WO2002077017A2 - Medical uses of intercellular communication facilitating compounds - Google Patents

Medical uses of intercellular communication facilitating compounds Download PDF

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WO2002077017A2
WO2002077017A2 PCT/US2002/005773 US0205773W WO02077017A2 WO 2002077017 A2 WO2002077017 A2 WO 2002077017A2 US 0205773 W US0205773 W US 0205773W WO 02077017 A2 WO02077017 A2 WO 02077017A2
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sar
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pro
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formulae
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PCT/US2002/005773
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English (en)
French (fr)
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WO2002077017A3 (en
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Bjarne Due Larsen
Jorgen Soberg Petersen
Eddie Meier
Anne Louise Kjolbye
Niklas Rye Jorgensen
Morten Schak Nielsen
Niels-Henrik Holstein-Rathlou
James B. Martins
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Zealand Pharma A/S
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Priority claimed from PCT/DK2001/000127 external-priority patent/WO2001062775A2/en
Priority claimed from US09/792,286 external-priority patent/US7250397B2/en
Priority to NZ527571A priority Critical patent/NZ527571A/en
Priority to JP2002576275A priority patent/JP2005506295A/ja
Priority to EA200300912A priority patent/EA007792B1/ru
Priority to KR10-2003-7011099A priority patent/KR20040004538A/ko
Priority to IL15744702A priority patent/IL157447A0/xx
Priority to DK02723240.4T priority patent/DK1370276T3/da
Priority to DE60239126T priority patent/DE60239126D1/de
Priority to BR0207476-1A priority patent/BR0207476A/pt
Application filed by Zealand Pharma A/S filed Critical Zealand Pharma A/S
Priority to AT02723240T priority patent/ATE497967T1/de
Priority to EP02723240A priority patent/EP1370276B1/en
Priority to AU2002254033A priority patent/AU2002254033B2/en
Priority to CA2439101A priority patent/CA2439101C/en
Priority to MXPA03007537A priority patent/MXPA03007537A/es
Publication of WO2002077017A2 publication Critical patent/WO2002077017A2/en
Priority to NO20033641A priority patent/NO20033641L/no
Priority to US10/646,294 priority patent/US7585839B2/en
Publication of WO2002077017A3 publication Critical patent/WO2002077017A3/en

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Definitions

  • the present invention relates to novel peptides including novel antiarrhythmic peptides of linear or cyclic structure having improved stability in vitro and/or in vivo, to compositions comprising said peptides, and to uses of said peptides for the preparation of medicaments.
  • the present invention also relates to the use of compounds that facilitate the intercellular communication for the preparation of medicaments for the treatment of a range of diseases characterised in impaired intercellular gap junctional communication.
  • the invention further relates to a method of treating diseases, such as bladder incontinence, disorders of alveolar tissue and bronchial tissue, impaired hearing due to diseases of the cochlea, endothelial lesions, diabetic retinopathy and diabetic neuropathy, ischemia of the central nervous system and spinal cord, dental tissue disorders including parodontal disease, kidney diseases such as impaired secernation from the juxtaglomarular apparatus leading to hypertension, and a method of preventing failures of bone marrow transplantation.
  • diseases such as bladder incontinence, disorders of alveolar tissue and bronchial tissue, impaired hearing due to diseases of the cochlea, endothelial lesions, diabetic retinopathy and diabetic neuropathy, ischemia of the central nervous system and spinal cord, dental tissue disorders including parodontal disease, kidney diseases such as impaired secernation from the juxtaglomarular apparatus leading to hypertension, and a method of preventing failures of bone marrow transplantation.
  • Gap junctions are specialized regions of the cell membrane with clusters of hundreds to thousands of densely packed gap junction channels that directly connect the cytoplasmic compartment of two neighboring cells.
  • the gap junction channels are composed of two hemichannels (connexons) provided by each of two neighboring cells.
  • Each connexon consists of six proteins called connexins (Cx).
  • the connexins are a large family of proteins all sharing the basic structure of four transmembrane domains, two extracellular loops, and a cytoplasmic loop. There is a high degree of conservation of the extracellular loops and transmembrane domains among species and connexin isoforms.
  • the length of the C- terminus varies considerably giving rise to the classification of the connexins on the basis of the molecular weight.
  • the gap junction channel can switch between an open and a closed state by a twisting motion. In the open state ions and small molecules can pass through the pore. The conduction of the electrical impulse and intercellular diffusion of signaling molecules take place through the gap junctions and normally functioning gap junctions are therefore a prerequisite for normal intercellular communication. Normal intercellular communication is requisite for for cellular homeostasis, proliferation and differentiation.
  • Gap junctions are one type of junctional complex formed between adjacent cells and consist of aggregated channels that directly link the interiors (cytoplasm) of neighbouring cells. In the adult mammal, gap junctions are found in most cell types with one known exception being circulating blood elements.
  • GJIC gap junction intercellular communication
  • Gap junctions are relatively non-selective and allow the passage of molecules up to about 1000 Daltons. Such substances are, i.a., ions, water, sugars, nucleotides, amino acids, fatty acids, small peptides, drugs, and carcinogens. Channel passage does not require ATP and appears to result from passive diffusion. This flux of materials between cells via gap junction channels is known as gap junctional intercellular communication (GJIC), which plays an important role in the regulation of cell metabolism, proliferation, and cell-to-cell signaling.
  • GJIC gap junctional intercellular communication
  • gap junction coupled cells within a tissue are not individual, discrete entities, but are highly integrated with their neighbors. This property facilitates homeostasis and also permits the rapid, direct transfer of second messengers between cells to co-ordinate cellular responses within the tissue.
  • GJIC The process of GJIC is regulated by a variety of mechanisms that can be broadly divided into major categories.
  • the cellular quantity of gap junctions is controlled by influencing the expression, degradation, cellular trafficking of connexins to the plasma membrane, or assembly of connexins into functional gap junctions.
  • Another type of regulation does not generally involve any gross alteration of the cellular levels of gap junctions or connexins, but induces opening or closure (gating) of existing gap junctions.
  • Extracellular soluble factors such as mitogens (e.g. DDT), hormones (e.g.
  • catecholamines catecholamines
  • anaesthetics e.g. halothane
  • intracellular biomolecules e.g. cA P
  • cell stress e.g. mechanical or metabolic stress
  • GJIC is regulated during the cell cycle and during cellular migration.
  • GJIC regulation or junctional gating has been widely studied for gap junctions especially gap junctions composed of Cx43.
  • These agents initiate complex signaling pathways consisting of the activation of kinases, phosphatases, and interacting proteins. Understanding the mechanisms of action of these GJIC modulators will not only define their respective signaling pathways responsible for junctional regulation, but will also provide experimental tools for characterising the biological functions of GJIC and connexins.
  • Changes in the phosphorylation of specific sites of the cytoplasmic carboxy terminal domain of Cx43 appear to be pivotal to the opening and closing of the gap junctional channel. Phosphorylation of the carboxy terminal domain may also be important to the process of bringing Cx43 gap junctional hemicomplex to the cell membrane, its internalisation and degradation. Connexins have half-lives (hours) that are much shorter than most plasma membrane proteins (days), e.g. the half-life of Cx43 in rat heart is less than I 1 /, hour. Thus, regulation of the turnover rate would be an important factor in regulating GJIC.
  • the carboxy terminal domain contains putative phosphorylation sites for multiple protein kinases (PKA, PKC, PKG, MAPK, CaMkll and tyrosine kinase). Phosphorylation of these sites of the carboxy terminal domain results in closure of gap junctional channels and various inhibitors of Cx43 gap junctional channels use different signalling pathways to induce phosphorylation of the carboxy terminal domain.
  • PKA protein kinases
  • PKC protein kinases
  • Other mechanisms regulating channel gating include intracellular levels of hydrogen and calcium ions, transjunctional voltage, and free radicals. Decreased pH or pCa induce channel closure in a cell- and connexin-specific manner.
  • GJIC permits the rapid equilibration of nutrients, ions, and fluids between cells. This might be the most ancient, widespread, and important function for these channels.
  • Electrical coupling Gap junctions serve as electrical synapses in electrically excitable cells such as cardiac myocytes, smooth muscle cells, and neurones. In these tissues, electrical coupling permits more rapid cell-to-cell transmission of action potentials than chemical synapses. In cardio yocytes and smooth muscle cells, this enables their synchronous contraction.
  • Second messengers such as cyclic nucleotides, calcium, and inositol phosphates are small enough to pass from hormonally activated cells to quiescent cells through junctional channels and activate the latter. Such an effect may increase the tissue response to an agonist.
  • Regulation of embryonic development Gap junctions may serve as intercellular pathways for chemical and/or electrical developmental signals in embryos and for defining the boundaries of developmental compartments. GJIC occurs in specific patterns in embryonic cells and the impairment of GJIC has been related to developmental anomalies and the teratogenic effects of many chemicals. The intercellular communication ensures that the activities of the individual cells happen in a co-ordinated fashion and integrates these activities into the dynamics of a working tissue serving the organism in which it is set.
  • the antiarrhythmic peptides are a group of peptides that exert their effect selectively on gap junctions and thus decrease cellular uncoupling and also reduce dispersion of action potential duration.
  • the native AAP as well as the synthetic AAP10 possess several undesired features, such as, low stability, high effective concentration etc. that has hitherto prevented their utilisation as drugs.
  • Grover and Dhein [ 1] have characterised two semi cyclic conformations of AAP10 using nuclear magnetic resonance spectroscopy. Therefore, one approach to obtaining a stable antiarrhytmic peptide could be the provision of cyclic derivatives of antiarrhythmic peptides.
  • DE19707854 discloses apparently cyclic CF 3 C(OH)-Gly-Ala-Gly-4Hy ⁇ -Pro-Tyr-CONH and cyclic CO-Gly-Ala-Gly-4Hyp-Pro-Tyr-CONH having the same antiarrhythmic properties as AAP and AAP10, but stated to have improved stability in aqueous solution and after repeated cycles of freezing and thawing.
  • the experimental conditions described in DE19707854 are insufficient for the preparation of said cyclic compounds, and the chemical identification data given therein using HPLC is not sufficient for identification of said cyclic compounds.
  • the peptides herein increase gap junction intercellular communication (GJIC) in vertebrate tissue, and specifically in mammalian tissue, and are useful in the treatment of a wide spectrum of diseases and ailments in vertebrates, such as mammals, relating to or caused by a decreased function of intercellular gap junction communication as is described below.
  • GJIC gap junction intercellular communication
  • diseases and medical conditions are characterised in reduced or impaired cellular communication, such as caused by impaired gap junctional intercellular communication or impaired coupling through gap junctions.
  • diseases and medical conditions are inflammation of airway epithelium, disorders of alveolar tissue, bladder incontinence, impaired hearing due to diseases of the cochlea, endothelial lesions, diabetic retinopathy and diabetic neuropathy, ischemia of the central nervous system and spinal cord, dental tissue disorders including periodontal disease, kidney diseases, and failures of bone marrow transplantation as mentioned above.
  • the purpose of the present invention is achieved with the present peptides including antiarrhythmic peptide compounds.
  • the present invention provided methods for preventing or treating diseases caused by impaired cellular communication or impaired gap junction function.
  • Illustrative diseases include those effecting the respiratory, circulatory or nervous systems, vision and hearing, dental tissues, smooth musculature, and transplantation of cells and tissues.
  • Such methods can be used alone as the sole therapeutic regimen or in combination with one or more other established protocols for addressing a particular disease or condition.
  • Preferred invention practice involves treatment of mammals e.g., primates, rodents (including mice, rats, hamsters, and lagomorphs, such as rabbits), dogs, pigs and goats.
  • a preferred primate is a human patient.
  • Compounds useful in the methods of the invention are characterised in functioning as facilitators of GJIC.
  • the present invention relates to a method of preventing or treating non-proliferative diseases caused by impaired gap junction function by facilitating (maintaining) the intercellular communication in the diseased cells and tissues occurring through gap junctions, preferably by administering a therapeutically effective amount of at least one compound which facilitates gap junction intercellular communication to a patient suffering from said disease.
  • the compounds that are useful in the present invention all share the feature of facilitating or mediating GJIC in cells and tissues.
  • the mechanisms through which this GJIC mediation is effected may vary since there are many cellular mechanisms that affect connexin functioning and/or mediate gap junction function. These mechanisms include, e.g. • control of the cellular quantity of gap junctions by upregulating or normalising the expression of connexins,
  • This mechanism can be described as a reversal of the gap junction closure effected by inhibitors of GJIC acting through a direct or indirect mechanism, such as e.g. hyperphosphorylation of the cytoplasmic carboxy terminal domain of the connexins, e.g. Cx43.
  • the carboxy terminal domain contains putative phosphorylation sites for multiple protein kinases (PKA, PKC, PKG, MAPK, CaMkll and tyrosine kinase). Phosphorylation of these sites of the carboxy terminal domain results in closure of gap junctional channels and various inhibitors of Cx43 gap junctional channels use different signalling pathways to induce phosphorylation of the carboxy terminal domain.
  • PKA protein kinases
  • PKC protein kinases
  • Other mechanisms regulating channel gating include intracellular levels of hydrogen and calcium ions, transjunctional voltage, low oxygen and glucose availability, and free radicals. Decreased pH or pCa induce channel closure in a cell- and connexin-specific manner.
  • peptides such as antiarrhythmic peptides, and preferably the peptides described below in detail (described in PCT/DK01/00127 and the USSN 09/792,286 both filed on 22 February 2001.
  • the USSN peptides, such as antiarrhythmic peptides, and preferably the peptides described below in detail (described in PCT/DK01/00127 and the USSN 09/792,286 both filed on 22 February 2001.
  • PA2000 00288 filed on February 23. 2000 and DK PA2000 00738 filed on May 4, 2000.
  • antiarrhythmic peptides disclosed in PCT/DK01/00127 and functional analogs thereof are useful in the present invention.
  • Said antiarrhythmic peptides include a group of peptides that exert their effect selectively on gap junctions and thus decrease cellular uncoupling and reduce dispersion of action potential duration similar to the effect described above for the antiarrhythmic peptide AAP10.
  • the molecular target or receptor for the antiarrhythmic peptides is presently unknown.
  • the structure of the binding site for AAP10 on a putative receptor has been hypothesised by R. Grover and S. Dhein (Peptides 2001, 22 1011- 1021).
  • a peptide which is useful in the present invention is an agonist of a receptor for an antiarrhythmic peptide, such as AAP10, and that the physiological effect of the interaction between peptide and receptor is an increased cellular coupling through gap junctions or a potentiation or mediation of GJIC.
  • AAP10 antiarrhythmic peptide
  • the present invention provides methods for the treatment of diseases and tissue disturbances caused by an excess of reactive oxygen species and/or free radicals and/or nitric oxide.
  • An example is diabetic neuropathy and wounds where free radicals cause a depletion of gluthation and consequently a reduction of gap junctions, or an uncoupling of the gap junction communication.
  • Low oxygen supply and/or high concentration of free radicals is significant in wounds with necrotic tissue, in diabetes, in arteriosclerosis, in surgery wounds, oedema, infection, burn wounds and in venous insufficiency will lower the gap junction communication.
  • Free radicals are of importance for nerve terminal destruction, decreased conductance, demyelination and increased inflammatory response.
  • Noise induced hearing loss, presbycusis is known to be associated with production of free radicals and is related to inhibition of gap junction coupling. Excess of free radicals may also reduce endothelial repair and capillary sprouting during angiogenesis.
  • the invention provides methods for treating or preventing airway inflammation.
  • Preferred methods include administering to a patient in need of such treatment a therapeutically effective amount of at least one compound which facilitates gap junctional intercellular communication.
  • Methods for treating or preventing bladder incontinence are also provided by the present invention.
  • the methods include administering to a patient in need of such treatment a therapeutically effective amount of at least one compound which facilitates gap junctional intercellular communication.
  • the invention also provides methods for treating or preventing impaired hearing due to diseases of the cochlea.
  • the methods include administering to a patient in need of such treatment a therapeutically effective amount of at least one compound which facilitates gap junctional intercellular communication.
  • the invention relates to the use of a compound which facilitates cellular communication, such as gap junctional intercellular communication for the manufacture of a pharmaceutical composition for the prevention or treatment of diseases and preferably non-proliferative diseases including, e.g. inflammation of airway epithelium, disorders of alveolar tissue, wounds, erectile dysfunction, urinary bladder incontinence, impaired hearing due to diseases of the cochlea, endothelial lesions, diabetic retinopathy and diabetic neuropathy, neuropathic pain, ischemia of the central nervous system, spinal cord injuries, dental tissue disorders including periodontal disease, kidney diseases, subchronic and chronic inflammation, cancer and failures of bone marrow and stem cell transplantation.
  • diseases and preferably non-proliferative diseases including, e.g. inflammation of airway epithelium, disorders of alveolar tissue, wounds, erectile dysfunction, urinary bladder incontinence, impaired hearing due to diseases of the cochlea, endothelial lesions, diabetic retin
  • Peptides useful in the present invention include compounds of the general formula — X-A-B-Y " 1 L ⁇ J where the dashed line indicates that formula I is optionally cyclic, and the bonds shown represent covalent bonds; and wherein A represents a chemical moiety having an amino group (radical) and a carboxy group (radical) that forms part of the peptide bond connecting A to X and B; B represents a chemical moiety having an amino group (radical) and a carboxy group (radical) that forms part of the peptide bond connecting B to A and Y; X represents a peptide sequence of from 1 to 3 amino acid residues which independently may be an L or D form when Y represents a C-terminal peptide sequence of from 2 to 5 amino acid residues which may independently be L- or D-forms; or X represents an N-terminal modification of the group A-B when Y represents a C-terminal peptide sequence of from 2 to 5 amino acid residues which may independently be L- or D- forms
  • CO-Gly-Ala-Gly-4Hyp-Pro-Tyr-CONH are not covered by said general formula. It is preferred that the covalent bonds are selected from peptide bonds, disulphide bonds, ester bonds, reduced amide bonds, alkoxy bonds, oxycarbonyl bonds, and acyloxyalkoxy bonds.
  • a and B include the formula Z
  • n is an integer having the value 3, 4, or 5
  • R represents an optional substituent, preferably selected from the group consisting of halogen, phenyl, hydroxy, NH2, and C(l-6)alkyl.
  • a and B each represents an amino acid or an amino acid derivative having functional amino and carboxylic acid groups. Further examples of A and B are represented by the formula Za
  • a and B include but are not limited to N- and C(O)- radicals of the following compounds: D/L-azetidin-3-carboxylic acid,
  • the chemical moiety of A and B each represents an amino acid residue having a saturated carbocyclic structure of 4, 5 or 6 members comprising one or more heteroatoms, such as N and S.
  • Said amino acids include L and D forms, natural and unnatural amino acids and derivatives thereof, such as a Prolin residue having one or more substituents in the 3, 4 or 5 position, said substituents being preferably selected from hydroxy, amino or phenyl; and N-substituted amino acids, such as Sarcosin, N- cyclohexylglycine, and N-phenylglycine.
  • the sequence A-B represents a dipeptide selected from the group consisting of Sar-Sar, Sar-Hyp, Hyp-Sar, Pro-Sar, Sar-
  • Pro, Pro-Hyp, Pro-Pro, Hyp-Pro, and Hyp-Hyp where Pro and Hyp independently may be an L or D form, where the ring structure of Pro and Hyp is optionally substituted with halogen, nitro, methyl, amino, or phenyl, and Hyp represents 3-hydroxyproline or 4- hydroxyproline, or one or both of the amino acid residues of A-B is a Sar, or N- cyclohexylglycine residue.
  • the general formula above may represents a linear peptide wherein said chemical modification of the N-terminal of X is an acylation with an optionally substituted C(l- 22)alkyl carboxylic acid, such as acetic acid, propionic acid, butyric acid and other fatty acids, or an optionally substituted C(2-22)alkenyl carboxylic acid, or an aryl carboxylic acid, such as benzoic acid, where the substitutent is selected from hydroxy, halogen, C(l- 6)alkyl, nitro or cyano and may be situated on the carbon chain or the aromatic moiety; or an alkylation with an optionally substituted C(l-22)alkyl, C(2-22)alkenyl, or aryl C(l- 22)alkyl, such as methyl, ethyl, propyl, butyl, phenylpropyl, 2-hydroxyphenylpropyl, and 4-hydroxyphenylpropyl, where the substitutent is selected from hydroxy,
  • X is selected from the group consisting of L-Tyr and D-Tyr optionally acylated with a C(l-4)carboxylic acid, preferably acetic acid, when Y represents a C- terminal peptide sequence of from 2 to 5 amino acid residues as defined above.
  • formula I represents a cyclic peptide wherein A-B is selected from the group consisting of
  • A-B represents unsubstituted L-Pro-L-4Hyp, L-4Hyp-L- Pro, D-Pro-D-4Hyp, or D-4Hyp-D-Pro.
  • X represents a single amino acid residue, preferably L-Tyr or D-Tyr optionally further substituted with halogen, phenyl, hydroxy, NH 2 , and C(l-6)alkyl optionally substituted with halogen, at its aromatic ring when Y represents a peptide of 3 or 4 amino acid residues being independently L- or D-forms, preferably having Asp or Glu at its C- terminal, and more preferably when Y represents a peptide sequence selected from the group consisting of Gly-L-Ala-L-Asn, Gly-D-Ala-L-Asn,
  • Or X represents a peptide sequence preferably selected from the group consisting of Gly-L-Ala-L-Asp,
  • Gly-D-Ala-Gly-D-Glu when Y represents a single amino acid residue, preferably L-Tyr or D-Tyr optionally further substituted with halogen, such as Cl, at its aromatic ring.
  • Formula I may represent a cyclic peptide sequence comprising all L-forms, all D-forms, or a sequence of mixed L- and D-forms of the amino acid residues.
  • Fig. 1 shows a general outline of seven different cyclic structures within the scope of the present invention.
  • formula I represents a cyclic compound where the groups X and Y are connected via an amino carbonyl bond, an alkoxy bond, an ester bond, a reduced amide bond, or a disulphide bond.
  • R' and R" each represents hydrogen or lower alkyl and/or lower aryl, preferably methyl and phenyl are listed below
  • R' and R" each represents hydrogen or lower alkyl and/or lower aryl, preferably methyl and phenyl, preferably R' ⁇ R", are listed below:
  • L may be derived from a hydroxy-carboxylic acid, such as a hydroxy C(3-6)alkyl carbocylic acid.
  • L is derived from an ⁇ - hydroxy-carboxylic acid preferably of the general formula HO-C(Rl)(R2)-COOH wherein Rl and R2 independently is H, C(l-6)-alkyl, C(2-6)-alkenyl, aryl, aryl-C(l-4)-alkyl, heteroaryl or heteroaryl-C(l-4)-alkyl; or Rl and R2 together with the carbon atom to which they are bound form a cyclopentyl, cyclohexyl, or cycloheptyl ring; where an alkyl or alkenyl group may be substituted with from one to three substituents selected from amino, cyano, halogen, isocyano, isothio
  • L is derived from a hydroxy aryl-C(3-6)-alkyl-carboxylic acid, or L is derived from a hydroxy C(2- 6)alkenyI-carboxylic acid, or L is derived from a hydroxy C(3-6)alkyl carboxylic acid. It is preferred that Rl and R2 represent different groups.
  • the group A-B is selected from the group consisting of Sar-Hyp, Hyp-Sar, Pro-Hyp, Pro-Pro, Hyp-Pro, and Hyp-Hyp where Pro and Hyp independently may be an L or D form and Hyp preferably represents 4- hydroxyproline. More preferably, A-B represents unsubstituted L-Pro-L-4Hyp, L-4Hyp-L- Pro, D-Pro-D-4Hyp, or D-4Hyp-D-Pro.
  • the invention relates to peptides and peptide derivatives of the general formula I
  • amino acid residues may be D- and/or L- forms, having the N-terminal at N* and the C-terminal at C* and being optionally cyclic via a covalent bond between N* and C* as shown by a broken line or between R d and C* as shown by the broken line U; and wherein
  • X represents an N-terminal moiety such as a photoprobe capable of being bond to the amino terminal N*, or an acyl group derived from a C(2-22)alkyl carboxylic acid, such as acetic acid, propionic acid, butyric acid and other fatty acids, such as behenic acid, optionally substituted with one or more substituents selected from the group consisting of hydroxy, halogen, C(l-6)alkyl, nitro and cyano; or X represents hydrogen;
  • R 7 represents OH, NH 2 , NHNH 2 or OR 8 when the bond between N* and C* is missing, or R 7 is absent when there is a bond between N* and C*;
  • Rs represents H or a straight or branched C(l-6)alkyl group, an aryl or an aralkyl group.
  • R a represents the amino acid side chain of Hyp or Pro
  • R b represents the amino acid side chain of Hyp or Pro
  • R c represents the amino acid side chain of Gly, Sar, an aromatic amino acid side chain optionally substituted with one or more hydroxy, halogen or lower alkoxy group in the aromatic ring or R c ;
  • R d represents the amino acid side chain of Ala, Gly, Glu, Asp, Dab, Dapa, Lys, Asn, Gin,
  • R e represents the amino acid side chain of Ala
  • R f represents the amino acid side chain of Ala, Sar or Gly
  • R g represents any amino acid side chain except the side chain of L-4Hyp or a moiety of formula Z or Za;
  • R represents the amino acid side chain of Ala, or R h represents a moiety of formula Z or
  • Ri represents the amino acid side chain of Gly or R, represents an aromatic amino acid optionally substituted with one or more halogen groups in the aromatic ring, preferably
  • R represents Asn, Gin, Asp, Glu, Cys, or Tyr; and each of j, k, I, m, n, p and q is independently 0 or 1; and the retro form, all D form, or retro all-D form of the peptide sequence of formula I, and salts and amides thereof.
  • X is preferably selected from the group consisting of photoprobes such as ASAL optionally iodinated in position 5, such as 2-hydroxy-4- azido-5-iodo benzoyl, and AB, and an acyl group such as Ac.
  • R 7 is preferably NH 2 .
  • R a is preferably the amino acid side chain of Pro.
  • R is preferably the amino acid side chain of Hyp.
  • R c is preferably the amino acid side chain of Gly or Tyr.
  • R d is preferably the amino acid side chain of Gly, Asp, Glu, Dapa, or Dab.
  • R e is preferably Ala.
  • R f is preferably the amino acid side chain of Gly or Ala.
  • R g is preferably the amino acid side chain of Asn, Gly, D-4Hyp or L-/D-Pro when formula I represents a linear peptide, or when formula I represents a peptide cyclised between N* and C* then R g represents the amino acid side chain of L-/D-4Hyp or L-/D-Pro.
  • R h is preferably the amino acid side chain of Ala when U is missing, or R h is Pro or Hyp when U is present.
  • Rj is preferably Tyr, Phe, Trp, Nal optionally substituted with one or more hydroxy or halogen group, preferably F or Cl, in the aromatic ring.
  • R j is preferably the amino acid side chain of Asp or Glu.
  • R 8 represents H, benzyl, tert-butyl or CH 3 .
  • j and k are preferably 0 when U is present, and j and k are preferably 1 when U is missing and formula I represents a cyclic peptide, m is preferably 0 when U is missing, p is preferably 1 when U is present, and q is preferably 0 when U is present.
  • Non-cyclic or linear peptides of formula I are preferably of the retro all-D form. When formula I represents a cyclic peptide, then the peptide preferably consists of between 3 and 9 amino acid residues, more preferably between 3 and 7 amino acid residues.
  • amino acid residues may be L and/or D forms, and wherein
  • X represents H or Ac; when all amino acid residues are L-forms then X represents Ac;
  • G' represents a glycine residue or a glycine analogue such as Sar, G' is preferably glycine; A represents alanine;
  • Px represents an amino acid residue of formula Z or Za such as Hyp or Pro, preferably proline;
  • Y' represents tyrosine or phenylalanine optionally substituted in the phenyl ring with halogen or hydroxy; Y' is preferably tyrosine; a and b are independently 0 or 1,
  • R 7 represents OH, NH 2 , NHNH 2 , Asn-NH 2 , or Gln-NH 2 ; and retro forms thereof having the formula Ila : X-(Y') b -(Px) 2 -G'-A-(G') a -R 7 wherein all amino acid residues preferably are D-forms and wherein all symbols have the same meaning as defined above for formula II; and peptide compounds of formula II wherein at least one Px residue is a D-amino acid and the rest are L-amino acids; and cyclic sequences of formula II wherein X represents H, R 7 represents Asn or Gin having a covalent bond to Y', b is 1, and a is 1; and salts thereof.
  • X represents H or an N-terminal moiety such as a photoprobe capable of to the N terminal or an acylation with a C(2-22)alkyl carboxylic acid, such as acetic acid, propionic acid, butyric acid and other fatty acids such as behenic acid, being optionally substituted with one or more substituents selected from the group consisting of hydroxy, halogen, C(l-
  • Ri represents H or CH 3 , preferably H;
  • R 2 and R 3 are different or the same and represent any possible amino acid side chain, preferably H or CH 3 ;
  • R 5 and R represent any possible amino acid side chain or when the optional bond is present
  • R 5 and R 4 represent together with the attached C and N atoms a proline ring which is optionally substituted with OH, preferably in the 4-position, or
  • R 5 and R 4 represent together with the attached C and N atoms a moiety of formula Z or Za above, preferably Pro or Hyp;
  • R 6 represents an aromatic amino acid side chain, preferably benzyl optionally substituted in the phenyl ring with one or more substituents selected from halogen, nitro and hydroxy, preferably R 6 represents Tyr; p is 0 or 1; n is 1, 2, 3 or 4; preferably n is 1; and salts thereof.
  • R 8 is the same as defined above, preferably H;
  • R and R 5 are different or the same and represent any possible amino acid side chain, preferably Gly or Ala;
  • R 2 and R 3 represent any possible amino acid side chain, or when the optional bond is present R 2 and R 3 represent together with the attached C and N atoms a proline ring which is optionally substituted with OH preferably in the 4-position or R 2 and R 3 represent a moiety of formula Z or Za;
  • Ri represents an aromatic amino acid side chain, preferably a Tyr side chain; p is 0 or 1; n is 1, 2, 3 or 4; preferably n is 1; and salts thereof.
  • a preferred embodiment of the invention relates to peptide compounds wherein the amino acid residues may be D- and/or L-forms, and having the general formula V
  • Ri represents an optional amide bond between the N and the C terminal of the peptide, H or Ac;
  • Aa x represents a peptide sequence, preferably of between 0 and 4 amino acid residues, when Aa ! represents a peptide sequence of from 1 to 4 amino acid residues
  • Aa x is preferably selected from the group consisting of Ala, Gly-Ala, Gly-Asn-Tyr, and Gly-Asn-
  • Ar represents an aromatic amino acid residue, such as a Tyr, Trp, Phe, His, or Nal, optionally substituted with one or more halogen, such as F, Cl, Br, I, OH, N0 2 , NH 2 , COOH,
  • halogen such as F, Cl, Br, I, OH, N0 2 , NH 2 , COOH
  • R 2 represents OH, NH 2 or is missing; and retro analogues, retro all-D analogues (retro-inverse analogues) and salts thereof.
  • Photo/thermo labile peptide derivatives Affinity labeling is a frequently used technique for studying the interactions of biologically active molecules. A photo or a thermo labile analogue of the compound is used for the investigation.
  • a photolabile analogue of the compound under investigation which is stable in the dark, is converted by illumination into a reactive intermediate that can participate in insertion reactions. This, by forming a covalent bond, stabilizes the interaction based on biological affinity.
  • aromatic azides and stabilized diazo compounds produce on photolysis very reactive and nonspecific intermediates, nitrenes and carbenes, respectively capable of participating in insertion reactions.
  • photo affinity labeling using aryl azides and stabilized diazo compounds as photo probes can be done on any binding site which contains carbon-hydrogen bonds and do not require the presence of a particular reactive functional group at the binding site.
  • Specificity of labeling therefore depends solely on the specific binding of the ligand to the receptor, which is then followed by a nonspecific covalent bond forming reaction that guarantees labeling of the binding site.
  • Photoaffinity probes is particularly useful for labeling hormone receptor sites where reactive functional groups may not be present, but which surely contains carbon-hydrogen bonds.
  • As photo active functionality the azido, diazirino, ⁇ -diazo ketones, thia- and selenodiazoles, benzophenone, nitrophenyl are especially useful.
  • thermo labile compound contains a reactive group which can form a covalent bond in a thermal controlled reaction with specificity towards amino or mercapto groups.
  • thermo probes aliphatic halides especially iodine and bromine, active esters such as N- hydroxysuccinimid, acid chlorides, pyridyldisulphides, isocyanates, isothiocyanates, carbodiimides, and maleimido can be used.
  • Labels for in vitro applications are most often chosen as radioactive isotopes such as Iodine-125 and 131, C-14 and tritium or fluorescence probes or biotin or haptens.
  • the influence of the label on the binding activity of the ligand needs to be investigated, in order to secure that the receptor affinity is maintained.
  • radioactive label Iodine-125 is often used for in-vitro applications, due to its 60 days half-life and low energy photon emissions. The long half-life permits the preparation and storage of labeled photoactive analogues and the resulting labeled protein products for extended periods prior to usage or analysis.
  • the incorporation of Iodine (1-125) into peptide ligands can easily be done if e.g.
  • tyrosine og histidine are present in the peptide sequence.
  • the influence of the labeling of the peptide on the biological activity of the ligand needs to be investigated, in order to secure that the biological activity is maintained.
  • Dhein et al. (W096/21674) have shown that a derivative of AAPlO where the phenyl ring of the Tyr residues carries an Iodine-125 substituent has biological activity.
  • the use of said AAPlO variant as an affinity probe is not possible due to the reversible binding to a possible ligand or receptor.
  • Photoaffinity labeling using aryl azides results generally in 50-60% peptide ligand non-reversibly attached to the target protein (receptor).
  • an antiarrhythmic peptide suitably modified with a photo or a thermo probe and optionally a radioactive label to be used in assays for the identification of possible ligands or receptors for the antiarrhythmic peptide.
  • Said purpose is achieved with a compound of formulae I, II or 9 herein, derivatised with one of the above mentioned photo probes, preferably 4-azidosalicyloyl (ASAL) and AB (4- azidobenzoyl).
  • said derivatised compound is further substituted with a radioactive label, such as Iodine-125.
  • the invention relates to peptide compounds selected from the group consisting of the general formulae
  • Pa is any amino acid residue or a moiety of formula Z or Za; at least one of Pa is a D amino acid; preferably Pa is Hyp, P, G or A;
  • Rl H, Ac, HAA, THAA(thiohydroxyacetic acid), Tfa, aroyl, acetyl
  • R3 the side chain of G, A, N, K, C, I would say any amino acid
  • R4 OH, N0 2 , Halogen (F, Cl,Br, I) NH 2 or H
  • R5 (4-hydroxyphenyl or 4-nitrophenyl or 4-Fluorophenyl or 4-Chlorophenyl or 4-Bromophenyl or 4-Iodophenyl or 4-aminophenyl or 4-alkoxyphenyl or H
  • R6 OH, N0 2 , Halogen (F, Cl,Br, I) NH 2 or H
  • R7 OH, N0 2 , Halogen (F, Cl,Br, I) NH 2 or H
  • Rl represents H or acetyl (Ac)
  • R2 represents a sidechain of one of the amino acids G, Y, D-Y, F and D-F, R3 represents O or H
  • R4 represents any amino acid sidechain
  • R5 represents O OR H
  • R6 represents a C(l-4)alkyl group, such as CH 2 , (CH 2 ) , (CH 2 ) 3 , and (CH 2 ) 4
  • R7 represents O OR H
  • R8 represents O OR H
  • R9 represents a sidechain of one of the amino acids G, Y, D-Y, F and D-F,
  • V 0 or 1
  • XI is 0, Ala, Gly, ⁇ -AJa, Tyr, D-Tyr, Asp, HAA
  • X2 is 0; Ala-Gly-T4c-Pro; Ala-Sar-Hyp-Pro; Ala-6ring-; Ala-Asn; D-Asn-D-Ala; D-Asn; yAbu; Gly, Ala; D-Ala; ⁇ -Ala; Pamh; Asn; or HAA;
  • X3 is Tyr; D-Tyr; Gly, Pamb, or Phe; and
  • Rl is H or Ac, with the proviso that XI and X2 are not both 0; and salts thereof.
  • compounds of the invention are used in the form of a pharmaceutically acceptable salt, an alkyl ester, an amide, an alkylamide, a dialkylamide or a hydrazide formed with the C-terminal carboxylic acid function of a linear compound or a free carboxylic acid function, if present, of a cyclic compound.
  • Amides and lower alkyl amides of linear compounds are among the preferred compounds of the invention.
  • Salts include pharmaceutically acceptable salts, such as acid addition salts and basic salts. Examples of acid addition salts are hydrochloride salts, sodium salts, calcium salts, potassium salts, etc.
  • Examples of basic salts are salts where the cation is selected from alkali metals, such as sodium and potassium, alkaline earth metals, such as calcium, and ammonium ions + N (R 3 ) 3 (R 4 ), where R 3 and R 4 independently designates optionally substituted C ⁇ _ 6 -alkyl, optionally substituted C 2 . 6 -alkenyl, optionally substituted aryl, or optionally substituted heteroaryl.
  • alkali metals such as sodium and potassium
  • alkaline earth metals such as calcium
  • R 3 and R 4 independently designates optionally substituted C ⁇ _ 6 -alkyl, optionally substituted C 2 . 6 -alkenyl, optionally substituted aryl, or optionally substituted heteroaryl.
  • R 3 and R 4 independently designates optionally substituted C ⁇ _ 6 -alkyl, optionally substituted C 2 . 6 -alkenyl, optional
  • the three letter code for natural amino acids is used as well as generally accepted three letter codes for other ⁇ -amino acids, such as Sarcosin (Sar), ⁇ -Amino-iso-butanoic acid (Aib), Naphthylalanine (Nal) including 1-naphthylalanine (INal) and 2-naphthyIalanine (2Nal), Phenylglycine Phg, 2,4-Diaminobutanoic acid (Dab), 2,3-Diaminopropanoic acid (Dapa), and Hydroxyproline (Hyp). Where nothing is specified Hyp represents 4-hydroxyproline.
  • the natural or essential amino acids are the amino acid constituents of proteins.
  • the aromatic amino acids are Phe, Tyr, Trp, INal, 2Nal and His. Where the L or D form has not been specified it is to be understood that the amino acid in question has the natural L form, cf. Pure & Appl. Chem. Vol. 56(5) pp595-624 (1984). Where nothing is specified it is to be understood that the C-terminal amino acid of a compound of the invention exists as the free carboxylic acid, this may also be specified as "-OH”.
  • the C-terminal amino acid of a compound of the invention may be shown to have the terminal function "-OH/NH2" which means that there are two preferred forms of the compound: the free carboxylic acid and the amidated derivative.
  • Hexapeptide compounds of the invention comprising the sequence Ala-Gly-Hyp and having an -NH2 group at the C- terminal do not contain a C-terminal Phe or Tyr or derivatives thereof having a halogen substitution in the phenyl ring.
  • analogues of antiarrhythmic peptides is meant any chemical entity or compound which has a structural conformation and/or binding properties that are sufficiently similar to the endogeneous AAP to provide one or more of the beneficial antiarrhythmic or antithrombotic properties of the endogeneous AAP.
  • heteroaryl includes 5- or 6-membered aromatic monocyclic heterocyclic groups containing 1-4 heteroatoms selected from nitrogen, oxygen and sulfur, such as pyrrolyl, fury!, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, triazolyl, pyridyl, and aromatic bicyclic heterocyclic groups containing 1-6 heteroatoms selected from nitrogen, oxygen and sulfur, such as quinolinyl.
  • halogen refers to F, Cl, Br, and I, where F and I are preferred.
  • alkyl refers to univalent groups derived from alkanes by removal of a hydrogen atom from any carbon atom: C n H 2n+ ⁇ -.
  • the groups derived by removal of a hydrogen atom from a terminal carbon atom of unbranched alkanes form a subclass of normal alkyl (n- alkyl) groups: H[CH 2 ] n -.
  • the groups RCH 2 -, R 2 CH- (R not equal to H), and R 3 C- (R not equal to H) are primary, secondary and tertiary alkyl groups respectively.
  • alkenyl refers to a straight or branched or cyclic hydrocarbon group containing one or more carbon-carbon double bonds.
  • C(2-22)alkenyl refers to any alkenyl group having from 1 to 22 carbon atoms and includes C(2-6)a)kenyl, vinyl, allyl, 1-butenyl, etc.
  • aralkyl refers to aryl C(l-22)alkyl, and the term “aryl” throughout this specification means phenyl or naphthyl.
  • 2HPP refers to 3-(2-hydroxyphenyl)propionyl
  • HAA refers to hydroxy acetic acid
  • 4HPPA refers to 4-hydroxyphenoxyacetic acid
  • 2HPPA refers to 2-hydroxyphenoxyacetic acid
  • 4HMPA refers to 4-(hydroxymethyl)phenoxyacetic acid
  • 4HPA refers to 4-hydroxyphenylacetic acid
  • 3HPA refers to 3-hydroxyphenylacetic acid
  • 3HBG refers to N-(3-hydroxybenzoyl)glycine
  • 2HBG refers to N-(2-hydroxybenzoyl)glycine
  • Tfa refers to trifluoroacetyl radical
  • T4c refers to L-thiazolidin-4-carboxylic acid radical
  • HOBt 4-azidobenzoyl radical
  • HOAt refers to l-Hydroxy-7-azabenzotriazole
  • Pd(PPh 3 ) 4 is tetrakis(triphenylphosphine)palladium(0)
  • DBF is defined as 2-aminoethyl-6-dibenzofuranpropionic acid
  • amino acid residue is meant a natural as well as an unnatural amino acid unit, which herein is represented by the generally accepted three letter codes for amino acids, such as Sarcosin (Sar), alpha-Amino-iso-butanoic acid (Aib), Naphthylalanine (Nal) including 1-naphthylalanine (INal) and 2-naphthylalanine (2Nal), Phenylglycine Phg, 2,4- Diaminobutanoic acid (Dab), 2,3-Diaminopropanoic acid (Dapa), and Hydroxyproline (Hyp) and beta-Ala for beta-alanine.
  • Sarcosin Sarcosin
  • Al alpha-Amino-iso-butanoic acid
  • Naphthylalanine Naphthylalanine
  • Naphthylalanine Naphthylalanine (Nal) including 1-naphthylalanine (INal) and 2-
  • Hyp or 4Hyp represents 4- hydroxyproline.
  • the natural or essential amino acids are the amino acid constituents of proteins and may be represented by the generally accepted one-letter code.
  • the aromatic amino acids are Phe, Tyr, Trp, INal, 2Nal and His.
  • the amino acid in question has the natural L form, cf. Pure & Appl. Chem. Vol. 56(5) pp595-624 (1984).
  • the C-terminal amino acid of a compound of the invention exists as the free carboxylic acid, this may also be specified as "-OH".
  • the C-terminal amino acid of a compound of the invention may be shown to have the terminal function "-OH/NH2" which means that there are two preferred forms of the compound: the free carboxylic acid and the amidated derivative. It is to be understood that this definition of amino acid residue includes compounds, such as DBF, T4c, Pc, DNP, and 3-amino-l- carboxymethylvalerolactam that are amino acid like.
  • DNP functions as a hapten for antibody recognition, and compounds of the invention that contain a DNP moiety may preferably be used as research tools.
  • peptide mimetic refers to compounds of both peptide and non-peptide nature.
  • the objective behind the creation of peptidomimetics is to create scaffolds which can replace the peptide backbone. It is assumed that the secondary amide bonds in peptides are responsible for instability and possibly poor peptide transport properties across cell membranes. Proper placement of amino acid side chains with appropriate trajectories is viewed as the key design tactic in peptide peptidomimetics to achieve biological activity.
  • peptoid refers to compounds that may be characterised by topological similarity between the structural formula of the peptoid and the parent peptide.
  • a peptoid may be a compound consisting of peptide-like chains of amino acids bearing side chains on the backbone nitrogen atom rather than on the alpha-carbon as in true peptides
  • Petidomimetics and peptoids may comprise amino acid units having modified side chains, such as Nal, Dab, and Dapa, or they may comprise D-amino acids.
  • the various modifications of peptide and peptidomimetic structure described by El Tayar, N et al. (Amino Acids (1995) 8: 125-139) are included in the definitions herein.
  • intercellular communication facilitating compound refers to a compound that facilitates or mediates GJIC irrespective of the particular mechanism behind the resulting improved or normalised GJIC.
  • gap junction opener may refer to a substance that upon stimulation of a cell which expresses connexins produces increased conductance of the gap junctional channel, which in turn results in an increased exchange of molecules that are able to pass through gap junctions between extracellular and intracellular space and/or increased GJIC.
  • agonist refers to an endogenous substance or a drug that can interact with a receptor and initiate a physiological or a pharmacological response characteristic of that receptor (contraction, relaxation, secretion, enzyme activation, etc.).
  • An "antiarrhythmic peptide receptor agonist” or” AAP-R agonist” as used herein may or may not be equivalent with a “gap junction opener” depending on the specific biological mechanism behind the effect of the compound.
  • Gap junctions are one type of junctional complex formed between adjacent cells and consist of aggregated channels that directly link the interiors (cytoplasm) of neighbouring cells. In the adult mammal, gap junctions are found in most cell types with one known exception being circulating blood elements.
  • the structural unit of the gap junction channel is the connexon or hemi-channel.
  • Each connexon is comprised of six connexin polypeptides (Cx) which oligomerise to form an aqueous pore that spans a single plasma membrane.
  • Cx connexin polypeptides
  • the gap junction channel-forming connexins comprise a multi-gene family with at least fourteen mammalian connexins discovered thus far.
  • Connexin expression is tissue and cell specific, with some cells expressing multiple connexin isoforms.
  • Experimental evidence suggests two different hybrid configurations are possible: heterotypic cell-to-cell channels in which each connexon or hemichannel consists of a specific connexin isoform; or heteromeric channels where each connexon is a mixture of the different connexin isoforms expressed in a particular cell type.
  • Connexins are expressed in a cell-, tissue-, and development-specific manner.
  • the process of GJIC is regulated by a variety of mechanisms that can be broadly divided into two major categories.
  • the first type of regulation controls the cellular quantity of gap junctions by influencing the expression, degradation, cellular trafficking of connexins to the plasma membrane, or assembly of connexins into functional gap junctions. Impaired GJIC caused by the down-regulation of connexin expression in tumour cells is an example of this mode of regulation.
  • the second type of regulation does not generally involve any gross alteration of the cellular levels of gap junctions or connexins, but induces opening or closure or gating of existing gap junctions.
  • Extracellular soluble factors such as mitogens (e.g. DDT), hormones (e.g. catecholamines), anaesthetics (e.g.
  • junctional permeability may be necessary for mediating the specific biological functions of the latter group.
  • These agents initiate complex signalling pathways consisting of the activation of kinases, phosphatases, and interacting proteins. Understanding the mechanisms of action of these GJIC modulators will not only define their respective signalling pathways responsible for junctional regulation, but will also provide experimental tools for characterising the biological functions of GJIC and connexins.
  • Changes in the phosphorylation of specific sites of the cytoplasmic carboxy terminal domain of Cx43 appear to be pivotal to the opening and closing of the gap junctional channel. Phosphorylation of the carboxy terminal domain may also be important to the process of bringing Cx43 gap junctional hemicomplex to the surface membrane, its internalisation and degradation. Connexins have half-lives (hours) that are much shorter than most plasma membrane proteins (days), e.g. the half-life of Cx43 in rat heart is less than 1V_ hour. Thus, regulation of the turnover rate would be an important factor in regulating GJIC.
  • the carboxy terminal domain contains putative phosphorylation sites for multiple protein kinases (PKA, PKC, PKG, MAPK, CaMkll and tyrosine kinase). Phosphorylation of these sites of the carboxy terminal domain results in closure of gap junctional channels and various inhibitors of Cx43 gap junctional channels use different signalling pathways to induce phosphorylation of the carboxy terminal domain.
  • PKA protein kinases
  • PKC protein kinases
  • Other mechanisms regulating channel gating include intracellular levels of hydrogen and calcium ions, transjunctional voltage, and free radicals. Decreased pH or pCa induce channel closure in a cell- and connexin-specific manner.
  • GJIC homeostasis. GJIC permits the rapid equilibration of nutrients, ions, and fluids between cells. This might be the most ancient, widespread, and important function for these channels.
  • Gap junctions serve as electrical synapses in electrically excitable cells such as cardiac myocytes, smooth muscle cells, and neurones. In these tissues, electrical coupling permits more rapid cell-to-cell transmission of action potentials than chemical synapses. In cardiomyocytes and smooth muscle cells, this enables their synchronous contraction.
  • GJIC Tissue response to hormones.
  • GJIC may enhance the responsiveness of tissues to external stimuli.
  • Second messengers such as cyclic nucleotides, calcium, and inositol phosphates are small enough to pass from hormonally activated cells to quiescent cells through junctional channels and activate the latter. Such an effect may increase the tissue response to an agonist.
  • Gap junctions may serve as intercellular pathways for chemical and/or electrical developmental signals in embryos and for defining the boundaries of developmental compartments.
  • GJIC occurs in specific patterns in embryonic cells and the impairment of GJIC has been related to developmental anomalies and the teratogenic effects of many chemicals.
  • the intercellular communication ensures that the activities of the individual cells happen in co-ordinated fashion and integrate these activities into the dynamics of a working tissue serving the organism in which it is set. It is therefore not very surprising that a wide variety of pathological conditions have been associated with decreased GJIC.
  • Reentry is the major cause of sustained ventricular fibrillation and sudden cardiac death.
  • Reentry occurs when the propagating impulse does not die out after complete activation of the heart, but persists to reexcite the heart after the end of the refractory period.
  • the induction of reentry is facilitated by slow conduction, increased dispersion of repolarization, non-uniform anisotropy and unidirectional conduction block.
  • the underlying disease responsible for the majority of cases of ventricular reentry is ischemic heart disease (e.g., acute myocardial infarction, chronic myocardial infarction, stable angina pectoris, and unstable angina pectoris).
  • ischemic heart disease e.g., acute myocardial infarction, chronic myocardial infarction, stable angina pectoris, and unstable angina pectoris.
  • Increased dispersion of action potential duration has long been known to facilitate the induction of ventricular fibrillation [23] .
  • the difference in action potential duration is smoothened due to the electrical coupling.
  • uncoupling will prevent this smoothening and contribute to an unmasking of dispersion of action potential duration and refractory period [2 ] . If ischemia is prolonged a reduced degree of Cx43 expression and a changed pattern of distribution can be observed.
  • Hypertrophic cardiomyopathy e.g. due to hypertension, aortic stenosis, congenital
  • hypertension e.g. due to hypertension, aortic stenosis, congenital
  • reentry arrhythmias due to the mismatch between the large amount of myocardial tissue and the relative small amount of conductive tissue which may lead to slow conduction, increased dispersion and unidirectional conduction block.
  • Congenital diseases e.g., the long-QT syndrome
  • drugs that prolong the QT interval e.g., antiarrhythmic drugs, antipsycotic drugs, antihistamines, antibacterial drugs etc.
  • drugs that prolong the QT interval e.g., antiarrhythmic drugs, antipsycotic drugs, antihistamines, antibacterial drugs etc.
  • drugs that prolong the QT interval e.g., antiarrhythmic drugs, antipsycotic drugs, antihistamines, antibacterial drugs etc.
  • Atrial fibrillation - the most common cardiac arrhythmia - is also caused by a reentrant mechanism. In this case multiple wavelets travel across the atria and re-excite the tissue that is no longer refractory. Atrial fibrillation can persist for years and will eventually lead to a remodelling of the atrias. An important part of the remodelling process is the changes in distribution of gap junctions. Thus, the Cx40 distribution pattern becomes increasingly heterogeneous. The time course of changes in the distribution and content of Cx40 gap junctions correlates with an increase in stability and complexity of AF and suggests that Cx40 gap junctional remodeling might be involved in the pathogenesis of sustained atrial fibrillation [27] . Moreover, several lines of evidence support the notion that during conditions with slowing of atrial conduction the susceptibility to atrial fibrillation is elevated.
  • the reentry is functional and manifests as ventricular fibrillation or polymorphic ventricular tachycardia.
  • reentry can become anatomically fixed, resulting in monomorphic ventricular tachycardia [29] .
  • Bradyarrhythmias Bradyarrhythmias can be caused by slowed conduction or conduction block of the sinoatrial node, atrioventricular node, bundle of His or right or left bundle branch.
  • the major connexin responsible for the conductance throughout the conductive system is Cx40. Mice homozygous for a knock-out of the Cx40 gene have significantly slower atrial, atrioventricular, and His-Purkinje conduction and are at increased risk of arrhythmias and bundle branch block [4"6] . Thus, normal functioning Cx40 gap junctions are essential for the maintenance of normal rhythm.
  • a substance, such as the compounds of the present invention which increases gap junction conductance is useful in the prevention and/or treatment of slowed conduction in the heart.
  • Reduced contractility is a common feature of many chronic heart diseases.
  • the contractility is reduced to a point where the ejection fraction is so low that the basal needs for organ perfusion can no longer be maintained.
  • Experimental as well as clinical evidence has shown that the expression and distribution of connexins in hearts from patients with endstage heart failure is changed.
  • Cx43 is significantly down-regulated with a highly irregular distribution in the abnormal tissue.
  • Cx45 expression which under normal conditions is very limited, is significantly increased in failing hearts; however, the conductive properties of Cx45 are inferior to the properties of Cx43 and therefore can not compensate for the reduction in Cx43.
  • the Tyrodes solution had the following composition in mM: Na + 135.33, K + 4, Cl " 145, P0 4 " 0.33, Mg 2+ 1, Ca 2+ 2, Hepes 10, Glucose 10, pH 7.4. All perfusion media were bubled by 100 % oxygen. After this the heart was perfused for two minutes with Tyrodes solution without Ca 2+ , followed by perusion for two minutes with a high K + solution containing in mM: Na + 20, K + 120, Cl " 22, glutamate 120, Mg 2+ 1, Ca 2+ 25 ⁇ M, Hepes 10, Glucose 10, pH 7.4.
  • the heart was perfused with high K + solution with 0.6 mg/ml collagenase, this was done for 10-15 minutes judged from the apperance of the heart.
  • the atria were cut off, the ventricles minced, whereafter the pieces were stirred in the collagenase solution by gently bubbling with 100 % oxygen.
  • the cells were then passed throug a sieve to isolate the liberated cells, and the collagenase was removed by centrifugation.
  • the cells were resuspended in Ca 2+ free Tyrodes solution and Ca 2+ was slowly increased to 0.65 mM. The cells were kept in this solution at room temperature until transferred to the experimental chamber.
  • Electrophysiology Cover slips are mounted in an open chamber on the stage of an inverted microscope, where the cells are superfused with Dulbeccos phosphate buffered saline (PBS) at 1 ml/min, 37°C.
  • the solution contain (in mM): Na + 152, K + 4.2, Cl " 141.5, P0 4 3" 9.5, Ca 2+ 0.9, Mg 2+ 0.5, pH 7.2.
  • Patch clamp pipettes are pulled from 1.5 mm glass capillaries (GC150F-15, Harvard Apparatus) on a Sutter Flaming-Brown P-87 microelectrode puller and fire polished to a resistance of 4-6 M ⁇ .
  • Pipettes are filled with an intracellular like solution containing in mM : K + 145, Na + 15, Cl “ 5, Gluconate " 153, Pyruvate 5, EGTA 1, HEPES 5, Ca 2+ 0.42 mM, Mg 2+ 1.6, pH 7.2.
  • amphotericin B 240 ⁇ g/ml is added from a 60 mg/ml stock solution (Solvent: DMSO).
  • the patch clamp set-up consists of two synchronised discontinuous amplifiers (SEC-05LX, NPI electronics) and data is digitised using an INT-10 interface (NPI electronics) and a PC1200 data acquisition board (National Instruments). Both current and voltage signals are low pass filtered at 1 kHz using the internal filters of the amplifiers and digitised at 10 kHz.
  • mice are excised from mice (Balb/cJ, 20 g), rinsed twice in ice-cold (0° C) 0.32 M sucrose and homogenized on ice in 10 volumes of sucrose with an Ultra Turrax homogeniser (1000 rpm) for 2 minutes. The homogenate is centrifuged at 1000 g mean for 10 minutes at 4° C and the supernatant collected and filtrated through 4 layers of gauze.
  • the filtrate is then centrifuged at 50,000 g mean for 45 min at 4° C and the pellet resuspended in 10 vol org , wet we ig ht ice-cold distilled water and incubated for 60 min at 0° C and re-centrifuged at 50,000 g mean at 45 min at 4° C.
  • the resulting pellet is resuspended in 2 vol o rg. wet w eigh t of PBS (Phosphate Buffered Saline) and stored at - 80° C until use.
  • PBS Phosphate Buffered Saline
  • CHO cells are seeded in 24-multi well dishes in a density of 7,900 cells/cm 2 ( ⁇ 15,000 cells/well) and grown for 3 Days In Vitro (DIV) in 1 ml/well of F-12K Nutrient Mixture supplemented with 10% Foetal Calf Serum (FCS) and 1000 units penicillin/1000 ⁇ g streptomycin (pen/strep) in an atmosphere of 5% C0 2 and 100% humidity at 37° C.
  • FCS Foetal Calf Serum
  • pen/strep penicillin/1000 ⁇ g streptomycin
  • Cells are incubated for 10 min in an N 2 -atmosphere in glucose free D-PBS (pH 7.2) pre- equilibrated with N 2 for at least 10 min at 37 °C. Control cells are incubated likewise for 10 min at 37 °C, only, at normal atmospheric conditions and in D-PBS containing glucose (6 mM).
  • Binding assay The in situ binding is performed by a modified protocol based on the description by Koenig [34] .
  • D-PBS is removed from the cell culture and 0.50 ml [ 125 I]AAP10 solution with or without unlabeled ligand or test compound is added. Cells incubate overnight at 4° C to reach equilibrium. Each well, one at the time, is then rinsed rapidly with 2 x 1 ml D-PBS and left to dry.
  • 0.25 ml of 0.5 % Triton-X-100 (v/v) is added to each well and cells left for at least 1 h to solubilize.
  • the extract is transferred to counting vials, the wells rinsed with 0.25 ml water and the rinse extract added to the corresponding vials.
  • the vials are counted in a ⁇ - counter.
  • CHO cells are seeded in 96-well microtiter plates in a density of 6,000 cells/cm 2 ( ⁇ 2,000 cells/well) and grown for 4 days in vitro in 200 ⁇ l/well of growth media as described in the previous section.
  • CHO cells are incubated at 37° C in D-PBS (pH 7.2) containing 6 mM glucose, 2.0 mM IBMX (phospodiesterase blocker), 10 ⁇ M forskoline (stimulates cAMP formation) and increasing concentrations of test peptide.
  • the reaction is stopped after 20 min by addition of 20 ⁇ l 0.5 M HCl and left for at least 20 min at room temperature.
  • the content of cAMP is analysed by mixing 20 ⁇ l of the acid cell extract into FlashPlateTM wells (NEN assay kit SMP001) containing 180 ⁇ l [ 125 I]cAMP tracer solution. FlashPlatesTM are incubated overnight at 4° C and plate bound radioactivity counted in TopCount
  • Neonatal Wistar rats (1-2 days old) are used.
  • Hank's calcium- and magnesium-free balanced salt solution buffered with 10 mM HEPES is used for washing during cell separation procedures.
  • the hearts are excised, the ventricles isolated and the tissue cut into small pieces.
  • the myocardial cells are isolated by stepwise enzymatic degradation with collagenase 0.05%, as described by t35] . After repeated rounds of centrifugation and washing, the precipitated cells are resuspended in culture medium M199 with Earle's salt, 10% NCS, penicillin (75 U/mL), and streptomycin (75 U/mL) and pre-plated in a Petri dish for 90 minutes.
  • the non-adherent cells are collected in the culture medium and plated in multidishes at 2.5*10 5 cells/well.
  • the cultures are kept in a water-saturated C0 2 -incubator at 37°C.
  • the cardiomyocyte cultures are used for analyses after 6-7 days.
  • Cardiomyocyte cultures are incubated for 48 hours in culture medium containing 4 ⁇ Ci/mL myo-[2- 3 H]inositol to label the inositol phospholipids.
  • the medium On the day of analysis the medium is replaced by a buffer solution containing lithium and incubated at 37°C, as described by Meier et al. [36] . After at least five minutes this buffer is replaced by the same volume of buffer containing test compound and incubated for exactly 20 minutes.
  • the reaction is stopped by rapid replacement of the buffer by ice cold 4%v/v perchloric acid (PCA) and incubation for at least 20 minutes at 0°C.
  • PCA-extract is neutralised and the
  • [ 3 H]inositol phosphates are separated by anion-exchange chromatography using AmprepTM columns containing 100 mg SAX Quaternary amine.
  • the [ 3 H]inositol mono-phosphates are eluted and radioactivity in the fraction measured by liquid scintillation counting.
  • the cells Before adding test substances to the cultures, the cells are depleted of glucose and oxygen by incubating them in a N 2 -atmosphere in glucose-free lithium-buffer for 10 minutes at 37°C. Control cells are incubated likewise only at normal atmospheric conditions and in a buffer containing glucose.
  • Noradrenaline stimulates phosphoinositol turnover in the cardiomyocyte cultures in a concentration-dependent manner.
  • the ability of noradrenaline (300 nM NA) to stimulate phosphoinositol turnover is considerably reduced in cultures following 10 minutes of glucose and oxygen deprivation as shown in Figure 3.
  • mice 25-30 g were anaesthetised with a neurolept anaesthetic combination (Hypnorm ® (fentanyl citrate 0.315 mg/ml and fuanisone 10 mg/ml) + midazolam (5 mg/ml)).
  • Hypnorm ® neurolept anaesthetic combination
  • Commercial solutions of hypnorm and midazolam were diluted 1:1 in distilled water and one part diluted Hypnorm ® is mixed with one part diluted midazolam.

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CA2439101A CA2439101C (en) 2001-02-22 2002-02-22 New medical uses of intercellular communication facilitating compounds
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AT02723240T ATE497967T1 (de) 2001-02-22 2002-02-22 Interzelluläre kommunikation erleichternden verbindungen und deren medizinischen verwendungen
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WO2003063891A1 (en) * 2002-01-29 2003-08-07 Wyeth Compositions and methods for modulating connexin hemichannels
WO2004048400A1 (en) * 2002-11-25 2004-06-10 Zealand Pharma A/S Peptide gap junction modulators
WO2005061437A3 (en) * 2003-12-23 2005-08-25 Zealand Pharma As Isopeptide gap junction modulators
DE102005005270A1 (de) * 2005-02-04 2006-08-10 Peter Heger Verwendung von Hydroxystilben-haltigen Wirkstoffen zur Behandlung leichter bis mittelschwerer Depressionen und Angstzustände
US7250397B2 (en) 2000-02-23 2007-07-31 Zealand Pharma A/S Antiarrhythmic peptides
WO2010026487A1 (en) * 2008-09-08 2010-03-11 University Of Concepcion Therapeutic methods and compositions
US7749969B2 (en) 2005-07-07 2010-07-06 Zealand Pharma A/S N- or C- terminally modified small peptides
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EP1370276A2 (en) 2003-12-17
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