Antagonistic analogues releasing hormone growth hormone (gh-rh), inhibiting insulin-like growth factors (igf-i and-ii)

 

The invention relates to a peptide selected from the group having the formula: X-R1-R2-Asp-Ala-R5-R6-Thr-R8-R9-R10-Arg-R12-R13-R14-R15-R16-Leu-R18-R19-Arg-R21-R22-Leu-Gln-Asp-Ile-R27-R28-R29-NH2where X means PhAc, IndAc or Nac, R1means Tyr or His, R2mean D-Arg, R5means Ile or Val, R6means Phe or Phe(Cl), R8means Asn, Gln, Ala or D-Asn, R9means Arg, Har, Lys, Orn, D-Arg, D-Har, D-Lys, D-Orn, Cit, Nle, Tyr(Me), Ser, Ala or Aib, R10means Tyr or Tyr(Me), R12means Lys, R13mean Val or Nle, R14means Leu or Nle, R15means Gly, Ala, Abu, Nle, or Gln, R16means Gln or Arg, R18means Ser or Nle, R19means Ala, R21means Lys, R22means Leu, Ala or Aib, R27means Met, Leu, Nle, Abu, or D-Arg, R28means Arg, D-Arg or Ser, R29means Arg, D-Arg, Har or D-Har, provided that when9and R28mean Ser, R29is Arg or Har, and its pharmaceutically acceptable salts. Compounds according to the invention are antagonists releasing hormone growth hormone and have a high affinity for the receptor. There is also described a method of suppressing excessive levels of the hormone and 7 C.p. f-crystals, 9 tab., 5 Il.

This invention was made with partial government support of Medical Research Service Department of veterans Affairs. The government has certain rights in this application.

The present invention relates to new synthetic peptides that inhibit the release of growth hormone from the pituitary in mammals, and to therapeutic compositions containing these peptides.

Background of invention

Growth hormone (GH) is a peptide with 191 amino acids, which stimulates the production of many different growth factors such as insulin-like growth factor I (IGF-I), and, thus, stimulates the growth of many tissues (skeleton, connective tissue, muscles and internal organs) and physiological activity (increase in the synthesis of nucleic acids and proteins and lipolysis and decrease the secretion of urea).

The release of GH is controlled by releasing and inhibiting factors secreted by the hypothalamus. The main releasing factor is a hormone-releasing hormone growth hormone (GH-RH); releasing hormone human growth hormone (hGH-RH) is a peptide, the residues from 1 to 29 (hGH-RH(1-29)NH2), i.e. analogs of a peptide having the amino acid sequence of:

Tyr-Ala-Asp-Ala-Ile5-Phe-Thr-Asn-Ser-Tyr10-Arg-Lys-Val-Leu-Gly15-

Gln-Leu-Ser-Ala-Arg20-Lys-Leu-Leu-Gln-Asp25-Ile-Met-Ser-Arg29-NH2

GH involved in some diseases. One of the diseases, which involved GH is acromegaly, in which there are excessive levels of GH. Pathologically exaggerated facial bones and the bones of the limbs in this disease can be treated by introducing an antagonist of GH-RH.

Other diseases involving GH are diabetic retinopathy and diabetic nephropathy. I believe that, accordingly, damage to the retina and kidneys in such diseases leading to blindness or decreased renal function, associated with GH. Such damage may be prevented or slowed down with the introduction of effective antagonist of GH-RH.

However, the main application of antagonists of GH-RH is assumed in the field of malignant tumors (A. V. Schally et al. in Growth Hormone Secretagogues in Clinical Practice, eds. Bercu, B. B. & Walker, R. F., Dekker, New York, pp. 145-162, 1998). IGF-I and-II are potent mitogens for a variety of malignant tumors. Suppressing the secretion of GH antagonists GH-RH reduce CIN/or IGF-II in various tumors. Under certain experimental malignant tumors treatment antagonists of GH-RH leads to a decrease in IGF-I and-II, concomitant inhibition of tumor growth.

When you try to influence the development of these diseases and other conditions, some researchers have attempted to control the levels of GH, using somatostatin, one of the inhibitors of the release of GH. However, somatostatin, entered separately, does not suppress the levels of GH or IGF-I to the desired degree. When introduced in combination with an antagonist of GH-RH, somatostatin would be much improved suppression of levels of IGF-I.

We investigated various modifications of GH-RH to determine communication patterns of GH-RH with its activity in an attempt to provide a synthetic related compounds with improved agonistic or antagonistic properties. So, earlier it was found that a fragment of GH-RH, comprising residues 1-29, or GH-RH (1-29), is the minimum sequence required for biological activity. This fragment retains 50% or more activity of natural GH-RH.

It was found that the first described antagonist of GH-RH [AU-Tight1D-Arg2]hGH-RH (1-29) NH2that is usually called in the literature "standard antagonist, prevents active GH-RH on its receptors in the pituitary and hypothalamus and inhibit the pulsating secretion of growth hormone.

In a significant number of patents and articles in the open literature describes analogs of GH-RH, which act either as agonists GH-RH (i.e., act as stimulators of GH release) or as antagonists of GH-RH (i.e., act as inhibitors of GH release). Most of these peptides derived from peptide sequences of GH-RH(1-29) with specific structural modifications, which are responsible for their increased agonistic or antagonistic properties.

Thus, U.S. patent No. 4659693 reveals antagonistic analogs of GH-RH, which contain certain N,N'-dialkyl--guanidino-aminoaniline residues in position 2 of the sequence GH-RH(1-29).

Published international patent application WO 91/16923 discusses early attempts to change the secondary structure of hGH-RH by modification of its amino acid sequence. Such taken earlier attempts include the replacement of Tight1Ala2Asp3or Asn8their D-isomers; the replacement of Asn8on L - or D-Ser, D-Arg, Asn, Thr, Gln or D-Lys; substitution of Ser9on Ala to increase amphiphiles plot and replacement of Gly15on Ala or Aib. When R2in the analogues is D-Arg, and R8, R9and R15for the waters are suitable for administration as pharmaceutical compositions for treating conditions associated with excessive levels of GH, such as acromegaly.

Antagonistic activity of an analog of hGH-RH [Ser9-[CH2-NH]-Tight10]hGH-RH(1-29) for U.S. patent No. 5084555 was defined as pseudopeptides communication (i.e., peptide bond, restored to [CH2-NH]-communication) between residues R9and R10. However, it was indicated that the antagonistic properties [Sr9-[CH2-NH]-Tight10] hGH-RH(1-29) were lower than the standard antagonist, [N-Ac-Tyr1D-Arg2]GH-RH(1-29)-NH2.

U.S. patent No. 5550212 and application for U.S. patent 08/642472 representing the same objects as the present application, describe the analogs of hGH-RH(1-29)NH2characterized by elevated antagonistic properties and prolonged time of action. It is believed that such properties are the result of the substitution of various amino acids and acylation of aromatic or non-polar acids at the N end of GH-RH(1-29)NH2. It should be noted that in the above U.S. patent and patent application U.S. R9always is Ser, R16is Gln or amino acid, forming a lactam bridge (i.e., Glu), R28is Ser, Asn, Asp, Ala or Abu and R29means Agm,and Orn).

Brief description of the invention

Presents a new series of synthetic analogs of hGH-RH(1-29)NH2. These analogs inhibit the activity of endogenous hGH-RH and thereby prevent the release of growth hormone. Stronger inhibiting effects of new analogues compared with the previously described are the result of substituting a different amino acid.

Specifically, the invention relates to peptides that are encompassed by the formula:

X-R1-R2-s-la-R5-R6-hr-R8-R9-R10-AGD-R12-R13-R14-R15-R16-Leu-R18-R19-Arg-R2l-R22-Leu-Gln-Asp-Ile-R27-R28-R29-NH2,

where X means PhAc, IndAc, Ibu, Nac, 1 - or 2-Npr or

Fpr,

R1means Tight or His,

R2mean D-Arg or D-Cit,

R5means Il or Val,

R6means Phe, Nal or Phe(Y), where Y=F, Cl, Br,

R8means Asn, Gln, Ser, Thr, Ala, D-Asn, D-Gln, D-Ser, D-Thr, Abu, D-Abu, or Aib,

R9means Arg, Har, Lys, Orn, D-Arg, D-Har, D-Lys, D-Orn, Cit, Nle, Tyr(Me), Ser, Ala or Aib,

R10means Tight or Phe(Y), where Y=H, F, Cl, Br or

Och3,

R12means Lys, D-Lys, or Orn,

R13mean Val or Nle,

R14means Leu or Nle,

R15means Gly, Ala, Abu, Nle, or Gln,

R16

R22means Leu, Ala or Aib,

R27means Met, Leu, Nle, Abu, or D-Arg,

R28means Arg, D-Arg, Ser, Asn, Asp, Ala or Abu,

R29means Arg, D-Arg, Naked or D-Har, and to their pharmaceutically acceptable salts.

In a preferred embodiment of the invention peptides are considered, in which X means PhAc, IndAc or Nac, R1means Tight or His, R2mean D-Arg, R5means Ile, R6means Phe(pCl), R8means Asn or Abu, R9means AGD or Naked, Lys, Orn, D-Arg, D-Har, D-Lys, D-Orn, Cit, Nle or Tight(Me), R10means Tight or Taut(Me), R12means Lys, R13mean Val or Nle, R14means Leu or Nle, R15means Abu, Ala, or Nle, R16means Gln or Arg, R18means Ser or Nle, R19means Ala or Abu, R21means Lys, R27means Nle or D-Arg, R28mean D-Arg, Arg or Ser, R29mean D-Arg, Naked or D-Har.

It is noted that amino acid residues from 30 to 44 natural molecules GH-RH, apparently, are not essential for activity; also is not critical to their identity. So it seems that the accession of some or all of these additional amino acid residues to the C-end of analogs of hGH-RH(1-29)-NH2according to the present invention will not affect the efficiency is once the analogs of hGH-RH(1-29)-NH2, the attached amino acid residues could be the same as the remains from 30 to 44 in the sequence of natural hGH-RH, or acceptable equivalents.

Synthetic methods.

Synthetic peptides receive suitable way, as, for example, exclusively solid-phase method, a partially solid-phase method, by condensation of fragments or by classical synthesis in solution.

When the analogs according to the invention are synthesized by solid-phase method, the C-terminal residue (here R29) properly associated (attached) with an inert solid carrier (resin), while at the same time, the protective group for your-amino group (and, if necessary, to a functional group of the side chain). After completing this stage protective group-amino group is removed with a fixed amino acid residue and add the following amino acid residue, R28with appropriately substitutedthe amino group (as well as any appropriate functional group of a side chain), and so on. The protective group of the N-Terminus is removed after attaching each residue, but at the same time, do not remove the eljnosti, the peptide is separated from the media and freed from all the protective groups of the side chain in conditions that are minimally destructive to residues in the sequence. Followed by a thorough cleaning and accurate identification of synthetic product to ensure that it really is obtained the desired structure.

Particularly, it is preferable to protect-amidofunctional group of amino acids during phase combination using sensitive to acid or base protective group. Such protective groups must have the property of preserving stability in the conditions of formation of peptide bonds at the same time should be easily removed without destruction of the growing peptide chain and without racemization of any of the information contained there chiral centers. Suitable protective groupsthe amino groups are BOC(tert-butoxycarbonyl) and Fmoc(9-fluorenylmethoxycarbonyl).

Application in medicine.

Which antagonists hGH-RH peptides or salts of these peptides can be prepared in the form of pharmaceutical dosage forms containing effective amounts of these compounds, and entered the human or animal therapeutic or diagnosticcriteria levels of GH, for example, diabetic retinopathy and nephropathy, and acromegaly. Also provided methods of treating these diseases by introducing the composition according to the invention to an individual in need of such treatment. The antagonists GH-RH is, however, to the field of malignant tumors, such as cancer of the breast, lung, colon, brain, pancreas and prostate man, where are the receptors for IGF-I or IGF-II.

Brief description of drawings

Fig.1 is a graphical dependence of volume change mouse pertaining to the breast malignant tumors Mat from the days of processing when processing certain antagonists GH-RH.

Fig.2 is a graphical dependence of volume changes in human malignant breast tumor MDA-MB-468 in Nude mice from days of processing when processing certain antagonists GH-RH.

Fig.3 is a graphical dependence of volume changes in human malignant tumors of the colon HT-29 in Nude mice from days of processing when processing certain antagonists GH-RH.

Fig.4 is a graphical dependence of the volume changes of the human g is aricescu dependence of volume changes in human malignant tumors of the prostate PC-3 in Nude mice from days of processing when processing certain antagonists GH-RH.

Detailed description of preferred embodiments of the invention

A. Abbreviations

The nomenclature used to define the peptides is that established by the Commissioner IUPAC-MBS (MBS international biochemical Union) on biochemical nomenclature, where in accordance with generally accepted view of the amino group at the N-end is on the left and the carboxyl group at the C-end is on the right. The term "natural amino acid" as used here, means one of the common, naturally occurring L-amino acids found in natural proteins: Gly, Ala, Val, Leu, Ile, Ser, Thr, Lys, Arg, Asp, Asn, Glu, Gln, Cys, Met, Phe, Tyr, Pro, Trp and His, When the remnant of the natural amino acids exist in isomeric forms, namely L-form amino acids are represented here, unless specifically stated otherwise.

FTA amino acids or amino acid analogues are also included in the antagonists GH-RH. ("FTA" amino acids are those amino acids that are not among about 20 natural amino acids found in natural peptides). To neodrepanis amino acids or amino acid analogs that can be used in the peptides, which are antagonists of hGH-RH chr/945.gif">-aminoadamantane acid, under Naked mean homoarginine under Nal mean 2-nafcillin under Nle mean norleucine and under the UCP mean ornithine. When these noncoding amino acids or analogs of amino acids exist in isomeric forms, namely L-form amino acids are represented here, unless specifically stated otherwise. Used here abbreviations:

Abu --aminobutyric acid;

AC is acetyl;

Asón - acetic acid;

AU2O - acetic anhydride;

Aib --aminoadamantane acid;

Vos - tert-butoxycarbonyl;

CMV - benzyloxyethyl;

2BrZ - 2-bromobenzyloxycarbonyl;

CNH - cyclohexyl;

Cit is citrulline (2-amino-5-freedomalliance acid);

2CIZ - 2-chlorobenzenesulfonyl;

DCM - dichloromethane (DHM);

DIC - N,N'-diisopropylcarbodiimide (DICD);

DIEA - diisopropylethylamine (DIEA);

DMF - dimethylformamide (DMF);

Fmoc - fluorenylmethoxycarbonyl;

Fpr - 3-phenylpropionyl;

GH - growth hormone;

GH-RH - releasing hormone growth hormone;

Naked - homoarginine;

HBTU - 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyl-Urania hexaphosphate;

hGH-RH - human GH-RH;

HOBt is 1-hydroxybenzotriazole;

HPLC - high performance fluid is);

Meon - methanol;

MeCN is acetonitrile;

Nac - 1-naphthylacetyl;

Nal - 2-nafcillin;

NMM is N-methylmorpholine;

Npr - naphtylamine;

RAM phenylacetamide;

Phe(pCl) - p-chlorophenylalanine;

PhAc - phenylacetyl;

rGH-RH - rat GH-RH;

RP-HPLC - HPLC with reversed phase;

TFA - triperoxonane acid (TFA);

Tos - p-toluensulfonyl;

Tight(Me) - simple tiresomely ether;

Z - benzyloxycarbonyl.

Century Analogs of GH-RH

Analogs of hGH-RH according to the present invention were obtained with the aim of increasing the affinity of the peptides to the receptor, increase metabolic stability and maximize amphiphiles secondary structure of molecules. Many of these analogues cause very effective and long-lasting inhibition of GH release stimulated hGH-RH(1-29)NH2in vitro and in vivo.

The following embodiments of the invention are particularly preferred as having excellent biological activity:

[PhAc0D-Arg2, Phe(pCl)6, Arg9Abu15, Nle27D-Arg28, Har29]hGH-RH(1-29)NH2peptide 1;

[IndAc0D-Arg2, Phe(pCl)6, Arg9Abu15, Nle27D-Arg28, Har29]hGH-RH(1-29)NH2peptide 2;

[PhAc0D-Arg2, Phe(pCl)6Naked2, Phe(pCl)6, Har9Abu15, Nle27D-Arg28, Har29]hGH-RH(1-29)NH2peptide 4;

[Nac0D-Arg3, Phe(pCl)6, Arg9Abu15, Nle27D-Arg28, Har29]hGH-RH(1-29)NH2peptide 5;

[PhAc0D-Arg2, Phe(pCl)6, Arg9, Tyr(Me)10Abu15, Nle27D-Arg28, Har29]hGH-RH(1-29)NH2peptide 6;

[PhAc0, His1D-Arg2, Phe(pCl)6, Arg9Abu15, Nle27D-Arg28, Har29]hGH-RH(1-29)NH2peptide 7;

[Nac0, His1D-Arg2, Phe(pCl)6, Arg9Abu15, Nle27D-Arg28, Har29]hGH-RH(1-29)NH2peptide 8;

[PhAc0D-Arg2, Phe(pCl)6, Arg9Abu15, Nle27D-Arg29]hGH-RH(1-29)NH2peptide 9;

[PhAc0D-Arg2, Phe(pCl)6Abu15, AGD16, Nle27D-Arg29]hGH-RH(1-29)NH2peptide 10;

[PhAc0D-Arg2, Phe(pCl)6Abu15, Nle27D-Arg28, Har29]hGH-RH(1-29)NH2peptide 11;

[PhAc0D-Arg2, Phe(pCl)6, Nle9Abu15, Nle27D-Arg29]hGH-RH(1-29)NH2peptide 12;

[PhAc0D-Arg2, Phe(pCl)6, Nle13, Nle14Abu15, Nle27D-Arg29]hGH-RH(1-29)NH2peptide 13;

[PhAc0D-Arg2, Phe(pCl)6, le18, Nle27D-Arg29]hGH-RH(1-29)NH2peptide 15;

[PhAc0D-Arg2, Phe(pCl)6, Tight(IU)10Abu15, Nle27D-Arg29]hGH-RH(1-29)NH2peptide 16;

[PhAc0D-Arg2, Phe(pCl)6Abu8, Tyr(Me)10Abu15, Nle27D-Arg29]hGH-RH(1-29)NH2peptide 17;

[PhAc0D-Arg2, Phe(pCl)6, D-Abu8, Tyr(Me)10Abu15, Nle27D-Arg29]hGH-RH(1-29)NH2peptide 18;

[PhAc0D-Arg2, Phe(pCl)6, Tyr(Me)10Abu15D-Arg27, Arg28D-Arg29]hGH-RH(1-29)NH2peptide 19;

[PhAc0D-Arg2, Phe(pCl)6, Tyr(Me)3Abu15D-Arg27, Arg28D-Arg29]hGH-RH(1-29)NH2peptide 20;

[PhAc0D-Arg2, Phe(pCl)6Abu15D-Arg27, Arg28D-Arg29]hGH-RH(1-29)NH2peptide 21;

[PhAc0D-Arg2, Phe(pCl)6Abu8, Tyr(Me)10Abu15D-Arg27, Arg28D-Arg29]hGH-RH(1-29)NH2peptide 22;

[PhAc0D-Arg2, Phe(pCl)6, D-Abu8, Tight(IU)10Abu15D-Arg27, Arg28D-Arg29]hGH-RH(1-29)NH2peptide 23;

[PhAc0D-Arg2, Phe(pCl)6, Lys9Abu15, Nle27D-Arg28, Har29]hGH-RH(1-29)NH2peptide 24;

[PhAc0D-Arg2, Phe(pCl)sup>, Phe(pCl)6D-Agde9Abu15, Nle27D-Arg28, Har29]hGH-RH(1-29)NH2peptide 26;

[PhAc0D-Arg2, Phe(pCl)6, D-Har9Abu15, Nle27D-Arg28, Har29]hGH-RH(1-29)NH2peptide 27;

[PhAc0D-Arg2, Phe(pCl)6, D-Lys9Abu15, Nle27D-Arg28, Har29]hGH-RH(1-29)NH2peptide 28;

[PhAc0D-Arg2, Phe(pCl)6, D-Orn9Abu15, Nle27D-Arg28, Har29]hGH-RH(1-29)NH2peptide 29;

(PhAc0D-Arg2, Phe(pCl)6, Cit9Abu15, Nle27D-Arg28, Har29]hGH-RH(1-29)NH2peptide 30.

Six very preferred embodiments of the invention have the formula:

PhAc0-Tyr1-D-Arg2-Asp3-Ala4-Ile5-Phe(pCl)6-Thr7-Asn8-Arg9-Tyr10-Arg11-Lys12-Val13-Leu14Abu15-Gln16-Leu17-Ser18-Ala19-Arg20-Lys21-Leu22-Leu23-Gln24-Asp25-Ile26-Nle-27-D-Arg28-Har29-NH2peptide 1;

lndAc0-Tyr1-D-Arg2-Asp3-Ala4-Ile5-Phe(pCl)6-Thr7-Asn8-Arg9-Tyr10-Arg11-Lys12Val13-Leu14Abu15-Gln16-Leu17-Ser18-A29
-NH2peptide 2;

PhAc0-Tyr1-D-Arg2-Asp3-Ala4-Ile5-Phe(pCl)6-Thr7-Asn8-Har9-Tyr(Me)10-Arg11-Lys12-Var13-Leu14Abu15-Gln16-Leu17-Ser18-Ala19-Arg20-Lys21-Leu22-Leu23-Gln24-Asp25-Ile26-Nle27-D-Arg28-Har29-NH2peptide 3;

PhAc0-Tyr1-D-Arg2-Asp3-Ala4-Ile5-Phe(pCl)6-Thr7-Asn8-Arg9-Tyr(Me)10-Arg11-Lys12-Val13-Leu14Abu15-Gln16-Leu17-Ser18-Ala19-Arg20-Lys21-Leu22-Leu23-Gln24-Asp25-Ile26-Nle27-D-Arg28-Har29-NH2peptide 6;

PhAc0-His1-D-Arg2-Asp3-Ala4-Ile5-Phe(pCl)6-Thr7-Asn8-Arg9-Tyr10-Arg11-Lys12-Val13-Leu14Abu15-Gln16-Leu17-Ser18-Ala19-Arg20-Lys21-Leu22-Leu23-Gln24-Asp25-Ile26-Nle27-D-Arg28-Har29-NH2peptide 7;

Nac0-His1-D-Arg2-Asp3-Ala4-Ile5-Phe(pCl)6-Thr7-Asnin-Arg9-Tyr10-Arg11-Lys12-Val13-Leu14Abu15-Gln1627-D-Arg28-Har29-NH2peptide 8.

As a General rule such compounds can be represented with a shorter name, as follows:

[PhAc0D-Arg2, Phe(pCl)6, Arg9Abu15, Nle27D-Arg28, Har29]hGH-RH(1-29)NH2peptide 1;

[IndAc0D-Arg2, Phe(pCl)6, Arg9Abu15, Nle27D-Arg28, Har29]hGH-RH(1-29)NH2peptide 2;

[PhAc0D-Arg2, Phe(pCl)6, Har9, Tyr(Me)10Abu15, Nle27D-Arg28, Har29]hGH-RH(1-29)NH2peptide 3;

[PhAc0D-Arg2, Phe(pCl)6, Arg9, Tyr(Me)10Abu15, Nle27D-Arg28, Har29]hGH-RH(1-29)NH2peptide 6;

[PhAc0, His1D-Arg2, Phe(pCl)6, Arg9Abu15, Nle27D-Arg28, Har29]hGH-RH(1-29)NH2peptide 7;

[Nac0, His1D-Arg2, Phe(pCl)6, Arg9Abu15, Nle27D-Arg28, Har29]hGH-RH(1-29) NH2peptide 8.

S. the Method of obtaining

1. Overview of synthesis.

The peptides are synthesized by suitable means, as, for example, solely by solid-phase peptide synthesis, in part by solid-phase synthesis by condensation of fragments or by classical synthesis is, .M. Stewart and J. D. Young, Pierce Chem. Company, Rockford, 111, 1984 (2nd. ed.), and M. Bodanszky, "Principles of Peptide Synthesis", Springer Verlag, 1984. Which antagonists hGH-RH peptides preferably receive when applying solid-phase synthesis, as described by Merrifield, J. Am. Chem. Soc., 85, R. 2149 (1963), although other equivalent chemical syntheses known in this field may also be applied, as indicated above.

The synthesis is performed with the amino acids that are protected bythe amino group. It is preferable to protect-use amino protective groups such as urethane (BOC or Fmoc). The preferred protecting group is BOC.

For solid-phase synthesis of protected nitrogen atom-the amino groups of the stretch of amino acids that forms aminoacyl group final peptide at the C-end, attached by a chemical bond to the bearer of the polymer resin. After completion of the binding reaction of the protective groupthe amino group for the subsequent coupling reaction to aminobenzo selectively removed, preferably by 50% triperoxonane acid (TFA) in dichloromethane (DHM), when N-protecting group is a BOC is Ino attached to the free amino group of the preceding amino acid on the resin to obtain the desired peptide sequence. Because amino acid residues associated with-amino group of the C-terminal residue, the growth of synthetic peptide analogs of hGH-RH starts With the end and moving towards the N end. When the desired sequence, the peptide acelerou N-end and it is removed from the polymer carrier.

Each protected amino acid is used in excess (2.5 or 3 equivalent), and of combination reaction is usually carried out in DHM, DMF or mixtures. The degree of completion of the binding reaction is monitored at every stage through reaction with ninhydrin. In cases when determining incomplete binding procedure repeat binding or reaction complete by acylation of unreacted amino groups prior to the removal of the protective group with-amino prior to binding of the next amino acid.

A typical cycle of the synthesis are presented in table 1.

After completion of the synthesis, cleavage of the peptide from the resin can be carried out using techniques well known in the chemistry of peptides.

Some of the amino acid residues of the peptides have such a functional group in the side chain, the side chain are present, such groups, suitable protective groups attached to these functional groups to prevent unwanted chemical reactions with the reactions used for production of peptides. When selecting suitable protective groups for the side chain adhere to the following General rules: (a) protective group preferably retains its protective properties and is not cleaved under the conditions of binding, (b) the protective group must be stable under the conditions of removal of the protective group with-amino group at each stage of the synthesis, (C) a protective group of the side chain must be able after completion of the synthesis of the desired amino acid sequences to be removed under reaction conditions that will not change unwanted way of the peptide chain.

The reactive functional group of the side chain preferably protects as follows: benzyl for Thr and Ser; 2-bromobenzyloxycarbonyl for Tight; p-toluensulfonyl or nitrogroup for AGD and Naked; 2-chlorobenzenesulfonyl or fluorenylmethoxycarbonyl for Lys, Orn; benzoyloxymethyl for His and cyclohexyl or fluorenylmethyl for Asp and Glu. The side chains of Asn and Gln are not protected.

3. Sequential binding amino acid residues with half the bearers, that is, the resin-based p-methylbenzhydrylamine (MBHA), Merrifield resin, resin-based phenylacetamide or on the Wang resin. When used for the synthesis of N--Boc-protected amino acids, the preferred resin is a resin-based MBA. In this case, receive the peptides with emitirovannykh With-end with the removal from the phase of the carrier.

First C-terminal amino acid is attached to neutralized MBHA-resin and then are subject to the following binding amino acids. Each protected amino acid is bound about with a threefold molar excess relative to the associated resin residues with free amino groups, and linking can be carried out in the environment, such as DMF: CH2Cl2(1:1) or in DMF or CH2Cl2. Choosing the right binding reagent is a competence of the specialist in this field. Particularly suitable as binding reagents are N,N-diisopropylcarbodiimide (DICD) or hexaphosphate 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea, mixed with 1-hydroxybenzotriazole. The success of the passage of the binding assays at each stage of the synthesis is preferably controlled using ning is knitted resin unreacted residues with amino groups will acetimidoyl with acetic anhydride in DHM before removal of the protective group-amino group.

The final acylation N-Terminus of the peptide is carried out in the same manner as the previous link, with the difference that instead of amino acids using the appropriate carboxylic acid.

4. Removal of the peptide from the polymer carrier.

When the synthesis is complete, the peptide otscheplaut from the phase of the carrier. Removal of the peptide from the resin is carried out at a processing reagent, such as liquid hydrogen fluoride, which is also it all of the remaining protective groups of the side chain.

Accordingly, dried and protected peptide-resin is treated with a mixture consisting of 1.0 ml of m-cresol and 10 ml of anhydrous hydrogen fluoride per gram of peptide-resin for 60 min at 0°C for cleavage of the peptide from the resin and removal of all protective groups of the side chain. After removal of the hydrogen fluoride in a stream of nitrogen and in vacuum free peptide precipitated in diethyl ether, filtered, washed with diethyl ether and ethyl acetate, extracted with 50% acetic acid and subjected to lyophilization.

5. The cleanup.

Cleaning technical peptides can be performed using techniques well known in the chemistry of peptides. For example, purification may be carried out using a system MacRabbit for HPLC (Rainin Rosenau phase (10250 mm, filled with silica gel, 18, pore size 300particle size 5 μm) (The Separations Group Inc., Hesperia, CA). Column elute with the solvent system consisting of (A) 0.1% aqueous TFA and (B) of 0.1% TFA in 70% aqueous MeCN with a linear gradient (for example, 30-55% In 120 min). Eluent is monitored at 220 nm and the fractions are analyzed by means of analytical HPLC using a liquid chromatograph model HP-1090 Hewlett-Packard and unite to ensure maximum purity. Analytical HPLC carried out on a Vydac column 218TP52 with reversed phase (2250 mm, 18, 300, 5 μm) using isocrates elution with solvent system consisting of (a) and (b) above. Peaks are recorded at 220 and 280 nm. The peptides according to analytical HPLC receive substantially pure (> 95%). The expected amino acid composition is also confirmed by amino acid analysis.

D. Pharmaceutical composition

The peptides according to the invention can be administered in the form of a pharmaceutically acceptable non-toxic salts, as, for example, additive salt of the acid. Examples of such additive salts of acids are the hydrochloride, hydrobromide, sulfate, phosphate, fumarate, gluconate, tannat, maleate, acetate, zithers, the agonists are salts with low solubility, for example, pamoate and the like. They exhibit prolonged activity.

Compounds of the present invention respectively administered to humans or animals subcutaneously, intramuscularly or intravenously, through the nose or by pulmonary inhalation, or in a form with a slow release depot-form) (e.g., microcapsules, microspheres or cylindrical stick-like implants), prepared from bioresidues a suitable polymer, such as D,L-lactide-coglycolide), the first two depot-forms are preferred. Other equivalent forms of introduction are also presented in the framework of this invention, i.e., continuous intravenous drip infusion, injection of substances slow suction, infusion pump and forms with release time, as, for example, microcapsules, and the like. Introduction valid in any physiologically acceptable injectable medium, saline, although other carriers known in this field can also be used.

The peptides are preferably administered parenterally, intramuscularly, subcutaneously or intravenously with a pharmaceutically acceptable carrier, such as isotonic saline solution. Alternative the inhalation. Appropriate introduction is depot-form made from bioresidues appropriate polymer, such as poly-D,L-lactide-coglycolide, in the form of microcapsules, microgranules or cylindrical implants containing dispersed antagonistic compounds.

The required amount of peptide depends on the method of administration and the intended result. Typically, the dose range is 1-100 mg/kg of body weight of the host body in a day.

E. Therapeutic use of antagonists of GH-RH.

Antagonists hGH-RH can be used in the treatment of conditions caused by an excess of growth hormone, such as acromegaly, which manifests itself in the form of abnormal growth of the bones of the face and extremities. Antagonists GH-RH can also be used for the treatment of diabetic retinopathy (a major cause of blindness in diabetics and diabetic nephropathy, in which damage to the eyes and kidneys, respectively, associated with GH.

Antagonists hGH-RH is designed to block the binding and, consequently, the actions of GH-RH, which stimulates the secretion of GH, which, in turn, stimulates the production of IGF-I. Antagonists GH-RH can be entered separately or together with somatostatin analogues, as a combination, to the one that antagonists of GH-RH can be applied in situations where sites targets do not have receptors for somatostatin.

However, the main antagonists of GH-RH used in the field of malignant diseases. It is based on the following considerations: antagonists of GH-RH is designed to block the binding and therefore the actions of GH-RH, which stimulates the secretion of GH, which, in turn, stimulates the production of insulin-like growth factor I (IGF-I), which is also known as somatomedin-C. Well-established involvement of IGF-I (somatomedin-C) in malignant disease of the breast, prostate, colon, bone tumors and other malignant diseases but some analogs of somatostatin does not adequately suppress the levels of GH and IGF-I. To the best of inhibition of tumor growth requires a complete suppression of IGF-I levels or allocation. Autocrine production of IGF-I in various tumors could also be under the control of GH-RH and therefore could also be inhibited by antagonists of GH-RH. Antagonists GH-RH could also inhibit the production of IGF-I. a More detailed theoretical background applications GH-RH in the field of Oncology (cancer) is the following: the receptors for IGF-I are present in Pervyy, malignant lung tumors, malignant tumors of the colon, brain tumors, malignant tumors of the pancreas and in hypernatraemic tumors of the kidney.

The presence of receptors for IGF-I in these tumors is associated with malignant transformation and proliferation of these malignant tumors. IGF-I may act as endocrine, paracrine or autocrine growth factor for various human malignant tumors, the growth of these tumors depends on IGF-I. Antagonists GH-RH, suppressing the secretion of GH, will reduce the production of IGF-I. Since IGF-I stimulates the growth of such various neoplasms (cancer), the decrease in levels of circulating IGF-I which should lead to inhibition of tumor growth. It is possible that antagonists of GH-RH could also reduce paracrine or autocrine production of IGF-I tumors, which should also lead to inhibition of proliferation of malignant neoplasms. These views are in line with modern concepts of clinical Oncology. Antagonists GH-RH should be given separately or in combination with somatostatin analogues, and the combination would lead to the achievement of a more complete suppression of IGF-I levels, to eliminate the levels of IGF-I in testfiles cancer and non-small cell lung cancer (non-SCLC).

The advantage antagonists GH-RH on somatostatin analogues is based on the fact that antagonists of GH-RH can be used to suppress tumors that do not have somatostatin receptors, such as human osteogenic sarcoma.

It was shown that antagonistic analogs of GH-RH inhibit the growth of various tumors in vivo. This effect manifests itself in part through inhibition system GH-RH-IGF-I. However, autocrine/paracrine control of cell proliferation using IGF-II is also a major factor in many tumors. Violation of such autocrine growth-enhancing metabolic pathways suggests an approach to the control tumors. Antagonistic analogs of GH-RH MZ-4-71 {[Ibu0, Tyr1D-Arg2Abu15, Nle27]hGH-RH(1-28) Agm} and MZ-5-156 {[PhAc0D-Arg2Abu15, Nle27]hGH-RH(1-28) Agm} significantly inhibited the rate of proliferation of the cell lines breast cancer (MDA-MB-468, ZR-75-1), prostate (PC-3 and DU-145) and pancreatic (MiaPaCa-2, SW-1990 and Capan-2) in vitro, as shown by colorimetric tests and tests for the inclusion of [3H]-thymidine, reduced the expression of mRNA for IGF-II in cells and the concentration of IGF-II secreted into the culture medium. The same antagonists GH-RH gave the test cancer (PC-3, DU-145), adenocarcinoma of the renal (Caki-I) and non-small cell lung cancer (N). These results suggest that antagonistic analogs of GH-RH can inhibit tumor growth not only by inhibiting system GHRH-GH-IGF-I, but also by reducing the production of IGF-II in some tumor cells, thereby disrupting its autocrine regulatory metabolic pathway.

The present invention is described in the following examples, which are presented only for illustrative purposes. In the examples used optically active protected amino acid in L-configuration, except for special instructions.

The following examples are appropriate ways for the synthesis of novel antagonists of GH-RH using solid-phase synthesis.

Example 1

PhAc0-Tyr1-D-Arg2-Asp3-Ala4-Ile5-Phe(pCl)6-Thr7-Asn8-Arg9-Tyr10-Arg11-Lys12-Val13-Leu14Abu15-Gln16-Leu17-Ser18-Ala19-Arg20-Lys21-Leu22-Leu23-Gln24-Asp25-Ile26-Nle27-D-Arg28-Har29-NH2(peptide 1) {[PhAc0D-Arg2, Phe(pCl)6, Arg9Abu15, Nle27D-Arg28Naked29] hGH-RH (1-29) NH2}

The synthesis of the implementation of the, resin-based MBGA (Bachem, CA) (720 mg, 0.5 mmol) neutralized 5% DIEA in CH2Cl2and washed according to the Protocol described in table 1. A solution of Boc-Har(NO2)-HE (500 mg, 1.5 mmol) in DMF-CH2CL2(1:1) shake with the neutralized resin and DICT (235 μl, 1.5 mmol) in the device manual for solid-phase peptide synthesis for 1 hour. After the binding reaction, as evidenced by a negative ninhydrin test, remove protection using 50% TFA in CH2Cl2and neutralization with 5% DIEA in CH2Cl2the peptide chain is built Paladino by binding to the resin following protected amino acids in order to obtain the desired peptide sequence: Boc-D-Arg(Tos)-HE, Boc-Nle-OH, Boc-Ile-OH, Boc-Asp(OcHx)-OH, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH, Boc-Lys(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH, Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH, Boc-Lys(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(2BrZ)-OH, Boc-Arg(Tos)-OH, Boc-Asn-OH, Boc-Thr(Bzl)-OH, Boc-Phe(pCl)-OH, Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Arg(Tos)-OH, Boc-Tyr(2BrZ)-OH.

These protected amino acid residues (also usually derived from the company Bachem Co.) indicated above in accordance with the well-known rule. A suitable protective group for the functional group of the side chain of certain amino acids of the TC of each residue free.

Protected amino acids (1.5 mmol each) associated with DID (235 μl, 1.5 mmol) with the exception of Boc-Asn-OH and BOC-Gln-HE who communicate with their previously formed esters with 1-hydroxybenzotriazole. After removal of-Boc-protective group from Tight1peptide acelerou using phenylacetic acid (PhAc) (272 mg, 2 mmol), using DICT (313 μl, 2 mmol).

In order to split the peptide from the resin and remove his protection, dried resin peptide (2,18 g) is stirred with 2 ml of m-cresol and 20 ml of hydrogen fluoride (HF) at 0within 1 hour. After evaporation of the HF under vacuum, the residue was washed with anhydrous diethyl ether and ethyl acetate. Split-off and deprived of the protection of the peptide is dissolved in 50% acetic acid and separated from the resin by filtration. After dilution with water and lyophilization obtain 1.51 g of the crude product.

The crude peptide is examined by analytical HPLC, using a liquid chromatograph model HP-1090 Hewlett-Packard with column Vydac 218TP52 with reversed phase (2250 mm, filled with silica gel C18, pore size 300particle size 5 μm) (The Separation Group Inc., Hesperia, CA) and linear gradient elution (e.g., 40-70%) is placed peptide in AcOH/H2O, stirred, filtered and applied to a column Beckman Ultraprep ODS (21,2150 mm, filled with silica gel C18, pore size 300particle size 10 μm). Column elute with the solvent system described above, with a linear gradient (for example, 30-55% In 120 min); flow rate 6 ml/min Eluent control at 220 nm and the fractions are examined by analytical HPLC. Fractions with purity higher than 95% unite and lyophilized, receiving 98 mg of the pure product. Analytical HPLC is carried out on a column of Vydac C18 reversed-phase, described above, using isocrates elution with solvent system described above, a flow rate of 0.2 ml/min Peaks are analyzed at 220 and 280 nm. According to analytical HPLC, the product is largely pure (> 95%). The molecular weight determined by mass spectrometry with electrodispersion, and the expected amino acid composition confirm amino acid analysis.

Example 2

PhAc0-Tyr1-D-Arg2-Asp3-Ala4-Ile5-Phe(pCl)6-Thr7-Asn8-Har9-Tyr(Me)10-Arg11-Lys12-Val13-Leu14Abu15-Gln16-Leu17-Ser18-Ala19-Arg20-Lys21-Leu D-Arg2, Phe(pCl)6, Har9, Tyr(Me)10Abu15, Nle27D-Arg28, Har29]hGH-RH(1-29)NH2}

The synthesis carried out Paladino using the equipment for solid-phase peptide synthesis with manual control, Briefly, the resin-based MBGA (Bachem, CA) (100 mg, 0,070 mmol) neutralized 5% DIEA in CH2Cl2and washed according to the Protocol described in table 1. A solution of BOC-Har(NO2)-OH (83 mg, 0.25 mmol) in DMF-CH2CL2(1:1) shake with the neutralized resin and DID (44 μl, 0,275 mmol) in unit with manual control for solid-phase peptide synthesis for 1 hour. After the binding reaction, as evidenced by a negative ninhydrin test, remove protection using 50% TFA in CH2Cl2and neutralization with 5% DIEA in CH2Cl2the peptide chain is built Paladino by binding to the resin following protected amino acids in order to obtain the desired peptide sequence: Boc-D-Arg(Tos)-HE, Boc-Nle-OH, Boc-Ile-OH, BOC-Asp(OcHx)-HE, Boc-Gln-OH, Boc-Leu-OH, Boc-Leu-OH, Boc-Lys(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH, Boc-Leu-OH, Boc-Gln-OH, Boc-Abu-OH, Boc-Leu-OH, Boc-Val-OH, Boc-Lys(2CIZ)-OH, Boc-Arg(Tos)-OH, Boc-Tyr(Me)-OH, Boc-Har(NO2)-OH, Boc-Asn-OH, Boc-Thr(Bzl)-OH, Boc-Phe(pCl)-OH, Boc-Ile-OH, Boc-Ala-OH, Boc-Asp(OcHx)-OH, Boc-D-Arg(Tos)-Once in accordance with well-known rule. A suitable protective group for the functional group of the side chain of certain amino acids is indicated in parentheses. Hydroxyl group in the above formulas indicate that the carboxyl end of each residue free.

Protected amino acid (0.25 mmol each) associated with DID (44 μl, 0,275 mmol) with the exception of Boc-Asn-OH and Boc-Gln-OH, which are associated with their previously formed esters with 1-hydroxybenzotriazole. After removal ofThe BOC-protective group from Tight1peptide acelerou using phenylacetic acid (PhAc) (54 mg, 0.4 mmol), using DICT (70 μl, 0.44 mmol).

In order to split the peptide from the resin and remove his protection, dried resin peptide (206 mg) was stirred with 0.5 ml of m-cresol and 5 ml of hydrogen fluoride (HF) at 0within 1 hour. After evaporation of the HF under vacuum, the residue was washed with anhydrous diethyl ether and ethyl acetate. Split-off and deprived of the protection of the peptide is dissolved in 50% acetic acid and separated from the resin by filtration. After dilution with water and lyophilization obtain 112 mg of the crude product.

The crude peptide is examined by analytical HPLC, using a liquid chromatograph model HP-1090 firmer then 300particle size 5 μm) (The Separation Group Inc., Hesperia, CA) and linear gradient elution (e.g., 40-70%) with a solvent system consisting of (A) 0.1% aqueous TFA and (B) of 0.1% TFA in 70% aqueous MeCN. Dissolve 80 mg of the crude peptide in the Asón/N2O, stirred, filtered and applied to a column Vydac TR (10250 mm) filled with silica gel With 8. Column elute with the solvent system described above, with a linear gradient (for example, 30-55% In 120 min); flow rate 2 ml/min Eluent control at 220 nm and the fractions are examined by analytical HPLC. Fractions with purity higher than 95% unite and lyophilized receive 9.6 mg of pure product. Analytical HPLC is carried out on a column of Vydac C18 reversed-phase, described above, using isocrates elution with solvent system described above, a flow rate of 0.2 ml/min Peaks are analyzed at 220 and 280 nm. According to analytical HPLC, the product is largely pure (>95%). The molecular weight determined by mass spectrometry with electrodispersion, and the expected amino acid composition confirm amino acid analysis.

Peptide 2 and peptide from 4 to 30 synthesized in the same manner as peptide 1 and peptide 3, ickler>, Arg9Abu15, Nle27D-Arg28, Har29]hGH-RH(1-29)NH, peptide 2;

[PhAc0D-Arg2, Phe(pCl)6Naked9Abu15, Nle27D-Arg28, Har29]hGH-RH(1-29)NH2peptide 4;

[Nac0D-Arg2, Phe(pCl)6, Arg9Abu15, Nle27D-Arg28, Har29]hGH-RH(1-29)NH2peptide 5;

[PhAc0D-Arg2, Phe(pCl)6, Arg9, Tyr(Me)10Abu15, Nle27D-Arg28, Har29]hGH-RH(1-29)NH2peptide 6;

[PhAc0, His1D-Arg2, Phe(pCl)6, Arg9Abu15, Nle27D-Arg28, Har29]hGH-RH(1-29)NH2peptide 7;

[Nac0, His1D-Arg2, Phe(pCl)6, Arg9Abu15, Nle27D-Arg28, Har29]hGH-RH(1-29)NH2peptide 8;

[PhAc0D-Arg2, Phe(pCl)6, Arg9Abu15, Nle27D-Arg29]hGH-RH(1-29)NH2peptide 9;

[PhAc0D-Arg2, Phe(pCl)6Abu15, Arg16, Nle27D-Arg29]hGH-RH(1-29)NH2peptide 10;

[PhAc0D-Arg2, Phe(pCl)6Abu15, Nle27D-Arg28, Har29]hGH-RH(1-29)NH2peptide 11;

[PhAc0D-Arg2, Phe(pCl)6, Nle9Abu15, Nle27D-Arg29]hGH-RHC(1-29)NH2

peptide 12;

[PhAc0D-Arg2, Phe(pCl)6, Nle13, Nle15, Nle27D-Arg29]hGH-RH(1-29)NH2peptide 14;

[PhAc0D-Arg2, Phe(pCl)6Abu15, Nle18, Nle27D-Arg29]hGH-RH(1-29)NH2peptide 15;

[PhAc0D-Arg2, Phe(pCl)6, Tyr(Me)10Abu15, Nle27D-Arg29]hGH-RH(1-29)NH2peptide 16;

[PhAc0D-Arg2, Phe(pCl)6Abu8, Tyr(Me)10Abu15, Nle27D-Arg29]hGH-RH(1-29)NH2peptide 17;

[PhAc0D-Arg2, Phe(pCl)6, D-Abu8, Tight(IU)10Abu15, Nle27D-Arg29]hGH-RH(1-29)NH2peptide 18;

[PhAc0D-Arg2, Phe(pCl)6, Tight(IU)10Abu15D-Arg27, Arg28D-Arg29]hGH-RH(1-29)NH2peptide 19;

[PhAc0D-Arg2, Phe(pCl)6, Tyr(Me)9Abu15D-Arg27, Arg28D-Arg29]hGH-RH(1-29)NH2peptide 20;

[PhAc0D-Arg2, Phe(pCl)6Abu15D-Arg27, Arg28D-Arg29]hGH-RH(1-29)NH2peptide 21;

[PhAc0D-Arg2, Phe(pCl)8Abu8, Tyr(Me)10Abu16D-Arg27, Arg28D-Arg29]hGH-RH(1-29)NH2peptide 22;

[PhAc0D-Arg2, Phe(pCl)6, D-Abu8, Tyr(Me)10Abu15D-Arg27, Arg28D-Arg29]hGH-RH(1-29)NH2peptide 23;

[PhAc0D-Arg2, Phe(pCl)6, Lys9, Orn9Abu15, Nle27D-Arg28, Har29]hGH-RH(1-29)NH2peptide 25;

[PhAc0D-Arg2, Phe(pCl)6D-Arg9Abu15, Nle27D-Arg28, Har29]hGH-RH(1-29)NH2peptide 26;

[PhAc0D-Arg2, Phe(pCl)6, D-Har9Abu15, Nle27D-Arg28, Har29]hGH-RH(1-29)NH2peptide 27;

[PhAc0D-Arg2, Phe(pCl)6, D-Lys9Abu15, Nle27D-Arg28, Har29]hGH-RH(1-29)NH2peptide 28;

[PhAc0D-Arg2, Phe(pCl)6, D-Orn9Abu15, Nle27D-Arg28, Har29]hGH-RH(1-29)NH2peptide 29;

[PhAc0D-Arg2, Phe(pCl)6, Cit9Abu15, Nle27D-Arg28, Har29]hGH-RH(1-29)NH2peptide 30.

Example 3

Biological activity

The peptides of the present invention were investigated in in vitro and in vivo tests for the ability to inhibit induced hGH-RH(1-29)NH3the release of GH.

Superlite hypophyseal system of the rat.

The analogues were investigated in vitro test described previously (S. Vigh and A. V. Schally, Peptides 5: 241-347, 1984) with modification (Rekasi z and A. V. Schally, P. N. A. S. 90:2146-2149, 1993).

Briefly, the cells were pre-incubated with peptides for 9 minutes (3 ml) at various concentrations. Immediately after incubation injected 1 h the economic effect of similar used 1 nm hGH-RH(1-29)NH2after 30, 60, 90 and 120 minutes for 3 minutes [response 30, 60, 90, 120 min]. Estimated net total value of GH responses. The GH responses are compared and expressed as a percentage of the initial GH response induced by 1 nm GH-RH(1-29)NH2. The effect of new antagonists compared with from [AC-Tight1D-Arg2]hGH-RH(1-29)NH2, "standard antagonist".

Radioimmunoassay analysis of growth hormone.

Levels of rat GH in aliquot samples of the undiluted and diluted superslick samples was measured using radioimmunoassay analysis with double antibody, using materials provided by the National Program on hormones and the pituitary gland (National Hormone and Pituitary Program), Baltimore, Maryland. The results of radioimmunoassay analysis was analyzed using a computer program developed at the Institute, where the authors work (V. Csernus and A. V. Schally, in Neuroendocrine Research Methods, Harwood Academic (Greenstein, B. D. ed., London, pp. 71-109, 1991), incorporated here by reference. The variation between tests was less than 15% and the variation within the series of tests was less than 10%.

Analysis of the binding of GH-RH. To characterize the binding of antagonists GH-RH was the analysis of the binding of sensitive radioreceptor (G. Halmos, A. V. Schally et al., Receptor 3, 87-97, 1993), here IMEI membranes of the anterior lobe of the pituitary gland of the rat. Iodirovannoye derivatives [His1, Nle27]hGH-RH(1-32)NH2get method with chloramine T (F. C. Greenwood et al., Biochemistry 89; 114-123, 1963), here referred to. Hypophysis male rats Sprague-Dawley (250-300 g) are used for preparation of crude membranes. For analysis of saturation binding membrane homogenates incubated with at least 6 concentrations [His1,125I-Tyr10, Nle27]hGH-RH(1-32)NH2in the range of 0.005 to 0.35 nm in the presence or absence of excess unlabeled peptide (1 μm).

The radioactivity of the precipitate counted in the count of gamma rays. The affinity of the peptide antagonist studied in relation to the receptors GH-RH pituitary of the rat, determined in experiments on competitive binding. The final affinity for binding estimate with Ki(the dissociation constant of the complex inhibitor-receptor) and is determined using computer programs Ligand PC and McPherson, developed by Munson and Rodbard (P. J. Munson and D. Rodbard, Anal. Biochem. 107, 220-239, 1980). The relative affinity compared with [AC-Tight1D-Arg2]hGH-RH(l-29)NH2standard antagonist, calculated as the ratio of Kistudied antagonist of GH-RH Kistandard antagonist.

The in vivo tests.

Antagonistic effect is isovale 5 groups, out of 7 animals each. Compound (80 mg/kg) and GH-RH(1-29)NH2(3 μg/kg) was dissolved in 5.5% of mannitol and injected intravenously into the jugular vein of rats under anesthesia with Nembutal. The time elapsing between the introduction of the antagonist and GH-RH ranged between groups in accordance with the following schedule. The first group of animals received an injection of GH-RH in 5 min after administration of the antagonist; for the second, third and fourth groups of animals time intervals between injection of the antagonist and the injection of GH-RH was 15, 30 and 60 min, respectively. The control group was first injected only solvent instead of the antagonist, then after 5 min did the injection of GH-RH.

Blood samples (0.4 ml were collected for radioimmunoassay analysis of GH before the introduction of the antagonist ("blood 0) and after 5 min after injection of GH-RH (blood 1"). The GH response in each group was calculated as GOS=(CHblood1/CHblood0), the average value ± standard error of the measurement of individual differences. The relative inhibition of the GH response (%) in each treated group was calculated as 100(CLOobrabotannaya-1)/(CLOcontrol-1).

The results of in vitro studies.

The antagonistic results in table 2 and table 3, respectively. As can be seen from these data, the substitution taking place in molecules, causing a huge increase in the binding of the receptor, and inhibition of GH release in vitro in comparison with the standard antagonist. The strongest antagonist in vitro, peptide 1 has caused a complete inhibition caused by GH-RH release of GH for 90 min under standard conditions analysis. The first sign of recovery reactivity GH-RH was determined after 120 min after exposure of this analogue.

The results of in vivo studies.

Table 4 presents the response of serum GH and their relative inhibition in rats pretreated with antagonists GH-RH. All of the studied analogues (peptide 1, peptide 2, peptide 3, peptide 4, peptide 8, peptide 9, peptide 11 and peptide 16) induce strong and long-lasting inhibition of GH release stimulated hGH-RH(1-29)NH2. Peptide 1 and peptide 2 are the most potent in a short time, inhibiting the GH responses to 95% and 91%, when administered 5 min before hGH-RH(1-29)NH2. The effects of these two peptides lasts at least 30 minutes. on the other hand, peptide 11 and peptide 3, which is slightly less active in t is.

Example 4

Cancer research.

Antitumor activity of the peptides of the present invention investigated in various cancer models. The antitumor effects of these new peptides were compared with those for early analogues (MZ-4-71 and MZ-5-156, the object of U.S. patent No. 5550212 and patent applications U.S. 08/642472).

The effect of antagonists of GH-RH in the mouse related to mammary gland tumors Mat.

Estrogen-independent tumor Mat transplanted subcutaneously to female mice BDF. One day after transplantation, the mice were divided into groups of 10 animals each and started processing. Mice in groups 1, 2, 3 and 4 did daily disposable injection of various antagonists GH-RH subcutaneously at a dose of 20 µg per day for 18 days. In groups 5 and 6, the peptides were administered via osmotic pumps Alzet, releasing the daily amount of peptide in 20 µg. Tumors were regularly measured and calculated tumor volume. Mice were killed on day 18 and measured the mass of tumors.

Results

Peptides, peptide 1 and peptide 3, had a similar strong inhibitory effect on murine related to mammary gland tumors Mat. About who was b weaker, what is the effect of peptide 1 or peptide 3 (see table 5 and Fig.1).

The effects of antagonists of GH-RH on xenografts of human breast cancer MDA-MB-468 in Nude mice.

Nude mice with xenografts gormonozawisimogo cancer human breast MDA-MB-468 were divided into groups of 10 animals each. Exposed treatment groups did daily subcutaneous injection of 20 μg antagonists GH-RH. One group was treated with peptide 1, the second group was treated with MZ-5-156 for comparison. The control group were injected with a carrier in the form of a solvent. Treatment continued for 5 weeks. Tumors were measured once a week and calculated tumor volume. At the end of the experiment, mice were killed and measured the mass of tumors.

Results

Both peptides had any abscopal significant tumor effects on xenografts of MDA-MB-468. In the group, injectable peptide 1, 4 tumors were found constant regression during the experiment. Similarly MZ-5-156 caused regression 3 tumors. After 5 weeks of treatment in these malignant tumors were observed regression to small, resembling scar tissue residues. Histological examination of these tissues found wholeway tissue. In contrast, all tumors in control animals steadily progressed. The final volume of tumors and masses in the groups treated, significantly decreased (see table 6 and Fig.2), peptide 1 showed a stronger effect.

The effect of antagonists of GH-RH on xenografts of human colon cancer HT-29 in Nude mice

Human malignant tumors of the colon HT-29 transplanted subcutaneously to male Nude mice. 19 days after transplantation, the mice were divided into groups each of 10 animals and started processing. Mice were done daily disposable injection of various antagonists GH-RH subcutaneously at a dose of 20 µg per day for 6 weeks. Regularly measured tumor and was calculated tumor volume. At the end of the experiment, mice were killed and measured the mass of tumors.

Results

Peptide 1 and MZ-5-156 had equally strong inhibitory effect on human malignant tumors of the colon HT-29. Treatment with peptide 9 was led to a smaller, but still significant inhibition of tumor growth. Peptide 11 and MZ-4-71 showed only a small effect. (The results are summarized in table 7 and Fig.3).

Mice implanted subcutaneously glioblastoma U87MG and when tumors reached a volume of approximately 70 mm3mice by random sampling were divided into 2 experimental groups. One group was treated with peptide 1 in the form of a single daily subcutaneous injection of 20 µg within 28 days, while the other group served as a control.

Results

Treatment with peptide 1 resulted in inhibition of tumor growth by 77% after four weeks of treatment versus the control group (see table 8 and Fig.4).

The effect of antagonists of GH-RH on xenografts of human prostate cancer PC-3 in Nude mice.

Male Nude mice were implanted subcutaneously in both flanks fragments of tissue in 3 mm3human gormonozawisimogo prostate cancer PC-3. When tumors reached a volume of about 40-50 mm3mice were divided into 3 experimental groups. The first and second groups were treated with peptides, peptide 3 and MZ-5-156, respectively, daily by one subcutaneous injection of 20 μg in 21 days, while the third group served as a control. The volumes of the tumors were measured at intervals of a week, the weight of the tumors was measured at the end of the exposure is caused stronger inhibition of growth (65% inhibition in tumor volume and 62% in the tumor weight), than MZ-5-156 (52% and 46%, respectively).

Claims

1. The peptide selected from the group having the formula:

X-R1-R2-Asp-Ala-R5-R6-Thr-R8-R9-R10-Arg-R12-R13-R14-R15-R16-Leu-R18-R19-Arg-R21-R22-Leu-Gln-Asp-Ile-R27-R28-R29-NH2,

where X means PhAc, IndAc or Nac;

R1means Tight or His;

R2mean D-Arg;

R5means Il or Val;

R6means Phe or Phe(Cl);

R8means Asn, Gln, Ala or D-Asn;

R9means Arg, Har, Lys, Orn, D-Arg, D-Har, D-Lys, D-Orn, Cit, Nle, Tyr(Me), Ser, Ala or Aib;

R10means Tight or Taut(Me);

R12means Lys;

R13mean Val or Nle;

R14means Leu or Nle;

R15means Gly, Ala, Abu, Nle, or Gln;

R16means Gln or Arg;

R18means Ser or Nle;

R19means Ala;

R21means Lys;

R22means Leu, Ala or Aib;

R27means Met, Leu, Nle, Abu, or D-Arg;

R28means Arg, D-Arg or Ser;

R29means Arg, D-Arg, Naked or D-Har, provided that when R9and R28mean Ser, R29is Arg or Naked

and its pharmaceutically acceptable salts.

up>15, Nle27D-Arg28, Har29]hGH-RH(l-29)NH2peptide 1

[IndAc0D-Arg2, Phe(pCl)6, Arg9Abu15, Nle27D-Arg28, Har29]hGH-RH(l-29)NH2peptide 2

[PhAc0D-Arg2, Phe(pCl)6, Har9, Tyr(Me)10Abu15, Nle27D-Arg28, Har29] hGH-RH(1-29)NH2peptide 3

[PhAc0D-Arg2, Phe(pCl)6, Arg9, Tyr(Me)10Abu15, Nle27D-Arg28, Har29] hGH-RH(1-29)NH2peptide 6

[PhAc0, His1D-Arg2, Phe(pCl)6, Arg9Abu15, Nle27D-Arg28, Har29]hGH-RH(l-29)NH2peptide 7

[Nac0, His1D-Arg2, Phe(pCl)6, Arg9Abu15, Nle27D-Arg28, Har29] hGH-RH(1-29)NH2peptide 8.

3. Connection on p. 1 having the formula [PhAc0D-Arg2, Phe(pCl)6, Arg9Abu15, Nle27D-Arg28, Har29]hGH-RH (1-29)NH2peptide 1.

4. Connection on p. 1 having the formula [IndAc0D-Arg2, Phe(pCl)6, Arg9Abu15, Nle27D-Arg28, Har29]hGH-RH (1-29)NH2peptide 2.

5. Connection on p. 1 having the formula [PhAc0D-Arg2, Phe(pCl)6, Har9, Tight(Me)10Abu15, Nle27D-Arg28, Har29]hGH-RH(1-29)NH2peptide 3.

6. Compounds , D-Arg28, Har29]hGH-RH(1-29)NH2peptide 6.

7. Connection on p. 1 having the formula [PhAc0, His1D-Arg2, Phe(pCl)6, Arg9Abu15, Nle27D-Arg28, Har29]hGH-RH(1-29)NH2peptide 7.

8. Connection on p. 1 having the formula [Nac0, His1D-Arg2, Phe(pCl)6, Arg9Abu15, Nle27D-Arg28, Har29]hGH-RH(1-29)NH2peptide 8.

9. The way to suppress excess levels of GH in a patient, in need thereof, which consists in the introduction indicated to the patient an effective amount of the compounds under item 1.

10. A method of treating a patient having a malignant tumor bearing receptors for IGF-I or-II, which is specified in the introduction to the patient an effective amount of the compounds under item 1.

11. Method of inhibiting levels of IGF-II in tumors (malignant tumors) and the expression of mRNA for IGF-II in the same tumors, which is mentioned in the introduction to the patient an effective amount of the compounds on p. 1.

 

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