Particle structures comprising sterols and saponins   

This application is a continuation of U.S. application Ser. No. 10/114,957, filed on Apr. 4, 2002, now allowed, which claims the benefit of U.S. provisional application Ser. No. 60/308,609 filed on Jul. 31, 2001; and of Danish application number PA 200100560, which was filed on Apr. 4, 2001, all incorporated herein by reference in their entireties. All patent and nonpatent references cited in the application, or in the present application, are also hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to complexes of sterols and saponin glycosides. The complexes are capable of interacting with bioactive agents including genetic determinants, such as e.g. polynucleotides. The complexes pertaining to the present invention can be used to facilitate the transport of polynucleotides, including DNA and derivatives thereof, across cellular membranes.

Acting as carriers of various bioactive agents and genetic determinants, the complexes provide a means for introducing e.g. a polynucleotide into a patient, a predetermined region of the patient, or a predetermined biological cell of the patient, in order e.g. to express a gene comprised by the polynucleotide and/or to regulate the expression of genes being expressed in the biological cell in vivo and/or in vitro.

More particularly, the present invention relates to novel methods for transfecting a biological cell, to complexes involved in such methods, and to diagnostic methods and therapeutic methods for treating a patient by e.g. gene therapy and DNA-vaccination.

BACKGROUND OF THE INVENTION

Several medical applications utilizing genetic determinants have evolved in recent years. In such applications, the introduction of whole genes or specific nucleic acids into cells is of central importance. This process is often referred to as gene transfection independently of the origin of the cells, the sequence and character of the nucleic acid, and irrespective of whether the transfer is performed in vivo or ex vivo.

To facilitate the process of gene transfection several different approaches have been developed. Such approaches include among others i) using biological vectors (including viral vectors), ii) associating a nucleic acid with a cationic liposome, iii) associating a nucleic acid with peptides covalently linked to a transfection agent, and iv) coating minute gold particles by nucleic acids and using the coated particles for a bio-ballistic transfer.

The major iscom constituents are quillaja saponins and cholesterol. The procedure for preparation of iscoms comprises solubilization of amphipathic polypeptides in preferably nonionic detergents, addition of Quillaja saponins, cholesterol, and possibly also phosphatidylcholine. In the presence of amphipathic proteins, iscom particles are formed on removal of the detergent.

Morein (see e.g. U.S. Pat. No. 4,578,269) was the first to describe that iscoms not only formed a very characteristic structural complex, but also possessed significant immunogenic properties when amphipathic antigens were inserted into this complex by hydrophobic interaction. Conventional iscoms (immunostimulating complexes) have since been used for vaccine formulations and combine a multimeric presentation of an antigen with an adjuvant functionality. (see e.g. U.S. Pat. No. 6,080,725 to Marciani).

SUMMARY

The present invention in one aspect relates to a complex comprising at least one sterol and/or at least one saponin, wherein the at least one sterol or the at least one saponin is capable of forming an electrostatic interaction or a hydrophobic interaction with at least one bioactive agent, including a genetic determinant, including a polynucleotide, including DNA and derivatives thereof. When the at least one sterol and the at least one saponin is incapable of forming an electrostatic interaction or a hydrophobic interaction with the at least one bioactive agent as described herein above, the complex comprises at least one contacting group capable of contacting the at least one bioactive agent, including a genetic determinant, including a polynucleotide, including DNA and derivatives thereof, by means of an interaction selected from an electrostatic interaction and a hydrophobic interaction and an interaction resulting from intercaation of the genetic determinant by the contacting group.

According, in one aspect of the invention there is provided a complex comprising

i) at least one first sterol and/or at least one second sterol,

wherein the at least one second sterol is capable of contacting a genetic determinant by means of an interaction selected from an electrostatic interaction and a hydrophobic interaction, and

wherein the at least one first sterol and/or the at least one second sterol is capable of forming a complex with at least one first saponin and/or at least one second saponin, and

ii) at least one first saponin and/or at least one second saponin,

wherein the at least one second saponin is capable of contacting a genetic determinant by means of an interaction selected from an electrostatic interaction and a hydrophobic interaction, and

wherein the at least one first saponin and/or the at least one second saponin is capable of forming a complex with at least one first sterol and/or at least one second sterol, and optionally

iii) at least one contacting group for contacting a genetic determinant by means of an interaction selected from an electrostatic interaction and a hydrophobic interaction,

with the proviso that the at least one contacting group is present when no second sterol and no second saponin is present in the complex.

The complexes according to the invention are useful for binding polynucleotides including naturally occurring nucleic acids including DNA and RNA, and derivatives thereof, including, but not limited to peptide nucleic acids (PNA) and locked nucleic acids (LNA). The bound polynucleotide can subsequently be transferred into a biological cell including any animal cell or human cell.

The complexes according to the present invention may in one preferred embodiment adopt a micro-particle structure in the form of a cage-like matrix similar to that known as an immune stimulating complex (iscom). Beside iscom structures, the interaction between sterols and saponins have been reported to result in a variety of different structural entities, including entities such as e.g. lattices, honeycombes, rods, and amorphic particles, all of which structural entities are covered by the present invention. However, other structures and matrix formations are also envisaged by the present invention which is in no way limited to iscom-like structures or matrixes.

In the case where the complexes according to the present invention do form iscoms, or iscom-like structures, such iscoms or structures may be prepared e.g. essentially as described in European patent EP 0 109 942 B1.

Accordingly, a glycoside solution, containing fx cholesterol, phospholipid, and one or more glycosides (fx Quillaja components) with hydrophobic and hydrophilic domains in a concentration of at least a critical micelle-binding concentration, is formed and a complex is generated. The complex may subsequently be isolated and/or purified.

Optionally, as a first step, the component to be inserted into the complex, fx a bioactive agent, including a polynucleotide, such as DNA and derivatives thereof, an immunogenic agent, an antigenic agent, a therapeutic agent, a diagnostic agent, and the like, can be mixed with one or more solubilizing agents, whereby complexes are formed between the component and solubilizing agents, after which the components are separated from the solubilizing agent and e.g. transferred directly to the glycoside solution.

In line with the present invention, the glycoside solution may initially be mixed with a polynucleotide. It is possible to proceed from a matrix that can be made by solubilizing at least one sterol in a solution agent, adding at least one glycoside or at least one saponin, and optionally the other lipophilic moieties, after which addition the solution agent may be removed, if it is proving unacceptable to the final product.

The matrix may be transferred to a water solution in which its separate parts are not soluble. The solubilizing agent can be removed through eg gel filtration, ultra filtration, dialysis, or electrophores. The matrix can then be purified from surplus of first sterol and saponin e.g. by ultracentrifugation, through a density gradient, or through gel filtration. The solubilizing agent may be any one of those mentioned in U.S. Pat. No. 5,679,354, which is incorporated herein by reference.

In one preferred embodiment, the complexes according to the invention are formed essentially as described in Example 1 herein.

Accordingly, in one preferred embodiment, there is provided a method for preparation of a complex comprising

i) at least one first sterol and/or at least one second sterol,

wherein the at least one second sterol is capable of contacting a genetic determinant by means of an interaction selected from an electrostatic interaction and a hydrophobic interaction, and

wherein the at least one first sterol and/or the at least one second sterol is capable of forming a complex with at least one first saponin and/or at least one second saponin, and

ii) at least one first saponin and/or at least one second saponin,

wherein the at least one second saponin is capable of contacting a genetic determinant by means of an interaction selected from an electrostatic interaction and a hydrophobic interaction, and

wherein the at least one first saponin and/or the at least one second saponin is capable of forming a complex with at least one first sterol and/or at least one second sterol, and optionally

iii) at least one contacting group for contacting a genetic determinant by means of an interaction selected from an electrostatic interaction and a hydrophobic interaction,

with the proviso that the at least one contacting group is present when no second sterol and no second saponin is present in the complex, and further optionally

iv) at least one lipophilic moiety,

wherein said method comprises the steps of

a) mixing a sterol composition comprising at least one first sterol and/or at least one second sterol, with

b) a saponin composition comprising at least one first saponin and/or at least one second saponin, and

c) at least one lipophilic moiety, and

d) at least one organic solvent,

wherein the steps a) to d) may be carried out simultaneously, or sequentially, in any order, and optionally

e) removing surplus reactants and/or purifying the prepared complexes.

The organic solvent is preferably selected from ethanol, DMSO, and DMF, and the solvent is preferably present in an amount of at the most 25% (vol/vol), such as at the most 20% (vol/vol), for example at the most 15% (vol/vol), such as at the most 10% (vol/vol), for example at the most 8% (vol/vol), such as at the most 6% (vol/vol), for example at the most 4% (vol/vol), such as at the most 2% (vol/vol), for example at the most 1% (vol/vol), such as at the most 0.5% (vol/vol), for example at the most 0.1% (vol/vol).

First and Second Sterols and Saponins

The following general distinction is made between first and second sterols. First and/or second sterols can be naturally occurring sterols synthesised as secondary metabolites by many organisms. They may also be synthetic, or they may be made by chemical synthesis or enzymatic synthesis either in vitro or in vivo. First sterols are generally incapable of forming an association with a genetic determinant as defined herein, whereas such an association is formed between second sterols and the genetic determinant.

Similarly, first and/or second saponins can be any saponin as defined herein comprising as an aglycone part either i) a triterpene part, ii) a steroid part, or iii) a steroid alkaloid part. The saponins may be naturally occulting or synthetic, or they may be made by chemical synthesis or enzymatic synthesis either in vitro or in vivo. First saponins are generally incapable of forming an association with a genetic determinant as defined herein, whereas such an association is formed between second saponins and the genetic determinant.

Consequently, any sterol will either be a first sterol, or a second sterol, and any saponin will either be a first saponin, or a second saponin. In principle, first and/or second sterols, as well as first and/or second saponins, may be either anionic, neutral, or cationic. The terms cationic sterols and cationic saponins shall denote sterols and saponins, respectively, carrying a net positive charge at pH 7.0. Second sterols and second saponins are preferably cationic sterols and cationic saponins, respectively, whereas first sterols and first saponins are preferably anionic or neutral sterols and saponins, respectively.

Accordingly, the second sterols and/or second saponins preferably comprise at least one positively charged moiety or reactive group at pH=7.0, and this positively charged moiety or reactive group is according to one preferred embodiment of the invention capable of contacting a bioactive agent, including a genetic determinant, by means of an electrostatic interaction.

In another embodiment, the second sterols and/or second saponins preferably comprise an uncharged moiety or non-polar reactive group, and this uncharged moiety or non-polar reactive group is according to another preferred embodiment of the invention capable of contacting a bioactive group, including a genetic determinant, by means of a hydrophobic interaction.

A combination of electrostatic interactions and hydrophobic interactions can also be used for generating an association between bioactive agents and second sterols and/or saponins.

When the complexes according to the present invention comprise no second sterol and no second saponin, the complexes comprise at least one contacting group capable of contacting a genetic determinant by means either of an electrostatic interaction, or a hydrophobic interaction.

The contacting group is necessary in order for such complexes to form the desired association with the genetic determinant. However, the contacting group may also be present in complexes comprising a second sterol and/or a second saponin. The teen “contacting group” as used herein will also refer to any moiety of a second sterol and/or a second saponin capable of forming an association with a genetic determinant, including an association generated by an electrostatic interaction and/or a hydrophobic interaction.

Preferred contacting groups comprise at least one lipophilic moiety capable of forming an association with the complex according to the invention made up by sterols (first and/or second) and/or saponins (first and/or second).

In addition to acting as a “docking group” or “anchor” for contacting groups according to the invention, lipophilic moieties may also serve to facilitate a saponin-sterol complex formation essentially without serving, during or after complex formation, as a “docking group” for a contacting group or any other functional group forming part of the saponin-sterol complex. The purpose of using lipophilic moieties may thus be two-fold: The lipophilic moieties according to the invention may act—during or after saponin-sterol complex formation, preferably during saponin-sterol complex formation—as a facilitator of complex formation, and they may, independently of this action, also act as a “docking group” for any functional group including contacting groups for contacting a genetic determinant and/or targeting ligands for targeting the complexes according to the invention.

Lipophilic moieties such as phospholipids are preferably present during the process of forming the complexes according to the invention. The presence of e.g. phospholipids facilitates complex formation over a broad concentration range. Accordingly, phosphatidyl choline may be added when mixing saponins and sterols during complex formation.

It is preferred to use a phospholipid with a positively charged headgroup such as phosphatidyl ethanolamine. One reason for this is the inclusion into the complex of an overall positive charge capable of facilitating or resulting in an interaction with negatively charged polynucleotides including ribonucleic acids. This interaction will facilitate the uptake of the polynucleotides across a cellular membrane and into a biological cell for subsequent integration and/or translation and/or polypeptide expression. Phosphatidyl choline is an example of another phospholipid capable of being used in connection with the present invention.

Additional moieties of a contacting group may be either lipophilic or hydrophilic, depending on the nature of the association formed with the genetic determinant. One preferred additional moiety of the contacting group is a ionic group, or a charged group, preferably a positively charged group, including a group comprising a positive charge at pH=7.0. Examples of such groups are illustrated for lipophilic moieties in FIGS. 6, 7 and 8. Such lipophilic moieties can be obtained from, among others, Avanti Polar Lipids, Inc. Alabaster, Ala.

It is thus possible to imagine a series of different compositions of the complexes according to the invention. The invention in preferred embodiments relates to

i) complexes comprises at least one first sterol and at least one first saponin and at least one contacting group,

ii) complexes comprising at least one first sterol and at least one second saponin,

iii) complexes comprising at least one first sterol and at least one second saponin and at least one contacting group,

iv) complexes comprising at least one first sterol and at least one first saponin and at least one second saponin,

v) complexes comprising at least one first sterol and at least one first saponin and at least one second saponin and at least one contacting group,

vi) complexes comprising at least one second sterol and at least one first saponin,

vii) complexes comprising at least one second sterol and at least one first saponin and at least one contacting group,

viii) complexes comprising at least one second sterol and at least one second saponin,

ix) complexes comprising at least one second sterol and at least one second saponin and at least one contacting group,

x) complexes comprising at least one second sterol and at least one first saponin and at least one second saponin,

xi) complexes comprising at least one second sterol and at least one first saponin and at least one second saponin and at least one contacting group,

xii) complexes comprising at least one first sterol and at least one second sterol and at least one first saponin,

xiii) complexes comprising at least one first sterol and at least one second sterol and at least one first saponin and at least one contacting group,

xiv) complexes comprising at least one first sterol and at least one second sterol and at least one second saponin,

xv) complexes comprising at least one first sterol and at least one second sterol and at least one second saponin and at least one contacting group,

xvi) complexes comprising at least one first sterol and at least one second sterol and at least one first saponin and at least one second saponin,

xvii) complexes comprising at least one first sterol and at least one second sterol and at least one first saponin and at least one second saponin and at least one contacting group,

including any composition comprising any combination of the complexes listed herein immediaterly above, i.e. any combination of complexes i) to xvii) Such combinations may be of value when attempting to direct or target different complexes to different regions of a patient.

The mechanism by which the contacting group interacts with nucleic acid depends upon the character of the contacting group in question. One example of a contacting group is a cationic (positively charged) derivative of a cholesterol which is capable of forming an interaction with a genetic determinant by e.g. forming an electrostatic interaction with the backbone of a polynucleotide including any DNA-backbone. Another example is a lipid-tailed acridin-compound that intercalates with double stranded DNA.

In addition to sterols (first and/or second), saponins (first and/or second), and contacting groups comprising a lipophilic moiety, the complexes according to the present invention may further comprise a targeting ligand for targeting a particular polynucletide, including a nucleic acid, to a specific cell surface or tissue. This may be accomplished by incorporation of specific targeting ligands and/or receptor binding molecules and/or ligands into the structure of the complexes according to the invention.

Accordingly, two different methods for the formation of the complexes according to the invention are provided, in one method the contacting group are incorporated during the formation of the particles, and in another method the contacting group is added to preformed particles, optionally in the form of iscom-matrices.

One principal aspect of the invention is thus the provision of a novel system that enhances the uptake of polynucleotides including nucleic acids by combining—in one preferred embodiment—i) the ability of iscom-structures to associate with, and penetrate into or through membranes containing cholesterol, with ii) the property of contacting or associating with polynucleotides including nucleic acids. In particular for the application of vaccination with naked DNA the complexes according to the invention is likely to reduce the required amount of DNA.

It should be noted that the invention is not limited to complexes capable of forming an association with polynucleotides. The complexes according to the invention in other preferred embodiments are capable of contacting or forming an association with polypeptides and/or polynucleotides.

Accordingly, the present invention in one embodiment pertains to complexes comprising components also forming a part of traditional iscoms. However, the complexes according to the present invention are different from conventional iscoms in a number of ways. Firstly, the complexes according to the present invention are capable of binding a bioactive agent including a genetic determinant in the form of e.g. a polynucleotide. Secondly, the complexes in one preferred embodiment are characterised by a zeta-potential which is less negative—or more positive—than about −50 mV.

Accordingly, there is provided a complex having a zeta-potential which is less negative or more positive than about −50 mV, such as a zeta-potential of about −45 mV, for example a zeta-potential of about −40 mV, such as a zeta-potential of about −37 mV, for example a zeta-potential of about −35 mV, such as a zeta-potential of about −32 mV, for example a zeta-potential of about −30 mV such as a zeta-potential of about −29 mV, for example a zeta-potential of about −28 mV, such as a zeta-potential of about −27 mV, for example a zeta-potential of about −26 mV, such as a zeta-potential of about −25 mV, for example a zeta-potential of about −20 mV, such as a zeta-potential of about −15 mV, for example a zeta-potential of about −10 mV, such as a zeta-potential of about −5 mV, for example a zeta-potential of about 0 mV, such as a zeta-potential of about 5 mV, for example a zeta-potential of about 10 mV, such as a zeta-potential of about 15 mV, for example a zeta-potential of about 20 mV, and preferably a zeta-potential of less than 50 mV.

The complexes in preferred embodiments may thus have a zeta-potential of from about −50 mV to about −40 mV, such as from about −40 mV to about −35 mV, for example of from about −35 mV to about −30 mV, such as from about −30 mV to about −25 mV, for example of from about −25 mV to about −20 mV, such as from about −20 mV to about −15 mV, for example of from about −15 mV to about −10 mV, such as from about −10 mV to about −5 mV, for example of from about −5 mV to about 0 mV, such as from about 0 mV to about 5 mV, for example of from about 5 mV to about 10 mV, such as from about 10 mV to about 15 mV, for example of from about 15 mV to about 20 mV, such as from about 20 mV to about 30 mV, for example of from about 30 mV to about 40 mV, such as from about 40 mV to about 50 mV.

Using as a standard reference point a complex comprising i) any given saponin(s) of interest and ii) cholesterol as a first sterol, complexes according to the invention comprising i) any given saponin(s) of interest, ii) cholesterol as a first sterol, and iii) any given second sterol of interest, preferably a cholesterol derivative as listed in FIG. 5 herein, said complexes according to the invention have a zeta-potential which is at least about 5 mV less negative or more positive than said reference complexes: such as a zeta-potential which is at least about 10 mV less negative or more positive than said reference complexes, for example a zeta-potential which is at least about 15 mV less negative or more positive than said reference complexes, such as a zeta-potential which is at least about 20 mV less negative or more positive than said reference complexes, for example a zeta-potential which is at least about 25 mV less negative or more positive than said reference complexes, such as a zeta-potential which is at least about 30 mV less negative or more positive than said reference complexes, for example a zeta-potential which is at least about 35 mV less negative or more positive than said reference complexes, such as a zeta-potential which is at least about 40 mV less negative or more positive than said reference complexes, for example a zeta-potential which is at least about 45 mV less negative or more positive than said reference complexes, such as a zeta-potential which is at least about 50 mV less negative or more positive than said reference complexes.

The values of the zeta-potentials for the complexes of the present invention are determined in part by the inclusion of e.g. cationic second saponins and/or cationic second sterols into the complexes according to the present invention.

The molar ratio between saponins (first and second) and sterols (first and second) in complexes according to the present invention is preferably from less than 1000:1 to preferably more than 1:1000. Preferred ratios are about 100:1, for example about 80:1, such as about 60:1, for example about 50:1, such as about 40:1, for example about 30:1, such as about 25:1, for example about 20:1, such as about 18:1, for example about 16:1, such as about 14:1, for example about 12:1, such as about 10:1, for example about 9:1, such as about 8:1, for example about 7:1, such as about 6:1, for example about 5:1, such as about 4:1, for example about 3:1, such as about 2:1, for example about 1.9:1, such as about 1.8:1, for example about 1.7:1, such as about 1.6:1, for example about 1.5:1, such as about 1.4:1, for example about 1.3:1, such as about 1.2:1, for example about 1.1:1, such as about 1:1, for example about 1:1.1, such as about 1:1.2, for example about 1:1.3, such as about 1:1.4, for example about 1:1.5, such as about 1:1.6, for example about 1:1.7, such as about 1:1.8, for example about 1;1.9, such as about 1:2.0, for example about 1:2.5, such as about 1:3, for example about 1:3.5, for example about 1:4, such. as about 1:4.5, for example about 1:5, for example about 1:5.5, such as about 1:6, for example about 1:7, such as about 1:10, for example about 1:20, such as about 1:40, for example about 1:60, such as about 1:80, for example about 1:100.

Without being bound by theory, the complexes according to the present invention may according to one presently preferred hypothesis adopt either an iscom-like structure, or a structure that does not resemble such a structure when contacting or being associated with either a polynucleotide and/or a polypeptide comprising e.g. natural or synthetic amino acids and variants thereof. When being connected by the complexes, the polynucleotides and/or polypeptides are according to one preferred hypothesis in a degradation-resistant conformation, or in a conformation less prone to degradation, as compared to the conformation of the polynucleotide or the polypeptide when it is not associated with the complex according to the invention The invention thus in one preferred embodiment relates to complexes comprising a polynucleotide and/or a polypeptide that is less likely to be degraded by nucleases and proteases, respectively, under practical circumstances.

Accordingly, there is provided in one preferred embodiment a method for administration of a polynucleotide and/or a polypeptide to an individual in need thereof, said polynucleotide and/or said polypeptide being administered in association with the complexes according to the invention in a conformation that is not susceptible to degradation under practical circumstances—or less susceptible to degradation under practical circumstances—as compared to the degradation taking place when the polynucleotide and/or the polypeptide is administered in the absence of the complexes under substantially similar conditions, including conditions wherein the polynucleotide and/or the polypeptide is administered in combination with conventional carriers or adjuvants.

Consequently, the present invention in one particular embodiment relates to complexes in the form of modified iscoms that are used as carriers for bioactive agents including polynucleotides and other genetic determinants which are desirably transfected into a biological cell by means of an interaction of the complex with the cellular membrane.

Complexes according to the invention may thus comprise one or more selected from the group consisting of bioactive agents, immunogenic determinants, genetic determinants, enzymes, adjuvants and medicaments. Hence, by way of example a complex according to the present invention may comprise both a genetic determinant and an antigenic determinant, such as a nucleic acid and a polypeptide.

The complexes according to the invention may further comprise—in addition to a bioactive agent and/or a genetic determinant, preferably a polynucleotide, i) a lipophilic moiety, optionally a lipophilic moiety comprising a contacting group and/or a targeting ligand, and/or ii) a saccharide moiety, preferably, but not limited to a saccharide moiety forming part of a naturally occurring saponin. It is particularly preferred that the complexes according to the invention comprise a saccharide moiety when it is intended to direct the complexes to cellular surfaces known to contain saccharide binding receptors.

The complexes according to the invention may thus further comprise a bioactive agent and/or a genetic determinant and/or an immunogenic determinant and/or an antigenic determinant and/or a medicament and/or a therapeutic agent and/or a diagnostic agent.

The complexes may in even further embodiments be encapsulated by an encapsulation agent including a liposome and/or a biodegradable microsphere. By analogy to the complexes, the liposome and/or the biodegradable microsphere may comprise a targeting ligand suitably associated with the microsphere for targeting the microsphere to a particular location in a human or animal body. Preferred targeting ligands include, but are not limited to, ligands having affinity to receptors on antigen presenting cells, including e.g. dendritic cells and macrophages. When used for therapeutic purposes, the ligands are targeted to e.g. vitamin receptors, folate receptors, high affinity IL-2 receptors, growth factor receptors, such as EGF-receptors, and the like.

First saponins and/or second saponins and/or first sterols and/or second sterols may also be present in e.g. the biodegradable microsphere. It is particularly preferred in one embodiment that the microsphere has an overall positive charge, i.e. contains more positively charged groups than negatively charged groups.

The present invention also pertains to pharmaceutical compositions and methods of treatment of an individual by therapy and/or surgery, methods of cosmetic treatment, and diagnostic methods practised on the human or animal body.

There is also provided a method for manufacturing the complexes according to the invention, and in even further aspects the present invention relates to using the complexes according to the invention, or compositions comprising such complexes, including pharmaceutical compositions, in the manufacture of a medicament for treating a condition in an individual in need of such treatment.

In addition, the present invention relates to a kit-of-parts, wherein said kit-of-parts comprises a complex according to the invention and at least one immunogenic determinant. The present invention also relates to a kit-of-parts, wherein said kit-of-parts comprises a complex according to the invention and at least one immunogenic determinant and at least one genetic determinant. For example the immunogenic determinant may comprise an antigenic determinant.

The kit-of-parts according to the invention—as well as the complexes themselves—can be used in a method for raising a desirable immune response in a patient. Said method comprise the steps of i) providing a complex according to the invention, and ii) providing a genetic determinant such as a polynucleotide such as DNA and/or iii) providing an immunogenic determinant such as antigenic determinant such as a peptide such as an epitope, iv) mixing said complex with said genetic determinant and/or said immunogenic determinant, v) administering said mixture to a patient in an amount effective to raise a desirable immune response in said patient, and vi) raising said desirable immune response.

In preferred embodiments, the method comprises the further step of administering, simultaneously or sequentially in any order, a) a complex comprising a genetic determinant, and b) a complex comprising an immunogenic determinant such as an antigenic determinant. The genetic determinant in one embodiment encodes the immunogenic/antigenic determinant, or part thereof. The immunogenic determinant Is preferably a lipid associated peptide (lipo-peptide, e.g. a lipo-protein or a part thereof). When the above method comprises the steps of sequentially administering a) a complex comprising a genetic determinant, and b) a complex comprising an immunogenic determinant, it is thus possible to administer initially a genetic determinant and then boost after a suitable time period any initial immune response by subsequently administering a complex comprising an immunogenic determinant, preferably an immunogenic determinant comprising an epitope encoded by the initially administered genetic determinant. Initial administration of an immunogenic determinant followed by administration after a suitable time period of a genetic determinant. Likewise, it is possible to administer to a patient—simultaneously or sequentially in any order—i) a complex comprising a genetic determinant, and ii) a peptide including an epitope administered according to any state of the art method, as well as administering to said patient—simultaneously or sequentially in any order—i) a complex comprising an immunogenic determinant, including a peptide epitope, and ii) a polynucleotide such as DNA administered according to any state of the art method.

The above complexes, or the individual components thereof, as well as the genetic and immunogenic determinants can be provided in individual vials ready for use in a kit-of-parts. In this way it is possible to mix complexes according to the invention with genetic determinants, such as polynucleotides, such as DNA, and/or with immunogenic determinants such as antigenic determinants, such as peptides, such as epitopes.

Aglycone: Part of a saponin glycoside, linked to saccharide part through a glycosidic bond.

Amphiphilic moiety: Any moiety, including a lipid, comprising a synthetic, semi-synthetic (modified natural) or naturally-occurring compound having a water-soluble, hydrophilic portion and a water-insoluble, hydrophobic portion. Preferred amphiphilic compounds are characterized by a polar head group, for example, a phosphatidylcholine group, and one or more nonpolar, aliphatic chains, for example, palmitoyl groups.

Antigenic determinant: Any determinant or substance with the capability of binding an antibody, or a binding fragment thereof, and inducing a specific antigen response. The antigenic determinant may comprise or essentially consist of an epitope that forms part of a polypeptide and elicits a specific antibody response when the whole polypeptide is used as an antigen. Such epitopes are confined to a single or a few loci on the molecule in question.

Bioactive agent: Any a substance which may be used in connection with an application that is therapeutic or diagnostic, such as, for example, in methods for diagnosing the presence or absence of a disease in a patient and/or methods for the treatment of a disease in a patient. “Bioactive agent” refers to substances, which are capable of exerting a biological effect in vitro and/or in vivo. The bioactive agents may be neutral, positively or negatively charged. Suitable bioactive agents include, for example, prodrugs, diagnostic agents, therapeutic agents, pharmaceutical agents, drugs, oxygen delivery agents, blood substitutes, synthetic organic molecules, proteins, peptides, vitamins, steroids, steroid analogs and genetic determinants, including nucleosides, nucleotides and polynucleotides.

Cationic lipid: Any lipid carrying a net positive charge at pH 7.0.

Cationic saponin: Any saponin carrying a net positive charge at pH 7.0.

Cationic sterol: Any sterol carrying a net positive charge at pH 7.0.

Contacting group: Group comprising a lipophilic moiety and a moiety capable of association with a genetic determinant by means of either i) electrostatic interaction, or ii) hydrophobic interaction.

Complex: Formation of saponins and sterols capable of interacting with genetic determinants or immunogenic determinants. The interaction can result from electrostatic interactions and/or hydrophobic interactions formed between on the one hand i) a saponin and/or a sterol, and on the other hand ii) a genetic determinant and/or an immunogenic determinant. When this is the case, the, saponin and/or the sterol is termed a second saponin and/or a second sterol, respectively. Complexes devoid of second saponins and second sterols form electrostatic and/or hydrophobic interactions with genetic determinants and/or immunogenic determinants by means of a contacting group. When the contacting group forms part of a saponin or a sterol, said saponin or sterol is by definition a second saponin or a second sterol. Contacting groups may be present in a complex independently of the presence of second saponins and/or second sterols. Accordingly, contacting groups can be present in a complex also comprising second saponins and/or second sterols without said contacting group forming part of said second saponins and/or second sterols. The overall charge of a second saponin or a second sterol can be neutral or even anionic, as long as the contacting group of the saponin or sterol in question comprises at least one positively charged moiety at pH=7 capable of forming an association with a genetic determinant. Contacting groups can also be neutral in which case predominantly hydrophobic interactions with a genetic determinant are formed.

Degenerated polynucleotide sequence: Polynucleotide encoding a polypeptide and comprising an altered sequence of nucleotides as compared to a polynucleotide comprising a predetermined sequence of nucleotides and encoding a predetermined polypeptide, wherein the polypeptide and the predetermined polypeptide have the same biological activity.

Diagnostic agent: Any agent which may be used in connection with methods for imaging an internal region of a patient and/or diagnosing the presence or absence of a disease in a patient. Diagnostic agents include, for example, contrast agents for use in connection with ultrasound imaging, magnetic resonance imaging (MRI), nuclear magnetic resonance (NMR), computed tomography (CT), electron spin resonance (ESR), nuclear medical imaging, optical imaging, elastography, radiofrequency (RF) and microwave laser. Diagnostic agents may also include any other agents useful in facilitating diagnosis of a disease or other condition in a patient, whether or not imaging methodology is employed. As defined herein, a “diagnostic agent” is a type of bioactive agent.

Diagnostic method: Any method involving an outcome aiding the medical practitioner in reaching a diagnosis of a clinical condition.

Dipole-dipole interaction: The attraction which can occur among two or more polar molecules. Thus, “dipole-dipole interaction” refers to the attraction of the uncharged, partial positive end of a first polar molecule to the uncharged, partial negative end of a second polar molecule. Dipole-dipole interactions are exemplified by the attraction between the electropositive head group, for example, the choline head group, of phosphatidylcholine, and an electronegative atom, for example, a heteroatom, such as oxygen, nitrogen or sulphur “Dipole-dipole interaction” also refers to intermolecular hydrogen bonding in which a hydrogen atom serves as a bridge between electronegative atoms on separate molecules and in which a hydrogen atom is held to a first molecule by a covalent bond and to a second molecule by electrostatic forces.

Electrostatic interaction: Any interaction occurring between charged components, molecules or ions, due to attractive forces when components of opposite electric charge are attracted to each other. Examples include, but are not limited to: ionic interactions, covalent interactions, interactions between a ion and a dipole (ion and polar molecule), interaction between two dipoles (partial charges of polar molecules), hydrogen bonds, i.e. hydrogen bonded to e.g. i) a nitrogen atom, an oxygen atom, or a fluor atom in one molecule, while at the same time being bonded to ii) a nitrogen atom, an oxygen atom, or a fluor atom in another molecule or the same molecule, interchelating interactions, and London dispersion bonds (induced dipoles of polarizable molecules). Thus, for example, “ionic interaction” or “electrostatic interaction” refers to the attraction between a first, positively charged molecule and a second, negatively charged molecule. Ionic or electrostatic interactions include, for example, the attraction between a negatively charged bioactive agent, for example, a genetic determinant, and one or more of i) a positively charged lipid, for example, a cationic lipid, ii) a positively charged saponin, for example a cationic saponin, and iii) a positively charged sterol, for example a cationic sterol.

Genetic determinant: Genetic determinant refers to nucleotides and polynucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The genetic determinant may be made by synthetic chemical methodology known to one of ordinary skill in the art, or by the use of recombinant technology, or by a combination thereof. The DNA and RNA may optionally comprise unnatural nucleotides or nucleotide derivatives including LNA (locked nucleic acids) and PNA (peptide nucleic acids), and it may be single or double stranded. “Genetic determinant” also refers to sense and anti-sense DNA and RNA, which are nucleotide sequences which are complementary to specific sequences of nucleotides in DNA and/or RNA.

Hydrogen bond: An attractive force, or bridge, which may occur between a hydrogen atom which is bonded covalently to an electronegative atom, for example, oxygen, sulfur, or nitrogen, and another electronegative atom. The hydrogen bond may occur between a hydrogen atom in a first molecule and an electronegative atom in a second molecule (intermolecular hydrogen bonding). Also, the hydrogen bond may occur between a hydrogen atom and an electronegative atom which are both contained in a single molecule (intramolecular hydrogen bonding).

Hydrophobic interaction: Any interaction occurring between essentially non-polar (hydrophobic) components located within attraction range of one another in a polar environment (e.g. water). As used herein, attraction range is on the scale of about 100 nm. A particular type of hydrophobic interaction is exerted by “Van der Waal\'s forces”, i.e. the attractive forces between non-polar molecules that are accounted for by quantum mechanics. Van der Waal\'s forces are generally associated with momentary dipole moments which are induced by neighboring molecules and which involve changes in electron distribution.

Immunogenic determinant: Any determinant or substance with the capability of raising an immune response, such as a specific or non-specific antibody response, or a cytolytic response. The immunogenic determinant may comprise or essentially consist of an epitope that forms part of a polypeptide and elicits an immune response when the whole polypeptide is used as an immunogen. Such epitopes are confined to a single or a few loci on the molecule in question.

Interaction: Transient or longer lasting attraction or binding of two or more moieties to one another, mediated by physical forces such as e.g. electrostatic interactions and hydrophobic interactions.

Intercalation: Intercalation specifically denotes the association between a contacting group and a genetic determinant wherein the contacting group form a complex with two adjacent layers of purine-pyrimidine base-pairs of the genetic determinant. The intercalating group is oriented in parallel to the base-pairs and interacts with the genetic determinant by hydrogen bond forces, electrostatic interactions and/or hydrophobic interactions.

Iscom structure: Rigid, cage-like matrix characterised by an icosahedral symmetry, about 30 to 40 nanometers in diameter, and composed of 12 nanometer ring-like subunits.

Essentially non-polar: Nature of compounds or domains capable of contacting or interacting with lipids or lipophilic moieties of a similar nature.

Lipophilic moiety: Moiety attached to or in contact with either i) any suitable functional group of one or more compounds that is essentially non-polar, or ii) moiety forming an essentially non-polar domain within the complexes according to the present invention. Preferred lipophilic moieties are a naturally-occurring, synthetic or semi-synthetic (modified natural) compound which is generally amphipathic. Lipids typically comprise a hydrophilic component and a hydrophobic component. The phrase semi-synthetic (modified natural) denotes a natural compound that has been chemically modified in some fashion. Lipids are also referred to herein as “stabilizing materials” or “stabilizing compounds.”

Liposome: A generally spherical or spheroidal cluster or aggregate of amphipathic compounds, including lipophilic moieties, typically in the form of one or more concentric layers, for example, monolayers, bilayers or multi-layers. They may also be referred to herein as lipid vesicles. The liposomes may be formulated, for example, from ionic lipids and/or non-ionic lipids. Liposomes formulated from non-ionic lipids may be referred to as niosomes. Liposomes formulated, at least in part, from cationic lipids or anionic lipids may be referred to as cochleates.

Patient: Any member, individual, or “animal body” of the subphylum chordata, including, without limitation, mammals such as cattle, sheep, pigs, goats, horses, and humans, preferably humans; domestic animals such as dogs and cats; and birds, including domestic, wild and game birds such as cocks and hens including chickens, turkeys and other gallinaceous birds. The term also covers fish, and it does not denote any particular age. Thus, both adult and newborn animals are intended to be covered.

Phospholipid; Any moiety consisting of a glycerol backbone, a hydrophobic part comprising a phosphate group, and a lipophilic part in the form of two fatty acids.

Polynucleotide: A molecule comprising at least two nucleic acids. The nucleic acids may be naturally occurring, or locked nucleic acids (LNA), or peptide nucleic acids (PNA). Polynucleotide as used herein generally pertain to

i) a polynucleotide comprising a predetermined coding sequence, or

ii) a polynucleotide encoding a predetermined amino acid sequence, or

iii) a polynucleotide encoding a fragment of a polypeptide encoded by polynucleotides (i) or (ii), wherein said fragment has at least one predetermined activity as specified herein; and

iv) a polynucleotide the complementary strand of which hybridizes under stringent conditions with a polynucleotide as defined in any one of (i), (ii) and (iii), and encodes a polypeptide, or a fragment thereof, having at least one predetermined activity as specified herein; and

v) a polynucleotide comprising a nucleotide sequence which is degenerate to the nucleotide sequence of polynucleotides (iii) or (iv);

or the complementary strand of such a polynucleotide.

Polypeptide: A molecule comprising at least two amino acids. The amino acids may be natural or synthetic.

Positively charged moiety: Any moiety comprising a positive electrical charge capable of attracting a negative electrical charged moiety. Positively charged moieties are typically found in cationic species, one of which is a species comprising at least one positively charged moiety at pH=7.0.

Quil A: Quillajabark Araloside A, a crude saponin mixture containing QA-7, QA-17, QA-18, and QA-21 as well as other saponins.

Region of a patient: A particular area or portion of the patient and in some instances to regions throughout the entire patient. Exemplary of such regions are the pulmonary region, the gastrointestinal region, the cardiovascular region (including, myocardial tissue), the renal region as well as other bodily regions, tissues, lymphocytes, receptors, organs and the like, including the vasculature and circulatory system, and as well as diseased tissue, including cancerous tissue. “Region of a patient” includes, for example, regions to be treated with a bioactive agent, regions to be targeted for the delivery of a bioactive agent, and regions to be imaged with diagnostic imaging. The “region of a patient” is preferably internal, although, if desired, it may be external. The phrase “vasculature” denotes blood vessels (including arteries, veins and the like). The phrase “gastrointestinal region” includes the region defined by the esophagus, stomach, small and large intestines, and rectum. The phrase “renal region” denotes the region defined by the kidney and the vasculature that leads directly to and from the kidney, and includes the abdominal aorta.

Receptor: A molecular structure within a cell or on the surface of a cell which is generally characterized by the selective binding of a specific substance. Exemplary receptors include cell-surface receptors for peptide hormones, neurotransmitters, antigens, complement fragments, immunoglobulins and cytoplasmic receptors.

Saccharide: Sugar part of saponins according to the present invention

Saponin: Any compound comprising a saccharide part linked by means of a glycosidic bond to one of i) a triterpene aglycone part, ii) a steroid aglycone part, and iii) a steroid alkaloid aglycone part. Saponin as used herein denotes either a substantially purified saponin, or one or more saponins comprised in a crude composition or a composition obtained by predetermined purification means. Saponin shall also denote any biologically active fragment of any saponin. The saponins pertaining to the present invention may be naturally occurring or synthetic, or they may be made by chemical synthesis, or enzymatic synthesis involving one or more enzyme catalysed steps, either in vitro or in vivo. First saponins are generally incapable of forming an association with a genetic determinant as defined herein, whereas such an association is formed between second saponins and the genetic determinant.

Second saponin: Second saponins may be either anionic, neutral, or cationic. Second saponins are capable of forming an electrostatic interaction and/or a hydrophobic interaction with a bioactive agent, including a genetic determinant. The term cationic saponin shall denote a saponin carrying a net positive charge at pH 7.0. Second saponins are preferably cationic saponins. Alternatively, second saponins preferably comprises at least one moiety carrying a positive charge at pH 7.0 regardless that the overall net charge is not positive. Accordingly, second saponins preferably comprise at least one positively charged moiety or reactive group at pH=7.0, and this positively charged moiety or reactive group is according to one preferred embodiment of the invention capable of contacting a bioactive group, including a genetic determinant, by means of an electrostatic interaction. In another embodiment, a second saponin preferably comprises an uncharged moiety or non-polar reactive group capable of contacting a bioactive group, including a genetic determinant, by means of a hydrophobic interaction. One group of preferred second saponins are saponins comprising as the aglycone part a synthetic or naturally occurring, cationic quillaic acid, or a quillaic acid comprising at least one positively charged group at pH=7.0.

Sterol: Any sterol, including any derivative thereof, comprising the characteristic skeleton structure of a steroid as depicted herein below in the form of gonane. All steroids are related to a characteristic molecular structure composed of 17 carbon atoms arranged in four rings conventionally denoted by the letters A, B, C, and D and bonded to 28 hydrogen atoms.

This skeleton structure (1) is named gonane and often referred to as the steroid nucleus. This skeleton structure may be modified in a practically unlimited number of ways by removal, replacement, or addition of a few atoms, moieties or reactive groups at a time. The skilled artisan will know how to isolate steroids from plants and animals, and optionally prepare derivatives by chemical and/or enzymatic treatment of natural steroids. The skilled artisan will also know how to synthesize synthetic steroids from simpler compounds, or natural precursors or parts thereof, and optionally prepare derivatives of such compounds by chemical and/or enzymatic treatment of steroids thus obtained. Accordingly, one preferred steroid compound according to the invention is a sterol and any cationic derivative thereof, in particular cationic derivatives obtained by linking a cationic moiety, or a cationic reactive group, to e.g. an OH-group of the sterol including an OH-group located at position 3 of the steroid skeleton, including the OH-group of cholesterol located at position 3 (C:3, or 3-OH). Consequently, preferred sterols according to the present invention are cholesterol (CAS (Chemical Abstract) accession no. 57-88-5) and any cationic derivative thereof, in particular cationic derivatives obtained by linking a cationic moiety, or a cationic reactive group, to the OH-group located at position 3 of the steroid skeleton (C3, or 3-OH). Preferred examples of such compounds are illustrated in FIG. 5. First sterols are generally incapable of forming an association with a genetic determinant as defined herein, whereas such an association is formed between second sterols and the genetic determinant.

Second sterol: Second sterols may be either anionic, neutral, or cationic. Second sterols are capable of forming an electrostatic interaction and/or a hydrophobic interaction with a bioactive agent, including a genetic determinant. The term cationic sterol shall denote a sterol carrying a net positive charge at pH 7.0. Second sterols are preferably cationic sterols. Alternatively, second sterols preferably comprises at least one moiety carrying a positive charge at pH 7.0 regardless that the overall net charge is not positive. Accordingly, second sterols preferably comprise at least one positively charged moiety or reactive group at pH=7.0, and this positively charged moiety or reactive group is according to one preferred embodiment of the invention capable of contacting a bioactive group, including a genetic determinant, by means of an electrostatic interaction. In another embodiment, the second sterol preferably comprises an uncharged moiety or non-polar reactive group, and this uncharged moiety or non-polar reactive group is according to another preferred embodiment of the invention capable of contacting a bioactive group, including a genetic determinant, by means of a hydrophobic interaction.

Steroid alkaloid glycoside: Saponin comprising a saccharide part linked by means of a glycosidic bond to a steroid alkaloid aglycone part.

Steroid glycoside: Saponin comprising a saccharide part linked by means of a glycosidic bond to a steroid aglycone part.

Stringent conditions: Stringent conditions as used herein shall denote stringency as normally applied in connection with Southern blotting and hybridization as described e.g. by Southern E. M., 1975, J. Mol. Biol. 98; 503-517. For such purposes it is routine practise to include steps of prehybridization and hybridization. Such steps are normally performed using solutions containing 6×SSPE, 5% Denhardt\'s, 0.5% SDS 50% formamide, 100 μg/ml denaturated salmon testis DNA (incubation for 18 hrs at 42° C.), followed by washings with 2×SSC and 0.5% SDS (at room temperature and at 37° C.), and a washing with 0.1×SSC and 0.5% SDS (incubation at 68° C. for 30 min), as described by Sambrook et al., 1989, in “Molecular Cloning/A Laboratory Manual”, Cold Spring Harbor), which is incorporated herein by reference.

Substantially pure saponin: Saponin substantially free from compounds normally associated with the saponin in its natural state, wherein the saponin exhibits a constant and reproducible chromatographic response and/or elution profile and/or biologic activity. The term “substantially pure” as used herein is not meant to exclude artificial or synthetic mixtures of the saponin with other compounds. Preferably, the substantially pure saponin is purified to one or more of the following standards: i) Appearing as only one major carbohydrate staining band on silica gel TLC (EM Science HPTLC Si60) in a solvent system of 40 mm acetic acid in chloroform/methanol/water (60/45/10, v/v/v), ii) Appearing as only one major carbohydrate staining band on reverse phase TLC (EM Science Silica Gel RP-8) in a solvent system of methanol/water (70/30, v/v), 3) Appearing as only one major peak upon reverse-phase HPLC on Vydac C4 (5 μm particle size, 330 Ångstrøm pore, 4.6 mm ID×25 cm L) in 40 mM acetic acid in methanol/water (58/42, v/v).

Targeting ligand: Any material or substance which, when comprised in a complex according to the invention, is capable of promote targeting of tissues and/or receptors in vivo or in vitro with the complexes of the present invention, or compositions such as e.g. biodegradable microsphere or liposomes comprising such complexes. In the latter case the compositions comprise the targeting ligand independently of whether or not the complexes also comprise the targeting ligand. The targeting ligand may be synthetic, semi-synthetic, or naturally-occurring. Materials or substances which may serve as targeting ligands include, for example, proteins, including antibodies, antibody fragments, hormones, hormone analogues, glycoproteins and lectins, peptides, polypeptides, amino acids, sugars, saccharides, including monosaccharides and polysaccharides, carbohydrates, vitamins, steroids, steroid analogs, hormones, cofactors, and genetic determinants, including nucleosides, nucleotides, nucleotide acid constructs, polynucleotides, optionally constructs or polynucleotides comprising derivatised nucleotides such as PNA (peptide nucleic acid) and/or LNA (locked nucleic acid). A “precursor” to a targeting ligand refers to any material or substance capable of being converted to a targeting ligand. Such conversion may involve, for example, anchoring a precursor to a targeting ligand. Exemplary targeting precursor moieties include maleimide groups, disulfide groups, such as ortho-pyridyl disulfide, vinylsulfone groups, azide groups, and α-iodo acetyl groups.

Therapeutic agent: The terms “pharmaceutical agent” or “drug” or “medicament” refers to any therapeutic or prophylactic agent which may be used in the treatment (including the prevention, diagnosis, alleviation, or cure) of a malady, affliction, condition, disease or injury in a patient. Therapeutically useful genetic determinants, peptides, polypeptides and polynucleotides may be included within the meaning of the term pharmaceutical or drug. As defined herein, a “therapeutic agent,” “pharmaceutical agent” or “drug” or “medicament” is a type of bioactive agent.

Tissue: Specialized cells which may perform a particular function. The term “tissue” may refer to an individual cell or a plurality or aggregate of cells, for example, membranes or organs. The term “tissue” also includes reference to an abnormal cell or a plurality of abnormal cells. Exemplary tissues include pulmonary tissue, myocardial tissue, including myocardial cells and cardiomyocytes, membranous tissues, including endothelium and epithelium, laminae, blood, connective tissue, including interstitial tissue, and tumors. “Cell” refers to any one of the minute protoplasmic masses which make up organized tissue, comprising a mass of protoplasm surrounded by a membrane, including nucleated and unnucleated cells and organelles.

Treatment: Method involving therapy including surgery of a clinical condition in an individual including a human or animal body. The therapy may be profylactic, ameliating or curative.

Triterpene glycoside: Saponin comprising a saccharide part linked by means of a glycosidic bond to a triterpene aglycone part.

Zeta potential: The parameter of zeta potential is a measure of the magnitude of the repulsion or attraction between particles. Its measurement relates to some extent to the overall charge of particles but also to the stability of particles in dispersion. The surface charge of particles in polar liquids dose not directly correlate to the electrical potential at the surface of the particle but to the potential that in the close vicinity of the particle.

DETAILED DESCRIPTION

The present invention is related to complexes which are capable of binding bioactive agents such as e.g. genetic determinants such as for example polynucleotides, therapeutic agents, e.g. polypeptides, diagnostic agents and imaging agents. When attached to the complexes according to the invention, the bioactive agents are capable of being taken up by a biological cell including a human or animal cell. The complexes according to the invention are thus useful as a means for transfecting a biological cell with e.g. a polynucleotide either in vivo or in vitro.

In other preferred embodiments there are provided methods of delivering bioactive agents to a patient and/or treating conditions in a patient comprising administering to the patient a complex according to the invention, or a composition comprising such a complex.

The present invention describes methods of delivering bioactive agents to a patient and/or treating conditions in a patient comprising administering to the patient a complex according to the invention, or a composition comprising such a complex.

The present invention also describes methods of diagnosing the presence of diseased tissue in a patient comprising administering to the patient a complex according to the invention, or a composition comprising such a complex.

The present invention also describes methods of providing an image of an internal region of a patient comprising administering to the patient a complex according to the invention, or a composition comprising such a complex.

FIG. 1 illustrates the carbon skeleton of the three main classes of saponins according to the invention. Depending on the type of genin present, the saponins according to the invention can be divided into three major classes: i) triterpene glycosides, ii) steroid glycosides, and iii) steroid alkaloid glycosides.

FIG. 2 illustrates the carbon skeleton of various pentacyclic triterpenes divided into three main classes, depending on whether they have a β-amyrin, α-amyrin or lupeol skeleton. In addition thereto, several minor classes also exist. The aglycone skeleton of the following triterpene glycosides are illustrated in the figure: Oleane (β-Amyrin), Ursane (α-Amyrin), Lupane, Taraxastane, Taraxerane, Friedelane, Glutinane, and Hopane.

FIG. 3 illustrates the olean-12-en skeleton of triterpene glycosides. A number of such aglycone variants pertaining to the present invention are listed in Table 1 herein.

FIG. 4 Illustrates other representative variants of triterpene aglycones according to the present invention and listed in Table 2 herein. The aglycones of FIG. 4 are representative of aglycones which do not have an olean-12-en skeleton. Examples are listed in Table 2 herein.

FIG. 5 illustrates preferred second sterols according to the invention and capable of being incorporated into the complexes according to the present invention. The listed compounds are ChoTB and ChoSC as described by Leventis, R. et. al. (1989) Biochim Biophys Acta 1023, 124; DC-Chol and TC-Chol as described by Avanti Lipids and further characterised herein; Lipid 67 (also known as GL-67), as described by Lee, E. R. et al. (1996) Human Gene Therapy 7, 1701; BGTC is an acronym for 3-beta[N′, N′-diguanidioethyl-aminoethane)carbamoyl]cholesterol; and BGSC is an acronym for 3-beta[4N-(1N,8N-diguanidino spermidine)-carbamoyl]cholesterol.

FIG. 6 illustrates preferred lipophilic moieties according to the invention and capable of being incorporated into the complexes according to the present invention. The listed compounds are DOTIM, or 1-[2-(9(Z)-octadecenoyloxy)ethyl]-2-(8(Z)-heptadecenyl)-3-(2-hydroxy-ethyl)-imidazoliniumchloride, DODAC is an acronym for dioleoyidimethylammonium chloride, and GAP-DLRIE is an acronym for (+/−)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminium bromide.

FIG. 7 illustrates yet further preferred lipophilic moieties capable of being incorporated into the complexes according to the present invention. The listed compounds are DOTMA, or N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride, DMRIE, or N,N-dimethyl-1,2-dimyristoyloxy-3-aminopropane, DOSPA, or 2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanammonium trifluoroacetate, and DOGS, or Transfectam®, as described by Behr, J. P. et al. (1989) Proc, Natl. Acad. Sci. USA 86,6982, U.S. Pat. No. 5,171,678.

FIG. 8 Illustrates even further preferred lipophilic moieties capable of being incorporated into the complexes according to the present invention. The listed compounds are DOTAP, or (N[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium-ethylsulfate; Tfx-50, or N,N,N′,N′-tetramethyl-N,N-bis(2-hydroxyethyl)-2,3-dioleoyloxy-1,4-butaned-iammoniumiodide; DDAB, or dimethyl-dioctadecylammonuim-bromide; and TM-TPS, or tetrapalmitylspermine,

FIG. 9 illustrates the influence of organic solvent on the formation of ISCOM-matrix as evaluated by visual inspection by electron microscopy (EM). The primary criterion was the shape and uniformity of the structures, secondary the number of ISCOM particles. The formation of ISCOM-matrix was unaffected by the presence of DMSO up to a concentration of 20% v/v as shown in panel A. Above this limit the yield was affected. With 25% DMSO intact structures could be observed but the number of ISCOMs were reduced. The same pattern was observed for DMF, as described in detail in Example 1 herein. Panel B in FIG. 9 demonstrates that EtOH was compatible with the process at concentrations up to approx. 15%. Some diverging structures appeared among intact structures prepared at this concentration.

FIG. 10 illustrates that undisrupted ISCOM complexes according to the present invention protect embedded peptides from hydrolysis during the harsh conditions of the acidic hydrolysis during an amino acid analysis. This result emphasizes the high stability of the ISCOM structure and the difference in chemical environment within ISCOMs.

FIG. 11 illustrates measurement of zeta potentials for ISCOMs containing no DC-cholesterol performed as described in example 4. Similar to the experiments described in FIG. 12, the Zeta-potential was measured five times. The average Zeta-potential was found to be approx. −50 mV (see table below), which is significantly different from the ISCOMs containing DC-cholesterol (DC-cholesterol to cholesterol ratio 1:1 or 1:2), as demonstrated in FIG. 12.

Recorded mean Conductivity value (mV) Width (mV) (mS/cm) Title 16 −51.6 6.3 0.300 Std. ISCOM 17 −45.0 6.2 0.362 Std. ISCOM

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