Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Patent
1987-02-12
1989-09-19
Schain, Howard E.
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
424 858, 424 86, 424 87, 436518, 436535, 436543, 530359, 530387, 514773, A61K 3702, C07K 1516
Patent
active
048681580
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a method for the preparation of a carrier for a biologically active substance, below called drug, based on the reconstitution of LDL (Low Density Lipoprotein).
The term "biologically active substance", or "drug", is used in its most wide definition and includes substances such as pharmaceutically active substances, enzymes, toxins, radiosensitizers, radioactive substances etc.
Many attempts have been made to increase the concentration 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.
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 well act as a slow release system for appropriate substances.
Krieger 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 solubilized in an aqueous buffer.
Even if the reconstituted LDL particle according to the method of Krieger et al shows LDL receptor mediated uptake in vitro, it can not be used in vivo since the reconstituted particles are rapidly taken up by the cells of the reticuloendothelial system in liver and spleen.
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 protecting 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 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 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 different 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 polysaccharide, a sugar alcohol or a mixture of these.
In order to investigate plasma clearance, hum
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Masquelier Michele
Peterson Curt
Ros Mikael
Rudling Mats
Vitols Sigurd
Lewen Bert J.
Oncholab AB
Schain Howard E.
Sternberg Henry
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