Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1999-10-07
2002-04-16
Ketter, James (Department: 1632)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C435S458000, C424S422000, C424S428000, C424S449000, C424S450000, C264S004100
Reexamination Certificate
active
06372714
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a composition for gene transfer into cells, as well as to a method for gene transfer into cells by use of the composition.
BACKGROUND ART
Plasma membranes have low permeability to some compounds which are used as drugs, and thus the drugs fail to exhibit sufficient intracellular pharmacological effect. Low plasma membrane permeability may be attributable to, for example, the compound having low lipid solubility or high molecular weight. A typical example of such a compound to which plasma membranes exhibit low permeability is a gene.
At present, therapeutic treatment by use of such drugs to which plasma membranes exhibit low permeability, in particular, a gene, is conducted by way of injection, etc. However, the permeation of such a drug into the inside of cells is so poor that it is unable to provide satisfactory therapeutic effect.
To solve this problem, there have been proposed various conventional techniques known as drug delivery systems (DDS). As such systems there are, for example, liposomes formed primarily of phospholipids, emulsions composed of surfactants and oils such as soybean oil, mixed micelles made of lipids and surfactants, and microcapsules/microspheres made of biodegradable or non-degradable polymers. However, the conventional techniques have failed to increase permeability of a drug through the membrane; rather, in vitro evaluation has revealed that conventional drug delivery systems have an effect of decreasing cell-membrane permeability of a drug. This is because the drug itself is encapsulated in the drug delivery system, and release of the drug from the system serves as a determining factor. Despite this drawback, drug delivery systems have been attracting close attention, and have been applied to many drugs. This is because when a drug is encapsulated in a drug delivery system, in vivo degradation of the drug can be suppressed, and in vivo kinetics of the drug can be controlled, thus eventually increasing the in viva drug concentration in the vicinity of the target tissue or cells. Even in the case of liposomes, one of the typical drug delivery systems, although suppression of drug degradation and controlling the in vivo kinetics can both be achieved with relative ease, particles of liposomes ultimately accumulate in the vicinity of the target tissue or cells at high concentration and release the drug, and the remainder of the drug delivery steps depend solely on the permeability of the drug through the plasma membrane. Thus, although liposomes can attain an increased drug concentration near target cells, they have no effect on the permeability of the drug through the plasma membrane.
In some in vitro cases, a drug delivery system increases the rate of drug delivery into cells. Example cases include use of phagocytic cells such as macrophages and monocytes. Phagocytic cells readily ingest microparticles, such as liposomes, by endocytosis, and therefore, transferability of the drug into cells may be increased if the drug is encapsulated in a drug delivery system rather than administered alone. In this case, permeability of the drug through the plasma membrane is not increased. However, if the drug can be temporarily incorporated into cellular vesicles, such as endosomes and lysosomes, together with the drug delivery system and happens to be stable in these microenvironments, the drug can further enter the cytoplasm, resulting in increased drug transferability into cells.
Also, in recent years, extensive research has been directed to gene transfer into non-phagocytic cells, which is achieved through formation of a complex with a gene and cationic lipids (or liposomes containing the cationic lipids) or through encapsulation of a gene into liposomes containing the cationic lipids, thereby allowing the gene to be expressed within the cells. Even though almost nothing is known about the gene transfer mechanism into cells, reagents related to the above-described research are widely commercialized (including reagents such as lipofectAMINE, lipofectACE, lipofectin, transfectam, and genetransfer). Presently, biological researchers are using these reagents on a daily basis as very useful tools for gene transfer into cells, and this method serves as a substitute for the virus and microinjection methods. However, these commercialized reagents have many drawbacks as described in the following a) to d). a) These reagents, as commercialized products, are not stable, and thus are not suitable for storage. Many of these commercialized products are sold in the form of a dispersion in water, and the pH of their aqueous solvents are usually very low pH (for example, the pH is 3.5 for lipofectAMINE and lipofectACE, and 4.3 for lipofectin). Because of this low pH, lipids tend to degrade during storage. It has often been pointed out that efficiencies of gene transfer into cells and of gene expression by use of liposomes, etc. do not have satisfactory reproducibility. One reason for this is the inherent instability of the products. (b) Another drawback is that those products are very unstable in the presence of fetal bovine serum added to a medium for cell culture. As a matter of fact, the commercialized products employ the following protocol for gene transfer: Before gene transfer, the cultured medium containing fetal bovine serum is replaced with serum-free medium; and then, after completion of the gene transfer, the serum-free medium is replaced by serum-containing medium. Recently, it has become clear that these commercialized products are also very unstable in blood as well as in vivo. (c) A further drawback is that those commercialized products are not suitably designed for easy handling. Many of the commercialized products, such as lipofectAMINE, lipofectACE, and lipofectin, are provided in the form of a dispersion in water. For gene delivery, aqueous solvents that contain gene samples are added to these products. However, this protocol only allows the products to form complexes with the genes where genes bind only to the outside of the liposomes. Therefore, these products cannot yield vesicles having the genes encapsulated inside the liposomes. (d) Moreover, a further drawback is very strong cytotoxicity originated from those products. As is well known, the primary purpose for which biological researchers use these commercialized gene transfer reagents is to obtain the cells that have been transformed with exogenous genes and are capable of expressing those genes, and to subsequently use the obtained cells in subsequent studies. For such a purpose, in most cases, whether or not a minor portion of cell population dies during the gene transfer step is immaterial, so long as such transformed cells can be obtained. Thus, there have been commercialized some reagents making use of cationic lipids alone or in combination with liposomes, for delivering into cells a drug such as gene which in nature cannot be permeated through the membrane. However, those commercialized reagents involve many problems, as mentioned above. Thus, it is no exaggeration to say that application of such reagents to human use (such as gene therapy) is unthinkable at present. (Note: Gene therapy includes ex vivo and in vivo methods. In the case of ex vivo treatment, in which cells are taken out of a patient, treated in vitro, and subsequently returned to the patient, cytotoxicity raises a great problem.)
As pointed out above, it would be no exaggeration to conclude that there is no conventional satisfactory method whereby a gene—which, except for special cases such as the case of phagocytic cells or some commercially available gene transfer reagents, is poorly permeated through the plasma membrane, is poorly delivered into the inside of cells, or encounters difficulty in manifesting its activity inside the cells—can be delivered into cells, after which the gene is allowed to exert its pharmacological efficacy.
Thus, an object of the present invention is to improve permeation through a plasma membrane, transmembrane delivery, and intracellular expression
Kikuchi Hiroshi
Suzuki Norio
Tanaka Ken'ichi
Daiichi Pharmaceutical Co. Ltd.
Ketter James
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
Schrizer Richard
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