U.S. patent application number 11/868184 was filed with the patent office on 2008-03-27 for polycationic sterol derivatives as transfection agents.
This patent application is currently assigned to Imperial College of Science Technology and Medicine. Invention is credited to Robert G. Cooper, Christopher J. Etheridge, Andrew D. Miller.
Application Number | 20080075763 11/868184 |
Document ID | / |
Family ID | 26309393 |
Filed Date | 2008-03-27 |
United States Patent
Application |
20080075763 |
Kind Code |
A1 |
Miller; Andrew D. ; et
al. |
March 27, 2008 |
Polycationic sterol derivatives as transfection agents
Abstract
A method for treating genetic disorders or conditions or
diseases in patients in need of such treatments. The method
includes administering an effective amount of a compound comprising
a cholesterol group or derivative thereof having linked thereto a
head group. The head group is more positive than the heat group of
DC-Chol. The heat group may be a straight chain polymeric group
having two or more amine groups separated by an ethylene group.
Inventors: |
Miller; Andrew D.; (London,
GB) ; Cooper; Robert G.; (London, GB) ;
Etheridge; Christopher J.; (London, GB) |
Correspondence
Address: |
FAY SHARPE LLP
1100 SUPERIOR AVENUE, SEVENTH FLOOR
CLEVELAND
OH
44114
US
|
Assignee: |
Imperial College of Science
Technology and Medicine
|
Family ID: |
26309393 |
Appl. No.: |
11/868184 |
Filed: |
October 5, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10770294 |
Feb 2, 2004 |
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11868184 |
Oct 5, 2007 |
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09194267 |
Mar 22, 1999 |
6756054 |
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PCT/GB97/01426 |
May 23, 1997 |
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10770294 |
Feb 2, 2004 |
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Current U.S.
Class: |
424/450 ;
514/169; 514/44R; 552/502 |
Current CPC
Class: |
A01K 2267/0393 20130101;
A61P 43/00 20180101; A01K 2227/105 20130101; C07J 41/0055 20130101;
A61K 9/1272 20130101; A61K 31/711 20130101; A01K 2207/05 20130101;
A61K 31/56 20130101; C07J 9/00 20130101 |
Class at
Publication: |
424/450 ;
514/169; 514/044; 552/502 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 31/56 20060101 A61K031/56; A61K 31/711 20060101
A61K031/711; C07J 9/00 20060101 C07J009/00; A61P 43/00 20060101
A61P043/00; A61K 48/00 20060101 A61K048/00 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 17, 1997 |
GB |
9705498.5 |
May 24, 1996 |
GB |
9610944.2 |
Claims
1. A compound capable of acting as a cationic lipid, the compound
comprising a cholesterol group having linked thereto a head group;
and wherein the head troup is more positive than the head group of
DC-Chol; but wherein the compound is not synthesised by reacting
spermidine and cholesterol chloroformate in CH.sub.2Cl.sub.2 in the
presence of N,N-diisoprophylethylamine.
2-22. (canceled)
23. A method for treating a genetic disorder, or condition or
disease in a patient in need of treatment, comprising:
administering an effective amount of a compound comprising a
cholesterol group or derivative thereof having linked thereto a
head group; wherein the head group is more positive than the head
group of DC-Chol; further wherein the head group is a straight
chain polyamine; further wherein two or more of the amine groups of
the polyamine group are separated by an ethylene group.
24. The method according to claim 23 wherein the cholesterol group
or derivative thereof is cholesterol.
25. The method according to claim 23 wherein the cholesterol group
is linked to the head group via a carbamoyl linkage.
26. The method according to claim 23 wherein the compound is
selected from compounds of the formula ##STR1## where Chol denotes
a group of the formula ##STR2##
27. The method according to claim 23 wherein the compound is
selected from compounds of the formula ##STR3##
28. The method according to claim 23 wherein the compound is
selected from compounds of the formula ##STR4##
29. The method according to claim 23 wherein the compound is of the
formula ##STR5##
30. The method according to claim 23 wherein the compound is of the
formula ##STR6##
31. The method according to claim 23 wherein the compound is of the
formula ##STR7##
32. The method according to claim 23, wherein the compound is a
cationic lipid compound.
33. The method according to claim 32, wherein the cationic lipid
compound is in admixture with or associated with a nucleotide
sequence.
34. The method according to claim 23, wherein the compound is a
cationic liposome formed from a cationic lipid compound.
35. The method according to claim 34, wherein the cationic liposome
is in admixture with or associated with a nucleotide sequence.
36. A method for treating a genetic disorder, or condition or
disease in a patient in need of treatment, comprising:
administering an effective amount of a compound selected from the
group consisting of cationic lipid compounds, cationic liposomes
formed from cationic lipid compounds, cationic lipid compounds in
admixture with or associated with a nucleotide sequence, cationic
liposomes, formed from a cationic lipid compound, in admixture with
or associated with a nucleotide sequence, and combinations thereof,
the compound comprising a cholesterol group or derivative thereof
having linked thereto a head group; wherein the head group is more
positive than the head group of DC-Choi; further wherein the head
group is a straight chain polyamine; further wherein two or more of
the amine groups of the polyamine group are separated by an
ethylene group.
37. The method according to claim 36 wherein the cholesterol group
or derivative thereof is cholesterol.
38. The method according to claim 36 wherein the cholesterol group
is linked to the head group via a carbamoyl linkage.
39. The method according to claim 36 wherein the compound is
selected from compounds of the formula ##STR8## where Chol denotes
a group of the formula ##STR9##
40. The method according to claim 36 wherein the compound is
selected from compounds of the formula ##STR10##
41. The method according to claim 36 wherein the compound is
selected from compounds of the formula ##STR11##
42. The method according to claim 36 wherein the compound is of the
formula ##STR12##
43. The method according to claim 36 wherein the compound is of the
formula ##STR13##
44. The method according to claim 36 wherein the compound is of the
formula ##STR14##
45. A method for treating a genetic disorder, or condition or
disease in a patient in need of treatment, comprising:
administering an effective amount of a composition, the composition
comprising: i. a compound selected from the group consisting of
cationic lipid compounds, cationic liposomes formed from a cationic
lipid compound, and combinations thereof, the compound comprising a
cholesterol group or derivative thereof having linked thereto a
head group; wherein the head group is more positive than the head
group of DC-Chol; further wherein the head group is a straight
chain polyamine; further wherein two or more of the amine groups of
the polyamine group are separated by an ethylene group, and ii. a
pharmaceutical, and optionally a pharmaceutically acceptable
diluent, carrier or excipient.
46. The method according to claim 45 wherein the cholesterol group
or derivative thereof is cholesterol.
47. The method according to claim 45 wherein the cholesterol group
is linked to the head group via a carbamoyl linkage.
48. The method according to claim 45 wherein the compound is
selected from compounds of the formula ##STR15## where Chol denotes
a group of the formula ##STR16##
49. The method according to claim 45 wherein the compound is
selected from compounds of the formula ##STR17##
50. The method according to claim 45 wherein the compound is
selected from compounds of the formula ##STR18##
51. The method according to claim 45 wherein the compound is of the
formula ##STR19##
52. The method according to claim 45 wherein the compound is of the
formula ##STR20##
53. The method according to claim 45 wherein the compound is of the
formula ##STR21##
54. A method for treating a genetic disorder or condition or
disease in a patient in need of treatment, comprising:
administering an effective amount of a composition comprising a
compound selected from the group consisting of cationic lipid
compounds, cationic lipid compounds in admixture with or associated
with a nucleotide sequence, cationic liposomes (formed from a
cationic lipid compound) in admixture with or associated with a
nucleotide sequence, and combinations thereof; the compound
comprising a cholesterol group or derivative thereof having linked
thereto a head group; wherein the head group is more positive than
the head group of DC-Chol; further wherein the head group is a
straight chain polyamine; further wherein two or more of the amine
groups of the polyamine group are separated by an ethylene
group.
55. The method according to claim 54 wherein the cholesterol group
or derivative thereof is cholesterol.
56. The method according to claim 54 wherein the cholesterol group
is linked to the head group via a carbamoyl linkage.
57. The method according to claim 54 wherein the compound is
selected from compounds of the formula ##STR22## where Chol denotes
a group of the formula ##STR23##
58. The method according to claim 54 wherein the compound is
selected from compounds of the formula ##STR24##
59. The method according to claim 54 wherein the compound is
selected from compounds of the formula ##STR25##
60. The method according to claim 54 wherein the compound is of the
formula ##STR26##
61. The method according to claim 54 wherein the compound is of the
formula ##STR27##
62. The method according to claim 54 wherein the compound is of the
formula ##STR28##
63. The method according to claim 54 wherein the composition
further comprises a pharmaceutical.
64. The method according to claim 63, wherein the composition
further comprises a pharmaceutically acceptable diluent, carrier or
excipient.
65. A method for the treatment of a genetic disorder or condition
or disease in a patient in need thereof, comprising administering a
cationic lipid compound, the compound comprising a cholesterol
group or derivative thereof having linked thereto a head group,
wherein the head group is more positive than the head group of
DC-Chol; further wherein the head group is a polyamine group which
is a straight chain polyamine group; further wherein two or more of
the amine groups of the polyamine group are separated by an
ethylene group.
66. A method for the treatment of a genetic disorder or condition
or disease in a patient in need thereof, comprising administering a
cationic liposome formed from a cationic lipid compound, the
compound comprising a cholesterol group having linked thereto a
head group; wherein the head group is more positive than the head
group of DC-Chol; further wherein the head group is a polyamine
group which is a straight chain polyamine group; further wherein
two or more of the amine groups of the polyamine group are
separated by an ethylene group.
67. A method for the treatment of a genetic disorder or condition
or disease in a patient in need thereof, comprising administering a
cationic lipid compound in admixture with or associated with a
nucleotide sequence, the compound comprising a cholesterol group or
derivative thereof having linked thereto a head group; wherein the
head group is more positive than the head group of DC-Chol; further
wherein the head group is a polyamine group which is a straight
chain polyamine group; further wherein two or more of the amine
groups of the polyamine group are separated by an ethylene
group.
68. A method for the treatment of a genetic disorder or condition
or disease in a patient in need thereof, comprising administering a
cationic liposome in admixture with or associated with a nucleotide
sequence, wherein the cationic liposome is formed from a cationic
lipid compound, the compound comprising a cholesterol group having
linked thereto a head group; wherein the head group is more
positive than the head group of DC-Chol; further wherein the head
group is a polyamine group which is a straight chain polyamine
group; further wherein two or more of the amine groups of the
polyamine group are separated by an ethylene group.
69. A method for the treatment of a genetic disorder or condition
or disease in a patient in need thereof, comprising administering a
pharmaceutical composition comprising (i) a cationic lipid
compound, the compound comprising a cholesterol group or derivative
thereof having linked thereto a head group; wherein the head group
is more positive than the head group of DC-Chol; further wherein
the head group is a polyamine group which is a straight chain
polyamine group; further wherein two or more of the amine groups of
the polyamine group are separated by an ethylene group; and (ii) a
pharmaceutical and, optionally, a pharmaceutically acceptable
diluent, carrier or excipient.
70. A method for treatment of a genetic disorder or condition or
disease in a patient in need thereof, comprising administering a
pharmaceutical composition comprising: (i) a cationic liposome
formed from a cationic lipid compound, the compound comprising a
cholesterol group having linked thereto a head group; wherein the
head group is more positive than the head group of DC-Chol; further
wherein the head group is a polyamine group which is a straight
chain polyamine group; further wherein two or more of the amine
groups of the polyamine group are separated by an ethylene group;
and (ii) a pharmaceutical and, optionally, a pharmaceutically
acceptable diluent, carrier or excipient.
Description
[0001] The present invention relates to a compound. In addition,
the present invention relates to processes for making the compound
and to the use of that compound in therapy, in particular gene
therapy (especially gene transfer).
[0002] One aspect of gene therapy involves the introduction of
foreign nucleic acid (such as DNA) into cells, so that its
expressed protein may carry out a desired therapeutic
function..sup.1
[0003] Examples of this type of therapy include the insertion of
TK, TSG or ILG genes to treat cancer; the insertion of the CFTR
gene to treat cystic fibrosis; the insertion of NGF, TH or LDL
genes to treat neurodegenerative and cardiovascular disorders; the
insertion of the IL-1 antagonist gene to treat rheumatoid
arthritis; the insertion of HIV antigens and the TK gene to treat
AIDS and CMV infections; the insertion of antigens and cytokines to
act as vaccines; and the insertion of .beta.-globin to treat
haemoglobinopathic conditions, such as thalassaemias.
[0004] Many current gene therapy studies utilise adenoviral gene
vectors--such as Ad3 or Ad5--or other gene vectors. However,
serious problems have been associated with their use..sup.2 This
has prompted the development of less hazardous, non-viral
approaches to gene transfer..sup.3
[0005] A non-viral transfer system of great potential involves the
use of cationic liposomes..sup.4 In this regard, cationic
liposomes--which usually consist of a neutral phospholipid and a
cationic lipid--have been used to transfer DNA.sup.4, mRNA.sup.5,
antisense oligonucleotides.sup.6, proteins.sup.7 and drugs.sup.8
into cells. A number of cationic liposomes are commercially
availabe.sup.4,9 and many new cationic lipids have recently been
synthesised.sup.10. The efficacy of these liposomes has be en
illustrated by both in vitro.sup.4 and in vivo.sup.11.
[0006] A neutral phospholipid useful in the preparation of a
cationic liposome is N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl
ammonium chloride, otherwise known as "DOTMA". The structure of
DOTMA is shown in FIG. 1.
[0007] One of the most commonly used cationic liposome systems
consists of a mixture of a neutral phospholipid
dioleoylphosphatidylethanolamine (commonly known as "DOPE") and a
cationic lipid,
3.beta.-[(N,N-dimethylaminoethyl)carbamoyl]cholesterol (commonly
known as "DC-Chol").sup.12. The structure of DOPE is shown in FIG.
2. The structure of DC-Chol is shown in FIG. 3.
[0008] A lipid has been synthesised by reacting spermidine and
cholesterol chloroformate in CH.sub.2Cl.sub.2 in the presence of
N,N-diisoprophylethylamine.sup.18. However, this resulted in a
mixture of the lipid and the corresponding regio-isomeric lipid,
which mixture proved inseparable by chromatography.
[0009] Despite the efficacy of the known cationic liposomes there
is still a need to optimise the gene transfer efficiency of
cationic liposomes in human gene therapy.sup.10.
[0010] According to one aspect of the present invention there is
provided a compound capable of acting as a cationic lipid, the
compound comprising a cholesterol group having linked thereto a
head group; and wherein the head group is more positive than the
head group of DC-Chol; but wherein the compound is not synthesised
by reacting spermidine and cholesterol chloroformate in
CH.sub.2Cl.sub.2 in the presence of N,N-diisoprophylethylamine.
[0011] As indicated above, the head group of DC-Chol is
Me.sub.2N(CH.sub.2).sub.2NH--.
[0012] According to another aspect of the present invention there
is provided a process of preparing a compound according to the
present invention comprising reacting a cholesterol group with a
head group.
[0013] According to another aspect of the present invention there
is provided a compound according to the present invention or a
compound when prepared by the process of the present invention for
use in therapy.
[0014] According to another aspect of the present invention there
is provided the use of a compound according to the present
invention or a compound when prepared by the process of the present
invention in the manufacture of a medicament for the treatment of a
genetic disorder or a condition or a disease.
[0015] According to another aspect of the present invention there
is provided a cationic liposome formed from the compound according
to the present invention or a compound when prepared by the process
of the present invention.
[0016] According to another aspect of the present invention there
is provided a method of preparing a cationic liposome comprising
forming the cationic liposome from the compound according to the
present invention or a compound when prepared by the process of the
present invention.
[0017] According to another aspect of the present invention there
is provided a cationic liposome according to the present invention
or a cationic liposome as prepared by the method of the present
invention for use in therapy.
[0018] According to another aspect of the present invention there
is provided the use of a cationic liposome according to the present
invention or a cationic liposome as prepared by the method of the
present invention in the manufacture of a medicament for the
treatment of genetic disorder or condition or disease.
[0019] According to another aspect of the present invention there
is provided a combination of a nucleotide sequence and any one or
more of: a compound according to the present invention, a compound
when prepared by the process of the present invention, a liposome
of the present invention, or a liposome as prepared by the method
of the present invention.
[0020] According to another aspect of the present invention there
is provided a combination according to the present invention for
use in therapy.
[0021] According to another aspect of the present invention there
is provided the use of a combination according to the present
invention in the manufacture of a medicament for the treatment of
genetic disorder or condition or disease.
[0022] According to another aspect of the present invention there
is provided a pharmaceutical composition comprising a compound
according to the present invention or a compound when prepared by
the process of the present invention admixed with a pharmaceutical
and, optionally, admixed with a pharmaceutically acceptable
diluent, carrier or excipient.
[0023] According to another aspect of the present invention there
is provided a pharmaceutical composition comprising a cationic
liposome according to the present invention or a cationic liposome
as prepared by the method of the present invention admixed with a
pharmaceutical and, optionally, admixed with a pharmaceutically
acceptable diluent, carrier or excipient.
[0024] It is believed that a key advantage of the compound of the
present invention is that it can be used as a cationic lipid
(amphiphile) in the preparation of a cationic liposome useful in
gene therapy, in particular the transfer of nucleic acids
(including genes and antisense DNA/RNA) into cells (in vitro and in
vivo) to derive a therapeutic benefit.
[0025] The cholesterol group can be cholesterol or a derivative
thereof. Examples of cholesterol derivatives include substituted
derivatives wherein one or more of the cyclic CH.sub.2 or CH groups
and/or one or more of the straight-chain CH.sub.2 or CH groups
is/are appropriately substituted. Alternatively, or in addition,
one or more of the cyclic groups and/or one or more of the
straight-chain groups may be unsaturated.
[0026] In a preferred embodiment the cholesterol group is
cholesterol. It is believed that cholesterol is advantageous as it
stabilises the resultant liposomal bilayer.
[0027] Preferably the cholesterol group is linked to the head group
via a carbamoyl linkage. It is believed that this linkage is
advantageous as the resultant liposome has a low or minimal
cytotoxicity.
[0028] Preferably the head group is a polyamine group. It is
believed that the polyamine group is advantageous because it
increases the DNA binding ability and efficiency of gene transfer
of the resultant liposome.
[0029] In one embodiment, preferably the polyamine group is a
naturally occurring polyamine. It is believed that the polyamine
head-group is advantageous because the increased amino
functionality increases the overall positive charge of the
liposome. In addition, polyamines are known to both strongly bind
and stabilise DNA.sup.14. In addition, polyamines occur naturally
in cells and so it is believed that toxicological problems are
minimised.sup.15.
[0030] Typical examples of suitable polyamines include spermidine,
spermine, caldopentamine, norspermidine and norspermine. These
polyamines are shown in FIG. 4.
[0031] Preferably the polyamine is spermidine or spermine as these
polyamines are known to interact with single or double stranded
DNA. An alternative preferred polyamine is caldopentamine.
[0032] Thus, a preferred compound is spermidine linked to
cholesterol via a carbamate linkage. This compound is shown in FIG.
5. It is believed that the polyamino head group is advantageous for
DNA condensation, the carbamate linkage is stable but biodegradable
and the cholesteryl group imparts bilayer rigidity. The carbamate
linkage may be part of, or an integral component of, the head
group.
[0033] Another preferred compound is spermine linked to cholesterol
via a carbamate linkage. Likewise, it is believed that the
polyamino head group is advantageous for DNA condensation, the
carbamate linkage is stable but biodegradable and the cholesteryl
group imparts bilayer rigidity.
[0034] Preferably the compound is in admixture with or associated
with a nucleotide sequence.
[0035] The nucleotide sequence may be part or all of an expression
system that may be useful in therapy, such as gene therapy.
[0036] Preferably the process comprises at least one step utilising
aza-Wittig methodology.
[0037] Preferably the process comprises the use of
trimethylphosphine.
[0038] Preferably the process comprises the use of a molecular
sieve.
[0039] Preferably, the cationic liposome is formed from the
compound of the present invention and a neutral phospholipid--such
as DOTMA or DOPE. Preferably, the neutral phospholipid is DOPE.
[0040] In another embodiment, preferably two or more of the amine
groups of the polyamine group of the present invention are
separated by one or more groups which are not found in nature that
separate amine groups of naturally occurring polyamine compounds
(i.e. preferably the polyamine group of the present invention has
unnatural spacing).
[0041] In summation, the present invention provides a compound
capable of acting as a cationic lipid, the compound comprising a
cholesterol group having linked thereto a head group; and wherein
the head group is more positive than the head group of DC-Chol; but
wherein the compound is not synthesised by reacting spermidine and
cholesterol chloroformate in CH.sub.2Cl.sub.2 in the presence of
N,N-diisoprophylethylamine.
[0042] A preferred embodiment of the present invention is a
compound capable of acting as a cationic lipid, the compound
comprising a cholesterol group having linked thereto a head group;
wherein the head group is more positive than the head group of
DC-Chol; wherein the cholesterol group is cholesterol; and wherein
the head group is a polyamine group; but wherein the compound is
not synthesised by reacting spermidine and cholesterol
chloroformate in CH.sub.2Cl.sub.2 in the presence of
N,N-diisoprophyl-ethylamine.
[0043] A more preferred embodiment of the present invention is a
compound capable of acting as a cationic lipid, the compound
comprising a cholesterol group having linked thereto a head group;
wherein the head group is more positive than the head group of
DC-Chol; wherein the cholesterol group is cholesterol; wherein the
head group is a polyamine group; and wherein the cholesterol group
is linked to the head group via a carbamoyl linkage; but wherein
the compound is not synthesised by reacting spermidine and
cholesterol chloroformate in CH.sub.2Cl.sub.2 in the presence of
N,N-diisoprophylethylamine.
[0044] A highly preferred embodiment of the present invention is a
compound capable of acting as a cationic lipid, the compound
comprising a cholesterol group having linked thereto a head group;
wherein the head group is more positive than the head group of
DC-Chol; wherein the cholesterol group is cholesterol; wherein the
head group is a polyamine group; wherein the cholesterol group is
linked to the head group via a carbamoyl linkage; and wherein the
polyamine group is a naturally occurring polyamine, such as any one
of spermidine, spermine or caldopentamine; but wherein the compound
is not synthesised by reacting spermidine and cholesterol
chloroformate in CH.sub.2Cl.sub.2 in the presence of
N,N-diisoprophylethylamine.
[0045] The present invention will now be described only by way of
examples, in which reference is made to the following Figures:
[0046] FIG. 1 which is a structure;
[0047] FIG. 2 which is a structure;
[0048] FIG. 3 which is a structure;
[0049] FIG. 4 which is a series of structures;
[0050] FIG. 5 which is a structure;
[0051] FIG. 6 which is a structure;
[0052] FIG. 7 which includes a reaction scheme;
[0053] FIG. 8 which includes a reaction scheme and a Table of
results;
[0054] FIG. 9 which includes a reaction scheme and a Table of
results;
[0055] FIG. 10 which includes a reaction scheme and a Table of
results;
[0056] FIG. 11 which includes a reaction scheme;
[0057] FIG. 12 which includes a reaction scheme and a Table of
results;
[0058] FIG. 13 which includes a reaction scheme and a Table of
results;
[0059] FIG. 14 which includes a reaction scheme and a Table of
results;
[0060] FIG. 15 which includes a reaction scheme and a Table of
results;
[0061] FIG. 16 which includes a reaction scheme and a Table of
results;
[0062] FIG. 17 which includes a reaction scheme;
[0063] FIG. 18 which includes a reaction scheme and a series of
results;
[0064] FIG. 19 which includes a reaction scheme and a series of
results;
[0065] FIG. 20 which includes a reaction scheme and a Table of
results;
[0066] FIG. 21 which includes a reaction scheme and a Table of
results;
[0067] FIG. 22 which presents a model;
[0068] FIG. 23 which presents data from some studies;
[0069] FIG. 24 which presents data from some studies;
[0070] FIG. 25 which presents data from some studies;
[0071] FIG. 26 which presents a reaction scheme;
[0072] FIG. 27 which presents a reaction scheme;
[0073] FIG. 28 which presents a reaction scheme; and
[0074] FIG. 29 which presents some formulae.
GENERAL COMMENTS
[0075] In these studies DC-Chol was used as a template for the
synthesis of more efficient gene transfer lipids, due to its good
gene transfer ability.sup.12 and low cytotoxicity.sup.13. In
particular, the head group of DC-Chol was changed to a series of
polyamine head-groups.
Initial Studies
[0076] Initially, a range of cationic lipids as shown in FIG. 6
(where m-=1-3, n=1-2; referenced as 2), with head groups based on
the naturally occurring polyamine, spermidine (m=3, n=3; referenced
as 3) were synthesised.
[0077] To prepare these compounds we used aza-Wittig
methodology.sup.16, and so we made and used a range of suitably
protected homologous azides and aldehydes (see Scheme 1 in FIG.
7).
[0078] Our initial studies suggested that benzyloxycarbonyl
protected aminoazides (see FIG. 8, referenced as 4) would be
suitable. These could be prepared in three steps and in excellent
yield from aminoalcohols (see FIG. 8, referenced as 3), by
sequential N-benzyloxycarbonylation, mesylation and azidation.
[0079] This process and the yields therefrom are shown in FIG. 8
(see Scheme 2 and Table 1).
[0080] The desired aldehyde could also be synthesised from
aminoalcohols (see FIG. 9, referenced as 5) in excellent yield,
this time by N-protection with cholesteryl chloroformate to give
alcohols (see FIG. 9, referenced as 8), followed by Swern-type
oxidation.sup.17 to aldehydes (see FIG. 9, referenced as 9).
[0081] This process and the yields therefrom are shown in FIG. 9
(see Scheme 3, Table 2).
[0082] The aldehydes (9) were isolated as white crystalline solids
which, in contrast with analogous aldehydes with different
protecting groups, proved to be extremely stable, easy to handle
and could be stored for long periods of time without any
discernible decomposition.
[0083] The aza-Wittig reaction between azides (4) and aldehydes (9)
occurred smoothly in THF to yield, after in situ reduction,
protected polyamines (10) in excellent yield. We found that the use
of trimethylphosphine rather than triphenylphosphine, led both to
higher yields and lower reaction times. The use of molecular sieves
also proved advantageous in obtaining consistently good yields, by
eliminating adventitious water from the reaction system. Finally,
removal of the benzyloxycarbonyl protecting group by hydrogenolysis
yielded the desired lipids (2) in quantitative yield. These
processes and the yields are shown in FIG. 10 (see Scheme 4, Table
3).
[0084] Liposomes containing the new lipids could be prepared using
the following general procedure which by way of example refers to
lipid (2) and DOPE.
[0085] Lipid (2) (6 .mu.mol) and DOPE (298 .mu.l of a 10 mg/ml
solution in CHCl.sub.3, 4 .mu.mol) were added via syringe to
freshly distilled CH.sub.2Cl.sub.2 (5 ml) under a nitrogen
atmosphere. 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid
(HEPES) buffer (5 ml of a 20 mM solution adjusted to pH 7.8) was
added. The two phase mixture was sonicated for 3 minutes and the
organic solvents were removed under reduced pressure. The resulting
liposome suspension was then sonicated for a further 30 min,
yielding liposomes with an average diameter of 150 nm (as
determined using a Coulter N4 MD photon correlation
spectrometer).
[0086] The new compounds yielded the correct analytical data by
.sup.1H NMR, .sup.13C NMR, 1R, MS and elemental analyses or
HRMS.
[0087] This process therefore yields spermidine containing DC-Chol
analogues from readily available starting materials, using
aza-Wittig methodology. For these studies reference can also be
made to FIGS. 11-21.
Further Studies
[0088] The further studies involved the inclusion of higher
homologue polyamines such as spermine and unusual polyamines such
as caldopentamine into a lipid framework. It is believed that these
lipids, when used within the confines of a liposome, provide a
DNA-condensing property that results in stronger, more stable
liposome/DNA conjugates. Thus, we believe that these properties
will improve the overall transfection of, for example, plasmid DNA
into, for example, cell lines.
[0089] For these further studies reference can also be made to
FIGS. 11-21.
Preparation of Methanesulfonate Esters
[0090] To a solution of dry alcohol (1.48 mmol) in dry
CH.sub.2Cl.sub.2 (15 ml) was added triethylamine (0.62 ml, 4.4
mmol) and the solution stirred under nitrogen and cooled to around
0.degree. C. At this point, methanesulfonyl chloride (0.29 ml, 3.7
mmol) in dry CH.sub.2Cl.sub.2 (5 ml) was added carefully and
dropwise to the solution of alcohol. After the addition stirring
was continued at 0.degree. C. for 10 minutes and then at RT for 10
minutes. Ice was then added carefully and the reaction quenched for
35 minutes. The CH.sub.2Cl.sub.2 was diluted five-fold and then
extracted with sat. ammonium chloride (70 ml), water (50 ml) and
brine (70 ml). The organic layer was then dried over anhydrous
sodium sulfate and the solvent removed in vacuo to give a
quantitative yield of the crude mesylate.
Preparation of Azides
[0091] To a stirred mixture of methanesulfonate ester (1.48 mmol),
sodium azide (0.48 g, 7.4 mmol, 5 eq.) and sodium iodide (0.222 g,
1.48 mmol) under N.sub.2, was added by syringe dry DMF (10 ml) and
the suspension heated to 80.degree. C. This was maintained for two
hours, and then allowed to cool to room temperature. The slurry was
then filtered over Celite.TM. and the DMF removed under high vacuum
to give a pale yellow oil.
[0092] The pale yellow oil was then redissolved in diethylether
(100 ml) and washed with water (3.times.60 ml) and brine (80 ml).
The organic layer was dried over anhydrous Na.sub.2SO.sub.4 and
evaporated in vacuo to give the colourless azide. The azide was
then purified further by flash column chromatography (ether/acetone
4-30%) to give the pure azide.
Preparation of Bromides
[0093] To a mixture of methanesulfonate ester (1 mmol) and sodium
bromide (0.515 g, 5 mmol, 5 eq.) was added dry DMF (10 ml) via
syringe and the solution heated at 80.degree. C. for two hours, and
then allowed to cool to room temperature and the slurry filtered
over Celite.TM., and the DMF removed in vacuo. The bromide was then
redissolved in diethylether (50 ml) and washed with water
(3.times.30 ml) and brine (50 ml). The organic extract was dried
over anhydrous Na.sub.2SO.sub.4, and the solvent removed in vacuo
to give a colourless oil. The bromide was then purified by flash
column chromatography (diethylether 20%/petrol 80%
.fwdarw.diethylether) to give pure bromide.
Mono-N-Alkylation with Bromides
[0094] To a stirred solution of bromide (1 mmol) and
4-amino-1-butanol (0.36 g, 4 mmol, 4 eq.) in anhydrous DMF (10 ml)
under N.sub.2, was added anhydrous K.sub.2CO.sub.3 (0.276 g, 2
mmol, 2 eq.) and sodium iodide (15 mg, 0.1 mmol, 0.1 eq.). The
reaction was stirred for 72 hours at room temperature, until
reaction complete. The DMF was evaporated in vacuo and dried
exhaustively under high vacuum. The resulting oily solid was
redissolved in CH.sub.2Cl.sub.2 (100 ml) and washed with water
(4.times.50 ml).
[0095] The combined aqueous extracts were then washed with
CH.sub.2Cl.sub.2 (40 ml) and the CH.sub.2Cl.sub.2 extracts dried
over anhydrous Na.sub.2SO.sub.4. The CH.sub.2Cl.sub.2 was
evaporated in vacuo to give a pale yellow oil.
Aza-Wittig Reaction
[0096] To a suspension of flame-dried 4 .ANG. molecular sieves (500
mg) and toluene-dried azide (1.08 mmol) in anhydrous THF (9 ml)
under a nitrogen atmosphere, was added dropwise a solution of
trimethylphosphine (1.2 ml of a 1.0 M solution in THF, 1.19 mmol,
1.1 eq.) via syringe.
[0097] After stirring at room temperature for one hour, the
aldehyde (1.19 mmol, 1.1 eq.) in THF (2 ml) was added dropwise via
cannula and the reaction mixture stirred for 18 hours. At this
point, the THF was evaporated under nitrogen, and the resulting
slurry resuspended in dry ethanol (7 ml). To this solution was
added sodium borohydride (4.32 ml of 0.5 M solution in diglyme,
2.16 mmol, 2 eq.) via syringe and the reaction stirred for a
further 24 hours at room temperature.
[0098] After this, the suspension was filtered over a short pad of
Celite.TM., and the diglyme removed in vacuo. Exhaustive drying
prior to work up was carried out, and the resulting oil redissolved
in CH.sub.2Cl.sub.2 (120 ml) and treated with sat. sodium
bicarbonate (100 ml), water (120 ml) and brine (120 ml). The
organic fraction was dried over anhydrous Na.sub.2SO.sub.4 and the
solvent removed in vacuo to give a pale yellow oil and the crude
product purified by flash column chromatography.
Hydrogenolysis
[0099] To a solution of Z-protected lipid (0.52 mmol, 1 eq.) in
ethanol (6 ml) was added cyclohexene (2.11 ml, 20.8 mmol, 40 eq.)
followed by palladium on activated carbon (10%, 277 mg, 0.5 eq.)
under nitrogen blanket. The solution was then refluxed under
nitrogen and the reaction followed by TLC
(CH.sub.2Cl.sub.2/MeOH/NH.sub.3 92:7:1).
[0100] After two hours, the catalyst was carefully filtered off
under nitrogen, washed with fresh ethanol and the solvent
evaporated in vacuo to give a white solid.
Preparation of Silanols
[0101] To a cooled, stirred solution of alcohol (0.93 mmol),
triethylamine (0.39 ml, 2.8 mmol) and DMAP (11.4 mg, 0.093 mmol,
0.1 eq.) in dry CH.sub.2Cl.sub.2 (1.5 ml) was added dropwise a
solution of tert-butylchlorodiphenylsilane (0.6 ml, 2.33 mmol, 2.5
eq.) in dry CH.sub.2Cl.sub.2 (1.5 ml). The solution was stirred at
room temperature for three to five hours at which point
CH.sub.2Cl.sub.2 (45 ml) was added to the reaction mixture and then
extracted with sat. sodium bicarbonate (70 ml) and brine (70
ml).
[0102] The CH.sub.2Cl.sub.2 extracts were dried over anhydrous
Na.sub.2SO.sub.4 and the solvent removed in vacuo to give a pale
yellow oil. This was purified by flash column chromatography.
N-Protection of Aminosilanols
[0103] To an ice-cooled aminosilanol (0.85 mmol) and triethylamine
(0.24 ml, 1.7 mmol, 2 eq.) in CH.sub.2Cl.sub.2 (4 ml) was added
dropwise a solution of phenylmethoxycarbonyl chloride (0.26 ml, 1.7
mmol, 2 eq.) in CH.sub.2Cl.sub.2 (1 ml). The resulting solution was
stirred at room temperature for 18 hours. The solution was then
diluted with CH.sub.2Cl.sub.2 (30 ml), washed with saturated
ammonium chloride (40 ml) and brine (40 ml). The CH.sub.2Cl.sub.2
extract was then dried over anhydrous sodium sulfate and evaporated
to dryness in vacuo to give a pale yellow oil. The compound was
purified by flash column chromatography.
Preparation of Aldehydes
[0104] To a stirred solution of ethanedioyl dichloride (0.80 ml,
9.3 mmol, 1.5 eq.) in CH.sub.2Cl.sub.2 (20 ml) at -78.degree. C.
under a nitrogen atmosphere was added a solution of DMSO (1.35 ml,
18.6 mmol, 3 eq.) in CH.sub.2Cl.sub.2 (20 ml) via cannula over a
time period of 15 minutes. The resulting solution was stirred for a
further 20 minutes after which time a solution of alcohol (6.2
mmol, 1 eq.) in CH.sub.2Cl.sub.2 (60 ml) was added dropwise via
cannula. After a further 30 minutes N,N,N-diisopropylethylamine
(3.25 ml, 18.6 mmol, 3 eq.) was added dropwise and the solution
warmed slowly to room temperature.
[0105] The pale yellow solution was then washed with saturated
ammonium chloride (150 ml), saturated sodium bicarbonate (80 ml)
and water (100 ml). The aqueous extracts were combined and washed
with CH.sub.2Cl.sub.2 (100 ml). The CH.sub.2Cl.sub.2 extracts were
combined, washed with brine (100 ml) and dried over anhydrous
sodium sulfate. The solvent was then evaporated in vacuo to give a
pale yellow solid. The crude aldehyde was then purified by flash
column chromatography (ether) to a white solid.
Deprotection of Silanols
[0106] To a solution of silanol (1 mmol) in THF (5 mmol) was added
carefully tetrabutyl ammonium fluoride (TBAF) (1.1 mmol) and the
solution stirred for two to three hours. The solution was then
diluted with CH.sub.2Cl.sub.2 (50 ml) and sat. sodium bicarbonate
(50 ml) added. The organic phase was collected, washed with water
(50 ml) and brine (60 ml). The organic layer was then dried over
anhydrous sodium sulfate, and the solvent removed in vacuo. The
pale yellow oil was then purified by flash column
chromatography.
Additional Studies
[0107] For these particular studies reference is made inter alia to
FIGS. 22-28.
[0108] In particular, FIG. 22 is the putative alignment of DC-Chol
1 and DOPE 2 in cationic liposome bilayer.
[0109] FIG. 23 is an example of in vitro optimisation of
pCF1-.beta.Gal plasmid transfection of CFT1 cells in vitro using
complexes of pCF1-.beta.Gal and cationic liposomes formulated from
DC-Chol analogue 8f' and DOPE 2 (1:1) molar ratio. CFT1 cells were
transfected with an array of different cationic liposome and
plasmid ratios in a 96-well plate. The extent of transfection in
each well was determined after 2 days by measuring the levels of
.beta.-galactosidase expression. Plasmid DNA concentration is
expressed as the concentration of nucleotides assuming an average
nucleotide FWt of 330. Cationic liposome concentration is expressed
in ter-Ms of the concentration of the constituent DC-Chol analogue
8f' alone.
[0110] FIG. 24 presents ran order of DC-Chol polyamine analogues
transfecting CFT1 cells in vitro with the pCF1-.beta.Gal plasmid.
Gene delivery activity is expressed as a proportion of the activity
measured with standard liposomes containing DC-Chol 1 and DOPE 2.
The data shown are the averages of four separate experiments, each
performed in triplicate.
[0111] The ratios in curved brackets refer to the DC-Chol
analogue:DOPE 2 molar ratio used to formulate the liposomes. The
numbers in square brackets refer to compound serial numbers. Where
appropriate DC-Chol analogue name abbreviations are also included
(see experimental).
[0112] FIG. 25 presents the rank order of DC-Chol polyamine
analogues transfecting the lungs of female BALB/c mice with the
pCF1--CAT plasmid. Mice were instilled with a solution of the
plasmid (4 mM nucleotide concentration) and the optimal quantity
and ratio of cationic liposome, in a total volume of 100 .mu.l.
Gene delivery activity was determined as a function of
chloramphenicol acetyl transferase activity in lung homogenates
after 2 days.
[0113] Data points were from separate experiments with each optimal
formulation tested in 4 BALB/c mice. The ratios in curved brackets
refer to the DC-Chol analogue:DOPE 2 molar ratio. The numbers in
square brackets refer to compound serial numbers. Where appropriate
DC-Chol analogue name abbreviations are also included (see
experimental).
[0114] Extra studies have shown that the gene delivery efficiency
of DC-Chol/DOPE liposomes is equivalent to the performance of
liposomes containing DC-Chol analogue 8c'..sup.[32]
[0115] In order to improve to DC-Chol/DOPE liposomes, a simple
model for the association of DC-Chol 1 and DOPE 2 in the bilayer of
a cationic liposome was devised (FIG. 22). This was based upon the
proposed behaviour of cholesterol in bilayer membranes.sup.[26] and
a liposome model proposed by Felgner and co-workers..sup.[27]
[0116] Carbon atoms C-1 to C-9 of the oleoyl side chains of DOPE 2
pack against the four fused cholesterol rings of DC-Chol 1 so that
the phosphate ester group of DOPE and the protonated tertiary amine
functionality of DC-Chol 2 are aligned and neutralise each other.
The positive charge of the liposome then derives from the
protonated ethanolamine side chain of DOPE 2. On the basis of the
recent transfection experiments in mouse and human.sup.[24, 25] it
was thought that increased transfection efficiency could best be
achieved by constructing either DC-Chol or DOPE polyamine analogues
which would be expected to interact with DNA more strongly. Since
DOPE is sensitive to oxidation of the oleoyl cis-double bonds, we
considered that it would be more appropriate to synthesise
polyamine analogues of DC-Chol instead. The model (FIG. 22)
suggested that the methylene group spacing between carbamoyl and
the first amine functional group of a given DC-Chol polyamine
analogue should be two or at most three, in order to maintain
charge complementation with DOPE 2. The preferred DC-Chol polyamine
analogues (i.e. polyamine analogues of
3DF-[N--(N',N'-dimethylaminoethane) carbamoyl]cholesterol) were
designed with this constraint in mind.
[0117] The syntheses of triamine analogues of DC-Chol were carried
out as follows.
[0118] Initially, two different
N-cholesteryloxycarbonylaminoaldehydes 3' were prepared by
N-protection of aminoalcohols 4' with cholesteryl chloroformate
followed by Swern-type oxidation.sup.[28] of protected alcohols 5'
to give the aldehydes 3' (see Scheme A in FIG. 26). Typically
speaking aminoaldehydes can be quite unstable and prone to
polymerisation but the steric stabilisation of the N-cholesteryl
moiety resulted in crystalline compounds which could be stored for
extended periods of time without any discernible decomposition.
[0119] N-Benzyloxycarbonyl protected aminoazides 6' were then
prepared in three steps from aminoalcohols 4' using sequential
N-benzyloxycarbonylation, mesylation and azidation (Scheme A).
Finally, azides 6' were coupled to aminoaldehydes 3' using
aza-Wittig methodology.sup.[29, 30] giving protected DC-Chol
triamine analogues 7' which were stored at this stage. In our
hands, aza-wittig coupling reactions were found to be more
efficient with trimethylphosphine rather than the customary
triphenylphosphine, in line with literature precedent..sup.[29]
Also, the elimination of adventitious water with activated
molecular sieves proved helpful to obtain consistently high
yields..sup.[29] Just prior to any gene delivery studies,
protecting groups were removed by catalytic transfer
hydrogenolysis, to give triamine analogues 8' (Scheme A) in 44-77%
overall yield. The syntheses of tetramine analogues of DC-Chol were
carried out in the following way. Several different N-protected
diaminoalcohols 9' were prepared by mono N-alkylation of
aminoalcohols 4' with N-benzyloxycarbonyl protected
aminoalkylbromides 10'. Bromides 10' were themselves prepared in
three steps from aminoalcohols by sequential
N-benzyloxycarbonylation, mesylation and bromination (see Scheme B
in FIG. 27). Customarily, mono-N-alkylations of primary amines are
considered to be difficult to control. Nevertheless, a combination
of steric crowding in the reactants and mild reaction conditions
are now being found increasingly to prevent over-N-alkylation
occurring..sup.[31] Temporary protection of the alcohol functional
groups of 9' as t-butyldiphenylsilylethers, followed by
N-benzyloxycarbonylation and fluoride promoted desilylation gave
bonafide di-N-benzyloxycarbonyl protected diaminoalcohols which
were then converted into diaminoazides 11' by mesylation followed
by azidation (Scheme B). Finally, protected DC-Chol tetramine
analogues 12' were formed by coupling azides 11' to
cholesteryl-aminoaldehydes 3' by means of the aza-Wittig procedure
once more (Scheme B). As for the preparation of 8' (Scheme A),
protecting groups were removed just prior to transfection studies
by catalytic transfer hydrogenolysis giving tetramine analogues 13'
(Scheme B) in 12-38% overall yield. Gratifyingly, we found that the
mono-N-alkylation procedure could be used equally well to prepare
N-cholesteryloxycarbonyl-diaminoaldehyde 14' from
N-cholesterylalcohol 5' via bromide 15' (see Scheme C in FIG. 28).
As a result, several fully protected DC-Chol pentamine analogues
16' could be prepared by aza-Wittig coupling of diaminoaldehyde 14'
to di-N-benzyloxycarbonyl protected diaminoazides 11'. DC-Chol
pentamine analogues 17' were then prepared by hydrogenolysis of the
protecting groups in the usual way (Scheme C).
[0120] The ability of cationic liposomes containing the different
DC-Chol polyamine analogues to mediate gene delivery was analysed
both in vitro and in vivo. Cationic liposomes were formulated by
hydrating a dried lipid film, containing DC-Cholanalogue and DOPE 2
in an appropriate molar ratio of either 1:0, 1:1, 1:2 or 2:1
respectively, and vortex mixing..sup.[32] Cationic liposome/plasmid
DNA complexes were then prepared by adding appropriately diluted
cationic liposome suspensions into equal volumes of aqueous plasmid
DNA solutions at 30.degree. C. and allowing the mixture to
equilibrate to ambient temperature over 15 min..sup.[32] In vitro
studies were then performed with immortalised cystic fibrosis
airway epithelial (CFT1) cells followed by in vivo studies in which
cationic liposome/plasmid DNA complexes were instilled intranasally
into the lungs of female BALB/c mice..sup.[32] Typically, cationic
liposome gene delivery was first optimised in vitro so as to
establish the best molar ratio of cationic liposome to plasmid DNA
(DNA concentration was expressed as nucleotide concentration) as
well as the best absolute quantities of both, as illustrated (FIG.
23). This optimised combination was then tested in vivo. The in
vitro results are shown (FIG. 24). Six DC-Chol analogue containing
liposomes proved to confer significant improvements on gene
delivery efficiency over and above DC-Chol/DOPE liposomes
formulated in a similar way. The analogues were 8a', 8b', 8e', 8f',
13a' and 17b', the first four being triamines, the fifth a
tetramine and the sixth a pentamine. With one exception, 8f', these
polyamine analogues contain inter-nitrogen methylene group spacings
not normally associated with the natural polyamines spermidine 18'
(see for example FIG. 29), spermine 19' (see for example FIG. 29)
and pentamine 20' (see for example FIG. 29) upon which these
structures are based. In vivo (FIG. 25), the best DC-Chol analogues
were 13c', 13f' and especially 17a'. Both 17a' and 13c' also
contain unnatural methylene group spacings.
[0121] Of the best in vivo analogues, 17a' works about 100-fold
more effectively in mouse lung than DC-Chol 1 and approx 500-fold
relative to plasmid DNA alone (FIG. 25). Only one other cytofectin
has been reported to function at this level of efficacy in vivo,
namely the lipid 67 21' (see FIG. 29), a "T" shaped tetramine
analogue of DC-Chol..sup.[32] None of the other reported
cytofectins appear to be close to this level of in vivo efficacy
with the possible exception of
(B1)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis-(dodecyloxy)-1-propanaminium
bromide (GAP-DLRIE) 22' (see FIG. 29) which has been reported to
work about 100 fold better than plasmid DNA alone..sup.[33] Other
cytofectins have been reported, but frequently have either not yet
been used in vivo or else with rather poor results..sup.[21, 34,
35]
[0122] Analogues 8f' and 13f' have been reported previously, either
without synthetic characterisation details.sup.[32] or else as
impure mixtures known as SpdC and SpC respectively..sup.[34] In the
former case, in vitro and in vivo gene delivery behaviours were
found to be comparable with our results reported here..sup.[32] In
the latter case, SpC was reported not to work well and to be
relatively toxic..sup.[34] Our data with analogue 13f' do not
support these observations.
Experimental Procedure
[0123] The synthetic procedures used to make the DC-Chol polyamine
analogues are documented below.
Scheme A
[0124] Reagents and conditions: i, CH.sub.2Cl.sub.2 (0.2M),
CholOC(O)Cl (0.45 eqv), 5 h, 98-99%; ii, (a) CH.sub.2Cl.sub.2
(0.1M) (COCl).sub.2 (1.5 eqv), DMSO (3 eqv), -78.degree. C., 15
min; (b) 5', 15 min; (c) i-Pr.sub.2NEt (3 eqv) to r.t., 93-97%;
iii, (a) CH.sub.2Cl.sub.2 (0.2M), PhCH.sub.2OC(O)Cl (0.45 eqv), 10
h; (b) CH.sub.2Cl.sub.2 (0.2M), Et.sub.3N (3 eqv),
CH.sub.3SO.sub.2Cl (2.5 eqv), 0.degree. C. to r.t., 15 min; (c) DMF
(0.15 M), NaN.sub.3 (5 eqv), NaI, 80.degree. C., 2 h, 68-87%; iv,
(a) 6', THF (0.5M), 4 .ANG. molecular sieves, PMe.sub.3 (1.15 eqv),
30 min; (b) 3' (1.1 eqv), 3h; (c) EtOH (0.5 M), NaBH.sub.4 (2 eqv),
20 h, 72-90%; v, EtOH (0.2M), c-C.sub.6H.sub.10 (20 eqv), 10% Pd(C)
(0.5 eqv), reflux, 30 min, 99%.
Scheme B
[0125] Reagents and conditions: i, (a) CH.sub.2Cl.sub.2 (0.2M),
PhCH.sub.2OC(O)Cl (0.45 eqv), 10 h; (b) CH.sub.2Cl.sub.2 (0.2M),
Et.sub.3N (3 eqv), CH.sub.3SO.sub.2Cl (2.5 eqv), 0.degree. C. to
r.t., 15 min; (c) DMF (0.15 M), NaBr (5 eqv), 80.degree. C., 2 h,
68-87%; ii, 10', DMF (0.1M), 4' (5 eqv), K.sub.2CO.sub.3 (2 eqv),
NaI (0.3 eqv), 24-72 h, 74-88%; iii, (a) CH.sub.2Cl.sub.2 (0.2M),
Et.sub.3N (1.5 eqv), TBDPSCl (1.2 eqv), DMAP (0.1 eqv), 09AC to
r.t., 2-4 h; (b) CH.sub.2Cl.sub.2 (0.2M), PhCH.sub.2OC(O)Cl (1.5
eqv), Et.sub.3N (1.5 eqv), 6-10 h; (c) THF (0.5M), TBAF (1.1 eqv),
4-5 h; (d) CH.sub.2Cl.sub.2 (0.2M), Et.sub.3N (3 eqv),
CH.sub.3SO.sub.2Cl (2.5 eqv), 0.degree. C. to r.t., 15 min; (e) DMF
(0.15M), NaN.sub.3 (5 eqv), NaI, 80.degree. C., 2 h, 37-70%; iv,
(a) 11', THF (0.5M), 4 .ANG. molecular sieves, PMe.sub.3 (1.15
eqv), 30 min; (b) 3' (1.1 eqv), 3h; (c) EtOH (0.5 M), NaBH.sub.4 (2
eqv), 20 h, 64-71%; v, EtOH (0.2M), c-C6H.sub.10 (20 eqv), 10%
Pd(C) (0.5 eqv), reflux, 30 min, 99%.
Scheme C
[0126] Reagents and conditions: i, (a) CH.sub.2Cl.sub.2 (0.2M),
Et.sub.3N (3 eqv), CH.sub.3SO.sub.2Cl (2.5 eqv), 0.degree. C. to
r.t., 15 min; (b) DMF (0.15 M), NaBr (5 eqv), 80.degree. C., 2h,
68-87%; ii, (a) 15', DMF (0.1M), 4' (5 eqv), K.sub.2CO.sub.3 (2
eqv), NaI (0.3 eqv), 24-72 h; (b) CH.sub.2Cl.sub.2 (0.2M),
Et.sub.3N (1.5 eqv), TBDPSCl (1.2 eqv), DMAP (0.1 eqv), 0.degree.
C. to r.t., 2-4 h; (c) CH.sub.2Cl.sub.2 (0.2M), PhCH.sub.2OC(O)Cl
(1.5 eqv), Et.sub.3N (1.5 eqv), 6-10 h; (d) THF (0.5M), TBAF (1.1
eqv), 4-5 h; (e) CH.sub.2Cl.sub.2 (0.1M) (COCl).sub.2 (1.5 eqv),
DMSO (3 eqv), -78.degree. C., 15 min; (f) di N-protected
diaminoalcohol, 15 min; (g) i-Pr.sub.2NEt (3 eqv) to r.t., 53-66%;
iii (a) 11', THF (0.5M), 4 .ANG. molecular sieves, PMe.sub.3 (1.15
eqv), 30 min; (b) 14' (1.1 eqv), 3h; (c) EtOH (0.5 M), NaBH.sub.4
(2 eqv), 20 h, 56-74%; iv, EtOH (0.2M), c-C.sub.6H.sub.10 (20 eqv),
10% Pd(C) (0.5 eqv), reflux, 30 min, 99%.
Data
[0127] Representative analytical data presented for the most
effective analogues in vitro (triamine 8e', tetramine 13a' and
pentamine 17b') and in vivo (tetramine 13f' and pentamine 17a')
4-aza-N'-cholesteryloxycarbonyl-1,7-heptanediamine (ACH) [B178 ]
8e'
[0128] v.sub.max (CH.sub.2Cl.sub.2) 3347, 2937, 2905, 2868, 1698,
1534, 1467, 1379 and 1264 cm.sup.-1; .delta..sub.H (300 MHz) 5.76
(1H, br s, NHCO), 5.22 (1H, m, H-6'), 4.33 (1H, m, H-3'), 3.08 (2H,
m, H-1), 2.65 (2H, t, J 6.5 Hz, H-7), 2.53 (2H, t, J 6.5 Hz, H-3),
2.41 (2H, m, H-5), 2.24-2.12 (2H, m, H-4'), 1.90-1.69 (5H, m, H-2',
H-7', H-8'), 1.55-0.93 (28H, m, H-2, H4, H-6, H-1', H-9', H-11',
H-12', H-14' to H-17', H-20', H-22' to H-25', NH.sub.2), 0.88 (3H,
s, H-19'), 0.79 (3H, d, J 6.5 Hz, H-21'), 0.73 (6H, dd, J 6.0 and
0.5 Hz, H-26' and H-27'), 0.55 (3H, s, H-18'); .delta..sub.C (75
MHz) 56.29 (NHC(O)O), 139.77 (C-5'), 122.24 (C-6'), 73.85 (C-3'),
56.59 (C-14'), 56.09 (C-17'), 49.92 (C-9'), 47.61 (C-1), 47.52
(C-3), 42.20 (C-4'), 40.22 (C-5), 39.66 (C-16'), 39.42 (C-24'),
38.57 (C-7), 36.94 (C-2), 36.44 (C-22'), 36.11 (C-8'), 35.71
(C-20'), 33.04 (C-7'), 31.78 (C-6), 28.14 (C-2'), 27.87 (C-25'),
24.19 (C-12'), 23.78 (C-15'), 22.74 (C-23'), 22.49 (C-26'), 20.96
(C-11'), 19.25 (C-19'), 18.65 (C-21'), 11.77 (C-18'); m/z (FAB) 544
(M+H).sup.+, 369 (Chol).sup.+, 95, 43 (Found: (M+H).sup.+,
544.4885. C.sub.34H.sub.62N.sub.3O.sub.2 requires (M+H).sup.+,
544.4842).
N.sup.1-cholesteryloxy-carbonyl-3,7-diaza-1,9-nonanediamine (CDAN)
[B198] 13a'
[0129] V.sub.max (CH.sub.2Cl.sub.2) 3584-3245, 2937, 2868, 1695,
1538, 1469, 1379, 1251, 1133 and 1014 cm.sup.1; .delta..sub.H (400
MHz) 5.82 (1H, br s, NHCO), 5.23 (1H, m, H-6'), 4.33 (1H, m, H-3'),
3.54-2.55 (16H, m, H-1 to H-4, H-6 to H-9, H.sub.2N), 2.21-2.09
(2H, m, H-4'), 1.97-1.73 (5H, m, H-2', H-7', H-8'), 1.55-0.99 (23H,
m, H-5, H-1', H-9', H-11', H-12', H-14' to H-17', H-20', H-22' to
H-25'), 0.88 (3H, s, H-19'), 0.78 (3H, d, J 6.0 Hz, H-21'), 0.74
(6H, d, J 6.5 Hz, H-26' and H-27'), 0.55 (3H, s, H-18');
.delta..sub.C(100 MHz) 156.38 (NHC(O)O), 139.70 (C-5'), 122.30
(C-6'), 73.99 (C-3'), 56.56 (C-14'), 56.05 (C-17'), 51.91 (C-1),
49.88 (C-9'), 47.95 (C-2), 42.18 (C-4'), 41.12 (C-8), 39.63
(C-16'), 39.40 (C-24'), 38.53 (C-2), 36.90 (C-1'), 36.42 (C-22'),
36.08 (C-8'), 35.69 (C-20'), 31.75 (C-7'), 29.62 (C-5), 28.12
(C-2'), 27.86 (C-25'), 24.17 (C-12'), 23.75 (C-15'), 22.73 (C-23'),
22.48 (C-26'), 20.94 (C-11'), 19.24 (C-19'), 18.62 (C-21'), 11.76
(C-18'); m/z (FAB) 573 (M+H).sup.+, 544, 513, 460, 369
(Chol).sup.+, 215, 95 (Found: (M+H).sup.+, 573.5139.
C.sub.35H.sub.65N.sub.4O.sub.2 requires (M+H).sup.+, 573.5108).
N.sup.10-cholesteryloxycarbonyl-4,8,13-triaza-1,16-hexadecanediamine(CTAH)-
[B222] 17b'
[0130] v.sub.max (CH.sub.2Cl.sub.2) 3344. 2936, 2855, 1700, 1536,
1468, 1379, 1265, 1122 and 1028 cm.sup.-1; .delta..sub.H (300 MHz)
5.69 (1H, br s, NHCO), 5.22 (1H, d, J 4.0 Hz, H-6'), 4.33 (1H, m,
H-3'), 3.20 (2H, m, H-1), 2.63 (2H, t, J 6.5 Hz, H-3), 2.55-2.35
(12H, m, H-5, H-8, H-10, H-12. H-14, H-16), 2.23-2.08 (7H, m, H-4,
H-9, H-13, H-4', NH.sub.2), 1.90-1.63 (5H, m, H-2', H-7', H-8'),
1.57-0.90 (31H, m, H-2, H-6, H-7, H-11, H-15, H-11', H-9', H-111',
H-12', H-14' to H-17', H-20', H-22' to H-25'), 0.87 (3H, s, H-19'),
0.78 (3H, d, J 6.5 Hz, H-21'), 0.73 (6H, d, J 6.5 Hz, H-26' and
H-27'), 0.55 (3H, s, H-18'); .delta..sub.C (75 MHz) 156.27
(NHC(O)O), 139.79 (C--S'), 122.24 (C-6'), 73.86 (C-3'), 56.58
(C-14'), 56.06 (C-17'), 49.92 (C-9'), 49.62 (C-1), 49.72 (C-3),
49.62 (C-12), 47.52 (C-14), 42.20 (C-4'), 39.65 (C-16'),39.42
C-24'), 38.56 (C-8), 36.93 (C-1'), 36.45 (C-22'), 36.09 (C-8'),
35.69 (C-20'), 31.78 (C-7'), 28.14 (C-2'), 27.88 (C-25'), 27.74
(C-2), 24.19 (C-12'), 23.74 (C-15'), 22.75 (C-23'), 22.49 (C-26'),
20.95 (C-11'), 19.26 (C-19'), 18.64 (C-21'), 11.77 (C-18'); m/z
(FAB) 672 (M+H).sup.+, 570, 539, 369 (Chol).sup.+, 84 (Found:
(M+H).sup.+, 672.6205. C.sub.41H.sub.78N.sub.5O.sub.2 requires
(M+H).sup.+, 672.6156).
N.sup.1-cholesteryloxy-carbonyl-4,9-diaza-1,12-dodecanediamine
(CDAD) [B185] 13f'
[0131] v.sub.max (CH.sub.2Cl.sub.2) 3349, 2937, 2868, 1697, 1468,
1378 and 1253 cm.sup.-1; .delta..sub.H (400 MHz) 5.70 (1H, br s,
NHCO), 5.27 (1H, m, H-6'), 4.38 (1H, m, H-3'), 3.2 8-3.14 (6H, m,
H-1, H-4, H-9, NH.sub.2), 2.71 (2H, t, J 6.5 Hz, H-12), 2.63-2.52
(8H, m, H-3, H-5, H-8, H-10), 2.33-2.17 (2H, m, H4'), 1.93-1.85
(5H, m, H-2', H-7', H-8'), 1.77-1.07 (29H, m, H-2, H-6, H-7, H-11,
H-1', H-9', H-11', H-12', H-14' to H-17', H-20', H-22' to H-25'),
1.03 (3H, s, H-19'), 0.97 (3H, d, J 6.0 Hz, H-21'), 0.77 (6H, dd, J
5.0 and 1.5 Hz, H-26' and H-27'), 0.59 (3H, s, H-18');
.delta..sub.C (100 MHz) 156.25 (NHC(O)O), 139.77 (C-5'), 122.26
(C-6'), 73.87 (C-3'), 56.57 (C-14'), 56.04 (C-17'), 49.90 (C-1),
49.60 (C-9'), 49.47 (C-3), 47.40 (C-10), 42.19 (C-4'), 39.63
(C-16'), 39.40 (C-24'), 38.55 (C-8), 36.92 (C-5), 36.44 (C-22'),
36.08 (C-8'), 35.69 (C-20'), 31.77 (C-7'), 28.13 (C-2'), 27.87
(C-25'), 27.53 (C-2), 24.18 (C-16), 23.74 (C-12'), 22.73 (C-23'),
22.48 (C-26'), 20.94 (C-11'), 19.25 (C-19'), 18.62 (C-21'), 11.76
(C-18'); m/z (FAB) 615 (M+H).sup.+, 539, 369 (Chol).sup.+, 57
(Found: (M+H).sup.+, 615.5626. C.sub.38H.sub.71N.sub.4O.sub.2
requires (M+H).sup.+, 615.5577).
N.sup.15-cholesteryloxycarbonyl-3,7,12-triaza-1,15-pentadecanediamine(CTAP-
)B[232] 17a'
[0132] v.sub.max (CH.sub.2Cl.sub.2) 3568-3295, 2937, 1690, 1537,
1467, 1380, 1130 and 1019 cm.sup.-1; .delta..sub.H (300 MHz) 5.76
(1H, br s, NHCO), 5.22 (1H, m, H-6'), 4.32 (1H, m, H-3'), 3.21 (2H,
m, H-15), 2.65 (2H, t, J 5.5 Hz, H-13), 2.56-2.45 (12H, m, H-1,
H-2, H-4, H-6, H-8, H-11), 2.18-2.05 (2H, m, H-4'), 1.97-1.67 (10H,
m, H-3, H-7, H-12, H-2', H-7', H-8', NH.sub.2), 1.59-0.91 (29H, m,
H1-5, H-9, H-10, H-14, H-1', H-9', H-11', H-12', H-14' to H-17',
H-20', H-22' to H-25'), 0.86 (3H, s, H-19'), 0.77 (3H, d, J 6.5 Hz,
H-21'), 0.72 (6H, dd, J 6.0 and 1.0 Hz, H-26' and H-27'), 0.53 (3H,
s, H-18'); .delta..sub.C (75 MHz) 156.24 (NHC(O)O), 139.77 (C-5'),
122.21 (C-6'), 73.81 (C-3'), 56.57 (C-14'), 56.05 (C-17'), 49.91
(C-9'), 49.67 (C-15), 48.20 (C-13), 42.19 (C-4'), 39.64 (C-16'),
39.40 (C-24'), 38.56 (C-2), 36.92 (C-1'), 36.43 (C-22'), 36.08
(C-8'), 35.68 (C-20'), 31.77 (C-7'), 28.12 (C-2'), 27.86 (C-25'),
27.69 (C-14), 27.63 (C-5), 24.17 (C-12'), 23.74 (C-15'), 22.73
(C-23'), 22.48 (C-26'), 20.94 (C-11'), 19.25 (C-19'), 18.63
(C-21'), 11.76 (C-18'); m/z (FAB) 658 (M+H).sup.+, 539, 369
(Chol).sup.+, 95, 84 (Found: (M+H).sup.+, 658.6056.
C.sub.40H.sub.76N.sub.5O.sub.2 requires (M+H).sup.+, 658.5999).
In Vitro and In Vivo Tests
[0133] For the in vitro and in vivo tests, a dried lipid film
containing the given DC-Chol analogue and DOPE 2 (in either a 1:0,
1:1, 1:2 or 2:1 respective molar ratio), was hydrated for 10 min in
sterile pyrogen-free water and then liposomes were produced by 2
min vortex mixing. The average diameter was between 200-400
nm..sup.[32] Cationic liposomes containing DC-Chol 1 and DOPE 2
were formulated in the same way described previously..sup.[24,
36]
[0134] Cationic liposome/plasmid DNA complexes were then prepared
as follows.
[0135] Both cationic liposome suspensions and DNA (either
pCF1-.beta.Gal plasmid expressing .beta.-galactosidase or pCF1-CAT
expressing chloramphenicol acetyl transferase).sup.[32] solutions
were separately pre-incubated for 5 min at 30.degree. C., before
being diluted to the appropriate final concentrations and then
combined. Usually, cationic liposome suspensions were added to an
approx. equal volume of plasmid DNA solutions. Complexes were
allowed to equilibrate for a minimum of 15 min at ambient
temperature and used within 2 h of preparation. In vitro and in
vivo gene delivery assays were then performed as described
previously using CFT1 cells and female BALB/c mice
respectively..sup.[32]
Discussion
[0136] In theory, genetic trait analysis will eventually be able to
identify all the genetic loci which cause or contribute towards
disease. With this information, corrective gene(s) may be
identified which if introduced into the appropriate organs and
cells of the body in vivo should correct the basic
pathophysiological defect of the disease. This is the basic concept
of gene therapy. Such a simple approach should be capable of curing
the disease in contrast to most conventional pharmaceutical
approaches which typically treat symptoms only.
[0137] However, introducing potentially corrective gene(s) is not
straightforward. Whilst naked DNA may be administered under certain
circumstances, for the most part a delivery vehicle or vector is
required to effect efficient gene delivery. Several physical,
chemical and virus-based vector systems are known but none are
sufficiently efficacious for general use in human gene therapy. In
spite of this, some vectors are showing some promise, in particular
cationic liposome-based gene transfer systems..sup.[21]
[0138] Cationic liposomes are heterogeneous, lipid vesicles
typically formed from either a single cationic amphiphile
(sometimes known as a cytofectin) or more commonly from a
combination of a cationic amphiphile and a neutral lipid. They
mediate gene delivery by interacting electrostatically with
negatively charged DNA sequences forming complexes which may enter
cells by endocytosis.sup.[221] or phagocytosisl.sup.[23] and then
release DNA for expression in the cell nucleus..sup.[21] We have
shown that cationic liposomes, formed from the cationic amphiphile
3.beta.-[N--(N',N-dimethyl-aminoethane)-carbamoyl]cholesterol
(DC-Chol) 1 and the neutral phospholipid dioleoyl
L-.alpha.-phosphatidylethanolamine (DOPE) 2, were able to transfect
the lungs of mice in vivo..sup.[24] Since then, some preparatory
human clinical trials have been performed using similar
DC-Chol/DOPE cationic liposomes..sup.[25]
[0139] Both sets of experiments represent a proof of principle
demonstrating that gene therapy with cationic liposomes is
possible. However, both sets of experiments also showed that
DC-Chol/DOPE cationic liposomes are unlikely to be efficient enough
at gene delivery for general use in human gene therapy. Moreover,
it is difficult to make improvements in the absence of any
understanding of cationic liposome structure/activity
relationships.
[0140] The present invention seeks to improve the earlier studies.
In addition, the present invention seeks to understand some of the
underlying chemical principles behind liposome-mediated gene
delivery.
[0141] In this regard, a systematic series of DC-Chol analogues
were made which could be incorporated into cationic liposomes and
evaluated for gene delivery.
[0142] In conclusion, we have developed a flexible synthetic route
to DC-Chol polyamine analogues which has allowed us to identify
analogues with optimised methylene group spacing between amine
functional groups for both in vitro and in vivo gene delivery On
the whole, so far we have found that unnatural spacing appears to
work better. Without wishing to be bound by theory, perhaps, such
polyamines have a slightly weakened interaction with DNA which
facilitates the release of DNA into the cytoplasm, after transfer
of the cationic liposome/DNA complex across the outer cell
membrane.
[0143] At present, evidence so far suggests that our preferred
DC-Chol analogue for in vivo studies and application is N.sup.
-cholesteryloxycarbonyl-3,7,12-triaza-1,15-penta-decane-diamine
(CTAP) 17a'. This compound is a novel pentamine of a type not
previously shown to transfect cells. The efficacy of this compound
appears to meet the necessary levels apparently required for a
cytofectin to have realistic potential clinical use..sup.[21,
24.25, 32]
SUMMATION
[0144] In summation, in the above examples liposomes were prepared
from compounds comprising a cholesterol component and a head group
component. The design of the compounds, which were used as cationic
lipids, concentrated on the direct manipulation of the head group.
With these compounds, the presence of the carbamoyl linkage is
believed to be advantageous for low cytotoxicity and the presence
of cholesterol is believed to be advantageous for the stabilisation
of the liposomal bilayer. In these studies, we were able to alter
the identity of the head group in order to increase the net
positive charge of the liposome. Increasing the net positive charge
is advantageous because it is believed to increase the DNA binding
ability and the efficiency of gene transfer of the resultant
liposome. Other modifications of the present invention will be
apparent to those skilled in the art.
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* * * * *