WO1994011396A1 - Agonistes de l'hormone de liberation de l'hormone de croissance - Google Patents

Agonistes de l'hormone de liberation de l'hormone de croissance Download PDF

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Publication number
WO1994011396A1
WO1994011396A1 PCT/US1993/011057 US9311057W WO9411396A1 WO 1994011396 A1 WO1994011396 A1 WO 1994011396A1 US 9311057 W US9311057 W US 9311057W WO 9411396 A1 WO9411396 A1 WO 9411396A1
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Prior art keywords
leu
ser
ala
abu
asp
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PCT/US1993/011057
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English (en)
Inventor
Jozsef Zsigo
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The Administrators Of The Tulane Educational Fund
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Priority to AU56688/94A priority Critical patent/AU5668894A/en
Publication of WO1994011396A1 publication Critical patent/WO1994011396A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • 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

Definitions

  • the present invention relates to peptides having influence on the function of the pituitary gland in humans and other animals.
  • the present invention is directed to a synthetic peptide which promotes the release of growth hormone by the pituitary gland .
  • hypothalamic releasing factors have been characterized for the pituitary hormone thyrotropin (the tripeptide TRF), for the pituitary gonadotropins luteinizing hormone and follicle stimulating hormone (the decapeptide LRF, LH-RH or GnRH) and for the pituitary hormone adrenocorticotropin (the 41 -amino acid polypeptide CRF).
  • GHRHs or GRFs growth hormone releasing hormones or GH-releasing factor
  • novel synthetic peptides of this invention are extremely potent in stimulating the release of pituitary GH in animals, including humans. These synthetic peptides retain their physiological activity in solution for an extended period of time and are resistant to enzymatic degradation in the body. Without in any way limiting the invention or its scope, Applicants wish to express their understanding that this retention of activity in vitro and resistance to in vivo degradation are due to the omega-guanidino lower alkyl group at the terminal 29- position of the peptide and the presence non-coded amino acids in the enzymic cleavage site(s) of the synthetic peptides.
  • the synthetic peptides (abbreviated [PeP]) have the sequence: O -CO-R 2 -R 3 -Ala 4 -lle 5 -Phe 6 -Thr 7 -R 8 -R 9 -R 10 -Arg 11 -R 12 -R 13 -R 14 -R 15 -Gln 16 -R 17 -R l 8 -Ala 19 - Arg 20 -R 21 -R 22 -R 23 -R 24 -R 25 -lle 2 ⁇ -R 27 -R 28 -NH-Q 2 I wherein Q 1 is 3-(5-hydroxyindolyl)-methyl or an omega or alpha-omega substituted alkyl of the structure: z j
  • Y is H, -NH 2 , CH 3 CONH- or CH 3 NH-;
  • Z is H or CH 3 ; m is 1 or 2; and n is 0, 1 , or 2;
  • R 2 is Ala, D-Ala, D-N-Methyl-Ala,Abu or Aib
  • R 3 is Asp, D-Asp, Glu or D-Glu;
  • R 8 is Asn, D-Asn, Ser, D-Ser, or Abu
  • R 9 is Ser, Ala, or Abu R ⁇ o s Tyr, D-Tyr,or 5-HTP
  • R 12 s Lys, D-Lys, Arg or Orn R 13 s Val, He, or Tbg R 14 s Leu or D-Leu R 15 s Gly, Ala, Abu or Tbg R 1 7 s Leu or D-Leu
  • R 21 s Lys, D-Lys, Arg or D-Arg
  • R 22 s Leu, D-Leu, Ala or Abu
  • R 23 s Leu or D-Leu
  • R 24 s Gin or His
  • R 25 s Asp, D-Asp, Glu or D-Glu
  • R 27 s Met, Nle, lie, Leu, Tga or Tba
  • at least one of R 2 , R 8 , R 9 and R 15 is Abu or Aib.
  • the synthetic peptides are synthesized by a suitable method such as by exclusively solid phase techniques, by partial solid-phase techniques, by fragment condensation or by classical solution phase synthesis.
  • the C-terminal residue (here, Q 2 ) is appropriately linked (anchored) to an inert solid support (resin) .
  • an amino acid bearing protecting groups for its alpha amino group (and, where appropriate, for its amino acid side chain) is coupled in the C- > N direction.
  • the alpha amino protecting group is removed from this newly added amino acid residue and the next amino acid (suitably protected) is added, and so forth.
  • the N-terminal protecting groups are removed after each residue is added, but the side chain protecting groups, e.g., X 2 , X 3 etc. in formula [PR][PeP] below are not yet removed.
  • the peptide is cleaved from the support and then freed from any side chain protecting group(s) under conditions that are minimally destructive towards residues in the sequence. This must be followed by a careful purification and scrupulous characterization of the synthetic product, so as to ensure that the desired structure is indeed the one obtained.
  • alpha amino function of the amino acids during the coupling step with an acid or base sensitive protecting group.
  • protecting groups should have the properties of being stable in the conditions of peptide linkage formation, while being readily removable without destruction of the growing peptide chain or racemization of any of the chiral centers contained herein.
  • Suitable alpha amino protecting group are Boc and Fmoc.
  • the synthetic peptides of Formula I may be formulated in pharmaceutical dosage form and administered to humans or animal for therapeutic or diagnostic purposes. More particularly, the peptides may be used to promote the growth of warm-blooded animals, as, in humans, to treat human growth deficiency by stimulating in vivo synthesis and/or release of endogenous GH; to treat certain physiological conditions such as severe growth retardation due to chronic renal insufficiency; to offset certain effects of aging, e.g., reducing loss of muscle and bone loss; to accelerate healing and tissue repair; to improve feed utilization, thereby increasing lean/fat ratio favoring muscle gain at the cost of fat; and also to enhance milk production in lactating cattle. Further, the synthetic peptides may be used in a method to ascertain endogenous physiological ability to produce hGH and a diagnostic kit for carrying out this method.
  • Figure 1 is a graphical representation of selected data from Table 4 in Example XX herein, where "A” indicates the peptide of Example XVI -- i.e., (Dat 1 , Abu 2 , Ser 8 , Abu 15 , NIe 27 , Agm 29 )hGH-RH( 1 -29) and "B” indicates the peptide of Example XI -- i.e., (Dat 1 , Aib 2 , Ser 8 , Ala 15 , NIe 27 , Agm 29 )hGH-RH( 1 -29) .
  • NIe norleucine
  • Nva norvaline
  • Abu alpha amino butyric acid
  • Aib alpha iso-butyric acid
  • Tba alpha-t-butylalanine
  • Tbg alpha-t-butylglycine
  • 5-Hiaa 5- Hydroxy-indolylacetic acid
  • C-MeTyr is meant 1 -methyl-DL-Tyrosine
  • N- MeAla and N-MeTyr are meant N-methyl-alanine and N-methyl-tyrosine respectively.
  • amino acid sequences of the synthetic peptides are numbered to in correspondence with the amino acid residues in hGH-RH( 1 -29) . That is, the Ala 4 and R 8 of the Formula I peptide occupy the same position in the sequence as the Ala 4 and R 8 residues in hGH-RH( 1 -29) .
  • N-terminal of a peptide is placed to the left, and the C-terminal to the right is also followed herein.
  • the synthetic peptides of Formula I have neither an N nor a C terminal since the Q 1 or Q 2 moieties, at what would otherwise be the N and C termini, lack an N- and a C- terminus respectively.
  • the terms N- and C-terminal used with respect to the synthetic peptides of Formula I mean Q 1 and Q 2 .
  • the synthetic peptides of the present invention have the sequence:
  • R 27 , R 28 and Q 2 are as defined above.
  • at least one of R 2 , R 8 , R 9 and R 15 is Abu or Aib.
  • Q 1 -CO moieties may when taken together form an amino acid residue: thus, when in Q 1 m is 1 , n is 1 and y is NH 2 , Q ⁇ CO forms tyrosyl ("Tyr-"). Even when the Q 1 functional groups do not cause Q 1 to form an amino acid with CO-, the two constituents of the Formula I peptide may together form a readily recognized group. Thus, when in m is 1 , n is 1 and Y and Z are H, Q -CO together form 3-(4-hydroxyphenyl)propionyl, known as des-amino-Tyr (or "Dat”) .
  • the Q 1 -CO moieties may be taken as a unitary moiety R 1 and replaced in the Formula I peptides by abbreviations such as Tyr 1 , Dat 1 , etc. as appropriate.
  • the NH-Q 2 moieties may be taken as a unitary moiety R 29 when together they form a single recognized functional group.
  • R 2 is Ala, D-Ala, Abu or Aib
  • R 8 is Asn, Ser, ,or Abu
  • R 9 is Ser, Ala, or Abu
  • R 15 is Abu,Ala or Gly
  • R 27 is Met, Tba, or NIe
  • R 28 is Ser or Asp
  • Especially preferred embodiments include peptides of Formula I in which Q 1 - CO is Dat, Ac-Tyr, N-Me-Tyr, C-Me-Tyr and Tyr; and NH-Q 2 is Agm. Further preferred embodiments include peptides of Formula I in which Q 1 -CO is Dat or Tyr; R 15 is Abu; R 27 is NIe; R 28 is Asp; and NH-Q 2 form Agm.
  • n 1
  • Y and Z are H, so that Q ⁇ CO is Dat;
  • R 2 is Aib,
  • R 8 is Ser
  • R 9 is Ser
  • R 15 is Ala
  • R 27 is NIe
  • R 28 is Ser;
  • Q 2 p is 4.
  • the peptide has the formula:
  • the peptides are synthesized by a suitable method such as by exclusively solid phase techniques, by partial solid-phase techniques, by fragment condensation or by classical solution phase synthesis.
  • a suitable method such as by exclusively solid phase techniques, by partial solid-phase techniques, by fragment condensation or by classical solution phase synthesis.
  • the techniques of exclusively solid-phase synthesis are set forth in the textbook "Solid Phase Peptide Synthesis", J.M. Stewart and J.D. Young, Pierce Chem. Company, Rockford, 1 1 1 . , 1984 (2nd. ed.), G. Barany and R.B. Merrifield, "The Peptides", Ch. 1 , 1 -285, pp. 1979, Academic Press, Inc., and M. Bodanszky, "Principles of Peptide Synthesis", SpringerVerlag, 1984.
  • the synthetic peptides of Formula I are preferably prepared using solid phase synthesis, such as that generally described by Merrifield, J.Am.Chem.Soc, 85, p. 2149 (1 963), although other equivalent chemical syntheses known in the art can also be used as previously mentioned.
  • the moiety which forms the C or N-terminal group of the resulting peptide is joined to a polymeric resin support phase via a chemical link.
  • Amino acid residues or oligopeptide fragments are then added sequentially to the alpha amino functional group of the C or N-terminal moiety until the desired peptide sequence is obtained . Because the amino acid residues or oligopeptide fragments are added to the C-terminal Q 2 group here, growth of the synthetic peptides of Formula I begins at the C terminal and progresses toward the N- terminal. When the desired sequence has been obtained, it is removed from the support phase.
  • the peptides of Formula I may be synthesized on a variety of support phases. These support phases may be amino or hydroxy resins such as amino- methyl resins (suitably 1 % cross linked), benzhydrylamine resins (suitably 2% cross linked) p-methylbenzhydrylamine resins (suitably 2% cross linked) and the like. It is generally preferred that the support phase [SP] is an amino type resin of the formula: H 2 N-CH 2 - ⁇ -[Pm] IX where [Pm] is a polymeric substrate.
  • the initial material joined to the support phase is suitably the C-terminal Q 2 group.
  • the adjacent NH functional group is already affixed to Q 2 .
  • Agm or 4-guanidino butylamine are especially preferred .
  • the NH-Q 2 group is joined to the support phase via a stable but readily cleavable bridging group. It has been found that such a bridge may be readily provided by a sulfonyl phenoxy acetyl moiety.
  • Boc-NH-Q 2 -S0 2 - ⁇ -(M ⁇ )O-CH 2 -CO-[SP] or Fmoc-NH-Q 2 -S0 2 -[ ⁇ ]-(M s )O-CH 2 -CO-[SP] constitute the material joined to the support phase for the synthetic sequence. They are prepared and linked to the support phase as follows.
  • the protected aminoalkyl guanidino-sulfonyl phenoxyacetic acid is then coupled to the support phase [SP] by the action of a diloweralkyl carbodiimide or BOP on:
  • Boc-NH-Q 2 -S0 2 - ⁇ -0-CH 2 -CO-[SP] which represents Q 2 linked to the polymeric substrate support phase via the sulfonyl phenoxy acetyl moiety, more fully expressed as:
  • the coupling is carried out using any of the known dialkyi carbodiimide coupling procedures.
  • diisopropyl-carbodiimidein the presence of 1 -hydroxybenzotriazolehydrate in dimethylformamide at ambient temperatures.
  • N,N'dicyclohexyl-carbodiimide there may be employed N,N'dicyclohexyl-carbodiimide.
  • the aforesaid Boc- aminoalkyl guanidino-sulfonyl-phenoxyacetic acid is mixed with the carbodiimide in 2 to 1 molar ratio, the formed N,N'dicyclohexyl urea is removed by filtration and the resulting solution is added to the support phase.
  • the protected aminoalkyl guanidinosulfonyl phenoxyacetic acid is mixed with equimoiar BOP and dissolved in DMF containing twice molar NMM in 10% concentration.
  • the activation can be enhanced in the presence of 1 -3 molar excess of HOBt yielding an efficient coupling rate to the solid support preferably to an aminomethyl resin at room temperature.
  • N- benzyloxycarbonyl chloride is used in the presence of an aqueous alkali, suitably sodium hydroxide, preferably at about 4N under agitation and cooling to between about 5 and about 1 5 °C.
  • an aqueous alkali suitably sodium hydroxide, preferably at about 4N under agitation and cooling to between about 5 and about 1 5 °C.
  • sodium hydrogen carbonate treatment the protected product is precipitated and thereafter is reacted with an aryl-sulfonyl chloride as previously.
  • the Fmoc procedure comprises the sequential steps of reacting N-benzyloxy carbonyl chloride with NH 2 -Q 2 in the presence of a base to form Cbz-NH-Q 2 , reacting said Cbz-NH-Q 2 with a substituted arylsulphonyl halide in the presence of a strong aqueous base and removing the Cbz group by catalytic hydrogenation.
  • the substituted arylsulphonyl halide has the formula:
  • Hal-S0 2 -[ ⁇ ](M s )-O-CH 2 -COOH wherein Hal is chloro or bromo; M s is lower alkyl or lower alkoxy of 1 to 5 carbon atoms located at the positions of the phenyl nucleus other than 1 and 4; and s is 1 -3.
  • the reaction yields H 2 N-Q 2 -S0 2 -[ ⁇ ](M s )-O-CH 2 -COOH.
  • the product is reacted with Fmoc-CI in the presence of a base to yield the corresponding Fmoc protected aminoalkyl guanidino-sulfonyl aryloxy acetic acid, i.e. Fmoc-NH-Q 2 -S02-[ ⁇ ](M ⁇ )-O-CH 2 -COOH.
  • the arylsulfonyl halide should preferably carry between 1 and 3 M substituents which may be alkyl or lower alkoxy, suitably methyl or methoxy.
  • substituents which may be alkyl or lower alkoxy, suitably methyl or methoxy.
  • Boc procedure it is desirable to substitute the phenyl moiety with up to three alkyl or alkoxy groups. These groups weaken the strength of sulfonamide bridge and enable it to be cleaved by trifluoroacetic acid, once all the amino acid residues have been added and one wishes to remove the intermediate from the support phase.
  • This reaction is carried out in the presence of a strong but dilute aqueous base, suitably about 1 N sodium hydroxide.
  • the reaction mixture is acidified and the product extracted, suitably with a water immiscible organic solvent such as ethyl acetate.
  • the extract is then concentrated and hydrogenated, suitably at atmospheric pressure and room temperature in the presence of a catalyst such as palladium on charcoal in a reduction inert solvent. If ethyl acetate is used as the extractant, this may be used as the hydrogenation solvent. 3. Stepwise Addition of Amino Acid Residues.
  • the peptide itself may then suitably be built up by solid phase synthesis in the conventional manner.
  • the alpha amino-protecting group of the next amino acid residue is protected by Boc or Fmoc to prevent reaction between the N-terminal of R 28 while this is being reacted with NH 2 -Q 2 .
  • Boc or Fmoc are attached to the NH 2 -Agm or H 2 N-Q 2 -SO 2 -[ ⁇ ](M ⁇ )- O-CH 2 -COOH respectively, as described above.
  • the amino acid residues of the synthetic peptides are added sequentially . Because the peptide is constructed from the C-terminal, the residues are added in reverse numerical order, i.e., R 28 , R 27 , and so forth.
  • the protected amino acids are coupled sequentially, with Q 1 finally being added.
  • some may be coupled to one another in a separate vessel and added as an oligopeptide in the solid phase reaction.
  • the selection of an appropriate coupling reagent is within the skill of the art. Particularly suitable as coupling reagents are N,N'-diisopropyl carbodiimide (DIC) or the BOP carboxyl activating reagent.
  • Each protected amino acid or amino acid sequence is introduced into the solid phase reaction in about a three-fold molar excess, with respect to NH 2 -Q 2 , etc., and the coupling may be carried out in as medium such as DMF: CH 2 CI 2 (1 : 1 ) or in DMF or CH 2 CI 2 alone. In cases where incomplete coupling occurs, the coupling procedure is repeated before removal of the alpha amino protecting group prior to the coupling of the next amino acid. The success of the coupling reaction at each stage of the synthesis is preferably monitored by the ninhydrin reaction.
  • I peptide is cleaved from the support phase at the sulfonyl group. If the Boc protocol is utilized, then the cleaving agent is anhydrous hydrofluoric acid; whereas, in the Fmoc protocol the cleaving agent is trifluoroacetic acid .
  • Removal of the intermediate peptide from the resin support is performed by treatment with a reagent such as liquid hydrogen fluoride or trifluoroacetic acid . This also cleaves all remaining side chain protecting groups X 3 , X 7 , X 1 1 , X 12 and and the anchoring guanidinosulfo bond, if present, and also the alpha amino or alpha-hydroxyl protecting group X 1 . These groups have different values depending on whether Boc or Fmoc protocols are used .
  • side chain amino protecting group is not critical except that generally one is chosen which is not removed during deprotection of the alpha amino groups during the synthesis.
  • the protecting group preferably retains its protecting properties and is not split off under coupling conditions
  • the protecting group should be stable to the reagent, is preferably stable under the coupling reaction conditions selected for removing the alpha amino protecting group at each step of the synthesis and
  • the side chain protecting group must be removable upon the completion of the synthesis of the desired amino acid sequence, under reaction conditions that will not undesirably alter the peptide chain.
  • the side chain protecting groups are attached to the amino acid residues by steps well known in the art.
  • M is hydrogen, lower alkyl or lower alkoxy of 1 to 5 carbon atoms, suitably methyl or methoxy, s is 1 -3
  • [SP] is an amino type resin.
  • [PR][PeP] has the structure:
  • X 1 is either hydroxyl, or an amino protecting group or nil, depending on the meaning of Y in Formula II (the formula for one of the Q 1 moieties).
  • X 3 is a suitabl ister-forming protecting group for the carboxyl group of Asp or
  • Glu In the Boc protocol, it may form cyclohexyl esters; in the Fmoc protocol tert- butyl esters are preferred.
  • X 7 is a suitable protecting group for the hydroxyl group of Thr or Ser, such as tert-butyl, Bzl.
  • the preferred protecting group in the Boc protocol is Bzl and in the
  • Fmoc protocol it is tert-butyl.
  • X 10 is a suitable protecting group for the phenolic hydroxyl group of Tyr or N- MeTyr, such as tert-butyl, Bzl, 4Br-Cbz and 2, 6-dichlorobenzyl (DCB) .
  • the preferred protecting group in the Boc protocol is 2, 6-dichlorobenzyl and in the Fmoc protocol it is tert-butyl.
  • X 1 1 is a suitable protecting group for the guanidino group of Arg, such as nitro, Tos, Pmc, Mtr; in the Boc protocol Tos is the preferred group and 2,2,5,7,8- pentamethyl chroman-6-sulfonyl in the Fmoc protocol.
  • X 12 is a suitable protecting group for the side chain amino group of Lys. Illustrative of suitable side chain amino protecting groups are 2-chloro- benzyloxycarbonyl (2-CI-CBz) and tert-butyloxycarbonyl (Boc). In the Boc protocol, 2-chlorobenzyloxycarbonyl is the preferred protecting group and in the Fmoc protocol Boc is thus utilized .
  • X 24 is hydrogen or a protecting group for the imidazole nitrogen of His, such as Tos in the Boc or Trt in the Fmoc protocol.
  • the products of the present invention may be utilized to promote the growth of warm-blooded animals (e.g., humans) and also enhance the milk production of females of milk producing mammals, suitably but not exclusively goats and cows, preferably cows.
  • warm-blooded animals e.g., humans
  • milk production of females of milk producing mammals suitably but not exclusively goats and cows, preferably cows.
  • the peptides of the invention may be administered in the form of pharmaceutically acceptable, nontoxic salts, such as acid addition salts.
  • acid addition salts are hydrochloride, hydrobromide, sulphate, phosphate, fumarate, gluconate, tannate, maleate, acetate, citrate, benzoate, succinate, alginate, pamoate, malate, ascorbate, tartrate, and the like.
  • the compounds of the present invention are suitably administered to the subject humans or animals s.c, i.m., or i.v; intranasally or by pulmonary inhalation; or in a depot form (e.g., microcapsules, microgranules, or cylindrical rod like implants) formulated from a biodegradable suitable polymer (such as D,L- lactide-co-glycolide), the former two depot modes being preferred .
  • a biodegradable suitable polymer such as D,L- lactide-co-glycolide
  • Other equivalent modes of administration are also within the scope of this invention, i.e., continuous drip, depot injections, infusion pump and time release modes such as microcapsules and the like.
  • Administration is in any physiologically acceptable injectable carrier, physiological saline being acceptable, though other carriers known to the art may also be used .
  • the dosage level should be between 0.2 and 2 micrograms/kg body weight per injection, except for depot form where the amount injected would be calculated to last from about 1 5 to about 30 days or longer. These dosage ranges are merely preferred . Administration of non-depot forms may be between 1 and 4 times per day. It is convenient to inject the animal whenever it is milked .
  • GH growth hormone
  • synthetic hGH is currently approved only for the treatment of growth failure due to a lack of adequate endogenous growth hormone, but hGH has also been used to treat short children who are not classically GH-deficient such as girls with Turner's syndrome; prepubertal children with chronic renal insufficiency and severe growth retardation; and children with non-GH deficient short stature.
  • hGH-RH a 50-100 microgram dose of GH-RH
  • a 50-100 microgram dose of a GH-RH analog of Formula I a 50-100 microgram dose of a GH-RH analog of Formula I
  • the assay means may be any conventional means which will indicate the quantitative amount of hGH present in a blood sample drawn from the patient.
  • the concentration of GH in serum is determined using standard radioimmunoassay
  • RIA procedures as set forth in e.g., Miles I.E.M. et al., Lancet jj, 492-493 (1968) or O'Dell W et al., J.Lab.Clin.Med. 70, 973-80 (1967) .
  • the test is used as follows. First, the GH-RH dose is administered. Thirty minutes later, a blood sample is taken for RIA of GH.
  • Various commercially available kits e.g., Nichols Institute of Diagnostics, San Juan Capistrano, CA
  • reference preparations of hGH e.g., NIAMDD-hGH-RP- 1
  • NIAMDD-hGH-RP- 1 can be used for RIA of
  • a diagnostic kit for testing endogenous physiological ability to produce GH may incorporate a 50-100 microgram dose of GH-RH; a 50-100 microgram dose of GH-RH analog peptide of Formula I; and means for assaying the GH response evoked by each dose.
  • short stature in children may result from many causes, none of which are immediately apparent.
  • Use of the diagnostic test on all children with this condition would greatly clarify the cause of short stature. Such a widespread screening test would also provide earlier indications for desirable treatment.
  • the invention further includes a method for ascertaining the endogenous physiological ability to produce hGH and a diagnostic kit for carrying out this method.
  • Glucocorticoids are potent inhibitors of linear growth in man and growth suppression is a well known risk of long term treatment of asthmatic children with steroids. Thus stunted growth is an important consequence of chronic administration of glucocorticoids in childhood.
  • the inhibition of GH secretion is due in some extent to the fact that chronic administration of glucocorticoids suppresses GHRH. This inhibition occurs at the level of the hypothalamus or above and in this situation only the treatment with GHRH agonists will stimulate linear growth.
  • Growth hormone is a potent anabolic hormone that enhances protein synthesis and nitrogen retention and chronic administration of agonistic analogs of GHRH increase the endogenous growth hormone secretion.
  • the therapy with GHRH agonistic analogs has uses in other areas of medicine such as catabolic states causing accelerated weight loss; tissue repair in patients with severe body surface burn, in accelerating healing of nonunion fractures and in some cases of cardiac failure.
  • GH-deficient children respond to GH-RH( 1 -40), GH-RH(1 -29) or GH-RH(1 - 44), with an increase in growth.
  • GH-RH(1-29)NH 2 given subcutaneously twice a day promoted linear growth in approximately 50% of a group of GH-deficient children (Ross et al, cited above). A small group of severely GH-deficient children will respond to
  • Geriatric Patients To reduce the loss of muscle, bone and skin mass and lessen the increase of body fat that normally accompanies the aging process. Catabolic states Wound healing Delayed healing of fractures Osteoporosis Obesity
  • GHRH agonists could be used during and after space flights to counteract the decrease in GH secretion. Weightlessness of space flight significantly decreases the release of growth hormone. This finding could explain the bone loss and muscle weakness many astronauts experience after prolonged space flights.
  • Deblocking and neutralization are preferably carried out in accordance with
  • the couplings are preferably carried out as set out in Schedule A for coupling: SCHEDULE A Coupling
  • BOC is used for the alpha amino protection.
  • Benzyl ether is used as the hydroxyl side chain protecting group for Ser and Thr.
  • the phenolic hydroxyl group of Tyr is protected with DCB and the side chain carboxyl group of Asp is protected in the form of the cyclohexyl ester.
  • Tos is used to protect the guanidino group of Arg and 2-CI-Cbz is used as the protecting group for the Lys side-chain. Due to the decreased reaction rate of the 3-(4-hydroxyphenyl)propionic acid N- hydroxy-succinimide ester, a prolonged coupling time 60 minutes was used.
  • cleave and deprotect the protected peptide-resin In order to cleave and deprotect the protected peptide-resin, it is treated with 1 ml m-cresol and 10 ml hydrogen fluoride (HF) per gram of peptide resin, at 0°C for 45 min. After elimination of the HF under high vacuum and evaporating the traces of the scavenger, the cleaved peptide and resin remainder is washed with dry diethyl ether and ethyl acetate. The peptide is then extracted with 50% aqueous acetic acid, separated from the resin by filtration and lyophilized.
  • HF hydrogen fluoride
  • the crude peptide is purified using either a Beckman Prep-350 preparative HPLC system (with a Beckman Type 1 63 variable wavelength UV detector) or a Beckman semipreparative HPLC system (with a Beckman 420 Gradient Controller, two 1 14M Solvent Delivery Modules, a 1 65 Variable Wavelength Detector and a Kipp and Zonen BD41 Recorder.
  • a Beckman Prep-350 preparative HPLC system with a Beckman Type 1 63 variable wavelength UV detector
  • a Beckman semipreparative HPLC system with a Beckman 420 Gradient Controller, two 1 14M Solvent Delivery Modules, a 1 65 Variable Wavelength Detector and a Kipp and Zonen BD41 Recorder.
  • separations are achieved on a 41 .5 x 1 50 mm.
  • DYNAMAX column packed with spherical C1 8 silica gel 300 A pore size, 1 2 um Particle size) (RAININ Inc., Co. , Woburn, MA
  • purification can be performed on a VYDAC C1 8 reversed phase column (10 x 250 mm., 300 A pore size, 5 um particle size) . Gradient elution is used .
  • Solvent A consists of 0.1 % aqueous TFA and solvent B is 0. 1 % TFA in 70% aqueous acetonitrile.
  • the column eluate is monitored at 220 nm and 280 nm.
  • the peptide is judged to be substantially (95%) pure by using a Hewlett-
  • Packard HP-1090 liquid chromatograph The peptides are chromatographed on a 4.6 x 250 mm. W-Porex 5 um C1 8 column (Phenomenex, Collinso Paios Verdes, CA) at a flow rate of 1 .2 ml/min. with a mixture of Solvent A and Solvent B using a gradient (from 40 to 70% B in 30 min) .
  • X 1 is t-butyl ether
  • X 3 is t-butyl ester
  • X 11 is 2,2,5,7,8-pentamethyl-chroman-6- sulphonyl
  • X 12 is Boc.
  • the protected peptide produced as above is utilized as starting material.
  • the cleavage of the peptide from the solid support and the removal of side chain protecting groups was carried out simultaneously by the reaction of the protected peptide resin with a mixture of TFA(90%)-thioanisol(5%)-ethandithiol (2.5%)- water(2.5%) at a temperature 35-40°C for 2-3 hours (10 ml liquid per each gram of resin was used).
  • the reagent was removed on centrifugation in high vacuo at room temperature. The remaining material was washed with ether.
  • the peptide is then extracted with 50% aqueous acetic acid, separated from the resin by filtration and lyophilized. After lyophilization the crude peptide was subjected to purification as described in Example VIII.
  • Dat 1 -Ala 2 -As p 3 (CO Hx )-Ala 4 -lle 6 -Phe 6 -Thr 7 (Bzl)-Abu 8 -Ser 9 (Bzl)-Tyr 10
  • DCB Arg 1 1 (Tos)-Lys 12 (2-CI-Cbz)-Val 13 -Leu 1 -Ala 15 -Gln 16 -Leu 17 -Ser 18 (Bzl)-Ala 19 -Arg 20
  • Tos)- Lys 21 (2-CI-Cbz)-Leu 2 -Leu 23 -Gln 24 -Asp 25 (OChx)-lle 6 -Nle 27 -Ser 28
  • Bzl)-NH-(CH 2 ) 4 -NH- C(NH 2 ) N-[SPA]-[SP] which is then similarly converted to the desired peptide in accordance with the procedure of Example VIII.
  • the compounds of the present invention were tested both in vitro and in vivo. Growth hormone releasing activities in vitro are summarized in Table 1 . in vivo effect of the compounds on the release of GH in rats after intravenous (i.v.) and subcutaneous (sc.) injections are given in Tables 2 and 3 and Tables 4 and 5, respectively.
  • Dawley rats were anesthetized with pentobarbital (6 mg./100/g., b.w.), injected i.p.; 20 minutes after the injection of pentobarbital, blood samples were taken from the jugular vein (pretreated level) and immediately thereafter hGH-RH ( 1 - 29)NH 2 (as a control) or hGH-RH(1 -29)NH 2 analogs were injected i.v. Blood samples were taken from the jugular vein 5 and 15 minutes after the injection. The blood samples were centrifuged, plasma was removed and the GH level was measured by RIA.
  • the analogs Following intravenous administration, the analogs gave growth hormone levels greater than those from hGHRH alone. The effect was long lasting which indicates that the analogs have not only higher receptor affinity but increased peptidase resistance too.
  • irj . vitro activity depends primarily on binding capacity of the peptide to its receptor, whereas .in vivo potency relies also on favorable transport properties, suitable binding to plasma proteins and metabolic stability.
  • the above findings therefore indicate that the analogs tested are resistant to local degradation at the injection site and they may also be less susceptible to enzyme degradation in the blood stream and/or have more affinity for GH-RH receptors than hGHRH(1-29)NH 2 .

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Abstract

Peptides synthétiques correspondant à la séquence: Q?1-CO-R2-Asp3-Ala4-Ile5-Phe6-Thr7-R8-R9-Tyr10-Arg11-Lys12-Val13-Leu14-R15-Gln16-Leu17-Ser18-Ala19-Arg20-Lys21-Leu22-Leu23-Gln24-Asp25-Ile26-R27-R28-NH-Q2¿ dans laquelle Q1 est un alkyle à substitution oméga ou alpha-oméga de structure (I), où: [Ζ] représente phényle; Y représente H, -NH¿2?, CH3CONH- or CH3NH; Z représente H ou CH3; m vaut 1 ou 2; n vaut 0, 1 ou 2; R?2¿ représente Ala, Abu ou Aib; R8 représente Asn, Ser, Ala, ou Abu; R9 représente Ser, Ala or Abu; R15 représente Gly, Ala, ou Abu; R27 représente Met, Tba or Nle; R28 représente Asp ou Ser; et Q2 représente un groupe oméga-guanidino-alkyle inférieur correspondant à la formule: -(CH¿2?)p-NH-C(NH2) = NH dans laquelle p vaut 2-6 et au moins un élément parmi R?2, R8, R9 et R15¿ représente Abu ou Aib. On décrit également les sels d'addition de ces peptides avec les bases ou acides organiques ou inorganiques acceptables en pharmacologie; une forme de dosage pharmaceutique comprenant ledit peptide et un excipient; des procédés de traitement des déficits de croissance chez l'homme, consistant à administrer ces peptides; un test diagnostique; et un procédé permettant de s'assurer de la capacité physiologique endogène de produire l'hormone hGH.
PCT/US1993/011057 1992-11-13 1993-11-10 Agonistes de l'hormone de liberation de l'hormone de croissance WO1994011396A1 (fr)

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996022782A1 (fr) * 1995-01-24 1996-08-01 The Administrators Of The Tulane Educational Fund Nouveaux agonistes extremement puissants de l'hormone liberant l'hormone de croissance
WO1997040071A1 (fr) * 1996-04-19 1997-10-30 Novo Nordisk A/S Composes capables de liberer une hormone de croissance
US5990084A (en) * 1996-04-19 1999-11-23 Novo Nordisk A/S Compounds with growth hormone releasing properties
US6610496B1 (en) * 1998-07-08 2003-08-26 University Of Maryland, College Park Prediction of growth performance and composition in animals, including cattle, from response to growth hormone releasing hormone
US8192719B2 (en) 2006-02-18 2012-06-05 Aeterna Zentaris Gmbh Methods and kits to diagnose growth hormone deficiency by oral administration of EP 1572 or EP 1573 compounds
WO2013095903A1 (fr) 2011-12-21 2013-06-27 University Of Miami Nouveaux analogues gh-rh ayant des effets agonistes puissants
EP2616095A2 (fr) * 2010-09-16 2013-07-24 University Of Miami Accélération de la cicatrisation des plaies par l'hormone de libération de l'hormone de croissance et ses agonistes
EP2664343A2 (fr) 2008-11-17 2013-11-20 Syntaxin Limited Suppression du cancer
US8980249B2 (en) 2010-06-03 2015-03-17 University Of Miami Agonists of growth hormone releasing hormone as effectors for survival and proliferation of pancreatic islets
US9393271B2 (en) 2012-12-21 2016-07-19 University Of Miami GHRH agonists for islet cell transplantation and function and the treatment of diabetes
US9855312B2 (en) 2012-12-21 2018-01-02 University Of Miami GHRH agonists for the treatment of ischemic disorders
EP3473643A1 (fr) 2008-06-12 2019-04-24 Ipsen Bioinnovation Limited Protéines de fusion pour leur utilisation dans le traitement du cancer
EP3590956A1 (fr) 2008-06-12 2020-01-08 Ipsen Bioinnovation Limited Suppression de maladies neuroendocrines
CN111407884A (zh) * 2019-06-24 2020-07-14 浙江大学 促生长激素释放激素激动剂ghrh-a在制备抗衰老药物中的用途

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Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996022782A1 (fr) * 1995-01-24 1996-08-01 The Administrators Of The Tulane Educational Fund Nouveaux agonistes extremement puissants de l'hormone liberant l'hormone de croissance
US5792747A (en) * 1995-01-24 1998-08-11 The Administrators Of The Tulane Educational Fund Highly potent agonists of growth hormone releasing hormone
WO1997040071A1 (fr) * 1996-04-19 1997-10-30 Novo Nordisk A/S Composes capables de liberer une hormone de croissance
US5990084A (en) * 1996-04-19 1999-11-23 Novo Nordisk A/S Compounds with growth hormone releasing properties
US6610496B1 (en) * 1998-07-08 2003-08-26 University Of Maryland, College Park Prediction of growth performance and composition in animals, including cattle, from response to growth hormone releasing hormone
US8192719B2 (en) 2006-02-18 2012-06-05 Aeterna Zentaris Gmbh Methods and kits to diagnose growth hormone deficiency by oral administration of EP 1572 or EP 1573 compounds
EP3473643A1 (fr) 2008-06-12 2019-04-24 Ipsen Bioinnovation Limited Protéines de fusion pour leur utilisation dans le traitement du cancer
EP3590956A1 (fr) 2008-06-12 2020-01-08 Ipsen Bioinnovation Limited Suppression de maladies neuroendocrines
EP3241560A1 (fr) 2008-11-17 2017-11-08 Ipsen Bioinnovation Limited Suppression du cancer
EP2664343A2 (fr) 2008-11-17 2013-11-20 Syntaxin Limited Suppression du cancer
US8980249B2 (en) 2010-06-03 2015-03-17 University Of Miami Agonists of growth hormone releasing hormone as effectors for survival and proliferation of pancreatic islets
EP2616095A4 (fr) * 2010-09-16 2014-03-19 Univ Miami Accélération de la cicatrisation des plaies par l'hormone de libération de l'hormone de croissance et ses agonistes
EP2616095A2 (fr) * 2010-09-16 2013-07-24 University Of Miami Accélération de la cicatrisation des plaies par l'hormone de libération de l'hormone de croissance et ses agonistes
US9079974B2 (en) 2011-12-21 2015-07-14 The University Of Miami GH-RH analogs with potent agonistic effects
WO2013095903A1 (fr) 2011-12-21 2013-06-27 University Of Miami Nouveaux analogues gh-rh ayant des effets agonistes puissants
US9393271B2 (en) 2012-12-21 2016-07-19 University Of Miami GHRH agonists for islet cell transplantation and function and the treatment of diabetes
US9855312B2 (en) 2012-12-21 2018-01-02 University Of Miami GHRH agonists for the treatment of ischemic disorders
US10555987B2 (en) 2012-12-21 2020-02-11 University Of Miami GHRH agonists for the treatment of ischemic disorders
CN111407884A (zh) * 2019-06-24 2020-07-14 浙江大学 促生长激素释放激素激动剂ghrh-a在制备抗衰老药物中的用途
CN111407884B (zh) * 2019-06-24 2021-12-07 浙江大学 促生长激素释放激素激动剂ghrh-a在制备抗衰老药物中的用途

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