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WO2000010589A9 - Analogues de la somatostatine - Google Patents

Analogues de la somatostatine

Info

Publication number
WO2000010589A9
WO2000010589A9 PCT/US1999/019090 US9919090W WO0010589A9 WO 2000010589 A9 WO2000010589 A9 WO 2000010589A9 US 9919090 W US9919090 W US 9919090W WO 0010589 A9 WO0010589 A9 WO 0010589A9
Authority
WO
WIPO (PCT)
Prior art keywords
cys
tyr
lys
thr
isocyanate
Prior art date
Application number
PCT/US1999/019090
Other languages
English (en)
Other versions
WO2000010589A1 (fr
Inventor
Jeffrey D White
Original Assignee
Jeffrey D White
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Jeffrey D White filed Critical Jeffrey D White
Priority to AU63829/99A priority Critical patent/AU6382999A/en
Publication of WO2000010589A1 publication Critical patent/WO2000010589A1/fr
Publication of WO2000010589A9 publication Critical patent/WO2000010589A9/fr

Links

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
    • C07K14/655—Somatostatins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Somatostatin is a cyclic tetradecapeptide which inhibits release of several pituitary and intestinal factors that regulate cell proliferation, cell motility, and/or secretion including growth hormone, adrenocorticotropin hormone, prolactin, thyroid stimulating hormone, insulin, glucagon, motilin, gastric inhibitory peptide, vasoactive intestinal peptide, secretin, cholecystokinin, bombesin, gastrin releasing peptide, gastrin, thyroid releasing hormone, pancreatic polypeptide, cytokines (e.g., interleukins, interferons), growth factors (e.g., epidermal growth factor, nerve growth factor), and vasoactive amines (e.g., serotonin).
  • cytokines e.g., interleukins, interferons
  • growth factors e.g., epidermal growth factor, nerve growth factor
  • vasoactive amines e.g., serot
  • Native somatostatin has a very short half life in vivo.
  • a large number of novel analogues have been prepared in order to enhance the duration of effect, biological activity and the selectivity of this hormone.
  • a variety of somatostatin peptide analogs have been produced by elimination of amino acids that are not absolutely required for activity and substitution of the native L-amino acids with the corresponding D-amino acid isomers. Thus, some of these analogs are long acting, more potent receptor agonists than native somatostatin, due in part to the resistance of D-amino acids to enzyme degradation.
  • the synthetic somatostatin analog octreotide acetate which has the amino acid sequence D-Phe-Cys-Phe-D-T ⁇ -Lys-Thr-Cys-Thr(ol) is more potent than native somatostatin in inhibition of growth factor release.
  • the present invention provides novel chemically modified somatostatin analogs, structural derivatives of native somatostatin which bind a somatostatin receptor. Analogs include both antagonists and agonists of somatostatin activity.
  • the present invention provides a somatostatin analog which comprises a chemically substituted heptapeptide sequence having the cysteine groups in the 1 and 6 position being linked together to form an disulfide bridge in the monocyclic configuration. Chemical modifications are made at the free amino group of cysteine of the peptide below
  • somatostatin analog peptide can be modified by the addition of isocyanates, isothiocyanates, acid chlorides, chloro formates and glycidyl ethers (epoxides) at the free amino group, at the terminal cysteine resulting in a measurable enhancement of the ability of the chemically modified compounds to bind somatostatin receptors.
  • Figure 1 is a graph which illustrates that peptide 3502 suppresses secretion of growth hormone.
  • Figure 2 is a graph which shows that orally administered peptide 3502 prevents
  • the compounds of this invention are cyclic heptapeptide analogs of somatostatin
  • RI is C1-C4 alkyl, adamantyl,
  • Preferred compounds of this invention include compounds of Formula I: wherein A is
  • Rl is C1-C4 alkyl, adamantyl,
  • X ] and X 2 are independently, flourine, chlorine, bromine, iodine, C1-C4 alkyl,
  • Y is CH 2
  • X is hydrogen
  • Rl is C1-C4 alkyl, adamantyl,
  • X, and X 2 are independently, flourine, chlorine, bromine, iodine, C1-C4 alkyl, O
  • Another prefened somatostatin analog of this invention is a compound of
  • X is hydrogen
  • Y is a bond
  • the invention features compounds, compositions and methods for the treatment of diseases in mammals associated with increased production or secretion of any factor or factors which can be regulated by somatostatin, including but not limited to growth hormone, insulin, glucagon and pancreatic exocrine secretion.
  • the compounds can be administered in the dosages used for somatostatin or, because of their greater potency, in smaller dosages.
  • the compounds of the invention can be used for the treatment of cancer, particularly growth hormone- or growth factor- dependent cancer (e.g., bone, cartilage, pancreas, prostate, or breast), acromegaly and related hypersecretroy endocrine states, or of bleeding ulcers and in those suffering from pancreatitis or dianhea.
  • growth hormone- or growth factor- dependent cancer e.g., bone, cartilage, pancreas, prostate, or breast
  • acromegaly and related hypersecretroy endocrine states or of bleeding ulcers and in those suffering from pancreatitis or dianhea.
  • the compounds can also be used in the management of diabetes and to protect the liver of patients suffering from ckrhosis or hepatitis.
  • the compounds can also be used to treat Alzheimer's disease and as gastric cytoprotective compounds for ulcer therapy.
  • the compounds will also be useful in treating diabetes-related retinopathy, nephropathy and vascular disease.
  • the anti-cancer activity of the compounds may be related to their ability to antagonize the actions of cancer-related growth factors such as epidermal growth factor, insulin-like growth factor (IGF-1), or vasoactive endothelial growth factor (NEGF).
  • cancer-related growth factors such as epidermal growth factor, insulin-like growth factor (IGF-1), or vasoactive endothelial growth factor (NEGF).
  • the analogs can be made available in the form of pharmaceutically acceptable salts or complexes.
  • therapeutically acceptable acids for the formulation of salts of the somatostatin analogs are inorganic acids, such as hydrochloric acid, sulfuric acid, phosphoric acid, and the organic lactic, maleic, citric, succinic, benzoic, salicylic, toluensulfonic acids.
  • Complexes are compounds of Formula I formed by the addition of organic salts or hydroxides such as Ca and Zn salts or the addition polymeric organic materials, such as tannic acid or carboxymethyl cellulose.
  • a therapeutically effective amount of the somatostatin analog or pharmaceutically acceptable salt or complex thereof are combined with a pharmaceutically acceptable carrier substance (e.g., magnesium carbonate, lactose, a phospholipid or mannitol) to form a pharmaceutical composition.
  • a pharmaceutically acceptable carrier substance e.g., magnesium carbonate, lactose, a phospholipid or mannitol
  • Examples of methods of administration of the therapeutic reagent of the pharmaceutical composition thereof include a pill, tablet, capsule or liquid for oral administration.
  • the pharmaceutical composition can also be administered as an ointment, gel, cream or lotion for application to the skin, or as a solution capable of being administered intravenously, parenterally, subcutaneously, transmucosally, intranasally or intraperitoneally in an appropriate buffer if necessary.
  • the solid forms of this therapeutic composition can be coated with a substance capable of protecting the modified peptide from digestion by gastric acid in the stomach for a period of time sufficient to allow the composition to pass undisintegrated into the small intestine.
  • the therapeutic composition can be administered via a sustained release formulation or a dermal patch. The descriptions are provided as examples and are not meant to limit the possibilities of therapeutic compositions or methods of administration of the somatostatin analogs. Examples
  • the cyclic heptapeptide NH 2 Cys-Tyr-D-Trp-Lys-Thr-Cys-D-Tyr-NH 2 was purchased from Polypeptide Laboratories. The reagents used to modify the heptapeptide are widely available from commercial sources.
  • the heptapeptide (as a trifluoroacetic acid salt) containing Boc-Lys was suspended in anhydrous acetonitrile to yield a 1 rnM concentration. Fifty to 100 ⁇ l of this mixture was placed in a microcentrifuge tube. One equivalent of triethylamine (100 mM in acetonitrile) was added with mixing, followed by the addition of 1 equivalent of the isocyanate (100 mM in acetonitrile). The reactions with the isocyanates were incubated at room temperature for 60 to 120 min. Ten ⁇ l of water was added. The solvent was removed by evaporation under vacuum. The chemically modified peptides were than purified by reverse phase HPLC.
  • the heptapeptide (as a trifluoroacetic acid salt) containing Boc-Lys was suspended in anhydrous acetonitrite to yield a 1 mM concentration. Fifty to 100 ⁇ l of this mixture was placed in a microcentrifuge tube. One equivalent of triethylamine (100 mM in a acetonitrile) was added with mixing, followed by the addition of one equivalent of isocyanate (100 mM in acetonitrile). The reactions with the isothiocyanates were incubated at room temperature for 18 hrs. The solvent was removed by evaporation under vacuum. The chemically modified peptides were then purified by reverse phase HPLC.
  • the heptapeptide (as a trifluoroacetic acid salt) containing Boc-Lys was suspended in anhydrous acetonitrile to yield a 1 mM concentration. Fifty to 100 ⁇ l of this mixture was placed in a microcentrifuge tube. Two equivalents of triethylamine (100 mM in acetonitrile) was added with mixing, followed by the addition of 1 equivalent of an acid chloride (100 mM in acetonitrile). The reactions with the acid chlorides were incubated at room temperature for 60-120 min. The solvent was removed by evaporation under vacuum. The chemically modified peptides were then purified by reverse phase HPLC.
  • the heptapeptide (as a trifluoroacetic acid salt) containing Boc-Lys was suspended in anhydrous methanol to yield a 1 mM concentration. Fifty to 100 ⁇ l of this mixture was placed in a microcentrifuge tube. One equivalent of triethylamine (100 mM in acetonitrile), followed by the addition of 1 equivalent of a glycidyl ether (100 mM in methanol). The reaction was incubated at 65° C for 6-8 hrs. The solvent was removed by evaporation under vacuum. The chemically modified desired peptides were purified by reverse phase HPLC. In the resulting modified peptides, R_ is C1-C4 alkyl, adamantyl,
  • X j and X 2 are independently, fluorine, chlorine, bromine, iodine, C1-C4 alkyl, NO 2 or
  • somatostatin receptors hsstl, hsst2, hsst3, hsst4, hsst5 have been identified and cloned.
  • Somatostatin receptor subtype 2 is
  • CHO-Kl cells were grown as monolayers in Dulbecco's Modified Eagle's medium (DMEM, Mediatech, Washington DC) supplemented with 10% fetal calf serum, non-essential amino acids, 2mM glutamine, ImM pyruvate and 500 mg/mL gentamycin in 5% CO2 at 37°C.
  • DMEM Dulbecco's Modified Eagle's medium
  • hsst expression of hsst in CHO-Kl cells.
  • CHO-Kl cell lines stably expressing hsst were created and propagated.
  • the predicted coding region of each hsst was generated by PCR from human genomic DNA and oligonucleotides corresponding to the coding region 5' and 3' ends as primers.
  • the DNA fragment generated by PCR contained a Hind III restriction site at the 5' end and a Not 1 restriction site at the 3' end. The fragment was digested with these two restriction enzymes and directionally subcloned into the Hind Ill/Not 1 sites of the mammalian expression vector pCDNAl . The identity of each insert was verified by DNA sequencing.
  • the construct was co-transfected with pSV2neo into a CHO-Kl cell line using the calcium phosphate protocol. Stable transfectants were selected using 400 mg/mL G418 and maintained in supplemented DMEM. After an initial ligand binding screen, one stable clone for each sst was chosen for all subsequent experiments.
  • Preparation of Plasma Membranes CHO/sst cells grown on 100mm tissue culture dishes were washed with ice cold PBS then scraped into 5ml of 50mM HEPES, pH 7.4-5mM MgCl 2 - 200 KIU/mL aprotinin - 2mg/mL PMSF and 2 mg/mL bacitracin (homogenization buffer).
  • the cells were homogenized on ice using a Brinkman Polytron (setting 5, 15 sec) then re- homogenized with a hand held homogenizer (6 strokes). After centrifugation at 500 x g for 5 min. at 4°C, the supernatant was centrifuged again at 12,000 x g for 25 minutes at 4°C. The final pellet was resuspended in homogenization buffer. Protein content was measured using the bicinchoninic acid protein assay using BSA as a standard.
  • Specific binding was defined as the difference between the amount of [ 125 I-Tyr ⁇ ] SS14 bound in the absence and presence of l ⁇ M unlabeled SSI 4. Ki was determined using software programs Ligand or Prism. The purified peptides were tested for binding to one or more of the five human somatostatin receptor subtypes. Table 1 lists the results of receptor binding studies for a number of peptides assayed against the five human somatostatin receptor subtypes. The data of Table 1 indicates the chemical modifications change the binding affinity of the 5 parent heptapeptide to the various receptor subtypes.
  • modified peptides were determined by evaluating their inhibitory potency on pituitary growth hormone (GH) release in sodium pentobarbital-anesthetized rats.
  • GH pituitary growth hormone
  • the pentobarbital treated rat is a well characterized and frequently used model for studying GH secretory dynamics (see K. Chihara, A. Axmura and AN Schally 1979 Endocrinology 104 1434).
  • Dose-Response Studies Adult male Sprague-Dawley rats weighing 250-300g with jugular vein cannulas were obtained from Zivic-Miller Labs, Zelienople, PA.
  • the rats were anesthetized with sodium pentobarbital (60mg/kg of body weight, administered i.p.). Thirty minutes later, the animals were injected iv. with saline or test compound at doses ranging from 0J to 30 ⁇ g/kg. Blood samples (250 ⁇ L) were drawn from the jugular vein cannula 10 min prior to test compound injection (baseline) and 5, 15, 30, 45 and 60 minutes after injection. The plasma was separated and assayed for GH by RIA using material supplied by the National Hormone and Pituitary Program, and for glucagon and glucose using commercially available reagents.
  • Figure 1 illustrates the dose response of peptide 3502 at three dosage levels 5 ⁇ g/kg, 2.5 ⁇ g/kg, and 1 ⁇ g/kg. At each dosage, the peptide was shown to be effective in suppressing production of growth hormone.
  • Time-Course Assay Groups of cannulated rats were treated with sodium pentobarbital as in the dose-response assay. Thirty minutes later animals were injected via the jugular cannula with saline or test compound at the minimum dose giving maximal GH inhibition. Sodium pentobarbital at half the initial dose was given at 60- to 90-minute intervals to maintain anesthesia. Blood (250 ⁇ L) was collected from the jugular vein at approximately 15, 30, 60, 120, 180, and 240 min. after the injection of test compound and treated as described above. Data from the time-course assay are shown in Table 2.
  • Oral activity Adult male Sprague-Dawley rats weighing 250-300g with jugular vein and gastric cannulas were obtained from Zivic-Miller Labs, Zelienople, PA. On the evening prior to assay, rats were given 5 gram of food to eat with free access to water. On the day of assay, the rats were anesthetized with sodium pentobarbital (60mg/kg of body weight, administered i.p.). Thirty minutes later, the animals were injected through the gastric cannula with saline or test compound at doses ranging from 0J to 30 ⁇ g/kg in a total volume of 200 ⁇ L.
  • the graph representing oral saline illustrates the cyclic increase and decrease of growth hormone levels during normal secretion.
  • the graph representing peptide 3502 shows that the peptide, orally administered, prevents the normal secretion of growth hormone.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Toxicology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Endocrinology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne des analogues de la somatostatine qui renferment une séquence d'heptapeptides chimiquement substitués dont les groupes cystéine sont situés aux emplacements 1 à 6 et qui sont reliés les uns aux autres pour former un pont bisulphure dans la configuration monocyclique. Des modifications d'ordre chimique sont apportées au groupe amino libre de cystéine du peptide (1). Il a été démontré que le peptide analogue de la somatostatine pouvait être modifié par adjonction d'isocyanates, d'isothiocyanates, de chlorures d'acide, de chloroformates et d'éthers de glycidyle (époxides) du groupe amino libre avec, au terminal cystéine, une amélioration marquée de l'aptitude des composés chimiquement modifiés à se lier à des récepteurs de la somatostatine.
PCT/US1999/019090 1998-08-24 1999-08-23 Analogues de la somatostatine WO2000010589A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU63829/99A AU6382999A (en) 1998-08-24 1999-08-23 Somatostatin analogs

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US9756298P 1998-08-24 1998-08-24
US60/097,562 1998-08-24

Publications (2)

Publication Number Publication Date
WO2000010589A1 WO2000010589A1 (fr) 2000-03-02
WO2000010589A9 true WO2000010589A9 (fr) 2000-12-14

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Country Status (2)

Country Link
AU (1) AU6382999A (fr)
WO (1) WO2000010589A1 (fr)

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4145337A (en) * 1977-10-11 1979-03-20 Hoffmann-La Roche Inc. Aminoethylglycine containing polypeptides
US5770687A (en) * 1995-06-07 1998-06-23 Peptor Limited Comformationally constrained backbone cyclized somatostatin analogs
US5846934A (en) * 1996-02-20 1998-12-08 American Cyanamid Company Pure somatostatin antagonist and methods of use thereof

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WO2000010589A1 (fr) 2000-03-02
AU6382999A (en) 2000-03-14

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