KR100691315B1 - Methods and compositions for lipidization of hydrophilic molecules - Google Patents

Abstract

The present invention relates to methods of delivering such compounds to mammalian cells using disulfide containing compounds (eg disulfide containing peptides or proteins) comprising complex products with fatty acids having disulfide bonds. According to the improved method of the present invention, it is possible not only to significantly increase the absorption rate of the compound by mammalian cells as compared to the absorption rate of the compound which does not form a complex, but also to extend the compound's blood and tissue retention. In addition, disulfide bonds in complexes are highly reactive in vivo, facilitating intracellular or extracellular release of the parent compound from fatty acid moieties.

Description

METHODS AND COMPOSITIONS FOR LIPIDIZATION OF HYDROPHILIC MOLECULES

The present invention relates generally to the field of biology and medicine. More specifically, the present invention relates to methods and compositions useful for increasing the uptake and retention of hydrophilic molecules, particularly peptides and proteins, in mammals.

Advances in biotechnology have enabled the mass production of active and pure proteins and peptides as therapeutic agents. At present, the therapeutic effect of most of these agents can only be achieved if they are administered via an aggressive route such as injection. Since most proteins have very short half-lives, effective concentrations of protein agents can only be achieved by frequent injections.

Administration of the protein by injection is the most effective means of in vivo delivery of the protein, but it is very difficult for the patient to withstand multiple injections. In addition, the injection of drugs requires training and skill, which is not always possible. If the protein drug functions as a life preserver, the patient can accept administration by injection. However, when protein drugs are only one of several possible treatments, it is difficult for patients to accept injections of proteins and peptides. Therefore, there is a need to develop alternative pathways for delivering proteins and peptides.

Such alternative routes include buccal, nasal, oral, pulmonary, rectal and intraocular routes. Of course, these routes are less effective than parenteral routes of administration, but are of more interest than parenteral administration, since they are simple and patient controllable for the patient. The oral route is particularly important because it is the simplest and easiest to adapt to the patient.

Mucosal barriers that separate the interior of the body from the outside (eg, gastrointestinal tract, eyes, lungs, rectum, and nasal mucosa) comprise layers of tightly bound cell monolayers that tightly control the transport of molecules. Each cell in the barrier is bound by tight connections that control entry into the intracellular space. Therefore, the mucosa is first a physical barrier, and the transport through the mucosa depends on the intercellular or paracellular pathway [Lee, VHL, CRC. Critical Rev. Ther. Drug Delivery Sys. , 5: 69-97 (1988).

Cell-side transport through water-filled connections is limited to small molecules (molecular weight <1 kDa) and is a diffusion process driven essentially by concentration gradients across the mucosa [Lee, (1988), supra, Arturrson, P. and Magnusson, C., J. Pharm. Sci., 79: 595-600 (1990)]. The tight junctions account for less than 0.5% of the total mucosal surface area [Gonzalez-Mariscal, LN et al, J. Membrane. Biol. 86: 113-125 (1985), Vetvicka, V., and Lubor, F., CRC Critical Rev. Ther. Drug Deliv. Sys. , 5: 141-170 (1988). Therefore, they only play a secondary function in the transport of protein drugs across the mucosa.

Transcellular transport of small drugs occurs effectively as long as the physicochemical properties of the drug are suitable for transport across hydrophobic cell barriers. However, transcellular transport of proteins and peptides is limited to the process of transcytosis [Shen, WC, et al., Adv. Drug Delivery Rev. , 8: 93-113 (1992). Transcytosis is a complex process in which proteins and peptides are taken up from one side of the cell into the vesicles and then travel through the cells to the other side of the cell, where they release the proteins and peptides from the intracellular vesicles [Mostov, KE, and Semister, NE, Cell , 43: 389-390 (1985). The cell membrane of the mucosal barrier is a hydrophobic lipid bilayer, having no affinity for hydrophilic charged macromolecules such as proteins and peptides. In addition, mucosal cells can secrete mucins that act as a barrier to the transport of large numbers of macromolecules [Edwards, P., British Med. Bull. , 34: 55-56 (1978). Therefore, unless there are specific transport mechanisms for proteins and peptides, it is almost impossible to transport them across mucosal barriers.

In addition to providing a solid physical barrier to the transport of proteins and peptides, mucosal barriers have enzymes that can degrade proteins and peptides before they pass through, after, and during passage. This barrier is called the enzyme barrier. Enzyme barriers consist of endopeptidase enzymes and exopeptidase enzymes that degrade proteins and peptides at their ends or within their structure. Several enzymatic activities of the mucosa have been studied, and as a result, there is substantial protease activity in homogenates of the buccal, nasal, rectal and vaginal mucosa of albino rabbits, which proved to be comparable to those present in the iliac bone. Lee, et al, (1988), supra. Therefore, without having to consider what is a mucosa, enzyme barriers will exhibit strong properties in the degradation of protein and peptide molecules.

The N and C termini of the peptide are charged and the charged side chains provide high hydrophilic properties on this macromolecule. In addition, the presence of charged side chains suggests that proteins and peptides have strong hydrogen bonding capacity, which proves to be a major factor in inhibiting the transport of even small peptides across cell membranes. [Conradi, RA et al., Pharm. Res., 8 : 1453-1460 (1991). Therefore, the size and hydrophilic properties of proteins and peptides together severely limit the transport of proteins and peptides across mucosal barriers.

One method used to change the physical properties of mucosal barriers is to use penetration enhancers. Methods of using penetration enhancers include disruption of cell membranes by low molecular weight agents capable of fluidizing cell membranes [Kaji, H .. et al., Life Sci. , 37: 523-530 (1985), opening the tight junctions [Inagaki, M., et al., Rhinology , 23: 213-221 (1985)], based on the formation of pores in the cell membrane [Gordon , S., et al., Proc. Natl. Acad. Sci. USA, 82: 7419-7423 (1985), Lee, VHL et al., Critical Reviews in Therapeutic drug Carrier Systems, CRC Press , 8: 91-192 (1991). Methods using such reagents can induce nonspecific loss of barrier integrity and induce the uptake of various macromolecules which may be toxic to cells in vivo.

Administration of protease inhibitors with proteins and peptides has been shown to exhibit somewhat limited activity in increasing uptake 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 such methods have yet to be studied closely.

The method of using prodrugs is based on modifying the peptides in a manner that protects the peptides from enzymatic degradation and recognition. This method is accomplished by substituting the D-form amino acids in the peptide structure and protecting the reactive groups by amidation and acylation, reversing the chirality of the peptide and introducing conformational constraints into the peptide structure. Synthesis of prodrugs is only applicable to small peptides that can readily identify active domains.

Reducing size is another possible way to increase the transport potential of proteins. However, before reducing the size, it is necessary to map the synthetic site of the protein. In general, this method is difficult to apply to most proteins.

Carrier ligands, due to their properties, can change the cellular uptake and transport properties of proteins and peptides. An essential feature of this method is the covalent attachment of a cell impermeable protein or peptide to a carrier that is well transported into the cell. The mechanism by which the carrier ligand causes endocytosis or transcytosis is important in determining the suitability of the carrier for increasing the transport of proteins and peptides. Macromolecular carriers are hydrophilic and do not distribute into the membrane. Therefore, the transport of large polymer carriers intracellularly is mediated by the affinity of the carriers for the cell membrane. In general, uptake of large molecule conjugates begins with binding to the cell membrane. The binding of the carrier to the cells is specific (eg binding of antibodies to cell surface antigens), nonspecific (binding of cationic ligands or lecithin to cell surface sugars), or mediated by receptors (transferrin Or binding of insulin to its receptor). Once the carrier binds to the cell surface, it is taken up into the vesicles. The vesicles can then be processed step by step to several paths. One route is that the vesicles are recycled back to the membrane from which they were derived. Another route is fusion with lysozomes, which is destructive to the complex. Another pathway and pathway leading to transcytosis of the complex is the fusion of the vesicle with the membrane on the side opposite to the side from which the vesicle is derived.

The exact equilibrium between endocytosis and transcytosis determines the delivery of the protein complex to the target. For example, endocytosis determines the extent to which the complex is taken up by target cells, while transcytosis determines whether the complex reaches its target [Shen, et al., (1992), supra] . For sufficient absorption through the gastrointestinal tract, the complex must bind to the leading membrane of the gastrointestinal mucosa, internalize within the mucosal cells, cross the cells, and finally release from the basal membrane.

Recent literature includes nonspecific carriers such as polylysine [Shen, WC and Ryser, HJP, Proc. Natl. Acad. Sci. USA , 78: 7589-7593 (1981)] and lectins [Broadwell, RD et al., Proc. Natl. Acad. Sci. USA , 85: 632-646 (1988), and specific carriers such as transferrin [Wan, J., et al. , J. Biol. Chem. , 267: 13446-13450 (1992), asialoglycoproteins (Seth, R. et al., J. Infect. Diseases, 168: 994-999 (1993)] and antibodies [Vitetta, ES, J. Clin. Immunol. , 10: 15S-18S (1990) have been reported to increase endocytosis of proteins into cells. There are fewer reports of transcytosic carriers, and very few studies have quantified the transport of protein complexes across cell barriers. Wheat germ aglutinin [Broadwell, et al., (1988) supra] and antitransferrin / methotrexate complex [Friden, PM and Waius, LR, Adv. Exp. Med. Biol., 331 : 129-136 (1993) have been shown to cause transcytosis across the blood brain barrier in vivo. In addition, polylysine complexes of horseradish peroxidase (HRP) and transferrin complexes of HRP have been found to cause transcytosis by crossing cell monolayers in vitro [Wan, J. and Shen, WC, Pharm. Res. 8: S-5 (1991), Taub, ME and Shen WC, J. Cell. Physiol. 150: 283-290 (1992), Wan, J. et al., J. Biol. Chem. , 267: 13446-13450 (1992), supra.

Fatty acids, which are components of phospholipids, make up the bulk of cell membranes. They are available as commercially available products and are relatively inexpensive. Due to its lipid properties, fatty acids can be readily distributed to and interact with the cell membrane in a nontoxic manner. Therefore, fatty acids are potentially the most useful carrier ligands for proteins and peptides. As a method which can use a fatty acid for protein and peptide delivery, the method of covalent-bond modification of a protein and a peptide, and the method of using a fatty acid emulsion are mentioned.

Some studies have reported examples of successful use of fatty acid emulsions for the delivery of peptides and proteins in vivo [Yoshikawa, H. et al., Pharm. Res. , 2: 249-251 (1985), Fix, JA et al, Am. J. Physiol. 251 : G332-G340 (1986)]. The mechanism by which fatty acid emulsions can promote the absorption of proteins and peptides is not yet known. Fatty acid emulsions can open tight junctions, solubilize membranes, conceal proteins and peptides from the gastrointestinal tract environment, and transport proteins and peptides across the gastrointestinal mucosa as part of their absorption [Smith, P. et al. , Adv. Drug Delivery Rev. , 8: 253-290 (1992). The latter mechanism has been proposed, but is inconsistent with the currently known knowledge about fat absorption mechanisms.

A more logical method for delivering proteins and peptides across the gastrointestinal epithelium is to use fatty acids as nonspecific membrane absorbers. Several studies have shown that nonspecific membrane binding agents bound to proteins can promote transcytosis of protein complexes that cross cells in vitro [Wan, J. et al., J. Cell. Physiol. , 145: 9-15 (1990), Taub and Shen (1992), supra. In addition, fatty acid complexes have been demonstrated to enhance the uptake of macromolecules into and across cell membranes [Letsinger, R., et al., Proc. Natl. Acad. Sci. USA , 86: 6553-6556 (1989), Kabanov, A. et al., Protein Eng. , 3: 39-42 (1989). However, there are the following problems in binding fatty acids to peptides and proteins. That is, (1) fatty acids cannot be dissolved in an aqueous solution used for complex formation reactions, (2) peptides and proteins lose biological activity after fatty acid acylation, and (3) peptides complexed with fatty acids are dissolved in aqueous solutions. Can't be. 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. , 8: 649-652 (1991), Robert, S., et al., Biochem. Biophys. Res. Commun. , 196: 447-454 (1993).

It is an object of the present invention to provide methods and compositions useful for binding fatty acids to hydrophilic molecules to increase the bioavailability of peptides and proteins.

Summary of the Invention

According to the present invention, a sulfhydryl containing compound comprising a fatty acid complex product having a disulfide bond (s) or a fatty acid derivative of a disulfide containing compound (eg, a peptide containing or modified to contain a sulfhydryl group) , Proteins or oligonucleotides) are used to deliver sulfhydryl containing or disulfide containing compounds to mammalian cells. According to this modification method, the uptake of the compound by mammalian cells is significantly increased compared to the absorption rate of the uncomplexed compound, as well as prolonging the blood and tissue retention of the compound. In addition, disulfide bonds in complexes are highly reactive in cells or in vivo, thus promoting intracellular or extracellular release of the parent compound from fatty acid moieties. The present invention also provides a method for producing such a fatty acid derivative.

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings.

1 is a bar graph showing the uptake of BBI, BBIssPal and BBIssOleic in Caco-2 cells.

2A to 2D are graphs showing the biodistribution of BBI and BBIssPal in blood, kidney, lung and liver of CF-1 mice after intravenous administration.

3A to 3D are graphs showing the biodistribution of BBI and BBIssOleic in blood, kidney, lung and liver of CF-1 mice after intravenous administration.

4A-4C are bar graphs showing the biodistribution of BBI and BBIssPal in CF-1 mice after intraperitoneal administration.

5A and 5B are graphs and bar graphs showing transcytosis and cumulative amounts of BBI, BBIssPal (2) and BBIssPal (4) into and across Caco-2 cells, respectively.

FIG. 6 is a graph showing the results of Sephadex® G50 gel filtration analysis of basal medium derived from Caco-2 cells containing transcytosed BBI, BBIssPal (2) and BBIssPal (4).

7A and 7B are graphs showing the results of subcutaneous administration of DP and DP-P at a dose of 3.3 μg / kg to rats with diabetes insipidus, and FIG. 7A shows urine output and FIG. 7B shows water uptake.

8 is a graph comparing the efficacy of one subcutaneous injection of DP and DP-P to treat rats with diabetes insipidus at various doses.

9 is a graph comparing the relationship between the structure and activity of the desmopressin-fatty acid complex according to the chain length of fatty acids under a subcutaneous injection dose of 0.5 μg / kg. "C-6" stands for caproic acid, "C-10" stands for capric acid, "C-16" stands for palmitic acid, and "C-18" stands for stearic acid.

10 is a graph showing plasma desmopressin concentrations after intravenous administration of desmopressin (DP) or its palmitic acid complex (DP-P) to mice.

11A and 11B are graphs showing plasma calcitonin and calcium concentrations after subcutaneous administration of calcitonin (CT) or its palmitic acid complex (CT-P) at a dose of 100 μg / kg, and FIG. 11A is CT-P. 11B shows the result of the treatment with CT.

According to the present invention, sulfhydryl containing compounds (e.g., biopolymers as described below) are bound to fatty acid derivatives via reversible biodegradable disulfide bonds. Such complexes are expected to bind to the proximal side of the cell membrane and be released into interstitial fluid as a result of the reduction of disulfide bonds after reaching the basal side membrane of the gastrointestinal epithelium as a result of cell membrane transport and conversion.

According to a first aspect of the present invention, there is provided a complex represented by the following formula (VI).

Wherein P is a residue derived from a sulfhydryl containing compound, R ¹ is a hydrogen atom, lower alkyl or aryl, R ² is a hydrophobic substituent as described below, R ³ is a hydroxy, hydrophobic substituent, or Or an amino acid chain comprising 1 or 2 amino acids and having -CO ₂ H or -COR ² as an end group. The complex is particularly useful for increasing the absorption of sulfhydryl containing compound PSH and prolonging blood and tissue retention of the sulfhydryl containing compound.

According to a second aspect of the present invention, there is provided a method for prolonging or increasing the uptake of mammalian tissue and blood in the sulfhydryl-containing compound represented by the formula PSH, in which the sulfhydryl-containing Compounds are formed from the compounds represented by Formula VI above, and then the complexes are administered to mammals (eg, as aqueous components or as components of pharmaceutical compositions in dosage unit dosage forms) in amounts effective to achieve the desired purpose.

According to a third aspect of the present invention, there is provided a compound represented by the following general formula (V).

ASS-CH ₂ -CR ¹ (NHCOR ² ) C (= O) R ³

In the above formula, A is an aromatic active moiety as described later, and R ¹ , R ² and R ³ are as defined above. The compounds of formula (V) are particularly useful for preparing complexes of formula (VI) from sulfhydryl containing compounds represented by formula (PSH).

According to a fourth aspect of the present invention, there is provided a method for preparing a complex represented by the formula (VI) from a sulfhydryl-containing compound represented by the formula (PSH), wherein the method of the present invention converts the compound represented by the formula (PSH) to the formula (V). Reacting with the compound represented. This reaction is usually carried out using an aqueous buffer solution (e.g., at a temperature of about 4 ° C to about 37 ° C for a period of about 1 hour to about 24 hours, using an excess (e.g., 2 to 10 times excess) of the compound of Formula V. : Phosphate, bicarbonate or borate buffer). This reaction is preferably carried out in a bicarbonate buffer at pH 8.

According to a fifth aspect of the present invention, a compound represented by the following general formula (III) is provided.

ASS-CH ₂ -CR ¹ (NH ₂ ) C (= O) R ^{3 '}

Wherein R ^{3 ′} is hydroxy or an amino acid chain comprising 1 or 2 amino acids and having —CO ₂ H as the end group, and A and R ¹ are as defined above. Compounds of formula III are useful for preparing compounds represented by formula V. Compounds of formula III can be prepared by reacting a compound represented by formula II with a compound represented by formula ASSA or ASS-A ', wherein A' is different from A and represents an aromatic active moiety.

HS-CH ₂ -CR ¹ (NH ₂ ) C (= O) R ^{3 '}

These reactants are either commercially available (eg 2,2'-dithiopyridine and 5,5'-dithiobis (2-nitrobenzoic acid)), or may be prepared according to conventional synthetic routes well known to those skilled in the art. .

According to a sixth aspect of the invention, the compound of formula III is represented by the formula XO ₂ CB or X-OC-B wherein X is a lipid active group as described below and B is part of a lipid group as described below ) To provide a compound of formula V wherein R ² is a hydrophobic substituent. The compound represented by the formula XO ₂ CB or X-OC-B can be easily prepared according to a method known per se.

In order to prepare the compound represented by the formula (III), the procedure of one embodiment generally involves mixing an equimolar amount of a compound of formula (II) with a compound of formula (SASA) or ASS-A 'in a polar organic solvent (e.g. ethanol) Can be. The product represented by formula III can then be suitably separated by crystallization from a nonpolar organic solvent such as benzene. Of course, other suitable procedures may be used.

If one wishes to prepare XO ₂ CB or X-OC-B, the fatty acids can be reacted with, for example, (a) N-hydroxysuccinimide and carbodiimide reagents to form H-hydroxysuccinimidyl active esters. Or (b) react with trifluoroacetic anhydride to form a fatty acid anhydride, or (c) react with thionyl chloride to form a fatty acid chloride. Other procedures can also be used to introduce such lipid active groups and other active groups.

In the present invention, the terms "hydrophobic substituent" and "lipid containing moiety" refer to the lipid group itself or a hydrocarbon-based group (specifically, one or more amino acids) including the lipid group. The number of carbon atoms of such hydrophobic substituents may be about 4 to 26, preferably about 5 to 19. Suitable hydrophobic groups with the carbonyl to which they are attached in the formula include myristyl (C ₁₃ H ₂₇ ), palmityl (C ₁₅ H ₃₁ ), oleyl (C ₁₅ H ₂₉ ), stearyl (C ₁₇ H ₃₅ ) And lyidyl (C ₁₇ H ₃₃ ), but are not limited to these, in addition to steroid moieties having a carboxyl group such as cholate, deoxycholate, 17-carboxyquilenin and 17-carboxystrone. .

The term "aromatic active moiety" is a moiety that forms the disulfide group of the compound represented by the formula (V), and the sulfide group is highly reactive to the substitution reaction by the sulfhydryl-containing compound represented by the formula (PSH). Acts as an excellent leaving group). Preferred aromatic active groups are 2-pyridyl, and other suitable aromatic active groups include 4-nitrophenyl.

As used herein, the term "lipid active group" means a moiety that provides reactivity to a compound of formula III with the carboxy lipid group to which it is attached. Preferred lipid active groups are N-hydroxysuccinimidyl esters, and other suitable lipid active groups include acid chlorides and acid anhydrides.

Although the present invention relates to methods of making and using complexes of formula VI comprising a wide range of compounds containing sulfhydryl groups, the methods and compositions of the present invention are particularly advantageous for use in preparing complexes comprising biopolymers. Do. Examples of the biopolymer include peptides, proteins and oligonucleotides (as described below). As is well known in the art, biopolymers or thiolated biopolymers containing sulfhydryl groups have a number of moieties corresponding to the structure for the complex of formula VI (ie, excluding the portion P in the structure of the compound of formula VI). Groups with structures.

In the present invention, the term "peptide" means an amino acid chain containing 2 to 50 amino acids, and the term "protein" means an amino acid chain comprising 50 or more amino acids. Proteins and peptides can be isolated from natural sources or prepared by means known to those skilled in the art, such as by recombinant DNA techniques or solid phase synthesis. Peptides and proteins used according to the invention may comprise natural L-amino acids, mixtures of L-amino acids with other amino acids (e.g., D-amino acids and modified amino acids) or only amino acids other than L-amino acids. . In order to form the complex represented by Formula (I), the peptide or protein must have at least one reactive thiol group. In many cases, the peptide or protein contains cysteine residues (amino acids including thiol groups). Peptides or proteins that do not contain a thiol group may be modified by methods known in the art, and specifically known thiolating agents (eg, N-succinimidyl-3- (2-pyridylthio) propionate (SPDP) and 2-iminothiolane (traut reagent) can be used conventionally for this purpose.

The term "oligonucleotide" refers to a chain comprising two or more natural or modified nucleic acids, such as natural or recombinant deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) sequences. To prepare complexes according to the present invention, the oligonucleotides must be modified to contain sulfhydryl groups used to bind the lipid containing moieties by thiolation reactions. Such modifications can usually be carried out in a known manner. For example, oligonucleotides can be bound to cystamine using carbodiimide, followed by reduction with dithiothreitol to generate free sulfhydryl groups. Such oligonucleotide complexes can be used to deliver therapeutically effective oligonucleotides in vivo or ex vivo. That is, the complex may be contacted with cells in vitro to cause transfection. The cells are then administered to the animal to achieve the therapeutic purpose. Alternatively, oligonucleotide complexes can be used to facilitate the transfection of mammalian cells in vitro to prepare valuable recombinant proteins in vitro.

Preferred oligonucleotides are antisense oligonucleotides. Antisense oligonucleotides are derivatives of DNA or RNA molecules containing nucleotide sequences complementary to DNA or RNA molecules or specific mRNAs. Antisense oligonucleotides bind to complementary sequences in specific mRNAs to inhibit transcription of the mRNA. Many antisense oligonucleotides and derivatives thereof are known. In this regard, for example, U.S. Pat. 5,264,523 and WO96 / 35706, WO96 / 32474, WO96 / 29337 (thioostere modified antisense oligodeoxynucleotide phosphorothioates), WO94 / 17093 (oligonucleotide alkylphosphonates and alkylphosphatides Oate), WO 94/08004 (oligonucleotide phosphorothioate, methyl phosphate, phosphoramidate, dithioate, crosslinked phosphorothioate, crosslinked phosphoramidate, sulfone, sulfate, keto, phosphate ester And phosphorobutylamine (van der Krol et al., Biotech. 6: 958-976 (1988), Uhlmann et al., Chem. Rev. 90 : 542-585 (1990)), WO 94/02499 (oligonucleotides). Alkylphosphonothio And arylphosphonothioates), and WO 92/20697 (3'-terminally capped oligonucleotides) As preferred antisense oligonucleotides, S-oligonucleotides (phosphorothioate derivatives or S-oligogoses) can be referred to. Derivatives, such as Jack Cohen, Oligodeoxynucleotides, Antisense Inhibitors of Gene Expression , CRC Press (1989)) S-oligo (nucleoside phosphorothioate) is an isoelectric analogue of oligonucleotide (O-oligo) Where the non-crosslinkable oxygen atom of the phosphate group is substituted with a sulfur atom, the S-oligo converts the corresponding O-oligo to the sulfur transfer agent 3H-1,2-benzodithiol-3-one-1,1-dioxide. It can be prepared by treatment, as described in Lyer et al., J. Org. Chem. 55: 4693-4698 (1990) and Lyer et al., J. Am. Chem. Soc. 112: 1253-1254 (1990).

In a preferred class of compounds represented by Formula VI, R ¹ is a hydrogen atom, R ² is a hydrophobic substituent or a lipid moiety, and R ³ is -OH. Complexes of this type are suitably derived from cysteine. In another preferred class of composites according to the invention, R ¹ is a hydrogen atom, R ² is —CH ₂ CH ₂ CH (NH ₂ ) CO ₂ H or —CH ₂ CH ₂ CH (NHCO-lipid) CO-lipid , R ³ is —NHCH ₂ CO ₂ H or —NHCH ₂ CO-lipid, wherein at least one of R ² and R ³ together with the carbonyl to which they are attached represent a lipid moiety. Complexes of this type are suitably derived from glutathione.

The synthesis method for an example of a compound represented by the formula (VI) is shown in Scheme I below, wherein P is a protein. Of course, as is known to those skilled in the art, various other synthetic routes may be used.

A)

B)

C)

According to another feature, the present invention provides a fatty acid complex represented by the following general formula (X).

Wherein P ¹ is a residue derived from a disulfide containing compound, which may optionally include a disulfide group bonded to a hydrophobic group. Examples of P ¹ include, but are not limited to, peptides and proteins, or derivatives of compound (N) containing disulfide bonds as described below. P and (N) are preferably drugs. It is preferable that complex (X) is an organic compound, n is an integer of 1-20, Preferably it is an integer of 10 or less, More preferably, it is an integer of 5 or less. R ¹ , R ² and R ³ are as defined above for Formula VI. R ⁴ is a hydrogen atom, lower alkyl or aryl, R ⁵ is a hydrophobic substituent as described above, R ⁶ comprises a hydroxy, hydrophobic substituent, or 1 or 2 amino acids and -CO ₂ H or -COR ² Amino acid chain which has a terminal group. R ¹ may be the same as or different from R ⁴ . R ² may be the same as or different from R ⁵ . R ³ may be the same as or different from R ⁶ .

R ² and R ⁵ together with the carbonyl to which they are attached, each comprise (1) a lipid containing moiety comprising a lipid group, or (2) an amino acid chain comprising 1 or 2 amino acids and having -CO ₂ H as a terminal group; A lipid containing moiety comprising a lipid group is shown. R ³ and R ⁶ each preferably represent (1) a hydroxy group, (2) a hydrophilic group, or (3) an amino acid. Examples of R ³ and R ⁶ include, but are not limited to, glycine. Examples of R ² and R ⁵ include, but are not limited to, glutamic acid derivatives, fatty acids and steroids such as deoxycholate and cholate.

The present invention is applicable to biologically active agents such as sulfhydryl containing or disulfide containing proteins and peptides. In general, such agents are difficult to transport across biological barriers, are quickly removed from plasma, and are prone to chemical proteolysis, thus their therapeutic use is limited. The present invention may overcome some or all of these limitations. Examples of sulfhydryl containing or disulfide containing proteins and peptides include insulin [Czech, M.P., Ann. Rev. Biochem., 46, 359 (1977)], calcitonin [Brown, E.M. Aurbach, G.D. Vitam, Horm, 38, 236 (1980)], desmopressin [Vavra et al., J. Pharmacol. Exp. Ther., 188, 241 (1974)], interferon alpha, interferon beta and interferon gamma [Stiem, E.R., Ann. Inter. Med., 96, 80-93 (1982)], interleukin-2, interleukin-3, interleukin-4, interleukin-6 and interleukin-11 [Kluth, DC, Rees, AJ, Semin, Nephrol., 16, 576- 82 (1996), Holyoake, TL, Blood Rev., 10, 189-200 (1996)], G-CSF [Spiekermann, K. et al., Leukemia, 11, 466-78 (1997), GM-CSF [ Jonuleit, H. et al., Arch. Dermatol. Res., 289, 1-8 (1996)], human growth hormone [Strobl, J.S., Thomas, M.J., Pharmcol. Rev., 46, 1-34 (1994)], erythropoietin [Spivak, J.L., Semin. Hematol., 30, 2-11 (1993)], Vasopressin [Schroder, E., Lubke, K. The Peptide, 2, 336-350 (1996)], Octreotide [Sheppard, MC, Stewart, PM, Metabolism : Clinical and Experimental, 45, 63-64 (1996)], Aprotinin [Haberland, G., McConn, R., Fed. Proc., 38, 2760-2767 (1979)], Oxytocin [Nachtmann, F. et al., Anal. Prof. Drug Subst., 10, 563-600 (Florey, K. Ed., Academic Press, New York, 1981)], beta-TGF [Moses, H. L., Serra, R., Curr. Opin. Genet. Dev., 6, 581-6 (1996)], BDNF [Apfel, S.C. Kessler, J.A., Bailleres. Clin. Neuronol., 4: 593-606 (1995)], bFGF [Bikfalvi, A. et al., Endocr. Rev., 18, 26-45 (1997)], PDGF [Hughes, A.D. et al., Gen. Pharmacol., 27, 1079-89 (1996)], TNF [Majno, P.E. et al., Swiss. Surg., 4, 182-5 (1995)], atrial sodium excretory increasing peptide [Nakao, K., Curr. Opin. Nephrol. Hypertens., 2, 45-50 (1993)], uterine relaxation factor [Schwabe, C. et al., Recent Progr. Horm. Res., 34, 123-211 (1978)], Amylin [Rink, T.J. et al., Trends. Pharmacol. Sci., 14, 113-8 (1993)], deoxyribonuclease [Laskowski, The Enzymes, 2, 289-311 (Boyer, PD, Ed .. Academic Press, New York, 1971)], EGF [ Carpenter, G., Curr. Opin. Cell. Biol., 5, 261-4 (1993)], hirudin (Markwardt, Methods Enzymol., 19, 924 (1970)), neocardinostatin [Dedon, P.C., Goldberg, I.H., Chem. Res. Toxicol., 311-32 (1992), blood regulatory peptides [Paukovits, W. R. et al., Cancer Treat. Rev. 17, 347-54 (1990) and somatostatin [Moss, R. L., Ann. Rev. Physiol., 41, 617 (1979), but is not limited to these.

Specific examples of the complex (X) include, but are not limited to the following compounds.

In the above formula, "Gly" represents glycine, "Glu" represents glutamic acid, and "n ¹ " is an even integer, preferably 10 or less.

Preferred complex (X) is represented by the following formula (wherein R ¹ = R ⁴ , R ² = R ⁵ and R ³ = R ⁶ ).

If the group P ¹ contains a sulfhydryl group, this may in accordance with the invention bind to another hydrophobic group via a disulfide bond to give a complex with an odd number of hydrophobic substituents (eg n ¹ is odd). .

Such complexes are particularly useful for the absorption of disulfide containing compounds and for prolonging blood and tissue retention.

According to another feature of the invention, in animals such as mammals, the absorption of the complex (X) administered to the animal as part of a pharmaceutical composition (eg, an aqueous solution or an oral dosage form), or blood and tissue retention is increased. A method of extending is provided.

According to another feature of the present invention, a method for producing the composite (X) is provided. Complex (X) is preferably prepared from compounds containing at least one disulfide bond and optionally at least one thiol group.

Synthetic methods for one example of a compound represented by formula (X) are shown in Schemes II, III, and IV below (complexes of formulas (D), (H) and (M), respectively, as examples of complex (X) Used). Of course, as is well known in the art, various other synthetic methods may be used.

The following schemes are represented by formula (X) from disulfide-containing compounds, such as proteins and peptides, or compounds of formula IV (N), represented by formula R-NH ₂ or R-OH, wherein R is an organic moiety The general method of making the difatty acid disulfide derivatives is shown. Specific examples of compound (N) include, but are not limited to, proteins, peptides, amino acids, nucleotides, nucleosides, carbohydrates and derivatives and drugs thereof. Examples of compound (N) as a drug include, but are not limited to, antibiotics and hydrophilic drug molecules such as acyclovir. According to this method, the difatty acid disulfide derivative, i.e., complex (X), can be reversibly modified from a disulfide containing peptide or protein or compound (N).

This method can be used to prepare a peptide, protein or compound (N) modified with a disulfide containing compound (e.g., compound (L) of Scheme IV), as described above, ie, ASS-CH ₂ -CR ¹ Reacting with (NHCOR ² ) C (= 0) R ³ to produce a complex (X). For example, the compounds of the formula (V) are illustrated as compounds (C) in Schemes II, III and IV. In accordance with the teachings herein, one skilled in the art will be able to prepare the desired complex (X) by reacting a suitable compound with a compound of Formula (V). If the disulfide containing compound also contains one or more sulfhydryl groups, these may be combined with hydrophobic groups to provide compounds with additional hydrophobic groups, which may be odd or even, depending on the number of sulfhydryl groups.

Complex (X) may be converted to compound (N) or parental disulfide molecule upon reaching blood or tissue. According to this method and complex (X), the peptides, polypeptides, proteins and organic compounds (by improving in vivo the permeability, stability and bioavailability of peptides, polypeptides, proteins and organic compounds (N) which can act as drugs) ( N) Overcome the major challenges facing drug formulations. Compared to unmodified peptides, proteins and organic compounds (N), complex (X) (e.g. fatty acid-peptide complex or fatty acid-drug complex) provides improved in vivo absorption, e.g. gastrointestinal absorption (e.g. For example, when administered orally) and sustained release in vivo (for example, when injected subcutaneously). Compared to the prior art, the present invention provides the following advantages: (1) the reaction can be carried out under aqueous conditions, (2) the product is generally water soluble, and (3) the product as a parent peptide in blood or tissue. Since it can be converted, it has the advantage of being able to regenerate a medicament active drug.

For example, this method can be used to prepare fatty acid-disulfide complexes of cyclic disulfide containing peptide drugs or hormones by using the compound represented by Formula (V). As shown in Scheme II, two free sulfhydryl groups (B) can be generated by reducing cyclic disulfide bonds in the peptide or polypeptide (A). Such sulfhydryl groups can be reacted with fatty acid-disulfide derivatives such as N-acysteine pyridine disulfide (C) to obtain difatty acid-disulfide complexed peptides or polypeptide derivatives represented by formula (D). Such derivatives (D) can enhance permeability to cells and prolong the retention time in tissues. It is preferred that it does not have biological activity. When the derivative (D) is administered in the patient's body to be reduced in vivo, the derivative (D) can be converted into the parent peptide or polypeptide (A).

Scheme II depicts the preparation of difatty acid disulfide complexes (D) from cyclic disulfide containing peptides (A). (D) can be converted back to the parent peptide (A) as soon as it is oxidized in blood or tissue, for example via intermediate (E).

Scheme III below illustrates another embodiment of the present invention. Two disulfide crosslinked chains produce fatty acid derivatives from the polypeptide to produce a complex represented by the formula (H).

Scheme III illustrates the preparation of a difatty acid disulfide complex (H) from a two chain polypeptide (F) having a cyclic structure consisting of two or more disulfide bonds. Similar to Scheme II, (H) can be converted back to the parent polypeptide (F) as soon as it is reduced.

Another method for preparing complex (X) is illustrated in Scheme IV below, where the necessary disulfide bonds are introduced into raw compound (N) having no disulfide bonds or rings, or easily accessible disulfide bonds or rings. Cyclodisulfide containing molecules, such as lipoic acid (J), are first complexed with compound (N) via ester bonds or amide bonds. After reduction, two disulfide fatty acid bonds may be formed (M). The difatty acid complex can be reduced in blood or tissue and converted back to the parent drug molecule (N) after hydrolysis by tissue enzymes. Examples of formula IV are shown in Examples 20 and 21 below.

Complex (X) is preferred in that it is water soluble and has lipophilic moiety (s) that makes it readily absorbable into the cell, wherein the lipophilic moiety (s) may delay release in vivo. Because it has a degradable bond (s). Peptides or polypeptides may have cyclic disulfide bonds with multiple amino acids. Modification of the peptide or protein is usually carried out in an aqueous solution such as PBS, or other buffers known in the art. In general, fatty acids that can be used are 4 to 26 carbon atoms, more commonly 5 to 19 carbon atoms. Examples of fatty acids include acetic acid (2 carbon atoms), caproic acid (6 carbon atoms), capric acid (10 carbon atoms), lauric acid (12 carbon atoms), myristic acid (14 carbon atoms), Palmitic acid (16 carbon atoms) and stearic acid (18 carbon atoms), but are not limited to these (see Example 11 below). The method of Scheme II is generally useful for peptides or polypeptides with one to five cyclic disulfide bonds.

The reaction can be carried out at room temperature. At the start of the reaction, fatty acids are generally present in excess in molar concentration relative to the peptide or polypeptide, and in general, the ratio between fatty acid and peptide or polypeptide for one cyclodisulfide is 3: 1. In complex (X), the weight of fatty acids is generally less than the weight of the peptide or polypeptide. Thus, since relatively small amounts of fatty acids are used, there is less risk of toxicity, unlike the case of administering drugs in lipid preparations, micelles or liposomes.

The reaction is generally carried out in an aqueous medium, preferably at pH 6-8, preferably pH 7.6. The reaction generally proceeds rapidly, completing within about 30 minutes to yield a stable complex (X). Complexes (X) can be purified using methods known in the art for protein and peptide purification. The presence of peptides or polypeptides in the complex is confirmed by methods known in the art, such as chromatography.

Examples of peptides or proteins that can be modified according to Scheme II include desmopressin (nanopeptides consisting of 9 amino acids and one cyclic disulfide ring, see Example 11 below), calcitonin (30 amino acids and one Shi) Peptides with click disulfide rings, see Example 17 below, octreotide (octapeptide with one disulfide ring), oxytocin (nanopeptide with one cyclic disulfide ring) and epidermal growth factor (53 amino acids and Single polypeptide chains consisting of three cyclic disulfide rings). Insulin is an example of a polypeptide that can be modified according to Scheme II or III, which consists of two chains (A and B chains) and one disulfide ring in the A chain (as used in the modification method according to Scheme II). Total of 51 amino acids are present, and there is one ring structure formed by two disulfide bonds (available for the modification method according to Scheme III) between the A chain and the B chain.

In another embodiment of the invention, the fatty acid moiety of complex (X) may be substituted with other lipids, such as steroids (see examples 15 and 16 below for examples). Group A of the reagent represented by the formula (V) (ie ASS-CH ₂ -CR ¹ (NHCOR ² ) C (= 0) R ³ ) is a good leaving group. Preferred leaving groups are defined as moieties that act as a good leaving group by acting to make the disulfide group of compound (A) represented by the formula (V) more reactive to the substitution reaction with the sulfhydryl containing compound represented by the formula (PSH). do. Examples of preferred leaving groups include, but are not limited to, p-nitro-o-carboxythiophenone and thiopyridine. Descriptions of leaving groups can be found in most organic chemistry textbooks. For example, on page 241 of Cram and Hammond's Organic Chemistry, McGraw-Hill Book Co. (Second Edition, 1964), the leaving group "L" and the "CL bond can be combined with L. Decompose in the way that they are. " Therefore, the part in the organic molecule is a good leaving group when it is capable of sucking electron pairs by high electronegativity or resonance stability.

The fatty acid complexes of the present invention are soluble in most buffered solutions in which proteins and peptides are soluble. Specifically, any free carboxylic acid group is charged at neutral pH, thus increasing the solubility of the complex. As such, the complex may be readily prepared using a pharmaceutically acceptable carrier or adjuvant suitable for administering the protein or peptide to the patient orally or via other routes.

When R ¹ is not equal to R ⁴ , R ² is not equal to R ^5, and R ³ is not equal to R ⁶ , the complex (X) is a compound represented by the formula (VIA). It can be prepared by forming a compound represented by the formula (X) by forming a complex with the compound represented by the formula (X). Formula (VIA) and Formula (VIB) are as follows.

Wherein P ² and P ³ are residues derived from a sulfhydryl containing compound, and R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ are as defined above. Those skilled in the art will appreciate that compounds of Formulas VIA and VIB are examples of compounds represented by Formula VI as described above. Upon formation of the complex, P ² and P ³ become P ¹ . P ¹ , P ² and P ³ are preferably proteins or peptides, and those skilled in the art can carry out the synthesis by modifying methods of peptide or protein complex formation known in the art.

According to the present invention, there is an advantage in that the disulfide bond between the fatty acid moiety and the peptide or protein can be easily reduced. Therefore, the active peptide or protein molecule is released in the parent form in the target tissue or cell. In addition, the fatty acid portion of the complex contains only amino acids and lipid molecules that are not toxic to mammals, especially humans.

The pharmaceutical composition of the present invention can be administered by any means that accomplishes a given purpose. For example, administration can be via the oral, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, intra- or intracranial routes. The dose administered depends on the age, health and weight of the recipient, the type of concomitant treatment (if concomitant treatment is present), the frequency of treatment and the nature of the desired effect.

Within the scope of the composition of the present invention, all compositions containing the compound of the present invention in an amount effective to achieve a predetermined object are included. Depending on the needs of the individual, one of ordinary skill in the art will readily recognize the optimum effective amount range for each compound.

In addition to administering the lipidated compounds of the present invention as raw chemicals in solution, the compounds of the present invention are suitable including excipients and auxiliaries that facilitate processing of the compounds into preparations that can be used in the pharmaceutical industry. It can be administered as part of a medicament containing a pharmaceutically acceptable carrier. Suitable parenteral dosage preparations include aqueous solutions of the compounds in water-soluble form. Moreover, suspension of a compound and oily injection suspension can be administered as needed. Suitable lipophilic solvents or excipients include fatty oils such as sesame oil or synthetic fatty acid esters such as ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, for example sodium carboxymethyl cellulose, sorbitol and / or dextran. Optionally, the suspension may further contain a stabilizer.

The compounds of the present invention may also be combined with lipophilic cationic compounds, which may be in the form of liposomes. Methods of injecting nucleotides into cells using liposomes are disclosed, for example, in US Pat. Nos. 4,897,355, 4,394,448 and 5,635,380. In addition, reference may be made to US Pat. Nos. 4,235,871, 4,231,877, 4,224,179, 4,753,788, 4,673,567, 4,247,411, 4,814,270 for general methods of preparing liposomes comprising biological materials. In a preferred embodiment, a methol solution of dimyristoyl phosphatidyl choline, cholesterol and stearylamine (7: 2: 1) is evaporated to obtain a dry film. The film is hydrated in Tris® buffer containing the appropriate amount of lipidated compound followed by probe sonication.

Hereinafter, the present invention will be described in accordance with embodiments, but the following embodiments are merely exemplary and do not limit the protection scope of the present invention as defined by the appended claims.

Example 1

Synthesis of N-palmityl-2-pyridyldithiocysteine (Pal-PDC)

An ice-cold solution of L-cysteine (I) (3.0 g) in ethanol (50 mL) was added dropwise to a stirred solution of 2,2-dithiopyridine (7.5 g) in ethanol (30 mL) before the reaction was carried out at 20 ° C. Continued for 18 hours. After the solution was centrifuged to remove the precipitate, the volume of the supernatant was reduced to 40 ml using a rotary evaporator. The reaction mixture was then added dropwise to 400 ml of ice cold benzene. PDC (III) was crystallized in benzene, filtered off, dissolved in 40 ml of ethanol and recrystallized in 400 ml of ice cold benzene in the same manner as described above. The recrystallized product was filtered off, dried under vacuum overnight, and finally stored in a desiccator at -20 ° C.

PDC (III) (100 mg) was dissolved in 5 ml of DMF and mixed with 100 μl of triethylamine, and the resulting suspension was dissolved N-hydroxysuccinimide ester (IV) of palmitic acid dissolved in DMF (5 ml). (250 mg) was reacted at 25 ° C. for 24 hours. In the meantime, the suspension became transparent. The solution was diluted with 40 ml of ice cold water at pH 3.0 and the precipitate (containing Pal-PDC (V) and palmitic acid) was separated by centrifugation at 10000 rpm for 30 minutes. Pal-PDC (V) was isolated from palmitic acid by a precipitate suspension in water (Pal-PDC (V) dissolved but not palmitic acid). Pal-PDC (V) was further purified using two or more acid precipitation steps in the manner described above.

Example 2

Synthesis of complex

Unless otherwise noted, all final reagents used in the complex formation steps (Pal-PDC and PDC) were analyzed using silica coated thin layer chromatography (TLC) plates containing fluorescent indicators. These plates were not activated by heating prior to analysis. For routine analysis of the synthetic reagents, 5 μl of ethanol-based solution (5 mg / ml) containing the reagents was applied to the plate. The plates were then developed in a solvent chamber in equilibrium with the mobile phase. After sufficient transfer of the forward solvent, the plates were separated, dried and irradiated under UV lamp. The positions of the points were marked directly on the plate to create a picture of the plate and the points. The Rf value of each point shown was calculated and recorded. The composition of the mobile phase used for analysis was adjusted to provide optimal separation of reagent points.

As an example, a complex of BBI was synthesized. BBI is a hydrophilic protein contained in small amounts in cells and has no oral bioavailability. In addition, BBI is stable in the gastrointestinal tract and resistant to degradation by intestinal mammalian proteolytic enzymes [Yavelow, J., et al . , Cancer. Res ., 43: 2454: s-2459s (1983). The use of BBI for chemical prophylaxis is acceptable only if an orally absorbable form of BBI can be developed.

BBI (20 mg) was dissolved in 1 ml of sodium bicarbonate solution (0.3 M, pH 8.0) and it was reacted with SPDP (5 mg / 100 μl (DMF)) at 25 ° C. for 2 hours. Purification of BBI-PDP using Sephadex® G50 gel filtration chromatography, followed by evaluation of the release of thiopyridine moiety after reaction of BBI-PDP with dithiothreitol (DTT) to evaluate PDP derivatization of BBI It was. Through this process, about 4 amino groups per molecule of BBI were modified by SPDP. The degree of derivatization of the BBI can be controlled by adjusting the pH of the reaction buffer, and the modification of the BBI can be modified from one amino group per molecule of BBI when the reaction is carried out at pH 7 to 4.5 when the reaction is carried out at pH 8.5. Up to amino groups can be controlled.

BBI-PDP (20 mg) in PBS (1 mL, pH 5.0) was reduced to DTT (25 mM) and then eluted from Sephadex® G50 column. The sulfhydryl-containing BBI fraction eluted in the column pore volume was identified using Elman's reagent, followed by a 3-fold excess of Pal-PDC (V) in PBS at pH 7.0 (per one sulfhydryl group on BBI). And reacted at 4 ° C. for 16 hours. The reaction mixture was then acidified to pH 3.0 with HCl (1 M) and placed on ice for 30 minutes. Supernatants were analyzed separately using a Sephadex® G25 gel filtration column. A precipitate containing palmithyl disulfide complex of BBI, BBIssPal (VI) and excess reagent was separated by centrifugation, dissolved in DMF (2 mL) and eluted from Sephadex® LH20 column using DMF. BBIssPal (VI) eluted with column pore volume was isolated, dialyzed three times with 500 times volume of water, and lyophilized. Using this procedure the yield of the composite was about 80% by weight. Complex formation of Pal-PDC and BBI was confirmed and quantitatively analyzed after complex formation of [3H] -labeled Pal-PDC (V) and BBI using the same complex formation conditions as described above. In addition, the same procedure was used to synthesize a complex of oleic acid and BBI (BBIssOleic).

Example 3

Delivery of the complex

Human colon carcinoma cells (Caco-2) were removed from a 25 cm 2 raw material flask by incubation with 0.5 ml of trypsin / EDTA solution (0.5% trypsin, 5.3 mM EDTA) at 37 ° C for 10 minutes. The cells are then suspended in Dulbecco's minimal essential medium and supplemented with 15% fetal calf serum (FBS), L-glutamine (1%) and essential amino acids (1%), followed by coulter counters. Counting using

Caco-2 cells suspended in 1.5 ml medium were seeded at a density of 500,000 cells per insert in the transwell tip chamber. Then 2.5 ml of the medium was added to the basal chamber of each transwell. The cells were allowed to attach for two days without shaking, and then injected every other day until the experiment was conducted. The cells were maintained for about 14 to 20 days before the experiment was performed and the cells were injected 24 hours before the experiment. The cell monolayer exhibited an electrical resistance (TEER) of about 500-600 mm <2> in the epithelium within one week after the incubation and maintained this resistance until 21 days after the incubation.

Chloramine-T method [McConahey, PC and Dixon, FJ, Meth. Enzymol., 70: 221-247 (1980)] was used to radioiodize BBI and BBIssPal. The 14-day-old confluent cell monolayers were washed once with Dulbecco's medium without serum and incubated in the medium at 37 ° C. for 30 minutes. The incubation medium was then replaced with a serum-free medium containing ¹²⁵ I-BBI (10 μl / ml) as natural-BBI or as BBIssPal or BBIssOleic and the monolayer was further incubated at 37 ° C. for 60 minutes. . The monolayer was then washed three times with ice cold PBS and then exposed to trypsin (0.5%, EDTA 5.3 mM) at 37 ° C. for 10 minutes. The detached cells were injected into tubes, centrifuged, washed three times with ice-cold PBS, and analyzed for accumulated radiation using a gamma counter, and finally known methods [Lowry, OH et al., J .Biol. Chem., 193: 265-275 (1951)].

Several experiments measured reduced ¹²⁵ I-BBIssPal uptake into cells. ¹²⁵ I-BBIssPal was reduced with DTT (50 mM) at 60 ° C. for 5 minutes and then further reduced at 37 ° C. for 25 minutes. In the control experiment, ¹²⁵ I-BBIssPal was exposed to the medium at the same temperature and not to DTT.

The ¹²⁵ I-BBIssPal uptake in the presence of BSA (fatty acid free) was measured as follows. ¹²⁵ I-BBIssPal was incubated with medium containing 0.1% BSA for 30 minutes at 37 ° C. Then it was added to the cell monolayer. In some uptake experiments, BSA was first mixed with a 3-fold molar excess of palmitic acid and incubated with the complex before testing. ^In the experiment when the ¹²⁵ I-BBIssPal uptake was measured in a medium containing FBS, the complex was simply added to the medium containing a predetermined amount of FBS.

First, a 2 to 3 week old confluent cell monolayer with a TEER of about 500 m 2 was incubated with Dulbecco's MEM containing 1% of FBS for 30 minutes at 37 ° C. The incubation medium was then separated and ¹²⁵ I-BBI (10 μg / mL) complex in 1.5 mL of the medium was added to the tip chamber of the transwell. To the basal chamber, 2.5 ml of medium was added and the transwells were incubated at 37 ° C. At a given time, the entire basal chamber medium (2.5 ml) obtained from each transwell was separated and counted for radioactivity using a gamma counter. In each experiment, seven samples were usually taken at 1, 2, 3, 4, 5, 6 and 24 hours after incubation. After 24 hours, samples were taken, the cell monolayers were washed three times with ice cold PBS, cut out of the inserts, and the accumulated radiation was counted using a gamma counter.

The intensity of ¹²⁵ I-BBI complexes traveling across monolayers was investigated using Sephadex® G50 gel filtration chromatography. Briefly, basal medium was sampled at 24 hours, then 1.0 mL of medium was centrifuged at 2000 rpm and eluted from a G50 column (10 mL) using PBS. 1 ml fractions were recovered and fraction total radiation was measured using a gamma counter. The original complex was eluted to the column pore volume and fractions smaller than 1 kDa were eluted to or above the column volume.

The results of measuring the uptake of ¹²⁵ I-BBI to palmitic acid in the presence of FBS added in different portions as a free protein or as a complex form are listed in Table 1 below. When the complex was incubated and incubated with cellularization in serum-free medium, the uptake of BBIssPal was about 140 times higher than the uptake of BBI. In the presence of medium containing 1% FBS, the internalization of BBIssPal increased by 35 times the degree of internalization of BBI. Increasing the serum concentration further up to 10%, the uptake of BBIssPal into cells increased by only 10 times more than the uptake of native BBI. Internalization of BBIssPal into Caco-2 cells rapidly decreased to 14% and 2.3% of serum-free internalization for 1% and 10% FBS containing media, respectively.

Uptake {ng BBI / mg (cell protein)} / hour Serum free 1% FBS 10% FBS BBI 3.9 ± 0.19 2.2 ± 0.17 1.3 ± 0.02 BBIssPal 540.0 ± 24.13 78.5 ± 3.41 12.9 ± 0.02

Cell monolayers were incubated at 37 ° C. for 60 min with 10 μg / ml of ¹²⁵ I-labeled complex. The results obtained are shown as mean ± SEM of the three monolayers. Studies on its uptake were performed in the presence and absence of FBS added in Dulbecco's medium.

Since BBIssPal uptake into cells is believed to be mediated by palmitic acid ligands on the complex, uptake of ¹²⁵ I-BBIssPal into Caco-2 cells before and after reduction with DTT was studied. Since the presence of serum in a constant temperature medium has the effect of inhibiting the uptake of the complex into cells, the absorption in the serum-free medium was studied. The results are listed in Table 2 below. Caco-2 uptake of untreated ¹²⁵ I-BBIssPal into the cells was 80 times higher than the uptake of ¹²⁵ I-BBI into cells. Exposure of ¹²⁵ I-BBI to DTT did not cause a decrease in uptake. In contrast, reduction of ¹²⁵ I-BBIssPal by DTT reduced uptake of the complex into cells by about 80%. Reduction of BBIssPal by DTT separated palmitic acid from the complex. Thus, the uptake of ¹²⁵ I-BBIssPal was mediated by hydrophobic palmitic acid ligands.

Uptake {ng BBI / mg (cell protein)} / hour Untreated DTT-treated BBI 4.8 ± 0.00 5.2 ± 0.00 BBIssPal 381.7 ± 0.03 46.5 ± 0.00

As a natural protein or as BBIssPal, cell uptake of ¹²⁵ I-BBI was measured before and after reduction by DTT (50 mM) for 5 minutes at 60 ° C and 25 minutes at 37 ° C.

Bovine serum albumin (BSA) is known as a carrier of fatty acids in vivo, which contains hydrophobic regions capable of tightly binding fatty acids. ^Since the uptake of ¹²⁵ I-BBIssPal decreases in the presence of serum, the probability of BBI-binding BBIssPal in FBS was investigated. Cell uptake of ¹²⁵ I-BBIssPal and ¹²⁵ I-BBI in the presence of medium containing BSA or fatty acid containing BSA was studied and the results are shown in Table 3. In the presence of medium without BSA, the ¹²⁵ I-BBIssPal uptake into cells was 80 times higher than the BBI uptake, as expected from the results obtained in the above study. When degreasing BSA (fatty acid-free BSA) (0.1%) was present in the medium, the uptake of ¹²⁵ I-BBIssPal was reduced by 82%, while the uptake of ¹²⁵ I-BBI was not affected. Even in the presence of fatty acid-containing BSA (0.1%) prepared by spiked nonfat BSA in 3 molar excess of palmitic acid, absorption of ¹²⁵ I-BBI was not affected. Therefore, ¹²⁵ I-BBIssPal binds strongly with BSA, which depends on the number of fatty acids already bound to BSA.

Uptake {ng BBI / mg (cell protein)} / hour Serum free BSA BSA / FA BBI 4.8 ± 0.00 4.8 ± 0.00 3.9 ± 0.00 BBIssPal 380.0 ± 0.03 69.7 ± 0.00 258.9 ± 0.00

Absorption studies in the presence and absence of added fatty acid free BSA (BSA) or fatty acid containing BSA (BSA / FA) were performed in Dulbecco's medium. Results of uptake studies for palmitic acid or oleic acid of ¹²⁵ I-BBI in Caco-2 cells in the presence of serum-free medium, either as native BBI or in the form of a complex, are shown in FIG. The results were recorded as BBI internalization mean value (ng) ± SEM (n = 3). ¹²⁵ I-BBIssPal intracellular uptake of approximately 100 times the absorption in the ¹²⁵ I-BBI cells. Similarly, ¹²⁵ I-BBIssOleic intracellular uptake of ¹²⁵ I-BBI is approximately 108 times the absorption amount within a cell. ^The difference between ¹²⁵ I-BBIssPal uptake and ¹²⁵ I-BBIssOleic uptake was not significant.

Example 4

Biodistribution analysis

Two to three week old female CF-1 mice with a body weight of 20 g to 25 g freely fed food and water before the experiment were used in animal experiments. ¹²⁵ I-BBI (3 mg / kg) as a native BBI or BBIssPal or BBIss oleic acid complex was injected into the animal via the tail vein. Three animals from each experimental group were killed at 0.5, 3 and 24 hours post-injection to extract their blood (200 μl), kidney, lung and liver, rinsed with ice-cold PBS and analyzed for accumulated radioactivity. . The weight of each organ was recorded and used to control the concentration of the complex in the organ.

In an intravenous biodistribution study, ¹²⁵ I-BBI (3 mg / kg) as natural BBI or BBIssPal was injected into the lower left quarter of the abdominal cavity of each animal. The animals were then treated by the methods described above in the study of intravenous biodistribution.

In vivo distribution results of BBI and BBIssPal after intravenous administration are listed in FIG. 2 as cumulative dose% / g (organ) ± SEM. As a result, BBIssPal accumulates in the blood at a relatively high concentration and is removed more slowly through circulation, while BBI is rapidly secreted from the body without increasing blood concentration. Kidney biodistribution results showed that while BBI accumulated rapidly in the kidney, BBIssPal did not. Hepatic accumulation of BBIssPal was about five times higher than that of BBI, and BBIssPal concentration remained high within the liver even 24 hours after injection. Lung accumulation of BBIssPal was also about 2 times higher than lung accumulation of BBI, but this result may be due to residual blood present in the trachea after extraction. Obviously, BBIssPal was maintained longer at high concentrations in the blood and liver. On the other hand, the elongation removal rate for BBIssPal was about four times lower than for native BBI.

Intravenous biodistribution of BBI and BBIssOleic was also studied in CF-1 mice. The result is 0.5. Cumulative dose% / g (organ) ± SEM at 3 and 24 hours is shown in FIG. 3. In vivo distribution of BBIssOleic was very similar to BBIssPal. As observed for BBIssPal, BBIssOleic was at a higher concentration than BBIss and apparently slowed down through circulation. The blood concentration of BBIssOleic was about four times higher than that of BBI at the same time point. The kidney removal rate of BBIssOleic was about 4 times lower, and liver accumulation was about 4 times higher than that of native BBI. The intrahepatic retention time of BBIssOleic was prolonged, and the concentration of the complex present in the liver was over 24 hours after injection. The lung concentration of BBIss oleic acid was about two times higher than the native BBI concentration, but this observation can be explained by the higher amount of residual blood in the lungs.

Intravenous biodistribution of ¹²⁵ I-BBIssPal in CF-1 mice was calculated at the dose% / g (organ) range (bar) accumulated at 0.5 hours (Figure 4A), 3 hours (Figure 4B) and 24 hours (Figure 4C) As shown in FIG. Kidney accumulation of ¹²⁵ I-BBIssPal was lower than four times the height of the accumulation of natural ¹²⁵ I-BBI for 0.5 and 3 hours. At 24 hours, ¹²⁵ I-BBIssPal was higher than ¹²⁵ I-BBI. ¹²⁵ blood concentration of I-BBIssPal were similar to the ¹²⁵ I-BBI from 0.5 hour, was the third time 1.5 times higher than that of BBI, the 24-hour was about 3 times greater than the BBI. ¹²⁵ I-BBIssPal accumulation of liver were 1.5 times higher than that of ¹²⁵ I-BBI in 0.5 hours, in three hours was 2.5 times higher than that of BBI, the 24-hour was about 4 times greater than the BBI. A relatively large amount of ¹²⁵ I-BBIssPal was present in the liver and kidney at 24 hours.

Example 5

In vitro transformation studies

Published in Reznikoff, CA et al . , Cancer. Res ., 33: 3239-3249 (1973); Reznikoff, CA, et al., Cancer. Res., 33: 3231-3238 (1973), were used for transformation using C3H 10T1 / 2 (clone 8) cells. Raw cultures of mycoplasma free cells were maintained by subcultured 50,000 cells / 75 cm 2 (flasks) every 7 days. Using this schedule, cells were always passaged about 2 days before reaching fusion. Raw cultures were grown in Eagle's basal medium supplemented with 10% FBS, penicillin (100 units) and streptomycin (100 μg) and used for transformation analysis at passage 9-9. The cells were treated with trypsin (0.1%) in PBS for 5 minutes and the cells were passaged by quenching trypsin with 5 ml of medium. This process minimizes spontaneous transformation in the raw culture and maximizes the smear efficiency in the Petri dish. The FBS mother liquor used in the culture was prescreened to confirm that the serum can assist in the expression and growth of transformed cells.

For transformation analysis, C3H 10T1 / 2 cells (1000 / dish) were seeded into 60 mm Petri dishes, which were supplemented with Eagle's basal medium supplemented with 10% FBS, penicillin (100 units) and streptomycin (100 μg). It was grown for 24 hours in an atmosphere of 5% humidity CO ₂ . Cells were then started by treating 25 μl of 3-methylchloranthrene (MCA) in acetone mother liquor to a final concentration of 1 μg / ml {MCA (5 μg / 5 ml)}. After growing the cells for 24 hours in the presence of calcinegen or solvent, each medium in the dish was replaced with fresh medium containing no calcinegen or solvent. The medium in the dish was changed twice weekly for the first two weeks of analysis and then once a week for the remaining four weeks of analysis. In experiments designed to measure the transformative inhibitory activity of the complex, cells were maintained in medium containing the complex (1 μg / ml) for the first 3 weeks after analysis. The cells were then maintained in medium without addition of the complex.

Six weeks after the calcinegen treatment, the cells were observed microscopically to see if they adhered to the culture dish, washed with PBS and fixed in 100% methanol. The fixed monolayer was then stained with Giemsa dye. In each experiment 20 dishes per group were treated. In addition to the test group, at least three other groups [negative control (not treated with calcinegen or solvent), acetone control (treated with 25 μl of acetone) and positive control [MCA in 25 μl of acetone (1 μg / ml ) Were also analyzed for transformation. The transformed spot on the plate (diameter> 3 mm) was studied under a microscope and classified according to published guideline type I, II or III [Landolph, JR, Transfomatin assay of established cell lines ; Mechanism and Application (ed. Kakunaga, T., and Yamasaki, H.) IARC Scientific Publications, Lyon, France pp. 185-201 (1985). Briefly, type III dots were cell growth regions that were dense, multi-layered, philophilic, stained dark blue by GM, and had rough crisscross edges. Type II viscosities are dense, multi-layered, dyed purple by the cell growth area or GM, and have smoother and more elaborate edges than type III dots. Type I points did not appear in the analysis.

The smear efficiency (PE) of the cells was also studied with each transformation assay. To measure PE of cells in different treatment groups, cells (200 cells / dish) were seeded in three 60 mm Petri dishes per experimental group and treated in the same manner as in the transfection assay cells. Analytical cells were terminated on day 10, fixed with 100% methanol and stained with GM. Thereafter, a population of 50 or more cells visible under the microscope was counted. Smear efficiency was defined as (colony count / cell culture count) × 100%.

The antitransformation activities of BBI, BBIssPal and BBIssOleic in vitro are listed in Table 4 below. Immediately after MCA treatment, BBI was added to the culture at a concentration of 1.0 μg / ml as the free protein or in the form of a complex to palmitic or oleic acid for the first three weeks of the transformation assay period. MCA treated cells were exposed to 3-methylchloranthrene and lysed in 25 μl of acetone at a concentration of 1 μg / ml for 24 hours. Acetone treated cells were only exposed for 24 hours. The test group was exposed to MCA for 24 hours and then to the complex for the first 3 weeks after analysis. Untreated cells were not exposed to MCA or acetone. Statistical analysis (Chi-square): group 4 vs group 3, p <0.05; Group 5 vs group 3, 0.05 <p <0.1; Group 6 vs group 3, p <0.05. About 14 days after seed culture, the untreated cells of the control that caused fusion in the dish formed a well adhered, contact inhibited monolayer. These dishes did not contain transformed points at the end of the assay. Acetone treated cells also caused fusion, forming well-adhering monolayers about 14 days after species culture, which did not include the transformed points. However, MCA treated dishes contained morphologically transformed dots. Six of the 19 dishes were shown to contain type III dishes. The BBI treated group did not include the transformed points, indicating that BBI can prevent MCA induced transformation in these cells. BBIssPal treated cells contained type II dots in one of 20 dishes, which were labeled in the analyte. BBIssOleic treated cells did not contain transformed points. All groups had a PE of 20% to 25% in this analysis. As demonstrated in Table 4, both BBIssPal and BBIssOleic retain the original biological activity of BBI.

Treatment group Smear Efficiency (%) Number of dishes containing transformed dots / number of dishes Fraction of dish containing transformed dots 1.Control group-untreated 23 ± 1.5 0/20 0 2. Negative Control-Acetone Treatment 22 ± 2.0 0/20 0 3. Positive Control-MCA Treatment 21 ± 3.0 6/19 0.32 4. TEST-MCA Treatment + BBI 24 ± 2.0 0/20 0 5. TEST-MCA Treatment + BBIssPal 23 ± 3.0 1/20 0.05 6. TEST-MCA Treatment + BBIssOleic 24 ± 3.5 0/20 0

Example 6

Delivery of single complexes and multiple complexes

Studies were conducted on delivery of tip membrane bound ¹²⁵ I-BBIssPal using transwell and 6 well plates. In a six-well plate experiment, ¹²⁵ I-BBI or ¹²⁵ I-BBIssPal (10 μg / ml) was incubated with Caco-2 cells in serum free medium at 37 ° C. for 1 hour. The cells were then washed three times with ice cold PBS and then divided into two groups. In the first group, internalization of the complexes was measured after trypsinization and cell separation. In the second group, cells were reincubated in serum-free medium, complex release from the cells was stopped for 24 hours, and the medium was separated every hour to count for radioactivity. At the end of the interruption period, cells were trypsinized, isolated and counted for accumulated radioactivity. The total count value (medium + cell cpms) in each experiment was measured and the percentage of total number of releases at various times was measured.

In transwell experiments, the complex was incubated at 37 ° C. for 1 hour with the tip side of the cells. The transwells were then rinsed three times with ice cold PBS and then reincubated with serum free medium. Release of the complex into the leading medium and basal medium was stopped for 24 hours by counting the total basal medium or the leading basal medium at various times. The total count values obtained at the end of the suspension period were added (Transwell + media count values) and the release rate (% of total release) of the composites at different times was calculated. To ensure that the counts obtained in the transwells at 24 hours were due to the presence of intracellular complexes and not due to nonspecific binding to plastics, the transwells were exposed to trypsin for 10 minutes and ice-cold PBS. After rinsing three times, counting was made for accumulated radiation.                 

BBI was modified with two or four palmitic acids and the delivery was measured in transwells. The cumulative deliveries of BBI modified with BBI, 4-palmitic acid [BBIssPal (4)] and BBI modified with 2-palmitic acid [BBIssPal (2)] in Caco-2 monolayers are listed in FIG. 5A. The results are expressed as BBI (ng / monolayer) ± SEM, n = 3. The order of delivery range was BBIssPal (4)> BBI> BBIssPal (2). The results of internalization of the complex into the same cell are listed in FIG. 5B as ng (internalized BBI) / monolayer. As expected, BBIssPal (4) had the highest uptake into cells, followed by BBIssPal (2) followed by BBI. Basal medium obtained 24 hours from the transwell was analyzed using a G50 column and the results are shown in FIG. 6. As observed earlier, neither BBI nor BBIssPal (4) traversed the fault. However, in small amounts, a significant amount of basal medium of BBIssPal (2) consists of the parent complex. This amount consists of about 10% to about 20% of the total radioactivity present in the basal medium.

Example 7

Skin Absorption of BBIssPal

Freshly formed skin from the hairless mice was placed on small rings. To each mounted skin, 5 μl of a ¹²⁵ I-labeled BBI or BBIssPal sample was applied to the 0.38 cm 2 area. Two pieces of skin were used in one treatment. The skin was kept at room temperature (23 ° C.) in a humid environment. After 30 minutes, the skin surface was first carefully washed with PBS, then the skin was removed and impregnated twice with 100 ml of PBS. The skin was then sucked into filter paper and counted with a gamma counter. The amount of PBS retained in the skin was calculated using specific radiation of BBI or BBIssPal. The uptake of BBI or BBIssPal into mouse cells was 0.14 and 1.6 μg / cm 2, respectively. This demonstrates that when Pal-PDC is used to modify the polypeptide, the amount of BBI uptake into the skin has increased by at least 10 times.

Example 8

Synthesis of Horseradish Peroxidase (HRPssPal)

10 mg of horseradish peroxidase (molecular weight 40,000; Cat. No. P 8375 of Sigma Chemical Company, St. Louis, MO) in 0.5 ml of PBS was mixed with 2 ml of SPDP in 0.1 ml of DMF at 25 ° C. for 2 hours. The reaction was terminated by diluting with 0.5 mL of PBS and dialyzed in 500 mL of PBS at 4 ° C. After 24 hours, the solution in the dialysis tube was removed and reduced by the addition of 50 μl of 1 M DTT, and separated using a Sephadex® G50 column. Fractions in the column pore volume were combined and mixed with 10-fold molar excess of PalPDC in borate buffer at pH 9.6 for 4 hours at 25 ° C. The reaction mixture was then dialyzed at 4 ° C. for 3 days without leaving and the final product was evaluated to contain 10 palmitic acid residues per molecule of HRP. The HRP molecule retained about 20% of the original enzyme activity.

Example 9

HRPssPal Cell Uptake

Confluent monolayers of mouse fibroblast L929 in 6 well culture cluster plates were incubated with 30 μg / ml (HRP) in serum-free medium either in its original form or as palmitic acid complex (HRPssPal). After 1 hour at 37 ° C., the monolayers were washed three times with PBS and then dissolved in 1 ml of 0.05% Triton-X100. Cell association HRP was measured by measuring enzymatic activity in each cell extract, and the results were converted to HRP (ng) / cell monolayer. The results indicated that cellular uptake of HRP and HRPssPal was 7 and 229 ng per cell monolayer, respectively. Therefore, cellular uptake increased 30-fold by modifying HRP with Pal-PDC.

Example 10

Lipidation of Oligonucleotides

Antisense 21mer oligonucleotides complementary to the mRNA of monoamine oxidase B were thiolated through the following procedure. Oligonucleotides were mixed with a 2-fold molar excess of cystamine in the presence of a water soluble carbodiimide reagent, EDC. The mixture was kept at 25 ° C. for 2 hours and the disulfide bonds were reduced by adding a 2 molar excess of cystamine of DTT. After separation of oligonucleotides from free cystamine and DTT using a Sephadex® G25 column, small amounts of thiolated oligonucleotides were reacted with Elman's reagent and the concentration of sulfhydryl groups was absorbed at 412 nm. (Ε is estimated to be 1.36 × 10 ⁻⁴ M ⁻¹ ). The number of sulfhydryl groups per molecule of oligonucleotide was then measured. Thiolated oligonucleotides were mixed in bicarbonate buffer (pH 8) with Pal-PDC twice the molar excess relative to the number of sulfhydryl groups in the oligonucleotides. Palmitylated oligonucleotides were purified using Sephadex® G25 column.

Example 11

Synthesis of Desmopressin-fatty Acid Complex

Desmopressin (DP, 4 mg) was dissolved in 2 mL of PBS (pH 7.4) and treated with 74.8 mL of dithiothreitol (DTT, 0.1 M) at 37 ° C. The reaction was monitored using TLC (solvent: butanol: water: acetic acid (4: 5: 1), upper). Reduction of disulfide bonds in the DP was completed within 30 minutes, which was shown by switching from DP (Rf = 0.15) to single UV (UV) absorption point (Rf = 0.20) in TLC. The complex was then subjected to DTT reduced DP (dithiodesmopressin) without further purification. The reduced DP solution was mixed with 2.24 mL of 10 mM Pal-PDC (10 mM, pH 7.6) at 25 ° C. for 30 minutes and then acidified to pH 3 with HCl (1 N). Formed in the acidified reaction mixture, a precipitate consisting of palmityl disulfide complex of desmopressin (DP-P) and excess reagent was separated by centrifugation and redissolved in 1 ml of dimethylformamide (DMF). DP-P was continuously purified using Sephadex® G15 column (40 mL) using DMF as eluent. Fractions containing DP-P solution at the pore volume of the column were confirmed by TLC analysis (DP-P: Rf = 0.24) and combined. After removing the solvent in vacuo, 3.8 mg of purified DP-P was obtained.

In vitro conversion from DP-P to DP was demonstrated in the presence of the reducing agent DTT. 10 ml of DP-P (1 mg / ml, PBS) was treated with 5 ml of DTT (0.1 M) and reacted at 37 ° C. TLC analysis of the obtained solution confirmed that DP-P (Rf = 0.24) gradually disappeared and DP (Rf = 0.15) was simultaneously regenerated. After 1 hour, the conversion of DP-P to DP was completed.

Similar procedures as described above were used to prepare DP complexes linked to acetic acid, caproic acid, capric acid, lauric acid, myristic acid or stearic acid. However, Pal-PDC was replaced with each fatty acid-PDC reagent.

Example 12

Effect of DP-P on Rats with Incontinence of Hereditary Hypothalamus

Brattleboro rats with hereditary hypothalamus insipidus were compared for the effects of DP and DP-P in alleviating disease symptoms, namely polyuria and next polydose. Three Brattleboro rat groups were placed in three metabolic cages each. Their body weight, water uptake and urine output were measured daily. DP and DP-P were dissolved in 10% Lyposyn® II (Abbott Laboratories, Abbott Park, IL. USA) and injected subcutaneously (sc) at doses ranging from 0.02 to 20 μg / kg for each rat. It was. FIG. 7 shows the response of a typical DP treatment at a dose of 3.3 μg / kg, which keeps rats asymptomatic for up to 1 day. On the other hand, the rats could remain asymptomatic for at least 3 days with the same dose of DP-P, as evidenced by both reduced water uptake and urine output without significant change in body weight.

To assess dose dependent responsiveness to DP and DP-P in Battleboro rats, the effect of diuretic inhibitory activity was defined as the length of time that could maintain at least 50% urine volume reduction in treated animals. As shown in FIG. 8, similar effects are obtained using DP-P 0.02 μg / kg and DP 5 μg / ml, so DP-P is 250-fold higher than DP when administered subcutaneously for the treatment of diabetes insipidus. It was more than effective.

Example 13

In vivo structural activity relationship of desmopressin-fatty acid complex

The antidiuretic effect of DP as well as its fatty acid derivatives on Brattleboro rats was tested. Three rats were injected subcutaneously at a dose of 0.5 μg / kg in 10% Lyposyn® II (Abbott Laboratories). The urine output of each rat was measured and averaged and plotted against the number of days. As shown in FIG. 9, to increase the effect of antidiuretic activity, at least 10 carbons were needed for the fatty acid moiety in the DP-complex.

Example 14

In vivo distribution of desmopressin-palmitic acid complex (DP-P) administered intravenously to mice

Both DP and DP-P were iodized to ¹²⁵ I by the chloramine T method. ¹²⁵ I-DP or ¹²⁵ I-DP-P was injected intravenously into CF mice at a dose of 1 × 10 ⁶ cpm / mouse. Three groups of treated mice were killed at different times and blood dose was measured by counting 0.2 ml of blood in a gamma counter. As shown in FIG. 10, the plasma half-life of DP-P was much longer than the plasma half-life of DP, resulting in an approximately 6-fold increase in AUC.

Example 15

Synthesis of N-decyclocycloyl 2-pyridylthiocysteine (DOC-PDC)

A mixture of deoxycholic acid (585.2 mg), N-hydroxysuccinimide (230.0 mg) and dicyclohexylcarbodiimide (412.6 mg) in 25 ml of ethyl acetate was stirred at 25 ° C. for 16 hours. The dicyclohexylurea precipitate was separated by filtration and the ethyl acetate filtrate was evaporated under reduced pressure to give the product, N-hydroxysuccinimide ester of deoxycholic acid.

The crude N-hydroxysuccinimide ester of deoxycholic acid was redissolved in 10 ml of DMF, 533 mg of PDC was added thereto, followed by the addition of 556.7 µl of triethylamine. The resulting suspension was stirred at 25 ° C. for 5 hours. The reaction mixture was then diluted with 80 mL of distilled water and acidified to pH 3 with HCl (6 N). The DOC-PDC precipitate was separated by centrifugation, resuspended in 40 ml of distilled water, and the pH of the final solution was adjusted to 8 with NaOH (5 N). In this condition, DOC-PDC was suspended in aqueous solution and insoluble impurities were separated by centrifugation. The supernatant was acidified again with HCl (6 N) and the DOC-PDC was recovered by centrifugation and dried under vacuum. TLC analysis confirmed that the final product was practically pure and was used for the preparation of the complex without further purification.

Example 16

Synthesis of Desmopressin-Deoxycholic Acid (DP-DOC) Complex

DP-DOC was prepared in a similar manner to Example 11. Briefly, 280 μl of 10 mM DOC-PDC (10 mM, pH 7.7) was added to a reduced DP [0.5 mL, 1 mg / mL (PBS)] solution. The mixture was stirred at 25 ° C. for 30 minutes, at which time TLC analysis confirmed that the complex reaction was complete. The reaction mixture was then acidified to pH 3 with HCl (IN) and the final product, DP-DOC, was isolated as a precipitate.

Example 17

Synthesis of Calcitonin-palmitic Acid Complex (CT-P)

Salmon calcitonin (CT, 4 mg) was dissolved in 2 ml of PBS pH 7.4 and treated with 46.6 μl of dithiothreitol (DTT, 0.1 M) at 37 ° C. for 30 minutes. Reduced calcitonin (dithiocalcytonin) proceeded as described above and was used in the subsequent complex formation step without separation. 1.4 mL of 10 mM Pal-PDC (10 mM, pH 7.6) was added to the reaction mixture. The mixture was stirred at 25 ° C. for 45 min and then acidified to pH 3 with HCl (1 N). A precipitate containing palmityl disulfide complex (CT-P) of calcitonin and excess reagent was dissolved in 1 ml of DMF. CT-P was purified by eluting on a Sephadex® LH20 column (40 mL) using DMF as the eluent. The CT-P containing fractions eluting at the column pore volume were identified by TLC and UV (280 nm) analysis and combined. After evaporation of the solvent under vacuum, 3.2 mg of final product CT-P was obtained.

Example 18

Sustained Effects of Calcitonin (CP) and Their Palmitate Complex (CTP) Subcutaneously Administered to Mice

CT and CT-P were iodinated to ¹²⁵ I using the chloramine T method. ¹²⁵ I-CT or ¹²⁵ I-CT-P were injected subcutaneously at a dose of 125 μg / kg in 10% Liposyn® II (Abott). Three groups of mice were killed at different times. The amount of calcium in the blood sample was measured using a commercially available calcium diagnostic kit (Sigma Chemical Company). Plasma obtained from each mouse was isolated and treated with 5% trichloroacetic acid (TCA) in an ice bath. Radioactivity in the TCA precipitate was considered the original CT in plasma. As shown in FIG. 11, CT plasma half-life of CT-P injected mice was significantly greater than CT plasma half-life in CT injected mice. In CT-P injected mice, the transient decrease in plasma calcium concentration indicates that CT-P retains the biological activity of CT. More importantly, the subcutaneously injected CT-P maintains a nearly constant CT or CT-P concentration for about 16 hours in the blood, as compared to the rapid plasma ablation of the subcutaneously injected CT.

Example 19

Gastrointestinal Uptake and Calcium Reduction Effects of Orally Administered Calcitonin-Palmite Complex (CT-P)

CF mice were orally administered CT and CT-P at a dose of 100 μg / kg in PBS using a feeding needle. Three mice in each group were killed one hour after treatment and plasma was separated from its blood. CT and calcium concentrations were measured using commercially available CT-RIA (Phoenix) and calcium diagnostic kit (Sigma Chemical Company). The results are listed in Table 5 below. Through oral administration of CT-P, the concentration of RIA sensed CT in mice was much higher than that of CT. In addition, at 1 hour, plasma calcium concentrations were lower in CT-P treated mice than in CT treated mice. This is consistent with findings at CT concentrations. Since the cross reactivity of anti-CT antibodies is only about 10%, the actual concentration of total CT in the plasma of CT-P treated mice can be much higher than the values listed in Table 5.

Plasma Calcitonin Concentration and Calcium-Reducing Effects in Mice After 1 Hour of CT and CT-P Administration at 100 ㎍ / kg CT (pg / 0.1 ml plasma) Calcium (% reduction) CT 9.2 ± 0.7 17.5 ± 3.6 CT-P 18.3 ± 4.0 28.9 ± 1.2

Example 20

Synthesis of Acyclovir-Lipoic Acid Ester Complex (ACV-LA)

The esterification between acyclovir (ACV) and lipophosphoric acid (LA) was catalyzed by using dicyclohexylcarbodiimide in the presence of p-toluenesulfonic acid (TsOH). The product, ACV-LA, was purified using preparative TLC.

Acyclovir (ACV, 25 mg), lipoic acid (LA, 229 mg) and dicyclohexylcarbodiimide (572. 2 mg) were dissolved together in DMF (2.5 mL). 10.8 mg of TsOH was added to the obtained solution. The mixture was stirred for 3 days at room temperature. Dicyclohexylurea precipitate was removed by filtration. DMF in the filtrate was evaporated in vacuo. By evaporation, the residue was suspended in 5 mL of deionized water and extracted with ethyl ether (2 x 3 mL). The aqueous layer was separated and lyophilized.

The lyophilized residue was redissolved in a small volume of methanol and loaded onto a preparative TLC plate. The plate was developed using methylene chloride: acetone: methanol (4: 1: 1) as the solvent system. UV absorbance bands (Rf = 0.41) of products different from the absorbance bands of ACV (Rf = 0.10) were identified and scraped from TLC plates and silica gel was extracted with 2 × 15 mL of the same solvent mixture as in TLC. The solvent extract was evaporated in a rotary evaporator to give 11.7 mg of the final product, ACV-LA.

Example 21

Synthesis of Acyclovir-Lipoic Acid-Dipalmitic Acid Complex (ACV-LA-DP)

The preparation of the title complex was similar to that of the desmopressin-palmitic acid complex (Example 11). Acyclovir-lipoic acid ester (ACV-LA, 0.6 mg) was dissolved in 0.5 ml of PBS (PH 7.4) and treated with dithiothritol (DTT, 0.1 M). The reduction reaction proceeded at 37 ° C. for 30 minutes. The reduced ACV-LA (dithiol compound) was used for the subsequent complex formation step without further purification. 531 μl of 10 mM Pal-PDC (10 mM, pH 7.7) was added to the reaction mixture. The mixture was stirred at 25 ° C. for 30 minutes. The reaction mixture was acidified to pH 3 with HCl (IN) and a precipitate appeared. A precipitate containing ACV-LA-DP was obtained. This product can be further purified by chromatographic methods.

Example 22

Effect of Liposomal Compositions on Antidiuretic Actions of Desmopressin-Palmite Complex (DP-P)

The diuretic inhibitory effect of DP-P in Tris® buffer as well as liposome DP-P was tested using Brattleboro rats at an oral dose of 37.5 μg / kg. To prepare liposome DP-P, a methol solution of dimyristoyl phosphatidyl choline, cholesterol and stearylamine (7: 2: 1) was evaporated to obtain a dry film. The film was hydrated in Tris® buffer (2 mL) containing an appropriate amount of DP-P (2 hours / 25 ° C.), followed by probe sonication (15 minutes / 37 ° C.). ). The resulting liposome preparation was diluted with Tris® buffer to bring the total volume to 5 ml. This was used immediately. In rats treated with DP-P solution, the total volume of urine recovered during the first 5 hours after oral administration was 53.3 ± 15.3 ml, which was not significantly different from that of the control (47.0 ± 3.5 ml). However, liposome DP-P showed a significant antidiuretic effect because the total volume of urine recovered during the first 5 hours was 27.0 ± 1.0 ml.

Through the foregoing description, those skilled in the art will be able to easily grasp the essential structural features of the present invention. In addition, various uses and conditions of the present invention can be modified without departing from the spirit and scope of the present invention. Modifications by equivalent forms of change and substitution are also included within the scope of the present invention, and the specific technical terms described in the above specific embodiments do not limit the protection scope of the present invention. In addition, all documents, patents, and patent applications described in this specification are incorporated herein by reference.

Claims (25)

Compound represented by the following formula:

Wherein P ¹ is a residue derived by reduction of a disulfide containing compound or derivative thereof selected from the group consisting of peptide, protein, amino acid, nucleotide, nucleotide, oligonucleotide and carbohydrate; R ¹ and R ⁴ are each selected from the group consisting of hydrogen atom, methyl; R <^2> and R <^5> is a hydrocarbon group containing a C4-C26 lipid group or lipid group, respectively; R ³ and R ⁶ each represent a hydroxy, a hydrocarbon group containing 4 to 26 carbon atoms or a lipid group including a lipid group, and an amino acid chain containing one or two amino acids and having -CO ₂ H or -COR ² as a terminal group. Selected from the group consisting of; n is an integer from 1 to 20. R ¹ is the same as R ⁴ , R ² is the same as R ^5, and R ³ is the same as R ⁶ . The compound according to claim 1, wherein P ¹ comprises a further disulfide group bonded to a C4-C26 lipid group or a hydrocarbon group comprising a lipid group of the formula

R ¹ is selected from the group consisting of hydrogen atom, methyl; R ² is a hydrocarbon group containing a lipid group or lipid group having 4 to 26 carbon atoms; R ³ is selected from the group consisting of hydroxy, a lipid group having 4 to 26 carbon atoms or a hydrocarbon group including a lipid group, and an amino acid chain containing one or two amino acids and having -CO ₂ H or -COR ² as an end group. do. The compound of claim 1, wherein each R ¹ is a hydrogen atom, each R ² is a hydrophobic moiety of a fatty acid or steroid, each R ³ is hydroxy, and n is 10 or less. The compound of claim 1, wherein each R ¹ is a hydrogen atom, each R ² is a hydrophobic moiety of a fatty acid or steroid, and each R ³ is hydroxy. The compound of claim 1, wherein the lipid group or hydrocarbon group including the lipid group is a fatty acid residue having 5 to 19 carbon atoms. 6. The compound of claim 5, wherein the fatty acid is palmityl, oleyl or stearyl. The compound of claim 1, wherein each R ² is a steroid residue. 8. The compound of claim 7, wherein said steroid is selected from the group consisting of deoxycholate and cholate. The compound of claim 1, wherein P ¹ is a peptide, protein, amino acid, nucleotide, nucleotide, oligonucleotide and carbohydrate; Or a derivative thereof. A pharmaceutical composition for promoting absorption of a compound comprising the compound of claim 1 and a pharmaceutically acceptable carrier, the compound selected from the group of peptides, proteins, and oligonucleotides by mammalian cells. 11. A pharmaceutical composition according to claim 10 in the form of a liposome. A method for enhancing in vivo absorption of disulfide-containing organic compounds by mammals other than humans, comprising administering the compound of claim 1 to mammals other than humans. 13. The method of claim 12, wherein R ¹ and R ⁴ are each hydrogen, R ² and R ⁵ are each a hydrophobic moiety of a fatty acid or steroid, and R ³ and R ⁶ are each hydroxy. A method of prolonging in vivo blood and tissue retention of disulfide containing organic compounds in a mammal, except humans, comprising administering the compound of claim 1 to a mammal, except humans 15. The organic compound of claim 14, wherein the disulfide group containing organic compound comprises: peptides, proteins, amino acids, nucleotides, nucleotides, oligonucleotides and carbohydrates; Or an extension method selected from the group consisting of derivatives thereof. 15. The method of claim 14, wherein R ¹ and R ⁴ are each hydrogen, R ² and R ⁵ are each a hydrophobic moiety of a fatty acid or a steroid, and R ³ and R ⁶ are each a hydroxy group. (a) reducing the disulfide containing compound to form a reduced disulfide containing compound and (b) reacting the reduced disulfide containing compound with a compound represented by the formula ASS-CH ₂ -CR ¹ (NHCOR ² ) C (= 0) R ³ , wherein A is an aromatic active moiety, according to claim 1 A method for producing a compound according to claim 1, comprising the step of obtaining a compound of Claim 1. 18. A process according to claim 17, wherein said disulfide containing compound contains at least one disulfide and said disulfide containing compound is partially reduced in step (a). A method for producing a compound according to claim ¹ , wherein R ¹ and R ⁴ are not the same, R ² and R ⁵ are not the same, and R ³ and R ⁶ are not the same. A method comprising: combining a compound represented by the following formula (V ¹⁾ with a compound represented by the following formula (V ² ) with a sulfhydryl-containing compound P ¹ to obtain a compound according to claim 1: ASS-CH ₂ -CR ¹ (NHCOR ² ) C (= O) R ³ V ¹ ASS-CH ₂ -CR ⁴ (NHCOR ⁵ ) C (= O) R ⁶ V ² Wherein A is an aromatic active moiety delete delete delete delete delete delete

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