WO2010127381A1 - Diagnostic, contrôle, pronostic, prévention et traitement des anévrysmes - Google Patents

Diagnostic, contrôle, pronostic, prévention et traitement des anévrysmes Download PDF

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Publication number
WO2010127381A1
WO2010127381A1 PCT/AU2009/000579 AU2009000579W WO2010127381A1 WO 2010127381 A1 WO2010127381 A1 WO 2010127381A1 AU 2009000579 W AU2009000579 W AU 2009000579W WO 2010127381 A1 WO2010127381 A1 WO 2010127381A1
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kks
substituted
gene
aneurysm
antagonist
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PCT/AU2009/000579
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English (en)
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Jonathan Golledge
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James Cook University
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Publication of WO2010127381A1 publication Critical patent/WO2010127381A1/fr

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    • 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
    • A61K38/55—Protease inhibitors
    • A61K38/57—Protease inhibitors from animals; from humans
    • 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
    • A61K38/043—Kallidins; Bradykinins; Related peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/329—Diseases of the aorta or its branches, e.g. aneurysms, aortic dissection

Definitions

  • This invention relates generally to methods for the diagnosis, monitoring, prognosis, prevention and treatment of aneurysms in individuals.
  • An aneurysm is a localized, blood-filled dilatation of a blood vessel being an artery, vein or heart.
  • Aortic aneurysms are classified by which site within the aorta they occur.
  • Abdominal aortic aneurysms (AAAs), the most common form of aortic aneurysm are found in the abdominal aorta, and thoracoabdominal aortic aneurysms involve both the thoracic and abdominal aorta.
  • An aortic root aneurysm occurs at the commencement of the aorta.
  • Thoracic aortic aneurysms are found within the thoracic aorta and are further classified as ascending, aortic arch, or descending aneurysms depending on the location within the thoracic aortic involved.
  • aneurysms Initially most intact aneurysms are asymptomatic. If left untreated, aneurysms tend to become progressively larger, although the rate of enlargement is unpredictable for any individual. As the size of an aneurysm increases, pain and numbness may be experienced, normally caused by nerve compression. Further, the risk of rupture increases as the size of an aneurysm enlarges. When rupture occurs, symptoms including a drop in blood pressure, increased heart rate, and loss of consciousness are often observed and can result in severe haemorrhage or other complications including sudden death. There is a low survival rate after rupture has occurred, even if surgery is performed. [0006] The result is that aneurysms place a large burden on the health system.
  • AAA for example, is the 10 most common cause of death in men aged over 60 years (Tilson, 2005). In 2004, 470 deaths (5/100,000/yr) and 2,169 hospitalisations (50/100,000/yr) as a result of AAA were recorded in New South Wales, Australia alone (Report by Population Health Division, 2007). This burden is growing as the numbers of identified early AAAs increases, caused by both the growth in the elderly population and an increase in the use of abdominal imaging (Lederle, et al. , 1997).
  • aneurysms are deficient in a number of areas. At this time, for example, there are no blood-based diagnostic tests available. Diagnosis (and monitoring) of intact aneurysms is usually achieved by medical imaging, normally ultrasound. Current imaging protocols simply provide anatomical information on the aorta, such as maximum aortic diameter. AAA 5 for example, is thus diagnosed most commonly using a definition of maximum infrarenal aortic diameter greater than or equal to 30mm. Whilst aortic diameter provides some predictive power with respect to the subsequent behaviour of the aorta, the information provided is incomplete. Some aortas measuring >30mm remain stable for many years with no progression in diameter or risk of rupture.
  • AAAs aneurysms
  • the available treatments for aneurysms are also limited.
  • the only treatments which are currently employed are open or endovascular surgery.
  • Open surgery involves a large laparotomy, clamping of the aneurysm above and below, and replacing the weakened artery with a plastic graft. This operation is associated with a significant mortality of around 5%.
  • Endovascular AAA repair is a more minimally invasive procedure whereby stent grafts are secured within the aneurysm from inside via a groin approach.
  • the technique is associated with a lower mortality of approximately 1% and a smaller rate of perioperative complications and faster recovery than for open surgery.
  • There is some concern over the long term outcome for this procedure since reintervention is not uncommon during long term follow up in up to 20% of patients.
  • AAAs with a maximum diameter ⁇ 55mm Patients with an AAA measuring ⁇ 50mm are thus usually treated conservatively, and simply monitored by regular imaging. Only large aneurysms or those that have ruptured are considered eligible for the surgical or endovascular treatment options. There is therefore an "untreated group" of patients with aneurysms, including those with small aneurysms and those for which these treatments are not an option for other reasons, for example if surgery is considered too high risk.
  • the kallikrein-kinin system represents a metabolic cascade from kininogens to kinins which exert their pharmacological activities by binding specific receptors.
  • the kallikrein-kinin system therefore consists of a large number of proteins and smaller polypeptides, and includes enzymes that activate and deactivate various other components in the system.
  • HMWK high molecular weight kininogen
  • LMWK low molecular weight kininogen
  • HMWK and LMWK have no activity in themselves but are precursors to kinins, including bradykinin and kallidin.
  • HMWK is produced by the liver, while
  • LMWK is protected locally by numerous tissues, and secreted together with tissue kallikrein.
  • the kallikreins of the kallikrein-kinin system are serine proteases that liberate the kinins from the kininogens. Tissue kallikrein is initially secreted as the inactive prokallikrein, and plasma kallikrein is initially secreted as the inactive prekallikrein. Both prokallikrein and prekallikrein require activation by factors XII or other stimuli.
  • tissue kallikrein 1 (KLKl) encoded by the gene KLKl, is the only tissue kallikrein associated with the kallikrein-kinin system.
  • the kinins are small peptides. Bradykinin is a nonapeptide with the amino acid sequence Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg, and is produced when plasma kallikrein releases it from HMWK.
  • Kallidin (Lys-bradykinin) is a decapeptide with the same amino acid sequence as bradykinin but with the addition of a lysine at the N-terminus. Kallidin is released from HMWK or LMWK by tissue kallikrein.
  • Bradykinin may also be generated by aminopeptidase-mediated cleavage of kallidin.
  • Alternative pathways of kinin formation involving enzymes other than kallikreins operate, for example in some disease states.
  • a proportion of kininogens are hydroxylated which leads to the formation of hydroxylated kinins which have similar biological activity to non-hydroxylated kinins.
  • Bradykinin and kallikrein are metabolised by carboxypeptidases.
  • the carboxypeptidases are present in two forms: N circulates and M is membrane-bound.
  • the carboxypeptidases remove arginine residues at the carboxy-terminus of bradykinin and kallidin.
  • Carboxypeptidase metabolites of the kinins include: des-Arg9-BK and Ly s- des-Arg9-BK.
  • kinin when used in reference to humans and most mammals refers to bradykinin and kallidin, the hydroxylated forms of each, and also to carboxy-terminal des-Arg metabolites of bradykinin, kallidin or the hydroxylated forms of each.
  • the kinins exert their pharmacological activities by binding specific kinin receptors: being type 1 (Bl) and type 2 (B2) receptors.
  • Kinin production in vivo is controlled, in part, by endogenous inhibitors of the kallikrein enzymes.
  • the main inhibitors of plasma kallikrein are the Cl inhibitor (Cl-INH - encoded in humans by the gene SERPINGl), ⁇ 2 -macroglobulin (encoded in humans by the gene A2M) and antithrombin III (encoded in humans by the gene SERPINCl).
  • Tissue kallikrein is inhibited by kallistatin which in humans is encoded by the gene SERPINA4.
  • ACE angiotensin converting enzyme
  • neutral endopeptidase deactivate certain peptide mediators, including kinins.
  • altering the levels of components in the kallikrein-kinin system can assist in preventing or treating aneurysms in an individual, and that detecting the presence, determining the prognosis or monitoring the progression of an aneurysm may be achieved by detecting aberrant expression of one or more genes in the kallikrein-kinin system (KKS), as described hereinafter.
  • the present invention provides methods for preventing or treating an aneurysm in an individual. These methods generally comprise administering to the individual an effective amount of a KKS antagonist.
  • KKS antagonists include small organic molecules, nucleic acids, peptides, polypeptides, proteins, proteoglycans, peptidomimetics, carbohydrates, sugars, lipids or other organic (carbon containing) or inorganic molecules, including those as further described herein.
  • the KKS antagonist modulates the expression of a gene or the level or functional activity of an expression product of the gene, wherein the gene is selected from a gene encoding a component of the KKS (e.g.
  • the agent reduces the expression of a gene (e.g. KNGl, KLKl, KLKBl, BDKRBl, BDKRB2) or the level or functional activity of an expression product of that gene (e.g. HMWK, LMWK 5 prekallikrein, prokallikrein, plasma kallikrein, tissue kallikrein, bradykinin, kallidin, des-Arg9-bradykinin, Lys-des- Arg9-bradykinin, the kinin Bl receptor, the kinin B2 receptor).
  • the agent increases the expression of a gene (e.g.
  • SERPINGl A2M, SERPINCl, SERPINA4
  • level or functional activity of an expression product of that gene e.g. Cl -INH, ⁇ 2 -macroglobulin, antithrombin III, kallistatin
  • the agent reduces or increases the expression of the gene or the level of functional activity of an expression product of that gene by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% relative to the expression, level or functional activity in the absence of the agent.
  • the aneurysm is an aortic aneurysm.
  • An illustrative example of this type of aneurysm includes AAA.
  • Another aspect of the present invention contemplates the use of an effective amount of an agent, which is optionally formulated with a pharmaceutically acceptable carrier or diluent, for preventing or treating an aneurysm in an individual, wherein the agent comprises a KKS antagonist as broadly described herein.
  • the aneurysm is an aortic aneurysm.
  • An illustrative example of this type of aneurysm includes AAA.
  • the present invention resides in the use of an effective amount of an agent in the manufacture of a medicament for preventing or treating an aneurysm in an individual, wherein the agent comprises a KKS antagonist as broadly described herein.
  • the aneurysm is an aortic aneurysm.
  • An illustrative example of this type of aneurysm includes AAA.
  • the present invention provides methods for detecting the presence of an aneurysm, determining the prognosis of an aneurysm, or monitoring the progression of an aneurysm in an individual.
  • One suitable method comprises detecting in the individual the presence of an aberrant gene encoding a component of the KKS or detecting in the individual aberrant expression of a gene encoding a component of the KKS.
  • the aberrant gene is selected from an aberrant KNGl, KLKl, KLKBl, BDKRBl, BDKRB2, SERPINGl, A2M, SERPINCl, or SERPINA4 gene.
  • Another suitable method comprises determining in the individual a level or functional activity of a component in the KKS, which is different than a reference or control level (e.g. no aneurysm, level measured in the same individual at an earlier time) or functional activity of the component.
  • the method comprises determining an increase or elevation in the level of functional activity of the component (e.g.
  • the method comprises determining a decrease in the level or functional activity of the component (e.g. Cl-INH, ⁇ 2 -macroglobulin, antithrombin III, kallistatin) relative to a normal (e.g. no aneurysm, level measured in the same individual at an earlier time) reference or control level or functional activity of the component.
  • the method is performed on a biological sample obtained from the individual.
  • the aneurysm is an aortic aneurysm.
  • An illustrative example of this type of aneurysm includes AAA.
  • the invention provides methods for identifying agents that antagonise the KKS.
  • One suitable method comprises contacting a preparation with a test agent, wherein the preparation comprises (i) a polypeptide comprising an amino acid sequence corresponding to at least a biologically active fragment of a polypeptide component of the KKS, or to a variant or derivative thereof; or (ii) a polynucleotide comprising at least a portion of a genetic sequence (e.g.
  • a transcriptional control element such as a promoter or a czs-acting sequence
  • a transcriptional control element that regulates a nucleotide sequence that encodes at least a biologically active fragment of a polypeptide component of the KKS, or a variant or derivative thereof, , which is operably linked to a reporter gene; or (iii) a polynucleotide comprising a nucleotide sequence that encodes a polypeptide according to (i).
  • a detected change in the level and/or functional activity of the polypeptide component, or an expression product of the reporter gene, relative to a reference and/or control level or functional activity in the absence of the test agent indicates that the agent modulates the KKS .
  • Another suitable method comprises contacting a sample of cells expressing a kinin receptor with a kinin and a test agent.
  • the agents identified using the methods broadly described above are useful for preventing or treating an aneurysm in an individual.
  • the aneurysm is an aortic aneurysm.
  • An illustrative example of this type of aneurysm includes AAA.
  • Still another aspect of the present invention provides methods of producing an agent comprising a KKS antagonist for preventing or treating an aneurysm in an individual. These methods generally comprise: testing a test agent as broadly described above; and synthesising the agent on the basis that it tests positive for antagonising the KKS.
  • the method further comprises derivatising the agent, and optionally formulating the derivatised agent with a pharmaceutically acceptable carrier or diluent, to improve the efficacy of the agent for treating or preventing the aneurysm.
  • the aneurysm is an aortic aneurysm.
  • An illustrative example of this type of aneurysm includes AAA.
  • Figure 1 shows some of the results of Example 2.
  • bands of the B2 results are shown in the top line for samples taken from the neck (left hand side) and the body (right hand side) of the aneurysm.
  • the bottom line of Figure 1 shows the bands of the control, ⁇ -actin.
  • Figure 2 also shows some of the results of Example 2.
  • the concentration of both the Bl kinin receptor and B2 kinin receptor are shown.
  • the left result for each of Bl and B2 shows the results from the sample from the aneurysm neck, and the right result for each of Bl and B2 shows the results from the sample from the aneurysm body.
  • Figure 3 shows photographs of an aorta taken from a normal mouse (A — left hand side) and one from a mouse with an aneurysm (B — right hand side) from the experiment described in Example 3.
  • Figure 4 shows the survival rates of the four mice groups from the experiment described in Example 3.
  • an element means one element or more than one element.
  • allelic expression refers to the overexpression or underexpression of a gene encoding a component of the KKS relative to the level of expression of a gene encoding a component of the KKS or a variant thereof in cells obtained from a healthy individual or from an individual with no aneurysm, and/or to a higher or lower level of an expression product of that gene (e.g., transcript or polypeptide) in a biological sample obtained from a healthy individual or from an individual with no aneurysm.
  • transcript or polypeptide e.g., transcript or polypeptide
  • a gene encoding a component of the KKS is aberrantly expressed if the level of expression of the gene is higher by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, or even an at least about 100%, 200%,
  • the reference to a level of expression of a gene of expression product of that gene in a healthy individual or an individual with no aneurysm may refer to an average level of expression in a group of healthy individuals or individuals with no aneurysm.
  • alkoxy alkenoxy
  • alkynoxy alkynoxy
  • aryloxy heteroaryloxy
  • heterocyclyloxy acyloxy
  • alkyl is intended to include both branched and straight-chain saturated aliphatic hydrocarbon group and may have a specified number of carbon atoms.
  • Ci-Cio as in “Ci-Cioalkyl” is defined to include groups having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbons in linear or branched arrangement.
  • Ci-C 10 alkyl specifically includes, but is not limited to, methyl, ethyl, n- propyl, i-propyl, n-butyl, t-butyl, i-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl.
  • Alkoxy represents either a cyclic or non-cyclic alkyl group attached through an oxygen bridge. "Alkoxy” therefore encompasses the definitions of alkyl and cycloalkyl above.
  • alkoxy groups include but are not limited to methoxy, oxy ethoxy, n-propyloxy, i-propyloxy, cyclopentyloxy and cyclohexyloxy.
  • alkenyl refers to a non-aromatic hydrocarbon radical, straight, branched or cyclic, containing from 2 to 10 carbon atoms and at least one carbon to carbon double bond. Preferably one carbon to carbon double bond is present, and up to four non-aromatic carbon-carbon double bonds may be present.
  • C2-C 6 alkenyl means an alkenyl radical having from 2 to 6 carbon atoms.
  • Alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, 2-methylbutenyl and cyclohexenyl.
  • the straight, branched or cyclic portion of the alkenyl group may contain double bonds and may be substituted if a substituted alkenyl group is indicated.
  • alkynyl refers to a hydrocarbon radical straight, branched or cyclic, containing from 2 to 10 carbon atoms and at least one carbon to carbon triple bond. Up to three carbon-carbon triple bonds may be present.
  • C 2 -C 6 alkynyl means an alkynyl radical having from 2 to 6 carbon atoms.
  • Alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, 3-methylbutynyl and so on.
  • the straight, branched or cyclic portion of the alkynyl group may contain triple bonds and may be substituted if a substituted alkynyl group is indicated.
  • substituents may be defined with a range of carbons that includes zero, such as (Co-C 6 )alkylene-aryl. If aryl is taken to be phenyl, this definition would include phenyl itself as well as, for example, -CH 2 Ph, -CH 2 CH 2 Ph, CH(CH 3 )CH 2 CH(CH 3 )Ph.
  • alkylene refers to a straight, branched or cyclic, preferably straight or branched, bivalent aliphatic hydrocarbon group, preferably having from 1 to about 20 carbon atoms, more preferably 1 to 12 carbons, even more preferably lower alkylene.
  • the alkylene group is optionally substituted with one or more "alkyl group substituents.” There may be optionally inserted along the alkylene group one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms, where the nitrogen substituent is alkyl as previously described.
  • exemplary alkylene groups include methylene (-CH 2 -), ethylene (-CH 2 CH 2 -), propylene (-(CH 2 ) 3 -), cyclohexylene (-C 6 H 10 -), methylenedioxy (-0-CH 2 -O-) and ethylenedioxy (-O-(CH 2 ) 2 -O-).
  • the term "lower alkylene” refers to alkylene groups having 1 to 6 carbons. Preferred alkylene groups are lower alkylene, with alkylene of 1 to 3 carbon atoms being particularly preferred.
  • alkenylene refers to a straight, branched or cyclic, preferably straight or branched, bivalent aliphatic hydrocarbon group, preferably having from 2 to about 20 carbon atoms and at least one double bond, more preferably 2 to 12 carbons, even more preferably lower alkenylene.
  • the alkenylene group is optionally substituted with one or more "alkyl group substituents.” There may be optionally inserted along the alkenylene group one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms, where the nitrogen substituent is alkyl as previously described.
  • the term "lower alkenylene” refers to alkenylene groups having 2 to 6 carbons. Preferred alkenylene groups are lower alkenylene, with alkenylene of 3 to 4 carbon atoms being particularly preferred.
  • alkynylene refers to a straight, branched or cyclic, preferably straight or branched, bivalent aliphatic hydrocarbon group, preferably having from 2 to about 20 carbon atoms and at least one triple bond, more preferably 2 to 12 carbons, even more preferably lower alkynylene.
  • the alkynylene group is optionally substituted with one or more "alkyl group substituents.” There may be optionally inserted along the alkynylene group one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms, where the nitrogen substituent is alkyl as previously described.
  • the term "lower alkynylene” refers to alkynylene groups having 2 to 6 carbons. Preferred alkynylene groups are lower alkynylene, with alkynylene of 3 to 4 carbon atoms being particularly preferred.
  • “Amplification product” refers to a nucleic acid product generated by a nucleic acid amplification technique.
  • antigen-binding molecule a molecule that has binding affinity for a target antigen. It will be understood that this term extends to immunoglobulins, immunoglobulin fragments and non-immunoglobulin derived protein frameworks that exhibit antigen-binding activity.
  • Antigenic or immunogenic activity refers to the ability of a polypeptide, fragment, variant or derivative according to the invention to produce an antigenic or immunogenic response in an animal, suitably a mammal, to which it is administered, wherein the response includes the production of elements which specifically bind the polypeptide or fragment thereof.
  • Alkyl means alkyl as defined above which is substituted with an aryl group as defined above, e.g., -CH2 ⁇ henyl, -(CH2)2phenyl, -(CH2)3phenyl, - H2CH(CH3)CH2phenyl, and the like and derivatives thereof.
  • aromatic or aryl is intended to mean any stable monocyclic or bicyclic carbon ring of up to 7 atoms in each ring, wherein at least one ring is aromatic.
  • aryl elements include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, biphenyl, phenanthryl, anthryl or acenaphthyl.
  • biologically active fragment is meant a fragment of a full-length parent polypeptide which fragment retains an activity of the parent polypeptide.
  • biologically active fragment includes deletion variants and small peptides, for example of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 contiguous amino acid residues, which comprise an activity of the parent polypeptide.
  • Peptides of this type may be obtained through the application of standard recombinant nucleic acid techniques or synthesized using conventional liquid or solid phase synthesis techniques. For example, reference may be made to solution synthesis or solid phase synthesis as described, for example, in Chapter 9 entitled “Peptide Synthesis” by Atherton and Shephard which is included in a publication entitled “Synthetic Vaccines” edited by Nicholson and published by Blackwell Scientific Publications.
  • peptides can be produced by digestion of a polypeptide of the invention with proteinases such as endoLys-C, endoArg-C, endoGlu- C and staphylococcus V8-protease.
  • the digested fragments can be purified by, for example, high performance liquid chromatographic (HPLC) techniques.
  • biological sample refers to a sample that may be extracted, untreated, treated, diluted or concentrated from a patient.
  • the biological sample is a urine, whole blood, serum or plasma sample.
  • cycloalkenyl means a monocyclic unsaturated hydrocarbon group and may have a specified number of carbon atoms.
  • cycloalkenyl includes but is not limited to, cyclobutenyl, cyclopentenyl, 1- methylcyclopentenyl, cyclohexenyl and cyclohexadienyl.
  • cycloalkyl or "aliphatic ring” means a monocyclic saturated aliphatic hydrocarbon group and may have a specified number of carbon atoms.
  • cycloalkyl includes, but is not limited to, cyclopropyl, methyl- cyclopropyl, 2,2-dimethyl-cyclobutyl, 2-ethyl-cyclopentyl, cyclohexyl.
  • derivative is meant a polypeptide that has been derived from the basic sequence by modification, for example by conjugation or complexing with other chemical moieties or by post-translational modification techniques as would be understood in the art.
  • derivative also includes within its scope alterations that have been made to a parent sequence including additions or deletions that provide for functional equivalent molecules.
  • an aneurysm in the context of treating or preventing an aneurysm is meant the administration of an amount of active ingredient to an individual in need of such treatment or prophylaxis, either in a single dose or as part of a series, that is effective for treatment or prophylaxis or improvement of that condition.
  • improvements in an individual include reducing the size of an aneurysm, preventing an increase in the size of an aneurysm in an individual, reducing the number of small aneurysms in an individual, and/or preventing or inhibiting the occurrence of aneurysms in an individual.
  • the effective amount will vary depending upon the health and physical condition of the individual to be treated, the formulation of the composition being administered, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
  • expression product refers to production of mRNA, translation of RNA message into proteins or polypeptides, or processed forms of those proteins or polypeptides.
  • function refers to a biological, enzymatic, or therapeutic function.
  • the term "gene” as used herein refers to any and all discrete coding regions of the cell's genome, as well as associated non-coding and regulatory regions.
  • the gene is also intended to mean the open reading frame encoding specific polypeptides, introns, and adjacent 5' and 3' non-coding nucleotide sequences involved in the regulation of expression.
  • the gene may further comprise control signals such as promoters, enhancers, termination and/or polyadenylation signals that are naturally associated with a given gene, or heterologous control signals.
  • the DNA sequences may be cDNA or genomic DNA or a fragment thereof.
  • the gene may be introduced into an appropriate vector for extrachromosomal maintenance or for integration into the host.
  • halo or halogen as used herein is intended to include chloro, fluoro, bromo and iodo.
  • Heteroaralkyl means alkyl as defined above which is substituted with a heteroaryl group, e.g., -CHbpyridinyl, -(CH 2 ) 2 pyrimidinyl, - (CH 2 ) 3 imidazolyl, and the like, and derivatives thereof.
  • heteroaryl or “heteroaromatic,” as used herein, represents a stable monocyclic or bicyclic ring of up to 7 atoms in each ring, wherein at least one ring is aromatic and contains from 1 to 4 heteroatoms selected from the group consisting of O, N and S.
  • Heteroaryl groups within the scope of this definition include but are not limited to: acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrrazolyl, indolyl, benzotriazolyl, furanyl, thienyl, benzothienyl, bezofuranyl, quinolinyl, isoquinolinyl, oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, tetrahydroquinoline.
  • heteroaryl is also understood to include the N-oxide derivative of any nitrogen-containing heteroaryl.
  • heteroaryiene refers to a bivalent monocyclic or multicyclic ring system, preferably of about 3 to about 15 members where one or more, more preferably 1 to 3 of the atoms in the ring system is a heteroatom, that is, an element other than carbon, for example, nitrogen, oxygen and sulfur atoms.
  • the heteroaryiene group may be optionally substituted with one or more, preferably 1 to 3, aryl group substituents.
  • Exemplary heteroaryiene groups include, for example, 1,4-imidazolylene.
  • heterocycle means a 5- to 10-membered nonaromatic heterocycle containing from 1 to 4 heteroatoms selected from the group consisting of O, N and S, and includes bicyclic groups.
  • heterocyclylalkyl means alkyl as defined above which is substituted with a heterocycle group, e.g., -CHbpyrrolidin-l-yl, -(CH 2 ) 2 piperidin-l-yl, and the like, and derivatives thereof.
  • Hybridization is used herein to denote the pairing of complementary nucleotide sequences to produce a DNA-DNA hybrid or a DNA-RNA hybrid.
  • Complementary base sequences are those sequences that are related by the base-pairing rules.
  • match and mismatch refer to the hybridization potential of paired nucleotides in complementary nucleic acid strands. Matched nucleotides hybridize efficiently, such as the classical A-T and G-C base pair mentioned above. Mismatches are other combinations of nucleotides that do not hybridize efficiently.
  • hydrocarbyl as used herein includes any radical containing carbon and hydrogen including saturated, unsaturated, aromatic, straight or branched chain or cyclic including polycyclic groups. Hydrocarbyl includes but is not limited to Ci-Cealkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 10 cycloalkyl, aryl such as phenyl and naphthyl, Ar (C 1 -C 8 )alkyl such as benzyl, any of which may be optionally substituted.
  • Reference herein to "immuno-interactive” includes reference to any interaction, reaction, or other form of association between molecules and in particular where one of the molecules is, or mimics, a component of the immune system.
  • Suitable animals that fall within the scope of the invention include, but are not restricted to, primates, livestock animals (e.g., sheep, cows, horses, donkeys, pigs), laboratory test animals (e.g., rabbits, mice, rats, guinea pigs, hamsters), companion animals (e.g., cats, dogs) and captive wild animals (e.g., foxes, deer, dingoes, avians, reptiles).
  • livestock animals e.g., sheep, cows, horses, donkeys, pigs
  • laboratory test animals e.g., rabbits, mice, rats, guinea pigs, hamsters
  • companion animals e.g., cats, dogs
  • captive wild animals e.g., foxes, deer, dingoes, avians, reptiles.
  • isolated is meant material that is substantially or essentially free from components that normally accompany it in its native state.
  • modulating is meant increasing or decreasing, either directly or indirectly, the level or functional activity of a target molecule.
  • an agent may indirectly modulate the level/activity by interacting with a molecule other than the target molecule.
  • indirect modulation of a gene encoding a target polypeptide includes within its scope modulation of the expression of a first nucleic acid molecule, wherein an expression product of the first nucleic acid molecule modulates the expression of a nucleic acid molecule encoding the target polypeptide.
  • a sample such as, for example, a polynucleotide extract or polypeptide extract is isolated from, or derived from, a particular source of the host.
  • the extract can be obtained from a tissue or a biological fluid isolated directly from the host.
  • oligonucleotide refers to a polymer composed of a multiplicity of nucleotide residues (deoxyribonucleotides or ribonucleotides, or related structural variants or synthetic analogues thereof) linked via phosphodiester bonds (or related structural variants or synthetic analogues thereof).
  • oligonucleotide typically refers to a nucleotide polymer in which the nucleotide residues and linkages between them are naturally occurring, it will be understood that the term also includes within its scope various analogues including, but not restricted to, peptide nucleic acids (PNAs), phosphoramidates, phosphorothioates, methyl phosphonates, 2-O-methyl ribonucleic acids, and the like. The exact size of the molecule can vary depending on the particular application.
  • PNAs peptide nucleic acids
  • phosphoramidates phosphoramidates
  • phosphorothioates phosphorothioates
  • methyl phosphonates 2-O-methyl ribonucleic acids
  • oligonucleotide is typically rather short in length, generally from about 10 to 30 nucleotide residues, but the term can refer to molecules of any length, although the term “polynucleotide” or “nucleic acid” is typically used for large oligonucleotides.
  • operably linked is meant that transcriptional and translational regulatory polynucleotides are positioned relative to a polypeptide-encoding polynucleotide in such a manner that the polynucleotide is transcribed and the polypeptide is translated.
  • pharmaceutically acceptable carrier or diluent is meant a solid or liquid filler, diluent or encapsulating substance that can be safely used in topical or systemic administration to a mammal.
  • Phenylalkyl means alkyl as defined above which is substituted with phenyl, e.g., -CH 2 phenyl, (CH 2 ) 2 phenyl, -(CH 2 ) 3 phenyl, CH 3 CH(CH 3 )CH 2 phenyl, and the like and derivatives thereof. Phenylalkyl is a subset of the aralkyl group.
  • polynucleotide or “nucleic acid” as used herein designates mRNA, RNA, cRNA, cDNA or DNA. The term typically refers to oligonucleotides greater than 30 nucleotide residues in length.
  • polynucleotide variant and “variant” refer to polynucleotides displaying substantial sequence identity with a reference polynucleotide sequence or polynucleotides that hybridize with a reference sequence under stringent conditions as known in the art (see for example Sambrook et al. , Molecular Cloning. A Laboratory Manual", Cold Spring Harbor Press, 1989).
  • polynucleotides in which one or more nucleotides have been added or deleted, or replaced with different nucleotides.
  • certain alterations inclusive of mutations, additions, deletions and substitutions can be made to a reference polynucleotide whereby the altered polynucleotide retains a biological function or activity of the reference polynucleotide.
  • polynucleotide variant and “variant” also include naturally-occurring allelic variants.
  • Polypeptide “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally occurring amino acid, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • polypeptide variant refers to polypeptides in which one or more amino acids have been replaced by different amino acids. It is well understood in the art that some amino acids may be changed to others with broadly similar properties without changing the nature of the activity of the polypeptide (conservative substitutions) as described hereinafter. These terms also encompass polypeptides in which one or more amino acids have been added or deleted, or replaced with different amino acids.
  • Probe refers to a molecule that binds to a specific sequence or subsequence or other moiety of another molecule. Unless otherwise indicated, the term “probe” typically refers to a polynucleotide probe that binds to another polynucleotide, often called the "target polynucleotide", through complementary base pairing. Probes can bind target polynucleotides lacking complete sequence complementarity with the probe, depending on the stringency of the hybridization conditions. Probes can be labeled directly or indirectly.
  • Pseudohalides are groups that behave substantially similar to halides. Such groups can be used in the same manner and treated in the same manner as halides (X, in which X is a halogen, such as Cl or Br). Pseudohalides include, but are not limited to cyanide, cyanate, thiocyanate, selenocyanate, trifluoromethyl and azide.
  • recombinant polynucleotide refers to a polynucleotide formed in vitro by the manipulation of a polynucleotide into a form not normally found in nature.
  • the recombinant polynucleotide can be in the form of an expression vector.
  • expression vectors include transcriptional and translational regulatory polynucleotide operably linked to the polynucleotide.
  • recombinant polypeptide is meant a polypeptide made using recombinant techniques, i.e., through the expression of a recombinant or synthetic polynucleotide.
  • reference or control level refers to any suitable reference or control level, including, but not limited to, a normal healthy individual, an individual without an aneurysm, a level measured in the same individual at a different time, and a level measured in a biological sample taken from a different tissue in the same individual or control.
  • reporter molecule as used in the present specification is meant a molecule that, by its chemical nature, provides an analytically identifiable signal that allows the detection of a complex comprising an antigen-binding molecule and its target antigen.
  • reporter molecule also extends to use of cell agglutination or inhibition of agglutination such as red blood cells on latex beads, and the like.
  • stereoisomers As used herein, it will also be recognized that the compounds described herein may possess asymmetric centers and are therefore capable of existing in more than one stereoisomeric form.
  • the invention thus also relates to compounds in substantially pure isomeric form at one or more asymmetric centers e.g., greater than about 90% ee, such as about 95% or 97% ee or greater than 99% ee, as well as mixtures, including racemic mixtures, thereof.
  • Such isomers may be naturally occurring or may be prepared by asymmetric synthesis, for example using chiral intermediates, or by chiral resolution.
  • vector is meant a polynucleotide molecule, preferably a DNA molecule derived, for example, from a plasmid, bacteriophage, yeast or virus, into which a polynucleotide can be inserted or cloned.
  • a vector preferably contains one or more unique restriction sites and can be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or tissue thereof, or be integrable with the genome of the defined host such that the cloned sequence is reproducible.
  • the vector can be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a linear or closed circular plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome.
  • the vector can contain any means for assuring self-replication.
  • the vector can be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.
  • a vector system can comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon.
  • the choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced.
  • the vector is preferably a viral or viral-derived vector, which is operably functional in animal and preferably mammalian cells.
  • Such vector may be derived from a poxvirus, an adenovirus or yeast.
  • the vector can also include a selection marker such as an antibiotic resistance gene that can be used for selection of suitable transformants.
  • resistance genes are known to those of skill in the art and include the nptll gene that confers resistance to the antibiotics kanamycin and G418 (Geneticin®) and the hph gene which confers resistance to the antibiotic hygromycin B.
  • wild-type and "normal” are used interchangeably to refer to the phenotype that is characteristic of most of the members of the species occurring naturally and contrast for example with the phenotype of a mutant.
  • underscoring or italicizing the name of a gene shall indicate the gene, in contrast to its protein product, which is indicated by the name of the gene in the absence of any underscoring or italicizing.
  • KLKl shall mean the KLKl (tissue kallikrein, human kallikrein 1, hKl) gene
  • KLKl shall indicate the protein product or products generated from transcription and translation and alternative splicing of the "KLKl” gene.
  • the present invention is predicated in part on the discovery that the modulation of levels of components in the KKS, can increase or decrease the size of an aneurysm and/or the number of aneurysms and/or the likelihood of aneurysm rupture. It is proposed, therefore, that KKS antagonists will be useful inter alia for the prevention or treatment of an aneurysm, including intact or ruptured aneurysms.
  • the aneurysm may be located in an artery, a vein or the heart. Suitable arterial aneurysms include, but are not limited to, arterial aneurysms that occur at the base of the brain (the circle of Willis) and aortic aneurysms.
  • Suitable aortic aneurysms include, but are not limited to, abdominal aortic aneurysms (AAAs), thoracoabdominal aortic aneurysms, aortic root aneurysms, and thoracic aortic aneurysms.
  • AAAs abdominal aortic aneurysms
  • thoracoabdominal aortic aneurysms thoracoabdominal aortic aneurysms
  • aortic root aneurysms aortic root aneurysms
  • thoracic aortic aneurysms thoracic aortic aneurysms.
  • the present invention provides methods for preventing or treating an aneurysm in an individual, the methods comprising administering to the individual an effective amount of a KKS antagonist.
  • Another aspect of the present invention contemplates the use of an effective amount of an agent, which is optionally formulated with a pharmaceutically acceptable carrier or diluent, for preventing or treating an aneurysm in an individual, wherein the agent comprises a KKS antagonist.
  • the present invention resides in the use of an effective amount of an agent in the manufacture of a medicament for preventing or treating an aneurysm in an individual, wherein the agent comprises a KKS antagonist.
  • KKS antagonist refers to any agent which directly or indirectly agonises or antagonises a component in the KKS so as to inhibit or otherwise reduce the metabolic cascade in the KKS.
  • the KKS antagonist modulates the expression of a gene or the level or functional activity of an expression product of the gene, wherein the gene encodes a component of the KKS.
  • Representative genes encoding a component of the KKS include KNGl, KLKl, KLKBl, BDKRBl, BDKRB2, SERPINGl, A2M, SERPINCl, and SERPINA4.
  • Representative expression products of genes encoding components of the KKS include kininogen (e.g. HMWK and LMWK), kallikrein precursors (e.g. prekallikrein and prokallikrein), kallikrein (e.g. plasma kallikrein and tissue kallikrein), kinin (e.g. bradykinin, kallidin, des-Arg9-bradykinin, and Lys-des-
  • kininogen e.g. HMWK and LMWK
  • kallikrein precursors e.g. prekallikrein and
  • Arg9-bradykinin kinin receptor (e.g. the kinin Bl receptor and the kinin B2 receptor), Cl-INH, ⁇ 2 -macroglobulin, antithrombin III and kallistatin.
  • the KKS antagonist modulates the expression of a gene or the level or functional activity of an expression product of the gene, wherein the gene encodes an expression product which modulates directly or indirectly the expression of a gene encoding a component of the KKS.
  • the KKS antagonist modulates the expression of a gene or the level or functional activity of an expression product of the gene, wherein the gene encodes an expression product which modulates directly or indirectly the expression product of a gene encoding a component of the KKS.
  • the agent suitably modulates the expression of the gene directly, or modulates an upstream regulator of the expression of the gene, or directly or indirectly modulates the level or functional activity of an expression product of such genes.
  • the agent reduces the expression of a gene (e.g. KNGl, KLKl, KLKBl, BDKRBl, BDKRB2) or the level or functional activity of an expression product of that gene (e.g. HMWK, LMWK 5 prekallikrein, prokallikrein, plasma kallikrein, tissue kallikrein, bradykinin, kallidin, des-Arg9-bradykinin, Lys-des- Arg9-bradykinin, the kinin Bl receptor, the kinin B2 receptor).
  • the agent increases the expression of a gene (e.g.
  • the agent reduces or increases the expression of the gene or the level of functional activity of an expression product of that gene by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% relative to the expression, level or functional activity in the absence of the agent.
  • Suitable agents for reducing or abrogating gene expression include, but are not restricted to, oligoribonucleotide sequences, including anti-sense RNA and
  • Anti- sense RNA and DNA molecules act to directly block the translation of mRNA by binding to targeted mRNA and preventing protein translation.
  • antisense DNA oligodeoxyribonucleotides derived from the translation initiation site, e.g., between -10 and +10 regions are preferred.
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by a endonucleolytic cleavage.
  • engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of target sequences.
  • Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences, GUA, GUU and GUC.
  • RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for predicted structural features such as secondary structure that may render the oligonucleotide sequence unsuitable.
  • the suitability of candidate targets may also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using ribonuclease protection assays.
  • RNA molecules and DNA molecules and ribozymes may be prepared by any method known in the art for the synthesis of RNA molecules. These include techniques for chemically synthesizing oligodeoxyribonucleotides well known in the art such as for example solid phase phosphoramidite chemical synthesis.
  • RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule.
  • DNA sequences may be incorporated into a wide variety of vectors which incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters.
  • RNA polymerase promoters such as the T7 or SP6 polymerase promoters.
  • antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.
  • DNA molecules may be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribo- or deoxy- nucleotides to the 5' or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the oligodeoxyribonucleotide backbone.
  • RNA molecules that mediate RNA interference (RNAi) of a target gene or gene transcript can be used to reduce or abrogate gene expression.
  • RNAi refers to interference with or destruction of the product of a target gene by introducing a single stranded, and typically a double stranded RNA (dsRNA) that is homologous to the transcript of a target gene.
  • dsRNA double stranded RNA
  • dsRNA ⁇ er se and especially dsRNA-producing constructs corresponding to at least a portion of a target gene may be used to reduce or abrogate its expression.
  • RNAi-mediated inhibition of gene expression may be accomplished using any of the techniques reported in the art, for instance by transfecting a nucleic acid construct encoding a stem-loop or hairpin RNA structure into the genome of the target cell, or by expressing a transfected nucleic acid construct having homology for a target gene from between convergent promoters, or as a head to head or tail to tail duplication from behind a single promoter. Any similar construct may be used so long as it produces a single RNA having the ability to fold back on itself and produce a dsRNA, or so long as it produces two separate RNA transcripts which then anneal to form a dsRNA having homology to a target gene.
  • Absolute homology is not required for RNAi, with a lower threshold being described at about 85% homology for a dsRNA of about 200 base pairs (Plasterk, R. H., et al, 2000). Therefore, depending on the length of the dsRNA, the RNAi- encoding nucleic acids can vary in the level of homology they contain toward the target gene transcript, i.e. , with dsRNAs of 100 to 200 base pairs having at least about 85% homology with the target gene, and longer dsRNAs, i.e., 300 to 100 base pairs, having at least about 75% homology to the target gene.
  • RNA-encoding constructs that express a single RNA transcript designed to anneal to a separately expressed RNA, or single constructs expressing separate transcripts from convergent promoters are preferably at least about 100 nucleotides in length.
  • RNA-encoding constructs that express a single RNA designed to form a dsRNA via internal folding are preferably at least about 200 nucleotides in length.
  • the promoter used to express the dsRNA-forming construct may be any type of promoter if the resulting dsRNA is specific for a gene product in the cell lineage targeted for destruction. Alternatively, the promoter may be lineage specific in that it is only expressed in cells of a particular development lineage. This might be advantageous where some overlap in homology is observed with a gene that is expressed in a non-targeted cell lineage.
  • the promoter may also be inducible by externally controlled factors, or by intracellular environmental factors.
  • RNA molecules of about 21 to about 23 nucleotides which direct cleavage of specific mRNA to which they correspond, as for example described by Tuschl et al. in U.S. Patent Application No. 20020086356, can be utilized for mediating RNAi.
  • Such 21-23 nt RNA molecules can comprise a 3' hydroxyl group, can be single-stranded or double stranded (as two 21-23 nt RNAs) wherein the dsRNA molecules can be blunt ended or comprise overhanging ends (e.g., 5', 3').
  • Suitable agents for modulating the level or functional activity of an expression product of a gene include, but are not restricted to small organic molecules, nucleic acids, peptides, polypeptides, proteins, proteoglycans, peptidomimetics, carbohydrates, sugars, lipids or other organic (carbon containing) or inorganic molecules, as further described herein.
  • antigen-binding molecules can be used to modulate the level or functional activity of an expression product of a gene.
  • binding or activation of the kinin Bl receptor and/or the kinin B2 receptor is the subject of the targeting.
  • inhibition or abrogation of receptor signalling is achieved through reduction in receptor expression, receptor mutation (in particular, but not exclusively, of phosphorylation sites), prevention of receptor aggregation or through approaches that interfere with ligand-receptor interaction including those via blockade of the active binding sites or relevant associated motifs.
  • Such strategies include blocking antibodies to the receptors and small molecule inhibitors of binding. Pharmacological strategies to impair receptor phosphorylation can also be effective.
  • the kinin receptor antagonist may inhibit both the kinin Bl receptor and kinin B2 receptor.
  • the kinin receptor antagonist is a selective kinin Bl receptor antagonist.
  • the kinin receptor antagonist is a selective kinin B2 receptor antagonist.
  • the kinin receptor antagonist is a mutant kinin that binds to and recognises a kinin Bl receptor and/or a kinin B2 receptor but is unable to induce the pharmacological activities induced when a wild-type kinin binds to a kinin receptor.
  • the mutant kinin varies from the corresponding wild-type kinin by the deletion or modification of all or part of the nuclear translocation sequence, which renders it inoperative.
  • the present invention also contemplates the use of receptor antagonistic antigen-binding molecules with varying blocking capacities.
  • Exemplary receptor antagonists include those disclosed herein, including B9430 (Gera et al, 1996), and those disclosed in Blakeney et al 2007, see pages 2967-2971.
  • the subject of the targeting is a component of post-receptor signal transduction.
  • the present invention also contemplates the use in the above method of gene or expression product inhibitors identified according to methods described herein.
  • the present invention provides methods for preventing or treating an aneurysm in an individual, the methods comprising administering to the individual a KKS antagonist.
  • Another aspect of the present invention contemplates the use of an agent, which is optionally formulated with a pharmaceutically acceptable carrier or diluent, for preventing or treating an aneurysm in an individual, wherein the agent comprises a KKS antagonist.
  • the present invention resides in the use of an agent in the manufacture of a medicament for preventing or treating an aneurysm in an individual, wherein the agent comprises a KKS antagonist.
  • KKS antagonists include small organic molecules, nucleic acids, peptides, polypeptides, proteins, proteoglycans, peptidomimetics, carbohydrates, sugars, lipids or other organic (carbon containing) or inorganic molecules, including those as further described herein.
  • Suitable small organic molecules include:
  • Z' is selected from O, S and NH;
  • Q is selected from the group consisting of -NR 4 R 5 , -OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, heterocyclic, substituted heterocyclic, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl and substituted heteroaryl;
  • R 1 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic;
  • R 2 and R 3 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic;
  • R 4 and R 5 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, heterocyclic, substituted heterocyclic and where R 4 and R 5 , together with the nitrogen atom pendent thereto are joined to form a heterocyclic, a substituted heterocyclic, a heteroaryl or a substituted heteroaryl group, provide that when R 4 or R 5 is a substituted alkyl group this group is not ⁇ CHR a ⁇ C(O)-NR
  • X is selected from the group consisting of hydrogen, halogen, alkyl, substituted alkyl, nitro, cyano, hydroxyl, alkoxy, substituted alkoxy, carboxy, carboxyl esters, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, acylamino, aminoacyl, and -C(O)NR 7 R 8 wherein R 7 and R 8 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocyclic and substituted heterocyclic, or R and R together with the nitrogen atom to which they are joined form a heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic;
  • provisos apply: [0146] A) when Z' is O; R 2 is H; R 3 is 5-(2, 4-dichlorophenyl)-imidazol-4- ylidene-[2-methyl-4-(N-(l-methylsufonyleth-2- -yl)-N-ethylamino)phenyl]-amine; X is H; and R 1 is l-(3-t-butyl-4-hydroxyphenyloxy)tridec-l-yl; then Q is not ethoxy;
  • R 1 is substituted aryl or substituted heteroaryl, the aryl or heteroaryl group is not substituted with -C(0)NH ⁇ W'-C(0)0R b or -C(O)NH ⁇ W' ⁇ C(O)NR b R c , where W is aryl, substituted aryl, heteroaryl or substituted heteroaryl;
  • Exemplary compounds include:
  • A is an acylating group, and preferably a hydrophobic acylating group, or an anti-inflammatory substituent selected from the group consisting of: Aaa,
  • B is an amino acid or substituted amino acid selected from the group consisting of: Bip, Ddip, F5F, F3MF, hPhe, MC2Y, NaI, NMF, OBPY 5 OBrZY, OC2Y,
  • R is a substituted amide, preferably having additional polar character, selected from the group consisting of: Abzp, Aem, Alp, Ambi, Apia, Apyr, AquR, Atmp,
  • Aic 2-Aminoindane-2-carboxylic acid
  • Aq2c Anthraquinone-2-carboxyl
  • AquR (R)-(+)-3-Aminoquinuclidine
  • Atfb 3-Amino-2,5,6-trifluorobenzoyl
  • 4Atfb 4-Aniino-2,3,5,6-tetrafluorobenzoyl
  • Atmp 4-Amino-2,2,6,6-tetramethylpiperidine
  • AtmpO 4-Amino-2,2,6,6-tetramethylpiperidinyloxy
  • B6 3-Hydroxy-5-(hydroxymethyl)-2-methyl-4-pyridylmethyl (Vitamin B6, Pyridoxamine)
  • B6P 3-Hydroxy-5-(hydroxymethyl)-2-methyl-4-pyridylmethyI- 5- phosphate
  • BapR (R)-(-)-l-Benzyl-3-aminopyrrolidine
  • BapS (S)-(+)-l-Benzyl-3-aminopyrrolidine
  • Bcpa bis(4-Chlorophenyl)acetyl
  • Bcpoa bis(4-Chlorophenoxy)acetyl
  • Bdbh (1 S,4S)-(+)-2-Benzyl-2,5-diazabicyclo[2.2. l]heptane
  • Bhp 1-Benzylhomopiperazine
  • Bipa 4-Biphenylacetyl
  • 4Bpc 4-Biphenylcarboxyl
  • Bzac 3-BenzoylacryIoyl
  • Cbp l-(4-Chlorobenzhydrylpiperazine)
  • Chbu 2-Cyclohexylbutyryl
  • Che ⁇ -Cyano-4-hydroxycinnamoyl
  • CHFB 4-Carboxy-hexafluorobutyryl
  • ChI Chlorambucil: 4-[p-(bis[2-Chloroethyl]amino)- phenyljbutyryl
  • Cmioc 3-(2-Chlorophenyl)-5-methylisoxazole-4-carbonyl
  • Daep l-(2-(Diallylamino)ethyl)piperazme
  • tDecl trans-4-(Diethylamino)cinnamyl
  • Dfc Diclofenac: 2-[(2,6-Dichlorophenyl)amino]phenylacetyl
  • cDmap cis-2,6-Dimethyl- 1 -allyl-piperazine
  • CDmm cis-2,6-Dimethylmorpholine
  • Dmmp cis-2,6-Dimethyl- 1 -(methoxycarbonylmethyl)piperazine
  • Esul Exisulindacyl: (Z)-5-Fluoro-2-methyl-[[4- (methylsulfonyl)phenyl]methylene]-lH-mdene-3-acetyl; (cis)
  • Fbhp l-(4-Fluorobenzyl)homopiperazine
  • F5bs Pentafluorobenzenesulfonyl
  • F5bz Pentafluorobenzoyl
  • F3c 2,3,5-Trifluorocinnamoyl
  • ⁇ Fcn ⁇ Fluorocinnamoyl
  • F5F Pentafluorophenylalanine
  • F3MF 4-Trifluoromethylphenylalanine
  • F5pa 2,3,4,5,6-Pentafluorophenylacetyl
  • Fpmp l-bis(4-Fluorophenyl)methylpiperazine
  • Fmpi (Z)-5-Fluoro-2-methyl-(4- ⁇ yridylidene)-3-indenylacetyl
  • F5po 2,3,4,5,6-Pentafluorophenoxyacetyl
  • F5Pt Pentafluorophenylthiocarbamyl
  • Fpdh (1 S,4S)-(-)-2-(4-Fluorophenyl)-2,5- diazabicyclo[2.2. ljheptane
  • HPhe Homo-phenylalanine
  • Indo Indomethacin: l-[p-Chlorobenzoyl]-5-methoxy-2- methylindole-3 acetyl
  • Inp Isonipecotic acid: hexahydroisonicotinic Isoquinolineacetyl
  • KtIc Ketorolac: (.+-.)5-benzoyl-2,3dihydro-lH- ⁇ yrrolizine-l- carboxyl: Toradol
  • Ktpf Ketoprofen: 2-(3-benzoylphenyl)propionyl
  • Matp 4-(Methylamino)-2,2,6,6-tetramethyl ⁇ iperidine
  • Mca 2-Methylcinnamoyl
  • ⁇ Mcn ⁇ -Methylcinnamoyl
  • Mcoa 7-Methoxycoumarin-4-acetyl
  • MC2Y N-Methyl-O-2,6-dichlorobenzyl-tyrosine
  • Mpz 1 -Methylpiperazine
  • MTPA ⁇ -Methoxy- ⁇ -trifluoromethylphenylacetyl
  • Napr Naproxen: ⁇ -Methoxy- ⁇ -methyl ⁇ -Naphthaleneacetyl
  • Nba Norbornane-2-acetyl
  • Nif Niflumic acid, 2-(3-[Trifluoromethyl]aniline)nicotinic acid
  • NMF N-Methylphenylalanine
  • OBPY O-Benzyl-phosphotyrosine
  • OCIY O-2,6-Dichlorobenzyl-3 5 5-diiodo-tyrosme
  • OC2Y 0-2,6-dichlorobenzyl tyrosine
  • Ocp 1-Octylpiperazine
  • Octe 2-Octenoyl
  • Pas p-Aminosalicyloyl
  • PBF p-Bromophenylalanine
  • PCNF p-Cyano-L-phenylalanine
  • Pep l-(2-Phenylethyl)piperazine
  • PFF p-Fluorophenylalanine
  • ⁇ Phc ⁇ -Phenylcinnamoyl
  • ⁇ Ptpa ⁇ -(Phenylthio)phenylacetyl
  • Pxa Pyridoxamine [4-(ammomethyl)-5-hydroxy-6-methyl- 3- pyridinemethanol]
  • CSsa cis-Styrenesulphonylacetyl
  • t ⁇ Sts trans- ⁇ -Styrenesulfonyl
  • Taa l,2,4-Triazole-acetyl
  • Tchc (1R,3R,4S,5R)-1 ,3,4,5-Tetrahydrocyclohexane-l -carboxyl
  • Tf2c trans-3,5-bis(Trifluoromethyl)cinnamoyl
  • 4Tfmb 4-(Trifluoromethoxy)benzoyl
  • Th2n 1 ,2,3 ,4-Tetrahydro-2-naphthoyl
  • Tic Tetrahydroisoquinoline-3-carboxylic acid
  • Tmb Trimethoxybenzoyl
  • Tmbp l-(2,4,6-Trimethylbenzyl)piperazine
  • Tmcc 2,2,3,3-Tetramethylcyclopropanecarboxyl
  • TmpC Carboxy-TEMPO: 4-carboxy-2,2,6,6- tetramethylpiperidinyloxy
  • Representative compounds include:
  • n is an integer from 0 to 4; [0399] p is zero or one; [0400] q is zero or one;
  • W is selected from the group consisting of O, S, and N, wherein:
  • R is selected from the group consisting of aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic;
  • R 1 and R 2 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic, or R 1 and R 2 together with the nitrogen atom to which they are attached form a heteroaryl, substituted heteroaryl, heterocyclic, or substituted heterocyclic;
  • each R 3 is independently selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, amino, substituted amino, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, aryloxy, substituted aryloxy, heteroaryl, substituted
  • R 7 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, acyl and acyloxy; [0412] or R 7 together with at least one of R 3 and the nitrogen and carbon atoms to which they are joined forms a fused ring heteroaryl, substituted heteroaryl, unsaturated heterocyclic or substituted unsaturated heterocyclic;
  • R 8 is selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, acyl and acyloxy;
  • alkylene-X a wherein alkylene is optionally substituted and X a is selected from the group consisting of -OH, cyano, and -NR b R b wherein each R b is independently as defined above;
  • Representative compounds include:
  • R 1 is phenyl, 1,2-dichlorophenyl, 3-methylphenyl, 3-bromophenyl, naphth-2-yl, indan-5-yl benzo[l,3]dioxol-5-yl or l,2,3,4-tetrahydronaphth-6-yl;
  • R 2 is hydrogen, halogen, unsubstituted Q-Qalkyl or substituted C 1 -
  • R 3 is hydrogen, halogen or d-C 4 alkyl
  • R 4 is hydrogen or C 1 -C 4 alkyl
  • R 5 is hydrogen or Cj-Qalkyl
  • R 6 is CH 2 OH; tetrazol-5-yl; 1 5 2,4-triazol-5-yl; 1 ,2,3-triazol-5-yl; C(O)OH, C(O)NH 2 ; or ZNH(CH 2 ) n CHR 7 R 8 , wherein Z is -C(O)- or -CH 2 -, n is zero, 1, 2,
  • R 7 is unsubstiruted or substituted Ci-C 4 alkyl, C(O)OH, C(O)OC r
  • R 8 is hydrogen, unsubstituted or substituted C]-C 4 alkyl, unsubstituted or substituted Cs-C 10 aryl or heteroC 5 -C 10 aryl or C 1 -C 4 alkylC 5 -C 10 aryl or Ci-C 4 alkyl- heteroCs- Cioaryl, heteroCs-CiQaryl comprising one or more heteroatoms selected from N, O, and S; and m is 2, 3 or 4,
  • Ci-C 4 alkyl, C 5 -C 10 aryl or heteroCs-Cioaryl, when substituted may be one or more substituents selected from OH, C(O)OH, halogen, Ci-C 4 alkyl, Ci-
  • FRl 73657 involves crystallographic polymorphism, of which crystal hydrates having high purity and good solid stability and easy to handle for formulation into medicines. Three types of crystals referred to as crystal type A, crystal type B and crystal type C, have been found (Japanese Patent Laid-Open No. 316677/1998). However, though having good solid stability and releasability, crystal type A is problematic in that it is often contaminated with crystal type C, making impossible production with stable quality.
  • Crystal type B is the most stable and there is no problem in producing it, but is problematic in that its releasability is much inferior to that of crystal type A.
  • Crystal type C is also problematic in that its solid stability is inferior to that of crystal type B and its releasability is inferior to that of crystal type A.
  • US 7,094,899 describes a vitreous form of FRl 73657 that overcomes these problems. The vitreous form has high purity, good solid stability, good solubility and good releasability and capable of production with stable quality.
  • FR167344 N-[N-[3-[3-bromo-2-methylimdazo[l,2-a]pyridin-8- yl)oxymethyl]2,4-dichloro phenyl]N-methylaminocarbonylmethyl]-4- (dimethylaminocarbonyl)cinnarnylamide hydrochlroide) (also discussed below);
  • WIN-64338 [4- ⁇ (2[ ⁇ bis(cyclohexylamino)methylene ⁇ amino]-3-[2- naphthyl]-l-oxopropyl)amino ⁇ phenyl]methyl)tributylphosphonium chloride monohydrochloride) (also discussed below);
  • Q is selected from the group consisting of -SO 2 R and -CH 2 C(O)R;
  • W is selected from the group consisting of O, S, and N, wherein when W is O or S, then q is zero and when W is N, then q is one;
  • R is selected from the group consisting of aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic;
  • R 1 and R 2 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic, or R 1 and R 2 together with the nitrogen atom to which they are attached form a heteroaryl, substituted heteroaryl, heterocyclic, or substituted heterocyclic;
  • each R 3 is independently selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, amino, substituted amino, acylamino, aminoacyl, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, aryloxy, substituted aryloxy, heteroaryl, substituted heteroaryl, heteroaryloxy, substituted heteroaryloxy, heterocyclic, substituted heterocyclic, heterocyclyloxy, substituted heterocyclyloxy, acyl, acyloxy, halogen, nitro, cyano, hydroxy, carboxy, and carboxyl esters;
  • R 7 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, acyl and acyloxy;
  • R together with at least one of R and the nitrogen and carbon atoms to which they are joined forms a fused ring heteroaryl, substituted heteroaryl, unsaturated heterocyclic or substituted unsaturated heterocyclic;
  • p is an integer of from 0 to 3;
  • Exemplary compounds include the following compounds and pharmaceutically acceptable salts thereof:
  • one of bonds characterized by is a double bond and the other two are single bonds;
  • n is an integer from 0 to 4.
  • W is selected from the group consisting of O, S, and N, wherein: [0510] when W is O or S, then q is zero; and when W is N, then q is one;
  • R is selected from the group consisting of aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic;
  • R 1 and R 2 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic, or R 1 and R 2 together with the nitrogen atom to which they are attached form a heteroaryl, substituted heteroaryl, heterocyclic, or substituted heterocyclic;
  • each R 3 is independently selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, amino, substituted amino, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, aryloxy, substituted aryloxy, heteroaryl, substituted
  • R 3 or two or more of R 3 together with the carbon atoms to which they are joined form a fused ring cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, unsaturated heterocyclic or substituted unsaturated heterocyclic;
  • R 7 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, acyl and acyloxy; [0516] or R 7 together with at least one of R 3 and the nitrogen and carbon atoms to which they are joined forms a fused ring heteroaryl, substituted heteroaryl, unsaturated heterocyclic or substituted unsaturated heterocyclic; [0517] R 8 is selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, acyl and acyloxy; [0518] and pharmaceutically acceptable salts
  • R 1 nor R 2 is selected from the following:
  • alkylene-X a wherein alkylene is optionally substituted and X a is selected from the group consisting of -OH, cyano, and -NR b R b wherein each R b is independently as defined above;
  • Representative compounds include: [0547] 2-[2-(R,S)-l-(2,4,6-trimethylbenzenesulfonyl)-3-oxo-l,2,3,4- tetrahydro-6,-7-dichloroquinoxalin-2-yl]-N-benzylacetamide;
  • R 5A is -X A -R 6A or -N(R 7A )R 8A , wherein X A is piperidinylere or piperazinylene, R 6A is H, C 3 -C 4 alkenyl, C 3 -C 4 alkinyl, Ci-C 4 (alkoxyalkyl), C 1 -C 4 (carboxyalkyl), a C 5 -C 7 heterocyclic group or phenylQ-Qalkyl; [0558] R 7A is amino-C 2 -C 4 alkyl or mono- or di-(Ci-C 5 alkyl)amino-C 2 -
  • R 8A is H, C 1 -C 4 alkyl or has the meanings as given for R 7A ;
  • X 1 is a divalent group of formula IA':
  • n is zero or 1
  • X 3 is CH or N
  • X 4 is a direct bond
  • R 3A is H, C r C 4 alkyl, C 3 -C 6 cycloalkyl, C 3 - C 6 alkenyl, C 3 -C 6 alkinyl, C 7 -C 10 aralkyl or C 6 -C 9 heteroaralkyl
  • R 4A is H and m is 1 or 2 or
  • X 4 is -CH(R 12 )-, R 3A is H and R 4A and R 12 together are propylene and m is 1 , or ethylene and m is 2;
  • X 2 is a divalent group of formula IA" :
  • R 1 ' is d-C 4 alkyl, C 3 -C 6 cloalkyl or -NR 1A R 2A , wherein R 1A and R 2A independently are Q-Qalkyl or, together with the N-atom to which they are attached, represent a 5 to 7 membered heterocyclic ring; and
  • R 9 and R 10 independently are a phenyl or pyddine ring; and salts thereof.
  • Representative compounds include:
  • X 1 and X 2 are independently halo or C ⁇ alkyl
  • R 1 and R 2 are independently hydrogen or C 1-4 alkyl
  • R 3 and R 4 are independently hydrogen or halo; and [0580] R 5 is
  • A is independently halo
  • Y is -(CH 2 )" 1 -, -C(O)- or -S(O)-;
  • R and R are independently C 1-4 alkyl
  • R 3 is selected from
  • Ci -6 alkyl-C 3-7 cycloalkyl the cycloalkyl moiety being optionally substituted with one, two or three substituents independently selected from cyano, amino-
  • R 4 is phenyl substituted at the 2-position with substituent selected from
  • Ci-4alkoxy or C 1-4 alkylthio the Ci ⁇ alkoxy or Ci -4 alkylthio being substituted with one, two or three substituents independently selected from amino, amino-
  • n 0, 1, 2, 3, 4 or 5;
  • Representative compounds include:
  • R 1 is a hydrogen atom or a C 1 -C 4 alkyl group
  • R 2 and R 3 which can be the same or different, are a C 1 -C 4 alkyl group, or R 2 and R 3 , together with the carbon atom which they are linked to, form a cyclic aliphatic group having 3 to 7 carbon atoms or a heterocyclic aliphatic group having 3 to 7 atoms, one or two of which are selected from the group N, O, S and the others being C atoms;
  • R 4 and R 5 which can be the same or different, are a hydrogen atom or a C 1 -C 4 alkyl group
  • X is selected from the group consisting of halogen, OR 1 , SRi, CN, Ci- C 4 alkyl;
  • B has at least one amino group with basic characteristics or a tetraalkylammonium group and can be selected from the group consisting of:
  • T is selected from the group of -NR 7 R 8 , -NR 14 Ri s Ri 9 , -OR 6 ;
  • R 7 and Rg which can be the same or different, are a hydrogen atom, a C 1 -C 4 alkyl group, a cyclohexyl group, or NR 7 R 8 together are a group selected from: i) guanidine optionally substituted with 1 or 2 C 1 -C 4 alkyl or cyclohexyl groups, ii) a 5-7 membered nitrogen heterocycle optionally containing another heteroatom selected from O 3 N 5 S;
  • Y 1 is selected from the group consisting OfNR 7 R 8 , NR 14 R 18 R 19 or from the following residues:
  • Z is selected from the group consisting of H, C 1 -C 6 alkyl, OR 6 , SR 6 , CF 3 , OCOR 65 COR 10 , NHCOR 6 , SO 2 R 6 , SOR 6 , CO 2 R 6 , N(Re) 2 , CI, Br, NO 2 , NH 2 , CN, F, imidazole, phenyl, amidine, guanidine, guanidyl-methyl; [0637] R 9 is selected from the group consisting of hydrogen, ⁇ (CH 2 ) q -L, wherein L is selected from the group of —OH, -NR 5 R 6 , -NR 14 R 1S Ri 9 , amidine optionally substituted with 1 or 2 C 1 -C 4 alkyl groups, guanidine optionally substituted with 1 or 2 Cj-C 4 alkyl groups;
  • R 10 is selected from the group consisting of OR 6 , NR 6 Ri 2 ;
  • Rn is selected from the group consisting of hydrogen, ⁇ (CH 2 ) q -L, — (CH 2 ) p ⁇ NR 4 ⁇ (CH 2 ) q -L;
  • R 12 is a hydrogen atom, Ci-C 6 alkyl, COR 6 ,
  • R 13 is selected from the group consisting of H, C 1 -C 6 alkyl, ⁇ (CH 2 ) p W(CH 2 ) q Y,, Y, -COY, -CH 2 -Y;
  • Rj 5 is selected from the group consisting of hydrogen or straight or branched C ] -C 4 alkyl groups
  • the -NR 16 Ri ? group is a 5-7 membered nitrogen aliphatic heterocycle optionally containing another heteroatom selected from O, S, N;
  • Representative compounds include:
  • fragment L represents a hydrophobic group such as a 5-membered unsaturated ring, 6-membered unsaturated ring, alkene, saturated 3-7 membered ring;
  • fragment E represents carbonyl derivative that in the presence of L, R and Q becomes an activated serine trap
  • fragment R represents a hydrophobic, aromatic ring system such as a 6-membered ring, fused 5-6 membered ring, or fused 6—6 membered ring;
  • fragment Q represents a basic group.
  • Suitable peptides, polypeptides, proteins, and peptidomimetics include: [0665] (A) Peptides having the following sequence:
  • r is any amino acid, preferably Arg;
  • p is any amino acid, preferably Pro;
  • g is amino acid, preferably GIy;
  • X is any amino acid
  • f is any amino acid, preferably Phe;
  • N-terminal amino acid residue is modified and the proline at the position third from the C-terminal end of this sequence is substituted with an aromatic amino acid of the D-configuration or another amino acid as described in
  • X 1 is a modified amino acid, preferably modified proline or modified serine, more preferably proline, and may be N-terminal;
  • X 2 is any amino acid, preferably proline or phenylalanine
  • X 3 is isoleucine, leucine, or valine, preferably isoleucine or valine;
  • X 4 is any amino acid, preferably valine, isoleucine, or leucine;
  • X 5 is any amino acid, preferably serine, threonine, or proline; and [0681] X 6 is any amino acid, preferably phenylalanine, leucine or arginine;
  • N-terminal amino acid residue is modified and the proline at the position third from the C-terminal end of this sequence is substituted with an aromatic amino acid of the D-configuration or another amino acid as described in US 4,801,613;
  • X 1 is N-terminal and is a modified amino acid, preferably modified proline or modified serine, more preferably modified proline;
  • n is a contiguous sequence of any amino acids of length n, where n can be up to about 50, up to about 30, up to about 10 amino acids or 0 amino acids;
  • X 3 is any amino acid, preferably proline or phenylalanine;
  • X 4 is isoleucine, leucine, or valine, preferably isoleucine or valine;
  • X 5 is any amino acid, preferably valine, isoleucine, or leucine;
  • X 6 is any amino acid, preferably serine, threonine, or proline;
  • X 7 is any amino acid, preferably phenylalanine, leucine, or arginine;
  • N-terminal amino acid residue is modified and the proline at the position third from the C-terminal end of this sequence is substituted with an aromatic amino acid of the D-configuration or another amino acid as described in US 4,801,613;
  • X 2 is a hydrophobic aliphatic residue, preferably alanine, valine, isoleucine, or leucine, but not glycine;
  • X 3 is a hydrophobic aliphatic residue
  • Representative sequences include:
  • modified means any modification at the N-terminal amino acid residue that blocks sequencing by the standard Edman degradation method. Such modifications include, but are not limited to acetylation, amidation, ⁇ -methylthiolation, biotin, carbamylation, citrullination, c-mannosylation, deamidation, n-acyl diglyceride cysteine (tripalmitate), dimethylation, fad, farnesylation, formylation, geranyl-geranyl, gamma-carboxyglutamic acid, o-glcnac, glucosylation (glycation), hydroxylation lipoyl, methylation, myristoylation, palmitoylation, phosphorylation, pyridoxal phosphate, phosphopantetheine, pyrrolidone carboxylic acid, sulfation and trimethylation. Modifications may be on the following modifications, amidation, ⁇ -methylthiolation, biotin, carbamylation, citrullination
  • Xaai is selected from D-arginyl, acetyl lysyl, D-lysyl, sarcosyl, acetyl tyrosyl epsilon-aminohexanoyl lysyl, sarcosyl tyrosyl epsilon-aminohexanoyl lysyl, and sarcosyl tyrosyl (3,5-iodine) epsilon-aminohexanoyl lysyl;
  • X a a2 is selected from prolyl and hydroxyprolyl; ⁇
  • X a a3 is selected from D-l,2,3,4,-tetrahydroisoquinoline-3-carboxyl and "D- ⁇ -(2-naphthyl) alanyl;
  • X a a4 is selected from leucine and isoleucine
  • X aa5 is selected from acetyl tyrosyl epsilon-aminohexanoyl lysyl, sarcosyl tyrosyl epsilon-aminohexanoyl lysyl, and sarcosyl tyrosyl (3,5-iodine) epsilon- aminohexanoyl lysyl;
  • X aa6 is selected from prolyl and hydroxyprolyl; [0717] X aa7 is propyl;
  • X aa g is selected from leucine and isoleucine
  • HOE 140 (or Icatibant) having the following sequence:
  • This peptide, methods for its preparation and its biological activity are discussed in Boa et al. 1991 and many other publications.
  • the peptide is described as being a highly potent, specific and long-acting bradykinin B2-receptor antagonist.
  • the peptide is described as a bradykinin antagonist.
  • NPC 17761 having the following sequence:
  • the peptide is described as being a potent bradykinin antagonist.
  • M is an amide bond
  • x and z are each independently an integer of 1 to 10
  • K is H or an acyl group.
  • Representative peptide analogs include: [0733] Ada-(D)Arg-Arg-cyclo(N ⁇ (l-(6-aminohexylene)Gly-Hyp-Phe-D-
  • Ada is the abbreviated designation for the protecting groups adamantane acetyl.
  • a 0 , B 1 , C 2 , D 3 , and E 4 are basic or neutral aromatic, aliphatic, heterocyclic, or alicyclic amino acids or A 0 is absent;
  • G 6 is an aromatic, aliphatic, heterocyclic, or alicyclic amino acid
  • F 5 , H 7 and J 8 are aromatic, aliphatic, aliphatic heterocyclic, or alicyclic amino acids, provided that at least one of F 5 , H 7 and J 8 is selected from cyclopentane-, cyclohexane- or indane-substituted glycine; and
  • Z is COOH.
  • Representative analogues include: [0747] DArg-Cys-Pro-Hyp-Gly-Cpg-Ser-DCpg-Cpg;
  • R is selected from the group consisting of Ci-C 6 alkyl, substituted C 1 - C 6 alkyl, C 2 -Cgalkenyl, Ca-Cscycloalkyl, C 3 -Cscycloalkyl substituted Ci-C 6 alkyl, an aryl group, a substituted aryl group, an arylalkyl group, and a group of the formula
  • Xj-Arg-Pro-Xa-Gly-Phe-Ser-Xa- ⁇ [0775] wherein [0776] X 1 is selected from D-arginyl, acetyl lysyl, D-lysyl, sarcosyl, acetyl tyrosyl epsilon-aminohexanoyl lysyl, sarcosyl tyrosyl epsilon-aminohexanoyl lysyl, and sarcosyl tyrosyl (3,5-iodine) epsilon-aminohexanoyl lysyl;
  • X 2 is selected from prolyl and hydroxyprolyl
  • X 3 is selected from D-l,2,3,4-tetrahydroisoquinolme-3-carboxyl and "D-beta-(2-naphthyl) alanyl;
  • X 4 is selected from leucine and isoleucine
  • X 1 is selected from acetyl tyrosyl epsilon-aminohexanoyl lysyl, sarcodyl tyrosyl epsilon-aminohexanoyl lysyl and sarcosyl tyrosyl (3,5-iodine) epsilon- aminohexanoyl lysyl;
  • X 2 is selected from prolyl and hydroxyprolyl;
  • X 3 is prolyl;
  • X 4 is selected from leucine and isoleucine.
  • NPC 17751 (described in Mak et al. 1991);
  • NPC 349 (described in Wirth et al. 1995 and Mak et al. 1991);
  • NPC- 1776 (described in Cheronis et al. 1993); [0792] WIN 64338 (described in Sawutz et al. 1995 and elsewhere herein);
  • NPC 18565 D-Arg(Hyp 3 ,D-Hyp 7 (transpropyl),Oic 8 )desArg 9 BK developed by Scios Inc;
  • NPC 18828 D-Arg(Hyp 3 ,D-Hyp 7 (trans thiophenyl),Oic 8 )desArg 9 BK also developed by Scios Inc;
  • KKS antagonists are also disclosed in these references.
  • Phe(4NH 2 ) 4-amino phenylalanine
  • the sequence comprises naturally occurring, non-naturally occurring, or chemically synthesized amino acids, amino acid derivatives, modified amino acids, or combinations thereof.
  • Extracts from at least one plant of the Rosaceae family as disclosed in US 6,586,020 may be prepared from any plant material (including whole plant, or from a plant portion such as the leaves, stems, flowers, petals, roots or from undifferentiated cells) derived from at least one plant of the Rosaceae family using the extraction techniques described in US 6,586,020, including aqueous or alcoholic extracts, or extracts obtained from an organic solvent; and
  • KKS antagonists are known in the art in addition to those specifically described above, including those described in Hirayama, Y. et al. (2002); Hall, J. M. (1997); Campbell et al. (2001); Regoli, et al. (1998); Altamura et al. (1999); Moreau et al. (2005); and Blakeney et al. (2007).
  • B 9430 D-Arg-[Hyp 3 ,Igl 5 ,D-Igl 7 ,Oic 8 ]-bradykinin (discussed herein);
  • HOE 140 (also discussed above) and desArg 9 derivatives of HOE 140;
  • R 493 D-Arg-[Hyp 3 , D-Phe 7 , Leu 8 ]bradykinin
  • NPC 567 D-Arg-[Hyp 3 ,D-Phe 7 ]-bradykinin
  • Martinelline a pyrroloquinoline alkaloid isolated from the roots of the tropical plant Martinella iquitosensis.
  • kinin B 1 -receptor ligands including many described in Regoli et al. (1998) and also including:
  • R-954 (Ac-Orn-[Oic 2 , ⁇ -MePhe 5 , D- ⁇ Nal 7 , Ile 8 ]des-Arg 9 bradykinin);
  • compound 12 (benzodiazepine-based structure); [0845] benzo-sulfonyalmide compounds including compound 12, compound l l and SSR240612;
  • kinin B 2 -receptor ligands including many described in Regoli et al. (1998) and Altamura et al. (1999) and also including: [0847] [Thi 5 ' 8 , D-Phe 7 ]-bradykinin;
  • KKS antagonists including aprotinin and aprotinin-like drugs including ulinastatina nd nafamostat;
  • agents that comprise KKS antagonists are useful for the prevention or treatment of aneurysms.
  • agents can be administered to an individual either by themselves, or in pharmaceutical compositions where they are mixed with a suitable pharmaceutically acceptable carrier or diluent.
  • the agents of the present invention may be conjugated with biological targeting agents which enable their activity to be restricted to particular cell types.
  • biological-targeting agents include substances which are immuno-interactive with cell- specific surface antigens.
  • the agents may be formulated and administered systemically or locally. Techniques for formulation and administration may be found in "Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition.
  • Suitable routes may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
  • the drugs of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • Intra-muscular and subcutaneous injection is appropriate, for example, for administration of immunogenic compositions, vaccines and DNA vaccines.
  • the agents can be formulated readily using pharmaceutically acceptable carriers or diluents well known in the art into dosages suitable for oral administration.
  • Such carriers or diluents enable the compounds of the invention to be formulated in dosage forms such as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
  • These carriers or diluents may be selected from sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline, and pyrogen-free water.
  • compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose.
  • the dose of agent administered to an individual should be sufficient to effect a beneficial response in the individual over time such as reducing the size of an aneurysm, preventing an increase in the size of an aneurysm, reducing the number of small aneurysms, and/or preventing or inhibiting the occurrence of aneurysms.
  • the quantity of the agent(s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof. In this regard, precise amounts of the agent(s) for administration will depend on the judgement of the practitioner.
  • the physician may evaluate the characteristics of the patient, their response to the drug and the safety profile of the drug. In any event, those of skill in the art may readily determine suitable dosages of the agents.
  • compositions for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or other components which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • compositions for oral use can be obtained by combining the active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, or polyvinylpyrrolidone (PVP).
  • disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association one or more drugs as described above with the carrier or diluent which constitutes one or more necessary ingredients.
  • the pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterise different combinations of active compound doses.
  • Pharmaceuticals which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticiser, such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • filler such as lactose, binders such as starches, or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added.
  • Dosage forms of the drugs of the invention may also include injecting or implanting controlled releasing devices designed specifically for this purpose or other forms of implants modified to act additionally in this fashion.
  • Controlled release of an agent of the invention may be effected by coating the same, for example, with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids and certain cellulose derivatives such as hydroxypropylmethyl cellulose.
  • controlled release may be effected by using ⁇ other polymer matrices, liposomes or microspheres.
  • the drugs of the invention may be provided as salts with pharmaceutically compatible counterions.
  • Pharmaceutically compatible salts may be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents that are the corresponding free base forms.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC50 as determined in cell culture (e.g., the concentration of a test agent, which achieves a half-maximal inhibition or enhancement in activity of a component in the KKS). Such information can be used to more accurately determine useful doses in humans.
  • Toxicity and therapeutic efficacy of such drugs can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio
  • LD50/ED50 Compounds that exhibit large therapeutic indices are preferred.
  • the data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilised.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See for example Fingl et al, 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 pi).
  • Dosage amount and interval may be adjusted individually to provide plasma levels of the active agent which are sufficient to maintain KKS antagonistic effects.
  • Usual patient dosages for systemic administration range from 1-2000 mg/day, commonly from 1-250 mg/day, and typically from 10-150 mg/day. Stated in terms of patient body weight, usual dosages range from 0.02-25 mg/kg/day, commonly from 0.02- 3 mg/kg/day, typically from 0.2-1.5 mg/kg/day. Stated in terms of patient body surface areas, usual dosages range from 0.5-1200 mg/m2/day, commonly from 0.5-150 mg/m2/day, typically from 5-100 mg/m2/day.
  • the liposomes will be targeted to and taken up selectively by the tissue.
  • the effective local concentration of the agent may not be related to plasma concentration.
  • the present invention also contemplates a method of gene therapy of a mammal.
  • a method of gene therapy of a mammal utilises a gene therapy construct which includes an isolated polynucleotide comprising a nucleotide sequence encoding a component of the kallikrein- kinin system, or a biologically active fragment thereof, wherein the polynucleotide is ligated into a gene therapy vector which provides one or more regulatory sequences that direct expression of the polynucleotide in the mammal.
  • gene therapy vectors are derived from viral DNA sequences such as adenovirus, adeno-associated viruses, herpes-simplex viruses and retroviruses. Suitable gene therapy vectors currently available to the skilled person may be found, for example, in Robbins et al, 1998.
  • Administration of the gene therapy construct to the mammal may include delivery via direct oral intake, systemic injection, or delivery to selected tissue(s) or cells, or indirectly via delivery to cells isolated from the mammal or a compatible donor.
  • An example of the latter approach would be stem-cell therapy, wherein isolated stem cells having potential for growth and differentiation are transfected with the gene therapy vector comprising a component of the KKS. The stem-cells are cultured for a period and then transferred to the mammal being treated.
  • a polynucleotide encoding a KKS antagonist may be used as a therapeutic or prophylactic composition in the form of a "naked DNA" composition as is known in the art.
  • an expression vector comprising the polynucleotide operably linked to a regulatory polynucleotide (e.g. a promoter, transcriptional terminator, enhancer etc) may be introduced into an animal, preferably a mammal, where it causes production of a KKS antagonist in vivo.
  • a regulatory polynucleotide e.g. a promoter, transcriptional terminator, enhancer etc
  • the step of introducing the expression vector into a target cell or tissue will differ depending on the intended use and species, and can involve one or more of non- viral and viral vectors, cationic liposomes, retroviruses, and adenoviruses such as, for example, described in Mulligan, R.C., (1993).
  • the present invention also provides methods for detecting the presence of an aneurysm, determining the prognosis of an aneurysm, or monitoring the progression of an aneurysm in an individual.
  • One suitable method comprises detecting in the individual the presence of an aberrant gene encoding a component of the KKS or detecting in the individual aberrant expression of a gene encoding a component of the KKS.
  • the aberrant gene is selected from an aberrant KNGl, KLKl, KLKBl, BDKRBl, BDKRB2, SERPINGl, A2M, SERPINCl, or SERPINA4 gene.
  • Another suitable method comprises determining in the individual a level or functional activity of a component in the KKS, which is different than a reference or control level (e.g. no aneurysm, level measured in the same individual at an earlier time) or functional activity of the component.
  • a reference or control level e.g. no aneurysm, level measured in the same individual at an earlier time
  • the level or functional activity of a component in the KKS, which is different than a reference or control level indicates the presence or risk of development of an aneurysm or a negative prognosis.
  • the method is performed in vivo. In other embodiments, the method is performed ex vivo, including in a biological sample obtained from the individual.
  • the method comprises determining an increase or elevation in the level of functional activity of the component (e.g. HMWK, LMWK, prekallikrein, prokallikrein, plasma kallikrein, tissue kallikrein, bradykinin, kallidin, des- Arg9-bradykinin, Lys-des-Arg9-bradykinin, the kinin B 1 receptor, the kinin B2 receptor) relative to a reference or control level (e.g. no aneurysm, level measured in the same individual at an earlier time) or functional activity of the component, .
  • the method comprises determining a decrease in the level or functional activity of the component (e.g.
  • the aneurysm is an aortic aneurysm.
  • An illustrative example of this type of aneurysm includes AAA.
  • individual is one that is suspected of having an aneurysm or being at risk of having an aneurysm.
  • determining the presence of an aneurysm, determining the prognosis of an aneurysm, or monitoring the progression of an aneurysm in an individual is achieved by using the methods described above in combination with medical imaging, including ultrasound.
  • the invention also features methods of screening for an agent that antagonises the KKS, including agents that modulate the expression of a gene or the level and/or functional activity of an expression product of that gene, wherein the gene is selected from a gene encoding a component of the KKS, a gene whose expression product modulates directly or indirectly the expression of a gene encoding a component of the KKS, and a gene whose expression product modulates directly or indirectly the expression product of a gene encoding a component of the KKS.
  • the methods comprise: (1) contacting a preparation with a test agent, wherein the preparation contains (i) a polypeptide comprising an amino acid sequence corresponding to at least a biologically active fragment of a polypeptide component of the KKS, or to a variant or derivative thereof; or (ii) a polynucleotide comprising at least a portion of a genetic sequence that regulates a nucleotide sequence that encodes at least a biologically active fragment of a polypeptide component of the KKS, or a variant or derivative thereof, which is operably linked to a reporter gene; or (iii) a polynucleotide comprising a nucleotide sequence that encodes a polypeptide according to (i) and (2) detecting a change in the level and/or functional activity of the polypeptide component, or an expression product of the reporter gene, relative to a reference or control level and/or functional activity in the absence of the test agent, which indicates that the agent modulates the KKS
  • the methods comprise contacting a sample of cells expressing a kinin receptor with a kinin and a test agent.
  • Candidate test agents encompass numerous chemical classes.
  • Candidate organic molecules comprise functional groups necessary for structural interaction with KKS components including proteins, particularly by way of hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups.
  • the candidate test agent often comprises cyclical carbon or heterocyclic structures or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • Candidate test agents are also found among biomolecules including, but not limited to: peptides, saccharides, fatty acids, steroids, purines, pyrrolidines, derivatives, structural analogues or combinations thereof.
  • small molecules are particularly preferred as candidate test agents because such molecules are more readily absorbed after oral administration, have fewer potential antigenic determinants, or are more likely to cross the cell membrane than larger, protein-based pharmaceuticals. Small organic molecules may also have the ability to gain entry into an appropriate cell and affect the expression of a gene
  • libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced.
  • natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries.
  • Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc to produce structural analogues.
  • Screening may also be directed to known pharmacologically active compounds and chemical analogues thereof.
  • Screening candidate test agents according to the invention can be achieved by any suitable method.
  • the method may include contacting a cell expressing a polynucleotide corresponding to gene encoding a component of the KKS, with an agent suspected of having the modulatory activity and screening for the modulation of the level or functional activity of a protein encoded by the polynucleotide, or the modulation of the level of a transcript encoded by the polynucleotide, or the modulation of the activity or expression of a downstream cellular target of the protein or of the transcript (hereafter referred to as target molecules).
  • target molecules a downstream cellular target of the protein or of the transcript
  • Detecting such modulation can be achieved utilizing techniques including, but not restricted to, ELISA, cell-based ELISA, inhibition ELISA, Western blots, immunoprecipitation, slot or dot blot assays, immunostaining, RIA, scintillation proximity assays, fluorescent immunoassays using antigen-binding molecule conjugates or antigen conjugates of fluorescent substances such as fluorescein or rhodamine, Ouchterlony double diffusion analysis, immunoassays employing an avidin-biotin or a streptavidin-biotin detection system, and nucleic acid detection assays including reverse transcriptase polymerase chain reaction (RT-PCR).
  • a polynucleotide from which a target molecule of interest is regulated or expressed may be naturally occurring in the cell which is the subject of testing or it may have been introduced into the host cell for the purpose of testing. Further, the naturally-occurring or introduced polynucleotide may be constitutively expressed — thereby providing a model useful in screening for agents which down-regulate expression of an encoded product of the sequence wherein the down regulation can be at the nucleic acid or expression product level - or may require activation - thereby providing a model useful in screening for agents that up-regulate expression of an encoded product of the sequence.
  • a polynucleotide may comprise the entire coding sequence which codes for a target protein or it may comprise a portion of that coding sequence (e.g., a binding domain) or a portion that regulates expression of a product encoded by the polynucleotide (e.g., a promoter).
  • a promoter e.g., the promoter that is naturally associated with the polynucleotide may be introduced into the cell that is the subject of testing.
  • detecting modulation of the promoter activity can be achieved, for example, by operably linking the promoter to a suitable reporter polynucleotide including, but not restricted to, green fluorescent protein (GFP), luciferase, ⁇ -galactosidase and catecholamine acetyl transferase (CAT). Modulation of expression may be determined by measuring the activity associated with the reporter polynucleotide.
  • GFP green fluorescent protein
  • CAT catecholamine acetyl transferase
  • the subject of detection could be a downstream regulatory target of the target molecule, rather than the target molecule itself or the reporter molecule operably linked to a promoter of a gene encoding a product the expression of which is regulated by the target protein.
  • These methods provide a mechanism for performing high throughput screening of putative modulatory agents such as proteinaceous or non-proteinaceous agents comprising synthetic, combinatorial, chemical and natural libraries. These methods will also facilitate the detection of agents which bind either the polynucleotide encoding the target molecule or which modulate the expression of an upstream molecule, which subsequently modulates the expression of the polynucleotide encoding the target molecule.
  • the present invention provides assays for identifying small molecules or other compounds (i.e. modulatory agents) which are capable of inducing or inhibiting the level and/or functional activity of target molecules according to the invention.
  • the assays may be performed in vitro using non-transformed cells, immortalised cell lines, or recombinant cell lines.
  • the assays may detect the presence of increased or decreased expression of genes or production of proteins on the basis of increased or decreased mRNA expression (using, for example, the nucleic acid probes disclosed herein), increased or decreased levels of protein products (using, for example, the antigen binding molecules disclosed herein), or increased or decreased levels of expression of a reporter gene (e.g., GFP 5 ⁇ -galactosidase or luciferase) operably linked to a target molecule-related gene regulatory region in a recombinant construct.
  • a reporter gene e.g., GFP 5 ⁇ -galactosidase or luciferase
  • recombinant assays are employed in which a reporter gene encoding, for example, GFP, ⁇ -galactosidase or luciferase is operably linked to the 5' regulatory regions of a target molecule related gene.
  • a reporter gene encoding, for example, GFP, ⁇ -galactosidase or luciferase
  • Such regulatory regions may be easily isolated and cloned by one of ordinary skill in the art.
  • the reporter gene and regulatory regions are joined in-frame (or in each of the three possible reading frames) so that transcription and translation of the reporter gene may proceed under the control of the regulatory elements of the target molecule related gene.
  • the recombinant construct may then be introduced into any appropriate cell type although mammalian cells are preferred, and human cells are most preferred.
  • the transformed cells may be grown in culture and, after establishing the baseline level of expression of the reporter gene, test compounds may be added to the medium.
  • the ease of detection of the expression of the reporter gene provides for a rapid, high throughput assay for the identification of agonists or antagonists of the target molecules of the invention.
  • Compounds identified by this method will have potential utility in modifying the expression of target molecule related genes in vivo. These compounds may be further tested in the animal models to identify those compounds having the most potent in vivo effects.
  • these molecules may serve as "lead compounds" for the further development of pharmaceuticals by, for example, subjecting the compounds to sequential modifications, molecular modelling, and other routine procedures employed in rational drug design.
  • methods of identifying agents that antagonise the KKS are provided in which a purified preparation of a component of the KKS is incubated in the presence and absence of a candidate agent under conditions in which the component is active, and the level of activity is measured by a suitable assay.
  • a KKS antagonist can be identified by measuring the ability of a candidate agent to decrease kinin - kinin receptor binding. An agent tests positive if it inhibits this activity.
  • methods of identifying agents that antagonise the KKS are provided in which a purified preparation of a component of the KKS is incubated in the presence and absence of a candidate agent under conditions in which kinin is able to bind a kinin receptor, and the level of binding is measured by a suitable assay. An agent tests positive if it inhibits this activity.
  • random peptide libraries consisting of all possible combinations of amino acids attached to a solid phase support may be used to identify peptides that are able to bind to a target molecule or to a functional domain thereof. Identification of molecules that are able to bind to a target molecule may be accomplished by screening a peptide library with a recombinant soluble target molecule.
  • the target molecule may be purified, recombinantly expressed or synthesised by any suitable technique.
  • Such molecules may be conveniently prepared by a person skilled in the art using standard protocols as for example described in Sambrook, et ah, (1989, supra) in particular Sections 16 and 17; Ausubel et ah, ("Current Protocols in Molecular Biology", John Wiley & Sons Inc, 1994-1998), in particular Chapters 10 and 16; and
  • a target polypeptide according to the invention may be synthesized using solution synthesis or solid phase synthesis as described, for example, in Chapter 9 of Atherton and Shephard ⁇ supra) and in Roberge et al. (1995).
  • target polypeptide may be conjugated to any suitable reporter molecule, including enzymes such as alkaline phosphatase and horseradish peroxidase and fluorescent reporter molecules such as fluorescein isothiocynate (FITC), phycoerythrin (PE) and rhodamine. Conjugation of any given reporter molecule, with target polypeptide, may be performed using techniques that are routine in the art.
  • suitable reporter molecule including enzymes such as alkaline phosphatase and horseradish peroxidase and fluorescent reporter molecules such as fluorescein isothiocynate (FITC), phycoerythrin (PE) and rhodamine.
  • target polypeptide expression vectors may be engineered to express a chimeric target polypeptide containing an epitope for which a commercially available antigen-binding molecule exists.
  • the epitope specific antigen- binding molecule may be tagged using methods well known in the art including labelling with enzymes, fluorescent dyes or coloured or magnetic beads.
  • the "tagged" target polypeptide conjugate is incubated with the random peptide library for 30 minutes to one hour at 22° C to allow complex formation between target polypeptide and peptide species within the library. The library is then washed to remove any unbound target polypeptide.
  • the whole library is poured into a petri dish containing a substrate for either alkaline phosphatase or peroxidase, for example, 5-bromo-4-chloro-3-indoyl phosphate (BCIP) or 3,3 ',4,4"- diamnobenzidine (DAB), respectively.
  • a substrate for either alkaline phosphatase or peroxidase for example, 5-bromo-4-chloro-3-indoyl phosphate (BCIP) or 3,3 ',4,4"- diamnobenzidine (DAB), respectively.
  • BCIP 5-bromo-4-chloro-3-indoyl phosphate
  • DAB 3,3 ',4,4"- diamnobenzidine
  • a chimeric target polypeptide having a heterologous epitope may be used, detection of the peptide/target polypeptide complex may be accomplished by using a labeled epitope specific antigen-binding molecule. Once isolated, the identity of the peptide attached to the solid phase support may be determined by peptide sequencing. 7. Methods of producing agents comprising a KKS antagonist
  • the present invention also provides methods of producing an agent comprising a KKS antagonist for preventing or treating an aneurysm in an individual. These methods generally comprise: testing a test agent as broadly described above; and synthesising the agent on the basis that it tests positive for antagonising the KKS.
  • the method further comprises derivatising the agent, and optionally formulating the derivatised agent with a pharmaceutically acceptable carrier or diluent, to improve the efficacy of the agent for treating or preventing the aneurysm.
  • the aneurysm is an aortic aneurysm.
  • An illustrative example of this type of aneurysm includes AAA.
  • Serum was obtained from archived serum from The Health In Men Study (HIMS) previously described (Norman et ⁇ l, 2009) and came from subjects from Western Australia, Australia. Ethical approval from the centre providing the samples and informed consent from all subjects was obtained.
  • HIMS Health In Men Study
  • Serum samples were assayed by ELISA for kallistatin (Duoset, R&D Systems) according to the manufacturer's instructions. Inter-assay coefficient of variation for the assay was ⁇ 10%.
  • Arg 9 -BK produce important physiological effects by stimulating Bl or B2 receptors (Leeb-Lundberg et al, 2005).
  • Protein extraction and Western blotting [0925] Proteins (30 ⁇ g) were extracted and quantitated from human aortic biopsies as previously described in Moran et al, 2005.
  • Proteins were separated using SDS gel electrophoresis (12% SDS; Gradipore) and Western analysis carried out using primary antibodies to Bl receptor (BDKRBl 3 Abeam) and to B2 receptor (BDKRB2, Abeam) and secondary antibody: goat anti-rabbit IgG (Abeam) that was HRP-labelled.
  • SDS gel electrophoresis 12% SDS; Gradipore
  • Western analysis carried out using primary antibodies to Bl receptor (BDKRBl 3 Abeam) and to B2 receptor (BDKRB2, Abeam) and secondary antibody: goat anti-rabbit IgG (Abeam) that was HRP-labelled.
  • mice Male 13 week old ApoE " ' " mice were anaesthetised by intraperitoneal injection of ketamine (150mg/kg) and xylazine (10mg/kg) and a dorsal incision made at the upper back. Alzet® Osmotic miniature pumps (Model 2004) were inserted and the incision sutured.
  • mice were divided into four groups. In three of the four groups the pumps were pre-loaded with human recombinant Angll (Sigma). In the last group, the pumps were pre-loaded with saline. The pumps were loaded so as to deliver 1.44mg/kg/day of Angll or saline over the course of the 28 day experiment. [0941] In addition, the three groups of mice receiving AngII received intraperitoneal injections (2mg/kg/dose) the day prior to pump insertion and every other day throughout the experimental period with a vehicle control, the bradykinin receptor B2 agonist B9972 or the B1/B2 antagonist B9430. [0942] At the conclusion of the experimental period, mice with euthanased by
  • Aortas were dissected from the aortic arch to the iliac bifurcation, taking care to remove adherent adipose tissue and then photographed intact for aortic morphometry. Photographs of an aorta taken from a normal mouse (A - left hand side) and one from a mouse with an aneurysm (B - right hand side) are shown in Figure 3.
  • mice The mean survival time of AngII treated mice was 23 ⁇ 1.5 days with aortic rupture and death occurring in 33% (9/27) mice.
  • mice receiving the B2 agonist B9972 and AngII showed a very high incidence of aortic rupture (68%, 17/25 mice) and mean survival time decreased to 14.5 ⁇ 2 days (p ⁇ 0.01 compared to AngII alone).
  • mice receiving AngII plus the Bl /B2 antagonist B9430 there was significantly less aortic ruptures (8%, 2/24 mice) and the mean survival of this group was 27 days which was significantly different to AngII group (p ⁇ 0.05).
  • Table 8 Maximum aortic diameter in relation to AngII and kinin administration.
  • AngII 1.5Id O. ,32** 1, ,24 ⁇ 0. ,18** 1.54 ⁇ 0.59* 0.79 ⁇ 0, .08 1.27 ⁇ 0.24** + B1/B2 antagonist

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Abstract

La présente invention concerne de manière générale la modification des taux des composants dans le système kallikréine-kinine (KKS) pour aider la prévention ou le traitement d'anévrysmes chez un individu, et également la détection de la présence, la détermination du pronostic ou le contrôle de la progression d'un anévrysme par la détection d'une expression aberrante d'un ou plusieurs gènes dans le système kallikréine-kinine (KKS). L'invention a également pour objet des méthodes permettant d'identifier des agents qui sont des antagonistes du KKS et des méthodes de production d'agents comprenant des antagonistes du KKS pour prévenir ou traiter des anévrysmes.
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WO2022187751A1 (fr) * 2021-03-05 2022-09-09 Moore Ella Antagonistes du récepteur de la bradykinine 1 et leurs utilisations pour la prévention et le traitement de complications respiratoires

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Publication number Priority date Publication date Assignee Title
EP3171173A1 (fr) 2015-11-19 2017-05-24 Medizinische Universität Innsbruck Procédé de diagnostic d'un anévrisme de l'aorte thoracique
WO2022187751A1 (fr) * 2021-03-05 2022-09-09 Moore Ella Antagonistes du récepteur de la bradykinine 1 et leurs utilisations pour la prévention et le traitement de complications respiratoires

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