Process for preparing functionalized polyalkyleneimines,...

Details Process for preparing functionalized polyalkyleneimines,... Process for preparing functionalized polyalkyleneimines,...

2001-02-16

2004-11-30

Epps-Ford, Janet L. (Department: 1635)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C525S054200, C435S455000

Reexamination Certificate

active

06825341

ABSTRACT:

The present invention relates to a process for preparing functionalized polyalkyleneimines which are useful for formulating nucleic acids intended to be transfected into cells.
With the development of biotechnologies, the possibility of effectively transferring nucleic acids into cells has become a fundamental technique with numerous biotechnological applications. This can involve the in vitro transfer of nucleic acids into. cells, for example for the production of recombinant proteins, or in the laboratory to study the regulation of gene expression, the cloning of genes or any other manipulation involving DNA. It can also involve the in vivo transfer of nucleic acids into cells, for example to prepare vaccines, for labelling studies or for therapeutic approaches. It can also involve the transfer of genes into cells taken from an organism, for the purpose of readministering them subsequently, for example for the creation of transgenic animals.
Currently, the means most commonly used for transferring genes into cells is the use of viral vectors. However, since these are not entirely free of risks, several other methods based on the use of synthetic vectors have been proposed. These synthetic vectors have two main functions: to complex and compact the nucleic acid to be transfected, and to promote its passage across the plasma membrane and optionally across the two nuclear membranes.
Several families of synthetic vectors have thus been proposed. Among these, cationic polymers such as polyalkyleneimines are particularly advantageous. The reason for this is that they have been found to be relatively effective during the transfection of nucleic acids, in particular in vivo, and they also show relatively low toxicity. It has also been observed that the complexes they form with nucleic acids (also known as “polyplexes”) diffuse relatively well out of the site of injection (J. S. Remy et al.,
Advanced Drug Delivery Reviews,
30, 1998, pp. 85-95).
Moreover, it appears at the present time to be essential to be able to provide vectors capable of targeting an appropriate nucleic acid toward an organ, a tissue, a cell type or a specific cell compartment, since it is important to be able to ensure that the nucleic acid transfected is interacting effectively with the target cells without unfavourably diffusing into the rest of the body. The intended aim is to avoid all nonspecific action of the nucleic acids on cells other than the target cells. It has thus been shown that galactosyl polyethyleneimine is an effective vector for the in vitro transfer of plasmids into cells bearing the cell receptor (lectin) corresponding to galactose (Zanta et al.,
Bioconj. Chem.,
8(2), 1997, p. 839; T. Bettinger et al.,
Bioconj. Chem.,
1999).
Hitherto, polymers such as galactosyl polyethyleneimine, for example, were obtained by the action of an oligosaccharide with the polyethyleneimine in the presence of sodium cyanoborohydride. However, this reagent has the drawback of being expensive and above all very toxic. The risk of potential presence of residual cyanide ions in the final product, which is relatively cationic, prohibits any possibility of pharmaceutical use. In addition, the search for an alternative preparation process has so far remained fruitless since polyalkyleneimines are polymers—and not small molecules—which are insoluble in many solvents, in particular apolar solvents. Thus, the use of an alternative process using sodium triacetoxyborohydride was not possible. The use of sodium borohydride in aqueous sulfuric acid and a pyridine/borane mixture was also found to be incompatible with cationic polymers. It thus appeared necessary to develop an alternative process which is compatible with cationic polymers such as polyalkyleneimines, and which involves only pharmaceutically acceptable reagents.
It has thus been found that it is possible to prepare functionalized polyalkyleneimines by treating a polyalkyleneimine with a functionalized hemiacetal in the presence of titanium (IV) isopropoxide and sodium borohydride.
Such a process has the advantage of being able to be carried out in a solvent which is compatible with polyalkyleneimines, for example such as alcohols, and only involves reagents that are both less expensive and relatively nontoxic.
Various articles from Bhattacharyya et al. (
J. Org. Chem.,
1995, 60, pp. 4928-4929;
Synlett,
1995, pp. 1079-1080;
J. Chem. Soc.
, Perkin Trans. 1, 1998, pp. 2527-2531) have disclosed the process below for preparing amines from ketones or aldehydes:
in which R and R′ represent, independently of each other, a hydrogen atom, an alkyl group or an aryl group, or together form a 5-, 6- or 7-membered cycloalkyl group optionally containing a hetero atom, and R
1
and R
2
represent, independently of each other, a hydrogen atom or an alkyl group optionally substituted with a hydroxyl or an ester, or alternatively R
1
and R
2
together form a 5- or 6-membered cycloalkyl group optionally containing a hetero atom. However, such a process was described only in the context of the preparation of small molecules, i.e. in particular nonpolymeric molecules, containing only one amine function, from the corresponding ketone or aldehyde.
According to the present invention, the starting polyalkyleneimine has the general formula:
in which R represents a hydrogen atom or a group of general formula:
n is an integer between 2 and 10 inclusive, and p and q are integers, it being understood that the sum p+q is such that the average molecular weight of the polymer is between 100 Da and 10
7
Da inclusive.
It is understood that, in the general formula (I), the value of n and the group R can vary between the various units —NR—(CH
2
)
n
— and —(CH
2
)
n
—NH—. Thus, the general formula I includes both linear polymers and branched polymers, as well as homopolymers and heteropolymers.
n is preferably between 2 and 5. Preferred polymers are, for example, polyethyleneimine (PEI) or polypropyleneimine (PPI). In addition, the polymers which are preferred for carrying out the present invention and which have been shown to be most particularly effective in transfection are those whose average molecular weight is between 10
3
and 5×10
6
. By way of example, mention may be made of the polyethyleneimine of average molecular weight 50,000 Da (PEI 50K), 25,000 Da (PEI 25K) or 22,000 Da (PEI 22K) or alternatively polypropyleneimine 800,000 Da (PPI 800K).
The polyalkyleneimines used in the present invention can be obtained according to various methods known to those skilled in the art. For example, they can be synthesized chemically from the corresponding monomer(s), under anionic polymerization conditions (for example polymerization of ethyleneimine), or by reduction of polyamides obtained by polycondensation of diacids or diamines, or alternatively by reduction of imines obtained by polycondensation of dialdehydes with diamines. In addition, many polyalkyleneimines are commercially available, for example such as PEI 25K, PEI 22K or PPI 800K.
For the purposes of the invention, the expression “functionalized polyalkyleneimine” means cationic polymers of polyalkylimine type onto which targeting elements are covalently bonded. These targeting elements direct the transfer of the nucleic acid toward certain desired cell types, certain desired tissues or certain desired cell compartments. The covalent bonding between the targeting element and the polyalkyleneimine is obtained by reaction with a functionalized hemiacetal, i.e. a hemiacetal in which one of the substituents is said targeting element, under the reaction conditions mentioned previously.
More specifically, for the purposes of the present-invention, the expression “functionalized hemiacetal” means any molecule having the general formula:
in which n is equal to 0 or 1, and R
1
, R
2
, R
3
and R
4
are identical or different and represent, independently of each other, a hydrogen atom, a group which is compatible with the reaction carried out or a targeting element, it being understood that one and only one of the sub

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