Drug – bio-affecting and body treating compositions – Preparations characterized by special physical form – Liposomes
Reexamination Certificate
1998-05-13
2001-01-09
Kishore, Gollamudi S. (Department: 1615)
Drug, bio-affecting and body treating compositions
Preparations characterized by special physical form
Liposomes
C424S121000, C424S009321, C424S009510, C514S04400A, C564S225000, C564S226000
Reexamination Certificate
active
06171612
ABSTRACT:
This application is a 371 of PCT/FR96/01774 filed Nov. 8, 1996.
The present invention relates to novel compounds similar to the family of lipopolyamines, to pharmaceutical compositions containing them, to their applications for the in vivo and/or in vitro transfection of nucleic acids and to a process for their preparation.
Many genetic diseases are associated with an expression defect and/or abnormal expression, that is to say deficient or excessive expression, of one or more nucleic acids. The main aim of gene therapy is to correct genetic abnormalities of this type by means of the in vivo or in vitro cellular expression of cloned genes.
Today, several methods are proposed for the intracellular delivery of this type of genetic information. One of them in particular is based on the use of chemical or biochemical vectors. Synthetic vectors have two main functions, to compact the DNA to be transfected and to promote its cellular binding as well as its passage across the plasma membrane and, if necessary, across the two nuclear membranes.
Considerable progress has been achieved in this mode of transfection, with the development of technology based on the use of a cationic lipid. It has thus been demonstrated that a positively charged cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), interferes spontaneously, in the form of liposomes or small vesicles, with DNA, which is negatively charged, to form lipid-DNA complexes, which are capable of fusing with cell membranes, and thus allows the intracellular delivery of DNA. However, although this molecule is effective in terms of transfection, it has the drawback of being non-biodegradable and of having a toxic nature with regard to cells.
Since DOTMA, other cationic lipids have been developed along the lines of this structural model: lipophilic group combined with an amino group via a so-called “spacer” arm. Among these, mention may be made more particularly of those comprising, as lipophilic group, two fatty acids or a cholesterol derivative, and additionally containing, if necessary, as amino group, a quaternary ammonium group. DOTAP, DOBT and ChOTB may be mentioned in particular as representatives of this category of cationic lipids. Other compounds, for instance DOSC and ChOSC, are characterized by the presence of a choline group in place of the quaternary ammonium group. In general, however, the transfecting activity of these compounds remains fairly low.
Another category of cationic lipids, lipopolyamines, has also been described. For the purposes of the present invention, the term lipopolyamine denotes an amphiphilic molecule comprising at least one hydrophilic polyamine region combined, via a so-called spacer region, with a lipophilic region. The polyamine region of lipopolyamines, which are cationically charged, is capable of combining reversibly with nucleic acid, which is negatively charged. This interaction compacts the nucleic acid greatly. The lipophilic region renders this ionic interaction insensitive to the external medium, by coating the nucleolipid particle formed with a lipid film. In compounds of this type, the cationic group may be represented by the L-5-carboxyspermine radical which contains four ammonium groups, two primary and two secondary. DOGS and DPPES are in particular among the compounds of this type. These lipopolyamines are most particularly effective for transfecting primary endocrine cells.
In point of fact, an ideal synthetic transfecting agent should display a high level of transfection, and should do so for a broad spectrum of cells, should have no toxicity or, failing that, a very minimal toxicity at the doses used, and, lastly, should be biodegradable so as to be rid of any side-effects on the cells treated.
The object of the present invention is, precisely, to propose novel lipopolyamines, which are original on the basis of their polyamine fraction, and which can be used effectively in the in vitro and/or in vivo transfection of cells and in particular for the vectorization of nucleic acids.
A first subject of the present invention is lipopolyamines, in D, L or D,L form and their salts, characterized in that they are represented by the general formula I
in which:
R1, R2 and R3 represent, independently of each other, a hydrogen atom or a group —(CH
2
) q—NRR′ with
q able to range between 1, 2, 3, 4, 5 and 6, and doing so independently between the various groups R1, R2 and R3 and
R and R′ representing, independently of each other, a hydrogen atom or a group —(CH
2
)q′—NH
2
, q being able to range between 1, 2, 3, 4, 5 and 6, and doing so is independently between the various groups R and R′,
m, n and p represent, independently of each other, an integer which may vary between 0 and 6 with, when n is greater than 1, m able to take different values and R3 able to take different meanings within the general formula I, and
R4 represents a group of general formula II
in which:
R6 and R7 represent, independently of each other, a hydrogen atom or a saturated or unsaturated C10 to C22 aliphatic radical with at least one of the two groups being other than hydrogen,
u is an integer chosen between 0 and 10 with, when u is an integer greater than 1, R5, X, Y and r able to have different meanings within the different units [X—(CHR5)r—Y]
X represents an oxygen or sulphur atom or an amine group which may or may not be monoalkylated,
Y represents a carbonyl group or a methylene group R5 represents a hydrogen atom or a side chain of a natural amino acid, which is substituted if necessary, and
r represents an integer ranging between 1 and 10 with, when r is equal to 1, R5 representing a side chain of a natural amino acid and, when r is greater than 1, R5 representing a hydrogen atom.
For the purposes of the invention, the expression side chain of a natural amino acid is understood in particular to denote chains containing amidinium units such as, for example, the side chain of arginine. As mentioned above, this chain may be substituted with saturated or unsaturated, linear, branched or cyclic C1 to C24 aliphatic groups such as, for example, cholesteryl, arachidonyl or retinoyl radicals and mono- or polyaromatic groups such as, for example, benzyloxycarbonyl derivatives, benzyl ester derivatives and substituted or unsubstituted rhodaminyl derivatives.
These novel products of general formula (I) may be in the form of non-toxic and pharmaceutically acceptable salts. These non-toxic salts comprise salts with inorganic acids (hydrochloric acid, sulphuric acid, hydrobromic acid, phosphoric acid and nitric acid) or with organic acids (acetic acid, propionic acid, succinic acid, maleic acid, hydroxymaleic acid, benzoic acid, fumaric acid, methanesulphonic acid and oxalic acid) or with inorganic bases (sodium hydroxide, potassium hydroxide, lithium hydroxide and lime) or organic bases (tertiary amines such as triethylamine, piperidine and benzylamine).
Representatives of the compounds according to the invention which may be mentioned more particularly are the compounds of the following general sub-formulae:
in which R4, R6 and R7 have the above definitions. Preferably, R4 represents therein an NR6R7 group with R6 and R7 appearing in subformulae III to XII as an identical group chosen from (CH
2
)
17
CH
3
, (CH
2
)
11
CH
3
, (CH
2
)
13
CH
3
or (CH
2
)
12
CH
3
.
In a particularly advantageous embodiment, the compounds claimed also comprise a targeting element which makes it possible to direct the transfer of the nucleic acid with which they are combined. This targeting element is preferably incorporated, on the compound of general formula I, in the amino acid side chain featured by the substituent R5. More preferably, the targeting element is attached, covalently or non-covalently, to the compound according to the invention.
This element may be an extracellular targeting element which makes it possible to direct the transfer of the nucleic acid towards certain desired cell types or certain desired tissues (tumour cells, liver cells, haematopoie
Byk G{acute over (e)}rardo
Dubertret Catherine
Scherman Daniel
Schwartz Bertrand
Aventis Pharma S.A.
Comolly, Bove, Lodge & Hutz LLP
Kishore Gollamudi S.
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