Transport vehicles for macromolecules

Details Transport vehicles for macromolecules Transport vehicles for macromolecules

1998-12-28

2004-04-27

Ceperley, Mary E. (Department: 1641)

Drug, bio-affecting and body treating compositions

Radionuclide or intended radionuclide containing; adjuvant...

Molecular bilayer structure

C424S001490, C424S178100, C424S450000, C514S332000, C514S358000, C514S356000, C546S263000, C546S321000, C546S347000

Reexamination Certificate

active

06726894

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention related to new compounds which are capable of introducing macromolecules into eucaryotic cells.
The introduction of macromolecules, including DNA, proteins and the like, into eucaryotic cells can be carried out in different ways, for instance by means of transport vehicles. Such vehicles introduce a molecule into the cell, for instance by means of endocytosis. The vehicles may bind, but for instance also encapsulate, the molecules to be transported. In the latter case the vehicles are referred to as vesicles. Known vesicles are liposomes which consist of a bilayer of phospholipids.
Liposomes are for instance used to introduce medicines into the cell. It appeared that liposomes are incorporated into the cell both in vivo and in vitro by means of endocytosis (Nandi, P. K. et al. (1986) J. Biol. Chem. 261:16722; Heath, T. D. (1987) Methods Enzymol. 149:111). This means that the largest portion of the material which is incorporated in the cell will ultimately appear in the lyposomal apparatus, where it will be decomposed. Particularly for substances which have their effect in the cytoplasm or the nucleus this is obviously very disadvantageous.
If the substances to be introduced are hydrophilic it will be difficult to introduce them into liposomes. The main portion of the material remains in the aqueous phase. Particularly in case of expensive substances, like probes and many medicines, this is an obvious disadvantage.
To prevent that the substances to be introduced into the cell end up in the cell by means of endocytosis, attempts have been made to use fusogenic phospholipids as transport vehicles. When fusogenic phospholipids have bound (hydrophilic) substances and have formed vesicles, the vesicles will introduce there substances into the cell after fusion with the cell membrane. However, such attempts have not proven to be very successful because fusogenic liposomes have a strong tendency to mutually merge instead of fusing with the cell membrane (Fonteijn, T. A. A., Ph.D. Thesis (1992)).
One of the most important applications in which molecules are introduced into a cell is transfection of the (eucaryotic) cell with DNA or RNA. Transfection is being used for studying the function and regulation of genes and proteins, but also for the genetic modification of micro-organisms, plants and animals. There is a large number of artificial techniques which allow DNA to be introduced into a cell, including DNA-micro-injection, DNA-coprecipitation within inorganic salts or with polycations, DNA-encapsulation in liposomes, and making the cell membrane permeable with the aid of chemical or physical means.
A more recent technique involves the use of cationic amphiphilic molecules as transport vehicles. One of the best-known amphiphiles is the quaternairy ammonium amphiphile DOTMA (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride) which in combination with dioleyol phosphatidyl ethanolamine (DOPE), is commercially available with the name Lipofectinem™. Both molecules are lipidic(analogues), which form liposomes, which will form complexes with the negatively charged nucleic acids. Supposedly, the liposomes merge with the plasma membrane and introduce in this way nucleic acids into the cell. However, it could also be done by means of endocytosis. The exact mechanism is yet unknown. With the aid of Lipofectinem™ the transfection efficiency may be enhanced by a factor of 30 with respect to other known systems, including the classical calcium phosphate precipitation method. However, the disadvantage of Lipofectinel™ is its toxicity and therefore it may be difficult or not possible to use it in vivo. Therefore, a demand still remains for other and better transfection methods.
SUMMARY OF THE INVENTION
It is the aim of the present invention to provide new cationic amphiphilic compounds, which allow high efficiencies, for the introduction into a cell of nucleic acids and other macromolecules, including for example proteins and medicines.
The aim of the invention is achieved by compounds of general formula I:
in which:
R
1
is a
(a) branched or linear (C
1
-C
5
)alkyl, or
(b) branched or linear ((C
1
-C
5
)alkyl)aryl, or
(c) branched or linear ((C
1
-C
5
)alkyl)N
+
(CH
3
)
3
, or
(d) branched or linear ((C
1
-C
5
)alkyl)
2
R
2
,R
3
,R
4
-pyridinium in compliance with formula I
X
−
is a halide counter ion chosen from Cl
−
, I
−
or Br, and in which
R
3
is hydrogen and R
2
and R
4
are identical or different and are selected from the group consisting of branched or linear: (C
10
-C
20
)alkyl, mono- or polyunsaturated (C
10
-C
20
)alkenyl, (C═O)—O—(C
10
-C
20
)alkyl, O—(C═O)—(C
10
-C
20
)alkyl, or ((C
10
-C
20
)alkyl)aryl, or
R
2
and R
4
are hydrogen and R
3
is CHR
5
R′
5
in which R
5
and R′5 are identical or different and are selected from the group consisting of hydrogen branched or linear: (C
10
-C
20
)alkyl, mono- or polyunsaturated (C
10
-C
20
)alkenyl, (C═O)—O—(C
10
-C
20
)alkyl, O—(C═O)—(C
10
-C
20
)alkyl, or ((C
10
-C
20
)alkyl)aryl.


REFERENCES:
patent: 4824850 (1989-04-01), Bodor
patent: 4957735 (1990-09-01), Huang
patent: 0 516 194 (1992-12-01), None
patent: 516194 (1992-12-01), None
Sudholter et al, JACS, vol. 102, No. 7, Communications to the Editor, 2467-2469, 1980.*
Sudholter et al, JACS, vol. 104, 1069-1072, 1982.*
Sudholter et al, J. Phys. Chem., vol. 86, 1908-1913, 1980.*
N.S. Bodor, “Preparation Of Dihydropyridine-Containing Prodrugs For Brain-Specific Drug Delivery,”Chemical Abstract, vol. 112:7383s, No. 1, 1990.
E.J.R. Sudholter et al., “Thermotropic Liquid-Crystalline Behavior of Some Single- and Double-Chained Pyridinium Amphiphiles,”Chemical Abstract, vol. 96:19111b, No. 22, 1982.
J.J.H. Nusselder et al., “Liquid-Crystalline And Thermochromic Behavior Of 4-Substituted 1-Methylpyridinium Iodide Surfactants,”Chemical Abstract, vol. 118:202541t, No. 20, 1993.
J.J.H. Nusselder et al., “Relation Between Surfactant Structure And Properties Of Spherical Micelles,”Chemical Abstracts, vol. 115:240420y, No. 22, 1991.
M. DeCuyper et al., “Exchangeability of Phospholipids Between Anionic, Zwitterionic and Cationic Membranes,”Chemical Abstracts, vol. 101:165887m, No. 19, 1984.
A.R. Katritzky et al., “Utilization Of Pyridinium Salts As Microsensor Coatings,”Chemical Abstracts, vol. 111:208460t, No. 22, 1989.
P.L. Felgner et al., “Lipofection: A Highly Efficient, Lipid-mediated DNA-transfection Procedure,”Proc. Natl. Acad. Sci. USA 84:7413-7417 (Nov. 1987).
Sudhölter et al.,JACS, 102(7):2467-2469 (1980).
Fadnavis et al.,JACS, 47:2923-2928 (1982).
Sudhölter et al.,J. Phys. Chem., 86:1908-1913 (1982).
Sudhölter et al.,J. Phys. Chem., 86:263-271 (1982).
Sudhölter et al.,J. Am. Chem. Soc., 104:1069-1072 (1982).
Nusselder et al.,J. Org. Chem., 53: 2423-2426 (1988); and.
Stryer,Biochemistry, W.H. Freeman and Company, New York, NY, pp. 207 and 212-213 (1981).

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