METHOD FOR THE PREPARATION OF A MACROMOLECULAR CARRIER LOADED WITH A BIOLOGICALLY ACTIVE SUBSTANCE, PRODUCT THUS OBTAINED AND USE THEREOF

Abstract: Method for the preparation of a carrier loaded with lipophilic biologically active substance based on reconstituted LDL (Low Density Lipoprotein), wherein (1) LDL is lyophilized in the presence of a protective agent; (2) the lyophilized LDL is extracted with an organic solvent; (3) the biologically active substance solubilized in a solvent is incubated with extracted LDL; the solvent is evaporated and the reconstituted LDL solubilized in an aqueous buffer; the non-incorporated biologically active substance is separated from the LDL-complex, characterized in that in step (1) the protective agent is a monosaccharide, a disaccharide, a water-soluble polysaccharide, a sugar alcohol or a mixture of these and in step (3) optionally the extract obtained during the extraction of the lyophilized LDL is mixed with lipophilic biologically active substance solubilized in an organic solvent and this mixture is then incubated with the LDL. Preferred protective agents are sucrose, glucose and fructose. The reconstituted LDL obtained by the claimed process has properties similar to native LDL in vitro and in vivo.

Description: METHOD FOR THE PREPARATION OF A MACROMOLECULAR CARRIER LOADED WITH A BIOLOGICALLY ACTIVE SUBSTANCE, PRODUCT THUS OBTAINED AND USE THEREOF The present invention relates to a method for the pre¬ paration of a carrier for biologically active substance, below called drug, based on the reconstitution of LDL (Low Density Lipoprotein) . The term biologically active substance, or drug, is in the present paper used in its most wide definition and includes substances such as pharmaceutically activ sub¬ stances, enzymes, toxins, radiosensitizers, radioactive substances etc. Many attemps have been made to increase the concentra¬ tion of a biologically active substance, for example an antitumoral drug, in a .certain organ or in certain target cells in order to increase the efficacy of the treatment and reduce the side effects. One way to accomplish this is to link the drug to a carrier, for example a macromolecule. The rationale is that the macromolecule should have a high uptake by the target cell or that the linkage carrier/drug in other aspects would give a better efficacy than would be the case with the free drug. A number of macromolecules have been investigated with respect to their use as carriers, such as DNA, liposomes, red blood ghost cells, lectines, different proteins such as antibodies, peptide hormones, and glucoproteins. Other molecules such as estrogens have as well been tried. An ideal carrier linked to a drug should fulfil the following criteria: SUBSTITUTE SHEET 1. It should permeat through the anatomical barriers separating the administrating site and the target cell, 2. The linkage between the carrier and the drug should be sufficiently stable in the blood, 3. It should not be rapidly cleared from the blood circulation by the reticuloendothelial system in liver and spleen, 4. It should be unable to cross the cell membranes through diffusion in order to prevent unspecific uptake by non-target cells, 5. It should interact as selectively as possible with receptors on the target cell surface, 6. Substances which are inactively linked to the carrier but biologically active in the cell shall be carried into the cell by the carrier where the linkage should be cleaved. 7. It should be non-immunogenic. Several proposed carriers, such as liposomes, do not fulfil the third criteria above, i.e. they are cleared rapidly from blood by the liver and spleen, and hence do not stay for a sufficient time to give the required effect. LDL, Low Density Lipoprotein, is a macromolecule that has several attractive properties. This lipoprotein is a sphericle particle with a diameter of approximately 220 A, a density between 1.019 and 1.063 and molecular weight of approximately 3 megadaltons, and stays in the circulation for 2-3 days in humans. Each LDL particle SUBSTITUTE SHEET contains a non-polar core composed of about 1500 cholesterol molecules esterified to long-chain fatty acids. The core is surrounded by a polar coat of cholesterol, phospholipid and apoprotein B, apo B. LDL is the major cholesterol transporter in human plasma, containing about 70% of plasma cholesterol. Mammalian cells have specific LDL receptors on the surface that recognises the apo B and binds the LDL-particle. The LDL particle is then endocytosed and transported to the lysosomes where it is degraded. If a biologically active substance could be linked or incorporated to the LDL particle in such a way that the reconstituted LDL behaves like native LDL with respect to LDL-receptor mediated uptake and biological half- life in plasma, the substance could be targetted to cells expressing high levels of LDL-receptors. Due to the long half-life of LDL the system could as weli act as a slow release system for appropriate substances. rieger et al (J. Biol. Chem. 253 (1978) 4093-4101 and J. Biol. Chem. 254 (1979) 3843-3853) have described a reconstitution procedure. Their method in brief is that LDL is lyophilized in the presence of unsoluble potato starch, and the neutral lipids are extracted with an organic solvent such as heptane. The substance to be incorporated is solubilized in an organic solvent and incubated with the lipid-depleted LDL. After evaporation of the organic solvent the reconstituted LDL is solubi- lized in an aqueous buffer. Even if the reconstituted LDL particle according to the method of Krieger et al shows LDL receptor mediated up¬ take in vitro, it can not be used in vivo since the re- constituted particles are rapidly taken up by the cells of the reticuloendothelial system in liver and spleen. SUBSTITUTE SHEET We have unexpectedly found a modified reconstitution procedure that gives a reconstituted LDL-particle with almost the same properties as native LDL both in vitro and in vivo. The present invention relates to a procedure for the preparation of.a carrier loaded with a biologically active substance on the basis of reconstituted LDL, where (1) LDL is lyophilized in the presence of a protec- ting agent; (2) the lyophilized LDL is extracted with an organic solvent; (3) the biologically active substance is solubilized in an solvent and incubated with the extracted LDL; (4) the solvent is evaporated; (5) the reconstituted LDL is solubilized in an aqueous buffer, and (6) the non-incorporated substance is separated from the LDL-complex. In the procedure in step (1) the protec¬ tive agent is a monosaccharide, a disaccha-ride, a water- soluble polysaccharide, a sugar alcohol or a mixture of these and in step (3) optionally the extract obtained during the extraction of the lyophilized LDL is mixed with the lipophilic biologically active substance solubilized in an organic solvent and this mixture is then incubated with the LDL. The method according to the present invention is diffe¬ rent from the method of Krieger et al mainly in the use of the protective substance. In step (1) Krieger is using potato starch as a protective agent. Potato starch is a water-insoluble polysaccharide with high molecular weight. In our method we are using as a protective agent a monosaccharide, a disaccharide, a water-soluble poly¬ saccharide, a sugar alcohol or a mixture of these. In order to investigate plasma clearance, human LDL was injected intravenously in mice (Balb/C) and the percen¬ tage of injected dose remaining in plasma was determined after 90 minutes. The percentage of nativ LDL remaining in plasma after 90 minutes is about 59. The percentage of LDL reconstituted according to the method of Krieger et al remaining in plasma was very low, less than 3. The percentage remaining in plasma according to the present invention have values of 57-58, which is a very surprising and technically important development. The plasma clearance in rabbits was determined from plasma decay curves for radiolabeled LDL: for nativ LDL, reconstituted LDL according to the method of Krieger et al and reconstituted LDL according to the method of the present invention. As can be seen from Figure 1 , native LDL and the reconstituted LDL according to the present invention had the same clearance, while the reconstituted LDL according to Krieger et al disappeared rapidly from plasma. The particle sizes of various preparations of LDL re- solubilized in buffer have been determined by means of quasielectric light scattering. It was found that the apparent mean hydrodynamic radius of lyophilized LDL without any protective substance was about 2.9 times larger than that for native LDL, and LDL lyophilized in the presence of potato starch as protective substance had a radius of about 2.7 times, while LDL lyophilized in the presence of sucrose had a radius about 1.2 times that of native LDL. The drug-LDL complex according to the method of Krieger had an apparent mean hydrodynamic radius of about 3.8 times the value for native LDL, while the reconstituted drug-LDL complex according to the present invention had a radius of about 1.3 times that of native LDL. The reconstitution method of Krieger et al is based on a method developed by A. Gustafson (J. Lipid Res. 1965, 6, 512-517) to extract the cholesteryl esters from the core of LDL. The key step in this procedure is to use SUBSTITUTE SHEET potato starch to stabilize the lipid-depleted apo- proteins during the lyophilization and heptaneextrac- tion of the neutral lipids from the core of the LDL. As shown above the method of Krieger et al will not give sufficient protection of the LDL. Important for the reconstitution of the LDL particle is to replace an appropriate amount of the neutral lipids in the core with the desired substance, or drug. If the drug- LDL complex should be used for targetting wia the LDL receptor pathway and too much of the drug is inserted into the LDL particle there is a risk for the drug leaking out when the LDL particle is colliding with different cells in the blood and the blood vessels. This gives an unspecific uptake of the drug and in this case the incorporated drug-LDL complex functions as a slow release system. A suitable protective substance should prevent aggregation of the LDL particles or other damages such as the falling apart of the particle and prevent too high a replacement of the lipids from the LDL particle. If the amount of protective substance used is to small, then the resulting reconstituted LDL particle will have a rapid clearance from plasma; this effect is not linear, and the amount of protective substance should be at least 1 mg/mg LDL, where LDL is measured as protein. On the other hand, if the amount of protective substance is too high then the amount of drug incorporated will be too low; this means that there exists a certain range for the amount of protective substance used. Protective substances fulfilling the above criterias are water-soluble βaccharides such as monosaccharides, disaccharides, trisaccharides, watersoluble poly- saccharides, sugar alcohols or mixtures of these. SUBSTITUT Examples of monosaccharides, that can be used according to the present invention are: glucose, mannose, glycer- aldehyde, xylose, lyxose, talose, sorbose, ribulose, xylulose, galactose and fructose. Examples of disaccha- rides are sucrose, trehalose, lactose and maltose. Examples of trisaccharides are raffinose. Among water- soluble polysaccharides certain watersoluble starches and celluloses can be mentioned. Examples of sugar alcohols are glycerol. Preferred protectiva agents in the present invention is sucrose, glucose or fructose which gives a LDL comp¬ lex behaving like nativ LDL. The organic solvent used in step (2) for the extraction of the lyophilized LDL is preferrably a non-polar solvent such as heptane or carbontetrachloride. Heptane is the preferred solvent. The biologically active substance that is incorporated into the lyophilized LDL shall be a lipophilic substance. Examples of such biologically active substances that can be incorporated into the LDL particle according to the present invention are anti-tumoral drugs, drugs affection the cholesterol synthesis, as well as radio- sensitizers, enzymes, ensymeinhibitors, radioactiv substances, toxins, etc. The separation of the non-incorporated biologically active substance from the complex consisting of the LDL and the biologically active substance is done after solubilization in aqueous buffer by known proce¬ dures, such as centrifugation, filtration etc. The invention is illustrated in the following examples, where the temperature is given in centrigrades. SUBSTITUTE The LDL used in the examples was human LDL (density 1.019 - 1.063), obtained by ultracentrifugation of plasma from healthy human volunteers. All LDL amounts refer to protein according to the method of Lowry et al, J. Biol. Chem. 193 (1951) 265-275 where bovine serum albumin is used as standard. The biologically active substance used is the anti-tumoral drug AD-32, N-trifluoroacetyladriamycin-14-valerate. The in vivo tests were performed on Balb/C mice. 0 Example 1 2 mg LDL (400 microliter of liquid) were after dialysis against 0,3 mM Na EDTA, transferred to a siliconized glass tube and 100 mg sucrose was added. The solution 5 was rapidly frozen at -50 C in ethanol and lyophilized for 5-6 hours. The dried preparation was extracted three times with 5 ml of heptane. The heptane phases were " ' pooled and evaporated. 2 mg of AD-32 in 0.5 ml of anhydrous dietyl ether were then added to the dried 0 extracts. The solution was mixed with the extracted LDL by gentle agitation, and incubated at 4 C for 15 minutes. The solvent was evaporated under nitrogen, the drug-LDL complex was solubilized by the addition of 1 ml 10 mM Tricine, pH 8,4. Insoluble, non-incorporated 5 drug was separated by centrifugation for 5 minutes in a Beckman microfuge. This gave a reconstituted LDL particle containing 110-130 molecules of AD-32 per LDL particle. The percentage remaining in plasma in Balb/C mice was about 58 after 90 minutes. 0 Example 2 Like example 1 , but the lyophilized LDL was not extracted by heptane, instead AD-32 solubilized in ether was added directly. This gave about 35 - 43 molecules AD-32 per 5 LDL particle and percentage ramaining in plasma of about 55. Example 3 Like example 1 , but instead of sucrose as a protective agent 80 microliters of glycerol was used. This gave about 50 molecules AD-32 per LDL and the percentage remaining in plasma was about 55. Example 4 - Comparative test I Like example 1 , but instead of sucrose 25 mg potato starch was used. The extract obtained after the extrac- tion of LDL was discarded and AD-32 in anhydrous ether was added directly to the extractet LDL. (Method of Krieger et al) . This gav about 400 molecules of AD-32 per LDL, but the percentage of injected dose remaining in plasma was below 3. Example 5 - Comparative test II Like example 1 but 25 mg of potato starch and 80 micro- liters of glycerol was used instead of sucrose. This gave about 50 molecules AD-32 per LDL particle and the percentage remaining in plasma was about 50. Therapeutic test 1 In this test the drug AD-32 was used. The amount AD-32 administrated in this test was well below the toxic level for the mice and the result is presented here to give an example of the targetting effect of the drug-LDL complex compared to free drug. Balb/C mice were injected intraperitonally (i.p.) with 1.0 million WEHI-3B cells, a urine monomyelocytic cell line. No difference (2%) was found in survival (ILS, Increased Life Span) between the controls and the group treated with 1.9 mgAD-32/kg/- day for five days. The AD-32 was administrered i.p. dissolved in a mixture of ethanol and castor oil. The mice that obtained the same dose of AD-32 incorporated into LDL according to the procedure in Example 1 ad¬ ministrated i.p. for five days had a 26% increase in life span. SUBSTITUTE SHEET Therapeutic test 2 Balb/C mice were injected i.p. with 1.0 million WEHI- 3B cells and an alkylating agent, 4-amino N.N.-bis (chloroethyl)-2-methyl-naphtalene, was incorporated according to the present method. The animals were treated for four consecutive days i.p. and the result is shown in Figure 2. The control group died during days 12-18, while in the treated group they died from day 25, with 50% still living on day 42 and 40% still surviving (on day 50) . SUBSTITUTE SHEET

Claims: CLAIMS 1. Method for the preparation of a carrier loaded with lipophilic biologically active substance based on reconstituted LDL, wherein (1) LDL is lyophilized in the presence of a protective agent; (2) the lyophilized LDL is extracted with an organic solvent; (3) the bio¬ logically active substance solubilized in a solvent is incubated with extracted LDL; (4) the solvent is evapo¬ rated and the reconstituted LDL solubilized in an aqueous buffer; (5) the non-incorporated biologically active substance is separated from the LDL-complex, characterized in that in step * (1) the protective agent is a monosaccharide, a disaccharid, a water-soluble polysaccharide, a sugar alcohol or a mixture of these and that in step (3) optionally the extract obtained during the extraction of the lyophilized LDL is mixed with the lipophilic biologically active substance and this mixture is then incubated with the LDL. 2. Method according to claim 1, wherein the protective agent is glyceraldehyde, xylose, lyxose, raffinose, glucose, mannose, galactose, talose, ribulose, xylulose, trehalose, fructose, sorbose, maltose, lactose, sucrose, glycerol or a mixture of any of these. 3. Method according to claims 1 and 2, wherein the protective agent is sucrose. 4. Method according to claims 1 and 2, wherein the protective agent is glucose. 5. Method according to claims 1 and 2, wherein the protective agent is fructose. 6. Method according to any of claims 1 to 5 wherein the organic solventused in step (2) is n-heptane. SUBSTITUTE SHEET 7. Method according to any of the claims 1 - 6 wherein the solvent used in step (3) for the solubulization of the biologically active substance is ethyl ether, heptane, carbontetrachloride or benzene. 8. Method according to claims 1 to 7 wherein the amount of protective agent is in the range of 0.5 to 100 mg/mg LDL, where LDL is measured as protein. 9. Use of the product obtained according to claims 1 to 8 for diagnostic and therapeutical purposes in humans and animals, preferably for cancer treatment in humans. 10. Product obtained by the process of claims 1 to 8. SUBSTITUTE SHEET

Inventors: MASQUELIER MICHELE, PETERSON CURT, ROS MIKAEL, RUDLING MATS, VITOLS SIGURD, MASQUELIER, MICHELE, PETERSON, CURT, ROS, MIKAEL, RUDLING, MATS, VITOLS, SIGURD
Applicants: ONCHOLAB AB
Application year: 1986
Main classification: A61K37
IPC codes: A61K9, A61K47, A61K49