JP2007524667A - Lipid composition and use thereof - Google Patents

Abstract

  The present invention provides a composition suitable for use as a transfection agent comprising a cationic cardiolipin analog and other lipid species. The compositions of the present invention can facilitate transfection of a wide variety of polynucleotide species (eg, oligodeoxyribonucleotides, plasmids, RNAi species, etc.). Furthermore, the transfection agent of the present invention is effective in promoting primary cell culture and transfection of transformed cells. Furthermore, the transfection agents of the present invention are suitable for use both in vitro and in vivo. The compositions of the present invention also have other uses such as, for example, delivery of various active agents, dermatological and cosmetic applications, and agricultural applications. The present invention further provides a method for introducing an active agent into a cell by contacting the cell with the composition of the present invention. The present invention further provides a method for inhibiting the growth of neoplastic cells and a method for treating a patient suffering from a neoplastic disease by using the composition of the present invention wherein the active agent is an antitumor agent. The present invention further examines (a) administering a composition containing cationic liposomes and SiRNAi to a cell to allow SiRNAi to enter the cell to suppress gene expression in the cell, and (b) gene suppression. A method for confirming a gene target is provided. The method also provides fluorescent / luminescent cationic cardiolipin analogs and compositions comprising such analogs. By using cationic cardiolipin analogs, the present invention provides a method for tracking the migration of lipid substances in animals.

Description

CROSS-REFERENCE TO RELATED APPLICATION This application claims priority of U.S. Provisional Patent Application No. 60 / 535,042 of the total filed Jan. 7, 2004, incorporated herein by reference. This application also claims priority to US Provisional Patent Application No. 60 / 556,843, filed Mar. 27, 2004, which is incorporated herein by reference in its entirety. This application also claims priority to US Provisional Patent Application No. 60 / 557,232, filed March 29, 2004, which is incorporated herein by reference in its entirety.

  The present invention relates to lipid compositions and in particular their use as transfection agents.

  Polynucleotides (eg, oligonucleotides, cDNAs, plasmids, RNAi, etc.) have potential uses as therapeutic agents. For example, antisense oligonucleotides targeted to oncogenes are expected in neoplastic diseases such as cancer. An antisense drug (AON) targeted to the mRNA of the human c-raf oncogene, which is an exemplified drug, is called c-rafAON. c-rafAON is an antineoplastic agent used for the treatment of radioresistant tumors and multiple myeloma. c-rafAON acts by hybridizing to mRNA and inhibiting translation, protein synthesis and tumor growth. Other examples of polynucleotides having therapeutic benefit include RNAi that can also suppress the expression of a gene of interest such as an oncogene. Plasmids or other vectors containing the gene of interest also have potential as therapeutic agents by being able to deliver the transgene of interest to the patient.

  While potential as a therapeutic agent is expected, due to its negative charge, the drug cannot efficiently penetrate the cell membrane and transfect cells. That is, a delivery system is required to improve cell transfection and increase the therapeutic effect of the polynucleotide. The use of cationic liposomes to enhance the delivery of oligonucleotides has been reported (US Pat. Nos. 6,559,129B1 and 6,333,314B1, Zelphati et al. J. Liposome Res., 7 ( 1): 31-49 (1997)). However, there are several difficulties with current cationic lipid transfection agents that limit their usefulness as transfection agents.

  For example, many cationic lipids have poor chemical stability. Furthermore, liposomes containing more cationic lipids are poor in polynucleotide carrying ability due to the inflexibility of the cationic bilayer membrane. Therefore, many currently available transfection agents are not suitable for transfection with various types of polynucleotides (DNA and RNAi). Furthermore, most available transfection agents such as LIPOFECTIN® are not effective when transfecting primary cell cultures. Furthermore, many cationic transfection agents, such as LIPOFECTIN®, exhibit a relatively high toxicity that makes them unsuitable for in vivo use. In view of the above, there is still a need for effective transfection agents.

  A problem faced by medical technology is the identification of targets for genetic suppression or therapeutic intervention. A typical method for identifying a target gene as the cause of or associated with a particular disease is to remove the gene of interest from the animal strain and create an animal model. A common way to achieve this is typically to create a rodent knockout animal. However, this method is burdensome, expensive, and takes time to become usable. Of course, it is not always possible to make a knockout because many genes that are important for a particular disease are important for the normal development of an animal. Furthermore, because many genes contribute to normal physiological conditions and the onset of disease, the “compensation effect” prevents the knockout animal from functioning as an appropriate model or system for target gene identification. There is. Therefore, there remains a need for effective methods of identifying target genes as contributing to disease or phenotype.

  The present invention provides a composition suitable for use as a transfection agent comprising a cationic cardiolipin analog and desirably other lipid species. The compositions of the present invention can facilitate transfection of a wide variety of polynucleotide species (eg, oligodeoxyribonucleotides, plasmids, RNAi species, etc.). Furthermore, the transfection agents of the present invention are effective in promoting transfection of primary cell cultures as well as transformed cells. The present invention is also suitable for use both in vitro and in vivo. The compositions of the present invention also have other uses such as delivery of various active agents, dermatological and cosmetic applications and agricultural applications.

  A preferred cationic cardiolipin analog (CCLA) is 1,3-bis- (1,2-bis-tetradecyloxy-propyl-3-dimethylethoxyammonium bromide) -propan-2-ol (PCL-2). More preferably, the composition is a lipid group consisting of cholesterol, dioleoylphosphatidylethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), alpha tocopheryl succinate and any other phosphatidylcholine. A lipid selected from The composition can include an active agent, eg, a polynucleotide. The present invention also includes pharmaceutical formulations of c-raf. Methods for making stable lamellar structure complexes of antisense oligonucleotides (c-rafAON) and anti-agents to minimize rapid hydrolysis of raf-AON in plasma are described in vivo clearance. , Improve transfection of cells, and increase the therapeutic effect of c-rafAON.

  The present invention further provides a method for introducing an active agent into a cell by contacting the cell with the composition of the present invention. The present invention further provides a method for inhibiting the growth of neoplastic cells and a method for treating a patient suffering from a neoplastic disease by using the composition of the present invention wherein the active agent is an antitumor agent. The present invention further tests (a) the composition containing cationic liposomes and RNAi to cells to cause RNAi to enter the cells and suppress gene expression in the cells, and (b) to test gene suppression. A method for confirming a gene target is provided.

  The present invention also provides fluorescent cationic cardiolipin analogs and compositions comprising such analogs. By using cationic cardiolipin analogs, the present invention provides a method for tracking the migration of lipid substances in animals. The invention further provides luminescent cationic cardiolipin analogs and compositions comprising such analogs, which are useful in measurements that require higher decay times than fluorescent analogs.

  These and other advantages of the invention, as well as other inventive features, will be apparent from the description of the invention provided herein.

  In one embodiment, the present invention provides a composition comprising a cationic cardiolipin analog (CCLA) and desirably another lipid species. Cationic cardiolipin analogs suitable for use in the compositions of the present invention are described in International Patent Application PCT / US03 / 33099, which is hereby incorporated by reference in its entirety. The most preferred cationic cardiolipin analog (CCLA) for inclusion in the composition of the present invention is 1,3-bis- (1,2-bistetradecyloxy-propyl-3-dimethylethoxyammonium bromide) -propane-2. -All (PCL-2), which is identified as compound 19 in PCT / US03 / 33099.

  The present invention also provides fluorescent and / or luminescent cationic cardiolipin analogs. For example, the fluorescent analog can comprise a cationic cardiolipin molecule as compound 19 conjugated to a fluorescent moiety (eg, as described in International Patent Application PCT / US03 / 33099). Examples of fluorescent moieties are fluorescein derivatives such as [N- (5-fluoresceinyl) -5-carbamylpentanoic acid], 2,1,3-benzoxadiazole derivatives, coumarin derivatives such as 3-bromoacetyl-6, 7-methylenedioxycoumarin, 4- (7-diethylaminocoumarin-3-yl) benzene isothiocyanate, 4- (4,5-diphenyl-1H-imidazo-2-yl) benzoyl chloride, 2-dansylethylchloro Formate, 5- (5,6-dimethoxy-2-phthalimidinyl) -2-methoxyphenylsulfonyl chloride and the like. A preferred fluorescent analog is the PCL-2 fluorescent analog. Fluorescent cationic cardiolipin analogs can be constructed by any suitable method for conjugating a fluorescent moiety to a molecule such as a cationic lipid, many of which are known in the art. Luminescent analogs can also be synthesized by those skilled in the art. Fluorescent or luminescent analogs can be used in the present invention.

  The fluorescent and luminescent cationic cardiolipins of the present invention can be used to track the passage / location of cationic cardiolipin liposomes. The fluorescent and luminescent cationic cardiolipins of the present invention can also be used to study liposome uptake and gene delivery distribution in various tissues. The fluorescent and luminescent cationic cardiolipins of the present invention can also be used to investigate the mechanism of intracellular gene delivery.

  Any suitable amount of cationic cardiolipin analog (CCLA) can be used herein in the composition, preferably the cationic cardiolipin analog PCL-2 is about 0.3 mM to about 20 mM, such as about 1 mM. Although present at ~ 15 mM, the composition can optionally include higher or lower amounts of cationic cardiolipin PCL-2. In certain preferred compositions, the cationic cardiolipin PCL-2 is present at about 0.35 mM.

  Desirably, the composition further comprises at least one lipid in addition to the cationic cardiolipin analog. Another lipid can be any desired lipid species suitable for forming the composition of interest, such as those described in PCT / US03 / 33099. Preferred lipids for inclusion in the compositions of the present invention are cholesterol, cholesterol derivatives, dioleoylphosphatidylethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), alpha tocopheryl succinate and Selected from the lipid group consisting of any other phosphatidylcholine. Typically, the total lipid concentration of the composition is from about 1.0 mg / mL to about 60 mg / mL (eg, at least about 5.0 mg / mL or at least about 10 mg / mL or even at least about 10 mg / mL and about 50 mg / mL). up to mL, or up to about 40 mg / mL or even up to about 30 mg / mL), but can be higher or lower than the above amounts if desired.

  The most preferred composition according to the invention comprises DOPC, cationic cardiolipin PCL-2 and cholesterol. In this composition, the cationic cardiolipin analog, DOPC and cholesterol can be present in any suitable ratio. However, such compositions preferably have a DOPC: PCL-2: cholesterol percent molar ratio of about (50-65) :( 25-35) :( 5-20). Furthermore, desirably such a composition further comprises D-α tocopheryl succinate. The D-alpha tocopheryl succinate can be present in any suitable amount in the composition, but is desirably present at about 0.1 wt% to about 1 wt%, most preferably about 0.2 wt%. However, the amount of D-alpha tocopheryl succinate can be higher or lower if desired.

  Another preferred composition of the invention comprises cationic cardiolipin PCL-2 and cholesterol. In such compositions, the cationic cardiolipin and cholesterol can be present in any suitable molar ratio. However, preferably the molar ratio of PCL-2 and cholesterol is 1: 3 to 6: 1, such as 1: 3 to 3: 1, or 2: 3 to 3: 2, or even 1: 2 to 2: 1. .

  Another preferred composition of the present invention comprises PCL-2 and DOPE. In such compositions, the cationic cardiolipin and DOPE can be present in any suitable molar ratio. Preferably, however, the molar ratio of PCL-2 and DOPE is 1: 3 to 6: 1, such as 1: 3 to 3: 1, or 2: 3 to 3: 2, or even 1: 2 to 2: 1. .

  In addition to cationic cardiolipin and other lipids, the composition can include stabilizers, absorption enhancers, antioxidants, phospholipids, biodegradable polymers and pharmaceutically active agents along with other ingredients. Suitable antioxidants include compounds such as ascorbic acid, tocopherol and deteroxime mesylate. Suitable absorption enhancers are Na-salicylate-chenodeoxycholate, Na deoxycholate, polyoxyethylene 9-lauryl ether, chenodeoxycholate-deoxycholate and polyoxyethylene 9-lauryl ether, monolein, Na tauro-24,25- Contains dihydrofusidate, Na taurodeoxycholate, Na glycochenodeoxycholate, oleic acid, linoleic acid and linolenic acid. Also included are polymer absorption enhancers such as polyoxyethylene ether, polyoxyethylene sorbitan ester, polyoxyethylene 10-lauryl ether, polyoxyethylene 16-lauryl ether and azone (1-dodecylazacycloheptan-2-one) be able to.

  In some embodiments, the compositions of the invention, particularly liposome compositions, are targeting agents such as carbohydrates or proteins, or other ligands that bind to specific substrates such as antibodies (or fragments thereof), or cell receptors. It preferably includes a ligand that recognizes. Include such substances (eg, carbohydrates or one or more proteins selected from the group consisting of antibodies, antibody fragments, peptides, peptide hormones, receptor ligands, eg, antibodies to cellular receptors and mixtures thereof) This facilitates targeting of the composition to a given tissue or cell type.

  In a preferred embodiment, the composition comprises at least one sugar or biodegradable polymer. Examples of suitable sugars and polymers include sucrose, lactose, trehalose, dextrose, epichlorohydrin, branched chain hydrophilic polymers of sucrose, polyethylene glycol, polyvinyl alcohol, methoxy polyethylene glycol, ethoxy polyethylene glycol, polyethylene oxide, Polyoxyethylene, polyoxypropylene, polyvinylpyrrolidone, polypyrrolidone, dextran, cellulose acetate, sodium alginate, N, N-diethylaminoacetate, block copolymer of polyoxyethylene and polyoxypropylene, polyvinylpyrrolidone, polyoxyethylene X-lauryl Ethers, where X includes 9-20, and polyoxyethylene sorbitan esters. A preferred sugar for inclusion in the composition of the present invention is sucrose. If a sugar or polymer is present, it typically represents from about 1% to about 20%, such as from about 5% to about 30%, by weight of the composition. Particularly when the sugar is sucrose, an appropriate amount to contain in the composition is 10-12% by weight. Furthermore, the composition can also include a carrier, such as a physiologically acceptable carrier. A suitable carrier for physiological (eg, medical, livestock, laboratory, etc.) applications is a physiologically compatible buffer (eg, phosphate buffered saline (PBS), HEPES, etc.), Many of these are known in the art.

  The composition typically has a pH of about 3 to about 8. However, the pH of the composition varies greatly depending on its desired use. The selection of a suitable pH for the desired application is well known to those skilled in the art. That is, in some embodiments, the pH of the composition can be adjusted to acidic (eg, from about 2 to about 6.9, such as from about 3 to about 6, or from about 4 to about 5). In other applications, it may be desirable for the pH of the composition to be alkaline (eg, about 7.1 to about 11, or about 8 to about 10, such as about 9). The pH of the composition can be adjusted using a suitable acidic or alkaline buffer as is known to those skilled in the art.

  The composition can be in any suitable form, such as a liposomal formulation, complex, emulsion, suspension, and the like. Such formulations can be prepared by any suitable technique depending on the type of composition, as is well known to those skilled in the art. Preferred compositions are liposome compositions or other compositions containing lipid vesicles. Such compositions comprise single layer lamellar or multilamellar vesicles, or mixtures thereof. Any suitable technique can be used to produce such liposome formulations. Suitable techniques include thin film hydration, reverse phase evaporation, ethanol injection and the like. For example, the lipophilic liposome-forming component is dissolved or dispersed in a suitable solvent or combination of solvents and dried. Suitable solvents include any non-polar or slightly polar solvent such as t-butanol, ethanol, methanol, chloroform or acetone that can be evaporated without leaving a pharmaceutically unacceptable residue. Drying can be by any suitable means such as freeze drying, use of a rotary dryer, and the like. Hydrophilic ingredients such as some drugs, preservatives and other drugs can be dissolved in a polar solvent such as water and mixed with the lipid phase before drying or upon reconstitution. Liposomes can be formed by mixing the dried lipophilic component with a hydrophilic mixture. Mixing of the polar solution and the dry lipid membrane can be done by any means to strongly homogenize the mixture. It can be homogenized by rotary mixing, magnetic stirring and / or sonication.

  If active agents are included in the liposomes, they can be dissolved or dispersed in a suitable solvent and added to the liposome mixture prior to mixing. Typically, the hydrophilic active agent is added directly to the polar solvent and the hydrophobic active agent is added to the nonpolar solvent used to dissolve the other components, but this is not required. After the active agent is dissolved in the third solvent or solvent mixture and added to the polar solvent and lipid membrane mixture, the mixture can be homogenized.

  The liposomes of the present invention can be vesicles of multilamellar or monolamellar lamella structure depending on the particular composition and the method of manufacture for producing it. Liposomes can be prepared to have a substantially homogeneous size within a selected size range, for example, about 1 micron or less, or about 500 nm or less, about 200 nm or less, or about 100 nm or less. Particle size has been found to play an important role in liposome biodistribution and cell entry pathways. Larger liposomes are mainly distributed in the reticuloendothelial cell (RES) system and are negligible in other tissues, but are localized in other smaller organs. Furthermore, the clearance of multilamellar vesicles with a heterogeneous particle size distribution shows a biphasic pattern, with larger liposomes having a faster clearance and smaller liposomes having a slower clearance. Information is limited regarding the biodistribution of cationic liposomes containing oligonucleotides. Letsinger et al. Previously reported that oligonucleotides complexed with cationic liposomes of about 2.0 microns in diameter were transiently taken up by the lung and later rapidly distributed to the liver. Recent studies have shown that the primary route for delivery of oligonucleotides via cationic liposomes is endocytosis. Preferably, in forming the liposome lamellar complex of the present invention, the clearance rate of the active agent is slowed by including small liposomes. One effective sizing method involves extruding an aqueous suspension of liposomes through a series of polycarbonate membranes having a selected uniform pore size; the pore size of the membrane is the maximum of liposomes produced by extruding through the membrane. It roughly corresponds to the size of For physiological use, the formulation is sterilized by extruding liposomes from a 0.22 μm or smaller filter. Desirably, the average size of the liposome is from about 50 nm to about 250 nm, such as from about 100 nm to about 200 nm, or from about 150 nm to 250 nm. Preferably, the liposome particle size distribution is substantially uniform. In addition, preferably about 99% of the liposomes have a diameter of less than about 50 nm per preparation (ie, D99 of less than about 500 nm), and desirably the D99 of the liposome composition of the present invention is from about 170 nm to about 500 nm, About 200 nm to about 350 nm, or about 250 nm to about 300 nm.

  Preferred manufacturing protocols are: i) lipid excipients in dehydrated ethanol, ie DOPC, cholesterol, PCL-2 and D-α tocopheryl succinate, and ii) active ingredient, ie polynucleotide (c-raf-1 in FIG. 1) And sucrose are dissolved in sterile water for injection. Ethanolic lipid solution is added to the aqueous active substance solution to form liposomes. Depending on the batch size, the liposomes are passed through a 0.2 μ pore size polycarbonate membrane filter three times to 0.1 μm according to a quantity (QS) that is the target batch weight to increase the weight of the product to the desired weight. The size specification is met by reducing the size by extruding 5 times through a polycarbonate membrane filter of pore size. The added ethanol is removed from the extruded liposomes by evaporation on a rotary evaporator under vacuum. After the product weight is adjusted to the weight prior to solvent removal using sterile water for injection, the product is filtered through a 0.22μ sterile filter, filled into sterile vials, capped and sealed. This method is used to produce formulations on the scale of 10-20 kilograms.

  The compositions of the present invention have been found to be stable. In this sense, the composition can be stored refrigerated and at room temperature for a long time. In general, the composition is stable under such conditions for at least about 2 months. Stability can be assessed by measuring changes in the components of the composition over a desired time frame. For example, the composition is stable if it contains at least about 70-110% of the amount of cationic cardiolipin (eg, PLC-2) present at the time the composition was initially formulated after 2 months at room temperature or refrigerated. Can be considered. A formulation is considered stable if less than 10% of the active part lost during the product's lifetime.

  The composition (or formulation) of the liposome (or other lipid) can be in any desired form. For example, in the case of use as a pharmaceutical, the composition can be directly administered to a patient. Where such compositions contain liposomes or other types of lipid vesicles, such formulations are typically in the form of vesicles in an aqueous medium such as ethanol and water for injection. Alternatively, the formulation can be in a dried or lyophilized form, in which case the composition preferably also includes a cryoprotectant. Suitable cryoprotectants include, for example, sugars such as trehalose, maltose, lactose, sucrose, glucose and dextran, and the most preferred sugars from a performance standpoint are trehalose and sucrose. Other more complex sugars such as aminoglycosides such as streptomycin and dihydrostreptomycin can also be used.

  The compositions of the present invention comprising the cationic cardiolipin of the present invention can be used in various applications, such as active agents for human or animal (typically vertebrate) patients, plants or cultured cells, such as peptides, polypeptides, proteins, nucleotides. , Polynucleotides, small molecules and other active agents can be used in vivo or in vitro delivery. In order to facilitate such use, the composition can include at least one such active agent in addition to the cationic cardiolipin species, other lipids and other components. The composition can be used in any procedure including, for example, the use of liposomes or lipid vesicles to deliver substances into cells either in vitro or in vivo (Felner et al., US Pat. No. 5,264,264). 618). The composition can also be used cosmetically, for example as a dermatological preparation. Thus, the composition can be formulated for use depending on the desired end use.

  The most preferred active agent for inclusion in the composition of the present invention is a polynucleotide, which can be DNA, RNA, or a mixture of DNA and RNA. Examples of therapeutic polynucleotides include ribozymes, interfering RNA (RNAi) antisense RNA or DNA sequences, which contain desired sequences in cells, such as genes associated with disease states (eg, oncogenes or viral genes). Can be targeted. Desirably, the polynucleotide for use in the compositions of the present invention hybridizes to specific human mRNA in the cell and further suppresses gene expression as a result of hybridization to the targeted mRNA. For example, the polynucleotide can be targeted to an oncogene such as ras, raf cot, mos, myc, etc., and is preferably targeted to the human c-raf gene. Other preferred genes for targeting with therapeutic ribozymes, interfering RNA (RNAi) antisense RNA or DNA sequences include viral genes, particularly HIV genes, such as rev transcription activators. In other embodiments, the therapeutic polynucleotide can be absent or mutated in the disease state, or can encode a gene product that is defective or absent in the disease state. Other polynucleotides can encode therapeutic polypeptides, such as immunogenic peptides (which can be used as vaccines), natural hormones or synthetic analogs of natural hormones.

  Preferred polynucleotides for use in the compositions of the present invention are 5-50mer antisense oligodeoxyribonucleotides, preferably 10-40mer sequences, more preferably 15-25mer sequences. US Pat. No. 6,126, which discloses 15-mer anti-c-raf-1 oligonucleotides that are targeted to a specific gene of interest (eg, the sequence 5′-GTGCTCCATTGATGC-3 ′ 965). Other suitable anti-raf oligodeoxyribonucleotide sequences are known in the art and can be used as appropriate in the context of the present invention (eg, US Pat. Nos. 6,090,626, 6,126,965, 6,333). , 314, 6,410,518 and International Patent Application Publication Nos. WO94 / 15645 and 94/23755). When oligonucleotides are included in the composition, they preferably contain one or more phosphothioate linkages, preferably two phosphothioate linkages. Most preferably, the oligonucleotides included in the composition of the invention contain one phosphothioate linkage at each end, but they are present at any position from one end of the oligonucleotide to the other (eg, between both ends). it can.

  The most preferred composition of the present invention is a 15-mer anti-c-raf-1 oligonucleotide having the sequence 5′-GTGCTCCATTGATGC-3 ′, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), a new positive Charged phospholipid 1,3-bis- (1,2-bis-tetradecyloxy-propyl-3-dimethylethoxyammonium bromide) -propan-2-ol (PCL-2) and cholesterol as stabilizer, Contains D-α tocopheryl succinate as oxidizing agent, sucrose as osmotic pressure regulator and ethanol as solvent and water for injection. The composition can be provided as a sterile liquid containing 2 mg / mL rafAON (50 mg rafAON per vial). Each vial is diluted 2-fold with 5% dextrose USP to give a 1 mg / mL rafAON liposome formulation. Prior to administration, it may be further diluted 8 times with 5% dextrose USP.

  Another preferred polynucleotide for use in the compositions of the present invention is an RNAi species. RNAi technology provides an effective method of suppressing cellular gene activity. SiRNA (RNAi) is an RNA molecule (dsRNA) having a double-stranded structure including a first complementary strand and a second complementary strand. Double stranded RNA comprises about 20-26 nucleotides in length, preferably 21 or 23 nucleotides. Each of the first duplexes of the dsRNA has a 5 'end and a 3' end. Each strand of dsRNA has 1 to 4 nucleotide hangovers, preferably 2 nucleotides at the 3 'end. Hangover nucleotides typically include thymidine or uridine. Within the siRNA, the first strand of double stranded RNA (dsRNA) has a nucleotide sequence corresponding to the target sequence in at least part of its sequence (eg, the target mRNA transcript portion of the target gene). The second strand of dsRNA is complementary to the first strand of dsRNA. Desirably the target sequence corresponds to an oncogene such as those described herein (preferably c-raf-1), but can be any desired target gene. Those skilled in the art are familiar with constructing appropriate RNAi for targeting specific genes. Preferably dsRNA is produced by chemical synthesis.

  The gene to be targeted with RNAi according to the present invention can be selected from any desired species depending on the desired end use. Thus, for example, the composition can be used in agricultural applications by targeting the genes of plants such as crop plants, or the genes of plants or mold parasites or pathogens. Similarly, RNAi is an animal gene, such as a pet (eg, cat, dog, etc.), a general livestock (eg, cow, pig, sheep, goat, poultry (eg, chicken, turkey or hunting bird) or fur animal, eg, Mink, sables, foxes, etc. can be targeted, and the gene to be targeted can also be such a parasite or pathogen (eg a bacterial or viral pathogen).

  The technology can also be used for medical applications by targeting human genes. In a preferred embodiment, the gene to be targeted (eg by oligonucleotide or siRNA) is a human or animal oncogene such as ras, raf cot, mos, myc, etc. Most preferably, the gene to be targeted is c-raf-1. The sequences of such genes are publicly available, and those skilled in the art can construct siRNAs targeting such genes without particular experimentation. Guidance in this regard is further provided by the ability to successfully suppress such genes with antisense RNA and oligonucleotides. Examples of siRNA molecules for use in the context of the present invention include:

  In some embodiments, the composition preferably includes a plurality of active agents as described herein or known in the art. For example, the compositions of the present invention desirably include at least one nucleic acid species (eg, oligonucleotide, dsRNA, etc.) as described above, although the composition can include more than one such species. For example, the composition of the invention can comprise a plurality of oligonucleotides, siRNA species or mixtures thereof. In this regard, in a preferred embodiment, at least two active agents present in the composition of the invention comprise a nucleic acid. For example, if desired, at least one of the nucleic acids can be a siRNA species, an oligonucleotide, or another species of nucleic acid. The nucleic acid targets the gene sequence (eg, for suppression, such as suppression of RNAi or antisense oligonucleotide), while the first and second nucleic acid species present in the composition target the individual gene sequences It can also be designed. Such individual target sequences can be present in individual genes, thereby facilitating suppression of multiple genes in the target cell. Alternatively or additionally, individual target sequences can be present in the same gene. In such embodiments, the combined effects of various species of repression of nucleic acids in the composition can enhance the ability to reduce target gene expression.

  Desirably, when a polynucleotide is included in the composition of the present invention, the ratio of the respective charges of cationic lipid: polynucleotide (eg, oligodeoxyribonucleotide, RNAi, etc.) is in the range of (1-4): 1; For example, 2: 1 to 3: 1. Because PCL-2 has two positive charges, the overall lipid: drug molar amount can be reduced in the present invention over previously reported cationic lipoformulations. Desirably the molar amount of each total lipid relative to the polynucleotide (eg, oligodeoxyribonucleotide, RNAi, etc.) is from about 10: 1 to about 200: 1, such as from about 20: 1 to about 150: 1 or from about 50: 1 to about 100. : 1. Where the composition is a liposome, desirably at least about 40% and more desirably at least about 50% of the polynucleotide (eg, oligodeoxyribonucleotide, RNAi, etc.) is complexed with a cationic lipid in the inner core of the liposome. . Typically, about 60% to about 80% of a polynucleotide (eg, oligodeoxyribonucleotide, RNAi, etc.) is complexed with a cationic lipid within the inner core of the liposome, and about 40 to about 20% of the polynucleotide (eg, Oligodeoxyribonucleotide, RNAi, etc.) on the outer surface of the membrane. However, in some embodiments, higher (eg, about 90% or even about 95%) polynucleotides (eg, oligodeoxyribonucleotides, RNAi, etc.) are complexed with cationic lipids in the inner core of the liposome. can do. High liposome capture enables c-rafAON release from the formulation.

  Where a polynucleotide (eg, RNAi or oligonucleotide) in the composition targets an oncogene, the present invention provides a method of inhibiting neoplastic cell growth using such a composition. According to the methods of the invention, the composition is administered to such cells under conditions that allow the polynucleotide (eg, RNAi or oligonucleotide) to enter the cell. Within a cell, a polynucleotide (eg, RNAi or oligonucleotide) suppresses the activity of the oncogene that it targets, thereby suppressing the growth of such neoplastic cells. In this regard, “suppression” does not require complete arrest of such cell growth or proliferation. It is sufficient that the method delays the growth of the neoplasm. However, it is desirable that neoplastic cell proliferation is substantially reduced or even disappeared.

  When used against neoplastic cells, the method can be used on cells in vivo or in vitro. For in vitro applications, the method can be used, for example, in studies examining neoplastic growth for proliferation. In vivo applications can also be used in the development of therapeutics based on the methods of the invention. For such applications, the composition can be added to the cell culture medium or used in a manner similar to common lipid-based transfection agents (eg, LIPOFECTIN®).

  When used in vivo, the methods of the invention can be used to treat patients suffering from infectious diseases (eg, bacterial infections, viral infections, etc.), autoimmune diseases (eg, cancer). Preferably, in such an embodiment, a composition comprising a cationic cardiolipin analog and a polynucleotide (eg, RNAi or oligonucleotide) targeting an oncogene, wherein the polynucleotide (eg, RNAi or oligonucleotide) is new to the patient. Delivered to the patient under conditions sufficient to enter the biological cell. Within a cell, a polynucleotide (eg, RNAi or oligonucleotide) suppresses oncogene expression or activity, thereby inhibiting neoplastic cell growth in the patient. The cell can be, for example, a cancer cell and can be within a tumor or can metastasize.

  As described above, it is sufficient for the method of the present invention to delay the growth or proliferation of such cells. The method of the present invention need not completely eliminate neoplastic cells from the patient. Rather, it is sufficient that the method of the present invention delays the growth or proliferation of neoplastic cells within the patient. Preferably, the methods of the invention substantially inhibit neoplastic cell growth, and in some embodiments, the methods of the invention may result in tumor regression or elimination of neoplastic cells from the patient. .

  According to the method of the present invention, a composition comprising a polynucleotide can be used in combination with a second antitumor agent. Such second anti-tumor drug can be, for example, a chemotherapeutic agent, a radiation source, a gene therapy vector, or other anti-tumor drug. Specific examples of such chemotherapeutic agents are camptothecin (eg SN-38, irinotecan, etc.), doxorubicin, daunorubicin, methotrexate, adriamycin, tamoxifen, toremifene, cisplatin, epirubicin, docetaxal, paclitaxel, gemzer, gencitabine hydrochloride, and mixotantron hydrochloride Including other known chemotherapeutic agents useful for the treatment of. When used in combination, the compositions of the invention can be delivered before, simultaneously with, or after the second anti-tumor agent. However, if used, the composition of the present invention can enhance the efficacy of the second antitumor agent. That is, the present invention can be used to make a tumor chemosensitive to the action of chemotherapeutic agents. Similarly, the present invention can be used to make a tumor radiosensitive to the effects of radiation. In this regard, the method of the present invention using a composition comprising a cationic cardiolipin analog and a polynucleotide (eg, RNAi or oligonucleotide) is sufficient if it contributes to the efficacy of the combined treatment method.

  In another embodiment, the present invention provides a method for identifying a target for gene suppression. According to this aspect of the invention, a composition of the invention comprising a polynucleotide targeted to a particular gene can be obtained under conditions that allow the polynucleotide (eg, RNAi or oligonucleotide) to enter the cell. Administer to expressing cells. Within a cell, a polynucleotide (eg RNAi or oligonucleotide) suppresses the activity of the gene to which it is targeted. Thereafter, the inhibitory effect of the gene is tested.

  Methods for identifying targets for gene suppression can be performed using cells in vitro or in vivo, depending on the nature of the study. Tests performed to assess the effect of target gene suppression can be quantitative, for example by probing the presence or level of RNA or expressed protein of the targeted gene. In this sense, a decrease in the level of the target gene RNA or a decrease in the expression of the encoded protein of interest can function to identify the target of the oligonucleotide, RNAi or other gene expression inhibitor. Alternative tests can be behavioral or phenotypic. For example, a change in behavior or phenotype can be assessed by administering the composition to an animal or tissue within the animal and monitoring the animal. In this sense, for example, by confirming a gene that mediates appetite by administering a composition containing a gene suppressor of a putative appetite preparation gene to an animal and observing the effect of suppression on the appetite of the animal. Can do. A positive correlation between suppression of gene expression and changes in animal behavior can serve as target confirmation. This method can quickly find and confirm targets for therapeutic intervention. Traditionally, verification methods are expensive and time consuming and require the creation of an animal model, such as a knockout animal. In contrast, the method of the present invention can suppress the expression of a putative target gene in a cell or animal much more quickly and easily, which accelerates and simplifies the process of target identification. be able to.

  Where the composition includes a fluorescent or luminescent cardiolipin analog (or other fluorescent or luminescent moiety), the present invention tracks the migration of the composition within an animal, plant or other desired system. Provide a method. According to this aspect of the invention, the composition of the invention comprising a fluorescent or luminescent moiety is introduced into an animal, plant or other desired system. Fluorescence or luminescence can be monitored by light, X-ray or other ionizing radiation exposure immediately after introduction or after a desired period of time. This can be measured by real-time observation or detection by photography. The method can be used to monitor the migration of lipid compositions within an animal, plant or other desired system. The method functions, for example, to identify the site where the lipid composition is concentrated. This has the function of evaluating the effectiveness of the targeting moiety if present in the composition. Furthermore, the method can be used to determine the local concentration of the composition within an animal, plant or other desired system. The local lipid concentration can also be detected by taking a sample of the organ tissue or blood of interest and treating it with an appropriate agent prior to fluorescence or luminescence. Such information is useful when designing and evaluating therapeutic agents where the local concentration of the therapeutic composition is of interest.

  Of course, the method can be used repeatedly. In this sense, after the initial detection of fluorescence, fluorescence is generated again in the portion, and the fluorescence is detected again. This can be used to account for positional changes in the composition over time. Such information can be used to track the progression of the tissue in which the composition is enriched, the migration of specific cell types. Alternatively, the method is useful when monitoring the clearance of the composition from an animal, plant or other desired system. Both fluorescence and luminescence methods are useful depending on the application. Luminescent applications include tests that require higher values of disintegration time, such as effective monitoring of the lateral diffusion of lipids through the membrane.

  In another embodiment, the present invention encompasses the use of the compositions described herein as transfection agents. That is, the composition can be mixed with the desired species or library of polynucleotides and delivered to the cells. The compositions of the present invention comprise a desired polynucleotide, and the present invention is such that by exposing the cells to the composition under conditions sufficient for the polynucleotide to enter the desired cells in vivo and in vitro. Methods of transfecting cells with a polynucleotide are provided. For in vitro applications, the composition can be added to the culture medium for the cells or used in a manner similar to common lipid-based transfection agents (eg LIPOFECTIN®). While the desired amount of the composition of the invention comprising cationic cardiolipin (especially PCL-2) is such that it can function as an effective transfection agent even in primary culture, it is commonly used. The transfection agents that are present are generally not effective in primary culture. Furthermore, the method of transfecting cells using the compositions of the present invention can be used in vivo. As described herein, the methods can be used to deliver polynucleotides that target specific genes, particularly oncogenes. However, the method can be used to transfect cells in vivo with any desired polynucleotide. The advantage of using the composition of the present invention for this purpose is that the polynucleotide of interest can be obtained without substantial toxicity as observed for example with LIPOFECTIN® and other lipid transfection agents. It can be delivered to cells. That is, the present invention provides transfection agents that can be used in vivo and in vitro, including primary and transformed cells.

  Many aspects of the invention involve delivering the composition to an animal, such as a livestock patient or human. For delivery to a patient, the composition can be supplied in any suitable manner. For delivery to the tumor, for example, the composition can be formulated for injection directly into the tumor mass or injected into the tumor's circulatory system. Alternatively, the composition can be delivered by injection (eg, parenteral, intravenous, etc.) or by absorption (eg, transdermal, transmucosal) as desired. Dermatological preparations suitable for skin or mucosal tissue can be applied.

  The following examples further illustrate the present invention and are not intended to limit the scope thereof.

Example 1
This example shows the construction of nine types of PCL-2 cationic liposomes for gene transfer. These formulations are suitable for mixing with any polynucleotide selected to assess transfection efficiency.

  Cationic cardiolipin analogs, PCL-2 and any equivalent of each formulation of DOPE or cholesterol (shown in Table 1A) were dissolved in chloroform in a round bottom flask. The solvent was removed under reduced pressure using a rotary evaporator to form a lipid membrane. The lipid membrane was further dried under vacuum overnight and hydrated with 20 mM HEPES buffer under 25-40 ° C. nitrogen. Agglomerated cationic liposomes were extruded through a 0.2 μm pore size polycarbonate filter three times and a 0.1 μm pore size polycarbonate filter five times. The size and pH of the extruded cationic liposomes were characterized (Table 1A).

  Cationic cardiolipin analogs, PCL-2 and any equivalent of each formulation of DOPE or cholesterol (shown in Table 1B) were dissolved in chloroform in a round bottom flask. The solvent was removed under reduced pressure using a rotary evaporator to form a lipid membrane. The lipid membrane was further dried under vacuum overnight and hydrated with water at 25-40 ° C. under nitrogen. Agglomerated cationic liposomes were extruded through a 0.2 μm pore size polycarbonate filter three times and a 0.1 μm pore size polycarbonate filter five times. The size and pH of the extruded cationic liposomes were characterized (Table 1B).

Example 2
This example shows the construction of a PCL-2- (CCLA) based cationic liposome composition containing antisense oligodeoxyribonucleotides.

  The composition is 55:27:17 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC): cholesterol: and 1,3-bis- (1,2-bistetradecyloxy-propyl-3-dimethyl Ethoxyammonium bromide) -propan-2-ol (PCL-2) in a molar ratio and 90: 1 total lipid: c-rafAON ratio and 2.2: 1 PCL-2: c-rafAON Is a complex of lamellar structure having c-rafAON (sequence 5′-GTGCTCCATTGATGC-3 ′) having a charge ratio of The formulation also contains 0.2% by weight D-alpha tocopheryl succinate and 10-12% by weight sucrose.

  c-rafAON (lot ALH-01J-003-M) was obtained from Avecia Laboratories (MA, USA). DOPC, cholesterol, and α-tocopheryl succinate were obtained from Avanti Polar Lipids (Alabaster, AL, USA). PCL-2 is available from NeoPharm Inc. Was synthesized. Sucrose was obtained from Mallinckrodt (St. Louis, MO, USA).

  To form this composition, a lamellar composite formulation was prepared by thin film hydration. Lipids (DOPC, PCL-2, cholesterol and D-α tocopheryl succinate) were dissolved in dehydrated alcohol. The lipid solution was evaporated to dryness on a rotary evaporator. After evaporation, the lipid residue was further dried overnight in a desiccator. Sucrose and c-rafAON were dissolved in deionized water. The dried lipid residue was then hydrated in a c-rafAON / sucrose solution to form a homogeneous suspension. The size of the particles in the suspension was further reduced by extrusion through 0.8, 0.4, 0.2 and 0.1 μm sized polycarbonate filters. Prototypes can also be prepared by reverse phase evaporation and ethanol injection methods.

  The form of the formulation was measured by freeze fracture microscopy. In other words, the sample was quenched using a sandwich method and liquid nitrogen cooled propane at a cooling rate of 10,000 Kelvin / second. The cleaving process was performed in a JEOL JED-9000 freeze-etching apparatus and the exposed cleaved plane was shaded with platinum at an angle of 25-35 degrees for 30 seconds and with carbon for 35 seconds (kV / 60-70 mA, 1 / 10-5 Torr). The platinum replicas were cleaned with concentrated fuming nitric acid for 24-36 hours and then shaken at least 5 times with fresh chloroform / methanol (1: 1 volume). These cleaned replicas were then examined with a JEOL 100CX electron microscope.

  The stability of the formulation was evaluated after storage at 2-8 ° C and 25 ° C for 1 month. The particles of the lamellar structure composite preparation were measured by a dynamic light scattering method (Nicomp Particle Sizer). The drug capture efficiency of the formulation was measured by ultrafiltration and / or passing through a Centricon filter with a 100,000 molecular weight cut-off. The content of free and captured drug and lipid was determined by HPLC method.

  The frozen cleaved replica showed a complex of unilamellar lamellar liposomes with an average particle size of about 50-200 nm. C-rafAON formulations stored at 2-8 ° C or 25 ° C were stable for 1 month. The average particle size, capture efficiency, c-rafAON and DOPC concentrations were not significantly different from the initial values after 1 month storage at 2-8 ° C and 25 ° C. These results are shown in Table 2.

Example 3
This example shows the construction of a PCL-2-based cationic liposome composition containing antisense oligodeoxyribonucleotides.

  The composition is 60: 31: 9 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC): 1 cholesterol: and 3-bis- (1,2-bistetradecyloxy-propyl-3-dimethylethoxy Ammonium bromide) -propan-2-ol (PCL-2) and has a total lipid: c-rafAON ratio of 195: 1 and a PCL-2: c-rafAON ratio of 2.4: 1. It is a lamellar composite having c-rafAON having a charge ratio complexed therein. The formulation also contains 0.2% by weight D-alpha tocopheryl succinate and 10% by weight sucrose.

  c-rafAON (lot ALH-01J-003-M) was obtained from Avecia Laboratories (MA, USA). DOPC and cholesterol were obtained from Avanti Polar Lipids (Alabaster, AL, USA), and α-tocopheryl succinate was obtained from Sigma. PCL-2 is available from NeoPharm Inc. Was synthesized. Sucrose was obtained from Mallinckrodt (St. Louis, MO, USA).

  The lamellar composite preparation was prepared by thin film hydration method. Lipids (DOPC, PCL-2, cholesterol and D-α tocopheryl succinate) were dissolved in dehydrated alcohol. Sucrose and c-rafAON were dissolved in deionized water. With constant stirring, the lipid solution was added to the c-rafAON solution to form a multilayer lamellar complex. The size of the particles in the suspension was further reduced by extrusion through 0.8, 0.4, 0.2 and 0.1 μm sized polycarbonate filters. The particles of the lamellar structure composite preparation were measured by a dynamic light scattering method (Nicomp Particle Sizer). Drug capture efficiency was measured by ultrafiltration and / or passing through a Centricon filter with a 100,000 molecular weight cut-off and subsequent analysis of free c-rafAON in the filtrate by HPLC method.

  Release of c-rafAON from the lamella complex preparation was examined by reverse dialysis. In other words, 20 mL of the preparation is placed in 180 mL of stirred phosphate buffered saline (PBS), pH 7.4 buffer, which contains a dialysis membrane tube (10,000 MW cut-off previously containing 2 mL of the same PBS buffer). ). At various time intervals, one dialysis tube was removed and analyzed for c-rafAON concentration.

  The stability of the formulation was evaluated after 2 months storage at 2-8 ° C. The particles of the lamellar structure composite preparation were measured by a dynamic light scattering method (Nicomp Particle Sizer). The drug capture efficiency of the formulation was measured by ultrafiltration and / or passing through a Centricon filter with a 100,000 molecular weight cut-off. The content of free and captured drug and lipid was determined by HPLC method. The lamellar complex preparation stored at 2-8 ° C. was found to be stable for 2 months. The average particle size, capture efficiency, c-rafAON and DOPC concentrations were not significantly different from the initial values after 2 months storage at 2-8 ° C. These results are shown in Tables 3A and 3B.

  The physical and chemical stability of 8-fold diluted formulation samples in 5% dextrose solution was evaluated after storage at room temperature and refrigerated temperature (2-8 ° C.) for up to 48 hours. All stability samples were analyzed for appearance, pH, particle size, c-rafAON capture efficiency, c-rafAON concentration, DOPC concentration, cholesterol concentration and PCL2 concentration. The average vesicle size was about 200 nm and the capture efficiency was> 99%. Less than 20 percent of c-rafAON was released from the formulation after 72 hours of dialysis. As shown in Table 3C, c-rafAON, DOPC, PCL-2, cholesterol test values, capture efficiency, pH and average vesicle size are not significantly different from initial values after storage at 2-8 ° C. or room temperature for 48 hours. It was. The preparation was diluted in 5% dextrose USP.

Example 4
This example demonstrates the in vivo efficacy and low toxicity of the compositions of the present invention containing c-raf-1 antisense oligonucleotides when compared to other liposomal antisense oligonucleotide formulations.

Formulation Test formulation (LErafAON-ETU) was prepared as described in FIG. In other words, lipid excipients, ie DOPC, cholesterol, PCL-2 and D-α tocopheryl succinate are dissolved in dehydrated ethanol and ii) active ingredient c-raf-1 antisense oligonucleotide (sequence 5′- GTGCTCCATTGATGC-3 ′) and sucrose were dissolved in sterile water for injection. Ethanolic lipid solution was added to the aqueous active solution to form liposomes. After filling the batch weight (QS), the size specification was satisfied by extruding the liposomes and the ethanol was removed on a rotary evaporator. After adjusting the batch weight to the weight before solvent removal, the product was filtered through a 0.22μ sterile filter, filled into sterile vials, capped and sealed.

  A comparative formulation (DDAB-LErafAON formulation) contained the same concentration of antisense oligonucleotide, but was formulated using dimethyldioctadecyl ammonium bromide (DDAB), a commercially available cationic lipid.

  In the manufacturing process of the non-sonicated 2 vial LErafAON formulation, separate batch preparations of lyophilized lipid and rafAON were performed. For lyophilized lipids, lipid excipients, ie egg phosphatidylcholine (egg PC), cholesterol and dimethyl dodecyl ammonium bromide (DDAB) are first dissolved in t-butyl alcohol and the solution is filtered through a 0.22 μm sterile filter and sterilized. Vials were filled and lyophilized. Lyophilized rafAON was prepared by dissolving the drug in sterile water for injection and then filtering through a 0.22 μm sterile filter, filling into sterile vials and lyophilizing. Immediately prior to administration, rafAON lyophilized vials were reconstituted with 0.9% sodium chloride USP. The diluted and reconstituted rafAON was then transferred to a freeze-dried lipid vial, hydrated and sonicated for 10 minutes.

Pharmacological test The in vivo efficacy of the PCL-2 formulation, LErafAON-ETU, was compared with the DDAB formulation, LErafAON in four studies.

1. In vivo Efficacy of LErafAON-ETU and LErafAON in SCID Mice with Human Prostate (PC-3) Tumors Multi-dose therapeutic efficacy of PCL-2 formulation LErafAON-ETU and control formulation LErafAON in a SCID mouse xenograft model of human prostate cancer evaluated. PC-3 cells in logarithmic growth phase were transformed into C.I. B. -17 SCID mice were implanted subcutaneously (sc). Tumor-bearing animals (50-125 mm3) were randomly assigned to different treatment groups (5-7 / group), and mice were given control vehicle, LErafAON 12.5 or 25 mg / kg / day, or LErafAON-ETU 12 Administered iv on days 1, 2, 4, 5, 7, 8, 10, 11, 13 and 14 at .5 or 25 mg / kg / day. The efficacy was evaluated by comparing the% initial tumor volume of the control (Day 1 = 100%) with the LErafAON and LErafAON-ETU groups on Day 20.

  Animals treated with LErafAON and LErafAON-ETU 12.5 mg / kg / day did not show any tumor growth inhibition. However, animals treated with LErafAON or LErafAON-ETU at 25 mg / kg / day showed tumor growth inhibition of 15% and 44%, respectively, compared to their respective controls (10% sucrose). Since there was no difference in weight loss, both LErafAON and LErafAON-ETU were well tolerated.

  Overall, the results of this study suggest that both the PCL-2 formulation (LErafAON-ETU) and the DDAB liposome formulation (LErafAON) have comparable therapeutic efficacy against the human prostate tumor model in SCID mice.

2. In vivo efficacy of LErafAON-ETU and LErafAON in SCID mice with human breast cancer (MDA-MB-231) tumors Multi-dose therapeutic efficacy of PCL-2 formulation LErafAON-ETU and control formulation LErafAON is shown in SCID mouse xenograft model of human breast cancer Evaluated. Logarithmically growing MDA-MB-231 cells were implanted subcutaneously in CB-17 female SCID mice. Tumor-bearing animals (50-100 mm ³ ) were randomly assigned to different dose groups (8 / group), and mice received control vehicle or 25 mg / kg / day LErafAON or LErafAON-ETU in the first, second, fourth, Administered iv on days 5, 7, 8, 10, 11, 13 and 14.

  Maximum tumor growth inhibition was 65% on day 12 and 59% on day 15 compared to the corresponding vehicle control in LErafAON and LErafAON-ETU, respectively. No weight loss was observed in any of the treatment groups.

  The results show that LErafAON-ETU has equal therapeutic efficacy against the MDA-MB-231 human breast cancer tumor model compared to the control LErafAON formulation.

3. In vivo efficacy of LErafAON-ETU and their combination with LErafAON and Taxol® in SCID mice with human ovarian (SKOV-3) tumors LErafAON-ETU and control formulation LErafAON alone and with Taxol® A combined multi-dose therapeutic efficacy study was evaluated in a SCID mouse xenograft model of human ovarian cancer. Logarithmically growing SKOV-3 cells were transformed into C.I. B. -17 SCID mice were implanted subcutaneously. Control or test substance treatment was initiated when the tumor volume reached 50-100 mm ³ . Mice were randomly assigned to groups of 5-7 animals, vehicle control or 25 mg / kg / day LErafAON and LErafAON-ETU at 1, 2, 4, 5, 7, 8, 10, 11, 12, and On day 13 it was administered intravenously. Taxol® (25 mg / kg) was also administered intravenously to certain groups on days 2, 5, and 9. Taxol (registered trademark) was administered to animals in the combination therapy group 2-3 hours after administration of the corresponding liposome preparation of rafAON.

LErafAON and LErafAON-ETU showed 38% and 45% maximum tumor growth inhibition on day 23, respectively. Taxol® showed 41% maximum tumor growth inhibition on day 19. Taxol® + LErafAON or Taxol® + LErafAON-ETU combinations significantly reduced tumor growth by 90%. Furthermore, tumor growth to a volume greater than 400 mm ³ was delayed for 13 days for Taxol® + LErafAON and 24 days for Taxol® + LErafAON-ETU.

  The results of this study show that the PCL-2 formulation LErafAON-ETU has the same therapeutic efficacy as the control LErafAON against the SKOV-3 human ovarian cancer tumor model in SCID mice when administered as a single drug Yes. However, in combined treatment with Taxol®, the PLC-2 formulation LErafAON-ETU resulted in a better tumor growth delay compared to the DDAB formulation (LErafAON).

4). In vivo efficacy of LerafAON-ETU and their combination with LErafAON and Taxotere® in SCID mice with human prostate (PC-3) tumors PCL-2 formulation LErafAON-ETU and control DDAB formulation, LErafAON alone, and Multi-dose therapeutic efficacy in combination with sub-therapeutic doses of Taxotere® was performed in a SCID mouse xenograft model of human prostate cancer. PC-3 cells in logarithmic growth phase were transformed into C.I. B. -17 SCID mice were implanted subcutaneously. Tumor-bearing animals (50-100 mm ³ ) were randomly assigned to various treatment groups (5-7 / group) and control or test substance (25 mg / kg / day, LErafAON or LErafAON-ETU) for 5 consecutive days Administered intravenously. Taxotere® was administered to a given group at 10 mg / kg on day 2 and 5 mg / kg on day 5. The efficacy was evaluated by comparing the% initial tumor volume of the control group (1st day = 100%) with the 21st day administration group.

  Tumor growth was suppressed by 7% and 40% in mice administered LErafAON and LErafAON-ETU, respectively, compared to their controls. Taxotere® alone resulted in 83% tumor growth inhibition, while in combination therapy LErafAON + Taxotere® resulted in 81% similar tumor growth inhibition. Animals administered the PCL-2 formulation, LErafAON-ETU + Taxotere® exhibited 99% tumor growth inhibition on day 21. These results are shown in FIGS. 4A and 4B.

  The results of this study suggest that the combination of PCL-2 formulation, LErafAON-ETU with Taxotere (R) has a higher therapeutic effect compared to the combination of the control DDAB formulation LErafAON + Taxotere (R) .

Pharmacokinetics The pharmacokinetics of the PCL-2 rafAON formulation and LErafAON-ETU were compared in the control (DDAB) liposome formulation, LErafAON and CD2F1 mice.

1. Bioanalytical methods Bioanalytical methods are developed to quantify the total rafAON of mouse plasma and mouse tissues containing LErafAON or LErafAON-ETU, and plasma from single pharmacokinetics and tissue distribution studies of administration in mice And used to quantify rafAON in tissue samples. RafAON was extracted from mouse plasma samples by solid phase extraction (SPE) and then quantified using LC-MS / MS. The method was found to be linear in the range of 25-5000 ng / mL in mouse crystals. The tissue was mechanically homogenized to form a 5% (w / v) homogenate; this was then subjected to protein precipitation for sample preparation. Next, the total rafAON in the sample was quantified by LC-MS / MS method with linearity of 50-10,000 ng / mL tissue homogenate.

2. Pharmacokinetic and tissue distribution studies of LErafAON-ETU and LErafAON in mice Single-dose pharmacokinetics and tissue distribution studies of control LErafAON and PLC-2 formulations (LErafAON-ETU) were administered to male CD2F1 mice at 30 mg / kg intravenously. Performed after dosing. Three mice / group / time point were sacrificed at 5, 15 and 30 minutes and 1, 2, 4, 8, 24 and 48 hours after formulation administration. Blood samples were processed from plasma and plasma and tissues were tested for rafAON concentrations.

Plasma C _{max of} animals administered LErafAON-ETU was about twice that of animals administered LErafAON. Total exposure (AUC0-48h and AUC0-∞) was 2.3 times higher in LErafAON-ETU than in LErafAON-treated mice. The clearance of LErafAON was 2.5 times faster than LErafAON-ETU, and the distribution volume (Vz) was about 4 times higher.

  rafAON was rapidly distributed in the tissues after administration. The increasing order of tissue exposure over 48 hours was lung> spleen> liver> kidney> heart for both formulations. The rafAON concentration was found to be higher in mice administered with the PCL-2 formulation (LErafAON-ETU) compared to the DDAB formulation (LErafAON).

Toxicity The toxicity of the PCL-2 rafAON formulation, LErafAON-ETU was compared to the control liposome formulation LErafAON in CD2F1 mice.

A comparative multi-dose toxicity study of multi-dose toxicity control (LErafAON) and PCL-2 formulation (LErafAON-ETU) in mice was performed in male and female CD2F1 mice. Animals were randomly assigned to treatment groups (10 / sex / group) and 35 mg / kg / day LErafAON or LErafAON-ETU were administered intravenously for 5 consecutive days.

  No significant weight loss or clinical signs of toxicity were observed in mice administered the PCL-2 formulation (LErafAON-ETU). The mortality of the animals administered LErafAON-ETU was 0% for both males and females, 100% for males administered LErafAON and 40% for females. Forty percent of all males and females receiving LErafAON died on day 5 and all showed clinical signs of toxicity (flexion posture, rough skin and dehydration). No statistically significant weight loss was observed in males, whereas females showed 12% weight loss on day 8 but recovered on day 17. Surviving females administered LErafAON for more than 5 days became comatose, but recovered by day 9. This test is shown in FIG. 4C.

  Based on histopathological evaluation, it was concluded that the death of mice administered LErafAON was caused by lung absence. The disease was observed in the spleen, lung and liver of LErafAON-administered mice, but not in PCL-2 preparation (LErafAON-ETU) -administered mice. According to the clinical pathology test, there was no significant difference in clinical laboratory values between the mice administered with the control and those administered with LErafAON or LErafAON-ETU.

  This test result showing the PCL-2 formulation (LErafAON-ETU) is less toxic to CD2F1 mice than the control formulation LErafAON.

Example 5
This example shows the transfection efficiency of the composition of the invention when transfecting cells. The examples focus on the ability of PCL-2 formulations to transfect various cell lines including primary cells, and the results show that the compositions of the present invention are like LIPOFECTIN® or Lipofectamine 2000 ™. It has been shown that it can mediate transfection of cells with higher efficiency in test cell lines than commonly used transfection reagents.

  A composition (referred to herein as “NeoPectin ™”) for use in the experiments reported in this example was prepared using the procedure described in Example 1. The formulation of the present invention contained PCL-2 and DOPE.

1. Transfection efficiency in CHO cells NeoPectin ™ was evaluated for transfection in Chinese hamster ovary (CHO) cells using CMV-luciferase reporter gene plasmid and compared to LIPOFECTIN®. 1 μg of DNA was added to CHO cells in the presence and absence of NeoPectin ™ or LIPOFECTIN® in 24-well plates. Cells were incubated for 4 hours in serum-free medium, then changed to 10% serum-containing medium and held for an additional 24 hours. Protein concentration was determined by the bicinchoninic acid (BCA) protein test using bovine serum albumin as a standard, and transfection efficiency was determined by measuring relative light units / mg cell protein. The results of this experiment (see FIG. 5A) indicate that NeoPectin ™ showed significantly higher transfection efficiency at all concentrations than LIPOFECTIN ™.

2. Transfection efficiency in COS-1 cells NeoPectin (TM) transfection in COS-1 cells was evaluated using the LacZ plasmid. 4 μg of plasmid pcDNA3.1 / His / LacZ was incubated with NeoPectin ™ or LIPOFECTIN® for 30 minutes at room temperature in serum-free medium. The reagent mixture was then added to 0.3 million cells per well and incubated in serum free medium for 6 hours. The mixture was replaced with 10% serum medium and incubated for an additional 48 hours. Transfection efficiency was measured by staining with a β-Gal staining kit and counting the number of transfected cells per field using an inverted microscope. The results of this experiment (see FIG. 5B) show that NeoPectin ™ showed significantly higher transfection efficiency than LIPOFECTIN® at all concentrations.

3. NeoPectin ™ mediated delivery of RNAi in primary rat lung microvascular endothelial cells (RLMVEC) NeoPectin ™ was evaluated for delivery of RNAi in primary rat lung microvascular endothelial cells (RLMVEC). RNAi was mixed with NeoPectin ™ and various concentrations were exposed to RLVEC (see FIG. 5C). The efficiency of transfection was tested by measuring RNAi oligo incorporation in relative units. The results of this experiment (see FIG. 5C) show that NeoPectin ™ efficiently delivered RNAi to RLMVCEC.

4). NeoPectin ™ Transfection Efficiency in BALB / 3T3 Cells NeoPectin ™ Transfection Efficiency was evaluated using the β-galactosidase reporter gene test. After overnight culture in 96-well plates, BALB / 3T3 cells were cultured for 6 hours with 0.2 μg / well of pSV-β-galactosidase vector and 0.375-6 μg / well of NeoPectin ™ or Lipofectamine 2000 ( (Trademark). Following transfection, the liposome-DNA complex was replaced with serum-containing medium. β-galactosidase activity was measured 24 hours after transfection and plotted against exogenous β-galactosidase standard. The results of this test (see FIG. 5D) revealed that NeoPectin ™ showed significantly higher transfection efficiency than Lipofectamine 2000 ™.

5. Inhibition of cytokine receptor (TbRII) expression by dsRNA interference using NeoPectin ™ in rat lung microvascular endothelial primary cell culture (PLMVEC) Rat lung microvascular endothelial cell (PLMVEC) primary cell culture for RNAi By using it, the production of cytokine receptors involved in tumorigenesis was suppressed. NeoPectin ™ was used to transfect PLMVEC cells with specific double stranded RNA (dsRNA). Cells were exposed to NeoPectin ™ and oligonucleotides at various times at 37 ° C. and 5% CO ₂ . Inhibition was observed within 48 hours, with inhibition increasing at 72 and 96 hours (see FIG. 5E).

6. Effect of NeoPectin ™ Mediated Delivery of RNAi (200 nM) Oligonucleotides in Primary Cells 48 hours later NeoPectin ™ was evaluated for delivery of RNAi in RLMEC primary cells. TbRII expression was detected using indirect immunofluorescence. The primary antibody was rabbit and anti-TbRII and the secondary antibody was goat anti-rabbit conjugated to FITC. RNAi was mixed with NeoPectin ™ and 200 nM was exposed to primary cells (see FIG. 5C). The effect of NeoPectin ™ mediated delivery of RNAi was measured by fluorescence. After 48 hours, NeoPectin ™ treated cells showed significantly reduced fluorescence compared to control cells (see FIG. 5F), indicating suppression of genes that mediate intracellular fluorescence. These results indicate that NeoPectin ™ efficiently delivers RNAi to primary cells and that RNAi can function in cells to suppress gene expression.

7). NeoPectin ™ (2.5 μg / mL) mediated delivery of RNAi oligonucleotides suppresses NeoPectin ™ suppresses T_RII gene expression from rat lung microvascular endothelial cells (RLMVEC), primary human umbilical vein endothelial cells (HUVEC) and primary RNAi delivery was evaluated in human pulmonary artery endothelial cells (HPAEC). RNAi targeted to the T_RII gene was mixed with NeoPectin ™ and 2.5 μg / mL was exposed to three cell types (see FIGS. 5G, 5H and 5I). The efficiency of transfection was tested by measuring percent gene expression by relative comparison with control cells. The results of this experiment (see FIGS. 5G, 5H and 5I) indicate that NeoPectin ™ efficiently delivered RNAi to cells.

Example 6
This example demonstrates that the composition of the present invention provides a new cationic cardiolipin platform for safe and enhanced in vitro and in vivo delivery.

A. Materials and methods
1. Preparation of NeoPectin-AT (TM) (PCL-2- (CCLA) -based liposome) Cholesterol and DOPE (dioleoylphosphatidylethanolamine) used as helper lipids were purchased from Avanti Polar Lipids, Inc (Alabaster, AL). . Several CCLA-based liposomes were prepared from CCLA and helper lipids at various molar ratios using the thin film hydration method. In short, CCLA, DOPE or cholesterol was dissolved in chloroform in a round bottom flask. The solvent was removed under reduced pressure by a rotary evaporator to form a lipid film and further dried under vacuum overnight. Lipid membranes were hydrated with 10% sucrose in RNase-free water under nitrogen at 25-40 ° C. RNase-free water was obtained from Sigma (St. Louis, MO). Agglomerated cationic liposomes were extruded three times through a 0.2 μm pore size polycarbonate filter and five times through a 0.1 μm pore size polycarbonate filter. The extruded liposomes were sterilized by filtration through a 0.22 μm sterilized filter device (Millipak® positive charge). The prepared liposomes (NeoPectin-AT ™) had an average particle size of 110-120 nm. The size of the liposomes was characterized using a light scattering particle sizer (Nicomp 380 model, Santa Barbara, CA).

2. Cell culture preparations A549 (human lung cancer), PC-3 (human prostate cancer), SK-OV-3 (human ovarian cancer), MX-1 and MDA-MB-231 (human breast cancer) cells were obtained from National Cancer Institute ( Obtained from Fredrick, MD) and maintained in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (HI-FBS). CHO (Chinese hamster ovary), BALB / 3T3 (murine embryo fibroblasts) and HepG ₂ (human hepatocellular carcinoma) were purchased from American Type Culture Collection (Manassas, VA). CHO cells were maintained in Kai's denaturing medium (F12K) in Ham's F12 medium containing 10% HI-FBS. BALB / 3T3 cells were maintained in Dulbecco's modified Eagle medium supplemented with 10% bovine serum. HepG ₂ cells were maintained in Eagle's minimal essential medium supplemented with 1.0 mM sodium pyruvate, 0.1 mM non-essential amino acids and 10% HI-FBS. All media and reagents were purchased from Invitrogen Life Technologies (Carlsbad, CA).

B. Liposome test
1. One day prior to in vitro efficiency transfection, CHO (25000 cells / well), BALB / 3T3 (25000 cells / well), MX-1 (50000 cells / well) and A549 (25000 cells / well) in a 96-well plate, HepG ₂ (200000 / well) were seeded in a 24-well plate and cultured overnight in a 5% CO ₂ incubator. The cells were then washed with phosphate buffered saline to remove residual serum and the transfection mixture was added.

The transfection efficiency of PCL-2- (CCLA) -based liposomes is the β-galactosidase (β-gal) reporter gene test for CHO, BALB / 3T3 and HepG ₂ cells and for MX-1 and A549 cells Was measured using a luciferase reporter gene. The pSV-β-galactosidase control vector was purchased from Promega (Madison, WI), and the gWIZ luciferase vector was purchased from Gene Therapy Systems (San Diego, Calif.). The total amount of DNA plasmid was 0.2 μg / well for the 96-well plate and 1 μg / well for the 24-well plate. The plasmid was diluted in cell culture test irrigated OptiMEMI reduced serum medium (Invitrogen, Carlsbad, CA). CCLA-based liposomes were diluted with an equal amount of OptiMEMI medium. Appropriate amount of CCLA-based liposomes is resuspended in OptiMEMI medium to 8: 1 CCLA / DNA (+/−) charge ratio and 4: 1, 2: 1, 1: 1 and 1: 2 in OptiMEMI medium. Serially diluted and incubated for 5 minutes at room temperature. The DNA was then added dropwise to the CCLA liposome-based dilution and incubated for an additional 30 minutes at room temperature. After incubation, a DNA / CCLA-based liposome complex was added to the cells and incubated for an additional 4-6 hours. After transfection, the remaining DNA / CCLA liposome complex was washed and the cells were supplemented with normal medium and incubated for up to 24 hours.

  The β-gal reporter gene test was performed according to the manufacturer's protocol using a β-gal reporter gene test system (Promega, Madison, WI). The optical density (OD) of the sample was read at 414 nm using a plate reader (Thermo Electron, Franklin, Mass.). The amount of β-gal expression was calculated based on exogenous β-gal standards. Luciferase expression was measured using a luciferase test system (Promega, Madison, WI). Test reagents were mixed with 20 μl of cell lysate and relative light units (RLU) were measured with a luminometer (Thermo Electron, Franklin, Mass.).

  Cholesterol and DOPE, the two most commonly used helper lipids, are formulated with CCLA in various molar ratios to determine the appropriate combination for CCLA-based liposome transfection, and the β-gal reporter gene test Were screened for their transfection efficiency in vitro. According to the evaluation results, it was found that a 1: 2 molar ratio CCLA: DOPE formulation resulted in high transfection in the cell lines tested.

In CHO, BALB / 3T3, A549 and MX-1 cells, peak gene expression from the reporter gene was obtained at a CCLA / DNA (+/−) charge ratio of 2: 1 whereas peak activity in HepG ₂ cells. Was observed at a charge ratio of 1: 1 (FIG. 6A).

2. The transfection efficiency of PCL-2- (CCLA) -based liposomes in in vivo male male Balb / c mice was measured using the luciferase reporter gene test. GWIZ luciferase vector (50 μg / animal) was gently mixed on the surface of CCLA-based liposome. The DNA / CCLA-based liposome complex was intravenously administered to mice via the tail vein within 8 hours of preparation. Balb / c mice were obtained from Charles River Laboratories (Portage, MI). Equal amounts of DNA were delivered by the DOTAP-based formulation In Vivo GeneSHUTTLE ™ (QBiogene, Carlsbad, Calif.) According to the manufacturer's recommended protocol. Mice were sacrificed 24 hours after injection. Lung and heart tissues were rapidly collected and frozen in an ethanol / dry ice bath. The tissue was then homogenized in cell culture lysis reagent (Promega, Madison, Wis.) Using an autogeiser (Tomtec, Hamden, CT). The homogenate was centrifuged at 12000 rpm (16000 g) for 10 minutes at 4 ° C. and the supernatant was transferred to another tube. Luciferase activity was measured using 20 μL of the supernatant. The protein concentration of each tissue sample was measured using the RC DC protein test according to the manufacturer's protocol (Bio-Rad Laboratories, Hercules, CA).

  Several formulations of different molar ratios of CCLA and cholesterol or DOPE were screened. Interestingly, a 1: 2 CCLA: DOPE molar ratio, ie the same composition as the in vitro formulation, showed better transfection activity than CCLA in combination with cholesterol. When different charge ratios of CCLA / DNA (+/−) were tested against 50 μg of luciferase reporter gene, maximum expression of luciferase was observed at 5: 1 (+/−) in both lung and heart tissues ( FIG. 6B). In contrast, no expression was observed in mice treated with DNA or CCLA-based liposomes alone.

  The transfection efficiency of CCLA-based liposomes was also compared with commercially available cationic liposomes (In Vivo GeneSHUTTLE ™) in male Balb / c mice. The DOTAP-based In Vivo GeneSHUTLE ™ is one of the very few commercially available liposomes used for in vivo experiments. The luciferase reporter gene (50 μg / animal) was delivered using either CCLA-based liposomes with an appropriate charge ratio (+/− 5: 1) or In Vivo GeneSHUTLE ™ using the manufacturer's recommended conditions. According to the results of the present inventors, the expression of the luciferase gene in the lung was about 7 times higher when delivered by CCLA liposomes than by In Vivo GeneSHUTLE ™ (FIG. 6C). The calculated charge ratio of DOTAP to DNA was still about 5: 1.

3. In Vivo Toxicity The toxicity of PCL-2- (CCLA) -based liposomes was measured in male Balb / c mice. Toxicity of a single dose of CCLA liposomes was evaluated by injecting 100 mg / kg of CCLA liposomes into mice via the tail vein. A DOTAP-based In Vivo GeneSHUTTLE ™ was used for comparative analysis. Multi-dose toxicity was examined by injecting mice with CCLA-based liposomes 50, 75 or 100 mg / kg via the tail vein for 3 consecutive days. Three animals were used in each group. Clinical signs of mortality, weight and toxicity were monitored daily for 14 days.

  No deaths were observed in the CCLA liposome administration group, whereas a 66.6% mortality was recorded in the In Vivo GeneSHUTTLE ™ administration group (Table 6). In multi-dose toxicity studies, mice were injected with CCLA liposomes 50, 75 or 100 mg / kg for 3 consecutive days. No deaths were observed in the groups injected with 50 and 75 mg / kg / day, but 33.3% of mice died 3 days after administration of 100 mg / kg / day CCLA liposomes (Table 6). No significant weight loss was observed in the 50 and 75 mg / kg dose groups, and only 10.1% weight loss in the 100 mg / kg / day mice administered with CCLA liposomes. Surviving mice recovered completely 10 days after the first dose. Our results showed that the toxicity of CCLA-based liposomes was significantly lower than In Vivo GeneSHUTLE ™.

C. siRNA delivery
1. In vitro efficacy of siRNA against c-raf The therapeutic efficacy of siRNA was transfected into A549, PC-3, MDA-MB-231 and SK-OV-3 cancer cells using PCL-2- (CCLA) liposomes. To test. The siRNA duplex was designed to target a specific sequence of human mRNA for the c-Raf gene (accession number X03484). The selected sequence was screened in a BLAST search to confirm that only c-Raf mRNA was targeted. Synthetic siRNA duplexes were synthesized exclusively in Dharmacon (Lafayette, CO) in 2'-deprotected desalted form. The double stranded sense and antisense sequences were AUUCCUUGCUCAAUGGAUUUdTdT and AAAUCCAUUGAGCAGGAAUdTdT, respectively. Mismatched double stranded siRNA sequences (5′-AGCUUGCCAUCCAUGCUAUdTdT-3 ′ and 5-AUAGCAUGGAUGGCCAAGCUdTdT-3 ′) were also obtained from Dharmacon (Lafayette, CO). The mismatch sequence was also screened by BLAST search, and it was confirmed that there was no homology with the c-raf mRNA sequence.

  A549, PC-3, MDA-MB-231 and SK-OV-3 cells were transfected with siRNA alone or CCLA-based liposome alone, or a combination of both. Approximately 10,000 cells per well were plated in 96-well plates and transfected with 400 nM c-rafsiRNA and / or 10 μg / mL CCLA-based liposomes for 6 hours. After transfection, the c-rafsiRNA / CCLA-based liposome complex was washed, the cells were replenished with fresh media and incubated for 72 hours. Cytotoxicity was measured 72 hours after transfection using the sulforhodamine B (SRB) test. The results show that 400 nM siRNA delivered by CCLA-based liposomes was 54%, 62%, 33% and A549, PC-3, MDA-MB-231 and SK-OV-3 cells, respectively, compared to control cells. Induced 34% cytotoxicity. Apparent cytotoxicity was not observed in cells administered free siRNA or liposomes alone (FIG. 6D).

2. In vivo efficacy The therapeutic efficacy of siRNA against c-raf was further tested against a human breast cancer xenograft model in SCID mice. MDA-MB-231 cells (2 × 10 ⁶ cells in 0.1 ml HBSS) were injected subcutaneously into CB-17SCID mice (4-5 weeks old, purchased from Harlan Sprague Dawley, Indianapolis, IN). When tumors reached a volume of 50-100 mm ³ , mice were randomly assigned into 4 groups. Three groups were administered with free RNase-free and DNase-free water, mismatched c-rafsiRNA / PCL-2- (CCLA) -based liposomes or c-rafsiRNA / CCLA-based liposomes, respectively. The injection was performed twice per site (bid) for 5 days by tail vein injection at siRNA 7.5 mg / kg / day. An equal volume of sucrose (10%, w / v) of liposome complex was injected into the fourth group as a control. Tumor size was measured twice a week with a caliper, and tumor volume was calculated as follows: ax (b / 2) ² xπ (where π = 3.14, a and b are the length and width of the tumor, respectively) Calculated using Mice were sacrificed 15 days after the initial treatment when the control group tumor reached maximum deposition (˜400 mm ³ ). All data are mean ± S. E. M.M. Displayed. Comparison between groups was performed by Student's t test (two-sided, unpaired). A p value <0.05 was considered statistically significant.

  Administration of c-rafsiRNA / CCLA-based liposome complex showed 73% tumor growth inhibition when compared with the free c-rafsiRNA group 8 days after the first administration (FIG. 6E). The difference was statistically significant (p <0.05 by t test). In contrast, 10% sucrose, free c-rafsiRNA or mismatched siRNA delivered by CCLA-based liposomes had no significant effect on tumor growth.

Example 7
This example shows that the composition of the present invention comprising DOPE, cholesterol, cardiolipin and rafsiRNA effectively knocks down the expression of raf-1 and cyclin D1 in (1) tumor tissue or PC-3 tumor-bearing SCID mice. And (2) increase the therapeutic index of combined administration of Taxotere.

A. Materials and methods
1. Design and synthesis of RAF siRNA The rafsiRNA sequence is designed from within the coding region of c-raf-1 mRNA (nt 600-618, Genbank Accession No. X03484) and is screened in the BLAST search to show significant sequence with c-raf-1 mRNA. Homology was confirmed. Private synthetic double-stranded siRNA for Raf-1 was obtained from Dharmacon (Lafayette, CO).

2. Preparation of liposomes PLC-2- (CCLA) -based liposomes (NeoPectin-AT ™) were prepared as described in Example 6 from PCL-2 and helper lipids using the thin film hydration method.

3. Cell culture human prostate cancer cells (PC-3) were obtained from Biological Testing Branch, Developmental Therapeutics Program of National Cancer Institute (Frederick, MD). Tumor cells were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 units / mL penicillin and 100 μg / mL streptomycin at 37 ° C. in a humidified atmosphere of 5% CO ₂ . Media and related reagents were purchased from Invitrogen Life Technologies (Carlsnad, Calif.).

4). Preparation of rafsiRNA-neophectin complex Lyophilized rafsiRNA was hydrated with nuclease-free water. This siRNA solution was slowly dispensed into a tube containing the appropriate volume of NeoPectin-AT ™, mixed gently, and maintained at room temperature for 15-20 minutes prior to mouse administration. The mismatch siRNA-NeoPectin complex was prepared as described above.

5. Animal and Tumor Transplantation Male CB-17 SCID mice (3-4 weeks old) were obtained from Harlan Sprague Dawley Laboratories (Indianapolis, IN). Mice were handled aseptically and housed in microisolators (Tecniplast, Italy). Animals were given free access to autoclaved TEKLAD 18% protein rodent diet (Harlan Teklad, Madison, Wis.) And autoclaved water. Logarithmically growing PC-3 cells were injected subcutaneously (5 × 10 6 cells / 0.1 ml / animal). Tumors were grown to about 120 mm3.

6). Western blot analysis Adslfj PC-3 tumor-bearing SCID mice in the following administration groups (administration once daily, QD, or twice daily, BID): mismatched siRNA-NeoPectin (7.5 mg / kg, BID, x5) , Free rafsiRNA (7.5 mg / kg, BID, x5), rafsiRNA-NeoPectin (7.5 mg / kg, QD or BID, x5), Taxotere®, free rafsiRNA (7.5 mg / kg, QD, x5) ) + Taxotere®, rafsiRNA-NeoPectin (7.5 mg / kg, QD, x5) + Taxotere®, and control (saline, BIDx5) were randomly assigned. In the Taxotere (registered trademark) single administration group and the combined administration group, Taxotere (registered trademark) was administered on the second day (10 mg / kg) and the fifth day (5 mg / kg). All administrations were intravenously via the tail vein. Tumor tissue was removed within 6-8 hours after the last dose. Raf-1 and cyclin D1 protein expression was examined in tumor tissue homogenates by immunoblotting using the corresponding antibodies (Santa Cruz Biotech, Santa Cruz, Calif.). The blot was reprobed with a polyclonal anti-GAPDH antibody (Trevigen Inc., Gaithersburg, MD). Raf-1 and cyclin D1 protein levels were normalized to GAPDH expression in corresponding lanes using Low, ImageQuant software (Amersham Pharmacia Biotech, Piscataway, NJ).

7). Antitumor efficacy The therapeutic efficacy of rafsiRNA-NeoPectin and the combination of rafsiRNA-NeoPectin and Taxotere® was evaluated in a human prostate tumor model (PC-3). Animals were administered intravenously with free rafsiRNA, rafsiRNA-NeoPectin, Taxotere® free rafsiRNA + Taxotere®, or rafsiRNA-NeoPectin + Taxotere® from the tail vein. The dose and administration schedule were the same as those described in the Western blot analysis. In the control group, physiological saline was intravenously administered on the same administration schedule as rafsiRNA-NeoPectin. Tumor size was monitored once or twice weekly by measuring the length and width with a caliper, and tumor volume was calculated using the following formula: length x (width / 2) ² xπ. The mean tumor volume ± SEM of each treatment group was calculated. Statistical significance between groups was determined by Student's t test.

B. Systemic administration of rafsiRNA-NeoPectin suppresses Raf-1 expression in PC-3 tumor tissue.
Administration of 7.5 mg / kg free rafsiRNA at BID for 5 consecutive days resulted in 63% expression of Raf-1 in PC-3 tumor tissue compared to the control saline group (100%) (N = 3). Animals administered rafsiRNA-NeoPectin on QD or BID schedule (7.5 mg / kg for 5 consecutive days) showed 21% and 11% Raf-1 expression, respectively (FIGS. 7A and 7B) (n = 2/3) .

  When 7.5 mg / kg mismatch siRNA-NeoPectin was administered as BID for 5 consecutive days, no inhibitory effect was observed on Raf-1 levels (n = 3). Taxotere® alone or a combination of free rafsiRNA and Taxotere showed 43% and 51% Raf-1 expression, respectively (n = 3).

  Notably, animals that received the combination of rafsiRNA-NeoPectin and Taxotere® only left 2% Raf-1 expression in the PC-3 tumor tissue compared to the control group ( n = 3) (FIGS. 7C and 7D).

C. RafsiRNA-NeoPectin-mediated suppression of Raf-1 expression is associated with decreased expression of PC-3 tumor tissue.
Cyclin D1 is an important regulator of cell cycle progression. Furthermore, cyclin D1 functions as an oncogene in several tumor types. We evaluated the effect of rafsiRNA-NeoPectin administration on cyclin D1 expression in tumor tissue. Cyclin in PC-3 tumor tissue in animals administered free rafsiRNA (7.5 mg / kg, BID, x5) and mismatched rafsiRNA-NeoPectin (7.5 mg / kg, BID, x5) (n = 3) There was no significant difference in D1 expression (FIGS. 7E and 7F).

  Administration of rafsiRNA-NeoPectin to mice at 7.5 mg / kg (QD, x5) reduced cyclin D1 expression by 58% compared to saline controls (100%) (n = 3). Furthermore, administration of rafsiRNA-NeoPectin at 7.5 mg / kg (BID, x5) resulted in a more pronounced suppression of cyclin D1 expression (75%). Taxotere® alone or a combination of Taxotere® and free rafsiRNA did not suppress cyclin D1 expression in tumor tissue. Consistent with this, combined administration of rafsiRNA-NeoPectin (7.5 mg / kg, QD, x5) and Taxotere® resulted in suppression of cyclin D1 expression comparable to rafsiRNA-NeoPectin alone (rafsiRNA- NeoPectin + Taxotere, 65%, siRNA-NeoPectin, 75%; n = 3) (FIGS. 7G and 7H).

D. rafsiRNA-NeoPectin suppresses tumor growth and improves the response to Taxotere in a PC-3 tumor xenograft model.
7.5 mg / kg (BID, x5) of free rafsiRNA has no effect on tumor growth. Animals administered Taxotere® alone or in combination with Taxotere in combination with free rafsiRNA resulted in 71% and 66% tumor growth inhibition, respectively, compared to the control group (p <0.05, 18 days after initiation of treatment). ). Animals administered rafsiRNA-NeoPectin 7.5 mg / kg QDx5 or 7.5 mg / kg BIDx5 resulted in 39% and 49% tumor growth inhibition, respectively, compared to the control group (p <0.05, 18 after administration). Day).

  Most notable was the effect of rafsiRNA-NeoPectin and Taxotere on tumors. On day 18 after the start of dosing, rafsiRNA-NeoPectin and Taxotere® combined administration resulted in 89% tumor growth inhibition and 18-50% enhanced inhibition over the single agent compared to the control group ( FIG. 7I).

  No morbidity or mortality was associated with rafsiRNA-NeoPectin administration. A marked knockdown of Raf-1 expression suggests that NeoPectin-AT ™ has enhanced rafsiRNA delivery and efficacy in vivo. Administration of free rafsiRNA during the BID schedule resulted in moderate suppression of Raf-1 expression and this may be due to rapid degradation or inactivation of free rafsiRNA or poor cellular uptake of free rafsiRNA It is done. Significant down-regulation of Raf-1 protein expression was observed in tumor tissues of animals administered rafsiRNA-NeoPectin on QD or BID schedule compared to animals receiving mismatched siRNA-NeoPectin, and rafsiRNA specific for Raf-1 Suggest targeting. Raf-1 RNA-mediated silencing of Raf-1 was found to be associated with reduced expression of cyclin D1, a cyclin involved in the regulation of cell cycle progression and neoplastic transformation. These data suggest that rafsiRNA can be researched and developed as a next generation therapeutic that can be administered alone or in combination with antitumor agents for the treatment of human prostate or other cancer types.

Manufacturing example
Example 8-A: Synthesis of cationic cardiolipin analog (11) [FIG. 8A] 1,3-bis [(2-ethoxytetrahydro-2H-pyran)]-2-benzyloxy-glycerol (7)

To a stirred suspension of sodium hydride (59.3 g, 1.48 mol, 60% in oil) in anhydrous DMF (300 mL) at 0 ° C. under an argon atmosphere was added 2-benzyloxy 1 in DMF (700 mL). , 3-propanediol (5) (90 g, 0.49 mol) was added over 2 hours while maintaining the temperature below 15 ° C. After stirring for 2 hours at room temperature, 2- (2-bromoethoxy) tetrahydro-2H-pyran (6) (310 g, 1.48 mol) was added at 0 ° C. over 3 hours while maintaining the temperature below 10 ° C. . The reaction mixture was stirred for 12 hours at room temperature. The reaction mixture was cooled to 0 ° C. and ice water was added very slowly to quench the excess sodium hydride. The reaction mixture was concentrated under reduced pressure to remove the maximum amount of DMF and the crude solution was diluted with water (1 L) and extracted with ethyl acetate (2 × 500 mL). The organic layer was washed with saturated aqueous sodium chloride (500 mL) and dried over sodium sulfate. The solvent was concentrated under reduced pressure. The crude compound was purified by column chromatography on silica gel (230-400 mesh) with 10-30% ethyl acetate in hexane to give 1,3-bis-[(2-ethoxytetrahydro-2H-- as a colorless oil. Pyran)]-2-benzyloxy-glycerol (7) (154 g, 71%) was obtained. TLC (SiO ₂₎ hexane / ethyl acetate _{(3: 2) R f ~0.40} .

メタノール（５００ｍＬ）中の１，３−ビス［（２−エトキシテトラヒドロ−２Ｈ−ピラン）］−２−ベンジルオキシ−グリセロール（７）（５０ｇ、０．１１モル）の溶液に、エーテル（５ｍＬ）中の１ＮＨＣｌを添加し、２時間室温で攪拌した。反応混合物を溶液が中和するまで重炭酸ナトリウムで中和した。反応混合物を濾過し、減圧下に濃縮した。粗製の化合物を酢酸エチル（１Ｌ）中に溶解し、水（１００ｍＬ）で洗浄し、硫酸ナトリウム上に乾燥した。有機層を減圧下に濃縮し、粗製の化合物を酢酸エチル次いで酢酸エチル中の５％メタノールを溶離剤とするシリカゲル（７０〜２３０メッシュ）上のカラムクロマトグラフィーにより精製し、無色の油状物として３，７−ジオキサ−５−ベンジルオキシ−１，９−ノナンジオール（８）（２７ｇ、８８％）を得た。ＴＬＣ（ＳｉＯ₂）酢酸エチルＲ_f〜０．１０。

3,7-Dioxa-5-benzyloxy-1,9-nonanediol (8)

To a solution of 1,3-bis [(2-ethoxytetrahydro-2H-pyran)]-2-benzyloxy-glycerol (7) (50 g, 0.11 mol) in methanol (500 mL) in ether (5 mL). Of 1N HCl was added and stirred at room temperature for 2 hours. The reaction mixture was neutralized with sodium bicarbonate until the solution was neutralized. The reaction mixture was filtered and concentrated under reduced pressure. The crude compound was dissolved in ethyl acetate (1 L), washed with water (100 mL) and dried over sodium sulfate. The organic layer is concentrated under reduced pressure and the crude compound is purified by column chromatography on silica gel (70-230 mesh) eluting with ethyl acetate and then 5% methanol in ethyl acetate to yield 3 as a colorless oil. , 7-Dioxa-5-benzyloxy-1,9-nonanediol (8) (27 g, 88%) was obtained. TLC (SiO ₂₎ ethyl acetate R _f to 0.10.

1,9-Dibromo-3,7-dioxa-5-benzyloxy-nonane (9)

To a solution of 3,7-dioxa-5-benzyloxy-1,9-nonanediol (8) (27 g, 0.1 mol) in anhydrous dichloromethane (400 mL) under an argon atmosphere at 0 ° C. was added triphenylphosphine ( 65.5 g, 0.25 mol) and then carbon tetrabromide (79.4 g, 0.24 mol) was added. The reaction mixture was stirred at 0 ° C. for 2 hours. The reaction mixture was diluted with water (300 mL) and the organic layer was separated and dried over sodium sulfate. The organic layer was concentrated under reduced pressure and the crude compound was purified by column chromatography on silica gel (70-230 mesh) with 20% ethyl acetate in hexane to give 1,9-dibromo-3, as a colorless oil. 7-Dioxa-5-benzyloxy-nonane (9) (36 g, 91%) was obtained. TLC (SiO ₂₎ hexane / ethyl acetate _{(3: 2) R f ~0.60} .

１，９−ジブロモ−３，７−ジオキサ−５−ベンジルオキシ−ノナン（９）（３６ｇ、９０．９０ミリモル）をエタノール（１１０ｍＬ）中に溶解し、５０ｐｓｉ気圧で２時間１０％Ｐｄ／Ｃ（３．６ｇ）とともに水素化した。触媒を濾過後、溶液を減圧下に蒸発させた。粗製の物質をシリカゲルカラムクロマトグラフィー（７０〜２３０メッシュ）に付し、ヘキサン中の６０％酢酸エチルで溶離して、無色の油状物として１，３−ビス−（２−ブロモエトキシ）プロパン−２−オール（１０）（２６ｇ、９４％）を得た。ＴＬＣ（ＳｉＯ₂）ヘキサン／酢酸エチル（１：１）Ｒ_f〜０．３０。

1,3-bis- (2-bromoethoxy) propan-2-ol (10)

1,9-Dibromo-3,7-dioxa-5-benzyloxy-nonane (9) (36 g, 90.90 mmol) was dissolved in ethanol (110 mL) and 10% Pd / C ( Hydrogenated with 3.6 g). After filtering the catalyst, the solution was evaporated under reduced pressure. The crude material was subjected to silica gel column chromatography (70-230 mesh) eluting with 60% ethyl acetate in hexane to give 1,3-bis- (2-bromoethoxy) propane-2 as a colorless oil. -All (10) (26 g, 94%) was obtained. TLC (SiO ₂₎ hexane / ethyl acetate _{(1: 1) R f ~0.30} .

無水エタノール（６００ｍＬ）中の１，２−ビス−テトラデシルオキシ−３−ジメチルアミノプロパン（２）（５０．２ｇ、９８．３６ミリモル）および１，３−ビス−（２−ブロモエトキシ）プロパン−２−オール（１０）（１０ｇ、３２．７８ミリモル）の溶液を５日間かけて７８〜８０℃で還流した。反応混合物を冷却し、溶媒を蒸発させ、粗製のワックス状固体を得た。ヘキサン（４００ｍＬ）を添加し、１時間攪拌した。固体を濾過し、ヘキサン（６ｘ１００ｍＬ）で洗浄した。化合物を再結晶［化合物／メタノール／アセトン比率（１：３：５０）］により精製し、一夜−２０℃を保持した。分離した固体を濾過し、冷アセトンで洗浄した。再結晶を２回繰り返し、分析上純粋な試料を得た。化合物を真空下に２４時間、次に３６時間Ｐ₂Ｏ₅上に乾燥し、白色固体としてカチオン性カルジオリピン類縁体（１１）（３０ｇ、６９％）を得た。ＴＬＣ（ＳｉＯ₂）メタノール／クロロホルム（１：９）Ｒ_f〜０．１１。

(R, S) -1,3-bis- (1,2-ditetradecyloxypropyl-3-N, N-dimethyl-3-ethoxyammonium bromide) propan-2-ol (11)

1,2-bis-tetradecyloxy-3-dimethylaminopropane (2) (50.2 g, 98.36 mmol) and 1,3-bis- (2-bromoethoxy) propane- in absolute ethanol (600 mL) A solution of 2-ol (10) (10 g, 32.78 mmol) was refluxed at 78-80 ° C. for 5 days. The reaction mixture was cooled and the solvent was evaporated to give a crude waxy solid. Hexane (400 mL) was added and stirred for 1 hour. The solid was filtered and washed with hexane (6 × 100 mL). The compound was purified by recrystallization [compound / methanol / acetone ratio (1: 3: 50)] and kept at −20 ° C. overnight. The separated solid was filtered and washed with cold acetone. The recrystallization was repeated twice to obtain an analytically pure sample. The compound was dried over P ₂ O ₅ under vacuum for 24 hours and then 36 hours to give the cationic cardiolipin analog (11) (30 g, 69%) as a white solid. TLC (SiO ₂₎ methanol / chloroform _{(1: 9) R f ~0.11} .

０℃でアルゴン雰囲気下に無水テトラヒドロフラン（５００ｍＬ）中の水素化ナトリウム（３０．３ｇ、０．７５モル、油中の６０％）の攪拌懸濁液に、内部温度を２０℃以下に維持しながら１時間かけてＲ（−）−２，２−ジメチル−１，３−ジオキソラン−４−メタノール（１２）（５０ｇ、０．３７モル）を添加した。１時間室温で攪拌後、臭化ベンジル（９７．１ｇ、０．５６モル）を１時間かけて０℃で添加した。添加完了後、反応混合物を室温で１４時間攪拌した。反応混合物を０℃に冷却し、冷水を非常にゆっくり添加し、この混合物を飽和塩化アンモニウム水（５００ｍＬ）で希釈した。水層を酢酸エチル（５００ｍＬ）で抽出し、水（３００ｍＬ）で洗浄した。有機層を減圧下に濃縮し、シロップとして（Ｒ）−４−ベンジルオキシメチル−２，２−ジメチル−１，３−ジオキソラン（１３）（１２１ｇ）粗生成物を得た。ＴＬＣ（ＳｉＯ₂）ヘキサン／酢酸エチル（１：９）Ｒ_f〜０．２６（粗製の物質を精製することなく次工程に付した）。

Example 8B: Synthesis of cationic cardiolipin analog (19) [Figure 8B]
(R) -4- (Benzyloxymethyl) -2,2-dimethyl-1,3-dioxolane (13)

A stirred suspension of sodium hydride (30.3 g, 0.75 mol, 60% in oil) in anhydrous tetrahydrofuran (500 mL) at 0 ° C. under an argon atmosphere while maintaining the internal temperature below 20 ° C. R (-)-2,2-dimethyl-1,3-dioxolane-4-methanol (12) (50 g, 0.37 mol) was added over 1 hour. After stirring for 1 hour at room temperature, benzyl bromide (97.1 g, 0.56 mol) was added at 0 ° C. over 1 hour. After the addition was complete, the reaction mixture was stirred at room temperature for 14 hours. The reaction mixture was cooled to 0 ° C., cold water was added very slowly and the mixture was diluted with saturated aqueous ammonium chloride (500 mL). The aqueous layer was extracted with ethyl acetate (500 mL) and washed with water (300 mL). The organic layer was concentrated under reduced pressure to obtain a crude product of (R) -4-benzyloxymethyl-2,2-dimethyl-1,3-dioxolane (13) (121 g) as a syrup. TLC (SiO ₂ ) hexane / ethyl acetate (1: 9) R _f ˜0.26 (crude material was taken to next step without purification).

メタノール（７００ｍＬ）中の（Ｒ）−４−ベンジルオキシメチル−２，２−ジメチル−１，３−ジオキソラン（１３）（１２０ｇ）の溶液に濃ＨＣｌ（２０ｍＬ）を添加し、１０時間室温で攪拌した。反応混合物を溶液が中和するまで重炭酸ナトリウムで中和した。反応混合物を濾過し、減圧下に濃縮した。粗製の化合物をジクロロメタン（６００ｍＬ）中に溶解し、有機層を分離し、硫酸ナトリウム上に乾燥した。有機層を減圧下に濃縮した。粗製の化合物を溶離剤としてヘキサン中の１０〜２０％酢酸エチル次いで酢酸エチル中の１０％メタノールでシリカゲル（２３０〜４００メッシュ）上のカラムクロマトグラフィーにより精製し、シロップとして（Ｓ）−（−）−３−ベンジルオキシ−１，２−プロパンジオール（１４）（６４ｇ、９３％）を得た。ＴＬＣ（ＳｉＯ₂）酢酸エチルＲ_f〜０．４４。

(S)-(−)-3-Benzyloxy-1,2-propanediol (14)

Concentrated HCl (20 mL) was added to a solution of (R) -4-benzyloxymethyl-2,2-dimethyl-1,3-dioxolane (13) (120 g) in methanol (700 mL) and stirred for 10 hours at room temperature. did. The reaction mixture was neutralized with sodium bicarbonate until the solution was neutralized. The reaction mixture was filtered and concentrated under reduced pressure. The crude compound was dissolved in dichloromethane (600 mL) and the organic layer was separated and dried over sodium sulfate. The organic layer was concentrated under reduced pressure. The crude compound was purified by column chromatography on silica gel (230-400 mesh) with 10-20% ethyl acetate in hexane followed by 10% methanol in ethyl acetate as eluent and (S)-(-) as syrup. -3-Benzyloxy-1,2-propanediol (14) (64 g, 93%) was obtained. TLC (SiO ₂₎ ethyl acetate R _f ~0.44.

０℃でアルゴン雰囲気下に無水ＤＭＦ（２２０ｍＬ）中の水素化ナトリウム（５４．５ｇ、１．３６モル、油中６０％）の攪拌懸濁液にＤＭＦ（４００ｍＬ）中の（Ｓ）−（−）−３−ベンジルオキシ−１，２−プロパンジオール（１４）（６２ｇ、０．３４モル）の溶液を内部温度を２０℃以下に維持しながら１時間かけて添加した。２時間室温で攪拌後、臭化テトラデシル（３７７．４ｇ、１．３６モル）を２時間かけて０℃で添加した。添加完了後、反応混合物を室温で２時間攪拌し、温度を徐々に７０℃に上昇し、次に５時間攪拌した。反応混合物を０℃に冷却し、冷水を非常にゆっくり添加し、飽和塩化アンモニウム水（５００ｍＬ）で希釈した。水層を酢酸エチル（１Ｌ）で抽出し、水（３ｘ１Ｌ）で洗浄した。有機層を減圧下に濃縮し、粗生成物をヘキサン中の２〜１０％酢酸エチルを溶離剤とするシリカゲル（７０〜２３０メッシュ）上のカラムクロマトグラフィーにより精製し、無色の油状物として（Ｓ）−１，２−ビス−テトラデジルオキシ−３−Ｏ−ベンジルプロパン（１５）（１４６ｇ、７５％）を得た。ＴＬＣ（ＳｉＯ₂）ヘキサン／酢酸エチル（１：９）Ｒ_f〜０．５３。

(S) -1,2-bis-tetradecyloxy-3-O-benzylpropane (15)

To a stirred suspension of sodium hydride (54.5 g, 1.36 mol, 60% in oil) in anhydrous DMF (220 mL) at 0 ° C. under an argon atmosphere, (S)-(− in DMF (400 mL). ) -3-Benzyloxy-1,2-propanediol (14) (62 g, 0.34 mol) was added over 1 hour keeping the internal temperature below 20 ° C. After stirring for 2 hours at room temperature, tetradecyl bromide (377.4 g, 1.36 mol) was added at 0 ° C. over 2 hours. After the addition was complete, the reaction mixture was stirred at room temperature for 2 hours, the temperature was gradually raised to 70 ° C. and then stirred for 5 hours. The reaction mixture was cooled to 0 ° C., cold water was added very slowly and diluted with saturated aqueous ammonium chloride (500 mL). The aqueous layer was extracted with ethyl acetate (1 L) and washed with water (3 × 1 L). The organic layer is concentrated under reduced pressure and the crude product is purified by column chromatography on silica gel (70-230 mesh) eluting with 2-10% ethyl acetate in hexane as a colorless oil (S ) -1,2-bis-tetradecyloxy-3-O-benzylpropane (15) (146 g, 75%) was obtained. TLC (SiO ₂₎ hexane / ethyl acetate _{(1: 9) R f ~0.53} .

（Ｓ）−１，２−ビス−テトラデシルオキシ−３−Ｏ−ベンジルプロパン（１５）（７０ｇ、０．１２ミリモル）の溶液を酢酸エチル（２８０ｍＬ）中に溶解し、５０ｐｓｉ気圧で１２時間１０％パラジウム（３ｇ）とともに水素化した。触媒を濾過後、溶液を減圧下に蒸発させた。残存物を熱エタノール（５００ｍＬ）中に溶解し、一夜−２０℃で保持した。分離した固体を濾過し、真空下に乾燥し、白色固体として（Ｒ）−１，２−ビス−テトラデシルオキシ−プロパン−３−オール（１６）（５４ｇ、９２％）を得た。ＴＬＣ（ＳｉＯ₂）ヘキサン／酢酸エチル（１：９）Ｒ_f〜０．１７。

(R) -1,2-bis-tetradecyloxy-propan-3-ol (16)

A solution of (S) -1,2-bis-tetradecyloxy-3-O-benzylpropane (15) (70 g, 0.12 mmol) was dissolved in ethyl acetate (280 mL) and 10 hours at 50 psi pressure for 10 hours. Hydrogenated with% palladium (3 g). After filtering the catalyst, the solution was evaporated under reduced pressure. The residue was dissolved in hot ethanol (500 mL) and kept at −20 ° C. overnight. The separated solid was filtered and dried under vacuum to give (R) -1,2-bis-tetradecyloxy-propan-3-ol (16) (54 g, 92%) as a white solid. TLC (SiO ₂₎ hexane / ethyl acetate _{(1: 9) R f ~0.17} .

０℃でアルゴン雰囲気下に無水ジクロロメタン（２８０ｍＬ）中の（Ｒ）−１，２−ビス−テトラデシルオキシ−プロパン−３−オール（１６）（５２ｇ、０．１モル）の溶液に、トリフェニルホスフィン（３５．１ｇ、０．１３モル）を添加した。ジクロロメタン（２４０ｍＬ）中の四臭化炭素（４６．２ｇ、０．１３モル）の溶液を反応混合物に１時間かけて滴下し、さらに３時間０℃で攪拌した。反応混合物を水（５００ｍＬ）で希釈し、有機層を分離し、硫酸ナトリウム上に乾燥した。有機層を減圧下に濃縮し、粗製の化合物をヘキサン中の１〜５％酢酸エチルでシリカゲル（２３０〜４００メッシュ）上のカラムクロマトグラフィーにより精製し、無色の油状物として（Ｓ）−１，２−ビス−テトラデシルオキシ−３−ブロモプロパン（１７）（５３ｇ、９０％）を得た。ＴＬＣ（ＳｉＯ₂）ヘキサン／酢酸エチル（１：９）Ｒ_f〜０．７２。

(S) -1,2-bis-tetradecyloxy-3-bromopropane (17)

To a solution of (R) -1,2-bis-tetradecyloxy-propan-3-ol (16) (52 g, 0.1 mol) in anhydrous dichloromethane (280 mL) at 0 ° C. under an argon atmosphere, triphenyl was added. Phosphine (35.1 g, 0.13 mol) was added. A solution of carbon tetrabromide (46.2 g, 0.13 mol) in dichloromethane (240 mL) was added dropwise to the reaction mixture over 1 hour and further stirred at 0 ° C. for 3 hours. The reaction mixture was diluted with water (500 mL) and the organic layer was separated and dried over sodium sulfate. The organic layer was concentrated under reduced pressure and the crude compound was purified by column chromatography on silica gel (230-400 mesh) with 1-5% ethyl acetate in hexane to give (S) -1, 2-Bis-tetradecyloxy-3-bromopropane (17) (53 g, 90%) was obtained. TLC (SiO ₂₎ hexane / ethyl acetate _{(1: 9) R f ~0.72} .

（Ｓ）−１，２−ビス−テトラデシルオキシ−３−ブロモプロパン（１７）（５０ｇ、０．０９モル）をネジ蓋圧力ビン中にジメチルアミンの２Ｍメタノール性溶液（４００ｍＬ）中に溶解した。圧力ビンを密封し、６０時間８８〜９０℃で攪拌しながらオイルバス中に加熱した。圧力ビンを冷却し、溶液を減圧下に濃縮した。粗製の残存物を酢酸エチル（５００ｍＬ）中に溶解し、水（５００ｍＬ）で洗浄した。有機層を減圧下に濃縮し、溶離剤としてヘキサン中の５〜２０％酢酸エチルでシリカゲル（２３０〜４００メッシュ）上のカラムクロマトグラフィーにより精製し、明色の油状物として（Ｒ）−１，２−ビス−テトラデシルオキシ−３−ジメチルアミノプロパン（１８）（４１ｇ、８８％）を得た。ＴＬＣ（ＳｉＯ₂）メタノール／クロロホルム（１：９）Ｒ_f〜０．５１。

(R) -1,2-bis-tetradecyloxy-3-dimethylaminopropane (18)

(S) -1,2-bis-tetradecyloxy-3-bromopropane (17) (50 g, 0.09 mol) was dissolved in a 2M methanolic solution of dimethylamine (400 mL) in a screw cap pressure bottle. . The pressure bottle was sealed and heated in an oil bath with stirring at 88-90 ° C. for 60 hours. The pressure bottle was cooled and the solution was concentrated under reduced pressure. The crude residue was dissolved in ethyl acetate (500 mL) and washed with water (500 mL). The organic layer was concentrated under reduced pressure and purified by column chromatography on silica gel (230-400 mesh) with 5-20% ethyl acetate in hexane as eluent to give (R) -1, 2-Bis-tetradecyloxy-3-dimethylaminopropane (18) (41 g, 88%) was obtained. TLC (SiO ₂₎ methanol / chloroform _{(1: 9) R f ~0.51} .

無水エタノール（４３０ｍＬ）中の（Ｒ）−１，２−ビス−テトラデシルオキシ−３−ジメチルアミノプロパン（１８）（３５．６ｇ、６９．８ミリモル）および１，３−ビス−（２−ブロモエトキシ）プロパン−２−オール（１０）（７．１ｇ、２３．２ミリモル）の溶液を５日間かけて７８〜８０℃で還流した。熱反応混合物をエルレンマイヤーフラスコに移送し、２時間かけてアセトン（４．３Ｌ）を滴下した。混合物を一夜−２０℃で保持した。固体を濾過し、冷アセトン（５００ｍＬ）で洗浄し、無色の白色固体（２８ｇ）を得た。粗製の固体を温メタノール（１４０ｍＬ）：アセトン（１．４Ｌ）の混合物中での再結晶により精製し、次に一夜−２０℃で貯蔵した。固体を分離し、濾過し、冷アセトン（３００ｍＬ）で洗浄した。再結晶を２回繰り返し、分析上純粋な試料を得た。化合物を２４時間、次に真空下にＰ₂Ｏ₅上に３６時間乾燥し、白色固体として（Ｒ）−カチオン性カルジオリピン類縁体（１９）（２４ｇ、７８％）を得た。ＴＬＣ（ＳｉＯ₂）メタノール／クロロホルム（１：９）Ｒ_f〜０．１３。

(R) -1,3-bis- (1,2-ditetradecyloxypropyl-3-N, N-dimethyl-3-ethoxyammonium bromide) propan-2-ol (19)

(R) -1,2-bis-tetradecyloxy-3-dimethylaminopropane (18) (35.6 g, 69.8 mmol) and 1,3-bis- (2-bromo) in absolute ethanol (430 mL) A solution of ethoxy) propan-2-ol (10) (7.1 g, 23.2 mmol) was refluxed at 78-80 ° C. over 5 days. The hot reaction mixture was transferred to an Erlenmeyer flask and acetone (4.3 L) was added dropwise over 2 hours. The mixture was kept at −20 ° C. overnight. The solid was filtered and washed with cold acetone (500 mL) to give a colorless white solid (28 g). The crude solid was purified by recrystallization in a mixture of warm methanol (140 mL): acetone (1.4 L) and then stored at −20 ° C. overnight. The solid was separated, filtered and washed with cold acetone (300 mL). The recrystallization was repeated twice to obtain an analytically pure sample. The compound was dried for 24 hours and then under vacuum over P ₂ O ₅ for 36 hours to give the (R) -cationic cardiolipin analog (19) (24 g, 78%) as a white solid. TLC (SiO ₂₎ methanol / chloroform _{(1: 9) R f ~0.13} .

０℃でアルゴン雰囲気下に無水テトラヒドロフラン（３５０ｍＬ）中の水素化ナトリウム（２２．７ｇ、０．５７モル、油中６０％）の攪拌懸濁液に内部温度を２０℃以下に維持しながら１時間かけてＳ（−）−２，２−ジメチル−１，３−ジオキソラン−４−メタノール（２０）（５０ｇ、０．３７モル）を添加した。１時間室温で攪拌後、臭化ベンジル（９７．１ｇ、０．５６モル）を１時間かけて０℃で添加した。添加完了後、反応混合物を室温で１８時間攪拌した。反応混合物を０℃に冷却し、氷水（２０ｍＬ）を非常にゆっくり滴下し、この混合物を飽和塩化アンモニウム水（５００ｍＬ）で希釈した。水層を酢酸エチル（７５０ｍＬ）で抽出した。有機層を減圧下に濃縮し、シロップとして粗生成物（Ｓ）−４−ベンジルオキシメチル−２，２−ジメチル−１，３−ジオキソラン（２１）（８３ｇ）を得た。ＴＬＣ（ＳｉＯ₂）ヘキサン／酢酸エチル（１：９）Ｒ_f〜０．２６。 Example 8C: Synthesis of cationic cardiolipin analog (27) [Figure 8C]
(S) -4- (Benzyloxymethyl) -2,2-dimethyl-1,3-dioxolane (21)

A stirred suspension of sodium hydride (22.7 g, 0.57 mol, 60% in oil) in anhydrous tetrahydrofuran (350 mL) at 0 ° C. under argon atmosphere for 1 hour while maintaining the internal temperature below 20 ° C. Over time, S (−)-2,2-dimethyl-1,3-dioxolane-4-methanol (20) (50 g, 0.37 mol) was added. After stirring for 1 hour at room temperature, benzyl bromide (97.1 g, 0.56 mol) was added at 0 ° C. over 1 hour. After the addition was complete, the reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was cooled to 0 ° C., ice water (20 mL) was added very slowly and the mixture was diluted with saturated aqueous ammonium chloride (500 mL). The aqueous layer was extracted with ethyl acetate (750 mL). The organic layer was concentrated under reduced pressure to obtain a crude product (S) -4-benzyloxymethyl-2,2-dimethyl-1,3-dioxolane (21) (83 g) as a syrup. TLC (SiO ₂₎ hexane / ethyl acetate _{(1: 9) R f ~0.26} .

メタノール（８００ｍＬ）中の（Ｓ）−４−（ベンジルオキシメチル）−２，２−ジメチル−１，３−ジオキソラン（２１）（８３ｇ）の溶液に、濃ＨＣｌ（２０ｍＬ）を添加し、１５時間室温で攪拌した。反応混合物を溶液が中和するまで重炭酸ナトリウムで中和した。反応混合物を濾過し、減圧下に濃縮した。粗製の化合物をジクロロメタン（２００ｍＬ）中に溶解し、有機層を分離し、硫酸ナトリウム上に乾燥し、減圧下に濃縮した。粗製の化合物を溶離剤としてヘキサン中の１０〜２０％酢酸エチル次いで酢酸エチル中の１０％メタノールでシリカゲル（２３０〜４００メッシュ）上のカラムクロマトグラフィーにより精製し、シロップとして（Ｒ）−（−）−３−ベンジルオキシ−１，２−プロパンジオール（２２）（６５ｇ、９４％）を得た。ＴＬＣ（ＳｉＯ₂）酢酸エチルＲ_f〜０．４４。

(R)-(−)-3-Benzyloxy-1,2-propanediol (22)

To a solution of (S) -4- (benzyloxymethyl) -2,2-dimethyl-1,3-dioxolane (21) (83 g) in methanol (800 mL) was added concentrated HCl (20 mL) for 15 hours. Stir at room temperature. The reaction mixture was neutralized with sodium bicarbonate until the solution was neutralized. The reaction mixture was filtered and concentrated under reduced pressure. The crude compound was dissolved in dichloromethane (200 mL) and the organic layer was separated, dried over sodium sulfate and concentrated under reduced pressure. The crude compound was purified by column chromatography on silica gel (230-400 mesh) with 10-20% ethyl acetate in hexane then 10% methanol in ethyl acetate as eluent and (R)-(-) as syrup. -3-Benzyloxy-1,2-propanediol (22) (65 g, 94%) was obtained. TLC (SiO ₂₎ ethyl acetate R _f ~0.44.

０℃でアルゴン雰囲気下に無水ＤＭＦ（２００ｍＬ）中の水素化ナトリウム（３８．４ｇ、０．９６モル、油中６０％）の攪拌懸濁液に、内部温度２０℃以下を維持しながら１時間かけてＤＭＦ（１５０ｍＬ）中の（Ｒ）−（−）−３−ベンジルオキシ−１，２−プロパンジオール（２２）（３５ｇ、０．１９モル）の溶液を添加した。２時間室温で攪拌後、臭化テトラデシル（２６６ｇ、０．９７モル）を２時間かけて０℃で添加した。添加完了後、反応混合物を２時間室温で攪拌し、温度を徐々に６０℃に上昇し、次に２０時間攪拌した。反応混合物を０℃に冷却し、氷水を非常にゆっくり滴下し、飽和塩化アンモニウム水（５００ｍＬ）で希釈した。水層をヘキサン（３ｘ５００ｍＬ）で抽出し、水（５００ｍＬ）および塩水（５００ｍＬ）で洗浄した。有機層を減圧下に濃縮し、粗生成物をヘキサン中の２〜１０％酢酸エチルを溶離剤とするシリカゲル（７０〜２３０メッシュ）上のカラムクロマトグラフィーにより精製し、無色の油状物として（Ｒ）−１，２−ビス−テトラデシルオキシ−３−Ｏ−ベンジルプロパン（２３）（８８ｇ、８０％）を得た。ＴＬＣ（ＳｉＯ₂）ヘキサン／酢酸エチル（１：９）Ｒ_f〜０．５３。

(R) -1,2-bis-tetradecyloxy-3-O-benzylpropane (23)

A stirred suspension of sodium hydride (38.4 g, 0.96 mol, 60% in oil) in anhydrous DMF (200 mL) under argon atmosphere at 0 ° C. for 1 hour while maintaining the internal temperature below 20 ° C. A solution of (R)-(−)-3-benzyloxy-1,2-propanediol (22) (35 g, 0.19 mol) in DMF (150 mL) was added. After stirring for 2 hours at room temperature, tetradecyl bromide (266 g, 0.97 mol) was added at 0 ° C. over 2 hours. After the addition was complete, the reaction mixture was stirred for 2 hours at room temperature, the temperature was gradually raised to 60 ° C. and then stirred for 20 hours. The reaction mixture was cooled to 0 ° C., ice water was added very slowly and diluted with saturated aqueous ammonium chloride (500 mL). The aqueous layer was extracted with hexane (3 × 500 mL) and washed with water (500 mL) and brine (500 mL). The organic layer is concentrated under reduced pressure and the crude product is purified by column chromatography on silica gel (70-230 mesh) eluting with 2-10% ethyl acetate in hexane as a colorless oil (R ) -1,2-bis-tetradecyloxy-3-O-benzylpropane (23) (88 g, 80%) was obtained. TLC (SiO ₂₎ hexane / ethyl acetate _{(1: 9) R f ~0.53} .

（Ｒ）−１，２−ビス−テトラデシルオキシ−３−Ｏ−ベンジルプロパン（２３）（８３．８ｇ、０．１４モル）を酢酸エチル（４５０ｍＬ）中に溶解し、５０ｐｓｉ気圧で１２時間１０％Ｐｄ／Ｃ（３．１ｇ）とともに水素化した。触媒を濾過後、溶液を減圧下に蒸発させた。残存物を熱ヘキサン（２００ｍＬ）中に溶解し、一夜−２０℃で保持した。分離した固体を濾過し、乾燥して白色固体として（Ｓ）−１，２−ビス−テトラデシルオキシ−プロパン−３−オール（２４）（６４．８ｇ、９２％）を得た。ＴＬＣ（ＳｉＯ₂）ヘキサン／酢酸エチル（１：９）Ｒ_f〜０．１７。

(S) -1,2-bis-tetradecyloxy-propan-3-ol (24)

(R) -1,2-bis-tetradecyloxy-3-O-benzylpropane (23) (83.8 g, 0.14 mol) was dissolved in ethyl acetate (450 mL) and 10 hours at 50 psi pressure for 10 hours. Hydrogenated with% Pd / C (3.1 g). After filtering the catalyst, the solution was evaporated under reduced pressure. The residue was dissolved in hot hexane (200 mL) and kept at −20 ° C. overnight. The separated solid was filtered and dried to give (S) -1,2-bis-tetradecyloxy-propan-3-ol (24) (64.8 g, 92%) as a white solid. TLC (SiO ₂₎ hexane / ethyl acetate _{(1: 9) R f ~0.17} .

０℃でアルゴン雰囲気下に無水ジクロロメタン（３７５ｍＬ）中の（Ｓ）−１，２−ビス−テトラデシルオキシ−プロパン−３−オール（２４）（３６．８ｇ、５９．０ミリモル）の溶液に、トリフェニルホスフィン（２７．９ｇ、１０６．０ミリモル）を添加した。ジクロロメタン（６０ｍＬ）中の四臭化炭素（３６．２ｇ、１０９．０ミリモル）の溶液を１時間かけて反応混合物に滴下した。反応混合物をさらに２時間０℃で攪拌し、次に水（３ｘ３００ｍＬ）で希釈した。有機層を分離し、硫酸ナトリウム上に乾燥し、減圧下に濃縮した。粗製の化合物をヘキサン中の１〜５％酢酸エチルでシリカゲル（２３０〜４００メッシュ）上のカラムクロマトグラフィーにより精製し、無色の油状物として（Ｒ）−１，２−ビス−テトラデシルオキシ−３−ブロモプロパン（２５）（３７．８ｇ、８７％）を得た。ＴＬＣ（ＳｉＯ₂）ヘキサン／酢酸エチル（１：９）Ｒ_f〜０．７２。

(R) -1,2-bis-tetradecyloxy-3-bromopropane (25)

To a solution of (S) -1,2-bis-tetradecyloxy-propan-3-ol (24) (36.8 g, 59.0 mmol) in anhydrous dichloromethane (375 mL) under an argon atmosphere at 0 ° C. Triphenylphosphine (27.9 g, 106.0 mmol) was added. A solution of carbon tetrabromide (36.2 g, 109.0 mmol) in dichloromethane (60 mL) was added dropwise to the reaction mixture over 1 hour. The reaction mixture was stirred for an additional 2 hours at 0 ° C. and then diluted with water (3 × 300 mL). The organic layer was separated, dried over sodium sulfate and concentrated under reduced pressure. The crude compound was purified by column chromatography on silica gel (230-400 mesh) with 1-5% ethyl acetate in hexane to give (R) -1,2-bis-tetradecyloxy-3 as a colorless oil. -Bromopropane (25) (37.8 g, 87%) was obtained. TLC (SiO ₂₎ hexane / ethyl acetate _{(1: 9) R f ~0.72} .

（Ｒ）−１，２−ビス−テトラデシルオキシ−３−ブロモプロパン（２５）（３６．７ｇ、０．０７モル）をネジ蓋圧力ビン中にジメチルアミンの２Ｍメタノール性溶液（４５０ｍＬ）中に溶解した。圧力ビンを密封し、７５時間９２℃で攪拌しながらオイルバス中に加熱した。圧力ビンを冷却し、溶液を減圧下に濃縮した。粗製の残存物を酢酸エチル（５００ｍＬ）中に溶解し、水（５００ｍＬ）で洗浄した。有機層を減圧下に濃縮し、溶離剤としてヘキサン中の５〜２０％酢酸エチルでシリカゲル（２３０〜４００メッシュ）上のカラムクロマトグラフィーにより精製し、明色の油状物として（Ｓ）−１，２−ビス−テトラデシルオキシ−３−ジメチルアミノプロパン（２６）（２８．７ｇ、８０％）を得た。ＴＬＣ（ＳｉＯ₂）メタノール／クロロホルム（２：８）Ｒ_f〜０．６６。

(S) -1,2-bis-tetradecyloxy-3-dimethylaminopropane (26)

(R) -1,2-bis-tetradecyloxy-3-bromopropane (25) (36.7 g, 0.07 mol) in a 2M methanolic solution of dimethylamine (450 mL) in a screw cap pressure bottle. Dissolved. The pressure bottle was sealed and heated in an oil bath with stirring at 92 ° C. for 75 hours. The pressure bottle was cooled and the solution was concentrated under reduced pressure. The crude residue was dissolved in ethyl acetate (500 mL) and washed with water (500 mL). The organic layer was concentrated under reduced pressure and purified by column chromatography on silica gel (230-400 mesh) with 5-20% ethyl acetate in hexane as eluent to give (S) -1, 2-Bis-tetradecyloxy-3-dimethylaminopropane (26) (28.7 g, 80%) was obtained. TLC (SiO ₂₎ methanol / chloroform _{(2: 8) R f ~0.66} .

無水エタノール（２２０ｍＬ）中の（Ｓ）−１，２−ビス−テトラデシルオキシ−３−ジメチルアミノプロパン（２６）（２２．５ｇ、４３．９ミリモル）および１，３−ビス−（２−ブロモエトキシ）プロパン−２−オール（１０）（４．４８ｇ、１４．６ミリモル）の溶液を５日間かけて７８〜８０℃で還流した。熱溶液をエルレンマイヤーフラスコに移送し、溶液を攪拌しながらアセトン（２．５Ｌ）を滴下した。フラスコを１５時間冷凍庫（−２５℃）中に貯蔵した。白色固体を濾過し、冷アセトン（１００ｍＬ）で洗浄した。生成物をクロロホルム（１００ｍＬ）中に溶解し、アセトン（１Ｌ）を添加した。フラスコを１５時間−２５℃で貯蔵した。分離した白色固体を濾過し、冷アセトン（１００ｍＬ）で洗浄した。再結晶工程を３回繰り返した。生成物をヘキサン（１Ｌ）で磨砕し、濾過し、２４時間高真空下にＰ₂Ｏ₅上に乾燥し、白色固体として（Ｓ）−カチオン性カルジオリピン類縁体（２７）（１５．５ｇ、８０％）を得た。ＴＬＣ（ＳｉＯ₂）メタノール／クロロホルム（１：９）Ｒ_f〜０．１３。

(S) -1,3-bis- (1,2-ditetradecyloxypropyl-3-N, N-dimethyl-3-ethoxyammonium bromide) propan-2-ol (27)

(S) -1,2-bis-tetradecyloxy-3-dimethylaminopropane (26) (22.5 g, 43.9 mmol) and 1,3-bis- (2-bromo) in absolute ethanol (220 mL) A solution of ethoxy) propan-2-ol (10) (4.48 g, 14.6 mmol) was refluxed at 78-80 ° C. over 5 days. The hot solution was transferred to an Erlenmeyer flask, and acetone (2.5 L) was added dropwise while stirring the solution. The flask was stored in a freezer (−25 ° C.) for 15 hours. The white solid was filtered and washed with cold acetone (100 mL). The product was dissolved in chloroform (100 mL) and acetone (1 L) was added. The flask was stored at -25 ° C for 15 hours. The separated white solid was filtered and washed with cold acetone (100 mL). The recrystallization process was repeated three times. The product was triturated with hexane (1 L), filtered and dried over P ₂ O ₅ under high vacuum for 24 hours to give (S) -cationic cardiolipin analog (27) (15.5 g, 80%). TLC (SiO ₂₎ methanol / chloroform _{(1: 9) R f ~0.13} .

  All publications, patent applications and patents cited herein are hereby incorporated by reference in their entirety as if specifically set forth herein.

  What is described in this specification (especially in the claims that follow) does not distinguish between the singular and the plural unless specifically stated otherwise. “Including”, “having”, “including” and “containing” mean open ranges unless otherwise specified (ie meaning “including but not limited”). The recitation of numerical ranges herein is intended only to serve as a convenient way to independently indicate that an individual numerical value belongs to that range, unless expressly stated otherwise, Independently incorporated herein, as described herein. All methods described herein can be performed in any suitable order, unless otherwise specified, or as long as the content does not conflict. The use of examples and illustrative notation (eg, “such as”) is intended to better explain the invention and is not intended to limit the scope of the invention unless otherwise indicated. The notation in the specification shall indicate that any element not described in the claims is essential for the practice of the present invention.

  Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of these preferred embodiments will be apparent to those skilled in the art upon reading this specification. The inventors anticipate that those skilled in the art will use such variations as appropriate and intend to practice the invention in addition to those specifically described herein. Accordingly, this invention includes all modifications and equivalents of the requirements recited in the appended claims as permitted by applicable law. Furthermore, any combination of the above-described elements in all possible variations is intended to be encompassed by the invention unless otherwise indicated or otherwise contradicted by content.

The manufacturing method of LEafAON-ETU is shown. Data presented graphically on the therapeutic effects of DDAB-liposomes or cationic cardiolipin liposome-based antisense oligonucleotides (AON) in combination with Taxotere® against human prostate (PC-3) tumor growth in SCID mice is there. Figure 6 shows the effect of DDAB-liposome or cationic cardiolipin liposome-based antisense oligonucleotide (AON) in combination with Taxotere® against human prostate (PC-3) tumor growth in SCID mice. FIG. 2 is a graph of data on toxicity of DDAB ^* -liposomes or cationic cardiolipin liposome-based antisense oligonucleotides (AON) in mice. It is the data displayed by the graph regarding the transfection efficiency of NeoPectin (trademark) in a CHO cell. It is the data displayed by the graph regarding the transfection efficiency of NeoPectin (trademark) in a COS-1 cell. FIG. 3 is a graphical representation of NeoPectin ™ -mediated delivery of RNAi in primary rat lung microvascular endothelial cells (RLMVEC). It is the data displayed by the graph regarding the transfection efficiency of NeoPectin (trademark) in a BALB / 3T3 cell. It is the data displayed by the graph regarding suppression of cytokine receptor (TbRII) expression by dsRNA interference using NeoPectin (trademark) in a rat lung microvascular endothelial primary cell culture (PLMVEC). FIG. 5 shows the effect of NeoPectin ™ mediated delivery of RNAi (200 nM) oligonucleotide in primary cells after 48 hours. FIG. 5 is a graphed data showing that NeoPectin ™ (2.5 μg / mL) mediated delivery of RNAi oligonucleotides suppresses the T_RII gene in primary human umbilical vein endothelial cells (HUVEC). FIG. 5 is a graphed data showing that NeoPectin ™ (2.5 μg / mL) mediated delivery of RNAi oligonucleotides suppresses the T_RII gene in primary human pulmonary artery endothelial cells (HPAEC). FIG. 3 is a graphed data showing that NeoPectin ™ (2.5 μg / mL) mediated delivery of RNAi oligonucleotides suppresses the T_RII gene in primary rat lung microvascular endothelial cells (RLMVEC). CHO, a data displayed in the graph relates to the transfection efficiency of PCL-2- (CCLA) based liposome in _{BALB / 3T3, HepG 2, MX} -1 and A549 cells. FIG. 2 is a graph of data on the transfection activity of PCL-2-CCLA-based liposomes in lung (upper panel) and heart (lower panel) tissues of Balb / c mice. FIG. 3 is a graph of data regarding the transformation efficiency of CCLA-based liposomes compared to in vivo GeneSHUTLE ™ in Balb / c mice. FIG. 5 is a graph of data on in vitro drag of siRNA delivered by CCLA-based liposomes against c-raf in A549, PC-3, MDA-MB-231 and SK-OV-3 cancer cells. FIG. 5 is a graphical representation of the in vivo drag of siRNA delivered by CCLA-based liposomes against c-raf using a human breast cancer tumor xenograft model in SCID mice. Figure 2 shows Raf-1 expression in animals administered rafsiRNA-NeoPectin ™ in a human prostate tumor model (PC-3) in CB-17SCID mice. It is the data displayed by the graph regarding the expression of Raf-1 in the animal which administered rafsiRNA-NeoPectin (trademark) in the human prostate tumor model (PC-3) in a CB-17SCID mouse. Figure 2 shows Raf-1 expression in animals administered rafsiRNA-NeoPectin ™ and Taxotere ™ in a human prostate tumor model (PC-3) in CB-17SCID mice. FIG. 5 is a graph of data regarding Raf-1 expression in animals administered rafsiRNA-NeoPectin ™ and Taxotere® in a human prostate tumor model (PC-3) in CB-17SCID mice. Figure 2 shows cyclin D-1 expression in animals administered rafsiRNA-NeoPectin ™ in a human prostate tumor model (PC-3) in CB-17SCID mice. It is the data displayed by the graph regarding the expression of cyclin D-1 in the animal which administered rafsiRNA-NeoPectin (trademark) in the human prostate tumor model (PC-3) in a CB-17SCID mouse. Figure 2 shows cyclin D-1 expression in animals administered rafsiRNA-NeoPectin ™ and Taxotere® in a human prostate tumor model (PC-3) in CB-17SCID mice. FIG. 5 is a graphical representation of cyclin D-1 expression in animals administered rafsiRNA-NeoPectin ™ and Taxotere® in a human prostate tumor model (PC-3) in CB-17SCID mice. FIG. 5 is a graph of data regarding the ability of rafsiRNA-NeoPectin ™ to suppress tumor growth in PC-3 tumor xenografts in CB-17SCID mice and improve response to Taxotere®. 1 shows the synthesis of a cationic cardiolipin molecule useful in the context of the present invention. 1 shows the synthesis of a cationic cardiolipin molecule useful in the context of the present invention. 1 shows the synthesis of a cationic cardiolipin molecule useful in the context of the present invention.

Claims (119)

  1,3-bis- (1,2-bistetradecyloxy-propyl-3-dimethylethoxyammonium bromide) -propan-2-ol (PCL-2) and further cholesterol, dioleoylphosphatidylethanolamine ( DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), alpha tocopheryl succinate and any other phosphatidylcholine-containing composition comprising at least one lipid.   The composition according to claim 1, which comprises DOPC, PCL-2 and cholesterol.   The composition of claim 2, wherein the percent molar ratio of DOPC: cholesterol: PCL-2 is about (50-65) :( 25-35) :( 5-20).   The composition according to claim 2 or 3, further comprising D-α tocopheryl succinate.   5. The composition of claim 4, wherein the D- [alpha] tocopheryl succinate is present from about 0.1% to about 1% by weight.   The composition according to claim 1, comprising PCL-2 and cholesterol.   The composition according to claim 6, wherein the molar ratio of PCL-2 to cholesterol is 1: 3 to 6: 1.   The composition of claim 1 comprising PCL-2 and DOPE.   The composition according to claim 8, wherein the molar ratio of PCL-2 to DOPE is 1: 3 to 3: 1.   The composition according to claim 9, wherein the molar ratio of PCL-2 to DOPE is 1: 2.   The composition according to any one of claims 6 to 10, wherein the total lipid concentration is about 1.0 mg / mL to about 60 mg / mL.   The composition according to claim 1, wherein the concentration of PCL-2 is about 10 mM.   Furthermore, the composition in any one of Claims 1-12 containing saccharide | sugar.   14. A composition according to claim 13, wherein the sugar is sucrose.   15. A composition according to claim 13 or 14, wherein the sugar is present from about 5% to about 30% by weight.   The composition according to claim 1, further comprising a carrier.   The composition of claim 16, wherein the carrier is water.   17. A composition according to claim 16, wherein the carrier is a physiologically compatible buffer.   The composition according to any one of claims 1 to 18, wherein a part of PCL-2 is an emulsion part.   The composition according to any one of claims 1 to 18, wherein a part of PCL-2 is present in the liposome.   21. The composition of claim 20, wherein the average size of the liposome is from about 50 nm to about 200 nm.   The composition of claim 20 or 21, wherein 99% of the liposomes have a diameter of less than about 170 nm to about 500 nm.   23. A composition according to any preceding claim having a pH of about 3 to about 8.   23. A composition according to any preceding claim having a pH of about 7 to about 8.   25. A composition according to any preceding claim, further comprising at least one active agent.   26. The composition of claim 25, wherein the active agent is a polynucleotide.   27. The composition of claim 26, wherein the polynucleotide encodes a therapeutic polypeptide.   28. The composition of claim 27, wherein the therapeutic polypeptide comprises an immunogenic peptide.   27. The composition of claim 26, wherein the polynucleotide is DNA.   30. The composition of claim 29, wherein the DNA is an oligodeoxyribonucleotide.   31. The composition of claim 30, wherein the corresponding charge ratio of cationic cardiolipin PCL-2: oligodeoxyribonucleotide is in the range of (1.0-4.0): 1.   31. The composition of claim 30, wherein the corresponding total lipid is in the range of (10-200): 1 relative to the molar amount of oligodeoxyribonucleotide.   32. The composition of claim 30, wherein the oligodeoxyribonucleotide contains at least one phosphothioate modification.   The composition according to claim 30, wherein the size of the oligodeoxyribonucleotide is 10 to 40 nucleotides.   35. The composition of claim 34, wherein the oligodeoxyribonucleotide size is 15-25 nucleotides.   36. A composition according to any of claims 30 to 35, comprising liposomes, wherein 60 to 80% of the oligodeoxyribonucleotides are complexed with a cationic lipid in the inner core of the liposome.   37. A composition according to any of claims 30 to 36, wherein the oligodeoxyribonucleotide has a dispensing agent comprising 5'-GTGCTCTCATTGATGC-3 '(SEQ ID NO: 1).   27. The composition of claim 26, wherein the polynucleotide is RNA.   40. The composition of claim 38, wherein the RNA is siRNA.   40. The composition of any of claims 26 to 39, wherein the polynucleotide hybridizes to human mRNA.   40. The composition of any of claims 26 to 39, wherein the polynucleotide hybridizes to plant mRNA.   40. The composition of any of claims 26 to 39, wherein the polynucleotide hybridizes to plant, parasite or mold mRNA.   43. The composition according to any one of claims 40 to 42, wherein the polynucleotide suppresses gene expression by binding to mRNA.   41. The composition of claim 40, wherein the mRNA is an oncogene.   45. The composition according to claim 44, wherein the oncogene is selected from the group of oncogenes consisting of ras, raf cot, mos, and myc.   The composition according to claim 44 or 45, wherein the oncogene is c-raf-1.   Use of the composition according to any one of claims 1 to 46 for cosmetics.   Use of the composition according to any of claims 1 to 46 for a vaccine.   Use of the composition according to any of claims 1 to 46 for agricultural purposes.   41. The neoplastic cell under conditions such that the polynucleotide enters the cell, thereby suppressing the expression of the oncogene that is itself targeted within the neoplastic cell. 46. A method for suppressing the growth of neoplastic cells, comprising administering the composition according to any one of 46.   51. The method of claim 50, wherein the cell is in vitro.   51. The method of claim 50, wherein the cell is in vivo.   Claiming the patient under conditions such that the polynucleotide enters the patient's neoplastic cells, whereby the polynucleotide suppresses expression of the oncogene that is itself targeted within the neoplastic cell. 47. A method of treating a patient suffering from a neoplastic disease comprising administering a composition according to any of 40, 43, 44, 45 or 46.   54. The method of claim 53, wherein the patient is a vertebrate.   55. The method of claim 54, wherein the vertebrate is a human.   56. The method according to any of claims 53 to 55, wherein the neoplastic disease comprises cancer.   57. The method according to any of claims 53 to 56, wherein the neoplastic cell is in a tumor.   57. The method according to any of claims 53 to 56, wherein the neoplastic cell is a metastatic cell.   59. A method according to any of claims 53 to 58, wherein the composition is administered in combination with a second antitumor agent.   60. The method of claim 59, wherein the composition is administered before, simultaneously with, or after the second anti-tumor agent.   61. The method according to claim 59 or 60, wherein the antitumor agent is radiation.   61. The method according to claim 59 or 60, wherein the antitumor agent is a chemotherapeutic agent.   61. The method according to claim 59 or 60, wherein the antitumor agent is docetaxel.   (A) administering a composition containing a cationic liposome and RNAi to a cell to cause RNAi to enter the cell to suppress gene expression in the cell, and (b) testing gene suppression. A method for confirming a target gene.   65. The method of claim 64, wherein the composition is a composition according to any of claims 1-46.   Fluorescent cationic cardiolipin analog.   68. The fluorescent cationic cardiolipin analog of claim 66 comprising a cationic cardiolipin molecule conjugated to a fluorescent / luminescent moiety.   68. The fluorescent cationic cardiolipin analog of claim 67, wherein the cationic cardiolipin molecule is PCL-2.   Luminescent cationic cardiolipin analogs.   70. The luminescent cationic cardiolipin analog of claim 69 comprising a cationic cardiolipin molecule conjugated to a fluorescent / luminescent moiety.   71. The luminescent cationic cardiolipin analog according to any one of claims 69 to 70, wherein the cationic cardiolipin molecule is PCL-2.   72. A composition comprising the fluorescent / luminescent cationic cardiolipin analog according to any of claims 66-71 and a physiologically acceptable carrier.   72. A liposome composition comprising the fluorescent / luminescent cationic cardiolipin analog according to any of claims 66-71.   74. The liposome composition of claim 73, further comprising another lipid.   75. The liposome composition according to claim 74, further comprising a physiologically acceptable carrier.   (A) introducing the composition of any of claims 72-75 into an animal, (b) making the fluorescent / luminescent cationic cardiolipin analog fluorescent or luminescent, and (c) fluorescent. Or observing the location of luminescence, and a method for tracking the migration of lipid substances in an animal.   77. The method of claim 76, wherein steps (b) and (c) are repeated one or more times.   47. Use of the composition according to any of claims 1-46 as a transfection agent.   49. Transfecting a cell with a polynucleotide comprising exposing the cell to the composition of any of claims 1-46 under conditions sufficient for the polynucleotide present in the composition to enter the cell. Method.   80. The method of claim 79, wherein the cell is in vitro.   80. The method of claim 79, wherein the cell is in vivo. The siRNA has the following sequence:

40. The composition of claim 39 comprising: The siRNA has the following sequence:

40. The composition of claim 39 comprising: The siRNA has the following sequence:

40. The composition of claim 39 comprising: The siRNA has the following sequence:

40. The composition of claim 39 comprising: The siRNA has the following sequence:

40. The composition of claim 39 comprising: The siRNA has the following sequence:

40. The composition of claim 39 comprising: The siRNA has the following sequence:

40. The composition of claim 39 comprising: The siRNA has the following sequence:

40. The composition of claim 39 comprising: The siRNA has the following sequence:

40. The composition of claim 39 comprising: The siRNA has the following sequence:

40. The composition of claim 39 comprising: The siRNA has the following sequence:

40. The composition of claim 39 comprising: The siRNA has the following sequence:

40. The composition of claim 39 comprising: The siRNA has the following sequence:

40. The composition of claim 39 comprising: The siRNA has the following sequence:

40. The composition of claim 39 comprising: The siRNA has the following sequence:

40. The composition of claim 39 comprising: The siRNA has the following sequence:

40. The composition of claim 39 comprising: The siRNA has the following sequence:

40. The composition of claim 39 comprising: The siRNA has the following sequence:

40. The composition of claim 39 comprising: The siRNA has the following sequence:

40. The composition of claim 39 comprising: The siRNA has the following sequence:

40. The composition of claim 39 comprising: The siRNA has the following sequence:

40. The composition of claim 39 comprising: The siRNA has the following sequence:

40. The composition of claim 39 comprising: The siRNA has the following sequence:

40. The composition of claim 39 comprising: The siRNA has the following sequence:

40. The composition of claim 39 comprising: The siRNA has the following sequence:

40. The composition of claim 39 comprising: The siRNA has the following sequence:

40. The composition of claim 39 comprising: The siRNA has the following sequence:

40. The composition of claim 39 comprising: The siRNA has the following sequence:

40. The composition of claim 39 comprising: The siRNA has the following sequence:

40. The composition of claim 39 comprising: The siRNA has the following sequence:

40. The composition of claim 39 comprising: The siRNA has the following sequence:

40. The composition of claim 39 comprising:   113. A composition according to any of claims 1-112, comprising a plurality of active agents.   114. The composition of claim 113, wherein the at least two active agents comprise nucleic acids.   115. The composition of claim 114, wherein the at least one nucleic acid is a siRNA species.   115. The composition of claim 114, wherein the at least one nucleic acid is an oligonucleotide.   117. A composition according to any one of claims 114 to 116, wherein the first and second nucleic acids target individual gene sequences.   118. The composition of claim 117, wherein individual gene sequences are present within individual genes.   118. The composition of claim 117, wherein the individual gene sequences are present within the same gene.

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