WO2010065572A1 - Analogues sélectifs pour sstr1 - Google Patents

Analogues sélectifs pour sstr1 Download PDF

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Publication number
WO2010065572A1
WO2010065572A1 PCT/US2009/066305 US2009066305W WO2010065572A1 WO 2010065572 A1 WO2010065572 A1 WO 2010065572A1 US 2009066305 W US2009066305 W US 2009066305W WO 2010065572 A1 WO2010065572 A1 WO 2010065572A1
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peptide
sstrl
srif
terminus
phe
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PCT/US2009/066305
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English (en)
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Judit Erchegyi
Jean E. F. Rivier
Jean Claude Reubi
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The Salk Institute For Biological Studies
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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
    • C07K14/655—Somatostatins
    • C07K14/6555—Somatostatins at least 1 amino acid in D-form
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/083—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins the peptide being octreotide or a somatostatin-receptor-binding peptide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/088—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins conjugates with carriers being peptides, polyamino acids or proteins

Definitions

  • This invention is directed to peptides related to somatostatin and to methods for pharmaceutical treatment of mammals using such peptides. More specifically, the invention relates to short receptor-selective somatostatin analogs which include particular amino acid substitutions that create receptor-selectivity and increased affinity to the selected receptor, to pharmaceutical compositions containing such peptides, to such peptides complexed with radioactive nuclides or conjugated to cytotoxins, and to methods of diagnostic and therapeutic treatment of mammals using such peptides and their conjugates, particularly peptides that are coupled to chelators and then complexed with radioactive nuclides or otherwise labeled.
  • SRIF cyclic tetradecapeptide somatostatin- 14
  • Somatostatin and many analogs of somatostatin exhibit activity in respect to the inhibition of growth hormone (GH) secretion from cultured, dispersed rat anterior pituitary cells in vitro; they also inhibit GH, insulin and glucagon secretion in vivo in the rat and in other mammals.
  • GH growth hormone
  • One suc Ih analog is [D-Trp ; ]-SRIF which has the amino acid i sequence: (cyclo 3-14)H-Ala-Gly-Cys-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH, which is disclosed in U.S. Patent No. 4,372,884 (2/8/83).
  • SRIF has also been found to inhibit the secretion of gastrin and secretin by acting directly upon the secretory elements of the stomach and pancreas, respectively, and SRIF is being sold commercially in Europe for the treatment of ulcer patients. SRIF is also known to inhibit the growth of certain tumors.
  • SRIF induces its biological effects by interacting with a family of membrane-bound structurally similar receptors.
  • Five SRIF receptors have been cloned and are referred to as SSTRl -5. All five receptors bind SRIF and SRIF-28 (an N-terminally extended version of SRIF) with high affinity; cell lines bearing these cloned receptors are available to test SRIF analogs for binding affinity, selectivity and functional effects. Studies have now shown that different receptor subtypes mediate distinct functions of SRIF in the body.
  • a cyclic SRIF analog variously termed SMS-201-995 and Octreotide, i.e. H-D-Phe- c[Cys-Phe-D-Trp-Lys-Thr-Cys]-Thr-ol is being used clinically to inhibit certain tumor growth.
  • Analogs complexed with 111 In or the like are also used as diagnostic agents to detect SRIF receptors expressed in cancers.
  • 7,019,109 disclose SRIF analogs which are selective for SSTRl. It has been reported that in medullary thyroid carcinoma, calcitonin secretion and gene expression can be reduced by treatment with SSTRl -selective agonists and that such may be able to inhibit endothelial activities, suggesting a potential therapeutic utility for administration of SSTRl -selective agonists in the proliferative diseases involving angiogenesis. Although numerous reports on the localization, physiological and therapeutic functions of SSTRl have been published, it is still not clear which is its main function and the main related pathology resulting from over- or under-expression.
  • One such peptide can be radioiodinated or otherwise radiolabeled while retaining its desirable biological properties.
  • These novel peptide agonists are useful in determining the tissue and cellular expression of the receptor SSTRl and its biological role in the endocrine, exocrine and nervous system, as well as in regulating certain pharmacological functions without the accompanying side effects which have heretofore been characteristic of administering the native releasing factor SRIF or an analog which binds significantly to two or more of SSTRl -5.
  • These short SRIF analog peptides when radiolabeled, can be used in scintigraphy in order to locate, i.e.
  • these analogs can be turned into radiopharmaceuticals which are suitable for radionuclide therapy in treatment of such tumors; alternatively, they can be covalently joined to a cytotoxic moiety using an appropriate covalent conjugating agent, e.g. glutaraldehyde or one which binds via a disulfide linkage.
  • an appropriate covalent conjugating agent e.g. glutaraldehyde or one which binds via a disulfide linkage.
  • Conjugating agent is used herein to broadly refer to this class of well known chelating, complexing or otherwise covalently bound agents that serve to link desired moieties to peptides.
  • These short SRIF agonists bind selectively and with high affinity to SSTRl; by selectively binding is meant that they exhibit an IC 50 (or K D ) with SSTRl that is about 10% or less of the IC 50 which they exhibit with respect to each of the other four SRIF receptors; by high affinity is meant an IC 50 of not greater than about 10 nanomolar. Of course, the greater the differentials, the more selective the analog is.
  • These short SRIF analogs can also be readily labeled and effectively used in radionuclide and cytotoxic therapy; for example, they are useful in localizing such receptor in the body and in diagnosing the locations of tumors, particularly prostate cancers, sarcomas and neuroendocrine tumors. As radionuclide therapeutic agents, they are considered to be particularly useful in destroying tumors expressing SSTRl receptors.
  • the invention provides novel cyclic short somatostatin (SRIF) analog peptide agonists which selectively bind the SRIF receptor SSTRl, said peptide comprising the cyclic amino acid sequence:
  • Cys-Phe(X)-D-Agl(NMe, 2Np)-IAmp-Xaa-Cys wherein Xaa is Phe, Tyr or iodotyrosine (ITyr) and the C-terminus is amidated, where X is H, 4Cl, 4F, 4NO 2 , 4NH 2 , 4NH-CONH 2 , 4NHCONHOCH 3 , 4NHCONHOCH 2 -CH 3 or 4NHC0NH0H;
  • D-AgI(NMe, 2Np) stands for N ⁇ Me, 2-naphthoyl aminoglycine and IAmp stands for 4-(N-isopropyl)- aminomethy lphenylalanine .
  • the invention provides pharmaceutical compositions comprising the novel peptides and a pharmaceutically acceptable carrier which can be administered in an amount effective to treat IH or another SSTRl -mediated physiopathology by reaching tissue having SSTRl receptors and activating said receptors, or administered in an effective amounts so as to selectively bind to cells having SSTRl cells and thereby provide a detectable signal at the location thereof, or administered in an amount which is effective to destroy cells containing SSTRl via a radioactive nuclide or a cytotoxin coupled to the novel peptide.
  • Cys residue at or adjacent the N-terminus may be in either the L- or D-isomer form without affecting binding affinity or function.
  • cyclo or “c” is meant that a cyclizing bond is present between the side chains of the two cysteine residues.
  • SRIF analog peptides are herein provided that have both a selective affinity for the SRIF receptor SSTRl and a high affinity for SSTRl, i.e. an IC 5O that is preferably less than 10 nM.
  • These short SRIF peptide analogs have a ring of six amino acid residues formed by a disulfide linkage between Cys residues at the 1 and 6-positions wherein the central two residues, i.e.
  • a D-isomer aminoglycine having a substituted ⁇ -amino group in the 3 -position and an L-isomer alkylated aminomethylphenylalanine (Amp), preferably (isopropyl)Amp, in the adjacent 4-position.
  • Amp L-isomer alkylated aminomethylphenylalanine
  • These two residues are flanked by a pair of amino acid (AA) residues having aromatic side chains, preferably either phenylalanine or tyrosine, substituted or unsubstituted. More preferably, Tyr or ITyr is present in the 5-position, and the C-terminus is amidated.
  • amidated is meant that the residue at the C-terminus carries an amide group, symbolized by -NH 2 , or a substituted amide group symbolized by -NHR where R is lower alky, preferably, methyl, ethyl or propyl.
  • R is lower alky, preferably, methyl, ethyl or propyl.
  • the residue in the 3- position is a D-isomer of a substituted aminoglycine, more particularly one where the ⁇ - amino group is both methylated and acylated, preferably with 2-naphthalene carboxylic acid.
  • the adjacent residue in the 4-position is an aminomethylphenylalanine (Amp) which is C 2 -C 5 alkylated, preferably with isopropyl, i.e. 4-(N-isopropyl)-aminomethyl Phe.
  • the C-terminus may be either amide or substituted amide, e.g. ethylamide.
  • a threonine or a naphthylalanine residue may be added at the C-terminus, e.g.
  • Thr-NH 2 or 2NaI-NH 2 to somewhat improve the potency of the analog as a result of its binding with a stronger affinity to SSTRl; however, such may cause some lessening of the differential between its affinity for SSTRl and for SSTR4.
  • Tyr may be slightly preferred over Phe for the 5 -position residue, and radioiodinated Tyr (ITyr) acts as a tracer without significantly lessening its selectivity for SSTRl or its binding affinity thereto.
  • L- or D-Tyr can be added at the N-terminus, which may also be radioiodinated; such addition may also improve potency of the SRIF agonist but may also exhibit a somewhat increased affinity for SSTR4.
  • a conjugating/complexing agent can be linked to the ⁇ -amino group of Cys at the N-terminus of the peptide analog, which agent is capable of joining thereto a radioactive nuclide or a cytotoxin.
  • conjugating/complexing agents may be any of those presently used in this art which covalently bond to an ⁇ -amino group. They may be designed to link, as by chelation, to a radioactive metal or to covalently bind to a cytotoxin, such as saporin, gelonin, ricin A chain, etc.
  • the effective SRlF cyclic analog agonists comprise the amino acid sequence: Cys-Phe-D- AgI(NMe, 2Np)-IAmp-Xaa-Cys wherein Xaa is Phe, Tyr or iodotyrosine (ITyr) and the C-terminus is amidated, where X is H, 4Cl, 4F, 4NO 2 , 4NH 2 , 4NH-CONH 2 , 4NHCONHOCH 3 , 4NHCONHOCH 2 -CH 3 or 4NHC0NH0H, where D-AgI(NMe, 2Np) stands for D-N ⁇ Me, 2-naphthoyl aminoglycine and where IAmp stands for 4-(N-isopropyl)- aminomethylpheylalanine.
  • Amp is meant (aminomethyl)phenylalanine where the methyl group with its amino substitution should be understood to be in the 4- or para-position on the phenyl ring.
  • IAmp is meant (N-isopropyl-aminomethyl)phenylalanine, where the 4-aminomethyl group is alkylated with an isopropyl group; whereas in EAmp, the alkylation is with an ethyl group.
  • lower alkyl refers to a straight or branched chain, saturated hydrocarbon group having from 1 to 6 carbon atoms such as, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, isopentyl, n-pentyl and n- hexyl.
  • Me is meant methyl.
  • Np is meant naphthoyl.
  • naphthoyl is inclusive of 1- and 2-naphthoyl, with 2-naphthoyl being preferred.
  • NaI 3-(2- naphthyl)-alanine.
  • SRIF is meant the 14-residue cyclic peptide, somatostatin. When Tyr appears in the 5-position, it may be radioiodinated or otherwise labeled; by ITyr is meant radioiodinated tyrosine.
  • the C-terminus is amidated as described hereinbefore.
  • the N-terminus may be modified in various ways without significantly adversely effecting the binding affinity, all of which modifications in these cyclic peptides are considered to be included as a part of the peptides of the overall invention.
  • a variety of additions may also be made to the N- terminal amino acid in the form of conjugating agents which can be then used to link a desired moiety to the peptide.
  • chelating agents such as DTPA, DOTA, HYNIC and P 2 S 2 -COOH may be attached; alternatively, a cytotoxin may be covalently linked thereto via a suitable linker well known in this art if desired.
  • Receptor binding assays are performed on cloned SRIF receptors as generally set forth in Reubi, J.C. et al., J. Clin. Endocrinol. Metab., 63, 433-438 (1986) and in Reubi, J. C. et al., Eur. J. Nucl. Med. 2000, 27, 273-282.
  • K D values which are indicative of the concentration of a ligand necessary to occupy one-half (50%) of the binding sites on a selected amount of a receptor or the like
  • competitive assays can generate IC 5O values which are indicative of the concentration of a competitive ligand necessary to displace a saturation concentration of a target ligand being measured from 50% of binding sites.
  • H-c[Cys-Phe-DAgl(NMe,2Np)-IAmp-Tyr-Cys]-NH 2 inhibits the binding to SSTRl of an iodinated SRIF-28 ligand that is known to have a strong affinity for all five receptors. Testing shows that it binds to the cloned human SSTRl with an IC 50 of about 6 nM, while this short SRIF analog peptide does not bind strongly to either human SSTR3 and SSTR4 and does not bind to SSTR2 or SSTR5 at a concentration below 1,000 nM.
  • the tyrosine residue in position-5 may be radioiodinated without significantly changing the selective affinity of the 125 I-Tyr analog so it serves as an excellent tracer for SSTRl.
  • I 1 analog H-c[Cys-Phe-DAgl(NMe,2Np)-IAmp-Phe-Cys]-Thr-NH 2 inhibits binding of iodinated SRIF-28 to SSTRl while itself binding even more strongly with an IC 50 of about 1.0 nM, while still not binding to receptors SSTR2 or SSTR5 at concentrations below 1,000 nM; however, it does show slightly higher affinity for SSTR4.
  • These SRIF analogs that selectively show high affinity to SSTRl are considered to be particularly useful in the treatment of SSTRl -mediated physiopathologies and in combating tumors by carrying radionuclides or cytotoxins to the sites of these receptors but not to other SRIF receptors.
  • SSTRl mRNA has been detected in a variety of tumors.
  • SSTRl plays a major role in tumor growth regulation and, if it does, whether it mediates stimulation or inhibition. Therefore, at this time, it is difficult to foretell whether a selective SSTRl antagonist would have a beneficial role for long-term treatment of tumors.
  • SRIF analogs selective for SSTRl that bind strongly thereto, and that are long-acting can be effectively used to kill such tumors via radionuclide or cytotoxic therapy.
  • Octreotide in the treatment of such tumors has not been considered to be satisfactorily effective particularly because of its selectivity to 3 of the 5 receptors, i.e. SSTR2, 3 and 5.
  • SSTRl has been reported to couple to a tyrosine phosphatase, and stimulation of this enzyme is believed to mediate anti-proliferative effects of SRIF via activation of this receptor.
  • SSTRl niRNA has been detected in a number of tumors. The ability of SSTRl to mediate anti-proliferative effects of SRIF renders SSTRl -selective SRIF agonists effective as therapeutic treatment agents for treating those cancers, such as prostrate cancers and sarcomas wherein the malignant tissues express this receptor.
  • SSTRl -selective agonists as anti-cancer agents may be their continued effectiveness after prolonged use.
  • continuous use of SMS-201-995 in the treatment of tumors is considered to be hindered by rapid desensitization of SSTR2, SSTR3 and SSTR5, the receptors with which this peptide can interact; in fact, all 3 of these receptors have been reported to rapidly desensitize.
  • the SSTRl -selective peptide agonists of the invention are considered to have prolonged anti-proliferative actions and should therefore exhibit improved effectiveness in treating SSTRl -mediated cancers, compared to the commercially available SRIF analogs presently used as anti-cancer agents that have low affinity for SSTRl.
  • the SRIF analogs of the present invention are considered to be useful in combating cancers which express SSTRl and in combating SSTRl -mediated physiopathologies. They are also considered to be most useful in scintigraphy to determine the distribution of cells and tissues expressing this receptor in the brain and in the endocrine and exocrine systems, and also in identifying selective functions of this receptor in the body.
  • Labeled SRIF analogs of the invention are also considered to be useful in drug- screening assays to screen for new effective peptide and nonpeptide agents which will bind with high affinity to SSTRl and which may be either highly effective agonists or antagonists for treating GI track motility.
  • a known ligand for the receptor SSTRl is in hand, one can obtain a baseline activity for the recombinantly produced receptor.
  • inhibitors or modifiers i.e. antagonists of the receptor function
  • the cyclic SRIF analogs described in the following examples are agonists which can be also employed to selectively stimulate the inhibitory activity of somatostatin at SSTRl .
  • the peptides of the present invention can be synthesized by classical solution-phase synthesis, but they are preferably synthesized by solid-phase technique, as described in United States Patent No. 5,750,499 issued May 12, 1998, the disclosure of which is incorporated herein by reference.
  • Boc-IAmp(Z) N ⁇ Boc-(4-aminomethyl)phenylalanine (Boc-Amp) (15.3 g, 52 mmol) is dissolved in acetone (200 mL), and molecular sieves (6.0 g, 4A) are added to the solution in a 500 mL Parr hydrogenation vessel.
  • Boc-N B MeAgl(2Np) or Boc-Agl(NMe,2Np)- OH
  • Boc-D/L-Agl(Fmoc)-OH prepared by the synthesis set forth in Qasmi, et al., Tetrahed, Lett, 34:3861-3862 (1993).
  • N ⁇ Fmoc-D/L- is synthesized using the procedure set forth in Jiang, et al., Orthogonally protected N ⁇ methyl-substituted-aminoglycine, Protein and Peptide Lett., 3:219-224 (1996).
  • the Fmoc protection can be removed from the monomer (or when a part of the peptide resin) with 20% piperidine in NMP, or the Boc protection can be removed from the monomer (or when a part of the peptide resin) with TFA in DCM and naphthoyl is then introduced by reaction of the free side chain secondary amino group with naphthoyl chloride.
  • a somatostatin agonist having the structure:
  • Couplings of the protected amino acids were mediated by diisopropylcarbodiimide (DIC) and (HOBt) in DMF for 1 h and monitored by the qualitative ninhydrin test. A 3 -equivalent excess of the protected amino acids based on the original substitution of the resin was generally used.
  • the monomer synthesized in Example 1 i.e. N ⁇ (Boc)-4-isopropylaminomethyl Phe(Z) is coupled into the chain.
  • the monomer synthesized in Example 2 is so coupled. Boc removal was achieved with trifluoroacetic acid (60% in CH 2 Cl 2 , 1-2% ethanedithiol or m-cresol) for 20 min.
  • the peptides were cleaved from the resin support with simultaneous side chain deprotection by anhydrous HF containing the scavengers anisole (10% v/v) and methyl sulfide (5% v/v) for 60 min at 0 0 C.
  • the diethyl ether precipitated crude peptides were cyclized in 75% acetic acid (200 mL) by addition of iodine (10% solution in methanol) until the appearance of a stable orange color. Forty minutes later, ascorbic acid was added to quench the excess iodine.
  • the collected fractions were screened by analytical RP-HPLC, and the fractions containing the product were pooled and subjected to lyophilization.
  • the purity of the final peptide was determined by analytical RP-HPLC performed with a linear gradient using 0.1 M TEAP pH 2.5 in water as eluent A and 60% CH 3 CN/40% A as eluent B on a Hewlett-Packard Series II 1090 Liquid Chromatograph connected to a Vydac Qg column (0.21 x 15 cm, 5 ⁇ m particle size, 300 A pore size).
  • the products were then analyzed by capillary zone electrophoresis (CZE), and mass spectra (MALDI-TOF-MS) were measured on an ABI-Perseptive DE-STR instrument.
  • Peptide 3A A portion of the product Peptide 3 above is taken and subjected to iodination, as well known in this art, to iodinate the Tyr residue in the 5-position and create a tracer.
  • the iodinated compound is thereafter referred to as Peptide 3A.
  • Example 3 The synthesis described in Example 3 is repeated with one change. Boc-Thr(Bzl) is coupled to the resin prior to the first Cys residue. Elongation of the chain is then carried out as in Example 3, and cleavage, deprotection, cyclization, purification, separation and assignment the stereochemistry of the L and D-AgI containing analogs are also carried out as in Example 3.
  • Example 4 The synthesis described in Example 4 is repeated with one change. N ⁇ Boc-Phe is used instead of N ⁇ Boc-Tyr(2BrZ) in position-5. Following removal of the Boc group at the N-terminus, HF cleavage, deprotection, cyclization, and purification are carried out as in Example 3.
  • the purified cyclic peptide has the formula:
  • Example 5 The synthesis described in Example 5 is repeated with one change. Elongation of the chain by one residue is carried out by coupling N ⁇ Boc-D-Tyr(2BrZ) at the N-terminus. Cleavage, deprotection, cyclization and purification are carried out as in Example 3. The purified, cyclic peptide has the formula:
  • Example 3 The synthesis described in Example 3 is repeated with one change.
  • DOTA (1,4,7,10- tetraazacyclododecane-l,4,7,10-tetraacetic acid), a polyaminopolycarboxylic chelator, is added to the N-terminus of the peptide.
  • This conjugation step is carried out (a) as the last step of the solid-phase peptide synthesis, or it is carried out (b) in solution phase after synthesis, cleavage, cyclization and purification of the peptide.
  • the reagent l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid mono(N-hydroxysuccinimide) ester.3CH 3 COOH.HPFg used in both procedures is purchased from Macrocyclics Inc. (Dallas, TX, USA).
  • Example 7 The synthesis described in Example 7 is repeated with one change.
  • a different polyaminopolycarboxylic chelator moiety namely DTPA (Diethylenetriamine pentaacetic acid), is added to the N-terminus of the peptide.
  • DTPA Diethylenetriamine pentaacetic acid
  • This conjugation step is carried out (a) as the last step of the solid-phase peptide synthesis, or it is carried out (b) in solution phase after synthesis, cleavage, cyclization and purification of the peptide.
  • the reagent DPTA-tetra-tert-butyl ester Diethyl enetriamine- N,N,N",N"-tetra-tert-butyl acetate-N'-acetic acid
  • DPTA-tetra-tert-butyl ester Diethyl enetriamine- N,N,N",N"-tetra-tert-butyl acetate-N'-acetic acid
  • Example 5 The synthesis described in Example 5 is repeated with two changes. N ⁇ Boc-2Nal is used instead of N ⁇ Boc-Thr(Bzl) as the first residue coupled to the resin, and D-Cys is used instead of L-Cys for the residue at the N-terminus. Following removal of the Boc group at the N-terminus, HF cleavage, deprotection, cyclization, and purification are carried out as in Example 3. The purified cyclic peptide has the formula:
  • Example 9 The synthesis described in Example 9 is repeated with one change. N ⁇ Boc- Tyr(2BrZ) is coupled to the D-Cys residue at the N-terminus. Following removal of the Boc group at the N-terminus, HF cleavage, deprotection, cyclization, and purification are carried out as in Example 3.
  • the purified cyclic peptide has the formula: (cyclo)H-Tyr-D-Cys-Phe-D-Agl(NMe,2Np)-IAmp-Phe-Cys-2Nal-NH 2 and is referred to as Peptide No. 10.
  • Example 3 The synthesis described in Example 3 is repeated with one change. N ⁇ Boc-4ClPhe is used instead of N ⁇ BocPhe in the 2-position. Following removal of the Boc group at the N-terminus, HF cleavage, deprotection, cyclization and purification are carried out as in Example 3. A purified cyclic peptide having the formula:
  • Peptide No. 11 (cyclo)H-Cys-4ClPhe-D-Agl(NMe,2Np)-IAmp-Tyr-Cys-NH 2 results and is referred to as Peptide No. 11.
  • the mass of Peptide No. 11 is 1023.35.
  • Peptide No. 11 binds strongly and selectively to SSTRl.
  • Example 3 The synthesis described in Example 3 is repeated with one change. N ⁇ Boc-4FPhe is used instead of N ⁇ Boc-Phe in the 2-position. Following removal of the Boc group at the N- terminus, HF cleavage, deprotection, cyclization and purification are carried out as in Example 3. A purified cyclic peptide having the formula:
  • Peptide No. 12 (cyclo)H-Cys-4FPhe-D-Agl(NMe,2Np)-IAmp-Tyr-Cys-NH 2 results and is referred to as Peptide No. 12.
  • the mass of Peptide No. 12 is 1007.38.
  • Peptide No. 12 binds strongly and selectively to SSTRl.
  • the short SRIF analogs were evaluated for their agonist/antagonist properties using a reporter gene assay that determines the biological activity of the human SSTRl in CCL39- SSTRl -Luci cells constitutively expressing the human SSTRl as well as the luciferase gene under the control of the serum response element (SRE).
  • SRE serum response element
  • the SRE is regulated by transcription factors and is activated by many extracellular signals including ligands acting at G-protein-coupled receptors. It has been shown that upon ligand binding SSTRs mediate the increase of luciferase expression via SRE in a reporter gene assay. Stimulating CCL39- SSTRl -Luci cells with somatostatin analogues activates the luciferase gene in a dose dependant manner.
  • This hexapeptide containing only 6 residues is a much smaller and appealing molecule than prior SSTRl -selective peptides.
  • the substitution of Cys with D-Cys has little effect on SSTRl binding affinity and function, as shown by testing results for Peptides Nos. 9 and 10.
  • Peptide No. 6 shows that the addition of a residue at the C-terminus and the N- terminus is tolerated. It exhibits a binding IC 5 Q of 0.19 nM and 5, 000-fold selectivity versus SSTR2 and SSTR5, 500-fold selectivity versus SSTR3, and 100-fold selectivity versus SSTR4; it has an EC 5O value of 0.37 nM in the luciferase reporter gene assay.
  • the peptides of the invention not only provide more selective ligands for binding SSTRl but the use of labeled peptides, for example, a radioiodinated version of one of Peptide Nos. 3, 6 and 10, facilitates drug screening for inhibitors for the receptor, e.g. antagonists that are more effective than those presently known.
  • Competitive binding assays with candidate compounds would first be carried out in this manner with SSTRl to search for high binding affinity; then by screening the multiple SRIF receptors, it could be confirmed whether there was selective binding to only this receptor, as is desired.
  • SRIF analog do not adversely affect the selective binding
  • these compounds can be complexed with a cytotoxic or a radioactive agent for the purpose of carrying that agent to a tumor or other tissue for which degradation is desired.
  • a dialdehyde linker such as glutaraldehyde may be used to link the SRIF analog to saporin or gelonin.
  • linkers such as DOTA or DTPA or other suitable chelating agents can be used to complex the SRIF analog with a highly radioactive element as indicated hereinbefore.
  • the solubility of the SRIF analogs can be improved by acylation of the N-terminal amino group using a hydrophilic compound, such as hydroorotic acid or the like, or by reaction with a suitable isocyanate, such as methylisocyanate or isopropylisocyanate, to create a urea moiety at the N-terminus.
  • a hydrophilic compound such as hydroorotic acid or the like
  • a suitable isocyanate such as methylisocyanate or isopropylisocyanate
  • Other agents can also be N-terminally linked that will increase the duration of action of the SRIF analog as known in this art.
  • SRIF analogs or nontoxic salts thereof may be administered to animals, including humans and other mammals, either intravenously, subcutaneously, intramuscularly, percutaneously, e.g. intranasally, intracerebrospinally or orally.
  • the peptides should be at least about 90% pure and preferably should have a purity of at least about 98%; however, lower purities are effective and may well be used with mammals other than humans. This purity means that the intended peptide constitutes the stated weight % of all like peptides and peptide fragments present.
  • Administration to humans should be under the direction of a physician to combat specific tumors and cancers or to mediate other conditions where the SSTRl receptors exert a control function, such as coupling to a tyrosine phosphatase so that stimulation of this enzyme can be carried out to mediate the anti-proliferative effects of SRIF.
  • the required dosage will vary with the particular condition being treated, with the severity of the condition and with the duration of desired treatment.
  • Such peptides are often administered in the form of pharmaceutically or veterinarily acceptable nontoxic salts, such as acid addition salts or metal complexes, e.g., with zinc, iron, calcium, barium, magnesium, aluminum or the like.
  • nontoxic salts such as hydrochloride, hydrobromide, sulphate, phosphate, tannate, oxalate, fumarate, gluconate, alginate, maleate, acetate, citrate, benzoate, succinate, malate, ascorbate, tartrate and the like.
  • the tablet may contain a binder, such as tragacanth, corn starch or gelatin; a disintegrating agent, such as alginic acid; and a lubricant, such as magnesium stearate.
  • a binder such as tragacanth, corn starch or gelatin
  • a disintegrating agent such as alginic acid
  • a lubricant such as magnesium stearate.
  • sweetening and/or flavoring may be used, and intravenous administration in isotonic saline, phosphate buffer solutions or the like may be effected.
  • a dosage form may contain a pharmaceutically acceptable non-toxic salt of the compound which has a low degree of solubility in body fluids, for example, an acid addition salt with a polybasic acid; a salt with a polyvalent metal cation; or combination of the two salts.
  • a relatively insoluble salt may also be formulated in a gel, for example, an aluminum stearate gel.
  • a suitable, slow-release depot formulation for injection may also contain an SRIF analog or a salt thereof dispersed or encapsulated in a slow degrading, non-toxic or non-antigenic polymer such as a polylactic acid/polyglycolic acid polymer, for example, as described in U.S. Pat. No. 3,773,919.
  • Therapeutically effective amounts of the peptides should be administered under the guidance of a physician, and pharmaceutical compositions will usually contain the peptide in conjunction with a conventional, pharmaceutically or veterinarily-acceptable carrier.
  • the SRIF analogs of the invention are generally effective at levels of less than 100 micrograms per kilogram of body weight. A therapeutically effective amount is considered to be a predetermined amount calculated to achieve the desired effect.
  • the required dosage will vary with the particular treatment and with the duration of desired treatment. Generally dosages between about 10 micrograms and about 1 milligram per kilogram of body weight per day will be used; however, for prolonged action, it may be desirable to use dosage levels of about 0.1 to about 2.5 milligrams per kilogram of body weight.
  • These analogs are soluble in water and thus can be prepared as relatively concentrated solutions for administration.
  • a therapeutically effective amount is typically an amount of an SRIF analog that, when administered peripherally, e.g. intravenously, in a physiologically acceptable composition, is sufficient to achieve a plasma concentration thereof from about 0.1 ⁇ g/ml to about 100 ⁇ g/ml, preferably from about 1 ⁇ g/ml to about 50 ⁇ g/ml, more preferably at least about 2 ⁇ g/ml and usually 5 to 10 ⁇ g/ml. In these amounts, they may be used for the prevention of IH, or in appropriate treatments for cardiovascular diseases and other SSTRl -mediated physiopathologies.

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Abstract

L'invention porte sur des analogues courts de SRIF qui sont sélectifs pour SSTR1 par rapport aux quatre autres récepteurs de SRIF, comprenant un noyau à 6 chaînons dans lequel D-Agl(NMe, 2Np) et IAmp sont flanqués par des résidus ayant une extrémité de chaîne latérale aromatique, son extrémité C étant amidée, lesquels inhibent la liaison d'un radioligand SRIF universel au récepteur humain cloné SSTR1, mais pas aux autres récepteurs. Ces analogues sélectifs courts de SRIF comprennent l'hexapeptide cyclique : formule (I) où X représente H, 4Cl, 4F, 4NO2, 4NH2, 4NHCONH2, 4NHCONHOCH3, 4NHCONHOCH2-CH3 ou 4NHCONHOH, où Xaa représente Phe, Tyr ou l'iodotyrosine (ITyr) et où l'extrémité C est amidée. La tyrosine iodée radioactive peut être l'un des deux résidus flanquants, ou à l'extrémité N, qui comportera également un agent de conjugaison ou une liaison à une cytotoxine.
PCT/US2009/066305 2008-12-04 2009-12-02 Analogues sélectifs pour sstr1 WO2010065572A1 (fr)

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US9957299B2 (en) 2010-08-13 2018-05-01 Aileron Therapeutics, Inc. Peptidomimetic macrocycles
US10213477B2 (en) 2012-02-15 2019-02-26 Aileron Therapeutics, Inc. Peptidomimetic macrocycles
US10227380B2 (en) 2012-02-15 2019-03-12 Aileron Therapeutics, Inc. Triazole-crosslinked and thioether-crosslinked peptidomimetic macrocycles
US10253067B2 (en) 2015-03-20 2019-04-09 Aileron Therapeutics, Inc. Peptidomimetic macrocycles and uses thereof
US10301351B2 (en) 2007-03-28 2019-05-28 President And Fellows Of Harvard College Stitched polypeptides
US10471120B2 (en) 2014-09-24 2019-11-12 Aileron Therapeutics, Inc. Peptidomimetic macrocycles and uses thereof

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US10301351B2 (en) 2007-03-28 2019-05-28 President And Fellows Of Harvard College Stitched polypeptides
US9957299B2 (en) 2010-08-13 2018-05-01 Aileron Therapeutics, Inc. Peptidomimetic macrocycles
US9096684B2 (en) 2011-10-18 2015-08-04 Aileron Therapeutics, Inc. Peptidomimetic macrocycles
US9522947B2 (en) 2011-10-18 2016-12-20 Aileron Therapeutics, Inc. Peptidomimetic macrocycles
US10308699B2 (en) 2011-10-18 2019-06-04 Aileron Therapeutics, Inc. Peptidomimetic macrocycles
US10213477B2 (en) 2012-02-15 2019-02-26 Aileron Therapeutics, Inc. Peptidomimetic macrocycles
US10227380B2 (en) 2012-02-15 2019-03-12 Aileron Therapeutics, Inc. Triazole-crosslinked and thioether-crosslinked peptidomimetic macrocycles
US9845287B2 (en) 2012-11-01 2017-12-19 Aileron Therapeutics, Inc. Disubstituted amino acids and methods of preparation and use thereof
US10669230B2 (en) 2012-11-01 2020-06-02 Aileron Therapeutics, Inc. Disubstituted amino acids and methods of preparation and use thereof
US10471120B2 (en) 2014-09-24 2019-11-12 Aileron Therapeutics, Inc. Peptidomimetic macrocycles and uses thereof
US10253067B2 (en) 2015-03-20 2019-04-09 Aileron Therapeutics, Inc. Peptidomimetic macrocycles and uses thereof

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