MXPA00008723A - Pharmacologically active peptide conjugates having a reduced tendency towards enzymatic hydrolysis - Google Patents

Abstract

The invention is directed to a pharmacologically active peptide conjugate having a reduced tendency towards enzymatic cleavage comprising a pharmacologically active peptide sequence (X) and a stabilising peptide sequence (Z) of 4-20 amino acid residues covalently bound to X.

Description

, • HND? N? ENCIA REDUCED TOWARDS ENZIMATGCA HYDROLYSIS. FIELD OF THE INVENTION The present invention relates to conjugates of pharmaceutically active peptides which have a reductive tendency towards enzymatic cleavages.

ANTECEDENTS OF L? INVENTION There is a large number of pharmaceutically active peptides, for example, occurring naturally in man or animals, or synthetic analogs of such peptides. An illustrative example of such a peptide is the analgesically active peptide enkephalin which gives an increase to a large number of synthetic analogues. However, due precisely to its peptide nature, the routes of administration of the same have to be rather limited. In addition, the peptides are rapidly and effectively degraded by enzymes, generally with half-lives in the range of minutes. Proteases and other proteolytic enzymes are ubiquitous, particularly in the gastro-intestinal zone, and therefore peptides are usually susceptible to degradation in multiple situations in oral administration, and extensions in the blood, liver, kidney, and vascular endothelium. In addition, a given peptide is usually susceptible to degradation in more than one bond PEF .: 122285 within the main structure; every situation of hydrolysis is mediated by a certain protease. Even if such obstacles are overcome, for neuropeptides in particular, the difficulties lie in their transportation through the blood-brain barrier.

A number of attempts have been made to protect the peptides against premature degradation (discussed in Prokai, 1997, Opin. Ther Pantent 7: 233-245, Tamai et al., 1996, Adv. Drug Deliver Rev. 19 : 401-424 and Zhou et al., 1991, Int. J. Pharm. 75: 97-115). An improvement osmotically includes the blood-brain barrier by the infusion of hypertonic solutions of mannitol, lactamide, saline, urea and radiographic contrast agents. However, there could be secondary toxic effects.

Another additional methodology involves the use of protease inhibitors (analyzed in Zhou et al., 1991, Int. J. Pharm. 75: 97-115). This improvement produces mixed results.

A third additional methodology involves the use of an increase in absorption in the peptide formulations analyzed in Zhou et al., 1991, Int. J. Pharm. 75: 97-115). Examples include fatty acids and bile salts. However, the variant results have been obtained considering efficiencies and the value of a particular increase depends on the administration route used.

Another additional methodology for increasing the absorption of peptides involves the chemical modification of the peptides by means of, for example, attaching a lifofilica moiety. What also finds that joining a pyroglutamyl residue at the end of the N-terminus can yield in a compound relatively resistant to hydrolysis. Tamai et al., 1996, Adv. Drug Delivery Rev. 19: 401.404, discloses E2078, an analogous dynorphin chemically modified to make it more stable to degradation of the enzyme by adding an N-methyl group to the amino-terminus of Arg and replacing D-Leu with L-Leu. and adding ethylamine to the carboxyl-terminus.

A different methodology involves the formation of chimeric peptides. This approach involves binding of the peptide that is not transported normally through the blood-brain barrier to the peptide or protein "vectors" that suffer from transcytosis mediated by the receptor or mediated by the absorber.

WO 98/22577 describes a method for increasing the resistance of a "core protein" to proteolytic degradation by binding or inserting a "stabilizing polypeptide" having the formula [(GLya) X (GLyb) Y [(GLyc ) Z] n, X, Y, and Z can be alanine, serine, valine, isoleucine, leucine, methionine, phenylalanine, proline, and threonine.

The U.S. patent No. 5,54,719 describes molecules comprising fragments of proteins homologous to an active region of protein fragments capable of stimulating nerve growth (neuronotrophic proteins such as epidermal growth factor, tubulin, nerve growth factor, laminin, fibronectin, ncam and ependymin) not greater than 80 large amino acids connected to a secondary molecule which can be fragments of a second protein derived from the original protein, from another protein or of a non-proteinic half. This secondary molecule facilitates the transport of the peptide through the blood-brain barrier. This is indicated in column 3, lines 3-7, "on entry to the central nervous system, the prodrug may remain intact or the chemical bond between the carrier and the protein fragment may be hydrolyzed accordingly by separating the fragment carrier to release nerve fragment stimulation to growth ". A preferred method for facilitating the binding of the secondary molecule to the protein fragment is via one or more basic amino acids, preferably a pair of Lys residues, a residue of Arg, or Arg-Lys.

Fa ell et al., 1994, Proc. Nati Acad. Sci USA 91: 664-668 describes the chemical cross-linking of several Tat peptide fragments chemically linked to β-galactosidase, seRNA A and field III of pseudomonas exotoxin A. These include Tat- (37-72), Tat- (37-58) and Tat- (47-58). All of these peptides appear to promote the uptake of galactosidase, seRNA and field III into cells. This announces that this is the basic region of Tat. Conjugates containing poly (L-lysine) or poly (L-arginine) are not taken from the cells.

WO 97/24445 discloses albumin and growth hormone fusion proteins or variants thereof. This is indicated in the specification that albumin variants could have oncotic, ligand-binding and non-immunogenic properties of total albumin length and that growth hormone variants might have their non-immunogenicity and ability to bind and activate the growth hormone receptor.

WO 98/28427 discloses an Fc-OB fusion protein. The Fc is a fragment and OB is leptin. It had been found that such conjugates are more stable than the OB alone. The fragment of Fc is 378 amino acids long. The fragment can be conjugated directly or via a linker for the OB or an OB fragment.

Another additional methodology involves the preparation of peptide analogs with stability and / or increased activity by adding a peptide tail. Greene et al., J. Pharm. Exp. Therap. 277: 1366-1375, discloses results of studies with several enkephalin-related prodrugs of [D-Pen2, D-Pen5] enkephalin (DPDPE) and [D-Pen2, L-Cys5] enkephalin (DPLCE), specifically DPLCE-Arg- Pro-Ala, DPDPE-Phe, DPLCE-Phe, DPDPE-Arg-Gly, DPLCE-Arg-Gly, DPDPE-Phe-Ala-NH-C6Hi3, DPDPE-Phe-Ala-CONH2. The half-lives of most analogues, except for DPDPE-Arg-Gly, are little less than the parent compounds. This is indicated on page 1372, column 2 that "the ideal prodrug targeted in CNS could have a long half-life in serum and a short half-life in the brain." In US Patent No. 4,724,229 describes antagonists vasopressin having a tripeptide side chain having three basic amino acids, such as arginine, lysine or ornithine which has potent antagonistic activity. In the U.S. patent No. 4,542,124, discloses vasopressin antagonists having a dipeptide side chain having two amino acids, one of which is basic that has potent vasopressin antagonistic activity.

In the international patent application PCT / DK97 / 00376 (Bjarne Due Larsen and ARNE Holm) the prodrugs of pharmacologically active peptides are described, here in the pharmacologically active peptide is coupled in its C-terminal to a pre-sequence of peptide via a linker, the linker is typically a carboxylic acid a-hydroxide. These special peptide derivatives were found to have a prolonged half-life in the presence of proteolytic enzymes such as carboxypeptidase A, leucine aminopeptidase, pepsin A and a-chymotrypsin. In sum, PCT / DK97 / 00376 discloses (as reference compounds) four different peptides equipped with a peptide pre-sequence but without a linker, particularly DSIP- (Lys-Glu) 3, DSIP- (Glu) 6, Leu-enkephalin- (Glu) 6 and Leu-enkephalin- (Lys) 6.

It is clear that there is a need for a peptide conjugate that contains a pharmacologically active peptide and a stabilizing protein that is relatively simple to synthesize, retain its activity even without moving the stabilizing peptide, is stable in plasma or serum and is relatively resistant to degradation of the enzyme. Accordingly, it is an object of the invention to provide a peptide conjugate comprising a pharmacologically active peptide and a stabilizing peptide that is relatively resistant to degradation of the enzyme.

BRIEF DESCRIPTION OF THE INVENTION It has now surprisingly been found that by conjugating a pharmacologically active peptide, for example, at its C-terminus, at its N-terminus or at its C- and N-terminus, with an appropriate stabilizing sequence of In the case of a peptide, it is possible to contain a peptide conjugate that is significantly less susceptible to degradation by proteases compared to the corresponding pharmacologically active free peptide. Without being linked to any model by this effect, it is considered that the presence of a stabilizing peptide sequence induces a degree of the structure, based on hydrogen bonds, of the pharmacologically active peptide, whereby the peptide conjugate is not very susceptible to proteases in contrast to peptides in random coil conformation. As a result of structuring, the peptide conjugate is much harder for a protease to degrade. In addition, the resulting peptide conjugate is still pharmacologically active, for example the conjugate possesses the ability to exert the pharmacological function of the pharmacologically active free peptide.

Thus, in a first aspect the invention relates to a pharmacologically active peptide conjugate having a reductive tendency towards an enzymatic cleavage, said peptide conjugate comprising: a pharmacologically active peptide sequence (X) and a stabilizing peptide sequence ( Z) of 4-20 amino acid residues covalently linked to X, each amino acid residue in said peptide-stabilizer sequence (Z) is independently selected from the group consisting of Ala, Leu, Ser, Thr, Tyr, Asn, Gln, Asp, Glu, Lys, Arg, His, Met, Orn, and amino acid residues of the general formula I -NH-C R1) (R2) -C (= 0) - (I) Where R1 and R2 are selected from the group consisting of hydrogen, C6-6-alkyl, phenyl, and phenyl-methyl, wherein C6-6-alkyl is optionally substituted with one to three substitutes selected from halogen, hydroxide, amino, cyano, nitro, sulfone, and carboxyl, and phenyl and phenyl-methyl is optionally substituted with one to three substitutes selected from C? -6-alkyl, C2-.6-alkene, halogen, hydroxide, amino, cyano, nitro, sulfone, and carboxyl, or R1 and R2 together with the carbon atom in which there is in the form of bonds a cyclopentyl, cyclohexyl, or cycloheptyl ring, for example, 2,4-diaminobutanoic acid (Dbu) and 2,3-diaminopropanoic acid (Dpr) ); Y Where the ratio between the half-life of said peptide conjugate and the half-life of the corresponding pharmacologically active peptide sequence, X, when treated with carboxypeptidase A or leucine aminopeptidase in about 50 nM phosphate buffer at about pH 7.4 at about 37 ° C or in a plasma or in a serum is at least about 2, preferably at least about 3, such as at least about 5, more preferably at least about 7, such as at least about 9, example, at least about 10 and the ratio between the half-life of said peptide conjugate or when the pharmacologically active peptide is not absorbed orally, said conjugate is orally absorbed or a salt thereof.

In one embodiment, the pharmacologically active peptide is not selected from the group consisting of H-Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu-Lys-GluJ-OH. H-Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu- (Glu) 6 ~ 0H, H-Tyr-Gly-Gly-Phe-Leu- (Glu) 6 ~ OH and H-Tyr- Gly-Gly-Phe-Leu- (Lys) 6 ~ 0H.

The present invention also relates to a composition, for example, a pharmaceutical composition, comprising said pharmacologically active peptide conjugate and a pharmaceutically acceptable carrier, to a pharmacologically active peptide conjugate for use in therapy, a method of treating a disorder and the use of a pharmacologically active peptide conjugate for the manufacture of a pharmaceutical composition for use in therapy. Especially, the invention is directed to a method for inhibiting neurons from transmitting pain impulses to the spinal cord, comprising administering to a subject in need thereof a conjugate comprising enkephalin and Z in an amount effective to inhibit the neurons at from transmitting pain impulses, as well as the use of said conjugate for manufacturing a pharmaceutical composition for use in the treatment of pain; A method for stimulating the release of growth hormone from the pituitary comprising the administration to a subject in need thereof of a conjugate comprising the growth hormone releasing hormone or a hormone releasing peptide. growth and Z in an amount effective to stimulate the release of growth hormone as well as the use of said conjugate for the manufacture of a pharmaceutical composition for use in stimulating the release of growth hormone; a method for increasing hemoglobin levels comprising administration to a subject in need thereof of a conjugate comprising EMP-1 (protein-1 erythropithin mimetic) and Z in an amount effective to increase hemoglobin levels as well as the use of said conjugate for the manufacture of a pharmaceutical composition for use in treating increasing anemia by increasing hemoglobin levels; a method for treating or preventing bone loss by altering the balance between osteoclastic (bone resorption) and osteoblast activity comprising administering to a patient in need thereof a conjugate comprising the hormone of the parathyroid and Z in an amount effective to treat or provide for bone loss as well as the use of said conjugate for the manufacture of a pharmaceutical composition for use in the treatment or prevention of osteoporosis; a method for reducing blood glucose levels comprising administering to a subject in need thereof a conjugate comprising glucide-like peptide-1 and Z in an amount effective to reduce blood sugar levels, as well as the use of said conjugate in the treatment of diabetes; a conjugate comprising peptide that induce delta sleep and Z in an amount effective to provide for seizures, act as if it were a neuroprotective during ischemia and act as if it were an opioid detoxification agent as well as the use of said conjugate for the manufacture of a pharmaceutical composition for use in treating sleep disorders; A method for regulating the production of sex hormones comprising administering to a subject in need thereof a conjugate comprising the hormone releasing gonadotropin and Z in an amount effective to regulate the production of sex hormones as well as the use of said hormone. conjugate for the manufacture of a pharmaceutical composition for use in the regulation of the level of sex hormones.

In another aspect of the present invention it relates to the use of a peptide conjugate, as herein defined, for the manufacture of a pharmaceutical composition for the treatment or prophylaxis of a condition or disorder, wherein the peptide sequence X, where it is not linked to Z, is able to interact with a receiver (or a receiver system) involved with the condition or disorder in question, and where the interaction between X, when not linked to Z, and the receiver (or receiver system) they have a therapeutic or prophylactic effect on the condition or disorder.

The present invention also relates to methods for the preparation of said pharmacologically active peptide conjugate, by means of the DNA recombination technology comprising - the steps of (a) introducing a nucleic acid sequence encoding said conjugate within a cell pattern and (b) cultivate the standard cell and (c) isolate said conjugate from the culture or (a) cultivating a recombinant standard cell comprising a nucleic acid sequence encoding conjugated ducho under conditions that allow the production of said conjugate and (b) isolate said conjugate from culture.

The method also relates to methods for the preparation of said pharmacologically active peptide conjugate wherein the pharmacologically active peptide X is obtained via DNA recombination methods by isolating said peptide or from commercial sources. The X is then conjugated to Z which is adhered to a solid support or have been prepared by synthetic solid phase methods.

In addition, the invention relates to the use of a stabilizing peptide (Z) sequence for the preparation of a pharmacologically active peptide conjugate.

DETAILED DESCRIPTION OF THE INVENTION Peptide conjugates In the present context, the term "amino acid residue" as used in the connection with X mediates some a, β, or α-amino acid occurring in nature or synthetic (if in the L-form or the D-form) as well as amino acids modified in its side chain such as tyrosines where the aromatic ring is further substituted, for example, one or more halogens, sulphono groups, nitro groups etc., and / or the phenol group is converted in an ester group, etc., including protected amino acids in the side chain, wherein the side chains of the amino acids are protected according to methods known to a person skilled in peptide chemistry, such as described in, example, M. Bodanszky and A. Bodanszky, "The Practice of Peptide Synthesis", Ed, Springer-Verlag, 1994, and J. Jones, "The Chemical Synthesis of Peptides", Clarendon Press, 1991.

In the present context, the term "pharmacologically active peptide sequence" or "free peptide" as applied to X indicates any peptide or structure that contains a peptide, whether naturally occurring or synthetic which is therapeutically or prophylactically active without the stabilizing Z sequence covalently linked to it. As defined herein, a peptide sequence is "therapeutically active" if it can be used for the treatment, remission, or attenuation of the disease state, physiological condition, symptoms or etiological indication (s) or evaluation or diagnosis thereof. A peptide sequence is "prophylactically active" if it can be used to predict a disease state, physiological condition, symptoms or etiological indications. A pharmacologically active agent is also physiologically or biologically active. Measure the effect of the pharmacological activity of a substance (peptide) on physiological or biological systems in vitro, in vivo or ex vivo and can be assayed using standard in vitro, in vivo or ex vivo assays known in the art for a particular peptide or a peptide with a similar physiological function.

Peptides are used in a number of processes, eg, cell-to-cell communication, some are present in the autonomic and central nervous system. Some of the more recent peptides, and a number of other peptides, exert important effects on vascular and other mild muscle. In a preferred embodiment, X has at most 75 amino acid residues (or a corresponding structure having at most 75 amino acid residues). Alternatively, X consisting of a maximum of 65, 60, 55, 53, 50, 45, 40, 35, 30, 25, 20, 15, or at most 10 amino acid residues and consisting of at least 2, preferably 5 and more preferably 10 amino acid residues.

In the present context, the X sequence of pharmacologically active peptide can be some peptide which in its natural form is present as the carboxylic acid with free C-terminal, such as Leu-enkephalin (H-Tyr-Gly-Gly-Phe- Leu-OH), or is present in its natural form as a C-terminal amide, such as oxytocin (Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2), or is present in its natural form as a C-terminal ester. In addition, the pharmacologically active peptide may also contain other special structural characteristics such as disulfide bridges as in the case of insulin.

The pharmacologically active peptide can be selected from the group consisting of enkephalin, Leu-enkephalin, Met-enkephalin, angiotensin I, angiotensin II, vasopressin, endothelin, vasoactive intestinal peptide, neurotensin, endorphin, insulin, gramicidin, para-celsin, peptide of delta-dream induction, gonadotropin releasing hormone, human parathyroid hormone (1-34), the analogous truncated erythropithine described in Wrighton et al., 1996, Science 273: 458-463), specifically in EMP- 1, Atrial natriuretic peptide (ANP, ANF), human brain natriuretic peptide (hBNP), cecropin, cinetensin, neurophysin, elephant, guamerin, atriopeptin I, atriopeptin II, atriopeptin III, deltorphine I, deltrophin II, vasotocin, bradykinin, dynorphin , dynorphin A, dynorphin B, growth hormone release factor, growth hormone release peptide, oxytocin, calcitonin, peptide related to the calcitonin gene, Peptide II related to the calcitonin gene, growth hormone release peptide, tacicinin, adrenocorticotropic hormone (ACTH), brain natriuretic polypeptide, cholecistocinin, corticotropin release factor, inhibitor fragment bound to diazepam, FMRF-amide, galanin, gastric-release polypeptide, gastric inhibitory polypeptide, gastrin, gastrin-releasing peptide, glucagon, glucagon-like peptide-1, glucagon-like peptide-2, LHRH, melanin concentration hormone, hormone (MSH) stimulation of melanocyte, alpha-MSH, modulation peptides of morphine, motilin, neurokinin A, neurokinin B, neuromedin B, neu * romedin C, neuromadine K, neuromedin N, neuromedin U, neuropeptide K, neuropeptide Y, pituitary adenylate cyclase activation polypeptide (PACAP), pancreatic polypeptide, YY peptide, histidine-methionine amide peptide (PHM), secretin, somatostanin, substance K, thyrotropin releasing hormone (TRH), ciotorphine, melanostatin (MIF-1), thrombopoietin analogues, in particular AF 12505 (Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala), factor 1 (57- 70) of insulin-like growth (Ala-Leu-Leu-Glu-Thr-Tyr-Cys-Ala-Thr-Pro-Ala-Lys-Ser-Glu), factor I (30-41) of insulin-like growth (Gly- Tyr-Gly- Ser-Ser-Ser-Arg-Arg-Ala-Pro-Gln-Thr). Factor I (24-41) of insulin-like growth (Tyr-Phe-Asn-Lys-Pro-Thr-Gly-Tyr-Gly-Ser-Ser-Ser-Arg-Arg-Ala-Pro-Gln-Thr) , factor II (33-40) of insulin-like growth (Ser-Arg-Val-Ser-Arg-Arg-Ser-Arg), factor II (33-40) [tyro] of insulin-like growth (Tyr) -Ser-Arg-Val-Ser-Arg-Arg-Ser-Arg), factor II (69-84) of insulin-like growth (Asp-Val-Ser-Thr-Pro-Pro-Thr-Val-Leu Pro-Asp-Asn-Phe-Pro-Arg-Tyr), Peptide-6 (GHRP-6) Release (GH) Growth Hormone (His-DTrp-Ala-Trp-Dphe-Lys-NH2), beta -Interleucine II (163-171) (Val-Gln-Gly-Glu-Glu-Ser-Asn-Asp-Lys), Beta-Interleukin II (44-56) (Ile-Leu-Asn-Gly-Ile-Asn- Tyr-Lys-Asn-Pro-Lys-Leu), Interleukin II (60-70) (Leu-Thr-Phe-Lys-Phe-Tyr-Met-Pro-Lys-Lys-Ala), Exedin-4 (analog of GLP-1) (His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Glu-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile -Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2). exendin-3 (GLP-1 analog) (His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg -Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser), [Cys (Acm) 20, 31] epidermal growth factor (20-31) Cys (Acm-Met-His-Ile-Glu-Ser-Leu-Asp-Ser-Tyr-Thr-Cys (Acm), bivalirudin ( Hirulog) (D-Phe-Pro-Arg-Pro- (Gly) 4-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu), hiruloga-1 D-Phe- Pro-Arg-Pro- (Gly) 4-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Tyr-Leu, C-type natriuretic peptide (1-53) (CNP) (Asp-Leu -Arg-Val-Asp-Thr-Lys-Ser-Arg-Ala-Ala-Trp-Ala-Arg-Leu-Leu-Gln-Glu-His-Pro-Asn-Ala-Arg-Lys-Tyr-Lys-Gly -Ala-Asn-Lys-Lys-Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Met-Ser -Gly-Leu-Gly-Cys; Disulfide bridge: Cys-37-Cys53), "Mini ANP" (Met-Cys-His-cyclohexylAla-Gly-Gly-Arg-Met-Asp-Arg-Ile-Ser-Tyr-Arg. Disulfide bridge cys2-cysl3 ), Melanotan-II (Also known as MT-II, alpha-MSH4-10-NH2, or Ac-Nle4-Asp5-His6-D-Phe7-Arg8-Trp9-Lys 10), thymosin alfa I (TA 1) ( Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-SerGlu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Glu-Val-Val-Glu-Glu-Ala Glu-Asn), omipresin (also known as 8-ornithine-vasopressin, (POR-8), [Phe2-Ile3-Orn8] vasopressin), Cys-Phe-Ile-Gln-Asn-Cys-Pro-Orn-Gly- NH2, Disulfide bridge: Cysl-Cys6), octreotide (201-995) (Dphe-Cys-phe-DTrp-Lys-Thr-Cys-Thr-ol; Disulfide bridge: Cys2-Cys7), eptifibatide (INTEGRILIN), Peptide referred to the calcitonin gene (CGRP) (Ala-Cys-Asp-Thr-Ala-Thr-Cys-Val-Thr-His-Arg-Leu-Ala-Gly-Leu-Leu-Ser-Arg-Ser-Gly -Gly-Val-Val-Lys-Asn-Asn-Phe-Val-Pro-Thr-Asn-Val-Gly-Ser-Lys-Ala-Phe-NH2; Disulfide bridge Cys2-Cys7), Endornorphine-1 Tyr- Pro-Trp-Phe-NH2; endornorphine-2 Tyr-Pro-Phe-Phe-NH2, nociceptin (also known as Orphanin FQ, Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-Leu-Ala -Asn-Gln), angiotensinogen (1-13) (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-His), adrenornodulin (1-12) (Tyr-Arg -Gln-Ser-Met-Asn-Asn-Phe-Gln-Gly-Leu-Arg), antiarrhythmic peptide (AAP) (Gly-Pro-Hyp-Gly-Ala-Gly), Antagonist G (Arg-DTrp- (nMe Phe-Gln-Gly-Leu-Met-NH2), indolicidin (Ile-Leu-Pro-Trp-Lys-Trp-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg-NH2), osteocalcin (37 -49) (Gly-Phe-Gln-Gln-Ala-Tyr-Arg-Arg-Phe-Tyr-Gly-Pro-Val), cortistatin 29 (1-13) (Glp) -Glu-Arg-Pro-Pro- Leu-Gln-Gln-Pro-Pro-His-Arg-Asp), Cortistatin 14 Pro-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Ser-Ser-Cys-Lys; disulfide bridge: Cys2-Cysl3, PD-145065 (Ac-D-Bhg-Leu-Asp-Ile-Ile-Trp), PD-142893 (Ac-D-Dip-Leu-Asp-Ile-Ile-Trp), inhibitor peptide bound to fibrinogen (His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val), leptin (93-105) (Asn-Val-Leu-Gln-Ile-Ser -Asn-Asp-Leu-Glu-Asn-Leu-Arg), GR 83074 (Boc-Arg-Ala-DTrp-Phe-Dpro-Pro-Nle-NH2) Tyr-W-MIF-1 (Tyr-Pro-Trp -Gly-NH2), peptide referred to the parathyroid hormone (107-111) (Thr-Arg-Ser-Ala-Trp), Anglotensinogen (1-14) Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Asn, Leupeptin (Ac- Leu-Leu-Arg-CHO) and some modified or truncated analog thereof.

It is well known that many pharmacologically active peptides also exert their desired effects when presented in a modified or truncated form. In the case of, for example, insulin, porcine insulin differs from human insulin by only one amino acid residue, the amino acid B30 in porcine insulin is Ala and the amino acid B30 in human insulin is Thr . Despite these differences, porcine insulin has been used as an effective drug for diabetes for many years. In a similar manner it has been found that the essential characteristics for heptadecapeptide porcine gastrin I activity are all contained in the C-terminal tetrapeptide and that essentially all the pharmaceutical effects of neurotensin are associated with the C-terminal hexapeptide. In addition, pharmacologically active peptides, wherein one or more amide bonds have to be modified, eg, reduced, often exhibiting a similar or even linked pharmaceutical activity; for example the Cys2í¿ [CHY2NH] Tyr3 analog of somatostatin found to be an even more potent growth hormone releasing agent than somatostatin itself, and also the analogous transition state Leu10 ^ [CH (OH) CH2] Val11 of Angiotensin has been found to demonstrate a strong inhibitory effect against the Renin protease of aspartic acid. Thus, the term "modified or truncated analogue thereof" indicates either that such peptides are modified to change and / or eliminate one or more amino acid residues in the natural peptide sequence, including modification of the side chain, stereochemistry , and the main structure in the individual residues of the amino acids, such as changing one or more peptide bonds (-C (= 0) -NH-) for example, reduced forms such as (-CH (OH) -N- ), (-CH2-N-), and other mimetics linked to peptides such as (-C (= 0) -N (CH3) -), (-C (= 0) -0), (-C (= 0 ) -CH2-), (-CH = CH-), (-P02-NH-), (S0-CH2-), (S02-N-), etc.

It has been said, that it could be understood that the X sequence of peptide in question could preferably be comprised of at least one peptide bond (preferably at least two peptide bonds (this does not of course apply to a dipeptide)) susceptible to degradation of the peptide. enzyme to take full advantage of the present invention.

In the present context, the term "C? _6-alkyl" used alone or as part of another group designates a group of straight, branched or cyclic saturated hydrocarbons having from one to six carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec. butyl, tert. butyl, n-pentyl, n-hexyl, cyclohexyl, etc.

In the present context, the term "C2-6 ~ alkene" designates a hydrocarbon group having from two to six carbon atoms, which may be straight, branched, or cyclic and may contain one or more double bonds, such as vinyl , allyl, 1-butenyl, 2-butenyl, isobutenyl, 1-pentenyl, 2-pentenyl, 4-pentenyl, 3-methyl-1-butenyl, 2-hexenyl, 5-hexenyl, cyclohexenyl, 2,3-dimethyl-2 -butenyl etc, which can have a cis and / or trans configuration.

The term "aryl" is intended to mean an aromatic, carbocyclic group such as phenyl or naphthyl.

The term "heteroaryl" includes 5- to 6- membered heterocyclic monocyclic aromatic groups containing from 1-4 heteroatoms selected from nitrogen, oxygen and sulfur, such as pyrrolyl, furyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl. , oxadizolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, triazolyl, pyridyl, and heterocyclic bicyclic aromatic groups containing 1-6 heteroatoms selected from nitrogen, oxygen and sulfur, such as quinolinyl.

The term "halogen" includes fluorine, chlorine, bromine, and iodine.

"The peptide Z sequence is the part of the peptide conjugate responsible for the introduction and / or stabilization of a certain secondary structure in the molecule that must yield the most stable compound towards degradation by proteases. minus 4 amino acid residues for introducing such a stabilizing structure element On the other hand it is also believed that a sequence of more than about 20 amino acid residues does not improve the stability further.So, Z is typically a peptide sequence of 4-20 amino acid residues, example, in the range of 4-15, more preferably in the range of 4-10 in particular in the range of 4-7 amino acid residues, eg, 4.5.6 or 7 amino acid residues When Z is conjugated to X, the half-life of said peptide conjugate when treated with carboxypeptidase A or aminopeptidase leucine in about 50 mM of a buffer solution the pH of about 7.4 at about 37 ° C or in a plasma or in a serum is at least about 2, preferably at least 3, such as at least about 5, more preferably at least 7, such as at least 9, example, at least about 10 more than the half-life of X when it is not conjugated to Z. Furthermore, when the pharmacologically active X-peptide is not orally absorbed, the conjugate is orally absorbed.

Each of the amino acid residues in the peptide Z sequence are independently selected from Ala, Leu, Ser, Thr, Tyr, Asn, Gln, Asp, Glu, Lys, Arg, His, Met, Orn, and amino acids of the formula I as defined herein such as diaminobutanoic acid or diaminopropanoic acid.

Preferably, the amino acid residues are selected from Ser, Thr, Tyr, Asn, Gln, Asp, Glu, Lys, Arg, His, Orn, and Met, more preferably from Glu, Lys, and Met, especially Lys. The amino acids mentioned above can thus have the D- or L- configuration, but preferably the amino acids mentioned above have an L- configuration. Since the pharmacologically active peptide X sequence usually consists exclusively of L-amino acids, this should be expected, to preserve a possible stability in the helix structure of the complete peptide conjugate, than a peptide Z sequence consisting of either solely or principally of L-amino acids should be advantageous compared to a peptide Z-sequence consisting solely or mainly of D-amino acids. Furthermore, it is envisioned that a peptide Z sequence consisting of only or mainly D-amino acids may exert toxicological effects due to the resistance of the D-peptides and D-amino acids towards biodegradation.

Thus, the illustrative examples of the Z sequence of peptides are: Lys-Lys-Lys-Lys, Xaa-Lys-Lys-Lys, Lys-Xaa-Lys-Lys, Lys-Lys-Xaa-Lys, Lys-Lys-Lys -Xaa, Xaa-Xaa-Lys-Lys, Xaa-Lys-Xaa-Lys, Xaa-Lys-Lys-Xaa, Lys-Xaa-Xaa-Lys, Lys-Xaa-Lys-Xaa, Lys-Lys-Xaa-Xaa , Xaa-Xaa-Xaa-Lys, Xaa-Xaa-Lys-Xaa, Xaa-Lys-Xaa-Xaa, Lys-Xaa-Xaa-Xaa, Xaa-Xaa-Xaa-Xaa, Lys-Lys-Lys-Lys-Lys , Xaa-Lys-Lys-Lys-Lys, Lys-Xaa-Lys-Lys-Lys, • Lys-Lys-Xaa-Lys-Lys, Lys-Lys-Lys-Xaa-Lys, Lys-Lys-Lys-Lys- Xaa, Xaa-Xaa-Lys-Lys-Lys, Xaa-Lys-Xaa-Lys-Lys, Xaa-Lys-Lys-Xaa-Lys, Xaa-Lys-Lys-Lys-Xaa, Lys-Xaa-Xaa-Iys- Lys, Lys-Xaa-Lys-Xaa-Lys, Lys-Xaa-Lys-Lys-Xaa, Lys-Lys-Xaa-Xaa-Lys, Lys-Lys-Xaa-Lys-Xaa, Lys-Lys-Lys-Xaa- Xaa, Lys-Lys-Xaa-Xaa-Xaa, Lys-Xaa-Lys-Xaa-Xaa, Lys-Xaa-Xaa-Lys-Xaa, Lys-Xaa-Xaa-Xaa-Lys, Xaa-Lys-Lys-Xaa- Xaa, Xaa-Lys-Xaa-Xaa-Lys, Xaa-Xaa-Lys-Lys-Xaa, Xaa-Xaa-Lys-Xaa-Lys, Xaa-Xaa-Xaa-Lys-Lys, Lys-Xaa-Xaa-Xaa- Xaa, Xaa-Lys-Xaa-Xaa-Xaa, Xaa-Xaa-Lys-Xaa-Xaa, Xaa-Xaa-Xaa-Lys-Xaa, Xaa-Xaa-Xaa-Xaa-Lys, Xaa-Xaa-Xaa-Xaa -Xaa, Ly s-Lys-Lys-Lys-Lys-Lys, Xaa-Lys-Lys-Lys-Lys-Lys, Lys-Xaa-Lys-Lys-Lys-Lys, Lys-Lys-Xaa-Lys-Lys-Lys, Lys- Lys-Lys-Xaa-Lys-Lys, Lys-Lys-Lys-Lys-Xaa-Lys, Lys-Lys-Lys-Lys-Lys-Xaa, Xaa-Xaa-Lys-Lys-Lys-Lys, Xaa-Lys-Xaa-Lys-Lys-Lys, Xaa-Lys-Lys-Xaa-Lys-Lys, Xaa -Lys-Lys-Lys-Xaa-Lys, Xaa-Lys-Lys-Lys-Lys-Xaa, Lys-Xaa-Xaa-Lys-Lys-Lys, Lys-Xaa-Lys-Xaa-Lys-Lys, Lys-Xaa -Lys-Lys-Xaa-Lys, Lys-Xaa-Lys-Lys-Lys-Xaa, Lys-Lys-Xaa-Xaa-Lys-Lys, Lys-Lys-Xaa Lys-Xaa-Lys, Lys-Lys-Xaa- Lys-Lys-Xaa, Lys-Lys-Lys-Xaa-Xaa-Lys, Lys-Lys-Lys-Lys-Xaa-Lys-Xaa, Lys-Lys-Lys-Lys-Xaa-Xaa, Xaa-Xaa-Xaa- Lys-Lys-Lys, Xaa-Xaa-Lys-Xaa-Lys-Lys, Xaa-Xaa-Lys-Lys-Xaa-Lys, Xaa-Xaa-Lys-Lys-Lys-Xaa, Xaa-Lys-Xaa-Xaa- Lys-Lys, Xaa-Lys-Xaa-Lys-Xaa-Lys, Xaa-Lys-Xaa-Lys-Lys-Xaa, Xaa-Lys-Lys-Xaa-Xaa-Lys, Xaa-Lys-Lys-Xaa-Lys- Xaa, Xaa-Lys-Lys-Lys-Xaa-Xaa, Lys-Lys-Lys-Xaa-Xaa-Xaa, Lys-Lys-Xaa-Lys-Xaa-Xaa, Lys-Lys-Xaa-Xaa-Lys-Xaa, Lys-Lys-Xaa-Xaa-Xaa-Lys, Lys-Xaa-Lys-Lys-Xaa-Xaa, Lys-Xaa-Lys-Xaa-Lys-Xaa, Lys-Xaa-Lys-Xaa-Xaa-Lys, Lys- Xaa-XaaLys-Lys-Xaa, Lys-Xaa-Xaa-Lys-Xaa-Lys, Lys-Xaa-Xaa-Xaa-Lys-Lys, Lys-Lys-Xaa-Xaa-Xaa-Xaa, Lys-Xaa-Lys- Xaa-Xaa-Xaa, Lys-Xaa-Xaa-Lys-Xaa-Xaa, Lys-Xaa-Xaa-Xaa-Lys-Xaa, Lys-Xaa-Xaa-Xaa-Xaa-Lys, Xaa-Lys-Lys-Xaa-Xaa-Xaa, Xaa-Lys-Xaa-Lys-Xaa-Xaa, Xaa- Lys-Xaa-Xaa-Lys-Xaa, Xaa-Lys-Xaa-Xaa-Xaa-Lys, Xaa-Xaa-Lys-Lys-Xaa-Xaa, Xaa-Xaa-Lys-Xaa-Lys-Xaa, Xaa-Xaa- Lys-Xaa-Xaa-Lys, Xaa-Xaa-Xaa-Lys-Lys-Xaa, Xaa-Xaa-Xaa-Lys-Xaa-Lys, Xaa-Xaa-Xaa-Xaa-Lys-Lys, Lys-Xaa-Xaa- Xaa-Xaa-Xaa, Xaa-Lys-Xaa-Xaa-Xaa-Xaa, Xaa-Xaa-Lys-Xaa-Xaa-Xaa, Xaa-Xaa-Xaa-Lys-Xaa-Xaa, Xaa-Xaa-Xaa-Xaa- Lys-Xaa, Xaa-Xaa-Xaa-Xaa-Xaa-Lys, Xaa-Xaa-Xaa-Xaa-Xaa-Xaa, wherein each Xaa is independently selected from the group consisting of Ala, Leu, Ser, Thr, Tyr , Asn, Gln, Asp, Glu, Arg, His, Met, Orn, and amino acids of the formula I as defined herein, for example, Dbu or Dpr.

The peptide stabilizing Z sequence can, in one embodiment, have a total charge in the range from about 0 to +15, preferably in the range from 0 to +10, example, from 0 to +8, in particular from about 0 to + 6, such as approximately 0 to +4, example, from 0 to +3, at a pH 7. Without being bound by any specific theory, this visualizes that the non-negative charge in the Z sequence of stabilizing peptide can also to some degree facilitate transport to and through cell membranes that have a negative power in the extracellular part. Thus, to ensure a non-negative total charge in the peptide stabilizing Z sequence, the peptide Z-sequence preferably comprises at least one amino acid residue Lys, more preferably at least two amino acid residues Lys, such as at least three amino acid residues Lys, eg, at least four amino acid residues Lys, even more preferably at least five amino acid residues, Lys such as at least six amino acid residues Lys.

As indicated above, the amino acid residues of Z may be of totally different course or be completely identical. However, in interesting embodiments of the present invention, the amino acid residues in Z are selected from two or three different amino acids, or are identical amino acids. Examples of suitable peptide sequences, where the amino acid residues in Z are identical are, for example, (Lys) n, where n is an integer in the range of 4 to 15, preferably in the range of 4 to 10, such as in the range of 4 to 8, example, in the range of approximately 4 to 6, example, Lys4, Lys5 or Lys6. Examples of peptide sequences, where the amino acid residues in Z are selected from about two different amino acids are, for example, (Lys-Xaa) mo (Xaa-Lys) m, where m is an integer in the range of about 2 to 7, preferably in the range of 2 to 5, such as in the range of 2 to 4, example, 3, and Xaa is independently selected from the group consisting of Ser, Thr, Tyr, Asn, Gln, Asp, Glu, Arg, His, Orn, 2,4-diaminobutanoic acid, 2,3-diaminopropanoic acid and Met. More preferably such peptide sequences are for example, (Lys-Xaa) 3 or (Xaa-Lys) 3, where -xaa is as defined above, such as (Lys-Glu) 3 or (Glu-Lys) 3. Other examples of suitable peptide sequences, where the amino acid residues in z are selected from approximately two amino acid residues are for example, Lysp-Xaaq or Xaap-Lysq, where p and q are integers in the range of 1 to 14. , with the proviso that p + q is in the range of 4 to 15, preferably in the range of 4 to 10, such as in the range of 4 to 8, for example, in the tango from 4 to 6, for example , 4,5 or 6, and Xaa is independently selected from the group consisting of Ser, Thr, Tyr, Asn, Gln, Asp, Glu, Arg, His and Met. More preferably such peptide sequences are for example, Lys3-Xaa3 or Xaa3-Lys3, where Xaa is as defined above, such as Lys3-Glu3 or Glu3-Lys3.

Examples of suitable peptide sequences, where the amino acid residues in Z are selected from three different amino acids are for example Xaa1- (Lys) x- (xaa2) y, Xaa1- (Xaa2) x- (Lys) and , (Lys) x- (Xaa2) y-Xaa1, (Xaax) x- (Lys) y-Xaa2, (LYS)? - Xaa1- (Xaa2) y, (Xaa1) X-Xaa2- (Lys) y, Xaa1 -Lys- Xaa2-Lys, Xaa1-Lys-Xaa2-Lys-Xaa2, Xaa1-Lys-Xaa2-Lys-Xaa2-Lys, XaaU? Aa2-Lys-Xaa2, Xaa1-Xaa2-Lys-Xaa-Lys, Xaa1-Xaa1 -Lys-Xaa2-Lys-Xaa2, Lys-Xaa2-Lys-Xaa1, Lys-Xaa2-LYS-Xaa2-Xaa1, Lys-Xaa2-Lys-Xaa2-Lys-Xaax, Xaa1-Lys-Xaa2-Xaa1, Xaa2-Lys -Xaa2-Lys-Xaax, Xaa2-Lys-Xaa1-Lys-Xaa2-Xaa1, etc., where x and y are integers in the range of approximately 1 to 4 with the condition that x + y is at most 5, and Xaa1 and Xaa2 is independently selected from approximately the group consisting of Ala, Leu, Ser, Thr, Tyr, Asn, Gln, Asp, Glu, Arg, His, Met, Orn, 2,3-diaminopropanoic acid, 2,4-diamonobutanoic acid and amino acids of formula I as defined herein.

With respect to the peptide sequence Z, it is visualized that the amino acid residues mentioned as constituents of the peptide Z sequence, eg, Ala, -Leu, Ser, Thr, Tyr, Asn, Gln, Asp, Glu, Lys, Arg, His, Met, Orn, 2, 3-diaminopropanoic acid (Dpr), 2,4-diaminobutanoic acid (Dbu) and the amino acid residues of formula I as defined herein, are amino acid residues which, due to its wide configurations around the a-carbon atom, and probably also due to a specific electronic configuration, it has certain preferences to participate in, or even stabilize or initiate, helix-like structures. The additional methodology of Chou-Fasman (Chou, P: Y: &Fasman, GD Ann. Rev. Biochem. 47, 251-276 (1978)) in an attempt to quantify (empirically) the possibility for an amino acid residue for being involved in an a-helix structure (expressed as the "conformational parameter Pa"). The Chou and Fasman studies and related studies have, however, to show that amino acid residues that have a low Pa parameter can be found in a-helix, but of course not when the amino acid residues have an elevated P a. Thus, in the peptide sequence Z, it is considered possible to include a proportion of amino acid residues that is not among the previously selected residues of Z, and in spite of obtaining the desired effects of the peptide Z sequence, in which the residues of Selected amino acids are believed to compensate for some negative or neutral effect of such alternative amino acid residues.

In a specific embodiment, Z is (Dbu) not (Dpr) n, where n is an integer in the range of about 4 to 15, preferably in the range of about 4 to 10, such as in the range of about 4 to 8 , for example, in the range of approximately 4 to 6. In a more specific embodiment, Z is Dprβ.

Thus, in embodiments that are within the scope of the present invention, it may be realistic to include up to 25% of amino acid residues that are not among the preferred amino acids as constituents of Z. (by "25% percent"). "refers to the number of amino acid residues, for example, non-alternative amino acid residues are allowed in di- and tripeptides, until an alternative amino acid residue is allowed in tetra-, penta-, hexa-, and heptapeptides , up to two amino acid residues are allowed in octapeptides, etc.). Such alternative amino acid residues can be selected from Val, Lie, Pro, Phe, Gly, Trp, as well as amino acid residues of N-methyl, however, preferably not Pro, Gly, and residues of N-methyl amino acids. In addition, the C-terminus of Z can be in the form of the free acid, the amide, or an ester, for example, methyl ester, ethyl ester, benzyl ester, etc., depending on the type of the solid support material and the cleavage conditions used in connection with the synthesis of the peptide conjugates as should be elucidated by a person skilled in the art. The C-terminal may be in the form of free amine or a lactam.

The stabilizing peptide Z sequences can be linked to the C-terminus or the N-terminus of the pharmacologically active peptide sequence, X, or two peptide sequences can be linked individually to both the N-terminal Cy of X. In In case that the pharmacologically active X-peptide possesses a free C-terminal carboxylic acid (as in the case of Leu-enkephalin), the peptide Z-sequence can be linked to either of the two C-terminus of the X-peptide or the N-terminal peptide X, or the C- and N-terminus of X can both be linked to each individual peptide Z sequence. Alternatively. Z can be linked to the nitrogen atom in the side chain of lysine, histidine or arginine or a carbonyl function in the side chain of glutamic acid or aspartic acid anywhere within the X sequence., Z can be linked to X within the sequence and to the N- one / or C-terminal of X. Both the sequence could be linked to the X sequence of peptide and its C-terminal, at its N-terminal, or in both, or depending on the sequence X of peptide in the specific peptide X and the pharmaceutical function that said peptide exerts and can be easily determined by a person skilled in the art. In some cases, the biological or physiological activation can depend crucially on the negative charge in the C-terminal of the pharmacologically active X-peptide. Consequently, in such cases, the activities and consequently the pharmacological effects of X may be obstructed by blocking the negative charge at the C-terminus of the pharmacologically active X-peptide and this may therefore be advantageous for linking the peptide Z-sequence to the N- terminal of the X peptide. In a similar manner, in cases where the pharmacologically active X-peptide is present in its natural form as a C-terminal amide (such as oxytocin) it may be advantageous to bind the Z sequence of stabilizing peptide to the -N -terminal of peptide X if it is believed that the amide group has an important pharmacological function. Thus, this could be understood as some peptide sequences corresponding to pharmacologically active X-peptides having a free C-terminal carboxyl group as well as peptides corresponding to pharmacologically active X-peptides having a C-terminal amide or an ester group which can be used in the conjugates of the invention. However, in an interesting embodiment of the invention the peptide Z sequence is linked to the C-terminus of the pharmacologically active X-peptide (both X in its natural form is a carboxylic acid, an amide or an ester).

It could be understood that "the peptide conjugates of the invention can also be in the form of a salt thereof." The salts include pharmaceutically acceptable salts, such as add-on salts and basic salts.Examples of addition salts are the hydrochloride salts. , sodium salts, calcium salts, potassium salts, etc. Examples of basic salts are salts where the cation is selected from alkali metals, such as sodium and potassium, alkaline earth metals, such as calcium ions and ammonium + N (R3) 3 (R4), where R3 and R4 independently designate optionally substituted C6_6alkyl, optionally substituted C2_6, optionally substituted aryl, or optionally substituted heteroaryl Other examples of pharmacologically acceptable salts are; for example, those described in "Remington's Pharmaceutical Sciences" 17. Ed. Alfonso R. Gennaro (Ed), Mark Publishing Company, Easton, PA, USA 1985 and in edition most recent, and in Encyclopedia of Pharmaceutical Technology.

In a more specific embodiment, the peptide conjugate is selected from the group consisting of H-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln- Asp-lle-Met-Ser-Arg-Gin-Gln-Gly-Glu-Ser-Asn-Gln-Glu-Arg-Gly-Ala-Arg-Ala-Arg-Leu-Lys6-NH2 (GHRH (1-44) (Human) -LYS6-NH2); H-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln- Asp-Ile-Met-Ser-Arg-Gln-Gln-Gly-Glu-Ser-Asn-Gln-Glu-Arg-Gly-Ala-Arg-Ala-Arg-Leu-Glu6-NH2 (GHRH (1-44) (Human) -Glu6-NH2); H-Lys6-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp- Leu-Ar-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe-OH (Lys6-PTH (1-34) (Human) -OH); In a specific embodiment, the peptide conjugate was selected from the group consisting of H-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln- Asp-Ile-Met-Ser-Arg-Gln-Gln-Gly-Glu-Ser-Asn-Gln-Glu-Arg-Gly-Ala-Arg-Ala-Arg-Leu-Lys6-NH2 (GHRH (1-44) (Human) -Lys6-NH2) (SEQ ID NO 88); H-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln- Asp-Ile-Met-Ser-Arg-Gln-Gln-Gly-Glu-Ser-Asn-Gln-Glu-Arg-Gly-Ala-Arg-Ala-Arg-Leu-Glu6-NH2 (GHRH (1-44) (Human) -Glu6-NH2) (SEQ ID NO: 89); H-Lys6-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys- His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp- Leu-Arg-Lys-Lys-Leu-.

Gln-Asp-Val-His-Asn-Phe-OH (Lys6-PTH (1-34) (Human) -OH) (SEQ ID NO. 90); - H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu -Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe-Lyse-OH (PTH (1-34) (Human) -Lys6-0H) (SEQ ID No. 91); H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala Trp-Leu-Val-Lys-Gly-Arg-Lys6-OH (GLP-1- (7-36) (Human) -Lys6-0H) (SEQ ID NO: 92); H-Gly-Gly-Thr-Tyr-Ser-Cys (Acm) -His-Phe-Gly-Pro-Leu-Thr-Trp- Val-Cys (Acm) -Lys-Pro-Gln-Gly-Gly-Lys6- OH (EMP-1-Lys6-0H) (SEQ ID NO 93); H-Lys6-Gly-Gly-Thr-Tyr-Ser-Cys (Acm) -His-Phe-Gly-Pro-Leu-Thr-Trp-Val-Cys (Acm) -Lys-Pro-Gln-Gly-Gly- OH (Lys6-EMP-1-OH) (SEQ ID NO 94); H-Tyr-Gly-Gly-Phe-Leu-Lys 6-OH (H-Leu-enkephalin-Lys6); H-Lys6-Tyr-Gly-Gly-Phe-Leu-Lys6-OH (H-Lyse-Leu-enkephalin-Lys6-OH); Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly- (Lys6-OH (GnRH-Lys6-OH); Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly- (Lys-Glu) 3-OH (GnRH- (Lys-Glu) 3-0H); Y H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe- (Lys-Glu) 3-OH (PTH 1-34 human- (Lys-Glu) 3-0H).

As explained above, the peptide Z sequence is the part of the peptide conjugate responsible for introducing a certain structure within the molecule that must yield the most stable compound towards the degradation catalyzed by the protease. Accordingly, the present invention also relates to the use of a stabilizing peptide (Z) sequence as defined above for the preparation of a pharmacologically active peptide conjugate as defined above.

As previously mentioned, routes of administration of pharmacologically active peptides are thus rather limited due to rapid biodegradation by proteases such as chymotrypsin, trypsin, carboxypeptidase A, pepsin, leucine aminopeptidase, etc. Thus, the requirements of the pharmacologically active peptide conjugates suitable for the claimed purpose is that on the one hand, the peptide conjugate could, at least for some, be capable of resisting protease-catalyzed hydrolysis, and on the one hand , the peptide conjugate could still, at least for some reach, be able to exert the desired pharmaceutical effect normally supplied by free X-peptide.

With these bases, in vi tro assays have to be developed to give an assessment of the ability of a peptide conjugate to exert the desired properties. Such tests, as well as the results thereof, are illustrated in the examples. These types of tests are excellent preliminary tests that can be easily performed by a person skilled in the art to assess the applicability of some given peptide conjugate prepared according to the principles disclosed herein.

Thus, the tendency of the pharmacologically active peptide conjugates of the invention to resist protease-catalyzed hydrolysis can be measured directly by the in vi tro enzyme assay shown in the examples. The tendency of the peptide conjugate to resist degradation can for example be expressed as a pseudo constant of first order and / or as the half-life of said peptide conjugates, which can then be compared with the corresponding values of the free X-peptide.

As it should be apparent from the examples provided herein, it has been found that it is possible to obtain a remarkable increase in the half-life (t? / 2) of a pharmacologically active peptide sequence by conjugating the peptide (X) in question with a sequence ( Z) of stabilizing peptide according to the invention.

Thus, in one embodiment of the invention, the ratio between the half-life of the peptide conjugate in question in the "Hydrolysis in the enzyme solution test", as defined herein, and the half-life of the peptide (X ) corresponding free, in the "Hydrolysis in the enzyme solution test", is at least about 2, preferably at least about 3, such as at least about 5, more preferably at least about 20 , such as at least about 50, example, at least about 100, when a carboxypeptidase A or leucine aminopeptidase is used.

Although carboxypeptidase A proteases and leucine aminopeptidase have been used in the tests described herein, it is visualized that the ability of peptide conjugates to resist protease degradation can also be examined in identical or similar test systems using other endo- or exopeptidases, such as trypsin, or mixtures of such peptidases, examples, artificial gastric juice.

In addition, the ability of the peptide conjugates of the invention to exert the desired pharmaceutical effect was examined in various in vitro and in vivo assay methods disclosed herein. Thus, the preferred peptide conjugates are such conjugates which exert some pharmaceutical effects, preferably a similar one and in some cases even an improved pharmaceutical effect compared to the pharmacologically active free peptide (X).

As it should be understood by the examples provided herein, the peptide conjugates of the invention are capable of "surviving" at various proteolytic barriers present in the gastrointestinal environment. Thus, as demonstrated here in the examples, it is possible to administer a pharmacologically active peptide conjugate (example, orally) as some fractions of the administered peptide conjugate (example., at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% , 75%, 80%, 85%, 90%, 95%, or even at least 99% of the total amount of peptide conjugate administered) is able to enter the bloodstream. Accordingly, the especially interesting peptide conjugates of the invention are such compounds as when administered orally at a pharmacologically effective dose (which of course depend on the current disease or disorder to be treated and the current peptide or peptide conjugate selected for said treatment ) is present in the bloodstream at a therapeutically or prophylactically active concentration after a period of about 0.1 minutes to 24 hours, 0.1 minutes to 5 hours, eg, after a period of about 0.5 minutes to 3 hours, such as about 1 minute to 2 hours, preferably after a period of about 3 minutes to 1 hour, such as about 5 minutes to 1 hour, example, about 10 minutes to 1 hour, 1 minute -16 hours, 0.1 minutes -12 hours. The relevant therapeutic concentrations of said peptide conjugates must, of course, depend on the current disease or disorder to be treated, and such concentrations must be known to a person skilled in the art.

In addition, the peptide conjugates of the invention are surprisingly stable in the example, serum and blood plasma. Thus, the peptide conjugates of the invention are such compounds having a half-life in the serum or plasma of human or mouse (which optionally contain a buffer to ensure a certain pH, for example, a pH of 7.4) at 37 ° C of at least about 10 minutes, such as about 15 minutes, for example, at least 0.5 hours, preferably at least about 1 hour, such as at least about 2 hours, for example, at least about 3 hours, more preferably at least about 4 hours, such as at least about 5 hours, for example, at least about 6 hours, in particular at least about 12 hours, such as at least about 24 hours, for example, at least about 36 hours. It is especially preferred where the ratio of the half-life of said peptide conjugate and the half-life of the corresponding pharmacologically active peptide sequence, X, in the plasma or in the serum is at least about 2, preferably at least about 3. , such as at least about 5, more preferably at least about 7, tai like at least about 9, for example at least about 10.

Compositions The invention also concerns a composition comprising a pharmacologically active peptide conjugate as defined above in combination with a pharmaceutically acceptable carrier. Such compositions may be in a form adapted for oral, subcutaneous, parenteral (intravenous, intraperitoneal), intramuscular, rectal, epidural, intranasal, dermal, vaginal, buccal, ocular, direct to the brain or pulmonary administration, preferably in a form adapted to an oral administration, and such compositions, can be prepared in a manner well known to a person skilled in the art, for example, as generally described in "Remington's Pharmaceutical Sciences," 17. ED. Alfonso R. Gennaro (Ed), Mark Publishing Company, Easton, PA, U.S.A., 1985 and in more recent editions and in the monograph in the "Drugs and the Pharmaceutical Sciences" series, Marcel Dekker. The compositions may appear in conventional forms, for example, capsules, tablets, aerosols, solutions, suspensions or typical applications.

The carriers or pharmaceutical solvents used can be a conventional solid or liquid carrier. Examples of carriers are lactose, alba earth, sucrose, cyclodextrin, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid or lower alkyl cellulose ethers. Examples of liquid carriers are syrup, peanut oil, olive oil, phospholopids, fatty acids, fatty acid amines, polyoxyethylene and water.

Similarly, the carrier or solvent may include some sustained release material known in the art, such as glyceryl monostearate or glyceryl distereate, alone or mixed with a wax.

Without a solid carrier advertised for oral administration, the preparation can be tableted, placed in a hard gelatin capsule in the form of a powder or a tablet or it can be in the form of a tablet or tablet. The amount of the solid carrier can vary widely. it should usually be from about 25 mg to about 1 gram.

A typical tablet that can be prepared by conventional tabletting techniques may contain: In the center: 100 mg of an active compound (as free compound or salt thereof); 1.5 mg of colloidal silicon dioxide (Aerosil); cellulose, 70 mg of microcrystals (Avicel); 7.5 mg of modified cellulose gum (Ac-Di-Sol); magnesium stearate.

In the coating: approximately 9 mg of HPMC; approximately 0.9 mg of * Muwacett 9-40T; * adhered monoglyceride .. used as a plasticizer for the coating film.

If a liquid carrier is used, the preparation can be in the form of a syrup, emulsion, soft gelatin capsule or a sterile injectable liquid such as a non-aqueous liquid suspension or a solution.

For nasal administration, the preparation may contain a conjugate of the present invention dissolved or suspended in a liquid carrier, in particular, in an aqueous carrier, for an aerosol application. The carrier may contain additives such as solubilizing agents, for example, propylene glycol, surfactants such as salts of bile acid or polyoxyethylene high in alcohol esters, absorption linkers such as lecithin (phosphatidylchloro) or cylcodextrin, or preservatives such as parabins The composition may also be in a form suitable for an injection or systemic or an infusion and may, as such, be formulated with sterile water or an isotonic saline or a glucose solution. The compositions can be sterilized by conventional sterilization techniques that are well known in the art. The resulting aqueous solutions can be packaged by use or filtered under aseptic conditions and lyophilized, the preparation of the lyophilized was combined with a previous sterile aqueous solution for administration. The composition may contain pharmaceutically acceptable excipients as required under the approximate physiological conditions, such as buffering agents, tonicity adjusting agents and the like, for example sodium acetate, sodium lactate, sodium chloride, potassium chloride. , calcium chloride, etc.

The invention also concerns a pharmacologically active peptide conjugate as defined herein for use in therapy, and the use of a pharmacologically active peptide conjugate as defined above for the manufacture of a pharmaceutical composition for use in therapy, by example, in the treatment of disorders in the central nervous system, in vaccine therapy, • and in HIV treatment, cancer, diabetes, incontinence, hypertension, amnesia, Alzheimer's disease, fever, depression, sexual hormone regulation , eating, schizophrenia, osteoporosis and insomnia, and as analgesics and antiseptics, and such indications known to be treated by therapy comprising the administration of pharmacologically active peptides.

In specific embodiments, a conjugate comprising enkephalin and Z can be used to inhibit neurons from transmitting pain impulses, a conjugate comprising the growth hormone releasing hormone or the growth hormone and Z release peptide. can be used to stimulate the release of growth hormone, for use in stimulating the release of growth hormone, a conjugate comprising AMP-1 and Z can be used to increase the levels of hemoglobin a conjugate that comprises the hormone of parathiodes and Z can be used to treat or prevent bone loss, a conjugate comprising glycine-like peptide-1 and Z can be used in the treatment of diabetes, a conjugate comprising the peptide induction to delta sleep and Z can be used to treat sleeping disorders and a conjugate comprising the gonadotropin releasing hormone and Z to be used to regulate sex hormones.

As mentioned above, a major obstacle to the application of peptides as clinically useful drugs is that their poor distribution characteristics of most peptides are rapidly metabolized by proteolysis at the most routes of administration. Accordingly, a very interesting aspect of the present invention is that it is possible to prepare pharmacologically active peptide conjugates for the treatment of mammals, such as humans, which are stabilized towards degradation by proteases and, at the same time, they are capable of exerting an effective effect in the environment in which the free (X) peptide must exhibit a pharmaceutical action. Accordingly, the present invention also relates to the use of a pharmacologically active peptide conjugate as defined above for the manufacture of a pharmaceutical composition for the treatment or prophylaxis of a condition or disorder, wherein the X peptide sequence, when not linked to Z, is able to interact with a receiver (or a receiver system) involved with the condition in question, and where the interaction between X, when not linked to Z, and the receiver (or receiver system) have a therapeutic or prophylactic effect in the condition of disorder. Thus, it could be understood that a peptide conjugate of the present invention can substitute free peptide (X) in for example, therapies where peptide X is administered intravenously since the peptide conjugates of the invention can be administered in a more convenient, for example, orally, as said peptide conjugates are able to overcome the predominant proteolytic barriers in the body. In a similar manner, the peptide conjugates of the invention can be used in therapies where it had not previously been possible to use the free peptide (X) as X had been readily degraded in or secreted from the body.

Preparation of conjugates The peptide conjugates of the present invention can be prepared by methods known per se in the art. Thus, the peptide X and Z sequences can be prepared by standard peptide preparation techniques such as synthesis of the solution or synthesis of the solid phase of the Merrifield type.

In a possible synthetic strategy, the peptide conjugates of the invention can be prepared by the synthesis of the solid phase by the first construction of the peptide Z sequence using the well-known standard protection, the deprotection binding procedures, after this sequentially linking the pharmacologically active X or Z sequence in a manner similar to the Z construct, and finally fractionating the entire peptide conjugate from the carrier. This strategy produces a peptide conjugate, wherein the Z sequence of stabilizing peptide is covalently linked to the pharmacologically active X peptide to the C-terminal carbonyl function of X. If the desired peptide conjugate, however, is a peptide conjugate, where two stabilizing Z sequences are covalently and independently linked to both the C- and the N-terminus of the pharmacologically active X-peptide, the above strategy is also applicable but, as understood by a person skilled in the art, it is necessary to sequentially link the second Z sequence of N-terminal stabilizing peptide of X in a manner similar to the above described procedure. This strategy can also be used to link Z to the carbonyl function in the side chain of Glu or Asp.

A possible strategy for the preparation of the peptide conjugates, wherein the Z-sequence of stabilizing peptide is covalently linked to the N-terminal nitrogen atom or covalently bonded to the nitrogen atom in the side chain of Lys, Arg, or His of X is analogous to the above described method, for example, peptide conjugates can be prepared by synthesis in the solid phase by the first construction of the pharmacologically active peptide X sequence using the well-known standard protection, the binding and deprotection procedures, after sequentially linking the Z or X sequence of stabilizing peptide in a manner similar to the construction of X, and finally cleaving the peptide conjugate from the carrier.

Another possible strategy to prepare one or both of the two sequences x and Z (or parts thereof) is separately by the synthesis of the solution, the synthesis in the solid phase, recombinant techniques, or enzymatic synthesis, followed by the binding of the two sequences by well-known segment condensation procedures, either in the solution or techniques in the solid phase or a combination thereof. In one embodiment, X can be prepared by the recombination methods of DNA and Z can be prepared by synthesis in the solid phase. The conjugation of X and Z can be carried out by using chemical ligation. This technique allows for the assembly of completely deprotected peptide segments in a highly specific manner (Liu et al., 1996, J. Am. Chem, Soc. 118: 307-312 and Dawson et al., 1996, 226: 776). The conjugation can also be performed by the formation of the peptide bond catalyzed by the protease, which offers a highly specific technique for combining peptide segments deprotected totally via a peptide bond (W. Kullmann, 1987, Enzymatic Peptide Synthesis, CRC Press, Boca Raton, Florida, pp. 41-59 The derivation of the side chain of Lys, Arg, His, Trp, Ser, Thr, Cys, Tyr, Asp and Glu with the stabilizing peptide sequence, Z can be carried out by the synthesis of the traditional convergent peptide using suitable orthogonal protection schemes known in the art, or by using the method of the generally known solid phase with adequate protection of the movable side chain.

In addition, it is envisioned that a combination of the aforementioned strategies may be especially applicable where a modified peptide sequence, e.g., from a pharmacologically active peptide comprising isosteric bonds such as reduced peptide bonds or Na-substituted peptide bonds. , to be linked to a peptide Z sequence. In this case, this may be advantageous for preparing the immobilized fragment of Z by the successive linkage of amino acids, and then linking a complete X sequence of pharmacologically active peptide (prepared in the solution or completely or partially using the solid phase techniques). or by means of recombination techniques) to the fragment.

Examples of suitable support materials (SSM) are for example, functionalized resins such as polystyrene, polyacrylamide, polymethylacrylamide, polyethylene glycol, cellulose, polyethylene, polyethylene glycol grafted to polystyrene, latex, dynaccounts, etc.

It could be understood that it may be necessary or desirable that the C-terminal amino acid of the peptide Z-sequence or the C-terminal amino acid of the pharmacologically active X-peptide is bound to the solid support material by means of a common linker such as zinc. , 4-dimethoxy-4'-hydroxy-benzophenone, 4- (4-hydroxy-methyl-3-methoxyphenoxy) -butyric acid, 4-hydroxy-methylbenzoic acid, 4-hydroxymethyl-phenoxyacetic acid, 3- (4-hydroxymethylphenoxy) propionic, and p- [(R, S) -a [1- (9H-fluoren-9-yl) methoxyformamido] -2,4-dimethoxybenzyl] -phenoxy-acetic acid.

The peptide conjugates of the invention can be fractionated from the support material by means of an acid such as in trifluoroacetic acid, trifluoromethanesulfonic acid, hydrogen bromide, hydrogen chloride, hydrogen fluoride, etc., optionally in the combination with one or more "scavengers" suitable for the purpose, for example, ethanedithiol, triisopropylsilane, phenol, thioanisole, etc., or the peptide conjugate of the invention can be fractionated by the solid support by means of a base such as, ammonia, hydrazine, an alkoxide, such as sodium ethoxide, a hydroxide, such as sodium hydroxide, etc.

Thus, the present invention also relates to a method for the preparation of a pharmacologically active peptide conjugate, wherein Z is covalently linked to X in the C-terminal function of X (X-Z), comprising the steps of: a) linking a protected N-a amino acid in the activated carbonyl form, or a protected N-a dipeptide in the activated c-terminal form to an immobilized peptide H-Z-SSM sequence, consequently forming an immobilized protected N-a peptide fragment, b) removing the Na group, consequently forming a fragment of immobilized peptide having an N-terminal deprotected end, c) linking an additional protected Na amino acid in the activated carbonyl form, or an additional protected Nape dipeptide in the C-form terminal activated at the N-terminal end of the immobilized peptide fragment, and repeat the process of extraction / binding steps in step b) and c) until the desired sequence of peptide X is obtained, and then d) cleaving the peptide conjugate from the solid support material.

In another aspect the present invention also relates to a method for the preparation of a pharmacologically active peptide conjugate, wherein Z is covalently linked to the N-terminal nitrogen atom of X (Z-X), which comprises the steps of: a) binding a protected Na amino acid, or a protected Na dipeptide to a solid support material (SSM), consequently forming an immobilized protected Na amino acid, or an immobilized protected Napin dipeptide fragment, b) extracting the protective Na group, consequently forming an immobilized amino acid or a fragment of peptide having an N-terminal deprotected end, c) linking an additional protected Na-amino acid in the activated carbonyl form, or an additional protected Nape dipeptide in the C-terminal form activated at the N-terminal end of the immobilized amino acid or peptide fragment, and repeating the step extraction procedure / binding in step b) and c) until the desired sequence of peptide X is obtained, d) linking an additional protected N-a amino acid in the activated carbonyl form, or an additional protected N-a dipeptide in the activated C-terminal form at the end of the N-terminus of the immobilized peptide fragment, and repeating the extraction / binding step procedure in step b) and d) until the desired peptide sequence Z is obtained, and then e) cleaving the peptide conjugate from the solid support material.

"In another aspect of the present invention relates to the method for the preparation of a pharmacologically active peptide conjugate, wherein a first sequence (Z) is covalently linked to X to the C-terminal function of X a second sequence (Z) is covalently bonded to the N-terminal nitrogen atom of X (ZXZ), comprising the steps of: a) binding protected N-a amino acid in the activated carbonyl form, or a protected N-a dipeptide in the activated C-terminal form to an immobilized peptide H-Z-SSM sequence, consequently forming an immobilized protected N-a peptide fragment, b) extracting the protective N-a group, consequently forming an immobilized peptide fragment having an N-terminal deprotected end, c) linking an additional protected Na-amino acid in the activated carbonyl form, or an additional protected Nape dipeptide in the C-terminal form activated to the N-terminal end of the immobilized peptide fragment, and repeating the extraction / binding step procedure in step b) and c) until the desired Z peptide sequence is obtained, and then d) linking an additional protected Na-amino acid in the activated carbonyl form, or an additional additional protected Nape dipeptide in the activated C-terminal form at the end of the N-terminus of the immobilized peptide fragment, and repeating the extraction step procedures / link in step b) and d) until the desired peptide sequence is obtained, and then e) cleaving the peptide conjugate from the solid support material.

The linking, extraction and fractionation steps are carried out by methods known to a person skilled in the art taking into account the consideration of the protection strategy and the material in the selected solid phase. In general, however, it is believed that protection strategies with Boc (tert.butyloxycarbonyl) as well as Fmoc (9-fluorenylmathyloxycarbonyl) are applicable and that peptide bonds can be formed using the various activation procedures known per se. the person skilled in the art, for example, by reacting an activated derivative (acid halide, acid anhydride, activated ester for example, Hobt ester, tec.) of the appropriate amino acid or of the peptide with the amino group of the amino Relevant acid or peptide known to a person skilled in peptide chemistry.

In addition, it may be necessary or desirable to include side chain protection groups when using amino acid residues carrying functional groups that react under the prevailing conditions. The necessary protection scheme must be known to the person skilled in the art (see, for example, M. Bodanszky and A. Bodanszky, "The Practice of Peptides Synthesis", 2. Ed, Springer-Verlaag, 1994, J. Jones, "The Chemical Synthesis of Peptides", Clarendon Press, 1991, and Dryland et al., 1986, J. Chemm. Soc., Perkin Trans. 1: 125-137).

The peptide conjugates can also be prepared by means of recombinant DNA technology using the general methods and principles known to the person skilled in the art. A nucleic acid sequence encoding the conjugate can be prepared by synthetically prepared by standard established methods, for example, the phosphoamidite method described by S.L. Beaucage ang M.H. Caruthers, Tetrahedron Letters 22, 1982, pp. 801-805, or the method described by Matthes et al., EMBO Journal 3, 1984. pp. 801-805. According to the phosphoamidite method, oliogonucleotides are synthesized, for example, in an automated DNA synthesizer, purified, hardened, ligated and cloned into suitable vectors.

The techniques used to isolate or clone a nucleic acid sequence encoding the pharmacologically active X-peptide are known in the art and include isolation of the genomic DNA, preparation of the cDNA, or a combination thereof. The cloning of the nucleic acid sequences of the present invention from the genomic DNA can be effected, for example, by using the well-known polymerase chain reaction (PCR) or the antibody filtrate of expression libraries for detect cloned DNA fragments with shared structural characteristics. See, for example, Innis et al., 1990, A. Guide to Methods and Application, Academic Press, New York. Other methods of nucleic acid amplification such as the ligase chain reaction (RCL), activated ligand transcription (TLA) and amplification based on the nucleic acid sequence (ABSAN) can be used. This can then be linked to a nucleic acid sequence encoding Z.

The nucleic acid sequence encoding the conjugate is then grafted into a recombinant expression vector which may be some vector which may conveniently be subjected to recombinant DNA procedures. The choice of a vector must often depend on the host cell into which it is introduced. Thus, the vector can be an autonomously duplicated vector, for example, a vector that exists as an extrachromosomal entity, the copy of which is independent of the copy of the chromosome, eg, a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the chromosome of the host cell and copied together with the chromosome (s) into which it has been integrated.

Examples of suitable promoters for directing the transcription of the nucleic acid sequence encoding the conjugate, especially in a host cell of a bacterium, are the promoters obtained from E. coli lac operon, the gene from the coelicolor agarase of the streptomycosis ( Adag), the levansucrase gene subtilis of the bacillus (Bsac), the amylase gene alpha liceniformis of the bacillus (Mamy), the amylase gene alpha amiloliquefaciens of the bacillus (Qamy), the gene of the penicillinase liceniformis of the bacillus (Ppen), the Axyl and Bxyl subtilis genes of the bacillus, and the prokaryotic beta lactamase gene (Villa-Kamaroff et al., 1978, Proceedings of the National Academy of Sciences USA 75: 3727-3731), as well as the tac promoter (DeBoer et al., 1983, Proceedings of the National Academy of Sciences USA 80: 21-25). Other promoters are described in "Useful proteins from recombinant bacteria" in Sciences American, 1980, 242: 74-94; and in Sambrook et al., 1989, supra.

Examples of promoters suitable for directing the transcription of the nucleic acid sequence encoding the conjugate in a filamentous fugitive host cell are promoters obtained from the genes that condition the alkalase TAKA Aspergillus orizae, aspartic proteinase Rhizomucor miehei, alpha- neutral Aspergillus niger amylase, stable alpha-amylase of Aspergillus niger acid, glucoamylase (glaA) Aspergillus awamori or Arperllius niger, Rhizomucor iehei lipase, Aspergillus orizae alkaline protease, Trperose isomerase Aspergillus orizae, Aspergillus nidulans acetamidase, Fussrium trypsin-like protease oxuysporum (as described in US Patent No. 4,288,627, which is incorporated herein by reference), and hybrids thereof. Particularly preferred promoters for use in the filament • host cells are the TAKA amylase, NA2-t? I (a hybrid of the promoters from the genes coding for the neutral amylase Aspergillus niger and the isomerase of phosphate triose Aspergillus orizae ), and the glaA promoters.

In a yeast host, the useful promoters are obtained by means of the gene (ENO-1) of the enolase Saccharomyces cerevisiae, the gene (GALI) - of the galacticinase Saccharomyces cerevisiae, the genes (ADH2 / GAP) of the dehydrogenase dehydrogenase / glyceraldehyde-3-phosphate alcohol Saccharomyces cerevisiae, and the 3-phosphoglycerate kinase gene Saccharomyces cerevisiae. Other useful promoters for the host cells of the ferment are described by Romanos et al., 1992, Yeast 8: 423-488.

The nucleic acid sequence encoding said conjugate can also be operable connected to a suitable terminator, such as a hormone terminator. human growth (Palmiter et al., op.cit.). Preferred terminators for the fungal filament host cells are obtained by means of the genes encoding the TAKA amylase Aspergillus orizae, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, anthranilate tape, alpha-glucosidase Aspergillus niger, and Fusarium trypsin-like protease oxisporum.

The terminators for the ferment host cells are obtained by means of the gene encoding Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), or Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other terminators useful for the host cells of the ferment are described by Romanos et al., 1992, supra.

The vector may also comprise elements such as the polyadenylation signals (for example, by means of SV 40 or the adenovirus 5 Elb region), the transcriptional linker sequences (e.g., the SV 40 linker). In addition, the preferred polyadenylation sequences for the fungal filament host cells are obtained by means of the genes encoding the TAKA amylase Aspergillus orizae, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate, and Aspergillus niger alpha-glucosidase. Useful sequences of polyadenylation for ferment host cells are described by Guo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

The recombinant expression vector can also comprise a DNA sequence providing the vector to be duplicated in the host cell in question. Examples of such a sequence (when the host cell is a cell of a mammal) is the SV 40 or the origin of the polyoma of the duplication. Examples of the bacterial origins of duplication are the origins of the duplication of plasmids PBR322, pUC19, pACYC177, pACYC184, pUBUO, pE194, pTA1060, and pAMßl. Examples of the origin of the duplications for use in a host cell of the ferment are the origins of 2 microns of the duplication, the combination of CEN6 and ARS4, and the combination of CEN3 and ARS1. The origin of the duplication can be one that has a mutation to make its function sensitive to temperature in the host cell (see, for example, Ehrlich, 1978, Proc Nati Acad Sci USA 75: 1433).

The vector may also comprise a selectable producer, for example, a product gene such complements a defect in the host cell, such as the gene encoding reductase dihydrofolate (DHFR) or one that queries the resistance of a drug, e.g. , neomycin, geneticin, ampicillin, or hydramycin. Suitable producers for the host cells of the ferment are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. A selectable producer for use in the fungal filament host cell may be selected from the group including, but not limited to, amdS (acetamidase), argB (ornithine carbamoitransferase), bar (phosphinothricin acetyltransferase), hygB (phosphotransferase hydromycin), niaD (nitrata reductase), pyrG (orotidine-5'-decarboxylase phosphate), sC (adenyltransferase sulfate), trpC (anthranilate tape), and glufosinate resistance producers, as well as equivalents from other species. Preferred for use in an Aspergillus cell are the amdS and pyrG producers of Aspergillus nidulans or Aspergillus oryzae and the producer bar of Streptomyces hygrodcopicus. In addition, the selection may be performed by cotransformation, for example, as described in WO 91/17243, where the selectable producer is on a separator vector.

The methods used to ligate the nucleic acid sequences encoded for the conjugate, the promoter and the terminator, respectively, and to insert them into suitable vectors containing the information necessary for duplication, are well known to persons skilled in the art ( see, for example, Sambrook et al., op.cit.).

The host cell within which the expression vector is introduced may be to some cell which is capable of producing the conjugate and is such a eukaryotic cell, such as the invertebrate cell (insect) or vertebrate cells, for example. , lavender or mammalian Xenopus cells, in a particular insect and in mammalian cells. Examples of mammalian cell lines are the COS (eg, ATCC CRL 1650), BHK (eg, ATCC CRL 1632, ATCC CCL 10) or CHO cell lines (for example, ATCC CCL 61).

Methods for transferring mammalian cells and expressed DNA sequences introduced into cells are described in, for example, Kaufman and Sharp, 1982, J. Mol. Biol. 159: 601-621; Southern and Berg, 1982, J. Mol. Appl. Genet 1: 327-341; Loyter et al., 1982, Proc. Netl. Acad. Sci.

USA 79: 422-426; Wingler et al., 1978, Cell 14: 725; Corsaro and Pearson, 1981, Somatic Cell Genetics 7: 603, Graham and van der Eb, 1073, Virology 52: 456; Fraley et al., 1980, JBC 225: 10431; Capecchi, 1980, Cell 22: 479; Wiberg et al., 1983, NAR 11: 7287; and Neumannet al., 1982, EMBO J. 1: 841,845.

The host cell may also be a unicellular pathogenic agent, for example, a prokaryote, or a non-unicellular pathogenic agent, e.g., a eukaryote. Useful unicellular cells are bacterial cells such as a gram of positive bacteria including, but are limited to, a Bacillus cell, for example, Bacillus alkalopphilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus ciscunlans, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus mega terium, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis; or a Streptomyces cell, for example, Streptomyces linidans or Streptomyces murinus, or a gram of negative bacteria such as E. Coli and Pseudomonas sp. The transformation of a bacterial host cell, for example, is effected by the protoplast transformation (see, for example, Chang and Cohen, 1979, Molecular General Genetic 168: 111-115), by using competent cells (see, for example, Yonug and Spizizin, 1961, Journal of Bacteriology 81: 823-829, or Dubnar and Davidoff Abelson, 1971, Journal of Molecular Biology 56: 209-221), by electroporation (see, for example, Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or by conjugation (see, for example, Koehier and Thorme, 1987, Journal of Bacteriology 169: 5771-5278).

The host cell can be a furigical cell. "Fungi" as used here includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (as defined by Hawksworth et al., En, Ainsworth and Bisby 's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press , Cambridge, UK) as well as the Oomycota (as cited in Hawsworth et al., 1995, supra, page 171) and all fungi mitosporicas (Hawksworth et al., 1995, supra). Representative groups of the Ascomycota include, for example, Neurospora, Eupenicillium, (= Penicillium), Emericella (= Aspergillus), Eurotium (= Arpergillus), and the true ferments listed above. The fleeting host cell can also be a ferment cell. "Ferment" as used here includes ascosporogenous ferment (Endomycetales), ferment basidiosporogenous, and the ferment belongs to the Fungi Imperfecti (Blastomycetes). The medium used to grow the cells may be some conventional medium suitable for mammalian growth cells, such as one containing serum or a serum-free medium containing suitable supplements, or a suitable medium for the growth of the insect, the ferment or fleeting cells. Suitable means are available from commercial suppliers or can be prepared according to published recipes (for example, in the catalogs of American Type Culture Collection).

The conjugate produced by the cells can then be recovered by the culture medium by conventional methods including separating the host cell by means of centrifugation or filtration, precipitating protein components of the supernatant or filtering by means of a salt, for example, sodium sulfate. ammonium, purification by a variety of chromatographic methods, for example, ion exchange chromatography, affinity chromatography, or the like.

The invention is illustrated by the following examples. EXAMPLES Synthesis of the peptide General procedures Apparatus and synthetic strategy The peptides are intermittently synthesized in polyethylene containers equipped with a polypropylene filter for filtration using 9-fluorenylmethyloxycarbonyl (Fmoc) as the protective group Na amino and suitable common protection groups for functionalities of the side chain (Dryland et al., 1986, J. Chem. Soc., Perkin Trans. 1: 125-137).

Solvents The DMF solvent (N / N-dimethylformamide, riedel de-Háen, Germany) was purified by passing through a column packed with hard cation exchange resin (Lewatit S 100 MB / H strong acid, .Bayer AG Leverkusen, Germany) and analyzed by free amines previously used for the addition of 3,4-dihydro-3-hydroxy-4-oxo-l, 2,3-denzotriazine (Dhbt-OH) giving an elevation to the yellow color (D? TO- anion) yes the amines, free are present. The solvent DCM (dichloromethane, analytical grade, Riedel de-Háen, Germany) was used directly without purification.

Amino Acids The Fmoc-protected amino acids were purchased either from Milligen (UK) and through PerSeptive Biosystems GMBH Hamburgd, Germany in the form of suitable protected side chains. The non-protein amino acids FmocOrn (Boc) -OH, Fmoc-2-D-Nal-OH, Fmoc-D-Phe-OH, Fmoc-Aib-OH were purchased via Bachem (Switzerland) and FmocDbu (Boc) - OH, FmocDpr (Boc) -OH by means of Neosystem, France. Linker The (4-hydroxymethylphenoxy) acetic acid (HMPA) from Novabiochem, Switzerland was bonded to the resin as well as a pre-formed 1-hydroxybenzotriazole ester (HObt) or generated in it by the DIC. Linkage Reagents The diisopropylcarbodiimide (DIC) linkage reagent was purchased by (Riedel de-Háen, Germany) and distilled before use, dicylohexylcarbodiimide (DDC) was purchased via Merck-Schuchardt, München, Germany, and it was purified by distillation. Solid supports Peptides synthesized according to the Fmoc strategy were synthesized with the following types of solid support using 0.05 M or higher concentrations of Fmoc-activated amino acid protected in DMF.l) PEAG-PS (polyethylene glycol grafted to polystyrene; 2) NovaSyn TG resin, 0.29 mmol / g, Novabiochem, Switzerland); 3) Tentagel S resins 0.22-0.31 mmol / g (TentaGel-S-NH2; TentaGel S-Ram, TentaGel S PHB-Lys (Boc) Fmoc, TentaGel S RAM-Lys (Boc) Fmoc, Rapp polymer, Germany ). catalysts and other reagents' Diisopropylethylamine (DIEA) was purchased by means of Aldrich, Germany, and ethylenediamine by means of Fluka, piperidine and pyridine by means of Riedel-de Háen, Frankfurt, Germany. 4- (N,, -dimethylamino) pyridine (DMAP) was purchased by means of Fluka, Switzerland, and used as a catalyst in the binding reactions involving the symmetric anhydrides. Ethanedithiol was purchased through Riedel-de Haen, Frankfurt, Germany. 3, 4-Dihydro-3-hydroxy-4-oxo-l, 2,3-benzotriazine (Dhbt-OH) and 1-hydroxybenzotrizole (Hobt) were obtained by means of Fluka, Switzerland. Enzymes Carboxypeptidase A (EC 3.4.17.1) type I of the bovine pancreas, leucine aminopeptidase (EC 3.4.11.1) type III-CP of the porcine kidney, a-chymotrypsin (EC 4.421.1) of the bovine pancreas , and pepsin A (EC 3.4.23.1) of the stomach mucosa of the porcine and the pancreas the bovine were obtained by means of Sigma, UK.

Bonding Procedures The first amino acid was linked as a symmetric anhydride in the DMF generated by the appropriate protected N-a amino acid via the DIC or DCC. The following amino acids were linked as preformed Hobt esters made by means of appropriate protected N-a amino acids and Hobt by means of DIC in DMF. Acylation was checked by the ninhydrin test performed at 80 ° C to provide for the deprotection of Fmoc during the test (Larsen, BD and Holm, A., 1994, Int. J. Peptide Protein Res. 43: 1-9) . The bond as an ester Hobt Method a. The amino acid N-a amino protected 3 eq. It was dissolved in DMF together with Hobt 3 eq. AND DEC 3 eq. The solution was left in r.t. for 10 minutes and then resin was added, which had been washed with a 0.2% solution of Dhbt-OH in DMF before the addition of the preactivated amino acid. Method b. The amino acid N-a amino protected 3 eq. It was dissolved in DMF together with Hobt 3 eq. DEC 3 eq. They were added just before use. The final solution was added to the resin. Preformed symmetric anhydride The protected amino N-a amino acid 6 eq. It was dissolved in DCM and cooled to 0 ° C. The DDC or the DIC (3 eq.) Were dissolved in the continuous reaction for 10 minutes. The solvent was removed in vacuo and the residue was dissolved in the DMF. The DMF solution was filtered in the case of using DDC and immediately added to the resin followed by 0.1 eq of DMAP. Estimation of the yield of the binding of the first protected N-amino amino acid The dried protected Fmoc peptide resin of 3-5 mg was treated with 5 ml of 20% piperidine in the DMF for 10 minutes in r.t. and the UV absorption dibenzofulvene-piperidine addition was estimated at 301 nm. The yield was determined using a coefficient e30? of calculated etinction based on a Fmoc-Ala-OH standard. Deprotection of the Fmoc Na amino protection group The deprotection of the Fmoc group was carried out by treatment with 20% piperidine in DMF (1 x 5 and 1 x 10 minutes), followed by washing with DMF until it could not be detected. yellow color (Dhbt-O-) after the addition of Dhbt-OH to the reduced DMF.

Fractionation of the peptide by means of the resin with acid Method a: The peptides were fractionated by means of the resins by the treatment with 95% trifluoroacetic acid (ATF, Riedel-de Háen, Frankfurt, Germany) -water v / vo with ATF and 5% ethanedithiol v / v in rt for 2 hours. The filtered resins were washed with 95% ATF-water and the filtrates and washes were diluted by adding 10% acetic acid. The resulting mixture was extracted 3 times with ether and finally dehydrated by freezing. The crude product of the dehydrated by freezing was analyzed by a high performance liquid chromatography (HPLC) and was identified by a mass spectrometry (MS). Method b. The peptides were fractionated from resins by treatment with 95% trifluoroacetic acid (TFA, Riedel-de Háen, Frankfurt, Germany) -water v / v or with 95% TFA and 5% ethanedithiol v / v in r.t. for 2 hours. The filtered resins were washed with 95% TFA-water and the filtrates and washes were diluted by the addition of 10% acetic acid. The resulting mixture was extracted 3 times with ether and finally dehydrated by freezing. The product dehydrated by freezing was analyzed by high performance liquid chromatography (HPLC) and identified by mass spectrometry (MS).

Method c. Peptides were fractionated from the resins by treatment with 95% trifluoroacetic acid and 5% triisopropylsilane (Sigma) v / v in r.t. for 2 hours. The filtered resins were washed with 95% TFA-water and the filtrates and the washings were evaporated under reduced pressure. The residue was washed with ether and dehydrated by freezing from acetic acid-water. The product dehydrated by freezing was analyzed by high performance liquid chromatography (HPLC) and identified by mass spectrometry (MS).

The disulfide bond formation The crude protected Acm peptide was dissolved in methanol / water 4: 1 and the pH was adjusted to 3.33 (by adding concentrated acetic acid) and the concentration of the peptide was approximately 10"3 M. 10-eq Iodine was dissolved in methanol (20 mg / ml) was added to the peptide solution in one portion.The reaction was continued for 4-5 days at -18 to -20 ° C and continued by HPLC. it was then diluted by adding an extra volume of water, and extracted 3 times with chloroform or tetrachloromethane.The clean water solution was then dehydrated by freezing and the product was purified by a means of preparing the HPLC as described above. interminently of the peptide with PEG-PS The NovaSyn TG resin (250 mg, 0.27.29 mmol / g) was placed in a polyethylene container equipped with a polypropylene filter for filtration.The resin was dilated in the DMF (5 ml), and treated with 20% piperidine in DMF to ensure the presence of non-protonated amino groups in the resin. The resin was reduced and washed with MDF until the yellow color could not be detected after the addition of Dhbt-OH to the reduced DMF. HMPA (3 eq.) Was ligated as a preformed Hobt ester as described above and continuous binding for 24 hours. The resin was reduced and washed with MDF (5 x 5 ml, 5 minutes each) and the acylation was verified with the ninhydrin test. The first amino acid was linked as a preformed symmetric anhydride as described above. The binding performance of the protected Fmoc first amino acids was estimated as described above. It was found in all cases better than- 60%. The following amino acids according to the sequence were ligated as preformed protected Fmoc, and if the protected side chain was needed, the Hobt esters (3 eq.) As described above. The links were continued for 3 hours, unless otherwise specified. The resin was reduced and washed with DMF (5 x 5 ml, 5 minutes each) to remove excess reagent. All the acylations were verified by means of the ninhydrin test performed at 80 ° C. After the completion of the synthesis, the peptide resin was washed with DMF (3x5 ml, 5 each), DCM (3x5 m, 1 minute each) and dried in vacuo overnight. The interminent synthesis of the peptide with TentaGel S-NH2 The resin of TentaGel S-NH2 (100-500 mg, 0.2-0.31 mmol / g) was placed in a polyethylene container equipped with a polypropylene filter for filtration. The resin was dilated in DMF (5-10 ml), and treated with 20% piperidine in the DMF to ensure the presence of non-pronotinated amino groups in the resin. The resin was reduced and washed with DMF until the yellow color could not be detected after the addition of Dhbt-OH to the reduced DMF. The HMPA (3 eq.) Was linked as a Hobt ester generated in situ by means of the DIC as described above and the continuous binding for 24 hours. The resin was reduced and washed with DMF (4 x 5-10 ml, 2 minutes each) and the acylation was verified by the ninhydrin test. The first amino acid was linked as a preformed symmetric anhydride as described above. The binding yields of the first protected Fmoc amino acid were estimated as described above. It was found that in all cases better than 60%. The following amino acids according to the sequence were linked as protected Hobt Fmoc esters (3 eq.) Generated in-house by the DIC as described above. The links continued for 3 hours, unless otherwise specified. The resin was reduced and washed with DMF (4 x 5-10 ml, 2 minutes each) to remove excess reagent. All the acylations were verified by the ninhydrin test performed at 80 ° C. After completing the synthesis, the peptide resin was washed with DMF (3 x 5-10 ml, 5 minutes each), DCM (3 x 5-10 ml, 1 minute each) and finally diethyl ether ( 3 x 5-10 ml, 1 minute each) and dried in vacuo.

Intermittent Synthesis of the Peptide with TentaGel S-RAM The TentaGel S-RAM resin (100-1000 mg, 0.22-0.31 mmol / g) was placed in a polyethylene container equipped with a polypropylene filter. The resin was dilated in DMF (5-10 ml), and the Fmoc group was removed according to the procedure described above. The following amino acids according to the sequence were linked as protected Hobt Fmoc esters (3 eq.) Generated i-n if you by means of the DIC as described above. The links were continued for 3 hours, unless otherwise specified. The resin was reduced and washed with DMF (4 x 5-10 ml, 2 minutes each) to remove excess reagent. All the acylations were verified by means of the ninhydrin test performed at 80 ° C. After completing the synthesis, the peptide resin was washed with DMF (3 x 5-10 ml, minutes each), the DMC (3 x 5-10 ml, 1 minute each) and finally the diethyl ether (3 x 5-10 ml, 1 minute each) and dried in vacuo.

HPLC conditions The isocratic HPLC analysis was performed with a Shimadzu system consisting of an LC-6A pump, a MERCK HITACHI L-400 UV detector operated at 215 nm and a Rheodyne 7125 injection valve with a 29 μl ring. The column used for the isocratic analysis was a Spherisorb ODS-2 (100 X 3 mm, 5-μm particles) (MicroLab, Aarhus, Denmark).

HPLC analysis using gradients was performed with a MERCK-HITACHI L-6200 smart pump, a MERCK HITACHI L-400 UV detector operated at 215 nm and an injection valve Rheodyne 7125 with a 20 μl ring, or with an instrument Waters 600 E equipped with a photodiode array detector. The columns used were one Rescorce ™ RPC 1 ml (Waters) or a LiChoroCART 125-4, LiChrospher 100 RP-18 (5 μm) (MERCK).

Buffer A was 0.1 vol% TFA and buffer B 90 vol% acetonitrile, 9.9 vol% water and 0.1 vol% TFA. The buffers were pumped through the columns at a flow rate of 1.3-1.5 ml / min using any of the following gradients for peptide analysis 1) The linear gradient of 0% -100% B (30 minutes) or 2) 0% of the linear gradient of B (2 minutes) of 0-50% B (23 minutes) 50-100% of B (5 minutes).

For preparative HPLC, purification was performed with an instrument equipped with a Waters 996 photodiode rearrangement detector. The column used was a Waters Delta-Pak C-18 15 μm, 100 ° A, 25 x 100 mm, Gradient "2 ) "was used with a flow rate of 9 ml / min.

Mass spectrometry The mass spectra were obtained with a Finnigan Mat LCQ instrument equipped with an electroro (ESI) probe (ES-MS) and with a TofSpec E, an Instrument Fisons (MALDI-TOF) using ß-cyano-p-hydroxycinnamic acid as a matrix.

Peptide synthesis of individual peptides 1. Peptide synthesis of H-TYR-Gly-Phe-Leu-Glus-OH (Leu-enkephalin-Glu6-OH) with NovaSyn TentaGel Dry resin of NovaSyn TG (0.29 mmol / g, 250 mg) was placed in a polyethylene container equipped with a polypropylene filter for filtration and treatment as described under "intermittently synthesizing peptide with PEG-PS" until the end of the Gluß peptide probe. The following amino acids forming the Leu-enkephalin sequence were ligated as a preformed protected Fmoc, if necessary the protected side chain, esters HObt (3 eq.) In DMF (5 ml) generated by means of the DIC. Before each of the last five bonds, the resin was washed with a solution of Dnbt-OH (80 mg in 25 ml), to continue with the disappearance of the yellow color as the binding reaction proceeding. When -the yellow color was no longer visible, the bonds were interrupted by washing the resin with DMF (5 x 5 ml, 5 minutes each). The acylations were then verified by the ninhydrin test performed at 80 ° C as described above. After completing the synthesis, the peptide resin was washed with DMF (3 x 5 ml, 1 minute each), DCM (3 x 5 ml, 1 minute each), diethyl ether (3 x 5 ml, 1 minute each) one) and dried in vacuo.

The peptide was cleaved. from the resin according to method a. The crude product of the freeze dehydration was analyzed by means of HPLC and the purity was found better than 90%. The identity of the peptide was confirmed by means of ES-MS. Performance of 76%. 2. Synthesis of H-Tyr-Gly-Gly-Phe-Leu-Lysg-OH (Leu-enkephalin-Lys6-0H) and NovaSyn TentaGel NovaSyn's dry resin (0.29 mmol / g, 250 mg) was placed in a container of polyethylene equipped with a polypropylene filter for filtration and treatment as described in "the intermittent synthesis of the peptide with PEG-PS" until the Lys6 peptide probe is terminated. The following amino acids that form the sequence of Leu encephephine were bound as protected Hobt Fmoc esters (3 eq.) In DMF (5 ml) generated by means of the DIC. Before each of the last five bonds, the resin was washed with a solution of Dhbt-OH (80 mg in 25 ml), to continue with the disappearance of the yellow color as the reaction of the binding. When the yellow color is no longer visible, the bonds are interrupted by washing the resin with DMF (5 x 5 ml, 5 minutes each). The acylations were then verified by the ninhydrin test performed at 80 ° C as described above. After the synthesis was complete, the peptide resin was washed with DMF (3 x 5 ml, 1 minute each), DMC (3 x 5 ml, 1 minute each), diethyl ether (3 x 5 ml, 1 minute each one) and dried in vacuo.

The peptide was cleaved by means of the resin by method a. The crude product of the freeze dehydration was analyzed by HPLC and the purity found is better than 98%. The identity of the peptide was confirmed by means of ES-MS. 84% yield. 3. Synthesis of the peptide of H-Lysß-Tyr-Gly-Gly-Phe-Leu-OH (H-Lys6-Leu-enkephalin) with NovaSyn TentaGel The dry resin of NovaSyn TG (0.29 mmol / g, 250 mg) was placed in a polyethylene container equipped with a polypropylene filter for filtration and treatment as described in "the intermittent synthesis of the peptide with PEG-PS" and the first amino acid leucine was ligated as described by binding procedures. The following amino acids forming the H-Lys6-enkephalin sequence were linked as protected Hobt Fmoc esters (3 eq.) In DMF (5 ml) generated by the DIC and the linkages were continued for at least 2 hours. The acylations were then verified by the ninhydrin test performed at 80 ° C as described above.

After completing the synthesis, the peptide resin was washed with DMF 8 3 5 ml, 1 minute each), DCM (3 x 5 ml, 1 minute each), diethyl ether (3 x 5 ml, 1 minute each one) and dried in vacuo.

The peptide was cleaved by means of the resin as described above using 95% TFA and 5% water (v / v) as a fractionated reagent and dehydrated by freezing from acetic acid. The crude product dehydrated by freezing was analyzed by HPLC and found to be homogeneous without removal and protected Fmoc sequences. The purity was found better than 89% and the identity of the peptide conjugate was confirmed by ES-MS. 89% yield. 4. Synthesis of the peptide of H-Lysß-Tyr-Gly-Gly-Phe-Leu-Lysß-Leu-OH (H-Lys6-Leu-enkephalin-Lys6-OH) with NovaSyn TentaGel The dry resin of NovaSyn TG (0.29 mmol / g, 250 mg) was placed in a polyethylene container equipped with a polypropylene filter for filtration and treatment as described in "intermittently synthesizing the peptide in PEG-PS" until the Lyse-Los peptide probe is terminated. following amino acids forming the sequence H-Lysß-enkephalin were linked as preformed Hobt Fmoc-protected esters (3 eq.) in DMF (5 ml) generated by means of DIC and the linkages were continued for at least 2 hours. The acylations were then verified by the ninhydrin test performed at 80 ° C as described above. After completing the synthesis, the peptide resin was washed with DMF (3 x 5 ml, each), DCM (3 x 5 ml, 1 minute each), diethyl ether (3 x 5 ml, 1 minute each) ) and dried in vacuo.

The peptide was cleaved by means of the resin according to method a. The crude product dehydrated by freezing was analyzed by HPLC and the purity was found better than 98%. The identity of the peptide was confirmed by ES-MS. 90% yield.

. The synthesis of the peptide of H-Tyr-Gly-Gly-Phe-Leu-Lys-Lys-Glu-Glu-Glu-Lys-OH (Leu-enkephalin-Lys-Lys-Glu-Glu-Glu-Lys-OH) in TentaGel S-PHB-Lys (Boc) Fmoc The dried resin of TentaGel S-PHB-Lys (Boc) Fmoc (0.22 mmol / g, 500 mg) was placed in a polyethylene container equipped with a polypropylene filter for filtration and dilation for two hours in the DMF (5 ml). The Fmoc group in the first lysine was removed as described above and the synthesis was continued until the end of the peptide sequence as in "Synthesis intermittently in TentaGel S-PHB-Lys (Boc) Fmoc". After completing the synthesis, the peptide resin was washed with DMF (3 x 5 ml, 1 minute each) m DCM (3 x 5 ml, 1 minute each), diethyl ether (3 x 5 ml, 1 minute each one) and dried in vacuo.

The peptide was cleaved by means of the resin according to method b as described above and dehydrated by freezing by means of acetic acid. The crude dehydrated freeze peptide was purified by preparative HPLC using the procedure described above. The purified product was found homogeneous and the purity was found better than 90%. The identity of the peptide was confirmed by means of ES-MS. Yield of 60%. 6. The synthesis of the peptide of H-Tyr-Gly-Gly-Phe-Leu-Lys-Glu-Glu-Glu-Glu-Lys-OH (Leu-enkephalin-Lys-Glu-Glu-Glu-Glu-Lys-OH) in TentaGel S-PHB-Lys (Boc) Fmoc The dried resin of TentaGel S-PHB-Lys (Boc) Fmoc (0.22 mmol / g, 500 mg) was placed in a polyethylene container equipped with a polypropylene filter for filtration and dilation for two hours in DMF (5 ml). The Fmoc group in the first lysine was removed as described above. And the synthesis was continued until the peptide sequence was finished 'as described in the intermittent synthesis of the peptide in TentaGel S-PHB-LYS (Boc) Fmoc. "After completion of the synthesis, the peptide resin was washed with DMF (3 x 5 ml, 1 minute each), DMC (3 x 5 ml, 1 minute each), diethyl ether (3 x 5 ml, 1 minute each) and dried in vacuo.

The peptide was cleaved by means of the resin according to method b as described above and dehydrated by freezing by means of acetic acid. The crude crude peptide was purified by preparative HPLC using the procedure described above. The purified product was found homogeneous and the purity was found better than 98%. The identity of the peptide was confirmed by ES-MSA. 65% yield. 7. The synthesis of the peptide of H-Tyr-Gly-Gly-Phe-Leu- (Orn) 6-OH (leu-enkephalin- (Orn) 6-OH) in TentaGel S-NH2 The dry resin of S-NH2 (0.31 mmol / g, 500 mg) was placed in a polyethylene container equipped with a polypropylene filter for filtration and dilation by two in DMF (5 ml). The peptide according to the sequence was assembled as described in "The synthesis of the peptide intermittently in resins' TentaGel S". After completing the synthesis, the peptide resin was washed with DMF (3 x 5 ml, 1 minute each) and dried in vacuo.

The peptide was cleaved by means of the resin according to method b as described above and dehydrated by freezing by means of acetic acid. The crude product of the dehydrated freeze was oxidized to make the disulfide link according to the procedure described above. The crude cyclised peptide was purified by preparative HPLC using the procedure described above. The purified product was found homogeneous and the purity was found better than 90%. The identity of the peptide was confirmed by means of ES-MS. 20% yield. 8. Synthesis of the peptide of H-Tyr-Gly-Gly-Phe-Leu- (Dbu) 6-OH (Leu-enkephalin- (Dbu) 6-OH) in TentaGel S-NH2 The dry resin of TentaGel S-NH2 (0.31 mmol / g, 500 mg) was placed in a polyethylene container equipped with a polypropylene filter for filtration and expansion for two hours in DMF (5 ml). The peptide according to the sequence was assembled as described in "The intermittent synthesis of the peptide in the TentaGel S resins". After completing the synthesis, the peptide resin was washed with DMF (3 x 5 ml, 1 minute each), DCM (3 x 5 ml, 1 minute each), diethyl ether (3 x 5 ml, 1 minute each one) and dried in vacuo.

The peptide was cleaved by means of the resin according to method b as described above and dehydrated by freezing by means of acetic acid. The crude product dehydrated by freezing was oxidized to make the disulfide bond according to the procedure described above. The cyclized crude peptide was purified by preparative HPLC using the procedure described above. The purified product was found homogeneous and the purity was found better than 90%. The identity of the peptide was confirmed by means of ES-MS. Performance of 22%. 9. Synthesis of the peptide of H-Tyr-Gly-Gly-Phe-Leu- (Dpr) 6-OH (Leu-enkephalin- (Dpr) 6-OH) in TentaGel S-NH2 The dry resin of TentaGel S-NH2 (0.31 mmol / g, 500 mg) was placed in a polyethylene container equipped with a polypropylene filter for filtration and dilation for two hours in DMF (5 ML). The peptide according to the sequence was assembled as described in "The intermittent synthesis of the peptide in the TentaGel S resins". After completing the synthesis, the peptide resin was washed with DMF (3 x 5 ml, 1 minute each), DCM (3 x 5 ml, 1 minute each), diethyl ether (3 x 5 ml, 1 minute each one) and dried in vacuo.

The peptide was cleaved by means of the resin according to method b as described above and dehydrated by freezing by means of acetic acid. The crude peptide was purified by preparative HPLC using the procedure described above. The product was found homogeneous and the purity was found better than 98%. The identity of the peptide was confirmed by ES-MS. 22% yield.

. The synthesis of the peptide of H-Tyr-Gly-Gly < -Phe-Leu-Lysip-OH (Leu-enkephalin-Lysip-OH) in TentaGel S-PHB-Lys) Boc) Fmoc The dry resin of S-PHB-Lys (Boc) Fmoc (0.22 mmol / g, 500 mg) was placed in a polyethylene container equipped with a polypropylene filter for filtration and dilation for two hours in DMF (5 ml). The Fmoc group in the first lysine was removed as described above and the synthesis was continued until the peptide sequence was completed as described in "The intermittent synthesis of the peptide in TentaGel S-PHB-Lys (Boc) Fmoc". After completing the synthesis, the peptide resin was washed with DMF (3 x 5 ml, 1 minute each), DMC (3 x 5 ml, 1 minute each), diethyl ether (3 x 5 ml, 1 minute each one) and dried in vacuo.

The peptide was cleaved by means of the resin according to method b as described above and dehydrated by freezing by means of acetic acid. The crude dehydrated freeze peptide was purified by preparative HPLC using the procedure described above. The purified product was found homogeneous and the purity was found better than 98%. The identity of the peptide was confirmed by ES-MS. 7.1% yield. 11. Synthesis of the peptide of H-Trp-Ala-Gly-Gly-Asp-Ala-Ser-Glu-Glu6-OH (DSIP-Glu6-OH) in NovaSyn TentaGel The dry resin of Nova Syn TG (0.29 mmol / g, 250 mg) was placed in a polyethylene container equipped with a polypropylene filter for filtration and treatment as described in "Intermittently synthesizing the peptide in PEG-PS" until the Glu6 peptide probe is terminated.

The following amino acids that form the DSIP sequence were linked as protected Hobt Fmoc esters (3 eq.) In DMF generated by means of DIC. Before each of the last nine bonds, the resin was washed with a solution of Dhbt-OH (80 mg in 25 ml), to continue with the disappearance of the yellow color as the binding reaction proceeding. When the yellow color was no longer visible, the bonds were interrupted by washing the resin with DMF (5 x 5 ml, 5 minutes each). The acylations were then verified by the ninhydrin test at 80 ° C as described above. After completing the synthesis, the peptide resin was washed with DMF (3 x 5 ml, 1 minute each) -, DCM (3 x 5 ml, 1 minute each), diethyl ether (3 x 5 ml, 1 minute each), and dried in vacuo.

The peptide was cleaved by means of the resin according to method a. The crude product was dehydrated by freezing and analyzed by HPLC and the purity was found to be better than 98%. The identity of the peptide was confirmed by ES-MS. 80% yield. 12. The synthesis of the peptide of H-Trp-Ala-Gly-Gly-Asp-Ser-Gly-Glu- (Lys-Glu) 3- (DISP- (Lys-Glu) 3-OH) in NovaSyn TentaGel The dry resin of NovaSyn TG (0.29 mmol / g, 250 mg) was placed in a polyethylene container equipped with a polypropylene filter for filtration and treatment as described in "Intermittently synthesizing the peptide in PEG-PS" until the probe is finished of the peptide (Lys-Glu) 3. The following amino acids forming the DSIP sequence were linked as protd HOBT Fmoc esters (3 eq.) in DMF (5 ml) generated by means of the DIC. Before each of the last nine bonds, the resin was washed with a solution of Dhbt-OH (80 mg in 25 ml), to continue with the disappearance of the yellow color as the binding reaction proceeding. When the yellow color was no longer visible, the bonds were interrupted by washing the resin with DMF (5 x 5 ml, 5 minutes each). The acylations were verified by the ninhydrin test performed at 80 ° C as described above. After completing the synthesis, the peptide resin was washed with DMF (3 x 5 ml, 1 minute each), DCM (3 x 5 ml, 1 minute each), diethyl ether (3 x 5 ml, 1 minute each) one), and dried in vacuo.

The peptide was cleaved with the resin according to method a. The crude product dehydrated by freezing was analyzed by HPLC and the purity was found better than 98%.

The identity of the peptide was confirmed by ES-MS. 91% yield. 13. The synthesis of the peptide of H-Trp-Ala-Gly-Gly-- Asp-Ala-Ser-Gly-Gly-Glu-OH (DSIP) in NovaSyn TentaGel (Reference) the dry resin NovaSyn TG (0.29 mmol / g, 250 mg) was placed in a polyethylene container equipped with a polypropylene filter for filtration and treatment as described in "The intermittent synthesis of the peptide in PEG-PS". The first amino acid was linked as a preformed symmetric anhydride as described above. The yields of the bonds of the first protd Fmoc amino acids were estimated as described above. The yields in all cases are better than 60%. The following amino acids forming the DSIP sequence were ligated as Hobt Fmoc protd esters (3 eq.) Preformed in DMF (5 ml) generated by means of the DIC. Before each of the last eight bonds, the resin was washed with a solution of Dhbt-OH (80 mg in 25 ml), to continue with the disappearance of the yellow color as the binding reaction proceeding. When the yellow color was no longer visible, the bonds were interrupted by washing the resin with DMF (5 x 5 ml, 5 minutes each). The acylations were verified by the ninhydrin test performed at 80 ° C as described above. After completing the synthesis, the peptide resin was washed with DMF (3 x 5 ml, 1 minute each), DMC (3 x 5 ml, 1 minute each), diethyl ether (3 x 5 ml, 1 minute each one), and dried in vacuo.

The peptide was cleaved by means of the resin according to method a. The crude product was dehydrated by freezing and analyzed by HPLC and the purity was found to be better than 98%. The identity of the peptide was confirmed by ES-MS. Performance of 78%. 14. The synthesis of the peptide of H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-Lysg-OH (substance P-Lys6-OH) in NovaSyn TentaGel The dry resin of NovaSyn TG ( 0.29 mmol / g, 250 mg) was placed in a polyethylene container equipped with a polypropylene filter for filtration and treatment as described in "Intermittently synthesizing the peptide in PEG-PS" until the peptide probe is finished Ly = e-The following amino acids forming the sequence of substance P were bound as protd Hobt Fmoc esters (3 eq.) Preformed in DMF (5 ml) generated by means of DIC and the links were continued for at least 2 hours . The acylations were then verified by means of the ninhydrin test performed at 80 ° C as described above. After completing the synthesis, the peptide resin. washed with DMF (3 x 5 ml, 1 minute each), DMC (3 x 5 ml, 1 min. each), diethyl ether (3 x 5 ml, 1 minute each) and dried in vacuo.

The peptide was cleaved by means of the resin according to method a. The crude product dehydrate by freezing was analyzed by HPLC and the purity was found better than 98%. The identity of the peptide was confirmed by means of ES-MS. 80% yield.

. The synthesis of the peptide of H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 (substansia-P-NH2) in TentaGel S-RAM. The TentaGel S-RAM dry resin (0.25 mmol / g, 500 mg) was placed in a polyethylene container equipped with a polypropylene filter for filtration and dilation for two hours in DMF (5 ml). The Fmoc group was removed according to the procedure described above, and the peptide according to the sequence was assembled as described in "The intermittent synthesis of the peptide in the TentaGel S-RAM resins". After completing the synthesis, the peptide resin was washed with DMF (3.5 ml, 1 minute each), DCM (3 x 5 ml, 1 minute each), diethyl ether (3 x ml, 1 minute each >);) and dried in vacuo.

The peptide was cleaved by means of the resin according to method b as described above and dehydrated by freezing by means of acetic acid. The crude peptide was purified by preparative HPLC using the procedure described above. The purified product was found homogeneous and the purity was found better than 98%. The identity of the peptide was confirmed by ES-MS. Yield of 12.3%. 16. The synthesis of the peptide of H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-Lys6-NH2 (substance-P-Lys6-NH2) in TentaGel S-RAM-Lys (Boc ) Fmoc The dry resin Tenta Gel S-RAM-Lys (Boc) Fmoc (0.22 mmol / g, 500 mg) was placed in a polyethylene container equipped with a polypropylene filter for filtration and dilation for two hours in DMF ( 5 ml). The Fmoc group in the first lysine was removed as described above and the synthesis was continued until the peptide sequence was finished as described in "The intermittent synthesis of the peptide in TentaGel-S-Ram-Lys (Boc) Fmoc".

After completing the synthesis, the peptide resin was washed with DMF (3 x 5 ml, 1 minute each), DCM (3 x 5 ml, minute each), diethyl ether (3 x 5 ml, 1 minute each) ) and dried in vacuo.

The peptide was cleaved by means of the resin according to method b as described above and dehydrated by freezing with acetic acid. The crude peptide freeze dehydrate was purified by preparative HPLC using the procedure described above. The purified product was found homogeneous and the purity was found better than 98%. The identity of the peptide was confirmed by ES-MS. Yield of 17.2%. 17. The synthesis of the peptide of H- (Lys6) -Arg- Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 (_K6- substance-P-NH2) in TentaGel S-RAM The resin Dry Tenta Gel S-RAM (0.25 mmol / g, 500 mg) was placed in a polyethylene container equipped with a polypropylene filter for filtration and dilation for two hours in DMF (5 ml). The Fmoc group was removed according to the procedure described above, and the peptide according to the sequence was assembled as described in "The intermittent synthesis of the peptide in the TentaGel-S-Ram resins". After completing the synthesis, the peptide resin was washed with DMF (3 x 5 ml, 1 minute each), DCM (3 x 5 ml, minute each), diethyl ether (3 x 5 ml, 1 minute each) one) and dried in vacuo.

The peptide was cleaved by means of the resin according to method b as described above and dehydrated by freezing by means of acetic acid. The crude peptide was purified by HPLC using the procedure described above. The purified product was found homogeneous and the purity was found better than 98%. The identity of the peptide was confirmed by ES-MS. Yield of 10.3%. 18. The peptide synthesis of H-Aib-His-2-D-Nal-D-Phe-Lys- (Lys) 6-NH2 (GHRP- (Lys) 6 ~ NH2) in TentaGel S-RAM-Lys (Boc) Fmoc Dry resin Tenta Gel S-RAM-Lys (Boc) Fmoc (0.22 mmol / g, 500 mg) was placed in a polyethylene container equipped with a polypropylene filter for fition and dilation for two hours in DMF (5 ml). The Fmoc group in the first lysine was removed as described above and the synthesis was continued until the peptide sequence was finished as described in "The intermittent synthesis of the 'peptide in TentaGel-S-Ram-Lys (Boc) Fmoc" . After completing the synthesis, the peptide resin was washed with DMF (3 x 5 ml, 1 minute each), DCM (3 x 5 ml, minute each), diethyl ether (3 x 5 ml, 1 minute each) one) and dried in vacuo.

The peptide was cleaved by means of the resin according to method b as described above and dehydrated by freezing by means of acetic acid. The crude product dehydrated by freezing was purified by preparative HPLC using the procedure described above. The purified product was found homogeneous and the purity was found better than 90%. The identity of the peptide was confirmed by ES-MS. Performance of 35%. 19. Peptide synthesis of H-Aib-His-2-D-Nal-D-Phe-Lys-NH2 (GHRP-NH2) in TentaGel S-RAM Dry resin Tenta Gel S-RAM (0.25 mmol / g, 500 mg ) was placed in a polyethylene container equipped with a polypropylene filter for filtration and dilation for two hours in DMF (5 ml). The Fmoc group was removed according to the procedure described above, and the peptide according to the sequence was assembled as described in "The intermittent synthesis of the peptide in the TentaGel-S-RAM resins". After completing the synthesis, the peptide resin was washed with DMF (3 x 5 ml, 1 minute each), DCM (3.5 ml each, one minute), diethyl ether (3 x 5 ml, 1 minute each) and dried in vacuo.

The peptide was cleaved by means of the resin according to method b as described above and dehydrated by freezing by means of acetic acid. The crude product dehydrated by freezing was found to be homogeneous and the purity was better than 95%. The identity of the peptide was confirmed by ES-MS. 21% yield.

. Peptide synthesis of H- (Lys) 6-Aib-His-2-D-Nal-D-Phe-Lys-NH2 (K6-GHRP-NH2) in TentaGel S-RAM Dry resin Tenta Gel S-RAM ( 0.25 mmol / g, 500 mg) was placed in a polyethylene container equipped with a polypropylene filter for filtration and dilation for two hours in DMF (5 ml). The Fmoc group was removed according to the procedure described above, and the peptide according to the sequence was assembled as described in "The intermittent synthesis of the peptide in the TentaGel-S-RAM resins". After completing the synthesis, the peptide resin was washed with DMF (3 x 5 ml, 1 minute each), DCM (3 x 5 ml, minute, each), diethyl ether (3 x 5 ml, 1 minute each one) and dried in vacuo.

The peptide was cleaved by means of the resin according to method b. as described above and dehydrated by freezing by means of acetic acid. The crude product dehydrated by freezing was found to be homogeneous and the purity was better than 95%. The identity of the peptide was confirmed by ES-MS. 19% yield 21. Peptide synthesis of Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Lys6-OH (GnRH-Lys6-QH) in NovaSyn TentaGel Dry resin NovaSyn TG (0.29 mmol / g, 250 mg) was placed in a polyethylene container equipped with a polypropylene filter for filtration and treatment as described in "Intermittently synthesizing the peptide in PEG-PS" until the Lys6 probe of the peptide is terminated. The following amino acids forming the GnRH sequence were ligated as preformed Hobt Fmoc protected esters (3 eq.) In DMF (5 ml) generated by DIC and the linkages were continued for at least 2 hours. The acylations were verified by the ninhydrin test performed at 80 ° C as described above. After completing the synthesis, the peptide resin was washed with DMF (3 x 5 ml, 1 minute each), DCM (3 x 5 ml, minute each), diethyl ether (3 5 ml, 1 minute each) and dried in vacuo.

The peptide was cleaved by means of the resin according to method c. The crude product dehydrated by freezing was analyzed by means of HPLC and found to contain white peptide together with some impurities. The crude product was purified by preparative HPLC in a regressive phase. The purity was found better than 98% and the identity of the peptide conjugate was confirmed by ES-MS. Yield of 37%. 22. Peptide synthesis of pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly- (Lys-Glu) 3-OH (GnRH- (Lys-Glu) 3-OH in NovaSyn TentaGel Dry resin NovaSyn TG (0.29 mmol / g, 250 mg) was placed in a polyethylene container equipped with a polypropylene filter for filtration and treatment as described in "Intermittently synthesizing the peptide in PEG-PS" until the probe (Lys-Glu) 3 is finished. of the peptide. The following amino acids forming the GnRH sequence were ligated as preformed Hobt Fmoc protected esters (3 eq.) In DMF (5 ml) generated by DIC and the linkages were continued for at least 2 hours. The acylations were verified by the ninhydrin test performed at 80 ° C as described above. After . to complete the synthesis / the peptide resin was washed with DMF (3 x 5 ml, 1 minute each), DCM (3 x 5 ml, minute each), diethyl ether (3 x 5 ml, 1 minute each) and dried in vacuo.

The peptide was cleaved by means of the resin according to method c. The crude product dehydrated by freezing was analyzed by means of HPLC and found to contain white peptide together with some impurities. The crude product was purified by preparative HPLC in a regressive phase. The purity was found better than 98% and the identity of the peptide conjugate was confirmed by ES-MS. Performance of 43%. 23. Peptide synthesis of pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 (GnRH-NH2) NH2 in TentaGel S-RAM Dry resin Tenta Gel S-RAM (0.25 mmol / g , 500 mg) was placed in a polyethylene container equipped with a polypropylene filter for filtration and dilation for two hours in DMF (5 ml). The Fmoc group was removed according to the procedure described above, and the peptide according to the sequence was assembled as described in "The intermittent synthesis of the peptide in the TentaGel-S-RAM resins". After completing the synthesis, the peptide resin was washed with DMF (3 x 5 ml, 1 minute each), DCM (3 x 5 ml, minute each), diethyl ether (3 x 5 ml, 1 minute each) ) and dried in vacuo.

The peptide was cleaved by means of the resin according to method b as described above and dehydrated by freezing by means of acetic acid. The crude product dehydrated by freezing was found to be homogeneous and the purity was better than 95%. The identity of the peptide was confirmed by ES-MS. 28% yield. 24. Peptide-synthesis of H- (Lys) 6 ~ Gln-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 (K6-GnRH-NH2) NH2 in TentaGel S-RAM The dry resin TentaGel S-RAM (0.25 mmol / g, 500 mg) was placed in a polyethylene container equipped with a polypropylene filter for filtration and dilation for two hours in DMF (5 ml). The Fmoc group was removed according to the procedure described above, and the peptide according to the sequence was assembled as described in "The intermittent synthesis of the peptide in TentaGel S-RAM resins". After completing the synthesis, the peptide resin was washed with DMF (3 x 5 ml, 1 minute each), DCM (3.5 ml each, one minute), diethyl ether (3 x 5 ml, 1 minute each) and dried in vacuo. The peptide was cleaved by means of the resin according to method b as described above by means of acetic acid. The crude product dehydrated by freezing was found to be homogeneous and the purity was better than 95%. The identity of the peptide was confirmed by ES-MS. 20% yield.

. Peptide synthesis of H-Gly-Gly-Thr-Tyr-Ser-Cys-His-Phe-Gly-Pro-Leu-Thr-Trp-Val-Cys-Lys-Pro-Gln-Gly-Gly-OH (EM lOH) in TentaGel S-NH2 The dry resin Tenta Gel S-NH2 (0.31 mmol / g, 500 mg) was placed in a polyethylene container equipped with a polypropylene filter for filtration and dilation for two hours in DMF (5 μm). ml). The peptide according to the sequence was assembled as described in "The intermittent synthesis of the peptide in the TentaGel S resins". After completing the synthesis, the peptide resin was washed with DMF (3 x 5 ml, 1 minute each), DCM (3 x 5 ml, minute each), diethyl ether (3 x 5 ml, 1 minute each) ) and dried in vacuo.

The peptide was cleaved by means of the resin according to method b as described above and dehydrated by freezing by means of acetic acid. The crude product dehydrated by freezing was oxidized without further purification, to make the disulfide bond according to the procedure described above. The crude cyclised peptide was purified by HPLC using the procedure described above. The purified product was found homogeneous and the purity was found better than 98%. The identity was confirmed by ES-MS. 22% yield. 26. Peptide synthesis of H-Gly-Gly-Thr-Tyr-Ser-Cys-His-Phe-Gly-Pro-Leu-Thr-Trp-Val-Cys-Lys-Pro-Gln-Gly-Lys6-OH (EMP -l-Lys6-OH) in TentaGel S-PHB-Lys (Boc) Fmoc The Tenta Gel Dry Resin S-PHB-Lys (boc) Fmoc (0.22 mmol / g, 500 mg) was placed in a polyethylene container equipped with a polypropylene filter for filtration and dilation for two hours in DMF (5 ml). The Fmoc group in the first lysine was removed according to the procedure described above and the synthesis was continued until the peptide sequence was completed as described in "The intermittent synthesis of the peptide in TentaGel S-PHB-Lys (Boc) Fmoc" . After completing the synthesis, the peptide resin was washed with DMF (3 x 5 ml, 1 minute each), DCM (3 x 5 ml, minute each), diethyl ether (3 x 5 ml, 1 minute each) ) and dried in vacuo.

The peptide was cleaved by means of the resin according to method b as described above and dehydrated by freezing by means of acetic acid. The crude product dehydrated by freezing was oxidized without further purification to make the disulfide bond according to the procedure described above. The crude cyclised peptide was purified by HPLC using the procedure described above. The purified product was found homogeneous and the purity was found better than 98%. The identity of the peptide was confirmed by ES-MS. Yield of 27%. 27. Peptide synthesis of H- (Lys) 6-Gly-Gly-Thr-Tyr-Ser-Cys-His-Phe-Gly-Pro-Leu-Thr-Trp-Val-Cys-Lys-Pro-Gln-Gly- Gly-OH (K6-EMP-l-OH) in TentaGel S-NH2 The dry resin Tenta Gel S-NH2 (0.31 mmol / g, 500 mg) was placed in a polyethylene container equipped with a polypropylene filter for filtration and dilation for two hours in DMF (5 ml). The peptide according to the sequence was assembled as described in "The intermittent synthesis of the peptide in the TentaGel S resins". After completing the synthesis, the peptide resin was washed with DMF (3 x 5 ml, 1 minute each), DCM (3.5 ml, one minute each), diethyl ether (3.5 ml, 1 minute each) and dried in vacuo.

The peptide was cleaved by means of the resin according to method b as described above and dehydrated by freezing by means of acetic acid. The crude product dehydrated by freezing was oxidized without further purification to make the disulfide bond according to the procedure described above. The cyclized crude peptide was purified by HPLC using the procedure described above. The purified product was found homogeneous and the purity was found better than 98%. The identity of the peptide was confirmed by ES-MS. Yield of 12%. 28. The synthesis of the peptide of H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe -Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-OH (GLP-I- (7-36) (Human) -OH) in TentaGel S-NH2 The dry resin Tenta Gel S-NH2 (Boc) Fmoc (0.31 mmol / g, 500 mg) was placed in a polyethylene container equipped with a polypropylene filter for filtration and dilation for two hours in DMF (5 ml). The peptide according to the sequence was assembled as described in "The intermittent synthesis of the peptide in the TentaGel S resins". After completing the synthesis, the peptide resin was washed with DMF (3 x 5 ml, 1 minute each), DCM (3 x 5 ml, minute each), diethyl ether (3 5 ml, 1 minute each) and dried in vacuo.

The peptide was cleaved by means of the resin according to method b as described above and dehydrated by freezing by means of acetic acid. The crude dehydrated freeze peptide was purified twice by preparative HPLC using the procedure described above. The purified product was found homogeneous and the purity was found better than 98%. The identity of the peptide was confirmed by ES-MS. Performance of 8.7%. 29. The synthesis of the peptide of H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu -Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Lysß-OH (GLP-1- (7-36) (Human) Lys6-OH) in TentaGel S-PHB-Lys (Boc) Fmoc The dried resin Tenta Gel S-PHB-Lys (Boc) moc (0.22 mmol / g, 500 mg) was placed in a polyethylene container equipped with a polypropylene filter for filtration and dilation for two hours in DMF (5 ml The Fmoc group in the first lysine was removed as described above and the synthesis was continued until the peptide sequence was completed as described in "The intermittent synthesis of the peptide in TentaGel S-PHB-Lys (Bhoc) Fmoc" . After completing the synthesis, the peptide resin was washed with DMF (3 x 5 ml, 1 minute each), DCM (3 x 5 ml, minute each), diethyl ether (3 x 5 ml, 1 minute each) one) and-dried in vacuo.

The peptide was cleaved by means of the resin according to method b as described above and dehydrated by freezing by means of acetic acid. The crude product dehydrated by freezing was purified twice by preparative HPLC using the procedure described above. The purified product was found homogeneous and the purity was found better than 98%. The identity of the peptide was confirmed by ES-MS. Yield of 11%.

. The synthesis of the peptide of H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu -Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe-OH- (PTH (1-34) (Human) -OH) in TentaGel S-NH2 The dry resin Tenta Gel S-NH2 (Boc) Fmoc (0.31 mmol / g, 500 mg) was placed in a polyethylene container equipped with a polypropylene filter for filtration and dilation for two hours in DMF (5 ml). The peptide according to the sequence was assembled as described in "The synthesis of intermittently, the peptide in the TentaGel S resins". After completing the synthesis, the peptide resin was washed with DMF (3 x 5 ml, 1 minute each), DCM (3 x 5 ml, minute each), diethyl ether (3 x 5 ml, 1 minute each) ) and dried in vacuo.

The peptide was cleaved by means of the resin according to method b as described above and dehydrated by freezing by means of acetic acid. The crude dehydrated freeze peptide was purified twice by preparative HPLC using the procedure described above. The purified product was found homogeneous and the purity was found better than 98%. The identity of the peptide was confirmed by ES-MS. Yield of 5.3%. 31. The synthesis of the peptide of H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu -Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe-Lys6-0H '(PTH (1-34) (Human) -Lyse-OH in TentaGel S-PH-Lys (Boc) Fmoc Dry resin Tenta Gel S-PHB-Lys (Boc) Fmoc (Boc) Fmoc (0.22 mmol / g, 500 mg) was placed in a polyethylene container equipped with a polypropylene filter for filtration and dilation for two hours in DMF (5 ml). The Fmoc group in the first lysine was removed as described above. And the synthesis was continued until the peptide sequence was finished as described in "The intermittent synthesis of the peptide in TentaGel S-PHB-Lys (Boc) Fmoc". After completing the synthesis, the peptide resin was washed with DMF (3 x 5 ml -, - 1 minute each), DCM (3 x 5 ml, minute each), diethyl ether (3 5 ml, 1 minute each) and dried in vacuo.

The peptide was cleaved by means of the resin according to method b as described above and dehydrated by freezing by means of acetic acid. The crude dehydrated freeze peptide was purified twice by preparative HPLC using the procedure described above. The purified product was found homogeneous and the purity was found better. than 98% -The identity of the peptide was confirmed by ES-MS. Performance of . 3%. 32. The synthesis of the peptide of H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu -Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe- (Lys-Glu) 3-OH (PTH 1-34 human- (Lys-Glu) 3-0H) in NovaSyn TentaGel Dry resin NovaSyn TG (Boc) Fmoc (0.29 mmol / g, 250 mg) was placed in a polyethylene container equipped with a polypropylene filter for filtration and treatment as described in "Intermittently synthesizing the peptide in PEG-PS" until the peptide probe is complete (Lys-Glu 3. The following amino acids forming the PTH sequence were linked as protected Hobt Fmoc esters (3 eq.) In DMF (5 ml) generated by means of DIC and the linkages were continued for at least two hours. The acylations were verified by the ninhydrin test performed at 80 ° C as described above. After completing the synthesis, the peptide resin was washed with DMF (3 x 5 ml, 1 minute each), DCM (3 x 5 ml, minute each), diethyl ether (3 x 5 ml, 1 minute each) ) and dried in vacuo.

The peptide was cleaved by means of the resin according to method c. The crude product dehydrated by freezing was analyzed by HPLC and found to have the white peptide together with impurities. The product was purified by preparative HPLC in a regressive phase. Purity was found better than 98%. The identity of the peptide was confirmed by ES-MS. 28% yield. 33. The synthesis of the peptide of H- (Lys) 6 ~ Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu- Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe-OH (Lys6-PTH (1-34) (Human) -OH) in TentaGel S- NH2 Dry resin Tenta Gel S-NH2 (0.31 mmol / g, 500 mg) was placed in a polyethylene container equipped with a polypropylene filter for filtration and dilation for two hours in DMF (5 ml). The peptide according to the sequence was assembled as described in "The intermittent synthesis of the peptide in the TentaGe S resins". After completing the synthesis, the peptide resin was washed with DMF (3 x 5 ml, 1 minute each), DCM (3 x 5 ml, minute each), diethyl ether (3 x 5 ml, 1 minute each) ) and dried in vacuo.

The peptide was cleaved by means of the resin according to method b as described above and dehydrated by freezing by means of acetic acid. The crude product was purified twice by preparative HPLC using the procedure described above. The purified product was found homogeneous and the purity was found better than 90%. The identity of the peptide was confirmed by ES-MS. 6.2% yield.

IN VITRO KINETIC MEASUREMENTS HPLC The HPLC gradient analysis of the samples of in vitro kinetic measurements performed as described above using •• a HP 1100 Hewlett Packard HPLC system consisting of HP 1100 Binary Pump, an HP 1100 Autosampler, an HP 1100 Column Thermostat and an HP 1100 Variable Wavelength Detector. A Merck LiChroCART column (125 x 4 mm I.D.) and a LiChroCART precolumn (4 x 4 mm I.D.) packed with LiChrospher RP-18 (5 μm particles) were used. The column was preserved at 25 ° C or 75 ° c and the column flux was measured by UV detection at 215 nm. Separation of the peptide conjugates or natural peptides from the degradation products and constituents of the reaction solutions was performed using a gradient elution of the column with mixtures of mobile phase A (0.1 vol% TFA in water) and mobile phase B (0.085 vol% TFA in acetonitrilp) at a flow rate of 1 ml / min. The following used linear gradients are shown in the following table 1: Table 1 Peptide gradient / peptide conjugate HPLC 25-40% B in H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu 15 minutes Gly-Hisd-Leu-Asn-Ser -Met-Glu-Arg-Val-Glu-Trp- Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe-OH (PTH (1-34) (human) -OH 25-40% B in H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu 15 minutes Gly-Lys-His- Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe- (Lys) 6-OH Peptide / HPLC Conjugate Gradient (PTH (1-34) (Human) - (Lys) 6-OH) 25-40% B in H- (Lys) 6-Ser-Val-Ser-Glu-Ile Gln-Leu-Met-His- 15 minutes Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln- Asp-Val-His-Asn-Phe-OH ((Lys) 6-PTH (1-34) (Human) -OH) Peptide Gradient / HPLC Peptide Conjugate (Substance P- (Lys) 6-NH2 25-40% B in H- (Lys) e-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly- 15 minutes Leu-Met-NH2 ((Lys) e-substance P-NH?) 40-100% B in H-Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu-OH 15 minutes (DISP) 40-100% B in H-Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu- (Lys- 15 minutes Glu) 3-OH (DISP- (Lys-Glu) 3 ~ OH) 5-30% B in H-Tyr-Gly-Gly-Phe-Leu-OH 15 minutes (Leu-Encef aliña) 5-30% B in H-Tyr-Gly -Gly-Phe-Leu- (Lys) 6-0H 15 minutes (Leu-Encef aliña- (Lys) ß ~ H) 10-35% B in H- (Lys) e-Tyr-Gly-Gly-Phe-Leu -OH 15 minutes ( (Lys) g-Leu-Encephalin-OH) 10-35% B in H- (Lys) e-Tyr-Gly-Gly-Phe-Leu- (Lys) 6-OH 15 minutes ((Lys) g-Leu- Encef aliña- (Lys) g-OH) 5-30% B in H-Tyr-Gly-Gly-Phe-Leu (Lys) i0-OH 15 minutes (Leu-Encef aliña- (Lys)? Or ~ OH) 5-30% B in H-Tyr-Gly-Gly-Phe-Leu- (Orn) 6.-OH 15 minutes (Leu-Encef ali a- (Orn) 6 ~ OH) 5-30% B in H-Tyr -Gly-Gly-Phe-Leu- (Dbu) 6-OH 15 minutes (Leu-Encef aliña- (Dbu) 6-OH) 5-30% B in H-Tyr-Gly-Gly-Phe-Leu- (Dpr ) 6-OH 15 minutes (Leu-Encef aliña- (Dpr) 6-OH) 5-30% B in H-Tyr-Gly-Gly-Phe-Leu-Lys (Glu) 4-Lys-OH 15 minutes Peptide / peptide conjugate gradient HPLC (Leu-Encef aline -Lys- (Glu) -Lys-OH) 5-30% B in H-Tyr-Gly-Gly-Phe-Leu-Lys- (Glu) 3- (Lys) 2-OH 15 minutes (Leu-Encef aline-Lys- (Glu) 3- (Lys)? -OH) KINETIC HYDROLYSIS IN THE SOLUTION OF ENZYME Degradation of the peptide conjugate and the corresponding natural peptide were studied at 37 ° C in a 50 nM phosphate buffer solution at pH 7.4 containing leucine aminopeptidase (25 U / ml) or carboxypeptidase A (1 or 25 U / ml). The experiments are initiated by the addition of an aliquot (10 μl) of a stock solution (1 mg / ml) of peptide or natural peptide conjugate to 900 μl of a solution of the preheated enzyme giving a final concentration of ~ 0.1 mg / ml (10"5 - 10" 4 M) of the peptide conjugate or the native peptide. The peptide / enzyme solution was stored at 37 ° C using a block heater SHT200D from Stuart Scientific. At appropriate time intervals, the 100 μl samples were removed from the peptide / enzyme solution, thoroughly mixed with 20 μl 25% TFA in acetonitrile to stop the process of enzymatic degradation and analyzed by HPLC as described above. The half-lives (t? / 2) for the peptide conjugate and the corresponding natural peptide in the enzyme solutions were calculated by plotting the natural logarithm of the residual peptide concentration (the HPLC peak) against time using the formula x ln (2), where Kobs is the apparent proportion constant of the first order proportion for the observed degradation.

H-Ser-Val-Ser-glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe-OH (PTH (1-34) (Human) -OH) Kinetic hydrolysis in the aminopeptidase of leucine Degradation of H-Ser-Val -Ser-glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys -Leu-Gln-Asp-Val-His-Asn-Phe-OH (~ 2.4 x 10"5 M) in 50 mM phosphate buffer solutions of pH 7.4 containing leucine aminopeptidase (25 U / ml) was studied As previously described, the constant of the proportion of pseudo first order for degradation was estimated at 2.1 x 10 ~ 3 min "1 and the corresponding half-life was calculated at 330 minutes as previously described.

Kinetic hydrolysis in carboxypeptidase A The degradation of H-Ser-Val-Ser-glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg -Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe-OH (~ 2.4 x 10"5 M) in 50 mM phosphate buffer solutions of pH 7.4 containing carboxypeptidase A (25 U / ml) was studied as described above.The constant of the proportion of pseudo first order for degradation was estimated at 5.2 min "1 and the corresponding half-life was calculated at 0.13 minutes as previously stated. he described.

H-Ser-Val-Ser-glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe-OH (PTH (1-34) (Human) - (Lys) 6-OH) Kinetic hydrolysis in leucine aminopeptidase Degradation of H-Ser-Val-Ser-glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu -Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe- (Lys) 6-OH (~ 2.0 x 10 ~ 5 M) in 50 mM pH 7.4 phosphate buffer solutions containing leucine aminopeptidase (25 U / ml) were studied as described above. The constant of the proportion of pseudo first order for degradation was estimated at 1.2 min "3 and the corresponding half-life was calculated at 578 minutes as previously described.

Kinetic hydrolysis in carboxypeptidase A The degradation of H-Ser-Val-Ser-glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg -Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe- (Lys) 6-OH (~ 2.0 x 10"5 M) in 50 mM of solutions of phosphate buffer of pH 7.4 containing aminopeptidase A (1 U / ml) was studied as described above.The constant of the proportion of pseudo first order for degradation was estimated at 1.5 x 10"2 min" 1 and the corresponding life average - it was calculated at 47 minutes as previously described.

H- (Lys) 6-Ser-Val-Ser-glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu -Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe-OH ((Lys) 6-PTH (1-34) (Human) - (Lys) 6-OH) Kinetic hydrolysis in leucine aminopeptidase Degradation of H- (Lys) e-Ser-Val-Ser-glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn- Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe-OH (~ 2.0 x 10 ~ 5 M) in 50 mM Phosphate buffer solutions of pH 7.4 containing aminopeptidase leucine (25 U / ml) were studied as described above. The constant of the proportion of pseudo first order for degradation was estimated at 3.5 x 10"3 min" 1 and the corresponding half-life was calculated at 198 minutes as previously described.

H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala Trp-Leu-Val-Lys-Gly-Arg-OH (GLP-1 (7-36) (Human) -OH) Kinetic hydrolysis in aminopeptidase of leucine Degradation of H-His-Ala-Glu-Gly-Thr- Phe-Thr- Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg- OH (-3.0 x 10"5 M) in 50 mM phosphate buffer solutions of pH 7.4 containing leucine aminopeptidase (25 U / ml) was studied as described above.The constant of the pseudo-first order ratio for the degradation was estimated at 3.1 x 10"2 min" 1 and the corresponding half-life was calculated at 22 minutes as previously described.

Kinetic hydrolysis in carboxypeptidase A Degradation of H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-OH (-3.0 x 10"5 M) in 50 mM phosphate buffer solutions of pH 7.4 containing carboxypeptidase A (1 U / ml) was studied as described above.The constant of the proportion of pseudo first order for degradation was estimated at 4.7 x 10"3 min" 1 and the corresponding half-life was calculated at 148 minutes as previously described.

H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala Trp-Leu-Val-Lys-Gly-Arg- (Lys) 6-OH (GLP-1 (7-36) (Human) - (Lys) 6-OH) Kinetic hydrolysis in aminopeptidase of leucine Degradation of H- His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp- Leu-Val-Lys-Gly-Arg- (Lys) 6-OH (-2.5 x 10"5 M) in 50 mM phosphate buffer solutions of pH 7.4 containing leucine aminopeptidase (25 U / ml) was studied as previously described, the constant of the proportion of pseudo first order for degradation was estimated at 1.3 x 10"2 min" 1 and the corresponding half-life was calculated at 53 minutes as previously described.

Kinetic hydrolysis in carboxypeptidase A Degradation of H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg- (Lys) 6-OH (-2.5 x 10"5 M) in 50 mM phosphate buffer solutions of pH 7.4 containing carboxypeptidase A (1 U / ml) was studied as described above The constant of the pseudo-first order proportion for degradation was estimated at 8 x 10"3 min" 1 and the corresponding half-life was calculated at 87 minutes as previously It was described.

H-Gly-Gly-Thr-Tyr-Ser-Cys-His-Phe-Gly-Pro-Leu-Thr-Trp-Val-Cys-Lys-Pro-Gln-Gly-Gly-OH (EMP-l-OH) Kinetic hydrolysis in aminopeptidase of leucine Degradation of H-Gly-Gly-Thr-Tyr-Ser-Cys-His-Phe-Gly-Pro-Leu-Thr-Trp-Val-Cys-Lys-Pro-Gln-Gly- Gly-OH (-4.8 x 10"5 M) in 50 mM phosphate buffer solutions of pH 7.4 containing leucine aminopeptidase (25 U / ml) was studied as described above.The constant of the proportion of pseudo first order for degradation it was estimated at 1.5 x 10"3 min" 1 and the corresponding half-life was calculated at 462 minutes as previously described.

Kinetic hydrolysis in carboxypeptidase A Degradation of H-Gly-Gly-Thr-Tyr-Ser-Cys-His-Phe-Gly-Pro-Leu-Thr-Trp-Val-Cys-Lys-Pro-Gln-Gly-Gly -OH (-4.8 x 10"5 M) in 50 mM Phosphate buffer solutions of pH 7.4 containing carboxypeptidase A (l / ml) was studied as described above.A half-life of more than 50 hours was estimated for degradation.

H-Gly-Gly-Thr-Tyr-Ser-Cys-His-Phe-Gly-Pro-Leu-Thr-Trp-Val-Cys-Lys-Pro-Gln-Gly-Gly- (Lys) 6-OH (EMP -1- (Lys) 6-OH) Kinetic hydrolysis in leucine aminopeptidase Degradation of H-Gly-Gly-Thr-Tyr-Ser-Cys-His-Phe-Gly-Pro-Leu-Thr-Trp-Val-Cys-Lys-Pro-Gln-Gly -Gly- (Lys) 6-0H (-3.5 x 10"5 M) in 50 mM phosphate buffer solutions of pH 7.4 containing leucine aminopeptidase (25 U / ml) was studied as described above. of more than 100 hours was estimated for degradation.

Kinetic hydrolysis in carboxypeptidase A Degradation of H-Gly-Gly-Thr-Tyr-Ser-Cys-His-Phe-Gly-Pro-Leu-Thr-Trp-Val-Cys-Lys-Pro-Gln-Gly-Gly - (Lys) 6-OH (-3.5 x 10"5 M) in 50 mM phosphate buffer solutions of pH 7.4 containing carboxypeptidase A (1 U / ml) was studied as described above. 20 hours was estimated, for degradation.

H- (Lys) 6-Gly-Gly-Thr-Tyr-Ser-Cys-His-Phe-Gly-Pro-Leu-Thr-Trp-Val-Cys-Lys-Pro-Gln-Gly-Gly-OH- ( Lys) 6 ((Lys) 6-EMP-1-OH) Kinetic hydrolysis in leucine aminopeptidase Degradation of H- (Lys) 6-Gly-Gly-Thr-Tyr-Ser-Cys-His-Phe-Gly-Pro-Leu-Thr-Trp-Val-Cys-Lys- Pro-Gln-Gly-Gly-OH (-3.5 x 10"5 M) in 50 mM phosphate buffer solutions of pH 7.4 containing leucine aminopeptidase (25 U / ml) was studied as described above. more than 24 hours was estimated for degradation.

H- (Lys) 6-Gly-Gly-Thr-Tyr-Ser-Cys-His-Phe-Gly-Pro-Leu-Thr-Trp-Val-Cys-Lys-Pro-Gln-Gly-Gly- (Lys) 6-OH ((Lys) 6-EMP-l- (Lys) 6-OH) Kinetic hydrolysis in leucine aminopeptidase Degradation of H- (Lys) 6-Gly-Gly-Thr-Tyr-Ser-Cys-His-Phe-Gly-Pro-Leu-Thr-Trp-Val-Cys-Lys- Pro-Gln-Gly-Gly- (Lys) 6-0H (-2.8 x 10"5 M) in 50 mM phosphate buffer solutions of pH 7.4 containing leucine aminopeptidase (25 U / ml) was studied as previously described, a half-life of more than 100 hours was estimated for degradation.

H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 (substance P) Kinetic hydrolysis in the aminopeptidase of leucine Degradation of H-Arg-Pro-Lys-Pro Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 (-7.4 x 10"5 M) in 50 mM phosphate buffer solutions of pH 7.4 containing leucine aminopeptidase (25 U / ml) was studied as previously described, the constant of the proportion of pseudo first order for degradation was estimated at 4.5 x 10 ~ 2 min "1 and the corresponding half-life was calculated at 16 minutes as previously described.

Kinetic hydrolysis in carboxypeptidase A Degradation of H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 (-7.4 x 10"5 M) in 50 mM buffer solutions phosphate pH 7.4 containing carboxypeptidase A (1 U / ml) was studied as described above.The constant of the proportion of pseudo first order for degradation was estimated at 2.0 x 10"2-min" 1 and the corresponding half-life it was calculated at 35 minutes -as previously described.

H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met- (Lys) 6 ~ NH2 (Substance P- (Lys) ß ~ NH2) Kinetic hydrolysis in the aminopeptidase of leucine degradation of H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met- (Lys) 6-NH2 (-4.7 x 10"5 M) in 50 mM phosphate buffer solutions pH 7.4 containing leucine aminopeptidase (25 ^ ü / ml) was studied as described above The constant of the pseudo-first order proportion for degradation was estimated at 1.1 x 10"min" 1 and the corresponding half-life was calculated to 66 minutes as previously described.

Kinetic hydrolysis in carboxypeptidase A The degradation of H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met- (Lys) 6-NH2 (-4.7 x 10"5 M) in 50 mM phosphate buffer solutions of pH 7.4 containing carboxypeptidase A (1 U / ml) were studied as described above.The constant of the pseudo-first order proportion for degradation was estimated at 5.5 x 10"3 min" 1 and The corresponding half-life was calculated at 126 minutes as previously described.

H- (Lys) 6-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 ((Lys) 6-Substance P-NH2) Kinetic hydrolysis in the aminopeptidase of leucine degradation of H- (Lys) 6-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 (-4.7 x 10"5 M) in 50 mM phosphate buffer solutions pH 7.4 containing leucine aminopeptidase (25 U / ml) was studied as described above.The constant of the proportion of pseudo first order for degradation was estimated at 2 x 10"3 min-1 and the corresponding half-life was calculated at 347 minutes as previously described.

H-Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu-OH (DSIP) Kinetic hydrolysis in the aminopeptidase of leucine Degradation of H-Trp-Ala-Gly-Gly-Asp-Ala-Ser -Gly-Glu-OH (-10-5 M) in 50 mM phosphate buffer solutions of pH 7.4 containing leucine aminopeptidase (25 U / ml) was studied as described above. The half-life was calculated just under 20 minutes.

H-Trp-Ala-Gly-Gly-Asp-Ala-Ser-'Gly-Glu- (Lys-Glu) 3-OH (DSIP- (Lys-Glu) 3-OH) Kinetic hydrolysis in leucine inopeptidase Degradation of H-Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu- (Lys-Glu) 3 -OH (-10"5 M) in 50 mM phosphate buffer solutions of pH 7.4 containing leucine aminopeptidase (25 U / ml) was studied as described above The constant of the pseudo first order ratio for degradation it was determined as described above and the half-life was calculated at 145 minutes.

H-Tyr-Gly-Gly-Phß-Leu-OH (Leu-enkephalin) Kinetic hydrolysis in the aminopeptidase of leucine Degradation of H-Tyr-Gly-Gly-Phe-Leu-OH (-1.8 x 10"4 M ) in 50 mM phosphate buffer solutions of pH 7.4 containing leucine aminopeptidase (25 U / ml) was studied as described above The constant of the pseudo-first order ratio for degradation was estimated 6.8 x 10"1 min "1 and the corresponding half-life was calculated at 0.7 minutes as described above.

Kinetic hydrolysis in carboxypeptidase A The degradation of H-Tyr-Gly-Gly-Phe-Leu-OH (-1.8 x 10 ~ 4 M) in 50 mM phosphate buffer solutions of pH 7.4 containing carboxypeptidase A (1U / ml) was studied as described above. The constant of the proportion of pseudo first order for degradation was estimated at 9.8 x 10"1 min" 1 and the corresponding half-life was calculated at 0.7 minutes as previously described.

H-Tyr-Gly-Gly-Phe-Lßu- (Lys) 6"" OH (Leu-encef aline- (Lys) 6-OH) Kinetic hydrolysis in the aminopeptidase of leucine Degradation of H-Tyr-Gly-Gly -Phe-Leu- (Lys) 6-OH (-1.8 x 10 ~ 4 M) in 50 mM phosphate buffer solutions of pH 7.4 containing leucine aminopeptidase (25 U / ml) was studied as described above. The constant of the proportion of pseudo first order for degradation was estimated 9.7 x 10"3 min" 1 and the corresponding half-life was calculated at 72 minutes as described above.

Kinetic hydrolysis in carboxypeptidase A Degradation, from H-Tyr-Gly-Gly-Phe-Leu- (Lys) 6-OH (-7.5 x 10 ~ 5 M) in 50 mM phosphate buffer solutions of pH 7.4 containing carboxypeptidase A (1 U / ml) was studied as described above. The constant of the proportion of pseudo first order for degradation was estimated at 7 x 10 ~ 4 min "1 and the corresponding half-life was calculated at 990 minutes as previously described.

H- (Lys) 6-Tyr-Gly-Gly-Phe-Leu-OH ((Lys) 6-Leu-en-aline-OH) Kinetic hydrolysis in the aminopeptidase. of leucine The degradation of H- (Lys) 6-Tyr-Gly-Gly-Phe-Leu-OH (-7.5 x 10"5 M) in 50 mM phosphate buffer solutions of pH 7.4 containing leucine aminopeptidase (25 U / ml) was studied as described above.The constant of the proportion of pseudo first order for degradation was estimated 2.6 x 10"2 min" 1 and the corresponding half-life was calculated at 27 minutes as described above.

H- (Lys) ß-Tyr-Gly-Gly-Phe-Leu- (Lys) 6-OH ((Lys) 6-Leu-encephali- nia- (Lys) e ~ OH) Kinetic hydrolysis in the aminopeptidase of leucine Degradation of H- (Lys) -E-Tyr-Gly-Gly-Phe-Leu- (Lys) 6-OH (-4.8 x 10"5 M) in 50 mM phosphate buffer solutions of pH 7.4 containing leucine aminopeptidase ( 25 U / ml) was studied as described above.A half-life of more than 100 was estimated for degradation.

H-Tyr-Gly-Gly-Phe-Leu- (Lys)? 0-OH (Leu-encephali- nia- (Lys) 10-OH) Kinetic hydrolysis in the aminopeptidase of leucine Degradation of H-Tyr-Gly-Gly- Phe-Leu- (Lys)? 0-OH (-5.4 x 10"5 M) in 50 mM phosphate buffer solutions of pH 7.4 containing leucine aminopeptidase (25 U / ml) was studied as described above.A half-life of more than 100 was estimated for degradation .

Kinetic hydrolysis in carboxypeptidase A The degradation of H-Tyr-Gly-Gly-Phe-Leu- (Lys)? 0-OH (-5.4 x 10"5 M) in 50 mM of phosphate buffer solutions of pH 7.4 that containing carboxypeptidase A (1 U / ml) was studied as described above The constant of the proportion of pseudo first order for degradation was estimated at 3 x 10"4 min" 1 and the corresponding half-life was calculated at 2310 minutes as previously described.

H-Tyr-Gly-Gly-Phe-Leu- (Orn) ß-OH (Leu-en-aline- (Orn) 6-OH) Kinetic hydrolysis in the aminopeptidase of leucine Degradation of. H-Tyr-Gly-Gly-Phe-Leu (Orn) 6-OH (-5.7 x 10"5 M) in 50 mM phosphate buffer solutions of pH 7.4 containing leucine aminopeptidase (25 U / ml) it was studied as described above.The constant of the proportion of pseudo first order for degradation was estimated at 6.4 x 10"3 min" 1 and the corresponding half-life was calculated at 108 minutes as previously described.

Kinetic hydrolysis in carboxypeptidase A The degradation of H-Tyr-Gly-Gly-Phe-Leu- (Orn) 6-OH (-5.7 x 10"5 M) in 50 mM of phosphate buffer solutions of pH 7.4 containing carboxypeptidase A (1 U / ml) was studied as described above.A half-life of more than 100 hours was estimated for degradation.

H-Tyr-Gly-Gly-Phe-Leu (Dbu) 6-OH (Leu-enkephalin- (Dbu) e-OH) Kinetic hydrolysis in the aminopeptidase of leucine Degradation of H-Tyr-Gly-Gly-Phe -Leu- (Dbu) 6 ~ OH (-6.0 x 10"5 M) in 50 mM phosphate buffer solutions of pH 7.4 containing leucine aminopeptidase (25) • U / ml) was studied as described above. The constant of the proportion of pseudo first order for degradation was estimated at 2.5 x 10"2 min" 1 and the corresponding half-life was calculated at 28 minutes as previously described.

Kinetic hydrolysis in carboxypeptidase A Degradation of H-Tyr-Gly-Gly-Phe-Leu- (Dbu) 6-0H (-6.0 x 10 ~ 5 M) in 50 mM pH 7.4 phosphate buffer solutions containing carboxypeptidase A (1 U / ml) was studied as described above. The constant of the pseudo-first-order ratio for degradation was estimated at 5 x 10"3 min-1 and the corresponding half-life was calculated at 1386 minutes as previously described H-Tyr-Gly-Gly-Phe-Leu (Dpr) 6-OH (Leu-enkephalin- (Dpr) 6-OH) Kinetic hydrolysis in leucine aminopeptidase Degradation of H-Tyr-Gly-Gly-Phe-Leu- (Dpr) 6-OH (- 6.3 x 10"5 M) in 50 mM phosphate buffer solutions of pH 7.4 containing leucine aminopeptidase (25 U / ml) was studied as described above. The constant of the proportion of pseudo first order for degradation was estimated at 1.7 x 10"1 min" 1 and the corresponding half-life was calculated at 4.2 minutes as previously described.

Kinetic hydrolysis in carboxypeptidase A Degradation of H-Tyr-Gly-Gly-Phe-Leu- (Dpr) 6 ~ 0H (-6.3 x 10"5 M) in 50 mM phosphate buffer solutions of pH 7.4 containing carboxypeptidase A (1 U / ml) was studied as described above.The constant of the pseudo-first order ratio for degradation was estimated at 2.4 x 10 ~ 2 min "1 and the corresponding half-life was calculated at 29 minutes as previously described.

H-Tyr-Gly-Gly-Phe-Leu-Lys (Glu) 4-Lys-OH (Leu-enkephalin-Lys- (Glu) 4-Lys-OH) Kinetic hydrolysis in the aminopeptidase of leucine The degradation of H- Tyr-Gly-Gly-Phe-Leu-Lys- (Glu) 4-Lys-OH (-7.5 x 10"5 M) in -50 mM phosphate buffer solutions of pH 7.4 containing leucine aminopeptidase (25 U / ml) was studied as described above. The constant of the proportion of pseudo first order for the degradation was estimated at 6.5 x 10"2 min" 1 and the corresponding half-life was calculated at 11 minutes as previously described.

Kinetic hydrolysis in carboxypeptidase A The degradation of H-Tyr-Gly-Gly-Phe-Leu-Lys- (Glu) 4-Lys-OH (-7.5 x 10"5 M) in 50 mM phosphate buffer solutions of pH 7.4 containing carboxypeptidase A (1 U / ml) was studied as described above The constant of the pseudo-first order proportion for degradation was estimated at 6 x 10"4 min" 1 and the corresponding half-life was calculated at 1155 minutes as previously described.

H-Tyr-Gly-Gly-Phe-Leu-Lys (Glu) 3- (Lys) 2-OH (Leu-enkephalin-Lys- (Glu) 3- (Lys) 2-OH) Kinetic hydrolysis in the aminopeptidase of Leucine Degradation of H-Tyr-Gly-Gly-Phe-Leu-Lys- (Glu) 3- (Lys) 2-OH (-7.5 x 10"5 M) in 50 mM phosphate buffer solutions of pH 7.4 containing leucine aminopeptidase (25 U / ml) was studied as described above.The constant of the pseudo-first-order ratio for degradation was estimated at 1.2 x 10"1 min" 1 and the corresponding half-life was calculated at 5.7 minutes as previously described.

Kinetic hydrolysis in carboxypeptidase A The degradation of H-Tyr-Gly-Gly-Phe-Leu-Lys- (Glu) 3- (Lys) 2-OH (-7.5 x 10"5 M) in 50 mM buffer solutions pH 7.4 phosphate containing carboxypeptidase A (1 U / ml) was studied as described above. The constant of the proportion of pseudo first order for -degradation was estimated at 8 x 10"4 min" 1 and the corresponding half-life was calculated at 866 minutes as previously described. Studies with Encephalin Analogs The bioaability of Leu-enkephalin-OH and Leu-enkephalin- (Lys) 6 ~ OH in mice Male mice weighing 20-25 g were given 50 mg of Leu-enkephalin- (Lys) 6_0H per kilo of body weight i.v. or p.o. The compound was dissolved in an isotonic NaCl solution. Mice treated with Leu-Encefaliña- (Lys) 6 ~ OH, 50 mg / kg po were sacrificed by means of decapitation at 0, 15, 30, 60, 90, 240, 480, 960, and 1380 minutes after the dose. Mice treated with Leu-enkephalin- (Lys) 6-OH, 50 mg / kg i.v. they were sacrificed by means of decapitation at 5, 15, 30, 60, 180, 240, 370, 720, 1080, and 1440 minutes after the dose. Animals treated with natural Leu-enkephalin-OH, mg / kg p.o or i.v. they were sacrificed by decapitation 30 minutes after the dose. The blood samples were centrifuged immediately (3000 g, 4 ° C) and the serum was isolated and used for activity determination.

The concentrations of Leu-enkephalin-OH or Leu-encephali- na (Lys> 6-OH in the serum were determined by a bioassay using the model of the vas deferens by means of the mice.) The experiments were carried out essentially as described. by Takemori and Porthogese, 1984, Eur. J. Pharmacol.104: 101-104 Next: The vas deferens were isolated from male mice weighing 20-30 g (breeding M011egaard, DK) and suspended through two electrodes in 10 ml baths at rest tension of 1 g The tissues were bathed with a solution of Krebs bicarbonate (physiological buffer) maintaining them at 36-37 ° C and continuously bubbling with 95% 02 and 5% C02. Tissues were stimulated electrically (70 V, duration of 1 ms, 0.1 Hz), and contractions were recorded isometrically in a chart recorder.After tissue equilibration for at least 20 minutes, drugs were added in the bath and the effects mid-highs The data were trained to the equation of% inhibition = MAX x (1 - [Inh] n / ([Inh] n + IC50n)) + Vaseline, where MAX is the maximum contraction of the muscle, [Inh] is the contraction of the inhibitor, n is the Hill slope of the curve and Vaseline is the insensitive contraction of the muscle to the compound. Thus, the calculated concentration is a reflection of the inhibitory activity in the preparation of the vas deferens bioassay and not an exact measurement of Leu-enkephalin- (Lys) 6-OH or Leu-enkephalin- (Lys) 6-OH in the serum .

The values for Leu-enkephalin-OH and Leu-enkephalin- (Lys) 6-OH are average values ± S.E.M. of at least 5 experiments. In assays where the concentration of Leu-enkephalin- (Lysd) s-OH in the serum was determined, 100 μl of serum was added to the tissue bath and 5 inhibition of response was determined. The results are shown in the table 2.

Table 2 Functional activity in serum after p.o or i.v administration of Leu-enkephalin- (Lys) 6-OH in mice (n = 6-8 serum samples per period of time; medium ± S.E.M. ).

N / A: Not applicable Following an injection i.v. of 50 mg per kg body-weight of Leu-encephalitis- (Lys) 6 ~ OH, a rapid increase in activity was observed in the serum after about 5 minutes. Then, the activity declines with the next 30 minutes, but between 240 minutes (4 hours) and 720 minutes (12 hours), the activity reached a second maximum level. The second maximum was possibly related to the entire hepatic circulation of the drug after i.v. administration. of Leu-enkephalin- (Lys) 6-OH. After po administration of Leu-enkephalin- (Lys) 6-OH, the activity in the serum reached a maximum in 90-240 minutes (1.5-4 hours) and the activity was detected after 8, and 16 hours, but not after 23 hours. The total high activities were observed in 30 minutes in the serum samples of the animals treated with p.o or i.v. Leu-enkephalin- (Lys) 6-OH, no activity was detected after 30 minutes of p.o. or i.v. of natural Leu-enkephalin-OH.

The results suggest that Leu-enkephalin- (Lys) 6 ~ OH, but not Leu-enkephalin-OH, is absorbed after p.o. administration and that the rate of elimination in the serum is substantially relative to that of the Natural Leu-enkephalin-OH in the mice.

The stability of Leu-enkephalin-OH and La Leu-encef lina- (Lys) e-OH in the mouse plasmas at 37 ° C.

The stability of Leu-enkephalin-OH, Leu-enkephalin- (Lys) 6 ~ OH, Leu-enkephalin - (Glu2-Lys-Glu3), Leu-enkephalin- (Lys-Glu4-Lys) -OH, Leu- enkephalin- (Orn) 6-OH, Leu-enkephalin- (Dbu) 6-OH, Leu-enkephalin- (Dpr) 6-OH, and Leu-enkephalin- (Lys)? o-OH in the plasma of the mouse at 37 ° C was examined in the vas deferens bioassay model as described above. Mainly by the addition of the plasma sample, a curve of the response to the standard dose was generated in each preparation to express the inhibitory activity as the concentration of each test substance. Thus, the calculated concentration is a reflection of the inhibitory activity in the preparation of the bioassay of the vas deferens. The data of the dose response were adapted to the equation: Answer = Initial value (1- (conc / EC50 + conc) + antecedent Where initial = is the initial principal force of helically induced contraction of the test substance; conc = is the concentration of the test substance; Ec50 = is the concentration of the test substance that produces a maximum average inhibition of electrically induced contraction; antecedent = is the contraction force during maximum relaxation.

All the enkephalin analogues were dissolved in the Krebs buffer at a concentration of 1 mM.

Sixty-six μl of each solution of the test substance (66 nmol of the enkephalin analogue) were incubated with 600 μl of plasma at 37 ° C. A different time point (2-120 minutes), 10 μl samples were isolated by functional activity analysis. The functional activity of each test substance in the plasma was expressed as the concentration of the test substance that was obtained from the same inhibition of the electrically induced concentration in the bioassay of the vas deferens. T was calculated by • adapting the time-concentration data in the equation: Conc (t) = conc (O) • e ("ln2 tl 2) Ft Where conc (O) = is the concentration in t The results are shown in table 3.

Table 3: EC50 and T values for various enkephalin analogues (n = 3-4 / test substance; medium).

ND: Not determined These data suggest that the modifications of Leu-enkephalin-OH increase the EC50 value and increase the stability of the mouse plasma at 37 ° C.

Connection of the μ-Receptor of analogs of Leu-enkephalin-OH Affinities for the μ opiode receptor were determined using [3H] (D-Ala2, N-Me-Phe4, Gly-ol5) enkephalin (DAMGO) (1 nM) as described by Christensen, 1993, Pharmacol . Toxicol 73: 344-345. Next: Bovine brains were placed on ice minutes after slaughter. The caudate nuclei were dissected "and homogenized in 20 vol of sucrose at 0.32 M. The homogenate was centrifuged at 2000 g for 10 minutes.The pill was re-suspended in 10 vol of buffer 50 mM Tris-HCl 7.4 and stored at -20 ° C until use The fraction of the synaptic membrane was incubated with 1 nM of [3 H] DAMGO in the presence of various concentrations of the test ligand The [3 H] -DAMGO link was not specifically established using 1 μM of naloxone After incubation for 15 minutes at 36 ° C the samples were filtered through Whatman GF / C filters and washed with a buffer.The radioactivity was determined using conventional techniques.

As shown in Table 4 below, all compounds were activated in this binding assay, indicating that the modification of Leu-enkephalin-OH affects receptor affinity.

Table 4: The affinity of the Leu-enkephalin-OH analogs in μ opiode receptors measured as 3H-DAMGO bonds (IC 50 values (medium and SD).

The relative low affinity of Leu-enkephalin- (Glu) 6-OH to the other test substances may be due to the very poor solubility of this compound. Thus, the value of IC5o of Leu-enkephalin- (Glu) 6 OH may be low if tested in a solvent in which the compound is more soluble.

In vivo experiments with EMP-1-K6 in mice To examine the biological efficiency of peroral treatment (p.o.) with EMP-1 and EMP-1K6, the hematological responses of a p.o. equimolar (956 nmol) of EMP-1 (2 mg.) and EMP-1-K6 (2.56 mg) were examined in male mice (n = 8 / groups) .To examine the course of time of hematological responses, a 10 μl vein blood sample was collected from the retroorbital plexuses on days 0, 2, and 4. The body weight (BW) and the plasma concentration of the hemoglobin (P-Hgb), the hematocrit value ( Het), the cell calculation of red blood (RBC), and the mean concentration of cellular hemoglobin (MCHC9 were determined before (day 0), and 2 and 4 days after administration of EMP-1 or EMP-l- Kß The results are shown in Table 5.

Table 5: Changes in body weight in haematological parameters after 4 days of p.o. of 956 nmol EMP-1 or EMP-1-K6. Relative changes are presented in parentheses (mean 1 SEM). p < 0.05 vs EMP-1 p.o.

These data show that the p.o. of 2.56 mg EMP-1-K6 produces a significantly large increase in P-hgb, Het, and RBC in the equimolar dose of EMP-1 P.O. None of the compounds affect growth or MCHC. These results suggest that EMP-1-Ke is absorbed after p.o. and that this produces a rapid stimulation of the, erythropoiesis in the mice.

STUDIES WITH THE ANALOGS OF PARATHYROID HORMONE (PTH) General Procedures Osteoblast Retraction Test The retraction tests were performed with an osteoblast prepared from calvary of the 1-day-old mice according to published protocols (Miller et al. , 1076, Scienc e 192: 13401343). Briefly, the osteoblasts were seeded in a minimal medium-μ (μMEM) essentially free of serum at a density of 3000 cells per cm 2 in 96-well tissue culture plates coated with 50 μg / ml of type I collagen in a phosphate buffered saline solution containing 0.1% bovine serum albumin (PBS). One day after the coating, the PTH compounds were added to the final concentration of 10 nM and the incubation was carried out for 1 hour. The cells were then fixed and stained with toluidine blue, and the number of cells retracted was counted by a visual inspection.

The same PTH is able to portray something like 64% of the cells compared to bleaching where only 10-12% of the cells are retracted.

The enzyme immunoassay (EIA9 for human PTH (1-34) This is a standard EIA assay (EIAS (h) -6101 from Peninsula Laboratories, Inc.) The biotinylated peptide and the peptide compete for binding to (1-) 34) - PTH antibody Horseradish peroxidase conjugated to streptavidin (SA-HRP) is allowed to bind to the primary antibody / biotinylated peptide complex The 3,3,3'-Tetramethyl Benzidine dihydrochloride (TMB) is allowed to react with the HRP link, the intensity of the color is used for the quantification.

"The specificity of the assay: hPTH (1-34) = 100%; hPTH (1-38) = 1.00% hPTH (1-44) = 0%; hPTH (39-68) = 0%; hPTH (1-84) ) = 0%, ratPTH (l-34) = 0%.

The results are shown in Table 6. In the Osteoblast retraction assay, hPTH (1-34) retains approximately 89% of the activity of the native, human parathyroid hormone. H-hPTH (1-34) -K6OH and H-K6PTH (1-34) -OH show 55 and 49%, respectively, of the activity of the parent compound hpth (1-34). The antibody with respect to hPTH (1-34) was used in the two well recognized EIA modifications. Table 6 The functional activity of the substance P-NH2 and the (Lys) e-substance P-NH2 The functional activity of the substance P-NH2 and the (Lys) 6-substance P-NH2 is characterized by using guinea pig ileus. The experiments were carried out essentially as described by Kristiansen et al., 1992, Br. J. Pharmacol. 58: 1150) with the modification of the ileus not electrically stimulated. After the application the induced contraction was measured. The data of the dose response were adapted to the equation: Response = Initial value • conc 7 (EC50 + conc) Where the initial value = is the force of the initial contraction induced electrically by the addition of the test substance; conc = is the "concentration of the test substance; EC50 = is the concentration of the test substance produced by the average maximum inhibition of the electrically induced contraction of the electrically induced contraction of the substance P-NH2 (EC50 = 40nM) and ( Lys) 6-substanceP-NH2 (EC50 = 5 nM) both act as agonists in the ileum of guinea pigs.

The invention described and claimed herein is not limited in scope to the specific embodiments disclosed herein, since these embodiments are intended to be within the various aspects of the invention. Any of the equivalent embodiments are intended to be within the scope of this invention. Indeed, several modifications of the invention in addition to those that are shown and described here will apparently reach those skilled in the art of the foregoing description. Such modifications are also intended to be within the scope of the appended claims.

Several references are cited, the declaration of such is incorporated by reference in their totalities .- It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the manufacture of the objects to which it refers.

Having described the invention as above, property is claimed as contained in the following:

Claims (49)

1. A pharmacologically active peptide conjugate that has a reductive tendency towards the enzymatic fraction comprising X and Z, characterized in that X is a pharmacologically active peptide sequence, and where Z is a stabilizing peptide sequence, 4-20 amino acid units covalently linked to X via a peptide bond where each amino acid unit in said peptide Z sequence is selected from a group consisting of Ser, Thr, Tyr, Asn, Gln, - Glu, Lys, Arg, Ills, Orn 2, 4-diaminobutanoic acid (Dbu), 2,3-diaminopropanoic acid (Dpr) and Met, and where the ratio between the half-life of said conjugate and the half-life of the corresponding pharmacologically active peptide sequence, X, when treated with carboxypeptidase A or leucine aminopeptidase in about 50 mM of a phosphate buffer at about pH of 7.4 at about 37 ° C or in a serum or plasma at least about 2, preferably at least about 3, such as at least about 5, more preferably at least about 7, such as at least about 9, for example, at least 10, or when said pharmacologically active peptide X is not absorbed orally, said conjugate is absorbed, to a salt thereof, with the proviso that said pharmacologically active peptide conjugate is not selected from H-Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu- (Lys-Glu) 3-OH, H-Trp-Ala -Gly-Gly-Asp-Ala-Ser-Gly-Glu- (Glu) 6-OH, H-Tyr-Gly-Gly-Phe-Leu- (Glu) 6-OH and H-Tyr-Gly-Gly-Phe -Leu- (Lys) 6-OH.

2. A peptide conjugate according to claim 1, characterized in that said peptide conjugate is covalently linked to the C-terminal carboxyl function of X.

3. A peptide conjugate according to claim 1, characterized in that Z is covalently linked to the N-terminal nitrogen atom of X.

4. A peptide conjugate according to claim 1, characterized in that the first sequence (Z) is covalently linked to X at the C-terminal carbonyl function of X and a second sequence (Z) is covalently linked to the nitrogen atom of X.

5. A peptide conjugate according to claim 1, characterized in that Z is covalently linked to a nitrogen atom in the side chain of a lysine, arginine or histidine residue or in a carbonyl function in the side chain of glutamic acid and the aspartic acid of X.

6. A peptide conjugate according to any of the preceding claims, characterized in that Z consists of 4-15, preferably 4-10, more preferably 4-7, such as 6 amino acid units.

7. A peptide conjugate according to claim 6, characterized in that each amino acid unit in Z is selected from the group consisting of Glu, Lys, and Met.

8. A peptide conjugate according to any of claims 6 or 7, characterized in that at least one Lys unit of acidic amide, preferably at least two Lys units of amino acid, such as at least three Lys units of amino acid, for example, at least four Lys units of amino acid, more preferably at least five Lys units of amino acid, such as six Lys units of amino acid.

9. A peptide conjugate according to claim 8, characterized in that Z is (Lys) n where n is an -one in the range of 4 to 15, preferably in the range of 4 to 10, such as in the range of 4. to 8, for example, in the range of 4 to 6.

10. A peptide conjugate according to claim 9, characterized in that Z is Lys4, Lys5 or Lys6.

11. A peptide conjugate according to claim 10, characterized in that Z is Lys6.

12. A peptide conjugate according to claim 6, characterized in that Z is (Lys-Xaa) mo (Xaa-Lys) m where m is an integer in the range of 2 to 7, each Xaa is independently selected from the group consisting of of Ser, Thr, Tyr, Asn, Gln, Asp, Glu, Arg, His, Orn, 2,4-diaminobutanoic acid, 2, 3-diaminopropanoic acid and Met.

13. A peptide conjugate according to claim 12, characterized in that Z is (Lys-Xaa) 3 0 (Xaa-Lys) 3 wherein each Xaa is independently selected from the group consisting of Ser, Thr, Tyr, Asn, Gln, Asp, Glu, Arg, His, Orn, 2,4-diaminobutanoic acid, 2,3-diaminopropanoic acid and Met.

14. A peptide conjugate according to claim 13, characterized in that Z is (Lys-Glu) 3 or (Glu-Lys) 3.

15. A peptide conjugate according to any of claims 6 or 8, characterized in that Z is Lysp-Xaaq where p and q are integers in the range of 1 to 14, with the proviso that p + q is in the range of 3- 15, and each Xaa is independently selected from the group consisting of Ser, Thr, Tyr, Asn, Gln, Asp, Glu, Arg, His, Orn, 2,4-diaminobutanoic acid, 2,3-diaminopropanoic acid and Met.

16. A peptide conjugate according to claim 15, characterized in that Z that Lys3-Xaa3 or Xaa3-Lys3, wherein each Xaa is independently selected from the group consisting of Ser, Thr, Tyr, Asn, Gln, Asp, Glu, Arg , His, Orn, 2,4-diaminobutanoic acid, 2,3-diaminopropanoic acid and Met.

17. A peptide conjugate according to any of claims 1 to 7, characterized in that Z consists only of L-amino acids.

18. A peptide conjugate according to any of claims 1 to 7, characterized in that Z consists only of L-amino acids.

19. A peptide conjugate according to any of claims 1-6, characterized in that Z is (Dbu) not (Dpr) n where n is an integer in the range of 4 to 15, preferably in the range of 4 to 10, such as in the range of 4 to 8, for example, in the range of 4 to 6.

20. A peptide conjugate according to claim 19, characterized in that Z is Dpr6.

21. A peptide conjugate according to any of the preceding claims, characterized in that said pharmacologically active peptide sequence (X) consisting of • at most 75 amino acid units, such as at most for example, at a lot of 60, preferably at a lot of 55, such as at most 52, for example, at a lot of 50.

22. The peptide conjugate according to claim 21, characterized in that X is selected from the group consisting of enkephalin, Leu-enkephalin, Met-enkephalin, angiotensin I, angiotensin II, vasopressin, endothelin, vasoactive intestinal peptide, neurotensin, endorphins, insulin, gramicidin, paracelsin, delta-inducing peptide, gonadotropin-releasing hormone, hormone (1-34) of the human parathyroid, analogs of the truncated critropoicytin, specifically EMP-1, peptide (ANP, ANF) natriuretic atrial, natriuretic peptide (hBNP) of the human brain, cecropin, cinetensin, neurophysins, elaphine, guamerin, atriopeptin I, atriopeptin II, deltorphine I, deltorphine II, vasotocin, bradykinin, dynorphin, dynorphin A, dynorphin B, factor releasing growth hormone, growth hormone, growth hormone release peptide, oxytocin, calcitonin, peptide referred to the calcitonin gene, peptide II refer gone to the calcitonin gene, growth hormone release peptide, tachykinin, adrenocorticotropic hormone (ACTH), natriuretic brain polypeptide, colecisticinin, corticotropin releasing factor, diazepam binding inhibitor fragment, FMRF amide, galanin, gastric release polypeptide, gastric inhibitor polypeptide, gastrin, gastrin release peptide, glucagon, glucagon-like peptide I, glucagon-like peptide II, LHRH, melanin concentration hormone, melanocyte stimulation hormone (MSH) , alpha-MSH, modulation peptides of morphine, motilin, neurokinin A, neurosinin B, nueromedin B, neuromedin C, neuromedin K, neuromedin N, neuromedin U, neuropeptide K, neuropeptide Y, polypeptide (PACAP), pancreas polypeptide, peptide YY, peptide histidine-methionine amide (PHM), secretin, somatoscatine, substance K, thyrotropin releasing hormone (TRH), quiotopropin, melanostantin (MTF-1), thrombopoietin analogues, in particular AF 12505 (Ile-Glu-Gly-Pro-Thr-Leu-Arg -Gln-Trp-Leu-Ala-Ala-Arg-Ala), factor 1 (57-70) of insulin-like growth (Ala-Leu-Leu-Glu-Thr-Tyr-Cys-Ala-Thr-Pro- Ala-Lys-Ser-Glu), factor I (30-41) of insulin-like growth (Gly-Tyr-Gly-Ser-Ser-Ser-Arg-Arg-Ala-Pro-Gln-Thr). Factor I (24-41) of insulin-like growth (Tyr-Phe-Asn-Lys-Pro-Thr-Gly-Tyr-Gly-Ser-Ser-Ser-Arg-Arg-Ala-Pro-Gln-Thr) , factor II (33-40) of insulin-like growth (Ser-Arg-Val-Ser-Arg-Arg-Ser-Arg), factor II (33-40) [tyro] of insulin-like growth (Tyr) -Ser-Arg-Val-Ser-Arg-Arg-Ser-Arg), factor II (69-84) of insulin-like growth (Asp-Val-Ser-Thr-Pro-Pro-Thr-Val-Leu Pro-Asp-Asn-Phe-Pro-Arg-Tyr), peptide-6 (GHRP-6) release (GH) growth hormone (HiS-DTrp-Ala-Trp-Dphe-Lys-NH2), beta -Interleucine II (163-171) (Val-Gln-Gly-Glu-Glu-Ser-Asn-Asp-Lys), Beta-Interleukin II (44-56) (Ile-Leu-Asn-Gly-Ile-Asn- Tyr-Lys-Asn-Pro-Lys-Leu), Interleukin II (60-70) (Leu-Thr-Phe-Lys-Phe-Tyr-Met-Pro-Lys-Lys-Ala), Exedin-4 (analog of GLP-1) (His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Glu-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile -Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2). exendin-3 (GLP-1 analog) (His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser), [Cys (Acm) 20, 31] growth factor ( 20-31) epidermal Cys (Acm-Met-His-Ile-Glu-Ser-Leu-Asp-Ser-Tyr-Thr-Cys (Acm), bivalirudin (Hirulog) (D-Phe-Pro-Arg-Pro- ( Gly) 4-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu), hiruloga-1 D-Phe-Pro-Arg-Pro- (Gly) 4-Asn-Gly -Asp-Phe-Glu-Glu-Ile-Pro-Glu-Tyr-Leu, natriuretic peptide type C (1-53) (CNP) (Asp-Leu-Arg-Val-Asp-Thr-Lys-Ser-Arg- Ala-Ala-Trp-Ala-Arg-Leu-Leu-Gln-Glu-His-Pro-Asn-Ala-Arg-Lys-Tyr-Lys-Gly-Ala-Asn-Lys-Lys-Gly-Leu-Ser Lys-Gly-Cys'-Phe-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Met-Ser-Gly-Leu-Gly-Cys; Disulfide bridge: Cys -37-Cys53), "Mini ANP" (Met-Cys-His-cyclohexylAla-Gly-Gly-Arg-Met-Asp-Arg-Ile-Ser-Tyr-Arg. Disulfide bridge cys2-cysl3), Melanotan-II (Also known as MT-II, alpha-MSH4-10-NH2, or Ac-Nle4-Asp5-His6-D-Phe7-A rg8-Trp9-Lys 10), thymosin alfa I (TA 1) (Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-SerGlu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Glu-Val-Val-Glu -Glu-Ala-Glu-Asn), omipresin (also known as 8-ornithine-vasopressin, (POR-8), [Phe2-Ile3-Orn8] vasopressin), Cys-Phe-Ile-Gln-Asn-Cys-Pro-0rn-Gly-NH2, Disulfide bridge: Cysl-Cys6), octreotide (201-995) ) (Dphe-Cys-phe-DTrp-Lys-Thr-Cys-Thr-ol; Disulfide bridge: Cys2-Cys7), eptifibatide (INTEGRILIN), Peptide referred to the calcitonin gene (CGRP) (Ala-Cys-Asp -Thr-Ala-Thr-Cys-Val-Thr-His-Arg-Leu-Ala-Gly-Leu-Leu-Ser-Arg-Ser-Gly-Gly-Val-val-Lys-Asn-Asn-Phe-Val -Pro-Thr-Asn-Val-Gly-Ser-Lys-Ala-Phe-NH2; Disulfide bridge Cys2-Cys7), Endornorphine-1 Tyr-Pro-Trp-Phe-NH2; endornorphine-2 Tyr-Pro-Phe-Phe-NH2, nociceptin (also known as Orphanin FQ, Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-Leu-Ala - Asn-Gln), angiotensinogen (1-13) (Asp-Arg-Val-Tyr-Ile-His- Pro-Phe-His-Leu-Val-Ile-His), adrenornodulin (1-12) (Tyr-Arg -Gln-Ser-Met-Asn-Asn-Phe-Gln-Gly-Leu-Arg), antiarrhythmic peptide (AAP) (Gly-Pro-Hyp-Gly-Ala-Gly), Antagonist G (Arg-DTrp- (nMe Phe-Gln-Gly-Leu-Met-NH2), indolicidin (Ile-Leu-Pro-Trp-Lys-Trp-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg-NH2), osteocalcin (37 -49) (Gly-Phe-Gln-Gln-Ala-Tyr-Arg-Arg-Phe-Tyr-Gly-Pro-Val), cortistatin 29 (1-13) (Glp) -Glu-Arg-Pro-Pro- Leu-Gln-Gln-Pro-Pro-His-Arg-Asp), Cortistatin 14 Pro-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Ser-Ser-Cys-Lys; Disulfide bridge: Cys2-Cysl3, PD-145065 (Ac-D-Bhg-Leu-Asp-Ile-Ile-Trp), • PD-142893 (Ac-D-Dip-Leu-Asp-Ile-IleTrp), peptide inhibitor bound to the fibrinogen (His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-GIY-Asp-Val), leptin (93-105) (Asn-Val-Leu-Gln-Ile-Ser- Asn-Asp-Leu-Glu-Asn-Leu-Arg), GR 83074 (Boc-Arg-Ala-DTrp-Phe-Dpro-Pro-Nle-NH2) Tyr * W «MIF-1 (Tyr-Pro-Trp- Gly-NH2), peptide referred to the parathyroid hormone (107-111) (Thr-Arg-Ser-Ala-Trp), angiotensinogen (1-14) Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Asn, Leupeptin (Ac- Leu-Leu-Arg-CHO) and some modified or truncated analog thereof.

23. A peptide conjugate according to some of the previous claims characterized in that the conjugate is H-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu- Gly- Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-Gln-Gln-Gly-Glu-Ser-Asn-Gln-Glu-Arg-Gly-Ala Arg-Ala-Arg-Leu-Lys6-NH2 (GHRH (1-44) (Human) -Lys6-NH2 H-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln- Asp-Ile-Met-Ser-Arg-Gln-Gln-Gly-Glu-Ser-Asn-Gln-Gln-Arg-Gly-Ala-Arg-Ala-Arg-Leu-Glu6-NH2 (GHRH (1-44) (Human) -Glu6-NH2 H-Lys6-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp- leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-OH (Lys6-TPH (1-34) (Human) -Lys6-OH); H-Ser-Val-Ser-Glu-Ile-Gln-Ile-Gln-Leui-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu Trp-Leu- Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe-Lys6-OH (PTH (1-34) (Human) -Lys6-OH H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala Trp-Leu-Val-Lys-Gly-Arg-Lys6-OH (GLP-1- (7-36) (Human) -Lys6-0H); H-Gly-GLy-Thr-Tyr-Ser-Cys- (Acm) -His-Phe-Gly-Pro-Leu-Thr-Trp-Val-Cys (Acm) -Lys-Pro-Gln-Gly-Lys6-OH (EMP-1-Lys6-0H) H-Lys6-Gly-Gly-Thr-Tyr-Ser-Cys (Acm) -His-Phe-Gly-Pro-Leu-Thr-Trp-Val-Cys (Acm) -Lys-Pro-Gln-Gly-Gly- OH (Lys6-EMP-1-0H] H-Lys6-Gly-Gly-Thr-Tyr-Ser-Cys (Acm) -His-Phe-Gly-Pro-Leu-Thr-Trp-Val-Cys (Acm) -Lys-Pr-Gln-Gly-Gly- Lys6-OH (Lys6-EMP-1-Lys6-OH) H-Aib-His-2-D-Nal-D-Phe-Lys- (Lys6) -NH2 (GHRP- (Lys6-NH2); H-Tyr-Gly-GLy-Phe-Leu-Lys-Lys-Glu-Glu-Glu-Lys-OH (Leu-enkephalin-Lys-Lys-Glu-Glu-Glu-Lys-OH); H-Tyr-Gly-Gly-Phe-Leu-Lys-Glu-Glu-Glu-Glu-Lys-OH (Leu-enkephalin-Lys-Glu-Glu-Glu-Glu-Lys-OH); H-Tyr-Gly-Gly-Phe-Leu-Lys-Glu-Glu-Glu-Glu-Lys-OH (Leu-enkephalin- (Lys-Glu) 3; H-Tyr-Gly-Gly-Phe-Leu- (Dpr) 6-OH (Leu-encephali- na- (Dpr) 6-OH); H-Lyse-Tyr-Gly-Gly-Phe-Leu-OH (H-Lyse-eu-encefaliña); H-Tyr-Gly-Gly-Phe-Leu-Lys6-OH (H-Leu-enkephalin-Lys6); H-Lys6-Tyr-Gly-Gly-Phe-Leu-Lys6-OH (H-Lys6-Leu-enkephalin-Lys6-OH); Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly- (Lys) 6-0H (GnRH-Lys6-OH); Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly- (Lys-Glu) 3 ~ OH (GnRH- (Lys-Glu) 3-OH); Y H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu ' -Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe- (Lys-Glu) 3-OH (PTH 1-34 human- (Lys-Glu) 3-OH):

24. A method for the preparation of a pharmacologically active peptide cojugate (X-Z) as defined in claim 2, characterized in that it comprises the steps of: a) coupling a protected N-a amino acid in the form of an activated carboxyl; a dipeptide N-a protected in the C-terminal form activated to a peptide H-Z-SSM sequence - immobilized, consequently forms a protected N-a peptide fragment. b) extracting the protective N-a group, consequently forming immobilized peptide fragment having an unprotected N-terminal end; c) coupling an additional protected Na-amino acid in the form of an activated carboxyl, or an additional protected Nape dipeptide in the C-terminal form activated at the N-terminal end of the immobilized peptide fragment, and repeating the procedure in the estraer step / link in b) and c) until the desired sequence of peptide X is obtained, and then d) cleaving the peptide conjugate by means of the solid support material

25. A method for the preparation of a pharmacologically active peptide (Z-X) conjugate (Z-X) as defined in claim 3, characterized in that it comprises 1-steps; a) binding a protected N-a amino acid, or a dipeptide to a solid support material (SSM), consequently forming a protected N-a amino acid, b) removing the protected N-a group, consequently forming an immobilized amino acid or a peptide fragment having an unprotected N-terminal end, c) linking a protected Na amino acid in the form of an activated carboxyl, or an additional protected Nape dipeptide in the C-terminal form activated at the N-terminal end of the immobilized amino acid or peptide fragment, and repeating the step of the extracting process / link in Step b) and c) until the desired sequence of peptide X is obtained, d) linking an additional protected N-a amino acid in the activated carboxyl form, or an additional protected N-a dipeptide in the activated C-terminal form at the end N-thermally den immobilized peptide segment, and repeat the step of the estraer / bind procedure in step b) and d) until the desired Z peptide sequence is obtained, and then e) cleaving the peptide conjugate by means of the solid support material.

26. A method for the preparation of a pharmacologically active peptide (Z-X-Z) conjugate as defined in claim 4, characterized in that it comprises the steps of: a) binding a protected N-a amino acid in the form of an activated carboxyl, or a protected N-a dipeptide in the activated C-terminal form, to an immobilized peptide H-Z-SSM sequence, consequently forming a "protected N-a peptide fragment, b) extracting the protected N-a group, consequently forming an immobilized peptide fragment having an unprotected N-terminal fianl, c) linking an additional protected Na-amino acid in the activated carboxyl form, or an additional protected Nape dipeptide in the C-terminal form activated at the N-terminal end of the immobilized peptide fragment, and repeating the procedure in the extract / bind step in step b) and d) until the desired X sequence of peptide is obtained, and then d) linking an additional protected Na-amino acid in the carboxyl form, or an additional protected Na-dipeptide in the C-terminal form activated at the N-terminal end of the immobilized peptide fragment, and repeating the procedure in the extract / bind step in step b) and d) until the desired Z sequence is obtained, and then e) cleaving the peptide conjugate by means of the solid support material.

27. A method for producing the peptide conjugate of claim 1, characterized in that it comprises a) introducing a nucleic acid sequence encoding said peptide conjugate within the cell; b) cultivating said host cell and c) isolating said conjugate by means of culture

28. A method for producing the peptide conjugate of claim 1, characterized in that it comprises - a) culturing a recombinant host cell comprising a nucleic acid sequence that cidifies said conjugate under conditions that allow the production of said conjugate; and b) isolating said conjugate from the culture.

29. The method according to claim 28 or claim 29, characterized in that the nucleic acid sequence encoding said conjugate is contained within a nucleic acid construct or a vector.

30. A composition characterized in that it comprises a pharmacologically active peptide conjugate as defined in any of claims 1-24, and a pharmaceutically active carrier.

31. A composition characterized by comprising H-Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu- (Lys-Glu) 3-OH, H-Trp-Ala-Gly-Gly-Asp-Ala-Ser -Gly-Glu- (Glu) 6-OH, H-Tyr-Gly-Gly-Phe-Leu- (Glu) 6-OH, or H-Tyr-Gly-Gly-Phe-Leu- (Lys) 6-OH , and a pharmaceutically acceptable carrier.

32. The use of a pharmacologically active peptide conjugate as defined in any of claims 1-23 characterized in that it is used for the manufacture of a pharmaceutical composition.

33. The use of a pharmacologically active peptide conjugate characterized in that it is selected from the group consisting of H-Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu- (Lys-Glu) 3-OH; H-Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu- (Glu) 6-0H; H-Tyr-Gly-Gly-Phe-Leu- (Glu) 6-0H, and H-Tyr-Gly-Gly-Phe-Leu- (Lys) 6-OH, for the manufacture of a pharmaceutical composition.

34. The use of H-Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu- (Lys-Glu) 3-OH or H-Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly- Glu- (Glu) 6-OH characterized in that they are used in the manufacture of a composition for the treatment of sleep disorders.

35. USE OF H-Tyr-Gly-Gly-Phe-Leu (Glu) 6-OH, or H-Tyr-Gly-Gly-Phe-Leu (Lys) 6-OH, characterized in that they are used in the manufacture of a composition for the treatment of pain.

36. The use of a peptide conjugate according to any of claims 1-23, characterized in that said pharmacologically active peptide X is enkephalin for the manufacture of a pharmaceutical composition for inhibiting neurons from transmitting pain impulses.

37. The suo of a peptide conjugate according to any of claims 1-23, characterized in that said pharmacologically active peptide X is enkephalin for the manufacture of a pharmaceutical composition for use in the treatment of pain.

38. The use of a peptide conjugate according to any of the claims, characterized in that said pharmacologically active peptide X is the growth hormone releasing hormone or the growth hormone releasing peptide for the manufacture of a composition Pharmaceutical for use in stimulating the release of growth hormone.

39. The use of a peptide conjugate according to any of claims 1-23, characterized in that said pharmacologically active peptide X is EMP-1 for the manufacture of a pharmaceutical composition for increasing hemoglobin levels.

40. The use of a peptide conjugate according to any of claims 1-23, characterized in that said pharmacologically active peptide X is EMP-1 for the manufacture of a pharmaceutical composition for use in treating anemia by increasing the levels of hemoglobin. - -

41. The use of a peptide conjugate according to any of claims 1-23, characterized in that said pharmacologically active peptide X is the parathyroid hormone for the manufacture of a pharmaceutical composition for use in preventing or treating bone loss. altering the equilibrium between the ostoclastic activity or the osteoblast activity.

42. The use of a peptide conjugate according to any of claims 1-23, characterized in that said pharmacologically active peptide X is the parathyroid hormone for the manufacture of a pharmaceutical composition for use in preventing or treating osteoporosis.

43. The use of a peptide conjugate according to any of claims 1-23, characterized in that said pharmacologically active peptide X is peptide 1 similar to glucagon. for the manufacture of a pharmaceutical composition to reduce the levels of glucose in the blood.

44. The use of a peptide conjugate according to any of claims 1-23, characterized in that said pharmacologically active peptide X is glucagon-like peptide 1 for the manufacture of a pharmaceutical composition for use in the treatment of diabetes .

45. The use of a peptide conjugate according to any of claims 1-23, characterized in that said pharmacologically active peptide X is the gonadotropin releasing hormone, for the manufacture of a pharmaceutical composition for regulating the production of sex hormones .

46. The use of a peptide conjugate according to any of claims 1-23, characterized in that said pharmacologically active peptide X is gonadotropin releasing hormone, for the manufacture of a pharmaceutical composition for use to regulate the level of hormones sexual

47. The use of a peptide conjugate according to any of claims 1-23, characterized in that said pharmacologically active peptide X is the delta-inducing peptide for the manufacture of a pharmaceutical composition for use in treating a disorder of the peptide. dream.

48. The use of a. peptide conjugate according to any of claims 1-23, characterized in that said pharmacologically active peptide X is the antirust peptide for the preparation of a pharmaceutical composition

49. The use of a stabilizing peptide sequence (2) as defined in claim 1 for the preparation of a pharmacologically active peptide conjugate as defined in any of claims 1-23 or a salt thereof.