Patent Grant
9133262
Native somatostatin is comprised of both a 14-amino acid isoform (somatostatin-14) and a 28-amino acid isoform (somatostatin-28). Heiman, et al., Neuroendocrinology, 45:429-436 (1987). Because of the short half-life of the native somatostatin, various somatostatin analogs have been developed, e.g., for the treatment of acromegaly. Raynor, et al., Molecular Pharmacol. 43:838 (1993). Five distinct somatostatin receptors have been identified and characterized. Hoyer, et al., Naunyn-Schmiedeberg's Arch. Pharmacol., 350:441 (1994). Somatostatin produces a variety of effects, including modulation of hormone release, e.g., growth hormone, glucagon, insulin, amylin, and neurotransmitter release. Some of these effects have been associated with its binding to a specific somatostatin receptor. For example, the inhibition of growth hormone has been attributed to the somatostatin type-2 receptor (“SSTR-2”) (Raynor, et al., Molecular Pharmacol. 43:838 (1993); Lloyd, et al., Am. J. Physiol. 268:G102 (1995)) while the inhibition of insulin has been attributed to the somatostatin type-5 receptor (“SSTR-5”) (Coy, et al. 197:366-371 (1993)). The following invention relates to a novel class of somatostatin analogs which are antagonists to somatostatin receptors.
The invention features a compound of the formula:
wherein
A1 is a D- or L-isomer of an aromatic amino acid, or is deleted;
A2 is a D-isomer selected from the group consisting of Cys, Pen, an aromatic amino acid, or an aliphatic amino acid;
A3 is an aromatic amino acid;
A4 is Trp or D-Trp;
A7 is Cys, Pen, or an aromatjkic or an aliphatic amino acid;
A8 is a D- or L-isomer selected from the group consisting of Thr, Ser, an aromatic amino acid, or an, aliphatic amino acid;
each of R1 and R2, is, independently, H or substituted (e.g., one to four times) or unsubstituted lower alkyl, aryl, aryl lower alkyl, heterocycle, heterocycle lower alkyl, E1SO2 or E1CO (where E1 is aryl, aryl lower alkyl, heterocycle, or heterocycle lower alkyl), where said substituent is halo, lower alkyl, hydroxy, halo lower alkyl, or hydroxy lower alkyl; and
R3 is OH, NH2, C1-12 alkoxy, or NH—Y—CH2—Z, wherein Y is a C1-12 hydrocarbon moiety and Z is H, OH, CO2H, or CONH2, or R3, together with the carbonyl group of A8 attached thereto, are reduced to form H, lower alkyl, or hydroxy lower alkyl; provided if A2 is D-Cys or D-Pen, and A7 is Cys or Pen, then a disulfide bond links the sidechains of A2 and A7, and if A1 is D-Phe or p-NO2-Phe; A2 is D-Cys; A3 is Phe or Tyr; A6 is Thr or Val; and A7 is Cys; then A8 is β-Nal.
In one embodiment, A2 is D-Cys, A7 is Cys, and A4 is D-Trp. In a further embodiment, A1 is an L-aromatic amino acid. In still a further embodiment, A1 and A3, independently, is 5-Nal, o-X-Phe (where X is H, OH, CH3, halo, OCH3, NH2, CN, or NO2), p-X-Phe (where X is H, OH, CH3, halo, OCH3, NH2, CN, or NO2), m-X-Phe (where X is H, OH, CH3, halo, OCH3, NH2, CN, or NO2), F5-Phe, Trp, Dip, 2-Pal, Tyr(Bzl), His, Igl, Tyr(I), Bta, Bip, Npa, or Pal; A6 is Thr, Ser, Tle, Thr(Bzl), Abu, Ala, Ile, Leu, Gly, Nle, β-Ala, Gaba, or Val; and A8 is the D- or L-isomer of Thr, Dip, F5-Phe, p-X-Phe (where X is H, OH, CH3, halo, OCH3, NH2, CN, or NO2), o-X-Phe (where X is H, OH, CH3, halo, OCH3, NH2, CN, or NO2), m-X-Phe (where X is H, OH, CH3, halo, OCH3, NH2, CN, or NO2), Igl, Tyr(Bzl), or β-Nal. In yet still another embodiment, A1 is β-Nal, Npa, Igl, Phe, p-F-Phe, Trp, p-Cl-Phe, or p-CN-Phe; A3 is Tyr, Tyr(I), or Pal; A6 is Val, Tle, Nle, Ile, or Leu; A8 is p-F-Phe, β-Nal, Tyr, Dip, p-Cl-Phe, Igl, or p-CN-Phe; R1 is H, CH3CO, 4-(2-hydroxyethyl)-1-piperazinylacetyl, or 4-(2-hydroxyethyl)-1-piperizineethanesulfonyl; R2 is H; and R3 is NH2.
In another further embodiment, A1 is a D-aromatic amino acid. In still another further embodiment, A1 is D-β-Nal, D-o-X-Phe (where X is H, OH, CH3, halo, OCH3, NH2, CN, or NO2), D-p-X-Phe (where X is H, OH, CH3, halo, OCH3, NH2, CN, or NO2), D-m-X-Phe (where X is H, OH, CH3, halo, OCH3, NH2, CN, or NO2), D-F5-Phe, D-Trp, D-Dip, D-2-Pal, D-Tyr(Bzl), D-His, D-Igl, D-Tyr(I), D-Bta, D-Bip, D-Npa, or D-Pal; A3 is β-Nal, o-X-Phe (where X is H, OH, CH3, halo, OCH3, NH2, CN, or NO2), p-X-Phe (where X is H, OH, CH3, halo, OCH3, NH2, CN, or NO2), m-X-Phe (where X is H, OH, CH3, halo, OCH3, NH2, CN, or NO2), F5-Phe, Trp, Dip, 2-Pal, Tyr(Bzl), His, Igl, Tyr(I), Bta, Bip, Npa, or Pal; A6 is Thr, Ser, Tle, Thr(Bzl), Abu, Ala, Ile, Leu, Gly, Nle, β-Ala, Gaba, or Val; and A8 is the D- or L-isomer of Thr, Dip, F5-Phe, p-X-Phe (where X is H, OH, CH3, halo, OCH3, NH2, CN, or NO2), o-X-Phe (where X is H, OH, CH3, halo, OCH3, NH2, CN, or NO2), m-X-Phe (where X is H, OH, CH3, halo, OCH3, NH2, CN, or NO2), Tyr(Bzl), Igl, or β-Nal. In yet still a further embodiment, A1 is D-β-Nal, D-Npa, D-Igl, D-Phe, D-p-F-Phe, D-Trp, D-p-Cl-Phe, or D-p-CN-Phe; A3 is Tyr, Tyr(I), or Pal; A6 is Val, Tle, Nle, Ile, or Leu; A8 is p-F-Phe, β-Nal, Tyr, Dip, p-Cl-Phe, Igl, or p-CN-Phe; R1 is H, CH3CO, 4-(2-hydroxyethyl)-1-piperazinylacetyl, or 4-(2-hydroxyethyl)-1-piperizineethanesulfonyl; R2 is H; and R3 is NH2.
In still another further embodiment, A1 is deleted, R1 is substituted or unsubstituted E1CO, and R2 is H. In still a further embodiment, R1 is substituted or unsubstituted E1CO (where E1 is phenyl, β-naphthylmethyl, β-pyridinylmethyl, or 3-indolylmethyl); A3 is β-Nal, o-X-Phe (where X is H, OH, CH3, halo, OCH3, NH2, CN, or NO2), p-X-Phe (where X is H, OH, CH3, halo, OCH3, NH2, CN, or NO2), m-X-Phe (where X is H, OH, CH3, halo, OCH3, NH2, CN, or NO2), F5-Phe, Trp, Dip, 2-Pal, Tyr(Bzl), His, Igl, Tyr(I), Bta, Bip, Npa, or Pal; A6 is Thr, Ser, Tle, Thr(Bzl), Abu, Ala, Ile, Leu, Gly, Nle, β-Ala, Gaba, or Val; and A8 is the D- or L-isomer of Thr, Dip, F5-Phe, p-X-Phe (where X is H, OH, CH3, halo, OCH3, NH2, CN, or NO2), o-X-Phe (where X is H, OH, CH3, halo, OCH3, NH2, CN, or NO2), m-X-Phe (where X is H, OH, CH3, halo, OCH3, NH2, CN, or NO2), Igl, Tyr(Bzl), or β-Nal. In yet still a further embodiment, R1 is E1CO (where E1 is 4-hydroxy-phenyl, β-naphthylmethyl, or phenyl); A3 is Tyr, Tyr(I), or Pal; A6 is Val, Tle, Nle, Ile, or Leu; A8 is p-F-Phe, β-Nal, Tyr, Dip, p-Cl-Phe, Igl, or p-CN-Phe; R3 is NH2.
In yet still a further embodiment, R3, together with the carbonyl group of A8 attached thereto, are reduced to form H, lower alkyl, or hydroxy lower alkyl. In still another further embodiment, A1 is the D- or L-isomer of β-Nal, o-X-Phe (where X is H, OH, CH3, halo, OCH3, NH2, CN, or NO2), p-X-Phe (where X is H, OH, CH3, halo, OCH3, NH2, CN, or NO2), m-X-Phe (where X is H, OH, CH3, halo, OCH3, NH2, CN, or NO2), F5-Phe, Trp, Dip, 2-Pal, Tyr(Bzl), His, Igl, Tyr(I), Bta, Bip, Npa, or Pal; A3 is β-Nal, o-X-Phe (where X is H, OH, CH3, halo, OCH3, NH2, CN, or NO2), p-X-Phe (where X is H, OH, CH3, halo, OCH3, NH2, CN, or NO2), m-X-Phe (where X is H, OH, CH3, halo, OCH3, NH2, CN, or NO2), F5-Phe, Trp, Dip, 2-Pal, Tyr(Bzl), His, Igl, Tyr(I), Bta, Bip, Npa, or Pal; A6 is Thr, Ser, Tle, Thr(Bzl), Abu, Ala, Ile, Leu, Gly, Nle, β-Ala, Gaba, or Val; and A8 is the D- or L-isomer of Thr, Dip, F5-Phe, p-X-Phe (where X is H, OH, CH3, halo, OCH3, NH2, CN, or NO2), o-X-Phe (where X is H, OH, CH3, halo, OCH3, NH2, CN, or NO2), m-X-Phe (where X is H, OH, CH3, halo, OCH3, NH2, CN, or NO2), Igl, Tyr(Bzl), or β-Nal. In yet still another further embodiment, A1 is the D- or L-isomer of β-Nal, Phe, p-F-Phe, Trp, p-Cl-Phe, or p-CN-Phe; A3 is Tyr, Tyr(I), or Pal; A6 is Val, Tle, Nle, Ile, or Leu; A8 is p-F-Phe, β-Nal, Tyr, Dip, p-Cl-Phe, Igl, or p-CN-Phe; R1 is H, CH3CO, 4-(2-hydroxyethyl)-1-piperazinylacetyl, or 4-(2-hydroxyethyl)-1-piperizineethanesulfonyl; R2 is H, and R3, together with the carboxy group of A8 attached thereto, are reduced to form H or CH3OH.
In another embodiment, A2 is a D-aromatic amino acid or a D-aliphatic amino acid, A7 is an aromatic amino acid or an aliphatic amino acid, and A1 is D-Trp. In a further embodiment, A1 is an L-amino acid and A2 is a D-aromatic amino acid. In still a further embodiment, A1, A3, and A7 independently, is β-Nal, o-X-Phe (where X is H, OH, CH3, halo, OCH3, NH2, CN, or NO2), p-X-Phe (where X is H, OH, CH3, halo, OCH3, NH2, CN, or NO2), m-X-Phe (where X is H, OH, CH3, halo, OCH3, NH2, CN, or NO2), F5-Phe, Trp, Dip, 2-Pal, Tyr(Bzl), His, Igl, Tyr(I), Bta, Bip, Npa, or Pal; A2 is D-β-Nal, D-o-X-Phe (where X is H, OH, CH3, halo, OCH3, NH2, CN, or NO2), D-p-X-Phe (where X is H, OH, CH3, halo, OCH3, NH2, CN, or NO2), D-m-X-Phe (where X is H, OH, CH3, halo, OCH3, NH2, CN, or NO2), D-F5-Phe, D-Trp, D-Dip, D-2-Pal, D-Tyr(Bzl), D-His, D-Igl, D-Tyr(I), D-Bta, D-Bip, D-Npa, or D-Pal; A6 is Thr, Ser, Tle, Thr(Bzl), Abu, Ala, Ile, Leu, Gly, Nle, β-Ala, Gaba, or Val; and A8 is the D- or L-isomer of Thr, Dip, F5-Phe, p-X-Phe (where X is H, OH, CH3, halo, OCH3, NH2, CN, or NO2), o-X-Phe (where X is H, OH, CH3, halo, OCH3, NH2, CN, or NO2), m-X-Phe (where X is H, OH, CH3, halo, OCH3, NH2, CN, or NO2), Tyr(Bzl), Igl, or β-Nal. In yet still a further embodiment, A1 is β-Nal or Phe, A2 is D-Cpa or D-Phe; A3 is Phe or Tyr; A6 is Abu, Thr, or Val; A7 is Phe; and A8 is Thr; R1 is H, CH3CO, 4-(2-hydroxyethyl)-1-piperazinylacetyl, or 4-(2-hydroxyethyl)-1-piperizineethanesulfonyl; R2 is H; and R3 is NH2.
In another further embodiment, A1 is a D-amino acid and A2 is a D-aromatic amino acid. In still a further embodiment, A1 and A2, independently, is D-β-Nal, D-o-X-Phe (where X is H, OH, CH3, halo, OCH3, NH2, CN, or NO2), D-p-X-Phe (where X is H, OH, CH3, halo, OCH3, NH2, CN, or NO2), D-m-X-Phe (where X is H, OH, CH3, halo, OCH3, NH2, CN, or NO2), D-F5-Phe, D-Trp, D-Dip, D-2-Pal, D-Tyr(Bzl), D-His, D-Igl, D-Tyr(I), D-Bta, D-Bip, D-Npa, or D-Pal; A3 and A7, independently, is β-Nal, o-X-Phe (where X is H, OH, CH3, halo, OCH3, NH2, CN, or NO2), p-X-Phe (where X is H, OH, CH3, halo, OCH3, NH2, CN, or NO2), m-X-Phe (where X is H, OH, CH3, halo, OCH3, NH2, CN, or NO2), F5-Phe, Trp, Dip, 2-Pal, His, Igl, Tyr(I), Bta, Bip, Npa, Tyr(Bzl), or Pal; A6 is Thr, Ser, Tle, Thr(Bzl), Abu, Ala, Ile, Leu, Gly, Nle, β-Ala, Gaba, or Val; and A8 is the D- or L-isomer of Thr, Dip, F5-Phe, p-X-Phe (where X is H, OH, CH3, halo, OCH3, NH2, CN, or NO2), o-X-Phe (where X is H, OH, CH3, halo, OCH3, NH2, CN, or NO2), m-X-Phe (where X is H, OH, CH3, halo, OCH3, NH2, CN, or NO2), Igl, Tyr(Bzl), or β-Nal. In yet still a further embodiments A1 is D-β-Nal or D-Phe; A2 is D-Cpa or D-Phe; A3 is Phe or Tyr; A6 is Thr or Val; A7 is Phe; and A8 is Thr; R1 is H, CH3CO, 4-(2-hydroxyethyl)-1-piperazinylacetyl, or 4-(2-hydroxyethyl)-1-piperizineethanesulfonyl; R2 is H; and R3 is NH2.
Examples of compounds of the present invention include the following:
With the exception of the N-terminal amino acid, all abbreviations (e.g., Ala or A2) of amino acids in this disclosure stand for the structure of —NH—CH(R)—CO—, wherein R is a side chain of an amino acid (e.g., CH3 for Ala). For the N-terminal amino acid, the abbreviation stands for the structure of ═N—CH(R)—CO—, wherein R is a side chain of an amino acid. Pen, β-Ala, Gaba, Nle, Nva, Pal, F5-Phe, 2,4-dichloro-Phe, Cpa, β-Nal, β-1-Nal, Abu, Dip, 2-Pal, Bip, Npa, Igl, Bta, Tle, Bpa, Iph, Cha, Thr(Bzl), Tyr(Bzl), and Aib are respective abbreviations of the following α-amino acids: penicillamine, 3-aminopropionic acid, 4-aminobutyric acid, norleucine, norvaline, β-[3-pyridyl]-alanine, β-[2,3,4,5,6-pentafluorophenyl]-alanine, β-[2,4-dichlorophenyl]-alanine, β-[4-chlorophenyl]-alanine, β-[2-napthyl]-alanine, β-[1-naphthyl]-alanine; 2-aminobutyric acid, 3,3′-diphenylalanine, β-[2-pyridyl]alanine, 4,4′-biphenylalanine, p-NO2-phenylalanine, 2-indanylglycine, 3-benzothienylalanine, α-[t-butyl]-glycine, 4-bromo-phenylalanine, 4-iodo-phenylalanine, β-(cyclohexyl)-alanine, O-benzyl-threonine, O-benzyl-tyrosine, and 2-aminoisobutyric acid. Tyr(I) refers to an iodinated tyrosine residue (e.g., 3-I-Tyr, 5-I-Tyr, 3,5-I-Tyr) wherein the iodine may be a radioactive isotope, e.g., I125, I127, or I131. An aliphatic amino acid is an α-amino acid having one or two side chains which, independently, are hydrocarbons, e.g., a straight or branched chain of 1-6 carbons. Examples of aliphatic amino acids include Ala, Aib, Val, Leu, Tle, Ile, Nle, Nva, or Abu. An aromatic amino acid is an α-amino acid the side chain of which has a neutral (e.g., not acidic or basic) aromatic substituent, e.g., a substituted or unsubstituted phenyl, naphthyl, or aromatic heterocycle group (e.g., pyridyl or indolyl). Examples of aromatic amino acids include Phe, p-X-Phe (where X is a halo (e.g., F, Cl, Br, or I), OH, OCH3, CH3, or NO2), o-X-Phe (where X is a halo, OH, OCH3, CH3, or NO2), m-X-Phe (where X is a halo, OH, OCH3, CH3, or NO2), His, Pal, Trp, β-Nal, 2,4-dichloro-Phe, Tyr(I), β-[3,4,5-trifluorophenyl]-alanine, Bta, β-[3-cyanophenyl]-alanine, β-[4-cyanophenyl]-alanine, β-[3′,4-difluorophenyl]-alanine, β-[3,5-difluorophenyl]-alanine, β-[2-fluorophenyl]-alanine, β-[4-thiazolyl]-alanine, Bip, Dip, Npa, Igl, Bpa, Iph, homophenylalanine, 2-Pal, β-[4-pyridyl]-alanine, β-[4-thiazolyl]-alanine, β-[2-thiazolyl]-alanine, para-(CF3)-phenylalanine, and F5-Phe. What is meant by an “Eaa” is an amino acid of the formula —NH—[CH(R)n—CO— (where n is 2-6 and R is H, lower alkyl, or hydroxy lower alkyl). Examples of an Eaa include β-Ala and Gaba.
As used herein, “lower alkyl” is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having 1-6 carbon atoms. Examples of lower alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, t-butyl, isobutyl, sec-butyl, and the like.
As used herein, “aryl” is intended to include any stable monocyclic, bicyclic, or tricyclic carbon ring(s) of up to 7 members in each ring, wherein at least one ring is aromatic. Examples of aryl groups include phenyl, naphthyl, anthracenyl, biphenyl, tetrahydronaphthyl, indanyl, phenanthrenyl, and the like.
The term “heterocyclyl”, as used herein, represents a stable 5- to 7-membered monocyclic or stable 8- to 11-membered bicyclic or stable 11-15 membered tricyclic heterocyclic ring which is either saturated or unsaturated, and which consists of carbon atoms and from one to four heteroatoms selected from the group consisting of N, O, and S, and including any bicyclic group in which any of the above-defined heterocyclic rings is fused to a benzene ring. The heterocyclic ring may be attached at any heteroatom or carbon atom which results in the creation of a stable structure. Examples of such heterocyclic elements include, but are not limited to, azepinyl, benzimidazolyl, benzisoxazolyl, benzofurazanyl, benzopyranyl, benzothiopyranyl, benzofuryl, benzothiazolyl, benzothienyl, benzoxazolyl, chromanyl, cinnolinyl, dihydrobenzofuryl, dihydrobenzothienyl, dihydrobenzothiopyranyl, dihydrobenzothio-pyranyl sulfone, furyl, imidazolidinyl, imidazolinyl, imidazolyl, indolinyl, indolyl, isochromanyl, isoindolinyl, isoquinolinyl, isothiazolidinyl, isothiazolyl, isothiazolidinyl, morpholinyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, piperidyl, piperazinyl, pyridyl, pyridyl N-oxide, quinoxalinyl, tetrahydrofuryl, tetrahydroisoquindlinyl, tetrahydro-quinolinyl, thiamorpholinyl, thiamorpholinyl sulfoxide, thiazolyl, thiazolinyl, thienofuryl, thienothienyl, thienyl, and the like.
The term “substituted” is meant to include the recited chemical group (e.g., lower alkyl, heterocycle, aryl, cycloalkyl, etc.) substituted with one to four of the recited substituents (e.g., halo, hydroxy, lower alkyl, etc.). The substituent may be attached to any atom in the chemical group.
The structure of 4-(2-hydroxyethyl)]-1-piperazinylacetyl and 4-(2-hydroxyethyl)]-1-piperizineethanesulfonyl are, respectively, as follows:
The compounds of this invention can be provided in the form of pharmaceutically acceptable salts. Acceptable salts include, but are not limited to, acid addition salts of inorganic acids such as hydrochloride, sulfate, phosphate, diphosphate, hydrobromide, and nitrate or organic acids such as acetate, maleate, fumarate, tartrate, succinate, citrate, lactate, methanesulfonate, p-toluenesulfonate, pamoate, salicylate, oxalate, and stearate. Also within the scope of the present invention, where applicable, are salts formed from bases such as sodium or potassium hydroxide. For further examples of pharmaceutically acceptable salts see, “Pharmaceutical Salts,” J. Pharm. Sci. 66:1 (1977).
Where the amino acid residue is optically active, it is the L-isomer that is intended unless otherwise specified. In the formulae set forth herein, the disulfide bond between the thiol group on the side chain of residue A2 (e.g., Cys, Pen, D-Cys, or D-Pen) and the thiol group on the side chain of residue A7 (e.g., Cys or Pen) is not shown.
The peptides of the invention can be used to promote the release of growth hormone or insulin in a subject (e.g., a mammal such as a human patient). Thus, the peptides are useful in the treatment of physiological conditions in which the promotion of the release of growth hormone or insulin is of benefit. The peptides of the invention can also be used in enhancing wound healing or promoting angiogenesis. Also, peptides of the invention having a Tyr(I) residue can be used to image cells containing somatostatin receptors. Such peptides of the invention can be used either in vivo to detect cells having somatostatin receptors (e.g., cancer cells) or in vitro as a radioligand in a somatostatin receptor binding assay. The peptide of the invention can also be used as vectors to target cells with radioactive isotopes.
A therapeutically effective amount of a peptide of this invention and a pharmaceutically acceptable carrier substance (e.g., magnesium carbonate, lactose, or a phospholipid with which the therapeutic compound can form a micelle) together form a therapeutic composition (e.g., a pill, tablet, capsule, or liquid) for administration (e.g., orally, intravenously, transdermally, pulmonarily, vaginally, subcutaneously, nasally, iontophoretically, or by intratracheally) to a subject in need of the peptide. The pill, tablet, or capsule can be coated with a substance capable of protecting the composition from the gastric acid or intestinal enzymes in the subject's stomach for a period of time sufficient to allow the composition to pass undigested into the subject's small intestine. The therapeutic composition can also be in the form of a biodegradable or nonbiodegradable sustained release formulation for subcutaneous or intramuscular administration. See, e.g., U.S. Pat. Nos. 3,773,919 and 4,767,628 and PCT Application No. WO 94/00148. Continuous administration can also be obtained using an implantable or external pump (e.g., INFUSAID™ pump) to administer the therapeutic composition.
The dose of a peptide of the present invention for treating the above-mentioned diseases or disorders varies depending upon the manner of administration, the age and the body weight of the subject, and the condition of the subject to be treated, and ultimately will be decided by the attending physician or veterinarian. Such an amount of the peptide as determined by the attending physician or veterinarian is referred to herein as a “therapeutically effective amount.”
Also contemplated within the scope of this invention is a peptide covered by the above generic formula for both use in treating diseases or disorders associated with the need to promote the release of growth hormone or insulin, and use in detecting somatostatin receptors, e.g., radioimaging.
Other features and advantages of the present invention will be apparent from the detailed description and from the claims.
It is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Also, all publications, patent applications, patents, and other references mentioned herein are incorporated by reference.
Synthesis
The synthesis of short peptides is well examined in the peptide art. See e.g., Stewart, et al., Solid Phase Peptide Synthesis (Pierce Chemical Co., 2d ed., 1984). The following describes the synthesis of D-β-Nal-Cys-Pal-D-Trp-Lys-Val-Cys-β-Nal-NH2 and D-β-Nal-Cpa-Tyr-D-Trp-Lys-Val-Phe-Thr-NH2. Other peptides of the invention can be prepared in an analogous manner by a person of ordinary skill in the art.
Benzhhydrylamine-polystyrene resin (Advanced ChemTech, Inc., Louisville, Ky.) (1.2 g; 0.5 mmole) in the chloride ion form was placed in the reaction vessel of an Advanced ChemTech™ peptide synthesizer programmed to perform the following reaction cycle: (a) methylene chloride; (b) 33% trifluoroacetic acid in methylene chloride (2 times for 1 and 25 min each); (c) methylene chloride; (d) ethanol; (e) methylene chloride; and (f) 10% triethylamine in chloroform.
The neutralized resin was stirred with Boc-O-β-naphthylalanine and diisopropylcarbodiimide (1.5 mmole each) in methylene chloride for 1 hr, and the resulting amino acid resin was then cycled through steps (a) to (f) in the above wash program. The following amino acids (1.5 mmole) were then coupled successively by the same procedure: Boc-S-methylbenzyl-Cys, Boc-Val, Boc-Ne-benzyloxycarbonyl-lysine, Boc-D-Trp, Boc-Pal, and Boc-S-methylbenzyl-D-Cys and Boc-β-Nal. After washing and drying, the completed resin weighed 2.0 g.
The completed resin described in (1) (1.0 g, 0.25 mmole) was mixed with anisole (5 ml), dithiothreitol (100 mg), and anhydrous hydrogen fluoride (35 ml) at 0° C. and stirred for 45 min. Excess hydrogen fluoride was evaporated rapidly under a stream of dry nitrogen, and the free peptide is precipitated and washed with ether. The crude peptide was then dissolved in 500 ml of 90% acetic acid to which was added a concentrated solution of I2/MeOH until a permanent brown color is observed. Excess I2 is removed by addition of ascorbic acid and the solution evaporated to a small volume which was applied to a column (2.5×90 cm) of Sephadex™ G-25, which was eluted with 50% AcOH. Fractions containing a major component by ultraviolet (UV) absorption and thin layer chromatography were then pooled, evaporated to a small volume, and applied to a column (1.5×70 cm) of Vydac™ octadecylsilane silica (10-15 μm). This was eluted with a linear gradient of acetonitrile in 0.1% trifluoroacetic acid in water. Fractions were examined by thin layer chromatography (TLC) and analytical high performance liquid chromatography (HPLC) and pooled to give maximum purity. Repeated lyophilization of the solution from water gave the desired product as a white, fluffy powder. The product was found to be homogeneous by HPLC and TLC. Amino acid analysis of an acid hydrolysate and matrix-assisted laser desorption (MALD) mass spectroscopy confirmed the composition of the octapeptide.
Benzhydrylamine-polystyrene resin (Advanced ChemTech™, Inc.) (1.2 g, 0.5 mmole) in the chloride ion form was placed in the reaction vessel of an Advanced ChemTech peptide synthesizer programmed to perform the following reaction cycle: (a) methylene chloride; (b) 33% trifluoroacetic acid in methylene chloride (2 times for 1 and 25 min each); (c) methylene chloride; (d) ethanol; (e) methylene chloride; and (f) 10% triethylamine in chloroform.
The neutralized resin was stirred with Boc-O-benzylthreonine and diisopropylcarbodiimide (1.5 mmole each) in methylene chloride for 1 hr and the resulting amino acid resin was cycled through steps (a) to (f) in the above wash program. The following amino acids (1.5 mmole) were then coupled successively by the same procedure: Boc-phenylalanine, Boc-Val, Boc-Ne-benzyloxycarbonyl-lysine, Boc-D-Trp, Boc-O-dichlorobenzyl-Tyr, and Boc-D-4-chlorophenylalanine, and Boc-β-D-Nal. After washing and drying, the completed resin weighed 2.1 g.
The peptide resin from (1) was subjected to HF cleavage as described above. Column purification as described yielded the desired compound as a white, fluffy powder (170 mg) which is found to be homogeneous by HPLC and TLC. Amino acid analysis of an acid hydrolysate and MALD mass spectroscopy confirms the composition of the peptide.
Peptides containing C-terminal substituted amides can be made by solid phase methods by displacing the appropriate peptide off the solid phase with the corresponding amine. Alternatively, these analogs may be synthesized by solution-phase peptide synthesis methods in which the growing peptide chain is maintained in solution in an organic solvent during synthesis and assembled by iterative coupling/deprotection cycles. Final removal of the side chain protecting groups yields the desired peptide after appropriate purification. Peptides containing N-terminal substitutions (e.g., where R1 is E, CO, or E1SO2 (where E1 is heterocycle lower alkyl) substituted with hydroxy lower alkyl and R2 is H such as 4-(2-hydroxyethyl)-1-piperazinylacetyl or 4-(2-hydroxyethyl)-1-piperidineethanesulfonyl) can be synthesized as described in PCT Application No. WO 95/04752.
Bioassay on the In Vitro Release of Growth Hormone
(a) Rat Pituitary Cell Dispersion
Pituitaries from adult Charles River CD male rats (Wilmington, Mass.) housed under controlled conditions were dispersed and cultured using aseptic technique by modification of previously described methods (Hoefer, M. T., et al., Mol. Cell. Endocrinol. 35:229 (1984); Ben-Jonathan, N., et al., Methods Enzymol. 103:249 (1983); and Heiman, M. L., et al., Endocrinology 116:410 (1985)). Pituitaries were removed from sacrificed rats, sectioned, and then placed into a siliconized, liquid scintillation vial containing 2 ml 0.2% trypsin (Worthington Biochemicals, Freehold, N.J.) in sterile-filtered Krebs-Ringer bicarbonate buffer supplemented with 1% bovine serum albumin, 14 mM glucose, modified Eagle medium (MEM) vitamin solution, and MEM amino acids (Gibco Laboratories, Grand Island, N.Y.) (KRBGA). All glassware was siliconized as described by Sayers, et al., Endocrinology 88:1063 (1971). The fragments were incubated in a water bath for 35 min at 37° C. with agitation. The vial contents then were poured into a scintillation vial containing 2 ml 0.1% DNase (Sigma Chemical Co., St. Louis, Mo.) in KRBGA and incubated for 2 min at 37° C. with agitation. After incubation, the tissue was decanted into a 15 ml centrifuge tube and allowed to settle. Medium was discarded, and pituitary sections were washed 3 times with 1 ml fresh-KRBGA. The cells were then dispersed in 2 ml 0.05% LBI (lima bean trypsin inhibitor, Worthington Biochemicals) by gently drawing the fragments into and expelling them out of a siliconized, fire polished Pasteur pipette. Dispersed cells were then filtered through a 630 μm diameter Nylon mesh (Tetko, Elmsford, N.Y.) into a fresh 15 ml centrifuge tube. An additional 2 ml of 0.05% LBI solution was used to rinse the first tube and was transferred to the second tube with filtering.
(b) Cell Culture
The dispersed cells were then further diluted with approximately 15 ml sterile-filtered Dulbecco's modified Eagle medium (GIBCO), which was supplemented with 2.5% fetal calf serum (GIBCO), 3% horse serum (GIBCO), 10% fresh rat serum (stored on ice for no longer than 1 hr) from the pituitary donors, 1% MEM non-essential amino acids (GIBCO), and gentamycin (10 ng/ml; Sigma) and nystatin (10,000 U/ml; GIBCO). The cells were poured into a 50 ml round-bottomed glass extraction flask with a large diameter opening and then randomly plated at a density of approximately 200,000 cells per well (Co-star cluster 24; Rochester Scientific Co., Rochester, N.Y.). The plated cells were maintained in the above Dulbecco's medium in a humidified atmosphere of 95% air and 5% CO2 at 37° C. for 4-5 days.
(c) Experimental Incubation and IC50 Determination
In preparation for a hormone challenge, the cells were washed 3 times with medium 199 (GIBCO) to remove old medium and floating cells. Each treatment well contained a total volume of 1 ml medium 199 containing 1% BSA (fraction V; Sigma) with treatments as described below. Each antagonist candidate was tested using a single 24-well cell culture plate. Each treatment was performed in triplicate. Each plate contained 8 treatment groups: one 1 nM growth hormone releasing factor (GRF) (1-29)NH2-stimulated control group; one 1 nM somatostatin-inhibited control group in the presence of 1 nM GRF(1-29)NH2; and 6 doses of a given antagonist in the presence of both 1 nM SRIF and 1 nM GRF per plate. After 3 hrs at 37° C. in a air/carbon dioxide atmosphere (95/5%), the medium was removed and stored at −20° C. until radioimmunoassayed for growth hormone content. IC50's of each antagonist versus 1 nM SRIF were calculated using a computer program (SigmaPlot, Jandel Scientific, San Rafael, Calif.) with the maximum response constrained to the value of the 1 nM GRF(1-29)NH2-stimulated control. These IC50's are presented in Table I.
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, that the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the claims.
This application is a continuation (and claims the benefit of priority under 35 USC 120) of U.S. application Ser. No. 10/712,081, filed Nov. 13, 2003 now U.S. Pat. No. 7,550,423 which is a divisional of U.S. application Ser. No. 09/670,249, filed Sep. 26, 2000, now U.S. Pat. No. 6,703,481, which is a continuation of U.S. application Ser. No. 08/855,204, filed May 13, 1997, now U.S. Pat. No. 6,262,229 that claims the benefit of provisional application No. 60/032,358, filed Dec. 4, 1996. The disclosures of the prior applications are considered part of (and are incorporated by reference in) the disclosure of this application.
| Number | Name | Date | Kind |
|---|---|---|---|
| 4395403 | Bauer et al. | Jul 1983 | A |
| 4435385 | Bauer et al. | Mar 1984 | A |
| 4650787 | Schally et al. | Mar 1987 | A |
| 4728638 | Bauer et al. | Mar 1988 | A |
| 4853371 | Coy et al. | Aug 1989 | A |
| 4904642 | Coy et al. | Feb 1990 | A |
| 5269175 | Chmiel et al. | Dec 1993 | A |
| 5462926 | Coy et al. | Oct 1995 | A |
| 5506339 | Coy et al. | Apr 1996 | A |
| 5597894 | Coy et al. | Jan 1997 | A |
| 5633263 | Coy et al. | May 1997 | A |
| 5846934 | Bass et al. | Dec 1998 | A |
| 5965744 | Weichert et al. | Oct 1999 | A |
| 6262229 | Coy et al. | Jul 2001 | B1 |
| 7862825 | Vranic et al. | Jan 2011 | B2 |
| 20070259811 | Degli Uberti et al. | Nov 2007 | A1 |
| 20090004195 | Vranic et al. | Jan 2009 | A1 |
| 20110064742 | Vranic et al. | Mar 2011 | A1 |
| Number | Date | Country |
|---|---|---|
| 0 029 579 | Nov 1980 | EP |
| 0 203 031 | Apr 1985 | EP |
| 0 187 622 | Dec 1985 | EP |
| 0 214 872 | Mar 1987 | EP |
| 0 389 180 | Mar 1990 | EP |
| 0 395 417 | Oct 1990 | EP |
| 0 215 171 | Nov 1990 | EP |
| 0 791 603 | Aug 1997 | EP |
| 217635 | Mar 2000 | HU |
| WO 8805052 | Jul 1988 | WO |
| WO 8904666 | Jun 1989 | WO |
| WO 9012811 | Nov 1990 | WO |
| WO 9317037 | Sep 1993 | WO |
| WO 9711962 | Apr 1997 | WO |
| Entry |
|---|
| Hoyer et al.; “Molecular Pharmacology of Somatostatin Receptors”; Naunn-Schmiedeberg's Arch. Pharmacol; 350; pp. 441-453; (1993). |
| Raynor et al.; “Cloned Somatostatin Receptors: Identification of Subtype-Selective Peptides and Demonstration of High Affinity Binding of Linear Peptides”, Molecular Pharmacology, 43; pp. 838-844; (1993). |
| Osapay et al.; “Lanthionine-Somatostatin Analogs: Synthesis, Characterization, Biological Activity, and Enzymatic Stability Studies”; J. Med. Chem.; 40(14); pp. 2241-2251; (1997). |
| Melacini et al.; “A Refined Model for the Somatostation Pharmacophore: Conformational Analysis of Lanthionine-Sandostatin Analogs”; J. Med. Chem. 40(14); pp. 2252-2251; (1997). |
| Lamberts et al.; “The Role of Somatostation and Its Analogs in the Diagnosis and Treatment of Tumors”; Endocrine Reviews, vol. 12 No. 4; pp. 450-482; (1991). |
| Reisine et al.; “Molecular Biology of Somatostation Receptors”; Endocrine Reviews; vol. 16 No. 4; pp. 427-442; (1995). |
| Patel et al.; “The Somatostation Receptor Family”; Life Sciences; vol. 57 No. 13; pp. 1249-1265; (1995). |
| Hofland et al.; “Somatostation Analogs”; Clinical Application in Relation to Human Somatostation Receptor Subtypes; Biomedical Pharmacology; vol. 50 No. 3; pp. 287-297; (1995). |
| Reisine et al.; “Molecular Properties of Somatostatin Receptor”; Neuroscience; vol. 67 No. 4; pp. 777-790; (1995). |
| Bass et al.; “Identification and Characterization of Novel Somatostatin Antagonists”; Molecular Pharmacology; 50 pp. 709-715; (1996). |
| Patel et al.; “Subtype Selectiveity of Peptide Analogs for All Five Cloned Human Somatostatin Receptors”; Endocrinology; vol. 135 No. 6; pp. 2814-2817; (1994). |
| Heiman et al.; “Differential Binding of Somatostatin Agonists to Somatostatin Receptors in Brain and Adenohypophysis”, Neuroendocrinology; 45 pp. 429-436; (1987). |
| Lloyd, et al.; “Activation of somatostatin receptor subtype 2 inhibits acid secretion in rats”, Am. J. Physiol; 268: pp. G102-G106; (1995). |
| Rossowski et al.; “Potent inhibitory effects of a type four receptor-selective somatostatin analog on rat insulin release”, Biochemical and Biophysical Research Comm.; 197: pp. 366-371; (1993). |
| Coy, D. H., et al., “Receptor-specific somatostatin analogs: Correlations with biological activity,” Metabolism, 1996, 45(8):21-23. |
| Fries, J. L., et al., “Somatostatin antagonist analog increases GH, insulin, and glucagons release in the rat,” Peptides, 1982, 3:811-814. |
| Schally, A. V., et al., “Somatostatin analogs as adjuncts to agonists of luteinizing hormone-releasing hormone in the treatment of experimental prostate cancer,” PNAS, 1987, 84:7275-7279. |
| Number | Date | Country | |
|---|---|---|---|
| 20090253638 A1 | Oct 2009 | US |
| Number | Date | Country | |
|---|---|---|---|
| 60032358 | Dec 1996 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 09670249 | Sep 2000 | US |
| Child | 10712081 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10712081 | Nov 2003 | US |
| Child | 12422625 | US | |
| Parent | 08855204 | May 1997 | US |
| Child | 09670249 | US |
