ES2365501T3 - MODIFIED PEPTIDE CONJUGATES IN THE N AND / OR C END WITH LOADED PEPTIDIC CHAINS. - Google Patents

Abstract

A peptide conjugate of the general formula I R1-ZX-Z'R2 X represents a hexapeptide with the amino acid sequence (RK) YY (RK) (WI) (RK), in which the alternative amino acid residues at positions 1, 4 , 5 and 6 are shown in parentheses and in which each amino acid residue in said hexapeptide can be in the L or D form; Z represents a peptide chain loaded from 4 to 20 amino acid residues with the D or L configuration or is absent; and Z 'represents a peptide chain loaded from 4 to 20 amino acid residues with the D or L configuration or is absent, taking into account that Z and Z' are not absent at the same time; R1 represents H or an acyl group; R2 represents NR3R4, in which each of R3 and R4 independently represent hydrogen, C (1-6) alkoxy, aryloxy or alkyl; or R2 represents OH; wherein the peptide conjugates of the formula I may be optionally linked to a transport fraction; or a salt, hydrate or solvate thereof or a derivative thereof esterified at the C-terminus with a suitable organic or inorganic acid.

Description

FIELD OF THE INVENTION

[0001] The present invention relates to new peptide conjugates with interesting pharmacological activities, to a process for the preparation of new peptide conjugates, to pharmaceutical compositions containing peptide conjugates and to the use of peptide conjugates for the preparation of a medicament. .

BACKGROUND

[0002] The endogenous opioid peptide nociceptin (also called orphan CF) was first described in the central nervous system and most of the research in this field has focused on the effects on the CNS. Nociceptin binds to a specific receptor, called an opioid receptor-like receptor (ORL1) with much greater affinity than the three classic subtypes of opioid receptors. The effects of nociceptin on the CNS include: hyperalgesia / hypoalgesia, appetite stimulation and stabbing, increase (in low doses) or decrease (in high doses) of locomotion, learning disorders and dysphoria. However, nociceptin also exerts significant effects outside the CNS. Thus, low doses of nociceptin increase renal excretion of water and decrease urinary excretion of sodium (that is, it produces a selective aqueous diuresis), which makes this compound interesting for the treatment of hyponatremia (Daniel R. Kapusta, Life Science, 60: 15-21, 1997) (U.S. Patent No. 5,840,696). If administered centrally (i.c.v.)

or by peripheral route at high doses (bolus or IV infusion), nociceptin lowers blood pressure, heart rate and peripheral sympathetic nerve activity.

[0003] Dooley et al. (The Journal of Pharmacology and Experimental Therapeutics, 283 (2): 735-741, 1997) have shown that a hexapeptide positively charged with the amino acid sequence Ac-RYY (RK) (WI) (RK) NH2, in which the parentheses show the permitted variation of amino acid residues, acts as a partial agonist of the ORL1 nociceptin receptor. Said hexapeptide was identified from a combinatorial peptide library and the sequence is unique, without homology or similarity with the nociceptin heptadecapeptide. However, said hexapeptide is too unstable to produce a satisfactory medication.

[0004] WO 99/44627 discloses the use of hexapeptides, including the hexapeptides discovered by Dooley et al. for the preparation of a pharmaceutical composition for the treatment of the following conditions: migraine, type II diabetes, septic shock, inflammations and vasomotor disorders. It is shown that the hexapeptide Ac-Arg-Tyr-Tyr-Arg-Trp-Lys-NH2 inhibits the depressive response to spinal cord stimulation.

[0005] WO 99/46283 discloses the use of a flanking peptide sequence of 4-20 amino acid residues for the stabilization of pharmacologically active peptides against enzymatic hydrolysis. Preferred stabilizing peptides are positively charged polylysine sequences and nociceptin is mentioned among the active peptides that are thus stabilized against enzymatic hydrolysis.

[0006] Whereas ENT1 agonists, including the hexapeptide discovered by Dooley et al., As well as true nociceptin analogues, are expected to have serious side effects on the CNS if exposed to the brain, which is observed at levels of dosage greater than those required to cause aqueous diuresis in rats, a main objective of the present invention is to provide new peptide conjugates with the nociceptin-like activity of said hexapeptide or with ORL1 receptor binding activity, but that act selectively outside the CNS . Another object of the present invention is to provide new peptide conjugates with the nociceptin-like activity of said hexapeptide or with ORL1 receptor binding activity and with better stability. Furthermore, it is an object of the invention to provide new peptide conjugates with the nociceptin-like activity of said hexapeptide or with binding activity to the ORL1 receptor and / or to an unidentified receptor in renal tissue.

[0007] This objective is achieved with the peptide conjugates of the present invention comprising modified hexapeptides at the C and / or N ends by conjugation with short charged peptide chains.

SUMMARY OF THE INVENTION

[0008] A peptide conjugate of the general formula I R1-Z-X-Z’-R2 (I) in which

X represents a hexapeptide with the sequence of formula (II) (RK) YY (RK) (WI) (RK), in which the parentheses indicate alternatives;

Z represents a peptide chain loaded from 4 to 20 amino acid residues with the D or L configuration

or is absent; and Z ’represents a peptide chain loaded from 4 to 20 amino acid residues with the D or L configuration

or is absent, taking into account that Z and Z ’are not absent at the same time; R1 represents H or an acyl group; R2 represents NR3R4, in which each of R3 and R4, independently represent hydrogen, alkoxy

C (1-6), aryloxy or a lower alkyl as defined herein; or R2 represents OH; and salts, hydrates and solvates thereof and derivatives thereof esterified at the C-terminus with organic acids or suitable inorganic.

[0009] The peptide conjugates of the invention can form acid addition salts, preferably with pharmaceutically acceptable acids and these salts are included within the scope of the invention. The compounds can serve as medications in their pure form. However, preferably the compounds are incorporated into solid or liquid medical formulations, including tablets, capsules, solutions and suspensions. Other components of such formulations may include a vehicle, a diluent, a buffering agent, an osmotic agent and a preservative. Solid formulations are particularly suitable for oral administration, while solutions are more useful for injection (i.v., i.m., i.p. or s.c.) or for intranasal administration.

[0010] The peptide conjugates of the invention with nociceptin-like activity or ORL1 receptor binding activity, but acting outside the CNS are especially useful in the treatment of peripheral diseases and diseases related to ORL1, such as diseases in which requires or prefers a selective renal effect. More specifically, the peptide conjugates of the invention show a significant aquatic saving effect of sodium and potassium, which is beneficial in the treatment of pathological conditions forming edemas associated with hyponatremia and / or hypokalemia. The peptide conjugates of the invention are useful for the medical treatment of humans and for veterinary use as diuretics during edema-forming states in cattle, for example, horses and cattle. A particular advantage of the compounds of the invention is their stability in plasma compared to the hexapeptides disclosed by Dooley et al. [one]

[0011] The new peptide conjugates of the invention are useful for the preparation of antibodies capable of specifically binding to said peptide conjugates. Antibodies produced against the peptide conjugates of the invention or fractions thereof are also disclosed.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012]

Figure 1 shows concentration response curves for the effects of cumulative addition of test substances (Ac-RYYRWK-NH2 and compound 1) on nociceptin-induced relaxation in the mouse vas deferens duct test.

Figure 2 shows the cardiovascular and renal responses to nociceptin administered as an infusion i.v. (11 nmol / kg / min); HR is the heart rate (beats per minute, bpm), MAP means blood pressure (mm Hg), V is the urinary flow rate (µl / min) and UNaV is the rate of sodium urinary excretion (µeq / min ).

Figure 3 shows the cardiovascular and renal responses to compound 1 administered as an infusion i.v. (1 and 0.1 nmol / kg / min), as well as renal sympathetic nerve activity as a percentage of the level of control (RNSA,%).

Figure 4 shows the decrease in osmolar clearance (COsm) and the increase in free water clearance (CH2O) observed during the infusion i.v. of compound 1 (1 and 0.1 nmol / kg / min).

Figure 5 illustrates the similar magnitude of changes in cardiovascular and renal responses (V and UNaV) caused by the i.v. of 1 nmol / kg / min of compound 1 and 11 nmol / kg / min of nociceptin.

Figure 6 illustrates the similar magnitude of changes in cardiovascular and renal responses (V and UNaV) caused by the i.v. 0.1 nmol / kg / min of compound 1 and 1.1 nmol / kg / min of nociceptin.

Figure 7 is an illustration of the relationship between changes in the urinary flow rate and the excretion of sodium and potassium after administration i.v. of compound 1, 65 nmol / kg i.v. (arrow) in normal rats. The data are expressed relative (percentage) to the level prior to administration i.v. of compound 1.

Figure 8 illustrates changes in the urinary flow rate, free water clearance, fractional excretion of potassium and fractional excretion of sodium after administration i.v. of compound 1, 65 nmol / kg i.v. in normal rats and cirrhotic rats.

Figure 9 is an illustration of the fractional excretion of water (V / GFR) during the i.v. infusion. continue with a maximum aquatic dose of the selective vasopressin receptor antagonist type 2, OPC-31260 (0.8 mg / kg / h; n = 8 (data from [2]) or compound 1 (12 nmol / kg / min) In order to avoid the activation of the homeostatic mechanisms compensatory of the volume, the experiments were carried out in conditions in which a reduction of volume was avoided by means of a computer-controlled and servo-controlled 150 mM glucose vollemia replenishment system. .

Figure 10 is a graph showing the degradation rate of compound 1 and Ac-RYYRWK-NH2 in mouse heparinized plasma.

DETAILED DESCRIPTION OF THE INVENTION

[0013] Coupling of a charged peptide fraction introduces an increase in polarity or an increase in the charge on the positively charged hexapeptide (RK) YY (RK) (WI) (RK), resulting in a peptide conjugate with higher stability and hydrophilicity It is also believed that such coupling reduces the probability that the peptide conjugate crosses the blood brain barrier. In addition, preliminary data suggests that modification of the C-terminus with K6 seems to induce the helix structure α in hexapeptide X, as determined by 1D NMR spectra. In the amino acid sequence (RK) YY (RK) (WI) (RK), the alternative amino acids at positions 1, 4, 5 and 6 are shown in parentheses.

[0014] In preferred embodiments of the invention Z represents a negatively charged peptide chain of 4 to 20 amino acid residues, Z 'represents a positively charged peptide chain of 4 to 20 amino acid residues, R1 represents Ac or Tfa and R2 represents NH2 or R2 represents NR3R4, in which each of R3 and R4 independently represent hydrogen, methyl or ethyl.

[0015] Preferably, said hexapeptide X is selected from the group consisting of KYYRWR, RYYRWR, RYYRWK, RYYRWK (all D), RWRIK, RYYRIR, RYYKIK, RYYKIR, RYYKWR and RYYKWK, more preferably RYR group R , RYYRIK, RYYKWR and RYYKWK, more preferably said hexapeptide X is RYYRWK or KYYRWK, in which the amino acid residues are in the L form, unless otherwise specified. In addition, the number of amino acid residues in each of Z and Z ’is preferably in the range of 4 to 10 or 5 to 10.

[0016] It is preferred that the amino acid residues of Z be selected from the group consisting of Q, T, S, P, N, E and D, with the D or L configuration, and that the N-terminal amino acid of Z is selected from the group consisting of Q, T, N and S, with the D or L configuration, and that the remaining amino acids be selected from the group consisting of P, D and E. More specifically, Z is selected from the group consisting, by example, from N (E) 7, N (E) 6, N (E) 5, N (E) 3, S (E) 7, S (E) 6, S (E) 5, S (E) 3 , NP (E) 4, NP (E) 5, N (D) 7, N (D) 6, N (D) 5, N (D) 3, Q (E) 7, Q (E) 5, Q (E) 3, QN (D) 7, Q (D) 6, Q (D) 5, and Q (D) 3, preferably Z is N (E) 5.

[0017] It is preferred that the amino acid residues of Z 'be selected from the group consisting of A, G, K and R, preferably K, with the D or L configuration, more preferably Z' is selected from the group consisting of A (K4) G, K5G, AK5, H6, K10, K8, K6, K5 and K4.

[0018] Some examples of ZX-Z 'conjugates are N (E) 5RYYRWKK6, N (E) 5RYYRWK, RYYRWKK6, N (E) 5RYYRWRK6, N (E) 5RYYRWR, RYYRWRK6, N (E) 5RYYRIKK6, N (E) 5RYYRIK and RYYRIKK6.

[0019] Some examples of compounds of the invention are: Ac-RYYRWKK6-NH2, Ac-K6RYYRWK-NH2, N (E) 5RYYRWKEC6-NH2 and salts, preferably pharmaceutically acceptable salts, hydrates, solvates and derivatives thereof, including derivatives C-terminals, such as free carboxylic acids.

[0020] A preferred embodiment of the invention is represented by the general formula III

R1-X-Z'-R2 (III) in which R1, X, Z 'and R2 have the same meanings defined above, and hydrate and solvate salts thereof and derivatives thereof esterified at the C-terminus with organic acids or suitable inorganic. In a preferred peptide conjugate of formula III, R1 represents Ac, Z ’represents Kn, where n is an integer selected from 5, 6 or 7 and R2 represents NH2. Preferably, X is selected from the group consisting of KYYRWK, RYYRWR, RYYRWK, RYYRWK (all D), RYYRIK, RYYKWR and RYYKWK. Optionally, the peptide conjugates of formulas I and III are further linked to a transport fraction or an affinity tag, such as H6, in which the binding between the peptide conjugate and said transport fraction or affinity tag can take place by any convenient covalent bond. Said transport fraction is preferably selected from the group consisting of residues 49-57 of the HIV tat peptide, residues 49-56 of the tat peptide of the

5 HIV, the tat sequence YGRKKRRQRRR, a polyarginine peptide with 6 to 20 residues, such as R6 and transducer peptide sequences, such as the following peptide sequences YARKARRQARR, YARAAARQARA, YARAARRAARR, YARAARRAARA, ARRRRRRRRR and YAAARR WOULD be disclosed in WOA 99/29721 and in US Pat. UU. No. 6,221,355 (SEQ ID No: 3 and 8).

[0021] The peptide sequence of a compound of formulas I and III is optionally in the form with

10 all D, the retro form or the retro form with all D, in which the form with all D is the most preferred. Optionally, the sequence X of formulas I and II is a form with all D, a retro form or a retro form with all D of the peptide sequence of the formula II or of the hexapeptide (RK) YY (RK) (WI) ( RK) as defined above, respectively.

[0022] Some examples of compounds of the invention are shown in Table 1 below:

Compound 1

Ac-RYYRWKKKKKKK-NH2 Ac-KKKKKKRYYRWK-NH2 H-NEEEEERYYRWKKKKKKK-NH2 Ac-RYYRWKKKKKKK-NH2 (all D) Ac-KYYRWKKKKKKK-NH2 Ac-RYYRIKKKKKKK-NH2 Ac-RYYRWKAKKKKK-NH2 Ac-RYYRWKKKKKK-NH2 Ac-RYYRWKKKKKKKC-NH2 Tfa -RYYRWKKKKKKK-NH2

Compound 2

Compound 3

Compound 8

Compound 9

Compound 10

Compound 11

Compound 12

Compound 13

Compound 14

and salts, hydrates, solvates and C-terminal derivatives thereof, ie free carboxylic acid. Other preferred compounds are:

20 Ac-KWRYYNKKKKKK-NH2 Ac-KWRYYRKKKKKK-NH2 and Ac-KWRYYRKKKKKK-NH2 (all D) and C-terminal salts, hydrates, solvates and derivatives thereof, i.e. free carboxylic acid.

[0023] Some examples of acid addition salts with an organic acid and an inorganic acid of the invention are: compound 1A: Ac-RYYRWKKKKKKK-NH2 x 9 CH3COOH compound 1C: Ac-RYYRWKKKKKKK-NH2 x 9 HCl.

[0024] The peptide conjugates of the invention are preferably prepared by the synthesis process disclosed in WO 98/11125 and, preferably by a method as described in example 15 in said document. Such synthesis procedures will result in a primary peptide product with trifluoroacetate as counterion that may be suitable for the preparation of a medicament. However, in many cases it may be advantageous to replace the trifluoroacetate counterion with an anion.

Pharmaceutically acceptable or preferred, such as acetate. This can be done by ion exchange chromatography. Alternatively, the primary peptide product can be lyophilized and dissolved in repeatedly diluted hydrochloric acid to obtain the purified hydrochloride.

[0025] The peptide conjugate of formula I or III or an epitope fragment thereof comprising a

Part or all of the sequence (RK) YY (RK) (WI) (RK) and / or a part or all of the Z sequence or the Z 'sequence, preferably coupled to a transporter through a terminal cysteine residue, can used to produce antibodies capable of specifically binding to said peptide conjugates or said X sequence. The remaining Cys residue is optionally obtained through the substitution by a Cys residue of one of the terminal amino acid residues of said peptide conjugate or said epitope fragment of the same or said peptide conjugate or said

The epitope fragment thereof comprises another Cys residue at one of its ends. Some examples of peptide fragments with a C terminal useful for the production of antibodies capable of specifically binding the peptide conjugates of the invention are RYYRWKC, KYYRWKC, CWKKKKKKK, CKKKKKK and CWKKKKKK. Furthermore, it is possible to use peptide fragments with considerable sequence homology with the peptide conjugates of the invention for the production of antibodies capable of specifically binding the conjugates.

50 peptides of the invention. Some examples are CAPPSKKKKKK, CAAPKKKKKK, CPPSKKKKKK, etc. Optionally, each of the above peptide conjugates with a Cys at the end may have at least one amino acid in form D.

[0026] As will be discussed in more detail below, the new peptide conjugates can be administered as an active agent alone or in combination with one or more medications such as those specifically indicated below.

[0027] Throughout the description and in the claims, the one-letter code for natural amino acids is used, as well as the three-letter code for natural amino acids and other three-letter codes generally accepted for other α-amino acids, such as ornithine (Orn), 2,4-diaminobutanoic acid (Dab) and 2,3-diaminopropanoic acid (Dap). If the L or D form is not specified, it is understood that the amino acid in question has the natural L form, see Pure & Appl. Chem. Vol. 56 (5), p. 595-624 (1984). If nothing is specified, it is understood that the C-terminal amino acid of a compound of the invention is found as free carboxylic acid, which can also be expressed as "-OH". See Biochem. J., 1984, 219, 345-373; Eur.

J. Biochem., 1984, 138, 9-37; 1985, 152, 1; 1993, 213, 2; Internat. J. Pept. Prot. Res., 1984, 24, p. 84 et seq .; J. Biol. Chem., 1985, 260, 14-42; Pure Appl. Chem., 1984, 56, 595-624; Amino Acids and Peptids, 1985, 16, 387-410; Biochemical Nomenclature and Related Documents, 2nd. edition, Portland Press, 1992, p. 39-69; copyright IUBMB and IUPAC. The term "all-D form" of a peptide conjugate of formulas I or III refers to a peptide when all amino acid units are in form D. Optionally, in a peptide conjugate of the invention, the sequence defined by the formula (RK) YY (RK) (WI) (RK) is in the form with all D. Daminoacids are stable unnatural amino acids in an environment rich in proteases. Therefore, a useful way to stabilize the peptide conjugates of the invention against proteolytic degradation is to replace the Lamino Acids with the corresponding D-amino acids. The term "epitope fragment" refers to a truncated or shortened section of a peptide sequence that has, for example, 5-8 amino acid units and is capable of eliciting an antigenic response.

[0028] The term "alkyl" refers to monovalent groups derived from alkanes by the removal of a hydrogen atom from any carbon atom: CnH2n + 1-. The groups derived from unbranched alkanes by removal of a hydrogen atom from a terminal carbon atom form a subclass of the normal alkyl groups (n-alkyl): H [CH2] n-. The groups RCH2-, R2CH- (R other than H) and R3C- (R other than H) are primary, secondary and tertiary alkyl groups, respectively. "Alkyl" refers to any alkyl group and includes C (1-6) alkyl, such as methyl, ethyl, propyl, isopropyl, butyl, pentyl and hexyl and any possible isomer thereof. By "lower alkyl" is meant C (1-6) alkyl, preferably C (1-4) alkyl, more preferably methyl and ethyl.

[0029] The term "C (1-6) alkoxy" refers to an ester group of the formula R-O-, wherein R represents C (1-6) alkyl. The term "aryloxy" refers to an ester group of the formula R-O-, wherein R represents phenyl or naphthyl, optionally substituted with a lower alkyl group.

[0030] The term "acyl," as used herein, includes acyl radicals that are formally derived from oxoacids RkE (= O) l (OH) m (l other than 0) by removal of a hydroxyl cation HO +, a hydroxyl radical HO or a hydroxyl anion HO, respectively and substitution analogs of such intermediates. Acyl radicals can be formally represented by canonical forms that have a missing electron or a positive charge on the element that generates the oxoacid acid. Examples of acyl radicals: RC (= O), RS (= O) 2.

[0031] The term "acylated", as used herein, indicates that the compound in question carries an acyl group. An acyl group is formed by the removal of one or more hydroxy groups from an oxo acid, such as a carboxylic acid, which has the general structure RkE (= O) 1 (OH) m (1 other than 0) and substitution analogues of such acyl groups. Examples: CH3C (= O) -, CH3C (= NR) -, CH3C (= S) -, PhS (= O) 2-, HP (N) -. In inorganic chemistry, an unspecified acyl group is normally a carboxylic acyl group. See, International Union of Pure and Applied Chemistry, Recommendations on Organic & Biochemical Nomenclature, Symbols & Terminology, etc., IUPAC Recommendations 1994. "Ac" indicates an acetyl group and "Tfa" indicates a trifluoroacetic group.

[0032] The term "peptide conjugate," as used herein, indicates a fusion of at least two peptide sequences by means of a peptide bond or an equivalent bioisosteric bond, such as the mimicry of the peptide bond described in Table 1. in Tayar et al., Amino Acids (1995) 8: 125-139.

[0033] The term "transport fraction", as used herein, indicates a chemical entity that serves as an escort to molecules such as polypeptides through biological membranes. Transport polymers are disclosed in WO 98/52612. The peptide conjugates of the invention are linked to said transport fraction by means of a covalent bond. The term "residues 49-57 of the HIV tat peptide" refers to a transport fraction with the sequence RKKRRQRRR as disclosed in WO 91/09958.

[0034] "Agonist" refers to an endogenous substance or a drug that can interact with a receptor and initiate a physiological or pharmacological response characteristic of such a receptor (contraction, relaxation, secretion, enzymatic activation, etc.).

[0035] "Antagonist" refers to a drug or compound that opposes the physiological effects of another. Speaking of receptors, it is a chemical entity that opposes the responses associated with the receptor normally induced by another bioactive agent.

[0036] "Partial agonist" refers to an agonist who is unable to induce maximum activation in a population of recipients, regardless of the amount of drug administered (see also intrinsic activity). A "partial agonist" may also be referred to as "agonist with intermediate intrinsic efficacy" in a given tissue. In addition, a partial agonist can antagonize the effect of a total agonist that acts on the same receptor.

[0037] "Receptor" refers to a molecule or a polymeric structure in or on a cell that specifically recognizes and binds to a compound that acts as a molecular messenger (neurotransmitter, hormone, lymphokine, lectin, drug, etc.).

[0038] The term "salt", as used herein, denotes acidic and / or basic salts formed with inorganic or organic acids and / or bases, preferably basic salts. While pharmaceutically acceptable salts are preferred, in particular when the compounds of the invention are used as medicaments, other salts are useful, for example, for the processing of these compounds or in cases where non-medicated uses are contemplated. The salts of these compounds can be prepared by procedures recognized in the art. Some examples of such pharmaceutically acceptable salts include, but are not limited to addition salts of inorganic and organic acid, such as hydrochlorides, sulfates, nitrates or phosphates and acetates, trifluoroacetates, propionates, succinates, benzoates, citrates, tartrates, smokers, maleates, methanesulfonates, isothionates, theophyllinacetates, salicylates, respectively, or the like. Lower alkyl quaternary ammonium salts and the like are also suitable. The term "pharmaceutically acceptable anions," as used herein, includes the group consisting of CH3COO-, CF3COO-, Cl-, SO32-, maleate and oleate.

[0039] The term "peripheral administration" includes all forms of administration that exclude the administration of the active substance directly in the central nervous system. "Central administration," as used herein, means administration directly to the central nervous system, such as intracerebroventricular administration (i.c.v. administration).

[0040] The term "hyponatremia," as used herein, includes, but is not necessarily limited to the following medical conditions:

pseudohyponatremia, characterized by a normal plasma osmolality associated, for example, with hyperlipidemia, hyperproteinemia or after transurethral resection of prostate / bladder tumors and an increased plasma osmolality associated, for example, with hyperglycemia and mannitol;

hypoosmolal hyponatremia, characterized by

primary loss of Na + (secondary water gain) associated, for example, with integumentary losses: sweat, burns; gastrointestinal losses: vomiting, drainage tubes, fistulas, obstructions, diarrhea; renal losses: diuretics, osmotic diuresis, hypoaldosteronism, nephropathy with salt loss, postobstructive diuresis, acute non-oliguric tubular necrosis;

primary water gain (secondary loss of Na +) associated, for example, with primary polydipsia; decreased absorption of solutes (for example, beer potomania); release of AVP (vasopressin) due to pain, nausea, drugs; syndrome of inappropriate secretion of AVP; glucocorticoid deficiency; hypothyroidism and chronic renal failure; Y

Primary Na + gain (outweighed by a secondary water gain) associated, for example, with heart failure, liver cirrhosis and nephrotic syndrome.

Congestive heart failure (CHF)

[0041] Congestive heart failure (CHF) is the pathophysiological state in which an abnormality in cardiac activity is responsible for the heart's inability to pump blood at a rate corresponding to the requirements of metabolizing tissues. Regardless of the underlying cause of CHF (for example, ischemic heart disease, high blood pressure, dilated cardiomyopathy, valvulopathy, anemia, etc.), the main clinical manifestation of heart failure is dyspnea, which occurs with less fatigued activities. As the disease progresses. According to the functional classification of the New York Heart Association (NYHA), CHF patients can be classified into one of four classes (class I, class II, class III and class IV). This functional classification is used worldwide and is closely associated with mortality, which is independent of the ventricular ejection fraction.

[0042] Congestive heart failure is characterized by a complex series of neurohumoral adjustments. The main mechanisms are the activation of the sympathetic nervous system, the stimulation of the reninaangiotensin-aldosterone axis and the stimulation of vasopressin release. These influences raise systemic vascular resistance and increase sodium and water retention, while potassium excretion increases due to hyperaldosteronism. Due to the activation of water retention mechanisms, patients with terminal CHF have a reduced water excretion capacity and, unless water intake is limited, these patients have a high risk of suffering from dilutional hyponatremia.

Current treatment of the ICC

[0043] The purpose of medical treatment is to relieve the debilitating symptoms of patients without compromising too much myocardial activity. During the progression of CHF, medical treatment is intensified by the addition of drugs with different points of action. Current treatment of heart failure can be divided into three categories: (1) reduction of cardiac work, including preload and afterload;

(2) control of excessive water and salt retention; (3) improvement of myocardial contractility. Normally, NYHA class IV patients are treated with maximum doses of vasodilators, such as ACE inhibitors, hydralazine, α-adrenergic inhibitors and nitrates. However, pulmonary congestion due to water and salt retention is still a fundamental problem in these patients. Despite intensive medical treatment with medications of all kinds, mortality and morbidity is still very high in patients with CHF.

[0044] Patients with severe heart failure (NYHA classes III and IV) normally require a combination of at least two different diuretics with different points of action in the renal tubules. The most commonly used is a combination of a loop diuretic (for example, furosemide, bumetanide) and a thiazide diuretic (for example, chlorothiazide, bendroflumethiazide). High doses of this combination are the most effective treatment to produce natriuresis (the maximum efficacy is approximately 30% of the filtered load for loop diuretics and approximately 10% for thiazides). However, blocking sodium reabsorption in the distal contoured tubules (i.e., in the diluent segment), which is sensitive to thiazide can produce hypertonic urine and contribute to dilutional hyponatremia. Hyperaldosteronism and the associated increase in the release of sodium in the collecting tubes stimulates the exchange between sodium and potassium in the collecting tubes, resulting in potassium. Therefore, a combined treatment with loop diuretics and thiazides is the most common cause of both hyponatremia and hypokalemia in patients with heart failure. Potassium-sparing diuretics (for example, spironolactone, amiloride) can be used in combination with thiazides or loop diuretics, but the low maximum efficacy of these compounds (maximum 3% of filtered water) makes these drugs weak.

[0045] In addition, due to hyperaldosteronism, patients with terminal CHF have a high risk of hypokalemia.

[0046] When symptoms such as dyspnea at rest, orthopnea and paroxysmal nocturnal dyspnea persist despite maximum medical treatment with loop diuretics (ICC resistant to loop diuretics or refractory ICC), additional diuretic treatment is necessary to avoid pulmonary congestion That can cause death. Frequently, thiazides are the first choice when the therapeutic effect of loop diuretics fails. The combination of loop diuretics and thiazides produces effective natriuresis, but inhibition of sodium reabsorption in the thiazide-sensitive nephron diluent segment often results in hypertonic urine, which can lead to severe electrolyte disorders. Therefore, the combination of loop diuretics and thiazides is the most common cause of hypokalemia and hyponatremia in patients with class IV heart failure according to the NYHA [3].

[0047] Hypokalemia is a fundamental mechanism of predisposition to arrhythmias and the prognosis of CHF is poor when the potassium concentration drops below 3.3 mM. In addition, in CHF patients treated with digoxin, hypokalemia is the most common trigger for digitalis intoxication, which is a serious and potentially fatal complication.

[0048] In patients with severe heart failure, combined treatment with loop diuretics and thiazides frequently results in hypotonic hyponatremia as a result of increased sodium losses and reduced water excretion. If hyponatremia develops too quickly to allow an adaptation of the brain, symptoms of cerebral edema occur (for example, headache, nausea, vomiting, weakness, lack of coordination, tremor, delirium and ultimately attacks and coma). Death occurs when the serum sodium concentration (S-Na) drops below 120 mM at a rate greater than 1 mmol / l / h. If hyponatremia develops more gradually, the symptoms are more subtle, vague or nonspecific and tend to occur at even lower concentrations of S-Na. Typically, symptoms include personality changes (depression, lack of cooperation, confusion), anorexia, nausea, muscle weakness and cramping. If S-Na <120 mM, gait disorders, stupor and seizures may occur.

[0049] Class IV represents the group of patients with terminal heart failure. Among the characteristic clinical manifestations, the most notable symptom is dyspnea at rest. As heart failure progresses, dyspnea appears with less and less tiring activities, however, during terminal heart failure, dyspnea is common even in the position of the decubitus (orthopnea). Patients have thickened pulmonary vessels and interstitial pulmonary edema, which is often evident in a radiological examination. Patients with orthopnea should lift their heads to relieve pulmonary congestion and are frequently awake at night, with intense coughing attacks and the feeling of shortness of breath (nocturnal dyspnea). This condition is quite scary and anxiety often worsens the symptoms of heart failure.

[0050] In patients of class IV, in addition to pulmonary congestion, reduced cardiac activity is associated with hepatic congestion and the portal vein system, resulting in anorexia, nausea and abdominal pain. Class IV patients have an extremely high mortality in one year (30-80%). The main causes of death are sudden death (40%) and worsening of congestive heart failure (40%). Factors associated with a poor prognosis include a low ejection fraction (<25%), reduced oxygen absorption, a reduced serum sodium concentration (<133 mM) and a reduced serum potassium concentration ( <3 mM) [3]. The peptide conjugates of the invention show an advantageous pharmacological profile that suggests an ability to relieve pulmonary congestion, decrease preload and afterload, increase cardiac output and oxygen absorption and prevent hyponatremia and hypokalemia.

[0051] When the cardiac output is reduced, a series of compensatory adjustments takes place that ultimately result in abnormal accumulation of fluid. In CHF patients in Classes I through III according to the NYHA, abnormal fluid accumulation and accompanying expansion of vollemia constitute an important compensatory mechanism that tends to maintain cardiac output and perfusion of vital organs. However, in the terminal state of heart failure (class IV according to the NYHA) the cardiac ventricles work with a depressed and flattened activity curve and in terminal heart failure, the patient ends in the descending part of the Starling curve of volume-cardiac output. Therefore, these patients need an effective diuretic treatment to control volemia and avoid pulmonary edema that can cause death. The advantageous effects of the peptide conjugates of the invention on heart failure, such as refractory heart failure, resistant to loop diuretics, are to decrease volemia and pulmonary congestion. If the patient is in the flattened part of the curve, the cardiac output decreases slightly and if the patient is in the descending part of the curve, the cardiac output may increase [3].

[0052] By having a unique pharmacological profile as potassium and sodium sparing aquarians, the peptide conjugates of the invention, such as compound 1, represent the ideal candidate drugs for the treatment of CHF symptoms in patients with refractory heart failure who already They do not respond sufficiently to high doses of loop diuretics. In these patients, the new peptide conjugates, as additional treatment or combined with loop diuretics, may favor an effective diuresis without the accelerated losses of sodium and potassium observed with other diuretics. This same unique pharmacological profile will also present an advantage in the treatment of hyponatremia, nephropathy and edema associated with nephrotic syndrome and liver cirrhosis.

Pharmacology

Test of the mouse vas deferens

[0053] The compounds of the present invention are pharmacologically active, for example, as agonists or partial agonists of nociceptin and a useful method for demonstrating their activity is that described by Hughes, J., Kosterlitz, HW, Leslie, FM: Effect of morphine on adrenergic transmission in the mouse vas deferens. Assesment of agonist and antagonist potencies of narcotic analgesics [4]. The preparation of the electrically stimulated isolated vas deferens has been widely used to evaluate the functional pharmacological activities of opioid agonists and antagonists in vitro [5]. The mouse vas deferens has all three types of opioid receptors and subsequent studies have described that the mouse vas deferens test is also sensitive to nociceptin [6].

[0054] The vas deferens of male NMRI albino mice weighing 30-40 g were placed in a SCHULER organ bath type 809, Hugo Sachs Elektronik, Germany, and perfused at 37 ° C with a modified Krebs solution ( NaCl 6.9 g / l, KCl 0.35 g / l, KH2PO4 0.16 g / l, NaHCO3 2.1 g / l, glucose H2O 2.0 g / l, CaCl2 2.52 ml / l). The solution was gassed with carcinogen (95% O2 and 5% CO2) throughout the experiment. The tissue was electrically stimulated by means of two silver electrodes directly in the organ bath by an HSE I stimulator, type 215. The tissue stimulation was performed by applying a supramaximal tension (15 V) in successions of 10 square pulses every 60 s, each 1 ms long with a frequency of 100 ms. Concentration response curves were performed by cumulative addition to the organ baths of test substances that relax the smooth muscles of the mouse vas deferens. The test substances used in the test were nociceptin in an isotonic saline solution, compound 1 (as trifluoroacetate in an isotonic saline solution), compound 2 (as trifluoroacetate in an isotonic saline solution) and Ac-RYYRWK-NH2 hexapeptide ( as trifluoroacetate in an isotonic saline solution). The peptide conjugates of the present invention are biologically active in the mouse vas deferens duct (MVD) assay, but the compounds inhibit the contraction of the electrically induced mouse vas deferens with a variable potency: nociceptin> compound 1 = compound 2 >> Ac -RYYRWK-NH2 (data not shown).

[0055] The effects of Ac-RYYRWK-NH2 and compound 1 on nociceptin-induced relaxation were also tested in the mouse vas deferens duct test. The results of these experiments are shown in Figure 1. These data demonstrate that both the partial agonist Ac-RYYRWK-NH2 [1] and the peptide conjugate called compound 1 inhibit the relaxation of smooth muscle induced by nociceptin. The pA2 values indicating the affinity of a compound as an antagonist were calculated using the Schild equation [7]. These values are presented in table 2:

Table 2

Ac-RYYRWK-NH2  Compound 1

PA2 value (number of experiments)

7.8 (n = 4) 9.5 (n = 4)

[0056] These data demonstrate that compound 1 is approximately 50 times more potent as an antagonist in the smooth muscle relaxation induced by nociceptin in the MVD assay than the unconjugated hexapeptide Ac-RYYRWK-NH2.

In vivo studies

[0057] Previous studies have shown that nociceptin produces a marked increase in the rate of urinary flow and a decrease in urinary sodium excretion (ie, an aquatic response) when administered centrally (intracerebroventricular, icv) or as iv infusion in conscious rats [8-10]. In the conscious rat model, the animal permanently carries catheters in the urinary bladder, the femoral artery and the femoral vein. The animal receives an infusion i.v. continue with isotonic saline solution at 50 µl / min. Urine is collected in vials in consecutive collection periods of 10 min each. After two control periods, the test compound is administered and fractional urine collection is continued (periods of 10 min) for at least 2 h. Urinary sodium and potassium concentrations are determined by flame photometry (Instrumentation Laboratory 943) with cesium as internal standard.

[0058] The diuretic effect of the peptide conjugates of the invention has been tested in conscious rats, in which compound 1 causes an intense and sustained diuretic response. The cardiovascular and renal responses produced by the infusion i.v. Nociceptin (Phoenix Pharmaceuticals) and compound 1 have been studied in conscious Sprague Dawley rats. The results are shown in Figures 2 to 8, in which HR is the heart rate, MAP is the mean blood pressure, V is the urinary flow rate, UNaV is the sodium urinary excretion rate, RSNA is the nerve activity renal sympathetic expressed as% of the control level, Cosm is the osmolar clearance and CH2O is the free water clearance.

[0059] Figure 2 shows the typical cardiovascular and renal responses to nociceptin administered as an infusion i.v. (11 nmol / kg / min). Nociceptin decreases MAP significantly, possibly due to relaxation of arterial smooth muscle [11].

[0060] Figure 3 shows the significant diuretic (V µl / min) and antinatriuretic (UNaV µeq / min) responses to compound 1 administered as an i.v. infusion. (1 and 0.1 nmol / kg / min). Similar to nociceptin i.v., compound 1 produced a slight but significant reduction in MAP, with little effect on HR. None of the rats to which compound 1 was administered showed signs of increased appetite, sedation or behavioral changes normally associated with nociceptin treatment.

[0061] Figure 4 shows the decrease in osmolar clearance and the increase in free water clearance observed during the infusion i.v. of compound 1 (1 and 0.1 nmol / kg / min).

[0062] Figure 5 illustrates the similar magnitude of the changes in V and UNaV caused by the i.v. of 1 nmol / kg / min of compound 1 and 11 nmol / kg / min of nociceptin.

[0063] Figure 6 illustrates the similar magnitude of the changes in V and UNaV caused by the i.v. 0.1 nmol / kg / min of compound 1 and 1.1 nmol / kg / min of nociceptin. Taken together, the data presented in Figures 5 and 6 suggest that, when compared to true nociceptin, compound 1 is approximately 10 times more potent in the production of diuretic and antinatriuretic responses of similar magnitude. The diuretic effect of

10 infusion of compound 1 tended to persist for longer than infusion i.v. of nociceptin.

[0064] In addition, the effects of the administration i.v. of nociceptin boluses (30 and 100 nmol / kg, Phoenix Pharmaceuticals) and nociceptin-NH2 (30 nmol / kg) on heart rate, blood pressure and RSNA in groups of six Sprague-Dawley rats. Bowling injection produced rapid bradycardia, a hypotensive response and the initial suppression of RSNA (data not shown). An i.v. Similar bowling of compound 1 (30 nmol / kg) produced no change in heart rate, blood pressure or RSNA. In addition, the rats that received this dose showed no sign of appetite stimulation or behavioral changes. I.v. Bowling of compound 1 (30 nmol / kg) in this same rat produced a remarkable diuretic (V) and antinatriuretic (UNaV) response (data not shown). A summary of the responses in vivo to

Compound 1 obtained in the experiments described above is shown in Table 3 below:

Table 3: In vivo responses of the series of experiments in which

Dose i.v. of compound 1

HR MAP V One V CH2O  Food intake Behavior changes

Infusion i.v. 0.1 nmol / kg / min

0 (↓) ↑  0 ↑  0 0

Infusion i.v. 1.0 nmol / kg / min

0 (↓) ↑↑ ↓ ↑↑  0 0

I.v. bowling 30 nmol / kg

0 0 ↑ ↓ ↑  0 0

0 indicates lack of response, (↓) indicates a very slight decrease, ↓ indicates a slight decrease, ↑ indicates a slight increase and ↑↑ indicates a strong increase.

[0065] From these studies, it is clear that compound 1 causes a significant diuretic response to infusion doses i.v. well below those required to cause changes in feeding or behavior (for example, sedation, increased scanning behavior, catalepsy, seizures, etc.).

25 In addition, after 120 min of infusion i.v. of compound 1 (1 nmol / kg / min) in the study presented in figure 3, the HR and MAP responses to an i.v. injection were examined. of nociceptin bowling. In these studies, typical bradycardia and hypotension (rapid onset and large-scale) were completely blocked in all rats tested (n = 6).

[0066] In an additional series of dose response studies cardiovascular and renal responses to compound 1 were investigated after an i.v. of bowling (10 (n = 6), 30 (n = 4), 100 (n = 6) or 300 (n = 4) nmol / kg i.v.) or during an infusion i.v. continuous (0.1 (n = 6), 1 (n = 6) or 10 nmol / kg / min (n = 6)). After stabilization of the urinary flow rate and urinary sodium excretion, urine was collected during a 20-minute control period. Then the i.v. injection was administered. of bowling or the infusion was changed i.v. by

An isotonic saline solution containing compound 1 (i.v. infusion experiments). From the moment of drug administration, experimental urine samples (consecutive 10 min periods) were taken for 80 min.

Comments on the feed response

[0067] One hour before the start of each experiment, a food pellet (standard rodent food) accessible to the rat was placed through a hole in the immobilization device. The number and timing of feeding responses (ie, the times at which the rat started eating the food pellet) was recorded before and after drug administration. If a response is observed from

In the positive diet, the immobilizer pellet was removed to avoid alterations in heart rate and / or average blood pressure induced by the food. The feed pellet was presented to the rat at 10 min intervals to determine the duration of the feeding response.

[0068] In this series of studies in conscious rats with permanent catheters we have shown that the 50 injection i.v. Bowling of compound 1 produces a selective aqueous diuresis. The data is presented in table 4 below:

Table 4: responses after administration i.v. bowling of compound 1

Parameters studied

Dose i.v. bowling (nmol / kg)

10 (n = 6)

30 (n = 4) 100 (n = 6) 300 (n = 4)

 urine flow (%)

+83  42 +207  11 +249  60 +127  47

 Na excretion (%)

-44  3 -51  7 -70  2 -85  6

 excretion of K (%)

-35  4 -20  7 -52  5 -79  6

 blood pressure (%)

-4  1 -8  2 -12  2 -15  3

Food intake

- (+) * (+) ** (+) ***

* Indicates that of 4 rats tested with 30 nmol / kg of i.v. boluses, one rat demonstrated a feeding response at several time points during the 80 min experimental period. ** Indicates that of 6 rats tested with 100 nmol / kg of i.v. boluses, 3 rats showed a feeding response within 30 min after i.v. of the bowling of ZP120C. In the remaining 3 rats, no feeding response was observed during the entire test period (80 min). *** Indicates that of 4 rats tested with 300 nmol / kg of bowls i.v., 2 rats demonstrated a feeding response at various time points in the experimental period of 80 min.

5 [0069] In administration as an infusion i.v. continuous, a selective aqueous diuretic response (diuresis associated with antinatriuresis) was observed for 1 and 10 nmol / kg / min (table 5).

Table 5: responses during infusion i.v. of compound 1

Parameters studied

Infusion dose i.v. (nmol / kg / min)

0.1 (n = 6)

1.0 (n = 6) 10 (n = 6)

 urine flow (%)

+99  21 210  51 160  68

 Na excretion (%)

-5  3 -64  7 -79  6

 excretion of K (%)

-16  6 -51  9 -68  15

 blood pressure (%)

-5  4 -10  2 -15  3

Food intake

- - (+) *

* Indicates that of 6 rats tested with 10 nmol / kg / min, 4 rats did not show a feeding response and 2 rats ate in the last 10 min period of the 80 min infusion study.

[0070] Collectively, these dose response studies demonstrate that compound 1 produces a significant aqueous diuretic response to infusion doses i.v. and injection i.v. of bowling that are 10 well below those required to cause considerable changes in cardiovascular activity. This is in contrast to the diuresis produced by endogenous nociceptin, which is followed by a notable bradycardic and hypotensive response (i.v. bowls and i.c.v. injection) and a simultaneous inhibition of renal sympathetic nerve activity (i.c.v injection) [8-10]. These observations suggest that, unlike the endogenous ORL1 ligand, nociceptin, compound 1 has clinical utility as an aquatic in patients at therapeutic doses substantially lower than

15 required to cause adverse systemic cardiovascular effects. Our speculation is that lower penetration of compound 1 into the CNS may explain the more selective renal effect of the new compound compared to endogenous nociceptin.

[0071] Rats with liver cirrhosis induced by ligation of the common bile duct develop an avid

20 sodium and water retention and ascites in 2-4 weeks. The alteration of the balance of sodium and water in cirrhotic rats is consistent with the fundamental clinical observations of edema and ascites in patients with liver cirrhosis. To examine the efficacy of compound 1 in an experimental model of an edema-forming state, the diuretic response to a single injection i.v. of bowling of compound 1 (65 nmol / kg) in rats with liver cirrhosis induced by ligation of the common bile duct four weeks earlier. Before the experiments

25 permanent catheters were placed in the urinary bladder and in the vein and femoral artery. The renal responses to compound 1 were compared with the responses in rats used as surgical controls. Compound 1 produced a marked increase in free water clearance (aqueous diuresis), associated with a decrease in urinary excretion of sodium and potassium in the absence of changes in the glomerular filtration rate in all animals. Renal responses were similar in cirrhotic rats and control rats. The changes

30 relative in the rate of urinary flow and excretion of sodium and potassium after administration i.v. of 65 nmol / kg in animals used as surgical control are summarized in Figure 7.

[0072] As illustrated in Figure 8, cirrhotic rats showed a fractional excretion of sodium before significantly lower drug administration compared to control animals. This indicates that the cirrhotic rats had intense sodium retention, which is consistent with the remarkable sodium retention that characterizes human liver cirrhosis. Compound 1 produces a noticeable increase in flow rate

Urinary and free water clearance (aqueous diuresis), associated with a decrease in fractional excretion of sodium and potassium in the absence of changes in the glomerular filtration rate in all animals. Renal responses were similar in cirrhotic rats and control rats. Changes in the rate of urinary flow, free water clearance, fractional excretion of sodium and fractional excretion of potassium after administration i.v. 65 nmol / kg in rats used as surgical control and in cirrhotic animals are summarized in Figure 8. These results in an animal model of a pathological state characterized by avid sodium retention indicate that the compounds of the present invention, Like compound 1, they are useful in the treatment of edema, hypokalemia and hyponatremia in edema-forming states such as liver cirrhosis, congestive heart failure, nephrotic syndrome and renal failure.

[0073] To assess whether acute tachyphylaxis develops during treatment i.v. with compound 1 a series of additional experiments were performed. Rats with permanent catheters were subjected to the infusion of a vehicle (150 mM glucose) or compound 1 (1 nmol / kg) for 12 h while the water balance was maintained by means of an i.v. replacement of computer-driven and servo-controlled vollemia [2]. Acute tachyphylaxis has been observed during dosing i.v. Repeated vasopressin antagonist SFK 101926 in conscious dogs [12] and tolerance has been described during chronic treatment for the selective V2 receptor antagonist, OPC-31260 [13].

[0074] Compound 1 caused a sustained aquatic response that lasted 12 hours after the infusion period, suggesting that acute tachyphylaxis does not develop during treatment with this compound. The infusion i.v. of 1 nmol / kg / min of compound 1 caused an aquatic response of similar magnitude to the response observed with the maximum doses of the selective vasopressin receptor antagonist type 2, OPC-31260 (Figure 9) when tested in the same model [ 2]. In this experiment, 1 nmol / kg / min of compound 1 inhibited tubular reabsorption of water without concomitant changes in blood pressure, heart rate, renal blood flow, glomerular filtration rate or proximal tubular reabsorption. These experiments demonstrated that compound 1 has a maximum aquatic efficacy similar to that obtainable with vasopressin type 2 antagonists. However, acute tachyphylaxis does not develop within the first 12 hours of treatment with compound 1 and this observation represents a further therapeutic advantage of compound 1 in relation to type 2 vasopressin receptor antagonists. In addition, type 2 vasopressin receptor antagonists do not affect renal treatment of electrolytes, while the compounds of the present invention decrease excretion. urinary sodium and potassium.

[0075] To examine the effect of the compounds of the present invention on an in vivo model of heart failure, a chronic model of heart failure of low cardiac output is used. Briefly, the rats are anesthetized in an inhalation chamber with 4% halothane in a 1: 1 N2O / O2 gas mixture. After inserting an endotracheal tube, the animal is subjected to assisted breathing with 1% halothane in a 1: 1 N2O / O2 gas mixture. The tidal volume and respiratory rate are adjusted to maintain arterial pH between 7.35 and 7.45. During the operation, the animal is placed on a heated table that maintains the rectal temperature at 37-38 ° C. The left coronary artery ligation, used to produce congestive heart failure, is performed through a parasternal thoracotomy, placing a 6-0 silk suture between the pulmonary trunk and the left atrium. A surgical control is performed without ligating the left coronary artery. To minimize postoperative pain, the rats are treated after the operation with buprenorphine, 0.2 mg / kg s.c. During two days. Two weeks after the heart operation, Tygon permanent catheters are inserted into the artery and femoral vein and a bladder catheter into the urinary bladder. To prevent coagulation in vascular catheters, these ducts are filled with 50% dextrose with 1,000 units / ml of heparin and 10,000 units / ml of streptokinase. The inventors of the present invention have described this model previously [14]. Physiological exams are performed three weeks after the heart operation, since functional impairment after left coronary artery ligation is usually maximal at this time. Renal activity studies are performed as described above. To assess the degree of heart failure, the rat is anesthetized with halothane in N2O / O2 1: 1, intubated and subjected to assisted breathing and a Tygon catheter is inserted into the left ventricle through the right carotid artery to measure the final diastolic pressure of the left ventricle (PDFVI). The exact measurement of the PDFVI is done by adjusting the concentration of halothane to keep the afterload during anesthesia at the same level as the average blood pressure recorded in the conscious state. This animal model shares the clinical features of low cardiac output, sodium and water retention and edema formation of the most common form of human heart failure [15].

[0076] To examine the effect of the compounds of the present invention on an in vivo model of nephrotic syndrome, a chronic model of nephrotic syndrome induced by adriamycin is used. Nephrotic syndrome is induced with a single intravenous injection of adriamycin (= doxorubicin, Sigma Chemical, San Luis, MO, USA); 7-8 mg / kg at a concentration of 10 mg / ml dissolved in isotonic saline. In this model, proteinuria begins four to five days after a single intravenous injection of 7.5 mg / kg. Total expression of the syndrome with glomerular changes, proteinuria, sodium and water retention and edema formation begins 13 to 15 days later [15; 16].

[0077] To examine the effect of the compounds of the present invention on an in vivo model of liver cirrhosis, a chronic model of biliary cirrhosis induced by ligation of the common bile duct is used [17]. After 28 days of obstruction, the rats show progressive cirrhosis, associated with sodium and water retention and ascites [15].

[0078] To examine the effect of the compounds of the present invention on an in vivo model of multiple organic insufficiency that includes acute renal failure, a model of organic insufficiency caused by bleeding during anesthesia and surgery is used. In this model, the rats receive an infusion of saline solution or the compounds of the present invention for 15 min before anesthesia. The animals are then anesthetized with isoflurane (3% in a mixture of O2 / N2O) and undergo periods of surgery (permanent catheterization in the bladder and vein and femoral artery; 30 min); hemorrhage (20 cm3 / kg body weight; 45 min) and recovery (blood replacement; 120 min). In the test, consecutive 10-minute urine samples are collected and the rats are allowed to recover for seven days. After bleeding, the collected urine and blood samples (for example, days 2, 4, and 6) are collected to assess recovery as determined by urine production and serum creatinine and urea concentrations. Finally, on day 7 the rats are sacrificed for histological examination of all organs. Using this model, Kapusta et al. Previously demonstrated that the  U-50,488H opioid agonist avoids multiple organ failure (kidney, lungs, intestine) and increases survival after bleeding caused during surgical anesthesia [18].

[0079] In summary, in vivo studies have shown that compound 1 is a potent sodium and potassium-saving aquarium. In addition to its peripheral effects, nociceptin causes a centrally mediated decrease in sympathetic nerve activity, blood pressure and heart rate [10; 19; 20]. In addition, nociceptin decreases spinal sympathetic flow by decreasing the activity of preganglionic neurons both in vitro and in vivo [21]. The rapid onset and the similar temporal relationship of the decrease in sympathetic nerve activity, blood pressure and heart rate in rats with synoortic baroreceptor denervation suggest that the cardiovascular effects of nociceptin are mainly due to the inhibition of sympathetic nerve activity of the cardiovascular system [10]. Therefore, the inhibitory effect of compound 1 on hypotension and bradycardia induced by nociceptin suggests that compound 1 is able to antagonize the effects of nociceptin in the neurogenic control of heart rate and blood pressure. The observation that compound 1 acts as a nociceptin antagonist in vivo is supported by the fact that compound 1 antagonizes the relaxation effect of nociceptin smooth muscle in the mouse vas deferens duct. Therefore, in contrast to endogenous nociceptin, compound 1 increases the rate of urinary flow and decreases the urinary excretion of sodium and potassium in doses that do not produce remarkable bradycardia or hypotension.

In vitro studies of the binding of compound 1 to ENT1

[0080] We have demonstrated in a series of binding assays that compound 1 binds to the human nociceptin receptor (ie, ENT1) with high affinity and high specificity.

[0081] Through the use of HEK293 cells transfected with the human ORL1 receptor we have demonstrated that compound 1 displaces the specific nociceptin binding labeled with 3H with a Kl = 0.26 nM.

[0082] In addition, compound 1 has been tested in five opioid receptor binding assays (non-selective opioid, , ,  and ORL1), three vasopressin receptor binding assays (V1a, V1b and V2) a concentrations of 0.1, 10 and 1,000 nM, as well as 40 different sites of receptor binding, absorption and ion channels (A1, A2, α1, α2, 1, NE absorption, AT1, BZD, B2 (h) , CCKA (~ CCK1) .D1, D2, ETA (h), GABA, NMDA, H1, M, NK1 (h). Y, N, opioid, PCP, 5-HAT, 5-HT1B- 5HT2A, absorption of 5 -HAT, , V1) at a concentration of 10 µM. These experiments demonstrated that compound 1 is a selective ligand of the ORL1 receptor with no or only slight affinity for receptors other than ORL1. In addition, in transfected HEK293 cells expressing the human ORL1 receptor, compound 1 inhibits forscholine-induced cAMP formation with an EC50 = 0.54 nM and with a relative maximum nociceptin of 92%. These results suggest that compound 1 is a selective ORL1 receptor agonist.

[0083] Data demonstrating the binding of the compounds of the invention to the human ORL1 receptor expressed in CHO cells, calculated as IC50 values (nM) (all based on one experiment) are shown in Table 6 below. The ORL1 receptor is negatively coupled to cAMP and, therefore, the effect of ORL1 agonists is detected as an inhibition of forscholine-induced cAMP formation. Six compounds of the invention have been tested for their efficacy on the hORL1 receptor expressed in CHO cells. All EC50 (nM) values are based on an experiment (data shown in table 6).

Table 6. Binding of ZS compounds (peptides) to the hORL1 receptor

Compound Number

IC50 union (nM) Efficiency EC50 (nM)

CE1

 0.31 0.41

one

0.32 0.58

3

235 83

4

ND 91

6

115 135

7

> 100 ND

8

78 59

9

3.7 0.035

10

 0.51 ND

eleven

 0.27 ND

12

 0.15 ND

13

 ND ND

[0084] Most of the published work on the human ORL1 receptor has focused on the effects of the CNS receptor. However, the work described in this document focuses on the effect mediated by compound 1 in the kidney. In order to determine if the ORL1 receptor is present in the human kidney,

5 We have constructed PCR primers homologous to the ORL1 receptor expressed in the human brain. Total RNA isolated from the human kidney was subjected to the first strand synthesis to obtain material for PCR analysis. This analysis showed a 449 bp fragment of the ORL1 receptor, indicating that the receptor is expressed in the human kidney and that, therefore, it is expected that the ORL1 ligands of the present invention have renal effects in humans.

10

In vitro stability

[0085] A degradation of the peptides is observed almost exclusively in the presence of enzymes, for example, in the bloodstream. An important characteristic of a potential drug is if it degrades

15 quickly in vivo or if it is capable of producing its effects for a longer period. In order to examine the stability of the peptide conjugates of the present invention, plasma degradation of heparin stabilized mice (obtained from Harlan Seralab, UK) was tested.

Procedures

[0086] Stability studies were performed in triplicate. A volume of 100 µl of a solution of the test compound (peptide conjugate) with a concentration of 1 mg / ml was added to an Eppendorf tube containing 900 µl of plasma incubated at 37 ° C. Immediately after the addition a 100 µl sample was extracted and added to an Eppendorf tube containing 10 µl of the extraction solution (TFA (trifluoroacetic acid)

25 to 50% in MeCN, v / v). This sample constitutes t = 0. Next samples were taken at t = 15, 30, 60, 120, 180, 240, 300 and 360 min which were treated in the same way as the sample at t = 0. The samples were centrifuged at

20,000 g and the supernatant was transferred to vials and analyzed by high efficiency liquid chromatography (HPLC).

[0087] The analysis was performed in an HP1100 HPLC apparatus with a quaternary pump. One was used

The Vydac 201SP5215 column and the apparatus comprised an automatic injector, a column oven, a variable UV detector and a fluorescence detector. The process was controlled by ChemStation software. Gradient elution consisted of two solvents A and B containing 0.02% n-heptafluorobutyric acid (HFBA) in MilliQ water (MQW) and 0.02% HFBA in 90% MeCN in MQW, respectively. The separation was carried out at 30 ° C by a linear gradient of 5-100% B in 15 min with a flow of 0.250 ml / min. The chromatograms used in

35 This study was obtained by UV detection at a wavelength of 254 nm. The peaks identified as the test peptides by comparison with known standards were integrated to obtain the peak area as a measure of the concentration for each time point. These values were transferred to Microsoft Excel, where the half-lives (t½) and the velocity constant (kobs) were determined for Ac-RYYRWK-NH2 and compound 1 by adjustment in the equation. As illustrated in Figure 10, the rate of degradation of the unconjugated substance

40 Ac-RYYRWK-NH2 was significantly higher than that of compound 1 (Ac-RYYRWK-NH2: t½ = 2-5 min (estimate) and compound 1: t½ = 358  36 min).

[0088] These data demonstrate that the peptide conjugate, compound 1, is significantly more stable in mouse heparinized plasma than the unconjugated hexapeptide Ac-RYYRWK-NH2. Therefore, the invention also relates to a process for improving the stability of a hexapeptide, preferably a hexapeptide of the formula II herein and, more preferably, the RYYRWK hexapeptide, by binding a peptide sequence Z ' as defined is this document, in which preferably Z 'is a K6 sequence, at

C-terminus of said hexapeptide by means of a peptide bond.

Stability in heparinized human plasma

[0089] The degradation of compound 1 of the invention has been further studied in heparinized human plasma. Table 7 shows the degradation half-life (T½) of compound 1 of the invention compared to Ac-RYYRWK-NH2 and nociceptin-NH2. From the table it is evident that the compound 1 of the invention with a half-life greater than 16 h is considerably more stable in plasma than Ac-RYYRWK-NH2 having a half-life less than 1 h and nociceptin-NH2, which has a 1.5 h half life.

Table 7. Results of the in vitro stability test in plasma and serum, T½ in min (mean  deviation)

COMPOUNDS

T½ in min (degradation in heparinized human plasma)

Compound 1

973.8 min  21.9%

Ac-RYYRWK-NH2

50.7 min  20.9%

Nociceptin-NH2

97.5 min  6.4%

* no reaction in more than 5 h

Procedure for in vitro plasma stability analysis

[0090] The stability of the peptides in human plasma is analyzed. The peptides are incubated at 37 ° C in plasma and samples are taken at approximately 8 regular intervals between t = 0 and t = 390 min for compound 1 and nociceptin-NH2 and at 9 intervals between t = 0 and t = 32 min for compound CE1 which are analyzed by HPLC.

[0091] The appropriate conditions (column, solvent, gradient and temperature) for HPLC analyzes are estimated to ensure that the drug peak and plasma peaks do not have the same retention time. This is done by subsequent injections of the drug, the plasma and a co-injection of the drug and the plasma, followed by the optimization of the parameters of the LC procedure until a satisfactory separation is obtained. Three parallel experiments are performed for each type of plasma. A volume of 100 µl of the peptide is mixed with 900 µl of plasma at t = 0 and incubated at 37 ° C (concentration of the drug mixture and

25 plasma 0.1 mg / ml). 100 µl samples are extracted from the drug and plasma mixture at appropriate intervals and degradation is stopped by precipitation of the sample with 10 µl of MeCN: TFA 50:50 (v / v). A plasma control sample is also taken without the drug being treated in the same way. Plasma samples are centrifuged at 12,000 rpm for 15 min (Eppendorf centrifuge) at room temperature. The resulting supernatant solution is transferred to 300 µl vials for an HP automatic injector and analyzed by HPLC. HPLC analyzes are performed as follows:

Ac-RYYRWK-NH2 and compound 1 Column: Vydac 201 SP5215, 991203, 033, S / N N 905550-1-3, 150 x 2.1 mm, flow: 0.200 ml / min. Temperature: 40 ° C Solvent A: 0.02% HFBA in MQW. Solvent B: 0.02% HFBA in MQW: MeCN 10:90

35 Chromatography time = 25 min. Injected volume: 20 µl. Detection: FLD1 A, excitation = 280

nm, emission = 335 nm Gradient (time in min;% B): 0; 5 5; 50 15; 100 15.5; 5 25; 5 Nociceptin-NH2 Column: Vydac 218MSP52, 000517, 095, S / NN 970520- 9-7, 250 x 2.1 mm, flow: 0.200 ml / min. Temperature: 40 ° C Solvent A: 0.1% TFA in MQW. Solvent B: 0.1% TFA in MQW: MeCN 10:90 Chromatography time = 25 min. Injected volume: 20 µl. Detection: MWD, signal = 215.16 nm,

ref. = 360, 100 nm Gradient (time in min;% B): 0; 1 5; 1 15; 40 16; 100 17; 1 25; 5

[0092] The samples are analyzed in the following order: blank, the 0.1 mg / ml peptide, the plasma without the peptide, the three parallel samples for t = 0, the three parallel samples for t = 5 min, the three parallel samples for t = 60 min, etc. And finally the three parallel samples are repeated for t = 0 to ensure that there has been no degradation or other failure during the analysis. The concentration of the samples (peak height in milliabsorbance units, mAU) is plotted against time and a function that describes a monoexponential degradation (Excel) is set. The half-lives of peptides in plasma are presented in table 7 as mean (n = 3)  standard deviation.

Compositions

[0093] The invention also relates to a composition comprising a pharmacologically active peptide conjugate as defined herein in combination with a pharmaceutically acceptable carrier and / or diluent. Such compositions may be in a form adapted to oral, subcutaneous, parenteral (intravenous, intraperitoneal), intramuscular, rectal, epidural, intratracheal, intranasal, dermal, vaginal, buccal, ocular or pulmonary administration, preferably in a form adapted to administration by a peripheral route or that is suitable for oral administration or suitable for parenteral administration. Other preferred routes of administration are the subcutaneous, intraperitoneal and intravenous routes and such compositions can be prepared in a manner well known to the 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, USA UU., 1985 and more recent editions and in the monographs of the series "Drugs and the Pharmaceutical Sciences" by Marcel Dekker. The compositions may appear in conventional forms, for example solutions and suspensions for injection, capsules and tablets, preferably in the form of enteric formulations, for example, as disclosed in US 5,350,741 for oral administration.

[0094] The pharmaceutical vehicle or diluent used may be a conventional solid or liquid vehicle. Some examples of solid carriers are lactose, kaolin, sucrose, cyclodextrin, talc, gelatin, agar, pectin, acacia gum, magnesium stearate, stearic acid or lower alkyl cellulose ethers. Some examples of liquid vehicles are syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene and water. Similarly, the carrier or diluent may include any sustained release material known in the art, such as glycerin monostearate or glycerol distearate alone or mixed with a wax.

[0095] When a solid vehicle is used for oral administration, the preparation can be formed into tablets, placed in a hard gelatin capsule in powder or pellet form, or it may be in the form of a sucking tablet. The amount of the solid carrier will vary widely but will usually be between approximately 25 mg and approximately 1 g.

[0096] A typical tablet that can be prepared by conventional tabletting techniques may contain: core: the active ingredient (as free compound or salt thereof), 100 mg; colloidal silicon dioxide (Aerosil), 1.5 mg; microcrystalline cellulose (Avicel), 70 mg; modified cellulose gum (Ac-Di-Sol), 7.5 mg; magnesium stearate. Coating: HPMC, approximately 9 mg: Mywacett 9-40T (acylated monoglyceride used as plasticizer for film coatings), approximately 0.9 mg.

[0097] If a liquid carrier is used, the preparation may be in the form of a syrup, emulsion, soft gelatin capsule or sterile injectable liquid, as a suspension or aqueous or non-aqueous liquid solution.

[0098] The composition may also be in a form suitable for local or systemic injection or infusion and, as such, may be formulated with sterile water or an isotonic saline solution or a glucose solution. It is preferred that the compositions of the invention be in a form adapted to peripheral administration only, with the exception of centrally administrable forms.

[0099] The compositions may be sterilized by conventional sterilization techniques that are well known in the art. The resulting aqueous solutions can be packaged for use or filtered under aseptic conditions and lyophilized, in which the lyophilized preparation is combined with the sterile aqueous solution before administration. The composition may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as buffering agents, osmotic agents and the like, for example sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc. .

Formulation of peptide conjugates for intravenous injection:

[0100] Multidose formulations can be prepared as a solution of a compound of the invention in sterile isotonic saline, stored in vials with a lid and if necessary with the addition of a preservative (for example, benzoates). Fixed dose formulations can be prepared as a solution of the compound in sterile isotonic saline solution, stored in glass ampoules and if necessary filled with an inert gas.

[0101] All doses of the compound are stored dry in ampoules or vials with a lid, if necessary, filled with an inert gas. The multidose formulation requires the highest degree of stability of the compound. If the stability of the compound is low, fixed dose formulations can be used.

[0102] For nasal administration, the preparation may contain a compound of the present invention dissolved or suspended in a liquid vehicle, in particular an aqueous vehicle for administration as an aerosol. The carrier may contain additives such as solubilizing agents, for example propylene glycol, surfactants such as salts of bile acids or ethers of higher polyoxyethylene alcohols, absorption enhancers such as lecithin (phosphatidylcholine) or cyclodextrin or preservatives such as parabens.

[0103] In a preferred embodiment of the invention, the compound of the formula I, II or III is administered in a dose in the range of from about 0.001 to about 10 g daily per patient, preferably from about 1 to about 1,000 mg daily per patient, more preferably from about 10 to about 100 mg daily per patient, about 50 mg daily per patient. Typically, a pharmaceutical composition adapted for oral administration contains in a unit dose an amount of a compound of formula I or II in the range from about 1 to about 100 mg. Typically, a pharmaceutical composition adapted for parenteral administration contains in a unit dose an amount of a compound of the formula I or II in the range from about 0.1 to about 10 mg.

[0104] The invention also relates to a pharmacologically active compound that is a peptide conjugate or a derivative or salt thereof as disclosed herein for therapeutic use and the use thereof as defined herein for the preparation of a pharmaceutical composition for therapeutic use, for example, in the treatment of diseases and peripheral ailments related to ENT1. Preferably, a pharmaceutical composition is suitable for oral administration. The therapeutic uses of the new peptide conjugates in this document are the increase in renal excretion of water and the decrease in urinary excretion of sodium and potassium (i.e. a selective aqueous diuresis) with relatively low doses of the compound, i.e. preferably less than 50 mg daily per patient, and decreased blood pressure in the absence of reflex tachycardia and increased appetite with higher doses of the active compound.

[0105] In specific embodiments, a peptide conjugate according to the present invention can be used to avoid side effects in the CNS in the treatment of specific diseases or conditions that can be treated with compounds with activity similar to that of nociceptin, due to the low penetration of the peptide conjugates in the central nervous system.

[0106] In specific embodiments, a peptide conjugate according to the present invention can be used as an agonist or partial agonist or as an inhibitor, depending on the clinical utility, acting on a nociceptin receptor such as ENT1, especially when said receptor is in the tissue. peripheral. Thus, the peptide conjugate of formula I or III is useful for the preparation of a medicament for use in the treatment of pathological conditions associated with a high nociceptin activity.

[0107] In specific embodiments, a peptide conjugate according to the present invention can be used for the preparation of a medicament for the treatment and / or prevention of hyponatremia and / or hypokalemia, as well as in a procedure for the treatment and / or prevention. of hyponatremia and / or hypokalemia.

[0108] In addition, a peptide conjugate according to the present invention can be used for the preparation of a medicament for the treatment and / or prevention of conditions with sodium and water retention and acute renal failure, as well as in a treatment procedure. and / or the prevention of multiple organ failure and states with sodium and water retention and acute renal failure. In addition, a peptide conjugate according to the present invention can be used for the preparation of a medicament for the treatment and / or prevention of states of organic insufficiency associated with the expansion of extracellular fluid volume including:

one.

Congestive heart failure, in which heart failure can be described as systolic or diastolic, high-cost or low-cost, acute or chronic, right or left and antegrade or retrograde. An example of a predictive model of heart failure in vivo for the study of the therapeutic effects of the peptides of the present invention is the low cardiac output model induced by left coronary artery ligation in conscious rats [15].

2.

 Liver cirrhosis in which cirrhosis may be related to alcoholic cirrhosis; postnecrotic cirrhosis (caused by infectious diseases, inherited metabolic disorders, drugs and toxins, inflammatory diseases and others); biliary cirrhosis (primary or secondary); cardiac cirrhosis due to right, severe and prolonged congestive heart failure; Metabolic, hereditary, drug-related cirrhosis or other types of cirrhosis. An example of a predictive model of liver cirrhosis in vivo for the study of the therapeutic effects of the peptides of the present invention is the model of liver cirrhosis induced by ligation of the common bile duct in conscious rats [15].

3.

 Nephrotic syndrome related to a renal and / or systemic disease induced by drugs or toxins. An example of a predictive model of nephrotic syndrome in vivo for the study of the therapeutic effects of the peptides of the present invention is the nephrotic syndrome model induced by the administration i.v. of adriamycin [15].

Four.

 Hypertension, in which the hypertension can be primary (idiopathic) or secondary by drugs, toxins or diseases of the endocrine glands, kidneys or central nervous system. Some examples of predictive models of hypertension in vivo for the study of the therapeutic effects of the peptides of the present invention are the rat model with spontaneous hypertension or the salt-sensitive Dahl rat.

5.

 Multiple organ failure caused during hemorrhagic shock that includes acute renal failure. An example of an in vivo predictive model of multiple organ failure (kidney, lungs, intestine) caused during anesthesia, surgery and hemorrhage is that described by Kapusta et al. [16].

6. Acute renal failure, in which the pathogenesis of the disease may be related to

intrinsic prerenal or renal causes: Prerenal azotemia, as in the following conditions:

 I. Hypovolemia

 A. Hemorrhages, burns, dehydration

B. Loss of gastrointestinal fluid: vomiting, surgical drainage, diarrhea

C. Loss of renal fluid: diuretics, osmotic diuresis (eg diabetes saccharin), hypoadrenalism

D. Abduction in the extravascular space: pancreatitis, peritonitis, trauma, burns, severe hypoalbuminemia;

II.

 Low cardiac output

TO.

Myocardial diseases, valves and pericardium, arrhythmias, tamponade

B.

 Others: pulmonary hypertension, massive pulmonary embolism, mechanical ventilation assisted with positive pressure;

III.

Impaired relationship between renal and systemic vascular resistance

TO.

Systemic vasodilation: septicemia, antihypertensives, afterload reducers, anesthesia, anaphylaxis

 B.

Renal vasoconstriction: hypercalcemia, norepinephrine, epinephrine, cyclosporine, amphotericin B

C. Cirrhosis with ascites (hepatorenal syndrome);

IV. Renal hypoperfusion with renal self-regulation response disorder, cyclooxygenase inhibitors, angiotensin converting enzyme inhibitors;

V. Hyperviscosity syndrome (rare) Multiple myeloma, macroglobulinemia, polycythemia.

Acute renal failure also includes: Intrinsic renal azotemia, as in the following conditions:

I. Renovascular obstruction (bilateral or unilateral with a functional kidney)

A. Renal artery obstruction: atherosclerotic plaque, thrombosis, embolism, dissecting aneurysm, vasculitis

B. Renal vein obstruction: thrombosis, compression;

II.

 Glomerulus or renal microvasculature disease

A. Glomerulonephritis and vasculitis

B.

Hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, disseminated intravascular coagulation, pregnancy toxemia, accelerated hypertension, radiation nephritis, systemic lupus erythematosus, scleroderma;

III.

Acute tubular necrosis

TO.

 Ischemia: as in prerenal azotemia (hypovolemia, low cardiac output, renal vasoconstriction, systemic vasodilation), obstetric complications (placental abruption, puerperal hemorrhage)

   B. Toxins

one.

 Exogenous: radiocontrasts, cyclosporine, antibiotics (eg aminoglycosides), chemotherapy (eg cisplatin), organic solvents (eg ethylene glycol), acetaminophen, illegal abortives

 2.

Endogenous: rhabdomyolysis, hemolysis, uric acid, oxalate, plasma cell dyscrasia (eg myeloma);

IV.

Interstitial nephritis

TO.

Allergic: antibiotics (eg -lactams, sulfonamides, trimethoprim, rifampin), nonsteroidal anti-inflammatory drugs, diuretics, captopril

B.

 Infection: bacterial (eg acute pyelonephritis, leptospirosis), viral (eg cytomegalovirus), fungal (eg candidiasis)

C. Infiltration: lymphoma, leukemia, sarcoidosis

 D. Idiopathic;

V.

 Deposition and intratubular obstruction

 A. Myeloma, uric acid, oxalate, acyclovir, methotrexate, sulfonamide proteins;

SAW. Rejection of renal allografts

[0109] Some examples of predictive models of prerenal azotemia in vivo for the study of the therapeutic effects of the peptides of the present invention are models of acute ischemic renal failure induced by norepinephrine or by renal artery clamping in rats, which may accelerate by hemorrhage [22].

[0110] An example of a predictive model of intrinsic renal azotemia in vivo for the study of the therapeutic effects of the peptides of the present invention is the model of acute renal failure induced by gentamicin [23].

References [0111]

[1.] C. T. Dooley, C. G. Spaeth, I. P. Berzetei-Gurske, K Craymer, I. D. Adapa, S. R. Brandt, R. A. Houghten, L. Toll,

J. Pharmacol. Exp. Ther. 1997, 283: 735-741.

[2.] T. E. Jonassen, S. Christensen, T. H. Kwon, S. Langhoff, N. Salling, S. Nielsen, Am. J. Physiol. Renal Physiol. 2000, 279: F1101-F1109. [3.] E. Braunwald, in Harrison’s Principles of Internal Medicine, eds .: A. S. Fauci, E. Braunwald, K J. Isselbacher, J.

D. Wilson, J. B. Martin, D. L. Kasper, S. L. Hauser, D. L. Longo, McGraw-Hill, New York 1998, p. 1287-1298. [4.] J. Hughes, H. W. Kosterlitz, F. M. Leslie, Br. J. Pharmacol. 1975, 53: 371-381. [5.] Smith, C. B. NIDA Research Monograph. [76], 288: 294. 1987. Reference: series (book, monograph). [6.] G. Calo, A. Rizzi, G. Bogoni, V. Neugebauer, S. Salvadori, R. Guerrini, C. Bianchi, D. Regoli, Eur. J. Pharmacol.

1996, 311: R3-R5.

[7.] Kenakin, T. Pharmacological Analysis of Drug-Receptor Interaction. 2nd. 1993. USA UU., Raven Press. Reference:

series (book, monograph).

[8.] D. R. Kapusta, S. F. Sezen, J. K Chang, H. Lippton, V. A. Kenigs, Life Sci. 1997, 60: L15-L21.

[9.] D. R. Kapusta, Peptides 2000, 21: 1081-1099.

[10.] D. R. Kapusta, V. A. Kenigs, Am. J. Physiol. 1999, 277: R987-R995.

[11.] H. C. Champion, R. L. Pierce, P. J. Kadowitz, Regul. Pept 1998, 78: 69-74.

[12.] G. G. Liversidge, C. G. Wilson, W. L. Stemson, L. B. Kinter, J. Appl. Physiol 1988, 64: 377-383.

[13.] T. E. N. Jonassen, S. Christensen, J. S. Petersen, J. Am. Soc. Nephrol. 1997, 8: 19A.

[14.] S. Nielsen, J. Terris, D. Andersen, C. Ecelbarger, J. Frokiaer, T. Jonassen, D. Marples, M. A. Knepper, J. S.

Petersen, Proc. Natl Acad. Sci. U.S.A. 1997, 94: 5450-5455. [15.] G. F. DiBona, P. J. Herman, L L Sawin, Am. J. Physiol. 1988, 254: R1017-R1024. [16.] T. Bertani, A. Poggi, R. Pozzoni, F. Delaini, G. Sacchi, Y. Thoua, G. Mecca, G. Remuzzi, M. B. Donati, Lab.

Invest. 1982, 46: 16-23. [17.] J. Kountouras, B. H. Billing. P. J. Scheuer, Br. J. Exp. Pathol. 1984, 65: 305-311. [18.] D. R. Kapusta, V. A. Kenigs, L A. Dayan, A. W. Dreisbach, S. Meleg-Smith, V. Batuman, K. J. Varner, J. Am.

Soc. Nephrol. 2000, 11: A3175. [19.] T. Shirasaka, T. Kunitake, K Kato, M. Takasaki, H. Kannan, Am. J. Physiol. 1999, 277: R1025-R1032. [20.] D. R. Kapusta, J. K Chang, V. A. Kenigs, J. Pharmacol. Exp. Ther. 1999, 289: 173-180.

[21.] C. C. Lai, S. Y. Wu, C. T. Chen, N. J. Dun, Am. J. Physiol. Regul. Integr. Comp. Physiol 2000, 278: R592-R597.

[22.] J. D. Conger, M. F. Schultz, F. Miller, J. B. Robinette, Kidney Int. 1994, 46: 318-323.

[23.] D. de Rougemont, A. Oeschger, L. Konrad, G. Thiel, J. Torhorst, M. Wenk, P. Wunderlich, F. P. Brunner, Nephron 1981, 29: 176-184.

Peptide Preparation

[0112] The peptides or peptide conjugates herein are preferably prepared by peptide synthesis procedures, but can also be prepared by recombinant DNA technology with the general procedures and principles known to the person skilled in the art. Therefore, the present invention also relates to a nucleic acid sequence encoding a polypeptide sequence comprising the peptide sequence of formula I or III; a vector containing said nucleic acid sequence and a host cell that comprises said nucleic acid sequence and is capable of expressing said polypeptide sequence.

[0113] A nucleic acid sequence encoding the present peptide conjugate can be synthetically prepared by standard procedures established, for example, the phosphoramidite method described by S. L. Beaucage and M. H. Caruthers, Tetrahedron Letters 22, 1981, p. 1859-1869 or the procedure described by Matthes et al., EMBO Journal 3, 1984, p. 801-805. According to the phosphoramidite method, oligonucleotides are synthesized, for example, in an automatic DNA synthesizer, purified, hybridized, ligated and cloned into suitable vectors. The techniques used to isolate or clone a nucleic acid sequence encoding the X peptide are known in the art and include genomic DNA isolation, cDNA preparation or a combination thereof. The cloning of the nucleic acid sequences of the present invention from such genomic DNA can be carried out, for example, by means of the well-known polymerase chain reaction (PCR), or by screening of antibody expression libraries. to detect cloned DNA fragments with common structural characteristics. See, for example, Innis et al., 1990, A Guide to Methods and Application, Academic Press, New York. Other nucleic acid amplification procedures such as ligase chain reaction (LCR), ligation activated transcription (LAT) and nucleic acid sequence amplification (NASBA) can be used. It can then be linked to a nucleic acid sequence that encodes

Z.

[0114] The nucleic acid sequence encoding the conjugate is then inserted into a recombinant expression vector that can be any vector that can conveniently undergo recombinant DNA procedures. Frequently, the choice of the vector will depend on the host cell into which it is to be introduced. Therefore, the vector can be an autonomous replication vector, that is, a vector that exists as an extrachromosomal entity and whose replication is independent of chromosomal replication, for example, a plasmid. Alternatively, the vector may be a vector that when introduced into a host cell is integrated into the genome of said host cell and replicated together with the chromosome (s) into which it has been integrated. In the vector, the nucleic acid sequence encoding the conjugate of the present invention must be operatively connected to a suitable promoter sequence. The promoter can be any nucleic acid sequence that shows transcriptional activity in the chosen host cell and can be derived from genes encoding homologous or heterologous proteins to those of the host cell. Some examples of promoters suitable for directing the transcription of the nucleic acid sequence encoding the conjugate, especially in a bacterial host cell, are promoters obtained from the E. coli lac operon, the Streptomyces coelicolor agarase (dagA) gene. and the prokaryotic lacta-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 of recombinant bacteria" in Scientific American, 1980, 242: 74-94; and in Sambrook et al., 1989, see above. When the host is a yeast, useful promoters are obtained from the enochase gene (ENO-1) of Saccharomyces cerevisiae, the alcohol dehydrogenase and glyceraldheido-3-phosphate dehydrogenase (ADH2 / GAP) genes from Saccharomyces cerevisiae and Saccharomyces cerevisiae 3-phosphoglycerate kinase gene. Other promoters useful for yeast host cells are those described by Romanos et al., 1992, Yeast 8: 423-488.

[0115] The nucleic acid sequence encoding said conjugate can also be operatively connected to a suitable terminator, such as the human growth hormone terminator (Palmiter et al., Science 222, 1983, pp. 809-814). Preferred terminators for yeast host cells are obtained from the genes encoding Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1) and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase.

[0116] The vector may further comprise elements such as polyadenylation signals (for example, from SV 40 or adenovirus 5 region), transcriptional enhancer sequences (eg, SV 40 enhancer) and translational enhancer sequences (for example, those encoding adenovirus VA RNAs). Some polyadenylation sequences useful for yeast host cells are described in Guo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

[0117] The recombinant expression vector may further comprise a DNA sequence that allows replication of the vector in the host cell in question. Some examples of such a sequence (when the host cell is a mammalian cell) is the origin of SV 40 or polyoma virus replication. Some examples of bacterial replication origins are the origins of replication of plasmids pBR322, pUC19, pACYC177, pACYC184, pUB110, pE194, pTA1060 and pAMB1. Some examples of origins of replication for use in yeast host cells are the 2 µm plasmid replication origin, the combination of CEN6 and ARS4 and the combination of CEN3 and ARS1. The origin of replication may have a mutation to make its activity temperature sensitive in the host cell (see, for example, Ehrlich, 1978, Proc. Natl. Acad. Sci. USA 75: 1433).

[0118] The vector may also comprise a selectable marker, for example, a gene whose product complements a defect in the host cell, such as the gene encoding dihydrofolate reductase (DHFR) or a gene that confers resistance to a drug, by example, neomycin, geneticin, ampicillin or hygromycin. Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1 and URA3.

[0119] The methods used to link the nucleic acid sequences encoding the conjugate, promoter and terminator, respectively and to insert them into suitable vectors containing the information necessary for replication are well known to those skilled in the art ( see, for example, the cited work of Sambrook et al.).

[0120] The host cell into which the expression vector is introduced can be any cell capable of producing the conjugate and can be a eukaryotic cell, such as invertebrate or vertebrate cells, for example, Xenopus laevis oocytes or mammalian cells , in particular insect cells and mammalian cells. Some examples of suitable mammalian cell lines are COS, BHK or CHO cell lines. Methods for transfection of mammalian cells and the expression of DNA sequences introduced into cells are described, for example, in 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. Natl Acad. Sci. USA 79: 422-426; Wigler et al., 1978, Cell

14: 725; Corsaro and Pearson, 1981, Somatic Cell Genetics 7: 603; Graham and van der Eb, 1973, Virology 52: 456; Fraley et al., 1980, JBC 225: 10431; Capecchi, 1980, Cell 22: 479; Wiberg et al., 1983, NAR 11: 7287; Neumann et al., 1982, EMBO J. 1: 841-845.

[0121] The host cell may also be a unicellular pathogen, for example, a prokaryotic or a multicellular pathogen, for example, a eukaryotic. Useful unicellular cells are bacterial cells such as gram-positive bacteria, including, but not limited to Bacillus cells or Streptomyces cells, or gram-negative bacteria such as E. coli and Pseudomonas sp. The transformation of a bacterial host cell can be effected, for example, by transformation of protoplasts (see, for example, Chang and Cohen, 1979, Molecular General Genetics 168: 111-115), by the use of competent cells (see, for example , Young and Spizizin, 1961, Journal of Bacteriology 81: 823-829 or Dubnar and Davidoff Abelson, 1971, Journal of Molecular Biology 56: 209221), by electroporation (see for example, Shigekawa and Dower, 1988, Biotechniques 6: 742- 751) or by conjugation (see, for example, Koehler and Thorne, 1987, Journal of Bacteriology 169: 5771-5278).

[0122] The host cell may be a fungal cell, for example, Neurospora, Eupenicillium (= Penicillium), Emericella (= Aspergillus), Eurotium (= Aspergillus). The fungal host cell can also be a yeast cell. "Yeast," as used herein, includes yeasts that produce ascospores (endomycetes), yeasts that produce basidiospores, and yeasts that belong to imperfect fungi (blastomycetes). The medium used to grow the cells can be any conventional medium suitable for the growth of mammalian cells, such as a serum or serum-free medium containing the appropriate supplements or a suitable medium for the growth of insect, yeast or fungal cells. Suitable media can be obtained from commercial suppliers or can be prepared according to published formulas (for example, in the catalogs of the American Type Culture Collection).

[0123] Therefore, the invention also relates to a process for the production of peptide conjugates of the formula I or III having a natural polypeptide sequence comprising:

the introduction of a nucleic acid sequence encoding a polypeptide sequence comprising the peptide sequence of formula I or III and a selectable marker contained within a nucleic acid construct or a vector, in a host cell to obtain a recombinant host cell ;

the selection of said recombinant host cell;

the culture of said recombinant host cells under conditions that allow the production of said polypeptide sequence;

the isolation of said polypeptide sequence from the culture;

optionally the cleavage of said polypeptide sequence by means of a suitable protease to obtain said peptide conjugate.

[0124] The peptide conjugate of the formula I or III with a natural polypeptide sequence prepared in this manner can be recovered after the culture medium by conventional procedures including separation of the host cells from the medium by centrifugation or filtration, precipitation of the Protein components of the supernatant or filtered by means of a salt, for example, ammonium sulfate, purification by various chromatographic procedures, for example, ion exchange chromatography, affinity chromatography or the like. Subsequently, chemical modification steps can be used to obtain unnatural derivatives.

[0125] The peptides of the invention can be prepared by methods known per se in the art. Therefore, peptide sequences X and Z can be prepared by standard peptide preparation techniques, such as solution synthesis or solid phase synthesis of the Merrifield type. Both the Boc (tert-butyloxycarbonyl) strategy and the Fmoc (9-fluoroenylmethyloxycarbonyl) strategy can be applied.

[0126] Preferred synthesis procedures include a process for the preparation of a peptide conjugate of formula IV (X-Z ’) comprising the steps of:

a) coupling of an amino acid or dipeptide with the appropriate protecting groups, including an Nα protecting group, in the form activated with an immobilized H-Z’-SSM peptide sequence, in which an immobilized Nα protected peptide fragment is formed,

b) removal of said Nα protecting group, whereby a protected and immobilized peptide fragment with an unprotected N-terminus is formed,

c) coupling of an additional amino acid or dipeptide with the appropriate protecting groups, including a Nα protecting group, in the form with the activated carboxyl group at the N-terminus of the immobilized peptide fragment and repetition of the process of elimination and coupling of steps b) and c) until obtaining the desired peptide sequence X and then

d) excision of the peptide conjugate from the solid support material;

and a process for the preparation of a peptide conjugate of the formula V (Z-X) comprising the steps of:

a) coupling of an amino acid or dipeptide with suitable protecting groups, including a protective group of Nα, in the activated form to a solid support material (SSM), thereby forming a protected amino acid or a protected immobilized dipeptide,

b) elimination of said Nα protecting group, whereby an amino acid or an immobilized peptide fragment with an unprotected N-terminus is formed,

c) coupling of an additional amino acid or dipeptide with suitable protecting groups, including a Nα protecting group, in the form with the activated carboxyl group to the N-terminus of the immobilized amino acid or peptide fragment and repeating the step removal and coupling procedure b) and c) until the desired peptide sequence X is obtained,

d) coupling of an additional amino acid or dipeptide with the appropriate protecting groups, including a Nα protecting group, in the form with the activated carboxyl group at the N-terminus of the immobilized peptide fragment and repetition of the process of elimination and coupling of steps b) and d) until obtaining the desired peptide sequence Z and then

d) excision of the peptide conjugate from the solid support material,

and, in addition, a process for the preparation of a peptide conjugate of the formula VI (Z-X-Z ’) comprising the steps of:

a) coupling of an amino acid or dipeptide with suitable protecting groups, including a protective group of Nα, in the form with the activated carboxyl group to an immobilized peptide sequence H-Z'-SSM, whereby a protected peptide fragment is formed in Nα,

b) removal of said in the Nα protecting group, whereby an immobilized peptide fragment with an unprotected N-terminus is formed,

c) coupling of an additional amino acid or dipeptide with the appropriate protecting groups, including a Nα protecting group, in the form with the activated carboxyl group at the N-terminus of the immobilized peptide fragment and repetition of the process of elimination and coupling of steps b) and c) until obtaining the desired peptide sequence X, and then

d) coupling of an additional amino acid or dipeptide with the appropriate protecting groups, including a Nα protecting group, in the form with the activated carboxyl end to the N-terminus of the immobilized peptide fragment and repetition of the process of elimination and coupling of steps b) and d) until obtaining the desired peptide sequence Z and then

d) excision of the peptide conjugate from the solid support material,

Experimental procedures

Devices and synthesis strategy

[0127] The peptides were synthesized in batches in a polyethylene container equipped with a polypropylene filter for filtration, with 9-fluoroenylmethyloxycarbonyl (Fmoc) as a protective group of the Nα-amino group and with common protective groups suitable for chain functionalities lateral.

Solvents

[0128] The DMF solvent (N, N-dimethylformamide, Riedel-de Häen, Germany) was purified by passage through a column filled with a strong cation exchange resin (Lewatit S 100 MB / H strong acid, Bayer AG , Leverkusen, Germany) and the presence of free amines was analyzed before use by the addition of 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (Dhbt-OH), which produces a yellow color (Dhbt-O- anion) if free amines are present. The DCM solvent (dichloromethane, analytical grade, Riedel-de Häen, Germany) was used directly without purification. Acetonitrile (quality for HPLC, Lab-Scan, Dublin, Ireland) was used directly without purification.

Amino acids

[0129] Fmoc protected amino acids were purchased from Advanced Chem Tech (ACT) in suitable forms with protected side chains.

Coupling reagents

[0130] Diisopropylcarbodiimide coupling reagent (DIC) was purchased from Riedel-de Häen, Germany.

Solid supports

[0131] Peptides were synthesized on TentaGel S resins at 0.22-0.31 mmol / g. TentaGel S-Ram, TentaGel S RAM-Lys (Boc) Fmoc (Rapp Polymere, Germany) were used in cases where an amidated peptide at the C-terminus was preferred, while TentaGel S PHB, TentaGel S PHB Lys (Boc ) Fmoc were used when a free carboxylic acid at the C-terminus was preferred.

Catalysts and other reagents

[0132] Diisopropylethylamine (DIEA) was purchased from Aldrich, Germany, piperidine and pyridine from Riedel-de Häen, Frankfurt, Germany. Ethanedithiol was purchased from Riedel-de Häen, Frankfurt, Germany. The compounds 3,4dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (Dhbt-OH) and 1-hydroxybenzotriazole (HOBt) (HOAt) were obtained from Fluka, Switzerland. Acetic anhydride was obtained from Fluka.

Coupling Procedures

[0133] Amino acids were coupled as HObt or HOAt esters generated in situ, prepared from the appropriate amino acids protected in Nα and HObt or HOAt by means of DIC in DMF. Acylations were checked by the ninhydrin test at 80 ° C to prevent Fmoc deprotection during the test (Larsen,

B. D. and Holm, A., Int. J. Peptide Protein Res. 43, 1994, 1-9).

Deprotection of the protective group of the Nα-amino group (Fmoc)

[0134] Fmoc group deprotection was performed by treatment with 20% piperidine in DMF (1 x 5 and 1 x 10 min), followed by washing with DMF (5 x 15 ml, 5 min each time) until it stopped yellow color detected after the addition of Dhbt-OH to the drained DMF.

Hobs ester coupling

[0135] A quantity of 3 eq of amino acid protected in Nα was dissolved in DMF together with 3 eq of HObt and 3 eq of DIC and then added to the resin.

Acetylation of the N-terminal amino group with acetic anhydride

[0136] An amount of 40 eq of acetic anhydride was dissolved in DMF together with 5 eq of pyridine and then added to the resin.

[0137] Acylation was checked by the ninhydrin test, as described above.

Trifluoroacetylation of the N-terminal amino group with ethyl trifluoroacetate

[0138] A quantity of 30 eq of ethyl trifluoroacetate was dissolved in dichloromethane together with 10 eq of triethylamine and then added to the resin. Acylation was checked by the ninhydrin test, as described above.

Excision of resin peptide with acid

[0139] The peptide was cleaved from the resins by treatment with 95% trifluoroacetic acid (TFA, Riedel-de Häen, Frankfurt, Germany) in water (v / v) or with 95% TFA and 5% ethanedithiol (v / v) at room temperature for 2 h. The filtered resins were washed with 95% TFA in water and the filtrates and the washings evaporated under reduced pressure. The residue was washed with ether and lyophilized from acetic acid-water. The crude lyophilized product was analyzed by high efficiency liquid chromatography (HPLC) and identified by mass spectrometry (MS).

Synthesis of peptides in batches on TentaGel resin (PEG-PS)

[0140] TentaGel resin (1 g, 0.23-0.24 mmol / g) was placed in a polyethylene container equipped with a polypropylene filter for filtration. The resin was expanded in DMF (15 ml) and treated with 20% piperidine in DMF in order to remove the initial Fmoc group, either in the TentaGel RAM linker or in the first amino acid in the TentaGel S RAM-Lys resin ( Boc) Fmoc. The resin was drained and washed with DMF until yellow color ceased to be detected after the addition of Debt-OH to the drained DMF. The amino acids according to the sequence were coupled as preformed HObt esters, protected with Fmoc (3 eq) as described above. Couplings continued for 2 h, unless otherwise specified. The resin was drained and washed with DMF (5 x 15 ml, 5 min each time) to remove excess reagents. All acylations were checked by the ninhydrin test as described above. After completing the synthesis, the resin with the peptide was washed with DMF (3 x 15 ml, 5 min each time), DCM (3 x 15 ml, 1 min each time) and finally with diethyl ether (3 x 15 ml, 1 min each time) and dried under vacuum. The peptide was cleaved from the resin as described above and the crude peptide product was purified and analyzed as described below.

HPLC conditions

[0141] HPLC gradient analysis was performed using an HP 1100 Hewlett Packard HPLC system, consisting of an HP 1100 quaternary pump, an HP 1100 autosampler, an HP 1100 thermostat column and an HP multiple wavelength detector 1100. Hewlett Packard Chemstation software for LC (rev. A.06.01) was used for instrumental control and data acquisition. The following column and buffer system for HPLC were used: Column: VYDAC 238TP5415, C-18, 5 mm, 300 Å, 150 x 4.6 mm.

Buffer: A: 0.1% TFA in MQW; B: 0.085% TFA in MQW, 10% MQW, 90% MeCN.

Gradient: 0-1.5 min, 0% of B

1.5-25 min, 50% B

25-30 min, 100% B

30-35 min, 100% B

   35-40 min, 0% of B

Flow: 1 ml / min, oven temperature: 40 ° C, UV detection: I = 215 nm

HPLC crude peptide purification

[0142] The crude peptide products were purified by a PerSeptive Biosystems VISION Workstation system. VISION 3.0 software was used for instrumental control and data acquisition. The following column and HPLC buffer system were used:

Column: Kromasil KR 100 Å, 10 mm C-8, 250 x 50.8 mm.

Buffer system: buffers: A: 0.1% TFA in MQW; B: 0.085% TFA in 10% of MQW, 90% of MeCN.

Gradient: 0-37 min, 0-40% of B

Flow: 35 ml / min, UV detection: l = 215 nm and 280 nm. Mass spectroscopy

[0143] Peptides were dissolved in higher grade grade methanol (Labscan, Dublin, Ireland), Milli-Q water (Millipore, Bedford, MA, USA) and formic acid (Merck, Darmstadt, Germany) (50 : 50: 0.1, v / v / v) to give concentrations between 1 and 10 mg / ml. Peptide solutions (20 ml) were analyzed in the positive polarity mode by ESI-TOF-MS using an LCT mass spectrometer (Micromass, Manchester, UK).

Preparation and use of antibodies

[0144] The antibodies of the invention can be prepared by techniques generally known in the field. Preferred antibodies are generated against a purified sample of a peptide or peptide conjugate as an antigen. Peptides or peptide conjugates suitable for generating such antibodies are disclosed throughout the application, including examples 22-23, below. Although most polyclonal antibodies of the invention exhibit exceptionally good and specific binding, for some applications the use of monoclonal antibodies may be equally useful or more useful. See generally Harlow, E. and D. Lane in Antibodies: A Laboratory Manual, Cold Spring Harbor, NY, USA. UU. (description of procedures for the production and use of polyclonal and monoclonal antibodies).

[0145] Generally, antibodies can be prepared by immunization of a mammal with a purified or semi-purified sample of a peptide or peptide conjugate as an antigen, as provided herein, alone or complexed with a vehicle. Suitable mammals include typical laboratory animals such as sheep, goats, rabbits, guinea pigs, rats and mice. Rats and mice, especially mice are preferred for obtaining monoclonal antibodies. The antigen can be administered to the mammal by any of a number of suitable routes, such as subcutaneous, intraperitoneal, intravenous, intramuscular or intracutaneous injection. The optimal immunization interval, immunization dose, etc. they can vary in relatively wide intervals and can be determined empirically, based on this description. Typical procedures involve the injection of the antigen several times over a series of months. The antibodies are collected from the serum of the animal immunized by standard techniques and are screened to find the specific antibodies for the peptide or peptide conjugate used as an antigen. Monoclonal antibodies can be produced in cells that produce antibodies and these cells can be used to generate monoclonal antibodies by standard fusion techniques for hybridoma cell formation. See G. Kohler et al., Nature, 256: 456 (1975). Typically, this involves the fusion of an antibiotic producing cell with an immortal cell line such as myeloma cells to produce a hybrid cell. Alternatively, monoclonal antibodies can be produced from cells by the method of Huse et al., Science, 256: 1275 (1989).

[0146] See also Harlow, E. and D. Lane, above, for additional information regarding the production and use of polyclonal and monoclonal antibodies. Various suitable strategies for the purification of antibodies that can be used according to this invention have been described.

[0147] In embodiments in which monoclonal antibodies are desired, a suitable protocol provides immunization of a mouse intraperitoneally with a composition comprising a purified peptide conjugate for a period of approximately two to seven months. Then, cells from the immunized mouse spleen can be removed. The immunized mouse serum is analyzed to determine the specific antibody titers for a particular peptide conjugate prior to excision of the spleen cells. The excised cells of the mouse spleen are then fused with an appropriate homogenous (preferably homogeneous) lymphocytic line that has a marker such as hypoxanthine-guanine phosphoribosyltransferase deficiency (HGPRT) or thymidine kinase (TK-) deficiency. Preferably, myeloma cells are used as lymphocytic cell line. Myeloma cells and spleen cells are mixed, for example in a ratio between myeloma cells and spleen cells 1 to 4. The cells can be fused by the polyethylene glycol (PEG) procedure. See G. Kohler et al., Nature, above. The hybridoma thus cloned is grown in a culture medium, for example RPMI-1640. See G. E. More et al., Journal of American Medical Association, 199: 549 (1967). Hybridomas grown after the fusion procedure are screened by radioimmunoassay or by enzyme immunoassay to detect the secretion of antibodies that specifically bind to the antigen used, for example, antibodies that bind to the purified peptide conjugate are selected, but not other unrelated control peptides. Preferably, ELISA or a related enzyme immunoassay is used for screening. Hybridomas that show positive results in said screening can be amplified and cloned by the limited dilution procedure. Preferably other screening is performed to select the antibodies that bind to the peptide conjugate in solution. Isolated antibodies can be subsequently purified by virtually any suitable immunological technique, including affinity chromatography.

[0148] The molecular weight of the antibodies of the invention may vary depending on several factors, such as the intended use and whether the antibody includes a toxin, a pharmaceutical substance or a detectable label or similar recombinantly conjugated or fused. In general, an antibody of the invention will have a molecular weight between about 20 and 150 kDa. Such molecular weights can be easily determined by molecular size determination procedures such as SDS-PAGE gel electrophoresis, followed by protein staining or by immunoblot analysis.

[0149] Preferred peptide conjugates for the production of polyclonal antibodies of the invention are disclosed, for example, in Examples 22-23.

[0150] With the expression "specific binding" or a related expression referring to the association between an antibody and a peptide or peptide conjugate as an antigen is meant that the antibody forms an immune complex with the particular antigen and not with other antigens (as conjugates related and unrelated peptides). Methods for detecting and optionally quantifying such specific binding include standard enzyme immunoassays, for example ELISA and antibody capture and antigen capture assays. Preferred antibodies of the invention specifically bind a peptide or peptide conjugate bound as an antigen. See, for example, example 23 below.

[0151] The following examples are provided to indicate preferred aspects of the invention and are not intended to be indicative of the scope of the invention.

Synthesis Examples

Example 1. Synthesis of compound 1: Ac-Arg-Tyr-Tyr-Arg-Trp-Lys-Lys-Lys-Lys-Lys-Lys-Lys-NH2 in TentaGel S RAM-Lys (Boc) Fmoc.

[0152] TentaGel S RAM-Lys (Boc) dry Fmoc resin (0.24 mmol / g, 1 g) was placed in a polyethylene container equipped with a polypropylene filter for filtration and treated as described in section "Synthesis of peptides in batches on the TentaGel resin" until the end of the coupling of the N-terminal arginine. All links were continued overnight. After deprotection of the Fmoc group, the N-terminal amino group was acetylated as described above. The coupling was continued overnight. Acylations were checked as described above. After completing the synthesis, the peptide was cleaved from the resin as described above. The yield of crude product was 390 mg. After purification by preparative HPLC, as described above, 210 mg of peptide product with a purity greater than 95% was collected and the identity of the peptide was confirmed by MS (M found 1,780.25, M calculated 1,780.11) .

Example 2. Synthesis of compound 1A: Ac-Arg-Tyr-Tyr-Arg-Trp-Lys-Lys-Lys-Lys-Lys-Lys-Lys-NH2 x 9 AcOH (acetate). Exchange of the trifluoroacetate counterion for acetate in compound 1.

[0153] The purified synthetic peptide product of compound 1 is isolated as trifluoroacetate, Ac-Arg-TyrTyr-Arg-Trp-Lys-Lys-Lys-Lys-Lys-Lys-Lys-NH2 x 9 TFA, due to the presence of trifluoroacetic acid (0.1% v / v) in buffers used in HPLC for purification of the crude synthetic peptide product.

[0154] In order to exchange the trifluoroacetate counterion for acetate, a solution of the peptide was passed through a column filled with a strong base ion exchange resin in the acetate (Dowex 1 x 8).

[0155] An amount of 1 g of compound 1 is dissolved in 40 ml of water. The solution is passed through a column containing 40 ml of a strong base ion exchange resin in the acetate (Dowex 1 x 8, capacity 1.33 meq / ml resin). The resin was then washed with 4 x 30 ml of water and the eluate was collected and lyophilized, with a result of 792 mg of acetate (hygroscopic) with a purity of 98% according to the HPLC analysis.

Example 3. Synthesis of compound 1C: Ac-Arg-Tyr-Tyr-Arg-Trp-Lys-Lys-Lys-Lys-Lys-Lys-Lys-NH2 x 9 HCl (chloride). Exchange of the trifluoroacetate (tfa) counterion by chloride (Cl-) in compound 1.

[0156] A 100 mg amount of compound 1 was dissolved in 50 ml of 0.1 M hydrochloric acid and the resulting solution was lyophilized. The rest was dissolved in 50 ml of water and lyophilized again, with a result of 74 mg of chloride with a purity of 97% according to HPLC.

Example 4: Synthesis of compound 2: Ac-Lys-Lys-Lys-Lys-Lys-Lys-Arg-Tyr-Tyr-Arg-Trp-Lys-NH2 in TentaGel S RAM-Lys (Boc) Fmoc. [0157] TentaGel S RAM-Lys (Boc) dry Fmoc resin (0.24 mmol / g, 1 g) was placed in a polyethylene container equipped with a polypropylene filter for filtration and treated as described in section "Synthesis of peptides in batches on the TentaGel resin" until the end of the coupling of the N-terminal lysine. All links were continued overnight. After deprotection of the Fmoc group, the N-terminal amino group was acetylated as described above. The coupling was continued overnight. Acylations were checked as described above. After completing the synthesis, the peptide was cleaved from the resin as described above. The yield of crude product was 231 mg. After purification by preparative HPLC, as described above, 126 mg of peptide product with a purity greater than 93% was collected and the identity of the peptide was confirmed by MS (M found 1,780.25, M calculated 1,780.11) .

Example 5. Synthesis of compound 3: H-Asn-Glu-Glu-Glu-Glu-Glu-Arg-Tyr-Tyr-Arg-Trp-Lys-Lys-Lys-Lys-Lys-Lys-LysNH2 in TentaGel S RAM- Lys (Boc) Fmoc.

[0158] TentaGel S RAM-Lys (Boc) dry Fmoc resin (0.24 mmol / g, 1 g) was placed in a polyethylene container equipped with a polypropylene filter for filtration and treated as described in section "Synthesis of peptides in batches on the TentaGel resin" until the end of the coupling of the N-terminal asparagine. All links were continued overnight. Acylations were checked as described above. After completing the synthesis, the peptide was cleaved from the resin as described above. The yield of crude product was 212 mg. After purification by preparative HPLC, as described above, 112 mg of peptide product with a purity greater than 98% was collected and the identity of the peptide was confirmed by MS (M found 2,497.25, M calculated 2,497.35) .

Example 6. Synthesis of compound 4: Ac-Arg-Tyr-Asn-Arg-Trp-Lys-Lys-Lys-Lys-Lys-Lys-Lys-NH2 in TentaGel S RAM-Lys (Boc) Fmoc.

[0159] The TentaGel S RAM-Lys (Boc) dry Fmoc resin (0.24 mmol / g, 1 g) was placed in a polyethylene container equipped with a polypropylene filter for filtration and treated as described in section "Synthesis of peptides in batches on the TentaGel resin" until the end of the coupling of the N-terminal arginine. All links were continued overnight. After deprotection of the Fmoc group, the N-terminal amino group was acetylated as described above. The coupling was continued overnight. Acylations were checked as described above. After completing the synthesis, the peptide was cleaved from the resin as described above. The yield of crude product was 502 mg. After purification by preparative HPLC, as described above, 272 mg of peptide product with a purity greater than 98% was collected and the identity of the peptide was confirmed by MS (M found 1.731.25, M calculated 1.731.09) .

Example 7. Synthesis of compound 5: Ac-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Trp-Arg-Tyr-Tyr-Asn-NH2 in TentaGel S RAM.

[0160] The dry TentaGel S RAM resin (0.24 mmol / g, 1 g) was placed in a polyethylene container equipped with a polypropylene filter for filtration and treated as described in the section “Synthesis of batch peptides on the TentaGel resin ”until the coupling of the N-terminal lysine is finished. All links were continued overnight. After deprotection of the Fmoc group, the N-terminal amino group was acetylated as described above. The coupling was continued overnight. Acylations were checked as described above. After completing the synthesis, the peptide was cleaved from the resin as described above. The yield of crude product was 586 mg. After purification by preparative HPLC, as described above, 365 mg of peptide product with a purity greater than 99% was collected and the identity of the peptide was confirmed by MS (M found 1.738.0, M calculated 1.738.05) .

Example 8. Synthesis of compound 6: Ac-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Trp-Arg-Tyr-Tyr-Arg-NH2 in TentaGel S RAM.

[0161] The dry TentaGel S RAM resin (0.23 mmol / g, 1 g) was placed in a polyethylene container equipped with a polypropylene filter for filtration and treated as described in the section “Synthesis of batch peptides on the TentaGel resin ”until the coupling of the N-terminal lysine is finished. All links were continued overnight. After deprotection of the Fmoc group, the N-terminal amino group was acetylated as described above. The coupling was continued overnight. Acylations were checked as described above. After completing the synthesis, the peptide was cleaved from the resin as described above. The yield of crude product was 437 mg. After purification by preparative HPLC, as described above, 257 mg of peptide product with a purity greater than 98% was collected and the identity of the peptide was confirmed by MS (M found 1,780.0, M calculated 1,780.11) . Example 9. Synthesis of compound 7: Ac-D-Lys-D-Lys-D-Lys-D-Lys-D-Lys-D-Lys-D-Lys-D-Trp-D-Arg-D-Tyr- D-TyrDArg-NH2 in TentaGel S RAM.

[0162] The dry TentaGel S RAM resin (0.23 mmol / g, 1 g) was placed in a polyethylene container equipped with a polypropylene filter for filtration and treated as described in the section “Synthesis of batch peptides on the TentaGel resin ”until the coupling of the N-terminal D-lysine is finished. All links were continued overnight. After deprotection of the Fmoc group, the N-terminal amino group was acetylated as described above. The coupling was continued overnight. Acylations were checked as described above. After completing the synthesis, the peptide was cleaved from the resin as described above. The yield of crude product was 410 mg. After purification by preparative HPLC, as described above, 263 mg of peptide product with a purity greater than 98% was collected and the identity of the peptide was confirmed by MS (M found 1,780.13, M calculated 1,780.11) .

Example 10. Synthesis of compound 8: Ac-D-Arg-D-Tyr-D-Tyr-D-Arg-D-Trp-D-Lys-D-Lys-D-Lys-D-Lys-D-Lys- D-LysD-Lys-NH2 in TentaGel S RAM.

[0163] The dry TentaGel S RAM resin (0.23 mmol / g, 1 g) was placed in a polyethylene container equipped with a polypropylene filter for filtration and treated as described in the section “Synthesis of batch peptides on the TentaGel resin ”until final coupling of the N-terminal D-arginine. All links were continued overnight. After deprotection of the Fmoc group, the N-terminal amino group was acetylated as described above. The coupling was continued overnight. Acylations were checked as described above. After completing the synthesis, the peptide was cleaved from the resin as described above. The yield of crude product was 419 mg. After purification by preparative HPLC, as described above, 205 mg of peptide product with a purity greater than 97% was collected and the identity of the peptide was confirmed by MS (M found 1,780.0, M calculated 1,780.11) .

Example 11. Synthesis of compound 9: Ac-Lys-Tyr-Tyr-Arg-Trp-Lys-Lys-Lys-Lys-Lys-Lys-Lys-NH2 in TentaGel S RAM-Lys (Boc) Fmoc.

[0164] TentaGel S RAM-Lys (Boc) Fmoc dry resin (0.24 mmol / g, 1 g) was placed in a polyethylene container equipped with a polypropylene filter for filtration and treated as described in section "Synthesis of peptides in batches on the TentaGel resin" until the end of the coupling of the N-terminal lysine. All links were continued overnight. After deprotection of the Fmoc group, the N-terminal amino group was acetylated as described above. The coupling was continued overnight. Acylations were checked as described above. After completing the synthesis, the peptide was cleaved from the resin as described above. The yield of crude product was 445 mg. After purification by preparative HPLC, as described above, 287 mg of peptide product with a purity greater than 96% was collected and the identity of the peptide was confirmed by MS (M found 1,752.0, M calculated 1,752.1) .

Example 12. Synthesis of compound 10: Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-Lys-Lys-Lys-Lys-Lys-Lys-NH2 in TentaGel S RAM-Lys (Boc) Fmoc.

[0165] TentaGel S RAM-Lys (Boc) Fmoc dry resin (0.24 mmol / g, 1 g) was placed in a polyethylene container equipped with a polypropylene filter for filtration and treated as described in section "Synthesis of peptides in batches on the TentaGel resin" until the end of the coupling of the N-terminal arginine. All 5 links were continued overnight. After deprotection of the Fmoc group, the N-terminal amino group was acetylated as described above. The coupling was continued overnight. Acylations were checked as described above. After completing the synthesis, the peptide was cleaved from the resin as described above. The yield of crude product was 376 mg. After purification by preparative HPLC, as described above, 134 mg of

10 peptide product with a purity greater than 97% and the identity of the peptide was confirmed by MS (M found 1,707.0, M calculated 1,707.11).

Example 13. Synthesis of compound 11: Ac-Arg-Tyr-Tyr-Arg-Trp-Lys-Ala-Lys-Lys-Lys-Lys-Lys-NH2 in TentaGel S RAM-Lys (Boc) Fmoc.

[0166] TentaGel S RAM-Lys (Boc) dry Fmoc resin (0.24 mmol / g, 1 g) was placed in a polyethylene container equipped with a polypropylene filter for filtration and treated as described in section "Synthesis of peptides in batches on the TentaGel resin" until the end of the coupling of the N-terminal arginine. All links were continued overnight. After deprotection of the Fmoc group, the amino group N

The terminal was acetylated as described above. The coupling was continued overnight. Acylations were checked as described above. After completing the synthesis, the peptide was cleaved from the resin as described above. The yield of crude product was 429 mg. After purification by preparative HPLC, as described above, 260 mg of peptide product with a purity greater than 96% was collected and the identity of the peptide was confirmed by MS (M found

25 1,723.0, M calculated 1,723.05).

Example 14. Synthesis of compound 12: Ac-Arg-Tyr-Tyr-Arg-Trp-Lys-Lys-Lys-Lys-Lys-Lys-NH2 in TentaGel S RAMLys (Boc) Fmoc.

[0167] TentaGel S RAM-Lys (Boc) Fmoc dry resin (0.24 mmol / g, 1 g) was placed in a polyethylene container equipped with a polypropylene filter for filtration and treated as described in section "Synthesis of peptides in batches on the TentaGel resin" until the end of the coupling of the N-terminal arginine. All links were continued overnight. After deprotection of the Fmoc group, the N-terminal amino group was acetylated as described above. The coupling was continued overnight. The

35 acylations were checked as described above. After completing the synthesis, the peptide was cleaved from the resin as described above. The yield of crude product was 426 mg. After purification by preparative HPLC, as described above, 266 mg of peptide product with a purity greater than 98% was collected and the identity of the peptide was confirmed by MS (M found 1,651.88, M calculated 1,652.01) .

Example 15. Synthesis of compound 13: Ac-Arg-Tyr-Tyr-Arg-Trp-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Cys-NH2 in TentaGel S RAM.

[0168] The dry TentaGel S RAM resin (0.24 mmol / g, 1 g) was placed in an equipped polyethylene container

45 with a polypropylene filter for filtration and treated as described in the section "Synthesis of batch peptides on the TentaGel resin" until final coupling of the N-terminal arginine. All links were continued overnight. After deprotection of the Fmoc group, the N-terminal amino group was acetylated as described above. The coupling was continued overnight. Acylations were checked as described above. After completing the synthesis, the peptide was cleaved from the resin as

50 described above. The yield of crude product was 465 mg. After purification by preparative HPLC, as described above, 313 mg of peptide product with a purity greater than 91% was collected and the identity of the peptide was confirmed by MS (M found 1,883.0, M calculated 1,883.12) .

Example 16. Synthesis of compound 15: Ac-Arg-Tyr-Tyr-Arg-Trp-Lys-Cys-NH2 in TentaGel S RAM.

[0169] The dry TentaGel S RAM resin (0.24 mmol / g, 1 g) was placed in a polyethylene container equipped with a polypropylene filter for filtration and treated as described in the section “Synthesis of peptides in batches on the TentaGel resin ”until the coupling of the N-terminal arginine is finished. All links were continued overnight. After deprotection of the Fmoc group, the N-terminal amino group was acetylated.

60 as described above. The coupling was continued overnight. Acylations were checked as described above. After completing the synthesis, the peptide was cleaved from the resin as described above. The yield of crude product was 249 mg. After purification by preparative HPLC, as described above, 190 mg of peptide product with a purity greater than 94% was collected and the identity of the peptide was confirmed by MS (M found 1,114.50, M calculated 1,114.55) .

Example 17. Synthesis of compound 16: H-Cys-Ala-Pro-Pro-Ser-Lys-Lys-Lys-Lys-Lys-Lys-NH2 in TentaGel S RAMLys (Boc) Fmoc.

[0170] TentaGel S RAM-Lys (Boc) dry Fmoc resin (0.24 mmol / g, 1 g) was placed in a polyethylene container equipped with a polypropylene filter for filtration and treated as described in section "Synthesis of peptides in batches on the TentaGel resin" until final coupling of the N-terminal cysteine. All links were continued overnight. Acylations were checked as described above. After the final deprotection of the Fmoc group, the peptide was cleaved from the resin as described above. The yield of crude product was 359 mg. After purification by preparative HPLC, as described above, 242 mg of peptide product with a purity greater than 98% was collected and the identity of the peptide was confirmed by MS (M found 1,240.76, M calculated 1,240.78) .

Example 18. Synthesis of compound 17: H-Cys-Lys-Lys-Lys-Lys-Lys-Lys-NH2 in TentaGel S RAM-Lys (Boc) Fmoc.

[0171] TentaGel S RAM-Lys (Boc) dry Fmoc resin (0.24 mmol / g, 1 g) was placed in a polyethylene container equipped with a polypropylene filter for filtration and treated as described in section "Synthesis of peptides in batches on the TentaGel resin" until final coupling of the N-terminal cysteine. All links were continued overnight. Acylations were checked as described above. After the final deprotection of the Fmoc group, the peptide was cleaved from the resin as described above. The yield of crude product was 287 mg. After purification by preparative HPLC, as described above, 205 mg of peptide product with a purity greater than 90% was collected and the identity of the peptide was confirmed by MS (M found 888.5, M calculated 888.61) .

Example 19. Synthesis of compound 18: Ac-Arg-Tyr-Tyr-Arg-Trp-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Cys-NH2 in TentaGel S RAM.

[0172] The dry TentaGel S RAM resin (0.24 mmol / g, 1 g) was placed in a polyethylene container equipped with a polypropylene filter for filtration and treated as described in the section “Synthesis of batch peptides on the TentaGel resin ”until the coupling of the N-terminal arginine is finished. All links were continued overnight. After deprotection of the Fmoc group, the N-terminal amino group was acetylated as described above. The coupling was continued overnight. Acylations were checked as described above. After completing the synthesis, the peptide was cleaved from the resin as described above. The yield of crude product was 465 mg. After purification by preparative HPLC, as described above, 313 mg of peptide product with a purity greater than 91% was collected and the identity of the peptide was confirmed by MS (M found 1,883.00, M calculated 1,883.12) .

Example 20. Synthesis of compound 14: Tfa-Arg-Tyr-Tyr-Arg-Trp-Lys-Lys-Lys-Lys-Lys-Lys-Lys-NH2 in TentaGel S RAM-Lys (Boc) Fmoc.

[0173] TentaGel S RAM-Lys (Boc) Fmoc dry resin (0.24 mmol / g, 1 g) was placed in a polyethylene container equipped with a polypropylene filter for filtration and treated as described in section "Synthesis of peptides in batches on the TentaGel resin" until the end of the coupling of the N-terminal arginine. All links were continued overnight. After deprotection of the Fmoc group, the N-terminal amino group was trifluoroacetyl as described above. The reaction was continued overnight. Acylations were checked as described above. After completing the synthesis, the peptide was cleaved from the resin as described above. The yield of crude product was 392 mg. After purification by preparative HPLC, as described above, 105 mg of peptide product with a purity greater than 96% was collected and the identity of the peptide was confirmed by MS (M found 1834.25, M calculated 1.834.08) .

Example 21: Synthesis of compound CE1: Ac-Arg-Tyr-Tyr-Arg-Trp-Lys-NH2 in TentaGel S RAM-Lys (Boc) Fmoc.

[0174] TentaGel S RAM-Lys (Boc) Fmoc dry resin (0.24 mmol / g, 1 g) was placed in a polyethylene container equipped with a polypropylene filter for filtration and treated as described in section "Synthesis of peptides in batches on the TentaGel resin" until the end of the coupling of the N-terminal arginine. All links were continued overnight. After deprotection of the Fmoc group, the N-terminal amino group was acetylated as described above. The coupling was continued overnight. Acylations were checked as described above. After completing the synthesis, the peptide was cleaved from the resin as described above. The yield of crude product was 143 mg. After purification by preparative HPLC, as described above, 52 mg of peptide product with a purity greater than 97% was collected and the identity of the peptide was confirmed by MS (M found 1.011.54, M

5 calculated 1,011.54).

Example 22. Production of immunogens.

[0175] Specific antibodies against compound 1 have been generated by conjugation of the peptides

10 corresponding to the N end (Ac-RYYRWKC-NH2), the end (H-CAPPSKKKKKK-NH2) and compound 13, which is a full length molecule with a Cys terminal moiety to adapt to coupling chemistry (AcRYYRV11KKKKKKKGNH2). Ac-RYYRWKC-NH2 and H-CAPPSKKKKKK-NH2 were coupled to California limpet hemocyanin (KLH) and a commercially available cationized bovine serum albumin (Supercarrier, Pierce Chemical). The resulting conjugates with KLH and Supercarrier were combined and injected into rabbits. He

Compound 13 was conjugated to the purified protein derivative (PPD) and injected into a goat preimmunized with BCG. Immunizations were performed following standard immunization initial immunization protocols in Freund's complete adjuvant, followed by a booster regimen with an immunogen emulsion in Freund's incomplete adjuvant and test bleeding until antibody titers reached levels. acceptable. The antibodies were purified in one step by affinity chromatography on a specific G column.

20 for immunoglobulins of the IgG subclass.

Example 23. Production of antibodies

[0176] Antibodies were prepared according to the general lines discussed previously. Four representative polyclonal antibodies against peptides that conform to the general formulas described in the claims have been generated. These comprise a peptide that incorporates the N-end sequence of compound 1, two peptides that incorporate the C-end sequence of compound 1, as well as a peptide that incorporates the complete sequence of compound 1. These peptides are designated compounds 15, 16 , 17 or 18, respectively and have the following sequences: Ac-RYYRWKKKKKKKC-NH2 (compound 15); CAPPSKKKKKK-NH2 (compound 16); CKKKKKK-NH2 (compound 17) and Ac-RYYRWKKKKKKKC-NH2 (compound 18). Polyclonal antibodies directed against compounds 15, 16 and 17 were generated in rabbits and against compound 18 in goat. These 30 polyclonal antibodies are very specific and produce the titers indicated in the accompanying table when the antibodies are evaluated in a standard ELISA analysis, with compound 1 as an antigen and with colorimetric detection of the antibody bound by an HRP conjugated antibody directed against IgG, commercially available. Preliminary tests with several of the antibodies have also indicated the absence of cross-reactions with antigens with little sequence similarity to compound 1 in the standard ELISA analysis described

35 above. Table 7 is shown below.

Table 7

Immunogenic

ELISA antigen Antibody titer

fifteen

 Compound 1 1: 750,000

16

 Compound 1 1: 200,000

17

 Compound 1 1: 350,000

17

 Glucagon reactionless

18

 Compound 1 1: 2,000

[0177] See the examples and discussion above for more information regarding the peptide conjugate compounds disclosed in this example.

[0178] Although the invention has been described with reference to specific embodiments, it is possible to construct modifications and variations of the invention without departing from the scope of the invention, which is defined in the following claims.

Claims (41)

1. A peptide conjugate of the general formula I R1-Z-X-Z’R2 (I) in which X represents a hexapeptide with the amino acid sequence (RK) YY (RK) (WI) (RK), in which the alternative amino acid residues at positions 1, 4, 5 and 6 are shown in parentheses and in which each amino acid residue in said hexapeptide it may be in the form L or D; Z represents a peptide chain loaded from 4 to 20 amino acid residues with the D or L configuration or is absent; Y Z ’represents a peptide chain loaded with 4 to 20 amino acid residues with the D or L configuration or is absent, taking into account that Z and Z’ are not absent at the same time; R1 represents H or an acyl group; R2 represents NR3R4, in which each of R3 and R4 independently represent hydrogen, C (1-6) alkoxy, aryloxy or alkyl; or R2 represents OH; wherein the peptide conjugates of the formula I may be optionally linked to a transport fraction; or a salt, hydrate or solvate thereof or a derivative thereof esterified at the C-terminus with a suitable organic or inorganic acid.

2.

A peptide conjugate according to any one of the preceding claims, wherein the number of amino acid residues in Z and Z ’is in the range of 5 to 10.

3.

A peptide conjugate according to any one of the preceding claims, wherein the amino acid residues in Z and Z 'have the L configuration.

Four.

A peptide conjugate according to any one of the preceding claims, wherein the amino acid residues of Z are selected from the group consisting of Q, T, S, P, N, E and D.

5.

A peptide conjugate according to any one of the preceding claims, wherein the N-terminal amino acid of Z is selected from the group consisting of Q, T, N and S and the remaining amino acid residues are selected from the group consisting of P, D and AND.

6.

A peptide conjugate according to any one of the preceding claims, wherein Z is selected from the group consisting of N (E) 7, N (E) 6, N (E) 5, N (E) 3, S (E) 7 , S (E) 6, S (E) 5, S (E) 3, NP (E) 4, NP (E) 5, N (D) 7, N (D) 6, N (D) 5, N (D) 3, Q (E) 7, Q (E) 5, Q (E) 3, QN (D) 7, Q (D) 6, C (D) 5, and Q (D) 3.

7.

A peptide conjugate according to claim 1 with the general formula III R1-Z’R2 (III).

8.

A peptide conjugate according to any one of the preceding claims further linked to a transport fraction, wherein said transport fraction is preferably selected from the group consisting of residues 49-57 of the HIV tat peptide, residues 49-56 of the peptide HIV tat, the tat sequence YGRKKRRQRRR, a polyarginine peptide with 6 to 20 residues, such as R6 and transducer peptide sequences, such as the following peptide sequences YARKARRQARR, YARAAARQARA, YARAARRAARR, YARAARRAARA, YARRRRRRRR and YAAAR.

9.

A peptide conjugate according to any one of the preceding claims, wherein Z ’represents a positively charged peptide chain.

10.

A peptide conjugate according to any one of the preceding claims, wherein R1 represents acetyl or trifluoroacetyl.

eleven.

A peptide conjugate according to any one of the preceding claims, wherein R2 represents NH2 or R2 represents NR3R4, wherein each of R3 and R4 independently represents hydrogen, methyl or ethyl.

12.

A peptide conjugate according to any one of the preceding claims, wherein said hexapeptide X is selected from the group consisting of KYYRWK, RYYRWR, RYYRWK, RYYRWK (all D), RYYRIK, RYYRIR, RYYKIK, RYYKIR, RYYKWR and RYYKWR.

A peptide conjugate according to any one of the preceding claims, wherein said hexapeptide X is KYYRWK or RYYRWK.

14.

A peptide conjugate according to any one of the preceding claims, wherein the amino acid residues of Z 'are selected from the group consisting of A, G, K, H and R, preferably K.

fifteen.

A peptide conjugate according to the preceding claim, wherein Z ’is selected from the group consisting of A (K4) G, K5G, AK5, K10, K9, K8, K7, K6, K5 and K4.

16.

A peptide conjugate according to any one of the preceding claims further comprising a terminal cysteine residue.

17.

A peptide conjugate according to claim 1 selected from the group consisting of

compound 1: Ac-RYYRWKKKKKKK-NH2 compound 2: Ac-KKKKKKRYYRWK-NH2 compound 3. H-NEEEEERYYRWKKKKKKK-NH2 compound 8: Ac-RYYRWKKKKKKK-NH2 (all D) compound 9: Ac-KYYRWKKKKKKK-NH2 compound 10: Ac-RYYRIKKKKKKK-NH2 compound 11: Ac-RYYRWKAKKKKK-NH2 compound 12: Ac-RYYRWKKKKKK-NH2 compound 13: Ac-RYYRWKKKKKKKC-NH2 compound 14: Tfa-RYYRWKKKKKKK-NH2 C-terminal free acid thereof, esterified derivatives thereof and pharmaceutically acceptable acid addition salts thereof.

18.

A peptide conjugate according to claim 17, which is compound 1A: Ac-RYYRWKKKKKKKNH2 x 9 CH3COOH or compound 1C: Ac-FtYYRWKKKKKKK-NH2 x 9 HCl.

19.

An X-Z ’peptide conjugate selected from the group consisting of RYYRWKAK5, KYYRWKK6, RYYRWKK6, RYYRWRK6, RYYRIKK6 and RYYRWK6 or a pharmaceutically acceptable salt or an acetylated derivative at the N or amidated or esterified end at the same C end.

twenty.

A peptide conjugate according to any one of claims 1 to 16 which is Ac-RYYRWKK6NH2 (compound 1) or a salt, hydrate or solvate thereof.

twenty-one.

A biologically active substance comprising a peptide conjugate according to any one of claims 1 to 18 that is positively charged and a negatively charged counterion selected from the group consisting of pharmaceutically acceptable anions, preferably CH3COO-, CF3COO-, Cl-, SO32-, maleate and oleate.

22

A pharmaceutical composition comprising as active compound a peptide conjugate according to any one of claims 1 to 18 or an active substance as defined in claim 21 and a pharmaceutically acceptable carrier or diluent.

2. 3.

A pharmaceutical composition according to claim 22 further comprising a liquid vehicle selected from the group consisting of syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene and water for parenteral administration.

24.

A pharmaceutical composition according to claim 22 or claim 23 containing in a unit dose an amount of said peptide conjugate in the range of about 0.1 to about 10 mg.

25.

A pharmaceutical composition according to claim 22 in a form adapted for administration only peripherally.

26.

A peptide conjugate according to any one of claims 1 to 20 or a biologically active substance according to claim 21 for use in a medical treatment procedure.

27.

Use of a peptide conjugate according to any one of claims 1 to 20 or a biological substance according to claim 21 for the preparation of a medicament for the treatment and / or prevention of hyponatremia or hypokalemia.

28.

Use according to claim 27, wherein said hyponatremia or hypokalemia is associated with heart failure.

29.

Use according to claim 28, wherein said hyponatremia or hypokalemia is associated with an intensive diuretic treatment with thiazides and / or loop diuretics.

30

Use of a peptide conjugate according to any one of claims 1 to 20 or a biologically active substance according to claim 21 for the preparation of a medicament for selective aqueous diuresis.

31.

Use of a peptide conjugate according to any one of claims 1 to 20 or a biologically active substance according to claim 21 for the preparation of a medicament for the treatment and / or prevention of a condition with water retention, such as congestive heart failure, liver cirrhosis, nephrotic syndrome and hypertension.

32

Use of a peptide conjugate according to any one of claims 1 to 20 or a biologically active substance according to claim 21 for the preparation of a medicament for the treatment and / or prevention of multiple organic insufficiency.

33.

Use of a peptide conjugate according to any one of claims 1 to 20 or a biologically active substance according to claim 21 for the preparation of a medicament for the treatment and / or prevention of acute renal failure.

3. 4.

Use of a peptide conjugate according to any one of claims 1 to 20 or a biologically active substance according to claim 21 for the preparation of a medicament for use in the treatment of pathological conditions associated with a high nociceptin activity.

35

Use of a peptide conjugate according to any one of claims 1 to 20 or a biologically active substance according to claim 21 for the preparation of a medicament for the treatment of edema, optionally in combination with a diuretic.

36.

Use according to claim 35, wherein the edema is associated with coronary heart failure.

37.

Use according to claim 35, wherein said diuretic is a loop diuretic.

38.

A nucleic acid sequence encoding a polypeptide sequence comprising the peptide sequence of formula I or III.

39.

A recombinant host cell comprising the nucleic acid sequence of claim 38 and is capable of expressing said polypeptide sequence.

40

A process for the production of a peptide conjugate according to any one of claims 1 to 20 with a natural polypeptide sequence comprising:

the introduction of a nucleic acid sequence according to claim 38 and a selectable marker, contained within a nucleic acid construct or a vector, in a host cell to obtain a recombinant host cell; the selection of said recombinant host cell; the culture of said recombinant host cells under conditions that allow the production of said polypeptide sequence; the isolation of said polypeptide sequence from the culture; optionally the cleavage of said polypeptide sequence by means of a suitable protease to obtain said peptide conjugate. 41. A method for synthesizing a peptide conjugate according to any one of claims 5 1 to 20 by means of dissolution or solid phase peptide synthesis techniques. 42. A procedure for improving the stability in the blood plasma of a hexapeptide with the sequence (RK) YY (RK) (WI) (RK), in which the alternative amino acid residues at positions 1, 4, 5 and 6 are show in brackets and in which each amino acid residue in said hexapeptide can be in the form L or D; in 10 that the method comprises ligation of a peptide sequence Z 'by means of a peptide bond to the C-terminus of said hexapeptide to form a conjugated peptide, wherein Z' represents a peptide chain charged with 4 to 20 amino acid residues in configuration D or L and in that said procedure is not performed in vivo. A method according to claim 42, wherein said Z ’sequence is selected from the group consisting of unbranched K5, K6 and K5C sequences.