WO2005011738A2

WO2005011738A2 - Polymeric conjugates containing phosphine - based chelating groups

Info

Publication number: WO2005011738A2
Application number: PCT/IT2004/000422
Authority: WO
Inventors: Francesco Veronese; Ulderico Mazzi; Gianfranco Pasut; Roberta Visentin
Original assignee: Universita degli Studi di Padova
Current assignee: Universita degli Studi di Padova
Priority date: 2003-07-31
Filing date: 2004-07-29
Publication date: 2005-02-10
Anticipated expiration: 2006-01-31
Also published as: ITPD20030174A1; WO2005011738A3

Abstract

Reactive polymers can be conjugated, directly or by means of linkers and/or others polymers to chelating agents comprising at least one phosphorous atom in the form of a phosphine or phosphine oxide (or precursors thereof) to form conjugates useful in diagnostic and therapeutic applications. In particular this invention provides conjugates comprising a hydrophilic polymer bound, directly or by means of other moieties, to at least one chelating group able to chelate metal radioisotopes comprising at least one phosphine or one phosphine oxide phosphorous. Such chelating groups can be conjugated to the hydrophilic polymer directly or via one or more linkers and/or one or more additional polymers. The use of additional polymers can provide increased loading of chelating agent. The linkers are preferably selected among alkyl groups or aromatic groups or cleavable peptides or other biodegradable sequences. Additionally, one or more targeting molecules can be linked to the hydrophilic polymer directly or by means of linkers and/or others polymers. Due to their polymeric structure, the conjugates according to the invention have enhanced specificity toward certain tissues, such as tumors, inflamed tissued and the liver. The specificity can be further increased by the additional provision of targeting moieties such as antibodies or sugars. These conjugates can be formulated for remaining in the blood circulation for a period for time suitable for diagnostic and therapeutic applications. Moreover they possess thermodynamic and kinetic stability, keeping the metal chelate intact under physiological conditions. The invention also provides a very simple and efficient method for the labelling of radiopharmaceuticals, which avoids the use of any additional reducing agent. Accordingly, metal ions like technetium or rhenium can be added as pertechnetate or pherrhenate to chelating agents comprising polymer and a phosphine and surprisingly it has been found that such chelating agents can act as reducing agents of the metal and the use of an additional reducing agent is not necessary. This allows the preparation of simple kits comprising a component (a) comprising the polymeric chelating agent and a component (b) comprising the metal ion in its highest oxidation state. These two components can be kept separately and combined together just before use to yield the metal complex without the need of further reducing step and purification.

Description

POLYMERIC CONJUGATES FOR DIAGNOSIS AND THERAPY

DESCRIPTION

TECHNICAL FIELD

This invention relates to novel chelating agents comprising at least one polymer and at least a phosphorous atom in the form of a phosphine or phosphine oxide, to compositions and kits comprising them and to their uses in diagnostic and therapeutic methods.

BACKGROUND ART

Patent Documents:

European Patent 0,659,764 Mazzi et al. United States Patent 5,746,998 Torchilin et al. United States Patent 5,756,069 Torchilin et al. Other Publications :

R. Visentin et al., "Synthesis and Characterization of Rhenium(N)-oxo Complexes withΝ-[Ν-(3- Diphenylphosphinopropionyl) glycyl]cysteine Methyl Ester. X-ray Crystal Structure of {ReO[Ph₂P(CH₂)₂C(O)-Gly-Cys-OMe₅(ENNS)]} ' Inorg. Chem., 42: 950-959 (2003) M. J. Roberts et al., "Chemistry for peptide and protein PEGylation," Adv. Drug Del. Rev., 54: 459-476 (2002)

F. M. Veronese, "Peptide and protein PEGylation: a review of problems and solutions," Biomaterials, 22: 405-417 (2001)

G. Riess, "Micellization of block copolymers," Prog. Polym. Set, 28: 1107-1170 (2003)

N. P. Torchilin, "PEG-based micelles as carriers of contrast agents for different imaging modalities," Adv. Drug Del Rev. , 54: 235-252 (2002)

Chelating agents are often used in diagnostic and in therapeutic applications since they can bind specific metal ions and carry them in specific sites of the body. Some metal ions, for example Gd, ^99mTc, ⁶⁷⁶⁸Ga, ^U1ln, ⁶²Cu, which can be revealed by spectroscopic or scintigraphic methods are used in diagnostic methods, others, for example ⁹⁰Y, ^{186 188}Re, ¹⁵³Sm, ^mLu, ^64/67Cu, can be used in therapeutic applications, like the treatment of tumours. The labelled chelating agents often suffer of some disadvantages, such as the lack of thermodynamic and/or kinetic stability and specificity in localisation. EP0659764 describes chelating agents comprising at least one phosphine. These chelating agents have been used to chelate metals ions like technetium. These complexes which are formed are very stable and can be used as diagnostic agents. These agents, however, as most of the compounds of the prior art suffer from unfavourable pharmacokinetic. Poly(ethylene glycols) (PEGs) and derivatives thereof are having increase interest in chemical, biomedical, and other industrial applications due to their useful properties, such as, amphiphilic behaviour, solubility in aqueous and organic solvents, high purity, low polidispersivety, biological compatibility and since they can be activated for conjugation to other compounds such polymers have been employed for example, as drug carriers, matrices for liquid phase peptide or polynucleotides synthesis and to prepare conjugate with peptide and protein. (See, e.g., Roberts M. J. et al., Adv. Drug Del. Rev., 54: 459-476, 2002; Veronese F. M., Biomaterials, 22: 405-417, 2001). With new discoveries in medical research and development of nanotechnology tools there is a growing demand for new and improved PEG derivatives which can be tailored to meet user requirements.

Inada et al, U.S. Patent No. 4,814,098 disclose a conjugate comprising a magnetic material and a physiologically active substance bound to each other through a poly(ethylene glycol) derivative. Mutter, Tetrahedron Letters, 31, 2839-2842 (1978) describes a procedure to convert the terminal hydroxyl groups of PEG to reactive primary amino groups and the preparation of a number of reagents bound to PEG-NH₂. However, there is no suggestion of a polymer linked to a chelating agent comprising at least one phosphine or one phosphine oxide phosphorous. Harris et al, J. Polymer Science, 22, 341-352 (1984) describe various PEG derivatives including PEG-amine. However, there is no mention of a polymer linked to a chelating agent comprising at least one phosphine or one phosphine oxide phosphorous.

Recently, substances substituted with PEG chains for the treatment and the diagnosis of tumours have been described in PCT/EP91/00992. However, there is no mention of a polymer linked to a chelating agent comprising at least one phosphine or one phosphine oxide phosphorous. Patent No. US5932188 describes poly(alkylene oxide) linked to a chelating agent. However, there is no suggestion to link a polymer to a chelating agents comprising at least one phosphine or one phosphine oxide phosphorous.

European Patent No. 0659764, mentioned above, describes chelating agents comprising at least one phosphine or one phosphine oxide phosphorous. However in EP0659764, there is no suggestion to link a polymer to these chelating agents. Also, the skilled person would not have expected that such a conjugation could change the pharmacokinetic properties and in the meantime maintain the chelating properties necessary for diagnostic and therapeutic applications. It is apparent that there is a need of providing new alternative classes of diagnostic and therapeutic compounds capable to accumulate in certain tissues, and to remain in the blood pool for long periods of time. A further problem concerning the preparation of radiopharmaceutical agents is the development of a suitable labelling procedure capable to afford the desired radiopharmaceuticals simply, possibly i n a o ne s pot r eaction b efore u se. M any radiochemicals are c haracterised b y a s hort half-life. For example, when technetium is used as pertechnetate, it is combined with the chelating agent and must then be reduced by the addition of a suitable reducing agent; the complex comprising the technetium in reduced form must then be purified. This requires several steps, which are expensive and time consuming. Also since technetium, like other radiolabelling metals, has a short half-life, these multistep preparation processes cause a loss of specific radioactivity.

DISCLOSURE OF INVENTION

The present invention provides novel chelating agents that can be efficiently labelled with metal ions to produce radiopharmaceuticals for both diagnostic and therapeutic purposes. The chelating agents according to the invention (also referred here as the "conjugates") comprise a hydrophilic polymer conjugated directly or by means of other moieties to a chelating group able to chelate metal ions comprising at least one phosphine or one phosphine oxide phosphorous. The conjugates have preferably the formula: [BFC_n-Poll]_x-Pol2-[Pol3-BM_m]_y wherein: x is 1 or 2, y is 0 or 1, n = 1-200 and m = 0-100.

BFC is the chelating group, which is able to chelate a metal radioisotope, comprising at least one phosphine or one phosphine oxide phosphorous.

Pol2 is a hydrophilic water-soluble polymer, synthetic or naturally derived. Poll, Pol3 and BM may be present or absent,

Poll, if present, is a polymer or a dendrimeric structure, which bears several side-chain fuctionalizable residues,

Pol3, if present, is a polymer or a dendrimeric structure, which bears several side-chain fuctionalizable residues, BM, if present, is a targeting molecule. and where the different moieties are conjugated, preferably via ester, amide, carbamate, ether, thioether, disulphide or other covalent bonds, directly or by means of one or more linkers, the linkers preferably selected among alkyl groups, such as NH₂-(CH )_n-NH₂ (n = 0-12) or HOOC-

(CH )_m-COOH (m = 0-12), or aromatic groups or cleavable peptides or other biodegradable sequence, such as H-GFLG-OH, H-GLFG-OH.

Preferably the chelating group has the formula: RlR2-P(O)r-(Alk-X-)-(Alk-X-)p-R' where: r is O or 1, p is 1 to 4, each group Alk is independently an alkyl group preferably comprising 1 to 4 carbons, optionally substituted by a one or more groups preferably selected among an oxy group, a hydroxy, an amino group, a carboxy group or a residue of an aminoacid,

X is selected among N, O, S, P , K_\ and R₂ are optionally substituted alkyl, optionally substituted aryl or optionally substituted aryl-alkyl groups such as Z-Ar-(CH₂)_a (a = 0-2 and Z = H, CH₃-, CH₃O-), CH₃-(CH₂)_b- (b = 2-9),

Ethyl, Isopropyl, Isobutyl, tert-Butyl, CH₃-, CH₃-(CH₂)_c-O-(CH₂)_d- (c and d = 0-4);

R' is H or optionally substituted alkyl, or optionally substituted aryl or optionally substituted aryl-alkyl groups, such chelating group being conjugated to the other parts of the conjugate by a bond at any position of the chelating group, and preferably at an amine or carboxylic group or thiol group, or at any of the carbons of the backbone of the chelating group.

Among the preferred chelating groups used in the present invention are those previously reported in the European Patent 0659764 and summarised in the formula I- VI wherein n = 1, 2 and R" is an amino acid residue, R_ls R₂ and R' have the meaning indicated above:

IV VI These chelating groups possess a tetradentate coordination set PN₂X where X = S, N, P which can form so-called "coordination-bonds" to bind a metal ion, such as technetium, rhenium and copper among others. These chelating groups comprising a phosphine phosphorous have reductive properties towards metals such as technetium, rhenium and copper when these are in their upper oxidation state (+7 for technetium and rhenium and +2 for copper, respectively). Alternatively, the chelating group may be a phosphine moiety (monodentate BFC) or a phosphine group conjugated to a single natural or non natural amino acid (bidentate or tridentate BFC) as summarised in the formula VII-XII:

:P-(CH_-)_m-COOH -CCH- H, R2^"

VII VIII

IX X CHR"- (CH-)_n-COOH

XI XII where preferably m and n = 0, 1 and R" = amino acid side chain, Ri, R₂ and R' have the meaning indicated above. These chelating agents may be suitable to bind metal ion moieties such as [Tc(CO)₃]⁺ and [Re(CO)₃]⁺.

Moreover, the phosphorous in polydentate chelating agents may be present as phosphine oxide as summarised in the formula XIII-XVII:

XIII XIV XV

XVI XVII where preferably n = 1 and R" = amino acid side chain, R_ls R₂ and R' have the meaning indicated above.

Among the preferred phosphines or phosphine oxides according to the invention are those disubstitued with liphophilic groups Ri and R₂, such as Z-Ar-(CH₂)_a (a = 0-2 and Z = H, CH₃-,

CH₃O-), CH₃-(CH₂)_b- (b = 2-9); Ethyl, Isopropyl, Isobutyl, tert-Butyl, or alternatively more hydrophilic groups such as CH₃-, CH₃-(CH₂)₀-O-(CH₂)_d- (c and d = 0-4);

Preferred chelating groups are N-[N-(3-Diphenylphosphinopropionyl)glycyl]cysteine (PN₂S) and

N-[N-(3-Diphenylphosphinoxidepropionyl)glycyl]cysteine [(P)ON₂S]):

PN₂S (P)ON₂S

The conjugation between the chelating group and the other moieties of the conjugate can be made at any position of the chelating group; preferably conjugation involves a bond at a carboxylic or amino group present on the chelating group or at one of the carbons of the backbone of the chelating group. The conjugation to Pol2 or to Poll, if present, may be direct or mediated by means of linker.

BM may be present or not, and if present it is a targeting molecule designed for the recognition of tumours, inflammatory sites and infection processes to exploit in diagnosis and therapy. Among the preferred targeting molecules of the invention are peptides, monoclonal antibodies and antibody fragments, biotin, chemotherapeutic drugs and sugars.

Poll, if present, is a polymer or a dendrimeric structure which bears several side-chain residues useful to attach covalently a plurality of BFC (n > 1, e.g., 2 to 200, and preferably 5 to 50), to obtain a polychelating compound. Preferred hydrophilic polifunctional polymers include polyaspartic acid, polyglutamic acid, polyhydroxyethylaspartamide, polyhydroxyethylglutamide, poly(hydroxypropylmetacrylamide) and polylisine. In preferred embodiments, the polifunctional polymer has a molecular weight of 3000 to 9000 dalton. When Poll is a dendrimer, the branching moiety is glutamic acid, β-glutamic acid, amino adipic acid or other amino bicarboxylic or tricarboxylic acids. Preferred lipophilic polifunctional polymers include polyhydroxyl acids such as polylactic acid or polymalic acid. Poll also possesses a terminal functional group, which can be used for the conjugation to Pol2 directly or by means of linker. Among the preferred functional groups are carboxylic, amino or hydroxyl groups. When Poll is directly bound to Pol2, the conjugation can be mediated by ester, amide, carbamate or other co alent bonds.

Pol3, if present, is a dendrimeric structure bearing several residues useful to covalently attach a plurality of BM (m>l, e.g., 2 to 200, and preferably 5 to 50), to obtain a polytargeting compound. The branching moiety is glutamic acid, β-glutamic acid, amino adipic acid or other amino bicarboxylic or tricarboxylic acids. Pol3 also possesses a terminal functional group for the conjugation to Pol2 directly or by means of linker. Among the preferred functional groups are carboxylic and amino groups. When Pol3 is directly bound to Pol2, the conjugation can be mediated by ester, amide, carbamate or other covalent bonds.

Pol2 is a hydrophilic water soluble polymer, synthetic or naturally derived, which possesses one or two terminal functional groups in order to be conjugated to Poll, Pol3, and BM (if they are present) and to at least one BFC. When Pol2 is a monofunctional polymer the only derivatizable group has to be conjugated to the block bearing the BFC moiety. Such conjugation may be directly or by means of linkers. When Pol2 has two terminal groups (bifunctional Pol2) one has to be involved in the conjugation with BFC block, with at least one BFC molecule, and the other one can alternatively be conjugated to hydrophilic block (in this case x = 1, y = 1) or to another lipophilic block (x = 2, y = 0). In any case these conjugations can be carried out directly or by means of linkers. Among the preferred mono and bifunctional Pol2 are poly(ethylene glycol), polyvmylpirrolidone or polyacriloylmorpholine. The polymer can have at least one reactive group suitable for conjugation. For instance the end group of the polymer can be activated according to know procedures (this holds for PEG-COOH, PEG-NH₂) and others (Sabine Herman, et al. J. of Bioactive and Compatible Polymers, Vol. 10 1995). The polymer Pol2 has preferentially an average molecular weight of at least 1000, preferably of at least 4000, more preferably at least 10000, and even more preferably of at least 20000. In preferred embodiments, Pol2 is a polyethylene glycol) (PEG) derivative having an average molecular weight ranging from 1000 to 40000 Da. Some preferred Pol2 are PEG5000 and PEG20000. The different moieties of the conjugates are conjugated, preferably via ester, amide, carbamate or other covalent bonds, directly or by means of one or more linkers, the linkers preferably selected among alkyl groups, such as NH₂-(CH₂)_n-NH₂ (n = 0-12) or .HOOC-(CH₂)_m-COOH (m = 0-12), or aromatic groups or cleavable peptides or other biodegradable sequence, such as H-GFLG-OH, H-GLFG-OH.

Among the preferred conjugates according to the invention are a conjugates, as following reported, wherein x is 1, y is 0, n = 1-200, and having the formula: BFC-Pol2 or, BFC_n-Poll-Pol2

Among more preferred conjugates are a conjugates wherein Pol2 is mPEG 5000 Da or mPEG 20000 Da, n = 1, Poll is absent and BFC is N-[N-(3-Diphenylphosphinoρropionyl) glycyl]cysteine [PN₂S] or N-[N-(3-Diphenylphosphinoxidepropionyl)glycyl]cysteine [(P)ON₂S)], having the following formula:

mPEG₅₀oo-PN₂S mPEG₂oooo-PN₂S

mPEG₅₀oo-(P)ON₂S mPEG₂₀ooo-(P)ON₂S

The compounds according to the invention are suitable to complex metal ions, which can be used in therapeutic and diagnostic applications. Among the metals suitable in diagnostic applications are the ones which can be revealed by scintigraphic and spectroscopic methods (SPECT, PET, MRI, and others methods). Metals used in diagnostic applications are for example Gd, ^99mTc,

67/68 G, a, 11 l Iτn-, 62 C, u. Among the metals used in therapy are radioisotopes such as 90 Y_v, 186/188 Re,

¹⁵³Sm, ¹⁷⁷Lu, ^64/67Cu.

A conjugate according to the invention can be prepared conjugating the chelating group to the other polymeric moieties of the conjugate according to know chemical routes such as activation of a carboxyl group of the chelating group through N-hydroxysuccinimid / N, N' dicyclohexyl- carbodiimid followed by coupling with a polymer comprising a reactive end-group such as an amine or an hydroxy and deprotection of any protecting group.

A typical labelling method for ^99mTc involves transchelation of the metal from its complex with an exchange ligand, such as gluconate, to the compounds according to the invention. ^99mTc- gluconate, is prepared following a reducing procedure of ^99mTcO₄ ^" using Sn²⁺ as reducing agent (B. Johannsen et al, Inorg. Chim. Acta, 210, 209-214, 1993).

In the case of compounds according to the invention comprising a phosphorous atom in the form of phosphine group the labelling method can avoid the use of an external reducing agent. It is assumed that the reducing properties of the phosphine derivatives are enhanced by the special supramolecular arrangement of these compounds, which have amphiphilic character. Surprisingly these phosphine compounds catalyse an intramolecular oxido-reduction in which the phosphine is oxidised to phosphine oxide and acts as a reducing agent in respect to pertechnetate. In a preferred method a solution of metal ion is added to the conjugate in a solid form.

EXAMPLES

Syntheses

Example 1: Preparation of mPEG₅ooo-PN₂S(Trt)

The carboxylic group of PN₂S(Trt) was activated by EDC/NHS and coupled with monomethoxy amino-poly(ethyleneglycol) (mPEG-NH₂) to obtain the desired product: mPEG₅ooo-PN₂S(Trt).

198,23 mg of PN₂S(Trt) (0,3 mmol; MW 660,76Da) dissolved in 10 ml of CH₂C1₂ were cooled at 4°C and 51,80 mg of N-hydroxysuccinimid (NETS) (0,45 mmol; MW 115,19Da) and 172,53 mg of N'-(3-dimethylaminopropyl)-N-ethylcarbodiimid-HCl (EDC) (0,9 mmol; MW 191,71Da) were added. The reaction was maintained under Argon atmosphere and allowed warming at room temperature. After 5 hours at constant stirring 41,8 μl of Et N (0.3 mmol; MW 101,19Da; d²⁰ 0,726) and 1000 mg of mPEG-NH₂ ( 0.2 mmol; MW 5000Da), previously dissolved in 4 ml of CH₂C1₂, were mixed with the activated PN S(Trt). Coupling reaction was let to react for 10 hours under Argon atmosphere. Reaction mixture was filtered and dropped into 150 mL of ethyl alcohol where the product, mPEG₅ooo-PN₂S(Trt), was allowed to precipitate at 0°C and the excess of PN₂S(Trt) remains in solution. The product was dried under vacuum and purified by recrystallisation from Et-OH. The yield was 91%.

Example 2: Preparation of mPEG₂oooo-PN₂S(Trt)

The carboxylic group of PN S(Trt) was activated by EDC/NHS and coupled with monomethoxy amino-poly(ethyleneglycol) (mPEG-NH₂) to obtain the wanted product: mPEG₂oooo-PN₂S(Trt). 49,56 mg of PN₂S(Trt) (0,075 mmol; MW 660,75Da) dissolved in 10 ml of CH₂C1₂ were cooled at 4°C and 12,95 mg of N-hydroxysuccinimid (NHS) (0,112 mmol; MW 115,19Da) and 43,13 mg of N'-(3-dimethylaminoproρyl)-N-ethylcarbodiimid-HCl (EDC) (0,225 mmol; MW 191,71Da) were added. After the same procedure reported for example 1, 0,45 μl di Et₃N (0,075 mmol; MW 101,19Da; d²⁰ 0,726) and 1000 mg of mPEG-NH₂ (0.05 mmol; MW 20000Da), previously dissolved in 4 ml of CH₂C1₂, were mixed with the activated PN₂S(Trt). The product, mPEG₂oooo-PN₂S(Trt), was recovered and purified as described for example 1 . The yield was 91%.

Example 3: Preparation of mPEG₅ooo-(P)ON₂S(Trt)

The carboxylic group of (P)ON₂S(Trt) was activated by EDC/NHS and coupled with monomethoxy amino-poly(ethyleneglycol) (mPEG-NH₂) to obtain the wanted product: mPEG₅ooo-(P)ON₂S. 203,03 mg of (P)ON₂S(Trt) (0,3 mmol; MW 676,76Da) dissolved in 10 ml of CH₂C1₂ were cooled at 4°C and 51,80 mg of N-hydroxysuccinimid (NHS) (0,45 mmol; MW 115,19Da) and 172,53 mg of N'-(3-dimethylaminoρropyl)-N-ethylcarbodiimid-HCl (EDC) (0,9 mmol; MW 191,71Da) were added. After 5 hours under stirring, 41,8 μl of Et₃N (0.3 mmol; MW 101,19Da; d²⁰ 0,726) and 1000 mg of mPEG-NH₂ (0.2 mmol; MW 5000Da), previously dissolved in 2 ml of CH₂C1₂, were mixed with the activated (P)ON₂S(Trt). The product, mPEG₅₀oo-(P)ON₂S(Trt), was recovered and purified as described for example 1. The yield was 94%.

Example 4: Preparation of mPEG₂₀ooo-(P)ON₂S(Trt)

The carboxylic group of (P)ON₂S(Trt) was activated by EDC/NHS and coupled with monomethoxy amino-poly(ethyleneglycol) (mPEG-NH₂) to obtain the wanted product: mPEG₂₀ooo-(P)ON₂S.

50,76 mg of (P)ON₂S(Trt) (0,075 mmol; MW 676,76Da) dissolved in 10 ml of CH₂C1₂ were cooled at 4°C and 12,95 mg of N-hydroxysuccinimid (NHS) (0,112 mmol; MW 115,19Da) and 43,13 mg of N'-(3-dimethylaminopropyl)-N-ethylcarbodiimid-HCl (EDC) (0,225 mmol; MW 191,71Da) were added. After 5 hours under stirring, 10,45 μl of Et₃N (0.075 mmol; MW 101,19Da; d²⁰ 0,726) and 1000 mg of mPEG-NH₂ (0.05 mmol; MW 20000Da), previously dissolved in 2 ml of CH₂C1₂, were mixed with the activated (P)ON₂S(Trt). The product, mPEG₅ooo-(P)ON₂S(Trt), was recovered and purified as described for example 1. The yield was 89%. Example 5: Preparation of PEG₁₀ooo-(L-2-aminoadipic)₂-[CONH-(CH₂)₆-PN₂S(Trt)]₄ Step 1. Activation of HO-PEG₁₀ooo-OH lg of bifunctional poly(ethyleneglycol) (HO-PEG₁₀ooo-OH; 0.1 mmol; MW 10 KDa) were dissolved in 20 mL of toluene and dehydrated by water-toluene azeotropic distillation. 8 mL of anhydrous CH₂C1₂ were added to the polymer solution, followed by 121 mg of p-nitrophenyl chloroformate (0.6 mmol; MW 201,56Da) and 83.6 μL of Et₃N (0.6 mmol; MW 101,19Da; d²⁰ 0,726), to give PEG₁oooo-(p-nitrophenylcarbonate)₂ (1). The mixture was stirred for 6 hours at room temperature, then the product was precipitated by dropping the reaction mixture into 300 mL of diethyl ether. The resulting white solid was purified by repeated dissolution in CH₂C1₂ and precipitation into diethyl ether. The degree of activation, evaluated on the basis of p-nitrophenol release after hydrolysis by 0.2N NaOH solution, was 97%. Step 2. Synthesis of PEG₁oooo-(L-2-aminoadipic)₂-(COOH)₄

83.8 mg of L-2-aminoadipic acid (AD) (0.52 mmol; MW 161,16Da) were dissolved in 10 mL of CH₃CN/H₂O (2:3) with 217 μL Et₃N (1.56 mmol; MW 101,19Da; d²⁰ 0,726) and 900mg of 1 (0.087 mmol; MW 10330Da). The mixture was maintained at room temperature and under constant stirring for 12 hours. Then the solution was acidified to pH 3.0 and p-nitrophenol was extracted from reaction mixture with diethyl ether (4 x 50 mL). The product PEG₁oooo-(AD)₂- (COOH)₄ (2) was repeatedly extracted from the aqueous solution with chloroform (5 x 50 mL). The organic phase was dried with Na₂SO₄, concentrated under vacuum to 5 mL, and added dropwise to 200 mL of diethyl ether. 2 was collected by filtration and dried under vacuum. The degree of functionalisation, evaluated by titration of the carboxylic groups with NaOH 0.01 N, was 95%.

Step 3. Activation of PEG₁oooo-(L-2-aminoadipic)₂-(COOH)₄ 750 mg of 2 (0.072 mmol; MW 10374,3Da), were dissolved in 10 mL of anhydrous CH₂C1₂ and the solution was cooled to 0°C. 24.9 mg of N-hydroxysuccinimide (0.216 mmol; MW 115,19 Da) and 44.6 mg of N,N'-dicyclohexylcarbodiimide (0.216 mmol; MW 206,33Da) were added under stirring. The mixture was allowed warm to room temperature and react for 12 hours. Dicyclohexylurea was removed by filtration and the solution, concentrated under vacuum, was dropped into 200 mL of diethyl ether. The product obtained, PEGioooo-(AD)₂-(OSu)₄ (3), was dried under vacuum. The degree of activation, evaluated on the basis of the amino group modification of H-Gly-Gly-OH as reported for Snaider's assay [Anal. Biochem. 64, 1975, 284- 288], was 91%.

Step 4. Preparation of PEG₁oooo-(L-2-aminoadipic)₂-[CONH-(CH₂)₆-NH2] 100 μl of mono t-butil 1,6 diamine exan (0,48 mmol; MW 202,3Da; d²⁰ 0,972) were dissolved in DMF and, under costant stirring, 650 mg of 3 (0,06 mmol; MW 10763 Da) was added. After 3 hours reaction mixture was treated with 10 ml o f a solution composed as follows: 50%TFA, 49%CH₂C1₂, 1% water. This treatment removed the t-BOC and gave the product with PEG-AD₂- [CONH-(CH₂)₆-NH₂]₄ (4), which was recovered by previous concentration to small volume in rotavapor following by precipitation in ethanol previously cooled to 0°C. The product was collected in a funnel by filtration. The degree of modification was evaluated by Snaider's assay [Anal. Biochem. 64, 1975, 284-288].

Step 5. Coupling of PN₂S(Trt) to PEG₁₀ooo-(L-2-aminoadipic)₂-[CONH-(CH₂)₆-NH₂]₄ Carboxylic group of PN₂S(Trt) were activated by EDC/NHS and coupled with PEG-AD₂- [CONH-(CH₂)₆-NH₂]₄ to obtain the wanted product: PEGι₀₀oo-(AD)₂-[CONH-(CH₂)₆- PN₂S(Trt)]₄.

158,6 mg of PN₂S(Trt) (0,24 mmol; MW 660,76 Da) dissolved in 5 ml of CH₂C1₂ were cooled at 4°C and 41,47 mg of N-hydroxysuccinimid (NHS) (0,36 mmol; MW 115,19 Da) and 138 mg of N'-(3-dimethylaminopropyl)-N-ethylcarbodiimid-HCl (EDC) (0,72 mmol; MW 191,71Da) were added. After the same procedure reported for example 1, 25,6 μl of Et₃N (0,184 mmol; MW 101,19Da; d²⁰ 0,726) and 500 mg of 5 (0,046 mmol; MW 10834,3 Da), previously dissolved in 2 ml of CH₂C1₂, were mixed with the activated PN₂S(Trt). The product, PEG₁₀ooo-(AD)₂-[CONH- (CH )₆-PN₂S(Trt)]₄, was recovered and purified as described for example 1.

Detritylation of the amphiphilic conjugates

The a mphiphilic c onjugates o f E xamples 1 -4 w ere d eprotected t o t he cysteine s ulphur b efore reactions with ^99mTechnetium or ^185/187Rhenium or to lead the analogous PEG5000-PN₂S, PEG₂oooo-PN₂S and PEGsooo-(P)ON₂S, respectively. The cleavage of the trityl group was achieved with trifluoroacetic acid and triethylsilane according to the procedure reported by Pearson D.A. et al. (Tetr. Lett. 30, 1989, 2739-2742).

Example 6: Preparation of PEG₅₀oo-PN₂S

100 mg of PEG₅₀oo-PN₂S(Trt) was dissolved in dichlorometane (1 mL) and TFA (7 mL). Triethylsilane was added till the solution became colorless and the final mixture was stirred for 45 min. TFA was then removed under vacuum, and the resulting residue was crystallized as a white powder upon addition of ethyl acetate. Yield: 92%.

Example 7: Preparation of PEG₂₀ooo-PN₂S

The same procedure was followed as for Example 6, except for the following change: TFA 15 mL instead of 7 mL. Yield: 90%. Example 8: Preparation of PEG₅ooo-(P)ON₂S

The same procedure was followed as for Example 6. Yield: 89%.

Evaluation of the self-association of the amphiphilic compounds in aqueous solutions

The tendency of the amphiphilic compounds PEG₅ooo-PN₂S and PEG₂oooo-PN₂S to self-associate in micellar aggregates in aqueous solutions was evaluated by light scattering technique. A solid sample of the amphiphilic compound was directly dissolved in phosphate buffer (pH 4, 7.4, 10) and the analysis was performed at 496 nm keeping the temperature constant at 25° C.

Example 9: Self-association in aqueous solution of PEG₅ooo-PN₂S

PEG₅ooo-PN₂S (20 mg) was dissolved in phosphate buffer (1 mL). At all considered pH values, the compound self-associates in micelles showing similar mean diameter at pH 7.4 and 10 (387.9 nm and 391.6 nm, respectively) and being significantly smaller at pH 4 (175.2 nm).

Example 10: Self-association in aqueous solution of PEG₂₀₀₀₀-PN₂S

PEG₂oooo-PN₂S (80 mg) was dissolved in phosphate buffer (1 mL). At all considered pH values, the compound self-associates in micelles showing similar mean diameter at pH 7.4 and 10 (2757.6 nm and 2591.9 nm, respectively) and slightly bigger at pH 4 (3029.1 nm).

Labeling with ^99mTc

As stated by the present invention, the detritylated amphiphilic conjugates of Examples 6 and 7 PEG₅ooo-PN₂S and PEG20₀₀₀-PN₂S can be labeled with ^99mTc by transchelation of the metal from its complex with an exchange ligand, such as gluconate. According to the invention ^99mTc- gluconate, prepared by adding 100 μL of freshly e luted ^99mTcO₄ ^" (5-10 mCi) to 10 μl di Na- gluconato 0.01 M and 1 μl di SnCl₂ 0.1 M (HC1 0.1M), must be purified by Sep-pak chromatography to eliminate undesirable hydrolized oxides which can be absorbed unspecifically by PEG chains.

Example 11 : Labeling of PEG₅₀₀o-PN₂S via ^99mTc-gluconate

To a sample of PEG₅₀₀₀-PN₂S (2 mg) was added a solution of purified ^99mTc-gluconate (50 μl, 1- 2 mCi) diluted with absolute EtOH (50 μL). The pH was increased to 9-10 by adding NaOH 0.1M and the mixture was kept at 37°C for 45 minutes. Labeling yield 95%. Example 12: Labeling of PEG₂₀ooo-PN₂S via ^99mTc-gluconate

The labeling of PEG₂₀₀₀₀-PN₂S (8 mg) was performed following the same procedure of Example 11. Labeling yield: 96%.

In the labeled species of Examples 11 and 12, the PN₂S BFC coordinates around the TcO³⁺ core producing TcO(PN₂S) pentaccordinated complex with the ligand acting as tetradentate in the equatorial plane in respect to M=O.

According to the present invention, the detritylated amphiphilic conjugates of Examples 6 and 7 PEG₅ooo-PN₂S and PEG₂₀ooo-PN₂S can be alternatively labeled with ^99mTc exploiting the reductive properties of phosphine phosphorous and avoiding the need of an external reductive agent. Considering that both compounds aggregate in micelles in aqueous solution, the pertechnetate s olution i s p referably a dded t o a so lid s ample o f t he a mphiphilic c ompound, t o favour the binding interaction between the reducing-coordinating BFC and TcO₄ ^" before micelles formation.

Example 13 : Labeling of PEG₅ooo-PN₂S via TcO₄ ^"

To PEG₅₀₀₀-PN₂S (2 mg) in a 1.5 mL Eppendorf vial were added 100 μl of ^{9 m}TcO₄ ^" solution freshly eluted from generator and eventually diluted with saline (1-2 mCi) and acidified to pH 2 (HO 0.1M). The final mixture was kept at room temperature for 10-15 minutes with a quantitative yield in a single labeled species.

Example 14: Labeling of PEG₂₀ooo-PN₂S with ^99mTc via TcO₄ ^"

The same procedure was followed as for Example 13, except for the following changes: PEG₂oooo-PN₂S 8 mg, incubation at 37° C. Quantitative yield in a single labeled species. According to the invention, the amphiphilic conjugates PEG₅ooo-PN₂S and PEG₂oooo-PN₂S reduce ^99mTcO ^" and coordinate reduced ^99mTc species in mild conditions, in a really short time and with quantitative yields.

The free BFC PN₂S by itself is able to reduce TcO ^" due to the redox potential of phosphine phosphorous, but the reaction is kinetically not favoured and it is not useful in the time scale of technetium decay. Instead, the reduction of TcO₄ ^" by the amphiphilic conjugates is fast and complete in a short time (10-15 minutes). Thus, the labeling procedure of the present invention is in agreement with a catalytic process mediated by micellar aggregate formation.

According to the invention, when the solution of a metal radioisotope in its upper oxidation state is added to the solid amphiphilic compound bearing the most suitable chelating agent, micelle formation catalyses metal reduction and coordination. Meanwhile the amphiphilic compound associates in micelles, the radioactive metal is loaded into the lipophilic core and it undergoes reduction reaction, mediated by phosphorous, and coordination reaction, mediated alternatively by the PN₂S or the (P)ON₂S set. The inclusion into micelle core enhances the reductive properties of phosphorous and favours coordination. Moreover, no radiolabeled impurities or free pertechnetate are detectable indicating that the reduction is followed immediately by coordination.

As stated by the present invention, the analogous amphiphilic compounds bearing a phosphine oxide instead of the phosphine phosphorous, thus missing reductive properties, can be labeled with ^99mTc via gluconate.

Example 15: Labeling of PEG_SOoo-(P)°N₂S via Tc-gluconate

The labeling of PEG₅ooo-(P)ON₂S (8 mg) was performed following the same procedure of Example 10, obtaining a labeling yield of 95% after 45 minutes.

According to the invention, in the labeled species of Example 15 the TcO³⁺ core is coordinated by the (P)ON₂S set with the phosphine oxide oxygen bound to the metal centre instead of the phosphine phosphorous.

According to the invention, the labeled amphiphilic conjugates of Examples 11-15 are very stable as demonstrated by the fact that when micellar aggregates dissociated into their unimers no radiolabeled impurities or free radiometal species are detectable.

In vivo studies

According to the invention, the labeled amphiphilic conjugates are intended to be use as radiopharmaceuticals for diagnostic and therapeutic purposes. In vivo studies were performed to evaluate the biodistribution and stability of labeled compounds of Examples 13 and 14 after intravenous administration.

Example 16: Biodistribution of ^99mTc-labeled PEG₅₀oo-PN₂S

A normal Swiss mouse was injected into the tail vein with a diluted solution of the labeled compound prepared in Example 13 (pH 7, NaOH 0.1N). Scintigraphic images of the mouse were collected for 40 minutes with a YAP-camera (F. Vittori, T. Malatesta, F. de Notaristefani, Transactions on Nuclear Science, Vol. 44, No. 1, 47-53, 1997) and showed a rapid and efficient clearance from t he b loodstream m ainly b y t he urinary s ystem, a lso c onfirmed b y t he ex- vivo counting of activity in organs and tissues. Collected data confirm that the in vivo PEGs₀₀o-PN₂S biodistributionof is analogous to that reported for free PEG₅ooo- Example 17: Biodistribution of ^99mTc-labeled PN₂S-PEG₂₀ooo

The biodistribution of the labeled compound prepared in Example 14 were evaluated under the same conditions as those for Example 16, except for the image acquisition time which was extended to 4 hours. Scintigraphic images of the mouse showed a slow clearance from the blood pool which correlated with a high retention of activity in all organs and tissues, also confirmed by the ex-vivo counting of activity in organs and tissues. Collected data confirm that the in vivo PEG₅₀₀₀-PN₂S biodistributionof is analogous to that reported for free PEG₂oooo- According to the invention, Examples 16 and 17 demonstrated that the labeling procedure does not modify the polymer and that when the tracer (^99mTcO[PN₂S] complex) is a low molecular weight c ompound the body fate of the linked polymer determines the fate of the amphiphilic conjugate.

The low activity accumulation in the stomach showed that ^99mTcO ^" is not produced in relevant amount. This is in accordance with the great stability of the labeled amphiphilic conjugates recovered unmodified from the urines, due to the high affinity of the coordination set toward technetium and due to a shielding effect afforded by PEG chain.

Complexation reactions with ^185/187Rhenium

Example 18: Complexation of PEG₅ooo-PN₂S with ^185/187Rhenium

To a solution of PEG₅ooo-PN S (200 mg) in degassed dichlorometane (3 ml), was added a solution of ReOCl₃(PPh₃)₂ (30 mg) in the same solvent (3 mL). To the mixture stirred under was added TEA till pH 9, obtaining a reddish-brown solution. The final complex was crystallized by adding to the solution, concentrated under vacuum, diethylether/petroleum ether. Yield: 75%.

Example 19: Complexation of PEG₂oooo-PN₂S with ^18S/187Rhenium The same procedure was followed as for Example 18, except for the following changes: PEG₂₀₀₀0-PN2S 400 mg in 6 mL of dichlorometane, ReOCl₃(PPh₃)₂ 16 mg. Yield: 68%.

Example 20: Complexation of PEG_Sooo-(P)ON₂S with ^185/187Rhenium

To a solution of PEG₅ooo-(P)ON₂S (200 mg) in degassed dichlorometane (3 ml), was added a solution of ReOCl₃(PPh₃)₂ (30 mg) in the same solvent (3 mL). To the mixture stirred under was added TEA till pH 7, obtaining a reddish-brown solution. The final complex was crystallized by adding to the solution, concentrated under vacuum, diethylether/petroleum ether. Yield: 46%.

Claims

1. A conjugate comprising a hydrophilic polymer bound directly or by means of other moieties to a chelating group [able to chelate a metal ions] comprising at least one phosphine or one phosphine oxide phosphorous.

2. The conjugate of claim 1 wherein the polymer has an average MW of at least 1000, preferably of at least 4000, more preferably at least 10000, and even more preferably of at least 20000.

3. A conjugate as in claims 1-2, additionally comprising one or more polifunctional polymers, preferably a dendrimer, and /or one or more targeting moieties.

4. A conjugate as in claims 1, 2 or 3 having the following formula: [BFC_n-Poll]_x-Pol2-[Pol3-BM_m]_y wherein: x is 1 or 2, y is 0 or 1, n = 1-200 and m = 0-100. BFC is a chelating group, which is suitable for chelating metal ions, comprising at least one phosphine or one phosphine oxide phosphorous. Pol2 is a hydrophilic water-soluble polymer, synthetic or naturally derived. Poll, Pol3 and BM may be present or absent, Poll, if present, is a polymer or a dendrimeric structure, which bears several side-chain fuctionalizable residues, Pol3, if present, is a polymer or a dendrimeric structure, which bears several side-chain fuctionalizable residues, BM, if present, is a targeting molecule, and where the different moieties are conjugated, preferably via ester, amide, carbamate, ether, thioether, sulfur, disulphide or other covalent bonds, directly or by means of one or more linkers, the linkers preferably selected among alkyl groups, such as NH₂-(CH₂)_n-NH₂ (n = 0-12) or HOOC-(CH₂)_m-COOH (m = 0-12), or aromatic groups or cleavable peptides or other biodegradable sequence, such as H-GFLG-OH, H-GLFG-OH.

5. A conjugate as in claims 1-4, wherein the chelating group able to bind metal ions has the formula: RlR2-P(O)r-(Alk-X-)-(Alk-X-)p-R' where: r is 0 or 1 p is 1 to 4, each group Alk is independently an alkyl group preferably comprising 1 to 4 carbons, optionally substituted by a one or more groups preferably selected among an oxy group, a hydroxy, an amino group, a carboxy group or a residue of an aminoacid,

X is selected among N, O, S, P,

Ri and R₂ are optionally substituted alkyl, optionally substituted aryl or optionally substituted aryl-alkyl groups such as Z-Ar-(CH₂)_a (a = 0-2 and Z = H, CH₃-, CH₃O-), CH₃-

(CH₂)_b- (b = 2-9), Ethyl, Isopropyl, Isobutyl, tert-Butyl, Methyl, CH₃-(CH₂)₀-O-(CH₂)_d- (c and d = 0-4);

R' is H or optionally substituted alkyl, or optionally substituted aryl or optionally substituted aryl-alkyl groups, such chelating group being conjugated to the other parts of the compound by a bond at any position of the chelating group, and preferably at an amine or carboxylic or thiol group, or at any of the carbons of the backbone of the chelating group.

A conjugate as in claims 1-5, wherein the chelating group able to chelate metal ions has the formula:

π πi

IV V VI

;p-(CH₂)_m-NH₂

VII VIII ^Rl _/-P- (CH₂)_m-CONH- (CH₂)_n- CHR"-COOH ^Rl ^-P- (CH₂)_m-CONH- CHR"- (CH₂)_n-COOH IX R. IP- (CH₂)_m-NHOC- (CH₂)_n- CHR"-COOH ' - (CH-)_m-NHOC- CHR"- (CH₂)_n-COOH R. XI XII

Ri and R₂ are optionally substituted alkyl, optionally substituted aryl or optionally substituted aryl-alkyl groups such as Z-Ar-(CH₂)_a (a = 0-2 and Z = H, CH₃~, CH₃O-), CH₃- (CH₂)_b- (b = 2-9), Ethyl, Isopropyl, Isobutyl, tert-Butyl, Methyl, CH₃-(CH₂)_c-O-(CH₂)_d- (c and d = 0-4); where R" is any amino acid residue; X is selected among N, O, S, P, R is H, or optionally substituted alkyl, optionally substituted aryl or optionally substituted aryl-alkyl groups, and where for formula I- VI n = 1, 2; for formula VII-XII m and n = 0, 1; for formula XIII-XV n = 1, with the provision that such chelating agents are conjugated to the polymer at any of the amine, thiol or carboxylic groups or at any of the position of the backbone of the chelating group.

7. A conjugate as in claims 1-6, wherein Pol2 is a hydrophilic water-soluble polymer, synthetic or naturally derived, having at least one terminal functional groups for conjugation, the polymer preferably being poly(ethylene glycol), polyvinylpirrolidone or polyacriloylmorpholine .

8. A conjugate as in claims 1-7, wherein Poll, if present, is a polymer or a dendrimeric structure, which bears several side-chain fuctionalizable residues, and is preferably polyaspartic acid, polyglutamic acid, poly(hydroxyethylaspartamide), poly(hydroxyethylglutamide), poly(hydroxypropyl-metacrylamide), polylisine polyhydroxyl acids such as polylactic acid or polymalic acid, and, where Poll is a dendrimer, the branching moiety is preferably glutamic acid, β-glutamic acid, amino adipic acid or other amino bicarboxylic or tricarboxylic acids.

9. A conjugate as in claims 1-8, wherein Pol3, if present, is a polymer or a dendrimeric structure, which bears several side-chain fuctionalizable residues, such as polyaspartic acid, polyglutamic acid, poly(hydroxyethylaspartamide), poly(hydroxyethylglutamide), poly(hydroxypropyl-metacrylamide), polylisine polyhydroxyl acids such as polylactic acid or polymalic acid and, when Pol3 is a dendrimer, the branching moiety is preferably glutamic acid, β-glutamic acid, amino adipic acid or other amino bicarboxylic or tricarboxylic acids.

10. A conjugate as in claims 1-9, wherein BM, if present, is a targeting molecule, preferably selected among peptide, monoclonal antibody and antibody fragment, biotin, chemotherapeutic drug and sugar.

11. A conjugate as in claims 4-10, wherein x is 1, y is 0, n = 1-200, and having the formula: BFC-Pol2 or, BFC_n-Poll-Pol2

12. A conjugate as in claim 1-11, where Pol2 is PEG 5000 Da or PEG 20000 Da, n = 1, Poll is absent and BFC is N- N-(3-Diphenylphosphinopropionyl)glycyl]cysteine [PN₂S] or N- [N-(3-Diphenylphosphinoxidepropionyl)glycyl]cysteine [(P)ON₂S], having the following formula:

mPEGgooo-PNzS mPEG2oooo-PN₂S

mPEG₅₀oo-(P)ON₂S mPEG2oooo-(P)ON₂S

13. A conjugate as in claims 1-10, wherein x is 2 and having the formulas: BFC-Pol2-BFC or, BFC_n-Poll-Pol2-Poll-BFC_n or, BFC_n-Poll-Pol2-BFC

14. A conjugate as in claims 1-10, wherein x is l, y is 1 m = 1-100 and having the general formulas: BFC-Pol2-BM or, BFC_n-Poll-Pol2-BM or, BFC-Pol2-Pol3-BM_m or, BFC_n-Poll-Pol2-Pol3-BM_m

15. A complex of any of the conjugates according to any of the preceding claims with a metal ion.

16. A complex as in claim 15, where the metal ion is a paramagnetic ion or a γ, β⁺, β^" or α emitter.

17. The complex of claim 16 with a metal ion selected among Gd, ^99mTc, ^{67 68}Ga, ^mIn, ⁶²Cu, ⁹⁰Y, ^{186 188}Re, ¹⁵³Sm, ¹⁷⁷Lu, ^64/67Cu. QQm 1 Sή 1 SR

18. A complex as in claims 17 wherein the metal ions are Technetium, Rhenium and Copper radioisotopes.

19. A pharmaceutical or diagnostic composition comprising a conjugate or a complex as defined in any of claims 1-18, optionally also comprising a pharmaceutically acceptable excipient.

20. The composition of claim 19 being for oral, parenteral, rectal, topical, vaginal, ophthalmic or inhalation use.

21. A kit comprising a first composition (a) of a conjugate as in any o f claims 1-14 and a separate composition (b) comprising a metal ion suitable to be used in therapy or diagnosis.

22. The kit of claim 21 wherein the metal is a metal radioisotope such as γ, β⁺, β^" or α emitter like ⁹⁰Y, ^186/188Re, ¹⁵³Sm, ¹⁷⁷Lu, ^64/67Cu, ^99mTc, ^67/68Ga, ^mhι, or a metal ion which can be spectroscopically revealed, such as Gd.

23. A method for the preparation of radiopharmaceuticals comprising mixing a metal ion with a conjugate comprising a polymer and a chelating group comprising a phosphine or phosphine oxide phosphorous and with an external reducing agent.

24. The method of claim 23 in which a solution comprising the metal ion is added to the conjugate in solid form.

25. A method, according to claim 23 comprising transchelation of PEG₅ooo-PN₂S or PEG₂oooo- PN₂S with ^99tnTc from its complex with an exchange ligand, such as gluconate, to eliminate undesirable hydrolysed oxides which can be absorbed unspecifically by PEG chains.

26. The method of claim 23 where the conjugate is any of the phosphine or phosphine oxide phosphorous conjugates defined in claims 1-14.

27. A method for the preparation of radiopharmaceuticals comprising mixing a metal ion and a conjugate comprising a polymer and a chelating group comprising a phosphine, such method not involving the addition of an external reducing agent.

28. The method of claim 27 in which a solution comprising the metal ion is added to the conjugate in solid form.

29. A method, according to any of claims 28 to 30 comprising the addition of a solution of TcO₄ ^" obtained diluting with saline a TcO₄ ^" solution (1-2 mCi), freshly eluted from generator, to PEG₅ooo-PN₂S or to PEG₂oooo-PN₂S and keeping the final mixture at room temperature for at least 10 minutes.

30. The method of claim 27 where the conjugate is any of the phosphine phosphorous conjugates defined in claims 1-14.

31. A method as in claims 27, 28, 29, or 30 wherein said conjugate acts as a reductive agent of said metal.

32. Use of a conjugate as defined in any of claims 1 - 18 for the manufacture of a diagnostic or a therapeutic composition.

33. A diagnostic or therapeutic method comprising the use of a conjugate, a composition or a kit as defined as in any of claims 1-22.

34. The diagnostic method of claim 33 for the diagnosis of cancer, angio genesis, inflammations, or other diseases such as arteriosclerosis, Parkinson, Alzheimer, bone and intestinal diseases or other medical test.

35. A method of treatment of cancer, angiogenesis, inflammations, or other diseases such as arteriosclerosis, Parkinson, Alzheimer, bone and intestinal diseases comprising the use of a conjugate, a composition, or a kit as in any of claims 1-34.