Method for modulating cell proliferation of medullary thyroid carcinoma

FIELD: medicine.

SUBSTANCE: the present innovation deals with applying agonists of somatostatin receptors in case of thyroid carcinoma and, it also, deals with the method for modulating the rate of cell proliferation of medullary thyroid carcinoma (MTC) due to combined impact upon cells with either one or several agonists of somatostatin receptors of type 2 (SSTR2)or either with one or several agonists of somatostatin receptors of type 5 (SSTR5). Moreover, the mentioned SSTR5 agonist can be represented with D-Phe-Phe-Trp-D-Trp-Lys-Thr-Phe-Thr-NH2 or its pharmaceutically acceptable salt. The present innovation enables to enhance suppression of MTC cell proliferation due to antagonism between mediated SSTR2 and SSTR5 effects to the proliferation mentioned.

EFFECT: higher efficiency of modulation.

1 cl, 8 dwg, 2 tbl

 

Background of the invention

It has been shown that somatostatin (SS), tetradecapeptide found Brazeau et al., has a strong inhibitory effect on different secretory processes in tissues in organs such as the pituitary, pancreas and gastrointestinal tract. Somatostatin is also a neuromodulator in the Central nervous system. These biological effects of somatostatin inhibitory nature mediated near associated with G-protein receptors, which are characterized five different subtypes (SSTR1-SSTR5) (Reubi JC, et al., Cancer Res 47: 551-558, Reisine T, et al., Endocrine Review 16: 427-442, Lamberts SW, et al., Endocr Rev 12: 450-482, 4 Patel YC, 1999 Front Neuroendocrinology 20: 157-198). Five of these subtypes have the same affinity for endogenous somatostatinomas ligands, but differently distributed in different tissues. Somatostatin binds to receptors (SSTR) five different subtypes with relatively high and equal affinity for each subtype.

There is evidence that somatostatin regulates cell proliferation by stopping the growth of cells through receptors SSTR subtypes 1, 2, 4, and 5 (Buscail L, et al., 1995 Proc Natl Acad Sci USA 92: 1580-1584; Buscail L, et al., 1994, Proc Natl Acad Sci USA 91: 2315-2319; Florio T, et al., 1999 Mol Endocrinol 13: 24-37; Sharma K, et al., 1999 Mol Endocrinol 13: 82-90) or inducing apoptosis through receptor SSTR subtype 3 (Sharma K, et al., 1996, Mol Endocrinol 10: 1688-1696). It has been shown that somatostatin and different will analoginput proliferation of normal and tumor cells in vitro and in vivo (Lamberts SW, et al., Endocr Rev 12: 450-482) via specific receptors SS (SSTR) (Patel YC, 1999 Front Neuroendocrinology 20: 157-198) and may result in different postreceptor action (Weckbecker G, et al., Pharmacol Ther 60: 245-264; Bell GI, Reisine T, 1993 Trends Neurosci 16: 34-38; Patel YC, et al., Biochem Biophys Res Commun 198: 605-612; Law SF, et al., Cell Signal 7: 1-8). In addition, there is evidence that the expression of receptors different SSTR subtypes in normal and neoplastic human tissues (Virgolini I, et al., Eur J Clin Invest 27: 645-647), which provides a different affinity for a tissue to various analogs of somatostatin and different clinical response to therapeutic effect.

Binding to somatostatin receptors of different subtypes associated with different treatment conditions and/or diseases. For example, inhibition of growth hormone is attributed to the somatostatin receptor type 2 ("SSTR2") (Raynor, et al., Molecular Pharmacol. 43:838 (1993); Lloyd, et al., Am. J. Physiol. 268:G102 (1995)), while inhibition of insulin is attributed to the somatostatin receptor type 5 ("SSTR5") (Coy, et al., 197: 366-371 (1993)). Activation of receptor types 2 and 5 suppresses growth hormone, in particular, adenoma secreting growth hormone (acromegaly) and adenomas secreting thyrostimulin hormone. Activation of the receptor type 2 but not type 5, allows to treat prolactinemia adenomas. Other indications associated with activation of somatostatin receptors of different subtypes include inhibition is of Sulina and/or glucagon in the treatment of diabetes, angiopathy, proliferative retinopathy, the phenomenon of awakening and nephropathy; inhibition of gastric secretion, in particular, in the treatment of peptic ulcers, intestinal and pancreatic fistula, irritable bowel syndrome, dumping syndrome, watery stools, AIDS-associated diarrhea, diarrhea caused by chemotherapy, acute or chronic pancreatitis, tumors secreting gastrointestinal hormone; treatment of malignant tumors, such as hepatoma; inhibition of angiogenesis; the treatment of inflammatory diseases such as arthritis, retinopathy, chronic allograft rejection; plastic vessels, the prevention of bleeding from vascular graft and gastrointestinal the bleeding. It is preferable to have analogue that selectively affects the somatostatin receptors specific subtype or subtypes responsible for the desired biological response, thus reducing the interaction with other receptors subtypes, which can cause unwanted side effects.

Somatostatin (SS) and its receptors (SSTR1-SSTR5) is expressed in normal parafollicular C-cells and cells of medullary carcinoma of the thyroid gland (MTS) person. MTS is a tumor derived from parafollicular C-cells of the shields is ne gland produces calcitonin (CT), somatostatin and several other peptides (Moreau JP, et al., Metabolism 45 (8 Suppl 1): 24-26). Mato et al. recently discovered that SS and SSTR expressed in MTS person (Mato E, et al., J. Clin Endocrinol Metab 83: 2417-2420). In the literature that SS and its analogues reduce the amount of calcitonin in plasma and improve the condition of subjects suffering from MTS. However, to date, has not been clearly demonstrated antiproliferative effects of SS analogues in tumor cells (Mahler C, et al., Clin Endocrinol 33: 261-9; Lupoli G, et al., Cancer 78: 1114-8; Smid WM, et al., Neth J Med 40: 240-243). Thus, the creation of analogues different SSTR subtypes, selectively affecting the growth of cells, MTS, allows to obtain useful tool for use in clinical settings. To date in the scientific literature were not presented evidence about the involvement of receptors SSTR certain subtypes in the regulation of cell growth MTS.

The present invention relates to the discovery that the line TT-cell MTS person, with characteristics of cells MTS (Zabel M, et al., 1992 Histochemistry 102: 323-327, 2 Gagel RF, et al., 1986 Endocrinology 118: 1643-1651, Liu JL, et al., 1995 Endocrinology 136: 2389-2396) and stably expressing the receptor SSTR all subtypes, responds to the activation of SSTR2 and SSTR5-selective subtype agonists in two different profiles enable [3H]thy and number of cells. Preferred agonists SSTR2 is considerably inhibit the incorporation of [ 3H]thy, that is, inhibit DNA synthesis and reduced cell proliferation. Selective agonists SSTR5 significantly increase the incorporation of [3H]thy in TT cells, that is, increase DNA synthesis but does not affect cell proliferation. In addition, the SSTR2 antagonist inhibit the effects of the preferred SSTR2 agonists on TT cells. Increasing the concentration of the selective agonist SSTR5 depending on the dose prevents suppression enable [3H]thy and proliferation of TT cells, called preferred SSTR2 agonist, and Vice versa, indicating that the antagonism between these agonists.

It was recently demonstrated the presence of hetero - and homodimeric interactions between subtypes families Sputnik (Jordan BA, et al., 1999 Nature 399: 697-700) and SS receptors (Rocheville M, et al., 2000 J. Biol. Chem. 275: 7862-7869). Studies of cultured cells of pituitary adenomas show that the SSTR receptor subtypes 2 and 5 have a synergistic effect in suppressing the secretion of growth hormone and prolactin (Shimon I, et al., 1997 J. Clinical Invest. 100: 2386-2392, Jaquet P, et al., 2000 J. Clin Endocrinol Metab. 85: 781-792). The discovery that activation of SSTR5 reduces antiproliferative activity, mediated by SSTR2, differs from the results obtained in the study of other tissues (Patel YC, 1999 Front Neuroendocrinology 20: 157-198, Buscail L, et al., 1995 Proc Natl Acad Sci USA 92: 1580-1584. Buscail L, et al., 1994 Proc Natl Acad Sci USA 91: 2315-2319, Sharma K, et al, 1996 Mol Endocrinol 10: 1688-1696). This is the first evidence that receptors SSTR subtypes 2 and 5 can act as antagonists in the regulation of cell growth.

Thus, the preferred agonists SSTR2 and SSTR5 has a different effect on the proliferation of medullary line TT-cells of the thyroid gland of a person depending on their specific selectivity in respect of SSTR. The proliferation of line TT-cells can be reduced by using selective SSTR2 agonists, which cannot be achieved with the help of SSTR5 antagonists and agonist SSTR5 can prevent anti-proliferative effect mediated SSTR2. The main inhibitory effect of SSTR2 on cell proliferation MTS suggests that analogues with high affinity and selectivity relative to SSTR2 unlike SSTR5 should be useful as antiproliferative agents at treatment of MTS.

The invention

The basis of the present invention is the discovery that agonists of somatostatin selectively affecting SSTR2, effective in reducing the rate of proliferation of cells of medullary carcinoma of the thyroid gland, and agonists of the somatostatin selectively affecting SSTR5, effectively weakening caused by SSTR2 agonist decrease in the rate of cell proliferation.

One object of the present invention is a method modules and speed of cell proliferation MTS, which involves the interaction of cells MTS with one or more SSTR2 agonists and one or more agonists of SSTR5, resulting in the specified SSTR2 agonist reduces the rate of cell proliferation MTS, and the specified agonist SSTR5 weakens caused by SSTR2 agonist decrease in the rate of cell proliferation.

In accordance with one embodiment this invention relates to the above method, wherein said agonist SSTR5 represents D-Phe-Phe-Trp-D-Trp-Lys-Thr-Phe-Thr-NH2(SEQ ID No.1) or its pharmaceutically acceptable salt.

In accordance with another embodiment this invention relates to a method of reducing the rate of proliferation of cells of medullary carcinoma of the thyroid gland, which involves the interaction of cells of medullary carcinoma of the thyroid gland with one or more SSTR2 agonist or its pharmaceutically acceptable salt.

In the preferred example above of a variant embodiment of the invention SSTR2 agonist is a selective agonist SSTR2. In a more preferred example, the SSTR2 agonist or its pharmaceutically acceptable salt has a value of Ki for SSTR5, which is at least 2 times greater than similar values for SSTR2, more preferably specified value at least 5 times greater than similar values for SSTR2, yet p is impactfulness the specified value is at least 10 times greater than similar values for SSTR2.

In another preferred example of the above variant of the invention, the SSTR2 agonist or its pharmaceutically acceptable salt has a Ki value of less than 5 nm, more preferably less than 1 nm.

In yet another preferred example of the above variant of the invention, the selective SSTR2 agonist is a compound selected from the group including

D-Nal-cyclo[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2(SEQ ID No.2),

cyclo[Tic-Tyr-D-Trp-Lys-Abu-Phe] (SEQ ID No.3),

4-(2-hydroxy-ethyl)-1-piperazinylmethyl-D-Phe-cyclo(Cys-Tyr-D-Trp-lys-Abu-Cys)-Thr-NH2(SEQ ID No.4),

4-(2-hydroxyethyl)-1-piperazine-2-econsultancy-D-Phe-cyclo(Cys-Tyr-D-Trp-Lys-Abu-Cys)-Thr-NH2(SEQ ID No.5)

or their pharmaceutically acceptable salts, where 4-(2-hydroxyethyl)1-piperazinylmethyl" has the structure:

and 4-(2-hydroxyethyl)-1-piperazine-2-econsultancy" has the structure:

In accordance with a third embodiment this invention relates to a method for the treatment of medullary carcinoma of the thyroid gland, which provides for the introduction of the needy in this patient an effective amount of SSTR2 agonist.

In a preferred example of the third variant embodiment of the invention SSTR2 agonist is a selective agonist SSTR2. In a more preferred example, the SSTR2 agonist or f is rmaceuticals acceptable salt is the value of Ki for SSTR5, which is at least 2 times greater than similar values for SSTR2, more preferably specified value at least 5 times greater than similar values for SSTR2, still preferable to the specified value at least 10 times greater than similar values for SSTR2.

In another preferred example of the third variant embodiment of the invention SSTR2 agonist or its pharmaceutically acceptable salt has a Ki value of less than 5 nm, more preferably less than 1 nm.

In yet another preferred example of the third variant of the invention, the selective SSTR2 agonist is a compound selected from the group including

D-Nal-cyclo[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2(SEQ ID No.2),

cyclo[Tic-Tyr-D-Trp-Lys-Abu-Phe] (SEQ ID No.3),

4-(2-hydroxyethyl)-1-piperazinylmethyl-D-Phe-cyclo (Cys-Tyr-D-Trp-Lys-Abu-Cys)-Thr-NH2(SEQ ID No.4),

4-(2-hydroxyethyl)-1-piperazine-2-econsultancy-D-Phe-cyclo(Cys-Tyr-D-Trp-Lys-Abu-Cys)-Thr-NH2(SEQ ID No.5)

or their pharmaceutically acceptable salts, where 4-(2-hydroxyethyl)-1-piperazinylmethyl" and "4-(2-hydroxyethyl)-1-piperazine-2-econsultancy" defined above.

It is important to note that, as is well known in this area, the usual treatment of radioactive iodine, such as the introduction of the patient radioactive iodine salt, is unacceptable for the treatment of medullary carcinoma of the thyroid gland, as parafollicular cells do not absorb iodine. T is thus, another object of this invention is a method for the treatment of medullary carcinoma of the thyroid gland, which provides for the introduction of the needy in this patient an effective amount of SSTR2 agonist or its pharmaceutically acceptable salt and the specified SSTR2 agonist or its pharmaceutically acceptable salt contains a Tyr residue(I), and the iodine atom of the specified residue Tyr(I) is a radioactive isotope of iodine. The specified isotope of iodine is preferably a125I127I or131I.

In one embodiment of the invention the cells of medullary carcinoma of the thyroid gland to form metastases outside the thyroid gland. In another embodiment of the invention, these metastases are lymph, lung, liver, brain or bones.

Brief description of drawings

Figure 1. Mediated by SSTR2 activation of intracellular calcium in vitro.

Cells Cho-K1 expressing SSTR2 person, collect in the manner described under "Materials and methods", and add the SS analogues (10-7-10-6M) to measure the activation of intracellular CA2+expressed as the ratio between the concentration of intracellular calcium measured after addition of SS analogues, and the concentration determined at the initial level. The wavelengths of excitation and emission are equal COO is responsible 340 and 510 nm. Data are presented as median values ± standard error of the mean (SEM).

Figure 2. Mediated SSTR5 activation of intracellular calcium in vitro.

Cells Cho-K1 expressing human SSTR5, collected by the method described in section "Materials and methods", and add the SS analogues (10-7-10-6M) to measure the activation of intracellular CA2+expressed as the ratio between the concentration of intracellular calcium measured after addition of SS analogues (compound 1, compound 5 and compound 6), and the concentration determined at the initial level. The wavelengths of excitation and emission, respectively 340 and 510 nm. Data are presented as median values ±standard error of the mean (SEM).

The connection shown in figure 2, have the following structure: compound 1: D-Nal-cyclo[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2(SEQ ID No.2);

connection 2: cyclo[Tic-Tyr-D-Trp-Lys-Abu-Phe] (SEQ ID No.3);

compound 3: 4-(2-hydroxyethyl)-1-piperazinylmethyl-D-Phe-cyclo(Cys-Tyr-D-Trp-Lys-Abu-Cys)-Thr-NH2(SEQ ID No.4);

compound 4: 4-(2-hydroxyethyl)-1-piperazine-2-econsultancy-D-Phe-cyclo(Cys-Tyr-D-Trp-Lys-Abu-Cys)-Thr-NH2(SEQ ID No.5);

connection 5: D-Phe-Phe-Trp-D-Trp-Lys-Thr-Phe-Thr-NH2(SEQ ID No.1) and

compound 6: CPA-cyclo(D-Cys-4-Pal-D-Trp-Lys-Thr-Cys)-Nal-NH2(SEQ ID No.6).

Figure 3. Inhibition of SS-stimulated activation of intracellular calcium SSTR2 antagonist in vitro.

-9-10-6M) and SS (10 nm) to measure the effect of compound 6 on the SS-stimulated (10-8M) activation of intracellular calcium, expressed in percent compared to the effect of one SS. The wavelengths of excitation and emission, respectively 340 and 510 nm. Data are presented as median values ± standard error of the mean (SEM).

Figure 4. The mRNA expression of somatostatin receptors in TT cells.

Extracted RNA (1 μg/reaction) is treated with desoksiribonukleaza and subjected to reverse transcription using oligo(dT) as primer. Samples inkubirovanie without reverse transcriptase served as control samples. Aliquots of the obtained cDNA and negative control samples subjected to further amplification SSTR using polymerase chain reaction (PCR)using the primers listed in table 1. The PCR products were cleaved by 2% agarose gel. Anticipated products SSTR obtained using PCR, shown in sector a (lane M, marker PCR; G, the product of GAPDH amplification using PCR).

Figure 5. The impact of SS analogues on the inclusion of [3H]thy in TT cells.

Cells are incubated in 24-hole tablets within 48 hours of cultural the Noah environment, containing analogues SS in different concentrations (10-9, 10-8, 10-7and 10-6M). Control wells treated with a solution of carrier and measure the incorporation of [3H]thy in the form of radioactivity precipitated THUC substances. Six separate experiments independently assessed for four identical samples in each experiment and expressed as the average percentage ±SEM inhibition of incorporation of [3H]thy compared with untreated control cells. *P<0.05 and **P<0,01 compared with the control sample.

6. The impact of SS analogues on proliferation of TT cells.

Cells are incubated in 96-well tablets within 48 hours in culture medium containing analogues SS in different concentrations (10-9, 10-8, 10-7and 10-6M). Control wells treated with a solution of the carrier. The proliferation of TT cells measured in each well in the form of optical density at 490 nm. Six separate experiments independently assessed for eight identical samples in each experiment and expressed as the average percentage ±SEM inhibition of cell proliferation compared with untreated control cells. *P<0.05 and **P<0,01 compared with the control sample.

7. The impact of selective SSTR2 antagonist on the inclusion of [3H]thy TT to EDI and cell proliferation in the processing of SSTR2 agonist.

The upper block. Cells are incubated in 24-hole tablets within 48 hours in culture medium containing 100 nm of compound 1 or compound 2 with compound 6 (10-7M) or without it. Control wells treated with a solution of the carrier. The inclusion of [3H]thy measure in the form of radioactivity precipitated THUC substances. Six separate experiments independently assessed for four identical samples in each experiment and expressed as the average percentage ±SEM inhibition of incorporation of [3H]thy compared with untreated control cells. *P<0.05 and **P<0,01 compared with the control sample.

The lower block. Cells are incubated in 96-well tablets within 48 hours in culture medium containing 10-7M compound 1 or compound 2 with compound 6 (10-7M) or without it. Control wells treated with a solution of the carrier. The proliferation of TT cells measured in each well in the form of optical density at 490 nm. Six separate experiments independently assessed for eight identical samples in each experiment and expressed as the average percentage ±SEM inhibition of cell proliferation compared with untreated control cells. *P<0.05 and **P<0,01 compared with the control sample.

Fig. Impact izbiratelni the th antagonist SSTR5 on the inclusion of [ 3H]thy TT-cell and cell proliferation in the processing of selective SSTR2 agonist.

The upper block. Cells are incubated in 24-hole tablets within 48 hours in culture medium containing the compound 2 (10-7M) and compound 5 with a constant or increasing concentrations (10-9, 10-8, 10-7and 10-6M), or in culture medium containing the compound 5 (10-7M) and compound 2 with a constant or decreasing concentration (10-6, 10-7, 10-8and 10-9M). Control wells treated with a solution of the carrier. The inclusion of [3H]thy measure in the form of radioactivity precipitated THUC substances. Six separate experiments independently assessed for four identical samples in each experiment and expressed as the average percentage ±SEM inhibition of incorporation of [3H]thy compared with untreated control cells. *P<0.05 and **P<0,01 compared with the control sample.

The lower block. Cells are incubated in 96-well tablets within 48 hours in culture medium containing the compound 2 (10-7M) and compound 5 with a constant or increasing concentrations (10-9, 10-8, 10-7and 10-6M), or in culture medium containing the compound 5 (10-7M) and compound 2 with a constant or decreasing concentration (10 -6, 10-7, 10-8and 10-9M). Control wells treated with a solution of media and the proliferation of TT cells measured in each well in the form of optical density at 490 nm. Six separate experiments independently assessed for eight identical samples in each experiment and expressed as the average percentage ±SEM inhibition of cell proliferation compared with untreated control cells. *P<0.05 and **P<0,01 compared with the control sample.

Detailed description of the invention

It is assumed that the person skilled in the art can, in the most full use of the advantages of the present invention based on the above description of the invention. Therefore, discussed below, specific embodiments of the invention are only illustrative and in no way limit the scope of the present invention.

Except where otherwise indicated, all technical and scientific terms used herein have the meanings are well known to specialists in this field. In addition, all listed in the description of the publication, applications for patent, patents, and other materials are fully incorporated in this description by reference.

Several of somatostatin receptors (SSTR), for example, SSTR1, SSTR, SSTR3, SSTR4 and SSTR5. Thus, the somatostatin agonist may represent one or more agonists SSTR1, SSTR2 agonists, agonists SSTR3, agonists or SSTR4 SSTR5 antagonists. Agonist of somatostatin receptor type 2 (SSTR2 agonist is a compound which (1) has a high affinity binding (e.g., Ki of less than 100 nm, preferably less than 10 nm, or less than 1 nm) with SSTR2 (for example, the results of the following analysis of the receptor binding) and (2) reduces the rate of cell proliferation medullary carcinoma of the thyroid gland (for example, the results described below biological analysis). Selective agonist of somatostatin receptor type 2 is an agonist of somatostatin receptor type 2, which has higher affinity binding (i.e. has a lower value of Ki) with SSTR2 than with SSTR5. Agonist of somatostatin receptor type 5 is an agonist of somatostatin, which (1) has a high affinity binding (e.g., Ki of less than 100 nm, preferably less than 10 nm, or less than 1 nm) with SSTR-5 (for example, the results of the following analysis of the receptor binding) and (2) weakening caused by SSTR2 agonist decreases the rate of proliferation of cells of medullary carcinoma of the thyroid gland (for example, the results described below biological analysis). Selective agonist recipe is RA somatostatin type-5 is an agonist of somatostatin receptor type 5, which has a higher affinity binding (i.e. has a lower value of Ki) with SSTR5 than SSTR2.

In one embodiment of the invention SSTR2 agonist is a selective agonist SSTR2. In another embodiment of the invention, selective SSTR2 agonist has the value of Ki for SSTR5, which is at least 2 times (for example, at least 5 times or 10 times) greater than the value of the agonist for the receptor SSTR2 (for example, the results of the following analysis of the receptor binding).

Examples of SSTR2 agonists that can be used when implementing the present invention, include, but are not limited to: D-Nal-cyclo[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2(SEQ ID No.2) (compound 1);

cyclo[Tic-Tyr-D-Trp-Lys-Abu-Phe] (SEQ ID No.3) (compound 2);

4-(2-hydroxyethyl)-1-piperazinylmethyl-D-Phe-cyclo(Cys-Tyr-D-Trp-Lys-Abu-Cys)-Thr-NH2(SEQ ID No.4) (compound 3), and

4-(2-hydroxyethyl)-1-piperazine-2-econsultancy-D-Phe-cyclo(Cys-Tyr-D-Trp-Lys-Abu-Cys)-Thr-NH2(SEQ ID No.5) (compound 4).

An example of agonist SSTR5, which can be used when implementing the present invention, includes, but is not limited to them:

D-Phe-Phe-Trp-D-Trp-Lys-Thr-Phe-Thr-NH2(SEQ ID No.1) (compound 5).

Other examples of somatostatin agonists include compounds that have the formula given in the following publications, or described in these publications on the findings in this description by reference.

The application for the grant of European patent No. P5 164 EU (inventor: G. Keri).

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It should be noted that in all the described agonists of the somatostatin any amino acid residue has the formula-NH-C(R)H-CO-, where R is a side chain (for example, CH3for Ala). The line between amino acid residues means of peptide bonds linking amino acids. In addition, optically active amino acid residue presumably has the configuration of the L-form unless otherwise specified D-form. Disulfide bond (e.g., a disulfide bridge)existing between the two free thiols of Cys residues that are not shown for ease of presentation. The usual abbreviations of amino acids are given in accordance with the recommendations of the International Union of pure and applied chemistry and International Union of biochemistry (IUPAC-IUB).

Synthesis of somatostatin agonists

Methods of synthesis of somatostatin agonists are well described in the scientific literature and should be known to specialists in this field.

The synthesis of short amino acid the sequence comprehensively described in the synthesis of peptides. For example, the above-described compound H-D-Phe-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-NH2be synthesized by the method according to example I provided in the application for the grant of the European patent 0395417 A1. Agonists of somatostatin with substituted N-end can be synthesized by the method described in patent application WO 88/02756, the application for the grant of European patent No. 0329295 and PCT publication no WO 94/04752.

Some compounds of the present invention can have at least one asymmetric center. In the molecule may be present additional asymmetric centers depending on the type of different substituents in the molecule. Each such asymmetric center forms two optical isomers, and all such optical isomers, representing separated, pure or partially purified optical isomers, racemic or diastereomeric mixtures, are included in the scope of the present invention.

Compounds of the present invention can be isolated in the form of a pharmaceutically acceptable acid additive salts, such as salts, resulting from the use of inorganic and organic acids. Examples of such acids are hydrochloric, nitric, sulfuric, phosphoric, formic, acetic, triperoxonane, propionic, maleic, succinic, D-tartaric, L-tartaric, malonic, methansulfonate and similar acids. In addition, some soy is inane, containing acid functional group such as carboxyl group, can be allocated in the form of inorganic salts, in which the counterion may be selected from sodium, potassium, lithium, calcium, magnesium and the like, and organic bases.

Pharmaceutically acceptable salts can be obtained by interacting approximately 1 equivalent of SSTR2 agonist, e.g. compound 1, from about 1 or more equivalents of the appropriate acid, forming the desired salt. Methods of processing and separation of the formed salt is well known to specialists in this field.

Compounds according to this invention can be designed for oral, parenteral (e.g. intramuscular, intraperitoneal, intravenous or subcutaneous injection, or implant), nasal, vaginal, rectal, sublingual or topical administration and is used together with pharmaceutically acceptable carriers for the received dosage forms appropriate for each route of administration. In the scope of the present invention includes pharmaceutical compositions containing as active ingredient at least one SSTR2 agonist in combination with a pharmaceutically acceptable carrier.

Solid dosage forms for oral administration include ka is Sula, tablets, pills, powders and granules. In such solid dosage forms the active compound is in a mixture with at least one inert pharmaceutically acceptable carrier such as sucrose, lactose or starch. These dosage forms, as a rule, can contain additional substances addition of inert fillers, for example, lubricants such as magnesium stearate. In the case of capsules, tablets and pills dosage forms can also contain buffer substances. On tablets and pills can additionally be applied intersolubility floor.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, elixirs containing inert diluents commonly used in this field, such as water. Besides inert diluents, compositions can also contain adjuvants, such as wetting agents, emulsifying and suspendresume agents, and sweeteners, corrigentov and fragrances.

Preparations according to this invention for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions or emulsions. Examples of nonaqueous solvents or diluents are propylene glycol, polyethylene glycol, rastitel the e oil, such as olive and corn oil, gelatin and complex injectable organic esters, such as etiloleat. Such dosage forms may also contain adjuvants, in particular preserving, wetting, emulsifying and dispersing agents. These dosage forms can be sterilized, for example, by filtration through inhibiting bacteria filter, the introduction of sterilizing agents, by irradiation or by heating the compositions. These dosage forms can also be obtained in the form of sterile solid compositions which can be dissolved in sterile water or some other sterile injectable medium immediately before use.

Compositions for rectal or vaginal injection are preferably suppositories which may contain, besides the active substance such excipients as cocoa butter or wax for suppositories.

Composition for introduction into the nose or under the tongue also get using standard excipients, well known in this field.

The effective dose of the active ingredient in the compositions of this invention can be different; however, it is necessary that the amount of active ingredient sufficient to obtain an acceptable dosage forms. The dose may be abrana a person skilled in the art depending on the desired result of treatment, the route of administration and duration of treatment. The required dose, administered to humans and other animals, for example mammals, usually from 0.0001 to 100 mg/kg of body weight per day.

The preferred dose is in the range from 0.01 to 10.0 mg/kg of body weight per day and can be entered as a single dose or divided into several doses.

Materials and methods

The expression of mRNA of receptors all five SSTR subtypes in line TT-cell MTS person determined by the analysis of RT-PCR. The ability of SS analogues with different affinity and specificity receptor subtypes 2 and 5 affect the proliferation of TT cells can be assessed by determining the incorporation of [3H]thy cells, representing an indirect method of measuring the activity of DNA synthesis, and the number of viable cells.

All preferred SSTR2 agonists are able to reduce significantly the number of TT cells at concentrations from 10-9M to 10-6M Compound 3 and compound 4 significantly (p<0,05) reduced the incorporation of [3H]thy at a concentration of 10-9M and do not have similar actions at concentrations of 10-8M and 10-7M when the maximum decrease in the number of cells. All tested compounds SSTR2 are less effective in increasing concentration, however, the bell-shaped curves inherent in all SS. Inga is licensing the inclusion of [ 3H]thy and reducing the number of TT cells by compound 1 and compound 2 at a concentration of 10-7M is not associated with any cytotoxic effect, as evidenced by staining Trifanova blue. In addition, the specified action is completely void of simultaneous processing of TT cells by compound 6, selective SSTR2 antagonist. The results show that the analogues of the SS with the preferred selectivity in respect of SSTR2 inhibit the proliferation of TT-cells as a result of specific interaction with the receptor SSTR2.

Culture line TT-cells

Line TT-cells obtained from the American type culture collection (ATS, Manassas, VA, USA). Line TT-cells includes aneuploid transformed ART-producing parafollicular cells, which are characterized by the presence of mutations TGC in TGG (with the substitution of Cys at Trp) in exon 11 codon 634 of the RET proto-oncogene (Cooley LD, et al., 1995 Cancer Genet Cytogenet 80: 138-149), which is confirmed by the authors of the present invention in the investigated cell lines. In addition, TT-cells have been disrupted expression of the suppressor gene p53 tumor (Velasco JA, et al., 1997 Int J Cancer 73:449-455). Immunohistochemical studies show that TT-cells Express ARTICLE and the receptor ARTICLE (Frendo JL, et al., 1994 FEBS Lett. 342: 214-216), the carcinoembryonic antigen (CEA), SS, neurotensin, gastrenterology peptide (GRP), Leu - and Met-EN is evalin, the peptide that stimulates parathyroid hormone (PTHrp), chromogranin A, SP-I, synaptophysin, neurospecific enolase (NSE), the receptor for 1,25-dihydroxyvitamin D3tyrosinekinase, α-tubulin and cytokeratin (Zabel M, et al., 1995 Histochemical J. 27: 859-868). TT-cells secrete a significant amount of ART and respond to changes in the content of ionized calcium (Zabel M, et al., 1992 Histochemistry 102: 323-327). Thus, the line TT-suitable cells for research parafollicular functions and reactions of endocrine and pharmacological stimulating factors.

Cells are introduced in nutrient mixture ham F12 with glutamine (EuroClone Ltd, Torquay, UK)containing 10% fetal calf serum (FBS, Life Technology, Milano, Italy), 100 units/ml penicillin, 0.1 mg/ml streptomycin and 100 µg/ml amphotericin b (EuroClone Ltd, Torquay, UK), at 37°C in a humid atmosphere with 5% CO2and 95% air.

Allocation RNA

Total RNA extracted from subconfluent TT-cells using trizol (TRIZOL) (Life Technologies, Milan, Italy). Method of using trizol is a modification of the extraction with guanidine/phenol. In accordance with the specified method of the medium containing cultured cells, sucked off and the cells washed once with PBS. Add reagent trizol and cells are lysed at room temperature for 10 minutes. To a mixture of trizol/cell lysate add chloroform, leave wystawa the change for 2-3 minutes and centrifuged with acceleration 12000× g for 15 minutes. Of the mixture was centrifuged to remove the water layer. RNA is precipitated by adding isopropanol, the precipitate is collected, washed with 75% ethanol and air-dried. Total RNA re-suspended in treated diethylpyrocarbonate (DEPC) water and conduct quantitative determination using UV spectrophotometry at 260 nm. To prevent contamination of DNA, RNA process not containing ribonuclease a desoksiribonukleaza (Promega, Milano, Italy).

Reverse transcription and polymerase chain reaction (RT-PCR)

Using the set for synthesis of single-stranded complementary DNA (cDNA) (system preamplification to synthesize single-stranded cDNA SuperScript, Life Technologies, Milan, Italy), 1 μg total RNA is subjected to reverse transcription according to the manufacturer's instructions. Back transcribed (RT) mixture in the tubes for PCR cover 50 μl of light white mineral oil (Sigma-Aldrich Corp., Milano, Italy); reverse transcription is carried out in interpolate for carrying out reactions (MJ Research Inc., Watertown, MA, USA)using the program with the following parameters: 10 min at 70°C, 1 minute at 4°C, 5 minutes at 4°C. After the introduction of SuperScript II reaction performed with 42°C for 50 minutes and then at 70°C for 15 minutes. The samples were cleaved by RNase H (Promega, Milano, Italy) at 37°C for 20 minutes and stored at -20°to perform p the pout PCR.

cDNA (1 µl back transcribed reaction mixture) amplified by PCR using 1 unit of DNA Taq polymerase (Life Technologies, Milan, Italy) featured suppliers conditions in 50 µl reaction mixture. After the initial denaturation was performed at 95°C for 5 minutes, conduct a PCR reaction using oligonucleotide primers, under conditions shown in table 1, which shows the expected fragments. The PCR products analyzed on 2% agarose gel and visualize, staining with ethidium bromide (ETB). To ensure the absence of contamination in the process of conducting RT-PCR, receive two negative control sample. The first negative control sample is obtained, using total RNA for RT. A second sample get, replacing the cDNA mixture with water when carrying out the PCR reaction. The PCR product is only useful if the negative control samples, analyzed by 2% agarose gel, there is no absorption band. Each PCR product digested with restriction enzymes and analyzed on 2% agarose gel to further confirm the correct identification of amplicons.

Selective agonists and antagonists SSTR

The SS analogues used in this study and their inherent indicators of affinity for various receptors SSTR are shown in table 2. Each link is, provided by the company Biomeasure Incorporated (Milford, MA, USA), re-suspended in 0.01 n acetic acid solution containing 0.1% bovine serum albumin (BSA), to achieve a homogeneous solubility and prevent non-specific binding to surfaces of different drugs. Specificity and selectivity analogues determined by analysis of binding of radioligand using cells Cho-K1, stably transfected receptors SSTR following subtypes.

The complete coding sequences of the genomic fragments of genes SSTR 1, 2, 3 and 4 and clone cDNA for SSTR-5 subcloning in expressing the vector pCMV mammalian (Life Technologies, Milan, Italy). Clonal cell line stably expressing the receptor SSTR 1-5, receive, transferira cells Cho-K1 (ATS, Manassas, VA, USA) by the method of coprecipitation calcium phosphate (Davis L, et al., 1994 In: Basic methods in Molecullar Biology, 2nd edition, Appleton & Lange, Norwalk, CT, USA: 611-646). As a selective marker is injected plasmid pRSV-neo (ATCC). Choose a clonal cell line in media RPMI 1640 containing 0.5 mg/ml G418 (Life Technologies, Milan, Italy), cloned into the ring and grown in culture.

To obtain a membrane for analysis of the receptor binding in vitro, the cells are Cho-K1 expressing receptors different SSTR subtypes, homogenized in 50 mm cooled ice buffer Tris-HCl and centrifuged twice with acceleration is 39000× g (10 minutes), producing an intermediate re-suspension in fresh buffer. The final precipitation re-suspended in 10 mm buffer Tris-HCl. For the analysis of receptor SSTR subtypes 1, 3, 4 and 5 aliquots of membrane preparations are incubated for 90 minutes at 25°from 0.05 nm [125I-Tyr11]SS-14 in 50 mm HEPES buffer (pH 7.4)containing 10 mg/ml BSA, 5 mm MgCl2, 200 cmed/ml of drug, 0.02 mg/ml bacitracin and 0.02 mg/ml phenylmethylsulfonyl. The final volume of the analyte 0.3 ml For analysis SSTR-2 as radioligand use of 0.05 nm [125I]MK-678, and the incubation time is 90 minutes at 25°C. Incubation stop, producing rapid filtration through filters CF/C (pre-soaked in 0.3% polyethylenimine) using a filtration device Brandela. All test tubes and filters are then washed three times with 5 ml aliquot of the cooled ice buffer. Specific binding is defined as total binding radioligand minus binding in the presence of 1000 nm SS-14 for SSTR 1, 3, 4 and 5, or 1000 nm MK-678 for SSTR2.

Evaluation of biological activity

Biological activity selective agonists and antagonists SSTR determined by the analysis of the activation of a calcium in cells of Cho-K1 expressing SSTR2 or SSTR5 person. Cells are incubated in a solution of 0.3% EDTA/ saline solution with phosphate buffer (25°C), Sobir the Ute and washed twice by centrifugation. The washed cells are re-suspended in buffered saline solution, Hank (HBSS) with the aim of introducing a fluorescent indicator Sa2+Fura-2AM. Cell suspension (approximately 106 cells/ml) incubated with 2 mm Fura-2AM for 30 minutes at 25°C. the Remaining reagent Fura-2AM remove, double-centrifuger in HBSS, and the final suspension is transferred into spectrofluorometer (Hitachi F-2000)equipped with a magnetic stirring mechanism and a camera for a ditch with adjustable temperature. The temperature was adjusted to 37°and add the SS analogues for measuring activation of intracellular CA2+. The wavelengths of excitation and emission respectively 340 and 510 nm. In cells expressing SSTR2, (1) compound 2 and compound 1 stimulate significant activation of intracellular CA2+(expressed as a ratio between stimulated and the original value), while compound 6 does not exert such actions at the tested concentrations. In addition, compound 4 and compound 3 also strongly stimulate the activation of CA2+. In cells expressing SSTR5, (2) compound 5 and compound 1 stimulate significant activation of intracellular CA2+, while compound 6 exhibits a slight agonistic activity in the range from 300 to 1000 nm. In cells expressing SSTR2, (3) connection 6 inhibit the em SS-induced activation of intracellular CA 2+in cells expressing SSTR2, depending on the dose, with full suppression actions SS at a concentration of about 10-7M Thus, the evaluation of the activation of intracellular CA2+show that the biological activity of different analogues fits their profile of the receptor binding.

DNA synthesis

The effects of selective agonists and antagonists SSTR on DNA synthesis TT-cells is determined taking into account the speed of the inclusion of [3H]thymidine ([3H]thy) previously described method (Davis L, et al., 1994 In: Basic methods in Molecular Biology, 2nd edition, Appleton & Lange, Norwalk, CT, USA: 611-646, degli Uberti EC, et al., 1991 J. Clin Endocrinol Metab 72: 1364-1371). TT cells were seeded on 24-well plates (105 cells/well) and incubated for 48 hours in medium containing 10% FBS, in the presence of [3H]thy (1,5 MX/ml; 87 Ci/mmol) similar SS or with the specified analog in concentrations from 10-6up to 10-9M. After incubation for 24 hours in the wells add fresh counterparts, without removing the environment.

After incubation, the cells three times washed with cooled PBS and ice double-cooled ice 10% trichloroacetic acid (THUK). Besieged THUC substance solubilizer in 500 μl of 0.2 mol/l sodium hydroxide and 0.1% SDS. The radioactivity of the cells counted in a scintillation spectrometer. The results (number of counts per minute per well) receive, determining the average value of Rainey least six experiments for four identical samples. The viability of TT cells in control and treated cultures appreciate, producing staining Trifanova blue after 24 and 48 hours, it was found that the number of viable cells is always 85-95%.

Cell proliferation

The effects of selective agonists and antagonists SSTR on the proliferation of TT cells is determined by the analysis of non-radioactive proliferation of cells in aqueous solution (Promega, Milano, Italy), which is a colorimetric method for determining the number of viable cells in assays of cell proliferation. In this analysis, we use solutions of the compounds of tetrazole (reagent Aries; MTS) and electronegative reagent (methosulfate phenazine; PMS). MTS biologically recovered cells with the formation of formazan, soluble in tissue culture medium. The optical density of formazan at 490 nm can be measured directly on 96-well plates (Zatelli MC, et al., 2000 J Clin Endocrinol Metab 85: 847-852; Cory AH, et al., 1991 Cancer Commun 3: 207-212). The conversion of MTS into water-soluble formazan occurs under the action of dehydrogenase enzymes found in metabolically active cells. The number of formazan measured considering the magnitude of optical density at 490 nm is directly proportional to the number of living cells in culture. In accordance with the specified method TT-cells visa is up on 96-well plates (2× 104 cells/well) and incubated for 48 hours in medium containing 10% FBS, without analogue SS or in the presence of the specified analog in concentrations from 10-6up to 10-9M. after the first 24 hours of incubation in the wells add fresh counterparts. At the end of the incubation with a pipette to each well add 20 μl of the combined solution of MTS/PMS and tablets incubated for another 4 hours at 37°C in a humid atmosphere with 5% CO2. The spectral density at 490 nm determined in a spectrophotometer to read the tablets ELISA (device for reading tablets EASIA, Medgenix, Camarillo, CA). The results of the spectral density at 490 nm) receive, determining an average value for at least eight identical samples in six experiments.

Results

The expression of SSTR in line TT-cell MTS person

To understand the specific role of receptor subtypes SSTR2 and SSTR5 in the regulation of proliferation of parafollicular C-cells need to figure out whether TT-cells can Express receptors SSTR, able to mediate responsiveness to compounds selectively acting on the individual receptor subtypes SSTR. To answer the question, isolated total RNA from cultured TT-cells and carry out the reaction RT-PCR under the conditions described in "Materials and methods". The integrity of cDNA determined by the presence of a signal is La GAPDH. The absence of genomic DNA contamination in the cDNA samples determined by the absence of amplification when carrying out a PCR reaction using irreversibly transcribed samples. Positive amplification of SSTR1, 2, 3, 4 and 5, found in the studied cell line (figure 4), suggests that these receptors are expressed in the line TT-cell MTS person. Detection of stable expression of TP-cell receptor different SSTR subtypes makes the specified model cell system suitable for evaluating the effect rezeptrisperdal SS analogues.

The impact of selective analogues SS on the inclusion of [3H]thy TT-cells

Values include [3H]thy obtained using the preferred SSTR2 agonists at concentrations from 10-9up to 10-6M (compound 1, compound 2, compound 3 and compound 4), the preferred agonist SSTR5 (compound 5) and preferred SSTR2 antagonist (compound 6), presented in figure 5. As shown in the given figure, compound 2 inhibited the incorporation of [3H]thy on 58-23% at concentrations ranging from 10-9up to 10-7M Compound 1 inhibits the incorporation of [3H]thy on 41-21% at concentrations ranging from 10-9up to 10-6M. the Inclusion of [3H]thy also significantly reduced compound 4 (-13%, p<0.05) and compound 3 (-17%, p<0.05) in 10-9 M. In contrast to what this connection 5 increases the incorporation of [ 3H]thy in TT cells to 80-175%. Selective SSTR2 antagonist, compound 6, does not change the value of enable [3H]thy TT-cells compared with untreated control cells.

The impact of electoral SS analogues on proliferation TT-cells

For deeper research activity SS analogues in the growth of TT cells analyze their impact on the number of viable cells. The effects of preferred SSTR2 agonists, the preferred agonist SSTR5 and preferred SSTR2 antagonist in the number of viable TT-cells at concentrations ranging from 10-9up to 10-6M presented on Fig.6. As shown in the given figure, all preferred SSTR2 agonists significantly inhibited cell proliferation compared with untreated control cells at all tested concentrations. Selective agonist SSTR5, compound 5, slightly increases the proliferation of TT cells (up to 11% at 10-8M), however, this result does not differ at a statistically significant level from the result obtained for untreated control cells. Selective SSTR2 antagonist, compound 6, apparently, has no effect on the growth of TT cells at the tested concentrations.

Countering selective SSTR2 antagonist effects of the preferred agonist STR2

To further clarify the specific role of SSTR2 in mediating antiproliferative activity preferred SSTR2 agonists determine the degree of inclusion of [3H]thy and the growth rate of TT-cells after 48-hour exposure to compound 1 and compound 2 used separately (at a concentration of 10-7M) or in combination with compound 6, selective SSTR2 antagonist, in equimolar concentrations (10-7M). Inhibition of incorporation of [3H]thy caused by connection 1 and connection 2, is suppressed by simultaneous processing of TT cells by compound 6 (7, upper block). The inhibition of proliferation of TT cells induced by compound 1 is reduced by 46%-10% simultaneous processing of compound 6. In addition, compound 6, apparently, completely blocks the antiproliferative activity of compound 2 (Fig.7, bottom block). Thus, clearly demonstrated the specific part SSTR2 in mediating an inhibitory effect of SSTR2 agonist on the proliferation of TT cells.

The combined effect of the preferred SSTR2 agonist and preferred agonist SSTR5 on the inclusion of [3H]thy and cell proliferation

For the analysis of joint actions of agonists SSTR2 and SSTR5 determine the incorporation of [3H]thy, and proliferation of TT cells, testing the connection 2 and connection 5 in combination with each other when the post is Noy concentrations (10 -7M) single connection and increasing concentrations of other compounds (10-9M to 10-6M). The results are shown in Fig. The use of agonist SSTR5 in increasing concentrations (from 10-9M to 10-6M) depending on the dose prevents suppression enable [3H]thy in TT cells (Fig, upper block) and proliferation (Fig. 8, the lower block), caused by SSTR2 agonist (10-7M). The obtained data testify to the antagonism between mediated and SSTR5 SSTR2 effects on cell proliferation.

It should be noted that detailed description of the invention is for illustration only and does not restrict the scope of the invention set forth in the accompanying claims. The formula of the invention includes all other objectives, advantages and modifications of the present invention.

Table 1.

Primers and conditions for PCR amplification of somatostatin receptors (SSR)
SSRPrimersDenaturationAnnealingExtensionCyclesThe expected fragment (BP)
1Upper: 5'-AGCCGGTTGACTATTACGCC-3'95°C 30 sec.60°72°45334
(SEQ ID No.7)1 minutes2 minutes
Lower: 5'-GCTCTCACTTCTACCATTGTC-3'
(SEQ ID No.8)
2Upper: 5'-GGTGAAGTCCTCTGGAATCC-3'95°30 sec.63°72°45461
(SEQ ID No.9)1 minutes2 minutes
Bottom; 5'-CCATTGCCAGTAGACAGAGC-3'
(SEQ ID No.10)
3Upper: 5'-TCATCTGCCTCTGCTACCTG-3'95°30 sec.65°72°45221
(SEQ ID No.11)1 minutes2 minutes
Lower: 5'-GAGCCCAAAGAAGGAGGCT-3'
(SEQ ID No.12)
4Upper: 5'-CGGCAGTCTTCGTGGTCTAC-3'94°30 sec.63°72°45247
(SEQ ID No.13)1 minutes2 minutes
Lower: 5'-GCATCAAGGTCGGTCACGAC-3'
(SEQ ID No.14)
5Upper:94°1 min60°72°40211
5'-AACACGCTGGTCATCTACGTGGT - 3' (SEQ1 minutes1 minutes
ID No.15)15 sec.
Bottom:
5'-AGACACTGGTGAACTGGTTGAC-3' (SEQ
ID No.16)
GAPDHUpper: 5'-ATGACCCCTTCATTGACCTC-3'95°30 sec.60°72°40820
(SEQ ID No.17)1 minutes2 sec.
Lower: 5'-AAGTGGTCGTTGAGGGCAAT-3'
(SEQ ID No.18)

Table 2.

The specificity of the subtypes of somatostatin receptors person (IC50, nm)
Subtype receptor
Connection12345
121290,7598 1826a 12.7
210000,344121000213,5
352100,35215753711,2
460160,1926,838979,8
51152166100016182,4
6 (antagonist)27576,44442386,5

The affinity of receptors of different subtypes is determined by the analyses of the binding of radioligand with the receptor on the membrane using ovary cells Chinese hamster expressing the gene SSR2 human cDNA or SSR5.

1. The modulation speed of cell proliferation medullary carcinoma of the thyroid gland (MTS), providing a combined effect on the cells of one or more agonists somatostatine the s receptor type 2 (SSTR2) and one or more agonists somatostatin receptor type 5 (SSTR5).

2. The method according to claim 1, where the specified agonist SSTR5 represents D-Phe-Phe-Trp-D-Trp-Lys-Thr-Phe-Thr-NH2or its pharmaceutically acceptable salt.



 

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FIELD: medicine.

SUBSTANCE: method involves administering Capecitabine during 14 days within 15 days long treatment course in everyday mode and Oxyplatin at a dose of 50 mg/m2 at the first, eighth and fifteenth treatment day. Pause between treatment courses is 2-3 weeks long.

EFFECT: enhanced effectiveness of treatment.

FIELD: medicine.

SUBSTANCE: method involves evaluating neurological status and crude protein and calcium level in cerebrospinal fluid after intrathecally introducing methotrexate in the amount of 5 mg in auto liquor in postoperative period before starting chemotherapy. 5 ml of liquor are taken in a day and methotrexate course dose is selected depending on neurological status and changes in crude protein and calcium level in cerebrospinal fluid in liquor. No changes in neurological status and crude protein and calcium level being observed, methotrexate dose is increased by 20% when compared to the test dose. No changes in neurological status and increased crude protein level by 100% or higher and reduced calcium level by a value between 20% and 40% being observed, methotrexate dose is reduced by 20% when compared to the test dose. Negative changes in neurological status and crude protein being increased more than by 200% and reduced calcium level by more than 40% and reduced crude protein and increased calcium content being observed, chemotherapy course is restricted to already introduced methotrexate test dose.

EFFECT: improved biochemical criteria.

1 tbl

FIELD: medicine, oncology.

SUBSTANCE: method involves autoblood sample taking off in the amount 200-250 ml into a sterile flask with hemopreserving agent "Glugitsir" followed by centrifugation at 1500 rev/min for 30 min. Then supernatant plasma is placed into another sterile flask with a hemopreserving agent and cellular precipitate is reinfused. A single dose of a cytostatic agent is mixed with 10-100 ml of autoplasma, incubated at temperature 37°C for 1 h and administrated through catheter in abdominal cavity by puncture in lower quadrant of abdomen. Procedure is repeated if necessary. Method provides decreasing toxicity of chemotherapy, simple method and uniform distribution of volume of injected preparation for all abdomen cavity. Invention can be used in carrying out chemotherapy of abdomen cavity tumors with ascites.

EFFECT: improved method for treatment.

1 ex

FIELD: organic chemistry, amino acids.

SUBSTANCE: invention claims new compounds that elicit both high affinity and selectivity with respect to neuromedin B and somatostatin receptors. Compounds has the following formula: wherein α-atom in each group among AA1, AA2, AA3, AA4, AA5, AA6, AA7 and AA8 is substituted optionally and independently with (C1-4)-alkyl-(C3-4)-alkenyl, (C3-4)-alkynyl or (C1-6))-alkyl-C(O)-; AA1 is absent or means Ac-D-Phe or D- or L-isomer of R11, Pip, Pro or aromatic α-amino acid taken among the group consisting of Cpa, Dip, Nal and Phe; AA2 is absent or means Pal, Phe, Tyr; AA3 means D- or L-isomer of Cys; AA4 means D- or L-isomer of Trp; AA5 means Lys; AA6 means D- or L-isomer of Cys; AA7 is absent or means A3c, A4c, A5c, A6c, Abu, Aic, β-Ala, Gaba, Nle, Pro, Sar, Thr, Thr(Bzl) or Val; AA8 is absent or means R11, Nal, Thr, Tyr, Phe or Nle; each among R1 and R2 represents independently hydrogen atom (H) or absent; R5 means -NH2,; R11 means D- or L-amino acid independently in each case and AA3 and AA6 are bound by disulfide bond.

EFFECT: valuable biological properties of compounds.

9 cl, 2 tbl, 1 ex

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