WO2005123761A1 - GnRH ANALOGS BACKBONE CYCLIZED THROUGH METAL COMPLEXATION - Google Patents

Abstract

The present invention provides novel diagnostic and therapeutic peptides, specifically GnRH analogs backbone cyclized though metal complexation. The backbone cyclized peptide analogs disclosed possess unique and superior properties over other analogs, such as chemical and metabolic stability, increased bioavailability and improved pharmacokinetics. Pharmaceutical compositions comprising the backbone cyclized GnRH analogs and radiolabelled analogs, kits and reagents for synthesizing same, and methods using such compositions for diagnostic and therapeutic purposes are also disclosed.

Description

GnRH ANALOGS BACKBONE CYCLIZED THROUGH METAL COMPLEXATION

FIELD OF THE INVENTION The present invention relates to N^α backbone cyclic analogs of gonadotropin releasing hormone (GnRH), peptide analogs which are cyclized through complexation with metal, to pharmaceutical compositions containing same, to reagents for synthesizing same, and to methods for using such compounds for diagnosis and therapy.

BACKGROUND OF THE INVENTION Gonadotropin releasing hormone (GnRH), otherwise known as gonadotropin releasing factor (GnRF), otherwise known as lutheinizing hormone-releasing hormone (LH-RH), is a decapeptide that is secreted into the pituitary portal circulation and plays a major role in the biology of reproduction, inducing the biosynthesis and release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). GnRH pulsatile secretion is crucial for the control of gonadal function and normal ovarian cycle. GnRH activity is mediated by receptors for GnRH found on the pituitary gonadotrophs. Continuous stimulation of the pituitary, either by natural GnRH or by GnRH agonists, causes desensitization of gonadotropin secretion, resulting in gonadal suppression (Stojilkovic and Cart, Recent Prog Horm Res, 50:161-205 (1995); Schally, A.V., Anticancer Drugs, 5:115-30 (1994)). GnRH antagonists produce a competitive blockade as well as a down-regulation of pituitary GnRH receptors and cause an immediate inhibition of the release of gonadotropins and sex steroids. GnRH receptors and GnRH mRNA are over expressed in several tumors such as breast, prostatic and ovarian cancer (Schally, A.V., Neuroendocrinol, 22:248-91 (2001)). Thus GnRH can be involved in the growth of these tumors, providing the basis for the clinical applications of GnRH analogs in gynecology and oncology diagnosis and treatment. The amino acid sequence of native GnRH, a decapeptide, is pGlu-His-Trp-Ser- Tyr-Gly-Leu-Arg-Pro-Gly-NH₂ (SEQ ID NO: 1), wherein pGlu is pyroglutamic acid. GnRH in cancer Clinical use of GnRH analogs in the therapy of prostate cancer can be characterized as two general approaches. The first involves the chronic administration of GnRH analogs, agonists such as Leuprolide, Decapeptyl and Buserelin, and antagonists such as Cetrorelix and Ganirelix, resulting in medical castration (Schally and Cumaru- Schally, Hvpothalmic and Other Peptide Hormones in Cancer Medicine. (Williams and Wilkins, Baltimore, Md., 1997, pp. 1067-86), Huirne and Lambalk, Lancet. 358:1793-803 (2001); Gonzalez-Barcena et al., Urology, 45:275-81 (1995)). The second approach utilizes the high affinity of GnRH analogs for GnRH receptors to target cytotoxic, anti- tumor agents. In this approach, cytotoxic drugs linked to GnRH analogs are transported directly to metastases. An example of this type of therapeutic agent is AN- 152, which consists of doxorubicin linked through a glutaric acid spacer to the free amino group of [D-Lys⁶]GnRH (Letsch et al., Clin Cancer Res. 9:4505-13 (2003)). Radiolabelled Peptides as diagnostic/therapeutic agents Scintigraphy using labelled analog tracers helps to localize tumors and to evaluate the potential for chronic treatment of patients with inoperable receptor-positive tumors. One method for using radiolabelled analogs is to label tyrosine containing analogs with iodine. International patent application WO 96/39161 discloses multi-tyrosinated analogs in which the N-terminal of the peptides is extended with tyrosine residues, for radioiodination and subsequent diagnosis and treatment. One application of radiolabelled analogs is radio-guided surgery. Surgical intervention can be optimized by intraoperative detection of tissue-bound (¹²⁵I-Tyr3)- Octreotide administered before operation. This technique has been successfully utilized in surgery of, for example, medullary thyroid cancer, carcinoids and islet cell tumors. High specific activity is achieved by the multi-tyrosinated analogs as a result of multiple sites for iodination provided by the additional tyrosines. Certain peptides can be labeled directly without a loss of functional specificity but others must be labeled using bifunctional chelating agents (BFCA), which are covalently coupled to the tumor targeting peptide via a linker on one hand and form a complex with radiometals on the other hand. Liu and Edwards, Chem Rev, 99:2235-68 (1999). Methods for labeling peptides with ^99mTc are described in U.S. Patent No. 5,716,596 and U.S. Patent No. 5,620,675. A series of patents on radiolabelled analogs, describes cyclic (US 5,932,189, WO 95/00553 and WO 96/04308) and linear (US 5,620,675, WO 95/03330) peptides with 10-16 residues and high affinity for receptors. A second approach involves binding the metal isotope through the thiol groups of cysteine residues not essential for receptor binding but present in the peptide sequence, to achieve cyclization of the peptide through metal coordination. (Kolan and Thakur, Peptide Res., 9:144 (1996)). Giblin et al., Proc Natl Acad Sci USA, 95:12814-8 (1998); Melendez-Alafort et al, Int J Pharm. 182:165-72 (1999). ¹⁸⁸Re and ⁹⁹ Tc cyclic peptides can have outstanding stability in competition with phosphate, cysteine, diethylene triamine pentaacetic acid (DTP A) and in serum. ^{9 m}Tc is the most commonly used radiometal for diagnostic imaging because of its physical properties (pure γ-emitter, tι_/2=6 h, E_max=140 KeV) and its availability (Jurisson and Lydon, Chemical Reviews. 99:2205-18 (1999)). When carrying out synthetic and characterization work, ⁹⁹Tc can be replaced by its third row congener, rhenium (a mixture of ¹⁸⁵Re and ¹⁸⁷Re), which is cheaper, and easier to obtain and handle. The cyclic peptides disclosed contain an N₂S₂ type chelating ligand formed by two cysteine amino acids attached via their carboxyls to two amino groups of the backbone cyclic peptide. An N₂S₂ type chelating ligand containing two nitrogen and two sulfur atoms for chelate formation, and use for cyclic and linear hexapeptide analogs, is disclosed in international applications WO 96/11954 and WO 96/11918. The compounds are stated to have improved tumor/kidney distribution ratios over conventional analogs, thus reducing kidney radiation exposure. International application WO 94/00489 and U.S. Patent No. 5,871,711 disclose derived peptide reagents for preparation of scintigraphic imaging agents. The analogs are labeled with ^99mTc, ¹⁸⁶Re and ¹⁸⁸Re through complexation. US Patent No. 5,382,654 describes aminothiol ligands (N₂S and N₃S) which can be conjugated to an analog peptide and can accommodate a metal ion, which can be a radiometal. For diagnostic purposes, ^99mTc and ⁶²Cu are suggested for complex formation, while ¹⁸⁶Re, ⁶⁷Cu, ¹⁸⁸Re and ⁶⁰Co ions can be used for radiotherapy. The effect of labeling methods and peptide sequence on ^99mTc analogs was reviewed by Decristoforo C. and Mather S. J. (Eur. J. Nucl. Med.. 26:869, 1999). It is concluded that the selection of the labeling approach as well as the right choice of the peptide structure are crucial for labeling peptides with ^99mTc to achieve complexes with favorable activity and biodistribution. A number of ^99mTc-labeled bioactive peptides have proven to be useful diagnostic imaging agents. Pearson et al. (J. Med. Chem.. 39:1361, 1996) describe the chemistry and biology of ^99raTc labeled analogs. A radiolabelled analog, ^mrn-DTPA-(D)Phe-Octreotide (OctreoScan,

Mallinkrodt), has high diagnostic capacity for neuroendocrine tumors and lymphomas while its applicability for other tumors such as melanomas is lower. Labeled Octreotide analogs bind to -R2 and -R5. Octreotide labelled with ^mIn has been shown to detect a variety of neuroendocrine tumors with high specificity and sensitivity and becomes a valuable tool in diagnosis, but it suffers from at least one major drawback: the cost. Vapreotide (RC-160) was labeled with ^99mTc directly and also by using a bifunctional chelating agent and was successfully evaluated in nude mice bearing experimental human prostate cancer. The compound ^99mTc-Depreotide was successfully used in the evaluation of solitary pulmonary nodules in phase II/III clinical trial (Blum et al., Chest 117:1232, 2000). Receptor imaging has been used successfully (utilizing ^1I1In-pentetreotide) for detection of cardiac allograft rejection (Aparici et al. Eur. J. Nuc. Med. 27:1754, 2000). Cardiac rejection process usually presents with lymphocyte infiltration, which indicates the severity of the rejection and the necessity of treatment. Activated lymphocytes express receptors thus receptor imaging could be used to target them. GnRH receptor imaging may predict impending rejection at least one week before the endomyocardial biopsy becomes positive and thus allow earlier intervention in the event of rejection. A variety of radionuclides are known to be useful for radioimaging, including ⁶⁷Ga, ⁶⁸Ga, ^99mTc, ^mIn, or ^I23I. The sensitivity of imaging methods using radioactively- labeled peptides is much higher than other techniques known in the art, since the specific binding of the radioactive peptide concentrates the radioactive signal over the cells of interest, for example, tumor cells. This is particularly important for endocrine-active gastrointestinal tumors, which are usually small, slow-growing and difficult to detect by conventional methods. Technetium-99m (^99mTc, t = 6 h, Eγ= 140 keV) is the radionuclide of choice by virtue of its cost-effectiveness, availability and desirable nuclear characteristics. It is a decay product of ⁹⁹Mo. Because of its short half-life, it does not induce unnecessary radiation burden to a patient long after examinations are carried out, and its gamma ray energy is highly efficient for external imaging. ^99mTc is used in over 90% of the diagnostic nuclear medicine procedures. Other radionuclides have effective half-lives, which are much longer (for example, ^ιπIn, which has a half-life of 60-70 h), are toxic (for example In with its Auger electron emission) or are expensive (^ιπIn which is a cyclotron-produced radionuclide). US Patent No. 4,980,147 discloses ^{9 m}Tc compounds used as radiopharmaceutical imaging agents and particularly for conducting renal function imaging procedures. The preferred compound claimed is ^99mTc-mercaptoacetyl-glycylglycylglycine (^99mTc- MAG3). This and related compounds are used without conjugation with another peptide analog. US Patent No. 4,883,862 discloses the compound mercaptosuccinyl- glycylglycylglycine and its complexes with ^99mTc for use as renal agents. The mercaptosuccinyl -glycylglycylglycine is made by coupling glycylglycylglycine with S- acetyl-mercapto succinic anhydride. WO 01/02022 disclosed linear alpha melanocyte stimulating hormone (MSH) analogs cyclized through oxorhenium(V) and oxotechnetium(V), providing stable complexes able to reach their target in vivo. These novel compounds are candidates for diagnostic imaging and targeted radiopeptide therapy of melanotropin receptor-expressing melanoma. Radiolabelled GnRH analogs as diagnostic/therapeutic agents GnRH analogs have high affinity to GnRH receptors thus can also serve as carriers to direct diagnostic or therapeutic substances to GnRH receptor-positive tumors. Thus, synthetic, labeled GnRH analogs can be used for the treatment, diagnosis and monitoring of prostatic, breast and ovarian cancers. An example of a radiolabeled analog of GnRH is ^99mTcO₂-P₂S₂-D-Lys⁶-LHRH described by Gali et al. (Bioconiug Chem. 12:354-63 (2001)). To date, this is the only radiolabeled analog of GnRH, reported, but no biological data were reported. Improved Peptide Analogs As a result of major advances in organic chemistry and in molecular biology, many bioactive peptides can now be prepared in quantities sufficient for pharmacological and clinical use. Thus in the last few years new methods have been established for the treatment and diagnosis of illnesses in which peptides have been implicated. However, the use of peptides as therapeutic and diagnostic agents is limited by the following factors: a) tissue penetration; b) low metabolic stability towards proteolysis in the gastrointestinal tract and in serum; c) poor absorption after oral ingestion, in particular due to their relatively high molecular mass or the lack of specific transport systems or both; d) rapid excretion through the liver and kidneys; and e) undesired side effects in non-target organ systems, since peptide receptors can be widely distributed in an organism. It would be desirable to achieve peptide analogs with greater specificity thereby achieving enhanced clinical selectivity. It would be most beneficial to produce conformationally constrained peptide analogs overcoming the drawbacks of the native peptide molecules, thereby providing improved therapeutic properties. A novel conceptual approach to the conformational constraint of peptides was introduced by Gilon, et al., (Biopolymers 31:745, 1991) who proposed backbone to backbone cyclization of peptides. The theoretical advantages of this strategy include the ability to affect cyclization via the carbons or nitrogens of the peptide backbone without interfering with side chains that may be crucial for interaction with the specific receptor of a given peptide. Further disclosures by Gilon and coworkers (WO 95/33765, WO 97/09344, US 5,723,575, US 5,811,392, US 5,883,293 and US 6,265,375), provided methods for producing building units required in the synthesis of backbone cyclized peptide analogs. The successful use of these methods to produce backbone cyclized peptide analogs of bradykinin analogs (US 5,874,529), and backbone cyclized peptide analogs having GnRH activity was also disclosed (WO 98/04583, WO 99/65508, US 5,770,687, US 6,051,554 and US 6,355,613). WO 02/062819 of one of the present inventors, discloses radiolabelled-backbone cyclized somatostatin analogs for diagnostic and therapeutic uses, and WO 2004/000204 discloses labelled somatostatin analogs backbone cyclized through metal complexation. All of these methods and analogs are incorporated herein in their entirety, by reference. None of the background art teaches or suggests GnRH analogs backbone cyclized via complexation with a metal, disclosed herein having improved diagnostic and therapeutic properties.

There is an unmet need for GnRH analogs, both for diagnostic and therapeutic use. There remains a need for synthetic analogs having increased in vivo stability, to be used therapeutically, as scintigraphic agents when labelled with Tc-99m or other detectable isotopes for use in imaging tumors in vivo, and as radiotherapeutic agents when radiolabelled with a cytotoxic radioisotope such as rhenium- 188. The present invention addresses this need by providing backbone cyclized analogs that specifically fulfill these needs and provides additional advantages as well.

SUMMARY OF THE INVENTION The present invention provides novel GnRH analogs comprising backbone cyclic peptides for therapeutic and diagnostic applications, including radio-therapeutic and radio-diagnostic applications. In particular the present invention provides analogs backbone cyclized through metal complexation useful for scintigraphic imaging. The novel analogs according to the present invention having high affinity to GnRH receptors associated with several types of cancers may be used for diagnosis and treatment of tumors by application of receptor-specific reagents. Specific embodiments comprise GnRH analog of four to twenty-four amino acids that incorporates at least one building unit, comprising N^α-ω-functionalized derivative of an amino acid, wherein a backbone cyclic structure is formed by metal complexation to a chelating moiety comprising the at least one building unit and a second moiety selected from the group consisting of a second building unit, a side chain of an amino acid residue of the GnRH and a terminal amino acid residue of said GnRH analog. Distinct from native and analogs known in the art, the cyclic peptides of the present invention are analogs whose backbones are cyclized through metal complexation. Accordingly, the analogs of the present invention possess unique and superior properties such as chemical and metabolic stability, selectivity, increased bioavailability and improved pharmacokinetics. These subject analogs can be labeled with isotopes, preferably radioisotopes, as the metal by which cyclization is achieved. According to the specific embodiment of the present invention, novel labeled peptide analogs which are characterized in that they incorporate novel building units with bridging groups attached to the alpha nitrogens of alpha amino acids, are disclosed. Specifically, these compounds are backbone cyclized GnRH analogs comprising a peptide sequence of four to twenty four amino acids, each analog incorporating at least one building unit, said building unit containing one nitrogen atom of the peptide backbone connected to a bridging group comprising a chelator-metal complex, preferably an N₂S₂ oxorhenium(V) or oxotechnetium(V) metal complex, wherein at least one building unit is connected via side bridging group to form a cyclic structure with a moiety selected from the group consisting of a second building unit, the side chain of an amino acid residue of the sequence or a terminal amino acid residue. Preferably, the peptide sequence incorporates 4 to 24 residues, more preferably 5 to 16 amino acids, most preferably 7-12 amino acids. The present invention provides GnRH analogs cyclized through site-specific metal complexation. The chelating of the metal to the peptide through binding to a chelating moiety coupled to at least one N^α substituted amino acid enables formation of a cyclic structure. In preferred embodiments, the metal binds the peptide through a N₂S₂ type chelator. In even more preferred embodiments, the chelator is built from two thiol and two alpha amino groups of cysteine residues. The diagnostic radiopharmaceutical comprising a peptide cyclized through a radionuclide has several distinct advantages over compounds known in the art that are already cyclic prior to metal complexation. In both cases the cold kit labeling process results in less than 10% of the kit peptide being complexed with metal. In the case of the cyclic non-metal/non-radioactive peptide form of the known compounds, the peptide is relatively stable metabolically. This results in administration of a relatively long- circulating pharmacologically active compound. According to the present invention, because the unlabelled linear peptide is expected to be unstable metabolically, approximately 90% of unlabelled material should be cleared from the body rapidly and is expected to exhibit little to no pharmacological activity in comparison to analogs that are cyclic species per se prior to labeling. According to the present invention, labelled GnRH analogs backbone cyclized through metal complexation, have high affinity to GnRH receptors. The invention relates both to agonists and antagonists of native GnRH. According to another embodiment of the present invention GnRH analogs may advantageously include bicyclic structures containing at least one backbone structure cyclized through metal complexation, wherein at least one building unit is involved in the cyclic structure, and a second cyclic structure which is selected from the group consisting of side-chain to side-chain, backbone to backbone and backbone to terminal. The invention further provides peptide reagents capable of being labeled to form backbone cyclic diagnostic and therapeutic agents. These reagents comprise a GnRH analog covalently linked to a binding moiety which is formed using at least one N^α-ω- functionalized derivative of an amino acid. The metal binds to the binding moiety to form a backbone cyclic structure. In preferred embodiments according to the present invention the chelating moiety comprises four donor atoms and the metal is a radioactive isotope. According to a specific embodiment of the present invention the chelator is built from two free thiols and two free nitrogens, which through complexation with a metal form a backbone cyclic structure. In more specific analogs the chelator is made from two cysteine residues. In preferred embodiments according to the present invention at least one of the cysteine residues is covalently connected to the bridging group of an N^α-ω- functionalized derivative of an amino acid. Preferred chelating moieties according to the present invention include those in which the four donor atoms are two nitrogens and two sulfurs (N₂S₂) and, through metal complexation, the peptide analog is cyclized and stable 5- to 6-membered rings are formed according to the general Formula No. 1 :

wherein the Ds represent the four donor atoms of N₂S₂; the half-circles represent two- or three-carbon bridges between the donor atoms; the R groups are independently selected from the group consisting of cyclic peptide, linear peptide, oxo, hydroxy, a hydrocarbon, hydrogen, a linking or spacing group connecting the peptide analog and the chelating moiety, and are located on a position selected from the donor atoms and the carbon bridges, wherein at least two of the R groups together with the chelating moiety form a cyclic peptide structure; and M is a metal atom preferably selected from Re and Tc in the +5 oxidation state. Chelators of the N₂S₂ type are, for example, constructs of two NS hemi-chelators: two Cys residues; one Cys and one amidomercaptoacetyl (AMA) residue, one Cys and one amidomercaptoethyl (AME) residue; two AMA residues; one AMA and one AME residue; or two AME residues. The Cys residues are selected from the D and L stereoisomers and interposition of dissimilar residues on the peptide provides a second, isomeric analog. In preferred analogs the peptide is coupled to one hemi-chelator via a linker and a second hemi-chelator via the peptide backbone, to form a structure of the general Formula No. 2: Z-Q-Y-X Formula No. 2

wherein Z is a first hemi-chelating moiety comprising two donor atoms, one N and one S, that through metal complexation form a five- to six-membered ring;

Q is absent or a linker moiety which can be coupled to a free functional group of the peptide;

Y denotes a GnRH analog comprising at least one N^α-ω-functionalized derivative of an amino acid; and X is a second hemi-chelating moiety comprising two donor atoms, one N and one S, that through metal complexation form a five- to six-membered ring, wherein the chelating moiety is linked through a lower alkyl chain comprising 1-6 carbon atoms, to the alpha nitrogen of the Y backbone or to a free functional group of the peptide. Preferably, the linker Q is connected to the N-terminal of the peptide, and X is connected to the peptide backbone or to a peptide side chain. More preferably the linker Q is absent or is selected from the group consisting of gamma amino butyric acid (GABA), Gly, βAla, amino caproic acid and amino valeric acid, and X is connected to the α- nitrogen of an N-building unit. Most preferably, Z and X are each independently selected from the group consisting of L cysteine and D cysteine. Some of the preferred analogs according to the present invention may comprise two or more isomers. The present invention includes such isomers either in combination or individually isolated. The invention provides radiolabelled backbone cyclic peptides that are suitable for use as scintigraphic imaging agents, radiodiagnostic agents and radiotherapeutic agents. Scintigraphic imaging agents of the invention comprise peptide reagents backbone cyclized through metal complexation with radionuclides, preferably ^99mTc, for use in diagnostic imaging (single photon emission computed tomography, gamma camera, planar detector probes or devices for intraoperative use, positron emission tomography). Radiotherapeutic agents of the invention comprise backbone cyclic peptide reagents radiolabelled with a cytotoxic radioisotope (having α or β emission). The most preferred cytotoxic radioisotopes according to the present invention are rhenium- 186 and rhenium- 188. Additional preferred radionuclides according to the invention are radioisotopes of indium, yttrium, lutetium, gallium and gadolinium. Combination embodiments, wherein a particular complex is useful both in scintigraphic imaging and in targeted radiotherapy, are also provided by the invention. Methods for making and using such backbone cyclic peptides, backbone cyclic reagents and radiolabelled embodiments thereof are also provided. The amino acid sequence of native GnRH, a decapeptide, is pGlu-His-Trp-Ser- Tyr-Gly-Leu-Arg-Pro-Gly-NH₂ (SEQ ID NO:l), wherein pGlu is pyroglutamic acid. The currently most preferred analogs backbone cyclized through metal complexation according to the present invention are now disclosed: Ser(Bu^l) is serine where the side chain oxygen is t-butylated, Pro-NHET means proline amide, where the amide is ethylated, and the η-amino group of Gly is -(CH )₆- NH- attached to the amino end of Gly. In one embodiment of the invention, Gly (in the sixth position of the native GnRH molecule) is replaced with a D-amino acid. More particularly, Gly can be replaced with D-Trp, D-Leu, D-Ser(Bu^l) or D-Ala. In a preferred embodiment, Gly is replaced by D- Ala. In another embodiment of the invention, Pro (in the ninth position) has its amino group ethylated. In a further embodiment of the invention, pGlu (in the first position) is replaced with Gly. In yet another embodiment, His (in the second position) can be absent. Accordingly, the subject invention encompasses a compound having the general

Formula No. 3 (SEQ ID NO:2):

Y-W-Trp-Ser-Tyr-Z-Leu-Arg-Pro-NGly-X - Cys¹-M-Cys²-NH-(CH₂)_n -

Formula No. 3 wherein n is 1 to 8; W is absent or is His;

Y is absent or is selected from Gly, β-Ala, GABA, valeric acid and caproic acid; Z is selected from D-Trp, D-Leu, D-Ser, D-Ser(Bu^l) and D-Ala;

X is a terminal carboxy acid, amide or alcohol group; Cys¹ and Cys² are each independently L or D isomers; and M is a metal.

Preferably: n is 2, 3, 4 or 6 and, most preferably is 6; 1 9

Cys and Cys are L-Cys; Z is D-Ala;

Y is Gly; W is His;

X is an amide; and

M is a radiometal selected from [^naRe]oxorhenium(V), [¹⁸⁶Re]oxorhenium(V), [¹⁸⁸Re]oxorhenium(V) or [^99mTc]oxotechnetium(V). The most preferred analogs according to Formula 3 are:

ReO-Cys^!i:-Gly-His-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-N(η-Cys*-aminohexyl))GlyNH₂ (SEQ ID NO:3); and TcO-Cys^-Gly-His-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-N^Cys^-aminohexy^GlyN^ (SEQ ID NO:4).

Also included in the invention is the analog of SEQ ID NO:5:

ReO-Cys*-Gly-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-N(ηCys*-aminohexyl)GlyNH₂ (SEQ ID NO:5). The asterisks denote the chelating groups used for cyclization through metal complexation. More specifically, one Cys is attached to the η-amino hexamethylene group of the Gly building unit at the tenth position. The other Cys is attached to the amino end of the peptide. These backbone cyclized peptide analogs are prepared by incorporating at least one N^α-ω-functionalized derivative of an amino acid (backbone cyclization building unit) into a peptide sequence, for example Gly with an η-aminohexamethylene group attached to its N alpha. Two hemi-chelating NS donor atom-containing moieties are added, one to the nitrogen of the N^α-ω-functionalized amino acid (for example through addition of Cys) and another to either the terminal N or to a straight-chain AA spacer at the N-terminus (for example through addition of Cys to the terminal N). Selective cyclization is accomplished through binding of a single metal or radiometal (preferably as oxorhenium(V) or oxotechnetium(V)) to both bidentate hemi-chelators to form a tetradentate N₂S₂ oxometal(V) cyclic peptide (or peptidomimetic) complex. The hemi- chelating moieties can alternatively be covalently bound to two N^α-ω-functionalizations, one or more amino acid side chain in the peptide sequence, or any combination of N^α-ω- functionalization, amino acid side chain, C- or N-terminus or linker or spacer group attached to any of the above. It is another advantage of the analogs provided by this invention that the N- alkylation moiety acts to protect the peptide from degradation by exopeptidases. GnRH analogs backbone cyclized through metal complexation of the present invention may be used as diagnostic compositions in methods for diagnosing cancer and imaging the existence of tumors or their metastases, The methods for diagnosis of cancer comprise administering to a mammal, including a human patient, a backbone cyclic analog or analogs labeled with a detectable tracer which is selected from the group consisting of a radioactive isotope and a non-radioactive tracer. The methods for the diagnosis or imaging of cancer using such compositions represent another embodiment of the invention. The pharmaceutical compositions comprising pharmacologically active labelled backbone cyclized agonists or antagonists and a pharmaceutically acceptable carrier or diluent represent another embodiment of the invention, as do the methods for the treatment of cancers in targeted radiotherapy using such compositions. The pharmaceutical compositions according to the present invention advantageously comprise at least one peptide analog backbone cyclized through metal complexation. These pharmaceutical compositions may be administered by any suitable route of administration, including orally, topically or systemically. Preferred modes of administration include but are not limited to parenteral routes such as intravenous and intramuscular injections, as well as via intra-nasal administration or oral ingestion. The invention further provides a method for treating or diagnosing GnRH-related diseases in animals, preferably humans, comprising administering a therapeutically effective amount of backbone cyclic analogs of the invention. In some preferred embodiments, the reagent is radioactively labeled with ¹⁸⁶Re or ¹⁸⁸Re. Another aspect of the present invention provides methods for preparing therapeutic and diagnostic agents, including preferably scintigraphic imaging agents. Each such reagent comprises an analog capable of being backbone cyclized through metal complexation. The invention further provides kits for making and labelling such compositions.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the activity portion in plasma of cyclo [^99mTc(O)-Gn-2], a labeled GnRH analog backbone cyclized through metal complexation, after incubation in blood.

Figure 2 describes stability results of cyc/ø[^99raTc(O)-Gn-2] as percentage intact compound in plasma after 4 hours of incubation in blood.

Figure 3 depicts percentage intact compound in blood after 4 hours of incubation. DETAILED DESCRIPTION OF THE INVENTION According to the present invention, peptide analogs are cyclized through metal complexation, via bridging groups attached to the alpha nitrogens of amino acids that permit novel non-peptidic linkages. In general, the procedures utilized to construct such peptide analogs from their building units rely on the known principles of peptide synthesis; most conveniently, the procedures can be performed according to the known principles of solid phase peptide synthesis. The methods for design and synthesis of backbone cyclized analogs according to the present invention are disclosed in US Patent Nos.: 5,811,392; 5,874,529; 5,883,293; 6,051,554; 6,117,974; 6,265,375, and international applications WO 95/33765; WO 97/09344; WO 98/04583; WO 99/31121; WO 99/65508; WO 00/02898; WO 00/65467, WO 02/062819 and WO 2004/000204. All of these methods are incorporated herein in their entirety, by reference. The most striking advantages of backbone cyclization are: 1) cyclization of the peptide sequence is achieved without compromising any of the side chains of the peptide thereby decreasing the chances of sacrificing functional groups essential for biological recognition (e.g. binding to specific receptors) and function.

2) optimization of the peptide conformation is achieved by allowing permutation of the bridge length, and bond type (e.g., amide, disulfide, thioether, thioester, urea, carbamate, or sulfonamide, etc.), bond direction, and bond position in the ring.

3) when applied to cyclization of linear peptides of known activity, the bridge can be designed in such a way as to minimize its interaction between the active region of the peptide and its cognate receptor. This decreases the chances of the cyclization arm interfering with recognition and function, and also creates a site suitable for attachment of tags such as radioactive tracers, cytotoxic drugs, photoactive substances, or any other desired label. Distinct from native and analogs known in the background art, the peptides of the present invention are analogs backbone cyclized through metal complexation, which possess unique and superior properties such as chemical and metabolic stability, selectivity, increased bioavailability and improved pharmacokinetics. These analogs are labeled with metal isotopes, preferably radioisotopes. The diagnostic radiopharmaceutical comprising a peptide cyclized through a radionuclide has several distinct advantages over compounds known in the art that are already cyclic prior to metal complexation. In both cases the cold kit labeling process results in less than 10% of the kit peptide being complexed with metal. In the case of a cyclic non-metal/non-radioactive peptide, the peptide is relatively stable metabolically; this results in administration of a relatively long-circulating pharmacologically active compound. According to the present invention, the unlabelled linear peptide is expected to be unstable metabolically, therefore the 90% of unlabelled material should be cleared from the body rapidly and is expected to exhibit little to no pharmacological activity in comparison to analogs that are unlabeled cyclic species. Terminology and definitions The term "agonist of GnRH" means a molecule capable of mimicking at least one of the actions of GnRH. The term "antagonist of GnRH" means a molecule capable of reducing or preventing at least one of the actions of GnRH. The term "linker" means a chemical moiety whose purpose is to link, covalently, a chelating moiety and a peptide, peptide analog or peptido-mimetic. The linker may be also used as a spacer whose purpose is to allow distance between the chelating moiety (thus the metal) and the peptide, peptide analog or peptido-mimetic. The term "chelating agent" means a chemical moiety whose purpose is to stably form a chelating agent (or chelator)-metal complex. The complex is formed through electron donation from certain electron-rich atoms on the chelating agent to the electron- poor metal. The chelating agent typically has four donor atoms. The preferred donor atom for oxorhenium(V) and oxotechnetium(V) is nitrogen and the most preferred donor atom is sulfur. The term "hemi-chelator" means a chemical moiety whose purpose is to form half of the metal-complex with two donor atoms as described above. A second hemi-chelator on the same compound can form the second half of the complex with the same metal. The term "scintigraphic imaging agent" encompasses a radiolabelled agent capable of being detected with a radioactivity detecting means. The term includes, but is not limited to, a planar camera, a gamma-camera, a single photon emission (computed) tomography (SPECT or SPET) or any hand-held probe (e.g. Geiger-Muller counter or a scintillation detector) or device for use intraoperatively or otherwise in the detection of tumors. The term "peptide" means a sequence of amino acids linked by peptide bonds. The peptides according to the present invention comprise a sequence of 4 to 24 amino acid residues, preferably 5 to 16 residues, and more preferably 7 to 16 amino acids. A peptide analog according to the present invention may optionally comprise at least one bond which is an amide-replacement bond such as urea bond, carbamate bond, sulfonamide bond, hydrazine bond, or any other covalent bond. The term "analog" means a molecule which has the amino acid sequence according to the invention except for one or more amino acid changes. The design of appropriate "analogs" may be computer assisted. Whenever "peptide of the invention" or "analog of the invention" or any composition of the invention is referred to herein, also included are salts and functional derivatives thereof, provided that the biological activity of the peptide, analog or composition is maintained. Salts of the peptides, analogs or compositions of the invention contemplated are physiologically acceptable organic and inorganic salts. Functional derivatives of the peptides, analogs or compositions of the invention covers derivatives which may be prepared from the functional groups which occur as side chains on the residues or the N- or C-terminal groups, by means known in the art, and are included in the invention as long as they remain pharmaceutically acceptable, i.e., they do not destroy the activity of the peptide and do not confer toxic properties on compositions containing it. These derivatives may, for example, include aliphatic esters of the carboxyl groups, amides of the carboxyl groups produced by reaction with ammonia or with primary or secondary amines, N-acyl derivatives of free amino groups of the amino acid residues formed by reaction with acyl moieties (e.g., alkanoyl or carbocyclic aroyl groups) or O- acyl derivatives of free hydroxyl group (for example that of seryl or threonyl residues) formed by reaction with acyl moieties. As used herein the term "backbone cyclic peptide" or "backbone cyclic analog" means an analog of a linear peptide comprising a peptide sequence of preferably 4 to 24 amino acids that incorporates at least one building unit, comprising N^α-ω-functionalized derivative of an amino acid, wherein i. said building unit containing one nitrogen atom of the peptide backbone connected to a bridging group comprising an amide, thioether, thioester, disulfide, urea, carbamate, or sulfonamide, wherein at least one building unit is connected via said bridging group to form a cyclic structure with a moiety selected from the group consisting of a second building unit, the side chain of an amino acid residue of the sequence or a terminal amino acid residue; or ii. a backbone cyclic structure is formed by metal complexation to a chelating moiety connected to at least one building unit and to a second moiety selected from the group consisting of a second building unit, the side chain of an amino acid residue of the sequence or a terminal amino acid residue.. More preferably, the peptide sequence incorporates 3-24 amino acids, still more preferably it incorporates 5-16 amino acids, even more preferably 8-14 amino acids, yet even more preferably 9-13 amino acids and most preferably 10-11 amino acids.

A "building unit" indicates an N^α derivatized amino acid of the general Formula No. 4: -N-CH ( R' ) -CO-

X

Formula No. 4

wherein X is a spacer group selected from the group consisting of alkylene, substituted alkylene, arylene, cycloalkylene and substituted cycloalkylene; R' is an amino acid side chain, optionally bound with a specific protecting group; and G is a functional group selected from the group consisting of amines, thiols, alcohols, carboxylic acids, sulfonates and esters, and alkyl halides; which is incorporated into the peptide sequence and subsequently selectively cyclized via the functional group G with one of the side chains of the amino acids in said peptide sequence or with another ω-functionalized amino acid derivative, via complexation with a metal or metal, through N₂S₂ donor chemistry. The methodology for producing the building units is described in international patent applications published as WO 95/33765 and WO 98/04583 and in US Patent Nos. 5,770,687 and 5,883,293 all of which are expressly incorporated herein by reference thereto as if set forth herein in their entirety. The building units are abbreviated by the three letter code of the corresponding modified amino acid followed by the type of reactive group (N for amine, C for carboxyl), and an indication of the number of spacing methylene groups. For example, GlyC2 describes a modified Gly residue with a carboxyl reactive group and a two carbon methylene spacer, and PheN3 designates a modified phenylalanine group with an amino reactive group and a three carbon methylene spacer. In generic formulae the building units are abbreviated as R with a superscript corresponding to the position in the sequence preceded by the letter N, as an indication that the backbone nitrogen at that position is the attachment point of the bridging group specified in said formulae. The compounds herein disclosed may have asymmetric centers. All chiral, diastereomeric, and racemic forms are included in the present invention. Many geometric isomers of double bonds and the like can also be present in the compounds disclosed herein, and all such stable isomers are contemplated in the present invention. By "stable compound" or "stable structure" is meant herein a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious diagnostic or therapeutic agent. The term, "substituted" as used herein and in the claims, means that any one or more hydrogen atoms on the designated atom is replaced with a selection from the indicated group, provided that the designated atom's normal valency is not exceeded, and that the substitution results in a stable compound. When any variable (for example R, X, Z, etc.) occurs more than one time in any constituent or in any Formula herein, its definition on each occurrence is independent of its definition at every other occurrence. Also, combinations of substituents or variables are permissible only if such combinations result in stable compounds. As used herein, the term "therapeutically effective amount" means that amount of novel backbone cyclized peptide analog or composition comprising same to administer to a host to achieve the desired results for the indications disclosed herein, such as but not limited to cancer, endocrine disorders, inflammatory diseases, and gastrointestinal disorders. Certain abbreviations are used herein to describe this invention and the manner of making and using it. For instance, Alloc refers to allyloxycarbonyl, Boc refers to the t- butyloxycarbonyl, CT refers to computed tomography, DCM refers to dichloromethane, DIEA refers to diisopropyl-ethyl amine, DMF refers to dimethyl formamide, DTPA refers to diethylenetriaminepentaacetic acid, EDTA refers to ethylenediaminetetraacetic acid, Fmoc refers to fluorenylmethoxycarbonyl, FSH refers to follicle-stimulating hormone, GABA refers to gamma aminobutyric acid, mCi refers to millicurie, GnRF refers to gonadotropin releasing factor, GnRH refers to gonadotropin releasing hormone, HPLC refers to high pressure liquid chromatography, LH refers to luteinizing hormone, LH-RH refers to lutheinizing hormone-releasing hormone, MS refers to mass spectrometry, NMP refers to l-methyl-2- -pyrolidonone, PET refers to positron emission tomography, PyBrOP refers to bromo-tris-pyrrolidino-phosphonium hexafluorophosphate, SPECT refers to single photon emission computed tomography, SPET refers to single photon emission tomography, , TFA refers to trifluoroacetic acid. The amino acids used in this invention are those which are available commercially or are available by routine synthetic methods. Certain residues may require special methods for incorporation into the peptide, and sequential, divergent and convergent synthetic approaches to the peptide sequence are useful in this invention. Natural coded amino acids and their derivatives are represented by three-letter codes according to IUPAC conventions. When there is no indication, the L isomer was used. The D isomers are indicated by "(D)" or "D" before the residue abbreviation. List of Non-coded amino acids: Abu refers to 2-aminobutyric acid, Dab refers to diaminobutyric acid, Dpr and Dap both refer to diaminopropionic acid, GABA refers to gamma aminobutyric acid, INal refers to 1-naphthylalanine, 2Nal refers to 2-naphtylalanine, and Nle refers to norleucine. Conservative substitution of amino acids as known to those skilled in the art is within the scope of the present invention. Conservative amino acid substitutions includes replacement of one amino acid with another having the same type of functional group or side chain e.g. aliphatic, aromatic, positively charged, negatively charged. These substitutions may enhance oral bioavailability, penetration into the central nervous system, targeting to specific cell populations and the like. One of skill will recognize that individual substitutions, deletions or additions to peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following six groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). Pharmacology Apart from other considerations, the fact that the novel active ingredients of the invention are peptides or peptide analogs, dictates that the formulation be suitable for delivery of this type of compounds. Clearly, peptides are less suitable for oral administration due to susceptibility to digestion by gastric acids or intestinal enzymes. The preferred routes of administration of peptides are intra-articular, intravenous, intramuscular, subcutaneous, intradermal, or intrathecal. A more preferred route is by direct injection at or near the site of disorder or disease. Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, grinding, pulverizing, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. For injection, the compounds of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants for example polyethylene glycol are generally known in the art. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses. Pharmaceutical compositions, which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration. For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner. For administration by inhalation, the variants for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the peptide and a suitable powder base such as lactose or starch. Pharmaceutical compositions for parenteral administration include aqueous solutions of the active ingredients in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable natural or synthetic carriers are well known in the art (Pillai et al., Curr. Opin. Chem. Biol. 5:447, 2001). Optionally, the suspension may also contain suitable stabilizers or agents, which increase the solubility of the compounds, to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient may be in powder form for reconstitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use. The compounds of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides. Pharmaceutical compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of a compound effective to prevent, alleviate or ameliorate symptoms of a disease of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art. Toxicity and therapeutic efficacy of the peptides described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the IC^Q (the concentration which provides 50% inhibition) and the LD50 (lethal dose causing death in 50 % of the tested animals) for a subject compound. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition (e.g. Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.l). Depending on the severity and responsiveness of the condition to be treated, dosing can also be a single administration of a slow release composition, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved. The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, and all other relevant factors. Preferred embodiments According to the present invention, novel labelled peptide analogs which are characterized in that they incorporate novel building units with bridging groups attached to the alpha nitrogens of alpha amino acids, are disclosed. Specifically, these compounds are backbone cyclized GnRH analogs comprising a peptide sequence of four to twenty four amino acids, each analog incorporating at least one building unit, said building unit containing one nitrogen atom of the peptide backbone connected to a bridging group comprising an N₂S₂ oxorhenium(V) or oxotechnetium(V) metal complex, wherein at least one building unit is connected via said bridging group to form a cyclic structure with a moiety selected from the group consisting of a second building unit, the side chain of an amino acid residue of the sequence or a terminal amino acid residue. Preferably, the peptide sequence incorporates 4 to 24 residues, more preferably 5 to 16 amino acids, even more preferably 8-14 amino acids, yet even more preferably 9-13 amino acids and most preferably 10-11 amino acids. According to the present invention it is now disclosed that preferred analogs are decapeptide analogs backbone cyclized through metal complexation, with improved affinity and selectivity to specific subtypes. Other preferred GnRH analogs according to the present invention may advantageously include bicyclic structures containing at least one backbone structure cyclized through metal complexation, wherein at least one building unit is involved in the cyclic structure, and a second cyclic structure which is selected from the group consisting of side-chain to side-chain; backbone to backbone and backbone to terminal. The invention further provides peptide reagents capable of being labelled to form backbone cyclic diagnostic and therapeutic agents. These reagents comprise a GnRH analog covalently linked to a metal-binding moiety which is formed using at least one N^α- ω-functionalized derivative of an amino acid. The metal binds to the metal-binding moiety to form a backbone cyclic structure. In preferred embodiments according to the present invention the chelating moiety comprises four donor atoms and the metal is comprises radioactive isotope. According to the present invention the chelator is built from two free thiols and two free nitrogens, which through complexation with a metal form a backbone cyclic structure. In most preferred analogs the chelator is made from two cysteine residues. In preferred embodiments according to the present invention at least one of the Cysteine residues is covalently connected to the bridging group of an N^α-ω- functionalized derivative of an amino acid. Preferred chelating moieties according to the present invention include those in which the four donor atoms are two nitrogens and two sulfurs (N₂S₂) and, through metal complexation, the peptide analog is cyclized and stable 5- to 6-membered rings are formed according to the general Formula No. 1 :

Formula No. 1

wherein the Ds represent the four donor atoms of N₂S₂; the half-circles represent two- or three-carbon bridges between the donor atoms; the R groups are independently selected from the group consisting of cyclic peptide, linear peptide, oxo, hydroxy, a hydrocarbon, hydrogen, a linking or spacing group connecting the peptide analog and the chelating moiety, and are located on a position selected from the donor atoms and the carbon bridges, wherein at least two of the R groups together with the chelating moiety form a cyclic peptide structure; and M is a metal atom preferably selected from Re and Tc in the +5 oxidation state. Additional preferred embodiments comprise chelating moieties to form oxorhenium(V) or oxotechnetium(V) complexes having -1, neutral, +1, or +2 electronic charges as described in the following table: Table No. 1 : N₂S₂ donor set description

The invention provides radiolabelled backbone cyclic peptides that are scintigraphic imaging agents, radiodiagnostic agents and radiotherapeutic agents. Scintigraphic imaging agents of the invention comprise backbone cyclic peptide reagents radiolabelled with gamma-radiation emitting isotopes, preferably ^99mTc for use in diagnostic imaging (single photon emission computed tomography, gamma camera, planar, detector probes or devices for intraoperative use). Any other technetium or rhenium radioisotopes having decay characteristics making them useful in radionuclide imaging (including positron emission tomography, PET), capable of complexation with the backbone cyclic analogs of the invention, are also encompassed by the present invention. Radiotherapeutic agents of the invention comprise backbone cyclic peptide reagents radiolabelled with a cytotoxic radioisotope (α or β emission). Most preferred cytotoxic radioisotopes according to the present invention are rhenium- 186 and rhenium- 188. Combination embodiments, wherein such a complex is useful both in scintigraphic imaging and in targeted radiotherapy, are also provided by the invention. Any other technetium or rhenium radioisotopes having decay characteristics making them useful in radiotherapy, capable of complexation with the backbone cyclic analogs of the invention, are also are also encompassed by the present invention. GnRH analogs backbone cyclized through metal complexation according to the invention may be also used as contrast agents for magnetic resonance imaging (MRI) of cancer. In proton MRI diagnostics, increased contrast of internal organs and tissues may be obtained by administrating compositions containing paramagnetic metal species, which increase the relaxivity of surrounding water protons. In addition, the compounds of the present invention may be used for computed tomography (CT) diagnostics wherein increased contrast of tumors is obtained by administering a contrast agent which is substantially radiopaque. GnRH antagonists produce a competitive blockade as well as a down-regulation of pituitary GnRH receptors and cause an immediate inhibition of the release of gonadotropins and sex steroids. GnRH receptors and GnRH mRNA are over expressed in several tumors such as breast, prostatic and ovarian. Backbone cyclized analogs of the present invention may be used as diagnostic compositions in methods for diagnosing cancer and imaging the existence of tumors or their metastases. The methods for diagnosis of cancer comprise administering to a mammal, including a human patient, a backbone cyclic analog or analogs labeled with a detectable tracer which is selected from the group consisting of a radioactive isotope and a non-radioactive tracer. The methods for the diagnosis or imaging of cancer using such compositions represent another embodiment of the invention. The imaging agents provided by the invention have utility for tumor imaging, particularly for imaging primary and metastatic neoplastic sites wherein said neoplastic cells express receptors and in particular such primary and especially metastatic tumor cells that have been clinically difficult to detect and characterize using conventional methodologies. The imaging reagents according to the present invention may be used for visualizing organs, and tumors, in particular gastrointestinal tumors, myelomas, small cell lung carcinoma and other APUDomas, endocrine tumors such as medullary thyroid carcinomas and pituitary tumors, brain tumors such as meningiomas and astrocytomas, and tumors of the prostate, breast, colon, and ovaries can also be imaged. The ^99mTc labeled diagnostic reagents are preferably administered intravenously in a single unit injectable dose. These reagents may be administered in any conventional medium for intravenous injection such as an aqueous saline medium. Generally, the unit dose to be administered has radioactivity of about 1 to 30 mCi. The solution to be injected at unit dosage is from about 0.1 to about 10 mL. After intravenous administration, imaging in vivo can be performed any time from immediately up to and including four physical decay half lives following administration. Any method of scintigraphic imaging such as gamma scintigraphy, can be utilized in accordance with the present invention. Radioactively-labeled scintigraphic imaging agents according to the present invention are provided having radioactivity in solution containing at concentrations of from about 1 mCi to 100 mCi per mL. The pharmaceutical compositions comprising pharmacologically active backbone cyclized agonists or antagonists and a pharmaceutically acceptable carrier or diluent represent another embodiment of the invention, as do the methods for the treatment of cancers in targeted therapy using such compositions. The pharmaceutical compositions according to the present invention advantageously comprise at least one backbone cyclized peptide analog disclosed herein. These pharmaceutical compositions may be administered by any suitable route of administration, including orally, topically or systemically. Preferred modes of administration include but are not limited to parenteral routes such as intravenous and intramuscular injections, as well as via intra-nasal administration or oral ingestion. The preferred doses for administration of such pharmaceutical compositions range from about 0.1 μg/kg to about 20 mg/kg body weight/day. The pharmaceutical compositions may preferably be used to promote regression of certain types of tumors, particularly those that express receptors. Furthermore, the pharmaceutical compositions can also be used to reduce the hormonal hypersecretion that often accompanies certain cancers such as breast, prostatic and ovarian cancers. The invention further provides a method for alleviating GnRH-related diseases in animals, preferably humans, comprising administering a therapeutically effective amount of backbone cyclic analogs of the invention to the animal. In some preferred embodiments, rhenium- 186 or rhenium- 188 may be used for radiotherapy of certain tumors if the reagent is radioactively labeled with cytotoxic radioisotopes such as ¹⁸⁶Re or ¹⁸⁸Re. In preferred embodiments, the amount of the analog administered is from about 0.1 μg/kg to about 20 mg/kg body weight/day. For this purpose, an amount of radioactive isotope from about 10 mCi to about 200 mCi may be administered via any suitable clinical route, preferably by intravenous injection. Another aspect of the present invention provides methods for preparing therapeutic and diagnostic pharmaceuticals, preferably scintigraphic imaging agents, and the reagents required to make them. Each such reagent is comprised of an analog covalently linked to a radiometal complexing moiety. For example, scintigraphic imaging agents provided by the invention comprise ^99mTc labeled complexes formed by reacting the reagents of the invention with ^9mTc in the presence of an agent capable of reducing [^99mTc]pertechnetate ion (+7 metal oxidation state, that elutes from the ⁹⁹Mo/^99mTc generator found commonly in the nuclear medicine clinic or nuclear pharmacy) to the oxo[^{9 m}Tc]technetium species (+5 metal oxidation state). Preferred reducing agents include but are not limited to dithionite, stannous and ferrous ions. Such ^99mTc complexes of the invention are also formed by labeling the peptide analogs of the invention with ^99mTc by ligand exchange of a prereduced ^99mTc complex. In this case, a weak chelator is present in the in situ reduction cocktail, but the reagents of this invention are not initially present. The reagents of this invention are then added to the solution containing the +5 oxidation state oxo[^99raTc]technetium "weak chelator" complex, forming the more stable oxo[^99mTc]technetium complex with the reagents of this invention. The invention further provides kits for labeling analogs backbone cyclized through metal complexation. In a preferred embodiment of the invention, a kit for preparing

[^99mTc]technetium-labeled peptide analogs is provided. An appropriate amount of the backbone cyclic analog is introduced into a vial containing a reducing agent, such as stannous chloride, in an amount sufficient to label the analog with ^99mTc. An appropriate amount of a transfer ligand (a weak oxo[^99mTc]technetium chelator such as tartrate, citrate, gluconate, 2,5-dihydroxybenzoate, glucoheptanoate or mannitol, for example) can also be included. The kit may also contain additives such as salts to adjust the osmotic pressure, buffers to adjust the pH or preservatives to allow longer storage of either the cold kid or the final diagnostic radiopharmaceutical. The components of the kit may be in liquid, frozen or in dry form. In a preferred embodiment, the kit components are provided in lyophilized form. Technetium-99m labeled imaging reagents according to the present invention may be prepared by the addition of an appropriate amount of ^99mTc or ^99mTc-complex into the vial containing the reagents according to the present invention, and reaction under appropriate conditions. Kits for preparing radiotherapeutic agents wherein the preferred radioisotopes are rhenium- 186 and rhenium- 188 are also provided. Most preferred embodiments The most preferred backbone cyclized analogs according to the present invention are now described. In preferred analogs the peptide is coupled to one hemi-chelator via a linker and a second hemi-chelator via the peptide backbone, to form a structure of the general Formula No. 2: Z-Q-Y-X Formula No. 2 wherein Z is a first hemi-chelating moiety comprising two donor atoms, one N and one S, that through metal complexation form a five- to six-membered ring; Q is absent or a linker moiety which can be coupled to a free functional group of the peptide; Y denotes a GnRH analog comprising at least one N^α-ω-functionalized derivative of an amino acid; and

X is a second hemi-chelating moiety comprising two donor atoms, one N and one S, that through metal complexation form a five- to six-membered ring, wherein the chelating moiety is linked through a lower alkyl chain comprising 1-6 carbon atoms, to the alpha nitrogen of the PTR backbone or to a free functional group of the peptide. Preferably, the linker Q is connected to the N-terminal of the peptide, and X is connected to the peptide backbone or to a peptide side chain, and X is connected to the α- nitrogen of an N-building unit. Most preferably, Z and X are selected from the group consisting of L and D cysteines. Accordingly, the subject invention encompasses a compound having the general

Formula No. 3 (SEQ ID NO:2):

Y-W-Trp-Ser-Tyr-Z-Leu-Arg-Pro-NGly-X

Cys^I-M-Cys²-NH-(CH₂)_n Formula No. 3

wherein n is 1 to 8; W is absent or is His;

Y is absent or is Gly, β-Ala, GABA, valeric acid or caproic acid;

Z is D-Trp, D-Leu, D-Ser, D-Ser(Bu^t) or D-Ala;

X is a terminal carboxy acid, amide or alcohol group;

Cys¹ and Cys² are each independently L or D isomers; and M is a metal.

Preferably: n is 2, 3, 4 or 6 and, most preferably, 6; Cys² is L-Cys; Z is D-Ala; W is His;

X is an amide; and

M is a radiometal selected from [^natRe]oxorhenium(V), [¹⁸⁶Re]oxorhenium(V),

[¹⁸⁸Re]oxorhenium(V) or [^99mTc]oxotechnetium(V).

The most preferred analogs according to Formula 3 are:

ReO-Cys^-Gly-His-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-N^Cys^-aminohexy^-GlyN^

(SEQ ID NO:3); and

TcO-Cys^!l!-Gly-His-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-N(ηCys*-aminohexyl)GlyNH₂ (SEQ ID NO:4).

ReO-Cys*-Gly-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-N(ηCys^:i!-aminohexyl)-GlyNH₂ (SEQ

ID NO:5).

The asterisks denote the chelating groups used for cyclization through metal complexation.

More specifically, one Cys is attached to the η-amino group of the Gly at the tenth position. The other Cys is attached to the amino end of the peptide. These backbone cyclized peptide analogs are prepared by incorporating at least one N^α-ω-functionalized derivative of an amino acid into a peptide sequence (for example

Gly with the η-amino group. Two hemi-chelating NS donor atom-containing moieties are added, one to the nitrogen of the N^α-ω-functionalized amino acid (for example through addition of Cys) and another to either the terminal N or to a straight-chain AA spacer at the N-terminus (for example through addition of Cys to the terminal N). Selective cyclization is accomplished through binding of a single metal or radiometal (preferably as oxorhenium(V) or oxotechnetium(V)) to both bidentate hemi-chelators to form a tetradentate N₂S oxometal(V) cyclic peptide (or peptidomimetic) complex. The hemi- chelating moieties can alternatively be covalently bound to two N^α-ω-functionalizations, one or more amino acid side chain in the peptide sequence, or any combination of N^α-ω- functionalization, amino acid side chain, C- or N-terminus or linker or spacer group attached to any of the above.

General methods for radiolabelling with technetium In forming a complex of radioactive technetium with the reagents of this invention, the ⁹⁹Mo/"^mTc generator eluent, preferably containing sodium [^99mTc]pertechnetate (+7 oxidation state), is reacted with the reagent in the presence of a reducing agent. The preferred reducing agent is stannous chloride, which reliably reduces Tc^VI to Tc^v. Means for preparing such complexes are conveniently provided in a kit form comprising a sealed vial containing a predetermined quantity of a reagent of the invention to be labeled and a sufficient amount of reducing agent to label the reagent with Tc-99m. Alternatively, the complex may be formed by reacting a reagent of this invention with a pre-formed labile complex of technetium and another compound known as a transfer ligand. This process is known as ligand exchange and is well known to those skilled in the art. The labile complex may be formed using such transfer ligands as tartrate, citrate, gluconate, 2,5-dihydroxybenzoate, glucoheptanoate or mannitol, for example.

General method for forming metal complexes with crude chelator-cyclic peptide conjugates: Crude chelator peptide conjugates can be complexed with oxorhenium(V). The post-cleavage crude is weighed and the molar amount is calculated, assuming the mass is 100% desired conjugate. Alternatively, the molar amount of conjugate is calculated based on the solid phase resin loading. The appropriate metal reagent is added at an equimolar amount. This strategy works with rhenium when the crude peptide is relatively pure. By avoiding a chromatographic purification step, time and resources are saved.

General method for in vitro screening of GnRH analogs The ability of the analogs of the invention to bind to receptors in vitro was demonstrated by assaying the ability of such analogs to bind to receptor-containing pituitary membranes, as described in Example 5 below.

In vivo models for evaluating the activity of GnRH analogs The radiolabelled compounds of the present invention are tested in vivo for tumor uptake in xenografts derived from cell lines such as pituitary, and cancer cell lines such as breast, prostate and ovary. Briefly, the cells are implanted intramuscularly in a suspension of 0.05 to 0.1 mL/animal, the tumors are allowed to grow to approximately 0.5 to 2 g, harvested, and used to implant a second, naive set of animals. Passaging in this fashion is repeated to generate successive generations of tumor-bearing animals. Third- to fifth-passage of tumor-bearing animals are injected intravenously with labeled compound. At selected times, the animals are sacrificed and harvested tissue samples are weighed and counted, along with an aliquot of the injected dose, in a gamma well-counter. Alternatively, the radiolabelled compounds are studied in normal or immuno-deficient-tumor-free animals. For example, in such in-vivo study, target uptake is monitored, and non-target organs are also monitored to ascertain each compound's clearance profile.

General in vivo imaging methods In vivo imaging of receptors expressed by animal tumor cells is performed essentially as described by Bakker et al. (1991, Life Sciences 49:1593-1601). Additional in vivo screening methods are described in details in the examples below. Conformationally constrained analogs constructed based in part on the sequences of a number of known biologically active peptides or based on previously unknown novel sequences are presented in the examples below. The following examples are intended to illustrate how to make and use the compounds and methods of this invention and are in no way to be construed as a limitation. Although the invention will now be described in conjunction with specific embodiments thereof, it is evident that many modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such modifications and variations that fall within the spirit and broad scope of the amended claims. EXAMPLES

The invention will now be illustrated in a non-limitative manner by the following examples: Materials and methods Protected amino acids, 9-fluorenylmethyloxycarbonyl-N-hydroxysuccinimide

(Fmoc-OSu), bromo-tris-pyrrolidone-phosphonium hexafluorophosphate (PyBrop) and supports for SPPS were purchased from Nova Biochemicals (Laufelfmgen, Switzerland). Trifluoroacetic acid (TFA) and HPLC solvents were purchased from BioLab (Jerusalem, Israel). Glyoxylic acid, 1,6-diamino hexane and silica gel were purchased from Merck (Darmstadt, Germany), solvents for organic chemistry were purchased from Frutarom (Haifa, Israel), ⁹⁹Mo/^99mTc generator was purchased from Nahal Soreq Nuclear Center (Israel), trichlorooxobis(triphenylphosphine)rhenium(V) and all other chemicals were purchased from Aldrich (Milwaukee, WI, USA). Example 1: Organic synthesis Synthesis of glycine derived building unit was performed. Boc-NH(CH₂)₆NH₂ (compound 1) - introducing of mono tert-butoxycarbonyl (Boc) protecting group. 1,6- Diamino hexane (86.49 g, 0.744 mol) were dissolved in 1 L Chloroform and cooled in an ice bath. To the cooled solution, a mixture of (Boc)₂O (21.86 g, 0.1 mol) in Chloroform (500 ml) were added at 0 °C dropwise over 5 h and then stirred overnight at room temperature. The reaction mixture was washed with water (500 ml X 4), dried over sodium sulfate and evaporated in vacuo. The crude product was further purified by flash chromatography on silica gel. The crude product was adsorbed on silica gel (50 g). The silica gel adsorbed product was added onto a column with silica gel (450 g). The column was eluted with a mixture of dichloromethane (DCM):methyl alcohol(MeOH) 4.3:1, and was monitored by TLC (DCM:MeOH 4.3:1). Yield after evaporation: 19.54 g, 90.3% (0.0903 mol). 1H nuclear magnetic resonance (NMR) (300 MHz, CDC1₃): δ 1.31 (s, 2H), 1.34 (m, 4H), 1.44-1.50 (m, 13H), 2.68 (t, 2H), 3.10 (q, 2H). Boc-NH(CH )6NH-Alloc (compound 2) - protection of the free amine with allyloxycarbonyl (Alloc) protecting group. The mono-Boc protected diamine, described above, (19.54 g, 0.0903 mol) was dissolved in 4M KOH (60 ml), and cooled in an ice bath. Allyl chloroformate (12.5 ml, 0.118 mol, 1.3 eq) was added dropwise to the solution and precipitate was formed immediately. 4M KOH (100 ml) was added and the solution was allowed to stir overnight. Petroleum ether (PE) (60 ml) and water (60 ml) were added to the white suspension. The solid was filtered, washed with PE (60 ml x 2) and dissolved in DCM (200 ml). The organic solution was washed with 4M KOH (60 ml x 3), and with water), dried over Na SO and evaporated yielding 23.57 g (86.7%) of colorless oil. 1H NMR (300 MHz, CDC1₃): δ 1.31-1.35 (m, 4H), 1.44-1.52 (m, 13H), 3.07-3.20 (m, 4H), 4.70 (d, 2H), 5.23 (dd, 2H), 5.93 (m, 1H). H₂N(CH₂)₆NH-Alloc (compound 3). TFA (60 ml) was added to compound 2 (23.57 g, 0.0785 mol) and the reaction was stirred at room temperature for 1.5 h. Three portions of ether (200 ml) were added and evaporated to get rid of excess TFA. The acidic residue was dissolved in DCM (300 ml) and the solution was brought to pH=9 with 4M NaOH (75 ml). The organic layer was separated, dried over Na₂SO and evaporated in vacuo yielding 9.1 g (0.0454 mol, 57.9%) of colorless oil. ^!H NMR (300 MHz, CDC1₃): δ 1.33 (m, ~4H), 1.50 (m, 4H), 2.77 (t, 2H), 3.16 (q, 2H), 4.55 (d,2H), 5.2 (d, 1H), 5.28 (d, 1H), 5.92 (m, 1H). Fmoc-N^α-[ω-NH(AUoc)-hexyl] glycine-OH (compound 4). NaCNBH₃ (3.25 g, 0.052 mol) was dissolved in MeOH (40 ml). Compound (3) (9.1 g, 0.0454 mol) was dissolved in MeOH (50 ml) and added to the NaCNBH₃ solution. While stirred, glyoxilic acid (3.43g, 0.0463 mol) was added and the reaction was stirred for 18 h. The MeOH was evaporated under reduced pressure, and the residue was dissolved in water (110 ml), and triethyl amine (11 ml, 0.079 mol) was added. Fmoc-OSu (9.82 g, 0.0291 mol) in AcCN (170 ml) was added, and the reaction was stirred for 4 h while the pH was kept alkaline with triethyl amine. The reaction mixture was washed with PE (180 ml x 3) and ether:PE 7:3 (180 ml x 3). The aqueous layer was acidified under cooling to pH=3-4 with 2M HC1 (-10 ml), and extracted with ethyl acetate (EA) (150 ml x 4). The organic layer was washed with 1M HC1 (100 ml x 2) and sat. KHSO₄ (100 ml x 2), dried over Na₂SO₄ and evaporated in vacou to yield: 5.50 g, 0.0115 mol (39.5%) of colorless oil that was later solidified. The product was used for SPPS without further purification. 1H NMR (300 MHz, CDC1₃): δ 1.16-1.52 (m, 8H), 3.18 (m, 3H), 3.36 (t, 1H), 3.98 (d, 2H), 4.27 (m, 1 H), 4.47 (d, 1H), 4.57 (m, 3H), 5.28 (dd, 2H), 5.95 (m, 1H), 7.37 (m, 4H), 7.60 (t, 2H), 7.78 (t, 2H).

Example 2: Peptide synthesis The synthetic scheme of an exemplary GnRH analog backbone cyclized through metal complexation is described in scheme 1. When "AA" is His, the analogs are SEQ ID NO:3 or SEQ ID NO:4. When "AA" is absent, the analog is SEQ ID NO:5..

Scheme 1 NH-Alloc NH-Alloc (CHJ_j BTC ₍cH₂)_β I 2,4,6-Colhdιne | _ 1 Fmoc deprotection Fmoc N — OH; — COOH + ^H!^N — Q ► Fmoc N — CH₂-OONH — J — - ; — j» DCM 2 Coupling of Fmoc-Pro-OH BTC + 2,4,6-Colhdιne in DBM 50 °C NH-Alloc Peptide elongation I 1 Fmoc deprotection Pbf (CH₂)₀ 2 Coupling of Fmoc-AA-OH 1 Fmoc deprotection I I (PyBrop, DIEA, NMP)

Fmoc Pro — N — CH₂-CONH — Q *- Fmoc Arg— Pro — N — CH₂-CONH — Q *- 2 Coupling of Fmoc-Arg(Pbf)-OH P Brop, DIEA, NMP NH Alloc OH₂ Trt Boc OtBu OtBu Pbf (C IH₂)„ OC His — Trp— Ser— Tyr-D-Ala-Leu— Arg— Pro — N — CH₂-CONH — Q

1 Alloc deprotection 2 Coupling of Boc-Cys(Trt)-OH (PyBrop, DIEA, NMP)

1 Fmoc deprotection 2 Coupling of Boc-Cys(Trt)-OH (PyBrop, DIEA, NMP)

TIS (25%)

Cyclization Tnchlorooxobιs(rtιphenylphosphιne)rhenιum (V)

The synthesis was performed in a reaction vessel equipped with a sintered glass bottom, following general Fmoc chemistry protocols: Rink amide methylbenzhydrilamine (MBHA) resin (0.4 g, 0.66 mmol/g) was pre-swollen in N- methylpyrrolidone (NMP) for 2 h. Fmoc deprotection step was carried out with 20% piperidine in NMP (2 x 30 min), followed by wash with NMP (5 x 2 min) and DCM (2 x 2 min). Couplings of the building unit to the resin and of Fmoc-amino-acid-OH (Fmoc-AA-OH) to the building unit were carried out according to the procedure published by Falb et al. (J. Peptide Res. 1999, 53, 507-517). Fmoc-GlyN₆(Alloc)-OH (5 eq., 1.32 mmol) and bis-(trichloromethyl) carbonate (BTC, triphosgene) (1.613 eq., 0.44 mmol) were suspended in DCM. 2,4,6-collidine (8 eq., 2.112 mmol) was added to the pre cooled suspension in an ice bath. After all the solids were dissolved (about 1 min), the solution was poured onto the resin and shaken for 1 h at room temperature. This coupling cycle was repeated once more. At the end of the second coupling cycle, the peptidyl-resin was washed with DCM (5 x 2 min). Capping was carried out after the first amino acid and was repeated twice by reaction of the peptidyl-resin with a mixture of acetic anhydride (1.1 ml, 0.5M), diisopropyl amine (DIEA) (0.5 ml, 0.125M) and N- hydroxybenzotriazole (HOBT) (0.05 g, 0.015M) in dimethyl formamide (DMF) (25 ml). Capping was followed with resin wash with DMF (5 x 2 min), DCM (2 x 2 min) and NMP (2 x 2 min). Two cycles of the difficult coupling of Fmoc-Pro-OH to the building unit were carried out at 50°C as follows: Fmoc-Pro-OH (5 eq., 1.32 mmol) and BTC (1.613 eq., 0.44 mmol) were suspended in dibromomethane (DBM) and cooled in an ice bath for 5 min. 2,4,6-collidine (8 eq., 2.112 mmol) was added after all the solids were dissolved (~ 1 min), the solution was added to the peptidyl-resin and the vessel was shaken at 50 °C for 3 h. Washing steps were carried out with DCM (5 x 2 min). From this point all coupling steps were carried out using PyBrop as the coupling agent. A sample of a coupling cycle: Fmoc-AA-OH (3 eq.) and PyBrop (3 eq.) were dissolved in NMP and DIEA (7 eq.) was added. The reaction mixture was preactivated for 10 min before added to the reaction vessel, and shaken for 2 hours. Coupling was followed with washing the peptidyl-resin with NMP (3 x 2 min) and DCM (2 x 2 min). After coupling of Fmoc-Gly-OH, the resin was dried overnight in a desiccator and removal of the Alloc protecting group from the building unit was performed with tetrakis(triphenylphosphine)Pd(0) (0.75 g per g resin) in DCM containing acetic acid (5%) and N-methyl morpholin (2.5%) under Argon. This step was carried out for 4 h with vigorous shaking in the dark. After Alloc deprotection, Boc-Cys-(Trt)-OH was coupled to the ω-nitrogen of the building unit using the PyBrop coupling protocol described above. After Fmoc-deprotection at the N-terminus, Boc-Cys(Trt)-OH was coupled using PyBrop as coupling agent. All Fmoc removal and couplings were monitored by chloranil test, and coupling was repeated if necessary. Chloranil test after the coupling of Fmoc-His(Trt)-OH was negative, but became blue (positive) after 1 hour. A small amount of resin was cleaved (small cleavage procedure) and analyzed by MS. Because Fmoc-His(Trt)-OH coupling was not complete, resulting in a mixture of the desired mass and a significant mass of the des-His sequence. The synthesis was continued, with separation between the two peptides after cleavage. After coupling of the second Boc-Cys(Trt)-OH the resin was split to four portions (each of ~0.11 g peptidyl-resin). The first portion (portion A=precyclic peptide) was cleaved from the resin applying TFA, as described below, and biological activity was tested without cyclization. The second portion (portion B=S-S bridged cyclic peptide) was washed with DMF (2 x 2 min), and on-resin oxidation was performed with I₂ (10 eq.) [31], in DMF:triρle-distilled water (TDW) 4:1 for 70 min. The resin was washed with DMF (2 x 2 min), 2% ascorbic acid in DMF (2 x 2 min), NMP (5 x 2 min) and DCM (2 x 2 min) and then cleaved from the resin. The third portion (portion C=Re cyclic peptide) and the forth portion (portion D=Tc cyclic peptide) were cleaved from the resin as the pre cyclic peptide and cyclization was performed as will be described later. Cleavage from the resin and removal of side chain protecting groups were carried out simultaneously using a pre cooled mixture of 95% TFA, 2.5% TDW and 2.5% triisopropylsilane (TIS). After the resin was added, the mixture was cooled for 30 min in an ice bath, and then was shaken for 2.5 h at room temperature. The resin was then removed by filtration, washed with a small amount of neat TFA, and the combined TFA filtrates were evaporated under nitrogen. The oily product was triturated with cold ether and the ether was decanted. The residue was dissolved in AcCN:TDW 1 : 1 and lyophilized. Analysis of the crude peptides by analytical HPLC and MS showed that in each portion, two peptides were the major products: the full peptide (SEQ ID NO:3, and a peptide with deletion of His² (named Des His; SEQ ID NO:5). The peptides were purified by preparative HPLC, and analyzed by analytical HPLC and by MS.

Example 3: Small cleavage procedure A small amount of the peptidyl-resin was treated with a pre cooled mixture of TFA (2 ml), H O (1 drop) and TIS (1 drop) for 30 min. The resin was removed by filtration. TFA was evaporated with a stream of nitrogen. The residue was dissolved in an AcCN:TDW 1 : 1 and analyzed by MS. Example 4: Procedure for peptide cyclization through Re coordination Lyophilized peptide (14.3 mg) was dissolved in TDW (2 ml), and a solution of trichlorooxobis(triphenylphosphine)rhenium(V) (1 eq.) in DMF (~2 ml) was added. DMF was further added to the mixture to form a peptide concentration of 1 mg/ml, and shaking was carried out for 2 h. Cyclization was monitored by analytical HPLC. The solvents were evaporated by centrifugal vacuum apparatus (Speed Vac) Cabam and the dried residue was analyzed by analytical HPLC and MS, and purified by preparative HPLC.

Example 5: Binding of peptides to the GnRH receptor on pituitary membranes The peptides were iodinated by the chloramine T method, applied to a Sephadex G-25 column and eluted with 10 mM acetic acid. Pituitary membranes (0.1 pituitary equivalent/tube, prepared from Wister-derived proestrous rats) were incubated for 90 min at 4 °C with 50,000-80,000 cpm ¹²⁵I-peptide alone or in the presence of various concentrations of the unlabeled peptides in a total volume of 0.5 ml of assay buffer (10 mM Tris-HCl containing 0.1% BSA). The reaction was terminated by rapid filtration through Whatman GF/C filters. The filters were washed three times with cold assay buffer and counted in a Packard Auto-Gamma Spectrometer. The experiments were performed in triplicates. Non-specific binding was calculated by subtracting the nonspecific binding from the maximal binding, determined in the absence of any competing peptide. See Liscovitch et al, Eur J Biochem. 140:191-7 (1984). As shown below in Table 1, the IC 50 activity values of SEQ ID NO: 3 were almost as active as native GnRH.

Table 1

Example 6: Peptide cyclization and radiolabeling through ⁹⁹Tc coordination To a 15 ml Corning polypropylene tube equipped with a magnetic stirrer, the following aqueous solution were added,: sodium glucoheptanoate (93.9 μmol, 0.5 ml of a 188 mM solution), ethylenediaminetetraacetic acid (EDTA) (1.35 μmol, 394 μL of a 3.43 mM solution), Gn-2 (0.3445 μmol, 50 μL of a 6.9 mM solution), saline eluent from a "Mo/^99mTc generator (927 μCi sodium [^99mTc] pertechnate, 0.5 ml) and stannous chloride (1.11 μmol, 210 μL of freshly prepared 5.27 mM solution). The tube was capped, mixed and then heated to 98°c in an oil bath with stirring for 10 min. After cooling to RT, the solution was sampled for determination of recovery and radiochemical yield/purity. HPLC analysis of the radiolabeled compounds were compared to the Re cyclic peptides. The HPLC retention times of the two cyclo [^99mTc(O)-Gn-2] isomers (27.22 and 27.86 min) were comparable to those of the cyclo [Re(O)-Gn-2] isomers (26.73 and 27.34 min).

Example 7: Reaction of crude metal-free peptides with rhenium to yield the oxorheniumfV^") complex Crude peptide is dissolved in water and trichlorooxobis(triphenylphosphine)- rhenium(V) is added in DMF and the mixture is shaken at room temperature for about 2 hours. Removal of DMF is achieved by vacuum centrifugation (sample at 40°C) for about 10 hours and the resulting product is purified by HPLC, yielding the oxorhenium(V) complex of the peptide.

Example 8: Improved procedure for the synthesis and purification of

Cvcfor^99mTc(OVGn-21; To a 15 ml Corning polypropylene tube equipped with a magnetic stirrer, the following aqueous solution were added: saline elute from a ⁹⁹Mo/^99mTc generator (38.8 mCi sodium [^99mTc] pertechnate, 2.0 ml), stannous chloride (1.11 μmol, 210 μL of freshly prepared 5.27 mM solution), sodium glucoheptanoate (93.9 μmol, 0.5 ml of a 188 mM solution, ethylendiaminetetraacetic acid (EDTA) (1.35 μmol, 395 μL of a 3.43 mM solution), Gn-2 (0.3445 μmol, 50 μL of a 6.9 mM solution). The tube was capped, mixed and then heated to 91-98 °c in an oil bath with stirring for 30 min. after cooling to RT, the solution was filtered on a 13 mm filter unit (0.45 μm, PTFE). 1.8 ml of the filtrate (18.15 mCi) was injected to a preparative HPLC. Cyc/ø[^99mTc(O)-Gn-2] (6.33 mCi) was collected at 23.5 min. The pH of the collected solution was checked (pH=2 ), and an equal amount of water was added. The solution was passed through an activated C-18 sep-pak (5 ml EtOH, dry, 10 ml of H₂O, dry) and the sample was dried under a stream of nitrogen for 5 min. the labeled peptide was eluted from the C18 sep-pak with 0.5 ml EtOH and 2.5 ml of citrate buffer pH=3.8. The activity was measured (4.27 mCi).

Example 9; Stability and extraction partition of cyclo f^99mTc(O)-Gn-21 in blood Cyclo [^99mTc(O)-Gn-2] was incubated in blood at 37°c, and samples were removed at t=0, 60 min, 120 min, 180 min and 240 min. The extraction partition was determined by measuring the activity of total blood, the activity of the pellets and the activity of the plasma. The stability of cyclo [^99mTc(O)-Gn-2] in blood was determined by TLC chromatography of the plasma. Experiments showed constant high activity in plasma, e.g. 70-80% of the total activity (Figure 1). The stability results indicated that about 75-80% of cyc/o [^99mTc(O)- Gn-2] remained intact in blood after 4 hours of incubation (Figure 2) in comparison to the native GnRH which degrades within minutes in blood. The composition of the two experiments indicate that after 4 hours of incubation 55% of the activity in blood corresponds to the intact peptide (Figure 3). While the present invention has been described for certain preferred embodiments and examples it will be appreciated by the skilled artisan that many variations and modifications may be performed to optimize the activities of the peptides and analogs of the invention. The examples are to be construed as non-limitative and serve only for illustrative purposes of the principles disclosed according to the present invention, the scope of which is defined by the claims which follow.

Claims

1. A gonadotropin releasing hormone (GnRH) analog of four to twenty-four amino acids that incorporates at least one building unit, comprising an N^α-ω- functionalized derivative of an amino acid, wherein a backbone cyclic structure is formed by metal complexation to a chelating moiety comprising the at least one building unit and a second moiety selected from the group consisting of a second building unit, a side chain of an amino acid residue of the GnRH analog and a terminal amino acid residue of said GnRH analog.

2. The GnRH analog of claim 1 wherein the chelating moiety comprises four donor atoms.

3. The GnRH analog of claim 2 wherein the chelating moiety is the N₂S₂ type comprising two donor nitrogens and two donor sulfor atoms.

4. The GnRH analog of claim 1 comprising an analog having the general Formula No. 3 (SEQ ID NO: 2):

Y-W-Trp-Ser-Tyr-Z-Leu-Arg-Pro-NGly-X - Cys¹-M-Cys²-NH-(CH₂)_n -

Formula No. 3 wherein n is 1 to 8; W is absent or is His; Y is absent or is Gly, β-Ala, GAB A, valeric acid or caproic acid; Z is D-Trp, D-Leu, D-Ser, D-Ser(Bu^t) or D-Ala; X is a terminal carboxy acid, amide or alcohol group; Cys¹ and Cys² are each independently L or D isomers; and M is a metal.

5. The GnRH analog of claim 4 wherein: n is 2, 3, 4 or 6; W is His; Y is Gly; Z is D-Ala; X is an amide; and M is a radiometal selected from the group consisting of [^natRe]oxorhenium(V), [¹⁸⁶Re]oxorhenium(V), [¹⁸⁸Re]oxorhenium(V) or [^99mTc]oxotechnetium(V).

6. The GnRH analog of claim 5 selected from the group consisting of SEQ ID NOS :3 and 4.

7. The GnRH analog of claim 4 of SEQ ID NO:5.

8. The GnRH analog of claim 1 wherein the chelating moiety comprises a complex with a radioisotope.

9. The GnRH analog according to claim 8 wherein the radioisotope is selected from 99m_χ 186_Re ^ 188_Re>

10. A radiolabelled peptide analog comprising the GnRH analog of claim 1.

11. The radiolabelled peptide analog of claim 10 suitable for use in imaging sites within a mammalian body.

12. The radiolabelled peptide analog of claim 10 suitable for use in diagnosis in vitro or ex vivo.

13. A pharmaceutical composition comprising a GnRH analog of four to twenty-four amino acids that incorporates at least one building unit, comprising an N -ω- functionalized derivative of an amino acid, wherein a backbone cyclic structure is formed by metal complexation to a chelating moiety comprising the at least one building unit and a second moiety selected from the group consisting of a second building unit, a side chain of an amino acid residue of the GnRH analog and a terminal amino acid residue of said GnRH analog, and a pharmaceutically acceptable carrier.

14. The pharmaceutical composition of claim 13 wherein the chelating moiety comprises four donor atoms.

15. The pharmaceutical composition of claim 14 wherein the chelating moiety is the N₂S₂ type comprising two donor nitrogens and two donor sulfor atoms.

16. The pharmaceutical composition of claim 13 comprising an analog having the general Formula No. 3 (SEQ ID NO: 2):

Y-W-Trp-Ser-Tyr-Z-Leu-Arg-Pro-NGly-X

-Cys¹-M-Cys²-NH-(CH₂)„ - Formula No. 3 wherein n is 1 to 8; W is absent or is His; Y is absent or is Gly, β-Ala, GABA, valeric acid or caproic acid; Z is D-Trp, D-Leu, D-Ser, D-Ser(Bu^l) or D-Ala; X is a terminal carboxy acid, amide or alcohol group; Cys¹ and Cys² are each independently L or D isomers; and M is a metal.

17. The pharmaceutical composition of claim 16 wherein: n is 2, 3, 4 or 6; W is His; Y is Gly; Z is D-Ala; X is an amide; and M is a radiometal selected from the group consisting of [^natRe]oxorhenium(V), [¹⁸⁶Re]oxorhenium(V), [¹⁸⁸Re]oxorhenium(V) or [^99mTc]oxotechnetium(V).

18. The pharmaceutical composition of claim 17 wherein the analog is selected from the group consisting of SEQ ID NOS:3 and 4.

19. The pharmaceutical composition of claim 16 wherein the analog having SEQ ID NO:5.

20. The pharmaceutical composition of claim 13 wherein the chelating moiety comprises a complex with a radioisotope.

21. The pharmaceutical composition of claim 20 wherein the radioisotope is selected from ^99mTc, ¹⁸⁶Re and ¹⁸⁸Re.

22. A method for diagnosing or treating cancer comprising administering to a subject in need thereof a pharmaceutical composition comprising a GnRH analog of four to twenty-four amino acids that incorporates at least one building unit, comprising an N^α-ω-functionalized derivative of an amino acid, wherein a backbone cyclic structure is formed by metal complexation to a chelating moiety comprising the at least one building unit and a second moiety selected from the group consisting of a second building unit, a side chain of an amino acid residue of the GnRH analog and a terminal amino acid residue of said GnRH analog, and a pharmaceutically acceptable carrier.

23. The method of claim 22 wherein the chelating moiety comprises four donor atoms.

24. The method of claim 23 wherein the chelating moiety is the N₂S₂ type comprising two donor nitrogens and two donor sulfor atoms.

25. The method of claim 22 comprising an analog having the general Formula No. 3 (SEQ ID NO: 2):

Y-W-Trp-Ser-Tyr-Z-Leu-Arg-Pro-NGly-X, -€ys¹-M-Cys²-NH-(CH₂)„ - Formula No. 3 wherein n is 1 to 8; W is absent or is His; Y is absent or is Gly, β-Ala, GABA, valeric acid or caproic acid; Z is D-Trp, D-Leu, D-Ser, D-Ser(Bu^t) or D-Ala; X is a terminal carboxy acid, amide or alcohol group; Cys¹ and Cys² are each independently L or D isomers; and M is a metal.

26. The method of claim 25 wherein: n is 2, 3, 4 or 6; W is His; Y is Gly; Z is D-Ala; X is an amide; and M is a radiometal selected from the group consisting of [^natRe]oxorhenium(V), [¹⁸⁶Re]oxorhenium(V), [¹⁸⁸Re]oxorhenium(V) or [^99mTc]oxotechnetium(V).

27. The method of claim 26 wherein the analog is selected from the group consisting of SEQ ID NOS:3 and 4.

28. The method of claim 25 wherein the analog having SEQ ID NO:5.

29. The method of claim 22 wherein the chelating moiety comprises a complex with a radioisotope.

30. The method of claim 29 wherein the radioisotope is selected from ^99mTc, ¹⁸⁶Re and ¹⁸⁸Re.

31. The method according to claim 22 wherein the GnRH analog is used for imaging the existence of metastases.

32. The method according to claim 22 wherein the GnRH analog is labeled with a detectable tracer.

33. Use of a GnRH analog of four to twenty-four amino acids that incorporates at least one building unit, comprising an N^α-ω-functionalized derivative of an amino acid, wherein a backbone cyclic structure is formed by metal complexation to a chelating moiety comprising the at least one building unit and a second moiety selected from the group consisting of a second building unit, a side chain of an amino acid residue of the GnRH analog and a terminal amino acid residue of said GnRH analog, for preparation of a pharmaceutical composition for diagnosis and treatment of cancer.

34. The use of claim 33 wherein the chelating moiety comprises four donor atoms.

35. The use of claim 34 wherein the chelating moiety is the N₂S₂ type comprising two donor nitrogens and two donor sulfor atoms.

36. The use of claim 33 comprising an analog having the general Formula No. 3 (SEQ ID NO: 2):

Y-W-Trp-Ser-Tyr-Z-Leu-Arg-Pro-NGly-X

-Cys¹-M-Cys -NH-(CH₂)_n - Formula No. 3 wherein n is 1 to 8; W is absent or is His; Y is absent or is Gly, β-Ala, GABA, valeric acid or caproic acid; Z is D-Trp, D-Leu, D-Ser, D-Serβu') or D-Ala; X is a terminal carboxy acid, amide or alcohol group; Cys¹ and Cys² are each independently L or D isomers; and M is a metal.

37. The use of claim 36 wherein: n is 2, 3, 4 or 6; W is His; Y is Gly; Z is D-Ala; X is an amide; and M is a radiometal selected from the group consisting of [^natRe]oxorhenium(V), [¹⁸⁶Re]oxorhenium(V), [¹⁸⁸Re]oxorhenium(V) or [^99mTc]oxotechnetium(V).

38. The use of claim 37 wherein the analog is selected from the group consisting of SEQ ID NOS :3 and 4.

39. The use of claim 36 wherein the analog having SEQ ID NO:5.

40. The use of claim 33 wherein the chelating moiety comprises a complex with a radioisotope.

41. The use of claim 40 wherein the radioisotope is selected from ^{9 m}Tc, ¹⁸⁶Re and ¹⁸⁸Re.