WO1994007519A1 - Peptides liberant l'hormone de croissance - Google Patents

Peptides liberant l'hormone de croissance Download PDF

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Publication number
WO1994007519A1
WO1994007519A1 PCT/US1993/008805 US9308805W WO9407519A1 WO 1994007519 A1 WO1994007519 A1 WO 1994007519A1 US 9308805 W US9308805 W US 9308805W WO 9407519 A1 WO9407519 A1 WO 9407519A1
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WIPO (PCT)
Prior art keywords
phe
ala
trp
lys
gln
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PCT/US1993/008805
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English (en)
Inventor
Anita Hong
William Francis Huffman
Michael Lee Moore
Tobias Oregon Yellin
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Smithkline Beecham Corporation
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Publication date
Application filed by Smithkline Beecham Corporation filed Critical Smithkline Beecham Corporation
Priority to EP93921654A priority Critical patent/EP0663834A4/fr
Priority to JP6509101A priority patent/JPH08502250A/ja
Publication of WO1994007519A1 publication Critical patent/WO1994007519A1/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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • This invention relates to compounds which cause enhanced release of growth hormone in animals, pharmaceutical compositions containing the compounds and methods of using the compounds.
  • GH Growth Hormone
  • a mediator released from the pituitary causes physiologic and metabolic changes in the tissues of the body. Effects such as tissue growth, increased rate of protein synthesis, increased mobilization and use of fatty acids for energy, and decreased use of carbohydrates, have been attributed to GH secretion.
  • a deficiency of GH has been associated with certain types of medical disorders, such as certain instances of dwarfism.
  • GH is released and regulated by several natural secretagogues. In particular,
  • GH is specifically released in response to growth hormone releasing hormone (GHRH), a 40-44 amino acid peptide which acts directly upon specific pituitary receptors to cause the release. Secretion of GH is inhibited by somatostatin (SRIF). GH is also released in vitro and in vivo by certain smaller exogenous peptides. Such peptides are described, for instance, in U.S. Patents 4,410,513; 4,839,344;
  • this invention is a peptide compound comprising a hexa- to nonapeptide as described hereinafter by formula (I).
  • This invention is also a pharmaceutical composition for causing the release of growth hormone which comprises a compound of formula (I) and a pharmaceutically acceptable carrier.
  • This invention is further a method for causing the release of growth hormone, in vivo or in vitro, which comprises administering an effective amount of a compound of formula (I).
  • This invention is also a method for treating pathologies caused by a deficiency in the release of growth hormone, or conditions in which the release of growth hormone is desirable.
  • This invention discloses novel peptides which cause the release of growth hormone (GH). Although the precise mechanism of action is not known, these peptides are believed to act directly upon the pituitary to cause this effect.
  • the compounds of the present invention are novel and generally show greater potency at promoting the release of GH due to the residues incorporated in the residues V and Y.
  • This invention comprises compounds of formula (I): A-B-E-F-Ala-G-(D-Phe)-U-V-Y-Z ⁇ ) wherein A is H or an amino protecting group, or is absent when R 1 is H or C ⁇ _ 4 alkyl;
  • B is Gly, Ala, ⁇ -Ala, PyrAla, Gin or Lys, or is absent, when R 1 is H or C ⁇ _
  • E is absent or Het-(CR , 2) m CHR 1 (CR' 2 ) p CO;
  • F is D-Trp(R 2 ), D-Nal or D-Phe(R 2 );
  • G is Nal(R 2 ), Trp(R 2 ) or Phe(R 2 );
  • U is Lys, Arg or Orn
  • V is D- or L- Gin, NH(CR'2) n CO or, when Y and Z are absent, NH(CR' 2 )n-OH;
  • Y is absent or Gly;
  • Z is OR" or NR'R";
  • R is H or Ci- 4 alkyl
  • R 2 is H, F, Cl, Br, I or NO2;
  • R' is H or C alkyl
  • R" is H or CMalkyl
  • m and n are 0 to 3
  • p is 0 or 1; or a pharmaceutically acceptable salt thereof.
  • Prodrugs are hydrates, complexes or prodrugs of the compounds of formula (I).
  • Prodrugs arc considered to be any covalentiy bonded carriers which release the active parent drug according to formula (I) in vivo.
  • E is His, PyrAla, ImAc or ImPr.
  • F is D-Trp, ⁇ -D-Nal or D-Phe.
  • V-Y-Z is Gln-NH or Gln-Gly-NH 2 .
  • A-B-E is ImAc, His or Ala-His.
  • U is Lys.
  • Representative compounds of this invention are : ImPr-D-Trp-Ala-Trp-D-Phe-Lys-Gln-Gly-NH2; ImAc-D-Trp-Ala-Trp-D-Phe-Lys-Gln-NH2; ImAc-D-Phe-Ala-Phe-D-Phe-Lys-Gln-NH2;
  • this invention includes each unique nonracemic compound or diastereomer which may be synthesized and resolved by conventional techniques, and all mixtures thereof.
  • the meaning of any substituent at any one occurrence is independent of its meaning, or any other substituent's meaning, at any other occurrence, unless specified otherwise.
  • amino acid means the D- or L- isomer of alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid
  • Nal may be 2-naphthylalanine ( ⁇ -Nal) or 1- naphthylalanine ( ⁇ -Nal).
  • Phe(R 2 ), T ⁇ p(R 2 ), Nal(R 2 ) and His(R 2 ) refer to the indicated amino acid substituted on the aromatic ring by the group R 2 .
  • Ci-4alkyl as applied herein is meant to include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl and t-butyl.
  • Ci ⁇ alkyl additionally includes pentyl, n- pentyl, isopentyl, neopentyl and hexyl and the simple aliphatic isomers thereof.
  • AT, or aryl, as applied herein means phenyl or naphthyl, or phenyl or naphthyl substituted by one to three moieties R 11 .
  • R 11 may be Ci ⁇ alkyl, C ⁇ _ 4 alkoxy, Ci ⁇ alkthio, trifluoroalkyl, OH, CI, Br or I.
  • Het indicates pyridyl, imidazolyl, pyrazolyl or thienyl.
  • t-Bu refers to the tertiary butyl radical
  • Boc refers to the t-butyloxycarbonyl radical
  • Fmoc refers to the fluorenylmethoxycarbonyl radical
  • Ph refers to the phenyl radical
  • Cbz refers to the benzyloxycarbonyl radical
  • BrZ refers to the o-bromobenzyloxycarbonyl radical
  • C1Z refers to the o-chlorobenzyloxycarbonyl radical
  • Bzl refers to the benzyl radical
  • 4-MBzl refers to the 4-methyl benzyl radical
  • Me refers to methyl
  • Et refers to ethyl
  • Ac refers to acetyl
  • Alk refers to C ⁇ _4alkyl
  • Nph refers to 1- or 2-naphthy
  • ImAc refers to 4-imidazolylacetyl
  • ImPr refers to 4-imidazolylpropanoyl
  • PyrAla refers to 4- pyridylalanine
  • PyrPr refers to 4-pyridylpropanoyl.
  • DCC refers to dicyclohexylcarbodiimide
  • DIC refers to diisopropylcarbodiimide
  • DMAP refers to dimethylamino-pyridine
  • DEEA refers to diisopropylethyl amine
  • EDC refers to N-ethyl-N'(dimethylaminopropyl)- carbodiimid ⁇ .
  • HOBt refers to 1-hydroxybenzotriazole
  • THF tetrahydrofuran
  • DIEA diisopropylethyla ine
  • DMF dimethyl formamide
  • Pd/C a palladium on carbon catalyst
  • PPA 1-propanephosphonic acid cyclic anhydride
  • DPPA diphenylphosphoryl azide
  • BOP refers to benzotriazol-1- yloxy-tris(dimethylamino)-phosphonium hexafluorophosphate
  • HF refers to hydrofluoric acid
  • TEA refers to triethylamine
  • TFA trifluoroacetic acid
  • PCC pyridinium chlorochromate
  • BHA a benzhydrylamine polystyrene resin
  • CMR chloromethyl resin.
  • Coupling reagents as used herein denote reagents which may be used to form peptide bonds. Typical coupling methods employ carbodiimides, activated anhydrides and esters and acyl halides. Reagents such as EDC, DCC, DPPA, PPA, BOP reagent and oxalyl chloride, optionally used in conjunction with HOBt and N- hydroxysuccinimide are typical.
  • the compounds of this invention are prepared by conventional techniques of chemistry and peptide bond formation by coupling the various residues B, E, F, G, U, V and Y together to form a linear peptide, and, if necessary, modifying the carboxy or amino terminal group to add the group A and Z.
  • Coupling methods to form peptide bonds are generally well known to the art and include both solid phase and solution phase synthesis. Typically, any sidechain reactive groups are protected as known in the art to prevent unwanted side reactions in the coupling process.
  • this invention includes a process for preparing a compound of the formula:
  • A, B, E, F, G, U, V, Y and Z are as defined for formula (I), which comprises a) coupling the protected amino acids corresponding to B, E, F, G, U, V, Y as defined in formula (I) to form a suitably protected compound of the formula: A-B'-E , -F-Ala-G , -(D-Phe)-U , -V , -Y , -Z' wherein
  • A, B', E', F, G', U', V and Y' are A, B, E, F, G, U, V and Y as defined in formula (I) with any sidechain reactive groups protected;
  • X is NR'R", OR", NH-T" or O-T";
  • T" is a chloromethyl, hydroxymethyl, benzhydrylamine or methylbenzhydryl amine resin; b) removing any protecting groups and, if Z' is NH-T" or O-T", cleaving the peptide from the resin; and c) optionally forming a pharmaceutically acceptable salt thereof.
  • the synthesis is commenced by attaching the required ( ⁇ -amino-protected) alpha-amino acid to the resin.
  • the ⁇ -amino protecting group is an acid labile group, such as the Boc group
  • the ⁇ -amino protecting group can be removed by a choice of acidic reagents, such as trifluoroacetic acid or hydrochloric acid solutions in an organic solvent, at room temperature.
  • the ⁇ -amino protecting group is a base labile group, such as the Fmoc group
  • the ⁇ -amino protecting group can be removed by a choice of a basic reagent, such as piperidine in an organic solvent, at room temperature.
  • the next ( ⁇ -amino-protected) amino acid is coupled via its free carboxyl group to the free amino group.
  • Each protected amino acid is reacted in about a three fold excess using an appropriate coupling reagent, typically in a solution of DMF or methylene chloride. Repeating the cycle of deprotection of the amino group and coupling, each of the remaining protected amino acids is coupled stepwise in the desired order.
  • the desired peptide can be cleaved from the resin support by treatment with a suitable reagent, such as an acid.
  • a suitable reagent such as an acid.
  • hydrogen fluoride is used, since this reagent both cleaves the peptide from the resin and removes most common side-chain protecting groups.
  • Modification of amino groups may be accomplished, if necerney, by alkylation, sulfonylation, cyanation or acylation as is generally known in the art.
  • the carboxy terminal group may be modified, if necessary, by coupling to an amine to form an amide or by esterification. Solution synthesis for the formation of amide or peptide bonds is accomplished using conventional methods used to form amide bonds.
  • the amine is coupled via its free amino group to an appropriate carboxylic acid substrate using a suitable carbodiimide coupling agent, such as N,N' dicyclohexyl carbodiimide (DCC), optionally in the presence of catalysts such as 1-hydroxybenzotriazole
  • a suitable carbodiimide coupling agent such as N,N' dicyclohexyl carbodiimide (DCC)
  • DCC dicyclohexyl carbodiimide
  • a protected Boc-amino acid or Cbz-amino acid is treated in an anhydrous solvent, such as methylene chloride or tetrahydrofuran (THF), in the presence of a base, such as N-methyl morpholine, DMAP or a trialkylamine, with isobutyl chloroformate to form the "activated anhydride", which is subsequently reacted with the free amine of a second protected amino acid.
  • the solution phase synthesis can proceed by sequential addition of the individual amino acid residues to form the linear peptide, or individual fragments may be synthesized and coupled together in a convergent fashion.
  • the sidechain protecting groups may be removed simultaneously by treatment with hydrofluoric acid.
  • the reactive functional groups of the sidechains of each synthetic fragment are suitably protected as known in the art. Suitable protective groups are disclosed in Greene, PROTECTIVE GROUPS IN ORGANIC CHEMISTRY, John Wiley and Sons, New York, 1981.
  • a phthaloyl or Fmoc group or a suitably substituted alkyloxycarbonyl, benzyloxycarbonyl or benzyl group may be used for protection of an amino group.
  • the Boc group is generally preferred for protection of an ⁇ -amino group.
  • a t-Bu, cHex or benzyl ester may be used for the protection of the side chain carboxyl.
  • a benzyl group or suitably substituted benzyl group is used to protect the mercapto group or the hydroxyl group.
  • the tosyl group may be used for protection of the imidazolyl group and tosyl or nitro group for protection of the guanidino group.
  • a suitably substituted benzyloxycarbonyl, silyl or benzyl group may also be used for the hydroxyl group. Suitable substitution of the benzyloxycarbonyl or benzyl protecting groups is ortho and/or para substitution with chloro, bromo, nitro or methyl, and is used to modify the reactivity of the protective group.
  • the hydroxyl group of a heterocyclic carbinol is coverted to a suitable leaving group, such as a halogen (eg., by treatment with thionyl chloride or bromide) and reacted with the anion of a malonate ester. Hydrolysis, followed by decarboxylation produces the desired heterocyclic acid.
  • R 1 is an amino group
  • the heterocyclic acid may be converted to the acid bromide, brominated alpha to the carbonyl with thionyl bromide, and hydrolyzed to the alpha bromo carboxylic acid. Amination of the bromide produces the ⁇ -amino acid.
  • the bromide may be converted to a hydroxyl group, oxidized and reductively aminated, for instance with ammonium chloride and sodium cyanoborohydride.
  • Acid addition salts of the peptides are prepared in a standard manner in a suitable solvent from the parent compound and an excess of an acid, such as hydrochloric, hydrobromic, sulfuric, phosphoric, acetic, trifluoroacetic, maleic, succinic or methanesulfonic.
  • the acetate salt form is especially useful.
  • Certain of the compounds form inner salts or zwitterions which may be acceptable.
  • Cationic salts are prepared by treating the parent compound with an excess of an alkaline reagent, such as a hydroxide, carbonate or alkoxide, containing the appropriate cation; or with an appropriate organic amine.
  • Cations such as Li+, Na+, K+, Ca++, Mg++ and NH4+ are specific examples of cations present in pharmaceutically acceptable salts.
  • This invention provides a pharmaceutical composition which comprises a compound according to formula (I) and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition includes an effective amount of the compound to stimulate release of growth hormone, however, the administration of multiple doses of a composition containing suboptimal amounts of the compound may also be useful.
  • Pharmaceutical acceptable carrier are well known in the art and are disclosed, for instance, in SPROWL'S AMERICAN PHARMACY, Dittert, L. (ed.), J.B. Lippincott Co., Philadelphia, 1974, and REMINGTON'S PHARMACEUTICAL SCIENCES, Gennaro, A. (ed.), Mack Publishing Co., Easton, Pennsylvania, 1985.
  • compositions of the compounds of formula (I) may be formulated as sterile solutions, suspensions or emulsions, or lyophilized powders for parenteral administration.
  • Powders may be reconstituted by addition of a suitable diluent or other pharmaceutically acceptable carrier prior to use.
  • the liquid formulation may be a buffered, isotonic, aqueous solution.
  • suitable diluents are normal isotonic saline solution, standard 5% dextrose in water or buffered sodium or ammonium acetate solution.
  • Such formulation is especially suitable for parenteral administration, but may also be used for oral administration or contained in a metered dose inhaler or nebulizer for insufflation.
  • compositions for nasal administration may additionally contain enhancers, such as bile acids.
  • compositions for topical administration may also contain permeation enhancers, such as DMSO or formamide.
  • these peptides may be encapsulated, tableted or prepared in a emulsion or syrup for oral administration.
  • Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition, or to facilitate preparation of the composition.
  • Solid carriers include starch, lactose, calcium sulfate dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin.
  • the amount of solid carrier varies but, preferably, will be between about 20 mg to about 1 g per dosage unit.
  • the pharmaceutical preparations are made following the conventional techniques of pharmacy involving milling, mixing, granulating, and compressing, when necessary, for tablet forms; or milling, mixing and filling for hard gelatin capsule forms.
  • liquid carrier When a liquid carrier is used, the preparation will be in the form of a syrup, elixir, emulsion or an aqueous or non-aqueous suspension. Such a liquid formulation may be administered directly p.o. or filled into a soft gelatin capsule.
  • Liquid carriers include syrup, peanut oil, olive oil, saline and water.
  • the carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax.
  • the peptides of this invention may also be combined with excipients such as cocoa butter, glycerin, gelatin or polyethylene glycols and molded into a suppository.
  • the compounds of this invention may be used to treat conditions in which growth hormone has been shown to exert a beneficial effect
  • this invention provides a method of treating: 1) disease states wherein an insufficiency of endogenous GH is a factor, 2) catabolic states in which it is desirable to increase the level of circulating GH, 3) conditions in which it is beneficial to increase anabolic metabolism, or 4) enhanced development or production in commercial livestock.
  • the method comprises administering a compound of formula (I) to a mammal in need thereof.
  • the compounds of formula (I) may be used in the manufacture of a medicament.
  • the peptide is administered either orally, parenterally, rectally or intranasally, to the patient, in a manner such that the concentration of drug in the plasma is sufficient to cause the release of growth hormone.
  • the peptide is administered one to four times daily as is indicated by the effect being sought and the condition of the patient.
  • the pharmaceutical composition containing the peptide is administered at a dose between about 0.01 to about 5000 ⁇ g/kg in a manner consistent with the condition of the subject, route of administration and species being treated. Typically, cows and humans require about the same dosages, while rats require a somewhat higher dose. About 0.1 to 20 ⁇ g/kg is a typical intravensous dose.
  • An intravenous infusion of the peptide in 5% dextrose in water or normal saline is most effective, although an intramuscular bolus injection may be sufficient.
  • a somewhat higher dose is used for oral administration, dosages in the range of 1 to 1000 ⁇ g/kg being useful, and in the range of 20 to 600 ⁇ g/kg being typical.
  • the compounds of this invention may also be combined with other compounds and compositions which cause the release of GH. Such combinations will generally allow the use of lower dosages of each component to cause an equivalent amount of GH to be released.
  • the compounds of this invention may be combined with growth hormone releasing hormone (GHRH) and its bioactive analogs and inhibitors of somatostatin (SRIF).
  • GHRHs and analogues are disclosed, for instance, in U.S. Patents 4,517,181; 4,585,756; 4,605,643; 4,610,976; and 4,801,456, which are incorporated herein by reference.
  • Other suitable growth hormone releasing components are disclosed in U.S. Patent No. 4,880,778, which is incorporated herein by reference.
  • EC50 in rats for the compounds of this invention arc in the range of 2-30 ⁇ g/kg.
  • Preferred compounds have EC50 less than 10 ⁇ g kg.
  • His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 has an EC50 of about 42 ⁇ g/kg in the assay described hereinafter.
  • 20 ⁇ L of plasma (or a rat Growth Hormone standard) was diluted to 500 ⁇ L with assay buffer, and incubated at room temperature overnight with 100 ⁇ L 125 I-rat- Growth Hormone (Chemicon Corp., Temecula, CA; -20,000 cpm/tube) and 200 ⁇ L of monkey anti-rat Growth Hormone antiserum (diluted 1 :4000 with Ab buffer; Pituitary Hormones and Antisera Center, Torranc ⁇ , CA). On the following day, 200 ⁇ L of goat anti-monkey antisera (1:10 dilution P4; Antibodies Inc., Davis, CA) was added to each tube and the tubes were incubated for 2-4 h at room temperature.
  • the assay tubes were then centrifuged (5000xG, 30 min), the supernatant was aspirated, and the resulting pellet was counted for radioactivity in a Packard Gamma Counter.
  • the Growth Hormone content was calculated from the rat Growth Hormone standard curve. The lower limit of detection was 0.1 ng-GH/mL-plasma.
  • ODS refers to an octadecylsilyl derivatized silica gel chromatographic support.
  • Sephadex® is a cross-linked poly(dextran) manufactured by Pharmacia Fine Chemicals, Piscataway, N.J.
  • PRP-1® is a polymeric (styrene- divinyl benzene) chromatographic support and is a registered trademark of Hamilton Co., Reno, Nevada.
  • BondElut® C18 is an octadecyl silica manufactured Varian Associates, Palo Alto, California.
  • Peptide amides were synthesized by solid phase peptide synthesis using benzhydrylamine resin as the support. Protected amino acids were added sequentially starting from the carboxyl terminus until the desired sequence was obtained. The t- butyloxycarbonyl (Boc) group was used for protection of the alpha-amino group.
  • Imidazoleacetic acid hydrochloride (3 eq) is dissolved in 15 mL DMF with warming. This is added to the neutralized, free amine peptidyl resin, followed by DIC (3 eq) in CH2CL2 (15 mL), HOBt (3 eq) in DMF (5 mL) and then DIEA (3 eq).
  • DIC 3 eq
  • HOBt 3 eq
  • DMF 5 mL
  • DIEA DIEA
  • coupling is carried out using ImAc»HCl in DMF followed by DIC (3 eq) and DMAP (3 eq) in CH 2 CI 2 (15 mL) and then DIEA (3 eq).
  • Boc-Ala-His(Cbz)-D-T ⁇ p-Ala-Trp-D-Phe-Lys(ClZ)-Gln-Gly-BHA resin (0.5 mmol) was treated with a mixture of HF (10 mL) and anisole (1 mL) at 0°C for 1 h. After removal of HF by a stream of N2 and drying under vacuum, the resin was washed with ether, air-dried and extracted with glacial acetic acid (3 x 30 mL). The acetic acid extracts were combined and lyophilized to give crude peptide (586 mg).
  • the crude peptide (480 mg) was dissolved in 0.1% TF A water (3 mL) and chromatographed (BondElut Ci8, prewashed with 0.1 % TFA/acetonitrile followed by 0.1% TFA water, step gradient, 0.1% TFA/water, 20% acetonitrile/water-0.1% TFA, 40% acetonitrile/water-0.1% TFA).
  • the 20% acetonitrile/water-0.1% TFA fraction was evaporated to dryness and lyophilized from 1% HOAc/water to yield a partially purified peptide (285 mg). An aliquot (100 mg) of the partially purified peptide was dissolved in 1 %
  • the crude peptide was dissolved in 0.1 % TFA/water (3 mL) and chromatographed (BondElut Cis, prewashed with 0.1% TFA acetonitrile followed by 0.1% TFA water, step gradient, 0.1% TFA/water, 20% acetonitrile/water-0.1% TFA, 40% acetonitrile/water-0.1% TFA).
  • the 20% acetonitrile/0.1% TFA fraction was evaporated to dryness and lyophilized from 1% HO Ac to yield partially purified peptide (171 mg).
  • Boc-His(Cbz)-D-Trp-Ala-Trp-D-Phe-Lys(ClZ)-Gln-Gly-BHA resin (1.0 mmol) was treated with a mixture of HF (10 mL) and anisole (1 mL) at 0°C for 1 h. After removal of HF by a stream of N2 and drying under vacuum, the resin was washed with ether, air-dried and extracted with glacial acetic acid (3 x 30 mL). The acetic acid extracts were combined and lyophilized to give a crude peptide (1.20 g).
  • ImAc-D-Trp-Ala-Trp-D-Phe-Lys(ClZ)-Gln-Gly-BHA resin (1.0 mmol) was treated with a mixture of HF (10 mL) and anisole (1 mL) at 0°C for 1 h. After removal of HF by a stream of N2 and drying under vacuum, the resin was washed with ether, air-dried and extracted with glacial acetic acid (3 x 30 mL). The acetic acid extracts were combined and lyophilized to give crude peptide (1.2 g).
  • Boc-His(Cbz)-D-Trp-Ala-Trp-D-Phe-Lys(ClZ)-Gln-BHA resin (0.5 mmol) was treated with a mixture of HF (10 mL) and anisole (1 mL) at 0°C for 1 h. After removal of HF by a stream of N2 and drying under vacuum, the resin was washed with ether, air-dried and extracted with glacial acetic acid (3 x 30 mL). The acetic acid extracts were combined and lyophilized to give crude peptide (579 mg). Crude peptide (480 mg) was dissolved in 0.1% TFA/water (3 mL) and chromatographed (BondElut Cis, prewashed with 0.1% TFA/acetonitrile followed by
  • the crude peptide was dissolved in 0.1% TFA water (6 mL) and aliquots (3 mL) were chromatographed (BondElut Cis, prewashed with 0.1% TFA/acetonitrile followed by 0.1% TFA/water, step gradient, 0.1% TFA/water, 20% acetonitrile/water- 0.1% TFA, 40% acetonitrile/water-0.1% TFA). The 20% acetonitrile/0.1% TFA fractions were combined, evaporated to dryness and lyophilized from 1% HOAc to yield partially purified peptide (376 mg).
  • a parenteral dosage form containing 100 ⁇ g/mL of drug is prepared as follows:
  • Example 1 1 mg of the compound of Example 1 is dissolved in 10 mL normal sterile saline solution and the pH is adjusted to 5.0 with either acetic acid or sodium hydroxide. This solution is filtered through a 0.5 ⁇ filter into a sterile ampoule, the head space is filled with nitrogen and the ampoule is capped with a rubber septum.

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Abstract

Les peptides de la formule générale (I): A-B-E-F-Ala-G-(D-Phe)-U-V-Y-Z dans laquelle A représente H ou un groupe de protection amino ou est absent; B représente Gly, Ala, β-Ala, PyrAla, Gln ou Lys, ou est absent; E est absent ou représente H représente Het(CR'2)nCHR1(CR'2)pCO; F représente D-Trp(R2), D-Nal ou D-Phe(R2); G représente Nal(R2), Trp(R2) ou Phe(R2); U représente Lys, Arg ou Orn; V représente D- ou L- Gln, VH(CR'¿2?)nCO ou NH(CR'2)n-OH; Y est absent ou représente Gly; Z représente OR'' ou NR'NR''; R, R' et R'' représentent H ou alkyle C1-4; R représente H, alkyle C1-4; R?1¿ représente H, alkyle C¿1-4? ou NR'; R?2¿ représente H, F, Cl, Br ou NO¿2?; m et n représentent 0 à 3; et p représente 0 ou 1; lesdits peptides stimulant la libération de l'hormone de croissance chez les mammifères.
PCT/US1993/008805 1992-09-25 1993-09-17 Peptides liberant l'hormone de croissance WO1994007519A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP93921654A EP0663834A4 (fr) 1992-09-25 1993-09-17 Peptides liberant l'hormone de croissance.
JP6509101A JPH08502250A (ja) 1992-09-25 1993-09-17 成長ホルモン放出ペプチド

Applications Claiming Priority (2)

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US95150092A 1992-09-25 1992-09-25
US07/951,500 1992-09-25

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WO1994007519A1 true WO1994007519A1 (fr) 1994-04-14

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995017423A1 (fr) * 1993-12-23 1995-06-29 Novo Nordisk A/S Composes possedant des proprietes de liberation de l'hormone de croissance
WO1996010040A1 (fr) * 1994-09-27 1996-04-04 Romano Deghenghi Composes polypeptides contenant d-2-alkyltryptophanne pouvant promouvoir la liberation de l'hormone de croissance
WO1997023508A1 (fr) * 1995-12-22 1997-07-03 Novo Nordisk A/S Composes avec des proprietes de liberation de l'hormone de croissance
US5872100A (en) * 1990-05-11 1999-02-16 Deghenghi; Romano Peptides containing D-2-Alkyl-Tryptophan
WO2000009537A2 (fr) * 1998-08-14 2000-02-24 Administrators Of The Tulane Educational Fund Composes possedant une activite de liberation d'hormone de croissance
US6451970B1 (en) 1996-02-21 2002-09-17 Novo Nordisk A/S Peptide derivatives
WO2017075535A1 (fr) 2015-10-28 2017-05-04 Oxeia Biopharmaceuticals, Inc. Méthodes de traitement de troubles neurodégénératifs
US10105416B2 (en) 2014-02-05 2018-10-23 The Regents Of The University Of California Methods of treating mild brain injury

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992001711A1 (fr) * 1990-07-24 1992-02-06 Polygen Holding Corporation Composes de polypeptides presentant une activite de liberation d'hormone de croissance

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992001711A1 (fr) * 1990-07-24 1992-02-06 Polygen Holding Corporation Composes de polypeptides presentant une activite de liberation d'hormone de croissance

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
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See also references of EP0663834A4 *

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5872100A (en) * 1990-05-11 1999-02-16 Deghenghi; Romano Peptides containing D-2-Alkyl-Tryptophan
US5955421A (en) * 1990-05-11 1999-09-21 Deghenghi; Romano Peptides containing D-2-alkyl-Tryptophan
WO1995017423A1 (fr) * 1993-12-23 1995-06-29 Novo Nordisk A/S Composes possedant des proprietes de liberation de l'hormone de croissance
WO1996010040A1 (fr) * 1994-09-27 1996-04-04 Romano Deghenghi Composes polypeptides contenant d-2-alkyltryptophanne pouvant promouvoir la liberation de l'hormone de croissance
US5795957A (en) * 1994-09-27 1998-08-18 Deghenghi; Romano Polypeptide compounds containing D-2-alkyltryptophan capable of promoting the release of growth hormone
WO1997023508A1 (fr) * 1995-12-22 1997-07-03 Novo Nordisk A/S Composes avec des proprietes de liberation de l'hormone de croissance
AU715856B2 (en) * 1995-12-22 2000-02-10 Novo Nordisk A/S Compounds with growth hormone releasing properties
US6451970B1 (en) 1996-02-21 2002-09-17 Novo Nordisk A/S Peptide derivatives
WO2000009537A3 (fr) * 1998-08-14 2001-09-20 Univ Tulane Composes possedant une activite de liberation d'hormone de croissance
WO2000009537A2 (fr) * 1998-08-14 2000-02-24 Administrators Of The Tulane Educational Fund Composes possedant une activite de liberation d'hormone de croissance
US6468974B1 (en) 1998-08-14 2002-10-22 The Administrators Of The Tulane Educational Fund Compounds having growth hormone releasing activity
US7250399B2 (en) 1998-08-14 2007-07-31 The Administrators Of The Tulane Educational Fund Compounds having growth hormone releasing activity
US10105416B2 (en) 2014-02-05 2018-10-23 The Regents Of The University Of California Methods of treating mild brain injury
US10617740B2 (en) 2014-02-05 2020-04-14 The Regents Of The University Of California Methods of treating mild brain injury
US11241483B2 (en) 2014-02-05 2022-02-08 The Regents Of The University Of California Methods of treating mild brain injury
WO2017075535A1 (fr) 2015-10-28 2017-05-04 Oxeia Biopharmaceuticals, Inc. Méthodes de traitement de troubles neurodégénératifs

Also Published As

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EP0663834A4 (fr) 1996-01-24
EP0663834A1 (fr) 1995-07-26
JPH08502250A (ja) 1996-03-12

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