Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
  • C07K14/60—Growth hormone-releasing factor [GH-RF], i.e. somatoliberin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/22—Hormones
  • A61K38/25—Growth hormone-releasing factor [GH-RF], i.e. somatoliberin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/02—Nutrients, e.g. vitamins, minerals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/04—Anorexiants; Antiobesity agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P5/00—Drugs for disorders of the endocrine system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P5/00—Drugs for disorders of the endocrine system
  • A61P5/02—Drugs for disorders of the endocrine system of the hypothalamic hormones, e.g. TRH, GnRH, CRH, GRH, somatostatin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P5/00—Drugs for disorders of the endocrine system
  • A61P5/06—Drugs for disorders of the endocrine system of the anterior pituitary hormones, e.g. TSH, ACTH, FSH, LH, PRL, GH
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P5/00—Drugs for disorders of the endocrine system
  • A61P5/10—Drugs for disorders of the endocrine system of the posterior pituitary hormones, e.g. oxytocin, ADH
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• Human GHRH (Growth Hormone-Releasing Hormone) is a 44-amino-acid peptide whose full biological activity resides in its first 29 amino acids ("GHRH 1-29"). GHRH binds to the GHRH receptor and stimulates pulsatile GH [Growth Hormone] secretion, and with this mechanism of action GHRH represents an alternative to GH therapy in patients with an intact pituitary that may minimize the side effects associated with long-term GH administration. Because the quantity of GH release induced by GHRH is limited by IGF-1 levels, which exert a negative feedback effect, the risk of side effects associated with excessive GH secretion may also be lower with GHRH therapy than with GH therapy.
• GHRH may result in the pituitary secretion of a broader set of GH proteins, and not just the 22-kDa form provided by recombinant human GH, which may also have beneficial effects.
• GHRH has been shown to be safe and effective in increasing GH levels in adults and children, and the growth-promoting effect of GHRH is correlated with the dose and frequency of administration.
• the half-life of GHRH after intravenous injection is only 10-12 min, which has significantly limited its use as a therapeutic agent.
• analogs of GHRH that possess extended half- life in vivo could provide greater therapeutic benefit with an improved (less frequent) dosing regimen.
• the present invention provides GHRH-derived peptidomimetic macrocycles that are designed to possess improved pharmaceutical properties relative to GHRH. These improved properties include enhanced chemical stability, extended in vivo half- life, increased potency and reduced immunogenicity. These peptidomimetic macrocycles are useful to increase circulating levels of GH as a treatment for muscle wasting diseases, lipodystrophies, growth hormone disorders, gastroparesis/short bowel syndrome, and other conditions for which an increase in GH would provide therapeutic benefit.
• stably cross-linked peptides derived from the GHRH peptide contain at least two modified amino acids that together form an intramolecular cross-link that can help to stabilize the alpha-helical secondary structures of a portion of GHRH that is thought to be important for agonist activity at the GHRH receptor.
• any amino acid which is not essential to the growth- hormone releasing activity of the peptide may be replaced with any other amino acids, while amino acids which are essential to the growth-hormone releasing activity of the peptide may be replaced only with amino acid analogs which do not substantially decrease said activity.
• a cross-linked polypeptide described herein can have improved biological activity relative to a corresponding polypeptide that is not cross-linked.
• the GHRH peptidomimetic macrocycles are thought to activate the GHRH receptor, thereby stimulating production and release of growth hormone, which can increase lean muscle mass or reduce adipose tissue (such as abdominal adipose tissue).
• adipose tissue can be reduced in subjects suffering from obesity, including abdominal obesity.
• peptidomimetic macrocycles described herein can be used therapeutically, for example, to treat muscle wasting diseases that include anorexias, cachexias (such as cancer cachexia, chronic heart failure cachexia, chronic obstructive pulmonary disease cachexia, rheumatoid arthritis cachexia) and sarcopenias, to treat lipodystrophies that include HIV lipodystrophy, to treat growth hormone disorders that include adult and pediatric growth hormone deficiencies, or to treat gastroparesis or short bowel syndrome.
• cachexias such as cancer cachexia, chronic heart failure cachexia, chronic obstructive pulmonary disease cachexia, rheumatoid arthritis cachexia
• sarcopenias to treat lipodystrophies that include HIV lipodystrophy
• growth hormone disorders that include adult and pediatric growth hormone deficiencies
• gastroparesis or short bowel syndrome or to treat gastroparesis or short bowel syndrome.
• Pediatric growth hormone deficiency may be, for example, linked with or associated to idiopathic short stature, SGA (infant small for gestational age), chronic kidney disease, Prader-Willi syndrome Turner syndrome, short stature homeobox (SHOX) gene deficiency, or primary IGF- 1 deficiency.
• SGA infant small for gestational age
• chronic kidney disease Prader-Willi syndrome Turner syndrome
• SHOX short stature homeobox
• the present invention provides a peptidomimetic macrocycle comprising an amino acid sequence which is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to GHRH 1 -44, GHRH 1 -29 and/or to an amino acid sequence chosen from the group consisting of the amino acid sequences in Table 1 , 2 or 4.
• an amino acid sequence of said peptidomimetic macrocycle is chosen from the group consisting of the amino acid sequences in Table 1 , 2 or 4.
• the peptidomimetic macrocycle may comprise one, two, three, four, five or more macrocycle-forming linkers, wherein each macrocycle-forming linker connects one amino acid to another amino acid within the peptidomimetic macrocycle.
• a peptidomimetic macrocycle comprises at least two macrocycle-forming linkers wherein wherein the first of said at least two macrocycle-forming linkers connects a first amino acid to a second amino acid, and the second of said at least two macrocycle-forming linkers connects a third amino acid to a fourth amino acid.
• the peptidomimetic macrocycle comprises exactly two macrocycle-forming linkers.
• the peptidomimetic macrocycle comprises exactly one macrocycle-forming linker.
• Macrocycle-forming linkers connect any two amino acids which can be crosslinked without impairing the activity of the peptidomimetic macrocycle.
• a macrocycle- forming linker connects one of the following pairs of amino acids (numbered with reference to any sequences aligned to GHRH 1 -29): 4 and 8; 5 and 12; 8 and 12; 8 and 15; 9 and 16; 12 and 16; 12 and 19; 15 and 22; 18 and 25; 21 and 25; 21 and 28; 22 and 29; 25 and 29.
• a macrocycle-forming linkers connects of the following pairs of amino acids: 4 and 8; 5 and 12; 12 and 19; 15 and 22; 18 and 25; 21 and 25; 21 and 28.
• a first macrocycle- forming linker connects amino acid pairs 4 and 8; 5 and 12; 8 and 12; 8 and 15; 9 and 16; 12 and 16; or 12 and 19; and a second macrocycle-forming linker connects amino acid pairs 15 and 22; 18 and 25; 21 and 25; 21 and 28; 22 and 29; or 25 and 29.
• the first macrocycle- forming linker connects amino acid pairs 4 and 8; 5 and 12; or 12 and 19; and the second macrocycle-forming linker connects amino acid pairs 15 and 22; 18 and 25; 21 and 25; or 21 and 28.
• the first macrocycle-forming linker connects amino acid pairs 4 and 8 and the second macrocycle-forming linker connects amino acid pairs 21 and 25.
• a peptidomimetic macrocycle comprises an amino acid sequence which is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to GHRH 1 -44, GHRH 1 -29 and/or to an amino acid sequence chosen from the group consisting of the amino acid sequences in Table 1 , 2 or 4, and further comprises a macrocycle-forming linker connecting a first amino acid to a second amino acid, wherein the first and second amino acids are selected from the following pairs of amino acids: 4 and 8; 5 and 12; 8 and 12; 8 and 15; 9 and 16; 12 and 16; 12 and 19; 15 and 22; 18 and 25; 21 and 25; 21 and 28; 22 and 29.
• the macrocycle-forming linker connects amino acids 12 and 19.
• a peptidomimetic macrocycle comprises a sequence chosen from the group consisting of the amino acid sequences in Tables 1 , 2 or 4, or the amino acid sequence of the peptidomimetic macrocycle is chosen from the group consisting of the amino acid sequences in Tables 1 , 2 or 4.
• the peptidomimetic macrocycle comprises a helix, such as an a-helix or a
• the peptidomimetic macrocycle comprises an ⁇ , ⁇ -disubstituted amino acid.
• at least one amino acid, or each amino acid, connected by the macrocycle-forming linker is an ⁇ , ⁇ -disubstituted amino acid.
• a peptidomimetic macrocycle of the invention comprises a crosslinker linking the a-positions of at least two amino acids.
• each A, C, D, and E is independently an amino acid
• B is an amino acid, H ⁇ , [-NH-L3-CO-], [-NH-L3-SO 2 -], or [-NH-L3-] ;
• Ri and R 2 are independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-;
• R3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R 5 ;
• L is a macrocycle- forming linker of the formula -Li-L 2 -;
• Li and L 2 are independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [-R4-K-R 4 -] n , each being optionally substituted with R 5 ;
• each R 4 is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;
• each K is O, S, SO, S0 2 , CO, C0 2 , or CONR 3 ;
• each R 5 is independently halogen, alkyl, -OR 6 , -N(R 6 ) 2 , -SR 6 , -SOR 6 , -S0 2 R 6 , -C0 2 R 6 , a fluorescent moiety, a radioisotope or a therapeutic agent;
• each R 6 is independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent;
• R 7 is -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl,
• heterocycloalkyl cycloaryl, or heterocycloaryl, optionally substituted with R 5 , or part of a cyclic structure with a D residue;
• Rg is -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl,
• heterocycloalkyl cycloaryl, or heterocycloaryl, optionally substituted with R 5 , or part of a cyclic structure with an E residue;
• v and w are independently integers from 1 -1000, for example 1 -500, 1 -200, 1 -100, 1 -50, 1 -40, 1 -
• u, x, y and z are independently integers from 0-10, for example u is 1 , 2, or 3;
• n is an integer from 1 -5.
• u is 2.
• the sum of x+y+z is 2, 3 or 6, for example 3 or 6.
• the peptidomimetic macrocycle of Formula (I) has the Formula:
• each A, C is independently an amino acid
• B is an amino acid, [-NH-L 3 -CO-], [-NH-L 3 -S0 2 -], or [-NH-L3-] ;
• L' is a macrocycle-forming linker of the formula -Li '-L 2 '-;
• Ri' and R 2 ' are independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-;
• Li ' and L 2 ' are independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [-R4-K-R 4 -] n , each being optionally substituted with R 5 ;
• each K is independently O, S, SO, S0 2 , CO, C0 2 , or CONR 3 ;
• R 7 ' is -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl,
• heterocycloalkyl cycloaryl, or heterocycloaryl, optionally substituted with R 5 , or part of a cyclic structure with a D residue;
• Rg' is -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl,
• heterocycloalkyl cycloaryl, or heterocycloaryl, optionally substituted with R 5 , or part of a cyclic structure with an E residue;
• v' and w' are independently integers from 1 -1000, for example 1 -500, 1 -200, 1 -100, 1 -50, 1 -40, 1 -25, 1 -20, 1 to 15, or 1 to 10;
• x', y' and z' are independently integers from 0-10;
• n is an integer from 1 -5.
• the sum of x'+y'+z' is 2, 3 or 6, for example 3 or
• each K is O, S, SO, S0 2 , CO, or C0 2 .
• the peptidomimetic macrocycle may comprise a crosslinker linking a backbone amino group of a first amino acid to a second amino acid within the peptidomimetic macrocycle.
• the invention provides peptidomimetic macrocycles of the Formula (II) or (Ha): u Formula (Ila)
• each A, C, D, and E is independently an amino acid
• B is an amino acid, H ⁇ , [-NH-L3-CO-], [-NH-L3-SO 2 -], or [-NH-L3-] ;
• Ri and R 2 are independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo- or part of a cyclic structure with an E residue;
• R3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R 5 ;
• Li and L 2 are independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [-R4-K-R 4 -] n , each being optionally substituted with R 5 ;
• each R 4 is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;
• each K is O, S, SO, S0 2 , CO, C0 2 , or CONR 3 ;
• each R 5 is independently halogen, alkyl, -OR 6 , -N(R 6 ) 2 , -SR 6 , -SOR 6 , -S0 2 R 6 , -C0 2 R 6 , a fluorescent moiety, a radioisotope or a therapeutic agent;
• each R 6 is independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent;
• R 7 is -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl,
• heterocycloalkyl cycloaryl, or heterocycloaryl, optionally substituted with R 5 ;
• v and w are independently integers from 1 -1000, for example 1 -500, 1 -200, 1 -100, 1 -50, 1 -40, 1 - 25, 1 -20, 1 to 15, or 1 to 10;
• u, x, y and z are independently integers from 0-10, for example u is 1 -3;
• n is an integer from 1 -5.
• peptidomimetic macrocycle comprising an amino acid sequence of Formula:
• XI is Tyr or His
• X2 is Ala, D-Ala, or Val
• X3 is Asp
• X4 is Ala or a crosslinked amino acid
• X6 is Phe
• X7 is Thr
• X8 is Gin, Asn, or a crosslinked amino acid
• X9 is Ser or a crosslinked amino acid
• Xl O is Tyr
• XI I is Arg, Ala or Gin
• XI 2 is Lys, Ala, Gin or a crosslinked amino acid
• XI 3 is Val or lie
• X14 is Leu
• XI 5 is Gly, Ala or a crosslinked amino acid
• XI 6 is Gin, Glu or a crosslinked amino acid
• XI 7 is Leu
• XI 8 is Ser, Tyr or a crosslinked amino acid
• XI 9 is Ala or a crosslinked amino acid
• X20 is Arg or Gin
• X21 is Lys, Gin or a crosslinked amino acid
• X22 is Leu, Ala, or a crosslinked amino acid
• X23 is Leu
• X24 is Gin, Glu or His;
• X25 is Asp, Glu or a crosslinked amino acid;
• X27 is Met, lie, Leu or Nle
• X28 is Ser or a crosslinked amino acid
• X29 is Arg, Ala, Gin or a crosslinked amino acid
• the peptidomimetic macrocycle comprises at least one macrocycle- forming linker connecting at least one pair of amino acids selected from X1 -X29;
• L is a macrocycle- forming linker of the formula -Li-L 2 -;
• Li and L 2 are independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [-R4-K-R 4 -] n , each being optionally substituted with R 5 ;
• each R 4 is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;
• each K is O, S, SO, S0 2 , CO, or C0 2 ;
• each R 5 is independently halogen, alkyl, -OR 6 , -N(R 6 ) 2 , -SR 6 , -SOR 6 , -S0 2 R 6 , -C0 2 R 6 , a fluorescent moiety, a radioisotope or a therapeutic agent;
• each R 6 is independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent.
• the polypeptide comprises at least one, or at least two, macrocycle- forming linkers which connect one of the following pairs of amino acids: X4 and X8; X5 and X12; X8 and X12; X8 and X15; X9 and X16; X12 and X16; X12 and X19; X15 and X22; X18 and X25; X21 and X25; X21 and X28; X22 and X29; X25 and X29.
• each macrocycle-forming linker connects one of the following pairs of amino acids: X4 and X8; X5 and X12; X12 and X19; X15 and X22; XI 8 and X25; X21 and X25; X21 and X28.
• peptidomimetic macrocycles comprise a macrocycle-forming linker of Formula -Li-L 2 -, wherein Li and L 2 are independently alkylene, alkenylene or alkynylene.
• Li and L 2 are independently C3-C 10 alkylene or alkenylene, or C3-C6 alkylene or alkenylene.
• Ri and R 2 are independently H or alkyl, for example methyl.
• the invention provides a method of increasing the circulating level of growth
• GH hormone
• a method of increasing lean muscle mass in a subject and a method of reducing adipose tissue (such as abdominal adipose tissue) in a subject comprising administering to the subject a peptidomimetic macrocycle of the invention.
• adipose tissue such as abdominal adipose tissue
• subjects suffering from obesity, including abdominal obesity are treated using a peptidomimetic macrocycle of the invention.
• the invention also provides a method of treating muscle wasting diseases that include anorexias, cachexias (such as cancer cachexia, chronic heart failure cachexia, chronic obstructive pulmonary disease cachexia, rheumatoid arthritis cachexia) and sarcopenias, a method of treating lipodystrophies that include HIV lipodystrophy, a method of treating growth hormone disorders that include adult and pediatric growth hormone deficiencies, or a method of treating gastroparesis or short bowel syndrome.
• cachexias such as cancer cachexia, chronic heart failure cachexia, chronic obstructive pulmonary disease cachexia, rheumatoid arthritis cachexia
• sarcopenias a method of treating lipodystrophies that include HIV lipodystrophy
• growth hormone disorders that include adult and pediatric growth hormone deficiencies
• gastroparesis or short bowel syndrome a method of treating gastroparesis or short bowel syndrome.
• Pediatric growth hormone deficiency may be, for example, linked with or associated to idiopathic short stature, SGA (infant small for gestational age), chronic kidney disease, Prader-Willi syndrome Turner syndrome, short stature homeobox (SHOX) gene deficiency, or primary IGF-1 deficiency.
• the invention also provides a method of treating muscle wasting diseases, lipodystrophies, growth hormone disorders or gastroparesis/short bowel syndrome in a subject by administering an agonist of the GHRH receptor, such as an analog of GHRH, wherein the agonist is administered no more frequently than once daily, no more frequently than every other day, no more frequently than twice weekly, no more frequently than weekly, or no more frequently than every other week.
• the invention also provides a method of increasing the circulating level of growth hormone (GH) in a subject by administering an agonist of the GHRH receptor, such as an analog of GHRH, wherein the agonist is administered no more frequently than once daily, no more frequently than every other day, no more frequently than twice weekly, no more frequently than weekly, or no more frequently than every other week.
• an agonist of the GHRH receptor such as an analog of GHRH
• FIGURES 1A and IB show improved stabilities to trypsin proteolysis of the peptidomimetic macrocycles of the invention.
• FIGURE 2 shows improved serum stabilities of the peptidomimetic macrocycles of the
• FIGURES 3 and 3a show GHRH receptor agonist activities measured by cAMP release and trypsin half- lives of the peptidomimetic macrocycles of the invention.
• cAMP values “+” represents values greater than 50 nM; "++” represents values between 10-50 nM; "+++” represents values between 1 -10 nM;
• FIGURE 4 shows the result of a plasma PK study performed with peptidomimetic macrocycle SP-1.
• FIGURE 5 shows the result of a plasma PK study performed with peptidomimetic macrocycle SP-8.
• FIGURE 6 shows the result of a plasma PK study performed with peptidomimetic macrocycle SP-6.
• FIGURE 7 shows the result of a plasma PK study performed with peptidomimetic macrocycle SP-21.
• FIGURE 8 shows the result of a plasma PK study performed with peptidomimetic macrocycle SP-32.
• FIGURE 9 shows the result of a plasma PK study performed with peptidomimetic macrocycles
• SP-1 SP-6, SP-8, SP-21 , and SP-32.
• FIGURE 10 shows stimulation of growth hormone production by peptidomimetic macrocycle
• FIGURE 11 shows growth hormone release (AUC) induced by sermorelin in comparison to peptidomimetic macrocycles SP-1 , SP-6, SP-8, SP-21 , and SP-32.
• microcycle refers to a molecule having a chemical structure including a ring or cycle formed by at least 9 covalently bonded atoms.
• peptidomimetic macrocycle or "crosslinked polypeptide” refers to a compound comprising a plurality of amino acid residues joined by a plurality of peptide bonds and at least one macrocycle- forming linker which forms a macrocycle between a first naturally- occurring or non-naturally-occurring amino acid residue (or analog) and a second naturally- occurring or non-naturally-occurring amino acid residue (or analog) within the same molecule.
• Peptidomimetic macrocycle include embodiments where the macrocycle-forming linker connects the a carbon of the first amino acid residue (or analog) to the a carbon of the second amino acid residue (or analog).
• the peptidomimetic macrocycles optionally include one or more non-peptide bonds between one or more amino acid residues and/or amino acid analog residues, and optionally include one or more non-naturally-occurring amino acid residues or amino acid analog residues in addition to any which form the macrocycle.
• a "corresponding uncrosslinked polypeptide" when referred to in the context of a peptidomimetic macrocycle is understood to relate to a polypeptide of the same length as the macrocycle and comprising the equivalent natural amino acids of the wild-type sequence corresponding to the macrocycle.
• the term “stability” refers to the maintenance of a defined secondary structure in solution by a peptidomimetic macrocycle of the invention as measured by circular dichroism, NMPv or another biophysical measure, or resistance to proteolytic degradation in vitro or in vivo.
• Non-limiting examples of secondary structures contemplated in this invention are a-helices, 3 io helices, ⁇ -turns, and ⁇ -pleated sheets.
• helical stability refers to the maintenance of a helical structure by a peptidomimetic macrocycle of the invention as measured by circular dichroism or NMR.
• the peptidomimetic macrocycles of the invention exhibit at least a 1.25, 1.5, 1.75 or 2-fold increase in a-helicity as determined by circular dichroism compared to a corresponding uncrosslinked macrocycle.
• amino acid refers to a molecule containing both an amino group and a carboxyl group. Suitable amino acids include, without limitation, both the D-and L-isomers of the naturally-occurring amino acids, as well as non-naturally occurring amino acids prepared by organic synthesis or other metabolic routes.
• amino acid as used herein, includes without limitation, a-amino acids, natural amino acids, non-natural amino acids, and amino acid analogs.
• a-amino acid refers to a molecule containing both an amino group and a carboxyl group bound to a carbon which is designated the a-carbon.
• ⁇ -amino acid refers to a molecule containing both an amino group and a carboxyl group in a ⁇ configuration.
• Naturally occurring amino acid refers to any one of the twenty amino acids commonly found in peptides synthesized in nature, and known by the one letter abbreviations A, R, N, C, D,
• Hydrophobic amino acids include small hydrophobic amino acids and large hydrophobic amino acids.
• Small hydrophobic amino acid are glycine, alanine, proline, and analogs thereof.
• Large hydrophobic amino acids are valine, leucine, isoleucine, phenylalanine, methionine, tryptophan, and analogs thereof.
• Poly amino acids are serine, threonine, asparagine, glutamine, cysteine, tyrosine, and analogs thereof.
• Chargeged amino acids are lysine, arginine, histidine, aspartate, glutamate, and analogs thereof.
• amino acid analog refers to a molecule which is structurally similar to an amino acid and which can be substituted for an amino acid in the formation of a peptidomimetic macrocycle.
• Amino acid analogs include, without limitation, ⁇ -amino acids and amino acids where the amino or carboxy group is substituted by a similarly reactive group (e.g., substitution of the primary amine with a secondary or tertiary amine, or substitution of the carboxy group with an ester).
• non-natural amino acid refers to an amino acid which is not one of the twenty amino acids commonly found in peptides synthesized in nature, and known by the one letter abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y and V.
• Non-natural amino wing :
• Amino acid analogs include ⁇ -amino acid analogs.
• ⁇ -amino acid analogs include, but are not limited to, the following: cyclic ⁇ -amino acid analogs; ⁇ - alanine; (R) - ⁇ - phenylalanine; (R) - 1,2,3,4 - tetrahydro - isoquinoline - 3 - acetic acid; (R) - 3 - amino - 4 - (1 - naphthyl) - butyric acid; (R) - 3 - amino - 4 - (2,4 - dichlorophenyl)butyric acid; (R) - 3 - amino - 4 - (2 - chlorophenyl) - butyric acid; (R) - 3 - amino - 4 - (2 - cyanophenyl) - butyric acid; (R) - 3
• Amino acid analogs include analogs of alanine, valine, glycine or leucine.
• Examples of amino acid analogs of alanine, valine, glycine, and leucine include, but are not limited to, the following: a - methoxy glycine; a - allyl - L - alanine; a - aminoisobutyric acid; a - methyl - leucine; ⁇ - (1 - naphthyl) - D - alanine; ⁇ - (1 - naphthyl) - L - alanine; ⁇ - (2 - naphthyl) - D - alanine; ⁇ - (2 - naphthyl) - L - alanine; ⁇ - (2 - naphthyl) - L - alanine; ⁇ - (2 - pyridyl) - D - alan
• Amino acid analogs include analogs of arginine or lysine.
• amino acid analogs of arginine and lysine include, but are not limited to, the following: citrulline; L - 2 - amino - 3 - guanidinopropionic acid; L - 2 - amino - 3 - ureidopropionic acid; L - citrulline; Lys(Me)2 - OH; Lys(N 3 ) - OH; ⁇ - benzyloxycarbonyl - L - ornithine; ⁇ - nitro - D - arginine; ⁇ - nitro - L - arginine; a - methyl - ornithine; 2,6 - diaminoheptanedioic acid; L - ornithine; ( ⁇ - 1 - (4,4 - dimethyl - 2,6 - dioxo - cyclo
• Arg(Me) 2 - OH (asymmetrical); Arg(Me)2 - OH (symmetrical); Lys(ivDde) - OH; Lys(Me)2 - OH ⁇ HC1; Lys(Me3) - OH chloride; ⁇ - nitro - D - arginine; and ⁇ - nitro - L - arginine.
• Amino acid analogs include analogs of aspartic or glutamic acids. Examples of amino acid
• analogs of aspartic and glutamic acids include, but are not limited to, the following: a - methyl - D - aspartic acid; a - methyl - glutamic acid; a - methyl - L - aspartic acid; ⁇ - methylene - glutamic acid; (N - ⁇ - ethyl) - L - glutamine; [N - a - (4 - aminobenzoyl)] - L - glutamic acid; 2,6
• Amino acid analogs include analogs of cysteine and methionine.
• amino acid analogs of cysteine and methionine include, but are not limited to, Cys(farnesyl) - OH, Cys(farnesyl) - OMe, a - methyl - methionine, Cys(2 - hydroxyethyl) - OH, Cys(3 - aminopropyl) - OH, 2 - amino - 4 - (ethylthio)butyric acid, buthionine, buthioninesulfoximine, ethionine, methionine methylsulfonium chloride, selenomethionine, cysteic acid, [2 - (4 - pyridyl)ethyl] - DL - penicillamine, [2 - (4 - pyridyl)ethyl] - L - cysteine, 4 - meth
• Amino acid analogs include analogs of phenylalanine and tyrosine.
• amino acid analogs of phenylalanine and tyrosine include ⁇ - methyl - phenylalanine, ⁇ - hydroxyphenylalanine, a - methyl - 3 - methoxy - DL - phenylalanine, a - methyl - D - phenylalanine, a - methyl - L - phenylalanine, 1 ,2,3,4 - tetrahydroisoquinoline - 3 - carboxylic acid, 2,4 - dichloro - phenylalanine, 2 - (trifluoromethyl) - D -phenylalanine, 2 - (trifluoromethyl) - L - phenylalanine, 2 - bromo - D - phenylalanine, 2 - bromo - L - - pheny
• Amino acid analogs include analogs of proline. Examples of amino acid analogs of proline
• Amino acid analogs include analogs of serine and threonine.
• Examples of amino acid analogs of serine and threonine include, but are not limited to, 3 - amino - 2 - hydroxy - 5 - methylhexanoic acid, 2 - amino - 3 - hydroxy - 4 - methylpentanoic acid, 2 - amino - 3 - ethoxybutanoic acid, 2 - amino - 3 - methoxybutanoic acid, 4 - amino - 3 - hydroxy - 6 - methylheptanoic acid, 2 - amino - 3 - benzyloxypropionic acid, 2 - amino - 3 - benzyloxypropionic acid, 2 - amino - 3 - ethoxypropionic acid, 4 - amino - 3 - hydroxybutanoic acid, and a-methylserine.
• Amino acid analogs include analogs of tryptophan. Examples of amino acid analogs of
• tryptophan include, but are not limited to, the following: a - methyl - tryptophan; ⁇ - (3 - benzothienyl) - D - alanine; ⁇ - (3 - benzothienyl) - L - alanine; 1 - methyl - tryptophan; 4 - methyl - tryptophan; 5 - benzyloxy - tryptophan; 5 - bromo - tryptophan; 5 - chloro - tryptophan;
• amino acid analogs are racemic.
• the D isomer of the amino acid analog is used.
• the L isomer of the amino acid analog is used.
• the amino acid analog comprises chiral centers that are in the R or S configuration.
• the amino group(s) of a ⁇ -amino acid analog is substituted with a protecting group, e.g., tert-butyloxycarbonyl (BOC group), 9- fluorenylmethyloxycarbonyl (FMOC), tosyl, and the like.
• a protecting group e.g., tert-butyloxycarbonyl (BOC group), 9- fluorenylmethyloxycarbonyl (FMOC), tosyl, and the like.
• the carboxylic acid functional group of a ⁇ -amino acid analog is protected, e.g., as its ester derivative.
• the salt of the amino acid analog is used.
• a "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of a polypeptide without abolishing or substantially abolishing its essential biological or biochemical activity (e.g., receptor binding or activation).
• An "essential” amino acid residue is a residue that, when altered from the wild-type sequence of the polypeptide, results in abolishing or substantially abolishing the polypeptide's essential biological or biochemical activity.
• a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
• Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., K, R, H), acidic side chains (e.g., D, E), uncharged polar side chains (e.g., G, N, Q, S, T, Y, C), nonpolar side chains (e.g., A, V, L, I, P, F, M, W), beta-branched side chains (e.g., T, V, I) and aromatic side chains (e.g., Y, F, W, H).
• basic side chains e.g., K, R, H
• acidic side chains e.g., D, E
• uncharged polar side chains e.g., G, N, Q, S, T, Y, C
• nonpolar side chains e.g., A, V, L
• a predicted nonessential amino acid residue in a polypeptide is replaced with another amino acid residue from the same side chain family.
• Other examples of acceptable substitutions are substitutions based on isosteric considerations (e.g. norleucine for methionine) or other properties (e.g. 2-thienylalanine for phenylalanine).
• capping group refers to the chemical moiety occurring at either the carboxy or amino terminus of the polypeptide chain of the subject peptidomimetic macrocycle.
• the capping group of a carboxy terminus includes an unmodified carboxylic acid (ie -COOH) or a carboxylic acid with a substituent.
• the carboxy terminus can be substituted with an amino group to yield a carboxamide at the C-terminus.
• substituents include but are not limited to primary and secondary amines, including pegylated secondary amines. Representative secondary amine c
• amy lam id e isoamylam ide hexylam ide 3, 3- d i me th yl b utyla m i de (-NHAm) (-NHiAm) (-N HHex) (-NHnBu3,3Me)
• the capping group of an amino terminus includes an unmodified amine (ie -NH 2 ) or an amine with a substituent.
• the amino terminus can be substituted with an acyl group to yield a carboxamide at the N-terminus.
• substituents include but are not limited to substituted acyl groups, including Ci-Ce carbonyls, C7-C30 carbonyls, and pegylated carbamates.
• the term "member” as used herein in conjunction with macrocycles or macrocycle-forming linkers refers to the atoms that form or can form the macrocycle, and excludes substituent or side chain atoms.
• cyclodecane, 1 ,2-difluoro-decane and 1 ,3-dimethyl cyclodecane are all considered ten-membered macrocycles as the hydrogen or fluoro substituents or methyl side chains do not participate in forming the macrocycle.
• the symbol " when used as part of a molecular structure refers to a single bond or a trans or cis double bond.
• amino acid side chain refers to a moiety attached to the a-carbon (or another
• the amino acid side chain for alanine is methyl
• the amino acid side chain for phenylalanine is phenylmethyl
• the amino acid side chain for cysteine is thiomethyl
• the amino acid side chain for aspartate is carboxymethyl
• the amino acid side chain for tyrosine is 4-hydroxyphenylmethyl, etc.
• Other non-naturally occurring amino acid side chains are also included, for example, those that occur in nature ⁇ e.g. , an amino acid metabolite) or those that are made synthetically (e.g., an ⁇ , ⁇ di-substituted amino acid).
• ⁇ , ⁇ di-substituted amino acid refers to a molecule or moiety containing both an amino group and a carboxyl group bound to a carbon (the a-carbon) that is attached to two natural or non-natural amino acid side chains.
• polypeptide encompasses two or more naturally or non-naturally-occurring amino acids joined by a covalent bond ⁇ e.g., an amide bond).
• Polypeptides as described herein include full length proteins ⁇ e.g. , fully processed proteins) as well as shorter amino acid sequences ⁇ e.g., fragments of naturally-occurring proteins or synthetic polypeptide fragments).
• microcyclization reagent or "macrocycle- forming reagent” as used herein refers to any reagent which may be used to prepare a peptidomimetic macrocycle of the invention by mediating the reaction between two reactive groups.
• Reactive groups may be, for example, an azide and alkyne
• macrocyclization reagents include, without limitation, Cu reagents such as reagents which provide a reactive Cu(I) species, such as CuBr, Cul or CuOTf, as well as Cu(II) salts such as Cu(C0 2 CH 3 ) 2 , CuSO i, and CuCl 2 that can be converted in situ to an active Cu(I) reagent by the addition of a reducing agent such as ascorbic acid or sodium ascorbate.
• Macrocyclization reagents may additionally include, for example, Ru reagents known in the art such as Cp*RuCl(PPh 3 ) 2 , [Cp*RuCl]4 or other Ru reagents which may provide a reactive Ru(II) species.
• the reactive groups are terminal olefins.
• the macrocyclization reagents or macrocycle- forming reagents are metathesis catalysts including, but not limited to, stabilized, late transition metal carbene complex catalysts such as Group VIII transition metal carbene catalysts.
• such catalysts are Ru and Os metal centers having a +2 oxidation state, an electron count of 16 and pentacoordinated.
• catalysts have W or Mo centers.
• the reactive groups are thiol groups.
• the macrocyclization reagent is, for example, a linker functionalized with two thiol-reactive groups such as halogen groups.
• halo or halogen refers to fluorine, chlorine, bromine or iodine or a radical thereof.
• alkyl refers to a hydrocarbon chain that is a straight chain or branched chain
• Ci-Cio indicates that the group has from 1 to 10 (inclusive) carbon atoms in it.
• alkyl is a chain (straight or branched) having 1 to 20 (inclusive) carbon atoms in it.
• alkylene refers to a divalent alkyl ⁇ i.e. , -R-).
• alkenyl refers to a hydrocarbon chain that is a straight chain or branched chain having one or more carbon-carbon double bonds.
• the alkenyl moiety contains the indicated number of carbon atoms.
• C2-C10 indicates that the group has from 2 to 10 (inclusive) carbon atoms in it.
• lower alkenyl refers to a C2-C6 alkenyl chain. In the absence of any numerical designation, “alkenyl” is a chain (straight or branched) having 2 to 20 (inclusive) carbon atoms in it.
• alkynyl refers to a hydrocarbon chain that is a straight chain or branched chain having one or more carbon-carbon triple bonds.
• the alkynyl moiety contains the indicated number of carbon atoms.
• C2-C10 indicates that the group has from 2 to 10 (inclusive) carbon atoms in it.
• lower alkynyl refers to a C2-C6 alkynyl chain.
• alkynyl is a chain (straight or branched) having 2 to 20 (inclusive) carbon atoms in it.
• aryl refers to a 6-carbon monocyclic or 10-carbon bicyclic aromatic ring system
• aryl groups include phenyl, naphthyl and the like.
• arylalkoxy refers to an alkoxy substituted with aryl.
• Arylalkyl refers to an aryl group, as defined above, wherein one of the aryl group's hydrogen atoms has been replaced with a C1-C5 alkyl group, as defined above.
• Representative examples of an arylalkyl group include, but are not limited to, 2-methylphenyl, 3-methylphenyl, 4- methylphenyl, 2-ethylphenyl, 3-ethylphenyl, 4-ethylphenyl, 2-propylphenyl, 3-propylphenyl, 4- propylphenyl, 2-butylphenyl, 3-butylphenyl, 4-butylphenyl, 2-pentylphenyl, 3-pentylphenyl, 4- pentylphenyl, 2-isopropylphenyl, 3-isopropylphenyl, 4-isopropylphenyl, 2-isobutylphenyl, 3- isobutylphenyl, 4-isobutylphenyl
• Arylamido refers to an aryl group, as defined above, wherein one of the aryl group's hydrogen atoms has been replaced with one or more -C(0)NH 2 groups.
• Representative examples of an arylamido group include 2-C(0)NH2-phenyl, 3-C(0)NH 2 -phenyl, 4-C(0)NH 2 -phenyl, 2- C(0)NH 2 -pyridyl, 3-C(0)NH 2 -pyridyl, and 4-C(0)NH 2 -pyridyl
• Alkylheterocycle refers to a C1-C5 alkyl group, as defined above, wherein one of the C1-C5 alkyl group's hydrogen atoms has been replaced with a heterocycle.
• alkylheterocycle group examples include, but are not limited to, -CH 2 CH2-morpholine, -CH 2 CH 2 - piperidine, -CH 2 CH 2 CH 2 -morpholine, and -CH 2 CH 2 CH2-imidazole.
• Alkylamido refers to a C1-C5 alkyl group, as defined above, wherein one of the C1-C5 alkyl group's hydrogen atoms has been replaced with a -C(0)NH 2 group.
• alkanol refers to a C1-C5 alkyl group, as defined above, wherein one of the C1-C5 alkyl group's hydrogen atoms has been replaced with a hydroxyl group.
• Representative examples of an alkanol group include, but are not limited to, -CH 2 OH, -CH 2 CH 2 OH, -CH 2 CH 2 CH 2 OH, - CH 2 CH 2 CH 2 CH 2 OH, -CH 2 CH 2 CH 2 CH 2 CH 2 OH, -CH 2 CH(OH)CH 3 , -CH 2 CH(OH)CH 2 CH 3 , - CH(OH)CH 3 and -C(CH 3 ) 2 CH 2 OH.
• Alkylcarboxy refers to a C1-C5 alkyl group, as defined above, wherein one of the C1-C5 alkyl group's hydrogen atoms has been replaced with a -COOH group.
• Representative examples of an alkylcarboxy group include, but are not limited to, -CH 2 COOH, -CH 2 CH 2 COOH, - CH 2 CH 2 CH 2 COOH, -CH 2 CH 2 CH 2 CH 2 COOH, -CH 2 CH(COOH)CH 3 , - CH 2 CH 2 CH 2 CH 2 COOH, -CH 2 CH(COOH)CH 2 CH 3 , -CH(COOH)CH 2 CH 3 and - C(CH 3 ) 2 CH 2 COOH.
• cycloalkyl as employed herein includes saturated and partially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons, preferably 3 to 8 carbons, and more preferably 3 to 6 carbons, wherein the cycloalkyl group additionally is optionally substituted.
• Some cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.
• heteroaryl refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 1 1- 14 membered tricyclic ring system having 1 -3 heteroatoms if monocyclic, 1 -6 heteroatoms if bicyclic, or 1 -9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1 -3, 1 -6, or 1 -9 heteroatoms of O, N, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1 , 2, 3, or 4 atoms of each ring are substituted by a substituent.
• heteroaryl groups include pyridyl, furyl or furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, quinolinyl, indolyl, thiazolyl, and the like.
• heteroarylalkyl or the term “heteroaralkyl” refers to an alkyl substituted with a
• heteroaryl refers to an alkoxy substituted with heteroaryl.
• heteroarylalkyl or the term “heteroaralkyl” refers to an alkyl substituted with a heteroaryl.
• heteroarylalkoxy refers to an alkoxy substituted with heteroaryl.
• heterocyclyl refers to a nonaromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 1 1- 14 membered tricyclic ring system having 1 -3 heteroatoms if monocyclic, 1 -6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1 -3, 1 -6, or 1 -9 heteroatoms of O, N, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1 , 2 or 3 atoms of each ring are substituted by a substituent.
• heterocyclyl groups include piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, and the like.
• substituted refers to a group replacing a second atom or group such as a hydrogen atom on any molecule, compound or moiety. Suitable substituents include, without limitation, halo, hydroxy, mercapto, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, thioalkoxy, aryloxy, amino, alkoxycarbonyl, amido, carboxy, alkanesulfonyl, alkylcarbonyl, and cyano groups.
• the compounds of this invention contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of these compounds are included in the present invention unless expressly provided otherwise.
• the compounds of this invention are also represented in multiple tautomeric forms, in such instances, the invention includes all tautomeric forms of the compounds described herein (e.g. , if alkylation of a ring system results in alkylation at multiple sites, the invention includes all such reaction products). All such isomeric forms of such compounds are included in the present invention unless expressly provided otherwise. All crystal forms of the compounds described herein are included in the present invention unless expressly provided otherwise.
• the terms “increase” and “decrease” mean, respectively, to cause a statistically significantly (i.e. , p ⁇ 0.1) increase or decrease of at least 5%.
• variable is equal to any of the values within that range.
• variable is equal to any integer value within the numerical range, including the end- points of the range.
• variable is equal to any real value within the numerical range, including the end-points of the range.
• a variable which is described as having values between 0 and 2 takes the values 0, 1 or 2 if the variable is inherently discrete, and takes the values 0.0, 0.1 , 0.01 , 0.001 , or any other real values > 0 and ⁇ 2 if the variable is inherently continuous.
• biological activity encompasses structural and functional properties of a macrocycle of the invention.
• Biological activity is, for example, structural stability, alpha-helicity, affinity for a target, resistance to proteolytic degradation, cell penetrability, intracellular stability, in vivo stability, or any combination thereof.
• the peptide sequences are derived from a GHRH peptide.
• the peptide sequences are derived from human GHRH (1 -29) or human GHRH (1 -44).
• GHRH peptides for use in the present invention is given in Table 1 and Table 2 below.
• Tables 1 and 2 all peptides possess a free amino terminus (shown as H-) and all peptides possess an carboxamide terminus (shown as -NH2).
• X residues form cross-links to one other X residue
• Z residues form cross-links to one other Z residue
• XX residues form cross-links with two other X residues.
• amino acid A2 is either L-Ala or D-Ala
• A8 is either L-Asn or L-Gln
• A15 is either L-Ala or Gly
• A27 is either L-Nle or L-Leu.
• each A, C, D, and E is independently an amino acid (including natural or non-natural amino acids and amino acid analogs) and the terminal D and E independently optionally include a capping group;
• amino acid including natural or non-natural amino acids and amino acid analogs
• [-NH-L3-CO-] amino acid (including natural or non-natural amino acids and amino acid analogs), [-NH-L3-CO-], [-NH-L3-SO 2 -], or [-NH-L3-] ;
• Ri and R 2 are independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-;
• R3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R 5 ;
• L is a macrocycle- forming linker of the formula -Li-L 2 -;
• Li and L 2 are independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [-R4-K-R 4 -] n , each being optionally substituted with R 5 ; each R4 is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;
• each K is O, S, SO, S0 2 , CO, C0 2 , or CONR 3 ;
• each R 5 is independently halogen, alkyl, -OR 6 , -N(R 6 ) 2 , -SR 6 , -SOR 6 , -S0 2 R 6 , -C0 2 R 6 , a fluorescent moiety, a radioisotope or a therapeutic agent;
• each R 6 is independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent;
• R 7 is -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl,
• heterocycloalkyl cycloaryl, or heterocycloaryl, optionally substituted with R 5 , or part of a cyclic structure with a D residue;
• Rg is -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl,
• heterocycloalkyl cycloaryl, or heterocycloaryl, optionally substituted with R 5 , or part of a cyclic structure with an E residue;
• v and w are independently integers from 1 -1000, for example 1 -500, 1 -200, 1 -100, 1 -50, 1 -40, 1 -
• u, x, y and z are independently integers from 0-10, for example u is 1 , 2, or 3;
• n is an integer from 1 -5, for example 1.
• u is 2.
• the sum of x+y+z is 2, 3 or 6, for example 3 or 6.
• the peptidomimetic macrocycle of Formula (I) has the Formula:
• each A, C, D, and E is independently an amino acid
• B is an amino acid, H ⁇ , [-NH-L 3 -CO-], [-NH-L 3 -S0 2 -], or [-NH-L3-] ;
• L' is a macrocycle-forming linker of the formula -Li '-L 2 '-;
• Ri' and R 2 ' are independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-;
• Li ' and L 2 ' are independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [-R4-K-R 4 -] n , each being optionally substituted with R 5 ;
• each K is independently O, S, SO, S0 2 , CO, C0 2 , or CONR 3 ;
• R 7 ' is -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R 5 , or part of a cyclic structure with a D residue;
• Rg' is -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl,
• heterocycloalkyl cycloaryl, or heterocycloaryl, optionally substituted with R 5 , or part of a cyclic structure with an E residue;
• v' and w' are independently integers from 1 -1000, for example 1 -500, 1 -200, 1 -100, 1 -50, 1 -40, 1 -25, 1 -20, 1 to 15, or 1 to 10;
• x', y' and z' are independently integers from 0-10;
• n is an integer from 1 -5.
• the sum of x'+y'+z' is 2, 3 or 6, for example 3 or
• each K is O, S, SO, S0 2 , CO, or C0 2 .
• At least one of Ri and R 2 is alkyl, unsubstituted or substituted with halo-. In another example, both Ri and R 2 are independently alkyl, unsubstituted or substituted with halo-. In some embodiments, at least one of Ri and R 2 is methyl. In other embodiments, Ri and R 2 are methyl.
• the sum of the sum of x+y+z is at least 3, and/or the sum of x'+y'+z' is at least 3.
• the sum of the sum of x+y+z is 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 (for example 2, 3 or 6) and/or the sum of x'+y'+z' is 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 (for example 2, 3 or 6).
• each occurrence of A, B, C, D or E in a macrocycle or macrocycle precursor of the invention is independently selected.
• a sequence represented by the formula [A] x when x is 3, encompasses embodiments where the amino acids are not identical, e.g. Gin-Asp-Ala as well as embodiments where the amino acids are identical, e.g. Gln-Gln-Gln. This applies for any value of x, y, or z in the indicated ranges.
• each compound of the invention may encompass peptidomimetic macrocycles which are the same or different.
• a compound of the invention may comprise peptidomimetic macrocycles comprising different linker lengths or chemical compositions.
• the peptidomimetic macrocycle of the invention comprises a secondary structure which is an a-helix and Rg is -H, allowing intrahelical hydrogen bonding.
• at least one of A, B, C, D or E is an ⁇ , ⁇ -disubstituted amino acid.
• B is an ⁇ , ⁇ -disubstituted amino acid.
• at least one of A, B, C, D or E is 2- aminoisobutyric acid.
• the length of the macrocycle-forming linker L as measured from a first Ca to a second Ca is selected to stabilize a desired secondary peptide structure, such as an a-helix formed by residues of the peptidomimetic macrocycle including, but not necessarily limited to, those between the first Ca to a second Ca.
• each Ri and R 2 is independently independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-.
• the peptidomimetic macrocycle comprises a structure of Formula (I) which i
• the peptidomimetic macrocycle of Formula (I) is a compound of any of the formulas shown below:
• AA represents any natural or non-natural amino acid side chain and " ⁇ " is [D] v , [E] w as defined above, and n is an integer between 0 and 20, 50, 100, 200, 300, 400 or 500.
• the substituent "n” shown in the preceding paragraph is 0. In other embodiments, the substituent “n” shown in the preceding paragraph is less than 50, 40, 30, 20, 10, or 5.
• Exemplary embodiments of the macrocycle- forming linker L are shown below.
• X, Y -CH 2 -, O, S, or NH
• X, Y -CH 2 -, O, S, or NH
• X, Y -CH 2 -, O, S, or NH
• X, Y -CH 2 -, O, S, or NH
• R H, alkyl, other substituent
• D and/or E in the compound of Formula I are further modified in order to facilitate cellular uptake.
• lipidating or PEGylating a peptidomimetic macrocycle facilitates cellular uptake, increases bioavailability, increases blood circulation, alters pharmacokinetics, decreases immunogenicity and/or decreases the needed frequency of administration.
• At least one of [D] and [E] in the compound of Formula I represents a moiety comprising an additional macrocycle-forming linker such that the peptidomimetic macrocycle comprises at least two macrocycle-forming linkers.
• a peptidomimetic macrocycle comprises two macrocycle-forming linkers.
• any of the macrocycle-forming linkers described herein may be used in any combination with any of the sequences shown in Tables 1 -3 and also with any of the R- substituents indicated herein.
• the peptidomimetic macrocycle comprises at least one a-helix motif.
• A, B and/or C in the compound of Formula I include one or more a-helices.
• a-helices include between 3 and 4 amino acid residues per turn.
• the a-helix of the peptidomimetic macrocycle includes 1 to 5 turns and, therefore,
• the a-helix includes 1 turn, 2 turns, 3 turns,
• the macrocycle-forming linker stabilizes an a-helix motif included within the peptidomimetic macrocycle.
• the length of the macrocycle-forming linker L from a first Ca to a second Ca is selected to increase the stability of an a-helix.
• the macrocycle-forming linker spans from 1 turn to
• the macrocycle-forming linker spans approximately 1 turn, 2 turns, 3 turns, 4 turns, or 5 turns of the a-helix. In some embodiments, the length of the macrocycle-forming linker is approximately 5 A to 9 A per turn of the a-helix, or approximately
• the length is equal to approximately 5 carbon-carbon bonds to 13 carbon- carbon bonds, approximately 7 carbon-carbon bonds to 1 1 carbon-carbon bonds, or
• the length is equal to approximately 8 carbon-carbon bonds to 16 carbon-carbon bonds, approximately 10 carbon-carbon bonds to 14 carbon-carbon bonds, or approximately 12 carbon-carbon bonds.
• the length is equal to approximately 14 carbon-carbon bonds to 22 carbon-carbon bonds, approximately 16 carbon-carbon bonds to 20 carbon-carbon bonds, or approximately 18 carbon-carbon bonds.
• the length is equal to approximately 20 carbon-carbon bonds to 28 carbon-carbon bonds, approximately 22 carbon-carbon bonds to 26 carbon-carbon bonds, or approximately 24 carbon-carbon bonds.
• the length is equal to approximately 26 carbon-carbon bonds to 34 carbon-carbon bonds, approximately 28 carbon-carbon bonds to 32 carbon-carbon bonds, or approximately 30 carbon-carbon bonds.
• the linkage contains approximately 4 atoms to 12 atoms, approximately 6 atoms to 10 atoms, or approximately 8 atoms. Where the macrocycle-forming linker spans approximately 2 turns of the a-helix, the linkage contains approximately 7 atoms to
• the linkage contains approximately 13 atoms to 21 atoms, approximately 15 atoms to 19 atoms, or approximately 17 atoms.
• the linkage contains approximately 19 atoms to 27 atoms, approximately 21 atoms to 25 atoms, or approximately 23 atoms.
• the linkage contains approximately 25 atoms to 33 atoms, approximately 27 atoms to 31 atoms, or approximately 29 atoms.
• the resulting macrocycle forms a ring containing approximately 17 members to 25 members, approximately 19 members to 23 members, or approximately 21 members.
• the resulting macrocycle forms a ring containing approximately 29 members to 37 members, approximately 31 members to 35 members, or approximately 33 members.
• the resulting macrocycle forms a ring containing approximately 44 members to 52 members, approximately 46 members to 50 members, or approximately 48 members.
• the macrocycle-forming linker spans approximately 4 turns of the a-helix
• the resulting macrocycle forms a ring containing approximately 59 members to 67 members, approximately 61 members to 65 members, or approximately 63 members.
• the resulting macrocycle spans approximately 5 turns of the a-helix, the resulting macrocycle forms a ring containing approximately 74 members to 82 members, approximately 76 members to 80 members, or approximately 78 members.
• ents, L is a macrocycle-forming linker of the formula
• the invention provides peptidomimetic macrocycles of Formula (II) or (Ila):
• each A, C, D, and E is independently an amino acid
• B is an amino acid, H ⁇ , [-NH-L3-CO-], [-NH-L3-SO 2 -], or [-NH-L3-] ;
• Ri and R 2 are independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo- or part of a cyclic structure with an E residue;
• R3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R 5 ;
• Li and L 2 are independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [-R4-K-R 4 -] n , each being optionally substituted with R 5 ;
• each R 4 is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;
• each K is O, S, SO, S0 2 , CO, C0 2 , or CONR 3 ;
• each R 5 is independently halogen, alkyl, -OR 6 , -N(R 6 ) 2 , -SR 6 , -SOR 6 , -S0 2 R 6 , -C0 2 R 6 , a fluorescent moiety, a radioisotope or a therapeutic agent;
• each R 6 is independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent;
• R 7 is -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl,
• heterocycloalkyl cycloaryl, or heterocycloaryl, optionally substituted with R 5 ;
• v and w are independently integers from 1 -1000, for example 1 -100;
• u, x, y and z are independently integers from 0-10, for example u is 1 -3;
• n is an integer from 1 -5.
• At least one of Ri and R 2 is alkyl, unsubstituted or substituted with halo-. In another example, both Ri and R 2 are independently alkyl, unsubstituted or substituted with halo-. In some embodiments, at least one of Ri and R 2 is methyl. In other embodiments, Ri and R 2 are methyl.
• the sum of x+y+z is at least 1. In other embodiments of the invention, the sum of x+y+z is at least 2. In other embodiments of the invention, the sum of x+y+z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
• Each occurrence of A, B, C, D or E in a macrocycle or macrocycle precursor of the invention is independently selected.
• a sequence represented by the formula [A] x when x is 3, encompasses embodiments where the amino acids are not identical, e.g. Gin-Asp-Ala as well as embodiments where the amino acids are identical, e.g. Gln-Gln-Gln. This applies for any value of x, y, or z in the indicated ranges.
• the peptidomimetic macrocycle of the invention comprises a secondary structure which is an a-helix and Rg is -H, allowing intrahelical hydrogen bonding.
• at least one of A, B, C, D or E is an ⁇ , ⁇ -disubstituted amino acid.
• B is an ⁇ , ⁇ -disubstituted amino acid.
• at least one of A, B, C, D or E is 2- aminoisobutyric acid.
• the length of the macrocycle-forming linker -Li-L 2 - as measured from a first Ca to a second Ca is selected to stabilize a desired secondary peptide structure, such as an a- helix formed by residues of the peptidomimetic macrocycle including, but not necessarily limited to, those between the first Ca to a second Ca.
• X, Y -CH 2 -, O, S, or NH
• X, Y -CH 2 -, O, S, or NH
• X, Y -CH 2 -, O, S, or NH
• X, Y -CH 2 -, O, S, or NH
• R H, alkyl, other substituent
• Examples of peptidomimetic macrocycles of Formula (II) are shown in Table 4 and include SP- 85, SP-86, SP-87, SP-88, SP-91, and SP-92.
• Peptidomimetic macrocycles of the invention may be prepared by any of a variety of methods known in the art.
• any of the residues indicated by "X", “Z” or “XX” in Tables 1, 2 or 4 may be substituted with a residue capable of forming a crosslinker with a second residue in the same molecule or a precursor of such a residue.
• the "S5-olefin amino acid” is (S)-a-(2'-pentenyl) alanine and the "R8 olefin amino acid” is (R)-a-(2'-octenyl) alanine.
• the terminal olefins are reacted with a metathesis catalyst, leading to the formation of the peptidomimetic macrocycle.
• the following amino acids may be employed in the synthesis of the
• x+y+z is 3, and and A, B and C are independently natural or non-natural amino acids. In other embodiments, x+y+z is 6, and and A, B and C are independently natural or non-natural amino acids.
• the contacting step is performed in a solvent selected from the group consisting of protic solvent, aqueous solvent, organic solvent, and mixtures thereof.
• the solvent may be chosen from the group consisting of H 2 0, THF, THF/H 2 0, tBuOH/H 2 0, DMF, DIPEA, CH 3 CN or CH 2 C1 2 , C1CH 2 CH 2 C1 or a mixture thereof.
• the solvent may be a solvent which favors helix formation.
• peptidomimetic macrocycles disclosed herein are made, for example, by chemical synthesis methods, such as described in Fields et al., Chapter 3 in Synthetic Peptides: A User's Guide, ed. Grant, W. H. Freeman & Co., New York, N. Y., 1992, p. 77.
• peptides are synthesized using the automated Merrifield techniques of solid phase synthesis with the amine protected by either tBoc or Fmoc chemistry using side chain protected amino acids on, for example, an automated peptide synthesizer ⁇ e.g. , Applied Biosystems (Foster City, CA), Model 430A, 431, or 433).
• SPPS solid phase peptide synthesis
• peptidomimetic precursors are produced, for example, by conjoining individual synthetic peptides using native chemical ligation. Alternatively, the longer synthetic peptides are biosynthesized by well known recombinant DNA and protein expression techniques. Such techniques are provided in well-known standard manuals with detailed protocols.
• To construct a gene encoding a peptidomimetic precursor of this invention the amino acid sequence is reverse translated to obtain a nucleic acid sequence encoding the amino acid sequence, preferably with codons that are optimum for the organism in which the gene is to be expressed.
• a synthetic gene is made, typically by synthesizing oligonucleotides which encode the peptide and any regulatory elements, if necessary.
• the synthetic gene is inserted in a suitable cloning vector and transfected into a host cell. The peptide is then expressed under suitable conditions appropriate for the selected expression system and host.
• the peptide is purified and characterized by standard methods.
• the peptidomimetic precursors are made, for example, in a high-throughput, combinatorial fashion using, for example, a high-throughput polychannel combinatorial synthesizer (e.g., Thuramed TETRAS multichannel peptide synthesizer from CreoSalus, Louisville, KY or Model Apex 396 multichannel peptide synthesizer from AAPPTEC, Inc., Louisville, KY).
• a high-throughput polychannel combinatorial synthesizer e.g., Thuramed TETRAS multichannel peptide synthesizer from CreoSalus, Louisville, KY or Model Apex 396 multichannel peptide synthesizer from AAPPTEC, Inc., Louisville, KY.
• the peptidomimetic macrocyles of the invention comprise triazole
• macrocycle-forming linkers For example, the synthesis of such peptidomimetic macrocycles involves a multi-step process that features the synthesis of a peptidomimetic precursor containing an azide moiety and an alkyne moiety; followed by contacting the peptidomimetic precursor with a macrocyclization reagent to generate a triazole-linked peptidomimetic macrocycle.
• a process is described, for example, in US Application 12/037,041 , filed on February 25, 2008.
• Macrocycles or macrocycle precursors are synthesized, for example, by solution phase or solid- phase methods, and can contain both naturally-occurring and non-naturally-occurring amino acids. See, for example, Hunt, "The Non-Protein Amino Acids" in Chemistry and Biochemistry of the Amino Acids, edited by G.C. Barrett, Chapman and Hall, 1985.
• an azide is linked to the a-carbon of a residue and an alkyne is attached to the ⁇ -carbon of another residue.
• the azide moieties are azido-analogs of amino acids L-lysine, D-lysine, alpha-methyl-L-lysine, alpha-methyl-D-lysine, L-ornithine, D- ornithine, alpha-methyl-L-ornithine or alpha-methyl-D-ornithine.
• the alkyne moiety is L-propargylglycine.
• the alkyne moiety is an amino acid selected from the group consisting of L-propargylglycine, D-propargylglycine, (S)-2-amino- 2-methyl-4-pentynoic acid, (R)-2-amino-2-methyl-4-pentynoic acid, (S)-2-amino-2-methyl-5- hexynoic acid, (R)-2-amino-2-methyl-5-hexynoic acid, (S)-2-amino-2-methyl-6-heptynoic acid, (R)-2-amino-2-methyl-6-heptynoic acid, (S)-2-amino-2-methyl-7-octynoic acid, (R)-2-amino-2- methyl-7-octynoic acid, (S)-2-amino-2-methyl-8-nonynoic acid and (R)-2-amino-2-methyl-8- nonynoic acid.
• each Ri, R 2 , R7 and Rg is -H; each Li is -(CH 2 )4- ; and each L 2 is -(CH 2 )-.
• Ri, R 2 , R 7 , R 8 , Li and L 2 can be independently selected from the various structures disclosed herein.
• Synthetic Scheme 1 describes the preparation of several compounds of the invention.
• Ni(II) complexes of Schiff bases derived from the chiral auxiliary (S)-2-[N-(N'- benzylprolyl)amino]benzophenone (BPB) and amino acids such as glycine or alanine are prepared as described in Belokon et al. (1998), Tetrahedron Asymm. 9:4249-4252.
• the resulting complexes are subsequently reacted with alkylating reagents comprising an azido or alkynyl moiety to yield enantiomerically enriched compounds of the invention. If desired, the resulting compounds can be protected for use in peptide synthesis.
• the peptidomimetic precursor contains an azide moiety and an alkyne moiety and is synthesized by solution-phase or solid-phase peptide synthesis (SPPS) using the commercially available amino acid N-a-Fmoc-L-propargylglycine and the N-a-Fmoc-protected forms of the amino acids (S)-2-amino-2-methyl-4-pentynoic acid, (S)-2-amino-6-heptynoic acid, (S)-2-amino- 2-methyl-6-heptynoic acid, N-methyl-e-azido-L-lysine, and N-methyl-e-azido-D-lysine.
• SPPS solution-phase or solid-phase peptide synthesis
• the peptidomimetic precursor is then deprotected and cleaved from the solid-phase resin by standard conditions (e.g., strong acid such as 95% TFA).
• the peptidomimetic precursor is reacted as a crude mixture or is purified prior to reaction with a macrocyclization reagent such as a Cu(I) in organic or aqueous solutions (Rostovtsev et al. (2002), Angew. Chem. Int. Ed. 41 :2596-2599; Tornoe et al. (2002), J. Org. Chem. 67:3057-3064; Deiters et al. (2003), J. Am. Chem. Soc.
• the triazole forming reaction is performed under conditions that favor a-helix formation.
• the macrocyclization step is performed in a solvent chosen from the group consisting of H 2 0, THF, CH 3 CN, DMF , DIPEA, tBuOH or a mixture thereof.
• the macrocyclization step is performed in DMF.
• the macrocyclization step is performed in a buffered aqueous or partially aqueous solvent.
• the peptidomimetic precursor contains an azide moiety and an alkyne moiety and is synthesized by solid-phase peptide synthesis (SPPS) using the commercially available amino acid N-a-Fmoc-L-propargylglycine and the N-a-Fmoc-protected forms of the amino acids (S)-2- amino-2-methyl-4-pentynoic acid, (S)-2-amino-6-heptynoic acid, (S)-2-amino-2-methyl-6- heptynoic acid, N-methyl-e-azido-L-lysine, and N-methyl-e-azido-D-lysine.
• SPPS solid-phase peptide synthesis
• the peptidomimetic precursor is reacted with a macrocyclization reagent such as a Cu(I) reagent on the resin as a crude mixture (Rostovtsev et al. (2002), Angew. Chem. Int. Ed. 41 :2596-2599; Tornoe et al. (2002), J. Org. Chem. 67:3057-3064; Deiters et al. (2003), J. Am. Chem. Soc. 125 : 1 1782-1 1783; Punna et al. (2005), Angew. Chem. Int. Ed. 44:2215-2220).
• the resultant triazole-containing peptidomimetic macrocycle is then deprotected and cleaved from the solid-phase resin by standard conditions ⁇ e.g. , strong acid such as 95% TFA).
• the a macrocyclization reagent such as a Cu(I) reagent
• macrocyclization step is performed in a solvent chosen from the group consisting of CH 2 CI 2 , CICH 2 CH 2 CI, DMF, THF, NMP, DIPEA, 2,6-lutidine, pyridine, DMSO, H 2 0 or a mixture thereof.
• the macrocyclization step is performed in a buffered aqueous or partially aqueous solvent.
• the peptidomimetic precursor contains an azide moiety and an alkyne moiety and is synthesized by solution-phase or solid-phase peptide synthesis (SPPS) using the commercially available amino acid N-a-Fmoc-L-propargylglycine and the N-a-Fmoc-protected forms of the amino acids (S)-2-amino-2-methyl-4-pentynoic acid, (S)-2-amino-6-heptynoic acid, (S)-2-amino- 2-methyl-6-heptynoic acid, N-methyl-e-azido-L-lysine, and N-methyl-e-azido-D-lysine.
• SPPS solution-phase or solid-phase peptide synthesis
• the peptidomimetic precursor is then deprotected and cleaved from the solid-phase resin by standard conditions (e.g. , strong acid such as 95% TFA).
• the peptidomimetic precursor is reacted as a crude mixture or is purified prior to reaction with a macrocyclization reagent such as a Ru(II) reagents, for example Cp*RuCl(PPh 3 ) 2 or [Cp*RuCl]4 (Rasmussen et al. (2007), Org. Lett. 9:5337-5339; Zhang et al. (2005), J. Am. Chem. Soc. 127: 15998- 15999).
• the macrocyclization step is performed in a solvent chosen from the group consisting of DMF, CH 3 CN and THF.
• the peptidomimetic precursor contains an azide moiety and an alkyne moiety and is synthesized by solid-phase peptide synthesis (SPPS) using the commercially available amino acidN-a-Fmoc-L-propargylglycine and the N-a-Fmoc-protected forms of the amino acids (S)-2- amino-2-methyl-4-pentynoic acid, (S)-2-amino-6-heptynoic acid, (S)-2-amino-2-methyl-6- heptynoic acid, N-methyl-e-azido-L-lysine, and N-methyl-e-azido-D-lysine.
• SPPS solid-phase peptide synthesis
• the peptidomimetic precursor is reacted with a macrocyclization reagent such as a Ru(II) reagent on the resin as a crude mixture.
• a macrocyclization reagent such as a Ru(II) reagent on the resin as a crude mixture.
• the reagent can be Cp*RuCl(PPh 3 ) 2 or [Cp*RuCl]4 (Rasmussen et al. (2007), Org. Lett. 9:5337-5339; Zhang et al. (2005), J. Am. Chem. Soc. 127: 15998-15999).
• the macrocyclization step is performed in a solvent chosen from the group consisting of CH 2 C1 2 , C1CH 2 CH 2 C1, CH 3 CN, DMF, and THF.
• the present invention contemplates the use of non-naturally-occurring amino acids and amino acid analogs in the synthesis of the peptidomimetic macrocycles described herein.
• Any amino acid or amino acid analog amenable to the synthetic methods employed for the synthesis of stable triazole containing peptidomimetic macrocycles can be used in the present invention.
• L-propargylglycine is contemplated as a useful amino acid in the present invention.
• other alkyne-containing amino acids that contain a different amino acid side chain are also useful in the invention.
• L-propargylglycine contains one methylene unit between the a-carbon of the amino acid and the alkyne of the amino acid side chain.
• the invention also contemplates the use of amino acids with multiple methylene units between the a- carbon and the alkyne.
• the azido-analogs of amino acids L-lysine, D-lysine, alpha-methyl- L-lysine, and alpha-methyl-D-lysine are contemplated as useful amino acids in the present invention.
• other terminal azide amino acids that contain a different amino acid side chain are also useful in the invention.
• the azido-analog of L-lysine contains four methylene units between the a-carbon of the amino acid and the terminal azide of the amino acid side chain.
• the invention also contemplates the use of amino acids with fewer than or greater than four methylene units between the ⁇ -carbon and the terminal azide. Table 3 shows some amino acids useful in the preparation of peptidomimetic macrocycles disclosed herein.
• Table 3 shows exemplary amino acids useful in the preparation of peptidomimetic
• the amino acids and amino acid analogs are of the D-configuration. In other embodiments they are of the L-configuration. In some embodiments, some of the amino acids and amino acid analogs contained in the peptidomimetic are of the D-configuration while some of the amino acids and amino acid analogs are of the L-configuration. In some embodiments the amino acid analogs are ⁇ , ⁇ -disubstituted, such as a-methyl-L-propargylglycine, a-methyl-D-propargylglycine, ⁇ -azido-alpha-methyl-L-lysine, and ⁇ -azido-alpha-methyl-D- lysine.
• amino acid analogs are N-alkylated, e.g. , N-methyl-L- propargylglycine, N-methyl-D-propargylglycine, N-methyl-e-azido-L-lysine, and N-methyl- ⁇ - azido-D-lysine.
• the -NH moiety of the amino acid is protected using a protecting group, including without limitation -Fmoc and -Boc.
• the amino acid is not protected prior to synthesis of the peptidomimetic macrocycle.
• aminoacid precursors are used containing an additional substituent R- at the alpha position.
• Such aminoacids are incorporated into the macrocycle precursor at the desired positions, which may be at the positions where the crosslinker is substituted or, alternatively, elsewhere in the sequence of the macrocycle precursor. Cyclization of the precursor is then performed according to the indicated method.
• a peptidomimetic macrocycle of Formula (II) is prepared as indicated:
• each AA 1; AA 2 , AA 3 is independently an amino acid side chain
• a peptidomimetic macrocycle of Formula (II) is prepared as indicated:
• each AA 1; AA 2 , AA 3 is independently an amino acid side chain.
• a peptidomimetic macrocycle is obtained in more than one isomer, for example due to the configuration of a double bond within the structure of the crosslinker E vs Z).
• Such isomers can or can not be separable by conventional chromatographic methods.
• one isomer has improved biological properties relative to the other isomer.
• an E crosslinker olefin isomer of a peptidomimetic macrocycle has better solubility, better target affinity, better in vivo or in vitro efficacy, higher helicity, or improved cell permeability relative to its Z counterpart.
• a Z crosslinker olefin isomer of a peptidomimetic macrocycle has better solubility, better target affinity, better in vivo or in vitro efficacy, higher helicity, or improved cell permeability relative to its E counterpart.
• peptidomimetic macrocycles of the invention are assayed, for example, by using the methods described below.
• a peptidomimetic macrocycle of the invention has improved biological properties relative to a corresponding polypeptide lacking the substituents described herein.
• polypeptides with a-helical domains will reach a dynamic equilibrium between random coil structures and ⁇ -helical structures, often expressed as a "percent helicity".
• alpha-helical domains are predominantly random coils in solution, with ⁇ -helical content usually under 25%.
• Peptidomimetic macrocycles with optimized linkers possess, for example, an alpha-helicity that is at least two-fold greater than that of a corresponding uncrosslinked polypeptide.
• macrocycles of the invention will possess an alpha-helicity of greater than 50%.
• peptidomimetic macrocyles of the invention the compounds are dissolved in an aqueous solution (e.g. 50 mM potassium phosphate solution at pH 7, or distilled H 2 0, to concentrations of 25-50 ⁇ ).
• Circular dichroism (CD) spectra are obtained on a spectropolarimeter ⁇ e.g. , Jasco J-710) using standard measurement parameters (e.g. temperature, 20°C; wavelength, 190-260 nm; step resolution, 0.5 nm; speed, 20 nm/sec; accumulations, 10; response, 1 sec; bandwidth, 1 nm; path length, 0.1 cm).
• the ⁇ -helical content of each peptide is calculated by dividing the mean residue ellipticity (e.g. [ ⁇ ]222obs) by the reported value for a model helical decapeptide (Yang et al. (1986), Methods Enzymol. 130:208)).
• a peptidomimetic macrocycle of the invention comprising a secondary structure such as an a- helix exhibits, for example, a higher melting temperature than a corresponding uncrosslinked polypeptide.
• peptidomimetic macrocycles of the invention exhibit Tm of > 60°C representing a highly stable structure in aqueous solutions.
• Tm is determined by measuring the change in ellipticity over a temperature range (e.g. 4 to 95 °C) on a
• spectropolarimeter ⁇ e.g., Jasco J-710 using standard parameters (e.g. wavelength 222nm; step resolution, 0.5 nm; speed, 20 nm/sec; accumulations, 10; response, 1 sec; bandwidth, 1 nm; temperature increase rate: l°C/min; path length, 0.1 cm).
• standard parameters e.g. wavelength 222nm; step resolution, 0.5 nm; speed, 20 nm/sec; accumulations, 10; response, 1 sec; bandwidth, 1 nm; temperature increase rate: l°C/min; path length, 0.1 cm.
• the amide bond of the peptide backbone is susceptible to hydrolysis by proteases, thereby
• the peptidomimetic macrocycles of the present invention may be subjected to in vitro trypsin proteolysis to assess for any change in degradation rate compared to a corresponding uncrosslinked polypeptide.
• the peptidomimetic macrocycle and a corresponding uncrosslinked polypeptide are incubated with trypsin agarose and the reactions quenched at various time points by centrifugation and subsequent HPLC injection to quantitate the residual substrate by ultraviolet absorption at 280 nm.
• the peptidomimetic macrocycle and peptidomimetic precursor (5 meg) are incubated with trypsin agarose (Pierce) (S/E -125) for 0, 10, 20, 90, and 180 minutes. Reactions are quenched by tabletop centrifugation at high speed; remaining substrate in the isolated supernatant is quantified by HPLC-based peak detection at 280 nm.
• Peptidomimetic macrocycles with optimized linkers possess, for example, an ex vivo half-life that is at least two-fold greater than that of a corresponding uncrosslinked polypeptide, and possess an ex vivo half-life of 12 hours or more.
• an ex vivo half-life that is at least two-fold greater than that of a corresponding uncrosslinked polypeptide, and possess an ex vivo half-life of 12 hours or more.
• assays may be used. For example, a peptidomimetic macrocycle and a corresponding uncrosslinked polypeptide (2 meg) are incubated with fresh mouse, rat and/or human serum (2 mL) at 37°C for 0, 1 , 2, 4, 8, and 24 hours.
• the samples are extracted by transferring 100 ⁇ of sera to 2 ml centrifuge tubes followed by the addition of 10 of 50 % formic acid and 500 ⁇ acetonitrile and centrifugation at 14,000 RPM for 10 min at 4 ⁇ 2°C. The supernatants are then transferred to fresh 2 ml tubes and evaporated on Turbovap under N 2 ⁇ 10 psi, 37°C. The samples are reconstituted in ⁇ ⁇ of 50:50 acetonitrile:water and submitted to LC-MS/MS analysis.
• FPA fluorescence polarization assay
• fluoresceinated peptidomimetic macrocycles (25 nM) are incubated with the acceptor protein (25- 1000 nM) in binding buffer (140 mM NaCl, 50 mM Tris-HCL, pH 7.4) for 30 minutes at room temperature. Binding activity is measured, for example, by fluorescence polarization on a luminescence spectrophotometer (e.g. Perkin-Elmer LS50B). Kd values may be determined by nonlinear regression analysis using, for example, Graphpad Prism software (GraphPad Software, Inc., San Diego, CA).
• a peptidomimetic macrocycle of the invention shows, in some instances, similar or lower Kd than a corresponding uncrosslinked polypeptide.
• FPA fluorescence polarization assay
• FITC-labeled peptides bound to a large protein emit higher levels of polarized fluorescence due to their slower rates of rotation as compared to fluorescent tracers attached to smaller molecules ⁇ e.g. FITC-labeled peptides that are free in solution).
• a compound that antagonizes the interaction between the fluoresceinated peptidomimetic macrocycle and an acceptor protein will be detected in a competitive binding FPA experiment.
• peptidomimetic macrocycle 25 nM are incubated with the acceptor protein (50 nM) in binding buffer (140mM NaCl, 50 mM Tris-HCL, pH 7.4) for 30 minutes at room temperature.
• Antagonist binding activity ismeasured, for example, by fluorescence polarization on a luminescence spectrophotometer (e.g. Perkin-Elmer LS50B). Kd values may be determined by nonlinear regression analysis using, for example, Graphpad Prism software (GraphPad Software, Inc., San Diego, CA).
• Any class of molecule such as small organic molecules, peptides, oligonucleotides or proteins can be examined as putative antagonists in this assay.
• an affinity-selection mass spectrometry assay is used, for example.
• Protein-ligand binding experiments are conducted according to the following representative procedure outlined for a system-wide control experiment using 1 ⁇ peptidomimetic macrocycle plus 5 ⁇ target protein.
• a 1 DMSO aliquot of a 40 ⁇ stock solution of peptidomimetic macrocycle is dissolved in 19 ⁇ of PBS (Phosphate-buffered saline: 50 mM, pH 7.5 Phosphate buffer containing 150 mM NaCl).
• PBS Phosphate-buffered saline: 50 mM, pH 7.5 Phosphate buffer containing 150 mM NaCl
• the resulting solution is mixed by repeated pipetting and clarified by centrifugation at 10 OOOg for 10 min.
• To a 4 aliquot of the resulting supernatant is added 4 of 10 ⁇ target protein in PBS.
• Each 8.0 experimental sample thus contains 40 pmol (
• Protein-ligand K d titrations experiments are conducted as follows: 2 ⁇ ⁇ DMSO aliquots of a serially diluted stock solution of titrant peptidomimetic macrocycle (5, 2.5, 0.098 mM) are prepared then dissolved in 38 ⁇ ⁇ of PBS. The resulting solutions are mixed by repeated pipetting and clarified by centrifugation at 10 OOOg for 10 min. To 4.0 ⁇ ⁇ aliquots of the resulting supernatants is added 4.0 ⁇ ⁇ of 10 ⁇ target protein in PBS. Each 8.0 ⁇ ⁇ experimental sample thus contains 40 pmol (1.5 ⁇ g) of protein at 5.0 ⁇
• an affiinity selection mass spectrometry assay is performed, for example.
• a mixture of ligands at 40 ⁇ per component is prepared by combining 2 aliquots of 400 ⁇ stocks of each of the three compounds with 14 ⁇ ⁇ of DMSO. Then, 1 ⁇ ⁇ aliquots of this 40 ⁇ per component mixture are combined with 1 ⁇ ⁇ DMSO aliquots of a serially diluted stock solution of titrant peptidomimetic macrocycle (10, 5, 2.5, 0.078 mM). These 2 ⁇ ⁇ samples are dissolved in 38 ⁇ ⁇ of PBS.
• the resulting solutions were mixed by repeated pipetting and clarified by centrifugation at 10 OOOg for 10 min.
• To 4.0 ⁇ ⁇ aliquots of the resulting supernatants is added 4.0 ⁇ ⁇ of 10 ⁇ target protein in PBS.
• Each 8.0 ⁇ ⁇ experimental sample thus contains 40 pmol (1.5 ⁇ g) of protein at 5.0 ⁇ concentration in PBS plus 0.5 ⁇ ligand, 2.5% DMSO, and varying concentrations (125, 62.5, 0.98 ⁇ ) of the titrant peptidomimetic macrocycle.
• Duplicate samples thus prepared for each concentration point are incubated at room temperature for 60 min, then chilled to 4 °C prior to SEC-LC-MS analysis of 2.0 ⁇ ⁇ injections.
• Extracts are centrifuged at 14,000 rpm for 15 minutes and supernatants collected and incubated with 10 ⁇ goat anti-FITC antibody for 2 hrs, rotating at 4°C followed by further 2 hrs incubation at 4°C with protein A/G Sepharose (50 ⁇ of 50% bead slurry). After quick centrifugation, the pellets are washed in lysis buffer containing increasing salt concentration ⁇ e.g., 150, 300, 500 mM). The beads are then re-equilibrated at 150 mM NaCl before addition of SDS-containing sample buffer and boiling. After centrifugation, the supernatants are optionally electrophoresed using 4%-12% gradient Bis-Tris gels followed by transfer into Immobilon-P membranes. After blocking, blots are optionally incubated with an antibody that detects FITC and also with one or more antibodies that detect proteins that bind to the peptidomimetic macrocycle. Cellular Penetrability Assays.
• uncrosslinked macrocycle intact cells are incubated with fluoresceinated peptidomimetic macrocycles or corresponding uncrosslinked macrocycle (10 ⁇ ) for 4 hrs in serum free media at 37°C, washed twice with media and incubated with trypsin (0.25%) for 10 min at 37°C. The cells are washed again and resuspended in PBS. Cellular fluorescence is analyzed, for example, by using either a FACSCalibur flow cytometer or Cellomics' KineticScan ® HCS Reader.
• the compounds are, for example,administered to mice and/or rats by IV, IP, PO or inhalation routes at concentrations ranging from 0.1 to 50 mg/kg and blood specimens withdrawn at 0', 5', 15', 30', 1 hr, 4 hrs, 8 hrs and 24 hours post-injection. Levels of intact compound in 25 of fresh serum are then measured by LC-MS/MS as above.
• peptidomimetic macrocycles of the invention are selected and separated in treatment and one or more control groups, wherein the treatment group is administered a peptidomimetic macrocycle of the invention, while the control groups receive a placebo or a known GHRH or GH drug.
• the treatment safety and efficacy of the peptidomimetic macrocycles of the invention can thus be evaluated by performing comparisons of the patient groups with respect to factors such as survival and quality-of-life.
• the patient group treated with a peptidomimetic macrocyle show improved long-term survival compared to a patient control group treated with a placebo.
• the peptidomimetic macrocycles of the invention also include pharmaceutically acceptable
• a "pharmaceutically acceptable derivative” means any pharmaceutically acceptable salt, ester, salt of an ester, pro-drug or other derivative of a compound of this invention which, upon administration to a recipient, is capable of providing (directly or indirectly) a compound of this invention.
• Particularly favored pharmaceutically acceptable derivatives are those that increase the bioavailability of the compounds of the invention when administered to a mammal (e.g., by increasing absorption into the blood of an orally administered compound) or which increases delivery of the active compound to a biological compartment (e.g., the brain or lymphatic system) relative to the parent species.
• Some pharmaceutically acceptable derivatives include a chemical group which increases aqueous solubility or active transport across the gastrointestinal mucosa.
• the peptidomimetic macrocycles of the invention are modified by
• Such modifications include those which increase biological penetration into a given biological compartment (e.g., blood, lymphatic system, central nervous system), increase oral availability, increase solubility to allow administration by injection, alter metabolism, and alter rate of excretion.
• Pharmaceutically acceptable salts of the compounds of this invention include those derived from pharmaceutically acceptable inorganic and organic acids and bases.
• suitable acid salts include acetate, adipate, benzoate, benzenesulfonate, butyrate, citrate, digluconate, dodecylsulfate, formate, fumarate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, lactate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, palmoate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, tosylate and undecanoate.
• Salts derived from appropriate bases include alkali metal (e.g., sodium), alkaline earth metal (e.g., magnesium), ammonium
• pharmaceutically acceptable carriers include either solid or liquid carriers. Solid form
• a solid carrier can be one or more substances, which also acts as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material. Details on techniques for formulation and administration are well described in the scientific and patent literature, see, e.g., the latest edition of Remington's Pharmaceutical Sciences, Maack Publishing Co, Easton PA.
• the carrier is a finely divided solid, which is in a mixture with the finely divided active component.
• the active component is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.
• Suitable solid excipients are carbohydrate or protein fillers include, but are not limited to sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium
• carboxymethylcellulose and gums including arabic and tragacanth; as well as proteins such as gelatin and collagen.
• disintegrating or solubilizing agents are added, such as the cross- linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
• Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water/propylene glycol solutions.
• liquid preparations can be formulated in solution in aqueous polyethylene glycol solution.
• the pharmaceutical preparation is preferably in unit dosage form. In such form the preparation is subdivided into unit doses containing appropriate quantities of the active component.
• the unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules.
• the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.
• compositions of this invention comprise a combination of a peptidomimetic
• both the compound and the additional agent should be present at dosage levels of between about 1 to 100%, and more preferably between about 5 to 95% of the dosage normally administered in a monotherapy regimen.
• the additional agents are administered separately, as part of a multiple dose regimen, from the compounds of this invention.
• those agents are part of a single dosage form, mixed together with the compounds of this invention in a single composition.
• compositions are present as unit dosage forms that can deliver, for example, from about 0.0001 mg to about 1 ,000 mg of the peptidomimetic macrocycles, salts thereof, prodrugs thereof, derivatives thereof, or any combination of these.
• the unit dosage forms can deliver, for example, in some embodiments, from about 1 mg to about 900 mg, from about 1 mg to about 800 mg, from about 1 mg to about 700 mg, from about 1 mg to about 600 mg, from about 1 mg to about 500 mg, from about 1 mg to about 400 mg, from about 1 mg to about 300 mg, from about 1 mg to about 200 mg, from about 1 mg to about 100 mg, from about 1 mg to about 10 mg, from about 1 mg to about 5 mg, from about 0.1 mg to about 10 mg, from about 0.1 mg to about 5 mg, from about 10 mg to about 1 ,000 mg, from about 50 mg to about 1 ,000 mg, from about 100 mg to about 1 ,000 mg, from about 200 mg to about 1 ,000 mg, from about 300 mg to about 1 ,000 mg, from about 400 mg to about 1 ,000 mg, from about 500 mg to about 1 ,000 mg, from about 600 mg to about 1 ,000 mg, from about 700 mg to about 1 ,000 mg, from about 800 mg to about 1 ,000
• compositions are present as unit dosage forms that can deliver, for example, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, or about 800 mg of peptidomimetic macrocycles, salts thereof, prodrugs thereof, derivatives thereof, or any combination of these.
• Suitable routes of administration include, but are not limited to, oral, intravenous, rectal, aerosol, parenteral, ophthalmic, pulmonary, transmucosal, transdermal, vaginal, otic, nasal, and topical administration.
• parenteral delivery includes intramuscular, subcutaneous, intravenous, intramedullary injections, as well as intrathecal, direct
• intraventricular, intraperitoneal, intralymphatic, and intranasal injections are intraventricular, intraperitoneal, intralymphatic, and intranasal injections.
• a composition as described herein is administered in a local rather than systemic manner, for example, via injection of the compound directly into an organ.
• long acting formulations are administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
• the drug is delivered in a targeted drug delivery system, for example, in a liposome coated with organ-specific antibody.
• the liposomes are targeted to and taken up selectively by the organ.
• the compound as described herein is provided in the form of a rapid release formulation, in the form of an extended release formulation, or in the form of an intermediate release formulation.
• the compound described herein is administered topically.
• compositions described herein are formulated for oral administration.
• compositions described herein are formulated by combining a peptidomimetic macrocycle with, e.g., pharmaceutically acceptable carriers or excipients.
• the compounds described herein are formulated in oral dosage forms that include, by way of example only, tablets, powders, pills, dragees, capsules, liquids, gels, syrups, elixirs, slurries, suspensions and the like.
• pharmaceutical preparations for oral use are obtained by mixing one or more solid excipient with one or more of the peptidomimetic macrocycles described herein, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
• Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as: for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methylcellulose, microcrystalline cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose; or others such as: polyvinylpyrrolidone (PVP or povidone) or calcium phosphate.
• disintegrating agents are optionally added.
• Disintegrating agents include, by way of example only, cross-linked croscarmellose sodium, polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
• dosage forms such as dragee cores and tablets, are provided with one or more suitable coating.
• concentrated sugar solutions are used for coating the dosage form.
• the sugar solutions optionally contain additional components, such as by way of example only, gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
• Dyestuffs and/or pigments are also optionally added to the coatings for identification purposes. Additionally, the dyestuffs and/or pigments are optionally utilized to characterize different combinations of active compound doses.
• Oral dosage forms include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
• push-fit capsules contain the active ingredients in admixture with one or more filler.
• Fillers include, by way of example only, lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
• soft capsules contain one or more active compound that is dissolved or suspended in a suitable liquid. Suitable liquids include, by way of example only, one or more fatty oil, liquid paraffin, or liquid polyethylene glycol.
• stabilizers are optionally added.
• therapeutically effective amounts of at least one of the peptidomimetic macrocycles described herein are formulated for buccal or sublingual administration.
• Formulations suitable for buccal or sublingual administration include, by way of example only, tablets, lozenges, or gels.
• the peptidomimetic macrocycles described herein are formulated for parenertal injection, including formulations suitable for bolus injection or continuous infusion.
• formulations for injection are presented in unit dosage form (e.g., in ampoules) or in multi-dose containers. Preservatives are, optionally, added to the injection formulations.
• pharmaceutical compositions are formulated in a form suitable for parenteral injection as a sterile suspensions, solutions or emulsions in oily or aqueous vehicles.
• Parenteral injection formulations optionally contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
• pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form.
• suspensions of the active compounds are prepared as appropriate oily injection suspensions.
• Suitable lipophilic solvents or vehicles for use in the pharmaceutical compositions described herein include, by way of example only, fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
• aqueous injection suspensions contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
• the suspension contains suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
• the active ingredient is in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
• compositions herein can be administered, for example, once or twice or three or four or five or six times per day, or once or twice or three or four or five or six times per week, and can be administered, for example, for a day, a week, a month, 3 months, six months, a year, five years, or for example ten years.
• the present invention provides novel peptidomimetic macrocycles that are useful in competitive binding assays to identify agents which bind to the natural ligand(s) of the proteins or peptides upon which the peptidomimetic macrocycles are modeled.
• labeled peptidomimetic macrocycles based on GHRH can be used in a binding assay along with small molecules that competitively bind to the GHRH receptor.
• Competitive binding studies allow for rapid in vitro evaluation and determination of drug candidates specific for the GHRH system. Such binding studies may be performed with any of the peptidomimetic macrocycles disclosed herein and their binding partners.
• the invention further provides for the generation of antibodies against the peptidomimetic
• these antibodies specifically bind both the peptidomimetic macrocycle and the precursor peptides, such as GHRH, to which the peptidomimetic macrocycles are related.
• Such antibodies for example, disrupt the native protein-protein interactions, for example, between GHRH and the GHRH receptor.
• the present invention provides methods to activate the GHRH receptor, thereby stimulating production and release of growth hormone, which in turn can increase lean muscle mass or reduce adipose tissue, for example visceral and/or abdominal adipose tissue.
• subject suffering from obesity for example abdominal obesity
• the present invention provides methods for treating muscle wasting diseases that include anorexias, cachexias (such as cancer cachexia, chronic heart failure cachexia, chronic obstructive pulmonary disease cachexia, rheumatoid arthritis cachexia) and sarcopenias, methods for treating lipodystrophies that include HIV lipodystrophy, methods for treating growth hormone disorders that include adult and pediatric growth hormone deficiencies, or methods for treating gastroparesis or short bowel syndrome. These methods comprise administering an effective amount of a compound of the invention to a warm blooded animal, including a human.
• a pharmaceutical composition provided herein used in the treatment of muscle wasting diseases is administered no more frequently than once daily, no more frequently than every other day, no more frequently than twice weekly, no more frequently than weekly, or no more frequently than every other week.
• provided herein are methods for treating adult growth hormone
• a pharmaceutical composition of the invention used in treatment of adult growth hormone deficiency is administered no more frequently than once daily, no more frequently than every other day, no more frequently than twice weekly, no more frequently than weekly, or no more frequently than every other week.
• provided herein are methods for treating pediatric growth hormone
• GHRHR GHRHR
• GH1 congenital malformations involving the pituitary (such as septo-optic dysplasia or posterior pituitary ectopia), chronic kidney disease, intracranial tumors (e.g. in or near the sella turcica, such as craniopharyngioma), damage to the pituitary from radiation therapy to the cranium (for cancers such as leukemia or brain tumors), surgery, trauma or intracranial disease (e.g.
• a composition of the invention used in treatment of pediatric growth hormone deficiency is administered no more frequently than once daily, no more frequently than every other day, no more frequently than twice weekly, no more frequently than weekly, or no more frequently than every other week.
• treatment is defined as the application or administration of a
• therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease or the predisposition toward disease.
• the invention provides peptidomimetic macrocycles and methods of use as described in the items below.
• Item 1 A peptidomimetic macrocycle comprising an amino acid sequence which is at least about 60% identical to an amino acid sequence chosen from the group consisting of the amino acid sequences in Tables 1 , 2 or 4.
• Item 2 The peptidomimetic macrocycle of item 1 , wherein the amino acid sequence of said peptidomimetic macrocycle is at least about 80% identical to an amino acid sequence chosen from the group consisting of the amino acid sequences in Tables 1 , 2 or 4.
• Item 3 The peptidomimetic macrocycle of item 1 , wherein the amino acid sequence of said peptidomimetic macrocycle is at least about 90% identical to an amino acid sequence chosen from the group consisting of the amino acid sequences in Tables 1 , 2 or 4.
• Item 4 The peptidomimetic macrocycle of item 1, wherein the amino acid sequence of said peptidomimetic macrocycle is chosen from the group consisting of the amino acid sequences in Tables 1, 2 or 4.
• Item 5 The peptidomimetic macrocycle of item 1, wherein the peptidomimetic macrocycle comprises a helix.
• Item 6 The peptidomimetic macrocycle of item 1, wherein the peptidomimetic macrocycle comprises an a-helix.
• Item 7 The peptidomimetic macrocycle of item 1, wherein the peptidomimetic macrocycle comprises an ⁇ , ⁇ -disubstituted amino acid.
• Item 8 The peptidomimetic macrocycle of item 1, wherein the peptidomimetic macrocycle comprises a crosslinker linking the a-positions of at least two amino acids.
• Item 9 The peptidomimetic macrocycle of item 8, wherein at least one of said two amino acids is an ⁇ , ⁇ -disubstituted amino acid.
• Item 10 The peptidomimetic macrocycle of item 8, wherein the peptidomimetic macrocycle has the
• each A, C, D, and E is independently a natural or non-natural amino acid
• B is a natural or non-natural amino acid, amino acid analog, Yn ⁇ * , [-NH-L3-CO-],
• Ri and R 2 are independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-;
• R3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R 5 ;
• L is a macrocycle- forming linker of the formula -Li-L 2 -;
• Li and L 2 are independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [-R4-K-R 4 -] n , each being optionally substituted with R 5 ; each R 4 is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;
• each K is O, S, SO, S0 2 , CO, C0 2 , or CONR 3 ;
• each R 5 is independently halogen, alkyl, -OR 6 , -N(R 6 ) 2 , -SR 6 , -SOR 6 , -S0 2 R 6 , -C0 2 R 6 , a fluorescent moiety, a radioisotope or a therapeutic agent;
• each R 6 is independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent;
• R 7 is -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl,
• heterocycloalkyl cycloaryl, or heterocycloaryl, optionally substituted with R 5 , or part of a cyclic structure with a D residue;
• Rg is -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl,
• heterocycloalkyl cycloaryl, or heterocycloaryl, optionally substituted with R 5 , or part of a cyclic structure with an E residue;
• v and w are independently integers from 1-1000;
• u, x, y and z are independently integers from 0-10;
• n is an integer from 1-5.
• Item 11 The peptidomimetic macrocycle of item 1 , wherein the peptidomimetic macrocycle comprises a crosslinker linking a backbone amino group of a first amino acid to a second amino acid within the peptidomimetic macrocycle.
• Item 12 The peptidomimetic macrocycle of item 11, wherein the peptidomimetic macrocycle has the
• each A, C, D, and E is independently a natural or non-natural amino acid;
• B is a natural or non-natural amino acid, amino acid analog, [-NH-L3-CO-],
• Ri and R 2 are independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo- or part of a cyclic structure with an E residue;
• R3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R 5 ;
• Li and L 2 are independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [-R4-K-R 4 -] n , each being optionally substituted with R 5 ;
• each R 4 is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;
• each K is O, S, SO, S0 2 , CO, C0 2 , or CONR 3 ;
• each R 5 is independently halogen, alkyl, -OR 6 , -N(R 6 ) 2 , -SR 6 , -SOR 6 , -S0 2 R 6 , -C0 2 R 6 , a fluorescent moiety, a radioisotope or a therapeutic agent;
• each R 6 is independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent;
• R 7 is -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl,
• heterocycloalkyl cycloaryl, or heterocycloaryl, optionally substituted with R 5 ;
• v and w are independently integers from 1 -1000;
• u, x, y and z are independently integers from 0-10;
• n is an integer from 1 -5.
• Item 13 A method of increasing the circulating level of growth hormone (GH) in a subject comprising administering to the subject a peptidomimetic macrocycle of item 1.
• GH growth hormone
• Item 14 A method of increasing lean muscle mass in a subject comprising administering to the subject a peptidomimetic macrocycle of item 1.
• Item 15 A method of reducing adipose tissue in a subject comprising administering to the subject a peptidomimetic macrocycle of item 1.
• Item 16 A method of treating muscle wasting diseases, including anorexias, cachexias (such as cancer cachexia, chronic heart failure cachexia, chronic obstructive pulmonary disease cachexia, rheumatoid arthritis cachexia) or sarcopenias in a subject comprising administering to the subject a peptidomimetic macrocycle of item 1.
• cachexias such as cancer cachexia, chronic heart failure cachexia, chronic obstructive pulmonary disease cachexia, rheumatoid arthritis cachexia
• sarcopenias in a subject comprising administering to the subject a peptidomimetic macrocycle of item 1.
• Item 17 A method of treating lipodystrophies, including HIV lipodystrophy, in a subject comprising administering to the subject a peptidomimetic macrocycle of item 1.
• Item 18 A method of treating growth hormone disorders, including adult growth hormone deficiency and pediatric growth hormone deficiency, in a subject comprising administering to the subject a peptidomimetic macrocycle of item 1.
• Item 19 A method of treating gastroparesis or short bowel syndrome in a subject comprising administering to the subject a peptidomimetic macrocycle of item 1.
• Item 20 A method of treating muscle wasting diseases, lipodystrophies, growth hormone disorders or gastroparesis/short bowel syndrome in a subject by administering an agonist of the GHRH receptor, wherein the agonist is administered no more frequently than once daily, no more frequently than every other day, no more frequently than twice weekly, no more frequently than weekly, or no more frequently than every other week.
• Item 21 A method of treating muscle wasting diseases, lipodystrophies, growth hormone disorders or gastroparesis/short bowel syndrome in a subject by administering a GHRH analog, wherein the GHRH analog is administered no more frequently than once daily, no more frequently than every other day, no more frequently than twice weekly, no more frequently than weekly, or no more frequently than every other week.
• Item 22 A method of increasing the circulating level of growth hormone (GH) in a subject by administering an agonist of the GHRH receptor, wherein the agonist is administered no more frequently than once daily, no more frequently than every other day, no more frequently than twice weekly, no more frequently than weekly, or no more frequently than every other week.
• Item 23 A method of increasing the circulating level of growth hormone (GH) in a subject by administering a GHRH analog, wherein the GHRH analog is administered no more frequently than once daily, no more frequently than every other day, no more frequently than twice weekly, no more frequently than weekly, or no more frequently than every other week.
• Item 24 The peptidomimetic macrocycle of item 10, wherein Li and L 2 are independently alkylene, alkenylene or alkynylene.
• Item 25 The peptidomimetic macrocycle of item 24, wherein Li and L 2 are independently C3-C 10 alkylene or alkenylene
• Item 26 The peptidomimetic macrocycle of item 24, wherein Li and L 2 are independently C3-C6 alkylene or alkenylene.
• Item 27 The peptidomimetic macrocycle of item 10, wherein Ri and R 2 are H.
• Item 28 The peptidomimetic macrocycle of item 10, wherein Ri and R 2 are independently alkyl.
• Example 1 Peptidomimetic macrocycles of the invention
• Peptidomimetic macrocycles were synthesized, purified and analyzed as previously described and as described below (Schafmeister et al., J. Am. Chem. Soc. 122:5891 -5892 (2000); Schafmeister & Verdine, J. Am. Chem. Soc. 122:5891 (2005); Walensky et al., Science 305 : 1466-1470 (2004); and US Patent No. 7, 192,713). Peptidomimetic macrocycles were designed by replacing two or more naturally occurring amino acids with the corresponding synthetic amino acids. Substitutions were made at i and i+4, and i and i+7 positions.
• Non-natural amino acids (4 equiv) were coupled with a 1 : 1 :2 molar ratio of HATU (Applied Biosystems)/HOBt/DIEA.
• the N-termini of the synthetic peptides were acetylated, while the C-termini were amidated.
• Table 4 shows a list of peptidomimetic macrocycles of the invention prepared.
• amino acids represented as "$" are alpha-Me S5-pentenyl-alanine olefin amino acids connected by an all- carbon i to i+4 crosslinker comprising one double bond.
• “%” are alpha-Me S5-pentenyl-alanine olefin amino acids connected by an all-carbon i to i+4 crosslinker comprising no double bonds (fully saturated alkylene crosslinker).
• Amino acids represented as "$r8” are alpha-Me R8- octenyl-alanine olefin amino acids connected by an all-carbon i to i+7 crosslinker comprising one double bond.
• Amino acids represented as “%r8” are alpha-Me R8-octenyl-alanine olefin amino acids connected by an all-carbon i to i+7 crosslinker comprising no double bonds (fully saturated alkylene crosslinker).
• the designation “isol” or “iso2” indicates that the peptidomimetic macrocycle is a single isomer.
• Amino acids designated as lower case “a” represent D-Alanine.
• Amino acids which are used in the formation of triazole crosslinkers are represented according to the legend indicated below. Stereochemistry at the alpha position of each amino acid is S unless otherwise indicated.
• azide amino acids the number of carbon atoms indicated refers to the number of methylene units between the alpha carbon and the terminal azide.
• alkyne amino acids the number of carbon atoms indicated is the number of methylene units between the alpha position and the triazole moiety plus the two carbon atoms within the triazole group derived from the alkyne.
• hGHRHR human GHRH receptor
• Example 4 Plasma PK/PD study in rats.
• peptidomimetic macrocycles of the invention were studied to determine pharmacokinetic and pharmacodynamic parameters in rats.
• Male Sprague-Dawley rats 300 g, non-fasted, cannulated) were used. The study had three arms: IV administration, SC administration, and SC administration (vehicle control).
• sermorelin a dose level of 3 mg/kg IV/SC bolus was used (dose volume of 3 mL/kg dose and dose concentration of 1 mg/mL).
• the vehicle used was: 10 wt% N, N-Dimethylacetamide, 10 wt% DMSO, 2 wt% Solutol HS 15 in water for injection containing 45 mg/mL (4.5 wt%) Mannitol and 25 mM (0.38 wt%) Histidine (pH 7.5; 320 mOsm/kg).
• the peptide was first dissolved at high concentration in DMA and DMSO before a second dilution in Solutol vehicle.

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