WO2015190903A1 - Peptides derived from the c-terminal domain of cetpi as molecules blocking the cytotoxic effect induced by lipopolysaccharides in septicaemia and septic shock - Google Patents

Peptides derived from the c-terminal domain of cetpi as molecules blocking the cytotoxic effect induced by lipopolysaccharides in septicaemia and septic shock Download PDF

Info

Publication number
WO2015190903A1
WO2015190903A1 PCT/MX2014/000087 MX2014000087W WO2015190903A1 WO 2015190903 A1 WO2015190903 A1 WO 2015190903A1 MX 2014000087 W MX2014000087 W MX 2014000087W WO 2015190903 A1 WO2015190903 A1 WO 2015190903A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
cetpi
lps
peptide
septic shock
Prior art date
Application number
PCT/MX2014/000087
Other languages
Spanish (es)
French (fr)
Inventor
Jaime Mas Oliva
Nadia GUTIÉRREZ QUINTANAR
Victor Guadalupe GARCÍA GONZÁLEZ
Original Assignee
Universidad Nacional Autonoma De Mexico
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universidad Nacional Autonoma De Mexico filed Critical Universidad Nacional Autonoma De Mexico
Priority to PCT/MX2014/000087 priority Critical patent/WO2015190903A1/en
Publication of WO2015190903A1 publication Critical patent/WO2015190903A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans

Definitions

  • the present invention relates to the design and use of peptides for therapeutic activities, more specifically it relates to the design and use of peptides derived from the C-terminal domain of CETPI as blocking molecules of the cytotoxic effect induced by lipopolysaccharides, to formulate a medicament for the treatment of septicemia and septic shock caused by Gram-negative bacteria.
  • the innate immunity system provides highly regulated defense mechanisms, however its excessive response may represent a risk for homeostasis of the organism.
  • Overproduction of signaling molecules such as cytokines and excessive release of damaged cell components can directly or indirectly affect cells and tissues, and in extreme cases lead to blood poisoning or favor sepsis (Rathinam VA & Fitzgerald KA , 2013, Nature 501 (7466): 173-175).
  • the primary factors that induce such reactions are lipopolysaccharides (LPS), phosphorylated glycolipids that form a structural part of the outer membrane of Gram-negative bacteria.
  • Lipid A is the most important component, it consists of a 1, 4-bisphosphorylated diglucosamine skeleton to which acyl chains are attached. It should be noted that all bacterial species have unique LPS, however the variants mainly reside in lipid A, which are related to the pattern of acylation, the length of fatty acids, the presence of the 4- amino-deoxy-L-arabinose and / or phosphoethanolamine groups attached to the phosphate groups of glucosamines, as well as in the number of fatty acids.
  • the inner core is made up of two or more carbohydrates of the 2-keto-3-deoxyoctonic acid (KDO) type that bind to the glucosamines of lipid A, as well as two or three heptase (L- glycerol-D-hand-heptosa) that are linked to KDO, both being unique carbohydrates of bacteria.
  • the outer core has a more variable composition and is composed of common carbohydrates.
  • the O antigen remains attached to the terminal carbohydrate of the outer nucleus, so that it protrudes from the bacterial wall, being highly immunogenic. Although it is composed of repeated units of common oligosaccharides, there are several variants between species and between bacterial strains in relation to the type of carbohydrates that comprise it.
  • Septic shock can be defined as the presence of sepsis with hypotension, considering sepsis as an infection with obvious systemic inflammation and two or more of the following characteristics: Increase of body temperature, as well as the number of leukocytes, presence of tachycardia and rapid breathing. It has been described that a series of pathogenic events are responsible for the transition from sepsis to septic shock, the initial reaction is the cellular activation of monocytes, macrophages and neutrophils that interact with endothelial cells through pathogen recognition receptors. Subsequently, the mobilization of plasma molecules is carried out as a result of cell activation and endothelial disruption.
  • cytokines such as tumor necrosis factor, interleukins, caspases, proteases, leukotrienes, reactive oxygen species, nitric oxide, arachidonic acid, eicosanoids and platelet activating factor.
  • vascular endothelium is the predominant white site of these interactions, and as a result there may be microvascular damage, thrombosis and loss of endothelial integrity, which triggers tissue ischemia.
  • This diffuse endothelial disruption is responsible in most cases for the loss of functioning of the organs and the presence of tissue hypoxia, which is a characteristic condition in patients with septic shock (Nguyen HB, 2006, Ann EmergMed. 48 ( 1): 28-54). It has been reported that about one third of cases of septic shock result from infections by Gram-negative bacteria, and all these cases show high mortality (Rathinam VA, Fitzgerald KA, 2013, Nature. 501 (7466): 173-175 ).
  • the endothelial reticulum system is made up of specific tissue macrophages, responsible for the primary response to microorganisms (Van Amersfoort ES, Van Berkel TJC, Kuiper J, 2003, Clinical Microbiology Reviews. 16 (3); 379-414). Genetic studies suggest that LPS are recognized by Toll-type 4 receptors (TLR-4), which are members of an ancestral family of receptors dedicated to the detection of infectious microorganisms (Meng J, Gong M, Bjórkbacka H, Golenbock DT , 2011, J Immunol. 187 (7): 3683-3693).
  • TLR-4 Toll-type 4 receptors
  • TLR-4s by themselves do not interact directly with LPS, as the MD-2 correceptor is required, and accessory proteins such as LBP and CD14 (Ohto U et al, 2012, PNAS 109 (9); 7421 -7426. Park BS, et al, 2009, Nature. 458 (7242): 1191-1195).
  • PLUNC pulmonary palatine and nasal epithelial protein clone
  • LBP lipopolysaccharide binding protein
  • BPI bactericidal / permeability enhancing protein
  • PLTP phospholipid transfer protein
  • CETP cholesterol ester transfer protein
  • CETP and PLTP are two key proteins in the transport of neutral lipids and phospholipids between lipoproteins in plasma, respectively (Chiang SC et al, 2011, DevCornplmmunol. 35 (3): 285-295); (Bingle CD & Craven CJ, 2002, Hum Mol Genet. 11 (8): 937-943).
  • CETP is a plasma protein that is synthesized primarily by the liver, and promotes the transfer of cholesterol esters and triglycerides between lipoproteins.
  • Deletion studies and site specific mutagenesis have shown that the C-terminal domain (helix-X) structured as a helix-to-amphipathic, corresponds to a critical region in lipid transfer (Wang S et al, 1993, J. BiolChem 268 (3): 1955-1959); (Wang S, Kussie P, Deng L, Tall A, 1995, J. BiolChem. 270 (2): 612-618).
  • CETP CETP isoform synthesized exclusively in the small intestine and which was called CETPI.
  • This isoform differs from CETP in that it does not contain exon 16 and 54 bases contained in intron 15 form part of the new CETPI characteristic mRNA, which consequently replaces 24 terminal carboxyl residues present in CETP, by a sequence of 18 residues with a high content of prolines and positively charged residues, which turn out to be key for the new CETPI function (Alonso AL, Zentella-Dehesa A, Mas-Oliva J, 2003, Mol CelIBiochem. 245 (1-2): 173 -182). This difference implies the substitution of the secondary helix- ⁇ structure with a disordered structure.
  • CETP belongs to the family of PLUNC proteins, of which BPI protein is the most studied, considering that it participates in the innate immune response through LPS binding.
  • BPI protein is the most studied, considering that it participates in the innate immune response through LPS binding.
  • CETP and BPI independently of sharing a very low homology in the primary amino acid sequence (less than 16%), share important secondary and tertiary structure elements, including a structural folding in the form of a boomerang, as well as the symmetry and architecture of the amino and carboxyl terminal domains (Qiu X et al, 2007, NatStruct Mol Biol. 14 (2): 106-113).
  • both BPI and the new CETPI isoform do not show the helix- ⁇ domain located in the terminal carboxyl and instead both proteins have a high content of positively charged amino acids, property that It is important for a function related to binding to molecules with predominantly negative charge, such as LPS.
  • Novel peptides useful for inhibiting binding of lipopolysaccharides (Ips) by lipopolysaccharide binding protein (Ibp)); (EP patent 0842666 A2. Combined use of anti-endotoxin synthetic peptides and of anti-endotoxin antibodies for the prophylaxis and treatment of endotoxicosis and septic shock) .However, treatment for secondary alterations caused by systemic infections is not yet available.
  • the present invention consists in the design of high stability peptides derived from the C-terminal of CETPI, (it being understood for purposes of this invention as high stability, that the peptides maintain their secondary structure in a wide range of pH and strength ionic), to be used as blocking molecules of the cytotoxic effect induced by LPS in the events that occur during the complications of septic shock.
  • the peptide used as a molecule for the treatment of septic shock comprises the C-terminal sequence of 18 amino acids of CETPI identified as SEQ ID NO: 3, which does not present changes in its secondary structure caused by pH and at the same time is highly soluble .
  • an immunodetection system that allows the CETPI protein to be identified in plasma by polyclonal antibodies, derived from the C-terminal CETPI sequence (SEQ ID NO: 3). This possibility facilitates by means of Western-blot immunodetection techniques, in vitro monitoring of CETPI and peptides derived from its C-terminal end, as well as in vivo monitoring and control by ELISA techniques of plasma CETPI levels, before and after treatment with the SEQ ID NO 3 peptide of the present invention.
  • the peptide identified as SEQ ID NO: 3 of the present invention has shown a high potential for protection against the cytotoxic effects of septic shock, without presenting toxicity phenomena in experimental animals.
  • Figure 3A It shows the Circular Dichroism spectra of the SEQ ID NO: 3 peptide in a pH range of 3.8, 4.8, 6, 7.2, 7.8, 8.6 and 9.5, registering one line for each pH range.
  • Figure 3B Shows the Circular Dichroism spectra of the SEQ ID NO: 2 peptide in a pH range of 3.8, 4.8, 6, 7.2, 7.8, 8.6 and 9.5, registering one line for each pH range.
  • Figure 4A It is a polyacrylamide gradient native gel electrophoresis (from 0.8% to 25%) of LPS samples where the binding capacity of SEQ ID NO: 3 and SEQ ID NO: 2 to LPS is demonstrated.
  • Figure 4B shows the immunoblot detection of SEQ ID NO: 2 and SEQ ID NO: 3 using the antibody of the present invention.
  • Figure 5 Shows the immunoblot detection of SEQ ID NO: 3 to three different serotypes of LPS, 0111: B4; 026.B6 and 055.B5.
  • Figure 6A Shows by the ELISA technique, the union between SEQ ID NO: 3 at two different concentrations of 2 and 4 ng and LPS at a constant dose of 0.032 pg.
  • Figure 6B Shows by the ELISA technique, the binding between LPS and SEQ ID NO: 3 at a constant amount of 0.004 pg and LPS at two different concentrations of 0.004 g and 0.032pg using the Polyclonal Anti- CETPI A481-P491 antibody of the present invention
  • Figure 6C Shows by the ELISA technique, the union between LPS at a constant amount of 0.32 g and SEQ ID NO: 3 at a constant amount of 0.02 pg.
  • Figure 7 It shows the increasing dose treatment of SEQ ID NO: 3 in macrophage cultures demonstrating a lower cytotoxicity to the presence of LPS at a constant dose in the supernatant medium.
  • Figure 8B Treatment with SEQ ID NO: 3 in macrophage cultures, where it is shown that they develop a lower cytotoxicity to the presence of LPS in the supernatant medium at a constant dose of 10 pg / ml and at an incubation time of 2 hours.
  • Mean values (n 6, X ⁇ SD), * p ⁇ 0.05, ** p ⁇ 0.01, #p ⁇ 0.001, p ⁇ 0.000 compared to the control group.
  • Figure 9A It demonstrates the in vivo protective activity of the SEQ peptide. ID. NO. 3 in experimental animals (rabbits) to whom LPS was administered, by recording the rectal temperature, for an interval of 90 min.
  • Figure 9B It demonstrates the in vivo protective activity of the SEQ peptide. ID. NO: 3 in experimental animals (rabbits) to which LPS was administered, by plasma measurement of tumor necrosis factor alpha (TNFa) at the end of the experiment (90 minutes).
  • TNFa tumor necrosis factor alpha
  • This invention requires the use of an antibody directed against the key LPS binding site of CETPI, located in the C-terminal domain.
  • the Polyclonal Anti-CETPI A481-P491 antibody represents one of the novel components of this invention, and is a fundamental part of the CETPI detection system based on the specific recognition of the C-terminal domain, allowing the control and monitoring of treatment with the peptide of SEQ ID NO: 3.
  • the SEQ ID NO: 3 peptide was also designed and synthesized with high purity and in amounts that reach tens of mg. For this, the physicochemical and evaluation properties of the secondary structure of different sequences derived from the C-terminal of CETPI were considered. Also the following conditions were taken into account: • That is included in the reason for joining LPS.
  • SEQ ID NO: 2 A first peptide referred to as SEQ ID NO: 2, (CETPI A481-P491) that encompasses the last 11 residues of the C-terminal of CETPI was synthesized. To direct its coupling, an additional cysteine residue was added at the N-terminal end and is referred to herein as SEQ ID NO: 1.
  • SEQ ID NO: 1 has no homology with other epitopes of the CETP protein or with other proteins, and because it is made up of eleven residues, it presents only one recognition window to the immune system.
  • SEQ ID NO: 1 is used to obtain the Polyclonal Anti-CETPI A481-P491 antibody for monitoring and control of treatment with SEQ ID NO: 3.
  • peptide SEQ ID NO: 1 was coupled to the KLH carrier protein.
  • the antibodies were obtained in hens of the white LEGHORN line, inoculating subcutaneously once a week, and using a standard protocol of 63 days.
  • the titer of the antibodies in the plasma was determined by an ELISA technique.
  • the detection level of the ELISA kit in plasma is from 0.0007 g to 0.004 pg, using as a gold standard SEQ ID NO: 3, obtaining the standard curve that can be seen in Figure 1, where the detection efficiency is checked and CETPI protein measurement, in a range between 1 and 4 ng using the anti-CETPI polyclonal antibody A481-P491, disclosed in the present invention.
  • the SEQ ID NO: 3 peptide can interact with several LPS serotypes, and possibly through binding prevents the interaction of the LPS to its target receptors that trigger the inflammatory response. So the peptide SEQ ID NO: 3 keeps the LPS inactive.
  • the LPS being highly negatively charged molecules
  • the sequence analysis of the C-terminal domains of CETP and CETPI indicates that at a neutral pH, the net electrostatic charge in CETP is close to -2, in contrast to a +2 value in SEQ ID NO: 2 and +3 in SEQ ID NO: 3 derived from CETPI, values that extend to a wide pH range as can be seen in Figure 2, where the distribution of electrostatic charge is shown net (ordinate axis) as a function of the pH (abscissa axis) of SEQ peptides. ID. NO. 2, line with pictures and SEQ. ID. NO.
  • Lane 1 control without peptide or LPS Lane 1 control without peptide or LPS, lane 2 only LPS, lane 3 only peptide SEQ ID NO: 2, lane 4 peptide SEQ ID NO: 2 (8pg) and LPS (32 pg), lane 5 only peptide SEQ ID NO: 3 , lane 6 peptide SEQ ID NO: 3 (8pg) and LPS (32 pg).
  • Figure 4B In immunoblot analysis of the peptides SEQ ID NO: 2 and SEQ ID NO: 3 using the polyclonal antibody of the present invention, Figure 4B, the experiment demonstrates that only when the peptide and the LPS are together (lanes 4 and 6) the mixture of the two is retained in the gel and detected by the antibody.
  • Lane 1 control without peptide or LPS lane 2 only LPS, lane 3 only peptide SEQ ID NO: 2, lane 4 peptide SEQ ID NO: 2 (8pg) and LPS (32 pg), lane 5 only peptide SEQ ID NO: 3 , lane 6 peptide SEQ ID NO: 3 (8pg) and LPS (32 pg).
  • LPS binding is a general property of SEQ ID NO: 3.
  • the ELISA system developed in the present invention was used to characterize the LPS-SEQ ID NO: 3 junction.
  • LPS 0.032 pg
  • OD optical density
  • the first column is the control and the second only LPS at a concentration of 0.032 pg in which there is no detection.
  • Column 3 corresponds to the baseline detection of the SEQ ID NO: 3 (2 ng) peptide by the Anti-CETPI A481-P491 polyclonal antibody of the present invention.
  • Column 4 same amount of peptide SEQ ID NO: 3 (2 ng) in the presence of LPS.
  • FIG. 6B shows the optimal detection of the SEQ ID NO: 3 peptide in the presence of LPS at amounts of 0.004 and 0.032 pg.
  • SEQ ID NO: 3 was coupled to the surface of the box and incubated with the LPS, identifying an increase in the D.O. only when the LPS are present, situation shown in Figure 6C. In this case, the D.O. it was obtained with the use of an antibody that recognizes lipid A.
  • the experiment demonstrates that at a higher concentration of SEQ ID NO: 3 peptide, a better blockade of the cytotoxic effect of LPS is obtained, therefore , improves cell viability.
  • SEQ ID NO: 3 The protective effect of SEQ ID NO: 3 was extended to a broader analysis, where the same macrophages in culture were stimulated with 10 ng / ml of LPS prior to treatment with SEQ ID NO: 3, through different incubation times as shown in Figures 8A, 8B and 8C.
  • Figure 8A represents the results at 45 minutes of incubation. Column 1 control where there was no stimulation. Column 2 incubation only with LPS and represents the lowest viability. Column 3 only SEQ ID NO: 3 at a dose of 100 pg / ml. Column 4 same dose of peptide added to the LPS incubation where the improvement in viability can be seen in relation to column 2. Column 5 only SEQ ID NO: 3 at a dose of 500 pg / ml. Column 6 same dose of peptide added to incubation with LPS and viability greater than 90% is demonstrated. Column 7 only SEQ ID NO: 3 at a dose of 1000 pg / ml. Column 8 same dose of peptide added to incubation with LPS with a viability close to 100%. The results confirm that the higher the concentration of the peptide, the less cytotoxicity.
  • Figure 8B shows the results at 2 hours of macrophage incubation with LPS before the addition of the peptide SEQ ID NO: 3.
  • Column 1 control Column 2 only LPS where the lowest viability is appreciated.
  • Column 3 only SEQ ID NO: 3 at a dose of 100 g / ml.
  • Column 4 same dose of peptide added to the incubation with LPS and the viability is recovered close to 90%.
  • Column 5 only SEQ ID NO: 3 at a dose of 500 pg / ml.
  • Column 6 same dose of peptide added to incubation with LPS and about 90% viability.
  • Column 8 same dose of peptide added to incubation with LPS and feasibility close to 95%. The higher the peptide concentration, the less cytotoxicity.
  • Figure 8C shows the results at 12 hours of incubation of the macrophages with LPS, before the addition of the peptide SEQ ID NO: 3. It can be seen that despite the elapsed time, the recovery of viability is optimal although It requires the highest concentration of peptide.
  • Column 4 same dose of peptide added to incubation with LPS where no improvement in viability is seen.
  • Column 5 only SEQ ID NO: 3 at a dose of 500 pg / ml.
  • Column 6 same dose of peptide added to incubation with LPS where improvement in viability is already seen.
  • liver-derived hepG2 cells (American Type Cell Culture, ATCC) there were no changes in cell viability by the unique treatment with SEQ ID NO: 3, likewise using the microglia EOC cell line (ATCC) ), which is very sensitive to various harmful stimuli, no adverse effects were recorded. So it is feasible to use SEQ ID NO: 3 in in vivo models, since it has the optimal characteristics to counteract the endotoxic effect produced by LPS and at the same time it is not toxic to cells.
  • Fever is the intrinsic characteristic of the presence of an infection, and consequently the record of temperature rise is widely used in septic shock models in animals as an initial physiological response of damage (Gonzalo S et al, 2010, Eur J Pharmacol. 648 (1-3): 171- 178); (Shibata M, Uno T, Riedel W, Nishimaki M, Watanabe K, 2005, Int J Biometeorol. 50 (2): 67-74); (Nguyenet al, 2006, Ann EmergMed. 48 (1): 28-54). So the protective effect of treatment with SEQ ID NO: 3 in male rabbits New Zealand was evaluated. The animals were kept under ad libitum feeding conditions and free access to water. After a quarantine period of 10 days in an environment with controlled temperature and humidity, the treatment was started.
  • Rectal temperature was measured in rabbits with 24 h fast. After 5 min, the animals were inoculated intravenously into the ear with the peptide SEQ ID NO: 3 (60 pg / kg), and 10 min later the LPS 0111: B4 (0.3 pg / kg) was administered by this same route. ). Rectal temperature was recorded every 30 min for a period of 90 min, and subsequently the animals were sacrificed with sodium pentobarbital.
  • FIG 9B the in vivo protective activity of the SEQ ID peptide is demonstrated.
  • NO: 3 in experimental animals (rabbits) to which LPS was administered, and tumor necrosis factor alpha (TNFa) was measured in plasma at the end of the experiment (90 minutes).
  • the scale shows in picograms per milliliter the amount of TNFa.
  • a vehicle was administered to the control animals (first column), SEQ ID NO: 3 alone (second column), LPS only (third column) which shows the largest amount of TNFa, and SEQ ID NO: 3 + LPS in the fourth column, where the decrease in TNFa when the LPS are in the presence of SEQ ID NO: 3, confirming the protective activity.

Abstract

The invention relates to synthetic peptides derived from the C-terminal of the isoform of the CETP protein, called CETPI, that have the capacity to be molecules blocking the cytotoxic effect induced by lipopolysaccharides both in vitro and in vivo. The invention also relates to immunoenzymatic methods for detecting and quantifying the CETPI protein, using the polyclonal anti-CETPI A481-P491 antibody allowing the control and verification of the blocking of the cytotoxic effect of the polysaccharides.

Description

PÉPTIDOS DERIVADOS DEL DOMINIO C-TERMINAL DE CETPI COMO MOLÉCULAS BLOQUEADORAS DEL EFECTO CITOTÓXICO INDUCIDO POR LIPOPOLISACÁRIDOS EN SEPTICEMIA Y CHOQUE SÉPTICO.  PEPTIDES DERIVED FROM THE CETPI C-TERMINAL DOMAIN AS BLOCKING MOLECULES OF THE CYTOTOXIC EFFECT INDUCED BY LIPOPOLISACARIDS IN SEPTICEMIA AND SEPTIC SHOCK.
DESCRIPCIÓN DE LA INVENCIÓN DESCRIPTION OF THE INVENTION
Campo de la invención: Field of the invention:
La presente invención se relaciona con el diseño y uso de péptidos para actividades terapéuticas, más específicamente se relaciona con el diseño y uso de péptidos derivados del dominio C-terminal de CETPI como moléculas bloqueadoras del efecto citotóxico inducido por lipopolisacáridos, para formular un medicamento para el tratamiento de la septicemia y choque séptico causado por bacterias Gram-negativas.  The present invention relates to the design and use of peptides for therapeutic activities, more specifically it relates to the design and use of peptides derived from the C-terminal domain of CETPI as blocking molecules of the cytotoxic effect induced by lipopolysaccharides, to formulate a medicament for the treatment of septicemia and septic shock caused by Gram-negative bacteria.
Estado del Arte: State of the Art:
El sistema de inmunidad innata provee mecanismos de defensa altamente regulados, sin embargo su excesiva respuesta puede representar un riesgo para la homeostasis del organismo. La sobreproducción de moléculas de señalización como citocinas y una excesiva liberación de componentes de células dañadas, pueden de forma directa o indirecta afectar células y tejidos, y en casos extremos conducir a la intoxicación de la sangre o favorecer la sepsis (Rathinam VA & Fitzgerald KA, 2013, Nature. 501 (7466): 173-175). Los factores primarios que inducen tales reacciones son los lipopolisacáridos (LPS), glicolípidos fosforilados que forman parte estructural de la membrana externa de bacterias Gram- negativas.  The innate immunity system provides highly regulated defense mechanisms, however its excessive response may represent a risk for homeostasis of the organism. Overproduction of signaling molecules such as cytokines and excessive release of damaged cell components can directly or indirectly affect cells and tissues, and in extreme cases lead to blood poisoning or favor sepsis (Rathinam VA & Fitzgerald KA , 2013, Nature 501 (7466): 173-175). The primary factors that induce such reactions are lipopolysaccharides (LPS), phosphorylated glycolipids that form a structural part of the outer membrane of Gram-negative bacteria.
La descripción de los LPS se puede realizar considerando cuatro componentes, el lípido A, un núcleo interno, un núcleo externo y el antígeno O. El lípido A es el componente más importante, consta de un esqueleto de diglucosamina 1 ,4-bifosforilada al cual se unen cadenas acilo. Cabe resaltar que todas las especies bacterianas tienen LPS únicos, sin embargo las variantes residen principalmente en el lípido A, las cuales están relacionadas con el patrón de acilación, la longitud de los ácidos grasos, la presencia de los grupos 4- amino-deoxi-L-arabinosa y/o fosfoetanolamina unidos a los grupos fosfato de las glucosaminas, así como en el número de ácidos grasos. Es importante considerar que en cualquier variante de LPS, los dos grupos fosfato son importantes para la actividad agonista, en experimentos que conducen a su modificación, la actividad endotóxica se encuentra reducida (Van Amersfoort ES, Van Berkel TJC, Kuiper J, 2003, ClinicalMicrobiologyReviews. 16 (3); 379-414); (Ohto U et al, 2012, PNAS 109(9);7421-7426). The description of the LPS can be done considering four components, the lipid A, an internal nucleus, an external nucleus and the O antigen. Lipid A is the most important component, it consists of a 1, 4-bisphosphorylated diglucosamine skeleton to which acyl chains are attached. It should be noted that all bacterial species have unique LPS, however the variants mainly reside in lipid A, which are related to the pattern of acylation, the length of fatty acids, the presence of the 4- amino-deoxy-L-arabinose and / or phosphoethanolamine groups attached to the phosphate groups of glucosamines, as well as in the number of fatty acids. It is important to consider that in any variant of LPS, the two phosphate groups are important for agonist activity, in experiments that lead to their modification, the endotoxic activity is reduced (Van Amersfoort ES, Van Berkel TJC, Kuiper J, 2003, ClinicalMicrobiologyReviews .16 (3); 379-414); (Ohto U et al, 2012, PNAS 109 (9); 7421-7426).
Dentro de la misma molécula de LPS, el núcleo interno está conformado por dos o más carbohidratos del tipo 2-ceto-3-ácido deoxioctónico (KDO) que se unen a las glucosaminas del lípido A, así como dos o tres heptosas (L-glicero-D- mano-heptosa) que se encuentran unidas al KDO, siendo ambos carbohidratos únicos de bacterias. Por el contrario, el núcleo externo tiene una composición más variable y está integrado por carbohidratos comunes. El antígeno O se mantiene unido al carbohidrato terminal del núcleo externo, de manera que sobresale de la pared bacteriana, siendo altamente inmunogénico. Aunque está compuesto por unidades repetidas de oligosacáridos comunes, existen diversas variantes entre especies y entre cepas bacterianas con relación al tipo de carbohidratos que lo conforman. Within the same LPS molecule, the inner core is made up of two or more carbohydrates of the 2-keto-3-deoxyoctonic acid (KDO) type that bind to the glucosamines of lipid A, as well as two or three heptase (L- glycerol-D-hand-heptosa) that are linked to KDO, both being unique carbohydrates of bacteria. On the contrary, the outer core has a more variable composition and is composed of common carbohydrates. The O antigen remains attached to the terminal carbohydrate of the outer nucleus, so that it protrudes from the bacterial wall, being highly immunogenic. Although it is composed of repeated units of common oligosaccharides, there are several variants between species and between bacterial strains in relation to the type of carbohydrates that comprise it.
Cuando los LPS se encuentran incorporados a la bacteria no son tóxicos, pero una vez que son liberados de la pared bacteriana hacia el torrente sanguíneo se les consideran como endotoxinas debido a la exacerbada respuesta inflamatoria que pueden desencadenar, ocasionando bajo ciertas condiciones el desarrollo de choque séptico que puede evolucionar en muchos casos a la muerte (Beamer LJ, Carroll SF, Eisenberg D, 1998, ProteinSci. 7(4):906-914). When the LPS are incorporated into the bacteria they are not toxic, but once they are released from the bacterial wall into the bloodstream they are considered as endotoxins due to the exacerbated inflammatory response that can trigger, causing under certain conditions the development of shock septic that can evolve in many cases to death (Beamer LJ, Carroll SF, Eisenberg D, 1998, ProteinSci. 7 (4): 906-914).
El choque séptico se puede definir como la presencia de sepsis con hipotensión, considerando a la sepsis como una infección con una evidente inflamación sistémica y dos o más de las siguientes características: Incremento de la temperatura corporal, así como del número de leucocitos, presencia de taquicardia y respiración acelerada. Se ha descrito que una serie de eventos patogénicos son responsables de la transición de sepsis a choque séptico, la reacción inicial es la activación celular de monocitos, macrófagos y neutrófilos que interaccionan con las células endoteliales a través de receptores de reconocimiento a patógenos. Posteriormente, se lleva a cabo la movilización de moléculas plasmáticas como resultado de la activación celular y de la disrupción endotelial. Estas moléculas incluyen citocinas como el factor de necrosis tumoral, interleucinas, caspasas, proteasas, leucotrienos, especies reactivas de oxígeno, óxido nítrico, ácido araquidónico, eicosanoides y el factor activador de plaquetas. Septic shock can be defined as the presence of sepsis with hypotension, considering sepsis as an infection with obvious systemic inflammation and two or more of the following characteristics: Increase of body temperature, as well as the number of leukocytes, presence of tachycardia and rapid breathing. It has been described that a series of pathogenic events are responsible for the transition from sepsis to septic shock, the initial reaction is the cellular activation of monocytes, macrophages and neutrophils that interact with endothelial cells through pathogen recognition receptors. Subsequently, the mobilization of plasma molecules is carried out as a result of cell activation and endothelial disruption. These molecules include cytokines such as tumor necrosis factor, interleukins, caspases, proteases, leukotrienes, reactive oxygen species, nitric oxide, arachidonic acid, eicosanoids and platelet activating factor.
El endotelio vascular es el sitio blanco predominante de estas interacciones, y como resultado puede haber un daño microvascular, trombosis y pérdida de integridad endotelial, lo cual desencadena isquemia tisular. Esta disrupción endotelial difusa es la responsable en la mayoría de los casos de la pérdida de funcionamiento de los órganos y de la presencia de hipoxia tisular, que es una condición característica en pacientes con choque séptico (Nguyen HB, 2006, Ann EmergMed. 48(1):28-54). Se ha descrito que cerca de una tercera parte de los casos de choque séptico resultan de infecciones por bacterias Gram-negativas, y todos estos casos muestran alta mortalidad (Rathinam VA, Fitzgerald KA, 2013, Nature. 501 (7466): 173-175). The vascular endothelium is the predominant white site of these interactions, and as a result there may be microvascular damage, thrombosis and loss of endothelial integrity, which triggers tissue ischemia. This diffuse endothelial disruption is responsible in most cases for the loss of functioning of the organs and the presence of tissue hypoxia, which is a characteristic condition in patients with septic shock (Nguyen HB, 2006, Ann EmergMed. 48 ( 1): 28-54). It has been reported that about one third of cases of septic shock result from infections by Gram-negative bacteria, and all these cases show high mortality (Rathinam VA, Fitzgerald KA, 2013, Nature. 501 (7466): 173-175 ).
El sistema retículo endotelial está conformado por macrófagos tejido específicos, responsables de la respuesta primaria ante microorganismos (Van Amersfoort ES, Van Berkel TJC, Kuiper J, 2003, ClinicalMicrobiologyReviews. 16 (3); 379-414). Estudios genéticos sugieren que los LPS son reconocidos por los receptores tipo-Toll 4 (TLR-4), los cuales son miembros de una familia ancestral de receptores dedicados a la detección de microorganismos infecciosos (Meng J, Gong M, Bjórkbacka H, Golenbock DT, 2011 , J Immunol. 187(7):3683-3693). Sin embargo, los TLR-4 por sí solos no interaccionan directamente con los LPS, ya que se requiere del correceptor MD-2, y de proteínas accesorias como LBP y CD14 (Ohto U et al, 2012, PNAS 109(9);7421-7426. Park BS, et al, 2009, Nature. 458 (7242): 1191-1195). En consecuencia, los macrófagos en presencia de moléculas como LPS, LTA (ácido lipoteicoico) u otros componentes bacterianos, inducen la liberación de una serie de mediadores de procesos inflamatorios tales como el TNF-α, IL-1 , IL-6, eicosanoides, PAF, NO y especies reactivas de oxígeno (Van Amersfoort ES, Van Berkel TJC, Kuiper J, 2003, ClinicalMicrobiologyReviews. 16 (3); 379-414). Sin embargo, con un enfoque farmacológico dirigido contra el TLR-4, en estudios clínicos empleando eritoran, una molécula inhibidora del TLR-4 altamente específica, se ha encontrado que su empleo en pruebas clínicas en humanos, no reduce las probabilidades de muerte por sepsis (Opal SM, 2013, JAMA. 309(11):1154-1162). The endothelial reticulum system is made up of specific tissue macrophages, responsible for the primary response to microorganisms (Van Amersfoort ES, Van Berkel TJC, Kuiper J, 2003, Clinical Microbiology Reviews. 16 (3); 379-414). Genetic studies suggest that LPS are recognized by Toll-type 4 receptors (TLR-4), which are members of an ancestral family of receptors dedicated to the detection of infectious microorganisms (Meng J, Gong M, Bjórkbacka H, Golenbock DT , 2011, J Immunol. 187 (7): 3683-3693). However, TLR-4s by themselves do not interact directly with LPS, as the MD-2 correceptor is required, and accessory proteins such as LBP and CD14 (Ohto U et al, 2012, PNAS 109 (9); 7421 -7426. Park BS, et al, 2009, Nature. 458 (7242): 1191-1195). Consequently, macrophages in the presence of molecules such as LPS, LTA (lipoteic acid) or other bacterial components, induce the release of a series of mediators of inflammatory processes such as TNF-α, IL-1, IL-6, eicosanoids, PAF, NO and reactive oxygen species (Van Amersfoort ES, Van Berkel TJC, Kuiper J, 2003, Clinical Microbiology Reviews. 16 (3); 379-414). However, with a pharmacological approach directed against TLR-4, in clinical studies using eritoran, a highly specific TLR-4 inhibitor molecule, it has been found that its use in clinical trials in humans does not reduce the chances of death from sepsis. (Opal SM, 2013, JAMA. 309 (11): 1154-1162).
Hace más de una década, con base en estudios filogenéticos, se caracterizó una familia de proteínas denominada PLUNC (clon proteico palatino pulmonar y del epitelio nasal) que incluye proteínas asociadas a funciones inmunológicas, así como relacionadas con la unión y transporte de lípidos. En esta familia se encuentran las proteínas LBP (proteína de unión a lipopolisacáridos), BPI (proteína bactericida/incrementadora de permeabilidad), PLTP (proteína transferidora de fosfolípidos) y CETP (proteína transferidora de ésteres de colesterol). Tanto LBP como BPI juegan un papel importante en los mecanismos regulatorios durante la presentación de los LPS a los neutrófilos y en la subsecuente respuesta inflamatoria. Por otra parte, CETP y PLTP son dos proteínas clave en el transporte de lípidos neutros y fosfolípidos entre lipoproteínas en el plasma, respectivamente (Chiang SC et al, 2011 , DevCornplmmunol. 35(3):285-295); (Bingle CD &Craven CJ, 2002, Hum Mol Genet. 11 (8):937-943). More than a decade ago, based on phylogenetic studies, a family of proteins called PLUNC (pulmonary palatine and nasal epithelial protein clone) was characterized that includes proteins associated with immunological functions, as well as related to lipid binding and transport. In this family are the LBP (lipopolysaccharide binding protein), BPI (bactericidal / permeability enhancing protein), PLTP (phospholipid transfer protein) and CETP (cholesterol ester transfer protein) proteins. Both LBP and BPI play an important role in regulatory mechanisms during the presentation of LPS to neutrophils and in the subsequent inflammatory response. On the other hand, CETP and PLTP are two key proteins in the transport of neutral lipids and phospholipids between lipoproteins in plasma, respectively (Chiang SC et al, 2011, DevCornplmmunol. 35 (3): 285-295); (Bingle CD & Craven CJ, 2002, Hum Mol Genet. 11 (8): 937-943).
CETP es una proteína plasmática que es sintetizada principalmente por el hígado, y promueve la transferencia de ésteres de colesterol y triglicéridos entre lipoproteínas. Estudios de deleción y mutagénesis sitio específica han mostrado que el dominio C-terminal (hélice-X) estructurado como una hélice-a-anfipática, corresponde a una región crítica en la transferencia de lípidos (Wang S et al, 1993, J. BiolChem. 268(3): 1955-1959); (Wang S, Kussie P, Deng L, Tall A, 1995, J. BiolChem. 270(2):612-618). En este sentido, resultados de nuestro laboratorio sugieren que el mecanismo de transferencia puede estar directamente conectado con la formación de un sistema micelar de lípidos, en donde la conservación de la estructura hélice-α en el extremo C-terminal de CETP es clave para llevar a cabo este proceso (García-González V, Gutiérrez-Quintanar N, Mendoza-Espinosa P, Bracos P, Piñeiro A, Mas-Oliva J, 2014, J Struc Biol. 186(1): 19-27). CETP is a plasma protein that is synthesized primarily by the liver, and promotes the transfer of cholesterol esters and triglycerides between lipoproteins. Deletion studies and site specific mutagenesis have shown that the C-terminal domain (helix-X) structured as a helix-to-amphipathic, corresponds to a critical region in lipid transfer (Wang S et al, 1993, J. BiolChem 268 (3): 1955-1959); (Wang S, Kussie P, Deng L, Tall A, 1995, J. BiolChem. 270 (2): 612-618). In this sense, results from our laboratory suggest that the transfer mechanism can be directly connected with the formation of a micellar lipid system, where the conservation of the helix-α structure at the C-terminal end of CETP is key to carrying To carry out this process (García-González V, Gutiérrez-Quintanar N, Mendoza-Espinosa P, Bracos P, Piñeiro A, Mas-Oliva J, 2014, J Struc Biol. 186 (1): 19-27).
Nuestro equipo de trabajo ha definido la expresión de una nueva isoforma de CETP sintetizada exclusivamente en el intestino delgado y a la cual se denominó como CETPI. Esta isoforma se diferencia de CETP en que no contiene el exón 16 y 54 bases contenidas en el intrón 15 forman parte del nuevo mRNA característico de CETPI, que en consecuencia sustituye a 24 residuos del carboxilo terminal presentes en CETP, por una secuencia de 18 residuos con un alto contenido en prolinas y residuos con carga positiva, los cuales resultan ser clave para la nueva función de CETPI (Alonso AL, Zentella-Dehesa A, Mas-Oliva J, 2003, Mol CelIBiochem. 245 (1-2):173-182). Esta diferencia implica la sustitución de la estructura secundaria hélice-α por una estructura desordenada. Our team has defined the expression of a new CETP isoform synthesized exclusively in the small intestine and which was called CETPI. This isoform differs from CETP in that it does not contain exon 16 and 54 bases contained in intron 15 form part of the new CETPI characteristic mRNA, which consequently replaces 24 terminal carboxyl residues present in CETP, by a sequence of 18 residues with a high content of prolines and positively charged residues, which turn out to be key for the new CETPI function (Alonso AL, Zentella-Dehesa A, Mas-Oliva J, 2003, Mol CelIBiochem. 245 (1-2): 173 -182). This difference implies the substitution of the secondary helix-α structure with a disordered structure.
Para que se lleve a cabo la unión péptido-LPS, son de gran importancia las interacciones electrostáticas e hidrofóbicas entre el lípido A presente en los LPS y el péptido, el cual tomando en cuenta su alto contenido catiónico lleva a cabo el reemplazo de los cationes bivalentes que mantienen la estructura y neutralizan la carga negativa de los LPS (Levy O, 2000, Blood. 96(8): 2664- 2672); (Pulido D, Nogués MN, Boix E, Torrent M, 2012, J Innatelmmun. 4(4):327- 336). De manera que, considerando las características de la proteína CETPI, se llevó a cabo la optimización de la secuencia restringida al dominio C-terminal como una molécula con propiedades de unión a LPS. In order for the peptide-LPS binding to take place, electrostatic and hydrophobic interactions between the lipid A present in the LPS and the peptide are of great importance, which taking into account its high cationic content, replaces the cations bivalents that maintain the structure and neutralize the negative charge of the LPS (Levy O, 2000, Blood. 96 (8): 2664-2722); (Polished D, Nogués MN, Boix E, Torrent M, 2012, J Innatelmmun. 4 (4): 327- 336). So, considering the characteristics of the CETPI protein, the optimization of the sequence restricted to the C-terminal domain was carried out as a molecule with LPS binding properties.
Como ha sido comentado, CETP pertenece a la familia de proteínas PLUNC, de las cuáles, la proteína BPI es la más estudiada, considerando que participa en la respuesta inmune innata a través de la unión a LPS. Particularmente, CETP y BPI independientemente de compartir una muy baja homología en la secuencia primaria de aminoácidos (menos del 16%), comparten elementos de estructura secundaria y terciaria importantes, incluyendo un plegamiento estructural en forma de boomerang, así como la simetría y arquitectura de los dominios amino y carboxilo terminales (Qiu X et al, 2007, NatStruct Mol Biol. 14(2): 106-113). Una característica importante consiste en que en la superficie cóncava tanto de BPI como de la nueva isoforma CETPI, no muestran el dominio hélice-α situado en el carboxilo terminal y en su lugar ambas proteínas presentan un alto contenido de aminoácidos con carga positiva, propiedad que es importante para una función relacionada con la unión a moléculas con carga predominantemente negativa, como son los LPS. As mentioned, CETP belongs to the family of PLUNC proteins, of which BPI protein is the most studied, considering that it participates in the innate immune response through LPS binding. Particularly, CETP and BPI independently of sharing a very low homology in the primary amino acid sequence (less than 16%), share important secondary and tertiary structure elements, including a structural folding in the form of a boomerang, as well as the symmetry and architecture of the amino and carboxyl terminal domains (Qiu X et al, 2007, NatStruct Mol Biol. 14 (2): 106-113). An important feature is that on the concave surface of both BPI and the new CETPI isoform, they do not show the helix-α domain located in the terminal carboxyl and instead both proteins have a high content of positively charged amino acids, property that It is important for a function related to binding to molecules with predominantly negative charge, such as LPS.
El mecanismo de acción que se ha propuesto para la función de las proteínas que interaccionan con LPS es a través del desplazamiento de cationes bivalentes como el calcio y el magnesio que proporcionan estabilidad a los LPS, lo cual puede ocasionar la ruptura y un cambio en el potencial transmembranal de las bacterias (Levy O, 2000, AntimicrobAgentsChemother. 44(11):2925-2931). Así mismo, estudios realizados con BPI sugieren que esta proteína en presencia de LPS en el torrente sanguíneo favorece su agregación, impidiendo que se lleve a cabo la cascada de señalización proinflamatoria (Balakrishnan A, 2013, Innatelmmun. 19(4): 339-347). En este sentido, cuando la proteína CETPI fue descubierta en nuestro laboratorio, se encontró que su expresión se limitaba a células de intestino delgado y la identificación incluía su importante presencia en el plasma humano. Recientemente, utilizando varios modelos celulares de intestino delgado se han caracterizado los mecanismos que regulan su expresión. Estos modelos celulares en presencia de concentraciones muy bajas de LPS, presentan una importante sobreexpresión de CETPI, condición altamente relacionada con su función de unión a LPS (García-González V, Gutiérrez-Quintanar N, Mas-Oliva J, 2014, Manuscrito en preparación). The mechanism of action that has been proposed for the function of the proteins that interact with LPS is through the displacement of bivalent cations such as calcium and magnesium that provide stability to the LPS, which can cause rupture and a change in the Transmembrane potential of bacteria (Levy O, 2000, AntimicrobAgentsChemother. 44 (11): 2925-2931). Likewise, studies conducted with BPI suggest that this protein in the presence of LPS in the bloodstream favors its aggregation, preventing the proinflammatory signaling cascade from being carried out (Balakrishnan A, 2013, Innatelmmun. 19 (4): 339-347 ). In this sense, when the CETPI protein was discovered in our laboratory, it was found that its expression was limited to small intestine cells and the identification included its important presence in human plasma. Recently, using various cellular models of the small intestine, the mechanisms that regulate their expression have been characterized. These cellular models, in the presence of very low concentrations of LPS, have an important overexpression of CETPI, a condition highly related to their LPS binding function (García-González V, Gutiérrez-Quintanar N, Mas-Oliva J, 2014, Manuscript in preparation ).
Se ha descrito que la unión de péptidos catiónicos a LPS puede ocasionar la disrupción de los agregados que forman los LPS, lo que está correlacionado con el efecto anti-endotóxico y la modulación de la respuesta del sistema inmune (Schmidtchen A, Malmeten M, 2013, CurrentOpinion in ColloidÁ Interface Science. 18:381-392), previniendo la unión de los LPS tanto a la proteína LBP como a los receptores que desencadenan la respuesta proinflamatoria, disminuyendo de esta forma la secreción de citosinas (Rosenfeld Y, Papo N, Shai Y, 2006, J BiolChem. 281 (3): 1636-1643). It has been described that the binding of cationic peptides to LPS can cause disruption of the aggregates that form the LPS, which is correlated with the anti-endotoxic effect and modulation of the immune system response (Schmidtchen A, Malmeten M, 2013, CurrentOpinion in ColloidÁ Interface Science. 18: 381-392), preventing the binding of LPS to both the LBP protein and a the receptors that trigger the proinflammatory response, thereby decreasing cytosine secretion (Rosenfeld Y, Papo N, Shai Y, 2006, J BiolChem. 281 (3): 1636-1643).
A pesar de que de forma natural los organismos presentan mecanismos finamente diseñados para la síntesis de anticuerpos que pueden ser efectivos para contrarrestar infecciones por bacterias Gram-negativas, éstos no tienen la capacidad de neutralizar los efectos patofisiológicos asociados a los LPS. De esta manera, la utilización de péptidos derivados del C-terminal de CETPI como eficientes moléculas bloqueadoras de los LPS, se presenta como núcleo principal de la presente invención al mostrar un alto potencial terapéutico. Although the organisms naturally present finely designed mechanisms for the synthesis of antibodies that can be effective in counteracting infections by Gram-negative bacteria, they do not have the ability to neutralize the pathophysiological effects associated with LPS. Thus, the use of peptides derived from the C-terminal of CETPI as efficient LPS blocking molecules, is presented as the main nucleus of the present invention to show a high therapeutic potential.
El uso de péptidos antimicrobianos se ha considerado como una opción terapéutica en una época con problemas crecientes asociados a la resistencia a antibióticos y la incidencia de problemas de inflamación aguda y crónica (Nguyen LT, Haney EF, Vogel HJ, 2011 , TrendsBiotechnol. 29(9): 464-472); (Schmidtchen A, Malmsten M, 2013, Current Opinión in Colloid & Interface Science. 18:381- 392). Al respecto, diversos péptidos involucrados en la unión a LPS han sido estudiados (United States Patent 6384188 BlLipopolysaccharide-binding and neutralizing peptides); (WO 1995008560 A1. Novel peptides useful for inhibiting binding of lipopolysaccharides (Ips) by lipopolysaccharide binding protein (Ibp)); (EP patent 0842666 A2. Combined use of anti-endotoxin synthetic peptides and of anti-endotoxin antibodies for the prophylaxis and treatment of endotoxicosis and septic shock).Sin embargo, aún no se dispone de un tratamiento contra las alteraciones secundarias causadas por infecciones sistémicas generadas por bacterias Gram-negativas, debido a que el uso de los diversos tipos de péptidos que han sido reportados en la literatura, han presentado problemas importantes en su estabilidad química y sensibilidad a la proteólisis, tanto en las etapas de síntesis como durante su administración (Schmidtchen A, Malmsten M, 2013, Current Opinión in Colloid & Interíace Science. 18:381-392); (WO patent 2001000655 A2. Therapeutic peptides derived from subsequences of bpi). The use of antimicrobial peptides has been considered as a therapeutic option at a time with increasing problems associated with antibiotic resistance and the incidence of acute and chronic inflammation problems (Nguyen LT, Haney EF, Vogel HJ, 2011, TrendsBiotechnol. 29 ( 9): 464-472); (Schmidtchen A, Malmsten M, 2013, Current Opinion in Colloid & Interface Science. 18: 381-392). In this regard, various peptides involved in LPS binding have been studied (United States Patent 6384188 BlLipopolysaccharide-binding and neutralizing peptides); (WO 1995008560 A1. Novel peptides useful for inhibiting binding of lipopolysaccharides (Ips) by lipopolysaccharide binding protein (Ibp)); (EP patent 0842666 A2. Combined use of anti-endotoxin synthetic peptides and of anti-endotoxin antibodies for the prophylaxis and treatment of endotoxicosis and septic shock) .However, treatment for secondary alterations caused by systemic infections is not yet available. generated by Gram-negative bacteria, because the use of the various types of peptides that have been reported in the literature, have presented important problems in their chemical stability and sensitivity to proteolysis, both in the stages of synthesis and during their administration (Schmidtchen A, Malmsten M, 2013, Current Opinion in Colloid & Interíace Science. 18: 381-392); (WO patent 2001000655 A2. Therapeutic peptides derived from subsences of bpi).
Considerando lo anterior, la presente invención consiste en el diseño de péptidos de alta estabilidad derivados del C-terminal de CETPI, (entendiéndose para fines de esta invención como alta estabilidad, que los péptidos mantienen su estructura secundaria en un amplio rango de pH y fuerza iónica), para ser utilizados como moléculas bloqueadoras del efecto citotóxico inducido por LPS en los eventos que se presentan durante las complicaciones del choque séptico. El péptido usado como molécula para el tratamiento del choque séptico, comprende la secuencia C-terminal de 18 aminoácidos de CETPI identificada como SEQ ID NO: 3, la cual no presenta cambios en su estructura secundaria ocasionados por el pH y a la vez es altamente soluble. De forma complementaria a la presente invención, se divulga un sistema de inmunodetección que permite identificar a la proteína CETPI en plasma mediante anticuerpos policlonales, derivados de la secuencia C-terminal de CETPI (SEQ ID NO: 3). Esta posibilidad facilita mediante técnicas de inmunodetección tipo Western-blot, el seguimiento in vitro de CETPI y péptidos derivados de su extremo C-terminal, así como el seguimiento y control in vivo mediante técnicas de ELISA de los niveles de CETPI en plasma, antes y después del tratamiento con el péptido SEQ ID NO 3 de la presente invención. El péptido identificado como SEQ ID NO: 3 de la presente invención, ha mostrado un potencial alto de protección contra los efectos citotóxicos del choque séptico, sin presentar fenómenos de toxicidad en animales de experimentación. Considering the above, the present invention consists in the design of high stability peptides derived from the C-terminal of CETPI, (it being understood for purposes of this invention as high stability, that the peptides maintain their secondary structure in a wide range of pH and strength ionic), to be used as blocking molecules of the cytotoxic effect induced by LPS in the events that occur during the complications of septic shock. The peptide used as a molecule for the treatment of septic shock, comprises the C-terminal sequence of 18 amino acids of CETPI identified as SEQ ID NO: 3, which does not present changes in its secondary structure caused by pH and at the same time is highly soluble . In addition to the present invention, an immunodetection system is disclosed that allows the CETPI protein to be identified in plasma by polyclonal antibodies, derived from the C-terminal CETPI sequence (SEQ ID NO: 3). This possibility facilitates by means of Western-blot immunodetection techniques, in vitro monitoring of CETPI and peptides derived from its C-terminal end, as well as in vivo monitoring and control by ELISA techniques of plasma CETPI levels, before and after treatment with the SEQ ID NO 3 peptide of the present invention. The peptide identified as SEQ ID NO: 3 of the present invention has shown a high potential for protection against the cytotoxic effects of septic shock, without presenting toxicity phenomena in experimental animals.
El antecedente de diversos trabajos de nuestro grupo de investigación relacionados con el diseño y síntesis de péptidos del extremo C-terminal de CETP y anticuerpos producidos en contra de ellos, ha sido clave en el desarrollo de la presente invención (Patente MX 246,945 Sistema para la Cuantificación de la Proteína Transferidora de Ésteres de Colesterol en Muestras Biológicas y Sintéticas); (United States Patent 7,749,721. System for the Quantification of the Cholesterol Ester Transfer Protein in Biological and Synthetic Samples); (EP patent 1 ,242,446 System for the Quantification of the Cholesterol Ester Transfer Protein in Biological and Synthetic Samples) y (Vacuna de aplicación nasal contra el desarrollo de la enfermedad ateroesclerótica y el hígado graso. Número de solicitud MX/a/2012/007682, PCT/MX2013/000078). The background of various works of our research group related to the design and synthesis of C-terminal C-terminal peptides and antibodies produced against them, has been key in the development of the present invention (MX Patent 246,945 System for the Quantification of the Cholesterol Esters Transferring Protein in Biological and Synthetic Samples); (United States Patent 7,749,721. System for the Quantification of the Cholesterol Ester Transfer Protein in Biological and Synthetic Samples); (EP patent 1, 242,446 System for the Quantification of the Cholesterol Ester Transfer Protein in Biological and Synthetic Samples) and (Nasal application vaccine against the development of atherosclerotic disease and fatty liver. Application number MX / a / 2012/007682, PCT / MX2013 / 000078).
Descripción de las figuras Description of the figures
Figura 1. Gráfica de un ELISA con los valores de la curva estándar obtenida mediante el uso del péptido SEQ ID NO: 3.  Figure 1. Graph of an ELISA with the values of the standard curve obtained by using the peptide SEQ ID NO: 3.
Figura 2. Análisis de la secuencia de los últimos 48 residuos del dominio C- terminal de CETPI. Figure 2. Sequence analysis of the last 48 residues of the C-terminal CETPI domain.
Figura 3A. Muestra los espectros de Dicroísmo Circular del péptido SEQ ID NO: 3 en un rango de pH de 3.8, 4.8, 6, 7.2, 7.8, 8.6 y 9.5, registrando una línea por cada rango de pH. Figure 3A It shows the Circular Dichroism spectra of the SEQ ID NO: 3 peptide in a pH range of 3.8, 4.8, 6, 7.2, 7.8, 8.6 and 9.5, registering one line for each pH range.
Figura 3B Muestra los espectros de Dicroísmo Circular del péptido SEQ ID NO: 2 en un rango de pH de 3.8, 4.8, 6, 7.2, 7.8, 8.6 y 9.5, registrando una línea por cada rango de pH. Figure 3B Shows the Circular Dichroism spectra of the SEQ ID NO: 2 peptide in a pH range of 3.8, 4.8, 6, 7.2, 7.8, 8.6 and 9.5, registering one line for each pH range.
Figura 4A Es una electroforesis en gel nativo de gradiente de poliacrilamida (de 0.8% a 25%) de muestras de LPS donde se demuestra la capacidad de unión de la SEQ ID NO: 3 y de SEQ ID NO: 2 a LPS. Figure 4A It is a polyacrylamide gradient native gel electrophoresis (from 0.8% to 25%) of LPS samples where the binding capacity of SEQ ID NO: 3 and SEQ ID NO: 2 to LPS is demonstrated.
Figura 4B muestra la detección por inmunotransferencia de la SEQ ID NO: 2 y de la SEQ ID NO: 3 usando el anticuerpo de la presente invención. Figure 4B shows the immunoblot detection of SEQ ID NO: 2 and SEQ ID NO: 3 using the antibody of the present invention.
Figura 5. Muestra la detección por inmunotransferencia de SEQ ID NO: 3 a tres serotipos diferentes de LPS, 0111 :B4; 026.B6 y 055.B5. Figura 6A Muestra mediante la técnica de ELISA, la unión entre la SEQ ID NO: 3 a dos concentraciones diferentesde 2 y 4 ng y LPS a una dosis constante de 0.032 pg. Figure 5. Shows the immunoblot detection of SEQ ID NO: 3 to three different serotypes of LPS, 0111: B4; 026.B6 and 055.B5. Figure 6A Shows by the ELISA technique, the union between SEQ ID NO: 3 at two different concentrations of 2 and 4 ng and LPS at a constant dose of 0.032 pg.
Figura 6B Muestra mediante la técnica de ELISA, la unión entre LPS y la SEQ ID NO: 3 a una cantidad constante de 0.004 pg y LPS a dos concentraciones diferentes de 0.004 g y 0.032pg utilizando el anticuerpo policlonal Anti-CETPI A481-P491 de la presente invención. Figure 6B Shows by the ELISA technique, the binding between LPS and SEQ ID NO: 3 at a constant amount of 0.004 pg and LPS at two different concentrations of 0.004 g and 0.032pg using the Polyclonal Anti- CETPI A481-P491 antibody of the present invention
Figura 6C Muestra mediante la técnica de ELISA, la unión entre LPS a cantidad constante de 0.32 g y la SEQ ID NO: 3 a una cantidad constante de 0.02 pg. Figure 6C Shows by the ELISA technique, the union between LPS at a constant amount of 0.32 g and SEQ ID NO: 3 at a constant amount of 0.02 pg.
Figura 7. Muestra el tratamiento a dosis creciente de la SEQ ID NO: 3 en cultivos de macrófagos demostrándose una menor citotoxicidad a la presencia de LPS a dosis constante en el medio sobrenadante. Figure 7. It shows the increasing dose treatment of SEQ ID NO: 3 in macrophage cultures demonstrating a lower cytotoxicity to the presence of LPS at a constant dose in the supernatant medium.
Figura 8A. Tratamiento con la SEQ ID NO: 3 en cultivos de macrófagos, donde se demuestra que desarrollan menor citotoxicidad a la presencia de LPS en el medio sobrenadante a dosis constante y a un tiempo de incubación de 45 min. Valores de las medias (n=6, X±D.E.), *p<0.05, **p<0.01 , #p<0.001 , flpO.OOO comparados respecto al grupo control. Figure 8A Treatment with SEQ ID NO: 3 in macrophage cultures, where it is shown that they develop less cytotoxicity to the presence of LPS in the supernatant medium at a constant dose and at an incubation time of 45 min. Mean values (n = 6, X ± SD), * p <0.05, * * p <0.01, #p <0.001, flpO.OOO compared to the control group.
Figura 8B Tratamiento con la SEQ ID NO: 3 en cultivos de macrófagos, donde se demuestra que desarrollan una menor citotoxicidad a la presencia de LPS en el medio sobrenadante a dosis constante de 10 pg/ml y a un tiempo de incubación de 2 horas. Valores de las medias (n=6, X±D.E.), *p<0.05, **p<0.01 , #p<0.001 , p<0.000 comparados respecto al grupo control. Figure 8B Treatment with SEQ ID NO: 3 in macrophage cultures, where it is shown that they develop a lower cytotoxicity to the presence of LPS in the supernatant medium at a constant dose of 10 pg / ml and at an incubation time of 2 hours. Mean values (n = 6, X ± SD), * p <0.05, ** p <0.01, #p <0.001, p <0.000 compared to the control group.
Figura 8C Tratamiento con la SEQ ID NO: 3 en cultivos de macrófagos, donde se demuestra que desarrollan una menor citotoxicidad a la presencia de LPS en el medio sobrenadantea dosis constante de 10 pg/ml y a un tiempo de incubación de 12 horas. Valores de las medias (n=6, X±D.E.), *p<0.05, **p<0.01 , #p<0.001 , Up<0.000 comparados respecto al grupo control. Figure 8C Treatment with SEQ ID NO: 3 in macrophage cultures, where it is shown that they develop a lower cytotoxicity to the presence of LPS in the supernatant medium at a constant dose of 10 pg / ml and at an incubation time 12 hours Mean values (n = 6, X ± SD), * p <0.05, * * p <0.01, #p <0.001, Up <0.000 compared to the control group.
Figura 9A. Demuestra la actividad protectora in vivo del péptido SEQ. ID. NO. 3 en animales de experimentación (conejos) a los que se les administró LPS, mediante el registro de la temperatura rectal, durante un intervalo de 90 min. Figure 9A It demonstrates the in vivo protective activity of the SEQ peptide. ID. NO. 3 in experimental animals (rabbits) to whom LPS was administered, by recording the rectal temperature, for an interval of 90 min.
Figura 9B. Demuestra la actividad protectora in vivo del péptido SEQ. ID. NO: 3 en animales de experimentación (conejos) a los que se les administró LPS, mediante la medición en plasma del factor de necrosis tumoral alfa (TNFa) al término del experimento (90 minutos). Figure 9B It demonstrates the in vivo protective activity of the SEQ peptide. ID. NO: 3 in experimental animals (rabbits) to which LPS was administered, by plasma measurement of tumor necrosis factor alpha (TNFa) at the end of the experiment (90 minutes).
Descripción detallada de la invención. Detailed description of the invention.
Diseño de los péptidos sintéticos identificados como SEQ ID NO: 1 , SEQ ID NO: 2, y SEQ ID NO: 3, y obtención del anticuerpo policlonal Anti-CETPI A481-P491. Design of the synthetic peptides identified as SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and obtaining the polyclonal Anti-CETPI A481-P491 antibody.
Esta invención requiere del uso de un anticuerpo dirigido contra el sitio clave de unión a LPS de la CETPI, localizado en el domino C-terminal. El anticuerpo policlonal Anti-CETPI A481-P491 representa uno de los componentes novedosos de esta invención, y es pieza fundamental para el sistema de detección de CETPI basado en el reconocimiento específico del dominio C- terminal, permitiendo el control y seguimiento del tratamiento con el péptido de la SEQ ID NO: 3. This invention requires the use of an antibody directed against the key LPS binding site of CETPI, located in the C-terminal domain. The Polyclonal Anti-CETPI A481-P491 antibody represents one of the novel components of this invention, and is a fundamental part of the CETPI detection system based on the specific recognition of the C-terminal domain, allowing the control and monitoring of treatment with the peptide of SEQ ID NO: 3.
Para la presente invención también se diseñó y sintetizó el péptido SEQ ID NO: 3 con alta pureza y en cantidades que llegan a las decenas de mg. Para esto se consideró las propiedades fisicoquímicas y de evaluación de la estructura secundaria de diferentes secuencias derivadas del C-terminal de CETPI. Asimismo las siguientes condiciones fueron tomadas en cuenta: • Que se encuentre incluido dentro del motivo de unión a LPS.For the present invention, the SEQ ID NO: 3 peptide was also designed and synthesized with high purity and in amounts that reach tens of mg. For this, the physicochemical and evaluation properties of the secondary structure of different sequences derived from the C-terminal of CETPI were considered. Also the following conditions were taken into account: • That is included in the reason for joining LPS.
• Que su tamaño permita el menor número posible de ventanas de reconocimiento por el sistema inmune. • That its size allows the smallest possible number of recognition windows by the immune system.
• Que la secuencia no tenga homología con otros epítopos de la proteína CETP o de otras proteínas expresadas en mamíferos.  • That the sequence has no homology with other epitopes of the CETP protein or other proteins expressed in mammals.
• Que tenga un residuo de cisteína en su extremo amino para dirigir su acoplamiento a la proteína acarreadora.  • Have a cysteine residue at its amino end to direct its coupling to the carrier protein.
• Que se encuentre en una región cuya respuesta inmune haya sido probada.  • That you are in a region whose immune response has been tested.
Se sintetizó un primer péptido referido como SEQ ID NO: 2, (CETPI A481- P491) que abarca los últimos 11 residuos del C-terminal de CETPI. Para dirigir su acoplamiento se le agregó un residuo adicional de cisteína en el extremo N- terminal y se le refiere en esta invención como SEQ ID NO: 1. La SEQ ID NO: 1 no tiene homología con otros epítopes de la proteína CETP ni con otras proteínas, y debido a que está conformado por once residuos, presenta sólo una ventana de reconocimiento al sistema inmune. La SEQ ID NO: 1 se utiliza para obtener el anticuerpo policlonal Anti-CETPI A481-P491 para el seguimiento y control del tratamiento con SEQ ID NO: 3. Brevemente; se acopló el péptido SEQ ID NO: 1 a la proteína acarreadora KLH. Los anticuerpos se obtuvieron en gallinas de la línea LEGHORN blanca, inoculando por vía subcutánea una vez a la semana, y usando un protocolo estándar de 63 días. El título de los anticuerpos en el plasma se determinó por una técnica de ELISA. A first peptide referred to as SEQ ID NO: 2, (CETPI A481-P491) that encompasses the last 11 residues of the C-terminal of CETPI was synthesized. To direct its coupling, an additional cysteine residue was added at the N-terminal end and is referred to herein as SEQ ID NO: 1. SEQ ID NO: 1 has no homology with other epitopes of the CETP protein or with other proteins, and because it is made up of eleven residues, it presents only one recognition window to the immune system. SEQ ID NO: 1 is used to obtain the Polyclonal Anti-CETPI A481-P491 antibody for monitoring and control of treatment with SEQ ID NO: 3. Briefly; peptide SEQ ID NO: 1 was coupled to the KLH carrier protein. The antibodies were obtained in hens of the white LEGHORN line, inoculating subcutaneously once a week, and using a standard protocol of 63 days. The titer of the antibodies in the plasma was determined by an ELISA technique.
Se obtuvieron niveles altos de anticuerpos a través de la titulación del IgY anti-CETPI, alcanzando detecciones con diluciones de hasta 1 :100,000, lo cual está asociado con una metodología óptima durante la obtención de los anticuerpos. Cuando se usó el mismo protocolo para la producción de anticuerpos contra CETP se obtuvieron títulos más bajos (1 :10,000), indicativo de una menor inmunogenicidad de este fragmento. Con el anticuerpo policlonal Anti-CETPI A481-P491 obtenido con el péptido SEQ ID NO: 1 , se desarrolló un kit basado en una técnica de inmunodetección tipo ELISA o Western Blot. Para este ejemplo se usó, sin que sea limitativo, la técnica de ELISA integrando en el kit el anticuerpo policlonal Anti-CETPI A481-P491 para la detección y cuantificación de CETPI completa, así como los diferentes péptidos derivados de la misma. Esto permite el seguimiento de las concentraciones tanto de CETPI como de los péptidos en plasma. Esto es importante porque da certeza de la eficacia del tratamiento con el péptido SEQ ID NO: 3. High levels of antibodies were obtained through the titration of anti-CETPI IgY, reaching detections with dilutions of up to 1: 100,000, which is associated with an optimal methodology during obtaining the antibodies. When the same protocol was used for the production of antibodies against CETP, lower titers (1: 10,000) were obtained, indicative of a lower immunogenicity of this fragment. With the polyclonal Anti-CETPI A481-P491 antibody obtained with the SEQ ID NO: 1 peptide, a kit was developed based on an ELISA or Western Blot immunodetection technique. For This example was used, without limitation, the ELISA technique integrating in the kit the anti-CETPI polyclonal antibody A481-P491 for the detection and quantification of complete CETPI, as well as the different peptides derived therefrom. This allows monitoring of the concentrations of both CETPI and peptides in plasma. This is important because it gives certainty of the efficacy of the treatment with the peptide SEQ ID NO: 3.
El nivel de detección del kit ELISA en plasma es de 0.0007 g hasta 0.004 pg, empleando como estándar de oro a SEQ ID NO: 3, obteniendo la curva estándar que se puede apreciar en la Figura 1 , donde se comprueba la eficiencia de detección y medición de la proteína CETPI, en un rango entre 1 y 4 ng utilizando el anticuerpo policlonal anti-CETPI A481-P491 , divulgado en la presente invención. The detection level of the ELISA kit in plasma is from 0.0007 g to 0.004 pg, using as a gold standard SEQ ID NO: 3, obtaining the standard curve that can be seen in Figure 1, where the detection efficiency is checked and CETPI protein measurement, in a range between 1 and 4 ng using the anti-CETPI polyclonal antibody A481-P491, disclosed in the present invention.
(Mg/100 μΙ) (Mg / 100 μΙ)
0.0005  0.0005
0.00075  0.00075
0.001  0.001
0.0015  0.0015
0.002  0.002
0.0025  0.0025
0.003  0.003
0.004  0.004
Una vez demostrado que se pude hacer seguimiento del tratamiento con el péptido SEQ ID NO: 3, se realizaron experimentos para demostrar la utilidad de la presente invención. Once it was demonstrated that the treatment with the peptide SEQ ID NO: 3 could be followed up, experiments were carried out to demonstrate the utility of the present invention.
La gran mayoría de péptidos antimicrobianos presentes en eucariontes actúan sobre la membrana bacteriana o los LPS que las componen, contrario a los antibióticos que actúan sobre proteínas específicas. Lo anterior es una ventaja ya que el desarrollo de resistencia microbiana por mutación génica es menos probable (Nguyen LT, Haney EF, Vogel HJ, 2011 , TrendsBiotechnol. 29(9): 464-472). Nuestros resultados demuestran que la SEQ ID NO: 3 es una molécula inocua que origina condiciones de protección ante los efectos citotóxicos causados por los LPS. De manera que la SEQ ID NO: 3 se empleó en estudios in vivo, mediante protocolos estandarizados para demostrar su uso en el tratamiento del choque séptico. The vast majority of antimicrobial peptides present in eukaryotes act on the bacterial membrane or the LPS that compose them, contrary to the antibiotics that act on specific proteins. The above is an advantage since the development of microbial resistance by gene mutation is less likely (Nguyen LT, Haney EF, Vogel HJ, 2011, TrendsBiotechnol. 29 (9): 464-472). Our results demonstrate that SEQ ID NO: 3 is a harmless molecule that creates protective conditions against the cytotoxic effects caused by LPS. So SEQ ID NO: 3 was used in in vivo studies, using standardized protocols to demonstrate its use in the treatment of septic shock.
El péptido SEQ ID NO: 3 puede interaccionar con varios serotipos de LPS, y posiblemente a través de la unión impide la interacción de los LPS a sus receptores blanco que desencadenan la respuesta inflamatoria. De manera que el péptido SEQ ID NO: 3 mantiene en una forma inactiva a los LPS. The SEQ ID NO: 3 peptide can interact with several LPS serotypes, and possibly through binding prevents the interaction of the LPS to its target receptors that trigger the inflammatory response. So the peptide SEQ ID NO: 3 keeps the LPS inactive.
A través del uso de conejos como modelo experimental, se evidenció el papel protector de la SEQ ID NO: 3. Los resultados muestran que el péptido de la presente invención funciona en un modelo in vivo, en donde la dosis óptima de protección fue registrada bajo a una concentración de 60 pg/kg. El tratamiento únicamente con la SEQ ID NO: 3 no generó algún efecto adverso.Si bien muchos estudios se han enfocado en el uso de péptidos para neutralizar los efectos de los LPS, es frecuente encontrar que en las investigaciones realizadas por otros grupos de trabajo se requieren de altos índices de molaridad LPS/péptido, ocasionando que su uso en humanos sea prácticamente imposible debido a la citotoxicidad intrínseca generada por los péptidos a estas concentraciones (Gutsmann T et al., 2010, Antimicrob Agents Chemother. 54(9):3817-3824). Through the use of rabbits as an experimental model, the protective role of SEQ ID NO: 3 was evidenced. The results show that the peptide of the present invention works in an in vivo model, where the optimum protection dose was recorded under at a concentration of 60 pg / kg. The treatment with only SEQ ID NO: 3 did not generate any adverse effects.While many studies have focused on the use of peptides to neutralize the effects of LPS, it is common to find that in research conducted by other work groups they require high rates of LPS / peptide molarity, causing their use in humans is practically impossible due to the intrinsic cytotoxicity generated by the peptides at these concentrations (Gutsmann T et al., 2010, Antimicrob Agents Chemother. 54 (9): 3817 -3824).
Siendo los LPS moléculas altamente cargadas negativamente, el análisis de la secuencia de los dominios C-terminal de CETP y CETPI, indica que en un pH neutro, la carga electrostática neta en CETP es cercana a -2, en contraste con un valor +2 en la SEQ ID NO: 2 y de +3 en la SEQ ID NO: 3 derivadas de CETPI, valores que se extienden a un amplio rango de pH como se puede apreciar en la Figura 2, donde se muestra la distribución de la carga electrostática neta (eje de las ordenadas) en función del pH (eje de las abscisas) de los péptidos SEQ. ID. NO. 2, línea con cuadros y SEQ. ID. NO. 3, línea con círculos. Conforme el tamaño de la secuencia incrementa en el C-terminal de CETPI, la carga electrostática disminuye, por lo que la carga positiva más alta está específicamente determinada de manera óptima a la SEQ ID NO: 3, la cual permanece como una región libre en la estructura global de la CETPI. Esta condición favorece la unión a LPS. The LPS being highly negatively charged molecules, the sequence analysis of the C-terminal domains of CETP and CETPI, indicates that at a neutral pH, the net electrostatic charge in CETP is close to -2, in contrast to a +2 value in SEQ ID NO: 2 and +3 in SEQ ID NO: 3 derived from CETPI, values that extend to a wide pH range as can be seen in Figure 2, where the distribution of electrostatic charge is shown net (ordinate axis) as a function of the pH (abscissa axis) of SEQ peptides. ID. NO. 2, line with pictures and SEQ. ID. NO. 3, line with circles As the sequence size increases in the C-terminal of CETPI, the electrostatic charge decreases, so that the highest positive charge is specifically determined optimally to SEQ ID NO: 3, which remains a free region in the global structure of CETPI. This condition favors binding to LPS.
El estudio estructural de la SEQ ID NO: 3 a través de Dicroísmo Circular revela que este segmento se mantiene con una estructura desordenada. Así mismo, no se encontraron cambios en la estructura secundaria dependientes del pH como se muestra en la Figura 3A en un rango de pH de 3.8, 4.8, 6, 7.2, 7.8, 8.6 y 9.5, registrando una línea por cada rango de pH, donde el dominio C- terminal de CETPI mantiene una estructura secundaria desordenada independientemente del pH del medio en el que esté disuelto, lo que indica que es estable. En el eje de las abscisas la longitud de onda en nanómetros. The structural study of SEQ ID NO: 3 through Circular Dichroism reveals that this segment is maintained with a disorderly structure. Likewise, no pH-dependent secondary structure changes were found as shown in Figure 3A in a pH range of 3.8, 4.8, 6, 7.2, 7.8, 8.6 and 9.5, registering one line for each pH range, where the C-terminal domain of CETPI maintains a disordered secondary structure regardless of the pH of the medium in which it is dissolved, indicating that it is stable. On the axis of the abscissa the wavelength in nanometers.
Al realizar el estudio estructural de la SEQ ID NO: 2, que corresponde a os últimos 11 residuos del C-terminal de CETPI, este péptido se mantuvo con jna estructura desordenada sin importar los cambios de pH, lo que indica su estabilidad, situación que se aprecia en la Figura 3B en un rango de pH de 3.8, 4.8, 6, 7.2, 7.8, 8.6 y 9.5, registrando una línea por cada rango de pH. Previamente, se han reportado cambios conformacionales desorden-al-orden en a estructura secundaria del C-terminal de CETP dependientes de la incubación Don lípidos como el ácido lisofosfatídico o la lisofosfatidilcolina (García-González & Mas-Oliva J, 2013, BiochemBiophys Res Commun. 434(1 ):54-59). No Dbstante, este fenómeno no se presenta en los péptidos SEQ ID NO: 3 y SEQ ID NO: 2 de la presente invención. When carrying out the structural study of SEQ ID NO: 2, which corresponds to the last 11 residues of the C-terminal of CETPI, this peptide remained with a disordered structure regardless of the changes in pH, indicating its stability, a situation that It can be seen in Figure 3B in a pH range of 3.8, 4.8, 6, 7.2, 7.8, 8.6 and 9.5, registering one line for each pH range. Previously, disorder-to-order conformational changes have been reported in a secondary structure of the C-terminal CETP dependent on Don Lipid incubation such as lysophosphatidic acid or lysophosphatidylcholine (García-González & Mas-Oliva J, 2013, BiochemBiophys Res Commun 434 (1): 54-59). However, this phenomenon does not occur in the peptides SEQ ID NO: 3 and SEQ ID NO: 2 of the present invention.
Con base en protocolos en los que se ha empleado la electroforesis SDS- PAGE en la caracterización del lípido A y el antígeno O de los LPS (Marolda CL, _ahiry P, Vinés E, Saldías S, Valvano MA, 2006, Methods Mol Biol. 347.237-252), >e desarrolló un protocolo en geles nativos de gradiente de poliacrilamida (0.8- 25%), que permite evaluar la unión péptido-LPS. Los resultados sugieren que los péptidos SEQ ID NO: 2 y SEQ ID NO: 3 se mantienen unidos a los LPS, y solamente en esta condición quedan retenidos en la matriz de poliacrilamida, como se muestra en la Figura 4A, donde los carriles que contienen péptido más LPS presentan un claro corrimiento dentro del gel de poliacrilamida, lo que demuestra la unión entre estas dos moléculas. Carril 1 control sin péptido ni LPS, carril 2 solamente LPS, carril 3 solo péptido SEQ ID NO: 2, carril 4 péptido SEQ ID NO: 2 (8pg) y LPS (32 pg), carril 5 solo péptido SEQ ID NO: 3, carril 6 péptido SEQ ID NO: 3 (8pg) y LPS (32 pg). En el análisis por inmunotransferencia de los péptidos SEQ ID NO: 2 y SEQ ID NO: 3 usando el anticuerpo policlonal de la presente invención, Figura 4B, el experimento demuestra que solamente cuando el péptido y los LPS están juntos (carriles 4 y 6) la mezcla de los dos se retiene en el gel y lo detecta el anticuerpo. Carril 1 control sin péptido ni LPS, carril 2 solamente LPS, carril 3 solo péptido SEQ ID NO: 2, carril 4 péptido SEQ ID NO: 2 (8pg) y LPS (32 pg), carril 5 solo péptido SEQ ID NO: 3, carril 6 péptido SEQ ID NO: 3 (8pg) y LPS (32 pg). Based on protocols in which SDS-PAGE electrophoresis has been used in the characterization of lipid A and LPS antigen O (Marolda CL, _ahiry P, Vinés E, Saldías S, Valvano MA, 2006, Methods Mol Biol. 347.237-252),> and developed a protocol on polyacrylamide gradient native gels (0.8-25%), which allows to evaluate the peptide-LPS binding. The results suggest that Peptides SEQ ID NO: 2 and SEQ ID NO: 3 remain attached to the LPS, and only in this condition are they retained in the polyacrylamide matrix, as shown in Figure 4A, where lanes containing peptide plus LPS have a clear shift within the polyacrylamide gel, which demonstrates the union between these two molecules. Lane 1 control without peptide or LPS, lane 2 only LPS, lane 3 only peptide SEQ ID NO: 2, lane 4 peptide SEQ ID NO: 2 (8pg) and LPS (32 pg), lane 5 only peptide SEQ ID NO: 3 , lane 6 peptide SEQ ID NO: 3 (8pg) and LPS (32 pg). In immunoblot analysis of the peptides SEQ ID NO: 2 and SEQ ID NO: 3 using the polyclonal antibody of the present invention, Figure 4B, the experiment demonstrates that only when the peptide and the LPS are together (lanes 4 and 6) the mixture of the two is retained in the gel and detected by the antibody. Lane 1 control without peptide or LPS, lane 2 only LPS, lane 3 only peptide SEQ ID NO: 2, lane 4 peptide SEQ ID NO: 2 (8pg) and LPS (32 pg), lane 5 only peptide SEQ ID NO: 3 , lane 6 peptide SEQ ID NO: 3 (8pg) and LPS (32 pg).
El análisis se amplió a la evaluación de la unión de la SEQ ID NO: 3 a varios serotipos de LPS, como el 0111 :B4, 026:B6 y el 055:B5, en todos los casos se detectó la señal de unión de la SEQ ID NO: 3 a los tres serotipos de LPS como se aprecia en la Figura 5, demostrándose la unión entre el péptido SEQ ID NO: 3 y diferentes tipos de LPS. Carril 1 control, carril 2 solamente SEQ ID NO: 3 (7.2pg), carril 3 LPS serotipo 0111 :B4 (29pg), carril 4 LPS serotipo 01 1 :B4 (29pg) mas SEQ ID NO:3 (7.2pg), carril 5 LPS serotipo 026:B6 (29pg), carril 6 LPS serotipo 026.B6 (29pg) mas SEQ ID NO:3 (7.2pg), carril 7 LPS serotipo 055.B5 (29pg), carril 8 LPS 055:B5 (29pg) mas SEQ ID NO:3 (7.2pg). De tal forma que la unión a LPS es una propiedad general de la SEQ ID NO: 3. The analysis was extended to the evaluation of the binding of SEQ ID NO: 3 to several LPS serotypes, such as 0111: B4, 026: B6 and 055: B5, in all cases the binding signal of the SEQ ID NO: 3 to the three LPS serotypes as seen in Figure 5, demonstrating the binding between the SEQ ID NO: 3 peptide and different types of LPS. Lane 1 control, lane 2 only SEQ ID NO: 3 (7.2pg), lane 3 LPS serotype 0111: B4 (29pg), lane 4 LPS serotype 01 1: B4 (29pg) plus SEQ ID NO: 3 (7.2pg), lane 5 LPS serotype 026: B6 (29pg), lane 6 LPS serotype 026.B6 (29pg) plus SEQ ID NO: 3 (7.2pg), lane 7 LPS serotype 055.B5 (29pg), lane 8 LPS 055: B5 ( 29pg) plus SEQ ID NO: 3 (7.2pg). Thus, LPS binding is a general property of SEQ ID NO: 3.
De igual forma, se utilizó el sistema de ELISA desarrollado en la presente invención, para caracterizar la unión LPS-SEQ ID NO: 3. Cuando se acoplaron los LPS (0.032 pg) a la superficie de la caja, solamente se identificó un aumento en la señal de densidad óptica (D.O.) bajo la incubación con 0.04 pg de SEQ ID NO: 3, indicativo de la unión y se aprecia en la Figura 6A. La primera columna es el control y la segunda solo LPS a concentración de 0.032 pg en las cuales no hay detección. La columna 3 corresponde a la detección basal del péptido SEQ ID NO: 3 (2 ng) por el anticuerpo policlonal Anti-CETPI A481-P491 de la presente invención. La columna 4 misma cantidad de péptido SEQ ID NO: 3 (2 ng) en presencia de LPS. Columna 5 detección basal del péptido SEQ ID NO: 3 (4ng) por el anticuerpo de la presente invención. La columna 6 muestra la detección del péptido SEQ ID NO: 3 (4ng) en presencia de LPS. Se demuestra la eficiencia de la detección del péptido en presencia de LPS, y a mayor concentración de péptido SEQ ID NO: 3 mayor detección. Similarly, the ELISA system developed in the present invention was used to characterize the LPS-SEQ ID NO: 3 junction. When the LPS (0.032 pg) was coupled to the surface of the box, only an increase in the optical density (OD) signal under incubation with 0.04 pg of SEQ ID NO: 3, indicative of binding and is shown in Figure 6A. The first column is the control and the second only LPS at a concentration of 0.032 pg in which there is no detection. Column 3 corresponds to the baseline detection of the SEQ ID NO: 3 (2 ng) peptide by the Anti-CETPI A481-P491 polyclonal antibody of the present invention. Column 4 same amount of peptide SEQ ID NO: 3 (2 ng) in the presence of LPS. Column 5 baseline detection of peptide SEQ ID NO: 3 (4ng) by the antibody of the present invention. Column 6 shows the detection of the SEQ ID NO: 3 (4ng) peptide in the presence of LPS. The efficiency of the detection of the peptide in the presence of LPS is demonstrated, and the higher concentration of peptide SEQ ID NO: 3, the greater the detection.
En otro ensayo paralelo, cuando se mantiene constante la cantidad de SEQ ID NO: 3 se evidenció un incremento en la D.O. bajo la adición de LPS, lo que se aprecia en la Figura 6B.La figura muestra la óptima detección del péptido SEQ ID NO: 3 en presencia de LPS a cantidades de 0.004 y 0.032 pg. Columna 1 control; columna 2 solo el péptido SEQ ID NO: 3; columna 3 solo LPS a cantidad de 0.004 pg y no hay detección; columna 4 péptido SEQ ID NO: 3 y LPS a cantidad de 0.004 pg; columna 5 solo LPS a cantidad de 0.032 pg y no hay detección, y; columna 6 SEQ ID NO: 3 mas LPS a cantidad de 0.32 pg. Se aprecia que la detección se da solamente cuando está el péptido SEQ ID NO: 3 de la presente invención y la densidad óptica es mayor cuando está unido a LPS. In another parallel trial, when the amount of SEQ ID NO: 3 is kept constant, an increase in the D.O. under the addition of LPS, which can be seen in Figure 6B. The figure shows the optimal detection of the SEQ ID NO: 3 peptide in the presence of LPS at amounts of 0.004 and 0.032 pg. Column 1 control; column 2 only the peptide SEQ ID NO: 3; column 3 only LPS at the amount of 0.004 pg and there is no detection; column 4 peptide SEQ ID NO: 3 and LPS in the amount of 0.004 pg; column 5 only LPS at an amount of 0.032 pg and there is no detection, and; column 6 SEQ ID NO: 3 plus LPS in the amount of 0.32 pg. It is appreciated that detection occurs only when the SEQ ID NO: 3 peptide of the present invention is present and the optical density is higher when bound to LPS.
De manera complementaria se acopló la SEQ ID NO: 3 a la superficie de la caja y se incubó con los LPS, identificándose un aumento en la D.O. solamente cuando están presentes los LPS, situación que se muestra en la Figura 6C.En este caso, la señal de D.O. se obtuvo con el uso de un anticuerpo que reconoce al lípido A. Columna 1 control y no hay detección; columna 2 solo el péptido SEQ ID NO: 3; columna 3 solo LPS; columna 4 péptido SEQ ID NO: 3 y LPS. Se aprecia mayor detección cuando están la SEQ ID NO: 3 y LPS. In addition, SEQ ID NO: 3 was coupled to the surface of the box and incubated with the LPS, identifying an increase in the D.O. only when the LPS are present, situation shown in Figure 6C. In this case, the D.O. it was obtained with the use of an antibody that recognizes lipid A. Column 1 control and no detection; column 2 only the peptide SEQ ID NO: 3; column 3 only LPS; column 4 peptide SEQ ID NO: 3 and LPS. Greater detection is seen when SEQ ID NO: 3 and LPS are present.
Con esta evidencia experimental se demuestra la propiedad de unión de la SEQ ID NO: 3de la presente invención sobre los LPS, situación que era importante demostrar para dar soporte para su uso terapéutico. Habiendo demostrado la propiedad de unión del péptido SEQ ID NO: 3 a LPS, se evaluó su efecto protector sobre macrófagos, que es un modelo celular ampliamente caracterizado dentro de los mecanismos patofisiológicos que producen los LPS. Cultivos celulares fueron incubados durante 45 min con concentraciones crecientes de la SEQ ID NO: 3 (0, 0.01 , 0.5 y 1 pg/ml), y posteriormente se estimularon con una concentración constante de 10 ng/ml de LPS por 24 h. Los resultados de viabilidad celular a través del ensayo con la molécula MTT (3-(4,5-dimetiltiazol-2-ilo) 2,5 bromuro difeniltetrazolio) indican que los LPS inducen una disminución en la viabilidad de los macrófagos cercana al 50%, sin embargo, el tratamiento con la SEQ ID NO: 3 permite que la viabilidad celular mejore de forma importante, como se identifica en la Figura 7,donde se aprecia resultados contrarios a lo reportado en el modelo de investigación citado (Singh S et al, 2013, Biomacromolecules 14(5):1482-1492). Así mismo, no se encontraron indicios de toxicidad debido al tratamiento solamente con la SEQ ID NO: 3. El experimento demuestra que a mayor concentración de péptido SEQ ID NO: 3 se obtiene un mejor bloqueo del efecto citotóxico de los LPS, por lo tanto, mejora la viabilidad celular. El gráfico muestra la viabilidad celular evaluada a través de MTT en células bajo un tratamiento con dosis crecientes de SEQ ID NO: 3, antes del estímulo por 24 h con 10 ng/ml de LPS, (n=6, X±D.E.), *p<0.05, k*p<0.001 comparado con el grupo control. Columna 1 control viabilidad 100%. Columna 2 solamente LPS con una viabilidad menor al 50%. Columna 3 solo SEQ ID NO: 3 a dosis de 0.01 pg. Columna 4 misma dosis der péptido agregándose LPS y se aprecia que la viabilidad sube cercana al 60%. Columna 5 solo SEQ ID NO: 3 a dosis de 0.1 pg. Columna 6 misma dosis de péptido agregándose LPS apreciándose que se incrementa la viabilidad arriba del 60%. Columna 7 solo SEQ ID NO: 3 a dosis de 0.5 pg. Columna 8 misma dosis de péptido agregándose LPS y la viabilidad rebasa el 80%. Columna 9 SEQ ID NO: 3 a dosis de 1 pg. Columna 10 misma dosis de péptido agregándose LPS y la viabilidad se mantiene por arriba del 80%. El efecto protector de la SEQ ID NO: 3 se extendió a un análisis más amplio, en donde los mismos macrófagos en cultivo se estimularon con 10 ng/ml de LPS de forma previa al tratamiento con la SEQ ID NO: 3, a través de diferentes tiempos de incubación como se muestra en las Figuras 8A, 8B y 8C. With this experimental evidence the binding property of SEQ ID NO: 3 of the present invention on LPS is demonstrated, a situation that was important to demonstrate in order to support its therapeutic use. Having demonstrated the binding property of the SEQ ID NO: 3 peptide to LPS, its protective effect on macrophages was evaluated, which is a cellular model widely characterized within the pathophysiological mechanisms that produce LPS. Cell cultures were incubated for 45 min with increasing concentrations of SEQ ID NO: 3 (0, 0.01, 0.5 and 1 pg / ml), and subsequently stimulated with a constant concentration of 10 ng / ml of LPS for 24 h. The results of cell viability through the test with the MTT molecule (3- (4,5-dimethylthiazol-2-yl) 2,5 diphenyltetrazolium bromide) indicate that LPS induces a decrease in macrophage viability close to 50% , however, treatment with SEQ ID NO: 3 allows cell viability to improve significantly, as identified in Figure 7, where results contrary to what is reported in the cited research model can be seen (Singh S et al , 2013, Biomacromolecules 14 (5): 1482-1492). Likewise, no indications of toxicity were found due to treatment only with SEQ ID NO: 3. The experiment demonstrates that at a higher concentration of SEQ ID NO: 3 peptide, a better blockade of the cytotoxic effect of LPS is obtained, therefore , improves cell viability. The graph shows the cell viability evaluated through MTT in cells under treatment with increasing doses of SEQ ID NO: 3, before stimulation for 24 h with 10 ng / ml of LPS, (n = 6, X ± DE), * p <0.05, k * p <0.001 compared to the control group. Column 1 100% feasibility control. Column 2 only LPS with a viability less than 50%. Column 3 only SEQ ID NO: 3 at a dose of 0.01 pg. Column 4 same dose of peptide adding LPS and it is seen that the viability rises close to 60%. Column 5 only SEQ ID NO: 3 at a dose of 0.1 pg. Column 6 same dose of peptide adding LPS, appreciating that viability is increased above 60%. Column 7 only SEQ ID NO: 3 at a dose of 0.5 pg. Column 8 same dose of peptide adding LPS and the viability exceeds 80%. Column 9 SEQ ID NO: 3 at a dose of 1 pg. Column 10 same dose of peptide adding LPS and the viability is maintained above 80%. The protective effect of SEQ ID NO: 3 was extended to a broader analysis, where the same macrophages in culture were stimulated with 10 ng / ml of LPS prior to treatment with SEQ ID NO: 3, through different incubation times as shown in Figures 8A, 8B and 8C.
La Figura 8A representa los resultados a los 45 minutos de incubación. La columna 1 control donde no hubo estimulación. Columna 2 incubación solo con LPS y representa la más baja viabilidad. Columna 3 sólo SEQ ID NO: 3 a dosis de 100 pg/ml. Columna 4 misma dosis de péptido agregado a la incubación con LPS donde se aprecia la mejora en la viabilidad con relación a la columna 2. Columna 5 solo SEQ ID NO: 3 a dosis de 500 pg/ml. Columna 6 misma dosis de péptido agregado a la incubación con LPS y se demuestra viabilidad mayor al 90%. Columna 7 solo SEQ ID NO: 3 a dosis de 1000 pg/ml. Columna 8 misma dosis de péptido agregado a la incubación con LPS con una viabilidad cercana al 100%. Los resultados confirman que a mayor concentración de péptido menor citotoxicidad. Figure 8A represents the results at 45 minutes of incubation. Column 1 control where there was no stimulation. Column 2 incubation only with LPS and represents the lowest viability. Column 3 only SEQ ID NO: 3 at a dose of 100 pg / ml. Column 4 same dose of peptide added to the LPS incubation where the improvement in viability can be seen in relation to column 2. Column 5 only SEQ ID NO: 3 at a dose of 500 pg / ml. Column 6 same dose of peptide added to incubation with LPS and viability greater than 90% is demonstrated. Column 7 only SEQ ID NO: 3 at a dose of 1000 pg / ml. Column 8 same dose of peptide added to incubation with LPS with a viability close to 100%. The results confirm that the higher the concentration of the peptide, the less cytotoxicity.
La Figura 8B muestra los resultados a las 2 horas de incubación de macrófagos con LPS antes de la adición del péptido SEQ ID NO: 3. La columna 1 control. Columna 2 solo LPS donde se aprecia la más baja viabilidad. Columna 3 solo SEQ ID NO: 3 a dosis de 100 g/ml. Columna 4 misma dosis de péptido agregado a la incubación con LPS y la viabilidad se recupera cercana al 90%. Columna 5 solo SEQ ID NO: 3 a dosis de 500 pg/ml. Columna 6 misma dosis de péptido agregado a la incubación con LPS y la viabilidad alrededor del 90%. Columna 7 solo SEQ ID NO: 3 a dosis de 1000 pg/ml. Columna 8 misma dosis de péptido agregado a la incubación con LPS y se aprecia viabilidad cercana al 95%. A mayor concentración de péptido menor citotoxicidad. Figure 8B shows the results at 2 hours of macrophage incubation with LPS before the addition of the peptide SEQ ID NO: 3. Column 1 control. Column 2 only LPS where the lowest viability is appreciated. Column 3 only SEQ ID NO: 3 at a dose of 100 g / ml. Column 4 same dose of peptide added to the incubation with LPS and the viability is recovered close to 90%. Column 5 only SEQ ID NO: 3 at a dose of 500 pg / ml. Column 6 same dose of peptide added to incubation with LPS and about 90% viability. Column 7 only SEQ ID NO: 3 at a dose of 1000 pg / ml. Column 8 same dose of peptide added to incubation with LPS and feasibility close to 95%. The higher the peptide concentration, the less cytotoxicity.
Finalmente la Figura 8C muestra los resultados a las 12 horas de incubación de los macrófagos con LPS, antes de la adición del péptido SEQ ID NO: 3. Se puede apreciar que no obstante el tiempo transcurrido, la recuperación de la viabilidad es óptima aunque se requiere la mayor concentración de péptido. Columna 1 control. Columna 2 solo LPS. Columna 3 solo SEQ ID NO: 3 a dosis de 100 pg/ml. Columna 4 misma dosis de péptido agregado a la incubación con LPS donde no se aprecia mejora en la viabilidad. Columna 5 solo SEQ ID NO: 3 a dosis de 500 pg/ml. Columna 6 misma dosis de péptido agregado a la incubación con LPS donde ya se aprecia mejora en la viabilidad. Columna 7 solo SEQ ID NO: 3 a dosis de 1000 pg/ml. Columna 8 misma dosis de péptido agregado a la incubación con LPS donde ya es claro la recuperación de la viabilidad celular cercana al 100%. Finally, Figure 8C shows the results at 12 hours of incubation of the macrophages with LPS, before the addition of the peptide SEQ ID NO: 3. It can be seen that despite the elapsed time, the recovery of viability is optimal although It requires the highest concentration of peptide. Column 1 control. Column 2 only LPS. Column 3 only SEQ ID NO: 3 at a dose of 100 pg / ml. Column 4 same dose of peptide added to incubation with LPS where no improvement in viability is seen. Column 5 only SEQ ID NO: 3 at a dose of 500 pg / ml. Column 6 same dose of peptide added to incubation with LPS where improvement in viability is already seen. Column 7 only SEQ ID NO: 3 at a dose of 1000 pg / ml. Column 8 same dose of peptide added to incubation with LPS where it is already clear the recovery of cell viability close to 100%.
En las tres condiciones se continuó el tratamiento por 24 h, y en todos los casos cuando se usó la concentración más alta de SEQ ID NO: 3 (1000 ng/ml), la viabilidad celular se presenta en niveles similares a los respectivos controles que no se incubaron con LPS. Se demostró que la SEQ ID NO: 3 ejerce una función protectora eficiente en la prevención de los efectos citotóxicos que inducen los LPS. In all three conditions the treatment was continued for 24 h, and in all cases when the highest concentration of SEQ ID NO: 3 (1000 ng / ml) was used, cell viability is presented at levels similar to the respective controls that They were not incubated with LPS. It was shown that SEQ ID NO: 3 exerts an efficient protective function in the prevention of cytotoxic effects induced by LPS.
Empleando otros modelos celulares como células hepG2 derivadas de hígado, (American Type Cell Culture, ATCC) no se registraron cambios en la viabilidad celular por el tratamiento único con la SEQ ID NO: 3, así mismo usando la línea celular EOC de microglia (ATCC), que es muy sensible a diversos estímulos nocivos, no se registraron efectos adversos. De manera que es factible el uso de SEQ ID NO: 3 en modelos in vivo, ya que reúne las características óptimas para contrarrestar el efecto endotóxico producido por los LPS y a la vez que no es tóxico para las células. Using other cellular models such as liver-derived hepG2 cells, (American Type Cell Culture, ATCC) there were no changes in cell viability by the unique treatment with SEQ ID NO: 3, likewise using the microglia EOC cell line (ATCC) ), which is very sensitive to various harmful stimuli, no adverse effects were recorded. So it is feasible to use SEQ ID NO: 3 in in vivo models, since it has the optimal characteristics to counteract the endotoxic effect produced by LPS and at the same time it is not toxic to cells.
Ejemplo de uso. Efecto protector del péptido SEQ ID NO: 3 en un modelo de choque séptico en conejos. Example of use. Protective effect of the peptide SEQ ID NO: 3 in a septic shock model in rabbits.
La fiebre es la característica intrínseca de la presencia de una infección, y en consecuencia el registro del aumento de temperatura es ampliamente utilizado en los modelos de choque séptico en animales como una respuesta fisiológica inicial de daño (Gonzalo S et al, 2010, Eur J Pharmacol. 648(1-3): 171- 178); (Shibata M, Uno T, Riedel W, Nishimaki M, Watanabe K, 2005, Int J Biometeorol. 50(2):67-74); (Nguyenet al, 2006, Ann EmergMed. 48(1): 28-54). De manera que el efecto protector del tratamiento con la SEQ ID NO: 3 en conejos machos Nueva Zelanda fue evaluado. Los animales se mantuvieron bajo condiciones de alimentación ad libitum y acceso libre al agua. Después de un periodo de cuarentena de 10 días en un ambiente con temperatura y humedad controladas, se inició el tratamiento. Fever is the intrinsic characteristic of the presence of an infection, and consequently the record of temperature rise is widely used in septic shock models in animals as an initial physiological response of damage (Gonzalo S et al, 2010, Eur J Pharmacol. 648 (1-3): 171- 178); (Shibata M, Uno T, Riedel W, Nishimaki M, Watanabe K, 2005, Int J Biometeorol. 50 (2): 67-74); (Nguyenet al, 2006, Ann EmergMed. 48 (1): 28-54). So the protective effect of treatment with SEQ ID NO: 3 in male rabbits New Zealand was evaluated. The animals were kept under ad libitum feeding conditions and free access to water. After a quarantine period of 10 days in an environment with controlled temperature and humidity, the treatment was started.
Cuatro grupos de animales experimentales fueron utilizados como se describe en la Tabla 1. La temperatura rectal se midió en los conejos con 24 h de ayuno. Después de 5 min, los animales se inocularon por vía intravenosa en la oreja con el péptido SEQ ID NO: 3 (60 pg/kg), y 10 min después se administró por esta misma vía los LPS 0111 :B4 (0.3 pg/kg). La temperatura rectal se registró cada 30 min durante un periodo de 90 min, y posteriormente los animales fueron sacrificados con pentobarbital sódico. A través de la realización de este experimento se detectó un menor aumento de temperatura corporal en el grupo 4, que recibió el tratamiento con el péptido SEQ ID NO: 3 y LPS (At=1.7°C), en comparación con el grupo 3 que solo recibió la administración de LPS (At=2.3°C). En el grupo 2, la administración únicamente del péptido SEQ ID NO: 3 no originó efectos adversos (At=0.2°C) como se aprecia en la Figura 9A que muestra las gráficas del registro de temperatura de los animales y en los animales control que recibieron únicamente vehículo, así como los que recibieron únicamente la SEQ ID NO: 3 la temperatura no mostró alteración, mientras que los animales que recibieron solo LPS fueron los que registraron una mayor temperatura y el grupo que recibió SEQ ID N0.3+LPS, el ascenso de temperatura fue menor con relación a aquellos animales que recibieron solo LPS. Four groups of experimental animals were used as described in Table 1. Rectal temperature was measured in rabbits with 24 h fast. After 5 min, the animals were inoculated intravenously into the ear with the peptide SEQ ID NO: 3 (60 pg / kg), and 10 min later the LPS 0111: B4 (0.3 pg / kg) was administered by this same route. ). Rectal temperature was recorded every 30 min for a period of 90 min, and subsequently the animals were sacrificed with sodium pentobarbital. Through the conduct of this experiment, a lower increase in body temperature was detected in group 4, which received the treatment with the peptide SEQ ID NO: 3 and LPS (At = 1.7 ° C), compared to group 3 which He only received the administration of LPS (At = 2.3 ° C). In group 2, the administration of only the SEQ ID NO: 3 peptide did not cause adverse effects (At = 0.2 ° C) as shown in Figure 9A which shows the graphs of the temperature record of the animals and in the control animals that they received only vehicle, as well as those who received only SEQ ID NO: 3 the temperature showed no alteration, while the animals that received only LPS were those that registered a higher temperature and the group that received SEQ ID N0.3 + LPS, the temperature rise was lower compared to those animals that received only LPS.
En la Figura 9B se demuestra la actividad protectora in vivo del péptido SEQ ID. NO: 3 en animales de experimentación (conejos) a los que se les administró LPS, y se midió en plasma el factor de necrosis tumoral alfa (TNFa) al término del experimento (90 minutos). La escala muestra en picogramos por mililitro la cantidad de TNFa. Se administró un vehículo a los animales control (primera columna), SEQ ID NO: 3 sola (segunda columna), LPS solo (tercera columna) la cual muestra la mayor cantidad de TNFa, y SEQ ID NO: 3+LPS en la cuarta columna, done se aprecia la disminución de TNFa cuando los LPS están en presencia de SEQ ID NO: 3, confirmando la actividad protectora. In Figure 9B the in vivo protective activity of the SEQ ID peptide is demonstrated. NO: 3 in experimental animals (rabbits) to which LPS was administered, and tumor necrosis factor alpha (TNFa) was measured in plasma at the end of the experiment (90 minutes). The scale shows in picograms per milliliter the amount of TNFa. A vehicle was administered to the control animals (first column), SEQ ID NO: 3 alone (second column), LPS only (third column) which shows the largest amount of TNFa, and SEQ ID NO: 3 + LPS in the fourth column, where the decrease in TNFa when the LPS are in the presence of SEQ ID NO: 3, confirming the protective activity.
En el protocolo experimental, los mejores resultados fueron obtenidos con una relación 1-200 (LPS-SEQ ID NO: 3) en el grupo 4, equivalente a una dosis única de SEQ ID NO: 3 (150pg). Esta dosis representa una condición intermedia, y refuerza la propuesta de una dosis efectiva sin que sea limitativo para la presente invención. In the experimental protocol, the best results were obtained with a 1-200 ratio (LPS-SEQ ID NO: 3) in group 4, equivalent to a single dose of SEQ ID NO: 3 (150pg). This dose represents an intermediate condition, and reinforces the proposal for an effective dose without being limiting for the present invention.
Tabla 1. Grupos de animales experimentales usados Table 1. Groups of experimental animals used
Figure imgf000024_0001
Figure imgf000024_0001
El experimento se realizó en el bioterio del Instituto de Fisiología Celular de la Universidad Nacional Autónoma de México. Esta instalación cumple con las características establecidas por la Norma Oficial Mexicana NOM-062-ZOO-1999: Especificaciones Técnicas para la Producción, Cuidado y Uso de Animales de Laboratorio. Adicionalmente, se siguió lo establecido en la Guía de Cuidados y Uso de Animales de Laboratorio avalada por los Institutos Nacionales de Salud de los Estados Unidos y la declaración de Helsinki. The experiment was conducted in the bioterium of the Institute of Cellular Physiology of the National Autonomous University of Mexico. This installation complies with the characteristics established by the Official Mexican Standard NOM-062-ZOO-1999: Technical Specifications for the Production, Care and Use of Laboratory Animals. Additionally, the provisions of the Laboratory Animal Care and Use Guide endorsed by the National Institutes of Health of the United States and the declaration of Helsinki were followed.

Claims

REIVINDICACIONES: CLAIMS:
1. Los péptidos sintéticos que consisten de las secuencias identificadas como SEQ ID NO: 1 , SEQ ID NO: 2 y SEQ ID NO: 3 derivados del C- terminal de la isoforma I de la proteína transferidora de ésteres de colesterol (CETPI). 1. Synthetic peptides consisting of the sequences identified as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 derived from the C-terminal of isoform I of cholesterol ester transfer protein (CETPI).
2. Uso de los péptidos sintéticos identificados como SEQ ID NO: 1 y SEQ ID NO: 2 de conformidad con la reivindicación 1 , para obtener mediante inmunización un anticuerpo policlonal Anti-CETPI. 2. Use of the synthetic peptides identified as SEQ ID NO: 1 and SEQ ID NO: 2 according to claim 1, to obtain a polyclonal Anti-CETPI antibody by immunization.
3. El péptido sintético identificado como SEQ ID NO: 1 de la reivindicación 2, caracterizado porque está fusionado en su extremo amino a la proteína acarreadora KLH. 3. The synthetic peptide identified as SEQ ID NO: 1 of claim 2, characterized in that it is fused at its amino terminus to the KLH carrier protein.
4. El anticuerpo policlonal de la reivindicación 2, caracterizado porque reconoce la región que comprende los aminoácidos A481-P491 de la isoforma de CETP denominada CETPI. 4. The polyclonal antibody of claim 2, characterized in that it recognizes the region comprising amino acids A481-P491 of the CETP isoform called CETPI.
5. El anticuerpo policlonal de la reivindicación 2, en donde dicho anticuerpo es de tipo IgY. 5. The polyclonal antibody of claim 2, wherein said antibody is of the IgY type.
6. El anticuerpo policlonal de la reivindicación 2, caracterizado porque, reconoce el dominio que comprende los aminoácidos V474-P491 de CETPI. 6. The polyclonal antibody of claim 2, characterized in that it recognizes the domain comprising amino acids V474-P491 of CETPI.
7. Uso del anticuerpo policlonal Anti-CETPI de las reivindicaciones 3 a 6, para acoplarlo a un inmunoensayo tipo ELISA o Western Blot para detección en muestras de mamíferos de la isoforma I de CETP denominada CETPI. 7. Use of the Anti-CETPI polyclonal antibody of claims 3 to 6, to couple it to an ELISA or Western Blot type immunoassay for detection in mammalian samples of CETP isoform I called CETPI.
8. El inmunoensayo de la reivindicación 7, caracterizado porque la detección de la isoforma I de CETP permite seguir la evolución de la neutralización de los efectos citotóxicos producidos por lipopolisacáridos en el choque séptico cuando se administra la SEQ ID NO 3. 8. The immunoassay of claim 7, characterized in that the detection of CETP isoform I allows the evolution of neutralization to be monitored of the cytotoxic effects produced by lipopolysaccharides in septic shock when SEQ ID NO 3 is administered.
9. El inmunoensayo de la reivindicación 7, caracterizado porque usa como estándar los péptidos de conformidad con la reivindicación 1. 9. The immunoassay of claim 7, characterized in that it uses as standard the peptides according to claim 1.
10. El inmunoensayo de la reivindicación 7, donde la muestra de mamífero es plasma. 10. The immunoassay of claim 7, wherein the mammalian sample is plasma.
11. Uso del péptido sintético identificado como SEQ ID NO. 3 de conformidad con la reivindicación 1 , para prevenir y neutralizar los efectos citotóxicos inducidos por los polisacáridos en un mamífero. 11. Use of the synthetic peptide identified as SEQ ID NO. 3 according to claim 1, to prevent and neutralize the cytotoxic effects induced by polysaccharides in a mammal.
12. El péptido sintético de la reivindicación 11 para formular un medicamento para prevenir y neutralizar los efectos citotóxicos inducidos por lipopolisacáridos durante el choque séptico en un mamífero. 12. The synthetic peptide of claim 11 for formulating a medicament for preventing and neutralizing the cytotoxic effects induced by lipopolysaccharides during septic shock in a mammal.
13. Un kit de tratamiento y seguimiento de los efectos secundarios del choque séptico producidos por lipopolisacáridos que consiste de las secuencias identificadas como— j, SEQ ID NO: 3 y el anticuerpo policlonal Anti CETPI que comprende los aminoácidos A481 -P491 de la ¡soforma de CETP denominada CETPI. 13. A kit for treating and monitoring the side effects of septic shock produced by lipopolysaccharides consisting of the sequences identified as— j, SEQ ID NO: 3 and the polyclonal Anti CETPI antibody comprising amino acids A481-P491 of the soforma of CETP called CETPI.
14. El kit de la reivindicación 13, que consiste de un inmunoensayo para detectar en muestras de mamífero los niveles de CETPI. 14. The kit of claim 13, which consists of an immunoassay for detecting CETPI levels in mammalian samples.
15. El kit de la reivindicación 14, donde el inmunoensayo es del tipo ELISA o Wstern Blot. 15. The kit of claim 14, wherein the immunoassay is of the ELISA or Wstern Blot type.
16. El kit de la reivindicación 14 donde las muestras de mamífero son plasma. 16. The kit of claim 14 wherein the mammalian samples are plasma.
17. El kit de la reivindicación 14 donde el los niveles de CETPI detectados permiten el seguimiento de la neutralización de los efectos tóxicos de los lipopolisacáridos en el choque séptico. 17. The kit of claim 14 wherein the CETPI levels detected allow the neutralization of the toxic effects of lipopolysaccharides in septic shock to be monitored.
18. El kit de la reivindicación 13, caracterizado por que la SEQ ID NO: 3 se usa para prevenir y neutralizar los efectos citotóxicos inducidos por los polisacáridos durante el choque séptico en un mamífero. 18. The kit of claim 13, characterized in that SEQ ID NO: 3 is used to prevent and neutralize the cytotoxic effects induced by polysaccharides during septic shock in a mammal.
19. El kit de la reivindicación 13, caracterizado porque la SEQ ID NO: 3 se encuentra formulada como un medicamento. 19. The kit of claim 13, characterized in that SEQ ID NO: 3 is formulated as a medicament.
PCT/MX2014/000087 2014-06-11 2014-06-11 Peptides derived from the c-terminal domain of cetpi as molecules blocking the cytotoxic effect induced by lipopolysaccharides in septicaemia and septic shock WO2015190903A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/MX2014/000087 WO2015190903A1 (en) 2014-06-11 2014-06-11 Peptides derived from the c-terminal domain of cetpi as molecules blocking the cytotoxic effect induced by lipopolysaccharides in septicaemia and septic shock

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/MX2014/000087 WO2015190903A1 (en) 2014-06-11 2014-06-11 Peptides derived from the c-terminal domain of cetpi as molecules blocking the cytotoxic effect induced by lipopolysaccharides in septicaemia and septic shock

Publications (1)

Publication Number Publication Date
WO2015190903A1 true WO2015190903A1 (en) 2015-12-17

Family

ID=54833901

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/MX2014/000087 WO2015190903A1 (en) 2014-06-11 2014-06-11 Peptides derived from the c-terminal domain of cetpi as molecules blocking the cytotoxic effect induced by lipopolysaccharides in septicaemia and septic shock

Country Status (1)

Country Link
WO (1) WO2015190903A1 (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995005393A2 (en) * 1993-08-18 1995-02-23 Morphosys Gesellschaft Für Proteinoptimierung Mbh Lipopolysaccharide-binding and neutralizing peptides
ES2292493T3 (en) * 1999-12-13 2008-03-16 Universidad Nacional Autonoma De Mexico IMMUNOENZYMATIC QUANTIFICATION METHOD.

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995005393A2 (en) * 1993-08-18 1995-02-23 Morphosys Gesellschaft Für Proteinoptimierung Mbh Lipopolysaccharide-binding and neutralizing peptides
ES2292493T3 (en) * 1999-12-13 2008-03-16 Universidad Nacional Autonoma De Mexico IMMUNOENZYMATIC QUANTIFICATION METHOD.

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ALONSO AL. ET AL.: "Characterization of a naturally occurring new version of the cholesterol ester transfer protein (CETP) from small intestine.", MOLECULAR AND CELLULAR BIOCHEMISTRY, vol. 245, 2003, pages 173 - 182 *
GUTSMANN T. ET AL.: "New Antiseptic Peptides To Protect against Endotoxin-Mediated Shock.", ANTIMICROBIAL AGENTS AND CHEMOTHERAPY., vol. 54, no. 9, 2010, pages 3817 - 3824, XP055242250 *

Similar Documents

Publication Publication Date Title
ES2306195T3 (en) THERAPEUTIC AGENTS FOR THE TREATMENT OF PATHOLOGIES RELATED TO HMGB1.
Lamkanfi et al. Nlrp3: an immune sensor of cellular stress and infection
Johansson et al. The gastrointestinal mucus system in health and disease
Caution et al. Caspase-11 mediates neutrophil chemotaxis and extracellular trap formation during acute gouty arthritis through alteration of cofilin phosphorylation
Arizmendi et al. Mucosal allergic sensitization to cockroach allergens is dependent on proteinase activity and proteinase-activated receptor-2 activation
ES2848066T3 (en) Acinetobacter baumannii vaccine based on lipopolysaccharide deficient cellular components
ES2738287T3 (en) Inhibitor of the formation of extracellular traps in leukocytes
ES2365639T3 (en) ECTOINE FOR THE TREATMENT OF VASCULAR LEAKS.
ES2658966T3 (en) Leucolectins and their uses
EA024253B1 (en) Lytic polypeptide with peptidoglycan hydrolase activity and use thereof against gram-negative bacterial infections
ES2875498T3 (en) Synthetic Peptide Compounds and Procedures for Use
CN102516357A (en) Polypeptide combined with A-beta amyloid and application thereof
WO2005061532A1 (en) Pathogenic infection detection compositions and methods
ES2546310T3 (en) Human leukolectins and their uses
Wang et al. The antimicrobial peptide MPX kills Actinobacillus pleuropneumoniae and reduces its pathogenicity in mice
Ahmed et al. Mycobacterium tuberculosis PPE18 protein reduces inflammation and increases survival in animal model of sepsis
Suckling et al. Synthetic analogues of the parasitic worm product ES-62 reduce disease development in in vivo models of lung fibrosis
Magnadottir et al. A novel ladder-like lectin relates to sites of mucosal immunity in Atlantic halibut (Hippoglossus hippoglossus L.)
Shen et al. TREM1 blockade ameliorates lipopolysaccharide-induced acute intestinal dysfunction through inhibiting intestinal apoptosis and inflammation response
Cai et al. Membrane-active nonivamide derivatives as effective broad-spectrum antimicrobials: Rational design, synthesis, and biological evaluation
Katsargyris et al. Toll-like receptor modulation: a novel therapeutic strategy in cardiovascular disease?
WO2015190903A1 (en) Peptides derived from the c-terminal domain of cetpi as molecules blocking the cytotoxic effect induced by lipopolysaccharides in septicaemia and septic shock
ES2618402T3 (en) Modified peptides and their use to treat autoimmune diseases
ES2618881T3 (en) Collagen V for use in the treatment of asthma
Zhao et al. iTRAQ proteome analysis of insight into TLR20. 2 functions through IFN1 signaling exerts regulatory effects on GCRV replication

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 14894658

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 14894658

Country of ref document: EP

Kind code of ref document: A1