WO2003104267A1

WO2003104267A1 - Kit formulation of a novel peptide c-aca-qalgnqwavghlmnh2

Info

Publication number: WO2003104267A1
Application number: PCT/GR2003/000023
Authority: WO
Inventors: Alexandra Varvarigou; Grigorios Evaggelatos; Spyridon Arhimandritis
Original assignee: Biomedica Life Sciences S.A.; N.C.S.R. "Democritos"
Priority date: 2002-06-10
Filing date: 2003-06-10
Publication date: 2003-12-18
Also published as: AU2003232945A1; GR1004600B; GR20020100277A; AU2003232945A8

Abstract

The present invention provides a novel Bombesin derivative BN1, as a preformed, lyophilized kit, labelled with the radionuclides Tc-99m and Re-188, so that the obtained radiopharmaceuticals can be easily applied in nuclear medicine for the diagnosis, for the scintigraphic detection and localization of primary and metastatic tumours and/or therapy of several tumours, such as tumours of the neuroendocrine system and specifically prostate, breast, and ovary cancer, glioblastomas and small cell lung carcinomas, tumours of the gastroenteropancreatic system and more specifically pancreatic and colon cancer and melanomas.

Description

KIT FORMULATION OF A NOVEL PEPTIDE C-Aca-QALGNQ AVGHLMNH2

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a preparation of a new decapentapeptide, Bombesin (BN) derivative, having the structure C-Aca-QALGNQWAVGHLMNH2 (derivative BNl), as a preformed lyophilized kit.

Further, it relates to a method of preparation of said kit and its clinical application for the diagnosis and/or therapy of several tumours. BACKGROUND OF THE INVENTION

Small neuropeptides, labelled with gamma and/or beta emitting radionuclides, are currently being investigated for their ability to bind to cell-surface receptors, which are expressed in some malignant tissues. These molecules are potentially useful for radionuclidic detection and/or therapy of tumours .

Attention has been lately focused on the amphibian peptide bombesin (BN) , originally isolated from frog skin and the molecularly related-gastrin releasing peptide (GRP) . Over expression of receptors for both BN and GRP has been encountered on the cell surface of several malignant tissues. These peptides act as neurotransmitters and endocrine cancer cell growth factors. As neurotransmitters they affect a series of complicated functions, for example they activate the hypothalamus-hypophysis-adrenal axis and they adjust cerebral and intestinal functions. Bombesin receptors are also involved in sleep function, as well as in the anger-feeling development. Bombesin-like peptides are active in normal tissues, where they are responsible for mitosis and development, as well as in tumour cells, of diverse origin. Thus, receptors for BN and GRP-like peptides have been isolated in hypothalamus, hypophysis and several ganglia, as well as in pathological situations as gliomas, small cell lung carcinomas, epithelial ovary cancer, colon and prcstaue cancer and several other types of tumours. More specifically, Bombesin receptors have been found in normal Central Nervous System, in smooth muscle cells, in epithelial cells of the gastrointestinal tract, but also in tumours as in breast, ovarian and lung cancer tissue. Bombesin acts as an antagonist to somatostatin and somatostatin receptors. Over expression of Bombesin receptors is a significant cancer marker, indicative of the aggressiveness of a developing malignant tumour. Consequently, the in vivo imaging of Bombesin receptors is very important for diagnosis, being also significantly potential for therapy.

Several research groups have labelled Bombesin derivatives with iodine radioisotopes , aiming to the early detection of primary and metastatic tumours. Thus, the radiopharmaceutical ^12ΞI-Tyr⁴-BN has been clinically applied and is already on the market.

In addition, another bombesin derivative is also known, namely meta-iodophenyl-Des-Met¹⁴-BN (mlP-bombesin) , which has been labelled with 1-125 and presents significant uptake in experimentally induced ovary cancer. Further, it is also known that bombesin-like peptides labelled with Rhenium (Re-188) get bind, in vi tro, to cancer cells and are proposed for prostate cancer therapy.

Some recent studies refer to Bombesin-like peptides, properly modified to be labelled with the radionuclide

Tc-99m. In this case either two diaminedithiol (DADT) groups get attached onto the Bombesin aminoacid sequence, so that Tc-99m gets directly bound into the peptide as described in text "Design, synthesis and initial evaluation of high- affinity technetium Bombesin analogues ^Xλ by Baidoo et al

(1998) published in Bioconjug. Chem.9, pages 218-225, or a dithio-diphosphonic ligand is introduced into the peptide backbone, on which the radionuclide gets bound, so that the peptide is labelled indirectly as described in text ^u99Tc- labelling and in vivo studies of bombesin analogue with novel water-soluble dithiadiphosphine-based bifunctional chelating agent" by Karra et al (1999) published in Bioconjug . Chem.10 , pages 254-260. In these documents it is mentioned that both types of substituted derivatives present high affinity for Bombesin receptors, however their synthesis requires many steps, is complicated and time- consuming .

In another study described in text "Preparation and preclinical evaluation of a new ^99mTc- labelled bombesin-like peptide" by Varvarigou et al (1998) published in Eur. J. Nucl . Med. 25, page 915, it is mentioned a bombesin-like peptide with the following aminoacid sequence: Cys-Aca-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met - NH2 , wherein Ac : 6-amino-n, hexanoic acid. This derivative, BNl, labeled with Tc-99m presents radiochemical and radiobiological characteristics suitable for application in nuclear medicine. Clinical studies have already been initiated in Greece and abroad with satisfactory results in the imaging of diverse tumours. The known Bombesin derivatives either are radioiodinated e.g. 1-125, 1-131, with the well-known difficulties of these radionuclides or in case they are synthetic derivatives, in order to be labelled with Tc-99m they require complicated in si tu synthesis and labelling, resulting in difficulties for their transport in hospitals.

SUMMARY OF THE INVENTION

It is, therefore, an object of the present invention to provide a preformed kit of a new peptide, which overcomes the deficiencies of the prior art and can be safely transported and clinically applied for the detection and therapy of cancer tumours. Another object of the present invention is to provide a method of preparation of BNl, as a preformed, lyophilized kit, labelled with the radionuclides Tc-99m and Re-188, so that said new radiopharmaceuticals can be easily applied in nuclear medicine.

A further aspect of the present invention is the use of the preformed kit of the radiolabelled derivative for the diagnosis and/or therapy of diverse tumours. In accordance with the above objects of the present invention, a method of preparation of a kit formulation, a kit formulation and use of said kit formulation are provided as defined in claims 1, 8 and 16 respectively. Further preferred embodiments of the present invention are defined in dependent claims 2 to 7 and 9 to 15.

These and other, further and more specific objects and advantages of the present invention will become apparent to those skilled in the art in view of the following detailed description.

DETAILED DESCRIPTION OF THE INVENTION Kit formulation

The new decapentapeptide , Bombesin (BN) derivative according to the present invention has the structure: C-Aca-QALGNQ AVGHLMNH2 (derivative BNl) , wherein Aca: 6-amino-n, hexanoic acid. The peptide BNl is a lyophilized, injectable powder in a penicillin vial. A preformed, lyophilized kit of the peptide BNl of the present invention is prepared, containing in each vial a quantity of equal or less than 0.20 mg of said peptide BNl, 0.06 mg of sodium methylene diphosphonate (MDP) , 2.88xl0^"3mg of stannous chloride (SnCl₂) , 0.0144 mg of ascorbic acid, and an amount of sugar, as stabilizer. A series of diverse sugars is under evaluation. The formulation contains ingredients, which ensure the in vi ro stability of the kit, allowing storage for a long period of time, making the kit transfer possible and facilitating clinical application. The method of preparation of the kit ensures the pharmaceutical grade, the safety and the effectiveness of the radiopharmaceutical produced upon reconstruction, which is sterile, pyrogen-free and suitable to be used in diagnosis and/or therapy.

^99mTc-labelling The labelling is performed by the reconstitution of the content of the lyophilized kit, by adding 2.0 ml of a pertechnetate solution, containing 370-1110 MBq, (10.0-30.0 Ci) ^99mTcNa04 in saline. Thus the complex ⁹⁹Tc-BNl is formed in a single step.

¹⁸⁸Re-labelling

A ¹⁸⁸ /¹⁸⁸Re generator is eluted with saline. A mixture of reducing agents such as SnCl₂, ascorbic acid and citric acid, is added in 2.0 ml of the eluate for perrhenate ions reduction. The reduced Rhenium-188, containing radioactivity of 185-740 MBq (5.0-20.0 mCi), is added to a mixture of SnCl₂, Sodium Gluconate and Sodium Bicarbonate. Within about 15 minutes, Rhenium Gluconate has been formed, which is then added to a water solution of the decapentapeptide BNl . The mixture is first warmed to 90° C for one hour and then left at room temperature. Thus the complex ^lδεRe-BNl is formed

Radiochemical study of the radiolabelled BNl derivatives

The labelling efficiency and the in vi tro stability of the radiolabelled product were determined by analytical High Performance Liquid Chromatography (HPLC) with a Waters C₁₈ reverse phase column μ-Bondapack C₁₈, (3.9mm I.D. x 300mm) . For the elution has been used a linear gradient system, comprising A: 0.05% Trifluoro Acetic Acid (TFA) in water and B: 60% Acetonitrile (ACN) in solution A. Eluate was passed through the UV detector, at 220 nm and through a sodium iodide scintillation detector, both connected to a computer for data analysis and storage. It was found that a single ^99mTc-labelled BNl derivative is formed, with a retention time of Rt=17.0-17.5 min at a _ 97% yield. Also ¹⁸⁸Re-labelling resulted to a single radioactive BNl derivative, with Rt=13.5 min.

After labelling and prior to clinical application, the radiopharmaceutical is controlled by Instant Thin Layer Chromatography (ITLC) , using silica gel strips, as stable phase and methanol 75% as mobile phase. In this developing system, ^99mTc-BNl derivative has an Rf=0.8, while colloidal Tc-99m remains on the spot.

The in vi tro stability of the radiolabelled species, kept in refrigerator, was evaluated by Instant Thin Layer Chromatography. It has been found that both the radioactive derivatives remain stable for 48 hours, at 4°C. This finding advocates for the suitability of the new peptide to be used in nuclear medicine .

The shelf-life of the kit is still under evaluation, however until now it has been found to be greater than five months. The radiopharmaceutical according to the present invention has been found sterile and pyrogen-free.

Radiobiological evaluation of the species derived by labelling the BNl kit

The in vivo behaviour of the Bombesin-like peptide ^99mTc-BNl of the present invention has been initially evaluated in normal rodents (Swiss mice and Wistar rats) . The radiopharmaceutical was injected intravenously into the tail vein. Animals were sacrificed at predetermined time intervals and the main organs, as well as bone, muscle and urine samples were removed, weighted and counted in a well- type counter. The percentage of the injected dose per organ and per gram of tissue was calculated, in comparison to a standard. In vivo studies were performed in compliance with the Greek and European legislation.

In both species the radiolabelled peptide presented fast blood clearance and elimination by both the hepatobiliary and the renal system. Radioactivity values in stomach were low, indicating satisfactory in vivo stability.

The biodistribution of the peptide derivatXe of the present invention was also studied in tumor-bearing animals, in which experimental tumours had been created by implantation of cancer cells of human origin. More specifically two diverse cell lines were injected expressing colon cancer and small cell lung carcinoma.

The evaluation was performed by scintigraphic detection in gamma camera and afterwards animal vivisection. The ratio of tumour versus normal tissue concentration, expressed as %dose per gram, was found to be 3.5 for colon cancer and 7.8 for small cell lung carcinoma. The results of both the in vi tro and the in vivo study are considered as very interesting and advocate for the clinical trial of the radiopharmaceutical of the present invention.

It will be appreciated from the foregoing that the present invention represents a significant advance in the nuclear medicine for the diagnosis, for the scintigraphic detection and localization of primary and metastatic tumours and/or therapy of several tumours, such as:

Tumours of the neuroendocrine system and specifically prostate, breast, and ovary cancer, glioblastomas and small cell lung carcinomas.

Tumours of the gastroenteropancreatic system and more specifically pancreatic and colon cancer. Mel nomas .

In particular, it provides a novel Bombesin derivative BNl, as a preformed, lyophilized kit, labelled with the radionuclides Tc-99m and Re-188, so that the obtained radiopharmaceuticals can be easily applied in nuclear medicine .

The radiopharmaceutical ^99tnTc-BNl is prepared from the preformed kit in a single step, by reconstituting the content of the kit with 2.0 ml of a pertechnetate solution, containing 370-1110 MBq (10.0-30.0 mCi ^99mTcNa04). The injectable saline solution of sodium pertechnetate-^99mTc , is obtained by the elution of a ⁹⁹Mo/^99mTc generator, under sterile conditions. The radiopharmaceutical ¹⁸⁸Re-BNl is prepared from the same preformed kit by former reduction of a sodium perrhenate solution, obtained by the elution of a ¹⁸⁸W/^18SRe generator with saline, under sterile conditions. The reduced Re-188 is properly modified to ¹⁸⁸Rhenium Gluconate, which is added to a water solution of the decapentapeptide, and the mixture is first warmed at 90° C for one hour and then left at room temperature .

The preformed kit contains ingredients ensuring the in vi tro stability, permitting thus kit transport and storage for a period of time more than five months, thus facilitating clinical use.

The method used for preparation of the kit ensures the pharmaceutical grade, the safeness and the suitability of the radioactive solution prepared after kit reconstruction. The obtained radiopharmaceutical is sterile ar.d pyrogen- free .

Although a particular embodiment of the invention has been described in detail, it will be appreciated that various modi ications may be made without departing from the spirit and scope of the invention, as defined in the appended claims .

Claims

CLAIMS 1. A method of preparation of a kit formulation wherein said kit formulation comprises a decapentapeptide, Bombesin derivative, having the structure of C-Aca-QALGNQWAVGHLMNH2 , derivative BNl and wherein Aca : 6-amino-n, hexanoic acid, said peptide is a lyophilized, injectable powder, in at least one vial, ready to use upon reconstitution by the addition of a sterile pertechnetate or perrhenate solution.

2. The method according to claim 1, wherein said at least one vial contains a quantity of equal or less than 0,20mg of the lyophilized peptide BNl, 0.06 mg of sodium methylene diphosphonate (MDP) , • 2.88xl0^"3 mg of stannous chloride (SnCl₂) , 0.0144 mg of ascorbic acid, and an amount of sugar, as stabilizer.

3. The method according to claim 2, wherein said Bombesin derivative BNl is labelled with radionuclides Tc-99m and/or Re-188.

4. The method according to claim 3 , wherein said radiopharmaceutical ^99mTc-BNl is prepared from the preformed kit in a single step, by reconstituting the content of the kit with 2.0 ml of a pertechnetate solution, containing 370- 1110 MBq (^99mTcNa04) .

5. The method according to claim 4, wherein the injectable saline solution of sodium pertechnetate-^99mTc, is obtained by the elution of a ⁹⁹Mo/^99mTc generator, under sterile conditions .

6. The method according to claim 3, wherein the radiopharmaceutical ¹⁸⁸Re-BNl is prepared from the same preformed kit by reduction of a sodium perrhenate solution, obtained by the elution of a ¹⁸⁸W/¹⁸⁸Re generator with saline, under sterile conditions.

7. The method according to claim 6, wherein the reduced Re- 188 is properly modified to ¹⁸⁸Rhenium Gluconate, which is added to a water solution of the decapentapeptide, and the mixture is first warmed at 90° C for one hour and then left at room temperature .

8. A kit formulation comprising a decapentapeptide, Bombesin derivative, wherein said peptide has the structure of C-Aca- QALGNQWAVGHLMNH2 , derivative BNl and wherein Aca: 6-amino-n, hexanoic acid, said peptide is a lyophilized, injectable powder, in at least one vial, ready to use upon reconstitution by the addition of a sterile pertechnetate or perrhenate solution.

9. The kit formulation according to claim 8 , wherein said at least one vial contains a quantity of equal or less than 0,20mg of the lyophilized peptide BNl, 0.06 mg of sodium methylene diphosphonate (MDP) , 2.88xl0^"3 mg of stannous chloride (SnCl₂) , 0.0144 mg of ascorbic acid, and an amount of sugar, as stabilizer.

10. The kit formulation according to claim 9, wherein said Bombesin derivative BNl is labelled with the radionuclides Tc-99m and/or Re-188.

11. The kit formulation according to claim 10, wherein said radiopharmaceutical ^99mTc-BNl is prepared from the preformed kit in a single step, by reconstituting the content of the kit with 2.0 ml of a pertechnetate solution, containing 370- 1110 MBq (^99mTcNa04) .

12. The kit formulation according to claim 11, wherein the injectable saline solution of sodium pertechnetate-^99mTc, is obtained by the elution of a ⁹⁹Mo/^99mTc generator, under sterile conditions.

13. The kit formulation according to claim 10, wherein the radiopharmaceutical ¹⁸⁸Re-BNl is prepared from the same preformed kit by reduction of a sodium perrhenate solution, obtained by the elution of a ¹⁸⁸W/¹⁸⁸Re generator with saline, under sterile conditions.

14. The kit formulation according to claim 13, wherein the reduced Re-188 is properly modified to ¹⁸⁸Rhenium Gluconate, which is added to a water solution of the decapentapeptide, and the mixture is first warmed at 90° C for one hour and then left at room temperature.

15. The kit formulation according to any preceding claim , wherein the obtained radiopharmaceutical is sterile and pyrogen-free .

16. Use of the kit formulation according to any preceding claim for the diagnosis, for the scintigraphic detection and localization of primary and metastatic tumours and/or therapy of several tumours, such as tumours of the neuroendocrine system and specifically prostate, breast, and ovary cancer, glioblastomas and small cell lung carcinomas, tumours of the gastroenteropancreatic system and more specifically pancreatic and colon cancer and melanomas.