Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
  • A61K38/35—Corticotropin [ACTH]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C

Definitions

• the invention relates to urotensin-II polypeptide agonists and methods of their use.
• Urotensin-II (“UII”) is a cyclic neuropeptide with potent cardiovascular effects. Originally isolated from the caudal neurosecretory system of teleost fish, the primary structure of UII has been established for several species of vertebrates, including various fish species, frogs, and humans. Sequence analysis of various UII peptides from different species has revealed that, while the N-terminal region is highly variable, the C-terminal cyclic region of UII is strongly conserved. Indeed, this cyclic region, which is responsible for the biological activity of UII, is fully conserved from fish to humans (Coulouran, et al., Proc. Natl. Acad. ScL USA (physiology), 95:15803-15808 (1998)). The fact that evolutionary pressure has acted to fully conserve the biologically active sequence of UII suggests that this polypeptide plays an important role in human physiology.
• the cyclic region of UII includes six amino acid residues (-Cys-Phe-Trp-Lys- Tyr-Cys- (SEQ ID NO: I)) and is structurally similar to the biologically important central region of somatostatin- 14 (-Phe-Trp-Lys-Thr- (SEQ ID NO:2)).
• SEQ ID NO: I amino acid residues
• SEQ ID NO:2 the biologically important central region of somatostatin- 14
• SEQ ID NO:2 molecular cloning and sequence analysis of the carp preprourotensin II gene suggests that UII and somatostatin are not derived from a common ancestor (Ohsako, S., et ah, J. Neurosci., 6:2730-2735 (1986)).
• UII peptides In fish, UII peptides have been shown to exhibit several activities, including general smooth muscle contracting activity, although responses vary between species and vascular beds (Davenport, A., and Maquire, J., Trends in Pharmacological
• Human UII is found within both vascular and cardiac tissue (including coronary atheroma) and effectively constricts isolated arteries from non-human primates (Ames, H., et al, supra).
• the potency of vasoconstriction of UII is substantially greater than that of endothelin-1, making human UII one of most potent mammalian vasoconstrictors currently known.
• human UII markedly increases total peripheral resistance in anaesthetized non-human primates, a response associated with profound cardiac contractile dysfunction (Ames, H., et al, supra).
• UII cardiovascular homeostasis and pathology ⁇ e.g., ischemic heart disease and congestive heart failure.
• UII may have additional activities, including modulation of central nervous system and endocrine function in humans (Ames, H., et al, supra).
• a number of maladies have been potentially linked to an excess or an under expression of UII activity, including ischemic heart failure, hypotension, portal hypertension, angina pectoris, variceal bleeding, myocardial infarction, ulcers, and certain psychological and neurological disorders.
• potent compounds capable of modulating UII activity.
• the polypeptides of the invention are UII agonist peptides having the general formula:
• R 1 -cyclo(AA' - AA 2 - AA 3 -AA 4 - AA 5 - AA 6 )-AA 7 -NH 2 , wherein AA 1 is Cys, N-Me-Cys, or Pen;
• AA 2 is Phe
• AA 3 is Tip or N-Me-Trp; AA 4 is Lys; AA 5 is Tyr or Cpa; AA 6 is Cys or Pen; AA 7 is VaI, Ala, or Thr; and R 1 is Asp, N-Me- Asp, or succinyl.
• the peptide of this invention according to the above Formula I has either one or more N-Me amino acid(s) or one or more Pen residue(s).
• polypeptides of the present invention are capable of altering UII activity and can affect the binding of UII to a receptor.
• these polypeptides may be administered to a subject as a means for preventing or treating medical or psychological conditions characterized by a deficiency or under expression of Urotensin-II activity.
• Such conditions include, but are not limited to, ischaemic heart disease, congestive heart failure, portal hypertension, variceal bleeding, hypotension, angina pectoris, myocardial infarction, ulcers, anxiety, schizophrenia, manic depression, delirium, dementia, mental retardation, and dyskinesias.
• the present invention also provides pharmaceutical compositions that include a therapeutically effective amount of a polypeptide of Formula I in combination with a pharmaceutically acceptable carrier.
• suitable carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
• the composition can be adapted for the mode of administration and can be in the form of a pill, tablet, capsule, spray, powder, or liquid.
• polypeptide any peptide (including cyclic peptides) or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds.
• Polypeptide refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins.
• Polypeptides may contain amino acids other than the 20 gene-encoded amino acids.
• Polypeptides include amino acid sequences modified either by natural processes, or by chemical modification techniques which are well known in the art. Modifications may occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini.
• the notations used herein for the polypeptide amino acid residues are those abbreviations commonly used in the art. The less common abbreviations Pen and Cpa stand for penicillamine and p-chlorophenylalanine, respectively.
• salt non-toxic acid addition salts or metal complexes which are commonly used in the pharmaceutical industry.
• acid addition salts include organic acids such as acetic, lactic, pamoic, maleic, citric, malic, ascorbic, succinic, benzoic, palmitic, suberic, salicylic, tartaric, methanesulfonic, toluenesulfonic, or trifluoroacetic acids or the like; polymeric acids such as tannic acid, carboxymethyl cellulose, or the like; and inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid phosphoric acid, or the like.
• Metal complexes include zinc, iron, and the like.
• variant is meant a polypeptide that differs from a reference polypeptide, but retains essential properties. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical.
• a variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, and/or deletions, in any combination.
• a substituted or inserted amino acid residue may or may not be one encoded by the genetic code.
• a variant of a polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polypeptides may be made by mutagenesis techniques or by direct synthesis.
• the variant differs from the reference polypeptide by conservative amino acid substitutions, whereby a residue is substituted by another with like characteristics (e.g., acidic, basic, aromatic, etc.).
• Typical substitutions are among Ala, VaI, Leu and He; among Ser and Thr; among the acidic residues Asp and GIu; among Asn and GIn; and among the basic residues Lys and Arg; or aromatic residues Phe and Tyr.
• subject an animal or human suffering from a UII-related physiological or psychological condition.
• the subject may be a mammal, including, but not limited to, humans and non-human mammals such as primates, dogs, cats, pigs, cows, sheep, goats, horses, rats, mice, and the like.
• pharmaceutically acceptable carrier is meant a carrier that is physiologically acceptable to an administered animal while retaining the therapeutic properties of the compound with which it is administered.
• One exemplary pharmaceutically acceptable carrier is physiological saline.
• physiologically acceptable carriers and their formulations are known to one skilled in the art and described, for example, in Remington's Pharmaceutical Sciences, (18 th edition), ed. A. Gennaro, 1990, Mack Publishing Company, Easton, PA, U.S.A.
• Some of these peptides were discovered to have from moderate to high affinity for UII receptors and were tested for their contractile activity on circular strips of rat upper thoracic aorta which undergo potent tonic contractions in response to UII agonist peptides.
• polypeptides of the invention are UII agonist peptides having the general formula: R 1 -CVcIo(AA 1 -AA 2 - AA 3 - AA 4 - AA 5 - AA 6 )-AA 7 -NH 2 , wherein
• AA 1 is Cys, N-Me-Cys, or Pen;
• AA 2 is Phe;
• AA 3 is Tip or N-Me-Trp; AA 4 is Lys;
• AA 5 is Tyr or Cpa
• AA 6 is Cys or Pen
• AA 7 is VaI, Ala, or Thr
• R 1 is Asp, N-Me-Asp, or succinyl.
• the UII analogs have agonist activities as evidenced by their ability to stimulate contractions of rat upper thoracic aorta muscle in vitro, as does UII itself.
• the polypeptides of the invention are capable of modulating UII activity and are, therefore, useful for treating physiological and psychological conditions related to a deficiency or under expression of UII activity within a subject.
• physiological and psychological conditions include, for example, acute heart failure, hypotension, hypertension, angina pectoris, variceal bleeding, myocardial infarction, ulcers, and certain psychological and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, mental retardation, and dyskinesias.
• one approach to treatment is to administer to a subject in need thereof a compound which activates UII (agonist), optionally in combination with a pharmaceutically acceptable carrier, in an amount effective to activate the function of UII.
• a therapeutically effective amount of a polypeptide of Formula I, or a variant or pharmaceutically acceptable salt-thereof can be administered orally, parenterally (e.g., intramuscular, intraperitoneal, intravenous or subcutaneous injection, or implant), nasally, vaginally, rectally, sublingually or topically, in admixture with a pharmaceutically acceptable carrier adapted for the route of administration.
• parenterally e.g., intramuscular, intraperitoneal, intravenous or subcutaneous injection, or implant
• nasally, vaginally, rectally, sublingually or topically in admixture with a pharmaceutically acceptable carrier adapted for the route of administration.
• compositions intended for oral use may be prepared in solid or liquid forms according to any method known to the art for the manufacture of pharmaceutical compositions.
• the compositions may optionally contain sweetening, flavoring, coloring, perfuming, and/or preserving agents in order to provide a more palatable preparation.
• Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid forms, the active compound is admixed with at least one inert pharmaceutically acceptable carrier or excipient.
• Tablets and pills can additionally be prepared with enteric coatings.
• inert diluents such as calcium carbonate, sodium carbonate, lactose, sucrose, starch, calcium phosphate, sodium phosphate, or kaolin.
• Binding agents, buffering agents, and/or lubricating agents e.g., magnesium stearate
• Tablets and pills can additionally be prepared with enteric coatings.
• Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and soft gelatin capsules. These forms contain inert diluents commonly used in the art, such as water or an oil medium. Besides such inert diluents, compositions can also include adjuvants, such as wetting agents, emulsifying agents, and suspending agents.
• Formulations for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions, or emulsions.
• suitable vehicles include propylene glycol, polyethylene glycol, vegetable oils, gelatin, hydrogenated naphalenes, and injectable organic esters, such as ethyl oleate.
• Such formulations may also contain adjuvants, such as preserving, wetting, emulsifying, and dispersing agents.
• Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds.
• parenteral delivery systems for the polypeptides of the invention include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
• Liquid formulations can be sterilized by, for example, filtration through a bacteria-retaining filter, by incorporating sterilizing agents into the compositions, or by irradiating or heating the compositions. Alternatively, they can also be manufactured in the form of sterile, solid compositions which can be dissolved in sterile water or some other sterile injectable medium immediately before use.
• compositions for rectal or vaginal administration are preferably suppositories which may contain, in addition to active substances, excipients such as coca butter or a suppository wax.
• Compositions for nasal or sublingual administration are also prepared with standard excipients known in the art.
• Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops or spray, or as a gel.
• the amount of active ingredient in the compositions of the invention may be varied.
• One skilled in the art will appreciate that the exact individual dosages may be adjusted somewhat depending upon a variety of factors, including the polypeptide being administered, the time of administration, the route of administration, the nature of the formulation, the rate of excretion, the nature of the subject's conditions, and the age, weight, health, and gender of the patient.
• the severity of the UII- related condition being treated will also have an impact on the dosage level.
• dosage levels of between 0.1 ⁇ g/kg to 100 mg/kg of body weight are administered daily as a single dose or divided into multiple doses.
• the general dosage range is between 250 ⁇ g/kg to 5.0 mg/kg of body weight per day.
• polypeptides of the invention can be administered in a sustained release composition, such as those described in, for example, U.S. Patent Nos. 5,672,659 and 5,595,760.
• the use of immediate or sustained release compositions depends on the type of condition being treated.
• polypeptides of the present invention can be prepared in any suitable manner.
• Example 1 Preparation of Asp-cyclo(Cys-Phe-N-Me-Trp-Lys-Tyr-Cys)- VaI-NH 2 SEQ ID NO:3 Methylbenzhydrylamine polystyrene resin (1.0 g, 0.5 mmol) in the chloride ion form was placed in the reaction vessel of a CS Biosystems automatic peptide synthesizer programmed to perform the following reaction cycle: (a) wash with methylene chloride; (b) neutralize with 10% diisopropylamine in DMF; (c) wash with methylene chloride; (d) the neutralized resin was shaken with Boc-Val and diisopropylcarbodiimide coupling reagent (1 mmol each) in methylene chloride for 1 hour followed by washing of the resin with methylene chloride; and (e) removal of the Boc protecting group by treatment with 33% trifluoractetic acid in methylene chloride.
• the following amino acids were then coupled successively by the procedures (a) through (d): Boc-Phe, Boc-Cys(MeBzl), Boc-Asp(Bzl).
• the peptide was cleaved from the resin support with simultaneous side-chain deprotection by acidolysis using anhydrous hydrogen fluoride containing the scavenger anisole (-30% v/v) for 45 minutes at 0 °C.
• the peptides were cyclized in 90% acetic acid (-600 mL) with a slight excess of I 2 (15 min). Excess I 2 was then removed by the addition of ascorbic acid, solvent was reduce in vacuo and the crude peptides was subjected to HPLC purification.
• Methylbenzhydrylamine polystyrene resin (1.0 g, 0.5 mmol) in the chloride ion form was placed in the reaction vessel of a CS Biosystems automatic peptide synthesizer programmed to perform the following reaction cycle: (a) wash with methylene chloride; (b) neutralize with 10% diisopropylamine in DMF; (c) wash with methylene chloride; (d) the neutralized resin was shaken with Boc-Val and diisopropylcarbodiimide coupling reagent (1 mmol each) in methylene chloride for 1 hour followed by washing of the resin with methylene chloride; and (e) removal of the Boc protecting group by treatment with 33% trifluoractetic acid in methylene chloride.
• the following amino acids were then coupled successively by the same procedure: Boc-Cys(MeBzl), Boc-Tyr(ClZ), Boc-Lys(ClZ), Boc-Trp, Boc-Phe, Boc- Pen(MeBzl), Boc-Asp(Bzl).
• the peptide was cleaved from the resin support with simultaneous side-chain deprotection by acidolysis using anhydrous hydrogen fluoride containing the scavenger anisole ( ⁇ 30% v/v) for 45 minutes at 0 °C.
• the peptides were cyclized in 90% acetic acid (-600 mL) with a slight excess of I 2 (15 min). Excess I 2 was then removed by the addition of ascorbic acid, solvent was reduce in vacuo and the crude peptides was subjected to HPLC purification as described.
• the cDNAs of rat and human UII receptors were cloned by polymerase chain reaction from rat brain and human placenta cDNA (Clontech, Palo Alto, CA, U.S.A.) respectively, using gene specific primers flanking full-length coding sequence (Rossowki, WJ, et al, Eur. J. Phamacol., 438:159-170 (2002)).
• the receptors were cloned into the mammalian expression vector pcDNA 3.1 (Invitrogen, Carlsbad, CA, U.S.A.).
• the plasmids were transfected into Chinese hamster ovary cell line, CHO- Kl (American Type Culture Collection, Rockville, MD, U.S.A.), by the calcium phosphate method.
• Single cell clones stably expressing the rat or the human UII receptor were obtained by selecting transfected cells grown in cloning rings in RPMl 1640 media supplemented with 10% fetal bovine serum and 1 mM sodium pyruvate containing 0.8 mg/ml G418 (Gibco BRL, Grand Island, NY, U.S.A.).
• Membranes were prepared for radioligand binding studies by homogenization of the recombinant cells in 20 ml of ice-cold 50 mM Tris-HCl with a Brinkmann Polytron (Westbury, NY, U.S.A.) (setting 6, 15 seconds). The homogenates were washed twice by centrifugation (39,000 x g, 10 minutes), and the final pellets were resuspended in 50 mM Tris-HCl, containing 2.5 mM MgCl 2 , 0.1 mg/ml bacitracin (Sigma, St. Louis, MO, U.S.A.) and 0.1% BSA.
• the filters were then washed three times with 5-ml aliquots of ice-cold 50 mM Tris-HCl and 0.1% bovine serum albumin and the bound radioactivity trapped on the filters was counted by gamma spectrometry (Wallac LKB, Gaithersburg, MD, U.S.A.). Specific binding was defined as the total [ 125 I]Goby-urotensin II bound minus that bound in the presence of 1000 nM Goby- urotensin II (Bachem, Torrance, CA, U.S.A.).
• the thoracic aorta was dissected, freed from connective tissue, and cut into rings of about 1.5 mm in width.
• the rings were suspended in a 15 ml organ bath containing high potassium Kreb's solution (9.15 g/L potassium chloride, 2.1 g/L sodium bicarbonate, 1.0 g/L glucose, 0.16 g/L potassium phosphate monobasic, 0.14 g/L magnesium sulfate (anhydr.), and 0.22 g/L calcium chloride (dihydr.))
• Optimal tension was applied (0.2 g) to the tissues and the bath medium was maintained at 37 0 C and bubbled with a mixture of 95% O 2 / 5% CO 2 .
• Peptides in the concentration ranges of 10 "6 to 10 "12 M/L in a final volume of 16-80 ⁇ L were direcly introduced into the tested organ bath containing Krebs buffer continuously gassed with 95% O 2 and 5% CO 2 and the aorta rings at an optimal resting tension (1 - 0.2 g).
• Peptide-induced changes in tension of the aorta rings were recorded by force- displacement transducers and processed by the computer system BIOPAC Inc., as described above. Each ring was exposed to one peptide concentration only.

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• 2009
  • 2009-01-20 WO PCT/US2009/000354 patent/WO2009094137A2/fr not_active Ceased

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