US 201701.21385A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2017/0121385 A1 Shah et al. (43) Pub. Date: May 4, 2017

(54) METHODS OF TREATING Publication Classification NEURODEGENERATIVE CONDITIONS (51) Int. Cl. Applicant: Oxeia Biopharmaceuticals, Inc., San C07K I4/575 (2006.01) (71) A6II 45/06 (2006.01) Diego, CA (US) A638/22 (2006.01) (72) Inventors: Kartik Kiran Shah, San Diego, CA (52) U.S. Cl. (US); Amit Dilip Munshi, Greenwich, CPC ...... C07K 14/575 (2013.01); A61K 38/22 NC (US) (2013.01); A61K 45/06 (2013.01); A6 IK 38/00 (2013.01) (73) Assignee: Oxeia Biopharmaceuticals, Inc., San Diego, CA (US) (57) ABSTRACT (21) Appl. No.: 15/338,159 (22) Filed: Oct. 28, 2016 The present disclosure provides methods for treating a neurodegenerative condition or situation in a subject in need Related U.S. Application Data thereof, comprising administering to the Subject an effective (60) Provisional application No. 62/247,680, filed on Oct. amount of a compound comprising ghrelin or a ghrelin 28, 2015. variant. US 2017/O121385 A1 May 4, 2017

METHODS OF TREATING said neurodegenerative condition. In one embodiment, the NEURODEGENERATIVE CONDITIONS ghrelin variant is administered in a non-endogenous carrier. 0008. In some embodiments, the ghrelin variants can be 0001. This application claims priority to U.S. Provisional a sequence that includes any of a number of modifications to Application Ser. No. 62/247,680, filed Oct. 28, 2015, the the wild type ghrelin sequence, which comprises a polypep entire content of which is hereby incorporated by reference tide having an amino acid sequence of Gly-Ser-Ser-Phe herein. Leu-Ser-Pro-Glu-His-Gln-Arg-Val-Gln-Gln-Arg-Lys-Glu Ser-Lys-Lys-Pro-Pro-Ala-Lys-Leu-Gln-Pro-Arg (SEQ ID FIELD OF THE INVENTION NO. 1). Non-limiting examples of potential modifications 0002 The present disclosure provides methods for treat include modifying the length (shorter or longer) of the ing a neurodegenerative condition in a Subject in need sequences, modifying the chemistry of the amino acids, thereof by administering to the subject an effective amount Substituting one or more of the amino acids with another of a composition comprising ghrelin or a ghrelin variant. amino acid, a synthetic amino acid or otherwise rare or non-naturally occurring amino acid, introducing protecting BACKGROUND groups at the N and/or C termini, etc. In some embodiments, the polypeptide is modified with one or more fatty acids. In 0003 Mild brain injuries (mBI), typically including con Some embodiments, the fatty acid is an octanoic acid. In cussions, may result in long-term neurocognitive deficits Some embodiments, the polypeptide is modified at serine at mimicking other neurodegenerative condition, Such as, amino acid position 2 and/or serine at amino acid position 3 Alzheimer's disease (AD) pathology. Indeed, Chronic Trau of SEQ ID NO. 1. matic Encephalopathy (CTE), observed after repetitive con 0009 For example, in some embodiments, ghrelin or the cussions, is characterized by neurofibrillary tangles of phos ghrelin variants include C-C acylation of the carboxyl phorylated Tau (p-Tau) a core finding in AD. Evidence group of one or both of the glutamic acid residues or of the linking concussions and AD is growing, most evident in C-terminus arginine group. In other embodiments, ghrelin or patients with AD symptomology following repeated con ghrelin variants include C-C acylation of one or more of cussions. Therefore, therapeutic targets for concussions may the hydroxyl groups of the serine residues. Yet, in other provide models to enhance AD drug development, and if embodiments, ghrelin or ghrelin variants include replacing Successful, potentially prevent new AD cases. one or more of the L-amino acids with a D-amino acid. 0004 Mild brain injuries (mBI) describe an insult to the Every amino acid with the exception of glycine can occur in brain that, in turn, can cause long term injury to the brain. two isomeric forms, which are called L- and D-forms, It most often occurs from direct contact to the head, but can analogous to left-handed and right-handed configurations. also result from indirect injury (e.g., whiplash injury or L-amino acids are the form commonly manufactured in cells violent shaking of the head). Individuals who have suffered and incorporated into proteins. As mentioned above, some one brain injury are more at risk for a second brain injury ghrelin variants can have one or more of L-amino acids and more susceptible to Subsequent injuries. The damage Substituted with D-amino acids. In some embodiments, one from successive mBIs is cumulative (Cantu, R. C. Second or more of the ghrelin variants listed above, can be specifi impact syndrome, Clinics in Sports Medicine, 17(1):37-44. cally excluded. 1998). 0010. In some embodiments, the ghrelin variant com cIt is a 28-amino acid and an endogenously produced prises or consists of a polypeptide having at least 80%, 81%, peptide predominantly secreted by gastric mucosa. It has 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, been referred to as the “hunger hormone.” due to its well 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence studied effects on appetite, but it also is believed to play a identity to the amino acid sequence of SEQ ID NO. 1 significant role in regulating the distribution and rate of use provided that in Some embodiments such variants retain at of energy. least 50% of the activity of native ghrelin. 0005. There is a significant unmet need for a therapy for 0011. In some embodiments, the ghrelin variant is a treating neurodegenerative conditions that does not impose ghrelin mimetic Such as a compound which is one or more undesirable costs and delays and is effective. of RM-131 (Rhythm Pharmaceuticals, Boston, Mass.) (or BIM-28.131 (Ipsen Group), Dln-101 (DiaIlean Ltd., Israel), SUMMARY OF THE INVENTION Growth hormone (GH) releasing hexapeptide (GHRP)-6, EP 1572, Ape-Ser(Octyl)-Phe-Leu-aminoethylamide, isolated 0006. As provided below, this invention is predicated ghrelin splice variant-like compound, ghrelin splice variant, upon the discovery of a profound and Surprising effect of growth hormone secretagogue receptor GHS-R la ligand, ghrelin variants and analogs in the treatment of neurode and a combination thereof. In some embodiments, one or generative conditions. Such treatments can be easily admin more of the ghrelin variants listed above can be specifically istered without delays for individuals suffering from a excluded. In some embodiments, those ghrelin variants neurodegenerative condition to ensure that they are capable which are a polypeptide have at least 80%, 85%, 89%, 90%, of performing certain tasks safely without risk to themselves 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or others. sequence identity to the amino acid sequence of one or more 0007. This invention provides for methods for treating a of the compounds described in the present disclosure. In a neurodegenerative condition in a patient wherein said Some embodiments, ghrelin variants, which comprise short method comprises administering to the Subject in need amino acid sequences, such as the RM-131 pentapeptide thereof Suffering from said neurodegenerative condition an molecule, can have a Substitution of one of its amino acids, effective amount of ghrelin or a ghrelin variant or a com for example, a conservative or other type of Substitution as position comprising ghrelin or a ghrelin variant so as to treat described herein with a natural or non-natural amino acids, US 2017/O121385 A1 May 4, 2017

as well as isomers of the same. Other chemical modifications acids are provided below in Table 3. In some embodiments, also are contemplated, such as those described elsewhere one or more of the substitutions listed above, can be spe herein (e.g., protecting groups, octanoylation, acylation, cifically excluded. etc.). In some embodiments, one or more of the Substitution 0015. In some embodiments, the substitution is between listed above, can be specifically excluded. 1 and 5 amino acids. Substitution of one amino acid for 0012. In some embodiments, the ghrelin variant is one or another can be based on accepted and art recognized Sub more of RM-131 (or BIM-28131), Dln-101, Growth hor stitution principles. In some embodiments, one or more mone (GH) releasing hexapeptide (GHRP)-6, EP 1572, amino acids can be substituted with an amino acid or Ape-Ser(Octyl)-Phe-Leu-aminoethylamide, isolated ghrelin synthetic amino acid that has a similar property or a different splice variant-like compound, ghrelin splice variant, growth property at its side chain or otherwise, such as charge, hormone secretagogue receptor GHS-R la ligand, polarity, hydrophobicity, antigenicity, propensity to form or LY444711, LY426410, hexarelin/examorelin, growth hor break C-helical structures or 3-sheet structures. Non-limit mone releasing hexapeptide-1 (GHRP-1), GHRP-2, ing examples of common Substitutions for various residues GHRP-6 (SK&F-110679), ipamorelin, MK-0677, NN703, can be found in the NCBI Amino Acid Explorer database, capromorelin, CP 464709, prailmorelin, macimorelin (ac which includes listings of common Substitutions for each etate), anamorelin, relamorelin, ulimorelin, ipamorelin, tabi amino acid, along other types of information on each amino morelin, ibutamoren, G7039, G7134, G7203, G-7203, acid as part of its BLOSUM62 matrix database (see Sub G7502, SM-130686, RC-1291, L-692429, L-692587, stitutes in BLOSUM62 on the worldwide web at: nobi.nlm. L-739943, L-163255, L-163540, L-163833, L-166446, nih.gov/Class/Structure/aafaa explorer.cgi. In SO CP-424391, EP-51389, NNC-26-0235, NNC-26-0323, embodiments the substitutions can be conservative substi NNC-26-0610, NNC 26-0703, NNC-26-0722, NNC-26 tutions, which are well known in the art (see for example 1089, NNC-26-1136, NNC-26-1137, NNC-26-1187, NNC Creighton (1984) Proteins W. H. Freeman and Company 26-1291, MK-0677, L-692,429, EP 1572, L-252,564, (Eds), which is incorporated herein by reference in its NN703, S-37435, EX-1314, PF-5190457, AMX-213, and a entirety). Table 2 below depicts non-limiting examples of combination thereof. In some embodiments, one or more of conservative Substitutions that can be made. In some the ghrelin variants listed above, can be specifically embodiments, one or more of the substitutions listed above, excluded. can be specifically excluded. 0016 Some embodiments include the deletion or substi 0013. In some embodiments, the ghrelin variant com tution of one or more amino acids from the sequences prises a polypeptide comprising the sequence of Gly Ser Ser described herein. Adeletion refers to removal of one or more Phe Leu Ser Pro Glu His Gln Arg Val Glin Val Arg Pro Pro amino acids from a sequence. An insertion refers to one or Lys Ala Pro His Val Val (SEQ ID No. 2). In some embodi more amino acid residues being introduced into a site in a ments, the ghrelin variant comprises a polypeptide compris sequence. Insertions may comprise N-terminal and/or C-ter ing the sequence of Gly Ser Xaa Phe Leu Ser Pro Glu His minal fusions as well as intra-sequence insertions of single Gln Arg Val Glin Val Arg Pro Pro His Lys Ala Pro His Val or multiple amino acids. Generally, insertions within the Val (SEQ ID No. 3), wherein the third position is a 2.3- amino acid sequence will be smaller than N- or C-terminal diaminopropionic acid (Dpr), with the Dpr in the third fusions, of the order of about 1 to 10 residues. In some position being optionally octanoylated. In some embodi non-limiting embodiments, N- or C-terminal fusion proteins ments, the ghrelin variant comprises a polypeptide compris or peptides can include linking another molecule of the same ing the sequence of Gly Xaa Xaa Phe Leu Ser Pro Glu His sequence or a completely different molecule, including any Gln Arg Val Glin Val Arg Pro Pro His Lys Ala Pro His Val of those described herein to the N- or C-terminus of a ghrelin Val (SEQ ID No. 4), wherein the second and third position variant. In some cases, the linker can be via an ester bond or are 2,3-diaminopropionic acid (Dpr) residues, with the Dpr other bond or linker that can rapidly degrade in the body to in the third position being optionally octanoylated. In some liberate both of the linked molecules. In some embodiments, embodiments, the ghrelin variant comprises a polypeptide natural ghrelin or a fragment thereof can be linked to a comprising the sequence of Gly Ser Ser Phe Leu Ser Pro Glu ghrelin variant as described herein. In still further embodi His Gln Arg Val Glin Val Arg Pro Pro His Lys Ala Pro His ments, the two or more molecules can be linked via a Val Val Pro Ala Leu Pro (SEQ ID No. 5). In some embodi cyclized linker. For example, a diacid Such as those repre ments, the ghrelin variant comprises a polypeptide having at sented by the formula R CH(COOH)(CH2)COOH where least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, R is a Saturated or unsaturated aliphatic group of from 1 to 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 20 carbon atoms and n is 0, 1, 2, 3, or 4, can be utilized. In 99% sequence identity to the amino acid sequence of one or some cases the cyclized linker can provide added benefit, for more of the compounds described in the present paragraph. example, resistance against protease degradation. In some In some embodiments, the ghrelin variant comprises a embodiments, one or more of the substitutions or modifi polypeptide comprising the sequence of Inp-D-2Nal-D-Trp cations listed above, can be specifically excluded. Thr-Lys-NH. (SEQID No. 6). In some embodiments, one or 0017. In some embodiments, the ghrelin variant is one or more of the ghrelin variants listed above, can be specifically more of LY444711 (2CR)-(2-Amino-2-methylpropana excluded. mido)-N-1-1 (R)-(4-methoxyphenyl)-1-methyl-2-oxo-2- 0014. In some embodiments, one or more of the amino (1-pyrrolidinyl)ethyl)-1H-imidazol-4-yl)-5-phenylpentana acids of the sequence are Substituted or replaced by another mide dihydrochloride, C.H.ClNO; Ely Lilly). amino acid or a synthetic or otherwise rare amino acid (e.g., MK-0677 (2-Amino-N-2-benzyloxy-(1R)-1-(methanesul 4-fluoroproline. 4-hydroxyproline, 4-ketoproline, fonyl)spiro indoline-3,4'-piperidin-1-ylcarbonylethyl HNCDCOOH, and the like). Some non-limiting examples isobutyramide methanesulfonate, CsPINOS, Merck & of potential molecules that can be substituted for amino Co., Inc.), L-692,429 (Merck Research Laboratories), Tabi

US 2017/O121385 A1 May 4, 2017

0021. In some embodiments, ghrelin or the ghrelin vari preferred embodiment, a ghrelin variant is administered ant reduces the expression level of Tau protein. In some passively such as by Sublingual, inner ear or pulmonary embodiments, ghrelin or the ghrelin variant reduces the delivery. phosphorylation level of Tau protein. In some embodiments, 0027. In some embodiments, the composition comprising ghrelin or the ghrelin variant prevents or reduces Tau ghrelin or a ghrelin variant is administered in combination deposition and/or development of neurofibrillary tangles. with a therapeutic agent. In some embodiments, the thera 0022. In some embodiments, the neurodegenerative con peutic agent is one or more of an anti-inflammatory agent, dition is caused by a reperfusion injury. In some embodi anti-pain medication, acetylsalicylic acid, an antiplatelet ments, the reperfusion injury is resulted by post cardiac agent, a thrombolytic enzyme, an aggregation inhibitor, a arrest, coronary artery bypass grafting (CABG), ischemia, glycoprotein IIb/IIa inhibitor, a glycosaminoglycan, a anoxia, or hypoxia. thrombin inhibitor, an anticoagulant, heparin, coumarin, warfarin, tpA, GCSF, streptokinase, urokinase, Ancrod, 0023. In some embodiments, the ghrelin variant is melatonin, a caspase inhibitor, NMDA receptor agonist or coupled to a protein that extends the serum half-life of the antagonist (e.g., amantadine or gacyclidine/OTO-311, ghrelin variant. In some embodiments, the protein is a long, 1-(1R,2S)-2-methyl-1-thiophen-2-ylcyclohexylpiperidine, hydrophilic, and unstructured polymer that occupies a larger CHNS), P7C3, P2Y receptor agonist, glucagon, GLP-1R Volume than a globular protein containing the same number agonists, GLP-1, GLP-1 analog, synthetic form of GLP-1. of amino acids. In some embodiments, the molecule that GLP-1 (7-36) amide, Exendin-4 (Ex-4), Ex-4 analog, syn extends the half-life can be a molecule set forth in WO thetic form of EX-4, Lixisenatide, Liraglutide, a molecule in 2013/130683 entitled “XTEN Conjugate Compositions and a biological pathway involving GLP-1R signaling pathway, Methods of Making the Same,” and U.S. Pat. No. 8,673,860, incretin, incretin mimetic, Gastric inhibitory polypeptide entitled Extended Recombinant Polypeptides and Compo (GIP), sulfonamide compounds, Ebselen (SPI-1005 or sitions Comprising the Same,” each of which is incorporated 2-phenyl-1,2-benzisoselenazol-3(2H)-one; Sound Pharma herein by reference in its entirety. One example of a mol ceuticals; Kil J, et al. Hear Res. 2007 April: 226 (1-2):44-51. ecule that incorporates such XTEN molecules is AMX-213 Lynch E D, et al. Laryngoscope. 2004 February; 114(2): (Ammunix). In some embodiments, the protein comprises 333-7), glutathione peroxidase, glutathione peroxidase mim the sequence of XTEN. Multiple XTEN sequences are ics and inducers, aducanumab (BIIB037, Biogen Idec), or a known in the art, for example those set forth in U.S. Pat. No. combination thereof. In some embodiments, one or more of 8,673,860, which is incorporated herein by reference in its the therapeutic agents listed above, can be specifically entirety. One non-limiting example XTEN sequence is SEQ excluded. ID NO. 7, GSEPATSGSETPGTSESATPESGPGSE 0028. In some embodiments, the therapeutic agent is a PATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSE biologically equivalent polynucleotide that has the specified PATSGSETPGSEPATSGSETPGSEPATSGSETPGT percent homology (for example, 60%. 65%, 70%, 75%, STEPSEGSAPGTSESATPESGPGSEPAT SGSETPGT 80%, 85%, 90%, 95%, 98% or 99%) and encoding a STEPSEGSAP. In some embodiments, the mild brain injury polypeptide having the same or similar biological activity as comprises a concussion. In some embodiments, the extender the therapeutic agent described above. sequence can have the chemical structure: 0029. In some embodiments, the therapeutic agent is not a SARM compound. In some embodiments, one or more therapeutic agents disclosed herein are excluded from the combination therapy with ghrelin or the ghrelin variant.

DETAILED DESCRIPTION 0030. It is to be understood that the present disclosure is not limited to particular embodiments described, as Such 0024. In some embodiments, the subject that undergoes may, of course, vary. It is also to be understood that the the method of treatment is a mammal. In some embodi terminology used herein is for the purpose of describing ments, the Subject is a human. particular embodiments only, and is not intended to be 0025. In some embodiments, the composition comprising limiting, since the scope of the present disclosure will be ghrelin or the ghrelin variant is administered via a powder or limited only by the appended claims. stable formulation, wherein the ghrelin variant is formulated 0031. The detailed description of the present disclosure is in a dosage form selected from the group consisting of divided into various sections only for the reader's conve liquid, beverage, medicated sports drink, powder, capsule, nience and disclosure found in any section may be combined chewable tablet, Swallowable tablet, buccal tablet, troche, with that in another section. Unless defined otherwise, all lozenge, soft chew, Solution, Suspension, spray, Suppository, technical and Scientific terms used herein have the same tincture, decoction, infusion, and a combination thereof. meaning as commonly understood by one of ordinary skill 0026. In some embodiments, the composition comprising in the art to which the present disclosure belongs. ghrelin or the ghrelin variant is administered via inhalation, DEFINITIONS oral, intravenous, parenteral, buccal, Subcutaneous (includ ing "EpiPens'), transdermal, patch, Sublingual, into the 0032. It must be noted that as used herein and in the inner ear, intramuscular, or intranasal. In some embodi appended claims, the singular forms “a”, “an', and “the ments, the ghrelin variant is administered in a single dose. In include plural referents unless the context clearly dictates Some embodiments, the ghrelin variant is administered at a otherwise. Thus, for example, reference to “a compound dosage from 10 ng/kg per day to 10 mg/kg per day. In one includes a plurality of compounds. US 2017/O121385 A1 May 4, 2017

0033. Unless defined otherwise, all technical and scien acid residues or a covalent bond to an amino-terminal group tific terms used herein have the same meaning as commonly Such as NH or acetyl or to a carboxy-terminal group Such understood by one of ordinary skill in the art to which the as COOH. present disclosure belongs. As used herein the following 0038. As used herein, the term “amino acid derivatives’ terms have the following meanings. include, for example, alkyl-substituted tryptophan, B-naph thylalanine, naphthylalanine, 3,4-dihydrophenylalanine, and 0034. As used herein, the term “about when used before a numerical designation, e.g., temperature, time, amount, methylvaline. The amino acids and amino acid derivatives concentration, and Such other, including a range, indicates include both of L forms and D-forms. approximations which may vary by (+) or (-) 10%, 5% or 0039. The term “amino acid side chain” refers to any one 19/6. of the twenty groups attached to the C-carbon in naturally occurring amino acids. For example, the amino acid side 0035. As used herein, the term “administration' can be chain for alanine is methyl, the amino acid side chain for effected in one dose, continuously or intermittently or by phenylalanine is phenylmethyl, the amino acid side chain for several Subdoses which in the aggregate provide for a single cysteine is thiomethyl, the amino acid side chain for aspar dose. Dosing can be conducted throughout the course of tate is carboxymethyl, the amino acid side chain for tyrosine treatment. Methods of determining the most effective means is 4-hydroxyphenylmethyl, etc. and dosage of administration are known to those of skill in 0040. As used herein, the term “acylated ghrelin variant” the art and will vary with the composition used for therapy, is a ghrelin variant, which contains an acyl group attached the purpose of the therapy, the target cell being treated and to any of its constituent amino acids. Acylation of a hydroxyl the Subject being treated. Single or multiple administrations containing amino acid group can be conducted on the native can be carried out with the dose level and pattern being peptide by conventional blocking of all reactive amino selected by the treating physician. Suitable dosage formu groups using conventional blocking agents such as T-Boc, lations and methods of administering the agents are known CBZ and benzyl groups. in the art. Route of administration can also be determined and method of determining the most effective route of 0041. Subsequently, reaction of the OH functionality of administration are known to those of skill in the art and will serine, threonine, tyrosine, and the like is accomplished by vary with the composition used for treatment, the purpose of reaction with an C-Co aliphatic carboxylic acid or its the treatment, the health condition or disease stage of the halide, anhydride or activated form thereof to form the acyl subject being treated and target cell or tissue. Non-limiting group —OC(O)C-Calkyl. Suitable aliphatic acids include, examples of route of administration include oral adminis by way of example, formic acid, acetic acid, propanoic acid, tration, vaginal, nasal administration, injection, topical butyric acid, and the like. Acylation of a carboxyl containing application, Sublingual, pulmonary, and by Suppository. amino acid group can be conducted to the native peptide by conventional blocking of all reactive amino groups using 0036) As used herein, the term “affinity” refers to the conventional blocking agents such as T-Boc, CBZ and strength of binding between receptors and their ligands, for benzyl groups. Subsequently, reaction of the —COOH func example, between an antibody and its antigen. tionality of glutamic acid or aspartic acid is accomplished by 0037. As used herein, the term “amino acid residue' converting the carboxylic acid group to its corresponding refers to an amino acid formed upon chemical digestion halide, or activated form followed by reaction with an (hydrolysis) of a polypeptide at its peptide linkages. Unless C-C aliphatic alcohol Such as methanol, ethanol, propanol otherwise specified, the amino acid encompasses L-amino and the like to form an acyl group of the formula —C(O) acid, including both natural amino acid and synthetic amino O—C-Co alkyl. Such acylated groups are converted in acid or the like as long as the desired functional property is Vivo to the corresponding amino acids by enzymatic pro retained by the polypeptide. NH refers to the free amino cesses using endogenous esterases. Alternatively, synthetic group present at the amino terminus of a polypeptide. ghrelin analogs can be made by Standardized amino acid COOH refers to the free carboxy group present at the coupling well known in the art. In Such cases, the C-terminus carboxy terminus of a polypeptide. Standard polypeptide amino acid is attached to a solid Support and each Successive abbreviations for amino acid residues are as follows: A (Ala amino acid (directionally from the C to the N terminus) is or Alanine); C (Cys or Cysteine); D (Asp or Aspartic Acid); added with the appropriate blocking groups. An alternative E (Glu or Glutamic Acid); F (Phe or Phenylalanine); G (Gly amino acid as described above can be introduced into the or Glycine); H (His or Histidine); I (lie or Isoleucine); K polypeptide at any point or points. (Lys or Lysine); L (Leu or Leucine); M (Met or Methionine); 0042. As used herein, the term “comprising or “com N (Asn or Asparagine); P (Pro or Proline); Q (Glin or prises' is intended to mean that the compositions and Glutamine); R (Arg or Arginine); S (Ser or Serine); T (Thr methods include the recited elements, but not excluding or Threonine); V (Val or Valine); W (Trp or Tryptophan): X others. “Consisting essentially of when used to define (Xaa or Unknown or Other); Y (Tyr or Tyrosine); and Z compositions and methods, shall mean excluding other (Glx/Gln/Glu or Glutamic Acid/Glutamine); and Dpr (2,3- elements of any essential significance to the combination for diaminopropionic acid). All amino acid residue sequences the Stated purpose. Thus, a composition consisting essen represented herein by formula have a left-to-right orientation tially of the elements as defined herein would not exclude in the conventional direction of amino terminus to carboxy other materials or steps that do not materially affect the basic terminus. The phrase “amino acid residue” is broadly and novel characteristic(s) of the present disclosure. “Con defined to include the naturally occurring and modified and sisting of shall mean excluding more than trace elements of non-naturally occurring amino acids. A dash at the begin other ingredients and Substantial method steps. Embodi ning or end of an amino acid residue sequence indicates a ments defined by each of these transition terms are within peptide bond to a further sequence of one or more amino the scope of the present disclosure. US 2017/O121385 A1 May 4, 2017

0043. As used herein, the term “concussion' is a form of to growth hormone secretagogue receptor la (i.e., GHSR), head injury that refers to an immediate or transient loss or however, the present disclosure is not limited to a specific perturbation of consciousness, optionally accompanied by a type of receptor. brief period of amnesia, generally after a blow to the head. 0049. As used herein, the term “growth hormone secre 0044 As used herein, the term “dissociation constant” or tion promoting Substance receptors' are a family of recep “Kd' is a measure describing the strength of binding (or tors including receptors called Type 1a and Type 1b discov affinity or avidity) between receptors and their ligands, for ered in experiments on binding to MK-0677 (Hormon Res., example an antibody and its antigen. The Smaller the Kd, the 1999, 51(suppl 3), 1, Science, 1996, 273, 974), receptors stronger the binding. called FM1, FM2 and FM3 (Hormon Res., 1999, 51(suppl 0045. As used herein, the term “fusion polypeptide' is a 3), 1, Endocrine Reviews, 1997, 18(5), 621), and receptors polypeptide comprised of at least two polypeptides and a called a hexarelin binding site (Hormon Res., 1999, linking sequence to operatively link the two polypeptides 51(suppl 3), 1, Endocrinology 1998, 139, 432, J. Clin. into one continuous polypeptide. Without limitation, fusion Endocrinol. Metab., 2000, 85, 3803). However, these pub peptides can refer to dimer compounds as well as to conju licly known receptors are not restrictive. gates. The fusion polypeptide can include, for example, two 0050. As used herein, the term “growth hormone releas linked polypeptides not normally found linked in nature. The ing peptides (GHRP) refer to peptides having the pharma two polypeptides linked in a fusion polypeptide can be cological activity that promotes growth hormone release. derived from two independent sources, or can be two of the Various derivatives (for example, derivatives formed by same molecule. One example of Such a fusion polypeptide Substitution of amino acids constituting the peptides, ester is ghrelin-L-ghrelin where L is a biologically acceptable derivatives) are also included, as long as these derivatives linker. Such linkers can be synthetic or naturally occurring. have functions equivalent to Such function. There are no Synthetic linkers can range from 1 to 20 carbon atoms in restrictions on the numbers and origins of the amino acid length with up to 5 carbon atoms being replaced by het residues or amino acid derivative residues in the peptides eroatoms such as —O— —S— —S(O), —S(O)2-, -NH (for example, the peptides or derivatives isolated or purified and the like. Other non-limiting examples of fusions include from human cells, synthetic products, semi-synthetic prod linking a ghrelin variant to another ghrelin variant, linking ucts, and those obtained by genetic engineering). a ghrelin variant to ghrelin or a ghrelin fragment (e.g., less 0051. As used herein, the term “GH secretagogues than all of the 28 amino acids of ghrelin), and so forth. (GHS) refer to non-peptide substances having the pharma 0046. As used herein, the term 'ghrelin' is a polypeptide cological activity that promotes growth hormone secretion. having 28 amino acid sequence as set forth in SEQID NO. Various derivatives (for example, ester derivatives) are also 1, and can include the octanoyl acylation as described above. included, as long as these derivatives have functions equiva Human ghrelin is a polypeptide having the amino acid lent to Such function. sequence as set forth in GenBank R. Accession No. 0.052 Details of the “growth hormone secretion promot NP 057446 or Swiss-Prot Identifier GHRL HUMAN. ing Substances' are disclosed, for example, in the following Human ghrelin preprotein has 117 amino acids. This pre patent specifications. Furthermore, all of those classified in protein undergoes the following post-translational process the following documents as growth hormone releasing pep ing. The signal peptide (amino acids 1-23) is removed and tides (GHRP), growth hormone releasing peptide (GHRP)- the remaining 94 amino acids are cleaved by a protease to like compounds, growth hormone releasing peptide-mimet provide a mature 28 amino acid ghrelin (amino acids 24-51) ics (GHRP-mimetics), and growth hormone secretagogues or a mature 27 amino acid ghrelin (amino acids 24-50) and (GH secretagogoes, GHS) are included in the growth hor a mature 23 amino acid obestatin (amino acids 76-98). The mone secretion promoting Substances. However, such clas 28 amino acid mature ghrelin peptide can be further modi sifications are not strict, and are not to be interpreted as fied at the serine at position 26 in the preprotein by either an restrictive. WO00/48623, WO99/09991, WO99/08699, O-octanoyl group or an O-decanoyl group. The obestatin WO98/58950, WO98/58949, WO98/58948, WO98/58947, mature peptide can be further modified at the lysine at WO98/51687, WO98/50036, WO98/46569, WO98/46220, position 98 of the preprotein by an amide group. An addi WO98/25897, WO98/25622, WO98/16527, WO98/10653, tional ghrelin preprotein is known, which lacks the gluta WO98/03473, WO97/42223, WO97/40071, WO97/40023, mine at position 37 of the preprotein. WO97/39768, WO97/34604, WO97/27298, WO97/25057, 0047. As used herein, the term “ghrelin variant” refers to WO97/24369, WO97/23508, WO97/22622, WO97/22620, any compound (e.g., peptides, Small molecule drugs) having WO97/22367, WO97/21730, WO97/18233, WO97/15574, at least about 50% of a functional activity of ghrelin. The WO97/15573, WO97/15191, WO97/11697, WO97/00894, functional activity includes, without limitation, feeding WO96/38471, WO96/35713, WO96/33189, WO96/32943, regulation, nutrient absorption, gastrointestinal motility, WO96/32126, WO96/24587, WO96/24580, WO96/22997, energy homeostasis, anti-inflammatory regulation, Suppres WO96/22782, WO96/15148, WO96/13265, WO96/10040, sion of inflammatory cytokines, activation of Gq/G11, accu WO96/05195, WO96/02530, WO95/34311, WO95/17423, mulation of inositol phosphate, mobilization of calcium WO95/17422, WO95/16707, WO95/16692, WO95/16675, from intracellular stores, activation or deactivation of MAP WO95/14666, WO95/13069, WO95/12598, WO95/09633, kinases, NFKB translocation, CRE driven gene transcription, WO95/03290, WO95/03289, WO94/19367, WO94/18169, reduction in reactive oxygen species (ROS), NAMPT WO94/13696, WO94/11397, WO94/11012, WO94/08583, enzyme activation, and/or binding of arrestin to ghrelin WO94/07519, WO94/07486, WO94/07483, WO94/05634, receptor. Examples of ghrelin variants are provided herein. WO93/04081, WO92/16524, WO92/01711, WO89/10933, 0048. As used herein, the term “ghrelin receptor refers WO89/07111, WO89/07110, WO83/02272, U.S. Pat. No. to any naturally occurring molecule to which ghrelin binds 5,936,089, U.S. Pat. No. 5,877,182, U.S. Pat. No. 5,872,100, and induces a biological activity. Ghrelin is known to bind U.S. Pat. No. 5,854,211, U.S. Pat. No. 5,830,433, U.S. Pat. US 2017/O121385 A1 May 4, 2017

No. 5,817,654, U.S. Pat. No. 5,807,985, U.S. Pat. No. cardiac arrest, coronary artery bypass grafting (CABG). 5,804,578, U.S. Pat. No. 5,798,337, U.S. Pat. No. 5,783,582, ischemia, anoxia, or hypoxia. Neurodegenerative conditions U.S. Pat. No. 5,777,112, U.S. Pat. No. 5,776,901, U.S. Pat. are debilitating, the damage that they cause can be irrevers No. 5,773,448, U.S. Pat. No. 5,773,441, U.S. Pat. No. ible, and the outcome in a number of cases is fatal. 5,767,124, U.S. Pat. No. 5,767,118, U.S. Pat. No. 5,767,085, 0056. As used herein, the term “non-acylated ghrelin U.S. Pat. No. 5,731,317, U.S. Pat. No. 5,726,319, U.S. Pat. variant' or “unacylated ghrelin variant' is a ghrelin variant, No. 5,726,307, U.S. Pat. No. 5,721,251, U.S. Pat. No. which does not contain an acyl group attached to any of its 5,721,250, U.S. Pat. No. 5,691377, U.S. Pat. No. 5,672,596, constituent amino acids. It should be understood that in U.S. Pat. No. 5,668,254, U.S. Pat. No. 5,663,171, U.S. Pat. Some embodiments, the ghrelin variant can be partially non No. 5,663,146, U.S. Pat. No. 5,656,606, U.S. Pat. No. or unacylated at one or more of it residues. 5,652,235, U.S. Pat. No. 5,646,301, U.S. Pat. No. 5,635,379, 0057. As used herein, the term “polypeptide' or “pep U.S. Pat. No. 5,583,130, U.S. Pat. No. 5,578,593, U.S. Pat. tide' is intended to encompass a singular “polypeptide' as No. 5,576,301, U.S. Pat. No. 5,559,128, U.S. Pat. No. well as plural “polypeptides,” and refers to a molecule 5,545,735, U.S. Pat. No. 5,536,716, U.S. Pat. No. 5,534,494, composed of monomers (amino acids) linearly linked by U.S. Pat. No. 5,506,107, U.S. Pat. No. 5,494.919, U.S. Pat. amide bonds (also known as peptide bonds). The term No. 5,492,920, U.S. Pat. No. 5,492,916, U.S. Pat. No. "polypeptide' refers to any chain or chains of two or more 5,486,505, U.S. Pat. No. 5,434,261, U.S. Pat. No. 5,430,144, amino acids, and does not refer to a specific length of the U.S. Pat. No. 5,416,073, U.S. Pat. No. 5,374,721, U.S. Pat. product. Thus, peptides, dipeptides, tripeptides, oligopep No. 5,317,017, U.S. Pat. No. 5,310,737, U.S. Pat. No. tides, “protein.” “amino acid chain,” or any other term used 5,284.841, U.S. Pat. No. 5,283,241, U.S. Pat. No. 5,206,235, to refer to a chain or chains of two or more amino acids, are U.S. Pat. No. 5,030,630, U.S. Pat. No. 4,880,777, U.S. Pat. included within the definition of polypeptide,” and the term No. 4,851,408, U.S. Pat. No. 4,650,787, U.S. Pat. No. "polypeptide' may be used instead of, or interchangeably 4,485,101, U.S. Pat. No. 4411,890, U.S. Pat. No. 4410,513, with any of these terms. The term “polypeptide' is also U.S. Pat. No. 4410,512, U.S. Pat. No. 4,228,158, U.S. Pat. intended to refer to the products of post-expression modi No. 4,228, 157, U.S. Pat. No. 4,228,156, U.S. Pat. No. fications of the polypeptide, including without limitation 4,228,155, U.S. Pat. No. 4,226,857, U.S. Pat. No. 4,224,316, glycosylation, acetylation, phosphorylation, amidation, acy U.S. Pat. No. 4,223,021, U.S. Pat. No. 4,223,020, and U.S. lation, acylation by fatty acid, fatty acid-modification, Pat. No. 4,223,019. The contents of these documents are derivatization by known protecting/blocking groups, pro incorporated by reference in their entireties. teolytic cleavage, or modification by non-naturally occur 0053 As used herein, the term “individual' is an animal ring amino acids. Modification may be fatty acid modifica or human Susceptible to a condition, in particular mBI or tion or triglyceride modification. Fatty acid modification concussion. In some embodiments, the individual is a mam may be a short to medium-chain fatty acid. The short fatty mal, including human, and non-human mammals such as acid may be a two-carbon fatty acid or acetic acid. Medium dogs, cats, pigs, cows, sheep, goats, horses, rats, and mice. chain fatty acid may be 14-carbon fatty acid or tetradecanoic 0054 As used herein, the term “mild brain injury” (mBI), acid. Modification with a fatty acid may be acylation of SEQ Sometimes referred as a "mild traumatic brain injury ID NO. 1 at serine amino acid position 2 and/or serine amino (mTBI), refers to a non-disease event commonly caused by acid position 3. Modification may be catalyzed by ghrelin an injury resulting in an insult to the brain. mBI may be O-acyl transferase (GOAT) of fatty acid thioester and ghre caused, for example, by impact forces, in which the head lin as Substrates. In one embodiment, post-translationally strikes or is struck by something, or impulsive forces, in modified ghrelin may be bound and/or recognized by growth which the head moves without itself being subject to blunt hormone secretagogue receptor type 1a (GHSR-1a) or ghre trauma (for example, when the chest hits something and the lin receptor. In one embodiment, post-translationally modi head Snaps forward; or as a result of rapid acceleration or fied ghrelin may be fatty acid-acylated ghrelin at serine deceleration of the head). mBI commonly results, for amino acid position 2 and/or serine amino acid position 3 example, from a sports-related injury, a motor vehicle acci bound and/or recognized by GHSR-1a or ghrelin receptor. A dent, an accidental fall, or an assault. Although the vast polypeptide may be derived from a natural biological Source majority of Such injuries improve through natural recovery, or produced by recombinant technology, but is not neces the damage caused by Such an injury or repetitive injuries sarily translated from a designated nucleic acid sequence. It can cause long term deficits in cognitive, and/or motor skill may be generated in any manner, including by chemical functions. mBI is different from and has a distinct pathology synthesis. The term “Polypeptide' or “Peptide' also refers to as compared to diseases such as acute traumatic brain insults a compound comprising a plurality of amino acids linked Such as strokes (ischemic or hemorrhagic), AVMs, brain therein via peptide linkages. Here, the amino acid (also tumors, and the like. called an amino acid residue) includes naturally occurring 0055 As used herein, the term “neurodegenerative con amino acids represented by formula: NH2-CH(R')—COOH, dition” refers to a degeneration of neurons in either the brain wherein R is a naturally occurring Substituent group, as well or the nervous system of an individual. Non-limiting as its D, L-optical isomers etc. There is also a peptide, examples include Amyotrophic lateral Sclerosis (ALS), wherein a certain naturally occurring amino acid is replaced Alzheimer's disease (AD), Parkinson's Diseases (PD), mul by a modified amino acid. The modified amino acid includes tiple sclerosis (MS), dementia, and frontotemporal demen the amino acids of the above formula wherein the substituent tia. Neurodegenerative conditions also include traumatic group R' is further modified, its D, L-optical isomers thereof, brain injury (TBI), mild brain injury (mBI), recurrent TBI, and non-natural amino acids wherein e.g. various Substituent and recurrent mBI. In some embodiments, the neurodegen groups are bound to the substituent group R' of the above erative condition is caused by a reperfusion injury. In some formula via or not via an ester, ether, thioester, thioether, embodiments, the reperfusion injury is resulted by post amide, carbamide or thiocarbamide linkage. The modified US 2017/O121385 A1 May 4, 2017

amino acid also includes non-natural amino acids whose of the family are broadly classified as follows: P2X recep amino groups are replaced by lower alkyl groups. Antibod tors are ligand-gated ion channels; P1 receptors are adenos ies can be covered by the above definition of peptide and ine-activated G protein-coupled receptors; and P2Y recep polypeptide, include antibody ghrelin variants. tors, which form the basis of this application, are nucleotide 0058 As used herein, the term "peptide analogue' refers activated G protein-coupled receptors. to a compound wherein at least one amino acid in a peptide 0063. As used herein, the term “P2Y receptor or “P2Y is replaced by a non-amino acid compound, and thus at least R’ generally refers to a class of G protein-coupled puriner one linkage of said Substituent compound to the peptide gic receptors that are stimulated by nucleotides such as ATP analogue is not a peptide linkage. (P2Y2, P2Y11), ADP, UTP (P2Y2, P2Y4), UDP (P2Y6) and 0059. As used herein, the term “homology” or “identity” UDP-glucose. To date, 8 P2Y receptors have been cloned in or “similarity” refers to sequence similarity between two humans: P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13 peptides or between two nucleic acid molecules. Homology and P2Y14. P2Y receptors are present in almost all human can be determined by comparing a position in each sequence tissues where they exert various biological functions based which may be aligned for purposes of comparison. When a on their G-protein coupling. The biological effects of P2Y position in the compared sequence is occupied by the same receptor activation depend on how they couple to down base or amino acid, then the molecules are homologous at stream signaling pathways, either via Gi. Gq or Gs G that position. A degree of homology between sequences is a proteins. Human P2Y receptors have the following G protein function of the number of matching or homologous positions coupling: Gq/11 coupled: P2Y1, P2Y2, P2Y6, P2Y14, Gi shared by the sequences. An “unrelated' or “non-homolo and Gq/11 coupled: P2Y4Gs and Gq/11 coupled. P2Y1; Gi gous' sequence shares less than 40% identity, or less than coupled: P2Y 12, P2Y13. 25% identity, with one of the sequences of the present 0064. As used herein, the term “receptor agonist' is disclosure. generally used to refer to a synthetic or naturally occurring 0060 A polynucleotide or polynucleotide region (or a molecule that mimics the action of an endogenous biochemi polypeptide or polypeptide region) has a certain percentage cal molecule (Such as hormone or neurotransmitter) when (for example, 60%. 65%, 70%, 75%, 80%, 85%, 90%, 95%, bound to the cognate receptor of that hormone or neurotrans 98% or 99%) of “sequence identity” to another sequence mitter. An agonist is the opposite of an antagonist in the means that, when aligned, that percentage of bases (or amino sense that while an antagonist also binds to the receptor, the acids) are the same in comparing the two sequences. This antagonist does not activate the receptor and actually blocks alignment and the percent homology or sequence identity it from activation by agonists. A partial agonist activates a can be determined using software programs known in the receptor, but only produces a partial physiological response art, for example those described in Ausubel et al. eds. (2007) compared to a full agonist. A co-agonist works with other Current Protocols in Molecular Biology. Biologically co-agonists to produce the desired effect together. Receptors equivalent polynucleotides are those having the above-noted can be activated or inactivated by endogenous (such as specified percent homology and encoding a polypeptide hormones and neurotransmitters) or exogenous (such as having the same or similar biological activity. drugs) agonists and antagonists, resulting in stimulating or 0061. As used herein, the term “secretagogue' is a sub inhibiting the cell. The term “P2Y receptor agonist” or “P2Y stance stimulating growth hormone release, such as ghrelin purinergic receptoragonist' generally refers to any molecule or a ghrelin variant. A secretagogue according to the present that binds to P2Y receptors and elicits at least a portion of disclosure may for example be selected from L-692-429 and the cellular responses typically associated with P2Y receptor L-692-585 (benzoelactam compounds; available from activation in that cell type. Merck & Co, Inc., Whitehouse Station, N.J.), MK677 0065. As used herein, the term “alkyl generally refers to (spiroindaner; available from Merck), G-7203, G-7039, Co inclusive, linear, branched, or cyclic, Saturated or G-7502 (isonipecotic acid peptidomimetic; available from unsaturated (i.e., alkenyl and alkynyl) hydrocarbon chains, Genentech, Inc., South San Francisco, Calif.), NN703 (Novo for example, methyl, ethyl, propyl, isopropyl, butyl, isobu Nordisk Inc., Princeton, N.J.), or ipamorelin. The growth tyl, tert-butyl, pentyl, hexyl, octyl, ethenyl, propenyl, bute hormone secretagogue may in one embodiment be non nyl, pentenyl, hexenyl, octenyl, butadienyl, propynyl, buty acylated, for instance a non-acylated form of ghrelin variant. nyl, pentynyl, hexynyl, heptynyl, allenyl and optionally In some embodiments, the ghrelin variant binds to the Substituted arylalkenyl and arylalkyny groups. As used growth hormone secretagogue receptor GHS-R 1a (GHSR). herein, the term “acyl refers to an organic acid group The ghrelin variant compounds described herein are active wherein the –OH of the carboxyl group has been replaced at the receptor for growth hormone secretagogue (GHS). with another substituent (i.e., as represented by RCO , e.g., the receptor GHS-R 1a. The compounds can bind to wherein R is an alkyl or an aryl group). As such, the term GHS-R 1a, and stimulate receptor activity. In some embodi “acyl specifically includes arylacyl groups. Specific ments, the compounds can bind other receptors and, option examples of acyl groups include acetyl and benzoyl. As used ally, stimulate their activity. In some embodiments, the herein, the term “aryl refers to 5 and 6-membered hydro ghrelin variant increases uncoupling protein-2 (UCP-2) carbon and heterocyclic aromatic rings. Examples of aryl expression. In some embodiments, the ghrelin variant groups include cyclopentadienyl, phenyl, furan, thiophene, increases UCP-2 expression in mitochondria. In some pyrrole, pyran, pyridine, imidazole, isothiazole, isoxazole, embodiments, the ghrelin variant prevents the metabolic pyrazole, pyrazine, pyrimidine, and the like. The term consequence of mBI and any associated chronic conditions. “alkoxyl as used herein refers to Co inclusive, linear, 0062. As used herein, the term “purinergic receptor branched, or cyclic, Saturated or unsaturated oxo-hydrocar generally refers to a family of cell surface receptors which bon chains, including for example methoxy, ethoxy, are activated by purine-containing compounds Such as propoxy, isopropoxy, butoxy, t-butoxy, and pentoxy. The adenosine and the nucleotides ATP and UTP. The members term “aryloxyl as used herein refers to aryloxy such as US 2017/O121385 A1 May 4, 2017 phenyloxyl, and alkyl, halo, or alkoxyl Substituted aryloxyl. bridged and fused ring systems. The term "heterocyclylal As used herein, the terms “substituted alkyl and “substi kyl refers to an alkyl substituted with a heterocyclyl. tuted aryl' include alkyl and aryl groups, as defined herein, (0071. The term “sulfinyl refers to a sulfur attached to in which one or more atoms or functional groups of the aryl two oxygen atoms through double bonds. An “alkylsulfo or alkyl group are replaced with another atom or functional nyl refers to an alkyl substituted with a sulfonyl. The term group, for example, halogen, aryl, alkyl, alkoxy, hydroxy, “amino acid refers to a molecule containing both an amino nitro, amino, alkylamino, dialkylamino, Sulfate, and mer group and a carboxyl group. Suitable amino acids include, capto. The terms “halo,” “halide,” or “halogen as used without limitation, both the D- and L-isomers of the 20 herein refer to fluoro, chloro, bromo, and iodo groups. naturally occurring amino acids found in peptides (e.g., A. 0066. The term “alkenyl refers to a hydrocarbon chain R, N, C, D, Q, E, G, H, I, L. K. M. F. P. S. T. W. Y. V (as that may be a straight chain or branched chain having one or known by the one letter abbreviations)) as well as unnatu more carbon-carbon double bonds. The alkenyl moiety rally occurring amino acids prepared by organic synthesis or contains the indicated number of carbon atoms. For other metabolic routes. example, C2-C10 indicates that the group may have from 2 0072 The term “substituents’ refers to a group “substi to 10 (inclusive) carbon atoms in it. The term “lower tuted on an alkyl, cycloalkyl, aryl, heterocyclyl, or het alkenyl refers to a C2-C8 alkenyl chain. In the absence of eroaryl group at any atom of that group. Any moiety any numerical designation, “alkenyl' is a chain (straight or described herein can be further substituted with a substitu branched) having 2 to 10 (inclusive) carbon atoms in it. ent. Suitable substituents include, without limitation, halo, 0067. The term “alkynyl refers to a hydrocarbon chain hydroxy, mercapto, OXo, nitro, haloalkyl, alkyl, aryl, aralkyl, that may be a straight chain or branched chain having one or alkoxy, thioalkoxy, aryloxy, amino, alkoxycarbonyl, amido, more carbon-carbon triple bonds. The alkynyl moiety con carboxy, alkanesulfonyl, alkylcarbonyl, and cyano groups. tains the indicated number of carbon atoms. For example, C2-C10 indicates that the group may have from 2 to 10 Alzheimer's and Concussions (inclusive) carbon atoms in it. The term “lower alkynyl 0073. Similar to concussions, the pathogenesis of AD refers to a C2-C8 alkynyl chain. In the absence of any involves mitochondrial and metabolic dysfunction resulting numerical designation, “alkynyl is a chain (Straight or in significant changes in energy utilization. The Sub-clinical branched) having 2 to 10 (inclusive) carbon atoms in it. nature of this early pathology may in part explain the 0068. The terms “cycloalkyl or “cyclyl as employed difficulty in developing AD treatments. Concussions, and herein includes saturated and partially unsaturated cyclic more specifically repetitive sports concussions, exhibit AD hydrocarbon groups having 3 to 12 carbons, preferably 3 to like symptomatology, now coined CTE. Post-mortem histo 8 carbons, and more preferably 3 to 6 carbons, wherein the pathological studies of brains from professional athletes cycloalkyl group may be optionally substituted. Preferred have revealed the presence of tau rich neurofibrillary tangles cycloalkyl groups include, without limitation, cyclopropyl. and amyloid plaque deposition. Similar to AD, concussions cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclo and the neurobiology thereof are a mitochondrial and meta hexenyl, cycloheptyl, and cyclooctyl. bolic mediated process; concussions disrupt neuronal elec 0069. The term “heteroaryl” refers to an aromatic 5-8 trolyte homeostasis resulting in mitochondrial hyper-oxida membered monocyclic, 8-12 membered bicyclic, or 11-14 tive phosphorylation and the generation of reactive oxygen membered tricyclic ring system having 1-3 heteroatoms if species (ROS). ROS accumulation (as in AD) damages brain monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms tissue initiating chronic degenerative pathology. By amelio if tricyclic, said heteroatoms selected from O, N, or S (e.g., rating the acute mitochondrial disruption following concus carbon atoms and 1-3, 1-6, or 1-9 heteroatoms if N, O, or S Sion, a powerful platform is created. if monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, 3, or 4 atoms of each ring may be substituted by a Ghrelin and Neuroprotection Substituent. Examples of heteroaryl groups include pyridyl, 0074 Discovered in 1998, the endogenous hormone furyl or furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, ghrelin is a 28 amino acid molecule produced mostly in the thiophenyl or thienyl, quinolinyl, indolyl, thiazolyl, and the stomach. The active form is octanoylated at the serine-3 like. The term "heteroarylalkyl or the term "heteroaralkyl position and freely crosses the blood brain barrier. Ghrelin refers to an alkyl substituted with a heteroaryl. The term has been tested in over 6000 patients over the last decade "heteroarylalkenyl refers to an alkenyl substituted with a (clinical Satiety and cachexia) with a strong safety and heteroaryl. The term "heteroarylalkynyl refers to an alky pharmacokinetic profile across a spectrum of dosing regi nyl substituted with a heteroaryl. The term "heteroarylal CS. koxy' refers to an alkoxy substituted with heteroaryl. 0075 Ghrelin addresses post-concussive mitochondrial 0070. The term “heterocyclyl or “heterocyclylalkyl dysfunction, prevents neuronal degeneration and attenuates refers to a nonaromatic 5-8 membered monocyclic, 5-12 p-Tau formation, thus affecting post-concussive neurode membered bicyclic, or 11-14 membered tricyclic ring sys generation and Alzheimer's like pathology. Ghrelin works tem having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms by binding to the GHSR1a receptor, which is located if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms throughout the hypothalamus, hippocampus and mid-brain. selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or Investigative efforts have shown ghrelin to be neuro-protec 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tive following both severe TBI and concussion in animal tricyclic, respectively), wherein 0, 1, 2 or 3 atoms of each models. Activation of brain ghrelin receptors upregulates ring may be substituted by a substituent. Examples of expression of Uncoupling Protein 2 (UCP-2), which in turn heterocyclyl groups include piperazinyl, pyrrolidinyl, dioxa increases mitochondrial genesis and decreases ROS genera nyl, morphonlinyl, tetrahydrofuranyl, and include both tion. Ghrelin also increases hippocampal density, an area US 2017/O121385 A1 May 4, 2017

damaged in both AD and TBI. Dr. Vishal Bansal has shown I0081. Severe TBI causes significant tissue necrosis and that ghrelin administration following rodent brain injury neuron loss secondary to edema and apoptosis. Ghrelin prevents neuronal apoptosis, decreases neuron tissue loss, therapy significantly decreased brain tissue loss following improves neuro-cognitive function and prevents ROS accu severe TBI as measured 7 days following initial injury. mulation (Bansal, 2012: Lopez, 2013) as described in U.S. Severe TBI induces apoptosis within the neocortex (TUNEL Pat. No. 9,119,832, which is incorporated by reference in its staining green). Ghrelin therapy (TBIG) significantly entirety. reduced neocortical apoptosis. Severe TBI caused signifi cant neurocognitive defects (as measured by foot faults on a Ghrelin Clinical Development History balance beam assay) on post-TBI Day 1, 3 and 7 compared to sham. Ghrelin (does only at Day 0) prevented neurocog 0076 Ghrelin's pharmacokinetics, pharmacodynamics, nitive decline to levels of sham. Importantly, mice only and clinical safety have been extensively characterized in received ghrelin therapy (20 ug/kg) at the time of injury in clinical research. In humans, ghrelin has been dosed in both two doses; 10 minutes following TBI and a second dose 1 IV and Sub-cutaneous regimens from 1 g/kg up to 40 ug/kg hour thereafter, indicating that early ghrelin therapy modu for as long as 84 days in studies relating primarily to appetite lates the consequence of TBI. and Satiety. In these dose ranges ghrelin is very safe. To-date only transient gastrointestinal symptoms (e.g. minor nausea, Mitochondrial Stabilization diarrhea, flushing) have been observed as side effects. I0082. Despite exciting data from severe TBI models, it 0077. To date, no serious adverse events have been was unknown whether ghrelin would have any benefit in a reported in patients treated with ghrelin, analogs, or mimet mild TBI model. As is well established, severe TBI and mild ics. Gherlin’s strong safety profile will allow accelerated TBI are two distinct injuries. If protection is conferred on clinical research once an optimal dose for concussion is one model, it may not be in the other. Therefore, several identified. experiments were conducted and designed to test ghrelin 0078. Initial pre-clinical experiments have shown ghrelin effectiveness in a mild TBI model. efficacy in mTBI at 50 lug/kg with two doses administered I0083. Following a concussion, massive depolarization within 24 hours of injury. Currently, our post-concussion causes significant neuronal electrolyte shifting and mito dosing model for ghrelin in human trials has not been chondrial hyper-reactivity inducing a temporary bioenergic optimized. In pre-clinical modeling, ghrelin has shown a state (note decreased glucose levels) generating massive significant decrease in post-severe TBI edema in as low as reactive oxygen species (ROS). Recent advances explaining 20 g/kg in two doses early after injury. It is postulated that, the pathophysiology of concussion aided in developing the given the known biology of post-concussive energy inertia, hypothesis. Following a concussion, areas of the brain ghrelin should be dosed within the first 24 hours following undergo massive depolarization with extreme flux and concussion at a dose of at least 10 g/kg (given current PK change in electrolyte homeostasis. This flux leads to over knowledge and Surrogate growth hormone response). Spe utilization of mitochondrial respiration and deviation from cific aims are designed to Solidify ghrelin's dose timing and homeostasis. In doing so, a bioenergic crisis is initiated as the response of novel serum biomarkers in a rodent mTBI CNS mitochondria, in hopes to maintain homeostasis, gen model. erate detrimental amounts of reactive oxygen species (ROS). which are cytotoxic to cells and axons. Furthermore, ROS Neuroenteric Axis generation is tied to neurodegeneration, dementia and “tauopathies, i.e. Alzheimer's disease. The generation of 007.9 The concept of the neuroenteric axis is recognized ROS from mitochondrial dysfunction induces not only as an important biological paradigm; direct CNS connectiv direct neuron damage but increases a potential cytotoxic ity and the indirect neuro-hormonal regulatory pathways inflammatory reaction. provide communication and messaging crucial to homeo I0084. The ghrelin receptor (GHSR-1a) is located Stasis. The Bansal lab has shown that vagus nerve stimula throughout the mid-brain, hypothalamus and hippocampus. tion has profound effects on preventing edema following Activation of GHSR-1a increases mitochondrial membrane severe TBI (Lopez, 2012). Despite the known anti-inflam protein UCP-2 expression. UCP-2 directly “uncouples' oxi matory effects of vagus nerve stimulation in other injury and dative phosphorylation thereby decreasing ROS generation. septic models, it was postulated that a secondary mediator Perhaps most importantly, UCP-2 increases mitochondrial beyond acetylcholine must be implicated. In this regard, generation, thwarting the post-concussive bioenergic crisis serum ghrelin levels are profoundly elevated following of the brain. Several models have shown the importance of vagus stimulation. Recently, ghrelin has been shown to be UCP-2 neuroprotection directly reducing ROS generation, neuroprotective following stroke and in Parkinson's models and increasing mitochondria biogenesis through a UCP-2 (Andrews, 2013: Ersahin 2010). directed mechanism. Furthermore, ghrelin directly increases the synaptic density of the hippocampus, an area of the brain The Protective Effect of Ghrelin Following Severe TBI exquisitely sensitive to concussions. Taken together, ghrelin 0080. In a series of publications, the Bansal laboratory therapy treats the pathology of concussions in all major has shown that exogenously dosed ghrelin confers signifi areas: 1) decreases existing mitochondrial ROS generation, cant neuroprotective benefits following severe TBI. Animals 2) increases mitochondria biogenesis, 3) increases hip receiving ghrelin had significantly less loss of brain Volume pocampus Volume, 4) decreasing secondary inflammation. (7 days following injury), significantly less apoptosis in the neurocortex, decreased invasion of inflammatory cells, and Ghrelin's Neuroprotective Effect Following Mild TBI perhaps most interestingly, improved neurocognitive scor I0085. In a mild TBI model, ghrelin therapy provided ing and outcomes (up to 7 days after injury). significant neuroprotective effects. Using a controlled cor US 2017/O121385 A1 May 4, 2017

tical impactor, with standardized settings (5.0 m/s, a 3 mm Pro-Arg (SEQID NO. 1), which has at least one change or diameter impounder tip, a depth of 1.0 mm, and a dwell time modification to the sequence selected from the various of 0.2 milliseconds), a mouse mild brain injury was elicited changes described herein such that the variant is different in three groups (n=5 in each group), sham, mTBI only, from the natural ghrelin molecule. In some embodiments, mTBI with 2 doses of ghrelin (50 g/kg) 10 minutes and 1 the ghrelin variant comprises a polypeptide having at least hour after injury. 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 0.086 Animals were sacrificed after 24 hours and whole 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%0 brain reactive oxygen species was assessed by an oxidative sequence identity to the amino acid sequence of SEQ ID burst assay as measured by flow cytometry. Sham animals NO. 1. In some embodiments, one or more of the modifi had a baseline oxidative burst of 4,600 MFI (SEM 1,600), cations or substitutions listed above can be specifically whereas mTBI nearly doubled to 8,000 MFI (SEM 2.900). excluded. Ghrelin therapy decreased mTBI oxidative burst to sham levels 3,300 MFI (SEM 1,000); p<0.05 mTBI compared to 0090. In some embodiments, in the amino acid sequence drug treated. set forth in SEQID NO: 1, an amino acid sequence of amino 0087 Ghrelin reduces oxidative burst (a measure of acids 1 to 4 refers to Gly Ser Ser Phe (SEQ ID NO. 11), an ROS) following mild TBI in animal models. Oxidative burst amino acid sequence of amino acids 1 to 5 refers to Gly Ser was determined 24 hours following mild TBI in three groups Ser Phe Leu (SEQ ID NO. 12), an amino acid sequence of via flow cytometry. Ghrelin therapy significantly reduced amino acids 1 to 6 refers to Gly Ser Ser Phe Leu Ser (SEQ oxidative burst. In separate experiments, using the same ID NO. 13), an amino acid sequence of amino acids 1 to 7 three animal groups, myeloperoxidase (MPO) and Gr-1 refers to Gly Ser Ser Phe Leu Ser Pro (SEQ ID NO. 14), an (neutrophil) staining were conducted in the hippocampus. amino acid sequence of amino acids 1 to 8 refers to Gly Ser Ghrelin treated animals had a significant reduction in both Ser Phe Leu Ser Pro Glu (SEQ ID NO. 15), an amino acid MPO staining and neutrophil infiltration. Myeloperoxidase sequence of amino acids 1 to 9 refers to Gly Ser Ser Phe Leu (MPO) is a potent enzyme in neutrophils and astrocytes that Ser Pro Glu His (SEQ ID NO. 16), and, an amino acid forms hypochlorous acid (ROS). Mild TBI significantly sequence of amino acids 1 to 10 refers to Gly Ser Ser Phe increased MPO staining in the hippocampus. Ghrelin Leu Ser Pro Glu His Gln (SEQ ID NO. 17). therapy reduced MPO staining. These observations built 0091. In some embodiments, the polypeptide includes upon previous work showing ghrelin's protective effect both acylated and non-acylated forms. One non-limiting following severe TBI models, stroke and other neurodegen example of a ghrelin variant is ASP-531 (Alize Pharma), erative diseases. which is a clinical stage, unacylated ghrelin molecule. In Some embodiments, maximum ghrelin variant activity Human Evidence of Ghrelin's Neuroprotective Effect requires acylation of the third residue of ghrelin. Naturally 0088. In search of a clinical biomarker for concussion occurring ghrelin is acylated with octanoic acid; however, outcomes, Xu et al. analyzed 118 consecutive, concussed any bulky hydrophobic group attached to the side chain of patients presenting to a large research hospital. Patients the third residue is sufficient for ghrelin variant function underwent blood collection for their biorepository at Day 0, (Matsumoto et al., 2001, Biochem. Biophys. Res. Commun. 1 and 2. Three months following concussion, all patients 287: 142-146). Non-limiting examples of such hydrophobic underwent a battery of neurocognitive testing including groups include n-lauroyl, palmitoyl, 3-octenoyl and 4-meth memory, reaction time, visual tracking and attention. These ylpentanoyl. Furthermore, ghrelin variants wherein the ester tests were retrospectively compared to serum analysis from bond between octanoic acid and Ser3 is more chemically the biorepository. Out of 8 endogenous hormones analyzed, stable, such as a thioether (Cys3(octyl)) or ether (Ser3(octyl) only ghrelin was shown to be an independent predictor of bond), are also useful. Ghrelin variants also include trunca three-month cognitive deterioration. Patients with higher tion mutants of ghrelin. There is a wealth of information endogenous ghrelin levels were less likely to have cognitive regarding the structure and function of ghrelin to guide a impairment compared to those patients with lower endog skilled artisan in preparing ghrelin variants useful in the enous ghrelin levels. Endogenous ghrelin levels are predic present disclosure. See, for example, Kojima et al., 2005, tive of 3-month cognitive deterioration. Patients without Physiol. Rev. 85: 495-522 and references cited therein. 3-month cognitive deterioration (blue) had higher levels of Structurally, ghrelin is a random coil in aqueous solution endogenous ghrelin on the day of injury, Day 1 and Day 2 (Silva Elipe et al., 2001, Biopolymers 59:489-501). Various compared to patients with cognitive deterioration. truncated ghrelin peptides also demonstrate random coil structure. The minimum active ghrelin core is the first four Ghrelin Variants amino acids with Ser3 acylated (Bednarek et al., 2000, J. Med. Chem. 43:4370-4376; Matsumoto et al., 2001, Bio 0089. As noted herein, embodiments generally relate to chem. Biophys. Res. Commun. 284:655-659). Thus aghre methods of using ghrelin variants for treating, reducing the lin variant comprising only the first four amino acids, e.g., severity of and in Some cases preventing mild brain injury, Gly-Ser-Ser(n-octanoyl)-Phe (SEQ ID NO. 41), is also related symptoms and sequelae. Some embodiments relate useful in the present disclosure. In some embodiments, to methods of reducing the risk of, or preventing the ghrelin variant is 5-aminopentanoyl-Ser(Octyl)-Phe-Leu development of symptoms or sequelae. Non-limiting aminoethylamide (Ser-Phe-Leu are residues 3-5 of SEQ ID examples of various ghrelin variants are described. For NO. 13), which was found to have potent ghrelin activity example, in Some embodiments, the ghrelin variant com (Matsumoto et al., 2001, Biochem. Biophys. Res. Commun. prises a polypeptide having an amino acid sequence of 284:655-659). Each reference described above is incorpo Gly-Ser-Ser-Phe-Leu-Ser-Pro-Glu-His-Gln-Arg-Val-Gln rated herein by reference in its entirety for all of its disclo Gln-Arg-Lys-Glu-Ser-Lys-Lys-Pro-Pro-Ala-Lys-Leu-Gln Sure, including all methods, materials, etc. In some embodi US 2017/O121385 A1 May 4, 2017

ments, one or more of the modifications or Substitutions group of from 1 to 20 carbon atoms. Diacylation will lead to listed above, can be specifically excluded. a ring structure which is contemplated to be more resistant 0092. In some embodiments, the side-chain hydroxyl to protease degradation. group of third serine from the N-terminus of the ghrelin or 0098. In some embodiments, ghrelin variants comprise a ghrelin variants has been acylated with fatty acid. In some basic amino acid bound to the carboxyl-terminal, wherein embodiments, the third serine from the N-terminus of the the amino-terminal is modified with a saturated or unsatu ghrelin or ghrelin variants has been replaced by threonine. rated alkyl or acyl group containing one or more carbon 0093. In some embodiments, at least one amino acid atoms, and/or a hydroxyl group of the carboxyl-terminal deleted, replaced and/or added in a part outside the amino carboxyl group is OZ or NR2R3 wherein Z is a pharma acid sequences of ghrelin or ghrelin variants. In some ceutically acceptable cation or a lower branched or linear embodiments, ghrelin or ghrelin variants is a peptide or alkyl group, and R2 and R3 are the same or different and compound having the activity of increasing the intracellular represent H or a lower branched or linear alkyl group. calcium ion concentration and the activity of inducing 0099. In some embodiments, ghrelin variants can also be secretion of growth hormone, and (a) constitutional amino modified using ordinary molecular biological techniques so acids are modified or not modified and (b) at least one amino as to improve their resistance to proteolytic degradation or acid is replaced or not replaced by a non-amino acid to optimize solubility properties or to render them more compound. Suitable as a therapeutic agent. Even natural ghrelin can be so modified in order to produce a ghrelin variant. Analogs of 0094. In some embodiments, ghrelin variants comprise a Such ghrelin variants include those containing residues other modified amino acid or modified amino acids in which (a) a than naturally-occurring L-amino acids, e.g., D-amino acids saturated or unsaturated alkyl chain containing one or more or non-naturally occurring synthetic amino acids. The ghre carbon atoms was introduced at the C. carbon atom of the lin variants are not limited to products of any of the specific amino acid via or not via an alkylene group containing one example processes listed herein. The ghrelin variants useful or more carbon atoms and via an ester, ether, thioether, may further be conjugated to non-amino acid moieties that amide or disulfide linkage, or (b) a saturated or unsaturated are useful in their therapeutic application. In particular, alkyl chain containing one or more carbon atoms was moieties that improve the stability, biological half-life, water introduced at the C. carbon atom of the amino acid, and the solubility, and immunologic characteristics of the peptide symbol D is an amino acid having a hydrophobic residue. are useful. A non-limiting example of Such a moiety is 0095. In some embodiments, the ghrelin variants com polyethylene glycol (PEG). In some embodiments, ghrelin prise a modified amino acid at the second position from the variants include peptide analogs. N-terminal residue of ghrelin. In some embodiments, ghrelin 0100. In some embodiments, covalent attachment of bio variants comprise a modified amino acid at the third position logically active compounds to water-soluble polymers is one from the N-terminal residue of ghrelin. In some embodi method for alteration and control of biodistribution, phar ments, ghrelin variants comprise modified amino acids at the macokinetics and toxicity for ghrelin variant compounds second and the third position from the N-terminal residue of (Duncan et al., 1984, Adv. Polym. Sci. 57:53-101). For ghrelin. In some embodiments, the amino acid in the modi example, natural ghrelin can be so modified to produce a fied amino acid is serine or cysteine. ghrelin variant. Many water-soluble polymers have been 0096. In some embodiments, ghrelin variants comprise a used to achieve these effects, such as poly(sialic acid), modified amino acid modified by conversion of a functional dextran, poly(N-(2-hydroxypropyl)methacrylamide) group in a side chain of said amino acid into an ester linkage. (PHPMA), polyvinylpyrrollidone) (PVP), poly(vinyl alco In some embodiments, ghrelin variants comprise an amino hol) (PVA), poly(ethylene glycol-co-propylene glycol), poly acid having a fatty acid bound via an ester linkage to a (N-acryloyl morpholine (PAcM), and poly(ethylene glycol) side-chain hydroxyl group of said amino acid. For example, (PEG) (Powell, 1980, Polyethylene glycol. In R. L. David the fatty acid can be bound in Such a manner to a residue of son (Ed.), Handbook of Water Soluble Gums and Resins, ghrelin. McGraw-Hill, New York, chapter 18). PEG possesses an 0097. In some embodiments, ghrelin variants comprise ideal set of properties: very low toxicity (Pang, 1993, J. Am. an amino acid having a fatty acid bound via an ester linkage Coll. Toxicol. 12: 429–456) excellent solubility in aqueous to a side-chain hydroxyl group of said amino acid or via a Solution (Powell, Supra), low immunogenicity and antige thioester linkage to a side-chain mercapto group of said nicity (Dreborg et al., 1990, Crit. Rev. Ther. Drug Carrier amino acid. In some embodiments, ghrelin variants com Syst. 6: 315-365). PEG-conjugated or “PEGylated” protein prise an amino acid to which a fatty acid containing 2 to 35 therapeutics, containing single or multiple chains of poly carbon atoms was bound. In some embodiments, ghrelin ethylene glycol on the protein, have been described in the variants comprise an amino acid to which a fatty acid scientific literature (Clark et al., 1996, J. Biol. Chem. 271: selected from the group consisting of fatty acids containing 2 1969-2 1977). Each reference in this paragraph is incorpo 2, 4, 6, 8, 10, 12, 14, 16 and 18 carbonatoms was bound. For rated in its entirety herein. example, the variant can be the natural ghrelin sequence that 0101. In some embodiments, ghrelin variants may incor is modified as Suggested. In some embodiments, ghrelin porate amino acid residues which are modified without variants comprise a fatty acid, which is octanoic acid, affecting activity. For example, the termini may be deriva decanoic acid, a monoene fatty acid thereof or a polyene tized to include blocking groups, i.e. chemical Substituents fatty acid thereof. In other embodiments, acylation can be suitable to protect and/or stabilize the N- and C-termini from accomplished by use of a diacid result in acylation of one or “undesirable degradation,” a term meant to encompass any both of the hydroxyl groups of Ser(2) and Ser(3). Example type of enzymatic, chemical or biochemical breakdown of diacids can be represented by the formula R CH(COOH) the compound at its termini which is likely to affect the (CH2)COOH where R is a saturated or unsaturated aliphatic function of the compound, i.e. sequential degradation of the US 2017/O121385 A1 May 4, 2017

compound at a terminal end thereof. Blocking groups XaaXaa Phe Leu Ser Pro Glu His Gln Arg Val Glin Val Arg include protecting groups conventionally used in the art of Pro Pro His Lys Ala Pro His Val Val (SEQ ID No. 4), peptide chemistry which will not adversely affect the in vivo wherein the second and third position are 2,3-diaminopro activities of the variants. For example, suitable N-terminal pionic acid (Dpr) residues, with the Dpr in the third position blocking groups can be introduced by alkylation or acylation being optionally octanoylated. In some embodiments, the of the N-terminus. ghrelin variant comprises a polypeptide comprising the 0102) Examples of suitable N-terminal blocking groups sequence of Gly Ser Ser Phe Leu Ser Pro Glu His Gln Arg include C-C branched or unbranched alkyl groups, acyl Val Glin Val Arg Pro Pro His Lys Ala Pro His Val Val Pro Ala groups such as formyl and acetyl groups, as well as Substi Leu Pro (SEQID No. 5). In some embodiments, the ghrelin tuted forms thereof, such as the acetamidomethyl (Acm) variant is a ghrelin splice variant, comprising a polypeptide group. Desamino analogs of amino acids are also useful comprising the sequence of Gly Ser Ser Phe Leu Ser Pro Glu N-terminal blocking groups, and can either be coupled to the His Gin Arg Val Glin Val Arg Pro Pro His Lys Ala Pro His N-terminus of the peptide or used in place of the N-terminal Val Val Pro Ala Leu Pro Leu (SEQ ID No. 9). In some reside. Suitable C-terminal blocking groups, in which the embodiments, one or more of the modifications or Substi carboxyl group of the C-terminus is either incorporated or tutions listed above, can be specifically excluded. not, include esters, ketones or amides. Ester or ketone 0105. In some embodiments, the ghrelin variant is forming alkyl groups, particularly lower alkyl groups such RM-131 (also known as BIM-28.131), which is a small as methyl, ethyl and propyl, and amide-forming amino peptide ghrelin agonist, and BIM-28163, which is a full groups such as primary amines (—NH2), and mono- and length ghrelin analog antagonist. In some embodiments, the di-alkylamino groups such as methylamino, ethylamino, ghrelin variant is a polypeptide comprises the sequence of dimethylamino, diethylamino, methylethylamino and the Inp-D-2Nal-D-Trp-Thr-Lys-NH. (SEQ ID NO. 6). One or like are examples of C-terminal blocking groups. Descar more of the amino acids of SEQID NO. 6 can be substituted boxylated amino acid analogues such as agmatine are also with a natural or non-natural amino acid. Additional Small useful C-terminal blocking groups and can be either coupled peptides are disclosed in U.S. Pat. Nos. 8.377,865 and to the ghrelin variant's C-terminal residue or used in place 7,456,253, each of which is incorporated herein by reference of it. Further, the free amino and carboxyl groups at the in its entirety. Additionally, one or more of the amino acids termini can be removed altogether from the ghrelin variant can be chemically modified, for example, with an octanoyl to yield desamino and descarboxylated forms thereof with or like group, with an acyl group, a protecting group, and the out effect on the ghrelin variant activity. like. 0103) In some embodiments, the ghrelin variant com 0106. In some embodiments, the ghrelin variant is an pound is one or more Dln-101, Growth hormone (GH) isolated ghrelin splice variant-like compound with the for releasing hexapeptide (GHRP)-6, EP 1572, Ape-Ser(Octyl)- mula Z1-(X 1)-(X2)-(X3)n-Z2, wherein Z is an optionally Phe-Leu-aminoethylamide, isolated ghrelin splice variant present protecting group; each X1 is independently selected like compound, ghrelin splice variant, growth hormone from a naturally occurring amino acid and a synthetic amino secretagogue receptor GHS-R la ligand, and a combination acid; X2 is selected from a naturally occurring amino acid thereof. In some embodiments, one or more of the variants and a synthetic amino acid, said amino acid being modified listed above, can be specifically excluded. In some embodi with a bulky hydrophobic group; each X3 is independently ments, the ghrelin variant comprises a polypeptide having at selected from a naturally occurring amino acid and a syn least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, thetic amino acid, wherein one or more of X1 and X3 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or optionally may be modified with a bulky hydrophobic 99% sequence identity to the amino acid sequence of one or group; Z2 is an optionally present protecting group; m is an more of the compounds described in the present disclosure. integer in the range of from 1-10; n is an integer in the range In some embodiments the ghrelin agonist can be a short of from 4-92; provided that the compound according to peptide, for example a pentapeptide such as of RM-131 (or formula Z1-(X 1)-(X2)-(X3)n-Z2 is 15-94 amino acids in BIM-28.131). In some embodiments, at least 1 amino acid of length, and has at least 80% homology to the sequence of Such a pentapeptide can be substituted with a natural or a Gly Ser Ser Phe Leu Ser Pro Glu His Gln Arg Val Glin Val non-natural amino acid Such as those described herein, Arg Pro Pro His Lys Ala Pro His Val Val Pro Ala Leu Pro removed, and/or chemically modified (e.g., octanoylated, Leu Ser Asn Gln Leu Cys Asp Leu Glu Gln Gln Arg His Leu acylated, etc.). In some embodiments, one or more of the Trp Ala Ser Val Phe Ser Gln Ser Thr Lys Asp Ser Gly Ser modifications or Substitutions listed above, can be specifi Asp Leu Thr Val Ser Gly Arg Thr Trp Gly Leu Arg Val Leu cally excluded. ASn Gin Leu Phe Pro Pro Ser Ser Arg Glu Arg Ser Arg Arg 0104. In some embodiments, the ghrelin variant is Dln Ser His Gln Pro Ser Cys Ser Pro Glu Leu (SEQID NO. 10). 101, which is a small peptide ghrelin agonist. In some 0107. In some embodiments, the ghrelin variant is embodiments, the ghrelin variant comprises a polypeptide EP1572 or UMV1843 (Aib-DTrp-DgTrp-CHO), which is a comprising the sequence of Gly Ser Ser Phe Leu Ser Pro Glu peptido-mimetic GH secretagogue with selective GH-releas His Gln Arg Val Glin Val Arg Pro Pro Lys Ala Pro His Val ing activity. In some embodiments, the ghrelin variant is a Val (SEQ ID No. 2). In some embodiments, the ghrelin growth hormone secretagogue receptor GHS-R la ligand, variant comprises a polypeptide comprising the sequence of which binds to growth hormone secretagogue receptor Gly Ser Xaa Phe Leu Ser Pro Glu His Gln Arg Val Glin Val GHS-R 1a. Arg Pro Pro His Lys Ala Pro His Val Val (SEQ ID No. 3), 0108. In some embodiments, the ghrelin variant is wherein the third position is a 2,3-diaminopropionic acid Growth hormone (GH) releasing hexapeptide (GHRP)-6, (Dpr), with the Dpr in the third position being optionally which is a compound of the chemical nomenclature: L-his octanoylated. In some embodiments, the ghrelin variant tidyl-D-tryptophyl-L-alanyl-L-tryptophyl-D-phenylalanyl comprises a polypeptide comprising the sequence of Gly L-Lysinamide. US 2017/O121385 A1 May 4, 2017

0109. In some embodiments, the ghrelin variant is a 0117. A concrete example of the ghrelin variant, which growth hormone releasing peptide (GHRP) including, but has the third and fourth amino acids from the N-terminal and not limited to, Pralmorelin (GHRP 2, GPA 748, growth in which the side chain of the third amino acid (Ser) from the hormone-releasing peptide 2, KP-102 D, KP-102 LN, N-terminal has been substituted, is the compound reported at KP-102D, KP-102LN; Kaken Pharma and Stella Pharma), the 37th Peptide Forum (Oct. 18 to 20, 2000), i.e., NH Examorelin/Hexarelin, GHRP-1, GHRP-6 (SK&F-110679), (CH) CO-Ser(octyl)-Phe-Leu-NH-(CH), NH, Ipamorelin (NNC-260161), NNC-260194, NNC-260235, and salts and esters thereof. In some embodiments, the ghrelin variant is Pralmorelin as described in U.S. Pat. No. 6,468,974, which is incorporated by reference in its entirety. In some embodiments, one or more of the variants listed above, can be specifically excluded. 0110. In some embodiments, the ghrelin variants of the r present disclosure include, but not limited to, molecules and H compounds described in U.S. Pat. Nos. 6,849,597, 7,452, N n1n NH2 862, 7,476,653, 7,521,420, 7,550,431, 7,491,.695, 7,589,058, and 8,088,733; US Patent Application Publication Nos. US 2010/0216706 and US 2012/0232001; and WO 2004/ O O O 0096.16, WO 2008/143835, and WO 2013/113916, which are incorporated by reference in their entireties. In some embodiments, one or more of the variants listed above, can be specifically excluded. 0118. The GH secretagogues (GHS) include compounds 0111 Examples of these salts are described below, and expressed by the following formulae: hydrochlorides are named as preferred examples (e.g., pral morelin dihydrochloride, hexarelin hydrochloride). (1) Compounds of the Following General Formula 0112 No restrictions are imposed on the origins of the 0119) amino acid residues or amino acid derivative residues in the peptides (for example, the peptides or derivatives may have been isolated or purified from human or rat cells, or may be synthetic products or semi-synthetic products, or may have been obtained by genetic engineering). 0113. The ghrelin variants of the present disclosure include, but not limited to, one of the amino acids that has been deficient are typified by des-Gln14-ghrelin, i.e., ghrelin N (Y)p CO with the 14th Gln residue deleted, and the compounds described in J. Med. 5 Chem. 2000, 43, 4370-4376. 0114 Examples of the ghrelin variants include, but not limited to, peptides and their derivatives which have the third and fourth amino acids from the N-terminal among the 28 amino acids of ghrelin (preferably, the four amino acids at the N-terminal) and in which the side chain of the third where amino acid (Ser) from the N-terminal has been substituted, 1 denotes 0, 1 or 2, the peptides and derivatives having a growth hormone I0120 X represents —CH2—, —O —S(O)r- (r--0, secretion promoting action. In some embodiments, one or 1 or 2), —C(O)— —C(S)— —CH=CH , —CH more of the modifications or substitutions listed above, can (OH)– or NR , be specifically excluded. 0121 R represents a hydrogen atom, a (C-C alkyl 0115 Examples of the side chain of the third amino acid group, a (Cs-Cs)cycloalkyl group, an acyl group or an from the N-terminal include, but not limited to, an acyl alkoxycarbonyl group, group and an alkyl group (the number of their carbon atoms 0.122 m denotes 0, 1 or 2, is preferably 6 to 18) other than octanoyl which is the side (0123 Y represents —C(O)— —C(S) , or a (C-C) chain of ghrelin. alkylene group which may be substituted by (C-C) 0116 Concrete examples of the side chain are as follows: alkyl group(S), —CH2(CH2)9CH, —CO-(CH2)CH, —CO— 0.124 p denotes 0, 1 or 2, CH-CH CH-CH CH-CH CH, CO CH 0.125 Z represents a substituted or unsubstituted (C- (CH2CH2CH), —CO-(CH2)CH, —CO-(CH2)CH, Cs) alkylene group, —NR— (R is a hydrogen atom, a —CO-(CH2)CHBr, CO-CH(CH),CONH(CH.) (C-Cs) alkyl group, a (C-C) cycloalkyl group, an CH, —COPh, and a group of the following formula acyl group or an alkoxycarbonyl group), or a group of the formula

-CO-CH2 US 2017/O121385 A1 May 4, 2017 15

0.126 where A is a 5- or 6-membered aromatic ring 0.142 X represents —CH2—, —O —S(O)r-, optionally containing at least one hetero atom, and C(O) , C(S)-, -CH=CH-, -CH(OH)– or I0127. A may further be substituted by a group selected - NR, from a halogen atom, a hydroxyl group, a (C-C) alkyl 0143 R represents a hydrogen atom, a (C-C) alkyl group, a (C-C) alkoxy group, a (C-Cs) perfluoro group, a (C-Cs) cycloalkyl group, an acyl group, or an alkyl group, a (C-Cs) perfluoroalkoxy group, a nitro alkoxycarbonyl group, group, a cyano group, an amino group, a substituted 0.144 r denotes 0, 1 or 2, amino group, a phenyl group and/or a substituted 0145 m denotes 0, 1 or 2, preferred being —CH2—as phenyl group, X and m as 2. 0128 n denotes 0 or 1, 0146 Y represents —C(O)— —C(S) , or a (C-Cs) I0129. D represents alkylene group which may be substituted by (C-C) alkyl group(S), 0147 p denotes 0, 1 or 2, preferred being—C(O)—as i R2 Y. and 1 as p, N N E N 0.148 Z represents a substituted or unsubstituted (C- 1N M21 NR3 1 NM21 NR3 Cs) alkylene group, —NR— (R is a hydrogen atom, a (C-C) alkyl group, a (C-C) cycloalkyl group, an 0.130 where R' represents a hydrogen atom, an alkyl acyl group or an alkoxycarbonyl group), or a 6-mem group, a Substituted alkyl group, a cycloalkyl group or beredered aromauctic ringri representedted by unethe Iormulaf 1 a Substituted cycloalkyl group, I0131) R' and Reach represent, independently of each other, a hydrogen atom, an alkyl group, a Substituted alkyl group, an acyl group, an amidino group or an alkoxycarbonyl group, or one of R and R, taken together with R', may constitute an alkylene group, (0132 further, R and R may together constitute an 0.149 N denotes 0 or 1, and 013alkylene S. i.group RNd or a hetero by ring,E. formula 0.150 D represents a groups of the formula

R5 R7 H OH

-(CH)x-C-(CH)-C-(CH)z- 1. N-N-N: k k i 0151. In the above formula, the asterisk (*) represents an -(CH2)x-C=C-(CH2)2- or asymmetric center, so that isolated pure optical isomers, -(CH)x-CEC-(CH2)2- partially purified optical isomers or racemic mixtures are included. 0.134 where x, y and Z each represent, independently (2) Compounds of the Following General Formula of each other, an integer of 0 to 4. 0.135 R. R. R7 and Reach represent, independently 0152 of each other, a hydrogen atom, a halogen atom, an alkyl group, a substituted alkyl group, —OR. —SR, NR'R'', NHC(O)R’, C(O)OR, OCOR, –OC(O)CR or -CONR'R'', or may constitute an alkylene group or a hetero ring taken together with R' or R, 10136 RandR'each represent, independently of each other, a hydrogen atom, an alkyl group or a substituted alkyl group, I0137 R may constitute an alkylene group taken together with R' or R. O 0138 Rand R', or Rand R may together constitute an alkylene group or a hetero ring, or 0.139 R and R, or R7 and R may constitute a car bonyl group, a thiocarbonyl group or an imino group 0.153 where taken together with the carbon atom to which R and I0154) R' represents a substituted or unsubstituted R, or R7 and R have been bound, and alkyl, a Substituted or unsubstituted cycloalkyl, a Sub 0140 E represents an oxygen atom or a sulfur atom. stituted or unsubstituted alkoxy, a substituted or unsub 0141. Of the compounds (1), preferred compounds are stituted aryl, or a substituted or unsubstituted amino, those of the above-mentioned formula: where 1 denotes 0, 1 0.155 X represents a single bond, —CO— or or 2, preferably 0. —SO , US 2017/O121385 A1 May 4, 2017

0156 D represents unsubstituted aryland/or a hydroxy; a C-C cycloalkyl which may be substituted by a substituted or unsubsti tuted alkyl, a substituted or unsubstituted alkoxy, a R1 R2 R2 substituted or unsubstituted aryl and/or a hydroxy; a C-C alkoxy which may be substituted by a substi 1 NYM21 N NR3 1 E NM21 N NR3 tuted or unsubstituted cycloalkyl, a substituted or unsubstituted alkoxy, a substituted or unsubstituted aryl and/or a hydroxy; an aryl which may be substituted by where R' represents a hydrogen atom, an alkyl group, a a substituted or unsubstituted cycloalkyl, a substituted Substituted alkyl group, a cycloalkyl group, or a Substituted or unsubstituted alkoxy, a substituted or unsubstituted cycloalkyl group, aryl and/or a hydroxy; or an amino which may be (O157 R and Reach represent, independently of each substituted by a substituted or unsubstituted alkyl or a other, a hydrogen atom, an alkyl group, a Substituted substituted or unsubstituted aryl. alkyl group, an acyl group, an amidino group or an (0171 More preferably, R is represented by any of the alkoxy-carbonyl group, or either R or R and R' may following formulas: together constitute an alkylene group, I0158 further, R and R may together constitute an alkylene group or a hetero ring, HC CH CH 0159 M is represented by the formula -CH3, )- --ch. —Hoch. R5 R7 H3C CH3 CH3 -cir-l-en-l-cis CH k k —Hochsch. HC s R5 R7 CH3 HC3 H3C -(CH2)x-C=C-(CH2)2- or \ H3C 1. -(CH3)x-CEC-(CH2)2- N-, N^ s HC-7 CH3 0160 where x, y and Z each represent, independently CH3 of each other, an integer of 0 to 4. CH3 CH3 (0161 R. R. R7 and Reach represent, independently of each other, a hydrogen atom, a halogen atom, an alkyl group, a substituted alkyl group, —OR. —SR, CH3 NR'R'', NHC(O)R’, C(O)OR, OCOR, CH3, s –OC(O)OR or -CONR'R'', or may constitute an alkylene group or a hetero ring taken together with R' or R, (0162 RandR'each represent, independently of each () ()--O- other, a hydrogen atom, an alkyl group or a substituted alkyl group, (0163 R may constitute an alkylene group taken together with R' or R, (0164 Rand R', or RandR may together constitute neo-O- C s an alkylene group or a hetero ring, or (0165 R and R, or R7 and R may constitute a car bonyl group, a thiocarbonyl group or an imino group taken together with the carbon atom to which R and R, or R7 and R have been bound, and 0166 E represents an oxygen atom or a sulfur atom, 0172 D is preferably represented by the following and formula: 0.167 the asterisk (*) represents an asymmetric center, So that the compounds (2) include isolated pure optical isomers, partially purified optical isomers, racemic OH mixtures or diastereomer mixtures (all such optical isomers are included in the Scope of the present dis 1. N--: 2 closure). 0.168. Of the compounds (2), preferred aspects are as follows: 0173. In some embodiments, the ghrelin variant is a GH (0169 X is preferably – CO-. secretagogues (GHS). Concrete compounds as the GH (0170 R is preferably a C-C alkyl which may be secretagogues (GHS) are exemplified by, but not limited to, substituted by a substituted or unsubstituted cycloalkyl, S-38855, S-37555, S-39 100, ibutamorelin (e.g., ibutamo a substituted or unsubstituted alkoxy, a substituted or relin mesylate (MK-0677), capromorelin (CP-424391),

US 2017/O121385 A1 May 4, 2017 20

-continued

Chemical Names and Structural Formulas of Compounds as Concrete Examples

General Name Chemical Name Chemical Structure

NNC-26O161 2-Methylalanyl-L-histidyl 3 (2 NH2 (ipamorelin) naphthalenyl)-D-alanyl-D- Me Me phenylalanyl-L-lysineamide O N uly N 1.

Ol NH O

N N 1. (CH2)4 n N1 r N O O1. NH2

L-163540 1-2(R)-(2-amino-2- methylpropionylamino)-3-(IH indo1-3-yl)propionyl 3 benzylpiperidine-3 (S)-carboxylic H acid ethyl ester N NH2

O N O 'N-1

O

L-168721 N-(6-aminohexyl)-2-(4-oxo-2- H phenethyl-6-pheny1-4H- O N-1 nu1\-1\ quinazolin-3-yl)acetamide O NH2 US 2017/O121385 A1 May 4, 2017 21

-continued

Chemical Names and Structural Formulas of Compounds as Concrete Examples

General Name Chemical Name Chemical Structure LY-426410 2-Amino-N-2-benzyloxy-(1R)-1- (1R)-(4-methoxyphenyl)-2-(4- H methylpiperidin 1 yl)-2-oxo-ethyl N 1H-imidazol-4-ylcarbamoylethyl O NH2 2-methylpropionamide O HN O

LY-444711 2-Methylalanyl-N-1-(112)-1-(4- methoxyphenyl)-1-methyl-2-oxo 2-(1-pyrrollidinypethyl)-1H imidazol-4-yl)-5-phenyl-D- norvalineamide

OCH

0175 Salts of the above-described compounds include ules, ointments and Suppositories, by publicly known phar but are not limited to, for example, salts with mineral acids maceutical manufacturing techniques, when used alone or Such as hydrochloric acid, Sulfuric acid or phosphoric acid, combined with pharmaceutically acceptable carriers, addi salts with organic acids such as methanesulfonic acid, ben tives, etc. The growth hormone secretion promoting Sub Zenesulfonic acid, malic acid, citric acid or Succinic acid, stance of the present disclosure can also be made into dosage salts with alkali metals such as Sodium or potassium, Salts forms, such as drug delivery systems (for example, slow with alkaline earth metals such as calcium or magnesium, release preparations). and salts with basic amino acids such as arginine. 0.178 The carriers and additives usable in the preventive/ 0176). In some embodiments, the growth hormone secre therapeutic agents of the present disclosure include, for tion promoting Substances and their salts may be used in example, those which are ordinarily used in preparing phar combination of two or more. maceuticals: aqueous vehicles such as physiological Saline, 0177. The growth hormone secretion promoting sub water (tap water, distilled water, purified water, water for stance used in the present disclosure can be formed into injection) and Ringer solution, nonaqueous vehicles Such as ordinary oral preparations and parenteral preparations, for oily solvents (vegetable oils) and water-soluble solvents example, liquids and solutions (injections, nasal drops, (propylene glycol, macrogol, ethanol, glycerin), bases Such syrups, dry syrups), tablets, troches, capsules (hard capsules, as cacao butter, polyethylene glycol, microcrystalline wax, Soft capsules, microcapsules), powder, Subtle granules, gran white beeswax, liquid petrolatum and white petrolatum, US 2017/O121385 A1 May 4, 2017 22 excipients such as Sucrose, starch, mannitol, Sorbitol, lac of performing the same function, i.e. a homologue may be tose, glucose, cellulose, talc, calcium phosphate and calcium envisaged as a functional equivalent of a predetermined carbonate, binders such as cellulose, methylcellulose, Sequence. hydroxypropylcellulose, polypropylpyrrolidone, gelatin, 0183. A homologue of any of the predetermined acacia, polyethylene glycol, Sucrose and starch, disintegra sequences herein may be defined as i) homologues compris tors such as starch, carboxymethylcellulose, hydroxypropyl ing an amino acid sequence capable of being recognized by starch, Sodium bicarbonate, calcium phosphate, calcium an antibody, said antibody also recognizing the ghrelin carboxymethylcellulose and calcium citrate, lubricants such variant, including the acylated ghrelin variant (also un as magnesium Stearate, talc and sodium lauryl Sulfate, taste acylated ghrelin variants in Some embodiments), and/or ii) correctives such as citric acid, menthol, glycine, Sorbitol and homologues comprising an amino acid sequence capable of orange powder, preservatives and antiseptics Such as para binding selectively to GHS-R 1a, and/or iii) homologues hydroxybenzoate esters, benzyl alcohol, chlorobutanol and having a substantially similar or higher binding affinity to quaternary ammonium salts (benzalkonium chloride, ben GHS-R 1a than the ghrelin variant described herein. The Zethonium chloride), stabilizers such as albumin, gelatin, antibodies used herein may be antibodies binding the N-ter Sorbitol and mannitol, Suspending agents such as methyl minal region of ghrelin variant or the C-terminal region of cellulose, polyvinylpyrrolidone and aluminum Stearate, ghrelin variant, the N-terminal region. The antibodies may plasticizers such as glycerin and Sorbitol, dispersing agents be antibodies as described in Ariyasu H. et al. Endocrinol Such as hydroxypropyl methylcellulose, solution adjuvants ogy 143:3341-50 (2002), which is incorporated herein by Such as hydrochloric acid and cyclodextrin, emulsifying reference in its entirety. agents such as sodium monostearate, electrolytes Such as 0184. In some embodiments, one or more of the amino Sodium chloride, and nonelectrolyte tonicity regulating acids of the sequence are Substituted or replaced by another agents and flavors. Such as Sugar alcohols, Sugars and amino acid or a synthetic amino acid. In some embodiments, alcohols. the substitution is between 1 and 5 substitutions. 0179. In the oral preparation, water-swellable cellulose 0185. Example homologues comprise one or more con (carboxymethylcellulose, calcium carboxymethylcellulose, servative amino acid Substitutions including one or more sodium croscarboxymethylcellulose, low substitution conservative amino acid Substitutions within the same group degree hydroxypropylcellulose). Such as microcrystalline of predetermined amino acids, or a plurality of conservative cellulose ("Avicel trade name, a product of Asahi Chemi amino acid Substitutions, wherein each conservative Substi cal Industry) as described in Japanese Patent Publication tution is generated by substitution within a different group of No. 1998-456194 can be incorporated for increasing absorb predetermined amino acids. Homologues may thus comprise ability. conservative substitutions independent of one another, 0180 Normally, the preparation of the present disclosure wherein at least one glycine (Gly) of said homologue is is administered to mammals (e.g., mouse, rat, hamster, Substituted with an amino acid selected from the group of rabbit, cat, dog, cattle, horse, sheep, monkey), including amino acids consisting of Ala, Val, Leu, and Ile, and humans, by the oral route or by Such means as Subcutaneous independently thereof, homologues, wherein at least one of injection, nasal dropping, intra-arterial injection (including said alanines (Ala) of said homologue thereof is Substituted drip infusion), intravenous injection, intra-spinal injection or with an amino acid selected from the group of amino acids local cerebral administration. consisting of Gly, Val, Leu, and Ile; and independently 0181. In some embodiments, the ghrelin variant com thereof, homologues wherein at least one valine (Val) of said prises a polypeptide having at least 80%, 81%, 82%, 83%, homologue thereof is substituted with an amino acid 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, selected from the group of amino acids consisting of Gly, 94%. 95%, 96%, 97%, 98%, or 99% sequence identity to the Ala, Leu, and Ile; and, independently thereof, homologues amino acid sequence of one or more of the compounds wherein at least one of said leucines (Leu) of said homo described in the present disclosure. The term “sequence logue thereof is substituted with an amino acid selected from identity” or “homology” shall be construed to mean the the group of amino acids consisting of Gly, Ala, Val, and Ile; percentage of amino acid residues in the candidate sequence and independently thereof, homologues wherein at least one that are identical with the residue of a corresponding isoleucine (Ile) of said homologues thereof is substituted sequence to which it is compared, after aligning the with an amino acid selected from the group of amino acids sequences and introducing gaps, if necessary to achieve the consisting of Gly, Ala, Val and Leu; and, independently maximum percent identity for the entire sequence, and not thereof homologues wherein at least one of said aspartic considering any conservative substitutions as part of the acids (Asp) of said homologue thereof is substituted with an sequence identity. Neither N- or C-terminal extensions nor amino acid selected from the group of amino acids consist insertions shall be construed as reducing identity or homol ing of Glu, ASn, and Gin; and independently thereof, homo ogy. Methods and computer programs for the alignment are logues wherein at least one of said phenylalanines (Phe) of well known in the art. Sequence identity may be measured said homologues thereof is Substituted with an amino acid using sequence analysis Software (e.g., Sequence Analysis selected from the group of amino acids consisting of Tyr, Software Package, Genetics Computer Group, University of Trp, His, and Pro, and selected from the group of amino Wisconsin Biotechnology Center, Madison, Wis.). This soft acids consisting of Tyr and Trp; and independently thereof, ware matches similar sequences by assigning degrees of homologues wherein at least one of said tyrosines (Tyr) of homology to various Substitutions, deletions, and other said homologues thereof is Substituted with an amino acid modifications. selected from the group of amino acids consisting of Phe, 0182 Aghrelin variant homologue of one or more of the Trp, His, and Pro, or an amino acid selected from the group sequences specified herein may vary in one or more amino of amino acids consisting of Phe and Trp; and, indepen acids as compared to the sequences defined, but is capable dently thereof, homologues wherein at least one of said US 2017/O121385 A1 May 4, 2017 arginines (Arg) of said fragment is substituted with an amino bicity, antigenicity, propensity to form or break C-helical acid selected from the group of amino acids consisting of structures or B-sheet structures. Lys and His; and, independently thereof, homologues 0188 A non-conservative substitution leading to the for wherein at least one lysine (LyS) of said homologues thereof mation of a functionally equivalent homologue of the is Substituted with an amino acid selected from the group of sequences herein would, for example, i) differ Substantially amino acids consisting of Arg and His; and, independently in polarity, for example a residue with a non-polar side chain thereof homologues wherein at least one of said asparagines (Ala, Leu, Pro, Trp, Val, Ile, Gly, Leu, Phe or Met) substi (Asn) of said homologues thereof is substituted with an tuted for a residue with a polar side chain such as Ser. Thr, amino acid selected from the group of amino acids consist Cys, Tyr, ASn, or Glin or a charged amino acid such as Asp, ing of Asp, Glu, and Gin; and, independently thereof, Glu, Arg, or Lys, or Substituting a charged or a polar residue homologues wherein at least one glutamine (Gin) of said for a non-polar one; and/or ii) differ substantially in its effect homologues thereof is Substituted with an amino acid on polypeptide backbone orientation Such as Substitution of selected from the group of amino acids consisting of Asp, or for Pro or Gly by another residue; and/or iii) differ Glu, and ASn; and, independently thereof homologues Substantially in electric charge, for example Substitution of wherein at least one proline (Pro) of said homologues a negatively charged residue such as Glu or Asp for a thereof is substituted with an amino acid selected from the positively charged residue Such as Lys, His or Arg (and vice group of amino acids consisting of Phe, Tyr, Trp, and His, versa); and/or iv) differ substantially in steric bulk, for and, independently thereof, homologues wherein at least one example substitution of a bulky residue such as His, Trp, Phe of said cysteines (CyS) of said homologues thereof is Sub or Tyr for one having a minor side chain, e.g. Ala, Gly or Ser stituted with an amino acid selected from the group of amino (and vice versa). acids consisting of Asp, Glu, Lys, Arg, His, ASn, Gin, Ser, 0189 Substitution of amino acids may in one embodi Thr, and Tyr. Non-limiting examples of common Substitu ment be made based upon their hydrophobicity and hydro tions for various residues can be found in the NCBI Amino philicity values and the relative similarity of the amino acid Acid Explorer database, which includes listings of common side-chain Substituents, including charge, size, and the like. Substitutions for each amino acid, along other types of Examples of amino acid substitutions which take various of information on each amino acid as part of its BLOSUM62 the foregoing characteristics into consideration are well matrix database (see Substitutes in BLOSUM62 on the known to those of skill in the art and include, for example, worldwide web at: ncbi.nlm.nih.gov/Class/Structure/aa/aa arginine and lysine; glutamate and aspartate; serine and explorer.cgi. threonine; glutamine and asparagine; and Valine, leucine, 0186 Conservative substitutions may be introduced in and isoleucine. any position of a predetermined sequence. It may however 0190. Some non-limiting examples of potential mol also be desirable to introduce non-conservative substitu ecules that can be substituted for amino acids are provided tions, particularly, but not limited to, a non-conservative below in Table 3. Substitution in any one or more positions. In some embodi ments the Substitutions can be conservative Substitutions, TABLE 3 which are well known in the art (see for example Creighton (1984) Proteins. W. H. Freeman and Company (Eds). Table Symbol Meaning 2 below depicts non-limiting examples of conservative A3c 1-amino-1-cyclopropanecarboxylic acid Substitutions that can be made: A4c 1-amino-1-cyclobutanecarboxylic acid A.Sc. 1-amino-1-cyclopentanecarboxylic acid TABLE 2 A6c 1-amino-1-cyclohexanecarboxylic acid Aad 2-Aminoadipic acid Residue Conservative Substitutions bAad 3-aminoadipic acid bAla beta-Alanine, beta-Aminopropionic acid Ala Ser Abu 2-Aminobutyric acid Arg Lys 4Abu 4-Aminobutyric acid, piperidinic acid ASn Gln: His Acc 1-amino-1-cyclo(C-Co.)alkyl carboxylic acid Asp Glu Acp 6-Aminocaproic acid Gln ASn Act 4-amino-4-carboxytetrahydropyran Cys Ser Ahe 2-Aminoheptanoic acid Glu Asp Aib 2-Aminoisobutyric acid Gly Pro bAib 3-Aminoisobutyric acid His ASn; Glin Apc amino piperidinylcarboxylic acid Ile Leu, Val Apm 2-Aminopimelic acid Leu Ile: Val hArg homoarginine Lys Arg: Glin Bal 3-Benzothienylalanine Met Leu: Ile Bip 4,4'-Biphenylalanine Phe Met; Leu: Tyr Bpa 4-Benzoylphenylalanine Ser Thr; Gly Cha 3-cyclohexylalanine Thr Ser; Val Dbu 2,4-Diaminobutyric acid Trp Tyr Des Desmosine Tyr Trp; Phe Dip B B-Diphenylalanine Wal Ile: Leu Dmt 5,5-dimethylthiazolidine-4-carboxylic acid Dpm 2,2-Diaminopimelic acid Dr 2,3-Diaminopropionic acid EtGly N-Ethylglycine 0187. In some embodiments, one or more amino acids EtASn N-Ethylasparagine can be substituted with an amino acid or synthetic amino 2Fua B-(2-furyl)-alanine acid that has a similar property or a different property at its Hyl Hydroxylysine side chain or otherwise, such as charge, polarity, hydropho US 2017/O121385 A1 May 4, 2017 24

TABLE 3-continued Ser-Arg-Ile (SEQ ID NO. 18). In another embodiment, a ghrelin variant comprises a polypeptide comprising at least Symbol Meaning one modification to the natural form of an amino acid aHyl allo-Hydroxylysine sequence of Gly-Ser-Ser-Phe-Leu-Ser-Pro-Glu-His-Gln 3Hyp 3-Hydroxyproline Lys-Ala-Gln-Gln-Arg-Lys-Glu-Ser-Lys-Lys-Pro-Pro-Ala 4Hyp 4-Hydroxyproline Ide Isodesmosine Lys-Leu-Gln-Pro-Arg (SEQID NO.19). In another embodi aIle allo-Isoleucine ment, a ghrelin variant comprises a polypeptide comprising Inc indoline-2-carboxylic acid at least one modification to the natural form of an amino acid Inp isonipecotic acid Ktp 4-ketoproline sequence of Gly-Ser-Ser-Phe-Leu-Ser-Pro-Glu-His-Gln hLeu homoleucine Lys-Val-Gln-Gln-Arg-Lys-Glu-Ser-Lys-Lys-Pro-Ala-Ala MeGly N-Methylglycine, sarcosine Lys-Leu-Lys-Pro-Arg (SEQID NO. 20). In another embodi MeIle N-Methylisoleucine MeLys 6-N-Methyllysine ment, a ghrelin variant comprises a polypeptide comprising MeVal N-Methylvaline at least one modification to the natural form of an amino acid 1Nal B-(1-Naphthyl)alanine sequence of Gly-Ser-Ser-Phe-Leu-Ser-Pro-Glu-His-Gln 2Nal B-(2-Naphthyl)alanine Arg-Ala-Gln-Gln-Arg-Lys-Glu-Ser-Lys-Lys-Pro-Pro-Ala Nwa Norvaline Nle Norleucine Lys-Leu-Gln-Pro-Arg (SEQID NO. 21). In another embodi Oic octahydroindole-2-carboxylic acid ment, a ghrelin variant comprises a polypeptide comprising Orn Ornithine at least one modification to the natural form of an amino acid 2Pal B-2-Pyridyl)-alanine 3Pal B-(3-Pyridyl)-alanine sequence of Gly-Ser-Ser-Phe-Leu-Ser-Pro-Thr-Tyr-Lys 4Pal B-(4-Pyridyl)-alanine Asn-Ile-Gln-Gln-Gln-Lys-Asp-Thr-Arg-Lys-Pro-Thr-Ala Pff pentafluorophenylalanine Arg-Leu-His (SEQID NO. 22). In yet another embodiment, hPhe homophenylalanine Pim 2'-(4-Phenyl)imidazolyl aghrelin variant comprises a polypeptide comprising at least Pip pipecolic acid one modification to the natural form of an amino acid Taz 3-(4-thiazolyl)alanine sequence of Gly-Ser-Ser-Phe-Leu-Ser-Pro-Glu-His-Gln 2T 3-(2-thienyl)alanine Lys-Leu-Gln-Gln-Arg-Lys-Glu-Ser-Lys-Lys-Pro-Pro-Ala 3Thi B-(3-thienyl)alanine ThZ thiazolidine-4-carboxylic acid Lys-Leu-Gln-Pro-Arg (SEQID NO. 23). In another embodi Tic 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid ment, a ghrelin variant comprises a polypeptide comprising Tle tert-leucine an amino acid sequence of Gly-Ser-Ser(O-n-octanoyl)-Phe Leu-Ser-Pro-Glu-His-Gln-Arg-Val-Gln-Gln-Arg-Lys-Glu 0191 In some embodiments, a ghrelin variant is des Ser-Lys-Lys-Pro-Pro-Ala-Lys-Leu-Gln-Pro-Arg (SEQ ID acyl-ghrelin of the primary amino acid sequence as provided NO. 24). In another embodiment, a ghrelin variant com in SEQ ID NO. 1. In some embodiments, a ghrelin variant prises a polypeptide comprising an amino acid sequence of binds to a receptor other than GHSR-1a or ghrelin receptor, Fluorescein-Ahx-Gly-Ser-Ser(O-n-octanoyl)-Phe-Leu-Ser and wherein binding to a receptor other than GHSR-1a or Pro-Glu-His-Gln-Arg-Val-Gln-Gln-Arg-Lys-Glu-Ser-Lys ghrelin receptor provides a therapeutic benefit following Lys-Pro-Pro-Aa-Lys-Leu-Gln-Pro-Arg mBI, for example, neuroprotection following mBI, repeated (Ahx=Aminohexanoic acid) (SEQ ID NO. 25). In another mBI or CTE (Chronic Traumatic Encephalopathy). The embodiment, a ghrelin variant comprises a polypeptide therapeutic benefit such as neuroprotection following mBI comprising an amino acid sequence of Gly-Ser-Ser(O-n- or repeated mBI or CTE may include reduced oxidative octanoyl)-Tyr-Leu-Ser-Pro-Glu-His-Gln-Arg-Val-Gln-Gln stress or reduced apoptosis. Arg-Lys-Glu-Ser-Lys-Lys-Pro-Pro-Ala-Lys-Leu-Gln-Pro 0.192 In some embodiments, a ghrelin variant binds to Arg (SEQ ID NO. 26). CA36 receptor. In some embodiments, a ghrelin variant 0194 Some embodiments relate to and can utilize ghrelin binds to CD36. CD36 (i.e., Cluster of Differentiation 36) is or ghrelin variant molecules that have a carbon 14 (C14) also known as FAT (fatty acid translocase), FAT/CD36, content less than found in endogenously produced ghrelin or (FAT)/CD36, SCARB3, GP88, glycoprotein IV (gpIV), and ghrelin variant molecules or in ghrelin or ghrelin variant that glycoprotein IIIb (gpIIIb). CD36 is an integral membrane has a C14 content about the same as atmospheric C14 levels. protein and is a member of the class B scavenger receptor For example, ghrelin or ghrelin variant molecules can have family of cell surface proteins. CD36 participates in inter at least one carbon atom or carbon containing moiety that is nalization of apoptotic cells, bacterial and fungal pathogens, from fossil derived reagents that have a C14 content less contributes to inflammatory responses, and facilitates long than found in endogenous molecules or less than atmo chain fatty acids transport into cells. CD36 is involved in, spheric levels. In some embodiments, the ghrelin or ghrelin but not limited to, muscle lipid utilization, adipose energy variant molecules can have all, Substantially all or at least a storage, gut fat absorption and the pathogenesis of metabolic Some carbon having a C14 content less than found endog disorders, such as diabetes and obesity. Hexarelin, a growth enously or less than atmospheric levels. For example, one or hormone-releasing peptide, has been shown to bind CD36 more of the amino acids of a sequence can include carbon and ghrelin receptor, GH secretagogue-receptor la to up and have a C14 contentless than found in endogenous amino regulates sterol transporters and cholesterol efflux in mac acids or less than atmospheric levels. In other cases an entire rophages through a peroxisome proliferator-activated recep sequence can include carbon and have a C14 content less tor gamma-dependent pathway. than found endogenously or less than atmospheric levels. 0193 In some embodiments, a ghrelin variant comprises Still, in other embodiments, a ghrelin or ghrelin variant a polypeptide comprising at least one modification to the molecule can be modified, for example to have an octanoyl natural form of an amino acid sequence of Gly-Ser-Ser-Phe or other like group, and that octanoyl group can have a C14 Leu-Ser-Pro-Ser-Gln-Lys-Pro-Gln-Asn-Lys-Val-Lys-Ser content less than endogenous ghrelin C14 levels or less than US 2017/O121385 A1 May 4, 2017

atmospheric levels. Further examples and embodiments are bioactivity, which occurs via interaction with the growth described below and elsewhere herein. hormone secretagogue receptor (GHSR). GOAT is respon 0.195. In some embodiments, ghrelin or ghrelin variant sible for this esterification. GOAT is a member of the can have a C14 content of less than 0.9 ppt, 0.95 ppt, 1.05 membrane-bound O-acyltransferase (mBOAT) family of ppt, 1.10 ppt. 1.15 ppt. 1.2 ppt or atmospheric content of membrane proteins. GOAT is a polytopic integral membrane C14. In some embodiments, ghrelin molecule can have a protein that octanoylates Ser3 of proghrelin in the endoplas C14 content that is from about 1% to 50% (or any value or mic reticulum (ER)lumen after signal peptide cleavage. In Sub range therein) less than the content of C14 in endog some embodiment, GOAT is up-regulated to increase the enous ghrelin or the content of atmospheric C14. For endogenous acylated ghrelin after mild brain injury or example, a molecule according to some embodiments can concussion. In some embodiments, the up-regulation of have about 5% to about 11% less C14 content. Ghrelin with GOAT is at the protein expression level. In some embodi C14 content of less than 0.9 ppt, 0.95 ppt, 1.0 ppt, 1.05 ppt. ments, the up-regulation of GOAT is at the mRNA expres 1.10 ppt, 1.15 ppt, 1.2 ppt or atmospheric content of C14, or sion level. with a lesser percentage of C14 as discussed herein, may be 0.197 In some embodiments, ghrelin or ghrelin variant obtained by peptide or chemical synthesis using reactants with C14 content of less than 0.9 ppt, 0.95 ppt, 1.0 ppt, 1.05 with carbons free of C14, less than 1 ppt C14 or deficient in ppt, 1.10 ppt. 1.15 ppt. 1.2 ppt or atmospheric content of C14 C14 relative to the atmospheric content of C14. Alterna (or having a percentage as discussed herein) may be tively, ghrelin or ghrelin variant with C14 content of less obtained following modification of the primary sequence of than 0.9 ppt, 0.95 ppt, 1.0 ppt, 1.05 ppt, 1.10 ppt, 1.15 ppt. ghrelin (SEQ ID NO. 1) with a fatty acid with a carbon 1.2 ppt or atmospheric content of C14 may be produced in content free of C14, substantially free of C14, less than 1 ppt vitro by enzymatic methods using starting materials with a C14 or deficient in C14 relative to the atmospheric content carbon content free of C14, substantially free of C14, less of C14. Such fatty acids may be chemically synthesized with than 1 ppt C14 or deficient in C14 relative to the atmospheric a carbon content free of C14, substantially free of C14, less content of C14. Such enzymatic methods may include than 1 ppt C14 or deficient in C14 relative to the atmospheric cell-free protein synthesis system or coupled in vitro tran content of C14 or produced in a cell cultured in a medium Scription-translation system based on cellular extracts pre wherein carbon source used to synthesize the fatty acid or pared from bacteria, yeast, wheat germ, insect and/or mam fatty acids is free of C14, substantially free of C14, less than malian cells using aminoacyl-tRNAS charged with amino 1 ppt C14 or deficient in C14 relative to the atmospheric acids with a carbon content free of C14, Substantially free of content of C14. In some embodiments, the fatty acid or fatty C14, less than 1 ppt C14 or deficient in C14 relative to the acids are conjugated to coenzyme A (CoA) and the fatty acid atmospheric content of C14. In an alternative method, in the resulting fatty acid-CoA thioesters is transferred to ghrelin or ghrelin variant with C14 content of less than 0.9 serine at amino acid position 3 of ghrelin by ghrelin O-acyl ppt, 0.95 ppt, 1.0 ppt, 1.05 ppt, 1.10 ppt, 1.15 ppt, 1.2 ppt or transferase (GOAT), so as to produce a fatty acid-modified atmospheric content of C14 may be produced by recombi ghrelin with a carbon content free of C14, substantially free nant methods in bacterial, yeast, insect and/or mammalian of C14, less than 1 ppt C14 or deficient in C14 relative to the cells following introduction of an expression system with a atmospheric content of C14. In some embodiments, fatty cDNA comprising ghrelin-encoded sequences and culturing acids are straight chain fatty acids with a carbon content of the cells in a medium with a carbon content free of C14, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, substantially free of C14, less than 1 ppt C14 or deficient in C15, C16, C17, C18, C19 or C20 and having a general C14 relative to the atmospheric content of C14. Alterna chemical formula of (CH)3-(CH2)n-2-COOH, wherein “n” tively, the medium may include glucose, galactose, Sugars, is the number of carbons in the fatty acid. In a preferred glycerol, pyruvate, acetates, metabolites, fatty acids, and/or embodiment, the fatty acid is a C8 octanoic acid or C14 amino acids with a carbon content free of C14, substantially tetradecanoic acid. In a more preferred embodiment, the free of C14, less than 1 ppt C14 or deficient in C14 relative fatty acid is octanoic acid and the fatty acid-modified ghrelin to the atmospheric content of C14. Methods for changing is octanoyl-modified ghrelin or ghrelin variant at serine stable isotopic content of proteins may be found in Becker amino acid position 3. et al., 2008 (G. W. Becker (2008) Stable isotopic labeling of 0.198. In some embodiments, fatty acid or fatty acids may proteins for quantitative proteomic applications. Briefings in be conjugated to ghrelin or ghrelin variant at serine amino Functional Genomics Proteins 7 (5): 371-382, which is acid position 3. In some embodiments, fatty acid or fatty incorporated herein by reference in its entirety) Ghrelin may acids may be conjugated to ghrelin or ghrelin variant at a be co-expressed with or exposed to ghrelin O-acyl trans position other than serine amino acid position 3. In some ferase (GOAT) to permit fatty acid modification of the embodiments, fatty acid or fatty acids may be conjugated to primary sequence of ghrelin or ghrelin variant at serine at ghrelin or ghrelin variant at serine amino acid position 2. In amino acid position 3 So as to produce a biologically active Some embodiments, fatty acid or fatty acids may be conju ghrelin or ghrelin variant capable of being bound and gated to ghrelin or ghrelin variant at serine amino acid activating the ghrelin receptor (GHSR-1a or growth hor position 2 and serine amino acid position 3. In some mone secretagogue receptor type 1a). The modification may embodiments, fatty acid or fatty acids may be conjugated to be an octanoic acid modification of ghrelin orghrelin variant ghrelin or ghrelin variant at one or more amino acids. So as to produce octanoyl-ghrelin with a carbon content free 0199. In some embodiments, fatty acid or fatty acids may of C14, substantially free of C14, less than 1 ppt C14 or be conjugated to immature ghrelin (such as preproghrelin or deficient in C14 relative to the atmospheric content of C14. proghrelin) and then fatty acid- or fatty acids-modified 0196. The biosynthesis of acyl-ghrelin involves a post ghrelin is processed to a mature ghrelin that can activate the translational octanoylation of the serine at the 3 position of ghrelin receptor (GHSR-1a). Processing of immature ghrelin the ghrelin peptide. This octanoylation is necessary for its may be in vitro or in vivo and may be carried out by US 2017/O121385 A1 May 4, 2017 26 proteolytic enzymes. In some embodiments, fatty acid or fatty acids may be conjugated to a mature ghrelin having the amino acid sequence as provided in SEQ ID NO. 1. NH 0200. In some embodiments, ghrelin with one or more O NH2 modifications is an isolated ghrelin with one or more modi CO O fications. In some embodiments, ghrelin with one or more modifications is an isolated ghrelin with one or more fatty N acid modifications. In some embodiments, ghrelin with one or more modifications is an isolated ghrelin acylated at serine 3 with octanoic acid, Such as an isolated octanoyl ghrelin. 0201 In some embodiments, C14-deficient starting mate rial used in the synthesis of ghrelin or ghrelin variant with a carbon content free of C14, substantially free of C14, less d than 1 ppt C14 or deficient in C14 relative to the atmospheric content of C14 may be obtained from carbon sources not 0205. In some embodiments, the ghrelin variant is L-692, participating in atmospheric carbon cycle or by fractionating 429, which is a compound of the formula: naturally occurring carbon isotope to obtain carbons free of C14, substantially free of C14, less than 1 ppt C14 or deficient in C14 relative to the atmospheric content of C14. Such carbons will be enriched in carbon-12 (C12) and/or NH NH2 carbon-13 (C13) and depleted of C14. Methods for isotope no 1)< fractionation, enrichment or depletion are known in the art N and may be based on diffusion, centrifugation, electromag O O netism, laser excitation, kinetic isotope effect, chemical CH methods, gravity, evaporation, and cryogenic distillation among many other methods of isotope fractionation. 0202 In some embodiments, other types of ghrelin vari N ants are mimetics, which include: peptidomimetics, small N e molecule mimetics and GHS-R agonists. A substantial num YM ber of ghrelin mimetics are known in the art. Non-limiting N examples of ghrelin mimetics include LY444711 and H LY426410 (Eli Lilly), hexarelin/examorelin (Diverse Aca demic), growth hormone releasing hexapeptide-1 (GHRP 1), GHRP-2, GHRP-6 (SK&F-110679), ipamorelin L-692,429 (MK-0731) (Helsinn), MK-0677, NN703, capromorelin(Pfizer), CP 464709 (Pfizer), pralmorelin (GHRP 2, GPA 748, growth hormone-releasing peptide 2, KP-102 D, KP-102 LN, 0206. In some embodiments, the ghrelin variant is NNC KP-102D, KP-102LN, Kaken Pharma, Sella Pharma), maci 26-0703 (or Tabimorelin, NN-703), which is a compound of morelin (Aeterna Zentaris), anamorelin (Helsinn), relamor the formula: elin (Rhythm), ulimorelin (Tranzyme), ipamorelin ((NNC 2601.61, Helsinn), Tabimorelin (Novo Nordisk), ibutamoren (Merck), G7039, G7134, G7203, G7502, SM-130686 (Sumitomo), RC-1291, L-692429, L-692587, L-739943, L-163255, L-163540, L-163833, L-166446, CP-424391, EP-51389, NNC-26-0235, NNC-26-0323, NNC-26-0610, NNC 26-0703, NNC-26-0722, NNC-26-1089, NNC-26 136, NNC-26-1137, NNC-26-1187, NNC-26-1291, O O MK-0677, L-692,429, EP 1572, L-252,564, NN703, S-37435, EX-1314, PF-5190457, AMX-213, and macrocy "> -sul clic compounds (U.S. Publication No. 20060025566, which N r HN1 is incorporated herein by reference in its entirety). See also O Smith, 2005, Endo. Rev. 26:346-360 (incorporated herein by reference in its entirety) for information on developing ghrelin mimetics. In some embodiments, one or more of the variants listed above, can be specifically excluded. 0203. In some embodiments, the ghrelin variant is 0207. In some embodiments, the ghrelin variant is Ape LY444711 (Eli Lilly), which is a compound of the chemical Ser(Octyl)-Phe-Leu-aminoethylamide. In some embodi nomenclature: 2-(2-Amino-2-methyl-propionylamino)-5- ments, the ghrelin variant is Capromorelin (CP-424,391), phenyl-pentanoic acid 1-1-(4-methoxy-phenyl)-1-methyl which is a compound of the chemical nomenclature: (3aR)- 2-oxo-2-pyrrolidin-1-yl-ethyl)-1H-imidazol-4-yl)-amide. 3a-benzyl-2-methyl-5-(2-methylalanyl-O-benzyl-D-seryl)- 0204. In some embodiments, the ghrelin variant is 3-oxo-3,3a,4,5,6,7-hexahydro-2H-pyrazol o4.3-cpyridine. MK-0677 (or L-163,191), which is a compound of the 0208. In some embodiments, the ghrelin variant is L-252, formula: 564, which is a compound of the chemical nomenclature: US 2017/O121385 A1 May 4, 2017 27

2-(4-3-(4,5-Dichloro-2-methylphenyl)-4,5-dihydro-1H pyrazol-1-yl)phenylsulfonyl)ethyl acetate, and the for mula:

H

NF-NH CEO O N N H N HN

0213 U.S. Pat. No. 8,192,719, which is incorporated 2. L-262,564 herein by reference in its entirety for all of its materials, compositions of matter, methods of use and methods of making, discloses various compounds, including macimo 0209. In some embodiments, the ghrelin variant is relin, which can be utilized in the embodiments herein. S-37435 (Kaken), which is a compound of the chemical 0214. In some embodiments, the ghrelin variant is nomenclature: N-1 (R)-N-(3-Amino-2-hydroxypropyl)car anamorelin, which has the molecular formula, bamoyl-2-naphthylethyl-4-(4-oxo-2,3,4,5-tetrahydro-1,5- CHCINO, and the following structure: benzothiazepin-5-yl)butyramide hydrochloride. In some embodiments, the ghrelin variant is G-7203 (Genentech). In some embodiments, the ghrelin variant is SM-130868 (Sumitomo). In some embodiments, the ghrelin variant is EX-1314. C -N2. O 0210. In some embodiments, the ghrelin variant is uli morelin which has the molecular formula, CoHFN.O. and the following structure: O N O

H-CI N HN H 21

Hn

0215. In some embodiments, the ghrelin variant is ipamo relin, which has the molecular formula, CsPNOs, and the following structure:

HN

O

O NH2 HN O NH

0211 U.S. Pat. No. 7,491.695, which is incorporated s H herein by reference in its entirety for all of its materials, NH N O CO N compositions of matter, methods of use and methods of H making, discloses various macrocyclic compounds, includ O ing ulimorelin. 1N 0212. In some embodiments, the ghrelin variant is maci \ NH morelin, which has the molecular formula, CHNO. and the following structure: US 2017/O121385 A1 May 4, 2017 28

0216. In some embodiments, the ghrelin variant is mitochondria. In some embodiments, the ghrelin variant PF-5190457, which has the following structure: prevents the metabolic consequence of mBI and any asso ciated chronic conditions. 0220. In some embodiments, the ghrelin variant has at least about 50% of the functional activity of ghrelin. In some embodiments, the functional activity comprises one or more of feeding regulation, nutrient absorption, gastrointestinal motility, energy homeostasis, anti-inflammatory regulation, Suppression of inflammatory cytokines, activation of s Gq/G11, accumulation of inositol phosphate, mobilization of calcium from intracellular stores, activation or deactiva tion of MAP kinases, NFKB translocation, CRE driven gene transcription, binding of arrestin to ghrelin receptor, reduc tion in ROS, NAMPT enzyme activation, or a combination thereof. 0221 Receptor activity can be measured using different techniques such as detecting a change in the intracellular conformation of the receptor, in the G-protein coupled activities, and/or in the intracellular messengers. One simple measure of the ability of a ghrelin variant-like compound to activate the ghrelin variant receptor is to measure its ECso i.e. the dose at which the compound is able to activate the 16h MW/eLogD/TPSA signaling of the receptor to half of the maximal effect of the 5121.5/95 compound. When measuring, e.g., ECso, the receptor can human ghrelin receptor either be expressed endogenously on primary cell cultures, binding pKi 8.36 for example pituitary cells, or heterologously expressed on GTPYS assay pKi 8.18 cells transfected with the ghrelin receptor. Whole cell assays (Inverse Agonist) or assays using membranes prepared from either of these cell types can be used depending on the type of assay. In 0217. In some embodiments, the ghrelin variant binds to Some embodiments, the ghrelin variant has an ECso potency the growth hormone secretagogue receptor GHS-R 1 a on the GHSR of less than 500 nM. In some embodiments, (GHSR). The ghrelin variant compounds described herein the ghrelin variant has a dissociation constant from the are active at the receptor for growth hormone secretagogue GHSR of less than 500 nM. (GHS), e.g., the receptor GHS-R 1a. The compounds can 0222 Aghrelin variant compound has at least about 50%, bind to GHS-R 1a, and stimulate receptor activity. In some at least about 55%, at least about 60%, at least about 65%, embodiments, the compounds can bind other receptors and, at least about 70%, at least about 75%, at least about 80%, optionally, stimulate their activity. at least about 85%, at least about 90%, or at least about 95%, 0218. Ghrelin variants, in some embodiments, can acti functional activity relative to the 28 amino acid human vate the GHS receptors and additional yet to be identified wild-type ghrelin as determined using an assay described receptors. These receptors are found on GH producing cells, herein, and/or an EC50 greater than about 1,000, greater in the hypothalamic centers and in a number of additional than about 100, or greater than about 50, or greater than places in the organism. In the CNS, these receptors are tuned about 10. Greater refers to potency and thus indicates a to receiving signals from neurons containing local molecules lesser amount is needed to achieve binding inhibition. (e.g., ghrelin variants). Peripherally-secreted or artificially 0223) In some embodiments, the ghrelin variant has administered ghrelin variants and combination products potency (EC50) on the GHS-R 1a of less than 500 nM. In (including fusion therapeutic products) as described herein Some embodiments, the ghrelin variant has a potency can reach Such sites and pass the blood brain barrier spe (EC50) on the GHS-R 1a of less than 100 nM, such as less cifically activating the appropriate receptors and triggering a than 80 nM, such as less than 60 nM, such as less than 40 specific pathway. GH secretagogues, which are Small nM, such as less than 20 nM, such as less than 10 nM, such organic compounds such as MK-0677 (Merck), generally as less than 5 nM. Such as less than 1 nM. Such as less than target to bind the GHS receptor will pass the blood brain 0.5 nM, such as less than 0.1 nM, such as less than 0.05 nM, barrier and also reach these sites, activating various GHS such as less than 0.01 nM. receptor related pathways and consequently having the 0224. In some embodiments, the dissociation constant danger of causing unwanted side effects Such as dizziness, (Kd) of the ghrelin variant is less than 500 nM. In some nausea, falling, elevated fasting serum glucose and insulin, embodiments, the dissociation constant (Kd) of the ghrelin and blurred vision. Such compounds which do have the variant is less than 100 nM, such as less than 80 nM, such advantage of being, for example, orally active. Other ghrelin as less than 60 nM, such as less than 40 nM, such as less than variants, or homologues thereof, can be administered periph 20 nM, such as less than 10 nM, such as less than 5 nM, such erally to ensure that only the relevant, appetite-regulating as less than 1 nM, such as less than 0.5 nM, such as less than ghrelin splice variant receptors and pathways are reached 0.1 nM, such as less than 0.05 nM, such as less than 0.01 and stimulated. nM 0219. In some embodiments, the ghrelin variant increases 0225 Binding assays can be performed using recombi uncoupling protein-2 (UCP-2) expression. In some embodi nantly-produced receptor polypeptides present in different ments, the ghrelin variant increases UCP-2 expression in environments. Such environments include, for example, cell US 2017/O121385 A1 May 4, 2017 29 extracts and purified cell extracts containing the receptor Subject is a monkey, cow, goat, sheep, mouse, rat, cat, dog, polypeptide expressed from recombinant nucleic acid or horse, hamster, pig, fish or chicken. naturally occurring nucleic acid, and also include, for 0231. In some embodiments, an intravenous injection of example, the use of a purified GHS receptor polypeptide ghrelin or the ghrelin variant is employed. The administra produced by recombinant means or from naturally occurring tion route must ensure that the non-degraded, bioactive form nucleic acid which is introduced into a different environ of the peptide will be the dominating form in the circulation, ment. which will reach and stimulate the ghrelin receptors in order 0226. Using a recombinant GHS receptor offers several to obtain the maximum effect of ghrelin/ghrelin variant advantages, such as the ability to express the receptor in a treatment on mBI. In some embodiments, ghrelin or the defined cell system, so that a response to a compound at the ghrelin variant is administered within about 30 minutes of receptor can more readily be differentiated from responses at the incident that results in the neurodegenerative condition. other receptors. For example, the receptor can be expressed In some embodiments, ghrelin or the ghrelin variant is in a cell line such as HEK 293, COS 7, and CHO not administered within about 30 minutes to about 2 hours of the normally expressing the receptor by an expression vector, incident that results in the neurodegenerative condition. In wherein the same cell line without the expression vector can Some embodiments, ghrelin or the ghrelin variant is admin act as a control. istered within about 30 minutes to about 6 hours of the 0227. In some embodiments, the ghrelin variant is incident that results in the neurodegenerative condition. In coupled to a protein that extends the serum half-lives of the Some embodiments, ghrelin or the ghrelin variant is admin ghrelin variant. In some embodiments, the protein is a long, istered within about 30 minutes to about 12 hours of the hydrophilic, and unstructured polymer that occupies a larger incident that results in the neurodegenerative condition. In Volume than a globular protein containing the same number Some embodiments, the ghrelin variant is administered of amino acids. within about 30 minutes to about 24 hours of the incident 0228. In some embodiments, the protein comprising the that results in the neurodegenerative condition. sequence of XTENTM (SEQID NO. 7). XTENTM, is a long, 0232 A typical dosage is in a concentration equivalent to hydrophilic, and unstructured polymer that occupies a much 10 ng to 10 mg ghrelin variant per kg bodyweight. The greater Volume than any globular protein containing the concentrations and amounts herein are given in equivalents same number of amino acids. When attached to molecules of of amount ghrelin variant, wherein the ghrelin variant is a 28 interest, XTENTM greatly increases their effective size, amino acid human ghrelin (SEQ ID NO: 1) and/or a 24 thereby prolonging their presence in serum by slowing amino acid human ghrelin splice variant having a Dpr kidney clearance in a manner analogous to that of polyeth residue at the third position (SEQ ID NO:3) and/or a 24 ylene glycol (PEG). In addition to slowing kidney clearance, amino acid human ghrelin splice variant having Dpr residues attachment to XTENTM can also inhibit receptor-mediated at the second and third positions (SEQ ID NO:4) and being clearance by reducing the ligands affinity for its receptor. optionally octanoylated on the Dpr residue in the third Such an effect is not accomplished by fusion to other position. half-life extension technologies like HSA or Fc. Thus, 0233. In some embodiments, ghrelin variants are admin XTENTM acts through multiple mechanisms to affect drug istered in a concentration equivalent to from about 0.1 ug to concentration, resulting in long half-lives and monthly dos about 1 mg ghrelin variant per kg bodyweight, Such as from ing. Proteins and peptides can be produced as recombinant about 0.5 g to about 0.5 mg ghrelin variant per kg body fusions with XTENTM, the length of which can be modified weight, such as from about 1.0 g to about 0.1 mg ghrelin to reach the desired pharmacokinetic properties. XTENTM variant per kg bodyweight, Such as from about 1.0 ug to also enhances the solubility of attached molecules typically about 50 ugghrelin variant per kg bodyweight, such as from permitting liquid formulation of drugs that otherwise would about 1.0 ug to about 10 ug ghrelin variant per kg body be lyophilized. weight. In some embodiments, about 10 ug ghrelin powder is reconstituted in about 100 uL of a sterile saline solution Treatment of a Neurodegenerative Condition and Other before administration. In some embodiments, the sterile Neurological Disorders saline Solution is contained in an IV bag for ease of delivery. 0234. In some embodiments, a ghrelin or ghrelin variant 0229. The present disclosure is directed to the identifi is used in an assay to assess its effect at reducing expression cation of a novel use for a ghrelin variant in treating a level of Tau protein, or reducing the level of Tau phospho neurodegenerative condition. The present disclosure pro rylation. vides for a method of treating a neurodegenerative condition 0235. In some embodiments, ghrelin is used as a control in a Subject, comprising administering to the Subject (e.g., a to determine the relative efficacy of the candidate compound Subject that has a mBI) an effective amount of a compound or compounds. Suitable assays include by way of example comprising a ghrelin variant. The ghrelin variant can be only competitive assays for binding of a candidate com administered for the purpose of treating a neurodegenerative pound or compounds to growth hormone secretagogue condition in a therapeutically effective amount. In some receptor la (i.e., GHSR) in the presence of ghrelin as well embodiments, the methods can further include selecting or as frontal affinity chromatography. identifying a Subject that has suffered, is at risk of Suffering, 0236 Any competitive binding assay known in the art is is prone to Suffer, and/or is about to participate in an activity applicable for binding of a candidate compound or com with a high risk for Suffering, a neurodegenerative condition, pounds to growth hormone secretagogue receptor in the prior to administration of the ghrelin variant. presence of ghrelin, using either heterogeneous or homoge 0230. In some embodiments, the subject that undergoes neous methods, with one or more reagents, and with labels the method of treatment is a mammal. In some embodi and detection methods. By way of non-limiting example, ments, the Subject is a human. In some embodiments, the detection methods may include radioactive methods; US 2017/O121385 A1 May 4, 2017 30 enzyme techniques using intact enzymes of many types JOURNAL OF NEUROTRAUMA 31:1072-1075 (Jun. 1, including, for example, B-galactosidase, glucose 6-phos 2014)), radiology imaging, self-reporting, accelerometers phate dehydrogenase, alkaline phosphatase, horseradish per (for example, in helmets). Following a diagnosis of mild oxidase, or glucose oxidase; techniques using enzyme frag brain injury, Such as a concussion, ghrelin or a ghrelin ments, such as B-galactosidase complementation assays: variant can be administered to the patient, preferably in not detection systems including chromogenic Substrates; fluo more than 72 hours initially. rescent methods detected by direct fluorescence, time-re 0241 This invention also provides for methods for mea Solved fluorescence, fluorescence polarization, or fluores Suring ghrelin levels before starting a sport or activity, for cence energy transfer, and chemical or bioluminescence example prior to the beginning of football season (or any detection systems. other sport or activity, including those listed elsewhere 0237. In some embodiments, frontal affinity chromatog herein), and monitoring ghrelin levels during the season to raphy (FAC) can be used for screening of compound librar ascertain if the player or participant is at a level not qualified ies. The basic premise of FAC is that continuous infusion of to play or participate. The methods can include the use of a compound will allow for equilibration of the ligand any suitable measurement or assay technique for measuring between the free and bound states, where the precise con ghrelin levels, such as from blood to determine if blood centration of free ligand is known. The detection of com levels correlate to brain levels. pounds eluting from the column can be accomplished using 0242. With the benefit of the instant embodiments, the methods such as fluorescence, radioactivity, or electrospray skilled artisan can select any suitable technique for measur mass spectrometry. The former two methods usually make ing ghrelin levels. A number of assays known in the art for use of either a labeled library, or use a labeled indicator measuring a protein or hormone level are applicable for compound, which competes against known unlabeled com measuring ghrelin levels. By way of non-limiting example, pounds, getting displaced earlier if a stronger binding ligand assays such as a blood Sugar test by extracting a drop of is present. blood and putting it into a device, can quantitatively assess 0238. In some embodiments, a patient suffering loss of the amount of ghrelin, or an assay involving measuring a cognitive or motor skills due to mBI and, in particular, range of Substances whereby a specific reaction chemistry is repetitive mBI, can be monitored for therapy or progression followed photo-metrically with time, for example by utiliz of such skills by correlating the ghrelin level in the patients ing an antibody specific to ghrelin that is coated onto latex brain over time. As the ghrelin levels decrease, there will be particles and measuring the increased turbidity that is pro an increased need for intervention. duced when ghrelin being measured promotes aggregation 0239. The present disclosure provides for a method of of the latex particles as the reaction between ghrelin and treating mBI in a subject, comprising administering to the anti-ghrelin antibody proceeds. This measurement of Subject an effective amount of a compound comprising the increasing turbidity can be achieved using a conventional ghrelin variant that is encoded by or administered as a photometer and using the associated Scientific principles of nucleic acid. In some embodiments, the nucleic acid is any photometric measurements. Such concentration dependent that encodes the sequence of SEQ ID NO.1. In some turbidity is then compared to that produced by standards embodiments, the nucleic acid sequence comprises 5'-ggctic which are established in the art. Cagct tcCtgagcCC tigaacaccag agagtCcagc agagaaagga gtC 0243 Further compatible, but non-limiting, methodolo gaagaag ccaccagcca agctgcagcc ccga-3' (SEQID NO. 8). In gies include carrying out a series of enzyme-linked reactions Some embodiments, the ghrelin variant is encoded by a in Solution, where ghrelin in the plasma fraction of a whole nucleic acid sequence comprising SEQ ID NO. 8 with one blood sample is altered by an enzyme-promoted reaction to or more mutations. In some embodiments, the mutation is ultimately derive a colored dye from colorless reaction selected from the group consisting of nucleic acid insertion, constituents. The color is developed in a time dependent way deletion, Substitution and translocation. In some embodi and monitored photo-metrically. This measurement of color ments, the mutation occurs at one or more positions. change can also be achieved using a conventional photom 0240 Some embodiments relate to methods of treating eter using the associated Scientific principles of photometric mild brain injury or concussion or reducing the severity or measurements. Such concentration dependent change in duration of one or more symptoms or characteristics of the transmission is then compared to that produced by stan injury by utilizing the methods and compounds described dards. herein in combination with one or more diagnostic devices 0244. In whole blood samples, hematocrit or percentage or protocols, or with one or more recovery protocols. For of red blood cells by volume in the whole blood sample is example, a potential brain injury can be diagnosed and/or a variable can be taken into account when analyzing ghrelin monitored utilizing the BTrackSTM System (http://balance levels that are present in the plasma component. As the trackingsystems.com/. Balance Tracking Systems Inc.), uti hematocrit of a patient’s blood rises, so the volume of lizing the NFL Concussion Tool, 'sports concussion assess plasma in a fixed volume sample, which is introduced into ment tool” (“SCAT-2: http://static.nfl.com/static/content/ the test device decreases and vice versa. Since it is the public/photo/2014/02/20/0ap2000000327062.pdf) or other plasma component which exclusively carries the ghrelin similar tools utilized by the NHL, the NBA, FIFA, Rugby levels being measured, then the lower the volume of plasma leagues and unions, boxing organizations, etc. Examples component added to the reaction mix, the lower the resulting include, SCAT-3, ImPACTICD-10, nPITEST, acute concus concentration of the Substance being measured in that reac sion evaluation (“ACE), King-Devick, and the like. Other tion mix and the resulting assayed value and vice versa. diagnostics or assessments can utilize serum biomarkers 0245 Any analysis that produces a concentration of a (Glial Fibrilliary Acid Protein (GFAP); see for example, plasma substance in whole blood may be corrected for Mannix et al., “Serum Biomarkers Predict Acute Symptom variations in hematocrit to give a true plasma concentration. Burden in Children after Concussion: A Preliminary Study.” It can be most useful in these situations to measure two US 2017/O121385 A1 May 4, 2017

Substances, one of which is ghrelin under investigation and cell myelin protein; Glia Scar: Tenascin; Hippocampus: the other which is considered to be a marker by which to Stathmin, Hippocalcin, SCG10; Cerebellum: Purkinje cell estimate or normalize the sample hematocrit. The hemoglo protein-2 (Pcp2), Calbindin D9K, Calbindin D28K (NP bin concentration of whole blood, after red blood cells are 114190), Cerebellar CaBP spot 35: Cerebrocortex: Cor lysed, is directly proportional to the red blood cell volume texin-1 (P60606), H-271 gene product; Thalamus: CD15 in the whole blood sample. (3-fucosyl-N-acetyl-lactosamine) epitope; Hypothalamus: 0246 Pharmacodynamic measurements of Protein Bio Orexin receptors (OX-1R and OX-2R)-appetite, Orexins markers and micro-RNA screening are performed. A blood (hypothalamus-specific peptides); Corpus callosum: MBP. sample is obtained to measure the levels of protein biomark MOG, PLP, MAG; Spinal Cord. Schwann cell myelin pro ers (e.g., SBDP150, S100, GFAP, and UCH-L1) that are tein; Striatum: Striatin, Rhes (Ras homolog enriched in derived from the cytosol of cells, such as but not limited to striatum); Peripheral ganglia. Gadd45a; Peripheral nerve neurons, astrocytes, and axons. Micro-RNA (mi-RNA) lev fiber (sensory+motor): Peripherin, Peripheral myelin protein els are also measured. The blood samples are obtained on 22 (AAH91499); Other Neuron-specific proteins: PH8 (S Days 0, 1, 2, 3, 4, 5, 6, 7, 14, 21, and 28 days. Serotonergic Dopaminergic, PEP-19, Neurocalcin (NC), a 0247 The present technology provides biomarkers that neuron-specific EF-hand Ca"-binding protein, Encephalop are indicative of mild brain injury or concussion, neuronal sin, Striatin, SG2NA, Zinedin, Recoverin, Visinin; Neu damage, neural disorders, brain damage, neural damage, and rotransmitter Receptors: NMDA receptor subunits (e.g. diseases associated with the brain or nervous system, Such as NR1A2B), Glutamate receptor subunits (AMPA, Kainate the central nervous system. In some embodiments, the receptors (e.g. GluR1, GluR4), beta-adrenoceptor subtypes biomarkers are proteins, fragments or derivatives thereof, (e.g. beta(2)), Alpha-adrenoceptors Subtypes (e.g. alpha(2c)), and are associated with neuronal cells, brain cells or any cell GABA receptors (e.g. GABA(B)). Metabotropic glutamate that is present in the brain and central nervous system. In receptor (e.g. mGluR3), 5-HT serotonin receptors (e.g. 5-HT Some embodiments, the biomarkers are neural proteins, (3)), Dopamine receptors (e.g. D4), Muscarinic Ach recep peptides, fragments or derivatives thereof. Examples of tors (e.g. M1), Nicotinic Acetylcholine Receptor (e.g. alpha neural proteins, include, but are not limited to axonal 7); Neurotransmitter Transporters: Norepinephrine proteins, amyloid precursor protein, dendritic proteins, Transporter (NET), Dopamine transporter (DAT), Serotonin Somal proteins, presynaptic proteins, post-synaptic proteins transporter (SERT), Vesicular transporter proteins (VMAT1 and VMAT2), GABA transporter vesicular inhibitory amino and neural nuclear proteins. acid transporter (VIAAT/VGAT), Glutamate Transporter 0248. In some embodiments, the biomarker is one or (e.g. GLT1), Vesicular acetylcholine transporter, Vesicular more of, but not limited to, Axonal Proteins: C. II spectrin Glutamate Transporter 1, VGLUT1, BNP1 and VGLUT2, (and SPDB)-1, NF-68 (NF-L)-2, Tau-3, C. II, III spectrin, Choline transporter, (e.g. CHT1); Cholinergic Biomarkers: NF-200 (NF-H), NF-160 (NF-M), Amyloid precursor pro Acetylcholine Esterase, Choline acetyltransferase (Ch.AT): tein, C. internexin: Dendritic Proteins: beta III-tubulin-1, p24 Dopaminergic Biomarkers: Tyrosine Hydroxylase (TH). microtubule-associated protein-2, alpha-Tubulin (P02551), Phospho-TH, DARPP32, Noradrenergic Biomarkers: Dop beta-Tubulin (P04691), MAP-2A/B-3, MAP-2C-3, Stath amine beta-hydroxylase (DbH); Adrenergic Biomarkers: min-4, Dynamin-1 (P21575), Phocein. Dynactin (Q13561). Phenylethanolamine N-methyltransferase (PNMT); Sero Vimentin (P31000), Dynamin, Profilin, Cofilin 12, Somal tonergic Biomarkers: Tryptophan Hydroxylase (TrEI); Glu Proteins: UCH-L1 (O00981)-1, Glycogen phosphorylase tamatergic Biomarkers: Glutaminase, Glutamine synthetase; BB-2, PEBP (P31044), NSE (P07323), CK-BB (P07335), Thy 1.1, Prion protein, Huntingtin, 14-3-3 proteins (e.g. GABAergic Biomarkers: GABA transaminase (GABAT)), 14-3-3-epsolon (P42655)), SM22-C., Calgranulin AB, alpha GABA-B-R2, or a combination thereof. Synuclein (P37377), beta-Synuclein (Q63754), HNP 22: 0249. In some embodiments, the biomarkers comprise at Neural nuclear proteins: NeuN-1, S/G(2) nuclear autoanti least one biomarker from each neural cell type including, but gen (SG2NA), Huntingtin; Presynaptic Proteins: Synapto not limited to, ö 11 spectrin, SPDB-1, NF-68, NF-L-2, physin-1, Synaptotagmin (P21707), Synaptojanin-1 Tau-3, BIII-tubulin-1, p.24 microtubule-associated protein-2, (Q62910), Synaptojanin-2, Synapsin1 (Synapsin-1a). Syn UCH-L1 (O00981)-1, Glycogen phosphorylase-BB-2, apsin2 (Q63537), Synapsin3. GAP43, Bassoon(NP NeuN-1, Synaptophysin-1, synaptotagmin (P21707), Syn 003449), Piccolo (aczonin) (NP 149015), Syntaxin, aptojanin-1 (Q62910), Synaptojanin-2, PSD95-1, NMDA CRMP1, 2, Amphiphysin-1 (NP 001626), Amphiphysin-2 receptor-2 and subtypes, myelin basic protein (MBP) and (NP 647477): Post-Synaptic Proteins: PSD95-1, NMDA fragments, GFAP (P47819), Iba1, OX-42, OX-8, OX-6, receptor (and all subtypes)-2, PSD93, AMPA-kainate recep ED-1, Schwann cell myelin protein, tenascin, stathmin, tor (all subtypes), mGluR (all subtypes), Calmodulin depen Purkinje cell protein-2 (Pcp2), Cortexin-1 (P60606), Orexin dent protein kinase II (CAMPK)-alpha, beta, gamma, receptors (OX-1R, OX-2R), Striatin, Gadd45a, Peripherin, CaMPK-IV. SNAP-25, a-?b-SNAP, Myelin-Oligodendro peripheral myelin protein 22 (AAH91499), and Neurocalcin cyte: Myelin basic protein (MBP) and fragments, Myelin (NC). proteolipid protein (PLP), Myelin Oligodendrocyte specific 0250 In some embodiments, the biomarkers are spectrin, protein (MOSP), Myelin Oligodendrocyte glycoprotein BII-spectrin and BII-spectrin breakdown products (BII-SE (MOG), myelin associated protein (MAG), Oligodendrocyte DPs) generated by calpain-2 and/or caspase-3 proteolysis. NS-1 protein; Glial Protein Biomarkers: GFAP (P47819), 0251. In some embodiments, at least one biomarker, such Protein disulfide isomerase (PDI) P04785, Neurocalcin as a protein, peptide, variant or fragment thereof, is used to delta, S100beta; Microglia protein Biomarkers: Iba1, detect a neural injury, neuronal disorder or neurotoxicity in OX-42, OX-8, OX-6, ED-1, PTPase (CD45), CD40, CD68, a subject, wherein said at least one biomarker is BII-spectrin, CD11b, Fractalkine (CX3CL1) and Fractalkine receptor BII-SBDP-80, BII-SBDP-85, B-SBDP-108, or BII-SBDP (CX3CR1), 5-d-4 antigen; Schwann cell markers: Schwann 110. US 2017/O121385 A1 May 4, 2017 32

0252. In some embodiments, a plurality of biomarkers, prion protein, 14-3-3 proteins; neural nuclear proteins, such Such as proteins, peptides, variant or fragment thereof, is as for example S/G(2) nuclear autoantigen (SG2NA), NeuN. used to detect a neural injury, neuronal disorder or neuro Thus, detection of specific biomarkers will determine the toxicity in a Subject, where said plurality biomarker is extent and Subcellular location of injury. BII-spectrin, BII-SBDP-80, BII-SEDP-85, BII-SBDP-108, 0257. In some embodiments, biomarkers specific for dif BII-SEBDP-110 or combinations thereof. In some embodi ferent anatomical regions, different cell types, and/or differ ments, the biomarks are as disclosed in US 2014/0024053, ent subcellular structures of a cell are selected to provide which disclosure is hereby incorporated by reference in its information as to the location of anatomical injury, the entirety. location of the injured cell type, and the location of injury at 0253. In some embodiments, at least one biomarker or a a subcellular level. A number of biomarkers from each set plurality of biomarkers, such as a protein, peptide, variant or can be used to provide highly enriched and detailed infor fragment thereof, is used to detect a neural injury, neuronal mation of mechanism, mode and Subcellular sites of injury, disorder or neurotoxicity in a Subject, wherein said at least anatomical locations of injury and status of different cell one biomarker is microtubule-associated proteins (MAPs), types in the nervous system (neuronal Subtypes, neural stem MAP-2 (e.g., MAP-2A, MAP-2B, MAP-2C, MAP-2D), cells, astro-glia, oligodendrocyte and microglia cell). MAP breakdown products (MAP-BDP) or combinations 0258. In some embodiments, subcellular neuronal bio thereof. In some embodiments, the biomarks are as disclosed markers for diagnosis and detection of mild brain injury or in US 2014/0018299, which disclosure is hereby incorpo concussion are at least one or more of axonal proteins, rated by reference in its entirety. dendritic proteins, Somal proteins, neural nuclear proteins, 0254. In some embodiments, an expanded panel of bio presynaptic proteins, post-synaptic proteins, or a combina markers is used to provide highly enriched information of tion thereof. mechanism of injury, modes of cell death (necrosis versus 0259. In some embodiments, axonal proteins identified as apoptosis), sites of injury, sites and status of different cell biomarkers for diagnosis and detection of mild brain injury types in the nervous system and enhanced diagnosis (better or concussion include, but not limited to, C. II spectrin (and selectivity and specificity). In some embodiments, the bio SPDB)-1, NF-68 (NF-L)-2, Tau-3, C.II, III spectrin, NF-200 markers are selected to distinguish between different host (NF-M), NF-160 (NF-M), Amyloid precursor protein, C. anatomical regions. For example, at least one biomarker can internexin, peptides, fragments or derivatives thereof. be selected from neural subcellular protein biomarkers, nervous System anatomical markers such as hippocampus 0260. In some embodiments, dendritic proteins identified protein biomarkers and cerebellum protein biomarkers. as biomarkers for diagnosis and detection of mild brain Examples of neural subcellular protein biomarkers are injury or concussion include, but not limited to, beta III NF-200, NF-160, and NF-68. Examples of hippocampus tubulin-1, p24 microtubule-associated protein-2, alpha-Tu protein biomarkers are SCG10 and stathmin. An example of bulin (P02551), beta-Tubulin (P04691), MAP-2A/B-3, a cerebellum protein biomarker is Purkinje cell protein-2 MAP-2C-3, Stathmin-4, Dynamin-1 (P21575), Phocein, (Pcp2). Dynactin (Q13561), Vimentin (P31000), Dynamin, Profilin, Cofilin 1.2, peptides, fragments or derivatives thereof. In 0255. In some embodiments, the biomarkers are selected Some embodiments, neural nuclear proteins identified as to distinguish between mild brain injury or concussion at the biomarkers for diagnosis and detection of mild brain injury cellular level, thereby detecting which cell type has been or concussion include, but not limited to, NeuN-1, S/G(2) injured. For example at least one biomarker protein is nuclear autoantigen (SG2NA), Huntingtin, peptides or frag selected from a representative panel of protein biomarkers specific for that cell type. Examples for biomarkers specific ments thereof. for cell types include myelin-oligodendrocyte biomarkers 0261. In some embodiments, somal proteins identified as such as myelin basic protein (MBP), myelin proteolipid biomarkers for diagnosis and detection of mild brain injury protein (PLP), myelin oligodendrocyte specific protein or concussion include, but not limited to, UCH-L1 (MOSP), oligodendrocyte NS-1 protein, myelin oligoden (Q00981)-1, Glycogen phosphorylase-BB-2, PEBP drocyte glycoprotein (MOG) Examples of biomarkers spe (P31044), NSE (P07323), CK-BB (P07335), Thy 1.1, Prion cific for Schwann cells include, but not limited to Schwann protein, Huntingtin, 14-3-3 proteins (e.g. 14-3-3-epsolon cell myelin protein. Examples of Glial cell protein biomark (P42655)), SM22-C., Calgranulin AB, alpha-Synuclein ers include, but not limited to GFAP (protein accession (P37377), beta-Synuclein (Q63754), HNP 22, peptides, number P47819), protein disulfide isomerase (PDI)— fragments or derivatives thereof. P04785. Thus, by detecting one or more specific biomarkers 0262. In some embodiments, presynaptic proteins iden the specific cell types that have been injured can be deter tified as biomarkers for diagnosis and detection of mild brain mined. injury or concussion include, but not limited to, Synapto 0256 In some embodiments, biomarkers specific for dif physin-1, Synaptotagmin (P21707), Synaptojanin-1 ferent subcellular structures of a cell can be used to deter (Q62910), Synaptojanin-2, Synapsin1 (Synapsin-1a). Syn mine the subcellular level of injury. Examples include but apsin2 (Q63537), Synapsin3. GAP43, Bassoon (NP not limited to neural Subcellular protein biomarkers such as, 003449), Piccolo (aczonin) (NP 149015), Syntaxin, NF-200, NF-160, NF-68; dendritic biomarkers such as for CRMP1, 2, Amphiphysin-1 (NP 001626), Amphiphysin-2 example, alpha-tubulin (P02551), beta-tubulin (P04691), (NP 647477), peptides, fragments or derivatives thereof: MAP-2A/B, MAP-2C, Tau, Dynamin-1 (P212575), Phoe 0263. In some embodiments, post-synaptic proteins iden cin, Dynactin (Q13561), p24 microtubule-associated pro tified as biomarkers for diagnosis and detection of mild brain tein, vimentin (P131000); somal proteins such as for injury or concussion include, but not limited to, PSD95-1, example, UCH-L1 (Q00981), S100, SBDP150, GFAP, NMDA-receptor (and all subtypes)-2, PSD93, AMPA-kain PEBP (P31044), NSE (P07323), CK-BB (P07335), Thy 1.1, ate receptor (all subtypes), mGluR (all subtypes), Calmo US 2017/O121385 A1 May 4, 2017 dulin dependent protein kinase II (CAMPK)-alpha, beta, 0267 Demyelination proteins identified as biomarkers gamma, CaMPK-IV. SNAP-25, a-?b-SNAP, peptides, frag for diagnosis and detection of mild brain injury or concus ments or derivatives thereof. sion are, but not limited to: myelin basic protein (MBP). 0264. In some embodiments, identified biomarkers dis myelin proteolipid protein, peptides, fragments or deriva tinguish the damaged neural cell Subtype such as, for tives thereof. In some embodiments, glial proteins identified example, myelin-oligodendrocytes, glial, microglial, as biomarkers for diagnosis and detection of mild brain Schwann cells, glial scar. In some embodiments, Myelin injury or concussion are, but not limited to: GFAP (P47819), Oligodendrocyte biomarkers are, but not limited to: Myelin protein disulfide isomerase (PDI-P04785), peptides, frag basic protein (MBP) and fragments, Myelin proteolipid ments and derivatives thereof. protein (PLP), Myelin Oligodendrocyte specific protein 0268. In some embodiments, cholinergic proteins identi (MOSP), Myelin Oligodendrocyte glycoprotein (MOG), fied as biomarkers for diagnosis and detection of mild brain myelin associated protein (MAG), Oligodendrocyte NS-1 injury or concussion are, but not limited to: acetylcholine protein; Glial Protein Biomarkers: GFAP (P47819), Protein esterase, choline acetyltransferase, peptides, fragments or disulfide isomerase (PDI) P04785, Neurocalcin delta, derivatives thereof. In some embodiments, dopaminergic S100beta, Microglia protein Biomarkers: Iba1, OX-42, proteins identified as biomarkers for diagnosis and detection OX-8, OX-6, ED-1, PTPase (CD45), CD40, CD68, CD11b, of mild brain injury or concussion are, but not limited to: Fractalkine (CX3CL1) and Fractalkine receptor (CX3CR1), tyrosine hydroxylase (TH), phospho-TH, DARPP32, pep 5-d-4 antigen; Schwann cell markers: Schwann cell myelin tides, fragments or derivatives thereof. protein; Glia Scar: Tenascin. 0269. In some embodiments, noradrenergic proteins 0265. In some embodiments, biomarkers identifying the identified as biomarkers for diagnosis and detection of mild anatomical location of neural injury or damage include, but brain injury or concussion are, but not limited to: dopamine not limited to: Hippocampus: Stathmin, Hippocalcin, beta-hydroxylase (DbH), peptides, fragments or derivatives SCG10; Cerebellum: Purkinje cell protein-2 (Pcp2), Calbin thereof. In some embodiments, serotonergic proteins iden din D9K, Calbindin D28K (NP 114190), Cerebellar CaBP. tified as biomarkers for diagnosis and detection of mild brain spot 35; Cerebrocortex: Cortexin-1 (p60606), H-271 gene injury or concussion are, but not limited to: tryptophan product; Thalamus: CD15 (3-fucosyl-N-acetyl-lactosamine) hydroxylase (TrH), peptides, fragments or derivatives epitope: Hypothalamus; Orexin receptors (OX-1R and thereof. In some embodiments, glutamatergic proteins iden OX-2R)-appetite, Orexins (hypothalamus-specific pep tified as biomarkers for diagnosis and detection of mild brain tides); Corpus callosum: MBP, MOG, PLP, MAG; Spinal injury or concussion are, but not limited to: glutaminase, Cord; Schwann cell myelin protein; Striatum: Striatin, Rhes glutamine synthetase, peptides, fragments or derivatives (Ras homolog enriched in striatum); Peripheral ganglia: thereof. In some embodiments, GABAergic proteins iden Gadd45a: Peripheral nerve fiber(sensory+motor): Periph tified as biomarkers for diagnosis and detection of mild brain erin, Peripheral myelin protein 22 (AAH91499); PH8 (S injury or concussion are, but not limited to: GABA transami Serotonergic Dopaminergic), PEP-19, Neurocalcin (NC), a aSC (4-aminobutyrate-2-ketoglutarate transaminase neuron-specific EF-hand Ca"-binding protein, Encephalop (GABAT)), glutamic acid decarboxylase (GAD25, 44, 65, sin, Striatin, SG2NA, Zinedin, Recoverin, Visinin, or a 67), peptides, fragments and derivatives thereof. combination thereof. 0270. In some embodiments, neurotransmitter receptors 0266. In some embodiments, biomarkers identifying identified as biomarkers for diagnosis and detection of mild damaged neural Subtypes include, but not limited to: Neu brain injury or concussion are, but not limited to: beta rotransmitter Receptors: NMDA receptor subunits (e.g. NR1 adrenoreceptor Subtypes, (e.g. beta (2)), alpha-adrenorecep A2B), Glutamate receptor subunits (AMPA, Kainate recep tor Subtypes, (e.g. (alpha (2c)), GABA receptors (e.g. GABA tors (e.g. GluR1, GluR4), beta-adrenoceptor Subtypes (e.g. (B)), metabotropic glutamate receptor. (e.g. mGluR3), beta(2)), Alpha-adrenoceptors Subtypes (e.g. alpha(2c)), NMDA receptor subunits (e.g. NR1 A2B), Glutamate recep GABA receptors (e.g. GABA(B)). Metabotropic glutamate tor Subunits (e.g. GluR4), 5-HT Serotonin receptors (e.g. receptor (e.g. mGluR3), 5-HT serotonin receptors (e.g. 5-HT 5-HT(3)), dopamine receptors (e.g. D4), muscarinic Ach (3)), Dopamine receptors (e.g. D4), Muscarinic Ach recep receptors (e.g. M1), nicotinic acetylcholine receptor (e.g. tors (e.g. M1), Nicotinic Acetylcholine Receptor (e.g. alpha alpha-7), peptides, fragments or derivatives thereof. In some 7); Neurotransmitter Transporters: Norepinephrine embodiments, neurotransmitter transporters identified as Transporter (NET), Dopamine transporter (DAT), Serotonin biomarkers for diagnosis and detection of mild brain injury transporter (SERT), Vesicular transporter proteins (VMAT1 or concussion are, but not limited to: norepinephrine trans and VMAT2), GABA transporter vesicular inhibitory amino porter (NET), dopamine transporter (DAT), serotonin trans acid transporter (VIAAT/VGAT), Glutamate Transporter porter (SERT), vesicular transporter proteins (VMAT1 and (e.g. GLT1), Vesicular acetylcholine transporter, Vesicular VMAT2), GABA transporter vesicular inhibitory amino acid Glutamate Transporter 1, VGLUT1: BNP1 and VGLUT2, transporter (VIAAT/VGAT), glutamate transporter (e.g. Choline transporter, (e.g. CHT1); Cholinergic Biomarkers: GLT1), vesicular acetylcholine transporter, choline trans Acetylcholine Esterase, Choline acetyltransferase ChaT; porter (e.g. CHT1), peptides, fragments, or derivatives Dopaminergic Biomarkers: Tyrosine Hydroxylase (TH). thereof. In some embodiments, other proteins identified as Phospho-TH, DARPP32; Noradrenergic Biomarkers: Dop biomarkers for diagnosis and detection of mild brain injury amine beta-hydroxylase (DbH); Adrenergic Biomarkers: or concussion are, but not limited to, vimentin (P31000), Phenylethanolamine N-methyltransferase (PNMT); Sero CK-BB (P07335), 14-3-3-epsilon (P42655), MMP2, tonergic Biomarkers: Tryptophan Hydroxylase (TrEI); Glu MMP9, peptides, fragments or derivatives thereof. tamatergic Biomarkers: Glutaminase, Glutamine synthetase; 0271 The markers are characterized by molecular GABAergic Biomarkers: GABA transaminase (GABAT), weight, enzyme digested fingerprints and by their known GABA-B-R2, or a combination thereof. protein identities. The markers can be resolved from other US 2017/O121385 A1 May 4, 2017 34 proteins in a sample by using a variety of fractionation for sports is the Zurich graduated return to play protocol, techniques, e.g., chromatographic separation coupled with which is the five stage or phase return process commonly mass spectrometry, or by traditional immunoassays. In some referenced in cases of NFL players rehabilitating from a embodiments, the method of resolution involves Surface recent concussion. It is based on a guideline recommended Enhanced Laser Desorption/Ionization (“SELDI) mass by the 2012 Zurich Consensus Statement on Concussion in spectrometry, in which the Surface of the mass spectrometry Sport. The guideline was written by a group of authors that probe comprises adsorbents that bind the markers. In some included Dr. Robert Cantu, a senior adviser to the NFL's embodiments, a plurality of the biomarkers are detected, at Head, Neck and Spine committee, and Dr. Margot Putakian, least two, or three, or four of the biomarkers are detected. who also serves on the committee and was involved in the 0272. In some embodiments, the amount of each bio development of the sideline assessment protocols. (http:// marker is measured in the Subject sample and the ratio of the Subscribers.footballguys.com/apps/article. amounts between the markers is determined. The amount of php?article-13bramel inside concussions 2) Thus, the each biomarker in the subject sample and the ratio of the ghrelin variant can be administered to a patient with a mild amounts between the biomarkers and compared to normal brain injury or concussion that needs a recovery protocol. healthy individuals. The increase in ratio of amounts of After administration of at least an initial dose, a recovery biomarkers between healthy individuals and individuals protocol can be administered and followed to determine Suffering from injury is indicative of the injury magnitude, when the patient can undertake or resume certain activity. disorder progression as compared to clinically relevant data. 0273. In some embodiments, biomarkers that are detected Pharmaceutical Compositions at different stages of injury and clinical disease are corre 0277 Ghrelin, ghrelin variants and the combinations lated to assess anatomical injury, type of cellular injury, described herein can be formulated as a pharmaceutical subcellular localization of injury. Monitoring of which bio composition, e.g., flash frozen or lyophilized for storage markers are detected at which stage, degree of injury in and/or transport. In some embodiments, the compound can disease or physical injury will provide panels of biomarkers be in a composition with sterile Saline, for example. In some that provide specific information on mechanisms of injury, embodiments, a ghrelin, ghrelin variant, or combination identify multiple subcellular sites of injury, identify multiple material can be reconstituted in Such saline or other accept cell types involved in disease related injury and identify the able diluent. In some embodiments, about 10 ug ghrelin anatomical location of injury. In some embodiments, one or powder is reconstituted in about 100 uL saline solution more of the biomarkers disclosed herein, can be specifically before administration. In addition, the composition can be excluded. administered alone or in combination with a carrier, Such as 0274. In some embodiments, a single biomarker is used a pharmaceutically acceptable carrier or a biocompatible in combination with one or more biomarkers from normal, scaffold. Compositions of the invention may be convention healthy individuals for diagnosing injury, location of injury ally administered parenterally, by injection, for example, and progression of disease and/or neural injury, or a plurality intravenously, Subcutaneously, or intramuscularly. Addi of the markers are used in combination with one or more tional formulations which are suitable for other modes of biomarkers from normal, healthy individuals for diagnosing administration include oral formulations. Oral formulations injury, location of injury and progression of disease and/or include Such normally employed excipients such as, for neural injury. In some embodiments, one or more protein example, pharmaceutical grades of mannitol, lactose, starch, biomarkers are used in comparing protein profiles from magnesium Stearate, sodium saccharine, cellulose, magne patients susceptible to, or Suffering from disease and/or sium carbonate and the like. These compositions take the neural injury, with normal Subjects. form of solutions, Suspensions, tablets, pills, capsules, Sus 0275. In some embodiments, detection methods include tained release formulations or powders and contain about use of a biochip array. Biochip arrays useful include protein 10% to about 95% of active ingredient, about 25% to about and nucleic acid arrays. One or more markers are immobi 70%. lized on the biochip array and Subjected to laser ionization 0278 Typically, compositions are administered in a man to detect the molecular weight of the markers. Analysis of ner compatible with the dosage formulation, and in Such the markers is, for example, by molecular weight of the one amount as will be therapeutically effective for the disease or or more markers against a threshold intensity that is nor condition by treated. The quantity to be administered malized against total ion current. In some embodiments, depends on the subject to be treated. Precise amounts of the logarithmic transformation is used for reducing peak inten composition to be administered depend on the judgment of sity ranges to limit the number of markers detected. In some the practitioner. Suitable regimes for initial administration embodiments, data is generated on immobilized subject and boosters are also variable, but are typified by an initial samples on a biochip array, by Subjecting said biochip array administration followed by Subsequent administrations. to laser ionization and detecting intensity of signal for 0279. In some embodiments, additional pharmaceutical mass/charge ratio; and transforming the data into computer compositions are administered to a Subject to Support or readable form; and executing an algorithm that classifies the augment the compositions as described herein. Different data according to user input parameters, for detecting signals aspects of the present invention involve administering an that represent markers present in injured and/or diseased effective amount of the composition to a subject. Addition patients and are lacking in non-injured and/or diseased ally, such compositions can be administered in combination Subject controls. with other agents. Such compositions will generally be 0276 A follow up or recovery protocol, optionally can dissolved or dispersed in a pharmaceutically acceptable then be used to assess the recovery of the patient, including carrier or aqueous medium. whether the patient can return to participate in certain 0280. The phrases “pharmaceutically acceptable' or activities such as a sport Once such recovery protocol used “pharmacologically acceptable” refer to molecular entities US 2017/O121385 A1 May 4, 2017

and compositions that do not produce an adverse, allergic, or 0284. A ghrelin variant can be administered subcutane other untoward reaction when administered to an animal, or ously in an amount allowing sufficient levels of the bioactive human. As used herein, "pharmaceutically acceptable car form of ghrelin variant, i.e., the acylated form, to reach the rier includes any and all solvents, dispersion media, coat receptors. ings, antibacterial and antifungal agents, isotonic and 0285. The present disclosure also provides a procedure absorption delaying agents, and the like. The use of Such for an optimal administration of ghrelin variants to patients media and agents for pharmaceutical active Substances is in order to obtain a maximal response and to avoid, for well known in the art. Except insofar as any conventional example, desensitization mechanisms. media or agent is incompatible with the active ingredients, 0286 The ghrelin receptor normally is exposed to short its use in immunogenic and therapeutic compositions is lived surges in ghrelin concentration. The GHS-R 1a recep contemplated. tor (growth hormone secretagogue receptor 1a) belongs to the class of G protein coupled receptors or 7TM receptors, 0281 Suitable pharmaceutical carriers include inert solid which upon continued exposure to an agonist will be desen diluents or fillers, sterile aqueous solution and various sitized, internalized, and down-regulated. These mecha organic solvents. Examples of solid carriers are lactose, terra nisms, which are inherent to the overall signal transduction alba, Sucrose, cyclodextrin, talc, gelatin, agar, pectin, acacia, system, involve processes such as receptor phosphorylation magnesium Stearate, Stearic acid or lower alkyl ethers of (which, in itself, decreases the affinity of the receptor for the cellulose. Examples of liquid carriers are syrup, peanut oil, agonist) and binding of inhibitory proteins such as arrestin olive oil, phospholipids, fatty acids, fatty acid amines, (which sterically block the binding of signal transduction polyoxyethylene or water. Nasal aerosol or inhalation for molecules such as G proteins). mulations may be prepared, for example, as solutions in 0287 Another part of the agonist-mediated desensitiza saline, employing benzyl alcohol or other Suitable preser tion process is receptor internalization (physical removal of Vatives, absorption promoters to enhance bioavailability, the receptor from the cell surface where it could bind the employing fluorocarbons, and/or employing other solubiliz agonist) as well as receptor down regulation (decreased ing or dispersing agents. production/expression of the receptor). Receptor internal ization could, after short-lived exposure of the receptor to 0282. The carrier may be a solvent or dispersion medium agonist, be followed by a re-sensitization process, where the containing, for example, water (e.g., hydrogels), ethanol, receptor is dephosphorylated and recycled to the cell surface polyol (for example, glycerol, propylene glycol, and liquid to be used again. Without being bound by theory, upon poly(ethylene glycol), and the like), Suitable mixtures prolonged stimulation which would occur for example dur thereof, and vegetable oils. The proper fluidity can be ing a long-lasting continuous infusion of the agonist, the maintained, for example, by the use of a coating, such as receptor down-regulation process ensures that the target cell lecithin, by the maintenance of the required particle size in is adjusted in its signal transduction system to this situation. the case of dispersion, and by the use of surfactants. The 0288 Ghrelin variant compositions can be produced prevention of the action of undesirable microorganisms can using techniques well known in the art. For example, a be brought about by various antibacterial and antifungal polypeptide region of a ghrelin variant can be chemically or agents, for example, parabens, chlorobutanol, phenol, Sorbic biochemical synthesized and modified. Techniques for acid, thimerosal, and the like. In many cases, isotonic agents chemical synthesis of polypeptides are well known in the art are included, for example, Sugars or sodium chloride. Pro (Lee V. H. L. in “Peptide and Protein Drug Delivery”, New longed absorption of the injectable compositions can be York, N.Y., M. Dekker, 1990). Examples of techniques for brought about by the use in the compositions of agents biochemical synthesis involving the introduction of a delaying absorption, for example, aluminum monostearate nucleic acid into a cell and expression of nucleic acids are and gelatin. provided in Ausubel F. M. et al., “Current Protocols in 0283 An effective amount of therapeutic composition is Molecular Biology”, John Wiley, 1987-1998, and Sambrook determined based on the intended goal. The term “unit dose' J. et al., “Molecular Cloning, A Laboratory Manual. 2d or “dosage” refers to physically discrete units suitable for Edition, Cold Spring Harbor Laboratory Press, 1989, each of use in a subject, each unit containing a predetermined which is incorporated herein by reference. Another example quantity of the composition calculated to produce the technique, described in U.S. Pat. No. 5,304,489, incorpo desired responses discussed above in association with its rated herein by reference, is the use of a transgenic mammal administration, i.e., the appropriate route and regimen. The having mammary gland-targeted mutations which result in quantity to be administered, both according to number of the production and secretion of synthesized ghrelin variant treatments and unit dose, depends on the result and/or in the milk of the transgenic mammal. protection desired. Precise amounts of the composition also 0289. The ghrelin variants can also be produced recom depend on the judgment of the practitioner and are peculiar binantly using routine expression methods known in the art. to each individual. Factors affecting dose include physical The polynucleotide encoding the desired ghrelin variant is and clinical state of the Subject, route of administration, operably linked to a promoter into an expression vector intended goal of treatment (alleviation of symptoms versus Suitable for any convenient host. Both eukaryotic and pro cure), and potency, stability, and toxicity of the particular karyotic host systems are used in forming recombinant composition. Upon formulation, Solutions will be adminis ghrelin variants. The ghrelin variant is then isolated from tered in a manner compatible with the dosage formulation lysed cells or from the culture medium and purified to the and in Such amount as is therapeutically or prophylactically extent needed for its intended use. Isolated ghrelin orghrelin effective. The formulations are easily administered in a variant may be modified further at serine amino acid posi variety of dosage forms, such as the type of injectable tion 2 and/or serine amino acid position 3 by fatty acid solutions described above. acylation in vivo or in vitro, with the latter in vitro acylation US 2017/O121385 A1 May 4, 2017 36 reaction condition comprising fatty acid thioester, ghrelin, organism in which the expression vector is introduced, as and microsomes comprising ghrelin O-acyl transferase explained in U.S. Pat. No. 5,082,767, which disclosure is (GOAT). In some embodiments, acyl ghrelin or ghrelin hereby incorporated by reference in its entirety. variant modified with fatty acid at serine amino acid position 0296. In some embodiments, additional nucleotide 2 and/or serine amino acid position 3 is isolated from sequencers, which codes for secretory or leader sequences, cellular or reaction components. pro-sequences, sequences which aid in purification, Such as 0290 Ghrelin compositions can include pharmaceuti multiple histidine residues, or an additional sequence for cally acceptable salts of the compounds therein. These salts stability during recombinant production, are added to ghre will be ones which are acceptable in their application to a lin variants or to ghrelin itself to produce a ghrelin variant. pharmaceutical use, meaning that the salt will retain the biological activity of the parent compound and the salt will 0297. In some embodiments, introduction of a polynucle not have untoward or deleterious effects in its application otide encoding a ghrelin variant into a host cell can be and use in treating diseases. Pharmaceutically acceptable affected by calcium phosphate transfection, DEAE-dextran salts are prepared in a standard manner. mediated transfection, cationic lipid-mediated transfection, 0291. In some embodiments, a DNA coding an amino electroporation, transduction, infection, or other methods. acid sequence of ghrelin variants described in the present Such methods are described in many standard laboratory disclosure, which comprises a nucleotide sequence coding a manuals, such as Davis et al., (1986) Basic Methods in peptide containing an amino acid sequence recognizing at Molecular Biology, ed., Elsevier Press, NY, which disclo least one modifiable amino acid in the amino acid sequence sure is hereby incorporated by reference in its entirety. encoded by said DNA. In some embodiments, a vector 0298 ghrelin variants can be recovered and purified from comprises a DNA described above. In some embodiments, recombinant cell cultures by well-known methods including cells comprise the vector described above. differential extraction, ammonium sulfate or ethanol precipi 0292. In some embodiments, a method for producing a tation, acid extraction, anion or cation exchange chroma ghrelin variant compound by genetic recombination tech tography, phosphocellulose chromatography, hydrophobic nology comprises transforming a vector containing a DNA interaction chromatography, affinity chromatography, described above into host cells capable of modifying a side hydroxylapatite chromatography and lectin chromatography chain of at least one amino acid in said peptide, then (“Methods in Enzymology: Aqueous Two-Phase Systems’. culturing the resulting transformed cells and recovering the Walter H et al. (eds.), Academic Press (1993), incorporated desired ghrelin variant compound from the culture. In some herein by reference, for a variety of methods for purifying embodiments, a method for producing a ghrelin variant proteins). In some embodiments, high performance liquid compound by genetic recombination technology comprises chromatography (“HPLC) is employed for purification. using cells having the activity of binding a fatty acid via an 0299. A recombinantly produced version of ghrelin vari ester linkage to a side-chain hydroxyl group of an amino ants can be substantially purified using techniques described acid or via a thioester linkage to a side-chain mercapto group herein or otherwise known in the art, such as, for example, of an amino acid in the ghrelin variant compound. by the one-step method described in Smith & Johnson, Gene 0293. In some embodiments, a method for producing a 67:31 40 (1988), which disclosure is hereby incorporated by ghrelin variant compound by genetic recombination tech reference in its entirety. Ghrelin variants also can be purified nology comprises using cells having the serine acylation from recombinant sources using antibodies directed against activity of binding a fatty acid via an ester linkage to a ghrelin variants, which are well known in the art of protein side-chain hydroxyl group of serine. In some embodiments, purification. a method for producing a ghrelin variant compound by genetic recombination technology comprises using cells 0300. In some embodiments, depending upon the host having the threonine acylation activity of binding a fatty employed in a recombinant production procedure, the ghre acid via an ester linkage to a side-chain hydroxyl group of lin variants may be glycosylated or may be non-glycosy threonine. lated. In addition, polypeptides of the invention may also 0294. In some embodiments, the present disclosure pro include an initial modified methionine residue, in some vides for a method of producing a ghrelin variant, said cases as a result of host-mediated processes. Thus, it is well method comprising the steps of: (a) providing a cDNA known in the art that the N-terminal methionine encoded by comprising a polynucleotide sequence encoding a ghrelin the translation initiation codon generally is removed with variant; (b) inserting said cDNA in an expression vector high efficiency from any protein after translation in all such that the cDNA is operably linked to a promoter; and (c) eukaryotic cells. While the N-terminal methionine on most introducing said expression vector into a host cell whereby proteins also is efficiently removed in most prokaryotes, for said host cell produces said ghrelin variant. Some proteins, this prokaryotic removal process is inefli 0295. In some embodiments, the method further com cient, depending on the nature of the amino acid to which the prises the step of recovering the ghrelin variant produced in N-terminal methionine is covalently linked. step (c). The expression vector is any of the mammalian, 0301 The present disclosure provides for a pharmaceu yeast, insect, or bacterial expression systems known in the tical composition comprising a mixture of at least two art. Commercially available vectors and expression systems different ghrelin variants, such as a mixture of a ghrelin are available from a variety of Suppliers including Genetics variant acylated with a Cs acyl and a ghrelin variant acylated Institute (Cambridge, Mass.), Stratagene (La Jolla, Calif.), with a Co acyl. Without being bound by theory, it is Promega (Madison, Wis.), and Invitrogen (San Diego, believed that such a mixture will have a longer half-life in Calif.). If desired, to enhance expression and facilitate plasma. In some embodiments, the pharmaceutical compo proper protein folding, the codon context and codon pairing sition comprises acylated ghrelin variants, optionally com of the sequence is optimized for the particular expression pounds having different acyl chain lengths selected from the US 2017/O121385 A1 May 4, 2017 37 group consisting of C7 acyl group, Co acyl group, and C1 ammonium salts. Acid addition salts include salts of inor acyl group, optionally in combination with a non- or un ganic acids as well as organic acids. acylated ghrelin variant. 0309. In some embodiments, compounds or pharmaceu 0302) In some embodiments, the pharmaceutical compo tical acceptable acid addition salts are any hydrates (hy sition comprising any secretagogue. Such as any ghrelin drated forms) thereof. Salts formed with the free carboxyl variant or a pharmaceutically acceptable salt thereof and groups can also be derived from inorganic bases such as, for pharmaceutical acceptable carriers, vehicles and/or excipi example, Sodium, potassium, ammonium, calcium or ferric ents; said composition further comprising transport mol hydroxides, and Such organic bases as isopropylamine, ecules. The transport molecules are primarily added in order trimethylamine, 2-ethylamino ethanol, histidine, procaine to increase the half-life of the acylated compound, prevent and the like. ing premature des-acylation, since the des-acylated ghrelin 0310. For parenteral administration, solutions of the pres variant might not be active at the GHS-R 1a. ent compounds in Sterile aqueous solution, aqueous propyl 0303 Transport molecules act by having incorporated ene glycol or sesame or peanut oil may be employed. Such into or anchored to it a compound disclosed herein. Any aqueous solutions should be suitably buffered if necessary, Suitable transport molecule known to the skilled person may and the liquid diluent first rendered isotonic with sufficient be used. Examples of transport molecules are those saline or glucose. The aqueous Solutions are particularly described in the conjugate section, Supra. Other examples Suitable for intravenous, intramuscular, Subcutaneous and are liposomes, micelles, and/or microspheres. intraperitoneal administration. The sterile aqueous media 0304. In some embodiments, the active ingredient can be employed are all readily available by standard techniques mixed with excipients and non-endogenous carriers, which known to those skilled in the art. are pharmaceutically acceptable and compatible with the 0311 Liquid compositions can also contain liquid phases active ingredient and in amounts suitable for use in the in addition to and to the exclusion of water. Examples of therapeutic methods described herein. Suitable excipients Such additional liquid phases are glycerin, vegetable oils are, for example, water, Saline, dextrose, glycerol, ethanol or Such as cottonseed oil, organic esters such as ethyl oleate, the like and combinations thereof. In addition, if desired, the and water-oil emulsions. composition can contain minor amounts of auxiliary Sub 0312 Suitable pharmaceutical carriers include inert solid stances such as wetting or emulsifying agents, pH buffering diluents or fillers, sterile aqueous solution and various agents and the like which enhance the effectiveness of the organic solvents. Examples of solid carriers are lactose, terra active ingredient. alba, Sucrose, cyclodextrin, talc, gelatine, agar, pectin, aca cia, magnesium Stearate, Stearic acid or lower alkyl ethers of 0305. In some embodiments, the formulation has a pH cellulose. Examples of liquid carriers are syrup, peanut oil, within the range of 3.5-8, such as in the range 4.5-7.5, such olive oil, phospholipids, fatty acids, fatty acid amines, as in the range 5.5-7. Such as in the range 6-7.5. Such as polyoxyethylene or water. Nasal aerosol or inhalation for about 7.3. However, as is understood by one skilled in the mulations may be prepared, for example, as solutions in art, the pH range may be adjusted according to the individual saline, employing benzyl alcohol or other Suitable preser treated and the administration procedure. For example, Vatives, absorption promoters to enhance bioavailability, certain ghrelin variants or ghrelin homologs, may be stabi employing fluorocarbons, and/or employing other solubiliz lized at a lower pH; thus in some embodiments, the formu ing or dispersing agents. lation has a pH within the range 3.5-7. Such as 4-6, Such as 0313 The present disclosure provides a pharmaceutical 5-6, such as 5.3-5.7., such as about 5.5. composition stably containing ghrelin or ghrelin variants 0306 Ghrelin variant compositions can include pharma and a method for preventing degradation of modifying ceutically acceptable salts of the compounds therein. These hydrophobic group of ghrelin or ghrelin variants in an salts will be ones which are acceptable in their application aqueous Solution. The modifying hydrophobic group of the to a pharmaceutical use, meaning that the salt will retain the ghrelin or ghrelin variants, is not limited to octanoyl (Cs) biological activity of the parent compound and the salt will group, and is a residue of fatty acid having 2 to 20, not have untoward or deleterious effects in its application preferably 4 to 12 carbon atoms, such as hexanoyl (C) and use in treating diseases. Pharmaceutically acceptable group, decanoyl (Co) group or dodecanoyl (C) group. The salts are prepared in a standard manner. If the parent hydrophobic group can also be a residue of branched, compound is a base, it is treated with an excess of an organic saturated or unsaturated fatty acid, a residue of fatty acid or inorganic acid in a Suitable solvent. If the parent com having an aromatic group Such as phenylpropionyl group, pound is an acid, it is treated with an inorganic or organic and an adamantane skeleton. base in a suitable solvent. 0314. In some embodiments, the ghrelin variants of the 0307 Ghrelin variant compositions may be administered present disclosure include the ghrelin or ghrelin variant in the form of an alkali metal or earth alkali metal salt peptides, in which the amino acid sequence is modified by thereof, concurrently, simultaneously, or together with a the insertion, addition and deletion of one or more amino pharmaceutically acceptable carrier or diluent, especially acid, and/or the Substitution by other amino acid to said and in the form of a pharmaceutical composition thereof, amino acid sequence, and is modified chemically if neces whether by various routes (e.g., oral, rectal, parenteral, sary. In some embodiments, the ghrelin variants include the Subcutaneous) in an effective amount. peptides in which modifying hydrophobic group is bonded 0308. Other suitable pharmaceutically acceptable salts to amino acid chain by ester bond and having same or include the acid addition salts (formed with the free amino similar physiologically activity and function as ghrelin. groups of the polypeptide). Other examples of salts include 0315. In some embodiments, the ghrelin or ghrelin vari pharmaceutically acceptable acid addition salts, pharmaceu ant is to be used in the pharmaceutical composition of the tically acceptable metal salts, ammonium salts and alkylated present disclosure includes free form peptides and salts US 2017/O121385 A1 May 4, 2017

thereof. The free form peptide and salt thereof can be 0321 Considering the stability of the ghrelin or ghrelin reciprocally converted. The free form peptide can be con variants in the aqueous solution, it is desired that the verted to a pharmaceutically acceptable salt by reacting with fluctuation of pH values of the solution have to be reduced an inorganic or an organic acid. The examples of the Therefore, the pharmaceutical composition of the present inorganic acid include, but are not limited to, carbonate, disclosure is the Solution having buffer capacity, that is, the bicarbonate, hydrochloride, Sulfate, nitrate, borate or a com buffer solution. bination thereof, and the examples of the organic acid 0322. In some embodiments, the buffer solution, having include, but are not limited to. Succinate, acetate, propionate, the pH range wherein the degradation of the ghrelin or trifluoroacetate, or a combination thereof. Other examples of ghrelin variants is inhibited, and the solution having the pH the salt include, but are not limited to, the salt with alkali range of 2 to 7, more preferably 3 to 6 is used. The suitable metal Such as Sodium salt or potassium salt; the salt with buffer solution include, but not limited to, glycine hydro alkali earth metal Such as calcium salt or magnesium salt; the chloride buffer, acetate buffer, citrate buffer, lactate buffer, salt with organic amine Such as triethylamine salt; and the phosphate buffer, citric acid-phosphate buffer (including salt with basic amino acid Such alginic acid salt, or a combination thereof. The ghrelin or ghrelin variant peptides McIlvaine buffer), phosphate-acetate-borate buffer (includ of the present disclosure can exist as metal complex Such as ing Britton-Robinson buffer), and phthalate buffer. The copper complex or Zinc complex. The form of the salt as examples of the components of each buffers include the mentioned above has a role is the stability of the ghrelin or buffer agents mentioned above. ghrelin variants. That is, pH values of the aqueous solution 0323. In some embodiments, the concentration of pH of the salts above are different from each other, and there adjuster is not limited and can be the concentration com fore, these salts play the role as pH adjuster for the aqueous monly used to adjust the Solution with the desired pH range, Solution of the ghrelin or ghrelin variants. and in general, the concentration of 0.01 to 100 mM is used. 0316. In some embodiments. The ghrelin or ghrelin vari In some embodiments, the concentration of buffer agent is ants to be used as raw materials for medicines are commonly also not limited and can be the concentration maintaining the supplied as lyophilized powder after purified by reverse buffer capacity. Generally, the concentration is about 0.01 to liquid chromatography and so on. The aqueous solution is about 100 mM, or about 0.1 to about 100 mM, or about 1 to the solution used water as the solvent; however, other about 100 mM. Solvent Such as ethanol, 2-propanol and the like can be used 0324. In some embodiments, the pharmaceutical compo Within a pharmaceutically acceptable range. sition stably containing the ghrelin or ghrelin variants in the 0317. The concentration of the ghrelin or ghrelin variants aqueous solution is provided. The composition contains in the pharmaceutical composition is not limited to, and is other additives in consideration of osmolality, solubility, low preferably within a pharmaceutically acceptable range. The irritation of the solution, as well as antisepsis effect and lower limit of concentration is the concentration wherein the prevention of absorption of the ingredient in the solution. ghrelin or ghrelin variants exhibit the pharmacologically 0325 In some embodiments, anti-adsorbents are used to activities, and the upper limit of concentration is the con prevent ghrelin or ghrelin variants peptide from absorbing to centration wherein the ghrelin or ghrelin variants can be glass vessels or polypropylene vessels. Examples of anti dissolve in the aqueous Solutions. In some embodiments, the adsorbent include, but not limited to, Surfactants, saccha concentration ghrelin or ghrelin variants used in the phar rides, amino acids and proteins. maceutical composition is about 0.01 nmol/mL to about 10 0326 Examples of the surfactant include, but not limited umol/mL, or about 0.03 nmol/mL to about 3 umol/mL. to, quaternary ammonium salts, polyoxyethylene Sorbitan 0318. In some embodiments, in the physiological com fatty acid esters, Sorbitan fatty acid esters, parabens, poly position of the present disclosure, containing ghrelin or ethylene glycols, phospholipids, bile acids, polyoxyethylene ghrelin variants, the pH value of the solution is in the range castor oils, polyoxyethylenes, polyoxyethylene polyoxypro of 2 to 7, more preferably 3 to 6 In some embodiments, the pylenes, polyalcohols, anionic Surfactant, synthetic or semi pH value of the solution containing the ghrelin or ghrelin synthetic polymers. variants that are stable is in the range of 2 to 7. The adjustment of pH of the solution containing the ghrelin or 0327. The suitable quaternary ammonium salts include, ghrelin variants is conducted with pH adjuster or buffer but not limited to, benzalkonium chloride, benzethonium agent. chloride and cetylpyridinium chloride. 0319. Examples of pH adjuster include, but not limited 0328. The suitable polyoxyethylene sorbitan fatty acid to, hydrochloric acid, Sulfuric acid, nitric acid, boric acid, esters include, but not limited to, polyoxyethylene sorbitan carbonic acid, bicarbonic acid, gluconic acid, sodium monolaurate (Polysorbate?R 20 or TweenR 20), polyoxyeth hydroxide, potassium hydroxide, aqueous ammonia, citric ylene sorbitan monopalmitate (Polysorbate R 40 or Tween(R) acid, monoethanolamine, lactic acid, acetic acid, Succinic 40), polyoxyethylene sorbitan monostearate (PolysorbateR) acid, fumaric acid, maleic acid, phosphoric acid, methane 60 or Tween R. 60), polyoxyethylene sorbitan tristearate Sulfonic acid, malic acid, propionic acid, trifluoroacetic acid, (Polysorbate(R) 65 or Tween R 65), polyoxyethylene sorbitan and salt thereof. monooleate (Polysorbate R 80 or Tween R. 80), and polyoxy 0320 Examples of buffer agent include, but not limited ethylene sorbitan trioleate (Polysorbate R 85 or Tween R. 85). to, glycine, acetic acid, citric acid, boric acid, phthalic acid, 0329. The suitable sorbitan fatty acid esters include, but phosphoric acid, Succinic acid, lactic acid, tartaric acid, not limited to, sorbitan monolaurate (SpanR20), sorbitan carbonic acid, hydrochloric acid, Sodium hydroxide, and the monopalmitate (Span R40), sorbitan monostearate (SpanR) salt thereof. In some embodiments, glycine, acetic acid or 60), sorbitan monooleate (Span R. 80), sorbitan trioleate Succinic acid are used as buffer agent. (Span R 85), and sorbitan sesquioleate. US 2017/O121385 A1 May 4, 2017 39

0330. The suitable parabens include, but not limited to, 0338. The pharmaceutical composition of the present methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl disclosure can contain further additives for any purpose, and paraoxybenzoate, butyl paraoxybenzoate, and isobutyl examples of the additives is selected from the “Handbook of paraoxybenzoate. PHARMACEUTICAL EXCIPIENTS 2000” (Japan Phar 0331. The suitable polyethylene glycols include, but not maceutical Excipients Council: Yakuji Nippoh Sha). These limited to, glycofurol (glycofurol 75), Mcrogol R 400 (poly include isotonizing agent Such as, but not limited to, Sodium ethylene glycol 400). Mcrogol R 600 (polyethylene glycol chloride and mannitol, antiseptic agent such as, but not 600), and Mcrogol R 9000 (polyethylene glycol 4000); the limited to, sodium benzoate; antioxidant Such as, but not suitable phospholipids include refined soybean lecithin and limited to, Sodium bisulfite, sodium pyrosultite and ascorbic refined yolk lecithin; and suitable bile acids include sodium acid; soothing agent Such as, but not limited to, lidocaine desoxycholic acid. hydrochloride and mepivacaine hydrochloride, as explained 0332 The suitable polyoxyethylene castor oils include, in U.S. Pat. No. 8,518,893, which disclosure is hereby but not limited to, polyoxyethylene castor oil, polyoxyeth incorporated by reference in its entirety. ylene hydrogenated castor oil, polyoxyethylene hydroge 0339. The manufacture of the pharmaceutical composi nated castor oil 50, and polyoxyethylene hydrogenated tion of the present disclosure is conducted by mean of the castor oil 60. Examples of other polyoxyethylenes include common procedure applied in the pharmaceutical field. For polyoxyethylene oleyl ether, polyoxyethylene stearyl ether, example, first, freeze dried ghrelin is dissolved in the polyoxyethylene cetyl ether, and polyoxyethylene lauryl purified water, and then, buffer agent, anti-adsorbent and sulfate salt. other additives are also dissolved in another purified water. 0333. The suitable polyoxyethylene polyoxypropylenes Then the resulting water solutions are combined and sterilize include, but not limited to, polyoxyethylene polyoxypropyl by filtration if necessary, and the obtained solution is filled ene glycol (Pluronic R) and polyoxyethylene polyoxypro in ampoules or vials to obtain the pharmaceutical composi pylene cetyl ether. tion containing the ghrelin or ghrelin variants of the present 0334. The suitable polyalcohols include, but not limited disclosure. to, glycerin (glycerol), propylene glycol, and monoglyceryl Stearate; and the Suitable anionic Surfactants include, but not Administration of Compositions limited to, alkyl ether sulfate such as sodium cetyl sulfate, 0340. The present disclosure provides for treating a neu sodium lauryl sulfate and sodium oleyl sulfate; alkyl sulfo rodegenerative condition in a Subject in need thereof, com succinate such as sodium lauryl Sulfosuccinate. The Suitable prising administering to the subject an effective amount of a synthetic or semi-synthetic polymers include, but not limited compound comprising ghrelin or a ghrelin variant. to, polyvinyl alcohol, carboxyvinyl polymer, polyvinyl pyr 0341. In some embodiments, an administration route for rolidone and Sodium polyacrylate. aghrelin variant is selected from: buccal delivery, Sublingual 0335 Examples of saccharides include, but not limited delivery, transdermal delivery, inhalation and needle-free to, monosaccharide such as mannitol, glucose, fructose, injection, such as using the methods developed by Powder inositol; Sorbitol, and Xylitol, disaccharide Such as lactose, Jet. For inhalation, the aghrelin variant can be formulated Sucrose, maltose, and trehalose; polysaccharide Such as using methods known to those skilled in the art, for example starch, dextran, pullulan, alginic acid, hyaluronic acid, pec an aerosol, dry powder or solubilized Such as in microdrop tinic acid, phytic acid, phytin, chitin, and chitosan Examples lets, in a device intended for Such delivery (such as com of dextrin include, but not limited to, C.-cyclodextrin, B-cy mercially available devices and formulation technologies clodextrin, Y-cyclodextrin, dextrin, hydroxypropyl Starch, from Aradigm Corp. (Hayward, Calif.), Alkermes, Inc. and hydroxyl starch. Examples of celluloses include, but not (Cambridge, Mass.), Nektar Therapeutics (San Carlos, limited to, methylcellulose, ethylcellulose, hydroxyethyl Calif.), or MannKind Corporation (Valencia, Calif.: e.g., cellulose, hydroxypropyl cellulose, and hydroxypropyl Technosphere(R), Dreamboat(R), and CricketTM technolo methylcellulose, sodium carboxymethyl cellulose. gies)). 0336. The suitable amino acids include, but not limited 0342. In some embodiments, the DANA mobile medical to, glycine and taurine; and polyamino acid Such as poly application (AnthroTronix, www.atinc.com) is utilized to glutamic acid, polyaspartic acid, polyglycine and polyleu determine the therapeutic effectiveness of ghrelin variant cine. The Examples of proteins include, but not limited to, compositions in treating mild brain injury or concussion. albumin and gelatin. DANA provides clinicians with objective measurements of 0337. Non-human serum albumin can be used as anti reaction time (speed and accuracy) to aid in the assessment adsorbent for the pharmaceutical composition of the present of an individual’s medical or psychological state. DANA is invention when the composition is used as a reagent for a phone or tablet-based app on Android or iOS operating examination or as Veterinary medicines; however, it is systems and is indicated for use as part of any clinical preferable to use human serum albumin when the compo assessment where concerns for changes in cognitive or sition is used for a medicine for treating human being. These psychological status are present. DANA's battery of cogni anti-adsorbents can be used in combination. The concentra tive and psychological tests are administered and the results tion of the anti-adsorbent is in the range wherein the amount are evaluated by a qualified health professional who can of the anti-adsorbent is pharmaceutically acceptable one and assess factors that may affect measurement of reaction time the adsorption of the ghrelin or ghrelin variants to the vessel Such as concussion, dementia, post-traumatic stress, depres is inhibited and the aggregation of the components does not Sion, stress, fatigue, prescription and non-prescription medi occur during the manufacturing process or the long-term cations, and some nutritional Supplements, among others. storage. For example, the concentration of the anti-adsorbent 0343. In some embodiments, the ghrelin variant is admin is in the range of about 0.001 to about 5%, or from about istered via a powder or stable formulation, wherein the 0.01 to about 1%. ghrelin variant is formulated in a dosage form selected from US 2017/O121385 A1 May 4, 2017 40 the group consisting of liquid, beverage, medicated sports limiting factor to nasal drug delivery. Nasal mucociliary drink, powder, capsule, chewable tablet, caplet, swallowable clearance severely limits the time allowed for drug absorp tablet, buccal tablet, troche, lozenge, soft chew, solution, tion to occur and may effectively prevent Sustained drug Suspension, spray, Suppository, tincture, decoction, infusion, administration. However, it has been documented that nasal and a combination thereof. administration of certain hormones has resulted in a more 0344. In some embodiments, the composition comprising complete administration. In some embodiments, the present ghrelin or the ghrelin variant is administered via inhalation, disclosure utilizes nasal delivery of ghrelin. oral, intravenous, parenteral, buccal, Subcutaneous (includ 0348. In some embodiments, a composition comprising ing "EpiPens'), transdermal, patch, Sublingual, intramuscu ghrelin or the ghrelin variant that is Suitable for nasal lar, or intranasal. In some embodiments, EpiPens is either administration may include one or more bioadhesive poly EpiPen 0.3 mg or EpiPen Jr.R (epinephrine) 0.15 mg Auto mers. Some polymers such as carbopol, can adhere onto the Injectors for people who have a history of life-threatening nasal mucosa for reasonably prolonged periods, preventing allergic reactions (anaphylaxis) to things like bee stings, rapid nasal clearance. In some embodiments, a composition peanuts or seafood, or are at increased risk for a severe Suitable for nasal administration, the percentage of bioad allergic reaction. EpiPen and EpiPen Jr are self-injectable hesive polymer in a suitable solution of ghrelin is about devices (auto-injectors) that contain epinephrine. 0.1%. In some embodiments, a composition suitable for 0345. In some embodiments, the composition comprising nasal administration, the percentage of bioadhesive polymer ghrelin or the ghrelin variant is administered via the auto in a suitable solution of ghrelin is about 0.5%. In some matic mixing device and delivery system by Windgap Medi embodiments, a composition Suitable for nasal administra cal Devices as described in U.S. Patent Application No. tion, the percentage of bioadhesive polymer in a Suitable 2013/0178823, which is incorporated by reference in its Solution of ghrelin is about 1%. In some embodiments, a entirety. In some embodiments, the automatic mixing device composition Suitable for nasal administration, the percent and delivery system by Windgap Medical Devices is a age of bioadhesive polymer in a suitable solution of ghrelin wet/dry auto-mixing injector having a mixing device con is about 5%. taining at least one microfluidic channel for mixing or 0349. In some embodiments, a composition comprising dissolving a dry component with a wet component stored in ghrelin or the ghrelin variant that is Suitable for nasal the injector device. In some embodiments, the automatic administration may include one or more surfactants. Sur mixing device and delivery system by Windgap Medical factants that may be used in the compositions of the present Devices is a mixing and/or automatic injection device hav invention include different polyethylene glycols (PEGS) or ing an interior chamber containing a wet component that polyethylene glycol-derivatives. In some embodiments, a may be pH optimized to be mixed with a dry component composition Suitable for nasal administration, the percent contained in a mixing assembly. The wet component being age of Surfactant in a suitable solution of ghrelin is about confined or sealed in the interior chamber by a seal or valve, 1%. In some embodiments, a composition Suitable for nasal where upon activation of the seal or valve the wet interior administration, the percentage of Surfactant in a Suitable chamber becomes in fluid communication with the mixing Solution of ghrelin is about 2%. In some embodiments, a assembly and dissolution of the dry component into the wet composition Suitable for nasal administration, the percent component occurs. The mixing assembly can contain at least age of Surfactant in a suitable solution of ghrelin is about one fluidic conduit, for example at least one fluidic channel. 5%. In some embodiments, a composition Suitable for nasal In some embodiments, the mixing assembly contains at least administration, the percentage of Surfactant in a Suitable one microfluidic channel. The mixing assembly is also solution of ghrelin is about 10%. configured to transfer the dissolved or reconstituted wet and 0350. In some embodiments, a composition comprising dry components into a needle assembly or other delivery ghrelin or the ghrelin variant that is Suitable for nasal assembly configured to inject or deliver said components administration may include one or more buffering agents for into a Subject, person or animal. controlling the pH of the composition. Buffering agents that 0346. In some embodiments, the composition comprising may be used in the compositions of the present invention ghrelin or the ghrelin variant is administered via inhalation, include citric acid and Sodium citrate dihydrate. In some oral, intravenous, parenteral, buccal, Subcutaneous, trans embodiments, a composition Suitable for nasal administra dermal, patch, Sublingual, intramuscular, or intranasal. In tion, the percentage of buffering agent in a suitable Solution Some embodiments, ghrelin is administered in a single dose. of ghrelin is about 0.001%. In some embodiments, a com In some embodiments, ghrelin is administered in multi position Suitable for nasal administration, the percentage of doses. In some embodiments, ghrelin is administered at a buffering agent in a suitable solution of ghrelin is about dosage from 10 ng/kg per day to 10 mg/kg per day (or any 0.005%. In some embodiments, a composition suitable for Sub Value or Sub range there between, e.g., 0.1 ug/kg per day nasal administration, the percentage of buffering agent in a to 5 mg/kg per day). In some embodiments, a dosing suitable solution of ghrelin is about 0.01%. In some embodi regimen (2 ug/kg per day, for example delivered intrave ments, a composition Suitable for nasal administration, the nously) is administered within 8 hours following injury. The percentage of buffering agent in a Suitable solution of ghrelin dosing is a one-time dose with possible recurrent dosing is about 0.1%. based on patient symptoms. 0351. In some embodiments, the osmolarity of the com 0347 Nasal delivery is a non-invasive route for thera position comprising ghrelin or the ghrelin variant may be peutics targeting the central nervous system because of controlled by propylene glycol. When a composition com relatively high permeability of nasal epithelium membrane, prising ghrelin or the ghrelin variant is a gel, the composi avoidance of hepatic first pass elimination. Nasal delivery is tion may include a gelling agent Such as hydroxylpropyl easy to administer and allows for self-medication by an cellulose, carbopols, carboxymethylcellulose, and ethylcel individual. Nasal mucociliary clearance is an important lulose. In some embodiments, the composition comprising US 2017/O121385 A1 May 4, 2017

ghrelin or the ghrelin variant may include a preservative about 0.45 g/cm3 to about 1.2 g cm3, at least about 0.55 such as ethylenediaminetetraacetic acid (EDTA) and ben g/cm3 to about 1.1 g cm3, or at least about 0.65 g cm3 to Zalkonium chloride. Non-limiting examples of Suitable sol about 1.0 g cm.J.; and a total content of therapeutic agent or vents for compositions of the present invention include agents of at least 25%, at least 35%, at least 50%, at least water, vegetable oil and ethanol. In some embodiments, the 65%, or at least 80% by weight (i.e., dry weight relative to use of a nasal inhalant reduces the concentration required to the total dry weight of dry powder). The powders can be treat mBI and prevent unwanted side effects. further characterized by an angle of repose of 50° or less, 0352. In some embodiments, nasal administration is a 40° or less, or 30° or less. The particles can be further more practical means of delivery in a military or sport characterized by a dispersibility ratio (1 bar/4 bar) of less setting. In some embodiments, the present invention pro than about 2 as measured by laser diffraction (RODOS/ vides a method for improving the standard of care for HELOS system), less than about 1.7, less than about 1.4, or preventing or treating mBI in military personnel or athletes less than about 1.2. The particles can be further character through a prophylactic and post-acute intranasal therapeutic. ized by a fine particle fraction (e.g., FPF<5.6, <5.0, <4.4 or In some embodiments, the active ingredient of the thera <3.4) of 30% or greater, 40% or greater, 50% or greater, or peutic is ghrelin. In some embodiments, ghrelin may be part 60% or greater. of a formulation that is delivered intranasally to facilitate 0355. In some embodiments, the respirable dry powders ease of access and use in the field and to minimize the dose comprising respirable dry particles, are “processable.” For required further limiting side effects. example, the dry powders can be deposited or filled into a 0353. In some embodiments, the composition comprising sealable receptacle that has a volume of about 12 cubic ghrelin or the ghrelin variant is administered via the millimeters (mm3) or less, a volume of about 9 mm3 or less, iSPERSE (inhaled small particles easily respirable and emit a volume of about 6 mm3 or less, a volume of about 3 mm3 ted) technology, which is a dry powder technology devel or less, a volume of about 1 mm3 or less, or a volume of oped by Pulmatrix. iSPERSE particles are engineered to be about 0.5 mm3 or less, preferably to substantially fill the Small, dense and easily dispersible. In some embodiments, volume of the receptacle. Alternatively or in addition, the the iSPERSE technology allows flexible drug loading for powders can be deposited or filled into a sealable receptacle delivery of microgram to tens of milligrams per dose: to provide a mass of about 1 mg or less, about 0.75 mg or iSPERSE particles do not require lactose or other carriers less, about 0.5 mg or less, about 0.3 mg or less, about 0.1 mg and can be engineered to include <1% to greater than 80% or less, or about 0.05 mg or less of powder in the receptacle. API to allow for dosing of low potency and high drug load 0356. The respirable dry powders consisting of respirable therapeutics. In some embodiments, the iSPERSE technol dry particles can be deposited into receptacles to provide a ogy allows reproducible and one-step manufacture: total dry powder mass of between about 5 mg to about 15 iSPERSE powders are manufactured by a scalable and mg, between about 5 mg and less than 10 mg, between about reproducible one-step spray drying process with high and 5 mg and about 9 mg, between about 5 mg and about 8 mg, consistent yields. Formulations are created independent of or between about 5 mg and about 8 mg. The receptacles that API physical chemistry in either crystalline or amorphous contain the dry powder mass can be sealed if desired. excipient matrices. In some embodiments, the iSPERSE 0357 The dry powders comprising respirable dry par technology allows Superior flow rate independent pulmo ticles can be deposited into receptacles to provide a total dry nary administration: iSPERSE formulations are dispersible powder mass of about 5 mg or less, about 4 mg or less, about across a range of flow rates with consistent emitted dose and 3 nig or less or about 2 mg or less, and provide about 1 mg particle size. Performance across flow rates provides reliable or more, wherein the total dry powder mass contains 1.5 mg dose delivery across patient populations and reduces patient or more, or about 2 mg or more of one or more therapeutic to-patient variability. In some embodiments, the iSPERSE agents. In Such embodiments, the receptacle will contain technology allows delivery of macromolecules and biolog between 1.5 mg and about 5 mg or less, or about 2 mg and ics: iSPERSE enables delivery of antibodies, peptides and about 5 mg or less of total dry powder mass. The receptacles nucleic acids across a range of drug loads and with robust that contain the dry powder mass can be sealed if desired. product performance. In some embodiments, the iSPERSE 0358. The total content of therapeutic agent or agents in technology allows homogenous combinations of multiple the respirable dry powder is at least 20%, at least 25%, at drugs: iSPERSE creates homogenous particles including least 35%, at least 50%, at least 65%, or at least 80% by excipients and API. Dual and triple iSPERSE combinations weight (i.e., dry weight relative to the total dry weight of dry have been manufactured to date. In some embodiments, the powder). The one or more metal cation salt can be present iSPERSE technology allows flexibility of patient interface. in the respirable dry particles in any desired amount. Such as iSPERSE dry powders are compatible with a range of about 3% by weight or more of the respirable particles, 5% inhalers, allowing for a products configuration to be tai by weight of the respirable particles, 10% by weight of the lored to the specific needs of a patient population. The respirable particles, 15% by weight of the respirable par iSPERSE technology is disclosed in U.S. Patent Application ticles, or in 20% by weight of the respirable particles. The No. 2015/0136130, the disclosure of which is incorporated one or more metal cation salt can independently be selected herein by reference in its entirety. from the group consisting of a sodium salt, a potassium salt, 0354. In some embodiments, the respirable dry powder a magnesium salt, and a calcium salt. comprises respirable dry particles that contain at least one 0359. The respirable dry powder is filled or deposited therapeutic agent and at least one metal cation salt, such as into receptacles using standard filling equipment such as a a sodium salt, a potassium salt, a magnesium salt, or a vacuum dosator, for example, a rotating drum vacuum calcium salt, and that have a volume median geometric dosator, e.g., the Omnidose TT (Harro Hofliger, Germany). diameter (VMGD) about 10 micrometers or less. These dry The volume of the receptacle into which the respirable dry particles can be further characterized by a tap density at least powder is filled can be 400 microliters or less, 330 micro US 2017/O121385 A1 May 4, 2017 42 liters or less, 250 microliters or less, 150 microliters or less, of the receptacles are filled within 80% to 120% of the target 70 microliters or less, 40 microliters or less, or 20 microliters fill weight, at least 80% of the receptacles are filled within or less. In one aspect, the respirable dry powder can be filled 85% to 115% of the target fill weight, or 85% of the into two or more receptacles that are physically attached to receptacles are filled within 90% to 110% of the target fill each other or in an array, for example, using an intercon weight. More preferably, at least 85% of the receptacles are nected blister piece comprising 30 blisters or more, 60 filled within 90% to 110% of the target fill weight. blisters or more, 90 blisters or more, or 120 blisters or more. 0362. In addition or alternatively to any of the forgoing Each receptacle or array of receptacles (e.g., an intercon processability characteristics, the processable powders can nected blister piece) can be filled at a rate of about every 10 further be filled into a receptacle for use in a DPI at a rate seconds or less, about every 8 seconds or less, about every of 300 receptacles or more every hour, 500 receptacles or 6 seconds or less, about every 4 seconds or less, about every more every hour, 750 receptacles or more every hour, 1100 2 seconds or less, or about every 1 second or less. Preferably, receptacles or more every hour, 1500 receptacles or more the relative standard deviation (RSD) is about 3% or less, every hour, 2000 receptacles or more every hour, 2500 about 2.5% or less, about 2% or less, or about 1.5% or less. receptacles or more every hour, or 3000 receptacles or more A dry powder inhaler (DPI) that contains the receptacles can every hour. Preferably, at least 70% of the receptacles are be any suitable DPI, such as a multi-dose blister DPI, a filled within 80% to 120% of the target fill weight, at least single-dose capsule DPI, or other DPI. The angle of repose 80% of the receptacles are filled within 85% to 115% of the of the respirable dry powder that is filled into the receptacles target fill weight, or at least 85% of the receptacles are filled can be 50° or less, 40" or less, or 30° or less. The processable within 90% to 110% of the target fill weight. In addition or powder may be essentially free of non-respirable carrier alternatively, the relative standard deviation of the fill weight particles, such as lactose, that have a VMGD that is greater is 3% or less, 2.5% or less, 2% or less, or 1.5% or less. than 10 micrometers, about 20 micrometers or greater, 30 Filling equipment that may be used include pilot scale micrometers or greater, or 40 micrometers or greater. equipment and commercial scale equipment. 0360. In other examples, the processable powders can be 0363. In some embodiments, the respirable dry powders metered in a multi-dose reservoir dry powder inhaler (DPI), are processable and dispersible. Such dry powders can the metering achieved by a dosing cup, disk, or other contain a proportionately large mass of one or more thera structure for dosing in the reservoir DPI itself. Unit doses peutic agents (e.g., 50% or more (w/w) by dry weight) and can be metered which are 100 cubic millimeters or less, 75 be administered to a subject to deliver an effective amount cubic millimeters or less, 50 cubic millimeters or less, 35 of the therapeutic agent to the respiratory tract. For example, cubic millimeters or less, 20 cubic millimeters or less, 10 a unit dosage form of the dry powder, provided as a small cubic millimeters or less, 5 cubic millimeters or less, or 2.5 Volume receptacle (e.g., capsule or blister) with the dry cubic millimeters or less. In some aspects, the metering powder disposed therein or as a reservoir-based DPI metered mechanism can possess one receptacle to measure a unit to dispense a small Volume, can be used to deliver an dose, and in other aspects, the metering mechanism can effective amount of the therapeutic agent to the respiratory possess multiple receptacles to measure a unit dose. Alter tract of a subject in need thereof. In one aspect, at least 20 natively or in addition, the processable powders can further milligrams of one or more therapeutic agent can be delivered be characterized as processable in that the mass of the to the respiratory tract from a small Volume unit dosage metered dose from a multi-dose reservoir DPI is within 80% form. For example, at least about 25 milligrams, at least to 120% of a target mass 85% or more of the time, or within about 30 milligrams, at least about 45 milligrams, at least 85% to 115% of a target mass 90% of the time, or within about 60 milligrams, at least about 80 milligrams, at least 90% to 110% of a target mass 90% of the time. Preferably, about 100 milligrams, at least about 130 milligrams, at least the mass of the metered dose from a multi-dose reservoir about 160 milligrams, or at least about 200 milligrams of DPI is within 85% to 115% of a target mass 90% or more of one or more therapeutic agent can be delivered to the the time, or within 90% to 110% of a target mass 90% or respiratory tract from a unit dosage form provided as a small more of the time. The processable dry powders can be volume receptacle (e.g., volume of about 400 microliters or further be characterized by an angle of repose of 50° or less, less, about 370 microliters or less, less than 370 microliters, 40° or less, 30° or less. Angle of repose is a characteristic about 300 microliters or less, less than about 300 microliters, that can describe both respirable dry powder as well as the preferably, about 370 microliters, or about 300 microliters) powder's processability. with the dry powder disposed therein. In some embodi 0361. In addition or alternatively to any of the forgoing ments, the receptacle is a size 2 or a size 3 capsule. processability characteristics, the processable powders can 0364. Features of the dry powders, receptacle and/or further be filled into a receptacle for use in a DPI at a rate inhaler can be adjusted to achieve the desired delivery of an of one receptacle about every 10 seconds or less, about every effective amount of the therapeutic agent to the respiratory 8 seconds or less, about every 6 seconds or less, about every tract of a subject in need thereof. Such features include 1) the 4 seconds or less, about every 2 seconds or less, about every therapeutic agent load in the dry particles or dry powder; 2) 1 second or less, or about every 0.5 seconds or less; and/or the bulk density of the dry powder, 3) the degree to which filled into receptacles for use in a DPI at a rate of 300 the receptacle is filled with the dry powder, and 4) the receptacles every hour, 500 receptacles every hour, 750 processability and dispersibility of the dry powder. The receptacles every hour. 1 100 receptacles every hour, 1500 therapeutic agent load in the dry powder is generally at least receptacles every hour, 2000 receptacles every hour, 2500 about 25%, at least about 35%, at least about 50%, at least receptacles every hour, or 3000 receptacles every hour. The about 65%, at least about 80%, or at least about 90% by rate of filling the receptacles can also be 800 receptacles or weight, on a dry basis. The bulk density of the dry powder more every hour, 1600 receptacles or more every hour or is generally greater than 0.1 g cc, between about 0.2 g/cc and 2400 receptacles or more every hour. Preferably, at least 700 about 0.9 g cc, and preferably, at least about 0.3 g/ml, at least US 2017/O121385 A1 May 4, 2017

about 0.4 g/ml, or at least 0.5 g/ml. The bulk density, also administration also particularly Suits drugs targeting the referred to as the apparent density, is a measure that indicates brain because certain drug Solutions can bypass the blood how much dry powder can be filled into a fixed volume brain barrier (BBB) and reach the central nervous system without the intense compaction experienced when determin (CNS) directly from the nasal cavity—uptake of these drugs ing the tap density of a dry powder. The receptacle is depends on their molecular weight and lipophilicity. The generally filled with dry powder to be at least 50% full, intranasal delivery increases brain levels of the drug while preferably, at least 60% full, at least 70% full, or at least 90% decreasing systemic concentrations and thus should have full. The processability and dispersibility of the dry powder less harmful side effects. can be altered, as desired, by including appropriate amounts 0368. In some embodiments, the compositions compris of one or more monovalent and/or divalent metal cation ing ghrelin or ghrelin variants are delivered by Precisa TM salts, (e.g., a Sodium salt, a potassium salt, a magnesium salt, Platform. Precisa TM is Edge Therapeutics proprietary, pro a calcium salt, or a combination thereof, total metal cation grammable, biodegradable polymer-based development salts less than about 75%, equal to or less than about 60%, platform. It allows creating polymer-based therapeutics about 50%, about 40%, about 300%, about 200%, about capable of delivering therapeutics directly to the site of 100%, about 5%), and optionally, one or more other excipi injury to potentially avoid serious systemic side effects often ents (e.g., carbohydrates, Sugar alcohols, and/or amino acids, associated with oral or intravenous delivery. Precisa TM total excipients equal to or less than about 700, about 55%, enables high and Sustained drug exposure with only a single about 40%, about 30%, about 20%, about 10%, about 5%) dose at the initial time of procedural or Surgical intervention. in the dry powders or dry particles. If desired, the therapeutic 0369 Precisa TM can be designed based on specific physi agent load may be at least about 20% by weight, on a dry cal and chemical properties (size, shape, Surface area) of the basis. Although it is preferable that the receptacle is filled at drug to be delivered that allow for one-time administration least 50%, the receptacle can be filled to any desired degree, at or near the targeted injured organ, Vessel or cell. In some such as at least 10% filled, at least 20% filled, at least 30% embodiments, the specific form of PrecisatM microparticles filled, or at least 40% filled. containing the compositions comprising ghrelin or ghrelin 0365. In some embodiments, the present invention pro variants that is Small enough to allow easy administration vides compositions comprising ghrelin or the ghrelin variant through a brain catheter, yet large enough to prevent mac that are applied as nasal drops, eye drops and nasal sprays. rophages from carrying the microparticles away from the For the nasal application, a solution or Suspension may be site of injury. used which is applied as spray, i.e., in the form of a fine 0370 PrecisatM is programmed with a specific blend of dispersion in air or by means of a conventional spray polymers in order to obtain the desired release profile of the Squeeze bottle or pump. Suitable nontoxic pharmaceutically selected therapeutic. This is accomplished by immersing the acceptable carriers for use in a drug delivery system for specified therapeutic in a matrix of clinically validated, intranasal administration of ghrelin may include, but not biodegradable and biocompatible polymers. The foundation limited to, carriers used for nasal pharmaceutical formula of PrecisatM is poly (DL-Lactic-co-glycolide), or PLGA, the tions for other steroids, such as estrogen. polymer in dissolvable sutures that has been used since the 0366. In some embodiments, formulations of the present 1970s. PLGA is biodegradable, has minimal toxicity in invention may contain a preservative and/or stabilizer. These humans, even when used intracranially, and is one of the few include, but not limited to, ethylene diamine tetraacetic acid matrix delivery systems where drug release can be sustained (EDTA) and its alkali salts (for example dialkali salts such over weeks. as disodium salt, calcium salt, calcium-sodium salt), lower 0371. Upon administration, the therapeutic that is on the alkyl p-hydroxybenzoates, chlorhexidine (for example in the Surface of the polymer immediately releases to provide high form of the acetate or gluconate) and phenyl mercury borate. initial concentrations of Such therapeutic. Subsequently, the Other suitable preservatives are: pharmaceutically useful therapeutic begins to diffuse through the polymer-based quaternary ammonium compounds, for example cetylpyri matrix and the polymer breaks down into lactic acid, a dinium chloride, tetradecyltrimethyl ammonium bromide, compound naturally found in the body, in order to deliver the generally known as “cetrimide', N-Benzyl-N,N-dimethyl therapeutic with the desired release profile. 2-2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy 0372. In some embodiments, the compositions compris ethoxyethanaminium chloride, generally known as “ben ing ghrelin or ghrelin variants are delivered by Quanterix's Zethonium chloride' and myristyl picolinium chloride. Each proprietary Simoa TM technology. SimoaTM technology is of these compounds may be used in a concentration of about based upon the isolation of individual immunocomplexes on 0.002 to 0.05%, for example about 0.02% (weight/volume in paramagnetic beads using standard ELISA reagents. The liquid formulations, otherwise weight/weight). In some main difference between Simoa and conventional immuno embodiments, preservatives among the quaternary ammo assays lies in the ability to trap single molecules in femto nium compounds are, but not limited to, alkylbenzyl dim liter-sized wells, allowing for a “digital' readout of each ethyl ammonium chloride and mixtures thereof, for individual bead to determine if it is bound to the target example, benzalkonium chloride. analyte or not. 0367 Intranasal (IN) administrations may have fewer 0373. In some embodiments, for acute treatments, nasal side effects than intraperitoneal (IP) administrations due to administration of the composition comprising the ghrelin a shift in pharmaceutical research to nasal sprays, drops and variant may reduce the time for uptake and increase the gels: the nasal route of drug administration continues to concentration of the ghrelin variant that reaches the blood or receive increasing attention from pharmaceutical Scientists brain. and clinicians because this route circumvents hepatic first 0374. In some embodiments, ghrelin or the ghrelin vari pass elimination associated with oral delivery, is easily ant is administered in a single dose. In some embodiments, accessible and Suitable for self-medication. Intranasal ghrelin or the ghrelin variant is administered in multi-doses. US 2017/O121385 A1 May 4, 2017 44

In some embodiments, ghrelin or the ghrelin variant is 0380. Other forms suitable for oral administration administered at a dosage from 10 ng/kg per day to 10 mg/kg include liquid form preparations including emulsions, Syr per day. ups, elixirs, aqueous solutions, aqueous Suspensions, tooth 0375. In some embodiments, ghrelin or the ghrelin vari paste, gel dentifrice, chewing gum, or Solid form prepara ant compositions may be formulated in an oral administra tions which are intended to be converted shortly before use tion dosage forms. The pharmaceutical compositions and to liquid form preparations. Emulsions may be prepared in dosage forms may comprise the compounds disclosed herein Solutions in aqueous propylene glycol solutions or may or their pharmaceutically acceptable salt or crystal forms contain emulsifying agents such as lecithin, Sorbitan thereof as the active component. monooleate, or acacia. Aqueous Solutions can be prepared by dissolving the active component in water and adding 0376. The pharmaceutical acceptable carriers can be Suitable colorants, flavors, stabilizing and thickening agents. either solid or liquid. Solid form preparations include pow Aqueous Suspensions can be prepared by dispersing the ders, tablets, pills, capsules, cachets, Suppositories, and finely divided active component in water with viscous dispersible granules. A Solid carrier can be one or more material. Such as natural or synthetic gums, resins, methyl Substances, which may also act as diluents, flavoring agents, cellulose, sodium carboxymethylcellulose, and other well solubilizers, lubricants, Suspending agents, binders, preser known Suspending agents. Solid form preparations include Vatives, wetting agents, tablet disintegrating agents, or an Solutions, Suspensions, and emulsions, and may contain, in encapsulating material. addition to the active component, colorants, flavors, stabi 0377 For oral administration, such excipients include, lizers, buffers, artificial and natural Sweeteners, dispersants, e.g., pharmaceutical grades of mannitol, lactose, starch, thickeners, Solubilizing agents, and the like. magnesium Stearate, Sodium saccharine, talcum, cellulose, 0381. The administration of ghrelin or ghrelin variants glucose, gelatin, Sucrose, magnesium carbonate, and the are based on Suitable dosing regimens that take into account like. In powders, the carrier is a finely divided solid, which factors well-known in the art including, e.g., type of Subject is a mixture with the finely divided active component. In being dosed; age, weight, sex and medical condition of the tablets, the active component is mixed with the carrier Subject; the route of administration; the renal and hepatic having the necessary binding capacity in Suitable propor function of the subject; the desired effect; and the particular tions and compacted in the shape and size desired. The compound employed. Optimal precision in achieving con powders and tablets contain from one to about seventy centrations of drug within the range that yields efficacy percent of the active compound. Suitable carriers are mag without toxicity requires a regimen based on the kinetics of nesium carbonate, magnesium Stearate, talc, Sugar, lactose, the drugs availability to target sites. This involves a con pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sideration of the distribution, equilibrium, and elimination of Sodium carboxymethylcellulose, a low melting wax, cocoa a drug. butter, and the like. The term “preparation' is intended to 0382. In some embodiments, ghrelin and ghrelin variants include a composition comprising an active compound dis are administered subcutaneously. In some embodiments, closed herein with encapsulating material as carrier provid ghrelin and ghrelin variants are administered as a bolus, ing a capsule in which the active component, with or without wherein the administration form may be any suitable par carriers, is Surrounded by a carrier, which is in association enteral form. with it. Similarly, cachets and lozenges are included. Tablets, 0383 Pharmaceutical compositions for parenteral admin powders, capsules, pills, cachets, and lozenges can be as istration include sterile aqueous and non-aqueous injectable solid forms suitable for oral administration. Solutions, dispersions, Suspensions or emulsions, as well as 0378 Drops may comprise sterile or nonsterile aqueous sterile powders to be reconstituted in sterile injectable or oil solutions or Suspensions, and may be prepared by Solutions or dispersions prior to use. Other Suitable admin dissolving the active ingredient in a suitable aqueous solu istration forms include Suppositories, sprays, ointments, tion, optionally including a bactericidal and/or fungicidal creams, gels, inhalants, dermal patches, implants, pills, agent and/or any other Suitable preservative, and optionally tablets, lozenges and capsules. including a Surface active agent. The resulting solution may 0384. A typical non-limiting dosage is in a concentration then be clarified by filtration, transferred to a suitable equivalent to from 10 ng to 10 mg ghrelin variant per kg container which is then sealed and sterilized by autoclaving bodyweight. The concentrations and amounts herein are or maintaining at 98-100° C. for half an hour. Alternatively, given in equivalents of amount ghrelin variant. For example, the solution may be sterilized by filtration and transferred to where the ghrelin variant is a 28 amino acid human ghrelin the container aseptically. Examples of bactericidal and fun (SEQ ID NO: 1) and/or a 24 amino acid human ghrelin gicidal agents suitable for inclusion in the drops are phe splice variant having a Dpr residue at the third position (SEQ nylmercuric nitrate or acetate (0.002%), benzalkonium chlo ID NO:3) and/or a 24 amino acid human ghrelin splice ride (0.01%) and chlorhexidine acetate (0.01%). Suitable variant having Dpr residues at the second and third positions Solvents for the preparation of an oily solution include (SEQ ID NO:4) and being optionally octanoylated on the glycerol, diluted alcohol and propylene glycol. Dpr residue in the third position. In some embodiments, the 0379 Also included are solid form preparations, which dosage can be the same for Smaller peptides (e.g., RM-131 are intended to be converted, shortly before use, to liquid pentapeptide) or other ghrelin mimetics, antagonists, or form preparations for oral administration. Such liquid forms agonists described herein. In some embodiments, a ghrelin include Solutions, Suspensions, and emulsions. These prepa or ghrelin variant is administered in a concentration equiva rations may contain, in addition to the active component, lent to from about 0.1 ug to about 1 mg ghrelin per kg colorants, flavors, stabilizers, buffers, artificial and natural bodyweight. Such as from about 0.5 g to about 0.5 mg Sweeteners, dispersants, thickeners, Solubilizing agents, and ghrelin per kg bodyweight, Such as from about 1.0 ug to the like. about 0.1 mg ghrelin per kg bodyweight, such as from about US 2017/O121385 A1 May 4, 2017

1.0 ug to about 50 ug ghrelin per kg bodyweight. Such as similar way as for cartridge growth hormone (GH) or from about 1.0 ug to about 10 ugghrelin per kg bodyweight. Insulin. The cartridge contains compounds disclosed herein 0385. In some embodiments, ghrelin or ghrelin variants in solvents. The pen, which is basically a needle, Syringe and are administered in a concentration equivalent to from 0.1 ug vial in one piece, is operated by a turning movement and to 1 mg ghrelin variant per kg bodyweight, such as from 0.5 allows different doses to be administrated. This device offers ug to 0.5 mg ghrelin variant per kg bodyweight, Such as from simplicity, convenience, and enhanced safety features for 1.0 ug to 0.1 mg ghrelin variant per kg bodyweight, such as compounds delivery. It provides a simple device design, few from 1.0 g to 50 ugghrelin variant per kg bodyweight, Such administration steps and one-step dial-back dose knob. Such as from 1.0 ug to 10 ug ghrelin variant per kg bodyweight. injection pen can be obtained by means known in art. 0386. In some embodiments, an intravenous injection of 0391 Ghrelin or ghrelin variant compositions may be ghrelin variant is employed. The administration route must formulated for parenteral administration (e.g., by injection, ensure that the non-degraded, bioactive form of the peptide for example bolus injection or continuous infusion) and may will be the dominating form in the circulation, which will be presented in unit dose form in ampules, pre-filled reach and stimulate the ghrelin variant receptors in order to Syringes, Small Volume infusion or in multi-dose containers obtain the maximum effect of ghrelin variant treatment on with an added preservative. The compositions may take Such mBI. In some embodiments, ghrelin or the ghrelin variant is forms as Suspensions, solutions, or emulsions in oily or administered within about 30 minutes of the incident that aqueous vehicles, for example solutions in aqueous poly results in mBI. In some embodiments, ghrelin or the ghrelin ethylene glycol. Examples of oily or nonaqueous carriers, variant is administered within about 30 minutes to about 2 diluents, solvents or vehicles include propylene glycol, hours of the incident that results in mBI. In some embodi polyethylene glycol, vegetable oils (e.g., olive oil), and ments, ghrelin or the ghrelin variant is administered within injectable organic esters (e.g., ethyl oleate), and may contain about 30 minutes to about 6 hours of the incident that results formulatory agents such as preserving, wetting, emulsifying in mBI. In some embodiments, ghrelin or the ghrelin variant or Suspending, stabilizing and/or dispersing agents. Alter is administered within about 30 minutes to about 12 hours of natively, the active ingredient may be in powder form, the incident that results in mBI. obtained by aseptic isolation of sterile solid or by lyophiliza 0387 Ghrelin variant compositions may also be formu tion from solution for constitution before use with a suitable lated for nasal administration. The solutions or Suspensions vehicle, e.g., Sterile, pyrogen-free water. Aqueous solutions are applied directly to the nasal cavity by conventional should be suitably buffered if necessary, and the liquid means, for example with a dropper, pipette or spray. The diluent first rendered isotonic with sufficient saline or glu compositions may be provided in a single or multi-dose cose. The aqueous solutions are particularly Suitable for form. In the latter case of a dropper or pipette, this may be intravenous, intramuscular, Subcutaneous and intraperito achieved by the patient administering an appropriate, pre neal administration. The sterile aqueous media employed are determined Volume of the Solution or Suspension. In the case all readily available by standard techniques known to those of a spray, this may be achieved for example by means of a skilled in the art. metering atomizing spray pump. 0392 Ghrelin or ghrelin variant compositions may be 0388 Ghrelin variant compositions may be formulated prepared in Solutions, such as water or saline, and optionally for aerosol administration, particularly to the respiratory mixed with a nontoxic Surfactant. Compositions for intra tract and including intranasal administration. The compound venous or intra-arterial administration may include sterile will generally have a small particle size, for example of the aqueous solutions that may also contain buffers, liposomes, order of 5 microns or less. Such a particle size may be diluents and other suitable additives. Oils useful in parent obtained by means known in the art, for example by eral compositions include petroleum, animal, vegetable, or micronization. The active ingredient is provided in a pres synthetic oils. Specific examples of oils useful in such Surized pack with a Suitable propellant Such as a hydrofluo compositions include peanut, soybean, Sesame, cottonseed, roalkane (HFA) for example hydrofluoroalkane-134a and corn, olive, petrolatum, and mineral. Suitable fatty acids for hydrofluoroalkane-227, carbon dioxide or other suitable gas. use in parenteral compositions include oleic acid, Stearic The aerosol may conveniently also contain a surfactant Such acid, and isostearic acid. Ethyl oleate and isopropyl as lecithin. The dose of drug may be controlled by a metered myristate are examples of Suitable fatty acid esters. valve. 0393. The parenteral compositions typically will contain 0389. Alternatively, the active ingredients may be pro from about 0.5 to about 25% by weight of the active vided in a form of a dry powder, for example a powder mix ingredient in solution. Preservatives and buffers may be of the compound in a suitable powder base Such as lactose, used. In order to minimize or eliminate irritation at the site starch, starch derivatives such as hydroxypropylmethyl cel of injection, Such compositions may contain one or more lulose and polyvinylpyrrolidine (PVP). The powder carrier nonionic Surfactants having a hydrophile-lipophile balance will form a gel in the nasal cavity. The powder composition (HLB) of from about 12 to about 17. The quantity of may be presented in unit dose form for example in capsules Surfactant in Such compositions will typically range from or cartridges of, e.g., gelatin or blister packs from which the about 5 to about 15% by weight. Suitable surfactants include powder may be administered by means of an inhaler. Com polyethylene Sorbitan fatty acid esters, such as Sorbitan positions administered by aerosols may be prepared, for monooleate and the high molecular weight adducts of eth example, as Solutions in Saline, employing benzyl alcohol or ylene oxide with a hydrophobic base, formed by the con other Suitable preservatives, absorption promoters to densation of propylene oxide with propylene glycol. The enhance bioavailability, employing fluorocarbons, and/or parenteral compositions can be presented in unit-dose or employing other solubilizing or dispersing agents. multi-dose sealed containers, such as ampules and vials, and 0390 Ghrelin or ghrelin variant compositions may also can be stored in a freeze-dried (lyophilized) condition be formulated for administration by injection pen in a requiring only the addition of the sterile liquid excipient, for US 2017/O121385 A1 May 4, 2017 46 example, water, for injections, immediately prior to use. homogeneously therein, for example, by stirring. The mol Extemporaneous injection Solutions and Suspensions can be ten homogeneous mixture is then poured into convenient prepared from sterile powders, granules, and tablets of the sized molds, allowed to cool, and to solidify. The active kind previously described. compound may be formulated into a Suppository compris 0394 The pharmaceutical dosage forms suitable for ing, for example, about 0.5% to about 50% of a compound injection or infusion can include sterile aqueous solutions or disclosed herein, disposed in a polyethylene glycol (PEG) dispersions comprising the active ingredient that are adapted carrier (e.g., PEG 1000 96% and PEG 4000 (4%). for administration by encapsulation in liposomes. In all cases, the ultimate dosage form must be sterile, fluid and Combination Therapies, Products and Compositions stable under the conditions of manufacture and storage. 0401 Some embodiments relate to products that com 0395 Sterile injectable solutions are prepared by incor prise two or more agents as discussed herein that can be porating a ghrelin variant or pharmaceutical acceptable salt utilized in combination. For example, the at least two agents thereof in the required amount in the appropriate solvent can be selected from ghrelin, a ghrelin variant, an anti with various of the other ingredients enumerated above, as inflammatory agent, anti-pain medication, acetylsalicylic required, followed by, e.g., filter sterilization. acid, an antiplatelet agent, a thrombolytic enzyme, an aggre 0396 Ghrelin or ghrelin variant compounds can also be gation inhibitor, a glycoprotein IIb/IIIa inhibitor, a gly delivered topically. Regions for topical administration cosaminoglycan, a thrombin inhibitor, an anticoagulant, include the skin Surface and also mucous membrane tissues heparin, coumarin, warfarin (Coumadin), tRA, GCSF, strep of the rectum, nose, mouth, and throat. Compositions for tokinase, urokinase, Ancrod, melatonin, a caspase inhibitor, topical administration via the skin and mucous membranes an NMDA receptor agonist or antagonist, an anti-TNF-C. should not give rise to signs of irritation, such as Swelling or compound, an antibody, erythropoietin/EPO, angiotensin II redness. lowering agent, selective androgen receptor modulator, lep 0397 Ghrelin or ghrelin variant compounds may include tin, an agonists of the renin-angiotensin System, an opioid a pharmaceutical acceptable carrier adapted for topical receptor agonist, progesterone, a peroxisome proliferator administration. Thus, the composition may take the form of activated receptor gamma agonist, P7C3, P2Y purinergic for example, a Suspension, Solution, ointment, lotion, cream, receptor agonist, UCP-2 agonists, glypromate, NNZ-2591 foam, aerosol, spray, Suppository, implant, inhalant, tablet, (Neuren), NNZ-2566 (Neuren), cyclosporine A (NeuroVive capsule, dry powder, syrup, balm or lozenge. Methods for Pharmaceutical), NTX-265 (Stem Cell Therapeutics), preparing such compositions are well known in the phar DP-b99 (D-Pharm), apomorphine, Epoetin Alfa (EPO), pro maceutical industry. gesterone, KN 38-727 1/BAY38-7271 (KeyNeurotek/Bayer 0398 Ghrelin or ghrelin variant compounds may be AG), VAS-203 (Vasopharm), SAR 127963 (Sanofi), formulated for topical administration to the epidermis as BHR100 (BHR Pharma), Oxycyte (Oxygen Biotherapeu ointments, creams or lotions, or as a transdermal patch. tics), Sulfonamide compounds, Ebselen (2-phenyl-1,2-ben Ointments and creams may, for example, beformulated with Zisoselenazol-3(2H)-one), SPI-1005 (Sound Pharmaceuti an aqueous or oily base with the addition of suitable thick cals), glutathione peroxidase, glutathione peroxidase mimics ening and/or gelling agents. Lotions may be formulated with and inducers, aducanumab (BIIB037, Biogen Idec), and any an aqueous or oily base and will in general also containing other compounds described herein, and the like. one or more emulsifying agents, stabilizing agents, dispers 0402 Glypromate is a naturally occurring peptide frag ing agents, Suspending agents, thickening agents, or coloring ment that is found in normal brain tissue. When injected agents. Compositions suitable for topical administration in intravenously, glypromate has been shown to act by multiple the mouth include lozenges, pastilles, and mouthwashes. pathways in protecting brain tissue from injury. NNZ-2591 0399. Ghrelin or ghrelin variant compounds may be (cyclo-L-glycyl-L-2-allylproline), a diketopiperazine, has administered transdermally, which involves the delivery of been shown to be neuroprotective after ischemic brain injury a pharmaceutical agent for percutaneous passage of the drug and also improves motor function in a rat model of Parkin into the systemic circulation of the patient. The skin sites son's disease. NNZ-2566, a synthetic analogue of neuro include anatomic regions for transdermally administering protective tripeptide glypromate, is an IGF-1 like neuropep the drug and include the forearm, abdomen, chest, back, tide that is a caspase-3 inhibitor. Cyclosporine (cyclosporin, buttock, and the like. Transdermal delivery is accomplished cyclosporine, cyclosporine A, or CSA) is an immunosup by exposing a source of the active compound to a patients pressant drug used in organ transplantation to prevent rejec skin for an extended period of time. Transdermal patches can tion that Suppresses the activity of the immune system by add advantage of providing controlled delivery of a com interfering with T cell activation and proliferation. KN pound complex to the body. Such dosage forms can be made 38-7271 is a CBI-2 agonist. VAS-203 is an allosteric NO by dissolving, dispersing, or otherwise incorporating a ghre synthase inhibitor. SAR127963 is a P75 receptor antagonist. lin variant compound in a proper medium, Such as an BHR100 is a progesterone receptor agonist. Oxycyte is a elastomeric matrix material. Absorption enhancers can also perfluorocarbon oxygen carrier. KN 38–727 1/BAY38-7271 be used to increase the flux of the compound across the skin. is a cannabinoid receptor agonist. DP-b99 is a Membrane The rate of such flux can be controlled by either providing Active Chelator (MAC) derivative of the known calcium a rate-controlling membrane or dispersing the compound in chelator, BAPTA. Apomorphine is a non-selective dopamine a polymer matrix or gel. agonist which activates both D1-like and D2-like receptors. 04.00 Ghrelin or ghrelin variant compounds may be NTX-265 is a drug comprising human Chorionic Gonado formulated for administration as Suppositories. A typical tropin (hCG) and Epoetin Alfa (EPO). Suppository is produced by providing a low melting wax, 0403. In some embodiments, at least two agents are Such as a mixture of fatty acid glycerides or cocoa butter, separate agents that can be administered as part of the that is first melted and the active component is dispersed therapy for the neurodegenerative condition, but not neces US 2017/O121385 A1 May 4, 2017 47 sarily at the same time or as part of the same composition, gamma/LXR inhibitor, an orphan nuclear receptor family although in some embodiments, the at least two agents are 4A (NR4A) inhibitor or modulator, an ERbeta inhibitor or part of the same composition and/or are administered con modulator, an inhibitor of STriatal-Enriched protein tyrosine currently or at the same time. In some embodiments, the at Phosphatase (STEP), a STEP-derived peptide, P7C3, P2Y least two agents are bound together. The at least two agents purinergic receptor agonist, glypromate, NNZ-2591 can form, for example, a dimer, a trimer, a tetramer, a (Neuren), NNZ-2566 (Neuren), cyclosporine A (NeuroVive pentamer, etc. In some cases, they can be conjugated, fused Pharmaceutical), NTX-265 (Stem Cell Therapeutics), or otherwise bound together. In some aspects the agents can DP-b99 (D-Pharm), apomorphine, Epoetin Alfa (EPO), pro be bound together in Such a manner that upon administration in vivo, the agents separate, for example, thereby releasing gesterone, KN 38-727 1/BAY38-7271 (KeyNeurotek/Bayer the two agents from each other. The agents can be bound AG), VAS-203 (Vasopharm), SAR 127963 (Sanofi), together via any suitable manner. A variety of kits are BHR100 (BHR Pharma), Oxycyte (Oxygen Biotherapeu commercially available and when taken in the context of the tics), glucagon, GLP-1R agonists, GLP-1, GLP-1 analog, instant disclosure, one of skill in the art also can utilize synthetic form of GLP-1, GLP-1 (7-36) amide, Exendin-4 various known methodologies for peptide-peptide, peptide (EX-4), EX-4 analog, synthetic form of EX-4, Lixisenatide, nonpeptide chemical, and peptide to pharmaceutical bind Liraglutide, a molecule in a biological pathway involving ing, fusion and conjugation. GLP-1R signaling pathway, incretin, incretin mimetic, Gas 0404 In some embodiments, at least one of the at least tric inhibitory polypeptide (GIP), sulfonamide compounds, two agents can be ghrelin. In some embodiments, at least Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), SPI two of the at least two agents are ghrelin molecules. The at 1005 (Sound Pharmaceuticals), glutathione peroxidase, glu least two ghrelin molecules further may include a pharma tathione peroxidase mimics and inducers, aducanumab ceutically acceptable excipient, such as for example, sterile (BIIB037, Biogen Idec) or a combination thereof. saline. In some embodiments, at least one of the at least two 0407. The present disclosure provides for a number of agents is a ghrelin variant, for example, a peptide of between GLP-1R agonists or derivatives, as well as incretin and 15 amino acids and 40 amino acids, a peptide of between 4 incretin mimetics to be used in a therapeutic combination amino acids and 14 amino acids, a small molecule pharma with ghrelin or ghrelin variants in treating mBI. GLP-1 is an ceutical, and the like. In some embodiments, the at least two endogenous metabolic hormone that stimulates insulin agents can include at least one compound from the catego secretion, which is a naturally-occurring peptide that is ries described herein (including, but not limited to those released after a meal. GLP-1 is known to suppress glucagon specific compounds and categories disclosed herein). secretion from pancreatic alpha cells and stimulate insulin 0405. In some embodiments, the ghrelin variant com secretion by pancreatic beta cells. GLP-1 receptor agonists pounds may be administered in combination with additional are in development as an add-on treatment for type 2 pharmacologically-active Substances or other pharmacologi diabetes. Glucagon-like peptide-1 (GLP-1) receptor signal cally-active material and/or may be administered in combi ing pathway in preclinical models of several CNS related nation with another therapeutic method, which is adminis neurological disorders such as Alzheimer's disease (AD), tered before, during (including concurrently with) and/or Parkinson's disease (PD), stroke, amyotrophic lateral scle after treatment of an individual with ghrelin or a ghrelin rosis (ALS) and Huntington's disease (HD). Studies have variant compound. The combination may be in the form of shown that a GLP-1 receptor (GLP-R) agonist, Exendin-4 kit-in-part systems, wherein the combined active substances (Ex-4), which readily crosses the blood-brain barrier, can be may be used for simultaneous, sequential or separate admin used to treat mild brain injury or concussion. The peptide istration. The combination therapies are administered in Ex-4 can be obtained from Bachem (Torrance, Calif.). pharmaceutically effective amounts, i.e., an administration Lixisenatide (intended trade name LyXumia) is a once-daily involving a total amount of each active component of the injectable GLP-1 receptor agonist. Liraglutide (NN2211) is medicament or pharmaceutical composition or method that a long-acting glucagon-like peptide-1 receptoragonist, bind is Sufficient to show a meaningful patient benefit. ing to GLP-1R as does GLP-1. Incretins are a group of 0406. In some embodiments, ghrelin or a ghrelin variant metabolic hormones that stimulate a decrease in blood can be used together or administered in combination with glucose levels. Incretins can increase in the amount of each other. In some embodiments, ghrelin and/or a ghrelin insulin released from pancreatic beta cells. Gastric inhibi variant can be administered in combination with a therapeu tory polypeptide (GIP), also known as the glucose-depen tic agent. In some embodiments, the therapeutic agent is one dent insulinotropic peptide, along with GLP-1, are members or more of an anti-inflammatory agent, anti-pain medication, of the incretin class. In some embodiments, the therapeutic acetylsalicylic acid, an antiplatelet agent, a thrombolytic agent is a GLP-1R agonist. In some embodiments, the enzyme, an aggregation inhibitor, a glycoprotein Ilb/IIIa therapeutic agent is GLP-1. In some embodiments, the inhibitor, a glycosaminoglycan, a thrombin inhibitor, an therapeutic agent is a GLP-1 analog, synthetic form of anticoagulant, heparin, coumarin, warfarin, tRA, GCSF, GLP-1, or GLP-1 (7-36) amide. In some embodiments, the streptokinase, urokinase, Ancrod, melatonin, a caspase therapeutic agent is Exendin-4 (EX-4), EX-4 analog, or inhibitor, an NMDA receptor agonist or antagonist (e.g. synthetic form of Ex-4. In some embodiments, the thera gacyclidine OTO-311), amantadine (e.g. ADS-5102), an peutic agent is Lixisenatide. In some embodiments, the anti-TNF-C. compound, an antibody, erythropoietin/EPO, therapeutic agent is Liraglutid. In some embodiments, the angiotensin II lowering agent, selective androgen receptor therapeutic agent is a molecule in a biological pathway modulator, leptin or leptin variants, an agonists of the involving GLP-1R signaling pathway. In some embodi renin-angiotensin System, an opioid receptor agonist, pro ments, the therapeutic agent is an incretin or incretin gesterone or progesterone mimetics and variants, a peroxi mimetic. In some embodiments, the therapeutic agent is a Some proliferator-activated receptor gamma agonist, a PPAR Gastric inhibitory polypeptide (GIP). US 2017/O121385 A1 May 4, 2017 48

0408. The present disclosure provides for a number of described in further detail below, the domain, termed the STEP-derived peptides or STEP derivatives to be used in a kinase specificity sequence (MS domain) includes two phos therapeutic combination with ghrelin or ghrelin variants in phorylation sites, Thr 59 (Thr 231 in STEP61) and Ser 72 treating mBI. The brain-enriched tyrosine phosphatase (Ser 244 in STEP61), which are important in regulating the STEP (also known as STriatal Enriched Phosphatase or stability of the STEP protein. PTPN5) is activated following stimulation of NMDARs and 0413 STEP61 and STEP46 contain the phosphatase is emerging as an important regulator of neuronal Survival domain, putative proteolytic sites (PEST), transmembrane and death. STEP is expressed specifically in neurons of the domain (TM), polyproline rich domain (PP), kinase inter striatum, neo-cortex and hippocampus. STEP61 and acting motif (KIM), kinase specificity sequence (KIS) and STEP46, the two STEP isoforms contain a highly conserved the above-mentioned phosphorylation sites involved in the Substrate-binding domain termed as the kinase interacting activation and subsequent degradation of STEP. Addition motif or KIM domain. Phosphorylation of a critical serine ally, the position of a cysteine residue (Cys 23 in STEP46/ residue within the KIM domain is mediated through dop Cys 195 in STEP61) that has been shown to be involved in amine/D1 receptor dependent activation of the Protein intermolecular dimerization and a threonine residue (Thr 18 Kinase A(PKA) pathway. Dephosphorylation of this residue in STEP46/Thr 190 in STEP61) that is known to be phos by Ca2+ dependent phosphatase calcineurin, following glu phorylatable by both ERK and p38 MAPKs. tamate/NMDA receptor stimulation, renders STEP active in 0414. In its active form STEP can modulate synaptic terms of its ability to bind to its substrates. Active STEP in plasticity by regulating the activity of extracellular regulated turn can bind to and modulate the activity of its substrate kinase 1/2 (ERK 1/2), a key protein involved in memory through tyrosine dephosphorylation of a regulatory tyrosine formation. Active STEP can also modulate NMDA receptor residue. Known substrates of STEP include ERK (extracel dependent long term potentiation by interfering with NMDA lular regulated kinase 1/2) and p38 MAPKs, Src family receptor trafficking to synaptic membrane, possibly through tyrosine kinases and NMDAR subunits, all of which are regulation of the upstream kinase Fyn and tyrosine dephos involved in neuronal survival and death. phorylation of NR2B-NMDA receptor subunits. Several 04.09 STEP is an intracellular protein tyrosine phos studies also indicate a role of active STEP in neuroprotection phatase (PTP) that is exclusively expressed in the central through its regulation of p38 MAPK. nervous system. STEP is preferentially expressed in neurons 0415 Because the STEP protein is known to interfere of the basal ganglia, hippocampus, cortex and related struc with NMDAR, a constitutively active peptide based on the tures. The STEP-family of PTPases includes both membrane STEP protein is a likely candidate for treatment, ameliora associated (STEP61) and cytosolic (STEP46) variants that tion and/or prevention of ischemic brain damage. A consti are formed by alternative splicing of a single gene. STEP61 tutively active STEP-derived peptide according to an differs from STEP46 by the presence of an additional 172 embodiment of the present disclosure. The peptide contains amino acids at its N-terminus. For the purposes of this the first 107 amino acids of STEP46 including the KIM and disclosure, specific amino acids residues within the STEP KIS domains. Furthermore, as shown in SEQID No 29, the protein are frequently referred to using the numbering from serine residue (Ser 49) which acts as a PKA phosphorylation the STEP46 variant. The 107 amino acid sequence of the site has been modified. In some embodiments, Ser 49 has STEP protein discussed herein is highly conserved between been converted, using standard point mutation techniques, to animal species with 95% sequence homology between rat alanine, which is non-phosphorylatable, resulting in a con (SEQ ID NO. 27) and human (SEQ ID NO. 28). stitutively active peptide. Modification of the PKA phos 0410 The present disclosure provides for a number of phorylation site addresses the problem of inactivation of the STEP-derived peptides containing mutations of the STEP STEP-derived peptide due to phosphorylation. protein. Where the described mutation sites are conserved 0416. Additional studies show that the active STEP pro between species, it will be well understood that the various tein is more susceptible to degradation. The study highlights STEP-based peptides described herein may be based on or the role of the KIS domain in regulation of the level of active derived from the STEP proteins from a variety of species STEP. Two SP/TP sites (i.e., Thr 59 and Ser 72) in the KIS and that Such peptides may or may not be derived from domain are phosphorylated primarily through the basal proteins that are endogenous to the specific species being activity of ERK and p38 MAPKs. Dephosphorylation of treated and/or studied. Accordingly, the peptides disclosed these two sites selectively results in ubiquitin-mediated herein should not be construed as being limited to the proteasomal degradation of the active form of STEP. These specific peptides sequences included in the sequence listing. findings imply that ubiquitin-mediated proteasomal degra 0411 STEP along with two other PTPs, PTPRR and dation of active STEP may also lead to secondary activation HePTP belongs to a family of PTPs that contains a highly of p38 MAPK. conserved 16-amino acid Substrate-binding domain termed 0417. In some embodiments, an active STEP-derived the kinase interacting motif (KIM domain). A regulatory peptide that remains stable and is not susceptible to ubiq serine residue, ser 49 (ser 221 in STEP61) lies in the middle uitin-mediated proteasomal degradation in vivo is desired. of the KIM domain and dephosphorylation of this residue In some embodiments, the STEP-derived peptide is where renders STEP active in terms of its ability to bind to its Ser49 has been converted to alanine, which is non-phos substrates. Phosphorylation of ser 49 is mediated by dop phorylatable and Thr 59 and Ser 72 are converted, to amine/D1 receptor dependent activation of the cAMP/PKA glutamic acid to mimic the phosphorylatable form (SEQ ID pathway while dephosphorylation is mediated by glutamate/ No. 30). It will be appreciated that other phophomimics may NMDA receptor induced activation of the Ca2+ dependent be used including, for example, Aspartic acid. phosphatase, calcineurin. 0418 Studies show that enzymatic activity of both 0412. A second conserved domain carboxy-terminal to STEP61 and STEP46 are also regulated by intermolecular the KIM domain is present in STEP, PTP-SL and HePTP. As dimerization. Dimerization of STEP involves intermolecular US 2017/O121385 A1 May 4, 2017 49 disulfide bond formation involving several cysteine residues 33, Thr18 mutated to Alanine). In some embodiments, SEQ and oxidative stress leads to increase in dimerization of ID No. 34 provides the amino acid sequence of the human STEP resulting in reduced activity. One such cysteine resi STEP Peptide including all the mutations points discussed due (Cys 23 in STEP46/Cys 195 in STEP61) that is involved above. in intermolecular dimerization is present in the STEP 0420 Constitutively active STEP-derived peptides derived peptide described herein. It is possible that oxidative including a TAT sequence at the N-terminal of the peptide. stress during an ischemic insult and/or reperfusion can lead In some embodiments, the phosphorylation site in the KIM to dimerization, at least in part, of the STEP derived peptide, domain has been altered (SEQID No. 35). In some embodi thereby reducing its therapeutic efficacy. Accordingly, the ments, the phosphorylation sites in both the KIM and MS domains have been altered (SEQID No. 36). TAT is an 11 present disclosure further provides a STEP peptide including amino acid peptide that renders peptide sequences cell a mutated Cys 23 residue (cysteine to alanine) (SEQID No. permeable and enables these peptides to cross the blood 31). brain barrier. The ability of the STEP-derived peptide to 0419. In some embodiments, the phosphorylation site in cross the blood brain barrier enables the peptide to be the STEP-derived peptide are (Thr 18 in STEP46/Thr 190 in delivered to a patient’s brain via the significantly less STEP61). In vitro studies show that this site is phosphory invasive mechanism of intravenous injection, for example latable by both ERK and p38 MAPKs. Based on the func via the femoral vein, rather than previous treatment mecha tional significance of phosphorylation, the present disclosure nisms that require direct Surgical access to the brain. Those further provides a STEP peptide including a nonphospho of skill in the art will be familiar with other suitable delivery rylatable or mimic phosphorylatable form of Thr 18 (SEQ mechanisms that could be employed including, for example, ID No. 32, Thr18 mutated to Glutamic Acid and SEQID No. known targeted and viral-based delivery systems.

SEO ID NO. MEEKVEDDFLDLDAVPETPWFDCVMDIKPETDPASLTVKSMGLOER 27 RGSNWSLTLDMCTPGCNEEGFGYLWSPREESAHEYLLSASRWLRAEE LHEKALDPFLLOAE

SEO ID NO. MEEKIEDDFLDLDPVPETPWFDCVMDIKPEADPTSLTVKSMGLOERR 28 GSNWSLTLDMCTPGCNEEGFGYLMSPREESAREYLLSASRVLOAEEL HEKALDPFLLOAE

SEQ ID NO. MEEKIEDDFLDLDPVPETPWFDCVMDIKPEADPTSLTVKSMGLOERR 29 GANVSLTIDMCTPGCNEEGFGYLMSPREESAREYLILSASRVLOAEE LHEKALDPFLLOAE

SEQ ID NO. EEKIEDDFLDLDPVPETPWFDCVMDIKPEADPTSLTVKSMGLOERR 3 O GANVSLTLDMCEPGCNEEGFGYLMEPREESAREYLLSASRVLOAEE LHEKALDPFLLOAE

SEQ ID NO. E E K I E D D LDLDPWPETPWFDAVMDIKIPEADPTSLTVKSMGLOERR 31 GSNWSLTLDMCTPGCNEEGFGYLMSPREESAREYLLSASRVLOAEEL HFKALDPFLLOAE

SEQ ID NO. E E K I E D D LDLDPWPEEPVFDCVMDIKIPEADPTSLINKSMGLOERR 32 GSNWSLTLDMCTPGCNEEGFGYLMSPREESAREYLLSASRVLOAEEL HEKALDPFLLOAE

SEQ ID NO. E E K I E D D LDLDPWPEAPVFDCVMDIKIPEADPTSLTVKSMGLOERR 33 GSNWSLTLDMCTPGCNEEGFGYLMSPREESAREYLLSASRVLOAEEL HEKALDPFLLOAE

SEQ ID NO. EEKIEDDFLDLDPVPEEPVFDAVMDIKPEADPTSLTVKSMGLOERR 34 GANVSLTIDMCEPGCNEEGFGYLMEPREESAREYLLSASRVLOAEE LHEKALDPFLLOAE

SEQ ID NO. MALYGRKKRRORRRGEEKIEDDFLDLDPWPETPWFDCVMDIKIPEAD 35 PTSLTVKSNIGLOERRGANVSLTLDMCEPGCNEEGFGYLMEPREESA REYLLSASRVLQAEELHEKALDPFLLOAE

SEQ ID NO. MALYGRKKRRORRRGEEKVEDDFLDLDAVPETPVIDCVMDIKPEI 36 DPASLTVKSMGLOERRGANVSLTLDMCEPGCNEEGFGYLVEPREES AHEYLLSASRVLRAEELHEKALDPFLLOAE

US 2017/O121385 A1 May 4, 2017

1-(3-azido-6-bromo-9H-carbazol-9-yl)-3-(3-methoxyphe CHFNOS: CH19N3O2S2, C15H1NOS; nylamino)propan-2-ol: 1-(3,6-dibromo-9H-carbazol-9-yl)- CHINOS: CHINO; CHBrNOS; 3-(4-methoxyphenoxy)propan-2-ol: 1-(3,6-dichloro-9H-car CHNOs, or CHCIFN.O. bazol-9-yl)-3-(phenylsulfonyl)propan-2-ol: 3,6-dibromo-9- (2-fluoro-3-(phenylsulfonyl)propyl)-9H-carbazole, (S)-1-(3. 0428. In some embodiments, ghrelin and/or a ghrelin 6-dibromo-9H-carbazol-9-yl)-3-(phenylsulfonyl) propan-2- variant, is administered in combination with anti-inflamma ol; (R)-1-(3,6-dibromo-9H-carbazol-9-yl)-3- tory compounds, such as an NSAID, indomethacin, COX1/ (phenylsulfonyl)propan-2-ol; 1-(3,6-dicyclopropyl-9H COX2 inhibitors, anti-TNF-C. compounds, infliximab, etan carbazol-9-yl)-3-(phenylamino) propan-2-ol. 1-(3,6-diiodo 9H-carbazol-9-yl)-3-(phenylamino)propan-2-ol. 1-(3,6- ercept, adalimumab, erythropoietin/EPO, angiotensin II diethynyl-9H-carbazol-9-yl)-3-(3-methoxyphenylamino) lowering agents, selective androgen receptor modulators, propan-2-ol; 9-(2-hydroxy-3-(3-methoxyphenylamino)pro leptin, agonists of the renin-angiotensin System, opioid pyl)-9H-carbazole-3,6-dicarbonitrile; N-(3-(3,6-dibromo receptor agonists, progesterone, amantadine (adamantan-1- 9H-carbazol-9-yl)-2-fluoropropyl)aniline; 3,6-dibromo-9- amine, CHN), peroxisome proliferator-activated recep (2,2-difluoro-3-phenoxypropyl)-9H-carbazole; N-(3-(3,6- dibromo-9H-carbazol-9-yl)-2-fluoropropyl)-4- tor gamma agonists, or combinations of the same. In some methoxyaniline; N-(2-bromo-3-(3,6-dibromo-9H-carbazol embodiments, ghrelin and/or a ghrelin variant, is adminis 9-yl)propyl)-N-(4-methoxyphenyl)-4- tered in combination with purinergic ligand 2-methylthi nitrobenzenesulfonamide: Ethyl 2-(4-(3-(3,6-dibromo-9H oladenosine 5' diphosphate (2MeSADP). carbazol-9-yl)-2-fluoropropylamino)phenoxy)acetate; and N-(3-(3,6-dibromo-9H-carbazol-9-yl)-2-fluoropropyl)-4-(2- 0429 P2Y purinergic receptor agonist can include uri (2-methoxyethoxy)ethoxy)aniline; or a pharmaceutically dine 5'-di- and triphosphate (UDP, UTP) and their analogs, acceptable salt thereof. adenosine 5'-diphosphate (ADP) and its analogs, cytidine 0423. In some embodiments, the therapeutic agent is a 5'-di- and triphosphate (CDP, CTP) and their analogs, and compound having formula 1-(3,6-dibromo-9H-carbazol-9- yl)-3-(phenylamino)propan-2-ol. In some embodiments, the dinucleoside polyphosphate compounds. Example though therapeutic agent is a compound having formula R-1-(3.6- non-limiting P2Y receptor agonists Suitable for use in com Dibromo-9H-carbazol-9-yl)-3-(3-methoxyphenylamino)- bination with ghrelin orghrelin variants may include uridine propan-2-ol. In some embodiments, the therapeutic agent is 5'-di'- and triphosphate (UDP, UTP) and their analogs (For a compound having formula R-1-(3,6-Dibromo-9H-carba mulae Ia and Ib), 5'-adenosine monophosphate (AMP) and Zol-9-yl)-3-(3-methoxyphenylamino)-propan-2-ol. its analogs, adenosine 5'-di- and triphosphate (ADP, ATP) 0424. In some embodiments, the therapeutic agent is a compound having formula S-1-(3,6-Dibromo-9H-carbazol and their analogs (Formulae IIa and IIb), and cytidine 5'-di 9-yl)-3-(3-methoxyphenylamino)-propan-2-ol. In some and triphosphate (CDP, CTP) and their analogs (Formulae embodiments, the therapeutic agent is a compound having IIIa and IIIb). P2Y receptor agonists also include dinucle formula S-1-(3,6-Dibromo-9H-carbazol-9-yl)-3-(3- otide polyphosphate compounds of general Formula (IV). methoxyphenylamino)-propan-2-ol. Examples of P2Y receptor agonists that may be useful to be 0425. In some embodiments, the therapeutic agent is a compound having formula S-1-(3,6-Dibromo-9H-carbazol used in a combination with ghrelin or ghrelin variants for 9-yl)-3-(3-methoxyphenylamino)-propan-2-ol. In some treatment and/or protection of brain injury include 2-Me embodiments, the therapeutic agent is a compound having SADP and N-methanocarba-2MeSADP (“MRS2365') as formula R-1-(3,6-Dibromo-9H-carbazol-9-yl)-3-(3- disclosed in U.S. Pat. No. 8,618,074, which is incorporated methoxyphenylamino)-propan-2-ol. In some embodiments, by reference herein. the therapeutic agent is a compound having formula (+) (dextrorotatory) enantiomer of 1-(3,6-Dibromo-9H-carba 0430. The present disclosure provides for methods of Zol-9-yl)-3-(3-methoxyphenylamino)-propan-2-ol. In some using a pharmaceutical composition comprising ghrelin or embodiments, the therapeutic agent is a compound having ghrelin variants and P2Y receptor agonists for purpose of formula (+) (dextrorotatory) enantiomer of 1-(3,6-Dibromo treating mild brain injury or concussion. P2Y receptor 9H-carbazol-9-yl)-3-(3-methoxyphenylamino)-propan-2-ol. agonists, including analogs, derivatives and pharmaceuti 0426 In some embodiments, the therapeutic agent is a cally acceptable salts thereofthat may find use in the present compound having formula (-) (levorotatory) enantiomer of 1-(3,6-Dibromo-9H-carbazol-9-yl)-3-(3-methoxyphe treatment methods include, but are not limited to, nucleoside nylamino)-propan-2-ol. In some embodiments, the therapeu mono-, di-, and triphosphates and dinucleoside polyphos tic agent is a compound having formula (-) (levorotatory) phates. Nucleoside monophosphates may include adenosine enantiomer of 1-(3,6-Dibromo-9H-carbazol-9-yl)-3-(3- 5'-monophosphate (AMP) and its derivatives such as 2-thio methoxyphenylamino)-propan-2-ol. In some embodiments, ether-substituted AMP, e.g., 2-hexylthio AMP. Nucleoside the therapeutic agent is a compound having formula (-) di- and triphosphates may include uridine 5'-di- and triphos (levorotatory) enantiomer of 1-(3,6-Dibromo-9H-carbazol 9-yl)-3-(3-methoxyphenylamino)-propan-2-ol. In some phate (UDP and UTP) and their analogs of general formulae embodiments, the therapeutic agent is a compound having Ia and Ib; adenosine 5'-di- and triphosphate (ADP and ATP) formula(+) (dextrorotatory) enantiomer of 1-(3,6-Dibromo and their analogs of general formulae IIa and IIb, and 9H-carbazol-9-yl)-3-(3-methoxyphenylamino)-propan-2-ol. cytosine 5'-di- and triphosphate (CDP and CTP) and their 0427. In some embodiments, the therapeutic agent is a analogs of general formulae IIIa and IIIb, and dinucleoside compound having molecular formula CHBrNO; polyphosphates of general formula IV. US 2017/O121385 A1 May 4, 2017 52

0431 UDP and its analogs are depicted by general for 0434 UTP and its analogs are depicted by general for mula Ia: mula Ib. (Ib) (Ia)

O O O O O | | | HO-P-R-P-O-P-O HO-P-R-P-O X X X3

wherein: X, X and X are each independently either —OH, —O. —SH, or - S. Y is H or OH: R. R. R. and Ra are wherein: X, and X are each independently either —OH, defined as above in Formula Ia. Preferably, X and X are O—, R is oxygen or imido, and R is H. Particularly –O, -SH, or -ST: Y is H or OH: R is selected from the preferred compounds of Formula Ib may include uridine group consisting of O, imido, methylene, and dihalometh 5'-triphosphate (UTP) and uridine 5'-O-(3-thiotriphosphate) ylene (e.g., dichloromethylene, difluoromethylene): R is (UTPS). selected from the group consisting of H, halogen, alkyl, 0435 ADP and its analogs are depicted by general For Substituted alkyl, alkoxyl. nitro and azido; R is selected mula IIa: from the group consisting of nothing, H, alkyl, acyl (includ ing arylacyl), and arylalkyl; and R is selected from the group consisting of —OR', SR', NR', and NRR", wherein R" and R" are independently selected from the group con sisting of H, alkyl, Substituted alkyl, aryl, Substituted aryl, arylalkyl, alkoxyl, and aryloxyl, and with the proviso that R' O O is absent when R is double bonded from an oxygen or sulfur HO-P-R-P-O atom to the carbon at the 4-position of the pyrimidine ring. X X 0432 Compounds illustrative of the compounds of For mula (Ia) may include, though are not limited to: uridine 5'-diphosphate (UDP); uridine 5'-O-(2-thiodiphosphate) wherein: R. X, X and Y are defined as in Formula Ia; Z (UDPBS), 5-bromouridine 5'-diphosphate (5-BrUDP); 5-(1- is H, Cl, or SR, wherein R is alkyl (C-C saturated or phenylethynyl)-uridine 5'-diphosphate (5-(1-phenylethynyl) unsaturated); R and Ra are H while R is nothing and there is a double bond between N-1 and C-6 (adenine), or R and UDP; 5-methyluridine 5'-diphosphate (5-methylUDP): Ra are H while R is nothing and Z is SR, or R and R are 4-hexylthiouridine 5'-diphosphate (4-hexylthioUDP); H while R is O and there is a double bond between N-1 and 4-mercaptouridine 5'-diphosphate (4-mercaptoUDP); C-6 (adenine 1-oxide), or R. R. and R taken together are 4-methoxyuridine 5'-diphosphate (4-methoxyl JDP): 4-(N- —CH=CH forming a ring from N-6 to N-1 with a morpholino)uridine 5'-diphosphate (4-(N-morpholino)UDP; double bond between N-6 and C-6 (1.N-ethenoadenine). Particularly preferred compounds of Formula IIa may 4-hexyloxyuridine 5'-diphosphate (4-hexyloxyl JDP); N.N- include 5'-adenosine diphosphate (ADP), 2-methyl-SADP dimethylcytidine 5'-diphosphate (N,N-dimethylCDP); and N-methanocarba-2MeSADP (“MRS2365”). N-hexylcytidine 5'-diphosphate (N-hexylCDP); and N-cy 0436 ATP and its analogs are depicted by general For clopentylcytidine 5'-diphosphate (N-cyclopentylCDP). mula IIb: 0433 Certain compounds of Formula Ia (e.g., UDP, (IIb) dUDP, UDPBS, and 4-mercaptoUDP) are known and may be

NRR4 made in accordance with known procedures or variations thereof, which will be apparent to those skilled in the art. For example, the identification and preparation of certain thio phosphate analogues of nucleoside diphosphates (such as O O O UTPBS) are set forth in U.S. Pat. No. 3,846,402 (Eckstein et | Ho----0--0 O al.), and in R. S. Goody and F. Eckstein, J. Am. Chem. Soc. X X X 93: 6252-6257 (1971). Alternatively, UDP, and other ana H H logs thereof are also commercially available from vendors OH Y Such as Sigma (St. Louis, Mo.) and Pharmacia (Uppsala, wherein: R. X. X, X: and Y are defined as in Formula Ib. Sweden). and R. R. R. and Z are defined as in Formula IIa. US 2017/O121385 A1 May 4, 2017 53

0437 CDP and its analogs are depicted by general For mula IIIa: (IIIb)

(IIIa)

O O O HO-P-R-P-O-P-O O O | X X X HO-P-R-P-O X X2 wherein: R. X. X, X and Y are defined as in Formula Ib, and Rs. R and R, are defined as in Formula IIIa. Preferred wherein: R. X, X and Y are defined as in Formula Ia; Rs compounds of Formula IIIb may include cytidine 5'-triphos and R are H while R, is nothing and there is a double bond phate (CTP) and 4-nitrophenyl ethenocytidine 5'-triphos between N-3 and C-4 (cytosine), or Rs. R and R, taken phate. together are —CH=CH forming a ring from N-3 to N-4 0439 For simplicity, Formulas I, II, and III, herein illus with a double bond between N-4 and C-4 (3.N-ethenocy trate the active compounds in the naturally occurring D-con tosine), optionally the hydrogen of the 4- or 5-position of the figuration, but it is to be understood that, unless otherwise etheno ring is substituted with alkyl, substituted alkyl, aryl; indicated, the present disclosure also encompasses com Substituted aryl (heteroaryl, etc.), alkoxyl. nitro, halogen, or pounds in the L-configuration, and mixtures of compounds azido. in the D- and L-configurations. 0438 CTP and its analogs are depicted by general For 0440 Dinucleoside polyphosphates are depicted by gen mula IIIb. eral Formula IV:

(IV) O O O O –0 -x- o- O- O B OH pi O O OH iii. S. Z. Yi

wherein: X is oxygen, methylene, difluoromethylene, imido; n=0, 1 or 2, m=0, 1 or 2; n+m=0, 1, 2, 3 or 4; B and B' are each independently a purine residue or a pyrimidine residue linked through the 9- or 1-position, respectively; Z—OH or N: Z—OH or N.; Y—H or OH; and Yi—H or OH. The ribosyl moieties are in the D configuration, as shown, but may be L-, or D- and L-. 0441. A preferred compound of Formula IV includes Formula IVa:

(IVa) O O O O

B O-P-O P-X-P O-P O B

OH < > h O O iii. O

Z Yi US 2017/O121385 A1 May 4, 2017 54 wherein: X=O; n+m=1 or 2: Z, Z, Y and Y-OH, B and 1o)-adenine, wherein the acyl group is chosen from among, B' are uracil, thymine, cytosine, guanine, adenine, Xanthine, but not limited to, acetyl, trifluororoacetyl, benzoyl, substi hypoxanthine or as defined in Formulas V and VI; or X—O; tuted-benzoyl, etc., or the carboxylic moiety is present as its n+m=3 or 4: Z, Z, Y and Y-OH B=uracil; B' is uracil, ester or amide derivative, for example, the ethyl or methyl thymine, cytosine, guanine, adenine, Xanthine, hypoxan ester or its methyl, ethyl or benzamido derivative. thine or as defined in Formulas V and VI; or X—O n+m=1 0443 Band B', can also be a pyrimidine with the general or 2: Z, Y and Z'-OH: Y—H; B=uracil; B' is uracil, formula of Formula VI, linked through the 1-position to thymine, cytosine, guanine, adenine, Xanthine, hypoxan ribosyl residue. thine or as defined in Formulas V and VI; or X—O; n+m=0, 1 or 2: Z and Y-OH: Z—N: Y—H; B–uracil;

B'-thymine; or X—O, n+m=0, 1 or 2: Z and Z'—N: Y and (VI) Y—H; B and B'=thymine; or X—CH, CF, or NH; n and m=1; Z, Z, Y and Y'—OH: B and B' are uracil, thymine, cytosine, guanine, adenine, Xanthine, hypoxanthine or as defined in Formulas V and VI:

(V) wherein: R is hydrogen, hydroxy, mercapto, amino, cyano, C7-2 arylalkoxy, C alkylthio, C alkoxy, C alky lamino or diC alkylamino, wherein the alkyl groups are optionally linked to form a heterocycle; R is hydrogen, acetyl, benzoyl, C. alkyl, phenyloxy, Cls alkanoyl, aroyl, or Sulphonate; R is hydroxy, mercapto, Ca. alkoxy, C7-12 arylalkoxy, C. alkylthio, amino, S-phenyl, Cs disubsti tuted amino, triazolyl, C. alkylamino, or di-C alky wherein R is hydrogen, Cs alkyl, C. cycloalkyl, phenyl, lamino wherein said dialkyl groups are optionally linked to or phenyloxy; wherein at least one hydrogen of said Cls form a heterocycle or linked to N to form a substituted ring: alkyl, phenyl, phenyloxy, is optionally substituted with a or Rs and R taken together form a 5-membered fused moiety selected from the group consisting of halogen, imidazole ring between positions 3 and 4 of the pyrimidine hydroxy, Calkoxy, Calkyl, Cao aryl, carboxy, cyano, ring and form a 3.N-ethenocytosine derivative, wherein nitro, Sulfonamido, Sulfonate, phosphate, Sulfonic acid, said etheno moiety is optionally Substituted on the 4- or amino, C alkylamino, di-C alkylamino wherein said 5-positions with C alkyl; phenyl; or phenyloxy; wherein alkyl groups are optionally linked to form a heterocycle, at least one hydrogen of said C. alkyl; phenyl or phenyloxy ()-A(alkyl)CONHCalkyl)-, and co-A(alkyl)NHCO(alkyl)- is optionally substituted with a moiety selected from the wherein A is amino, mercapto, hydroxy or carboxyl; R is 0 group consisting of halogen, hydroxy, C. alkoxy, Ca or is absent; or R and R taken together form a 5-membered alkyl, Co-o aryl, C7-12 arylalkyl, carboxy, cyano, nitro, fused imidazole ring optionally Substituted on the 4- or Sulfonamido, Sulfonate, phosphate, Sulfonic acid, amino, 5-positions of the etheno moiety with C alkyl, phenyl or Calkylamino, and di Ca. alkylamino wherein said dialkyl phenyloxy, wherein at least one hydrogen of said C. alkyl, groups are optionally linked to form a heterocycle; R, is phenyl, phenyloxy, is optionally Substituted with a moiety hydrogen, hydroxy, cyano, nitro, or Cs alkenyl; wherein selected from the group consisting of halogen, hydroxy, Ca said alkenyl moiety is optionally linked through an oxygen alkoxy, C. alkyl, Co-o aryl, C7-12 arylalkyl, carboxy, to form a ring, wherein at least one hydrogen of said alkenyl cyano, nitro, Sulfonamido, Sulfonate, phosphate, Sulfonic moiety on the carbon adjacent to said oxygen is optionally acid, amino, Calkylamino, and di-C alkylamino wherein Substituted with Ce alkyl, phenyl, Substituted Cls alkynyl, said dialkyl groups are optionally linked to form a hetero halogen, Substituted C. alkyl, CF, C2- alkenyl, C cycle; and R. is hydrogen, NH, Cs alkyl, C. cycloalkyl, alkynyl, allylamino, bromovinyl, ethyl propenoate, or pro phenyl; or phenyloxy; wherein at least one hydrogen of said penoic acid; or R and R, together form a 5 or 6-membered NH, Cs alkyl, phenyl, or phenyloxy, is optionally substi saturated or unsaturated ring bonded through N or O at R. tuted with a moiety selected from the group consisting of Such ring optionally contains Substituents that themselves halogen, hydroxy, Calkyl, Cao aryl, C7-12 arylalkyl, Ca contain functionalities; provided that when Rs is amino or alkoxy, C7 arylalkyloxy, C alkylthio, phenylthio. C Substituted amino, R, is hydrogen, and Rs is hydrogen, arylalkylthio, carboxy, cyano, nitro, Sulfonamido, Sulfonate, amino or di-C alkylamino, Calkoxy, C7 arylalkoxy, phosphate, sulfonic acid, amino, C alkylamino, phe C. alkylthio, C-2 arylalkylthio, carboxamidomethyl, car nylamino, C-2 arylalkyamino, di-C alkylamino wherein boxymethyl, methoxy, methylthio, phenoxy or phenylthio. said dialkyl groups are optionally linked to form a hetero 0444. In the general structure of Formulae I, II, III, V, and cycle, ()-A(alkyl)CONHCalkyl).B—, and co-A(alkyl)NHCO VI above, the dotted lines in the 2- to 6-positions are (alkyl).B—, wherein A and B are independently amino, intended to indicate the presence of single or double bonds mercapto, hydroxy or carboxyl. in these positions; the relative positions of the double or 0442. The substituted derivatives of adenine (Formula V) single bonds being determined by whether the R. Rs and R. may include adenine 1-oxide, 1.N6-(4- or 5-substituted Substituents are capable of keto-enol tautomerism. In the etheno) adenine: 6-substituted adenine; or 8-substituted general structures of Formula V and VI above, the acyl aminoadenine, 6-aminohexylcarbamoylmethyl-adenine; groups comprise alkanoyl or aroyl groups. The alkyl groups and (O-acylated-amino(hydroxy, thiol and carboxy)alkyl (C- contain 1 to 8 carbon atoms, particularly 1 to 4 carbon atoms US 2017/O121385 A1 May 4, 2017 optionally Substituted by one or more appropriate Substitu 0449 The compounds, including stereoisomers thereof, ents, as described below. The aryl groups including the aryl can be created either singly, in Small clusters, or in a moieties of Such groups as aryloxy are preferably phenyl combinatorial fashion to give structurally diverse libraries of groups optionally Substituted by one or more appropriate compounds. substituents, as described below. The above-mentioned alk 0450. In one aspect, the invention features a compound of enyl and alkynyl groups contain 2 to 8 carbon atoms, formula (I) particularly 2 to 6 carbon atoms, e.g., ethenyl or ethynyl, optionally Substituted by one or more appropriate Substitu ents as described below. formula (I) 0445 Appropriate substituents on the above-mentioned alkyl, alkenyl, alkynyl, and aryl groups are selected from, but not limited to, halogen, hydroxy, Calkoxy, C alkyl, C-2 aryl, C-2 arylalkoxy, carboxy, cyano, nitro, Sulfona mido, Sulfonate, phosphate, Sulfonic, amino and Substituted amino wherein the amino is singly or doubly Substituted by a C alkyl, and when doubly Substituted, the alkyl groups optionally being linked to form a heterocycle. Dinucleoside wherein, polyphosphate compounds useful in this disclosure are P", R" is hydrogen, halo (e.g., fluoro), aryl, heteroaryl, arylalkyl, P-di (urdine-5')-tetraphosphate, dOPU, U.P., U.Ps, heteroarylalkyl, cyclyl cyclylalkyl, heterocyclyl, heterocy dCPU, CPU, IP51, APA, CPU, UPA and AP. clylalkyl, alkyl, alkenyl, alkynyl, or R' can be taken together 0446. Some compounds of Formula I, II and III can be with R or R to form a ring; each of which is optionally made by methods known those skilled in the art; some substituted with 1-4 R: compounds are commercially available, for example, from k' is a bond, O, C(O), C(O)O, OC(O), C(O)NR, NRC(O), Sigma Chemical Co. (St. Louis, Mo. 631 78). Compounds of S, SO, SO, CR-CR, or C=C; Formulae Ia (UDP and its analogs) can be prepared accord n is 0-6, preferably 1-3: ing to WO99/09998. Compounds of Formulae Ib, IIb and R is hydrogen, halo (e.g., fluoro), C-C alkyl, C-C, IIIb (UTP ATP, CTP and their analogs) can be prepared alkenyl, or C-C alkynyl: or R can be taken together with according to U.S. Pat. No. 5,763,447 Compounds of For R" to form a ring: mula IV can be made in accordance with known procedures R is hydrogen, C-C alkyl, C-C alkenyl, or C-C alky described by Zamecnik, et al., Proc Natl. Acad. Sci. USA89, nyl, or R can be taken together with R', 838-42 (1981); and Ng and Orgel, Nucleic Acids Res. R", or R to form a ring; each of which can be optionally 15:3572-80 (1987), Pendergast et al., U.S. Pat. No. 5,837, substituted with 1-2 R: 861, or variations thereof. 0447 The compounds of the present disclosure also A is encompass their non-toxic pharmaceutically acceptable 0451 salts, such as, but not limited to, an alkali metal salt Such as Sodium or potassium; an alkaline earth metal salt such as manganese, magnesium or calcium, or an ammonium or tetraalkyl ammonium salt, i.e., NX+ (wherein X is C.). Pharmaceutically acceptable salts are salts that retain the (CH2).- -(CH), s desired biological activity of the parent compound and do not impart undesired toxicological effects. The present dis R7a closure also encompasses the acylated prodrugs of the compounds disclosed herein. Those skilled in the art recog -cis--city s nize various synthetic methodologies that may be employed to prepare non-toxic pharmaceutically acceptable salts and R8 R7b acylated prodrugs of the compounds. (CH), -M- co- Or Sulfonamide Compounds 0448. The present disclosure provides for a number of Sulfonamide compounds to be used in a therapeutic combi - (CH), -M- co-- nation with ghrelin or ghrelin variants in treating mBI, for R8 example heteroaryl Sulfonamide compounds, and other Sul fonamide compounds having cyclic moieties. Examples of X and y are each independently 0-6: heteroaryl compounds include oxadiazole and triazole com M is aryl, heteroaryl, cyclyl, or heterocyclyl, each of which pounds. The compounds can be used in therapeutic appli is optionally substituted with 1-4 R: cations, including modulation of disorders, diseases or dis R" and Rare each independently hydrogen, alkyl, alkenyl, ease symptoms in a Subject (e.g., mammal, human, dog, cat, haloalkyl, cyclyl, or heterocyclyl, or R and R can be taken horse). The compounds include useful GHS-R antagonists. together to form a heterocyclic ring, or R and R can be Additional compounds are disclosed in U.S. Pat. No. 7,829, taken together to form an azido moiety, or one or both of R' 589, which is incorporated herein by reference in its entirety and Rican independently be joined to one or both or R'' and for all of its disclosure, including all methods, materials, etc. R” to form one or more bridges between the nitrogen to US 2017/O121385 A1 May 4, 2017 56 which the R and Rare attached and R'' and R', wherein each R' is independently -(CH.(N(R')C(O)R'?, each bridge contains 1 to 5 carbons; or one or both of R and -(CH2)CN, (CH,), (N(R')C(O)CR', —(CH.)N Rican independently be joined to one or both of R'' and R' (R')C(O)NR'R'', (CH.)N(R)SO.R', -(CH.) to form to form one or more heterocyclic rings including the SO.NR'R'', -(CH.),C(O)NR'R'', -(CH.),C(O) nitrogen to which the Rand Rare attached, or one or both OR', -(CH)OC(O)CR°, -(CH)OC(O)R., (CH.) of R and R' can independently be joined to R to form a OC(O)NR'R 2, (CHN(RSO.NR'R'', —(CH) ring, or one or both of RandR can independently be joined OR 12 (CHOC(O)N(R (COH, —(CH2) to R to form a ring; wherein each R and Rare optionally SOR', (C)SOR , -(CH.)NR'R' or -(CH.) independently substituted with 1-5 halo, 1-3 hydroxy, 1-3 OCHC(O)N(R')(CH), OH: alkyl, 1-3 alkoxy, 1-3 amino. 1-3 alkylamino. 1-3 dialky each R' is independently halo, alkyl, alkenyl, alkynyl, lamino. 1-3 nitrile, or 1-3 haloalkyl; alkoxy, -(CH.)NR'C(O)NR'R'', -(CH.),C(O) Y is a monocyclic aryl or monocyclic heteroaryl; each of NR'R'', (CH)NR'C(O)R'', CN, (CH.) which is optionally substituted with 1-4 R': NR'SO.R'', (CH)OC(O)R'', -(CH.)SO.NR'R'', each R and R are independently halo, alkyl, alkenyl, -(CH.)SOR', -(CH.)COOH or -(CH.),C(O)OR'; alkynyl, cyclyl, heterocyclyl, aryl, heteroaryl, alkoxy, X is CR'R'', O, S, S(O), S(O), or NR'': haloalkyl, haloalkyloxy, haloalkylthio, acetyl, cyano, nitro, m is an integer between 1 and 6: hydroxy, oxo, C(O)CR, OC(O)R, N(R), C(O)N(R), p is an integer from 0 to 5: NRC(O)R’, or SR; q and S are each independently an integer between 1 and 3; R" and R'' are each independently hydrogen, alkyl, alk and enyl, haloalkyl, cyclyl cyclylalkyl, or heterocyclyl; or one w is an integer between 0 and 5. or both of R'' and R' can independently be joined to one 0452. In some embodiments, formula (I), comprises an or both of R and R to form one or more bridges between enriched preparation of formula (I) the nitrogen to which the RandR are attached and R7 and R’, wherein each bridge contains 1 to 5 carbons; or one or both of R" and R' can independently be joined to one or formula (I) both or R and R to form to form one or more heterocyclic R1 rings including the nitrogen to which the R' and R are attached, or one or both of R'' and R' can independently be k joined with R to form a ring; wherein each R" and R' can . AR4 be independently optionally substituted with 1-5 halo, 1-3 C-N-SO-A-N hydroxy, 1-3 alkyl, 1-3 alkoxy, 1-3 amino, 1-3 alkylamino, \,: 1-3 dialkylamino. 1-3 nitrile, or 1-3 haloalkyl; R2 R3 R. R is hydrogen or C-C alkyl, or R can be joined with R', R. R' or R' to form a ring: 0453. In some embodiments, formula (I), comprises an R is halo, alkyl, cyclyl, heterocyclyl, aryl, heteroaryl, enriched preparation of formula (I") alkoxy, haloalkyl, haloalkyloxy, haloalkylthio, acetyl, cyano, nitro, hydroxy, oxo, C(O)CR, OC(O)R’, N(R), C(O)N(R), NR°C(O)R’, SR; formula (I") each R" is independently alkyl, alkenyl, alkynyl, halo, cyano, carbonyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, cyclyl cyclylalkyl, alkoxy, alkoxyalkyl, aryloxy, aryloxy alkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroary lalkyl, -OR'', NR'R'', CF, -SOR', SOR, OC(O)R'', SONR'R'', (CH) R' or R'; each of which is optionally independently substituted with 1-3 R': R'' and R'' are each independently hydrogen, alkyl, alk enyl, alkynyl, cyclyl, heterocyclyl, aryl or heteroaryl; 0454. In some embodiments, n is 1; k is a bond or O; and R'' and R'' are each independently hydrogen, alkyl, alk R" is aryl, heteroaryl, arylalkyl, or heteroarylalkyl. enyl, alkynyl, alkylthioalkyl, alkoxyalkyl, aryl, arylalkyl, 0455. In some embodiments, n is 1; k is O; and R' is heterocyclyl, heteroaryl, heteroarylalkyl, heterocycloalkyl arylalkyl. For example, R' can be phenylmethyl. or cyclyl, cyclylalkyl, or R'' and R' taken together can be 10456. In some embodiments, n is 2: k is a bond; and R' cyclized to form -(CH2).X(CH2), ; wherein each R'' and is aryl. R* may independently optionally be substituted with 1 to 3 0457. In some embodiments, n is 0 or 1; k is a bond; and Substituents selected from the group consisting of halogen, R" is alkyl, for example unsubstituted or substituted with one OR'', alkoxy, heterocycloalkyl, - NR'C(O)NR'R''. R. For example, R' can be a branched alkyl such as one of C(O)NR'R'', NRC(O)R'', - CN, OXO, the following. NR''SOR'', OC(O)R', SONR'R'', SOR, —S(O).R', -COOH and C(O)OR'; each R' is inde pendently alkyl, aryl, arylalkyl, heteroaryl, or heteroarylal kyl, each of which may optionally be substituted with —(CH), OH: each R'' is independently alkoxy, alkoxycarbonyl, -C(O) Xu NR2R12, NR'R'', C(O)R, NRC(O)NR'R'' or —N-heteroaryl; US 2017/O121385 A1 May 4, 2017 57

0458. In some embodiments, R is hydrogen or C-C, I0469. In some embodiments, R'' and R'' are each inde alkyl. pendently alkyl; R and Rare each independently hydrogen 0459. In some embodiments n is 0 and k is a bond. or alkyl; and X and y are each independently 0 or 1; Example R' moieties include methyl, and ethyl. Preferred R' 0470. In some embodiments, moieties include methyl. In some embodiments R' is unsub stituted methyl or methyl or ethyl substituted with C(O)N (R). 0460. In some embodiments n is 0 and k is a bond, and R" and Rare both methyl. 0461. In some embodiments, n is 0; k is a bond; and R' is hydrogen. 0462. In some embodiments R is hydrogen. taken together is 0463. In some embodiments, R' and R together from a heterocyclic ring Such as a pyrrolidine or an aZetidine ring. The heterocyclic ring can be unsubstituted or substituted, for example, with 1-2 R. 0464) In some embodiments, R' and R together form a ring. 0465. In some embodiments, A is

R7a

(CH2).-C-(CH2) O R7b R7a - (CH),- o co-- R8 R7b

For example. A can be 0471. In some embodiments,

R4 M A-N V (CH),--(CH2) R5, R7b taken together is or A can be 0466

R7a (CH),-C-(CH2) R7b wherein R7 and R7 are H: X is 1; and N1 y is 0 or 1. 0467. In some embodiments, A is CHCH or CHCH-CH; and each RandR is independently alkyl, or R" and R, when taken together, form a heterocyclic ring. In some embodiments, R'' and R' can each be H. 0468. In some embodiments, at least one of R' or R' is taken together with at least one or R' or R to form a Vol.H N 1 heterocyclic ring including the nitrogen to which the R and Rare attached. US 2017/O121385 A1 May 4, 2017 58

0472. In some embodiments, 0474. In some embodiments,

taken together is taken together is

/c/c NH2 N 7 0473. In some embodiments, 0475. In some embodiments, Y is a monocyclic het R4 eroaromatic moiety, for example a nitrogen containing het M eraromatic moiety Such as nitrogen containing five mem A-N V bered heteroaromatic moiety. R5, 0476. In some embodiments, Y is a heterocyclic moiety containing at least two heteroatoms, for example, a five membered heterocyclic moiety containing at least two het taken together is eroatoms or at least three heteroatoms. 0477. In some embodiments Y is substituted with one R''. R' can be positioned, for example, 1.3 relative to the point of attachment of Y to the adjacent chain carbon or 1.2 relative to the point of attachment of Y to the adjacent chain carbon. 0478. In some embodiments, R' is aryl or heteroaryl, for example a monocyclic aryl or monocyclic heteroaryl Such as phenyl, pyridyl, oxazolyl, thiazolyl, or thiophenyl. In some embodiments, R' is substituted with 1-3 R''. In some embodiments, R' is halo, alkyl, or alkoxy, for example chloro, fluoro, methyl, cyano, or methoxy. 0479. In some embodiments, R' is a bicyclic heteroaryl, for example indolyl, benzimidazolyl, benzoxazolyl, benzo furanyl, benzothiophenyl, or benzthiazolyl. In some embodi vo, ( ments, R' is substituted with 1-3 R''. In some embodiments, R' is halo, alkyl, or alkoxy, for example chloro, fluoro, methyl, cyano, or methoxy. 0480. In some embodiments, R' is arylalkyl or het eroarylalky, for example a monocyclic or bicyclic arylalkyl vo 2. or monocyclic or bicyclic heteroaryalkyl. In some embodi ments, R' is substituted with 1-3 R''. In some embodi ments, R' is halo, alkyl, or alkoxy, for example chloro, fluoro, methyl, cyano, or methoxy. US 2017/O121385 A1 May 4, 2017 59

0481. In some embodiments, R' includes an unsaturated 0494. In some embodiments, one of Q, Q, or Q is CR. or partially unsaturated cyclic moiety, for example a cyclyl 0495. In some embodiments, Q is CR'. or heterocyclyl moiety. The cyclic moiety can either be 0496. In some embodiments, Q is CR. directly attached to Y or attached via a linker such as an 10497. In some embodiments, Q', Q, Q and Q together alkylenyl linker. In some embodiments, R" is substituted form with 1-3 R''. In some embodiments, R' is halo, alkyl, or alkoxy, for example chloro, fluoro, methyl, cyano, or methoxy. RQ N 0482. In some embodiments Y is oxadiazole or triazole. 0483. In another aspect, the invention features a com pound of formula (II), No. 0498. In some embodiments, Q is NR. formula (II) 10499. In some embodiments, Q', Q, Q and Q together form

R2 N N s NNo. wherein, Q', Q, Q and Q together with the carbon to which they are (0500. In some embodiments, Q is NR. attached form a heteroaryl moiety, and each Q', Q, Q and 0501. In another aspect, the invention features a com Q is independently S, O, N, CR, CR'', NR, or NR'. pound of formula (III), 0484. In some embodiments, the compound of formula (II), comprises an enriched preparation of formula (II)

formula (II)

wherein, Z", Z, Z, Z, and Z together form an aryl or heteroaryl moiety, and each Z, Z, Z, Z, and Z is independently N. CR 0, or CR2. 0485. In some embodiments, the compound of formula 0502. In some embodiments, the compound of formula (II), comprises an enriched preparation of formula (II") (III), comprises an enriched preparation of formula (III)

formula (II") RI formula (III) y'N),k R4 o-Q : / 2 s--so-A-N Qso R2 R3 R5.

0486 In some embodiments, Q and Q are each inde 0503. In some embodiments, the compound of formula pendently S, O, N, or NR'. (III), comprises an enriched preparation of formula (III) 0487. In some embodiments, Q' and Q are each inde pendently S, O, N, or NR'. 0488. In some embodiments, Q is CR or CR'. formula (III) 0489. In some embodiments, Q is S, O, N, or NR'. 0490. In some embodiments, at least one of Q or Q is CR' or CR. 0491. In some embodiments, at least two of Q', Q, Q, or Q is S, O, N, or NR'. 0492. In some embodiments, Q', Q, and Q are each independently S, O, N, or NR'. 0493. In some embodiments, Q is NR. US 2017/O121385 A1 May 4, 2017 60

0504. In some embodiments, one of Z, Z, Z, Z, and Z alkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroary is N. lalkyl, OR'', NR'R'', CF, SOR', SOR, 0505. In some embodiments, two of Z, Z, Z, Z, and OC(O)R'', SONR'R'', (CH) R' or R'; each of Z are N. which is optionally independently substituted with 1-3 R': 0506. In some embodiments, three of Z, Z, Z, Z, and R'' and R'' are each independently hydrogen, alkyl, alk Z is N. enyl, alkynyl, cyclyl, heterocyclyl, aryl or heteroaryl; 0507. In some embodiments, two of Z' and Z are N. R'' and R'' are each independently hydrogen, alkyl, alk 0508. In some embodiments, two of Z' and Z are N. enyl, alkynyl, alkylthioalkyl, alkoxyalkyl, aryl, arylalkyl, 0509. In some embodiments, two of Z' and Z are N. heterocyclyl, heteroaryl, heteroarylalkyl, heterocycloalkyl 0510. In some embodiments, two of Z, Z, and Zare N. or cyclyl, cyclylalkyl, or R'' and R' taken together can be 0511. In some embodiments, the compound is a com cyclized to form (CH,)x(CH.) : wherein each R', and pound of formula (I), wherein Y is substituted with a single r' may independently optionally be substituted with 1 to 3 substituent R". For example, R' can be aryl or heteroaryl, Substituents selected from the group consisting of halogen, optionally substituted with up to three independent R. OR', alkoxy, heterocycloalkyl, -NR'C(O)NR'R'', 0512. In some embodiments, R' is aryl or heteroaryl, for C(O)NR'R'', NRC(O)R'', - CN, OXO, example a monocyclic aryl or monocyclic heteroaryl Such as NRSOR'', OC(O)R'', SONR'R'', SOR, phenyl, pyridyl, oxazolyl, thiazolyl, or thiophenyl. In some —S(O) R', -COOH and C(O)CR'; each R' is inde embodiments, R' is substituted with 1-3 R''. In some pendently alkyl, aryl, arylalkyl, heteroaryl, or heteroarylal embodiments, R' is halo, alkyl, or alkoxy, for example kyl, each of which may optionally be substituted with chloro, fluoro, methyl, cyano, or methoxy. —(CH), OH: 0513. In some embodiments, R' is a bicyclic heteroaryl, each R'' is independently alkoxy, alkoxycarbonyl, -C(O) for example indolyl, benzimidazolyl, benzoxazolyl, benzo NR2R12, NRIRI, C(O)R', NRC(O)NR'R''' furanyl, benzothiophenyl, or benzthiazolyl. In some embodi or —N-heteroaryl; ments, R' is substituted with 1-3 R''. In some embodi each R" is independently -(CH)N(R')C(O)R', ments, R' is halo, alkyl, or alkoxy, for example chloro, CSN. -(CH.)N(R')C(OOR'? (CH.)N(R') fluoro, methyl, cyano, or methoxy. C(O)NR2R12, (CH.)N(R')SOR', —(CH2) 0514) In some embodiments, R' is arylalkyl or het SONR'R'', -(CH.),C(O)NR'R'', -(CH.),C(O) eroarylalky, for example a monocyclic or bicyclic arylalkyl OR', -(CH)OC(O)CR', -(CH)OC(O)R., (CH.) or monocyclic or bicyclic heteroaryalkyl. In some embodi OC(O)NR'R 2, -(CH.)N(R)SO.NR'R'', —(CH2) ments, R' is substituted with 1-3 R''. In some embodi OR', -(CH)OC(O)N(R')(CH), OH, -(CH.) ments. R' is halo, alkyl, or alkoxy, for example chloro, SOR, (C)SOR, —(CH.)NR'R' or -(CH.) fluoro, methyl, cyano, or methoxy. OCHC(O)N(R')(CH.), OH: 0515. In some embodiments, R' includes an unsaturated each R' is independently halo, alkyl, alkenyl, alkynyl, or partially unsaturated cyclic moiety, for example a cyclyl alkoxy, -(CH.)NR'C(O)NR'R'', -(CH2)C(O) or heterocyclyl moiety. The cyclic moiety can either be NR'R'', -(CH.)NR'C(O)R'', CN, (CH.) directly attached to Y or attached via a linker such as an NR'SOR, —(CH)OC(O)R', —(CH2) alkylenyl linker. In some embodiments, R" is substituted SO.NR'R'', (CH.)SOR', -(CH.)COOH or with 1-3 R''. In some embodiments, R' is halo, alkyl, or (CH.),C(O)OR': alkoxy, for example chloro, fluoro, methyl, cyano, or X is CR'R'', O, S, S(O), or NR'; methoxy. m is an integer between 1 and 6: 0516. In some embodiments, R' is R'. p is an integer from 0 to 5: 0517. In some embodiments, Y is substituted with a q and S are each independently an integer between 1 and 3; second R', for example an alkyl, halo or alkoxy. and 0518. In some embodiments, R' is aryl, heteroaryl, ary w is an integer between 0 and 5. lalkyl, or heteroarylalkyl; k is a bond or O; n is 1 or 2: R 0520 For example, in some embodiments, n is 1: k" is a and Rare both hydrogen; bond or O; and R' is aryl, heteroaryl, arylalkyl, or heteroary lalkyl. In some embodiments, n is 1; k is O; and R' is A is arylalkyl, for example phenylmethyl. In some embodiments, n is 2; k is a bond; and r" is aryl. 0519 0521 For example, in some embodiments, R7 and R' are H; x is 1; and y is 0 or 1. In some embodiments, A is CHCH or CHCHCH. R7a 0522. In some embodiments, each R and R is indepen dently alkyl, for example, methyl or ethyl, preferably ethyl. -- (CH2).- -(CH2), s 0523. In some embodiments, Y is a monocyclic het R7b eroaromatic moiety, for example a nitrogen containing het eraromatic moiety Such as nitrogen containing five mem X and y are each independently 0-6: bered heteraromatic moiety. R" and Rare each independently hydrogen or alkyl: 0524. In some embodiments, Y is a heterocyclic moiety Y is a monocyclic aryl or monocyclic heteroaryl; each of containing at least two heteroatoms, for example, a five which is optionally substituted with 1-4 R': membered heterocyclic moiety containing at least two het each R" is independently alkyl, alkenyl, alkynyl, halo, eroatoms or at least three heteroatoms. cyano, carbonyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, 0525. In some embodiments Y is substituted with one cyclyl cyclylalkyl, alkoxy, alkoxyalkyl, aryloxy, aryloxy R". R' can be positioned, for example, 1.3 relative to the US 2017/O121385 A1 May 4, 2017

point of attachment of Y to the adjacent chain carbon or 1.2 each R" is independently alkyl, alkenyl, alkynyl, halo, relative to the point of attachment of Y to the adjacent chain cyano, carbonyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, carbon. cyclyl cyclylalkyl, alkoxy, alkoxyalkyl, aryloxy, aryloxy 0526. In some embodiments, R' is aryl or heteroaryl, for alkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroary example a monocyclic aryl or monocyclic heteroaryl Such as lalkyl, OR'', NR'R'', CF, SOR', SOR, phenyl, pyridyl, oxazolyl, thiazolyl, or thiophenyl. In some OC(O)R'', -SONR'R'',-(CH) R' or R'; each of embodiments, R' is substituted with 1-3 R'. In some which is optionally independently substituted with 1-3 R': embodiments, R' is halo, alkyl, or alkoxy, for example R'' and R'' are each independently hydrogen, alkyl, alk chloro, fluoro, methyl, cyano, or methoxy. enyl, alkynyl, cyclyl, heterocyclyl, aryl or heteroaryl; 0527. In some embodiments, R' is a bicyclic heteroaryl, R'' and R'' are each independently hydrogen, alkyl, alk for example indolyl, benzimidazolyl, benzoxazolyl, benzo enyl, alkynyl, alkylthioalkyl, alkoxyalkyl, aryl, arylalkyl, furanyl, benzothiophenyl, or benzthiazolyl. In some embodi heterocyclyl, heteroaryl, heteroarylalkyl, heterocycloalkyl ments, R' is substituted with 1-3 R''. In some embodi or cyclyl, cyclylalkyl, or R'' and R' taken together can be ments, R' is halo, alkyl, or alkoxy, for example chloro, cyclized to form (CH,)x(CH.) : wherein each R', and fluoro, methyl, cyano, or methoxy. R" may independently optionally be substituted with 1 to 3 0528. In some embodiments, R' is arylalkyl or het Substituents selected from the group consisting of halogen, eroarylalky, for example a monocyclic or bicyclic arylalkyl OR'', alkoxy, heterocycloalkyl, - NR'C(O)NR'R''. or monocyclic or bicyclic heteroaryalkyl. In some embodi C(O)NR'R'', NRC(O)R'', - CN, OXO, ments, R' is substituted with 1-3 R''. In some embodi NRSOR'', OC(O)R', SONR'R'', SOR, ments, R' is halo, alkyl, or alkoxy, for example chloro, —S(O) R', -COOH and –C(O)CR; each R' is inde fluoro, methyl, cyano, or methoxy. pendently alkyl, aryl, arylalkyl, heteroaryl, or heteroarylal 0529. In some embodiments, R' includes an unsaturated kyl, each of which may optionally be substituted with or partially unsaturated cyclic moiety, for example a cyclyl —(CH), OH: or heterocyclyl moiety. The cyclic moiety can either be each R'' is independently alkoxy, alkoxycarbonyl, —C(O) directly attached to Y or attached via a linker such as an NR'2R12, NR'R'', C(O)R2, NRC(O)NR'R'' alkylenyl linker. In some embodiments, R' is substituted or —N-heteroaryl; with 1-3 R''. In some embodiments, R' is halo, alkyl, or each R' is independently —(CH)N(R')C(O)R', alkoxy, for example chloro, fluoro, methyl, cyano, or CSN. -(CH.)N(R')C(O)OR' (CH.)N(R') methoxy. C(O)NR2R12, (CH.)N(R')SOR', —(CH) 0530. In some embodiments Y is oxadiazole or triazole. SONR'R'', -(CH.),C(O)NR'R'', -(CH.),C(O) 0531. In some embodiments, Y is OR', -(CH)OC(O)CR', -(CH.)OC(O)R, (CH.) OC(O)NR'R'', -(CH.)N(R')SO.NR'R'', -(CH.) OR, -(CH)OC(O)N(R)(CH), OH, -(CH.)SOR, RQ N (CH.).S.O.R. —(CH.)NR'R' or -(CH)OCHC N s (O)N(R')(CH), OH: each R" is independently halo, alkyl, alkenyl, alkynyl, NNo. alkoxy, -(CH.)NR'C(O)NR'R'', -(CH2)C(O) NR'R'', (CH.)NR'C(O)R'', CN, (CH.) wherein Q1 is O or NR, preferably O or NH. In some NR'SO.R'', (CH)OC(O)R'', -(CH.)SO.NR'R'', embodiments, R' is aryl, arylalkyl, heteroaryl, or heteroary (CH.)SOR', -(CH2)COOH or -(CH.),C(O)OR': lalkyl, for example optionally substituted with one or more X is CR'R'', O, S, S(O), OR NR'; R". In some embodiments, R' is substituted with one R', m is an integer between 1 and 6: Such as halo (e.g., fluoro or chloro) or alkoxy. p is an integer from 0 to 5: 0532. In some embodiments, the compounds has a for q and S are each independently an integer between 1 and 3; mula (Ia) and w is an integer between 0 and 5. For example in some embodiments, n is 1; k is a bond or O: formula (Ia) and R' is aryl, heteroaryl, arylalkyl, or heteroarylalkyl. In R1 some embodiments, n is 1; k is O; and R' is arylalkyl, for k example phenylmethyl. In some embodiments, n is 2; k is )n R4 a bond; and R' is aryl. 0534. In some embodiments, A is CHCH or t-so-A-( CHCHCH, preferably CHCHCH. R5 0535. In some embodiments, each R and R is indepen dently alkyl, for example, methyl or ethyl, preferably ethyl. 0533. In some embodiments, R is aryl, heteroaryl, ary 0536. In some embodiments, Y is a monocyclic het lalkyl, or heteroarylalkyl: eroaromatic moiety, for example a nitrogen containing het k' is a bond or O: eraromatic moiety Such as a nitrogen containing five mem n is 1 or 2; bered heteraromatic moiety. A is CH, CHCH, or CHCHCH: 0537. In some embodiments, Y is a heterocyclic moiety R" and Rare each independently hydrogen or alkyl: containing at least two heteroatoms, for example, a five Y is a monocyclic aryl or monocyclic heteroaryl; each of membered heterocyclic moiety containing at least two het which is optionally substituted with 1-4 R': eroatoms or at least three heteroatoms. US 2017/O121385 A1 May 4, 2017 62

0538. In some embodiments Y is substituted with one Y is a monocyclic aryl or monocyclic heteroaryl; each of R''. R' can be positioned, for example, 1.3 relative to the which is optionally substituted with 1-4 R': point of attachment of Y to the adjacent chain carbon or 1.2 each R" is independently alkyl, alkenyl, alkynyl, halo, relative to the point of attachment of Y to the adjacent chain cyano, carbonyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, carbon. cyclyl cyclylalkyl, alkoxy, alkoxyalkyl, aryloxy, aryloxy 0539. In some embodiments, R' is aryl or heteroaryl, for alkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroary example a monocyclic aryl or monocyclic heteroaryl Such as lalkyl, OR'', NR'R'', CF, SOR', SOR, phenyl, pyridyl, oxazolyl, thiazolyl, or thiophenyl. In some OC(O)R'', SONR'R'', -(CH),R or R'; each of embodiments, R' is substituted with 1-3 R'. In some which is optionally independently substituted with 1-3 R': embodiments, R' is halo, alkyl, or alkoxy, for example R'' and R'' are each independently hydrogen, alkyl, alk chloro, fluoro, methyl, cyano, or methoxy. enyl, alkynyl, cyclyl, heterocyclyl, aryl or heteroaryl; (0540. In some embodiments, R' is a bicyclic heteroaryl, R'' and R'' are each independently hydrogen, alkyl, alk for example indolyl, benzimidazolyl, benzoxazolyl, benzo enyl, alkynyl, alkylthioalkyl, alkoxyalkyl, aryl, arylalkyl, furanyl, benzothiophenyl, or benzthiazolyl. In some embodi heterocyclyl, heteroaryl, heteroarylalkyl, heterocycloalkyl ments, R' is substituted with 1-3 R''. In some embodi or cyclyl, cyclylalkyl, or R'' and R' taken together can be ments, R' is halo, alkyl, or alkoxy, for example chloro, cyclized to form (CH,)x(CH.) : wherein each R', and fluoro, methyl, cyano, or methoxy. R" may independently optionally be substituted with 1 to 3 (0541. In some embodiments, R' is arylalkyl or het Substituents selected from the group consisting of halogen, eroarylalky, for example a monocyclic or bicyclic arylalkyl OR'', alkoxy, heterocycloalkyl, - NR'C(O)NR'R''. or monocyclic or bicyclic heteroaryalkyl. In some embodi C(O)NR'R'', NRC(O)R'', - CN, OXO, ments, R' is substituted with 1-3 R''. In some embodi NRSOR'', OC(O)R'', SONR'R'', SOR, ments, R' is halo, alkyl, or alkoxy, for example chloro, S(O).R, COOH and C(O)CR': fluoro, methyl, cyano, or methoxy. each R is independently alkyl, aryl, arylalkyl, heteroaryl, (0542. In some embodiments, R' includes an unsaturated or heteroarylalkyl, each of which may optionally be substi or partially unsaturated cyclic moiety, for example a cyclyl tuted with —(CH), OH: or heterocyclyl moiety. The cyclic moiety can either be each R'' is independently alkoxy, alkoxycarbonyl, —C(O) directly attached to Y or attached via a linker such as an NR'2R12, NR'R'', C(O)R2, NRC(O)NR'R'' alkylenyl linker. In some embodiments, R' is substituted or —N-heteroaryl; with 1-3 R''. In some embodiments, R' is halo, alkyl, or alkoxy, for example chloro, fluoro, methyl, cyano, or methoxy. 0543 In some embodiments Y is oxadiazole or triazole. SONR'R'', -(CH.),C(O)NR'R'', -(CH.),C(O) 0544 In some embodiments, Y is OR', -(CH)OC(O)CR°, -(CH)OC(O)R., (CH.) OC(O)NR'R'', -(CH.)N(R)SO.NR'R'', -(CH.) OR', -(CH)OC(O)N(R')(CH), OH, -(CH.) RQ N SOR*, (C)SOR, —(CH.)NR'R' or -(CH.) N s OCHC(O)N(R')(CH), OH: each R' is independently halo, alkyl, alkenyl, alkynyl, NNo. alkoxy, -(CH.)NR'C(O)NR'R'', -(CH.),C(O) NR'R'', -(CH.)NR'C(O)R'', CN, (CH.) wherein Q1 is O or NR, preferably O or NH. In some NR'SOR, —(CH)OC(O)R'', —(CH) embodiments, R' is aryl, arylalkyl, heteroaryl, or heteroary SO.NR'R'', (CH.)SOR', -(CH.)COOH or lalkyl, for example optionally substituted with one or more (CH.),C(O)OR': R". In some embodiments, R' is substituted with one R', X is CR'R'', O, S, S(O), S(O), or NR'': Such as halo (e.g., fluoro or chloro) or alkoxy. m is an integer between 1 and 6: (0545. In some embodiments, R' is hydrogen or alkyl, for p is an integer from 0 to 5: example unsubstituted or substituted with one R: q and S are each independently an integer between 1 and 3; n is 0 or 1; and k' is a bond; and w is an integer between 0 and 5. R° and Reach independently hydrogen or C-C alkyl: (0547. In some embodiments, n is 0 or 1; k is a bond; and R" is alkyl, for example unsubstituted or substituted with one A is R. 0548. In some embodiments n is 0 and k is a bond. 0546 Example R' moieties include methyl, and ethyl. Preferred R' moieties include methyl. In some embodiments R' is unsub stituted methyl or methyl or ethyl substituted with C(O)N (R). (CH),-C-(CH2), s 0549. In some embodiments, n is 0 or 1; k' is a bond; and R" is alkyl, for example unsubstituted or substituted with one X and y are each independently 0-6: R. For example, R' can be a branched alkyl such as one of R" and Rare each independently hydrogen or alkyl: the following US 2017/O121385 A1 May 4, 2017

0562. In some embodiments. Y is

A-lis - RQ N No wherein Q1 is O or NR, preferably O or NH. In some embodiments, R' is aryl, arylalkyl, heteroaryl, or heteroary lalkyl, for example optionally substituted with one or more 0550. In some embodiments n is 0 and k is a bond, and R''. In some embodiments, R' is substituted with one R', R" and R are both methyl. Such as halo (e.g., fluoro or chloro) or alkoxy. 0551. In some embodiments, n is 0; k is a bond; and R' 0563. In some embodiments, the compounds has a for is hydrogen. mula (Ib) 0552. In some embodiments, A is CHCH or CHCHCH preferably CHCHCH. formula (Ib) 0553. In some embodiments, each RandR is indepen RI R4 dently alkyl, for example, methyl or ethyl, preferably ethyl. N-SOA 0554. In some embodiments, Y is a monocyclic het O-H-so-( eroaromatic moiety, for example a nitrogen containing het R2 R5 eraromatic moiety Such as a nitrogen containing five mem bered heteraromatic moiety. 10564) In some embodiments, R' is hydrogen or alkyl: 0555. In some embodiments, Y is a heterocyclic moiety A is CH, CHCH, or CHCHCH: containing at least two heteroatoms, for example, a five R’ is hydrogen or C-C alkyl: membered heterocyclic moiety containing at least two het R" and Rare each independently hydrogen or alkyl: eroatoms or at least three heteroatoms. Y is a monocyclic aryl or monocyclic heteroaryl; each of 0556. In some embodiments Y is substituted with one which is optionally substituted with 1-4 R': R". R' can be positioned, for example, 1.3 relative to the each R' is independently alkyl, alkenyl, alkynyl, halo, point of attachment of Y to the adjacent chain carbon or 1.2 cyano, carbonyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, relative to the point of attachment of Y to the adjacent chain cyclyl cyclylalkyl, alkoxy, alkoxyalkyl, aryloxy, aryloxy carbon. alkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroary lalkyl, OR'', NR'R'', CF, SOR, SOR, 0557. In some embodiments, R' is aryl or heteroaryl, for OC(O)R'', SONR'R'', (CH) R' or R'; each of example a monocyclic aryl or monocyclic heteroaryl Such as which is optionally independently substituted with 1-3 R': phenyl, pyridyl, oxazolyl, thiazolyl, or thiophenyl. In some R'' and R'' are each independently hydrogen, alkyl, alk embodiments, R' is substituted with 1-3 R'. In some enyl, alkynyl, cyclyl, heterocyclyl, aryl or heteroaryl; embodiments, R' is halo, alkyl, or alkoxy, for example R'' and R'' are each independently hydrogen, alkyl, alk chloro, fluoro, methyl, cyano, or methoxy. enyl, alkynyl, alkylthioalkyl, alkoxyalkyl, aryl, arylalkyl, 0558. In some embodiments, R' is a bicyclic heteroaryl, heterocyclyl, heteroaryl, heteroarylalkyl, heterocycloalkyl for example indolyl, benzimidazolyl, benzoxazolyl, benzo or cyclyl, cyclylalkyl, or R'' and R' taken together can be furanyl, benzothiophenyl, or benzthiazolyl. In some embodi cyclized to form (CH,)x(CH.) : wherein each R', and ments, R' is substituted with 1-3 R''. In some embodi R" may independently optionally be substituted with 1 to 3 ments, R' is halo, alkyl, or alkoxy, for example chloro, Substituents selected from the group consisting of halogen, fluoro, methyl, cyano, or methoxy. OR', alkoxy, heterocycloalkyl, -NR'C(O)NR'R'', 0559. In some embodiments, R' is arylalkyl or het C(O)NR'R'', NRC(O)R'', - CN, OXO, eroarylalky, for example a monocyclic or bicyclic arylalkyl NR''SOR'', OC(O)R', SONR'R'', SOR, or monocyclic or bicyclic heteroaryalkyl. In some embodi —S(O) R', -COOH and C(O)CR'; each R' is inde ments, R' is substituted with 1-3 R''. In some embodi pendently alkyl, aryl, arylalkyl, heteroaryl, or heteroarylal ments, R' is halo, alkyl, or alkoxy, for example chloro, kyl, each of which may optionally be substituted with fluoro, methyl, cyano, or methoxy. —(CH), OH: each R'' is independently alkoxy, alkoxycarbonyl, —C(O) 0560. In some embodiments, R' includes an unsaturated NR'2R12, NR'R'', C(O)R12, NRC(O)NR'R'', or partially unsaturated cyclic moiety, for example a cyclyl or —N-heteroaryl; or heterocyclyl moiety. The cyclic moiety can either be each R' is independently —(CH)N(R')C(O)R, directly attached to Y or attached via a linker such as an CSN. -(CH.)N(R')C(O)OR' (CH.)N(R') alkylenyl linker. In some embodiments, R" is substituted C(O)NR2R12, (CH.)N(R')SOR', —(CH) with 1-3 R''. In some embodiments, R' is halo, alkyl, or SONR'R'', -(CH.),C(O)NR'R'', -(CH.),C(O) alkoxy, for example chloro, fluoro, methyl, cyano, or OR', -(CH)OC(O)CR', -(CH)OC(O)R., (CH.) methoxy. OC(O)NR'R'2, -(CH.)N(R)SO.NR'R'', -(CH.) 0561. In some embodiments Y is oxadiazole or triazole. OR', -(CH)OC(O)N(R')(CH.),OH, -(CH.) US 2017/O121385 A1 May 4, 2017 64

(0575. In some embodiments, Y is OCHC(O)N(R')(CH),SORCH, SOR(CH.),NR'R' OH: or -(CH.) each R" is independently halo, alkyl, alkenyl, alkynyl, alkoxy, —(CH.)NR'C(O)NR'R'', -(CH2)C(O) RQ N NR'R'', (CH)NR''C(O)R'', CN, (CH.) NRSOR, (CH2)9C(OR, —(CH2) SO.NR'R'', (CH.)SOR", -(CH2)COOH or NNo. (CH,)(C(O)CR; wherein Q1 is O or NR, preferably O or NH. In some X is CR'R'', O, S, S(O), S(O), or NR'': embodiments, R' is aryl, arylalkyl, heteroaryl, or heteroary m is an integer between 1 and 6: lalkyl, for example optionally substituted with one or more R". In some embodiments, R' is substituted with one R', p is an integer from 0 to 5: Such as halo (e.g., fluoro or chloro) or alkoxy. q and S are each independently an integer between 1 and 3; 0576. In some embodiments, R' and R together form a and heterocyclic ring Such as a pyrrolidine or an aZetidine ring (The heterocyclic ring can be unsubstituted or substituted, w is an integer between 0 and 5. for example, with 1-2 R); 0565. In some embodiments, A is CHCH or n is 0 or 1; CHCHCH preferably CHCHCH. k' is a bond; 0566) In some embodiments, each RandR is indepen Rhydrogen or C-C alkyl, preferably hydrogen; dently alkyl, for example methyl or ethyl, preferably ethyl. A is 0567. In some embodiments, Y is a monocyclic het 0577 eroaromatic moiety, for example a nitrogen containing het eraromatic moiety Such as a nitrogen containing five mem bered heteraromatic moiety. 0568. In some embodiments, Y is a heterocyclic moiety (CH),-C-(CH2) s containing at least two heteroatoms, for example, a five membered heterocyclic moiety containing at least two het eroatoms or at least three heteroatoms. X and y are each independently 0-6: 0569. In some embodiments Y is substituted with one R" and Rare each independently hydrogen or alkyl; R". R' can be positioned, for example, 1.3 relative to the Y is a monocyclic aryl or monocyclic heteroaryl; each of point of attachment of Y to the adjacent chain carbon or 1.2 which is optionally substituted with 1-4 R': relative to the point of attachment of Y to the adjacent chain each R" is independently alkyl, alkenyl, alkynyl, halo, carbon. cyano, carbonyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, (0570. In some embodiments, R' is aryl or heteroaryl, for cyclyl cyclylalkyl, alkoxy, alkoxyalkyl, aryloxy, aryloxy example a monocyclic aryl or monocyclic heteroaryl Such as alkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroary phenyl, pyridyl, oxazolyl, thiazolyl, or thiophenyl. In some lalkyl, OR'', NR'R'', CF, SOR', SOR, embodiments, R' is substituted with 1-3 R''. In some OC(O)R'', -SONR'R'',-(CH) R' or R'; each of embodiments, R' is halo, alkyl, or alkoxy, for example which is optionally independently substituted with 1-3 R': chloro, fluoro, methyl, cyano, or methoxy. R'' and R'' are each independently hydrogen, alkyl, alk enyl, alkynyl, cyclyl, heterocyclyl, aryl or heteroaryl; 0571. In some embodiments, R' is a bicyclic heteroaryl, R'' and R'' are each independently hydrogen, alkyl, alk for example indolyl, benzimidazolyl, benzoxazolyl, benzo enyl, alkynyl, alkylthioalkyl, alkoxyalkyl, aryl, arylalkyl, furanyl, benzothiophenyl, or benzthiazolyl. In some embodi heterocyclyl, heteroaryl, heteroarylalkyl, heterocycloalkyl ments, R' is substituted with 1-3 R''. In some embodi or cyclyl, cyclylalkyl, or R'' and R' taken together can be ments, R' is halo, alkyl, or alkoxy, for example chloro, cyclized to form -(CH2).X(CH2), ; wherein each R'' and fluoro, methyl, cyano, or methoxy. R" may independently optionally be substituted with 1 to 3 0572. In some embodiments, R' is arylalkyl or het Substituents selected from the group consisting of halogen, eroarylalky, for example a monocyclic or bicyclic arylalkyl OR', alkoxy, heterocycloalkyl, -NR'C(O)NR'R'', or monocyclic or bicyclic heteroaryalkyl. In some embodi C(O)NR'R'', NRC(O)R'', - CN, OXO, ments, R' is substituted with 1-3 R''. In some embodi NR''SOR''': OC(O)R'', SONR'R'', SOR, ments, R' is halo, alkyl, or alkoxy, for example chloro, —S(O), R', -COOH and C(O)CR'; each R' is inde fluoro, methyl, cyano, or methoxy. pendently alkyl, aryl, arylalkyl, heteroaryl, or heteroarylal kyl, each of which may optionally be substituted with 0573. In some embodiments, R' includes an unsaturated —(CH), OH: or partially unsaturated cyclic moiety, for example a cyclyl each R'' is independently alkoxy, alkoxycarbonyl, -C(O) or heterocyclyl moiety. The cyclic moiety can either be NR12R12, NR'R'', C(O)R'?, NRC(O)NR'R'' directly attached to Y or attached via a linker such as an or —N-heteroaryl; alkylenyl linker. In some embodiments, R" is substituted each R' is independently —(CH.)N(R')C(O)R’’, with 1-3 R''. In some embodiments, R' is halo, alkyl, or -(CH2)CN, -(CH.)N(R)')C(O)OR', -(CH.)N alkoxy, for example chloro, fluoro, methyl, cyano, or (R')C(O)NR'R'', (CH.)N(R)SO.R', -(CH.) methoxy. SONR'R'', -(CH.),C(O)NR'R'', -(CH.),C(O) 0574) In some embodiments Y is oxadiazole or triazole. OR', -(CH)OC(O)OR°, -(CH)OC(O)R’, (CH.) US 2017/O121385 A1 May 4, 2017

wherein Q1 is O or NR, preferably O or NH. In some OR, (CHOC(O)NRCHOH, -(CH.)SOR, embodiments, R' is aryl, arylalkyl, heteroaryl, or heteroary (CH),SO.R., -(CH.)NR'R' or -(CH)OCHC lalkyl, for example optionally substituted with one or more (O)N(R')(CH), OH: R". In some embodiments, R' is substituted with one R', each R' is independently halo, alkyl, alkenyl, alkynyl, Such as halo (e.g., fluoro or chloro) or alkoxy. alkoxy, —(CH.)NR'C(O)NR'R'', -(CH2)C(O) 0588. In some embodiments, the compounds has a for NR'R'', -(CH.)NR'C(O)R'', CN, (CH.) mula (Ic) NR'SOR, —(CH)OC(O)R', —(CH2) SO.NR'R'', (CH.)SOR', -(CH.)COOH or (CH.),C(O)OR': formula (Ic) X is CR'R'', O, S, S(O), S(O), or NR'': m is an integer between 1 and 6: p is an integer from 0 to 5: q and S are each independently an integer between 1 and 3; and w is an integer between 0 and 5; n is 0, 1, 2, 3, or 4: preferably 1 or 2: 0578. In some embodiments, A is CHCH or A is CH, CHCH, or CHCHCH: CHCHCH preferably CHCHCH. R" and Rare each independently hydrogen or alkyl: (0579. In some embodiments, each RandR is indepen Y is a monocyclic aryl or monocyclic heteroaryl; each of dently alkyl, for example, methyl or ethyl, preferably ethyl. which is optionally substituted with 1-4 R': 0580. In some embodiments, Y is monocyclic heteroaro each R' is independently alkyl, alkenyl, alkynyl, halo, matic moiety, for example a nitrogen containing heteraro cyano, carbonyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, matic moiety Such as a nitrogen containing five membered cyclyl cyclylalkyl, alkoxy, alkoxyalkyl, aryloxy, aryloxy heteraromatic moiety. alkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroary 0581. In some embodiments, Y is a heterocyclic moiety lalkyl, OR'', NR'R'', CF, SOR', SOR, containing at least two heteroatoms, for example, a five OC(O)R'', -SONR'R'',-(CH) R' or R'; each of membered heterocyclic moiety containing at least two het which is optionally independently substituted with 1-3 R': eroatoms or at least three heteroatoms. R'' and R'' are each independently hydrogen, alkyl, alk 0582. In some embodiments, R' is aryl or heteroaryl, for enyl, alkynyl, cyclyl, heterocyclyl, aryl or heteroaryl; example a monocyclic aryl or monocyclic heteroaryl Such as R'' and R'' are each independently hydrogen, alkyl, alk phenyl, pyridyl, oxazolyl, thiazolyl, or thiophenyl. In some enyl, alkynyl, alkylthioalkyl, alkoxyalkyl, aryl, arylalkyl, embodiments, R' is substituted with 1-3 R'. In some heterocyclyl, heteroaryl, heteroarylalkyl, heterocycloalkyl embodiments, R' is halo, alkyl, or alkoxy, for example or cyclyl, cyclylalkyl, or R'' and R' taken together can be chloro, fluoro, methyl, cyano, or methoxy. cyclized to form -(CH2).X(CH2), ; wherein each R'' and 0583. In some embodiments, R' is a bicyclic heteroaryl, R" may independently optionally be substituted with 1 to for example indolyl, benzimidazolyl, benzoxazolyl, benzo 3 Substituents selected from the group consisting of halogen, furanyl, benzothiophenyl, or benzthiazolyl. In some embodi OR', alkoxy, heterocycloalkyl, -NR'C(O)NR'R'', ments, R' is substituted with 1-3 R''. In some embodi C(O)NR'R'', NRC(O)R'', - CN, OXO, ments, R' is halo, alkyl, or alkoxy, for example chloro, NR''SOR'', OC(O)R', SONR'R'', SOR, fluoro, methyl, cyano, or methoxy. —S(O), R', -COOH and C(O)CR'; each R' is inde 0584) In some embodiments, R' is arylalkyl or het pendently alkyl, aryl, arylalkyl, heteroaryl, or heteroarylal eroarylalky, for example a monocyclic or bicyclic arylalkyl kyl, each of which may optionally be substituted with or monocyclic or bicyclic heteroaryalkyl. In some embodi —(CH), OH: ments, R' is substituted with 1-3 R''. In some embodi each R'' is independently alkoxy, alkoxycarbonyl, -C(O) ments, R' is halo, alkyl, or alkoxy, for example chloro, NR12R12, NR'R'', C(O)R'?, NRC(O)NR'R'' fluoro, methyl, cyano, or methoxy. or —N-heteroaryl; 0585. In some embodiments, R' includes an unsaturated each R" is independently -(CH)N(R')C(O)R', or partially unsaturated cyclic moiety, for example a cyclyl or heterocyclyl moiety. The cyclic moiety can either be directly attached to Y or attached via a linker such as an alkylenyl linker. In some embodiments, R' is substituted with 1-3 R''. In some embodiments, R' is halo, alkyl, or OC(O)NR'R''. CHNIR)SO.NR'R'', —(CH2) alkoxy, for example chloro, fluoro, methyl, cyano, or methoxy. E.p s E.N.E.R.2. 2 s 2 p. E.2 In some embodiments Y is oxadiazole or triazole. OCHC(O)N(R')(CH), OH: 0586 each R' is independently halo, alkyl, alkenyl, alkynyl, 0587 In some embodiments, Y is alkoxy, -(CH.)NR'C(O)NR'R'', -(CH.),C(O) NR'R'', -(CH.)NR'C(O)R'', CN, (CH.) NR'SOR, —(CH)OC(O)R', —(CH2) RQ N SO.NR'R'', (CH.)SOR', -(CH.)COOH or N s (CH),C(O)OR; NNo. X is CR'R'', O, S, S(O), S(O), or NR'': m is an integer between 1 and 6: p is an integer from 0 to 5: US 2017/O121385 A1 May 4, 2017 66 q and S are each independently an integer between 1 and 3; 0600. In another aspect, the invention features a com and pound of formula (IV) formula (IV) w is an integer between 0 and 5. 0589. In some embodiments, A is CHCH or formula (IV) CHCHCH preferably CHCHCH. R1 0590. In some embodiments, each RandR is indepen dently alkyl, for example, methyl or ethyl, preferably ethyl. k 0591. In some embodiments, Y is a monocyclic het eroaromatic moiety, for example a nitrogen containing het eraromatic moiety Such as nitrogen containing five mem O---so bered heteraromatic moiety. 0592. In some embodiments. Y is a heterocyclic moiety containing at least two heteroatoms, for example, a five wherein, membered heterocyclic moiety containing at least two het R" is hydrogen, aryl, heteroaryl, arylalkyl, heteroarylalkyl, eroatoms or at least three heteroatoms. cyclyl cyclylalkyl, heterocyclyl, heterocyclylalkyl, alkyl, 0593. In some embodiments Y is substituted with one alkenyl, alkynyl, or R' can be taken together with R". R' can be positioned, for example, 1.3 relative to the R° or R to form a ring; each of which is optionally point of attachment of Y to the adjacent chain carbon or 1.2 substituted with 1-4 R: relative to the point of attachment of Y to the adjacent chain k' is a bond, O, C(O), C(O)O, OC(O), C(O)NR, NRC(O), carbon. S, SO, SO, CR-CR, OR C=C; n is 0-6, preferably 1-3: 0594. In some embodiments, R' is aryl or heteroaryl, for R is hydrogen, C-C alkyl, C-C alkenyl, or C-C alky example a monocyclic aryl or monocyclic heteroaryl Such as nyl: phenyl, pyridyl, oxazolyl, thiazolyl, or thiophenyl. In some A' is heterocyclyl: optionally substituted with 1-3 R: embodiments, R' is substituted with 1-3 R'. In some Y is a monocyclic aryl or monocyclic heteroaryl; each of embodiments, R'halo, alkyl, or alkoxy, for example chloro, which is optionally substituted with 1-4 R': fluoro, methyl, cyano, or methoxy. each R is independently halo, alkyl, alkenyl, alkynyl, cyclyl, heterocyclyl, aryl, heteroaryl, alkoxy, haloalkyl, 0595. In some embodiments, R' is a bicyclic heteroaryl, haloalkyloxy, haloalkylthio, acetyl, cyano, nitro, hydroxy, for example indolyl, benzimidazolyl, benzoxazolyl, benzo oxo, C(O)OR, OC(O)R, N(R), C(O)N(R), NRC(O) furanyl, benzothiophenyl, or benzthiazolyl. In some embodi R°, or SR; ments, R is substituted with 1-3 R''. In some embodiments, R is halo, alkyl, cyclyl, heterocyclyl, aryl, heteroaryl, R" is halo, alkyl, or alkoxy, for example chloro, fluoro, alkoxy, haloalkyl, haloalkyloxy, haloalkylthio, acetyl, methyl, cyano, or methoxy. cyano, nitro, hydroxy, oxo, C(O)CR, OC(O)R, N(R), 0596) In some embodiments, R' is arylalkyl or het C(O)N(R), NR°C(O)R’, SR; eroarylalky, for example a monocyclic or bicyclic arylalkyl each R" is independently alkyl, alkenyl, alkynyl, halo, or monocyclic or bicyclic heteroaryalkyl. In some embodi cyano, carbonyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, ments, R' is substituted with 1-3 R''. In some embodi cyclyl cyclylalkyl, alkoxy, alkoxyalkyl, aryloxy, aryloxy ments. R' is halo, alkyl, or alkoxy, for example chloro, alkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroary fluoro, methyl, cyano, or methoxy. lalkyl, OR'', NR'R'', CF, SOR', OC(O) R'', -SONR'R'',-(CH) R' or R'; each of which is 0597. In some embodiments, R' includes an unsaturated optionally independently substituted with 1-3 R'; or partially unsaturated cyclic moiety, for example a cyclyl R'' and R'' are each independently hydrogen, alkyl, alk or heterocyclyl moiety. The cyclic moiety can either be enyl, alkynyl, cyclyl, heterocyclyl, aryl or heteroaryl; directly attached to Y or attached via a linker such as an R'' and R'' are each independently hydrogen, alkyl, alk alkylenyl linker. In some embodiments, R" is substituted enyl, alkynyl, alkylthioalkyl, alkoxyalkyl, aryl, arylalkyl, with 1-3 R''. In some embodiments, R' is halo, alkyl, or heterocyclyl, heteroaryl, heteroarylalkyl, heterocycloalkyl alkoxy, for example chloro, fluoro, methyl, cyano, or or cyclyl, cyclylalkyl, or R'' and R' taken together can be methoxy. cyclized to from -(CH2)X(CH.) : wherein each R and R" may independently optionally be substituted with 1 to 0598 In some embodiments Y is oxadiazole or triazole. 3 Substituents selected from the group consisting of halogen, 0599 In some embodiments, Y is OR', alkoxy, heterocycloalkyl, -NR'C(O)NR'R'', C(O)NR'R'', NRC(O)R'', - CN, OXO, NR''SOR'', OC(O)R', SONR'R'', SOR, RQ N S(O).R, COOH and C(O)CR': each R" is independently alkyl, aryl, arylalkyl, heteroaryl, N s or heteroarylalkyl, each of which may optionally be substi tuted with —(CH), OH: NNo. each R'' is independently alkoxy, alkoxycarbonyl, —C(O) NR'2R12, NR'R'', C(O)R2, NRC(O)NR'R'' wherein Q1 is O or NR, preferably O or NH. In some or —N-heteroaryl; embodiments, R" aryl, arylalkyl, heteroaryl, or heteroary each R" is independently heterocycloalkyl, heteroaryl, lalkyl, for example optionally substituted with one or more —CN, -(CH.)N(R')C(O)R', -(CH2)CN, -(CH.)N R". In some embodiments, R' is substituted with one R', (R'2)C(O)OR, -(CH.)N(R')C(O)NR'R'', -(CH.) Such as halo (e.g., fluoro or chloro) or alkoxy. N(Ri)SO.R'°, -(CH.)SO.NR'R'', -(CH.),C(O) US 2017/O121385 A1 May 4, 2017 67

NR'R'', -(CH.)N(R')SO.NR'R'', -(CH.) OR', 0608. In some embodiments, Y is a monocyclic het —(CH)OC(O)N(R')(CH),OH, -(CH.)SOR or eroaromatic moiety, for example, a nitrogen containing —(CH.).OCHC(O)N(R')(CH), OH: heteraromatic moiety, Such as a nitrogen containing 5 mem each R" is independently halo, alkyl, alkenyl, alkynyl, bered heteraromatic moiety. alkoxy, -(CH.)NR'C(O)NR'R'', -(CH2)C(O) NR'R'', -(CH.)NR'C(O)R'', CN, (CH.) 0609. In some embodiments, Y is a heterocyclic moiety NR'SOR, —(CH.)OC(O)R'', —(CH) containing at least two heteroatoms, for example, a 5 mem SO.NR'R'', (CH.)SOR', -(CH.)COOH or bered heterocyclic moiety containing at least two heteroa (CH.),C(O)OR': toms or a heterocyclic moiety containing at least 3 heteroa X is CR'R'', O, S, S(O), S(O), or NR'': tOmS. m is an integer between 1 and 6: 0610. In some embodiments, Y is substituted with 1 R'. p is an integer from 0 and 5. The R' can be positioned, for example, 1.3 relative to the q and S are each independently an integer between 1 and 3; point of attachment of Y to the adjacent chain carbon or can and be positioned, for example, 1.2 relative to the point of w is an integer between 0 and 5. attachment of Y to the adjacent chain carbon. 0601. In some embodiments, the compound of formula 0611. In some embodiments, R' is aryl or heteroaryl, for (IV), comprises an enriched preparation of formula (IV). example a monocyclic aryl or monocyclic heteroaryl Such as phenyl, pyridyl, or thiophenyl. In some embodiments, R' is substituted with 1-3 R''. In some embodiments, R' is halo, formula (IV) alkyl, or alkoxy, for example chloro, fluoro, methyl, or RI methoxy. k I0612. In some embodiments, R' is a bicyclic heteroaryl, for example indolyl, imidazolyl, benzoxazolyl, or benzthi O---so azolyl. In some embodiments, R' is substituted with 1-3 R2 A R''. In some embodiments, R' is halo, alkyl, or alkoxy, for example chloro, fluoro, methyl, or methoxy. 0613 In some embodiments, Y is oxadiazole or triazole. 0602. In some embodiments, the compound of formula 0.614. In another aspect, the invention features a com (IV), comprises an enriched preparation of formula (IV") pound of formula (V), formula (V)

formula (IV") formula (V) R1 g- S )n i--so

0603. In some embodiments, A' is a 5 or 6 membered heterocyclyl. wherein, 0604. In some embodiments, the 5 or 6 membered het Q', Q, Q and Q together with the carbon to which they are erocyclyl includes at least two nitrogen atoms. attached form a heteroaryl moiety, and each Q', Q, Q and 0605. In some embodiments, A' is Q is independently S, O, N, CR, CR', NR, or NR'. 0615. In some embodiments, the compound of formula O O (V), comprises an enriched preparation of formula (V) V/

formula (V)

0606. In some embodiments, A' is substituted with one R. for example, N(R). 0607. In some embodiments, n is 1; k is a bond or O; and R" is aryl, heteroaryl, arylalkyl, or heteroarylalkyl. In some embodiments, n is 1; k is O; and R' is arylalkyl. For example, R' can be phenylmethyl. In some embodiments, in is 2; k is a bond; and R' is aryl. US 2017/O121385 A1 May 4, 2017 68

0616) In some embodiments, the compound of formula 0624. In some embodiments, the compound of formula (V), comprises an enriched preparation of formula (V") (IV), comprises an enriched preparation of a compound of formula (VI'). formula (V") formula (VI)

0617. In some embodiments, Q' and Q are each inde pendently S, O, N, or NR'. 0625. In some embodiments, the compound of formula 0618. In some embodiments, Q' and Q are each inde (VI), comprises an enriched preparation of a compound of pendently S, O, N, or NR". In some embodiments, Q’ is CR or CR''. In some embodiments, Q is S, O, N, or NR'. formula (VI"). In some embodiments, at least one of Q or Q is CR or CR''. In some embodiments, at least two of Q', Q, Q, or formula (VI") Q is S, O, N, or NR''. In some embodiments, Q, Q, and RI Q are each independently S, O, N, or NR''. In some embodiments, Q' is NR". In some embodiments, one of Q, /Nsk Q, or Q is CR. In some embodiments, Q is CR''. In some A s embodiments, Q is CR. Z3 -N-SO2 0619. In some embodiments, Q', Q, Q and Q together form Y-z R2 U

RQ N 0626. In some embodiments, one of Z, Z, Z, Z, and Z is N. In some embodiments, two of Z, Z, Z, Z, and Zare N. In some embodiments, three of Z', Z, Z, Z, and Z is NNo. N. In some embodiments, two of Z' and Z are N. In some embodiments, two of Z' and Z are N. In some embodi 0620. In some embodiments, Q is NR. ments, two of Z' and Z are N. In some embodiments, two 0621. In some embodiments, Q', Q, Q and Q together of Z, Z, and Z are N. form 0627. In some embodiments, the compound is a com pound of formula (IV), wherein Y is substituted with a single substituent R". For example, R' can be aryl or heteroaryl, optionally substituted with up to three independent R. R2 N (0628. In some embodiments, R' is aryl or heteroaryl, for y example a monocyclic aryl or monocyclic heteroaryl Such as NNo. phenyl, pyridyl, or thiophenyl. In some embodiments, R' is substituted with 1-3 R''. In some embodiments, R' is halo, alkyl, or alkoxy, for example chloro, fluoro, methyl, or 0622. In some embodiments, Q is NR". methoxy. 0623. In another aspect, the invention features a com (0629. In some embodiments, R' is a bicyclic heteroaryl, pound of formula (VI), for example indolyl, imidazolyl, benzoxazolyl, or benzthi azolyl. In some embodiments, R' is substituted with 1-3 R''. In some embodiments, R' is halo, alkyl, or alkoxy, for formula (VI) R1 example chloro, fluoro, methyl, or methoxy. 0630. In some embodiments, R' is R''. In some embodi k ments, Y is substituted with a second R', for example an Z-Z / S. alkyl, halo or alkoxy. In another aspect, the invention A features a pharmaceutically acceptable salt comprising a Z3 )---so compound of any of the formulae described herein. Y- Zl R2 U 0631. In some embodiments, the compound is an enantiomerically enriched isomer of a stereoisomer described herein. For example, the compound has an wherein enantiomeric excess of at least about 100, 15%, 20%, 25%, Z", Z, Z, Z, and Z together form an aryl or heteroaryl 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, moiety, and each Z, Z, Z, Z, and Z is independently N. 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%. CR'', or CR. Enantiomer, when used herein, refers to either of a pair of US 2017/O121385 A1 May 4, 2017 69 chemical compounds whose molecular structures have a the Table below. By enriched is meant at least 600/o, e.g., of mirror-image relationship to each other. the molecules of compound in the preparation have a 0632. In some embodiments, a preparation of a com selected stereochemistry of a selected stereocenter. In pre pound disclosed herein is enriched for an isomer of the ferred embodiments it is at least 65%, 70%, 75%, 80%, 85%, compound having a selected Stereochemistry, e.g., R or S. 90%. 95%, 96%, 97%, 98%, or 99%. Enriched refers to the level of a Subject molecule(s) and does not connote a process corresponding to a selected Stereocenter, e.g., the position limitation unless specified. corresponding to the carbon alpha to the Sulfonamide nitro 0635. In some embodiments, a preparation of a com gen in formula (I). Example R/S configurations can be those pound disclosed herein, is enriched for isomers (subject provided in an example described herein, e.g., those isomers) which are diastereromers of the compound described in the Table below, or the configuration of the described herein. For example, a compound having a majority or minority species in a synthetic scheme described selected Stereochemistry, e.g., R or S. corresponding to a herein. For example, the compound has a purity correspond selected Stereocenter, e.g., the position corresponding to the ing to a compound having a selected Stereochemistry of a carbon alpha to the Sulfonamide nitrogen of a formula selected stereocenter of at least about 60%, 65%, 70%, 75%, described herein e.g., formula (I), (II), (III), (IV), (V), or 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%. (VI). Example R/S configurations can be those provided in 0633. In some embodiments, a compound described an example described herein, e.g., those described in the herein includes a preparation of a compound disclosed Table below, or the configuration of the majority or minority herein that is enriched for a structure or structures having a species in a synthetic scheme described herein. For example, selected Stereochemistry, e.g., R or S, at a selected Stereo the compound has a purity corresponding to a compound center, e.g., the carbon alpha to the Sulfonamide nitrogen of having a selected Stereochemistry of a selected Stereocenter a formula described herein e.g., formula (I), (II), (III), (IV), of at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, (V), or (VI). Example R/S configurations can be those 95%, 96.0%, 97%, 98%, or 99%. Diastereromer, when used provided in an example described herein, e.g., those herein, refers to a stereoisomer of a compound having two described in the Table below, or the configuration of the or more chiral centers that is not a mirror image of another majority or minority species in a synthetic scheme described Stereoisomer of the same compound. herein. For example, the compound has a purity correspond 0636. In one embodiment, the compound has a molecular ing to a compound having a selected Stereochemistry of a weight less than D-Lys-3-GHRP6 or H(2)N-D-arg-Pro selected stereocenter of at least about 60%, 65%, 70%, 75%, Lys-Pro-d-Phe-Gln-d-Trp-Phe-d-Trp-Leu-Leu-NH(2) (L 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%. 756,867) or within 2, 1.5, 14, 1.2, 1.1, 0.8, 0.6, or 0.5 fold 0634. An "enriched preparation,” as used herein, is that of D-Lys-3-GHRP-6 or L 756,867. enriched for a selected Stereoconfiguration of one, two, three 0637. In another aspect, the invention features a com or more selected Stereocenters within the Subject compound. pound listed in Table 4. Representative compounds of the Example selected Stereocenters and example stereoconfigu invention are depicted below in Table 4. Other example rations thereof can be selected from those provided, herein, compounds are within the scope set forth in the Summary or e.g., in an example described herein, e.g., those described in are described elsewhere herein. TABLE 4 Examples of GHS-R Modulating Compounds Number Name Activity* 1 Die ny onic acid (R)-2-benzyloxy-1-(3- C - )-ethyl-amide 2 ny ulfonic acid (R)-2-benzyloxy-1-3- ich oxadiazol-5-yl)-ethyl-amide ny onic acid (R)-1-(5-(2,3-dichloro -3-phenyl-propyl-amide ny onic acid (R)-3-phenyl-1-(3-o- 2 propyl-amide ny O onic acid (R)-1-(5-(4-fluoro )-2H-1.2.4 -3-phenyl-propyl-amide hylamino-pro onic acid (R)-3-phenyl-1-(3- A. -1,2,4-oxadi )-propyl-amide hylamino onic acid (R)-3-phenyl-1-(5- en-3-yl-2H -3-yl)-propyl-amide ny amino onic acid (R)-3-phenyl-1-(5- Eri luoro-p 2.4 triazol-3-yl)-propyl-amide amino-pro onic acid (R)-1-3-(3-methyl -yl)-1.2.4 5-yl)-3-phenyl-propyl-amide 10 amino-pro onic acid (R)-1-3-(2-chloro-6- henyl)-1,2,4-oxadiazol-5-yl)-3-phenyl-propyl-amide 11 hylamino propane-1-sulfonic acid (R)-1-(5- 1,3dioxo -5-yl-2H-1,2,4-triazol-3-yl)-3-phenyl-propyl 12 ny amino-propane-1-Su onic acid (R)-1-3-(2,6-dichloro )-1,2,4-oxadiazol-5-yl -3-phenyl-propyl-amide 13 hylamino-ethanesulfonic acid (R)-3-phenyl-1-(3-o-tolyl oxadiazol-5-yl)-propyl -amide

US 2017/O121385 A1 May 4, 2017 74

TABLE 4-continued Examples of GHS-R Modulating Compounds Number Name Activity* 154 3-Diethylamino-propane-1-sulfonic acid {1-3-(4-bromo-benzyl)- A. 1,2,4-oxadiazol-5-yl)-1-methyl-ethyl-amide 155 3-Diethylamino-propane-1-sulfonic acid {1-3-(4-methoxy B phenyl)-1,2,4-oxadiazol-5-yl)-1-methyl-ethyl-amide 156 3-Diethylamino-propane-1-sulfonic acid 5-(4-methoxy-phenyl)- 2H-1,2,4-triazol-3-ylmethyl-amide 157 3-Diethylamino-propane-1-sulfonic aci 5-(4-chloro-benzyl)-2H 1,2,4-triazol-3-ylmethyl-amide 158 3-Diethylamino-propane-1-sulfonic aci 3-(4-fluoro-phenyl)- 1,2,4-oxadiazol-5-ylmethyl-amide 159 (S)-3-Diethylamino-propane-1-sulfonic acid {1-5-(4-methoxy phenyl)-2H-1,2,4-triazol-3-yl)-3-methyl-butyl-amide 160 (R)-3-Diethylamino-propane-1-sulfonic acid {1-5-(4-methoxy phenyl)-2H-1,2,4-triazol-3-yl)-3-methyl-butyl-amide *A refers to a compound having antagonist activity with a Ki <100 nM in a cell based assay. B refers to a compound having antagonist activity with a Kibetween 100 nM and 500 nM in a cell based assay. Crefers to a compound having antagonist activity with a Kibetween 500 nM and 1000 nM in a cell based assay. D refers to a compound having antagonist activity with Ki, s1000 nM in a cell-based assay, E refers to other example compounds,

0638 Representative compounds that modulate GHS-R -continued include the compounds of formulas (I), (II), (III). (IV), (V), formula (V) and (VI) below, where all variables are as described herein. R1 k (), formula (I) Q -Q RI QNo)---so R2 A. k ), R4 M formula (VI) -N-SOA-N R1 R2 R3 \,:R k

formula (II) RI k

In some preferred embodiments, Y is a 5 membered het No. R2 R3 R5 eroaromatic moiety substituted with 1 or 2 substituents as described herein. Example Y moieties are reproduced below. formula (III) RI k ) Z4-75 pi MR4 Z3 )---soa-X Y-A R R5 y y Y formula (IV) RI O N O k -y N1 N N1 N n ), NNN l l O- -N-SO H H R2 A. US 2017/O121385 A1 May 4, 2017 75

-continued -continued N N R1

S S NN's ), Rg N N - PG 0639. In another aspect, the invention features a com- Sk H pound, including the hydrogens depicted on the nitrogen N-O atoms, can be substituted with R''. In some preferred embodiments, the heteroaryl moiety includes 1 or 2 R' substituents. In some preferred embodiments, R' is aryl, 0644. In the Schemes provided herein, all variables are arylalkyl, or R5. When two R0 substituents are included, in defined as herein and PG is a nitrogen protecting group. The some embodiments, one R' is R'' and the second R' is a nitrogen protected amino acid is reacted with a N-hydroxy imidamide (amidoxime) moiety (which is prepared by react different Substituent, such as alkyl, alkoxy, halo, etc. ing a cyano containing moiety with hydroxylamine) to (0640. In certain instances, R' is an aryl moiety such as a produce an oxadiazole containing moiety. The resulting phenyl moiety, for example unsubstituted or substituted aryl compound can be further manipulated to form a compound moiety. In some instances, R' is a heteroaryl moiety such as of formula (I) by removing the nitrogen protecting group an indole moiety. In many instances where R' is aryl or and reacting the resulting moiety with an activated Sulfone, heteroaryl (or other lipophilic moiety such as alkyl), K is an such as a sulfonyl chloride as depicted below. oxygen or a bond. A and Rand R can be chosen to vary the compounds type of interaction with GHS-R. For example, in some instances where R and Rare both hydrogen, the i compound is an agonist of GHS-R. In other instances where 'k R4 R" and Rare both independently alkyl, the compound is an ), V / antagonist of GHS-R. PG C11 n-Na R5 0641. Other aspects of this invention relate to a compo- Rg N N1 H sition having a compound of any of the formulae described Sk O herein and a pharmaceutically acceptable carrier, or a com- N pound of any of the formulae described herein, an additional therapeutic compound (e.g., an anti-hypertensive compound 'k or a cholesterol lowering compound), and a pharmaceuti- ), O O R4 cally acceptable carrier; or a compound of any of the \/ l formulae described herein, an additional therapeutic com- Rg N N1,N1 NRs pound, and a pharmaceutically acceptable carrier. \ O H 0642 Combinations of substituents and variables envi N sioned by this invention are only those that result in the formation of stable compounds. The term “stable', as used Scheme B below depicts the formation of a triazole con herein, refers to compounds which possess stability Sufi taining moiety which can be further reacted in a manner cient to allow manufacture and which maintains the integrity similar to the oxadiazole containing moiety to form a of the compound for a sufficient period of time to be useful compound of formula (I). for the purposes detailed herein (e.g., therapeutic or prophy lactic administration to a Subject). 0643. The compounds described herein can be made Scheme B using a variety of synthetic techniques. In some embodi- RI NH ments, a Y moiety, or other ring corresponding to a Y moiety, N1 can be synthesized onto an amino acid or amino acid type k ) l starting material as depicted in schemes A and B and B' pi R10 NH 2 below. HO PG - e N1 formula (XI) H O Scheme A R1

RI -OH k 'k N ), ), R10 l NH Rg N N1 PG HO PG - - H N1 formula (X) M H NH US 2017/O121385 A1 May 4, 2017 76

0645 Scheme B' below depicts an alternative method of forming a triazole containing moiety which can be further Scheme C. reacted in a manner similar to the oxadiazole containing moiety to form a compound of formula (I). R1 O O R4 'k \/ ), C1 N1 NR5 + HO -e- P-OAlkyli NH2 HS 1. N OAlkyl O RI Y 2 R10 'k S ), O O R4 HO \/ l R10 ls NH, - AlkylatinT - N1 N1 NRs agent H Alkyl O s1 0648. The free carboxyl moiety can then be further a?s manipulated to produce a compound of formula (I). For R1 example, the free carboxyl moiety can be reacted with a 'k compound of formula (X) or (XI) above to form an oxadi ), azole or triazole containing compound of formula (I) in a O PG NHNH2-HCI manner similar to that described in schemes A, B and B' Alky1 N1 Her above. H 0649. As can be appreciated by the skilled artisan, further O methods of synthesizing the compounds of the formulae R1 herein will be evident to those of ordinary skill in the art. S - Alkyl Additionally, the various synthetic steps may be performed 'k in an alternate sequence or order to give the desired com H ), O 1. pounds. Synthetic chemistry transformations and protecting -N - PG - N - group methodologies (protection and deprotection) useful in HN N H synthesizing the compounds described herein are known in O the art and include, for example, those such as described in RI R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts, Pro 'k tective Groups in Organic Synthesis, 2d. Ed., John Wiley and ), Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons RQ N N1 PG (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and sub \{N - NH H sequent editions thereof. Additionally, the compounds dis closed herein can be prepared on a solid Support or using a Solid phase peptide synthesis. 0646. The triazole precursor moiety can be prepared in a 0650. The term "solid support” refers a material to which variety of manners, for example, by reacting a cyano con a compound is attached to facilitate identification, isolation, taining moiety with a hydrazine hydrate (to form the inter purification, or chemical reaction selectivity of the com pound. Such materials are known in the art and include, for mediate amidraZone) as depicted in Scheme B. Alternatively, example, beads, pellets, disks, fibers, gels, or particles Such the triazole precursor moiety can be prepared as shown in as cellulose beads, pore-glass beads, silica gels, polystyrene scheme B' by reacting a nitrile moiety (e.g., an arylnitrile or beads optionally cross-linked with divinylbenzene and benzylnitrile) with a dialkyl dithiophosphate moiety such as optionally grafted with polyethylene glycol, poly-acrylam diethyl dithiophosphate to provide anthioimidate, which is ide beads, latex beads, dimethylacrylamide beads optionally cross-linked with N,N'-bis-acryloyl ethylene diamine, glass reacted with a acyl hydrazide moiety to provide the triazole particles coated with hydrophobic polymer, and material precursor. The acyl hydrazide moiety can be prepared, for having a rigid or semi-rigid Surface. The Solid Supports example, by reacting a carboxylic acid or derivative thereof optionally have functional groups such as amino, hydroxy, with hydrazine. carboxy, or halo groups, (see, Obrecht, D. and Villalgrodo. J. M., Solid-Supported Combinatorial and Parallel Synthe 0647. In other embodiments, a compound of formula (I) sis of Small-Molecular-Weight Compound Libraries, Perga can be prepared by first reacting an activated Sulfone moiety mon-Elsevier Science Limited (1998), and include those (e.g., a Sulfonyl chloride) with an amino acid moiety or useful in techniques such as the “split and pool” or “parallel protected amino acid, as depicted in Scheme C below. synthesis techniques, Solid-phase and solution-phase tech US 2017/O121385 A1 May 4, 2017 77 niques, and encoding techniques (see, for example, Czarnik, quaternization of any basic nitrogen-containing groups of A. W., Curr. Opin. Chem. Bio., (1997) 1, 60). the compounds disclosed herein. Water or oil-soluble or 0651. The term “solid phase peptide' refers to an amino dispersible products may be obtained by Such quaterniza acid, which is chemically bonded to a resin (e.g., a solid tion. Support). Resins are generally commercially available (e.g., from Sigma Aldrich). Some examples of resins include Rink Glutathione Peroxidase Mimics/Mimetics and Inducers resins. Tentagel S RAM, MBHA, and BHA-resins. 0656. In some embodiments, the composition comprises 0652 The compounds of this invention may contain one glutathione peroxidase mimics/mimetics and inducers. Glu or more asymmetric centers and thus occur as racemates and tathione peroxidase is one of the enzymes which are actively racemic mixtures, single enantiomers and enantiometric involved in the regulation of the concentration of oxygen mixtures, individual diastereomers and diastereomeric mix derived free radicals formed during various physiological or tures. All Such isomeric forms of these compounds are pathological processes. As used herein, the term "gluta expressly included in the present invention. The compounds thione peroxidase designates any enzyme having gluta of this invention may also be represented in multiple tau thione peroxidase activity. By way of illustration of these tomeric forms, in Such instances, the invention expressly enzymes, there may in particular be mentioned as gluta includes all tautomeric forms of the compounds described thione peroxidase 1 (GPX1), glutathione peroxidase 2 herein (e.g., alkylation of a ring system may result in (GPX2), glutathione peroxidase 3 (GPX3), glutathione per alkylation at multiple sites, the invention expressly includes oxidase 4 (GPX4), glutathione peroxidase 5 (GPX5), glu all Such reaction products). All Such isomeric forms of Such tathione peroxidase 6 (GPX6), glutathione peroxidase 7 compounds are expressly included in the present invention. (GPX7), and glutathione peroxidase 8 (GPX8). GPX1 and All crystal forms of the compounds described herein are GPX4 are expressed in most tissues with a clear predomi expressly included in the present invention. nance in the erythrocytes, the liver and the kidneys for 0653. As used herein, the compounds of this invention, GPX1 (Chambers et al; EMBO J 5: 1221-1227 (1986)) and including the compounds of formulae described herein, are in the testicles for GPX4 Roveri et al; J. Biol. Chem. defined to include pharmaceutically acceptable derivatives 267:6142–6146 (1992). GPX3 is produced in the kidneys, or prodrugs thereof. A "pharmaceutically acceptable deriva the lungs, the heart, the breast, the placenta as well as in the tive or prodrug” means any pharmaceutically acceptable liver (Chu et al. Blood 79: 3233-3238 (1992)) as for GPX2, salt, ester, Salt of an ester, or other derivative of a compound it has mainly been demonstrated in the gastrointestinal of this invention (for example an imidate ester of an amide), tissues and in the liver Chu et al. J. Biol. Chem. 268: which, upon administration to a recipient, is capable of 2571-257 (1993). The DNA sequence encoding glutathione providing (directly or indirectly) a compound of this inven peroxidase may be a cDNA, a genomic DNA (gDNA), or a tion. Particularly favored derivatives and prodrugs are those hybrid construct consisting for example of a cDNA into that increase the bioavailability of the compounds of this which one or more introns would be inserted. The nucleic invention when such compounds are administered to a sequence of the cDNA encoding human glutathione peroxi mammal (e.g., by allowing an orally administered com dase has been described by Mullenbach et al., Oxy-Radicals pound to be more readily absorbed into the blood) or which in Molecular Biology and Pathology, 313-326, (1988). It enhance delivery of the parent compound to a biological may also be synthetic or semisynthetic sequences. compartment (e.g., the brain or lymphatic system) relative to 0657 Example proteins that intended to be encompassed the parent species. Preferred prodrugs include derivatives by the term “glutathione peroxidase' include those having where a group, which enhances aqueous solubility or active amino acid sequences disclosed in GenBank with accession transport through the gut membrane is appended to the number for Homo sapiens (Human)—NP 000572, structure of formulae described herein. NP 002074, NP 002075, NP_001034936, NP 001500, 0654 The compounds of this invention may be modified NP 874360, NP 056511, NP_001008398, or Mus musculus by appending appropriate functionalities to enhance selec (Mouse) NP 032186, NP 109602, NP 032187, tive biological properties. Such modifications are known in NP 001032830, NP-034473, NP 663426, NP 077160, the art and include those which increase biological penetra NP 081403. Example nucleic acid molecules that encode tion into a given biological compartment (e.g., blood, lym glutathione peroxidase are those disclosed in GenBank with phatic system, central nervous system), increase oral avail accession number for Homo sapiens (Human)—NM ability, increase solubility to allow administration by 000581, NM_002083, NM_002084, NM_00103.9847, injection, alter metabolism and alter rate of excretion. NM_001509, NM 182701, NM 015696, NM_001008397, 0655 Pharmaceutically acceptable salts of the com or Mus musculus (Mouse) NM_008160, NM 030677, pounds of this invention include those derived from phar NM_001083929, NM_001037741, NM 010343, maceutically acceptable inorganic and organic acids and NM 145451, NM 024198, NM_027127. bases. Examples of Suitable acid salts include acetate, adi 0658. The present disclosure provides for a number of pate, benzoate, benzenesulfonate, butyrate, citrate, diglu Glutathione peroxidase mimics to be used in a therapeutic conate, dodecylsulfate, formate, fumarate, glycolate, combination with ghrelin or ghrelin variants in treating mBI. hemisulfate, heptanoate, hexanoate, hydrochloride, hydro Representative non-limiting examples of glutathione peroxi bromide, hydroiodide, lactate, maleate, malonate, methane dase mimics include: 2-phenyl-1,2-benzoisoselenazol-3 Sulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, pal (2H)-one (ebselen); 6A,6B-diseleninic acid-6A, 6B"-sele moate, phosphate, picrate, pivalate, propionate, Salicylate, nium bridged B-cyclodextrin (6-diseCD); and 2,2'-diseleno Succinate, Sulfate, tartrate, tosylate and undeconoate. Salts bis-Beta-cyclodextrin (2-diSeCD) as disclosed in U.S. Pat. derived from appropriate bases include alkali metal (e.g., No. 7.923,442, which is incorporated herein by reference in Sodium), alkaline earth metal (e.g., magnesium), ammonium its entirety for all of its disclosure, including all methods, and N-(alkyl) salts. This invention also envisions the materials, etc. US 2017/O121385 A1 May 4, 2017

0659. In one embodiment, the glutathione peroxidase 0672. In another embodiment, the glutathione peroxidase mimic/mimetic or its isomer, metabolite, and/or salt thereof mimetic or its isomer, metabolite, and/or salt thereof is is represented by the compound of formula (A): represented by the compound of formula (B):

R R5 R RI C N st R2 MS N )n yea S1 NR3 R R4

0660 wherein R' and Rare independently hydrogen; 0673 wherein, lower alkyl; OR: -(CH)NR'R'': —(CH)NH: 0674 X is O or NH —(CH), NHSO(CH)NH; —NO; CN: 0675 M is Se or Te - SOH: N*(R)-O; F: Cl; Br; I; (CH), R: 0676 n is 0-2 -(CH2)COR: S(O)NR'R'; SONR'R'': CO 0677 R is oxygen; and forms an oxo complex with (CH.)COR. R. M; or 10661) R'-hydrogen; lower alkyl: aralkyl; substituted 0678 R is oxygen or NH; and aralkyl, -(CH2)COR; -(CH2)R. —CO(CH) 0679 forms together with the metal, a 4-7 member COR: —(CH), SO.R. —(CH.).S(O)R. ring, which optionally is Substituted by an oxo or amino 10662) R'lower alkyl: aralkyl; substituted aralkyl: group; or —(CH.)COR: —(CH.) R. F; 0680 forms together with the metal, a first 4-7 member 0663 R-lower alkyl: aralkyl; substituted aralkyl: ring, which is optionally Substituted by an oxo or amino 10664) R-lower alkyl: aralkyl; substituted aralkyl: group, wherein said first ring is fused with a second 4-7 —(CH.),COR: —(CH.) R. member ring, wherein said second 4-7 member ring is 0665 R=lower alkyl; aralkyl; substituted aralkyl; optionally substituted by alkyl, alkoxy, nitro, aryl, -(CH2)COR: cyano, hydroxy, amino, halogen, Oxo, carboxy, thio. 0666 R-lower alkyl: aralkyl; substituted aralkyl: thioalkyl, or NH(C=O)R', C(=O)NR'R'', aryl; substituted aryl; heteroaryl; substituted heteroaryl; - NR'R' or -SOR where R and R are indepen hydroxy: lower alkoxy: dently H, alkyl or aryl; and 0681 R, R and Rare independently hydrogen, alkyl, 0667 R’ is represented by any structure of the follow alkoxy, nitro, aryl, cyano, hydroxy, amino, halogen, ing formulae: oxo, carboxy, thio, thioalkyl, or -NH(C=O)R', C(=O)NR'R'', NR'R' or SOR where R and R° are independently H, alkyl or aryl; or R, R or R together with the organometallic ring to which two of SOHO the substituents are attached, form a fused 4-7 member OH HN1 N1 3 N-1 n.1 Y ring system wherein said 4-7 member ring is optionally Substituted by alkyl, alkoxy, nitro, aryl, cyano, hydroxy, amino, halogen, oxo, carboxy, thio, thioalkyl, or —NH (C—OR, C(=O)NR'R'', NR'R' or SOR O OH where R and R are independently H, alkyl or aryl; wherein R is not an alkyl, and 0682 wherein if R. R. and Ra are hydrogen and R. forms an oxo complex with M., n is 0 then M is Te; or 0683 if R. R. and Ra are hydrogen and R is an oxygen that forms together with the metal an unsub stituted, saturated, 5 member ring, n is 0 then M is Te; O 0684 if R is an oxo group, and n is 0, R and R form together with the organometallic ring a fused benzene ring, R is hydrogen, then M is Se; or HO O 0685 if R is an oxo group, and R and R form together with the organometallic ring a fused benzene ring, R is oxygen, n is 0 and forms together with the 0668 R'-hydrogen; lower alkyl: aralkyl or substi metal a first 5 membered ring, substituted by an oxo tuted aralkyl: aryl or substituted aryl; group a to R, and said ring is fused to a second 0669 Y" represents the anion of a pharmaceutically benzene ring, then M is Te. acceptable acid; 0686. In another embodiment, the glutathione peroxidase 0670) n-O, 1; m=O, 1, 2; p=1, 2, 3: q=2, 3, 4; and mimetic or its isomer, metabolite, and/or salt thereof is 0671 r–0, 1. represented by the compound of formula (C): US 2017/O121385 A1 May 4, 2017 79

R O R3 M No NH R M R2 2 YNH R4

R3 O 0687 wherein, M. R. and Ra are as described above. 0688. In another embodiment, the glutathione peroxidase (0695 wherein, M. R. and Rs are as described above. mimetic or its isomer, metabolite, and/or salt thereof is 0696. In another embodiment, the glutathione peroxidase represented by the compound of formula (D): mimetic or its isomer, metabolite, and/or salt thereof is represented by the compound of formula (H):

R4 R4

Rsb-Rsb R2 R2 R4 R4 R3 R3 0689 wherein, M. R. R. and Ra are as described above; 0697 wherein, 0690. In another embodiment, the glutathione peroxidase 0698 M is Se or Te; mimetic or its isomer, metabolite, and/or salt thereof is 0699 R, R or R are independently hydrogen, alkyl, represented by the compound of formula (E): alkoxy, nitro, aryl, cyano, hydroxy, amino, halogen, oxo, carboxy, thio, thioalkyl, or -NH(C=O)R', C(O)NR'R'', NR'R' or SOR where RandR R4 R3 are independently H, alkyl or aryl; or R, R or R together with the organometallic ring to which two of the substituents are attached, is a fused 4-7 member O ring system, wherein said 4-7 member ring is option R2 MC R2 ally Substituted by alkyl, alkoxy, nitro, aryl, cyano, O hydroxy, amino, halogen, oxo, carboxy, thio, thioalkyl, or NH(C–O)R, C(=O)NR'R'', NR'R' or —SOR where R and Rare independently H, alkyl or R3 R4 aryl; and 0700 Rs or Rs, is one or more oxygen, carbon, or nitrogen atoms and forms a neutral complex with the 0691 wherein, M. R. R. and R are as described above: chalcogen. 0692. In another embodiment, the glutathione peroxidase 0701. In another embodiment, the glutathione peroxidase mimetic or its isomer, metabolite, and/or salt thereof is mimetic or its isomer, metabolite, and/or salt thereof is represented by the compound of formula (F): represented by the compound of formula (I):

0693 wherein, M. R., and R are as described above. 0702 or their combination. 0694. In another embodiment, the glutathione peroxidase 0703. In another embodiment, the glutathione peroxidase mimetic or its isomer, metabolite, and/or salt thereof is mimetic or its isomer, metabolite, and/or salt thereof is represented by the compound of formula (G): represented by the compound of formula (J): US 2017/O121385 A1 May 4, 2017 80

-continued R R2

--- A. Ar ---- S1 B (O) -N O NH OS. (0704 in which: Rhydrogen; lower alkyl: optionally o2noS 1CCOO. N- X"; substituted aryl: optionally substituted lower aralkyl: Rhydrogen; lower alkyl: optionally substituted aryl; optionally substituted lower aralkyl; A=CO; (CRR): B—NRs, O: S: Ar-optionally substituted phenyl or an optionally substituted radical of formula:

OS. o2 S YNH N-so X"; y– Z in which: Z=O; S: NRs: Rhydrogen; lower alkyl: option ally substituted aryl: optionally substituted lower aralkyl Rhydrogen; lower alkyl: optionally substituted aryl; O -O optionally substituted lower aralkyl; Rs-hydrogen; lower alkyl: optionally substituted aryl: optionally substituted lower aralkyl: optionally substituted heteroaryl: optionally substituted lower heteroaralkyl: CO(lower alkyl); CO(aryl); m=0 or 1, n=0 or 1: X" represents the cation of a pharma SO (lower alkyl); SO(aryl); Rhydrogen; lower alkyl; ceutically acceptable base; and their pharmaceutically optionally substituted aryl: optionally substituted lower acceptable salts of acids or bases. aralkyl: optionally substituted heteroaryl: optionally substi 0705. In other embodiments compounds useful for the tuted lower heteroaralkyl; trifluoromethyl: purposes herein include 4.4-dimethyl-thieno-3.2-e-isosel enazine, 4.4-dimethyl-thieno-3-2-e-isoselenazine-1-oxide, 4,4-dimethyl-thieno-2,3-e-isoselenazine, and 4.4-dim ethyl-thieno-2,3-e-isoselenazine-1-oxide. 0706. In another embodiment, the glutathione peroxidase mimetic or its isomer, metabolite, and/or salt thereof is represented by the compound of formula (K): ---r

- N

O 1n-N O O COO. in which: R-hydrogen; —C(RR)-A-B; R=lower alkyl: optionally substituted aryl: optionally sub stituted lower aralkyl; US 2017/O121385 A1 May 4, 2017

R=lower alkyl: optionally substituted aryl: optionally sub -continued stituted lower aralkyl; A=CO; (CRR): S O NH-aminoacyle CH3 B represents NRR: NRRR,Y: ORs: SRs: 1. NH-aminoacyle Aran optionally Substituted phenyl group or an optionally O N substituted radical of HN s1 O w O X S HN O NH-aminoacyle HN s O HO r NH2 Me Me Q J. J. O N S NH-aminoacyle HN in which Z represents O; S: NRs; when Rhydrogen; lower alkyl: optionally substituted aryl, optionally substituted lower aralkyl: Ra-hydrogen; lower alkyl: optionally substituted aryl: optionally substituted lower aralkyl: or Ara radical of formula Rs hydrogen; lower alkyl: optionally substituted aryl: optionally substituted lower aralkyl: optionally substituted heteroaryl: optionally substituted lower heteroaralkyl: CO(lower alkyl); CO(aryl); SO(lower alkyl); SO (aryl); Rhydrogen; lower alkyl: optionally substituted aryl; optionally substituted lower aralkyl: optionally substituted ( Z ) Rs heteroaryl: optionally substituted lower heteroaralkyl: R7-hydrogen; lower alkyl: optionally substituted aryl: in which Z—O; S: NRs; when R is hydrogen; X—Ar(R)— optionally substituted lower aralkyl: optionally substituted Se—; —S-glutathione. —S-N-acetylcysteine; —S-cyste heteroaryl: optionally substituted lower heteroaralkyl: ine; —S-penicillamine; —S-albumin; —S-glucose: Rs hydrogen; lower alkyl: optionally substituted aryl; optionally substituted lower aralkyl: optionally substituted heteroaryl: optionally substituted lower heteroaralkyl; trif luoromethyl: S O OR3 CH3

O1. N OR HN s1 O w R3: O s S r 3 HN O O 1N1 N - OR3 HN s O HO r NH2 Me Me Q O Ns OR3 N COO. O 1N1'N, O O HN ? 1C US 2017/O121385 A1 May 4, 2017 82

-continued of a ghrelin variant or a salt thereof equivalent to from about 0.3 g to about 600 mg ghrelin, Such as from about 2.0 ug to about 200 mg ghrelin, such as from about 5.0 ug to about 100 mg ghrelin, such as from about 10 ug to about 50 mg ghrelin, Such as from about 10 ug to about 5 mg ghrelin, such as from about 10 ug to about 1.0 mg ghrelin. 0709. The kit contains instructions indicating the use of -N the dosage form to achieve a desirable affect and the amount NH of dosage form to be taken over a specified time period. OS. Accordingly, in one embodiment the kit comprises instruc oanoS 1CCOO. O N-so X"; tions for administering the pharmaceutical composition. In particular said instructions may include instructions refer ring to administration of said pharmaceutical composition after mBI or concussion, or at the most about 12 hours after the incident causing mBI or concussion, such as at the most about 6 hours after the incident causing mBI or concussion, On Such as at the most about 3 hours after the incident causing Sn mBI or concussion, Such as at the most about 1 hours after O2 N H the incident causing mBI or concussion, such as at the most about 30 minutes after the incident causing mBI or concus N- SO X"; Sion, such as at the most about 10 minutes after the incident causing mBI or concussion, Such as at the most about 5 minutes after the incident causing mBI or concussion. Examples 0710. The following Examples are intended to further illustrate certain embodiments of the disclosure and are not Cu intended to limit its scope. Specific Aim 1: Establish Ghrelin Dosing, Time Course and n=0 or 1; X" represents the cation of a pharmaceutically Biomarker Signaling. acceptable base; 0711. Rationale: Y represents the anion of a pharmaceutically acceptable 0712. The following series of experiments characterize acid; an effective ghrelin concentration and a Subsequent time and their salts of pharmaceutically acceptable acids or bases. course of ghrelin dosing following mTBI. These experi Additional compounds are disclosed in U.S. Patent Appli ments delineate not only optimal dosing of ghrelin following cation No: 2008/0207679, which is incorporated herein by mTBI, but as importantly, characterize the time course of reference in its entirety for all of its disclosure, including all ghrelin efficacy following mTBI. methods, materials, etc. 0713 Feasibility: 0707. The present disclosure provides for a number of 0714. The experimental work-plan is based on standard glutathione peroxidase inducers to be used in a therapeutic neurological protocols and methodologies, with well-estab combination with ghrelin or ghrelin variants in treating mBI. lished assays, read outs and end-points and is performed in Representative non-limiting examples of glutathione peroxi partnership with Charles River Laboratories Inc. dase inducers include: selenium and retinoic acid (Brigelius 0715. The doses to tested in these experiments range Flohe, R., 1999, Free Radicals in Biology and Medicine, 27. from 0.1-100 ug/kg, which is an efficacious range as prior 951-965; Chu et al., 1999, Journal of Nutrition 129, 1846 mTBI experiments at 50 ug/kg (Sc) have shown efficacy. 1854). 0716 A. Dosing concentration model: Four groups of animals (n=7 per group): Sham (no injury), TBI (con Kits trolled cortical impact (CCI) injury mild BI), TBI+Drug 0708 Ghrelin variant compositions may be administered (CCI mild BI+substrate), Sham+Drug. For the (CCI) alone or in combination with pharmaceutically acceptable model in male 10-12 week old C57BL/6. carriers or excipients, in either single or multiple doses. The 07.17 1) Mice anesthetized with isoflurane and the body formulations may conveniently be presented in unit dosage temperature maintained with a heating pad and thermo form by methods known to those skilled in the art. The static rectal probe in a BSL2 hood. The skull skin cut and compounds can be provided in a kit. Such a kit typically using a dental drill with a trephine bit a 3 mm craniotomy contains an active compound in dosage forms for adminis over the right frontal cortex (1 mm anterior and 1 mm tration. The kit comprises an amount of dosage units corre lateral to the bregma) is performed just before the dura. sponding to the relevant dosage regimen. In some embodi The animal is placed in the stereotactic frame, which will ment, the kit comprises a pharmaceutical composition restrain its head and keep the animal in place and con comprising a ghrelin variant compound or a pharmaceuti tinuously exposed to isoflurane. cally acceptable salt thereof and pharmaceutically accept 0718, 2) A CCI injury device (Leica Microsystems Inc., able carrier, vehicles and/or excipients, said kit having Buffalo Grove, Ill.) is used for mild BI (Washington P. et. multiple dosage units. The dosage units comprise an amount al. Journal of Neurotrauma, 2012). This device uses US 2017/O121385 A1 May 4, 2017 83

electromagnetic force to produce an impact velocity with tical impact (CCI) injury mild BI), TBI+Drug (CCI mild speed, and dwell time all being individually manipulated BI+substrate), Sham+Drug. For the (CCI) model in male to produce the mild BI. The impact is delivered at a 10-12 week old C57BL/6. Ideal dosing strategy will be velocity of 5.0 m/s, a 3 mm diameter impounder tip, a optimized with experiment A and depth of 1.0 mm, and a dwell time of 200 milliseconds. 0733 B. Time course for dosing will be assessed in D. After the injury, the wound is glued using a tissue 0734 1) See Specific Aim 1 section A (Steps 1-4) adhesive that polymerizes in seconds after contact with 0735. 2) Ghrelin dosing and time course: Ghrelin is tissue. The mice respiratory rate and spontaneous move administered to randomized animals and given at two ment, tail and foot pinch will be monitored during the doses following injury at a selected concentration. The entire procedure to determine any discomfort or the need second dose at each increment is given 1 hour after the for additional analgesic. The animal’s body temperature is first. Dose times are structured as follows: Time 0 (TBI)+ maintained using a water circulating heating pad. 30 minutes; Time 0 (TBI)+90 minutes; Time 0 (TBI)+120 0719. 3) Ghrelin dosing: Ghrelin is administered to ran minutes; Time 0 (TBI)+240 minutes; Time 0 (TBI)+480 domized animals and given at two doses, 10 minutes minutes; Time 0 (TBI)+1.440 minutes; Time 0 (TBI)+2, following injury and at 1 hour following injury in varying 880 minutes concentrations (See Step 6). 0736. 3) Following incubation in a dedicated post-surgi 0720 4) Dosing concentrations: Begin first experiment at cal quarantine area, mice are sacrificed by inhalation of 0.1 ug/kg. Then increments increased accordingly to CO2 and the brains removed at 24 hours post treatment achieve doses of 0.1 ug/kg, 1 ug/kg, 10 ug/kg, and 100 for oxidative burst (See B). Lig/kg. 0737 E. Chronicity of Dosing Regimen: Four groups of 0721 5) Following incubation in a dedicated post-surgi animals (n=7 per group): Sham (no injury), TBI (con cal quarantine area, mice are sacrificed by inhalation of trolled cortical impact (CCI) injury mild BI), TBI+Drug CO2 and the brains removed at 24 hours post injury for 1-day dosing strategy (CCI mild BI+substrate), TBI+ oxidative burst. Importantly, whole brain oxidative burst Drug multi-day dosing strategy. For the (CCI) model in serves as a relatively simple and reproducible read-out for male 10-12 week old C57BL/6. Ideal dosing concentra ghrelin CNS ROS reduction following injury. tion is optimized with experiment A and B. Time course 0722 6) Data analysis compare relative oxidative burst from TBI dosing is assessed in D. Benefits of chronic reduction as a function of ghrelin dosing concentration, dosing is assessed in E. 1) See A (Steps 1-4) determining lowest effective dose. 0738 2) Drug dosing and time course: Ghrelin is admin 0723 B. Measurement of oxidative burst: (References: istered to randomized animals and given at two doses Chen Yet al. Methods in Molecular Biology, 2012, Banati injury at a selected concentration (See A and B) and a R B, et al. Neuropathology and Applied Neurobiology, selected time post injury (See C). The second dose each 1991) day is given 1 hour following the first. Dose regimen are 0724. 1) Isolation of brain cells (incorporating the structured as follows: injury site, 1/8 of a 2 mm slab at injury site: Collage (0739 Day 0 (Day of injury): 1st Ghrelin dose. 2nd nase Dispase (final 1 mg/ml, stored at 20° C.)+DNase Ghrelin dose 1 hour post 1st dose. (NEB 4C antibodies box deli fridge, add 50 ul into 0740 Day 1: 1 st Ghrelin dose. 2nd Ghrelin dose 1 20-50 ml) at 37° C. for 20 minutes, then add 4 uL of hour post 1st dose. DHR (15 ng/mL) or 4 uL of DMSO to 96 uL of the 0741. Day 2: 1st Ghrelin dose. 2nd Ghrelin dose 1 brain cell samples (5 minutes at 37°C.) (protected from hour post 1st dose. light) 0742 Day 3: 1st Ghrelin dose. 2nd Ghrelin dose 1 0725 2) Measure respiratory burst using flow cytom hour post 1st dose. etry (FL 1 channel with 488-nm laser within 10 min 0743, 3) Following incubation in a dedicated post-surgi utes) cal quarantine area, mice are sacrificed by inhalation of 0726 C. Biomarker analysis with dosing concentration CO2 and the brains removed at Day 7 post-injury for model: Experiment A and B inform optimal drug dosing. following brain histopathology analyses: B-amyloidosis/ Selected drug dosing are four groups of animals (n=7 per Tau deposition; Neurodegeneration (Fluoro-Jade); Hip group): Sham (no injury), TBI (controlled cortical impact pocampal Volume; Brain Morphology (CCI) injury mild BI), TBI+Drug (CCI mild BI--sub 0744. 4) Data analysis assesses relative accumulation of strate), Sham+Drug. 3-amyloidosis/Tau deposition, alterations in neurodegen 0727 1) See Step 1-6 (only 1 dosing level) eration and hippocampal Volume between experimental 0728, 2) Following incubation in a dedicated post-surgi groups. cal quarantine area, mice will have 1.3 ml of blood extracted through cardiac puncture and sacrificed by Specific Aim 2: Verify Ghrelin's Effect on Neurocognitie inhalation of CO2. Function Following Mild Brain Injury 0729) 3) Blood will be centrifuged at 3000 rpm at 4° C.: 0745. Rationale: plasma will be separated and stored at -20° C. 0746 Expectedly, single impact mild BI does not confer 0730 4) Plasma analysis will be conducted for tau pro significant neurocognitive dysfunction compared to severe tein (Quanterix Inc. Lexington, Mass.). TBI. However, repeat mild BI has been shown to induce 0731 5) Data analysis will compare tau protein, relative neurocognitive defects and induce both amyloid deposition rise following TBI and reduction following ghrelin as well as early tauopathy. These experiments provide clarity therapy. whether ghrelin dosing lead to decreased cognitive defects, 0732 D. Dosing Time Course: Four groups of animals and improved histopathology following repeat injury. (n=7 per group): Sham (no injury), TBI (controlled cor Assessment of hippocampal Volume further assess the pro US 2017/O121385 A1 May 4, 2017 84 tective effects of ghrelin thereof. Specific cognitive studies includes relative accumulation of B-amyloidosis/Tau depo will be determined by Morris-Water Maze (MWM) testing. sition, alterations in neurodegeneration and hippocampal 0747 A. Injury and treatment model: Three groups of Volume between experimental groups. animals (n=15 per group): Sham (no injury), TBI (con (0757. It is to be understood that while the present dis trolled cortical impact (CCI) injury mild BI), TBI+Drug closure has been described in conjunction with the above (CCI mild BI--substrate). For the (CCI) model in male embodiments, that the foregoing description and examples 10-12 week old C57BL/6. are intended to illustrate and not limit the scope of the 07:48 1) See Specific Aim 1 section A (Steps 1-4) present disclosure. Other aspects, advantages and modifica 0749 2) Ghrelin dosing: Ghrelin is administered to ran tions within the scope of the present disclosure will be domized animals at two doses, 10 minutes following apparent to those skilled in the art to which the present injury and at 1 hour following injury. The dosing strategy disclosure pertains. is optimized from data in specific aim 1. 0758. The present disclosure is not to be limited in scope 0750 3) Repeat injury trial: Injury produced same brain by the specific embodiments described which are intended region, same cortex, and identical CCI settings: as single illustrations of individual aspects of the present 0751 Day 0 CCI perform cognitive testing Day 2 disclosure, and any compositions or methods, which are 0752 Day 3 CCI perform cognitive testing Day 4 functionally equivalent are within the scope of this disclo (0753 Day 6 CCI perform cognitive testing Day 7 sure. It will be apparent to those skilled in the art that various 0754 Day 7 euthanize for brain examination modifications and variations can be made in the methods and 0755 4) At the time of sacrifice animals are perfused with compositions of the present disclosure without departing cold heparinized saline, brains extracted and preserved in from the spirit or scope of the disclosure. Thus, it is intended formalin, fixated and embedded in paraffin for 7 um that the present disclosure cover the modifications and coronal sections. Brain histopathologic analysis is per variations of this disclosure provided they come within the formed for: B-amyloidosis/Tau deposition; Neurodegen Scope of the appended claims and their equivalents. eration (Fluoro-Jade); Hippocampal volume: Brian Mor 0759 All publications and patent applications mentioned phology in this specification are herein incorporated by reference to 0756 Data analysis includes relative differences between the same extent as if each individual publication or patent groups at all-time points for latency to platform, Swim application was specifically and individually indicated to be speed, and number of platform crossing. Further analysis incorporated by reference.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 41

SEO ID NO 1 LENGTH: 28 TYPE PRT ORGANISM: Homo sapiens <4 OOs SEQUENCE: 1 Gly Ser Ser Phe Leu Ser Pro Glu. His Glin Arg Val Glin Glin Arg Llys 1. 5 1O 15 Glu Ser Llys Llys Pro Pro Ala Lys Lieu. Glin Pro Arg 2O 25

SEO ID NO 2 LENGTH: 23 TYPE PRT ORGANISM: Artificial Sequence FEATURE; OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs SEQUENCE: 2 Gly Ser Ser Phe Leu Ser Pro Glu. His Glin Arg Val Glin Val Arg Pro 1. 5 1O 15 Pro Lys Ala Pro His Val Val 2O

SEO ID NO 3 LENGTH: 24 TYPE PRT ORGANISM: Artificial Sequence FEATURE; OTHER INFORMATION: Description of Artificial Sequence: Synthetic US 2017/O121385 A1 May 4, 2017 85

- Continued peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: 2, 3-diaminopropionic acid; optionally octanoylated

<4 OOs, SEQUENCE: 3 Gly Ser Xaa Phe Leu Ser Pro Glu. His Glin Arg Val Glin Val Arg Pro 1. 5 1O 15 Pro His Lys Ala Pro His Val Val 2O

<210s, SEQ ID NO 4 &211s LENGTH: 24 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: 2, 3-diaminopropionic acid 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: 2, 3-diaminopropionic acid; optionally octanoylated

< 4 OO SEQUENCE: 4 Gly Xaa Xala Phe Lieu. Ser Pro Glu. His Glin Arg Val Glin Val Arg Pro 1. 5 1O 15 Pro His Lys Ala Pro His Val Val 2O

<210s, SEQ ID NO 5 &211s LENGTH: 28 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 5 Gly Ser Ser Phe Leu Ser Pro Glu. His Glin Arg Val Glin Val Arg Pro 1. 5 1O 15 Pro His Lys Ala Pro His Val Val Pro Ala Leu Pro 2O 25

<210s, SEQ ID NO 6 &211s LENGTH: 4 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic

22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: Isonipecotic acid-D-2Nal 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: D-Trp 22 Os. FEATURE: 223 OTHER INFORMATION: C-term. NH2 US 2017/O121385 A1 May 4, 2017 86

- Continued

<4 OOs, SEQUENCE: 6 Xaa Trp Thir Lys 1.

SEO ID NO 7 LENGTH: 144 TYPE : PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic polypeptide

<4 OOs, SEQUENCE: 7

Gly Ser Glu Pro Ala Thir Ser Gly Ser Glu Thir Pro Gly Thir Ser Glu 1. 5 15

Ser Ala Thir Pro Glu Ser Gly Pro Ser Glu Pro Ala Thir Ser Gly

Ser Glu Thir Pro Gly Ser Pro Ala Ser Pro Thir Ser Thir Glu Glu 35 4 O 45

Gly Thir Ser Thir Glu Pro Ser Glu Ser Ala Pro Gly Ser Glu Pro SO 55 6 O

Ala Thir Ser Gly Ser Glu Thir Pro Ser Glu Pro Ala Thir Ser Gly 65 70

Ser Glu Thir Pro Gly Ser Glu Pro Thir Ser Gly Ser Glu Thir Pro 85 90 95

Gly Thir Ser Thir Glu Pro Ser Glu Ser Ala Pro Gly Thir Ser Glu 11 O

Ser Ala Thir Pro Glu Ser Gly Pro Ser Glu Pro Ala Thir Ser Gly 115 12 O 125

Ser Glu Thir Pro Gly Thir Ser Thir Pro Ser Glu Gly Ser Ala Pro 13 O 135 14 O

<210s, SEQ ID NO 8 &211s LENGTH: 84 &212s. TYPE: DNA <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 8 ggct coagct tcc tagg cc talacaccag agagt cc agc agagaaagga gtcgaagaag ccaccagc.ca agctgcagcc ccga

SEO ID NO 9 LENGTH: 29 TYPE : PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

SEQUENCE: 9 Gly Ser Ser Phe Leu Ser Pro Glu. His Glin Arg Val Glin Val Arg Pro 1. 5 15

Pro His Lys Ala Pro His Val Val Pro Ala Leu Pro Leu 25

<210s, SEQ ID NO 10 &211s LENGTH: 94 US 2017/O121385 A1 May 4, 2017 87

- Continued

212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223s OTHER INFORMATION: Description of Artificial Sequence: Synthetic polypeptide

<4 OOs, SEQUENCE: 10

Gly Ser Ser Phe Leul Ser Pro Glu His Glin Arg Val Glin Wall Arg Pro 1. 5 15

Pro His Lys Ala Pro His Wall Wall Pro Ala Leul Pro Lell Ser Asn Glin 2O 25

Lell Asp Lieu. Glu Glin Glin Arg His Luell Trp Ala Ser Wall Phe Ser 35 4 O 45

Glin Ser Thr Asp Ser Gly Ser Asp Luell Thir Wall Ser Gly Arg Thir SO 55 6 O

Trp Gly Luell Arg Wall Lieu. Asn Glin Luell Phe Pro Pro Ser Ser Arg Glu 65 70 7s 8O

Arg Ser Arg Arg Ser His Glin Pro Ser Cys Ser Pro Glu Luell 85 90

<210s, SEQ ID NO 11 &211s LENGTH: 4 212. TYPE: PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 11 Gly Ser Ser Phe 1.

<210s, SEQ ID NO 12 &211s LENGTH: 5 212. TYPE: PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 12

Gly Ser Ser Phe Lell 1.

<210s, SEQ ID NO 13 &211s LENGTH: 6 212. TYPE: PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 13

Gly Ser Ser Phe Luell Ser 1.

SEQ ID NO 14 LENGTH: 7 TYPE PRT ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 14

Gly Ser Ser Phe Leul Ser Pro 1. 5

<210s, SEQ ID NO 15 &211s LENGTH: 8 212. TYPE: PRT &213s ORGANISM: Homo sapiens US 2017/O121385 A1 May 4, 2017 88

- Continued

<4 OOs, SEQUENCE: 15 Gly Ser Ser Phe Leu Ser Pro Glu 1. 5

<210s, SEQ ID NO 16 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 16 Gly Ser Ser Phe Leu Ser Pro Glu. His 1. 5

<210s, SEQ ID NO 17 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 17 Gly Ser Ser Phe Leu Ser Pro Glu. His Glin 1. 5 1O

<210s, SEQ ID NO 18 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 18 Gly Ser Ser Phe Leu Ser Pro Ser Glin Llys Pro Glin Asn Llys Val Lys 1. 5 1O 15 Ser Ser Arg Ile 2O

<210s, SEQ ID NO 19 &211s LENGTH: 28 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 19 Gly Ser Ser Phe Lieu. Ser Pro Glu. His Gln Lys Ala Glin Glin Arg Llys 1. 5 1O 15 Glu Ser Lys Llys Pro Pro Ala Lys Lieu. Glin Pro Arg 2O 25

<210s, SEQ ID NO 2 O &211s LENGTH: 28 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 2O Gly Ser Ser Phe Leu Ser Pro Glu. His Glin Llys Val Glin Glin Arg Lys 1. 5 1O 15

Glu Ser Lys Llys Pro Ala Ala Lys Lieu Lys Pro Arg US 2017/O121385 A1 May 4, 2017 89

- Continued

2O 25

<210s, SEQ ID NO 21 &211s LENGTH: 28 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 21 Gly Ser Ser Phe Lieu. Ser Pro Glu. His Glin Arg Ala Glin Glin Arg Llys 1. 5 1O 15 Glu Ser Lys Llys Pro Pro Ala Lys Lieu. Glin Pro Arg 2O 25

<210s, SEQ ID NO 22 &211s LENGTH: 26 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 22 Gly Ser Ser Phe Leu Ser Pro Thr Tyr Lys Asn Ile Glin Glin Glin Lys 1. 5 1O 15 Asp Thr Arg Llys Pro Thr Ala Arg Lieu. His 2O 25

<210s, SEQ ID NO 23 &211s LENGTH: 28 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 23 Gly Ser Ser Phe Lieu. Ser Pro Glu. His Glin Llys Lieu. Glin Glin Arg Llys 1. 5 1O 15 Glu Ser Lys Llys Pro Pro Ala Lys Lieu. Glin Pro Arg 2O 25

<210s, SEQ ID NO 24 &211s LENGTH: 28 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Ser (O-n-octanoyl)

<4 OOs, SEQUENCE: 24 Gly Ser Ser Phe Leu Ser Pro Glu. His Glin Arg Val Glin Glin Arg Lys 1. 5 1O 15

Glu Ser Lys Llys Pro Pro Ala Lys Lieu. Glin Pro Arg 2O 25

<210s, SEQ ID NO 25 US 2017/O121385 A1 May 4, 2017 90

- Continued

&211s LENGTH: 29 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: Fluorescein-Ahicx 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: Ser (O-n-octanoyl) <4 OOs, SEQUENCE: 25 Xaa Gly Ser Ser Phe Leu Ser Pro Glu. His Glin Arg Val Glin Glin Arg 1. 5 1O 15 Lys Glu Ser Lys Llys Pro Pro Ala Lys Lieu Gln Pro Arg 2O 25

<210s, SEQ ID NO 26 &211s LENGTH: 28 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222 LOCATION: (3) . . (3) <223> OTHER INFORMATION: Ser (O-n-octanoyl)

<4 OOs, SEQUENCE: 26 Gly Ser Ser Tyr Lieu. Ser Pro Glu. His Glin Arg Val Glin Glin Arg Llys 1. 5 1O 15 Glu Ser Lys Llys Pro Pro Ala Lys Lieu. Glin Pro Arg 2O 25

<210s, SEQ ID NO 27 &211s LENGTH: 107 212. TYPE: PRT <213> ORGANISM: Rattus sp. <4 OOs, SEQUENCE: 27 Met Glu Glu Lys Val Glu Asp Asp Phe Lieu. Asp Lieu. Asp Ala Val Pro 1. 5 1O 15 Glu Thr Pro Val Phe Asp Cys Val Met Asp Ile Llys Pro Glu. Thir Asp 2O 25 3O Pro Ala Ser Lieu. Thr Val Llys Ser Met Gly Lieu. Glin Glu Arg Arg Gly 35 4 O 45 Ser Asn Val Ser Lieu. Thir Lieu. Asp Met Cys Thr Pro Gly Cys Asn Glu SO 55 6 O Glu Gly Phe Gly Tyr Lieu Val Ser Pro Arg Glu Glu Ser Ala His Glu 65 70 7s 8O Tyr Lieu. Lieu. Ser Ala Ser Arg Val Lieu. Arg Ala Glu Glu Lieu. His Glu 85 90 95

Lys Ala Lieu. Asp Pro Phe Lieu. Lieu. Glin Ala Glu 1OO 105

<210s, SEQ ID NO 28 &211s LENGTH: 107 US 2017/O121385 A1 May 4, 2017 91

- Continued

212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 28 Met Glu Glu Lys Ile Glu Asp Asp Phe Lieu. Asp Lieu. Asp Pro Val Pro 1. 5 1O 15 Glu Thr Pro Val Phe Asp Cys Val Met Asp Ile Llys Pro Glu Ala Asp 2O 25 3O Pro Thir Ser Lieu. Thr Val Lys Ser Met Gly Lieu Gln Glu Arg Arg Gly 35 4 O 45 Ser Asn Val Ser Lieu. Thir Lieu. Asp Met Cys Thr Pro Gly Cys Asn Glu SO 55 6 O Glu Gly Phe Gly Tyr Lieu Met Ser Pro Arg Glu Glu Ser Ala Arg Glu 65 70 7s 8O Tyr Lieu. Lieu. Ser Ala Ser Arg Val Lieu. Glin Ala Glu Glu Lieu. His Glu 85 90 95 Lys Ala Lieu. Asp Pro Phe Lieu. Lieu. Glin Ala Glu 1OO 105

<210s, SEQ ID NO 29 &211s LENGTH: 107 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic polypeptide

<4 OOs, SEQUENCE: 29 Met Glu Glu Lys Ile Glu Asp Asp Phe Lieu. Asp Lieu. Asp Pro Val Pro 1. 5 1O 15 Glu Thr Pro Val Phe Asp Cys Val Met Asp Ile Llys Pro Glu Ala Asp 2O 25 3O Pro Thir Ser Lieu. Thr Val Lys Ser Met Gly Lieu Gln Glu Arg Arg Gly 35 4 O 45 Ala Asn Val Ser Lieu. Thir Lieu. Asp Met Cys Thr Pro Gly Cys Asn. Glu SO 55 6 O Glu Gly Phe Gly Tyr Lieu Met Ser Pro Arg Glu Glu Ser Ala Arg Glu 65 70 7s 8O Tyr Lieu. Lieu. Ser Ala Ser Arg Val Lieu. Glin Ala Glu Glu Lieu. His Glu 85 90 95 Lys Ala Lieu. Asp Pro Phe Lieu. Lieu. Glin Ala Glu 1OO 105

<210s, SEQ ID NO 3 O &211s LENGTH: 107 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic polypeptide

<4 OOs, SEQUENCE: 30 Met Glu Glu Lys Ile Glu Asp Asp Phe Lieu. Asp Lieu. Asp Pro Val Pro 1. 5 1O 15

Glu Thr Pro Val Phe Asp Cys Val Met Asp Ile Llys Pro Glu Ala Asp 2O 25 3O

Pro Thir Ser Lieu. Thr Val Lys Ser Met Gly Lieu Gln Glu Arg Arg Gly US 2017/O121385 A1 May 4, 2017 92

- Continued

35 4 O 45

Ala Asn Val Ser Lieu. Thir Lieu. Asp Met Cys Glu Pro Gly Cys Asn Glu SO 55 6 O

Glu Gly Phe Gly Tyr Lieu Met Glu Pro Arg Glu Glu Ser Ala Arg Glu 65 70 7s 8O

Tyr Lieu. Lieu. Ser Ala Ser Arg Val Lieu. Glin Ala Glu Glu Lieu. His Glu 85 90 95

Lys Ala Lieu. Asp Pro Phe Lieu. Lieu. Glin Ala Glu 1OO 105

SEQ ID NO 31 LENGTH: 1. Of TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic polypeptide

SEQUENCE: 31

Met Glu Glu Lys Ile Glu Asp Asp Phe Lieu. Asp Lieu. Asp Pro Val Pro 1. 15 Glu Thr Pro Val Phe Asp Ala Val Met Asp Ile Llys Pro Glu Ala Asp 2O 25 3O Pro Thir Ser Lieu. Thr Val Lys Ser Met Gly Lieu. Glin Glu Arg Arg Gly 35 4 O 45

Ser Asn. Wal Ser Lieu. Thir Lieu. Asp Met Cys Thr Pro Gly Cys Asn Glu SO 55 6 O

Glu Gly Phe Gly Tyr Lieu Met Ser Pro Arg Glu Glu Ser Ala Arg Glu 65 70 7s

Tyr Lieu. Lieu. Ser Ala Ser Arg Val Lieu. Glin Ala Glu Glu Lieu. His Glu 85 90 95

Lys Ala Lieu. Asp Pro Phe Lieu. Lieu. Glin Ala Glu 1OO 105

SEQ ID NO 32 LENGTH: 1. Of TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic polypeptide

SEQUENCE: 32

Met Glu Glu Lys Ile Glu Asp Asp Phe Lieu. Asp Lieu. Asp Pro Val Pro 1. 15 Glu Glu Pro Val Phe Asp Cys Val Met Asp Ile Llys Pro Glu Ala Asp 2O 25 3O

Pro Thir Ser Lieu. Thr Val Lys Ser Met Gly Lieu. Glin Glu Arg Arg Gly 35 4 O 45

Ser Asn. Wal Ser Lieu. Thir Lieu. Asp Met Cys Thr Pro Gly Cys Asn Glu SO 55 6 O

Glu Gly Phe Gly Tyr Lieu Met Ser Pro Arg Glu Glu Ser Ala Arg Glu 65 70 7s 8O

Tyr Lieu. Lieu. Ser Ala Ser Arg Val Lieu. Glin Ala Glu Glu Lieu. His Glu 85 90 95

Lys Ala Lieu. Asp Pro Phe Lieu. Lieu. Glin Ala Glu US 2017/O121385 A1 May 4, 2017 93

- Continued

1OO 105

<210s, SEQ ID NO 33 &211s LENGTH: 107 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic polypeptide

<4 OOs, SEQUENCE: 33 Met Glu Glu Lys Ile Glu Asp Asp Phe Lieu. Asp Lieu. Asp Pro Val Pro 1. 5 1O 15 Glu Ala Pro Val Phe Asp Cys Val Met Asp Ile Llys Pro Glu Ala Asp 2O 25 3O Pro Thir Ser Lieu. Thr Val Lys Ser Met Gly Lieu Gln Glu Arg Arg Gly 35 4 O 45 Ser Asn Val Ser Lieu. Thir Lieu. Asp Met Cys Thr Pro Gly Cys Asn Glu SO 55 6 O Glu Gly Phe Gly Tyr Lieu Met Ser Pro Arg Glu Glu Ser Ala Arg Glu 65 70 7s 8O Tyr Lieu. Lieu. Ser Ala Ser Arg Val Lieu. Glin Ala Glu Glu Lieu. His Glu 85 90 95 Lys Ala Lieu. Asp Pro Phe Lieu. Lieu. Glin Ala Glu 1OO 105

<210s, SEQ ID NO 34 &211s LENGTH: 107 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic polypeptide

<4 OOs, SEQUENCE: 34 Met Glu Glu Lys Ile Glu Asp Asp Phe Lieu. Asp Lieu. Asp Pro Val Pro 1. 5 1O 15 Glu Glu Pro Val Phe Asp Ala Val Met Asp Ile Llys Pro Glu Ala Asp 2O 25 3O Pro Thir Ser Lieu. Thr Val Lys Ser Met Gly Lieu Gln Glu Arg Arg Gly 35 4 O 45 Ala Asn Val Ser Lieu. Thir Lieu. Asp Met Cys Glu Pro Gly Cys Asn. Glu SO 55 6 O Glu Gly Phe Gly Tyr Lieu Met Glu Pro Arg Glu Glu Ser Ala Arg Glu 65 70 7s 8O Tyr Lieu. Lieu. Ser Ala Ser Arg Val Lieu. Glin Ala Glu Glu Lieu. His Glu 85 90 95

Lys Ala Lieu. Asp Pro Phe Lieu. Lieu. Glin Ala Glu 1OO 105

<210s, SEQ ID NO 35 &211s LENGTH: 121 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic polypeptide

<4 OOs, SEQUENCE: 35 US 2017/O121385 A1 May 4, 2017 94

- Continued

Met Ala Lieu. Tyr Gly Arg Llys Lys Arg Arg Glin Arg Arg Arg Gly Glu 1. 5 1O 15 Glu Lys Ile Glu Asp Asp Phe Lieu. Asp Lieu. Asp Pro Val Pro Glu Thir 2O 25 3O Pro Val Phe Asp Cys Val Met Asp Ile Llys Pro Glu Ala Asp Pro Thr 35 4 O 45 Ser Lieu. Thr Val Llys Ser Met Gly Lieu. Glin Glu Arg Arg Gly Ala Asn SO 55 6 O Val Ser Lieu. Thir Lieu. Asp Met Cys Glu Pro Gly Cys Asn. Glu Glu Gly 65 70 7s 8O Phe Gly Tyr Lieu Met Glu Pro Arg Glu Glu Ser Ala Arg Glu Tyr Lieu. 85 90 95 Lieu. Ser Ala Ser Arg Val Lieu. Glin Ala Glu Glu Lieu. His Glu Lys Ala 1OO 105 11 O Lieu. Asp Pro Phe Lieu. Lieu. Glin Ala Glu 115 12 O

<210s, SEQ ID NO 36 &211s LENGTH: 121 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic polypeptide

<4 OOs, SEQUENCE: 36 Met Ala Lieu. Tyr Gly Arg Llys Lys Arg Arg Glin Arg Arg Arg Gly Glu 1. 5 1O 15 Glu Lys Val Glu Asp Asp Phe Lieu. Asp Lieu. Asp Ala Val Pro Glu Thir 2O 25 3O Pro Val Phe Asp Cys Val Met Asp Ile Llys Pro Glu Thr Asp Pro Ala 35 4 O 45 Ser Lieu. Thr Val Llys Ser Met Gly Lieu. Glin Glu Arg Arg Gly Ala Asn SO 55 6 O Val Ser Lieu. Thir Lieu. Asp Met Cys Glu Pro Gly Cys Asn. Glu Glu Gly 65 70 7s 8O Phe Gly Tyr Lieu Val Glu Pro Arg Glu Glu Ser Ala His Glu Tyr Lieu. 85 90 95 Lieu. Ser Ala Ser Arg Val Lieu. Arg Ala Glu Glu Lieu. His Glu Lys Ala 1OO 105 11 O Lieu. Asp Pro Phe Lieu. Lieu. Glin Ala Glu 115 12 O

<210s, SEQ ID NO 37 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: 223 OTHER INFORMATION: C-term. NH2

<4 OO > SEQUENCE: 37 Gly Ile Gly Llys Phe Lieu Lys Lys Ala Lys Llys Phe Gly Lys Ala Phe 1. 5 1O 15 US 2017/O121385 A1 May 4, 2017

- Continued Val Lys Ile Lieu Lys Llys 2O

<210s, SEQ ID NO 38 &211s LENGTH: 28 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Ser (O - (C=O) - (CH2)6- CH3)

<4 OOs, SEQUENCE: 38 Gly Ser Ser Phe Lieu. Ser Pro Glu. His Gln Lys Ala Glin Glin Arg Llys 1. 5 1O 15 Glu Ser Lys Llys Pro Pro Ala Lys Lieu. Glin Pro Arg 2O 25

<210s, SEQ ID NO 39

<4 OOs, SEQUENCE: 39

OOO

<210s, SEQ ID NO 4 O

<4 OOs, SEQUENCE: 4 O

OOO

<210s, SEQ ID NO 41 &211s LENGTH: 4 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Ser (n-octanoyl)

<4 OOs, SEQUENCE: 41 Gly Ser Ser Phe 1.

What is claimed is: 5. The method of claim 4, wherein the fatty acid is an 1. A method of treating a neurodegenerative condition in octanoic acid. a Subject in need thereof, comprising administering to the 6. The method of claim 2, wherein the polypeptide is Subject a therapeutically effective amount of grehlin or a modified at serine at amino acid position 2 and/or serine at ghrelin variant. amino acid position 3 of SEQ ID NO. 1. 2. The method of claim 1, wherein the ghrelin variant 7. The method of claim 1, wherein the ghrelin variant comprises a polypeptide comprising at least one modifica comprises a polypeptide having at least 80%, 81%, 82%, tion to the natural form of an amino acid sequence of 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, Gly-Ser-Ser-Phe-Leu-Ser-Pro-Glu-His-Gln-Arg-Val-Gn 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity Gln-Arg-Lys-Glu-Ser-Lys-Lys-Pro-Pro-Ala-Lys-Leu-Gln to the amino acid sequence of SEQ ID NO. 1. Pro-Arg (SEQ ID NO. 1). 8. The method of claim 1, wherein the ghrelin variant is 3. The method of claim 2, wherein the polypeptide one or more of RM-131 (or BIM-28131), Dln-101, Growth comprises at least one acylated and at least one non-acylated hormone (GH) releasing hexapeptide (GHRP)-6, EP 1572, amino acid. Ape-Ser(Octyl)-Phe-Leu-aminoethylamide, isolated ghrelin 4. The method of claim 2, wherein the polypeptide is splice variant-like compound, ghrelin splice variant, growth modified with one or more fatty acids. hormone secretagogue receptor GHS-R la ligand, US 2017/O121385 A1 May 4, 2017 96

LY444711, LY426410, hexarelin/examorelin, growth hor activation or deactivation of MAP kinases, NFKB translo mone releasing hexapeptide-1 (GHRP-1), GHRP-2, cation, CRE driven gene transcription, binding of arrestin to GHRP-6 (SK&F-110679), ipamorelin, MK-0677, NN703, ghrelin receptor, reducing ROS, NAMPT enzyme activation, capromorelin, CP 464709, prailmorelin, macimorelin (ac or a combination thereof. etate), anamorelin, relamorelin, ulimorelin, ipamorelin, tabi 22. The method of claim 1, wherein ghrelin or the ghrelin morelin, ibutamoren, G7039, G7134, G7203, G-7203, variant reduces the expression level of Tau protein. G7502, SM-130686, RC-1291, L-692429, L-692587, 23. The method of claim 22, wherein ghrelin or the L-739943, L-163255, L-163540, L-163833, L-166446, ghrelin variant reduces the phosphorylation level of Tau CP-424391, EP-51389, NNC-26-0235, NNC-26-0323, protein. NNC-26-0610, NNC 26-0703, NNC-26-0722, NNC-26 24. The method of claim 1, wherein ghrelin or the ghrelin 1089, NNC-26-1136, NNC-26-1137, NNC-26-1187, NNC variant prevents or reduces Tau deposition and/or develop 26-1291, MK-0677, L-692,429, EP 1572, L-252,564, ment of neurofibrillary tangles. NN703, S-37435, EX-1314, PF-5190457, AMX-213, and a 25. The method of claim 1, wherein the neurodegenera combination thereof. tive condition is caused by a reperfusion injury. 9. The method of claim 1, wherein the ghrelin variant 26. The method of claim 25, wherein the reperfusion comprises a polypeptide comprising the sequence of Gly Ser injury is resulted by post cardiac arrest, coronary artery Ser Phe Leu Ser Pro Glu His Gln Arg Val Glin Val Arg Pro bypass grafting (CABG), ischemia, anoxia, or hypoxia. Pro Lys Ala Pro His Val Val (SEQ ID No. 2). 27. The method of claim 1, wherein ghrelin or the ghrelin 10. The method of claim 1, wherein the ghrelin variant variant is coupled to a protein that extends the serum comprises a polypeptide comprising the sequence of Gly Ser half-life of the ghrelin variant. Xaa Phe Leu Ser Pro Glu His Gln Arg Val Glin Val Arg Pro 28. The method of claim 27, wherein the protein is a long, Pro His Lys Ala Pro His Val Val (SEQ ID No. 3), wherein hydrophilic, and unstructured polymer that occupies a larger Xaa is a 2,3-diaminopropionic acid (Dpr) and optionally Volume than a globular protein containing the same number octanoylated. of amino acids. 11. The method of claim 1, wherein the ghrelin variant 29. The method of claim 27, wherein the protein com comprises a polypeptide comprising the sequence of Gly prising the sequence of XTEN (SEQ ID NO. 7). XaaXaa2 Phe Leu Ser Pro Glu His Gln Arg Val Glin Val Arg 30. The method of claim 1, wherein the subject is a Pro Pro His Lys Ala Pro His Val Val (SEQ ID No. 4), mammal. wherein Xaa is a 2,3-diaminopropionic acid (Dpr) residue, 31. The method of claim 30, wherein the subject is a and Xaa2 is Dpr and is optionally octanoylated. human. 12. The method of claim 1, wherein the ghrelin variant 32. The method of claim 1, wherein ghrelin or the ghrelin comprises a polypeptide comprising the sequence of Gly Ser variant is administered via a powder or stable formulation, Ser Phe Leu Ser Pro Glu His Gln Arg Val Glin Val Arg Pro wherein the ghrelin variant is formulated in a dosage form Pro His Lys Ala Pro His Val Val Pro Ala Leu Pro (SEQ ID selected from the group consisting of liquid, beverage, No. 5). medicated sports drink, powder, capsule, chewable tablet, 13. The method of claim 1, wherein the ghrelin variant hydrogel, Swallowable tablet, buccal tablet, troche, lozenge, comprises a polypeptide comprising the sequence of Inp-D- Soft chew, Solution, Suspension, spray, Suppository, tincture, 2Nal-D-Trp-Thr-Lys-NH. (SEQ ID No. 6). decoction, infusion, and a combination thereof. 14. The method of claim 1, wherein one or more of the 33. The method of claim 32, wherein ghrelin or the amino acids of the sequence are Substituted or replaced by ghrelin variant is administered via inhalation, oral, intrave another amino acid or a synthetic amino acid. nous, parenteral, buccal, Subcutaneous (including 15. The method of claim 14, comprising between 1 and 5 "EpiPens'), transdermal, patch, Sublingual, intramuscular, Substitutions. intratympanic injection or placement, or intranasal. 16. The method of claim 1, wherein the neurodegenera 34. The method of claim 1, wherein ghrelin or the ghrelin tive condition is traumatic brain injury (TBI), mild brain variant is administered in a single dose, in two doses, in injury (mBI), recurrent TBI, recurrent mBI, Alzheimer's three doses, in four doses, in five doses or in multiple doses. disease, Parkinson's disease, demential, or frontotemporal 35. The method of claim 1, wherein ghrelin or the ghrelin dementia. variant is administered at a dosage from 10 ng/kg per day to 17. The method of claim 1, wherein the ghrelin variant 10 mg/kg per day. binds to the growth hormone secretagogue receptor GHS-R 36. The method of claim 1, wherein ghrelin or the ghrelin 1a (GHSR). variant is administered in combination with a therapeutic 18. The method of claim 17, wherein the ghrelin variant agent. has an ECso potency on the GHSR of less than 500 nM. 37. The method of claim 36, wherein the therapeutic agent 19. The method of claim 17, wherein the ghrelin variant is one or more of an anti-inflammatory agent, anti-pain has a dissociation constant from the GHSR of less than 500 medication, acetylsalicylic acid, an antiplatelet agent, a nM. thrombolytic enzyme, an aggregation inhibitor, a glycopro 20. The method of claim 1, wherein the ghrelin variant has tein IIb/IIIa inhibitor, a glycosaminoglycan, a thrombin at least about 50% of a functional activity of ghrelin. inhibitor, an anticoagulant, heparin, coumarin, warfarin, 21. The method of claim 20, wherein the functional tPA, GCSF, Streptokinase, urokinase, Ancrod, melatonin, a activity comprises one or more of feeding regulation, nutri caspase inhibitor, an NMDA receptor agonist or antagonist ent absorption, gastrointestinal motility, energy homeostasis, (e.g. OTO-311), an anti-TNF-C. compound, an antibody, anti-inflammatory regulation, Suppression of inflammatory erythropoietin/EPO, angiotensin II lowering agent, selective cytokines, activation of Gq/G11, accumulation of inositol androgen receptor modulator, leptin or leptin mimetics and phosphate, mobilization of calcium from intracellular stores, variants, an agonists of the renin-angiotensin System, an US 2017/O121385 A1 May 4, 2017 97 opioid receptor agonist, progesterone or progesterone mimetics and variants, a peroxisome proliferator-activated receptor gamma agonist, P2Y purinergic receptor agonists (e.g., 2-MeSADP, MRS2365), amantadine (e.g. ADS-5102), P7C3, or a combination thereof. k k k k k