Novel antiarrhythmic peptides

ES 2 228 807 T3

DESCRIPTION

New antiarrhythmic peptides.

The present invention relates to new peptides, including new antiarrhythmic peptides of linear or cyclic structure, exhibiting improved stability in vitro and / or in vivo, to compositions comprising said peptides, and to the use of said peptides for the preparation of medicaments. .

Background of the invention

Sudden death due to cardiac arrhythmias is one of the leading causes of death in the Western world. The most common disease, responsible for sudden death, is ischemic heart disease, but inherited diseases such as hypertrophic cardiomyopathy and long QT syndrome are also important in younger patients.

Cardiac arrhythmias can arise from abnormalities in impulse formation, impulse conduction, or a combination of both. The regulation of impulse formation and conduction involves a complex interaction between the autonomic nervous system, cardiac ion channels, and cardiac cleft junctions.

The results of pharmacological prevention, especially of ischemia-induced arrhythmias, have been disappointing. Thus, clinical trials have shown that various class I and class III antiarrhythmic drugs increase mortality in patients with ischemic heart disease (1). A common characteristic of all currently used antiarrhythmics is that they interfere with cardiac ion channels (sodium, potassium, and calcium channels), or with the autonomic nervous system, thus interfering with the generation of the action potential. This is probably the reason why they not only produce an antiarrhythmic action, but also a pro-arrhythmic effect, with the potential to induce lethal arrhythmias, especially in patients with reduced left ventricular function, congestive heart failure or a history of sustained ventricular tachy-arrhythmia. Examples of antiarrhythmic drugs are flecainide, encainide, moricizine, and quinidine. Antiarrhythmic drugs that prolong cardiac repolarization, such as amiodarone and sotalol, are associated with the potential development of a striking and specific arrhythmia, torsades de pointes. Torsades, a very fast ventricular arrhythmia, probably occurs when a set of associated features: hypokalemia, bradycardia, and possibly delayed conduction, alter membrane stability, causing oscillations. Amiodarone, like sotalol, is indicated only for life-threatening arrhythmias. The drug blocks sodium channels and, to some extent, calcium channels, and also has beta-blocking effects. In early trials, side effects (which are dose related) led to drug discontinuation in up to 20% of patients within a year. Cardiac toxicities include sinus bradycardia, atrioventricular block, congestive heart failure, and ventricular arrhythmias.

In summary, currently available antiarrhythmic drugs have not been able to prevent sudden death caused by cardiac arrhythmias. Therefore, there is a great and unmet need for new safe and effective antiarrhythmic drugs for the treatment of life-threatening arrhythmias. Due to the serious side effects that limit the use of current antiarrhythmic drugs, a new class of antiarrhythmic drugs is desirable, with a completely different mechanism of action. As mentioned above, the regulation of impulse formation and conduction is a complex interaction between the autonomic nervous system, cardiac ion channels, and cardiac cleft junctions. To date, antiarrhythmic development has focused on the autonomic nervous system and cardiac ion channels, and no currently available drug works by opening cardiac cleft junctions. However, recently, various lines of research have revealed the important role of cleft junctions in the development of arrhythmias, which is why modulation of cleft junctions represents a very interesting new target in the treatment of arrhythmias.

Cleft junctions are specialized regions of the cell membrane with clusters of hundreds to thousands of tightly packed cleft junction channels that directly connect the cytoplasmic compartment of two adjacent cells. The gap junction channels are composed of two hemichannels (connexons) provided by each of the two adjacent cells. Each connexon consists of six proteins called connexins. Connexins are a broad family of proteins, all of which share the basic structure of four transmembranous domains, two extracellular loops, and a cytoplasmic loop. There is a high degree of conservation of extracellular loops and transmembranous domains among species and isoforms of connexins. However, the length of the C-terminus varies considerably, leading to the classification of connexins on the basis of molecular weight. The distribution of the different types of connexins (Cx) varies throughout the heart. The Cx43 isoform is the predominant type in the ventricles, while Cx40 is the most abundant isoform in the atria and conduction system. The slit bond channel can vary between an open and a closed state by twisting motion. In the open state, ions and small molecules smaller than about 1000 D can pass through the pore. The conduction of the electrical impulse takes place through the gap junctions and, therefore, normal functioning gap junctions are a prerequisite for normal conduction and therefore a normal heart rate.

The development of "knockout" mice, free of different types of connexins, has provided a growing understanding of the important role of cleft junctions in abnormal conduction. From these studies,

ES 2 228 807 T3 mice homozygous for a targeted deletion of the Cx43 gene have been shown to die shortly after birth from cardiac and pulmonary malformations, whereas heterozygous mice survive. However, the heterozygous genotype exhibits significantly slowed conduction compared to wild-type mice (2). In adult mice (6-9 months of age), ventricular epicardial conduction of beat markets is slowed by 44% and the QRS complexes of ECG recordings are significantly prolonged, compared to wild-type mice. The reduced expression of Cx43 is directly related to an increased incidence of ventricular arrhythmias during ischemia in mice heterozygous for the deletion of the Cx43 gene (3). Thus, the incidence of spontaneous ventricular tachycardia after induction of regional ischemia in pre-fused isolated hearts from heterozygous mice is twice that of wild-type hearts. Additionally, mice with a cardiospecific loss of Cx43 develop spontaneous ventricular arrhythmias and sudden cardiac death, with 100% mortality by two months of age. The deletion of the Cx43 gene is not fatal, although atrial, atrioventricular and His-Purkinje conduction is significantly slower in Cx40 - / - mice compared to Cx40 + / + mice, with Cx40 - / - mice exhibiting a increased risk of arrhythmias and bundle branch block (4-6).

The relationship between connexin abnormalities and heart disease in humans has also been established. An example is Chagas disease, caused by the protozoan parasite Trypanosoma cruzi. This disease is an important cause of cardiac dysfunction in Latin America. An altered distribution of Cx43 has been observed in cells infected with Trypanosoma cruzi and this alteration may be involved in the genesis of the conduction disorders characterized by the disease (7). Several studies on the expression and distribution of Cx43 in chronically ischemic, hibernating or hypertrophic hearts also describe a reduced degree of Cx43 expression and a modified pattern of distribution (8-10). In fact, the expression and / or distribution of the connexins have been altered in all the pathological states of the hearts investigated to date.

In summary, there are multiple evidences that relate the malfunction or the absence of cleft junctions with an increased risk of arrhythmias, and multiple evidences that show an altered expression / distribution of connexins in chronic heart disease. As mentioned above, no currently available antiarrhythmic drug works by increasing cleft junction function. However, a group of peptides (antiarrhythmic peptides) capable of increasing the conductance of cleft junction has been described in the past.

Antiarrhythmic peptides

In 1980, Aonuma et al. (11) isolated a hexapeptide with a molecular weight of 470 D from the bovine atrium. In newborn rat cardiomyocytes, it was shown that 0.1 pg / ml of this peptide could convert fibrillation induced by uabain, high calcium (3 mM) or low potassium (0.7 mM) levels into a normal rhythm. Furthermore, 2.5-5.0 pg / ml of this peptide could convert the arrhythmic movement of isolated rat atria, induced by the combination of low levels of potassium (0.3 mM) and acetylcholine in normal rhythm. Thus, this peptide was named antiarrhythmic peptide (AAP) (Comparative Example 1 below (CE1)). When added to a cell culture medium, AAP increased the number of beat centers, the relative content of disseminating cells, and protein synthesis (12). In 1982, the amino acid sequence of AAP was determined as H-Gly-Pro-4Hyp-Gly-Ala-Gly-OH (13). In subsequent in vivo studies, the antiarrhythmic effect of AAP observed in vitro was confirmed. AAP, 10 mg / kg, was shown to be effective against CaCl-induced arrhythmia₂-, uabain and acotinin in the mouse (14). Several synthetic derivatives of AAP have been tested, which have been shown to be more potent than endogenous AAP against experimentally induced arrhythmias in mice and rats (15-17). The most extensively investigated synthetic derivative is AAP10 (H-GlyAla-Gly-4Hyp-Pro-Tyr-NH2) (Comparative Example 2, below (CE2)). In the isolated and pre-fused rabbit heart, 0.1 nmol / l to 10 nmol / l of this peptide reduced the dispersion of the activation-recovery intervals measured in 256 ventricular epicardial electrodes under normal conditions (18). AAP10 has no effect on mean action potential duration, left ventricular end diastolic pressure, coronary flow, QRS duration, or PQ interval. If the hearts were subjected to regional ischemia due to occlusion of the descending branch of the left coronary artery for 30 min, pretreatment with 10 nmol / L AAP10 led to a significant reduction in the ischemia-induced alterations of the activation patterns and reduced the dispersion of the activation-recovery intervals (18). Additional studies demonstrated that AAP10 did not affect the action potential of papillary muscles isolated from guinea pig hearts at concentrations up to 1 pmol / l (18). These findings are consistent with those of Argentieri et al. (19), who investigated the mechanism of the antiarrhythmic properties of AAP by examining the effect on the action potential in isolated canine Purkinje fibers. In this model, AAP did not affect inotropy or any of the electro-physiological parameters measured (maximum diastolic potential, action potential amplitude, maximum depolarization rate, and action potential duration with repolarization of 50% and 95%. ). It was therefore concluded that aAp does not affect transmembrane ion currents. In guinea pig papillary muscles, the effect on coupling time, that is, the time interval between electrostimulation and the onset of action potential, was examined (20). It was found that high concentrations of AAP10 (1 pM) could decrease the stimulus-response interval by approximately 10% under normoxic conditions. Additionally, during hypoxia and glucose-free perfusion, an increase in the stimulus-response interval, indicating uncoupling, was prevented by means of 10 nmol / l of AAP10. Since the effect of AAP10 on docking time was very pronounced in poorly coupled cells, the authors suggested that AAP10 preferentially acts on poorly coupled cells. The effect on coupling time indicated that AAP exerts its actions through conductance at gap junctions. To test this theory, the authors examined the effect of AAP10 on conductance in cleft junctions.

ES 2 228 807 T3 in mature guinea pig ventricular cardiomyocytes using the double cell voltage stapling technique. These studies demonstrated that 10 nmol / L AAP10 produced a rapid and reversible increase in conductance at gap junctions. In this way, the antiarrhythmic properties of AAP10 were explained by an improvement in the coupling of the cleft junctions, thus reducing the dispersion of the action potential and preventing the slowing of conduction.

In summary, antiarrhythmic peptides are a group of peptides that exert their effect selectively on cleft junctions, thereby reducing cell uncoupling and reducing the spread of action potential duration or shape. Therefore, antiarrhythmic peptides can be expected to lack the proarrhythmic effects that limit the use of many currently available antiarrhythmic agents. This makes antiarrhythmic peptides extremely interesting as a potentially new and safer class of antiarrhythmic compounds. However, both native AAP and synthetic AAP10 possess various undesirable characteristics such as low stability, high effective concentration, etc., which, until now, have prevented their use as drugs. Grover and Dhein (21) have characterized two semi-cyclic conformations of AAP10, using nuclear magnetic resonance spectroscopy. Accordingly, one method of obtaining a stable antiarrhythmic peptide could be the provision of cyclic derivatives of antiarrhythmic peptides. DE19707854 describes CF₃C (OH) -Gly-Ala-Gly-4HypPro-Tyr-CONH apparently cyclic and CO-Gly-Ala-Gly-4Hyp-Pro-Tyr-CONH cyclic that have the same antiarrhythmic properties as AAP and AAP10, but of which they are claimed to have improved stability in aqueous solution and after repeated freeze-thaw cycles. However, the experimental conditions described in document DE19707854 are insufficient for the preparation of said cyclic compounds, and the chemical identification data indicated in said document, using HPLC, are not sufficient to identify the mentioned cyclic compounds. US 4,775,743 describes HP5, a peptide derivative possessing the sequence N-3- (4-hydroxyphenyl) -propionyl-Pro-4Hyp-Gly-Ala-Gly-OH and which is active against platelet agglutination. Dhein and Tudyka (22) have reviewed the literature regarding activity and concentration of peptides, including peptide derivatives belonging to the group of antiarrhythmic peptides, see Table 1, finding only 7 active compounds and another 4, which were weakly active. However, none of these peptides or peptide derivatives have been shown to be stable enough to be effective in a therapeutic regimen.

Additionally, Japanese Patent Application No. 08281636 and Japanese Patent Application No. 09308589 describe cyclic depsipeptides with antiarrhythmic action, but possessing an ester bond that exhibits lability towards endogenous esterases. Furthermore, WO96 / 21674 describes AAP10 derivatives in which a hydrogen on the phenyl ring of the tyrosine residue has been substituted with halogen. These AAP10 derivatives have antiarrhythmic properties and a reduced proarrhythmic risk compared to lidocaine and flecainide.

The following AAP peptides and AAP-like compounds are described in the literature:

(AAP) H-Gly-Pro-4Hyp-Gly-Ala-Gly-OH,

H-Gly-Pro-4Hyp-OH,

H-Gly-Pro-OH,

H-Gly-Pro-Leu-OH,

H-Gly-Pro-4Hyp-Gly-OH,

H-Gly-Pro-Leu-Gly-Pro-OH,

H-4Hyp-Gly-OH,

H-Gly-Ala-Gly-OH,

H-Gly-Gly-Gly-OH,

H-Pro-Pro-Gly-OH,

H-Pro-4Hyp-Gly-Ala-Gly-OH,

H-Pro-4Hyp-OH,

H-Pro-4Hyp-Gly-OH,

H-Pro-4Hyp-Gly-Ala-OH, (HP5) N-3- (4-hydroxyphenyl) -propionyl-Pro-4Hyp-Gly-Ala-Gly-OH,

N-3-phenylpropionyl-Pro-4Hyp-Gly-Ala-Gly-OH,

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N-3-phenylpropyl-Pro-4Hyp-Gly-Ala-Gly-OH,

N-3- (4-hydroxyphenyl) -propionyl-Pro-4Hyp-Gly-Ala-OH,

N-3- (4-hydroxyphenyl) -propionyl-Pro-4Hyp-Gly-OH,

N-3- (4-hydroxyphenyl) -propionyl-Pro-4Hyp-OH,

N-3- (3-Hydroxyphenyl) -propionyl-Pro-Pro-Gly-Ala-Gly-OH, (AAP10) H-Gly-Ala-Gly-4Hyp-Pro-Tyr-NH₂,

H-Gly-Ala-Gly-4Hyp-Pro-Tyr-OH,

H-Ala-Gly-4Hyp-Pro-Tyr-NH₂,

H-Gly-Sar-Pro-Gly-Ala-Gly-OH,

H-Gly-Pro-Sar-Gly-Ala-Gly-OH,

H-Gly-Sar-Sar-Gly-Ala-Gly-OH,

H-Gly-Ala-Gly-Hyp-Pro-Tyr (3-I) -NH2,

H-Gly-Ala-Gly-Hyp-Pro-Tyr (3-F) -NH2,

H-Gly-Ala-Gly-Hyp-Pro-Tyr (3-Cl) -NH2,

H-Gly-Ala-Gly-Hyp-Pro-Tyr (3-Br) -NH₂,

H-Arg-Ala-Gly-Hyp-Pro-Tyr-NH₂,

H-Val-Ala-Gly-Hyp-Pro-Tyr-NH₂,

H-Ala-Ala-Gly-Hyp-Pro-Tyr-NH₂,

H-Gly-Ala-Gly-Hyp-His-Tyr-NH₂,

H-Gly-Ala-Gly-Hyp-Pro-Phe-NH₂,

Cycle- (CF₃C (OH) -Gly-Ala-Gly-4Hyp-Pro-Tyr-CONH), and

Cyclo- (CO-Gly-Ala-Gly-4Hyp-Pro-Tyr-CONH).

The following compounds:

H-Gly-Pro-4Hyp-Gly-Ala-Gly-OH (AAP),

H-Gly-Pro-4Hyp-Gly-Ala-Gly-OH,

H-Gly-Ala-Gly-4Hyp-Pro-Tyr-NH₂ (AAP10),

H-Gly-Ala-Gly-4Hyp-Pro-Tyr-OH,

H-Gly-Ala-Gly-Hyp-Pro-Tyr (3-I) -NH2,

H-Gly-Pro-Sar-Gly-Ala-Gly-OH,

N-3- (4-hydroxyphenyl) -propionyl-Pro-4Hyp-Gly-Ala-Gly-OH (HP5),

N-3-phenylpropionyl-Pro-4Hyp-Gly-Ala-Gly-OH,

N-3- (4-hydroxyphenyl) -propionyl-Pro-Pro-Gly-Ala-Gly-OH,

Cycle- (CF₃C (OH) -Gly-Ala-Gly-4Hyp-Pro-Tyr-CONH), and

Cyclo- (CO-Gly-Ala-Gly-4Hyp-Pro-Tyr-CONH)

ES 2 228 807 T3 have shown activity or weak activity in test models, see, for example, Dhein and Tyduka (1995).

Although active antiarrhythmic peptides have been provided, none of them have led to the development of a much sought after antiarrhythmic drug. The purpose of the present invention is to provide additional antiarrhythmic peptides and functional analogs thereof, useful in the treatment of various coronary heart diseases and useful in preparing medicaments. Additionally, the new peptides described herein increase cleft junction intercellular communication (GJIC) in vertebrate tissue and, specifically, mammalian tissue, and are useful in the treatment of a broad spectrum of diseases. and disorders in vertebrates such as mammals, related to or caused by a decrease in the communication function of the intercellular cleft junction, as described below.

Summary of the invention

The purpose of the present invention is achieved with the present peptides, including antiarrhythmic peptide compounds, which are distinguished by having the following general formula I, as established in claim 1, and with pharmaceutical compositions comprising these compounds, and with their medical use , especially for the treatment of arrhythmias.

Brief description of the drawings

Figure 1 is an illustration of the different principles useful in the cyclization of peptide sequences.

Figure 2 shows the relative changes of the intercellular conductance GJ as a function of time, before and during stimulation with Compound 2 (10 ⁸ M), or vehicle, on isolated guinea pig myocytes. The change in conductance is expressed as a percentage change in relation to the conductance immediately prior to perfusion with Compound 2.

Figure 3 shows the phosphoinositol (PI) metabolic cycle as a function of norepinephrine concentration in cultures of cardiomyocytes isolated from newborn Wistar rats, after 10 minutes of glucose and oxygen deprivation.

Figure 4 shows the effect of Compound 2 on the attenuated norepinephrine-induced increase in the phosphoinositol metabolic cycle during ischemia-induced metabolic stress and glucose deficiency when added to cardiomyocyte culture.

Figure 5 shows measurements of the standard deviation of APD₉₀ as a measure of electrical dispersion (APD dispersion₉₀) during four conservative infusion protocols. * indicates p <0.05 versus vehicle treated group.

Figure 6 is an activation map of a canine heart, in which the Purkinje layer is stimulated approximately two hours after a coronary artery occlusion with an epicardial activation plane (EPI) in the upper left and sub-planes. epicardial (S-EPI), midwall, sub-endocardial (S-ENDO), endocardial (ENDO), and Purkinje (PURK) plotted down to the right of the last premature stimulus.

Figure 7 illustrates epicardial electrograms (E-) in the same dog whose examples are presented in Figures 6, 7, 8 and 9, recorded with a surface lead of ECGII and V5R during the second to the fifth extra-premature stimulus (seen better in EL), with 4 VT insurance complexes. Electrograms are recorded from the lateral, border (L) marking, and east (E), north (N), central (C), sub-epicardial (SE), below EC, as well as south (S) and northwest (NW) and southwest (SW) of EC.

Figure 8 illustrates the epicardial activation of the first ventricular tachycardia complex, which begins at -44 msec before the onset of the surface QRS, and which corresponds to the electrogram recorded in EC in Figure 7. The activation takes place in a double-loop reentry that fires first at -17 msec and then continues for up to 57 msec on the northwest loop. The southeast loop is activated first at 2 msec, 31 msec, and then at 57 msec.

Figure 9 shows the same leads from the same dog (s) (?) That are presented in Figure 7. This figure illustrates the epicardial electrograms (E-) recorded during stimulation of the same site used in Figure 7, but after stimulation. iv administration of Compound 2. After 30 minutes, a second dose of Compound 2 was administered and, after an additional 30 minutes, a third dose was administered. After administration of any of these doses, VT was not induced until one and a half hours after administration of the antiarrhythmic peptide.

Figure 10 shows the short-term effect of 1 x 10 ⁸M of Compound 2 on the propagation of the intercellular calcium wave in human osteoblasts. The number of cells in the wave is plotted before (1) and 10 minutes after adding Compound 2 (2) to the bath solution.

Figure 11 shows the number of cells in the calcium wave traced before (1) and 10 minutes after the addition of 1 x 10^-8 M of Compound 2 (2) to ROS 17 / 2.8 cells, cultured under hypoxic conditions (5% CO2).

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Figure 12 illustrates the staining blot of ROS 17 / 2,8 cells, cultured under hypoxic conditions (O₂3-6%). The number of coupled cells is plotted before (1) and 10 minutes after adding 1 x 10^-8 M of Compound 2 (2) to the bath solution.

Figure 13 illustrates the short-term effect of 1 x 10 ⁸ M of Compound 2 on the propagation of the intercellular calcium wave in human osteoblasts under hypoxic conditions. The Figure shows the number of cells in the wave during hypoglycemia (1) and 10 minutes after adding Compound 2 to the hypoglycemic bath solution (2).

Figure 14 shows alkaline phosphatase (ALP) activity in human osteoblast cell cultures. ALP activity is a measure of osteoblastic activity. ALP activity was measured during a 4-day stimulation with 10^-13-10^-6 M of Compound 2 in each culture, and compared to untreated controls. The relationship between ALP activity in the treated and untreated cultures is plotted for each concentration of the compound. Compound 2 stimulated ALP activity and consequently osteoblastic activity at all concentrations, in a concentration range of 10¹³ to 10 ⁷ M.

Figure 15 shows the effect of Compound 2 on Lucifer Yellow (LY) staining transfer in human osteoblast cells treated with 13 pM DDT, the compound 1,1-bis- (p-chlorophenyl) -2,2,2-trichloroethane . Incubation for 10 minutes with Compound 2 10^-8M produced an increase in the number of cells coupled to staining in all experiments (1 indicates before and 2, after the addition of Compound 2 to the bath).

In the detailed description and Examples, reference is made to compounds not within the scope of claim 1 by way of comparison.

Detailed description of the invention

In preferred embodiments of the invention, the covalent bonds are selected from peptide bonds, disulfide bonds, ester bonds, reduced amide bonds, alkoxy bonds, oxycarbonyl bonds, and acyloxy-alkoxy bonds.

Examples of Px are amino acids represented by formula II

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wherein n is an integer with a value of 3, 4 or 5, and R represents an optional substituent, preferably selected from the group consisting of halogen, phenyl, hydroxy, NH2 and C (1-6) alkyl.

Preferably, the sequence (Px) 2A-B represents a dipeptide selected from the group consisting of Sar-Sar, Sar-Hyp, Hyp-Sar, Pro-Sar, Sar-Pro, Pro-Hyp, Pro-Pro, Hyp-Pro, and Hyp-Hyp, where Pro and Hyp, where the Pro and Hyp ring structure is optionally substituted with halogen, nitro, methyl, amino, or phenyl, and Hyp represents 3-hydroxyproline or 4-hydroxyproline, or one of the two amino acid residues of Px is a Sar or N-cyclohexylglycine residue,

Ac-D-Tyr-D-Pro-D-4Hyp-Gly-D-Ala-Gly-OH,

Ac-D-Tyr-D-Pro-D-4Hyp-Gly-D-Ala-Gly-NH2 (Compound 2), and its pharmaceutically acceptable salts.

The present invention provides compounds of the general formula (I)

X - (Y¹)_b- (Px) 2-G'-A- (G ')_to-R7 which specifies a peptide sequence in which the amino acid residues are independently D-forms and in which

X represents H or Ac;

G 'represents a glycine or Sar residue, G' is preferably glycine;

A represents alanine;

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Px represents an amino acid residue of formula II such as Hyp or Pro, preferably proline;

Y 'represents tyrosine or phenylalanine, optionally substituted on the phenyl ring with halogen or hydroxy; Y 'is preferably tyrosine;

a and b are independently 0 or 1,

R7 represents OH, NH₂-NHNH₂-Asn-NH₂, or Gln-NH₂; and its salts.

Photo / thermolabile peptide derivatives

Affinity tagging is a frequently used technique to study the interactions of biologically active molecules. A photo- or heat-labile analog of the compound is used for research.

A photolabile analog of the test compound, which is stable in the dark, is converted by illumination into a reactive intermediate that can participate in insertion reactions . This, by forming a covalent bond, stabilizes the interaction based on biological affinity. As photosamples, aromatic azides and stabilized diazo compounds produce, with photolysis, highly reactive and nonspecific intermediates, nitrenes and carbenes, respectively capable of participating in insertion reactions. In this way, photoaffinity labeling using stabilized aryl azides and diazo-compounds as photo-samples can be carried out at any point of attachment that contains carbon-hydrogen bonds and does not require the presence of a particular reactive functional group in the Union Point. The specificity of the label therefore depends on the specific binding of the ligand to the receptor, which is then followed by a reaction that forms a nonspecific covalent bond that guarantees the label of the binding site. Photoaffinity samples are particularly useful for labeling hormone receptor sites where reactive functional groups may not be present, but which certainly contain carbon-hydrogen bonds. As photoactive functionality, azides, diazirino, α-diazido ketones, thia- and selenodiazoles, benzophenones and nitrophenyl are especially useful. The labeling process using aryl azides includes photolysis at = 300-320 nm for about 0.5-2 h at room temperature of an aqueous solution containing the photolabile peptide analog and receptor.

A thermolabile compound contains a reactive group that can form a covalent bond in a thermally controlled reaction, with specificity for amino or mercapto groups. Aliphatic halides, especially iodine and bromine, active esters such as N-hydroxy-succinimide, acid chlorides, pyridyl disulfides, isocyanates, isothiocyanates, carbodiimides and maleimido can be used as thermal samples.

Labels for in vitro applications are most often selected as radioactive isotopes such as Iodine 125 and 131, C-14 and tritium, or fluorescence samples, or biotin or haptens. It is necessary to investigate the influence of the label on the binding activity of the ligand in order to ensure that the affinity of the receptor is preserved. As a radioactive brand, Iodine-125 is often used for in vitro applications, due to its 60-day half-life and low-energy photon emissions. The long half-life allows the preparation and storage of labeled photoactive analogs and the resulting labeled protein products for extended periods prior to use or analysis. Incorporation of iodine (I-125) into peptide ligands can be easily accomplished if, for example, tyrosine or histidine are present in the peptide sequence. It is necessary to investigate the influence of the peptide label on the biological activity of the ligand to ensure the maintenance of biological activity. Dhein et al. (WO96 / 21674) have shown that a derivative of AAP10, in which the phenyl ring of the Tyr residues carries an Iodine-125 substituent has biological activity. However, the use of said AAP10 variant as an affinity sample is not possible due to the reversible binding to a possible ligand or receptor. Photoaffinity tagging using aryl azides typically results in 50-60% of the peptide ligand non-reversibly bound to the target protein (receptor). Therefore, an aim of the present invention is to additionally provide an antiarrhythmic peptide suitably modified with a photo- or thermosample and, optionally, a radioactive label, for use in assays for the identification of possible ligands or receptors for the antiarrhythmic peptide. Said purpose is achieved with a compound of the formulas I, XII, XII or 9 of the present document, derivatized with one of the aforementioned photo-samples, preferably, 4-azido-salicyloyl (ASAL) and AB- (4-azido- benzoyl). Preferably, said derivatized compound is further substituted with a radioactive label such as Iodine-125.

You go out

It is preferred that the compounds of the invention are used in the form of a pharmaceutically acceptable salt, an alkyl ester, an amide, an alkylamide, a dialkylamide or a hydrazide formed with the C-terminal carboxylic acid function of a linear compound, or a function free carboxylic acid, if present, of a cyclic compound. Linear compound lower alkyl amides and amides are among the preferred compounds of the invention. The salts include pharmaceutically acceptable salts such as acid addition salts and basic salts. Examples of acid addition salts are hydrochloride salts, sodium salts, calcium salts, potassium salts, etc. Examples of basic salts are salts in which the cation is selected from alkali metals such as sodium and potassium,

ES 2 228 807 T3 alkaline earth metals such as calcium, and ammonium ions + N- (R³)₃(R⁴), where R³ and R⁴ independently designate optionally substituted C 1-6 alkyl, optionally substituted C 2-6 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^to edition, Alfonso R. Genaro (comp ..), Mark Publishing Company, Easton, PA, USA, 1985 and newer editions, and in the Encyclopedia of Pharmaceutical Technology.

Definitions

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), aamino-iso-butanoic acid (Aib) , naphthyl-alanine (Nal), including 1-naphthyl-alanine (1Nal) and 2-naphthyl-alanine (2Nal), phenylglycine (Phg), 2,4-diamino-butanoic acid (Dab), 2,3-diamino acid -propanoic (Dapa) and hydroxyproline (Hyp). If not specified otherwise, Hyp represents 4-hydroxyproline. Natural or essential amino acids are the amino acid constituents 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 &amp; Appl. Chem. Vol. 56 (5), pp. 595-624 (1984).

Unless otherwise specified, it is to be understood that the C-terminal amino acid of a compound of the invention exists as a free carboxylic acid, which may also be specified as "-OH". It can be shown that the C-terminal amino acid of a compound of the invention has the terminal function "-OH / NH₂", Which means that there are two preferred forms of the compound: the free carboxylic acid and the amidated derivative. The hexapeptide compounds of the invention, which comprise the sequence Ala-Gly-Hyp and possess a -NH2 group at the C-terminus, do not contain a C-terminal Phe or Tyr, nor derivatives thereof that have a halogen substitution at the phenyl ring.

By "functional analogs" of antiarrhythmic peptides is meant any entity or chemical compound that has a structural conformation and / or binding properties sufficiently similar to that of endogenous AAP to provide one or more of the beneficial antiarrhythmic or antithrombotic properties of endogenous AAP.

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.

The expression "retro-analog" is meant to mean a peptide whose sequence is the reverse of the mentioned peptide.

The term "halogen" refers to F, Cl, Br and I, with F and I being preferred.

The term "alkyl" refers to univalent groups derived from alkanes by the removal of a hydrogen atom from any carbon atom: C_nH_2n+₁-. Groups derived by removal of a hydrogen atom from a terminal carbon atom of unbranched alkanes form a subclass of alkyl (n-alkyl) groups: H [CH₂]_n-. The RCH groups₂-, R₂CH- (R is different from H) and R₃C- (R different from H) are, respectively, primary, secondary and tertiary alkyl groups. 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, isopropyl, butyl, pentyl, and hexyl and all thereof. possible isomers. By "lower alkyl" is meant C (1-6) alkyl, preferably C (1-4) alkyl, and more preferably methyl and ethyl.

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

The term "aralkyl" refers to aryl-C (1-22) alkyl, and the term "aryl" means, throughout this specification, phenyl or naphthyl.

HPP refers to hydroxyphenyl-propionyl,

4HPP refers to 3- (4-hydroxyphenyl) -propionyl,

2HPP refers to 3- (2-hydroxyphenyl) -propionyl,

4HPPA refers to 4-hydroxy-phenoxyacetic acid,

2HPPA refers to 2-hydroxy-phenoxyacetic acid, 4HMPA refers to 4- (hydroxymethyl) -phenoxyacetic acid, 4HPA refers to 4-hydroxy-phenylacetic acid,

ES 2 228 807 T3

3HPA refers to 3-hydroxy-phenylacetic acid,

2HPA refers to 2-hydroxy-phenylacetic acid,

4HBG refers to N- (4-hydroxybenzoyl) -glycine,

3HBG 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,

Tfa refers to the trifluoroacetyl radical,

ASAL refers to the 4-azido-salicyloyl radical,

AB refers to the 4-azido-benzoyl radical,

HOBt refers to 1-hydroxybenzotriazole,

HOAt refers to 1-hydroxy-7-aza-benzotriazole,

Acm refers to the acetamidomethyl radical,

Pd (PPh₃)₄ is tetrakis- (triphenylphosphin) -palladium (0)

Stability of the compounds of the invention

Additionally, the compounds of the present invention are distinguished by being stable against enzymatic degradation, and / or by being stable against degradation in plasma, and / or by having an improved half-life in vivo.

It is preferred that the compounds including the antiarrhythmic compounds of the present invention are stable against enzymatic degradation, and / or stable in plasma. The various derivatives and chemical modifications of the native peptide sequence of AAP as presented by the invention, for example, C-terminal amidation or esterification, use of D-amino acids and natural amino acid derivatives, N-terminal modifications, and the cyclic analogs represent, in their entirety, modifications designed to enhance stability, while preserving the essential antiarrhythmic and / or antithrombotic properties of native AAP.

The following Table 1 shows the degradation half-life (T_1/2) of various compounds of the invention, compared to AAP10, AAP and HP5. From the table, it appears that compounds 2, 3, 27, 48 and 49 of the invention, with half-lives of 3 hours or more, are considerably more stable in plasma and serum than AAP10, which has a half-life of less than 10 minutes. and HP5, which has a half-life of less than 12 minutes.

(Table goes to next page)

ES 2 228 807 T3

TABLE 1

Results of the in vitro stability test in plasma and serum, T_1/2 in min and h

<td rowspan="2">MEDIA AND COMPOUNDS</td><td colspan="3">PLASMA, KEPARIN</td><td colspan="2">SERUM</td>

<td>RAT</td><td>RABBIT</td><td>HUMAN</td><td>RABBIT</td><td>HUMAN</td>

<td>Compound CE1 AAP</td><td></td><td>4.4 min ± 12%</td><td>7.6 min ± 6%</td><td></td><td></td>

<td>Compound CE2 AAP10</td><td>8.2 min ± 13%</td><td>9.5 min ± 12%</td><td></td><td>2.7 min ± 4%</td><td></td>

<td>Compound CE3 KP5</td><td></td><td>3.7 min ± 1%</td><td>11.9 min ± 11%</td><td></td><td></td>

<td>Compound 3</td><td></td><td>* &gt; 5h</td><td></td><td></td><td>&gt; 5h</td>

<td>Compound 2</td><td></td><td>* &gt; 5h</td><td>« &gt; 5h</td><td></td><td>« &gt; 5h</td>

<td>Compound 27</td><td></td><td>3.8 h ± 13%</td><td></td><td></td><td>3.1 h ± 6%</td>

<td>Compound 49</td><td></td><td>30.4 h ± 28%</td><td>13.1 h ± 3%</td><td></td><td></td>

<td>Compound 48</td><td></td><td>13.6 h ± 17%</td><td>14.8 h ± 3%</td><td></td><td></td>

* no reaction for 5 hours

In vitro plasma stability analysis method

The stability of the peptides is analyzed in different types of plasma and serum. Peptides are incubated at 37 ° C in plasma and sampled at approximately 9 regular intervals between t = 0 and t = 156. The analysis is carried out by HPLC.

Appropriate conditions (column, solvent, gradient, and temp.) For HPLC analyzes are believed to ensure that the drug peak and plasma peaks do not have the same retention time. This is accomplished by means of subsequent injections of the drug, plasma, and a co-injection with the drug and plasma, followed by optimization of the LC method parameters until satisfactory separation is obtained. Three parallel experiments are carried out for each type of plasma. 100 µl of peptide is mixed with 900 µl of plasma at t = 0 and incubated at 37 ° C (concentration of the drug-plasma mixture 0.1 mg / ml). 100 µl samples are withdrawn from the drug-plasma mixture at suitable intervals and degradation is stopped by precipitation of the sample with 10 µl MeCN: TFA 50:50 by volume. Likewise, a control plasma sample is 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 vials of HP autosampler and analyzed by HPLC. HPLC analyzes are carried out as follows:

Compound CE1

Column: Vydac 218MS52, 250 x 2.1 mm, flow rate: 0.200 ml / min. Temp .: 40 ° C.

Solvent: MeCN / MQW / TFA (0.1%). Travel time: 25 min.

Iny Vol .: 15 μΕ Detection: DAD1 A, 214.5 nm.

ES 2 228 807 T3

Compound CE2

Column: Kromasil KR100-10C8, 250 x 4.6 mm, flow rate: 1 ml / min. Temp .: 40 ° C.

Solvent: MeCN / MQW / TFA (0.1%). Travel time: 20 min.

Iny Vol .: 25 pl. Detection: VWD 1 A, 214.5 nm.

Except for rabbit serum: DAD1 A, 214.5 nm.

Except for rat plasma: Solvent: MeOH / MQW / TFA (0.1%). Detection: VWD1 A, 210 nm.

Compound CE3

Column: Vydac 218MS52, 250 x 2.1 mm, flow rate: 0.200 ml / min. Temp .: 40 ° C.

Solvent: MeCN / MQW / TFA (0.1%). Travel time: 35 min.

Iny Vol .: 15 pl. Detection: DAD1 A, 214.5 nm.

Compound 3

Column: Kromasil KR100-10C8, 250 x 4.6 mm, flow rate: 1 ml / min. Temp .: 40 ° C.

Solvent: MeCN / MQW / TFA (0.1%). Travel time: 25 min.

Iny Vol .: 25 pl. Detection: VWD 1 A, 214.5 nm.

Compound 2

Column: Luna 3u C18 (2), 150 x 2 mm, flow rate: 0.250 ml / min. Temp .: 40 ° C.

Solvent: MeCN / MQW / HFBA (0.02%). Travel time: 25 min.

Iny Vol .: 25 pl. Detection: VWD 1 A, 214.5 nm.

Except for human plasma: Column: Luna 5u C18, 150 x 2 mm, Temp .: 10 ° C.

Except for human serum: Column: Kromasil KR100-10C8, 250 x 4.6 mm, flow rate: 1 ml / min. Solvent: MeCN / MQW / TFA (0.1%).

Compound 27

Column: Kromasil KR100-10C8, 250 x 4.6 mm, flow rate: 1 ml / min. Temp .: 40 ° C.

Solvent: MeCN / MQW / TFA (0.1%). Travel time: 20 and 25 min.

Iny Vol .: 25 pl. Detection: VWD 1 A, 214 nm.

Compound 49

Column: Kromasil KR100-10C8, 250 x 4.6 mm, flow rate: 1 ml / min. Temp .: 40 ° C.

Solvent: MeCN / MQW / TFA (0.1%). Travel time: 25 min.

Iny Vol .: 25 pl. Detection: VWD 1 A, 214.5 nm.

Compound 48

Column: Kromasil KR100-10C8, 250 x 4.6 mm, flow rate: 1 ml / min. Temp .: 40 ° C.

Solvent: MeCN / MQW / TFA (0.1%). Travel time: 25 min.

Iny Vol .: 25 pl. Detection: VWD 1 A, 214.5 nm.

The samples are analyzed in the following order: blank, the peptide at 0.1 mg / ml, the plasma without the peptide, the three parallel samples for t = 0, the three parallel samples for t = 5 min, the three parallel samples for t = 10 min, etc.

ES 2 228 807 T3

And finally, the three parallel samples are repeated for t = 0 to ensure that there has been no degradation or other error during the analyzes. Sample concentrations (peak height in mAU) are plotted against time and fitted to a function that describes a mono-exponential degradation (Excel). The half-lives of the peptides in the different types of plasma are presented in Table 1 as mean (n = 3) ± standard deviation.

General Background on Splint Joints

In a multicellular organism, the coordination between cells is of enormous importance. Among the various means of cellular cross-interaction, cleft junctions provide the most direct pathway. Gap junctions are a type of junction complex formed between adjacent cells and consist of aggregated channels that directly connect the interiors (cytoplasm) of adjacent cells. In the adult mammal, cleft junctions are found in most cell types, with one known exception, which is the circulating elements of the blood.

The structural unit of the gap junction channel is the connexon or hemi-channel. Each connexon is composed of six connexin (Cx) polypeptides that oligomerize to form an aqueous pore that spans a single plasma membrane. To form a complete cleft junction channel, two adjacent cell connexons are aligned and linked together to form a continuous channel, which joins the cytoplasms of the two cells.

The connexins that form the cleft junction channel comprise a multi-gene family, with fourteen mammalian connexins having been discovered to date. Expression of connexins is tissue and cell specific, with some cells expressing multiple isoforms of connexins. Experimental evidence indicates that two different hybrid configurations are possible: heterotypic intercellular channels, in which each connexon or hemichannel consists of a specific connexin isoform; or heteromeric channels, in which each connexon is a mixture of the various isoforms of connexins expressed in a particular cell type. Connexins are expressed cell, tissue, and developmentally specific.

Relatively little is known about the gene structure of connexins. The results reported for mouse Cx43 have revealed that Cx43 contains two exons and one intron located in the 5 'untranslated region. Additional analyzes have demonstrated the start point of Cx43 transcription in both embryos and adult tissues. Several putative transcription factor binding sites have been identified in the proximal 5 'promoter. In vitro studies have shown that permeable channels could be produced by hemi-channels composed of different pairs of Cx. For example, Cx43 can produce functional channels with endogenous oocyte Cx32, Cx37, and Cx (Cx38), but not with Cx26 oocytes. However, very 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 single cells are capable of expressing several different Cx. Permeable gap junctions can form between cells expressing different types of Cx. Thus, cleft junction intracellular communication (GJIC) in tissues appears to be very important for maintaining tissue integrity. It appears that several genes generate the equivalent products to prevent the loss of GJIC due to the mutation of one of the genes.

The pore diameter of the formed gap junction channel has been established in the range of 0.8-1.4 nm. Cleavage junctions are relatively non-selective and allow the passage of molecules of up to about 1,000 daltons. These substances are, among others, ions, water, sugars, nucleotides, amino acids, fatty acids, small peptides, drugs and carcinogens. Passage through the channel does not require ATP and appears to be the result of passive diffusion. This flow of materials between cells, through gap junction channels, is known as gap junction intercellular communication (GJIC), which plays an important role in the regulation of cell metabolism and proliferation, and in signaling. cell to cell. One of the most important physiological implications of GJIC is that cleft junction-coupled cells within tissue are not individual discrete entities, but are highly integrated with their neighbors. This property facilitates homeostasis and also allows the rapid and direct transfer of second messengers between cells to coordinate cellular responses within the tissue.

The GJIC process is regulated by a variety of mechanisms that can be divided into two main categories. The first type of regulation controls the number of cells in the cleft junctions, influencing the expression, degradation, cellular trafficking of connexins to the plasma membrane, or the assembly of connexins into functional cleft junctions. Altered GJIC, caused by down-regulation of connexin expression in tumor cells is an example of this mode of regulation. The second type of regulation does not generally involve any overt alteration of the cellular levels of cleft junctions or connexins, but instead induces the opening, closing, or barrier action of existing cleft junctions. Soluble extracellular factors such as mitogens (eg, DDT), hormones (eg, catecholamines), anesthetics (eg, halothane), intracellular biomolecules (eg, cAMP), and cellular stress (eg, mechanical or metabolic stress) can result in this type of regulation. Additionally, GJIC is regulated during the cell cycle and during cell migration.

The GJIC or barrier binding mode of regulation has been extensively studied for cleft junctions, especially cleft junctions composed of connexin 43 (Cx43) and, therefore, it has been used as representative of all connexins. Some factors exert their inhibitory effects on GJIC indirectly, altering, for example, the lipid environment and the fluidity of cell membranes, while other inhibitors

GJIC ES 2 228 807 T3 include oncogenes, growth factors and tumor promoters, which induce various modifications on Cx43. Binding permeability disruption may be necessary to mediate the specific biological functions of the latter group. These agents initiate complex signaling pathways, consisting of the activation of kinases, phosphatases, and interacting proteins. Understanding the mechanisms of action of these GJIC modulators will not only define their respective signaling pathways, responsible for regulating junctions, but will also provide experimental tools to characterize the biological functions of GJIC and connexins.

Variations in site-specific phosphorylation of the carboxy-terminal domain of the cytoplasm of Cx43 appear to be essential for the opening and closing of the cleft junction channel. Phosphorylation of the carboxy-terminal domain may also be important for the process of driving the hemicomplex of cleavage junctions of Cx43 to the surface membrane, its internalization and degradation. Connexins have much shorter half-lives (hours) than most plasma membrane proteins (days); for example, the half-life of Cx43 in the rat heart is less than 1.5 hours. Thus, the regulation of metabolic rate would be an important factor in the regulation of GJIC.

The carboxy-terminal domain contains putative phosphorylation sites for multiple protein kinases (PKA, PKC, PKG, MAPK, CaMkII, and tyrosine kinase). Phosphorylation of these carboxy-terminal domain sites results in the closure of cleft junction channels, and various Cx43 cleft junction channel inhibitors use different signaling pathways to induce phosphorylation of the carboxy-terminal domain. The cell type and the particular inhibitor determine which signaling pathway to use, and the type of protein kinase involved indicates the intracellular messenger system employed. Therefore, it has been reported that the activation of PKA requires the involvement of the cAMP second messenger system, whereas PKC requires the involvement of the intracellular phosphoinositol signaling system.

Other mechanisms that regulate the channel barrier system include intracellular levels of hydrogen and calcium ions, the voltage between junctions, and free radicals. A lowered pH or pCa induces channel closure specifically for the cell and connexin.

In addition to growth control, many physiological functions have been proposed for GJIC:

Homeostasis. GJIC enables rapid balance of nutrients, ions, and fluids between cells. It could be the oldest, most widespread, and most important feature for these channels.

Electrical coupling. The cleft junctions act as electrical synapses in electrically excitable cells such as cardiac myocytes, smooth muscle cells, and neurons. In these tissues, electrical coupling allows for faster cell-to-cell transmission of action potentials than chemical synapses. In cardiomyocytes and smooth muscle cells, this allows their synchronous contraction.

Tissue response to hormones. GJIC can enhance the responsiveness of tissues to external stimuli. Secondary messengers such as cyclic nucleotides, calcium, and inositol phosphates are small enough to pass from hormonally activated cells to quiescent cells through junction channels and activate the latter. This effect can increase the response of tissues to an agonist.

Regulation of embryonic development. The cleft junctions can act as intercellular pathways for developmental chemical and / or electrical signals in embryos, and to define the boundaries of developmental compartments. GJIC occurs in specific patterns in embryonic cells, altered GJIC has been linked to developmental abnormalities and the teratogenic effects of many chemicals.

Intercellular communication ensures that the activities of individual cells take place in a coordinated manner and the integration of these activities in the dynamics of a functional tissue that serves the organism to which it belongs. Therefore, it is not too surprising that a wide variety of pathological disorders have been associated with decreased GJIC.

Pharmacology

Cardiac indications

As noted in the description of the background of the invention, there is ample evidence supporting an important role for GJIC in cardiomyocytes under normal and pathological conditions. Specific cardiac disorders associated with a GJIC disorder are discussed below, and in vitro and in vivo evidence is presented showing that compounds that increase GJIC in the heart are useful for the prevention and / or treatment of a number of disorders. pathological disorders of the heart.

Reentry arrhythmias

Cardiac arrhythmias are caused by abnormal impulse onset or abnormal impulse conduction. Among arrhythmias with abnormal impulse conduction, those caused by a reentry mechanism are the most serious.

ES 2 228 807 T3

Ventricular reentry

Reentry is the main cause of sustained ventricular fibrillation and sudden cardiac death. Reentry occurs when the propagation impulse does not end after complete activation of the heart, but persists to re-excite the heart after the end of the refractory period. Reentry induction is facilitated by slow conduction, increased dispersion of repolarization, nonuniform anisotropy, and unidirectional conduction block. The underlying disease responsible for most cases of ventricular reentry is ischemic heart disease (eg, acute myocardial infarction, chronic myocardial infarction, stable angina pectoris, and unstable angina pectoris). During acute ischemia, cleft junction channels close, leading to uncoupling of neighboring cells. Heterogeneous changes in ion channel function and cleft junction lead to increased dispersion of action potential duration and effective refractory period, especially in the borderline zone separating the ischemic zone from the normal myocardium. Increased dispersion of action potential duration has long been known to facilitate induction of ventricular fibrillation (23). Normally, in well-coupled cells, the difference in action potential duration is smoothed out by electrical coupling. However, decoupling will prevent such a smoothing effect and contributes to the unmasking of the dispersion of the duration of the action potential and the refractory period (24). If ischemia is prolonged, a reduced degree of Cx43 expression and an altered pattern of distribution can be observed. Cleft junctional channel closure during acute ischemia, as well as variations in the expression and distribution pattern in chronic ischemia, can lead to slow conduction, increased dispersion, non-uniform anisotropy, and unidirectional block conduction, thus facilitating the induction of reentry arrhythmias. Thus, experimental studies have revealed a correlation between the site of abnormal expression and distribution of connexins and the location of the reentrant ventricular tachycardia circuits (25).

Conditions that favor the development of reentry, that is, slow conduction, increased dispersion of repolarization, non-uniform anisotropy, and unidirectional conduction block, are present in various measures in a number of additional heart diseases. Therefore, in infectious or autonomic cardiomyopathies, the inflammation that occurs can lead to the deposition of fibrous tissue in the myocardium, thus creating foci of slow conduction-increased dispersion and, possibly, unidirectional conduction block . Hypertrophic cardiomyopathy (eg, due to hypertension, aortic stenosis, congenital) can result in reentry arrhythmias due to improper pairing of the large amount of myocardial tissue and the relatively small amount of conductive tissue, which can lead to slow conduction, increased dispersion and unidirectional conduction block. Congenital diseases (for example, long QT syndrome) and drugs that prolong the QT interval (for example, antiarrhythmic drugs, antipsychotics, antihistamines, antibacterials, etc.) also increase the dispersion of the duration of the action potential, possibly due to the heterogeneity of the distribution of ion channels throughout the different layers of the myocardium, and they are an important cause of sudden reentry-induced death in younger subjects (26).

Headphone reentry

Atrial fibrillation - the most common cardiac arrhythmia - is also caused by a reentry mechanism. In this case, multiple waves travel through the atrium and re-excite tissue that is no longer refractory. Atrial fibrillation can persist for years and will eventually lead to remodeling of the atria. An important part of the remodeling process is constituted by changes in the distribution of the cleft joints. In this way, the Cx40 distribution pattern becomes increasingly heterogeneous. The evolution over time of the changes in the distribution and content of the Cx40 cleft junctions correlates with an increase in the stability and complexity of AF, and indicates that remodeling of the Cx40 cleft junctions could play a role in the pathogenesis of sustained atrial fibrillation (27). Additionally, various lines of research confirm the opinion that during slowing conditions of atrial conduction, susceptibility to atrial fibrillation increases.

Alternating repolarization

The appearance of alternating electrocardiographic T waves with a high heart rate or metabolic insult has been observed for about a century. Macroscopic alternating T waves are frequently recorded as a harbinger of sudden death from arrhythmia. Recent research suggests a common mechanism that may relate the presence of discordant alternating repolarization to the onset of various reentry arrhythmias, depending on the anatomical nature of the substrate (28). Under chronotropic or metabolic stress, the repolarization phase of the myocardial action potential develops an alternation in morphology and duration. With additional stress, or in the presence of structural barriers, the alternating repolarization becomes discordant in space. The discordant alternation gives rise to sufficiently wide repolarization gradients capable of producing unidirectional blocking and reentry. Without a structural barrier, reentry is functional and manifests as ventricular fibrillation or polymorphic ventricular tachycardia. In the presence of a structural barrier, reentry can be anatomically fixed, resulting in monomorphic ventricular tachycardia (29).

In summary, it appears that a substance such as the compounds of the present invention, which increases the conductance of the cleft junction and makes the anisotropy more uniform, will prevent one-way block and reentry arrhythmias. This substance will be useful in patients with reentry circuits of both ventricular origin

ES 2 228 807 T3 as a headset. Patients with alternating T wave are prone to reentry arrhythmias, and a substance capable of increasing the coupling of the cleft junctions and reducing anisotropy may be helpful in the prevention of fatal ventricular arrhythmias in these patients.

Bradyarrhythmias

Bradyarrhythmias can be caused by slow conduction or a conduction block in the sinoatrial node, atrioventricular node, bundle of His, and right or left bundle. The main connexin responsible for the conductance throughout the conduction system is Cx40. Mice homozygous for a deletion of the Cx40 gene show significantly slower atrial, atrioventricular and His-Purkinje conduction and are at increased risk of arrhythmias and bundle branch block. (4-6). Therefore, the normally functioning Cx40 gap junctions are essential for maintenance of normal rhythm.

A substance such as the compounds of the present invention, which increases the conductance of the gap junctions, is useful in the prevention and / or treatment of slow conduction in the heart.

Reduced contractility

Reduced contractility is a common feature of many chronic heart diseases. In the worst case scenario (ie, end-stage heart failure), contractility is reduced to a point where the ejection fraction is so low that the baseline perfusion requirements of the organ can no longer be maintained. Both experimental and clinical evidence have shown that the expression and distribution of connexins in the heart of patients with final heart failure are modified. Thus, Cx43 undergoes significant down-regulation with a highly irregular distribution in abnormal tissue. The expression of Cx45, which, under normal conditions, is very limited, is significantly increased in heart failure; however, the conduction properties of Cx45 are inferior to those of Cx43 and are therefore unable to compensate for the reduction in Cx43. Recent evidence indicates that some regulatory ion channels and receptors are concentrated in intercellular binding sites, so it is highly probable that changes in the expression and distribution of Cx43 may affect excitation-contraction coupling and, therefore, the contractility (30). Strong evidence of a relationship between cleft junction function and contractility is the fact that chimeric mice, formed from Cx43-free embryonic stem cells and wild-type blasts, and therefore express a loss heterogeneous of Cx43, they develop serious contraction defects (31).

The present inventors suggest that a substance capable of increasing the conductance of the cleft junctions will improve intercellular communication of mediators involved in excitation-contraction coupling, thereby improving contractility.

Experimental example 1

Effect of Compound 2 on GJIC in cardiomyocytes

Cell preparation: Cells were isolated from guinea pig hearts by collagenase perfusion, according to the Langendorf method. Briefly, 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 section of the dorsal column at the neck. The thorax was opened and the aorta cannulated. The cannula was then attached to the aorta by ligation, excised, and prefused 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 sparged with 100% oxygen. The heart was then pre-infused for two minutes with Ca-free Tyrodes solution.²+, followed by perfusion for two minutes with a solution with a high K + content, which contained in mM: Na + 20, K + 120, Cl^- 22, glutamate 120, Mg²+1, Ca²+25 pM, Hepes 10, glucose 10, pH 7.4.

The heart was then pre-fused with a solution with a high K + content with 0.6 mg / ml of collagenase, which was carried out for 10-15 minutes depending on the appearance of the heart. The atria were sectioned, the ventricles were ground, after which the pieces were shaken in the collagenase solution, gently bubbling with 100% oxygen. The cells were then sieved to isolate the released cells and the collagenase was removed by centrifugation. Cells were resuspended in Ca-free Tyrodes solution.²⁺ and the Ca content was slowly increased²⁺ up to 0.65 mM. The cells were kept in this solution at room temperature until their transfer to the experimental chamber.

Electrophysiology: Coverslip plates are mounted in an open chamber on an inverted microscope, where cells are perfused with Dulbecco's phosphate-buffered saline (PBS) at 1 ml / min, 37 ° C. The solution contains (in mM): Na + 152, K + 4.2, Cl^- 141.5, PO₄^3- 9.5, Ca²+ 0.9, Mg²+ 0.5, pH 7.2. Clamp pipettes (GCF150F-15, Harvard Apparatus) are cut from 1.5 mm glass capillaries on a Sutter Flaming-Brown P-87 microelectrode device and fire polished to a strength of 4-6 MQ. The pipettes are filled with a solution similar to the intracellular one that contains, 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 pg / ml) is added to this solution from a 60 mg / ml stock solution (solvent: DMSO).

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The patch clamp assembly consists of two synchronized discontinuous amplifiers (SEC-05LX, NPI Electronics) and the data is digitized using an INT-10 interface (NPI Electronics) and a PC1200 data acquisition table (National Instruments). Both current and voltage signals are filtered by low pass at 1 kHz, using the internal filters of the amplifiers, and digitized at 10 kHz.

One cell of a pair is approximated with an electrode, using a PatchMan 5173 micro-manipulator (Eppendorf). When contact with the cell is obtained (seen as a sudden increase in input resistance), suction is applied until the Giga seal setting is established. This procedure is then repeated on the other cell. The membrane under the pipettes is then ruptured by a brief application of suction and the cell interior is set at -70 mV, which is close to the spontaneous membrane potential of the cells. For 10 seconds, each cell is consecutively hyperpolarized with 10 mV for 1 second, and then the resulting current change in the other cell can be used to calculate intercellular conductance (Gj) using the formula:

ΔΙ_Ρ

p. press

-I

p.rest

AUj

Up - Ua

Equation 1 in which I_p._press and I_p._rest represent the current in the passive cell during the pulse and before the pulse, respectively, and U_p and U_to represent the voltage of the passive and active cell. This type of experiment does not allow the comparison of absolute values ​​of Gj due to differences in the contact between cells and, consequently, the number of functional gap junction channels. However, the variation of the Gj value compared to a standardized intervention, such as a drug, can be analyzed by comparing the relative changes of Gj.

Results: Figure 2 summarizes the results of 9 successful experiments. This figure shows the relative Gj as a function of time before and during stimulation with Compound 2 (10^-8M). In the five experiments in which the cells were treated with Compound 2, it produced a significant increase in Gj, which reached a steady state level after approximately 400 seconds of stimulation (AG, = +120 ± 46%). The conductance remained unchanged in the four vehicle-treated preparations (AGj = -3 ± 5%).

These findings are consistent with experiments reported in the literature, which used the synthetic analog of AAP, AAP10, and which showed increased electrical coupling between cardiomyocytes after stimulation (32). However, in the study by Müller et al. (32), the conductance of the cleft junctions was not stable during the control conditions. Thus, in three of six experiments, the application of AAP10 did not increase the conductance, but prevented the decrease in the conductance of the cleft junctions and, in two of six experiments, the conductance of the cleft junctions actually increased during the control period. In the experiments presented herein, Compound 2 increased the conductance of the cleft junctions in preparations with stable control conditions.

Experimental example 2

Binding of Compound 2 to mouse heart histological preparations

Preparation

Mouse hearts (Balb / cJ, 20 g) are separated, rinsed twice in ice cold (0 ° C) 0.32 M sucrose 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 g_half for 10 minutes at 4 ° C and the supernatant is collected and filtered through 4 layers of cheesecloth. The filtrate is then centrifuged at 50,000 g_half for 45 min at 4 ° C and the granulate is resuspended in 10 vol_org._{wet weight} of ice distilled water, and incubate for 60 min at 0 ° C, and centrifuge again at 50,000 g_half for 45 min at 4 ° C. The resulting granules are resuspended in 2 vol_org._{wet weight} of PBS (Phosphate Buffered Saline) and stored at -80 ° C until use.

Previous treatment

On the day of analysis, cells are removed from the incubator and each well is washed twice with, depending on the experiment, 2 ml of prewarmed (37 ° C) or ice (0 ° C) D-PBS to separate the serum. It is important to keep to a minimum the period during which cells remain without physiological solutions, to avoid drying out during washing procedures. Cold washed cells are used directly for fixation assays, while hot washed cells are used for glucose and oxygen deprivation experiments.

Glucose and oxygen deprivation

The cells are incubated for 10 min in an atmosphere of N₂ in glucose-free D-PBS (pH 7.2), pre-equilibrated with N₂ for at least 10 min at 37 ° C. Control cells are similarly incubated for 10 min at 37 ° C only, under normal atmospheric conditions and in D-PBS containing glucose (6 mM).

ES 2 228 807 T3

Fixation test

Fixation in situ is carried out according to a modified protocol, based on the description by Koenig (34). The D-PBS is separated from the cell culture and 0.50 ml of [¹²⁵I] AAP10 with or without unlabeled ligand or test compound. The cells are incubated overnight at 4 ° C until equilibrium is reached. Each well, and one at a time, is then rinsed rapidly with 2 x 1ml D-PBS and allowed to dry.

0.25 ml of 0.5% Triton-X-100 (by volume) is added to each well and the cells are allowed to solubilize for at least 1 h. The extract is transferred to counting vials, the wells are washed with 0.25 ml of water, and the rinse extract is added to the corresponding vials. The vials are counted in a gamma counter.

TABLE 3

Fixation in place, CI₅₀ (nM)

<td>Test compounds</td><td>IC50 (nM)</td>

<td>AAP (CE1)</td><td> 0,8</td>

<td>AAP10 (CE2)</td><td> 130</td>

<td>Compound 2</td><td> 0,5</td>

<td>Compound 32</td><td> 0,5</td>

<td>Compound 24</td><td> 65</td>

These results reveal a high affinity binding to CHO cells of different substances of the present invention, comparable to that of the peptides of the prior art.

Displacement experiments with Compound 2

40-250 pg of filtrate or membrane material are incubated in a total volume of 100 µl of D-PBS (Dulbecco's Phosphate Buffered Saline containing 1 g / l MgCl₂-6H₂O and CaCl₂) containing [¹²⁵I] AAP10 0.8 nM and increasing concentration of test compounds AAP and Compound 2. Nonspecific binding is determined with AAP10 10 pM (CE2).

Calculations

The data from the displacement experiments fit the equation:

f = (Total - ns) / (1 + s / CI₅₀) + ns where Total is the total bound radioactivity at the concentration s of the labeled ligand, ns is nonspecific binding, and CI₅₀ is the concentration of test compound that reduces specific binding (Total-ns) to 50% of maximum specific binding.

Results

TABLE 2

Displacement of f²⁵I] AAP10 0.8 nM mouse cardiac tissue preparations (ne = not tested)

<td>Test compounds</td><td>CI filtering₅₀ (nM)</td><td>IC membranes₅₀ (nM)</td>

<td>AAP</td><td> 1,2</td><td>ne</td>

<td>AAP10 (CE2)</td><td> 1,2</td><td>ne</td>

<td>Compound 2</td><td> 3,6</td><td> 1,2</td>

The values ​​indicated in Table 2 above are of the same order of magnitude (0.2 nM) as that indicated for AAP10 by Dhein et al. (33), using rabbit heart membranes.

In situ fixation method in intact cells

CHO cell cultures

CHO cells are seeded in 24-unit multi-well plates at a density of 7900 cells / cm² (~ 15.000

ES 2 228 807 T3 cells / well) and cultured for 3 days in vitro (DIV) on 1 ml / well of Nutrient Mix F-12K supplemented with 10% Fetal Calf Serum (FCS) and 1000 units of penicillin / 1000 μg of streptomycin (pen./estrep) in a CO atmosphere₂ at 5% and humidity from 100% at 37 ° C. Cell density had so far increased to 295,000 cells / cm² (152 pg_prot/ cell ~ 85 μgp_rot / well).

Experimental example 3

Effect of Compound 2 on cAMP formation in CHO cells

CHO cell culture

CHO cells are seeded in 96-well microtiter plates at a density of 6,000 cells / cm² (~ 2,000 cells / well) and cultured for 4 days in vitro in 200 µl / well of growth medium, as described in the previous section.

Previous treatment

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

CAMP efficacy trial

CHO cells are incubated at 37 ° C in D-PBS (pH 7.2) containing 6 mM glucose, 2.0 mM IBMX (phosphodiesterase blocker), 10 μM forskolin (stimulates cAMP formation) and increasing concentrations of the peptide of testing. The reaction is stopped after 20 min by adding 20 µl of 0.5 M HCl and left for at least 20 min at room temperature.

The cAMP content is analyzed by mixing 20 μl of the acidic cell extract in FlashPlate® wells (NEN SMP001 assay kit) containing 180 μl of [¹²⁵I] cAMP. FlashPlates® are incubated overnight at 4 ° C and radioactivity bound to the plate is counted on TopCount® (Packard Instruments). The data is calculated as described in the previous section.

Results

The inhibition of forskolin-stimulated cAMP formation caused by AAP-like compounds in CHO cells indicates that AAP receptors are negatively coupled to the second messenger system of cAMP. Additionally, it demonstrates the presence of functional AAP receptors on CHO cells.

TABLE 4

Inhibition of forskolin-stimulated cAMP formation in CHO cells

<td>Test compounds</td><td>EC50 (nM)</td>

<td>AAP</td><td> 53</td>

<td>AAP10 (CE2)</td><td> 11</td>

<td>Compound 2</td><td> 6,2</td>

Experimental example 4

Analysis of phosphoinositol in rat primary cardiomyocytes

Primary cardiomyocyte culture

Newborn (1-2 days old) Wistar rats are used. Hank's balanced salt solution, calcium and magnesium free, buffered with 10 mM HEPES is used for washing during cell separation procedures. The hearts are removed, the ventricles are isolated, and the tissue is divided into small pieces. Myocardial cells are isolated by gradual enzymatic degradation with 0.05% collagenase, as described in (35). After repeated cycles of centrifugation and washing, the precipitated cells are resuspended in M199 culture medium with Earle's salt, 10% NCS, penicillin (75 U / ml) and streptomycin (75 U / ml) and are pre-seeded in a Petri dish for 90 minutes. Non-adherent cells are collected in the culture medium and seeded in multi-plates at 2.5 · 10⁵ cells / well. The cultures are kept in a CO incubator.₂ saturated with water at 37 ° C. Cardiomyocyte cultures are used for analysis after 6-7 days.

ES 2 228 807 T3

Analysis of the metabolic cycle of phosphoinositol

Cardiomyocyte cultures are incubated for 48 hours in culture medium containing 4 pCi / ml myo- [2^-3H] -inositol to label inositol phospholipids. On the day of analysis, the medium is replaced by a lithium-containing buffer solution and incubated at 37 ° C, as described by Meier et al. (36). After at least five minutes, this buffer is replaced with the same volume of buffer that contains the test compound, and incubated for exactly twenty minutes. The reaction is stopped by rapid replacement of the medium with ice cold 4% by volume perchloric acid (PCA), and incubation for at least 20 minutes at 0 ° C. The PCA extract is neutralized and the phosphates of [³H] -inositol are separated by anion exchange chromatography, using Amprep® columns containing 100 mg of SAX Quaternary amine. Monophosphates of [³H] -inositol are eluted and the radioactivity in the fraction is measured by liquid scintillation counting.

Glucose and oxygen deprivation

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

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

Under normal atmospheric and nutritional conditions, the present inventors obtained a value of E_max 3852 ± 266 cpm and an EC value₅₀ 203 nM (SD_R = 1.2), while in cells subjected to an atmosphere of N₂ and with glucose depletion, a value of E_max 2248 ± 702 cpm and an EC value₅₀ 303 nM (SDr = 1.7).

To examine the effect of substances of this invention on the attenuated and norepinephrine-induced increase of the phosphoinositol metabolic cycle during ischemia-induced cell stress and glucose deprivation, Compound 2 or AAP10 (CE2) were added to the cardiomyocyte cultures. Both substances strongly potentiated the phosphoinositol metabolic cycle, Compound 2 being the most powerful. As shown in the following Table 5, the value of CE₅₀ for AAP10 (CE2) was 200 times higher during normoxia and 10 times higher during metabolic stress induced by anoxia and glucose deprivation than the EC value₅₀ for Compound 2.

TABLE 5

Potentiation of the phosphoinositol metabolic cycle during anoxia-induced metabolic stress and glucose deprivation, using Compound 2 and AAP10

<td></td><td>EC50 (nM) AAP10 (CE2)</td><td>EC50 (nM) Compound 2</td>

<td>Normal conditions</td><td> 2000</td><td> 10</td>

<td>Glucose and oxygen deprivation</td><td> 100</td><td> 10</td>

The addition of Compound 2 (100 nM) had no additional effect on the norepinephrine (300 nM) -induced increase of the phosphoinositol metabolic cycle in neonatal rat cardiomyocytes during control conditions, but in cells subjected to anoxia and deprivation. glucose (metabolic tension), the addition of Compound 2 (100 nM) + norepinephrine (300 nM) normalized the altered phosphoinositol metabolic cycle, as shown in Figure 4, with an increase that was approximately 70% greater than the increase effected by norepinephrine alone.

Experimental example 5

Calcium-induced arrhythmia model in mice

The antiarrhythmic effects of the compounds of this invention were tested in an in vivo model of calcium-induced arrhythmias, according to the model of Lynch et al (37). Mice (25-30 g) were anesthetized with a neuroleptic-anesthetic combination (Hypnorm® (0.315 mg / ml fentanyl citrate and 10 mg / ml fluanisone) + midazolam (5 mg / ml)). The commercial solutions of Hypnorm® and midazolam were diluted 1: 1 in distilled water and one part of diluted Hypnorm® was mixed with one part of diluted midazolam.

Anesthesia was induced by sc administration in a dose of 0.05-0.075 µl / 10 grams of mouse. A

ES 2 228 807 T3 IV cannula in the tail vein. Lead II ECG signal was continuously recorded by placing a stainless steel ECG electrode on the right foreleg and left hindlimb. The ground electrode was placed on the right hind leg. The signal was amplified (x 5,000-10,000) and filtered (0.1-150 Hz) through a Hugo Sachs Electronic model 689 ECG module. The 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. Vehicle pretreated mice were tested as a measure of the level of control in untreated animals. The injection volume was 100 µl in all experiments. CaCl infusion₂ (30 mg / ml, 0.1 ml / min -100 mg / kg / min (calcium chloride-2 hydrate, Riedel-de Haen, Germany)) was started 3 min after iv administration of drug or vehicle. The time to the 2nd degree of AV block was determined as the time elapsed since the start of the CaCl infusion.₂ until the appearance of the first arrhythmic event. A 2nd degree AV block event was defined as intermittent AV conduction failure, characterized by a P wave without the concomitant QRS complex.

Responses were expressed in relation to the time to the appearance of 2nd degree AV block in vehicle-treated mice. The maximum effect of each of the substances tested is summarized in the following Table 6.

TABLE 6

In vivo antiarrhythmic activity of the compounds of the invention +++ refers to a&gt; 60% increase in time to arrhythmia; ++ refers to a 3050% increase in time to arrhythmia; + refers to a 15-29% increase in time to arrhythmia; (+) refers to &lt;15% increase in time to arrhythmia, and nd indicates “not determined”.

<td>Comp. n °</td><td>Compound name</td><td>Activity in alive</td>

<td>Group 1</td><td>Comparative examples</td><td></td>

<td>CE-1</td><td>H-Gly-Pro-Hyp-Gly-Ala-Gly-OH (AAP)</td><td> ++</td>

<td>CE-2</td><td>H-Gly-Ala-Gly-Hyp-Pro-Tyr-NH₂ (AAP10) 3- (4-hydroxyphenyl) -propyl-Pro-Hyp-Gly-Ala-Gly-OH</td><td> +++</td>

<td>CE-3</td><td>(HPS) H-GAG- (Pa) 2-NH2: Pa is any amino acid residue or a moiety of formula II or lia; at least one of Pa is</td><td></td>

an amino acid D; preferably, Pa is Hyp, P, G, or A;

<td colspan="3">Group 2</td>

<td>Formula 2</td><td></td><td></td>

<td> 5</td><td>H-Gly-Ala-Gly-D-Hyp-Pro-Tyr-N H₂</td><td> ++</td>

<td> 6</td><td>H-Gly-Ala-Gly-D-Pro-Pro-Tyr-NH₂</td><td>nd</td>

<td> 7</td><td>H-Gly-Ala-Gly-D-Pro-Ala-Tyr-NH₂</td><td>nd</td>

<td> 8</td><td>H-Gly-Ala-Gly-Gly-D-Pro-Tyr-NH₂</td><td>nd</td>

<td> 9</td><td>H-Gly-Ala-Gly-D-Hyp-Ala-Tyr-NH₂</td><td> +</td>

<td> 10</td><td>H-Gly-Ala-Gly-D-Hyp-D-Pro-Tyr-NH₂H-GAG- (Px)_rY-NH₂: Px is a remainder of formula II or lia, where a Px is a remainder of formula II, lia and the another Px is P or Hyp.</td><td> +++</td>

ES 2 228 807 T3

Group 3 Formula 3 11 12

H-Gly-Ala-Gly-NCG-Pro-Tyr-NHj

H-Gly-Ala-Gly-T4C-Pro-Tyr-NH₂

H-Gly-Ala-Gly-A2C-Pro-Tyr-NH₂

H-Gly-Ala-Gly-PC-Pro-Tyr-NH₂ na ++ na +

Group 4 Formula 4 1

Group 5 Formula 5 16

19

Group 6 Formula 6 22

2

Ac-Y '- (Px)₂-GAG-OH: Y 'is Y or F; Px is po Hyp

Ac-Tyr-Pro-Hyp-Gly-Ala-Gly-OH

Ac-Tyr-Pro-Hyp-Gly-Ala-Gly-NH₂ +

nd

Cys (Acm) -AAP10 * / retroAAP10 * -Cys (Acm)

H-Cys (Acm) -Gly-Ala-Gly-Hyp-Pro-Tyr-Cys (Acm) -NH₂ +

H-Cys (Acm) -Gly-Hyp-Pro-Tyr-Cys (Acm) -NH₂ nd

H-Cys (Acm) -Tyr-Pro-Hyp-Gly-Ala-Gly-Cys (Acm) -NH₂ nd

H-Cys (Acm) -Tyr-Pro-Hyp-Gly-Cys (Acm) -NH₂ nd

XGDAG- (D-Px) 2-DY-NH₂: X is H, Ac; Px is a moiety of formula II, preferably Hyp or P; optionally has one or more C or N isotopes

H-Gly-D-Ala-Gly-D-Hyp-D-Pro-D-Tyr-NH₂ nd

H-Gly-D-Ala-Gly-D-Hyp-D-Pro-D-Tyr-D-Asp-OH nd

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

Ac-D-Tyr (3,5-di-l) -D-Pro-D-Hyp-Gly-D-Ala-Gly-NH₂ nd

Ac-D-Tyr (mono-iodo-substituted phenyl ring) -D-Pro-DHyp-Gly-D-Ala-Gly-NH₂ nd

Ac-D-Tyr-D-Pro-D-Hyp- (1,2¹'C, ^,5N-Gly) -D-Ala- {1,2¹³C, ¹⁵N-Gly) -NH₂ nd

H- (Px)_n-Y (N / Q) G-AG- (Px)_m-NH₂: Px is P or Hyp, n is 1 or 2; m is 0 or 1; preferably, m = 0 when n = 2 and m = 1 when n = 1

ES 2 228 807 T3

Group 7 Formula 7

H-Pro-Tyr-Asn-Gly-Ala-Gly-Hyp-NH₂H-Hyp-Pro-Tyr-Asn-Gly-Ala-Gly-N H₂

Group 8 H-G'-A-G '- (Px) 2-Y-NH₂: G 'is Sar or Gly and at least one

Formula 8 G 'is Sar; Px is P or Hyp

H-Sar-Ab-Sar-Hyp-Pro-Tyr-NK₂

H-Gly-Ala-Sar-Kyp-Pro-Tyr-NH₂

X- (Y) p- (Px) 2-GAG-NI-I₂: X is ASAL or AB; p is 0 or 1; the phenyl ring of Y optionally has one or more

Group 9 halogen substituents, preferably I; Px is P or Formula 9 Hyp

AAL-Pro-Hyp-Gly-Ala-Gly-NH₂

ASAL (mono-iodo-substituted) -Pro-Hyp-Gly-Ala-Gly-NH₂

AB-Tyr-Pro-Hyp-Gly-Ala-Gly-NH₂

AB-Tyr (3,5-di-l) -Pro-Hyp-Gly-Ala-Gly-NH₂

Group 10 Formula 10

Group 11 Formula 11 na (+) +

++ na +++ na na

Cyclo- (GAG- {Px) rY-N / Q-): Px is P or Hyp cyclo- (Gly-Ala-Gly-Hyp-Pro-Tyr-Gln-) ++ cyclo- (Gly-Ala-Gly- Hyp-Pro-Tyr-Asn-) +++ dclo- {Gly-Ala-Gly-Pro-Pro-Tyr-Asn-) nd

Cyclo- (Y- (Px)₂-GA- (G)_what-N / Q-): q is 0 or 1, the phenyl ring of Y optionally has one or more halogen substituents, preferably I; Px is P or Hyp dclo- (Tyr-Pro-Hyp-Gly-Ala-Gly-Asn-) +++ cyclo- (Tyr-Pro-Hyp-Gly-Ala-Asn-) nd cyclo- (Tyr ( 3-l. 5-l) -Pro-4Hyp-Gly-Ala-Gly-Asn) nd

Group 12 X-Zd-G (N / Q) Y-NH₂: Zd is a sequence of 0, 1 or 2

Formula amino acid residues selected from G or A; X is H,

Ac

H-Gly-Ala-Gly-Asn-Tyr-NH₂ +++

ES 2 228 807 T3

<td> 40</td><td>Ac-Gty-Asn-Tyr-N H₂</td><td> ++</td>

<td> 41</td><td>K-Gly-Asn-Tyr-N H₂</td><td> ++</td>

<td> 42</td><td>Ac-Aia-Gly-Asn-Tyr-NH₂</td><td>nd</td>

<td> 43</td><td>H-Ala-Gly-Asn-Tyr-N H₂</td><td>nd</td>

As can be seen from the results shown in Table 6, a wide range of new compounds of the present invention show antiarrhythmic activity comparable to that of the prior art compounds AAP, AAP10 and HP5.

Experimental example 6

Effects of Compound 2 on isolated rabbit precast hearts

Langendorff technique principle

The Langendorff technique provides a method of maintaining the adequate metabolic requirements of an isolated heart, thus allowing in vitro experiments to be performed on the entire heart for several hours. In the Langendorff method, the heart is retrograde perfused through a cannula inserted into the aorta. When the perfusion solution enters the aorta, the resulting pressure in the aorta closes the aortic valves, thereby preventing fluid from entering the cardiac chambers. Instead, the infusion solution enters the coronary circulation that supplies the heart. Therefore, in the Langendorff technique, the total flow in the aorta equals the coronary flow. The Langendorff experiments are carried out using the Type 833 apparatus for INSULATED HEART SIZE 5, manufactured by Hugo Sachs Elektronik, Germany. The central component of this apparatus is the aortic block to which the heart is attached by means of a cannula. The aortic block is directly connected to an artificial flow resistor, operated by a rotary knob that therefore allows adjustments of the back load and, consequently, of the perfusion pressure. The perfusion fluid is supplied from a thermostatted reservoir to the aortic block through tubes connected to a roller pump. The volume delivered by the pump can be adjusted to suit different needs. The excess fluid flows back from the aortic block to the reservoir. Below the aortic block there is a cardiac chamber with a thermostat that can be raised to cover the heart. This arrangement allows continuous recordings of coronary flow, left ventricular pressure (LVP), perfusion pressure, a 12-lead ECG and 8 monophasic action potentials (MAPs). The result of these multiple records is analyzed using the nOtOCORD HEM 3.3 software. This software allows the calculation of a wide range of electro-physiological and hemodynamic parameters of the heart.

Perfusion technique and perfusion media

The experiments are carried out in the constant pressure perfusion mode. The pump flow rate is set to deliver 70 ml / min and the post load is set at 50 mm Hg, ensuring a perfusion pressure of approximately 60 mm Hg. Unless otherwise specified, hearts are perfused with a prewarmed (38 ° C) modified Krebs-Henseleit solution with the following composition (mmol / l): NaCl: 118, KCl: 4.7, CaCl2,2H2O : 2.52, KH2PO4: 1.18, MlgStíJII-O: 1.64, sodium pyruvate: 2.0, NaHCO3: 24.88, glucose: 5.55. The solution is filtered through a 45 pm bottleneck filter before use.

A pH of approximately 7.4 and a suitable oxygen content of the solution are obtained by means of continuous bubbling with carbogen (O₂ 95% / cO₂ 5%). Volumes of 2 or more liters are allowed to equilibrate with carbogen for at least 20 min, while volumes less than 1 liter are allowed to equilibrate for 10 min.

Anesthesia, surgery, and experimental procedures

Male Ssc: CPH rabbits (2.5-4.0 kg), purchased from Hvidesten, Aller ^ d, Denmark are used. They are sedated with 1.2 ml of Hypnorm® (fentanyl citrate 0.315 mg / ml and fluanisone 10 mg / ml), im Ten min later, anesthesia is induced by slow iv administration of 0.55 ml of Dormicum® ( midazolam 5 mg / ml). Additionally, they receive 500 IU of heparin intravenously to prevent clotting.

Rabbits are fixed on the back, with the front legs fixed to the sides, and an incision is made to expose the trachea. Tracheostomy is performed and rabbits are ventilated with oxygen using a Ugo Basile rodent ventilator (tidal volume: 18 ml, rate: 60 bpm). The abdominal cavity is opened from the caudal end to the xiphoid process and the abdominal muscles are laterally divided on both sides. To gain access to the thoracic cavity, the diaphragm is opened below the sternum and the cut is extended bilaterally along the curvature of the rib. The mediastinum is sectioned closest to the sternum and the ribs are cut on both sides, parallel to the sternum, to allow the chest wall to be raised in the direction of the skull. Wall

ES 2 228 807 T3 raised thoracic is fixed on the head of the rabbit to offer a complete view 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 cranially to the liver to reduce return flow to the heart, and the cranial vena cava and pulmonary artery are opened to reduce volume overload to the heart. The aorta is opened and the cannula is immediately inserted, connected to the aortic block by means of an extension tube filled with perfusion fluid, to allow artificial perfusion. The ligature is tightened and the heart is removed, which is transferred to the perfusion machine. The time elapsed from clamping the caudal vena cava to insertion of the cannula is approximately 30 seconds.

The MAP's are sampled at 2000 Hz and the pressure and flow parameters at 500 Hz. The mean duration of the action potential is calculated from the 8 MAP records as the mean duration from the moment of maximum depolarization (time dV / dt Max) until the moment of 90% repolarization. This duration is designated as APD₉₀ and the dispersion of APD₉₀ is measured as the standard deviation of the 8 APD measurements₉₀.

Results

As illustrated in Figure 5, three groups were studied. Rabbit hearts were perfused with Krebs-Henseleit buffer only (vehicle; n = 11 experiments), 10^-10 mol / l of Compound 2 (n = 10 experiments), or 10^-10mol / l AAP10 (CE2; n = 3 experiments). Increased dispersion of APD₉₀ observed during acute myocardial ischemia due to hypokalemia in vehicle-treated rabbit hearts was prevented with 10^-10 mol / l of Compound 2, but not with 10^-10 mol / l of AAP10 (CE2). These findings demonstrate that Compound 2 prevents increased electrical leakage during ischemia, and suggest that the antiarrhythmic properties of Compound 2 are related to this mechanism. It has been previously reported that AAP10 (CE2) is capable of reducing the dispersion of the epicardial activation-recovery interval and diminishing the alterations of epicardial activation patterns induced by regional ischemia in the rabbit, with a maximum effect at a concentration of 10^-8 mol / l (39). In our experiments, Compound 2 effectively prevented the increase in electrical scattering 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 the myocardial infarction, because the reduction in coronary flow during ischemia and the risk zone 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 Ventricular Reentry Arrhythmias in Dogs

The influence of cleft junctions in arrhythmias has been clarified in studies on the influence of connexin 43 (Cx43) on the conduction properties of the ventricle (33). In a suppressed heterozygous mouse, deficient in Cx43, there is twice the frequency of spontaneous VT with coronary artery occlusion (CAO) (3). Ischemia negatively regulates the effect of Cx43 after 6 hours in the dog, showing a 60% reduction in Cx43 from end to end and a 49% reduction in Cx43 from side to side (40), probably secondary to the dephosphorylation. In subacute ischemia in the dog, epicardial reentry is facilitated in areas where Cx43 is reduced (25). Thus, reentry mechanisms may be critically dependent on ischemia-mediated down-regulation of Cx43 and, presumably, cleft junction strength, leading to heterogeneity of recovery and predisposing conduction properties. to VT and VF.

When the heart has been transferred to the machine, an incision is made in the left atrium to allow insertion of a fluid-filled balloon (size 12) into the left ventricle to measure the pressure in the left ventricle. The volume of the balloon is adjusted to give a final diastolic pressure of approximately 10 mm Hg. The ring of electrodes for 12-lead ECG measurements is placed around the heart, at the level of the coronary sulcus, leaving the tip of the left atrium between the 5^to and 6^to precordial shunt. The 8 MAP electrodes are placed on the heart, in direct contact with the epicardium. MAP5 and MAP6 are located over the right ventricle, while the other MAP electrodes are evenly distributed over the left ventricle. This method is similar to that used by Zabel et al. (38). When all the electrodes are in place, the cardiac chamber is raised to ensure that the heart is immersed in Krebs-Henseleit solution at 38 ° C at all times.

Before starting the experiment, a ligature is placed around the major branch of the circumflex artery, which supplies a large part of the left ventricle. The two ends of the ligature are passed through a small plastic tube that allows the induction of an ischemia by pressing the plastic tube against the heart, and clamping the ends of the ligature. All hearts are allowed to equilibrate for 15 min before starting the experiment.

The program at the time of the experiments is as follows:

1. 15 min perfusion with normal Krebs-Henseleit buffer (equilibration period).

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

ES 2 228 807 T3

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

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

At the end of the experiment, the hearts are perfused with Evans blue stain to assess the area of ​​risk of infarction. The atria and the right ventricle are divided and the remaining left ventricle is separated into the area stained with Evans blue and the area that does not stain, that is, the area of ​​risk. The two areas are dried with paper towels and weighed to determine the percentage risk zone for heart attack.

Records

The following parameters are continuously recorded: coronary flow, left ventricular pressure, perfusion pressure, 12-lead ECG, and 8 MAP records. ECG and ...

In the studies described below, the present inventors examined the effect of Compound 2 on reentry arrhythmias during CAO-induced myocardial ischemia of the anterior descending artery. Preparation of the animals

Three anesthetized dogs were studied, with the thoracic cavity open to facilitate the placement of electrodes for mapping. Α-Chloralose was administered as a bolus (200 mg / kg) and then as a constant infusion of 8 mg / kg / h (dissolved in polyethylene glycol, MW = 200). The femoral vein and artery were cannulated for administration of fluid and drugs, and for measurement of ascending aortic pressure, respectively.

Electro-physiological methods

The sinus node was clamped and the atrial appendage activated with a programmable stimulator with constant current outputs at twice the diastolic threshold. The activation rate was&gt; 200 beats / min to control heart rates. Ventricular single pole placement of a multipolar needle in the normal zone used an anode (7 cm² stainless steel) in the abdominal muscle. The Endocardial Effective Refractory Period (ERP) was measured by the conventional technique of extra stimuli. The late ventricular diastolic threshold was measured during each intervention; the trigger current was four times the threshold.

Electrogram record

Test sites were selected along the body of 16 polar needles (J. Kassell, Fayetteville, NC); each pole completely surrounds the body of the needle to prevent the directionality of the needle orientation from registering adjacent Purkinje fibers. Six bipolar electrograms (1 mm spaced) were recorded sequentially across the needle body, amplifying up to 1000 times, filtering from 3-1300 Hz, and recording through the oscilloscope during atrial activation. Four intramural electrograms are recorded on each multipolar needle. Epicardial electrograms are activated last on each needle. An arrangement of 23 multipolar electrodes was used, with 17 of them located in the area of ​​risk of infarction in the anterior descending coronary artery, and 6 in the surrounding normal area, as described in detail by Xing and Martins (41). The distance between needles measured at the epicardium varies by 6-10 mm in dogs weighing 12-16 kg.

Induction of arrhythmias

The endocardium was activated at the base, apical septum, and on the free lateral wall, immediately outside the risk zone. After determining the ERP, the S1-S2 interval was prolonged by 4 msec&gt; ERP and an S3 was added to the protocol initially, with an S2-S34 interval equal to 50 msec&gt; S1-S2. The intervals were shortened until it was impossible to detect them. If ventricular tachycardia was not induced at any trigger site, an extra third (S4) and fourth (S5) stimulus were added. The present inventors performed a complete ventricular tachycardia induction protocol prior to OAC to exclude artificial ventricular tachycardias due to needle mass or ischemia due to needles compromising blood flow. After confirming physiological blood gases and adequate anesthesia, the anterior descending CAO was ligated. After 60 minutes, the infarct size is approximately 75% of the risk zone and a further increase in the infarct zone is negligible. Ventricular tachycardia was then induced at least twice before the interventions. The tests were repeated every 20 minutes and continued up to 3 hours after OAC. With each intervention, the ERP of normal heart muscle was recorded.

Arrhythmia mapping

Epicardial mapping was performed using a computerized system from BARD Electrophysiology Inc. The software encompasses 64 data channels with a 12-bit resolution, with a sample rate of 1 kHz / channel. Filtering was 30-300 Hz. Eight second windows are triggered externally, including up to 8 seconds of data.

ES 2 228 807 T3 before the trigger signal. This system is used to record from the 2-3 outer epicardial bipoles on each recording electrode.

A customized computer software system was used to resolve the Purkinje signals from the 3 internal bipoles in each endocardial multipolar electrode by sampling at 3 kHz per channel. The filters incorporate the Purkinje frequency (3-1300 Hz). The sampling frequency was 235 kHz. The PC was interfaced with an amplifier consisting of an analog signal multiplexer and 64 instrument amplifier circuits. Each of them had selectable gain (up to 1000) and bandwidth cuts. The acquisition, processing and visualization of the electro-physiological data were carried out by means of the software. High speed acquisition allowed the present inventors 14 sec of data, including up to 8 sec before the trigger signal.

Mapping analysis

The mapping analysis was carried out offline. The computer selects activation times using the first maximum dv / dt. Electrograms were considered impossible to interpret only if they were not reproducible with stimuli; there was no exclusion based on the voltage of the electrograms. Electrotonic or far field potentials are considered to be present when substantial voltage and dv / dt loss occurs in a complex, with smaller coupling ranges of refractory capacity. Isochrons are drawn manually. The mechanisms of ventricular tachycardia are defined as follows: reentry ventricular tachycardia occurs when the lead that registers the earliest activity, which occurs after unidirectional block, is located immediately adjacent to the site of the last activation of the anterior complex and is records diastolic activity between complexes. Epicardial reentry is almost always recorded in acute ischemia, with retrograde (epicardial to endocardial) activation of the wall.

Experimental protocol

After heart instrumentation and after one hour of OAC, activation protocols to induce ventricular tachycardia were carried out to confirm reproducible induction capacity (double induction of ventricular tachycardias with similar surface morphologies), or induction failure. (activating the three sites twice without ventricular tachycardia for one hour). In three dogs with re-inducible ventricular tachycardia, a reentry mechanism was identified. In these three dogs, Compound 2 was administered as an iv bolus injection, followed by constant infusion for 30 min at three dose levels in two dogs, while the third was treated with saline. The extra stimulus test was then repeated throughout the entire protocol at all sites to determine whether or not ventricular tachycardia was present. Compound 2 was administered iv at three dose levels to produce plasma concentrations of 10^-10 M (bolus: 0.1 pg / kg; infusion: 2 ng / kg / min), 10 ⁹ M (bolus: 1.1 pg / kg; infusion: 21 ng / kg / min), and 10 ⁸ M (bolus: 11 pg / kg; infusion: 210 ng / kg / min), respectively.

Results

The first animal, of which Figures 6-9 are attached, was studied after the induction of sustained monomorphic VT. Induction was carried out only from the lateral ventricular activation site, two successive times that took place at 2 hours and 10 minutes and which were repeated at 2 hours and 20 minutes after OAC. An activation map after septal activation, which did not cause VT, is presented in Figure 6. This shows the normal orthograde activation pattern, with early activation of the PURK activation site, activated at 6 msec after stimulation, and late activation of the activated epicardial site, at the latest, at 107 msec. Note that the activation time adjacent to 86 msec, immediately east and south of the last activation in the epicardium is ES in Figure 7. The epicardial activation of the first VT complex, which begins at -44 msec before the onset of the superficial QRS, and which corresponds to the electrogram recorded in EC in Figure 7.

In Figure 7, sustained monomorphic ventricular tachycardia (VT) is shown, induced by stimulation at the lateral epicardial ventricular activation site causing a reentry circuit. Activation occurs in a double loop reentry, activating first at -17 msec and then at 57 msec in the northwestern loop. The southeast loop is activated first at 2 msec, 31 msec, and then at 57 msec. The protocol that induced VT was S1-S2 = 150, S1-S3 = 280, S1-S4 = 390, S1-S5 = 490 msec. The figure illustrates epicardial electrograms (E-) recorded with surface lead ECG II and RSV during the second to fifth extra-premature stimulus (best seen in EL), ensuring 4 VT complexes. Electrograms are recorded from the activation site of the border (L), lateral, and east (E), north (N), central (C), subepicardial (SE), low EC, as well as south (S) and Northwest (NW) and Southwest (SW) of EC. EC shows gradually dissociated electrograms, the latter showing a blockage of the second component (perpendicular lines). The adjacent conduction delay in ES allowed conduction to proceed around and back to the central site (EC), continuing reentry excitation between EC and ES (straight line and arrow line).

Figure 8 illustrates the activation map during epicardial activation of the first ventricular tachycardia complex, which starts at -44 msec before the onset of surface QRS and corresponds to the electrogram recorded in EC in Figure 7. The activation has place in a double-loop reentry that activates first at -17 msec and then continues up to 57 msec in the northwestern loop. This activation map also illustrates retrograde activation of the ventricular wall during reentry arrhythmia.

ES 2 228 807 T3

Compound 2 was administered in three increasing iv doses, which did not alter mean arterial pressure (MAP = 80 mm Hg). The effective refractory period in control was 150 msec, 154 msec after the lowest dose and 148 msec with the last and highest dose. The VT that could be induced was the typical epicardial reentry tachycardia shown in Figures 7 and 8. After the first dose of Compound 2 (bolus: 0.1 pg / kg; infusion: 2 ng / kg / min), it was no longer possible to induce a VT, despite the fact that the induction protocols that induced VT before the administration of Compound 2; the protocol that induced VT before drug administration was S1-S2 = 150, S1-S3 = 280, S1-S4 = 390; S1-S5 = 490 msec, and during the infusion of Compound 2 the intervals were 150, 270, 370 and 470 msec, respectively. Vt was not induced until an hour and a half after starting the infusion of the minimum dose of Compound 2. In Fig. 9 the electrocardiographic recordings are shown after iv administration of the lowest dose of Compound 2. These results demonstrate that Compound 2 effectively blocked reentry VT in this dog.

A second dog with inducible VT was studied, this time from two activation sites in the border areas, located laterally and in the septum. Also on this occasion, Compound 2 did not produce any change in MAP, which started at 90 mm Hg and ended at 90 mm Hg. The effective refractory period at the two induction sites was maintained at 163 and 144 msec, respectively, throughout the Compound 2 test period, which began 85 minutes after OAC and continued for an additional 2 hours. After the minimal dose of Compound 2, the VT induced from 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 VT induced from the septal site was also epicardial reentry before the administration of Compound 2, but after the administration of Compound 2, the epicardial reentry was completely blocked. Thus, in these two experiments, it was possible to induce epicardial reentry VT before the administration of the lowest dose of Compound 2 and, after administration of the substance, it was not possible to re-induce any reentry with any dose.

Finally, an additional animal was subjected to electro-physiological tests during the time period used in the two experiments described above, without introduction of Compound 2, but using saline. Epicardial reentry was induced one hour after OAC and the same VT morphology and reentry mechanism was induced 1.5-2.5 hours as OAC. Thus, the reproducibility of reentry VT in this time-controlled experiment is consistent with the notion that Compound 2 is an effective antiarrhythmic compound under the conditions of reentry arrhythmias.

These experiments demonstrate that Compound 2 is effective in the prevention and / or treatment of fatal reentry arrhythmias. Consequently, it is the purpose of the present invention to offer 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 fulfilled with the present peptide compounds such as the compounds of the formulas I, XII, XIII, XIIIa, XIV, XV and XVI and of the formulas 2-12 of the present invention, more specifically the compounds of the Synthesis Examples 1-47 of this document.

Experimental example 8

Effect of gap junction openers on bone tissue cells

Background

Osteoblasts, which are bone tissue-forming cells, and osteocytes are well connected. Osteoblast-osteoblast, osteoblast-osteocyte and osteocyte-osteocyte connections have been found in bone preparations examined under electron microscopy (42). The most interesting connexin in relation to bone tissue is Cx43, as it happens in the heart. In bone cells, the expression of these proteins is associated with the expression of some specific osteoblast proteins. Calciotropic hormones can also regulate the expression of cleft junction proteins.

Human osteoblasts (HOB) and bone marrow-derived stromal cells (BMSC) have been shown to express Cx43 and Cx45. As demonstrated by the Lucifer Yellow (LY) staining transfer technique, they are functionally coupled (43). Rat osteoblast cell lines differ from human primary cultures; ROS 17 / 2,8 cells express only Cx43 and are well coupled, whereas UMR 10601 predominantly express Cx45 and are poorly coupled to staining (44). The two rat osteoblastic cell lines are electrically coupled. Transfection of Cx43 into UMR cells results in cells highly coupled to staining. Thus, Cx43 allows the transfer of LY and other larger molecules, while Cx45 does not allow this step. In contrast, introduction of Cx45 q cells expressing Cx43 reduces coupling to staining. In osteoblast differentiation, the expression of Cx43 is modified; thus, the more mature the osteoblasts, the greater the expression of Cx43 (45).

The effect of different stimuli on bone cells and the relationship with variations in the communication of the cleft junctions has been investigated. It is well known that moderate mechanical stress on bone increases bone density. To limit this situation, ROS 17 / 2.8 cells were exposed to cyclic stress, which resulted in increased staining coupling of the cells. Cyclic stress applied to loosely coupled UMR 10601 cells also resulted in increased coupling to staining, but less intense compared to ROS cells. No increase in mRNA was found for Cx43, but rather

ES 2 228 807 T3 plus phosphorylated forms of Cx43, indicating that cyclic stress on osteoblasts increases cleft junction communication between cells by modulating the intracellular localization of the Cx43 cleft junction protein. The same group has shown that transfection of Cx43 to loosely coupled UMR 106-01 cells not only increases the coupling to staining (46), but also increases the expression of the products of mature osteoblasts, osteocalcin and bone sialoprotein ( BSP). Decreased coupling between osteoblast cells (ROS) through transfection of Cx45 into cells reduces the expression of osteocalcin and BSP, genes that are essential for the formation and calcification of the bone matrix. A recent study has shown that Cx43-free mice exhibit poor bone tissue formation and development compared to wild-type mice (47). Thus, an intercellular communication network is required for the complete elaboration of a differentiated osteoblastic phenotype, as well as for the formation and metabolic cycle of bone tissue. Therefore, poor gap junction communication can result in increased bone loss.

Similarly, cleft junctions have been shown to be partially responsible for the propagation of intercellular calcium signals in bone cells. The mechanical stimulation of a human osteoblast is a cellular monolayer, in vitro, it induces a pulse of calcium that propagates towards a series of adjacent cells. The propagation of this signal involves the passage of a messenger molecule through the cleft junctions, with subsequent activation of adjacent cells (48,49). These signals probably propagate through the cellular network of bone tissue in vivo, in response to mechanical stimuli, and could be responsible for the increase in bone tissue formation in response to mechanical load on the bone.

The communication of the cleft junctions and the effect of the calciotropic hormones are linked. Stimulation with 1,25- (OH) has been shown to₂-vit-D₃ of fibroblasts in human skin enhance communication through cleft junctions, as well as increase the levels of Cx43 protein and mRNA (50), but only in the presence of functional vitamin D receptors (VDRs). Loss of Cx43 expression has been shown to decrease the responsiveness of cells to PTH, with no variations in the number of PTH receptors or the cAMP response (51). In contrast, PTH and PGE2 enhance communication between cleft junctions in osteoblast cell cultures through two mechanisms: a rapid initial redistribution of Cx43 to the cell membrane, and a subsequent stimulation of the expression of the Cx43 gene (52). In this way, the modulation of intercellular communication represents a mechanism by which osteotropic factors regulate the activity of bone tissue-forming cells.

It is quite possible that cleft junction intercellular communication is shown to be one of the most important mechanisms by which bone cells coordinate their activities and responses to mechanical and hormonal stimuli. In this way, if it were possible to increase the communication through cleft junctions between bone cells by pharmacological means, the activity of osteoblasts could be increased, enhancing the formation of bone tissue in vivo.

Cardiac myocytes are also connected by cleft junctions and, as in osteoblasts, the predominant connexin is Cx43. Certain compounds have been shown to increase gap junction communication between cardiac myocytes, of which artificially synthesized AAP10 (CE2) is the best investigated. Cardiac myocytes respond to ischemia with decreased cell coupling. In in vitro experiments, the addition of AAP10 (CE2) to cardiac myocytes exposed to ischemia reestablished part of the lost cellular coupling. If cardiac myocytes are able to respond to this group of compounds with increased cleft junction coupling, osteoblasts could do the same. In this case, it is evident that the increase in cell coupling could well be accompanied by an increase in osteoblast maturation and activity, and the subsequent increase in bone tissue formation. To investigate this hypothesis, the present inventors have examined the effect of Compound 2 on GJIC in human osteoblasts and rat osteosarcoma cells. Additionally, the inventors have studied the effect of Compound 2 on a label (ie, alkaline phosphatase) for human osteoblast activity and bone tissue formation.

Methods

Cell culture

Human osteoblasts (hOB): cells were isolated from human bone marrow obtained by puncturing the posterior iliac crest of healthy volunteers (ages 20-36 years: 10-15 ml of marrow material were collected in 15 ml of PBS + Ca, Mg (Life Technologies, Cat. No. 14040) with 100 U / ml heparin (Sigma, Cat. No. H-3149). The mononuclear fraction of the marrow was isolated on a Lymphoprep gradient (Nycomed Pharma, No. Cat. 1001967), by centrifugation at 2200 rpm for 30 min. After harvesting, the mononuclear fraction was washed once with culture medium and centrifuged at 1800 rpm for 10 min. The cells were then counted and plated with culture medium at 8 x 10⁶ 100 mm cells / plate. HOB Medium (all reagents sourced from Life Technologies): MEM are Phenol Red with Glutamax (Cat. # 041-93013), supplemented with 10% heat-inactivated fetal calf serum (Cat. # 10106 ) and 0.1% Penicillin / Streptomycin (Cat # 15140). The medium was changed the next day and the cells were cultured at 37 ° C in 5% CO2, with a change of medium every 7 days. After 3-4 weeks of culture, the cells had reached 70% confluence. The medium was then supplemented with 100 nM dexamethasone (Sigma, Cat. No. D-4902) for 7 days. The cells were then plated for video imaging experiments: a 25mm glass coverslip # 1 was placed on a 35mm plate

ES 2 228 807 T3 (or each well of a 6-well multiple plate), cells were plated at 2.5 x 10⁵ cells / coverslips and cultured for 2-3 days before use.

ROS 17 / 2.8 cells: cells were grown in 100 mm plates at 37 ° C with CO₂ at 5% and change of medium every 2-3 days. ROS medium (all reagents sourced from Life Technologies): MEM (Cat. # 31095) supplemented with 10% heat-inactivated fetal calf serum (Cat. # 16170), 1% NEAA (# 11140), 1% Sodium Pyruvate (Cat. No. 11360), 1% L-Glutamine (Cat. No. 25030), and 0.1% Penicillin / Streptomycin (Cat. 15140). For video imaging experiments, cells were seeded on coverslips at 2-3 x 10⁵cells / coverslips and cultured for 2-3 days before use.

Calcium wave measurement

Cells grown on coverslips were loaded with 5 pM fura-2-AM (Molecular Probes, Cat. No. F-1221), for 30 minutes at 37 ° C, and incubated in fresh medium for 20 minutes. Next, the coverslips were fixed in a PDMI-2 culture chamber (Medical Systems Corp.), kept at 37 ° C with CO perfusion.₂, on a Zeiss Axiovert microscope. Intercellular calcium waves were induced by mechanical stimulation of a single cell, using a borosilicate glass micro-pipette attached to an Eppendorf 5171 micro-manipulator. Images were obtained using a MetaMorph imaging system (Universal Imaging). Excitation light (340 and 380 nm) was provided by means of a Monochromator (TILL Photonics GmbH). Images were acquired with an intensified CCD camera (Dage MTI) and digitized with a Matrox MVP imaging plate.

Microinjection

Cells grown on coverslips were placed under the microscope as previously described. Microinjections were made using the Eppendorf 5171 micro-manipulator and the Eppendorf Transjector 5346 system. A micro-pipette was loaded with 10 mM Lucifer Yellow (LY) solution (Sigma, Cat. No. L-0259). A cell from the monolayer was carefully injected with LY for 30 seconds, the cell was micro-pipetted and, after 30 seconds, the number of cells showing staining blotting was counted. The excitation light for LY was 430 nm and the images were acquired as previously described.

Alkaline phosphatase assay

Day 1: cells were seeded 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 in the cells was changed.

Day 4: (Day 3 for ROS): cells were washed with 200 µl MEM, 0.1% BSA (Sigma, Cat. No. A-9418), 200 µl MEM, BSA were added to the cells 0.1% containing various concentrations of Compound 2, and the culture was continued for 4 days (2 days for ROS cells).

Day 8: (Day 5 for ROS): The alkaline phosphatase (ALP) assay is a colorimetric method to measure enzyme activity, and was carried out using the Alkaline Phosphatase kit (Sigma, Cat. No. 104-LL ): 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. The absorbance was measured using a plate reader at 405 nm.

Effects of Compound 2 on GJIC

To assess the ability of cleft junction modifiers to increase communication through intercellular calcium signals, mediated by cleft junctions, human osteoblast monolayers were loaded onto fura-2 glass coverslips. During real-time imaging, mechanical stimulation was performed with a glass micro-pipette. There was an increase in intracellular calcium, with subsequent diffusion of the signal to surrounding cells. The mean number of cells in the wave was 6.5 cells. Next, 100 pM adenosine tri-phosphate (ATP) was added to desensitize the purinergic receptors. After desensitization, the propagation of the calcium wave depends exclusively on the GJIC. With ATP stimulation, an increase in intracellular calcium was observed in most cells in the field of view. Once again, a single cell was mechanically stimulated. Now the wave propagation was limited to an average of 4.5 cells in the wave. Compound 2 was added at a concentration of 10^-8 mol / l the bath solution. An increase in intracellular calcium concentrations was observed in most cells in the field of view. After a 10 minute incubation with Compound 2, a single cell was mechanically stimulated again. Again, the intracellular calcium concentration increased in the stimulated cell, with subsequent wave propagation. In this case, the wave extended to an average of 6.2 cells (Figure 10), with a significant increase compared to that reached before the addition of Compound 2.

In order to test the ability of the compound to restore the suppressed coupling of cleavage junctions30

ES 2 228 807 T3 ra, similar experiments were performed with the ROS 17 / 2.8 (ROS) osteoblast cell line, but after incubating the cells for 48 hours under hypoxic conditions, with only 3-6% O2 , that is, conditions known to reduce cell coupling. ROS cells in monolayers were loaded with fura-2 and, under the same previous conditions, a mechanical stimulation was performed. Since ROS cells do not express purinergic receptors, pretreatment with ATP was not performed. Following stimulation, the intracellular calcium concentration increased in the stimulated cell and a wave was initiated, which spread 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^-8M. After 10 minutes, mechanical stimulation was repeated. Now, the wave propagated to an average of 5.4 cells (n = 18) (Figure 11), which represents a significant increase compared to that obtained before adding the compound. Consequently, Compound 2 effectively increases cleft junction mediated intercellular calcium waves.

To evaluate the effect of the compound on direct cell coupling, microinjection experiments were carried out according to the method described above. Lucifer Yellow (LY) stain was injected into a single human osteoblast in a monolayer. After 30 seconds, the number of cells containing the stain was evaluated. Under physiological conditions, the staining spread to a mean of 14 cells (n = 19). To suppress cell coupling, the cells were now incubated under hypoxic conditions (3-6% O₂) for 48 hours. Cell coupling was then re-evaluated by LY microinjections, and in this case, the staining happened only to a mean of 7 cells (n = 10). Compound 2 was added to the medium and, after 10 minutes, the staining coupling was re-evaluated. Already after 10 minutes of incubation with Compound 2, cell coupling increased, with transfer of staining to 9 cells (n = 11).

Similar experiments were carried out with ROS cells. The basic coupling under physiological conditions in ROS cells was 12 cells (n = 19). After 48 hours of incubation in 3-6% O₂, a reduction in staining transfer was observed at 9 cells (n = 27). Once again, Compound 2 was added to the bath solution, and cell coupling was restored to pre-hypoxia levels, with a mean transfer of staining to 12 cells (n = 27) (Figure 12). Therefore, Compound 2 is capable of increasing communication between the cleft junctions and restoring hypoxia-induced reductions in cell coupling.

Hypoglycemia-induced metabolic stress is also known to decrease communication between cleft junctions. Therefore, the present inventors decided to evaluate whether Compound 2 could reverse the reduction in cell coupling induced by hypoglycemia. Human osteoblasts were grown in monolayers on glass coverslips and loaded with fura-2. After desensitization 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 spanned a mean of 3.2 cells (n = 19). The medium was changed to medium without glucose and, after 8 minutes, another mechanical stimulation was performed. Now, the wave was practically blocked, with a wave propagation of only 1.4 cells (n = 20). Compound 2 was added to the medium in a final concentration of 10^-8 M. A final stimulation was performed and the waveform was almost completely restored, with a mean extension of 2.9 cells (n = 18) (Figure 13). Therefore, Compound 2 is capable of restoring hypoglycemia-induced cell uncoupling.

Finally, to evaluate the effect of Compound 2 on bone tissue formation and osteoblast activity, the present inventors measured the effect of the compound on alkaline phosphatase (ALP) activity of cells. Human osteoblasts were stimulated with different concentrations of Compound 2, from 1 x 10^-13up to 1 x 10^-6, and were compared with untreated controls. Under normal culture conditions, Compound 2 increased Alp activity with most of the concentrations tested, except the maximum (10⁶ mol / l), which can be toxic (Figure 14).

Additionally, the effect of the compound on ALP activity under hypoxic conditions was also tested. Human osteoblasts were cultured for four days in 5% O2. The medium was enriched with different concentrations of Compound 2, and they were compared with the responses during normal conditions. During hypoxia, the stimulation of ALP activity induced by Compound 2 was about 15% greater than during normoxia at all concentrations in the range of 10 ¹¹ to 10 ⁸ mol / l (Figure 15).

In summary, these results demonstrate that Compound 2 is capable of normalizing attenuated GJIC among human osteoblasts during hypoxia. Additionally, Compound 2 stimulates the production of alkaline phosphatase, suggesting that Compound 2 is capable of stimulating osteoblast activity and therefore bone tissue formation. Thus, Compound 2 may be useful in treating bone diseases with altered bone formation with respect to bone resorption. The effect of Compound 2 on inter-cell coupling during hypoxia indicates 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 of bone tissue.

From these experiments, it can be concluded that the substances of the present invention, which 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 other diseases such as Cushing's syndrome or osteogenesis imperfecta. In most cases of osteoporosis, however, no other disease is evident. One form manifests in children or young adults of both sexes and with normal gonadal function, which is often referred to as idiopathic osteoporosis, although most of the remaining forms are also of unknown pathogenesis. Type I osteoporosis occurs in a subset of postmenopausal women between the ages of 51 and 75, and is characterized by accelerated and disproportionate bone loss

ES 2 228 807 T3 trabecular. Common complications are vertebral body and distal forearm fractures. A decrease in parathyroid function may compensate for increased bone resorption. Type II osteoporosis occurs in women and men older than 70 years of age and is associated with fractures of the femoral neck, proximal humerus, proximal tibia, and pelvis, that is, sites containing both cortical and trabecular bone tissue. In addition to osteoporosis, substances that increase GJIC can also increase bone tissue formation in metabolic bone diseases such as rickets and osteomalacia, and in osteoporosis due to chronic administration of glucocorticoids or chronic renal failure. Therefore, one purpose of the present invention is to offer compounds for the preparation of medicaments useful in the prevention and / or treatment of osteoporosis. This objective is achieved with the present peptide compounds.

Effects of gap junction openers on cartilage

Articular cartilage is a tissue designed to resist compression during joint movement and, in vivo, is subjected to a wide range of mechanical loading forces. Mechanical sensitivity has been shown to influence chondrocyte metabolism and cartilage homeostasis. In many cell types, mechanical stimulation induces increases in the cytosolic concentration of Ca²⁺, which spreads from one cell to another in the form of an intercellular Ca wave²+. Communication between cells through gap junctions underlies tissue coordination of metabolism and sensitivity to extracellular stimuli. The permeability of the cleft junctions to intracellular second messengers allows transduction pathways to be shared among several cells, ultimately resulting in coordinated tissue responses. Ca signaling has been investigated²+ mechanically induced in chondrocytes, and communication between cleft junctions has been shown to be essential for Ca signaling²⁺ mechanically induced in chondrocytes (53). Additionally, mechanical stimulation activates phospholipase C, thus leading to an increase in intracellular inositol 1,4,5-triphosphate. The second messenger, through the cleft junctions, stimulates the release of Ca²⁺ intracellular in neighboring cells and this system is considered to be very important for coordinated signaling in chondrocytes during mechanical stress, being able to provide a mechanism for coordinated metabolic activity during metabolic stress in chondrocytes (53,54). The predominant connexin in cartilage is Cx43, which, in addition to its role in inter-cell regulation of metabolism and signaling, is essential for normal chondrogenesis (47,55).

Thus, it appears that the substances of this invention, which increase CJIC, can be used for the prevention and / or treatment of arthropathies involving an altered coupling between cells.

As the present inventors have demonstrated in human osteoblasts, these inventors also suggest that substances that increase GJIC can be used for the prevention and / or treatment of joint diseases involving metabolic stress. These would include any form of arthritis associated with reduced vascularization or scarring of the fractured chondral tissue. Accordingly, it is an object of the present invention to provide compounds for the preparation of medicaments useful in the prevention and / or treatment of joint diseases, including arthritis. This objective is achieved with the present peptide compounds.

Effects of gap junction openers on cancer

Cleft junction permeability and GJIC regulation occur at different levels in the cell. The reduction or absence of GJIC can be the consequence of variations in the expression of Cx during transcription and translation, alteration of post-translational processing and alteration of the assembly and insertion of connexons in the plasma membrane. An unusual feature of Cx is its short half-life compared to other membrane proteins. The rapid metabolic cycle of connexins has been shown to be between 1.5 and 2 h. The degradation of Cx has been shown to be dependent on phosphorylation, leading to destabilization of some connexin subtypes. The rapid rate of the metabolic cycle provides an additional mechanism by which GJIC can be regulated by substances that affect Cx mRNA half-life, translation, intracellular transport, and Cx assembly at cleft junctions.

Yet another way of regulating the permeability of the gap junctions is the complete or partial closure of the channels of the gap junctions under certain circumstances, by means of mechanical torsion of the six subunits or connexion. Tumor promoters, which reduce GJIC, are known to cause barrier formation at cleft junctions. Tumor promoters are agents that enhance or accelerate carcinogenesis when administered repeatedly after tumor initiation. The mechanisms by which tumor promoters modulate GJIC are not fully understood, but there is evidence to confirm that tumor promoters can affect GJIC by altering Cx phosphorylation and / or inhibiting Cx expression and assembly. Recent results have shown that in vivo retrovirus-mediated gene transfer of connexin 43 in malignant processes with low GJIC capacity significantly reduces the capacity for tumor formation (56). As further confirmation of an essential role for normal GJIC in cancer prevention, Cx32-deficient mice have been shown to exhibit a very high incidence of spontaneous liver tumors, as well as an increased susceptibility to developing chemically-induced liver tumors (57). Additionally, the tumor-promoting action of phenobarbital requires functional Cx32 for tumor progression (58). This indicates that GJIC uncoupling is important for the oncogenic actions of phenobarbital (58).

Carcinogenesis is characterized by the progressive alteration of growth control mechanisms in

ES 2 228 807 T3 involving growth factors, oncogenes and tumor suppressor genes. Since the alteration of the GJIC could result in the alteration of growth control, the effect of growth factors and oncogenes on the GJIC could be crucial for the genesis of tumors. Several oncogenes have been shown to mediate down-regulation of GJIC (59). It is shown that pp60^v-be mediates the closure of the Cx43 cleft junction by means of a ball-and-chain mechanism, involving phosphorylation of the C-terminal serine residue by MAP-kinase (59). Interestingly, in some cases, cells transfected with oncogenes could communicate with each other, but lack heterologous communication with adjacent normal cells.

The permeability of cleft junctions in tumor cells, using the stain transfer assay, was lower than that of GJIC in surrounding liver tissue. Interestingly, many tumors are encapsulated in an extracellular matrix-like structure, and are physically separated from normal tissue.

Neoplastic transformation in normal human tissues occurs as a consequence of an accumulation of genetic alterations. However, a general theme in carcinogenesis and tumor genesis is the down-regulation of GJIC. The various connexins are expressed in a tissue-specific manner. Cx43, Cx26, Cx32 have been detected in normal breast tissue. The expression level of Cx43 was analyzed in a set of human breast cancers. Cx43 cleft junctions were not seen in ductal carcinomas in situ, infiltrating ductal carcinomas, and infiltrating lobular carcinomas, and appear to be independent of estrogen, progesterone, and erbB2 receptor status. On the contrary, human mammary cancer cell lines and rodent mammary carcinoma tissues have shown a negative regulation of Cx43, which was shown to be found at the mRNA level, suggesting the existence of a transcriptional mechanism for protein descent. Cx43 in breast cancer (60). Another example of the connection between cancer and GJIC is hepatocellular carcinoma, in which the removal of connexin 32 has been shown to be prone for this specific type of cancer (57). Studies with oval cells have indicated that they can differentiate into hepatocytes and that neoplastic derivatives of oval cells can produce both hepatocellular and biliary neoplasms. The specific connexin expressed by the differentiating oval cells determines whether it communicates with hepatocytes or biliary epithelial cells. This communication may be necessary for further differentiation and regulated growth of differentiating oval cells, and GJIC alteration may contribute to the formation of hepatocellular and cholangiocellular neoplasms. Thus, GJIC may be the key factor in oval cell differentiation, and a blockage of GJIC may promote its neoplastic transformation. Additionally, in vitro analyzes of tumor invasion in malothylate-treated rat lung endothelial cells have shown that malothylate stimulated the development of inter-cell adhesion via cleft junctions, resulting in inhibition of tumor cells (61). Taken together, these findings largely confirm the hypothesis that disruption of GJIC is a critical event in carcinogenesis and that the substances of the present invention, which increase GJIC, could be beneficial in cancer therapy. Accordingly, a further purpose of the invention is to offer new compounds that increase GJIC. The present inventors suggest that the peptide compounds of the invention may be especially advantageous as drugs for the treatment of cancer due to their low effective concentration and consequently low toxicity.

Experimental example 9

Effect of Compound 2 on DDT-induced cleft junction reduction in human osteoblasts

Protocol and results

The compound 1,1-bis- (p-chlorophenyl) -2,2,2-trichloroethane, also known as the insecticide DDT, is an inhibitor of gap junction communication and exhibits tumor-promoting ability. It inhibits cell-to-cell communication by reducing the number and size of cleft junctions, as well as by reduced cellular levels of phosphorylated (active) forms of the cleft junction protein Cx43. These actions are considered fundamental to the oncogenic properties of the compound (62-64). Therefore, compounds with the ability to prevent tumor promoter-induced GJIC decline may be potential candidates for use in protection against tumor promotion and in cancer treatment (65). To examine whether the substances of this invention prevent tumor promoter-induced reduction of GKIC, the present inventors have studied the effects of Compound 2 on DDT-induced uncoupling in human osteoblasts.

Methods

Cell culture

Human osteoblasts: human bone marrow cells were isolated, obtained by puncturing the posterior iliac crest of healthy volunteers (20-36 years of age): 10-15 ml of marrow material were collected in 15 ml of PBS + Ca, Mg (Life Technologies, Cat. No. 14040), with 100 U / ml Heparin (Sigma, Cat. No. H-3149). The mononuclear fraction of the marrow was isolated on a Lymphoprep gradient (Nycomed Pharma, Cat. No. 1001967), by centrifugation at 2200 rpm for 30 min. After harvesting, the mononuclear fraction was washed with culture medium and centrifuged at 1800 rpm for 10 min. The cells were then counted and plated.

ES 2 228 8 07 T3 in culture medium, at 8 x 10⁶ 100 mm cells / plate. HOB Medium (all reagents purchased from Life Technologies): MEM without Phenol Red with Glutamax (Cat. # 041-93013), supplemented with 10% heat-inactivated fetal calf serum (Cat. 10106) and 0.1% Penicillin / Streptomycin (Cat. # 15140). The medium was changed the next day and the cells were cultured at 37 ° C in CO₂ at 5%, with a change of medium every 7 days. After 3-4 weeks of culture, the cells reached 70% confluence. The medium was then supplemented with 100 nM Dexamethasone (Sigma, Cat. No. D-4902) for 7 days. The cells were then plated for video imaging experiments: a 25 mm glass coverslip # 1 was placed in a 35 mm plate (or each well of a 6-well plate), the cells were seeded at 2.5 x 10⁵ cells / coverslips and cultured for 2-3 days before use.

Microinjection

The cells were cultured on coverslips and fixed in a PDMI-2 culture chamber (Medical Systems Corp.), kept at 37 ° C with CO perfusion.₂, on a Zeiss Axiovert microscope. Microinjections were carried out using an Eppendorf 5171 micro manipulator and the Eppendorf Transjector 5346 system. A micropipette was loaded with 10 mM Lucifer Yellow (LY) solution (Sigma, Cat. No. L-0259). A cell from the monolayer was carefully injected with LY for 30 seconds, the cell was micro-pipetted, and after 30 seconds the number of cells showing staining blotting was counted. Excitation light (430 nm) was provided by a monochromator (TILL Photonics GmbH). Images were acquired with an intensified CCD camera (Dage MTI) and digitized with a Matrox MVP imaging plate, using MetaMorph (Universal Imaging) imaging software.

Results

To evaluate the ability of cleft junction modifiers to prevent tumor promotion, the present inventors wanted to analyze whether cleft junction modifiers could reverse the decrease in gap junctional communication induced by a well-known tumor promoting agent, DDT. Therefore, human osteoblast monolayers were incubated on glass coverslips at 37 ° C in a humid atmosphere containing CO.₂ at 5%. DDT was added to the medium in a final concentration of 13 pM and left for 60 minutes.

To evaluate the effect of Compound 2 on direct cell coupling after DDT treatment, microinjection experiments were carried out according to the method described above. Lucifer Yellow (LY) stain was injected into a single human osteoblast in a monolayer. After 30 seconds, the number of cells containing the stain was evaluated. Under control conditions (no DDT treatment), the staining spread to a median of 14.5 cells (n = 12). The same experiment was performed with cells exposed to DDT. These cells showed reduced cell coupling, with a median of 7 (n = 13). Compound 2 was added to the bath solution to a final concentration of 10^-8 mol / l and after 10 minutes another microinjection was made. Compound 2 produced an increase in intercellular transfer in all preparations, with a median of 8.3 cells (Figure 15). This increase is highly significant, with p &lt;0.01, using the Wilcoxon non-parametric statistical test. Consequently, cleft junction openers are capable of reversing decreased intercellular coupling, related to tumor promotion, suggesting that the substances of this invention may be useful in the chemoprevention and / or treatment of cancer. . The compounds of the present invention are useful for the preparation of drugs for the chemo-prevention and / or treatment of cancer. The compounds of this invention can also be used in combination therapy with other anticancer agents. Therefore, it is the purpose of the present invention to offer compounds for the preparation of drugs useful in the prevention and / or treatment of cancer. This objective is achieved with the present peptide compounds.

Effects of Slit Joint Openers on Wound Healing

A wound is a discontinuation of the normal anatomy that affects the skin, and it can be a surgical or traumatic wound, or it can be secondary to various diseases such as diabetes, arteriosclerosis, malnutrition, etc. Normal wound healing is a gradual, systemic process that includes hemostasis and inflammation. Remodeling follows these processes, which can last for years, and is responsible for the formation of scar tissue. Fibrin hemostasis provides a surface under which migrations and movements of the wound edges take place. Epithelialization, fibroplasia, and capillary proliferation in the healing wound begin immediately. Angiogenic capillary buds invade the fibrin clot in the wound and, after a few days, organize into a microvascular network along the granulation tissue, also consisting of leukocytes and phagocytic mononuclear cells. There is a very dynamic interaction between the various tissue components involved in the wound healing process. The angiogenic process is essential for effective healing. Intercellular communication and cleft junctions are essential for the creation of a fibroblast syncytium and proliferation of the capillary network. The normal distribution of connexin 43 is necessary for this growth of the different tissue components.

Various local factors often seen during pathological conditions, such as edema, ischemia, low oxygen tension, and infections, can delay the wound healing process. Scarring involves the interactions of many cell types and intercellular communication, mediated by junctions by

ES 2 228 807 T3 cleft, plays an important role in the coordination of cellular metabolism during the growth and development of tissues and organs (66-68).

The present inventors point out that the substances of this invention, which increase GJIC, can be used for the treatment of wounds and, in particular, to accelerate wound healing. Taking into consideration that experiments with cardiac and bone tissues suggest that these substances exhibit enhanced efficacy during metabolic stress (eg, hypoglycemia, hypoxia, ischemia), it can be deduced that these substances may be especially useful in the treatment of ulcers due to ischemia. Therefore, it is an aim of the present invention to offer compounds for the preparation of medicaments useful in the treatment of wounds and, in particular, of ischemic ulcers. This objective is achieved with the present peptide compounds.

Effects of gap junction openers on gastric and duodenal ulcer healing

Mine et al. have shown that normal human gastric mucosa contains connexin 32 and connexin 43 (69,70). In contrast, the gastric mucosa surrounding a chronic gastric ulcer lesion contains a lower amount of connexin 32 and connexin 43. In the 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 were decreased in the active ulcer phase, practically returned to the levels observed in normal gastric mucosa. These data indicate that the disappearance of both connexins, 32 and 43, is closely related to the phase of chronic gastric ulcer lesions. Additionally, using a rat model of acetic acid-induced chronic gastric ulcer, the same group of researchers demonstrated that the clinical effect of the anti-ulcer drug cimetidine was closely related to the reappearance of connexin 32 (69).

Therefore, the substances of this invention, which increase GJIC, can stimulate the healing of gastric and duodenal ulcers. Accordingly, it is an object of the present invention to provide compounds for the preparation of medicaments useful in the treatment of gastric and duodenal ulcers. This objective is achieved with the present peptide compounds.

Role of cleft junctions in vascular biology

Coordination of cellular responses at the endothelial interface between blood and underlying tissues is mediated by multiple signaling mechanisms, including direct intercellular communication through cleft junctions. Among the functions in which intercellular communication by cleft junctions has been implicated is the migratory behavior of endothelial cells after injury, in angiogenesis, endothelial growth and senescence, and the coordination of vasomotor responses (71).

Regulation of blood flow requires, in a highly dynamic range, coordinated resistance responses and feeding arteries. This coordination between vessels can be achieved by the vascular effects of the shear stress exerted by the bloodstream or by the conduction of vasomotor signals along the cells of the vascular wall. In fact, the local application of certain vasoactive compounds such as acetylcholine (ACh) or norepinephrine (NE) induces not only local dilation or constriction, but also vasomotor responses several millimeters upstream or downstream (71). Vasomotor responses can also be driven from capillaries to arterioles, and may contribute to matching demands for tissue and blood supply. This has been demonstrated as follows: by stimulating the contraction of isolated muscle fibers, dilation of arterioles upstream of the capillaries feeding these fibers was observed (72).

The high conduction velocity is consistent with the electrotonic transmission of a signal along the vascular wall. In fact, locally induced hyperpolarizations and depolarizations have been shown to be conducted several millimeters upstream in vascular smooth muscle and endothelial cells. The conduction of the electrical signal requires the coupling of vascular cells by means of gap junctions that provide conduits of low electrical resistance between cells. In vascular tissue, at least three different connexin (Cx) proteins (Cx37, Cx40 and Cx43) are expressed that form cleft junctions. Cx40 appears to be the predominant connexin isoform in aortic endothelial cells, whereas Cx43 expression is abundant in smooth muscle.

Studies in mice deficient in Cx40 (Cx40 - / -) have shown that the diffusion of vasodilation induced by local application of acetylcholine or bradykinin is strongly attenuated in Cx40 - / - animals, compared to normal wild-type animals ( Cx40 + / +) (73). Additionally, blood pressure is significantly increased in Cx40 - / - animals compared to normal wild-type mice (Cx40 + / +). These results confirm an important role for Cx40 in vascular intercellular communication, and indicate that altered communication between the cleft junctions in the vascular wall is associated with decreased transmission of endothelium-dependent vasodilation responses which, in turn, increases vascular resistance and causes hypertension. Recent in vivo studies indicate that normal pressure fluctuations in the kidney are extremely important for the regulation of blood pressure (74). In this way, altered vasomotor responses, due to poor coupling between cells, may contribute to the development of hypertension in animals deficient in Cx40.

Down-regulation of Cx43 mRNA and protein levels in senescent endothelial cells suggests

ES 2 228 807 T3 that altered intercellular communication between cleft junctions could play a role in the vascular aging process (75).

Based on the available information on the role of cleft junctions in vascular responses, it is likely that a drug compound capable of increasing the coupling of cleft junctions in the vascular wall could facilitate the conduction of vascular responses and improve the delivery of blood during situations in which there is an increased metabolic demand (for example, physical exercise, tachycardia) and during ischemia. Furthermore, such a substance could prevent and / or treat hypertension. Accordingly, it is a further purpose of the invention to provide compounds that increase the coupling of cleft junctions and / or GJIC in the vascular wall, thus being useful for the prevention or treatment of hypertension. This objective is achieved with the present peptide compounds.

Effects of gap junction openers on nerve tissue

Eight different connexins are expressed in the CNS (Cx26, 30, 32, 37, 40, 43, 45, 46). Additionally, Cx36 appears to be preferentially expressed in neurons. Different connexins allow communication between different cell populations or segregate cells into isolated compartments, according to their pattern of connexin expression. There are compartmental interfaces, in which heterotypic coupling could have functional importance, between oligodendrocytes (Cx32, Cx45) and astrocytes (Cx43, Cx45, Cx40, Cx30) or neurons (Cx26, Cx32, Cx43) (76).

It is possible that a specific set of connexins provides a functional advantage in certain compartments of the brain; that is, a greater or lesser unit conductance could functionally facilitate or limit the synchronization of neural inputs or the speed of conduction.

Extensive cleft junction mediated intercellular coupling has been documented in immature neuroblasts and postnatal neurons (76,77). The postnatal increase in neuronal cleft junctions and their cortical organization suggest an essential role of these junctions in morphogenetic events underlying the critical phase of corticogenesis. The involvement of cleft junctions in neuronal trafficking is enhanced by the fact that neurotransmitters are capable of modifying cleft junction coupling.

Therefore, the present inventors suggest that the substances of this invention, known to increase GJIC, could accelerate repair after nerve injury or during engraftment of immature cells (progenitor cells) into brain tissue. Among the technologies currently undergoing experimental evaluation for cellular repair of the central nervous system are grafts of progenitor cells, fetal tissue and viral vectors used for the treatment of diseases such as Parkinson's, Huntington's and other neurodegenerative brain diseases.

Axon injury rapidly activates microglial and astroglial cells near axotomized neurons. Following motor axon injury, astrocytes upregulate connexin 43 cleft junction protein (GFAP) within one day and upregulate glial fibrillary acidic protein (GFAP) within one hour (s). Simultaneously, microglial cells proliferate and migrate towards the perikaryion of the axotomized neuron. A hypothetical scheme of glial cell activation after axon injury assumes that damaged neurons initially interact with adjacent astrocytes through the GJIC. Next, neighboring resting microglial cells are activated. These glial reactions are amplified by paracrine and autocrine mechanisms, in which cytokines appear to play an important role as mediators. The specific functional properties of activated glial cells will determine their influence on neuronal survival, axon regeneration, and synaptic plasticity. Therefore, controlling the induction and progression of these responses are possibly critical for the outcome of, for example, neurological trauma, cerebral ischemia, and chronic neurodegenerative diseases (78).

Cleft junctions are believed to provide molecular bonding for coordinated long-range signaling between individual members of the glial compartment. In the same way, astrocytes are specially adapted for the metabolic support of neurons, since they are functionally polarized, one end touching the vascular bed and the other pole approaching the neuronal parenchyma (76). In this way, the malfunction of these support mechanisms can be instrumental for the malfunction of the integrated neural pathways and, therefore, the development of diseases in the central nervous system. Accordingly, the present inventors suggest that the substances of this invention, which have been shown to increase GJIC, can prevent ischemic injury in the brain by increasing metabolic support between glial cells and neurons. Additionally, the substances of the invention can be of great importance in patients with organic psychoses that can present with signs such as depression, anxiety, learning and memory deficits, phobias and hallucinations. Therefore, it is a purpose of the present invention to offer compounds for the preparation of drugs useful in the prevention of ischemic injury to the brain, and for the treatment of organic psychoses, including depression, anxiety, learning and memory deficits, phobias. and hallucinations. This objective is achieved with the peptide compounds of the invention, when they are selected or formulated so that they are available to the central nervous system. Effects of cleft joint openers on cataract

The ocular lens of vertebrates is a solid cyst of cells that grows throughout life by the addition of new cells to the surface. Older cells, buried by new generations, differentiate

ES 2 228 807 T3 in long prismatic fibers, lose their cellular organelles and fill their cytoplasms with high concentrations of soluble proteins, the crystallines. Long-lasting lenticular fibers are interconnected by gap junctions, both with each other and with the anterior layer of single cuboidal epithelial cells, on the lens surface. This cleft junction network brings together the lens cells into 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 capable of providing the metabolic energy necessary to maintain correct concentrations of ions and metabolites within the cytoplasm of lenticular fibers, so that crystallines they are kept in solution and do not aggregate (cataract). Three classes of connexins are present in the lens: Cx43, Cx46, and Cx50, and mutations in each of these cleft junction proteins have been associated with cataract (79-81). These findings demonstrate that GJIC is essential for normal lens metabolism and function. Therefore, the present inventors suggest that the substances of this invention, known to increase GJIC, can be used in the prevention and / or treatment of cataract. Accordingly, it is an object of the present invention to provide compounds for the preparation of medicaments useful in the prevention and / or treatment of cataract. This objective is achieved with the present peptide compounds.

Effects of cleft joint openers on ear disease

Many different Cx32 mutations have been found in hereditary peripheral neuropathy - Charcot-Marie-Tooth X-associated deafness syndrome, with multiple Cx25 and Cx31 mutations detected in deafness (80). Thus, the present inventors suggest that the substances of this invention, which are known to increase GJIC, can be used in the prevention and / or treatment of certain types of deafness associated with impaired GJIC in the ear. Therefore, it is an object of the present invention to provide compounds for the preparation of medicaments useful in the prevention and / or treatment of deafness associated with impaired GJIC. This objective is achieved with the present peptide compounds.

Function of gap junction openers on the intestines

Both Cx43 and Cx45 are expressed in the wall of the small intestine (82). Cells expressing Cx45 along the deep muscular plexus of the small intestine are thought to act as probably constituents of a pacemaker system, which may include a conductive system, forming a cellular network that operates through specific types of junctions by cleft. In the intestine and colon, the interstitial cells of Cajal (ICC) are pacemaker cells located between the intestinal smooth muscles; they generate spontaneous slow waves from the smooth muscle layers and mediate neurotransmission. The three-dimensional cell network of ICC is connected by Cx43 cleft junctions both between ICC and between ICC and smooth muscle cells (83). In patients with Hirschsprung's disease, the absence of Cx43 expression in the aganglionic intestine suggests that impaired intercellular communication between ICC and smooth muscle cells may be partially responsible for motility dysfunction in this disease (83). Patients with Chagas disease (caused by infection with the protozoan Trypanosoma cruzii) exhibit a marked reduction in Cx expression, which is considered responsible for both cardiomyopathy and the severely dilated megacolon seen in these patients (7). Therefore, normal gap junction communication between ICC and between ICC and smooth muscle cells is considered essential for normal motility of the small intestine and colon. Accordingly, it is a further purpose of the invention to provide a substance capable of increasing the conductance between gap junctions in the intestine and which may therefore be useful in the treatment of gastrointestinal motility disorders.

Reproductive organs and cleft unions

Ovaries

The gap junctions between granulosa cells and between the oocyte and the surrounding granulosa cells play an important role during the development of the ovarian follicle. At birth, the ovary contains primordial follicles consisting of meiosis-arrested oocytes, surrounded by a single layer of supporting cells (granulosa). Periodically, subsets of primordial follicles undergo further development, during which the oocyte increases in size and the granulosa cells proliferate, stratify, and develop a fluid-filled antrum. After ovulation, the oocytes resume meiosis and the granulosa cells retained in the follicle differentiate into steroid cells, forming the corpus luteum.

The cleft junctions directly connect adjacent cells, allowing diffusion movements of ions, metabolites, and other potentially signaling molecules of importance in the regulation of the ovarian cycle and female fertility. As confirmation of the essential role of cleft junctions for normal ovarian function, Cx37-deficient mice have been shown to lack mature (Graafian) follicles, do not ovulate, and develop numerous inappropriate corpora lutea. Furthermore, oocyte development stops before being competitive in meiosis. In this way, intercellular signaling through the corresponding channels critically regulates the highly coordinated set of cellular interactions necessary for efficient oogenesis and ovulation (84).

Follicle-stimulating hormone (FSH) is the main regulator of growth and development of the ovarian follicle.

ES 2 228 807 T3

Along with its various actions on follicle maturation, FSH improves inter-cell coupling between granulosa cells and enhances the expression of the Cx43 gene and possibly the formation of new cleft junctions (85). In contrast, luteinizing hormone (LH) disrupts cell-to-cell communication within the ovarian follicle, leading to a decrease in intra-oocyte cAMP concentrations, followed by a resumption of meiosis (86).

These data demonstrate that the presence of normal gap junction communication through Cx37 and Cx43 is essential for normal follicular growth and ovulation. Thus, certain forms of female infertility may be due to poor inter-cell coupling in the ovaries. Therefore, a substance that increases inter-cell coupling could be used for the treatment of female infertility in women with altered expression and / or regulation of the function of the ovarian cleft junctions. The compounds of the present invention, having the ability to increase GJIC, are useful for the treatment of female infertility due to poor inter-cell coupling in the ovaries.

Uterus

The powerful synchronous contractions of the uterus during labor depend on the electrical coupling of the smooth muscle cells of the myometrium through cleft junctions. In humans and other mammals, cleft junctions are rare in the myometrium of the non-pregnant uterus, but they become abundant at term and / or with the onset of labor. The cleavage junction protein predominantly expressed by smooth muscle cells of the human myometrium is Cx43, but Cx26, Cx40, and Cx45 have also been identified in the human myometrium (87,88).

Due to the great importance of coordinated muscle contractions during labor, it is a further purpose of the invention to provide a substance capable of increasing inter-cell coupling in the myometrium, which is expected to have a positive influence on the synchronization of the cells. muscle contractions. This substance can be used together with oxytocin for the induction and facilitation of labor. This objective is achieved with the present peptide compounds and the invention further relates to the use of the peptide compounds of the invention for the preparation of a medicament for the induction and facilitation of labor.

Male reproductive organs

Cx43 is the most abundant connexin in the testes and it is interesting to note that rat strains with reduced Cx43 expression exhibit altered spermatogenesis (ebo / ebo, jun-d - / -, Cx43 +/- mice). Additionally, previous work suggests that patients with hypospermia or aspermia have reduced cleft junctions in the testes (90). These data confirm the suggestion that reduced inter-cell coupling in the testes can lead to male infertility and, therefore, it is a further purpose of the invention to provide a substance capable of increasing inter-cell coupling and thus can be a useful therapeutic agent in the treatment of male infertility associated with an altered coupling between cells.

Function of cleft junctions in the pancreas

The cleft junction channels, formed by Cx43, functionally couple the glucose-sensitive cells of pancreatic islets and a rat insulinoma cell line (91). In contrast, cells from several cell lines with abnormal insulin secretion do not express Cx43, have poor cleft junctions, and are poorly coupled. After correction of these defects by stable transfection of Cx43 cDNA, cells expressing modest levels of Cx43 and coupling, as observed in native beta cells, show markedly high insulin gene expression and insulin content, in comparison with those observed both in wild-type (uncoupled) cells and in transfected cells that overexpress Cx43. These findings indicate that adequate coupling of Cx43 is required for adequate insulin production and storage (91). Additionally, in vivo stimulation of insulin release by glibenclamide is associated with increased Cx43 expression and increased inter-cell coupling between adjacent β cells within the pancreatic islet (92).

These observations indicate an important role for cleft junction coupling between pancreatic islet β cells for insulin production and release. Therefore, it is still a further purpose of the present invention to provide a substance capable of increasing the electrical conductance of the cleft junctions and thus improving glucose tolerance in subjects with non-insulin-dependent diabetes mellitus. Said objective is achieved with the peptide compounds of the invention.

Effects of cleft junction openers (antiarrhythmic peptides) on thrombosis

Two peptides closely related to the substances of the present invention have previously been shown to have antithrombotic activity. Thus, Dikshit et al. (15) found that the peptides Gly-Pro-PrpGly-Ala-Gly and Gly-Pro-Gly-Gly-Ala-Gly prevent the development of pulmonary embolism in mice when an iv dose of collagen and epinephrine is administered. US 4,775,743 describes HP5, an AAP-derived peptide having the sequence N-3- (4-hydroxyphenyl) -propionyl-Pro-4Hyp-Gly-Ala-Gly-OH and which is active against platelet agglutination. The compounds of the present invention exhibit striking similarity and it is likely that

ES 2 228 807 T3 show similar effects on thrombosis. Thus, the substances of this invention can be used in the prevention of thrombosis.

Compositions

The invention also relates to a composition comprising a pharmacologically active antiarrhythmic peptide, as defined herein, in combination with a pharmaceutically acceptable carrier and / or diluent. These compositions may be in a form adapted for direct oral, subcutaneous, parenteral (intravenous, intraperitoneal), intramuscular, rectal, epidural, intratracheal, intranasal, dermal, vaginal, buccal, ocular, cerebral or pulmonary administration, in a form adapted for subcutaneous, intravenous or oral administration, and these compositions can be prepared in a manner well known to the person skilled in the art, for example, as generally described in "Remington's Pharmaceutical Sciences", 17^to edition, Alfonso R. Gennaro (compiler), Mark Publishing Company, Easton, PARTICLE, USA, 1985 and newer editions, and in the monographs in the "Drugs and the Pharmaceutical Sciences" series, Marcel Dekker. The compositions may be present in conventional forms, for example, solutions and suspensions for injection, including concentrates for iv infusions, capsules and tablets, preferably in the form of enteric formulations, for example as described in US 5,350,741, for oral administration.

The pharmaceutical carrier or diluent employed can be a conventional solid or liquid carrier. Examples of solid carriers are lactose, white clay, sucrose, cyclodextrin, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid, or lower alkyl cellulose ethers. Examples of liquid carriers are syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene, and water.

Similarly, the carrier or diluent may contain any sustained release material known in the art such as glyceryl monostearate or glyceryl distearate, alone or in admixture with a wax.

If a solid carrier is used for oral administration, the preparation can be compressed, included in powder or granule form in a hard gelatin capsule, or it can be in troche or lozenge form. The amount of solid carrier will vary widely, but will normally be from about 25 mg to about 1 g.

A typical tablet, which can be prepared by conventional compression techniques, may contain: Core: active compound (as free compound or as its salt), 100 mg; colloidal silicon dioxide (Aerosil), 1.5 mg; cellulose, microcrystalline (Avicel), 70 mg; modified cellulose gum (Ac-Di-Sol), 7.5 mg; magnesium stearate.

Coating: HPMC, approximately 9 mg; * Mywacett 9-40T, about 0.9mg; * acylated monoglyceride used as a plasticizer for film coating.

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 an aqueous or nonaqueous liquid solution or suspension.

Also, the composition may be in a form suitable for local or systemic injection or infusion and, as such, may be formulated with sterile water or an isotonic glucose or saline solution. The compositions can be sterilized by conventional sterilization techniques, 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 prior to administration. The composition may contain pharmaceutically acceptable auxiliary substances, as necessary to approximate physiological conditions, such as buffering agents, tonicity adjusting agents, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride. , etc.

Formulation of peptides for intravenous administration

Multi-dose formulations can be prepared as a solution of a compound of the invention in sterile and isotonic saline, packaged in stoppered vials and, if necessary, a preservative (eg benzoates) is added. Fixed-dose formulations can be prepared as a solution of the compound in sterile and isotonic saline, packed in glass ampoules and, if necessary, packed with an inert gas. Each dose of the compound is packaged dry in ampoules or stoppered vials, packed, if necessary, with inert gas. The multi-dose formulation demands the maximum 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 iv infusion.

For nasal administration, the preparation may contain a compound of the present invention dissolved or suspended in a liquid vehicle, in particular an aqueous vehicle, for aerosol application. The carrier may contain additives such as solubilizing agents, for example propylene glycol, surfactants such as bile acid salts, or higher alcohol ethers of polyoxyethylene, absorption enhancers such as lecithin (phosphatidyl choline) or cyclodextrin, or preservatives such as parabens. .

Additionally, the small size of the peptide compounds of the invention may be an advantage for oral or nasal administration, due to the relatively rapid absorption through the mucous membranes, in

ES 2 228 807 T3 compared to larger peptides, minimizes enzymatic degradation, especially in the duodenum and ileum.

Preparation of enteric tablets containing Compound 2

400 mg of L-tartaric acid and 40 mg of hydrogenated castor oil-polyethylene glycol are dissolved in 5 ml of methanol. The solution is deposited in a mortar previously heated to 30 ° C. 1.5 mg of Compound 2 are added to the solution. Immediately after the addition of Compound 2, the mixture is stirred with the pestle under a stream of hot air of 40 ° C and then placed in vacuum desiccator overnight to remove solvent. The resulting solid mass is pulverized with the pestle and kneaded with 30 mg of sodium bicarbonate and a small amount of 70% ethanol. The mixture is then divided into tablets and dried. The dry tablets are coated with hydroxypropyl methylcellulose phthalate to obtain an enteric tablet.

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

In specific embodiments, an antiarrhythmic peptide according to the present invention can be used to treat and / or prevent bradyarrhythmias (eg, due to diseases of the sinus node, AV node, bundle of His, right or left bundle branch), and tachyarrhythmias associated with reentry (for example, premature atrial complexes, AV junction complexes, premature ventricular complexes, atrial fibrillation, atrial flutter, paroxysmal supraventricular tachycardia, sinus node reentrant tachycardia, AV node reentrant tachycardia, and nonsustained ventricular tachycardia), either alone or in combination with other antiarrhythmic agents such as class I agents (for example, lidocaine), class II agents (for eg metoprolol or propranolol), class III agents (eg amiodarone or sotalol), or class IV agents (eg verapamil).

In specific embodiments, an antiarrhythmic peptide according to the present invention can be used to prevent thrombotic events in patients with diseases of the vascular wall (eg, atherosclerosis), increased platelet production (universal polycythemia), and / or reduced blood flow (heart disease. , vasculopathy), either alone or in combination with GP IIb / IIIa inhibitors (eg, c7E3; abciximab), cyclooxygenase inhibitors (eg, aspirin), thromboxane A2 antagonists, coumarin derivatives (eg, warfarin), or with the synthetic peptide intergryllin.

In specific embodiments, an antiarrhythmic peptide according to the present invention can be used, due to its effect on the channels of the intercellular cleft junctions, to treat and / or prevent loss of bone tissue and accelerate the healing of bone fractures (93); treat and / or prevent diseases in cartilage and joints with poor vascularization (94); treat and / or prevent cataracts (81); treat and / or prevent the vascularization of the cornea in pathological states with malnutrition of the cornea and increase the healing of corneal lesions (95); treating and / or preventing the growth and spread of cancer cells such as cancer cells derived from epithelial cell lines (96); treat and / or prevent hypertension due to increased vasomotion (74); prevent rejection of grafts such as cells and organs in an organism.

Peptide synthesis

A preferred general procedure is described below. However, more detailed descriptions of solid phase peptide synthesis are found in WO98 / 11125.

Synthesis apparatus and strategy

The peptides were synthesized batchwise in a polyethylene vessel equipped with a polypropylene filter for filtration, using 9-fluorenylmethyl-oxycarbonyl (Fmoc) as the Na-amino-protecting group and suitable common protecting groups for the side chain functionalities.

Solvents

The solvent DMF (N, N-dimethylformamide, Riedel de-Haen, Germany) was purified by passing it through a column packed with a powerful ion exchange resin (Lewatit S 100 MB / H strong acid, Bayer AG, Leverkusen, Germany ), and the free amines were analyzed before use by adding 3,4-dihydro-3-hydroxy-4-oxo1,2,3-benzotriazine (Dhbt-OH), giving rise to a yellow color (anion Dhbt-O^-) if free amines are present. Solvent DCM (dichloromethane, analysis category, Riedel de-Haen, Germany) was used directly, without purification. Acetonitrile (HPLC category, Lab-Scan, Dublin, Ireland) was used directly without purification.

ES 2 228 807 T3

Amino acids

Fmoc protected amino acids were purchased from Advanced ChemTech (ACT) in suitable side chain protection forms. Otherwise protected amino acids (Fmoc-Glu (OH) -O-allyl; Fmoc-Asp (OH) -O-allyl were purchased from NovaBiochem (Switzerland), Fmoc-4-Hyp (OtBu) -OH from Bachem (Switzerland) .

Coupling reagents

Diisopropyl-carbodiimide coupling reagent (DIC) was purchased from Riedel de-Haen, Germany, PyBop from Advanced ChemTech.

Linkers

(4-Hydroxymethyl-phenoxy) -acetic acid (HMPA) was purchased from Novabiochem, Switzerland and coupled with the resin as a preformed 1-hydroxybenzotriazole ester (HOBt), generated by DIC.

Solid supports

The peptides synthesized according to the Fmoc strategy on 0.22-0.31 mmol / g TentaGel S resins (TentaGel-SNH2; TentaGel S RAM, TentaGel S-RAM-Lys (Boc) -Fmoc; Rapp polymer, Germany);

Catalysts and other reagents

Diisopropyl ethylamine (DIEA) was purchased from Aldrich, Germany, and ethylenediamine from Fluka, piperidine and pyridine from Riedel de-Haen, Frankfurt, Germany. 4- (N, N-dimethylamino) -pyridine (DMAP) was purchased from Fluka, Switzerland and used as a catalyst in coupling reactions involving symmetric anhydrides. Ethanedithiol was purchased from Riedel de-Haen, Frankfurt, Germany. 3,4-dihydro-3-hydroxy-4-oxo-1,2,3benzotriazine (Dhbt-OH), 1-hydroxybenzotriazole (HOBt) (HOAt) were purchased from Fluka, Switzerland.

Docking procedures

The first amino acid was coupled as a symmetric anhydride in DMF generated from the appropriate Nα-protected amino acid and DIC. The following amino acids were coupled as in situ generated HOBt or HOAt esters prepared from suitable Na-protected amino acids and HOBt or HOAt by means of DIC in DMF. The acylations were analyzed by means of the ninhydrin test performed at 80 ° C to prevent the deprotection of Fmoc during the test (97).

Na-amino-protecting group (Fmoc) deprotection

The 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 a wash with DMF (5 x 15 ml, 5 min each) until no yellow color was detected after the addition of Dhbt-OH to the drained DMF.

Allyl deprotection

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

Coupling of HOBt esters

3 eq. of Na-amino-protected amino acids in DMF along with 3 eq. of HOBt and 3 eq. of DIC, then adding it to the resin.

Preformed symmetric anhydride

6 eq. Na-amino-protected amino acid 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 remainder was dissolved in DMF. The solution was immediately added to the resin, followed by 0.1 eq. of DMAP.

Cyclization of the peptide on the resin

1.5 eq. of PyBop in DMF along with 1.5 eq. of HOBt and 3 eq. of NMM to the peptide resin. The reaction was continued overnight.

Cleavage of the peptide from the resin with acid

The peptides were cleaved from the resins by treatment with 95% trifluoroacetic acid (TFA, Riedel de41

ES 2 228 807 T3

Haen, Frankfurt, Germany) -water v / v, or with 95% TFA and 5% v / v ethanedithiol, at room temperature for 2 h. The filtered resins were washed with 95% TFA-water and the filtrates and washes were evaporated under reduced pressure. The residue was washed with ether and lyophilized from acetic acid-water. The crude lyophilized product was analyzed by high performance liquid chromatography (HPLC) and identified by electrospray ionization mass spectrometry (ESMS).

Discontinuous synthesis of peptides on TentaGel resin (PEG-PS)

TentaGel resin (1 g, 0.22-0.31 mmol / g) was deposited into 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 on 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 h. The resin was drained and washed with DMF (5 x 5 ml, 5 min each time) and acylation was checked by the ninhydrin test. The first amino acid was coupled as a preformed symmetric anhydride, as described above. The following amino acids, based on sequence, were coupled as preformed Fmoc-protected HOBt esters (3 eq.) As described above. Couplings were continued for 2 h, unless otherwise specified. The resin was stopped and washed with DMF (5 x 15 ml, 5 min each time) to remove excess reagent. All acylations were verified by the ninhydrin test carried out at 80 ° C. After completion of the synthesis, the peptide resin was washed with DMF (3 x 15 ml, 5 min each time), DCM (3 x 15 ml, 1 min each time) and finally diethyl ether (3 x 15 ml , 1 min each time) and dried under vacuum.

HPLC conditions

HPLC gradient analysis was carried out 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. used the Hewlett Packard Chemstation for LC software (rev. A.06.01) for instrument control and data acquisition.

The following columns and HPLC buffer systems were used:

Column

Kromasil, Phenomenex 00F-3033-EO, 329889 (new); 5 pm C-18, 100A 150 x 4.6 mm; Lot n ° 5243-10. Buffer system: A: 0.1% TFA in MQV; B: 0.085% TFA, 10% MQV, 90% MeCN.

Gradient:

- 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 rate: 1.5 ml / min Oven temperature: 40 ° C

UV detection: λ = 215 nm

Mass spectra were obtained on an LCT Micro-mass instrument.

The invention is further illustrated by the following specific synthesis examples.

ES 2 228 807 T3

Peptide synthesis of individual peptides

Synthesis example 1

Peptide synthesis of Ac-Tyr-Pro-4Hyp-Gly-Ala-Gly-OH (Compound 1) on TentaGel-S-NH₂; Rapp polymer, Germany

First batch: dry TentaGel-S-NH2 (0.27 mmol / g, 1 g) was deposited in a polyethylene container equipped with a polypropylene filter for filtration and treated as described in "discontinuous synthesis of peptides on resin TentaGel ”until completion of the N-terminal tyrosine coupling. All couplings were continued overnight. After deprotection of the Fmoc group, the N-terminal amino group was acetylated with acetic acid anhydride (1 ml, 10.5 mmol), along with 100 µl of pyridine dissolved in 2 ml of DMF. Coupling was continued overnight. Acylations were checked by the ninhydrin test carried out at 80 ° C, as described above. After completion of 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). time), and dried under vacuum.

The peptide was cleaved from the resin in the manner described above and lyophilized from acetic acid. The crude lyophilized product was analyzed by HPLC and the purity was shown to be greater than 70%, and the identity of the peptide was confirmed by ES-MS (found MH + 619.24, calculated MH + 619.26). The yield of crude material was 137.7 mg. After purification using preparative HPLC, as described above, 59 mg of peptide product was collected, with a purity greater than 95%. The total yield of purified peptide product was 35%.

Second batch: dry TentaGel-S-NH2 (0.27 mmol / g, 1 g) was deposited in a polyethylene container equipped with a polypropylene filter for filtration and treated as described in "discontinuous synthesis of peptides on resin TentaGel ”until completion of the N-terminal tyrosine coupling. All couplings were continued overnight. After deprotection of the Fmoc group, the N-terminal amino group was acetylated with acetic acid anhydride (1 ml, 10.5 mmol), along with 100 µl of pyridine dissolved in 2 ml of DMF. Coupling was continued overnight. Acylations were checked by the ninhydrin test carried out at 80 ° C, as described above. After completion of 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). time), and dried under vacuum.

The peptide was cleaved from the resin in the manner described above and lyophilized from acetic acid. The crude lyophilized product was analyzed by HPLC and the purity was shown to be greater than 70%, and the identity of the peptide was confirmed by ES-MS (found MH +: 619.25; calculated MH +, 619.26). The yield of crude material was 137.3 mg. After purification using preparative HPLC in the manner described above, 27.9 mg of peptide product was collected with a purity greater than 91%. Total yield of purified peptide product: 15.5%.

Synthesis example 2

Ac-D-Tyr-D-Pro-D-4Hyp-Gly-D-Ala-Gly-NH Peptide Synthesis₂ (Compound 2) on TentaGel-S-Ram; Rapp polymer, Germany

First batch: dry TentaGel-S-Ram (0.23 mmol / g, 1 g) was deposited in a polyethylene container equipped with a polypropylene filter for filtration and treated as described in "discontinuous synthesis of peptides on resin TentaGel ”until the end of the coupling of the N-terminal D-tyrosine. All couplings were continued overnight. After deprotection of the Fmoc group, the N-terminal amino group was acetylated with acetic acid anhydride (1 ml, 10.5 mmol), along with 100 µl of pyridine dissolved in 2 ml of DMF. Coupling was continued overnight. Acylations were checked by the ninhydrin test carried out at 80 ° C, as described above. After completion of 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). time), and dried under vacuum.

The peptide was cleaved from the resin in the manner 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 618.25, 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 purified peptide product was 30%.

Second batch: dry TentaGel-S-Ram (0.23 mmol / g, 1 g) was deposited in a polyethylene container equipped with a polypropylene filter for filtration and treated as described in "discontinuous synthesis of peptides on resin TentaGel ”until completion of the coupling of the N-terminal D-tyrosine. All couplings were continued overnight. After deprotection of the Fmoc group, the N-terminal amino group was acetylated with acetic acid anhydride (1 ml, 10.5 mmol), along with 100 µl of pyridine dissolved in 2 ml of DMF. Coupling was continued overnight. Acylations were checked by the ninhydrin test carried out at 80 ° C, as described above. After completion of 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). time), and dried under vacuum.

ES 2 228 807 T3

The peptide was cleaved from the resin in the manner 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 (found MH +: 618.29; calculated MH +, 618.28). After purification using preparative HPLC in the manner described above, 100 mg of peptide product was collected with a purity greater than 99%. Total yield of purified peptide product: 71%.

Synthesis example 3

Cyclo- (Tyr-Pro-4Hyp-Gly-Ala-Gly-Asn) peptide synthesis (Compound 3) on TentaGel-S-Ram; Rapp polymer, Germany

First batch: dry TentaGel-S-Ram (0.23 mmol / g, 1 g) was deposited in a polyethylene container equipped with a polypropylene filter for filtration and treated as described in "discontinuous synthesis of peptides on resin TentaGel ”. The first amino acid Fmoc-Asp (OH) -O-All was connected to the TentaGel-S-Ram resin through the side chain carboxylic acid, which will end up being amidated (Asn) finally, after cleavage. The procedure described under "discontinuous synthesis of peptides on TentaGel resin" was followed until completion of the N-terminal tyrosine coupling. All couplings were continued overnight. After deprotection of the Fmoc group and the allyl group (according to the procedure described above), the peptide bound to the resin was cyclized using PyBop as the coupling reagent, as described above, and the coupling was continued overnight. Acylations were checked by the ninhydrin test carried out at 80 ° C, as described above. After completion of 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). time), and dried under vacuum.

The peptide was cleaved from the resin in the manner described above and lyophilized from acetic acid in a crude product yield of 57 mg. After purification using preparative HPLC, as described above, 2.7 mg of cyclic peptide product was collected, with a purity greater than 95%. The total yield of purified peptide product was 1.3%. The identity of the peptide was confirmed by ES-MS (MH + found 673.32; MH⁺ calculated, 673.28).

Second batch: dry TentaGel-S-Ram (0.23 mmol / g, 1 g) was deposited in a polyethylene container equipped with a polypropylene filter for filtration and treated as described in "discontinuous synthesis of peptides on resin TentaGel ”. The first amino acid Fmoc-Asp (OH) -O-All was connected to the TentaGel-S-Ram resin through the side chain carboxylic acid, which will end up being amidated (Asn) finally, after cleavage. The procedure described under "discontinuous synthesis of peptides on TentaGel resin" was followed until completion of the N-terminal tyrosine coupling. All couplings were continued overnight. After deprotection of the Fmoc group and the allyl group (according to the procedure described above), the peptide bound to the resin was cyclized using PyBop as the coupling reagent, as described above, and the coupling was continued overnight. Acylations were checked by the ninhydrin test carried out at 80 ° C, as described above. After completion of 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). time), and dried under vacuum.

The peptide was cleaved from the resin in the manner described above and lyophilized from acetic acid in a crude product yield of 57 mg. After purification using preparative HPLC, as described above, 10 mg of cyclic peptide product was collected, with a purity greater than 99%. The total yield of purified peptide product was 7%. The identity of the peptide was confirmed by ES-MS (MH + found 673.30; MH⁺ calculated, 673.29).

Synthesis example 4

Cyclo- (Tyr-Pro-4Hyp-Gly-Ala-Asn) peptide synthesis (Compound 4) on TentaGel-S-Ram; Rapp polymer, Germany

First batch: dry TentaGel-S-Ram (0.23 mmol / g, 1 g) was deposited in a polyethylene container equipped with a polypropylene filter for filtration and treated as described in "discontinuous synthesis of peptides on resin TentaGel ”. The first amino acid Fmoc-Asp (OH) -O-All was connected to the TentaGel-S-Ram resin through the side chain carboxylic acid, which will end up being amidated (Asn) finally, after cleavage. The procedure described under "discontinuous synthesis of peptides on TentaGel resin" was followed until completion of the N-terminal tyrosine coupling. All couplings were continued overnight. After deprotection of the Fmoc group and the allyl group (according to the procedure described above), the peptide bound to the resin was cyclized using PyBop as the coupling reagent, as described above, and the coupling was continued overnight. Acylations were checked by the ninhydrin test carried out at 80 ° C, as described above. After completion of 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). time), and dried under vacuum.

The peptide was cleaved from the resin in the manner described above and lyophilized from acetic acid to give the crude product. After purification using preparative HPLC, as described above, a cyclic peptide product was collected.

ES 2 228 807 T3

Second batch: dry TentaGel-S-Ram (0.23 mmol / g, 1 g) was deposited in a polyethylene container equipped with a polypropylene filter for filtration and treated as described in "discontinuous synthesis of peptides on resin TentaGel ”. The first amino acid Fmoc-Asp (OH) -O-All was connected to the TentaGel-S-Ram resin through the side chain carboxylic acid, which will end up being amidated (Asn) finally, after cleavage. The procedure described under "discontinuous synthesis of peptides on TentaGel resin" was followed until completion of the N-terminal tyrosine coupling. All couplings were continued overnight. After deprotection of the Fmoc group and the allyl group (according to the procedure described above), the peptide bound to the resin was cyclized using PyBop as the coupling reagent, as described above, and the coupling was continued overnight. Acylations were checked by the ninhydrin test carried out at 80 ° C, as described above. After completion of 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). time), and dried under vacuum.

The peptide was cleaved from the resin in the manner described above and lyophilized from acetic acid, to give 58.6 mg of crude product.

After purification using preparative HPLC, as described above, 5.7 mg of cyclic peptide product was collected, with a purity greater than 98%. The total yield of purified peptide product was 4.4%. The identity of the peptide was confirmed by ES-MS (MH + found 616.25; MH + calculated 616.27). Synthesis example 5

H-Gly-Ala-Gly-D-Hyp-Pro-Tyr-NH Peptide Synthesis₂ (Compound 5) on TentaGel-S-Ram; Rapp polymer, Germany

Dry TentaGel-S-Ram (0.23 mmol / g, 1 g) was deposited in a polyethylene container equipped with a polypropylene filter for filtration, and treated as described in "discontinuous synthesis of peptides on TentaGel resin" until completion of the coupling of the N-terminal glycine. All couplings were continued overnight. Acylations were checked by the ninhydrin test carried out at 80 ° C, as described above. After deprotection of the Fmoc group and the N-terminal amino group, the peptide resin was washed with DMF (3 x 15 ml, 1 min each time), DCM (3 x 15 ml, 1 min each time), diethyl ether (3 x 15 ml, 1 min each time) and dried in vacuo.

The peptide was cleaved from the resin in the manner described above and lyophilized from acetic acid. After purification using preparative HPLC, as described above, 46.6 mg of peptide product was collected, with a purity greater than 99%. The total yield of purified peptide product was 28.6%.

The identity of the peptide was confirmed by ES-MS (MH⁺ found 576.27, MH⁺ calculated 576.26).

Synthesis example 6

H-Gly-Ala-Gly-D-Pro-Pro-Tyr-NH Peptide Synthesis₂ (Compound 6) on TentaGel-S-Ram; Rapp polymer, Germany

Dry TentaGel-S-Ram (0.23 mmol / g, 1 g) was deposited in a polyethylene container equipped with a polypropylene filter for filtration, and treated as described in "discontinuous synthesis of peptides on TentaGel resin" until completion of the coupling of the N-terminal glycine. All couplings were continued overnight. Acylations were checked by the ninhydrin test carried out at 80 ° C, as described above. After deprotection of the Fmoc group and the N-terminal amino group, the peptide resin was washed with DMF (3 x 15 ml, 1 min each time), DCM (3 x 15 ml, 1 min each time), diethyl ether (3 x 15 ml, 1 min each time) and dried in vacuo.

The peptide was cleaved from the resin in the manner described above and lyophilized from acetic acid. After purification using preparative HPLC, as described above, 26 mg of peptide product was collected, with a purity greater than 98%. The total yield of purified peptide product was 16.3%.

The identity of the peptide was confirmed by ES-MS (MH⁺ found 560.25, MH⁺ calculated 560.28).

Synthesis example 7

H-Gly-Ala-Gly-D-Pro-Ala-Tyr-NH Peptide Synthesis₂ (Compound 7) on TentaGel-S-Ram; Rapp polymer, Germany

Dry TentaGel-S-Ram (0.23 mmol / g, 1 g) was deposited in a polyethylene container equipped with a polypropylene filter for filtration, and treated as described in "discontinuous synthesis of peptides on TentaGel resin" until completion of the coupling of the N-terminal glycine. All couplings were continued overnight. Acylations were checked by the ninhydrin test carried out at 80 ° C, as described above. After deprotection of the Fmoc group and the N-terminal amino group, the peptide resin was washed with DMF (3

ES 2 228 807 T3 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.

The peptide was cleaved from the resin in the manner described above and lyophilized from acetic acid. After purification using preparative HPLC, as described above, 18.9 mg of peptide product was collected, with a purity greater than 98%. The total yield of purified peptide product was 12.2%.

The identity of the peptide was confirmed by ES-MS (MH + found 534.25, MH + calculated 534.26).

Synthesis Example 8

H-Gly-Ala-Gly-Gly-D-Pro-Tyr-NH Peptide Synthesis₂ (Compound 8) on TentaGel-S-Ram; Rapp polymer, Germany

Dry TentaGel-S-Ram (0.23 mmol / g, 1 g) was deposited in a polyethylene container equipped with a polypropylene filter for filtration, and treated as described in "discontinuous synthesis of peptides on TentaGel resin" until completion of the coupling of the N-terminal glycine. All couplings were continued overnight. Acylations were checked by the ninhydrin test carried out at 80 ° C, as described above. After deprotection of the Fmoc group and the N-terminal amino group, the peptide resin was washed with DMF (3 x 15 ml, 1 min each time), DCM (3 x 15 ml, 1 min each time), diethyl ether (3 x 15 ml, 1 min each time) and dried in vacuo.

The peptide was cleaved from the resin in the manner described above and lyophilized from acetic acid. Yield of crude material 130 mg. After purification using preparative HPLC, as described above, 70.1 mg of peptide product was collected, with a purity greater than 94%. The total yield of purified peptide product was 48.2%.

The identity of the peptide was confirmed by ES-MS (MH⁺ found 520.25, MH⁺ calculated 520.56).

Synthesis example 9

H-Gly-Ala-Gly-D-Hyp-Ala-Tyr-NH Peptide Synthesis₂ (Compound 9) on TentaGel-S-Ram; Rapp polymer, Germany

Dry TentaGel-S-Ram (0.23 mmol / g, 1 g) was deposited in a polyethylene container equipped with a polypropylene filter for filtration, and treated as described in "discontinuous synthesis of peptides on TentaGel resin" until completion of the coupling of the N-terminal glycine. All couplings were continued overnight. Acylations were checked by the ninhydrin test carried out at 80 ° C, as described above. After deprotection of the Fmoc group and the N-terminal amino group, the peptide resin was washed with DMF (3 x 15 ml, 1 min each time), DCM (3 x 15 ml, 1 min each time), diethyl ether (3 x 15 ml, 1 min each time) and dried in vacuo.

The peptide was cleaved from the resin in the manner described above and lyophilized from acetic acid. Yield of crude material 131 mg. After purification using preparative HPLC, as described above, 72.4 mg of peptide product was collected, with a purity greater than 92%. The total yield of purified peptide product was 49%.

The identity of the peptide was confirmed by ES-MS (MH⁺ found 550.28, MH⁺ calculated 550.59).

Synthesis Example 10

H-Gly-Ala-Gly-D-Hyp-D-Pro-Tyr-NH Peptide Synthesis₂ (Compound 10) on TentaGel-S-Ram; Rapp polymer, Germany

Dry TentaGel-S-Ram (0.23 mmol / g, 1 g) was deposited in a polyethylene container equipped with a polypropylene filter for filtration, and treated as described in "discontinuous synthesis of peptides on TentaGel resin" until completion of the coupling of the N-terminal glycine. All couplings were continued overnight. Acylations were checked by the ninhydrin test carried out at 80 ° C, as described above. After deprotection of the Fmoc group and the N-terminal amino group, the peptide resin was washed with DMF (3 x 15 ml, 1 min each time), DCM (3 x 15 ml, 1 min each time), diethyl ether (3 x 15 ml, 1 min each time) and dried in vacuo.

The peptide was cleaved from the resin in the manner described above and lyophilized from acetic acid. Yield of crude material 150.8 mg. After purification using preparative HPLC, as described above, 93.1 mg of peptide product was collected, with a purity greater than 99%. The total yield of purified peptide product was 58%.

The identity of the peptide was confirmed by ES-MS (MH⁺ found 576.63, MH⁺ calculated 576.63).

ES 2 228 807 T3

Synthesis example 11

H-Gly-Ala-Gly-NCG-Pro-Tyr-NH Peptide Synthesis₂ (Compound 11) on TentaGel-S-Ram; Rapp polymer, Germany

Dry TentaGel-S-Ram (0.23 mmol / g, 1 g) was deposited in a polyethylene container equipped with a polypropylene filter for filtration, and treated as described in "discontinuous synthesis of peptides on TentaGel resin" until completion of the coupling of the N-terminal glycine. All couplings were continued overnight. Acylations were checked by the ninhydrin test carried out at 80 ° C, as described above. After deprotection of the Fmoc group and the N-terminal amino group, the peptide resin was washed with DMF (3 x 15 ml, 1 min each time), DCM (3 x 15 ml, 1 min each time), diethyl ether (3 x 15 ml, 1 min each time) and dried in vacuo.

The peptide was cleaved from the resin in the manner described above and lyophilized from acetic acid. Yield of crude material 24.3 mg. After purification using preparative HPLC, as described above, 10.2 mg of peptide product was collected, with a purity greater than 91%. The total yield of purified peptide product was 4%.

The identity of the peptide was confirmed by ES-MS (MH + found 602.23, MH + calculated 602.32).

Synthesis example 12

H-Gly-Ala-Gly-T4C-Pro-Tyr-NH Peptide Synthesis₂ (Compound 12) on TentaGel-S-Ram; Rapp polymer, Germany

Dry TentaGel-S-Ram (0.23 mmol / g, 1 g) was deposited in a polyethylene container equipped with a polypropylene filter for filtration, and treated as described in "discontinuous synthesis of peptides on TentaGel resin" until completion of the coupling of the N-terminal glycine. All couplings were continued overnight. Acylations were checked by the ninhydrin test carried out at 80 ° C, as described above. After deprotection of the Fmoc group and the N-terminal amino group, the peptide resin was washed with DMF (3 x 15 ml, 1 min each time), DCM (3 x 15 ml, 1 min each time), diethyl ether (3 x 15 ml, 1 min each time) and dried in vacuo.

The peptide was cleaved from the resin in the manner described above and lyophilized from acetic acid. Yield of crude material 29.9 mg. After purification using preparative HPLC, as described above, 19 mg of peptide product was collected, with a purity greater than 97%. The total yield of purified peptide product was 50%.

The identity of the peptide was confirmed by ES-MS (found MH + 578.18, calculated MH + 578.23).

Synthesis example 13

H-Gly-Ala-Gly-A2C-Pro-Tyr-NH Peptide Synthesis₂ (Compound 13) on TentaGel-S-Ram; Rapp polymer, Germany

Dry TentaGel-S-Ram (0.23 mmol / g, 1 g) was deposited in a polyethylene container equipped with a polypropylene filter for filtration, and treated as described in "discontinuous synthesis of peptides on TentaGel resin" until completion of the coupling of the N-terminal glycine. All couplings were continued overnight. Acylations were checked by the ninhydrin test carried out at 80 ° C, as described above. After deprotection of the Fmoc group and the N-terminal amino group, the peptide resin was washed with DMF (3 x 15 ml, 1 min each time), DCM (3 x 15 ml, 1 min each time), diethyl ether (3 x 15 ml, 1 min each time) and dried in vacuo.

The peptide was cleaved from the resin in the manner described above and lyophilized from acetic acid. Yield of crude material 27.3 mg. After purification using preparative HPLC, as described above, 12.7 mg of peptide product was collected, with a purity greater than 97%. The total yield of purified peptide product was 34%.

The identity of the peptide was confirmed by ES-MS (MH + found 546.28, MH + calculated 546.55).

Synthesis Example 14

Peptide synthesis of H-Gly-Ala-Gly-PC-Pro-Tyr-NH2 (Compound 14) on TentaGel-S-Ram; Rapp polymer, Germany

Dry TentaGel-S-Ram (0.23 mmol / g, 1 g) was deposited in a polyethylene container equipped with a polypropylene filter for filtration, and treated as described in "discontinuous synthesis of peptides on TentaGel resin" until completion of the coupling of the N-terminal glycine. All couplings were continued overnight. I know

ES 2 228 807 T3 checked the acylations by the ninhydrin test carried out at 80 ° C, as described above. After deprotection of the Fmoc group and the N-terminal amino group, the peptide resin was washed with DMF (3 x 15 ml, 1 min each time), DCM (3 x 15 ml, 1 min each time), diethyl ether (3 x 15 ml, 1 min each time) and dried in vacuo.

The peptide was cleaved from the resin in the manner described above and lyophilized from acetic acid. Yield of crude material 23.4 mg. After purification using preparative HPLC, as described above, 13.5 mg of peptide product was collected, with a purity greater than 97%. The total yield of purified peptide product was 34.6%.

The identity of the peptide was confirmed by ES-MS (MH⁺ found 574.32, MH⁺ calculated 574.29).

Synthesis Example 15

Ac-Tyr-Pro-Hyp-Gly-Ala-Gly-NH Peptide Synthesis₂ (Compound 15) on TentaGel-S-Ram; Rapp polymer, Germany

Dry TentaGel-S-Ram (0.23 mmol / g, 1 g) was deposited in a polyethylene container equipped with a polypropylene filter for filtration, and treated as described in "discontinuous synthesis of peptides on TentaGel resin" until completion of the coupling of the N-terminal glycine. All couplings were 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. Acylations were checked by the ninhydrin test carried out at 80 ° C, as described above. After completion of 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). time) and dried under vacuum.

After deprotection of the Fmoc group and 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 in vacuo.

The peptide was cleaved from the resin in the manner described above and lyophilized from acetic acid. Yield of crude material 89.9 mg. After purification using preparative HPLC, as described above, 80.1 mg of peptide product was collected, with a purity greater than 99%. The total yield of purified peptide product was 58.9%.

The identity of the peptide was confirmed by ES-MS (MH + found 618.30, MH + calculated 618.28).

Synthesis Example 16

Peptide synthesis of H-Cys (Acm) -Gly-Ala-Gly-Hyp-Pro-Tyr-Cys (Acm) -NH₂ (Compound 16) on TentaGel-S-Ram; Rapp polymer, Germany

Dry TentaGel-S-Ram (0.23 mmol / g, 1 g) was deposited in a polyethylene container equipped with a polypropylene filter for filtration, and treated as described in "discontinuous synthesis of peptides on TentaGel resin" until completion of the coupling of the N-terminal Cystine (Acm). All couplings were continued overnight. Acylations were checked by the ninhydrin test carried out at 80 ° C, as described above. After deprotection of the Fmoc group and the N-terminal amino group, the peptide resin was washed with DMF (3 x 15 ml, 1 min each time), DCM (3 x 15 ml, 1 min each time), diethyl ether (3 x 15 ml, 1 min each time) and dried in vacuo.

The peptide was cleaved from the resin in the manner described above and lyophilized from acetic acid. Yield of crude material 47.3 mg. After purification using preparative HPLC, as described above, 29.1 mg of peptide product was collected, with a purity greater than 97%. The total yield of purified peptide product was 12.9%.

The identity of the peptide was confirmed by ES-MS (MH⁺ found 924.50, MH⁺ calculated 924.36).

Synthesis example 17

H-Cys (Acm) -Gly-Hyp-Pro-Tyr-Cys (Acm) -NH Peptide Synthesis₂ (Compound 17) on TentaGel-S-Ram; Rapp polymer, Germany

Dry TentaGel-S-Ram (0.23 mmol / g, 1 g) was deposited in a polyethylene container equipped with a polypropylene filter for filtration, and treated as described in "discontinuous synthesis of peptides on TentaGel resin" until completion of the coupling of the N-terminal Cystine (Acm). All couplings were continued overnight. Acylations were checked by the ninhydrin test carried out at 80 ° C, as described above. After deprotection of the Fmoc group and the N-terminal amino group, the peptide resin was washed with DMF (3 x 15 ml, 1 min each time), DCM (3 x 15 ml, 1 min each time), diethyl ether (3 x 15 ml, 1 min each time) and dried in vacuo.

ES 2 228 807 T3

The peptide was cleaved from the resin in the manner described above and lyophilized from acetic acid. Yield of crude material 45.67 mg. After purification using preparative HPLC, as described above, 29.15 mg of peptide product was collected, with a purity greater than 94%. The total yield of purified peptide product was 14.9%.

The identity of the peptide was confirmed by ES-MS (MH + found 796.25, MH + calculated 796.30).

Synthesis Example 18

Peptide synthesis of H-Cys (Acm) -Tyr-Pro-Hyp-Gly-Ala-Gly-Cys (Acm) -NH₂ (Compound 18) on TentaGel-S-Ram; Rapp polymer, Germany

Dry TentaGel-S-Ram (0.23 mmol / g, 1 g) was deposited in a polyethylene container equipped with a polypropylene filter for filtration, and treated as described in "discontinuous synthesis of peptides on TentaGel resin" until completion of the coupling of the N-terminal Cystine (Acm). All couplings were continued overnight. Acylations were checked by the ninhydrin test carried out at 80 ° C, as described above. After deprotection of the Fmoc group and the N-terminal amino group, the peptide resin was washed with DMF (3 x 15 ml, 1 min each time), DCM (3 x 15 ml, 1 min each time), diethyl ether (3 x 15 ml, 1 min each time) and dried in vacuo.

The peptide was cleaved from the resin in the manner described above and lyophilized from acetic acid. The lyophilized crude product was analyzed by HPLC and compound 17 was similarly purified and characterized.

Synthesis Example 19

H-Cys (Acm) -Tyr-Pro-Hyp-Gly-Cys (Acm) -NH Peptide Synthesis₂ (Compound 19) on TentaGel-S-Ram; Rapp polymer, Germany

Dry TentaGel-S-Ram (0.23 mmol / g, 1 g) was deposited in a polyethylene container equipped with a polypropylene filter for filtration, and treated as described in "discontinuous synthesis of peptides on TentaGel resin" until completion of the coupling of the N-terminal Cystine (Acm). All couplings were continued overnight. Acylations were checked by the ninhydrin test carried out at 80 ° C, as described above. After deprotection of the Fmoc group and the N-terminal amino group, the peptide resin was washed with DMF (3 x 15 ml, 1 min each time), DCM (3 x 15 ml, 1 min each time), diethyl ether (3 x 15 ml, 1 min each time) and dried in vacuo.

The peptide was cleaved from the resin in the manner described above and lyophilized from acetic acid. After purification using preparative HPLC, as described above, 2.76 mg of peptide product was collected, with a purity greater than 94%. The total yield of purified peptide product was 17.9%.

The identity of the peptide was confirmed by ES-MS (MH⁺ found 796.25, MH⁺ calculated 796.30).

Synthesis Example 20

Synthesis of H-Cys-Tyr-Pro-Hyp-Gly-Cys-NH₂ (Compound 20)

19 mg of the H-Cys-Tyr-Pro-Hyp-Gly-Cys-NH peptide are oxidized₂ dissolving the peptide in 1.5 ml of 5% acetic acid in water and DMSO, 4: 1 v / v, pH ~ 6. The mixture is placed in the refrigerator for 6 days.

After purification using preparative HPLC, in the manner described above, 91 mg of peptide product was collected with a purity greater than 97%. The total yield of purified peptide product was 47%. The identity of the peptide was confirmed by ES-MS (found MH + 652.29, calculated MH + 652.21).

Synthesis Example 21

Synthesis of H-Cys-Gly-Hyp-Pro-Tyr-Cys-NH₂ (Compound 21)

32 mg of the peptide H-Cys-Gly-4Hyp-Pro-Tyr-Cys-NH are oxidized₂ dissolving the peptide in 1.5 ml of 5% acetic acid in water and DMSO, 4: 1 v / v, pH ~ 6. The mixture is placed in the refrigerator for 6 days.

After purification using preparative HPLC, in the manner described above, 6.13 mg of peptide product was collected with a purity greater than 99%. The total yield of purified peptide product was 3%.

The identity of the peptide was confirmed by ES-MS (found MH + 652.23, calculated MH + 652.21).

ES 2 228 807 T3

Synthesis Example 22

Peptide synthesis of H-Gly-D-Ala-Gly-D-Hyp-D-Pro-D-Tyr-NH2 (Compound 22) on TentaGel-S-Ram; Rapp polymer, Germany

Dry TentaGel-S-Ram (0.23 mmol / g, 1 g) was deposited in a polyethylene container equipped with a polypropylene filter for filtration, and treated as described in "discontinuous synthesis of peptides on TentaGel resin" until completion of the coupling of the N-terminal glycine. All couplings were continued overnight. Acylations were checked by the ninhydrin test carried out at 80 ° C, as described above. After deprotection of the Fmoc group and the N-terminal amino group, the peptide resin was washed with DMF (3 x 15 ml, 1 min each time), DCM (3 x 15 ml, 1 min each time), diethyl ether (3 x 15 ml, 1 min each time) and dried in vacuo.

The peptide was cleaved from the resin in the manner described above and lyophilized from acetic acid. After purification using preparative HPLC, as described above, 47 mg of peptide product was collected, with a purity greater than 94%. The total yield of purified peptide product was 30%.

The identity of the peptide was confirmed by ES-MS (MH⁺ found 576.26, MH⁺ calculated 576.26).

Synthesis example 23

Peptide synthesis of H-Gly-D-Ala-Gly-D-Hyp-D-Pro-D-Tyr-D-Asn-OH (Compound 23) on TentaGel-S-Ram; Rapp volume, Germany

Dry TentaGel-S-Ram (0.23 mmol / g, 1 g) was deposited in a polyethylene container equipped with a polypropylene filter for filtration, and treated as described in "discontinuous synthesis of peptides on TentaGel resin" until completion of the coupling of the N-terminal glycine. All couplings were continued overnight. Acylations were checked by the ninhydrin test carried out at 80 ° C, as described above. After deprotection of the Fmoc group and the N-terminal amino group, the peptide resin was washed with DMF (3 x 15 ml, 1 min each time), DCM (3 x 15 ml, 1 min each time), diethyl ether (3 x 15 ml, 1 min each time) and dried in vacuo.

The peptide was cleaved from the resin in the manner described above and lyophilized from acetic acid. Yield of crude material 93.7 mg. After purification using preparative HPLC, as described above, 60.7 mg of peptide product was collected, with a purity greater than 93%. The total yield of purified peptide product was 47.5%.

The identity of the peptide was confirmed by ES-MS (MH⁺ found 690.32, MH⁺ calculated 690.30).

Synthesis Example 24

Synthesis of Ac-D-Tyr (3,5-di-I) -D-Pro-D-Hyp-Gly-D-Ala-Gly-NH₂ (Compound 24)

40.6 mg (64 pinol) of the peptide (Compound 2) are dissolved in 10 ml of 0.1 M phosphate buffer, pH 6.5 (solution A).

75.6 mg of KI (400 pinol) are dissolved in 10 ml of phosphate buffer at pH 6.5 and 120 iodine beads (IODO-BEADS, N-chloro-benzenesulfonamide, oxidative capacity 0.55 pmol / bead; PIERCE 28665ZZ), and the solution is left at room temperature for 10 min (solution B).

Solutions A and B are combined and shaken gently for 15 min. The iodinated peptide was isolated and purified using preparative HPLC, as described above. 39.5 mg of peptide product were collected with a purity greater than 90%. The identity of the peptide was confirmed by ES-MS (MH⁺ found 870.09, MH⁺ calculated 870.08).

Synthesis Example 25

Synthesis of Ac-D-Tyr (mono-iodo) -D-Pro-D-Hyp-Gly-D-Ala-Gly-NH2 (Compound 25)

40.6 mg (64 pinol) of the peptide (Compound 2) are dissolved in 10 ml of 0.1 M phosphate buffer, pH 6.5 (solution A).

75.6 mg of KI (400 pinol) are dissolved in 10 ml of phosphate buffer at pH 6.5 and 120 iodine beads (IODO-BEADS, N-chloro-benzenesulfonamide, oxidative capacity 0.55 pmol / bead; PIERCE 28665ZZ), and the solution is left at room temperature for 10 min (solution B).

Solutions A and B are combined and shaken gently for 15 min. The iodinated peptide was isolated and purified using preparative HPLC, as described above. 3.3 mg of peptide product were collected with

ES 2 228 807 T3 has a purity greater than 90%. The identity of the peptide was confirmed by ES-MS (MH + found 744.19, MH + calculated 744.18).

Synthesis Example 26

Ac-D-Tyr-D-Pro-D-4Hyp- (1,2¹³C,¹⁵N-Gly) -D-Ala- (1,2¹³C,¹⁵N-Gly-NH₂ (Compound 26) on TentaGel-S-Ram; Rapp polymer, Germany

Dry TentaGel-S-Ram (0.23 mmol / g, 1 g) was deposited in a polyethylene container equipped with a polypropylene filter for filtration, and treated as described in "discontinuous synthesis of peptides on TentaGel resin" until the end of the coupling of the N-terminal D-tyrosine. All couplings were 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. Coupling was continued overnight. Acylations were checked by the ninhydrin test carried out at 80 ° C, as described above. After completion of 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). time) and dried under vacuum.

The peptide was cleaved from the resin in the manner described above and lyophilized from acetic acid. Yield of crude material, 142.4 mg. After purification using preparative HPLC, as described above, 79.7 mg of peptide product was collected, with a purity greater than 99%. The total yield of purified peptide product was 50%.

The identity of the peptide was confirmed by ES-MS (MH + found 624.25, MH + calculated 624.26).

Synthesis Example 27

H-Pro-Tyr-Asn-Gly-Ala-Gly-Hyp-NH Peptide Synthesis₂ (Compound 27) on TentaGel-S-Ram; Rapp polymer, Germany

Dry TentaGel-S-Ram (0.23 mmol / g, 1 g) was deposited in a polyethylene container equipped with a polypropylene filter for filtration, and treated as described in "discontinuous synthesis of peptides on TentaGel resin" until completion of the coupling of the N-terminal proline. All couplings were continued overnight. Acylations were checked by the ninhydrin test carried out at 80 ° C, as described above. After deprotection of the Fmoc group and the N-terminal amino group, the peptide resin was washed with DMF (3 x 15 ml, 1 min each time), DCM (3 x 15 ml, 1 min each time), diethyl ether (3 x 15 ml, 1 min each time) and dried in vacuo.

The peptide was cleaved from the resin in the manner described above and lyophilized from acetic acid. After purification using preparative HPLC, as described above, 135.7 mg of peptide product was collected, with a purity greater than 98%. The total yield of purified peptide product was 82.7%.

The identity of the peptide was confirmed by ES-MS (MH + found 690.38, MH + calculated 690.31).

Synthesis Example 28

H-Hyp-Pro-Tyr-Asn-Gly-Ala-Gly-NH Peptide Synthesis₂ (Compound 28) on TentaGel-S-Ram; Rapp polymer, Germany

Dry TentaGel-S-Ram (0.23 mmol / g, 1 g) was deposited in a polyethylene container equipped with a polypropylene filter for filtration, and treated as described in "discontinuous synthesis of peptides on TentaGel resin" until completion of the coupling of the N-terminal 4-hydroxyproline. All couplings were continued overnight. Acylations were checked by the ninhydrin test carried out at 80 ° C, as described above. After deprotection of the Fmoc group and the N-terminal amino group, the peptide resin was washed with DMF (3 x 15 ml, 1 min each time), DCM (3 x 15 ml, 1 min each time), diethyl ether (3 x 15 ml, 1 min each time) and dried in vacuo.

The peptide was cleaved from the resin in the manner described above and lyophilized from acetic acid. After purification using preparative HPLC, as described above, 127 mg of peptide product was collected, with a purity greater than 98%. The total yield of purified peptide product was 69.8%.

The identity of the peptide was confirmed by ES-MS (found MH + 690.25, calculated MH + 690.31).

Synthesis Example 29

H-Sar-Ala-Sar-Hyp-Pro-Tyr-NH Peptide Synthesis₂ (Compound 29) on TentaGel-S-Ram; Rapp polymer, Germany

Dry TentaGel-S-Ram (0.23 mmol / g, 1 g) was placed in a polyethylene container equipped with a filter of

ES 2 228 807 T3 polypropylene for filtration, and was treated as described in "discontinuous synthesis of peptides on TentaGel resin" until completion of the N-terminal sarcosine coupling. All couplings were continued overnight. Acylations were checked by the ninhydrin test carried out at 80 ° C, as described above. After deprotection of the Fmoc group and the N-terminal amino group, the peptide resin was washed with DMF (3 x 15 ml, 1 min each time), DCM (3 x 15 ml, 1 min each time), diethyl ether (3 x 15 ml, 1 min each time) and dried in vacuo.

The peptide was cleaved from the resin in the manner described above and lyophilized from acetic acid. Yield of crude material, 150 mg. After purification using preparative HPLC, as described above, 85.5 mg of peptide product was collected, with a purity greater than 93%. The total yield of purified peptide product was 57%.

The identity of the peptide was confirmed by ES-MS (MH⁺ found 604.33, MH⁺ calculated 604.30).

Synthesis Example 30

H-Gly-Ala-Sar-Hyp-Pro-Tyr-NH Peptide Synthesis₂ (Compound 30) on TentaGel-S-Ram; Rapp polymer, Germany

Dry TentaGel-S-Ram (0.23 mmol / g, 1 g) was deposited in a polyethylene container equipped with a polypropylene filter for filtration, and treated as described in "discontinuous synthesis of peptides on TentaGel resin" until completion of the coupling of the N-terminal glycine. All couplings were continued overnight. Acylations were checked by the ninhydrin test carried out at 80 ° C, as described above. After deprotection of the Fmoc group and the N-terminal amino group, the peptide resin was washed with DMF (3 x 15 ml, 1 min each time), DCM (3 x 15 ml, 1 min each time), diethyl ether (3 x 15 ml, 1 min each time) and dried in vacuo.

The peptide was cleaved from the resin in the manner described above and lyophilized from acetic acid. Yield of crude material, 124 mg. After purification using preparative HPLC, as described above, 64.8 mg of peptide product was collected, with a purity greater than 96%. The total yield of purified peptide product was 41.6%.

The identity of the peptide was confirmed by ES-MS (found MH + 590.19, calculated MH + 590.29).

Synthesis Example 31

ASAL-Pro-Hyp-Gly-Ala-Gly-NH Peptide Synthesis₂ (Compound 31) on TentaGel-S-Ram; Rapp polymer, Germany

Dry TentaGel-S-Ram (0.23 mmol / g, 1 g) was deposited in a polyethylene container equipped with a polypropylene filter for filtration, and treated as described in "discontinuous synthesis of peptides on TentaGel resin" until completion of the coupling of the N-terminal proline. All couplings were continued overnight. After deprotection of the Fmoc group, the N-terminal amino group was acetylated with Azido-salicylic acid using the standard coupling procedure, as described above. Coupling was continued overnight. Acylations were checked by the ninhydrin test carried out at 80 ° C, as described above. After completion of 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). time) and dried under vacuum.

The peptide was cleaved from the resin in the manner described above and lyophilized from acetic acid. After purification using preparative HPLC, as described above, 15.9 mg of peptide product was collected, with a purity greater than 94%.

The identity of the peptide was confirmed by ES-MS (MH⁺ found 575.23, MH⁺ calculated 575.56).

Synthesis Example 32

Peptide Synthesis of ASAL (mono-iodine) -Pro-Hyp-Gly-Ala-Gly-NH2 (Compound 32)

10.3 mg of the peptide (Compound 31) is dissolved in 2.5 ml of 0.1 M phosphate buffer, pH 6.5 (solution A).

18.9 mg of KI (100 // mol) are dissolved in 2.5 ml of phosphate buffer at pH 6.5 and 30 iodine beads are added (IODO-BEADS, N-chloro-benzenesulfonamide, oxidative capacity 0.55 jumol / perla; PIERCE 28665ZZ), and the solution is left at room temperature for 10 min (solution B).

Solutions A and B are combined and shaken gently for 1 hour. The iodinated peptide was isolated and purified using preparative HPLC, as described above. 4.4 mg of peptide product were collected with a purity greater than 99%. The identity of the peptide was confirmed by ES-MS (MH + found 701.13, MH + calculated 701.46).

ES 2 228 807 T3

Synthesis Example 33

Peptide synthesis of AB-Tyr-Pro-Hyp-Gly-Ala-Gly-NH2 (Compound 33) on TentaGel-S-Ram; Rapp polymer, Germany

Dry TentaGel-S-Ram (0.23 mmol / g, 1 g) was deposited in a polyethylene container equipped with a polypropylene filter for filtration, and treated as described in "discontinuous synthesis of peptides on TentaGel resin" until completion of the coupling of the N-terminal tyrosine. All couplings were continued overnight. After deprotection of the Fmoc group, the N-terminal amino group was acetylated with Azido-benzoic acid using the standard coupling procedure, as described above. Coupling was continued overnight. Acylations were checked by the ninhydrin test carried out at 80 ° C, as described above. After completion of 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). time) and dried under vacuum.

The peptide was cleaved from the resin in the manner described above and lyophilized from acetic acid. After purification using preparative HPLC, as described above, 20.5 mg of peptide product was collected, with a purity greater than 90%.

The identity of the peptide was confirmed by ES-MS (MH + found 721.28, MH + calculated 721.26).

Synthesis Example 34

AB-Tyr (3,5-di-iodo) -Pro-Hyp-Gly-Ala-Gly-NH Peptide Synthesis₂ (Compound 34)

10.3 mg of the peptide (Compound 33) is dissolved in 2.5 ml of 0.1 M phosphate buffer, pH 6.5 (solution A).

18.9 mg of KI (100 pinol) are dissolved in 2.5 ml of phosphate buffer at pH 6.5 and 30 iodine beads (IODO-BEADS, N-chloro-benzenesulfonamide, oxidative capacity 0.55 pmol / pearl; PIERCE 28665ZZ), and the solution is left at room temperature for 10 min (solution B).

Solutions A and B are combined and shaken gently for 1 hour. The iodinated peptide was isolated and purified using preparative HPLC, as described above. 1.2 mg of peptide product were collected with a purity greater than 90%. The identity of the peptide was confirmed by ES-MS (MH + found 973.08, MH + calculated 973.46).

Synthesis Example 35

Cyclo- (Gly-Ala-Gly-Hyp-Pro-Tyr-Gln) peptide synthesis (Compound 35) on TentaGel-S-Ram; Rapp polymer, Germany

Dry TentaGel-S-Ram (0.23 mmol / g, 1 g) was deposited into a polyethylene container equipped with a polypropylene filter for filtration and treated as described in "batch peptide synthesis on TentaGel resin." The first amino acid Fmoc-Glu (OH) -O-All was connected to the TentaGel-S-Ram resin through the side chain carboxylic acid, which will end up being amidated (Asn) finally, after cleavage. The procedure described under "discontinuous synthesis of peptides on TentaGel resin" was followed until completion of the N-terminal glycine coupling. All couplings were continued overnight. After deprotection of the Fmoc group and the allyl group (according to the procedure described above), the peptide bound to the resin was cyclized using PyBop as the coupling reagent, as described above, and the coupling was continued overnight. Acylations were checked by the ninhydrin test carried out at 80 ° C, as described above. After completion of 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). time), and dried under vacuum.

The peptide was cleaved from the resin in the manner described above and lyophilized from acetic acid. Yield of crude material, 135.3 mg. After purification using preparative HPLC, as described above, 19.1 mg of peptide product was collected, with a purity greater than 98%. The total yield of purified peptide product was 6.6%.

The identity of the peptide was confirmed by ES-MS (MH⁺ found 687.38; MH⁺ calculated 687.32).

Synthesis Example 36

Cyclo- (Gly-Ala-Gly-Hyp-Pro-Tyr-Asn) peptide synthesis (Compound 36) on TentaGel-S-Ram; Rapp polymer, Germany

Dry TentaGel-S-Ram (0.23 mmol / g, 1 g) was deposited into a polyethylene container equipped with a polypropylene filter for filtration and treated as described in "batch peptide synthesis on TentaGel resin." The first amino acid Fmoc-Asp (OH) -O-All was connected to the TentaGel-S-Ram resin through the side chain carboxylic acid, which will end up being amidated (Asn) finally, after cleavage. The procedure described was followed

ES 2 228 807 T3 under "discontinuous synthesis of peptides on TentaGel resin" until the end of the N-terminal glycine coupling. All couplings were continued overnight. After deprotection of the Fmoc group and the allyl group (according to the procedure described above), the peptide bound to the resin was cyclized using PyBop as the coupling reagent, as described above, and the coupling was continued overnight. Acylations were checked by the ninhydrin test carried out at 80 ° C, as described above. After completion of the synthesis, the peptide resin was washed with DMF (3 x 15 ml, 1 min each time), DCM (3 x 15 ml, 1 min each time), diethyl ether (3 x 15 ml, min each time ), and dried under vacuum.

The peptide was cleaved from the resin in the manner described above and lyophilized from acetic acid. Yield of crude product, 63.4 mg. After purification using preparative HPLC, as described above, 13.2 mg of peptide product was collected, with a purity greater than 97%. The total yield of purified peptide product was 6.2%.

The identity of the peptide was confirmed by ES-MS (MH⁺ found 673.38; MH⁺ calculated, 673.30).

Synthesis Example 37

Cyclo- (Gly-Ala-Gly-Pro-Pro-Tyr-Asn) peptide synthesis (Compound 37) on TentaGel-S-Ram; Rapp polymer, Germany

Dry TentaGel-S-Ram (0.23 mmol / g, 1 g) was deposited into a polyethylene container equipped with a polypropylene filter for filtration and treated as described in "batch peptide synthesis on TentaGel resin." The first amino acid Fmoc-Asp (OH) -O-All was connected to the TentaGel-S-Ram resin through the side chain carboxylic acid, which will end up being amidated (Asn) finally, after cleavage. The procedure described under "discontinuous synthesis of peptides on TentaGel resin" was followed until completion of the N-terminal glycine coupling. All couplings were continued overnight. After deprotection of the Fmoc group and the allyl group (according to the procedure described above), the peptide bound to the resin was cyclized using PyBop as the coupling reagent, as described above, and the coupling was continued overnight. Acylations were checked by the ninhydrin test carried out at 80 ° C, as described above. After completion of 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). time), and dried under vacuum.

The peptide was cleaved from the resin in the manner described above and lyophilized from acetic acid. Yield of crude product, 85.1 mg. After purification using preparative HPLC, as described above, 9.8 mg of peptide product was collected, with a purity greater than 98%. The total yield of purified peptide product was 3.5%.

The identity of the peptide was confirmed by ES-MS (MH + found 657.38; MH + calculated, 657.31).

Synthesis Example 38

Synthesis of cyclo- (Tyr (3,5-diiodo) -Pro-4Hyp-Gly-Ala-Gly-Asn) (Compound 38)

10.8 mg of the peptide (Compound 3) is dissolved in 2.5 ml of 0.1 M phosphate buffer, pH 6.5 (solution A).

18.9 mg of KI (400 pinol) are dissolved in 2.5 ml of phosphate buffer at pH 6.5 and 30 iodine beads (IODO-BEADS, N-chloro-benzenesulfonamide, oxidative capacity 0.55 pmol / pearl; PIERCE 28665ZZ), and the solution is left at room temperature for 10 min (solution B).

Solutions A and B are combined and shaken gently for 2 hours. The iodinated peptide was isolated and purified using preparative HPLC, as described above. 9.8 mg of peptide product were collected with a purity greater than 95%. The identity of the peptide was confirmed by ES-MS (MH + found 925.10, MH + calculated 925.30).

Synthesis Example 39

Peptide synthesis of H-Gly-Ala-Gly-Asn-Tyr-NH2 (Compound 39) on TentaGel-S-Ram; Rapp polymer, Germany

Dry TentaGel-S-Ram (0.23 mmol / g, 1 g) was deposited in a polyethylene container equipped with a polypropylene filter for filtration, and treated as described in "discontinuous synthesis of peptides on TentaGel resin" until completion of the coupling of the N-terminal glycine. All couplings were continued overnight. Acylations were checked by the ninhydrin test carried out at 80 ° C, as described above. After deprotection of the Fmoc group and the N-terminal amino group, the peptide resin was washed with DMF (3 x 15 ml, 1 min each time), DCM (3 x 15 ml, 1 min each time), diethyl ether (3 x 15 ml, 1 min each time) and dried in vacuo.

The peptide was cleaved from the resin in the manner described above and lyophilized from acetic acid. Yield of crude material, 124 mg. After purification using preparative HPLC, as described

ES 2 228 807 T3 above, 26.5 mg of peptide product was collected, with a purity greater than 96%. The total yield of purified peptide product was 20.5%.

The identity of the peptide was confirmed by ES-MS (MH + found 480.24, MH + calculated 480.50).

Synthesis Example 40

Ac-Gly-Asn-Tyr-NH Peptide Synthesis₂ (Compound 40) on TentaGel-S-Ram; Rapp polymer, Germany

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 "batch peptide synthesis on TentaGel resin" until finalize the 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), along with 100 µl of pyridine dissolved in 2 ml of DMF. Coupling was continued overnight. Acylations were checked by the ninhydrin test carried out at 80 ° C, 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 time), and dried under vacuum.

The peptide was cleaved from the resin in the manner described above and lyophilized from acetic acid. Yield of crude material, 90.4 mg. After purification using preparative HPLC, in the manner described above, 63.4 mg of peptide product was collected with a purity greater than 99%. The total yield of purified peptide product was 65.1.

The identity of the peptide was confirmed by ES-MS (found MH + 394.16, calculated MH + 394.20).

Synthesis Example 41

Peptide synthesis of H-Gly-Asn-Tyr-NH2 (Compound 41) on TentaGel-S-Ram; Rapp polymer, Germany

Dry TentaGel-S-Ram (0.23 mmol / g, 1 g) was deposited in a polyethylene container equipped with a polypropylene filter for filtration, and treated as described in "discontinuous synthesis of peptides on TentaGel resin" until completion of the coupling of the N-terminal glycine. All couplings were continued overnight. Acylations were checked by the ninhydrin test carried out at 80 ° C, as described above. After deprotection of the Fmoc group and the N-terminal amino group, the peptide resin was washed with DMF (3 x 15 ml, 1 min each time), DCM (3 x 15 ml, 1 min each time), diethyl ether (3 x 15 ml, 1 min each time) and dried in vacuo.

The peptide was cleaved from the resin in the manner described above and lyophilized from acetic acid. Yield of crude material 91.4 mg. After purification using preparative HPLC, as described above, 62.1 mg of peptide product was collected, with a purity greater than 95%. The total yield of purified peptide product was 54.5%.

The identity of the peptide was confirmed by ES-MS (MH + found 352.16, MH + calculated 352.18).

Synthesis Example 42

Peptide synthesis of Ac-Ala-Gly-Asn-Tyr-NH2 (Compound 42) on TentaGel-S-Ram; Rapp polymer, Germany

Dry TentaGel-S-Ram (0.23 mmol / g, 1 g) was deposited into a polyethylene container equipped with a polypropylene filter for filtration and treated as described in "batch peptide synthesis on TentaGel resin" until terminate the coupling of the N-terminal alanine. After deprotection of the Fmoc group, the N-terminal amino group was acetylated with acetic acid anhydride (1 ml, 10.5 mmol), along with 100 µl of pyridine dissolved in 2 ml of DMF. Coupling was continued overnight. Acylations were checked by the ninhydrin test carried out at 80 ° C, 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 time), and dried under vacuum.

The peptide was cleaved from the resin in the manner described above and lyophilized from acetic acid. Yield of crude material, 105 mg. After purification using preparative HPLC, as described above, 52 mg of peptide product was collected with a purity greater than 98%. The total yield of purified peptide product was 45%.

The identity of the peptide was confirmed by ES-MS (MH + found 465.22, MH + calculated 465.30).

ES 2 228 807 T3

Synthesis example 43

H-Ala-Gly-Asn-Tyr-NH Peptide Synthesis₂ (Compound 43) on TentaGel-S-Ram; Rapp polymer, Germany

Dry TentaGel-S-Ram (0.23 mmol / g, 1 g) was deposited in a polyethylene container equipped with a polypropylene filter for filtration, and treated as described in "discontinuous synthesis of peptides on TentaGel resin" until completion of the coupling of the N-terminal alanine. All couplings were continued overnight. Acylations were checked by the ninhydrin test carried out at 80 ° C, as described above. After deprotection of the Fmoc group and the N-terminal amino group, the peptide resin was washed with DMF (3 x 15 ml, 1 min each time), DCM (3 x 15 ml, 1 min each time), diethyl ether (3 x 15 ml, 1 min each time) and dried in vacuo.

The peptide was cleaved from the resin in the manner described above and lyophilized from acetic acid. Yield of crude material, 104.5 mg. After purification using preparative HPLC, as described above, 77.8 mg of peptide product was collected, with a purity greater than 96%. The total yield of purified peptide product was 58.8%.

The identity of the peptide was confirmed by ES-MS (MH + found 423.19, MH + calculated 423.28).

Synthesis Example 44

Cyclo- (Tyr-Ala-Ser-Ala-Gly-Asn) peptide synthesis (Compound 44) ​​on TentaGel-S-Ram; Rapp polymer, Germany

Dry TentaGel-S-Ram (0.23 mmol / g, 1 g) was deposited into a polyethylene container equipped with a polypropylene filter for filtration and treated as described in "batch peptide synthesis on TentaGel resin." The first amino acid Fmoc-Asp (OH) -O-All was connected to the TentaGel-S-Ram resin through the side chain carboxylic acid, which will end up being amidated (Asn) finally, after cleavage. The procedure described under "discontinuous synthesis of peptides on TentaGel resin" was followed until completion of the N-terminal tyrosine coupling. All couplings were continued overnight. After deprotection of the Fmoc group and the allyl group (according to the procedure described above), the peptide bound to the resin was cyclized using PyBop as the coupling reagent, as described above, and the coupling was continued overnight. Acylations were checked by the ninhydrin test carried out at 80 ° C, as described above. After completion of 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). time), and dried under vacuum.

The peptide was cleaved from the resin in the manner described above and lyophilized from acetic acid. Yield of crude product, 60.2 mg. After purification using preparative HPLC, as described above, 5.0 mg of peptide product was collected with a purity greater than 87%. The total yield of purified peptide product was 4.3%.

The identity of the peptide was confirmed by ES-MS (MH⁺ found 564.25, MH⁺ calculated 564.57).

Synthesis Example 45

Cyclo- (Tyr-Gly-Asn-Tyr-Gly-Asn) peptide synthesis (Compound 45) on TentaGel-S-Ram; Rapp polymer, Germany

Dry TentaGel-S-Ram (0.23 mmol / g, 1 g) was deposited into a polyethylene container equipped with a polypropylene filter for filtration and treated as described in "batch peptide synthesis on TentaGel resin." The first amino acid Fmoc-Asp (OH) -O-All was connected to the TentaGel-S-Ram resin through the side chain carboxylic acid, which will end up being amidated (Asn) finally, after cleavage. The procedure described under "discontinuous synthesis of peptides on TentaGel resin" was followed until completion of the N-terminal tyrosine coupling. All couplings were continued overnight. After deprotection of the Fmoc group and the allyl group (according to the procedure described above), the peptide bound to the resin was cyclized using PyBop as the coupling reagent, as described above, and the coupling was continued overnight. Acylations were checked by the ninhydrin test carried out at 80 ° C, as described above. After completion of 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). time), and dried under vacuum.

The peptide was cleaved from the resin in the manner described above and lyophilized from acetic acid. Yield of crude product, 79.1 mg. After purification using preparative HPLC, as described above, 20 mg of peptide product was collected with a purity greater than 90%. The total yield of purified peptide product was 14%.

The identity of the peptide was confirmed by ES-MS (MH⁺ found 569.25, MH⁺ calculated 569.67).

ES 2 228 807 T3

Synthesis Example 46

Cyclo- (Tyr-Gly-Asn-Tyr-Ala-Gly-Asn) peptide synthesis (Compound 46) on TentaGel-S-Ram; Rapp polymer, Germany

Dry TentaGel-S-Ram (0.23 mmol / g, 1 g) was deposited into a polyethylene container equipped with a polypropylene filter for filtration and treated as described in "batch peptide synthesis on TentaGel resin." The first amino acid Fmoc-Asp (OH) -O-All was connected to the TentaGel-S-Ram resin through the side chain carboxylic acid, which will end up being amidated (Asn) finally, after cleavage. The procedure described under "discontinuous synthesis of peptides on TentaGel resin" was followed until completion of the N-terminal tyrosine coupling. All couplings were continued overnight. After deprotection of the Fmoc group and the allyl group (according to the procedure described above), the peptide bound to the resin was cyclized using PyBop as the coupling reagent, as described above, and the coupling was continued overnight. Acylations were checked by the ninhydrin test carried out at 80 ° C, as described above. After completion of 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). time), and dried under vacuum.

The peptide was cleaved from the resin in the manner described above and lyophilized from acetic acid. Yield of crude product, 58.9 mg. After purification using preparative HPLC, as described above, 15.9 mg of peptide product was collected with a purity greater than 98%. The total yield of purified peptide product was 11%.

The identity of the peptide was confirmed by ES-MS (MH + found 740.31, MH + calculated 740.75).

Synthesis example 47

Cyclo- (Tyr-Val-Ser-Gly-Ala-Gly-Asn) peptide synthesis (Compound 47) on TentaGel-S-Ram; Rapp polymer, Germany

Dry TentaGel-S-Ram (0.23 mmol / g, 1 g) was deposited into a polyethylene container equipped with a polypropylene filter for filtration and treated as described in "batch peptide synthesis on TentaGel resin." The first amino acid Fmoc-Asp (OH) -O-All was connected to the TentaGel-S-Ram resin through the side chain carboxylic acid, which will end up being amidated (Asn) finally, after cleavage. The procedure described under "discontinuous synthesis of peptides on TentaGel resin" was followed until completion of the N-terminal tyrosine coupling. All couplings were continued overnight. After deprotection of the Fmoc group and the allyl group (according to the procedure described above), the peptide bound to the resin was cyclized using PyBop as the coupling reagent, as described above, and the coupling was continued overnight. Acylations were checked by the ninhydrin test carried out at 80 ° C, as described above. After completion of 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). time), and dried under vacuum.

The peptide was cleaved from the resin in the manner described above and lyophilized from acetic acid. Yield of crude product, 54.1 mg. After purification using preparative HPLC, as described above, 19.6 mg of peptide product was collected with a purity greater than 95%. The total yield of purified peptide product was 15%.

The identity of the peptide was confirmed by ES-MS (MH + found 649.10, MH + calculated 649.68).

Synthesis Example 48

Peptide synthesis of H-Gly-Pro-Hyp-Gly-Ala-Gly-OH (Compound CE-1) on TentaGel-S-NH2; Rapp polymer, Germany

Dry TentaGel-S-NH2 (0.27 mmmol / g, 1 g) was deposited in a polyethylene container equipped with a polypropylene filter for filtration, and treated as described in "discontinuous synthesis of peptides on TentaGel resin" until completion of the coupling of the N-terminal glycine. All couplings were continued overnight. Acylations were checked by the ninhydrin test carried out at 80 ° C, as described above. After deprotection of the Fmoc group and the N-terminal amino group, the peptide resin was washed with DMF (3 x 15 ml, 1 min each time), DCM (3 x 15 ml, 1 min each time), diethyl ether (3 x 15 ml, 1 min each time) and dried in vacuo.

The peptide was cleaved from the resin in the manner described above and lyophilized from acetic acid. After purification using preparative HPLC, as described above, 16.9 mg of peptide product was collected, with a purity greater than 92%. The total yield of purified peptide product was 10.1%.

The identity of the peptide was confirmed by ES-MS (MH + found 471.22, MH + calculated 471.21).

ES 2 228 807 T3

Synthesis Example 49

Peptide synthesis of H-Gly-Ala-Gly-Hyp-Pro-Tyr-NH2 (Compound CE-2) on TentaGel-S-Ram; Rapp polymer, Germany

Dry TentaGel-S-Ram (0.23 mmol / g, 1 g) was deposited in a polyethylene container equipped with a polypropylene filter for filtration, and treated as described in "discontinuous synthesis of peptides on TentaGel resin" until completion of the coupling of the N-terminal glycine. All couplings were continued overnight. Acylations were checked by the ninhydrin test carried out at 80 ° C, as described above. After deprotection of the Fmoc group and the N-terminal amino group, the peptide resin was washed with DMF (3 x 15 ml, 1 min each time), DCM (3 x 15 ml, 1 min each time), diethyl ether (3 x 15 ml, 1 min each time) and dried in vacuo.

The peptide was cleaved from the resin in the manner described above and lyophilized from acetic acid. Yield of crude material, 159 mg. After purification using preparative HPLC, as described above, 101 mg of peptide product was collected, with a purity greater than 98%. The total yield of purified peptide product was 60%.

The identity of the peptide was confirmed by ES-MS (MH⁺ found 576.26, MH⁺ calculated 576.26).

Synthesis Example 50

Peptide synthesis of 3- (4-hydroxyphenyl) -propionyl-Pro-Hyp-Gly-Ala-Gly-NH2 (Compound CE-3) on TentaGel-SRam; Rapp polymer, Germany

Dry TentaGel-S-Ram (0.23 mmol / g, 1 g) was deposited in a polyethylene container equipped with a polypropylene filter for filtration, and treated as described in "discontinuous synthesis of peptides on TentaGel resin" until completion of the coupling of the N-terminal proline. All couplings were continued overnight. After deprotection of the Fmoc group, the N-terminal amino group was acetylated with 3- (4-hydroxyphenyl) propionic acid, using the conventional coupling procedure described above. Coupling was continued overnight. Acylations were checked by the ninhydrin test carried out at 80 ° C, as described above. After completion of 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). time) and dried under vacuum.

The peptide was cleaved from the resin in the manner described above and lyophilized from acetic acid. Yield of crude material, 143 mg. After purification using preparative HPLC, as described above, 73.7 mg of peptide product was collected, with a purity greater than 95%. The total yield of purified peptide product was 50%.

The identity of the peptide was confirmed by ES-MS (MH + found 561.30, MH + calculated 561.24).

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