JP2002518313A - Cationic amphiphilic micelle complex - Google Patents

Abstract

(57) Abstract The efficient introduction of foreign genes and other bioactive molecules into target mammalian cells is a challenge still faced by those skilled in the art. For example, gene therapy requires successful transfection of a patient's target cells. The present invention relates to novel micellar complexes of cationic amphiphilic compounds that facilitate delivery of bioactive molecules to target cells in mammals. This novel micellar complex consists of a cationic amphiphile, a bioactive molecule, a derivative of polyethylene glycol (PEG), and optionally a co-lipid. A further aspect of the present invention is the use of a targeting agent in a method to facilitate delivery of a bioactive molecule to a mammalian cell. Targeting agents are typically molecules, peptide sequences, or large proteins that preferentially target or bind to a particular mammalian cell.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to novel micellar complexes of cationic amphiphilic compounds that facilitate delivery (and / or transfection) of biologically active molecules to mammalian target cells. More specifically, the present invention relates to the unique properties of these micellar complexes, as well as methods and methods for making micelles of cationic amphiphiles to enhance delivery of bioactive molecules to desired cells of a mammal. About the method. The goal of the present invention is to provide new conjugates that can be used for gene therapy. The invention also relates to the use of targeting agents to facilitate the delivery of bioactive molecules to certain types of mammalian cells.

[0002] Efficient introduction of foreign genes and other bioactive molecules into target mammalian cells is a challenge still faced by those skilled in the art. Gene therapy requires successful transfection of the patient's target cells. Transfection, which is itself useful in practice, is generally defined as the process of introducing an expressible polynucleotide (eg, a gene, cDNA, or mRNA) into a cell. Successful expression of the transfected encoding polynucleotide produces a normal protein in the cell. The goal is, of course, to obtain sufficient expression of the abnormal gene to lead to the correction of the associated condition.

[0003] Examples of diseases that are targets for gene therapy include genetic diseases such as cystic fibrosis, Gaucher disease, Fabry disease, and muscular dystrophy. Representative acquired target diseases are (1) in cancer-multiple myeloma, leukemia, melanoma, ovarian cancer and small cell lung cancer; (2) in cardiovascular conditions-progressive heart failure, restenosis, and And (3) in neurological symptoms-there is traumatic brain injury.

[0004] Cystic fibrosis, a common lethal genetic disease, is a specific example of a disease that is a target for gene therapy. This disease is a cystic fibrosis transmembrane modulator (cystic fibrosi
It is caused by the presence of one or more mutations in the gene encoding the protein known as transmembrane conductance regulator ("CFTR"). Cystic fibrosis is characterized by chronic sputum production, recurrent infections, and lung destruction (Boat, TF, McGraw-Hill (McGr.
aw-Hill, Inc.), 1989, p. 2649-2680). It is not known exactly how mutations in the CFTR gene cause clinical symptoms (Welsh, MJ et al., Cell 73: 1251-1254.
, 1993), incomplete Cl ^- secretion and increased Na ⁺ absorption (Welsh, M., et al.).
J. (Welsh, MJ) et al., Cell 73: 1251-1254, 1993;
Quinton, PM, FASEB Lett. 4:27
09-2717, 1990) has been extensively described. Furthermore, these changes in ion transport result in altered fluid transport across the surface and glandular epithelium (Jiang, C. et al., Science 262: 424-427,19).
93; Jiang, C. et al. Physiol. (London), 5
01.3: 637-647, 1997; Smith, JJ
) Et al. Clin. Invest. , 91: 1148-1153, 1993;
And Zhang, Y. et al., Am. J. Physiol 270: C
1326-1335, 1996). Do these changes in the water and salt content of airway surface fluid (ASL) reduce the activity of bactericidal peptides secreted from epithelial cells (Smith, JJ, Cell, 85: 229-2
36, 1996), and / or impairs mucociliary clearance, thus promoting recurrent lung infection and inflammation.

[0005] Some evidence suggests that the submucosal gland contributes to the pathophysiology of CF lung disease. Maintaining mucociliary clearance requires coordinated regulation of pili motility, ASL depth, and mucin content. The amount and composition of ASL is controlled by both epithelium and submucosal glands and is based on estimates of cell volume, the latter being thought to be a more important cause of mucosal secretion. Recent studies also indicate that the serous cells of the secretory tubules of the submucosal gland are the predominant site of CFTR expression in human bronchi, and that the fluid secreted from the serous cells sheds mucin secreted by the mucosal cells It is shown that. Additional evidence suggesting that the submucosal gland contributes to the pathophysiology of CF lung disease includes (1) that CFTR is predominantly expressed in submucosal gland serous cells (
Engelhardt, JF et al., Nat. Genet.
2: 240-248, 1992), (2) that the bronchial submucosal gland culture from CF patients cannot secrete Cl ⁻ (Finkbeine, Finkbeine
r, WE) et al., Am. J. Physiol. 267: L-206-L-210,
1996; Yamaya, M. et al., Am. J. Physiol. 261
: L-485-L-490, 1991; Yamaya, M. et al., Am.
J. Physiol. 261: L-491-L-494, 1991), (3) More than 60% of submucosal gland cultures from non-CF subjects showed baseline secretion, whereas cultures from CF patients did not. , Exclusively that they absorbed body fluids (Jean,
Jiang, C. et al. Physiol. (London), 501.3: 63
7-647, 1997), (4) Obstruction of the submucosal gland duct is the first pulmonary symptom in CF patients, followed by marked hyperplasia and hypertrophy (Oppenheimer,
Ophheimer, EH, et al., New York: Year Book M
electronic Publishers, 1975, p. 241-278).

[0006] Evidence suggesting the involvement of submucosal glands in the pathogenesis of CF suggests that effective gene therapy for CF lung disease should target these structures. CF
Many attempts have been made to carry the TR gene into the embryonic epithelium, but little attention has been paid to submucosal gland cells. Furthermore, it has been demonstrated that low levels of β-galactosidase expression were detectable in submucosal glands following intratracheal administration of adenovirus vectors (Pilewski, JM et al., Am.
J. Physiol. 268: L657-665, 1995), gland transfection levels were lower than in embryonic epithelium and decreased significantly in proportion to distance from the airway lumen.

[0007] Efficient introduction of many types of bioactive molecules is difficult, and not all methods that have been developed have been able to efficiently deliver the appropriate amount of the desired molecule into target cells. . The complex structure, behavior and environment presented by intact tissue targeted for intracellular delivery of bioactive molecules often often substantially impede such delivery. Viral vectors, DNA encapsulated in liposomes, lipid delivery vehicles,
Many methods have been utilized to deliver DNA into mammalian cells, including and naked DNA. To date, delivery of DNA in vitro, ex vivo, and in vivo has been demonstrated using many of the methods described above.

[0008] While virus transfection is relatively efficient, the host's immune response is often a significant problem. Specifically, viral proteins are cytotoxic T
Activate lymphocytes (CTLs), which terminate gene expression in the lungs of the tested in vivo model by destroying virus-infected cells. Other issues are
Repeated administration of a viral vector, due to the generation of neutralizing antiviral antibodies, reduces gene transfer. These issues are currently being investigated to modify both the vector and the host immune system. In addition, non-viral and non-protein vectors have received attention as an alternative approach.

[0009] Compounds designed to facilitate the intracellular delivery of biologically active molecules can interact with nonpolar and polar environments (eg, in or on the plasma membrane, tissue fluid, intracellular compartments, and the biologically active molecule itself). Because they must interact, such compounds are typically designed to contain both polar and non-polar domains. Compounds with both such domains, called amphiphiles, have been disclosed for use in facilitating such intracellular delivery (whether in vitro or in vivo applications). Many lipids and synthetic lipids meet this definition. A particularly promising group of amphiphilic compounds for efficient delivery of bioactive molecules are cationic amphiphiles. Cationic amphiphiles have polar groups that are positively chargeable near physiological pH, a property that many amphiphiles include, for example, negatively charged polynucleotides such as DNA. It is understood in the art that it is important to define how to interact with a type of bioactive molecule.

Examples of cationic amphiphilic compounds that have been described as being useful in intracellular delivery of bioactive molecules can be found, for example, in the following references, the disclosures of which are incorporated by reference: It is essentially a part of this specification. Many of these references also describe the nature of cationic amphiphiles, which are understood in the art to be suitable for such applications, and such amphiphiles for intracellular delivery. Includes a useful discussion of the nature of structures understood in the art to be formed by complexing a substance with a therapeutic molecule.

(1) Felgner et al., Proc. Natl. Acad. Sci.
USA, 84, 7413-7417 (1987) describes N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride ("
It discloses the use of positively charged synthetic cationic lipids, including DOTMA "), to form lipid / DNA complexes suitable for transfection. Felgner et al., The Journal of Biological.
See also Chemisty, 269 (4), 2550-2561 (1994). (2) Behr et al., Proc. Natl. Acad. Sci. USA 8
6,692-6986 (1989) discloses a number of amphiphiles, including dioctadecylamidogloglycylspermine ("DOGS"). (3) U.S. Pat. No. 5,283,185 to Epand et al.
Additional classes and species have been described, including 3β- [N- (N ¹ , N ¹ -dimethylaminoethane) carbamoyl] cholesterol, referred to as “ol”. (4) Additional compounds that enhance the transport of bioactive molecules into cells are disclosed in Felgner et al., US Pat. No. 5,264,618. "DM
RIE, for disclosure of additional compounds, including 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (discussed below), Felgner et al., The Journal of Bio.
biological Chemistry 269 (4), pp. 2550-256
1 (1994). (5) References to amphiphiles suitable for intracellular delivery of bioactive molecules can also be found in Gebeyehu et al., US Pat. No. 5,334,761 and Felgner.
ner) et al., Methods (Methods in Enzymology),
5, 67-75 (1993). (6) Brigham, KL, B. Meyric
k), B. Christman, M. Magnuson
, G. King and LC Berry. "In vivo transfection of murine lung with a functional prokaryotic gene using a liposome vehicle". Am. J. Med. Sci. 298: 278-281,1
989. (7) Gao, XA and L. Huang. "A novel cationic liposome reagent for efficient transfection of mammalian cells". Biochem Biophys Res Commun 179: 28
0-285, 1991. (8) Yoshimura, K., MA Rosenfeld (MA
Rosenfeld), H. Nakamura, E.M.
M. Scherer), A. Pavirani, Jay P. Lecock (JP)
Lecocq) and RG Crystal. "Expression of human cystic fibrosis transmembrane conductance regulator gene in mouse lung after in vivo endotracheal plasmid-mediated gene transfer". Nucl. Acids Res. 20: 3233-3240, 1992. (9) Zhu, N., D. Liggitt, Y. Liu, and R. Debs. "Intravenous DNA into adult mice
Systemic Gene Expression After Delivery ". Science 261: 209-211,19
93. (10) Solodin, I., CS Brow
n), MS Bruno, CY Chow
, E. Jang, RJ Debs and TD Heath. "A new series of amphiphilic imidazolinium compounds for in vitro and in vivo gene delivery". Biochem. 3
4: 13537-13544, 1995. (11) Lee, ER, J. Marshall
), CS Siegal, C. Jiang, NS Yew, MR Nichols, JB Nietupski , Earl Jay Ziegler (RJ
Ziegler), M. Lane, KX Wang,
NC Wan, RK Scheule, DJ Harris, DJ Smith and AE Smith and SH Cheng. "Detailed analysis of the structure and formulation of cationic lipids for efficient gene transfer to the lung." Hum. Gene Ther. 7
: 1701-1717, 1996.

In addition, several recently issued US patents, the disclosure of which is hereby incorporated by reference in their entirety, describe cations for delivering polynucleotides to mammalian cells. Describes the usefulness of sex amphiphiles. (Briam (
U.S. Pat. No. 5,676,954 to Brigham et al., And Felgner
U.S. Pat. No. 5,703,055. )

Although the compounds referred to in the above references have been shown to facilitate the transfer of bioactive molecules into cells, the uptake efficiency provided thereby is improved,
It is believed that it could support many therapeutic applications, especially gene therapy. Furthermore, there is a need to improve the activity so that the required amount of such compounds is lower and the concerns regarding the toxicity of such compounds or their metabolites can be reduced.

Another class of cationic amphiphiles with enhanced activity is, for example, US Pat. No. 5,747,471 to Siegel et al., Issued May 5, 1998;
Harris et al., U.S. Pat. No. 5,650, issued Jul. 22, 1997.
No. 096 and PCT publication WO 98/02191 published Jan. 22, 1998.
(The disclosures of which are hereby incorporated by reference in their entirety). These patents also disclose methods of preparing cationic amphiphiles relevant to the practice of the present invention.

[0015] Although there are many cationic amphiphiles and viral vectors that cause enhanced activity, new methods of binding and targeting lipid and non-lipid delivery vehicles to specific mammalian cells are still being explored. . For gene therapy and other applications of in vivo, in vitro and ex vivo delivery of bioactive molecules,
A highly desirable factor in using cationic amphiphiles and viral vectors is the ability to effectively target and bind to specific mammalian cells. To date, effective methods of targeting specific cell types are lacking. The ability to target specific cells reduces the dose of cationic amphiphile, virus or other delivery vehicle complex required to effectively treat a particular condition,
Thereby reducing the problem of toxicity, which is a function of the high dose. As a result, methods to improve the efficiency and quantity of bioactive molecules delivered to a desired mammalian cell may require the survival of cationic amphiphilic complexes, viral vectors, and other delivery vehicles as an effective therapeutic treatment. Desired to enhance activity.

[0016] Accordingly, the present invention relates to novel micellar complexes that facilitate delivery of bioactive molecules to mammalian cells. This novel micellar complex consists of a cationic amphiphile, a bioactive molecule, a derivative of polyethylene glycol (PEG), and optionally a neutral, positive or negative co-lipid. These novel micellar complexes may have unique properties not observed with conventional cationic amphiphilic complexes. For example, the novel micellar complex allows one skilled in the art to preferentially bind the micellar complex to airway epithelial cells. Also, one skilled in the art can preferentially bind the micellar complex to other specific cell types, or target it to particular mammalian cells for delivery by the micellar complex.

The present invention provides the use of a cationic amphiphile to form a mixed micellar complex with a PEG derivative and optionally a co-lipid. Any cationic amphiphile that can facilitate intracellular delivery of a bioactive molecule is useful in the practice of the present invention. Although the present invention is not limited to the disclosed amphiphiles, many examples of cationic amphiphiles useful in the practice of the present invention are described in the previously referenced publications.

In the practice of the present invention, there is provided a micelle complex, wherein the complex is effective for binding to airway epithelial cells. Without being limited by theory, it is believed that the present micelle complexes demonstrate preferential binding when compared to conventional lipid complexes due to the difference in charge density of the micelle complexes.

The micellar complexes of the present invention can also be provided wherein the complexes are substantially more homogeneous as compared to conventional lipid complexes. That is, the micelle complex of the present invention has a narrower particle size distribution curve than the lipid complex prepared by conventional means.

[0020] Preferred micellar complexes of the present invention are also more stable than conventional lipid complexes. Micellar preparations can be prepared the day before and stored overnight without any deleterious effects.

In yet another aspect, the invention provides for improved binding efficiency between a cationic amphiphile and a bioactive molecule. The improved binding efficiency results in more "loading" of DNA per lipid present in the preparation. PEG derivatives are known in the art to stabilize conventional lipid: bioactive molecule conjugates and prevent precipitation. However, micelle conjugates containing PEG derivatives can load more bioactive molecules without precipitation than conventional lipid bilayer conjugates, which also contain PEG derivatives. That is, as compared to the cationic amphiphile in the conventional cationic amphiphilic complex, more bioactive molecules are
It is associated with each cationic amphiphile in the micellar complex.

In yet another aspect, the invention relates to a cationic amphiphile, a bioactive molecule, a PE
A method for preparing a micellar lipid complex comprising a G derivative and, optionally, a co-lipid. The resulting complex is homogeneous, stable, and effective for binding to airway epithelial cells. In a preferred embodiment, the conjugate is effective for systemic delivery of a bioactive molecule.

The present invention also provides a method of administering a micelle complex to deliver a biologically active molecule to a mammalian cell. Further, a method is provided for facilitating transfection of a gene into a mammalian cell by administration of a micelle complex.

[0024] In yet another aspect of the invention, the micelle complex may include a targeting agent that facilitates delivery of a bioactive molecule to a particular type of mammalian cell. This targeting substance
Effective for both lipid and non-lipid methods, the present invention is directed to viral vectors, including adenoviruses, and other methods utilized in the art to achieve delivery of bioactive molecules into mammalian cells. In addition to the use of targeting substances in
Provided is the use of targeting agents in all lipid complexes, including both conventional and micellar cationic amphiphiles.

The present invention also provides drug compositions of micelle complexes, and other lipid and non-lipid complexes with targeting agents. Micellar complexes are active ingredients in pharmaceutical compositions, including carriers, fillers, bulking agents, dispersants, creams, gels, solutions, and other excipients common in the field of pharmaceutical formulation. May be.

[0026] Additional features and advantages of the invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learned by practice of the invention. The objects and other advantages of the invention will be realized and attained by the method particularly pointed out in the written description and claims hereof as well as the appended drawings.

In the present invention, prior art cationic amphiphilic compounds are used in a preparation comprising a PEG derivative and optionally a co-lipid. The resulting preparation is complexed with one or more bioactive molecules. This new preparation exhibits unique and surprising properties not found in conventional cationic amphiphilic preparations, other cationic amphiphilic preparations, and lipid carriers. An additional aspect of the invention is the use of the targeting agent in a new preparation. Targeting agents facilitate delivery to certain mammalian cells. The practice of the present invention is not limited by theory.

Conventional conjugates of a cationic amphiphile, a PEG derivative, and optionally a co-lipid, are well known in the art. These conventional conjugates are formed by preparing a lipid film of a cationic amphiphile, a PEG derivative, and optionally a co-lipid. The lipid film is then hydrated in an aqueous medium to form a lipid bilayer, and then complexed with the bioactive molecule. Conventional cationic amphiphilic complexes formed by this method are usually 400-500 nm (nanometers) in diameter and vary in particle size by 50% or more.

A preferred embodiment of the present invention is a small, homogeneous and stable mixed micelle preparation or micelle complex. One embodiment of the present invention contemplates a micellar complex that exhibits binding to airway epithelial cells, a property not found in conventional cationic amphiphilic complexes. In the practice of the present invention, micelle complex preparations that can have unique and surprising properties are prepared by new methods. The micelle complex is preferably prepared by hydrating a cationic lipid and adding the hydrated cationic lipid to a PEG derivative, which is also hydrated to form a micellar lipid suspension. can do. The micelle cationic lipid: PEG: bioactive molecule conjugate is prepared by adding a micelle cationic lipid: PEG derivative solution to the bioactive molecule. The molar ratio of lipid: bioactive molecule and cationic lipid: PEG derivative is
It can vary over a wide range and will depend on the cationic lipid, PEG derivative, and bioactive molecule used. This ratio can also vary significantly as a function of the site of administration and the target disease. In one embodiment, the molar ratio of lipid: bioactive molecule is 1: 8. In another preferred embodiment, the biologically active molecule is DNA.

[0030] Micellar complexes can also be prepared with neutral, positive, or negative colipids as part of a preparation. Co-lipids can be prepared with PEG lipids as lipid films and hydrated as a single solution, or co-lipids can be prepared alone as lipid films and hydrated with PEG lipids. The cationic lipid is then added to a solution of the PEG lipid and the co-lipid in hydrated form to form a micellar lipid, which is then used to form a micellar complex with the bioactive molecule. In one embodiment, the molar ratio of lipid: bioactive molecule is 1: 8. In another preferred embodiment, the biologically active molecule is DNA.

In the practice of the present invention, there is provided a micellar lipid complex, wherein the complex is effective for binding to airway epithelial cells. Without being limited by theory, it is believed that the present micelle lipid complex demonstrates preferential binding when compared to conventional lipid complexes due to the difference in charge density of the micelle complex.

[0032] Also provided are micelle complexes of the invention wherein the complex is substantially more homogeneous as compared to conventional lipid complexes. That is, in this embodiment, the micelle complex of the present invention comprises:
It has a narrower particle size distribution curve than lipid complexes prepared by conventional means. For example, the particle size distribution of a conventional lipid complex can vary by more than 50%, depending on the lipid, DNA, and lipid: DNA ratio. In comparison, the particle size distribution of the micellar composite according to this embodiment varies only up to about 20%.

In addition to being significantly more homogeneous, the preferred micellar conjugates of the present invention do not change significantly in particle size when more bioactive molecules are added to the conjugate. For example, a constant ratio of cationic lipid to pDNA and cationic lipid to PEG derivative to co-lipid in the micellar complex, while pDNA being part of the micellar complex (ie,
Experiments were performed to increase the amount of pDNA not released in solution). The size of the preferred micellar complex and its size distribution did not change significantly with increasing amounts of pDNA in the micellar complex.

[0034] Preferred micellar complexes of the present invention are also more stable than conventional lipid complexes. Many conventional lipid complexes require special storage methods or mixing of the preparation with the DNA to be delivered immediately prior to administration to mammalian or in vitro cells.
There are storage and stability issues. For example, conventional cationic lipids are known to degrade by acyl exchange reactions unless stored under special conditions. Many lipid: DNA complexes are also known to precipitate out of solution shortly after complex formation, so it is necessary to delay preparation of the complex until just before use. Pharmaceutical products that require the preparation to be prepared shortly before use are not very practical. The micelle complexes of the present invention preferably do not precipitate out of solution shortly after preparation. For example, micelle preparations can be prepared the day before and stored overnight without any deleterious effects. One skilled in the art can also vortex the micelle preparation without observing significant precipitation.

When a micellar lipid solution is added to the bioactive molecule, a minimum amount of PEG lipid is preferred to form a stable and homogeneous complex. The minimum amount of PEG required will depend on the particular combination of cationic lipid and PEG lipid selected. The method for determining the minimum amount of PEG required to form a micellar complex is not particularly limited. 1) To confirm that the suspension is clear to opaque and free of particles, Observe the lipid: bioactive molecule complex after addition of the micellar lipid to the bioactive molecule; 2) after preparation using a sizing machine to determine if the particle population is substantially homogeneous with respect to particle size Sizing of the micelle lipid: bioactive molecule complex; and 3) analysis of the behavior of the bioactive molecule in the micelle complex in agarose gel electrophoresis. Details regarding the above-described method can be found in the examples attached herein.

Another embodiment of the present invention relates to micelle conjugates that are smaller in diameter than conventional cation-regulated amphiphilic conjugates and remain small and stable over a wide range of lipid: DNA and lipid: PEG ratios. To form small, homogeneous micellar complexes, a minimum amount of PEG derivative is preferred. In a preferred embodiment of the invention, the micellar complex prepared by one or more cationic amphiphiles, one or more PEG derivatives, bioactive molecules, and optionally co-lipids, Is about 25-250 nanometers on average. However, the particle size of the micellar complex depends on the amount and size of the cationic lipid, PEG lipid, DNA used, and co-lipid (if present).

Without being limited by theory, it is believed that the charge density of the micellar complex is responsible for the favorable unique and surprising properties of the complex that bind to airway epithelial cells. High charge densities are believed to cause high affinity of some cell membranes. Thus, many of the micellar complexes bind to airway epithelial cells. Simple in vitro fluorescence experiments demonstrated that the micelle complex bound strongly to exposed airway epithelial cells. Conventional complexes of cationic amphiphiles (usually 400-
500 nm) does not show strong binding in the same experiment.

[0038] The micellar conjugates of the present invention are also believed to be less toxic than conventional cationic lipid conjugates when administered to mammals. For example, when a mouse is injected intravenously,
The micelle complex prepared using the cationic lipid GL-67 is also a GL-67
Was less toxic than conventional cationic lipid conjugates prepared using The low toxicity of the micellar complex does not significantly affect the ability of the complex to deliver to tumor cells.
In a preferred embodiment, the micellar complex maintains equivalent deposition on tumor cells and especially on tumor endothelial cells, while having low toxicity for other cells of the mammal.

In another embodiment of the present invention, the micellar complex is coated with lipids or other compositions used to coat the compositions and preparations in the field of formulation. For example, mixing the micellar complex with yet another hydrophobic species (eg, a neutral lipid mixture) can coat the outside of the complex without disturbing the complex. Without being limited by theory, it is desirable to use a cationic lipid: PEG derivative to efficiently concentrate DNA. The resulting small, highly concentrated package of DNA (eg, micellar complex) is then co-lipid or other PE
Surrounded by other species and lipids, such as G derivatives, and
A may interact with the charged surface of A. This protects and / or masks the cationic lipid: DNA from the immune system. Coating would be an effective means of reducing toxicity, as most of the toxicity of the lipid: DNA complex is due to the recognition of the sequence by the bacteria. Further, the coating facilitates the encapsulation of the targeting substance and allows for delivery of the complex to a particular tissue or cell type. In a preferred embodiment, the coated conjugate is delivered systemically.

In addition, the coating is useful to extend the residence time of the micelle complex in the bloodstream or as a long-lasting mechanism. The use of other coatings or coatings known in the field of formulation is included in the practice of the invention.

Cationic Amphiphiles Used in Micellar Complexes The present invention is directed to any cationic amphiphile or cationic lipid compound useful for facilitating delivery of a bioactive molecule to a cell. And compositions comprising them. Particularly useful amphiphiles facilitate the transport of biologically active polynucleotides into cells, and especially into the cells of patients for gene therapy purposes.

Some suitable cationic amphiphiles according to the practice of the present invention are described in US Pat.
, 747,471 and 5,650,096 and PCT publication WO 98/0.
2191, the disclosures of which are hereby incorporated by reference in their entirety. In addition to the cationic amphiphilic compound, these 2
One patent discloses many suitable co-lipids, bioactive molecules, preparations, procedures, routes of administration, and dosages.

In connection with the practice of the present invention, cationic amphiphiles tend to carry one or more positive charges in a solution near physiological pH. Representative cationic amphiphiles useful in the practice of the present invention include: And other amphiphiles known in the art, including those described in US Pat. No. 5,747,471, the disclosure of which is hereby incorporated by reference in its entirety. It is.

PEG Derivatives As discussed above, surprisingly, the stability of cationic amphiphilic compositions (both conventional and micelles) is such that even smaller amounts of one or more It has been confirmed that the above-mentioned derivatized polyethylene glycol compound can be substantially improved by adding the compound. Such enhanced performance is particularly evident as measured by the stability of the cationic amphiphilic preparation to storage and handling.

PEG derivatives were originally used to stabilize conventional cationic amphiphilic preparations. Without being limited by theory, the use of PEG and PEG derivatives allows higher ratios of lipid to DNA to be used. Previous attempts to prepare more concentrated lipid: pDNA complexes using conventional preparations have shown that lipid: pDN, especially where the majority of the pDNA is bound to the lipid.
At the A ratio, the complex precipitated. The precipitation observed at high concentrations in conventional preparations was thought to be related to the phase separation of the cationic lipid component from the non-bilayer lipid component. In an effort to maintain conventional lipid preparations in a bilayer structure, PEG-containing lipids have been found to be effective in preventing complex precipitation at high pDNA concentrations.

To form stable conventional preparations that do not precipitate with high concentrations of lipids and DNA,
Only a small molar fraction of the PEG-containing lipid was used. For example, 1.6 mol% PE
In G-DMPE, the cationic lipid: pDNA complex has more than 20 mM pDNA
The concentration could be stabilized. For more information regarding the use of PEG derivatives, the following references are hereby incorporated by reference in their entirety. Simon J. Eastman et al., Human Gene
Therapy, 8, pp. 765-773 (1997); Simon Jay
Eastman (Simon J. Eastman) et al., Human Gene Therapy.
, P. 8, pp. 313-322 (1997).

Subsequently, PEG derivatives could also be used to prepare new micelle preparations. PEG-containing preparations of micellar conjugates may exhibit unique properties that are not found in conventional preparations of cationic amphiphiles, including PEG derivatives, including improved affinity of the preparation for bioactive molecules.

[0048] The improved binding efficiency results in greater or greater "loading" of DNA per lipid present in the preparation. In the art, PEG
Derivatives are known to stabilize the lipid: bioactive molecule complex and prevent precipitation. However, micelle conjugates containing PEG derivatives can load more bioactive molecules without precipitation than conventional lipid bilayer conjugates, which also contain PEG derivatives. That is, more bioactive molecules are associated with each cationic amphiphile in the micellar complex as compared to the cationic amphiphiles in the conventional cationic amphiphile complex. For example, amphiphile: pDNA 0
. At a molar ratio of 125: 1, all pDNA appears to be associated with the micellar complex. Conventional cationic amphiphilic complexes require a 0.75: 1 to 1: 1 molar ratio of amphiphile: pDNA to completely bind all pDNA. The high affinity of micellar systems for pDNA allows more pDNA to be delivered using less cationic amphiphile.

With respect to the micellar complex, a minimal amount of PEG lipid can form a stable and homogeneous complex when the micellar lipid solution is added to the bioactive molecule. In the practice of the present invention, any derivative of polyethylene glycol may be part of a preparation for preparing a micelle complex. Conjugates were prepared using a variety of PEG derivatives, but all of the PEG derivatives were able to form small, homogeneous conjugates with some minimum cationic amphiphile: PEG derivative ratio. Was. The micellar complex remains stable and homogeneous over a wide range of cationic lipid: PEG and cationic lipid: DNA ratios once the minimum amount of PEG lipid to form a small, homogeneous complex is determined. is there. The minimum amount of PEG to form a stable and homogeneous conjugate can be routinely determined by one skilled in the art.

The minimum amount of PEG used will depend on the particular combination of cationic lipid and PEG lipid selected. For example, cationic lipids containing an acyl chain (GL-89) are less likely to precipitate upon mixing with bioactive molecules than cholesterol-based lipids such as GL-67. This does not suggest that cholesterol-based lipids such as GL-67 are ineffective, but merely use different ratios of cationic lipid: PEG derivatives to form stable and homogeneous micellar complexes. Implies that As a result, a cationic lipid can be selected that is sufficiently stable to form a micelle preparation without the presence of a PEG derivative.

Derivatives of polyethylene glycol useful in the practice of the present invention include any PEG polymer having a hydrophobic group attached to the PEG polymer. For example, P
EG-DSPE, PEG-PE, PEG-DMPE, PEG-DOPE, PEG
-Including DPPE or PEG-ceramide. While not being limited by theory, it is believed that suitable PEG-containing lipids are any PEG polymer derivatives attached to a hydrophobic group capable of stabilizing or interacting with the cationic lipid. Two highly preferred species of this, dimyristoyl phosphatidyl ethanolamine (di C ₁₄₎ ( "DMPE"); and a distearoylphosphatidylethanolamine (di C ₁₈₎ ( "DSPE").

With regard to the choice of PEG polymer, the polymer is linear, from 1,000 to 1
Those having a molecular weight in the range of 000 are preferred embodiments of the present invention. Suitable species of this include those having a molecular weight of 1500-7000, with 2000 and 5000 being examples of useful and commercially available sizes. In the practice of the present invention, it is convenient to use derivatized PEG species provided by vendors, and the molecular weight assigned to PEG in such products often represents a molecular weight average, and Note that there are both short and long molecules. Such molecular weight ranges are typically the result of the synthetic methods used, and the use of any such product is included in the practice of the present invention.

Also, derivatization that comprises (1) two or more linked phospholipids, (2) a branched PEG sequence, or (3) a combination of both modifications (1) and (2). The use of PEG species is also included in the practice of the invention.

Thus, a preferred species of derivatized PEG is: (a) polyethylene glycol 5000, also referred to as PEG ₍₅₀₀₀₎ -DMPE
-Dimyristoyl phosphatidylethanolamine; (b) PEG ₍₂₀₀₀₎ -polyethylene glycol 2000, also called DMPE
-Dimyristoyl phosphatidylethanolamine; (c) PEG ₍₅₀₀₀₎ -polyethylene glycol 5000, also called DSPE.
-Distearoyl phosphatidylethanolamine; (d) PEG ₍₂₀₀₀₎ -polyethylene glycol 2000, also called DSPE.
-Distearoyl phosphatidylethanolamine;

Some phospholipid derivatives of PEG are available from commercial suppliers. For example, the following species: diC14: 0, diC16: 0, diC18: 0, diC18: 1, and 1
6: 0/18: 1 is Avanti Polar Lipids
) (Alabaster, Alabama, USA) as PEG derivatives with an average of 2000 or 5000 MW, catalog numbers 880150, 880
160, 880 120, 880 130, 880 140, 880 210, 8802
00, 880220, 880230, and 880240.

Colipid Selection The use of colipids is optional. Including a neutral, positive, or negative colipid in the micelle complex, depending on the preparation, may substantially enhance delivery and transfection capabilities. Representative co-lipids are dioleoyl phosphatidylethanolamine ("DOPE"), diphytanoyl phosphatidylethanolamine, lyso-phosphatidylethanolamine, other phosphatidyl-ethanol, the most commonly used species in the art. Includes amines, phosphatidylcholine, lyso-phosphatidylcholine, phosphatidyl-inositol and cholesterol. Typically, the preferred molar ratio of cationic amphiphile to co-lipid is about 1: 1. However, varying this ratio, including exceeding the range to be considered, is included in the practice of the invention, but a ratio of 2: 1 to 1: 2 is usually preferred.
The use of diphytanoyl phosphatidylethanolamine, like the use of "DOPE", is highly preferred for the practice of the present invention.

In the practice of the present invention, suitable preparations are also defined in terms of the molar ratio of the PEG derivative, but the preferred ratio will vary with the cationic amphiphile chosen.
A typical preferred micellar preparation for practicing the invention has a molar composition ratio of cationic amphiphile GL-67: colipid DOPE: PEG ₅₀₀₀ of about 1: 1: 0.25. In this preferred example, the co-lipid is diphytanoyl phosphatidylethanolamine, or DOPE, and the PEG derivative is PEG ₂₀₀₀ or P
It is a DMPE or DSPE conjugate of EG _5000.

Biologically Active Molecules Biologically active molecules that may be useful in the practice of the present invention include, for example, genomic DNA
, CDNA, mRNA, antisense RNA or DNA, oligodeoxynucleotides, polypeptides and low molecular weight drugs or hormones. In practicing the present invention, one of ordinary skill in the art can determine, as a routine experiment, which molecules will be effectively delivered to mammalian cells. In the art, once the delivery of a bioactive molecule to a mammalian cell by a cationic amphiphilic complex (or other lipid or non-lipid carrier) has been demonstrated, the selection of other molecules for delivery is It is well known that it becomes a routine task.

Targeting and Targeting Complexes Yet another aspect of the present invention is the use of a targeting agent in any method that achieves delivery of a biologically active molecule into mammalian cells. In a preferred embodiment, the targeting agent is used with conventional and micellar cationic amphiphilic preparations or viral preparations such as viral vectors and adenoviruses. Targeting agents are typically molecules that preferentially target or bind to a particular mammalian cell,
A peptide sequence, or a large protein. Many targeting agents are molecules well known in the art. For example, Pertactin, a peptide containing the RGD sequence, preferentially targets and binds to airway epithelial cells. In the practice of the present invention, the targeting agent or ligand is bound to a carrier complex. It is well known in the art that a single targeting agent targets a particular cell, but binding of the targeting agent to another unit often alters or abolishes the targeting activity of the molecule. However, binding of the targeting substance to the PEG derivative does not always eliminate the activity of the targeting substance. Thus, binding of the targeting agent to the micelle complex preserves the activity of the agent, and binding the targeting agent to the micelle complex is a preferred embodiment of the present invention.

[0060] Binding of the targeting agent to a cationic amphiphilic complex, adenovirus, or other carrier can allow specific targeting to the desired mammalian cells. Targeting to the desired mammalian cells advantageously allows for more efficient delivery of the bioactive molecule, thus increasing transfection of the target mammalian cells.

The lipid complex bound to the targeting agent can be any of the cationic amphiphiles described above,
It may include a PEG derivative, a bioactive molecule, and optionally a co-lipid. In addition, there are preparations where no PEG derivative is required and the targeting agent is directly conjugated to a cationic amphiphile / bioactive molecule complex, optionally including a co-lipid. The lipid complex may be a micelle or a conventional lipid preparation. The targeting substance also
It may be added directly to the PEG derivative, ie PEG-DMPE. In yet another embodiment, the targeting agent binds to a hydrophobic residue, such as a cationic polymer or lipid.

[0062] Any targeting agent known in the art may be useful in the practice of the present invention. Suitable targeting agents are pertactin, a peptide containing an RGD sequence that targets airway epithelial cells; UDP / UTP, which targets P2U receptors, including P2U receptors on cells of the airways; endogenous airways and liver Lactose targeting lectins; and cyclic RGD peptides targeting tumor endothelial cells. Other suitable targeting agent, penetratin amphipathic peptides (Penetratin), the LDL receptor is a substance that targets lectin, the mannose -6-PO ₄ receptor targeting mannose-6-phosphate, And airway-specific single-chain antibodies.

In a preferred aspect of the invention, the peptide ligand is incorporated into a lipid: bioactive molecule complex to increase the transfection activity of the gene transfer system,
Alternatively, it can improve binding to airway epithelial cells.

[0064] Other examples of peptide targeting agents include HAV peptides and CNP-22 peptides. HAV peptides are histidines that regulate cadherin-mediated cell adhesion;
A series of peptides containing the alanine and valine sequences. Without being limited by theory, cadherin complexes maintain tissue integrity by forming cell-cell adhesions and create physical and permeable barriers in the body. Cadherin has been shown to regulate epithelial, endothelial cell, neural and tumor cell adhesion. Cell adhesion involves the extracellular domain of cadherin between cells and intracellular catenin (
catenin) achieved by interaction between the protein and the cytoplasmic domain of cadherin, including the actin cytoskeleton. Histidine, alanine and valine tripeptides (HAV) are located in the extracellular and cytoplasmic domains of cadherin. HAV peptides are critical for the hemophilic interaction between cadherins and play an important role in the catenin protein-mediated interaction with the actin cytoskeleton. The HAV peptide may be linear or cyclic.

In the practice of the present invention, the HAV peptide preferably binds to and is internalized in epithelial cells of the respiratory tract, and thus can be used as a targeting substance for delivering bioactive molecules to the following cells. 1) as a targeting substance for delivering the cystic fibrosis transmembrane conductance regulator (CFTR) gene to airway epithelial cells of CF lung; 2) endothelial cells such as Inhibits angiogenesis around tumors by delivering genes that can cause apoptosis to endothelial cells; 3) nerve cells; and 4) tumor cells. HA
V can also be, for example, 1) poly-L-lysine or linear / branched polyethyleneimine or other polypeptide or (X) -phosphatidylethanolamine (X-PE) by a pDNA complex with or without linker and lipid. For example, N
-MPB-PE); or 2) by chemically linking to a viral vector via a linker, to more efficiently deliver the bioactive molecule to the target cells. It is also possible to use the positively charged HAV of the conjugate for pre-treatment before administration of the negatively charged non-viral or viral vector, or to combine the positively charged non-viral or viral vector with the negatively charged non-viral or viral vector. A mixed complex of charged HAV may be co-administered.

The HAV peptide may also be used as a cell adhesion regulator. A certain HAV
Peptides strongly destroy cell-cell adhesions, and some strongly prevent the formation of cell-cell adhesions. Based on these functions, this peptide alone,
Or tight junction disrupting substances (eg, EGTA or palmitoyl-
L-carnitine or dimethyl β-cyclodextrin or methyl β-cyclodextrin or α-cyclodextrin)
Improved delivery of bioactive molecules to the following cells: 1) CFTR through cell-cell permeable barrier in airway epithelium of CF lung to enhance gene uptake into epithelial cells, eg, basolateral membrane Gene delivery; 2) delivery of genes across the blood-brain barrier to endothelial cells, eg, tumor cells; 3) to increase translocation of nerve cells, eg, vectors; 4) tumor cells, especially solid tumors (eg, Melanomas), because many solid tumors spread inside the barrier, thereby limiting gene delivery to central cells or cells distant from the injection site; and 5) to increase vector transfer Muscle, liver or all other organs by local injection, including the vector.

A C-type natriuretic peptide containing 22 amino acids (CNP-22) binds to the guanylate cyclase-B (GC-B) receptor, a transmembrane receptor containing an intracellular guanylate cyclase domain And activate it. Without being limited by theory, the regulatory pathway for this peptide is cyclic GMP (cG
MP). In the lung, CNP-22 binding and function is predominant in airway epithelial cells. CN to airway epithelial cells in vivo
Specific binding of P-22 has been demonstrated by the functional ability of CNP-22 to increase cGMP levels, active CFTR-dependent chloride transport, and stimulate ciliary beat frequency. Furthermore, CNP-22 bound to 16 lysines (K16-CNP-
22) binds to certain types of epithelial cells in the trachea and other airways of mice.

In practicing the present invention, CNP-22 may be used as a targeting agent.
For example, adenoviral vectors (AdV) into the mouse trachea, other airways, and lungs
) -Mediated gene transfer is increased in mice treated with K16-CNP-22. Cationic lipid to lung: enhancement of pDNA-mediated gene transfer was also demonstrated by K16-
Observed in mice treated with CNP-22. Since CNP-22 and / or GC-B receptors have also been identified in the brain, uterus / tubal tubule, small intestine, colon and kidney, CNP-22 peptides may also be used to deliver bioactive molecules to these organs. Used to target

Also, CNP can be 1) linked to poly-L-lysine, linear / branched polyethyleneimine or other polypeptides or (X) -phosphatidylethanolamine (X-PE; eg N-MPB-PE) The present invention also provides for the more efficient delivery of genes to target cells by chemically linking to a pDNA to create pDNA with or without lipids, or by chemically linking CNP to a viral vector 2) with a linker. Included in the implementation. Also, the bound positively charged CNP can be used for pre-treatment prior to administration of a negatively charged non-viral or viral vector, or can be used to bind a positively charged non-viral or viral vector Mixed complexes of charged CNPs may be co-administered.

In the practice of the present invention, conventional and micellar conjugates containing the targeting agent may be prepared and administered in the same manner and using the same methods as the conjugate without the targeting agent. . Similarly, the ratio of cationic amphiphile: PEG derivative and cationic amphiphile: bioactive molecule depends on the type of lipid used.

Methods of Preparing and Administering Pharmaceutical Compositions The present invention provides pharmaceutical compositions that facilitate delivery and / or transfection of bioactive molecules. The pharmaceutical compositions of the present invention facilitate the delivery of bioactive molecules to tissues and organs such as gastric mucosa, heart, lung, liver, and tumor vasculature, and solid tumors. Further, the compositions of the present invention facilitate the transfer of bioactive molecules into cells maintained in vitro, such as in tissue culture.

The bioactive molecules that can be delivered into cells in therapeutic amounts using the amphiphiles of the invention include (a) genomic DNA encoding therapeutically useful proteins known in the art.
Polynucleotides such as NA, cDNA, and mRNA; (b) ribosomal RNA; (c) useful for inactivating gene transcripts, for example, useful as therapeutics to regulate the growth of malignant cells Antisense polynucleotides (RNA or DNA); (d) ribozymes; and (e) low molecular weight bioactive molecules such as hormones and antibiotics.

The cationic amphiphilic species, PEG derivatives, and co-lipids of the present invention can be used to target two or more species of cationic amphiphiles or PEG derivatives or co-lipids in combination with target cells. And / or to facilitate the transfer of bioactive molecules into their subcellular compartments. The cationic amphiphiles of the invention can also be formulated for use with amphiphiles known in the art. Further, the targeting agent is conjugated to any combination of cationic amphiphile, PEG derivative, and co-lipid.

The dosage of the pharmaceutical composition of the present invention may be determined by the half-life of the bioactive molecule, the potency of the bioactive molecule,
Half-life of the amphiphile, possible adverse effects of the amphiphile or its degradation products,
It will vary according to factors such as the route of administration, the condition of the patient and the like. Such factors can be measured by one skilled in the art.

Various methods of administration can be used to provide very precise dosages of the micelle conjugates and pharmaceutical compositions containing the micelle conjugates of the invention. Such formulations may be orally, intravenously, parenterally, topically, transmucosally, or by injection of the formulation into the body cavity of a patient, or by using a sustained release formulation comprising a biodegradable material, or Administration can be by on-site delivery using additional micelles, gels and liposomes. Nebulizers, powder inhalers, and aerosolized solutions are typical methods used to administer such formulations to the respiratory tract. The use of micelle complexes for systemic delivery is also included in the practice of the invention.

In addition, the therapeutic compositions of the present invention generally contain an excipient (eg, a carbohydrate lactose,
Trehalose, sucrose, mannitol, maltose or galactose, and inorganic or organic salts) and can be lyophilized (and then rehydrated) in the presence of such excipients before use. You may. The conditions of the optimized formulation for each conjugate of the present invention can be determined by those skilled in the field of formulation. Selecting the optimal concentration of a particular excipient for a particular formulation requires experimentation,
One skilled in the art can determine for each formulation.

[0077] Yet another aspect of the invention relates to the cationic amphipathic nature of the conjugates of the present invention prior to contact with the biologically active molecule for delivery or before the exoconjugate contacts the biological fluid. Regarding the protonated state of a substance. Providing a fully protonated, partially protonated, or free base form of the amphiphile to form or maintain such a therapeutic composition is an object of the present invention. Included in implementation.

EXAMPLES Example 1-Preparation of Micelle and Conventional Cationic Lipid: Bioactive Molecule Complex The following example outlines a typical procedure used to prepare a cationic lipid micelle complex. FIG. 1 is a schematic representation of the procedure for the preparation of a conventional cationic lipid complex (a) compared to a cationic lipid micelle complex (b) with and without a co-lipid (c). . The practice of the present invention is not limited to the procedures disclosed herein.

Preparation of cationic lipid: PEG lipid: pDNA micelle complex (1) A micelle cationic lipid: PEG lipid solution was prepared as follows. Cationic lipids were hydrated at four times the desired final cationic lipid concentration of the lipid: pDNA complex (typical but not exclusive range of 0.25-16 mM cationic lipid). The PEG-containing lipid was hydrated at four times the desired final PEG lipid concentration of the lipid: pDNA complex (typical but not exclusive range is 0.25-16 mM cationic lipid). (For PEG lipids, the maximum range of lipid anchors is utilized, and the PEG head group can be any of a variety of sizes.)
Once hydrated, the cationic lipid was added to the PEG lipid in a 1: 1 (volume: volume) ratio. While this is a typical method, it is not essential if the desired ratio of cationic lipid: PEG lipid is finally achieved. Plasmid DNA contains lipid: p
The DNA complex was diluted to twice the desired final pDNA concentration. Next, the cationic lipid: PEG lipid: pDNA complex is converted into a micellar cationic: PEG lipid solution by pDN.
A was prepared by adding to A in a 1: 1 ratio (volume: volume).

(2) A micellar cationic lipid solution was also prepared using the co-lipid as part of the preparation. This was done as described above in (1), except that the co-lipid was combined with the PEG lipid as a lipid film and hydrated as a single solution, or, alternatively, the co-lipid was formulated as a lipid film and Hydrate with lipids. PEG: The co-lipid solution is then
Lipids can be substituted.

Analysis of Micellar Complexes When a micellar lipid solution was added to a bioactive molecule, preferably using a minimal amount of PEG lipid, a stable and homogeneous complex was formed. Furthermore, the minimum amount of PEG required was dependent on the particular combination of cationic lipid and PEG lipid selected. Methods for determining the minimum amount of PEG used to form the micelle complex include, but are not limited to, the following: 1) After adding the micelle lipid to the bioactive molecule, the lipid: bioactive molecule complex Body was observed. If micelle complexes were observed after preparation, the suspension was clear to opaque and free of particles. When particles were observed, the preparation was lacking the minimum amount of PEG to form a suitable stable and homogeneous micellar complex. In comparison, the conventional cationic lipid: pDNA complex was an opaque solution that was entirely free of particles. 2) The micelle lipid: bioactive molecule complex was prepared using a granulator and then sized. When the micelle complex was sized after preparation, the particle population was substantially homogeneous in size and more preferably small (in a preferred embodiment, about 25-250 nm in diameter).
). If the size population contained large heterogeneous particles, minimal amounts of PEG lipids were not present in the preparation. By comparison, the conventional cationic lipid: pD
In the case of the NA complex, particles having a diameter of about 200 to 800 nm were obtained as a whole. These suspensions tended to be very heterogeneous in particle size, and the particle size of the complex was highly dependent on the cationic lipid used in the preparation. The conventional cationic lipid complex showing the small, homogeneous properties observed in the micelle preparation was not observed throughout. 3) The behavior of bioactive molecules in the micelle complex was analyzed by agarose gel electrophoresis. Using the minimum amount of PEG lipids to form stable, homogeneous micellar complexes, the bioactive molecules migrated into the agarose gel in a different manner than the free bioactive molecules (but not to the complexed plasmid). In addition, it was possible to visualize the population of "free" plasmids). If the minimum amount of PEG lipid was not used, the majority of the plasmid visualized in the gel would have: 1) free pDNA
Or 2) retained in the wells of the gel and therefore not visualized on the gel. Two or more of these tests were performed to be confident that the minimum amount of PEG lipid was used.

Analysis of the micelle lipid: pDNA complex by agarose gel electrophoresis was performed by preparing a 0.7% agarose gel in Tris-borate EDTA buffer (pH 8.0). A volume of micelle lipid: pDNA complex containing 0.25-1 μg of pDNA per well was loaded. The gel was run at 100 V for about 1 hour. The gel was then stained overnight in 1 × SYBR Green Nucleic Acid Stain (Molecular Probes) or another stain to visualize the pDNA in the gel.

Example 2 Particle Size Distribution of Micelle Complex In FIGS. 2 and 3, the particle size distribution of a conventional cationic lipid: pDNA complex (FIG.
A and 3A) are compared to the particle size distribution of cationic lipid: pDNA complexes prepared by the micellar method (FIGS. 2B, 2C, 3B and 3C).

The particle size distribution of the composite was measured using Malvern Zeta-Sizer 4.
Measured by quasi-elastic light scattering according to 4). The conjugate was sized within one hour of preparation and measured at the manufacturer's recommended count rate of 50-250 kilocounts per second (KCPS). If necessary, the sample count rate was adjusted to the desired range of 50-250 KCPS by dilution with water.

For the results shown in FIG. 2A, a cationic lipid: pDNA complex was prepared using the cationic lipid GL-67 in a conventional manner. US Patent No. 5,747,4
Nos. 71 and 5,650,096, the disclosures of which are hereby incorporated by reference in their entirety. Briefly, cationic lipid preparations containing cationic lipids, co-lipids, and PEG lipids were either 1) dried from chloroform to lipid films or 2) t-butanol: water (9: 1,
(Volume: volume). The resulting preparation was then hydrated using distilled water to twice the desired final concentration of the three lipids in the complex. Cationic lipid: pDN
The A complex is prepared by adding an equal volume of lipid to pDNA and then gently mixing.
For the particle size distribution shown in FIG. 3A, the same procedure was followed, replacing GL-67 with GL-89.

In FIGS. 2B and 2C, the cationic lipid: p-
DNA complexes were prepared by the micellar method. First, micellar cationic lipid: P
An EG lipid solution was prepared as follows. GL-89 is a cationic lipid: pDN
A conjugate was hydrated at 4 times the desired final cationic lipid concentration. The PEG-containing lipid was also hydrated at four times the desired final PEG lipid concentration of the cationic lipid: pDNA complex. Once hydrated, 1: 1 cationic lipid (volume: volume)
The ratio was added to the PEG lipid. Next, the plasmid DNA is converted into a cationic lipid: pDNA.
The complex was diluted to twice the desired final pDNA concentration. Next, a micelle cationic lipid: PEG lipid solution is added to the pDNA in a 1: 1 ratio (volume: volume),
A cationic lipid: PEG lipid: pDNA complex was prepared. The same procedure was followed for the particle size distributions shown in FIGS. 3B and 3C, except that GL-67 was replaced by GL-89.

As can be seen in FIGS. 2A and 3A, the particle size distribution of conventional cationic lipid complexes varies considerably, for example, from 200 nm to 1000 nm for GL-67. The particle size distribution of the conventional complex does not vary significantly as a function of the cationic lipid: pDNA ratio. 2B and 3B, micelle complexes are formed, but there is no minimum amount of PEG lipid to form a suitable stable, homogeneous micelle complex. As a result, the particle size distribution in FIGS. 2B and 3B has expanded to a particle size greater than 400 nm. Finally, FIGS. 2C and 3C show the particle size distribution of a suitable micellar conjugate prepared with a sufficient amount of PEG lipid. The particle size distribution of a preferred micellar complex is significantly more homogeneous than a conventional cationic lipid complex and significantly more homogeneous than a micellar lipid complex lacking an effective amount of PEG lipid.

Another example of the difference in particle size distribution between a micellar conjugate lacking an effective amount of PEG lipid and a preferred micellar conjugate of the present invention that includes an effective amount of PEG lipid is shown in FIG. Upon addition of an effective amount of PEG lipid, the particle size distribution becomes significantly homogeneous.

Example 3 Binding of Conventional and Micellar Cationic Lipid Complexes to Airway Epithelial Cells In the following example, conventional and micellar cationic lipid: pDNA complexes bind to the surface of unequalized normal human bronchial airway cells Consider both body abilities.

Propagation of unequalized normal human epithelial cells at the air-liquid interface Human collagen (Sigma, human placental collagen, # 7521)
. Cell culture flasks were coated with human collagen at 50 mg / 100 mL in 2% glacial acetic acid. Once dissolved, the collagen solution is filtered through a 0.45 μm filter. This concentrated sterile stock can be stored at 4 ° C. for 6 months. Solutions were prepared for flask coating by diluting the collagen stock 1: 5 (vol: vol) with sterile distilled water for several minutes before use. Place the appropriate solution of diluted collagen in the flask (12 mL for T75 flask, 24 mL for T150 flask)
And 400 μl for each Millicell-PCF insert
L), left at 4 ° C for at least 2 hours (preferably overnight). After incubation at 4 ° C., the collagen was removed from the flask / insert and placed in a sterile draft to dry for 6-12 hours. The flask / insert was washed twice with sterile phosphate-buffered saline (pH 7.4) containing penicillin / streptomycin.
These flasks can be kept at room temperature for up to 6 months.

One vial of normal human bronchial epithelial cells (Clonetics) was quickly thawed and distributed into five T150 flasks (pre-coated with human collagen as described above). DMEM cells in flasks (Gibco (Gibco / BRL)): BEGM of (Clonetics (Clonetics)) 1: 1 (vol: vol) were grown at 5% CO ₂ environment by medium. Cells were grown to 80-90% confluence. The cells were then pre-coated with human collagen as described above with a Millicell-PCF insert (200).
μl, 2 × 10 ⁵ cells / 200 μl medium). Twenty-four hours after inoculation, the medium was removed from inside the insert and the medium outside the insert was replaced with fresh medium.
The cells were then maintained at gas-liquid interface conditions by changing the outer media every other day. Approximately 5-7 days after switching to gas-liquid interface conditions, cells developed high trans-epithelial resistance.

Investigation of Conventional and Micellar Complex Binding to Unequalized Normal Human Bronchial Airway Epithelial Cells Normal human bronchial airway epithelial cells were grown at the air-liquid interface as described above. Cells were maintained at the air-liquid interface for about 5 days or until trans-epithelial resistance of about 1000 Ω / cm ² developed. The cells are now ready for use in binding experiments.

The plasmid DNA was prepared using the fluorescent probe Toto-1-iodide.
Labeled non-covalently by (Molecular Probes) at a 1: 200 molar ratio of toto-1-iodide to pDNA according to the manufacturer's instructions. Micelles and conventional lipid: pDNA complexes were prepared at 10 times the desired lipid: pDNA concentration for use in experiments using toto-1 labeled plasmids.
The micelle complex was prepared as described above. Conventional conjugates were prepared by hydrating a conventional lipid film with water to 20 times the desired final experimental lipid concentration. Labeled pDNA was prepared at 20 times the desired final experimental DNA concentration. Lipids were added to the labeled pDNA and allowed to form complexes for 15 minutes. The complex was then diluted 1:10 (volume: volume) with Optimem.

The complex (about 300 μl) was added to the apical membrane of airway epithelial cells inside the insert and allowed to bind for 1 hour at 37 ° C. under a 5% CO ₂ atmosphere. The complexes were then aspirated from the cell surface; the cell surface was washed three times with 0.5 mL cold PBS; fixed with 2% paraformaldehyde in PBS for 15 minutes; and washed once with 0.5 mL cold PBS. The insert is then cut from the insert housing, mounted on a slide, covered with a coverslip, and placed on an Immunomount (Shandon-Lipshaw) containing 2 μg / mL DAPI as a nuclear counterstain. Mounted.

At a cationic lipid: pDNA ratio of 50:50, 75:50 μM, strong binding of the micelle complex to the apical surface of airway cells was observed throughout. 50:50, 75:
At a cationic lipid: pDNA ratio of 50 μM, there was little binding of the conventional complex in the same environment. This method will be applicable to essentially any adherent cell line.

[Brief description of the drawings]

FIG. 1 compares the procedure for the formulation of a conventional lipid complex (a) with a micelle complex (b) with and without a co-lipid (c).

FIG. 2 shows a conventional cationic lipid GL-67: pDNA complex (a) (GL-6).
7: pDNA (0.5: 0.5) and GL-67: DOPE: DMPE-PE
The particle size distribution of G5000 (1: 2: 0.05) was measured using the micelle complex ((b) and (c)).
) Is compared with the particle size distribution. (B) (GL-67: DMPE-PEG5
000: pDNA (1.5: 0.5: 2)) represents the particle size distribution of the micellar complex lacking the minimum amount of PEG required to form a suitable homogeneous complex, while (c
) (GL-67: DMPE-PEG5000: pDNA (1.5: 0.75: 2
)) Shows the particle size distribution of the micelle conjugate prepared with a sufficient amount of PEG.

FIG. 3 shows a conventional cationic lipid GL-89: pDNA complex (a) (GL-8
9: pDNA (2: 2) and GL-67: DOPE: DMPE-PEG500
The particle size distribution of 0 (1: 1: 0.005) was measured using the micelle complex ((b) and (c)).
Is compared with the particle size distribution. (B) (GL-89: DMPE-PEG50
00: pDNA (1.5: 0.0025: 2)) represents the particle size distribution of the micellar complex lacking the minimum amount of PEG required to form a suitable homogeneous complex, while (c) (GL-67: DMPE-PEG5000: pDNA (1.5: 0.25
: 2)) shows the particle size distribution of the micelle conjugate prepared with a sufficient amount of PEG.

FIG. 4 shows the change in the particle size distribution of the micelle cationic lipid GL-67: pDNA complex as the amount of co-lipid and PEG (DOPE: DMPE-PEG ₅₀₀₀ ) increases. A minimum amount of PEG is required to form a small, homogeneous and stable micellar complex.

[Procedural Amendment] Submission of translation of Article 34 Amendment of the Patent Cooperation Treaty

[Submission date] June 30, 2000 (2000.6.30)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 47/34 A61K 47/34 47/42 47/42 47/44 47/44 48/00 48/00 C12N 15/00 C12N 15/00 (72) Inventor Eastman, Simon, J. United States of America Massachusetts, Hudson, Birchwood Road 7 (72) Inventor of New Tapskiy, Ginifer, Be United States of America Massachusetts, Millbury, Victoria 1 (72) Inventors Lee, Edward, Earl Massachusetts, United States, Natick, Bacon Street 56 (72) Inventor Chu, Quimin United States Massachusetts, Melrose, Florence Street 110 (72) Inventor , Ronald, Kay United States Massachusetts, Hopkinton, East Street 25 (72) Inventor Marshall, John United States Massachusetts, Hopedale, Northrop Street 25 F-term (reference) 4B024 AA01 AA20 CA01 HA17 4C076 AA19 AA95 CC15 DD15 DD16 DD53 DD63 EE23 EE41 4C084 AA01 AA13 AA17 BA03 MA24 NA13 ZA592 4C085 AA21 BB31 EE01 4C086 AA01 EA16 MA03 MA05 MA24 NA13 ZA59

Claims (30)

[Claims] 1. A process for producing a micelle complex, comprising: a) combining at least one cationic lipid with a sufficient amount of a PEG derivative in an amount suitable to produce a substantially homogeneous micellar lipid; b) combining a substantially homogeneous micellar lipid and at least one bioactive molecule to form a micelle complex. 2. The method according to claim 1, wherein the PEG derivative is conjugated to a co-lipid before step a). 3. The method for producing a micelle complex according to claim 1, wherein the bioactive molecule is DNA. 4. The method of claim 4, wherein the at least one cationic lipid and the DNA are present at a lipid: DNA ratio of 1: 8. 5. The method for producing a micelle composite according to claim 1, wherein the particle size distribution of the micelle composite group varies less than 20% with respect to the average particle size of the composite in the micelle composite group. 6. The method for producing a micelle complex according to claim 1, further comprising a step of coating the micelle complex with at least one hydrophobic species. 7. The method for producing a micelle complex according to claim 1, further comprising adding a substance targeting a mammalian cell. 8. The substance for targeting is a peptide containing RGD, UDP / U
TP, lactose, cyclic RGD peptide, penetratin, lectin, substance targeting LDL receptor, mannose-6-phosphate, HAV peptide, CNP-2
The method for producing a micelle complex according to claim 7, which is selected from two peptides and an airway-specific single-chain antibody. 9. A micelle complex produced according to claim 1. 10. The micelle complex produced according to claim 2. 11. The micelle complex according to claim 9, wherein the micelle complex further comprises a substance targeting a mammalian cell. 12. The substance for targeting is a peptide containing RGD, UDP /
UTP, lactose, cyclic RGD peptide, penetratin, lectin, LDL
Receptor targeting substance, mannose-6-phosphate, HAV peptide, CNP-
The micelle complex of claim 11, wherein the micelle complex is selected from 22 peptides, and an airway-specific single chain antibody. 13. The micelle complex of claim 9, wherein the micelle complex further comprises a hydrophobic species for coating the micelle complex. 14. The micelle complex according to claim 9, wherein the bioactive molecule is DNA. 15. The micelle complex of claim 14, wherein said at least one cationic lipid and DNA are present in a lipid: DNA ratio of 1: 8. 16. The micelle complex according to claim 9, wherein the particle size distribution of the micelle complex group varies by less than 20% with respect to the average particle size of the complex in the micelle complex group. 17. A method of delivering a biologically active molecule to a cell, comprising contacting a mammalian cell with a complex comprising a micelle complex, wherein the micelle complex comprises at least one cation A method as described above, comprising a sex lipid; at least one bioactive molecule; and at least one PEG derivative. 18. The micelle complex of claim 17, further comprising a co-lipid.
A method for delivering a biologically active molecule to mammalian cells as described. 19. The method of claim 1, wherein the at least one bioactive molecule is DNA.
8. The method for delivering a biologically active molecule to mammalian cells according to 7. 20. The at least one cationic lipid and DNA comprise 1: 8
20. The method of delivering a biologically active molecule to mammalian cells according to claim 19, wherein said bioactive molecule is present in a lipid: DNA ratio. 21. The method for delivering a biologically active molecule to mammalian cells according to claim 17, wherein the micelle complex further comprises a hydrophobic species for coating the micelle complex. 22. The method for delivering a biologically active molecule to mammalian cells according to claim 17, wherein the micelle complex further comprises a substance that targets mammalian cells. 23. A substance for targeting is a peptide containing an RGD sequence, UD
P / UTP, lactose, cyclic RGD peptide, penetratin, lectin, L
Substance targeting DL receptor, mannose-6-phosphate, HAV peptide, CN
23. The method of delivering a biologically active molecule to mammalian cells according to claim 22, wherein the method is selected from a P-22 peptide and an airway-specific single chain antibody. 24. The method of claim 17, wherein the cell is an airway epithelial cell. 25. A particle size of a micellar complex comprising at least one cationic lipid; at least one PEG derivative; and at least one bioactive molecule, the micelle complex comprising said micellar complex. Such a composite, wherein the distribution has a substantially homogeneous particle size distribution. 26. The micelle complex further comprises a co-lipid.
A micellar complex as described. 27. The micelle composite of claim 25, wherein the substantially homogeneous particle size distribution of the micelle composites varies by less than 20% relative to the average particle size of the composites in the micelle composites. 28. The micelle complex according to claim 25, wherein the bioactive molecule is DNA. 29. The micelle complex according to claim 25, wherein the micelle complex further comprises a substance targeting a mammalian cell. 30. A substance for targeting is a peptide comprising an RGD sequence, UD
P / UTP, lactose, cyclic RGD peptide, penetratin, lectin, L
Substance targeting DL receptor, mannose-6-phosphate, HAV peptide, CN
30. The micelle complex of claim 29, wherein the micelle complex is selected from a P-22 peptide and an airway-specific single chain antibody.