WO2010116016A1

WO2010116016A1 - Use of peptides of the e1 protein of the hepatitis g virus in order to inhibit the activity of the hiv virus

Info

Publication number: WO2010116016A1
Application number: PCT/ES2010/070210
Authority: WO
Inventors: Isabel Haro Villar; María José GÓMARA ELENA
Original assignee: Consejo Superior De Investigaciones Científicas (Csic)
Priority date: 2009-04-06
Filing date: 2010-04-05
Publication date: 2010-10-14
Also published as: ES2351027B1; ES2351027A1

Abstract

The invention relates to the use of peptides of the E1 protein of the hepatitis G virus in order to inhibit the activity of the HIV virus. More specifically, the invention relates to the use of at least one peptide, or a derivative of same, contained in the sequence of the E1 protein of the hepatitis G virus (GBV-C/HGV), capable of inhibiting the activity of the HIV virus, impeding or repressing the function and/or activity of the HIV virus (human immunodeficiency virus), in a temporary or permanent manner, or additionally capable of interacting with an amino acid sequence of glycoprotein gp41 of the HIV virus, for the production of a pharmaceutical composition. Said peptide can impede the inhibition of the function of the fragment of protein gp41 of the HIV virus, impede cell fusion or impede the replication of HIV in a host cell. In addition, the invention relates to the aforementioned pharmaceutical composition and to the method for the production thereof.

Description

USE OF HEPATITIS G VIRUS PROTEIN E1 PEPTIDES TO INHIBIT HIV VIRUS ACTIVITY

The present invention relates to the use of at least one peptide, or derivative thereof, contained in the sequence of the E1 protein of the hepatitis G virus (GBV-C / HGV), capable of inhibiting the activity of the HIV virus, preventing or repressing the function and / or activity of the HIV virus (Human Immunodeficiency Virus), temporarily or permanently, or also capable of interacting with an amino acid sequence of the glycoprotein gp41 of the HIV virus, for the elaboration of a pharmaceutical composition. Said peptide is capable of preventing the inhibition of the function of the fragment of the gp41 protein of the HIV virus, preventing the cell fusion or preventing the replication of HIV in a host cell. Likewise, the present invention also refers to said pharmaceutical composition and the method for its manufacture.

STATE OF THE PREVIOUS TECHNIQUE

When a supposed new hepatitis virus was discovered in the mid-1990s, the GB virus C (GBV-C), also called hepatitis G virus (HGV) (Simons et al., 1997. Nature Medicine 1: 564 -569; Linnen et al., 1996 Science 271, 505-508), many research groups tried to correlate it with liver inflammation or other associated diseases. However, no influence on health could be identified (Mphahlele et al., 1998. Liver 18: 143-155; Theodore et al., 1997. Hepatoloqy 25: 1285-1286), until Prof. Tillmann discovered the Unexpected benefit of GBV-C / HGV coinfection in the course of the disease of patients who had been infected with HIV (Human Immunodeficiency Virus 1) (Heringlake eí a /., 1998. J. Infect. Dis. 177: 1723-1726).

Although in some occasions the results have not been clearly significant (Zhang et al., 2006. HIV Med. 7: 173-180), a lower progression of the disease and an increase in survival in GBV-C / HGV positive individuals compared to GBV-C / HGV negative individuals, while the loss of GBV-C / HGV viremia is closely related to increased mortality as well as a greater progression of AIDS (Van der Bij et al ., 2005. J. Infect. Dis. 191: 678-685; Williams et al., 2004. N. Enαl. J. Med. 350: 981-990).

The use of synthetic peptides as HIV-1 inhibitors has been the subject of research in recent years. They have been studied and are currently being, for their clinical application in the fight against HIV-1 infection, a large number of peptides that target the different stages of the life cycle of HIV-1.

HIV-1 inhibitor peptides have been identified and / or developed by different methods. Some therapeutic peptides such as Enfuvirtide, already approved for clinical use (Burton, 2003. Lancet Infec. Dis. 3, 260), are derived from HIV-1, while others are natural peptides such as chemokines, defensins or the "Inhibitory Virus" Peptide "(VIRIP) (Munch et al., 2007, CeII 129: 263-275) or have been designed and synthesized from crystallographic data of HIV-1 proteins or from peptide libraries (Boggiano et al., 2006 Biochem. Biophvs. Res. Common. 347: 909-915).

The HIV replicative cycle has been described in detail in numerous reviews (Greene and Peterling, 2002. Nat. Med. 8: 673-680; Freed and Mouland, 2006. Retrovirology 3: 77). The entry of the virus into the host cell has been described as a process that requires three stages: 1) approximation and binding of HIV to the CD4 molecules (primary receptor) through the viral glycoprotein gp120; 2) conformational change in gp120, which allows the chemokine coreceptor binding (CCR5 or CXCR4) and 3) fusion of the HIV envelope with the cell membrane through a conformational change in the membrane glycoprotein gp41, which has place only after the productive union of gp120 to CD4. Each of these stages represents a potential target in the inhibition of viral replication. Thus, the use of inhibitors of viral fusion or entry and / or virus replication would be useful, to prevent the action of the HIV virus in host cells, this fusion and / or entry phase being a suitable point for the action of inhibitors. , since the virus has not yet introduced its genetic material into the cellular interior.

EXPLANATION OF THE INVENTION

The present invention relates to the use of at least one peptide contained in the sequence of the GB1-C / HGV virus E1 protein, capable of inhibiting the activity of the HIV virus, preventing or repressing the function and / or activity of the HIV virus ( Human Immunodeficiency Virus), temporarily or permanently, or also capable of interacting with an amino acid sequence of the gp41 glycoprotein of the HIV virus (Human Immunodeficiency Virus 1), for the elaboration of a pharmaceutical composition. Said peptide is capable of inhibiting the function of the fragment of the gp41 protein of the HIV virus, preventing cell fusion or preventing the replication of HIV in a host cell.

In the examples of the present invention, 58 peptides composed of 18 amino acids are synthesized that encompass the complete sequence of the GB1-C / HGV virus E1 protein (SEQ ID NO: 1). Said sequences overlap in 15 amino acids, that is, a specific peptide has three different amino acids with respect to the peptide that precedes or follows.

Next, the interaction of any of the peptides of the sequence SEQ ID NO: 1 of the GBV-C / HGV virus with an amino acid sequence (SEQ ID NO: 2) of the gp41 membrane glycoprotein of the HIV-1 virus (Virus) is determined of Human Immunodeficiency 1) through the use of biophysical techniques that allow to determine the inhibition of vesicle contents induced by SEQ ID NO: 2, and assessing the potential character Inhibitory of HIV-1 by cellular assays directed to determine the inhibition of cell fusion or the inhibition of virus replication.

The main advantages provided by the synthetic peptides of the present invention are:

- Its low systemic toxicity.

- The possibility of making structural modifications in them and consequently the ability they present to mimic certain substrates or epitopes. - Its applicability in combination therapies or when resistance to other antiretroviral drugs appears, or

- That they can act before the virus enters the cell. In this sense, any of the peptides of the present invention could have the same potential as the induction of immunity provided by a vaccine.

Thus, one aspect of the present invention is the use of at least one peptide, or derivative thereof, contained in an amino acid sequence that has at least 60% identity with the sequence SEQ ID NO: 1, capable of inhibiting Ia HIV virus activity, for the elaboration of a pharmaceutical composition.

That is, the pharmaceutical composition of the present invention is used for the treatment or prevention of acquired immunodeficiency syndrome (AIDS) caused by any type of HIV, either of the HIV-1 type, of the HIV-2 type or any other type of HIV

Amino acid sequences that have at least 60% identity with SEQ ID NO: 1 are homologous sequences of different GBV-C / HGV virus genotypes. This percentage of identity has not been chosen arbitrarily but after obtaining the identity of the known genotypes of the GeneBank database and comparing them by means of a Blast analysis of the sequence SEQ ID NO: 1. In this comparative analysis it has been found one sequence corresponding to a fragment of a polyprotein of the variant Troglodites of the GBV-C / HGV virus. The complete sequence (of 2942 amino acids) can be found in the accession number AAD31543 of the GeneBank. In this comparative analysis, it was possible to determine an identity of 66% of the fragment of the GBV-C / HGV Troglodites virus with respect to SEQ ID NO: 1.

The term "derivative thereof", as understood in the present invention refers to the peptide resulting from variations in the amino acid composition of its sequence so that a peptide having its origin in the original sequence is obtained without losing the functional characteristic which characterizes it in the present invention. By way of example, a peptide derived from any of the peptides that come from a sequence that has at least 60% identity with the sequence SEQ ID NO: 1, can have different amino acids in the same position of the sequence provided that those Changes do not affect the efficacy in the function defined for them in the present invention and taking into account that said derived peptides have at least 60% identity with the sequence SEQ ID NO: 1. The term "inhibition of the activity of the HIV virus "as understood in the present invention refers to preventing or suppressing the function and / or activity of the HIV virus, temporarily or permanently.

SEQ ID NO: 1 is an amino acid sequence of the envelope protein E1 of the hepatitis G virus (GBV-C / HGV). The peptide, contained in the sequence SEQ ID NO: 1, is capable of inhibiting the activity of the HIV virus by acting in one or several stages of the infection process of said virus.

A preferred embodiment refers to the use of at least one peptide, or derivative thereof, contained in an amino acid sequence that has at least 80% identity with the sequence SEQ ID NO: 1 capable of inhibiting the activity of the HIV virus, to Ia elaboration of a pharmaceutical composition. According to a more preferred embodiment, the peptide, or derivative thereof is contained in Ia amino acid sequence SEQ ID NO: 1.

Another preferred embodiment refers to the use of at least one peptide, wherein said peptide is also capable of interacting with a fragment of the HIV virus gp41 protein.

According to another more preferred embodiment, the HIV virus is the HIV-1 virus. Another more preferred embodiment refers to the use of at least one peptide, where the fragment of the gp41 protein is the sequence SEQ ID NO: 2 of the HIV-1 virus.

SEQ ID NO: 2 is the amino acid sequence comprised between amino acids 1 and 23 Ia membrane glycoprotein gp41 of HIV-1 virus (HIV - 1) (N ⁰ ACJ04095 GeneBank access). The envelope of the HIV-1 virus is composed of spicules, which are protein complexes integrated in the membrane. Each spicule is formed by the gp41 protein, integral in the membrane, and an external head formed by the gp120 protein, essential for the coupling with the outside of certain cells prior to its invasion. One possibility to inhibit the binding of the virus to healthy cells, such as, but not limited to, lymphocytes is to act against the gp41 protein.

The interaction of any of the peptides with the sequence SEQ ID NO: 2, as understood in the present invention, refers to the action that is reciprocally exerted between said sequences. Thus, for example, but not limited to, the interaction refers to any type of chemical bond, Van der Waals forces or hydrophobic interactions.

To refer to the pharmaceutical composition, the term "medicament" can also be used. The pharmaceutical composition comprises any peptide contained in the sequence SEQ ID NO: 1, as described in previous paragraphs. Said composition may comprise one or more peptides as well as any combination of said peptides. Any of the Peptides of the present invention, are formulated in an appropriate pharmaceutical composition, in the therapeutically effective amount, together with one or more pharmaceutically acceptable carriers, adjuvants or excipients.

Hereinafter, reference may be made to any of the peptides defined in the preceding paragraphs as "peptide / s of the present invention" or "peptide / s of the invention".

The peptides of the present invention can be linear or cyclic, can incorporate non-natural residues such as D-amino acids and, also, can be modified by conjugation with fatty acids. In addition, the peptides of the present invention can be part of a system to generate anti-peptide antibodies such as, but not limited to, MAPS (Multiple

Antigen Peptide System). These different strategies of peptide presentation allow to increase the antiviral activity of synthetic peptides and, in addition, can improve the stability and selectivity of this type of molecules in their clinical application.

A preferred embodiment refers to the use where the peptide of the invention has between 15 and 20 amino acids. The order occupied by each amino acid of any of the peptides of the invention is the same as the order it occupies in the sequence SEQ ID NO: 1, in this way, the peptides of the present invention are partial sequences of the sequence SEQ ID NO : 1 having an extension of between 15 and 20 amino acids or sequences derived from the above that incorporate a substituted amino acid.

Another preferred embodiment refers to the use of the pharmaceutical composition according to any of claims 1 to 6, wherein the inhibition of the activity of the HIV virus is produced by preventing the entry of genetic material of said virus into the host cell.

The term "entry of the genetic material of the virus" refers to the entry of viral RNA protected or not by capsid and / or nucleocapsid as well as the entry of viral proteins that are a consequence of the translation of some viral RNA sequences into the virus itself.

The possible host cells into which the genetic material of the virus could enter are selected, but not limited to, from the list comprising, CD4 + T lymphocytes, monocytes, macrophages, dendritic cells, Langerhans cells or microglia cells of the brain.

According to a more preferred embodiment, the entry of the genetic material into the host cell is prevented by the inhibition of cell fusion. Another even more preferred embodiment refers to the use of the pharmaceutical composition, where the inhibition of cell fusion is carried out by means of a pharmaceutical composition comprising at least one peptide contained in any of the amino acid fragments SEQ ID NO: 3 or SEQ ID NO: 4.

The term "inhibition of cell fusion" as understood in the present invention refers to preventing or permanently or temporarily suppressing the fusion of the envelope of the HIV virus with the membrane of the healthy cell (host cell to which it is binds the HIV virus). At least in some cases, this inhibition is probably based on a conformational change in the gp41 membrane glycoprotein of the HIV-1 virus that prevents fusion with the host cell.

The evaluation of the degree of inhibition of cell fusion is carried out by means of the observation of syncytiums under a microscope. Syncytium formation is the cytopathic effect of HIV in some cell lines and consists of the formation of cellular accumulations (due to the accumulation of various nuclei) that lose the ability to separate the membrane from the infected cell after mitosis occurs, that is, , The cell loses the ability to divide. Another even more preferred embodiment refers to the use of the pharmaceutical composition, wherein the peptide, or any combination thereof, is selected from the list comprising SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16. The list of peptides presented belongs to the fragments SEQ ID NO: 3 and SEQ ID NO: 4 in the manner presented in Table 1.

Table 1. Correspondence of the peptides involved in the inhibition of cell fusion, with the fragments of SEQ ID NO: 1.

Another preferred embodiment refers to the use of the pharmaceutical composition where the inhibition of the activity of the HIV virus is produced by inhibiting the function of a fragment of the gp41 protein of the HIV virus. Preferably the fragment of the gp41 protein is SEQ ID NO: 2. The fragment of the gp41 protein is an N-terminal fusion peptide of the HIV virus. The function of the cell membrane fusion peptide causes the virus envelope to adhere and penetrate the virus or its genetic material into the cytoplasm of the host cell. That is, the inhibition of the function of the fragment of the gp41 protein of the HIV virus can be experienced by the inhibition of the release of vesicular contents. A more preferred embodiment refers to the use of the pharmaceutical composition, where the inhibition of the function of the fragment of the gp41 protein of the HIV-1 virus is carried out by means of a pharmaceutical composition comprising at least one peptide contained in the fragment amino acid SEQ ID NO: 3.

The term "inhibition of the release of vesicular contents" as understood in the present invention refers to permanently or temporarily preventing or suppressing the lysis of lipid vesicles.

The sequence SEQ ID NO: 3 is an amino acid fragment that corresponds to a partial sequence of SEQ ID NO: 1. The fragment is composed of 69 amino acids. Thus, by way of example, the fragment may contain 55 peptides of 15 amino acids or 50 peptides of 20 amino acids in addition to any peptide derived therefrom.

An even more preferred embodiment refers to the use of the pharmaceutical composition, wherein the peptide, or any combination thereof, is selected from the list comprising SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 11. The above list of peptides belongs to the fragment SEQ ID NO: 3.

The pharmaceutical composition of the present invention could be used in the inhibition of the replication of the HIV-1 virus and the efficacy of said inhibition can be assessed by means of the measurement of the concentration of the p24 antigen of the HIV-1 virus. The detection of the p24 protein is a direct marker of the presence of the virus in the organism.

The term "inhibition of replication" as understood in the present invention refers to permanently or temporarily preventing or suppressing the generation of new genetic material from the genetic material of the virus.

HIV-1, new viruses or virions. The term "virion" refers to the viral particle Infective composed of at least viral nucleic acid and / or viral proteins. Viral proteins are structural proteins such as those that form the outer shell or capsid, or they can also be another type of enzymatic proteins (for example POL, VIF, VPR, TAT, etc.). Virions are released into the extracellular environment.

Another aspect of the present invention relates to a pharmaceutical composition comprising at least one peptide, or derivative thereof, contained in an amino acid sequence that has at least 60% identity with the sequence SEQ ID NO: 1, capable of inhibiting The activity of the HIV virus.

A preferred embodiment refers to the pharmaceutical composition where the amino acid sequence has at least 80% identity with SEQ ID NO: 1. According to another more preferred embodiment, the amino acid sequence is SEQ ID NO: 1.

Another preferred embodiment refers to the pharmaceutical composition, where at least one peptide, or derivative thereof, capable of inhibiting the activity of the HIV virus, is also capable of interacting with a fragment of the gp41 protein of the HIV virus. According to another more preferred embodiment, the fragment of the gp41 protein is the sequence SEQ ID NO: 2 of the HIV-1 virus.

Another preferred embodiment relates to the pharmaceutical composition, where the peptide has between 15 and 20 amino acids.

Another preferred embodiment refers to the pharmaceutical composition which further comprises pharmaceutically acceptable excipients.

The term "excipient" refers to a substance that helps the absorption of any of the peptides of the present invention, stabilizes said peptides or helps the preparation of the pharmaceutical composition in the sense of giving consistency or providing any other desirable characteristic for such preparation. Thus, the excipients could have the function of keeping the ingredients together, such as starches, sugars or cellulose, sweetening function, coloring function, protection function of the medicine such as to isolate it from air and / or moisture, function filling a tablet, capsule or any other form of presentation such as dibasic calcium phosphate, a disintegrating function to facilitate the dissolution of the components and their absorption in the intestine, without excluding other types of excipients not mentioned in this paragraph.

The term "pharmaceutically acceptable" excipient refers to the excipient being allowed and evaluated so as not to cause damage to the organisms to which it is administered.

In addition, the excipient must be pharmaceutically suitable, that is, an excipient that allows the activity of the active principle or of the active principles, that is, that it is compatible with the active principle, in this case, the active principle is any of the peptides of the present invention.

Another preferred embodiment relates to a pharmaceutical composition that further comprises at least one pharmaceutically acceptable carrier.

The vehicle, like the excipient, is a substance that is used in the pharmaceutical composition to dilute any of the compounds of the present invention to a certain volume or weight. The pharmaceutically acceptable carrier is an inert substance or action analogous to any of the compounds of the present invention. The function of the vehicle is to facilitate the incorporation of other compounds, allow a better dosage and administration or give consistency and form to the medication. When the form of presentation is liquid, the pharmaceutically acceptable carrier is the diluent. Another preferred embodiment refers to the pharmaceutical composition which also comprises another active substance. This active substance must allow the activity of any of the peptides of the invention, that is, it must be compatible.

Hereinafter, any of the pharmaceutical compositions described as "pharmaceutical composition / s of the present invention" or "pharmaceutical composition / s of the invention" may be referred to.

Another aspect of the present invention relates to a method for manufacturing the pharmaceutical composition of the invention, which comprises:

a) Synthesize at least one peptide, or derivative thereof, and b) prepare the product obtained in section (a) in a form adapted to oral or parenteral administration.

As described in previous paragraphs, the peptide, or derivative thereof, is contained in an amino acid sequence that has at least 60% identity with the sequence SEQ ID NO: 1. Preferably, the peptide, or derivative thereof. , is contained in an amino acid sequence with at least 80% and even more preferably the peptide, or derivative thereof, is contained in the sequence SEQ ID NO: 1. Any of the peptides of the present invention is capable of inhibiting the activity of the HIV virus, preferably the virus is HIV-1 and in addition, it may be able to interact with a fragment of the gp41 protein of the HIV virus, preferably the fragment of the gp41 protein is the sequence SEQ ID NO: 2 of the HIV-1 virus .

The synthesis of any of the peptides of the present invention can be carried out by means of any technique known in the state of the art. The synthesis technique can, but is not limited to, a macromolecular synthesis technique, such as, but not limited to, solid phase peptide synthesis. Any of the peptides of Ia can also be obtained invention by recombinant DNA techniques, that is, by adapted techniques of molecular and biotechnological biology, in microorganisms or genetically modified plants.

The synthesis of any of the peptides of the present invention by recombinant DNA techniques is carried out by insertion into an expression vector of the nucleotide sequence encoding the amino acid sequence of any of the peptides of the present invention.

The host cell is transfected by means of techniques known in the state of the art, such as but not limited to, with electroporation, with biolistics, Agrobacterium tumefaciens or any other technique that allows the integration of any of the nucleic acids of the invention into the DNA of the host cell, either genomic, chloroplast or mitochondrial.

The expression of the nucleic acid in the cell of the invention gives rise to a peptide that can be purified by techniques known in the state of the art.

In each case the form of presentation of the pharmaceutical composition will be adapted to the type of administration used, therefore, the composition of the present invention can be presented in the form of solutions or any other form of clinically permitted administration and in a therapeutically effective amount. .

The form adapted to oral administration refers to a physical state that can allow oral administration. The form adapted to oral administration is selected from the list comprising, but not limited to, drops, syrup, tea, elixir, suspension, extemporaneous suspension, drinkable vial, tablet, capsule, granulate, seal, pill, tablet, tablet, tablet or lyophilized. The form adapted to parenteral administration refers to a physical state that can allow its injectable administration, that is, preferably in a liquid state. Parenteral administration can be carried out by intramuscular, intraarterial, intravenous, intradermal, subcutaneous or intraosseous administration but not limited to these types of parenteral administration routes. Any of the peptides of the invention can be associated, for example, but not limited to liposomes or micelles. A liposome is a spherical vesicle with a phospholipid membrane. The liposome contains a core of aqueous solution. The micelle is a spherical lipid that contains non-aqueous material. Both liposomes and micelles can be used as carriers of various substances between the exterior and interior of the cell.

On the other hand, any of the peptides of the present invention can be linked to one or more lipids. The lipid may be formed by saturated or unsaturated, linear aliphatic chains, or constituting one or more aromatic rings. In this case they are called lipopeptides. In addition, any of the peptides of the invention can be associated with particles such as gold particles.

Another possibility is that the pharmaceutical composition is present in a form adapted to the sublingual, nasal, intracatecal, bronchial, lymphatic, rectal, transdermal or inhaled administration. The form adapted to the rectal administration is selected from the list comprising, but not limited to, suppository, rectal capsule, rectal dispersion or rectal ointment. The form adapted to transdermal administration is selected from the list comprising, but not limited to, transdermal patch or iontophoresis.

Another preferred embodiment refers to the method where the peptide is selected from the list comprising SEQ ID NO: 5-16, that is, SEQ ID NO: 5, SEQ ID

NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16.

Throughout the description and the claims, the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For the person skilled in the art, other objects, advantages and characteristics of the invention will be derived partly from the description and partly from the practice of the invention. The following figures and examples are provided by way of illustration, and are not intended to be limiting of the present invention.

DESCRIPTION OF THE FIGURES

FIG. 1. It shows the peptides capable of inhibiting the release of vesicular contents induced by the fusion peptide SEQ ID NO: 2 of HIV-1.

In brackets the number of the sequence that has been assigned in the present invention to each of the peptides of the figure is shown: P7 (SEQ ID NO: 6), P8 (SEQ ID NO: 7), P10 (SEQ ID NO: 8), P18 (SEQ ID NO: 10), P22 (SEQ ID NO: 11).

EXAMPLES

Next, the invention will be illustrated by tests carried out by the inventors that describe the synthesis of the peptides of the present invention as well as the inhibition of certain activities of the HIV-1 virus. The following specific examples provided in this patent document serve to illustrate the nature of the present invention. These examples are included for illustrative purposes only and should not be construed as limitations to the invention claimed herein. Therefore, the examples described below illustrate the invention without limiting its scope of application. EXAMPLE 1. Materials and methods.

1.1. Peptide Synthesis

58 peptides of the envelope protein E1 of the GBV-C / HGV virus (SEQ ID NO: 1) have been obtained by Solid Phase Peptide Synthesis methodology in a semi-automatic peptide synthesizer (SAM, Multisyntech, Germany), in form of terminal carboxamides, using a Tentagel RAM resin (Rapp Polymere GmbH, Germany) (100 mg, 0.28 meq / g) and following an Fmoc / tBut strategy. The protection of amino acids has been carried out in the following way:

Triphenylmethyl (Trt) for glutamine, asparagine, histidine and cysteine; terbutyl (tBu) for aspartic acid, glutamic acid, serine, threonine and tyrosine; 2,2,5,7,8-pentametill-chroman-6-sulfonyl (Pmc) for arginine and tert-butyloxycarbonyl (Boc) for lysine and tryptophan.

The coupling reaction between amino acids was carried out in duplicate using threefold excesses of the Fmoc-amino acids. Amino acid activation was performed with diisopropylcarbodiimide (DIPCDI) and 1- hydroxybenzotriazole (HOBt) or with 2- (1 H-9-azobenzotriazol-1-yl) -1, 1, 3,3-tetramethylaminium (HATU) hexafluorophosphate in the presence of N-ethyldiisopropylamine (DIEA). Sequential removal of the Fmoc protecting group was carried out with 20% piperidine in dimethylformamide (DMF). The completion of these reactions was evaluated by colorimetric reaction (ninhydrin test). Once the synthesis was completed, the final stage of de-anchoring the resin peptide and deprotection of the functional groups was performed. For this, peptidyl resin was treated with 95% trifluoroacetic acid (TFA) in the presence of carbocation captors, essentially 2.5% water and 2.5% triisopropylsilane (TIS), for four hours. The final peptides were isolated by precipitation with diethyl ether, centrifuged and lyophilized in 10% acetic acid. The characterization of the peptides was carried out by analytical HPLC chromatography on a reverse phase column (Kromasil, C-18, 5 μm, 25x0.46 cm) using acetonitrile and water as eluents 0.05% in TFA. The identity of the peptides obtained with a degree of purity greater than 90% by analytical HPLC at a wavelength of 215 nm, was confirmed by MALDI-Tof mass spectrometry.

1.2. Study of the inhibition of HIV-1 by GBV-C / HGV virus peptides

The possible inhibitory role of peptide sequences related to the GBV-C / HGV virus, designed, synthesized, purified and conveniently characterized, has been proven by means of the following tests:

to. Vesicle content inhibition assay induced by HIV-1 FP, b. Inhibition assay of cell fusion.

a) Test of inhibition of vesicle content release induced by the sequence SEQ ID NO: 2 (amino acid fragment belonging to the sequence of the glycoprotein of gp41 membrane of HIV-1).

The biophysical vesicle content release assay described by Ellens has been performed (Ellens et al., 1984. Biochemistrv 23: 1532-1538). For this, unilamellar lipid vesicles (LUVs) containing the fluorescent probes have been prepared: sodium salt of 8-aminonaphthalene-1, 3,6-trisulfonic acid (ANTS) and p-xylene-bis (pyridinium) -bromide (DPX), according to the protocol described by our working group (Larios et al., 2005. Arch. Biochem. Biophvs. 442: 149-159). The different peptide constructs have been mixed with SEQ ID NO: 2 in DMSO prior to their addition to the suspension of LUVs liposomes, registering the emission of the ANTS probe at a wavelength of 520 nm. The possible lithic effect of each of the GBV constructions- C / HGV has been subtracted from the release observed in each case. The tests have been performed on a PTI QM4CW (Photon Technology International) spectrofluorimeter.

b) Test of inhibition of cell fusion.

This assay was carried out using two cell lines:

HeLa-env, which expresses the protein of the HIV-1 envelope and integrates into its genome the LTR promoter of HIV-1, and TZM-bl (AIDS reagents Cat. No 8129), which expresses the lymphocyte membrane receptor CD4 and CCR5 and CXCR4 co-receptors and integrates the luciferase and β-galactosidase genes into its genome. When the two cells fuse, the expression of the luciferase gene is activated, which produces the oxidation of the luciferin, forming an intermediate peroxide that degrades rapidly and gives rise to the oxyluciferin, which when passing from the excited state to the fundamental emits photons that they can be detected in a luminescence reader (indicative of the degree of cell fusion).

The cell fusion inhibition assay induced by GBV-C / HGV virus peptides consists of the incubation of approximately 2,500 cells

HeLa-env per well (Cultek plates Ref: 3912) for 1 hour with increasing concentrations of the peptides to be tested (in the present invention concentrations of between 100 and 1000 μM have been used), followed by the addition of about 10 times (approximately of about 25,000 cells) of TZM-bl and incubation for 24 hours. To control cell fusion wells were reserved in the absence of peptides, and a known inhibitor of cell fusion, C-34 (AIDS reagents Cat. No. 9824), was used as a positive control.

The qualitative assessment of the degree of inhibition of cell fusion was carried out by means of the observation of syncytium formation under a microscope.

Quantitative values have been obtained by reading the luminescence (kit commercial Britelite, Perkin Elmer Cat. No. 6016761) in a SpectraMax M5 microplate reader (Molecular Devices).

1.3. Study of the cell viability of GBV-C / HGV virus peptides

The cellular toxicity of the synthesized peptides of the GBV-C / HGV virus was determined according to the assay using 3- (4,5-dimethyltriazol-2-yl) -2,5-diphenyltetrazolium (MTT) bromide (Mosmann, 1983. J.lmmunol.Methods 65: 55-63).

EXAMPLE 2. Results of inhibition of the function of the HIV-1 fusion peptide (SEQ ID NO: 2) and inhibition of cell fusion, induced by different peptides of the sequence SEQ ID NO: 1.

In order to analyze the interaction of the envelope protein of the GBV-C / HGV virus with the fusion peptide of the gp41 glycoprotein of the HIV-1 virus (SEQ ID NO: 2), the synthesis of peptides covering the Ia complete sequence of the GBV-C / HGV virus E1 protein (SEQ ID NO: 1) by means of the synthesis of peptide sequences composed of 18 amino acids overlapped in 15 residues. The 58 peptides obtained were characterized by HPLC and HPLC-MS showing a purity greater than 90%.

The study of its ability to inhibit the process of destabilization of lipid vesicles induced by the HIV-1 fusion peptide (SEQ ID NO: 2) was carried out following the procedure detailed in the experimental part. As shown in FIG. 1, it has been possible to select a series of peptides capable of inhibiting the function of SEQ ID NO: 2. These selected peptides are SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 11. To verify the specificity of the interaction observed between the GBV-C / HGV virus peptides and the SEQ ID NO: 2 virus peptide of HIV-1 virus, melitin was used as a control peptide. It was found that the selected peptides did not inhibit the lytic capacity of Ia Melitin after performing the same test but in the presence of melitin instead of the peptide SEQ ID NO: 2, so that the specificity of the interaction described above was confirmed.

Table 2 shows the peptides corresponding to the envelope protein E1 of the GBV-C / HGV virus (SEQ ID NO: 1) that at different concentrations, have shown the ability to inhibit syncytium formation according to the fusion test performed mobile. The inhibition values achieved by the control inhibitor peptide are also shown. After carrying out the toxicity tests, following the MTT test (example 1.3), with the GBV-C / HGV virus peptides it was possible to verify a cell viability of 100%.

The calculation of the percentage of inhibition for each peptide has been obtained according to the equation:

% peptide inhibition = 100- {[(Lum (P) - Lum o) / (Lum 100- Lum o)] x 100}

being: Lum (P) = luminescence of the cells treated with the Lum peptide o = luminescence of the non-fused control cells Lum ioo = luminescence of the fully fused control cells

The studies carried out indicate that several of the sequences synthesized, belonging to the envelope protein E1 of the GBV-C / HGV virus, significantly decrease the fusion of cell membranes, which indicates an inhibitory effect of these peptides against the HIV-1 virus.

Table 2. Results of the inhibition of cell fusion induced by different peptides of the sequence SEQ ID NO: 1 (envelope protein E1) of the GBV-C / HGV virus at different concentrations. The inhibition values achieved by the control inhibitor (C34) are shown. Peptide% inhibition% inhibition% inhibition% inhibition

(100 μM) (500 μM) (1000 μM) C34

(1.34 μM)

SEQ ID NO: 5 46 62 70

SEQ ID NO: 9 46 70

SEQ ID NO: 10 14 65 53

SEQ ID NO: 12 50 72

SEQ ID NO: 13 70 81 72

SEQ ID NO: 14 57 69 60 72

SEQ ID NO: 15 68 47 23 72

SEQ ID NO: 16 93 88 80 74

Claims

1. Use of at least one peptide, or derivative thereof, contained in an amino acid sequence that has at least 60% identity with the sequence SEQ ID NO: 1, capable of inhibiting the activity of the HIV virus, to

Ia elaboration of a pharmaceutical composition.

2. Use of at least one peptide according to claim 1, wherein the amino acid sequence has at least 80% identity with SEQ ID NO: 1.

3. Use of at least one peptide according to any one of claims 1 or

2, where the amino acid sequence is SEQ ID NO: 1.

4. Use of at least one peptide according to any of claims 1 to

3, where said peptide is also capable of interacting with a fragment of the gp41 protein of the HIV virus.

5. Use of at least one peptide according to any one of claims 1 to 4, wherein the HIV virus is of the HIV-1 type.

6. Use of at least one peptide according to claim 5, wherein the fragment of the gp41 protein is the sequence SEQ ID NO: 2 of the HIV-1 virus.

7. Use according to any one of claims 1 to 6, wherein the peptide has between 15 and 20 amino acids.

8. Use of the pharmaceutical composition according to any of claims 1 to 7, wherein the inhibition of the activity of the HIV virus is produced by preventing the entry of the genetic material thereof into the host cell.

9. Use of the pharmaceutical composition according to claim 8, wherein the entry of the genetic material into the host cell is prevented by the inhibition of cell fusion.

10. Use of the pharmaceutical composition according to claim 9, wherein the inhibition of cell fusion is carried out by means of a pharmaceutical composition comprising at least one peptide contained in any of the amino acid fragments SEQ ID NO: 3 or SEQ ID NO: 4.

11. Use of the pharmaceutical composition according to claim 10, wherein the peptide, or any combination thereof, is selected from the list comprising SEQ ID NO: 5, SEQ ID NO: 9-10, SEQ ID NO: 12-16 .

12. Use of the pharmaceutical composition according to any of claims 1 to 7 wherein the inhibition of the activity of the HIV virus is produced by inhibiting the function of a fragment of the gp41 protein of the HIV virus.

13. Use of the pharmaceutical composition according to claim 12, wherein the inhibition of the function of a fragment of the HIV virus gp41 protein is carried out by means of a pharmaceutical composition comprising at least one peptide contained in the amino acid SEQ fragment ID NO: 3.

14. Use of the pharmaceutical composition according to claim 13, wherein the peptide, or any combination thereof, is selected from the list comprising SEQ ID NO: 6-8, SEQ ID NO: 10-11.

15. Pharmaceutical composition comprising at least one peptide, or derivative thereof, contained in an amino acid sequence having at least 60% identity with the sequence SEQ ID NO: 1, capable of inhibiting the activity of the HIV virus.

16. Pharmaceutical composition according to claim 15, wherein the amino acid sequence has at least 80% identity with SEQ ID

NO: 1.

17. Pharmaceutical composition according to any of claims 15 or

16, where the amino acid sequence is SEQ ID NO: 1.

18. Pharmaceutical composition according to any of claims 15 to

17, where at least one peptide, or derivative thereof, is capable of interacting with a fragment of the gp41 protein of the HIV virus.

19. Pharmaceutical composition according to claim 18, wherein the fragment of the gp41 protein is the sequence SEQ ID NO: 2 of the HIV-1 virus.

20. Pharmaceutical composition according to any of claims 19 to 22, wherein the peptide has between 15 and 20 amino acids.

21. Pharmaceutical composition according to any of claims 15 to

20, which further comprises pharmaceutically acceptable excipients.

22. Pharmaceutical composition according to any of claims 15 to

21, which further comprises at least one pharmaceutically acceptable vehicle.

23. Pharmaceutical composition according to any of claims 15 to 22, which further comprises another active substance.

24. Method for manufacturing the pharmaceutical composition according to any of claims 15 to 23 comprising:

25. Method for manufacturing the pharmaceutical composition according to claim 24, wherein the peptide is selected from the list comprising SEQ ID NO: 3-5, SEQ ID NO: 7, SEQ ID NO: 10-11, SEQ ID

NO: 15, SEQ ID NO: 16-41.