U.S. patent application number 11/521857 was filed with the patent office on 2007-05-10 for amphoteric liposomes.
Invention is credited to Gerold Endert, Yvonne Kerwitz, Silke Lutz, Steffen Panzner, Una Rauchhaus.
Application Number | 20070104775 11/521857 |
Document ID | / |
Family ID | 37631866 |
Filed Date | 2007-05-10 |
United States Patent
Application |
20070104775 |
Kind Code |
A1 |
Panzner; Steffen ; et
al. |
May 10, 2007 |
Amphoteric liposomes
Abstract
A serum-stable mixture of lipids capable of encapsulating an
active agent to form a liposome, said mixture comprising
phosphatidylcholine and phosphatidylethanolamine in a ratio in the
range of about 0.5 to about 8. The mixture may also include pH
sensitive anionic and cationic amphiphiles, such that the mixture
is amphoteric, being negatively charged or neutral at pH 7.4 and
positively charged at pH 4. Amphoteric liposomes comprising such a
mixture may be used for encapsulating nucleic acid therapeutics,
such as oligonucleotides and DNA plasmids. The drug/lipid ratio may
be adjusted to target the liposomes to particular organs or other
sites in the body.
Inventors: |
Panzner; Steffen; (Halle,
DE) ; Kerwitz; Yvonne; (Nordhausen, DE) ;
Rauchhaus; Una; (Halle, DE) ; Lutz; Silke;
(Halle, DE) ; Endert; Gerold; (Halle, DE) |
Correspondence
Address: |
MINTZ LEVIN COHN FERRIS GLOVSKY & POPEO
666 THIRD AVENUE
NEW YORK
NY
10017
US
|
Family ID: |
37631866 |
Appl. No.: |
11/521857 |
Filed: |
September 15, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60717199 |
Sep 15, 2005 |
|
|
|
60717291 |
Sep 15, 2005 |
|
|
|
Current U.S.
Class: |
424/450 ;
435/458; 514/44A; 977/907 |
Current CPC
Class: |
A61P 37/00 20180101;
A61K 9/0019 20130101; A61P 29/00 20180101; C12N 2310/11 20130101;
C12N 15/88 20130101; A61P 1/04 20180101; A61P 5/14 20180101; A61K
31/713 20130101; A61K 48/00 20130101; A61P 1/16 20180101; A61P
25/00 20180101; A61P 3/10 20180101; A61P 37/06 20180101; A61P 5/16
20180101; A61P 19/02 20180101; A61P 35/00 20180101; C12N 15/1138
20130101; A61K 9/1272 20130101; A61P 11/06 20180101; A61K 9/127
20130101; A61P 1/00 20180101; A61P 25/28 20180101; C12N 2320/32
20130101; A61K 9/1271 20130101; A61P 17/06 20180101 |
Class at
Publication: |
424/450 ;
514/044; 977/907; 435/458 |
International
Class: |
A61K 48/00 20060101
A61K048/00; A61K 9/127 20060101 A61K009/127; C12N 15/88 20060101
C12N015/88 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 15, 2005 |
EP |
05020218.3 |
Sep 15, 2005 |
EP |
05020217.5 |
Nov 4, 2005 |
EP |
PCT/EP05/11905 |
Nov 4, 2005 |
EP |
PCT/EP05/11908 |
May 10, 2006 |
EP |
06113784.0 |
Nov 21, 2005 |
EP |
05090322.8 |
Sep 15, 2005 |
EP |
05020216.7 |
Claims
1. A mixture of lipids capable of encapsulating an active agent to
form a liposome, said mixture comprising phosphatidylcholine and
phosphatidylethanolamine in a ratio of phosphatidylethanolamine to
phosphatidylcholine in the range of about 0.5 to about 8.
2. The mixture as claimed in claim 1, wherein said ratio is in the
range of about 0.75 to about 5.
3. The mixture as claimed in claim 1, wherein said ratio is in the
range of about 1 to about 4.
4. The mixture as claimed in claim 1, wherein said
phosphatidylcholine is selected from DMPC, DPPC, DSPC, POPC, DOPC,
soy bean PC or egg PC.
5. The mixture as claimed in claim 1, wherein said
phosphatidylethanolamine is selected from DOPE or DMPE or DPPE.
6. The mixture as claimed in claim 1, wherein said mixture is
neutral.
7. Neutral liposomes comprising a mixture of lipids as claimed in
claim 1.
8. The mixture as claimed in claim 1, further comprising one or
more charged amphiphiles.
9. The mixture as claimed in claim 8, wherein said one or more
charged amphiphiles are amphoteric, being negatively charged or
neutral at pH 7.4 and positively charged at pH 4.
10. The mixture as claimed in claim 9, wherein said mixture
comprises a plurality of charged amphiphiles which in combination
with one another have amphoteric character.
11. The mixture as claimed in claim 10, wherein said one or more
charged amphiphiles comprise at least one pH sensitive anionic
lipid and at least one pH sensitive cationic lipids.
12. The mixture as claimed in claim 11, wherein said anionic lipid
is selected from DOGSucc, POGSucc, DMGSucc, DPGSucc and CHEMS.
13. The mixture as claimed in claim 11, wherein said cationic lipid
is selected from MoChol, H is Chol and CHIM.
14. The mixture as claimed in claim 11, wherein the ratio between
the cationic and the anionic lipids (the charge ratio) is in the
range of 4:1 to 1:4.
15. The mixture as claimed in claim 11, wherein the ratio of
cationic lipids to anionic lipids is in the range of 3:1 to 2:1,
and said mixture comprises 5 to 95 mol. % charged lipids and 95 to
5 mol % phosphatidylcholine and phosphatidylethanolamine.
16. The mixture as claimed in claim 11, wherein the ratio of
cationic lipids to anionic lipids is about 1:1, and said mixture
comprises 5 to 75 mol. % charged lipids and 95 to 25 mol %
phosphatidylcholine and phosphatidylethanolamine.
17. The mixture as claimed in claim 11, wherein the ratio of
cationic lipids to anionic lipids is in the range of 1:3 to 1:2,
and said mixture comprises 40 to 75 mol. % charged lipids and 60 to
25 mol % phosphatidylcholine and phosphatidylethanolamine.
18. The mixture as claimed in claim 14, wherein said mixture
comprises: 70 to 20 mol. % of POPC and DOPE in a ratio in the range
of 1:1 to 1:4; and 30 and 80 mol. % of an amphoteric pair of
charged lipids, said pair being selected from MoChol and CHEMS,
MoChol and DMGSucc, MoChol and DOGSucc, CHIM and CHEMS or CHIM and
DMGSucc, wherein the ratio of cationic to anionic lipid is in the
range of 3:1 to 1:1.
19. The mixture as claimed in claim 18, wherein said mixture
consists of a formulation selected from: TABLE-US-00025
POPC/DOPE/MoChol/CHEMS 6:24:47:23 (mol. %) POPC/DOPE/MoChol/CHEMS
15:45:20:20 (mol. %) POPC/DOPE/MoChol/CHEMS 10:30:30:30 (mol. %)
POPC/DOPE/MoChol/DMGSucc 6:24:47:23 (mol. %)
POPC/DOPE/MoChol/DMGSucc 16:24:30:30 (mol. %)
20. The mixture as claimed in claim 11, wherein said mixture
comprises: 70. to 20 mol. % of POPC and DOPE in a ratio in the
range of 1:1 to 1:4; and and 80 mol. % of MoChol and DMGSucc or
DOGSucc, wherein the molar amount of DMGSucc or DOGSucc exceeds the
molar amount of MoChol.
21. The mixture as claimed in claim 20, wherein the ratio of
cationic to anionic lipid is in the range of 1:3 to 1:2, and said
mixture comprises 30 to 50 mol. % POPC and DOPE and 70 to 50 mol. %
charged lipids.
22. The mixture as claimed in claim 21, wherein said mixture
consists of a formulation selected from: TABLE-US-00026
POPC/DOPE/MoChol/DMGSucc 6:24:23:47 (mol. %)
POPC/DOPE/MoChol/DMGSucc 10:30:20:40 (mol. %)
23. Amphoteric liposomes comprising a mixture of lipids as claimed
in claim 9.
24. The amphoteric liposomes as claimed in claim 23, wherein said
liposomes have a size in the range of 50 to 500 mm.
25. The amphoteric liposomes as claimed in claim 23, wherein said
liposomes encapsulate at least one active agent.
26. The amphoteric liposomes as claimed in claim 25, wherein said
active agent comprises a nucleic acid that is capable of being
transcribed in a vertebrate cell into one or more RNAs, said RNAs
being mRNAs, shRNAs, miRNAs or ribozymes, said mRNAs coding for one
or more proteins or polypeptides.
27. The amphoteric liposomes as claimed in claim 26, wherein said
nucleic acid is a circular DNA plasmid, a linear DNA construct or
an mRNA.
28. The amphoteric liposomes as claimed in claim 25, wherein said
active agent is an oligonucleotide.
29. The amphoteric liposomes as claimed in claim 28, wherein said
oligonucleotide is a decoy oligonucleotide, an antisense
oligonucleotide, a siRNA, an agent influencing transcription, an
agent influencing splicing, Ribozymes, DNAzymes or Aptamers.
30. The amphoteric liposomes as claimed in claim 28, wherein said
oligonucleotides comprise modified nucleosides such as DNA, RNA,
locked nucleic acids (LNA), peptide nucleic acids (PNA), 2'O-methyl
RNA (2'Ome), 2' O-methoxyethyl RNA (2'MOE) in their phosphate or
phosphothioate forms.
31. The amphoteric liposomes as claimed in claim 28, wherein said
oligonucleotide is an antisense oligonucleotide of 15 to 30
basepairs length.
32. The amphoteric liposomes as claimed in claim 28, wherein said
oligonucleotide is a siRNA of 15 to 30 basepairs length.
33. The amphoteric liposomes as claimed in claim 28, wherein said
oligonucleotide is a decoy oligonucleotide of 15 to 30 basepairs
length.
34. The amphoteric liposomes as claimed in claim 28, wherein said
oligonucleotide is an agent influencing the transcription of 15 to
30 basepairs length.
35. The amphoteric liposomes as claimed in claim 28, wherein said
oligonucleotide is a DNAzyme of 25 to 50 basepairs length.
36. The amphoteric liposomes as claimed in claim 28, wherein said
oligonucleotide is a Ribozyme of 25 to 50 basepairs length.
37. The amphoteric liposomes as claimed in claim 28, wherein said
oligonucleotide is a Aptamer of 15 to 60 basepairs length.
38. The amphoteric liposomes as claimed in claim 28, wherein said
oligonucleotide is adapted to target a nucleic acid encoding CD40
gene, its sense or antisense strand, any exons or introns or
untranslated regions thereof thereby to modulate expression of CD40
in mammalian cells.
39. The amphoteric liposomes as claimed in claim 38, wherein said
oligonucleotide is directed against any mRNA of CD40, wherein such
mRNAs include pre-mRNA and their subsequently matured forms.
40. The amphoteric liposomes as claimed in claim 38, wherein said
mixture of lipids consists of a formulation selected from:
TABLE-US-00027 POPC/DOPE/MoChol/CHEMS 6:24:47:23 (mol. %)
POPC/DOPE/MoChol/CHEMS 15:45:20:20 (mol. %) POPC/DOPE/MoChol/CHEMS
10:30:30:30 (mol. %) POPC/DOPE/MoChol/DMGSucc 6:24:47:23 (mol. %)
POPC/DOPE/MoChol/DMGSucc 16:24:30:30 (mol. %)
POPC/DOPE/MoChol/DMGSucc 6:24:23:47 (mol. %)
POPC/DOPE/MoChol/DMGSucc 10:30:20:40 (mol. %)
41. The amphoteric liposomes as claimed in claim 28, wherein at
least 80 wt. % of said oligonucleotide is disposed inside said
liposomes.
42. The amphoteric liposomes as claimed in claim 28, wherein said
liposomes comprise non-encapsulated oligonucleotides.
43. A pharmaceutical composition comprising active agent-loaded
amphoteric liposomes as claimed in claim 25 and a pharmaceutically
acceptable vehicle therefor.
44. The pharmaceutical composition as claimed in claim 43, wherein
said liposomes have a size of greater than about 150 nm.
45. The pharmaceutical composition as claimed in claim 43, wherein
said liposomes have a size of less than about 150 nm.
46. The pharmaceutical composition as claimed in claim 43, said
composition further comprising empty liposomes having a similar
composition and size to said active agent-loaded amphoteric
liposomes.
47. A method of using the amphoteric liposomes as claimed in claim
28 for preventing or treating an inflammatory, immune or autoimmune
disorder of a human or non-human animal.
48. The method as claimed in claim 28 for preventing or treating
graft rejection, graft-versus-host disease, diabetes type I,
multiple sclerosis, systemic lupus erythematosous, rheumatoid
arthritis, asthma, inflammatory bowel disease, psoriasis or
thyroiditis, wherein said amphoteric liposomes are formulated for
systemic administration.
49. A method as claimed in claim 28 for preventing or treating
graft rejection, graft-versus-host disease, inflammatory bowel
disease, asthma, Crohn's disease or ulcerative colitis, wherein
said amphoteric liposomes is formulated for local
administration.
50. A method of treating a human or non-human animal by
administering systemically thereto at a low lipid dose a
pharmaceutical composition as claimed in claim 43, wherein said
liposomes have a size of greater than 150 nm, thereby targeting
said active agent to the liver.
51. A method of treating a human or non-human animal by
administering systemically thereto at a high lipid dose a
pharmaceutical composition as claimed in claim 43, wherein said
liposomes have a size of greater than 150 nm, thereby targeting
said active agent to the spleen, sites of infections and
inflammations or solid tumours.
52. A method of treating a human or non-human animal by
administering systemically thereto at a low lipid dose a
pharmaceutical composition as claimed in claim 43, wherein said
liposomes have a size of less than 150 mm, thereby targeting the
active agent to the liver.
53. A method of treating a human or non-human animal by
administering systemically thereto at a high lipid dose a
pharmaceutical composition as claimed in claim 43, wherein said
liposomes have a size of less than 150 nm, thereby targeting the
active agent to sites of infections and inflammations or solid
tumours, excluding the spleen
54. The method as claimed in claim 51 or claim 53, further
comprising lowering the drug/lipid ratio to the desired lipid
concentration.
55. The method as claimed in claim 51 or claim 53, further
comprising including in said composition empty liposomes having a
similar size and composition to said active agent-loaded liposomes.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from U.S. Provisional
Application Ser. No. 60/717,199, filed on Sep. 15, 2005; European
Application No. 05020218.3, filed on Sep. 15, 2005; European
Application No. 05020217.5, filed on Sep. 15, 2005; PCT Application
No. PCT/EP2005/011905, filed on Nov. 4, 2005; PCT Application No.
PCT/EP2005/011908, filed on Nov. 4, 2005; U.S. application Ser. No.
11/266,999, filed on Nov. 4, 2005; U.S. application Ser. No.
11/267,423, filed on Nov. 4, 2005; European Application No.
06113784.0, filed on May 10, 2006; and European Application No.
05090322.8, filed on Nov. 21, 2005.
[0002] Each of the applications and patents cited in this text, as
well as each document or reference cited in each of the
applications and patents (including during the prosecution of each
issued patent; "application cited documents"), and each of the U.S.
and foreign applications or patents corresponding to and/or
claiming priority from any of these applications and patents, and
each of the documents cited or referenced in each of the
application cited documents, are hereby expressly incorporated
herein by reference. More generally, documents or references are
cited in this text, either in a Reference List before the claims,
or in the text itself; and, each of these documents or references
("herein-cited references"), as well as each document or reference
cited in each of the herein-cited references (including any
manufacturer's specifications, instructions, etc.), is hereby
expressly incorporated herein by reference. Documents incorporated
by reference into this text may be employed in the practice of the
invention.
FIELD OF THE INVENTION
[0003] The present invention relates to amphoteric liposomes and
has particular reference to such liposomes having improved
stability in human or animal serum. The present invention also
comprehends mixtures of lipids capable of encapsulating active
agents or ingredients such, for example, as drugs to form liposomes
and pharmaceutical compositions comprising such liposomes.
BACKGROUND OF THE INVENTION
[0004] Oligonucleotides represent a novel class of drugs that can
very specifically down-regulate or interfere with protein
expression. Such oligonucleotides include antisense, locked nucleic
acids (LNA), peptide nucleic acids (PNA), morpholino nucleic acids
(Morpholinos), small interfering RNAs (siRNA) and transcription
factors decoys of various chemistries. A detailed description of
the different mechanisms of action of such oligonucleotide
therapeutics can be found in the literature (e.g., Crooke in BBA
(1999), 1489(1), 3144; Tijsterman, et al. in Cell (2004), 117(1),
1-3; and Mann, et al. in J Clin Invest, (2000), 106(9),
1071-5).
[0005] The use of oligonucleotides for gene repair applications
(see, e.g., Richardson, et al. in Stem Cells (2002), 20, 105-118)
and micro RNAs are other examples from this rapidly growing
field.
[0006] It is known in the art that nucleic acid therapeutics,
irrespective of their actual chemical origin, may lack therapeutic
efficacy owing to their instability in body fluids or because of
inefficient uptake into cells, or both. Chemical modifications of
such oligonucleotide, including the above-mentioned variants, as
well as the formation of conjugates with ligands or polymers,
represent one strategy to overcome such practical limitations.
[0007] A second set of strategies involves the use of carrier
systems, in particular liposomes, for protecting, targeting and
affording enhanced uptake into cells. Liposomes are artificial
single, oligo or multilamellar vesicles having an aqueous core and
being formed from amphiphilic molecules having both hydrophobic and
hydrophilic components (amphiphiles). The cargo may be trapped in
the core of the liposome, disposed in the membrane layer or at the
membrane surface. Such carrier systems should meet an optimum score
of the following criteria: high encapsulation efficiency and
economical manufacture, colloidal stability, enhanced uptake into
cells and of course low toxicity and immunogenicity.
[0008] Anionic or neutral liposomes are often excellent in terms of
colloidal stability, as no aggregation occurs between the carrier
and the environment. Consequently their biodistribution is
excellent and the potential for irritation and cytotoxicity is low.
However, such carriers lack encapsulation efficiency and do not
provide an endosomolytic signal that facilitates further uptake
into cells (Journal of Pharmacology and experimental Therapeutics
(2000), 292, 480-488 by Klimuk, et al.).
[0009] A great many of publications deal with cationic liposomal
systems; see, e.g., Molecular Membrane Biology (1999), 16, 129-140
by Maurer, et al.; BBA (2000) 1464, 251-261 by Meidan, et al.;
Reviews in Biology and Biotechnology (2001), 1(2), 27-33 by Fiset
& Gounni. Although cationic systems provide high loading
efficiencies, they lack colloidal stability, in particular after
contact with body fluids. Ionic interactions with proteins and/or
other biopolymers lead to in situ aggregate formation with the
extracellular matrix or with cell surfaces. Cationic lipids have
often been found to be toxic as shown by Filion, et al. in BBA
(1997), 1329(2), 345-356; Dass in J. Pharm. Pharmacol. (2002),
54(5), 593-601; Hirko, et al. in Curr. Med. Chem., 10(14),
1185-1193.
[0010] These limitations were overcome by the addition of
components that provide a steric stabilisation to the carriers.
Polyethylenglycols of various chain length, for example, are known
to eliminate aggregation problems associated with the use of
cationic components in body fluids, and PEGylated cationic
liposomes show enhanced circulation times in vivo (BBA (2001) 1510,
152-166 by Semple, et al.). However, the use of PEG does not solve
the intrinsic toxicity problems associated with cationic lipids. It
is also known that PEG substantially inhibits the productive entry
of such liposomes into the cells or their intracellular delivery
(Song, et al. in BBA (2002), 1558(1), 1-13). Quite recently,
Morrissey, et al. (Nature Biotechnology (2005), 23 (8), 1002-1007)
described a diffusible PEG-lipid for a cationic vector that is able
to transfer siRNA into liver cells in vivo. However, the huge
demand for such solutions and the given attrition rate of clinical
development more than motivates the development of conceptually
independent solutions.
[0011] Amphoteric liposomes represent a recently described class of
liposomes having an anionic or neutral charge at pH 7.5 and a
cationic charge at pH 4. WO 02/066490, WO 02/066012 and WO
03/070735, all to Panzner, et al. and incorporated herein by
reference, give a detailed description of amphoteric liposomes and
suitable lipids therefor. Further disclosures are made in WO
03/070220 and WO 03/070735, also to Panzner, et al. and
incorporated herein by reference, which describe further pH
sensitive lipids for the manufacture of such amphoteric
liposomes.
[0012] Amphoteric liposomes have an excellent biodistribution and
are very well tolerated in animals. They can encapsulate nucleic
acid molecules with high efficiency.
[0013] The use of amphoteric liposomes as carriers for drugs for
the prevention or treatment of different conditions or diseases in
mammals requires stability of the liposomes after their injection
into the bloodstream. For systemic applications especially, the
drug must be stably encapsulated in the liposomes until eventual
uptake in the target tissue or cells. The FDA's guidelines
prescribe specific preclinical tests for drugs comprising liposomal
formulations (http://www.fda.gov/cder/guidance/2191dft.pdf). For
example, the ratio of encapsulated drug to free drug must be
determined during the circulation time in the bloodstream.
[0014] After the injection of liposomes into the bloodstream, serum
components interact with the liposomes and may lead to
permeabilisation of the liposomal membrane. However, the release of
a drug that is encapsulated by the liposome also depends upon the
molecular dimensions of the drug. This means that a plasmid drug
with a size of thousands of base pairs, for example, may be
released much more slowly than smaller oligonucleotides or other
small molecules. For liposomal delivery of drugs it is essential
that the release of the drug during the circulation of the
liposomes is as low as possible.
OBJECTS OF THE INVENTION
[0015] An object of the present invention therefore is to provide
liposomes and mixtures of lipids capable of forming such liposomes
having improved stability upon contact with human or animal
serum.
[0016] In particular, an object of the present invention is to
provide amphoteric liposomes having such improved serum
stability.
[0017] Another object of the invention is to provide pharmaceutical
compositions comprising such liposomes as a carrier for the
targeted delivery of active agents or ingredients, including drugs
such as nucleic acid drugs, e.g., oligonucleotides and
plasmids.
[0018] A particular object of the present invention is to provide
such a pharmaceutical composition for the treatment or prophylaxis
of inflammatory, immune or autoimmune disorders of humans or
non-human animals.
[0019] Yet another object of the present invention is provide
methods for the treatment of human or non-human animals in which a
pharmaceutical composition comprising an active agent is targeted
to a specific organ or organs, tumours or sites of infection or
inflammation.
SUMMARY OF THE INVENTION
[0020] According to one aspect of the present invention therefore
there is provided a mixture of lipids capable of encapsulating an
active agent to form a liposome, said mixture comprising
phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in a
ratio of phosphatidylethanolamine to phosphatidylcholine in the
range of about 0.5 to about 8.
[0021] Suitably, said ratio range from about 0.75 to about 5,
preferably from about 1 to about 4.
[0022] In some embodiments, said phosphatidylcholine may be
selected from DMPC, DPPC, DSPC, POPC or DOPC, or from
phosphatidylcholines from natural sources such, for example, as soy
bean PC and egg PC.
[0023] Said phosphatidylethanolamines may be selected from DOPE,
DMPE and DPPE.
[0024] Preferred neutral lipids include DOPE, POPC, soy bean PC and
egg PC.
[0025] It is known that cholesterol may stabilise
phosphatidylcholine bilayers against serum attack. However, neither
POPC nor DOPE form serum stable structures by themselves. It has
now been found surprisingly that mixtures of DOPE and POPC may form
serum stable liposomes.
[0026] Accordingly, in a particular aspect of the present
invention, said mixture of lipids may be neutral. In some
embodiments said mixture may consist or consist essentially of
phosphatidylcholine and phosphatidylethanolamine in a ratio in the
aforementioned range.
[0027] In another aspect of the present invention there are
provided neutral liposomes comprising a mixture of lipids in
accordance with the invention. Such liposomes may be used as a
serum-stable excipient or carrier for active agents such as
drugs.
[0028] In a different aspect of the present invention however, said
mixture may further comprise one or more charged amphiphiles.
[0029] Preferably said one or more charged amphiphiles are
amphoteric, being negatively charged or neutral at pH 7.4 and
positively charged at pH 4.
[0030] By "amphoteric" herein is meant a substance, a mixture of
substances or a supra-molecular complex (e.g., a liposome)
comprising charged groups of both anionic and cationic character
wherein: [0031] (i) at least one of the charged groups has a pK
between 4 and 8, [0032] (ii) the cationic charge prevails at pH 4,
and [0033] (iii) the anionic charge prevails at pH 8, resulting in
an isoelectric point of neutral net charge between pH 4 and pH 8.
Amphoteric character is by this definition different from
zwitterionic character, as zwitterions do not have a pK in the
range mentioned above. In consequence, zwitterions are essentially
neutrally charged over a range of pH values; phosphatidylcholines
and phosphatidylethanolamines are neutral lipids with zwitterionic
character.
[0034] Suitably therefore, said mixture may comprise a plurality of
charged amphiphiles which in combination with one another have
amphoteric character. Preferably said one or more charged
amphiphiles comprise a pH sensitive anionic lipid and a pH
sensitive cationic lipid. Herein, such a combination of a
chargeable cation and chargeable anion is referred to as an
"amphoteric II" lipid pair. Said chargeable cation may have a pK
value of between about 4 and about 8, preferably between about 5.0
or 5.5 and about 7.0 or 7.5. Said chargeable anion may have a pK
value of between about 3.5 and about 7, preferably between about 4
or 4.5 and about 6.0 or 6.5. Examples include MoChol/CHEMS,
DPIM/CHEMS and DPIM/DGSucc.
[0035] An "amphoteric I" lipid pair comprises a stable cation
(e.g., DDAB/CHEMS, DOTAP/CHEMS and DOTAP/DOPS) and a chargeable
anion, while an "amphoteric III" lipid pair comprises a stable
anion and a chargeable cation (e.g., MoChol/DOPG and
MoChol/Chol-SO.sub.4).
[0036] It is of course possible within the scope of the present
invention to use amphiphiles with multiple charges such, for
example, as amphipathic dicarboxylic acids, phosphatidic acid,
amphipathic piperazine derivatives and the like. Such multi-charged
amphiphiles may be pH sensitive amphiphiles or stable anions or
cations, or they may have "mixed" character.
[0037] Suitably, said anionic lipid may be selected from DOGSucc,
POGSucc, DMGSucc, DPGSucc and CHEMS.
[0038] Said cationic lipid may be selected from MoChol, H is Chol
and CHIM.
[0039] In yet another aspect of the present invention there are
provided amphoteric liposomes comprising phosphatidylcholine and
phosphatidylethanolamine in a ratio in the aforementioned range, a
pH sensitive anionic lipid and a pH sensitive cationic lipid.
[0040] Said amphoteric liposomes may be negatively or neutrally
charged at pH 7.4 and cationic at pH4.
[0041] In another particular aspect of the present invention, said
liposomes encapsulate at least one active agent. Said active agent
may comprise a drug. In some embodiments said active agent may
comprises a nucleic acid such, for example, as an oligonucleotide
or DNA plasmid that is capable of being transcribed in a vertebrate
cell into one or more RNAs, said RNAs being mRNAs, shRNAs, miRNAs
or ribozymes, said mRNAs coding for one or more proteins or
polypeptides.
[0042] Said oligonucleotide or other nucleic acid based drug may be
encapsulated in said amphoteric liposomes. A substantial portion or
all of said oligonucleotides may be physically entrapped in the
amphoteric liposomes. The serum stable amphoteric liposomal
formulations can be used for the intracellular delivery of drugs or
for the prevention or treatment of a condition and/or disease in
mammals or part of mammals, especially humans or their organs.
[0043] In some embodiments, said oligonucleotide may be adapted to
target a nucleic acid encoding CD40, thereby to modulate expression
of CD40 in mammalian cells. Suitably, said oligonucleotide may be
directed against the mRNA of CD40.
[0044] In yet another aspect of the present invention there is
provided a pharmaceutical composition comprising active
agent-loaded amphoteric liposomes in accordance with the present
invention and a pharmaceutically acceptable vehicle therefor.
[0045] Said composition may be formulated for high or low lipid
doses, and suitably therefore the drug/lipid ratio may be adjusted
to a desired lipid concentration. In some embodiments, said
composition may further comprise empty liposomes to decrease said
drug/lipid ratio, said empty liposomes having the same or similar
size and composition to said active agent-loaded liposomes. Said
empty liposomes may comprise a mixture of lipids according to the
present invention.
[0046] In yet another aspect, the present invention comprehends the
use of a pharmaceutical composition according to the present
invention for the prevention or treatment of an inflammatory,
immune or autoimmune disorder of a human or non-human animal,
wherein said composition comprises an oligonucleotide adapted to
target a nucleic acid encoding CD40 for modulating the expression
of CD40 in mammalian cells.
[0047] Said composition may be formulated for systemic or local
administration. When used systemically, the present invention
comprises the use of said composition inter alia for the prevention
or treatment of graft rejection, graft-versus-host disease,
diabetes type I, multiple sclerosis, systemic lupus erythematosous,
rheumatoid arthritis, asthma, inflammatory bowel disease, psoriasis
or thyroiditis.
[0048] When formulated for local application, the invention
comprises the use of said composition inter alia for the prevention
or treatment of graft rejection, graft-versus-host disease,
inflammatory bowel disease, asthma, Crohn's disease or ulcerative
colitis.
[0049] These and other embodiments are disclosed or are obvious
from and encompassed by the following Detailed Description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0050] The following Detailed Description, given by way of example,
but not intended to limit the invention to specific embodiments
described, may be understood in conjunction with the accompanying
Figures, incorporated herein by reference, in which:
[0051] FIG. 1 is a graph of carboxyfluorescein (CF) release from
the MoChol/CHEMS formulations of Table 1 below after incubation in
full human serum for 4 hours. CF release is expressed as % of the
unquenched CF signal. The x-axis shows the total amount of charged
lipid at a 1:1 ratio between MoChol and CHEMS.
[0052] FIG. 2 is a graph of CF release from the MoChol/DMGSucc
formulations of Table 4 below after incubation in full human serum
for 4 hours. CF release is expressed as % of the unquenched CF
signal. The x-axis shows total amount of charged lipid at a 1:1
ratio between MoChol and DMGSucc.
[0053] FIG. 3 is graph of CF release from liposomes containing
MoChol/CHEMS or MoChol/DMGSucc after incubation in full human serum
at 37.degree. C. CF release is expressed as % of the unquenched CF
signal. Excess cation stabilises the liposomes against serum
attack. DMGSucc is notably more stable then the CHEMS
counterpart.
[0054] FIG. 4 is a graph of CF release from the MoChol/CHEMS and
MoChol/DMGSucc formulations of Tables 3 and 6 below after
incubation in full human serum at 37.degree. C. The formulations
have DOPE/POPC ratios of 2 and 4 and the ratio cationic to anionic
lipids is less than 1. Release is expressed as % of the unquenched
CF signal.
[0055] FIG. 5 is a bar chart showing the biodistribution of the
formulation POPC/DOPE/MoChol/CHEMS 15:45:20:20 having a size
>150 nm when administered at low and high lipid doses in rat
liver and spleen (see Example 7 below)
[0056] FIG. 6 is a bar chart showing the biodistribution of the
formulation POPC/DOPE/MoChol/CHEMS 15:45:20:20 having a size
<150 nm when administered at low and high lipid doses in rat
liver and spleen (see Example 7 below)
[0057] FIG. 7 is a set of photographs of the limbs of sacrificed
collagen-induced arthritic mice obtained by NIR-imaging and showing
the biodistribution of amphoteric liposomes encapsulating Cy5.5
labelled CD40 antisense (see Example 8 below)
[0058] FIG. 8 is a graph showing the effect of treatment with
amphoteric liposomes containing CD40 antisense on the paw swelling
of inflamed mice.
[0059] FIG. 9 is a graph of the assessed clinical score of mice
treated with amphoteric liposomes containing CD40 antisense.
[0060] FIG. 10 is a porcine CD40 cDNA sequence (SEQ ID NO:4) for
targeting in accordance with the present invention
DETAILED DESCRIPTION OF THE INVENTION
[0061] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the invention pertains. Although
a number of methods and materials similar or equivalent to those
described herein can be used in the practice of the present
invention, the preferred materials and methods are described
herein.
[0062] As mentioned above, the amphoteric liposomes of the present
invention may comprise anionic and cationic components, wherein
both components are pH-sensitive, as disclosed in WO 02/066012, the
contents of which are incorporated herein by reference.
[0063] Cationic lipids that are sensitive to pH are disclosed in WO
02/066489 and WO 03/070220, and in the references made therein, in
particular Budker, et al. 1996, Nat. Biotechnol. 14(6):760-4, the
contents of all of which are incorporated herein by reference.
[0064] Preferred cationic components are MoChol, H is Chol and
CHIM, especially MoChol.
[0065] Preferred anionic lipids are selected from the group
comprising: DOGSucc, POGSucc, DMGSucc, DPGSucc and CHEMS,
especially DOGSucc, DMGSucc and CHEMS.
[0066] The following abbreviations for lipids are used herein, the
majority of which abbreviations are in standard use in the
literature: [0067] PC Phosphatidylcholine, unspecified membrane
anchor [0068] PE Phosphatidylethanolamine, unspecified membrane
anchor [0069] DMPC Dimyristoylphosphatidylcholine [0070] DPPC
Dipalmitoylphosphatidylcholine [0071] DSPC
Distearoylphosphatidylcholine [0072] POPC
Palmitoyl-oleoylphosphatidylcholine [0073] DOPC
Dioleoylphosphatidylcholine [0074] DOPE
Dioleoylphosphatidylethanolamine [0075] DMPE
Dimyristoylphosphatidylethanolamine [0076] DPPE
Dipalmitoylphosphatidylethanolamine [0077] CHEMS
Cholesterolhemisuccinate [0078] CHIM Cholesterol-(3-imidazol-1-yl
propyl)carbamate [0079] DDAB Dimethyldioctadecylammonium bromide
[0080] DOTAP (1,2-dioleoyloxypropyl)-N,N,N-trimethylammonium salt
[0081] DOPS Dioleoylphosphatidylserine [0082] DOPG
Dioleoylphosphatidylglycerol [0083] Chol-SO.sub.4 cholesterol
sulfate [0084] MoChol
4-(2-Aminoethyl)-Morpholino-Cholesterolhemisuccinate: ##STR1##
[0085] His Chol Histaminyl-Cholesterolhemisuccinate: ##STR2##
[0086] DGSucc 1,2-Dipalmitoyglycerol-3-hemisuccinate (&
Distearoyl-, dimyristoyl-Dioleoyl or palmitoyl-oleoylderivatives)
(in the structure below the acyl chain is shown schematically)
##STR3##
[0087] It has been found that the ratio between the cationic and
anionic lipids (the charge ratio) not only determines the
isoelectric point, but may also affect the serum stability of the
composition. Accordingly, said charge ratio may vary from 4:1 to
1:4, preferably between 3:1 and 1:3 (cation:anion).
[0088] In some embodiments of the invention, the cation may be
present in excess over the anion. Preferably said charge ratio is
between 3:1 and 2:1. The total amount of charged lipids may vary
from 5 to 95 mol. % of the lipid mixture, preferably from 30 to 80
mol. %, and more preferably from 45 or 50 mol. % to 75 mol. %, with
the remaining lipids being formed from the neutral phospholipids PC
and PE.
[0089] Alternatively, the cation and anion may be present in
substantially equal amounts. The total amount of charged lipids may
vary from 5 to 75 mol. % of the lipid mixture, preferably from 20
to 65 mol. %, with the remaining lipids being formed from the
neutral phospholipids PC and PE.
[0090] In another alternative, the anion may be present in excess
over the cation. Said charge ratio may be between 1:3 and 1:2,
preferably about 1:2 (cation:anion). The total amount of charged
lipids may vary from 40 mol. % to 75 or 80 mol. % of the lipid
mixture, preferably from 45 or 50 mol. % to 70 or 75 mol. %, with
the remaining lipids being formed from the neutral phospholipids PC
and PE.
[0091] A number of different combinations of cations and anions may
be selected from the lists of suitable components given above.
Advantageously, the invention may be practised using MoChol or CHIM
as a chargeable cation and CHEMS, DMGSucc or DOGSucc as a
chargeable anion.
[0092] Presently preferred liposomes are made from a mixture of
lipids comprising POPC and DOPE in a ratio between 1:1 and 1:4 and
an amphoteric lipid pair selected from MoChol and CHEMS, MoChol and
DMGSucc, MoChol and DOGSucc, CHIM and CHEMS, and CHIM and DMGSucc,
in a ratio between 3:1 and 1:1, wherein the amount of charged
lipids is between 30 and 80 mol. % of the lipid mixture.
[0093] Specific examples of such liposomes in accordance with the
present invention include, but are not limited to: TABLE-US-00001
POPC/DOPE/MoChol/CHEMS 6:24:53:17 POPC/DOPE/MoChol/CHEMS 6:24:47:23
POPC/DOPE/MoChol/CHEMS 15:45:20:20 POPC/DOPE/MoChol/CHEMS
10:30:30:30 POPC/DOPE/MoChol/CHEMS 24.5:35.5:20:20
POPC/DOPE/MoChol/CHEMS 16:24:30:30 POPC/DOPE/MoChol/DMGSucc
6:24:53:17 POPC/DOPE/MoChol/DMGSucc 6:24:47:23
POPC/DOPE/MoChol/DMGSucc 15:45:20:20 POPC/DOPE/MoChol/DMGSucc
10:30:30:30 POPC/DOPE/MoChol/DMGSucc 24.5:35.5:20:20
POPC/DOPE/MoChol/DMGSucc 16:24:30:30 POPC/DOPE/MoChol/DOGSucc
12.5:37.5:33:17 POPC/DOPE/MoChol/DOGSucc 7.5:22.5:47:23
POPC/DOPE/CHIM/CHEMS 12.5:37.5:33:17 POPC/DOPE/CHIM/CHEMS
7.5:22.5:47:23 POPC/DOPE/CHIM/DMGSucc 12.5:37.5:33:17
POPC/DOPE/CHIM/DMGSucc 7.5:22.5:47:23
[0094] Further presently preferred liposomes comprise a mixture of
lipids comprising POPC and DOPE in a ratio between 1:1 and 1:4,
DMGSucc or DOGSucc, and MoChol, wherein the molar amount of DMGSucc
or DOGSucc exceeds the molar amount of MoChol and the amount of
charged lipids is between 30 and 80 mol. %. Preferably, the charge
ratio is between 1:2 and 1:3 and charged components constitute
between 45 or 50 mol. % and 70 or 75 mol. % of the lipid
mixture.
[0095] Specific examples of such further liposomes include, but are
not limited to: TABLE-US-00002 POPC/DOPE/MoChol/DMGSucc 6:24:23:47
POPC/DOPE/MoChol/DMGSucc 8:32:20:40 POPC/DOPE/MoChol/DMGSucc
10:40:17:33 POPC/DOPE/MoChol/DMGSucc 10:20:23:47
POPC/DOPE/MoChol/DMGSucc 13:27:20:40 POPC/DOPE/MoChol/DMGSucc
10:30:20:40 POPC/DOPE/MoChol/DMGSucc 17:33:17:33
POPC/DOPE/MoChol/DOGSucc 12.5:37.5:17:33
[0096] Without being limited to such use, the materials described
in the present invention are well suited for use as carriers for
nucleic acid-based drugs such for example as oligonucleotides and
DNA plasmids. These drugs are classified into nucleic acids that
encode one or more specific sequences for proteins, polypeptides or
RNAs and into oligonucleotides that can specifically regulate
protein expression levels or affect the protein structure through
inter alia interference with splicing and artificial
truncation.
[0097] In some embodiments of the present invention, therefore, the
nucleic acid-based therapeutic may comprise a nucleic acid that is
capable of being transcribed in a vertebrate cell into one or more
RNAs, which RNAs may be mRNAs, shRNAs, miRNAs or ribozymes, wherein
such mRNAs code for one or more proteins or polypeptides. Such
nucleic acid therapeutics may be circular DNA plasmids, linear DNA
constructs, like MIDGE vectors (Minimalistic Immunogenically
Defined Gene Expression) as disclosed in WO 98/21322 or DE
19753182, or mRNAs ready for translation (e.g., EP 1392341).
[0098] In another embodiment of the invention, oligonucleotides may
be used that can target existing intracellular nucleic acids or
proteins. Said nucleic acids may code for a specific gene, such
that said oligonucleotide is adapted to attenuate or modulate
transcription, modify the processing of the transcript or otherwise
interfere with the expression of the protein. The term "target
nucleic acid" encompasses DNA encoding a specific gene, as well as
all RNAs derived from such DNA, being pre-mRNA or mRNA. A specific
hybridisation between the target nucleic acid and one or more
oligonucleotides directed against such sequences may result in an
inhibition or modulation of protein expression. To achieve such
specific targeting, the oligonucleotide should suitably comprise a
continuous stretch of nucleotides that is substantially
complementary to the sequence of the target nucleic acid.
[0099] Oligonucleotides fulfilling the abovementioned criteria may
be built with a number of different chemistries and topologies.
Oligonucleotides may be single stranded or double stranded.
[0100] The mechanisms of action of oligonucleotides may vary and
might comprise effects on inter alia splicing, transcription,
nuclear-cytoplasmic transport and translation.
[0101] In a preferred embodiment of the invention single stranded
oligonucleotides may be used, including, but not limited to,
DNA-based oligonucleotides, locked nucleic acids, 2'-modified
oligonucleotides and others, commonly known as antisense
oligonucleotides. Backbone or base or sugar modifications may
include, but are not limited to, Phosphothioate DNA (PTO),
2'O-methyl RNA (2'Ome), 2' O-- methoxyethyl-RNA (2'MOE), peptide
nucleic acids (PNA), N3'-P5' phosphoamidates (NP), 2'fluoroarabino
nucleic acids (FANA), locked nucleic acids (LNA), Morpholine
phosphoamidate (Morpholino), Cyclohexene nucleic acid (CeNA),
tricyclo-DNA (tcDNA) and others. Moreover, mixed chemistries are
known in the art, being constructed from more than a single
nucleotide species as copolymers, block-copolymers or gapmers or in
other arrangements. In addition to the aforementioned
oligonucleotides, protein expression can also be inhibited using
double stranded RNA molecules containing the complementary sequence
motifs. Such RNA molecules are known as siRNA molecules in the art
(e.g., WO 99/32619 or WO 02/055693). Again, various chemistries
were adapted to this class of oligonucleotides. Also, DNA/RNA
hybrid systems are known in the art.
[0102] In another embodiment of the present invention, decoy
oligonucleotides can be used. These double stranded DNA molecules
and chemical modifications thereof do not target nucleic acids but
transcription factors. This means that decoy oligonucleotides bind
sequence-specific DNA-binding proteins and interfere with the
transcription (e.g. Cho-Chung, et al. in Curr. Opin. Mol. Ther.,
1999).
[0103] In a further embodiment of the invention, oligonucleotides
that may influence transcription by hybridizing under physiological
conditions to the promoter region of a gene may be used. Again
various chemistries may adapt to this class of
oligonucleotides.
[0104] In a still further alternative of the invention, DNAzymes
may be used. DNAzymes are single-stranded oligonucleotides and
chemical modifications therof with enzymatic activity. Typical
DNAzymes, known as the "1023" model, are capable of cleaving
single-stranded RNA at specific sites under physiological
conditions. The 10-23 model of DNAzymes has a catalytic domain of
15 highly conserved deoxyribonucleotides, flanked by 2
substrate-recognition domains complementary to a target sequence on
the RNA. Cleavage of the target mRNAs may result in their
destruction and the DNAzymes recycle and cleave multiple
substrates.
[0105] In yet another embodiment of the invention, ribozymes can be
used. Ribozymes are single-stranded oligoribonucleotides and
chemical modifications thereof with enzymatic activity. They can be
operationally divided into two components, a conserved stem-loop
structure forming the catalytic core and flanking sequences which
are reverse complementary to sequences surrounding the target site
in a given RNA transcript. Flanking sequences may confer
specificity and may generally constitute 14-16 nt in total,
extending on both sides of the target site selected.
[0106] In a still further embodiment of the invention, aptamers may
be used to target proteins. Aptamers are macromolecules composed of
nucleic acids, such as RNA or DNA, and chemical modifications
thereof that bind tightly to a specific molecular target and are
typically 15-60 nt long. The chain of nucleotides may form
intramolecular interactions that fold the molecule into a complex
three-dimensional shape. The shape of the aptamer allows it to bind
tightly against the surface of its target molecule including but
not limited to acidic proteins, basic proteins, membrane proteins,
transcription factors and enzymes. Binding of aptamer molecules may
influence the function of a target molecule.
[0107] All of the above-mentioned oligonucleotides may vary in
length between as little as 10, preferably 15 and even more
preferably 18, and 50, preferably 30 and more preferably 25,
nucleotides. The fit between the oligonucleotide and the target
sequence is preferably perfect with each base of the
oligonucleotide forming a base pair with its complementary base on
the target nucleic acid over a continuous stretch of the
abovementioned number of oligonucleotides. The pair of sequences
may contain one or more mismatches within the said continuous
stretch of base pairs, although this is less preferred. In general,
the type and chemical composition of such nucleic acids is of
little impact for the performance of the inventive liposomes as
vehicles be it in vivo or in vitro, and the skilled artisan may
find other types of oligonucleotides or nucleic acids suitable for
combination with the inventive liposomes.
[0108] In a preferred embodiment of the invention however,
oligonucleotides may used that are adapted to target a nucleic acid
encoding the CD40 gene, its sense or antisense strand, any exons or
introns or untranslated regions thereof, thereby to modulate
expression of CD40 in mammalian cells.
[0109] In another preferred embodiment of the invention, said
oligonucleotides may directed against any mRNA of CD40, wherein
such mRNAs include pre-mRNA and their subsequently matured
forms.
[0110] Protein expression can be specifically down-regulated using
oligonucleotides such, for example, as antisense, locked nucleic
acids (LNA), peptide nucleic acids (PNA), morpholino nucleic acids
(Morpholinos) and small interfering RNAs (siRNA) of various
chemistries.
[0111] CD40 was first described by Pauli, et al. 1984 (Cancer
Immunol. Immunotherapy 17: 173-179). The protein is primarily
expressed on dendritic cells, endothelia cells and B-cells and
interacts with its ligand (CD40 ligand or CD154) on T-cells. The
signalling between CD40 and CD154 is crucial for the development of
a humoral immune response. Over-stimulation of the pathway may lead
to a variety of immune-associated disorders, including graft
rejection, graft-versus-host disease, multiple sclerosis, systemic
lupus erythematosous, rheumatoid arthritis, asthma, inflammatory
bowel disease, psoriasis and thyroiditis. CD40 over-expression
might also be involved in tumour growth (Gruss, et al. 1997, Leuk.
Lymphoma. 24(5-6): 393-422) and enhanced levels of a soluble form
of CD40 were reported to be associated with Alzheimers disease
(Mocali et al. 2004, Exp Gerontol. 39(10):1555-61. CD40 signals
into the NF-.kappa.B pathway, consequently leading to activation of
the transcription factor and the eventual release of cytokines such
as IL-1, TNF.alpha. and IFN.gamma., which in turn activate other
cells, thus promoting inflammation using a positive feedback
mechanism.
[0112] Inhibition of the early events in the pathway described
above has been proposed as an effective strategy to inhibit immune
disorders or inflammation processes. Examples include the
competitive binding of TNF.alpha. using antibodies, receptor
blocking using antibodies against the TNF.alpha.-receptor and
competitive inhibition of NF-.kappa.B binding. Since CD40 signals
through its interaction with the trimeric ligand, CD154, inhibition
of the signalling event with small molecule inhibitors is unlikely
and therapeutic developments have therefore focused on the use of
blocking antibodies. More specifically, the CD40/CD154 interaction
may be blocked using antibodies targeted against one of the
components, as described by Holstager, et al. 2000 (J. Biol. Chem.
275:15392-15398) or Baccam & Bishop 1999 (Eur. J. Immunol. 29:
3855-3866). However, the CD40 antibodies under development give
rise to side reactions, and there is therefore an need for
alternative means to cut the inflammatory feedback loop at this
point.
[0113] A number of oligonucleotide sequences targeted against CD40
mRNA have been validated in vitro so far. U.S. 2004/0186071 and
U.S. Pat. No. 6,197,584, both to Bennett, et al., for example, give
a detailed description of such oligonucleotides based on antisense
mechanisms. Pluvinet, et al. in Blood, 2004 first described the
down-regulation of CD40 using siRNA against the human target.
Further, WO 2004/090108 to Manoharan describes the applicability of
novel oligonucleotides to inhibit the expression of CD40 protein.
Indirect means to down-regulate the CD40 expression are described
in DE 10049549 to Hecker and Wagner, using the inhibition of
transcription factor IFR-1. Suitable specific nucleic acids for
modulating the expression of CD40 are set forth in Example 11
below.
[0114] In a particular aspect of the present invention therefore
there is provided a pharmaceutical composition comprising an
oligonucleotide directed against CD40 as an active agent and an
amphoteric liposome of the present invention as an excipient. Such
formulations have been found to be therapeutically active in the
treatment of inflammations and autoimmune disorders, and
accordingly the invention further comprehends the use of the
composition of the invention for the prevention or treatment of
inflammations, immune or autoimmune disorders, including graft
rejection, graft-versus-host disease, multiple sclerosis, systemic
lupus erythematosous, rheumatoid arthritis, asthma, asthma
bronchiale, inflammatory bowel disease, psoriasis, thyroiditis,
Morbus Crohn, Colitis ulcerosa, COPD and atopic dermatitis.
[0115] The pharmaceutical composition of the present invention may
also be used for topical treatments, for example the treatment of
inflamed mucosa. In particular, the composition of the invention
may be used for the treatment or prophylaxis of inflammatory bowel
disease or graft rejection. The composition of the present
invention may also be adapted for topical application to the skin
or lungs.
[0116] Liposomes have been widely used to alter the pharmacokinetic
and biodistribution profile of encapsulated drugs in vivo. The
liposomes of the present invention, together with their cargo, may
be cleared rapidly and to a great extent by the liver. However, the
pharmacokinetic parameters as well as the biodistribution pattern
may be controlled by adjusting the size of the liposomes and/or the
lipid dose as illustrated in the examples below.
[0117] In some embodiments, the liposomes of the present invention
may have a size greater than about 150 nm. Such liposomes may be
administered at a low lipid dose. Said liposomes may be
unilamellar, oligolamellar or multilamellar. Such a dosing scheme
allows for effective and rapid targeting to the liver and avoids
the accumulation of liposomes and drug in other organs, such as the
spleen.
[0118] Alternatively, such liposomes having a size greater than
about 150 nm may be administered at a high lipid dose, leading to
saturation of the liver and an alteration of the biodistribution
pattern to an accumulation of the liposomes in the spleen and more
distal sites in the circulation, such as sites of infection or
inflammation or tumours. These areas of the body have fenestrated
or incomplete capillaries through which liposomes may be filtered
out. Furthermore, it is known that the spleen and such other areas
of infection or inflammation and many tumors often have high
contents of macrophages which can remove the liposomes from the
circulation.
[0119] Said pharmaceutical composition according to the present
invention may be provided with a high lipid dose by different
methods. In some embodiments, the drug/lipid ratio of the
composition can be lowered to achieve the desired lipid
concentration. Alternatively, the lipid concentration of the
pharmaceutical composition may be controlled by adding empty
liposomes of comparable composition and size to the drug loaded
liposomes.
[0120] In some embodiments, the liposomes according to the present
invention may have a size of less than about 150 nm. Said liposomes
may be unilamellar, oligolamellar or multilamellar. The spleen acts
as a filter which removes unwanted red blood cells and particles
from the blood. Large liposomes are also retained by the reticular
filter in the same way. However, small liposomes may escape and
thus do not accumulate in spleen. Accordingly, liposomes according
to the present invention, having a size of less than 150 nm may
circumvent the spleen as an organ.
[0121] Such liposomes having a size of less than 150 nm may be
administered at a low lipid dose in order to target liver cells.
Such liposomes are particularly well adapted to penetrate fully the
entire liver and to reach a substantial portion of the parenchymal
cells of the liver such as hepatocytes.
[0122] Alternatively, said liposomes having a size of less than 150
nm may be administered at a high lipid dose to target more distal
sites in the circulation, such as areas of infection or
inflammation or solid tumours, and simultaneously to circumvent the
spleen.
[0123] In general, the pharmacokinetic profile and the
biodistribution of the liposomes of the present invention may
depend upon many factors. Next to the lipid composition of the
liposomes, the size and lipid dose determine the in vivo fate of
the liposomes. The liposomes of the invention may be unilamellar,
oligolamellar or multilamellar, irrespective of their size.
[0124] In some embodiments, the liposomes of the present invention
may be used to target an inflamed lung by systemic administration
to a human or non-human animal patient.
[0125] Starting from the data presented herein, those skilled in
the art will be able to establish appropriate dosage regimens for
other species, in particular for other mammals or humans.
Specifically, whether a lipid dose in another species (e.g. human)
is "low" or "high" can be determined by pharmacokinetic data. The
pharmacokinetic of liposomes follows a two compartment model. As
mentioned above, high lipid doses lead to a saturation of the liver
and an alteration of the biodistribution pattern. This leads to
enhanced Cmax values in the terminal part of the pharmacokinetic
curve.
[0126] The pharmaceutical composition of the present invention may
be formulated for use as a colloid in a suitable pharmacologically
acceptable vehicle. Vehicles such as water, saline, phosphate
buffered saline and the like are well known to those skilled in the
art for this purpose.
[0127] In some embodiments, the composition of the present
invention may be administered at a physiological pH of between
about 7 and about 8. To this end, the composition comprising the
active agent, excipient and vehicle may be formulated to have a pH
in this range.
[0128] Methods for manufacturing liposomes are known to those
skilled in the art. They include, but are not limited to, extrusion
through membranes of defined pore size, injection of lipid
solutions in ethanol into the water phase containing cargo or high
pressure homogenisation.
[0129] Also, it is known in the art that nucleic acid therapeutics
can be contacted with the lipids at neutral pH, resulting in volume
inclusion of a certain percentage of the solution containing the
nucleic acid. High concentrations of lipids ranging from 50 mM to
150 mM are preferred to achieve substantial encapsulation of the
drug.
[0130] In contrast to such standard procedures, amphoteric
liposomes offer the distinct advantage of binding nucleic acids at
or below their isoelectric point, thereby concentrating the drug at
the liposome surface. Such a process is described in WO 02/066012
in more detail. Upon elevating the pH of the liposomes to
physiological pH (about pH 7.4) the negatively charged nucleic
acids dissociate from the liposomal membrane. Irrespective of the
actual production process, the non-encapsulated active drug can be
removed from the liposomes after the initial production step,
wherein liposomes are formed as tight containers. Again, the
technical literature and the references included here describe such
methodology in detail and suitable process steps may include, but
are not limited to, size exclusion chromatography, sedimentation,
dialysis, ultrafiltration, diafiltration and the like.
[0131] In some embodiments of the invention, more than 80 wt. % of
the drug may be disposed inside said liposomes.
[0132] However, such removal of non-encapsulated material is not
mandatory and in some embodiments the composition may comprises
entrapped as well as free drug.
[0133] The particle size of the liposomes may be between 50 and 500
nm, preferably between 50 and 300 nm.
[0134] Following is a description by way of example only with
reference to the accompanying drawings of embodiments of the
present invention.
EXAMPLES
Example 1
Preparation of Carboxyfluorescein (CF) Loaded Liposomes with the
Amphoteric Ii Lipids MoChol and CHEMS
[0135] Stock solutions of lipids in chloroform were mixed and
finally evaporated in a round bottom flask to dryness under vacuum.
Lipid films were hydrated with 100 mM CF in PBS pH 7.5. The
resulting lipid concentration was 20 mM. The suspensions were
hydrated for 45 minutes in a water bath at room temperature,
sonicated for 5 minutes following by three freeze/thaw cycles at
-70.degree. C. After thawing the liposomal suspensions were
extruded 15 times through polycarbonate membranes with a pore size
of 100 nm. Non-encapsulated CF was removed by gel filtration,
whereas the liposomes were diluted by a factor three. Lipid
recovery and concentration was analysed by organic phosphate assay.
Particle size was measured by dynamic light scattering on a Malvern
Zetasizer 3000 HSA. TABLE-US-00003 TABLE 1 Variation of the ratio
DOPE/POPC and the total amount of charged components Lipids
Composition DOPE/MoChol/CHEMS 60:20:20 DOPE/MoChol/CHEMS 50:20:30
DOPE/MoChol/CHEMS 40:30:30 DOPE/MoChol/CHEMS 20:40:40
POPC/MoChol/CHEMS 60:20:20 POPC/MoChol/CHEMS 40:30:30
POPC/MoChol/CHEMS 20:40:40 POPC 100 POPC/DOPE 20:80
POPC/DOPE/MoChol/CHEMS 10:50:20:20 POPC/DOPE/MoChol/CHEMS
7:35:30:30 POPC/DOPE/MoChol/CHEMS 3:17:40:40 POPC/DOPE 25:75
POPC/DOPE/MoChol/CHEMS 15:45:20:20 POPC/DOPE/MoChol/CHEMS
10:30:30:30 POPC/DOPE/MoChol/CHEMS 5:15:40:40 POPC/DOPE 40:60
POPC/DOPE/MoChol/CHEMS 24.5:35.5:20:20 POPC/DOPE/MoChol/CHEMS
16:24:30:30 POPC/DOPE/MoChol/CHEMS 8:12:40:40 POPC/DOPE 57:43
POPC/DOPE/MoChol/CHEMS 34:26:20:20 POPC/DOPE/MoChol/CHEMS
22.8:17.2:30:30 POPC/DOPE/MoChol/CHEMS 11.4:8.6:40:40
[0136] TABLE-US-00004 TABLE 2 Variation of the ratio MoChol/CHEMS
Lipids Composition POPC/DOPE/MoChol/CHEMS 6:24:53:17
POPC/DOPE/MoChol/CHEMS 6:24:47:23 POPC/DOPE/MoChol/CHEMS 6:24:35:35
POPC/DOPE/MoChol/CHEMS 6:24:23:47
[0137] TABLE-US-00005 TABLE 3 Variation of ratio DOPE/POPC and the
total amount of charged components Lipids Composition
POPC/DOPE/MoChol/CHEMS 4:16:27:53 POPC/DOPE/MoChol/CHEMS 6:24:23:47
POPC/DOPE/MoChol/CHEMS 8:32:20:40 POPC/DOPE/MoChol/CHEMS
10:40:17:33 POPC/DOPE/MoChol/CHEMS 7:13:27:53
POPC/DOPE/MoChol/CHEMS 10:20:23:47 POPC/DOPE/MoChol/CHEMS
13:26:20:40 POPC/DOPE/MoChol/CHEMS 17:33:17:33
Example 2
Preparation of Carboxyfluorescein (CF) Loaded Liposomes with the
Amphoteric H Lipids MoChol and DMGSucc
[0138] Liposomes were prepared as described in Example 1.
TABLE-US-00006 TABLE 4 Variation of the ratio DOPE/POPC and the
total amount of charged components Lipids Composition
POPC/DOPE/MoChol/DMGSucc 15:45:20:20 POPC/DOPE/MoChol/DMGSucc
10:30:30:30 POPC/DOPE/MoChol/DMGSucc 5:15:40:40
POPC/DOPE/MoChol/DMGSucc 24.5:35.5:20:20 POPC/DOPE/MoChol/DMGSucc
16:24:30:30 POPC/DOPE/MoChol/DMGSucc 8:12:40:40
POPC/DOPE/MoChol/DMGSucc 34:26:20:20 POPC/DOPE/MoChol/DMGSucc
22.8:17.2:30:30 POPC/DOPE/MoChol/DMGSucc 11.4:8.6:40:40
[0139] TABLE-US-00007 TABLE 5 Variation of the ratio MoChol/DMGSucc
Lipids Composition POPC/DOPE/MoChol/DMGSucc 6:24:53:17
POPC/DOPE/MoChol/DMGSucc 6:24:47:23 POPC/DOPE/MoChol/DMGSucc
6:24:35:35 POPC/DOPE/MoChol/DMGSucc 6:24:23:47
[0140] TABLE-US-00008 TABLE 6 Variation of ratio DOPE/POPC and the
total amount of charged components Lipids Composition
POPC/DOPE/MoChol/DMGSucc 4:16:27:53 POPC/DOPE/MoChol/DMGSucc
6:24:23:47 POPC/DOPE/MoChol/DMGSucc 8:32:20:40
POPC/DOPE/MoChol/DMGSucc 10:40:17:33 POPC/DOPE/MoChol/DMGSucc
7:13:27:53 POPC/DOPE/MoChol/DMGSucc 10:20:23:47
POPC/DOPE/MoChol/DMGSucc 13:26:20:40 POPC/DOPE/MoChol/DMGSucc
17:33:17:33
Example 3
Preparation of Carboxyfluorescein (CF) Loaded Liposomes with the
Amphoteric Ii Lipids MoChol and DOGSucc
[0141] Liposomes were prepared as described in Example 1.
TABLE-US-00009 TABLE 7 Variation of the ratio MoChol/DOGSucc and
the total amount of charged components Lipids Composition Serum
stability POPC/DOPE/MoChol/DOGSucc 12.5:37.5:17:33 +
POPC/DOPE/MoChol/DOGSucc 12.5:37.5:33:17 + POPC/DOPE/MoChol/DOGSucc
7.5:22.5:23:47 - POPC/DOPE/MoChol/DOGSucc 7.5:22.5:47:23 +
Example 4
Preparation of Carboxyfluorescein (CF) Loaded Liposomes with the
Amphoteric Ii Lipids CHIM and CHEMS
[0142] Liposomes were prepared as described in Example 1.
TABLE-US-00010 TABLE 8 Variation of the ratio CHIM/CHEMS and the
total amount of charged components Lipids Composition Serum
stability POPC/DOPE/CHIM/CHEMS 12.5:37.5:17:33 -
POPC/DOPE/CHIM/CHEMS 12.5:37.5:33:17 + POPC/DOPE/CHIM/CHEMS
7.5:22.5:23:47 - POPC/DOPE/CHIM/CHEMS 7.5:22.5:47:23 +
Example 5
Preparation of Carboxyfluorescein (CF) Loaded Liposomes with the
Amphoteric H Lipids CHIM and DMGSucc
[0143] Liposomes were prepared as described in Example 1.
TABLE-US-00011 TABLE 8 Variation of the ratio CHIM/DMGSucc and the
total amount of charged components Lipids Composition Serum
stability POPC/DOPE/CHIM/DMGSucc 12.5:37.5:17:33 -
POPC/DOPE/CHIM/DMGSucc 12.5:37.5:33:17 + POPC/DOPE/CHIM/DMGSucc
7.5:22.5:23:47 - POPC/DOPE/CHIM/DMGSucc 7.5:22.5:47:23 +
Example 6
Serum Stability Test of CF-Loaded Amphoteric Liposomes of Examples
1 and 2
[0144] Carboxyfluorescein (CF) was used as model drug to determine
the serum stability of amphoteric liposomes. As well as
oligonucleotides, CF is negatively charged. 25 .mu.l of the
CF-loaded liposomes were mixed with 100 .mu.l pre-warmed full human
serum or PBS, respectively and incubated at 37.degree. C. At
defined time points 5 .mu.l sample was transferred into a 96-well
microtiter plate to 20 .mu.l PBS, pH 7.5 or 20 .mu.l 20% Triton
X-100. Finally 275 .mu.l PBS were added to each well and
fluorescence intensity was measured at 475/530 nm.
[0145] The serum stability was observed over a period of 4 hours by
determining the release of CF from the liposomes via the
fluorescence measurement. The released amount of CF (in %) is
measured at defined time points as well as after a treatment of the
liposomes with a detergent (Triton X-100) to get a 100% release
value.
Results:
[0146] Mixtures of POPC and DOPE are stable in serum. POPC itself
does not form liposomes that withstand attack from serum. In
addition, DOPE does not form liposomes at all. Quite surprisingly,
mixtures from both components were found to be very stable and
resistant against serum attack. In this example, DOPE/POPC ratios
from 0.75 to 5 were found to form stable structures with a broad
optimum between 1.5 and 5 (see also FIGS. 1 and 2).
[0147] Charged components and neutral lipids are independent
variables. Serum sensitivity for a 1:1 ratio of both MoChol/CHEMS
or MoChol/DMGSucc is low to very low and stable particles are
formed over a wide range of mixtures. At least 60 or 70 mol. % of
total charged components was required to affect significantly the
bilayer stability.
[0148] The serum stability of lipid mixtures containing 70% of
charged components (see Tables 2 and 5) is shown in FIG. 3. In
general, an excess of MoChol has a stabilising effect.
[0149] The formulations of Tables 3 and 6 that were tested for
serum stability have DOPE and POPC in a ratio of either 2:1 or 4:1.
The total amount of the charged lipids was titrated from 80% down
to 50%. The results are shown in FIG. 4.
Example 7
Biodistribution of Serum Stable Amphoteric Liposomes
[0150] Stock solutions of lipids (+/-1% 14C-DPPC) in chloroform
were mixed and finally evaporated in a round bottom flask to
dryness under vacuum. Lipid films were hydrated with 1.5 ml
3H-Inulin in PBS pH 7.5 or 5 ml PBS alone. The resulting lipid
concentration was 100 mM. The suspensions were hydrated for 45
minutes in a water bath at room temperature, sonicated for 30
minutes following by three freeze/thaw cycles at -70.degree. C.
After thawing the liposomal suspensions were extruded 15 times
through polycarbonate membranes with an appropriate pore size.
Liposomes were separated from non-encapsulated 3H-Inulin by
ultracentrifugation (twice).
[0151] Lipid recovery and concentration was analysed by organic
phosphate assay and in case of radiolabelled particles, the
encapsulation efficiency was measured by liquid scintillation.
Particle size was measured by dynamic light scattering on a Malvern
Zetasizer 3000 HSA. The resulting unlabelled and radiolabelled
preparations were mixed up and diluted with PBS to the final lipid
concentrations.
[0152] Formulations: TABLE-US-00012 3H 14C Size Lipid [kBq/ [kBq/
Number Formulation [nm] [mM] ml] ml] LD-1 POPC/DOPE/ 229 12.3 332
52 MoChol/CHEMS 15:45:20:20 HD-2 POPC/DOPE/ 231 54.8 453 70
MoChol/CHEMS 15:45:20:20 LD-3 POPC/DOPE/ 148 10 173 53 MoChol/CHEMS
15:45:20:20 HD-4 POPC/DOPE/ 140 50 182 58 MoChol/CHEMS
15:45:20:20
Biodistribution Study
[0153] 39 male Wistar rats (Charles River) were divided into five
groups and injected intravenously via the tail vein. At specific
time points blood samples (for PK) and/or tissue samples (for BD)
were collected and analysed by catalytic oxidation under high
temperature. Percentage of carry over between samples was
determined and included into the analysis of the data set.
TABLE-US-00013 Study group Formulation Number Animals 1
POPC/DOPE/MoChol/CHEMS LD-1 9 15:45:20:20 2 POPC/DOPE/MoChol/CHEMS
HD-2 9 15:45:20:20 3 POPC/DOPE/MoChol/CHEMS LD-3 9 15:45:20:20 4
POPC/DOPE/MoChol/CHEMS HD-4 9 15:45:20:20 5 PBS PBS 3
[0154] The results of the biodistribution study is shown in FIGS.
5-6 wherein biodistribution of the different liposomal formulations
in liver and spleen is shown. The accumulation of the liposomes in
other organs did not exceed 5% and is therefore not shown. FIG. 5
clearly demonstrates that amphoteric liposomes of the present
invention having a size >150 nm accumulate solely in the liver
when administered in low lipid doses. In contrast, by administering
the same liposomal formulation in a high lipid dose it could be
shown that the biodistribution pattern is changed. Next to the
liver the liposomes with a size >150 nm accumulate in spleen as
well.
[0155] FIG. 6 shows the biodistribution of amphoteric liposomes of
the present invention prepared in a size <150 nm. Whereas the
biodistribution of these liposomes administered at low lipid dose
does not differ from the liposomes with a size >150 nm, it can
be demonstrated that an administration of the liposomes having a
size <150 nm in high lipid dose does not lead to an accumulation
in spleen.
Example 8
Biodistribution of Amphoteric Liposomes Encapsulating Cy5.5
Labelled CD40 Antisense in Collagen Induced Arthritic Mice
[0156] Stock solutions of lipids in chloroform were mixed and
finally evaporated in a round bottom flask to dryness under vacuum.
Lipid film was hydrated with Cy5.5 labelled CD40 antisense in 10 mM
NaAc, 50 mM NaCl, pH 4.5. The resulting lipid concentration was 20
mM. The suspensions were hydrated for 45 minutes in a water bath at
50.degree. C., sonicated for 5 minutes following by a freeze/thaw
cycle at -70.degree. C. After thawing the liposomal suspensions
were extruded 19 times through 200 nm polycarbonate membranes.
After the extrusion process the pH of the liposomal suspension was
shifted to pH 7.5 by adding 1/10 Vol. 1M HEPES, pH 8.
Non-encapsulated Cy5.5 labelled CD40 antisense was removed by high
speed sedimentation (twice) and discarding the supernatant.
[0157] Lipid recovery and concentration was analysed by organic
phosphate assay. Encapsulation efficiency was measured by
fluorescence spectroscopy. Particle size was measured by dynamic
light scattering on a Malvern Zetasizer 3000 HSA.
[0158] Empty liposomes were produced by injecting 10 Vol-% of an
ethanolic lipid solution (a mixture of 15 mol. % POPC, 45 mol. %
DOPE, 20 mol. % MoChol and 20 mol. % CHEMS) into 10 mM NaAc 50 mM
NaCl pH 4.5. The resulting lipid concentration was 2 mM. The pH of
this solution was immediately shifted with 1/10 volume 1M Hepes pH
8. To concentrate the diluted liposomes the suspension was
diafiltered. TABLE-US-00014 Encapsu- lation Size Lipid effi-
Formulation [nm[ [mM] Cargo ciency POPC/DOPE/ 192 19 Cy5.5 CD40-ODN
77% MoChol/CHEMS 15:45:20:20 POPC/DOPE/ 104 195 empty --
MoChol/CHEMS 15:45:20:20
[0159] For the biodistribution study in mice the filled and empty
liposomes were mixed as follows:
200 .mu.l Cy5.5 liposomes and 41 .mu.l empty liposomes
[0160] DBA/1 mice were immunized by subcutaneous injections of type
II collagen (200 .mu.g/mouse) emulsified in complete Freund's
adjuvant. Mice were injected intravenously with the liposomal
suspension (241 .mu.l) at day 1 of arthritis induction (around day
21 after single immunization with collagen type II). Day one was
defined as the day where the inflammation was obvious (clinical
score after R.O. Williams of at least 2).
[0161] Mice were sacrificed ten hours after the injection of the
liposomal suspension. Organs and paws were removed and immediately
freezed in liquid nitrogen. The biodistribution of the Cy5.5
labelled CD40 antisense encapsulated in the liposomes was assessed
by NIR-Imaging and compared with tissue samples of untreated mice.
Specific enrichment was found for inflamed paws in mice with active
disease. More specifically, accumulation of the amphoteric
liposomes coincides with the highly active sites of the disease on
individual paws or even toes or fingers (see FIG. 7).
Example 9
Preparation of CD40-ODN-Containing Liposomes with the Advanced
Loading Procedure
[0162] Liposomes were produced by injecting 10 Vol-% of an
ethanolic lipid solution (a mixture of 15 mol. % POPC, 45 mol. %
DOPE, 20 mol. % MoChol and 20 mol. % CHEMS) into 10 mM NaAc 50 mM
NaCl pH 4.5 containing 60 .mu.g/ml of a 18 bp antisense against
CD40.
[0163] The resulting lipid concentration was 2 mM. The pH of this
solution was immediately shifted with 1/10 volume 1M Hepes pH 8. To
concentrate the diluted liposomes the suspensions were sedimented
for 2 h and 5 min at 65.000 rpm at 20.degree. C. in a T865 rotor
(Sorvall Ultra Pro 80). Afterwards the formulation was sterile
filtered through 0.45 .mu.m. TABLE-US-00015 TABLE 9 example for
Smarticles formulation which encapsulate CD40 ODN Polydisp. Lipid
Mol. % size Index POPC/DOPE/MoChol/CHEMS 15:45:20:20 178.5
0.317
[0164] The amount of encapsulated ODN was measured by checking the
optical density (OD) by 260 nm. The following amount of ODN was
encapsulated in the Smarticles formulation. TABLE-US-00016 TABLE 10
encapsulated amount of ODN in the Smarticles formulation .mu.g
Encapsu- ODN/.mu.mol lation Lipid Mol. % lipid efficacy
POPC/DOPE/MoChol/CHEMS 15:45:20:20 8.87 29.58%
Example 10
Therapeutic Efficacy in Arthritis
[0165] DBA/1 mice were immunized by subcutaneous injections of type
II collagen (200 .mu.g/mouse) emulsified in complete Freund's
adjuvant. Treatment with Smarticles or controls was initiated at
day 1 of arthritis induction (around day 21 after single
immunization with collagen type II) and repeated at day 3 and 5.
Day one was defined as the day where the inflammation was obvious
(clinical score after R.O. Williams of at least 2).
[0166] For the treatment studies the liposomal CD40-ODN was
injected intravenously into the tail vein of rats with established
inflammation. Each dosage contains 4 mg CD40-ODN per kg bodyweight
(encapsulated CD40-ODN).
[0167] During the experiment the swelling of paws were observed and
the clinical arthritis score were determined.
[0168] As evidenced by FIGS. 8 and 9, there was a significant
reduction of the swelling of the paws after a treatment with
CD40-ODN encapsulated in the amphoteric liposomes. Also the
clinical score was significant reduced after treatment with
CD40-ODN encapsulated in such liposomes.
Example 11
Materials
[0169] This example provides non-limiting examples of CD40
nucleotide sequences that may be targeted by oligonucleotides that
modulate the expression of CD40 and that are suitable for use in
the compositions in accordance with the present invention.
Human CD40 mRNA (GenBank Accession no. X60592)
[0170] Human CD40 mRNA sequence for targeting in accordance with
the present invention is presented in SEQ ID NO: 1. Related
sequence information is found in published patent application
number U.S. 2004/0186071 (i.e., SEQ ID NO: 85) to Bennett, et al.
and in U.S. Pat. No. 6,197,584 (i.e., SEQ ID NO: 85) to Bennett, et
al. and in Pluvinet, et al., Blood, 2004, 104(12), 3642-3646, the
contents of which are incorporated by reference herein.
TABLE-US-00017 (SEQ ID NO: 1): 1 gcctcgctcg ggcgcccagt ggtcctgccg
cctggtctca cctcgccatg gttcgtctgc 61 ctctgcagtg cgtcctctgg
ggctgcttgc tgaccgctgt ccatccagaa ccacccactg 121 catgcagaga
aaaacagtac ctaataaaca gtcagtgctg ttctttgtgc cagccaggac 181
agaaactggt gagtgactgc acagagttca ctgaaacgga atgccttcct tgcggtgaaa
241 gcgaattcct agacacctgg aacagagaga cacactgcca ccagcacaaa
tactgcgacc 301 ccaacctagg gcttcgggtc cagcagaagg gcacctcaga
aacagacacc atctgcacct 361 gtgaagaagg ctggcactgt acgagtgagg
cctgtgagag ctgtgtcctg caccgctcat 421 gctcgcccgg ctttggggtc
aagcagattg ctacaggggt ttctgatacc atctgcgagc 481 cctgcccagt
cggcttcttc tccaatgtgt catctgcttt cgaaaaatgt cacccttgga 541
caagctgtga gaccaaagac ctggttgtgc aacaggcagg cacaaacaag actgatgttg
601 tctgtggtcc ccaggatcgg ctgagagccc tggtggtgat ccccatcatc
ttcgggatcc 661 tgtttgccat cctcttggtg ctggtcttta tcaaaaaggt
ggccaagaag ccaaccaata 721 aggcccccca ccccaagcag gaaccccagg
agatcaattt tcccgacgat cttcctggct 781 ccaacactgc tgctccagtg
caggagactt tacatggatg ccaaccggtc acccaggagg 841 atggcaaaga
gagtcgcatc tcagtgcagg agagacagtg aggctgcacc cacccaggag 901
tgtggccacg tgggcaaaca ggcagttggc cagagagcct ggtgctgctg ctgcaggggt
961 gcaggcagaa gcggggagct atgcccagtc agtgccagcc cctc
Mus musculus CD40 mRNA
[0171] Murine CD40 mRNA sequence for targeting in accordance with
the present invention is presented in SEQ ID NO: 2. Related
sequence information is found in published patent application
number U.S. 2004/0186071 (i.e. SEQ ID NO: 132) to Bennett, et al.,
the contents of which are incorporated by reference herein.
TABLE-US-00018 (SEQ ID NO: 2): gcctcctggc ccttcagctg tggtctttcc
cgttttctga ctttgcggtg acactgggga 60 cttccttaga cctctctgga
gacgctttcg gttctgcaga gattcccagg ggtattgtgg 120 gtggggtggg
gtaacaatag tgtccctgtg gcgctcccag tccctatagt aatccttcac 180
ccctctgcta tcttgcaatc aggagagtcc ttagccctgc tataggtggc ttttgaggtc
240 ctggatgcga ggagggggac tggggggtgg gtcgggtaat gtaagaaaag
ggctcctttt 300 gggaccctgg ctcctccagc caccttggtg cccatccctt
aaactcttgg ggacaatcag 360 actcctggga aggtcctggg gaaatccctg
ctcagtgact agccataggc ccaccgcgat 420 tggtgcccga agaccccgcc
ctcttcctgg gcgggactcc tagcagggac tttggagtga 480 cttgtggctt
cagcaggagc cctgtgattt ggctcttctg atctcgccct gcgatggtgt 540
ctttgcctcg gctgtgcgcg ctatggggct gcttgttgac agcggtgagt ggcttgtgtt
600 ctaacctcca agggagttag ggcttagaga gtgagagatg gaaagaggaa
agaggagaca 660 agactttgga gatgagagat cttcctactg gaagcggcgg
ttagtaggat gggcaagatc 720 tctcgcgtct tgacacacac acacacacac
acaaatgagg tgggctgctc ctctttcctt 780 ccagaaggtc ggggttctgt
tccacgaagc ccacagggaa ccttagggag ggcattcctc 840 cacagcggtg
cctggacagc tttgtctgac ccaagccttg ctccggagct gactgcagag 900
actggaaagg gttagcagac aggaagcctg gctggggg 938
Rat CD40 mRNA (GenBank Accession No. AF 241231)
[0172] Rat CD40 mRNA sequence for targeting in accordance with the
present invention is presented in SEQ ID NO: 3. (See, Gao, Ph.D.
thesis, Goettingen 2003). TABLE-US-00019 (SEQ ID NO: 3): 1
tgggacccct gtgatctggc tgctctgatc tcgctctgca atgctgcctt tgcctcagct
61 gtgcgcgctc tggggctgct tgttgacagc ggtccatcta ggacagtgtg
ttacgtgcag 121 tgacaaacag tacctccaag gtggcgagtg ctgcgatttg
tgccagccgg gaaaccgact 181 agttagccac tgcacagctc ttgagaagac
ccaatgccaa ccgtgcgact caggcgaatt 241 ctcagctcac tggaacaggg
agatccgctg ccaccagcac cgacactgcg aactcaatca 301 agggcttcag
gttaagaagg agggcaccgc ggtntcagac actgtttgta cctgcaagga 361
agggcagcac tgcgccagca aggagtgcga gacgtgcgct cagcacaggc cctgtggccc
421 tggctttgga gtcgtgcaga tggccactga gactactgat accgtctgcc
aaccctgccc 481 ggtcggattc ttctccaatg ggtcatcact ttttgaaaag
tgtcatccat ggacaagctg 541 tgaagat
Porcine CD40 cDNA
[0173] Porcine CD40 cDNA sequence for targeting in accordance with
the present invention is presented in SEQ ID NO: 4. (FIG. 10).
Related sequence information is found in Rushworth, et al.,
Transplantation, 2002, 73(4), 635-642, the contents of which are
incorporated by reference herein.
[0174] In addition, the following provide non-limiting examples of
anti-CD40 oligonucleotides, e.g., antisense CD40 nucleic acid
sequences, that are suitable for use in the present invention:
Oligonucleotides Against Human CD40
[0175] Examples of human antisense CD40 oligonucleotides are
presented below. Further sequence information is found in published
patent application number U.S. 2004/0186071 and U.S. Pat. No.
6,197,584 to Bennett, et al., the contents of which are provided by
reference herein. The SEQ ID NOS. referred to by Bennett, et al.
are provided to the right. TABLE-US-00020 SEQ ID NO: 5 ccaggcggca
ggaccact Seq ID No: 1 of Bennett et al. SEQ ID NO: 6 gaccaggcgg
caggacca Seq ID No.:2 of Bennett et al. SEQ ID NO: 7 aggtgagacc
aggcggca Seq ID No: 3 of Bennett et al. SEQ ID NO: 8 gcagaggcag
acgaacca Seq ID No: 5 of Bennett et al. SEQ ID NO: 9 gcaagcagcc
ccagagga Seq ID No: 6 of Bennett et al. SEQ ID NO: 10 ggtcagcaag
cagcccca Seq ID No.:7 of Bennett et al. SEQ ID NO: 11 gacagcggtc
agcaagca Seq ID No: 8 of Bennett et al. SEQ ID NO: 12 gatggacagc
ggtcagca Seq ID No: 9 of Bennett et al. SEQ ID NO: 13 tctggatgga
cagcggtc Seq ID No.:10 of Bennett et al. SEQ ID NO: 14 ggtggttctg
gatggaca Seq ID No: 11 of Bennett et al. SEQ ID NO: 15 gtgggtggtt
ctggatgg Seq ID No: 12 of Bennett et al. SEQ ID NO: 16 gcagtgggtg
gttctgga Seq ID No: 13 of Bennett et al. SEQ ID NO: 17 ctggcacaaa
gaacagca Seq ID No: 15 of Bennett et al. SEQ ID NO: 18 gtgcagtcac
tcaccagt Seq ID No: 20 of Bennett et al. SEQ ID NO: 19 attccgtttc
agtgaact Seq ID No: 23 of Bennett et al. SEQ ID NO: 20 ttcaccgcaa
ggaaggca Seq ID No: 25 of Bennett et al. SEQ ID NO: 21 ctctgttcca
ggtgtcta Seq ID No: 26 of Bennett et al. SEQ ID NO: 22 ctggtggcag
tgtgtctc Seq ID No: 27 of Bennett et al. SEQ ID NO: 23 ggtgcccttc
tgctggac Seq ID No: 31 of Bennett et al. SEQ ID NO: 24 ctgaggtgcc
cttctgct Seq ID No: 32 of Bennett et al. SEQ ID NO: 25 gtgtctgttt
ctgaggtg Seq ID No: 33 of Bennett et al. SEQ ID NO: 26 acaggtgcag
atggtgtc Seq ID No: 35 of Bennett et al. SEQ ID NO: 27 gtgccagcct
tcttcaca Seq ID No: 37 of Bennett et al. SEQ ID NO: 28 tgcaggacac
agctctca Seq ID No: 40 of Bennett et al. SEQ ID NO: 29 gagcggtgca
ggacacag Seq ID No: 41 of Bennett et al. SEQ ID NO: 30 aatctgcttg
accccaaa Seq ID No: 43 of Bennett et al. SEQ ID NO: 31 gctcgcagat
ggtatcag Seq ID No: 46 of Bennett et al. SEQ ID NO: 32 gcagggctcg
cagatggt Seq ID No: 47 of Bennett et al. SEQ ID NO: 33 gactgggcag
ggctcgca Seq ID No: 49 of Bennett et al. SEQ ID NO: 34 gcagatgaca
cattggag Seq ID No: 52 of Bennett et al. SEQ ID NO: 35 tcgaaagcag
atgacaca Seq ID No: 53 of Bennett et al. SEQ ID NO: 36 gtccaagggt
gacatttt Seq ID No: 54 of Bennett et al. SEQ ID NO: 37 caggtctttg
gtctcaca Seq ID No: 57 of Bennett et al. SEQ ID NO: 38 ctgttgcaca
accaggtc Seq ID No: 58 of Bennett et al. SEQ ID NO: 39 gtttgtgcct
gcctgttg Seq ID No: 59 of Bennett et al. SEQ ID NO: 40 gtcttgtttg
tgcctgcc Seq ID No: 60 of Bennett et al. SEQ ID NO: 41 caccaccagg
gctctcag Seq ID No: 64 of Bennett et al. SEQ ID NO: 42 gggatcacca
ccagggct Seq ID No: 65 of Bennett et al. SEQ ID NO: 43 gtcgggaaaa
ttgatctc Seq ID No: 71 of Bennett et al. SEQ ID NO: 44 ggagccagga
agatcgtc Seq ID No: 73 of Bennett et al. SEQ ID NO: 45 tggagccagg
aagatcgt Seq ID No: 74 of Bennett et al. SEQ ID NO: 46 tggcatccat
gtaaagtc Seq ID No: 77 of Bennett et al. SEQ ID NO: 47 ggtgcagcct
cactgtct Seq ID No: 81 of Bennett et al. SEQ ID NO: 48 aactgcctgt
ttgcccac Seq ID No: 82 of Bennett et al.
[0176] The following siRNA sequences are suitable for use in the
present invention. (See, e.g., Pluvinet, et al., Blood, 2004,
104(12), 3642-3646), the contents of which are incorporated by
reference herein. TABLE-US-00021 (SEQ ID NO: 49):
5_-GCGAAUUCCUAGACACCUGUU-3.sub.-- (siRNA-2 of Pluvinet
3_-UUCGCUUAAGGAUCUGUGGAC-5.sub.-- et al.) (SEQ ID NO: 50):
5_-CUGGUGAGUGACUGCACAGUU-3.sub.-- (siRNA-6 of Pluvinet
3_-UUGACCACUCACUGACGUGUC-5.sub.-- et al.) (SEQ ID NO: 51):
5_-UACUGCGACCCCAACCUAGUU-3.sub.-- (siRNA-8 of Pluvinet
3_-UUAUGACGCUGGGGUUGGAUC-5.sub.-- et al.)
[0177] All siRNA contain a 2 nucleotide overhang at 3'ends.
Oligonucleotides Against Murine CD40
[0178] Examples of murine antisense CD40 oligonucleotides are
presented below. Further sequence information is found in published
patent application number U.S. 2004/0186071 to Bennett, et al., the
contents of which are hereby incorporated by reference herein. The
SEQ ID NOS. referred to by Bennett, et al. are provided to the
right. TABLE-US-00022 Murine SEQ ID NO: 52 agacaccatc gcag Seq. ID
No. 116 of Bennett et al. SEQ ID NO: 53 gcgagatcag aagag Seq. ID
No. 117 of Bennett et al. SEQ ID NO: 54 cgctgtcaac aagca Seq. ID
No. 118 of Bennett et al. SEQ ID NO: 55 ctgccctaga tggac Seq. ID
No. 119 of Bennett et al. SEQ ID NO: 56 ctggctggca caaat Seq. ID
No. 120 of Bennett et al. SEQ ID NO: 57 cttgtccagg gataa Seq. ID
No. 123 of Bennett et al. SEQ ID NO: 58 cacagatgac attag Seq. ID
No. 124 of Bennett et al. SEQ ID NO: 59 tgatatagag aaaca Seq. ID
No. 125 of Bennett et al. SEQ ID NO: 60 ctcattatcc tttgg Seq. ID
No. 127 of Bennett et al. SEQ ID NO: 61 ggttcagacc agg Seq. ID No.
128 of Bennett et al. SEQ ID NO: 62 tttatttagc cagta Seq. ID No.
130 of Bennett et al. SEQ ID NO: 63 agccccacgc actgg Seq. ID No.
131 of Bennett et al. SEQ ID NO: 64 tctcactcct atcccagt Seq. ID No.
134 of Bennett et al. SEQ ID NO: 65 attagtctga ctcgt Seq. ID No.
138 of Bennett et al. SEQ ID NO: 66 acattagtct gactc Seq. ID No.
139 of Bennett et al. SEQ ID NO: 67 cagatgacat tagtc Seq. ID No.
142 of Bennett et al. SEQ ID NO: 68 ctggactcac cacag Seq. ID No.
143 of Bennett et al. SEQ ID NO: 69 ggactcacca cagat Seq. ID No.
144 of Bennett et al. SEQ ID NO: 70 actcaccaca gatga Seq. ID No.
145 of Bennett et al. SEQ ID NO: 71 tcaccacaga tgaca Seq. ID No.
146 of Bennett et al. SEQ ID NO: 72 accacagatg acatt Seq. ID No.
147 of Bennett et al. SEQ ID NO: 73 agatgacatt ag Seq. ID No. 153
of Bennett et al. SEQ ID NO: 74 cagatgacat tag Seq. ID No. 154 of
Bennett et al. SEQ ID NO: 75 acagatgaca ttag Seq. ID No. 155 of
Bennett et al. SEQ ID NO: 76 ccacagatga cattag Seq. ID No. 156 of
Bennett et al. SEQ ID NO: 77 accacagatg acattag Seq. ID No. 157 of
Bennett et al. SEQ ID NO: 78 caccacagat gacattag Seq. ID No. 158 of
Bennett et al. SEQ ID NO: 79 tcaccacaga tgacattag Seq. ID No. 159
of Bennett et al. SEQ ID NO: 80 ctcaccacag atgacattag Seq. ID No.
160 of Bennett et al.
Oligonucleotides Against Rat CD40
[0179] Examples of rat antisense CD40 oligonucleotides are
presented below. (See, Gao, Ph.D. thesis, 2003, University of
Gottingen, Germany). TABLE-US-00023 SEQ ID NO: 81
accgctgtcaacaagcagc (rAS2 of Gao) SEQ ID NO: 82 tcctagatggaccgctgt
(rAS3 of Gao) SEQ ID NO: 83 taacacactgtcctag (rAS4 of Gao)
Oligonucleotides against porcine CD40
[0180] Examples of porcine antisense CD40 oligonucleotides are
presented below. See, Rushworth, et al., Transplantation, 2002,
73(4), 635-642, the contents of which are incorporated by reference
herein. TABLE-US-00024 SEQ ID NO: 84 gctgatgacagtgtttct (Aso3 of
Rushworth et al.) SEQ ID NO: 85 gcctcactctcgctcctg (Aso8 of
Rushworth et al.) SEQ ID NO: 86 ggactgtatctggactgc (Aso9 of
Rushworth et al.) SEQ ID NO: 87 gtggacagtcatgtatat (Aso10 of
Rushworth et al.)
[0181] The present invention therefore provides formulations of
amphoteric liposomes that exhibit improved stability upon contact
with mammalian serum, releasing less or no encapsulated drugs. Such
liposomal formulations may be useful in the delivery of drugs after
a systemic administration into the blood stream. The invention
especially suits the delivery of oligonucleotides, a new class of
drugs that is currently under development, and DNA plasmids,
without being limited to such uses. The majority of such compounds
have an intracellular site of action. Carrier systems are used to
overcome the poor uptake of such substances and are sometimes an
indispensable prerequisite.
[0182] Other embodiments and uses of the invention will be apparent
to those skilled in the art from consideration of the specification
and practice of the invention disclosed herein. All patents, patent
applications, and other references noted herein for whatever reason
are specifically incorporated by reference. The specification and
examples should be considered exemplary only with the true scope
and spirit of the invention indicated by the following claims.
Sequence CWU 1
1
87 1 1004 DNA Homo sapiens 1 gcctcgctcg ggcgcccagt ggtcctgccg
cctggtctca cctcgccatg gttcgtctgc 60 ctctgcagtg cgtcctctgg
ggctgcttgc tgaccgctgt ccatccagaa ccacccactg 120 catgcagaga
aaaacagtac ctaataaaca gtcagtgctg ttctttgtgc cagccaggac 180
agaaactggt gagtgactgc acagagttca ctgaaacgga atgccttcct tgcggtgaaa
240 gcgaattcct agacacctgg aacagagaga cacactgcca ccagcacaaa
tactgcgacc 300 ccaacctagg gcttcgggtc cagcagaagg gcacctcaga
aacagacacc atctgcacct 360 gtgaagaagg ctggcactgt acgagtgagg
cctgtgagag ctgtgtcctg caccgctcat 420 gctcgcccgg ctttggggtc
aagcagattg ctacaggggt ttctgatacc atctgcgagc 480 cctgcccagt
cggcttcttc tccaatgtgt catctgcttt cgaaaaatgt cacccttgga 540
caagctgtga gaccaaagac ctggttgtgc aacaggcagg cacaaacaag actgatgttg
600 tctgtggtcc ccaggatcgg ctgagagccc tggtggtgat ccccatcatc
ttcgggatcc 660 tgtttgccat cctcttggtg ctggtcttta tcaaaaaggt
ggccaagaag ccaaccaata 720 aggcccccca ccccaagcag gaaccccagg
agatcaattt tcccgacgat cttcctggct 780 ccaacactgc tgctccagtg
caggagactt tacatggatg ccaaccggtc acccaggagg 840 atggcaaaga
gagtcgcatc tcagtgcagg agagacagtg aggctgcacc cacccaggag 900
tgtggccacg tgggcaaaca ggcagttggc cagagagcct ggtgctgctg ctgcaggggt
960 gcaggcagaa gcggggagct atgcccagtc agtgccagcc cctc 1004 2 938 DNA
Mus musculus 2 gcctcctggc ccttcagctg tggtctttcc cgttttctga
ctttgcggtg acactgggga 60 cttccttaga cctctctgga gacgctttcg
gttctgcaga gattcccagg ggtattgtgg 120 gtggggtggg gtaacaatag
tgtccctgtg gcgctcccag tccctatagt aatccttcac 180 ccctctgcta
tcttgcaatc aggagagtcc ttagccctgc tataggtggc ttttgaggtc 240
ctggatgcga ggagggggac tggggggtgg gtcgggtaat gtaagaaaag ggctcctttt
300 gggaccctgg ctcctccagc caccttggtg cccatccctt aaactcttgg
ggacaatcag 360 actcctggga aggtcctggg gaaatccctg ctcagtgact
agccataggc ccaccgcgat 420 tggtgcccga agaccccgcc ctcttcctgg
gcgggactcc tagcagggac tttggagtga 480 cttgtggctt cagcaggagc
cctgtgattt ggctcttctg atctcgccct gcgatggtgt 540 ctttgcctcg
gctgtgcgcg ctatggggct gcttgttgac agcggtgagt ggcttgtgtt 600
ctaacctcca agggagttag ggcttagaga gtgagagatg gaaagaggaa agaggagaca
660 agactttgga gatgagagat cttcctactg gaagcggcgg ttagtaggat
gggcaagatc 720 tctcgcgtct tgacacacac acacacacac acaaatgagg
tgggctgctc ctctttcctt 780 ccagaaggtc ggggttctgt tccacgaagc
ccacagggaa ccttagggag ggcattcctc 840 cacagcggtg cctggacagc
tttgtctgac ccaagccttg ctccggagct gactgcagag 900 actggaaagg
gttagcagac aggaagcctg gctggggg 938 3 547 DNA Rattus sp.
modified_base (334) a, c, g, t, unknown or other 3 tgggacccct
gtgatctggc tgctctgatc tcgctctgca atgctgcctt tgcctcagct 60
gtgcgcgctc tggggctgct tgttgacagc ggtccatcta ggacagtgtg ttacgtgcag
120 tgacaaacag tacctccaag gtggcgagtg ctgcgatttg tgccagccgg
gaaaccgact 180 agttagccac tgcacagctc ttgagaagac ccaatgccaa
ccgtgcgact caggcgaatt 240 ctcagctcac tggaacaggg agatccgctg
ccaccagcac cgacactgcg aactcaatca 300 agggcttcag gttaagaagg
agggcaccgc ggtntcagac actgtttgta cctgcaagga 360 agggcagcac
tgcgccagca aggagtgcga gacgtgcgct cagcacaggc cctgtggccc 420
tggctttgga gtcgtgcaga tggccactga gactactgat accgtctgcc aaccctgccc
480 ggtcggattc ttctccaatg ggtcatcact ttttgaaaag tgtcatccat
ggacaagctg 540 tgaagat 547 4 1284 DNA Sus sp. 4 gcctcgccat
ggttcgtctg cctctgaagt gtctcctctg gggctgcttt ttgaccgccg 60
tccacccaga accacccact tcatgcaaag aaaaccaata cccaacaaac agccggtgct
120 gtaatttgtg cccgccagga cagaaactgg tgaaccactg cacagaggtc
actgaaacag 180 aatgccttcc ttgcagttcc agcgaattcc tagccacctg
gaatagagag aaacactgtc 240 atcagcacaa atactgcgac cccaacctag
gtctccaggt ccagagggag ggcacctcga 300 aaacagacac cacttgtgtg
tgcagtgaag gccatcactg taccaacagc gcctgtgaaa 360 gttgcacctt
gcacagcttg tgcttccctg gcctcggggt caagcagatg gcgacagagg 420
tttctgacac tatctgtgaa ccctgcccag ttggcttctt ctccaatgta tcatctgctt
480 cagaaaagtg tcagccttgg acaagctgcg agagcaaagg cctggtggaa
caacgtgcgg 540 ggactaacaa gaccgatgtt gtctgtggtt tccagagtcg
gatgagagcc ctggtggtta 600 tccccatcac gctggggatc ctgtttgccg
tcctgttggt atttctctgt atcagaaagg 660 tgaccaagga gcaggagact
aaggccctgc accctaagac tgaaaggcag gatcccgtgg 720 agacgattga
tctggaggat tttcccgact ccaccgctcc ggtgcaggag accttacatt 780
ggtgccagcc cgtcacccag gaggatggca aagagagccg catctccgtg caggagcgag
840 agtgaggctg tgcgtggcca ggagcgtgga ggcacgggca caggggcatg
tgactggaga 900 gcccggggcg gctgctgctg ctgtggcggt ggtgagaggg
tggtgctggg cacagcccct 960 tctgcctgca cccctgcagt ccagatacag
tccacctcga ggagcttctc accccagccc 1020 tggagcccat tcaatctcag
tttgctttta aagatggaga caaaactttg gggagtcaca 1080 gccacagtaa
taaccaccag agcttccaac ccagaggttc agtacctgca gatgcaaggg 1140
atggcgtcta ggagcccagg aggcatatac atgactgtcc accactgcat tgttcgtgac
1200 agtgagtgac tggaaactgc ttaactgtcc atcaacaggg gactggctaa
ataaaattgt 1260 aacatgttta tgcaaaaaaa aaaa 1284 5 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 5 ccaggcggca ggaccact 18 6 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 6 gaccaggcgg caggacca 18 7 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 7 aggtgagacc aggcggca 18 8 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 8 gcagaggcag acgaacca 18 9 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 9 gcaagcagcc ccagagga 18 10 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 10 ggtcagcaag cagcccca 18 11 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 11 gacagcggtc agcaagca 18 12 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 12 gatggacagc ggtcagca 18 13 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 13 tctggatgga cagcggtc 18 14 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 14 ggtggttctg gatggaca 18 15 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 15 gtgggtggtt ctggatgg 18 16 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 16 gcagtgggtg gttctgga 18 17 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 17 ctggcacaaa gaacagca 18 18 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 18 gtgcagtcac tcaccagt 18 19 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 19 attccgtttc agtgaact 18 20 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 20 ttcaccgcaa ggaaggca 18 21 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 21 ctctgttcca ggtgtcta 18 22 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 22 ctggtggcag tgtgtctc 18 23 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 23 ggtgcccttc tgctggac 18 24 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 24 ctgaggtgcc cttctgct 18 25 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 25 gtgtctgttt ctgaggtg 18 26 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 26 acaggtgcag atggtgtc 18 27 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 27 gtgccagcct tcttcaca 18 28 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 28 tgcaggacac agctctca 18 29 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 29 gagcggtgca ggacacag 18 30 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 30 aatctgcttg accccaaa 18 31 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 31 gctcgcagat ggtatcag 18 32 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 32 gcagggctcg cagatggt 18 33 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 33 gactgggcag ggctcgca 18 34 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 34 gcagatgaca cattggag 18 35 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 35 tcgaaagcag atgacaca 18 36 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 36 gtccaagggt gacatttt 18 37 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 37 caggtctttg gtctcaca 18 38 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 38 ctgttgcaca accaggtc 18 39 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 39 gtttgtgcct gcctgttg 18 40 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 40 gtcttgtttg tgcctgcc 18 41 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 41 caccaccagg gctctcag 18 42 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 42 gggatcacca ccagggct 18 43 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 43 gtcgggaaaa ttgatctc 18 44 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 44 ggagccagga agatcgtc 18 45 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 45 tggagccagg aagatcgt 18 46 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 46 tggcatccat gtaaagtc 18 47 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 47 ggtgcagcct cactgtct 18 48 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 48 aactgcctgt ttgcccac 18 49 21 RNA Artificial
Sequence Description of Artificial Sequence Synthetic siRNA
sequence 49 gcgaauuccu agacaccugu u 21 50 21 RNA Artificial
Sequence Description of Artificial Sequence Synthetic siRNA
sequence 50 cuggugagug acugcacagu u 21 51 21 RNA Artificial
Sequence Description of Artificial Sequence Synthetic siRNA
sequence 51 uacugcgacc ccaaccuagu u 21 52 14 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 52 agacaccatc gcag 14 53 15 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 53
gcgagatcag aagag 15 54 15 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 54 cgctgtcaac aagca
15 55 15 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 55 ctgccctaga tggac 15 56 15 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 56 ctggctggca caaat 15 57 15 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 57 cttgtccagg gataa 15 58 15 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 58 cacagatgac attag 15 59 15 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 59 tgatatagag aaaca 15 60 15 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 60 ctcattatcc tttgg 15 61 13 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 61 ggttcagacc agg 13 62 15 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 62
tttatttagc cagta 15 63 15 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 63 agccccacgc actgg
15 64 18 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 64 tctcactcct atcccagt 18 65 15 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 65 attagtctga ctcgt 15 66 15 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 66 acattagtct gactc 15 67 15 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 67 cagatgacat tagtc 15 68 15 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 68 ctggactcac cacag 15 69 15 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 69 ggactcacca cagat 15 70 15 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 70 actcaccaca gatga 15 71 15 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 71 tcaccacaga tgaca 15 72 15 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 72 accacagatg acatt 15 73 12 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 73 agatgacatt ag 12 74 13 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 74
cagatgacat tag 13 75 14 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 75 acagatgaca ttag 14
76 16 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 76 ccacagatga cattag 16 77 17 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 77 accacagatg acattag 17 78 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 78 caccacagat gacattag
18 79 19 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 79 tcaccacaga tgacattag 19 80 20 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 80 ctcaccacag atgacattag 20 81 19 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 81 accgctgtca acaagcagc 19 82 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 82 tcctagatgg accgctgt 18 83 16 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 83 taacacactg tcctag 16 84 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 84 gctgatgaca gtgtttct 18 85 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 85 gcctcactct cgctcctg 18 86 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 86 ggactgtatc tggactgc 18 87 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 87 gtggacagtc atgtatat 18
* * * * *
References