WO2017011890A1

WO2017011890A1 - Pharmaceutical compositions including a peptide capable of preventing or treating platelet aggregation disorders

Info

Publication number: WO2017011890A1
Application number: PCT/BR2016/050170
Authority: WO
Inventors: Antonio Marcus DE ANDRADE PAES; Adriana LEANDRO CAMARA; Elyjany MORAIS LIMA SENA; Samira ABDALLA DA SILVA; Hiran REIS SOUSA; João Lucas LIMA FONTELLES; Renato SIMÕES GASPAR; Francisco Rafael MARTINS LAURINDO; Andres Ezequiel TROSTCHANSKY VASCONCELLOS
Original assignee: Universidade Federal Do Maranhão
Priority date: 2015-07-23
Filing date: 2016-07-22
Publication date: 2017-01-26
Also published as: BR102015018076A2

Abstract

This invention relates to pharmaceutical compositions containing the peptide sequence of CxxC peptide designed based on the linear sequence of the dithiol active sites of PDI (protein disulfide isomerase) or structures derived therefrom, while maintaining the same active chemical group. Therefore, these pharmaceutical compositions contain a peptide capable of preventing or treating diseases or conditions associated with the pathological increase of platelet aggregation.

Description

"PHARMACEUTICAL COMPOSITIONS INCLUDING A PEPTIDE CAPABLE OF PREVENTING OR TREATING PLATELET AGGREGATION DISORDERS"

I) TECHNICAL FIELD OR STATE OF THE ART

[001 ] This invention relates to pharmaceutical compositions containing the CxxC Peptide (VEFYAPWCGHCK) or structures derived therefrom, which are capable of preventing or treating diseases or conditions associated with the pathological increase of platelet aggregation.

II) BACKGROUND OF THE INVENTION

[002] Thromboembolic disorders are one of the most common causes of morbidity and mortality around the world (Kasper, L. D et. al. Harrison's McGraw-Hill, 17^th ed. 2008). In Brazil, between January and November 2013, the number of hospital admissions for pulmonary embolism, embolism and arterial thrombosis, phlebitis thrombophlebitis, embolism and venous thrombosis was 59,938, at a total cost of R$ 61 ,683,409.54. In a broader analysis, circulatory system diseases represent 7.28% of the causes of death, one of the highest rates of mortality in the country, with 2.38% being directly caused by thromboembolic disorders (Ministry of Health - Hospital Information Systems of SUS - SIH/SUS, DAT ASUS, Brazil, 2010. Available at www.saude.gov.brhttp://tabnet.datasus. gov.br/cgi/tabcgi. exe?sih/cnv/niuf.def.

[003] Thromboembolism consists of the acute obstruction of venous or arterial circulation caused by blood clots (ALVARES F.; PADUA A.; TERRA FILHO J. Medicina, April/December, v. 36, p. 214-240, 2003). According to the affected site, thromboembolic arterial obstruction can be clinically expressed by acute myocardial infarction, angina pectoris, ischemic stroke or peripheral arterial obstruction (BIZZACCHI, J.M.A. In: Zago, M.A. (Org.). Hematologia: Fundamentos e Pratica. Sao Paulo: Atheneu, p. 879-88, 2004). The venous thrombus has a higher composition of fibrin and red blood cells, and the arterial thrombus has a higher composition of platelets. These aspects have therapeutic implications, since the fibrinolytic agents constitute the therapy of choice in venous thrombosis, while the blood platelet antiaggregants are best used in arterial processes. Platelets have a major role in arterial thrombus formation, because they bind to the injured endothelium regardless of blood flow and trigger the whole process of local aggregation and consequent thrombus formation (MORELLI, V.M. In: Zago, M.A. A., Falcao, R. P., Pasquini, R. Hematologia: Fundamentos e Pratica. Sao Paulo: Atheneu, p.731 -38, 2004).

[004] The major receptors involved in the initial activation of platelets include the thrombin receptors, PARI and PAR4, the GPVI collagen receptor and the ADP, P2Y1 and P2Y12 receptors, as well as the thromboxane A2 receptors (ESSEX, D.W. Antioxid Redox Signal, v.1 1 , n. 5, p. 1 191 -225, 2009). Activation results in conformational change of GP allbp3 membrane integrin. This conformational change is a response to intracellular events involving the spreading and conformational change of integrin cytoplasmic domain, leading the exposure of the active site for binding to plasma-soluble fibrinogen (SHATTIL, S. J.; NEWMAN P. J. Blood, v. 104, p. 1606-15, 2004).

[005] Several studies have demonstrated that part of this process of stimulating the platelet responses, including secretion and aggregation, has been attributed to rearrangements involving thiol and disulfide groups exposed on platelet membrane (ESSEX, D. W. Antioxid Redox Signal, v.1 1 , n. 5, p. 1 191 -225, 2009; ESSEX, D.W. Antioxid Redox Signal, v. 6, p. 736^16, 2004; ESSEX D.W., et al. Blood, v. 86, p. 2168-73, 1995). The αΙΙ β3, α2β1 , GPIb, P2Y12, and GPVI receptors have free thiol groups and are, therefore, potential sites for redox regulation performed by thiol proteins. Such group includes proteins with disulfide reductase activity, such as thioredoxin, glutaredoxin and disulfide isomerase protein - (PDI) (ESSEX, D.W. Antioxid Redox Signal, v. 6, p. 736-46, 2004).

[006] PAES, A. M. A. et al., J. of Leukocyte Biology, v. 90, p. 799-810, 201 1 , have observed that the PDI activity both in vitro and in vivo can be inhibited by the use of peptides whose sequence mimics the catalytic site of the protein. They have shown that these peptides are able to inhibit the reductase activity of PDI, thus contributing to decrease the production of reactive oxygen species by NADPH oxidase from human neutrophils. Data from literature have shown that platelet aggregation can be inhibited with the use of thiol alkylating agents or PDI inhibitors (thus, these peptides could also inhibit platelet aggregation. DTNB- 5,5'-dithiobis- (2-nitrobenzoic acid) and anti-PDI antibodies, respectively).

Ill) DETAILED DESCRIPTION OF THE INVENTION

[007] This invention relates to pharmaceutical compositions containing the peptide sequence of CxxC peptide (VEFYAPWCGHCK) designed based on the linear sequence of the dithiol active sites of PDI (protein disulfide isomerase) or structures derived therefrom, while maintaining the same active chemical group. CxxC peptide (VEFYAPWCGHCK) contains exactly the CGHC redox motif of PDI, as the chemical group responsible for the biological activity.

[008] This invention further relates to pharmaceutical compositions containing associations of CGHC peptide (VEFYAPWCGHCK) with various pharmaceutically acceptable excipients or inert vehicles.

[009] Among the pharmaceutical compositions, according to the invention, it may be particularly mentioned those that are more convenient for oral, parenteral, nasal administration, simple tablets or dragees,, sublingual tablets and capsules.

[010] The dosage is adaptable according to the nature and severity of the disease, the route of administration and the weight and age of the patient.

[01 1 ] These pharmaceutical compositions can have varying concentrations of peptide CxxC (VEFYAPWCGHCK), since the achieved plasma concentration, that is, the bioavailable concentration, reaches levels between 1 and 100 μΜ.

CHARACTERIZATION OF ASSOCIATION BETWEEN CXXC PEPTIDES AND FREE THIOLS EXPOSED ON THE PLATELET SURFACE

[012] In order to examine the possible association of the CxxC peptide with PDI thiols and other thiol proteins exposed on the platelet surface, resting platelets, pre- incubated or not with the peptide, were labeled with MPB, which is a thiol-specific membrane-impermeable biotinylated reagent. As a result, we verified that CxxC peptide did not modify the labeling of total free thiols exposed on the surface of the platelet membrane. However, when the specific PDI labeling is analyzed, its reduction is observed. This fact is indicative that the CxxC peptide specifically binds to PDI and it is not a nonspecific ligand of free thiols exposed on the platelet surface.

[013] Burgess, J.K., et al., J BiolChem, 2000.275(13): p. 9758-66, has found that the activation of platelet aggregation with thrombin increased the labeling of free thiols with MPB to 460%. However, when the platelets were prevented from aggregating, this increase was only 60%. More recently, it was demonstrated that successive thiol-disulfide exchanges between critical cysteines, more than the disulfide reduction itself, comprise a key event in the platelet activation-aggregation, in which PDI is an indispensable component (ESSEX, D.W. Antioxid Redox Signal, v.1 1 , n. 5, p. 1 191 -225, 2009; ESSEX, D.W. Antioxid Redox Signal, v. 6, p. 736^16, 2004).

[014] Predictive computational analysis of molecular interaction between peptides and PDI has shown a close association of CxxC and Scr peptides with PDI through the formation of a disulfide bridge. For this reason, it was further investigated the possibility of a specific binding of this peptide with PDI by precipitation of proteins labelled with MPB. The results showed that the reduction of disulfides with TCEP (1 imM) has doubled the PDI labeling. This data is supported by already described evidences that the active sites of the domains a and a' coexist in the reduced and oxidized forms (Appenzeller-Herzog, C. and L.EIIgaard. Antioxid Redox Signal, 2008. 10(1 ): p. 55-64. In addition, reduction of surface thiols did not interfere in the content of intracellular PDI, analyzed in the supernatant obtained after lysis of platelets and precipitation of membrane PDI labeled with MPB.

[015] When the platelets were incubated with the peptides, it was observed that the CxxC peptide (25 μΜ) promoted a slight, but important labeling reduction of PDI free thiols with MPB, resulting in less protein precipitation. These data reinforce the possibility of formation of a disulfide bridge between CxxC peptide and PDI, maybe by a mechanism similar to that described for bacitracin. Although the mechanism of PDI inhibition by bacitracin is under discussion, it has been shown that it acts via the formation of disulfide bridges with Cys314 and Cys345, located in domain b' and the binding region x, respectively. In addition, more hydrophobic types of bacitracin, such as bacitracin F and H, promote greater inhibitory effect of reductase activity of PDI (Dickerhof, N., et al., FEBS J, 201 1 . 278(12): p. 2034-43). In this context, it is remarkable that our data show that the CxxC peptide binds to the PDI via hydrophobic interactions and also inhibits the reductase activity of this enzyme (de, A. P. A.M., et al., J LeukocBiol, 201 1 . 90(4): p. 799-810).

EVALUATION OF THE EFFECT OF CXXC PEPTIDES ON ADP-INDUCED PLATELET AGGREGATION.

[016] In face of the recent findings that short PDI inhibitory peptides and phenolic molecules are potential antithrombotic agents [16], we tested the ability of CxxC peptide to inhibit ADP-induced platelet aggregation (5 μΜ) in a platelet-rich plasma (PRP). The choice of ADP as an agonist relies on the fact of being an agent of great physiological relevance, whose biphasic aggregation response pattern is fully characterized in the PRP. The first stage occurs through the binding of the agonist to its receptor, leading to the activation of allbp3. The second occurs through the binding of allbp3 to fibrinogen, promoting a more stable and irreversible aggregation [8, 10]. 45

[017] Initially, the role of redox state modulators of thiol proteins and the surface PDI itself on platelet aggregation was characterized. The addition of 3 imM TCEP and DTT reducing agents promoted a slow and steady platelet aggregation, with maximum effect of 50% of the ADP-induced aggregation. The similarity of aggregation profiles showed that the permeability of DTT to the membrane did not promote an additional effect on platelet aggregation induced by disulfide reduction of platelet surface by TCEP. On the other hand, the aggregating activity of ADP was drastically reduced by the addition of DTNB (2.5 imM), a thiol alkylating agent, or totally eliminated by PAO (30 μΜ), a vicinal thiol blocker. Vicinal thiols means the thiol groups that are close enough to carry out thiol-disulfide exchanges, such as those performed by the CGHC active site of PDI. The addition of both bacitracin (5 mM) and the RL90 anti-PDI antibody (1 :250) resulted in the reduction of about 50% of the maximum ADP-induced aggregation. These data are supported by other authors that showed that the RL90 antibody only partially inhibits platelet aggregation induced by this agonist. Furthermore, it has been recently shown that the RL90 antibody inhibits only the reductase activity of PDI, undermining the isomerase activity without interfering with the oxidase activity.

[018] When the ability of peptides to interfere with ADP-induced platelet aggregation was analyzed, it was found that the CxxC peptide reduced the maximum aggregate in 14%, 27% and 30% at concentrations of 3, 10 and 30 μΜ, respectively. Higher concentrations resulted in additional reduction.

[019] This data set herein presented suggests that the CxxC peptide binds to the surface PDI and partially inhibits the platelet aggregation via mechanisms mediated by thiol-disulfide exchanges. However, there are further analyses to be carried out in future studies. Especially, there is a need to assess the possibility of direct interaction of CxxC peptide with critical thiols of allbp3. In addition, it must be carried out molecular docking studies of peptides with the PDI oxidized form, which is predominant in resting platelets. In any case, this work reinforces the potential use of CxxC peptide, or similar molecules, as platelet antiaggregant agent.

Claims

1. Pharmaceutical compositions containing the peptide sequence of CxxC peptide (VEFYAPWCGHCK), CHARACTERIZED in that the CxxC peptide (VEFYAPWCGHCK) contains the CGHC redox motif of PDI.

2. Pharmaceutical compositions containing the peptide sequence of CxxC peptide (VEFYAPWCGHCK), according to claim 1 , CHARACTERIZED in that it contains the peptide sequence CGHC (VEFYAPWCGHCK) in varying concentrations, being preferentially used in concentrations from 1 to 100 μΜ.

3. Pharmaceutical compositions containing the peptide sequence of CxxC peptide (VEFYAPWCGHCK), according to claim 2, CHARACTERIZED in that it contains associations of CGHC peptide (VEFYAPWCGHCK) with various pharmaceutically acceptable excipients or inert vehicles.

4. Pharmaceutical compositions containing the peptide sequence of CxxC peptide (VEFYAPWCGHCK), according to claim 3, CHARACTERIZED in that they are prepared for oral, parenteral, nasal administration, simple tablets or dragees, sublingual tablets and capsules.

5. Pharmaceutical compositions containing the peptide sequence of CxxC peptide (VEFYAPWCGHCK), according to claim 4, CHARACTERIZED in that they are capable of reducing platelet aggregation by inhibiting the isomerase ability of the protein disulfide isomerase (PDI) and its active isomers.

6. Pharmaceutical compositions containing the peptide sequence of CxxC peptide (VEFYAPWCGHCK), according to claim 4, CHARACTERIZED in that they are effective for treating cardiovascular diseases related to atherosclerosis, hypertension, diabetes and heart failure, and for preventing and treating thromboembolic disorders associated with atherosclerosis and invasive surgical procedures in cardiology and neurology.