACACC; Insulin Rv: GAGGGAGCAGATGCTGGT Glucagon Fw: CCACTCACAGGGCACATTCA; Glucagon Rv:
• Mouse pancreas or human islets were fixed in 10% buffered formalin or Bouin's solution, paraffin embedded and 5 ⁇ m sections were prepared. After dewaxing, rehydration, and antigen retrieval in citrate buffer, immunostaining was performed with guinea pig anti-insulin (Abeam), rabbit anti-ghrelin (Abeam) and chicken anti-GHSR (Chemicon). Appropriate fluorescence-tagged secondary antibodies (Donkey anti guinea pig Cy3, Donkey anti -rabbit FITC, Jackson Immunoresearch) were used for antigen localization. Nuclei were counterstained using DAPI contained in mounting medium (Vector Biolabs). A Zeiss Axioskop equipped with a CCD digital camera and an Apotome optical sectioning device connected to a digital image processor for pseudocoloring were used for image preparation.
• Ghrelin-deficient mice were generated by using the high-throughput VelociGene gene-targeting system, as described previously (42). All animals were maintained on a 12:12-h light-dark cycle at 22°C and fed a diet enriched in medium chain triglycerides (MCT)-(10% MCT, 40% sucrose, Teklad TD 08622) with free access to food and water. 40 mg/kg Go-CoA- TAT was administered to WT and KO mice and vehicle was administered to WT mice. Whole body composition (fat and lean mass) was measured using NMR technology (EchoMRI, Houston, TX) every 3-4 days and daily weight measurements were taken. Results are presented in Figure 11.
• MCT medium chain triglycerides
• Example 18 Cross-Linking of Photoactivatable GO-CoA-Tat analogs and solubilized GOAT
• FLAG-GOAT enzyme was overproduced in SF9 cells, solubilized with detergent, and treated with UV light in the presence of a benzoyl-phenylalanine- and biotin-containing analog of GO-CoA-Tat (5 uM). These led to crosslinking as shown in the Biotin blot ( Figure 13A), which could be blocked with standard GO-CoA-Tat (100 uM). FLAG ( Figure 13B) and Coomassie ( Figure 13C) gels indicate even loading in these lanes. [00132] Example 19: GOAT Inhibitor Effects on IGF-I
• Plasma samples were collected from transcardial puncture using EDTA-coated Microvette tubes and immediately chilled on ice. After 15 min of centrifugation at 3,000 g and 4°C, plasma was stored at -80 0 C.
• a commercially available assay was used (R&D Systems, Minneapolis, MN). This assay is validated for the measurement of IGF-I in nonacidified plasma samples and does not require protein extraction. The assay was performed according to the assay manufacturer's instructions. Results are presented in Figure 14.

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