WO1993018797A1

WO1993018797A1 - Method of intraoperatively detecting and locating tumoral tissues

Info

Publication number: WO1993018797A1
Application number: PCT/US1993/002772
Authority: WO
Inventors: Geert Jacob Ensing; Karel Jan Panek; Bareld Jan Doedens
Original assignee: Mallinckrodt Medical, Inc.
Priority date: 1992-03-25
Filing date: 1993-03-24
Publication date: 1993-09-30
Also published as: JPH07505621A; EP0636032A1; AU3967593A; CA2131315A1

Abstract

The invention relates to a method of intraoperatively detecting and locating tumoral tissues in the body of a warm-blooded living being, comprising (a) parenterally administering to said being a pharmaceutical composition comprising, in a quantity sufficient for detection by a gamma detecting probe, a peptide compound labelled with a low-energy gamma photon emitting radionuclide, and then (b), after allowing the active substance to be taken up in the tumoral tissues and after blood clearance of radioactivity, subjecting said being to a radioimmunodetection technique by using a gamma detecting probe. The invention further relates to a method of radioguided surgery.

Description

Method of intraoperativelv detecting and locating i

The invention relates to a method of intraoperativt; / detecting and locating tumoura tissues in the body of a warm-blooded living being, to a method of radioguide surgery of said being, and to a radiopharmaceutical composition to be used for th latter method. The invention further relates to a labelled peptide compound to b used in said composition and to a kit for preparing said composition.

The accurate staging of tumours, in particular malignant tumours, in general remain one of the most important clinical challenges. Often such tumours or thei me astases are extremely small «1 cm), and because of this small size they are no readily detectable and distinguishable using conventional imaging techniques. Even the use of advanced imaging techniques, such as SPECT acquisition techniques, in combination with tumour-selective imaging agents, is frequently unable to show all lesions, because of disturbing background activity that makes accurate image interpretation difficult. Especially in the abdominal area it is often difficult t distinguish benign for malignant tissues using conventional imaging methods. An example of such a subgroup of tumours are gastro-enteropancreatic tumours that produce hormones, which result in sometimes life-threatening symptoms, e.g. massive diarrhea. The obvious therapy in these cases is to surgically remove thes tumours. However, their often small size makes imaging techniques not reliable i accurately locating the lesion, while the surgeon is simply not capable of finding th tumours quickly and, moreover, is in fact not sure that aJi lesions are operativel removed.

A relatively new technique provides surgical aid: a gamma detecting probe, e.g. Neoprobeζ that can be used to detect sources of gamma radiation that are ver small. After parenteral administration of a radiolabelled substance, the surgeon can intraoperatively, use this probe to find the lesions in which uptake of this substanc has taken place. E.W. Martin and coworkers have investigated this new technique e.g. Amer. J. Surgery 1^6, 1988, 386-392; Antibody Immunocon. Radiopharm. 4 1991 , 339-358. These investigators have observed that antibodies or antibod fragments, labelled with iodine-125, a low-energy gamma photon emittor, ar promising substances to be used in this technique. They indicate that this techniqu may successfully target 80% of colorectal cancer and detect occult tumours in th abdomen in 20% of the surgical cases involving cancer of the colon. Although it i generally recognized that this improvement in diagnosing enables the surgeon t better resect tumour deposits, in particular those tumours and metastases which cannot be seen or palpated, and so contributes to the chance of curing cancer patients, the results of this technique are not yet satisfactory. The known radiolabelled substances generally show an insufficiently selective tumour uptake and, in particular, a not sufficiently fast blood clearance, so that the tumour to background ratio is often inadequate for accurate detection.

It is the object of the invention to provide a method of intraoperatively detecting and locating tumoural tissues in the body of a warm-blooded living being by using a radiolabelled substance showing an improved and more selective tumour uptake and a much faster blood clearance.

This object can be achieved according to the present invention by a method as mentioned above, comprising (a) parenterally administering to said being a pharmaceutical composition comprising, in a quantity sufficient for detection by a gamma detecting probe, a peptide compound labelled with a low-energy gamma photon emitting radionuclide, said peptide compound being derived from a peptide selected from the following groups: (i) peptides having a selective neurokinin 1 receptor affinity and having the general formula

R₁-(A₁)_m-A₂)_n-A₃)₀-Pro)_p-A₄)_<l-A_s-Phe-A₆-A₇-A₈-NH-CH-R₂

Re

(I)

wherein all of the symbols m, n, o, p and q are 1 , or all but one of the symbols m, n, o, p and q are 1 , and the remaining symbol is 0;

R, is a hydrogen atom or a C^C* alkylcarbonyl group; R₂ is a carbamoyl group, a carboxy group, a C,-C₄ alkoxycarbonyl group, a hydroxymethyl group or a C^C* alkoxymethyl group;

A, is Arg, Gly or 5-oxo-Pro (pGlu);

A₂ is Pro or β-Ala;

A₃ is Lys or Asp; A« is Gin, Asn or 5-oxo-Pro;

A₅ is Gin, Lys, Arg, N-acγlated Arg or 5-oxo-Pro; or wherein A₅ together with A₃ forms a cystine moiety; A_β is Phe or Tyr; A₇ is Gly, Sar or Pro; A₈ is Leu or Pro; and R_β is a straight or branched C₂-C₄ alkyl group, which group may be inter rupted by thio, sulphinyl or sulphonyl; and their Tyr⁰ derivatives;

(ii) peptides having a selective somatostatin receptor affinity and having th general formula

(II)

wherein R, and R₂ have above meanings,

B, and B₂ are each independently Phe, MePhe, EtPhe, Tyr, Trp and Nal,

B₃ is Lys or MeLys,

B₄ is Thr or Val, and R₇ is a 1 -hydroxyethyl group or an indol-3-ylmethyl group; and their Tyr⁰ derivatives;

and —

(iii) peptides selected from cytokines, growth factors and hormones, as well as thei derivatives and analogues;

and then (b), after allowing the active substance to be taken up in the tumour tissues and after blood clearance of radioactivity, subjecting said being to radioimmunodetection technique in the relevant area of the body of said being, b using a gamma detecting probe.

In the above description of the invention the symbol Nal means a naphthylalan group, and Sar means a sarcosyl group.

Suitable examples of substituent R_β are (CH₂)₂S(0),CH₃, wherein s is 0,1 or 2, an CH₂CH(CH₃)₂.

Suitable examples of cytokines are tumour necrosis factor (TNF), in particular TN o , interleukines (IL), in particular IL-1 , IL-2, IL-4, IL-5 and IL-6, and interferons. Suitable examples of growth factors are epidermal growth factor (EGF), insulin-like growth-factor (IGF), in particular IGF-I (somatedin C) and IGF-II, bombesin, transforming growth factor (TGF), in particular TGF-oCand TGF- i, platelet-derived growth factor, fibroblast growth factor and nerve growth factor. Suitable examples of hormones are luteinizing hormone-releasing hormone (LHRH), gastrin, gastrin-releasing peptide, angiotensin, thyroid-stimulating hormone, vasoactive intestinal polypeptide, prolactin, thyrotropin-releasing hormone, insulin, adrenocorticotropic hormone (ACTH), in particular 0 -MSH (melanocyte-stimulating hormone) and i -(methγlsulfonyl)-L- oi-aminobutyryl-L- < -glutamyl-L-histidyl-L- phenylalanyl-D-lysyl-L-phenylalanine, cholecystokinin, corticotropin-releasing hormone (CRH), growth hormone-releasing hormone (GRH), arginine and lysine vasopressin, oxytocin, glucagon, secretin, parathyroid hormone (PTH) and PTH related peptide.

By using the above method of the invention, virtually all malignant tumours can be detected and located and can be distinguished from benign tissues, because these tumours contain substantially large numbers of receptors for binding the above peptide compound.

The above-defined peptides are composed of amino acids, of which at least one may have the D-configuration. The peptides may also comprise so-called pseudo peptide bonds, viz. -CH₂-NH- bonds, in addition to the natural amide bonds, viz. -CO-NH- bonds.

Labelled peptide compounds for external imaging according to conventional imaging techniques and consequently labelled with radioisotopes suitable for this purpose, such as Ga-67, ln-1 1 1 and Tc-99m, are described in literature. Peptide compounds, labelled in this manner and derived from peptides mentioned sub (i) above, are the subject of the non-prepublished European patent application no. 91200955.2 in the name of Applicants. Equally labelled peptide compounds derived from peptides mentioned sub (ii) and sub (iii) above are known from the published international patent applications WO 90/06949 and WO 91/01 144, respectively.

Suitable examples of peptides sub (i) above, which can be used as indicated above, are: (1 ) H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH₂ (Substance P), (2) H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Sar-Leu-Met(0₂)-NH₂,

(3) H-β-Ala-Gln-Gln-Phe-Phe-Sar-Leu-Met(0₂)-NH₂,

(4) H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Tyr-Gly-Leu-Met-NH ₂,

(5) H-Arg-Pro-Cys-Pro-Gln-Cys-Phe-Tyr-Pro-Leu-Met-NH₂, and Tyr⁰ derivatives thereof.

Suitable examples of peptides sub(ii) above, which can be used as indicated above, are:

6) H-(D)Phe-C Iys-Phe-(D)Trp-Lys-Thr-Cy 1s-Thr-ol (Octreotide), l 1

(7) H-(D)Phe-Cys-Thr-(D)Trp-Lys-Val-Cys-Thr-NH₂,

(8) H-(D)Phe-C Iys-Tyr-(D)Trp-Lys-Val-C 1ys-Trp-NH₂,

(9) H-(D)Trp-Cys-Phe-(D)Trp-Lys-Thr-Cys-Thr-NH₂ ,

(10) H-(D)Phe-Cys-Phe-(D)Trp-Lys-Thr-Cys-Thr-NH₂,

(1 1 ) H-(D)Nal-Cys-Tyr-(D)Trp-Lys-Val-Cys-Thr-NH_a ,

(12) H-(D)Nal-Cys-Tyr-(D)Trp-Lys-Val-Cys-Nal-NH₂,

(13) H-(D)Nal-Cys-Nal-(D)Trp-Lys-Val-Cys-Thr-NH₂,

I I

(14) H-(D)Phe-Cys-Phe-(D)Trp-Lys-Thr-Cys-Nal-NH ₂,

(15) H-(D)Phe-Cys-Tyr-(D)Trp-Lγs-Thr-Cγs-Thr-ol, and Tyr⁰ derivatives thereof.

Suitable examples of peptides sub (iii) above, which can be used as indicated above, are: EGF, TGF-c , gastrin, bombesin and derivatives of these peptides.

Suitable low-energy gamma photon emitting radionuclides which can be used as labels for the peptide compounds to be used in the method of the present invention should have a gamma energy of approx. 80 keV at most. Such radionuclides are well tuned to the gamma detecting microprobe to be manipulated by the surgeon and intended to register the emitted gamma radiation. Such a hand-held microprobe, e.g. Neoprobe 1000®, is at present equipped with a miniature cadmium telluride crystal detector. Such a detector requires for optimum detection properties gamma energies in the range of approx. 30-80 KeV. Higher energies may cause excessive scattering so that the accuracy of the detection is considerably decreased. After uptake of the labelled peptide in the tumoural tissues and after blood clearance of radioactivity to avoid disturbing background activity, the surgeon can use his gamma detecting probe during operation to be sure that he/she does not overlook small-sized tumours. The well-tuned label enables the surgeon to accurately detect and locate such small tumours with the aid of the hand-held microprobe in order to guide the surgery treatment. Examples of suitable radionuclides for labelling the above peptide compounds are 1-125, As-73, Sb- 1 19, Cs-131 , Dy-159, W-181 and Hg-197.

The desired radioisotope should be firmly attached to the peptide molecule to reduce the chance of detaching this label after administration to the living being. The peptide can be labelled with the desired isotope directly or indirectly, i.e. via a so-called linker. Direct labelling may be carried out, for example, by introducing a halogen atom or radioactive halogen atom, i.e. iodine-125, into an activated aromatic group (e.g. tyrosyl or imidazolyl) .present in the peptide, into the peptide molecule in a manner known per se, if desired followed by exchange with 1-125. Tyrosine and histidine are suitable amino acids which, if present in the peptide molecule, allow an easy substitution with (radioactive) halogen. Often, however, the labelling procedure is performed via a suitable linker, being capable of reacting with an amino group, preferably a terminal amino group, of said peptide, and having a functional group for binding said radioisotope. By using a suitable linker, the desired isotope can generally better be introduced into the peptide molecule.

It is of advantage to attach the linker to a terminal amino group of the peptide molecule, in order to affect the biological properties of this peptide as least as possible.

Suitable linkers for labelling the peptide with metal radionuclides are discussed in detail hereinafter. A suitable linker for labelling the peptide with 1-125 is derived from tyrosine or from Bolton-Hunter reagent, i.e. N-succinimidyl-3-(4-hydroxy-3- halo*phenyl)propionate, wherein halo* means iodine-125. Preferably, however, the peptide is first reacted with tyrosine or with a "halo-deprived" Bolton-Hunter reagent, viz. N-succinimidyl-3-(4-hydroxyphenyl)propionate, after which the derivatised peptide, thus obtained, is substituted by the desired halogen radioisotope by an appropriate reaction. Both by using the latter, more convenient reaction route and by employing the former method, the peptide can be labelled with the desired radioactive haiogen isotope without affecting its biological properties.

The above radioiodinating reaction is preferably performed by reacting the peptide in question with a solution of an alkali metal radionuclide from 1-125 iodide, under the influence of a halide-oxidizing agent, such as chloramine T or iodogen. Alternatively, the above substitution reaction can be carried out with a non-radioactive halogenide, after which halo-exchange with radioactive halogen is performed, e.g. as described in European patent 165630.

In general, the tumours and metastases which can be detected and located by using the method of the present invention, are small soft tissue tumours which are firmly attached to the surrounding benign tissues. This makes the surgical removal of such tumours after their detection often difficult. It is another object of the present invention to facilitate the therapeutic treatment of such tumoural tissues and consequently to improve the radioguided surgery.

It is a particular merit of the present invention to combine detection and improved therapy. Consequently, the present invention also and in particular relates to a method of radioguided surgery of a warm-blooded living being, which method, in addition to the method as discussed hereinbefore and intended to detect and locate tumoural tissues, comprises (i) parenterally administering to said being a pharmaceutical composition comprising, in a quantity sufficient for at least partial necrosis of tumoural tissues, a peptide compound derived from a peptide as defined hereinbefore and labelled with an isotope with sufficiently high specific activity and emitting corpuscular radiation, preferably selected from the group consisting of radionuclides as reviewed by Schubiger et al. (in 6th Int. Symp. Radiopharm. Chem., Boston 1986, paper no. 149) or by Volkert et al. (in J. Nucl. Med., 1991 , Vol. 32 (1 ), 174-185), such as the radionuclides selected from the group consisting of P- 32, S-35, As-77, Y-90, Rb-105, Ag-1 1 1 , Sn-121 , Te-127, Re-186, Re-188, Au-198, Au-199 and radionuclides of the lanthanide group emitting corpuscular radiation; and then (ii), after allowing the active substance to be taken up in the tumoural tissues and to cause an at least partial necrosis of said tissues, subjecting said being to a surgical treatment.

Suitable examples of the last-mentioned lanthanide radionuclides are Pr-142, Pr-143, Pm-149, Pm-151, Sm-153, Gd-159, Tb-161 , Dy-165, Ho-166, Er- 169, Tm-172, Yb-169, Yb-175 and Lu-177.

Because the above isotopes have sufficient corpuscular emissions to be useful for therapeutic purposes, an injected dose has a cell killing effect due to the uptake in the tumoural tissues in question, leading to an at least partial necrosis of the tumour cells. This enables the surgeon to more easily remove these tumours by excision during surgery. The overall result can be a treatment schedule, wherein an optimum use is made of this combination of detection and therapy. After uptake of the "detecting" peptide compound in the tumoural tissues, the gamma emissions permit the accurate detection of these tissues with a microprobe. These tissues, however, have already been

"attacked" by the corpuscular radiation caused by uptake of the peptide compound labelled with one of the above corpuscular radiation emitting isotopes. The surgical treatment of these malignant tissues, i.e. the excision of the already at least partially necrotic tissues, is therefore highly facilitated.

It is of advantage to use for both detection and for therapy the same pharmaceutical composition, comprising a peptide compound labelled with a low-energy gamma photon emitting radionuclide and a corpuscular radiation emitting isotope as defined hereinbefore. In this manner, viz. by using exactly the same peptide as a starting material, the surgeon can be sure that the specific affinity to the tumoural tissues to be removed is exactly the same for the diagnostic agent as for the therapeutic agent.

Surprisingly it has been found, that certain lanthanide radionuclides are very suitable to perform both functions at the same time, viz. have a suitable gamma energy for detection and have a corpuscular radiation emitting effectiveness sufficient for tumour cell necrosis. By combining these both functions in one and the same radionuclide, it is guaranteed that the uptake in the tumour cells is optimal for both intended effects. In this connection terbium-1 61 (Tb-161 ) is pre-eminently suitable. This lanthanide radionuclide combines an optimum gamma energy for microprobe detection, viz. in the range of 25-74 keV, with a beta emission suitable for therapeutic treatment of tumour cells, viz. in the range of 250-590 keV. In addition, the half-life of terbium-161 is extremely appropriate for the intended purpose, viz. 6.91 days. This means that sufficiently long after injection the blood clearance of radioactivity is sufficiently complete to permit accurate detection of the tumoural tissues, while at the same time necrosis of the malignant tissue cells has advanced sufficiently to allow easy excision of the detected tumours. Finally, this preferred lanthanide radionuclide is readily accessible and can be produced carrier-free by irradiation of highly enriched gadolinium Gd-160 in a nuclear reactor.

Preferably the labelled peptide compound to be used in the method of the invention, for detecting purposes or both for detection and for therapy, is provided, directly or through a spacing group, with a chelating group. This chelating group is attached by an amide bond to an amino group of said peptide and is derived from ethylene diamine tetra-acetic acid (EDTA), di¬ ethylene triamine penta-acetic acid (DTPA), ethyleneglycol-0,0'-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'"-tetra-acetic acid (DOTA), 1 ,4,8,1 1 -tetra-azacyclotetradecane-N,N',N",N'"-tetra-acetic acid

(TETA), 1 ,2-diaminocγclohexane tetra-acetic acid (DCTA), substituted DTPA, substituted EDTA, or from a compound of the general formula

- S — Y — (III)

wherein R is a branched or non-branched, optionally substituted hydrocarbyl radical, which may be interrupted by one or more hetero-atoms selected from N, 0 and S and/or by one or more NH groups, and Y is a group which is capable of reacting with an amino group of the peptide and which is preferably selected from the group consisting ofcarbonyl,carbimidoyl,N-(C,-C_β)alkylcarbimidoyl, N-hydroxycarbi- midoyl and N-(C,-C₆)-aIkoxycarbimidoyl; and wherein said optionally present spacing group has the general formula

-NH— R„

wherein R₅ is a C,-C₁₀ alkylene group, a C,-C₁₀ alkylidene group or a C₂-C₁₀ alkenylene group, and X is a thiocarbonyl group or a methylcarbonylgroup.

Examples of suitable chelators of the general formula HI are unsubstituted or substituted 2-iminothioIanes and 2-iminothiacyclohexanes, in particular 2- ϊmino-4-mercaptomethylthiolane.

It has been observed, that the above preferred peptide compound, provided with a chelating group, shows a very fast clearance. This is of great advantage, because the use of this preferred peptide compound after labelling allows surgery a very short time after administration, if desired from medical considerations.

The invention further relates to a radiopharmaceutical composition to be used for the method of radioguided surgery as defined above, which composition comprises in addition to a pharmaceutically acceptable carrier and, if desired, at least one pharmaceutically acceptable adjuvant, as the active substance a peptide compound labelled with a low-energy gamma photon emitting radionuclide and a corpuscular radiation emitting isotope as defined hereinbefore. If desired, the composition can be brought into a form more suitable for parenteral administration, e.g. by adding a pharmaceutically acceptable liquid carrier material. For parenteral administration the solution should of course be in a sterile condition.

The above composition preferably comprises as the active substance a peptide compound provided with a chelating group as defined above, said chelating group chelating a metal radionuclide. The preferred radionuclides of the lanthanide group have excellent characteristics for being chelated with the above aminoacetic acid based chelating agents, thus assuring a good and stable binding to the chelating group of the peptide compound. In this chelating or complex-forming reaction, the radioisotope is presented to the chelating group comprising peptide compound in the form of a salt of a suitable acid, e.g. a mineral acid or acetic acid. The complex-forming reaction can generally be carried out in a simple manner and under conditions which are not detrimental to the peptide.

The invention further relates to a labelled peptide compound to be used as an active ingredient in the above composition, said peptide compound having a selective affinity to endocrinically active tumours and being labelled with a low-energy gamma photon emitting radionuclide and a corpuscular radiation emitting isotope, as defined hereinbefore, preferably with a suitable lanthanide radionuclide having the above-defined radiation characteristics.

The invention finally relates to a so-called cold kit for preparing a radiopharmaceutical composition, comprising (i) a peptide provided with a chelating group as defined hereinbefore, to which substance, if desired, an inert pharmaceutically acceptable carrier and/or formulating agent(s) and/or adjuvant(s) is/are added, (ii) a solution of a salt of a lanthanide radionuclide having the above-defined radiation characteristics, and (iii) instructions for use with a prescription for reacting the ingredients present in the kit.

Such a kit as described above can be delivered to the user. The user can easily perform the labelling of the chelated peptide with the lanthanide radionuclide by himself or herself in the clinical hospital or laboratory. The labelling procedure is simple and does not require complicated manipulations, so that the user is able to prepare the labelled composition from the kit ingredients by using the facilities that are at his or her disposal. The radionuclide is the only ingredient in the kit having a restricted shelf life. Therefore, if desired, the lanthanide radionuclide may be delivered separately and substituted after its expiration date.

The invention will now be described in greater detail with reference to the following specific Examples. EXAMPLE 1.

A. Preparation of DTPA-Octreotide kit

The DTPA-Octreotide kit formulated on basis of sodium acetate buffer with the final composition

3.89 mg sodium acetate 0.029 mg acetic acid 10 μg DTPA-Octreotide

per vial is prepared as follows:

First the following solutions are made:

acetic acid solution 0.06M, by diluting 35.9 mg glacial acetic acid to 100.0 ml with water;

- sodium acetate solution 0.286M, by dissolving 3.89 g of sodium acetate 3H₂0 in 100 ml water.

To formulate the kit, 0.5 mg of DTPA-Octreotide is dissolved in 4 ml of acetic acid solution, and 5 ml of sodium acetate solution are added.

To this mixture are added 16 ml water to make 25 ml of final solution, which is subsequently filtered through a 0.22μ bacterial filter. The filtrate is then dispensed in 0.5 ml portions per vial and vials are lyophilized. The final freeze dried product is stored at 4C. In a similar way, starting from 2.5 mg DTPA-Octreotide was also prepared and a kit containing 50μg DTPA-Octreotide per vial.

B. Preparation of Tb-161 solution

2 mg of enriched (98.1%) 160-Gd₂O₃ is irradiated for 48 hours in a nuclear reactor with thermal neutron flux 2 x 10¹⁴ n/cm² sec. After ca 30 hours of cooling the sample is dissolved in 2 portions of 1 ml warm (70C) ION HCI directly in the irradiation quartz ampoule, the solutions are transferred to a 20 ml quartz beaker and combined with 3 portions of 1 ml washing water. The solution is 2 times evaporated with ION HCl till dry and the dry rest is taken up in few ml of

0.02N HCl and diluted to 10 ml with 0.02N HCl, Irradiation yield is ca 11.5 Ci Tb-161.

For the separation of Tb-161, 5 ml of the Gd/Tb stock solution in 0.02N HCl is evaporated to dry and taken up in 200μl of 0.02N HCl. This solution is loaded on a 0.8 x 12 cm column of SCX BioRad® 50 W-X8, 200-400 mesh, in NH₄+ form. As an eluent is used a 0.2 M solution of α-hydroxy- isobutyric acid, adjusted to pH 4.1 with ammonia. Fractions of 1 ml of eluate are collected for radiodnuclide identification. Combined fractions containing Tb-161 are made of 0.5N in HCl and run over a second small column of BioRed® 50 -X8 in H⁺ form. The loaded column is then washed with 0.5 and 1.5N HCl, followed with water to remove the excess of α-hydroxy-isobutric acid. Tb-161 is finally stripped from the column with 6N HCI. The strip solution is again evaporated to dryness. The residue is taken up into 0.001N HCI (4 ml) and used for analysis and labelling experiments. Analysis :

Radionuclide purity determined by γ spectrometry (ND 66 γ spectrometer Ga/Li detector) : substantiallyl 100%; no other radionuclide detected. Radiochemical purity: Thin layer chromatography -ITL SG (Gelman) plates, ascending, solvent, 1M sodium acetate pH 5. Result: single peak on front, 98.9% Tb-161 activity.

C. Labelling of DTPA-Octreotide kit with Tb-161.

Several kits of DTPA-Octreotide, prepared according to Example 1 containing 10 or 50 μg DTPA-Octreotide, are labelled by addition of 0.5 ml of Tb-161 solution obtained under B. The mixture is incubated for 30 min. at room temperature.

Analysis:

ITLC as described above,

Tb-161-DTPA-Octreotide Rf ca 0.5-0.6 Free Tb-161 Rf ca 0.9-1.0

Hydrolysed Tb-161 Rf ca 0.0-0.1

HPLC: Column: μBondapak®C 18 10μm, 3.9 x 300 mm

Eluent: 0.07M acetate buffer pH 5.5 (a), 100% MeOH (b) , a and b mixed in ratio 6:4 v/v. Gradient: 40-80% b in 20 min. Operation: Flow rate 1 ml/min., temperature 35C. Detection: Dual, Nal crystal, UV detector at

280nm.

Results of labelling experiment: (at time interval between addition of Tb-161 activity and analysis) LY = Labelling yield. Time (h) LY 50μ*

0.5 >92%

3 >92%

24

>93%

challenge experiment with serum (bovine) , added at 24 h

48 76.4% >95%

* Free Tb-161 was not detectable in any kit containing 50 μg DTPA-Octreotide.

Radiochemical purity - 50 μg at 3 h - HPLC 96.2%

HPLC identification positive, because UV spectrum and activity peaks of Tb-161 are found identical with^' those for In-IIl labelled DTPA-Octreotide used as control.

To obtain an injectable preparation with radiochemical purity > 98%, the labelled solution is purified over a Sep- Pak^RC18 cartridge, which after loading is washed with water (5 ml) and eluted with methanol (5 ml), the latter fraction containing Tb-161 Octreotide. Evaporation and dissolution of the residue in physiologic saline solution give after sterilization (membrane filtration) the desired injectable preparation.

EXAMPLE II Labelling of DTPA-Octreotide kit with Yb-175 and its use in combination with detecting agent DTPA-125-I-Tyr³-Octreotide

A. Labelling of DTPA-Octreotide kit with Yb-175.

Ca 1 mg of enriched (97.8%) 174-Yb₂0₂ is irradiated for 48 hours in a nuclear reactor with thermal neutron flux 2 x 10¹⁴ n/cm².sec.

After 30 hours cooling time the sample is dissolved directly in the irradiation quartz ampoule in 2 x 1-ml portions of warm (70C) concentrated HCl.

The obtained solution is withdrawn and trasferred to a small quartz beaker, the ampoule is washed with 3 x 1-ml portions of water and the washings are combined with the active solution. The solution of Yb-175 chloride is twice evaporated to dryness with concentrated HCl and the rest is taken up into 2 x 5-ml of 0.02N HCl, transferred to a 20 ml volumetric flask and diluted to the desired volume with 0.02N HCl.

A 1.0 ml aliquot of this solution is diluted to 50.0 ml with 0.02N HCl to obtain a stock solution of Yb-175 chloride, used for the labelling experiments. This solution (1 ml) has a specific activity of 400 μCi/μg Yb- 175, a radionuclide purity of > 99% and a radiochemical purity of >_^ 99.9%; both values are determined by the methods described in Example 1.

Several kits containing 10 μg of DTPA-Octreotide prepared according to Example 1 are labelled by addition of 1 ml of the Yb-175 stock solution. The mixture is let to incubate 30 min. at room temperature. Samples for analysis are taken at time intervals indicated by the results. Used analytical methods are described in Example 1.

Results:

Radiochemical purity Yb-175:ITLC, IM Na-acetate Rf 0.9-1.0 99.5%

Yb-175 Octreotide: LY at 30 min. ITLC Rf 0.5-0.6 98.1%

SepPak® 98.8%

LY at 75 min. HPLC 90.7%

Identity of Yb-175 labelled Octreotide is confirmed as described above for the Tb-161 labelled peptide compound.

Challenge experiment with added (at 75 min.) serum (bovine) :

Yb-175 Octreotide: LY at 3 h. ITLC Rf 0.5-06 91.2% at 24 h. ITLC Rf 0.5-06 91.7%

B. Preparation of DTPA-1-125-Tyr³-Octreotide.

DTPA-Tyr³-Octreotide of the formula

DTPA-(D)Phe-Cys-Tyr^*-(D)Trp-Lys-Thr-Cys-Throl

is prepared from Tyr³-Octreotide in a corresponding manner as described in Int. Pat. Appln. WO 90/06949, Example 1, and further iodinated with 1251 sodium iodide, dissolved in phosphate buffer in the presence of chloramine T. The molar ratio of DTPA-Tyr³-Octreotide; chlora ine T: 125-1 is 1:4,6:0.6 The reaction is terminated with 10% BSA solution. The labelled product of the above formula wherein Tyr^* = 125-I-Tyr, is purified by HPLC.

C. Combined use for detection and therapy.

To combine the therapeutical effect with the radioguided surgery are used both preparations; Yb-175-Octreotide for the desired therapeutic effect and DTPA-125-I-Tyr³- Octreotide as the "detecting" agent.

Depending on the conditions, they can be used separately, in this case by administering Yb-175-Octreotide first to cause partial or deep tumour necrosis, followed by administration of DTPA-125-I-Tyr³-Octreotide to guide the tumours removal, or they can be administered simultaneously as a mixture in an appropriate ratio. Such a mixture is obtained by mixing both agents in the proper ratio. In this case the difference in IPA of Yb-175 and 1-125, viz. 4.2 and 60.2 days respectively, gives sufficient time for therapeutic effect while at the moment of surgery the background radiation, originating from Yb-175 (having also very low γ abundance) , is already sufficiently low as not to diminish the sensitivity of the microprobe.

EXAMPLE III

Labelling of DTPA-Octreotide kit with Ho-166 and its use in combination with Octreotide labelled with Tb-161. A. Labelling of DTPA-Octreotide kit with Ho-156-

Ca 1 mg of natural (monσisotopic) 165-Ho₂0₃ is irradiated for 48 hours in nuclear reactor with a thermal neutron flux 2 x 10¹⁴ n/cm².sec.

After 30 hours cooling time the sample is treated in exactly the same way as described for 175-Yb in Example II.A.

Obtained Ho-166 stock solution (1 ml) has a specific activity 525 μCi/μg Ho-166, a radionuclide purity > 99.9% and a radiochemical purity > 99.9%, both values determined by the ιethods described in Example I.

Several kits, containing lOμg of DTPA-Octreotide prepared according to Example I., are labelled by addition of 0.5 or 1 ml of Ho-166 stock solution. The mixture is let to incubate 30 min. at room temperature. Samples for analysis at time intervals indicated by the results. Used analytical methods are described in Example I.

Results for labelling with 0.5 ml Ho-166 stock solution:

Time (h) LY (%) Free-Ho-166 (%)

0.5 99.3% < 1

20 99.7 < 1

48 99-100 < 1

72 98.9 1.1 Radiochemical purity at 72 h - HPLC 99-100%, identity confirmed.

Results for labelling with 1.0 ml of Ho-166 stock solution:

Time (h) LY (%) Free Ho-166 (%)

1 94.3 5.7

8 92.6 7.4

challenge test with addition of serum (bovine) at 8 h.

32 (S24) 89.0 11

56 (S48) 88.7 11.3

Radiochemical purity at 56 h- HPLC:

Labelled Ho-166-Octreotide 91.1% Free Ho-166 8.9%

B. Preparation of DTPA-Tb-161-Octreotide as described in Example I., with kit containing 50 lig DTPA-Octreotide.

C. Combined use for detection and therapy

Similarly as described in Example II. is used a combination of both preparations, Ho-166-Octreotide and Tb-161-

Octreotide, particularly when larger tumours are suspected. Since the maximum range of beta particles of Ho-166 in tissue is about 0.85 cm (vs. 0.15 cm for Yb-175) and the deposited energy is for Ho-166 (g.rad/μCi.h - 1,42) five times higher than for Yb-175 (g.rad/μCi.h - 0,27), then the process of tumour necrotization proceeds more rapidly. At the same time the favourable ratio of half-lives (Ho-166 26.9 h, Tb-161 d) guarantees that after one VA of Tb-161 no more than 1.3% of the originally bound Ho-166 activity remains at the site of the tumour so that the sensitivity of the microprobe cannot be influenced, particularly not since the range of gamma rays emitted by Ho-166 (48-80 keV) is comparable to that of Tb-161.

Claims

Claims.

1.A method of intraoperatively detecting and locating tumoural tissues in the body of a warm-blooded living being, comprising (a) parenterally administering to said being a pharmaceutical composition comprising, in a quantity sufficient for detection by a gamma detecting probe, a peptide compound labelled with a low-energy gamma photon emitting radionuclide, said peptide compound being derived from a peptide selected from the following groups:

(i) peptides having a selective neurokinin 1 receptor affinity and having the general formula

R (A,)_m-A₂)_n-A₃)₀-Pro)_p-A₄)_q-A₅-Phe-A_β-A₇-A₈-NH-CH-R₂ R_β

(I)

wherein all of the symbols m, n, o, p and q are 1 , or all but one of the symbols m, n, o_r p and q are 1 , and the remaining symbol is 0;

R, is a hydrogen atom or a C,-C₄ alkylcarbonyl group;

R₂ is a carbamoyl group, a carboxy group, a C,-C₄ alkoxycarbonyl group, a hydroxymethyl group or a C,-C₄ alkoxymethyl group; A, is Arg, Gly or 5-oxo-Pro (pGlu);

A₂ is Pro or β-Ala;

A₃ is Lys or Asp;

A₄ is Gin, Asn or 5-oxo-Pro;

A₅ is Gin, Lys, Arg, N-acylated Arg or 5-oxo-Pro; or wherein A₅ together with A₃ forms a cystine moiety;

A_e is Phe or Tyr;

A₇ is Gly, Sar or Pro;

A₈ is Leu or Pro; and

R_e is a straight or branched C₂-C₄ alkyl group, which group may be interrupted by thio, sulphinyl or sulphonyl; and their Tyr⁰ derivatives; (ii) peptides having a selective somatostatin receptor affinity and having the general formula

R,-B,-Cys-B₂-(D)Trp-B₃-B₄-Cys-NH-CH-R₂ (II)

wherein R, and R₂ have above meanings,

B, and B₂ are each independently Phe, MePhe, EtPhe, Tyr, Trp and Nal, B₃ is Lys or MeLys,

B₄ is Thr or Val, and

R₇ is a 1 -hydroxyethyl group or an indol-3-γlmethyl group; and their Tyr⁰ derivatives;

and

(iii) peptides selected from cytokines, growth factors and hormones, as well as their derivatives and analogues;

and then (b), after allowing the active substance to be taken up in the tumoural tissues 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.

2. A method as claimed in Claim 1 , wherein said peptide compound is labelled with a radionuclide having a gamma energy of approx. 80 keV at most, said radionuclide being preferably selected from the group consisting of 1-125, As- 73, Sb-1 19, Cs-131 , Dy-159, W-181 and Hg-197.

3. A method as claimed in Claim 1 or 2, wheren said peptide compound comprises a functional group, derived from tyrosine or imidazoline or from N- succinimidyl-3-(4-hydroxγphenyl)propionate, said group being substituted with 1-1 25.

4. A method of radioguided surgery of a warm-blooded living being, which method, in addition to the method as claimed in any of the preceding Claims, comprises (i) parenterally administering to said being a pharmaceutical composition comprising, in a quantity sufficient for at least partial necrosis of tumoural tissues, a peptide compound derived from a peptide as defined in Claim 1 and labelled with an isotope, having sufficiently high specific activity and emitting corpuscular radiation, and then (ii), after allowing the active substance to be taken up in the tumoural tissues and to cause an at least partial necrosis of said tissues, subjecting said being to a surgical treatment.

5. A method as claimed in claim 4, wherein the isotope is selected from the group consisting of P-32, S-35, As-77, Y-90, Rb-105, Ag-1 1 1 , Sn-121 , Te- 127, Re-186, Re-188, Au-198, Au-199 and radionuclides of the lanthanide group emitting corpuscular radiation.

6. A method as claimed in Claim 4 or 5, comprising administering both for detection and for therapy the same pharmaceutical composition, comprising a peptide compound labelled with a low-energy gamma photon emitting radionuclide and an isotope as defined in Claim 4 or 5.

7. A method as claimed in Claim 6, wherein said peptide compound is labelled with a lanthanide radionuclide, having both a suitable gamma energy for detection and a corpuscular radiation emitting effectiveness sufficient for tumour cell necrosis, preferably with Tb-161.

8. A method as claimed in any of the preceding Claims, wherein said peptide compound is provided directly or through a spacing group, with a chelating group, said chelating group being attached by an amide bond to an amino group of said peptide and being derived from ethylene diamine tetra-acetic acid

(EDTA), diethylene triamine penta-acetic acid (DTPA), ethγleneglycol-0,0'- bis(2-aminoethyI)-N,N,N',N'-tetra-aceticacid(EGTA),N,N-bis(hydroxybenzyl)- ethylenediamine-N,N'-diacetic acid (HBED), triethylene tetramine hexa-acetic acid (TTHA), 1 ,4,7,10-tetraazacγclododecane-N,N',N",N'"-tetra-acetic acid (DOTA), 1 ,4,8,11-tetra-azacyclotetradecane-N,N',N",N'"-tetra-acetic acid

(TETA), 1 ,2-diaminocyclohexane tetra-acetic acid (DCTA), substituted DTPA, substituted EDTA, or from a compound of the general formula

, - — R .

(

- — S — Y — ' (VI) wherein R is a branched or non-branched, optionally substituted hydrocarbyl radical, which may be interrupted by one or more hetero-atoms selected from N, 0 and S and/or by one or more NH groups, and

Y is a group which is capable of reacting with an amino group of the

5 peptide and which is preferably selected from the group consisting

V ofcarbonyl,carbimidoyl, N-(C,-C₆)alkyIcarbimidoyl,N-hydroxycarbi- midoyl and N-(C,-C₆)alkoxycarbimidoyl; and wherein said optionally present spacing group has the general formula

wherein R₅ is a C,-C₁₀ alkylene group, a C,-C₁₀ alkylidene group or a C₂-C,₀ 15 alkenylene group, and X a thiocarbonyl group or a methylcarbonyl group.

9. A radiopharmaceutical composition to be used for the method as claimed in Claim 6 or 7, comprising in addition to a pharmaceutically acceptable liquid

20 carrier material and, if desired, at least one pharmaceutically acceptable adjuvant, as the active substance a labelled peptide compound as defined in Claim 6 or 7.

10. A composition as claimed in Claim 9, wherein said peptide compound is 25 provided with a chelating group as defined in Claim 8, said chelating group chelating a metal radionuclide.

1 1. Use of a peptide compound as defined in any of Claims 1 to 8 for the manufacture of an agent for intraoperatively detecting and locating tumoural

30 tissues in the body of a warm-blooded living being.

1 2. A labelled peptide compound to be used as an active ingredient in the composition as claimed in Claim 9 or 10, said peptide compound having a selective affinity to endocrinically active tumours and being labelled with at

35 least one isotope as defined in Claim 6 or 7.

13. A kit for preparing a radiopharmaceutical composition, comprising (i) a peptide as defined in Claim 1 , provided with a chelating group derived from an aminoacetic acid based chalating agent as defined in Claim 8, to which substance, if desired, an inert pharmaceutically acceptable carrier and/or formulating agent(s) and/or adjuvant(s) is/are added, (ii) a solution of a salt of a lanthanide radionuclide as defined in Claim 7, and (iii) instructions for use with a prescription for reacting the ingredients present in the kit.