WO1997010854A1

WO1997010854A1 - (radio) labelled biotin derivatives

Info

Publication number: WO1997010854A1
Application number: PCT/US1996/014883
Authority: WO
Inventors: Helmut Mäcke
Original assignee: Mallinckrodt Medical, Inc.
Priority date: 1995-09-18
Filing date: 1996-09-17
Publication date: 1997-03-27
Also published as: IL123725A0; CZ75498A3; EP0857071A1; CA2232411A1; JPH11513374A; MX9802107A; NO981202D0; NO981202L

Abstract

The invention relates to a labelled biotin compound, wherein said biotin compound is represented by general formula (I) wherein: n is 1 or 2, R is a chelating group for chelating a metal atom, and Sp is a spacing group having at least 4 atoms spacing NH from R; and wherein said biotin compound is labelled with a metal atom selected from (a) the group consisting of the radioactive isotopes ?99mTc, 203Pb, 66Ga, 67Ga, 68Ga, 72As, 111In, 113mIn, 114mIn, 97Ru, 62Cu, 64Cu, 52Fe, 52mMn, 51Cr, 186Re, 188Re, 77As, 90Y, 67Cu, 169Er, 117mSn, 121Sn, 127Te, 142Pr, 143Pr, 198Au, 199Au, 149Tb, 161Tb, 109Pd, 165Dy, 149Pm, 151Pm, 153Sm, 157Gd, 166Ho, 172Tm, 169Yb, 175Yb, 177Lu, 105Rh and 111¿Ag or (b) the group consisting of the paramagnetic metal atoms Cr, Mn, Fe, Co, Ni, Cu, Pr, Nd, Sm, Yb, Gd, Tb, Dy, Ho and Er; said metal atom being attached to the biotin compound by means of said chelating group. The invention further relates to a pharmaceutical composition comprising said labelled biotin compound, to the use of said composition for diagnosis and therapy, and to a kit for preparing a radiopharmaceutical composition.

Description

(RADIO) LABELLED BIOTIN DERIVATIVES

The present invention relates to labelled biotin compounds, to a method of preparing these compounds, to a pharmaceutical composition comprising these compounds, to the use of this composition for diagnosis and therapy, and to a kit for preparing a radiopharmaceutical composition.

It is well-known in the art, that various polypeptides or proteins, such as monoclonal antibodies (MAb's), bind with high affinity to tumour associated antigens. Consequently, radiolabelled monoclonal antibodies (MAb*'s), have been successfully used for the in vivo localization of tumours in nuclear medicine. One of the major drawbacks in the use of MAb*'s is their prolonged blood clearance. As a consequence, poor tumour/background ratios are usually achieved and the radiation dose to normal tissues is high.

A promising development in the application of MAb*'s in nuclear diagnosis is the pretargeting approach. According to this method the administration of the tumour-specific protein (antibody) and of the radioactivity take place at separate time points. This methodology is possible by use of the avidin/biotin system. There are two different basic protocols which can be used for tumour localization wih this avidin/biotin pretargeting system (Paganelli et al., Nucl. Med. Commun. 12, 1991, 211-234), viz. the two-step approach and the three-step approach. In the two-step approach an in vitro prepared conjugate of (strepf)avidine and the tumour-seeking polypeptide, e.g. the antibody, is injected first, two or three days later followed by injection with radiolabelled biotin. By the three-step pretargeting approach, biotinylated MAb (lst step), streptavidin

(2nd step) and radiolabelled biotin (3rd step) are injected successively (in about 24 h time intervals) to the patient. The first two steps, as well as the first step in the two-step approach, result in the avidination of the tumour. In addition, the biotinylated MAb's, which have not localized on the tumour but are still circulating in the blood, are macroaggregated by the excess of avidin. These avidin/MAb adducts of high molecular weight are rapidly taken up and catabolized by the liver. The 3rd step involves the in vivo administration of radiolabelled biotin. Due to the fast blood clearance of the non-tumour-bound biotin derivative, good tumour/background ratios are expected to be reached rapidly.

For the visualization of tumours, biotin. labelled with a radionuclide, preferably a metal-radionuclide, or with a paramagnetic metal ion are required. Since biotin is lacking Q is a group which is capable of reacting with an amino group

of the peptide and which is preferably selected from the group consisting of carbonyl, carbimidoyl, N-(C₁-C₆)alkylcarbimidoyl, N-hydroxycarbimidoyl and N-(C₁-C₆)alkoxycarbimldoyl.

Suitable examples of N₁P_qS_(4-t-q) tetradentate chelating agents are selected from

The above-defined spacing group Sp is preferably a group of the general formula

wherein A is a biradical of the formula

-NH-(CH₂)_m- or -NH-C(=X)-(CH₂)_x-Y-(CH₂)_m-C(CO₂H)H- wherein x and y are each independently 0 or 1;

m is an integer from 1 to 4;

p is an integer from 0 to 4;

X is O or S; and

Y is NH, CO or S.

The above-defined chelating group R is preferably selected from the group consisting of N_tP_qS_(4-t-q) tetradentate chelating agents, wherein t+q=2-4, or groups derived from ethylene diamine tetra-acetic acid (EDTA), diethylene triamine penta-acetic acid (DTPA), cyclohexyl 1,2-diamine tetra-acetic acid (CDTA), ethyleneglycol-O,O'-bis(2-aminoethyl)-N,N,N',N'-tetra-acetic acid (EGTA), N,N-bis(hydroxybenzyl)-ethylenediamine-N,N'-diacetic acid (HBED), triethylene tetramine hexa-acetic acid (TTHA), 1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tri and tetra-acetic acid (DO3A and DOTA), 1,4,7,10-tetraazacyclotridecane-N,N',N",N"'-tri and tetraacetic acid (TRI3A and TRITA), hydroxyethyldiamine triacetic acid (HEDTA), 1,4,8,11-tetra-azacyclotetradecane-N,N',N",-N"'-tetra-acetic acid (TETA), substituted DTPA substituted EDTA or from deferoxamine (desferrioxamine), or from a compound of the general formula

wherein R₁ is a branched or non-branched, optionally substituted

hydrocarbyl radical, which may be interrupted by one or more hetero-atoms selected from N, O and S and/or by one or more NH groups, and The new metal-labelled biotin compounds of the invention can be prepared in a manner known per se for related compounds. For this purpose the biotin molecule is derivatized with the desired chelating agent as defined hereinbefore, e.g. a N_tP_qS_(4-†-q) tetradentate chelating agent, or EDTA

DTPA etc., after Introduction of the spacing group Sp as defined above, after which the compound obtained, having the general formula

wherein:

n is i or 2,

R is a chelating group for chelating a metal atom, and

Sp is a spacing group having at least 4 atoms spacing NH from R;

is reacted with a metal, as defined hereinbefore, in the form of a salt or of a chelate bound to a comparatively weak chelator, in order to form a complex.

Suitable examples of salts or chelates of the desired metal are: ¹¹¹Irvcitrate and acetate, ^99mTc-tartrate, etc. The complex-forming reaction can generally be carried out in a simple manner and under moderate conditions. The invention also relates to a biotin compound of the general formula I, as defined above, which compound can be used for the above-described method of preparing the labelled biotin compound.

The present invention further relates to a pharmaceutical composition, comprising in addition to a pharmaceutically acceptable carrier material and, if desired, at least one pharmaceutically acceptable adjuvant, as the active substance a labelled biotin compound as defined hereinbefore.

The invention also relates to a method for detecting and localizing tumours in the body of a warm-blooded living being, which comprises (i) administering to said being a composition comprising (strept)avidin conjugated with a polypeptide having a selective affinity to said tumour, (ii) thereupon, after avidination of said tumour, administering to wherein:

R₆-R₂₀ are each individually hydrogen atoms or (C₁-C₄)alkyl groups, with the proviso that at least one of C₆ to C₉ is the symbol Y;

R₂₁ is a hydrogen atom or a CO₂(C₁-C₄)alkyl group;

R₂₂ and R₂₃ are each individually (C₁-C₄)alkylcarbonyl, benzoyl or benzyl groups;

v is 0 or 1;

s is 2 or 3;

R₂₄ is CH₂COOH or a functional derivative thereof;

A is (C₁-C₄)alkylene, if desired substituted with CO₂alkyl, CH₂COalkyl, CONH₂,

CONHCH₂CO₂alkyl; phenylene, phenylene substituted by CO₂alkyl, wherein the alkyl groups have 1 to 4 carbon atoms;

G is NH or S;

Y is a valence bond or a functional group capable of binding with the spacing group;

and Z is S or O.

If Y is a functional group, Y preferably comprises isocyanato, isothiocyanato, formyl, o-halonitrophenyl, diazonium, epoxy, trichloro-s-triazinyl, ethyleneimino, chlorosulfonyl, alkoxycarbimidoyl, (substituted or unsubstituted) alkylcarbonyloxycarbonyl, alkylcarbonylimidazolyl, succinimido-oxycarbonyl, which group is prferably attached to a

(C₁-C₁₀)hydrocarbon biradical.

Suitable examples of hydrocarbon biradicals are biradicals derived from benzene, (C₁-C₆)alkanes, (C₂-C₆)alkenes and (C₁-C₄)alkyibenzenes. Examples of suitable chelators of the general formula III are described in the international patent application WO 89/07456, such as unsubstituted or substituted 2-iminothiolanes and 2-iminothiacyclohexanes, in particular 2-imino-4-mercaptomethytthiolane.

The above labelled biotin compounds have been tested in a number of suitable model experiments that are predictive for in vivo application. These experiments are described in the Examples. From the results of these experiments it will be evident, that the labelled biotin compounds of the present invention have properties which make them suitable for diagnostic and therapeutic purposes. If labelled with a suitable atom for diagnostic purposes, the labelled biotin compound remains sufficientiy long intact after administration to permit imaging of the target tumour without presenting a disturbing background. If labelled with a suitable radioisotope for therapy, such-labelled biotin compounds are promising therapeutic agents for the treatment of a number of malignant tumours. the body of α warm-blooded living being, which comprises (i) administering to said being a composition comprising (strept)avidin conjugated with a polypeptide having a selective affinity to said tumour, (ii) thereupon, after avidination of said tumour, administering to said being a composition comprising,, in a quantity effective for combating or controling tumours, a labelled biotin compound as defined hereinbefore, said biotin compound being labelled with a metal isotope selected from the group consisting of ^114mIn. ¹⁸⁶Re, ¹⁸⁸Re, ⁷⁷As, ⁹⁰Y, ⁶⁶Ga, ⁶⁷Cu, ¹⁶⁹Er, ^117mSn, ¹²¹Sn, ¹²⁷Te, ¹⁴²Pr, ¹⁴³Pr, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁴⁹Tb, ¹⁶¹Tb, ¹⁰⁹Pd, ¹⁶⁶Dy, ¹⁴⁹Pm, ¹⁵¹Pm, ¹⁵³Sm, ¹⁵⁷Gd,¹⁵⁹Gd, ¹⁶⁶Ho, ¹⁷²Tm, ¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁷⁷Lu. ¹⁰⁶Rh and ¹¹¹Ag.

It will be clear, that instead of the above two-step protocol, the three-step pretargeting method can be used as discussed hereinbefore.

In case a radioactive labelled biotin compound is used as a diagnostic agent, it is frequently impossible to put the ready-for-use composition at the disposal of the user, in connection with the often poor shelf life of the radiolabelled compound and/or the short half-life of the radionuclide used. In such cases the user will carry out the labelling reaction with the radionuclide in the clinical hospital or laboratory. For this purpose the various reaction ingredients are then offered to the user in the form of a so-called "kir. It will be obvious that the manipulations necessary to perform the desired reaction should be as simple as possible to enable the user to prepare from the kit the radioactive labelled composition by using the facilities that are at his disposal. Therefore the invention also relates to a kit for preparing a radiopharmaceutical composition.

Such a kit according to the present invention may comprise (a) a composition comprising (strept)avidin conjugated with a polypeptide having a selective affinity to tumours, (b) a biotine compound of the general formula I, shown above, wherein the symbols have the above meanings, to which compound, if desired, an inert pharmaceutically acceptable carrier and/or formulating agents and/or adjuvants is/are added, (c) a solution of a salt or chelate of a metal isotope selected from the group consisting of ²⁰³Pd, ⁶⁶Ga. ⁶⁷Ga, ⁶⁸Ga ⁷²As, ¹¹¹In, ^113mIn. ^114mIn, ⁹⁷Ru, ⁶⁷Cu, ^66mTc , ¹⁸⁶Re, ¹⁸⁸Re, ⁶⁴Cu, ⁵²Fe, ^52mMn, ⁵¹Cr, ⁷⁷As, ⁹⁰Y. ⁶⁷Cu, ¹⁶⁹Er, ^117mSn, ¹²¹Sn, ¹²⁷Te, ¹⁴²Pr, ¹⁴³Pr, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁴⁹Tb, ¹⁶¹Tb, ¹⁰⁹Pd,

¹⁶⁵Dy, ¹⁴⁹Pm, ¹⁵1Pm, ¹⁵³Sm, ¹⁵⁷Gd, ¹⁶⁶Ho, ¹⁷²Tm, ¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁷⁷Lu, ¹⁰⁶Rh and ¹¹¹Ag, and (d) instructions for use with a prescription for reacting the ingredients present in the kit.

Preferably the biotin compound to be used as an ingredient of the above kit has been modified by a spacing group Sp and a chelating agent R as defined hereinbefore. The resulting biotin compound provides a facility for firmly attaching the radionuclide in a simple manner. Suitable chelating agents for modifying the biotin molecule are said being a composition comprising, in a quantity sufficient for external imaging, a labelled biotin compound as defined hereinbefore, said biotin compound being labelled with (a) a radioactive metal isotope selected from the group consisting of ^99mTn, ²⁰³Pd, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ¹¹¹In, ^113mln, ⁹⁷Ru, ⁶²Ga, ⁶⁴Ga, ⁵²Fe, ^52mMn and ⁵¹Cr, or (b) with a paramagnetic metal atom selected from the group consisting of Cr, Mn, Fe, Co, Ni, Cu,

Pr, Nd, Sm, Yb, Gd, Tb, Dy, Ho and Er, and (iii) finally subjecting said being to external imaging to determine the targeted sites in the body of said being in relation to the background activity.

Instead of the above two-step protocol, the three-step pretargeting method can be used as discussed hereinbefore. In this method, the following protocol is used: (i-α) administering to said being a composition comprising a biotinylated polypeptide having a selective affinity to said tumour, (i-b) then administering a composition comprising (strept)avidin; both steps in substitution for the above step (i). The invention also relates to a method of intraoperatively detecting and localizing tumours in the body of a warm-blooded living being, which comprises (i) administering to said being a composition comprising (strept)avidin conjugated with a polypeptide having a selective affinity to said tumour, (ii) thereupon, after avidination of said tumour, administering to said being a composition comprising, in a quantity sufficient for detection by a gamma detecting probe, a labelled biotin compound as defined hereinbefore, said biotin compound being labelled with ¹⁶¹Tb, and (iii) finally, after allowing the active substance to be bound by and taken up in said tumour and after blood clearance of radioactivity, subjecting said being to a radioimmunodetection technique in the relevant area of the body of said being, by using a gamma detecting probe.

The above radioisotope, viz. ¹⁶¹Tb, allows the use of a such-labelled peptide compound in the technique of radioguided surgery, wherein relevant tissues in the body of a patient can be detected and located intraoperatively by means of a gamma detecting probe. The surgeon can, intraoperatively, use this probe to find the lesions in which uptake of the compound labelled with said radioisotope, which is a low-energy gamma photon emittor, has taken place.

It will be clear, that instead of the above two-step protocol, the three-step pretargeting method can be used as discussed hereinbefore. The biotin compounds of the present invention, provided they are radiolabelled with isotopes suitable for such purpose, can be used for the therapeutic treatment of tumours. So the invention further relates to a method for the therapeutic treatment of tumours in excellently suitable. Examples of suitable Sn(II)-cornpounds are SnCl₂, Sn(II)-tartrate, Sn(II)-phosphonate or -pyrophosphate, and Sn(II)-glucoheptonate. The modified biotin constituent of the above-mentioned kits, i.e. preferably the biotin compound, may be supplied as a solution, for example, in the form of a physiological saline solution, or in some buffer solution, or may be present in a dry condition, for example, in a lyophilized condition. When used as a component for an injection liquid it should be sterile, in which, when the constituent is in the dry state, the user should preferably use a sterile physiological saline solution as a solvent. If desired, the abovementioned constituent may be stabilized in the conventional manner with suitable stabilizers, for example, ascorbic acid, gentisic acid or salts of these acids, or it may comprise other auxiliary agents, for example, fillers, such as glucose, lactose, mannitol, and the like.

The invention will now be described in greater detail with reference to the following specific Examples.

described in detail hereinbefore. N_tP_qS_(4-t-q) tetradentate chelating agents or N-containing di- or polyacetic acids or their derivatives, such as the compounds mentioned before, have proved to be pre-eminently suitable for attaching various metal radionuclides, such as ln-111 and ln-113m, to the biotin molecule. The Wt to be supplied to the user may also comprise the ingredient(s) defined sub (a) and (b) above, together with instructions for use, whereas the solution of a salt or chelate of the radionuclide, defined sub (c) above, which solution has a limited shelf life, may be put to the disposal of the user separately.

The above Wt is destined for use in a two-step protocol. If, instead thereof, the three-step pretargeting method is to be used, as discussed hereinbefore, the following Wt is suitable:

(a-i) a composition comprising a biotinylated polypeptide having a selective affinity to said tumour, and (a-ii) a composition comprising (strept)avidin; both compositions in substitution for the above composition (a). In case the kit serves to prepare a radiopharmaceutical composition labelled with Tc- 99m, Re-186 or Re-188, such a Wt according to the present invention may comprise, in addition to the ingredient(s) defined sub (a) and (b) above, (c) a reducing agent and, if desired, a chelator, and (d) instructions for use with a prescription for reacting the ingredients of the Wt with Tc-99m in the form of a pertechnetate solution, or with Re-186 or Re-188 in the form of a perrhenate solution. If desired, certain ingredients of the kit may be combined, provided they are compatible. The pertechnetate or perrhenate solution can simply be obtained by the user from a suitable generator.

When the radionuclide is present in the Wt itself, the complex forming reaction with the biotin compound of the general formula I above can simply be produced by combining the components in a neutral or buffered medium and causing them to react. For that purpose the radionuclide may be presented to said biotin compound in the form of a chelate bound to a comparatively weak chelator, as described hereinbefore. When the Wt comprises a biotin compound as defined hereinbefore and is intended for the preparation of a radiopharmaceutical composition, labelled with Tc-99m, Re-186 or Re-188, the radionuclide will preferably be added separately in the form of a pertechnetate or perrhenate solution. In that case the kit will comprise a suitable reducing agent and, if desired, a chelator, the former to reduce the pertechnetate or the perrhenate. As a reducing agent may be used, for example, a dithionite or a metallic reducing agent. As a reducing agent for the above-mentioned kits is preferably used a metallic reducing agent, for example, Sn(II), Ce(III), Fe(II), Cu(I), Ti(III) or Sb(III); Sn(II) is o-Ar-H-), 8.04 (t, 2H, CH₂-NH-CO), 7.46 (d, 2H, m-Ar-H), 3.45 (t, 1H, CH-CH₂-Ar), 3.15 (d, 2H, CH₂-Ar), 3.05 (m, 4H, CH₂-NH-), 2.50 (m, 4H, CH-NH₂); ¹³C-NMR (90 MHz, d₆-DMSO): 168.25 (2C, C=O), 147.71 (1C, C(Ar)-NO₂), 145.86 (1C, CH₂-C(Ar)), 129.95 (2C, Ar), 123.06 (2C, Ar), 54.00 (1C, CH-CH₂-Ar), 41.93 (2C, CH₂-NH(CO)), 40.86 (2C, CH₂-NH₂), 34.48 (1C, CH₂-Ar). Elemental analysis corresponds to C₁₄H₂₁N₅O₄

6-(p-Nitrobenzyl)-1,4,8,11-tetraazaundecane (2) The crude (1) (480 mg, 1 ,48 mmol) is reacted at RT with 1 M BH₃/THF solution (30 ml, 20 eq) and the reaction mixture is refluxed for 60 h. For decomposing the excess BH₃, MeOH is added dropwise with caution to the reaction mixture, which is precooied at 0° C. The MeOH is evaporated in vacuum and the residue is suspended in EtOH and sonicated for several minutes. The undissolved part is filtered off and HCl gas is introduced for 20 min to the filtrate. The formed precipitate is collected and further purified by column chromatography (SiO₂;CHCl₃/MeOH/25% NH₃ 5/5/2). The resulting purified amine is dissolved in EtOH and the corresponding hydrochloric salt is obtained as a white solid by passing HCl gas to this solution (460 mg). Alternatively, the precipitate is directly recrystallized from HCl gas saturated MeOH, without previous purification by chromatography.

Yield = 70%; Rf: 0.36 (SiO₂; CHCl₃/MeOH/25% NH₃ 5/5/2); MS (FAB) m/z (relative intensity): 296 (MH⁺, 100); ¹H-NMR (360 MHz, d₆-DMSO) (hydrochloride): 8.21 (d, 2H;O-Ar-NO₂), 7.68 (d, 2H, m-Ar-NO₂), 3.50-264 (m, 15H); ¹³C-NMR(90 MHz, d₆-DMSO): 146.29 (2C, C(Ar)-NO₂, CH₂-Ar), 130.46 (2C, Ar), 123.36 (2C, Ar), 47.56, 44.68, 35.04, 34.97.

The elemental analysis is consistent with C₁₄CI₄H₂₉N₅O₂.

N,N,N',N"'-Tetrabutyloxycarbonyl-6-(p-Nitrobenzyl)-1,4,8,11-tetraazaundecane (3)

1.5 g (3.5 mMol) (2) is suspended in 5 ml dioxane and 1N NaOH is added until pH12. At ice temperature 4.5 ml (21 m.Mol/6 eq) di-tert-butyl-dicarbonate in 30 ml dioxane and 40 ml 1N NaOH are added. The mixture is stirred at RT over night. To this mixture 100 ml diethylether and 50 ml H₂O are added. The ether layer is separated and extracted four times with 30 ml water, dried with Na_jSO^ and evaporated. The resulting oil cannot be crystallized but shows a structure confirming FAB-MS (m/z: 696) and is pure on TLC (Silica gel, RF = 0.5, acetic acid ethyl ester: hexane = 9: 1 ). Yield: 2.2 g, 90%. FAB-MS: m/z: 696. Η

NMR and IR are consistent with the structure. Example I

Synthesis of 6-(N-(Biotinylsulfoxide)-p-aminobenzyl)-1,4,8,11-tetraazaundecane(6) (Fig. 1)

Biotinsulfoxide is synthesized according to Melville (D.B. Melville, J. Biol. Chem., 1954, 208, 495). The sulfoxide exists in two isomeric forms:α-(+)-biotinsulfoxide and ß-(-)-biotinsulfoxide (Fig.2)

They can be separated by fractional crystallization and/or silica gel chromatography.

6-(p-Nitro-benzyl)-1,4,8,11-tetraaza-5,7-dioxoundecane (1)

Four mMol (1.07 g) p-nitrobenzylmalonic acid diethylester (G. Ruser et al., Bioconj. Chem. 1990, 2 345) is suspended in 40 ml methanol; 5.3 ml (80 mmol) ethylenediamine are added and the mixture is heated under reflux for 12 h. Solvent and excess ethylenediamine are evaporated and the crude product purified by silica gel chromatography (CHCl₃/MeOH/NH₃(25%) = 5:5:1, R_f= 0.3). Yield 1.4 g.

MS (FAB) m/z (relative intensity): 324 (MH⁺, 100); ¹H-NMR (300 MHz; d₆-DMSO). 8.14 (d. 2H, solution of sodium citrate dihydrate (5 mg). To this solution 200-400 μl (1000-2000 MBq) ^99mTcO₄ generator eluate and 20 μl (20 μg) of a freshly prepared N₂-purged SnCI₂. 2 H₂O solution (10mg/10 ml 0.1 M HCl) are added. The pH is adjusted to 10 with naOH. Incubation is done at room temperature for 30 min. Complexing yield is better than 97%. On a Hamilton PRP-1 (10 μm, 4.1 × 150 mm) column and isocratic elution with 100% 10 mM phosphate buffer (pH 7, 0-5 min), followed by a linear gradient (5-10 min; 20% MeCN/80% phosphate buffer) and isocratic elution up to 15 min the ^99mTc complex elutes at 10'04" (flow: 1.5 ml min). Under the same conditions (^99mTc)6-(p-aminobenzyl)-1,4,8,1 1-tetraazaundecane elutes at 8'45". The complex is stable in citrate and phosphate buffer for at least 10 h.

Example III

Binding of (^99mTc)-(6) to avidin

(^99mTc)-(6), obtained as described in Example II, is loaded onto a column of avidin coupled to beaded agarose which was preconditioned with 3 times 1.5 ml phosphate buffer (pH 8.9). The column is washed with 5 times 1.5 ml phosphate buffer. Activity retained on the column is 97+3% (n = 3). If the column is presaturated with cold biotin less than 2% of the activity is retained. So the conclusion is that the binding to avidin is specific.

Example IV

Stability of (^99mTc)-(6) in human serum

Biotinidase is an enzyme found in different organs and in human serum which functions as a biotin-amide amidohydrolase; e.g. it hydrolyzes (N-(d-biotinyl)-p-aminobenzoic acid and biocytin. The stability of the amide bond in fresh human serum is studied. Within 4 h of incubation the amount of intact (^99mTc)-(6), obtained according to Example II, decreases by about 15±2% which is by far superior to the corresponding conjugate with biotin. Considering in fact that the optimal time of scintigraphy with a ^99mTc-labelled biotin conjugate is within 2 h post injection, the stability of ^99mTc(6) in human serum is adequate for human studies. N,N',N",N'"-Tetrabutyloxycarbonyl-6-(p-aminobenzyl)-1,4,8,11-tetraazaundecane (4)

1.3 g (1.9 mmol) (3) is dissolved in 40 ml 80% MeOH; 150 mg Pd/C (10%) are added and hydrogenation is performed for 3 h at room temperature. The catalyst is removed by filtration, the mixture evaporated and pure product obtained by silica gel chromatography (Silica gel 60, 32 cm × 2 cm, eluent: hexane: acetic acid ethylester = 2:3). Yield 0,53 g (43%). The compound is pure on TLC and HPLC and is characterized by FAB-MS (m/z = 666) and ¹H-NMR.

6-(N-(blotinylsulfoxide)-p-amino-benzy)--,N",N'"-tetrabutyloxycarbonyl-1,4,8, 11-tetroazaundecane (5)

34.4 mg a-(+)-biotinsulfoxide are dissolved in 700 μl DMF; 42.5 mg HATU (O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorphosphate) and 23 μl diisopropylethylamine (Hünigbase) are added and the mixture left at room temperature for 10 min. Afterwards 88 mg of (4) and 23 μl Hünigbase in 1 ml DMF are added. Stirring is continued for 20 min. The reaction mixture is added to 2 ml ethylacetate and 2 ml 5% NaHCO₃ solution. The organic layer is washed twice with sodium bicarbonate and saturated sodium chloride solution, dried with sodium sulfate, evaporated and crystallized from diethylether/isopropylether/hexane. Yield: 96 mg (80%). The product is chromatographically pure and characterized by FAB-MS (m/z = 908).

6-{N-(biotinylsutfoxide)-p-aminobenzyl)-1,4,8,11-tetraazaundecane (6)

(Fig. 1)

96 mg (5) is dissolved in 2 ml trifluoracetic acid: thioanisole: water = 96:2:2 and left for 2 h at room temperature. Thereafter the product is precipitated by the addition of 2 ml diethylether and dried under high vacuum. Yield: 96 mg. To remove small impurities and remaining thioanisole the compound is purified by C18-HPLC. FAB-MS: m/z = 508.

Example II ^99mTc-Labelling of (6)

Fifty μg of (6) obtained as described in Example I, are dissolved in 300 μl of an aqueous 3. A labelled biotin compound as claimed in claim 1 or 2, wherein said chelating group R is selected from the group consisting of N_tP_qS_(4-t-q) tetradentate chelating agents, wherein t + q = 2-4, or groups derived from ethylene diamine tetra-acetic acid (EDTA), diethylene triamine penta-acetic acid (DTPA), cyclohexyl 1,2-diamine tetra-acetic acid (CDTA), ethyleneglycol-O,O'-bis(2-aminoethyl)-N,N,N',N'-tetra-acetic acid (EGTA), N,N-bis(hydroxybenzyl)-ethylenediamine-N,N'-diacetic acid (HBED), triethylene tetramine hexoacetic acid (TTHA), 1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tri and tetra-acetic acid (DO3A and DOTA), 1,4,7,10-tetraazacyclotridecane-N,N',N",N,"-tri and tetra-acetic acid

(TRI3A and TRITA), hydroxyethyldiamine triacetic acid (HEDTA), 1,4,8,11-tetra-azacyclotetradecane-N,N',N",N'"-tetra-acetic acid (TETA), substituted DTPA substituted EDTA or from deferoxamine, or from a compound of the general formula

wherein R₁ is a branched or non-branched, optionally substituted

hydrocarbyl radical, which may be interrupted by one or more hetero-atoms selected from N, O and S and/or by one or more NH groups, and

Q is a group which is capable of reacting with an amino group

of the peptide and which is preferably selected from the group consisting of carbonyl, carbimidoyl, N-(C₁-C₆)alkylcarbimidoyl, N-hydroxycarbimidoyl and

N-(C₁-C₆)alkoxycarbimidoyl.

4. A labelled biotin compound as claimed in claim 3, wherein said chelating group R is a group is selected from

Claims

1. A labelled biotin compound, wherein said biotin compound is represented by the general formula

wherein:

n is 1 or 2,

R is a chelating group for chelating a metal atom, and

Sp is a spacing group having at least 4 atoms spacing NH from R; and

wherein said biotin compound is labelled with a metal atom selected from (a) the group consisting of the radioactive isotopes ^99mTc, ²⁰³Pb, ⁶⁶Ga ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ¹¹¹In, 1^13mIn, ^114mIn ⁹⁷Ru, ⁶²Cu, ⁶⁴Cu, ⁵²Fe, ^52mMn, ⁵¹Cr, ¹⁸⁶Re, ¹⁸⁸Re, ⁷⁷As, ⁹⁰Y, ⁶⁷Cu, ¹⁶⁹Er, ^117mSn, ¹²¹Sn, ¹²⁷Te, ¹⁴²Pr, ¹⁴³Pr. ¹⁹⁸Au, ¹⁹⁹Au, ¹⁴⁹Tb, ¹⁶¹Tb. ¹⁰⁹Pd, ¹⁶⁵Dy, ¹⁴⁹Pm, ¹⁵¹Pm, ¹⁵³Sm, ¹⁵⁷Gd, ¹⁶⁶Ho, ¹⁷²Tm, ¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁷⁷Lu, ¹⁰⁶Rh and ¹¹¹Ag or (b) the group consisting of the paramagnetic metal atoms Cr, Mn, Fe, Co, Ni, Cu, Pr, Nd, Sm, Yb, Gd, Tb, Dy, Ho and Er; said metal atom being attached to the biotin compound by means of said chelating group.

2. A labelled biotin compound as claimed in claim 1 , wherein said spacing group Sp is a group of the general formula

wherein A is a biradical of the formula

m is an integer from 1 to 4;

p is an integer from 0 to 4;

X is O or S; and

Y is NH, CO or S. 18

Y is α valence bond or a functional group capable of binding with the spacing group; and

Z is S or O.

5. A method of preparing a labelled biotin compound as claimed in claim 1,

characterized in that a biotin compound of the general formula I, as shown in claim 1, wherein the symbols have the meanings given in claim 1, is reacted with a metal atom as defined in claim 1 in the form of a salt or of a chelate bound to a comparatively weak chelator, in order to form a complex.

6. A biotin compound to be used for the method according to claim 5, having the general formula

wherein:

n is 1 or 2,

R is a chelating group for chelating a metal atom, and

Sp is a spacing group having at least 4 atoms spacing NH from R.

7. A pharmaceutical composition, comprising in addition to a pharmaceutically acceptable carrier material and, if desired, at least one pharmaceutically acceptable adjuvant, as the active substance a labelled biotin compound as claimed in claim 1, 2, 3 or 4.

8. A method for detecting and localizing tumours in the body of a warm-blooded living being, which comprises (i) administering to said being a composition comprising

(strept)avidin conjugated with a polypeptide having a selective affinity to said tumour, (ii) thereupon, after avidination of said tumour, administering to said being a composition comprising, in a quantity sufficient for external imaging, a labelled biotin compound as claimed in claim 1, 2, 3 or 4, said biotin compound being labelled with (a) a radioactive metal Isotope selected from the group consisting of ^99mTc, ²⁰³Pb, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ¹¹¹In, ^113mIn, ⁹⁷Ru, ⁶²Cu, ⁶⁴Cu, ⁵²Fe, ^52mMn and ⁵¹Cr, or (b) with a paramagnetic metal atom selected from the group consisting of Cr, Mn, Fe, Co, Ni, Cu, Pr, Nd, Sm, Yb, Gd, Tb, Dy, Ho and Er, and

wherein:

R₂, is a hydrogen atom or a CO₂(C₁-C₄)alkyi group;

v is 0 or 1;

s is 2 or 3;

R₂₄ is CH₂COOH or a functional derivative thereof;

G is NH or S;

12. A method for the therapeutic treatment of tumours in the body of a warm-blooded living being, which comprises (i) administering to said being a composition comprising (strept)avidin conjugated with a polypeptide having a selective affinity to said tumour, 00 thereupon, after avidination of said tumour, administering to said being a composition comprising,, in a quantity effective for combating or confroiing tumours, a labelled biotin compound as claimed in claim 1, 2, 3 or 4, said biotin compound being labelled with a metal isotope selected from the group consisting of ^114mln ¹⁸⁶Re, ¹⁸⁸Re, ⁷⁷As, ⁹⁰Y, ⁶⁶Ga, ⁶⁷Cu,¹⁶⁹Er, ^117mSn, ¹²¹Sn, ¹²⁷Te, ¹⁴²Pr, ¹⁴³Pr, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁴⁹Tb, ¹⁶¹Tb, ¹⁰⁹Pd, ¹⁶⁶Dy, ¹⁴⁹Pm, ¹⁵¹Pm, ¹⁵³Sm, ¹⁵⁷Gd, ¹⁵⁹Gd, ¹⁶⁶Ho, ¹⁷²Tm. ¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁷⁷Lu, ¹⁰⁶Rh and ¹¹¹Ag.

13. A method for the therapeutic treatment of tumours in the body of a warm-blooded living being, which comprises (i) administering to said being a composition comprising a biotinylated polypeptide having a selective affinity to said tumour, (ii) then administering a composition comprising (strept)avidin, (iii) thereupon, after avidination of said tumour, administering to said being a composition comprising, in a quantity effective for combating or confroiing tumours, a labelled biotin compound as claimed in claim 1, 2, 3 or 4, said biotin compound being labelled with a metal isotope selected from the group consisting of ^114mln, ¹⁸⁶Re, ¹⁸⁸Re, ⁷⁷As, ⁹⁰Y, ⁶⁶Ga, ⁶⁷Cu, ¹⁶⁹Er, ^117mSn, ¹²¹Sn, ¹²⁷Te, ¹⁴²Pr, ¹⁴³Pr, ¹⁹⁸Au, ¹⁹⁹Au. ¹⁴⁹ Tb. ¹⁶¹Tb, ¹⁰⁹Pd, ¹⁶⁶Dy, ¹⁴⁹Pm, ¹⁵¹Pm, ¹⁵³Sm. ¹⁵⁷Gd, ¹⁵⁹Gd, ¹⁶⁶Ho, ¹⁷²Tm, ¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁷⁷Lu, ¹⁰⁵Rh and

¹¹¹Ag.

14. A Wt for preparing a radiopharmaceutical composition, comprising (a) a composition comprising (sfrept)avidin conjugated with a polypeptide having a selective affinity to tumours, (b) a biotine compound of the general formula I, shown in claim 1, wherein the symbols have the meanings given in claim 1, to which compound, if desired, an inert pharmaceutically acceptable carrier and/or formulating agents and/or adjuvants is/are added, (c) a solution of a salt or chelate of a metal isotope selected from the group consisting of ²⁰³Pb, ⁶⁶Ga, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ^{1 11}In, ^113mIn, ^114mIn, ⁹⁷Ru, ⁶²Ga, ^99mTc, ¹⁸⁶Re, ¹⁸⁸Re, ⁶⁴Cu, ⁵²Fe, ^52mMn, ⁵¹Cr, ⁷⁷As, ⁹⁰Y, ⁶⁷Cu, ¹⁶⁹Er, ^117mSn, ¹²¹Sn, ¹²⁷Te, ¹⁴²Pr, ¹⁴³Pr, ¹⁹⁸Aa ¹⁹⁹Au, ¹⁴⁹Tb, ¹⁶¹Tb, ¹⁰⁹Pd,

¹⁶⁶Dy, ¹⁴⁹Pm, ¹⁵¹Pm, ¹⁵³Sm, ¹⁵⁷Gd, ¹⁶⁶Ho, ¹⁸²Tm, ¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁷⁷Lu, ¹⁰⁶Rh and ¹¹¹Ag, and (d) instructions for use with a prescription for reacting the ingredients present in the kit.

15. A kit for preparing a radiopharmaceutical composition, comprising (a) a composition comprising a biotinylated polypeptide having a selective affinity to tumours, (b) a composition comprising (strept)avidine, (c) a biotine compound of the general formula I, shown in claim 1, wherein the symbols have the meanings given in claim 1, to which (iii) finally subjecting said being to external imaging to determine the targeted sites in the body of said being in relation to the background activity.

9. A method for detecting and localizing tumours in the body of a warm-blooded living being, which comprises (i) administering to said being a composition comprising a biotinylated polypeptide having a selective affinity to said tumour, (ii) then administering a composition comprising (strept)avidin, (iii) thereupon, after avidination of said tumour, administering to said being a composition comprising, in a quantity sufficient for external imaging, a labelled biotin compound as claimed in claim 1, 2, 3 or 4, said biotin compound being labelled with (a) a radioactive metal isotope selected from the group consisting of ^99mTc, ²⁰³Pb, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ¹¹¹In, ^113mln. ⁹⁷Ru, ⁶²Cu, ⁶⁴Cu, ⁵²Fe, ^52mMn and ⁵¹Cr, or (b) with a paramagnetic metal atom selected from the group consisting of Cr, Mn, Fe, Co, Ni, Cu, Pr, Nd, Sm, Yb, Gd, Tb, Dy, Ho and Er, and (iv) finally subjecting said being to external imaging to determine the targeted sites in the body of said being in relation to the background activity.

10. A method of intraoperatively detecting and localizing tumours in the body of a warm-blooded living being, which comprises (i) administering to said being a composition comprising (strept)avidin conjugated with a polypeptide having a selective affinity to said tumour, (ii) thereupon, after avidination of said tumour, administering to said being a composition comprising, in a quantity sufficient for detection by a gamma detecting probe, a labelled biotin compound as claimed in claim 1, 2, 3 or 4, said biotin compound being labelled with ¹⁶¹Tb, and (iii) finally, after allowing the active substance to be bound by and taken up in said tumour and after blood clearance of radioactivity, subjecting said being to a radioimmunodetection technique in the relevant area of the body of said being, by using a gamma detecting probe.

11. A method of intraoperatively detecting and localizing tumours in the body of a warmblooded living being, which comprises (i) administering to said being a composition comprising a biotinylated polypeptide having a selective affinity to said tumour, (ii) then administering a composition comprising (sfrept)avidin, (iii) thereupon, after avidination of said tumour, administering to said being a composition comprising, in a quantity sufficient for detection by a gamma detecting probe, a labelled biotin compound as claimed in claim 1, 2, 3 or 4, said biotin compound being labelled with ¹⁶¹Tb, and (iv) finally, after allowing the active substance to be bound and taken up in said tumour and after blood clearance of radioactivity, subjecting said being to a radioimmunodetection technique in the relevant area of the body of said being, by using a gamma detecting probe. compound, if desired, an inert pharmaceutically acceptable carrier and/or formulating agents and/or adjuvants is/are added, (d) a solution of a salt or chelate of a metal isotope selected from the group con-sisting of ²⁰³Pb, ⁶⁶Ga, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ¹¹¹In, ^113mln, ^114mIn, ⁹⁷Ru, ⁶²Cu, ^99mTc, ¹⁸⁶Re, ¹⁸⁸Re, ⁶⁴Cu, ⁵²Fe, ^52mMn, ⁵¹Cr, ⁷⁷As, ⁹⁰Y, ⁶⁷Cu, ¹⁶⁹Er, ^117mSn, ¹²¹Sn, ¹²⁷Te, ¹⁴²Pr, ¹⁴³Pr, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁴⁹Tb, ¹⁶¹Tb, ¹⁰⁹Pd, ¹⁶⁵Dy, ¹⁴⁹Pm, ¹⁵¹Pm, ¹⁵³Sm, ¹⁵⁷Gd, ¹⁶⁶Ho, ¹⁷²Tm, ¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁷⁷Lu, ¹⁰⁵Rh and ¹¹¹Ag, and (e) instructions for use with a prescription for reacting the ingredients present in the Wt.

16. A Wt for preparing a radiopharmaceutical composition, comprising (a) a composition comprising (sfrept)avidin conjugated with a polypeptide having a selective affinity to tumours, (b) a biotine compound of the general formula I, shown in claim 1, wherein the symbols have the meanings given in claim 1, to which compound, if desired, an inert pharmaceutically acceptable carrier and/or formulating agents and/or adjuvants is/are added, (c) a reducing agent, and, if desired, a chelator, and (d) instructions for use with a prescription for reacting the ingredients of the kit with ^99mTc in the form of a pertechnetate solution or with ¹⁸⁶Re or ¹⁸⁸Re in the form of a perrhenate solution.

17. A Wt for preparing a radiopharmaceutical composition, comprising (a) a composition comprising a biotinylated polypeptide having a selective affinity to tumours, (b) a composition comprising (strept)avidine, (c) a biotine compound of the general formula I, shown in claim 1, wherein the symbols have the meanings given in claim 1, to which compound, if desired, an inert pharmaceutically acceptable carrier and/or formulating agents and/or adjuvants is/are added, (c) a reducing agent, and, if desired, a chelator, and (d) instructions for use with a prescription for reacting the ingredients of the kit with^99mTc in the form of a pertechnetate solution or with ¹⁸⁶Re or ¹⁸⁸Re in the form of a perrhenate solution.