Somastotatin analogs containing chelating groups and their radiolabeled compositions

Abstract: Compounds of formula I wherein M is a cation equivalent and A is Phe or Tyr, in free form or in salt form are useful as radiopharmaceuticals when complexed with an α- or β-emitting radionuclide or a nuclide with Auger-e - -cascades.

Description: The present invention relates to somatostatin peptides, process for their production, pharmaceutical preparations containing them and their use as a radiopharmaceutical, e.g. for radiotherapy of somatostatin receptor positive tumors. Radiotherapy of tumors with radioactive compounds has the advantages of selectively targeting tumors and their metastases, thus limiting the radiation dose to normal tissue. A radiotherapeutic agent should have a fast accumulation and high retention at the target organs, e.g. tumor, and a fast clearance from the circulation to limit the whole body radiation dose. A radiotherapeutic agent based on a chelated radiometal should also be thermodynamically and/or kinetically stable against loss of the radiometal. A radiotherapeutic agent being administered repeatedly should not be immunogenic. GB-A-2,225,579 discloses somatostatin peptides bearing at least one chelating group which can be radiolabelled for in vivo diagnostic and therapeutic applications. These compounds are capable of binding to somatostatin receptors, e.g. expressed or overexpressed by tumors or metastases.The compound of Example 1 of EP-A2-515,313 comprises a chelating group DOTA attached by one of its -CH 2 - via a p-isothioxyanatobenzyl spacer to the terminal amino groups of octreotide(somatostatin octapeptide). EP-A2-607,103 describes somatostatin peptides comprising a bifunctional polyaminopolycarboxylic acid chelating group attached to their terminal amino group by means of a spacer group. However, even though the presence of a bifunctional chelating group (octadentate) and of a spacer group lead to the improvement of certain properties of the resulting conjugate, there is still a compelling need for a radiopharmaceutical with further improved properties, particularly a high target/kidney ratio to minimize the radiation dose in the kidneys. It has now been found that somatostatin peptides as disclosed hereinafter comprising a monofunctional DOTA (1,4,7,10-tetra-azacyclododecane-1,4,7,10-tetraacetic acid) conjugated directly, i.e. in the absence of a spacer group, to the terminal amino group of the somatostatin peptides have improved properties. Particularly they have an improved tumor/kidney ratio hence a lower radiation dose delivered to the kidneys. According to the invention there is provided a compound of formula I wherein M is a cation and A is Tyr, in free form, salt form or complexed with a radionuclide. As it will be appreciated, each bond connecting 2 nitrogen atoms in the DOTA formula above represents ethylene. When A is Phe, the peptide moiety of the compound of formula I corresponds to octreotide. When A is Tyr, the peptide moiety corresponds to [Tyr 3 ]-octreotide. M may be H + or any salt forming cation for a carboxy group, e.g. a monovalent or one equivalent of a polyvalent cation, for example an alkali metal ion such as sodium, potassium or a substituted or unsubstituted ammonium ion. Compounds of formula I may also exist e.g. in the form of an internal salt when M is H + or in the form of acid addition salts. Acid addition salts include e.g. salts obtained by addition of an organic, polymeric or inorganic acid, for example chlorhydrate, acetate, trifluoroacetate or lactate. By radionuclide is meant an α- or β-emitting nuclide or a nuclide with Auger-e - -cascades. Suitable nuclides include e.g. 64 Cu, 67 Cu or a radiolanthanide, particularly 90 Y, 140 La, 161 Tb, 169 Er, 153 Sm, 177 Lu, 166 Dy, 166 Ho or 175 Yb, more preferably 161 Tb and 90 Y, most preferably 90 Y. The present invention also includes a process for the production of the compounds of formula I. They may be produced by analogy to known methods. The compounds of formula I may be produced for example as follows: a) removing at least one protecting group which is present in a compound of formula I in protected form, or b) linking together by an amide bond two peptide units, one of them containing at least one amino alcohol in protected or unprotected form and the other of them containing the DOTA group, wherein the amide bond is in such a way that the desired amino acid sequence of formula I is obtained, and stage a) of the process is then optionally effected, or c) linking together DOTA and [Tyr 3 ]- octreotide in protected or unprotected form in such a way that the group derived from DOTA is fixed on the terminal amino group of the peptide, and stage a) is then optionally effected or, d) oxidising a DOTA-peptide having the amino acid sequence as stated in formula I wherein the mercapto groups of the Cys radicals exist in free form so as to produce a compound of formula I in which the 2 Cys radicals are joined by an S-S-bridge, and recovering the compounds of formula I thus obtained in free form, in salt form or in complexed form with a radionuclide. The above reactions may be effected in analogy with known methods, e.g. as described in the following examples. Where desired, in these reactions, protecting groups which are suitable for use in peptides or for the DOTA chelating group may be used for functional groups which do not participate in the reaction. The term protecting group may also include a polymer resin having functional groups. DOTA may be used in process step (c) in the free acid form, as an anhydride or as an activated ester, e.g. with N-hydroxysuccinimide. The complexation with the radionuclide may be performed at room temperature in accordance with methods known in the art, e.g. by reacting an uncomplexed compound of formula I with a salt yielding the desired radionuclide. Preferably the complexation of the uncomplexed compound of formula I with the radionuclide-yielding salt is effected at a temperature of from 60° to 120° C, more preferably from 80 to 100° C. When the complexation is performed at a temperature ≥ 100° C, it may be effected in an autoclave. The complexation under heating may conveniently be carried out for a short time period, e.g. from 10 to 20 minutes. When the radionuclide is 90 Y, preferred 90 Y-yielding salt is 90 YCl 3 . The above mentioned reactions may conveniently be effected under conditions avoiding trace metal contamination. Preferably distilled de-ionized water, ultrapure reagents, no carrier-added radioactivity etc. are used to reduce the effects of trace metal. The compounds of formula I, when complexed with a radionuclide, exhibit pharmaceutical activity and are therefore useful as a radio- pharmaceutical for the in vivo treatment of somatostatin receptor positive tumors and metastases as indicated by standard tests. In particular, the compounds of formula I possess affinity for somatostatin receptors which can be assessed in vitro in binding assays performed as disclosed by J.C. Reubi, Life Sc. 36, 1829 (1985) and by C. Bruns et al. in Biochem. J., 265, 39 (1990). It is observed that the compounds of formula I, e.g. a compound of formula I complexed with 90 Y or 161 Tb, bind with a good affinity and specificity to somatostatin receptors with pK D values of from about 8.0 to 10.0. Compound of Example 1 binds with high affinity to somatostatin receptors expressed in rat cortex or AR42J pancreatic tumor cells: it has a pKi of 8.9 ± 0.1 using [ 125 I] [Tyr 3 ]-octreotide as specific ligand. Compound of Example 2 is a somatostatin receptor specific ligand that can be displaced from the somatostatin receptors by octreotide: pIC 50 = 9.0 ± 0.3. The affinity of the complexed compounds of formula I for somatostatin receptors can also be shown by in vivo testing, according to standard test methods, e.g. as disclosed in GB-A-2, 225,579. For example, in one trial, the compound of Example 2 gives a significant tumor accumulation 2 hours following i.p. injection at a dose of 5 µCi into mice bearing an exocrine pancreatic tumor: 9.53 ± 0.70 %ID/g present in the somatostatin receptor positive tumor whereas significantly less radioactivity is accumulated in somatostatin receptor positive normal tissues, e.g. in the pancreas it amounts 1.52 ± 0.08 %ID/g. By % ID/g is meant the percentage of the injected radioactivity dose per g tissue. Not only does Compound of Example 2 provide 24 hours after injection a high tumor/kidney ratio but also a high tumor/liver and tumor/femur ratio. Bone marrow being regarded as the most radiosensitive organ, the low radioactivity accumulated in bones is particularly advantageous. The compounds of formula I complexed with a radionuclide have an antiproliferative effect on tumor cells bearing somatostatin receptors, e.g. as indicated in nude mice tests. AR42J rat pancreatic tumor cells are trypsinized and 1x10 7 tumor cells (in 0.2 ml) are injected subcutaneously into both flanks of nude mice. When tumors have reached a significant volume of 0.1 to 2 ml animals are randomized into control and treatment groups. Control animals, receive either an uncomplexed compound of formula I or the corresponding cold complexed compound of formula I by i.p. injections at doses corresponding to the highest dose of the treatment groups. Doses from 0.8 mCi up to 40 mCi/kg/100 µl are given per mouse. The size of the tumors is determined with a caliper. To calculate the tumor volume in ml the equation "volume (ellipsoid) = length x depth x height x 0.52" is used. For statistical calculations Student's t-test is applied. In this test, transient tumor shrinkage is observed after one week and tumor growth is delayed for two weeks upon a single application of the compound of Example 2. In contrast the control groups showed continuous tumor growth with a volume doubling time about seven days. The survival rate of the treated groups is significantly increased. Similar results are obtained when treating mice or rats bearing other tumor types, e.g. mammary carcinomas or small cell lung cancer, with Compound of Example 2. Accordingly, in a series of specific or alternative embodiments, the present invention also provides the use of a compound of formula I or a pharmaceutically acceptable salt thereof in the manufacture of a radiopharmaceutical composition. Dosages employed in practising the radiotherapeutic use of the present invention will of course vary depending e.g. on the particular condition to be treated, for example the known radiosensitivity of the tumor type, the volume of the tumor and the therapy desired. In general, the dose is calculated on the basis of radioactivity distribution to each organ and on observed target uptake. A β-emitting complex of formula I may be administered at several time points e.g. over a period of 1 to 3 weeks or longer. An indicated dosage range is of from 5 to 100 µg compound of formula I complexed with e.g. 10 to 100 mCi radionuclide, e.g. 90 Y or 161 Tb, the higher radioactivity range being preferred for 161 Tb. The compounds of formula I complexed with a radionuclide may be administered by any conventional route, in particular intravenously, e.g. in the form of injectable solutions or suspensions. They may also be administered advantageously by infusion, e.g. an infusion of 30 to 60 min. Depending on the site of the tumor, they may be administered as close as possible to the tumor site, e.g. by means of a catheter. They may also be administered repeatedly in divided doses. Tumors which may be treated with Compounds of formula I complexed with a radionuclide are e.g. pituitary, gastro-enteropancreatic, carcinoids, central nervous system, breast, prostatic, ovarian or colonic tumours, small cell lung cancer, paragangliomas, kidney cancer, skin cancer, neuroblastomas, pheochromocytomas, medullary thyroid carcinomas, myelomas, lymphomas, Hodgking and non-Hodgkins disease, bone tumors and metastases thereof. Excretion of the radioactive compounds essentially takes place through the kidneys. Further protection of the kidneys from radioactivity accumulation may be achieved by administration of lysine or arginine or an amino acid solution having a high content of lysine and/or arginine, e.g. a commercially available amino acid solution such as Vamina R 18EF or Synthamin R -14 or -10, prior to the injection of or together with the complexed compound of formula I. Compounds of formula I complexed with a radionuclide may exist in free acid form or as pharmaceutically acceptable salts which exhibit the same order of activity as the complexes in free acid form. Compound of Example 1 complexed with 90 Y is preferred. According to a further aspect of the invention, there is provided a pharmaceutical composition comprising a compound of formula I or a compound of formula I complexed with a radionuclide, or a pharmaceutically acceptable salt thereof, together with one or more pharmaceutically acceptable carriers or diluents therefor. Such compositions may be manufactured in conventional manner. According to a preferred embodiment of the invention, the pharmaceutical compositions based on a compound of formula I complexed with a radionuclide further comprise a stabilizer, e.g. a free radical scavenger, which inhibits autoradiolysis of the peptide moiety. Suitable stabilizers include e.g. serum albumine, ascorbic acid, retinol, gentisic acid or a derivative thereof, or an amino acid infusion solution, e.g. as used for parenteral protein feeding, preferably free from electrolyte and glucose, for example a commercially available amino acid infusion such as Proteinsteril R KE Nephro. Ascorbic acid is preferred. The stabilizer may conveniently be used in a weight ratio of 1'000 - 10'000 : 1, preferably 4000 - 7000 : 1 to the uncomplexed compound of formula I. The pharmaceutical compositions may comprise further additives, e.g. an agent to adjust the pH between 7.2 and 7.4, e.g. Na or ammonium acetate or Na 2 HPO 4 . Preferably the stabilizer is added to the uncomplexed compound of formula I and the complexation with the radionuclide is performed in the presence of the stabilizer, either at room temperature or, preferably, at a temperature of from 60 to 120°C. The complexation may conveniently be performed under air free conditions, e.g. under N 2 or Ar. Further stabilizer may be added to the composition after complexation. Compounds of formula I complexed with a radionuclide may also be used in the treatment of autoimmune or inflammatory disorders exhibiting somatostatin receptors, e.g. rheumatoid arthritis. The compounds of formula I complexed with a radionuclide may be employed as adjunct or adjuvant to cytostatic therapy, e.g. in combination with a cytostatic agent, preferably a cytostatic agent which enhances the radiotherapy effects by acting as a radiosensitizer. Examples of suitable cytostatic or radiosensitizing agents include e.g. cisplatin, 5-fluorouracil, cyclophosphamide and doxorubicin. In accordance with the foregoing, the present invention provides in a yet further aspect a kit, for use in treating tumor invasiveness or symptoms associated with tumor growth, said kit including a pharmaceutical composition comprising a compound of formula I complexed with a radionuclide or a pharmaceutically acceptable salt thereof and further including at least one pharmaceutical composition comprising a cytostatic agent. The cytostatic agent may be used in any amount known to be an effective cytostatic or radiosensitizing amount. The cytostatic agent may be added concomitantly or at a sequenced regimen with the radiopharmaceutical of formula I. The following examples are illustrative of the invention. All temperatures are in °C. Following abbreviations are employed: Fmoc = 9-fluorenylmethoxycarbonyl Boc = tert-butoxycarbonyl DOTA = 1,4,7,10-tetra-azacyclododecane-1,4,7,10-tetraacetic acid DMF = dimethylformamide Octreotide = EXAMPLE 1: 6 g of DOTAx2H 2 O (free acid), is dissolved in 50 ml of water (pH ∼ 3.7). After diluting with 60 ml of DMF, 1 g of N-hydroxysuccinimide, 2.7 g of N,N-dicyclohexylcarbodiimide (DCCI) and 3 g of [Tyr 3 ,ε -Boc-Lys 5 ]-octreotide is added immediately. The reaction is kept at room temperature for 72 hours under continuous stirring. The ε-Lys 5 protected compound is isolated by purification on a silica gel 60 column using methylene chloride/methanol/acetic acid 50% (9/1/0.125) as mobile phase. Subsequent cleavage of the Lys 5 protecting group with trifluoroacetic acid (TFA, 100%) over a period of 10 minutes at room temperature and purification on a silica gel 60 column (mobile phase: methylene chloride/methanol/acetic acid 50% (7/3/1 to 7/5/2)) and on a RP18-HPLC column, using water/acetonitrile/acetic acid 1% as buffer system, results in a pure and homogenous title compound in its acetate salt form. FAB-MH + : 1421 [α] D 22 = -14.75° (95 % AcOH; c = 0.52) EXAMPLE 2 : 90 Y labelled compound of Example 1 The 90 Y labelled CHELATE is prepared by adding 20 µl of 90 Y (1.2 mCi, 0.04 M HCl) to 20 µL of 50 µM compound of Example 1 (0.15 M NH 4 OAc, 0.3 % BSA, pH 4.5). This solution is incubated at 100° for 15 minutes. An aliquot is removed and diluted with 4 mM DTPA (pH 4.5) before being analyzed by C18 reverse phase HPLC to ascertain the amount of free unchelated 90 Y in the reaction mixture (as indicated by the presence of [ 90 YDTPA] 2- ). The radiochemical purity is typically >99.5 % and the produced chelate is kinetically stable in 4 mM DTPA (4 nM compound of Example 1, pH 4.5) over a seven day period. EXAMPLE 3: 90 Y-labelled compound of Example 1 120 µl of 0.23 M ascorbic acid, 0.15 M ammonium acetate (pH 4.8), 6 µl of 1 mM compound of Example 1 and 120 µl of 90 Y (85 mCi/ml, 0.04 M HC1) are heated in a vial for 15 minutes in a boiling water bath. The resulting solution is diluted to 1 ml with either 0.23 M ascorbic acid and 0.15 M NH 4 OAc or Proteinsteril R KE Nephro amino acid infusion solution and stored at ambient temperature for long term. The Proteinsteril R KE Nephro amino acid solution comprises: 11.4 g/l Leu, 9.63 g/l Lys, 9.53 g/l Val, 7.76 g/l Phe, 7.52 g/l Ile, 6.78 g/l Thr, 6.59 g/l Met, 4.9 g/l His and 2.91 g/l Trp. EXAMPLE 6: 161 Tb labelled compound of Example 1 The labelling procedure of Example 2 is repeated using a 161 Tb yielding salt instead of the 90 Y yielding salt, thus giving the 161 Tb chelate.

Claims: A compound of formula I wherein M is a cation, A is Tyr, in free form, salt form or complexed with a radionuclide. A compound according to claim 1, wherein the compound is complexed with 90 Y. A compound according to claim 1, wherein the compound is complexed with 161 Tb. A compound according to any one of the preceding claims wherein M is H + . Use of a compound of formula I according to claim 1 or a pharmaceutically acceptable salt thereof, in the manufacture of a radiopharmaceutical composition. A pharmaceutical composition comprising a compound of formula I optionally complexed with a radionuclide, according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, together with one or more pharmaceutically acceptable carriers or diluents therefor. A composition according to claim 6 comprising additionally a stabilizer. A composition according to claim 7, wherein the stabilizer is selected from the group consisting of serum albumine, ascorbic acid, retinol, gentisic acid or a derivative thereof, and an amino acid solution. A kit for use in treating somatostatin receptor positive tumor invasiveness or symptoms associated with tumor growth, said kit including a pharmaceutical composition comprising a compound of formula I according to any one of claims 1 to 4, complexed with a radionuclide or a pharmaceutically acceptable salt thereof and further including at least one pharmaceutical composition comprising a cytostatic agent. A pharmaceutical composition for use in treating somatostatin receptor positive tumor invasiveness or symptoms associated with tumor growth including a compound of formula I according to any one of claims 1 to 4, complexed with a radionuclide or a pharmaceutically acceptable salt thereof to be administered together or subsequently to an amino acid solution having a high content of lysine and/or arginine. A pharmaceutical composition according to claim 10, to be administered together or subsequently to an amino acid solution having a high content of lysine.

Inventors: ALBERT RAINER, BRUNS CHRISTIAN, SMITH-JONES PETER, STOLZ BARBARA, WECKBECKER GISBERT, ALBERT, RAINER, BRUNS, CHRISTIAN, SMITH-JONES, PETER, STOLZ, BARBARA, WECKBECKER, GISBERT
Applicants: NOVARTIS AG, NOVARTIS ERFIND VERWALT GMBH, NOVARTIS-ERFINDUNGEN VERWALTUNGSGESELLSCHAFT M.B.H.
Application year: 1995
Main classification: C07K14
IPC codes: A61P35, A61K38, C07B59, A61K51, C07K14, C07K7