RECEPTOR-SELECTIVE SOMATOSTATIN ANALOGS

Abstract: Analogs of SRIF which are selective for SSTR1 in contrast to the other cloned SRIF receptors. These analogs are useful in determining the tissue and cellular expression of the receptor SSTR1 and its biological role in the endocrine, exocrine and nervous system, as well as in regulating tumor growth. SRIF analog peptides, such as des-AA<1,2,5>[D-Trp<8>, IAmp<9>]-SRIF inhibit the binding of [<125>I-Tyr<11>]SRIF to the cloned human receptor SSTR1 but do not bind to mouse SSTR2 and SSTR3, human SSTR4 or rat SSTR5. By incorporating an iodinated tyrosine in position-2 in these selective SRIF analogs, a labelled compound useful in drug-screening methods is provided.

Description: RECEPTOR-SELECTIVE SOMATOSTATIN ANALOGS 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 shortened somatostatin analogs, to pharmaceutical compositions containing such peptides, to methods of diagnosis and treatment of mammals using such peptides and to drug screening methods using such peptides. BACKGROUND OF THE INVENTION The cyclic tetradecapeptide somatostatin-14 (SRIF) was originally isolated from the hypothalamus and characterized as a physiological inhibitor of growth hormone release from the anterior pituitary. It was characterized by Guillemin et al. and is described in U.S. Patent No. 3,904,594 (September 9, 1975). This tetradecapeptide has a bridging or cyclizing bond between the sulfhydryl groups of the two cysteinyl amino acid residues in the 3- and 14-positions. SRIF was found to also regulate insulin, glucagon and amylase secretion from the pancreas, and gastric acid release in the stomach. SRIF is also expressed in intrahypothalaπic regions of the brain and has a role in the regulation of locomotor activity and cognitive functions. SRIF is localized throughout the central nervous system, where it acts as a neurotransmitter.- In the central nervous system, SRIF has been shown to both positively and negatively regulate neuronal firing, to affect the release of other neurotransmitters, and to modulate motor activity and cognitive processes. 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. One such analog is [D-Trp 8 ]-SRIF which has the amino acid 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). Somatostatin 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 somatostatin is being sold commercially in Europe for the treatment of ulcer patients. The powerful inhibitory effects of somatostatin on the secretion not only of GH but also of insulin and glucagon have led to studies of a possible role of somatostatin in the management or treatment of juvenile diabetes and have proved useful in studying the physiological and pathological effects of these hormones on human metabolism. . Pradayrol , et al. in FEBS Letters 109. January 1980, pages 55-58, reported the isolation and characterization of somatostatin-28 (SRIF-28) from porcine upper small intestine. SRIF-28 is an N-terminally extended version of SRIF which has an additional 14 amino acid residues and which shows some increased potency when administered in vivo. SRIF affects multiple cellular processes. Studies have shown that SRIF is an inhibitory regulator of adenylyl cyclase in different tissues. SRIF also regulates the conductance of ionic channels, including both K * and Ca 2* channels. These actions of SRIF are mediated via pertussis toxin-sensitive guanine nucleotide-binding proteins. SRIF also regulates the activity of tyrosine phosphatases, the Na + /H + antiport, and cellular proliferation through pertussis toxin-insensitive mechanisms. SRIF induces its biological effects by interacting with a family of membrane-bound structurally similar receptors. Five SRIF receptors have fairly recently been cloned and are referred to as SSTR1-5. Human SSTRl, mouse SSTR2 and mouse SSTR3 are described in Raynor et al. , Molecular Pharmacology, 43. 838-844 (1993) . Human SSTRl, 2 and 3 are disclosed in U.S. Patent No. 5,436,155 (7/25/95) . All five receptors, including the human SSTR4 and the rat SSTR5, bind SRIF and SRIF-28 with high affinity. Selective agonists at SSTR2 and SSTR5 have been identified and used to reveal distinct functions of these receptors. SSTR2 is believed to mediate the inhibition of growth hormone, glucagon and gastric acid secretion. In contrast, SSTR5 5 appears to be primarily involved in the control of insulin and amylase release. SSTR3 is believed to mediate inhibition of gastric smooth muscle contraction by SRIF. These findings indicate that different receptor subtypes appear to mediate distinct functions of SRIF in the body. 10 SRIF has also now been found to inhibit tumor cell proliferation, and a cyclic SRIF analog, termed SMS-201- 995, i.e. D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol is being used clinically to inhibit tumor growth and as a diagnostic agent to detect SRIF receptors expressed in cancers. SMS- 15 201-995 and other clinically used SRIF analogs interact significantly with three of the receptor subtypes, i.e. SSTR2, SSTR3 and SSTR5. SSTR2 and SSTR5 have recently been reported to mediate antiproliferative effects of SRIF on turner cell growth; therefore, they may mediate the clinical 0 effects of SMS-201-955 in humans. SSTRl was the first SRIF receptor cloned. It has high affinity for SRIF and SRIF-28, but it has very low affinity for most synthetic analogs of SRIF. SSTRl mRNA is expressed in the central nervous system as well as in a 5 number of endocrine and exocrine organs, and SSTRl mRNA is particularly high in a number of tumors. The cloned SSTRl has been reported to mediate antiproliferative effects of SRIF possibly by modulating the activity of a Na+/H+ ion exchanger or through the stimulation of a tyrosine 0 phosphatase. SSTRl is therefore considered to be an important target both for the physiological actions of SRIF and for the therapeutic actions of SRIF analogs to inhibit tumor cell growth. The search has continued for somatostatin analogs 5 which are more potent than somatostatin and/or exhibit dissociated inhibitory functions, and particularly for analogs which are selective for SSTRl. However, no ligand has been available that selectively binds to SSTRl, and efforts to determine the precise localization of SSTRl in the body and to identify its biological actions have been hindered by the lack of selective SSTRl ligands. SUMMARY OF THE INVENTION Certain modifications have now been discovered which are effective to create analogs of SRIF that are selective for SSTRl in contrast to the other cloned SRIF receptors. SRIF agonist peptides have been created that bind specifically to cloned SSTRl, and analogs of these peptides can be iodinated while retaining their desirable biological properties. These novel peptides 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 tumor growth and certain other pharmacological functions for which SRIF has previously Been used without the accompanying side effects heretofore characteristic of administering SRIF. The SRIF analog peptides of the invention inhibit the binding of [ 125 I-Tyr 11 ]SRIF to the cloned human receptor SSTRl, but they do not bind to mouse SSTR2 and SSTR3, human SSTR4 or rat SSTR5. Additional of these SRIF analogs which incorporate an iodinated tyrosine in position-2 alεo do not bind to SSTR2-5 but still bind potently and saturably to SSTRl. The binding of these SRIF analogs to SSTRl expressed in COS-7 cells is reduced by GTPgS and pertussis toxin treatment, whereas the binding of [ 125 I-Tyr 11 ]SRIF to SSTRl (to which it also binds strongly) is not affected by these treatments, indicating that these SRIF analogs bind to SSTRl in a different manner than the native peptide SRIF. Many of these SRIF analogs not only selectively bind to SSTRl, but they bind thereto with high affinity. By selectively binding is meant that they exhibit a K d or an IC 50 with SSTRl of about one-tenth or less of that with respect to at least 3 of the other SRIF receptors. They are useful in localizing such receptor in the body and in diagnosing for tumors which express the SSTRl receptor. Moreover, they are particularly useful in combatting tumor growth which is mediated by the SSTRl receptors because such can be carried out without the side effects that would otherwise accompany administration of currently available SRIF analogs which interact with a plurality of SRIF receptors. Certain of these SRIF analogs can be readily labelled and used in effective drug screening methods. They can alεo be used in the selective purification of SSTRl receptors that can then be employed in drug-screening tests. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. The nomenclature used to define the peptides is that specified by Schroder & Lubke, "The Peptides", Academic Press (1965) wherein, in accordance with conventional representation, the amino group appears to the left and the carboxyl group to the right. The standard 3-letter abbreviations to identify the alpha-amino acid residues, and where the amino acid residue has isomeric forms, it is the L-form of the amino acid that is represented unless otherwise expressly indicated, e.g. Ser = L-serine. By D,L is meant a mixture of the D- and L- isomers of a particular α-amino acid. SRIF analog peptides are provided having a selective affinity for the SRIF receptor SSTRl; the preferred analogs also have a high affinity for SSTRl, i.e. equal to a K d of about 10 nanomolar or less. These peptides broadly encompass known analogs of SRIF or obvious variations thereof which have a D-isomer, preferably an aromatic D- isomer, and more preferably D-Trp or D-Nal, in the position corresponding to the 8-position of the native peptide and an aπinomethylPhe residue, which may be alkylated, in the adjacent position that corresponds to the 9-position of the native peptide. So long as the basic molecule exhibits SRIF properties by binding to SRIF receptors, insertion of this combination of residues in the corresponding 8- and 9- positions will create a molecule which is highly selective for the SSTRl receptor. Preferably the 5-position residue is also deleted to increase binding affinity to SSTRl. Since the characterization of SRIF, a large number of SRIF analogs have been synthesized having increased potency in some respect, which can be rendered selective for the SSTRl receptor by the incorporation of the modification of the present invention, and the following U.S. patents are illustrative of such SRIF analogs: Re. 30,548; 4,133,782; 4,211,693; 4,316,891; 4,372,884; 4,393,050; 4,061,608; 4,081,433; 4,182,707; 4,190,575; 5,185,010; 4,215,039; 4,230,617; 4,238,481; 4,253,998; 4,282,143; 4,328,214; 4,358,439; 4,209,441; 4,210,636; 4,316,890; and 5,073,541. Examples of some representative peptides exhibiting desired features of the invention include those having the following amino acid sequence, which is based upon a numbering system consistent with the 14-residue sequence of native mammalian SRIF: (cyclo 3-14 ) Xaa 1 -Xaa 2 -Cys-Lys-Xaa 5 -Phe-Phe-D-Xaa 8 -Xaa 9 -Thr- Phe-Thr-Xaa 13 -Cys wherein Xaa, is des-Xaa or Ala; Xaa ∑ is Tyr, D-Tyr or des-Xaa or Gly; Xaa 5 is des-Xaa or Asn; D- Xaa 8 is a D-amino acid; Xaa 9 is an aminomethyl Phe; and Xaa 13 is Ser or D-Ser. Xaa 5 is preferably des-Xaa. D-Xaa 8 should be aromatic and may be D-Trp or a substituted D-Trp, or an equivalent reεidue such as D-Nal. Xaa 9 includes a Phe residue that has an aminomethyl substitution on the phenyl ring, i.e. Amp, preferably in the 3- or 4-position on the ring, and most preferably Xaa 9 includes (C)4Amp where C is hydrogen or lower alkyl (C, to C 5 ) group. More preferably C is methyl, ethyl, isopropyl or isobutyl, and most preferably C is isopropyl. A preferred subgenus of SRIF agonists has the amino acid sequence: (cyclo 3-14) Xaa,-Xaa 2 -Cys-Lys-Xaa 5 -Phe-Phe-D-Xaa 8 -Xaa 9 -Thr- Phe-Thr-Xaa, 3 -Cys wherein Xaa, is des-Xaa; Xaa 2 is Tyr, D- Tyr or des-Xaa; Xaa 5 is des-Xaa; D-Xaa 8 is either D-Trp or a substituted D-Ala wherein one hydrogen on the 3-carbon is replaced by: (a) a carbocyclic aryl-containing radical selected from the group consisting of phenyl substituted with three or more straight chain lower alkyl groups, naphthyl, pyridyl, anthryl , fluorenyl, phenanthryl, biphenylyl and benzhydryl; or (b) a saturated carbocyclic radical selected from the group consisting of cycloalkyl substituted with three or more straight chain lower alkyl groups, perhydronaphthyl , perhydrobiphenylyl , perhydro- 2 , 2-diphenylmethyl , and adamantyl; Xaa, is an aminomethyl Phe that is optionally (C, to C 5 ) alkylated; and Xaa, 3 is Ser or D-Ser. Another preferred subgenus of SRIF agonist peptides haε the amino acid sequence: (cyclo 3-14 ) Xaa 1 -Xaa 2 -Cys-Lys-Xaa 5 -Phe-Phe-D-Xaa 8 ~Xaa 9 -Thr- Phe-Thr-Xaa, 3 -Cys wherein Xaa, is des-Xaa; Xaa 2 is Tyr, D- Tyr or des-Xaa; Xaa 5 is des-Xaa; D-Xaa 8 is either D-Trp or a D-Ala wherein one hydrogen on the /3-carbon is replaced by: (a) a carbocyclic aryl-containing radical selected from the group consiεting of phenyl substituted with three or more straight chain lower alkyl groups, naphthyl, pyridyl, anthryl, fluorenyl, phenanthryl, biphenylyl and benzhydryl; or (b) a saturated carbocyclic radical selected from the group consisting of cycloalkyl substituted with three or more straight chain lower alkyl groups, perhydronaphthyl, perhydrobiphenylyl, perhydro- 2 , 2-diphenylmethyl, and adamantyl. Xaa 9 is a (C 3 or CJ alkylated aminomethyl Phe; and Xaa, 3 is Ser or D-Ser. An additional preferred subgenus of SRIF agonist peptides has the amino acid sequence: (cyclo 3-14)Xaa,-Xaa 2 -Cys-Lys-Xaa 5 -Phe-Phe-D-Xaa 8 -Xaa 9 -Thr- Phe-Thr-Xaa, 3 -Cyε wherein Xaa, is des-Xaa; Xaa 2 is Tyr, D- Tyr or des-Xaa; Xaa 5 is des-Xaa; D-Xaa 8 is (A)D-Trp where A is H, F, Cl or Br, or (B)D-Ala or (B)D,L-Ala where B is naphthyl, pyridyl, fluorenyl, adamantyl, anthryl, biphenyl, tri (lower alkyl) phenyl , pentamethylphenyl, phenanthryl, trialkylcyclohexyl , perhydronaphthyl or perhydrobiphenyl; Xaa 9 is a (C 3 or CJ alkylated aminomethyl Phe; and Xaa, 3 is Ser or D-Ser. Still another preferred εubgenuε of SRIF agoniεt peptides has the amino acid sequence: (cyclo 3-14)Xaa,-Xaa 2 -Cys-Lys-Xaa 5 -Phe-Phe-D-Xaa 8 -Xaa 9 -Thr- Phe-Thr-Xaa, 3 -Cys wherein Xaa, is deε-Xaa; Xaa 2 iε Tyr, D- Tyr or deε-Xaa; Xaa 5 iε des-Xaa; D-Xaa 8 is D-Trp, D-lNal, D-Me5-Phe, D-TMP, D-BIA, D-TBA, -D-anthryl-Ala, D- fluorenyl-Ala or D-adamantyl-Ala; Xaa 9 is isopropyl-4- aminomethyl Phe; and Xaa, 3 is Ser or D-Ser. By 9-D-Nal is meant the D-isomer of alanine which is substituted by naphthyl on the 0-carbon atom; it is alternatively referred to as 3-D-Nal. Preferably 3-D-2Nal is employed wherein the attachment to naphthalene is at the 2-position on the ring structure; however, 3-D-lNal may also be used. Pal representε alanine which is substituted by pyridyl on the 3-carbon atom; preferably the linkage is to the 3-position on the pyridine ring. When εubstituted D-Trp is employed, single substitutionε for hydrogen are preferably made in either the 5- or 6-position, which are selected from chloro, fluoro, bromo and nitro, with chloro, fluoro and nitro being preferred. Alternatively, the indole nitrogen may be acylated with formyl (N ,n For- or lFor-) . By Amp is meant (aminomethyl)phenylalanine; unless otherwise specified, the methyl group with its amino substitution should be understood to be in the 4- or para- position on the phenyl ring. By IAmp is meant (N- isopropyl-aminomethyl)phenylalanine, where the amino group is alkylated with an isopropyl group. By D-Me5Phe is meant 3-(2 , 3,4 ,5, 6-pentamethylphenyl) -D-Ala. By D-TMP is meant 3-(2,4 , 6-trimethylphenyl)-D-Ala. By D-BIA is meant 3- (benzimidazol-2-yl) -D-Ala. By D-TBA is meant 3-(4, 5,6,7- tetrahydrobenzimidazol-2-yl) -D-Ala. As used herein, the term "lower alkyl" refers to a straight or branched chain, saturated hydrocarbon group having from 1 to 5 carbon atomε εuch as, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec- butyl, tert-butyl, isopentyl and n-pentyl ; the term "cycloalkyl group" refers to a cyclic saturated hydrocarbon group having from 4 to 6 carbon atoms, e.g. cyclobutyl, cyclopentyl and cyclohexyl. As used herein, "naphthyl" is inclusive of 1- and 2-naphthyl; "anthryl" is inclusive of 1-, 2- and 9-anthryl; "fluorenyl" is inclusive of 2-,3-,4- and 9-fluorenyl; "phenanthryl" is inclusive of 2-, 3- and 9- phenanthryl ; and "adamantyl" iε inclusive of 1- and 2- adamantyl . Although SSTRl was the first somatostatin receptor cloned, identification of its biological and pharmacological properties has lagged behind the other SRIF receptors because of the lack of ligands which are significantly selective for SSTRl. The peptides of the invention are believed to be the first truly SSTR1- selective ligands, which will accordingly be most useful in determining the functional roles of this receptor and in selectively activating only this SRIF receptor and not the others. Selectivity for binding of the peptides of the invention to SSTRl was demonstrated by testing their interaction with the five different cloned SRIF receptors. Recombinant cells expressing the receptor may be washed and homogenized to prepare a crude protein homogenate in a suitable buffer as known in the art. In a typical assay, 5 an amount of protein from the cell homogenate is placed into a small volume of an appropriate assay buffer at an appropriate pH. Candidate substances, such as potential agonists and antagonists, are added to the admixture in convenient concentrations and the interaction between the 0 candidate substance and the receptor polypeptide is monitored. The peptides of the invention bound substantially only to SSTRl, and their binding was of high affinity. Receptor binding assays were performed on cloned SRIF 5 receptors as generally set forth in Raynor et al. supra to generate K d values which are indicative of the concentration of a ligand necesεary to occupy one-half (50%) of the binding sites on a εelected amount of a - receptor or the like. Alternatively, IC 50 values 0 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 can also be calculated in such competitive assays. The peptide des-AA 1 - ' 5 -[D-Trp 8 , IAmp°]SRIF inhibits the binding of [ 125 I- 5 Tyr 11 ]SRIF to the cloned human SSTRl with an IC 50 of 1.8 + 0.7 nM. This SRIF analog peptide does not bind to mouse SSTR2 , mouse SSTR3 , human SSTR4 or rat SSTR5 at concentrations below 100 nM. An analog of des-AA 1,2,5 -[D-Trp 8 , IAmp°]SRIF having a 0 tyrosine residue in position-2 was iodinated and tested for binding to the cloned SRIF receptors. ^I-Tyr^des-AA 1 ' 5 - [D-Trp s , IAmp°]SRIF likewise did not bind to SSTR2-5, but it bound potently and εaturably to SSTRl with a Kd of 0.5 ± 0.1 nM and a B max of 226 ± 56 fmol/mg protein. The B |ηaχ 5 value represents the highest number of specific sites which can be occupied at a given time, i.e. the saturation point. The binding of 125 I-Tyr 2 -des-AA 1 - 5 -[D-Trp β f IAmp 9 ]SRIF to SSTRl was potently inhibited by SRIF, [D-Trp 8 ]SRIF and SRIF-28, with IC 50 values of 0.8 ± 0.4, 0.2 ± 0.1 and 0.7 + 0.2 nM, respectively. Furthermore the SRIF analog des- AA 1 ' 2 - 5 -[D-Trp 8 , IAmp 9 , D-Ser 13 ]SRIF potently bound to SSTRl with an IC 50 value of 5.3 + 0.8 nM compared to 125 I-Tyr 2 -des- AA 1<5 -[D-Trp s , IAmp'jSRIF. In contrast, SRIF analogs that selectively bind to SSTR2 and SSTR5 were impotent in displacing 125 I-Tyr 2 -des-AA 1<5 -[D-Trp 8 , IAmp°]SRIF from SSTRl. The binding of 125 I-Tyr 2 -des-AA 1 - 5 -[D-Trp 8 , IAmp°]SRIF to SSTRl expressed in COS-7 cells was reduced by GTPgS and pertussis toxin treatment; in contrast, the binding of [ 125 I-Tyr 11 ] SRIF to SSTRl was not affected by these treatments. Theεe findings indicate that des-AA 1,2,5 -[D- Trp ε , IAmp 9 ]SRIF and 125 I-Tyr 2 -des-AA 1 < 5 -[D-Trp 8 , IAmp°]SRIF may bind to SSTRl in a different manner than the native peptide SRIF. des-AA 1 ' 2 - 5 -[D-Trp 8 , IAmp°]SRIF and the other SRIF analogs that selectively bind to SSTRl with high affinity are found to activate the SSTRl receptor. Accordingly, these SRIF analogs are considered to be useful in combatting tumor growth which is mediated by the receptor without activating the other SRIF receptors. Although the binding of the SRIF analogs of the invention to SSTRl is potently inhibited by native SRIF and by SRIF-28, it is not inhibited by many other synthetic SRIF analogs. Moreover, the lack of binding of the peptides of the invention to SSTR4 is of particular interest because SSTRl and SSTR4 have approximately 68% amino acid sequence identity, which constituteε higher εequence εimilarity than for any other two SRIF receptors. Furthermore, SSTRl and SSTR4 both exhibit very low affinity for a large number of other synthetic analogs of SRIF which bind potently to SSTR2 , SSTR3 and SSTR5. The high amino acid sequence similarity and similar ligand binding characteristicε would uεually have predicted that these two SRIF receptors would share some common structural similaritieε in ligand-binding domainε; however, the fact that the peptides of the invention selectively bind only to SSTRl indicates that there may be considerable differences in ligand-binding properties between these two otherwise structurally similar receptors. 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 mRNA 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 wherein the malignant tissues expreεε thiε receptor. A particularly important advantage of SSTRl-selective agonistε aε anti-cancer agentε may be their continued effectiveneεs after prolonged use. Continuous use of SMS- 201-995 in the treatment of tumors is considered to be hindered by rapid deεenεitization of SSTR2, SSTR3 and SSTR5, the receptorε thiε peptide can interact with; in fact, all 3 of theεe receptorε have been reported to rapidly desensitize. In contrast, εtudieε suggest that SSTRl may be more resistant to agonist-induced regulation than the other receptors. As a result, the SSTRl-selective peptide agonists of the invention are considered to have prolonged anti-proliferative actions and should therefore exhibit improved effectiveness in treating cancers, compared to the commercially available SRIF analogs presently used as anti-cancer agentε. Furthermore, an analog of SMS-201-995 has recently been employed to detect human tumors having high expression of SRIF receptors through the use of positron-emission tomography. Thiε SRIF analog does not distinguish among SSTR2 , SΞTR3 or ΞSTR5, and it is unlikely to be able to detect SSTRl expression. Labelled SRIF analogs of the present invention can be employed for similar purposes and are considered to be specifically useful in identifying tumorε expreεεing SSTRl, which tumorε would then be therapeutic targets for treatment with SSTRl-selective ligands. While SSTRl has the typical structure of other G protein-linked receptors, controversy exists over whether this receptor truly associates with G proteins and effectively couples to adenylyl cyclase. Some investigators have reported that SRIF binding to this receptor is not affected by GTP analogs or pertussis toxin and does not effectively couple to adenylyl cyclase, whereas others have reported opposite findings. Furthermore, some investigators have failed to show GTP analog regulation of agonist binding to the receptor but have found that receptor mediateε the inhibition of cAMP formation by SRIF; however, thiε may have been a function of the particular cell εystems that were used. Previouε εtudieε have now been confirmed that the binding of [ 125 I-Tyr 11 ]SRIF to SSTRl waε not significantly effected by GTP analogs nor pertusεiε toxin treatment. In contraεt, the binding of the radiolabelled SRIF analogs of the present invention is reduced by GTPgS and the effects of GTPgS are diminished by pertussis toxin treatment. Because pertussiε toxin treatment aboliεhed the GTPgS regulation of binding, SSTRl appears to be able to associate with G ia and/or G oa so that the SRIF analogs of the preεent invention will be agonists at this receptor. The regulation of the binding of the present analogs to SSTRl by GTPgS and effect of pertusεis toxin, compared to the lack of effect of theεe agents on SRIF binding to this receptor, suggest that SRIF and the present analogs may induce different conformational changes in SSTRl. As such, the SRIF analogs of the present invention are believed to cause an effective association of the receptor with G proteins, while SRIF may be less effective in inducing such coupling. These results imply that SRIF interacts with SSTRl in a fundamentally distinct manner from that of the SRIF analogs of the present invention. Some results alεo suggest that the SRIF analogs of the present invention are able to induce responses in cells expressing SSTRl that even SRIF is incapable of producing. The SRIF analogs of the present invention are the first εelective agonists at SSTRl and are useful in combatting cancers which express SSTRl. They are also considered to be most useful in localizing cells and tissues expressing this receptor in the brain and in the endocrine and exocrine systems and in identifying selective functions of this receptor in body. They are further useful in selectively carrying out certain of the pharmacological effects mediated by SSTRl for which SRIF has been found useful over the past 2 decades. Labelled SRIF analogs of the invention are also considered to be useful in drug-screening assayε to screen for new effective peptide and nonpeptide agents that will bind with high affinity to SSTRl and which may be either highly effective agonists or antagonists. Having a known ligand for the receptor SSTRl, one can obtain a baseline activity for the recombinantly produced receptor. Then, to test for inhibitors or modifiers, i.e. antagonists of the receptor function, one can incorporate into a test mixture a candidate εubεtance whoεe effect on the receptor is to be tested. By comparing reactions which are carried out in the presence or absence of the candidate substance, one can then obtain information regarding the effect of the candidate substance on the normal function of the receptor. The peptides of the preεent invention can be εyntheεized by clasεical solution syntheεis, but they are preferably synthesized by solid-phase technique. A chloromethylated resin or a hydroxy ethylated resin is preferably used. For example, these peptides having a free carboxyl C-terminus are preferably synthesized as taught in United States Patent No. 4,816,438 issued March 28, 1989. Solid-phase synthesis is conducted in a manner to stepwise add amino acids in the chain beginning at the C-terminus in the manner set forth in that U.S. patent. Side-chain protecting groups, which are well known in the art, are preferably included as a part of any amino acid which has a particularly reactive side chain, and optionally may be used in the case of others such as Trp, when such amino acids are coupled onto the chain being built upon the resin. Such synthesis provides the fully protected intermediate peptidoresin. Chemical intermediates used to synthesize certain preferred SRIF analogs generally in accordance with the invention may be represented by the formula: X 1 -Xaa 2 (X 2 ) -Cys(X 3 ) -Lys(X*)-Phe-Phe-D-Xaa 8 (X 5 ) -Xaa 9 (X 3 ) - Thr(X 6 ) -Phe-Thr(X 6 )-Xaa 13 (X 6 ) -Cys(X 3 )-X 7 wherein X 1 is an α-amino protecting group of the type known to be uεeful in the art in the εtepwiεe εyntheεis of polypeptides. When G in the desired peptide composition is a particular acyl group, it may be appropriate to employ such group as the protecting group. Among the classes of α-amino protecting groups covered by X 1 are (1) acyl-type protecting groups, such aε formyl (For) , trifluoroacetyl, phthalyl, p-toluenesulfonyl (Tos) , benzoyl(Bz) , benzeneεulfonyl , dithiasuccinoyl (Dts) o-nitrophenylsulfenyl (Nps) , tritylsulfenyl, o-nitrophenoxyacetyl, acrylyl (Aer) , chloroacetyl, acetyl (Ac) and y-chlorobutyryl ; (2) aromatic urethan-type protecting groupε, e.g., benzyloxycarbonyl (Z) , fluorenylmethyloxycarbonyl(Fmoc) and εubεtituted benzyloxycarbonyl, εuch aε p-chlorobenzyloxy-carbonyl (C1Z) , p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl and p-methoxybenzyloxycarbonyl; (3) aliphatic urethane protecting groupε, εuch aε tertbutyloxycarbonyl(Boc) , diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl and allyloxycarbonyl; (4) cycloalkyl urethane-type protecting groups, such as cyclopentyloxycarbonyl, adamantyloxycarbonyl and cyclohexyloxycarbonyl; (5) thiourethane-type protecting groups, such aε phenylthiocarbonyl; (6) alkyl-type protecting groupε, such as allyl (Aly), triphenylmethyl (trityl) and benzyl(Bzl) ; (7) trialkylsilane groups, such as trimethylsilane. The preferred α-amino protecting group is Boc. X 2 is hydrogen or a protecting group for the phenolic hydroxyl group of Tyr selected from the group consisting of tetrahydropyranyl, tert-butyl, trityl, benzyl, 2, 2-bromobenzyloxycarbonyl (2BrZ) and 2, 6-dichlorobenzyl (DCB) . 2BrZ is preferred. X 3 is a protecting group for Cys preferably selected from the class consisting of p-methoxybenzyl (Mob) , p- methylbenzyl , acetamidomethy, trityl and Bzl. The most preferred protecting group is p-methoxybenzyl. X 3 can also be hydrogen, meaning that there is no protecting group on the sulfur. X 4 iε a protecting group for an amino side chain group, primary or secondary amino, such as that of Lys. Illustrative of suitable εide chain amino protecting groupε are Z, 2-chlorobenzyloxycarbonyl (2-C1-Z) , Toε, t- amyloxycarbonyl and Boc. The selection of a side chain amino protecting group is not critical except that it must be one which is not removed during deprotection of the a- amino groups during the synthesis. Hence, the α-amino protecting group and the side chain amino protecting group cannot be the same. X 5 is hydrogen or a protecting group for the indole nitrogen of Trp, such as Bz, Ac or For. In many syntheεeε there is no need to protect Trp. X fc iε hydrogen or a protecting group for the hydroxyl εide chain of Thr or Ser, e.g. Ac, Bz, trityl, DCB or benzyl ether(Bzl) and iε preferably Bzl. X 7 is selected from the class consisting of OH, OCH 3 and esters, including a benzyl ester or a hydroxymethyl ester anchoring bond used in solid-phase synthesis for linking to a solid resin support, represented by the formulae: -0-CH 2 -polystyrene resin support and 0-CH 2 -benzyl-polystyrene resin support Such a polystyrene polymer is preferably a copolymer of styrene with about 0.5 to 2% divinyl benzene as a cross¬ linking agent, which causes the polystyrene polymer to be completely insoluble in certain organic solvents. The criterion for selecting certain side chain protecting groups for X 2 -X 6 is that the protecting group should be stable to the reagent under the reaction conditions selected for removing the α-amino protecting group (preferably Boc) at each step of the synthesis. These protecting groups generally should not be split off under coupling conditions but should be removable upon completion of the synthesis of the desired amino acid sequence under reaction conditions that will not alter the peptide chain. A protecting group initially employed for a certain 8-position reεidue may be removed prior to cleavage from the reεin to carry out a reaction at that poεition, aε explained hereinafter. Similarly, the 9-poεition reεidue in the form of the aminomethylPhe (Amp) may be introduced in its final desired form, or it may be deprotected and alkylated, if desired, while part of the resin-peptide. Thus, there is broadly provided a method for making a SRIF analog peptide having the formula: (cyclo 3-14)H-Xaa 2 -Cys-Lys-Phe-Phe-D-Xaa 8 -Xaa 9 -Thr-Phe-Thr- Xaa 13 -Cyε-0H wherein Xaa 2 , D-Xaa g , Xaa 9 and Xaa. 3 are aε set forth hereinbefore, which method comprises (a) forming an intermediate peptide having the formula: X 1 -Xaa 2 (X 2 ) -Cys(X 3 ) -Lyε(X 4 ) -Phe-Phe-D-Xaa 8 (X 5 ) -Xaa,(X 3 ) - Thr(X 6 )-Phe-Thr(X 6 )-Xaa 13 (X 3 )-Cyε wherein X 1 is hydrogen or an α-amino protecting group; X 5 is hydrogen or a protecting group for an indole nitrogen; X 6 is a protecting group for a hydroxyl group of Thr or Ser; X 2 is hydrogen or a protecting group for a phenolic hydroxyl group of Tyr; X 3 is either hydrogen or a protecting group for a sulfhydryl side chain; X 4 is a protecting group for an amino side chain, X 7 is -0-CH 2 -[resin support], or a protecting group or OH, (b) splitting off any remaining groups X 1 to X 6 and/or cleaving from any resin support included in X 7 ; (c) oxidizing to create a disulfide bond between the Cys side chains; and (d) converting to a nontoxic salt, if desired. Purification of the peptide is effected by ion exchange chromatography on a CMC column, followed by partition chromatography using the elution system: n-butanol ;0.IN acetic acid (1:1 volume ratio) on a column packed with Sephadex G-25, or by using HPLC, as known in the art and specifically set forth in J. Rivier, et al. J. Chromatography. 288. 303-328 (1984) . The SRIF analogs of the invention are effective at levelε of less than 100 micrograms per kilogram of body weight. For prolonged suppression of cancerous tumors having SSTRl receptors, it may be necessary to use dosage levels in the range of from about 0.1 to about 2.5 milligrams per kilogram of body weight. These analogs are particularly soluble at physiological pHs and thuε can be prepared as relatively concentrated solutions for administration. The following Examples illuεtrate the εyntheses of a number of SRIF analog peptides embodying various features of the invention, together with the syntheses of a protected amino acids for use in such peptide syntheses. All of these peptides include at leaεt one D-isomer amino acid reεidue, and although the preferred SRIF analogε do not include 14 amino acid residues, to permit ready comparison with the native SRIF sequence, the peptide analogs are described by making reference to the comparable poεitionε in the native SRIF sequence having positions 1 through 14, with the Cys residues in positions 3 and 14 being joined by the cyclizing disulfide bond. EXAMPLE 1 The synthesis of L-N a -Boc-N 4 -Cbz-(4- isopropylaminomethyl)phenylalanine, which is referred to by the shorthand nomenclature as Boc-IAmp(Z) , is carried out as followε: L-N α -Boc-(4-aminomethyl)phenylalanine (Boc-A p) (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. The mixture is purged with N 2 for 10 minutes; then Pd/C 10% (600 mg) is added. A reductive alkylation reaction occurs and is monitored by HPLC; it is carried out for about 26 hours. After filtration to remove the catalyst and molecular sieves and evaporation of the solvent, the deεired intermediate L-N a - Boc-(4-isopropylaminomethyl)phenylalanine is obtained aε a viεcouε liquid. The product iε then Cbz-protected uεing benzyl chloroformate (Z-Cl, 8.6 mL, 60 mmol) in a mixture of THF/H 2 0(1: 1,200 mL) at pH = 9.5. A good yield of L-N a -Boc- N 4 -Cbz-(4-isopropylaminomethyl)phenylalanine is obtained as a foam: 17.5 g (37 mmol, 71.4%) ; m.p. 39-42°C; [α] 0 = +5.2° (c = 1, MeOH, t = 20"C) . EXAMPLE 2 The somatostatin agonist des-AA 1 ' 2 - 5 [D-Trp 8 , IAmp°]-SRIF having the structure: (cyclo)H-Cys-Lys-Phe-Phe-D-Trp-IAmp-Thr-Phe-Thr-Ser- Cys-OH is syntheεized by the following εolid phase methodology in a stepwise manner on a chloromethylated resin. The reεin is composed of fine beads (20-70 microns in diameter) of a synthetic resin prepared by copolymerization of styrene with one to two percent divinylbenzene. The benzene rings in the resin are chloromethylated in a Friedel-Crafts reaction with chloromethyl methyl ether and stannic chloride. The chlorine thus introduced creates a reactive benzyl chloride type of linker. The Friedel-Crafts reaction is continued until the resin contains 0.5 to 2 millimoles of chlorine per gram of resin. The tert-butyloxycarbonyl-S-paramethoxybenzyl derivative of Cys, i.e. Boc-Cys(Mob) , is linked to the resin by a known method, such as: (1) reflux in ethanol in presence of triethylamine, (2) cesium salt of the Boc- protected amino acid is kept at 50"C in dimethylformamide (DMF) overnight or (3) Boc-protected amino acid is kept at 80"C in N-methypyrrolidone (NMP) for 24 hours in the KF. One milliequivalent of the protected Cys per milliequivalent of Cl on the resin is used. Deprotection, neutralization and addition of each amino acid is performed in accordance with the following εchedule: MIX STEP REAGENTS AND OPERATIONS TIMES (MIN. ) 1 CH ? Cl 7 wash-80 ml. (2 times) 3 2 Methanol (MeOH) wash-30 ml. (2 times) 3 3 CH-.C1., wash-80 ml. (3 times) 3 4 50% TFA plus 5% meta-cresol in 20 CH ? Cl ? -70 ml. (2 times) 5 Isopropyl alcohol + 1% meta-cresol 3 waεh-80 ml. (2 timeε) 6 TEA 12.5% in CH,Cl,-70 ml. 5 7 MeOH wash-40 ml. (2 times) 2 8 TEA 12.5% in CH ? Cl 7 -70 ml. (2 timeε) 2 9 CH ? C1, wash-80 ml. (3 timeε) 3 10 Boc-amino acid (5 mmoles) in 30 ml. of CH ∑ C1 2 30-300 (DCM) or dimethylformamide(DMF) : DCM or NMP: DCM, depending upon the εolubility of the particular protected amino acid, plus DIC or DCC (5 mmoles) in CH 7 C1 7 11 MeOH wash-40 ml. (2 times) 3 12 Triethylamine (TEA) 12.5% in CH 7 Cl 7 -70 ml. 3 13 MeOH wash-30 ml. (2 times) 3 14 DCM wash-80 ml. (2 times) 3 The Boc derivative of each amino acid is used. After α-amino deprotection of the first residue, i.e., Boc-Cys(Mob) , according to the above schedule, the N a Boc derivative of Ser is added along with the coupling agent, dicyclohexylcarbodiimide (DCC) , the side chain of Ser being protected with benzyl ether (Bzl) . The N β Boc derivative of Thr(Bzl) is next added along with DCC. After coupling N a Boc-Phe and then N β Boc-Thr(Bzl) , N a Boc-4- isopropylaminomethyl Phe(Z) is similarly coupled. N α Boc-Lys(2-C1Z) is next added, and then the remaining protected Cys is coupled to extend the peptide chain to the N-terminus. This creates the intermediate: Boc-Cys(Mob) - Lys (2-C1Z) -Phe-Phe-D-Trp-IAmp(Z) -Thr(Bzl) -Phe-Thr(Bzl) - Ser(Bzl) -Cys(Mob) -0-CH 2 -resin support. Cleavage of the peptide from the resin and deprotection of the εide chain protecting groups are performed in hydrofluoric acid(HF) (25 ml) in the presence of 2 ml of anisole and 2 ml of di ethylsulfide for 1.5 hours at 0°C after 20 minutes at ambient temperature. After elimination of hydrofluoric acid under high vacuum, the reεin-peptide iε washed with anhydrous diethyl ether. The resin is immediately extracted with 75% acetic acid (200 ml) . The extract is filtered into a 500 milliliter round-bottom flask and is then oxidized to create the disulfide cyclic linkage by stirring vigorously while rapidly adding a 10 weight percent solution of iodine in methanol until the resultant solution remains orange- colored. It is then stirred for 10-20 additional minutes and quenched with 10% ascorbic acid in water until the yellow color iε gone. Concentration under vacuum is carried out to reduce the volume to about 50 milliliters, followed by dilution to about 300 milliliters with water. The solution is then applied to a 4 centimeter by 7 centimeter pad of C, 8 silica in a coarse-fritted funnel that was previously equilibrated with 5% CH 3 CN in 0.1% trifluoroacetic acid (TFA) in water. Following vacuum filtration, the eluate is diluted to 500 milliliters and reapplied to the pad. The eluate is collected and diluted to about 800 ml, and filtration is repeated. Thereafter, the pad is washed with about 300 milliliters of 5% acetonitrile in 0.1% TFA, and the peptide is eluted using about 250 milliliters of 60% CH j CN in water. The resultant solution is diluted to about 500 milliliters with distilled water, frozen and lyophilized. The lyophilized material is then purified by subjection to analytical HPLC on a C, 8 column. Peaks are located which are then individually purified using buffer systems as disclosed in Hoeger et al., Biochromatoαraphv. 2 , 134-142 (1987) . Final analytical HPLC is carried out using a gradient of Buffer B from 30% to 60% over 30 minuteε with Buffer A being 0.1% aqueous TFA and Buffer B being 60% CH 3 CN in Buffer A. The desired cyclic peptide (cyclo)H-Cys-Lys-Phe-Phe-D-Trp-IAmp-Thr-Phe-Thr-Ser- r 1 Cys-OH lε obtained which appears to be greater than 97% pure on capillary zone electrophoresis. MS analysis shows the expected mass of 1485.7 Da. The specific optical rotation measures [ a f*= -10.3 " + 1° (c = 0.5 in 50% acetic acid) The peptide iε hereinafter referred to as Peptide No. 2. EXAMPLE 2A The εyntheεiε described in Example 2 iε repeated with one change. N a Boc-N 4 -CBZ-(4-aminomethyl) phenylalanine, i.e. Boc-Amp, iε used to provide the 9-position residue. Further elongation of the chain then proceeds aε in Example 2, and cleavage, deprotection, cyclization and purification are carried out aε in Example 2. The purified cyclic peptide haε the formula: (cyclo l-ll)H-Cys-Lys-Phe-Phe-D-Trp-Amp-Thr-Phe-Thr- Ser-Cys-OH and is referred to as Peptide No. 2A. EXAMPLE 2B The initial synthesis described in Example 2A is repeated with one change; N a Boc-N 4 -Fmoc-(4-aminomethyl) phenylalanine, i.e. Boc-Amp(Fmoc) , is used to provide the 9-position residue. Further elongation of the chain then proceeds as in Example 2A, until the final coupling with Boc-Cys(Mob) is effected. With the N°Boc group still in place at the N-terminus, the side chain of Amp is alkylated. The Fmoc protecting group is selectively removed by treatment with 20 percent piperidine in NMP or DMF(10 ml.) for about 15 minutes; the intermediate is preferably washed with NMP and then treated with more piperidine/NMP for another 15 minutes. After preferably washing the peptidoresin with NMP, the newly freed aminomethyl groups are treated with (MeO-phenyl) 2 - CHC1, which is referred to as Dod-Cl, in NMP plus diiεopropyl ethylamine (DIEA) for 1-2 hourε. After waεhing, the peptidoresin (about 1-2 g) is treated for 20 minutes with 6 mis of 36% formaldehyde and 10 mis of 1% acetic acid in NMP, adding 0.35 gram of cyanoborohydride (NaBH 3 CN) ; at the end of 20 minutes, this treatment is repeated one more time. This reaction adds a single methyl group to the aminomethylPhe side chain. Details of the reaction are found in Kaljuεte and Unden, Int. J. Peptide Protein Res. , 42 , 118-124 (1993). The usual washing is carried out, and then the Dod-Cl protecting group and the Boc group at the N-terminus are removed by treatment with 60% TFA in DCM for 20 minutes. Cleavage, deprotection, cyclization and purification are then carried out as in Example 2. The purified cyclic peptide has the formula: (cyclo l-ll)H-Cys-Lys-Phe-Phe-D-Trp-MAmp-Thr-Phe-Thr- Ser-Cyε-OH and is referred to as Peptide No. 2B. EXAMPLE 2C The synthesis described in Example 2B is repeated with one change. Instead of reacting the protected aminomethyl moiety with 36% formaldehyde, the reaction is carried out for 20 minutes with 15 mis of a solution of 40% acetaldehyde in 1% acetic acid in NMP, plus 0.35 gm. of cyanoborohydride and repeated once. This results in the (MeO-phenyl) 2 -CHCl (4-ethyl aminomethyl) phenylalanine as the 9-position residue. Removal of the Boc and Dod-Cl groups, cleavage, deprotection, cyclization and purification are carried out as in Example 2B. The purified cyclic peptide has the formula: (cyclo l-ll)H-Cys-Lys-Phe-Phe-D-Trp-EAmp-Thr-Phe-Thr- Ser-Cys-OH and is referred to as Peptide No. 2C. EXAMPLE 2D The synthesis described in Example 2B is repeated with one change. Inεtead of reacting the protected aminomethyl moiety with 36% formaldehyde, the reaction iε carried out for 20 minuteε with 15 mis of a solution of 40% iεobutyraldehyde in 1% acetic acid in NMP, pluε 0.35 gm. of cyanoborohydride and repeated once. Thiε results in the (MeC-phenyl) 2 -CHCl (4-isobutyl aminomethyl) phenylalanine as the 9-positιon residue. Removal of the Boc and Dod-Cl groups, cleavage, deprotection, cyclization and purification are carried out as in Example 2B. The purified cyclic peptide haε the formula: (cyclo l-ll)H-Cys-Lyε-Phe-Phe-D-Trp-iBAmp-Thr-Phe-Thr- ι ■> Ser-Cys-OH and is referred to as Peptide No. 2D. Peptide Nos. 2A, 2B, 2C and 2D bind εelectively to SSTRl. EXAMPLE 3 The εynthesiε deεcribed in Example 2 is repeated with one change. Inεtead of coupling Boc-D-Trp aε what is referred to as the 8-position residue, N a Boc-D-2Nal is used. Further elongation of the chain then proceeds as in Example 2. Cleavage, deprotection, cyclization and purification are carried out as in Example 2. Amino acid analysis showε the expected ratio for the different amino acids. The purified cyclic peptide has the formula: (cyclo 1-11)H-Cys-Lys-Phe-Phe-D-2Nal-IAmp-Thr-Phe-Thr- Ser-Cys-OH and is referred to as Peptide No. 3A. The synthesis is repeated, substituting N α Boc-D-lNal for N α Boc-D-2Nal to obtain the following purified cyclic peptide, which is referred to as Peptide No. 3B: (cyclo l-ll)H-Cys-Lys-Phe-Phe-D-lNal-IAmp-Thr-Phe-Thr- Ser-Cys-OH. Both Peptideε Noε. 3A and 3B bind εelectively to human SSTRl. EXAMPLE 4 The synthesis described in Example 2 is repeated with one change. Instead of coupling Boc-Ser(Bzl) as what is referred to aε the 13-position residue, N a Boc-D-Ser(Bzl) is used. Elongation of the chain is then carried out as in Example 2. Cleavage, deprotection, cyclization and purification are carried out as in Example 2. HPLC and CZE show the peptide to have a purity of greater than 97%. The specific optical rotation measureε [ cx f = +20.0' ± 1° (c = 0.5 in 50% acetic acid) Amino acid analyεiε shows the expected ratio for the different amino acids. MS analysis shows the expected mass of 1485.7 Da. The purified cyclic peptide haε the formula: (cyclo)H-Cys-Lys-Phe-Phe-D-Trp-IAmp-Thr-Phe-Thr-D-Ser- ι ι Cyε-OH and lε referred to as Peptide No. 4. EXAMPLE 5 The synthesis described in Example 2 is again repeated with one change. Elongation of the chain by one residue is carried out by coupling N a Boc-Tyr(2BrZ) at the N-terminus. Cleavage, deprotection, cyclization and purification are carried out as in Example 2. The specific optical rotation measures l a f ϋ ~ -9-6° ± 1" (c = 0.5 in 50% acetic acid) Amino acid analysis shows the expected ratio for the different amino acids. MS analysis shows the expected mass of 1648.7 Da. The purified cyclic peptide has the formula: (cyclo)H-Tyr-Cys-Lys-Phe-Phe-D-Trp-IAmp-Thr-Phe-Thr- Ser-Cys-OH and iε referred to as Peptide No. 5. EXAMPLE 6 The syntheεiε described in Example 2 is repeated with one change. Instead of coupling Boc-D-Trp aε what iε referred to aε the 8-position residue, N α Boc-D-3-Pal is used. Elongation of the chain is then carried out as in Example 2. Cleavage, deprotection, cyclization and purification are carried out as in Example 2. The purified cyclic peptide haε the formula: (cyclo)H-Cys-Lyε-Phe-Phe-D-3Pal-IAmp-Thr-Phe-Thr-Ser- ι j Cyε-OH and iε referred to as Peptide No. 6A. The above syntheεis is then repeated 22 timeε, each time substituting a different amino acid at position 8, as follows: N a Boc-D-5ClTrp ; N a Boc-D-6FTrp ; N a Boc-D-5N0 2 Trp ; N a Boc-D-6BrTrp ; N a Boc-D-N 1 n ForTrp ; N α Boc-3- (9-fluorenyl) -D,L-Ala; N a Boc-3-(1-adamantyl) -D,L-Ala; N a Boc-3-(4-biphenyly1)-D,L-Ala; N α Boc-3-(2-anthryl)-D,L-Ala; N a Boc-3-(2,4 , 6-triethylphenyl) -D,L-Ala; N a Boc-3-(9-anthryl)-D,L-Ala; N a Boc-3-(2-fluorenyl)-D,L-Ala; N a Boc-3-(l-anthryl)-D,L-Ala; N α Boc-D,L-Me5-Phe; N a Boc-D,L-TMP; N a Boc-D,L-BIA; N a Boc-D,L-TBA; N a Boc-3-(3-phenanthryl)-D,L-Ala; N a Boc-3-(2, 4,6-trimethylcyclohexyl) -D,L-Ala; N a Boc-3-(perhydro-4-biphenylyl) -D,L-Ala; N a Boc-3-(1-perhydronaphthyl)-D,L-Ala; and N a Boc-3-(2,2-diphenylmethyl)-D,L-Ala The resultant SRIF analogs are referred to as peptides 6B through 6W. Peptides 6A-6W bind selectively to SSTRl. EXAMPLE 7 A betide somatoεtatin agonist des- AA 1 - 2 ' 5 [Agl (indolecarboxyl) 8 , IAmp°]-SRIF having the structure: (cyclo)H-Cys-Lys-Phe-Phe-Agl (indolecarboxyl) - IAmp-Thr-Phe-Thr-Ser-Cyε-OH iε εyntheεized by the following solid phase methodology described in Example 2 using a chloromethylated resin. The Boc derivative of each amino acid is used. After deprotection of the first residue, i.e., Boc-Cys (Mob), according to the schedule set forth in Example 2, the N a Boc derivative of Ser iε added along with the coupling agent, dicyclohexylcarbodiimide(DCC) , the side chain of Ser being protected with O-benzyl ether (Bzl) . The N a Boc derivative of Thr(Bzl) is next added along with DCC. After coupling N α Boc-Phe and then N a Boc-Thr(Bzl) , N a Boc-4-iεopropylamino Phe(Z) iε εimilarly coupled. Then Boc-D/L-Agl (Fmoc) iε coupled in the 8-position, followed by the coupling of 2 residues of N a Boc-Phe. Benzyloxycarbonyl-2Cl, i.e. 2C1Z, is used as the protecting group for the Lys side chain in the 4-position of SRIF. The final protected Cys is coupled to extend the peptide chain to the N-terminus. With the N a Boc group still in place at the N-terminus, the side chain of Agl is modified. The Fmoc protecting group is selectively removed by treatment with 20 percent piperidine in NMP or DMF(10 ml.) for about 15 minutes; the intermediate is preferably washed with DMF and then treated with more piperidine/DMF for another 15 minutes. After preferably washing the peptidoresin with DMF, the newly freed amino groups are reacted with ιndole-3-carboxyl acid uεing a suitable coupling agent such as TBTU or HBTU or BOP for about 10 hourε. Cleavage of the betide from the reεin and deprotection of the εide chain protecting groups of the betide are performed in hydrofluoric acid(HF) (25 ml) in the presence of 2 ml of anisole and 2 ml of di ethylsulfide for 1.5 hours at 0°C after 20 minutes at ambient temperature. After elimination of HF under high vacuum, the resin-betide is waεhed with anhydrous diethyl ether. The resin is then extracted, cyclized and lyophilized aε generally described in Example 2. The lyophilized material is then purified by subjection to analytical HPLC on a C, β colurn. Peaks are located which are then individually purified using similar buffer systems. Final analytical HPLC is carried out using a gradient of Buffer B from 30% to 601 over 30 minutes, with Buffer A being 0.1% aqueous TFA and Buffer B being 60% CH 3 CN in Buffer A. The desired cyclic betides (cyclo 1-11)H-Cys-Lys-Phe-Phe- (indole-3-amido) Gly-IA p-Thr-Phe-Thr-Ser-Cyε-OH are obtained in the form of two εeparate diaεtereomers which appear to be greater than 97% pure on capillary zone electrophoresis. Both the cyclic betides bind to SSTRl. EXAMPLE 8 A synthesis generally as described in Example 2 is carried out to synthesize [IAmp 5 ]-0DT8 which has the formula: i 1 (cyclo)H-Cys-Phe-Phe-D-Trp-IAmp-Thr-Phe-Cys-OH. Cleavage, deprotection, cyclization and purification are carried out as in Example 2. HPLC and CZE show the peptide to have a purity of greater than 97%. The specific optical rotation measures [ a f*= -35° ± 1° (c = 1 in 50% acetic acid) Amino acid analysiε shows the expected ratio for the different amino acids. EXAMPLE 9 The synthesis described in Example 2 is repeated with one change. Before coupling Boc-Lys (2C1Z) in the chain, BocAsn(Xan) is coupled as the 5-position residue. Further elongation of the chain then proceeds as in Example 2. Cleavage, deprotection, cyclization and purification are carried out as in Example 2. Amino acid analysis shows the expected ratio for the different amino acids. The purified cyclic peptide has the formula: (cyclo l-12)H-Cyε-Lyε-Aεn-Phe-Phe-D-Trp-IAmp-Thr-Phe- Thr-Ser-Cys-OH and is referred to as Peptide No. 9. The synthesiε iε then repeated, adding N a Boc-Gly and N a Boc-Ala at the N-terminus to obtain the following purified cyclic peptide, which is referred to aε Peptide No. 9A: (cyclo 3-14)H-Ala-Gly-Cyε-Lyε-Aεn-Phe-Phe-D-Trp-IAmp- Thr-Phe-Thr-Ser-Cyε-OH. It iε [D-Trp 8 , IAmp 9 ]SRIF. Both Peptides Nos. 9 and 9A bind selectively to human SSTRl. In vitro Bioassay: The effects of the various somatostatin analogs are tested in vitro for their ability to bind to isolated cloned receptorε expressed on COS and CHO cells. The molecular cloning of the genes encoding multiple somatostatin receptor subtypes permits the individual expression of these receptors in mammalian cells and the characterization of their respective pharmacological profileε. Five such receptor subtypes, termed SSTRl through SSTR5, have been cloned and are reported and described in Raynor et al., Molecular Pharmacology. 43. 838-844 (1993) and in Raynor et al., Molecular Pharmacology. 44 , 385-392 (1993) . These references describe binding assays that can be used to determine whether particular SRIF analogs bind selectively to one or more of the 5 receptor types and also whether they bind to such receptor types with high or low affinity. Because these receptor typeε have now been characterized with regard to their pharmacological profileε, knowledge of the resultε of εuch binding studies along with knowledge of the unique patterns of distribution of these receptors in the body indicate that each receptor subtype may mediate distinct but overlapping physiological effects of SRIF; therefore, compounds which bind selectively to receptors SSTRl, for example, can be used to modulate a particular physiological function of SRIF without potentially having an undesired effect resulting from another physiological function of SRIF which is mediated by other SRIF receptors. To test the selectivity of Peptide No. 2 to bind to the SRIF receptors, the ability of thiε peptide to inhibit the binding of [ 125 I-Tyr 11 ]SRIF to the five cloned SRIF receptorε waε first tested. Previous studies had shown that [ 125 I-Tyr 1l ]SRIF bindε to all five cloned SRIF receptorε with εimilar affinitieε. At concentrationε of 1 μM, Peptide No. 2 had minimal effectε on the binding of [ 125 I- Tyr 11 ]SRIF to mouse SSTR2 and SSTR3 , to human SSTR4 and to rat and human SSTR5. In contrast, it potentially diεplaced the binding of [ 125 I-Tyr 11 ]SRIF to human SSTRl with an IC 50 value of 1.8 + 0.7 nM. Similarly, Peptide No. 4 containing the D-Ser 13 modification selectively bound to SSTRl, and Peptide No. 5, the tyrosine analog, also εelectively interacts with SSTRl. To investigate whether an iodinated analog of Peptide No. 5 may serve as a selective SSTRl radioligand, the peptide was iodinated and tested for binding to the five cloned SRIF receptors. No specific binding of Peptide No. 5 to SSTR2-5 was detectable. In contrast, Peptide No. 5 effectively bound to SSTRl to about the same extent as [ 125 I-Tyr 11 ]SRIF. The binding of Peptide No. 5 to SSTRl reached equilibrium by 30 minutes. The binding waε εaturable with a Kd of 0.5 + 0.1 nM and a Bmax of 226 + 56 fmol/mg protein. SRIF, [D-Trp 8 ]SRIF and SRIF-28 all potently inhibited binding of Peptide No. 5 to SSTRl with IC 50 valueε of 0.8 ± 0.4, 0.2 ± 0.1 nM and 0.7 ± 0.2 nM, respectively. The SRIF analog Peptide No. 4 also potently binds to SSTRl exhibiting an IC 50 value of 5.3 + 0.8 nM, based upon itε ability to inhibit the binding of iodinated Peptide No. 5. Previouε εtudieε with SSTRl expreεεed in COS-07 cellε have εhown that the binding of [ 125 I-Tyr n ]SRIF to SSTRl iε not significantly effected by FTPgS nor pertussiε toxin pretreatment, suggesting that this receptor may not be effectively coupled to G proteins. Similarly, GTPgS .and pertusεis toxin treatment did not diminish the binding of [ 125 I-Tyr 11 ]SRIF binding to the cloned SSTRl. However, GTPgS significantly reduced the binding of Peptide No. 5 to SSTRl, in a concentration-dependent manner. Overnight pertussiε toxin treatment diminiεhed the effect of GTPgS, i.e. GTPgS waε no longer able to εignificantly affect binding to pertuεsis toxin-pretreated SSTRl. These findings suggeεt that SSTRl can couple to pertussis toxin- sensitive G proteins and that differences in association of the receptor with G proteins occur following SRIF binding to the receptor versus the binding of one of the peptides of the present invention. Because the binding of Peptide No. 5 was GTP regulated, the ability of the peptide to modulate cAMP accumulation in COS-7 cells expressing SSTRl was tested. Neither 1 μM of Peptide No. 2, nor SRIF itself, inhibits forskolin-sti ulated cAMP formation in COS-7 cells expressing SSTRl. Therefore, while Peptide No. 2 can induce an apparent association of SSTRl with pertussis toxin G proteins, it does not appear to regulate adenylyl cyclase activity in COS-7 cells. Screening assays as are well known in the art which employ the receptor polypeptide SSTRl directly from the recombinant host can be used to identify agents useful in mimicking the desirable aspectε of εomatostatin while eliminating the undeεirable aspects of the hormone which may arise from activation of other receptors. The potencies of certain SRIF analogs, including the prior art compound ODT8, (cyclo 1-8)H-Cys-Phe-Phe- D-Trp-Lyε-Thr-Phe-Cys-OH, to inhibit radioligand binding of [ 125 I-Tyr 11 ]SRIF to the various cloned mouse SRIF receptors, relative to SRIF and SRIF-28, are shown in the following table wherein the IC 50 values are given in nanomolar concentration. Additional the meaεurementε are in progress. TABLE The peptides of the invention not only provide more selective ligands for binding SSTRl but the use of a labelled Peptide No. 5, e.g. with 125 I, facilitates drug screening for even more effective agonists. Competitive binding assays with candidate compounds would first be carried 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. These SRIF analogs or nontoxic salts thereof, combined with a pharmaceutically or veterinarily acceptable carrier to form a pharmaceutical composition, may be administered to animals, including humans and other mammalε, either intravenously, subcutaneously, intramuscularly, percutaneouεly, e.g. intranaεally, intracerebroεpinally or orally. The peptideε should be at leaεt 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. Thiε purity means that the intended peptide constitutes the stated weight % of all like peptides and peptide fragments preεent. Adminiεtration to humanε 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 doεage 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 aε acid addition salts or metal complexes, e.g., with zinc, iron, calcium, barium, magnesium, aluminum or the like. Illuεtrative of such nontoxic saltε are hydrochloride, hydrobromide, εulphate, phoεphate, tannate, oxalate, fumarate, gluconate, alginate, maleate, acetate, citrate, benzoate, succinate, malate, ascorbate, tartrate and the like. If the active ingredient is to be administered in tablet form, 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. If administration in liquid form is deεired, sweetening and/or flavoring may be used, and intravenous administration in isotonic saline, phosphate buffer solutions or the like may be effected. It may also be desirable to deliver these SRIF analogε over prolonged periods of time, for example, for periods of one week to one year from a single administration, and slow release, depot or implant doεage forms may be utilized aε well known in this art. For example, a dosage form may contain a pharmaceutically acceptable non-toxic salt of the compound which has a low degree of εolubility 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 saltε. A relatively inεoluble salt may also be formulated in a gel, for example, an aluminum stearate gel. A εuitable, slow-release depot formulation for injection may alεo contain an SRIF analog or a salt thereof disperεed or encapεulated 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 peptideε εhould be ad iniεtered under the guidance of a physician, and pharmaceutical compositionε will usually contain the peptide in conjunction with a conventional, pharmaceutically or veterinarily-acceptable carrier. 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; however, it is anticipated that doεages between about 10 micrograms and about 1 milligram per kilogram of body weight per day will be used for therapeutic treatment. It may be particularly advantageous to administer such compounds in depot or long-lasting form as earlier described. 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 μg/ml to about 100 μg/ml, preferably from about 1 μg/ml to about 50 μg/ml, more preferably at least about 2 μg/ml and usually 5 to 10 μg/ml . Although the invention has been described with regard to its preferred embodiments, which conεtitute the best mode presently known to the inventors, it should be understood that various changeε and modificationε aε would be obvious to one having the ordinary skill in thiε art may be made without departing from the scope of the invention which iε εet forth in the claims appended hereto. Although the claims variously define the invention in terms of a peptide sequence, it should be understood that such is intended to include nontoxic salts thereof which are well known to be the full equivalent thereof and which are most frequently administered. Instead of the simple free acid at the c-terminus, a lower alkyl eεter may be incorporated aε well known in the peptide art. Cyclic peptides having an amino acid residue sequence substantially identical to the sequence of the SRIF analogs specifically shown herein, in which one or more residues have been conservatively subεtituted with a functionally εimilar amino acid reεidue, are considered to be equivalents so long as they selectively bind to SSTRl. Aε previouεly indicated, these specified modifications can be incorporated in previously disclosed SRIF analogs to create SSTRl-selectivity. Incorporation of a residue in the 5-position is optional, but not preferred, because it generally detracts from binding affinity although selectivity remains. Likewise inclusion of residues in the 1- and 2-position iε optional, but except for Tyr or D-Tyr, is not considered worthwhile. Broadly it is considered that cyclic somatostatin analog peptides can be created having specific affinity for the SRIF receptor SSTRl by modifying the amino acid sequence of existing SRIF analogs known in the art to have SRIF biological activity. The modified peptide should have an amino acid sequence at least 8 residueε in length, contain a Cys-Cys disulfide bond with a sequence of at least 6 residues located between said Cys residues as a ring, and contain D-Trp or its equivalent centrally of such sequence of at least 6-residue sequence, preferably spaced by 2 or 3 residues from the Cys residue at or near the N-terminus, which analog peptide is characterized by the preεence of (C)Amp joined immediately C-terminally to said D-Trp, with C being H or lower alkyl. The diεclosed cyclic SRIF analogε may also be modified by N-methylation of the α-amino group on one (or two) of the residueε, preferably thoεe in the 7-10 poεitions of SRIF, e.g. des-AA 1 ' 2 ' 5 [N α CH 3 D-Trp 8 , IAmp°]-SRIF. The resulting SRIF analogs retain their specificity for SSTRl, and in some instanceε have greater binding affinity. Such peptideε and εalts thereof are conεidered aε being within the εcope of the claimed invention. The diεclosureε of all patentε and publicationε set forth hereinbefore are expresεly incorporated herein by reference. As used herein, all temperatures are °C. and all ratios are by volume. Percentages of liquid materials are also by volume. Various features of the invention are emphasized in the claimε which follow.

Claims: WHAT IS CLAIMED IS: 1. A cyclic somatostatin (SRIF) analog peptide having specific affinity for the SRIF receptor SSTRl, which peptide has the amino acid sequence (cyclo 3-14)Xaa 1 -Xaa 7 - i r Cys-Lys-Xaa 5 -Phe-Phe-D-Xaa 8 -Xaa 9 -Thr-Phe-Thr-Xaa, 3 -Cys wherein Xaa, is des-Xaa or Ala; Xaa 2 is Tyr, D-Tyr or des- Xaa or Gly; Xaa 5 is des-Xaa or Asn; D-Xaa 8 is an aromatic D-amino acid; Xaa 9 is (C)aminomethyl Phe with C being H or lower alkyl; and Xaa 13 is Ser or D-Ser. 2. A peptide according to claim 1 wherein Xaa 8 is (A) D-Trp, (B) D-Ala, or (B)D,L-Ala wherein A is H, Cl, F, Br, N0 2 or formyl and B is naphthyl, fluorenyl, adamantyl, anthryl, biphenyl, tri (lower alkyl)phenyl , penta- methylphenyl , phenanthryl, trialkylcyclohexyl, perhydronaphthyl or perhydrobiphenyl . 3. A peptide according to claim 2 wherein Xaa,, Xaa 2 and Xaa 5 are des-Xaa and C is methyl, ethyl, isopropyl or isobutyl . 4. A peptide according to claim 2 wherein Xaa 9 is (isopropylaminomethyl) Phe, i.e. IAmp. 5. A peptide according to claim 4 wherein Xaa 13 is D- Ser. 6. A peptide according to claim 4 wherein Xaa 2 is Tyr or D-Tyr. 7. A peptide according to claim 4 wherein D-Xaa 8 iε (A)Trp and A iε H or F. 8. A peptide according to claim 4 wherein D-Xaa 8 iε (B)D-Ala or (B)D,L-Ala and B is 3-(1-naphthyl) . 9. A peptide according to claim 4 wherein D-Xaa 8 is (B)D-Ala or (B)D,L-Ala and B is 3-(9-fluorenyl) . 10. A peptide according to claim 4 wherein D-Xaa 8 is (B)D-Ala or (B)D,L-Ala and B is 3-(1-adamantyl) . 11. A peptide according to claim 4 wherein D-Xaa 8 is (B)D-Ala or (B)D,L-Ala and B is 3- (4-biphenyl) . 12. A peptide according to claim 4 wherein D-Xaa 8 is (B)D-Ala or (B)D,L-Ala and B is 3-(2-anthryl) . 13. A peptide according to claim 4 wherein D-Xaa 8 is (B)D-Ala or (B)D, L-Ala and B is 3- (2 , 4 , 6-trimethylphenyl) . 14. A peptide according to claim 1 having the amino acid sequence: (cyclo) Cys-Lyε-Phe-Phe-D-Trp-IAmp-Thr-Phe- Thr-Xaa 13 -Cys, wherein Xaa, 3 is Ser or D-Ser. 15. A pharmaceutical compoεition compriεing a mixture of a peptide according to claim 1 and at least one pharmaceutically acceptable carrier. 16. A method of arresting tumor growth in a patient, which method compriseε adminiεtering an effective amount of a composition according to claim 15, which amount iε effective to at leaεt slow the growth of a tumor having SSTRl receptorε. 17. A method for screening for ligandε which bind with high affinity to SSTRl, which method compriseε carrying out a competitive binding assay with SSTRl, a labelled peptide according to claim 1 and a candidate ligand, and determining the ability of the said candidate ligand to displace said labelled peptide. 18. A cyclic somatostatin (SRIF) analog peptide having the amino acid sequence: (cyclo)Tyr-Cys-Lys-Phe- ι • ' Phe-D-Trp-IAmp-Thr-Phe-Thr-Ser-Cys. 19. The peptide of claim 18 wherein Tyr iε iodinated. 20. A cyclic somatostatin (SRIF) analog peptide having specific affinity for the SRIF receptor SSTRl, which peptide haε an amino acid sequence at least 8 residues in length, contains a Cys-Cys disulfide bond with a seguence of at least 6 residueε located between said Cys residues as a ring, and contains D-Trp or its equivalent centrally of said at least 6-residue sequence, which analog peptide is characterized by the presence of (C)Amp joined immediately C-terminally to said D-Trp, with C being H or lower alkyl.

Inventors: HOEGER CARL A, RIVIER JEAN E F, HOEGER, CARL, A., RIVIER, JEAN, E., F.
Applicants: SALK INST FOR BIOLOGICAL STUDI, THE SALK INSTITUTE FOR BIOLOGICAL STUDIES
Application year: 1996
Main classification: C07K14
IPC codes: A61K31, A61K38, A61K51, A61P35, C07K14