NL9301690A - Compounds with adjuvant activity. - Google Patents

Abstract

The invention relates to saponin-type compounds with adjuvant activity obtained by the separation into fractions of an extract of relatively young plant cell material, originating for example from Quillaja species, Aeculus species or Gypsophilia species. The plant cell material is formed by the bark of trees not more than 15 years old, or cambium cells, or cells from a suspension cell culture or a tissue culture. The invention also relates to a method for preparing essentially pure saponin-type compounds with adjuvant activity, involving the subjection of a crude extract of relatively young plant cell material to a preparative HPLC treatment in order to obtain at least one purified fraction; and various products which can be manufactured on the basis of the compounds.

Description

LINKS THE ADJUVANS ACTIVITY

The present invention relates to compounds with adjuvant activity, in particular saponins. The invention relates to compounds in very pure form, hydrolysed derivatives of these pure compounds and modified derivatives thereof. The invention further relates to methods of making the compounds, the use of the compounds in ISCOM matrices, ISCOMS, adjuvants and vaccines and the ISCOM matrices, ISCOMS, adjuvants and vaccines containing the compounds, as such, and a method for controlling the immune response as well as the targeted delivery of drugs. In addition, the compounds can be used for the careful and selective hemolysis of erythrocytes.

Saponins are substances that occur in plants of, for example, the genera Saponaria, Gvpsophilia and in particular Quillaia. Although the chemical structure of these saponins is not fully elucidated, they in any case contain gypsogenin or the gypsoginic acid or Ouillaia acid derived therefrom. all triterpenoids, and a number of sugar chains. In addition, in many cases there is a fatty acid residue, which is linked to a sugar residue on the triterpenoid structure.

Saponins are known to exhibit adjuvant activity. They are therefore regularly used as adjuvants in vaccines against, for example, foot-and-mouth disease. It has been found that the adjuvant activity is linked to certain components from the crude extract of plants, especially those of the genus Quillaia.

It has previously been shown that a component obtained after purifying a dialyzed extract from the bark of Quillaia saponaria Molina on an ion exchange column of type DE-52, followed by Sepha-dex G50 gel filtration, yields a fraction, which QUIL A is called. This QUIL A shows strong adjuvant activity (Dalsgaard, K., Arch. Ges. Virusforsch. 44, 243-254 (1974)). However, QUIL A itself is still not a pure substance, as has been shown after separation on reverse phase HPLC (RP-HPLC). The QUIL A appears to consist of a large number of components. Also, the use of a dialyzed, methanol-soluble extract does not give a better result and still appears to be very heterogeneous even when fractions thereof are further purified (WO 88/09336).

The drawback of such an impure substance is that both the activity and the side effects, such as irritation, allergy and the like, of the substance cannot be predicted and controlled. Each QUIL A fraction from each extract will have a different composition and must be tested for activity before use, for example.

The need therefore exists to obtain the components from the QUIL A or from other saponin-containing extracts or fractions thereof, in a very pure form, in order to better control the activity and the like. This is particularly important when one wishes to use the saponins as adjuvants in human vaccines. Strict purity requirements are imposed on such adjuvants.

It has now been found that compounds isolated from fresh plant material, consisting of mainly living cells, can be obtained in very pure form. The invention therefore provides compounds obtained by fractionally separating an extract of fresh plant material consisting of mainly living cells from saponin-containing plants.

The fact that the compounds according to the invention can be obtained very purely can probably be explained by the fact that in fresh plant material consisting of mainly living cells no oxidation or degradation of the compounds present therein has yet occurred. Until now it has been assumed that only using the bark of 50 to 100 year old trees could yield sufficient saponins. The use of young material was therefore not considered. However, only very heterogeneous mixtures of saponins can be obtained from the bark of old trees. In addition, in current practice, the bark, after it has been removed from the trees, is exposed for a long time to the weather conditions, as it turns out, the active compounds can be broken down or oxidized. An additional drawback of using the bark of old trees is that there is a risk of a shortage of old trees (Hoffman, J., A.E., Flores Silvestre de Chile, p. 102, Ediciones Fundacion Claudio Goy (1991)).

In this specification, the term "saponins" refers to those compounds which can be obtained directly from a plant extract and do not undergo subsequent hydrolysis or modification treatment. "Saponin-like compounds" are compounds which do have such hydrolysis or modification. The general term "compounds" includes both saponins and saponin-like compounds.

When the text refers to "fresh plant material" consisting of mainly living cells, the material is isolated from parts of a plant that are undifferentiated or recently differentiated, such as the cambium, young wood and phloem, as well as the roots. By "fresh" it is further meant that extraction is carried out substantially immediately after harvesting, without exposing the material to the weather conditions, so that oxidative and degradation-stimulating conditions can take place as little as possible.

The term "ISCOM matrix" stands for a matrix for immunostimulatory complexes as described in WO 90 / 031-84.

By "ISCOMS" is meant an ISCOM matrix having at least one antigen included therein, as described in U.S. Patent 4,578,269.

The sources from which the saponin-like compounds of the present invention can be obtained are very diverse. However, an important condition is that fresh plant material consisting of mainly living cells is used. In addition, it is important that the material is processed immediately after harvesting.

Suitable sources are, for example, the trunks of trees up to 15 years old of the species Ouillaia saponaria and the roots of such trees. In principle, but less desirable because of the scarcity of older trees, older trees could also use the layer of living cells, consisting of growing wood, cambium and phloem, and the roots. Furthermore, tissue cultures and suspension cell cultures, in particular cells originating from the cambium, are also very suitable sources, as described in Dutch patent application 93.01404, the contents of which are incorporated herein by reference, as well as root cultures and the like.

After separation on an RP-HPLC column, two different compounds are found, which are called QUADRI-1 and QUADRI-2. Analytical RP-HPLC shows that in this way very pure substances of 99.99% pure material can be obtained.

The invention further relates to hydrolysis products derived from the saponins. It has been found that by selective hydrolysis of the pure saponins, the adjuvant activity can be varied. Different compounds were found by different methods of alkaline hydrolysis. Based on the hydrophobicity of the products and based on the structure proposed by Higuschi et al. (In Phytochemistry 26, 229-235 (1987)) and shown in Figure 12, the acyl group with the arabino is first - and any other sugar residue split off to obtain product A. Starting from QUADRI-1 and QUADRI-2, respectively, these compounds are characterized by their relatively strong hydrophilicity characterized by a retention time of approximately 7,968 minutes (in Figure 8; 8,645 in Figure 11) , respectively, about 9.6 minutes (in Figure 9; 10.36 in Figure 11).

These compounds are further characterized by their sugar content, as shown in Table 2. Subsequently, after more intensive hydrolysis, the fucose with attached sugars is also split off, resulting in compounds QUADRI-1B and QUADRI-2B, which appear to be identical and at about 13 , 1 (figure 9 and figure 11) come from the column (see also table 2).

The compounds according to the invention, and in particular the hydrolysed derivatives thereof, can also serve as the basis for modified compounds. For example, it is possible to couple a peptide to the acid group in position 23 of product B or the fucose of product A, respectively. The peptide-modified compound, itself or in combination with other normal saponins or saponin-like compounds, may be used as an adjuvant and also in the formation of ISCOMS.

Another possible application of the compound according to the invention is the site-directed delivery of drugs. The hydrolysed compounds in particular could be useful for this.

The saponins of the present invention can be prepared by a method known per se. Such a method has been described, for example, by Dalsgaard (supra). Separation of the product obtained by this method (QUIL A) into fractions can take place by means of preparative HPLC such as, for example, RP-HPLC. However, any separation of an extract into its separate components can be used in the preparation of the compounds of the invention.

The compounds of the invention exhibit to a greater or lesser extent adjuvant activity. They can be used alone or in combination in, for example, vaccines.

It is possible to produce ISCOM matrix from compounds of the invention or combinations thereof together with, for example, cholesterol and phosphatidylcholine. Including one or more antigens in the matrix provides ISCOMS.

The present invention is illustrated in this specification with reference to saponins and saponin-like compounds from Ouillaia saponaria. but is not limited thereto.

A number of ISCOMS have been shown to induce a hitherto unique MHC class 1 response in addition to the MHC class 2 response common to normal adjuvants leading to increased T helper cell concentration. Furthermore, it has been shown that the T-helper cells induced are mainly of the type TH-1 and thus provide protection, and to a lesser extent or not at all of the type TH-2, which lead to a hypersensitivity reaction.

QUADRI-1 and QUADRI-2, by themselves and as part of ISCOMS, have such an adjuvant function and thus can also induce cytolytic T lymphocytes (CTLs). The hydrolysed compounds also exhibit these properties, but not to the same extent. they may help to dissolve the antigens and appear to induce a lower MHC class 2 immune response.

Since the studies leading to the invention provide a much clearer understanding of how the various components, which are part of the crude extract, arise, in particular through partial hydrolysis of QUADRI-1 and QUADRI-2, much better understanding where the differences in adjuvant activity come from. QUADRI-1 and QUADRI-2 are surrounded by much more sugar residues than the derivatives QUADRI-1A, -1B, -1D and QUADRI-2A, -2B, -2D generated by selective hydrolysis, which will affect hydrophobicity, among other things and the three-dimensional structure. This allows the presentation of antigens to be selectively influenced.

Saponins can also be used for the hemolysis of erythrocytes. A well-known method that uses hemolysis is the counting of white blood cells using a Coulter counter. The accuracy of the count is normally greatly affected by the presence of the red blood cells. Lysing the red blood cells removes the disturbing background. Saponins can be used to lyse erythrocytes. It has been found that existing saponin preparations are not reliable enough to standardize this method of white blood cell counting.

There is a demand from both industry and legislation for a pure preparation with which this white blood cell determination can be carried out reproducibly. The saponins, and optionally saponin-like compounds, according to the invention meet this requirement. In addition, it appears that different types of erythrocytes, such as, for example, normal erythrocytes or sickle cells, react differently to the saponin preparations according to the invention. That is, the hemolysis of the different types of erythrocytes requires different concentrations of the saponins. The specificity of the pure saponins, respectively the mixture of QUADRI-1 and QUADRI-2 before RP-HPLC thereof, gives entirely new applications in the field of hemolysis.

Another field of application of the compounds of the invention is the site-directed delivery of drugs. An amount of medicine is included in an ISCOM matrix. The ISCOMS can be transported to the desired location in the body by means of molecules with a site-oriented function, such as, for example, antibodies, which are included in the matrix.

The invention will be further elucidated on the basis of a number of examples, which, however, are not intended to limit the invention in any way.

EXAMPLE 1

Purification and analysis of saponins according to the invention.

QUIL A is extracted from plant cell material, freshly extracted from approximately 15-year-old Quillaia trees, according to the method of Dalsgaard (supra). The Dalsgaard method essentially consists of a purification of a dialyzed extract on an ion exchanger column of the type DE-52, followed by Sephadex G50 gel filtration. It is also possible to use ultrafiltration instead of gel filtration.

The QUIL A thus obtained is subjected to an RP-HPLC (VYDAC®, C4 column, eluted with 30% -45% acetonitrile in a 0.15% aqueous TFA solution). The HPLC elution pattern shows two major peaks, which are called QUADRI-1 AND QUADRI-2, respectively (see Figure 1).

The same procedure is performed on plant cell material obtained by tissue culture or suspension cell culture. Figure 2 shows the HPLC elution pattern of QUIL A isolated from a suspension cell culture. In addition to the two peaks characteristic of QUADRI-1 and QUADRI-2, a number of peaks can also be distinguished, which are later shown to be deacylated versions of QUADRI-2.

For comparison, Figure 3 shows the HPLC cartridge of QUIL A from old bark. The arrows in Figure 3 indicate the positions of QUADRI-1 and QUADRI-2 and of deacylated QUADRI-2. It can be seen from this figure that the material from the old bark is much more heterogeneous than the material obtained from young plant cells according to the invention.

Figure 4 shows the RP-HPLC elution pattern of a compound disclosed in patent application WO 90/03184, designated "B3" therein. WO 90/03184 assumes that "B3" is a pure substance. Figure 4 shows that this is clearly not the case.

This figure illustrates that although it was previously assumed that pure components were used in the preparation of ISCOMS, it now appears that even these "pure" components have a very heterogeneous character. This again underlines the importance of a method for the recovery of saponin-like compounds which can be classified as "pure".

In Figure 15, the HPLC elution pattern of a component known as B4b is further shown for comparison. B4b is known to have a very high ISCOM-forming ability, but a low adjuvant activity (WO 90/03184). The HPLC pattern lacks the peaks QUADRI-1 and QUADRI-2 and precisely the deacylated QUADRI-1A and QUADRI-2A are present. This shows, among other things, that B4b is by no means a pure substance. Furthermore, the absence of QUADRI-1 and QUADRI-2 can now explain why B4b does give ISCOMS but does not have normal adjuvant activity for ISCOMS.

Figures 5 and 6 show RP-HPLC elution patterns for the compounds QUADRI-1 and QUADRI-2, respectively. The figures show that both fractions are more than 99.99% pure.

Tables 1 and 2 show the composition of the sugars present, which was determined according to the method of Kamerling, J.P. et al., Carbohydrates 175-263, (1989), A.M. Lawson (ed.), Clin. Biochem. 1, Mass Spectrometry, W. de Gruyter, New York. For QUADRI-1, 0.4 mg was used with 250 nmol mannitol as an Internal Standard (IS). The results are shown in the top part of Table 1. Of QUADRI-2, 0.3 mg was used with 250 nmol IS. The results are shown in the lower part of Table 2. 250 μΐ solution of 20 nmol IS of QUADRI-1A was used. The results are shown in the top part of Table 2. 250 μΐ solution of 20 nmol IS of QUADRI-1B was also used. The results are shown in the lower part of Table 2.

Table 1 shows that QUADRI-1 contains 415.9 μg of sugars per mg. This corresponds to 41.6% carbohydrates. QUADRI-2 is less glycosylated and contains 359.6 μg per mg (35.9% carbohydrates).

* No reference is included for this sugar. The remaining peak is believed to be apiosis.

** The amount of this sugar is set at 1 in the molecular ratio.

TABLE 2

The meaning of * and ** is as in table 1.

Table 2 shows that the void volume after strong hydrolysis contains as good as pure the sugar portion, which has been cleaved from QUADRI-1A in position 23 of the triterpeno ideation.

The UV absorption spectra of QUADRI-1 and QUADRI-2 (see Figure 7 for the QUADRI-1 spectrum) show a clear absorption peak at 292 nm. The patent applications WO 88/09336, WO 93/05789 and WO 92/06710 also show UV spectra of mixtures of saponin-like compounds extracted from old bark. However, said spectra only peak at about 210 nm. It is therefore expected that the compounds according to the invention deviate in a certain respect from the compounds already described in said patent applications. This presumably points to a clearly more unsaturated character of the trillapenoid of Ouillaia acid. isolated from fresh material. The saturation of the material from the old bark is probably caused by oxidation of the Ouillaia acid during the storage of the bark.

EXAMPLE 2

Toxicity of QUADRI-1 and QUADRI-2

Groups of 6 mice each received 50, 100, 200, 400 or 800 βg QUADRI-1 and QUADRI-2, respectively. Even the maximum dose of 800 μg QUADRI-2 per mouse of 20 g was found to have no lethal consequences for any of the 6 test animals. This means that the LD 50 of QUADRI-2 is greater than 40 mg / kg animal. In the case of QUADRI-1, an LD50 of about 7.5 mg / kg was found. QUIL A® from Superfos was used as reference material. The LD50 of this preparation was about 25 mg / kg. It should be noted, however, that the amount of effective substance in the case of QUADRI-1 and QUADRI-2 is much higher than in QUIL A®. QUIL A® contains only 5% of effective saponins. A dose of 1 mg / kg QUADRI-1 therefore corresponds to 20 mg / kg QUIL A. This shows that the toxicity of the pure QUADRI-1 and QUADRI-2 compounds is much lower than the toxicity of the commercially available QUIL A preparation .

EXAMPLE 3

Hydrolysis of QUADRI-1 and QUADRI-2

In order to prove the structure of QUADRI-1 and QUADRI-2, hydrolysis tests are performed with the pure substances QUADRI-1 and QUADRI-2. QUADRI-1 is dissolved in an alkaline NaOH solution with a pH of 12 at room temperature (so-called "mild hydrolysis"). After 30 minutes, the hydrolysis is stopped and the reaction mixture is analyzed on RP-HPLC (VYDAC, C4 column, eluted with a 0.15% aqueous trifluoroacetic acid solution, combined with an acetonitrile gradient of 30% to 45%). It appears that QUADRI-1 has been largely converted into a new compound, which is characterized by a retention time of approximately 8 minutes (see figure 8). This compound is the hydrolysis product QUADRI-1A. Then 30% acetonitrile is added to an alkaline solution (pH 12) of QUADRI-1, which solution is heated at 100 ° C for 16 hours (so-called "strong hydrolysis"). The elution pattern then shows two other peaks in addition to the "void volume" peak (see Figure 10), the first of which, with a retention time of approximately 8.7 minutes, corresponds to the peak created after the mild hydrolysis (compare Figure 8), and the second corresponds to QUADRI-1B.

Corresponding experiments are performed with QUADRI-2. The product resulting from QUADRI-2 after mild hydrolysis is characterized by a retention time of about 9.6 minutes, while after strong hydrolysis the product QUADRI-2B exhibits the same retention time as QUADRI-1B (results not shown). To compensate for the differences in the elution times, the strong alkaline hydrolysates of QUADRI-1 and QUADRI-2 are mixed and placed on a column. Figure 11 shows that only three peaks can be seen. These peaks are: QUADRI-1A (8,645 minutes), QUADRI-2A (10,363 minutes) and QUADRI-1B and -2B together (13,109 minutes). The results found are consistent with the fact that QUADRI-1 has one glucose more on a part that is not cleaved off after mild hydrolysis, but after strong hydrolysis. The residual products after strong hydrolysis are therefore identical, while the products differ from each other after mild hydrolysis.

A sugar analysis of the hydrolysis products of QUADRI-1 was carried out. The results are shown in Table 2 (above).

The fact that these compounds, also found in the QUIL A extracted from old bark, can be generated from two compounds QUADRI-1 and QUADRI-2 was previously unknown.

EXAMPLE 4 ISCOM formation

A solution of one or more of the compounds of the present invention was prepared by dissolving several mg of the compound in a corresponding number of ml of water (this is solution A).

Phosphatidylcholine was dissolved in water with 20% of the detergent Mega-10® (Sigma) to a concentration of 10 mg / ml. 10 mg of cholesterol was dissolved therein per ml by stirring at 50 ° C. This is solution B.

An antigen solution was obtained by dissolving a given number of mg of antigen in a corresponding number of ml of water. This is solution C.

20 μΐB per ml A is added to form an ISCOM matrix. ISCOMS are prepared from 1 ml A, 1 ml C and 20 μΐB. The mixtures are shaken on a tumbler for 3 to 4 hours. If necessary, the Mega-10® is removed by dialyzing against physiological saline (0.85% NaCl, 0.01 M phosphate buffer, pH 6.5-7.2).

Figure 13 is an electron microscopic photograph illustrating the ISCOM matrix-forming properties of QUADRI-1. Figure 14 shows the ISCOM matrices formed with one of the two hydrolysis products obtained after mild hydrolysis of QUADRI-i (the product which showed a value of 2.405 in the sedimentation experiments of Figure 19). The hydrolysis products appear to form smaller ISCOM matrices than the starting products.

The so-called Zeta potential is measured using the "Zetamaster" from Malvern Instruments Ltd., England. The Zeta potential represents the stability of a particle in a solution. Using the ISCOM matrices with compounds according to the invention in a physiological saline solution (0.85% NaCl, 0.01 M phosphate buffer pH 7.2), a value of -22 mV is obtained. This means that a very stable micelle (ISCOM) has been obtained.

Ultracentrifuge experiments are conducted in which the sedimentation rates of the various ISCOM matrices are observed in a sucrose gradient, in a well-known manner (Figures 17 and 18). QUADRI-1 and QUADRI-2 form homogeneous ISCOM matrices. The products which have been alkaline hydrolysed for only half an hour are not homogeneous (see Figures 8 and 10) and therefore show two peaks in their sedimentation rates (Figures 19 and 20). The absolute value of the various sedimentation rates indicates that ISCOMS can be made of very different sizes, which is another means of influencing the presentation of antigens in a controlled manner. This also offers the opportunity to interact selectively with various types of cells or cell compartments. It is noteworthy that the ISCOM matrices prepared with hydrolysed saponins also have the same high stability, as characterized by their Zeta potential of -22 mV.

The availability of highly pure saponins, and hydrolysed and / or modified derivatives thereof, makes it possible to control the immune response. Depending on the choice of connection, a broad spectrum of responses, for example, ranging from a fast, but possibly less broad and persistence, to slower but wider and longer lasting protection may be possible. The formation of antibodies and cytotoxic T lymphocytes, respectively, can also be influenced. The separate connections can also be used in combination with ISCOMS. In addition, the ISCOMS can be used on their own.

In summary, it can be stated that the hydrolysed and / or modified compounds of the invention, whether or not hydrolysed, can be used independently as adjuvants, or may be involved in the formation of the ISCOM matrix or ISCOMS. In addition, the compounds are suitable for use in hemolysis and can be used for the site-directed delivery of drugs. In all cases, the substances have the advantage of being identifiable as pure components and therefore likely to be registered as an acceptable adjuvant for use in, for example, human vaccines.

The high degree of purity will further lead to low toxicity and fewer side effects (such as allergy, irritation and the like). This is due in part to the fact that due to the greater purity, a lower concentration can be used, but also because impurities which can induce such reactions in the existing compositions are not present in the compositions of the present invention.

Claims (32)

1. Compounds with adjuvant activity obtained by separating into its components an extract of fresh plant material consisting of mainly living cells from saponin-producing plants. Compounds according to claim 1, characterized in that the plant cell material comes from plants of the genera Quillaia. Saoonaria or Gvosoohilia. Compounds according to claim 1 or 2, characterized in that the plant cell material comes from Ouilla-1 ... saponaria Molina. Gypsophilia paniculata. Gvpsophilia pacifica, Gvpsophilia arrostii. Gvpsophilia struthium or Saponaria officinalis. Compounds according to claim 1, 2 or 3, characterized in that the plant cell material consists of cambium cells or meristem cells. Compounds according to claim 1, 2 or 3, characterized in that the plant cell material consists of cells from a suspension cell culture. Compounds according to claim 1, 2 or 3, characterized in that the plant cell material is constituted by cells from a tissue culture. Compounds according to claim 1, 2 or 3, characterized in that the plant cell material originates from a root culture. 8. QUIL A, isolated from fresh plant material consisting of mainly living cells. 9. QUADRI-1. 10. QUADRI-2. Compounds according to any one of claims 1 to 10, characterized in that they are at least partially hydrolysed derivatives of the starting product. 12. QUADRI-1A. 13. QUADRI-1B. 14. QUADRI-2A. 15. QUADRI-2B. Compounds according to any one of claims 1-15, characterized in that they are modified derivatives of the starting product. A process for preparing substantially pure compounds with adjuvant activity according to any one of claims 1-10, comprising fractionally separating a crude extract of fresh plant material consisting of essentially living cells, which are derived from is from saponin producing plants to provide at least one purified component. A method for preparing substantially pure saponin-like compounds having adjuvant activity according to any one of claims 11-15, comprising fractionally separating a crude extract of fresh plant cell material consisting of substantially living cells for obtaining at least one purified component and then at least partially hydrolyzing the purified component (s). A method for preparing substantially pure saponin-like compounds with adjuvant activity according to claim 16, comprising fractionally separating a crude extract of fresh plant cell material consisting of substantially living cells to obtain at least one purified component, optionally hydrolyzing the purified component (s) and then at least partially modifying them. A method according to claim 19, characterized in that the modification consists of coupling an antigen to the compound. Method according to any one of claims 17-20, characterized in that the fraction separation is carried out by preparative HPLC. ISCOM matrix, comprising at least one lipid, at least one detergent and at least one compound according to any one of claims 1 to 16, or combinations thereof. ISCOMS, comprising the ISCOM matrix of claim 22 in combination with at least one antigen. An adjuvant comprising at least one of the compounds of any one of claims 1 to 16 and / or an ISCOM matrix of claim 22 and / or one or more ISCOMS of claim 23. Vaccine comprising an adjuvant according to claim 24 and at least one vaccine. Use of compounds according to any one of claims 1 to 16 in an ISCOM matrix. Use of compounds according to any one of claims 1-16 in ISCOMS. Use of compounds according to any one of claims 1 to 16 as an adjuvant. Use of vaccinations according to any one of claims 1-16 in a vaccine. A method for controlling the degree of enhancement and type of the immune response by choosing a compound according to any one of claims 1-16 depending on the desired response, optionally in combination with one or more ISCOM matrices according to claim 22 and / or one or more ISCOMS according to claim 23. A method for the hemolysis of, for example, red blood cells by adding at least one compound according to any one of claims 1-16 to the cells. A method for delivering a pharmaceutical agent to a particular location in the body by administering to a patient a pharmaceutical agent included in an ISCOM matrix according to claim 22, wherein optionally in and / or on the ISCOM matrix also a molecule directed to the particular location in the body is included.