MINI-GASTRIN ANALOGUE, IN PARTICULAR FOR USE IN CCK2 RECEPTOR POSITIVE TUMOUR DIAGNOSIS AND/OR TREATMENT

Abstract

A gastrin analogue shows high uptake in CCK-2 receptor positive tumors and simultaneously a very low accumulation in the kidneys. This is achieved by a mini-gastrin analogue PP-F11 having the formula: PP-F11-X-DGlu-DGlu-DGlu-DGlu-DGlu-DGlu-Ala-Tyr-Gly-Trp-Y-Asp-Phe-NH.sub.2, wherein Y is an amino acid replacing methionine and X is a chemical group attached to the peptide for diagnostic and/or therapeutic intervention at CCK-2 receptor relevant diseases. Very suitable compounds with respect to a high tumor to kidney ratio are mini-gastrin analogues with six D-glutamic acids or six glutamines. These compounds still possess a methionine which can be oxidized easily which is a disadvantage for clinical application under GMP due to the forms which may occur. The elimination of the methionine leads to a lower affinity to oxidation which in general favors the tumor-kidney-ratio. Ideally, the methionine is replaced by norleucine. This PP-F11N mini gastrin exhibits currently the best tumor-kidney-ratio and is the most promising candidate.

Claims

1-9. (canceled)

10. A pharmaceutical composition comprising a therapeutically effective amount of a mini-gastrin analogue having the formula:
X-DGlu-DGlu-DGlu-DGlu-DGlu-DGlu-Ala-Tyr-Gly-Trp-Y-Asp-Phe-NH.sub.2, wherein Y is norleucine and X is a chemical group attached to the peptide for the purpose of therapeutic intervention at CCK-2 receptor associated diseases, and a pharmaceutically acceptable carrier.

11. The pharmaceutical composition of claim 10, wherein the therapeutically effective amount is effective for treating CCK-2 receptor positive tumors.

12. The pharmaceutical composition of claim 11, wherein the CCK-2 receptor positive tumor is selected from the group consisting of medullary thyroid carcinomas (MTC), small cell lung cancers (SCLC), astrocytomes and stromal ovarial tumors

13. The pharmaceutical composition of claim 10, wherein X is a chelator for radiometals complexed with a radionuclide.

14. The pharmaceutical composition of claim 13, wherein the chelator for radiometals is DOTA.

15. The pharmaceutical composition of claim 13, wherein the radionuclide is selected from the group consisting of .sup.177Lu, .sup.90Y and .sup.111In.

16. The pharmaceutical composition of claim 14, wherein the mini-gastrin analogue is labelled with .sup.177Lu.

17. The pharmaceutical composition of claim 10, wherein X is an optically active chemical compound.

18. The pharmaceutical composition of claim 10, wherein X is a chemotherapeutic active compound.

19. The pharmaceutical composition of claim 10, wherein X is a nanoparticle or liposome which has a therapeutic function by itself or which is loaded with an active compound.

20. The pharmaceutical composition of claim 19, wherein X is a nanoparticle or liposome which is an optically active agent.

21. The pharmaceutical composition of claim 10, which is formulated for i.v. injection.

22. The pharmaceutical composition of claim 16, wherein the isotope:peptide ratio is 1:47.

23. The pharmaceutical composition of claim 14, wherein the radionuclide is selected from the group consisting of .sup.177Lu, .sup.90Y and .sup.111In.

Description

[0012] Preferred embodiments of the present invention are hereinafter described in more detail with respect to the attached drawings which depict in:

[0013] FIG. 1 the structural design of PP-F11N starting from a mini-gastrin analogue PP-F11 being arised from the COST initiative;

[0014] FIG. 2 the biodistribution of PP-Flu, PP-F11N and PP-Flu ox (oxydized PP-F11) after four hours in the CD1 nu/nu mice modell;

[0015] FIG. 3 the bio diversion of PP-F10, PP-F10N and PP-F10 ox (oxydized PP-F10) after four hours in the CD1 nu/nu mice modell; and

[0016] FIG. 4 the stability of diverse mini-gastrin analogues with the course of the time.

[0017] FIG. 1 illustrates the mini-gastrin analogue PP-Flu that has been derived from the COST initiative mentioned above. The modified mini-gastrin analogue PP-F11N has been achieved by the exchange of the oxidizable amino acid methionine with norleucin. DOTA stands for 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid which is an organic compound with the formula (CH.sub.2CH.sub.2NCH.sub.2CO.sub.2H).sub.4. The molecule consists of a central 12-membered tetraaza (i.e., containing four nitrogen atoms) ring. DOTA is used as a complexing agent, especially for lanthanide ions. Its complexes have medical applications as contrast agents and cancer treatments.

[0018] PP-F11N has been investigated according to the CD1 nu/nu mice model. As compared to PP-F11, the mini-gastrin analogue PP-F11N exhibited a significant higher tumor uptake which also leads to a very favorable tumor-noise ratio with very few accumulation in the kidneys. FIG. 2 illustrates the biodistribution of PP-F11, PP-F11N and oxidized PP-F11 ox (oxidized PP-F11) after 4 hours in the athymic CD1 nu/nu mice model. Tumour positive: with human CCK-2 receptor transfected A431 cells on one side of the mouse; tumor negative: CCK-2 receptor negative A431 cells on the other side of the mouse. The effect of the higher tumor uptake by the exchange of methionine with norleucine is specifically apparent with compounds having the DGlu6 sequence, different from compounds having a sequence with DGLn6 which is referred to as PP-F10.

[0019] FIG. 3 shows the results for PP-F10 and PP-F10N and PP-F10 ox (oxidized PP-F10) in comparison to the results shown in FIG. 2. The structural formula of the PP-F10's is given below:

[0020] PP-F10: DOTA-DGln-DGln-DGln-DGln-DGln-DGln-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH.sub.2

[0021] PP-F10N: DOTA-DGln-DGln-DGln-DGln-DGln-DGln-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH.sub.2;

[0022] PP-F10 ox: DOTA-DGln-DGln-DGln-DGln-DGln-DGln-Ala-Tyr-Gly-Trp-Met(ox)-Asp-Phe-NH.sub.2.

[0023] A promising tumor uptake can therefore not be seen in FIG. 3 since other organs, such as the pancreas, the kidney and the bones take partially even higher doses than the targeted tumor.

[0024] FIG. 4 illustrates the pharmacologic stability of PP-F11N as compared to PP-F11, PP-F10N and PP-F10 (all radio-labelled with .sup.177Lu. The stability has been checked in human serum; the measurements of the metabolites have been executed by means of HPLC. The mini-gastrin analogue PP-F11N according to the present invention shows the highest pharmacologic stability of all probands.

[0025] Materials: The peptides (PP-F10, PP-F10N, PP-F11 and PP-11N) were synthesized by PLS (Heidelberg, Germany) by the Merrifield method. All the chemicals were purchased from Sigma-Aldrich (Buchs, Switzerland). A431 cells (cell line squamous cell carcinoma) were stably transfected with cDNA encoding for CCK2R or with empty vector (‘mock-transfected’).sup.1 and were a kind gift from L. Aloj (Naples). Lu-177 was purchased from ITG (Germany, Munich). The peptide conjugates were complexed with natural .sup.natLu.

[0026] The labeling of the mini-gastrins has been executed under the following circumstances:

[0027] System for HPLC Analysis:

[0028] System: Pump Varian Prostar 2030.01, Diode Array 330.71, Autosampler 410, Packard Radiomatic Flow-One\

[0029] Column: Stability 120 BS-C23 3 μm 150*4.6 mm, Dr. Maisch

[0030] Gradient:

TABLE-US-00001 % H2O + 0.1% % ACN + 0.1% Min TFA TFA 0 90 10 3 90 10 15 5 95

[0031] System for the Purification:

[0032] Pump 1: Waters 515, Pump 2: Hitachi L-7000, Knauer UV Detector K2510, Radio-Monitor Eberline, interface SS420X, EZstart

[0033] Rheodyne Manuel Injector

[0034] Column 1: Stability 120 BS-C23 3 μm 10*4.6 mm, Dr. Maisch

[0035] Column 2: Stability 120 BS-C23 3 μm 150*4.6 mm, Dr. Maisch

TABLE-US-00002 % H2O + 0.1% % ACN + 0.1% Min TFA TFA Valve1 Valve2 0 72 28 Inject Load 3 72 28 Inject Inject 20 60 40 25 5 95 35 5 95

[0036] Products:

[0037] Lu-177: lot Lu-12-052-01/121042, Activity 2 GBg/200 μl 0.04M HCL, itg (ITM AG)

[0038] PP-F11N: 0.25 mM H.sub.2O solution

[0039] Ammonium solution: Sigma-Aldrich metal free

[0040] Na Ascorbate: Sigma-Aldrich

[0041] HCl 30%: sigma-Aldrich nmetal free

[0042] H2O: from Milipore system Biotel

[0043] Labelling of the Peptide PPF11N with Lu-177:

[0044] The Labelling of Lu-177 with the PPF-11N was made with an isotope:peptide ratio of 1:47.

[0045] Mixture of Lu-177 with peptide to an eppendorf 1.5 ml lw binding: [0046] 20 μl Lu-177 (190MBq) [0047] 5 μl Ammonium ascorbate 0.7M [0048] 50 μl PPF-11N 0.25 mM [0049] 5 μl HCl 0.04M

[0050] The mixture was heated for 20 minutes at 95° C.

[0051] Afterwards the complex was purified and checked with HPLC methods.

[0052] Two syntheses were achieved in parallel [0053] Purification of labelling peptide with HPLC

[0054] The two labellings reaction was injected into a 2 D HPLC. [0055] Description of 2D HPLC: [0056] First step: inject the product into the loop with a rheodyne manual injector and push the product with a first pump through the column 1. The product is transferred from loap to the column 1 and is washed with H.sub.2O+.sup.0.1TFA. [0057] Second Step: start the gradient with a second pump and change the position of the valve to connect in serial the column 1 and column 2. [0058] The product is push from column 1 in column 2. The excess of peptide is separed of the labeled Lu-177-PPF11N in the column 2 and is collected with a fraction size of 500 ul. The collected tube contains still 5 mg Na-ascorbate. [0059] Result of purification with HPLC

TABLE-US-00003 Activity Collection [MBq] % time [min] Volume [μl] Injected 378 100 80 activity Remainder 30 8 eppendorf tube Fraction 3 120 31 12.5-13.0 500 Fraction 4 180 48 13.0-13.5 500

[0060] Preparation of the labeled peptide for mouse i.v. injection

[0061] The two fractions have been fused and the solvent was evaporated during 35 min.

[0062] Afterwards 600 ul PBS 1× with 10 ul 5 mM DTPA solution was added.

[0063] Final solution: 295 MBq/610 ul.

[0064] Stability

[0065] 12 MBq of the radiolabelled compound was incubated in 2 mL fresh human plasma. A 40 μL probe was taken after 0, 1, 2, 18, 24, 48 and 72 h and added 200 μL (50% Methanol and 50% Acetonitril) in a Mini-UniPrep Filter. The solution is filtered after vortexing. 40 μL of the filtered solution is analyzed by HPLC.

[0066] Biodistribution Studies

[0067] CD1 nu/nu mice were injected with 5×10.sup.6 A431 cells. CCK-2 receptor positive A431 cells' were injected into one flank and mock cells on the other as an unspecific control. The tumors reach a weight of about 80 to 120 mg after about 10 days. 150-200 kBq (5 pmol) of the radiolabelled peptides were injected into the tail vein. Mice were killed by CO.sub.2 asphyxiation after defined time points post injection. The organs were dissected, weighted and the activity was measured. The % injected activity per gram (% i.A./g) was calculated. The animal experiments were approved by the local animal welfare committee and performed according to national regulations.

DISCUSSION

[0068] 1. Aloj L, Caraco C, Panico M, Zannetti A, Del Vecchio S, Tesauro D, De Luca S, Arra C, Pedone C, Morelli G, Salvatore M. In vitro and in vivo evaluation of 111In-DTPAGlu-G-CCK8 for cholecystokinin-B receptor imaging. J Nucl Med. 2004; 45(3):485-494. [0069] 2. Lehenberger S, Barkhausen C, Cohrs S, Fischer E, Grunberg J, Hohn A, Koster U, Schibli R, Turler A, Zhernosekov K. The low-energy beta(-) and electron emitter (161)Tb as an alternative to (177)Lu for targeted radionuclide therapy. Nucl Med Biol. 38(6):917-924.