CA2131315A1 - Method of intraoperatively detecting and locating tumoral tissues - Google Patents

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

W~ 93/1X797 PCT~/U~93/02772 Metho~ of intraoPerativelv detectina ~nd locatinp tumoural tissues.

S The invention relates to a method of intraoperatively detectin~ and locating tumoural tissues in the body of a warm-blooded living being, tD a method of radioguid3d sursery of said being, and to a radiopharmaceutical composition to be used for ths latter rnethod. The invention ~urther relates to a labslled peptide compound to be used in said composition and to a kit for preparing said composition.
The accurate staging of tumours, in particular malignant tumours, in general remains -.; one of the most important clinical challenges. Often such tumours or their me~astases are ex~rernely srnall ~<1 cm), and because o1 this small size they are not readily det~ctable and distinguishable using conventional imaging techniqLIes. Even the us~ of advanced imaging t~chniques, such as SPECT acquisition techniques, incornbination with tl~mour-selective imaging agents~ is frequentlv unable to show all l~sions, b2cause of disturbing background activity that makes accuraee image interpretation difficuît. Especially in the abdominal area it is often difficult ~o distin~uish benign for mali9nant tissues usin~ conwntional irnagin~ methods. An example of such a subgroup of tumours are gastro-enteropancreatic tumours that produce hormones, whi~h result in sometimes life-threatening symptoms, e.g.
massive diarrhea. The obvious thetapy in these cases is to surgicalty remove these tumours. However, their often small si~e makes imaging techniques no~ relia~le in accurately locating the lesion, while the surgeon is simply not capable of findin~ the tumours quickly and, mo!eover~ is in fact not sure that all lesions are operatively removed.

A relatively new technigue provides surgical aid: a gamma detecting probe, e.g.
Neoprobe~, that can b~ used to detect sourc~s o~ 0amma radiation that are v0ry small. After parenteral adrninistration of a radiolabelled substance, the surgeon can, intraoperatively, USB this probe to find the lesions ih which uptake of ~his substanc~
has taken place. E.W. Martin and coworkers have investi~ated this new technique:e.g. Amer. J. Surgcty 156, 1988, 386-392; Antibody Immunocon. Radiopharm. 4, 1991, 339-358. These investigators have observed that antibodies or antibody fr~gmentS, labelled with iodine-125, a low-energy gamm~ photon ernittor, are promising substances to be used in th;s technique- They indicate that this technique may successfullY target 80% of colorectal cancer and detect occult turnours in ~h~
abdomen in 20% of the surgical cases involving can~er of the colon. Although it is generally reco~nized that this improvement in diagnosing enables the surgeon to W~ 93/1~797 ~ ~ ~ PCr/US93/02772 .t 3 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 radioiabelled 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 inttaoperativsly detecting and locating ~umoural tissues in the body of a warm-blooded liYing being by using a radiolabelled substance showing an improved and more selective tumour uptake anda much faster blood clearance.
,~ , . This object can be achieved according to the present invention by a method as mentioned ~bove, 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 emit~ing radionuclide, said peptide compound being derived ~rom a peptide selectsd from the following groups:
(i) peptidcs having ~ selective neurokinin 1 receptor affinity and having the ~eneral formula R~~~A~)m~A2)n~A3)0-pro)p-A4~-As-phe-A6-A7-A8-NH-c~ H-F~2 O
~1) wherein all of the symbols rn, 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 a~om or a C.,-(::4 alkyicarbonyl ~roup;
R2 is a carbamoyl group, a carboxy group, a C:~-C4 alkoxycarbonyl ~roup, a hydroxymethyl group or a C,-C~ alkoxymethyl group;
A, is Arg, Gly or 5-oxo-Pro (pGlu);
A2 is Pro or ~-AIa;
A3 is Lys or Asp;
A4 is Gln, Asn or 5-oxo-Pro;
A5 is Gln, Lys, Arg, N-acylated Arg or 5-oxo-Pro;

WO 93/18797 ~ PCI/US93/02772 or wherein As together with A3 forms a cystine moiety;
AQ jS Phe or Tyr;
A7 is Gly, Sar or Pro;
AB jS Leu or Pro; and RB jS a straight or branched C2-C4 alkyl group, which group may be inter-rupted by thio, suiphinyl or sulphonyl;
and their Tyr derivatives;

(ii) peptides having a selective somatostatin receptor affinity ~nd having the general formula , R1-B,-Cys-B2-~D)Trp-B3-B,,-Cys-NH-CH-R2 ~11) t5 :: wherein R, and. R2 have above meanings, B, and B2 are e~ch independently Ph~, MePhe, EtPhe, Tyr, Trp and Nal, E33 is L~s or MeLys, B, is Thr or Val, and R~ is a 1-hy~roxyethyl group or an indol-3-ylmethyl group;
and their Tyr detivatives:

and iii) peptides selected from cytokines, growth factors and hormones, as well as their derivatives and analogues;
~: ~
and then ~b~, after allowing the activ~ substance to be taken up in the ~umoutaltissues and a~ter blood clearanc~ of radioactivity, subjectin~ saicl being to a radioimmunodetectlon techniqu~ in the relevant area of the body of said being, by using a gamma detecting probe.

In ~he above ~escription of the invention the symbol Nal means a naphthylalanyl :: ~ group, and Sar means a sarcosyl ~roup.
Sui~able examples of substituent R~ are (CH~)2S~0)5(:H3, wherein s is 0,1 or 2, and CH2CH~CH3~
SL~itable examples of cytokines are tumour necrosis factor ~TNF~, in partic~llar TNF-o~, interleukines (IL), in particular IL~ L-2, IL-4, IL-5 and IL-6, and interferons.

Wo 93/18797 PC~/US93fO?772 .. . . .

Suitable examples of growth factors are epiderrnal growth factor ~E5F), insulin-like growth-factor (IGF~, in particular IGF-I ~somatedin C) and IGF-II, bombesin, transformin~ growth factor (TGF), in particular TGF-~and TGF-J~, platelet-derived growth factor, fibroblast grow~h factor and nerve growth factor.
Suitable examples of hormones are luteinizing hormone-releasing hormone (LttRH),gastrin, gasttin-releasitlg pep~ide, angiotensin, thyroid-stimulating hormone, vasoactive intestinal polypeptide, prolactin, thyrotropin-releasing hormone, insulin, adrenoeortico~ropic hormone ~ACTH), in particular ~-MSH ~melanocyte-stimulating hormone) and J~-~methylsulfonyl)-L- o~-aminobutyryl-L- o~-9lutamyl-L-histidyl-L
phenylalanyl-D-lysyl-L-phenylalanine, cholecystokinin, corticotropin-releasing hormona ~CRtl), ~rowth hormone-releasing hormone (GRH), arginine and Iysine vasopressin, ox~ocin, glucagon, secretin, parathyroid hormone (PTH) and PTH
relat~d peptide.

By usin~ the above method of the invention, virtually all malignant turnours can be detected and located and can be distinguished ~rom benign tiss~es, because ~hesetumours contain substantially large numbers of receptors for binding the above peptide comp~und.
The above-defined peptides are composed of amino acids, of which at least one may have the D-configuration. The peptidss may also comprise so-called pseudo peptide bonds, viz. -CH2-NH bonds, in addition to the natural amide bonds, viz. -C0-NH-bonds.
Labelled peptide compounds for external imaging aceording to conventional imaging teehniques and consequently labelled with radioisotop~s suitable for ~his purpose, such as Ga-6~, In-~ 11 and Tc-99m, are describ~d in literature. Peptide oompounds, labelled in this manner and derived from peptides mentioned sub ~i) above, are the subjeot of the non-prepublished European patent application no. ~1200955.2 in the name of Applicants. Equaily labelled peptide compounds derived from peptides mentioned sub (ii) and sub (iii) above are known ~rorn the published intsrnational patent applicatiGns W0 90/06949 and W0 9t/01144, resp~c~ively.

3~ Suitable ~xamples 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 lSubstanoe P), W O 93/18797 ~ ~ P ~ IUS43/02772 (2) H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Sar-Leu-Met(02)-NH2, (3~ H-13-Ala-Gln-Gln-Phe-Phe-Sar-Leu-Met(O~)-NH2, (4) H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Tyr-Gly-Leu-Met-NH 2~

~51 H-Arg-Pro-Cys-Pro-Gln-Cys-Phe-Tyr-Pro-Leu-Met-NH2, and Tyr~ deriva~ives thereof.

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

6) H-(D~Phe-C ys-Phe-(l))Trp-Lys-Thr-Cys-Thr-ol (Octreotide), ~7) H-(D)Phe-Cys-Thr~ )Trp-Lys~Val-Cys-Thr-NH~, (8~ H-(D)Ph~-Cys-Tyr-(D)Trp-Lys-Val-Cys-Trp-NH2, : ~9) H-(D)Trp-Cys-Phe ~D)Trp-Lys-Thr-Cys-Thr-NHa, (10) H-(D)Phe-Cys-Phe-~D)Trp-Lys-Thr-Cys-Thr-NH;?, (11) H-(D)Nal-Cys-Tyr-(D)Trp-Lys-Val-Cys-Thr-NH2, '- i (t2) H-(D)Nal-Cys-Tyr-(D)Trp-Lys-Val-Cys-Nal-NH2, i13) H-(D)Nal-~ys-Nal-~DjTtp-Lys-VaJ-Cys-Thr-NH2, ( 14~ H-(D3Phe-Cys-Phe-(D)Trp-Lys-Thr-Cys-Nal-NH 2, ( 1 5~ H-~D~Phe-C ys-Tvr-~D)Trp-Lys-Thr-Cys-Thr-ol, : 30 and Tyr derivatives thereof.

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

Suitable low-energy gamm~ photon emitting radionuciides which can be used as labels for the peptide compounds to be used in the method vf the present invention should have a gannma energy of approx. 80 keV at most. Such W0 ~3/18797 ~ , PC~r/US93J02772 radionuclides ar~ well tuned to the gamma detecting microprobe to be manipulated by the surgeon and intended to register the emitted ~amma radiation. Such a hand-held microprobe, e.g. Neoprobe 100d0, is at present equipped with a rniniature cadmium telluride crystal detector. Such a detector requires for optinnurn detection properties gamma energies in the range of approx. 30-~0 KeV. Higher energies may cause excessive scattering so that the accuracy of the detection is considerably decreased. After uptake of the labelled peptide in ths tumoural tissues and after blood clearance of radioactivity to avoid disturbing background activity, the surgeon can use his ~amma detecting probe during operation to be sure that he/she does not overlook srnail-sized tumours. The weli-tuned label enables the surgeon to accura~ely detec~ and locate such smalt tumours with the aid of ~he hand-held microprobe in otder to guide the surgery treatment. Examptes of suitable radionuclides fot labelling the above peptide compounds are 1-125, As-73, Sb-119, Cs-131, Dy-159, W-181 and Hg-197.

The desir~d radioisotope should be firmly attached to the peptide molecule to reducs l;he chance of detaching this label after adrninistratisn to ~he living being. The peptide can be labelled with the desired ;sotope directly or indirectly, i.e. via a so-called linker. Direct labelling 1113y be carried out, for example, b~ introducing a halogen atom or radioactivs halogen atom, i.c.
iodine-125, in~o an activated aromatic group te.g. tyrosyl or irnidazolyl) ,present in the peptida, into the peptide molecule in a manner known per se, if desired followed by exchange with 1-125. Tyrosine and histidine are suitable amino aoids which, if present in ~h~ peptid~ molecule, allow an easy substitution with ~radioactive) halogen. Often, however, the labelling procedureis performed via a suitable linker, bein~ capabie of reacting with an amino group, pre~erably a terminal amino ~roup, of said peptide, and having a ~unctional group for binding said radioisotope. By using a suitable linker, the desired isotope can ~eneratly better be introduced into the peptide rnolecule.
It is of advantage to attach the linker to ~ terminal amino group of the peptidemolecule, în order to affect the biological properties of this peptide as least as possibl~.

Suitabla linkers ~Ot labelling the peptide with metal radionuclides are discussed in detaii hereinafter.

WO g3/18797 ~ " PCr/US93/02772 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- 1 25. 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 haJogen radioisotope by an appropriate reaction. Both by using the latter, more conveni2nt r~action route and by employing the former method, the peptide can be labelled with the desired radioactive halogen isotope without affecting its biolo~ical properties.

- The above radioiodinating reaction is preferably performed by reacting th2 peptide in question with a solution o~ an alkali metal radionuclide from 1-125 iodide, under the influence of a halide-oxidizing agent, such as chlotarnine T
or iodo~en. Alternatively, the above substi~ution reaction can be carried out with a non-radioactive halogenide, after which halo-exchange with radioactive halogen is performed, eØ as described in European patent 165630.

In geneta3i the tumour~ and metastases which can be detected and located by using the method of the present invention, are small soft tissue tumours which are firmly a~ached to the surrounding benign tissues. This makes the surgical remova3 of such tumours after their detection often difficult. It is another o~lect of the present invention to facilitate the therapeutic treatment of such tumoural tissues and consequently to improve ~he radioguided surgery.
"~
1~ 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 warrn-blooded living being, which method, in addition to the method as discussed hereinbafore and intended to detect and locate tumoural tissues, comprises (i~ parenterally administering to said bein~ a pharmaceutical composition comprising, in a quantity suff;cient ~or at least partial necrosis of turnoural tissues, a pep~ide compound derived {rom a peptide as defined herein~efore and labelled with an isotope with suffici~ntly high specific activity and ernitting Gorpuscular radiation, preferably selected from the group consisting of Tadionuclides as reviewed by Schubigsr et al. (in 6th Int. Symp. Rad;opharm. Chem., Boston 1986, paper no. t49) or by Volkert et al. (in ~). Nuc3. Med., 1991, Vol. 32 (1), WO 93/187g7 PCI~ S93~02772 174-185), such as the radionuclides selected from the group consisting of P-32, S-35, As-77, Y-90, Rb-105, Ag-111, 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 treatmen~.

Suitable examples of the last-mentioned lanthanide radionuclides are Pr-142, Pr-143, Pm-î49, Pm-151, Srn-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 treatmsnt schedule, wherein an optimum use is made of this combination ~f detection and therapy. After uptake of the "detecting" peptide compound i the tumoural tissues, the gamma emissions permit the aecurate detection of thess tissues with a microprobe. These tissues, however, have already been nat~ackedn 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.~. the excision of the already at least partially necrotic tissues, is therefore highly facilitated.
It is of advantage to use for both detection and ~or therapy th~ same pharmaceuticai composition, comprising a peptide compound labelled with a : low-ener~y 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 bs sure that the specific affinity to the tumoural tissuss to be removed is exactly the same for the diagnostic a~ent as for ~he therapeutic agent.
: : :
Surprisingly it has been found, that certa;n lanthanide radionuclides are very suitabl~ to perform both functions at the same time, viz. have a suitable gam~na energy f or detection and havo a corpuscular radiation emitting effectiveness sufficient for tumour cell necrosis. By combining these both WO 93/l B797 PCl /US93/02772 functions in one and the same radionuclids, it is guaranteed that the uptake in ths tumour oells is optimal for both intended effects. In this connection terbium-161 (Tb-161~ is pre-erninently suitable. This ianthanide 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 ofterbium- 1 61 is extremely appropriate for the intended purpose, viz. 6.91 days.This means that sufficiently long after injection the blood clearance of r2dioactivi~y is sufficiently complete to permit accurate detsction of the tumoural tissu~s, while at the same time necrosis of the malignant tissue cells has advanced sufficiently to allow easy excision of the detec~ed 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 labeiled peptide cornpound to be used in the method of the invention, for de~ecting purposes or both for detection and for therapy, i~
provided, directly or through a spacing group, with 3 chelatin~ gtoup. This chelating group is attached by an amide bond to an amino group of said p0ptide and is derived from ethylene diamine tetra-acetic acid ~FDTA), di-ethylene triamine penta-acetic acid (DTPA~, ethyleneglycol-0,0'-bis(2-aminoe~hyl)-N,N,N',N'-tetra-acetlc acid (EGTA), N,N-bislhydroxybenzyll-sthyienediamine-N,N'-diacetic acid (HBED), triethylene tetramine hexa-acetic acid (TTHA), 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetra-ace~ic acid ~D~A), 1,4,8,11~tetra-azacyclotetradecane-N,N',N",N"'-tetra-acetic acid ~TETA), 1 ,2-di~minocyclohexane t~tra-acetic acid ~DCTA), substituted DTPA, substituted EDTA, or ~rom a compound of the general formula ,_ ~R-- --`~--S--Y ~

wherein R is a b~anched or non-branched, optionally substituted hydrocarbyl radioal, which rnay be interrupted by one or more hetero-atoms selected from N, 0 and S andlor by ons or more NH groups, and WO 93/18797 P~lUS93/02772 Y is a group which is capable of reacting with an amino group of the peptide and which ;s preferably seiected from the group consis~ing of carbonyl, carbimidoyl, N-~C,-C~)alkylcarbimidoyl, N-hydroxycarbi-n~idoyl and N-(Cl-C6)-alkoxycarbimidoyl;
and wherein said optionally present spacing group has the general formula ~NH--R5 eo-- or ~H2-e3NH-X--~lV) (V) lQ
- wherein Rs is a C~ o alkylene group, a C,-C,0 alkylidene ~roup or a C2-C,O
, alkenylene group, and X is a thiocarbonyl group or a methylcarbonyigroup.

Examples of sui~able chela~ors of the general formula 111 are unsubstituted or t S . subs~i~uted 2-iminothiolanes and 2-iminothiacyclohexanes, in particular 2-imino-4-rnerca ptomethylthiolane .

It has be~n observed, that the 2bove preferred peptide compound, provided ~; ~ with a chelating group, shows a very fast clearance. This is of great ; ~ ~ 20 advantage, because the use of this preferred peptide compound after labelling allows surgery a very short time after administration, if desired ftom medical considerations.

~: The invention further relates to a radiopharmaceutical composition to be used ~or the me~hod of radioguided sur~ery as defined abov~, which composition comprises in addition to a pharmaceutically acceptable carrier and, if desired, : at least one pharnnaceutically acceptable adjuvant, as îhe activ~ substance a peptide compound labelled with a iow-energy gamma photon emitting radionuclids and a corpuscuiar radiation emitting isotope as defined hereinbe~ore. If desired, th0 composition can b~ brought into a form more suitable for parenteral administra~ion, e.~. by adding a pharmaceutically acceptable liquid catrier material. For parenteral administration the solution should of co~rse be in a sterile condition.

The above oomposi~ion preferably comprises as the active substance a pep~ide compound provided with a chelating ~roup as defined above, said chela~ing group chelating a metal radionuclide. The preferred radionuciides of the WO 93/18797 ,, . ! A. j ~; ) PCI'/US93/02772 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 chelatin~ gro~.p comprising peptide compound in the form of a salt o~ a suitable acid, e.g. a rnineral acid or acetic acid. The complex-formin~ reactioncan ~enerally be carried out in a simple manner and under conditions which are not detrimental to the peptide.

The invention further relates to a labelled pep~ide 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 emi~ting radionuclide and a corpuscular radiation emitting iso~ope, as defined hereinbefore, preferably with a suitable lanthanideradionuclid~ having the above-defined radiation characteristics.

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

~uch a kit a5 described above can be delivered to the user. The user can easily perform the labelling of the chelated peptide with the ianthanide radionuelide by himself or herself in the clinical hospital or laboratory. The labelling procedure is simple and does not require complicated manipulations, so that the ussr is able to prepare the labelled composition ~rom the kit ingredients byusin~ 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, thelan~hanide radionuclide may be delivered separately and substituted af~er its expiration date.

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

WO93/18797 PCT/US93/Q~772 t) I .~` 12 ~X~P~ 1 .

A. Pre~aratlon of DTP~-Octreo~ida 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 ~g DTPA-Octreotide per vial is prepared as follows:

~ 15: 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;

2;0 ~ - sodium acetate solution 0.286M, by dissolving 3.B9 g of sodium acetate 3H20 in 100 ml water.

To formulate the kit, 0.5 mg of DTPA-Octreotide is :: ~
d:issolved in 4 ml of acetic acid solution, and 5 ml of 25~ sodium acetate solutîon are added.
:
To this mixture are added 16 ml water to make 25 ml of ~ final solution, which is subsequently filtered through a :~ : 0.22~ bacterial filter. The filtrate is then dispensed in 0.5 ml portions per vial and ~ial5 are lyophiliæed. The final freeze dried product is stored at 4C.

WO93/18797 ~ PC~/US93/0277 In a similar way, startin~ from 2.5 mg DTPA-Octreotide was also prepared and a kit containing 50~g DTPA-Octreotide per vlal .

S B. P~3L__n of Tb-161 solution 2 mg of enriched (98.1%) 160-Gd2O3 is irradiated for 48 hours in a nuclear reactor with thPrmal neutron flux 2 x 10l4 n/cm2 sec.
After ca 30 hours of cooling the sample is dissolved in 2 portions of 1 ml warm (70C) lON HCI directly in the irradiation quartz ampo~le, 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 lON ~Cl till dry and the dry rest is taken up in few ml of O.O~N HCl and diluted ~o 10 ml with 0.02N HCl, Irradiation : yield is ca 11.5 mCi Tb-161.

For the separation of Tb-1610 5 ml of the Gd/Tb s~ock solu~ion in 0.02N HCl is evaporated to dry and taken up in ~, 200~1 of 0.02N HCl. Thi~ solution is loaded on a 0.8 x 12 ; : : cm column of 5CX BioRad~ 50 W-X8, 200-40C mesh, in NH4t fo~m. As an eluent is used a 0.2 M solution of a-hydroxy-isobutyric acid, adjusted to pH 4.1 with ammonia.
Frac~ions ~f 1 ml of eluate are collected for radiodnuclide identification. ~ombined fractions containing Tb-161 are : made of 0.5N in HCl and run over a second small columQ of BioRed~ 50W-X8 in H~ form. The loaded column is then washed wi~h 0.5 and 1.5~ HCl, followed with water to re~ove the exce~s of a-~ydroxy-isobutric acid. Tb 161 is finally stripped from the column with 6N H~I. The strip solution is again evaporated to dryness. The residue is taken up into O.OOlN HCI (4 ml) and u~ed for analy~is and labelling experiments.
:~ 35 W~3/t8797 - PCT/US93/02772 .~, ; 14 Analysis:

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

C. Labellinq of_DTPA-Octreotide kit with Tb-161.
.
~; Several kiks of DTPA-Octreotide, prepared according to Example 1 containing 10 or 50 ~g DTPA-Octreotide, are labelled by addition of 0.5 ml of Tb-161 solution obtained under B. The mixture is incuba~ed for 30 min. at room temperature.

~nalysis:

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 ~; 25 : HPLC: Column:: ~Bondapak~C 18 10~m, 3.9 x 300 m~
: Eluent: 0.07M acetate buffer pH 5.5 ~a), ~,, ~:: 100% MeOH ~b~, a and b mixed in ratio 6:4 vtv.
~:
Gradient: 40-80% b in 20 min.
Operation: Flow rate 1 ml/min., temperature 35C.
Detection: Dual, NaI crystal, W detec~or~at 280nm.

Results of labelling experiment: (at time interYal between addition of Tb-161 activlty and analysis) LY = Labelling yield.

WO93/1~797 ~ PCT/US93/02772 Time (h) LY 10 ~g LY 50 0.5 33% >92%
3 46% >92%
24 78.4% >93%

challenge exper}ment with serum (bovine), added at 24 h .

4~ 76.4~ >95%
,, ' * Free Tb-161 was not detectable in any kit containing 50 ~g DTPA-Octreotide.

Radiochemical purity - 50 ~g at 3 h - HPLC 96.2 ' ~20 HPLC identification positivet because W spectrum and ~ : activity peaks of Tb-161 are found identical with those for ; In~ labelled DTPA-Oc~reotide used as control.

T~ obtain an injectab~e preparation with radiochemical 2:5 ~ puri~y > 98%,:the labelle~ soluti~n is purified over a Sep-PakRCl~ cartridge, which after loading is ~a~hed with water ~(5~ml) and eluted~with methanol (5 ml), the latter fraction containing T~-161 Octreotide. Evaporation and dissolution of the residue in physiologic saline solution give after ; :30 sterilization ~membrane filtration) the desired injectable preparation.
:: :

~ ~XAMP~
. .

WO 93fl8797 PCT/US93/02772 Labellinq of DTPA-Qctreotide kit with Yb-175 and its use ir.
combination with detectinq a~ent DTPA-125-I-Tyr3-Octreotide Labellinq of DTPA-Octreotide kit with Yb-175.

Ca 1 mg of enriched (97.8%) 174-Yb2O2 is irradiated for 48 hours in a nuclear reactor with thermal neutron flux 2 x 10l4 / m2 C
n c .~e .

After 30 hours cooling time the sample is dissol~ed directly in the irradiation quartz ampoule in 2 X 1-ml .- portions of warm (70C) concentrated HC1.

The obtained solution is withdrawn ~nd trasferred to a small ~uartz 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 chlori~e is twice evaporated to dryness with concentrated HCl and the res't is taken up into 2 x 5-ml of 0.02N HCl, transferred to a 20 ml volumetric fl sk and dilu~ed to the desired volume with 0.02N ~C'.

A 1.O ml aliquot of this solution is diluted t~ 50.0 ml with 0.02N HCl to`obtain a stock solution of Yb-175 :~ 25 chloride, used for the labelling experiments. This ;~ solution (1 ml) has a specific activity of A00 ~Ci/~g 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 ~g 9f DTPA-Octreotide prepared a cording 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 w~93/l~7s7 `~ PCT/VS93/02772 analytical methods are described in Example 1.

Results:

Radiochemical purity Yb-175:ITLC, lM Na-acetate Rf 0.9-1~0 99.5% :

Yb-17~ Octreotide: LY at 30 min. ITLC Rf 0.5-0.6 98.1%
SepPak~ 98.8%
: LY at 75 min. HPLC 90.7%

Id~ntity of Yb-175 labelled Octreotide is confirmed as described abo~e for the Tb-161 labelled peptide compound.

Cha}lenge 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-l-lZ5=~L~ eot~ide.

~:
~ DTPA-Tyr3-Octreotide of the fo~mula :

DTPA- (D) Phe-~s-l~rr - (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 125I sodium iodide, dissolved in phosphate buffer in the presence of chloramine T. The W093/~797 PCT/US93/0~772 ~ i ,l 3 18 molar ratio of DTPA-Tyr3-Octreotide; chloramine T: 125-I is 1:4,6:0.6 The reaction is terminated with 10% BSA
solution. The labelled product of the abo~e formula wherein Tyr = 125-I-Tyr, is p~rified 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 e~fect 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-17S-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 mixin~ both agents in the proper ratio. In this case the difference in ~h of Yb-175 and I-125, viz.
: :~ 4.2 and 60.2 days respectiYely, gives sufficient time for ~: : therapeutic effect while at the moment of surgery the : background radiation, originating from Yb-175 (having also very low ~ abundance), is already sufficiently low as not to diminish the sensitivity of the microprobe.

;
:
EXAMPLE III

Labellinq of DTPA-Octreotide kit with Ho-166 and its use in ombination with Qctreotide labelled with Tb~161.

WO93/18797 ~ PCT/~S93/02772 A. Labellinq of DTPA-Octreotide kit with Ho 16~

Ca 1 mg of natural ~monoisotopic) 165-Ho2O3 is irradiated for 48 hours in nuclear reactor with a thermal neutron flux 2 x 10l4 n/cm2.sec.

After 30 hours cooling time the sample is treated in exactly the same w2y as described for 175-Yb in Example II.A.
, 10 Ob~ained Ho-166 stock solution (1 ml) has a specific activity 525 ~Ci/~ Ho-166, a radionuclide purity > 99.9~
and a radiochemica~ purity > 99.9%, both values determined by the methods described in Example I.
Several kits, containing 10~g 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 roo~ 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 (%) : 30 0.5 99.3% < 1 : 20 99.7 ~ 1 48 9~-100 ~ 1 72 98.9 ~.1 ~093/1~7~7 PCT/US93/02772 ~ 20 Radiochemical purity at 72 h - HPLC 99-100%i, 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 ~ 92.6 7.
challenge tast with addition of serum lbovine) a~ 8 h.
,, 32 (S24) 89.0 11 56 (S48~ 88.7 11.3 Radiochemical purity at 56 h- HPLC:

: Labelled Ho-166-Oc~reotide 91.1%
2~0 Free Ho-166 B.9%

:
.

:: :
C. Combined use for_detection_and thera~

Similarly as described in Example II. is used a combination o~ both preparations, Ho-166-Octreotide and Tb-161-Oc~reo~ide, 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 dep~sited energy is for Ho-166 ~s.ràd/~lci.h - 1,42) five 3S times higher than or Yb-175 (~.rad~Ci.h - 0,27~, then the WO93/18797 ,~ PCT/US93/02772 process of tumour necrotization proceeds more rapidly. At the same time the favourable ratio of half-lives (Ho-166 26.9 h, Tb-~61 d) guarantees that after one T~ 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 (13)

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 (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 O;
R1 is a hydrogen atom or a C1-C4 alkylcarbonyl group;
R2 is a carbamoyl group, a carboxy group, a C1-C4 alkoxycarbonyl group, a hydroxymethyl group or a C1-C4 alkoxymethyl group;
A1 is Arg, Gly or 5-oxo-Pro (pGlu);
A2 is Pro or .beta.-Ala;
A3 is Lys or Asp;
A4 is Gln, Asn or 5-oxo-Pro;
A5 is Gln, Lys, Arg, N-acylated Arg or 5-oxo-Pro;
or wherein A5 together with A3 forms a cystine moiety;
A6 is Phe or Tyr;
A7 is Gly, Sar or Pro;
A8 is Leu or Pro; and R? is a straight or branched C2-C4 alkyl group, which group may be interrupted by thio, sulphinyl or sulphonyl;
and their Tyro derivatives;

(ii) peptides having a selective somatostatin receptor affinity and having the general formula (II) wherein R1 and R2 have above meanings, B1 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 Tyro 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 l-125, As-73, Sb-119, 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-hydroxyphenyl)propionate, said group being substituted with l-125.

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 3 peptide as defined in Claim 1 and labelled with an isotope, having sufficiently high specific activityand 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-111, 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), 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), 1,4,8,11-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 (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, O 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 of carbonyl,carbimidoyl, N-(C1-C6)alkylcarbimidoyl, N-hydroxycarbi-midoyl and N-(C1-C6)alkoxycarbimidoyl;
and wherein said optionally present spacing group has the general formula -NH-R5-CO- or (IV) (V) wherein R5 is a C1-C10 alkylene group, a C1-C10 alkylidene group or a C2-C10 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 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 provided with a chelating group as defined in Claim 8, said chelating group chelating a metal radionuclide.

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

12. 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 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.