COMPOSITION FOR THERAPEUTIC OR DIAGNOSTIC USE, PROCESS FOR ITS PREPARATION AND ITS USE

Abstract: The present invention concerns a composition comprising a carrier selected from the group consisting of lipoproteins, in particular reconstituted LDL (Low Density Lipoprotein), other microemulsion particles, liposomes and micelles, containing a therapeutically or diagnostically active, lipo- or amphiphilic agent, and associated with a suitable ligand recognizable by its specific (complementary) cell receptor, together with a lysosomotropic agent, for targeting of the active agent to a site of interest, such as cancerous tissue.

Description: Composition for therapeutic or diagnostic use, process for its preparation and its use FIELD OF INVENTION The present invention is based on the use of a carrier selected from the group consisting of lipoproteins, in particular reconstituted LDL (Low Density Lipoprotein) , other types of microemulsion particles, liposomes and micelles, and containing a therapeutically or diagnostical- ly active, lipo- or amphiphilic agent, and associated with a suitable ligand recognizable by its specific (complemen¬ tary) cell receptor, together with a lysosomotropic agent, for targeting of the active agent to the site of interest, such as cancerous tissue, and to optimize the effect thereof at the said site. BACKGROUND OF THE INVENTION Ligand specific receptors on the cell surface mediate both information on the environment as well as nutrients into the cell. Such ligands to be recognized include a range of molecular structures, e.g. growth factors (for example platelet derived growth factor, endothelial cell growth factor, tumor growth factor, nerve growth factor) , hormones (e.g. insulin, vasopressin etc.), extracellular matrix protein (e.g. collagen) and lipoproteins (e.g. low density lipoprotein) . In many cases the binding of the ligand to the cell surface receptor is followed by a clustering of the ligand-receptor complex and subsequent internalization via coated pits, ultimately locating the ligand in ly- sosomes. This pathway is utilized for example by viruses in their entry into cells. It has been known for a long time that people suffering from cancer exhibit a cholesterol level in the serum which is lower than normal. Cholesterol circulates in the blood in the form of a lipid-protein complex, primarily as the so-called Low Density Lipoprotein, LDL. LDL is a spherical macromolecule having a diameter of about 180 A. LDL is surrounded by a surface film consisting of phospholipids, cholesterol and sphingomyelin, an of protein, i.e. apolipoprotein B (apo B) . The interior of LDL is formed by cholesterol in esterified form (compounds of fatty acids and cholesterol) , triacylglycerol, retinol, as well as other hydrocarbon soluble compounds. Cells contain on their surfaces specific LDL-receptors which bind to the LDL-particle by specifically recognizing the protein component of the LDL-surface, i.e. apolipopro¬ tein B. The density of LDL-receptors is much higher on cancerous cells than on normal cells and cancer cells bind LDL more effectively. It is evident that the lower serum cholesterol level of cancer patients is due to enhanced uptake of LDL by the cancer cells. This is easy to ra¬ tionalize in terms of the increased requirement of com- ponents such as cholesterol for the growth of the tumor cells. The use of reconstituted LDL containing cytotoxic compounds which replace the natural apolar lipids in the particle core has been described. The lipids are removed from the core of LDL by first lyophilizing the lipoprotein in the presence of starch (Gustafson, A (1965) J. Lipid Res. 20, 254-264) . A slightly modified technique has been described by Krieger (Krieger, M. (1986) Methods Enzymol. 128, 608- 613) . LDL is reconstituted by lyophilizing in the presence of potato starch and extracting the neutral lipids with an organic solvent, such as heptane. The agent to be introdu¬ ced is dissolved in an organic solvent and incubated with the extracted LDL. The organic solvent is evaporated and the reconstituted LDL is dissolved in a buffered aqueous solution. According to an improvement to this method, described in the O-publication 86/07540, the starch is replaced by a saccharide or a sugar alcohol, and according to this publication, a reconstituted LDL is obtained having similar properties as natural LDL, in vitro and in vivo. However, when using e.g. reconstituted LDL as a carrier for e.g. drugs, in the form of an injectable aqueous solution, retro-endocytosis has been observed. Thus after an initial decrease of the drug level in the blood, indicating uptake of LDL by the cells, an increase in the level of the drug is observed, indicating expulsion of the drug from the cells. Thus it is difficult to achieve a significant accumulation of the drug in the target tissue, and conse- quently the action of the active agent cannot be fully taken advantage of. It is also prior known to use, for drug delivery, li¬ posomes wherein the active agent is incorporated a) in a core surrounded by a bilayer of a lipid substance, typical¬ ly a phospholipid (together with e.g. cholesterol and sphingomyelin) or b) if the desired therapeutic compound is amphiphilic (e.g. an anti-cancer phospholipid) , it is incorporated in the bilayer itself. For the said purpose also microemulsions, for example an emulsion of active agent, such as a lipophilic drug surrounded by a monolayer of phosphatidyl choline, are used. SUMMARY OF THE INVENTION The present invention relates to an improvement to the known methods by means of which it is possible to achieve an enrichment of the active agent at the target site. This is made possible by using, in combination, (a) a carrier selected from the group consisting of lipoproteins, other types of microemulsion particles, liposomes and micelles, and containing a therapeutically or a diagnostically effective amount of a lipo- or amphiphilic active agent, associated with at least one ligand which is complementary to and recognizable by specific receptor in the living cells, and (b) a lysosomotropic agent. The invention concerns, on the one hand, a composition for diagnostic or therapeutic use which contains the said combination, and on the other hand a method for the preparation of the said composition. Especially the invention concerns a composition and a method wherein the carrier is specifically reconstituted LDL, as well as its use on humans and non-human animals. DETAILED DESCRIPTION OF THE INVENTION The composition according to the invention thus contains at least two components, i.e. a carrier selected from the group of lipoproteins, other types of microemulsion parti¬ cles, liposomes and micelles, and which is associated to a suitable ligand. The carrier components are known as such, as is their manner of manufacture. Common for these carriers is that they can contain a therapeutically or diagnostically active agent. The carrier is associated with a ligand which is characte¬ rized in that it is complementary to and can be recognized by a specific cell receptor. The term "associated with" is intended to include also the possibility that the carrier incorporates the said ligand, such as in the case of LDL, where the ligand can be the apo B moiety included in the surface film of the LDL. The ligand is thus typically one of the substances mentioned earlier, such as a protein, e.g. growth factor, a hormone, apolipoprotein, viral protein, synthetic peptide etc. As stated, when the carrier is a reconstituted LDL, the ligand is the apo B-moiety, or a corresponding receptor recognizable synthetic peptide present on its surface. The ligand may also in all cases be a synthetic ligand made lipophilic or amphiphilic to allow for its incorporation with the carrier, into the lipid surface of the carrier particle, provided that the ligand carries the elements recognizable by the cell receptors. Such substances are also well known in the art. The second central component in the composition is the lysosomotropic agent which according to the invention is a lysosomotropic amphiphile, and is characterized as having both lysosomotropic and detergent activity. The lysosomot¬ ropic agents exert lysosomal blocking activity and are well known in the art. They may in addition be amphiphilic such as Triton WR-133 (American Roland Corporation, USA, "tyloxapol") , weak amines (e.g..pK 5 to 9) such as imidazo- les or morpholines containing longer hydrocarbon alkyl or acyl chains, e.g. N-dodecyl imidazole, or chloroquine, see e.g. Miller et al., J. Cell Biol. 97 (1983) 1841-1851; Wilson et al.. Cancer Research 49, (1989), 507-510; and deDuve et al., Bioche . Pharmacol. 23 (1974) 2495, which are all included here for reference. When used together with the carrier and associated ligand, the lysosomotropic amphiphile due to its hydrophobic nature readily accomoda- tes onto the carrier/particle surface. Lysosomotropic amines may in themselves have cytotoxic properties. According to the invention it has been observed that the presence of the lysosomotropic amphiphile does not interfe¬ re with the recognition of the ligand by the receptor, but the carrier with active agent is taken up from the extra- cellular space into the cells according to their contents of receptors and the activity of the endocytic pathway. However, once the carrier particles carrying the lysosomot¬ ropic agent enters the lysosomes, the processing of the particles is blocked. In other words, the cycling of the active agent containing particle back to the cell surface (retro-endocytosis) does not take place, and an accumu¬ lation of the active agent in the target site is obtained. The composition according to the invention is preferably an aqueous solution for parenteral use, such as for injection purposes (iv) , containing a therapeutically or diagnosti- cally effective amount of active agent containing carrier particles together with the lysosomotropic agent. The ratio (weight) of carrier particles to the lysosomotropic agent is suitably such that in each particle approximately 2 to 40 mole-% of its surface active compounds is constituted by the said lysosomotropic agents. The composition according to the invention is made by combining the active agent containing carrier with the lysosomotropic agent in a pharmaceutically suitable vehicle, together with possible pharmaceutically acceptable adjuvants. The vehicle is normally sterile water. The adjuvants may be any substances known per se and suitable for the purpose, the choice thereof being within the knowledge of a person skilled in the art. The dosage used naturally depends on the drug or diagnostic agent used, as well as the condition to be treated or diagnozed. The amount to be administered for any specific purpose can readily be determined by a person skilled in the art. As stated above, according to a preferred embodiment, the carrier is comprised of reconstituted LDL. Such reconstitu¬ ted LDL is made by a method comprising the steps of lyophilizing LDL in the presence of a protecting substan¬ ce, extracting the lyophilized LDL with an organic solvent, incubating a therapeutically or diagnostically effective lipo- or amphiphilic agent in an organic solvent with the extracted LDL, evaporating the solvent and dissolving the product in an aqueous buffered solution to remove any non- included active agent and separating the LDL e.g. by ultracentrifugation. The LDL to be reconstituted is usually isolated from human serum by differential ultracentrifugational flotation in the density range of 1.019 < d < 1.063 g/ml. Alternative techniques include, for instance, rate-zonal ultracentrifu- gation. The protecting substance may be any of the substances known for the purpose, such as starch, in particular potato starch, but also a sugar derivative as is disclosed in the WO-publication 86/07540. The solvent used for the extraction of the neutral lipids from the lyophilized LDL is suitably a non-polar solvent, such as heptane, hexane, pentane, petroleum ether, octane, or their mixtures. The solvent used for dissolving the active agent and for incubating with the lyophilized and extracted LDL, is also preferably a non-polar solvent such as the ones listed above. According to another mode of the invention, the carrier particle can be a liposome. The techniques for making liposomes and for including active agents, such as a drug therein, are very well known in the art. In this case the ligand can comprise a suitable substance recognizable by a specific cell surface receptor, such as those listed earlier, and which is incorporated in the liposome shell during the manufacture thereof. According to the invention also a microemulsion can be used, the preparation of which is known in the art (e.g. Walsh, M. T. et al. Methods in Enzymology, vol. 128 582 (1986) . The same ligands as those mentioned above may be used. The active agent is an agent for diagnostic or therapeutic purposes. It can be lipophilic or amphiphilic, although the border line between these two types is diffuse. As a diagnostic agent e.g. light sensitizers, such as hemato- porphyrins, radiosensitizers, such as boronated fatty acid esters, or x-ray contrast agents can be used. As a thera¬ peutic agent, preferably anti-cancer drugs are used, such as doxorybicin, daunomycin, l-hexadecyl-2-methyl-3-phospho- choline etc. The following examples illustrate the invention without limiting the same. Example LDL was isolated by differential ultracentrifugational flotation in the density range 1.1019 < d < 1.063 g/ml. Subsequently, KBr used for the adjustment of appropriate densities was removed by extensive dialysis against 0.3 mM EDTA, pH 7.0 at 4 °C. Thereafter the lipoprotein was concentrated by ultrafiltration using an A icon XM-300 membrane and the concentration of cholesterol was measured. Pure potato starch (60 mg from Sigma) was dissloved in the LDL-solution containing 16 μmoles of cholesterol in a total volume of 1.5 ml. The resulting solution was then frozen in liquid nitrogen and lyophilized. The core lipids of the lyophilized LDL were then extracted with 4 x 10 ml of ice- cold heptane. The core lipids were replaced with a cytoto- xic compound, namely cholesteryl ester of chlorambucil (15 mg dissolved in 1.5 ml of heptane) which contained a 3 H- cholesteryl moiety as a marker for the quantitation of tissue distribution of the cytotoxic compound. The solution was first incubated at -10 °C for 90 minutes, whereafter the solvent was removed under a gentle stream of nitrogen while keeping the sample on an ice-water bath. The dry residue was then dissolved in 3.5 ml of 10 mM Tricine buffer, pH 8.4, and incubated at + 4 °C for 48 hours. In order to separate the cholesteryl chlorambucil loaded LDL, the solution was centrifuged at 2000 rpm and the superna¬ tant collected. To the supernatant solution containing the cytostat-LDL particles, was then added 50 μL of Triton WR- 1339, and the mixture was incubated at + 4 °C overnight. In this manner an injectable preparation was obtained. This can be used as such, or if necessary, it can be irradiated by bath type sonication to speed up the dispersion into small particles. EXPERIMENTAL The preparation obtained in the Example above was used as such and injected i.v. into rats bearing Rous sarcoma inoculated in their peritoneum. The average weight of the developed tumors was 0.9 g. The animals were killed after 0.5, 1.0 and 3 hrs from the injection and the radioactivi¬ ties in different tissues were determined. In the following table, test animals designated A to E are compared to the values from a control rat (killed at 1 hr) without the implanted sarcoma. From the results it can be seen that the LDL-chlorambucil ester accumulates in the tumor, and that the degree of expulsion is low, whereas its level in the blood decreases rapidly to a low level. In the appended drawing. Fig. 1 illustrates similar tests and it shows the level of chlorambucil ester-LDL in the blood and in the tumor, respectively, when injected as described above but in the absence of a lysosomotropic agent (Triton WR 1339) . In Fig. 1 the abscissa indicates the time and the ordinate the amount (in promille) in blood and tumor respectively, calculated from that injected. Fig.2 shows in graphic form the mean values for the levels obtained above, when the same LDL has been injected in the presence of the lysosomotropic agent. From the Figures it is evident that in the absence of a lysosomotropic agent, after an initial transient decrease, the level in the blood increases rapidly, whereas the opposite is true for the level in the tumour. On the other hand, when using a preparation according to the invention, the level of reconstituted LDL in the blood decreases, and the level thereof in the tumour increases, indicating that retro-endocytosis is prevented by the incorporation of the lysosomotropic agent according to the invention.

Claims: Claims 1. Composition for therapeutical or diagnostical use comprising, in combination, (a) a carrier selected from the group consisting of lipoproteins, other types of microemul¬ sion particles, liposomes and micelles, and containing a therapeutically or a diagnostically effective amount of a lipo- or amphiphilic active agent, associated with at least one ligand which is complementary to and recognizable by a specific cell receptor, and (b) a lysosomotropic agent. 2. Composition according to claim 1, wherein the carrier is reconstituted Low Density Lipoprotein (LDL) , the ligand being an apolipoprotein B moiety on its shell surface. 3. Composition according to claim 1 or 2, wherein the lysosomotropic agent is Triton WR 1339 ethyl oleate. 4. Composition according to claim 1 or 2, wherein the therapeutically or diagnostically active agent is selected from the group consisting of light sensitizers, radiosensi- tizers, x-ray contrast agents and anti-cancer drugs. 5. Composition according to claim 4, wherein the anti- cancer drug is chlorambucil cholesteryl ester. 6. Composition for therapeutical use comprising, in combination, a reconstituted Low Density Lipoprotein (LDL) containing chlorambucil cholesteryl ester and Triton WR 1339 ethyl oleate. 7. Process for the preparation of a composition for therapeutic or diagnostic use comprising the steps of combining a carrier selected from the group consisting of lipoproteins, other types of microemulsion particles, liposomes and micelles, containing a therapeutically or a diagnostically effective amount of a lipo- or amphiphilic active agent, and associated with at least one ligand which is complementary to and recognizable by a specific cell receptor, with a lysosomotropic agent and a pharmaceutical¬ ly suitable vehicle and optional further adjuvants. 8. Process for the preparation of a composition according to claim 2, comprising lyophilizing LDL in the presence of a protecting substance, extracting the lyophilized LDL with an organic solvent, incubating the therapeutically or diagnostically active agent in an organic solvent with the extracted LDL, removing the solvent, dissolving the reconstituted LDL in an aqueous buffered solution, and recovering the reconstitiuted LDL and combining a thera¬ peutically or diagnostically effective amount of the reconstituted LDL with a lysosomotropic agent and a suitable vehicle and optionally further adjuvants. 9. Process according to claim 8, wherein the lysosomot¬ ropic agent is Triton WR 1339 ethyl oleate. 10. Process according to claim 8 or 9, wherein the thera¬ peutically or diagnostically active agent is selected from the group consisting of light sensitizers, radiosensiti- zers, x-ray contrast agents and anti-cancer drugs. 11. Process according to claim 10, wherein the anti-cancer drug is chlorambucil cholesteryl ester. 12. Process according to claim 8, wherein the therapeuti- cally active agent is chlorambucil cholesteryl ester and the lysosomotropic agent is Triton WR 1339 ethyl oleate. 13. Process according to claim 9, wherein the protecting substance is starch. 13. Process according to claim 9 wherein the solvent used for extraction of the LDL is heptane. 14. Use of a composition according to claim 1 or a composi¬ tion prepared according to claim 7 for therapeutic or diagnostic treatment of humans and non-human animals.

Inventors: KINNUNEN PAAVO KAI JOHANNES, KINNUNEN, PAAVO, KAI, JOHANNES
Applicants: KINNUNEN PAAVO KAI JOHANNES, KINNUNEN, PAAVO, KAI, JOHANNES
Application year: 1993
Main classification: A61K9
IPC codes: A61K9, A61K47, A61K49