Peptides for the treatment of diseases associated with the involvement of 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 treating a disease associated with the involvement of apolipoprotein A1 (Apo-A1) or transthyretin (TTR). In particular, the peptide may be useful in the treatment of diseases characterized by the involvement of lipid homeostasis or TTR stability. The present invention also relates to a method for treating a disease associated with the involvement of Apo-A1 or TTR, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising the peptide identified as SEQ ID NO: 1.

Description

[Technical Field]

[0001] The present invention relates to the field of medicine, particularly to modified APL-type peptides derived from HSP60 (abbreviated as Altered Peptide Ligands (APL)) and their use to ensure lipid homeostasis, prevent cellular oxidative stress, and avoid amyloidosis caused by inappropriate modification of apolipoprotein AI (Apo-AI) and transthyretin (TTR). The peptides are also useful for treating patients with impaired lipid transport and processing. [Background technology]

[0002] Lipids are essential for the functioning of the human organism; these molecules are important for energy acquisition and storage. In addition, lipids are fundamental components of lipid membranes, various hormones, and bile salts. Disorders in lipid concentrations, either due to excess or deficiency, are directly related to the development of cardiovascular diseases such as coronary artery disease, cerebrovascular diseases, 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 based on their size and density. Lipoproteins are hydrophobic globular structures with surface proteins (apoproteins or apolipoproteins) that can bind to enzymes responsible for lipid processing. (Rosenson, RS et al. (2016). Nat. Rev. Cardiol. 13, 48-60)

[0004] High-density lipoproteins (HDL) transport excess cholesterol from extrahepatic tissues to the liver, where it is metabolized. HDL is associated with reduced cardiovascular risk, while low-density lipoproteins (LDL) and very-low-density lipoproteins (VLDL) increase it. There is an inverse correlation between HDL-associated cholesterol levels and atherosclerosis risk. (Lee, M., PT et al. (2003). J. Lipid Res. 44:539-546).

[0005] HDL is the densest lipoprotein because it has a higher concentration of protein than lipid. These lipoproteins are composed of cholesterol, triglycerides, and a number of apolipoproteins: Apo-AI, Apo-AII, Apo-AIV, Apo-AV, Apo-C1, Apo-CII, Apo-CIII, and Apo-E. Apo-AI is the major protein component of HDL and is the primary apolipoprotein responsible for maintaining HDL structure (Jonas, A., K. E. Kezdy, and J. H. Wald. (1989). J. Biol. Chem. 264:4818-4824).

[0006] Apo-AI activates the enzyme lecithin-cholesterol-acyltransferase, which catalyzes the conversion of cholesterol to its ester form, thereby increasing the ability of HDL to transport lipids to the liver (Gordon, T. et al. (1977). Am. J. Med. 62, 707-714). Blood levels of Apo-AI increase during certain physiological conditions, such as pregnancy, liver disease, and estrogen use, and decrease during sepsis and atherosclerosis. High concentrations of Apo-AI and low concentrations of Apo-B (the main protein component of low-density lipoprotein) significantly correlate with a reduced risk of cardiovascular disease. HDL has anti-atherogenic and anti-inflammatory properties, as it contributes to the reduction of atherosclerotic plaques by transporting cholesterol accumulated in the plaques to the liver (TM Forte et al. (2002) J Lipid Res 43:477-485).

[0007] Although the mechanisms of atherosclerotic plaque formation are not fully understood, LDL and HDL lipoproteins play a fundamental role in these mechanisms. To date, no effective treatment has been found to prevent the formation of atheroma and the resulting damage to the human body. Therefore, it is important to establish treatments that ensure lipid homeostasis and proper processing of lipoproteins.

[0008] On the other hand, it has been reported that TTR forms part of HDL through binding to Apo-AI (Liz, MA, CM Gomes, MJ Saraiva, and MM Sousa. (2007) J Lipid Res 48:2385-2395). TTR is a tetrameric protein present in plasma, synthesized primarily in the liver, and responsible for the transport of thyroxine and retinol-binding protein. This protein can decompose into monomers and dimers, which can form fibers and deposit in tissues.

[0009] Age and mutations (over 100 identified) may increase predisposition to aggregation. Two forms of TTR amyloidosis have been described: wild-type (wt ATTR, also known as senile systemic amyloidosis) and hereditary (h-ATTR, hereditary transthyretin cardiac amyloidosis). In the wild-type form, TTR synthesis is normal, but in the hereditary form, the presence of mutations affects the conformation of TTR for its synthesis in hepatocytes (Kittleson MM, Maurer MS, Ambardekar AA et al. (2020) Circulation 142:e7-22).

[0010] Among the diseases caused by TTR amyloidosis is amyloid cardiomyopathy, a cardiac disease resulting from the extracellular deposition of abnormal insoluble fibrils. The prevalence of this disease is estimated to be between 13% in elderly patients hospitalized with preserved ejection fraction and 16% in those with severe aortic stenosis requiring intervention (Coelho T, Maurer MS, Suhr OB. (2013) Curr Med Res Opin;29:63-76).

[0011] Additionally, post-translational modifications of TTR affecting its tetrameric conformation and the presence of its oligomers have been strongly associated with Parkinson's disease (Ando Y, Nakamura M, and Araki S. (2005) Arch Neurol 62:1057-1062) and pathologies related to oxidative stress (Sharma M, Khan S, Rahman S, and Singh LR (2019) Front. Physiol. 10:5. doi:10.3389 / fphys.2019.00005).

[0012] It has been reported that a portion of plasma TTR circulates in HDL through binding to Apo-AI. TTR can cleave the carboxyl terminus of lipid-free Apo-AI. Cleavage of Apo-AI by TTR induces the formation of Apo-AI amyloid fibrils. Furthermore, modified HDL particles have a reduced ability to promote proper cholesterol turnover. In addition, Apo-AI cleaved by TTR has a high preference for forming aggregated particles, which affects HDL turnover, reduces cholesterol metabolism, and increases the amyloidogenic potential of Apo-AI, thereby favoring the development of atherosclerosis (Liz, MA, CM Gomes, MJ Saraiva, and MM Sousa. (2007) J. Lipid Res. 48:2385-2395).

[0013] The search for treatments that stabilize HDL is important for ensuring adequate lipid exchange, especially cholesterol, and for preventing or reducing the formation of atherosclerotic plaques that promote atherosclerosis. At the same time, treatments that stabilize the conformation of TTR and prevent its modification and aggregation may be effective in preventing TTR-mediated amyloidosis and Parkinson's disease. Summary of the Invention

[0014] The present invention solves the above problem by providing a peptide identified as SEQ ID NO: 1 for use in the manufacture of a medicament for the treatment of diseases affecting Apo-AI or TTR. The modified peptide identified as SEQ ID NO: 1 can stabilize the supramolecular structure of HDL through binding to Apo-AI. In this way, it promotes proper lipid metabolism and the reduction of atherosclerotic plaques associated with atherosclerosis. As shown in the present invention, surprisingly, this peptide interacts only with Apo-AI and TTR in human plasma. This interaction allows the stability of both proteins.

[0015] In the context of the present invention, "diseases affecting Apo-AI or TTR" are defined as diseases in which there is an increase or decrease in the plasma concentration of Apo-AI or TTR, or in which alterations occur in the interaction of these proteins in HDL particles, or in which the structure of Apo-AI and TTR is affected, thereby affecting their biological function.

[0016] Previously, the peptide identified as SEQ ID NO: 1 was described to induce immune response regulation in different experimental systems. The peptide increased the frequency of regulatory T cells (Tregs) in ex vivo assays using peripheral blood mononuclear cells from rheumatoid arthritis (RA) patients, but not from healthy donors. These cells have suppressive activity (Barbera A, Lorenzo N, van Kooten P et al. (2016) Cell Stress and Chaperones.;21:735-744). Similarly, the peptide induced a significant increase in the Treg cell population in BALB / c mice. Furthermore, this peptide efficiently inhibited RA in two animal models (Lorenzo N, Altruda F, Silengo L and Dominguez MC. (2017) Clin Exp Med.;17:209-216). Treatment with the peptide identified as SEQ ID NO: 1 in RA patients has proven safe and had good therapeutic effects (Dinorah Prada, Jorge Gomez, 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).

[0017] These results were disclosed in International Patent Applications Nos. PCT / CU2005 / 000008 and PCT / CU2009 / 000009, which claim a peptide identified as SEQ ID NO: 1 and its use for treating RA and Crohn's disease, ulcerative colitis, and type 1 diabetes mellitus, respectively.

[0018] Subsequently, International Patent Application No. PCT / CU2018 / 050007 claimed highly stable pharmaceutical compositions comprising the peptide of SEQ ID NO: 1 for the treatment of diseases characterized by increased citrullination and neutrophil counts. In particular, the invention relates to the treatment of RA, ankylosing spondylitis, juvenile idiopathic arthritis, hepatic and pulmonary fibrosis, and Alzheimer's disease.

[0019] Meanwhile, the peptide identified as SEQ ID NO: 1 has been used to treat patients with hyperinflammation, such as COVID-19 patients, and has been shown to reduce inflammation in such patients (Hernendez-Cedeno M et al. (2021). Cell Stress and Chaperones 26:515-525; Dominguez Horta MC et al. (2022). Annals of the Academy of Sciences of Cuba; 12(1):e1072. http: / / www.revistaccuba.cu / index.php / revacc / article / view / 1072). This fact was first revealed in International Patent Application No. PCT / CU2021 / 050001, which claims the use of the peptide for the treatment of hyperinflammation.

[0020] The present invention demonstrates for the first time the ability of the peptide of SEQ ID NO: 1 to bind strongly to APO-AI and TTR proteins, which contributes to ensuring stable lipid homeostasis and reducing the amyloidosis process mediated by said proteins.In addition, the present invention shows the benefit received by patients who had reached a critical state due to diseases related to lipid metabolism and were treated with the peptide identified as SEQ ID NO: 1.

[0021] In one embodiment of the present invention, disease progression exhibiting alterations in Apo-AI or TTR is characterized by deterioration of lipid homeostasis or TTR stability. In a specific embodiment, the disease is selected from the group consisting of atherosclerosis, ischemic heart disease, type II diabetes, secondary dyslipidemia, and metabolic syndrome. In another specific embodiment, the disease is TTR amyloidosis or Parkinson's disease.

[0022] The peptide identified as SEQ ID NO: 1 reduces oxidative stress and significantly improves the condition of patients who are clinically severe due to cardiovascular diseases such as ischemic heart disease and hypertension. Treatment with this peptide in patients with ischemic heart disease and type II diabetes was very safe, as it did not cause any adverse effects.

[0023] The same peptide is known to be useful for treating type I diabetes patients.However, this use is based on regulating the uncontrolled immune response that persists in type I diabetes patients.Type II diabetes is not considered an autoimmune disease.Surprisingly, treatment of decompensated type II diabetes patients with said peptide allows for a very rapid recovery compared to patients who receive standard treatment alone.Unexpectedly, the present invention has shown that treatment with the peptide identified as SEQ ID NO: 1 produces a very beneficial effect in type II diabetes patients, and this is attributed to its effect on lipid homeostasis and intermediary metabolism.

[0024] The present invention also provides a method for treating a disease associated with an effect on Apo-AI or 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. 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.

[0025] In one embodiment of the invention, the pharmaceutical composition is administered by systemic route. In a preferred embodiment, the composition is administered by subcutaneous or intravenous route. In one embodiment of the invention, the pharmaceutical composition is administered concurrently with standard therapy during the treatment. [Brief explanation of the drawings]

[0026] [Figure 1] 1 is an SDS-PAGE electrophoresis showing proteins in human plasma that specifically bind to the peptide identified as SEQ ID NO: 1. Lane 1: molecular weight standards. Lane 2: matrix-bound protein - no peptide (control). Lane 3: protein that specifically binds to the peptide identified as SEQ ID NO: 1 bound to a matrix. [Figure 2] Mass spectrum (MS) of the band identified as Apo-AI by SDS-PAGE. Boxes indicate peptides identified by the MASCOT program in the UniProtKB database as components of Apo-AI. [Figure 3] MS of the band identified as TTR on SDS-PAGE. Dots indicate peptides identified as components of TTR by the MASCOT program in the UniProtKB database. [Figure 4] Figure 1 is a Western blot showing proteins in human plasma that specifically bind to the peptide identified as SEQ ID NO: 1. Lane 1: molecular weight standards. Lane 2: plasma not incubated with peptide. Lane 3: plasma incubated with peptide. [Figure 5A] Structural superposition of the 10 highest scoring peptide-protein complex models predicted by the CABS-dock method. [Figure 5B] 1 is a model of the complex structure of the peptide identified as SEQ ID NO: 1 bound to the "idc" site of the TTR protein. [Figure 5C] Representation of peptides bound to the TTR protein, revealing the surface of the protein with hydropathic properties. [Figure 5D]Similar to FIG. 5C, but does not depict the electrostatic properties of the protein's surface. [Figure 5E] FIG. 1 is a diagram of the interactions observed in the environment of the Leu18 residue of the peptide. [Figure 6] Contact map between residues of the peptide identified as SEQ ID NO: 1 (vertical axis) and residues of the TTR protein (horizontal axis) from the 3D structure prediction of the complex. Boxes indicate atoms whose corresponding residues are located at a distance of 4.5 Å or less. Residue types are indicated by a three-letter code next to the chain identifier (chains B and D correspond to the TTR protein, and chain E corresponds to the peptide identified as SEQ ID NO: 1) and the residue number in the amino acid sequence. [Figure 7] Cholesterol levels in patients with ischemic heart disease and hypertension treated with the peptide identified as SEQ ID NO: 1. Time 0: cholesterol levels before starting peptide treatment, Day 7: cholesterol levels 7 days after starting peptide treatment. [Figure 8] Cholesterol levels of five diabetic type II patients treated with a peptide identified as SEQ ID NO: 1 compared to five patients not treated with the peptide. The dashed line marks the normal limit for blood cholesterol levels (5.20 mmol / L). Time 0: cholesterol levels before starting peptide treatment; Day 8: cholesterol levels 8 days after starting peptide treatment. DETAILED DESCRIPTION OF THE INVENTION [Example]

[0027] Example 1. Identification of apolipoprotein A (Apo-AI) as the protein that binds to the peptide identified as SEQ ID NO: 1 in human plasma. To investigate the binding ability of the peptide identified as SEQ ID NO: 1 to plasma proteins, affinity chromatography was performed using a chromatography resin to which the synthetic peptide identified as SEQ ID NO: 1 was attached. First, blood was collected from a healthy donor (female, 24 years old) with a normal lipid profile without any pathological association. The blood was diluted 1:2 with PBS 1X, added to 3 mL of Ficoll-Paque™, and centrifuged at 1200 rpm for 30 minutes. Plasma was collected and incubated with 100 μL of chromatography matrix. This matrix was a pearl-shaped resin called ChemMatrix™ (Quebec, Canada), to which the peptide identified as SEQ ID NO: 1 had been pre-attached (hereinafter, this linkage is referred to as matrix-peptide).

[0028] Prior to incubation, the matrix-peptide was treated with 70% methanol solution and RPMI culture medium. The matrix-peptide was incubated with plasma for 3 hours. Elution was then 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 to 95°C for 5 minutes and analyzed by SDS-PAGE electrophoresis (15% gel). As a control, the same procedure was repeated using ChemMatrix™ without the peptide identified as SEQ ID NO: 1 attached. The gel was stained with Coomassie Blue solution. This staining is compatible with protein identification by mass spectrometry.

[0029] Figure 1 shows the results of SDS-PAGE electrophoresis of the fractions collected after elution. The gel was subjected to silver staining. As can be clearly seen, two plasma proteins were detected that bound to the peptide identified as SEQ ID NO: 1, and these were designated proteins X1 and X2.

[0030] To identify the X1 protein observed on SDS-PAGE, the corresponding band was excised and 1 mL of an aqueous solution containing 250 mM ammonium bicarbonate (BCA), 30% acetonitrile (ACN), and 1% dithiothreitol (DTT) was added. The sample was shaken for 15 minutes. This solution was then removed, and 1 mL of an aqueous solution containing 250 mM BCA, 30% ACN, and 2.5% acrylamide was added. The sample was shaken for 15 minutes. This solution was then removed, and 1 mL of an aqueous solution containing 250 mM BCA, 30% ACN, and 2.5% acrylamide was added. The sample was shaken for 15 minutes. Immediately, this solution was removed, and 1 mL of an aqueous solution containing 250 mM BCA and 30% ACN was added. The sample was shaken for 15 minutes. Next, the band was washed three times with water for 10 minutes under agitation, and 1 mm was added. 3 The gel was cut into cubes. 400 mL of pure ACN was added, homogenized using a vortex, and the supernatant was removed. The sample was then dried in a Speed Vac for 5 minutes, and the gel was hydrated with a 50 mM BCA solution containing trypsin at a concentration of 12.5 ng / mL. Incubation with trypsin was carried out for 16 hours at 37°C. The gel was then rehydrated with water for 30 minutes at 37°C.

[0031] The sample was then desalted using a ZipTip C18™ microcolumn (Millipore, USA) and eluted with 3.5 μL of 60% ACN in water containing 1% formic acid. The sample was applied to a borosilicate capillary coated with conductive material (Thermo Scientific, USA) and analyzed by nanoESI-MS. ESI-MS spectra were acquired on a QTOF-2 (Micromass, UK) orthogonal hybrid configuration spectrometer equipped with a Z-spray electrospray ionization source in positive mode (nanoESI+). The analyzer was calibrated over a wide mass range (50–2000 Th) using a mixture of sodium iodide and cesium iodide (Sigma, USA) as a standard. ESI-MS spectra were acquired by applying voltages of 1200 and 30 volts to the capillary and input cone of the mass spectrometer, respectively. MassLynx version 4.1 (Micromass, UK) was the software used for mass spectral acquisition and processing. ESI-MS / MS spectra were obtained by applying collision energies ranging from 20 to 40 eV to induce fragmentation that contained sufficient information for the structural elucidation of the analyzed peptides. Argon was used as the collision gas (Gases Industriales, Cuba).

[0032] The ESI-MS spectrum of the tryptic digest of the protein corresponding to the band identified as X1 showed multiple charged ions (2+) corresponding to five peptides (m / z 620.43, 626.81, 700.85, 806.90, and 967.47), which were selected and fragmented to obtain ESI-MS / MS spectra. These species are peptide fragments of the protein Apo-AI, identified by MASCOT software searching the UniProtKB database. 37 DLATVYVDVLK 48 , 53 DYVSQFEGSALGK 66 , 72 LLDNWDSVTSTFSK 86 , 89 EQLGPVTQEFWDNLEK 104 and123 VQPYLDDFQK 133 This corresponds to (Figure 2).

[0033] Example 2. Identification of transthyretin as a protein that binds to the peptide identified as SEQ ID NO: 1 in human plasma. To identify protein X2 observed on SDS-PAGE, we proceeded as in Example 1. The ESI-MS spectrum of the protein in the upper band showed multiply charged ions (3+) corresponding to two peptides (m / z 819.07 and 817.45, respectively) selected for fragmentation using CID (collision-induced dissociation).

[0034] These species are peptide fragments of the TTR protein identified by the MASCOT program searching the UniProtKB database. 36 KTSESGELHGLTTEEEFVEGIYKV 59 and 68 KALGISPFHEHAEVVFTANDSGPRR 92 The ESI-MS spectrum of the protein is shown in Figure 3.

[0035] Example 3. Immune recognition of the peptide identified as SEQ ID NO: 1 bound to apolipoprotein-AI or transthyretin. To confirm that the peptide identified as SEQ ID NO:1 binds to Apo-AI and TTR proteins present in plasma, a Western blot-type immunological identification experiment was performed using a polyclonal antibody against this peptide. The serum of a rabbit pre-immunized with the peptide identified as SEQ ID NO:1 conjugated to the protein KLH (antibody vs. peptide) was used as the primary antibody. During biological characterization of the polyclonal serum used, it was confirmed that it did not recognize Apo-AI or TTR. First, SDS-PAGE electrophoresis (12.5% gel) was performed, with healthy donor plasma applied to two lanes and molecular weight standards applied to the other lane. Proteins present in the gel were then transferred to a nitrocellulose membrane, stained with Red Ponceau solution, and bands corresponding to the molecular weight standards were marked. The membrane was cut according to the lane to which the plasma had been applied. The membrane fragments were treated overnight at 4°C with a 1% bovine serum albumin solution containing 0.05% Tween 20.

[0036] Next, one of the membrane fragments was incubated with a peptide solution identified as SEQ ID NO: 1 at a concentration of 200 μg / mL in PBS 1X + Tween 20 (0.05%). This incubation was carried out for 3 hours at 37°C. The remaining membrane fragment was treated equally, but without the addition of peptide. The membrane fragment was then washed with a solution consisting of PBS 1X + Tween 20 (0.05%). The membrane fragment was then incubated with polyclonal serum paired with peptide as the primary antibody. The polyclonal serum was diluted 1 / 2500 in the washing solution, and the incubation was carried out for 2 hours at 37°C. After repeated washing, a secondary antibody consisting of a mixture of G-type immunoglobulins recognizing the constant region of rabbit antibodies conjugated to horseradish peroxidase (Sigma, USA) was added. The incubation with the secondary antibody was carried out for 1 hour at 37°C. Then, washing was performed and development was carried out with 0.01% hydrogen peroxide and 3.3'-diaminobenzidine (1 mg / mL).

[0037] The results are shown in Figure 4. Immune recognition by antibody versus peptide is 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 confirms that the peptide identified as SEQ ID NO: 1 interacts with these two proteins present in human plasma.

[0038] Example 4. Modeling the structure of the complex formed between the peptide identified as SEQ ID NO: 1 and the protein transthyretin. Monomeric TTR adopts a beta-sandwich fold consisting of two antiparallel beta sheets. Native TTR protein is characterized by a quaternary tetrameric structure established by the association of two dimers of the protein. TTR dimers are initially constructed by the interaction of strands at one end of the beta sheets of the monomeric protein, resulting in the formation of two extended antiparallel beta sheets. The convex surface of one sheet of each dimer interacts face-to-face with the similar extended sheet of the other dimer, resulting in a tetramer. The space between these sheets forms a cavity or tube that accommodates the natural ligand (T4) as well as other drug-like small molecules (Cotrina et al. (2021). European Journal of Medicinal Chemistry. Vol. 226, p. 113847). The dimer-dimer interaction is relatively unstable, and dissociation of the tetramer is associated with several pathologies related to the TTR protein, such as TTR amyloidosis (Ando Y, Nakamura M, and Araki S. (2005) Arch Neurol 62:1057-1062). Binding sites present in the space between the dimers are targets for drugs being investigated and / or developed for some of these diseases.

[0039] The CABS-dock flexible peptide-protein docking method (Kurcinski M et al. (2020) Protein Science, 29:211-222) was used to model the structure of the complex formed between the peptide identified as SEQ ID NO: 1 and the TTR protein. The crystallographic structures of the TTR protein (unliganded) and the wild-type sequence at 1.6 Å resolution were used (Protein Data Bank file 3CFM, PDB). Because the file contains a dimer of TTR protein in the asymmetric unit, a tetramer was obtained by applying the symmetry operations of BIOMOLECULE described in the pdb file. The two chains symmetric to chains A and B (the original chains in the pdb file) were designated C and D, respectively.

[0040] The docking protocol used is "blind," meaning that all patches on the protein's surface constitute potential binding sites. Figure 5A shows the 10 superimposed best models proposed by the CABS-dock algorithm. The arrow indicates the position of the peptide, identified as SEQ ID NO:1, bound to the protein in the resulting models. All models indicate that the most likely binding site is located in one of two tetramer clefts: a) a cleft formed between the monomers of the dimer, delineated by the extended beta sheets of the concave exposed surface (intermonomer cleft, "imc"), or b) a cleft formed in the space between the dimers, delineated by the antiparallel convex beta sheets (dimer cleft, "idc"). The "idc" cleft forms the mouth of the channel or binding site for the native T4 ligand of the TTR protein. This result suggests that these surface patches have favorable stereochemical properties relative to the rest of the protein surface, such that they constitute potential "attractors" for the interaction of the peptide, identified as SEQ ID NO:1. The two "imc" sites are related by a C2 symmetry operation and are therefore identical to each other (as are the two "idc" sites). The results show that 60% of the models position the peptide identified as SEQ ID NO: 1 attached to the "idc" site, and the remaining 40% are at the "imc" site.

[0041] Figure 5B shows a model of the peptide identified as SEQ ID NO: 1 attached to the "idc" site. In this model, the most hydrophobic segment of the peptide identified as SEQ ID NO: 1 (residues Leu15-Ala20) is projected into the channel mouth through interactions with hydrophobic residues of the TTR protein, which favorably contribute to the interaction energy (and binding affinity) through the "hydrophobic effect." As shown in Figure 5C, the deepest region of the "idc" site in contact with the peptide identified as SEQ ID NO: 1 is essentially hydrophobic. As shown in Figure 5D, electrostatic complementarity between the peptide identified as SEQ ID NO: 1 and the protein is also plausible. The residue of the peptide identified as SEQ ID NO: 1 further projected into the cleft is Leu18. Figure 5E shows that this leucine residue of the peptide has close atomic contact with residues Leu17, Leu103, and Ala108 of the protein. Hydrophobic interactions are characteristically observed in the binding of the peptide identified as SEQ ID NO: 1 to proteins.

[0042] On the other hand, Figure 6 shows how the peptide segment containing the most hydrophobic residues, Ile11-Ala20, is the segment making the greatest number of contacts with the TTR protein, highlighting the importance of the hydrophobic effect for the fundamental energy contribution in peptide-protein interactions.

[0043] From a functional perspective, binding of the peptide to the "idc" site may contribute to tetramer structural stabilization, thus inhibiting and / or modulating the negative effects of diseases associated with destabilization of the native quaternary structure of the TTR protein. Meanwhile, models are consistent with the central role of the Leu18 residue in the interaction, which is due to the substitution of the Asp18 residue present in the native sequence of the T epitope of the HSP60 protein (Barbera A, Lorenzo N, van Kooten P et al. (2016) Cell Stress and Chaperones.;21:735-744). The interaction of the Leu18 residue described herein may form 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.

[0044] Example 5. Treatment of patients with ischemic heart disease with a pharmaceutical composition comprising the peptide identified as SEQ ID NO:1. Patients with low HDL levels and high blood pressure often develop ischemic heart disease, which is associated with the formation of atheromatous plaques, a condition known as atherosclerosis. These heart diseases often lead to severe conditions in patients, and treatment with the peptide identified as SEQ ID NO: 1 may contribute to their recovery.

[0045] The results of six patients are illustrated. They presented with ischemic heart disease associated with hypertension, which resulted in a critical condition. They were treated in the Intermediate Intensive Care Unit (IMCU) of the "Luis Diaz Soto" Hospital in Havana. These patients were treated with a pharmaceutical composition containing the peptide identified as SEQ ID NO: 1, administered intravenously at a dose of 1 mg of peptide every 12 hours for 7 days. In addition, they received the standard treatment required for these cases.

[0046] Figure 7 shows the cholesterol levels in these six patients with ischemic heart disease and hypertension who were treated with the peptide. As can be seen, blood cholesterol levels decreased seven days after treatment. These patients recovered and were discharged from the hospital. These results indicate that treatment with the peptide identified as SEQ ID NO: 1 can stabilize HDL and promote sufficient cholesterol turnover, thereby reducing atherosclerotic plaques, improving atherosclerosis, and avoiding related complications. Treatment with this peptide was very safe, as verified by monitoring blood biochemistry parameters and white blood cell percentages. No side effects were observed in the treated patients based on clinical and imaging evaluations.

[0047] In contrast, five patients with similar characteristics who did not receive peptide treatment for the same period stayed longer in the IMCU, and cholesterol levels in these cases exceeded 5 mmol / L at the end of treatment.

[0048] Example 6. Treatment of patients with type II diabetes with a pharmaceutical composition comprising the peptide identified as SEQ ID NO:1. Obese and hypertensive patients often develop type II diabetes, which is associated with an imbalance in lipid metabolism. These conditions can be severe, and treatment with the peptide identified as SEQ ID NO: 1 may contribute to their reversal.

[0049] This is illustrated by 10 patients with diabetic decompensation who reached a critical state and were treated in the ICU of the Hospital "Luis Diaz Soto" in Havana. Five of these patients were treated with a pharmaceutical composition containing the peptide identified as SEQ ID NO: 1, administered intravenously at a dose of 1 mg of peptide every 12 hours for 8 days. In addition, they received the standard treatment required for these cases.

[0050] Figure 8 shows the cholesterol levels in type II diabetes patients treated with peptides compared with patients who received standard treatment without peptides. As can be seen, the cholesterol levels in patients treated with peptides remain constant compared with patients not treated with peptides, whose cholesterol levels exceed 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 promote sufficient cholesterol turnover, which contributes to the stability of blood glucose metabolism. Treatment with this peptide is very safe and was verified by monitoring blood biochemistry parameters and white blood cell percentage. No side effects were observed in treated patients based on clinical and imaging evaluations.

Claims

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

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

3. 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. 3. 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 associated with effects on 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. 6. The method of 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. 6. The method of claim 5, wherein the disease is TTR amyloidosis or Parkinson's disease.

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

9. 6. The method of claim 5, wherein such pharmaceutical composition is administered concurrently with standard therapy during the course of said treatment.

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