NZ276975A

NZ276975A - Guanidine based amphiphiles and their use in liposomes for delivering genetic material intracellularly

Info

Publication number: NZ276975A
Application number: NZ276975A
Authority: NZ
Inventors: Timothy D Heath; Igor Solodin
Original assignee: Megabios Corp
Priority date: 1993-11-24
Filing date: 1994-11-17
Publication date: 1998-04-27
Also published as: EP0730405A1; NO962074D0; EP0730405A4; KR960705771A; CA2176715A1; WO1995014381A1; AU691226B2; AU1184295A; NO962074L; JPH09505808A

Description

<div class="application article clearfix" id="description"> New Zealand Paient Spedficaiion for Paient Number £76975 New Zealand No. 276975 International No. PCT/US94/13428 TO BE ENTERED AFTER ACCEPTANCE AND PUBLICATION Priority dates: 24.11.1993;24.05.1994; Complete Specification Filed: 17.11.1994 Classification:^) C07C279/04; C12N15/88 Publication date: 27 April 1998 Title of Invention: Amphiphilic derivatives of guanidine Name, address and nationality of applicant(s) as in international application form: MEGABIOS CORPORATION, 863A Mitten Road, Burlingame, California 94010, United States of America Journal No.: 1427 NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION WO 95/14381 PCT/OS94/13428 276975 AMPHIPHILIC DERIVATIVES OF QUA CI I »J I CI CROSS-REFERENCE TO RELATED APPLICATIONS 5 This application is a continuation-in-part of USSN 08/157,727, filed November 24, 1993, which is a continuation-in-part of USSN 07/991,935, filed December 17, 1992, which disclosures are herein incorporated by reference. INTRODUCTION io fiem> pf the Invention This invention relates to nitrogen containing amphiphiles for use in the preparation of liposomes and other lipid-containing carriers of pharmaceutical substances, including nucleic acids used in gene therapy. IS Background of the Invention Liposomes are one of a number of lipid-based materials used as biological carriers and have been used effectively as carriers in a number of pharmaceutical and other biological situations, particularly to introduce drugs, radiotherapeutic agents, enzymes, viruses, transcriptional factors and other cellular vectors into a 20 variety of cultured cdl lines and animals. Successful clinical trials have examined the effectiveness of liposome-mediated drug delivery for targeting liposome-entrapped drugs to specific tissues and specific cell types. See, for example. U.S. patent No. 5,264,618, which describe. ... number of techniques for using lipid carriers, including the preparation of liposomes and pharmaceutical 25 compositions and the use of such compositions in clinical situations. However, WO 95/14381 PCT/US94/13428 2 while the basic methodology for using liposome-mediated vectors is well developed, improvements in the materials used in the methods, both in terms of biocompatability and in terms of effectiveness of the carrier process, are still desirable. 5 In particular, the expression of exogenous genes in humans and/or various commercially important animals will ultimately permit the prevention and/or cure of many important diseases and the development of animals with commercially important characteristics. Genes are high molecular weight, polyanionic molecules for which carrier-mediated delivery usually is required for DNA transfection of 10 cells either in vitro or in vivo. Therefore it is of interest to develop lipid transfection vectors which will enhance both the delivery and the ultimate expression of the cloned gene in a tissue or cell of interest. Since in some instances a treatment regimen will involve repeated administration of a gene (or other pharmaceutical product), it also is of interest that the lipid carriers be 15 nontoxic to the host, even after repeated administration. Relevant literature Literature describing the use of liposomes as carriers for DNA include the following: (Friedmann (1989), supra; Brigham, et al., (1989) Am. J. Med. Sci., 20 298:278-281; Nabel, et al. (1990) Science, 249:1285-1288; Hazinski, et al. (1991) Am. J. Resp. Cell Molec. Biol., 4:206-209; and Wang and Huang (1987) Proc. Natl. Acad. Sci. (USA), 84:7851-7855); coupled to ligand-specific, cation-based transport systems (Wu and Wu (1988) J. Biol. Chem., 263:14621-14624) or the use of naked DNA expression vectors (Nabel et al. (1990), supra; Wolff et al. wo 95/14381 27 6 9 7 5 pct/us94/13428 3 (1990) Science, 247:1465-1468). Direct injection of transgenic material into tissue produced only localized expression (Rosenfeld (1992) supra); Rosenfeld et al. (1991) supra). Brigham et al. (1989) supra; Nabel (1990) supra; and Hazinski et al. (1991) supra). The Brigham et aL group (Am. J. Med. Sci. (1989) 5 298:278-281 and Clinical Research (1991) 39 (abstract) have reported in vivo transfection restricted to lungs of mice following either intravenous or intratracheal administration of a DNA liposome complex. S& also Stxibling et aL Proc. Natl. Acad. ScL (USA) 89:11277-11281 (1992) which reports the use of liposomes as carriers for aerosol delivery of transgaes to the lungs of mice and Yoshimura et 10 al. Nucleic Acids Research (1992) 20:3233-3240. Cationic lipid camera have been shown to mediate intracellular delivery of plasmid DNA (Feigner, et aL, Proc. NatL Acad. Sci. USA (1987) 84:7413-7416); mRNA (Malooe, et aL, Proc. NatL Acad. ScL USA (1989) 86:6077-6081); and purified transcription factors (Debs, et aL, J. BioL Chem. (1990) 15 265:10189-10192), in functional form. 4 SUMMARY OF THE INVENTION Non-toxic, novel, amphiphilic derivatives of guanidine are provided as are the methods of their use and specified uses. The amphiphiles are capable of forming complexes with nucleic acids, end other biological 20 compounds, and the nucleic acid complexes are capable of transforming mammalian cells in vitro. The amphiphiles of the invention are non-toxic even when subjected to endogenous enzymatic processes. ' 9 t:S3 - Inland 276975 4 DESCRIPTION OF SPECIFIC EMBODIMENTS Metabolizable amphophilic derivatives of guanidine are provided which are wefiil as carriers for biologically active molecules, such as aotibiodcs or nucleic acids used in in vitro cell transformation processes. The use of the amphiphilic materials as nucleic acid carriers is described in detail, since the compositions prepared using the amphiphiles are particul?iriy efficacious for this purpose. However, the amphiphiles are also useful in the preparation of pharmaceutical compositions for use in standard drug delivery regimens, such as for the delivery of antibiotics to the lungs of a patient. In particular, complexes of the amphiphiles with DNA (for the transformation of cells in mammalian tissues) give rise to reduced amounts of toxic cleavage products when subject to the metabolic degradation process. The invention in r*^1" is directed to awpMpi'iHe derivatives of guanidine which are nontoxic themselves and which yield by-products, such as those produced by enzymatic cleavage, which are ncwtadc to a host organism or t which are identical to substances endogenous to a host organism. The: 15 amphiphiles gf the invention thus offer the advantage that they can readily be used for the preparation of pharmaceutical compositions suitable for administration in humans, since they can be used repeatedly without the accumulation of toxic by-products. It will be apparent that the cations of the invention must be present in a rendition with one or more anions, e.g., hydroxide, chloride, or bromide ions or 20 mare complex organic anions off bases. The particular anion associated with an amphiphilic cation is not critical to the formation or utility of the amphiphilic cation and may exchange (in whole or part) for other anions during use of the composition. Accordingly, the amphiphilic compounds of the invention are described in this specification generally in terms of the cation without reference to 2769" wo 95/14381 pct/us94/13428 5 any particular annion. However, a number of specific examples are given, as well as general guidance for selection of anions. For the preparation of pharmaceutical compositions for human administration, chloride is the preferred anion; also acceptable are bromide or other physiologically acceptable anions including acetate, succinate and citrate. Hie cations are either 5 nontoxic themselves, and/or they yield by-products, for example, enzymatic cleavage products, which are nontoxic to a host organism or which are endogenous to a host organism. Generally, both the original lipids and their degradation products are nontoxic to a host organism. The invention particularly relates to novel nitrogen-containing amphiphilic 10 compounds having the formula (I): NH I {R-|X-O.CH2.(CH2)n|-Jn-}2N.C.NH2 (I) « wherein each R independently is a straight-chain, aliphatic hydrocaibyl group of 5 15 to 29 carbon atoms inclusive, each X is -CBj- or -CO-, each m is an integer from 0 to 7 inclusive and each n is zero or 1, with the proviso that when n is 1, the total number of carbon atoms in R and is at least 10, and when n is zero, each R independeatiy is a straight-chain, aliphatic hydrocarbyl group of at least 11 carbon atoms inclusive. Preferred derivatives of the above formula I are those 20 wherein n is 1. Also preferred are those compounds of formula I wherein m is from 1 to 5 inclusive, particularly 1. Also preferred are those derivatives wherein each R independently has from 13 to 23 carbon atoms inclusive. The R groups are saturated or are unsaturated having one or more ethylenically unsaturated linkages and are suitably the same or are different from each other. Also WO 95/14381 PCT/US94/13428 6 preferred are those derivatives wherein X is -CO-, in which case illustrative R groups together with the -CO- group to which it is attached (i.e., R-CO-) include lauroyl, myristoyl, palmitoyl, stearoyl, linoleoyl, eicosanoyl, tricosanoyl and nonacosanoyl (derived from the fatty acids of the corresponding name: lauric, 5 myristic, etc.). Alternatively, X can be -CH2-. When given system names for the R groups alone, the corresponding names of the hydrocarbyl group derived from lauric acid is undecyl; from myristic acid, tridecyl; from palmitic acid, pentadecyl; from stearic acid, heptadecyl; from linoleic acid, cis,cis-8,11-heptadecydienyl; from eicosanoic acid, nonadecyl; from tricosanoic acid, dicosanyl; and from 10 hemicosanoyl, nonacosanyl. This grouping of R groups is preferred when n is 1. When n is 0, R is preferably the entire hydrocarbyl portion of a fatty alcohol, such as a lauryl, stearyl, or myristyl group. In the modification of the amphiphilic compounds of formula I wherein n is zero, the compounds are of the formula wherein R has the previously stated meaning. Illustrative of such compounds are 20 N,N-distearylguanidine, alternatively named amidinodioctadecylamine, N,N-dilaurylguanidine, alternatively named amidinodidodecylamine, and N,N-dimyristylguanidine, alternatively named amidinodi(tetradecyl)amine. Other illustrative compounds of the above formula n will be apparent from the formula and the above meaning of R. 15 (II) NH ir WO 95/14381 PCT/US94/13428 7 In the modification of the amphiphiles of formula 1 wherein n is 1, the compounds are of the formula (III) nh nh, [r - x - o - ch2 -(ch2)„-]2n - c - wherein R, X and m have the previously stated meanings. Such compounds are illustrated by N,N-di[2-(palmitoyloxy)ethyl]guanidine, alternatively named amidino-di[2-(hecadecanoyloxy)ethyl]amine, by N,N-di[2 (oleoyloxy)ethyl]guanidine, alternative named 10 amidino[2-(9-octadecenoyloxy)ethyl]amine, and by N,N-di[6-(stearoyloxy)hexyl]guanidine, alternatively named amidinodi[6-(octadecanoyloxy)hexyl]amine. Other illustrative compounds of formula m will be apparent from the formula and the above meanings of R and m. For convenience, the amphiphilic compounds of the invention represented 15 by formula II can be viewed as N,N-di-R-guanidine derivatives and the compounds represented by formula in can be visualized as N,N-di(R-carboxyalkyl)guanidine derivatives, wherein R has the previously stated meaning, although as previously stated the derivatives are not necessarily prepared from guanidine. In general, the compounds of formula HI are preferred over the compounds of formula H 20 There are a number of synthetic techniques in the art that have been developed for the synthesis of guanidinium compounds. A general synthesis that can be used to produce compounds of the invention involves the conversion of a dialkanolamine to a diacyl derivative (after protecting the amine), deprotection of the amine, and reaction of the resulting secondary amine with cyanamide in base WO 95/14381 PCT/OS94/13428 8 to provide the desired product. The initial dialkanolamine can be obtained commercially (diethanolamine is readily available in quantity and is inexpensive) or can be synthesized by standard dialkylation reactions for the production of secondary amines from hydroxy-protected omega-hydroxyalkylhalides. Omega-5 hydroxyalkylhalides are themselves available from the corresponding alpha,omega-dihydroxyalkanes, which can be readily prepared from cycloalkenes by oxidation (e.g., with ozone) and reduction. The acyl groups are available from the acid halides (or anhydrides) of the corresponding carboxylic acids, which, as previously indicated, are preferably fatty acids and thus available commercially. 10 On the other hand, several of the general methods are not suitable for producing the amphiphiles of the present invention. For example, the reaction of secondary aliphatic amines with bromocyanide will form the corresponding cyanidamine, which then has been reported to form a substituted guanidine on reaction with ammonium chloride; however, the second step of this procedure did IS not work for making the compounds described herein. It has also been reported that aliphatic amines can also be reacted with cyanamide in either acetic acid or n-butanol to produce the corresponding guanidine derivative, although the reaction in acetic acid did not work for making the compounds described herein. Finally, reaction of primary or secondary amines with 3,5-dimethylpyrazole-l— 20 carboxamidine nitrate in aqueous solution for several days has been shown to form guanidines, although this method did not work for making the compounds described herein. Nevertheless, either the method outlined in general above or detailed examples provided in more detail in the Examples section below will be sufficient to produce any of the guanidinium compounds within the scope of the 276975 wo 95/14381 pct/ds94/1342s 9 invention. The cadonic lipids of the invention are typically used as carriers for various biological molecules, such as antibiotics or nucleic acids. In particular, the cadonic lipids can be used alone or combined with other lipids in formulations for 5 the preparation of lipid vesicles or liposomes for use in intracellular delivery systems in vitro. Uses contemplated for the lipids of the invention include in vitro transfection procedures corresponding to those presently known that use amphiphilic lipids, including those using commercial cationic lipid preparations, such as Lipofection ™, and various other published techniques using conventional cationic lipid technology and methods. The cationic " lipids of the invention can be used in the preparation of pharmaceutical formulations to deliver therapeutic agents by various routes and to various sites in an animal body to achieve a desired therapeutic effect. Such pharmaceutical formulations may also be used in transforming or transfecting cells in one or more tissues of a mammal. The pharmaceutical formulations may further comprise an expression cassette or a transcription cassette respectively. Bccame such techniqurs are generally known in the art, background 15 * inftimarim and *»«"» f»rhTtiqn»« for the preparation of pharmaceutical compositions lipids will not be repeated at this time. A reader unfamiliar with thi« background infarmarinn jj ttftiitd to the publications under the heading Relevant Literature above and further to U.S. Patent No. 5,264,618; This last-cited patent describes a number of therapeutic formulations and methods 20 in iiyimHug examples of the use of cationic lipids (different from those described here) that can be followed in detail by substituting die cationic iipidf of the invention for thow described in the patent. Compositions of the present invention will minimally be useable in the mamvr described in the pa»»ntt although operating parameters may need to be modified in order to achieve optimum results, using the information provided for compounds of the 276975 wo 95/14381 pct/us94s13428 10 invention in this specification along with the knowledge of a person skilled in the arts of lipid preparation and use. The lipids of the present invention axe particularly useftil and advantageous in the transfection of animal cells in vitro by genetic material. Additionally, since these compositions are non-toxic even when subjected to host enzymatic reactions, the compositions provide a number of advantages in the area of low toxicity when compared to previously known cationic lipids. These and other advantages of the invention axe discussed in detail below. The remainder of tids discussion is directed principally to selection, production, and use parameters for the cadonic t 10 lipids of die present invention that may not immediately be apparent to one of ordinary skill in the art Particularly where it is desirable to target a lipid-DNA complex to a particular cell or tissue in vitro, a lipid mixture used as a carrier can be modified in a . variety of ways. By a lipid mixture is intend a formulation prepared from the * • ■ • •• 15 cationic amphiphile of the invention, with or without additional agents such as steroids, and includes liposomes, interleaved bilayers of lipid, and the like. Steroids, e.g. cholesterolor crgosterol, can be used in combination with the cationic amphiphiles when used to prepare mixtures. In some embodiments, the lipid mixture will have from 0-67 mole percexit steroid, preferably about 33 to 50 20 mole percent steroid. A lipid-DNA complex is the composition obtained following combination of DNA and a lipid mixture. Noo-lipid material (raeh as biological molecules being delivered to an animal or plant cell in vitro or target-specific ' moieties) can be conjugated through a linking group to one or more hydrophobic groups, e.g. using alkyl chains containing from about 12 to 20 carbon atoms, either prior WO 95/14381 PCT/US94/13428 11 or subsequent to vesicle formation. Various linking groups can be used for joining the lipid chains to the compound. Functionalities of particular interest include thioethers, disulfides, carboxamides, alkylamines, ethers, and the like, used individually or in combination. The particular manner of linking the compound to 5 a lipid group is not a critical part of this invention, as the literature provides a great variety of such methods. Alternatively, some compounds will have hydrophobic regions or domains, which will allow for their association with the lipid mixture without covalent linking to one or more lipid groups. For the most part, the active compounds to be bound to the lipid mixture 10 are ligands or receptors capable of binding to some biological molecule of interest that is present in the target cell. A ligand can be any compound of interest which can specifically bind to another compound, referred to as a receptor, the ligand and receptor forming a complementary pair. The active compounds bound to the lipid mixture can vary widely, from small haptens (molecular weights of about 125 15 to 2,000) to antigens which will generally have molecular weights of at least about 6,000 and generally less than about 1 million, more usually less than about 300,000. Of particular interest are proteinaceous ligands and receptors that have specific complementary binding partners on cell surfaces. Illustrative active compounds include chorionic gonadotropin, encephalon, endorphin, luteinizing 20 hormone, morphine, epinephrine, interferon, ACTH, and polyiodothyronines and fragments of such compounds that retain the ability to bind to the same cell-surface binding partners that bind the original (non-fragment) molecules. The numbe' A targeting molecules (either ligand or receptor) bound to a lipid mixture will vary with the size of the liposome, the size of the molecule, the 276975 wo 95/14391 pct/us94/13423 12 binding affinity of the molecule to the target cell receptor or ligand, and the like. Usually, the bound active molecufcs will be present in the lipid mixture in from about 0.05 to 2 mole percent, more usually from about 0.01 to 1 mole percent based cm the percent of bound molecules to the total number of molecules 5 available in the mixture for binding. The surface membrane proteins which bind to specific effector molecules (usually soluble molecules in the external environment of the cdl) are referred to as receptors. In the present context, receptors include antibodies and immunoglobulins since these molecules are found on the surface of certain cells. -10 However, since antibodies are generally used to bind liposomes to receptor molecules on target cells, the antibodies and immunoglobulins bound to a liposome containing a cationic lipid of the invention can also be considered to be ligands. .The immunoglobulins may be monoclonal or polyclonal, preferably monoclonal Usually the immunoglobulins will be IgG and IgM, although the other 4 15 immunoglobulins may also find use, such as IgA, IgD, and IjgE. The intact immunoglobulins may be used or only fragments thereof, such as Fab, F(ab')j,F4, or F, fragments as well as a complete light or heavy chain. For antibodies used as cell-targeting tigands, antibodies of interest are those that bind to surface membrane antigens such as those antigens comprising die 20 major histocompatibility complex, particularly die HLA-A, -B, -C and -D. Other surface antigens include thy-l,leu-5, and la. The cationic amphiphiles are particularly useftil as carriers for anionic compounds, particularly polyanionic macromolecules such as nucleic acids. Disclosed but not claimed are methods where the amphiphiles are intended for use in vivo, particularly in vivo in 276975 wo 95/14381 pct/ds94/13428 13 humans, or where it is necessary to use the amphiphiles repeatedly, it is important to screen the carriers for those which are metabolized to non-toxic by-products and which themselves axe not toxic or those which are eliminated from the body without degradation. The elimination of such cationic amphiphiles from tissues 5 can be demonstrated in animal experiments. An animal, such as a mouse, can be administered one or more doses of material containing between 0.5 and 10 pinole of the lipid to be tested, complexed with an active component (such as DNA) if desired. At various times after administration, the animals are sacrificed, tissues taken, total lipids extracted using an appropriate solvent extraction system, and the •*» 10 total lipid analyzed for the particular cationic lipid or its partial degradation product using, for example, HPLC. The cationic amphiphiles axe positively charged, and a tight charge complex can be formed between a cationic lipid carrier and a polyamooic nucleic acid, resulting in a lipid carrier-nucleic acid complex or pharmaceutical composition which can be used directly 15 for systemic delivery to a mammal or mammalian cell. Where delivery is via aerosolization, the charge complex will withstand both the forces of nebulization and the environment within the lung airways and be capable of transacting lung cells after the aerosolized DNA:lipid carrier complex has been deposited in the lung following intrananl or intraoral delivery of the aerosolized complex. 20 To evaluate die efficacy of a particular cationic amphiphilfi for use as a nucleic acid carrier in a pharmaceutical composition in an aerosolization process, as well as to determine the optimum concentrations of lipid carrier-nucleic add complexes, involves a two-step process. The first step is to identify lipid carriers and the concentration of lipid carrier-nucleic add complexes that do not aggregate when the components 276975 wo 95/14331 pct/us94/1342s 14 axe combined or during the significant agitation of the mixture that occurs during the nebulization step. The second step is to identify among those lipids that do not aggregate those complexes that provide for a high level of transfection and transcription of a gene of interest in target cells in the lung. These techniques are 5 described in WO/US PCT/US92/11008 filed December 17, 1992, which disclosure is hereby incorporated by reference. As an example, a reporter gene CAT (which encodes chloramphenicol acetyltransferase) can be inserted in an expression cassette and used to evaluate each lipid carrier composition of interest The DNA; lipid carrier complexes are -- • * 10 mixed in solutions which do not themselves induce aggregation of the DNA:lipid carrier complexes, such as sterile water. The expression cainffn (DNA) is mixed together with each of the lipid carriers to be tested in multiple different ratios, ranging as an example from 4:1 to 1:10 (micrograms of DNA to nanomoles of cationic lipid or total lipid, if a lipid mixture is present). Examination of die * 15 stability of the' resulting mixtures or pharmaceutical compositions provides information concerning which ratios result in aggregation of the DNAilipid carrier complexes and are therefore not useful for use in vivo, and which complexes remain in a form suitable for aerolization. The ratios which do not result in aggregation are tested in animal models to determine which of the DNA:lipid carrier rations make useful pharmaceutical compositions which confer the highest level 20 of transgeae cjipicsilon in vivo. For example, for aenxol-based transfection, optimal DNA:lipid carrier ratios for lipid mixtures va/is as N-[l-<2,3-dioleyloxy)- propyl]-N,N,N-triethylammonium chlaride(DGTMA):dioleoylpbosphatidylethanol- amine(DOPE) (the components of this mixture being present in a 1:1 weight ratio) and dimethyl dioctadecyl ammonium bromide (DDAB): Cholesterol (1:1) are 1 to 276975 wo 95/14381 pct/us94/1342s 15 1. For O-ethyl egg phosphatidylcholine (E-EPC) or especially Oethyl dimyristoylphosphatidylcholine (E-DMPC) in a 1:1 weight ratio with cholesterol, the DNA:lipid carrier ratio is preferably in the range of from 1.5:1 to 2:1. If the cationic amphiphile is used for tne preparation of a j pharmaceutical composition for injection, then it need be evaluated only for whether it is effective for transfection of a target cell. Particular cells can be targeted by the use of particular cationic lipids for preparation of the lipid-mixture carriers or pharmaceutical compositions, for example, by the use of E-DMPC to target lung cells preferentially, or by modifying the amphiphiles to direct them to particular types of cells using site-directing motecutes. Thus antibodies or 10 for particular receptors may be employed, to target a cell associated with a particular surface protein. A particular ligend or antibody can be conjugated to the cationic amphiphile in accordance with conventional wtitHgiM*, either by conjugating the site-directing molecule to a lipid for incorporation into the lipid bilayer or by providing a linking group on a lipid present in die bilayer for linking 15 * to a functionality of the site-directing compound. Such techniques are well known to those skilled in the art. The various lipid carrier-nucleic acid complexes wherein the lipid carrier is a liposome are prepared using methods well known in die art Mixing conditions can be optimized by visual examination of the resultant lipid-DNA mixture to 20 ftittahliih that no precipitation occurs. To make die lipid-DNA complexes more visible, the complexes can be stained with a dye which does not itself cause aggregation, but which will stain either the DNA or the lipid. For example, Sudan black (which stains lipid) can be used as an aid to examine the lipid-DNA mixture to determine if aggregation has occurred. Particle size also can be studied WO 95/14381 PCT/US94/13428 16 with methods known in the art, including electron microscopy, laser light scattering, Coulter™ counting/sizing, and the like. Standard-size beads can be included as markers for determining the size of any liposomes or aggregates that form. By "lipid carrier-nucleic acid complex" is meant a nucleic acid sequence as 5 described above, generally bound to the surface of a lipid carrier preparation, as discussed below. The lipid carrier preparation can also include other substances, such as enzymes necessary for integration, transcription and translation or cofactors. Furthermore, the lipid carrier-nucleic acid complex can include targeting agents to deliver the complex to particular cell or tissue types. 10 Generally, the nucleic acid material is added to a suspension of preformed liposomes which may be multi-lamellar vesicles (MLVs) or small unilamellar vesicles (SUVs), usually SUVs formed by sonication. The liposomes themselves are prepared from a dried lipid film that is resuspended in an appropriate mixing solution such as sterile water or an isotonic buffer solution such as lOmM 15 Tris/NaCl or 5 % dextrose in sterile water and sonicated to form the liposomes. Then the preformed lipid carriers are mixed directly with the DNA. Mixing and preparing of the lipid-DNA complex can be critically affected by the sequence in which the lipid and DNA are combined. Generally, it is preferable (to minimize aggregation) to add the lipid to the DNA at ratios of 20 DNA:lipid of up to 1:2 inclusive (microgram DNA:nanomoles cationic lipid). Where the ratio of DNA:lipid is 1:4 or higher, better results are generally obtained by adding the DNA to the lipid. In either case, mixing should be rapidly achieved by shaking or vortexing for small volumes and by use of rapid mixing systems for large volumes. The lipid carrier and DNA form a very stable wo 95/14381 2ZM.75 17 complex due to binding of the negatively charged DNA to the cationic lipid carriers. SUVs find use with small nucleic acid fragments as well as with large regions of DNA (%250kb). In preparing the lipid carrier-nucleic acid complex for a pharmaceutical composition for nebulization, care should be taken to exclude ^ any compounds from the mixing solution which promote the formation of aggregates of the lipid canier-nucleic add complexes. Large particles generally will not be aerosolized by the nebulizer, and even if aerosolized would be too large to penetnte beyond the large airways. Aggregation of the lipid carrier-nucleic add complex is prevented by controlling the ratio of 10 DNA to lipid carrier, minimizing the overall concentration of DNA:lipid carrier '* complex in solution, usually less than 5 mg DNA/8 ml solution, and avoiding the use of chelating agents such as EDTA and/or significant amounts of salt, either of which tends to promote macro-aggregation. The preferred exdpient is water, « dextrose/water or another solution having low or zero ionic strength. Further, the 15 volume should be adjusted to the minimum necessary for deposition in the lungs of the host mammal, while at the same time taking care not to make the solution too concentrated so that aggregates form. Increasing the volume of the solution is to be avoided if possible due to the need to increase the inhalation time for die host animal to accommodate the increased volume. In sooo cases, it may be preferable 20 to lyophilize the lipid carrier-nudtic acid complexes for inhalation. Such materials are prepared as complexes as described above, except that a cryoprotectant such as mannitol or trehalose is induded in the buffer solution which is used for preparation of the lipid cairier-DNA complexes. Any glucose generally induded in such a buffer is preferably omitted. The lipid carrier 27 6 9 7 wo 95/14391 pct/os94/1342s 18 complex is rapidly fxteze-dried following mixing of the lipid and DNA. The mixture can be reconstituted with sterile water to yield a composition which is ready for administration to a host animal. Where the amphiphiles of the invention used to prepare a pharmaceutical composition are in the form of liposomes, the liposomes may ^ be sized in accordance with conventional techniques, ^rtHling upon the desired size. In some instances, a large liposome injected into the bloodstream of an animal has higher affinity for lung cells as compared to liver cells. Therefore, the particular size range may be evaluated in accordance with any intended target tissue by 10 administering lipid-micleic acid complexes of varying particle sizes to a host animal and determining the size of particle which provides the desired results. The caAwie amphiphile with acid of this invention can be used to prepare a pharmaceutical composition which may be administered in a variety of ways to a host, such as intravenously, intramuscularly, subcutaneoosly, transdermal^, topically, intxaperitoneilly, IS intravascularly,. by. aerosol, following nebuliation, and the like. Normally, die amphiphiles will be injected in solution whets the concentration of compound bound to or entrapped in the liposome will the amount to be administered. This amount will vary with the effectiveness of the compound being administered, die required <vwwin-aHf>n for die desired effect, the number of administrations, 20 and the like. In some intfanm, particularly for aerosol administration, die lipid-DNA complexes can be administered in the form of a lyophilized powder. Upon administration of the amphiphiles, when a targeting moiety is used, the amphiphiles preferentially bind to a cell surfece factor complmmimy to the compounds bound to the liposome. If no targeting moiety is bound to the liposome, thea it binds to cell surfece by lipophilic interactions. The liposomes 2769 wo 95/14381 pct/us94/i3428 19 normally are transferred into the cell by eadocytosis. The cationic amphiphiles of the present invention find use for complexing with nucleic acid or protein for transporting these macromolecules in vitro. The nucleic acid can include DNA, RNA, antisense RNA or other antisense molecules. Cationic amphiphiles that form 5 liposomes also find use in drug delivery in vitro, where the drug can be entrapped within the liposome or bound to the outside. Drug delivery and delivery of macromolecules in vivo using the cationic amphiphiles of the present invention is disclosed but not claimed in the present invention. The following examples are offered by way of illustration and not by way of limitation. WO 95/14381 20 Example 1 (a) Preparation of N-amidino-O.O-diacvldiethanolamine OH boc2o bocn PCT/US94/13428 rcoci/net BOCN OCOR OCOR HCI/Dy hci • hn OCOR h n-cn 2 OCOR HCI HN-C-N 2 II . NH OCOR OCOR Commercially available diethanolamine was protected on nitrogen using di-tert-butyl-pyrocaibonate, acylated with appropriate acyl chloride, N-BOC protection group was cleaved with 4M HCI in dioxane, resulting hydrochloride salt was reacted with cyanamide in n-BuOH to afford the desired 20 N-amidino-0,0-diacyldiethanolamine. Example: Synthesis of N-amidino-0,0-dipalmitoyldiethanolamine5. N-BOC diethanolamine 2. To a solution of 10 ml (0.1 mol) diethanolamine in 150 ml of acetonitiile were added 22.8 g (0.105 mol) of di-tert-butyl-pyrocarbonate and mixture was SUBSTITUTE SHEET (RULE 26) WO 95/14381 PCT/US94/13428 21 stirred at T. overnight. The resulting solution was evaporated, the residue dissolved in ethyl acetate/hexane (7/3) and passed through a plug of silica. After evaporation of solvent to get 16.4 g (80%) of N-BOC diethanolamine. 5 N-BOC ester 2. To a solution of 2.0 g (0.0097 mol) of 2 in 100 ml of CH2C12 at 0°C were added 3.4 ml (0.024 mol) of triethylamine, then in 10 min with stirring were added 6.2 ml (0.02 mol) of palmitoyl chloride. The mixture was stirred at 0°C for 30 min, then at R.T. for 45 min. The resulting solution was washed with 10% 10 citric acid (50 mlx2), with 10% aqueous solution of sodium bicarbonate (50 mlx2), dried over MgS04, filtered, filtrate evaporated on rotavapor and the rest was chromatographed on silica gel using 0-15% EtOAc/Hexane to get 6.4 g (95%) of N-BOC ester 2- 15 Amino ester ±. To 3.5 g (0.086 mol) of N-BOC ester 2 were added 20 ml of 4M solution of HQ in dioxane and mixture was stirred at R.T. for 2 hrs. The resulting suspension was evaporated on rotavapor, diluted with ether (50 ml), filtered, washed with ether (25 mlx2) and dried in vacuum to get 3.1 g (97%) of amino 20 ester 4. Amidino ester 5. To 0.22 g (0.00036 mol) of amino ester 4 was added 0.5 ml of n-BuOH and 0.022 g (0.00054 mol) of cyanamide. The mixture was stirred at 110°C for 1 WO 95/14381 PCT/US94/13428 22 hr, diluted with chloroform (10ml) and evaporated on rotavapor. White precipitate formed was washed with ether (10ml x 2), then with water (10ml x 2) on filter and dried in vacuum to get 0.190 g (79%) of amidino ester £. 5 (b) Transfection using cationic liposomes containing N-anridino-Q.Q- dipalroitoyldiethanolamine (APPPE), N-amidino-Q, Q^ioieoyldigthanolamine (ADODE). and N-amidinodistearovlamine (ADS). Liposomes containing ADODE, ADPDE or ADS in a 1:1 molar ratio with cholesterol were tested as DNA carriers for gene transfer and expression in mice. 10 The plasmid used was pZN51. The methods and plasmids used are described in more detail in W093/24640. The liposomes were in a lOmM stock in 5% dextrose. The liposome:plasmid DNA ratios were screened for the presence of aggregation. Ratios from 1:2 to 1:7 (jig plasmid DNA to nanomoles cationic lipid) were screened. DNA:liposome ratios that did not produce aggregation were IS then tested in mice. 100 fig of pZNSl was complexed to 500 nanomoles of DDAB: cholesterol liposomes as a positive control and an uninjected mouse served as the negative control (N). ICR female mice (25 g) were used for the in vivo studies. A dose of 100 fig plasmid DNA in 200 n 15% dextrose in water was injected by tail vein per 20 mouse. The lung, heart, liver, kidney and spleen were removed after 24 hours. Each organ was homogenized in 0.3 ml of 0.25 M Tris-HCl pH7.8, 5 mM EDTA, and the resulting extract was centrifuged and then subjected to 3 cycles of freeze-thaw and then treated to 65 °C for 20 min. The protein concentration of </div>

Claims

<div class="application article clearfix printTableText" id="claims"> WO 95/114381 PCT/US94/13428 23 lung, heart, liver and kidney extracts were quantitated using a ninhydrin-based protein assay (Bio-Rad, Berkeley, CA), and same amount of total protein from each tissue extract was added in the CAT assay, together with 10 pi of 20 mM acetyl CoA+12 pi of I4C-chloramphenicol (25 pCi/ml, 55 mCi/mmole, 5 Amersham)), at 37 'C for 13 hrs. ADODE:CHOL liposomes in a 1:6 ratio produced the highest levels of CAT activity in the lung, liver and heart. However, CAT activity was lower than that produced by DDAB:CHOL in a 1:5 ratio. There was almost no CAT activity in the kidney and spleen in ADODE: CHOL-treated mice. 10 ADPDE:CHOL liposomes in a 1:6 ratio produced the highest levels of CAT activity in the lung, heart, liver, kidney and spleen. The level of CAT activity was similar to DDAB:CHOL in a 1:5 ratio in these organs. ADS:CHOL liposomes in a 1:5 ratio produced the highest levels of CAT activity in the lung and liver. The CAT activity was lower than that produced by 15 DDAB:CHOL at the 1:5 ratio. In heart, spleen and kidney ADS:CHOL produces little to no CAT activity. All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual 20 publication or patent application was specifically and individually indicated to be incorporated by reference. The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims. 276975 wo 95/14381 pct/us94/13428 24 WHAT IS CLAIMED IS: 1. A nitrogen -containing amphiphile of the fbimula (i): IT {R-[ X - G - CH2 - (CHj)^" In ") 2 ^ ^ " ^*2 Formula (I);wherein each R independently Is .1 straight-chain, aliphatic hydrocaibyl group of 5 to 29 carbon atoms inclusive, each X is -CH2- or -CO-, each m is an integer from 0 to 7 inclusive and each n is zero or 1, with the proviso that when n is !, the total number of carbon atoms in Rand-(CHz)n,- is at least 10, and when n is zero, each ^independently is a straight-chain, aliphatic hydrocarbyl group of at least 11 carbon atoms inclusive.;2. The amphiphile of claim 1 wherein a is zero.;3. • The amphiphile of claim 2 wherein each R independently has iroro 14 to 24 carbon atoms inclusive.;a;4. The amphiphile of claim 3 wherein R is octadecyl.;5. The ampbrphile of claim 3 wherein R is tetradecyl. _;6. The umphiphile of claim 1 wherein n is 1.;7. The amphiphile of claim 6 wherein m is 1.;3. The amphiphile of claim 7 wherein X is -CO-.;9. The amphiphile of claim 7 wherein each R independently has from 13 to 23 carbon atoms inclusive.;10. Hie amphiphile of claim 9 wherein R is pentadecyi.;11. The amphiphile of claim 9 wherein R is 8-heptadecenyL;2769?5;wo 93/14381 pct/us94/i342!;25;12. The amphiphile of claim 6 wherein m is 5.;13. The amphiphile of claim 12 wherein R is heptadecyL;14. A method of transforming cells in vitro in one or more tissues of a mammal, comprising:;5 contacting said cells with a plurality of complexes comprising an expression cassette and a nitrogen-containing amphiphile of Claim 1, wherein said complexes provide lor transmission of cells in at least one tissue of said mammal and are susceptible to endogenous enzymatic cleavage to non-torac products.;15. A method for transfecting a mammalian cell in vitro comprising contacting said cell with a complex comprising a transcription cassette or an expression cassette and a nitrogen-containing amphiphile of Claim 1.;16. A use of a nitrogen-containing amphiphile as claimed in any one of claims I to 13;in the preparation of a pharmaceutical composition for use in transforming cells in one or more tissues of a. mammal.;17., A use cf a nitrogca-cotiiauung ainphiphile as claimcd in aay one of claims 1 to 13;^ . •;in the preparation of a pharmaceutical composition for use in transfecting a mammalian cell.;18. The use of ciaixry 16 or claim Y1 wherein the pharmaceutical composition further comprises an expression, cassette.;19. The use of claim 18 wherein the pharmaceutical composition ftaher comprises a transcription cassette.;20. A use of a plurality of complexes comprising a nitrogen-containing amphiphile as claimed in any one of claims 1 to 13 and an expression cassette in the preparation of a pharmaceutical composition for use in transfecting a mammalian cell.;26;276975;A use of a plurality of complexes comprising a nitrogen-containing amphiphile as r.,jy ay one of claims 1 tc 13 and m expression cassette in the preparation of a;. ;rical composition for \ise in transforming cells in a rnarnraal.;22. A nitrogen-containing amphiphile of the formula (I) as defined in claim 1 substantially as herein described with reference to any example thereof.;23. A method as claimed in claim 14 of transforming cells in one or more tissues of a mammal substantially as herein described with reference to any example thereof.;24. A method as claimed in claim 15 for transfecting a mammalian cell substantially as herein described with reference to any example thereof.;25. Use according to any one of claims 16, 17, 20 and 21 for transfecting a mamnalian cell substantially as herein described with reference to any example thereof.;, END OF CLAIMS;' ^ Office;<>f -K'aw -Zealand* </div>