AU657770B2 - Radiolabelled somatostatin derivatives, their preparation and use - Google Patents

Description

UPI UAIE 08/01/93 AOJP DATE 25/02/93 APPLN. ID 20174/92 PCT NUMBER PCT/US92/04559 I1111111 1111111 AU92201741 1 I ii AU9220174 (51) International Patent Classification 5 (11) International Publication Number: WO 92/21383 A61K 49/02, 43/00 Al (43) International Publication Date: 10 December 1992 (10.12.92) (21) International Application Number: PCT/US92/04559 (81) Designated States: AT (European patent), AU, BE (European patent), CA, CH (European patent), DE (Euro- (22) International Filing Date: 2 June 1992 (02.06.92) pean patent), DK (European patent), ES (European patent), FR (European patent), GB (European patent), GR (European patent), IT (European patent), JP, LU (Euro- Priority data: pean patent), MC (European patent), NL (European pa- 9100954 3 June 1991 (03.06.91) NL tent), SE (European patent), US.

(71) Applicant (for all designated States except US): MAL- Published LINCKRODT MEDICAL, INC. [US/US]; 675 With international search report.

McDonnell Blvd., P.O. Box 5840, St. Louis, MO 63134 Before the expiration of the time limit for amending the claims and to be republished in the event of the receipt of amendments.

(72) Inventor; and Inventor/Applicant (for US only) COX, Peter, Henry [GB/ NL]; Boksdoorn 3, NL-2923 EA Krimpen ad Yssel (74) Agents: KLOSTERMANN, Roy, J. et al.; Mallinckrodt J Medical, Inc., 675 McDonnell Blvd., P.O. Box 5840, St.

Louis, MO 63134 (US).

(54) Title: RADIOLABELLED SOMATOSTATIN DERIVATIVES, THEIR PREPARATION AND USE (57) Abstract Radioactive-technetium-labelled and radioactive-rhenium-labelled somatostatin derivatives are disclosed for the selective detection/localisation or for the selective therapeutic treatment of tumours with somatostatin receptors. The labelled polypeptides are characterised by the general formula: RI-SI-AI-A2-(D)Trp-A 3 -A-A4-A-Thr-S-R 2 wherein R 1 is a hydrogen atom or a

CI-C

4 alkylcarbonyl group, R, is an amino group, a hydroxy group or a CI-C 4 alkoxy group, A, and A s each independently are Phe, MePhe, EtPhe, Tyr, Trp, Nal (naphthylalanyl), or Cys, A, is Phe, MePhe, EtPhe, Tyr, Trp or Nal, A 3 is Lys, MeLys or (c-Me)Lys, A 4 is Thr or Val, S I is an amino acid sequence of 1 to 6 amino acids, selected from the group consisting of Ala, Cys, Asn, Phe, MePhe, EtPhe, Tyr, Trp, Nal, Gly, Lys, MeLys, (e-Me)Lys, Thr, Val, Asn and Ser, and S, is an amino acid sequence of 0 to 3 amino acids selected from the group mentioned sub SI, with the proviso that the polypeptide comprises two cysteine amino acid radicals, the metal-radionuclide being selected from a radioactive Tc or Re isotope which, as a cation, is bound covalently to the mercapto groups of the cysteine amino acid radicals.

,i SWO 92/21383 PCT/ US92/04559 1 RADIOLABELLED SOMATOSTATIN DERIVATIVES, THEIR PREPARATION AND USE The invention relates to a metal radionuclide-labelled polypeptide intended for diagnostic or therapeutic applications.

Radionuclide-labelled compounds may be used for diagnostic examinations, for example, into deviations in shape and functions of internal organs and into the presence and location of pathological processes in the body. For this purpose a composition in which the radioactive compound is present is administered to the patient, for example, in the form of an injection liquid. By means of a suitable detection apparatus, for example, a gamma camera, images of, for example, the organ or the pathological process in which the active compound is incorporated can be obtained by detecting the emitted radiation ("scanning").

Radioactive-labelled biological macromolecules, in particular polypeptides, present interesting perspectives for diagnostic applications. Certain polypeptides have a very large target organ specificity and, att&r having been introduced into the body of the patient, can react very selectively with biological macromolecules present therein.

Binding studies have demonstrated that certain Pndocrinerelated tumours comprise large numbers of binding sites with a high affinity to somatostatin and somatostatin-related polypept4 es. Examples of such tumours having large numbers of somatostatin-receptors are pituitary tumours, tumours of PCT/US92/04559 2 the central nervous system, malignant breast tumours, gastro entero-pancreatic tumours, and metastases hereof.

Various metal radionuclides, provided they are bound to tumour-selective polypeptides, may successfully be used for controlling tumours and hence form a powerful tool in radiotherapy. The polypeptides used hence serve as vehicles to transport the desired radiation dose, viz. the metal radionuclide, to the tumour to be exposed to the radiation.

The direct labelling of a polypeptide with a metal radionuclide has two disadvantages. First, the biologically active site of the peptide necessary for a good target organ specificity or selectivity, is easily blocked by this reaction, so that the normal behaviour of the biological macromolecule is disturbed. In addition, the affinity between metal-radionuclide and macromolecule often is unsatisfactory, so that the formed bond is not sufficiently stable to remain intact under physiological conditions. The administered material then is no longer useful neither as a diagnostic -it cannot be detected any more how the polypeptide behaves in the body nor as a therapeutic the radiation dose is no longer transported to the desired target but causes an undesired radiation burden elsewhere! In order to mitigate these disadvantages it is suggested in European Patent Specification 237150 to treat proteinaceous materials which comprise dizulphide bonds first with a disulphide reducing agent, for example, dithiothreitol, and to react then the reduced proteinaceous substance which now comprises free mercapto groups, specifically with radionuclide species, for example, with Tc-99m-tartrate or glucoheptonate. It has been found, however, that in this reductive treatment of the protein, in which the protein i "unfolded" by cleavage of the disulphide bonds to the desired mercapto groups, damage to F- WO 92/21383 PC/US92/04559 3 the protein molecules may easily occur, as a result of which the selectivity is disturbed.

In the past few years a large number of publications have appeared in which biological macromolecules, usually proteins or proteinaceous substances, are described which comprise chelating groups for a bond with the desired metalradionuclide. For example, International Patent Application WO 90/06949 describes somatostatin analogues which comprise chelating groups, preferably derived from TDPA and the like, for a bond with a detectable element. As an example is described DTPA-modified octreotide which may be labelled radioactive with In-111 or with Y-90, for diagnostic or therapeutic purposes, respectively.

However, better suitable isotopes for these applications are radioactive technetium, in particular Tc-99m, and radioactive rhenium, in particular Re-186 and Re-188, because these radioisotopes have better radiation characteristics and are more readily available. However, it has so far not succeeded to label the somatostatin analogues mentioned hereinbefore with these radioisotopes to compositions which are suitable for in vivo applications and which are sufficiently stable. Somatostatin itself is rapidly biologically converted in the body and is therefore generally considered not suitable for radioactive labelling.

Therefore, one has so far resorted to stabilised somatostatin derivatives in which the two cysteine amino acid radicals, which apparently are responsible for the instability, are oxidised together to a cystine group. Both the commercially available somatostatin, and the octreotide described in Patent Application WO 90/06949 mentioned hereinbefore, comprise cystine bridges, formed by oxidation from two cysteine amino acid radicals.

r r 'I PCT/US92/04559 4 It is the object of the present invention to provide an easily accessible radioactive-technetium-labelled or radioactive-rhenium-labelled polypeptide for the selective detection/localisation or for the selective therapeutic treatment of tumours with somatostatin receptors, which is sufficiently stable for in vivo application. This object can be achieved with a labelled polypeptide which according to the present invention is characterised by the general formula

R

1

-S

1

-A

1

-A

2 -(D)Trp-A 3

-A

4

-A

5 -Thr-S 2

-R

2

(I)

wherein

R

1 is a hydrogen atom or a C 1

-C

4 alkylcarbonyl group,

R

2 is an amino group, a hydroxy group or a Cl-C 4 alkoxy group,

A

1 and A 5 each independently are Phe, MePhe, EtPhe, Tyr, Trp, Nal or Cys,

A

2 is Phe, MePhe, EtPhe, Tyr, Trp or Nal,

A

3 is Lys, MeLys or (E-Me)Lys, A4 is Thr or Val,

S

1 is an amino acid sequence of 1 to 6 amino acids, selected from the group consisting of Ala, Cys, Asn, Phe, MePhe, EtPhe, Tyr, Trp, Nal, Gly, Lys, MeLys, (£-Me)Lys, Thr, Val, Asn and Ser, and

S

2 is an amino acid sequence of 0 to a' v acids, selected from the group m- r-iicv ri sub A with the proviso that the polypeptide comprises two cysteine amino acid radicals, the metal-radionuclide being selected from a radioa. jve Tcisotope or Re-isotope which as a cation is bound covalently to the mercapto groups of the cysteine amino acid radicals.

(Nal naphthylalanyl).

It has been found surprisingly that the labelled polypeptide can simply be prepared and is sufficiently stable in vivo WO 92/21383 PCT/US92/04559 for performing the desired examinations and the desired therapeutic treatment, respectively. It has been established that the labelled compound remained stable at least one hour after injection. The selectivity is not adversely influenced by the labelling with radioactive technetium or rhenium. For example, Tc-99m-labelled polypeptide according to the invention is bound specifically to somatostatin receptorsites.

Suitable labelled polypeptides according to the invention are derived from the previously mentioned octreotide and analogues thereof, and may then be represented by the general formula

R

1

-A

6 -Cys-A 2 -(D)Trp-A 3

-A

4 -Cys-Thr-R 4

(II)

wherein R1, R 2

A

2

A

3 and A 4 have the meanings given hereinbefore, and

A

6 is Phe, MePhe, EtPhe, Tyr, Trp or Nal, the metal radionuclide being selected from the group consisting of Tc-99m, Re-186 and Re-188 which as a cation is bound covalently to the mercapto groups of the cysteine amino acid radicals.

Other excellently suitable labelled polypeptides according to the invention are derived from somatostatin and analogues thereof, and may be represented by the general formula

R

1 -S'l-A' 1

-A

2 -(D)Trp-A 3

-A

4

-A'

5 -Thr-S' 2

-R

2

(III)

wherein R 1

A

2

A

3

A

4 and R 2 have the meanings given hereinbefore,

A'

1 and A' 5 each independently represent Phe, MePhe, EtPhe, Tyr, Trp or Nal,

S'

1 is an amino acid sequence of 1 to 6 amino acids, preferably of 5 amino acids, selected from the group PCr/US92/04559 6 consisting of Ala, Cys, Asn, Phe, MePhe, EtPhe, Tyr, Trp, Nal, Gly, Lys, MeLys, (6-Me)Lys, Thr, Val, Asn and Ser, with the proviso that S' 1 comprises a cysteine amino acid radical, and S'2 is an amino acid se .ance of 1 to 3 amino acids, preferably of 2 amino acids, selected from the group mentioned sub S1', with the proviso that S 2 comprises a cysteine amino acid radical, the metal radionuclide being .0 selected from the group consisting of Tc-99m, Re-186 and Re- 188 which as a cation is bound covalently to the mercapto groups of the cysteine amino acid radicals.

In connection with the excellent properties of the labelled product and the ready availability of somatostatin as a starting peptide, a labelled polypeptide is preferred of the general formula

R

1 -Ala-Gly-Cys-Lys-Asn-Phe-Phe-(D)Trp-Lys-Thr-Phe- 0O Thr-Ser-Cys-R 2

(IV)

the metal radionuclide being selected from the group consisting of Tc-99m, Re-186 and Re-188 which as a cation is bound covalently to the mercapto groups of the cysteine amino acid radicals.

The invention also relates to a method of preparing a metalradionuclide-labelled polypeptide according to the invention by starting from a cyclised polypeptide, in which the cysteine amino acid radicals together are oxidised to a cystine group. Examples of such cyclised polypeptides are the already mentioned somatostatin commercial product and analogues thereof. Analogues are to be understood to mean polypeptides having corresponding biological activity, i.e.

specific somatostatin-receptor binding affinity, but with modifications in the amino acid sequence. It has been found '1- WO 92/21383 PCT/US92/04559 7 that the said cyclised polypeptides can be excellently reduced and may then be labelled under reducing conditions without the polypeptide molecule being attacked.

As a reduc:.ng agent are preferably chosen zinc ions or metallic zinc, the latter, for example, in the form of zinc powder, because such reducing agents are suitable both for the preparation of the polypeptide from the cyclised material, and also for the reduction of pertechnetate or perrhenate. An excellent example of use of the metallic zinc powder in the so-called SPED (Solid Phase Electron Donor) technique, in which the cyclised polypeptide is incubated by means of zinc powder, for example, on a 0.22 4 filter, after which addition of a Tc-99m pertechnetate solution immediately provides a solution of the pure, Tc- 99m-labelled polypeptide in the filtrate. Contaminations and non-reacted starting material remain on the filter, so that the filtrate is immediately ready for use. Metallic zinc may also be provided excellently as a zinc mirror on the inner wall of a tube or other reaction vessel and thus produce the desired conversions in the tube or reaction vessel.

The invention further relates to a pharmaceutical composition which comprises the metal-radionuclide-labelled polypeptide according to the invention, and to the use of the said composition for diagnostic or therapeutic purposes. For diagnostic purposes, i.e. for detecting and localising certain tumour tissues, as defined hereinbefore, the active substance should be labelled with radioactive technetium, for therapeutic purposes, the active substance should be labelled with radioactive rhenium. All this is defined in more detail in Claims 8 and 9.

The invention finally relates to a kit for preparing a ,773o7 PCT/US92/04559 8 radiopharmaceutical composition, comprising, in an optionally dry condition, a cyclised pcl'peptide, as defined hereinbefore, a reducing agent, preferably metallic zinc or zinc ions, and directives for reacting the ingredients of the kit and of the resulting product with Tc-99m pertechnetate or with Re-186 or Re-188 perrhenate.

In this manner the user of the kit himself can prepare in a clinical laboratory the labelled polypeptide according to the invention in the form of a composition to be administered: reduction of the cyclised polypeptide tc che polypeptide to be labelled, as well as the required labelling. The use of one reaction agent for the two reactions simplifies the method of preparation.

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

Example 1 Commercially available somatostatin is treated for minutes at a pH of 8 on a 0.224 filter by means of the socalled SPED technique as described hereinbefore. A freshly eluted sodium pertechnetate solution is then added and the mixture is incubated at room temperature for 15 minutes.

The labelled polypeptide may be obtained ac a filtrate.

Precipitate and liquid can be separated without a filter by decanting and extracting the liquid by means of a syringe.

A labelling efficiency of 90% is obtained. Free technetium is bound to thp SPED and cannot contaminate the product.

Labelling is confirmed by means of thin-layer chromatography and ion exchange column chromatography. The labelled compound is stable in vitro up to 4 hours.

A quantity of 22 MBq of the labelled compound is

P.-

WO 92/21383 PCT/US92/04559 9 administered to rats suffering from colorectal carcinoma and the biodistribution is determined by dynamic gamma camera scintigraphy up to one hour after injection.

The tumour take-up reaches a maximum at approximately 4 minutes after injection. During the determination period no significant activity reduction from the tumour can be observed. The labelled compound is stable in vivo during the whole of the determination period, as appears from the absence of thyroid gland and stomach activities.

Scintigrams of the tumours are made four minutes after injection. The uptake ratios tumour:muscle tissue are favourable, namely 5:1.

That the tumour accummulation of technetium is related to the somatostatin binding to receptors in the tur.our has been checked by treating one group of experimental animals having tumours prior to administration of the labelled compound with Sursamine® to block the receptor sites. No tumour uptake is observed in these animals, so that it is confirmed that the binding takes place at somatostatin receptors in the tumour.

Example 2 A group of adult female rats were implanted with CC531 Colon Carcinoma which is known to have somatostatin receptors. When the subcutaneous tumour implants had reached a size of ±1.5cm diameter, one group was injected with Technetium Somatostatin complex by intravenous injection and a second group was injected in a similar manner with Indium-111 Octreotide complex for comparison.

The animals were anaesthetised by means of nembutal and serial scintigrams were made. The Technetium Somatostatin PCT/US92/04559 was clearly visible in the tumour within 4-5 minutes post injection. At six minutes post injection the animals were sacrificed by cervical dislocation and tissue samples were taken and counted. Approximately 11% of the injected dose was found in the total tumour tissue with a tumour to soft tissue uptake ratio of 4.2/1. The blood concentration was relatively high tumour to blood ratios being 0.23. The bulk of the activity was recovered in the stomach, liver, spleen and kidneys.

In the case of animals injected with indium Octreotide complex the visualisation of the tumour was similar in pattern to that of the technetium complex. At 24 hours post injection, the optimal scanning time in humans, the animals were sacrificed and tissue samples were counted.

An average concentration of 4.9% of the injected dose was found in the total tumour with a tumour to soft tissue ratio of 9.8/1 this higher value in relation to the technetium somatostatin was primarily due to the low blood concentration. As with the technetium complex the bulk of the activity was recovered from the liver, spleen, GI tract and kidneys.

The results show a slight difference in the biodistribution characteristics of the two complexes but the kinetics during the first 15 minutes post injection were similar, and during this period good scintigraphic images of the tumour could be obtained with the technetium complex using a gamma camera.

Claims (14)

1. A metal-radionuclide-labelled polypeptide of the general formula R 1 -Sl-A 1 -A 2 -(D)Trp-A 3 -A 4 -A 5 -Thr-S 2 -R 2 (I) wherein R 1 is a hydrogen atom or a C 1 -C 4 alkylcarbonyl group, R 2 is an amino group, a hydroxy group or a C1-C 4 alkoxy group, Al and A 5 each independently are Phe, MePhe, EtPhe, Tyr, Trp, Nal or Cys, A 2 is Phe, MePhe, EtPhe, Tyr, Trp or Nal, A 3 is Lys, MeLys or (£-Me)Lys, A4 is Thr or Val, Si is an amino acid sequence of 1 to 6 amino acids, selected from the group consisting of Ala, Cys, Asn, Phe, MePhe, EtPhe, Tyr, Trp, Nal, Gly, Lys, MeLys,(£-Me)Lys, Thr, Val, Asn and Ser, and S 2 is an amino acid sequence of 0 to 3 amino acids, selected from the group mentioned sub Sl, with the proviso that the polypeptide comprises two cysteine amino acid radicals, the metal-radionuclide being selected from a radioactive Tc- isotope or Re-isotope which as a cation is bound covalently to the mercapto groups of the cysteine amino acid radicals.

2. A labelled polypeptide as claimed in Claim 1, having general formula R 1 -A 6 -Cys-A 2 -(D)Trp-A 3 -A 4 -Cys-Thr-R 4 (II) wherein R 1 R 2 A 2 A 3 and A 4 have the meanings given in Claim 1, and 3o PCT/US92/04559 32 A 6 is Phe, MePhe, EtPhe, Tyr, Trp or Nal, the metal radionuclide being selected from the group consisting of Tc-99m, Re-186 and Re-188 which as a cation is bound covalently to the mercapto groups of the cysteine amino acid radicals.

3. A labelled polypeptide as claimed in Claim 1, having the general formula Rl-Sl'-A 1 '-A 2 -(D)Trp-A 3 -A 4 -A 5 '-Thr-S2'-R 2 (III) wherein R 1 A 2 A 3 A 4 and R2 have the meanings given in Claim 1, A l and A 4 each independently are Phe, MePhe, EtPhe, Tyr, Trp or Nal, S 1 is an amino acid sequence of 1 to 6 amino acids, preferably of 5 amino acids, selected from the group consisting of Ala, Cys, Asn, Phe, MePhe, EtPhe, Tyr, Trp, Nal, Gly, Lys, MeLys, (£-Me)Lys, Thr, Val, Asn and Ser, with the proviso that S1' comprises a cys- teine amino acid radical, and S2' is an amino acid sequence of 1 to 3 amino acids, preferably of 2 amino acids, selected from the group mentioned sub Sl', with the proviso that S 2 comprises a cysteine amino acid radical, the metal-radionuclide being selected from the group consisting of Tc-99m, Re-186 and Re-188 which as a cation is bound covalently to the mercapto groups of the cysteine amino acid radicals.

4. A labelled polypeptide as claimed in Claim 3, having the general formula R 1 -Ala-Gly-Cys-Lys-Asn-Phe-Phe-(D)Trp-Lys-Thr-Phe-Thr- -Ser-Cys-R 2 (IV) F, 1 WO 92/21383 PCI/US92/04559 13 the metal-radionuclide being selected from the group consisting of Tc-99m, Re-186 and Re-188 which as a cation is bound to the mercapto groups of the cysteine amino acid radicals.

A method of preparing a metal-radionuclide- labelled polypeptide as claimed in any of the preceding Claims, characterised in that a polypeptide of the general formula I, wherein the symbols have the meanings given in Claim 1, is prepared by reducing the corresponding cyclised polypeptide, in which the cysteine amino acid radicals have been oxidized together to a cystine group, with a suitable reducing agent, and the resulting polypeptide is reacted under reducing conditions with Tc-99m in the form of a pertechnetate solution or with Re-186 or Re-188 in the form of a perrhenate solution.

6. A method as claimed in Claim 5, characterised in that metallic zinc or zinc ions is/are used as a reducing agent for the cyclised polypeptide.

7. A pharmaceutical composition which, in addition to a pharmaceutically acceptable liquid carrier material and optionally one or more equally pharmaceutically acceptable adjuvants, comprises a metal-radionuclide-labelled polypep- tide as claimed in any of the Claims 1 to 4 as the active substance.

8. A method of detecting and localising tumour Stissues in the body of a warmblooded living being, charac- terised in that a composition as claimed in Claim 7 optionally after dilution with a pharmaceutically acceptable liquid, is administered to the being in a quantity which is sufficient for external imaging and that the being is then subjected to a technique of external imaging to determine I -j 14 the radioactive-labelled sites in the body in relation to the background-activity.

9. A method of treating tumours in the body of a warmblooded living being, characterised in that a composition as claimed in Claim 7, optionally after dilution with a pharmaceutically acceptable liquid, is administered to the being in a quantity effective for controlling tumours.

A kit for the preparation of a radiopharmaceutical composition comprising in an optionally dry condition a cyclised polypeptide of the general formula I, wherein the symbols have the meanings given in Claim 1 but the cysteine- amino acid radicals together are oxidis-d to a cystine group, a reducing agent and directives for reacting the ingredients mentioned sub and and the resulting reaction product with Tc-99m in the form of a pertechnetate solution or with Re-186 or Re-188 in the form of a perrhenate solution.

11. A polypeptide according to any one of Claims 1 to 4 substantially as hereinbefore described.

12. A method according to any one of Claims 5, 6, 8 or 9 substantially as hereinbefore described.

13. A composition according to Claim 7 substantially as hereinbefore described.

14. A kit according to Claim 10 substantially as hereinbefore described. DATED: 21 December 1994 CARTER SMITH BEADLE Patent Attorneys for the Applicant: MALLINCKRODT MEDICAL, INC. 21 December 1994 A 1- PCT/US 92/04559 International Application No I. CL4SSIFICATION OF SUBJECT MATTER (if several classification symbols apply, indicate all) 6 Accoraing to International Patent Classification (IPC) or to both National Classification and IPC Int.Cl. 5 A61K49/02; A61K43/00 n. FIELDS SEARCIhED Minimum Documentation Searched 7 Classification System Classification Symbols Int.C1. 5 A61K C07K Documentation Searched other than Minimum Documentation to the Extent that such Documents are Included in the Fields Searched 8 II. DOCUMENTS CONSIDERED TO BE RELEVANT 9 Category Citation of Document, it with indication, where appropriate, of the relevant passages 2 Relevant to Claim No. 13 Y WO,A,9 101 754 (B.A.RHODES) 21 February 1991 1-10 see the whole document Y THE LANCET 1-10 vol. 1, no. 8632, 4 February 1989, LONDON pages 242 244; E.P.KRENNING ET AL: 'Localisation of endocrine-related tumours with radioiodinated analogue of somatostatin' see the whole document Y WO,A,9 006 949 (SANDOZ) 28 June 1990 1-10 see the whole document Special categories of cited documen.s 0 later document published after the international filing date or priority date and not in conflict with the application but document defining the general state of the art which is not cited to understand the principle or theory unerlying the considered to be of particular relevance invention earlier document but published on or after the international document of particular relevance; the claimed invention filing date cannot be considered novel or cannot be considered to 'L document which may throw doubts on priority claim(s) or involve an inventive step which is cited to establish the publication date of another document of particular relevance; the claimed invention citation or other special reason (as specified) cannot be considered to involve an inventive step when the document referring to an oral disclosure, use, exhibition or document is combined with one or more other such docu- other means ments, such combination being obvious to a person skilled document published prior to the international filing date but in the art. later than the priority date claimed document member of he same patent family IV. CERTIFICATION Date of the Actual Completion of the International Search Date of Mailing of this International Search Report 09 SEPTEMBER 1992 International Searching Authority Signature of Authorized Officer EUROPEAN PATENT OFFICE GROENENDIJK M.S.M. Fore PCT/ISA/210 (lecond s.tcl) (Jalnary 1985) d PCT/US 92/04559 International Application No PCT/US 92/04559 ll. DOCUMENTS CONSIDERED TO BE RELEVANT (CONTINUED FROM THE SECOND SHEET) Category o Citation of Document, with indication, where appropriate, of the relevant passage Relevant to Claim No. Y CHEMICAL ABSTRACTS, vol. 112, no. 16, 6 16 April 1990, Columbus, Ohio, US; abstract no. 150685, C.KREMER ET'AL: 'Solid-phase reduction of 99mTc04 with zinc: a method for the preparation of difficult Tc-99m complexes' page 797 ;column 2 see abstract P,X EUROPEAN JOURNAL OF NUCLEAR MEDICINE 1-5,7-10 vol. 18, no. 8, August 1991, page 558; P.H.COX: 'TECHNETIUM LABELLED SOMATOSTATINE FOR IN VIVO TUMOUR LOCALISATION' P,Y see the whole document 6 A GB,A,2 021 121 (C.A.MEYERS ET AL) 28 November 1-4 1979 see the whole document A WO,A,8 912 068 (SOCIETE D'ETUDES DE PRODUITS 1-3 CHIMIQUES) 14 December 1989 see the whole document Fm Frm PCT/ISA/ZO textr shecl) tJuay t9O~ I~ I CNATIONAL SEARCH REPORT .YPCT/.LUS 92/P n No. PCT/ US 92/ 04559 Box I Observations where certain claims were found unsearchable (Continuation of item I of first sheet) This international search report has not been established in respect of certain claims under Article 17(2)(a) for the following reasons: 1. Claims Nos.: because they relate to subject matter not required to be searched by this Authority, namely: Although claim 9 is directed to a method of treatment of the human/ animal body the search has been carried out and based on the alleged effects of the compound/composition 2. D Claims Nos.: because they relate to parts of the international application that do not comply with the prescribed requirements to such an extent that no meaningful international search can be carried out, specifically: 3. D Claims Nos.: because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4(a). Box II Observations where unity of invention is lacking (Continuation of item 2 of first sheet) This International Searching Authority found multiple inventions in this international application, as follows: 1. As all required additional 'search fees were timely paid by the applicant, this international search report covers all searchable claims. 2. As all searchable claims could be searches without effort justifying an additional fee, this Authority did not invite payment of any additional fee. 3. As only some of the required additional search fees were timely paid by the applicant, this international search report covers only those claims for which fees were paid, specifically claims Nos.: 4. No required additional search fees were timely paid by the applicant. Consequently, this international search report is restricted to the invention first mentioned in the claims; it is covered by claims Nos.: Remark on Protest O The additional search fees were accompanied by the applicant's protest. m No protest accompanied the payment of additional search fees. Form PCT/ISA/210 (col..muation of first sheet (July 1992) i1111113~ I ANNEX TO THE INTERNATIONAL SEARCH REPORT ON INTERNATIONAL PATENT APPLICATION NO. US SA 9204559 60720 This annex lists the patent family members relating to the patent documents cited in the above-mentioned international search report. The members are as contained in the European Patent Office EDP file on The European Patent Office is in no way liable for these particulars which are merely given for the purpose of information. 09/09/92 Patent document Publication Patent family Publication cited in search report date member(s) date WO-A-9101754 21-02-91 US-A- 5078985 07-01-92 US-A- 5102990 07-04-92 AU-A- 6543490 11-03-91 EP-A- 0486622 27-05-92 WO-A-9006949 28-06-90 AU-A- 4587189 14-06-90 BE-A- 1002296 20-11-90 CA-A- 2004532 05-06-90 CH-A- 678329 30-08-91 FR-A- 2639947 08-06-90 GB-A- 2225579 06 06-90 JP-A- 2184698 19-07-90 NL-A- 8902981 02-07-90 SE-A- 8904087 05-06-91 GB-A-2021121 28-11-79 CA-A- 1107273 18-08-81 US-A- 4224199 23-09-80 WO-A-8912068 14-12-89 EP-A- 0383856 29-08-90 JP-T- 3500660 14-02-91 0 o For more details about this annex see Official Journal of the European Patent Office, No. 12182 0 1