WO2010116015A1

WO2010116015A1 - Use of peptides of the e2 protein of the hepatitis g virus in order to inhibit the activity of the hiv virus

Info

Publication number: WO2010116015A1
Application number: PCT/ES2010/070209
Authority: WO
Inventors: Isabel Haro Villar; Maria José GÓMARA ELENA
Original assignee: Consejo Superior De Investigaciones Cientificas (Csic)
Priority date: 2009-04-06
Filing date: 2010-04-05
Publication date: 2010-10-14
Also published as: ES2351026A1; ES2351026B1

Abstract

The invention relates to the use of peptides of the E2 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 E2 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 peptides of the E2 protein of the hepatitis G virus to inhibit the activity of the HIV virus

The present invention relates to the use of at least one peptide, or derivative thereof, contained in the sequence of the E2 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), 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. Hepatoloαv 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 et al., 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 individuals have been observed. HGV positive compared to GBV-C / HGV negative individuals, while the loss of viremia

GBV-C / HGV is closely related to increased mortality as well as greater progression of AIDS (Van der Bij et al., 2005. J. Infect. Dis. 191: 678-685; Williams et al., 2004. N Engl. 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, CeN 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. Retroviroloqy 3: 77). The entry of the virus to the

The host cell as a process that requires three stages: 1) approach and binding of HIV to CD4 molecules (primary receptor) through the gp120 viral glycoprotein; 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 GBV-C / HGV virus E2 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), transiently or permanently, or also capable of interacting with an amino acid sequence of the gp41 glycoprotein of the HIV virus (Human Immunodeficiency Virus), for the preparation 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, 124 peptides composed of 18 amino acids that comprise the complete sequence of Ia are synthesized GB2-C / HGV virus E2 protein (SEQ ID NO: 1). These 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) by means of the use of biophysical techniques that allow to determine the inhibition of vesicle contents induced by SEQ ID NO: 2 that allow to determine the ability to inhibit the function of the fragment of the gp41 protein of the HIV-1 virus, and evaluating the potential inhibitory character of HIV-1 through cellular assays aimed at determining 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 the activity of the HIV virus, 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 an analysis

Blast of the sequence SEQ ID NO: 1. In this comparative analysis a sequence corresponding to a fragment of a polyprotein of the variant Troglodites of the GBV-C / HGV virus was found. 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 efficiency 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 HIV virus activity" 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 E2 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 the 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 between amino acids 1 and 23 Ia gp41 membrane glycoprotein of HIV-1 virus

(Human Immunodeficiency Virus 1) (N ⁰ of access ACJ04095 GeneBank). 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 is 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 in 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 Ia 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: 4- 8, SEQ ID NO: 10, SEQ ID NO: 13-14 or SEQ ID NO: 15

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 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: 4-8 (SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8), SEQ ID NO: 10, SEQ ID NO: 13-14 (SEQ ID NO: 13 or SEQ ID NO: 14) or SEQ ID NO: 15. 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: 4-5 (SEQ ID NO: 4, SEQ ID NO: 5 ), SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 15-23 (SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23), SEQ ID NO: 35, SEQ ID NO: 37 or SEQ ID NO: 41. The list of peptides that It is presented belongs to the fragments SEQ ID NO: 4-8, SEQ ID NO: 10, SEQ ID NO: 13-14 or SEQ ID NO: 15 in the manner presented in Table 2.

Table 2. 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.

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.

An even more preferred embodiment refers to the use of the pharmaceutical composition, where the inhibition of the function of a fragment of the gp41 protein of the HIV virus is carried out by means of a pharmaceutical composition comprising at least one peptide contained in any of amino acid fragments SEQ ID NO: 9, SEQ ID NO: 12-14 (ie, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14).

The sequences SEQ ID NO: 9, SEQ ID NO: 12-14 are fragments of amino acid sequences corresponding to the partial sequence of SEQ ID NO: 1. The fragments are composed of 18 amino acids or more, as the case may be. Thus, by way of example, fragments having 18 amino acids may contain peptides of between 15 and 18 amino acids. 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: 24-40. The list of peptides presented belongs to the fragments SEQ ID NO: 9, SEQ ID NO: 12-14 in the manner presented in Table 1.

Table 1. Correspondence of the peptides involved in the inhibition of the release of vesicular contents (inhibition of the function of the fragment of the gp41 protein), 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 replication of the genetic material of said virus.

The term "inhibition of replication" as understood in the present invention refers to permanently or temporarily preventing or repressing the generation of new genetic material from the genetic material of the HIV virus, new viruses or virions. The term "virion" refers to the infective viral particle 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. In the embodiments of the present invention, the degree of inhibition of HIV virus replication is assessed by measuring the concentration of the p24 antigen of the HIV-1 virus. The detection of the HIV-1 antigen, usually the p24 protein, is a direct marker of the presence of the virus in the organism.

A more preferred embodiment refers to the use of the pharmaceutical composition, where the inhibition of the replication 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: 3-11 ( SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11), SEQ ID NO: 13-14 (SEQ ID NO: 13 or SEQ ID NO: 14).

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: 3-5 (SEQ ID NO: 3,

SEQ ID NO: 4 or SEQ ID NOP: 5), SEQ ID NO: 7, SEQ ID NO: 10-11 (SEQ ID NO: 10 or SEQ ID NO: 11), SEQ ID NO: 16-24 (SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24), SEQ ID NO: 35, SEQ ID NO: 37 or SEQ ID NO: 41. The list of peptides presented belongs to the fragments SEQ ID NO: 3-11, SEQ ID NO: 13-14 in the manner presented in Table 3.

Table 3. Correspondence of the peptides involved in the inhibition of replication, with the fragments of SEQ ID NO: 1.

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, function of dye, protection function of the medicine such as for example to isolate it from air and / or moisture, filling function of a tablet, capsule or any other form of presentation such as dibasic calcium phosphate, 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, reference may be made to any of the pharmaceutical compositions described as "pharmaceutical composition / s of the present invention" or "pharmaceutical composition / s of the invention".

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 of the invention, 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 technique of macromolecular synthesis, such as, but not limited to, solid phase peptide synthesis. Any of the peptides of the invention can also be obtained by recombinant DNA techniques, that is, by means of 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 Ia Oral administration is selected from the list comprising, but not limited to, drops, syrup, herbal tea, elixir, suspension, extemporaneous suspension, drinkable vial, tablet, capsule, granulate, seal, pill, tablet, tablet, trochco 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 Ia 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: 3-5, SEQ ID NO: 7, SEQ ID NO: 10-11, SEQ ID NO: 15, SEQ ID NO: 16 -41.

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: P1 (SEQ ID NO: 42), P40 (SEQ ID NO: 43), P41 (SEQ ID NO: 44), P45 (SEQ ID NO: 21), P47 (SEQ ID NO: 22), P59 (SEQ ID NO: 24), P60 (SEQ ID NO: 25), P80 (SEQ ID NO: 26), P82 (SEQ ID NO: 28), P88-91 (SEQ ID NO: 30-33), P96 (SEQ ID NO: 34), P97 (SEQ ID NO: 35), P105-109 (SEQ ID NO: 37- 41) and P124 (SEQ ID NO: 15). FIG. 2. It shows the inhibition of cell fusion mediated by peptides of the GB2-C / HGV E2 protein selected as potential HIV-1 inhibitors.

The percentage of inhibition of the dose-dependent fusion of peptides P11 (SEQ ID NO: 4), P19-21 (SEQ ID NO: 16-18), P25 (SEQ ID NO: 20), P45 (SEQ ID NO: 21), P46 (SEQ ID NO: 22), P47 (SEQ ID NO: 23), P59 (SEQ ID NO: 24), P97 (SEQ ID NO: 35), P109 (SEQ ID NO: 41) and P124 (SEQ ID NO: 15).

FIG. 3. It shows microscopy images of the inhibition of cell fusion mediated by the gp41 protein after incubation with the P45 peptide.

The cells are grown in 96-well plates where they are treated with: A) C34 inhibitor (1.2 μM); B) control; C) 500 μM P45; D) 250 μM P45; E) 125 μM P45 and F) 62.5 μM P45.

FIG. 4. Shows the inhibitory activity of different doses of the P45 peptide of the cell fusion induced by the gp41 protein.

Each data represents the average of the percentage of inhibition mediated by the P45 peptide (SEQ ID NO: 21) ± standard error (bars).

FIG. 5. Shows the inhibition of HIV-1 infection of C E M 174 cells by the peptides of GB2-C E2 protein.

The percentages of inhibition of HIV-1 virus replication by peptides P2, P11, P15, P19-21, P23, P25, P34, P45-47, P59, P60, P67, P77, P89, P97 are shown and P109 administered at doses of 250 and 500 μM. EXAMPLES

The invention will now 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 HIV-1 virus activities. 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

124 peptides of the envelope protein E2 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 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. Test of inhibition of vesicle content release induced by the sequence SEQ ID NO: 2 of HIV-1 b. Inhibition assay of cell fusion c. HIV-1 replication inhibition assays

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 a /., 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-C / HGV constructions 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 in the incubation of approximately 2,500 HeLa-env cells 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 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 were obtained by reading the luminescence (Britelite commercial kit, Perkin Elmer Cat. No. 6016761) in a SpectraMax M5 microplate reader (Molecular Devices).

c) HIV-1 virus replication inhibition assays. In order to study the possible inhibitory role of the GBV-C / HGV virus peptides, the quantification of the concentration of the HIV p24 antigen in the cell culture supernatant was carried out. For this, serial concentrations of the peptides have been incubated with HIV-1 isolates of tropism X4 and R5 for two hours. Isolated viruses can come from infected patients. After the incubation period, CEM-174 cells (AIDS reagents Cat. No. 272) were incorporated and incubated with the virus and peptides for 3 hours. After this time, the HIV-1 virus that was still in the middle was removed by washing with PBS buffer. Finally, the cell pellet was resuspended in culture medium and a portion of the cell supernatant was collected every 12 hours for a week. The quantification assay of the p24 antigen was performed on the post-infection supernatants collected using the commercial Innotest HIV antigen mAb p24 kit (Innogenetics Diagnostica Iberia Cat. No. 80563). As a negative control, the cell culture supernatant was used in the absence of viruses and peptides; The culture supernatant derived from the incubation of the cells with the virus was used as a positive infection control. Finally, as a positive control of the inhibition, the supernatant from the incubation of the cells with the virus and the C34 inhibitor (AIDS reagents Cat. No. 9824) was used.

1.3. Study of the cellular viability of the GBV-C / HGV 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), inhibition of cell fusion and inhibition of replication, 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 complete sequence of the GB2-C / HGV virus E2 protein (SEQ ID

NO: 1) by means of the synthesis of peptide sequences composed of 18 amino acids overlapped in 15 residues. The 124 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. To verify the specificity of the interaction observed between the GBV-C / HGV virus peptides and the SEQ ID NO: 2 peptide HIV-1 virus, melitin was used as a control peptide. It was found that the selected peptides did not inhibit the lithic capacity of melitin after performing the same test but in the presence of melitin instead of the peptide SEQ ID NO: 2, so the specificity of the interaction described above was confirmed.

Table 4 shows the peptides that correspond to the envelope protein E2 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. Table 5 shows the most relevant results regarding the inhibition of p24 antigen production. The values of inhibition achieved by the control inhibitor peptide. 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:

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

being:

Lum (P) = luminescence of cells treated with the Lum peptide or = luminescence of non-fused control cells

Lum ioo = luminescence of fully fused control cells

To carry out the calculation of the percentages of inhibition of the replication of the HIV-1 virus (inhibition of p24 antigen production) the same equation was used but using the absorbance values instead of luminescence.

The studies carried out indicate that several of the sequences synthesized, belonging to the envelope protein E2 of the GBV-C / HGV virus, significantly decrease the fusion of cell membranes and the production of the p24 antigen, which indicates a possible inhibitory effect of these peptides against the HIV-1 virus.

Table 4. Results of the inhibition of cell fusion induced by different peptides of the sequence SEQ ID NO: 1 (envelope protein E2) of 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: ~ 57 84 78

4

SEQ ID NO: 88 95

5

SEQ ID NO: 62 92

16

SEQ ID NO: 48 45 92

17

SEQ ID NO: 21 59 100

18

SEQ ID NO: 32 61 95

19

SEQ ID NO: 64 91 97

twenty

SEQ ID NO: 8 36 95

7

SEQ ID NO: 21 48 95

twenty-one

SEQ ID NO: 39 98 96 100

22

SEQ ID NO: 13 56 70 92

2. 3

SEQ ID NO: 79 88 91

10

SEQ ID NO: 91 90 100 35

SEQ ID NO: 32 70 72 90

37

SEQ ID NO: 34 91

41

SEQ ID NO: 20 62 68 95

fifteen

Table 5. Results of the inhibition of the production of p24 antigen induced by different peptides of the sequence SEQ ID NO: 1 (envelope protein E2) of the GBV-C / HGV virus at different concentrations. The inhibition values achieved by the control inhibitor (C34) are shown.

Peptide% I% l% I% l% I C34

(50 (100 μM) (250 μM) (500 μM) (1.34μM) μM)

SEQ ID NO: 3 98 100

SEQ ID NO: 4 73 96 99

SEQ ID NO: 5 35 98 99

SEQ ID NO: 16 47 45 99

SEQ ID NO: 17 48 100 99

SEQ ID NO: 18 67 97 95 99 97

SEQ ID NO: 19 10 96 99 100 97

SEQ ID NO: 20 30 100 100

SEQ ID NO: 7 48 100 100

SEQ ID NO: 21 29 96 94

SEQ ID NO: 22 75 97 98 95 98

SEQ ID NO: 23 42 98 94

SEQ ID NO: 24 99 98

SEQ ID NO: 10 94 98

SEQ ID NO: 11 60 100 99

SEQ ID NO: 35 51 98 100

SEQ ID NO: 37 43 100

SEQ ID NO: 41 50 99 99

% I:% inhibition. EXAMPLE 3. Anti-HIV-1 activity of GB2-C / HGV virus E2 peptides in a dose-dependent manner.

The effect of E2 peptides on cell fusion was examined in cell-cell fusion assays analyzing their ability to block the formation of syncytia between HeLa cells that express the envelope protein of the HIV-1 virus and the TZM-bl cells that express the human CD4 receptor CXCR4 or CCR5 in the presence of various amounts of each peptide.

The following 18 amino acid peptides: P11 (SEQ ID NO: 4), P19-21 (SEQ ID NO: 16-18), P23 (SEQ ID NO: 19), P25 (SEQ ID NO: 20), P45-47 (SEQ ID NO: 21-23), P59 (SEQ ID NO: 24), P97 (SEQ ID NO: 35), P109 (SEQ ID NO: 41) and P124 (SEQ ID NO: 15) inhibited cell cell fusion -gp41 protein-induced cell in a dose-dependent manner, showing IC50 values between 142 and 832.9 µM (FIG. 2 and FIG. 5 on day 7 after infection). As a positive control, C34 peptides were used (IC50 = 0.023 μM; 95% CI = 0.021-0.025). In Figures 3 and 4 it can be seen how the p45 peptide inhibits syncytium formation in a dose dependent manner.

Table 3. Inhibitory activity of the cell fusion mediated by the gp41 protein by the E2 peptides.

Residue

Peptide Sequence IC ₅₀ "(μM) 95% Cl ^c no. ^A

P11 31-48 SEQ ID NO: 4 439.7 (361.4 - 535.0)

P19 55-72 SEQ ID NO: 16 369.5 (314.8 - 433.7)

P20 58-75 SEQ ID NO: 17 347.6 (305.1 - 396.0)

P21 61-78 SEQ ID NO: 18 832.9 (754.5 - 919.4)

P23 67-84 SEQ ID NO: 19 508.8 (496.9 - 520.9)

P25 73-90 SEQ ID NO: 20 304.4 (260.9 - 355.3) P45 153-170 SEQ ID NO: 21 141.2 (129.1 - 154.4)

P46 156-173 SEQ ID NO: 22 428 .8 (373.1 - 429.7)

P47 159-176 SEQ ID NO: 23 330 .8 (277.4 - 394.5)

P59 195-212 SEQ ID NO: 24 529 .6 (514.0 - 545.8)

P97 289-306 SEQ ID NO: 35 537 .6 (476.9 - 606.0)

P109 325-342 SEQ ID NO: 41 687 .1 (616.3 - 766.1)

P124 370-387 SEQ ID NO: 15 332 .7 (265.4 - 417.1)

The number of the residue corresponds to its position in the E2 protein (U45966_usa) of GBV-C. b IC ₅ O '. concentration of 50% peptide inhibitor of cell fusion inhibition (μM)

FIG. 2 and 5 and Table 4 show the dose-dependent inhibition of peptides P2 (SEQ ID NO: 3), P11 (SEQ ID NO: 4), P15 (SEQ ID NO: 5), P19-21 (SEQ ID NO: 16-18), P23 (SEQ ID NO: 19), P25 (SEQ ID NO: 20), P34 (SEQ ID NO: 7), P45-47 (SEQ ID NO: 21-23), P59 (SEQ ID NO: 24), P60 (SEQ ID NO: 25), P67 (SEQ ID NO: 10), P77 (SEQ ID NO: 11), P89 (SEQ ID NO: 81), P109 (SEQ ID NO: 41) and P124 (SEQ ID NO: 15). The inhibitory effect was differentially efficient between strains X4 and R5. The IC50 obtained by peptides P11, P19, P20 and P21 for HIV-1 HXB2 was <HIV-169/7 <HIV-I BaL; However, the IC50 obtained for peptides P34, P45, P46, P47 and P109 for HIV-169/7 was <HIV-1 HXB2 <HIV-I BaL. In general, all the peptides analyzed showed a higher IC50 log for the R5 virus than for the X4 or R5X4 DM virus (Table 4).

Table 4. Phenotypic susceptibilities of HIV-1 to E2 peptides in TZM-bl cells.

A replicate for each peptide in this test b IC ₅₀ : μM concentration of peptide causing 50% inhibition of infection, obtained from two independent experiments c 95% Cl: 95% Confidence Interval of IC50 ^d Linear mathematical model, not sigmoidal nd: not determined

P124 peptide was effective only for the HIV-I BaL viral strain. New experiments carried out in TZM-bl cells where the peptides were incubated with the cell for two hours before the adsorption of the virus, did not prevent the prevention of viral infection. The inhibitory effect of the infection of PBMCs cells was also observed by means of peptides P11, P19-P21, P34, P45, P46, P47, P109 and P124. In this case, the qualitative analysis of the production of p24 antigen produced in these cell cultures showed: 1) that the concentrations until the concentration of the virus was undetectable was lower than the IC50 observed in TZM-bl cell cultures, 2) that those concentrations were lower in HIV-1 HXB2 (R4) than in HIV-I BaL (R5), and 3) that peptides P19-P21, P45-P47 and P109 were more efficient in inhibiting the virus than peptides P11 and P34 (Table 5).

Table 5. Concentrations of E2 peptides (μM) above which no infection of PBMCs by HIV-1 was detected (after 7 days of cell culture and subsequent qualitative analysis of the p24 antigen).

HXB2 BAL peptide

P11 (SEQ ID NO: 4) 3T2 6Z5

P19 (SEQ ID NO: 16) 15.6 15.6

P20 (SEQ ID NO: 17) 7.8 15.6

P21 (SEQ ID NO: 18) 3.9 62.5

P34 (SEQ ID NO: 7) 31.2 62.5

P45 (SEQ ID NO: 21) 7.8 31.2

P46 (SEQ ID NO: 22) 7.8 7.8

P47 (SEQ ID NO: 23) 7.8 15.6

P109 (SEQ ID NO: 41) 7.8 15.6 P124 (SEQ ID NO: 15) 62.5 7.8

In short, this example demonstrates how the interaction of the peptides tested interferes with the HIV-1 virus, specifically in the peptide-vesicle interaction, producing a notable reduction in cell fusion interfering with the infectivity of the HIV-1 virus of a dose dependent form.

3.1. Inhibition assay of cell fusion.

Two cell lines were used: HeLa-env that expresses the envelope protein of the HIV-1 virus and that includes the LTR promoter in its genome, and the TZM-bl cell line (AIDS reagents Cat. No 8129), which expresses the receptor of CD4 membrane lymphocytes and the CCR5 and CXCR4 co-receptors and includes the luciferase and β-galactosidase genes in its genome.

These cell lines were cultured in DMEM (Dulbecco's Modified Eagle Medium) containing L-Glutamine and sodium pyruvate supplemented with 10% heat-inactivated fetal bovine serum (FBS),

100 μg / ml penicillin and 100 μg / ml streptomycin. The cell cultures were maintained in an incubator at 37 ⁰ C in an atmosphere

5% CO2 When the two cell types are co-cultured, the fusion of the membranes takes place and the luciferase is activated producing the oxidation of the luciferin. The oxyluciferin level was quantified using the kit

Britelite (Perkin Elmer) and SpectraMax M5 microplate reader.

The cell binding inhibition test induced by the E2 peptides consisted of the incubation of 2,500 cells / well of HeLa-env (Nunc Cat. plates No. 136101) for 1 hour with serial dilutions (5-1000 μM) of the peptides, followed by the addition of about 25,000 TZM-bl cells / well and incubated for 24 hours.

For the control of cell binding, wells without peptides were reserved and the C-34 inhibitor (AIDS reagents Cat. No. 9824) was used as a positive control. The level of inhibition of cell binding was also observed under the microscope by syncytium formation.

3.2. Assay for inhibition of HIV-1 virus infection.

3.2.1 Susceptibility test in TZM-bl cells

The set of peptides with potential capacity for HIV-1 virus inhibition demonstrated by biophysical tests and cell-cell fusion assays, were investigated in greater depth in a susceptibility test with TZM-bl cells and dependence on Ia was demonstrated. inhibition of the dose of peptide supplied. Briefly, triplicates of 2 serial concentrations per peptide (0-500 μM) were pre-incubated along a predetermined volume of each HIV-1 BaL strain (Tropism R5, AIDS Reagent) or HIV-1 HXB2 (R4 tropism, obtained from pHXB2) or an HIV-1 primary isolate called 69/7 (dual R5X4 or mixed DM tropism, isolated from an HIV-1 infected patient), in a final volume of 100 μL / DMEM post with 10% FBS in plates 96 well, for 2 hours at 37 ⁰ C and 5% CO2. In addition, 2 serial concentrations of T-20 (AIDS Reagent, # 9409) or C34 or Amphotericin B (Bristol-Myers Squibb, SL) were used in a dose-dependent inhibition of the high control dose of the assay. Subsequently, 100 μL of cell medium containing 15,000 TZM-bl cells were added to each well. Under these conditions, the final moi (multiplicity of infection) of each viral strain was 0.02 for HIV-I BaL and 0.01 for HIV-1 HXB2 and HIV-169/7, respectively. Each HIV-1 infected TZM-bl without peptides was analyzed in triplicate, as well as for the positive and negative controls. After 72 hours after the infection, the supernatant from each well was removed and 50 µl_ of lysis buffer (0.1% Triton X-100, 1OmM MgCI2, 0.5 mM dithiothreitol, DTT, in phosphate buffer) were added together with 50 μl_ of 1.5mM CPRG. The β-galactosidase activity was analyzed by spectrophotometry (570 nm, SpectraMax M5 microplate reader). The optical density obtained was transformed into a percentage of inhibition and the sigmoidal curves were analyzed by nonlinear regression (prísm Graph pad software v.5). In parallel, the effect of the toxicity on the TZM-bl cells of the concentration of each peptide was analyzed using the MTT assay described below. A modification of this procedure was analyzed to rule out whether the inhibitory effect of the peptides was due to the direct interaction with the receptor or co-receptor of the viral entry. In this case, the TZM-bl cells were incubated with peptides for 2 hours at 37 ⁰ C and 5% CO2, and washed 3 times with PBS before performing the viral adsorption for 2 hours. Subsequently, the cells were washed 3 times and incubated for 72 hours in the absence of peptides and analyzed as described above.

3.2.2. Susceptibility assay in PBMCs cells

A quantitative analysis of the susceptibility of the HIV-1 virus based on the HIV-1 p24 antigen was carried out. Said analysis was carried out by infecting PBMCs cells with HIV-1 HXB2 and HIV-I BaL, to confirm the inhibitory effect of the HIV-1 virus of the peptides of the present invention.

PBMCs cells were obtained from leukocyte layers of healthy donors by means of centrifugation gradient (ACCUSPIN System-Histopaque-λOU, Sigma Diagnostics) and activated with 5 μg / mL phytohemagglutinin (PHA, Sigma-Aldhrichl) in RPMI-1640 medium (Lonza- BioWithaker) and 10% of FBS, for 24 to 72 hours before the viral infection.

Serial concentrations of peptides were prepared (0-125 μM) in RPMI-1640 medium supplemented with 10% FBS and 10 U / mL recombinant interleukin-2 (rlL-2, Roche Diagnostic Systems) and pre-incubated with a predetermined volume of HIV -1 HXB2 or HIV-I BaL, for 2 hours at 37 ⁰ C and 5% of CO2. Subsequently, 8-10 ⁵ activated PBMCs cells were added and incubated for an additional 2 hours to achieve viral adsorption. Finally, the infected cells were washed 3 times with PBS and incubated for 7 days, together with the corresponding concentration of peptide, in 96-well plates containing 200-10 ³ PBMC cells in 200 μl of RPMI-1640, 20% of FBS and 10 U / mL of rlL-2 in the absence of peptides. The final moi for each viral strain was 0.001. Virus release was analyzed by HIV-1 p24 ELISA and Antigen-HIV-1 p24 ELISA (Ag HIV, Innogenetics # K1048).

3.3. MTT cell viability test.

The cellular toxicity of E2 peptides was analyzed in HeLa, TZM-bl and PBMCs using an assay (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT). Cells were cultured with DMEM (15,000 cells / well) in 96 - well plates and incubated with serial dilutions of each peptide at 37 ⁰ C for 72 h. Subsequently was added MTT to a final concentration of 7.5 mg / mL and incubated for 2 h at 37 ⁰ C The medium was removed and added

100 μL of DMSO to dissolve the formazan precipitate. The absorbance was measured at 570 nm after 45 minutes. Cell viability was determined by the ratio between the absorbance value of the treated cells and untreated cells. The cytotoxic concentration (CC50) was analyzed by non-linear regression.

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, for the 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 of claims 1 or 2, wherein the amino acid sequence is SEQ ID NO: 1.

4. Use of at least one peptide according to any of claims 1 to 3, wherein said peptide is also capable of interacting with a fragment of the HIV virus gp41 protein.

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: 4-8, SEQ ID NO: 10, SEQ ID NO: 13-14 or SEQ ID NO: 15.

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: 4-5, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 15-23, SEQ ID NO: 35, SEQ ID NO: 37 or SEQ ID NO: 41.

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 any of the amino acid fragments SEQ ID NO: 9, SEQ ID NO: 12-14.

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: 24-40.

15. 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 replication of the genetic material of the virus.

16. Use of the pharmaceutical composition according to claim 15, wherein the inhibition of replication 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: 3-11, SEQ ID NO: 13-14.

17. Use of the pharmaceutical composition according to claim 16 wherein the peptide, or any combination thereof, is selected from the list comprising SEQ ID NO: 3-5, SEQ ID NO: 7, SEQ ID NO: 10-11, SEQ ID NO: 16-24, SEQ ID NO: 35, SEQ ID NO: 37 or SEQ ID NO: 41.

18. 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.

19. Pharmaceutical composition according to claim 18, wherein the amino acid sequence has at least 80% identity with SEQ ID NO: 1.

20. Pharmaceutical composition according to claim 19, wherein the amino acid sequence is SEQ ID NO: 1.

21. Pharmaceutical composition according to any of the claims

18 to 20, where at least one peptide, or derivative thereof, is capable of interacting with a fragment of the HIV virus gp41 protein.

22. Pharmaceutical composition according to claim 21, wherein the fragment of the gp41 protein is the sequence SEQ ID NO: 2 of the HIV-1 virus.

23. Pharmaceutical composition according to any of the claims

19 to 22, where the peptide has between 15 and 20 amino acids.

24. Pharmaceutical composition according to any of the claims

19 to 23, which further comprises pharmaceutically acceptable excipients.

25. Pharmaceutical composition according to any of claims 19 to 24, further comprising at least one pharmaceutically acceptable carrier.

26. Pharmaceutical composition according to any of claims 19 to 25, further comprising another active substance.

27. Method for the manufacture of the pharmaceutical composition according to any of claims 18 to 26 comprising: 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.

28. Method for manufacturing the pharmaceutical composition according to claim 27, 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.