Drug – bio-affecting and body treating compositions – Preparations characterized by special physical form – Liposomes
Reexamination Certificate
1998-09-11
2003-09-02
Siew, Jeffrey (Department: 1632)
Drug, bio-affecting and body treating compositions
Preparations characterized by special physical form
Liposomes
C536S023100, C530S350000
Reexamination Certificate
active
06613351
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to a compound capable of introducing at least one molecule into a cell.
The invention also relates to a positively charged vesicle whose membrane comprises this compound, to a vector containing at least one molecular combined with the vesicle according to the invention; to the cell, animal and/or plant transformed by said compound or said vector as well as to the pharmaceutical or cosmetic composition, comprising said vector and/or the cell transformed by said vector.
The invention also relates to the process for the synthesis of said compound and to the process for the production of said vector.
Another aspect of the invention relates to the use of the compound and/or vector for the introduction in vitro and/or in vivo of at least one molecule into a cell.
TECHNOLOGICAL BACKGROUND AND PRIOR STATE OF THE ART WHICH FORM THE BASIS OF THE INVENTION
Various processes are used in genetic engineering and/or in pharmacy to introduce molecules such as nucleic acids or active therapeutic agents into cells.
Transfection is a method which is widely used for introducing genetic material into cells, for studying the expression of genes, and for developing strategies for gene therapy.
Numerous experimental procedures using physical modifications (microinjection, electroporation and the like) or chemical modifications (dextran phosphate, calcium phosphate and the like) of membranes have been developed with varying success for introducing nucleic acids into cells using this method.
Another axis of research relates to the development of new amphiphilic cationic vectors which have demonstrated their effectiveness and their ease of use for causing the genetic material to penetrate into cells in vitro (P. L. Felgner et al., Proc. Natl. Acad. Sci., USA, 84, pp. 7413-7417 (1987)).
Some of these vectors such as the Lipofectin® N-(2,3-dioleyloxy)propyl-N,N,N-trimethylammonium chloride (DOTMA) from GIBCO BRL or the Transfection-reagent® N-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium methyl sulfate (DOTAP) from Boehringer Mannheim GmbH, have been commercialized.
Patent Application WO91/15501 (YALE UNIVERSITY) also describes a positively charged reagent consisting of a neutral phospholipid such as dioleoylphosphatidylethanolamine and a cationic lipid such as stearylamine, a tertiary amine or a benzothorium salt for the transfection of nucleic acids.
Nevertheless, these products require large quantities of reagents in order to obtain an effective transfection of cells and therefore increase the cytotoxicity of the products and their costs (thus, the price of the “Lipofectin®” product (DOTMA) in U.S.$ 145/ml, equivalent to U.S. $10 per transfection).
In addition, Lipofectin® (DOTMA) has the additional disadvantage of not being capable of being added to a serum.
The document “Vesicle Formation by double long-chain Amidines” (Fabienne Defrise-Quertain et al.) (J. Chem. Soc., Chem. Commun., 1986, p. 1060 to 1062) describes the formation of vesicles consisting of 3-tetradecylamino-N-tert-butyl-N′-tetradecylpropionamidine.
However, this document does not contain sufficient description for the preparation of these compounds. Indeed, this document does not indicate the reaction temperatures to be used or the fact that it is necessary to use a basic aqueous solution in order to form the compound of formula
in which OR is the residue of an alcohol of formula ROH and R
2
is selected from the group consisting of hydrogen atom, saturated alkyl chains, unsaturated alkyl chains and substituted alkyl chains; an intermediate compound which is necessary for the production of the compounds according to the invention.
AIMS OF THE INVENTION
The aim of the present invention is to produce a new compound and/or a new vector capable of introducing at least one molecule into a cell, without exhibiting the disadvantages of the prior state of the art.
The invention is also intended for producing a pharmaceutical or cosmetic composition comprising said vector and/or said cell transformed by said vector.
An additional aim of the present invention is intended for producing a vector which can be used in a serum.
CHARACTERISTIC ELEMENTS OF THE INVENTION
The present invention relates to a compound of the general formula:
in which:
A is selected from the group consisting of the radicals comprising a hydrophilic group or a CH
2
group and a hydrophobic group;
n is a positive integer (1<n<10; more preferably n=2); and
R
2
, R
3
and R
4
are selected from the group consisting of a hydrogen atom, saturated alkyl chains, unsaturated alkyl chains and substituted alkyl chains.
Preferably, A represents a radical or residue of formula:
R
1
—X—
in which
R
1
is selected from the group consisting of a hydrogen atom, saturated alkyl chains, unsaturated alkyl chains and substituted alkyl chains, and
X represents a divalent atom or a divalent group of atoms.
R
1
—X represents a residue selected from the group consisting of:
Preferred compounds according to the invention are the nitrogen derivatives above-described.
Advantageously, R
1
and R
3
or R
1
and R
4
are selected from the group consisting of hydrocarbon chains containing 2 or more carbon atoms, preferably 12, 14, 16 or 18 carbon atoms chains.
According to a preferred embodiment of the invention, the compound according to the invention corresponds to the following formula:
in which 12≦m≦18 (m=12, 14, 16 or 18), and X represents the divalent atoms or divalent groups of atoms above-described.
According to another preferred embodiment of the present invention, the compound according to the invention has the following formula:
wherein m=12, 14, 16 or 18.
The invention also relates to a positively charged vehicle having a membrane comprising a compound according to the invention as well as a vector consisting of said vesicle associated with at least one molecule to be introduced into a cell, said molecule being advantageously selected from the group consisting of nucleic acids (nucleic acids oligomers or nucleic polymers), antigens, polypeptides or optionally glycosylated proteins and/or active therapeutic or cosmetic agents.
Said molecule is introduced in a cell by a process wherein said cell is brought into contact with said molecule and a compound and/or a vesicle according to the invention. Likewise, the introduction process can be achieved by bringing said cell into contact with a vector according to the invention.
A “nucleic acid according to the invention” means any kind of nucleic acids polymer of oligomer such as a plasmid, messenger RNAs, antisense RNAs, cDNAs, synthetic oligonucleotides and the like which are capable of genetically transforming a cell and which may express a specific antigen, polypeptide or protein as well as an active therapeutic or cosmetic agent into said cell.
According to a first embodiment of the process of the invention, a cell is treated in vitro in order to produce plants or animals which are transgenic or for gene therapy, in particular for the treatment of cellular disorders such as cancer or infections such as viral or bacterial infections or the like.
According to the second preferred embodiment of the process of the invention, a cell is treated in vivo in order to produce recombinant microorganisms, plants and/or animals which are transgenic or in for ex vivo or in vivo gene therapy.
Another aspect of the invention concerns a new composition comprising a molecule to compact DNA, preferably a protamine, a polylysine or a histone derived peptide, and the compound, the vesicle or the vector according to the invention.
The composition according to the invention may also comprise another compound which increases cell endocytosis, preferably a microtubule inhibitor.
Another aspect of the present invention is related to a transfection kit comprising said composition, compound(s), vesicle or vector according to the invention, for the transfection of cells, plants or animals.
The present invention also relates to the cell, the animal or the plant transformed by the vector
Fuks Robert
Ruysschaert Jean-Marie
Vandenbranden Michel
Biotech Tools S.A.
Merchant & Gould P.C.
Schnizer Richard
Siew Jeffrey
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