Peptides for treating diseases associated with impairment in apolipoprotein AI or transthyretin

Abstract

The present invention relates to a peptide identified as SEQ ID NO: 1 for use in the manufacture of a medicament for the treatment of diseases involving impairment in apolipoprotein A-I (ApoAI) or transthyretin (TTR). In particular, the peptides may be useful for the treatment of diseases characterized by impairment in intra-lipid environmental stability or TTR stability. The invention also relates to a method of treating a disease involving impairment in ApoAI or TTR, characterized in that a therapeutically effective amount of a pharmaceutical composition comprising a peptide identified as SEQ ID NO: 1 is administered.

Description

Technical Field

[0001] The present invention relates to the field of medicine, in particular to modified peptides of the APL (Altered Peptide Ligand, abbreviated APL) type derived from HSP60 and their use in ensuring lipid homeostasis, preventing cellular oxidative stress and avoiding amyloidosis caused by improper modification of apolipoprotein AI (Apo-AI) and transthyretin (TTR). The peptides are also useful for treating patients with impairment in lipid transport and improper processing of lipids. Background Art

[0002] Lipids have been described to be essential in the normal functioning of the human body and to play an indispensable role in the acquisition and storage of energy. In addition, they are essential components of lipid membranes, various hormones and bile salts. An imbalance in lipid concentrations (whether excessive or insufficient) is directly related to the development of cardiovascular diseases such as coronary artery disease, cerebrovascular disease or dyslipidemia (Krahmer, N. et al., (2013) EMBO Mol. Med. 5, 973-983).

[0003] Lipids are hydrophobic biomolecules, and most of them are insoluble in blood, so their transport is driven by lipoproteins. Lipoproteins are classified according to their size and their density. Lipoproteins are hydrophobic spherical structures with proteins (apoproteins or apolipoproteins) on their surfaces that can bind to enzymes responsible for lipid processing (Rosenson, RS et al., (2016). Nat. Rev. Cardiol. 13, 48-60).

[0004] High-density apolipoprotein (HDL) transports excess cholesterol from extrahepatic tissues to the liver, where it is metabolized. HDL is associated with a reduction in cardiovascular risk, while low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) increase cardiovascular risk. There is a negative correlation between cholesterol concentrations associated with HDL and the risk of atherosclerosis (Lee, M., PT et al., (2003). J. Lipid Res. 44: 539-546).

[0005] HDL is a lipoprotein with the highest density because it has a higher protein concentration than lipids. These lipoproteins are composed of cholesterol, triglycerides and many apolipoproteins (Apo-AI, Apo-AII, Apo-AIV, Apo-AV, Apo-C1, Apo-CII, Apo-CIII and Apo-E). Apo-AI is the main protein component of HDL and is the primary apolipoprotein responsible for maintaining the structure of HDL (Jonas, A., KE Kezdy and JH Wald. (1989). J. Biol. Chem. 264: 4818-4824). Apo-AI activates phosphatidylcholine-cholesterol-acyltransferase, which catalyzes the conversion of cholesterol into its ester form, thereby improving the ability of HDL to transport lipids to the liver (Gordon, T. et al., (1977). Am. J. Med. 62, 707-714). The blood concentration of Apo-AI increases during certain physiological conditions (e.g., pregnancy, liver disease, use of estrogen), and decreases during sepsis and atherosclerosis. High concentrations of Apo-AI and low concentrations of Apo-B (the main protein component of low-density lipoprotein) are significantly associated with a reduction in the risk of cardiovascular disease. HDL has anti-atherosclerotic and anti-inflammatory properties because they transport cholesterol accumulated in atherosclerotic plaques to the liver, thereby contributing to the reduction of the plaques (TM Forte et al., (2002) J Lipid Res, 43: 477-485).

[0006] The mechanism of formation of atherosclerotic plaques is not fully understood, but the lipoproteins LDL and HDL play a fundamental role in the mechanism. To date, no effective treatment has been found to avoid atherosclerotic plaque formation and the attendant damage to the human body. Therefore, it is crucial to obtain a treatment that ensures lipid homeostasis and proper processing of lipoproteins.

[0007] On the other hand, it has been described that TTR constitutes part of HDL by binding to Apo-AI (Liz, MA, CM Gomes, MG Saraiva and MM Sousa. (2007) J. Lipid Res. 48: 2385-2395). TTR is a tetrameric protein present in plasma, mainly synthesized in the liver, and is responsible for the transport of thyroxine and retinol binding protein. These proteins can be dissolved into monomers and dimers, which can be coupled in fibers and deposited in tissues. Age and point mutations (more than one hundred have been described) can increase the tendency to aggregate. Two forms of TTR amyloidosis have been described: natural form (wt ATTR, which is an abbreviation for wild type transthyretin cardiac amyloidosis (wild type transthyretin cardiac amyloidosis), called senile systemic amyloidosis) and hereditary form (h-ATTR, which is an abbreviation for hereditary transthyretin cardiac amyloidosis (hereditary transthyretin cardiac amyloidosis)). In the natural form, the synthesis of TTR is normal, while in the inherited form, the presence of point mutations causes TTR to be affected, from its synthesis in hepatocytes to its conformation (Kittleson MM, Maurer MS, Ambardekar AA et al., (2020) Circulation; 142: e7-22).

[0008] Among the diseases caused by TTR amyloidosis is amyloid cardiomyopathy, which refers to a cardiac disorder caused by extracellular deposition of insoluble abnormal fibrils. The prevalence of the disease is estimated to be between 13% (in elderly hospitalized with preserved ejection fraction) and 16% (severe aortic stenosis requiring intervention) (Coelho T, Maurer MS, Suhr OB. (2013) Curr Med Res Opin, 29: 63-76).

[0009] Furthermore, post-translational modifications of TTR that affect its tetrameric conformation as well as the presence of its oligomers are strongly associated with Parkinson's disease (Ando Y, Nakamura M, and Araki S. (2005) Arch Neurol 62:1057-1062), and pathological conditions associated with oxidative stress (Sharma M, Khan S, Rahman S, and Singh LR (2019) Front. Physiol. 10:5. doi:10.3389 / fphys.2019.00005).

[0010] It has been described that a portion of plasma TTR circulates in HDL by binding to Apo-AI, and it has been demonstrated that TTR can cleave the carboxyl terminus of lipid-free Apo-AI. TTR cleavage of Apo-AI induces the formation of Apo-AI amyloid fibrils. In addition, it also causes the modified HDL particles to have a reduced ability to promote the proper conversion of cholesterol. On the other hand, TTR cleavage of Apo-AI has a high preference for forming aggregated particles, thereby affecting the conversion of HDL, and by reducing cholesterol metabolism and increasing the amyloidogenic potential of Apo-AI, it is conducive to the development of atherosclerosis (Liz, MA, CM Gomes, MG Saraiva and MM Sousa. (2007) J. Lipid Res. 48: 2385-2395).

[0011] Therapies that seek to stabilize HDL are crucial to ensure proper lipid (especially cholesterol) exchange and to avoid the formation of atherosclerotic plaques or to reduce atherosclerotic plaques. At the same time, therapies that stabilize the conformation of TTR (which avoids its modification and its aggregation) would be effective in preventing amyloidosis and Parkinson's disease mediated by TTR. Summary of the invention

[0012] The present invention solves the above-mentioned problems by providing a peptide whose sequence is identified as SEQ ID NO: 1, which is used in the preparation of a medicament for treating a disease involving impairment in Apo-AI or TTR. The modified peptide identified as SEQ ID NO: 1 is able to stabilize the supramolecular structure of HDL by binding to Apo-AI. In this way, it promotes the proper metabolism of lipids and the reduction of atherosclerotic plaques associated with atherosclerosis. As shown in the present invention, surprisingly, the only proteins that interact with the peptide in human plasma are Apo-AI and TTR. This interaction allows both Apo-AI and TTR to be stabilized.

[0013] In the context of the present invention, "diseases involving impairments in Apo-AI or TTR" are defined as those diseases in which there is an increase or decrease in the plasma concentration of Apo-AI or TTR or in which there are changes in the interaction of these proteins in HDL particles, or those diseases in which the structure of Apo-AI and TTR and ultimately their biological function are affected.

[0014] Previously, the peptide identified as SEQ ID NO: 1 has been described to induce a regulatory mechanism of immune response in different experimental systems. The peptide increases the frequency of regulatory T cells (Treg) in an in vitro assay with peripheral blood mononuclear cells from patients with rheumatoid arthritis (RA), but not in healthy donors; these cells have suppressive activity (Barberá A, Lorenzo N, van Kooten P et al., (2016) Cell Stress and Chaperones., 21: 735-744). Similarly, the peptide induces a significant increase in Treg cell populations in BALB / c mice. In addition, the peptide effectively inhibits RA in animal models (Lorenzo N, Altruda F, Silengo L and Dominguez MC. (2017) Clin Exp Med, 17: 209-216). Treatment of patients with RA with the peptide identified as SEQ ID NO: 1 has been shown to be safe and have good therapeutic effects (Dinorah Prada, Jorge Gómez, Norailys Lorenzo, Oreste Corrales et al., (2018) Journal of Clinical Trials, 8:2167-0870; Cabrales-Rico, A., Ramos, Y., Besada, V., Del Carmen, DM, Lorenzo, N. et al., (2017) J Pharm Biomed Anal 143:130-140).

[0015] These results are disclosed in international patent applications Nos. PCT / CU2005 / 000008 and PCT / CU2009 / 000009. These patent applications claim protection for the peptide identified as SEQ ID NO: 1 and its use for treating RA and Crohn's disease, ulcerative colitis and type I diabetes, respectively.

[0016] Subsequently, International Patent Application No. PCT / CU2018 / 050007 claims a very stable pharmaceutical composition comprising a peptide of SEQ ID NO: 1 for use in the treatment of diseases characterized by citrullination and an increase in the number of neutrophils. In particular, the invention relates to the treatment of RA, ankylosing spondylitis, juvenile idiopathic arthritis, liver and lung fibrosis, and Alzheimer's disease.

[0017] On the other hand, the peptide identified as SEQ ID NO: 1 has been used to treat patients with excessive inflammation, such as in the case of patients with COVID-19; and it was demonstrated to reduce inflammation in said patients (Hernandez- M et al., (2021). Cell Stress and Chaperones 26:515-525; Domínguez Horta MC et al., (2022). Anales de la Academia de Ciencias de Cuba, 12(1): e1072. http: / / www.revistaccuba.cu / index.php / revacc / article / view / 1072. This fact was first disclosed in international patent application number PCT / CU2021 / 050001, which claims the use of the peptide for the treatment of excessive inflammation.

[0018] The ability of the peptide of SEQ ID NO: 1 to bind strongly to Apo-AI and TTR has been demonstrated for the first time in the present invention, which contributes to ensuring a stable lipid homeostasis and reducing the amyloidogenic process mediated by said protein. In addition, the present invention demonstrates the benefits received by patients treated with the peptide identified as SEQ ID NO: 1 who have reached a serious state due to diseases related to lipid metabolism.

[0019] In one embodiment of the invention, the disease involving impairment in Apo-AI or TTR is characterized by impairment in lipid homeostasis or TTR stability. In a particular embodiment, the disease is selected from the group consisting of atherosclerosis, ischemic heart disease, type II diabetes, secondary dyslipidemia and metabolic syndrome. In another particular embodiment, the disease is TTR amyloidosis or Parkinson's disease.

[0020] The peptide identified as SEQ ID NO: 1 reduces oxidative stress and significantly improves the status of patients who are clinically severe due to cardiovascular diseases (e.g., ischemic heart disease and hypertension). The treatment of patients with ischemic heart disease and type II diabetes with the peptide is very safe because it does not cause side effects.

[0021] The same peptide is known to be useful for treating patients with type I diabetes. However, the use is based on the regulation of the uncontrolled immune response that persists in patients with type I diabetes. Type II diabetes is not considered to be an autoimmune disease. Surprisingly, the treatment of patients with decompensated type II diabetes with the peptide allows them to recover very quickly compared to patients who only receive standard treatment. In the present invention, it is unexpectedly demonstrated that a very beneficial effect is produced in patients with type II diabetes, wherein the treatment with the peptide identified as SEQ ID NO:1 contributes to lipid homeostasis and its effect on intermediary metabolism.

[0022] The present invention also provides a method for treating a disease involving impairment in Apo-AI or TTR in an individual in need thereof. The method of treatment is characterized by administering a therapeutically effective amount of a pharmaceutical composition comprising a peptide identified as SEQ ID NO: 1. In one embodiment of the present invention, the disease is selected from the group consisting of: atherosclerosis, ischemic heart disease, type II diabetes, secondary dyslipidemia and metabolic syndrome. In another embodiment, the disease is TTR amyloidosis or Parkinson's disease.

[0023] In one embodiment of the invention, in the method of treatment, the pharmaceutical composition is administered by a systemic route. In a preferred embodiment, the composition is administered by a subcutaneous or intravenous route. In one embodiment of the invention, the pharmaceutical composition is administered simultaneously with standard therapy during the same treatment process. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 .SDS-PAGE electrophoresis showing proteins in human plasma that specifically bind to the peptide identified as SEQ ID NO: 1. Lane 1: Molecular weight standard. Lane 2: Proteins bound to a matrix without peptide (control). Lane 3: Proteins that specifically bind to the peptide identified as SEQ ID NO: 1 bound to the matrix.

[0025] Figure 2 Mass spectra (MS) of bands identified as Apo-AI in SDS-PAGE. Boxes indicate peptides identified as components of Apo-AI in the UniProtKB database using the MASCOT program.

[0026] Figure 3 MS of bands identified as TTR in SDS-PAGE. Dots indicate peptides identified as components of TTR in the UniProtKB database using the MASCOT program.

[0027] Figure 4 .Western blot showing proteins in human plasma that specifically bind to the peptide identified as SEQ ID NO: 1. Lane 1: Molecular weight standard. Lane 2: Plasma not incubated with the peptide. Lane 3: Plasma incubated with the peptide.

[0028] Figure 5 A, Superposition of the structures of the ten peptide-protein complex models predicted by the CABS-dock method with the highest scores. B, Structural model of the complex of the peptide identified as SEQ ID NO: 1 bound to the TTR protein via the "idc" site. C, Image of the peptide bound to the TTR protein, which presents the surface of the protein according to its hydrophilic characteristics. D, A similar image to C, but without presenting the electrostatic features of the surface of the protein. E, View of the interactions observed in the vicinity of residue Leu18 of the peptide.

[0029] Figure 6 .Contact map between the peptide identified as SEQ ID NO: 1 (vertical axis) and the residues of the TTR protein (horizontal axis) as predicted by the 3D structure of the complex. Squares mean that the corresponding residue contains a position equal to or less than The type of residue is shown in a three-letter code, followed by the chain identifier (chains B and D correspond to TTR protein, and chain E corresponds to the peptide identified as SEQ ID NO: 1) and the residue number of the amino acid sequence.

[0030] Figure 7 . Cholesterol levels in patients with ischemic heart disease and arterial hypertension treated with the peptide identified as SEQ ID NO: 1. Time 0: cholesterol level before starting treatment with said peptide; Day 7: cholesterol level 7 days after starting treatment with said peptide.

[0031] Figure 8 . Cholesterol levels in patients with type II diabetes, five of whom were treated with the peptide identified as SEQ ID NO: 1, compared to five patients who were not treated with the peptide. The discontinuous line marks the normal limit of cholesterol levels in the blood (5.20 mmol / L). Time 0: cholesterol level before starting treatment with the peptide; Day 8: cholesterol level 8 days after starting treatment with the peptide. DETAILED DESCRIPTION

[0032] Example 1. Apolipoprotein A (Apo-AI) was identified as a protein in human plasma that binds to the peptide identified as SEQ ID NO: 1.

[0033] In order to investigate the ability of the peptide identified as SEQ ID NO: 1 to bind to plasma proteins, affinity chromatography was performed by binding to a chromatographic resin on which the synthesized peptide identified as SEQ ID NO: 1 was bound. First, blood was drawn from a 24-year-old female healthy donor without any pathological disease and with a normal lipid profile. The blood was diluted 1:2 with 1X PBS and added to 3 mL of Ficoll-Paque TM The plasma was collected and incubated with 100 μL of chromatographic matrix. The matrix was ChemMatrix TM A resin in the form of beads from ELISA (Quebec, Canada) to which the peptide identified as SEQ ID NO: 1 had previously been coupled (this combination is hereinafter designated as substrate-peptide).

[0034] Before incubation, the matrix-peptide was treated with a 70% methanol solution and RPMI medium. The incubation of the matrix-peptide with the plasma was performed for 3 hours. Then, elution was performed with a solution containing 20% ​​glycerol, 62.5 mM TRIS-HCl, 2.5% bromophenol blue, 2% SDS and 5% β-mercaptoethanol; and the eluted fractions were collected. They were heated at 95°C for 5 minutes and analyzed by SDS-PAGE electrophoresis (15% gel). As a control for this experiment, ChemMatrix TM The matrix (without the peptide identified as SEQ ID NO: 1 coupled) was subjected to the procedure described previously. The gel was stained with a Coomassie blue solution, which is compatible with the identification of proteins by mass spectrometry.

[0035] For its part, Figure 1 The results of SDS-PAGE electrophoresis analysis of the fractions collected after elution are shown. The gel was subjected to silver staining. As can be clearly seen, two plasma proteins that bind to the peptide identified as SEQ ID NO: 1 were identified, which were named proteins X1 and X2.

[0036] In order to identify the protein X1 observed in SDS-PAGE, the band corresponding to this was cut off, and 1 mL of a solution of 250 mM ammonium bicarbonate (BCA), 30% acetonitrile (ACN), 1% dithiothreitol (DTT) contained in water was added. The sample was stirred for 15 minutes. Then, the solution was removed, and 1 mL of a solution of 250 mM BCA, 30% ACN, 2.5% acrylamide contained in water was added. The sample was stirred for 15 minutes. The solution was removed immediately, and 1 mL of a solution of 250 mM BCA and 30% ACN contained in water was added. The sample was stirred for 15 minutes. Then, three washes (lasting 10 minutes) with water were performed under agitation, and the band was cut into 1 mm 3 . Add 400 μL of pure ACN, homogenize using vortex oscillation, and remove the supernatant. Then, dry the sample in Speed ​​Vac for 5 minutes, and hydrate the gel with 50mM BCA solution containing trypsin at a concentration of 12.5ng / mL. Incubation with trypsin was performed at 37°C for 16 hours. Subsequently, the gel was rehydrated with water at 37°C for 30 minutes.

[0037] Next, place the sample in a ZipTip C18 TM Desalination was carried out in miniature column (Millipore, USA), and eluted with 60% ACN in water comprising 1% formic acid of 3.5 μ L. Sample was applied in borosilicate capillary (Thermo Scientific, USA) coated with conductive material, and analyzed by nanoESI-MS. ESI-MS mass spectrum was obtained in the orthogonal hybrid configuration spectrometer QTOF-2 (Micromass, United Kingdom) with the Z-spray electrospray ionization source (nanoESI+) of positive mode. The analyzer was calibrated as a reference in a wide mass range (50-2000Th) with a mixture of sodium and cesium iodide salt (Sigma, USA). ESI-MS spectrum was obtained by applying 1200 volts and 30 volts of voltage in capillary and in the input cone of mass spectrometer respectively. The mass spectrum adopted was obtained and the processing program was MassLynx 4.1 version (Micromass, United Kingdom). ESI-MS / MS spectra were obtained by applying collision energies in the range of 20-40 eV to induce fragmentation, which contained sufficient information for the structural elucidation of the analyzed peptides. The collision gas employed was argon (Gases Industriales, Cuba).

[0038] The ESI-MS spectrum of the tryptic digest of the protein corresponding to the band identified as X1 showed multiply charged ions (2+) corresponding to five peptides (m / z 620.43, 626.81, 700.85, 806.90 and 967.47, respectively), which were selected for fragmentation and obtaining their ESI-MS / MS spectra. The species corresponded to peptide fragments of the Apo-AI protein, 37 DLATVYVDVLK 48 , 53 DYVSQFEGSALGK 66 , 72 LLDNWDSVTSTFSK 86 , 89 EQLGPVTQEFWDNLEK 104 and 123 VQPYLDDFQ 133 , which was identified by searching the UniProtKB database using the MASCOT program ( Figure 2 ).

[0039] Example 2. Transthyretin was identified as a protein in human plasma that binds to the peptide identified as SEQ ID NO: 1.

[0040] To identify the protein X2 observed in SDS-PAGE, it was carried out as in Example 1. The ESI-MS spectrum of the analyzed protein corresponding to the upper band showed multiply charged ions (3+) corresponding to two peptides (m / z 819.07 and 817.45, respectively), which were selected for fragmentation by CID (abbreviation for Collision-Induced Dissociation) and obtaining their ESI-MS / MS spectra, which are presented in Figure 3 The species correspond to peptide fragments of TTR protein 36 KTSESGELHGLTTEEEFVEGIYKV 59 and 68 KALGISPFHEHAEVVFTANDSGPRR 92 , which was identified by the MASCOT program. This was confirmed by searching in the UniProtKB database.

[0041] Example 3. Immune recognition of the peptide identified as SEQ ID NO: 1 bound to apolipoprotein-AI or transthyretin

[0042] In order to confirm that the peptide identified as SEQ ID NO: 1 binds to the Apo-AI and TTR proteins present in the plasma, a Western blot type immunological characterization experiment was performed with a polyclonal antibody against the peptide. The serum of rabbits previously immunized with the peptide identified as SEQ ID NO: 1 conjugated to the KLH protein was used as the primary antibody (antibody against the peptide). During the biological characterization of the polyclonal serum used, it was confirmed that it recognized neither Apo-AI nor TTR. First, SDS-PAGE electrophoresis (12.5% ​​gel) was performed, in which the plasma of healthy donors was applied in two lanes and the molecular weight standard was applied in another lane. Then, the proteins present in the gel were transferred to a nitrocellulose membrane, which was stained with a Ponceau solution and the bands corresponding to the molecular weight standard were marked. The membrane was cut according to the lane in which the plasma was applied. The membrane fragments were treated overnight at 4° C. with a 1% bovine serum albumin solution containing 0.05% Tween 20.

[0043] Subsequently, one of the membrane fragments was incubated with a solution of the peptide identified as SEQ ID NO: 1 at a concentration of 200 μg / mL in 1X PBS plus Tween 20 (0.05%). The incubation was carried out at 37°C for 3 hours, and the remaining membrane fragments were treated in the same way, but without adding the peptide to the solution. The membrane fragments were then subjected to washing with a solution consisting of 1X PBS plus Tween 20 (0.05%). Next, the membrane fragments were incubated with a polyclonal serum against the peptide as a primary antibody. The polyclonal serum was diluted 1 / 2500 in the washing solution and incubated at 37°C for 2 hours. After this period of time, the washing was repeated and a secondary antibody (Sigma, USA) was added. The latter consists of a mixture of G-type immunoglobulins that recognize the constant region of rabbit antibodies conjugated to horseradish peroxidase. The incubation with the secondary antibody was carried out at 37°C for 1 hour. Subsequently, washing was performed and revealing was performed with 0.01% hydrogen peroxide and 3,3'-diaminobenzidine (1 mg / mL).

[0044] The results are shown in Figure 4 Immune recognition by antibodies against the peptide was observed at the height of the bands corresponding to Apo-AI and TTR. This experiment confirms the results shown in Examples 1 and 2 and therefore recognizes that the peptide identified as SEQ ID NO: 1 interacts with these two proteins present in human plasma.

[0045] Example 4. Modeling of the structure of the complex formed between the peptide identified as SEQ ID NO: 1 and transthyretin.

[0046] Monomeric TTR adopts a sandwich β-type fold formed by two antiparallel β sheets. The natural TTR protein is characterized by adopting a tetrameric quaternary structure, which is established by means of the association of two dimers of the protein. The dimer of TTR initially appears through the interaction of the strands at one of the ends of the β sheet of the monomeric protein, thereby forming two extended antiparallel β sheets. The convex surface of one of the sheets of each dimer interacts face-to-face with a similar extended sheet of another dimer to obtain a tetramer. In the space between the sheets, a cavity or channel is generated, which accommodates small molecules of natural ligands (T4) and other drug types (Cotrina et al., (2021). European Journal of Medicinal Chemistry. Vol226, p.113847). Inter-dimer interactions are relatively unstable, and dissociation of tetramers is associated with a number of pathological conditions associated with TTR proteins (e.g., TTR amyloidosis) (Ando Y, Nakamura M, and Araki S. (2005) Arch Neurol 62: 1057-1062). The described binding site located in the space between dimers is the target of several drugs being studied and / or developed for such diseases.

[0047] To model the structure of the complex formed between the peptide identified as SEQ ID NO: 1 and the TTR protein, the CABS-dock peptide-protein flexible connection method (Kurcinski M et al., (2020). Protein Science, 29: 211-222) was used. The crystallographic structure of the protein data bank PDB (3CFM file) with a resolution of 1.5 Å. The file contains a dimer of the TTR protein in an asymmetric unit, so a tetramer is obtained by applying the symmetry operation of BIOMOLECULE, as described in files of type pdb. The two chains symmetrical to chains A and B (the original chains of the pdb file) are called C and D, respectively.

[0048] The linkage scheme employed is "blind", i.e. all patches on the protein surface are considered as potential binding sites. Figure 5A shows a superposition of the ten best models proposed by the CABS-dock algorithm. The arrows indicate the positions of the peptide identified as SEQ ID NO: 1 that bind to the protein in the models obtained. All models indicate that the most likely binding site is located in one of the two clefts of the tetramer: a) the cleft formed between the monomers of the dimer (inter-monomer cleft: "imc"), which is delimited by an extended β sheet of an exposed concave surface, or b) the cleft formed in the space between the dimers (inter-dimer cleft: "idc"), which is delimited by a convex antiparallel β sheet. The cleft "idc" forms the channel mouth or binding site for T4, the natural ligand of the TTR protein. This result suggests that these surface patches have favorable stereochemical properties relative to the rest of the surface of the protein, and therefore they constitute potential interaction "attractors" for the peptide identified as SEQ ID NO: 1. The two "imc" sites are identical to each other (just like the two "idc" sites) because they are related by the symmetry operator C2. The results show that 60% of the models locate the peptide identified as SEQ ID NO: 1 in conjunction with the "idc" site, while the remaining 40% locate in the "imc" site.

[0049] Figure 5 B shows a model of the peptide identified as SEQ ID NO: 1 bound to the "idc" site. In the model, the most hydrophobic segment of the peptide identified as SEQ ID NO: 1 (residues Leu15-Ala20) protrudes to the interior of the channel mouth through interactions with the hydrophobic residues of the TTR protein, which favorably provides interaction energy (and binding affinity) through the "hydrophobic effect". Figure 5 As observed in C, the deepest region of the "idc" site that contacts the peptide identified as SEQ ID NO: 1 has a hydrophobic character. Electrostatic complementarity between the peptide identified as SEQ ID NO: 1 and the protein is also plausible, as shown in Figure 5 D. The residue of the peptide identified as SEQ ID NO: 1 that protrudes most into the cleft is Leu18. Figure 5 This leucine residue of the peptide is in close atomic proximity with the Leu17, Leu103 and Ala108 residues of the protein as seen in E. Hydrophobic interactions characteristic of the binding of the peptide of SEQ ID NO: 1 to the protein were observed.

[0050] On the other hand, Figure 6It was observed that the Ile11-Ala20 segment of the peptide, which contains the largest number of hydrophobic residues, is the segment that makes the most contact with the TTR protein. The importance of the hydrophobic effect in its energy provision is reinforced, which is fundamental in protein-protein interactions.

[0051] From a functional perspective, the binding of the peptide to the "idc" site contributes to the structural stabilization of the tetramer, and thus can lead to the inhibition and / or regulation of the negative effects of diseases associated with the destabilization of the native quaternary structure of the TTR protein. On the other hand, the model is consistent with the central role of the Leu18 residue in the interaction, which is the result of the replacement of the Asp18 residue present in the native sequence of the T epitope of the HSP60 protein (Barberá A, Lorenzo N, van Kooten P et al., (2016) Cell Stress and Chaperones., 21: 735-744). The interaction of the Leu18 residue described here may be the structural basis for the specific biological activity of the peptide identified as SEQ ID NO: 1, which is not observed in the native peptide.

[0052] Example 5. Treating a patient with ischemic heart disease with a pharmaceutical composition comprising a peptide identified as SEQ ID NO: 1.

[0053] Patients with low levels of HDL and high blood pressure often develop ischemic heart disease, which is associated with the formation of atherosclerotic plaques that are characteristic of a disease called atherosclerosis. These heart diseases often lead patients to serious conditions, and treatment with the peptide identified as SEQ ID NO: 1 can help patients recover.

[0054] Take the results of six patients, for example, who had ischemic heart disease associated with hypertension, which produced a serious condition. They were cared for in the Transitional Care Unit (IMCU) of the "Luis Díaz Soto" Hospital in Havana. These patients were treated with a pharmaceutical composition comprising a peptide identified as SEQ ID NO: 1, administering a dose of 1 mg peptide / 12 hours by intravenous route during 7 days. In addition, they received standard therapy necessary for these cases.

[0055] Figure 7Cholesterol levels in these six patients with ischemic heart disease and arterial hypertension treated with the peptide are shown. As can be observed, the cholesterol level in the blood decreased after seven days of treatment. The patients recovered and were discharged from the hospital. These results show that treatment with the peptide identified as SEQ ID NO: 1 can stabilize HDL and facilitate the proper conversion of cholesterol, thereby reducing atherosclerotic plaques, improving atherosclerosis and avoiding related complications. Treatment with the peptide is very safe, which is confirmed by monitoring blood biochemical parameters and blood cell differential counts. According to clinical and imaging evaluations, no side effects were observed in the treated patients.

[0056] In contrast, five patients with similar characteristics who did not receive treatment with the peptide during the same period of time stayed in IMCU longer and cholesterol levels in them exceeded 5 mmol / L at the end of treatment.

[0057] Example 6. Treatment of a patient with type II diabetes using a pharmaceutical composition comprising a peptide identified as SEQ ID NO: 1.

[0058] Obese and hypertensive patients often develop type II diabetes associated with an imbalance in lipid metabolism. These conditions can lead to serious conditions, and treatment with the peptide identified as SEQ ID NO: 1 can aid in the patient's recovery.

[0059] Take, for example, ten patients who had diabetic decompensation and reached a severe state that led them to be cared for in the IMCU of the "Luis Díaz Soto" Hospital in Havana. Five of these patients were treated with a pharmaceutical composition comprising a peptide identified as SEQ ID NO: 1, administering a dose of 1 mg peptide / 12 hours by intravenous route during 8 days. In addition, they received the standard therapy necessary for these cases.

[0060] Figure 8 Cholesterol levels in patients with type II diabetes treated with the peptide are shown, compared to patients receiving standard treatment for these cases but not including the peptide. As can be observed, cholesterol levels remain unchanged in patients treated with the peptide compared to patients not treated with the peptide, in which cholesterol levels exceeded normal values ​​at the end of the 8-day evaluation period. These results indicate that treatment with the peptide identified as SEQ ID NO: 1 can stabilize HDL and facilitate proper conversion of cholesterol, which contributes to the stabilization of blood glucose metabolism. Treatment with the peptide is safe, which is confirmed by monitoring blood biochemical parameters and blood cell differential counts. No side effects were observed in the treated patients based on clinical and imaging evaluations.

Claims

1. A peptide identified as SEQ ID NO: 1 for use in the preparation of a medicament for treating a disease involving impairment in apolipoprotein AI (Apo-AI) or transthyretin (TTR).

2. The peptide for use according to claim 1, wherein the disease is characterized by an impairment in lipid homeostasis or TTR stability.

3. The peptide for use according to claim 2, wherein the disease is selected from the group consisting of atherosclerosis, ischemic heart disease, type II diabetes, secondary dyslipidemia and metabolic syndrome.

4. The peptide for use according to claim 2, wherein the disease is TTR amyloidosis or Parkinson's disease.

5. A method for treating a disease involving impairment of apolipoprotein AI (Apo-AI) or transthyretin (TTR) in an individual in need thereof, characterized in that A therapeutically effective amount of a pharmaceutical composition comprising the peptide identified as SEQ ID NO: 1 is administered.

6. The method of treatment according to claim 5, wherein the disease is selected from the group consisting of atherosclerosis, ischemic heart disease, type II diabetes, secondary dyslipidemia and metabolic syndrome.

7. The method of claim 5, wherein the disease is TTR amyloidosis or Parkinson's disease.

8. The method of treatment according to claim 5, wherein the pharmaceutical composition is administered by a systemic route, preferably by a subcutaneous or intravenous route.

9. The method of treatment according to claim 5, wherein the pharmaceutical composition is administered concurrently with standard therapy during the same course of treatment.

Images