Note: Descriptions are shown in the official language in which they were submitted.
, CA 022132S7 1997-08-18
,
RAN 4600/074
alpha,gamma-Diaminobutyric acid (DAB) cont~inin~ Oligopeptide
Derivatives
Gene transfer technology has become a field of considerable interest.
Introduction of an exogenous gene into a cell (i.e. transfection) bears many
5 important scientific and medical applications, going from gene regulation
and the production of recombinant proteins to gene therapy.
Viruses have evolved to bypass the different cellular barriers to gene transfer
and have indeed become vectors of choice for transfection. Many viruses,
including retrovirus, adenovirus or herpes virus, are now engineered to
0 carry therapeutic genes and used in human clinical trials for gene therapy.
However, there remains a risk of infectious and immunologic reaction and
the large scale production of viruses is difficult and time consuming.
For these various reasons non viral systems have been developed to carry
DNA into cells, e.g., the transfection technique based on a cationic lipid, the
15 dioleoyloxypropyl trimethylammonium (Felgner et al., Proc. Natl., Acad.
Sci. USA, 84, 7413-7417, 1987) commercialised as LipofectinTM. Since the
discovery of this transfection technique, many more cationic lipids have been
synthesised and some are commercially available as transfecting reagent for
laboratory use: DOGS (TransfectamTM), DOSPA (LipofectamineTM), DOTAP
20 (DOTAPTM)
Nevertheless, despite an important progress in the formulation of non-viral
gene delivery systems, there remains a need for more efficient techniques,
since the transfection efficiency of synthetic systems is usually below that of
viral vectors. Furthermore, still many problems arise in vivo and the poor
25 stability of the non-viral systems in biological fluids does not allow high and
reproducible levels of transfection in vivo.
In accordance with the present invention, it has been found that
alpha,gamma-~ minobutyric acid (DAB) cont~ining oligopeptide
Grn/Ul 21.7.97
CA 022132~7 1997 - 08 - 18
derivatives bind polynucleotides and anionic macromolecules and can be
used for transfecting cells.
Thus, in one aspect~ the present invention relates to novel alpha,gamma-
rli~minobutyric acid (DAB) cont~ining oligopeptide derivatives of formula
R'-NH-A
wherein
R' is a cell recognition agent, a membrane
permeabilizing agent, a subcellular localization agent
or a masking agent;
0 A is an oligopeptide devoid of one amino group and
cont~ining 1 to 200 amino acids, wherein at least one of
the amino acid is alpha,gamma-~ minobutyric acids
(DAB) and the terminal carboxyl group of the
oligopeptide is converted into an amide, lower alkyl
amide, di-lower alkyl amide or hydrazide.
The oligopeptides A may be linear or branched. The amino acids may belong
to the L- or D- series or may be racemic. Compounds of formula I, wherein A
is an oligopeptide devoid of one amino group cont~ining 1 to ~0 amino acids
are preferred. Most preferred are compounds of formula I, wherein the
oligopeptide A contains 1 to 20 amino acids.
Another preferred embodiment are compounds of formula I wherein the
terminal carboxyl group of the oligopeptide A is converted into a hydrazide.
The "cell recognition agent" as used herein refers to a molecule capable of
recogni~ing a component on the surface of a targeted cell. Cell recognition
25 components include antibodies to cell surface antigens, ligands for cell
surface receptors including those involved in receptor-mediated endocytosis,
peptide hormones, etc. Specific ligands contemplated by this invention
include carbohydrate ligands such as galactose, mannose, mannosyl 5-
phosphate, fucose, sialic groups, N-acetylglucosamine or combinations of
30 these groups as complex carbohydrates such as those found on glycolipids of
the blood groups or on various secreted proteins. Other ligands include
folate, biotin, various peptides that can interact with cell surface or
intracellular receptors such as the chemoattractant peptide N-formyl-met-
leu-phe (SEQ ID NO:2, WO-A-0 2397), peptides cont~ining the arg-asp-gly
35 sequence or cys-ser-gly-arg-glu-asp-val-trp (SEQ ID NO:3, ibid.) peptides,
peptides that contain a cystine residue or that interact with cell surface
protein such as the human immunodeficiency virus GP-120, and peptides
CA 022132S7 1997-08-18
-3-
that interact with CD-4. Other ligands include antibodies or antibody
fragments such as those described by Hertler and Frankel (Hertler, A., and
Frankel, A., J. Clin. Oncol. 7: 1932 (1989)). The specificity of the antibodies
can be directed against a variety of epitopes that can be expressed on cell
5 surfaces including histocompatibility macromolecules, autoimmune
antigens, viral, parasitic or bacterial proteins. Other protein ligands include
hormones such as growth hormone and insulin or protein growth factors
such as GM-CSF, G-CSF, erythropoietin, epidermal growth factor, basic and
acidic fibroblast growth factor, and the like. Other protein ligands include
10 various cytokines that work through cell surface receptors such as
interleukin 2, interleukin 1, interleukin 12, tumor necrosis factor, and
suitable peptide fragments from such macromolecules.
In another embodiment, the cell recognition molecule is an integrin-binding
peptide or derivatives thereof. In a more preferred embodiment the integrin-
binding peptide is GGCRGDMFGCGG.
The "membrane permeabilizing agent" as used herein refers to a molecule
that aids in the passage of a polynucleotide or anionic macromolecule across
a membrane. Examples of membrane permeabilizing agents are melittin,
hemolysin, mastoparan, bombolitin, crabrolin, pardaxin, gramicidin,
20 alamethicin, apidaecin, bactenecin, cecropins, defensins, dermaseptin,
indolicidin, magainins, bombinin, brevinin, esculentin, seminalplasmin
and derivatives thereof (G. Saberwal and R. Nagaraj. BBA 1197:109-131
(1994)). Other membrane permeabilizing agents are peptides cont~ining the
amino-terminal amino acid sequence of the influenza virus hemagglutinin
25 and synthetic derivatives thereof as described in the Journal of Biological
Chemistry, Vol. 269 (17), 12918-12924 (1984) (Table I); or HIV and SIV fusion
peptides and synthetic derivatives thereof as described in Biochemica et
Biophysica Acta 1240, 95-100 (1995) (Fig. 1). Another class of cell
permeabilizing agents are detergent molecules such as bile salts. Other
30 examples of detergent molecules are sucrose monolaurate, n-octylglucoside
and tocopheryl PEG 1000 succinate. However, other detergent molecules are
also suitable
In another embodiment the membrane permeabilizing agents is a conjugate
between a membrane permeablili7ing agent and a lipid. An example of such
35 a conjugate is phe-glu-ala-ala-leu-ala-glu-ala-leu-ala-glu-ala-leu-ala with
an NH2 -terminal myristic acid (C. Puyal et al. BBA 1195:259-266 (1994)).
CA 022132~7 1997-08-18
- 4 -
The "subcellular localization agent" as used herein refers to a molecule
capable of recogni~ing a subcellular component in a targeted cell. Particular
subcellular components include the nucleus, ribosomes, mitochondria, and
chloroplasts.
5 In a preferred embodiment of this invention, the subcellular-localization
component is a nuclear-localization component. The nuclear-localization
components include known peptides of defined amino acid sequences, and
longer sequences cont~ining these peptides. One known peptide sequence is
the SV 40 large T antigen heptapeptide pro-lys-lys-lys-arg-lys-val (SEQ ID
0 NO:1, WO-A-0 2397). Other peptides include the influenza virus
nucleoprotein decapeptide ala-ala-phe-glu-asp-leu-arg-val-leu-ser (SEQ ID
No:4, ibid.), and the adenovirus Ela protein segment lys-arg-pro-arg-pro
(SEQ ID NO:5, ibid.). Other sequences may be discerned from Dingwall et al.
(Dingwall, C., et al., TIBS 16:478 (1991)).
5 In another embodiment, the subcellular-localization component is a
lysosomal-localization component. A known component for targeting the
lysosome is a peptide cont~ining the lys-phe-glu-arg-gln (SEQ ID NO:6,
WO-A-0 2397) segment.
In yet another embodiment, the subcellular-localization component is a
ao mitochondrial-localization component. A known component for targeting
mitochondria is a peptide cont~ining the sequence met-leu-ser-leu-arg-gln-
ser-ile-arg-phe-phe-lys-pro-ala-thr-arg (SEQ ID NO:7, ibid.). However, other
subcellular-localization components or agents are also suitable.
The "masking agent" as used herein refers to a molecule capable of masking
25 all or part of the polynucleotide or anionic macromolecule, thereby
increasing its circulatory half-life by inhibiting attack by degrading reagents
present in circulation or by blocking uptake by the reticuloendothelial
system. An example of such a masking agent is polyethylene glycol (PEG).
The PEG may have a molecular weight of about 700 to 20,000 Daltons,
30 preferably about 1800 to 6000 Daltons.
The compounds of formula I can be prepared by methods known in the art,
for example, in analogy to the method described in Gene Therapy (1995) 2,
562-554. This invention relates thus to a process for preparing the novel
compounds of formula I.
CA 022132~7 1997-08-18
The invention further relates to the use of a compound of formula I as
carrier for transfecting a cell with a polynucleotide or any other anionic
macromolecule .
Examples of polynucleotides that can be transfected into cells by means of the
5 novel compounds of this invention are deoxyribonucleic acids (DNA) and
ribonucleic acids (RNA). Examples of anionic macromolecules other than
polynucleotides are proteins, such as ribonucleoproteins and proteins used
for immunisation, e.g. viral proteins.
Examples of DNA that can be transfected into cells by means of the novel
10 compounds of this invention are plasmids and genes, especially those for
which gene therapy protocols have been launched such as cystic fibrosis
transmembrane regulator (CFTR), adenosine de~min~e (ADA), thymidine
kinase (tk) and HLA B7; as well as reporter genes such as beta-
galactosidase, luciferase, chloramphenicol acetyl transferase and alpha-1
15 antitrypsin. Other examples of DNA are oligodeoxynucleotides and their
analogues used as antisense, aptamer or "triple-helix" agents. Examples of
RNA are ribozymes or oligoribonucleotides antisense molecules.
The nature of the cell which is to be transfected is not narrowly crucial. The
cell can be a procaryotic or eucaryotic cell, a rn~mm~ n or a plant cell.
~o The transfection procedure using a compound of this invention can be
carried out according to methods known per se. For transfecting a cell, e.g.,
with DNA or RNA, the +/- charge ratio between the positively charged
compound of formula I and the negatively charged DNA or RNA is in the
range of 0.1 to 50, more preferably from 0.1 to 10.
25 The transfection may be carried out in the presence of a helper lipid, a short
chain phospholipid and/or another known transfection competent molecule.
Examples of helper lipids are phospholipids, such as phosphatidylcholine or
phosphatidylethanolamines or mixtures thereof. Preferred helper lipids are
phosphatidylethanolamines, such as dioleoylphosphatidylethanolamine.
30 Examples of short chain phospholipids are phosphatidylcholines that carry
two C6 12 fatty acid residues. Preferred short chain phospholipids are
dicapryl- and dicapryloyl phosphatidylcholine. The helper lipid and/or short
chain phospholipid is suitably in the form of a liposome, mixed micelle,
organic solution, or aqueous dispersion.
35 Examples of transfection competent molecules include cationic lipids as
described by J.B. Behr in Bioconjugate Chem. 5:382-389 (1994) and X. Gao
CA 022132S7 1997-08-18
- 6 -
and L. Huang in Gene Ther. 2:710-722 (1995); polycations as described by
A.V. Kabanov and V.A: Kabanov in Bioconjugate Chem. 6:7-20 (1995);
peptides and polymers and other nonviral gene delivery systems as described
by F.D. Ledley in Human Gene Therapy 6:1129-1144 (1995). Other
5 transfection competent molecules are those described by Legendre et al. (EP-
A-95 118 338.3 and EP-A-96 100 603.8).
For transfection, an appropriate amount of at least one compound of formula
I, suitably as an aqueous or organic solution, or in form of liposomes, mixed
micelles or micelles, is added to an aqueous solution of the molecule to be
10 transfected (e.g., plasmid DNA) or vice versa. Optionally, a cationic lipid or
any other transfection-competent molecule, a helper lipid and, if desired, a
short chain phospholipid is then added, either as an aqueous solution or
dispersion, as an organic solution or dispersion, or in form of liposomes,
mixed micelles or micelles. Alternatively, the molecule to be transfected may
15 be added to a composition comprising at least one compound in accordance
with this invention and, if desired, a cationic lipid or any other transfection-competent molecule, a helper lipid and a short chain phospholipid or vice
versa. Suitably, the composition is an aqueous or organic solution, an
aqueous or organic dispersion, or a liposome, a mixed micelle or a micelle.
ao The final composition may be in liquid, solid (freeze-dried, controlled
evaporative-dried), semisolid or aerosol form.
The optimal ratio between the components, i.e., the compound of formula I,
the molecule to be transfected and, optionally, additional constituents,
depends on the cell to be transfected. The optimal +/- charge ratio between
25 the compound of formula I and the molecule to be transfected varies between
0.1-50, preferably between 0.1-10. The optimal molar ratio between the
compound of formula I and additional constituents such as a cationic lipid,
and/or another transfection competent molecule, and/or a helper lipids
and/or a short chain phospholipid is 0.1-50, more preferably 0.1 to 10.
30 For transfecting cells in an animal or human patient the composition can be
a-lmini~tered by oral, parenteral (i.v., i.m., s.c., i.d., i.p.) transdermal,
pulmonary, nasal, rectal, ocular, ventricular, vascular (catheter) and
intratumoral route. Furthermore, the composition can be a-lmini~tered by
high velocity impaction a-lmini.~tration to the skin surface. The progress of
35 transfection can be measured by appropriate testing protocols which are
known to those skilled in the art.
CA 022132S7 1997-08-18
In another aspect, this invention relates to compositions comprising at least
one compound of formula I, and optionally, a cationic lipid or any other
known transfection competent molecule, a helper lipid and/or a short chain
phospholipid, and optionally, a polynucleotide or any other anionic
5 macromolecule.
The following examples which are not delimiting illustrate the invention
further.
Example 1
10 Preparation of a compound of formula I wherein Rl is an integrin bindung
peptide and A is Dab-Dab-NHNH2
The synthesis of compound of formula II (RGD-(Dab)2NHNH2)
Gly-Gly-Cys-Arg-Gly-Asp-Met-Phe-Gly-Cys-Gly-Gly-Dab-Dab-N2H3 (II)
was performed on a Peptide Synthesizer SP 650 (Labortec AG) using Wang
6 resin by a FMOC protocol as described in Gene Therapy (1995), 2, 552-554.
The peptide was cleaved from the resin by hydrazinolysis. The resulting
product was deprotected and subjected to oxidative cyclization to yield the
compound of formula II.
The homogeneity of the purified peptide was confirmed by analytical RP-20 HPLC and the molecule weight was determined by I~P MS: 1329.5 [M+H3f
calc. for CsoHglN2lol6s3 (1328.5)
Example 2
10 ~Lg of plasmid DNA (pGL3-CMV) is diluted with 250 ,ul of 10 mM Tris Cl
buffer pH 8.5. Then an appropriate amount of the compound obtained in
2~ Example 1, RGD-(Dab)2NHNH2, is mixed with the diluted DNA so that the
+/- charge ratio between RGD-(Dab)2NHNH2 and DNA is 2/1. An appropiate
volume of the resulting mixture cont~ining 5 ~lg of plasmid DNA is then
added per well of CaCo-2 cells grown in 6-well plate. 24h later, luciferase
activity is measured. Results are shown in Table 1.
Example 3
10 llg of plasmid DNA (pGL3-CMV) is diluted with 250 ~l of 10 mM Tris Cl
buffer pH 8.5. Then an appropriate amount of the compound obtained in
Example 1, RGD-(Dab)2NHNH2, is mixed with the diluted DNA so that the
+/- charge ratio between RGD-(Dab)2NHNH2 and DNA is 2/1. Then, dioleoyl
. CA 022132S7 1997-08-18
phosphatidylethanolamine (DOPE) dispersion is added to the mixture so that
the molar ratio between DOPE and RGD-(Dab)2NHNH2 is 5/1. An appropiate
volume of the resulting mixture conhining five ~lg of plasmid DNA is then
added per well of CaCo-2 cells grown in 6-well plate. 24h later, luciferase
5 activity is measured. Results are shown in Table 1.
TaJ~le 1
T~nefecling agent T.ll~iff~rase ac~ivity
(relative LIJ) per well
example 2 RGD-(Dab)2NHNH2 503
example 3 RGD-(Dab)2NHNH2 +DOPE 11412
Example 4
7 nmoles of the compound obtained in Example 1, RGD-(Dab)2NHNH2, is
10 mixed with 11 nmoles of dioleoyl-melittin, (DO-melittin, EP-A-96 100 603.8).
Then, the mixture is added to 10 ~lg of plasmid DNA pGL3-CMV in a total
volume of 200 1ll 10 mM Tris Cl buffer pH 8.5. This corresponds to a +/-
charge ratio between the cationic charge of the two transfection agents and
the anionic charge of the DNA of 4/1. An appropiate volume of the resulting
~5 mixture cont~inin~ 5 ~lg of plasmid DNA is then added per well of CaCo-2
cells grown in 6-well plate. 24h later, luciferase activity is measured. Table 2shows the transfection efficiency of the RGD-(Dab)2NHNH2/DO-melittin
mixture along with that of RGD-(Dab)2NHNH2 and DO-melittin alone.
Table 2
l~an~ec~on agent T.ll-.iff~rase ac~vi~ (R.LU)
RGD-(Dab)2NHNH2 250
DO-melittin* 6 X 104
RGD-(Dab)2NHNH2/ DO-melittin 4 X 105
* prepared according to example 4
Example 5
1.75 nmoles of the compound obtained in Example 1, RGD-(Dab)2NHNH2, is
mixed with 5.25 nmoles of the lipopeptide N~-palmitoyl-D-(a,r-rliiqmino-
butyryl)-L-(a,y-~ minobutyric acid) hydrazide (Palm-(Dab)2NHNH2). Then,
25 the mixture is added to 10 ,ug of plasmid DNA pGL3-CMV in a total volume of
200 ~ul 10 mM Tris Cl buffer pH 8.5. This corresponds to a +/- charge ratio
between the cationic charge of the two transfection agents and the anionic
CA 02213257 1997-08-18
_ 9 _
charge of the DNA of 2/1. Then, dioleoyl phosphatidylethanolamine (DOPE)
dispersion is added to the mixture so that the molar ratio between DOPE and
the two transfection agents is 5/1. An appropiate volume of the resulting
mixture cont~inin~ five ~g of plasmid DNA is then added per well of CaCo-2
5 cells grown in 6-well plate. 24h later, luciferase activity is measured. Table 3
shows the transfection efficiency of the RGD-(Dab)2NHNH2/Palm-
(Dab)2NHNH2 mixture along with that of RGD-(Dab)2NHNH2 and Palm-
(Dab)2NHNH2 alone.
Table 3
Tran~ection agent Luciferase activit y (RLIJ)
RGD-(Dab)2NHNH2* 1 x 104
Palm-(Dab)2NHNH2 * 1 x 105
RGD-(Dab)2NHNH2/Palm-(Dab)2NHNH2 1 x 106
0 * prepared according to example ~
