Peptide-based gemini compounds

Details Peptide-based gemini compounds Peptide-based gemini compounds

2000-06-08

2004-02-17

Low, Christopher S. F. (Department: 1653)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

8 to 10 amino acid residues in defined sequence

C530S329000, C530S330000, C530S331000, C514S015800, C514S016700, C514S017400, C514S018700, C514S019300

Reexamination Certificate

active

06693167

ABSTRACT:

This invention relates to newly identified peptide-based gemini surfactant compounds, to the use of such compounds and to their production. The invention also relates to the use of the peptide-based gemini compounds to facilitate the transfer of compounds into cells for drug delivery.
Surfactants are substances that markedly affect the surface properties of a liquid, even at low concentrations. For example surfactants will significantly reduce surface tension when dissolved in water or aqueous solutions and will reduce interfacial tension between two liquids or a liquid and a solid. This property of surfactant molecules has been widely exploited in industry, particularly in the detergent and oil industries. In the 1970s a new class of surfactant molecule was reported, characterised by two hydrophobic chains with polar heads which are linked by a hydrophobic bridge (Deinega,Y et al.,
Kolloidn. Zh
. 36, 649, 1974). These molecules, which have been termed “gemini” (Menger, FM and Littau,CA,
J.Am.Chem.Soc
. 113, 1451, 1991), have very desirable properties over their monomeric equivalents. For example they are highly effective in reducing interfacial tension between oil and water based liquids and have a very low critical micelle concentration.
Cationic surfactants have been used inter alia for the transfection of polynucleotides into cells in culture, and there are examples of such agents available commercially to scientists involved in genetic technologies (for example the reagent Tfx™-50 for the transfection of eukaryotic cells available from Promega Corp. WI, USA).
The efficient delivery of DNA to cells in vivo, either for gene therapy or for antisense therapy, has been a major goal for some years. Much attention has concentrated on the use of viruses as delivery vehicles, for example adenoviruses for epithelial cells in the respiratory tract with a view to corrective gene therapy for cystic fibrosis (CF). However, despite some evidence of successful gene transfer in CF patients, the adenovirus route remains problematic due to inflammatory side-effects and limited transient expression of the transferred gene. Several alternative methods for in vivo gene delivery have been investigated, including studies using cationic surfactants. Gao,X et al. (1995)
Gene Ther
. 2, 710-722 demonstrated the feasibility of this approach with a normal human gene for CF transmembrane conductance regulator (CFTR) into the respiratory epithelium of CF mice using amine carrying cationic lipids. This group followed up with a liposomal CF gene therapy trial which, although only partially successful, demonstrated the potential for this approach in humans (Caplen, N J. et al.,
Nature Medicine
, 1, 39-46, 1995). More recently other groups have investigated the potential of other cationic lipids for gene delivery, for example cholesterol derivatives (Oudrhiri,N et al.
Proc.Natl.Acad.Sci
. 94, 1651-1656, 1997). This limited study demonstrated the ability of these cholesterol based compounds to facilitate the transfer of genes into epithelial cells both in vitro and in vivo, thereby lending support to the validity of this general approach.
These studies, and others, show that in this new field of research there is a continuing need to develop novel low-toxicity surfactant molecules to facilitate the effective transfer of polynucleotides into cells both in vitro for transfection in cell-based experimentation and in vivo for gene therapy and antisense treatments. The present invention seeks to overcome the difficulties exhibited by existing compounds.
The invention relates to the peptide-based gemini compounds comprising two linked chains:
each chain having:
(1) a positively charged hydrophilic head, Q
1
or Q
2
, formed from one or more amino acids and/or amines;
(2) a central portion, P
1
or P
2
, having a polypeptide backbone; and
(3) a hydrophobic tail, R
1
or R
2
; the central sections of each chain being linked together by bridge Y through residues in P
1
and P
2
.
Preferably the central portion is made up of two or three amino acids, P
a
(optional), P
b
and P
c
, in which:
P
a
is a D- or L- amino acid, preferably hydrophilic, such as threonine or serine,
P
b
is preferably D- or L- cysteine, serine or threonine, and
P
c
is preferably D- or L- serine or threonine and is linked to R
1
or R
2
.
Preferred compounds of the present invention include compounds of the formula (I):
where:
A
1
and A
5
which may be the same or different, is a positively charged group formed from one or more amino acids or amines joined together in a linear or branched manner and preferably bonded by an amide (CONH) bond;
A
2
/A
6
CH(NH)CO, which may be the same or different, is derived from an amino acid, preferably serine;
p and q, which may be the same or different, is 0 or 1;
X
1
/X
2
CH
2
CH(NH)CO, which may be the same or different, is derived from cysteine (X
1
/X
2
═S), serine or threonine (X
1
/X
2
═O);
A
4
/A
8
CH(NH)CO, which may be the same or different, is derived from serine or threonine;
Y is a linker group, preferably (CH
2
)
m
where m is an integer from 1 to 6, most preferably 2, and may be a disulphide bond when X
1
and X
2
is each S;
R
1
and R
2
are C
(10-20)
saturated or unsaturated alkyl groups, and
W and Z are NH, O, CH
2
or S; or
a salt, preferably a pharmaceutically acceptable salt thereof.
Preferably, the compound is symmmetrical, that is A
1
and A
5
are the same, A
2
and A
6
are the same, A
4
and A
8
are the same, R
1
and R
2
are the same, and W and Z are the same.
Representative examples of A
1
/A
5
include D- or L-amino acids selected from arginine, lysine, ornithine and histidine, preferably lysine, or amines such as spermine and spermidine. Up to seven amino acids and/or amines may be linked in a linear or branched chain. Prefered examples include groups having two or three lysines or ornithines or a combination of lysine, ornithine, arginine and histidine, for instance:
COCH(NHR)(CH
2
)
4
NHCO(NH
2
)(CH
2
)
4
NH
2
or
COCH(NHR)(CH
2
)
3
NHCO(NH
2
)(CH
2
)
3
NH
2
or
COCH(NHR)(CH
2
)
4
NHCO(NH
2
)(CH
2
)
3
NH
2
in which R is H or NHCO(NH
2
)(CH
2
)
4
NH
2
or NHCO(NH
2
)(CH
2
)
3
NH
2
Preferably, —X
1
—Y—X
2
— is —SCH
2
CH
2
S— or —OCH
2
CH
2
O—
Preferably, R
1
and R
2
is each a C
12
-C
20
alkyl group, for instance C
12
.
Preferably, W and Z is NH, thereby forming a further amide (CONH) bond.
Compounds of the present invention may be prepared from readily available starting materials using synthetic peptide chemistry well known to the skilled person. For prefered compounds of the present invention a useful intermediate is the compound:
which is synthesised in a multi-stage process beginning, for instance, with the construction of the di-cysteine part and subsequently building up the hydrophilic head by attaching a serine moiety at the carboxyl group of each cysteine moiety, using standard peptide chemistry, and then attaching the hydrocarbon chains to the carboxyl group of the serine moiety using a standard amide forming reaction well known to those skilled in the art. This intermediate can then be taken through to compounds of formula (I) by further reaction at the nitrogens of the cysteine residues.
Another aspect of the invention relates to methods for using the peptide-based gemini compounds. Such uses include facilitating the transfer of oligonucleotides and polynucleotides into cells for antisense, gene therapy and genetic immunisation (for the generation of antibodies) in whole organisms. Other uses include employing the compounds of the invention to facilitate the transfection of polynucleotides into cells in culture when such transfer is required, in, for example, gene expression studies and antisense control experiments among others. The polynucleotides can be mixed with the compounds, added to the cells and incubated to allow polynucleotide uptake. After further incubation the cells can be assayed for the phenotypic trait afforded by the transfected DNA, or the levels of mRNA expressed from said DNA can be determined by Northern blotting or by us

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