MXPA01005746A - REVERSIBLE AQUEOUS pH SENSITIVE LIPIDIZING REAGENTS, COMPOSITIONS AND METHODS OF USE - Google Patents

Abstract

The present invention provides lipidized conjugates comprising an amino group-containing biologically active substance and a lipophilic group capable of penetrating a biological membrane. Under neutral or mildly acidic conditions, including those found in vivo, the free amino group-containing biologically active substance is released from the conjugate by hydrolysis of an amide bond. The present invention is also directed to methods of preparing lipidizing agents and lipidized conjugates, pharmaceutical compositions comprising lipidized conjugates and methods of increasing the delivery of amino group-containing substances into a cell. Preferred amino group-containing substances include peptides, proteins and derivatives thereof.

Description

REVERSIBLE AQUEOUS REAGENTS OF LJPIDIZATION, pH SENSITIVE, COMPOSITIONS AND METHODS OF USE BACKGROUND OF THE INVENTION Field of the Invention The present invention relates generally to the fields of biology and medicine. More particularly, the invention is directed to compounds, methods and compositions useful in increasing in mammals the transport and distribution of hydrophilic molecules having an amino group, in particular peptides and proteins.

Related Technique Advances in biochemistry have made possible the production of large quantities of therapeutically active and pure proteins and peptides. Currently, the therapeutic effects of most of these agents can be achieved only when they are administered via invasive routes, such as by injection. Since most proteins have very short half-lives, effective concentrations of these agents can be Rsf: 129946 maintained only when administered by frequent injections. Although administration of the protein by injection is the most effective means of its administration in vivo, the patient's tolerance of multiple injections is very poor. In addition, drug injection requires training and experience that may not always be transferable to patients. In cases where protein drugs have a role to save life, administration by injection may be acceptable to patients. However, in cases where protein drugs are only one of several possible therapies, injections of peptides and proteins are not likely to be accepted by patients. Therefore, alternative routes of protein and peptide administration need to be developed. Such alternative routes may include the oral, nasal, oral, pulmonary, rectal and ocular routes. Without exception, these routes are less effective than parenteral routes of administration, but they are still much more attractive than parenteral routes because they offer convenience and control to patients. The oral route is particularly attractive because it is the most convenient and with which the patient most meets.

The mucosal barriers, which separate the internal part of the body from the outside (for example, the gastrointestinal, ocular, pulmonary, rectal and nasal mucosa), comprise a layer of tightly bound cellular monolayers which strictly regulate the transport of the molecules. The individual cells in the barriers are joined by strong bonds that regulate the entry into the intercellular space. Therefore, the mucosa is at the first level a physical barrier, the transport through which it depends either on the transcellular or paracellular pathways (Lee, VHL, Critical Rev. Ther. Drug Delivery Sys. 5: 69- 97 (1988)). Paracellular transport through strong junctions filled with water is restricted to small molecules (molecular weight less than 1 kDa) and is essentially a diffusion process driven by a concentration gradient across the mucosa (Lee, VHL, Cri). Rev. Ther, Drug Delivery Sys. 5: 69-97 (1988), Artursson, P. and Magnusson, C, J. Pharm. Sci. 79: 595-600 (1990)). Strong junctions comprise less than 0.5% of the total surface area of the mucosa (González Mariscal, LM, et al., J. Membrane biol.86: 113-125 (1985); Vetvicka, V. and Lubor, F., cri tical. Rev. Ther.Drug Deliv Sys. 5: 141-170 (1988), therefore, they play only a minor role in the transport of protein drugs through the mucosa.The transcellular transport of small drugs occurs efficiently with the condition that the physical or chemical properties of the drug are adequate to transport it through the hydrophobic cell barriers, however, the cellular transport of proteins and peptides is restricted to the process of transcytosis (Shem., W.C. and collaborators, Adv. Drug. Deliv. Rev. 8: 93-113 (1992)). Transcytosis is a complex process in which proteins and peptides are picked up in vesicles from one side of a cell, and are subsequently fired through the cell to the other side of the cell, where they are discharged from the endocytic vesicles (Mostov, KE and Semister, NE, Cell 43: 389-390 (1985)). The cellular membrane of mucosal barriers in a hydrophobic lipid bilayer which has no affinity for charged, hydrophilic macromolecules, such as proteins and peptides. In addition, mucosal cells can secrete mucin which can act as a barrier to the transport of many macromolecules (Ed ards, P., Bri tish Med. Bull. 34: 55-56 (1978)). Therefore, unless there are specific transport mechanisms for proteins and peptides, their immediate transport through mucosal barriers is almost negligible. In addition to providing a tight or strong physical barrier for the transport of proteins and peptides, mucosal barriers possess enzymes that can degrade proteins and peptides before, after or during their passage through the mucosa. This barrier is referred to as the enzymatic barrier. The enzymatic barrier consists of endo- and exopeptidase enzymes which break proteins and peptides at their ends or within their structure. The enzymatic activity of several mucous membranes has been studied and the results showed that there is substantial protease activity in the homogenate of buccal, nasal, rectal and vaginal mucosa of albino rabbits and that these activities are comparable to those present in the ileum (Lee, VHL, Critical, Rev. Ther, Drug Delivery, Sys. 5: 69-97 (1988)). Therefore, notwithstanding the mucosa that is considered, the enzymatic barrier present will strongly characterize the degradation of the protein and peptide molecules. The N and C ends of the peptides are charged and the presence of the charged side chains imparts highly hydrophilic characteristics on these macromolecules. In addition, the presence of charged side chains means that proteins and peptides have strong hydrogen bonding capabilities; this hydrogen bonding capacity has been demonstrated, which plays a major role in inhibiting the transport of even small peptides through cell membranes (Conradi, R.A., et al., Pharm. Res. 8: 1453-1460 (1991)). Therefore, the size and hydrophilic nature of the proteins and peptides combine to severely restrict their transport through mucosal barriers. A procedure that has been used to alter the physical nature of mucosal barriers is the use of penetration enhancers. The use of penetration enhancers is based on the breakdown of cellular barriers by low molecular weight agents which can fluidize cell membranes (Kaji, H., et al., Life Sci. 37: 523-530 (1985)). ), open the tight joints (Inagaki, M., and collaborators, Rhinology 23: 213-221 (1985)), and create pores in the cell membrane (Gordon, S ,, and collaborators, Proc. Na ti. Acad. Sci USA 82: 7419-7423 (1985), Lee, VHL, Critical Rev. Ther, Drug Delivery Syst. 8: 91-192 (1991)). The use of these agents leads to a non-specific loss of the integrity of the barrier and can lead to the absorption of a variety of large molecules which can be toxic to cells in vivo.

Protease inhibitors have been co-administered with proteins and peptides and have shown some limited activity in enhancing or enhancing the absorption of these macromolecules in vivo (Kidron, M, et al., Life Sci. 31: 2837-2841 (1982); Takaroi; , K., et al., Biochem. Biophys. Res. Comm. 137: 682-687 (1986)). The safety and long-term effects of this procedure have yet to be fully investigated. The prodrug process is based on the modification of the peptides in a manner that will protect them from enzymatic degradation and recognition. This has been achieved by blocking the vulnerable groups on the peptides by amidation and acylation. The prodrug procedure has thus proven to be very useful only for small peptides which have easily identifiable activity domains. The reduction in size is another feasible procedure to increase the transport potential of proteins. Nonetheless, active protein sites need to be mapped before size reduction can be attempted. In general, this procedure is difficult to apply to most proteins.

Carrier ligands, by virtue of their properties, can alter the cellular uptake and the transport characteristics of proteins and peptides. The essence of this method is that a protein or peptide impermeable to the cells is covalently bound to a carrier that is highly transported in the cells. L03 mechanisms through which the carrier ligands become subject to endocytosis and transcytosis, are important in deciding the adequacy of the carrier to increase the transport of proteins and peptides. The macromolecular carriers are hydrophilic and do not divide in the membrane. Therefore, the transport of large polymeric carriers within the cells is mediated by the affinity of the carrier for the cell membrane. In general, uptake of the macromolecular conjugate begins with the binding to the cell membrane. The binding of the carrier to the cells can be specific (for example, the binding of antibodies to cell surface antigens), nonspecific (the binding of the cationic ligand or the lectins to the sugars on the cell surface), or mediated by the receptor (the binding of transferrin or insulin to its receptors). Once the carrier is bound to the cell surface, it is picked up inside the vesicles. These vesicles are then processed gradually and can be routed to several ways. One way is the recycling of the vesicle back to the membrane. Another way, which is destructive to the conjugate, is fusion with lysosomes. An alternative route, and one which leads to transcytosis of the conjugate, is the fusion of the vesicle with the membrane opposite the side from which it was derived. The correct balance between the processes of endocytosis and transcytosis determines the distribution of a protein conjugate towards its target. For example, endocytosis can determine the extent to which a conjugate is picked up by the target cell, but transcytosis determines whether a conjugate reaches its target or not (Shen, WC, et al., Adv. Drug, Deliv. Rev. 8: 93-113 (1992)). For successful absorption through the gastrointestinal tract, a conjugate must bind to the apical membrane of the gastrointestinal mucosa, become internalized within the mucosal cells, be distributed through the cells, and finally be released from the basolateral membrane. Current literature contains many reports showing that non-specific carriers such as polylysines (Shen, WC and Ryser, HJP, Proc. Na ti, Acad. Sci USA 78: 7589-7593 (1981)) and lectins (Broadwell, RD, et al, Proc. Na ti, Acad. Sci. USA 85: 632-646 (1988)), and specific carriers such as transferrin (Wan, J., et al., J. Biol. Chem. 257: 13446-123450 (1992)), asialoglycoprotein (Seth, R., et al., J. Infect. Diseases 168: 994-999 (1993)), and antibodies (Vietta, ES, J. Clin. Immunol. 15S-18S (1990)) can increase or improve the endocytosis of the proteins towards the cells. The reports that have to do with the transcytotic carriers for proteins are less, and very few studies have quantified the transport of protein conjugates through cellular barriers. Wheat germ agglutinin (Broad ell, R.D. and collaborators, Proc. Na ti. Acad. Sci.

USA 85: 632-646 (1988) and an antitransferrin / methotrexate conjugate (Friden, PM and Walus, LR, Adv. Exp. Med. Biol. 331: 129-136 (1993)) have shown that they are subjected to transcytosis through of the blood-brain barrier in vivo. Also, conjugates of horseradish peroxidase polylysine (HRP) and a transferrin conjugate of HRP have been shown to undergo transcytosis through the cell monolayers in vi tro (Wan, J. and Shen, WC Pharm. Res. : S-5 (1991), Taub, ME and Shen, WC, J. Cell Physiol., 150: 283-290 (1992), Wan, J., et al., Biol. Chem. 267: 13446-13450 (1992). )).

Fatty acids, as constituents of phospholipids, constitute the largest volume of cell membranes. These are commercially available and relatively cheap. Due to their lipid nature, fatty acids can be easily divided within and interact with the cell membrane in a non-toxic manner. Therefore, fatty acids potentially represent the most useful carrier ligand for the distribution of proteins and peptides. Strategies that can use acid degrees in the distribution of proteins and peptides include the covalent modification of proteins and peptides and the use of acid-grade emulsions. Some studies have reported the successful use of fatty acid emulsions to distribute the peptide and proteins in vivo (Yoshika a, H., et al., Pharm. Res. 2: 249-251 (1985); Fix, JA; collaborators, Am. J. Physiol. 251: G332-G340 (1986)). The mechanism through which fatty acid emulsions influence the absorption of proteins and peptides is still unknown. Fatty acid emulsions can open tight junctions, solubilize membranes, remove proteins and peptides from the gastrointestinal environment, and carry proteins and peptides through the gastrointestinal mucosa as part of their absorption (Smith, P., et al. Adv. Drug. Delivery Rev. 8: 253-290 (1992)). The last mechanism has been proposed, but it is inconsistent with current knowledge regarding the mechanism of fat absorption. A more logical strategy to distribute the proteins and peptides through the gastrointestinal epithelium is to make use of the fatty acids as non-specific membrane adsorbing agents. Several studies have shown that a non-specific membrane binding agent linked to a protein can promote the transcytosis of a protein conjugate through the cells in vi tro (Wan, J., et al., J. Cell. Physiol. 145: 9-15 (1990); Taub, ME and Shen, WC, J. Cell. Physiol. 150: 283-290 (1992)). It has also been shown that the conjugation of fatty acid improves the uptake of macromolecules in and through cell membranes (Letsinger, R., and collaborators, Proc. Na ti. Acad. Sci. USA 86: 6553-6556 (1989); Kabanov, A., and collaborators, Protein Eng. 3: 39-42 (1989)). However, there have been difficulties in conjugating the fatty acids to the peptides and proteins, including: (1) the lack of solubility of the fatty acids in the aqueous solution for the conjugation reaction; (2) the loss of the biological activity of the peptides and the proteins after the acylation of the fatty acid; and (3) the lack of solubility of the conjugated fatty acid peptides in aqueous solutions (see, for example, Hashimoto, M. et al., Pharm. Res. 6: 171-176 (1989); Martins, MBF, et al. Biochimie 72: 671-675 (1990); Muranishi, S., et al., Pharm. Res. 6: 171-176 (1989); Martins, MBF, et al. (1991); Robert, S., and collaborators, Biochem. Biophys. Res. Commun. 196: 447-454 (1993)). Once distributed within the cells, the peptides and proteins must be released from their carrier. Published PCT Applications Nos. WO 96/22773 and WO 98/13007 describe the transcellular distribution and release of peptides and proteins containing sulfhydryl group. The cellular absorption of the hydrophilic molecules containing sulfhydryl groups can be increased by conjugation with a fatty acid through a disulfide bond. The labile disulfide bond is easily reduced, providing a mechanism for the release of the hydrophilic compounds from the fatty acid portion once inside the body. In addition to the reduction of the disulfide bond, other mechanisms for the release of the biologically active hydrophilic compounds from the carrier systems include hydrolysis and photolytic cleavage of the bond (see for example U.S. Patent No. 5,505,931 and references cited in these). Distribution systems based on hydrolysis are known in which a biologically active amine is conjugated with an organic acid that incorporates a monoclonal antibody or other substrate for targeting the specific cells. (See U.S. Patents Nos. 4,764,368, 4,618,492, 5,505,931 and 5,563,250). After specific binding to the target cell, these conjugates distribute the active amine (typically in the form of an amide) within the cell where the hydrolysis (of the amide) releases the free amide into the cell. The success of hydrolysis-based administration or delivery systems of the prior art has inspired the search for improved drug-carrier conjugates capable of delivering a biologically active amino-containing compound to the interior of cells. Improved synthetic strategies and treatment techniques are currently being developed.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to the new drug-carrier conjugates and to the synthetic strategies suitable for their production. Accordingly, the present invention is directed to synthetic methods, intermediates and end products useful for the uptake and release of biologically active amino-containing compounds. In particular, the invention relates to the compounds of General Formula I wherein R2 is selected from the group consisting of hydrogen, halo, alkyl or aryl, wherein the alkyl or aryl groups are optionally substituted with one or more alkoxy, alkoxyalkyl, alkanoyl, nitro, cycloalkyl, alkenyl, alkynyl, alkanoyloxy groups, alkyl or halogen atoms; R3 is a lipophilic group; one of R4 and R5 is a biologically active amino group containing the selected substance, of the group consisting of a drug containing the amino group, a natural or unnatural amino acid, a peptide and a protein and the other of R4 and R5 is OR6 wherein R6 is hydrogen, an alkali metal or a negative charge; X is oxygen or sulfur; And it is a natural amino acid or non-natural bridge-forming agent; n is zero or 1; and m is an integer from zero to 10, The present invention also relates to the compounds of General Formula II wherein R is hydrogen, halo, alkyl, aryl, wherein the alkyl and aryl groups are optionally substituted with one or more of alkoxy, alkoxyalkyl, alkanoyl, nitro, cycloalkyl, alkenyl, alkynyl, alkanoyloxy, alkyl or halogen atoms; R3 is a lipophilic group; X is oxygen or sulfur; And it is a natural amino acid or non-natural bridge-forming agent; n is zero or 1; and m is an integer from zero to 10, The present invention also relates to the compounds of General Formula III or a pharmaceutically acceptable salt thereof, wherein R 2 is hydrogen, halo, alkyl, or aryl, wherein the alkyl and aryl groups are optionally substituted with one or more of alkoxy, alkoxyalkyl, alkanoyl, nitro, cycloalkyl, alkenyl, alkynyl, alkanoyloxy, alkyl or halogen atoms; R3 is a lipophilic group; X is oxygen or sulfur; And it is a natural amino acid or non-natural bridge-forming agent; n is zero or 1; and m is an integer from zero to 10. The present invention also relates to methods for forming the conjugates of General Formula I from General Formula II and a substance containing the biologically active amino group.

The present invention also relates to methods for forming the compounds of General Formula II from maleic acid derivatives and the corresponding thiols or alcohols. The present invention also relates to methods for increasing the absorption or prolongation of blood and tissue retention in a mammal of a substance containing the biologically active amino group, in which a conjugate of General Formula I is administered to the mammal in a pharmaceutically acceptable form. The present invention also relates to methods for increasing the distribution of hydrophilic amine-containing compounds to the inner part of the cell having a mucosal barrier, in which a conjugate of General Formula I is contacted with the cell , whereby the conjugate penetrates the mucosal barrier of the cell and the free amine is released by hydrolysis of an amide bond. The present invention also relates to pharmaceutical compositions comprising a compound of General Formula I. The foregoing and other advantages, features, modalities, aspects and objectives of the present invention will be clear to those skilled in the relevant art areas, Based on the description, teaching and guidance presented in this.

BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1 shows the pH dependence of the release of tyramine from a lipidizing carrier reagent (REAL-tyramine) according to the invention. The data show the mean and the standard deviation of 3 experiments. FIGURE 2 shows the cumulative urine production of diabetic rats after subcutaneous injection of 5 μg / kg AVP (arginine-vasopressin), palmitil-AVP and REAL-AVP according to the invention. The data show the mean and the standard deviation of the measurements of 3 rats. FIGURE 3 shows the cumulative urine production of diabetic rats in 24 hours after subcutaneous injection of 5 μg / kg dß AVP, palmityl-AVP and REA1-AVP according to the invention. The data show the mean and the standard deviation of 3 experiments. FIGURE 4 shows the change in blood glucose level in diabetic rats subjected to fasting, after subcutaneous injection of 0.35 U / kg of insulin, compared to the subcutaneous injection of 0.35 'U / kg of REAL-insulin from the invention. The data show the mean and the standard deviation of the measurement with 2 rats. FIGURE 5 shows the prolonged effect on blood glucose levels in fasting diabetic rats of the subcutaneous injection of 0.5 U / kg of insulin, compared to the subcutaneous injection of 0.5 U / kg of REAL-insulin of the invention. The data show the mean and the standard deviation of measurements with 2 rats. FIGURE 6 shows the short-term effect on the blood glucose level of diabetic fasting rats after oral administration of 10 U / kg of REAL-insulin, insulin and placebo. The data show the mean and the standard deviation of the measurements with 4 rats.

DETAILED DESCRIPTION OF THE INVENTION According to the present invention, a compound containing the biologically active amine (for example an amino acid, peptide or protein) is coupled to a lipophilic derivative via a reversible amide bond. The lipophilic group of such a conjugate is linked to the apical side of a cell membrane and facilitates the transport of the conjugate through the cell membrane. Once inside the cell membrane, the compound containing the biologically active amine is released into the interstitial fluid as a result of the hydrolysis of the amide bond. According to one aspect of the present invention, the conjugates of General Formula I are provided wherein R 2 is hydrogen, halo, alkyl, or aryl, wherein the alkyl and aryl groups are optionally substituted with one or more alkoxy, alkoxyalkyl, alkanoyl, nitro, cycloalkyl, alkenyl, alkynyl, alkanoyloxy, alkyl or halogen groups; R3 is a lipophilic group; one of R4 and R5 represents a biologically active amino group containing the substance selected from the group consisting of drugs containing the amino group, natural or unnatural amino acids, peptides and proteins and the other of R4 and R5 is OR6 wherein R6 represents hydrogen, an alkali metal or a negative charge; X is oxygen or sulfur; And it is a natural amino acid or non-natural bridge-forming agent; n is zero or 1; and m is an integer from zero to 10, According to another aspect of the present invention, the compounds of General Formula II are provided wherein R2 is hydrogen, halo, alkyl, or aryl, wherein the alkyl and aryl groups are optionally substituted with one or more of alkoxy, alkoxyalkyl, alkanoyl, nitro, cycloalkyl, alkenyl, alkynyl, alkanoyloxy, alkyl or halogen atoms; R3 is a lipophilic group; X is oxygen or sulfur; And it is a natural amino acid or non-natural bridge-forming agent; n is zero or 1; and m is an integer from zero to 10, According to yet another aspect of the present invention, the compounds of Formula III are provided or a non-toxic pharmaceutically acceptable salt thereof, wherein R 2 is hydrogen, halo, alkyl, or aryl, wherein the alkyl and aryl groups are optionally substituted with one or more of alkoxy, alkoxyalkyl, alkanoyl, nitro, cycloalkyl, alkenyl, alkynyl, alkanoyloxy, alkyl or halogen atoms; R3 is a lipophilic group; X is oxygen or sulfur; And it is a natural amino acid or non-natural bridge-forming agent; n is zero or 1; and m is an integer from zero to 10. Typical alkyl groups include alkyl groups of 1 to 6 carbon atoms including methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl groups , 2-pentyl, 3-pentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, and the like.

Typical alkoxy groups include oxygen substituted with any of the alkyl groups mentioned above. Typical alkoxyalkyl groups include any of the aforementioned alkyl groups, substituted with an alkoxy group, such as methoxymethyl, ethoxymethyl, propoxymethyl, butoxymethyl, pentoxymethyl, hexoxymethyl, methoxyethyl, methoxypropyl, methoxybutyl, methoxypentyl, methoxyhexyl, and the like. Preferred aryl groups are aryl groups of 6 to 14 carbon atoms and typically include phenyl, naphthyl, fluorenyl, phenanthryl, and anthracyl. Typical alkoxy-substituted aryl groups include the above aryl groups substituted with one or more of the above alkoxy groups, for example, 3-methoxyphenyl, 2-ethoxyphenyl, and the like. Typical alkyl substituted aryl groups include any of the above aryl groups substituted with any of the alkyl groups of 1 to 6 carbon atoms, including the group Ph (CH2) n, where n is 1-6, for example, tolyl, o-, m-, and p-xylyl, ethylphenyl, 1-propylphenyl, 2-propylphenyl, 1-butylphenyl, 2-butylphenyl, t-butylphenyl, 1-pentylphenyl, 2-pentylphenyl, 3-pentylphenyl.

Typical alkenyl groups include alkenyl groups of 2 to 6 carbon atoms, for example ethenyl, 2-propenyl, isopropenyl, 2-butenyl, 3-butenyl, 4-pentenyl, 3-pentenyl, 2-pentenyl, 5-hexenyl, 4-hexenyl, 3-hexenyl, and 2-hexenyl. Typical alkynyl groups include the groups alkynyl of 2 to 6 carbon atoms, for example, ethynyl, 2-propenyl, 2-butynyl, 3-butynyl, 4-pentynyl, 3-pentynyl, 2-pentynyl, 5-hexynyl, 4-hexynyl, 3-hexynyl, and 2-hexynyl. Typical alkenyl or alkynyl substituted aryl groups include any of the aforementioned aryl groups of 6 to 14 carbon atoms, substituted with any of the alkenyl groups of 2 to 6 carbon atoms or alkynyl of 2 to 6 carbon atoms above mentioned, for example, the ethynylphenyl, 1-propenylphenyl, 2-propenylphenyl, 1-butenylphenyl, 2-butenylphenyl, 1-pentenylphenyl, 2-pentenylphenyl, 3-pentenylphenyl, 1-hexenylphenyl, 2-hexenylphenyl, 3-hexenylphenyl, ethynylphenyl, 1-propynylphenyl, 2-propynylphenyl, 1-butynylphenyl, 2-butynylphenyl, 1-pentinylphenyl, 2-pentinylphenyl, 3-pentinylphenyl, 1-hexinylphenyl, 2-hexinylphenyl, 3-hexinylphenyl.

Typical halo groups include fluorine, chlorine, bromine, and iodine. Typical halo substituted alkyl groups include alkyl groups of 1 to 6 carbon atoms substituted by one or more fluorine, chlorine, bromine, or iodine atoms, for example fluoromethyl, difluoromethyl, trifluoromethyl, pentafluoroethyl, 1, 1- difluoroethyl, and trichloromethyl. Typical alkanoyl groups include C (O-alkanoyl having 1 to 5 carbon atoms, for example, the acetyl, propionyl, butanoyl, pentanoyl, and hexanoyl groups, or an arylalkanoyl group, for example, a C (0) -alkanoyl group from 1 to 5 carbon atoms substituted with any of the above aryl groups Typical cycloalkyl groups include cycloalkyl groups of 3 to 8 carbon atoms including cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl groups. In yet another aspect of the present invention, methods are provided for the formation of the conjugates of General Formula I from the compounds of General Formula II and a substance containing the amino group. present invention, methods for forming the compounds of General Formula II from maleic acid derivatives and thiols and alcohols are provided. In accordance with the present invention, methods are provided for increasing the absorption or prolongation of blood and tissue retention in a mammal, of a substance containing the biologically active amino group, in which a conjugate of General Formula I is administered to the mammal (e.g., in the form of emulsions, nanoparticles (e.g., solid lipid nanoparticles), liposomes, microspheres, microcapsules, aerosols, through inhalation, and transdermal dosage forms). According to still another aspect of the present invention methods are provided for increasing the distribution of the compounds containing the hydrophilic amine into a cell having a mucosal barrier, in which a conjugate of General Formula I is placed in contact with the cell, whereby the conjugate penetrates the mucosal barrier of the cell and the free amine is released by hydrolysis of an amide bond. The term "lipophilic group" as used herein refers to either a lipid of natural origin per se, a hydrophobic branched or unbranched hydrocarbon comprising about 4 to about 26 carbon atoms, preferably about 5 to about 19 carbon atoms, a fatty acid or ester thereof, or a surfactant. Suitable lipophilic groups include, but are not limited to, long chain alkanoyl groups including: palmityl (C? 5H3?), oleyl (C? 5H29), stearyl (C? 7H35), lauryl (CnH23), colyl, and myristyl (C? 3H27). The term "natural or non-natural amino acid" as used herein refers to any of the 21 naturally occurring amino acids as well as the amino acids of the D form, the blocked amino acids of the L and D forms such as those blocked by amidation or acylation, substituted amino acids (for example, those substituted with a spherically hindered alkyl group or a cycloalkyl group such as cyclopropyl or cyclobutyl) in which the solution introduces a conformational restriction on the amino acid. The amino acids of natural origin, preferred for use in the present invention as amino acids or components of a peptide or protein are alanine, arginine, asparagine, aspartic acid, citrulline, cysteine, cystine, α-glutamic acid, glutamine, glycine, histidine, isoleucine, norleucine, leucine, lysine, methionine, ornithine, phenylalanine, proline, hydroxyproline, serine, threonine, tryptophan, tyrosine, valine,? -carboxyglutamate, or O-fosfoserin. The amino acids of non-natural origin preferred for use in the present invention as amino acids or components of peptides or proteins are any of the β-amino acids, for example, β-alanine, α-aminobutyric acid, α-aminobutyric acid, α-amino acid. (aminophenyl) butyric, -aminoisobutyric acid, e-aminocaproic acid, 7-aminoheptanoic acid, aminobenzoic acid, aminophenylacetic acid, aminophenylbutyric acid, cysteine (ACM), methionisulfone, phenylglycine, norvaline, ornithine, d-ornithine, p-nitrophenylalanine, acid 1, 2, 3, 4-tetrahydroisoquinoline-3-carboxylic acid and thioproline. Also contemplated are the amino acid derivatives of the Formula: where p is 1 to 10. The term "substance qu? = =" contains the biologically active amino group "as used herein, refers to any substance that has biological activity when introduced into a cell, and that includes in its structure a primary or secondary amino group capable of forming an amine bond through acylation. Substances that do not include a primary or secondary amine may be suitably derivatized to be suitable for conjugation with compounds of General Formula II or III. For example, compounds having carboxyl groups can be reacted with a suitable diamine, for example, a diamine of 2 to 10 carbon atoms such as ethylene diamine, propylene diamine, 1,4-diaminobutane, spermine or spermidine and the like, in the presence of a compound of General Formula II or III and a water-soluble carbodiimide (e.g., EDC) as a coupling reagent. In this manner, the diamine serves as a means for coupling a biologically active compound, which does not include a primary or secondary amine, to a compound of the General Formula II or III via the formation of the amide bond. Preferred amine-containing drugs include, but are not limited to, tyramine, arginine, vasopressin, insulin (Czech, MP, Ann.Rev. Biochem. 46: 359 (1977)), calcitonin (Brown, EM, and Aurbach, GD. , Vi tam Horm 38: 236 (1980)), desmopressin (Vavra, et al, J. Pharmacol. Exp. Ther 188: 241 (1974)), interferon-a, -β and -? (Stiem, ER, Ann, Rev. inter.Med. 96: 80-93 (1982)), interleukin-2, -3, -4, -6 and -11 (Kluth, DC and Ress, AJ, Semin. Nephrol, 16: 576-582 (1996)); Holyoake, T.L. Blood Rev. 10: 169-200 (1996)), G-CSF (Spiekermann, K. and collaborators, Leukemia 11: 466-478 (1997)), GM-CSF (Jonuleit, H., et al., Arch.

Dermatol. Res. 289: 1-8 (1996), human growth hormone (Strobl, J.S. and Thomas, M.J., Pharmacol. Rev. 46: 1-34 81994)), erythropoietin (Spivak, J.L., Semin.

Hematol. 30: 2-11 (1993)), vasopressin (Schrober, E. and Lubke, K., The peptide 2: 336-350 (1996)), octreotide (Sheppard, M.C. and Steward, P.M., Metabolism, Clinical and Experimental 45: 63-64 81996)), aprotinin (Haderland, G.

MacConn, R., Fed Proc. 38: 2760-2767 (1979)), oxytocin (Nachtmann, F., and collaborators, in Anal, Prof. Drug.

Subst., Vol. 10, Florey, K., ed., Academic Press, New York.

NY (1981), pp. 563-600), β-TGF (Moses, HL and Serra, R., Curr Opin Genet, Dev 6: 581-586 (1996)), BDNF (Apfel, SC and Kessler, JA, Baillieres, Clin. Neurol, 4: 593-606 (1995)), b-FGF (Bikfalvi, A., et al., Endocr. Rev. 18: 26-45 (1997)), PDGF (Hughes, AD, et al., Gen. Pharmacol 27: 1079-1089 (1996)), TNF (Makhno, PE, et al Swiss Swiss Surg, 4: 182-185 (1995)), atrial natriuretic peptide (Nakao, K., Curr Opin, Nephrol, Hypertens. 2: 45-50 (1993)), reline (Schwabe, C, et al., Recent Progr. Horm Res. 34: 123-211 (1978)), amyrin (Rink, TJ, et al., Trends, Pharmacol. 14: 113-118 (1993)), deoxyriburanouclease (Laskowski, in The Enzymes, Vol. 2, Boyer, PD, ed., Academic Press, New York, NY (1971), pp. 289-311), EGF ( Carpenter, G., Curr, Opin, Cell, Biol. 5: 261-264 (1993)), hirudin (Markwardt, Methods, Enzymol 19: 924 (1970)), neocarzinostatin (Dedon, PC and Goldberg, IH, Chem. Res. Toxicol 311-332 (1992), hemorrhagic peptide (Paukovi ts, W.R., and collaborators, Cancer Trea t. Rev. 17: 347-354 (1990)), and somatostatin (Moss, R.L., Ann. Rev. Physiol. 41: 617 (1979)). For purposes of the present invention, the term "peptide" refers to natural or unnatural amino acid chains comprising 2 to 100 amino acids and the term "protein" to natural or unnatural amino acid chains comprising more than 100 amino acids. The proteins and peptides can be isolated from natural sources or prepared by means well known in the art, such as recombinant DNA technology or solid state synthesis. It is contemplated that the peptides and proteins used in accordance with the present invention may comprise only L-amino acids of natural origin, combinations of L-amino acids and other amino acids (including D-amino acids and modified amino acids), or only amino acids other than L-amino acids. In order to form a conjugate of General Formula I, the peptide or protein must possess at least one reactive amino group. The reactive amino group can be part of a side chain of amino acids, or a terminal amino group of the peptide or protein backbone, or introduced by chemical modification of the functional groups in the peptide or protein molecules. The peptides can be homo- or heteropropeptides and can include natural amino acids, synthetic amino acids, or any combination thereof. Also included within the scope of the present invention are the pharmaceutically acceptable, non-toxic salts of the compounds of the invention. In particular, alkali metal carboxylates, formed by known methods such as the addition of an alkali metal halide to the corresponding carboxylic acid, are also contemplated. Such salts include the sodium, potassium, lithium and ammonium salts. The term "negative charge" as used herein refers to any single solvated, solvated or complexed pair of electrons capable of providing the anionic character to a carboxylate group. The term "alkali metal" as used herein refers to any of the Group I or Group II metals, for example, sodium, potassium, lithium, calcium, and magnesium.

The preferred animal subject of the present invention is a mammal. The term "mammal" refers to an individual that belongs to the Mammalia class. The invention is particularly useful in the treatment of human patients. The term "treatment" refers to the administration to subjects of a lipidization conjugate for purposes which may include the prevention, amelioration, or cure of a disease or condition. It is considered that the medications are provided "in combination" with another if they are provided to the patient concurrently or if the time between the administration of each medication is such, to allow an overlap of the biological activity. In a preferred embodiment, at least one conjugate is present or is administered as part of a pharmaceutical composition. The pharmaceutical compositions for administration according to the present invention may comprise at least one conjugate according to the present invention in a pharmaceutically acceptable form optionally combined with a pharmaceutically acceptable carrier. These compositions can be administered by any means that achieves their intended purposes. For example, administration can be by the oral, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, intrathecal, intracranial or intranasal routes. The dose administered will depend on the age, health, and weight of the patient; the type of concurrent treatment, if any, the frequency of treatment, and the nature of the desired effect. The amounts and regimens for administration according to the present invention can be readily determined by those of ordinary skill in the art of clinical treatment. The form of administration may also include emulsions, nanoparticles (e.g., solid lipid nanoparticles), liposomes, microspheres, microcapsules, aerosols, through inhalation, and transdermal dosage forms. Formulations suitable for parenteral administration include aqueous solutions of the compounds in water-soluble form. In addition, suspensions of the compounds as appropriately oily suspensions for injection may also be administered. Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides. Suspensions for aqueous injection may contain substances that increase the viscosity of the suspension and include, for example, sodium carboxymethylcellulose, sorbitol and / or dextran. Optionally, aqueous solutions and / or suspensions may also contain stabilizers and / or buffers, such as borate buffer and the like. The pharmaceutical preparations of the present invention are manufactured in a manner that is known per se, for example, by means of conventional mixing, granulation, dragee-making, dissolution, or lyophilization processes. Thus, pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipients, optionally comminuting the resulting mixture, and processing the mixture of the granules, after the addition of suitable auxiliary materials, if you want or need, to obtain tablets or cores for dragee. Suitable excipients are, for example, fillers such as saccharides, lactose, sucrose, mannitol or sorbitol; cellulose and / or calcium phosphate preparations such as calcium triphosphate or calcium acid phosphate; as well as binders such as starch paste, using, for example, corn starch, wheat starch, potato starch, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone. If desired, disintegrating agents such as the aforementioned starches and also carboxymethyl starch can be added., crosslinked polyvinylpyrrolidone, agar or alginic acid or a salt thereof, such as sodium alginate. Auxiliaries are, above all, flow regulating agents and lubricants, for example, silica, talc, stearic acid or salts thereof, such as magnesium stearate or calcium stearate, and / or polyethylene glycol. The dragee cores are provided with suitable coatings which, if desired, are resistant to gastric juices. Concentrated saccharide solutions can be used for this purpose, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and / or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. In order to produce coatings resistant to gastric juices, solutions of suitable cellulose preparations are used such as acetylcellulose phthalate or hydroxypropylmethylcellulose phthalate. The coatings may also be provided to protect the lipidization conjugates of the present invention from premature exposure to an acidic environment sufficient to hydrolyze the amide bond formed between the active drug, the peptide or the protein and the carrier. See U.S. Patent Nos. 4,786,505 and 4,853,230 for methods of preparing unit doses with nuclei that are protected from gastric acid. Preferably, the core is neutral or basic. The basic cores contain one or more alkaline reaction compounds such as those described in U.S. Patent Nos. 4,786,505 and 4,853,230. The dyes or pigments can be added to the tablets or to the dragee coatings, for example, for identification in order to characterize the combinations of the doses of the active compound. Other pharmaceutical preparations that can be used include, but are not limited to, double-capped oral capsules, made of gelatin, rectal suppositories, inhalation formulations for oral and / or nasal administration, nasal or rectal creams, ointments optionally combined with a carrier pharmaceutically acceptable, penetration enhancer, excipient, and / or filler. Penetration enhancers or enhancers suitable for use include cationic, anionic, amphoteric and neutral penetration enhancers such as benzalkonium chloride, chlorbutanol, AZONE and others known in the art. The synthesis of the exemplary compounds of General Formula I is illustrated in reaction schemes 1 and 2. In general, a bromomethylmaleic anhydride derivative, or its maleate salt, is allowed to react with an alcohol or lipophilic group possessing thiol, to form an ether or a thiol ether of General Formula III. The lipophilic group containing alcohol or thiol optionally includes a natural or unnatural amino acid portion, bridging, which binds the oxygen or sulfur atom and the carbonyl linked to the lipophilic group. The natural or unnatural bridge-forming amino acid portion can be connected either to the oxygen or sulfur atom or to the carbonyl bonded to the lipophilic group at the amino terminus, the carboxyl terminus or the side chain of the amino acid. With reference to Reaction Scheme 1, Pal-cysteine effectively includes a glycine bridge bonded to the carbonyl at the amino terminus and to the sulfur atom via the side chain. The use of hexadecanethiol as in Reaction Scheme 2 represents the formation of the compounds of Formula III without the natural or unnatural amino acid forming the bridge. The product of General Formula III is then subjected to dehydration conditions to reform the maleic anhydride now substituted via the ether or thioether linkage, with a lipophilic group, giving compounds of General Formula II. Those skilled in the art will appreciate the variety of alternative synthetic schemes capable of reaching the desired compounds.

Reaction Scheme 1 Synthesis of Reagent A C- (CHJM-CH3 Br-D A Pal-Cistßlna Filter I Aqueous phase Acidify White precipitate Extraction with ether Evaporated ether Ito vac Brown solid Dehydrate? Ion uti li-J zando DCC in anhydrous tetrahydrofuran Y Reagent A Reaction Scheme 2 - Synthesis of Reagent B Filt? Rar- I u evaporated Residue Reagent B Reaction schemes 3-5 describe the synthesis of exemplary pH sensitive lipid conjugates according to the present invention. In general, a drug containing amine, amino acid, peptide or protein is allowed to react with a compound of Formula II to form an amide of Formula I. The amide bond is formed under alkaline conditions, preferably in a buffered aqueous solution. At lower pH, including the pH typically found in vivo, the amide bond is hydrolyzed releasing the free amine and a compound of Formula III. The reversible formation of the amide bond provides a mechanism for the conjugation of a hydrophilic amine with a lipidization reagent at a pH and the release of that amine from the lipidization reagent at a lower pH.

Reaction test 3 - Tyramine Lipidation CREAL-Tyramine) Reagent A low pH "hydrolysis Tyramine Reaction Scheme 4 Lipidization of Arg. Vasopreein (REAL-AVP) CH;, - (CHJJM- Arg Reagent A Gly NH2 Arg Gly NH2 AVP Reaction Scheme 5 - Insulin Lipidization (REAL-Insulin) Insulin Reagent Insulin Examples Example 1. Synthesis of 3-S- (N-Palmityl-cysteinyl) methyl-2-methylmaleic anhydride, Reagent A (Reaction Scheme 1) The pyridine disulfide derivative of N-palmityl-cysteine (Pal-CPD) was obtained by known methods. Pal-CPD was synthesized according to the procedure of Ekrami et al., FEBS Letters 371: 283-286 (1995). Pal-CPD (0.7 g, 0.0015 mol) was dissolved in 10 ml of NaOH pH 11. Dithiothreitol (DTT) (0.9 g, 0.006 mol) was dissolved in 5 ml of water. The Pal-CPD solution was added dropwise to the DTT solution under continuous stirring at room temperature. After 2 hours, the reaction was terminated. The weight of the mixture was adjusted to 3 using 0.01 N HCl where a white precipitate appeared (Pal-cysteine). The precipitate was washed 5 times using diluted HCl to remove the excess amount of DTT. The initial 3-bromomethyl-2-methylmaleic anhydride material (Br-DMMA) was obtained by the addition of one equivalent of the bromide radical to 2,3-dimethylmaleic anhydride (DMMA). Consequently, DMMA (1.5 g, 0.012 mol), NBS (2.3 g, 0.013 mol), benzoyl peroxide (0.3 g, 0.0012 mol) and magnesium oxide (0.02 g, 0.0005 mol) were heated in 40 ml of low chloroform. reflux for 4 hours. The mixture was filtered and the chloroform was evaporated under reduced pressure. To the brown residue, 40 ml of carbon tetrachloride was added and filtered. The filtrate was collected and the solvent was removed under reduced pressure. A light oil with a slight greenish color was obtained which solidified after storage at 4 ° C. With reference to Reaction Scheme 1, BrDMMA is reacted with Pal-cysteine to provide the thiol ether of Pal-cysteine III where R1 is hydrogen, R2 is methyl, R3 is palmityl (C? 5H31), X is sulfur, And it is a glycine radical (-NHCH (C02H) -), n = ly and M = l. The reaction is carried out by the addition of Br-DMMA (0.3 g, 0.0014 mol) directly to a d pal-cysteine suspension in 30 ml of dilute hydrochloric acid at room temperature. The pH of the mixture was gradually adjusted to 7, 9 and finally 11, using 1 N sodium hydroxide. The pH of the mixture was stabilized after 2 hours at pH 11. After 16 hours of stirring at 25 ° C, the The mixture was filtered and the filtrate was acidified using 1 N HCl. A white precipitate appeared which was extracted with ether. The ether was evaporated under reduced pressure. The remaining greenish oil was dried at high vacuum. 3-S- (N-Palmityl cysteinyl) -methyl-2-methylmaleic acid (420 mg, 0.84 mmol) with a melting point of 60-63 ° C was obtained. The molar yield was 56% 3-S- (N-Palmitylcysteinyl) -methyl-2-methylmaleic acid (420 mg, 0.84 mmol) was dissolved in 5 mL of anhydrous THF. N, N-dicyclohexylcarbodiimide (DCC) was dissolved (692 mg, 3.36 mmol) in 1 ml of anhydrous THF and added to the above solution in an ice bath. The reaction was stirred in an ice bath for 5 hours and then filtered.

The filtrate was collected and the THF was removed under reduced pressure. The residue (brown solid) was dissolved in 1.5 ml of anhydrous dioxane and filtered. The filtrate was added to 30 ml cold anhydrous hexane and kept at 4 ° C for 16 hours.

The precipitate obtained was washed using cold anhydrous hexane and applied to high vacuum in order to remove the solvent. A light brown product (reagent A) with a melting point of 46 to 49 ° C was obtained. The molar yield was 54%.

Example 2. Synthesis of 3-S- (hexadecanyl) -methyl-2-methylmaleic anhydride, Reagent B (Reaction scheme 2) With reference to Reaction Scheme 2, Br-DMMA is reacted with hexadecanethiol to provide a thiol ether of Formula II where R2 is methyl, R3 is hexadecane (Ci6H33), n = 0 and m = 0. Under hydration conditions the anhydride reagent B of Formula II is obtained wherein R2, R3, n and m are as described above. Accordingly, as described in Reaction Scheme 2, Br-DMMA (0.5 g, 0.0025 mol) was hydrolysed in 10 ml of water at pH 8 and added to 0.63 g of hexadecanethiol (0.0025 mol) disueltq in 50 ml of THF. 1 ml of triethylamine was added and stirred for 16 hours at room temperature. The reaction mixture was filtered and the filtrate was evaporated under reduced pressure. The obtained residue was dissolved in dilute sodium hydroxide solution (pH 11) and washed with ether (3 x 20 ml). The filtrate was adjusted to pH 2 using 1 N hydrochloric acid and a white precipitate appeared. The precipitate was extracted into ether and this was removed under reduced pressure. The final product, 3- (hexadecanylthio) methyl-2-methylmaleic acid was dried under a high vacuum. 3- (Hexadecanylthio) methyl-2-methylmaleic acid was dehydrated to give reagent B using the same procedure as previously described for reagent A. Reagent B was dissolved in hot DMF and kept at 4 ° C for 16 hours . The white precipitate appeared which was washed using cold DMF. The solvent was removed under a high vacuum. 73 mg of a white powder with a molar yield of 16% are obtained.

Example 3. Preparation of Reversibly Lipidized Tyramine (REAL-tyramine) Using Reagent A (Reaction Scheme 3) Reagent A (2 mg, 0.00426 mmol) was dissolved in 60 μl of anhydrous DMF and added to 0.2 mg, (0.00146 mmol) of tyramine in 200 μl of USP borate buffer (pH 10, 0.1 M) in a water bath. ice. The reaction was carried out for 4 hours in an ice bath and 16 hours at 4 ° C.

Example 4. Determination of the pH Sensitivity of REAL-Tyramine The pH dependence of the amide bond formation was determined by periodically checking the free tyramine concentration. The 1 M phosphate buffers at pH 6, 7 and 8 were prepared. The REAL-tyramine was diluted 1: 2 using these buffers. The stock solution of thiramines was also diluted to have the same concentration of REAL-tyramine and was used as a control. The samples were incubated at 37 ° C. The fluorescence of the tyramine released from the REAL-tyramine was determined at different time points using the fluorescamine reaction.

After lipidization of tyramine, the concentration of free tyramine decreases to 15% of the original concentration. Incubation of the REAL-tyramine at low pH resulted in an increase in free tyramine concentration, indicating the breakdown of the amide bond. The hydrolysis rate of the amide bond was pH dependent (pH 6> pH 7> pH 8). After 1 hour of incubation of the REAL-tyramine at pH 6, the amide bond was almost completely hydrolyzed however, at pH 7 about 45% and at pH 8 only 7% of the amide bond of the REAL-tyramine was hydrolyzed (Figure 1 ).

Example 5. Preparation of Reversible Lipidized AVP (REAL-AVP) Using Reagent A (Reaction Scheme 4) Arginine-vasopressin (AVP) (0.5 mg) was dissolved in 1 ml of buffer. borate (pH 10, 0.1 M). An aliquot of 0.5 ml (0.25 mg, 0.207 μmol) of this solution was reacted in an ice bath with 1 mg (2.1 μmol) of reagent A dissolved in 50 μl of anhydrous dimethylformamide (DMF). The mixture was stirred for 16 hours at 4 ° C. The final concentration of the REAL-AVP was 0.455 mg / ml.

Example 6. In Vivo Effect of REAL-AVP in Brattleboro Rats Deficient in Vasopressin REAL-AVP was injected subcutaneously into animals (5 μg / kg) and the urine was collected at different time points. Figure 2 shows the cumulative volume of urine during the first 8 hours after injection. REAL-AVP and Pal-AVP have similar effects with a delay in the expression of urine of 4 hours. A longer delay in the excretion of urine, up to 6 hours, was observed after the injection of REAL-AVP. The direct lipidization of AVP to palmitic acid, Pal-AVP, was not as effective as REAL-AVP. The amount of urine excretion returned to the original 24 hours after injection of Pal-AVP and AVP. However, the effect of REAL-AVP lasted for 3 days (Figure 3). It can be concluded that the pH sensitive lipidization of AVP prolongs the biological activity of AVP.

Example 7. Preparation of Reversible Lipidized Insulin (REAL-Insulin) Using Reagent A (Reaction Scheme 5) 2 mg of insulin were dissolved in 2 ml of borate buffer (pH 10, 0.1 M), Reagent A (1 mg, 2.1 μmol) was dissolved in 100 μl of DMF and reacted with 1 ml (1 mg, approximately 0.14 μmol) of insulin solution in an ice bath. The reaction mixture was stirred for 24 hours at 4 ° C and then dialyzed against 500 ml of borate buffer (pH 10, 0.01 M) for 24 hours at 4 ° C. The volume of dialyzed REAL-insulin was adjusted to 2 ml using borate buffer (pH 10, 0.1 M) to give a concentration of 0.5 mg / ml of REAL-insulin. The volume of 1 ml of insulin reserve solution was also adjusted to 2 ml to give a concentration of 0.5 mg / ml.

Example 8. The Effect of REAL-Insulin in Hyperglycemic Rats Diabetes was induced in Sprague Dawley rats using intravenous injection of 60 mg / kg of streptozotocin. Solutions of 0.5 Units / ml of insulin or REAL-insulin in borate buffer (pH 10, 0.1 M) were prepared. The rats were fasted for 16 hours before the experiment, and were injected subcutaneously with 0.5 Units / kg of insulin or REAL-insulin. The blood glucose level of the rats was checked periodically at different time points for 9 hours. After this time, the rats were fed and the blood glucose level was measured after 15 hours of feeding. The rats were again fasted and the blood glucose level was measured after 16 hours. The period of fasting and feeding was continued for 3 days. The blood glucose level of the rats increased one week after inducing diabetes from an average of 100 mg / dl to 420 mg / dl (rats not fasted). In rats treated with insulin, a significant drop in blood glucose level was observed within the first hour. However, in rats treated with REAL-insulin there were no changes in the blood glucose level within the first hour, and a significant drop in blood glucose was observed for the first two hours after the injection (Figure 4). This is possibly due to the time required for the REAL-insulin to be hydrolyzed and to release the free insulin. After the insulin injection, the fasting blood glucose level of the rats was again in the original within 24 hours. However, in the case of the rats treated with REAL-insulin, the effect of the drug on the fasting blood glucose level lasted for 3 days (Figure 5). Diabetic rats submitted to fasting were also administered orally with 10 U / kg of insulin, REAL-insulin and placebo. The rats were fasted for 16 hours before oral administration. A water / oil microemulsion was used as the carrier drug. Figure 6 shows that no significant reduction in blood glucose level was observed after oral administration of insulin or placebo. However, in rats treated with REAL-insulin, a 28% reduction in blood glucose level was observed in 9 hours. It can be concluded that through the use of REAL-insulin, the biological activity of insulin can be prolonged. Using an appropriate formulation, REAL-insulin can be administered orally to reduce blood glucose levels. Having now fully described this invention, it can be understood by those skilled in the art that it can be performed with a broad and equivalent range of conditions, formulations and other parameters without affecting the scope of the invention or any modality thereof. All patents and publications cited herein are fully incorporated by reference herein in their entirety.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (26)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. A compound of General Formula I cara_ ± eriza ± > because R 2 is selected from the group consisting of hydrogen, lower alkyl, or aryl, wherein the alkyl or aryl groups are optionally substituted with one or more alkoxy, alkanoyl, nitro, cycloalkyl, alkenyl, alkynyl, groups. acyloxy, lower alkyl or halogen atoms; R3 is a lipophilic group; one of R4 and R5 is a biologically active amino group containing the substance selected from the group consisting of a drug containing the amine group, a natural or unnatural amino acid, a peptide and a protein and the other of R4 and R5 is OR6 wherein R6 is selected from the group consisting of hydrogen, an alkali metal and a negative charge; X is oxygen or sulfur; And it is a natural amino acid or non-natural bridge-forming agent; n is zero or 1; and m is an integer from zero to 10.

2. A compound according to claim 1, characterized in that R2 is methyl and X is sulfur.

3. A compound according to claim 1, characterized in that n = 0, m = 0 and R3 is a straight or branched chain hydrocarbon having from 4 to 26 carbon atoms.

4. A compound according to claim 3, characterized in that the straight or branched chain hydrocarbon has from 5 to 19 carbon atoms.

A compound according to claim 4, characterized in that the straight or branched chain hydrocarbon together with the carbonyl group is selected from the group consisting of palmityl, oleyl, stearyl, lauryl, myristyl, cholate and deoxycholate.

6. A compound according to claim 1, characterized in that the natural or non-natural amino acid is an amino acid of natural origin.

7. A compound according to claim 1, characterized in that the amine-containing drug is tyramine.

8. A compound according to claim 1, characterized in that the peptide is selected from the group consisting of Arg-Vasopressin and insulin.

9. A compound of General Formula II characterized pacche R2 is selected from the group consisting of hydrogen, lower alkyl, or aryl, wherein the lower alkyl or aryl groups are optionally substituted with one or more of alkoxy, alkanoyl, nitro, cycloalkyl, alkenyl, alkynyl, acyloxy, lower alkyl or halogen atoms; R3 is a lipophilic group; X is oxygen or sulfur; And it is a natural amino acid or non-natural bridge-forming agent; n is zero or 1; and m is an integer from zero to 10.

10. A compound according to claim 9, characterized in that R2 is methyl and X is sulfur.

11. A compound according to claim 9, characterized in that n = 0, m = 0 and R3 is a straight or branched chain hydrocarbon having from 4 to 26 carbon atoms.

12. A compound according to claim 11, characterized in that the straight or branched chain hydrocarbon has from 5 to 19 carbon atoms.

13. A compound according to claim 11, characterized in that the straight or branched chain hydrocarbon together with the neighboring carbonyl group is selected from the group consisting of palmityl, oleyl, stearyl, cholate and deoxycholate.

14. A compound according to claim 9, characterized in that the natural or non-natural amino acid is an amino acid of natural origin.

15. A compound of General Formula III or a pharmaceutically acceptable salt thereof; charsriza ± because R 2 is selected from the group consisting of hydrogen, lower alkyl, or aryl, wherein the lower alkyl or aryl groups are optionally substituted with one or more of alkoxy, alkanoyl, nitro, cycloalkyl, alkenyl, alkynyl, acyloxy, lower alkyl or halogen atoms; R3 is a lipophilic group; X is oxygen or sulfur; And it is a natural amino acid or non-natural bridge-forming agent; n is zero or 1; and m is an integer from zero to 10.

16. A compound according to claim 15, characterized in that R2 is methyl and X is sulfur.

17. A compound according to claim 15, characterized in that n = 0, m = 0 and R3 is a straight or branched chain hydrocarbon having from 4 to 26 carbon atoms.

18. A compound according to claim 17, characterized in that the straight or branched chain hydrocarbon has from 5 to 19 carbon atoms.

19. A compound according to claim 18, characterized in that the straight or branched chain hydrocarbon together with the neighboring carbonyl group is selected from the group consisting of lauryl, myristyl, palmityl, oleyl, stearyl, cholate and deoxycholate.

20. A compound according to claim 15, characterized in that the natural or non-natural amino acid is an amino acid of natural origin.

21. A method for increasing the cellular uptake of an amine-containing substance selected from the group consisting of a drug containing amine, a peptide and a protein, characterized in that it comprises administering a compound according to claim 1 to the cells.

22. A method for prolonging blood and tissue retention in a mammal of a biologically active amine-containing compound selected from the group consisting of drugs containing amine, peptides and proteins, characterized in that it comprises administering to said mammal A compound according to claim 1.

23. A method for the formation of a compound according to claim 1, characterized in that it comprises reacting a substance containing the biologically active amino group, selected from the group consisting of a drug. containing amine, a peptide, and a protein with a compound according to claim 9, under conditions whereby the compound of claim 1 is obtained.

24. A method for distributing a substance containing the biologically active amino group to the inside of a cell, characterized the method because it comprises: the expo The cell according to claim 1, wherein the compound is absorbed by the cell and is exposed to a pH within the cell low enough to hydrolyze an amide bond and release the substance contained in the group. functional biologically active amino.

25. A pharmaceutical composition, characterized in that it comprises: (a) an effective amount of a compound according to claim 1; and (b) a pharmaceutically acceptable carrier.

26. A pharmaceutical preparation according to claim 25, characterized in that the composition comprises an enteric coating that protects the compound from the hydrolysis of the amide bond, thereby preventing the release of the substance containing the biologically active amino group, until said coating is removed or dissolved. ,: * 'REVERSIBLE AQUEOUS REAGENTS OF LJPIDIZATION, pH SENSITIVE, COMPOSITIONS AND METHODS OF USE SUMMARY OF THE INVENTION The present invention provides lipidized conjugates comprising a biologically active substance that contains the amino group and a lipophilic group capable of penetrating a biological membrane. Under mildly acidic or neutral conditions, including those found in in vivo tests, the biologically active substance containing the free amino group is 10 released from the conjugate by hydrolysis of an amide bond. The present invention is also directed to methods for preparing the lipidizing agents and the lipidized conjugates, the pharmaceutical compositions comprising the lipidized conjugates and the methods for 15 increase the distribution of substances that contain the amino group within a cell. Preferred substances containing the amino group include peptides, proteins and derivatives thereof.