New peptides

 

(57) Abstract:

Describes the oligopeptides, which have the amino acid sequence of from 6 to 100 amino acids recognized and proteoliticeski split free of the specific antigen of the prostate (PSA) in enzymatically active form. Also described tests in which these oligopeptides can be used for in vitro and in vivo determination by the activity of free PSA. In addition, the disclosed therapeutic agents containing conjugates of these oligopeptides represented by the General formula I, and known cytochemical agents. 4 C. and 19 C.p. f-crystals, 7 ill., table 2.

This patent application is a partial continuation of simultaneously applying reg. N 08/404833 filed March 15, 1995, which, in turn, is a partial continuation of simultaneously applying reg. N 08/267092 filed June 28, 1994.

Background of the invention

According to preliminary data, in 1994 in the USA, the number of diagnosed cases of cancer of the prostate in men is 200,000, while the mortality from this disease can be 38000 people (Garnick, M. C. (1994) The Dilemmas of Prostate Cancer. Scien ukovich diseases (besides skin cancer) among men in the United States, and ranks second in mortality from cancer (after lung cancer).

Prostatic specific antigen (PSA) is a single-chain glycoprotein 33 kDa, which is produced almost exclusively by the epithelium of the prostate man, and is present in human seminal in the amount of from 0.5 to 2.0 mg/ml (Nadji, M., Taber, S. Z., Castro, A., et al. (1981) Cancer 48: 1229; Papsidero, L., Kuriyama, M., Wang, M. et al. (1981). JNCI 66:37; Qui, S. D., Young, C. Y. F., Bihartz, D. L., (1990), J. Urol. 144:1550; Wang, M. C. , Valenzuela, L. A., Murphy, G. P., et al. (1979), Invest. Urol. 17:159). This glycoprotein has a single carbohydrate unit, which is attached to the asparagine residue 45, which is 2 to 3 kDa of the total molecular weight. PSA is a protease with chymotrypsinogen specificity (Christensson, A. , Laurell, C. B., Lilja, N. (1990). Eur. J. Biochem. 194: 755-763). It has been shown that PSA is responsible, mainly, for the dissolution of the gel structure formed by ejaculation by proteolysis of the main (major) proteins contained in the gel of sperm, such as simanagaon I and simanagaon II, and fibronectin (Lilja, N. (1985). J. Clin. Invest. 76: 1899; Lilja, H., Oldbring, J., Rannevik, G., et al. (1987). J. Clin. Invest. 80:281; McGee. R. S., Herr, J. C. (1988). Biol. Reprod. 39:499). PSA-mediated proteolysis gelling protein promotes formation of multiple rastvoronasosom more motile sperm (Lilja, N., Laurell, C. B. (1984), Scand. J. Glin. Lab. Invest. 44: 447; McGee, R. S. Herr, J. C. (1987); Biol. Reprod. 37: 431). In addition, prostatic specific antigen (PSA) has the ability to proteolytic cleavage of IGFBP-3 (protein, to bind to the insulin-like growth factor), which allows IGF (insulin-like growth factor) to specifically stimulate the growth of PSA-secreting cells (Cohen et al., (1992) J. Clin. Endo. & Meta., 75: 1046-1053).

The predominant molecular form of serum PSA is a complex formed with PSA-alpha 1-antichymotrypsin, and up to 95% of the total detectable serum PSA (Christensson, A., Bjork, T., Nilsson, 0., et. al. (1993). J. Uro. 150: 100-105; Lilja, H., Christensson, A., Danlen, U. (1991). Clin. Chem. 37: 1618-1625; Stenman, U. H., Leinoven, J., Alfthan, H. , et. al. (1991). Cancer Res. 51: 222-226). Prostatic tissue (normal tissue, benign hyperplastic tissue and malignant tissue) releases predominantly Mature enzymatically active PSA, because this form is necessary for formation of a complex with alpha 1-antichymotrypsin (Mast, A. E., Enghild, T. J., Pizzo, S. V., et. al. (1991), Biochemistry 30: 1723-1730; Perlmutter, D. H., Glover, G. I., Rivetna, M. , et. al. (1990). Proc. Natl. Acad. Sci. USA 87: 3753-3757). Therefore, in the microenvironment PSA-secreting cells of the prostate PSA, obviously, processed and secreted in his Mature is Abilene complexes with alpha 2-macroglobulin, but, since this leads to the encapsulation of PSA and total loss of the PSA epitopes, the meaning of in vivo formation of such a complex is not yet clear. Free unconjugated form of PSA is a minor fraction of serum PSA (Christensson, A., Bjork, T., Nilsson, 0., et. al. (1993). J. Urol. 150: 100-150; Lilja, H., Christensson, A. , Dahlen, U. (1991) Clin. Chem. 37: 1618-1625). The size of this form of serum PSA is similar to the size of the form PSA present in the seminal fluid (Lilja, H. , Christensson, A. , Dahlen, U. (1991) Clin. Chem. 37: 1618-1625), but it is not known whether the free form of serum PSA to be imagenum; internal hydrolyzed inactive Mature form of PSA; or PSA, with enzymatic activity. However, it seems unlikely that the free form of serum PSA has enzymatic activity as present in the serum molar excess of both unreacted alpha 1-antichymotrypsin and alpha 2-macroglobulin significantly exceeds (100-1000) times detected serum levels of free 33 kDa form of PSA (Christensson, A., Bjork, T., Nilsson, O., et. al. (1993). J. Urol. 150: 100-105; Lilja, H., Christensson, A., Danlen, U. (1991). Clin. Chem. 37: 1618-1625).

Measurement of PSA levels in serum may be useful when monitoring the development of adenocarcinoma of the prostate (Duffy, M. S. (1989). Ann is s', increasing the concentration of PSA in serum is also observed in benign prostatic hyperplasia and after surgery on the prostate gland (Lilja, N., Christensson, A., Dahlen, U. (1991). Clin. Chem. 37: 1617-1625). In addition, it is known that metastasis of the prostate gland secrete immunoreactive PSA, as in patients with a remote prostate gland and with widespread metastatic prostate cancer detected high levels of serum PSA (Ford, T. F., Butcher, D. N., Masters, R. W., et. al. (1985). Brit. J. Of Urology 57: 50-55). Therefore, the cytotoxic compound that is activated by the proteolytic activity of PSA, should be specific to the cells of the prostate and PSA-secreting metastasis of the prostate.

In line with this, the aim of the present invention to provide a new oligopeptides, which selectively and enzymatic split active free prostatic antigen (PSA).

Another objective of the present invention is to develop a quantitative analysis of the enzymatic activity of PSA using the new oligopeptides.

The next objective of the present invention to provide a new action which includes new oligopeptides, conjugated with a cytotoxic agent. Another purpose of this invention is the provision of a method for the treatment of prostate cancer, providing for the introduction of new anti-cancer composition.

Brief description of the invention

Identified several sites of cleavage in which simanagaon I selectively proteoliticeski hydrolyzed to free PSA. Describes the oligopeptides with amino acid sequences that are recognized and proteoliticeski split free prostatic antigen (PSA). Such oligopeptides can be used in the analysis of in vivo and in vitro to determine by the activity of the free prostate specific antigen (PSA). In addition, these oligopeptides can be incorporated into therapeutic agents, which contain conjugates of these oligopeptides and known cytotoxic agents, and which can be used to treat prostate cancer.

Brief description of drawings

Fig. 1a and 1b. The primary amino acid sequence semenovaelena I.

Shows the primary amino acid sequence semenovaelena I (SEQ ID No. 1). Also shown are the sites of proteolytic races against PSA-hydrolysis), fragments of the protein are numbered starting from aminocore.

Fig. 2. The affinity towards hydrolysis of synthetic oligopeptides.

Received nesting many synthetic oligopeptides, and these oligopeptides were subjected to enzymatic hydrolysis of the active free PSA during different periods of time. The results presented in table (ND = not determined). To denote amino acids was used one-letter code, where A=Ala, E=Glu, G=Gly, H=His, I=Ile, K=Lys, L= Leu, N=Asn, Q=Gln, P=pro, S=Ser, T=Thr, Y=Tyr.

Fig. 3a and 3b. The affinity towards hydrolysis of synthetic oligopeptides.

Received synthetic oligopeptides that were subjected to enzymatic hydrolysis of the active PSA within four (4) hours. In Fig. 3 shows the percentage of oligopeptides, hydrolyzed during this period of time.

Fig. 4. Data on cytotoxicity darassalam conjugates "Oligopeptide - doxorubicin".

The data presented in Fig. 4, illustrate the comparative cytotoxicity of doxorubicin and conjugate of doxorubicin covalently linked to the Oligopeptide (Compound 12b), which does not contain the site of proteolytic cleavage prostatitis the private Oligopeptide, has more than 300 times smaller IC50. This conjugate has no HPLC-detectable impurities unmodified doxorubicin. Oligopeptide, taken separately, has no appreciable cytotoxic activity.

Fig. 5. The affinity in vitro cleavage free PSA oligopeptides, conjugated with doxorubicin.

Received conjugates of oligopeptides with doxorubicin and these conjugates were subjected to enzymatic hydrolysis of the active free PSA within four (4) hours. The picture shows the percentage of the conjugate, the enzyme cleaved the Oligopeptide during this period of time.

Fig. 6. Affinity cleavage of oligopeptides, conjugated with doxorubicin, air-conditioned cell medium.

Conjugates of oligopeptides with doxorubicin was subjected to reaction for four (4) hours from the culture medium, conditioned LNCaP cells (which are known to secrete free PSA) or cells DuPRO (which do not secrete free PSA). In Fig. 6 shows the percentage of the conjugate, hydrolyzed in the Oligopeptide during this period of time.

Fig. 7. Data on cytotoxicity hydrolyzed conjugates of oligopeptides and doxorubicin.

Dan is an Oligopeptide, contains the site of proteolytic cleavage free PSA, directed against a cancer cell line, which, as you know, secretes a free PSA. For some conjugates shown that they possess cytotoxicity against cell lines (DuPRO), which does not secretes free PSA.

Detailed description of the invention

The present invention relates to new oligopeptides, which are specifically recognized by the free prostate antigen (PSA), and which can be proteoliticeski obtained through the enzymatic activity of free static antigen. Such oligopeptides are oligomers having the amino acid sequence selected from the following sequences:

a) AsnLysIleSerTyrGln Ser (SEQ ID No. 13),

b) LysIleSerTyrGln Ser (SEQ ID No. 14),

c) GlyGluAsnGlyValGlnLysAspValSerGlnxaaseriletyr SerGln ThrGlu (SEQ ID No. 15),

d) GlyLysGlyIleSerSerGlnTyr SerAsnThrGluGluArgLeu (SEQ ID No 2),

e) AsnLysIleSerTyrTyr Ser (SEQ ID No. 127),

f) AsnLysAlaSerTyrGln Ser (SEQ ID N 128),

g) SerTyrGln SerSer (SEQ ID N 129),

h) LysTyrGln SerSer (SEQ ID N 140),

i) hArgTyrGln SerSer (SEQ ID No. 141);

where hArg is homoarginine, a Xaa is any natural amino acid.

In one of the embodiments of the present invention, oligop is lnasty:

a) AsnLysIleSerTyrGln SerSer (SEQ ID No. 16),

b) AsnLysIleSerTyrGln SerAla (SEQ ID N 130),

c) AsnLyslleSerTyrGln SerSerSer (SEQ ID N 17),

d) AlaAsnLysIleSerTyrGln SerSerSer (SEQ ID No. 18),

e) LysIleSerTyrGln SerSerSerThrGlu (SEQ ID No. 19),

f) GlyGluAsnGlyValGlnLysAspValSerGlnargseriletyr SerGln ThrGlu (SEQ ID No. 4),

g) GlyGluAsnGlyValGlnLysAspValSerGlnserseriletyr SerGln ThrGlu (SEQ ID No. 5),

h) AlaAsnTysIleSerTyrTyr Ser (SEQ ID N 131),

i) AlaAsnLysAlaSerTyrGln Ser (SEQ ID No 132),

j) SerTyrGln SerSerThr (SEQ ID N 133),

k) SerTyrGln SerSerSer (SEQ ID N 134),

l) LysTyrGln SerSerSer (SEQ ID N 142),

m) hArg TyrGln SerSerSer (SEQ ID N 143),

n) SerTyrGln SerSerLeu (SEQ ID N 135),

In a more preferred embodiment of the present invention the oligopeptides are oligomers having the amino acid sequence selected from the following sequences:

a) AsnLysIleSerTyrGln SerSerSerThr (SEQ ID No. 10),

b) AlaAsnLysIleSerTyrGln SerAla (SEQ ID No. 136),

C) AsnLysLleSerTyrGln SerSerSerThrGlu (SEQ ID No. 3),

d) AlaAsnLysIleSerTyrGln SerSerSerThrGlu (SEQ ID No. 11),

e) GlyGluAsnGlyValGlnLysAspValSerGlnargseriletyr SerGln ThrGlu (SEQ ID No. 4),

f) AlaAsnLysIleSerTyrTyr SerSer (SEQ ID No. 137),

g) AlaAsnLysIleSerTyrTyr SerAla (SEQ ID N 138),

h) AlaAsnLysAlaSerTyrGln SerAla (SEQ ID N 139),

i) AlaSerTyrGln SerSerLeu (SEQ ID No. 94).

In an even more preferred embodiment of the present invention the oligopeptides are oligomers having the following amino acid sequence selected:

a) GlyArgLysAlaAsnLysIleSerTyrGln SerSerSerThrGluGluArg ArgLeuHisT is tnou sequence" refers to oligomers, consisting of about 6 to 100 amino acid residues, and containing in its amino acid sequence described specific amino acid sequence that proteoliticeski cleaved free PSA. For example, the oligomer GlnLeuAspAsnLysLleSerTyrGln SerSerSerThrHisGlnSerSer (SEQ ID No. 20) contains the amino acid sequence AsnLysIleTyrGln SerSerSerThr (SEQ ID No. 10), and therefore included in the scope of the present invention. It should be noted that these oligomers are not semenovaelena I and semenovaelena II. In addition, it should be noted that the present invention also relates to oligomers, where N-terminal amino acid or the C-terminal amino acid, or both of the end amino acids are modified. Such modifications include, but are not limited to, acylation of the amino group at the N-Terminus and the formation of amide groups to replace the carboxyl group at the C-end. Adding these groups can be carried out in the process of solid-phase synthesis of the oligomer. For example, the accession of the C-terminal amino acid to the solid phase resin can be carried out using the amine, which leads to the formation of amide groups after acid cleavage of the oligomer from the resin. Thus, as used above is by them:

AlaAsnLysIleSerTyrGln SerSerSerThrGlu-amide (SEQ ID No. 11),

Ac-AlaAsnLysLleSerTyrGln SerSerSerThrLeu (SEQ ID N 70),

Ac-AlaAsnLysIleSerTyrGln SerSerSerThrGlu-amide (SEQ ID No. 11),

AC-AlaAsnLysIleSerTyrGln SerSerSerThrLeu-amide (SEQ ID N 70),

Ac-AlaAsnLsIleSerTrGln SerAlaSerThrGlu-amide (SEQ ID N 73),

Ac-AlaAsnLysIleSerTyrGln SerSerLysThrGlu-amide (SEQ ID N 74),

Ac-AlaAsnLsIleSerTrGln SerSerThrGlu-amide (SEQ ID N 75),

Ac-AlaAsnLysIleSerTyrGln SerSerGlnThrGlu-amide (SEQ ID N 78),

Ac-AlaAsnLysIleSerTyrGIn SerAlaLysThrGlu-amide (SEQ ID No. 79),

Ac-AlaAsnLysIleSerTyrGln SerThrGlu-amide (SEQ ID N 81),

Ac-AlaAsnLysSerTyrGln SerSerThrGlu-amide (SEQ ID No. 82),

Ac-AlaAsnLysAlaSerTyrGln SerAlaSerThrGlu-amide (SEQ ID N 84),

Ac-AlaAsnGluIleSerTyrGln SerAlaSerThrGlu-amide (SEQ ID N 85),

Ac-AsnLysIleSerTyrGln SerSer-amide (SEQ ID No. 16),

Ac-LysIleSerTyrGln SerSer-amide (SEQ ID No. 86),

Ac-SerTyrGln SerSerThrGlu-amide (SEQ ID No. 87),

Ac-AlaSerTyrGln SerSerThrGlu-amide (SEQ ID No. 89),

Ac-AlaAsnLysIleSerTyrTyr SerSerSerThrGlu-amide (SEQ ID N 92),

Ac-AlaAsnLysIleSerTyrTyr SerAlaSerThrGlu-amide (SEQ ID No. 93),

Ac-AlaSerTyrGln SerSerLeu-amide (SEQ ID No. 94),

Ac-AlaAsnSerTyrGln SerSerSerThrGlu-amide (SEQ ID N 95),

Ac-AlaSerTrGln SerSerSerThGlu-amide (SEQ ID N 96).

Ac-SerTyrGln SerSerSerThrGlu-amide (SEQ ID N 97),

Ac-AlaAsnLysAlaSerTyrGln SerAlaSerCys-amide (SEQ ID No. 98).

For each specialist peptide chemistry, it is clear that some amino acids in biologically active Oligopeptide may be replaced by homologous, isostere and/or isoelectronic amino acids, but the biological activity Taco is the second list of amino acid substitutions is purely illustrative, and non-limiting character:

The original amino acid - Substituting amino acid

Ala - Gly

Arg - Lys Ornithine

Asn - Gln

Asp Glu

Glu - Asp

Gln - Asn

Gly - Ala

Ile - Val, Leu, Met, Nle

Leu - Ile, Val, Met, Nle

Lys - Aro, Ornithine

Met - Leu, Ile, Nle, Val

Ornithine - Lys, Arg

Phe - Tyr, Trp

Ser - Thr

Thr - Ser

Trp - Phe, Tyr

Tyr - Phe, Trp

Val - Leu, Ile, Met, Nle

For example, the following oligopeptides can be synthesized by methods well known to any specialist, and should, as expected, proteoliticeski to split

free PSA:

AsnArgIleSerTyrGln Ser (SEQ ID No. 21)

AsnLysValSerTyrGln Ser (SEQ ID No. 22)

AsnLysMetSerTyrGln SerSer (SEQ ID N 23)

AsnLysLeuSerTyrGln SerSer (SEQ ID N 24)

AsnLysIleThrTyrGln SerSerSer (SEQ ID No. 25)

AsnLysIleSerPheGln SerSerSer (SEQ ID No. 26)

AsnLysIleSerTrpGln SerSerSerThr (SEQ ID No. 27)

AsnLysIleSerTyrAsn SerSerSerThr (SEQ ID No. 28)

AsnLysIleSerTyrGln ThrSerSerThr (SEQ ID No. 29)

AsnLysIleSerTyrGln Ser (SEQ ID No. 30)

GlnLysIleSerTyrGln SerSer (SEQ ID No. 31)

AsnArgIleThrTyrGln SerSerSer (SEQ ID no 32)

AsnArgIleSerPheGln SerSerSerThr (SEQ ID No. 33)

AsnArgIleSerTrpGln SerSerSerThr (SEQ ID No. 35)

AsnArgIleSerTyrGln ThrSerSerThr (SEQ ID No. 36)

AsnLysIleThrTyrGln ThrSerSerThr (SEQ ID N 37)

AsnLysLeuSerTyrGln ThrSerSerThr (SEQ ID no 38)

GlnLysLeuSerTyrGln SerSerSerThr (SEQ ID No. 39)

AsnArgLeuSerTyrGln ThrSerSerThr (SEQ ID No. 40)

AsnLysValSerPheGln SerSerSerThr (SEQ ID No. 41)

AsnArgValSerTrpGln SerSerSerThr (SEQ ID No. 42)

GlnLysValSerTyrGln the oligopeptides can be synthesized by the methods well known to any specialist, and should, as expected, proteoliticeski to split free PSA:

GlyGluGlnGlyValGlnLysAspValSerGlnserseriletyr SerGlnThr Glu (SEQ ID No. 45),

GlyGluAsuGlyLeuGlnLysAspValSerGlnserseriletyr SerGlnThr Glu (SEQ ID N 47),

GlyGluAsnGlyValAsnLysAspValSerGlnserseriletyr SerGlnThr Glu (SEQ ID No. 48),

GlyGluAsnGlyValGlnArgAspValSerGlnargseriletyr SerGlnThr Glu (SEQ ID No. 49),

GlyGluAsnGlyValGlnLysAspValSerGlnlysseriletyr SerGlnThr Glu (SEQ ID No. 50),

GlyGluAsnGlyValGlnLysAspLeuSerGlnthrseriletyr SerGlnThr Glu (SEQ ID No. 51),

GlyGluAsnGlyValGlnLysAspValSerGlnserserilephe SerGlnThr Glu (SEQ ID No. 52),

GlyGluAsnGlyValGlnLysAspMetSerGlnserseriletyr ThrGlnThr Glu (SEQ ID N 53),

GlyGluAsnGlyValGlnLysAspValSerGlnargseriletyr ThrGlnThr Glu (SEQ ID No. 54),

GlyGluAsnGlyValGlnLysAspValSerGlnserseriletyr SerGlnSer Glu (SEQ ID No. 55),

GlyGluAsnGlyValGlnLysAspValSerGlnargseriletyr SerAsnThr Glu (SEQ ID No. 56),

GlyLysAlaIleSerSerGlnTyr SerAsnThrGluGluArgeLeu (SEQ ID No. 57),

GlyArgGlyIleSerSerGlnTyr SerAsnThrGluGluArgLeu(SEQ ID No. 59),

GlyLysGlyIleThrSerGlnTyr SerAsnThrGluGluArgLeu (SEQ ID N 60),

GlyLysGlyIleSerThrGlnTyr SerAsnThrGluGluArgLeu (SEQ ID N 61),

GlyLysGlyIleSerSerAsnTyr SerAsnThrGluGluArgLeu (SEQ ID No. 62),

AlaLysGlyIleSerSerGlnTyr SerAsnThrGluGluArgLeu (SEQ ID No. 63),

GlyLysGlyIleSerSerGlnPhe SerAsnThrGluGluArgLeu (SEQ ID N 64),

GlyLysGlyIleSerSerGlnTyr ThrAsnThrGluGluArgLeu (SEQ ID N 65),

GlyLysGlyIleSerSerGlnTyr SerAsnSerGluGluArgLeu (SEQ ID N 58 and

GlyLysGlyIleSerSerGlnTyr SerAsnThrAspGluArgLeu (SEQ ID no 46), etc.

The symbol included in the amino acid sequence indicates the point of proteolytic cleavage of oligopeptides free PSA.

Nastojashiu way is one of the important aspects of the present invention lies in the use of the analytical system, which allows to quantify the amount of free PSA present in some physiological fluids and tissues. In addition, this analysis not only allows you to control the isolation and purification of free PSA, but can also be used as the basis of a method of screening to identify inhibitors of the proteolytic activity of free PSA. In General, this method is simply to determine the ability of the composition, which is assumed to contain enzymatically active free PSA, proteoliticeski split Oligopeptide.

Basically the assay procedure is performed with the use of one of the oligonucleotides described above. However, it may be preferable to develop such an analysis, in which the Oligopeptide containing the cleavage site would be marked, for example, a radioactive label, the presence of which in the unsplit Oligopeptide, and part of oligopeptides remaining after cleavage and contains the label, could be measured.

In addition, the present invention relates to a method of identifying compounds (hereinafter referred to as compounds-candidates) that inhibit the proteolytic activity is entifically any connection candidate, which is inhibitor, regardless of whether or not the connection candidate protein or pettigrove structure.

Thus, the present invention also relates to a method for determining the ability of test compounds to inhibit the proteolytic activity of the free PSA; and the method includes:

(a) the reaction of the substrate having the amino acid sequence recognized and selectively proteoliticeski split free prostatic antigen, free prostate antigen in the presence of the test compound; and

(b) determining whether cleavage of the substrate, where the ability of the test compound to inhibit the proteolytic activity indicates a decrease in the level of cleavage of the substrate compared to the cleavage of the substrate in the absence of the test compound.

In its design and implementation analysis of screening candidate compounds is quite simple, and in many respects similar to the above analysis for the determination of proteolytic activity. For example, after receiving a relatively purified preparation of free PSA, preferably just smout to carry out its function splitting, but in the absence of inhibitory compounds. For example, it is desirable to include in the mixture a certain amount of oligopeptides having a specific cleavage site of PSA, such as oligopeptides described above. Thus, it is possible to measure the ability of the test compound to reduce the relative level of cleavage of oligopeptides in the presence of the test compound.

Accordingly, to assess the relative inhibitory ability of the test compound, it is desirable to measure or otherwise determine the activity of the free PSA in the absence of added test compound compared to the activity in the presence of the test compound.

The present invention also relates to anti-cancer compositions that can be used to treat prostate cancer. Such compositions contain the oligopeptides of the present invention, covalently linked to a cytotoxic agent, either directly or through a chemical linker. This combination of oligopeptides and cytotoxic agent may be defined as the conjugate. In the ideal case, the cytotoxic activity of a cytotoxic agent is very low or have is whether using a chemical linker to the cytotoxic agent and is intact. In addition, in the ideal case, the cytotoxic activity of a cytotoxic agent significantly increases or returned to the level of activity of unmodified cytotoxic agent after proteolytic hydrolysis linked oligopeptides in the site of cleavage. Most preferred, though optional, the embodiment of the present invention to provide a conjugate, where the Oligopeptide and the chemical linker, if present, are separated from the cytotoxic agent under the action of proteoliticeski active free PSA and any other native proteolytic enzymes present in the surrounding tissue, releasing, thereby unmodified cytotoxic agent in the internal environment of the body in place proteolytic cleavage.

It should be noted that the Oligopeptide of the present invention conjugated with a cytotoxic agent, regardless of whether it is covalently associated with the agent directly or through a chemical linker does not need to be an Oligopeptide, most recognizable and most easily cleaved free PSA. For example, the choice of oligopeptides for inclusion in protivorahiticheskimi agent and the cytotoxic activity of the cytotoxic agent conjugate and the proteolytic balance (or, in the ideal case, unmodified cytotoxic agent), which is the consequence of this cleavage.

Since the conjugates of the present invention can be used for modifying a given biological response, a cytotoxic agent should not be restricted to the classical types of chemical therapeutic agents. For example, the cytotoxic agent can be a protein or polypeptide possessing a desired biological activity. Such proteins can be, for example, a toxin such as abrin, ricin a, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor tissue, platelet-derived growth factor, tissue plasminogen activator; or biological response modifiers, such as lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), factor, stimulating the formation of colonies of granulocytes and macrophages (GM-CSF), factor colony-granulocytes (G-CSF) or other growth factors.

Preferred cytotoxic agents are, basically, alkylating agents, antiproliferative agents, tubulin binding agents, etc., Preferred kewannee means, related to Vinca compounds; mitomycin; bleomycin; cytotoxic nucleosides; family peridynamic medicines; deeney and podophyllotoxin. Particularly preferred compounds of the above classes are, for example, doxorubicin, karminomitsin, daunorubicin, aminopterin, methotrexate, methopterin, dichloromethotrexate, mitomycin C, porfiromycin, 5-fluorouracil inside the body, 6-mercaptopurine, citizenoriented, podophyllotoxin or its derivatives such as etoposide or etoposide; melphalan, vinblastine, vincristine, leirosidin, vindesine, Larsen, etc. Other suitable cytotoxic agents are estramustin, cisplatin and cyclophosphamide. While every person is able to implement appropriate chemical modification with the formation of the desired compounds, which is more suitable for carrying out reactions in order to obtain conjugates of the present invention.

Especially preferred group of cytotoxic agents of the present invention is a medicinal product having the following formula:

A group of metotrexato formulas (1)

< / BR>
where R12is an amino - or hydroxy-group;

R7is with whom is a hydroxy-group, or a group forming a salt of carboxylic acid.

Group mitomicina formula (2)

< / BR>
where R10represents hydrogen or methyl.

Group bleomycin formula (3)

< / BR>
where R11represents a hydroxy-group, the amino group;

C1-C3-alkylamino, di(C1-C3alkyl)amino group, a C4-C6-polietileniminov;

< / BR>
Melphalan formula (4)

< / BR>
6-Mercaptopurine formula (5)

< / BR>
Citizenries formula (6)

< / BR>
Podophyllotoxin formula (7)

< / BR>
where R13represents hydrogen or methyl;

R14represents methyl or thienyl; or phosphate salt.

Medicines group vinylchloride formula (8)

< / BR>
where R15represents H, CH3or OHO, when

R17and R18taken separately;

R18represents H, and one of R16I and R17is ethyl and the other is H or OH; and in the case when R17and R18taken together with the carbon atoms to which they are linked, they form oxirane ring, where R16is ethyl;

R19represents hydrogen, (C1/BR>< / BR>
where R21represents a base, having one of the formulas:

< / BR>
< / BR>
< / BR>
< / BR>
< / BR>
where, R22represents hydrogen, methyl, bromine, fluorine, chlorine or iodine

R23represents-OH or-NH2;

R24represents hydrogen, bromine, chlorine or iodine; or

Anthracycline antibiotics of the formula (10)

< / BR>
where R1represents-CH3CH2OH, -CH2OCO(CH2)3CH3or-CH2OCOCH(OC2H5)2;

R3represents-OCH3, -OH or H;

R4represents-NH2, -NHCOCF3, 4-morpholinyl, 3-cyano-4-morpholinyl, 1-piperidinyl, 4-methoxy-1-piperidinyl, benzylamine, dibenzylamine, cyanomethylation or 1-cyano-2-methoxyethylamine;

R5represents-OH, -OTHP or-H and

R6represents-OH or H, provided that:

R6is not-OH, if R5= -OH or-OTHP.

Estramustin (11)

< / BR>
Cyclophosphamide (12)

< / BR>
The most preferred drugs are anthracycline antibiotics of the formula (10) described previously. It should be noted that the above structural formulas describe compounds that are leasve. Table 1 presents some of anthracycline drugs (and their generic or trivial names) that are particularly preferred for use in the present invention.

Of the compounds represented in table 1, the most preferred drug is doxorubicin. Doxorubicin (also denoted "DOX") is anthracyclin formula (10), where R1is-CH2OH, R3is-OCH3, R4is-NH2, R5is-OH, a R6is-H.

Oligopeptides, peptide subunit and derived peptides (also called peptides) of the present invention can be synthesized from their constituent amino acids using standard peptide synthesis, preferably by the method of solid-phase synthesis. Then the peptides are purified using reverse-phase high-performance liquid chromatography (HPLC).

Standard methods of peptide synthesis are described, for example, in the following activities: Schroeder et al., "The Peptides", Vol. 1, Academic Press 1965; Bodansky et al., "Peptide Synthesis", Interscience Publishers, 1966; McOmie (ed. ) "Protective Groups in Organic Chemystry", Plemim Press, 1973; Barany et al. "The Peptides: Analysis, Synthesis, Biology" 2, Chapter 1, Academic Press, and Stewart et al., "Solid Phase Peptide "ptx2">

The conjugates of the present invention containing Oligopeptide having the PSA website-splitting and cytotoxic agent may be similarly synthesized by methods well known to the specialists in clinical biochemistry. For example, the free amine part in the cytotoxic agent can be covalently attached to the Oligopeptide at the carboxyl end, resulting in the formation of amide. Similarly, the amide bond can be formed by covalent binding of the amino group of oligopeptides with a carboxyl group of the cytotoxic agent. For these purposes may be used such reagent, as a combination of hexaflurophosphate 2-(1H-benzotriazol-1-yl)-1,3 .3m - tetramethylurea (known as HBTU) and hydrate of 1-hydroxybenzotriazole (known as HOBT), dicyclohexylcarbodiimide (DCC), N-ethyl-N-(3-dimethylaminopropyl)- carbodiimide (EDC), diphenylphosphinite (DPPA), hexafluorophosphate benzotriazol-1-yl-oxy-Tris (dimethylamino)phosphonium (BOP), etc.

In addition, the conjugate of the present invention can be formed by dipeptidyl communication between the site of cleavage of PSA and a cytotoxic agent. For example, the cytotoxic agent can be covalently attached those which formed ester bond. For these purposes may be used such agent, as the combination of HBTU and HOBT, the combination of a THIEF and imidazole, the combination of DCC and DMAP, etc., Carboxylic acid may be activated by education nitrophenylamino ether or etc., and subjected to reaction in the presence of DBU (1,8-diazabicyclo 5,4,0 undec-7-ene).

The conjugate of the present invention can also be formed by joining of oligopeptides to the cytotoxic agent via a linker unit. Such linker units can contain, for example, biharmonicity biradical through which the amine part of the cytotoxic agent is combined with the linker unit, resulting in an amide bond, and aminocore of oligopeptides connect with the other end of the linker unit, resulting also formed amide bond. Can also be used for other linker units, which are stable in physiological environment in the absence of free PSA, but which can be broken down after hydrolysis into the corresponding site of proteolytic cleavage under the action of the PSA. Additionally, there may be used such linker units, which after PSA-hydrolysis in the site of proteolytic cleavage of the Ghent, formed after cleavage, is not significantly reduced compared to the unmodified cytotoxic agent.

It is obvious that in the process of synthesis of compounds of the present invention, it may be necessary to protect or block various reactive functional groups present in the source compounds or in the intermediate compounds, and in this case, the desired reaction is carried out on other parts of the molecule. After completion of the desired reactions or upon expiration of the appropriate period of time such groups are usually removed, for example, by hydrolysis or hydrogenolysis. These under lock and release are the traditional procedures of organic chemistry. For selection of suitable protective groups, which can be used to obtain compounds of the present invention, the specialists can refer to the works of Protective Groups in Organic Chemistry, McOmie, ed. Plenum Press, NY (1973); and Protective Groups In Organic Synthesis, Green, ed., John Wiley & Sons, NY, NY (1981).

Examples of suitable aminosidine groups can serve as a C1-C10alcoholnye groups, such as formyl, acetyl, dichloroacetyl, propionyl, hexanoyl, 3,3-diethylhexyl, -chloro-botril etc.; C1-C10alkoxybenzyl, allyloxycarbonyl, 4-nitrobenzenesulfonyl, fluorenylmethoxycarbonyl and cinnamoylcocaine; halogen-(C1-C10)-alkoxycarbonyl group, such as 2,2,2-trichlorocyanuric; and (C1-C15arylalkyl and alkeneamine groups such as benzyl, phenethyl, allyl, trityl, etc. Other commonly used aminosidine groups are groups in the form of enamines derived by using the-ketoesters such as methyl - or ethylacetoacetate.

Suitable carboxyamide groups can be, for example, C1-C10alkyl groups such as methyl, tert-butyl, decyl; halogen-(C1-C10alkyl groups such as 2,2,2-trichloroethyl and 2-Iodate;5-C15arylalkyl groups such as benzyl, 4-methoxybenzyl, 4-nitrobenzyl, triphenylmethyl, diphenylmethyl; C1-C10alkanolamine groups, such as acetoxymethyl, propionoxy and so on ; and such groups as phenacyl, 4-halogenfrei, allyl, dimethylallyl, three-(C1-C3alkyl), silyl, for example, trimethylsilyl, -p-toluensulfonate, -p-nitrophenyl-thioethyl, 2,4,6-trimethylbenzyl-methylthioethyl, phthalimidomethyl, 2,4-dinitro-phenylsulfonyl, 2-nitrobenzamide and related groups.

group, benzyl group, benzydamine group, triticina group, 4-nitrobenzyl group, trimethylsilyl group, penicilina group, tert-bucilina group, methoxymethyl group, tetrahydropyranyl group, etc.

The preferred option of oligopeptides, conjugated with an anthracycline antibiotic with doxorubicinol summarized in the following Reaction schemes, illustrated by the synthesis of conjugates of the present invention.

Reaction scheme VI illustrates obtaining conjugates of the oligopeptides of the present invention with vinyltoluene cytotoxic agent vinblastine. Shows the connection of the N-Terminus of oligopeptides with vinblastine (S. P. Kandukuri et al., J. Med. Chem. 28: 1079-1088 (1985)). However, it can be expected that conjugation of oligopeptides in other provisions and functional groups of vinblastine and the C-Terminus of oligopeptides will also lead to the formation of compounds that can be used to treat prostate cancer.

In addition, it should be noted that there may be also obtained conjugates, where the N-Terminus of oligopeptides of the present invention covalently linked to one cytotoxic agent such as vinblastine, and C is the end of this oligopo the cytotoxic agent, such as doxorubicin. This poliziotteschi conjugate may have greater benefits than the conjugate containing only one cytotoxic agent.

Conjugate "Oligopeptide - cytotoxic agent of the present invention, in which the cytotoxic agent is the preferred cytotoxic agent doxorubicin can be represented by the General formula:

< / BR>
where "Oligopeptide" is an Oligopeptide which is specifically recognized by the free prostate antigen (PSA), which can be proteoliticeski split under the action of the enzymatic activity of the free prostate specific antigen;

XLis absent or is an amino acid chosen from:

(a) phenylalanine,

b) leucine,

(C) valine,

d) isoleucine,

(e) (2-naphthyl)alanine,

f) diphenylalanine,

g) Norvaline and

h) norleucine;

R represents hydrogen or -(C=O)R1;

R1represents a C1-C6is alkyl or aryl.

In a preferred embodiment, the conjugate "Oligopeptide-cytotoxic agent":

"Oligopeptide" is an oligomer, which has the amino acid sequence (SEQ ID No. 15),

d) GlyLysGlyIleSerSerGlnTyr SerAsnThrGluGluArgLeu (SEQ ID No. 2),

e) AsnLysIleSerTyrTyr Ser (SEQ ID No. 127),

f) AsnLysAlaSerTyrGln Ser (SEQ ID N 128),

g) SerTyrGln SerSer (SEQ ID N 129),

h) hArgTyrGln SerSer (SEQ ID No. 141);

where Xaa is any natural amino acid;

X1is absent or is an amino acid chosen from:

a) leucine,

b) isoleucine,

C) norleucine and

d) valine; and

R represents acetyl, pivaloyl or benzoyl.

Specific examples of conjugate "Oligopeptide - cytotoxic agent of the present invention are the following compounds.

< / BR>
where X is a:

< / BR>
< / BR>
< / BR>
< / BR>
< / BR>
< / BR>
< / BR>
< / BR>
< / BR>
< / BR>
< / BR>
< / BR>
< / BR>
< / BR>
< / BR>
< / BR>
As is well known in the art, and as is evident from the present invention, peptidase therapeutic agents, such as conjugates "Oligopeptide-cytotoxic agent of the present invention preferably have a terminal amino group of any Oligopeptide Deputy, which are sealed by an appropriate protective group such as acetyl, benzoyl, pivaloyl, etc., Such protection terminal amino groups reduces or eliminates the enzymatic degradation of these peptidergic therapeutic is 2">

In accordance with the present invention, conjugates of oligopeptides and a cytotoxic agent is administered to the patient in the form of a pharmaceutical composition comprising the conjugate of formula (1) and a pharmaceutically acceptable carrier, excipient or diluent. Used in the present description, the term "pharmaceutically acceptable" refers to those agents that can be used in the treatment or diagnosis of warm-blooded animals such as humans, horses, pigs, cows, mice, dogs, cats or other mammals, and birds or other warm-blooded animals. This is preferred parenteral route of administration, particularly intravenous, intramuscular, subcutaneous, intraperitoneal or WinUtilities introduction. These drugs can be obtained using carriers, diluents or fillers, which are usually used for this purpose. In this regard, see , for example. Remington''s Pharmaceutical Sciences, 16th ed., 1980, Mack Publishing Company, edited by Osol et al. These compositions may include proteins, such as serum proteins, such as albumin human serum; buffers or tabularasa compounds such as phosphates, other salts or electrolytes, etc., Suitable solvents are the alcohols such, for example, glycerol, propylene glycol, polyethylene glycol, etc., These compositions can contain preservatives, such as finitely alcohol, methyl - and propylparaben, thimerosal, etc., If necessary, the composition may be included from about 0.05 to about 0.20, the wt.% antioxidant, such as metasulfite sodium or sodium bisulfite.

Composition for intravenous preferably made so that the number of input patient conjugate was from about 0.01 to about 1, the Preferred amount of the conjugate is in the range from about 0.2 to about 1, the Conjugates of the present invention can be effective in a wide range of doses, depending on such factors as the severity of the disease, which is directed treatment, modifiable biological effect, which is desirable to achieve a method of administration of the conjugate, the age, weight and condition of the patient, and other factors that should be taken into account by the treating physician. Thus, the number of conjugate required for the introduction of a given patient must be determined individually for each patient.

It should be noted that although the examples below are described konkretniot and their various modifications. Therefore, the following procedures and examples are merely illustrative and should not be construed as a limitation of the invention.

EXAMPLES

Example 1

Identification of the site PSA-mediated cleavage semenovaelena

The liquefaction of the seminal gel with proteolytic fragmentation semenovaelena (Lilja, N., Laurell, C. B., (1984) Scand. J. Clin. Clab. Invest. 44, 447-452). I believe that proteolytic fragmentation caused mainly by the activity of prostate specific antigen (Lilja, N., (1985) J. Clin. Invest. 76, 1899-1903). Using published sequence semenovaelena I (Lilja, N., Abrahamsson, P. A., Lundwall, A., (1989)). Biol. Chem. 264, 1894-1900) (Fig. 1), we designed PCR primers (PCR - polymerase chain reaction) cloning of cDNA semenovaelena from commercially available cDNA library that encodes the proteins of the prostate (Clonetech, Palo Alto, CA.). Purified cDNA semenovaelena embedded in the bacterial expressing vector pTAC (Linemeyer, D. L., Kelli, I. J., Minke, J. G. , Gimenez-Gallego, G., DeSalvo, J. & Thomas, K. A., (1987) Bio/ Technology 5, 960-965). cDNA semenovaelena designed in such a way that the tubulin epitope was located at the carboxyl end of the protein semenovaelena. Simanagaon, expressed using a bacterial vector was purified on chotoviny industrial way (York Biologicals International, Stony Brook, NY)), in a molar ratio of 100/1 (simanagaon 1/PSA) in 12 mm Tris, pH 8.0, 25 mm NaCl, 0.5 mm CaCl2and then were incubated for various periods of time. The hydrolysate was fractionally using polyacrylamide gel electrophoresis and transferred by electrophoresis on filter paper ProBlott (Applied Biosystems, Inc., Foster City, CA.) in CAPs buffer (Matsudaira, P. , (1987) J. Biol. Chem. 252, 10035-10038). Filter paper ProBlott were stained with Kumasi blue to identify new PSA-produced fragments semenovaelena I. New fragments cut out from the filter with a scalpel and determined their sequences. After identification of proteolytic fragments by hydrolysis in different periods of time were in the hydrolysis reaction for 10 minutes. Then determined the relative affinity of PSA for 5 potential cleavage sites in the sequence semenovaelena I, namely: site 349/350 > website 375/376 > website 289/290 > website 315/316 > website 159/160. The relative affinity were obtained based on the intensity of the color Kumasi blue each PSA-produced peptide fragment. These rates had an approximate ratio of 3:1:0,6:0,3.

Example 2

Getting oligopeptides containing the PSA website-mediated Rasse the Birmingham dual connection for automatic peptide synthesizer, Applied Biosystems model 430A. Release and separation of oligopeptides from the polymer carrier was carried out by treatment with liquid hydrofluoric acid. Oligopeptides was purified using preparative liquid chromatography high-pressure treatment-phase column with silica C18 using a gradient of aqueous 0.1% of triperoxonane acid/acetonitrile. The identity and homogeneity of the oligopeptides were confirmed through the analysis of amino acids in liquid chromatography high pressure and mass spectrometric analysis by fast atom bombardment. Oligopeptides obtained by this method is shown in Fig. 2.

Example 3

Assessment recognition of oligopeptides free PSA

Oligopeptides obtained as described in example 2 were separately dissolved in buffer for PSA-hydrolysis (12 mm Tris (hydroxymethyl)aminomethane, pH 8.0. 25 mm NaCl, 0.5 mm CaCl2) and the resulting solution was added to PSA at a molar ratio of 100:1. The reaction was stopped after different periods of time by adding triperoxonane acid (TFA) to a final volume of 1% (volume/volume). Quenched reactions were analyzed using jhud on reversed-phase C18 column using a gradient of 0.1% TFA/acetone is analyzed in the same way, except that the reaction was suppressed after 4 hours. The evaluation results are shown in Fig. 3. Remove aspartic residue from the amino-Terminus of oligopeptides leads to a significant weakening of the PSA-mediated hydrolysis of the peptide and the presence of the glutamic acid residue at the carboxyl end of the peptide, obviously, does not play a decisive role in the detection of prostatic antigen.

Example 4

Getting darassalam conjugates of oligopeptides and doxorubicin

Derivatives of doxorubicin, shown in table 2, were obtained in accordance with the following General reaction:

To a mixture of doxorubicin (Sigma) and the corresponding peptide (obtained by solid-phase synthesis or purchased ready-made (Sigma) in DMSO was added HBTS and HOBT together with diisopropylethylamine and the reaction mixture stirred over night. Then the crude reaction mixture was purified using preparative HPLC on reversed-phase column (C-18, using a gradient of 0.1% triperoxonane acid (TFA) in acetonitrile/ 0.1% of TFA in water.

Example 5

In vitro analysis of cytotoxicity peptidergic derivatives of doxorubicin

Cytotoxicity darassalam conjugates of oligopeptides and doxorubicin (half the m unmodified doxorubicin, evaluated by analysis using almurabaha blue. For this cell culture tumor cells of the prostate LNCaP, which represent metastatic cells adenocarcinoma prostate cancer selected from the puncture biopsy of lymph node (LNCaP. FGC: American type culture collection, ATSS CRL 1740), or cells DuPRO in 96-well tablets were diluted with medium containing various concentrations of the given conjugate (final volume of the wells is 200 ál). Then cells were incubated for 3 days at 37oC, after which the well was added 20 μl almurabaha blue. After that, cells are again incubated and analytical tablets were read on an ELISA reader EI-310 at two wavelengths 570 and 600 nm after 4 and 7 hours after adding almurabaha blue. Then calculate the relative percentage of cell viability at various concentrations of the tested conjugates compared to control cultures (in the absence of conjugate). Evaluated the cytotoxicity of unmodified doxorubicin and unmodified oligopeptides. In Fig. 3 presents data cytotoxicity of typical compounds (Compound 12d).

Example 6

OC is aktivnosti was demonstrated by incubation with conditioned LNCaP cells serum-free medium (approximately 200-fold concentrated) with recombinant protein semenovaelena I. Then approximately 0.5 μg of immunoreactive PSA in concentrated conditioned medium (determined using ELISA HYBPIDTECH (Tandem E)) was mixed with approximately 3 μg of recombinant semenovaelena I and incubated for 4 hours at 37oC. after incubation gidralizovanny mixture was analyzed using Western blot analysis. The results showed that the purified PSA from the sperm and PSA from LNCaP-air-conditioned environment gave identical proteolytic card recombinant protein semenovaelena I. Thus LNCaP cells produce enzymatically active PSA. The LNCaP cells are oncogenic in naked (Nude) mice and produce detected levels of circulating PSA.

Example 7

Obtaining fissionable conjugates of oligopeptides-doxorubicin

Derivatives of doxorubicin in which the Oligopeptide, proteoliticeski cleaved free PSA, covalently linked to the amine of the carbohydrate part of doxorubicin were obtained in accordance with the following General reaction: to a mixture of doxorubicin (Sigma) and the corresponding peptide (obtained by solid-phase synthesis as described in example 2) in DMSO was added HBTS and HOBT together with diisopropylethylamine and the reaction mixture re the new column C-18, using a gradient of 0.1% triperoxonane acid (TFA) in acetonitrile /0.1% of TFA in water. If the peptide was present reactive amino groups, these functional groups are usually blocked fluorenylmethoxycarbonyloxy protective group, which was then removed by treatment with a secondary amine such as piperidine, etc., and then conjugatively with doxorubicin. The conjugates of the present invention have the structure of General formula:

< / BR>
and can be labeled as "AC-peptide-DOX (3')". The conjugates obtained in this way is shown in Fig. 5 (the table)/

Example 8

Assessment recognition conjugates "Oligopeptide-doxorubicin" free PSA

The conjugates obtained as described in example 7, was separately dissolved in the buffer for PSA-hydrolysis (12 mm Tris(hydroxymethyl)aminomethane, pH 8.0; 0.25 mm NaCl, 0.5 mm CaCl2), and the resulting solution was added to PSA at a molar ratio of 100: 1. The reaction is extinguished through different periods of time by adding triperoxonane acid (TFA) to a final concentration of 1% (volume/volume). After quenching the reaction, the reaction mixture was analyzed by HPLC on a reversed-phase column (C-18 using water gradient with 0.1% TFA/acetonitrile. The results of the assessment shall bicin in conditioned cell medium

Air-conditioned cells of serum-free MEM medium (containing no phenol red) were collected 3 days after they are added to either cell line LNCaP or cell lines Dupro (obtained as described in J. of Urology, 146: 915-919 (1991)). These environments 20-fold concentrated using an Amicon concentratorwith a molecular weight cutoff of 10,000. LNCaP-conditioned medium containing free PSA is a protein in an average concentration of approximately 100 ng/ml, as determined using the set of immunodetection Tandem-E PSA (Hybritech) . This air-conditioned cells Dupro environment of free prostate specific antigen was not detected.

100 μl - portion of the concentrated conditioned medium was mixed with 35 μg of the conjugate of oligopeptides-doxorubicin, obtained as described in example 7 and the mixture is incubated at 37oC for time periods of 0, 4 and 24 hours. Then the reaction was stopped by addition of ZnCl2(to final concentration 0.01 M) and analyzed by HPLC on a reversed-phase column with CD18 and using a gradient of water to 1% TFA/acetonitrile in order to determine the percentage of hydrolyzed peptide conjugate is a cytotoxic agent. tigrovyh derivatives of doxorubicin

Cytotoxicity split conjugates of oligopeptides-doxorubicin (obtained as described in example 7) against cell line, which are known to be susceptible to cytolysis under the action of unmodified doxorubicin, was evaluated by means of analysis using almurabaha blue as described in example 5. For this cell culture tumor cells of the prostate LNCaP or cells Dupro in 96-well tablets were diluted with medium containing various concentrations of the given conjugate (final volume in the wells was 200 μl). Then cells were incubated for 3 days at 37oC, and analytical well was added 20 μl almurabaha blue. After that, cells are again incubated and analytical tablets were read on an ELISA reader EL-310 at two wavelengths: 570 and 600 nm after 4 and 7 hours after adding almurabaha blue. Then calculate the relative percentage of cell viability at various concentrations of the tested conjugates compared to control cultures (in the absence of conjugate). In this same analysis also evaluated the cytotoxicity of the conjugates compared to the cytotoxicity of unmodified doxorubicin and unmodified oligopeptides. Raza is, the which is recognized and proteoliticeski selectively cleaved free specific antigen prostate enzymatically active form, not forming a complex with any inhibitory molecule and which contains a relationship Gln-Ser, or the relationship Tyr-Gln, or the relationship between homologous them, isostere and/or isoelectronic amino acids, split specific antigen prostate.

2. Oligopeptide under item 1, containing from 6 to 12 amino acids.

3. Oligopeptide under item 1, having the amino acid sequence shown in the graphics part,

where hArg is homoarginine;

XAA is any natural amino acid.

4. Oligopeptide under item 3, having the amino acid sequence shown in the graphics part.

5. Oligopeptide under item 3, having the amino acid sequence shown in the graphics part.

6. Oligopeptide under item 3, having the amino acid sequence shown in the graphics part.

7. Oligopeptide under item 1, which is selected from (see graphic part).

8. Oligopeptide under item 1, which is (see graphic part).

9. The method of determining proteolytic is: (a) the reaction of the substrate, where the substrate is an Oligopeptide containing from 6 to 100 amino acids that is recognized and proteoliticeski selectively cleaved free specific antigen prostate enzymatically active form, not forming a complex with any inhibitory molecule and which contains a relationship Gln-Ser, or the relationship Tyr-Ser, or the relationship between homologous them, isostere and/or isoelectronic amino acids, split specific antigen prostate; and (b) the registration of cleavage of the substrate.

10. The method according to p. 9, wherein the Oligopeptide contains from 6 to 12 amino acids.

11. The method according to p. 9, in which the stage of registration of the cleavage of the substrate includes analysis of the reaction mixture using liquid chromatography high resolution.

12. A method for identifying compounds that inhibit the proteolytic activity of a specific antigen of the prostate, comprising the following stages: (a) the reaction of the substrate, where the substrate is an Oligopeptide containing from 6 to 100 amino acids that is recognized and proteoliticeski selectively cleaved free specific antigen prostate enzymatically active form, not forming comological them, isostere and/or isoelectronic amino acids, split specific antigen prostate, with free specific antigen prostate in the presence of the test compound; and (b) the registration of cleavage of the substrate by which the ability of the test compound to inhibit the proteolytic activity specific antigen prostate enzymatically active form, not forming a complex with any inhibitory molecule, indicates the decline of cleavage of the substrate compared to the cleavage of the substrate in the absence of the test compound.

13. The method according to p. 12, wherein the Oligopeptide contains from 6 to 12 amino acids.

14. The method according to p. 12, at which stage the Desk cleavage of the substrate provides for the analysis of analytical mixtures using liquid chromatography high resolution.

15. Conjugate, which can be used to treat prostate cancer and which contains a cytotoxic agent associated with the Oligopeptide containing from 6 to 100 amino acids that is recognized and proteoliticeski selectively cleaved free specific antigen prostate enzymatically active form, not forming comological them, isostere and/or isoelectronic amino acids, split specific antigen prostate, and indicated the cytotoxic agent is associated with the specified Oligopeptide via a covalent bond or a chemical linker, and a cytotoxic agent is selected from doxorubicin and vinblastine or cytotoxic derivatives.

16. Conjugate under item 15, in which the Oligopeptide contains from 6 to 12 amino acids.

17. Conjugate under item 15, in which the cytotoxic agent is doxorubicin or its cytotoxic derivative.

18. Conjugate under item 17 having a formula I

< / BR>
in which "Oligopeptide" is an Oligopeptide containing from 6 to 100 amino acids that is recognized and proteoliticeski selectively cleaved free specific antigen prostate enzymatically active form, not forming a complex with any inhibitory molecule and which contains a relationship Gln-Ser, or the relationship Tyr-Ser, or the relationship between homologous them, isostere and/or isoelectronic amino acids, split specific antigen prostate;

XLis absent or is an amino acid selected from the following amino acids: (a) phenylalanine, (b) leucine,and;

R represents hydrogen or -(C=O)R1;

R1represents a C1- C6is alkyl or aryl.

19. Conjugate under item 18, in which the Oligopeptide contains from 6 to 12 amino acids.

20. Conjugate under item 18, in which the Oligopeptide is an oligomer containing an amino acid sequence selected from (see the graphical part),

where hArg is homoarginine;

XAA is any natural amino acid;

XLis absent or is an amino acid selected from: (a) leucine, (b) isoleucine and valine;

R represents acetyl, pivaloyl or benzoyl.

21. Conjugate under item 17, which is selected from

< / BR>
where X represents (see graphic part).

22. Conjugate under item 15, having the formula II

< / BR>
in which "Oligopeptide" is an Oligopeptide containing from 6 to 100 amino acids that is recognized and proteoliticeski selectively cleaved free specific antigen prostate and which contains a relationship Gln-Ser, or the relationship Tyr-Ser, or the relationship between homologous them, isostere and/or isoelectronic amino acids, split specific antigen prostate.

BR> 28.06.1994 on PP.6, 7, 11, 14 and 17;

15.03.1995 on PP.1, 2, 5, 8, 9, 10, 12, 13, 15, 16, 18 and 19;

07.06.1995 on PP.3, 4, 20, 21 and 22.

 

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