Somatostatinoma peptides, radiopharmaceutical agent, a method of treatment of tumors, the pharmaceutical composition

 

(57) Abstract:

Describes the new somatostatinoma peptides of General formula I, where M Is H or a cation which forms an acid additive salt; And is Phe or Tyr, in free form or in the form of an acid additive salt or complex with the radionuclide. They can be used as a radiopharmaceutical funds, provided that they are s - or - emitting nuclides or nuclide with the AU-ger-s-cascades. 4 c. and 8 C.p. f-crystals.

The object of the present invention are somatostatinoma peptides, the method of production thereof, containing pharmaceutical preparations and their use as radiopharmaceuticals means, for example, for the radiotherapy of tumors positive for somatostatin receptors.

Radiotherapy of tumors with radioactive compounds have the advantage that selective targeting of tumors and their metastases, which limits the radiation dose received by normal tissue. Radiotherapy agent would quickly accumulate and well maintained in the target organs, such as tumors, and quickly removed from circulation, in order to limit the dose glad the ll, capable of forming chelate complexes should also be thermodynamically and/or kinetically stable against loss of radioactive metal. Radiotherapy agent, intended for re-introduction should not be immunogenic.

Described [1] somatostatinoma peptides bearing at least one chelating group, which may be mecena radioactive isotope for in vivo diagnostic and therapeutic applications. These compounds are able to contact somatostatinoma receptors, for example, expressed or sverkhekspressiya tumors or metastases. Described [2] somatostatinoma peptides containing bifunctional polyaminopolycarboxylic acid chelating group attached to the terminal amino group by means of spacer elements of the group. However, even if the presence of a bifunctional chelating group (octodentata) and spacer elements group leads to the improvement of some properties of the resulting conjugate, there remains a need for a radiopharmaceutical product with improved properties, in particular high value target/kidney that will allow you to minimize the radiation dose to the kidneys.

It was found that somat araucana acid), conjugatesand directly, i.e. in the absence of spacer elements of the group, with the terminal amino group somatostatinoma peptides possess improved properties. In particular, they have the best ratio of tumor/kidney and therefore provide a lower radiation dose attributable to the kidneys.

According to the invention proposed compound of the formula I

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where M is a cation and

A is Phe or Tyr,

in free form, in salt form or in the form of a complex with the radionuclide.

As will be hereinafter defined, each link connecting 2 nitrogen atom in the formula DOCK above represents ethylene.

If A is h, the peptide portion of the compounds of formula I corresponds to octreotide. If A is Tyr, the peptide portion of the compounds of formula I corresponds to [Tyr3]-octreotide.

A is preferably Tyr.

M can be H+or any salt-forming cation for the carboxyl group, for example a monovalent cation or one equivalent of a polyvalent cation, such as alkali metal ion, such as sodium, potassium or substituted or unsubstituted ammonium ion.

The compounds of formula I can also be a veritable who's salts. Acid additive salts include, for example, salts obtained by adding an organic, polymeric or inorganic acids, such as hydrochloride, acetate, triptorelin or lactate.

Under radionucleotides imply - or - emitting nuclide with Auger - cascades. Among the acceptable nuclides include, for example,64Cu67Cu or radiolytic, in particular90Y140La,161Tb169Er153Sm177Lu,166Dy,166Ho or175Yb, more preferred161Tb and90Y, the most preferred 90Y

The present invention also includes a method of preparing compounds of formula I. They can be obtained by analogy with known methods. The compounds of formula I can be obtained, for example, as follows:

a) removing at least one protective group present in protected form the compounds of formula I, or

b) connect the amide bond two peptide units, one of which contains at least one aminoplast in protected or unprotected form, and the other contains DOD group, and amide bond offer so that you get the desired amino acid sequence of formula I, and then vozmozhnostnoi and unsubstituted form, thus that group derived from DOD, fixed on the terminal amino group of the peptide, and then may carry out stage (a), or

g) oxidizes DOCK-peptide having the amino acid sequence indicated in formula I, in which the mercapto group of Cys radicals exist in unbound form, which results in obtaining the compounds of formula I, in which 2 Cys radical connected S-S-bond,

and allocate the thus obtained compounds of formula I in unconjugated form, in salt form or in the form of a complex with the radionuclide.

The above reaction can be carried out by analogy with known methods, for example, as described in the following examples. If necessary, these reactions can be used protecting groups for functional groups that do not participate in the reaction, suitable for use in peptides or DOCK chelating groups. The term protective group can also include a polymer resin having functional groups. DOCK can be applied at the stage of the method (C) unrelated acidic form, in the form of an anhydride or activated, for example, N-hydroxysuccinimide complex ether.

The formation of the complex with the radionuclide can be realized when Y of formula I with salt, to give the desired radionuclide.

It is preferable to carry out the formation of the complex non-complex compounds of the formula I with forming radionuclide salt at a temperature of from 60 to 120oC, more preferably from 80 to 100oC. In the case where the formation of the complex is carried out at a temperature of 100oC, the process can be carried out in an autoclave. The formation of the complex by heating efficiently performed within a short period of time, for example from 10 to 20 minutes. In that case, when the radionuclide is90Y, preferably90Y-boathouse salt was 90YCl3.

The above reaction is conveniently carried out under conditions to avoid contamination of trace amounts of metals. It is preferable to reduce the impacts of trace quantities of metal to use distilled deionized water, ultrapure reagents, radioactivity without adding media, etc.

The compounds of formula I in the form of a complex with the radionuclide show pharmaceutical activity and are therefore promising as a radiopharmaceutical for the treatment of in vivo tumors and metastases, positive on somatostatinomas reobligate affinity to somatostatin receptors, which can be evaluated in in vitro assays binding conducted as described [3,4].

It is noted that the compounds of formula I, e.g. compound of formula I in the form of a complex with90Y or161Tb, bind with high affinity and specificity with somatostatinoma receptors with the value of the pKDapproximately ranging from 8.0 to 10.0.

Connection Example 1 binds with high affinity with somatostatinoma receptors expressed in cells of the cortex of the brain or pancreatic AR42J tumor cells: it has the pKiequal 8,90,1 with [125I][Tyr3]-octreotide as a specific ligand. The compound of Example 2 is specific in relation to somatostatin receptor ligand, which can be brewed in somatostatinoma receptors with octreotide: IR50= 9,0 0,3.

The affinity of the complex compounds of the formula I to somatostatin receptors can be demonstrated also when tested in vivo according to standard methods of testing, for example, as described [1]. For example, in one test compound from Example 2 detects significant tumor accumulation 2 hours after intraperitoneal injection dose of 5 Kw inovem receptors, while a much smaller amount of radioactivity accumulated in normal tissues, positive on somatostatinomas receptors, for example in the pancreas it is 1,520,08% ID/g Under the % ID/g understand the percentage of the injected dose of radioactivity per 1 g of tissue. The compound from Example 2 after 24 hours after the injection gives not only a high ratio of tumor/kidney, but also a high ratio of tumor/liver and tumor/thigh. Bone marrow is considered to be the most radiosensitive organ, so the low level of radioactivity accumulated in the bones, is very usefull.

The compound of formula I in the form of a complex with the radionuclide has an antiproliferative effect on tumor cells bearing somatostatin receptors, as shown, for example, in tests with hairless mice.

AR42J cells of pancreatic tumors in rats trypsinized and 1107tumor cells (in 0.2 ml) is injected subcutaneously in both flanks hairless mice. When the tumors reach a significant volume of about 0.1-2 ml, animals are randomly assigned to control and experimental groups. Control animals receive either non-complex soedinenii I by intraperitoneal injection in doses corresponding to the highest dose treated groups. Mice injected dose of 0.8 MCI to 40 MCI/kg/100 µl per mouse. The size of the tumors was determined by using Vernier caliper. To calculate the tumor volume in ml use equality "volume (ellipsoid) = length depth height 0,52". For statistical processing using a t-test t-test. Using this test, note the temporary tumor compression in one week and delayed tumor growth in two weeks after a single injection of the compound from Example 2. The control group, in contrast, showed a continuous tumor growth, in which the doubling time was approximately seven days. The survival rate in the treated groups was significantly increased.

Similar results were obtained when the processing of mice or rats, with other types of tumors, such as carcinomas of the breast cancer or small cell lung, the compound of Example 2.

Accordingly, in some specific or possible implementations of the present invention also offers:

1. The use of the compounds of formula I in the form of a complex with the radionuclide or its pharmaceutically acceptable salt as tools radiopharmaceutical for in vivo treatment of tumors and metastatically on somatostatinomas receptors, for example for the treatment of invasiveness of these tumors or symptoms associated with the growth of such tumors in a patient in need of such treatment, which includes the introduction of the indicated patient a therapeutically effective amount of the compounds of formula I in the form of a complex with the radionuclide or its pharmaceutically acceptable salt.

3. The use of the compounds of formula I or its pharmaceutically acceptable salts in the manufacture of radiopharmaceutical composition.

Doses used in the implementation of a radiotherapy application of the present invention will of course vary, in particular depending on the particular condition to be treated, for example the well-known radiosensitivity of tumor type, tumor volume and the desired therapy. Usually the dose is calculated based on the distribution of radioactivity in each organ and the observed target acquisitions. -radiant complex of formula I can be introduced at different points in time, for example after a period of 1-3 weeks or more. The nominal range of doses ranging from 5 to 100 μg of the compounds of formula I in the form of a complex with, for example, 10-100 MCI of the radionuclide, for example,90Y or161Tb, the higher the range the house can be entered by any standard method, in particular intravenously, for example, in the form of solutions or suspensions for injection. They can also be successfully introduced by infusion, for example by infusion over 30-60 minutes. Depending on the tumor location, they can be entered as close to the site of localization of the tumor as possible, for example, through a catheter. They can be entered in several separate doses.

Tumors that can be treated by compounds of the formula I in the form of a complex with the radionuclide are, for example, pituitary, gastro-enteropancreatic, carcinoid tumors, tumors of the Central nervous system, breast, prostatic, ovarian tumors or tumor, colon cancer, small cell lung, paraganglioma, kidney cancer, skin cancer, neuroblastoma, pheochromocytoma, medullary carcinoma of the thyroid gland, myeloma, lymphoma, disease Hodgking'and a non-Hodgkins'a, bone tumors and metastases.

Excretion of radioactive compounds occurs mainly through the kidneys. Additional protection of the kidney from the accumulation of radioactivity can be implemented by introducing a lysine or arginine, any amino acid solution having a high content of lysine and/or AI -10, before injection of the complex compounds of formula I or with him.

The compound of formula I in the form of a complex with a radionuclide may be in free acid form or in the form of pharmaceutically acceptable salts, having the same order of activity as the complexes in the free acid form.

Preferred is the compound of Example 1 in the form of a complex with90Y

According to further aspect of the invention features a pharmaceutical composition containing the compound of formula I or the compounds of formula I in the form of a complex with a radionuclide, or its pharmaceutically acceptable salt together with one or more pharmaceutically acceptable carrier or solvent. Such compositions can be prepared in a standard way.

According to a preferred implementation of the invention, the pharmaceutical compositions based on compounds of formula I in the form of a complex with the radionuclide include, in addition, a stabilizer, for example the recovery of free radicals, which inhibits autoradios peptide part. The number of valid stabilizers include, for example, serum albumin, ascorbic acid, retinol, gentisic acid or their derivatives, doctitle free from electrolyte and glucose, for example, commercially available amino acid infusion, such as ProteinsterilRKE Nephro. Preferred is ascorbic acid. Can be used stabilizer in a weight ratio of 1'000-10'000 : 1 for the uncomplexed compounds of formula I, preferably 4000-7000 : 1. Pharmaceutical compositions may also contain additional additives, such as an agent to maintain the pH between 7.2 and 7.4, for example the Na acetate or ammonium or Na2HPO4. It is preferable to add a stabilizer to non-complex to the compound of formula I, and complexing with a radionuclide to perform in the presence of the stabilizer either at room temperature or, preferably, at a temperature of from 60 to 120oC. the Reactions are conveniently carried out in vacuum conditions, for example under the N2or Ar. After complexation to the composition may be added a stabilizer.

The compounds of formula I in the form of a complex with a radionuclide can also be used for the treatment of autoimmune or inflammatory disorders associated with somatostatinoma receptors, such as rheumatoid arthritis.

According to the following implementation of the invention the compounds of formula I in the form of a complex is a cytostatic agent, preferably with a cytostatic agent, a reinforcing radiotherapy effect by acting as radiosensibility. Examples of natural cytotoxic or radio-sensitizing agents include, for example, cisplatin, 5-fluorouracil, cyclophosphamide and doxorubicin.

According to the above, the present invention provides another aspect.

4. The method of treatment invasiveness of tumors positive for somatostatin receptors, or symptoms associated with such tumor growth in a patient in need of such treatment, and this method includes the introduction of a specified patient an effective amount of (a) compounds of formula I in the form of a complex with the radionuclide or its pharmaceutically acceptable salt and (b) a second drug substance, and the specified drug is a cytotoxic agent.

5. therapeutic set, such as China, for use in the treatment of tumor invasiveness or symptoms associated with tumor growth, with a specified set includes a pharmaceutical composition containing the compound of formula I in the form of a complex with the radionuclide or its pharmaceutically acceptable Sol is CLASS="ptx2">

The cytotoxic agent can be applied in any number of known that it has an effective cytostatic or radio-sensitizing effect. The cytotoxic agent can be administered concurrently or sequentially with radiopharmaceutical agent of formula I.

The invention is illustrated by the following examples. All temperatures are given inoC. Use the following abbreviations:

Fmoc = 9-fluorenylmethoxycarbonyl

Side = tert-butoxycarbonyl

DOCK = 1,4,7,10-Tetra-azacyclopentadecan-1,4,7,10-tetraoxa acid

DMF = dimethylformamide

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EXAMPLE 1:

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6 g DOCK H2O (free acid) dissolved in 50 ml of water (pH of 3.7). After diluting 60 ml DMF immediately add 1 g of N-hydroxysuccinimide, 2.7 g of N,N-dicyclohexylcarbodiimide (DCCI) and 3 g of [Tyr3, -Boc-Lys5] -octreotide. The reaction of the support at room temperature for 72 hours under stirring. -Lys5-secure connection allocate by purification on a column of silica gel 60 (mobile phase: 50% mixture of methylene chloride/methanol/acetic acid (9/1/0,125).

After removal of Lys5-protective group using triperoxonane acid (TFU, 10 is telengard/methanol/acetic acid (7/3/1 to 7/5/2) and RP18-GHUR column using as buffer systems 1% mixture of water/acetonitrile/acetic acid pure and homogeneous connection, specified in the header, in the form of its acetate salt.

FAB-MH+:1421 [D22/= -14,75o(95% AcOH; C = 0,52)

(FAB - irradiation quick anions)

EXAMPLE 2:90Y-labeled compound from Example 1.

90Y-labeled CHELATE obtained by adding 20 ál90Y (1.2 MCI, 0.04 M HCl) to 20 μl of 50 μm compound of Example 1 (0.15 M NH4OAc, 0.3% BSA, pH 4.5). This solution is incubated for 15 minutes at 100oC. Take an aliquot and dilute it 4 mm DTPA (pH 4.5) before analysis by GHWR with reverse phase C18, which allows you to set the amount of free neklinovskogo 90Y in the reaction mixture (which is determined by the presence of [90YDTPA]2-). Radiochemical purity is typically > 99.5%, and the resulting chelate is kinetically stable in 4 mm DTPA (4 nm of the compound from Example 1, pH 4.5) for seven days.

EXAMPLE 3:90Y-labeled compound from Example 1.

120 μl of 0.23 M ascorbic acid, 0.15 M ammonium acetate (pH of 4.8), 6 μl of 1 mm of compound from Example 1 and 120 ál90Y (85 MCI/ml, 0.04 M HCl) is heated in the vial for 15 minutes in a boiling water bath. The resulting solution was diluted to 1 ml or 0,23 M ascorbic acid and 0.15 M NH4OAc or Prot is a long period of time.

ProteinsterilRKE Nephro amino acid solution contains:

11.4 g/l Leu, 9,63 g/l Lys, at 9.53 g/l Val, 7,76 g/l Phe, 7,52 g/l'ile, 6,78 Thr, 6,59 g/l Met, 4.9 g/l His, 2,91 r/l Trp.

EXAMPLE 4:

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The connection specified in the header, get by repeating the method of Example 1, but using Boc-Lys5- -octreotide.

MC (ion irradiation): 1403 (M - H).

EXAMPLE 5:90Y-labeled compound from Example 4.

Repeat the process for the introduction of labels of the Example 2, which allows to obtain90Y chelate.

EXAMPLE 6:161Tb-labeled compound from Examples 1 or 4.

Repeat the process of introducing the label of Example 2 using161Tb-forming salt instead of90Y-forming salts, resulting in161Tb chelate.

Literature

1. GB-A-2, 225, 579, C 07 K 7/26.

2. EP-A2-607, 103.

3. J. C. Reubi, Life Sc. 36, 1829 (1985).

4. C. Bruns et al., Biochem. J., 265, 39 (1990).

1. Somatostatinoma peptides of the formula I

< / BR>
where M is a cation;

A is Phe or Tyr;

in free form or in the form of an acid additive salt or complex with the radionuclide.

2. Connection on p. 1, wherein A is Tyr.

3. Connection PP.1 and 2,2, characterized in that the specified connection is complex with161TV.

5. Connection PP.1 to 4, wherein M is H+.

6. Radiopharmaceutical agent, representing a compound of formula I in complex with the radionuclide or its pharmaceutically acceptable salt.

7. A method of treating tumors, positive with respect to somatostatin receptors, or metastases, characterized in that use radiopharmaceuticals tool under item 6.

8. Pharmaceutical composition having anti-proliferative activity against tumor cells bearing somatostatin receptors containing the compound of the formula I according to any one of paragraphs.1 - 5 or its pharmaceutically acceptable salt together with one or more pharmaceutically acceptable carriers or diluents.

9. The composition according to p. 8, further comprising a stabilizer.

10. The composition according to p. 9, in which the stabilizer is selected from the group consisting of whey albumen, ascorbic acid, retinol, hentaimovi acid or derivatives thereof, and amino acid solution.

11. The composition according to p. 8, which additionally contains a solution of lysine, the information under item 8, characterized in that it is used in combination with lysine or arginine.

 

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