MXPA97007319A - Alpha acid, gamma-diaminobutirico (dab) containing oligopeptidi derivatives - Google Patents

Abstract

Alpha, gamma-diaminobutyric acid (DAB) containing oligopeptide derivatives of formula R'-NH-A wherein R 'is a cell recognition agent, a permeabilizing membrane agent, a subcellular localizing agent or a masking agent; A is an oligopeptide devoid of an amino group, containing 1 to 200 amino acids, wherein at least one of the amino acids, wherein at least one of the amino acids is alpha, gamma-diaminobutyric acid (DAB), and the group The terminal carboxyl of the oligopeptide is converted to an amide, lower alkyl amide, lower dialkyl amide or hydrazide is useful as a carrier for the transfection of cells with a polynucleotide or any other anionic macromolecule.

Description

, - OLIGOPEP? DIOOS " Field of the Invention The technology of gene transfer has become a field of considerable interest. The introduction of an exogenous gene into a cell (ie, transfection) leads to many important scientific and medical applications ranging from gene regulation and the production of recombinant proteins to gene therapy.

Background of the Invention Viruses have evolved to be a way to bypass the different cellular barriers to transfer genes and have actually become selective vectors for transfection. Many viruses, including retroviruses, adenoviruses or herpes viruses, are currently under genetic engineering to transport therapeutic genes and are used in human clinical trials for gene therapy. However, there is a risk of an infectious and immunological reaction and the large-scale production of viruses is difficult and time-consuming.

REF: 25447 For these various reasons, non-viral systems have been developed to transport DNA into cells, e.g. ex. the technique of transfection based on a cationic lipid, dioleoyloxypropyl trimethylammonium (Felgner et al., Proc. Nati., Acad. Sci. USA, 84, 7413-7417, 1987) marketed as Lipofectin ™. Since the discovery of this transfection technique, many more cationic lipids have been synthesized and some are commercially available as transfection reagents for use in the laboratory: DOGS (Trans-fectam ™), DOSPA (Lipofectamine ™, DOTAP (DOTAP ™).

However, despite the important progress made in the formulation of non-viral systems for gene delivery, the discovery of more efficient techniques continues to be necessary, given that the efficiency of the transfection of synthetic systems is normally less than that of vectors. viral In addition, many problems arise in the in vivo application and the poor stability of the non-viral systems in the biological fluids does not allow high and reproducible levels of transfection in vivo.

Description of the invention In accordance with the present invention, it has been discovered that alpha, gamma-diaminobutyric acid (DAB) containing oligopeptide derivatives binds to anionic polynucleotides and macromolecules, and can be employed for transfection of cells.

Thus, in one aspect, the present invention relates to the new alpha, gamma-diaminobutyric acid (DAB) with-having oligopeptide derivatives, of formula wherein p -NH-A 1 R 'is a cell recognition agent, a permeabilizing membrane agent, a subcellular localization agent or a masking agent; A is an oligopeptide devoid of an amino group, containing from 1 to 200 amino acids, wherein at least one of the amino acids is alpha, gamma-diaminobutyric acid (DAB), and the terminal carboxylic group of the oligopeptide is converted into a amide, lower alkyl amide, lower dialkyl amide or hydrazide.

The oligopeptides A can be linear or branched. The amino acids can belong to the L- or D- series or they can be racemic. Preferred are compounds of formula I, wherein A is an oligopeptide devoid of an amino group containing from 1 to 50 amino acids. Most preferred are the compounds of formula I, wherein oligopeptide A contains from 1 to 20 amino acids. Another preferred embodiment are the compounds of formula I wherein the terminal carboxyl group of oligopeptide A is converted to a hydrazide. The term "cell recognition agent" as used herein, refers to a molecule capable of recognizing a component of the surface of a predetermined cell. Cell recognition components include antibodies to cell surface antigens, ligands for cell surface receptors, including those involved in endocytosis induced by a receptor, peptide hormones, etc. The specific ligands contemplated by this invention include carbohydrate-based ligands such as galactose, mannose, mannosyl-5-phosphate, fucose, sialic groups, N-acetylglucosamine or combinations of these groups as complex carbohydrates such as those they are found in glycolipids of blood groups or in several secreted proteins. Other ligands include folate, biotin, various peptides that can interact with the cell surface or intracellular receptors such as the chemoattracting peptide N-formyl-met-leu-phe (SEQ ID NO: 2, WO-A-0 2397 ), peptides containing the sequence arg-asp-gly or peptides cys-ser-gly-arg-glu-asp-val-trp (SEQIDN0: 3., idem), peptides which contain a cystine residue or that interact with Protein of the cell surface such as the human immunodeficiency virus GP-120, and peptides that interact with CD-4. Other ligands include antibodies or antibody fragments such as those described by Hertler and Frankel (Hertler, A., and Frankel, A., J. Clin. Oncol. 7: 1932 (1989)). The specificity of the antibodies can be induced against a variety of epitopes that can be expressed on the surfaces of cells including histocompatibility macromolecules, autoimmune antigens, viral, parasitic or bacterial proteins. Other protein ligands include hormones such as growth hormone and insulin or growth factors proteins, such as GM-CSF, G-CSF, erythropoietin, epidermal growth factor, fibroblast growth factor, basic and acid , and similar. Other protein ligands include various cytokines that act through receptors on the cell surface such as interleukin 2, interleukin 1, interleukin 12, tumor necrosis factor, and suitable peptide fragments from such macromolecules. In another version, the cell recognition molecule is an integrating binding peptide or derivatives thereof. In a more preferred version, the integrating binding peptide is GGCRGDMFGCGG.

The term "membrane permeabilization agent" as used herein, refers to a molecule that aids the passage of an anionic polynucleotide or macromolecule through a membrane. Examples of permeabilizing agents membranes are melittin, hemolysin, mastoparan, bombolitina, crabrolina, pardaxina, gramicidin, alamethicin, apidaecin, bactenecin, cecropins, defensins, dermaseptin, indolicidin, magainins, bombinina, brevinina, Esculentin, seminalplasmina, and derivatives themselves (G. Saberwal and R. Nagaraj, BBA 1197: 109-131 (1994)). Other membrane permeating agents are peptides that contain the amino terminal sequence of the amino acids of influenza virus hemagglutinin and synthetic derivatives thereof as described in the Journal of Biological Chemistry, vol. 269 (17), 12918-12924 (1984) (table I): or HIV and SIV fusion peptides, and derivatives thereof, as described in Biochemica et Biophysica Acta 1240, 95-100 (1995) (fig. . 1) . Another class of cellular permeabilizing agents are detergent molecules such as bile salts. Other examples of detergent molecules are sucrose monolaurate, n-octylglucoside and tocopheryl succinate PEG 1000. However, other detergent molecules are also suitable. In another version, the membrane waterproofing agent is a conjugate between a membrane permeating agent and a lipid. An example of said conjugate is phe-glu-ala-ala-leu-ala-glu-ala-leu-ala-glu-ala-leu-ala with a myristic acid in the terminal NH2 (C. Puyal et al., BBA 1195: 259- 266 (1994)). The term "subcellular localization agent" as used herein refers to a molecule capable of recognizing a subcellular component in a predetermined cell. Particular subcellular components include the nucleus, ribosomes, mitochondria and chloroplasts. In a preferred embodiment of this invention the subcellular localization component is a nuclear localization component. Nuclear localization components include known peptides of defined amino acid sequences, and longer sequences containing these peptides. A known peptide sequence is the heptapeptide pro-lys-lys-lys-arg-lys-val (SEQ ID N0: 1, WO-A-0 2397) of the large SV-T antigen. Other peptides include the decapeptide nucleoprotein of influenza virus, ala-ala-phe-glu-asp-leu-arg-val-leu-ser (SEQ ID NO:. 4, idem) and protein segment Adenovirus ela, lys-arg-pro-arg-pro (SEQ ID NO: 5, idem.).

Other sequences can be found in Ding all and col. , (Dingwall, C, et al., TIBS 16: 478 (1991)). In another version, the subcellular localization component is a lysosome localization component. A known component for lysosome localization is a peptide that contains the lys-phe-glu-arg-gln segment (SEQ ID NO: 6, WO-A-0 2397). In yet another version, the subcellular localization component is a component of mitochondrial localization. A known component for the localization of mitochondria is a peptide that contains the sequence met-leu-ser-leu-arg-gln-ser- ile-arg-phe-phe-lys-pro-ala-thr-arg (SEQ IDN0: 7, idem.). However, other components or subcellular localization agents are also suitable. The term "masking agent" as used herein, refers to a molecule capable of masking all or part of the polynucleotide or anionic macromolecule, thereby increasing its circulatory half-life by inhibiting the attack of degradation reagents present in the circulation or by the blocking absorption of the reticuloendothelial system. An example of such a masking agent is polyethylene glycol (PEG). The PEG can have a molecular weight of about 700 to 20,000 daltons, preferably about 1800 to 6000 daltons. The compounds of formula I can be prepared by methods known in the art, for example, by analogy to the method described in Gene Therapy (1995) 2, 552-554. This invention also relates to a process for the preparation of new compounds of formula I. The invention also relates to the use of a compound of formula I as a carrier for the transfection of a cell with a polynucleotide or any other anionic macromolecule.

Examples of polynucleotides that can be transfected into cells by means of the novel compounds of this invention are deoxyribonucleic acids (DNA) and ribonucleic acids (RNA). Examples of anionic macromolecules other than polynucleotides are proteins, such as ribonucleoproteins and proteins used for immunization, e.g. ex. the viral proteins.

Examples of the DNA that can be transfected into cells by means of the novel compounds of this invention are plasmids and genes, especially those for which gene therapy protocols such as the transmembrane cystic fibrosis regulator (CFTR) have been developed. ), adenosine deaminase (ADA), thymidine kinase (tk) and HLA B7 / as well as reporter genes such as beta-galactosidase, luciferase, chloramphenicol acetyl transferase and alpha-1 antitrypsin. Other examples of DNA are oligodeoxynucleotides and their analogs used as desensitizers, aptamers or "triple helix" agents. Examples of RNA are ribozymes or oligoribonucleotide insensitizing molecules. The nature of the cell to be transfected is not strictly crucial. The cell can be a prokaryotic or eukaryotic cell, a mammalian cell or a plant cell. The transfection method using a compound of this invention can be carried out according to the methods known per se. To transfect a p cell ex. With DNA or RNA, the charge ratio +/- between the positively charged compound of formula I and the negatively charged DNA or RNA is in the order of 0.1 to 50, more preferably 0.1 to 10. Transfection it can be carried out in the presence of an auxiliary lipid, a short phospholipid chain and / or another known molecule competent for transfection. Examples of auxiliary lipids are phospholipids, such as phosphatidylcholine or the phosphatidylethanolamines or mixtures thereof. Preferred auxiliary lipids are phosphatidylethanolamines such as dioleoylphosphatidylethanolamine. Examples of short chain phospholipids are the phosphatidylcholines bearing two fatty acid radicals of 6 to 12 carbon atoms. The preferred short chain phospholipids are dicapril and dicapryloyl phosphatidylcholine. The auxiliary lipid and / or the short chain phospholipid are suitable in the form of a liposome, mixed micelles, organic solution or aqueous dispersion. Examples of competent transfection molecules include the cationic lipids as described by J.B. Behr in Bioconjugate Chem. 5: 382-389 (1994) and X. Gao and L. Huang in Gene Ther. 2: 710-722 (1995); polycations as described by A.V. Kabanov and V.A. Kabanov in Bioconjugate Chem. 6: 7-20 (1995); peptides and polymers and other non-viral systems of gene delivery as described by F.D. Ledley at Human Gene Therapy ("Human gene therapy") 6: 1129-1144 (1995). Other competent transfection molecules are those described by Legendre et al., (EP-A-95 118 338.3 and EP-A-96 100 603.8). For transfection, an appropriate amount of at least one compound of formula I, suitable in the form of an aqueous or organic solution, or in the form of liposomes, mixed micelles or micelles, is added to an aqueous solution of the molecule which is going to be transfected (eg the DNA of a plasmid) or vice versa. Next, a cationic lipid or any other molecule competent for transfection, an auxiliary lipid and optional nally, a short chain phospholipid, either as an aqueous solution or dispersion, as an organic solution or dispersion or in the form of liposomes, mixed micelles or micelles. Alternatively, the molecule to be transfected can be added to a composition comprising at least one compound according to this invention and, if desired, a cationic lipid or any other molecule competent for transfection, an auxiliary lipid and a phospholipid. short chain or vice versa. It is convenient that the solution be an aqueous or organic solution, an aqueous or organic dispersion, or a liposome, a mixed micelle or a micelle. The final composition may be in the form of liquid, solid (lyophilized, controlled evaporation), semi-solid or aerosolized. The optimal ratio between the components, ie, the compound of formula I, the molecule to be transfected and optionally, the additional constituents, depends on the cell to be transfected. The optimum charge ratio +/- between the compound of formula I and the molecule to be transfected varies between 0.1-50, preferably between 0.1-10. The optimum molar ratio between the compound of formula I and additional constituents such as a cationic lipid and / or another molecule competent for transfection and / or auxiliary lipids and / or a short chain phospholipid is 0.1-50, with higher Preference 0.1 to 10. For the transfection of cells in an animal or human patient, the composition can be administered orally, parenterally (iv, im, sc, id, ip), transdermally, pulmonary, nasal, rectal, ocular, ventricular, vascular (catheter) and intratumoral. In addition, the composition can be administered by high-speed impact administration on the surface of the skin. The transfection procedure can be measured by appropriate analysis protocols that are already known to those skilled in the art. In another aspect, this invention relates to compositions comprising at least one compound of formula I, and optionally a cationic lipid or any other known transfection competent molecule, an auxiliary lipid and / or a short chain phospholipid, and optionally a polynucleotide or any other anionic macromolecule. The following examples, which are not limiting, illustrate the invention further. Example 1 Preparation of a compound of formula I, wherein R1 is an integrating binding peptide and A is Dab-Dab-NHNH2 The synthesis of the compound of formula II (RGD- (Dab) -NHNH2) Gly-Gly-Cys-Arg-Gly-Asp-Met-Phe-Gly-Cys-Gly-Gly-Dab-Dab-N2H3 (II) was performed on a peptide synthesizer SP 650 (Labortec AG) using the Wang resin using an FMOC protocol as described in Gene Therapy (1995), 2, .552-554. The peptide was cleaved from the resin by hydrazinolysis. The resulting product was deprotected and subjected to an oxidative cyclization, obtaining the compound of formula II. The homogeneity of the purified peptide was confirmed by analytical RP-HPLC and the weight of the molecule was determined by ISP MS: 1329.5 [M + H] + calculated for CSoHßlN210lßS3 (1328.5). Example 2 10 μg of plasmid DNA (pGL3-CMV) is diluted with 250 μl of 10 mM Tris Cl buffer and pH 8.5. Then an appropriate amount of the compound obtained in Example 1 is mixed, RGD- (Dab) 2NHNH2, with the DNA diluted so that the charge ratio +/- between the RGD- (Dab) 2NHNH2 and the DNA is 2/1. Next, an appropriate volume of the resulting mixture containing 5 μg of the plasmid DNA is added per well of CaCo-2 cells grown in 6-well plates. 24 hours later, the activity of luciferase is measured. The results are indicated in Table 1. EXAMPLE 3 10 μg of plasmid DNA (pGL3-CMV) is diluted with 250 μl of 10 mM Tris Cl buffer and pH 8.5. Then an appropriate amount of the compound obtained in Example 1, RGD- (Dab) 2NHNH2, is mixed with the DNA diluted so that the charge ratio +/- between the RGD- (Dab) 2NHNH2 and the DNA is 2. /1. A dispersion of dioleoylphosphatidylethanolamine (DOPE) is then added to the mixture so that the molar ratio between DOPE and RGD- (Dab) 2NHNH2 is 5/1. Next, an appropriate volume of the resulting mixture containing five μg of plasmid DNA is added per well of CaCo-2 cells grown in 6-well plates. 24 hours later, the activity of luciferase is measured. The results are indicated in table 1.

Table 1 Example 4 7 nmoles of compound obtained in Example 1, RGD- (Dab) 2NHNH2 are mixed with 11 nmoles of dioleoyl-melittin, (D0-melittin, EP-A-96 100 603.8). The mixture is then added to 10 μg of plasmid pGL3-CMV DNA in a total volume of 200 μl of 10 mM Tris Cl buffer pH 8.5. This corresponds to a charge ratio +/- between the cationic charge of the two transfection agents and the anionic charge of the 4/1 DNA. Next, an appropriate volume of the resulting mixture containing 5 μg of plasmid DNA is added per well of CaCo-2 cells grown in 6-well plates. 24 hours later, the activity of luciferase is measured. Table 2 shows the efficiency of the transfection of the mixture RGD- (Dab) 2NHNH2 / DO-melittin together with that of the RGD- (Dab) 2NHNH2 and that of the DO-.melitin alone. Table 2 prepared according to example 4 EXAMPLE 5 1.75 nmoles of the compound obtained in example 1, RGD- (Dab) 2NHNH2 are mixed with 5.25 nmoles of the lipopeptide N? -palmitoyl-D- (a,? -diamino-butyryl) -L- ( acid, p-diaminobutyric) hydrazide (Palm- (Dab) 2NHNH2). The mixture is then added to 10 μg of plasmid pGL3-CMV DNA in a total volume of 200 μl of 10 mM TrisCl buffer pH 8.5. This corresponds to a charge ratio +/- between the cationic charge of the two transfection agents and the anionic charge of the DNA, of 2/1. Next, the phosphatidylethanolamine (DOPE) dispersion is added to the mixture such that the molar ratio between the DOPE and the two transfection agents is 5/1. Next, an appropriate volume of the resulting mixture containing five μg of the plasmid DNA is added per well of the CaCo-2 cells grown in 6-well plates. 24 hours later, the activity of luciferase is measured. Table 3 shows the transfection efficiency of the mixture RGD- (Dab) 2NHNH2 / Palm- (Dab) 2NHNH2 together with that of RGD- (Dab) 2NHNH2 and that of Palm- (Dab) 2NHNH2 alone. Table 3 * prepared according to the example 5 *****

Claims (14)

1. CLAIMS 1. Alpha, gamma-diaminobutyric acid (DAB) characterized in that they contain oligopeptide derivatives of formula R '-NH-A wherein R' is a cell recognition agent, a membrane permeabilizing agent, a subcellular localization agent or an agent masking, - A is an oligopeptide devoid of an amino group, containing from 1 to 200 amino acids, wherein at least one of the amino acids is alpha, gamma-diaminobutyric acid (DAB), and the terminal carboxyl group of the oligopeptide is converts to an amide, lower alkyl amide, lower dialkyl amide or hydrazide.
2. 2. Compounds according to claim 1, characterized in that the cell recognition agent is an antibody or antibody fragment, a hormone, a protein growth factor, a cytokine and / or a ligand directed to a cell surface receptor .
3. 3. Compounds according to claims 1 or 2, characterized in that the subcellular localization agent is a nuclear localization molecule capable of delivering a polynucleotide or an anionic macromolecule from the cytoplasm to the nucleus of the cell.
4. 4. Compounds according to claims 1 to 3, characterized in that the membrane permeabilization agent is a molecule that assists the passage of a polynucleotide or an anionic macromolecule through a membrane.
5. 5 . Compounds according to claims 1 to 4 characterized in that the masking agent is a molecule capable of increasing the circulating half-life of the polynucleotide or the anionic macromolecule. 6 Compounds according to any one of the r-claims 1 to 5, characterized in that the carbonyl terminal group of the oligopeptide is converted to a hydrazide. 7. The use of at least one compound of formula I according to claims 1 to 6 as a carrier for the transfection of cells with a polynucleotide or any other anionic macromolecules. 8. The use according to claim 6 in the presence of a cationic lipid or any other molecule competent for transfection. 9. The use according to any one of claims 7 and 8 in the presence of an auxiliary lipid. 10. Use according to any one of claims 7 to 9 in the presence of a short chain phospholipid. 11. A composition characterized in that it comprises at least one compound of formula I according to any one of claims 1 to 6, and optionally an auxiliary lipid, a short chain phospholipid and / or another molecule competent for transfection. 12. A composition as in claim 11, characterized in that the components are in the form of an aqueous or organic solution, an aqueous or organic dispersion or a liposome or micelle. 13 A composition as in claims 11 or 12, characterized in that the composition has a solid, liquid, semisolid or aerosol form. 14 A composition as in claims 11 to 13 characterized in that it comprises a polynucleotide or any other anionic macromolecule.