FIELD OF THE INVENTION The present invention relates to novel medical uses of the hexapeptide having the amino acid sequence (I) X-Hexapeptide-Y and the use of the hexapeptide for the preparation of a medicament.

BACKGROUND The endogenous opioid-like peptide, nociceptin (also referred to as orphanin FQ), was first described in the central nervous system, and most research in this field has focused on the CNS effects. Nociceptin binds to a specific receptor named opioid receptor-like one (ORL1) with much greater affinity than to the three classical subtypes of opioid receptors. Effects of nociceptin in the CNS include : hyperalgesia/hypoalgesia, stimulation of appetite and gnawing, increased (low doses) or decreased (high doses) locomotion, impaired learning, and dysphoria. However, nociceptin also exerts important effects outside the CNS. Thus, low doses of nociceptin increase the renal excretion of water and decreases urinary sodium excretion (i. e., produces a selective water diuresis) which render this compound interesting for the treatment of hyponatremia (Daniel R. Kapusta, Life Science, 60: 15-21, 1997) (US patent No. 5,840,696). When administered centrally (i. c. v.) or at high doses peripherally (i. v. bolus or infusion), nociceptin decreases blood pressure, heart rate and peripheral sympathetic nerve activity.

Dooley et al. (The Journal of Pharmacology and Experimental Therapeutics, 283 (2): 735-741,1997) have shown that a hexapeptide having the amino acid sequence Ac-RYY (RK) (WI) (RK)-NH2, where the brackets show allowable variation of amino acid residue, acts as a partial agonist of the nociceptin receptor ORL1. Said hexapeptide was identified from a combinatorial peptide library and the sequence is unique without homology or similarity to the nociceptin heptadecapeptide. WO 99/44627 discloses the use of hexapeptides including the hexapeptides discovered by Dooley et al. for the manufacture of a pharmaceutical composition for the treatment of the following conditions: Migraine, type 11 diabetes, septic shock, inflammation and vasomotor disturbances. The pharmacological rationale for using said hexapeptides in the treatment of said conditions is the shown inhibition of depressor response to spinal cord stimulation by the hexapeptide Ac-Arg-Tyr- Tyr-Arg-Trp-Lys-NH2. However, the present inventors have shown that the hexapeptides disclosed by Dooley et al. exhibit a peripheral effect that is distinctly different from the effect shown in WO 99/44627 and which is useful in the treatment of disease states, such as hyponatremia, acute renal failure and medical conditions that can be ameliorated by treatment with a selective water diuretic agent.

It is therefore an objective of the present invention to provide a new use of said hexapeptides for the preparation of a medicament for selective water diuresis and/or the treatment and/or prevention of hyponatremia and/or acute renal failure.

This objective is achieved with the use of said hexapeptide for the preparation of a medicament for treatment and/or prevention of said disease states and as described herein (below).

Summary of the invention The present invention relates to the use of a compound having the general formula I

(I) X-Hexapeptide-Y wherein X represents H or acyl ; Y represents OH or NH2; Hexapeptide represents an amino acid sequence of the formula 11 (II)A1-A2-A3-A4-A5-A6 wherein A1 represents Arg, Lys or His, A2 represents Tyr, Trp, or Phe, A3 represents Tyr, Asn, Trp or Phe, A4 represents Lys, Arg or His, A5 represents Phe, Tyr, Trp or lle, and A6 represents Arg, Lys or His and wherein each amino acid residue in said hexapeptide may be in the L or D form; and pharmaceutical acceptable salts, hydrates and solvates thereof for the manufacture of a pharmaceutical composition for selective water diuresis and/or for the treatment of hyponatremia and/or acute renal failure and/or sodium and water retaining conditions.

Detailed description of the invention In preferred embodiments of the invention the group X of formula I represents acetyl (Ac) or trifluoroacetyl (Tfa), most preferably X represents acetyl ; Y represents NH2, and Hexapeptide represents the amino acid sequence (RK) YY (RK) (WI) (RK) wherein alternative amino acid residues at positions 4,5 and 6 are shown in brackets. Preferably (RK) YY (RK) (WI) (RK) is selected form the group consisting of RYYRWR, RYYRWK, RYYRIK, RYYRIR, RYYKIK, RYYKIR, RYYKWR, and RYYKWK, more preferably from the group consisting of RYYRWR, RYYRWK, RYYRIK, RYYKWR, and RYYKWK ; and most preferably (RK) YY (RK) (WI) (RK) represents RYYRWK.

Preferably, all amino acid residues of the compounds of formula I and 11 are in the L-form.

A specific compound of formula I to be used in the present invention is Compound 1 Ac-RYYRWK-NH2, and pharmaceutical acceptable salts, hydrates and solvates thereof.

The invention also concerns a pharmacologically active compound of formula I or 11 or a derivative thereof including C-terminally esterified derivatives or C-terminal secondary and tertiary amidated derivatives or a salt, hydrate or solvate thereof for use in the treatment and/or prevention of hyponatremia and acute renal failure and for use in the manufacture of a medicament or pharmaceutical composition for selective water diuresis and/or treatment and/or prevention of hyponatremia and/or acute renal failure. In specific embodiments, a hexapeptide according to the present invention may be used for the manufacture of a medicament for treatment and/or prevention of hyponatremia.

The term hyponatremia includes but is not necessarily limited to the following medical conditions: 1. Pseudohyponatremia characterised by Normal plasma osmolality associated with, e. g., hyperlipidemia, hyperproteinemia or posttransurethral resection of prostate/bladder tumor, and Increased plasma osmolality associated with, e. g., hyperglycemia and mannitol ; 2. Hypoosmolal hyponatremia characterised by Primary Na+ loss (secondary water gain) associated with, e. g., integumentary loss : sweating, burns; gastrointestinal loss : vomiting, tube drainage, fistula, obstruction, diarrhea; renal loss : diuretics, osmotic diuresis, hypoaldosteronism, salt-wasting nephropathy, postobstructive diuresis, nonoligouric acute tubular necrosis; Primary water gain (secondary Na+ loss) associated with, e. g. primary polydipsia ; decreased solute intake (e. g., beer potomania); AVP release due to pain, nausea, drugs; syndrome of inappropriate AVP secretion; glucocorticoid deficiency; hypothyroidism and chronic renal insufficiency; and

Primary Na+ gain (exceeded by secondary water gain) associated with, e. g. heart failure, hepatic cirrhosis and nephrotic syndrome; or drug induced.

Furthermore, a hexapeptide according to the present invention may be used for the manufacture of a medicament for the treatment and prevention of sodium and water retaining conditions as well as acute renal failure and multiple organ failure. Sodium and water retaining conditions include diseases, such as 1. Congestive heart failure in which the heart failure may be described as systolic or diastolic, high-output or low-output, acute or chronic, right-sided or left-sided, and forward or backward.

2. Liver cirrhosis in which the cirrhosis may be related to alcoholic liver disease; postnecrotic cirrhosis (caused by infectious diseases, inherited metabolic disorders, drugs and toxins, inflammatory and other diseases); biliary cirrhosis (primary or secondary); cardiac cirrhosis due to prolonged, severe right-sided congestive heart failure ; metabolic, hereditary, drug-related or other types of cirrhosis.

3. Nephrotic syndrome related to systemic and/or renal disease, drug-or toxin-induced.

4. Hypertension in which the hypertension may be primary (idiopathic) or secondary to other eliciting causes, such as drugs, toxins or diseases in endocrine glands, kidneys, or in the central nervous system.

5. Multiple organ failure elicited during hemorrhagic shock including acute renal failure. An example of a predictive in vivo model of multiple organ failure (kidney, lungs, intestines) elicited during anaesthesia, surgery and hemorrhage is described by Kapusta et al. (J. Am. Soc. Nephrol. 2000,11 A3175) which is incorporated by reference.

6. Acute renal failure in which the pathogenesis of the disease may be related to either prerenal or intrinsic renal causes.

Acute renal failure include : Prerenal azotemia, such as the following conditions: f.Hypovolemia A. Hemorrhage, burns, dehydration B. Gastrointestinal fluid loss : vomiting, surgical drainage, diarrhea C. Renal fluid loss : diuretics, osmotic diuresis (e. g., diabetes mellitus), hypoadrenalism D. Sequestration in extravascular space: pancreatitis, peritonitis, trauma, burns, severe hypoalbuminemia ; II. Low cardiac output A. Diseases of myocardium, valves, and pericardium, arrhythmias, tamponade B. Other: pulmonary hypertension, massive pulmonary embolus, positive pressure mechanical ventilation ; III. Altered renal systemic vascular resistance ratio A. Systemic vasodilatation : sepsis, antihypertensives, afterload reducers, anesthesia, anaphylaxis B. Renal vasoconstriction: hypercalcemia, norepinephrine, epinephrine, cyclosporine, amphotericin B C. Cirrhosis with ascites (hepatorenal syndrome); IV. Renal hypoperfusion with impairment of renal autoregulatory responses Cyclooxygenase inhibitors, angiotensin-converting enzyme inhibitors; V. Hyperviscosity syndrome (rare) Multiple myeloma, macroglobulinemia, polycythemia.

Acute renal failure further include : Intrinsic renal azotemia, such as the following conditions: I. Renovascular obstruction (bilateral or unilateral with one functioning kidney) A. Renal artery obstruction: atherosclerotic plaque, thrombosis, embolism, dissecting aneurysm, vasculitis B. Renal vein obstruction: thrombosis, compression; II. Disease of glomeruli or renal microvasculature A. Glomerulonephritis and vasculitis B. Hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, disseminated intravascular coagulation, toxemia of pregnancy, accelerated hypertension, radiation nephritis, systemic lupus erythematosus, scleroderma ; III. Acute tubular necrosis A. Ischemia : as for prerenal azotemia (hypovolemia, low cardiac output, renal vasoconstriction, systemic vasodilatation), obstetric complications (abruptio placentae, postpartum hemorrhage) B. Toxins 1. Exogenous: radiocontrast, cyclosporine, antibiotics (e. g., aminoglycosides), chemotherapy (e. g :, cisplatin), organic solvents (e. g., ethylene glycol), acetaminophen, illegal abortifacients 2. Endogenous: rhabdomyolysis, hemolysis, uric acid, oxalate, plasma cell dyscrasia (e. g., myeloma) ; IV. Interstitial nephritis A. Allergic : antibiotics (e. g.,-lactams, sulfonamides, trimethoprim, rifampicin), nonsteroidal anti-inflammatory agents, diuretics, captopril B. Infection : bacterial (e. g., acute pyelonephritis, leptospirosis), viral (e. g., cytomegalovirus), fungal (e. g., candidiasis) C. Infiltration : lymphom, leukemia, sarcoidosis D. Idiopathic ;

V. Intratubular deposition and obstruction A. Myeloma proteins, uric acid, oxalate, acyclovir, methotrexate, sulphonamides ; Vi. Renal allograft rejection Hypokalemia is a major predisposing mechanism for the development of arrhythmias and the prognosis of chronic heart failure (CHF) is poor when serum potassium levels fall below 3.3 mM. Furthermore, in CHF patients treated with digoxin, hypokalemia is the most common precipitating cause of digitalis intoxication, which is a serious and potentially fatal complication. In specific embodiments, a hexapeptide according to the present invention may be used for the manufacture of a medicament for treatment and/or prevention of hypokalemia.

Preferred pharmaceutical compositions of the invention comprise a pharmacologically active hexapeptide of formula I or 11 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, ocularly, or pulmonary administration, preferably in a form adapted for administration by a peripheral route, or is suitable for oral administration or suitable for parenteral administration. Other preferred routes of administration are subcutaneous, intraperitoneal and intravenous, and such compositions may be prepared in a manner well-known to the person skilled in the art, e. g., as generally described in"Remington's Pharmaceutical Sciences", 17. Ed. Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, PA, U. S. A., 1985 and more recent editions and in the monographs in the"Drugs and the Pharmaceutical Sciences"series, 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, e. g. as disclosed in US 5,350,741, for oral administration.

The pharmaceutical carrier or diluent employed may be a conventional solid or liquid carrier. Examples of solid carriers are lactose, terra alba, sucrose, cyclodextrin, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid or lower alkyl ethers of cellulose. Examples of liquid carriers 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 glyceryl monostearate or glyceryl distearate, alone or mixed with a wax.

When a solid carrier is used for oral administration, the preparation may be tabletted, placed in a hard gelatin capsule in powder or pellet form or it can be in the form of a troche or lozenge. The amount of solid carrier will vary widely but will usually be from about 25 mg to about 1 g. When a liquid carrier is used, the preparation may be in the form of a syrup, emulsion, soft gelatin capsule or sterile injectable liquid such as an aqueous or non-aqueous liquid suspension or solution.

The composition may also be in a form suited for local or systemic injection or infusion and may, as such, be formulated with sterile water or an isotonic saline or glucose solution. Administration forms which exclude direct delivery into the central nervous system are preferred. The compositions may be sterilized by conventional sterilization techniques which are well known in the art. The resulting aqueous solutions may be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with the sterile aqueous solution prior to administration. The composition may contain pharmaceutical acceptable auxiliary substances as required to approximate physiological conditions, such as buffering agents, tonicity adjusting agents and the like, for instance sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.

For nasal administration, the preparation may contain a compound of the present invention dissolved or suspended in a liquid carrier, in particular, an aqueous carrier, for aerosol application. The carrier may contain additives

such as solubilizing agents, e. g., propylene glycol, surfactants such as bile acid salts or polyoxyethylene higher alcohol ethers, absorption enhancers such as lecithin (phosphatidylcholine) or cyclodextrin, or preservatives such as parabines.

In a preferred embodiment of the invention the compound of formula I or II is administered as a dose in the range from about 0.001 to about 10 g per patient per day, preferably from about 1 to about 1000 mg per patient per day, more preferably from about 10 to about 100 mg per patient per day, about 50 mg per patient per day.

In further aspects the invention relates to a method for providing selective water diuresis in a patient, a method for treatment and/or prevention of hyponatremia, e. g. as defined above, a method for the treatment and/or prevention of sodium and water retaining conditions, e. g. as defined above, and a method for the treatment and/or prevention of acute renal failure, e. g. as defined above, comprising administering an effective dose of a pharmacologically active compound having the formula I or 11.

Definitions Throughout the description and claims the one letter code for natural amino acids is used as well as the three letter code for natural amino acids and generally accepted three letter codes for other a-amino acids, such as Ornithine (Orn), 2,4-Diaminobutanoic acid (Dab) and 2,3-Diaminopropanoic acid (Dapa), Sarcosin (Sar), a-Amino-iso-butanoic acid (Aib), and Hydroxyproline (Hyp). Where the L or D form has not been specified it is to be understood that the amino acid in question has the natural L form, cf. Pure & Appl. Chem. Vol. 56 (5) pp595-624 (1984). Where nothing is specified it is to be understood that the C-terminal amino acid of a compound of the invention exists as the free carboxylic acid, this may also be specified as"-OH". Cf.

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, following p 84; J.

Biol. Chem., 1985,260,14-42; Pure Appl. Chem., 1984,56,595-624; Amino Acids and Peptides, 1985,16,387-410; Biochemical Nomenclature and Related Documents, 2nd edition, Portland Press, 1992, pages 39-69; Copyright IUBMB and IUPAC.

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, particularly when employing the compounds of the invention as medicaments, other salts find utility, for example, in processing these compounds, or where non- medicament-type uses are contemplated. Salts of these compounds may be prepared by art-recognized techniques. Examples of such pharmaceutically acceptable salts include, but are not limited to, inorganic and organic addition salts, such as hydrochloride, sulphates, nitrates or phosphates and acetates, trifluoroacetates, propionates, succinates, benzoates, citrates, tartrates, fumarates, maleates, methane-sulfonates, isothionates, theophylline acetates, salicylates, respectively, or the like. Lower alkyl quaternary ammonium salts and the like are suitable, as well.

The term"acyl"as used herein include acyl radicals which are formally derived from oxoacids RkE (=O) I (OH) m (I not equal to 0) by removal of a hydroxyl cation HO+, a hydroxyl radical HO. or a hydroxyl anion HO-, respectively, and replacement analogues of such intermediates. Acyl radicals 9 can formally be represented by canonical forms having an unpaired electron or a positive charge on the acid-generating element of the oxoacid. Acyl radicals, e. g. RC. (=O), RS. (=0) 2.

The term"acylated"as used herein indicates that the compound in question carries an acyl group. An acyl group is formed by removing one or more hydroxy groups from an oxoacid, such as a carboxylic acid, that has the general structure RkE (=O) I (OH) m (I not equal to 0), and replacement analogues of such acyl groups. E. g. CH 3C (=O)-, CH3C (=NR)-, CH3C (=S)-, PhS (=0) 3-, HP (=N)-. In organic chemistry an unspecified acyl group is

commonly a carboxylic acyl group. Cf. International Union of Pure and Applied Chemistry, Recommendations on Organic & Biochemical Nomenclature, Symbols & Terminology etc. IUPAC Recommendations 1994."Ac"indicates the acetyl group and"Tfa"indicates the trifluoroacetyl group.

"Agonist"refers to an endogenous substance or a drug that can interact with a receptor and initiate a physiological or a pharmacological response characteristic of that receptor (contraction, relaxation, secretion, enzyme activation, etc.)."Partial agonist"refers to an agonist which is unable to induce maximal activation of a receptor population, regardless of the amount of drug applied. A"partial agonist"may also be termed"agonist with intermediate intrinsic efficacy"in a given tissue. Moreover, a partial agonist may antagonize the effect of a full agonist that acts on the same receptor.

"Antagonist"refers to a drug or a compound that opposes the physiological effects of another. At the receptor level, it is a chemical entity that opposes the receptor-associated responses normally induced by another bioactive agent.

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

Pharmacology The pharmaceutical compositions of the present invention are useful as nociceptin agonists or partial agonists as described below.

The diuretic effect of a compound of formula I has been tested in rats, where Compound 1 in an isotonic saline solution elicits a significant diuretic response in the i. v. dose 1 nmol/kg/min. Cardiovascular and renal responses produced by i. v. infusion of nociceptin (Phoenix Pharmaceuticals, also in an isotonic saline solution) and Compound 1 have been studied in conscious Sprague Dawley rats. The results are shown in Fig. 1 and Fig. 2 herein, where

HR is heart rate (bpm), MAP is mean arterial pressure (mmHg), V is urine flow rate (ul/min), UNaV is urinary excretion rate of sodium (ueq/min), and n is number of animals.

Figure 1 shows cardiovascular and renal responses produced by the i. v. bolus injection of isotonic saline vehicle (100 ml) or Compound 1 as the trifluoroacetate dissolved in isotonic saline (100 nmol/kg) in conscious Sprague-Dawley rats with intact renal nerves. For these studies concious rats were continuously infused i. v. (55 ml/min) with isotonic saline for the duration of the study. * indicates P<0.05 versus control (C) value. A stock solution of Compound 1 (10 mg/ml distilled deionized water) was prepared, divided into aliquots, and frozen until use in each animal. These findings demonstrate the cardiovascular and renal excretory responses to i. v. bolus injection of Compound 1. The i. v. injection of Compound 1 produced a slight, but significant decrease in MAP. Compound 1 also produced a significant increase in urine flow rate 20 and 30 minutes after injection.

Figure 2 shows Cardiovascular and renal responses produced by the i. v. infusion of Compound 1 (1 nmol/kg/min) or nociceptin (0.11 nmol/kg/min) in concious Sprague-Dawley rats. * indicates P<0.05 versus control (C) value within group of animals treated with Compound 1. t indicates P<0.05 versus control (C) value within group of animals treated with nociceptin. This figure shows the cardiovascular and renal responses produced by the i. v. infusion of Compound 1 and low dose nociceptin. The i. v. infusion of nociceptin (0.11 nmol/kg/min) produced a significant increase in V without altering HR, MAP or UNaV. As compared to these nociceptin-induced responses, the i. v. infusion of Compound 1 (1 nmol/kg/min) elicited a similar time course and magnitude diuresis. Although Compound 1 did not significantly alter HR or UNaV, this compound produced a slight, but significant reduction in MAP.

The invention also concerns a pharmacologically active compound of formula I or 11 or a derivative thereof including C-terminally esterified derivatives or C-terminal secondary and tertiary amidated derivatives or a salt

thereof as disclosed herein for use in therapy, and the use thereof as defined herein for the manufacture of a medicament or pharmaceutical composition for use in therapy. Preferably, a pharmaceutical composition is suitable for oral administration or injection. Therapeutic uses of the compounds of formula I or 11 herein are to increase the renal excretion of water and to decrease urinary sodium excretion (i. e., a selective water diuresis). In specific embodiments, a pharmaceutical composition according to the present invention may be used to treat and/or prevent acute renal failure.

Yao K. et al. (Jpn J Pharmacol 1994 Jun; 65 (2): 167-70) describe an animal model of drug-induced (non-ischemic) acute renal failure. In this model the renal protective effect of a hexapeptide of the invention against gentamicin (GM)-induced acute renal failure (ARF) in rats may be investigated. ARF is induced by subcutaneous injection of GM at 80 mg/kg/day for 12 days.

Compound 1 will be administered to show attenuation of the increases of serum creatinine and urea nitrogen and the decrease of creatinine clearance in rats treated with GM. The results may suggest that Compound 1 can ameliorate the GM-induced ARF.

Additional causes of acute renal failure include renal failure due to trauma, or anaesthesia and surgery induced impairment of renal function.

Severe trauma may be associated with hemorrhage, ischemia, and release of toxic substances that may result in acute renal failure as well as multiple organ failure. The present inventors believe that the hexapeptides of the invention may exert their organ protective action through a protection against ischemia.

Fernandez-Llama P. et al. ( J Am Soc Nephrol 1999 Aug; 10 (8): 1658- 68) describe an animal model of ischemic acute renal failure suitable for showing the protective effects of the hexapeptide of the invention against ischemic acute renal failure

Examples of predictive in vivo models of prerenal azotemia for the study of therapeutic actions of peptides of the present invention are the norepinephrine and renal artery clamp rat ischemic acute renal failure models, that may be accelerated by hemorrhage (J. D. Conger, M. F. Schutz, F.

Miller, J. B. Robinette, Kidney Int. 1994,46 318-323) which is incorporated by reference.

An example of a predictive in vivo model of intrinsic renal azotemia for the study of therapeutic actions of peptides of the present invention is the gentamicin-induced acute renal failure model (D. de Rougemont, A.

Oeschger, L. Konrad, G. Thiel, J. Torhorst, M. Wenk, P. Wunderlich, F. P.

Brunner, Nephron 1981,29 176-184) which is incorporated by reference.

To examine the effect of compounds of the present invention in an in vivo model of multiple organ failure including acute renal failure, a model of organ failure elicited by hemorrhage during anesthesia and surgery will be used. In this model, rats are infused with isotonic saline or compounds of the present invention for 15 min prior to anesthesia. Then the animals are anestetized with isoflurane (3% in O2/N2O mixture) and subjected to periods of surgery (chronic bladder catheterization + femoral vein and artery; 30 min); hemorrhage (20 cc/kg b. w.; 45 min), and recovery (blood replacement; 120 min). Consecutive 10 min urine samples are collected throughout, and rats are allowed to recover for 7 days. Following the hemorrhagic event, urine collections and blood samples are collected (e. g., days 2,4, and 6) to evaluate the recovery as determined by urine production, and serum concentrations of creatinine and urea. Finally on day 7, rats are sacrified for histological examination of all organs. Using this model, Kapusta et al. previously demonstrated that the kappa opioid agonist U-50, 488H prevents multiple organ failure (kidney, lungs, intestines) and increases survival after a hemorrhagic event elicited during surgical anaesthesi (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).

Experimental procedures The peptides of the invention may be prepared by methods known per se in the art including recombinant techniques. It is preferred to prepare the peptide sequences of formula I herein by standard peptide-preparation techniques such as solution synthesis or Merrifield-type solid phase synthesis.

Both the Boc (tert. butyloxycarbonyl) as well as the Fmoc (9- fluorenylmethyloxycarbonyl) strategies are applicable.

Apparatus and synthetic strategy Peptides were synthesized batchwise in a polyethylene vessel equipped with a polypropylene filter for filtration using 9- fluorenylmethyloxycarbonyl (Fmoc) as N-a-amino protecting group and suitable common protection groups for side-chain functionalities.

Solvents Solvent DMF (N, N-dimethylformamide, Riedel de-Haen, Germany) was purified by passing through a column packed with a strong cation exchange resin (Lewatit S 100 MB/H strong acid, Bayer AG Leverkusen, Germany) and analyzed for free amines prior to use by addition of 3,4-dihydro-3-hydroxy-4- oxo-1,2,3-benzotriazine (Dhbt-OH) giving rise to a yellow color (Dhbt-O- anion) if free amines are present. Solvent DCM (dichloromethane, analytical grade, Riedel de-Haen, Germany) was used directly without purification.

Acetonitril (HPLC-grade, Lab-Scan, Dublin Ireland) was used directly without purification.

Amino acids Fmoc-protected amino acids were purchased from Advanced ChemTech (ACT) in suitabel side-chain protected forms.

Coupling reagents Coupling reagent diisopropylcarbodiimide (DIC) was purchased from Riedel de-Haen, Germany.

Solid supports Peptides were synthesized on TentaGel S resins 0,22-0,31 mmol/g.

TentaGel S-Ram, TentaGel S RAM-Lys (Boc) Fmoc (Rapp polymere, Germany) were used in cases where a C-terminal amidated peptide was preferred, while TentaGel S PHB, TentaGel S PHB Lys (Boc) Fmoc were used when a C-terminal free carboxylic acid was preferred.

Catalysts and other reagents Diisopropylethylamine (DIEA) was purchased from Aldrich, Germany, piperidine and pyridine from Riedel-de Häen, Frankfurt, Germany. Ethandithiol was purchased from Riedel-de Haen, Frankfurt, Germany. 3,4-dihydro-3- hydroxy-4-oxo-1,2,3-benzotriazine (Dhbt-OH), 1-hydroxybenzotriazole (HOBt) (HOAt) were obtained from Fluka, Switzerland. Acetic anhydride was obtained from Fluka.

Coupling procedures The amino acids were coupled as in situ generated HObt or HOAt esters made from appropriate N-a-protected amino acids and HObt or HOAt by means of DIC in DMF. Acylations were checked by the ninhydrin test performed at 80 oC in order 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 N-a-amino protecting group (Fmoc).

Deprotection of the Fmoc group was performed by treatment with 20% piperidine in DMF (1x5 and 1x10 min.), followed by wash with DMF (5 x 15 ml, 5 min. each) until no yellow color could be detected after addition of Dhbt-OH to the drained DMF.

Coupling of HOBt-esters 3 eq. N-a-amino protected amino acid was dissolved in DMF together with 3 eq. HObt and 3 eq DIC and then added to the resin.

Acetylation of the N-terminal amino group with acetic anhydride 40 eq acetic anhydride was dissolved in DMF together with 5 eq pyridine and then added to the resin.

The acylation was checked by the ninhydrin test as described above.

Trifluoroacetylation of the N-terminal amino group with ethyl trifluoroacetate 30 eq ethyl trifluoroacetate was dissolved in dichloromethane together with 10 eq triethyl amine and then added to resin. The acylation was checked by ninhydrin test as described above.

Cleavage of peptide from resin with acid.

Peptides were cleaved from the resins by treatment with 95% triflouroacetic acid (TFA, Riedel-de Haen, Frankfurt, Germany)-water v/v or with 95% TFA and 5% ethandithiol v/v at r. t. for 2 h. The filtered resins were washed with 95% TFA-water and filtrates and washings evaporated under reduced pressure. The residue was washed with ether and freeze dried from acetic acid-water. The crude freeze dried product was analyzed by high- performance liquid chromatography (HPLC) and identified by mass spectrometry (MS).

Batchwise peptide synthesis on TentaGel resin (PEG-PS).

TentaGel resin (1g, 0.23-0.24 mmol/g) was placed in a polyethylene vessel equipped with a polypropylene filter for filtration. The resin was swelled in DMF (15mi), and treated with 20% piperidine in DMF in order to remove the initial Fmoc group either on the linker TentaGel S RAM or on the first amino acid on the resin TentaGel S RAM-Lys (Boc) Fmoc. The resin was drained and washed with DMF until no yellow color could be detected after addition of Dhbt-OH to the drained DMF. The amino acids according to the sequence were coupled as preformed Fmoc-protected HObt esters (3 eq.) as described above. The coupling were continued for 2 h, unless otherwise specified. The resin was drained and washed with DMF (5 x 15 ml, 5 min each) in order to remove excess reagent. All acylations were checked by the ninhydrin test as

described above. After completed synthesis the peptide-resin was washed with DMF (3x15 ml, 5 min each), DCM (3x15 ml, 1 min each) and finally diethyl ether (3x15 ml, 1 min each) and dried in vacuo. The peptide was cleaved from the resin as described earlier and the crude peptide product was analysed and purified as described below.

HPLC conditions Gradient HPLC analysis was done using a Hewlett Packard HP 1100 HPLC system consisting of a HP 1100 Quaternary Pump, a HP 1100 Autosampler a HP 1100 Column Thermostat and HP 1100 Multiple Wavelength Detector. Hewlett Packard Chemstation for LC software (rev.

A. 06.01) was used for instrument control and data acquisition. The following columns and HPLC buffer system was used: Column : VYDAC 238TP5415, C-18, 5mm, 300A 150x4. 6mm.

Buffers: A: 0,1% TFA in MQV; B: 0,085% TFA, 10% MQV, 90% MeCN.

Gradient: 0-1,5 min. 0% B 1,5-25 min 50% B 25-30 min 100% B 30-35 min 100% B 35-40 min 0 % B Flow 1, ml/min, oven temperature 40oC, UV detection: I = 215 nm.

HPLC purification of the crude peptide The crude peptide products were purified PerSeptive Biosystems VISION Workstation. VISION 3.0 software was used for instrument control and data acquisition. The following column and HPLC buffer system was used: Column : Kromasil KR 100A, 10mm C-8,250 x 50.8mm.

Buffer system: Buffers: A: 0,1% TFA in MQV; B: 0,085% TFA, 10% MQV, 90% MeCN.

Gradient: 0-37 min. 0-40% B Flow 35 ml/min, UV detection : I = 215 nm and 280 nm.

Mass spectroscopy The peptides were dissolved in super gradient methanol (Labscan, Dublin, Ireland), milli-Q water (Millipore, Bedford, MA) and formic acid (Merck, Damstadt, Germany) (50: 50: 0.1 v/v/v) to give concentrations between 1 and 10 mg/ml. The peptide solutions (20 ml) were analysed in positive polarity mode by ESI-TOF-MS using a LCT mass spectrometer (Micromass, Manchester, UK).

Synthesis of Compound 1 Ac-Arg-Tyr-Tyr-Arg-Trp-Lys-NH2 on TentaGel S RAM-Lys (Boc) Fmoc.

Dry TentaGel S RAM-Lys (Boc) Fmoc (0.24 mmol/g, 1g) was placed in a polyethylene vessel equipped with a polypropylene filter for filtration. and treated as described under"batchwise peptide synthesis on TentaGel resin" until finishing the coupling of the N-terminal Arginine. All couplings were continued over night. After deprotection of the Fmoc group the N-terminal amino group was acetylated as described above. The coupling was continued over night. The acylations were checked as earlier described. After completed synthesis the peptide was cleaved from the resin as described above. Yield of crude product 143 mg. After purification using preparativ HPLC as earlier described, 52 mg peptide product was collected with a purity better than 97 % and the identity of the peptide was confirmed by MS (found M 1011.54, calculated M 1011.54).

Formulation examples Example 1. A tablet prepared by conventional tabletting techniques may contain: Core: Compound 1 (as the trifluoroacetate) 100 mg; colloidal silicon dioxide (Aerosil) 1.5 mg; cellulose, microcryst. (Avicel) 70 mg; modified cellulose gum (Ac-Di-Sol) 7.5 mg; magnesium stearate. Coating: HPMC approx. 9 mg; acylated monoglyceride used as plasticizer for film coating (Mywacett 9-40T) approx. 0.9 mg.

Example 2. Formulation of Compound 1 for intravenous injection: A multi-dose formulation is prepared as a solution of a compound of the invention in sterile, isotonic saline, stored in capped vials, and if necessary a preservative is added (e. g. benzoates).

Fixed dose formulations are prepared as a solution of the compound in sterile, isotonic saline, stored in glass ampoules, and if necessary filled with an inert gas. Each dose of the compound is stored dry in ampoules or capped vials, if necessary filled with inert gas. The multi-dose formulation demands the highest degree of stability of the compound. A fixed dose formulation may be preferred when the stability of the active compound is low.