ES2360599T3 - COMPOUNDS THAT FACILITATE THE INTERCELLULAR COMMUNICATION AND ITS MEDICAL USE. - Google Patents

Abstract

Compound which is acetyl-Asn-Tyr-NH2, hydroxyacetyl-Asn-Tyr-OH / NH2, or hydroxyacetyl-Gly-Tyr-OH; or a salt thereof.

Description

FIELD OF THE INVENTION

[0001] The present invention relates to novel peptides including novel antiarrhythmic peptides of improved linear 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 intercellular communication for the preparation of medicaments for the treatment of a number of diseases characterized by intercellular communication by damaged gap junctions.

BACKGROUND OF THE INVENTION

[0002] Gap links are specialized regions of the cell membrane with groups of hundreds to thousands of densely packed gap channels that directly connect the cytoplasmic compartment of two neighboring cells. The gap link channels are composed of two hemichannels (conneonas) provided by each of the two neighboring cells. Each conneone consists of six proteins called connexins (Cx). The connexins are a broad family of proteins that share the entire basic structure of four transmembrane domains, two extracellular loops and a cytoplasmic loop. There is a high degree of conservation of extracellular loops and transmembrane domains between connexin species and isoforms. The length of the C-terminal end, however, varies considerably resulting in the classification of connexins based on molecular weight. The gap link channel can be connected between an open and a closed state by a rotational movement. In the open state ions and small molecules can pass through the pore. The conduction of the electrical impulse and the intercellular diffusion of signaling molecules take place through gap links and therefore the normally functioning gap links are a prerequisite for normal intercellular communication. Normal intercellular communication is essential for homeostasis, proliferation and cell differentiation.

[0003] The relationship between abnormalities in connexins and diseases has been established in humans as it will appear in the next section. An example is Chagas disease caused by the protozoan parasite Trypanosoma cruzi. This disease is the main cause of cardiac dysfunction in Latin America. The damaged distribution of Cx43 has been observed in cells infected by Trypanosoma cruzi and this alteration may be involved in the genesis of the conduction abnormalities that characterize the disease [7].

[0004] In a multicellular organism, coordination between cells is of paramount importance. Among the various means of cellular intercommunication, gap links provide the most direct route. Gap bonds are a type of binding complex formed between adjacent cells and consist of aggregate channels that bind directly to the inside (cytoplasm) of neighboring cells. In the adult mammal, gap bonds are found in most cell types with a known exception that are circulating blood elements.

[0005] Relatively little is known about the gene structure of connexins. The results described for mouse Cx43 revealed that Cx43 contains two exons and an intron located in the 5 ’untranslated region. Several suspected transcription factor binding sites have been identified in the 5 ’proximal promoter. In vitro studies have shown that permeable channels could be produced by means of hemichannels composed of different pairs of connexins. For example, Cx43 can produce functional channels with endogenous Cx32, Cx37, Cx40 and Cx45 and Cx of oocytes (Cx38), but not with oocytes with Cx26. however, very little is known about their properties, as well as about the permeability regulation of these heterochannels. Cx are expressed in the vast majority of tissues and individual cells are capable of expressing several different Cx. Permeable gap bonds can be formed between cells that express different types of Cx. Thus, intercellular communication through gap links (GJIC) in tissues seems to be very important for maintaining tissue integrity. It seems that several genes produce equivalent products to prevent the loss of GJIC due to a mutation in one of the genes.

[0006] It has been described that the pore diameter of the gap channel formed is in the range of 0.81.4 nm. The gap bonds are relatively non-selective and allow the passage of molecules of up to approximately 1000 Daltons. These substances are, among others, ions, water, sugars, nucleotides, amino acids, fatty acids, small peptides, drugs and carcinogens. The passage of the channels does not require ATP and seems to be the result of passive diffusion. This flow of materials between cells through gap link channels is known as intercellular communication through gap links (GJIC), which plays an important role in regulating cell-to-cell metabolism, proliferation and signaling. One of the most important physiological implications for GJIC is that the cells coupled by gap bonds in a tissue are not individual, discrete entities, but are very integrated with their neighbors. This property facilitates homeostasis and also allows rapid and direct transfer of second messengers between cells to coordinate cellular responses in tissue.

[0007] The GJIC process is regulated by a set of mechanisms that can be broadly divided into main categories. In one type of regulation, the cellular amount of gap links is controlled by the influence on expression, degradation, cellular traffic of connecins to the plasma membrane, or assembly of connexins in functional gap bonds. The damaged GJIC caused by the disadvantage of the expression of connexins, for example, in tumor cells, is an example of this mode of regulation. Another type of regulation does not imply in general any major alteration of the cellular levels of gap or connection links, but induces the opening or closing ("gating") of the existing gap links. Soluble extracellular factors, such as mitogens (for example, DDT), hormones (for example, catecholamines), anesthetics (for example, halothane), intracellular biomolecules (for example, cAMP), and cellular stress (for example, mechanical stress or metabolic) can lead to this type of regulation. Additionally, GJIC is regulated during the cell cycle and during cell migration.

[0008] The mode of regulation or "gating" of GJIC links has been extensively studied for gap links, especially Cx43 composite gap links. Some factors exert their inhibitory effects on GJIC indirectly, for example, by altering the lipid medium and cell membrane fluidity, while other GJIC inhibitors include oncogenes, growth factors and tumor promoters, which induce various modifications. from Cx43. Altered link permeability may be necessary to mediate the specific biological functions of the last group. These agents initiate complex signaling mechanisms that consist of the activation of kinases, phosphatases and interaction proteins. The understanding of the mechanisms of action of these GJIC modulators will not only define their respective signaling mechanisms responsible for link regulation, but also provide tools to characterize the biological functions of GJIC and connexins.

[0009] Changes in the phosphorylation of specific sites of the cytoplasmic terminal carboxy domain of Cx43 appear to be fundamental for the opening and closing of the gap link channel. Phosphorylation of the carboxy terminal domain may also be important for the process of bringing the hem complex of Cx43 gap bonds to the cell membrane, its internalization and degradation. The connexins have a half-life (hours) that are much shorter than most plasma membrane proteins (days), for example, the half-life of Cx43 in the rat heart is less than 1 hour. Thus, the regulation of the renewal rate would be an important factor in the regulation of GJIC.

[0010] The carboxy terminal domain contains possible phosphorylation sites for multiple protein kinases (PKA, PKC, PKG, MAPK, CaMkII and tyrosine kinase). Phosphorylation of these sites in the carboxy terminal domain results in the closure of the gap link channels and several Cx43 gap link channel inhibitors use different signaling mechanisms to induce phosphorylation of the carboxy terminal domain. The type of cell and the particular inhibitor determine which signaling mechanisms to use and the type of protein kinase involved points to the intracellular messenger system used. Thus, PKA activation requires the involvement of the cAMP second messenger system while PKC requires the involvement of the intracellular phosphoinositol signaling system.

[0011] Other mechanisms for regulating channel "gating" include intracellular levels of hydrogen and calcium ions, voltage between bonds ("transjuctional") and free radicals. A decreased pH or pCa induces a closure of the channel in a specific way of cell and connexin.

[0012] Many physiological functions have been proposed in addition to growth control for GJIC. Homeostasis: GJIC allows the rapid balance of nutrients, ions and fluids between cells. This could be the oldest, extended and important function for these channels. Electrical coupling: The gap bonds serve as electrical synapses in electrically excitable cells, such as cardiac myocytes, smooth muscle cells, and neurons. In these tissues, the electrical coupling allows a faster cell-to-cell transmission of action potentials than the chemical synapse. In cardiomyocytes and smooth muscle cells, this allows synchronous contraction. Tissue response to hormones: GJIC can increase tissue response to external stimuli. The second messengers, such as cyclic nucleotides, calcium and inositol phosphates are small enough to pass from hormonally activated cells to inactive cells through link channels and activate the latter. Such an effect may increase the tissue response to an agonist. Embryonic development regulation: gap links can serve as intercellular mechanisms for signals of chemical and / or electrical development in embryos and to define the boundaries of development compartments. GJIC takes place in specific patterns in embryonic cells and the deterioration of GJIC has been linked to developmental anomalies and teratogenic effects of many chemicals.

[0013] Intercellular communication ensures that the activities of individual cells take place in a coordinated manner and integrate these activities into the dynamics of a working tissue that acts in the organism in which it is established. Therefore, it is not very surprising that a wide variety of pathological conditions have been associated with a decrease in GJIC. The relationship between abnormalities in connexins and a set of pathological conditions has been established both in vitro and in vivo. An example is the regulation of the communication of gap links by a pro-inflammatory cytokine in the epithelium of the nasal passages, where Chanson M, Berclaz PY, Scerri I, Dudez T, Wernke-Dollries K, Pizurki L, Pavirani A, Fiedler MA, Suter S. (Am J Pathol 2001 May; 158 (5): 1775-84) found that a decrease in intercellular communication induced by TNF-alpha progressively led to inflammation.

[0014] In summary, there is sufficient evidence relating dysfunction, such as "gating" or closure or even the absence of gap links with an increased risk of disease. There are currently no drugs available for the treatment of such diseases that act as a facilitator of intercellular communication facilitating or increasing the function of gap bonds. However, in the past a group of peptides (antiarrhythmic peptides) capable of increasing the conductance of gap bonds has been described. A summary is presented in WO01 / 62775.

[0015] Antiarrhythmic peptides are a group of peptides that exert their effect selectively on gap bonds and, thus, decrease cell decoupling and also reduce the duration dispersion of the action potential. However, native AAPs, as well as synthetic AAP10, have several unwanted characteristics, such as low stability, low effective concentration, etc. which until now have avoided its use as drugs. Grover and Dhein [21] have characterized two semi-cyclic conformations of AAP10 using nuclear magnetic resonance spectroscopy. Therefore, a strategy to obtain a stable antiarrhythmic peptide could be the provision of cyclic derivatives of antiarrhythmic peptides. DE19707854 apparently describes CF3C (OH) -Gly-Ala-Gly-4Hyp-Pro-Tyr-CONH cyclic and CO-Gly-Ala-Gly-4Hyp-Pro-Tyr-CONH cyclic having the same antiarrhythmic properties as AAP and AAP10, but stated that it presented greater stability in aqueous solution and after repeated cycles of freezing and thawing. However, the experimental conditions described in DE19707854 are insufficient for the preparation of said cyclic compounds and the chemical identification data provided therein using HPLC is not sufficient for the identification of said cyclic compounds. US 4,775,743 describes HPS, a peptide derivative having the sequence N-3- (4-hydroxyphenyl) propionyl-Pro-4Hyp-Gly-Ala-Gly-OH and that is active against platelet agglutination. Dhein and Tudyka [22] have reviewed the literature on peptides that include peptide derivatives belonging to the group of antiarrhythmic peptides for activity and concentration, see table 1 therein, and found that only 7 compounds were active and, in addition, 4 compounds were weakly active. However, it has been observed that none of these peptides or peptide derivatives are stable enough to be effective in a therapeutic regimen.

[0016] The peptides of the present invention increase intercellular communication by gap bonds (GJIC) in vertebrate tissues and specifically in mammalian tissues, and are useful in the treatment of a broad spectrum of diseases and conditions in vertebrates, such as mammals. , in relation to or caused by a diminished function of intercellular communication by gap links as described below.

[0017] Thus, the present invention relates to preventing or treating diseases and medical conditions that are characterized by reduced or damaged intercellular communication, such as that caused by intercellular communication by damaged gap links or deteriorated coupling by gap links. . Examples of diseases and medical conditions are inflammation of the airway epithelium, alveolar tissue disorders, bladder incontinence, hearing impairment due to diseases of the cochlea, endothelial lesions, diabetic retinopathy and diabetic neuropathy, central nervous system ischemia and spinal cord, dental tissue disorders, including periodontal disease, kidney disease and bone marrow transplant deficiencies as mentioned above.

SUMMARY DESCRIPTION OF THE INVENTION

[0018] In one aspect, the present invention provides a compound that is acetyl-Asn-Tyr-NH2 hydroxyacetyl-AsnTyr-OH / NH2 or hydroxyacetyl-Gly-Tyr-OH; and you leave them. These compounds are useful as gap link modulators. A particularly preferred compound is hydroxyacetyl-Asn-Tyr-NH2, or a salt thereof.

[0019] In a further aspect, the present invention provides a pharmaceutical composition comprising a compound of the present invention, or a salt thereof, and a pharmaceutically acceptable carrier or diluent.

[0020] In a further aspect, the present invention provides a compound of the present invention for use in therapy.

[0021] In a further aspect, the present invention provides the use of a compound of the present invention for the preparation of a medicament for the treatment of a disease characterized by a damaged intercellular communication by gap junctions (GJIC). Examples of such diseases are provided in the detailed description and are indicated in the claims.

[0022] The compounds that are useful in the present invention all share the characteristic of facilitating or mediating GJIC in cells and tissues. The mechanisms through which this GJIC mediation is performed may vary as there are many cellular mechanisms that affect the functioning of connexins and / or mediate the function of gap bonds. These mechanisms include, for example

•

control of the cellular quantity of gap bonds by promoting normalization of connexin expression,

•

inhibition of the degradation of gap and connexin bonds that include the regulation of the rate of renewal of connexins by increasing the half-life,

•

increase cellular traffic of connexins to the plasma membrane,

•

mediate the assembly of connexins in the functional gap links,

•

induce the opening of existing gap links, for example when they have been closed or “gated” by inhibitors. This mechanism can be described as the inversion of the closure of the gap link made by GJIC inhibitors that act through a direct or indirect mechanism, such as, for example, hyperphosphorylation of the cytoplasmic terminal carboxy domain of connexins, for example Cx43.

[0023] The carboxy terminal domain contains possible phosphorylation sites for multiple protein kinases (PKA, PKC, PKG, MAPK, CaMkII and tyrosine kinase). Phosphorylation of these sites in the carboxy terminal domain results in the closure of the gap link channels and several Cx43 gap link channel inhibitors use different signaling mechanisms to induce phosphorylation of the carboxy terminal domain. The type of cell and the particular inhibitor determine which signaling mechanisms are used and the type of protein kinase involved points to the intracellular messenger system used. Thus, it has been described that PKA activation requires the involvement of the cAMP second messenger system while PKC requires the involvement of the intracellular phosphoinositol signaling system.

[0024] Other mechanisms for regulating channel "gating" include intracellular levels of hydrogen and calcium ions, link voltage ("transjuctional") and free radicals. A decreased pH or pCa induces a closure of the channel in a specific way of cell and connexin.

[0025] Such antiarrhythmic peptides include a group of peptides that exert their effect selectively on gap bonds and thereby decrease cell decoupling and reduce the dispersion of the duration of the action potential similar to the effect described above for the AAP10 antiarrhythmic peptide. . The target or molecular receptor for antiarrhythmic peptides are currently unknown. However, a hypothesis has been made about the structure of the binding site for AAP10 in a supposed recipient by R. Grover and S. Dhein (Peptides 2001, 22 1011-1021). It is assumed that a peptide that 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 or gap bonds. an empowerment or mediation of the GJIC. However, there are many more theoretical signaling mechanisms that can regulate the operation of gap links and the present inventors do not wish to link to any specific theory following the biological action of GJIC modulation.

[0026] In general, the present invention provides compounds for the treatment of diseases and alterations in tissues caused by an excess of reactive species of oxygen and / or free radicals and / or nitric oxide. An example is diabetic neuropathy and wounds in which free radicals cause glutathione depletion and consequently a reduction in gap bonds, or a decoupling of gap link communication. The low oxygen supply and / or a high concentration of free radicals are significant in wounds with necrotic tissue, in diabetes, in arteriosclerosis, in surgical wounds, edema, infection, burn wounds and in venous insufficiency will decrease communication by gap links. Free radicals are important for the destruction of nerve terminals, decreased conductance, demyelination and increased inflammatory response. Noise-induced hearing loss, presbiacusia, is known to be associated with the production of free radicals and is related to the inhibition of gap link coupling. Excess free radicals can also reduce endothelial repair and the appearance of capillaries during angiogenesis.

[0027] Specifically, the present invention relates to the use of a compound that facilitates the

cellular communication, such as intercellular communication through gap links, for the manufacture of a composition

pharmaceutical for the prevention or treatment of diseases and preferably non-proliferative diseases,

5 which include, for example, inflammation of the airway epithelium, alveolar tissue disorders, wounds,

erectile dysfunction, urinary bladder incontinence, hearing impairment due to diseases of the cochlea,

endothelial lesions, diabetic retinopathy and diabetic neuropathy, neuropathic pain, nervous system ischemia

central, spinal cord injuries, dental tissue disorders, including periodontal disease,

kidney diseases, subchronic and chronic inflammation, cancer and bone marrow transplant deficiencies 10 stem cell transplantation.

DETAILED DESCRIPTION OF THE INVENTION

[0028] Peptides useful in the present invention include some of the compounds described in Table 1:

Table 1. Compounds of the invention as defined in claim 1 and reference compounds

Gly-Ala-6-ring-Tyr,

Gly-Ala-Asn-Tyr,

D-Tyr-D-Asn-D-Ala-Gly,

D-Tyr-D-Asn-Gly,

Gly-γAbu-Tyr,

Gly-γAbu-D-Tyr,

Gly-Gly-Tyr,

Gly-Ala-Tyr,

D-Tyr-D-Ala-Gly,

Gly-D-Asn-Tyr,

Gly-βAla-Tyr,

βAla-βAla-Tyr,

Gly-γAbu-Tyr,

βAla-γAbu-Tyr,

βAla-γAbu-D-Tyr,

Gly-βAla-Phe,

Gly-Pamh-Tyr,

Gly-Pamh-D- Tyr,

D-Tyr-Pamh-Gly,

βAla-Pamh-Tyr,

βAla-Pamh-D-Tyr,

Gly-Asn-Phe,

Gly-Ala-Gly-Pamb,

Asn-Tyr,

Ac-Gly-Tyr,

Ac-Ala-Tyr,

HAA-NY,

HAA-GY,

(Gly reduced) -Gly-Tyr (H2N-CH2-CH2-NH-CH2-C (O) -Tyr), 15

[0029] The compound Gly-Ala-6-ring-Tyr has the formula shown below [0030] It is preferred that the compounds of the invention be used in the form of a pharmaceutically acceptable salt

image 1

or an amide. The amides of linear compounds are among the preferred compounds of the invention. The salts include pharmaceutically acceptable salts, such as acid addition salts and basic salts. Examples of salts by acid addition are hydrochloride salts, sodium salts, calcium salts, potassium salts, etc. Examples of basic salts include salts in which the cation is selected from alkali metals, such as sodium and potassium, alkaline earth metals, such as calcium, and ions + N (R3) 3 (R4), where R3 and R4 designate independently C1-6-optionally substituted alkyl, optionally substituted C2-6-alkenyl, optionally substituted aryl or optionally substituted heteroaryl. Other examples of pharmaceutically acceptable salts are, for example, those described in "Remington's Pharmaceutical Sciences" 17. Ed. Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, PA, USA, 1985 and more recent editions and in the Encyclopedia of Pharmaceutical Technology.

Definitions

[0031] Throughout the description and claims, the three-letter code for natural amino acids is used, as well as the generally accepted three-letter codes for other α-amino acids, such as Sarcosine (Sar), α-Amino- acid iso-butanoic acid (Aib), Naphthylalanine (Nal), including 1-naphthylalanine (1Nal) and 2naphthylalanine (2Nal), Phenylglycine Phg, 2,4-Diaminobutanoic acid (Dab), 2,3-Diaminopropanoic acid (Dapa), and Hydroxyproline (Hyp). When nothing is specified, Hyp represents 4-hydroxyproline. Natural or essential amino acids are the constituent amino acids of proteins. The aromatic amino acids are Phe, Tyr, Trp, 1Nal, 2Nal and His. When the L or D form is not specified, it should be understood that the amino acid in question has the natural L form, see Pure & Appl. Chem. Vol. 56 (5) pp595-624 (1984). When nothing is specified, it should be understood that the C-terminal amino acid of a compound of the present invention exists as a free carboxylic acid, this can also be specified as "-OH". The C-terminal amino acid of a compound of the present invention can be shown to have the "-OH / NH2" terminal function, which means that there are two preferred forms of the compound: the free carboxylic acid and the amidated derivative.

[0032] "Functional analogs" of antiarrhythmic peptides means any chemical entity or compound that has a structural conformation and / or binding properties that are sufficiently similar to endogenous AAPs to provide one or more of the beneficial antiarrhythmic or antithrombotic properties of endogenous AAPs.

[0033] The term "heteroaryl" includes 5- or 6-membered aromatic monocyclic heterocyclic groups containing 1-4 heteroatoms selected from nitrogen, oxygen and sulfur, such as pyrrolyl, furyl, 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.

[0034] The term "retro analog" is intended to mean a peptide whose sequence is the inverse of the mentioned peptide.

[0035] The term "halogen" refers to F, Cl, Br, and I, where F and I are preferred.

[0036] The term "alkyl" refers to univalent groups derived from alkanes by removing a hydrogen atom from any carbon atom: CnH2n + 1-. The groups derived by removing a hydrogen atom from a terminal carbon atom of unbranched alkanes form a subclass of normal alkyl groups (n-alkyl): H [CH2] n-. The groups RCH2-, R2CH- (R is not equal to H), and R3C- (R is not equal to H) are primary, secondary and tertiary alkyl groups, respectively. C (1-22) alkyl refers to any alkyl group having 1 to 22 carbon atoms and includes C (1-6) alkyl, such as methyl, ethyl, propyl, iso-propyl, butyl, pentyl and hexyl and all possible isomers thereof. By "lower alkyl" is meant C (1-6) alkyl, preferably C (1-4) alkyl, more preferably, methyl and ethyl.

[0037] The term "alkenyl" refers to a linear or branched or cyclic hydrocarbon group containing one or more carbon carbon double bonds. C (2-22) alkenyl refers to any alkenyl group having 1 to 22 carbon atoms and includes C (2-6) alkenyl, vinyl, allyl, 1-butenyl, etc.

[0038] The term "aralkyl" refers to aryl C (1-22) alkyl and the term "aryl" throughout this specification means phenyl

or naphthyl. HPP refers to hydroxyphenylpropionyl 4HPP refers to 3- (4-hydroxyphenyl) propionyl 2HPP refers to 3- (2-hydroxyphenyl) propionyl HAA refers to hydroxyacetic acid 4HPPA refers to 4-hydroxyphenoxyacetic acid 2HPPA refers to 2- 4HMPA hydroxyphenoxyacetic refers to 4- (hydroxymethyl) phenoxyacetic acid 4HPA refers to 4-hydroxyphenylacetic acid 3HPA refers to 3-hydroxyphenylacetic acid 2HPA refers to 2-hydroxyphenylacetic acid 4HBG refers to N- (4-hydroxybenzoyl) glycine 3G refers to N- (3-hydroxybenzoyl) glycine 2HBG refers to N- (2-hydroxybenzoyl) glycine 4HPG refers to N- (4-hydroxyphenyl) glycine Ac refers to the acetyl radical Pc or PC refers to the acidic radical L- pipecolic Tfa refers to the trifluoroacetyl radical T4c refers to the radical L-thiazolidin-4-carboxylic acid ASAL refers to the radical 4-azidosalicyloyl AB refers to the radical 4-azidobenzoyl HOBt refers to 1-hydroxybenzotriazole HOAt refers to 1-Hydroxy - 7-azabenzotriazole Acm refers to the radical Acetamidomethyl Pd (PPh3) 4 is tetrakis (triphenylphosphine) palladium (0) DNP refers to dinitrophenyl. Pamh refers to 4-amino-6-methylheptanoic acid Pamb refers to 4-aminomethyl benzoic acid DBF is defined as 2-aminoethyl-6-dibenzofuranpropionic acid "6-ring" is used for 3-amino-1-carboxymethylvalerolactam andAbu refers to gamma aminobutyric acid

[0039] By the phrase "amino acid residue" is meant a natural amino acid unit, as well as a non-natural amino acid unit, which is here represented by generally accepted three-letter codes for amino acids, such as Sarcosine (Sar), acidic alpha-Amino-isobutanoic (Aib), Naphthylalanine (Nal) including 1-naphthylalanine (1nal) 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. When nothing is specified Hyp or 4Hyp represents 4-hydroxyproline. Natural or essential amino acids are the constituent amino acids of proteins and can be represented by the code of a generally accepted letter. The aromatic amino acids are Phe, Tyr, Trp, 1Nal, 2Nal and His. When the L or D form is not specified, it should be understood that the amino acid in question has the natural L form, see Pure & Appl. Chem. Vol. 56 (5) pp595-624 (1984). When nothing is specified, it should be understood that the C-terminal amino acid of a compound of the present invention exists as a free carboxylic acid, this can also be specified as "-OH". The C-terminal amino acid of a compound of the present invention can 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 should be understood that this Amino acid residue definition includes compounds, such as DBF, T4c, Pc, DNP, and 3-amino-1-carboxymethylvalerolactam which are of the amino acid type.PDP functions as a hapten for antibody recognition and can preferably be used as research tools compounds of the present invention that contain a DNP group.

[0040] The term "mimetic peptide" refers to compounds of a peptide and non-peptide nature. The objective after the creation of peptidomimetics is to create scaffolds that can replace the peptide skeleton. It is assumed that secondary amide linkages in peptides are responsible for instability and possibly poor peptide transport properties between cell membranes. Proper placement of amino acid side chains with appropriate trajectories is observed as the key design tactic in peptide peptidomimetics to achieve biological activity. Modifications in the skeleton include reduced amide bonds and alkylated amide bonds and the use of isosteric bonds, such as thioamide bonds, CH2-CH2, CH = CH, etc.

[0041] The term "peptoid" refers to compounds that can be characterized by a topological similarity between the structural formula of the peptoid and the parental peptide. Thus, a peptoid can be a compound consisting of peptide-like chains of amino acids with side chains in the nitrogen atom of the skeleton instead of in the alpha carbon as in the true peptides. Peptidomimetics and peptoids may comprise amino acid units that have modified side chains, such as Nal, Dab, and Dapa, or may comprise D-amino acids. The various modifications of the structure of the peptides and peptidomimetics described by El Tayar, N et al. (Amino Acids (1995) 8: 125-139) are included in the definitions of the present invention.

[0042] The terms "compound that facilitates intercellular communication", "gap link facilitator", "compound that facilitates communication through gap links" and "gap link opener", etc., all refer to a compound that facilitates or average in the GJIC regardless of the specific mechanism after the resulting improved or normalized GJIC. More specifically, the term "gap link opener" may refer to a substance that, after stimulation of a cell that expresses connexins, produces a greater conductance of the gap link channel, which, in turn, results in an increase in the exchange of molecules that are capable of passing through gap links between extracellular and intracellular space and / or an increased GJIC.

[0043] The term "agonist" refers to an endogenous substance or a drug that can interact with a receptor and initiate a physiological or pharmacological response characteristic of that receptor (contraction, relaxation, secretion, activation of enzymes, etc.). An "antiarrhythmic peptide receptor agonist" or "AAP-R agonist", as used herein, may or may not be equivalent to a "gap bond opener" depending on the specific biological mechanisms after the effect of the compound.

General background on gap links

[0044] In a multicellular organism, coordination between cells is of paramount importance. Among the various means of cellular intercommunication, gap links provide the most direct route. Gap links are a type of complex of bonds formed between adjacent cells and consists of aggregate channels that directly bind the interior (cytoplasm) of neighboring cells. In the adult mammal, gap bonds are found in most cell types with the known exception of circulating blood elements.

[0045] The structural unit of the gap link channel is the semiconnal connection. Each connection is comprised of six connexin polypeptides (Cx) that oligomerize to form an aqueous pore that encompasses a single plasma membrane. To form a complete gap link channel. Two adjacent cell connections align and couple together to form a continuous channel that joins the cytoplasm of the two cells.

[0046] The connexins that form gap link channels comprise a family of multiple genes with at least fourteen mammalian connexins to date. The expression of connexins is tissue and cell specific, some cells expressing multiple isoforms of connexins. Experimental evidence suggests that two different hybrid configurations are possible: heterotypic cell-to-cell channels in which each connection or semichannel consists of a specific connexin isoform; or heteromeric channels in which each connection is a mixture of the different isoforms of connexins expressed in a particular cell type. The connexins are expressed in a specific form of cell, tissue and development.

[0047] Relatively little is known about the gene structure of connexins. The results provided for the Cx43 mouse revealed that Cx43 contains two exons and an intron located in the 5 ’untranslated region. A subsequent analysis showed that the initial point of transcription of Cx43 in both embryos and adult tissues. Several possible transcription factor binding sites have been identified in the proximal 5 ’promoter. In vitro studies have shown that permeable channels could be produced by semiannual compounds of different Cx pairs. For example, Cx43 can produce functional channels with Cx32, Cx 37 and endogenous Cx of oocytes (Cx38), but with Cx26 oocytes. However, little is known about their properties, as well as about the regulation of the permeability of these heterochannels. Cx are expressed in the vast majority of tissues and individual cells are able to express several different Cx. Permeable gap bonds can be formed between cells expressing different types of Cx. Thus, intracellular communication by gap links (GJIC) in tissues seems to be very important to maintain tissue integrity. It seems that several genes produce the equivalent products in order to prevent the loss of GJIC due to a mutation in one of the genes.

[0048] It has been described that the pore diameter of the gap channel formed is in the range of 0.8 to 1.4 nm. The gap bonds are relatively non-selective and allow the passage of molecules up to approximately 1000 Daltons. These substances are, among others, ions, water, sugars, nucleotides, amino acids, fatty acids, small peptides, drugs and carcinogens. The passage of the channel does not require ATP and seems to result from passive diffusion. This flow of materials between cells through gap link channels is known as intercellular communication through gap links (GJIC), which plays an important role in the regulation of cell metabolism, cell-to-cell proliferation and signaling. One of the most significant physiological implications for GJIC is that cells coupled by gap bonds in a tissue are not individual discrete entities, but are highly integrated with their neighbors. This property facilitates homeostasis and also allows rapid and direct transfer of second messengers between cells to coordinate cellular responses in the tissue.

[0049] The GJIC process is regulated by a series of mechanisms that can be broadly divided into two main categories. The first type of regulation controls the cellular amount of gap bonds by influencing the expression, degradation, cellular traffic of connecins to the plasma membrane or assembly of connexins in functional gap bonds. The damaged GJIC caused by the disadvantage of the expression of connexins in tumor cells is an example of this mode of regulation. The second type of regulation does not generally imply any significant alteration of the cellular levels of gap or connexin links, but induces the opening or closing or "gating" of existing gap links. Soluble extracellular factors, such as mitogens (for example, DDT), hormones (for example, catecholamines), anesthetics (for example, halothane), intracellular biomolecules (for example, cAMP) and cellular stress (for example, mechanical or metabolic stress ) may lead to this type of regulation. Additionally, GJIC is regulated during the cell cycle and during cell migration.

[0050] The regulation mode of GJIC or "gating2 of links has been studied extensively for gap links, especially gap connections composed of connexin 43 (Cx43) and thus used as a representative of all connexins. Some factors exert their inhibitory effects on GJIC indirectly, for example, alternating lipid media and cell membrane fluidity, while other GJIC inhibitors include oncogens, growth factors and tumor promoters, which induce various modifications of the Cx43. The alteration of the permeability of bonds may be necessary to mediate the specific biological functions of the last group. These agents initiate complex signaling mechanisms that consist of the activation of kinases, phosphatases and interaction proteins. Understanding the mechanisms of action of these GJIC modulators will not only define their respective signaling mechanisms responsible for link regulation, but also provide experimental tools to characterize the biological functions of GJIC and connexins.

[0051] changes in the phosphorylation of specific sites of the cytoplasmic terminal carboxy domain of Cx43 appear to be fundamental in the opening and closing of the gap link channel. Phosphorylation of the carboxy terminal domain can also be important for the process to take the half-complex of Cx43 gap links with the surface membrane, its internalization and degradation. The connexins have half-lives (hours) that are much shorter than most plasma membrane proteins (days), for example, the half-life of Cx43 in rat heart is less than 1 hour. Thus, the regulation of the renewal rate would be important in the regulation of GJIC.

[0052] The carboxy terminal domain contains possible phosphorylation sites for multiple protein kinases (PKA, PKC, PKG, MAPK, CaMkII and tyrosine kinase). Phosphorylation of these sites in the carboxy terminal domain results in the closure of gap link channels and several Cx43 gap link channel inhibitors use different signaling mechanisms to induce phosphorylation of the carboxy terminal domain. The type of cell and the particular inhibitor determine which signaling mechanisms to use and the type of protein kinase involved points to the intercellular messenger system used. Thus, it has been described that PKA activation requires the involvement of the cAMP second messenger system, while PKC requires the involvement of the phosphoinositol intracellular signaling system.

[0053] Other mechanisms that regulate the "gating" of channels include intracellular levels of hydrogen ions and calcium ions, voltage between bonds ("transjuctional") and free radicals. A decreased pH or pCa induces the closure of the channel in a specific manner of cell and connexin.

[0054] Many physiological functions have been proposed in addition to growth control for GJIC: Homeostasis. The GJIC allows the rapid balance of nutrients, ions and fluids between cells. This could be the oldest, extended and important function for these channels.

[0055] Electrical coupling. The gap bonds serve as electrical synapses in electrically excitable cells, such as cardiac myocytes, smooth muscle cells, and neurons. In these tissues, the electrical coupling allows a faster cell-to-cell transmission of action potentials than chemical synapses. In cardiomyocytes and smooth muscle cells, this allows synchronous contraction.

[0056] Tissue response to hormones. GJIC can increase the response of tissues to external stimuli. The second messengers, such as cyclic nucleotides, calcium, and inositol phosphates are small enough to pass from hormonally activated cells to inactive cells through link channels and activate the latter. Such an effect can increase the tissue response to an agonist.

[0057] Embryonic development regulation. Gap links can serve as intercellular mechanisms for chemical and / or electrical development signals in embryos and to define the boundaries of development compartments. The GJIC appears in specific patterns in embryonic cells and the deterioration of the GJIC has been linked to developmental anomalies and teratogenic effects of many chemicals.

[0058] Intercellular communication ensures that the activities of individual cells occur in a coordinated manner and integrate these activities into the dynamics of a working tissue that acts in the organism in which it is established. Therefore, it is not very surprising that a wide variety of pathological conditions have been associated with a lower GJIC.

Pharmacology

Cardiac indications

[0059] As indicated in the description of the background of the invention, there is ample evidence supporting an important role of GJIC in cardiomyocytes under normal and pathological conditions. Specific cardiac conditions are described below and evidence is presented in vitro and in vivo to demonstrate that compounds that increase GJIC in the heart are useful for the prevention and / or treatment of a number of pathological conditions in the heart.

Arrhythmias for reentry

[0060] Cardiac arrhythmia is caused by an abnormal impulse onset or abnormal impulse conduction. Among arrhythmias with abnormal impulse conduction, arrhythmias caused by a reentry mechanism are the most serious.

Ventricular Reentry:

[0061] Reentry is the leading cause of prolonged ventricular fibrillation and sudden cardiac death. Reentry takes place when the propagation of impulses does not end after the complete activation of the heart, but persists to reexcite the heart after the end of the refractory period. Re-entry induction is facilitated by slow conduction, greater repolarization dispersion, non-uniform anisotropy and unidirectional conduction block. The underlying disease responsible for the majority of ventricular reentry cases is ischemic heart disease (for example, acute myocardial infarction, chronic myocardial infarction, stable angina pectoris and unstable angina pectoris). During acute ischemia, the gap link channels close leading to decoupling of neighboring cells. The heterogeneous changes in the function of the ion channels and the gap bonds lead to an increase in the dispersion of the duration of the action potential and the effective refractory period especially in the boundary zone that separates the ischemic area of the normal myocardium. It has long been known that an increase in the dispersion of the duration of the action potential facilitates the induction of ventricular fibrillation [23]. Normally, in well-duplicated cells, the difference in the duration of the action potential is stabilized due to the electrical coupling. However, decoupling will prevent this stabilization and will contribute to an unmasking of the dispersion of the duration of the action potential and the refractory period [24]. If the ischemia is prolonged, a reduced degree of Cx43 expression and a changed pattern of distribution can be observed. The closing of the gap link channels during acute ischemia, as well as changes in the pattern of expression and distribution in chronic ischemia can lead to slow conduction, greater dispersion, non-uniform anisotropy and unidirectional conduction block, thus facilitating the induction of arrhythmias by reentry. Thus, experimental studies have shown a correlation between the expression site and distribution of abnormal connexins and the location of reentrant ventricular tachycardia circuits [25].

[0062] Conditions that favor the development of reentry, ie slow conduction, increased repolarization dispersion, non-uniform anisotropy and unidirectional conduction blockage are present to varying degrees in many other heart diseases. . Thus, in infectious or autonomic cardiomyopathy, the inflammation that occurs can lead to the deposition of fibrous tissue in the myocardium, thus creating foci of slow conduction, greater dispersion and possibly blocking of unidirectional conduction. Hypertrophic cardiomyopathy (for example, due to hypertension, congenital, aortic stenosis) can lead to re-entry arrhythmias due to the mismatch between the large amount of myocardial tissue and the relatively small amount of conductive tissue that can lead to slow conduction, an increase in dispersion and a block of unidirectional conduction. Congenital diseases (for example, long-QT syndrome) and drugs that prolong the QT interval (for example, antiarrhythmic drugs, antipsychotic drugs, antihistamines, antibacterial drugs, etc.) also increase the dispersion of the potential duration of action possibly due to the heterogeneity of the distribution of ion channels along the different layers of the myocardium and is a leading cause of sudden death induced by reentry in younger subjects [26].

Earpiece reentry:

[0063] Atrial fibrillation - the most common cardiac arrhythmia - is also caused by a reentrant mechanism. In this case, multiple wavelets travel through the atria and reexcite tissue that is no longer refractory. Atrial fibrillation can persist for years and eventually lead to a remodeling of the atria. An important part of the remodeling process is the changes in the distribution of gap links. In this way, the distribution pattern of Cx40 becomes increasingly heterogeneous. The evolution over time of changes in the distribution and content of Cx40 gap links correlate with an increase in the stability and complexity of AF and suggests that remodeling of Cx40 gap links could be involved in the pathogenesis of fibrillation prolonged earpiece [27]. In addition, several lines of evidence support the notion that during conditions with slowed atrial conduction, susceptibility to atrial fibrillation increases.

Alternating repolarization

[0064] The appearance of an alternation of electrocardiographic T waves with elevated heart pumping or metabolic attack has been observed for almost a century. Alternation of macroscopic T waves is often indicated as a precursor to sudden arrhythmic death. Recent work suggests a common mechanism that can relate the presence of a discordant repolarization alternation at the onset of various reentrant arrhythmias, depending on the anatomical nature of the substrate [28]. Under chronotropic or metabolic stress, the repolarization phase of myocardial action potential develops an alternation in morphology and duration. With additional stress or in the presence of structural barriers, the repolarization alternation becomes spatially discordant. Discordant alternation leads to repolarization gradients wide enough to produce a unidirectional and reentry block. Without a structural barrier, reentry is functional and manifests as ventricular fibrillation or polymorphic ventricular tachycardia. In the establishment of a structural barrier, reentry can be anatomically fixed, resulting in monomorphic ventricular tachycardia [29].

[0065] In summary, it seems that a substance, such as the compounds of the present invention, that increases the conductance by gap bonds and makes anisotropy more uniform, will prevent arrhythmias by unidirectional and reentry block. This substance will be useful in patients with reentry circuits of both atrial and ventricular origin. Patients with an alternation of T waves are prone to re-entry arrhythmias and a substance that increases the coupling of gap bonds and decreases anisotropy may be useful in preventing lethal ventricular arrhythmias in these patients.

Brady arrhythmias

[0066] Brady arrhythmias may be caused by a slowed conduction or a blockage of the sinoatrial nodule conduction, atrioventricular nodule, His bundle or the right or left bundle branch. The main connexin responsible for conductance through the conductive system is Cx40. Homozygous mice for inhibition of the Cx40 gene have significantly slower atrial, ventricular and His-Purkinje conduction and have an increased risk of arrhythmias and bundle branch block. [4-6]. Thus, the Cx40 gap links of normal operation are essential for maintaining the normal rhythm.

[0067] A substance, such as the compounds of the present invention, that increases the conductance of gap bonds is useful in the prevention and / or treatment of a slowed conduction in the heart.

Reduced contractility

[0068] Reduced contractility is a common characteristic of many chronic coronary diseases. During the worst case scenario (i.e., a terminal phase heart failure), contractility is reduced to a point where the ejection fraction is so low that the baseline needs for organic perfusion cannot be maintained any more. Experimental evidence, as well as the clinic, it has shown that the expression and distribution of connexins in the hearts of patients with end-stage heart failure changes. In this way, its expression of Cx43 is significantly disadvantaged with a very irregular distribution in the abnormal tissue. The expression of Cx45, which is very limited under normal conditions, increases significantly in heart failure; although, the conductive properties of Cx45 are lower than the properties of Cx43 and, therefore, cannot compensate for the reduction in Cx43. Recent evidence indicates that some ion and receptor regulatory channels are concentrated at intercellular junction sites and, therefore, it is highly likely that changes in the expression and distribution of Cx43 may affect excitation-contraction coupling and thus , to contractility [30]. Strong evidence of the relationship between the function of gap bonds and contractility is the fact that chimeric mice formed from embryonic cells without Cx43 and wild-type blasts, thus expressing a heterogeneous loss of Cx43, develop several contractile defects [31].

[0069] It is suggested that a substance, which increases the conductance of gap links, will improve the intercellular communication of the mediators involved in the excitation-contraction coupling and, thus, improve contractility.

[0070] Some of the following examples are comparative examples. However, the techniques described in the examples are applicable to test compounds of the present invention.

Experimental Example 1

Effect of Compound 2 on GJIC in cardiomyocytes

[0071] Cell preparation: Guinea pig heart cells were isolated by perfusion with collagenase according to the Langendorf procedure. In summary, guinea pigs were heparinized with an intraperitoneal injection of heparin (1000 IU / kg). After 30 minutes the animal was sacrificed by a blow to the neck followed by the cutting of the spine by the neck. The thorax opened and the aorta was cannulated. Next, the cannula was fixed to the aorta by ligation, cut and perfused with Tyrodes solution for a couple of minutes.

-

The Tyrodes solution had the following composition in mM: Na + 135.33, K + 4, Cl-145, PO4 0.33, Mg2 + 1, Ca2 + 2, Hepes 10, Glucose 10, pH 7.4. All perfusion media were bubbled by 100% oxygen. After that, the heart was perfused for two minutes with Tyrodes solution without Ca2 +, followed by perfusion for two minutes with a high solution in K + containing mM: Na + 20, K + 120, Cl-22, glutamate 120, Mg2 + 1, Ca2 + 25 µM, Hepes 10, Glucose 10, pH 7.4.

[0072] Next, the heart was perfused with a solution high in K + with collagenase 0.6 mg / ml, this was done for 10-15 minutes to judge for the appearance of the heart. The atria were cut, the ventricles were chopped, then the pieces were stirred in the collagenase solution by gentle bubbling 100% oxygen. The cells were then passed through a sieve to isolate the released cells, and the collagenase was removed by centrifugation. The cells were resuspended in Tyrodes solution without Ca2 + and Ca2 + slowly increased to 0.65 mM. The cells were stored in this solution at room temperature until transfer to the experimental chamber.

[0073] Electrophysiology: coverslips are mounted in an open chamber on the stage of an inverted microscope, in which the cells are superfused with phosphate buffered saline (PBS) from Dulbecco at 1 ml / min, 37 ° C. The solution contains (in mM): Na + 152, K + 4.2, Cl-141.5, PO43-9.5, Ca2 + 0.9, Mg2 + 0.5, pH 7.2. Pipettes are stretched for a patch clamp from 1.5 mm glass capillaries (GC150F-15, Harvard Apparatus) in a Sutter Flaming-Brown P-87 microelectrode stretcher and polished with fire until a resistance of 4-6 MΩ. The pipettes are filled with an intracellular solution containing in mM: K + 145, Na + 15, Cl-5, Gluconate-153, Pyruvate 5, EGTA 1, HEPES 5, Ca2 + 0.42 mM, Mg2 + 1.6, pH 7.2. Amphotericin B (240 µg / ml) is added to this solution from a stock solution of 60 mg / ml (Solvent: DMSO).

[0074] Patch clamp preparation consists of two synchronous discontinuous amplifiers (SEC-05LX, NPI electronics) and the data is digitized using an INT-10 interface (NPI electronics) and a PC1200 data acquisition panel (National Instruments) Both current and voltage signals at 1 kHz are filtered at a low rate using the amplifier's internal filters and digitized at 10 kHz.

[0075] A pair cell with an electrode is approached using a PatchMan 5173 micromanipulator (Eppendorf). When contact with the cell is obtained (seen as a sudden increase in input resistance), suction is applied until the Giga seal configuration is established. Then, this procedure is repeated in the other cell. Next, the membrane is broken under the pipettes by a brief application of suction and the potential of the cell interior is set at –70 mV, which is close to the spontaneous membrane potential of the cells. Each cell is consecutively hyperpolarized every 10 seconds by 10 mV for 1 second and the resulting current change in the other cell can be used to calculate the intercellular conductance (Gj) using the formula:

Ip Ip, pulse - Ip, rest

Gj = -------- = ---------------------- (Equation 1)

Uj Up - Ua

[0076] Where Ip, pulse and Ip, remainder represent the current in the passive cell during the pulse and before the pulse respectively, and Up and Ua represent the passive and active cell voltage. This type of experiments does not allow comparison in absolute Gj values due to differences in cell-to-cell contact and therefore the number of functional gap link channels. However, the change in the value of Gj to a standardized intervention as a drug can be analyzed by comparing the relative changes in Gj.

[0077] Results: Figure 2 summarizes the results of nine successful experiments. This figure shows the relative Gj as a function of time before and during stimulation with Compound 2 (10-8 M). In the five experiments in which the cells were treated with Compound 2, the compound produced a significant increase in Gj, which reached a stable level after approximately 400 seconds of stimulation (ΔGj = +120 ± 46%). The conductance did not change in the four preparations treated with vehicles (ΔGj = -3 ± 5%).

[0078] These findings are consistent with the experiments described in the art using the synthetic AAP analog AAP10, which show an increased electrical coupling between cardiomyocytes after stimulation [32]. However, in the study by Müller et al. [32], the conductance of the gap links was not stable during the control conditions. Thus, in three of the six experiments the application of AAP10 did not increase the conductance, but prevented the depletion of the conductance of gap links and in two of the six experiments the conductance of gap links was actually increased during the control period. In the experiments presented in the present invention, Compound 2 increased the conductance of gap bonds in preparations with stable control conditions.

Experimental Example 2

Binding of Compound 2 to murine heart tissue preparations

Preparation

[0079] The hearts of the mice (Balb / cJ, 20 g) are removed, rinsed twice in frozen 0.32 M sucrose (0 ° C) and homogenized on ice in 10 volumes of sucrose with an Ultra Turrax homogenizer (1000 rpm) for 2 minutes. The homogenate is centrifuged at 1000 gmedia for 10 minutes at 4 ° C and the supernatant is collected and filtered through 4 layers of gauze. Then, the filtrate is centrifuged at 50,000 gmediate for 45 min at 4 ° C and the residue is resuspended in 10 volorg. by wet weight of frozen distilled water and incubated for 60 min at 0 ° C and recentrifuged at 50,000 gmedia for 45 min at 4 ° C. The resulting residue is resuspended in 2 vol org. by wet weight of PBS (phosphate buffered saline) and stored at -80 ° C until use.

Substitution experiments with Compound 2

[0080] 40-250 µg of filtrate or membrane material is incubated in a total volume of 100 µl of D-PBS (Dulbecco Phosphate Buffered Saline Solution containing MgCl2 • 6H2O 1 g / l and CaCl2) containing [125I] 0.8 nM AAP10 and an increasing concentration of the AAP test compounds and Compound 2. Non-specific binding to 10 µM AAP10 (CE2) is determined.

Calculations

[0081] Substitution experiment data fits the equation:

f = (Total - ns) / 1 + s / IC50) + ns

in which Total is the total binding radioactivity at concentration s of labeled ligand, ns is non-specific binding and IC50 is the concentration of the test compound that reduces the specific binding (Total-ns) up to 50% of maximum specific binding.

Results [0082] Table 2. Substitution of [125I] AAP10 0.8 nM of murine heart tissue preparations (n.t .: not tested).

Test Compounds

IC50 filtering (nM) IC50 membranes (nM)

AAP AAP10 (CE 2) Compound 2

1.2 1.2 3.6 n.t. n.t. 1.2

[0083] The values given in Table 2 above are in the same order of magnitude (0.2 nM) as those given for AAP10 by Dhein et al. [33] using rabbit heart membranes.

In situ binding method in intact cells

10 CHO cell cultures

[0084] CHO cells are plated in 24-well plates at a density of 7,900 cells / m2 ( 15,000 cells / well) and grown for 3 days in vitro (IVD) in 1 ml / well of Nutrient Mix F- 12K

15 supplemented with 10% Fetal Calf Serum (FCS) and 1000 units of penicillin / 1000 g streptomycin (pen / strep) in an atmosphere of 5% CO2 CO2 and 100% humidity at 37oC. The cell density increases at that point to 295,000 cells / cm2 (152 pgprot / cell  85 gprot / well).

Pretreatment

[0085] On the day of the analysis, the cells are removed from the incubator and each well is washed twice with, depending on the experiment, 2 ml of preheated D-PBS (37 ° C) or ice-cold (0 ° C) To eliminate the serum. It is important to keep the period to a minimum during which the cells are left without physiological solutions to prevent them from drying out during the washing procedures. Cold-washed cells are used directly for assays of

25 binding while hot-washed cells are used for experiments with glucose and oxygen deprivation.

 Deprivation of glucose and oxygen

[0086] The cells are incubated for 10 min under an atmosphere of N2 in glucose-free D-PBS (pH 7.2)

30 pre-equilibrated with N2 for at least 10 min at 37oC. Control cells also incubate for 10 min, only, under normal atmospheric conditions and in D-PBS containing glucose (6 mM).

Binding test

[0087] In situ binding is performed using a modified protocol based on the description of Koenig [34]. D-PBS is removed from the cell culture and 0.50 ml of a [125I] AAP10 solution with or without unlabeled ligand is added

or test compound. The cells are incubated overnight at 4 ° C until equilibrium is reached. Each well, one at a time, is then quickly rinsed with 2 x 1 ml D-PBS and allowed to dry.

[0088] 0.25 ml of 0.5% Triton-X-100 (v / v) is added to each well and the cells are allowed to solubilize for at least one hour. The extract is transferred to counting vials, the wells are rinsed with 0.25 ml of water and the rinse extract is added to the corresponding vials. The vials are counted with a counter 

Table 3. Union in situ, IC50 (nM)

Test compounds

AAP (CE1) AAP10 (CE2) Compound 2 Compound 32 Compound 24 IC50 (nM) 0.8 130 0.5 0.5 65 [0089] These results demonstrate high affinity binding to CHO cells by various substances different from the present invention comparable to peptides of the prior art.

Experimental Example 3

5

Effect of compound 2 on cAMP formation in CHO cells

CHO cell cultures [0090] CHO cells are seeded in 96-well microtiter plates at a density of 6,000 cells / cm2 (

2,000 cells / well) and were grown for 4 days in vitro in 200 µl / well of growth medium as described in the previous section.

Pretreatment

[0091] On the day of the analysis, the cells are removed from the incubator and washed twice with 200 µl of preheated D-PBS (37 ° C) (pH 7.2) to remove the serum. The cells are incubated for 10 minutes in glucose-free D-PBS and an N2 atmosphere as described in the previous section.

CAMP efficacy test

[0092] CHO cells are incubated at 37 ° C in D-PBS (pH 7.2) containing 6 mM glucose, 2.0 mM IBMX (phosphodiesterase blocker), forskolin (stimulates cAMP formation) 10 M and increasing concentrations of the test peptide. The reaction is stopped after 20 minutes by the addition of 20 µl of 0.5 M HCl and left

25 for at least 20 minutes at room temperature.

[0093] The cAMP content is analyzed by mixing 20 µl of the acid cell extract in FlashPlate ™ wells (test kit NEN SMP001) containing 180 µl of tracking solution [125 I] cAMP. FlashPlates ™ wells are incubated overnight at 4 ° C and plate-bound radioactivity was counted in TopCount (Packard Instrument). The data is calculated as described in the previous section.

Results

[0094] Inhibition of forskolin-stimulated cAMP formation of APP-type compounds in CHO 35 cells indicates that AAP receptors are negatively coupled to the cAMP second messenger system. In addition, the presence of functional AAP receptors in CHO cells is demonstrated.

Table 4. Inhibition of forskolin stimulated cAMP formation in CHO cells

Test compounds AAP AAP10 (CE2) Compound 2

Experimental Example 4

EC50 (nM) 53 eleven 6.2

Analysis of phosphoinositol in primary rat cardiomyocytes

Cultivation of primary cardiomyocytes

[0095] Neonatal Wistar rats (1-2 days of life) are used. A balanced Hank saline solution free of calcium and magnesium, buffered with 10 mM HEPES is used to wash during cell separation procedures. The hearts are removed, the ventricles are isolated and the tissue is cut into small pieces. Myocardial cells are isolated by stepwise enzymatic degradation with 0.05% collagenase as described in [35]. After repeated rounds of centrifugation and washing, the precipitated cells are resuspended in M199 culture medium with Earle salt, 10% NCS, penicillin (75 U / mL), and streptomycin (75 U / mL) and preplated in a Petri dish for 90 minutes. Non-adherent cells are collected in the culture medium and placed in multiplates 2.5 * 105 cells / well. The cultures are kept in a CO2 incubator saturated in water at 37 ° C. Cardiomyocyte cultures are used for analysis after 6-7 days.

Analysis of phosphoinositol replacement

[0096] Cardiomyocyte cultures are incubated for 48 hours in culture medium containing 4 Ci / mL myo- [23 H] inositol to label the inositol phospholipids. On the day of the 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 the test compound and incubated for exactly 20 minutes. The reaction is stopped by rapid replacement of the buffer with 4% v / v ice-cold perchloric acid (PCA) and incubation for at least 20 minutes at 0 ° C. The PCA-extract is neutralized and the [3 H] inositol phosphates are separated by anion exchange chromatography using Amprep ™ columns containing 100 mg of SAX quaternary amine. The [3 H] inositol mono-phosphates are eluted and the radioactivity in the fraction is measured by liquid scintillation counting.

Deprivation of glucose and oxygen

[0097] Before adding the test substances to the cultures, the cells are depleted in glucose and oxygen by incubation in an atmosphere of N2 in glucose-free lithium buffer for 10 minutes at 37 ° C. Likewise, control cells are incubated only in normal atmospheric conditions and in a buffer containing glucose.

[0098] Norepinephrine (NA) stimulates the turnover of phosphoinositol in cardiomyocyte cultures in a concentration-dependent manner. However, the ability of norepinephrine (300 nM of NA) to stimulate phosphoinositol turnover is greatly reduced in cultures after 10 minutes of glucose and oxygen deprivation as shown in Figure 3.

[0099] Under normal atmospheric and nutritional conditions, an Emax value of 3852  266 cpm and an EC50 value of 203 nM (SDR = 1.2) were obtained, while in cells subjected to an atmosphere of N2 and depleted in glucose, an Emax value of 2248  702 cpm and an EC50 value of 303 nM (SDR = 1.7) were observed.

[0100] To examine the effect of the substances of the present invention on the attenuated norepinephrine-induced increase in phosphoinositol turnover during ischemia-induced cell stress and glucose deprivation, compound 2 or AAP10 (CE2) was added to the cultures of cardiomyocytes. Both substances potently increased the phosphoinositol turnover, with compound 2 being the most potent. As shown in Table 5 below, the EC50 value for AAP10 (CE2) was 200 times higher during normoxia and 10 times higher during anoxia-induced metabolic stress and glucose deprivation than the EC50 value for compound 2. .

Table 5. Increase in phospho-inositol turnover during anoxia-induced metabolic stress and glucose deprivation by the compound and AAP10

EC50 (nM) AAP10 (CE2) EC50 (nM) Compound 2 Normal conditions 2000 10 Deprivation of glucose and oxygen 100 10

[0101] The addition of compound 2 (100 nM) did not have an additional effect on the increase induced by norepinephrine (300 nM) in the turnover of phosphoinositol in cardiomyocytes of neonatal rats during control conditions, but in cells undergoing anoxia and glucose deprivation (metabolic stress), the addition of compound 2 (100 nM) + norepinephrine (300 nm) normalized the altered phosphoinositol exchange as shown in Figure 4, an increase that was approximately 70% higher than the increase performed by norepinephrine alone.

Experimental Example 5

Calcium-induced arrhythmia model in mice

[0102] The antiarrhythmic effects of compounds of the present invention were analyzed in an in vivo model of calcium-induced arrhythmias according to the model of Lynch et al. [37]. The mice (25-30 g) were anesthetized with a neuroleptic anesthetic combination (Hypnorm® (fentanyl citrate 0.315 mg / ml and fuanisone 10 mg / ml) + midazolam (5 mg / ml)). Commercial solutions of hypnorm and midazolam were diluted 1: 1 in distilled water and a part of diluted Hypnorm® is mixed with a part of diluted midazolam.

[0103] Anesthesia was induced by administration s.c. at a dose of 0.05- 0.075 µl / 10 grams of mouse. An i.v. cannula was inserted in the tail vein. The bypass ECG signal II was continuously recorded by placing stainless steel ECG electrodes on the right front leg and on the left rear leg. The ground electrode was placed on the right rear leg. The signal was amplified (5,000-10,000 x) and filtered (0.1-150 Hz) using an ECG 689 module of the Hugo Sachs Electronic model. An analog signal was digitized through a 12-bit data acquisition board (Data Translation model DT321) and sampled at 1000 Hz using Notocord HEM 3.1 software for Windows NT. After a 10 min equilibration period, the drug test sample was injected into the tail vein. Mice pretreated with the vehicle were analyzed as a measure of the level of control in untreated animals. The injection volume was 100 µl in all

10 experiments Infusion of CaCl2 (30 mg / ml, 0.1 ml / min ≈ 100 mg / kg / min (2-hydrated calcium chloride, Riedel-de Haën, Germany)) was started 3 min after i.v. of the drug or the vehicle. The period of time for the start of the second degree AV block was determined as the time from the beginning of the infusion of CaCl2 until the onset of the first arrhythmic event. A second degree AV block event was defined as an intermittent insufficiency of AV conduction characterized by a P wave without the concomitant QRS complex.

[0104] Responses were expressed in relation to the time elapsed until second degree AV block in vehicle treated mice. The maximum effect of each of the analyzed substances is summarized in table 6 below.

Table 6. In vivo antiarrhythmic activity of compounds of the invention. +++ refers to a> 60% increase in time until arrhythmia; ++ refers to a 30-50% increase in time to arrhythmia; + refers to a 15-29% increase in time until arrhythmia; (+) refers to an increase ≤ 15% in time until the

arrhythmia, and nd to "not determined."

Compound No.

Compound Name In vivo activity

Group 1

Comparative examples

CE-1

H-Gly-Pro-Hyp-Gly-Ala-Gly-OH ++

(AAP)

CE-2

 H-Gly-Ala-Gly-Hyp-Pro-Tyr-NH2 +++

(AAP10)

3- (4-hydroxyphenyl) propionyl-Pro

Hyp-Gly-Ala-Gly-OH

CE-3 (HP5) ++ H-GAG- (Pa) 2-NH2: Pa is any amino acid residue

25 Experimental example 6

Effects of Compound 2 on isolated perfused rabbit hearts

The principle of Langendorff technique

[0105] The Langendorff technique provides a maintenance procedure for the metabolic requirements suitable for an isolated heart, thereby allowing in vitro experiments on the entire heart for several hours. In the preparation of Langendoorff the heart is perfused retrogradely through a cannula inserted into the aorta. When the infusion solution enters the aorta, the resulting pressure in the aorta closes the

35 aortic valves, thereby preventing fluid from entering the chambers of the heart. Instead, the perfusion solution enters the coronary circulation that supplies the heart. Thus, in the Langendorff technique the total flow in the aorta equals the coronary flow. Langendorff experiments are performed using the ISOLATED HEART SIZE 5, Type 833 apparatus manufactured by Hugo Sachs Elektronik, Germany. The central component of this device is the aortic block to which the heart is attached through a cannula. The aortic block is directly connected to

40 an artificial flow resistance directed by a rotary knob, thereby allowing afterload adjustments and consequently perfusion pressure. The perfusion fluid is released from a thermostated reservoir to the aortic block by tubes connected to a roller pump. The volume released by the pump can be adjusted to meet different needs. Excess fluid flows backward from the aortic block to the reservoir. Under the aortic block is a thermostatized heart chamber that can be raised to cover the heart. This

The preparation allows continuous records of coronary flow, left ventricular pressure (LVP), perfusion pressure, a 12-lead ECG, and 8 single-phase action potentials (MAP). The output of these multiple records is analyzed using the NOTOCORD HEM 3.3 software. This software allows calculations of a wide range of cardiac electrophysiological and hemodynamic parameters.

Perfusion technique and perfusion media

[0106] Experiments are performed in the constant pressure perfusion mode. The flow pump is set to provide 70 ml / min and the afterload is set at 50 mmHg, ensuring an infusion pressure of approximately 60 mmHg. The hearts are, unless otherwise specified, perfused with a preheated modified Krebs-Henseleit solution (38 ° C) with the following composition (mmol / l): NaCl: 118, KCI: 4.7, CaCl2, 2H2O: 2 , 52, KH2PO4: 1.18, Mg2SO4, 7H2O: 1.64, sodium pyruvate: 2.0, NaHCO3: 24.88, glucose: 5.55. The solution is filtered through a 45 "bottletop" filter before use.

[0107] A pH of approximately 7.4 and an adequate oxygen content of the solution are obtained by continuous bubbling with carbon (95% O2 / 5% CO2). Volumes of 2 or more liters are allowed to equilibrate with carcinogen for at least 20 min, while volumes of less than 1 liter are allowed to equilibrate for 10 min.

Anesthesia, surgery, and experimental procedures

[0108] Ssc rabbits are used: male CPH (2.5-4.0 kg) obtained from Hvldesten, Allerød, Denmark. They are sedated with 1.2 ml of Hypnorm® (fentanyl citrate 0.315 mg / ml and fluanisone 10 mg / ml) I.m. Ten minutes later anesthesia is induced by i.v. 0.55 ml of Dormicum® (midazolam 5 mg / ml). In addition, 500 IU of heparin I.v. to prevent clotting

[0109] The rabbits are placed on the back with the front legs fixed to the sides and an incision is made to expose the trachea. The tracheotomy is performed and the rabbits are ventilated with oxygen using a rodent fan Ugo Basile (tidal volume: 18 ml, frequency: 60 pr. Min).

[0110] The abdominal cavity is opened caudal to the xiphoid process and the abdominal muscles are cut laterally on both sides. To gain access to the thoracic cavity, the diaphragm is opened substernally and the cut extends bilaterally along the costal curvature. The mediastinum is cut as close as possible to the sternum and the ribs are cut on both sides in a line parallel to the sternum to allow the chest wall to rise in the cranial direction. The raised chest wall is fixed over the rabbit's head to provide a complete overview of the thoracic cavity. The pericardial sac is opened and the aorta is exposed. A loose ligature is placed around the aorta. The caudal vena cava is clamped with clamps to the liver to reduce the flow back to the heart and the cranial vena cava and pulmonary artery are opened to reduce the volume overload of the heart. The aorta is opened and the cannula, connected to the aortic block by an extension tube filled with perfusion fluid, is immediately inserted into the aorta to allow artificial perfusion. The ligation is squeezed and the heart is removed and transferred to the perfusion apparatus. The time from clamping the caudal vena cava to the insertion of the cannula is approximately 30 seconds.

[0111] When the heart has been transferred to the device, an incision is made in the left atrium to allow the insertion of a fluid-filled balloon (size 12) into the left ventricle for measurements of the left ventricular pressure. The volume of the balloon is adjusted to produce a final diastolic pressure of approximately 10 mmHg. The electrode ring is placed for measurements of a 12-lead ECG around the heart at the level of the coronary sulcus, with the tip of the left atrium between the 5th and 6th precordial leads. The 8 MAP electrodes are placed in the heart in direct contact with the epicardium. MAP5 and MAP6 are placed in the right ventricle, while the other MAP electrodes are distributed evenly over the left ventricle. This procedure is similar to that used by Zabel et al. [38] When the electrodes are placed, the heart chamber is raised to ensure that the heart is immersed in Krebs-Henseleit solution at 38 ° C all the time.

[0112] Before the experiment begins, a ligation is placed around a major branch of the circumflex artery that supplies a large part of the left ventricle. Both ends of the ligation are passed through a small plastic tube that allows the induction of ischemia by pressing the plastic tube against the heart and moistening the ends of the ligation. All hearts are allowed to equilibrate for 15 min before the start of the experiment.

[0113] The time programming for the experiment is as follows:

one.

 15 min infusion with normal Krebs-Henseleit buffer (the equilibration period)

2.

 15 min infusion with compound added to the normal Krebs-Henseleit buffer (the normokalemic control period; t = 0-15 min).

3.

 15 min of prefusion with compound added to the Krebs-Henseleit solution containing a reduced K + concentration (2.5 mM) (the hypokalemic control period: t = 15-30 min).

Four.

 Induction of regional ischemia followed by 30 min of perfusion with compound added to the Krebs-Henseleit solution containing a reduced K + concentration (2.5 mM) (the hypokalemic control period; t = 30-60 min).

[0114] At the end of the experiment, hearts are perfused with Evans blue dye to assess the area at risk of heart attack. The atria and the right ventricle are cut and the remaining left ventricle separates into the area stained by Evans blue and the area that is not stained, that is, the area at risk. The two areas are transferred dry using absorbent paper and weighed to determine the percentage area at risk of heart attack.

Logs

[0115] The following parameters are continuously recorded: coronary flow, left ventricular pressure, perfusion pressure, a 12-lead ECG and 8 MAP records. ECG and MAP are sampled at 2000 Hz, and the pressure and flow parameters at 500 Hz. The duration of the average action potential is calculated from the 8 MAP records as the average duration from the moment of maximum depolarization (dV / dt Max time) up to 90% repolarization. This duration is referred to as APD90 and the APD90 dispersion is measured as the standard deviation of the 8 APD90 measurements.

Results

[0116] As shown in Figure 5, three groups were studied. Rabbit hearts were perfused with Krebs-Henseleit buffer alone (vehicle; n = 11 experiments), Compound 2 10-10 mol / l, (n = 10 experiments),

or 10-10 mol / l) or AAP10 (CE2; n = 3 experiments). The increase in the dispersion of APD90 observed during acute hypokalemic myocardial ischemia in rabbit hearts treated with vehicle was avoided by 10-10 mol / l of Compound 2, but not by 10 -10mol / l of AAP10_ (CE2). These findings demonstrate that Compound 2 prevents the increase in electrical dispersion during ischemia and suggests that the antiarrhythmic properties of Compound 2 are related to this mechanism. It has been previously described that AAP10 (CE2) is able to reduce the dispersion of the epicardial activation-recovery interval and decrease alterations in epicardial activation patterns induced by regional ischemia in the rabbit with maximum effect at a concentration of 10-8 mol / l [39]. In our experiments, Compound 2 effectively avoided the increase in electrical dispersion induced during ischemia at a concentration of 10-10 mol / l while AAP10 (CE2) was ineffective at this concentration. These differences were not due to differences in the size of myocardial infarction since the decrease in coronary flow during ischemia and the risk area were similar in all groups. These results indicate that Compound 2 is more potent than AAP10 (CE2).

Experimental Example 7

Effect of Compound 2 on arrhythmias due to ventricular reentry in dogs

[0117] The influence of gap bonds in arrhythmias has been clarified in studies on the influence of connexin 43 (Cx43) on ventricular conduction properties [33]. In a heterozygous knockout mouse deficient in Cx43, there is twice the frequency of spontaneous VT with coronary artery occlusion (CAO) [3]. Ischemia distorts the effect of Cx43 after 6 hours in the dog showing a 60% decrease in Cx43 from end to end and a 49% decrease in Cx43 from side to side [40], probably secondary to dephosphorylation. In subacute ischemia in dogs, epicardial reentry is facilitated in areas where Cx43 is decreased [21]. Thus, the reentrant mechanisms can critically depend on the disadvantage mediated by the ischemia of Cx43 expression and presumably the resistance of the gap bonds makes the heterogeneity of the recovery and conduction properties predispose to VT and VF.

[0118] In the studies described below, the effect of Compound 2 on re-entry arrhythmias during myocardial ischemia obtained by CAO of the anterior descending artery is examined.

Animal preparation

[0119] Three dogs under anesthesia with an open chest were studied to facilitate electrode placement for mapping. Α-Chloralose was administered as a bolus (200 mg / kg) and then a constant Infusion at 8 mg / kg / h (dissolved in polyethylene glycol, MW = 200). The femoral vein and artery were cannulated for the administration of fluid and drugs and for the measurement of ascending aortic pressure, respectively.

Electrophysiological procedures

[0120] The sinus node was fixed and the ventricular appendix was controlled over time with a programmable stimulator with constant current outputs at twice the diastolic threshold. The control index was ≥200 b / min to control the heart rate. A ventricular control pole of a multipolar needle in the normal area used an anode (7 cm2 stainless steel) in the abdominal muscle. The Endocardial Effective Refractory Period (ERP) was measured by the standard extra-stimulus procedure. The late ventricular diastolic threshold was measured during each intervention; the control current was four times the threshold.

Electrogram Record

[0121] Test sites along the axis of 16-pin needles were chosen (J. Kassell, Fayetteville, NC); Each pole completely surrounds the needle axis to avoid the directionality of the needle orientation from the registration of adjacent Purkinje fibers. Six bipolar electrograms (1 mm spaced) were sequentially down the needle axis by amplification up to 1000 times, filtering from 3-1300 Hz and recording by the oscilloscope during atrial control. Four intramural electrograms are recorded on each multipolar needle. The latest epicardial electrograms on each needle are activated. A set of 23 multipolar electrodes was used with 17 in the risk zone of anterior descending coronary artery infarction and 6 in the surrounding normal zone as described in detail by Xing and Martins [43]. The distance between needles measured on the epicardium varies between 6-10 mm in dogs weighing 12-16 kg.

Arrhythmia induction

[0122] The rhythm of the endocardium at the base, the apical septum and the lateral free wall was controlled just outside the risk zone. After determining the ERP, the S1-S2 interval was extended by 4 ms> ERP and an S3 was initially added to the protocol with an S2-S3 interval equal to 50 ms> S1-S2. The intervals were shortened until it could not be captured. If ventricular tachycardia was not induced at any control site, a third (S4) and a fourth (S5) extrastimulus were added. An induction protocol of complete ventricular tachycardia was performed before the CAO to exclude ventricular tachycardia from artifact due to needle mass or ischemia due to needles that compromise blood flow. After confirming the physiological blood gases and adequate anesthesia, the anterior descending CAO was linked. After 60 minutes, the size of the infarction is almost 75% of the risk zone and the subsequent enlargement of the infarction zone is insignificant. Next, ventricular tachycardia was induced at least twice before the interventions. The test was repeated every 20 minutes and continued until 3 hours after the CAO. The normal heart muscle ERP was recorded with each intervention.

Arrhythmia mapping

[0123] Epicardial mapping was performed using a BARD Electrophysiology Inc. computer system. The software requires 64 data channels at a resolution of 12 bits with a sampling frequency of 1kHz / channel. The filtering was 30-300 Hz. Eight-second windows are triggered externally including up to 8 s of data before the trigger signal. This system is used to record from 2-3 external epicardial 2-3 bipoles on each recording electrode.

[0124] The usual software system was used to separate the Purkinje signals from the 3 internal bipoles in each endocardial multipolar electrode by sampling at 3 kHz per channel. The filters incorporate Purkinje frequency (3-1300 Hz). The sampling rate was 235 kHz. The PC was interconnected with an amplifier consisting of an analog signal multiplexer and 64 instrument amplifier circuits. Each had a selectable gain (up to 1000), and cuts in bandwidth. The acquisition, processing and visualization of electrophysiological data were performed by the software. The high-speed acquisition allowed 14 s of data that included up to 8 s before a trigger signal.

Mapping Analysis

[0125] Offline mapping analysis was done. The computer selects the activation times using the first maximum dv / dt. Electrograms were considered uninterpretable and were excluded from maps only if they were not reproducible with stimuli; there was no exclusion based on the electrogram voltage. Electrotonic or far-field potentials are considered present when substantial voltage and loss of dv / dt take place in a complex with shorter coupling intervals than refractoriness. Isochrones are drawn by hand. The mechanisms of ventricular tachycardia are defined as follows: reentrant ventricular tachycardia occurs when the electrode that records the earliest activity, which occurs after the unidirectional block is located immediately adjacent to the site of the later activation of the complex prior and diastolic activity between complexes is recorded. Almost always, epicardial reentry is recorded in acute ischemia, so that retrograde (epicardial to endocardial) activation of the wall is observed.

Experimental protocol

[0126] After the instrumentation of the heart and one hour since the CAO took place, heart rate control protocols were performed to induce ventricular tachycardia to confirm reproducible inducibility (two-fold induction of ventricular tachycardias with similar surface morphologies ) or inducibility failure (rhythm control at all three sites twice without ventricular tachycardia for one hour). A reentry mechanism was identified in the three dogs with reinducible ventricular tachycardia. In these three dogs, compound 2 was administered as a bolus injection i.v. followed by a constant 30 min infusion at three dose levels in two dogs, while the third dog was treated with saline. The tests were then repeated with extra stimuli through the complete protocol at all sites to determine whether ventricular tachycardia was present or not. Compound 2 was administered i.v. in 3 dose levels in order to produce plasma concentrations of 10-10 M (bolus: 0.1 g / kg; infusion: 2 ng / kg / min), 10-9 M (bolus: 1.1  g / kg; infusion: 21 ng / kg / min), and 10-8 M (bolus: 11 Pg / kg; infusion: 210 ng / kg / min), respectively.

Results

[0127] The first animal, of which figures 6-9 are included, was studied after induction of prolonged monomorphic VT only from the site of the lateral ventricular control twice in succession occurring at 2 hours and 10 minutes and They were repeated at 2 hours and 20 minutes after CAO. In Figure 6, an activation map is presented after septal stimulation that failed to produce VT. This demonstrates the pattern of normal spelling activation with early activation of the PURK control site activated at 6 ms after stimulation and late activation of the activated epicardial site later at 107 ms. It should be noted that the activation time adjacent to 86 ms immediately east and south of the last activation of the epicardium is ES in Figure 7. The epicardial activation of the first VT complex, which begins at -44 ms before the start of the QRS of surface and corresponding to the electrogram recorded in EC in Figure 7.

[0128] In Figure 7, prolonged monomorphic ventricular tachycardia (VT) induced by stimulation at the lateral epicardial ventricular control site causing a reentry circuit is shown. Activation proceeds in a double loop reentry that is activated first at -17 ms and then proceeds to 57 ms in the northwest loop. The southeast loop that activates first up to 2 ms, 31 ms and then up to 57 ms. The protocol that induced VT was S1-S2 = 150, S1-53 = 280, S1-S4 = 390, S1-S5 = 490 ms. The figure illustrates epicardial (E-) electrograms recorded with ECG II and V5R surface electrode during the second to fifth premature extrastimulus (best observed in E-L) ensuring 4 VT complexes. Electrograms are recorded from the control site of the lateral boundary zone (L) and east (E), north (N), centrally (C), subepicardially (SE), below EC, as well as south (S) and northwest (NW) and southwest (SW) of EC. E-C shows electrograms gradually dissociated with the last premature that shows a blockage of the second component (perpendicular lines). The delay of the adjacent conduction in ES allowed the conduction to proceed around and behind the central site (EC) with the reentrant excitation that continues between EC and ES (straight line and arrow line).

[0129] Figure 8 illustrates the activation map during epicardial activation of the first ventricular tachycardia complex, which begins at ms -44 before the onset of the surface QRS and corresponds to the electrogram recorded in EC in Figure 7. Activation proceeds in a double loop reentry that is activated first at -17 ms and then proceeds to 57 ms in the northwest loop. The southeast loop that activates first up to 2 ms, 31 ms and then up to 57 ms. This activation map illustrates the retrograde activation of the ventricular wall during reentry arrhythmia.

[0130] Compound 2 was administered in three incremental IV doses that did not alter the average blood pressure (MAP = 80 mm Hg). The effective refractory period in the control was 150 ms, 154 ms after the lowest dose and was 148 ms in the last and highest dose. The VT that was inducible was by typical epicardial reentry shown in Figures 7 and 8. After the first dose of compound 2 (bolus: 0.1 g / kg; infusion: 2 ng / kg / min), the VT already it was not inducible despite the fact that the protocols that induced VT before administration of compound 2 were achieved in a reproducible manner; the protocol that induced VT before drug admission was S1-S2 = 150, S1-S3 = 280, S1-S4 = 390, S1-S5 = 490 ms and during the infusion of compound 2, the intervals were 150, 270, 370 and 470 ms, respectively. VT was not inducible until an hour and a half after starting the infusion of the lowest dose of compound 2. Electrocardiographic records after administration i.v. of the lowest dose of compound 2 are shown in Figure 9. These results demonstrate that compound 2 effectively blocked VT by reentry in this dog.

[0131] A second dog with inducible VT was studied, this time from two border control points located laterally and septally. Again, compound 2 did not produce changes in MAP that started at 90 mm Hg and ended at 90 mmHg. The effective refractory period at the two induction sites remained at 163 and 144 ms, respectively, throughout the test period of compound 2, which began 85 minutes after CAO and continued for a further 2 hours. After the lowest dose of compound 2, the induced VT of the side wall was no longer inducible; The mechanism of this VT was epicardial reentry, very similar to that shown in Figures 7-9. The induced VT of the septal point was also of epicardial re-entry before the administration of compound 2, but after the administration i.v. of compound 2 epicardial reentry was completely blocked. Thus, in these two experiments, epicardial reentrant CT was inducible before induction of the lowest dose of compound 2 and after administration of the substance no reentry was reinducible at any dose.

[0132] Finally, an additional animal was subjected to an electrophysiological test during the time frame used in the two experiments described above without the introduction of compound 2 without saline. Epicardial reentry was induced one hour after CAO and the same VT morphology and 1 Yz-2 1/2 hour reentry mechanism was induced. Thus, the reproducibility of reentrant VT in this time-controlled experiment agrees that compound 2 is an effective antiarrhythmic compound during conditions with reentry arrhythmias. [0133] These experiments demonstrate that compound 2 is effective in the prevention and / or treatment of lethal reentry arrhythmias. Thus, an objective of the present invention is to provide compounds for the preparation of medicaments useful in the prevention and / or treatment of cardiac reentry arrhythmias of supraventricular or ventricular origin. This objective is achieved with the present peptide compounds, such as the compounds of formulas I to VIII, formulas 2 to 12 and the compounds of tables 1 to 8 of the present invention, more specifically the compounds of Synthesis Examples 1-55 of the present invention.

Experimental Example 8

Effect of gap link openers in bone cells

Background

[0134] Osteoblasts, which are bone-forming cells, and osteocytes are well connected. Osteoblast-osteoblast, osteoblast-osteocyte, and osteocyte-osteocyte connections have been observed in bone portions examined by electron microscopy [42]. The most interesting connexin in relation to bones is Cx43, as in the heart. In bone cells, the expression of these proteins is linked to the expression of some specific osteoblast proteins. Calciotropic hormones can also regulate the expression of gap bond proteins.

[0135] It has been observed that human osteoblasts (HOB) and bone marrow derived stromal cells (BMSC) express Cx43 and Cx45. They are functionally coupled as demonstrated by the Yellow Lucifer (LY) dye transfer technique [43]. Rat osteoblastic cell lines differ from primary human cultures; ROS 17 / 2.8 cells only express Cx43 and are very well coupled, while UMR 106-01 predominantly express Cx45 and are poorly coupled with the dye [44]. Both rat osteoblastic cell lines are electrically coupled. Transfection of Cx43 in UMR cells gave rise to cells closely coupled with the dye. Thus, Cx43 allows the transfer of LY and other larger molecules, while Cx45 does not allow this step. In contrast, the introduction of Cx45 into cells expressing Cx43 decreases coupling with the dye. In the differentiation of osteoblasts, the expression of Cx43 changes; therefore, the more mature the osteoblasts, the greater the expression of Cx43 [45].

[0136] The effect of different stimuli on bone cells and the relationship with changes in communication through gap links has been investigated. It is known that moderate mechanical stress in the bones increases bone density. To mimic this situation, ROS 17 / 2.8 cells were exposed to cyclic stress, which resulted in an increase in cell dye coupling. The cyclic stress applied to poorly coupled UMR 106-01 cells also resulted in an increase in dye coupling, but less markedly compared to ROS cells. No increase in mRNA for Cx43 was observed, but more phosphorylated forms of Cx43 were observed, indicating that cyclic stress on osteoblastic cells increases communication by gap bonds between cells by modulating the intracellular location of the Cx43 gap bond protein. The same group has shown that transfection of Cx43 into poorly coupled 106-01 UMR cells not only increases coupling with dye [46], but also increases the expression of mature osteoblast, osteocalcin and bone sialoprotein (BSP) products. The decrease in the coupling between osteoblastic cells (ROS) through the transfection of Cx45 in the cells decreases the expression of osteocalcin and BSP, fundamental genes for bone matrix formation and calcification. A recent study showed that mice lacking Cx43 have poor bone formation and development compared to wild-type mice [47]. In this way, a communicating intercellular network is required for the complete elaboration of a differentiated osteoblastic phenotype, as well as the formation and normal bone turnover. Poor gap link communication can therefore lead to an increase in bone loss.

[0137] It has also been observed that gap bonds are partially responsible for the propagation of intercellular calcium signals in bone cells. The mechanical stimulation of a human osteoblast in a cell monolayer induces in vitro a pulse of calcium that propagates to a set of surrounding cells. The propagation of this signal involves the passage of a messenger molecule through gap links with subsequent activation of neighboring cells [48; 49]. These signals probably propagate through the cellular network in bones in vivo in response to mechanical stimuli and may be responsible for the increase in bone formation in response to mechanical loading in the bone.

[0138] Communication by gap links and the effect of calciotropic hormones are related. It has been observed that stimulation with 1.25 (OH) 2 vit.D3 of human skin fibroblasts increases communication through gap links, and also increases levels of Cx43 protein and mRNA [50], but only in the presence of functional vitamin D receptors (VDR). It is observed that the loss of Cx43 expression decreases the sensitivity of cells to PTH, without any change in the number of PTH receptors or cAMP response [51]. Conversely, PTH and PGE2 increase communication by gap links in osteoblast cell cultures through two mechanisms; an initial rapid redistribution of Cx43 to the cell membrane and a subsequent stimulation of Cx32 gene expression [52]. Thus, the modulation of intercellular communication represents a mechanism by which osteotropic factors regulate the activity of bone-forming cells.

[0139] Intercellular communication by gap links may probably be one of the most important mechanisms by which bone cells coordinate their activities and responses to mechanical and hormonal stimuli. Thus, if communication by gap links between bone cells could be pharmacologically increased, the activity of osteoblasts could be increased, increasing bone formation in vivo.

[0140] Cardiac myocytes are also connected by gap links and, as in osteoblasts, the predominant connexin is Cx43. It has been observed that certain compounds increase the communication by gap links between cardiac myocytes, of which the artificially synthesized AAP10 (CE2) is the best investigated. Cardiac myocytes respond to ischemia with a decrease in cell coupling. In in vitro experiments, with the addition of AAP10 (CE2) to cardiac myocytes exposed to ischemia, part of the lost cell coupling was restored. If cardiac myocytes can respond to this group of compounds with a greater gap link coupling, osteoblasts could do the same. In this case, it is evident that the increase in cell coupling could be accompanied very well by an increase in the maturation and activity of osteoblasts and the subsequent increase in bone formation. To investigate this hypothesis, the effect of compound 2 on GJIC in human osteoblasts and rat osteosarcoma cells was examined. In addition, the effect of compound 2 on a marker (ie alkaline phosphatase) for human osteoblast activity and bone formation was studied.

Methods

Cell culture

[0141-2] Human osteoblast cells (hOB): Human bone marrow cells obtained by puncturing the posterior iliac spine from healthy volunteers (age 20 to 36) were isolated: 10-15 ml of bone marrow material was collected in 15 ml PBS + Ca, Mg (Life Technologies, Cat.No. 14040) with Heparin 100 U / ml (Sigma, Cat.No. H3149). The mononuclear fraction of the marrow was isolated in a gradient of Lymphoprep (Nycomed Pharma, Cat.No. 1001967), by centrifugation at 2200 rpm for 30 min. After collection, the mononuclear fraction was washed once with culture medium and centrifuged at 1800 rpm for 10 min. Subsequently, the cells were counted and placed in a culture medium at 8x106 cells / 100 mm plate. HOB medium (all reagents obtained from Life Technologies): MEM without Phenol Red with Glutamax (Cat.No. 041-93013) supplemented with heat-inactivated 10% fetal calf serum (Cat.No. 10106) and Penicillin / Streptomycin 0.1% (Cat.No. 15140). The medium was changed the next day and the cells were grown at 37 ° C in 5% CO2 with medium change every 7 days. After 3-4 weeks of culture, the cells had reached a confluence of 70%. The medium was then supplemented with 100 nM Dexamethasone (Sigma, Cat.No. D-4902) for 7 days. Next, the cells were placed for video imaging experiments: a 25 mm glass cover slip # 1 was placed on a 35 mm plate (or each well of a 6-well multi-plate), the cells were placed at 2.5x105 cells / coverslip and were cultured for 2-3 days before use.

[0143] ROS 17 / 2.8 cells: cells were cultured in 100 mm plates at 37 ° C with 5% CO2 and the medium was changed every 2-3 days. ROS medium (all reagents obtained from Life Technologies): MEM (Cat.No. 31095) supplemented with heat-inactivated 10% calf serum (Cat.No. 16170), 1% NEAA (Cat.No. 11140) , 1% sodium pyruvate (Cat.No. 11360), 1% L-glutamine (Cat.No. 25030) and Penicillin / Streptomycin 0.1% (Cat.No. 15140). For imaging experiments, the cells were placed on coverslips at 2-3 x 105 cells / coverslips and cultured for 2-3 days before use.

Measurement of calcium waves

[0144] Cells grown on coverslips were loaded with 5 uraM fura-2-AM (Molecular Probes, Cat.No. F1221) for 30 minutes at 37 ° C and incubated in fresh medium for 20 minutes. The coverslips were then fixed to a culture chamber with PDMI-2 (Medical Systems Corp.), kept at 37 ° C with a CO2 superfusion, in a Zeiss Axiovert microscope. Intercellular calcium waves were induced by mechanical stimulation of a single cell using a borosilicate glass micropipette attached to an Eppendorf 5171 micromanipulator. Imaging was performed using a MetaMorph (Universal Imaging) imaging system. The excitation light (340 and 380 nm) was provided by a monochromator (T.I.L.L. Photonics GmbH). The images were obtained with an intensified CCD camera (Dage MTI) and digitized with a Matrox MVP image processing board.

Microinjection

[0145] Cells grown on coverslips were placed under a microscope as described above. Microinjections were performed using an Eppendorf 5171 micromanipulator and the Eppendorf Transjector 5346 system. A micropipette was loaded with a 10 mM Yellow Lucifer (LY) solution (Sigma, Cat.No. L-0259). A cell was carefully injected into the monolayer with LY for 30 seconds, the micropipette was removed from the cell and, after 30 seconds, the number of cells showing dye transfer was counted. The excitation light for LY was 430 nm and the images were obtained as described above.

Alkaline Phosphatase Assay

[0146] Day 1: The cells were placed in 96-well plates at a concentration of 8000 cells / well (hOB) or 3000 cells / well (ROS) in 200 µl of normal culture medium Day 2: The medium was changed in the cells Day 4: (Day 3 for ROS): The cells were washed with 200 µl of MEM, 0.1% BSA (Sigma, Cat.No. A-9418). Several concentrations of compound 2 containing 200 µl of MEM, 0.1% BSA were added to the cells, and culture was continued for 4 days (2 days for ROS cells). Day 8: (Day 5 for ROS): The alkaline phosphatase (ALP) test is an end-point colorimetric method for measuring enzyme activity and was performed using an alkaline phosphatase kit (Sigma, Cat.No. 104-LL) : The cells were washed once with 200 µl of PBS + Ca, Mg. 100 µl of alkaline buffer solution was added to each well and the plate was placed at 37 ° C for 10 min. 100 µl of substrate solution was added to each well and the plate was incubated at 37 ° C for 30 min. 100 µl of 2.0 N NaOH was added to each well to stop the reaction. Absorbance was measured using a plate reader at 405 nm.

Effects of compound 2 on GJIC

[0147] In order to assess the ability of the gap link modifiers to increase communication by means of intercellular calcium signals mediated by gap links, monolayers of human osteoblast cells were loaded onto glass coverslips with fura-2. During real-time imaging, mechanical stimulation was performed with a glass micropipette. An increase in intracellular calcium appeared with a subsequent extension of the signal to surrounding cells. The average number of cells in the wave was 6.5 cells. Next, 100 µM adenosine triphosphate (ATP) was added in order to desensitize purinergic receptors. After desensitization, the calcium propagation wave depends exclusively on GJIC. After stimulation with ATP, an increase in intracellular calcium was observed in most of the cells in the field of vision. Again, a single cell was mechanically stimulated. Now the propagation of the wave was limited to an average of only 4.5 cells in the wave. Compound 2 was added in a concentration of 10-8 mol / l to the bath solution. An increase in intracellular calcium concentrations was observed in most cells in the field of vision. After 10 minutes of incubation with compound 2, a single cell was mechanically stimulated. Again, in the stimulated cell, the concentration of intracellular calcium was increased with subsequent wave propagation. Now the wave extended to an average of 6.2 cells (Figure 10), which is a significant increase compared to before the addition of compound 2.

[0148] In order to evaluate the ability of the compound to restore the coupling by suppressed gap bonds, similar experiments were performed on the osteoblast cell line ROS 17 / 2.8 (ROS), but after incubation of the cells for 48 hours under hypoxic conditions, with only O2 to 3-6 &, conditions known to decrease cell coupling. ROS cells were loaded in monolayers with fura-2 and, under the same conditions above, mechanical stimulation was performed. Since ROS cells do not express purinergic receptors, pretreatment with ATP was not performed. After stimulation, the concentration of intracellular calcium in the stimulated cells increased and a wave was started that extended to a total average of 2.2 cells (n = 18). Next, compound 2 was added to the bath solution in a final concentration of 10-8 M. After 10 minutes, the mechanical stimulation was repeated. Now, the wave propagated to an average of 5.4 cells (n = 18) (Figure 11), which is a significant increase compared to before adding the compound. Thus, compound 2 effectively increases intercellular calcium waves mediated by gap bonds.

[0149] To assess the effect of the compound on direct cell coupling, microinjection experiments were performed according to the method described above. The Yellow Lucifer (LY) dye was injected into a single human osteoblast in a monolayer. After 30 seconds, the number of cells containing dye was evaluated. Under physiological conditions, the dye extended to an average of 14 cells (n = 19). To eliminate cell coupling, the cells were incubated during hypoxia (O2 at 3-6 &) for 48 hours. Then, the cell coupling was reassessed by microinjection of LY and, at this point, the dye only went to an average of 7 cells (n = 10). Compound 2 was added to the medium and, after 10 minutes, the dye coupling was reassessed. After 10 minutes of incubation with compound 2, cell coupling was increased with the transfer of dye to 9 cells (n = 11).

[0150] Similar experiments were performed with ROS cells. The basic coupling under physiological conditions in ROS cells was 12 cells (n = 19). After a 48-hour incubation in 3-6% O2, a reduction in dye transfer to 9 cells was observed (n = 27). Again, compound 2 was added to the bath solution and the cell coupling was restored to prehypoxic levels with an average transfer of dye to 12 cells (n = 27), (Figure 12). In this way, compound 2 is capable of increasing communication through gap links and restoring hypoxia-induced reduction in cell coupling.

[0151] Hypoglycemia-induced metabolic stress is also known to decrease communication by gap links. Therefore, it was intended to assess whether compound 2 could reverse hypoglycemia-induced reduction in cell coupling. Human osteoblastic cells were cultured in monolayers on glass coverslips and loaded with fura-2. After desensitization with ATP as described above, a single cell was mechanically stimulated and the number of cells in the wave was recorded. In this group of experiments, the wave extended to an average of 3.2 cells (n = 19). The medium was changed to a medium without glucose and, after 8 minutes, another mechanical stimulation was performed. Now, the wave was almost blocked with a wave propagation of only 1.4 cells (n = 20). Compound 2 was added to the medium at a final concentration of 10-8 M. A final stimulation was performed and now the wave was almost restored with an average extension of 2.9 cells (n = 18) (Figure 1 ·). In this way, compound 2 is able to restore hypoglycemia-induced cell decoupling. Finally, to assess the effect of compound 2 on bone formation and the activity of osteoblasts, the effect of the compound on the alkaline phosphatase (ALP) activity of the cells was measured. Human osteoblasts were stimulated with different concentrations of compound 2 from 1 x 10-13 to 1 x 10-6, and compared with untreated controls. Under normal culture conditions, compound 2 increased ALP activity at most of the concentrations analyzed, except for the highest concentration (10-6 mol / l), which can be toxic (Figure 14). In addition, the effect of the compound on ALP activity during hypoxic conditions was also analyzed. Human osteoblasts were cultured for four days in 5% O2. The medium was enriched with compound 2 in different concentrations and compared with the responses during normoxic conditions. During hypoxia, the stimulation of ALP activity induced by compound 2 was 15% higher than during normoxia at all concentrations in the range of 10-11 to 10-8 mol / l, (Figure 15).

[0152] In summary, these results demonstrate that compound 2 is able to normalize attenuated GJIC among human osteoblasts during hypoxia. In addition, compound 2 stimulates the production of alkaline phosphatase, suggesting that compound 2 is capable of stimulating the activity of osteoblasts and, therefore, bone formation. Thus, compound 2 can be useful in the treatment of bone diseases with a damaged bone formation in relation to bone resorption. The effect of compound 2 on cell-to-cell coupling during hypoxia suggests that the substances of the present invention may be useful in the treatment and / or prevention of bone diseases associated with poor vascularization, hypoxia and ischemia in bone tissue.

[0153] From these experiments, it can be concluded that the substances of the present invention that increase GJIC may be useful for the preparation of medicaments for the prevention and / or treatment of osteoporosis. In some cases, osteoporosis is a manifestation of another disease, such as Cushing's syndrome or osteogenesis imperfecta. In most cases of osteoporosis, however, no other disease is evident. One form appears in childhood or in young adults of both sexes and with a normal gonadal function and is often called idiopathic osteoporosis, although most of the other forms are also of unknown pathogenesis. Type I osteoporosis appears in a subgroup of postmenopausal women who are between 51 and 75 years old and is characterized by an accelerated and disproportionate loss of trabecular bone. Fractures of the vertebral bodies and the distal forearm are common complications. A diminished function of the parathyroid gland may be compensatory for greater bone resorption. Type II osteoporosis appears in women and men above 70 and is associated with fractures of the femoral neck, proximal humerus, proximal tibia and pelvis, points that contain both cortical and trabecular bones. In addition to osteoporosis, substances that increase GJIC can also increase bone formation in bone metabolic diseases, such as rickets and osteomalacia and in osteoporosis due to chronic administration of glucocorticoids or chronic renal failure. Thus, the present invention provides compounds for the preparation of medicaments useful in the prevention and / or treatment of osteoporosis.

Effects of gap link openers on cartilage

[0154] Articular cartilage is a tissue designed to withstand compression during joint movement and, in vivo, is subjected to a wide range of mechanical loading forces. Mechanosensitivity has been shown to influence chondrocyte metabolism and cartilage homeostasis. In many types of cells, mechanical stimulation induces increases in the cytosolic concentration of Ca2 + that propagates from cell to cell as an intercellular Ca2 + wave. Cell-to-cell communication through gap links underlies the tissue coordination of metabolism and sensitivity to extracellular stimuli: the permeability of gap links to second intracellular messengers allows signal transduction mechanisms to be shared between several cells, eventually leading to responses tissue coordinates. Chondrocyte signaling has been mechanically induced Ca2 + signaling and it has been shown that gap link communication is essential for mechanically induced Ca2 + signaling in chondrocytes [53]. In addition, mechanical stimulation activates phospholipase C, thus leading to an increase in intracellular 1,4,5-trisphosphate inositol. The second messenger, through the permeation of gap bonds, stimulates the release of intracellular Ca2 + to neighboring cells and this system is considered very important for coordinated chondrocyte signaling during mechanical stress and can provide a mechanism for the coordination of metabolic activity during Metabolic stress in chondrocytes [53; 54]. The predominant connexin in cartilage is Cx43 and, in addition to its role in cell-to-cell regulation of metabolism and signaling, Cx43 is essential for normal chondrogenesis [47; 55].

[0155] In addition, the cytoarchitecture of the meniscal cells depends partially on the communication by gap links. The fibrocartilage part of the meniscus, as well as the fibrocartilage structure of the tendons, depends on intercellular communication. During injuries, gap link openers will improve repair speed.

[0156] Thus, it seems that the substances of the present invention that increase GJIC can be used for the prevention and / or treatment of joint diseases that involve an altered cell-to-cell coupling. As demonstrated in human osteoblastic cells, it is suggested that substances that increase GJIC can be used for the prevention and / or treatment of joint diseases that involve metabolic stress. These would include any form of arthritis associated with less vascularization or healing of fractured cartilaginous tissue. The effect of compound 2 and compound 40 on decreasing communication by DDT-induced gap bonds in human chondrocytes will be analyzed in the same manner as described below for osteoblast cells. The test compounds will be used in a concentration range of 10-10 -10-6 mol / kg and the test compounds are expected to reverse the decrease in gap link communication induced by the tumor inducing agent, DDT. Thus, the present invention provides compounds for the preparation of medicaments useful in the prevention and / or treatment of joint diseases, including arthritis. The administration will be an oral, parenteral or intraarticular administration.

Effects of gap link openers in cancer

[0157] The permeability of gap bonds and the regulation of the GJIC happens at different levels in the cell. A decrease or absence of GJIC may be the result of changes in the expression of Cx during transcription and translation, the alteration of post-translational processing and the alteration of the connection assembly and insertion into the plasma membrane. An unusual feature of Cx is its short half-life compared to other membrane proteins. It has been observed that the rapid replacement of the connexins is between 1.5 and 2 hours. It has been observed that the degradation of Cx depends on phosphorylation, which leads to the destabilization of some subtypes of connexins. The fast replacement speed provides an additional mechanism by which the GJIC can be quickly regulated by substances that affect the half-life of Cx mRNA, translation, intracellular transport and Cx assembly in gap bonds.

[0158] Another way to regulate the permeability of gap links is the complete or partial closure of the gap link channels under certain circumstances by mechanical screwing of the six connection units. It is known that the "gating" of gap links is performed by tumor inducers that decrease GJIC. Tumor inducers are agents that increase or accelerate carcinogenesis when given repeatedly after the onset of the tumor. The mechanisms by which tumor inducers modulate GJIC are not fully understood, but there is evidence to support that tumor inducers can affect GJIC by altering Cx phosphorylation and / or inhibiting Cx expression and assembly. . Recent results have shown that in vivo gene transfer mediated by connexin retrovirus 43 in tumors with a low GJIC capacity significantly reduced tumorigenicity [56]. As additional support for the essential role of normal GJIC in cancer prevention, it has been observed that CX32-deficient mice have a very high incidence of spontaneous liver tumors and an increased susceptibility to develop chemically induced liver tumors [57]. In addition, the tumor-inducing action of phenobarbital requires functional Cx32 for tumor progression [58]. This suggests that the decoupling of GJIC is important for the oncogenic actions of phenobarbital [58].

[0159] Carcinogenesis is characterized by the progressive inability of growth control mechanisms in which growth factors, oncogenes and tumor suppressor genes are involved. Since the alteration of GJIC could lead to the alteration of growth control, the effect of growth factors and oncogenes on the GJIC could be crucial for tumorigenesis. It has been observed that several oncogenes mediate in the disadvantage of GJIC [59]. It is observed that pp60v-src mediates the closure of Cx43 gap bonds by means of a ball and chain mechanism that involves phosphorylation of a C-terminal serine residue by MAP kinase [59]. Notably, in some cases, cells transfected with oncogenes may communicate with each other, but may lack heterologous communication with adjacent normal cells.

[0160] The permeability of the gap bonds in the tumor cells using the dye transfer assay was lower than the GJIC in the surrounding liver tissue. Notably, many tumors are encapsulated in an extracellular matrix type structure and physically separated from normal tissue.

[0161] Neoplastic transformation in normal human tissues takes place as a result of an accumulation of genetic alterations. However, a general point in carcinogenesis and tumorigenesis is the disadvantage of GJIC. The various connexins are expressed in a specific manner of tissue. Cx43, Cx26, Cx32 have been detected in normal breast tissue. A panel of human breast cancers was analyzed for the level of Cx43 expression. No Cx43 gap links were observed in ductal carcinomas in situ, infiltrating ductal carcinomas, and infiltrating lobular carcinomas and appear to be independent of the state of estrogen, progesterone and erbB2 receptor. In contrast, human breast cancer cell lines and rodent mammary carcinoma tissues showed a disadvantage of Cx43 expression and turned out to be mRNA level, suggesting a transcriptional mechanism for the decrease of Cx43 protein in breast cancer [60]. ]. Another example in the connection between cancer and GJIC is hepatocellular carcinoma, where it has been observed that the lack of connexin 32 is prone to this specific type of cancer [57]. Studies with oval cells have indicated that they can be differentiated into hepatocytes and that neoplastic derivatives of oval cells can produce both hepatocellular and biliary neoplasms. The specific connexin expressed by the differentiating oval cell determines whether it communicates with hepatocytes or biliary epithelial cells. This communication may be necessary for the subsequent differentiation and regulated growth of the differentiating oval cells and the deterioration of the GJIC may contribute to the formation of hepatocellular and cholangiocellular neoplasms. Thus, the GJIC can be a key factor in the differentiation of oval cells and a blocked GJIC can induce its neoplastic transformation. In addition, in vitro analysis of tumor invasion in rat lung endothelial cells treated with malotylate showed that the malotylate induced the development of cell-to-cell adhesion through gap bonds that resulted in the inhibition of tumor cell invasion [61]. ]. Together, these findings strongly support the hypothesis that the alteration of GJIC is a critical event in carcinogenesis and that the substances of the present invention that increase GJIC could be beneficial in cancer therapy. Therefore, the present invention provides new compounds that increase GJIC as medicaments for the treatment of cancer due to its low effective concentration and consequently low toxicity.

[0162] Specific uses of the peptides of the present invention include the treatment of the following medical conditions related to cancer: Tumor progression: during tumorigenesis, disruption of the physiological interaction of normal cells with their neighboring cells and the Loss of differentiation characteristics are a common denominator in tumor progression. It is believed that the alteration in communication by gap links is among the earliest changes during cell tumorigenesis (Wolburg H, Rohlmann A. Int Rev Cytol. 1995; 157: 315-73), Klaunig JE, Ruch RJ. 1990; 135-46)). Kyung-Sun Kang, Jun-Won Yun, ByoungSu Yoon, Yoon-Kyu Lim and Yong-Soon Lee (Cancer Letters 166 (2001) 147-153) have shown that preincubation and co-incubation with GeO2 in epithelial cells of rat liver treated with TPA eliminated the disadvantage of GJIC by TPA, suggesting that a substance that recovers GJIC inhibition can be used in the prevention or inhibition of tumor induction. Suzuki J, Na HK, Upham BL, Chang C_C and Trosko JE (Nutrition and Cancer, vol 36 No. 1 p. 1228) have shown that the lambda-carrageen food additive inhibits GJIC in rat liver epithelial cells similarly to that of the well-documented tumor promoter, forbol ester (TPA) and, therefore, could play a role in carcinogenesis as a tumor inducing agent. Thus, the compounds of the present invention can be used in the prevention or treatment of cancer caused by tumor inducing agents, such as TPA and lambda-carrageenan. Drug-sensitive resistance: an increase in communication by gap links improves the microenvironment in tumors and Carystinos GD, Alaoui_jamali MA, Phipps J, Yen L, Batist G. Metastasis: The loss of intercellular communication by gap links is associated with high potential Metastatic in all cancers with metastatic potential. (Saunders MM, Seraj MJ, Li Z, Zhou Z, Winter CR, Welch DR Donahue HJ. (Cancer Res. 2001; 61: 1765-1767), Nicolson GI, Dulski KM, Trosko JE, Porc Natl Acad Sci USA. 1988 ;

85: 473-6)). The prevention of metastasis is established by treatment with a gap link opener that will retain communication by gap links in tumors.

[0163] Treatment is an additional treatment to conventional chemotherapy.

Experimental Example 9

The effect of compound 2 on the decrease in communication by DDT-induced gap bonds in human osteoblast cells.

Protocol and results

[0164] The 1,1-bis (p-chlorophenyl) -2,2,2-trichloroethane compound, also known as the DDT insecticide, is an inhibitor of gap bond communication and has the capacity to induce tumors. It inhibits cell-to-cell communication by reducing the number and size of gap bonds, as well as decreased cell levels of phosphorylated (active) forms of the Cx43 binding protein and these actions are considered fundamental for the oncogenic properties of the compounds. [62-64]. [0165] Thus, compounds with the ability to prevent the decrease of GJIC induced by tumor inducers may be potential candidates for use in protection against tumor induction and cancer treatment [65]. To examine whether the substances of the present invention prevent the decrease in GJIC induced by tumor inducers, the effects of compound 2 on DDT-induced decoupling in human osteoblast cells were examined.

Methods

Cell culture

[0166] Human osteoblast cells: Human bone marrow cells obtained by puncturing the posterior iliac spine were isolated from healthy volunteers (age 20 to 36): 10-15 ml of marrow material was collected in 15 ml PBS + Ca, Mg (Life Technologies, Cat.No. 14040) with Heparin 100 U / ml (Sigma, Cat.No. H-3149). The mononuclear fraction of the marrow was isolated in a gradient of Lymphoprep (Nycomed Pharma, Cat.No. 1001967), by centrifugation at 2200 rpm for 30 min. After collection, the mononuclear fraction was washed once with culture medium and centrifuged at 1800 rpm for 10 min. Subsequently, the cells were counted and placed in a culture medium at 8x106 cells / 100 mm plate. HOB medium (all reagents obtained from Life Technologies): MEM without Phenol Red with Glutamax (Cat.No. 041-93013) supplemented with heat-inactivated 10% fetal calf serum (Cat.No. 10106) and Penicillin / Streptomycin 0.1% (Cat.No. 15140). The medium was changed the next day and the cells were grown at 37 ° C in 5% CO2 with medium change every 7 days. After 3-4 weeks of culture, the cells had reached a confluence of 70%. The medium was then supplemented with 100 nM Dexamethasone (Sigma, Cat.No. D-4902) for 7 days. Next, the cells were placed for video image recording experiments: a 25 mm glass cover slip # 1 was placed on a 35 mm plate (or each well of a 6-well multi-plate), the cells were placed at 2.5x105 cells / coverslip and were cultured for 2-3 days before use.

Microinjection

[0167] Cells were grown on coverslips and fixed to a culture chamber with PDMI-2 (Medical Systems Corp.), maintained at 37 ° C with a CO2 superfusion, in a Zeiss Axiovert microscope. Microinjections were performed using the Eppendorf 5171 micromanipulator and the Eppendorf Transjector 5346 system. A micropipette was loaded with a 10 mM Yellow Lucifer solution (Sigma, Cat.No. L-0259). A cell was carefully injected into the monolayer with LY for 30 seconds, the micropipette was removed from the cell and, after 30 seconds, the number of cells showing dye transfer was counted. The excitation light (430 nm) was provided by a monochromator (T.I.L.L. Photonics GmbH). The images were obtained with an intensified CCD camera (Dage MTI) and digitized with a Matrox MVP image processing board, using the MetaMorph (Universal Imaging) image software.

Results

[0168] In order to assess the ability of gap link modifiers to prevent tumor induction, it was wanted to analyze whether gap link modifiers could reverse communication by gap links, induced by a well-known tumor inducing agent, the DDT Therefore, they were incubated at 37 ° C in an atmosphere containing 5% CO2 monolayers of human osteoblast cells on glass coverslips. DDT was added to the medium in a final concentration of 13 µM and left for 60 minutes.

[0169] To assess the effect of compound 2 on direct cell coupling after treatment with DDT, microinjection experiments were performed according to the method described above. The Yellow Lucifer (LY) dye was injected into a single human osteoblast in a monolayer. After 30 seconds, the number of cells containing the dye was evaluated. Under control conditions (without DDT treatment), the dye was extended to a median of 14.5 cells (n = 12). The same experiment was performed with cells exposed to DDT. These cells showed a lower cell coupling with a median of 7 (n = 13). Compound 2 was added to the bath solution in a final concentration of 10-8 mol / l, and after 10 minutes, another microinjection was performed. Compound 2 produces an increase in cell-to-cell dye transfer in all preparations with a median of 8.3 cells (Figure 15). This increase is highly significant with p <0.01, using the non-parametric Wilcoxon statistical test. Thus, gap link openers are capable of reversing the lower intercellular coupling related to tumor induction, suggesting that the substances of the present invention may be useful in chemoprevention and / or cancer treatment. The compounds of the present invention are useful for the preparation of medicaments for chemoprevention and / or cancer treatment. The compounds of the present invention can also be used in a combination therapy with other anticancer agents.

Additional Pharmacological Methods

[0170] The utility of the peptides described in the present invention in therapeutic treatment methods will appear clear from the following additional examples.

Effects of gap link openers on wound healing

[0171] A wound is a discontinuation of the normal anatomy that involves the skin and can be a surgical or traumatic wound, or it can be secondary to various diseases, such as diabetes, atherosclerosis, malnutrition etc. Normal wound healing is a systematic process that takes place in stages and includes hemostasis and inflammation. After these processes, remodeling takes place that can last for years and is responsible for the formation of scar tissue. Hemostasis with fibrin provides a surface below which migrations and movements of the edge of the wound take place. Epithelialization, fibroplasia and capillary proliferation in wound healing begin immediately. Angiogenic capillary expansion invades the wound fibrin clot and in a few days organizes a microvascular network through granulation tissue that also consists of leukocytes and phagocytic mononuclear cells. A very dynamic interaction between the various tissue components involved in the wound healing process takes place. The angiogenetic process is essential for a satisfactory wound healing. The gap links of intercellular communication are essential for the creation of fibroblast syncytium and the proliferation of the capillary network. The normal distribution of connexin 43 is necessary for this growth of the different tissue components.

[0172] Several local factors often observed during pathological conditions, such as edema, ischemia, low oxygen tension and infection, can delay the wound healing process. Wound healing involves the interactions of many types of cells and gap-mediated intercellular communication is considered to play an important role in the coordination of cell metabolism during the growth and development of tissues and organs. [66-68].

[0173] It is suggested that the substances of the present invention that increase GJIC can be used for wound treatment and, in particular, to accelerate wound healing. Considering that experiments on cardiac and bone tissue suggest that these substances are more effective during metabolic stress (for example, hypoglycemia, hypoxia, ischemia), it can be deduced that these substances may be particularly useful in the treatment of ischemic ulcers. Thus, the present invention provides compounds for the preparation of medicaments useful in the treatment of wounds and, in particular, ischemic ulcers.

Wound healing processes

[0174] The healing processes are a series of overlapping phases that begin with hemostasis (coagulation). The second phase of the healing process is a cascade of inflammatory responses in which microphages accumulate in the wound part and the formulation of granulation tissue that involves, among other components, fibroblasts and lymphocytes begins. The epithelial cells will then begin to migrate from the edge of the wound to cover the area. Capillary expansion from normal tissue to the wound is also involved in order to ensure the supply of nutrients, oxygen and the different cells. All cells and cells of the capillary endothelium have active intercellular communication through gap bonds (Abdullah KM, Luthra G, Bilski JJ, Abdullah SA, Ryenolds LP, Grazul-Bilska AT. (Endocrine. 1999; 10: 35-41). Areas with a low oxygen supply and / or a high concentration of free radicals often observed in wounds with necrotic tissue, in diabetes, in arteriosclerosis, in surgical wounds, edema, infection, bum wounds and in venous insufficiency will decrease communication by gap links (Nagy JI, Hossain MZ, Lynn BD, Cupern GE, Yang S, Turley EA. Cell Growth Diff. 1996; 7: 745-51)).

[0175] The effect of the gap link opener is analyzed in an in vitro fibroblast culture. Fibroblasts are collected from human gums as described by Arora KK, Lee W, McCullock C. Am J Physiol Cell Physiol. 2000; 279: C147-57). The cell culture is exposed to 10-10-10-8 nM of the gap link opener and significantly faster cell growth will be observed. Growth is analyzed by conventional methods that measure thymidine nuclear uptake over time.

[0176] The stimulation of the gaps in the growth gap and endothelial cell formation of the endothelial tube is studied before and after exposure to the compound described by Ashton AW, Yokota R, John G, Zhao S, Suadicani SO, Spray DC, Ware JA. (J Biol Chem. 1999; 274: 35562-70).

[0177] The gap link openers stimulate healing processes in the oral mucosa. Hara A, et al. (J Gastroenterol 1999 Feb 34: 1-6) identified connexins 26 and 32 in the human oral mucosa, an indication of the presence of gap bonds in this tissue. However, the immunofluorescence study found no significant differences in the expression of connexins between patients with aphthous stomatitis and controls. Irsogladin maleate, which strengthens intercellular communication by gap links in vitro, was effective for the treatment of transient and recurrent aphthous stomatitis, as well as for symptomatic and drug-induced aphthous stomatitis. It was also useful for the prevention of recurrent aphthous stomatitis episodes with a daily administration that prevents the recurrence of stomatitis. The peptides of the present invention can be used in the same manner to accelerate the healing process of wounds in oral mucosa by strengthening intercellular communication by gap bonds between cells of the oral mucosa; and the peptides of the present invention are also useful for the treatment and prevention of aphthous stomatitis.

[0178] To examine wound healing in vivo, compound 2 and compound 40 are administered topically (concentration range 10-9-10-6 mol / l in aqueous gel) and parenterally (10-10-10-6 mol / kg) two to four times daily to mice. Two round wounds are created by excision below the panniculus camosus with a 6 mm biopsy penetrating the skin of each mouse's back. After a 5-day treatment with compound 2 and compound 40, the effect of the skin is evaluated histologically by microscopy of the biopsies and wound healing is measured by daily measurements of the wound diameter. It is predicted that compound 2 and compound 40 will not affect the structure of the skin alone, but that both compounds will accelerate wound healing after biopsies.

[0179] The treatment with a gap link opener will ensure maximum communication through gap links between the different cells considered to play an important role in the complicated repair process and, thus, improve wound repair. The compound will be administered parenterally, topically, systemically or orally.

Effects of gap link openers in the healing of gastric and duodenal ulcers

[0180] Gap links also play an important role in intercellular communication, proliferation and differentiation in the gastric mucosa cell. The gap link opener will stimulate regenerative processes after I-induced injury (Endo K, Watanabe S, Nagahara A, Hirose M, Sato N. (J Gastroenterol Hepatol. 1995; 10: 589-94)).

[0181] Mine et al. have shown that the normal gastric mucosa contains both connexin 32 and connexin 43 [69; 70]. In contrast, the gastric mucosa around a chronic gastric ulcer lesion contains a smaller amount of connexin 32 and connexin 43. In studies by Mine et al. The relationship between the appearance of connexins and ulcer healing was investigated. When ulcer healing was observed, connexins 32 and 43, which decreased in the active ulcer stage, had returned almost to levels observed in the normal gastric mucosa. These data indicate that the disappearance of connexin 32 and connexin 43 is closely related to the phase of chronic gastric ulcer lesions. In addition, using a rat model of gastric ulcer induced by acetic acid, the same group of researchers demonstrated that the clinical effect of the antiulcer drug cimetidine was closely related to the recurrence of connexin 32 [69].

[0182] Gap links are important in the gastric mucosa defense system and the return of acid-induced injury. Takahashi N, Joh T, Yokoyama Y, Sinus K, Nomura T, Ohara H, Ueda F, Itoh M. (J Lab Clin Med 2000 Aug; 136 (2): 93-9). Evidence has accumulated that intercellular communication through gap links (GJIC) determined whether GJIC mediates a process of restitution in the gastric mucosa. The Srague-Dawley male rats fasted and were anesthetized. Gastric lesion was induced by luminal perfusion with 0.2 N HCl for 10 minutes. The integrity of the mucosa was continuously monitored by measuring the purification of chromium-labeled ethylenediaminetetraacetic acid 51, which was used to analyze the recovery of the lesion. Perfusion with 0.25% octanol (OCT; GJIC inhibitor) was initiated after acid injury to assess its effect on restitution. The effect of irsogladin (IG; activator of GJIC) was also evaluated. The GJIC of the gastric mucosa was immunohistochemically evaluated with a gap binding protein (connexin 32) with monoclonal antibody. Recovery of the acid-induced mucosa lesion happened quickly when the perfusion with acid was discontinuous (in approximately 60 minutes). OCT, which did not cause any injury to normal gastric mucosa, significantly inhibited restitution. The IG reversed this inhibition in a dose-dependent manner. In an immunohistochemical study, the OCT-induced damage of the gap bond was demonstrated, but not after IG pretreatment. These findings suggest that GJIC may play a critical role in restoring the rat gastric mucosa and the peptides of the present invention are useful in the treatment of ulcers, such as gastric and duodenal ulcers. To substantiate this statement, experiments can be performed on rats using the general experimental design of Takahashi N et al. 2000, above, with the administration of COMPOUND 2 and COMPOUND 40 which are stable in acid solution in concentrations in the range of 10-11-10-7 M in rats. These experiments are expected to show the facilitating effect of COMPOUND 2 and COMPOUND 40 in the coupling of gap bonds and counteract the effect of cerulein resulting in the healing of gastric ulcer.

[0183] The administration of the peptides will be orally or parenterally, for example intravenously.

[0184] Therefore, the substances of the present invention that increase GJIC can induce the healing of gastric and duodenal ulcers. Thus, the present invention provides compounds for the preparation of medicaments useful in the treatment of gastric and duodenal ulcers.

Role of the gap links in vascular biology

[0185] The coordination of cellular responses at the endothelial interface between blood and underlying tissues is mediated by multiple signaling mechanisms, including direct intercellular communication through gap links. Among the functions that have involved endothelial intercellular communication by gap links are migratory behavior of endothelial cells after injury, angiogenesis, endothelial growth and senescence, and the coordination of vasomotor responses [71].

[0186] Regulation of blood flow over a wide dynamic range requires coordinated responses of resistance arteries and feeding arteries. Such coordination between the vessels can be achieved through the vascular effects of shear stress exerted by the bloodstream or by the conduction of vasomotor signals along cells of the vascular wall. In fact, the local application of certain vasoactive compounds, such as acetylcholine (ACh) or norepinephrine (NE), induced not only local dilation or constriction, but also vasomotor responses a few millimeters up and down [71]. Vasomotor responses can also be conducted from capillaries to arterioles and can contribute to the correlation between tissue demand and blood supply. This has been demonstrated as follows: When they stimulate the fibers of a muscle for contraction, it was observed that the arterioles above the capillaries that supply these fibers dilated [72].

[0187] The high conduction velocity corresponds to an electrotonic transmission of a signal along the vascular wall. In fact, it has been shown that locally induced hyperpolarizations and depolarizations lead several millimeters up into endothelial and vascular smooth muscle cells. Conduction of the electrical signal requires the coupling of vascular cells through gap links that provide conduits of low electrical resistance between the cells. In vascular tissue, at least three different connexin proteins (Cx) (Cx37, Cx40, and Cx43) that form gap bonds are expressed. Cx40 appears to be the predominant connexin isoform in aortic endothelial cells, while in smooth muscle, Cx43 expression is abundant.

[0188] Studies in mice deficient in Cx40 (Cx40 - / -) have shown that the expansion of vasodilation induced by the local application of acetylcholine or bradyquine is severely attenuated in Cx40 - / - animals compared to normal wild-type animals (Cx + / +) [73]. In addition, blood artery pressure increases significantly in Cx40 - / - animals compared to normal wild-type mice (Cx + / +). These results support the significant role of Cx40 in vascular intercellular communication and indicate that communication by gap bonds damaged in the vascular wall is associated with reduced transmission of endothelial-dependent vasodilatory responses, which, in turn, increases vascular resistance and It causes hypertension. Recent in vivo studies suggest that oscillations in normal kidney pressure are extremely important for blood pressure regulation [74]. Thus, damaged vasomotor responses due to poor cell-to-cell coupling can contribute to the development of hypertension in Cx40-deficient animals.

[0189] Disadvantage of mRNA expression and Cx43 protein levels in senescent endothelial cells suggests that intercellular communication by damaged gap bonds could play a role in the progression of vascular aging [75].

[0190] Based on the information available on the role of gap bonds in vascular responses, it is likely that a pharmacological compound that increases coupling with gap bonds in the vascular wall could facilitate targeted vascular responses and improve blood supply during conditions with increased metabolic demand (for example, physical exercise, tachycardia), and during ischemia. In addition, said substance is likely to prevent and / or treat hypertension. Accordingly, the present invention provides compounds that increase coupling by gap and / or GJIC bonds in the vascular wall and, thus, are useful in the prevention or treatment of hypertension.

Experimental procedure

[0191] For all experiments, isolated resistance arteries (internal diameter of approximately 200 mm) from the rat mesentery are used. The arteries are third order branches of the mesenteric artery and dissect from the mesenterium of male Wistar rats aged 14-18 weeks. The arteries are mounted on a myograph to measure the isometric force and passively stretched for maximum strength. The tissue bath is divided into two and an artery is mounted in each of the two halves. The arteries are bathed in a physiological solution of bicarbonate buffered salt and, unless otherwise indicated, was gassed with 5% CO2 in 21% O2.

[0192] For the evaluation of vasomotion, the arteries are activated with norepinephrine in a submaximal concentration. This is done after the endothelial extraction and with increasing concentrations of cGMP, which is known to increase the degree of vasomotion and intercellular communication.

[0193] For the evaluation of endothelial function, arteries are activated with an almost maximum norepinephrine concentration and relax in the presence of norepinephrine with increasing concentrations of acetylcholine, which is known to relax the arteries dependently on the endothelium in these arteries , through a mechanism partially dependent on NO and partially dependent on EDHF.

[0194] The effect of 10-8 M and 10-6 M of compound 2 and compound 40 is assessed on vasomotion and responses to acetylcholine. When the drug is present, a preincubation of at least 5 min is used.

[0195] To assess vasomotion, one artery in the tissue bath serves as a control and the other artery is treated with one between compound 2 and compound 40.

[0196] In experiments with hypoxia, tissues are exposed to 5% CO2 in N2 for at least 5 minutes before performing the experiments. This procedure decreases the PO2 of the bath to approximately 5 mm Hg.

[0197] It is expected that the compounds of the present invention will increase vasomotion and acetylcholine-induced vasodilation. Consequently, these substances will be useful in the treatment of hypertension and vascular diseases associated with vasoconstriction. The mode of administration will be oral or parenteral.

Smooth muscles

[0198] Vascular system. Intercellular communication through gap link channels plays a fundamental role in the regulation and modulation of myocyte vascular tone along the vascular tree (Christ GJ, DC Spray, Moore LK, El-Sabban ME, Brink PR. ( Circ Res. 1996; 79: 631-636)). Another important role of gap link communication is the expansion of hyperpolarization between smooth muscle cells involved in the response to vascular relaxation (Benny JL, Paicca C. Am J Physiol Heart Circ Physiol 1994; 266: H1465-72)) .

[0199] The specialized functions of the endothelium require intercellular communication by gap links between endothelial cells in the monolayer and between the endothelium and other cells present in the vessel wall. Communication between these different cell types through gap bonds in coronary capillaries, as well as in other vessels, has been documented in several studies. Evidence of involvement in adaptive arteriogenesis has also been demonstrated (Cai WJ, Koltai S, Kocsis E, Scholz D, Shaper W, Schaper J (J Mol Cell Cardiol 2001; 33: 957-67), Wang HZ, Day N, Valcic M, Hsieh K, Serels S, Brink PR, Christ GJ. (Am J Physiol Cell Physiol. 2001; 281: C75-88), Schuster A, Oishi H, Benny JL, Stergiopulos N, Meisater JJ. (Am J Physiol Heart Circ Physiol. 2001; 280: H1088-96)).

[0200] In different vascular pathophysiological situations in which the endothelial monolayer is altered as in diet-induced hypercholesterolemic lesions, communication by gap bonds decreases in vascular smooth muscles (Polacek D, Bech F, McKinsey JF, Davies PF. ( J Vasc Res 1997; 34: 19-30) Lesion in the endothelial cell layer is observed during venous stasis and when thrombophlebitis develops Kwak BR, Pepper MS, Gros DB, Meda P (Molec Biol Cell 2001; 12: 831- 845) have clearly demonstrated that gap link communication serves to coordinate cell migration during cell repair and is also important for capillary expansion during angiogenesis.

[0201] Treatment with compounds that facilitate communication by gap links will improve damaged intercellular communication in the affected vascular areas and will be particularly useful during organic ischemia, for example claudicatio intermittens and myocardial infarction.

[0202] However, after the injury with balloon catheter in the carotid of the rat, the vascular healing process is characterized by an increase in communication by gap links. (Yeh HI, Lupu F, Dupont E, Severs NJ, (Arterioscle Thromb Vasc Biol 1997; 17: 3174-84). The compound will be administered before balloon intervention and is preferably an additional therapy to conventional medical treatment of this pathology. The administration of the compound will be parenterally.

[0203] The effect will be analyzed in tissue samples before and at different time points after injury with the balloon catheter. Faster healing of the endothelial surface will be observed using conventional microscopy. There was also an improvement in communication through gap links.

[0204] (Arterioscle Thromb Vasc Biol 1997; 17: 3174-84). Treatment with gap link openers will increase the healing process.

[0205] The prophylactic effect of treatment with a gap link opener, such as compound 2 and compound 40, will be analyzed on an experimental basis described by Yeh HI, Lupu F, Dupont E, Severs NJ, (Arterioscle Thromb Vasc Biol 1997; 17: 3174-84). Compound 2 or compound 40 will be administered prior to balloon intervention using doses in the range of 10-11 to 10-8 depending on the biological kinetics of the compound, for example, as determined in the chloride-induced arrhythmia model. Calcium described above. Tissue samples will be taken before and at different time points after injury with the balloon catheter. Faster healing of the endothelial surface will be observed using conventional microscopy. There was also an improvement in communication through gap links.

[0206] The administration of the compound will be, for example, parenterally.

[0207] In other diseases, communication by gap links between smooth muscle cells is disturbed. In the cavernous body, a syncytial cellular network is established through gap links and is critical for erectile function and ensures that the body's and arterial smooth muscle cells of the penis respond in a uniform and coordinated manner. (Christ GJ. (Int J Impot Res. 2000; 12 suppl. 4: S15-25), Melman A, Christ JC. (Urolog Clin North America. 2001; 28: 217-31)). A damaged erectile function is observed in diabetes, arteriosclerosis, different neurological diseases and many chronic diseases. From studies in diabetes, a correlation between neuronal innervation and intercellular coupling points to the potential functional plasticity of the body environment although functional intercellular communication is not established through gap links.

[0208] Treatment with a compound that facilitates the opening of gap bonds will improve communication through gap links and thus normalize complex coordination between smooth muscle cells in the corpora cavernosa and vessels.

[0209] Isolated body smooth cells of rats are established as described by Christ GJ, Moreno AP, Melman A, DC Spray. (Am J Physiol 1992; 263: C373-83). The communication of gap links with Lucifer Yellow and another fluorescent dye is measured using the microinjection technique as described above or using a FACS method, such as that described by Juul MH et al. Cell Adhes Commun 2000; 7 (6): 501-12. The number of fluorescent cells is counted for several gap bond inhibitors with or without different doses of the described gap bond openers, for example, compound 2 or compound 40, in the range of 10-10-10-8 nM. An improvement of more than 25-50% of the communication of gap links will be identified after exposure to the gap link openers with a compound concentration in the indicated range.

[0210] In vivo pharmacological analysis of the erectile function of the compounds will be performed 10 weeks after streptozotocin-induced diabetes (35 mg / kg ip) in rats (8 weeks of life) as described by Rehman J, Cheven E, Brink P, Peterseon B, Walcott B, Wen YP, Melman A, Christ G. (Am J Physiol 1997; 272: H1960-71). Penile reflexes and intracavernous pressure are measured during the local and systemic administration of different doses of the different gap link openers with measurements and techniques described by the same research group. An increase in penile reflexes and intracavernous pressure of 25% or higher will be observed.

[0211] The treatment of erectile dysfunction can be administered locally in the penile body, as a subcutaneous or oral injection. The treatment will be a monotherapy or an additional treatment to a conventional treatment of this pathology.

[0212] Diabetic retinopathy can be diagnosed very early after the onset of the disease by identifying changes in blood flow velocity (Bursell SV, Clermont AC, Shiba T, King GL. (Curr Eye Res. 1992 ; 11: 287-95), rupture of the retinal-saguineal barrier (Cunha-Vaz JG, Faria de Abrue JR, Campos AJ, Figo GM. (Br J Ophthalmol. 1975; 59: 649-56), Do Carmo A, Ramos P, Reis A, Proenca R, Cunha-Vaz JG (Exp Eye Res. 1998; 67: 569-75)) and / or loss of self-regulation (Kohner EM, Patel V, Rassam SMB. (Diabetes 1995; 44: 603-607)). Using tracer transport techniques and double clamp of cell patch, Oku H, Koda T, Sakagami K, Puro DG (Invest Ophthalmol Vis Sci. 2001; 42: 1915-1920) have demonstrated a wide cell-to-cell coupling The closure of the gap junction pathways alters the multicellular organization of the retinal microvessels and contributes to diabetic retinal vascular dysfunction. Zhou ZY, Sugawara K, Hashi R, Muramoto K, Mawatari K, Matsukawa T, Liu ZW, Devadas M, Kato S. (Neuroscience. 2001; 102: 959-67) also demonstrated that reactive oxygen is involved in decoupling retinal gap bonds and in re-coupling when glutathione is supplied.

[0213] The effect of gap link openers on diabetic retinopathy will be studied in vitro using the streptozotocin-induced diabetic rat model as described above. New isolated retinal microvass coverslips (Sakagami K, Wu DM, Puro DG. J Physiol (Lond). 1999; 521: 637-50) will be transferred as described by Oku H, Koda T, Sakagami K, Puro DG. (Invest Ophthalmol Vis Sci. 2001; 42: 1915-1920). In this preparation, intercellular communication between the cells in the vascular wall will be measured with dye or tracer. Different concentrations in the 10-10 to 10-7 M range of gap, compound 2 or compound 40 link openings will be analyzed and a significant increase in intercellular communication compared to the baseline will be observed in the diabetic retina. A similar improvement will be observed when compared to controls (healthy animals). Treatment with a gap link opener will stop or slow the progression of the pathology. The treatment will be systemic, local or oral.

[0214] The therapy is preferably additional to a conventional antidiabetic treatment.

[0215] Not only diabetic retinopathy, but also other vascular abnormalities in the retina, such as arteriosclerosis, will benefit from improved communication by gap bonds by treatment with a gap link opener. It has been shown that gap links connect horizontal cells with each other and are responsible for electrical coupling between neurons (Raviola E, Gilula NB. (Proc Natl Acad Sci USA. 1975; 65: 192-222), Ravlola E, Dacheux RF (J Neurocytol. 1990; 19: 731-36), Schneeweis DM, Schnapf JL (Science 1995; 268: 1053-56)). The transmission of scotopic signals between rods and cones through gap links is also indicated (Bloofield SA, Dacheux RF. (Retinal Eye Res. 2001; 20: 351-384)). Therefore, a gap link opener will increase communication between not only neurons, but will also be able to avoid less vital rods or cones and still carry the scotopic signal to the ophthalmic nerve.

[0216] The effect of gap bond openers in diet-induced arteriosclerotic retinopathy will be studied in vitro using the rat model (non-diabetic) as described above. Intercellular communication between cells in the vascular wall will be measured with the Yellow Lucifer dye transfer method after microinjection or with the FACS method.

[0217] The compound will be administered parenterally.

[0218] The smooth muscles in the urinary bladder are characterized by physical contractions and show spontaneous physical contractions. However, the bladder is in the healthy condition capable of containing several hundred milliliters of urine without showing an increase in intravesical pressure. Unlike the normal bladder, unstable bladders develop spontaneous increases in intravesical pressure in relation to emergency incontinence (Turner WH, Brading AF. (Pharmacol Therap. 1997; 75: 77-110). Compared to smooth muscle gastrointestinal, smooth bladder muscles do not spontaneously generate coordinated contractions (Stevens RJ, Weinert JS, Publcover NG. (Am J Physiol. 199; 2777: C448-60), Hashitani H, Fukuta H, Takano H, Klemm MF , Suzuki H. (J Physiol. 2001; 530: 273-86)) Both electrical and morphological communications have recently been demonstrated by means of gap links between smooth muscle cells in the bladder (Hashitani H, Fukuta H, Takano H, Klemm MF, Suzuki H. (J Physiol. 2001; 530: 273-86), Wang HZ, Lee SW, Day NS, Christ GJ. (Urology. 2001; Suppl 6A: 111).) The importance of these gap links is demonstrated by specific inhibition of communication.The waves of spontaneous excitation in the muscle Bladder smooth spread through gap links.

[0219] Uncontrolled emergency incontinence will therefore be regulated by treatment with a gap link opener.

[0220] The improvement of communication by gap links after treatment with a gap link opener is studied in a cell culture of smooth muscle cells collected from the urinary bladder using FACS analysis. The compound will be dosed in concentrations ranging from 10-10 to 10-7 M and a significant increase in communication will be observed in cell cultures exposed to low oxygen or oxygen stress.

 [0221] Intravesical pressure will be measured after pretreatment and acute treatment with a gap link opener, preferably compound 2 or compound 40, in normal guinea pigs and in animals with the function of the experimentally damaged bladder. The animals are anesthetized with phenobarbital and the bladder is catheterized with a urine catheter that allows the entry and exit of water and a catheter with a "tip" transducer. A gap link opener will not change a normal volume-pressure ratio, while this ratio will normalize in the damaged bladder.

[0222] Administration will be parenterally, orally or in the urinary bladder. Administration will preferably be as an addition to a treatment with drugs intended to normalize muscle contraction in the urinary bladder.

[0223] Myoepithelial cells that occur in the submandibular glandular ducts, in the urethra, in the bile ducts, pancreatic ducts, lachrymal ducts, connect with gap bonds and intercellular communication is essential for synchronizing the contractile function of the Myoepithelial cells (Taugner R, Schiller A. (Cell Tissue Res. 1980; 206: 65-72) Damaged contractility in these ducts can be normalized by treatment with a gap bond opener administered parenterally or orally.

[0224] Intercellular communication in cardiac av nodule is maintained through gap links. A diminished function leads to less conduction and can lead to a total a-v block. AV block is observed in acute myocardial infarction, in ischemic heart disease, digital poisoning, calcium channel blocker intoxication and a gap link opener will improve av conduction.

[0225] Intravenous infusion of CaCl2 (100 mg / kg / min) in neuroleptic anesthetized mice induced a second degree av block. When pretreated with a gap link opener in doses of 10-11 at 10-6 mol / kg i.v., the dose of CaCl2 was significantly higher before observing the av blockage. Another measure of the effect was the 30-65% increase in the absence time until the CaCl2-induced second degree av block was observed. CaCl2 induction of av blockade is described Ronsberg M, Saunders TK, Chan PS, Cervoni

P. Med Sci. 1986; 14: 350-51).

[0226] The variation in the degree of communication by gap links increased by the av blockade will normalize the av conductance and the normal sinusoidal rhythm will be restored.

[0227] The administration of gap link openers will be parenterally or orally.

Effects of gap link openers in the falls

[0228] Vertebrate eye lenses are a solid cyst of cells that grow during life by adding new cells on the surface. Older cells, buried by the newer generations, differ in long prismatic fibers, losing their cellular organelles and filling their cytoplasms with high concentrations of soluble proteins, the crystalline ones. The long-life lens fibers are interconnected by gap links, both with themselves and with an anterior layer of simple cuboidal epithelial cells on the lens surface. This gap network links the lens cells in a syncytium with respect to small molecules, allowing metabolic cooperation: intercellular diffusion of ions, metabolites and water. In contact with nutrients on the surface of the lens, epithelial cells retain their cellular organelles and are able to provide metabolic energy to maintain the correct concentrations of ions and metabolites in the cytoplasms of the lens fibers, so that the crystalline they remain in solution and are not added (cataracts). Three types of connexins are present in the lenses: Cx43, Cx46 and Cx50 and mutations in each of these gap junction proteins have been linked to cataracts [79-81]. These findings demonstrate that GJIC is essential for the normal metabolism and function of the lenses. Therefore, it is suggested that the substances of the present invention, which are known to increase GJIC, can be used in the prevention and / or treatment of cataracts. The gap link channels formed by the Cx46 and Cx50 connexins provide avenues for communication between the fiber cells in normal transparent lenses. Mice with a lack of a "knockout" gene devoid of these connexins develop nuclear cataracts that are associated with the proteolysis of crystalline lenses. These studies have established the importance of gap links in the normal maintenance of lens transparency through the provision of a cell-to-cell signaling mechanism or structural component for the correct organization of the lens membrane and cytoplasmic proteins (Gong et al., Cell 1997 Dec 12; 91 (6): 833-43). The increase in intracellular calcium concentration is a major stimulus for the activation of the calcium-dependent cysteine protease Lp82 which is a key initiator of the cataractgenesis process (Baruch et al., J Biol Chem 2001; 276 (31): 28999 -9006). To examine the ability of compounds 2 and 40 of the present invention to avoid cataracts, the effect of said compounds (10-10-10-6 mol / l) on the cultured sheep lens cell model described by Churchill is analyzed. et al. (J Cell Sci 1996; 109 (Pt 2): 355-65)). Briefly, cell-to-cell Ca2 + signaling in primary cultures of ovine epithelial cells is investigated using the Ca2 + fura-2 informed dye and fluorescence microscopy. Mechanical stimulation of a single cell with a micropipette initiates a propagated increase in cytosolic free Ca2 + that extends from the stimulated cell through 2-8 levels of surrounding cells. Compounds 2 and 40 of the invention are expected to increase cell-to-cell coupling between the lens fiber cells and avoid cataracts.

[0229] The mode of administration will be topical.

[0230] Thus, the present invention provides compounds for the preparation of medicaments useful in the prevention and / or treatment of cataracts.

Effects of gap link openers in ear diseases

[0231] Many different mutations of Cx32 have been observed in hereditary peripheral neuropathy-Charcot-Marie-Tooth syndrome linked to deafness X and several mutations of Cx26 and Cx31 have been detected in deafness [80]. Thus, it is suggested that the substances of the invention, which are known to increase GJIC, can be used in the prevention and / or treatment of certain types of deafness that are associated with a damaged GJIC in the ear. Thus, the present invention provides compounds for the preparation of medicaments useful in the prevention and / or treatment of deafness associated with a damaged GJIC.

Function of the gap link openers in the intestines

[0232] Both Cx43 and Cx45 are expressed in the wall of the small intestine [82]. It is believed that cells expressing Cx45 along the deep muscular plexus of the small intestine are likely to act as a constituent of a rhythm control system, which may include a conductive system, by forming a cellular network that acts at through specific types of gap links. In the intestine and colon, the Cajal interstitial cells (ICC) are pacemaker cells located between the intestinal smooth muscles; they generate spontaneous slow waves of smooth muscle layers and mediate neurotransmission. The three-dimensional cellular network of ICC is connected by Cx43 gap link between ICC and between ICC and smooth muscle cells [83]. In patients with Hirschsprung's disease, the lack of Cx43 expression in the aganglionic intestine suggests that damaged intercellular communication between CHF and smooth muscle cells may be partially responsible for motility dysfunction in this disorder [83]. Patients with Chaga disease (due to infection with the trypanosoma Cruzii protozoan) experience a marked reduction in CX expression that is considered responsible for the cardiomyopathy and severely dilated megacolon observed in these patients [7]. Thus, normal communication by gap links between ICC and between ICC and smooth muscle cells is considered essential for normal motility in the small intestine and colon. Therefore, a further objective of the invention is to provide a substance that increases the conductance by gap bonds in the intestine and, therefore, may be useful in the treatment of gastrointestinal motility disorders.

Reproductive organs and gap links

Ovaries

[0233] The gap links between granular cells and between oocytes and the surrounding granular cells play an important role during the development of the ovarian follicle. At birth, the ovary contains primordial follicles consisting of melotically arrested oocytes surrounded by a single layer of support cells (granulosa). Periodically, the subgroups of primordial follicles undergo further development during which the oocyte increases in size and the granular cells proliferate, stratify and develop a fluid-filled antrum. After ovulation, the oocytes restart meiosis and the granular cells retained in the follicle differentiate into steroidogenic cells, forming the corpus luteum.

[0234] The gap links directly connect adjacent cells that allow movement by diffusion of ions, metabolites and other potential signaling molecules important for the regulation of the ovarian cycle and female fertility. In support of the essential role of gap bonds in normal ovarian function, it has been shown that Cx37-deficient mice lack mature follicles (Graafian), fail to ovulate and develop numerous inappropriate lutea corpora lutea. In addition, it is possible to stop the development of oocytes before the meiotic competition. Thus, cell-to-cell signaling through intercellular channels critically regulates the highly coordinated group of cellular interactions required for satisfactory oogenesis and ovulation [84].

[0235] Follicle stimulating hormone (FSH) is the main regulator of ovarian follicle growth and development. Throughout its many actions in follicular maturation, FSH improves cell-to-cell coupling between granular cells and increases the expression of the Cx43 gene, and possibly the formation of new gap bonds. [85]. In contrast, luteinizing hormone (LH) disrupts cell-to-cell communication in the ovarian follicle, leading to a decrease in intra-oocyte cAMP concentrations after the resumption of meiosis [86].

[0236] These data illustrate that the presence of communication through normal gap links through Cx37 and Cx43 is essential for normal follicular growth and ovulation. Thus, it is likely that certain forms of female infertility are due to little cell-to-cell coupling in the ovaries. Therefore, a substance that increases cell-to-cell coupling can be used for the treatment of female infertility in women with impaired expression and / or regulation of the ovarian function of gap bonds. The compounds of the present invention that have the ability to increase GJIC are useful for the treatment of female infertility which is due to the little cell-to-cell coupling in the ovaries.

Uterus

[0237] The powerful synchronous contractions of the uterus in the part depend on the electrical coupling of the smooth muscle cells of the myometrium through gap bonds. In humans and other mammals, the gap links are scarce in the myometrium of the non-pregnant wombs, but they are abundant in pregnancy and / or with the onset of labor. The predominant gap bond protein expressed by myometrial smooth muscle cells in humans is Cx43, but Cx26, Cx40 and Cx45 have also been identified in the human myometrium [87; 88].

[0238] Due to the great importance of coordinated muscle concentrations during childbirth, it is an additional objective of the invention to provide a substance that increases cell-to-cell coupling in the myometrium that is expected to have a positive influence on the timing of muscle contractions and this substance can be used together with oxytocin for induction and facilitation of labor. Said objective is achieved with the present compounds.

[0239] Huldobro-Toro JP, Gonzalez R, Varas JA, Rahmer A, Gonzalez R. (Rev Med Chil 2001 Oct; 129 (10): 1105-12) assessed the existence of rhythm control mechanisms related to motor activity rhythmic blood vessels of human placenta, and found that blocking the gap bonds decreased the frequency and amplitude of spontaneous contractions. They concluded that rhythmic contractions in the circular layer of chorionic and umbilical vessels are triggered by pacemaker cells located in the circular layer of the smooth muscle of blood vessels and spread through gap bonds; They probably contribute to blood flow control. Thus, the present invention provides a substance that increases cell-to-cell coupling in placental blood vessels that is expected to have a positive influence on the blood circulation of the placenta and the development of the fetus.

Male reproductive organs

[0240] Cx43 is the most abundant connexin in the testicles and, notably, rat strains with a decreased level of Cx43 expression have a damaged spermatogenesis (ebo / ebo mice, jun-d - / -, Cx43 +), [89]. In addition, in initial studies it was suggested that hypospermic or aspermic patients have lower gap links in the testicles [90]. These data support the suggestion that lower cell-to-cell coupling in the testicles can lead to male infertility and, therefore, it is a further objective of the invention to provide a substance that increases cell-to-cell coupling and, thus, can be therapeutically useful in the treatment of male infertility associated with a damaged cell-to-cell coupling.

Function of the gap links in the pancreas

[0241] The gap junction channels made of Cx43 functionally attach to glucose sensitive cells of pancreatic islets and a rat insulinoma cell line [91]. In contrast, cells in several abnormally secreting insulin cell lines do not express Cx43, have few gap links, and are poorly coupled. After correction of these defects by stable transfection of Cx43 cDNA, cells expressing moderate levels of Cx43 and coupling, as seen in native beta cells, show an expression of the insulin gene and an insulin content that It is markedly elevated, compared to those observed in wild-type (uncoupled) cells and in transfected cells that overexpress Cx43. These findings indicate that adequate coupling of Cx43 is required for proper production and storage of insulin [91]. In addition, in vivo stimulation of insulin release by glibenclamide is associated with an increase in Cx43 expression and an increase in cell-to-cell coupling between neighboring β cells in the pancreatic islet [9].

[0242] To examine the effect of compound 2 and compound 40 on non-insulin dependent diabetes mellitus, db / db of 6-16 weeks of age are used. Animals are caged (3 mice / cage) under controlled ambient conditions (20 ° C, humidity 55-75%) after 12/12-h light / dark cycles with the light on at 6 am. They find a standard Altromin No. 1324 diet with free access to running water. All animals acclimatize for at least one week and are treated for two days before the first oral glucose tolerance test. In addition, to reduce stress-induced glucose oscillations, animals are subjected to at least one oral glucose tolerance test without compound as described below before the experiment.

[0243] The peptides are dissolved in 0.1 M phosphate buffered saline (PBS) with 0.1% bovine albumin, where the pH was adjusted to 7.4 by adding 5M NaOH. All solutions are prepared new in the morning immediately before the experiment. The compounds are administered parenterally in doses 10-10-10-6 mol / kg. Vehicle-treated animals are given PBS with 0.1% albumin alone.

[0244] Animals were fasted for 17 hours before the glucose tolerance test. Starting at 9.00 am, blood is drawn from the tip of the tail (t = -15 min) and blood glucose is measured. The overall blood glucose concentration (mM) is analyzed by the immobilized glucose oxidase method using a drop of blood (<5 ml, Elite Autoanalyser, Bayer, Denmark). Animals with a severely elevated blood glucose level in the morning of the experiment (> 10.5 mM) are excluded. Immediately after the initial blood sample, the animals receive an i.p. of vehicle or different doses of compound. Fifteen minutes after administration i.p. of the substance, p.o. or i.p. a dose of glucose 1 g / kg dissolved in water (200 ml / 50 g body weight) and the animals are returned to their cages (t = 0). Blood glucose levels are measured at t = 30 min, t = 60 min, t = 120 min and t = 240 min. Animals were fasted during the observation period.

[0245] In order to analyze the effects of the compounds on glucose tolerance, the absolute and relative difference in blood glucose from the baseline (t = 0) is calculated for each time point after glucose loading . The area under the curve (AUC) for the entire experiment (AUC0-240 min) is determined using the trapezoid method. Thus, two groups of values of AUC0-240 min are generated, one based on the absolute values of blood glucose (unit: mM x min) and one based on the relative changes of blood glucose (unit:% x min)

[0246] These observations indicate an important role of the coupling of gap bonds between the β cells of the pancreatic islet for the production and release of insulin. Thus, the present invention provides a substance that increases intercellular communication by gap links and / or the electrical conductance of gap links and, thus, improves glucose tolerance in subjects with non-insulin dependent diabetes mellitus.

[0247] In addition, Ito T, Ogoshi K, Nakano I, Ueda F, Sakai H, Kinjo M, Nawata H (Pancreas 1997 Oct 15: 297-303) found the effect of Irsogladin on the gap bonds in acute pancreatitis induced by Cerulein in rats. The ability for intercellular communication (HF) through gap links is found in normal pancreatic acinar cells and the role of HF in cerulein-induced acute pancreatitis (Cn) in rats using irsogladin, an enhancer of HF is investigated. through gap links. Acute edematous pancreatitis was induced in rats by two intraperitoneal injections of 40 micrograms / kg Cn. The rats orally received several doses (25, 50 or 100 mg / kg body weight) of irsogladin, 15 and 2 h before the first injection with cn. The normal control group received only the vehicle. The severity of pancreatitis was evaluated enzymatically and histologically 5 h after the first injection with Cn. In acute Cn-induced pancreatitis, irsogladin significantly decreased serum amylase level, wet pancreatic weight, and pancreatic amylase and DNA content, in a dose-dependent manner. Particularly, the amylase content improved to the level of normal controls. Histologically, the severity of pancreatitis was significantly reduced by treatment with irsogladin and no distinguishable vacuolization was observed in the group with an irsogladin treatment of 100 mg / kg. By immunofluorinating the pancreas with anti-connexin 32 antibody (Cx32; a gap binding protein), it was observed that pancreatic acini spread positively for Cx32 in the control group, but the number of positive granules in Cx32 decreased markedly , up to 19%, in the group with pancreatitis. With a treatment with irsogladin of 100 mg / kg, the number of Cx32 granules was recovered up to 70% of the normal control value. These findings indicate that IC through the gap link is damaged in Cn-induced pancreatitis, which may lead to the breakdown of tissue homeostasis and the progression of acute pancreatitis.

[0248] Thus, the peptides described herein are useful in the treatment of pancreatitis. To substantiate this statement, experiments can be performed on rats using the general experimental design of Ito T et al. 2001, prior, with the administration of COMPOUND 2 and COMPOUND 40 in concentrations in the range of 10-11

- 10-8 M to rats. These experiments are expected to show the facilitating effect of COMPOUND and COMPOUND 40 on the coupling of gap bonds and counteract the effect of cerulein.

[0249] Peptide administration will be intravenously.

Effects of gap bond openers (antiarrhythmic peptides) on thrombosis.

[0250] The antithrombotic activity of two peptides closely related to the substances of the present invention has been shown to have antithrombotic activity. Thus, Dikshit et al. [15] found that the Gly-Pro-Prp-Gly-Ala-Gly and Gly-Pro-Gly-Gly-Ala-Gly peptides prevented the development of a pulmonary embolism in mice administered with an i.v. of collagen and adrenaline. The patent. US 4,775,743 describes HP5, a peptide derived from AAP having the sequence N-3- (4-hydroxyphenyl) proplonyl-Pro-4Hyp-Gly-Ala-Gly-OH, and which is active against platelet agglutination. The compounds of the present invention have surprising similarity and are likely to have similar effects on thrombosis. Thus, the substances of the present invention can be used in the prevention of thrombosis.

Immunology

[0251] Cell-to-cell interactions are crucial for maturation and lymphocyte activation. A wide range of membrane molecules ensure intercellular adhesion and allow cell-cell signaling during cell migration and activation in the immune system. Circulating human T, B, and NK lymphocytes express Cx43 and active gap bonds between cells have been shown using dye methods as previously described. It has also been shown that the decrease in intercellular coupling by gap links significantly decreased the secretion of IgM, IgG and IgA indicating that intercellular signaling through gap links is an important component of the mechanisms underlying metabolic cooperation in the immune system (Oviedo-Orta E, Hoy T, Evans WH. (Immunology. 2000; 99: 578-90), Oviedo-Orta E, Gasque P, Evans WH. (FASEB. 2001; 15: 768-774)).

[0252] In subchronic or chronic inflammation, a local increase in the synthesis of immunoglobulins regardless of the etiology is desirable. During inflammation, the tissue is often different from normal healthy tissue and low oxygen tension results in the decoupling of intercellular communication by gap links (The importance of low oxygen for the decoupling of GJIC has been demonstrated in several different cellular systems suggesting that oxygen tension is a universal regulator of GJIC.

[0253] In primary cultures of ventricular cardiomyocytes of neonatal rats, oxygen and glucose deprivation leads to a decrease in norepinephrine-induced stimulation of the phosphoinositol (PI) turnover at approximately 50% of the level in normal atmospheric and nutritional conditions. It has been observed that the gap link modifier, COMPOUND 2, normalizes the alteration of norepinephrine-induced stimulation of PI turnover during oxygen and glucose deprivation by increasing the PI turnover up to approximately 90% of the normal level . Furthermore, it has been observed that COMPOUND 2 does not alter the level of noradrenaline-induced PI turnover during normal atmospheric and nutritional conditions (Meier, E and Beck, MM: ZS42-0123 enhances norepinephrine (NE) - induced phosphoinositol (PI) turnover in cultured cardiomyocytes during metabolic stress 2001 International Gap Junction Conference, August 4-9, 2001, Hawaii, USA, summary no. 132). Likewise, in cultured human osteoblast cultures and in osteoblastic cell lines of rat osteosarcoma, hypoxia decreased the spread of intracellular calcium waves measured as the transfer of dyes after injection with Lucifer Yellow. This decrease could be completely reversed by treatment with COMPOUND 2 (Teilmann, SC, Henriksen, Z, Meier, E, Petersen, JS, Sørensen, OH and Jørgensen, NR: The gap junction opener ZS42-0123 enhances intercellular communication in osteoblastic cells 2001 International Gap Junction Conference, Aug 4-9, 2001, Hawaii, USA, summary no. 176).

[0254] Due to cell decoupling during inflammation, a gap link opener will improve the synthesis of immunoglobulins during inflammation.

[0255] The in vitro test of the effect of gap bond openers after immunoglobulin synthesis will be analyzed in stimulated and non-stimulated T and B lymphocytes isolated from purified and human tonsils as described by Oviedo-Orta E, Gasque P, Evans WH. (FASEB. 2001; 15: 768-774)). Immunoglobulins will be measured by ELISA and gap links by FACS analysis. The gap link openers will be analyzed in concentrations of 10-10 to 10-7 M.

[0256] Pharmacological tests will be performed in vivo in experimental inflammatory models in non-infectious and infectious models. In vivo pharmacological tests can be carried out experimentally in a series of animal models: 1) inhibition of carragen-induced rat hind leg (leg volume), 2) attenuation of carrageenan-induced cell recruitment in an air pocket in rats (leukocyte recruitment and exudate volume), 3) attenuation of streptococcal cell wall-induced arthritis (SCW) in the tibiotarsian joint (ankle swelling) and 4) attenuation of the progression of collagen-induced arthritis in rats (clinical signs and joint swelling).

[0257] Ye P, Chapple CC, Kumar RK, and Hunter N (J Pathol 192: 58; 2000 Sep) have shown that there was a surprising reduction in connexins 26 and 43 in the epithelium of inflamed gum lining supporting the concept that the ability of the epithelium to act as an effective barrier against microbial products in tissues is severely compromised in periodontitis. Thus, the treatment of inflamed gums with a gap link opener, for example, in combination with an antibiotic, can be advantageous in the recovery of GJIC and healing of the epithelium.

Peripheral neuropathy and neuropathic pain

[0258] It has been described that neuropathy and peripheral pain as seen in diabetes, during dialysis, liver cirrhosis and many other pathologies involve somatic and autonomic nerves. The exact mechanisms of peripheral nerve injury in the various pathologies are still speculative, although the destruction of terminal nerves, reduced conductance, demyelination and increased inflammatory response have been described. Common elements for various pathologies in the experimental protocol are that an increase in free radicals, an increase in nitric oxide, stress with oxygen and lack of free radical scavengers is observed and a reduction in gap link communication is recorded (Pitre DA , Seifert JL, Bauer JA (Neurosci Lett. 2001; 303: 67-71), Bolanos JP, Medina JM. (J Neurochem. 1996; 66: 2019-9), Low PA, Nickander, KK. (Diabetes. 1991; 40: 873-7), Levy D, Hoke A, Zochone DW (Neurosci Lett. 1999; 260: 207-9), Bruzzone R, Ressot C. J Eur Neurosci. 1997; 9: 1-6)).

[0259] In vitro studies will be performed on astrocyte cultures or rat Schwann cells and gap gap openers, such as compound 2 and compound 40, will be analyzed in cells stressed with nitric oxide as described by Bolanos JP, Medina JM. (J Neurochem. 1996; 66: 2019-9) using sodium nitroprusside as a nitric oxide donor (Blasits S, Maune S, Santos-Sacchi J. (Phlugers Arch. 2000; 440: 710-12)). The concentrations of the compounds will be in the range of 10-10 and 10-7 M and the dose dependent gap opening will be measured using the FACS analysis.

[0260] The administration will be parenterally.

Auditory deficit

[0261] The noise-induced hearing loss, presbiacusia, known to be associated with the production of free radicals, is related to the inhibition of the coupling of gap bonds between Hensen cells and Corti organ Deiters cells in the cochlea (Todt I , Ngezahayo A, Ernst A, Kolb HA. (J Membrane Biol. 2001; 181: 107114), Blasits S, Maune S, Santos-Sacchl J. (Phiugers Arch. 2000; 440: 710-12) Lagostena L, Ashmore JF , Kachar B. (J Physiol. 2001; 531: 693-707)). The communication by gap links between these cochlear support cells provides important homeostasis for sensory cells and, thus, normal neuronal activity of external hair cells (Johnstone BM, Pantuzzi R, Syka J, Sykova E. (J Physiol 1989; 408: 77-92)). This communication is damaged during oxidative stress (Todt I, Ngezahayo A, Ernst A, Kolb HA. (J Membrane Biol. 2001; 181: 107-114). The loss of the acquired or age-dependent ear will be avoided when deal with a compound that can maintain communication through gap links in support cells.

[0262] In vitro tests of gap link openers in guinea pig Hensen cells will be performed as described by Todt I, Ngezahayo A, Ernst A, Kolb H-A. (J Membrane Biol. 2001; 181: 107-114). Compound 2 or compound 40 in the concentration range of 10-10-10-8 M will be investigated and its effects studied under conditions stressed by oxygen and mechanically stressed. The compounds will significantly antagonize the induced decoupling of gap bonds.

[0263] The rats that were given an infusion i.v. of compound 2 were subjected to tests of otoacoustic emissions per dispersion product (DPOAE). The ear was subjected to two tones at the same time of near sine wave frequency (f1 and f2). The sound emitted from the inner ear consists of distortion products produced by the outer hair cells. The strongest of these distortion products is usually at the frequency of 2f1-f2. For example, if the tones used are at a frequency of 1000 Hz (f1) and 1200 Hz (f2), the strongest distortion product will be at 2x1000 - 1200, or 800 Hz. The relative intensity of the distortion product, in comparison With the two sine waves, it can be used to assess the integrity of the cells of the outer hairs. kHz

[0264] The compound will be administered parenterally.

[0265] Melanocytes in the dark cell area of the vestibular organ communicate importantly through gap links and can play a role in the transport of material between endolymph and perlinfa and may also be important in maintaining the Homeostasis of the microenvironment in the inner ear (Masuda M, Usami SI, Yamazaki K, Takumi Y, Shinkawa H, Kurashima K. (Anat Rec. 2001; 262; 137-146)). Endolymphatic hydrops is related to several clinical conditions characterized by vertigo and a reduced ear. A lower capacity for communication through gap links may be important in the regulation of transmembrane transport of several substances originally secreted or excreted through specific types of transporters.

Anemia and age-dependent bone marrow transplant

[0266] The existence of functional gap links between hematopoietic progenitor cells and stromal cells of the hematopoietic microenvironment was controversial for many years, but some studies have now demonstrated the existence of a gap link communication in humans (Rosendaal M, Gregan A, Green C Tissue Cell. 1991; 23: 457-470), Dürig J, Rosenthal C, Halfmeyer K, Wiemann M, Novotny J, Bingmann D, Dührsen U, Schirrmacher K. (Brit J Haematol. 2000; 111: 416-25) ). It has also been shown that communication is bidirectional, favoring the hypothesis that stromal cells control the proliferative behavior of hematopoietic progenitor cells, but also their functional status may be regulated by immature hematopoietic cells (Gupta P, Blazar B, Gupta K, Verfaillie C. (Blood. 1998; 91: 3724-3733)).

[0267] With age, the functionality of hematopoietic tissue decreases and anemia is often observed in the elderly.

[0268] The reduced capacity of hematopoietic tissue is also observed in hematologic tumors and after chemotherapy treatment. The donor bone marrow transplant is used to prevent pancytopenia.

[0269] The effect of a compound that facilitates communication through gap bonds in pretreated rats exposed to high dose cyclophosphamide will be studied. In these animals, the bone marrow has stopped the production of mature hematopoietic cells. The number of reticulocytes at different time intervals after cyclophosphamide will be significantly higher in animals pretreated with the gap link opener, compound 2, which uses doses of approximately 100 microliters of compound 10-10-10 M at approximately 108 M compared With animals not pretreated.

[0270] The administration of the drug will be parenterally.

Pituitary and hypothalamic hypofunction

[0271] The hormones of the anterior pituitary gland show a circadian variation in the secretion of minutes, hours, days and seasons. The part of the nervous system responsible for most of the circadian rhythm is located in a pair of structures in the hypothalamus known as the suprachiasmatic nucleus. In this center, this biological clock is intrinsic in individual cells. However, the coordinated electrical activity is mediated by neighboring cells through the communication of gap links (Colwell CS. (J Neurobiol. 2000; 43: 37988)). Also because the anterior pituitary lacks direct innervations, cell-to-cell communication mediated by gap links in the gland must be indispensable for proper cell-to-cell coordination and the synchronization required to ensure proper and programmed hormonal secretion (Vitale ML, Cardin J, Gilula NB, Carbajal ME, Pelletier RM. (Biol Reporo. 2001; 64: 625-633)). Guerineau NC, Bonnefont X, Stoeckel L, Mollard

P. (J Biol Chem. 1998; 273: 10389-95) concluded that spontaneously active endocrine cells are individual units or are arranged in assemblies coupled by synchronized gap bonds dispersed throughout the anterior pituitary gland. Synchrony between spontaneously excitable cells can help shape basal secretion patterns. From the anterior pituitary gland, growth hormone, prolactin, adrenocorticoid hormone, thyroid hormone, and gonadotropin hormones are synthesized under the control of hypothalamus-stimulating hormones. Therefore, one of the mechanisms is complicated hypothalamic endocrine-pituitary gland dysrhythmia in one of the axes is also related to reduced communication through gap links. The diseases are diabetes insipidus, hypogonadotropic hypogonadism, myxoedema, adrenocorticoid hypofunction and dwarfism. Treatment with a gap link opener will improve symptoms.

[0272] In addition, neurons in the suprachiasmatic nucleus of the hypothalamus depend on the optimal communication of gap bonds. On the axis mentioned above, the gap link opener with mode of action in this region will also benefit patients with an altered circadian rhythm (Shinohara K, Funabashi T, Mitsishiba D, Kimura F. (Neusosci Lett. 2000; 286: 107 -10).

Renovascular hypertension and nephrotoxicity

[0273] The specific kidney and endothelial gap links are widely distributed in the kidney and are found in the glomerulus, tubule and vasculature, including intraglomerular capillaries and juxtaglomerular arterioles (Haefliger JA, Demotz S, Braissant O, Suter E. (Kidney Int. 2001 ; 60: 190-201)). In that study, the authors demonstrated the presence of gap bonds connecting the cells that secrete renin from the afferent arteriole. The role of the gap link could contribute to the detection and propagation of blood transmission signals, such as those caused by increased blood pressure. In the kidney, these signals need to be converted into autocrine, paracrine and endocrine stimuli by the afferent arteriole endothelial cells and transmitted to renin-secreting cells. Communication through gap links thus plays an important role in the formation of the interconnected juxtaglomerular apparatus. The rapid transitions from opening to closing of the gap link channels also imply a rapid response to local vascular changes ensuring the continuous feedback required to adapt the glomerular and tubular function, as well as renin secretion to physiological needs. Diseases characterized by renal communication by damaged gap links will benefit from treatment with a specific gap linker administered orally or parenterally. Past metals are nephrotoxic and cause kidney damage. It has been shown that toxic metals such as cadmium (Fukumoto M, Kujiraoka T, Hara M, Shibasaki T, Hosoya T, Yoshida M. (Life Sciences. 2001; 69: 247-54)), as well as mercury (Yoshida M Kujiraoka T, Hara M, Nakazawas H, Sumi Y. (Arch Toxicol. 1998; 72: 192-96)) in rat proximal tubule primary cell cultures decouple the gap links and both groups suggest that renal dysfunction is related with reduced intercellular communication.

[0274] The heavy metal poisoning treatment of a gap link opener will reduce tissue damage and prevent progressive tissue devastation.

[0275] The in vitro test will be performed on cell cultures of tubule cells and the prevention of the compounds (compound 2 or compound 40 at a concentration of approximately 10-10-10-7 M) from the decoupling of gap bonds will be investigated when be exposed to heavy metals. Communication by gap links will be analyzed with the Lucifer dye method described previously.

[0276] After systemic administration of heavy metals to experimental animals (rats), renal function was measured using 3H-insulin as a clearance marker for glomerular filtration rate, 14C-labeled tetraethylammonium as a clearance marker for flow in Renal and lithium plasma as a marker for proximal tubular function (Petersen JS, Schalmi M, Lam HR, Christensen S, J. Pharmacol. Esp. Ther. 1991, 258: 1-7) before and after different times of chronic treatment with heavy metals Chronic treatment with a specific gap link opener, such as Compound 2, will begin when renal function is compromised and a significant improvement in renal function parameters (glomerular filtration rate and blood pressure) will be observed after treatment.

[0277] Administration of the compound will be parenterally.

[0278] Non-infectious inflammation, as well as infections with different microbes, induces significant non-specific chronic changes in renal function also characterized by a reduced glomerular filtration rate, reduced excretion of electrolytes and water and changes in blood pressure. Some of these symptoms will also be treated with a specific gap link opener and the symptoms will decrease.

STABILITY

Stability of the compounds of the invention

[0279] In addition, the compounds of the present invention are characterized by being stable against enzymatic degradation, and / or being stable against plasma degradation and / or having an improved in vivo half-life. It is preferred that compounds that include antiarrhythmic compounds of the present invention be stable against enzymatic degradation and / or stable in plasma. The various derivatives and chemical modifications of the AAP native peptide sequence presented by the present invention, for example, C-terminal amidation or N-terminal modifications, represent modifications that are designed to increase stability while maintaining antiarrhythmic properties and / or native AAP antithrombotic.

[0280] Peptides normally degrade very easily by proteolytic enzymes present in the gastrointestinal system and living tissues and body fluids. Therefore, it is preferred in the present invention to use peptides that have been modified to transmit greater stability. It is preferred that the compounds that include the antiarrhythmic compounds of the present invention be stable against enzymatic degradation and / or stable in plasma. Preferred peptides for use in the method of the present invention have a solution half-life measured in a standard stability test of more than 50 minutes and preferably more than 4 hours. As is evident in the following tables 7 and 8, a series of peptides of the present invention have a degradation half-life of more than 5 hours in a standard stability test. Stability is an important parameter for the efficacy of the drug and a prolonged half-life, such as a T1 / 2 of more than 300 min, of the peptides of the present invention is preferred. A standard stability test as used herein refers to the in vitro plasma stability test described below.

METHOD OF STABILITY ANALYSIS IN PLASMA IN VITRO

[0281] The stability of the peptides is analyzed in serum and plasma. Peptides are incubated at 37oC in plasma or serum and samples taken at approximately 9 regular intervals between t = 0 and t = 156 min are analyzed by HPLC.

[0282] The appropriate conditions (column, solvent, gradient and temperature) for HPLC analyzes are estimated to ensure that the drug and plasma peaks do not have the same retention time. This is done by subsequent injections of the drug, plasma and a co-injection with the drug and plasma, followed by optimization of the LC method parameters until a satisfactory separation is obtained. Three experiments are performed in parallel for each type of plasma. 100 ml of peptide are mixed with 900 ml of plasma at t = 0 and incubated at 37 ° C (drug-plasma mixture at 0.1 mg / ml concentration). 100 ml samples of the drug-plasma mixture are extracted at the appropriate intervals and degradation is stopped by precipitation of the sample with 10 ml of MeCN: TFA 50:50 v / v. A control plasma sample without the drug is also taken in the same way. Plasma samples are centrifuged for 15 min at 12,000 rpm (Eppendorf centrifuge) at room temperature. The resulting supernatant solution is transferred to 300 ml HP autowatch vials and analyzed by HPLC. The samples are analyzed in the following order: blank, 0.1 mg / mL peptide, plasma without peptide, the three samples in parallel for t = 0, the three samples in parallel for t = 5 min., The three samples in parallel for t = 10 min. etc. Finally, the three samples are repeated in parallel for t = 0 to ensure there has been no degradation or other failure during the analysis. Sample concentrations (peak height in mAU) are plotted against time and fit a function that describes a monoexponential drop (Excel). The degradation half-life (T1 / 2) (min.) Of various compounds of the invention compared to AAP10, AAP and HP5 in human plasma are presented in the following Table 7 as average (n = 3)  standard deviation, Compounds 2, 3, 27, 48 and 49 are considerably more stable in plasma and serum than AAP10 that has a half-life of less than 10 minutes and HP5 that has a half-life of less than 12 minutes.

Table 7. Results of the in vitro stability test in plasma and serum, T1 / 2 in min and h

MEDIUM AND COMPOUNDS

PLASMA  HEPARINE SERUM

RAT

RABBIT HUMAN RABBIT HUMAN

AAP

4.4 min 12%  7.6 min 6%

AAP10

8.2 min  13% 9.5 min 12% - 2.7 min  4% -

HP5

3.7 min 1%  11.9 min 11%

Cycle (retro-AAP10-Asn)

- * BR> 5 h - - *> 5 h

Ac-retro (AAP-10) (allD) NH2

- *> 5 h *> 5 hrs - *> 5 h

CF3C (O) -AAP10NH2

- 3.8 h  0.5% - - 3.1 h  10%

Cycle (GAG-Hyp-PYN)

- 30 h  8% 9 h  1% - -

Cycle (GAG-Hyp-PYQ)

- 2 pm  2% 15 h  1% - -

H-D-Y-D-N-GNH2

- 296 min  34%

[0283] The following Table 8 shows the activity of the compounds in a calcium chloride-induced arrhythmia model and the half-life.

TABLE 8 CaCl2 mice, in vivo% CaCl2 mice, Assessment Half life in human plasma, min

HPP-5-OH 50 +/- 11 2 12

HPP-PHypGAGKKKKKK-OH 80 +/- 17 3 2

H-AAP-10-NH2 (H-GAG-4Hyp-76 +/- 21 3 13

PYNH2)

H-AAP-10-K6-OH 64 +/- 10 3 87 * Cycle (retro-AAP-10-Asn) 59 +/- 9 2> 300 Ac-Retro (AAP-10) - (all D) -65 +/- 7 3> 300 NH2 Ac-Retro (AAP-10) -OH 50 +/- 20 1 CF3C (O) -AAP10-NH2 48 +/- 13 3 240 HPP-PHypGAGKKKKKK-NH2 21 +/- 17 1> 300 [Pro4] AAP10-NH2 62 +/- 9 3 5 AAP 35 +/- 7 2 8 H- [D-Hyp4] AAP-10-NH2 28 +/- 10 1 H- [D-Pro4, Ala5] AAP-10-NH2 29 +/- 12 1 AAP-10-K6-NH2 33 +/- 17 2 H- (Acm) GAGHypPYC (Acm) -23 +/- 10 1 NH2 H-AAP-10-Asn-NH2 31 +/- 6 2 Cycle (GAGHypPYN) 57 +/- 8 2 780 Cycle (GAGHypPYQ) 48 +/- 14 2 900 H-HypPYNGAG-NH2 34 +/- 10 2 H-GAG-T4c-PY-NH2 32 +/- 6 2 H-GA-Sar-Hyp-PY-NH2 46 +/- 11 2 H-Sar-A-Sar-Hyp-PY-NH2 24 +/- 7 1 H-GAG-Pc-PY-NH2 21 +/- 11 1 H-GAGGPY-NH2 32 +/- 9 2 H-GAG-DHypAY-NH2 29 +/- 9 1 H-GAG-DHyp-DProY-NH2 49 +/- 6 2 des-Hyp4- [Asn5] AAP-10-NH2 53 +/- 15 2 7 AcGNY 46 +/- 9 2 2140 GNY 58 +/- 10 2 63 H-GANY-NH2 21 +/- 7 1 H-DY-DN-G-NH2 25 +/- 9 1 296 H-YNG-NH2 34 +/- 9 2 H-GGY-NH2 39 +/- 9 2 H-G-DN-Y-NH2 37 +/- 10 2 H-Y-DN-G-OH 39 +/- 9 2 Ac-Y-DN-G-OH 44 +/- 10 2 Ac-G-D-N-Y-NH2 19 +/- 8 1 Ac-Y-D-N-G-NH2 17 +/- 8 1 H-GK (DNP) Y-NH2 25 +/- 7 1 * Half-life in human plasma measured in plasma with EDTA, HPP refers to the radical 3- (4-hydroxyphenyl) propionyl

Table 9

Analysis of in vitro plasma stability of AAP10 and compound 2 under sterile conditions

The objective of the study is to estimate the in vitro half-life of the test drugs (peptides) in plasma of different species

The drugs are incubated under sterile conditions at 37oC in plasma of various species and the degradation of the drugs is followed. The samples are analyzed by HPLC.

Drug

Lot No. PM (g / mol) Peptide content Solvent

Bx 24.53 B

575.63 83.47 MQW

Sequence:

H-GAG-4Hyp-PY-NH2, H-AAP-10-NH2

Test average Study groups (n = 3 / group)

Rat plasma, heparin

Plasma conc.

Peptide Conc. Analysis time

90%

0.2 mM t = 0, 1, 2, 3, 5, 7, 10, 15 and 20 min

Test average Study groups (n = 3 / group)

Human plasma, heparin

Plasma conc.

Peptide Conc. Analysis time

90%

0.2 mM t = 0, 1, 2, 3, 5, 7, 10, 15 and 20 min

Drug

Lot No. Pm (g / mol) Peptide content Solvent

 Bx 20.17B-2A

617.66 100% MQW

Sequence:

Ac-D-Tyr-D-Pro-D-Hyp-Gly-D-Ala-Gly-NH2

Test average Study groups (n = 3 / group)

Rat plasma, heparin

Plasma conc.

Peptide Conc. Analysis time

90%

0.2 mM t = 0, 390, 1370, 1865, 2800, 3240, 4242, 4702 and 10007 min

Test average Study groups (n = 3 / group)

Human plasma, heparin

Plasma conc.

Peptide Conc. Analysis time

90%

0.2 mM t = 0, 390, 1368, 1863, 2798, 3238, 4240, 4700 and 10005 min

Method and

Method:

analysis

Appropriate conditions (column, solvent, gradient and temperature) for HPLC analyzes are estimated to ensure that the drug peak and plasma peaks do not have the same retention time. This is done by subsequent injections of the drug, plasma and a co-injection with the drug and plasma, followed by optimization of the LC method parameters until a satisfactory separation is obtained. Three experiments are performed in parallel for each type of plasma under sterile conditions. 100 µl of the test peptides (2 mM in MQW) will be mixed with 900 µl of plasma at t = 0 and incubated at 37oC (drug-plasma mixture at 0.2 mM concentration). 100 µl samples are extracted from the drug-plasma mixture at the appropriate intervals and degradation is stopped by precipitation of the sample with 10 µl of MeCN: TFA 50:50 v / v. A control plasma sample is also taken without the drug treated in the same way. Plasma samples are centrifuged for 15 min at 12,000 rpm (Eppendorf centrifuge) at room temperature. The resulting supernatant solution is transferred to 300 µl of HP autowatch vials and analyzed by HPLC. HPLC analysis is performed as follows: Detection: DAD1, 214.5 nm. Flow: 0.200 ml / min. Injection volume 10 l. 30oC temperature AAP10: Column: Vydac 218MS52, # 000517, 250 mm x 2.1 mm. Solvents; A: 0.1% TFA in MQW B. 0.1% TFA in MQW: MeCN 10:90 Gradient (time,% B): 0; 0 2; 0 14; 25 15; 100 16; 100 17; 0 30; 0 Method file: TJE_63A.M Sequence file: 010712T1 (HPLC 2) Compound 2 Column: Luna 3u, C18 (2), No. 296440, 150 x 2 mm. Solvents; A: 0.02% HFBA in MQW B. 0.02% HFBA in MQW: MeOH 10:90 Gradient (time,% B): 0; 0 5; 30 15; 30 16; 95 17; 95 18; 5 35; 5 Method file: TJE_123A.M Sequence file: 010712T2 (HPLC 2) The samples are analyzed in the following order: blank, the 0.2 mM drug, the plasma without the drug, the three samples in parallel for t = 0, the three samples in parallel for t1, the three samples in parallel for t2, etc. Finally, the three samples are repeated in parallel for t = 0 to ensure there has been no degradation or other failures during the analysis.

Calculations and

Plasma concentrations (peak height in mAU) will be plotted against time and

statistics

will fit a function that describes a monoexponential descent (Excel). The half-life of the test drug in the different types of plasma will be presented as mean + / standard deviation

Stability in plasma H-AAP10-NH2

3.8 min +/- 0.1 min (rat); 1.8 min +/- 1.0 min (human); incubation 20 min

Ac-Retro (AAP10) - (all D) NH2

10.3 days +/- 1.2 days (rat); 14.1 days +/- 1.5 days (human); 7 day incubation

FORMULATIONS AND COMPOSITIONS

[0284] Formulations containing a compound as described herein for the treatment of the diseases and medical conditions mentioned above may be in any suitable form that can be administered by medical personnel or by the patient as necessary. Examples are injection formulations for administration i.v., formulations for oral administration, including tablets and capsules and suppositories. The compounds of the present invention can be administered as an independent medicament.

or in a combination treatment with other medications suitable for the treatment of the particular disease. The compounds described herein are relatively low molecular weight peptides that may have a relatively low oral bioavailability, in which case non-oral formulations will be preferred, for example, formulations for administration by injection or for administration through the nasal or rectal epithelium or through the skin, for example, aided by iontophoresis.

[0285] In the therapeutic methods of the present invention, a compound for treatment can be administered to a subject in any of several ways including intracorporeally or topically.

[0286] A compound for treatment can be administered to a subject, either alone or in combination with one or more therapeutic agents, as a pharmaceutical composition in admixture with conventional excipients, that is, pharmaceutically acceptable organic or inorganic carrier substances suitable for the treatment. parenteral, enteral or intranasal application that do not react in a harmful way with the active compounds and are not harmful to the receptor thereof. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, saline solutions, alcohol, vegetable oils, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscose paraffin, perfume oil, monoglycerides and acid diglycerides fatty, esters of petroetral fatty acids, hydroxymethylcellulose, polyvinylpyrrolidone, etc. Pharmaceutical preparations can be sterilized and, if desired, mixed with auxiliary agents, for example, lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts to influence osmotic pressure, buffers, dyes, flavoring substances and / or aromatic substances and the like, which do not react detrimentally with the active compounds.

[0287] Such compositions may be prepared for use in parenteral administration, particularly in the form of liquid solutions or suspensions; for oral administration, particularly in the form of tablets or capsules; intranasally, particularly in the form of powders, nasal drops, or aerosols; vaginally; topically, for example, in the form of cream; rectally, for example, as a suppository; etc.

[0288] Pharmaceutical agents can be conveniently administered in unit dosage form and can be prepared by any of the methods known in the pharmaceutical sector, for example, as described in Remington's Pharmaceutical Sciences (Mack Pub. Co., Easton, PA, 1980). Formulations for parenteral administration may contain as usual excipients, sterile water or saline, polyalkylene glycols, such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes, and the like. In particular, lactide polymers, lactide / glycolide copolymer or biodegradable and biocompatible polyoxyethylene-polyoxypropylene copolymers may be useful excipients for controlling the release of certain compounds of the invention.

[0289] Other potentially useful parenteral release systems include ethylene vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems and liposomes. Formulations for administration by inhalation contain as excipients, for example, lactose, or they may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasally. Formulations for parenteral administration may also include glycocholate for oral administration, methoxysalicylate for rectal administration, or citric acid for vaginal administration. Other release systems will administer the therapeutic agent or agents directly to a surgical site, for example, administration through the use of stents.

[0290] The concentration of one or more treatment compounds in a therapeutic composition will vary depending on a number of factors, including the dose of the compound of the invention to be administered, the chemical characteristics (eg, hydrophobicity) of the composition used, and the mode and route of administration intended. One or more compounds of the invention may be arranged in an aqueous physiological buffer solution containing approximately 0.1 to 10% w / v of a compound for parenteral administration.

[0291] It will be understood that the really preferred amounts of active compounds used in a given therapy will vary according to, for example, the specific compound to be used, the specific composition formulated, the mode of administration and the characteristics of the subject, for example, the species , sex, weight, general health and age of the subject. The optimal administration rates for a given administration protocol can be easily determined by those skilled in the art using conventional dose determination tests performed with respect to the above guidelines. Suitable dose ranges may include from about 1mg / kg to about 100 mg / kg of body weight per day.

[0292] The therapeutic compounds of the present invention are suitably administered in a protonated and water soluble form, for example, as a pharmaceutically acceptable salt, usually an acid addition salt, such as an inorganic acid addition salt. , for example, a hydrochloride, sulfate, or phosphate salt, or as an organic acid addition salt, such as an acetate, maleate, fumarate or citrate salt. Pharmaceutically acceptable salts of therapeutic compounds of the present invention may also include metal salts, particularly alkali metal salts, such as a sodium salt or a potassium salt; alkaline earth metal salts, such as a magnesium or calcium salt; ammonium salts, such as an ammonium salt or teteramethylammonium; or an amino acid addition salt, such as a lysine, glycine or phenylalanine salt.

Compositions

[0293] The present invention also relates to a composition comprising a pharmacologically active antiarrhythmic peptide as defined in the present invention in combination with a pharmaceutically acceptable carrier and / or diluent. Such combinations may be in a form adapted to oral, subcutaneous, parenteral (intravenous, intraperitoneal), intramuscular, rectal, epidural, intratracheal, intranasal, dermal, vaginal, buccal, ocular, directly to the brain or lung administration, preferably in a form adapted to subcutaneous, intravenous or oral administration, and said compositions may be prepared in a manner known to the person skilled in the art, for example as generally described in "Remington's Pharmaceutical Sciences," 17. Ed. Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, PA, USA, 1985 and more recent and monographic editions in the series "Drugs and the Pharmaceutical Sciences", Marcel Dekker. The compositions may appear in conventional forms, for example, solutions and suspensions for injection, including IV infusion concentrates, capsules and tablets, preferably in the form of enteric formulations, for example, as described in US 5,350,741, for oral administration.

[0294] The pharmaceutical carrier or diluent used may be a conventional solid or liquid carrier. Examples of solid carriers are lactose, terra alba, sucrose, cyclodextrin, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid or lower alkyl ethers of cellulose. Examples of liquid carriers are syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene and water.

[0295] Similarly, the carrier or diluent may include any prolonged release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax.

[0296] If a solid carrier is used for oral administrations, the preparation may be compressed, placed in a hard gelatin capsule powder or in the form of a pellet or it may be in the form of a troccus or tablet. The amount of solid carrier will vary widely, but will usually be from about 25 mg to about 1

g.

[0297] A usual tablet that can be prepared by usual tabletting techniques may contain: Core: 100 mg of active compound (as free compound or a salt thereof); 1.5 mg colloidal silicon dioxide (Aerosil); 70 mg of microcrystalline cellulose. (Avicel); 7.5 mg of modified cellulose gum (Ac-Di-Sol); magnesium stearate.

[0298] Coating: HPMC approx. 9 mg; * Mywacett 9-40T approx. 0.9 mg; * acylated monoglyceride used as a plasticizer for film coating.

[0299] If a liquid carrier is used, the preparation may be in the form of a syrup, emulsion, soft gelatin capsule or sterile injectable liquid, such as a suspension or aqueous or non-aqueous liquid solution.

[0300] The composition may also be in a form suitable for local or systemic injection or infusion and, therefore, may be formulated with sterile water or an isotonic saline solution or a glucose solution. The compositions can be sterilized by conventional sterilization techniques that are well known in the art. The resulting aqueous solutions can be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with the sterile aqueous solution before administration. The composition may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, chloride. calcium, etc.

Formulation of peptides for intravenous injection

[0301] Multi-dose formulations can be prepared as a solution of a compound of the present invention in a sterile isotonic saline solution stored in capped vials and, if necessary, a preservative (eg, benzoates) is added. The fixed dose formulations can be prepared as a solution of the compound in sterile isotonic saline, stored in glass ampoules, and if necessary filled with inert gas. Each dose of the compound is stored dry in sealed ampoules or vials, and if necessary filled with an inert gas. The multi-dose formulation requires the highest degree of stability of the compound. When the stability of the compound is low, fixed dose formulations can be used. The peptide can also be formulated as a concentrate for infusion i.v.

[0302] For nasal administration, the preparation may contain a compound of the present invention dissolved

or suspended in a liquid carrier, in particular, an aqueous carrier, for aerosol application. The carrier vehicle may contain additives such as solubilizing agents, for example, propylene glycol, surfactants, such as salts of bile acids or ethers of higher polyoxyethylene alcohols, absorption enhancers such as lecithin (phosphatidylcholine) or cyclodextrin, or preservatives such as parabines .

[0303] In addition, the small size of the peptide compounds of the present invention may be an advantage for oral and nasal administration, since relatively rapid absorption through mucous membranes compared to larger peptides minimizes enzymatic degradation. , especially the duodenum and theium.

[0304] The present invention also relates to an antiarrhythmic peptide or peptide derivative or a pharmacologically active functional analog thereof as described in the present invention for use in therapy, and the use thereof as defined herein. for the manufacture of a pharmaceutical composition for use in therapy, for example, in the treatment of arrhythmias and thrombotic complications during cardiovascular disorders, such as acute ischemic heart disease (eg, stable angina pectoris, unstable angina pectoris, infarction of acute myocardium), congestive heart failure (for example, systolic, diastolic, hyperdynamic, hypodynamic or right or left side heart failure), congenital heart disease, cor pulmonale, cardiomyopathy, myocarditis, hypertensive heart disease and during coronary revascularization.

[0305] In specific embodiments, an antiarrhythmic peptide according to the present invention can be used to treat and / or prevent brady arrhythmias (eg, due to a sinus node disease, AV node, His bundle, or the right branch or left of the beam) and tachyarrhythmias associated with reentry (for example, premature atrial complexes, AV joint complexes, premature ventricular complexes, atrial fibrillation, atrial flutter, paroximal supraventricular tachycardia, sinus node reentry tachycardia, AV node reentry tachycardia , non-prolonged ventricular tachycardia, either alone or in combination with other antiarrhythmic compounds, such as class I agents (for example, lidocaine), class II agents (for example, metoprolol or propranolol), class III agents (for example, amiodarone or sotalol) or class IV agents (for example, verapamil).

[0306] In specific embodiments, an antiarrhythmic peptide according to the present invention can be used to prevent thrombotic events in patients with vessel wall diseases (eg, atherosclerosis), increased production of platelets (universal polycythemia), and / or a smaller flow (heart disease, vascular disease), either alone or in combination with GP IIb / IIIa inhibitors (for example, c7E3 Fab; abciximab), cyclooxygenase inhibitors (for example, aspirin), thromboxane A2 antagonists, derivatives coumadin (for example, warfarin), or the synthetic peptide, integrilin.

[0307] In specific embodiments, an antiarrhythmic peptide according to the present invention can be used, due to the effect on the intercellular channels of gap bonds, to treat and / or prevent bone loss and increased healing of bone fractures [93] ; treat and / or prevent disease in cartilage and poorly vascularized joints [94]; treat and / or prevent cataracts [81]; treat and / or prevent corneal vascularization in atological states with poor corneal nutrition and increased healing of corneal lesions [95]; treat and / or prevent the growth and expansion of cancer cells, such as cancer cells derived from epithelial cell lines [96]; treat and / or prevent hypertension by increasing vasomotion [74]; avoid rejection of implants, such as cells and organs, in an organism.

SYNTHESIS OF PEPTIDES

[0308] A preferred general procedure is described below. However, more detailed descriptions of solid phase peptide synthesis are found in WO 98/11125.

[0309] Apparatus and synthetic strategy

[0310] The peptides were synthesized in batches in a polyethylene container equipped with a polypropylene filter for filtration using 9-fluorenylmethyloxycarbonyl (Fmoc) as an N-α-amino protective group and suitable usual protection groups for side chain functions .

Solvents

[0311] The DMF solvent (N, N-dimethylformamide, Riedel de-Häen, Germany) was purified by passing it through a column filled with a strong cation exchange resin (strong acid Lewatit S 100 MB / H, Bayer AG Leverkusen , Germany) and free amines were analyzed before using 3,4-dihydro-3-hydroxy-4oxo-1,2,3-benzotriazine (Dhbt-OH) which gives a yellow color (anion Dhbt-O-) if free amines are present. The DCM solvent (dichloromethane, analytical grade, Riedel de-Häen, Germany) was used directly without purification. Acetonitrile (HPLC grade, Lab-Scan, Dublin, Ireland) was used directly without further purification.

Amino acids

[0312] Fmoc protected amino acids were purchased from Advanced ChemTech (ACT) in suitably protected forms in the side chain. Amino acids otherwise protected (Fmoc-Glu (OH) -OAlilo; Fmoc-Asp (OH) -OAlilo from NovaBiochem (Switzerland), Fmoc-4-Hyp (OtBu) -OH; from Bachem (Switzerland).

Coupling reagents

[0313] Diisopropylcarbodiimide coupling reagent (DIC) was purchased from (Riedel de-Häen, Germany), PyBop from Advanced ChemTech.

Linkers

[0314] (4-Hydroxymethylphenoxy) acetic acid (HMPA) was purchased from Novabiochem, Switzerland; and the resin was coupled as a preformed 1-hydroxybenzotriazole (HOBt) ester generated by DIC.

Solid supports

[0315] The peptides were synthesized according to the Fmoc strategy in TentaGel S 0.22-0.31 mmol / g resins (TentaGelSNH2; TentaGel S-Ram, TentaGel S RAM-Lys (Boc) Fmoc; Rapp polymere, Germany).

Catalysts and other reagents

[0316] Diisopropylethylamine (DIEA) was purchased from Aldrich, Germany, and ethylenediamine from Fluka, piperidine and pyridine from Riedel-de Häen, Frankfurt, Germany. 4- (N, N-dimethylamino) pyridine (DMAP) was purchased from Fluka, Switzerland and was used as a catalyst in coupling reactions involving symmetric anhydrides. Ethanditiol was purchased from Riedel-de Häen, Frankfurt, Germany. 3,4-Dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (Dhbt-OH), 1-hydroxybenzotriazole (HOBt) (HOAt) were obtained from Fluka, Switzerland.

Coupling Procedures

[0317] The first amino acid was coupled as a symmetric anhydride in DMF generated from the appropriate N-α protected amino acid and DIC. The following amino acids were coupled as in situ generated HOBt or HOAt esters produced from appropriate N-α-protected amino acids and HOBt or HOAt by DIC in DMF. The acylations were checked by the ninhydrin test performed at 80 ° C in order to avoid Fmoc deprotection during the test [97].

Deprotection of the N-α-amino protecting group (Fmoc).

[0318] Deprotection of the Fmoc group was carried out by treatment with 20% piperidine in DMF (1 x 5 and 1 x 10 min.), Followed by washing with DMF (5 x 15 ml, 5 min. Each) until no yellow color could be detected after the addition of Dhbt-OH to drained DMF.

Allyl deprotection

[0319] A solution of 3 eq. of Pd (PPh3) 4 dissolved in 15-20 ml of CHCl3, AcOH, NMM (37: 2: 1) to the peptide resin. The treatment continued for three hours at room temperature accompanied by the bubbling of a stream of N2 through the mixture.

HOBt ester coupling

[0320] 3 eq. of amino acid N-α-amino protected in DMF together with 3 eq. of HOBt and 3 eq. of DIC and then added to the resin.

Preformed symmetric anhydride

[0321] 6 eq. of amino acid N-α-amino protected in DCM and cooled to 0 ° C. DIC (3 eq.) Was added and the reaction continued for 10 min. The solvent was removed in vacuo and the rest was dissolved in DMF. The solution was immediately added to the resin, followed by 0.1 eq. of DMAP.

Cycle of the peptide in the resin

[0322] 1.5 eq. PyBop in DMF together with 1.5 eq. of HOBt and 3 eq. from NMM to the peptide resin. The reaction continued overnight.

Separation of the resin peptide with acid

[0323] The peptides were separated from the resins by treatment with 95% trifluoroacetic acid (TFA, Riedel-de Häen, Frankfurt, Germany) -water v / vo with 95% TFA and 5% ethandithiol temperature ambient for 2 h. The filtered resins were washed with 95% TFA-water and the filtrates and washes evaporated under reduced pressure. The residue was washed with ether and lyophilized from acetic acid-water. The lyophilized crude product was analyzed by high performance liquid chromatography (HPLC) and identified by electrospray ionization mass spectrometry (ESMS).

Synthesis of batch peptides in TentaGel resin (PEG-PS)

[0324] TentaGel resin (1g, 0.22-0.31 mmol / g) was placed in a polyethylene container equipped with a polypropylene filter for filtration. The resin was swollen in DMF (15 ml) and treated with 20% piperidine in DMF to ensure the presence of non-protonated amino groups in the resin. The resin was drained and washed with DMF until no yellow color could be detected after the addition of Dhbt-OH to the drained DMF. HMPA (3 eq.) Was coupled as a preformed HOBt ester as described above and the coupling continued for 24 hours. The resin was drained and washed with DMF (5 x 5 ml, 5 min each) and the acylation was checked by the ninhydrin test. The first amino acid was coupled as a preformed asymmetric anhydride as described above. The following amino acids according to the sequence were coupled as preformed HOBt esters protected with Fmoc (3 eq.) As described above. Couplings continued for 2 hours, unless otherwise specified. The resin was drained and washed with DMF (5 x 15 ml, 5 min each) in order to remove excess reagent. All acylations were checked by the ninhydrin test performed at 80 ° C. After completing the synthesis, the peptide-resin was washed with DMF (3 x 15 ml, 5 min each), DCM (3 x 15 ml, 1 min each) and finally diethyl ether (3x15 ml, 1 min each ) and dried under vacuum.

HPLC conditions

[0325] Gradient HPLC analysis was performed using a Hewlett Packard HP 1100 HPLC system consisting of an HP 1100 Quaternary Pump, an HP 1100 Autosampler, an HP 1100 Column Thermostat and an HP 1100 Multiple Wavelength Detector. The Hewlett Packard Chemstation for LC software (rev. A.06.01) was used for instrument control and data collection.

[0326] The following columns and HPLC buffer systems were used: Column Kromasil, Phenomenex 00F-3033-E0, 329889 (new); 5 Cm C-18, 100Å 150 x 4.6 mm; Lot nr. 5243-10. Buffer system: A: 0.1% TFA in MQV; B: 0.085% TFA, 10% MQV, 90% MeCN. Gradient:

1-1.5 min. 25% B 1.5-13.5 min 25-50% B 13.5-14.5 min 50-100% B 14.5-15.5 min 100% B 15.5-17.5 min 100 - 25% B 17.5-20 min 25% B Flow 1.5 ml / min Oven temperature 40 ° C UV detection: λ = 215 nm Mass spectrum obtained in a Micro-mass LCT instrument.

[0327] Returning to the present invention, it is generally applicable for the treatment or prevention of diseases associated with decreased or damaged intercellular communication. Intercellular communication by gap links (GJIC) is vital for the normal functioning of mammalian cells and tissues, and the closure or "gating" of the gap links is often correlated with pathological conditions. Several examples of intercellular communication by gap links associated with pathological conditions have been described in the literature. Although the substances that block the gap bonds are known, descriptions about the use of compounds that facilitate or mediate communication by gap links or increase GJIC in the treatment of non-proliferative diseases are limited to the use of the compound irsogladin (6- (2 , 5-dichlorophenyl) -2,4-diamino-1,3,5-triazine) which has been described that activates intercellular communication by gap bonds through the M1 acetylcholine muscarinic receptor, where GJIC has been inhibited, but the Irsogladin 10-10 to 10-6 alone did not affect GJIC (Ueda, F. et al. J Pharmacol Exp Ther 1995 Aug; 274 (2): 815-9).

Peptide synthesis of individual peptides

Synthesis Example 1. Peptide synthesis of Ac-Tyr-Pro-4Hyp-Gly-Ala-Gly-OH (Compound 1) in TentaGel-S-NH-2; Rapp polymere, Germany.

[0328] First batch: TentaGel-S-NH2 dry (0.27 mmol / g, 1 g) was placed in a polyethylene container equipped with a polypropylene filter for filtration and treated as described in "synthesis of peptides in batches in TentaGel resin "until the end of the N-terminal tyrosine coupling. All links continued overnight. After deprotection of the Fmoc group the N-terminal amino group was acetylated with acetic acid anhydride (1 ml, 10.5 mmol) together with 100 µl of pyridine dissolved in 2 ml of DMF. The coupling continued overnight. The acylations were checked by the ninhydrin test performed at 80 ° C as described above. After completing the synthesis, the peptide-resin was washed with DMF (3 x 15 ml, 1 min each), DCM (3 x 15 ml, 1 min each), diethyl ether (3 x 15 ml, 1 min each one) and dried under vacuum.

[0329] The peptide was separated from the resin as described above and lyophilized from acetic acid. The lyophilized crude product was analyzed by HPLC and it was found that the purity was greater than 70% and the identity of the peptide was confirmed by ES-MS (MH + found 619.24, MH + calculated 619.26). The yield of the crude material was 137.7 mg. After purification using preparative HPLC as described above, 58 mg of peptide product with a purity greater than 95% was collected. The total yield of the purified peptide product was 35%.

[0330] Second batch: TentaGel-S-NH-2 dry (0.27 mmol / g, 1 g) was placed in a polyethylene container equipped with a polypropylene filter for filtration and treated as described in "synthesis of batch peptides in TentaGel resin" until the end of the N-terminal tyrosine coupling. All links continued overnight. After deprotection of the Fmoc group, the N-terminal amino group was acetylated with acetic acid anhydride (1 ml, 10.5 mmol) together with 100 µl of pyridine dissolved in 2 ml of DMF. The coupling continued overnight. The acylations were checked by the ninhydrin test performed at 80 ° C as described above. After completing the synthesis, the peptide-resin was washed with DMF (3 x 15 ml, 1 min each), DCM (3 x 15 ml, 1 min each), diethyl ether (3 x 15 ml, 1 min each one) and dried under vacuum.

[0331] The peptide was separated from the resin as described above and lyophilized from acetic acid. The lyophilized crude product was analyzed by HPLC and it was found that the purity was greater than 70% and the identity of the peptide was confirmed by ES-MS (MH + found 619 25, MH + calculated 619.26). The yield of the raw material is 137.3 mg. After purification using preparative HPLC as described above, 27.9 mg of peptide product with a purity greater than 91% was collected. The total yield of the purified peptide product was 15.5%.

Synthesis Example 2. Peptide synthesis of Ac-D-Tyr-D-Pro-D-4Hyp-Gly-D-Ala-Gly-NH2 (Compound 2) in TentaGelS-Ram; Rapp polymere, Germany

[0332] First batch: Dry TentaGel-S-Ram (0.23 mmol / g, 1 g) was placed in a polyethylene container equipped with a polypropylene filter for filtration and treated as described in "synthesis of peptides in batches in TentaGel resin "until the end of the coupling of the N-terminal D-tyrosine. All links continued overnight. After deprotection of the Fmoc group, the N-terminal amino group was acetylated with acetic acid anhydride (1 ml, 10.5 mmol) together with 100 µl of pyridine dissolved in 2 ml of DMF. The coupling continued overnight. The acylations were checked by the ninhydrin test performed at 80 ° C as described above. After completing the synthesis, the peptide-resin was washed with DMF (3 x 15 ml, 1 min each), DCM (3 x 15 ml, 1 min each), diethyl ether (3 x 15 ml, 1 min each one) and dried under vacuum.

[0333] The peptide was separated from the resin as described above in lyophilized form of acetic acid. The yield of lyophilized crude product was 119.7 mg. The identity of the peptide was confirmed by ES-MS (MH + found 618.25, MH + calculated 618.28). After purification using preparative HPLC as described above, 42 mg of peptide product was collected with a purity greater than 95%. The total yield of the purified peptide product was 30%.

[0334] Second batch: Dry TentaGel-S-Ram (0.23 mmol / g, 1 g) was placed in a polyethylene container equipped with a polypropylene filter for filtration and treated as described in "synthesis of peptides in batches in TentaGel resin "until the end of the coupling of the N-terminal D-tyrosine. All links continued overnight. After deprotection of the Fmoc group, the N-terminal amino group was acetylated with acetic acid anhydride (1 ml, 10.5 mmol) together with 100 µl of pyridine dissolved in 2 ml of DMF. The coupling continued overnight. The acylations were checked by the ninhydrin test performed at 80 ° C as described above. After completing the synthesis, the peptide-resin was washed with DMF (3 x 15 ml, 1 min each), DCM (3 x 15 ml, 1 min each), diethyl ether (3 x 15 ml, 1 min each one) and dried under vacuum.

[0335] The peptide was separated from the resin as described above and lyophilized from acetic acid. The yield of lyophilized crude product was 119.7 mg. The identity of the peptide was confirmed by ES-MS (MH + found 618.29, MH + calculated 618.28). After purification using preparative HPLC as described above, 100 mg of peptide product with a purity greater than 99% was collected. The total yield of the purified peptide product was 71%.

Example 3: Solid phase synthesis of Ac-Asn-Tyr-NH2 (Compound 56), Ac-Gly-Tyr-NH2 (Compound 57), Hydroxyacetyl-Asn-Tyr-NH2 (Compound 58).

[0336] PCT application WO 01/98324 entitled Conjugates of new peptides from Larsen, B.D et al describe general methods of solid phase synthesis.

[0337] Peptide synthesis of Ac-Asn-Tyr-NH2 (Compound 56) in TentaGel-S-Ram; Rapp polymere, Germany.

[0338] TentaGel-S-Ram (0.23 mmol / g, 1 g) dried was placed in a polyethylene container equipped with a polypropylene filter for filtration and treated as described in "peptide synthesis by TentaGel resin batches "until final coupling of the N-terminal asparagine. After deprotection of the Fmoc group the N-terminal amino group was acetylated with acetic acid anhydride (1 ml, 10.5 mmol) together with 100 ml of pyridine dissolved in 2 ml of DMF. The coupling continued overnight. Acylations were checked by the ninhydrin test performed at 80oC as described above. After acylation of the N-terminal amino group, the peptide-resin was washed with DMF (3 x 15 ml, 1 min each), DCM (3 x 15 ml, 1 min each), diethyl ether (3 x 15 ml, 1 min each) and dried under vacuum.

[0339] The peptide was separated from the resin as described above and lyophilized from acetic acid.

[0340] Peptide synthesis of Ac-Gly-Tyr-NH2 (Compound 57) in TentaGel-S-Ram; Rapp polymere, Germany.

[0341] TentaGel-S-Ram (0.23 mmol / g, 1 g) dried was placed in a polyethylene container equipped with a polypropylene filter for filtration and treated as described in "peptide synthesis by TentaGel resin batches "until final coupling of the N-terminal glycine. After deprotection of the Fmoc group the N-terminal amino group was acetylated with acetic acid anhydride (1 ml, 10.5 mmol) together with 100 ml of pyridine dissolved in 2 ml of DMF. The coupling continued overnight. Acylations were checked by the ninhydrin test performed at 80oC as described above. After acylation of the amino group

N-terminal, the peptide-resin was washed with DMF (3 x 15 ml, 1 min each), DCM (3 x 15 ml, 1 min each), diethyl ether (3 x 15 ml, 1 min each) and dried in vacuo.

[0342] The peptide was separated from the resin as described above and lyophilized from acetic acid.

[0343] Peptide synthesis of Hydroxyacetyl-Asn-Tyr-NH2 (Compound 58) in TentaGel-S-Ram; Rapp polymere, Germany.

[0344] TentaGel-S-Ram (0.23 mmol / g, 1 g) dried was placed in a polyethylene container equipped with a filter Polypropylene for filtration and treated as described in "synthesis of resin batch peptides TentaGel "until the coupling of the N-terminal asparagine is finished. After the Fmoc group is checked out, the N-terminal amino group was acetylated with hydroxyacetic acid using the standard coupling procedure. previously described. The coupling continued overnight. Acylations were checked by the test of ninhydrin made at 80oC as described above. After acylation of the amino group N- terminal, the peptide-resin was washed with DMF (3 x 15 ml, 1 min each), DCM (3 x 15 ml, 1 min each), diethyl ether (3 x 15 ml, 1 min each) and dried under vacuum.

[0345] The peptide was separated from the resin as described above and lyophilized from acetic acid.

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Claims (34)

one.

Compound which is acetyl-Asn-Tyr-NH2, hydroxyacetyl-Asn-Tyr-OH / NH2, or hydroxyacetyl-Gly-Tyr-OH; or a salt thereof.

2.

 Compound according to claim 1, which is hydroxyacetyl-Asn-Tyr-NH2, or a salt thereof.

3.

 Compound according to claim 1 or claim 2, wherein the salt is an acid addition salt or a basic salt.

Four.

 Pharmaceutical composition comprising a compound according to any one of claims 1 to 3, or a salt thereof, and a pharmaceutically acceptable carrier or diluent.

5.

 Pharmaceutical composition according to claim 4 which is selected from the group consisting of an enteric tablet and a preparation for injection.

6.

 Compound according to any one of claims 1 to 3 for use in therapy.

7.

 Use of a compound according to any one of claims 1 to 3 for the preparation of a medicament for the treatment of a disease characterized by a damaged intercellular communication by gap junctions (GJIC).

8.

 Use according to claim 7, wherein the medicament is for the treatment of arrhythmia.

9.

 Use according to claim 8, wherein the arrhythmia is a re-entry arrhythmia of atrial or ventricular origin, including alternating repolarization arrhythmia where both supraventricular and ventricular tachyarrhythmias can occur as tachycardia, flutter or fibrillation.

10.

 Use of a compound according to any one of claims 1 to 3 for the preparation of a medicament for the prevention and / or treatment of a slowed conduction in the heart.

eleven.

 Use of a compound according to any one of claims 1 to 3 for the preparation of a medicament for the improvement of contractility of the heart.

12.

 Use of a compound according to any one of claims 1 to 3 for the preparation of a medicament for the treatment of pathological conditions associated with an intercellular communication by gap links (GJIC) damaged during metabolic stress, including the deprivation of glucose and oxygen.

13.

 Use of a compound according to any one of claims 1 to 3 for the preparation of a medicament for antithrombotic treatment.

14.

 Use of a compound according to any one of claims 1 to 3 for the preparation of a medicament useful in the prevention and / or treatment of osteoporosis.

fifteen.

 Use of a compound according to any one of claims 1 to 3 for the preparation of a medicament useful in the prevention and / or treatment of joint diseases including arthritis.

16.

 Use of a compound according to any one of claims 1 to 3 for the preparation of a medicament useful in the prevention and / or treatment of a disease in cartilage and poorly vascularized joints including arthritis.

17.

 Use of a compound according to any one of claims 1 to 3 for the preparation of a medicament useful in the prevention and / or treatment of bone loss and the increase in healing of bone fractures.

18.

 Use of a compound according to any one of claims 1 to 3 for the preparation of a medicament useful in the prevention and / or treatment of corneal vascularization is pathological conditions with poor corneal nutrition and to increase the healing of lesions. in the cornea

19.

 Use of a compound according to any one of claims 1 to 3 for the preparation of a medicament useful in the treatment of wounds and / or ischemic ulcers.

twenty.

 Use of a compound according to any one of claims 1 to 3 for the preparation of a medicament useful in the treatment of gastric ulcers and duodenal ulcers.

twenty-one.

 Use of a compound according to any one of claims 1 to 3 that increases the coupling of gap and / or GJIC bonds in the vascular wall for the preparation of a medicament for the prevention and / or treatment of hypertension.

22

 Use of a compound according to any one of claims 1 to 3 for the preparation of medicaments useful in the prevention of ischemic damage in the brain and for the treatment of organic psychoses.

2. 3.

 Use according to claim 22, wherein the organic psychoses are depression, anxiety, learning and memory deficits, phobias or hallucinations.

24.

 Use of a compound according to any of claims 1 to 3 for the preparation of a medicament useful in the prevention and / or treatment of cataracts.

25.

 Use of a compound according to any one of claims 1 to 3 for the preparation of a medicament useful in the prevention and / or treatment of deafness associated with a damaged intercellular communication by gap gap (GJIC).

26.

 Use of a compound according to any one of claims 1 to 3 for the preparation of a medicament useful in the prevention and / or treatment of gastrointestinal motility disorders.

27.

 Use of a compound according to any one of claims 1 to 3 for the preparation of a medicament useful in the treatment of female infertility that is due to a poor cell-to-cell coupling in the ovaries.

28.

 Use of a compound according to any one of claims 1 to 3 for the preparation of a medicament useful together with oxytocin for induction and facilitation of labor.

29.

 Use of a compound according to any one of claims 1 to 3 for the preparation of a medicament useful in the treatment of male infertility associated with an altered cell-to-cell coupling.

30

 Use of a compound according to any one of claims 1 to 3 for the preparation of a medicament useful in improving glucose tolerance in a subject with non-insulin dependent diabetes mellitus due to a damaged GJIC between β cells.

31.

 Use of a compound according to any one of claims 1 to 3 for the preparation of a medicament for the treatment of cancer in tissue of endodermal, mesodermal or ectodermal origin, carcinomas, hepatocellular or cholangiocellular neoplasms or bone cancer.

32

 Use of a compound according to any one of claims 1 to 3 for the preparation of a medicament for the treatment of wounds or lesions on the skin or oral mucosa.

33.

 Use of a compound according to any one of claims 1 to 3 for the preparation of a medicament for the treatment or prevention of the growth or expansion of cancer cells.

3. 4.

 Use of a compound according to any one of claims 1 to 3 for the preparation of a medicament for the treatment of pancreatitis.