CZ372999A3 - Synthetic compound binding calcitonin receptor, reaction agent, scintigraphic representation agent, preparation and radiotherapeutic agent containing thereof, complex and system for preparing a radioactive preparation, the reaction agent labeling method and process of its preparation and use - Google Patents

Abstract

The synthetic calcitonin receptor compound has a molecular weight of less than 10,000 and is covalently a radioactive metal chelator to form a reaction metal an agent having the same or greater binding affinity to calcitonin receptor than radioactive binding affinity iodine-labeled natural calcitonin on said receptor. The reagent it contains the compound, the scintigraphic imaging agent it contains this reagent, a composition comprising the reaction reagent an agent, and a radiotherapeutic agent comprising the same a reagent and a cytotoxic radioisotope, a complex that is by reacting the reagent of any one from 1 to 20 with technetium-99m, rhenium-186 or rheniem-188 in the presence of a reducing agent, and an assembly for preparing a radiopharmaceutical composition containing it a sealed container containing a predetermined amount of the aforementioned reagent and sufficient amount of a reducing agent to indicate the reagent technetium-99m, rhenium-186 or rhenium-188. Way designating said reagent and method of manufacture; its use for the manufacture of a medicament for displaying the body a mammal and for the manufacture of a medicament for the treatment of the diseases for which it is characteristic of the presence of calcitonin receptor. Synthetic compound that binds calcitonin receptor, reagent, scintigraphic an imaging agent, a composition and a radiotherapeutic agent containing it; radioactive formulation preparation, reagent labeling method a how it is manufactured and used

Description

A synthetic compound that binds to a calcitonin receptor, a reagent, a scintigraphic imaging agent, a composition and a radiotherapeutic agent containing it, a complex and an assembly for preparing a radioactive preparation, a method for labeling a reagent, and a method for its manufacture and use

Technical field

The present invention relates to a synthetic compound that binds to the calcitonin receptor and is covalently bound to a radioactive metal chelator to form a reagent having the same or greater binding affinity to the calcitonin receptor than the binding affinity of radioactive iodine labeled natural calcitonin to said receptor. Described is a scintigraphic imaging agent comprising the reagent, the composition and a radiotherapeutic agent comprising the reagent and a cytotoxic radioisotope, and a complex formed by reacting the reagent with a cytotoxic radioisotope in the presence of a reducing agent. Said reagent may be used for the manufacture of a medicament, particularly in the treatment of cancer.

BACKGROUND OF THE INVENTION

Over the past 60 years, great progress has been made in reducing long-term mortality from some cancers, especially stomach cancer. However, over the same period, mortality trends for other types of cancer remain unchanged or even increase. For example, lung cancer is the most common type of cancer in the world, which today is the leading cause of cancer mortality in men and women. Breast cancer is the most common cancer in women and the second leading cause of cancer mortality in women. In some countries, ovarian cancer mortality is increasing. Lymphatic gland cancers in both children and adults, such as leukemia and non-Hodgkin's lymphomas, are also continuing to be important causes of cancer mortality. Today, all these cancers are targeted at early diagnosis and effective treatment.

9

9 99 9

9

9,999,999

9 9

9 9 9 9 9

Several years ago, locally directed diagnosis and therapy have been proposed that allow in vivo targeting of relevant disease sites in the animal's body. Generally, a site-directed diagnosis or therapy uses targeted particles, such as a disease-specific antibody or an organism that causes onemonium, which is associated with a label for a diagnostic agent or a cytotoxic agent for a therapeutic agent. There is very much literature on radiolabeled antibodies or antibody fragments for diagnostic imaging purposes. Similarly, numerous site-directed therapeutic agents have been proposed over the years using monoclonal antibodies and a variety of radioisotopes, e.g., as disclosed in U.S. Patent Nos. 4,454,106, 4,472,509, 4,828,991, 5246,691, 5,355,394, and 5641 471, European patents 429624 and 585 986, WO 90/15 625 and the like. These antibody-based agents provide side effects that relate to the immune response of the treated animal to the antibody, although antibody fragments or humanized antibodies are used as targeting particles.

Disadvantages of site-based antibody-based diagnostic and therapeutic agents can be avoided when targeting moieties having lower molecular weights, such as receptor-specific peptides or small molecules, are used. However, condensation of a peptide or small molecule with a label or cytotoxic agent, while maintaining the specificity of the compound at the receptor, may be technically difficult. Methods for radiolabeling peptides and other small molecules that retain the ability of a compound to bind specifically to a receptor are described in commonly-known U.S. Patent Nos. 5,251,580, 5,405,597, 5,443,815, 5,508,020, 5,552,525, 5,561,220 5,620,675, 5,645,815, 6,654,272, 5,711,931, 5,716,596, 5,720,934, and 5,736,122, in the abandoned U.S. Patent Application Nos. 07/955,466 and WO 92/13,572, WO 93 / 10,747, WO 93/17,719, WO 93/21962, WO 92/23 085, WO 93/25 244, WO 94/00489, WO 94/07 918 and WO 94/28 942. patents and publications are particularly suitable for the manufacture of locally directed diagnostic imaging agents. U.S. Patent Nos. 5,620,675 and 5,716,596 and WO 94/00489, WO 95/03330, WO 95/00 553, WO 95/31 221 and WO 96/04 308 φφ ··

Φ Φ 4

Φ Φ «

• Φ • 4 • 4 φ Φ Φ Φ describe somatostatin peptide analogues that can be used for site-directed radiotherapy. WO 95/33 497 discloses somatostatin analogues, gpIIb / IIIa receptor binding peptides and leukocyte binding peptides, which can be used for site-directed radiodiagnosis or radiotherapy. WO 96/30 055 discloses vasoactive intestinal peptide (VIP) receptor binding peptides, which can be used for site-directed radiodiagnosis or radiotherapy.

Tumor cells often occasionally express or overexpress a particular receptor or receptor subtype, as shown by binding receptor studies. In some cancers, tumor cell markers may vary as the disease progresses, possibly because it reflects the disease state and hence the prognosis for the patient. The type of receptor that the tumor cell expresses may be characteristic of the etiology of the tumor and may thus provide a relatively specific marker for the tumor. For example, radiolabeled self -ostatin analogs have been shown to specifically bind to neuroendocrine tumors, melanomas, lung cancer, and some breast cancers. One such analog, ¹¹¹ ln-Octreoscan, has received commercial approval for use in imaging neuroendocrine tumors. The second radiolabeled somatostatin analogue, ^m Tc-Depreotide, has completed the third phase of clinical trials for use in imaging lung tumors. ¹²³ L-vasoactive intestinal peptide has been shown to target colon and stomach adenocarcinomas.

Calcitonin (CT) is a 32 amino acid peptide that is secreted from the thyroid in response to elevated serum calcium levels. Calcitonin has numerous biological effects that are mediated by calcitonin receptors present on the cell surface of the target organ. Receptors with high affinity for CT have been identified in the bones, kidneys, lungs and central nervous system. In bone, CT inhibits bone resorption by osteocblasts, in kidney CT increases calcium excretion, and in the central nervous system, this peptide induces analgesia, gastric acid secretion, and taste inhibition. Small amounts of CT were administered to animals and humans without toxic effects. Salmon CT is clinically used to treat bone disorders such as Paget's disease, malignant hypercalcemia and osteoporosis. Intra-venous calcitonin disappears rapidly from the blood and is primarily excreted in the urine. The main sites of localization of administered CT are kidneys, liver and long bone epiphyses.

Circulating CT levels are considered to be a marker of some types or conditions of cancer, such as medullary thyroid cancer, small cell lung cancer, carcinoids, breast cancer, and gastrointestinal cancer. High affinity CT receptors have been identified in lymphatic cells, human lung cancer cell lines, human breast cancer cell lines, and in primary breast cancer tissue. Findlay et al., Biochem. J. 1981, 196, 513-520, disclose that CT receptors are overexpressed in some breast, lung, ovarian and lymphatic cancer cell lines.

The amino acid sequences of CTs of some species (human, salmon and eel) are listed below:

hCT CGNLSTCMLGTYTQDFNKFHTFPQTAIGVG.AP.amid (SEQ ID NO: 1), cCT CSNLSTCVLGKLSQELHKLQTYPRTNTGSG.TP.amid (SEQ ID NO: 2), eCT CSNLSTCVLGKLSQ amino acid sequence SEQ ID NO: 3). are as set forth in Zubay: Biochemistry, Second Edition, MacMillan Publishing, New York 1988, p. 33, and wherein the underlined amino acids between the two cysteine residues in the amino terminal portion of the peptide represent a disulfide bond). Nine residues are retained in these species, including the carboxyl terminal proline amide and disulfide-linked cysteine residues at positions 1 and 7. Salmon and eel CT are more effective than human CT both in vitro and in vivo.

CT peptide analogues have been developed in which the chemically labile disulfide is replaced by stable carbon-carbon bonds formed between the 2-aminocork acid, as described in U.S. Patent No. 4,086,221. CT analogs in which the amino terminus regions are omitted, the middle region or the carboxyl terminus of the molecule show only weak binding to CT receptors. Many amino acid substitutions can be made between residues 8 and 22 of the CT molecule, so that biologically active CTs are generated, and they are generated. 44 logos. Some CT analogs with only minimal sequence homology to the natural form of CT have biological activity similar to that of salmon CT. Modified CT peptide derivatives (such as Cbz-LHKLQY-OMe) also retain substantial receptor binding activity.

Since tumors can express or overexpress various receptors, a variety of radiodiagnostic and radiotherapeutic agents are required to achieve optimal diagnostic and therapeutic modalities for cancer.

SUMMARY OF THE INVENTION

CT receptors on the surface of lung and ovarian adenocarcinoma cells, breast cancer and lymphoma can be used as markers for the location, identification and treatment of these tumors in vivo. The present inventors have for the first time developed small, synthetic compounds, including CT-derived peptides, which have the ability to bind with high affinity to CT receptors and are favorable pharmacokinetics, thereby allowing efficient in vivo localization of diagnostic and therapeutic agents at tumor sites. These reagents of the invention provide the basis for rapid, cost-effective, non-invasive diagnostic imaging procedures that are useful for detecting tumors, detecting disease states, and evaluating metastatic spread of tumors, characterized by the expression or overexpression of CT receptors. The reagents of the invention also provide a basis for evaluating the therapeutic efficacy of other treatment modalities, for example, the placement of CT-receptor expressing tumor cells followed by surgical therapy, radiotherapy or chemotherapy. The reagents of the invention may also be used as target particles for topical radiotherapy.

The present invention provides CT receptor binding reagents comprising CT receptor binding compounds, preferably CT peptides, CT derivatives or CT analogs that are covalently linked to a radioactive metal chelator. The CT receptor binding compounds used in the reagents of the invention have a molecular weight of less than 10,000. In some embodiments, the reagents of the invention are characterized as peptides, due to the presence of a peptide bond in either part of the reagent that it binds to the CT receptor, or by the presence of a peptide bond in a radioactive metal chelator. The reagents of the invention have an affinity for binding to a CT receptor that is not less than one-tenth the affinity of radioactive iodine labeled natural CT for said receptor when compared in standardized assays such as those described in Example 4 below. In preferred embodiments the reagents of the present invention have a binding affinity for CT receptors equal to or higher than the natural CT or radiolabeled portions of the natural CT to said receptor when compared in said standardized assay.

The radiopharmaceutical agents of the invention may be used as site-specific diagnostic or therapeutic agents. When labeled with technetium-99m, iodine-123 and iodine-131, the reagents of the invention can be used as scintigraphic imaging agents. When labeled with a magnetic, paramagnetic, supermagnetic or superparamagnetic metal, the reagents of the invention can be used as magnetic resonance contrast agents. When labeled with cytotoxic radionuclides, the reagents of the invention may be used for site-directed radiotherapy. The present invention also provides pharmaceutical compositions comprising the radiolabeled CT receptor binding compounds of the invention and a pharmaceutically acceptable carrier. Also provided by the invention are methods for making the CT receptor binding reagents of the invention and radiolabelled embodiments thereof.

The invention also provides kits for preparing radiolabeled CT receptor binding compounds from the reagents of the invention. The kits of the invention comprise a sealed container containing a predetermined amount of the reagent of the invention and optionally a sufficient amount of a reducing agent to radiolabel the reagent.

«·»

The present invention provides methods of using the radiolabeled CT receptor binding reagents of the invention for diagnosis and therapy. In one embodiment, methods are provided for using the reagents of the invention in a labeled form for imaging sites in the mammalian body by obtaining an IV vivo imaging. These methods comprise the steps of administering an effective diagnostic amount of labeled reagents of the invention and detecting a marker positioned at the site of the mammal's body.

The present invention also provides methods for alleviating a disease characterized by the expression or overexpression of CT receptors, comprising the step of administering to an animal a therapeutically effective amount of a radiolabeled CT receptor binding reagent of the invention.

Other aspects and advantages of the present invention will become apparent from the following detailed description of the preferred embodiments set forth below.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides CT receptor binding reagents that are useful for the preparation of pharmaceutical CT receptor binding agents for diagnosis and therapy. For purposes of this invention, the term CT receptor binding compound is intended to include naturally occurring CT, CT fragments, CT analogs and CT derivatives that specifically bind to the CT receptor expressed by a variety of cell types recognized by those skilled in the art. Compounds that are designed to mimic the properties of a CT binding receptor are also included in this definition and are included in the invention.

For purposes of this invention, the term CT receptor binding affinity is intended to mean binding affinity measured by any method known to one skilled in the art, including, but not limited to, those that measure binding affinity by dissociation constant, inhibitory constant, or IC 50 value.

A term having a binding affinity for the CT receptor of at least one-tenth of the affinity of a radioactive iodine labeled CT to said receptor is meant that the term " CT " Means that the dissociation constant (Kd) of the reagent is not less than one-tenth of the Kd of the radiolabelled CT, when measured by direct binding of CT receptors or by competitive inhibition of CT receptors, or that the inhibitory constant (K i) or IC 50 of the reagent is not ten times greater than radiolabeled CT when measured by competitive inhibition of CT receptors.

A term having a CT receptor binding affinity equal to or greater than natural CT or radioactive iodine labeled CT particles to said CT receptor is meant to include those reagents having a dissociation constant (K d) equal to or greater than the dissociation constant of unlabeled CT or radioactive iodine labeled CT when measured by direct CT receptor binding or competitive CT receptor inhibition assay. The term can also be interpreted to include reagents having an inhibition constant (K) or IC 50 of the reagent equal to or less than that of natural CT or radiolabeled CT when measured by a competitive CT receptor inhibition assay.

According to the invention, the reagents of the invention have a binding affinity for CT receptors of at least ten times greater than the affinity of radiolabeled natural CT for said receptor when compared in standardizing the assay as described below. In preferred embodiments, the reagents of the invention have a binding affinity for CT receptors equal to or greater than the natural CT or radiolabelled portions of the natural CT for said receptor when compared in said standardized assay.

The dissociation constants or binding inhibition constants of the radiopharmaceutical agents of the invention with respect to CT receptors, as well as comparing the affinity or avidity of such binding with binding of ^{125 I} -labeled CT alone can be determined using known methods such as those listed in Receptors, A Quantitative Approach , A. Levitzki, The Benjamin, Cummings Publishing Company (California 1984). Preferably, standardized CT receptor binding assays, such as

The assays set forth in Example 4 below use the CT receptor to measure the binding affinity of the reagents of the invention.

In addition to the CT receptor binding compounds, the reagents obtained by the present invention comprise a radioactive metal chelator that is covalently attached to the compound in such a way that the binding specificity of the compound to the CT receptor is not substantially altered. Any radiolabeled chelator may be covalently bound to a CT receptor binding compound to provide a reagent of the invention. For example, a CT receptor binding compound, preferably a peptide, can be covalently linked to a chelator of the formula

C (pgp) ^s - (aa) -C (pgp) ^s wherein (pgp) ^s is a hydrogen atom or a thiol protecting group and (aa) is any non-thiol-containing primary α- or β-amino acid. In a preferred embodiment, the amino acid is glycine.

Another example - a radioactive metal chelator useful in the reagents of the invention contains a single thiol group of the formula

A-CZ (B) - {C (R ^a R ^b)} "- X wherein A is H, HOOC, H ₂ NOC, (peptide) -NHOC, (peptide) -OOC-, R ⁶ 2 NCO or R ^d , B is hydrogen, SH, -NHR ^C , -RNC (peptide) or R ^d , Z is hydrogen or R ^d , X is SH, -NHR ^C , -N (R ^c ) - (peptide) or R ^d , R ^a , R ^b , R ^c and R ^d independently represent a hydrogen atom or a straight or branched chain or cyclic lower alkyl, n is 0, 1 or 2, R ^c is an alkyl of 1 to 4 carbon atoms, an amino acid or a peptide of 2 to 10 amino acids and 1) when B is -NHR ^C or -NR- (peptide), X is SH and n is 1 or 2, 2) if X is -NHR ^C or -N (R) ^c ) - (peptide), B stands for SH group and n stands for 9 9 9 9 999 999 999

9 9

9 9 9 9 9 number 1 or 2, 3) if B is hydrogen or R ^d , A is HOOC, H2NOC, (peptide) -NHOC or (peptide) -OOC, X is SH and n is 0 or 1 (4) if A represents a hydrogen atom or R ^d , then if B represents a SH group, X represents a -NHR ^C group or -N (R °) - (peptide), and if X represents a SH group, B represents a -NHR ^C group or -N (R ^c ) - (peptide) and n is 1 or 2, 5) when X is hydrogen or R ^d , A is HOOC, H2NOC, (peptide) -NHOC or (peptide) -OOC and B is SH, 6) when Z is methyl, X is methyl, A is HOOC, H ₂ NOC, (peptide) -NHOC or (peptide) -OOC, B is SH and n is 0 and 7) if B is SH, X is not SH, and when X is SH and B is not SH.

A preferred embodiment of the radioactive metal chelator is a compound of the general chemical formula

R ¹ -CO- (amino acid) ^1- (amino acid) ² -Z, wherein each (amino acid) ¹ and (amino acid) ² independently represents any primary α- or β-amino acid not containing a thiol group, Z represents a thiol group which is cysteine, homocysteine, isocysteine, penicillamine, 2-mercaptoethylamine or 3-mercaptoethylamine, and R ¹ is C 1 -C 4 lower alkyl, amino acid or peptide of 2 to 10 amino acids. When Z is cysteine, homocysteine, isocysteine or penicillamine, the carbonyl group of said moiety is covalently bonded to a hydroxyl group, NR ³ R ⁴ , wherein R ³ and R ⁴ independently represent hydrogen or C 1 -C 4 lower alkyl, amino acid or a petid of 2 to 10 amino acids, or

Y- (amino acid) ^1- (amino acid) ² -NHR ² , wherein Y is a thiol-containing group that is cysteine, homocysteine, isocysteine, penicillamine, 2-mercaptoacetate or 3-mercaptopropionate, each (amino acid) 9 98 9 9

9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 99 99 999 9 9 9 9

99a 9 · 99a 99a) ^1a (amino acid) ² independently means any primary α- or β-amino acid that does not contain a thiol group, and R ² is hydrogen or C 1 -C 4 lower alkyl, amino acid or a peptide of 2 to 10 amino acids. When Y is cysteine, homocysteine, isocysteine or penicillamine, the amino group of said moiety is covalently attached to a hydrogen atom, an amino acid or a peptide of 2 to 10 amino acids.

In particular embodiments of this aspect of the invention, the formula of the radioactive metal chelator is selected from the group consisting of compounds of the following formulas

- (amino acid) ^1- (amino acid) ^2- {A-CZ (B) - {C (R ¹ R ² )} nX},

- {A-CZ (B) - {C (R ¹ R ² )} nX} - (amino acid) ^1- (amino acid) ² ,

- (primary αω- or B, a> -diamino acid) - (amino acid) ^1- {A-CZ (B) - {C (R ¹ R ² )} _n -X} -a

- {A-CZ (B) - {C (R ¹ R ² )} nX} - (amino acid) ^1- (primary α, β- or α, γ-diamino acid), each of which (amino acid) ¹ a (amino acid) ¹² independently represents any naturally occurring, modified, substituted or altered α- or β-amino acid that does not contain a thiol group, A represents a hydrogen atom, a HOOC, H2NOC, (amino acid or peptide) -NHOC, (amino acid or peptide) -OOC- or R ⁴ , B is hydrogen, SH, -NHR ³ , -N (R ³ ) - (amino acid or peptide) or R ⁴ , Z is hydrogen or R ⁴ , X is SH, -NHR ³ , -NtR 4 -amino acid or peptide) or R ⁴ , R ¹ , R ² , R ³ and R ⁴ independently represent a hydrogen atom or a straight or branched chain or cyclic lower alkyl, n is 0, 1 or 2, (peptide 1) is a peptide of 2 to 10 amino acids, and 1) when B is -NHR ³ or -R (N-amino acid or peptide), X is SH and n is 1 or 2 (2) when X is -NHR ³ or -NR (amino acid or peptide), B is SH and n is 1 or 2; 3) if B is hydrogen or R ⁴ , A is HOOC, H2NOC, ( amino acid or peptide) -NHOC or

9,999,9 (amino acid or peptide) -OOC, X is SH (and n is 0 or 1,4) if A is hydrogen or R ⁴ , then if B is SH, X is -NHR ³ or -N (R ³ ) - (amino acid or peptide), and when X is SH, B is -NHR ³ or -N (R ⁴ ) (amino acid or peptide) and n is 1 or 2, 5) if X is hydrogen or R ⁴ , A is HOOC, H ₂ NOC, (amino acid or peptide) -NHOC or (amino acid or peptide) -OOC and B is SH, 6) when Z is methyl, X is methyl, A is HOOC, H ₂ NOC, (amino acid or peptide) -NHOC or (amino acid or peptide) -OOC, B is SH and n is 0, and 7) when B is SH, X is not SH and when X is SH and B is not SH.

Specific preferred embodiments of this aspect of the invention include radioactive metal chelators selected from the group consisting of compounds of the following formulas -Gly-Gly-Cys-, Cys-Gly-Gly-, -Gly-Gly-Cys-, - (s-Lys) -Gly -Cys-, (6-Om) -Gly-Cys-, - (Y-Dab) -Gly-Cys-, - (B-Dap) -Lys-Cys- and - (B-Dap) -Gly-Cys- (In these formulas, it is to be understood that ε-Lys means a lysine residue in which an ε-amino group, rather than a typical α-amino group, is covalently attached to the carboxyl group of an adjacent amino acid to form a peptide bond; δ-Om means an omithine residue in which the δ-amino group, rather than a typical α-amino group, is covalently attached to the carboxyl group of an adjacent amino acid to form a peptide bond, γ-Dab means a 2,4-diaminobutyric acid residue in which the γ-amino group is covalently linked to the carboxyl group of an adjacent amino acid to form a peptide bond, β-Dap means a 1,3-d residue aminopropionic acid in which the β-amino group is covalently linked to the carboxyl group of an adjacent amino acid to form a peptide bond.).

In another embodiment, the raidoactive metal chelator of the reagent of the invention is a bisamino-bisthiol chelator of formula

NH

I (CRÁ

S- (pgp) ^p

9999

99

I 9 9 9> 9 9 9

99 · 999

9

9 9 9 (CR _2:

N-A-CO-peptide

I (CRÁ

S- (pgp) ⁵ wherein each R independently represents a hydrogen atom, methyl or ethyl, each (pgp) ^s independently represents a thiol protecting group or a hydrogen atom, m, n and p independently represent 2 or 3, A represents a linear or cyclic lower alkyl , aryl, heterocyclyl, a combination thereof, or substituted derivatives thereof, and X is a peptide. The bisaminobisthiol chelator in this embodiment of the invention is also a compound of formula

wherein each R independently represents hydrogen, methyl or ethyl, m, n, and p independently represent 2 or 3, A represents linear or cyclic lower alkyl, aryl, heterocyclyl, combinations thereof or substituted derivatives thereof, V represents hydrogen atom or a group A CO-peptide, R 'is a hydrogen atom or a peptide, and wherein when V is a hydrogen atom, R ¹ is a peptide, and when R' is a hydrogen atom, V is a CO-peptide group. For the purposes of the present invention, chelating particles having these structures will be referred to as BAT particles.

φ ΦΦΦΦ Φ · · · φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ • φ φ • • • • • φ

The radioactive metal chelator used in the reagent of the invention may be selected from the group consisting of diethylenetriaminepentaacetic acid (DTPA) of the general formula (HOOCCH ₂ ) 2N (CR ₂ ) (CR ₂ ) N (CH ₂ COOH) (CR ₂ ) (CR ₂ ) N (CH ₂ COOH) wherein each R is independently hydrogen, C 1 -C 4 alkyl or aryl and one R is covalently attached to a divalent linker of ethylenediaminetetraacetic acid (EDTA) of the general formula (HOOCCH ₂ ) 2N (CR 2) (CR 2) N (CH _{2 (} COOH) wherein R is independently hydrogen, C 1 -C 4 alkyl or aryl and one R is covalently bonded to a divalent linker,

1,4,7,10-tetraazododecantetraacetic acid, a compound of formula

(CRA

wherein n is 2 or 3 and each R is independently hydrogen, C 1 -C 4 alkyl or aryl and one R is covalently bonded to a calcitonin receptor binding compound and desferrioxamine.

Most of the radioactive metals can be chelated with the reagents of the invention, including the above-mentioned chelating agents for radioactive metals.

· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

9 · 99

The reagents of the invention may also contain a radioactive metal chelator selected from the group consisting of

(i) groups of the general formula I

(1) and (ii) a group of formula (II)

wherein n, m and p are independently 0 or 1, each R 'independently is hydrogen, lower alkyl, hydroxyalkyl (C 2 -C 4) or alkoxyalkyl (C 2 -C 4), and each R independently is hydrogen or R, wherein R is a substituted or unsubstituted lower alkyl or phenyl not containing a thiol group, and one of R or R 'is L, wherein L is a divalent linker linking group attached to a metal chelator on the target particle, provided that one R group 1 is L, NR ₂ is amine.

In preferred embodiments, L is a linear or branched chain of 1 to 6 carbon atoms or a cyclic alkyl group, carboxylic ester, carboxamide, sulfonamide, ether, thioether, amine, alkene, alkyne, 1,2-, 1,3- or 1,4 connected or 4 4444, respectively

4 * 44 • 4 4 4 4 4444 4 4 4 4 4 4 4 4

44,444,444,444,444 44,444,444 44,444 substituted benzene ring, amino acid, peptide of 2 to 10 amino acids or combinations thereof.

In preferred embodiments, R is a linear, branched or cyclic alkyl group having 1 to 6 carbon atoms, -C _q OC _r -, -C _q NHC _r - or -C _q SCr, wherein _{q and} r are independently 1 to 5, at wherein the sum of q + r is not greater than 6, alkyl (C 1 -C 6) -X, wherein X is hydroxyl, substituted amine, quanidine, amidine, substituted thiol, carboxylic acid, ester, phosphate or sulfate, phenyl halo, hydroxyl, substituted amine, guanidine, amidinium, substituted thiol, ether, phosphate or sulfate, indole, C 1 -C 6 heterocyclic group containing 1 to 3 nitrogen, oxygen or sulfur atoms, or a combination thereof, or a phenyl group .

According to the present invention, the radioactive metal chelator of the CT receptor binding reagent may have the formula III

R ⁶ .o ^R SM

Axis NH NH ⁴ -X. 2 _R 3 NR'R ² HS

O

LZ (III) wherein R ¹ and R ² independently of one another are hydrogen, lower alkyl, hydroxyalkyl (C 2 -C 4) or alkoxyalkyl (C 2 -C 4), R ³ , R ⁴ , R ⁵ and R ⁶ independently represent hydrogen, substituted or unsubstituted lower alkyl or phenyl, which does not contain a thiol group, R ⁷ and R ⁸ independently represent hydrogen, lower alkyl, lower hydroxyalkyl or lower alkoxyalkyl; L is a divalent group kinekru and Z is a CT peptide.

in 9 9 9 99 99 999

9

Other preferred metal chelators of the present invention include chelators of formula IV, 7 ° C

θγΝΗ HfA

R ⁴ 'Zx,. _R 2 NR 3 _{'is R j;} (IV) wherein R ¹ and R ² independently represent hydrogen, lower alkyl, hydroxyalkyl (2 to 4 carbon atoms) or alkoxyalkyl (C 2 -C 4 alkyl), R ^3, R ^4, R ^5, and R ^{6 are} independently hydrogen, substituted or unsubstituted lower alkyl or phenyl that does not contain a thiol group, and one of R ³ , R ⁴ , R ⁵ and R ⁶ is ZL-HN (CH ₂ ) _n - wherein L represents a divalent linker group, Z represents a targeting moiety, n represents a number from 1 to 6, R ⁷ and R ⁸ independently represent a hydrogen atom, a lower alkyl, a lower hydroxyalkyl or a lower alkoxyalkyl and X represents an amino group, a substituted amino group or -NR ^1- Y, wherein Y is an amino acid, an amino acid amide, or a peptide of 2 to 10 amino acids.

More preferred metal chelators of the invention include those of formula V

(V) ·· 99

9 · 9 9 9 9 9 9 9 9

9 9 9 9 9

9,999 9,999,999

9 9 9 9 9 9

Wherein R ¹ and R ² independently of one another are hydrogen, lower alkyl, lower hydroxyalkyl or lower alkenylalkyl, R ³ and R ⁴ independently of each other are hydrogen, substituted or unsubstituted lower alkyl or phenyl that does not contain a thiol group, n is a number from 1 to 6, L is a divalent linker group, and Z is a CT peptide particle.

Other more preferred chelating particles include chelators of Formula VI z-1.

r ~ ^ ⁰

O 2 / NH HN?

NH,

NH,

HS '(VI), wherein L is a divalent linker group and Z is a CT peptide particle.

The most preferred chelating particles of the invention include chelators of the following formulas (amino acid) ^1- (amino acid) ² -cysteine, (amino acid) ^1- (amino acid) ² -isocysteine, (amino acid) ^1- (amino acid) ² -homocysteine, (amino acid) ^1- (amino acid) ² -penicillamine, (amino acid) ^1- (amino acid) ² -2-mercaptoethylamine, (amino acid) ^1- (amino acid) ² -2-mercaptopropylamine, (amino acid) ^1- (amino acid) ² -2-mercapto -2-methylpropylamine-α (amino acid) ^1- (amino acid) ² -3-mercaptopropylamine- in which the (amino acid) in the primary and / or β-amino acid does not contain a thiol group and in which the chelator is attached to either the target particle or linker group by a covalent bond with a carboxyl terminus of a chelator or a side chain at one of the amino acid groups.

• »β b b b b b b b b b b

The most preferred chelators also include the chelators of the above formula wherein (amino acid) ¹ is either an α, ω- or β, ω-amino acid in which the α- or β-amino group is a free amine and α, ω- or β, ω the amino acid is covalently linked by a ω-amino group.

Other most preferred chelators include those selected from the group consisting of compounds of the following formulas

-cysteine- (amino acid) - (a, B- or β, γ-diamino acid),

-isocysteine (amino acid) - (a, B- or β, γ-diamino acid),

-homocysteine- (amino acid) - (a, B- or β, γ-diamino acid),

-penicillamine- (amino acid) - (a, B- or β, γ-diamino acid),

2-mercaptoacetic acid- (amino acid) - (a, B- or β, γ-diamino acid),

2- or 3-mercaptopropionic acid- (amino acid) - (a, B- or β, γ-diamino acid) and

2-mercapto-2-methylpropionic acid- (amino acid) - (a, B- or β, γ-diamino acid) in which the (amino acid) in the primary α- or β-amino acid does not contain a thiol group and in which the chelator is attached to either a target particle or linker group by a covalent bond with the amino terminus of a chelator or side chain at one of the amino acid groups.

Particularly preferred metal chelators are selected from the group consisting of compounds of the following formulas: -Gly-Gly-Cys-, -Arg-Gly-Cys-, - (e-Lys) -Gly-Cys- (6-Om) -Gly-Cys -, - (γ-Dab) -Gly-Cys-, - (B-Dap) -Lys-Cys-, and - (B-Dap) -Gly-Cys (The amino acid designations in these formulas are the same as above.).

An example of a radioactive metal chelator of formula III above is Gly-Gly-Cys-, wherein the chelating particle has the structure VII.

9 9 99 99

9 9 9 9 9

9 9 9 9 9 9 999 999

9 9 9 9 9 9

99 99 99 99 99

a calcitonin receptor binding compound (VII).

The chelating ligands of structure VII form oxotechneci complexes of the VIIIa structure

a calcitonin receptor binding compound (VIII).

An example of a radioactive metal chelator of formula (V) above is Lys - ((α-peptide) -Gly-Cys.amide, which forms the chelator of formula IX)

Chelating ligands of formula IX form oxotechneci complexes of formula X

99 «9

999 9 9 9 9 9 9 9 9 9 9

9 9 9

998 99 «• 9

99

An example of a reagent of the invention containing a radioactive metal chelator of formula II above is (target particle) -Cys-Gly-α, β-diaminopropionamide, which forms the chelator of formula XI a calcitonin-HN receptor-binding compound (o

SH ₂ N (XI).

The radiodiagnostic agents of formula XI form the oxotechnetic complexes of formula XII a calcitonin-HN receptor binding compound

NH ₂ (XH).

In radioactive metal chelators and CT receptor binding reagents obtained according to the invention, which contain a thiol covalently bound to a thiol protecting group {(pgp) ⁸ }, the thiol protecting group may be the same or different and may include, but are not limited to, the following groups :

• φ ·> ·> · φ ·

-CH ₂ -aryl (aryl means phenyl or alkyl or alkoxy substituted phenyl), -CH- (aryl) ₂ (aryl means phenyl or alkyl or alkoxy substituted phenyl),

-C- (aryl) ₃ (aryl means phenyl or alkyl or alkoxy substituted phenyl), -CH ₂ - (4-methoxyphenyl),

-CH- (4-pyridyl) (phenyl) ₂ ,

-C (CH _{3) 3,}

-9-phenylfluorenyl,

-CH ₂ NHCOR (R is unsubstituted or substituted alkyl or aryl),

-CH ₂ -NHCOOR (R represents unsubstituted or substituted alkyl or aryl),

-CONHR (R represents unsubstituted or substituted alkyl or aryl) or

-CH ₂ -S-CH ₂ -phenyl.

Preferred protecting groups are of the formula -CH ₂ -NHCOR wherein R is lower alkyl of 1 to 8 carbon atoms, phenyl or phenyl substituted with lower alkyl, hydroxyl, lower alkoxy, carboxy or lower alkoxycarbonyl. The most preferred protecting group is acetamidomethyl.

If the reaction of the invention comprises a CT receptor binding compound which is a peptide, the peptide preferably comprises the amino acid sequence of SEQ ID NO: 2. No. 10

CH 2 CO.SNLSTX- (SEQ ID NO 10), wherein X is selected from the group consisting of a cysteine residue, a homocysteine residue, and a homohomocysteine residue. The peptide may also comprise amino acid sequence id. No. 11 • · · · · · · · ·

CH? C0.1NLSTX ² - (SEQ ID NO: 11) wherein X ¹ is selected from the group consisting of alanine residue, glycine residue and serine residue, and X ² is selected from the group consisting of cysteine residue, homocysteine residue, and homohomocysteine residue. These peptides include naturally occurring human CT and CT peptide analogues, such as those specifically listed in the following amino acid sequences:

CH? CO.SNLST.Hhc.VLGKLSCELHKLQTYPRTNTGSGTP.amid (SEQ ID NO 5),

CH? CO.SNLST.Hcv.VLGKLSCELHKLQTYPRTNTGSGTP.amid (SEQ ID NO: 6),

CHpCO.SNLST.Cs.VLGKLSCELHKLQTYPRTNTGSGTP.amid (SEQ ID NO 7) and

SNLST.Asu.VLGKLSCELHKLQTYPRTNTGSGTP.amid (SEQ ID NO: 8).

Particularly preferred embodiments of the reagents of the invention include:

CHpCO.SNLST.Hhc.VLGKLSCiBAOELHKLQTYPRTNTGSGTP.amid (SEQ ID NO 4),

CH _? CO.SNLST.Hhc.VLGKLSCELHKLQTYPRTNTGSGTPfe-K) GC.amide.

CH? CO.SNLST.Hhc.VLGKLSC (CH? CQ.GGCK.amid)) ELHKLQTYPRTNTGSGTP.

.amid,

CH _? CO.SNLST.Hhc.VLGKLSC (CH _? CO.fB-Dap) KCK.amid) ELHKLQTYPRTNTGS ·

GTP.amid,

CH? CO.SNLST.Hhc.VLGKLSC (CHpCO.fe-K) GCE.amidúELHKLQTYPRTNTGSGTP.a mid,

CHpCO.SNLST.Hcv.VLGKLSC (CHpCO.GGCK.amid) ELHKLQTYPRTNTGSGTP.amid

CH? CO.SNLST.Hcv.VLGKLSC (CHpCO. (B-Dap) KCK.amid) ELHKLQTYPRTNTGS.

• · · · · ·

.GTP.amid,

CH _? CO.SNLST.Hcv.VLGKLSC (CH, CO.fs-K) GCE.amid) ELHKLQTYPRTNTGSGTP.

.amid,

CH _? CO.SNLST.Cs.VLGKLSC (CH, CO.GGCKamid) ELHKLQTYPRTNTGSGTP.amid

CH _? CQ.SNLST.Cs.VLGKLSC (CH, CO. (B-DaD) KCK.amid) ELHKLQTYPRTNTGS.

.GTP.amid,

CH ₂ CO ₁ SNLSLCys ₁ VLGKLSC (CH ₂ CO. (8-K) GCE.amide) ELHKLQTYPRTNTGSGTP.

.amid,

SNLSIAs _i VLGKLSC (CH ₂ CO. (B-Dap) KCK.amid)) ELHKLQTYPRTNTGSGTP.amida

SNLST ₁ Acu.VLGKLSC (CH ₂ CO. (B-Dap) KCK.amid) ELHKLQTYPRTNTGSGTP.amid.

All naturally occurring amino acids are abbreviated using standard abbreviations (which can be found in G. Zubay: Biochemistry, Second Edition, MacMillen Publishing, New York 1988, p. 33). For the purposes of this invention, naturally occurring amino acids are characterized as lipophilic (alanine, isoleucine, leucine, methionine, phenylalanine, tyrosine, proline, tryptophan and valine as well as S-alkylated cysteine derivatives), hydrophilic (asparagine, glutamine, threonine, serine), acidic (glutamic acid and aspartic acid), basic (arginine, histidine and lysine), ε-Κ, δ-Om, γ-Dab and β-Dab have the meanings given above. (BAT) means N ⁶ , N ⁹ -bis (2-mercapto-2-methylpropyl) -6,9-diazanonanoic acid, K. (BAT) and Lys. (BAT) mean the amino acid lysine acylated on the ε-amine group amino acid side chains of k (BAT), C (BAT) and Cys (BAT) mean S- (N ⁶ , N ⁹ -bis (2-mercapto-2-methylpropyl) -6,9-diazanonan-1-yl) cysteine, (BAM) means (N ¹ , N ⁴ -bis (2-mercapto-2-methylpropyl) -1,4,10-triazadecane, (BAT-BM) means N- {2- (N ', N'-bis ( 2-maleimidoethyl) aminoethyl] -N ⁹ - (tert -butoxycarbonyl) - (N ⁶ , N ⁹ -bis (2-methyl-2-triphenylmethylthiopropyl) -6,9-diazanonanamide, (BAT-BS) means N- {2- (N ⁶ , N ⁹ -bis (2-succinimidoethyl) aminoethyl] -N ', N'-bis (2-mercapto-2-methylpropyl) -6,9-diazanonanamide, (BMH) means bis-maleimidohexane, (BSH) means bis-succinimidohexane, (BMME) means bis-maleimidomethyl ether, (BSEE) means bis-succinimidoethyl ether, (BMEE) means bis-maleimidoethyl ether, and (BSME) means bis-succinimidomethyl ether As used herein, the following amino acids and amino analogs acids are indicated by the following abbreviations:

• · · «· · · ·

Acm stands for mercaptan protecting group acetamidomethyl, Pen stands for penicillamine, Aca stands for 6-aminocaproic acid, Hly stands for homolysin, Apc stands for LS- (3-aminopropyl) cysteine, F _D stands for D-phenylalanine, W _D stands for D-tryptophan, Y _D stands for D-tyrosine, Cpa is L- (4-chlorophenyl) alanine, Thp is 4-amino-tetrahydrothiopyran-4-carboxylic acid, D-Nal is D-2-naphthylalanine, Dpg is dipropylglycine, Nle is norleucine, Hcy is homocysteine, Hhc stands for homohomocysteine, Aib stands for aminoisobutyric acid, Nal stands for 2-naphthylalanine, D-Nasled stands for 2-naphthylalanine, Ain stands for 2-aminoindan-2-carboxylic acid, Achxa stands for 4-aminocyclohexylalanine, Amf stands for 4-aminomethyl- phenylalanine, Aec is S- (2-aminoethyl) cysteine, Apc is S- (3-aminopropyl) cysteine, Aes is O- (2-aminoethyl) serine, Aps is O- (3-aminopropyl) serine, Abu is 2- aminobutyric acid, Nva means norvaline and Asu means 2-aminocork acid elinua, wherein the amino terminal amino acid group of peptides containing the Asu residue is cyclized by an amide bond between the amino terminal amino group and the carboxyl group of the Asu side chain.

According to the present invention, CT receptor binding peptides may comprise one or more amino acid derivatives having a radioactive metal chelator linked to an amino acid side chain. The radioactive metal chelator is preferably incorporated into the peptide at the carboxyl terminus of the CT receptor binding peptide. More preferably, the radioactive metal chelator is incorporated into the synthetic CT receptor binding peptide to the sulfur atom of the cysteine side chain, corresponding to position 14 of the native peptide. Most preferably, the radioactive metal chelator is incorporated into the amino acid side chain of the CT receptor binding peptide of sequence id. No. 9

CH2CO.SNLST.Hhc.VLGKLSCELHKLQTYPRTNTGSGTP.amide (SEQ ID NO 9).

• ·

Most preferably, the radioactive metal chelator is incorporated into the CT receptor binding peptide to the sulfur atom of the cysteine side chain at position 13 of the peptide of SEQ. id. 9 (differentiated by the amplification in SEQ ID NO 9).

Another embodiment of the reagents of the invention comprises at least two synthetic CT receptor binding compounds, each compound covalently attached to a radioactive metal chelator, and a polyvalent linker forming covalent bonds, selected from the group consisting of binding to each compound, bonds to each chelator, and bonds to one compound and a chelator of another compound. Other permutations of this embodiment may also occur according to the invention. The polyvalent linkers suitable for use in this embodiment of the invention contain at least two identical functional groups capable of covalently binding to CT analogs, CT receptor binding compounds, CT peptides or radioactive metal chelators, or capable of binding to both CT receptor binding compounds and radioactive chelators. metal. Preferred functional groups include, without limitation, primary amines, secondary amines, hydroxyl groups, carboxylic acid groups, or thiol reactive groups. In preferred embodiments, the polyvalent linkers comprise bis-succinimidyl methyl ether (BSME), bis-succinimidyl ether (BSEE), 4- (2,2-dimethylacetyl) benzoic acid (DMBA), N- {2- (N ', N'-bis (2 -succinimido-ethyl) aminoethyl)} - (N ⁶ , N ⁹ -bis (2-methyl-2-mercaptopropyl) -6,9-diazonanamide (BAT-BS), tris (succinimidyl ethyl) amine (TSEA), bissuccinimidohexane (BSH) ), 4- (O-CH ₂ CO-Gly-Gly-Cys., Amide) -2-methylpropiophenone (ETAC), tris (acetamidoethyl) amine, bis-acetamidomethyl ether, bis-acetamidoethyl ether, α, ε- bis-acetyllysine, lysine and 1,8-bis-acetamido-3,6-dioxa-octane or derivatives thereof.

The CT receptor binding compounds obtained according to the present invention can be chemically synthesized in vitro using any suitable method of synthesis. Preferably, CT peptides can be synthesized according to the invention by recombinant means. More preferably, CT peptides, CT peptide derivatives, and CT peptide analogs can generally be conveniently prepared according to the present invention using a peptide synthesizer. The CT peptides of the invention are preferably synthesized by covalent attachment of a radioactive metal chelator to a peptide during in vitro chemical synthesis using methods well known to those skilled in the art such as solid peptide synthesis. stage. In this way, radioactive metal chelators can be incorporated into the peptide in a locally selective manner at virtually any position of the peptide, thereby avoiding reducing the affinity and specificity of the peptide for the CT receptor.

According to the present invention, CT receptor binding peptides are prepared having a thiol protected amino acid, typically a cysteine residue, included in the peptide. Subsequent cleavage of the peptide from the synthetic resin and cyclization of the amino-terminus residues deprotects the thiol protecting group to yield a prosthetic group containing a radioactive metal chelator and a thiol reactive group.

As mentioned above, the CT receptor binding reagents of the invention may be radiolabeled to provide radiodiagnostic or radiotherapeutic agents. An example of a radiodiagnostic application using the radiolabeled reagents of the invention is scintigraphic imaging in which the location and extent of CT receptor-bearing tumors can be determined. The term scintigraphic imaging agent, as used herein, includes a radiolabeled reagent that has the ability to be detected by means of detecting radioactivity (including but not limited to γ-cameras or scintillation detector probes). In radiotherapeutic embodiments of the invention, CT receptor binding reagents are labeled with cytotoxic radionuclides and are useful in the treatment of diseases or other diseases of animals, preferably humans, such as those characterized by the expression or overexpression of CT receptors. CT related diseases or diseases include, but are not limited to, breast cancer, ovarian cancer, lung cancer, lymphoma, and other diseases characterized by growth of malignant or benign tumors binding CT receptor binding compounds. These tumors contain CT or its derivatives or analogs by expressing CT receptors on the cell surface.

• <9 · · · · ·

9 9 · 9 9

Any radioactive metal may complex with the reagents of the invention to form a radiodiagnostic or radiotherapeutic agent. For example, the reagents of the invention may be radiolabeled with technetium-99m, iodine-125 or iodine-123 to provide a scintigraphic imaging agent. The present invention also provides CT receptor binding reagents capable of complexing with a magnetic, paramagnetic, supermagnetic or superparamagnetic atom, ion, or metal particle. Preferably, the CT receptor binding agents of the invention may also be radiolabeled with a cytotoxic radioisotope selected from the group consisting of scandia-47, copper-67, gallium-72, yttria-90, tin-117m, iodine-125, iodine-131, samaria -153, gadolinium-159m, dysprosia-165, holmia-166, yterbium-175, lutecia-177, rhenia-186, rhenia-188, astatu-211, vismutu-212 and vismutu-213 to obtain a radiotherapeutic agent.

When the reagents of the invention are used to form a complex of radioactive technetium or rhenium, a complex with technetium, preferably technetium-99m pertechnetate or a rhenium salt in the form of rhenate, is reacted with the reagent in the presence of a reducing agent. The preferred reducing agent is stannous or ferrous dithionite, the most preferred reducing agent is stannous chloride. The complex may also be formed by reacting a reagent of the invention with a preformed labile complex of technetium or rhenium and other compounds known as a ligand transporter. This method is known as ligand exchange and is well known to those skilled in the art. The labile complex may be formed using ligand transporters such as tartrate, citrate, gluconate or mannitol. Technetium-99m pertechnetate and rhenium salts useful in the present invention include alkali metal salts such as sodium salt, ammonium salts or lower alkyl ammonium salts.

The invention also encompasses a kit for the manufacture of radiolabelled reagents for use as radiopharmaceutical compounds. The kit of the invention comprises a sealed container containing a predetermined amount of a CT receptor binding agent, and optionally, if the radioactive metal is those. Necium-99m, rhenium-186 or rheniiim-188, reducing agent. For example, an appropriate amount of the reagent of the invention is introduced into a container containing a reducing agent such as stannous chloride in an amount sufficient to label the reagent with technetium-99m, rhenium-186 or rhenium-188. An appropriate amount of a ligand carrier as described above (such as tartrate, citrate, gluconate, glucoheptanate or mannitol) may also be included in the kit. The kit may also include conventional pharmaceutical excipients such as pharmaceutically acceptable salts for adjusting the osmotic pressure, buffers, preservatives and the like. These components may be in liquid, frozen or dry form. In a preferred embodiment, the kit components are prepared in lyophilized form. The technetium-99m, rhenium-186 and rhenium-188 radio-pharmaceutical compositions of the present invention can be prepared by adding appropriate amounts of technetium-99m, rhenium-186 or rhenium-188 or complexes of these radionuclides to flasks and reactions under the conditions described in the Examples below.

The kit of the invention may also be prepared in a form suitable for diagnostic imaging or as a therapeutic agent using an iodine radioisotope, including iodine-123 and iodine-131, preferably iodine-123. In this embodiment, the assembly comprises a sealed container containing a predetermined amount of a CT receptor binding agent capable of being radiolabelled with an iodine isotope.

CT receptor binding reagents suitable for use in this embodiment include CT alone, a CT derivative, a CT analog, CT mimetics, and CT peptidomimetics that specifically bind to the CT receptor. When peptide and peptidomimetic CT receptor binding reagents are used in this embodiment, the tyrosine residue in the reagent may be radiolabeled. The tyrosine residue may be naturally present in the peptide or peptidomimetic, or the tyrosine residue may be added to the peptide or peptidomimetic at a position that does not interfere with the binding of the reagent to the CT receptors. Dosage, sites and routes of administration, formulation compositions, and specific radioactivity administered using the kit of this embodiment are described herein for technetium and rhenium labeled reagents for scintigraphic and therapeutic uses.

• φ φ φ · · · • φ φ φ φ φ · · · · · · · · · · · · φ

The imaging agents obtained according to the invention are used for imaging of tumors, in particular for imaging primary and metastatic neoplastic sites characterized by neoplastic cells that express or overexpress CT receptors, and in particular those primary and particularly metastatic cells derived from breast, lung and ovarian tumors that have been clinically unmanageable by detection using conventional methods. The imaging agents obtained according to the present invention may also be used to visualize organs such as the kidneys or bones for diagnosing disorders in these organs.

For diagnostic purposes, an effective diagnostic amount of a diagnostic or radiodiagnostic agent of the invention is administered, preferably intravenously. In radiodiagnostic embodiments, the localization of the radiolabel is detected by conventional means such as γ-scintigraphy. In non-radioactive diagnosis embodiments, the localization of the accumulation sites of the paramanic-labeled diganostic agents of the invention is performed by magnetic resonance imaging methods.

According to the invention, the technetium-99m-labeled scintigraphic imaging reagents of the invention are administered in a single unit injectable dosage form. The technetium-99m labeled reagents obtained according to the invention can be administered intravenously in any conventional intravenous injection medium, such as aqueous saline or blood plasma medium. Generally, the unit dose to be administered has a radioactivity of 0.01 mCi to 100 mCi, preferably 1 mCi to 20 mCi. The amount of solution to be injected in a unit dose is from 0.01 to 10 ml. After intravenous administration, in vivo imaging is performed within minutes. However, imaging may also be performed, if desired, hours or even longer than the time when the radiolabeled reagent was injected into the patient. In most cases, a sufficient amount of administered dose accumulates in the area to be imaged within 0.1 hour, so that a scintigraphic photograph can be obtained. Any conventional scintigraphic imaging method for diagnostic purposes can be used according to the present invention.

• · ···

For the purposes of the present invention, radiotherapy includes any therapeutic effect ranging from pain relief to removal of the tumor or remission of symptoms associated with the particular cancer being treated. When used for therapeutic purposes, the reagents of the invention are radiolabelled with an effective amount of a cytotoxic radioisotope. For this purpose, the cytotoxic radioisotope may be administered in an amount of from 10 mCi to 200 mCi by any suitable clinical route, preferably by intravenous injection.

According to the present invention, effective radiodiagnostic or radiotherapeutic agents can be identified as follows. Reagents of the invention containing CT receptor binding compounds, including CT fragments, CT peptide analogs, and CT derivatives, are synthesized by the methods of the invention and the radioactive metal chelator is covalently bound to the compound. The reagents are then complexed with a radioactive metal or non-radioactive isotope having a chelating property similar to the desired radioactive metal, and the binding of the CT receptors is then evaluated by in vitro camping binding assays as described herein using radioactive iodine-labeled CT. As an example of this methodology, ReO is used to evaluate the suitability of CT receptor binding peptides for the use of 99m-technetium radiolabeled scintigraphic imaging agents as described in Example 4 below.

Methods for making and labeling these compounds are more fully illustrated in the following examples, which illustrate some aspects of the above-described invention and preferred results, and are given by way of illustration, not limitation.

• 9 99

9 9 9

999 999

9

99

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Synthesis of BAT chelators

BAT chelators, particularly S-cysteine and ε-aminolysine derived BAT chelators, are prepared according to the methods of co-owned and co-pending U.S. Patent Application Serial No. 08 / 414,424, which is incorporated herein by reference.

Example 2

Solid phase peptide synthesis

Solid phase peptide synthesis (SPPS) is performed on a 0.25 millimole (mmole) scale using an Applied Biosystems Model 431A peptide synthesizer and using the 9-fluorenylmethyloxycarbonyl (Fmoc) amino terminus protection, condensation with dicyclohexylcarbodiimide / hydroxybezotriazole or 2- (1) H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate / hydroxybenzotriazole (HBRU / / HOBT) and using p-hydroxymethyl-henoxymethyl-polystyrene (HMP resin, Sasrin ™, chlorotrityl carboxylic acid end resin) or Rink amide resins for the carboxyl end of the amides.

Where appropriate, Fmoc-Cys (BAT) and Na-Fmoc-N8- (BAT) Lys are synthesized as described in co-owned and co-pending U.S. patent application Ser. No. 08/414 424, which is incorporated herein by reference as link.

Where appropriate, the 2-chloroacetyl, 2-bromoacetyl and 2-bromo-3-phenylpropionyl groups are introduced either by using the corresponding 2-halo-acid as the last residue condensed during SPPS or by processing the free amino acid at the end of the peptide bound to either 2 -halogenic acid / diisopropyl • carbodiimide / N-hydroxysuccinimide / NMP or 2-halosuccinic anhydride / diisopropylethylamine • og♦φφ·φodhalod odφod karb karbodod / NMP.

Where appropriate, HPLC-purified 2-haloacylated peptides are cyclized by stirring a 0.1 to 1.0 mg / ml solution in phosphate or bicarbonate t buffer or dilute ammonium hydroxide (pH 8.0), optionally containing 0.5 to 1 0.1 mM EDTA, or acetonitrile or THF for 1 to 48 hours. Acetic acid acidification, lyophilization and HPLC purification followed.

Where appropriate, thiol-containing peptides are reacted with thiol-protected technetium-99m complexing particles containing a chloroacetyl group at pH 10 for 0.5 to 4 hours at room temperature. Acidification with acetic acid followed by evaporation of the solution yielded the corresponding peptide-sulfide adduct. Deprotection and purification are routinely performed as described. A chelator-peptide conjugate is obtained.

Where appropriate, BSME, BSEE and BSH adducts are prepared by reacting peptides containing one thiol (5-50 mg / ml in DMF buffered to pH 7 with N-methylmorpholine or N-ethylmorpholine or 50 mM sodium phosphate buffer, pH 7-8, optionally containing 0.5 mM EDTA, or DMF, THF or acetonitrile) with 0.5 molar equivalents of BMME (bis-maleimidomethyl ether), BMEE (bis-maleimidoethyl ether) or BMH (bis-maleimidohexane), previously dissolved in acetonitrile, at room temperature for about 1 to 18 hours. This solution was concentrated and the product purified by HPLC.

Where appropriate, TSEA adducts are prepared by reacting a single thiol containing peptide (at concentrations of 10-100 mg / ml peptide in DMF buffered to pH 7 with N-methylmorpholine or N-ethylmorpholine, or 5-50 mg / ml peptide in 50 mM sodium phosphate) , pH 7-8, optionally containing 0.5 mM EDTA, or DMF, THF or acetonitrile) with 0.33 molar equivalents of TMEA (tris (2-maleimidoethyl) amine) previously dissolved in acetonitrile or DMF with or without 1 molar equivalent

4

4 4 • ·· • 44 fl «

triethanolamine at room temperature for 1 to 18 hours. These reaction mixtures containing the adducts are concentrated and the adducts are then purified using HPLC.

Where appropriate (BAM) (N ¹ , N ⁴ -bis (2-mercapto-2-methylpropyl) -1,4,10-triazadecane) is condensed with the peptide by first activating the peptide carboxylate with a diisopropylcarbodiimide / N-hydroxysuccinimide or HBTU mixture / HOBt in DMF, NMP or methylene chloride. This is followed by condensation in the presence of diisopropylethylamine. After condensation, the protection of the conjugates is removed as described above.

Where appropriate, (BAT) (N ⁶ , N ⁹ -bis (2-mercapto-2-methylpropyl) -6,9-diazanonanoic acid) is incorporated into peptides as protected amino acid derivatives such as (Na (Fmoc-Ns) (N-Boc) -S, S'-bistrityl-BAT) lysine (prepared from Na (Fmoc) -lysine and Ns (N-Boc) -S, S'-bistrityl-BAT) as described in Example 2 co-owned and co-pending U.S. patent application Ser. No. 08/044 825, which is incorporated herein by reference) or as (N (Fmoc) -S, S'-bistrityl-BAT) cysteine (prepared as described above in Example 1F co-owned and accompanying US Patent Application No. 08 / 814,424, which is incorporated herein by reference) during peptide synthesis and then deprotection after cleavage of the complete peptide from the synthetic resin.

Where appropriate, BAT-BS (N- {2- (N ', N'-bis (2-succinimidoethyl) aminoethyl)} - N ⁶ , N ⁹ -bis- (2-methyl-2-mercaptopropyl)) is prepared. -6,9-diazanonanamide adducts by reacting a single thiol-containing peptide (at concentrations of 2-50 mg / ml peptide in DMF buffered to pH 7 with N-methylmorpholine or N-ethylmorpholine or in 50 mM sodium phosphate (pH 7-8), optionally containing 0.5 mM EDTA, or DMF, THF or acetonitrile) with 0.5 molar equivalents of BAT-BM (N- {2- (N ', N'-bis (2-maleimidoethyl) aminoethyl)} - N ⁹ - ( tert-butoxycarbonyl) -N ⁶ , N ⁹ -bis (2-methyl-2-triphenylmethylthiopropyl) -6,9-diazanonanamide) previously dissolved in acetonitrile or THF at room temperature for about 1 to 18 hours. This solution was then evaporated to dryness and the protection of the (BAT-BS) -peptide conjugates was removed by treatment with 10 ml of TFA and 0.2 ml of triethylsilane for 1 hour. The solution is concentrated, the product adducts are precipitated with ether and then purified by HPLC.

· »···· φ φ φ φ φ φ

Where appropriate, peptide precursors are cyclized (between amino and carboxyl termini) by reaction of the protected side chain, the N-terminal free amine and the C-terminal free acid with diphenylphosphoryl azide.

The Sasrin ™ resin-bound peptide is cleaved with a solution of 1% (w / w) TFA in dichloromethane. A protected peptide is obtained. Where appropriate, protected peptide precursors are cyclized between the amino and carboxyl termini by reaction of the protected side chain, N-terminal free amine, and free acid at the carboxyl terminus using diphenylphosphoryl azide.

The products bound to the HMP or Rink amide resin are routinely cleaved and the protected cyclized peptides are removed using a solution containing trifluoroacetic acid (TFA) or TFA and methylene chloride, optionally containing water, thioanisole, ethanedithiol and triethylsilane or triisopropylsilane in ratios of 100: 5: 5: 2.5: 2 at room temperature for 0.5 to 3 hours. Where appropriate, the products are re-S-tritylated in triphenol-methanol / TFA and the N-Boc groups are reintroduced into the peptide using (Boc) ₂ O.

Where appropriate, thiol functions in the peptide or peptidometric sequence designed for further treatment with the prosthetic group were protected using a compound such as S-tert. butyl (to form mixed tert-butyl disulfides) or p-methoxybenzyl. S-tert-butyl groups are removed by treatment with a solution of dithiothreitol or mercaptoethanol, wherein the p-methoxybenzyl groups are removed using boron trifluoride etherate in trifluoroacetic acid in the presence of a free radical scavenger such as m-cresol. Prosthetic peptides containing radioactive metal-binding groups are prepared by SPPS terminated with an N-terminal 2-haloacetyl group. The prosthetic group is removed from the resin and any thiol groups are protected, for example by a trityl group. The halo-acylated sequence is then condensed with a thiol-containing peptide under substantially the same conditions as described above for 99 99

9 9 9 9 9 9

9

9,999,999

9 9 9

9,999 99 9 9 • · «

9 9

9 9

9 9 9

99 preparation of cyclic thioethers. Removal of the remaining protecting groups is then achieved using the methods described herein to give the final product.

The crude peptides were purified by preparative high pressure liquid chromatography (HPLC) using a Waters-Pak C18 column and gradient elution with 0.1% trifluoroacetic acid (TFA) in acetonitrile-treated water. The acetonitrile was evaporated from the eluted fractions and lyophilized. The identity of each product was confirmed by FAB-MS or ES (ES-MS) mass spectrometry.

CT receptor binding peptides, synthesized derivatives and analogs obtained herein, as well as products of these syntheses identified by ES-MS or FAB-MS, are listed in Table I below.

Table I peptide peptide ReO complex

CH? CO.SNLST.Hhc.VLGKLSC (BAT) ELHKLQTYPRT

NTGSGTP.amid 3679 3877

2. CH ^ CO.SNLST.Hhc.VLGKLSCELHKLOTYPRTNTGS

GTP ( _s- K) GC.amide 3687 * 3886

3. CH? CO.SNLST.Hhc.VLGKLSCELHKLQTYPRTNTGS.

GTP.amid 3373 * NA

4. CH _? CO.SNLST.Hhc.VLGKLSC (CH, CO.GGCK.amid)) ·

ELHKLQTYPRTNTGSGTP.amid 3873 * 4078

5. CH ₇ C0.SNLST.Hhc.VLGKLSC (CH _? C0 · (B-Dap) KCK).

amide) ELHKLQTYPRTNTGSGTP.amid 3777 * 3975

7. CH, C0.SNLST.Hhc.VLGKLSC (CH9C0.fe-K) ·

GCE.amid)) ELHKLQTYPRTNTGSGTP.amid 3849 * 4047

7. CH9CO.SNLST.Hcv.VLGKLSCELHKLQTYPRTNTGS.

·· φ φ φ φ φ »» · · · ·

ΦΦ · · ·

ΦΦ · · · · Φ Φ Φ Φ Φ

ΦΦ ΦΦ

GTP.amid 3360 *

8. CH _? CO.SNLST.Hcv.VLGKLSC (CH, CO.GGCK.amide).

ELHKLQTYPRTNTGSGTP.amid 3762 *

9. CH? CO.SNLST.Hcv.VLGKLSC (CH? CO. (B-Dap) KCK ·, amide)) ELHKLQTYPRTNTGS.GTP.amid 3862 *

10. CH _? CO.SNLST.Hcv.VLGKLSC · CH · CO.fe-K ·

GCE.amid) ELHKLQTYPRTNTGSGTP.amid 3835

11- CH, CO.SNLST.Cvs.VLGKLSCELHKLQTYPRTNTGS.

.GTP.amid 3346 *

12. CH _? CO.SNLST.Cys.VLGKLSC (CH, CO.GGCCamid).

ELHKLQTYPRTNTGSGTP.amid 3748 *

13. CHyCO.SNLST.Cvs.VLGKLSCfCHoCOTB-DapY

KCK.amid) LHKLQTYPRTNTGSGTP.amid 3848 *

14. CHpCO.SNLST.Cvs.VLGKLSCfCHoCOTs-K).

GCE.amid) ELHKLQTYPRTNTGSGTP.amid 3820

15. SNLST.Asu.VLGKLSCELHKLQTYPRTNTGSGTP.

.amid 3356

16. SNLST.Asu.VLGKLSCfCH, CO. (B-Dap) KCK.amid)) ELHKLQTYPRTNTGSGTP.amid 3858

17. SNLST.Asu.VLGKLSCELHKLQTYPRTDVGAGTP.

.amid 3338

18. SNLST.Acu.VLGKLSC (CH, CO. (B-Dap) KCK).

, amide) ELHKLQTYPRTNTGSGTP.amid 3841

ON

3962

4063

4033

ON

3947

4048

4019

ON

4057

ON

4040 * means M ⁺ as determined by mass spectrometry (ES) For all other peptides, M ^{+ was} determined by FAB MS.

9 9 9 9 9 999 999 999

9 9 9

999 99 99 99

Example 3

General method of radioactive labeling with technetium-99m

0.1 mg of the peptides prepared as in Example 2 are dissolved in 0.1 ml or 0.2 ml of water or 0.9% (w / w) saline. Technetium-99m Gluceptate was prepared by filling a Glucoscan vial (E.I. Du Pont de Nemours, Inc., Wilmington, De.) With 0.25 ml of technetium-99m sodium pertechnetate containing up to 200 mCi and allowed to stand at room temperature for 15 minutes. 25 μΙ of technetium-99m gluceptate are then added to the peptides and the reaction is allowed to proceed at room temperature for 15 to 60 minutes or at 100 ° C for 10 to 30 minutes. It is then filtered through a 0.2 µm filter.

The purity of the technetium-99m labeled peptides was determined by reverse phase HPLC under the following conditions: a Waters-Pak C-18, 5µηη, 3.9mm x 150mm analytical column was loaded with each radiolabeled peptide and the peptides eluted with solvent at a flow rate of 1 ml / min (Δ-Pak). The gradient elution is carried out with a gradient of 20 to 50% (w / w) solvent B / solvent A (solvent A means 0.1% CF ₃ COOH in water and solvent B means 0.1% CF ₃ COOH in 90/10 CH ₃ CN / H ₂ O) for 20 minutes, followed by a 100% B / A mixture for 3 minutes.

Radioactive components are detected using an in-line radiometric detector connected to an integrating recorder. Under these conditions, technetium-99m gluconate and technetium-99m sodium pertechnetate elute between 1 and 4 minutes, whereas the technetium-99m-labeled peptides elute for a much longer time. Peptides were detected by in-line spectrophotometric detection at 220 nm.

Non-radioactive rhenium complexes were prepared by co-dissolving each peptide reagent of the invention with one molar equivalent of tetrabutylammonium oxotetrabromorate (+5), prepared according to Cotton et al. (Jnorg. Chem. 1996, 5, 9-16) in dimethylformamide or in a mixture.

acetonitrile / water and stirring for 0.5 to 5 days. The rhenium complexes were isolated by reverse phase HPLC as described above for the technetium-99m-labeled peptides and were characterized by FABMS or ESMS. Non-radioactive peptides are detected as peptides by in-line spectrophotometric detection at 220 nm.

Radioactive rhenium complexes, using, for example, Re-186 or Re-188, are prepared from the corresponding perhenate salts using the same procedure as for technetium-99m labeling or by adding a reducing agent to the peptide and rhenistan solution, or optionally using a ligand transfer agent such as citrate, and incubating the reaction at a temperature between room temperature and 100 ° C for between 5 and 60 minutes.

The results of HPLC purification of peptides, technetium-99m -peptides, and peptides complexed with ReO are shown in Table II.

Table II peptide peptide ReO complex

1. CH? CO. SNLST. Hhc.VLGKLSCfBAOELHKLQTYPRT.

NTGSGTP.amid 13.7 15.6 2. CH, CO.SNLST.Hhc.VLGKLSCELHKLQTYPRTNTGS. GTP (e-K) GC 15.0 13.5 3. CH, CO.SNLST.Hhc.VLGKLSCELHKLQTYPRTNTGS. GTP.amid 19.2 ON 4. CH7CO.SNLST.Hhc.VLGKLSCfCH9CO.GGCK.amide). ELHKLQTYPRTNTGSGTP.amid 21.2 17.8 5. CH7CO.SNLST.Hhc.VLGKLSCfCH7CO.fB-Dao). KCK.amid) ELHKLQTYPRTNTGSGTP.amid 12.1 12.9 6. CH9CO. SNLST. Hhc.VLGKLSCfCH, CO. ίε-Κ). GCE.amid) ELHKLQTYPRTNTGSGTP.amid 11.9 12.4

7. CH? CO.SNLST.Hcv.VLGKLSCELHKLQTYPRTNTGS.

· To to to to toto toto toto toto toto toto toto toto

GTP.amid 13.1 ON 8. CH, CO.SNLST.Hcv.VLGKLSCCH, CO.GGCK.amide). ELHKLQTYPRTNTGSGTP.amid 12.4 13.5 9. CH, CO.SNLST.Hcv.VLGKLSCCH, CO.IB-DaD1KCK. , amide) ELHKLQTYPRTNTGS.GTP.amid 11.0 11.8 10. CH _? CO.SNLST.Hcv.VLGKLSC (CH, CO.fe-K). GCE.amid) ELHKLQTYPRTNTGSGTP.amid 12.5 13.1 11. CH, CO.SNLST.Cvs.VLGKLSCELHKLQTYPRTNTGS. .GTP.amid 10.7 ON 12. CH9CO.SNLST.Cvs.VLGKLSCiCH7CO.GGCK.a- mid) ELHKLQTYPRTNTGSGTP.amide 9.98 11.1 13. CH ₇ CO.SNLST.Cs.VLGKLSCřCH, CO.ÍB-DaDY KCK.amid) LHKLQTYPRTNTGSGTP.amid 8.23 9.2 14. CH, CO.SNLST.Cvs.VLGKLSCrCH, CO.Is-Kl GCE.amid) ELHKLQTYPRTNTGSGTP.amid 9.98 10.7 15. SNLST.Asu.VLGKLSCELHKLOTYPRTNTGSGTP. .amid 12.7 ON 16. SNLST.Asu.VLGKLSCCH, CO.IB-DaD) KCK.a- mid) ELHKLQTYPRTNTGSGTP.amide 10.7 11.4 17. SNLST.Asu.VLGKLSCELHKLQTYPRTDVGAGTP. .amid 13.2 ON 18. SNLST.Acu.VLGKLSCÍCH, CO. (B-DaD) KCK.- mid) ELHKLQTYPRTNTGSGTP.amide 11.1 11.7

Data are HPLC retention times in minutes.

• «

Example 4

Biological tests

A. Binding Inhibition Assays

Peptide reagents within the scope of this invention or embodiments thereof complexed with ReO were tested using in vitro assays that measure their ability to inhibit specific binding of ¹²⁵ I-CT to the CT receptor using rat brain or breast tumor cell lines and whole cell from the cell line. breast tumor lines as detailed below.

Assays using rat brain brain and T-47D membrane fractions (obtained from the American Type Culture Collection, Rockville, Md., ATCC Accession No. HTB-133) were used to identify analogs with high affinity for the CT receptor by according to Fisher et al. (British J. Cancer 1977, 35, 777-784). In brief, the tissue is chopped into small pieces and homogenized. The membranes are washed several times, analyzed for protein content and used in the binding assay. Mebrane protein was incubated with 0.1 μϋβ -losose CT (Amersham, Cleveland, OH) in the presence or absence of various concentrations of reagents to be tested. Both uncomplexed reagents and reagents complexed with ReO were tested. The 100% specific binding of ¹³⁵ I-CT to the CT receptor was defined as the difference between full ¹³⁵ I-CT binding and non-specific binding of ¹²⁵ I-CT measured in the presence of a saturating receptor concentration (1 μΜ) of excess unlabeled salmon CT (Sigma, St. Louis, Mo .). The concentration at which the test reagents inhibited specific binding of ¹²⁵ I-CT by 50% was defined as IC 50. The results of binding to the CT receptor membrane compositions for each of the reagents tested are shown in Table III. These results show that the peptide reagents within the scope of the invention and the ReO complexes of these reactions are 4 &apos; 4 &apos; 4 &apos; The topical agents bind with high affinity to tumor cells and brain membranes expressing the CT receptor.

Similar experiments were performed with other peptide reagents within the scope of the invention using whole T-47D cells and MCF-7 cells (ATCC No. HTB-22). Cell binding assays were performed essentially according to the method of Findlay et al. (J. Endocrinol. 1990, 130, 321-326). Briefly, the cells were washed in brine and resuspended in Hank's balanced salt solution. One to two million cells were incubated with 0.1 μθΐ of ¹²⁵ L-salmon CT in the absence and in the presence of 1 μΜ of unlabeled salmon CT to determine the value of 100% specific binding. Cells are incubated with 0.1 pCi of ¹²⁵ I-CT in the presence or absence of various concentrations of reagents to be tested in uncomplexed and / or complexed (with ReO) form. IC 50 values were determined as described above. The results for each of the reagents tested are shown in Table IV.

Table III

Replacement of ¹²⁵ I-CT from CTR in T-47D and Rat Brain Membranes CT-Mimetic Peptides T-47D Peptide Brain Structure

IC50 Kj IC50 K

CH _? CO.SNLST.Hhc.VLGKLSCfBAT) ELHKLQ7YPRT.

NTGSGTP.amid 0.53 0.41 2.6 ND

CH _? CO.SNLST.Hhc.VLGKLSCELHKLQTYPRTNTGS.

(E-K) GC.amid 16 12 18 ND

CH? CO.NLST.Hhc.VLGKLSC (BAT) ELHKLQTYPRT.

NTGSGTP.amid (ReO)

CH ^ CO.SNLST.Hhc.VLGKLSCELHKLQTYPRTNTGS.

1.5 1.2 3.0 ND

GTP (e-K) GC.amide (ReO)

22 22 ND

Table IV

Replacement of ¹²⁵ I-CT from T47D (cell and membrane) and MCF-7 cells sequence

IC 50 IC 50 (nM) ^x (nM) ^y (nM) ^z

3. CHgCO.SNLST.Hhc.VLGKLSCELHKLQTYPRT.

NTGSGTP.amid 1.6 nd nd

4. CH _? CO.SNLST.Hhc.VLGKLSC (CH, CO.GGCK.amid)) ELHKLQTYPRTNTGSGTP.amid 2.6 nd

5. CH _? CO.SNLST.Hhc.VLGKLSC _(CH? CO. (Ft-Dady

KCK.amid) ELHKLQTYPRTNTGSGTP.amid.ReO nd 0,42 5,8

6. CH 2 CO.SNLST.Hhc.VLGKLSC (CK> CO. (E-K)).

GCE.amid) .ELHKLQTYPRTNTGSGTP.amid.ReO nd 0.33 3.3 8. CH _? CO.SNLST.Hcv.VLGKLSC (CH, CQ.GGCK.amid) ELHKLQTYPRTNTGSGTP.amid.ReO nd 0.83 4.8

9- CH _? CO.SNLST.Hcv.VLGKLSC (CH, CO. (B-Dap)).

KCKamid) ELHKLQTYPRTNTGS.GTP.amid.ReO nd 0.84 nd 10. CH ^ CO.SNLST.Hcy.VLGKLSCfCHoCQ.fe-KY

GCE.amid) ELHKLQTYPRTNTGSGTP.amid.ReO nd 0.89 nd

12. CH _? CO.SNLST.Cvs.VLGKLSC (CH _? CQ.GGCK.amid) ELHKLQTYPRTNTGSGTP.amid.ReO nd 1.0 5.2 13-CHgCO.SNLST.Cvs.VLGKLSC (CH, CQ. (Α-DaD)).

KCK.amid) LHKLQTYPRTNTGSGTP.amid.ReO nd 2.9 nd 14. CH? CO.SNLST.C vs. VGGKLSC (CH> CQ.fe-K).

GCE.amid) ELHKLQTYPRTNTGSGTP.amid.ReO nd 2,6 1,9 16. SNLST.Asu.VLGKLSC (CH, CQ. (B-Dap) KCK.amid) ELHKLQTYPRTNTGSGTP.amid.ReO nd 2,7 8,0 • · · · · · · · · • • • • • • • • • • • «•

18. SNLST.Acu.VLGKLSC (CH, CO. (B-Dap) KCK.amid) ELHKLQTYPRTNTGSGTP.amid.ReO nd 0.55 7.0

CSNLSTCVLGKLSQELHKLQIYPRTNTGSGTP-NH, (salmon calcitonin) nd nd 12.3 nd = not done while x T-47D cell merrtranes y whole T-47D cells from whole MCF-7 cell

These results indicate that the peptide reagents within the scope of this invention and the ReO complexes of these peptides were capable of specifically binding to whole cell CT receptors and that the reagents were potent inhibitors of CT binding in two different CT receptor expressing breast tumor cell lines.

B. Expression of CT receptor in breast cancer cell lines

Binding of ¹² I-CT to whole cells was used to evaluate the density of the CT receptor in seven different human breast tumor cell lines. The local cell density was determined for MCF-7 cells in the presence of various concentrations of CT to achieve receptor saturation. The data was then linearized according to the method of Scatchard et al. (/ VY Acad. Sci. 1949, 51, 600-672) to evaluate receptor density. Briefly, the saturation curves were linearized, the Kd value was calculated as -1 / slope, and the B _max value was equal to the intersection of the x-axis curve. Other cell lines (each obtained from ATTC, Rockville, Md.) Were compared to MCF-7 cells at a single concentration of ¹²⁵ I-CT to assess their CT receptor density. The results are summarized in Table V.

Table V

cell line CT receptors / cells.10 · ³ DU4475 21.5 ZR75 39.5 MDA-MB-157 51.0 MCF-7 1,000 Bt474 166 HS78T 0 T-47D 360

These data show that 86% (6 out of 7) of the breast cancer cell lines tested were positive for CT receptors. Thus, the target receptor for the reagents of the invention is present in most of the breast cancer cell lines tested. Recently, it has been discovered that the target receptor for the reagents of the invention is overexpressed in primary breast cancers (Gillespie et al., Int. J. Cancer 1997, 73, 812-815). These results indicate that the CT peptides of the invention are useful for preparing useful site-specific reagents for diagnosing and treating tumors in humans.

C. In vivo assay

Breast cancer cell lines were tested for receptor expression. The best cell lines were selected for implantation of the graft in immune deficient mice. These tumor models were used to evaluate the tumor target potential in vivo of the high affinity peptides identified in the above in vivo assays.

CT receptor-expressing breast cancer cells (T47D and MCF-7) were implanted in immune-deficient mice or Sprague-Dawley rats and tumors were allowed to grow. To test new Tc-CT peptides, approximately 0.025 mCi at approximately 6 mCi / 10 nmoles of peptides were injected into tumor-bearing mice or rats. Animals were cervically shifted and statically imaged for 5 minutes with a γ-camera. The biological distribution of Tc-CT peptides was then determined by evaluation of blood, tumor, target organs and muscles by γ-counter along with standard injected dose fractions. The time points for biodistribution were selected to represent the early, middle and late phase of Tc-CT analog disappearance. Biological distribution studies have shown that optimum signal to noise ratios are within 90 minutes of injection. To assess the tumor-displaying potential of selected analogues, the ratio of radioactivity in blood to various tissues was compared to that in the tumor. Representative biodegradation data after 90 minutes are shown in Table VI below.

• I · • · 1 ··· · · «

Table VI

biological distribution tumor blood lungs Gl liver kidney. PK (min)

ΡθΡ- tid kind (% ID / g) ki rev srv. (% ID g) (% id) % (% (% ID) ID) ID) £ 1 / 2a 8 mice ^b 0.42 2.8 2.8 0.16 1.23 / g 8 rat 0.026 0.1 / g 4.8 3.7 45.6 1.6? 20? 2 mice ^and 1.6 3,3 10 0.24 5,4 5,518 4 rat 0.027 0.53 2.0 4.0 35 1.4 19 Dec 5 rat 0.048 0.56 4.5 5.8 31 1.6 17 6 rat 0.044 0.10 1,82,5 32,2 1.6 21 6 rabbit 0.012 0.20 5.3 2,314.1 6 mice ^c 2,0 9.9 25 0.23 2.5 / g 3.1 25.9 6 mice ⁰ 3.9 4,2 2,5 0.94 6.5 / g 2,7 53 6 mice ^{* 3} 1.5 1,6 2,1 0.91 2.3 / g 1,5 27

The numbering of peptides corresponds to the numbering in Tables I to IV.

³ T-47D tumor in nude mice ^b MCF-7 breast cancer graft in nude mice ⁰ various means

It is to be understood that the foregoing description includes some specific embodiments of the invention and that all modifications or alternatives to them are equivalent within the spirit and scope of the invention as set forth in the appended claims.

• *

and:

SEQUENCE LIST (1) General information:

(i) Applicants: Dean Richard T.

Bush Larry R.

Pearson Daniel A.

Lister-James John (ii) Title of the Invention: A synthetic compound that binds a calcitonin receptor, a reagent, a scintigraphic imaging agent, a composition and a radiotherapeutic agent containing it, a complex and assembly for preparing a radioactive preparation, (iii) Number of sequences: 11 (iv) Mailing address:

(A) Addressee: Diatide, Inc.

(B) Street: 9 Δ Drive (C) City: Londonderry (D) State: Nh.

(E) country: USA (F) postal code: 03053 (v) Computer read form:

(A) media type: floppy disk (B) computer: IBM PC compatible (C) operating system: PC-DOS / MS-DOS (D) computer program (software): Patentln Release # 1.0, version # 1.30 (vi) about the current application:

(A) Application number:

(B) filing date:

(C) Classification:

(vii) Previous Application Details:

(A) Application Number: USA 08/847 007 (B) Filing Date: May 1, 1997 (viii) Agent Information:

(A) Name: McDaniels Patricia A.

(B) Registration number: 33 194 (C) Reference / File number: DITl 125.1 PCT (ix)

(A) telephone: (603) 437-8970 (C) fax: (603) 437-8977 (2) Sequence information id. no. 1:

(i) Sequence characteristics:

(A) length: 32 amino acids (B) type: amino acid (C) fiber type:

(D) topology: both (ii) Molecule type: peptide (vi) Original source:

(A) organism: calcitonin - human * (ix) (ix) Properties:

(A) name / key: disulfide bond (B) location: 1..7 (ix) Properties:

(A) name / key: locally modified (B) location: 32 (D) additional information: / product = PROLIN 32 / label = amide (xi) sequence description: sequence id. no. 1:

Cys Gly Asn द्वारा किया Leu Ser Thr Cys Gly Thr Tyr Thr Gin 1 5 10

Asp Phe Asn Lys Phe His Thr Phe

Val Gly Ala Pro 30 (2) Sequence Information id. No. 2:

(i) Sequence characteristics:

(A) length: 32 amino acids (B) type: amino acid (C) fiber type:

(D) topology: both (ii) Molecule type: peptide •• 4444 Β 4 4 4 4 · · * 44 4 4 4 4 · · 4 4 44 · 4

4 44 · 4 · 444444 • 444 44 44 • 44 444 444 44 44 (vi)

(A) organism: calcitonin - salmon (ix)

(A) name / key: disulfide bond (B) location: 1..7 (ix) Properties:

(A) name / key: locally modified (B) location: 32 (D) additional information: / product = PROLIN 31 / label = amide (xi) sequence description: sequence id. No. 2:

Cys Ser Asn Leu Ser Thr Cys Val Leu

Glu Leu His Lys Gin Thr Tyr Pro Arg Thr Asn Thr Gly 15 20 25

Ser Gly Thr Pro

9 9 9 9 9

9 9

999 99 99 (2) Sequence information id. No. 3:

(i) Sequence characteristics:

(A) length: 32 amino acids (B) type: amino acid (C) fiber type:

(D) topology: both (ii) Molecule type: peptide (vi) Original source:

(A) organism: calcitonin - eel (ix)

(A) name / key: disulfide bond (B) location: 1..7 (ix) Properties:

(A) name / key: locally modified (B) location: 32 (D) additional information: / product = PROLIN 3Γ / label = amide • · (xi) sequence description: sequence id. No. 3:

Cys Ser Asn Leu Ser Thr Cys Val Leu

Glu Leu His Lys Gin Thr Tyr Pro Arg Thr Asp Val Gly 15 20 25

Ala Gly Thr Pro 30 (2) Sequence Information id. 4:

(i) Sequence characteristics:

(A) length: 31 amino acids (B) type: amino acid (C) fiber type:

(D) topology: both (ii) Molecule type: peptide (ix) Properties:

(A) name / key: region (B) location: 1..6 (D) additional information: / product = S1 is acetylated and X 6 stands for Hhc / label = cyclized / note = The peptide is cyclized by a thioether bond between the N-terminal acetyl group of lilac 1 and homocysteine 6 thiol.

I * ··· (ix) Features:

(A) name / key: locally modified (B) location: 13 (D) additional information: / product = C 13 / label = BAT / note = “The thiol group of cysteine 13 is attached to the bisamino-bisthiol chelator (ix) Properties:

(A) name / key: locally modified (B) location: 31 (D) additional information: / product = PROLIN 31 / label = amide (xi) sequence description: sequence id. 4:

Ser Asn Leu Th Th a Le ly ly ly ly ly ly C C C

Leu His Lys Leu Gin Thr Tyr Pro Thr Asn Thr Gly Ser 15 20 25

Gly Thr Pro (2) Sequence information id. No 5:

(i) Sequence characteristics:

(A) length: 31 amino acids (B) type: amino acid (C) fiber type:

(D) topology: both (i i) Molecule type: peptide (ix) Properties:

(A) name / key: region (B) location: 1..6 (D) additional information: / product = S1 is acetylated and X6 stands for Hhc / label = cyclized / note = Peptide is cyclized by thioether bond between N-terminal acetyl with serine 1 and homohomocysteine 6 thiol (ix)

(A) name / key: locally modified (B) location: 31 (D) additional information: / product = PROLIN 31 / label = amide • * 909 ·

(xi) sequence description: sequence id. No 5:

Ser Asn Leu Th Th a Le ly ly ly ly ly ly C C C

Leu His Lys Leu Gin Thr Tyr Pro Thr Asn Thr Gly Ser 15 20 25

Gly Thr Pro 30 (2) Sequence Information id. No. 6:

(i) Sequence characteristics:

(A) length: 31 amino acids (B) type: amino acid (C) fiber type:

(D) topology: both (ii) Type of molecule: peptide (ix) Properties:

(A) name / key: region (B) location: 1..6 (D) additional information: / product = S1 is acetylated and X6 stands for Hcy / label = cyclized / note = Peptide is cyclized by thioether bond between N-terminal acetyl group 1 and thiol homocysteine 6 • φ φ φ φ φ φ • φ φ φ φ φ · · · · φ φ φ φ φ φ

(A) name / key: locally modified (B) location: 31 (D) additional information: / product = PROLIN 31 / label = amide (xi) sequence description: sequence id. No. 6:

Ser Asn Leu Th Th a Le ly ly ly ly ly ly C C C

Leu His Lys Leu Gin Thr Thr Pro Arg Thr Asn Thr Gly Ser

Gly Thr Pro 30 (2) Sequence Information id. No 7:

(i) Sequence characteristics:

(A) length: 31 amino acids (B) type: amino acid (C) fiber type:

(D) topology: both (ii) Molecule type: peptide peptid φ «φ φ φ φ φ φ φ φ φ φ φ φ φ · · · φ φ φ φ φ φ φ φ φ φ · φ φ φ® · * φφ (ix) Features:

(A) name / key: region (B) location: 1..6 (D) additional information: / product = S1 is acetylated / label = cyclized / note = The peptide is cyclized by a thioether bond between the N-terminal acetyl group of lilac 1 and cysteine 6 thiol (ix)

(A) name / key: locally modified (B) location: 31 (D) additional information: / product = PROLIN 31 / label = amide (xi) sequence description: sequence id. No 7:

Ser Asn Leu Th Th ys ys ys ys ys ly ly ly ly

Leu His Lys Leu Gin Thr Tyr Pro Thr Asn Thr Gly Ser 15 20 25

Gly Thr Pro # · • ly ly ly ly ly ly ly ly ly ly ly ly ly ly ly ly ly ly ly ly <(2) Sequence information id. No. 8:

(i) Sequence characteristics:

(A) length: 31 amino acids (B) type: amino acid (C) fiber type:

(D) topology: both (ii) Type of molecule: peptide (ix) Properties:

(A) name / key: region (B) location: 1..6 (D) additional information: / product = X6 means Asu / label = cyclized / note = The peptide is cyclized by a bond between serine 1 and 2-aminocork 6 ( (ix) Features:

(A) name / key: locally modified (B) location: 31 (D) additional information: / product = PROLIN 31 / label = amide

(xi) sequence description: sequence id. No. 8:

Ser Asn Leu Th Th a Le ly ly ly ly C C C C

Leu His Lys Leu Gin Thr Tyr Pro Thr Asn Thr Gly Ser 15 20 25

Gly Thr Pro 30 (2) Sequence Information id. No. 9:

(i) Sequence characteristics:

(A) length: 31 amino acids (B) type: amino acid (C) fiber type:

(D) topology: both (ii) Type of molecule: peptide (ix) Properties:

(A) name / key: region (B) location: 1..6 (D) additional information: / product = S1 is acetylated and X6 stands for Hhc / label = cyclized / note = Peptide is cyclized by thioether bond between N-terminal acetyl with serine 1 and homohomocysteine 6 thiol (ix)

(A) name / key: locally modified (B) location: 31 (D) additional information: / product = PROLIN 31 / label = amide (xi) sequence description: sequence id. No. 9

Ser Asn Leu Th Th a Le ly ly ly ly C C C C

Leu His Lys Leu Gin Thr Thr Pro Arg Thr Asn Thr Gly Ser

Gly Thr Pro 30 (2) Sequence Information id. No. 10:

(i) Sequence characteristics:

(A) length: 6 amino acids (B) type: amino acid (C) fiber type:

(D) Topology: Circular (ii) Molecule Type: Peptide (ix) Properties:

(A) name / key: region (B) location: 1..6 (D) additional information: / product = S1 is acetylated and X6 stands for C, Hcy or Hhc / label = cyclized / note = The peptide is cyclized by a thioether bond between N-terminal acetyl group 1 and thiol X6 (xi) sequence description: sequence id. No. 10

Ser Asn Leu

5 (2) Sequence information id. No. 11:

(i) Sequence characteristics:

(A) length: 6 amino acids (B) type: amino acid (C) fiber type:

(D) topology: circular (ii) Molecule type: peptide (ix) Properties:

(A) name / key: area (B) location: 1..6 (D) additional information: / product = X1 means A, G or S, X6 means C, Hcy or Hhc / label = cyclized / note = Peptide is cyclized by a thioether bond between N-terminal methylene and thiol X6 (xi) sequence description: sequence id. No. 11

Xaa Asn Leu Ser Thr Xaa

Claims (36)

A synthetic calcitonin receptor binding compound having a molecular weight of less than 10,000 and is covalently attached to a radioactive metal chelator to form a reagent characterized in that it has the same or greater binding affinity to the calcitonin receptor than the radioactive binding affinity iodine-labeled natural calcitonin at said receptor. 2. A synthetic calcitonin receptor binding compound having a molecular weight of less than 10,000 and covalently bound to a radioactive metal chelator to form a reagent characterized in that it has a calcitonin receptor binding affinity of not less than one tenth of the radioactive binding affinity iodine-labeled natural calcitonin at said receptor. 3. The reagent of any one of claims 1 or 2, wherein the chelator is selected from the group consisting of: (a) compounds of formula C (pgp) ^s - (aa) -C (pgp) ^s wherein (pgp) ^s is hydrogen or a thiol protecting group and (aa) is any primary α- or β-amino acid, b) a chelator containing a group containing a single thiol of the formula A-CZ (B) - {C (R ¹ R ² )} n X wherein A is hydrogen, HOOC, H ₂ NOC, (amino acid or peptide) -NHOC, (amino acid or peptide) -OCO or R ⁴ , B is hydrogen, SH, -NHR ³ , -N (R ⁴ -amino acid or peptide) or R ⁴ , X represents a hydrogen atom, a SH, -NHR ³ , -NR ³ (amino acid or peptide) or R ^{4 group} ; Z is hydrogen or R ⁴ , R ¹ , R ² , R ³ and R ⁴ independently represent a hydrogen atom or a lower straight or branched chain or a cyclic alkyl, n represents a number of 0,1 or 2, (amino acid) means any primary α- or β-amino acid not containing group, (peptide) means a peptide of 2 to 10 amino acids, and when B is -NHR ³ or -N (R ³ ) - (amino acid or peptide), X is SH and n is 1 or 2, when X is -NHR ³ or -NfR (amino acid or peptide), B is SH and n is 1 or 2 when B is hydrogen or R ⁴ , A is HOOC, H ₂ NOC, (amino acid or peptide) -NHOC or (amino acid or peptide) -OOC, X is SH and n is 0 or 1, if A is hydrogen or R ⁴ , then if B is SH, X is -NHR ³ or -NR ³ (amino acid or peptide), and if X is SH, B is -NHR ³ or -NR ³ (amino acid or peptide) and n is 1 or 2 when X is hydrogen or R ⁴ , A is HOOC, H ₂ NOC, (amino acid or peptide) -NHOC or (amino acid or peptide) -OOC and B is SH if Z is methyl X is methyl, A is HOOC, H ₂ NOC, (ami acid or peptide) -NHOC or (amino acid or peptide) -OOC, B is SH and n is 0 if B is SH, X is not SH and if X is SH and B is not SH, and wherein the thiol group exists in a reduced form, ·· ·· »· 9 · • · · 99 · 999 · · · · · · · · · · c) compounds of the general formula NH N-A-CO-peptide II (CR ₁ ) _m (CR 1) _P S- (pgp) ^with S- (pgp) ^s wherein each R independently represents a hydrogen atom, methyl or ethyl, each (pgp) ^s independently represents a thiol protecting group or a hydrogen atom, m, n and p independently represent a number 2 or 3, A represents a linear or cyclic lower alkyl, aryl, heterocyclyl, a combination thereof or a substituted derivative thereof, d) compounds of the general formula NH | N-A-CH (V) NHR ' (CRX (CK) _P 1 SH 1 SH wherein each R is independently hydrogen, methyl or ethyl, m, n, and p are independently 2 or 3, A represents a linear or cyclic lower alkyl, aryl, heterocyclyl, a combination thereof or a substituted derivative thereof, V represents a hydrogen atom or a -CO-peptide group, R 'represents a hydrogen atom or a peptide, and wherein if V represents a hydrogen atom, R' represents a peptide and if R 'is hydrogen, V is -CO-peptide, e) compounds of the formula wherein m, n and p independently represent 0 or 1, each R 'independently represents hydrogen, lower alkyl, hydroxyalkyl (2-4 carbon atoms) or alkoxyalkyl (2-4 carbon atoms), each R' independently is hydrogen or R, wherein R is substituted or unsubstituted lower alkyl or phenyl not containing a thiol group, one of R or R 'is L, wherein when R' is L, -NR ' ₂ is amine, and L is a divalent group that binds a chelator to a compound, f) compounds of the general formula R R R R ·· «» φ φ Β Φ Φ ΦΦΦ <1 wherein m, η and ρ independently represent 0 or 1, each R 'independently represents a hydrogen atom, a lower alkyl, a hydroxyalkyl (C2-C4) or an alkoxyalkyl (C2-C4), each R' independently is hydrogen or R, wherein R is substituted or unsubstituted lower alkyl or phenyl not containing a thiol group, one of R or R 'is L, wherein when R' is L, -NR ' ₂ is amine, and L is a divalent group that binds a chelator to a compound, g) compounds of the formula (HOOCCH ₂ ) ₂ N (CR ₂ ) (CR ₂ ) N (CH ₂ COOH) (CR ₂ ) (CR ₂ ) N (CH ₂ COOH) wherein R independently represents a hydrogen atom, 1-4 carbon atoms or aryl and one R is covalently attached to a divalent linker, h) compounds of formula (HOOCCH ₂ ) ₂ N (CR ₂ ) (CR ₂ ) N (CH ₂ COOH), wherein R is independently hydrogen, C 1 -C 4 alkyl or aryl and one R is covalently bonded to bivalent linker, (i) 1,4,7,10-tetraazododecantetraacetic acid; (j) compounds of the formula wherein n is 2 or 3 and each R is independently hydrogen, C 1 -C 4 alkyl or aryl and one R is covalently bound to a calcitonin receptor binding compound, and k) desferrioxamine. The reagent of claim 3, wherein the chelator is selected from the group consisting of compounds of the following formulas - (amino acid) ^1- (amino acid) ² - {A-CZ (B) - {C (R ¹ R ²⁾ )} nX} - {A-CZ (B) - {C (R ¹ R ² )} _n -X} - (amino acid) ^1- (amino acid) ² , - (primary α, β- or B, γ-diamino acid) - (amino acid) ^1- {A-CZ (B) - {CfR ^^ - XJ-a - {A-CZ (B) - {C (R ¹ R ² )} _n -X} - (amino acid) ^1- (direct α, β- or α, γ-diamino acid), each (amino acid) ¹ and (amino acid) ² independently means any naturally occurring, modified, substituted or altered α- or β-amino acid that does not contain a thiol group. 5. The reagent of claim 4, wherein the chelator is selected from the group consisting of compounds of the following formulas (amino acid) ^1- (amino acid) ^2- cysteine, (amino acid) ^1- (amino acid) ^2- isocysteine, (amino acid) ^1- (amino acid) ² -homocysteine, (amino acid) ^1- (amino acid) ² -penicillamine, (amino acid) ^1- (amino acid) ² -2-mercaptoethylamine, (amino acid) ^1- (amino acid) ² -2-mercaptopropylamine, (amino acid) ^1- (amino acid) ² -2-mercapato-2-methylpropylamine-α (amino acid) ^1- (amino acid) ² -3-mercaptopropylamine, wherein the carboxyl end or side chain of said chelator is covalently linked to compound. 9 9 9 999 < The reagent of claim 5, wherein (amino acid) ¹ is selected from the group consisting of α, β-diamino acid having free α-amine and β, γ-diamino acid having free β-amine. The reagent of claim 4, wherein the chelator is selected from the group consisting of compounds of the following formulas -cysteine- (amino acid) - (a, B- or β, γ-diamino acid), -isocysteine- (amino acid) - (a, B- or β, γ-diamino acid), -homocysteine- (amino acid) - (a, B- or β, γ-diamino acid), -penicillamine- (amino acid) - (a, B- or β, γ- diamino acid), 2-mercaptoacetic acid (amino acid) - (a, B- or β, γ-diamino acid), 2- or 3-mercaptoacetic acid- (amino acid) - (a, ft- or β, γ-diamino acid) and 2-mercapto-2-methylpropionic acid- (amino acid) - (a, B- or β, γ-diamino acid), wherein the amino terminus or side chain of said chelator is covalently linked to the compound. The reagent of claim 1 or 2, wherein the chelator is selected from the group consisting of compounds of the following formulas -Gly-Gly-Cys-Ala-Gly-Cys- - (s-Lys) -Gly-Cys- (6-Om) -Gly-Cys-, - (γ-Dab) -Gly-Cys- (B-Dap) -Lys-Cys-, - (B-Dap) -Gly-Cys- and -Cys (BAT). Reagent according to claim 3, characterized in that the chelator is a compound of the formula R ⁵ R ⁵ R ' L-Z Ft in nemz R ¹ and R ^{2 are each} independently hydrogen, lower alkyl, hydroxyalkyl (C 2 to 4 carbon atoms) or alkoxyalkyl (C 2 to 4 carbon atoms), R ³ , R ⁴ , R ⁵ and R ^{6 are} independently a hydrogen atom, substituted or unsubstituted lower alkyl or phenyl, which does not contain a thiol group, R ⁷ and R ^{8 are each} independently hydrogen, lower alkyl, lower hydroxyalkyl or lower alkoxyalkyl, L is a bivalent linking group and Z is a peptide. 10. The reagent of claim 3 wherein the chelator is a compound of formula R o n / o Cl MU UU ll X in which R ¹ and R ² independently of one another are hydrogen, lower alkyl, hydroxyalkyl (2 to 4 carbon atoms) or alkoxyalkyl (2 to 4 carbon atoms), φφ φφφφφφφφφφφφφφφφ φ ® • • • • • • • φ φ φ φ φ R ³ , R ⁴ , R ⁵ and R ⁶ independently represent a hydrogen atom, a substituted or unsubstituted lower alkyl or phenyl not containing a thiol group, and one of R ³ , R ⁴ , R ⁵ and R ⁶ represents a group ZL- (CR ₂ ) _n R ⁷ and R ⁸ independently of one another are hydrogen, lower alkyl, lower hydroxyalkyl or lower alkoxyalkyl, L is a divalent linking group, Z is peptide a X is -NH ₂ , -NR ¹ R ^2, or -NR ¹ -Y, wherein Y is an amino acid, an amino acid amide, or a peptide of 2 to 20 amino acids. The reagent of claim 3, wherein the chelator is a compound of the formula wherein: a R ¹ and R ^{2 are each} independently hydrogen, lower alkyl, hydroxyalkyl (C 2 to 4 carbon atoms) or alkoxyalkyl (C 2 to 4 carbon atoms), R ³ , R ⁴ , R ⁵ and R ^{6 are} independently a hydrogen atom, substituted or unsubstituted lower alkyl or phenyl, which does not contain a thiol group, n represents a number from 1 to 6, L is a bivalent linking group and Z is a peptide. 49 4449 • 9 · · 4 99 94 99 9 9 4 9 9 9 444 499 4 4 49 44 The reagent of claim 3 wherein the chelating agent is a compound of the formula Z-L NH in which L is a linking group and Z is a peptide. 13. The reagent of any one of claims 1 or 2, wherein the compound is a peptide. 14. The reagent of claim 13, wherein the peptide has an amino acid sequence CHyCO.SNLST.Hhc.VLGKLSCELHKLQTYPRTNTGSGTP.amide (SEQ ID NO: 9) and the chelator is included in the peptides in the amino acid side chain of the indicated sequence. The reagent of claim 13, wherein the peptide comprises a calcitonin receptor binding domain that is cyclized with a thioether. 16. The reagent of claim 15, wherein the peptide comprises an amino acid sequence. CH 2 CO.SNLSTX (SEQ ID NO: 10), wherein X is selected from the group consisting of a cysteine residue, a homocysteine residue, and a homohomocysteine residue. 17. The reagent of claim 15, wherein the peptide comprises an amino acid sequence. CH _? CO.X ¹ SNLSTX ² (SEQ ID NO 11), wherein X ¹ is selected from the group consisting of alanine residue, glycine residue and serine residue, and X ² is selected from the group consisting of cysteine residue, homocysteine residue, and homohomocysteine residue. 18. A reagent having a formula selected from the following group CH _? CO.SNLST.Hhc.VLGKLSCiBAT) ELHKLQTYPRTNTGSGTP.amid (SEQ ID NO 4), CH _? CO.SNLST.Hhc.VLGKLSCELHKLQTYPRTNTGSGTPfe-K) GC.amid _: CH, CO.SNLST.Hhc.VLGKLSC (CH, CO.GGCK.amid)) ELHKLQTYPRTNTG. .SGTP.amid, CH _? CO.SNLST.Hhc.VLGKLSC (CH, CO. (B-Dap) KCK.amid) ELHKLQTYPRT · .NTGSGTP.amid CH _? CO.SNLST.Hhc.VLGKLSCfCH _? CQ.fe-K) GCE.amid)) ELHKLQTYPRT · .NTGSGTP.amid, CH _? CO.SNLST.Hcy.VLGKLSC (CH, CO.GGCK.amid) ELHKLQIYPRTNTG · .SGTP.amid, CH _? CO.SNLST.Hcv.VLGKLSC (CH, CO. (B-Dap) KCamid)) ELHKLQTYPRT · .NTGS.GTP.amid, CH _? CO.SNLST.Hcv.VLGKLSCfCHoCO. (8-K) GCE.amid) ELHKLQTYPRTNT · .GSGTP.amid, • 4 4444 44 44 4 5 444 444 4 4 44 44 CH _? CO.SNLST.Cs.VLGKLSC (CH _? CQ.GGCKamid) ELHKLQTYPRTNTG · .SGTP.amid CHgCO.SNLST.Cvs.VLGKLSC (CH _? CO. (B-Dap) KCKamid) LHKLQPfPRT. .NTGSGTP.amid, CH _? CO.SNLST.Cvs.VLGKLSC (CH _? CO.fe-K) GCE.amid) ELHKLQIYPRTNTGS · .GTP.amid, SNLSTAS _and VLGKLSC (CH ₂ CO. (B-Dap) KCK.amide)) ELHKLQTYPRTNTGSGTP. amid, SNLSTAC _and VLGKLSC (CH ₂ CO. (B-Dap) KCK.amide) ELHKLQTYPRTNTGSGTP. .amid, CH ₂ CO.SNLST.Hhc.VLGKLSCELHKLQTYPRTNTGSGTP.amid (SEQ ID NO 5), CH ₂ CO.SNLST.Hcy.VLGKLSCELHKLQTYPRTNTGSGTP.amid (SEQ ID NO 6), CH ₂ CO.SNLST.Cs.VLGKLSCELHKLQTYPRTNTGS.GTP.amid (SEQ ID NO 7) and SNLST.Asu.VLGKLSCELHKLQTYPRTNTGSGTP.amid (SEQ ID NO: 8). The reagent of claim 18, having the formula CH ₂ CO.SNLST.Hhc.VLGKLSC (CH, CO. (EK) GCE.amide) ELHKLQTYPRTNT · GSGTP.amide. 20. A reagent comprising (a) at least two synthetic calcitonin receptor binding compounds, each of which is covalently bound to a radioactive metal chelator, and b) a polymeric linker forming a covalent bond, selected from the group consisting of a bond to each compound, a bond to each chelator, and a bond to one compound, and a chelator of another compound, wherein: 9 99 99 98 9 99 9 9 9,999,999 9 9 9 ··· 99 99 ·· · * ·· g> the reagent has a molecular weight of less than 10,000, and if the chelator is chelated to the radioactive metal, the reagent has a calcitonin receptor binding affinity of not less than one tenth of the binding activity of radioactive iodine labeled natural calcitonin to said receptor. A scintigraphic imaging agent comprising a reagent according to any one of claims 1 to 20 and technetium-99m. A composition comprising a reagent according to any one of claims 1 to 20 and a stannous ion. A radiotherapeutic agent comprising a reagent according to any one of claims 1 to 20 and a cytotoxic radioisotope. 24. The radiotherapeutic agent of claim 23, wherein the radioisotope is selected from the group consisting of scandia-47, copper-67, gallium-72, yttria-90, tin-117m, iodine-125. iodine-131, samaria-153, gadolinium-159, dysprosia-165, holmia-166, yterbia-175, lutecia-177, rhenia-186, rhenia-188, astatu-211, vismutu-212 and vismutu-213. A complex comprising forming a reagent according to any one of claims 1 to 20 with technetium-99m, rhenium-186 or rhenium-188 in the presence of a reducing agent. 26. The complex of claim 25 wherein the reducing agent is stannous. 27. A complex comprising forming a reagent label according to any one of claims 1 to 20 with technetium-99m by exchanging a ligand of the complex previously reduced with technetium-99m. • φ 9 99 9 ΦΦ ΦΦ 9 9 · · · 9 9 9 9 ΦΦΦΦ 9 9 9 Φ · · ΦΦΦΦΦΦ ΦΦΦΦ Φ Φ φφ ·· ΦΦΦ ΦΦΦ ΦΦ ΦΦ A complex comprising forming a reagent label according to any one of claims 1 to 20 with rhenium-186 or rhenium-188 by exchange of a ligand of the complex previously reduced by rhenium. A kit for preparing a radiopharmaceutical composition comprising a sealed container containing a predetermined amount of the reagent of any one of claims 1 to 20 and a sufficient amount of a reducing agent to label the reagent with technetium-99m, rhenium-186 or rhenium- 188 An assembly for preparing a radioactive preparation comprising a sealed container containing a predetermined amount of the reagent of any one of claims 1 to 20. A method for labeling a reagent according to any one of claims 1 to 20, comprising the step of reacting the reagent with technetium-99m, rhenium-186 or rhenium-188 in the presence of a reducing agent. 32. The method of claim 30 wherein the reducing agent is stannous. 33. A method for producing a reagent according to any one of claims 1 to 20, characterized in that it is carried out chemically by in vitro synthesis. 34. The process for producing a reagent according to claim 33, wherein the synthesis is solid phase peptide synthesis. 99 99 9 9 999 999 9 9 99 99 ·· ·· ♦ · * · 9 9 · · · · · · · · · · · · · · · · · · · · · · 9 9 9 9 · 99 99 99 9 999 35. A method of producing a reagent according to any one of claims 33 or 34, wherein the chelator is covalently bound to the compound during synthesis. Use of a reagent according to any one of claims 1 to 20 in the manufacture of a medicament for imaging a site in a mammal's body. The use of a reagent according to any one of claims 1 to 20 for the manufacture of a medicament for the treatment of diseases characterized by the presence of calcitonin receptors.