U.S. patent application number 11/254920 was filed with the patent office on 2006-06-22 for antiviral oligonucleotides.
Invention is credited to Jean-Marc Juteau, Andrew Vaillant.
Application Number | 20060135458 11/254920 |
Document ID | / |
Family ID | 31997929 |
Filed Date | 2006-06-22 |
United States Patent
Application |
20060135458 |
Kind Code |
A1 |
Vaillant; Andrew ; et
al. |
June 22, 2006 |
Antiviral oligonucleotides
Abstract
Random sequence oligonucleotides that have antiviral activity
are described, along with their use as antiviral agents. In many
cases, the oligonucleotides are greater than 40 nucleotides in
length and include chemical modifications, such as modified
internucleotidic linkages and modifications at the 2'-position of
the ribose moieties. Also described are methods for the prophylaxis
or treatment of a viral infection in a human or animal, and a
method for the prophylaxis treatment of cancer caused by
oncoviruses in a human or animal. The methods typically involve
administering to a human or animal in need of such treatment, a
pharmacologically acceptable, therapeutically effective amount of
at least one oligonucleotide that act by a sequence complementary
mode of action.
Inventors: |
Vaillant; Andrew; (Roxboro,
CA) ; Juteau; Jean-Marc; (Blainville, CA) |
Correspondence
Address: |
CHOATE, HALL & STEWART LLP
TWO INTERNATIONAL PLACE
BOSTON
MA
02110
US
|
Family ID: |
31997929 |
Appl. No.: |
11/254920 |
Filed: |
October 19, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10969812 |
Oct 19, 2004 |
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11254920 |
Oct 19, 2005 |
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10661402 |
Sep 12, 2003 |
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10969812 |
Oct 19, 2004 |
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10661088 |
Sep 12, 2003 |
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10969812 |
Oct 19, 2004 |
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10661099 |
Sep 12, 2003 |
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10969812 |
Oct 19, 2004 |
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10661403 |
Sep 12, 2003 |
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10969812 |
Oct 19, 2004 |
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10661097 |
Sep 12, 2003 |
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10969812 |
Oct 19, 2004 |
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10661415 |
Sep 12, 2003 |
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10969812 |
Oct 19, 2004 |
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PCT/IB03/04573 |
Sep 11, 2003 |
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10969812 |
Oct 19, 2004 |
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60430934 |
Dec 5, 2002 |
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60410264 |
Sep 13, 2002 |
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60430934 |
Dec 5, 2002 |
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60410264 |
Sep 13, 2002 |
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60430934 |
Dec 5, 2002 |
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60410264 |
Sep 13, 2002 |
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60430934 |
Dec 5, 2002 |
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60410264 |
Sep 13, 2002 |
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60430934 |
Dec 5, 2002 |
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60410264 |
Sep 13, 2002 |
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60430934 |
Dec 5, 2002 |
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60410264 |
Sep 13, 2002 |
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60668983 |
Apr 7, 2005 |
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Current U.S.
Class: |
514/44A ;
536/23.1 |
Current CPC
Class: |
Y02A 50/30 20180101;
A61P 31/20 20180101; C12N 2310/321 20130101; Y02A 50/393 20180101;
A61P 31/16 20180101; A61P 31/14 20180101; Y02A 50/387 20180101;
C12N 2310/315 20130101; C12N 2310/3125 20130101; A61K 31/7088
20130101; A61K 45/06 20130101; C12N 15/11 20130101; A61K 38/00
20130101; C12N 15/115 20130101; C12N 2310/351 20130101; A61P 31/22
20180101; A61P 25/28 20180101; A61P 31/18 20180101; A61P 35/00
20180101; A61P 25/00 20180101; Y02A 50/385 20180101; A61P 31/12
20180101; A61K 31/7088 20130101; A61K 2300/00 20130101; C12N
2310/321 20130101; C12N 2310/3521 20130101 |
Class at
Publication: |
514/044 ;
536/023.1 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C07H 21/02 20060101 C07H021/02 |
Claims
1. An oligonucleotide, having at least 50% of its nucleotides in
said oligonucleotide modified at the 2'-position of the ribose
moiety and having at least 50% of its internucleotidic linkages
modified, wherein said oligonucleotide has an antiviral activity
against a target virus, said activity operating predominantly by a
sequence independent mode of action.
2. The oligonucleotide according to claim 1, wherein said
oligonucleotide has at least 50% of its nucleotides in said
oligonucleotide modified at the 2'-position of the ribose moiety
and has at least 80% of its internucleotidic linkages modified,
wherein said oligonucleotide has an antiviral activity against a
target virus, said activity operating predominantly by a sequence
independent mode of action.
3. The oligonucleotide according to claim 1, wherein said
oligonucleotide has at least 80% of its nucleotides in said
oligonucleotide modified at the 2'-position of the ribose moiety
and has at least 80% of its internucleotidic linkages modified,
wherein said oligonucleotide has an antiviral activity against a
target virus, said activity operating predominantly by a sequence
independent mode of action.
4. The oligonucleotide according to claim 1, wherein said
oligonucleotide has at least 90% of its nucleotides in said
oligonucleotide modified at the 2'-position of the ribose moiety
and having at least 90% of its internucleotidic linkages modified,
wherein said oligonucleotide has an antiviral activity against a
target virus, said activity operating predominantly by a sequence
independent mode of action.
5. The oligonucleotide according to claim 1, wherein said
oligonucleotide has at least 100% of its nucleotides in said
oligonucleotide modified at the 2'-position of the ribose moiety
and having at least 100% of its internucleotidic linkages modified,
wherein said oligonucleotide has an antiviral activity against a
target virus, said activity operating predominantly by a sequence
independent mode of action.
6. The oligonucleotide of claim 1, wherein the modified linkages
are selected from the group consisting of phosphorothioate
linkages, phosphorodithioate linkages, and boranophosphate
linkages.
7. The oligonucleotide of claim 5, wherein the modified linkages
are selected from the group consisting of phosphorothioate
linkages, phosphorodithioate linkages, and boranophosphate
linkages.
8. The oligonucleotide of claim 1, wherein at least 50% of the
nucleotides in said oligonucleotide comprises 2'-OMe moieties at
the 2'-position of the ribose moiety.
9. The oligonucleotide of claim 5, wherein at least 100% of the
nucleotides in said oligonucleotide comprises 2'-OMe moieties at
the 2'-position of the ribose moiety.
10. The oligonucleotide of claim 1, wherein at least 50% of the
nucleotides in said oligonucleotide comprise 2'-methoxyethoxy
substitutions at the 2'-position of the ribose moiety.
11. The oligonucleotide of claim 5, wherein at least 100% of the
nucleotides in said oligonucleotide comprise 2'-methoxyethoxy
substitutions at the 2'-position of the ribose moiety.
12. The oligonucleotide of claim 1, wherein said oligonucleotide is
at least 30 nucleotides in length.
13. The oligonucleotide of claim 1, wherein said oligonucleotide is
at least 40 nucleotides in length.
14. The oligonucleotide of claim 1, comprising a homopolymer
sequence of at least 10 contiguous nucleotides selected from the
group consisting of A, T, U, C, G, and I.
15. The oligonucleotide of claim 1, comprising a sequence of at
least 10 nucleotides in length selected from the group consisting
of polyAT, polyAC, polyAG, polyAU, polyAI, polyGC, polyGT, polyGU,
polyGI, polyCT, polyCU, polyCI, and polyTI.
16. The oligonucleotide of claim 1, wherein at least 15% of the
nucleotides in said oligonucleotide comprise 2'-methoxyethoxy or
2'OMe substitutions at the 2'-position of the ribose moiety.
17. The oligonucleotide of claim 1, wherein said oligonucleotide is
a concatemer consisting of two or more oligonucleotide sequences
joined by a linker.
18. The oligonucleotide of claim 1, wherein said oligonucleotide is
linked or conjugated at one or more nucleotide residues, to a
molecule modifying the characteristics of the oligonucleotide to
obtain one or more characteristics selected from the group
consisting of higher stability, lower serum interaction, higher
cellular uptake, higher viral protein interaction, an improved
ability to be formulated for delivery, a detectable signal, higher
antiviral activity, better pharmacokinetic properties, specific
tissue distribution, lower toxicity.
19. The oligonucleotide of claim 1, wherein said oligonucleotide is
double stranded.
20. The oligonucleotide of claim 1, wherein said oligonucleotide
targets a DNA virus or a RNA virus.
21. The oligonucleotide of claim 1, wherein said oligonucleotide
targets a member of the group consisting of herpesviridae, HSV-1,
HSV-2, CMV Varicella Zoster Virus, Epstein Barr Virus, Human
Herpesvirus 6A and 6B, hepadnaviridae, HBV, parvoviridae,
poxviridae, papillomaviridae, adenoviridae, retroviridae, HIV-1,
HIV-2, paramyxoviridae, RSV, parainfluenza virus, human
metapneumovirus, bunyaviridae, hantavirus, Rift Valley fever virus,
Crimean Congo Hemorrhagic Fever virus, picornaviridae,
coxsackievirus, rhinovirus, flaviviridae, yellow fever virus,
dengue virus, West Nile virus, hepatitis C virus, filoviridae,
Ebola virus, Marburg virus, orthomyxoviridae, influenza virus,
togaviridae, Western Equine Encephalitis virus, coronaviridae,
reoviridae rhabdoviridae, arenaviridae, lassa fever virus and
calciviridae.
22. An oligonucleotide as set forth in any one of REP 1001, REP
2001, REP 3007, REP 2004, REP 2005, REP 2006, REP 2007, REP 2008,
REP 2017, REP 2018, REP 2020, REP 2021, REP 2024, REP 2036, A20,
G20, C20, REP 2029, REP 2031, REP 2030, REP 2033, REP 2055, REP
2056, REP 2057, REP 2060 and REP 2107.
23. An oligonucleotide mixture comprising a mixture of at least two
different antiviral oligonucleotides of claim 1.
24. An oligonucleotide mixture comprising a mixture of at least ten
different antiviral oligonucleotides of claim 1.
25. An antiviral pharmaceutical composition comprising a
therapeutically effective amount of at least one pharmacologically
acceptable, antiviral oligonucleotide as defined in claim 1; and a
pharmaceutically acceptable carrier.
26. A kit comprising at least one antiviral oligonucleotide as
defined in claim 1, in a labeled package, wherein the antiviral
activity of said oligonucleotide occurs principally by a
non-sequence complementary mode of action and the label on said
package indicates that said antiviral oligonucleotide can be used
against at least one virus.
27. The kit of claim 26, wherein said kit contains a mixture of at
least two different antiviral oligonucleotides.
28. A method for the prophylaxis or treatment of a viral infection
in a subject, comprising administering to a subject in need of such
a treatment a therapeutically effective amount of at least one
pharmacologically acceptable oligonucleotide as defined in claim
1.
29. A method for the prophylaxis or treatment of a viral infection
in a subject, comprising administering to a subject in need of such
a treatment a therapeutically effective amount of at least one
pharmacologically acceptable oligonucleotide mixture as defined in
claim 23.
30. A method for the prophylaxis or treatment of a viral infection
in a subject, comprising administering to a subject in need of such
a treatment a therapeutically effective amount of at least one
pharmacologically acceptable antiviral pharmaceutical composition
as defined in claim 25.
31. A method for the prophylactic treatment of cancer caused by
oncoviruses in a human or a non-human animal, comprising
administering to a subject in need of such a treatment a
therapeutically effective amount of at least one pharmacologically
acceptable oligonucleotide as defined in claim 1.
32. A method for the prophylactic treatment of cancer caused by
oncoviruses in a human or a non-human animal, comprising
administering to a subject in need of such a treatment a
therapeutically effective amount of at least one pharmacologically
acceptable oligonucleotide mixture as defined in claim 23.
33. A method for the prophylactic treatment of cancer caused by
oncoviruses in a human or a non-human animal, comprising
administering to a subject in need of such a treatment a
therapeutically effective amount of at least one pharmacologically
acceptable antiviral pharmaceutical composition as defined in claim
25.
34. An antiviral pharmaceutical composition comprising a
therapeutically effective amount of at least one pharmacologically
acceptable, polypyrimidine oligonucleotide and a pharmaceutically
acceptable carrier, wherein the antiviral activity of said
oligonucleotide occurs principally by a sequence independent mode
of action.
35. The antiviral pharmaceutical composition of claim 34, wherein
the oligonucleotide comprises at least one modified
internucleotidic linkage.
36. The composition of claim 34, wherein said composition is
formulated for administration to an acidic in vivo site.
37. The composition of claim 34, wherein said composition is
adapted for oral, vaginal, or topical administration.
38. The composition of claim 34, wherein said composition comprises
at least one polyC oligonucleotide.
39. The composition of claim 34, wherein said composition comprises
at least one polyT oligonucleotide.
40. The composition of claim 34, wherein said composition comprises
at least one polyCT oligonucleotide.
41. A method for the prophylaxis or treatment of a viral infection
in an acidic environnement in a subject, comprising administering
to a subject in need of such a treatment a therapeutically
effective amount of at least one pharmacologically acceptable
antiviral pharmaceutical composition as defined in claim 34, said
composition being adapted for administration to an acidic in vivo
site.
42. An oligonucleotide, having at least 50% of its internucleotidic
linkages modified, wherein said oligonucleotide has an antiviral
activity against a target virus, said activity operating
predominantly by a sequence independent mode of action, said
oligonucleotide comprising at least 80% of pyrimidine residues.
43. The oligonucleotide of claim 42, wherein said oligonucleotide
has at least 80% of its internucleotidic linkages modified.
44. The oligonucleotide of claim 42, wherein said oligonucleotide
has at least 80% of its internucleotidic linkages modified and has
100% of pyrimidine residues.
45. The oligonucleotide of claim 42, wherein said oligonucleotide
has 100% of its internucleotidic linkages modified and has at least
80% of pyrimidine residues.
46. The oligonucleotide of claim 42, wherein said oligonucleotide
has 100% of its internucleotidic linkages modified and has 100% of
pyrimidine residues.
47. The oligonucleotide of claim 42, wherein the modified linkages
are selected from the group consisting of phosphorothioate
linkages, phosphorodithioate linkages, and boranophosphate
linkages.
48. The oligonucleotide of claim 46, wherein the modified linkages
are selected from the group consisting of phosphorothioate
linkages, phosphorodithioate linkages, and boranophosphate
linkages.
49. The oligonucleotide of claim 42, wherein the modified linkages
are phosphorothioate linkages
50. The oligonucleotide of claim 48, wherein the modified linkages
are phosphorothioate linkages.
51. The oligonucleotide of claim 42, wherein the pyrimidine
residues are cytosine residues.
52. The oligonucleotide of claim 46, wherein the pyrimidine
residues are cytosine residues.
53. The oligonucleotide of claim 42, wherein the pyrimidine
residues are thymine residues.
54. The oligonucleotide of claim 46, wherein the pyrimidine
residues are thymine residues.
55. The oligonucleotide of claim 42, wherein the pyrimidine
residues are cytosine or thymine residues.
56. The oligonucleotide of claim 46, wherein the pyrimidine
residues are cytosine or thymine residues.
57. The oligonucleotide of claim 42, wherein said oligonucleotide
is at least 30 nucleotides in length.
58. The oligonucleotide of claim 46, wherein said oligonucleotide
is at least 30 nucleotides in length.
59. The oligonucleotide of claim 42, wherein said oligonucleotide
is at least 40 nucleotides in length.
60. The oligonucleotide of claim 46, wherein said oligonucleotide
is at least 40 nucleotides in length.
61. The oligonucleotide of claim 42, wherein at least 15% of the
nucleotides in said oligonucleotide comprise 2'-methoxyethoxy or
2'-OMe substitutions at the 2'-position of the ribose moiety.
62. The oligonucleotide of claim 42, wherein said oligonucleotide
is a concatemer consisting of two or more oligonucleotide sequences
joined by a linker.
63. The oligonucleotide of claim 46, wherein said oligonucleotide
is a concatemer consisting of two or more oligonucleotide sequences
joined by a linker.
64. The oligonucleotide of claim 42, wherein said oligonucleotide
is linked or conjugated at one or more nucleotide residues, to a
molecule modifying the characteristics of the oligonucleotide to
obtain one or more characteristics selected from the group
consisting of higher stability, lower serum interaction, higher
cellular uptake, higher viral protein interaction, an improved
ability to be formulated for delivery, a detectable signal, higher
antiviral activity, better pharmacokinetic properties, specific
tissue distribution, lower toxicity.
65. The oligonucleotide of claim 46, wherein said oligonucleotide
is linked or conjugated at one or more nucleotide residues, to a
molecule modifying the characteristics of the oligonucleotide to
obtain one or more characteristics selected from the group
consisting of higher stability, lower serum interaction, higher
cellular uptake, higher viral protein interaction, an improved
ability to be formulated for delivery, a detectable signal, higher
antiviral activity, better pharmacokinetic properties, specific
tissue distribution, lower toxicity.
66. The oligonucleotide of claim 42, wherein said oligonucleotide
is double stranded.
67. The oligonucleotide of claim 46, wherein said oligonucleotide
is double stranded.
68. The oligonucleotide of claim 42, wherein said oligonucleotide
targets a DNA virus or a RNA virus.
69. The oligonucleotide of claim 46, wherein said oligonucleotide
targets a DNA virus or a RNA virus.
70. The oligonucleotide of claim 42, wherein said oligonucleotide
targets a member of the group consisting of herpesviridae, HSV-1,
HSV-2, CMV Varicella Zoster Virus, Epstein Barr Virus, Human
Herpesvirus 6A and 6B, hepadnaviridae, HBV, parvoviridae,
poxviridae, papillomaviridae, adenoviridae, retroviridae, HIV-1,
HIV-2, paramyxoviridae, RSV, parainfluenza virus, human
metapneumovirus, bunyaviridae, hantavirus, Rift Valley fever virus,
Crimean Congo Hemorrhagic Fever virus, picornaviridae,
coxsackievirus, rhinovirus, flaviviridae, yellow fever virus,
dengue virus, West Nile virus, hepatitis C virus, filoviridae,
Ebola virus, Marburg virus, orthomyxoviridae, influenza virus,
togaviridae, Western Equine Encephalitis virus, coronaviridae,
reoviridae rhabdoviridae, arenaviridae, lassa fever virus and
calciviridae.
71. The oligonucleotide of claim 46, wherein said oligonucleotide
targets a member of the group consisting of herpesviridae, HSV-1,
HSV-2, CMV Varicella Zoster Virus, Epstein Barr Virus, Human
Herpesvirus 6A and 6B, hepadnaviridae, HBV, parvoviridae,
poxviridae, papillomaviridae, adenoviridae, retroviridae, HIV-1,
HIV-2, paramyxoviridae, RSV, parainfluenza virus, human
metapneumovirus, bunyaviridae, hantavirus, Rift Valley fever virus,
Crimean Congo Hemorrhagic Fever virus, picornaviridae,
coxsackievirus, rhinovirus, flaviviridae, yellow fever virus,
dengue virus, West Nile virus, hepatitis C virus, filoviridae,
Ebola virus, Marburg virus, orthomyxoviridae, influenza virus,
togaviridae, Western Equine Encephalitis virus, coronaviridae,
reoviridae rhabdoviridae, arenaviridae, lassa fever virus and
calciviridae.
72. An oligonucleotide mixture comprising a mixture of at least two
different antiviral oligonucleotides of claim 42.
73. An oligonucleotide mixture comprising a mixture of at least two
different antiviral oligonucleotides of claim 46.
74. An oligonucleotide mixture comprising a mixture of at least ten
different antiviral oligonucleotides of claim 42.
75. An oligonucleotide mixture comprising a mixture of at least ten
different antiviral oligonucleotides of claim 46.
76. An antiviral pharmaceutical composition comprising a
therapeutically effective amount of at least one pharmacologically
acceptable, antiviral oligonucleotide as defined in claim 42; and a
pharmaceutically acceptable carrier.
77. An antiviral pharmaceutical composition comprising a
therapeutically effective amount of at least one pharmacologically
acceptable, antiviral oligonucleotide as defined in claim 46; and a
pharmaceutically acceptable carrier.
78. The composition of claim 76, wherein said composition is
formulated for administration to an acidic in vivo site.
79. The composition of claim 77, wherein said composition is
formulated for administration to an acidic in vivo site.
80. The composition of claim 76, wherein said composition is
adapted for oral, vaginal, or topical administration.
81. The composition of claim 77, wherein said composition is
adapted for oral, vaginal, or topical administration.
82. A kit comprising at least one antiviral oligonucleotide as
defined in claim 42, in a labeled package, wherein the antiviral
activity of said oligonucleotide occurs principally by a
non-sequence complementary mode of action and the label on said
package indicates that said antiviral oligonucleotide can be used
against at least one virus.
83. A kit comprising at least one antiviral oligonucleotide as
defined in claim 46, in a labeled package, wherein the antiviral
activity of said oligonucleotide occurs principally by a
non-sequence complementary mode of action and the label on said
package indicates that said antiviral oligonucleotide can be used
against at least one virus.
84. The kit of claim 78, wherein said kit contains a mixture of at
least two different antiviral oligonucleotides.
85. The kit of claim 83, wherein said kit contains a mixture of at
least two different antiviral oligonucleotides.
86. A method for the prophylaxis or treatment of a viral infection
in a subject, comprising administering to a subject in need of such
a treatment a therapeutically effective amount of at least one
pharmacologically acceptable oligonucleotide as defined in claim
42.
87. A method for the prophylaxis or treatment of a viral infection
in a subject, comprising administering to a subject in need of such
a treatment a therapeutically effective amount of at least one
pharmacologically acceptable oligonucleotide as defined in claim
46.
88. A method for the prophylaxis or treatment of a viral infection
in a subject, comprising administering to a subject in need of such
a treatment a therapeutically effective amount of at least one
pharmacologically acceptable oligonucleotide mixture as defined in
claim 72.
89. A method for the prophylaxis or treatment of a viral infection
in a subject, comprising administering to a subject in need of such
a treatment a therapeutically effective amount of at least one
pharmacologically acceptable oligonucleotide mixture as defined in
claim 73.
90. A method for the prophylaxis or treatment of a viral infection
in a subject, comprising administering to a subject in need of such
a treatment a therapeutically effective amount of at least one
pharmacologically acceptable antiviral pharmaceutical composition
as defined in claim 76.
91. A method for the prophylaxis or treatment of a viral infection
in a subject, comprising administering to a subject in need of such
a treatment a therapeutically effective amount of at least one
pharmacologically acceptable antiviral pharmaceutical composition
as defined in claim 77.
92. A method for the prophylactic treatment of cancer caused by
oncoviruses in a human or a non-human animal, comprising
administering to a subject in need of such a treatment a
therapeutically effective amount of at least one pharmacologically
acceptable oligonucleotide as defined in claim 42.
93. A method for the prophylactic treatment of cancer caused by
oncoviruses in a human or a non-human animal, comprising
administering to a subject in need of such a treatment a
therapeutically effective amount of at least one pharmacologically
acceptable oligonucleotide as defined in claim 46.
94. A method for the prophylactic treatment of cancer caused by
oncoviruses in a human or a non-human animal, comprising
administering to a subject in need of such a treatment a
therapeutically effective amount of at least one pharmacologically
acceptable oligonucleotide mixture as defined in claim 72.
95. A method for the prophylactic treatment of cancer caused by
oncoviruses in a human or a non-human animal, comprising
administering to a subject in need of such a treatment a
therapeutically effective amount of at least one pharmacologically
acceptable oligonucleotide mixture as defined in claim 73.
96. A method for the prophylactic treatment of cancer caused by
oncoviruses in a human or a non-human animal, comprising
administering to a subject in need of such a treatment a
therapeutically effective amount of at least one pharmacologically
acceptable antiviral pharmaceutical composition as defined in claim
76.
97. A method for the prophylactic treatment of cancer caused by
oncoviruses in a human or a non-human animal, comprising
administering to a subject in need of such a treatment a
therapeutically effective amount of at least one pharmacologically
acceptable antiviral pharmaceutical composition as defined in claim
77.
98. An antiviral pharmaceutical composition comprising a
therapeutically effective amount of at least one pharmacologically
acceptable, polypyrimidine oligonucleotide and a pharmaceutically
acceptable carrier, wherein the antiviral activity of said
oligonucleotide occurs principally by a sequence independent mode
of action; and a pharmaceutically acceptable carrier.
99. The antiviral pharmaceutical composition of claim 98, wherein
the oligonucleotide comprises modified internucleotidic
linkages.
100. The composition of claim 98, wherein said composition is
formulated for administration to an acidic in vivo site.
101. The composition of claim 98, wherein said composition is in
the form of a powder.
102. The composition of claim 98, wherein said composition is
adapted for oral, vaginal, or topical administration.
103. The composition of claim 98, wherein said composition
comprises at least one polyC oligonucleotide.
104. The composition of claim 98, wherein said composition
comprises at least one polyT oligonucleotide.
105. The composition of claim 98, wherein said composition
comprises at least one polyCT oligonucleotide.
106. A method for the prophylaxis or treatment of a viral infection
in an acidic environnement in a subject, comprising administering
to a subject in need of such a treatment a therapeutically
effective amount of at least one pharmacologically acceptable
antiviral pharmaceutical composition as defined in claim 42, said
composition being adapted for administration to an acidic in vivo
site.
107. A method for the prophylaxis or treatment of a viral infection
in an acidic environnement in a subject, comprising administering
to a subject in need of such a treatment a therapeutically
effective amount of at least one pharmacologically acceptable
antiviral pharmaceutical composition as defined in claim 46, said
composition being adapted for administration to an acidic in vivo
site.
Description
RELATED APPLICATIONS
[0001] This application is related to Ser. No. 10/969,812 which is
a continuation-in-part of Vaillant & Juteau U.S. application
Ser. No. 10/661,402, filed Sep. 12, 2003, Ser. Nos. 10/661,088,
10/661,099, 10/661,403, 10/661,097 and 10,661/415, each of these
are a continuation-in-part of Vaillant & Juteau, PCT
application Ser. No. PCT/IB03/04573, filed Sep. 11, 2003, entitled
ANTIVIRAL OLIGONUCLEOTIDES; said application Ser. Nos. 10/661,402,
10/661,088, 10/661,099, 10/661,403, 10/661,097 and 10,661/415, also
claim the benefit of Vaillant & Juteau, U.S. Provisional Appl.
60/430,934, filed Dec. 5, 2002 and of Vaillant & Juteau, U.S.
Provisional Appl. 60/410,264, filed Sep. 13, 2002, all of which are
incorporated herein by reference in their entireties, including
drawings.
FIELD OF THE INVENTION
[0002] The present invention relates to oligonucleotides having
antiviral activities and their use as therapeutic agents in viral
infections caused by human and animal viruses and in cancers caused
by oncogene viruses and in other diseases whose etiology is
viral-based.
BACKGROUND OF THE INVENTION
[0003] The following discussion is provided solely to assist the
understanding of the reader, and does not constitute an admission
that any of the information discussed or references cited
constitute prior art to the present invention.
[0004] Many important infectious diseases afflicting mankind are
caused by viruses. Many of these diseases, including rabies,
smallpox, poliomyelitis, viral hemaoragghic fevers, hepatitis,
yellow fever, immune deficiencies and various encephalitic
diseases, are frequently fatal. Others are significant in that they
are highly contagious and create acute discomfort such as
influenza, measles, mumps and chickenpox, as well as respiratory or
gastrointestinal disorders. Others such as rubella and
cytomegalovirus can cause congenital abnormalities. Finally there
are viruses, known as oncoviruses, which can cause cancer in humans
and animals.
[0005] Among viruses, the family of Herpesviridae is of great
interest. The Herpesviridae are a ubiquitous class of icoshedral,
double stranded DNA viruses. Of over 100 characterized members of
Herpesviridae (HHV), only eight infect humans. The best known among
these are Herpes simplex type 1 (HSV-1), Herpes simplex type 2
(HSV-2), Varicella zoster (chicken pox or shingles),
cytomegalovirus (CMV) and Epstein-Barr virus (EBV). The prevalence
of Herpes viruses in humans is high, affecting at least one third
of the worldwide population; and in the United States, 70-80% of
the population have some kind of Herpes infection. While the
pathology of Herpes infections are usually not dangerous, as in the
case of HSV-1 which usually only causes short lived lesions around
the mouth and face, these viruses are also known to be the cause of
more dangerous symptoms, which vary from genital ulcers and
discharge to fetal infections which can lead to encephalitis (15%
mortality) or disseminated infection (40% mortality).
[0006] Herpes viruses are highly disseminated in nature and highly
pathogenic for man. For example, Epstein-Barr virus (EBV) is known
to cause infectious mononucleosis in late childhood or adolescence
or in young adults. The hallmarks of acute infectious mononucleosis
are sore throat, fever, headache, lymphadenopathy, enlarged tonsils
and atypical, dividing lymphocytes in the peripheral blood. Other
manifestations frequently include mild hepatitis, splenomegaly and
encephalitis. EBV is also associated with two forms of cancer:
Burkitt's lymphoma (BL) and the nasopharyngeal carcinoma (NPC). In
endemic areas of equatorial Africa, BL is the most common childhood
malignancy, accounting for approximately 80% of cancers in
children. While moderately observed in North American Caucasians,
NPC is one of the most common cancers in Southern China with age
incidence of 25 to 55 years. EBV, like the cytomegalovirus, is also
associated with post-transplant lymphoproliferative disease, which
is a potentially fatal complication of chronic immunosuppression
following solid organ or bone marrow transplantation.
[0007] Other diseases are also associated with HSV, including skin
and eye infections, for example, chorioretinitis or
keratoconjunctivitis. Approximately 300,000 cases of HSV infections
of the eye are diagnosed yearly in the United States.
[0008] AIDS (acquired immunodeficiency syndrome) is caused by the
human immunodeficiency virus (HIV). By killing or damaging cells of
the body's immune system, HIV progressively destroys the body's
ability to fight infections and certain cancers. There are
currently approximately 42 million people living with HIV/AIDS
worldwide. A total of 3.1 million people died of HIV/AIDS related
causes in 2002. The ultimate goal of anti-HIV drug therapy is to
prevent the virus from reproducing and damaging the immune system.
Although substantial progress has been made over the past fifteen
years in the fight against HIV, a cure still eludes medical
science. Today, physicians have more than a dozen antiretroviral
agents in three different drug classes to manage the disease.
Typically, drugs from two or three classes are prescribed in a
variety of combinations known as HAART (Highly Active
AntiRetroviral Treatment). HAART therapies typically comprise two
nucleoside reverse transcriptase inhibitors drugs with a third
drug, either a protease inhibitor or a non-nucleoside reverse
transcriptase inhibitor. Clinical studies have shown that HAART is
the most effective means of reducing viral loads and minimizing the
likelihood of drug resistance.
[0009] While HAART has been shown to reduce the amount of HIV in
the body, commonly known as viral load, tens of thousands of
patients encounter significant problems with this therapy. Some
side effects are serious and include abnormal fat metabolism,
kidney stones, and heart disease. Other side effects such as
nausea, vomiting, and insomnia are less serious, but still
problematic for HIV patients that need chronic drug therapy for a
lifetime.
[0010] Currently approved anti-HIV drugs work by entering an HIV
infected CD4+ T cell and blocking the function of a viral enzyme,
either the reverse transcriptase or a protease. HIV needs both of
these enzymes in order to reproduce. However, HIV frequently
mutates, rendering reverse transcriptase or protease inhibitor
drugs ineffective against these resistant strains. Once resistance
occurs, viral loads increase and dictate the need to switch the
ineffective agent for another antiretroviral agent. Unfortunately,
when a virus becomes resistant to one drug in a class, other drugs
in that class may also become less effective. This phenomenon,
known as cross-resistance, occurs because many anti-HIV drugs work
in a similar fashion. The occurrence of drug cross-resistance is
highly undesirable because it reduces the available number of
treatment options for patients.
[0011] There is therefore a great need for the development of other
antiviral agents effective against HIV that work through other
mechanisms of action against which the virus has not developed
resistance. This is becoming especially important in view of recent
data showing that 1 out of 10 patients newly diagnosed with HIV in
Europe, is infected with a strain of HIV already resistant to at
least one of the approved drugs on the market.
[0012] Respiratory syncytial virus (RSV) causes upper and lower
respiratory tract infections. It is a negative-sense, enveloped RNA
virus and is highly infectious. It commonly affects young children
and is the most common cause of lower respiratory tract illness in
infants. RSV infections are usually associated with
moderate-to-severe cold-like symptoms. However, severe lower
respiratory tract disease may occur at any age, especially in
elderly or immunocompromised patients. Children with severe
infections may require oxygen therapy and, in certain cases,
mechanical ventilation. According to the American Medical
Association, an increasing number of children are being
hospitalized for bronchiolitis, often caused by RSV infection. RSV
infections also account for approximately one-third of
community-associated respiratory virus infections in patients in
bone marrow transplant centers. In the elderly population, RSV
infection has been recently recognized to be very similar in
severity to influenza virus infection.
[0013] Influenza (INF), also known as the flu, is a contagious
disease that is caused by the influenza virus. It attacks the
respiratory tract in humans (nose, throat, and lungs). An average
of about 36,000 people per year in the United States die from
influenza, and 114,000 per year require hospitalization as a result
of influenza. Influenza has recently become a more serious concern
with the emergence of highly pathogenic strains previously only
found in animals (e.g. avian flu).
[0014] In all infectious diseases, the efficacy of a given therapy
often depends on the host immune response. This is particularly
true for herpes viruses, where the ability of all herpes viruses to
establish latent infections results in an extremely high incidence
of reactivated infections in immunocompromised patients. In renal
transplant recipients, 40% to 70% reactivate latent HSV infections,
and 80% to 100% reactivate CMV infections. Such viral reactivations
have also been observed with AIDS patients.
[0015] The hepatitis B virus (HBV) is a DNA virus that belongs to
the Hepadnaviridae family of viruses. HBV causes hepatitis B in
humans. It is estimated that 2 billion people have been infected (1
out of 3 people) in the world. About 350 million people remain
chronically infected and an estimated 1 million people die each
year from hepatitis B and its complications. HBV can cause lifelong
infection, cirrhosis of the liver, liver cancer, liver failure, and
death. The virus is transmitted through blood and bodily fluids.
This can occur through direct blood-to-blood contact, unprotected
sex, use of unsterile needles, and from an infected woman to her
newborn during the delivery process. Most healthy adults (90%) who
are infected will recover and develop protective antibodies against
future hepatitis B infections. A small number (5-10%) will be
unable to get rid of the virus and will develop chronic infections
while 90% of infants and up to 50% of young children develop
chronic infections when infected with the virus. Alpha-interferon
is the most frequent type of treatment used. Significant side
effects are related to this treatment including flu-like symptoms,
depression, rashes, other reactions and abnormal blood counts.
Another treatment option includes 3TC which also has many side
effects associated with its use. In the last few years, there have
been an increasing number of reports showing that patients treated
with 3TC are developing resistant strains of HBV. This is
especially problematic in the population of patients who are
co-infected with HBV and HIV. There is clearly an urgent need to
develop new antiviral therapies against this virus.
[0016] Hepatitis C virus (HCV) infection is the most common chronic
bloodborne infection in the United States where the number of
infected patients likely exceeds 4 million. This common viral
infection is a leading cause of cirrhosis and liver cancer, and is
now the leading reason for liver transplantation in the United
States. Recovery from infection is uncommon, and about 85 percent
of infected patients become chronic carriers of the virus and 10 to
20 percent develop cirrhosis. It is estimated that there are
currently 170 million people worldwide who are chronic carriers.
According to the Centers for Disease Control and Prevention,
chronic hepatitis C causes between 8,000 and 10,000 deaths and
leads to about 1,000 liver transplants in the United States alone
each year. There is no vaccine available for hepatitis C. Prolonged
therapy with interferon alpha, or the combination of interferon
with Ribavirin, is effective in only about 40 percent of patients
and causes significant side effects.
[0017] Today, the therapeutic outlook for viral infections in
general is not favourable. In general, therapies for viruses have
mediocre efficacies and are associated with strong side effects
which either prevent the administration of an effective dosage or
prevent long term treatment. Three clinical situations which
exemplify these problems are herpesviridae, HIV and RSV
infections.
[0018] In the case of herpesviridae, there are five major
treatments currently approved for use in the clinic: idoxuridine,
vidarabine, acyclovir, foscarnet and ganciclovir. While having
limited efficacy, these treatments are also fraught with side
effects. Allergic reactions have been reported in 35% of patients
treated with idoxuridine, vidarabine can result in
gastrointestional disturbances in 15% of patients and acyclovir,
foscarnet and ganciclovir, being nucleoside analogs, affect DNA
replication in host cells. In the case of ganciclovir, neutropenia
and thrombocytopenia are reported in 40% of AIDS patients treated
with this drug.
[0019] While there are many different drugs currently available for
the treatment of HIV infections, all of these are associated with
side effects potent enough to require extensive supplemental
medication to give patients a reasonable quality of life. The
additional problem of drug resistant strains of HIV (a problem also
found in herpesviridae infections) usually requires periodic
changing of the treatment cocktail and in some cases, makes the
infection extremely difficult to treat.
[0020] The treatment of RSV infections in young infants is another
example of the urgent need for new drug development. In this case,
the usual line of treatment is to deliver Ribavirin by inhalation
using a small-particule aerosol in an isolation tent. Not only is
Ribavirin only mildly effective, but its use is associated with
significant side effects. In addition, the potential release of the
drug has caused great concern in hospital personnel because of the
known teratogenicity of Ribavirin.
[0021] It is clear that for any new emerging antiviral drug being
developed, it would be highly desirable to incorporate the three
following features: 1--improved efficacy; 2--reduced risks of side
effects and 3--a mechanism of action which is difficult for the
virus to overcome by mutation.
[0022] Several attempts to inhibit particular viruses by various
antisense approaches have been made.
[0023] Zamecnik et al. have used ONs specifically targeted to the
reverse transcriptase primer site and to splice donor/acceptor
sites (Zamecnik, et al (1986) Proc. Natl. Acad. Sci. USA 83:4143-)
(Goodchild & Zamecnik (1989) U.S. Pat. No. 4,806,463).
[0024] Crooke and coworkers. (Crooke et al. (1992) Antimicrob.
Agents Chemother. 36:527-532) described an antisense against
HSV-1.
[0025] Draper et al. (1993) (U.S. Pat. No. 5,248,670) reported
antisense oligonucleotides having anti-HSV activity containing the
Cat sequence and hybridizing to the HSV-1 genes UL13, UL39 and
UL40.
[0026] Kean et al. (Biochemistry (1995) 34:14617-14620) reported
testing of antisense methylphosphonate oligomers as anti-HSV
agents.
[0027] Peyman et al. (Biol Chem Hoppe Seyler (1995) March;
376:195-198) have reported testing specific antisense
oligonucleotides directed against the IE110 and the UL30 mRNA of
HSV-1 for their antiviral properties.
[0028] Oligonucleotides or oligonucleotide analogs targeting CMV
mRNAs coding for IE1, IE2 or DNA polymerase were reported by
Anderson et al (1997) (U.S. Pat. No. 5,591,720)
[0029] Hanecak et al (1999) (U.S. Pat. No. 5,952,490) have
described modified oligonucleotides having a conserved G quartet
sequence and a sufficient number of flanking nucleotides to
significantly inhibit the activity of a virus such as HSV-1.
[0030] Jairath et al (Antiviral Res. (1997) 33:201-213) have
reported antisense oligonucleotides against RSV.
[0031] Torrence et al (1999) (U.S. Pat. No. 5,998,602) have
reported compounds comprising an antisense component complementary
to a single stranded portion of the RSV antigenomic strand (the
mRNA strand), a linker and a oligonucleotide activator of RNase
L.
[0032] Qi et al. (Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi
(2000) 14:253-256) have reported testing antisense
PS-oligonucleotides (PS-ONs) in Coxsackie virus B3.
[0033] International publication WO9203051 (Roizman and Maxwell)
describes methylphosphonate antisense oligomers which are
complementary to vital regions of HSV viral genome or mRNA
transcripts thereof which exhibit antiviral activity.
[0034] Guanosine/thymidine or guanosine-rich phosphorothioate
oligodeoxynucleotides (GT-PS-ONs) have been reported to have
antiviral activity. The article stated that "several different
PS-containing GT-rich ONs (B106-140, I100-12, and G106-57) all 26
or 27 nt in length, were just as effective at reducing HIV-2 titers
as GT-rich ONs consisting of 36 (B106-96, B106-97) or 45 nt (Table
4)." (Fennewald et al., Antiviral Res. (1995) 26:37-54).
[0035] In U.S. Pat. No. 6,184,369, anti-HIV, anti-HSV, and anti-CMV
oligonucleotides containing a high percentage of guanosine bases
are described. In preferred embodiments, the oligonucleotide has a
three dimensional structure and this structure is stabilized by
guanosine tetrads. In a further embodiment, the oligonucleotide
compositions of the invention have two or more runs of two
contiguous deoxyguanosines. The patent claims a G-rich
oligodeoxynucleotide (ODN) that includes at least two G residues in
at least two positions.
[0036] Cohen et al. (U.S. Pat. Nos. 5,264,423 and 5,276,019)
described the inhibition of replication of HIV, and more
particularly to PS-ODN analogs that can be used to prevent
replication of foreign nucleic acids in the presence of normal
living cells. Cohen et al describe antiviral activity of antisense
PS-ODNs specific to a viral sequence. They also describe testing
polyA, polyT and polyC PS-ODN sequences of 14, 18, 21 and 28-mers
and indicate an antiviral effect of those PS-ODNs.
[0037] Matsukura et al. (Matsukura et al (1987) Proc Natl Acad Sci
USA 84:7706-7710) later published the result described in Cohen et
al, U.S. patents above.
[0038] Gao et al (Gao et al (1989) J Biol Chem 264 :11521-11526),
describe the inhibition of replication of HSV-2, by PS-ODNs by
testing of polyA, polyT and polyC PS-ODN sequences in sizes of 7,
15, 21 and 28 nucleotides.
[0039] Archambault, Stein and Cohen (Archambault et al (1994) Arch
Virol 139:97109) report that a PS-ODN polyC of 28 nucleotides is
not effective against HSV-1.
[0040] Stein et al (Stein et al. (1989) AIDS Res Hum Retrovir
5:639-646), published results concerning additional data on
anti-HIV ODNs, generally of 21-28 nucleotides in length.
[0041] Marshal et al. (Marshall et al. (1992) Proc. Natl. Acad.
Sci. USA 89:6265-6269) describe anti-HIV-1 effect of
phosphorothioate and phosphorothioate poly-C oligos of 4-28
nucleotides in length.
[0042] Stein & Cheng (Stein et al. (1993) Science 261
:1004-1012), in a review article, mention the antiviral activity of
non specific ODNs of 28 nucleotides, stating that "the anti-HIV
properties of PS oligos are significantly influenced by
non-sequence-specific effects, that is, the inhibitory effect is
independent of the base sequence."
[0043] In a review article Lebedeva & Stein (Lebedeva et al
(2001) Annul Rev Pharmacol 41:403-419) report a variety of
non-specific protein binding activity of PS-ODNs, including viral
proteins. They state that "these molecules are highly biologically
active, and it is often relatively easy to mistake artifact for
antisense".
[0044] Rein et al. (U.S. Pat. No. 6,316,190) reported a GT rich ON
decoy linked to a fusion partner and binding to the HIV
nucleocapsid, which can be used as an antiviral compound.
Similarly, Campbell et al. (Campbell et al (1999) J. Virol. 73
:2270-2279) reported PO-ODN with a TGTGT motif binding specifically
to the nucleocapsid of HIV but with no references to an antiviral
activity.
[0045] Feng at al. (Feng et al. (2002) J. Virol. 76 :11757-11762)
described A(n) and TG(n) PO-ODNs binding to the recombinant HIV
nucleocapsid but with no data nor references to an anti-HIV
activity.
[0046] Antisense ODNs developed as anticancer agents, antiviral
agents, or to treat others diseases are typically approximately 20
nucleotides in length. In a review article (Stein, C A, (2001) J.
Clin. Invest. 108:641-644), it is affirmed that "the length of an
antisense oligonucleotide must be optimized: If the antisense
oligonucleotide is either too long or too short, an element of
specificity is lost. At the present time, the optimal length for an
antisense oligonucleotide seems to be roughly 16-20 nucleotides".
Similarly, in another review article (Crooke, S T (2000) Methods
Enzymol. 313:3-45) it is stated that "Compared to RNA and RNA
duplex formation, a phosphorothioate oligodeoxynucleotide has a
T.sub.m approximately -2.2.degree. lower per unit. This means that
to be effective in vitro, phosphorothioate oligodeoxynucleotides
must typically be 17- to -20-mer in length . . . ".
[0047] Caruthers and co-workers (Marshall et al. (1992) Proc. Natl.
Acad. Sci. USA 89:6265-6269) reported anti-HIV activity of
phosphorodithioate ODNs (PS2-ODNs) for a 12mer polycytidine-PS2-ODN
and for a 14mer PS2-ODN. No other sizes were tested for anti-HIV
activity. They also reported the inhibition of HIV reverse
transcriptase (RT) for 12, 14, 20 and 28mer polycytidine-PS2-ODNs.
Later, this group (Marshal et al (1993) Science 259:1564-1570)
reported results showing sequence specific inhibition of the HIV
RT. The same group published data for PS2-ODNs in several patents.
In U.S. Pat Nos. 5,218,103 and 5,684.148, PS2-ODN structure and
synthesis is described. In U.S. Pat. Nos. 5,452,496, 5,278,302, and
5,695,979 inhibition of HIV RT is described for PS2-ODNs not longer
than 15 bases. In U.S. Pat. Nos. 5,750,666 and 5,602,244, antisense
activity of PS2-ODNs is described.
[0048] Oligonucleotides modified at the 2' position of the ribose
and their uses in antisense strategies have been evaluated, e.g.,
as described in the references cited below.
[0049] Inoue and coworkers (Inoue et al. (1985) Nucleic Acids Res.
16:165168) describe the synthesis and properties of oligos
(2'-O-methylribonucleotides). The same group (Inoue et al. (1987)
FEBS Letter 215:327-330) reported that no RNAse H mediated mRNA
cleavage occurs when the oligonucleotide contains all
2'-O-methylribonucleotides. With mixed oligonucleotides i.e.
oligonucleotides having unmodified and 2'-O-methylribonucleotides,
they report sequence specific RNAse H hydrolysis of the nucleic
acid complex formed by RNA and 2'-O-methylribonucleotides.
[0050] Fully 2'-O-methylated and phosphorothioated oligonucleotides
which do not support RNase H-mediated cleavage of target mRNA, were
used to determine if active antisense oligonucleotides inhibited
ICAM-1 expression by an RNase H-dependent mechanism (Chiang et al.,
(1991) J. Biol. Chem. 266:18162-18171). They stated that these
antisense oligonucleotides may be useful as therapeutic agents.
[0051] Oligonucleotides with 2'-sugar modifications including
2'-O-methyl, 2'-O-propyl, 2'-O-pentyl, and 2'-fluoro were analyzed
for antisense activity. Evaluation of antisense activities of
uniformly 2'-modified oligonucleotides revealed that these
compounds were completely ineffective in inhibiting gene
expression. Activity was restored if the compound contained a
stretch of at least five 2'-deoxyribonucleotide residues. This
minimum deoxyribonucleotide length correlated perfectly with the
minimum length required for efficient RNase H activation in vitro.
(Monia et al., 1993, J. Biol. Chem. 268:14514.)
[0052] Yu et al. ((1996) Bioorganic. Med. Chem. 4:1685-1692)
reported that hybrid antisense oligonucleotides having
phosphorothioate, phosphodiester, or mixed backbones with a portion
of 2'-O-methyl modified sugars have a specific anti-HIV activity
measured by p24 ELISA quantification.
[0053] It is reported that correct splicing was efficiently
restored when phosphorothioated 2'-O-methyl-oligoribonucleotides
were targeted to the aberrant splice sites of IVS2-654 pre-mRNA
expressed in mammalian cells stably transformed with this mutated
human beta-globin gene. (Sierakowska, et al (1996) Proc. Natl.
Acad. Sci. USA 93:12840-12844.)
[0054] A review article, Agrawal ((1999) Biochim. Biophys. Acta
1489:53-68) suggests that for optimum activity, antisense
oligonucleotides should have a combination of various properties,
instead of only increased stability toward nucleases or high
affnity to target RNA. Such properties include RNAse H activation.
In a later review, Agrawal and Kandimalla ((2000) Mol. Med. Today
6:72-81) say that mixed backbone oligonucleotides, including
2'-O-methyl modifications, have become the choice for
second-generation antisense oligonucleotides for their improved
characteristics including RNAse H activation. An antisense oligo
should posses certain important characteristics such as the ability
to activate RNAse H upon binding to the target RNA. (Agrawal and
Kandimalla, 2001, Current Cancer Drug Target 1:197-209.) For most
antisense approaches target RNA cleavage by RNAse H is desired in
order to increase antisense potency. (Kurreck, 2003, Eur. J.
Biochem. 270:1628-1644.)
[0055] Many studies describe the use of the 2'-O-methoxyethyl
modification in antisense oligonucleotides. An example is a study
using a gapped 2' modified oligonucleotide antisense described in
Zellweger et al. ((2001) J. Pharmacol. Experimental Therapeutics
298:934-940). Another example shows inhibition of the formation of
the translation initiation complex using RNase H independent
2'-O-methoxyethyl antisense. (Baker et al. 1997) J. Biol. Chem. 272
:1994-12000.)
[0056] Kuwasaki et al. (2003) J. Antimicrob. Chemother. 51:813-819,
describes the design of a highly nuclease-resistant, dimeric
hairpin guanosine-quadruplex containing 2'-O-methyl groups on the
nucleosides and sulphur groups on the internucleotidic bonds, and
its anti-HIV-1 activity in cultured cells.
[0057] Mou and Gray (2002) (Nucleic Acids Res. 30:749-758),
indicates that, compared with typical phosphorothioate-DNA
oligomers, the addition of the 2'-O-methyl modification lowers the
non-specific protein binding property. The protein binding
affinities of g5p for a 36mers oligonucleotide increased in the
order of
dA.sub.36<rA.sub.36<2'-O-MeA.sub.36<S-rA.sub.36<<S-2'-O-Me-
A.sub.36<S-dA.sub.36 (where d=deoxy, r=ribo,
2'-O-Me=2'-O-methyl, S=phosphorothioate). This order was in
agreement with the order of S-RNA<<S-2'-O-MeRNA<S-DNA
reported in Kandimalla et al. ((1998) Bioorganic Med Chem Lett.
8:2103-2108) for the non-specific binding of plasma proteins, such
as human serum albumin, .gamma.-globulin and fibrinogen for these
oligomer modifications.
[0058] U.S. Pat. Nos. 5,591,623 and 5,514,788 describe compositions
and methods for the treatment and diagnosis of diseases amenable to
treatment through modulation of the synthesis or metabolism of
intercellular adhesion molecules. In accordance with preferred
embodiments, oligonucleotides are described which are specifically
hybridizable with nucleic acids encoding intercellular adhesion
genes. The invention describes the synthesis of 2'-O-methyl
phosphorothioate oligonucleotides and their use as antisense.
[0059] U.S. Pat. Nos. 5,652,355, 6,143,881 and 6,346,614 describe
hybrid oligonucleotides (containing segments of deoxy- and ribo
nucleotides) that resist nucleolytic degradation, form stable
duplexes with RNA or DNA, and activate RNase H when hybridized with
RNA. It is indicated that one property of phosphorothioate
2'-O-methyl-oligonucleotide is the non-activation of RNAse H. In
one aspect, the invention provides hybrid oligonucleotides that are
effective in inhibiting viruses, pathogenic organisms, or the
expression of cellular genes. A feature of oligonucleotides
according to this aspect of the invention is the presence of
deoxyribonucleotides. Oligonucleotides according to the invention
contain at least one deoxyribonucleotide. The nucleotide sequence
of oligonucleotides according to this aspect of the invention is
complementary to a nucleic acid sequence that is from a virus, a
pathogenic organism or a cellular gene.
[0060] U.S. Pat. Nos. 5,591,721 and 6,608,035 describe a method of
down-regulating the expression of a gene in an animal by the oral
administration of an oligonucleotide whose nucleotide sequence is
complementary to the targeted gene. Thus, because of the properties
described in the patent, such oligonucleotides are said to be
useful therapeutically by their ability to control or down-regulate
the expression of a particular gene in an animal. The hybrid
DNA/RNA oligonucleotides useful in the method of the invention
resist nucleolytic degradation, form stable duplexes with RNA or
DNA, and preferably activate RNase H when hybridized with RNA. The
oligonucleotides according to the invention are reported to be
effective in inhibiting the expression of various genes in viruses,
pathogenic organisms, or in inhibiting the expression of cellular
genes. Thus, oligonucleotides according to the method of the
invention have a nucleotide sequence which is complementary to a
nucleic acid sequence that is from a virus, a pathogenic organism
or a cellular gene.
[0061] U.S. Pat. No. 6,608,035 presents data indicating that a
phosphorothioate oligonucleotide is not stable in the stomach after
6 hours but a hybrid phosphorothioate oligonucleotide containing
2'-O-methyl ribonucleotide at the 3' and 5' ends and a
deoxyribonucleotide interior is more stable in the stomach but
partially degraded.
SUMMARY OF THE INVENTION
[0062] The present invention involves the discovery that
oligonucleotides (ONs), e.g., oligodeoxynucleotides (ODNs),
including highly modified oligonucleotides, can have a broadly
applicable, sequence independent antiviral activity. Advantageous
modifications include modified internucleotidic linkages and
2'-modifications. It is not necessary for the oligonucleotide to be
complementary to any viral sequence or to have a particular
distribution of nucleotides in order to have antiviral activity.
Such an oligonucleotide can even be prepared as a randomer, such
that there will be at most a few copies of any particular sequence
in a preparation, e.g., in a 15 micromol randomer preparation 32 or
more nucleotides in length.
[0063] In addition, the inventors have discovered that different
length oligonucleotides have varying antiviral effect. For example,
present results indicate that the length of antiviral
oligonucleotide that produces maximal antiviral effect when
modified with phosphorothioate internucleotidic linkages is
typically in the range of 40-120 nucleotides. In view of the
present discoveries concerning antiviral properties of
oligonucleotides, this invention provides oligonucleotide antiviral
agents that can have activity against numerous different viruses,
and can even be selected as broad-spectrum antiviral agents. Such
antiviral agents are particularly advantageous in view of the
limited antiviral therapeutic options currently available.
[0064] Therefore, the ONs, e.g., ODNs, of the present invention are
useful in therapy for treating or preventing viral infections or
for treating or preventing tumors or cancers induced by viruses,
such as oncoviruses (e.g., retroviruses, papillomaviruses, and
herpesviruses), and in treating or preventing other diseases whose
etiology is viral-based. Such treatments are applicable to many
types of patients and treatments, including, for example, the
prophylaxis or treatment of viral infections in immunosuppressed
human and animal patients.
[0065] A first aspect of the invention concerns antiviral
oligonucleotides, e.g., purified oligonucleotides, where the
antiviral occurs principally by a sequence independent, e.g.,
non-sequence complementary, mode of action, and formulations
containing such oligonucleotides.
[0066] Oligonucleotides useful in the present invention can be of
various lengths, e.g., at least 6, 10, 14, 15, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 35, 38, 40, 45, 50, 60, 70, 80, 90, 100,
110, 120, 140, 160, or more nucleotides in length. Likewise, the
oligonucleotide can be in a range, e.g., a range defined by taking
any two of the preceding listed values as inclusive end points of
the range, for example 10-20, 20-30, 20-40, 30-40, 30-50, 40-50,
40-60, 40-80, 50-60, 50-70, 60-70, 70-80, 60-120, and 80-120
nucleotides. In particular embodiments, a minimum length or length
range is combined with any other of the oligonucleotide
specifications listed herein for the present antiviral
oligonucleotides.
[0067] The antiviral nucleotide can include various modifications,
e.g., stabilizing modifications, and thus can include at least one
modification in the phosphodiester linkage and/or on the sugar,
and/or on the base. For example, the oligonucleotide can include
one or more phosphorothioate linkages, phosphorodithioate linkages,
and/or methylphosphonate linkages. Different chemically compatible
modified linkages can be combined, e.g., modifications where the
synthesis conditions are chemically compatible. While modified
linkages are useful, the oligonucleotides can include
phosphodiester linkages, e.g., include at least one phosphodiester
linkage, or at least 5, 10, 20, 30% or more phosphodiester
linkages. Additional useful modifications include, without
restriction, modifications at the 2'-position of the sugar, such as
2'-O-alkyl modifications such as 2'-O-methyl modifications,
2'-amino modifications, 2'-halo modifications such as 2'-fluoro;
acyclic nucleotide analogs. Other modifications are also known in
the art and can be used. In particular embodiments, the
oligonucleotide has modified linkages throughout, e.g.,
phosphorothioate; has a 3'- and/or 5'-cap; includes a terminal
3'-5' linkage; the oligonucleotide is or includes a concatemer
consisting of two or more oligonucleotide sequences joined by a
linker(s).
[0068] The present invention further provides an oligonucleotide,
wherein said oligonucleotide is linked or conjugated at one or more
nucleotide residues, to a molecule modifying the characteristics of
the oligonucleotide to obtain one or more characteristics selected
from the group consisting of higher stability, lower serum
interaction, higher cellular uptake, higher viral protein
interaction, an improved ability to be formulated for delivery, a
detectable signal, higher antiviral activity, better
pharmacokinetic properties, specific tissue distribution, lower
toxicity.
[0069] In certain embodiments, the oligonucleotide includes at
least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 100% modified
linkages, e.g., phosphorothioate, phosphorodithioate, and/or
methylphosphonate.
[0070] In certain embodiments, at least 10, 20, 30, 40, 50, 60, 70,
80, 90, or 95%, or all of the nucleotides are modified at the
2'-position of the ribose, e.g., 2'-OMe, 2'-F, 2'-amino.
[0071] In certain embodiments modified linkages are combined with
2'-modifications in oligonucleotides, for example, at least 30%
modified linkages and at least 30% 2'-modifications; or
respectively at least 40% and 40%, at least 50% and 50%, at least
60% and 60%, at least 70% and 70%, at least 80% and 80%, at least
90% and 90%, 100% and 100%. In certain embodiments, the
oligonucleotide includes at least 30, 40, 50, 60, 70, 80, 90, or
100% modified linkages and at least 30, 40, 50, 60, 70, 80, 90, or
100% 2'-modifications where embodiments include each combination of
listed modified linkage percentage and 2'-modification percentage
(e.g., at least 50% modified linkage and at least 80%
2'-modifications, and at least 80% modified linkages and 100%
2'-modifications). In particular embodiments of each of the
combinations percentages described, the modified linkages are
phosphorothioate linkages; the modified linkages are
phosphorodithioate linkages; the 2'-modifications are 2'-OMe; the
2'-modifications are 2'-fluoro; the 2'-modifications are a
combination of 2'-OMe and 2'-fluoro; the modified linkages are
phosphorothioate linkages and the 2'-modifications are 2'-OMe; the
modified linkages are phosphorothioate linkages and the
2'-modifications are 2'-fluoro; the modified linkages are
phosphorodithioate linkages and the 2'-modifications are 2'-OMe;
the modified linkages are phosphorodithioate linkages and the
2'-modifications are 2'-fluoro; the modified linkages are
phosphorodithioate linkages and the 2'-modifications are a
combination of 2'-OMe and 2'-fluoro. In certain embodiments of
oligonucleotides as described herein that combine a particular
percentage of modified linkages and a particular percentage of
2'-modifications, the oligonucleotide is at least 15, 20, 25, 30,
35, 40, 50, 60, 70, 80, 90, 100, 110, or 120 nucleotides in length,
or is in a length range defined by taking any two of the specified
lengths as inclusive endpoints of the range.
[0072] In certain embodiments, all but 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10 of the internucleotidic linkages and/or 2'-positions of the
ribose moiety are modified, e.g., with linkages modified with
phosphorothioate, phosphorodithioate, or methylphosphonate linkages
and/or 2'-OMe, 2'-F, and/or 2'-amino modifications of the ribose
moiety.
[0073] In some embodiments, the oligonucleotide includes at least
1, 2, 3, or 4 ribonucleotides, or at least 10, 20, 30, 40, 50, 60,
70, 80, 90%, or even 100% ribonucleotides.
[0074] In particular embodiments, the oligonucleotide includes
non-nucleotide groups in the chain (i.e., form part of the chain
backbone) and/or as side chain moieties, e.g., 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, or even more, or up to 5, 10, 20% or more of the chain
moieties and/or side chain moieties.
[0075] In certain embodiments, the oligonucleotide is free of
self-complementary sequences longer than 5, 8, 10, 15, 20, 25, 30
nucleotides; the oligonucleotide is free of catalytic activity,
e.g., cleavage activity against RNA; the oligonucleotide does not
induce an RNAi mechanism.
[0076] In particular embodiments, the oligonucleotide binds to one
or more viral proteins; the sequence of the oligonucleotide (or a
portion thereof, e.g., at least 20, 30, 40, 50, 60, 70% or more) is
derived from a viral genome; the activity of an oligonucleotide
with a sequence derived from a viral genome is not superior to a
randomer oligonucleotide or a random oligonucleotide of the same
length; the oligonucleotide includes a portion complementary to a
viral sequence and a portion not complementary to a viral sequence;
the sequence of the oligonucleotide is derived from a viral
packaging sequence or other viral sequence involved in an aptameric
interaction; unless otherwise indicated, the sequence of the
oligonucleotide includes A(x), C(x), G(x), T(x), U(x), I(x), AC(x),
AG(x), AT(x), AU(x), CG(x), CT(x), CU(x), GT(x), GU(x), TU(x),
AI(x), IC(x), IG(x), IT(x) IU(x) where x is 2, 3, 4, 5, 6, . . . 60
. . . 120 (in particular embodiments the oligonucleotide is at
least 15, 20, 25, 29, 30, 32, 34, 35, 36, 38, 40, 45, 46, 50, 60,
70, 80, 90, 100, 110, 120, 140, or 160 nucleotides in length or is
in a range defined by taking any two of the listed values as
inclusive endpoints, or the length of the specified repeat sequence
is at least a length or in a length range just specified); the
oligonucleotide includes a combination of repeat sequences (e.g.,
repeat sequences as specified above), including, for example, each
combination of the above monomer and/or dimer repeats taken 2, 3,
or 4 at a time; the oligonucleotide is single stranded (RNA or
DNA); the oligonucleotide is double stranded (RNA or DNA); the
oligonucleotide includes at least one Gquartet or CpG portion; the
oligonucleotide includes a portion complementary to a viral mRNA
and is at least 29, 37, or 38 nucleotides in length (or other
length as specified above); the oligonucleotide includes at least
one non-Watson-Crick oligonucleotide and/or at least one nucleotide
that participates in non-Watson-Crick binding with another
nucleotide and/or at least one nucleotide that cannot form base
pairs with other nucleotides; the oligonucleotide is a random
oligonucleotide, the oligonucleotide is a randomer or includes a
randomer portion, e.g., a randomer portion that has a length of at
least 5, 10, 15, 20, 25, 30, 35, 40 or more contiguous
oligonucleotides or a length as specified above for oligonucleotide
length or at least 10, 20, 30, 40, 50, 60, 70, 80, 90% or all the
nucleotides are randomer; the oligonucleotide is linked or
conjugated at one or more nucleotide residues to a molecule that
modifies the characteristics of the oligonucleotide, e.g. to
provide higher stability (such as stability in serum or stability
in a particular solution), lower serum interaction, higher cellular
uptake, higher viral protein interaction, improved ability to be
formulated for delivery, a detectable signal, improved
pharmacokinetic properties, specific tissue distribution, and/or
lower toxicity.
[0077] It was also discovered that phosphorothioated ONs containing
only (or at least primarily) pyrimidine nucleotides, including
cytosine and/or thymidine and/or other pyrimidines are resistant to
low pH and polycytosine oligonucleotides showed increased
resistance to a number of nucleases, thereby providing two
important characteristics for oral administration of an antiviral
ON. Thus, in certain embodiments, the oligonucleotide has at least
80, 90, or 95, or 100% modified internucleotidic linkages (e.g.,
phosphorothioate or phosphorodithoiate) and the pyrimidine content
is more than 50%, more than 60%, more than 70%, more than 80%, more
than 90%, or 100%, i.e.; is a pyrimidine oligonucleotide or the
cytosine content is more than 50%, more than 60%, more than 70%,
more than 80%, morethan 90% or 100% i.e. is a polycytosine
oligonucleotide. In certain embodiments, the length is at least 29,
30, 32, 34, 36, 38, 40, 45, 50, 60, 70, or 80 nucleotides, or is in
a range of 20-28, 25-35, 29-40, 30-40, 35-45, 40-50, 45-55, 50-60,
55-65, 60-70, 65-75, or 70-80, or is in a range defined by taking
any two of the listed values as inclusive endpoints of the range.
In particular embodiment, the oligonucleotide is at least 50, 60,
70, 80, or 90% cytosine; at least 50, 60, 70, 80, or 90% thymidine
(and may have a total pyrimidine content as listed above). In
particular embodiments, the oligonucleotide contains a listed
percentage of either cytosine or thymidine, and the remainder of
the pyrimidine nucleotides are the other of cytosine and thymidine.
Also in certain embodiments, the oligonucleotide includes at least
10, 12, 14, 16, 18, 20, 25, 30, 35, 40, or more contiguous
pyrimidine nucleotides, e.g., as C nucleotides, T nucleotides, or
CT dinucleotide pairs. In certain embodiments, the pyrimidine
oligonucleotide consists only of pyrimidine nucleotides; includes
at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-pyrimidine moieities;
includes 1-5, 6-10, 11-15, or at least 16 non-pyrimidine backbone
moieties; includes at least one, 1-20, 1-5, 6-10, 11-15, or 16-20
non-nucleotide moieties; includes at least one, 1-20, 1-5, 6-10,
11-15, or 16-20 purine nucleotides. Preferably, in embodiments in
which non-nucleotide moieities are present, the linkages between
such moieties or between such moieties and nucleotides are at least
25, 35, 50, 70, 90, or 100% as resistant to acidic conditions as PS
linkages in a 40-mer polyC oligonucleotide as evaluated by gel
electrophoresis under conditions appropriate for the size and
chemistry of the oligonucleotide.
[0078] Oligonucleotides can also be used in combinations, e.g., as
a mixture. Such combinations or mixtures can include, for example,
at least 2, 3, 4, 5, 10, 20, 50, 100, 1000, 10000, 100,000,
1,000,000, or more different oligonucleotides, e.g., any
combination of oligonucleotides are described herein. Such
combinations or mixtures can, for example, be different sequences
and/or different lengths and/or different modifications and/or
different linked or conjugated molecules. In particular embodiments
of such combinations or mixtures, a plurality of oligonucleotides
have a minimum length or are in a length range as specified above
for oligonucleotides. In particular embodiments of such
combinations or mixtures, at least one, a plurality, or each of the
oligonucleotides can have any of the other properties specified
herein for individual antiviral oligonucleoties (which can also be
in any consistent combination).
[0079] In certain embodiments, the sequence of the oligonucleotide
is not perfectly complementary to any equal length portion of the
genome sequence of the target virus, or has less than 95, 90, 80,
70, 60, or 50% complementarity to any equal length portion of the
genomic sequence of the target virus, the oligonucleotide sequence
does not consist essentially of polyA, polyC, polyG, polyT,
Gquartet, or a TG-rich sequence.
[0080] As used in connection with the present oligos, the term
"TG-rich" indicates that the sequence of the antiviral
oligonucleotide consists of at least 50 percent T and G
nucleotides, or if so specified, at least 60, 70, 80, 90, or 95% T
and G, or even 100%.
[0081] In a related aspect, the invention provides a mixture of
antiviral oligonucleotides that includes at least two different
antiviral oligonucleotides as described herein, e.g., at least 2,
3, 4, 5, 7, 10, 50, 100, 1000, 10,000, 100,000, 1,000,000, or even
more.
[0082] As used herein in connection with oligonucleotides or other
materials, the term "antiviral" refers to an effect of the presence
of the oligonucleotides or other material in inhibiting production
of viral particles, i.e., reducing the number of infectious viral
particles formed, in a system otherwise suitable for formation of
infectious viral particles for at least one virus. In certain
embodiments of the present invention, the antiviral
oligonucleotides will have antiviral activity against multiple
different viruses.
[0083] The term "antiviral oligonucleotide formulation" refers to a
preparation that includes at least one antiviral oligonucleotide
that is adapted for use as an antiviral agent. The formulation
includes the oligonucleotide or oligonucleotides, and can contain
other materials that do not interfere with use of the formulation
as an antiviral agent in vivo. Such other materials can include
without restriction diluents, excipients, carrier materials, and/or
other antiviral materials.
[0084] As used herein, the term "pharmaceutical composition" refers
to an antiviral oligonucleotide formulation that includes a
physiologically or pharmaceutically acceptable carrier or
excipient. Such compositions can also include other components that
do not make the composition unsuitable for administration to a
desired subject, e.g., a human.
[0085] In the context of the present invention, unless specifically
limited the term "oligonucleotide (ON)" means oligodeoxynucleotide
(ODN) or oligodeoxyribonucleotide or oligoribonucleotide. Thus,
"oligonucleotide" refers to an oligomer or polymer of ribonucleic
acid (RNA) and/or deoxyribonucleic acid (DNA) and/or analogs
thereof. This term includes oligonucleotides composed of
naturally-occurring nucleobases, sugars and covalent
internucleoside (backbone) linkages as well as oligonucleotides
having non-naturally-occurring portions which function similarly.
Such modified or substituted oligonucleotides are often preferred
over native forms because of desirable properties such as, for
example, enhanced cellular uptake, enhanced affinity for nucleic
acid target and increased stability in the presence of nucleases.
Examples of modifications that can be used are described herein.
Oligonucleotides that include backbone and/or other modifications
can also be referred to as oligonucleosides.
[0086] In the present context, the phrase "modified
internucleotidic linkage" refers to a linkage between nucleotides
or nucleotide analogs in an oligonucleotide that differs from the
phosphodiester linkage generally found in naturally-occurring
polynucleotides. Examples include phosphorothioate linkages,
phosphorodithioate linkages, and methylphosphonate linkages.
[0087] Specification of particular lengths for oligonucleotides,
e.g., at least 20 nucleotides in length, means that the
oligonucleotide includes at least 20 linked nucleotides. Unless
clearly indicated to the contrary, the oligonucleotide may also
include additional, non-nucleotide moieties, which may form part of
the backbone of the oligonucleotide chain. Unless otherwise
indicated, when non-nucleotide moieities are present in the
backbone, at least 10 of the linked nucleotides are contiguous.
[0088] As used in connection with an antiviral formulation,
pharmaceutical composition, or other material, the phrase "adapted
for use as an antiviral agent" indicates that the material exhibits
an antiviral effect and does not include any component or material
that makes it unsuitable for use in inhibiting viral production in
an in vivo system, e.g., for administering to a subject such as a
human subject.
[0089] As used herein in connection with antiviral action of an
antiviral oligonucleotide, "sequence independent mode of action"
indicates that the particular biological activity (e.g., antiviral
activity) is not dependent on a particular oligonucleotide sequence
in the oligonucleotide. For example, the activity does not depend
on sequence dependent hybridization such as with antisense
activity, or a particular sequence resulting in a sequence
dependent aptameric interaction. Similarly, the phrase
"non-sequence complementary mode of action" indicates that the
mechanism by which the material exhibits an antiviral effect is not
due to hybridization of complementary nucleic acid sequences, e.g.,
an antisense effect. Conversely, a "sequence complementary mode of
action" means that the antiviral effect of a material involves
hybridization of complementary nucleic acid sequences or sequence
specific aptameric interaction. Thus, indicating that the antiviral
activity of a material is due to a sequence independent mode of
action" or that the activity is "not primarily due to a sequence
complementary mode of action" means that the the activity of the
oligonucleotide satisfies at least one of the 4 tests provided
herein (see Example, 10). In particular embodiments, the
oligonucleotide satisfies test 1, test 2, test 3, test 4, or test
5; the oligonucleotide satisfies a combination of two of the tests,
i.e., tests 1 & 2; tests 1 & 3; tests 1 & 4, tests 1
& 5, tests 2 & 3, tests 2 & 4, test 2 & 5, tests 3
& 4, tests 3 & 5, or tests 4 & 5; the oligonucleotide
satisfies a combination of 3 of the tests, i.e., tests 1, 2, and 3,
tests 1, 2, and 4, test 1, 2, & 5, tests 1, 3, and 4, tests 1,
3, & 5, tests 2, 3, and 4, tests 2, 3, & 5, tests 3, 4,
& 5; the oligonucleotide satisifies all of tests 1, 2, 3, and
4.
[0090] As used herein in connection with administration of an
antiviral material, the term "subject" refers to a living higher
organism, including, for example, animals such as mammals, e.g.,
humans, non-human primates, bovines, porcines, ovines, equines,
dogs, and cats; birds (Aves),; and plants, e.g., fruit trees.
[0091] A related aspect concerns an antiviral oligonucleotide
randomer or randomer formulation that contains at least one
randomer, where the antiviral activity of the randomer occurs
principally by a sequence independent, e.g., non-sequence
complementary mode of action. Such a randomer formulation can, for
example, include a mixture of randomers of different lengths, e.g.,
at least 2, 3, 5, 10, or more different lengths, or other mixtures
as described herein.
[0092] As used herein in connection with oligonucleotide sequences,
the term "random" characterizes a sequence or an ON that is not
complementary to a viral mRNA, and which is selected to not form
hairpins and not to have palindromic sequences contained therein.
When the term "random" is used in the context of antiviral activity
of an oligonucleotide toward a particular virus, it implies the
absence of complementarity to a viral mRNA of that particular
virus. The absence of complementarity may be broader, e.g., for a
plurality of viruses, for viruses from a particular viral family,
or for infectious human viruses.
[0093] In the present application, the term "randomer" is intended
to mean a single stranded DNA having a wobble (N) at every
position, such as NNNNNNNNNN. Each base is synthesized as a wobble
such that this ON actually exists as a population of different
randomly generated sequences of substantially the same size. It is
recognized that preparation of such a randomer will normally
generate a distribution of sizes around a particular length
(primarily shorter lengths); unless clearly indicated to the
contrary, in the present context such a preparation is regarded as
a randomer of the particular length.
[0094] The phrase "derived from a viral genome" indicates that a
particular sequence has a nucleotide base sequence that has at
least 70% identity to a viral genomic nucleotide sequence or its
complement (e.g., is the same as or complementary to such viral
genomic sequence), or is a corresponding RNA sequence. In
particular embodiments of the present invention, the term indicates
that the sequence is at least 70% identical to a viral genomic
sequence of the particular virus against which the oligonucleotide
is directed, or to its complementary sequence. In particular
embodiments, the identity is at least 80, 90, 95, 98, 99, or
100%.
[0095] The invention also provides an antiviral pharmaceutical
composition that includes a therapeutically effective amount of a
pharmacologically acceptable, antiviral oligonucleotide or mixture
of oligonucleotides as described herein, e.g., at least 6
nucleotides in length or other length as listed herein, where the
antiviral activity of the oligonucleotide occurs principally by a
sequence independent, e.g., non-sequence complementary, mode of
action, and a pharmaceutically acceptable carrier. In particular
embodiments, the oligonucleotide or a combination or mixture of
oligonucleotides is as specified above for individual
oligonucleotides or combinations or mixtures of oligonucleotides.
In particular embodiments, the pharmaceutical compositions are
approved for administration to a human, or a non-human animal such
as a non-human primate.
[0096] In particular embodiments, the pharmaceutical composition is
adapted for the treatment, control, or prevention of a disease with
a viral etiology; adapted for treatment, control, or prevention of
a prion disease; is adapted for delivery by intraocular
administration, oral ingestion, enteric administration, inhalation,
cutaneous, subcutaneous, intramuscular, intraperitoneal,
intrathecal, intratracheal, or intravenous injection, or topical
administration.
[0097] In particular embodiments, the pharmaceutical composition
can be formulated for delivery by a mode selected from the group
consisting of but not restricted to oral ingestion, oral mucosal
delivery, intranasal drops or spray, intraocular injection,
subconjonctival injection, eye drops, ear drops, by inhalation,
intratracheal injection or spray, intrabronchial injection or
spray, intrapleural injection, intraperitoneal injection perfusion
or irrigation, intrathecal injection or perfusion, intracranial
injection or perfusion, intramuscular injection, intravenous
injection or perfusion, intraarterial injection or perfusion,
intralymphatic injection or perfusion, subcutaneous injection or
perfusion, intradermal injection, topical skin application, by
organ perfusion, by topical application during surgery,
intratumoral injection, topical application, gastric injection
perfusion or irrigation, enteral injection or perfusion, colonic
injection perfusion or irrigation, rectal injection perfusion or
irrigation, by rectal suppository or enema, by urethral suppository
or injection, intravesical injection perfusion or irrigation, or
intraarticular injection.
[0098] In particular embodiments, the composition includes a
delivery system, e.g., targeted to specific cells or tissues; a
liposomal formulation, another antiviral drug, e.g., a
non-nucleotide antiviral polymer, an antisense molecule, an siRNA,
or a small molecule drug.
[0099] In particular embodiments, the antiviral oligonucleotide,
oligonucleotide preparation, oligonucleotide formulation, or
antiviral pharmaceutical composition has an in vitro IC.sub.50 for
a target virus (e.g., any of particular viruses or viruses in a
group of viruses as indicated herein) of 10, 5, 2, 1, 0.50, 0.20,
0.10, 0.09. 0.08, 0.07, 0.75, 0.06, 0.05, 0.045, 0.04, 0.035, 0.03,
0.025, 0.02, 0.015, or 0.01 .mu.M or less.
[0100] In particular embodiments of formulations, pharmaceutical
compositions, and methods for prophylaxis or treatment, the
composition or formulation is adapted for treatment, control, or
prevention of a disease with viral etiology; is adapted for the
treatment, control or prevention of a prion disease; is adapted for
delivery by a mode selected from the group consisting of
intraocular, oral ingestion, enterally, inhalation, or cutaneous,
subcutaneous, intramuscular, or intravenous injection delivery;
further comprises a delivery system, which can include or be
associated with a molecule increasing affinity with specific cells;
further comprises at least one other antiviral drug in combination;
and/or further comprises an antiviral polymer in combination.
[0101] In particular embodiments, the pharmaceutical composition
contains at least one polypyrimidine oligonucleotide as described
herein. In view of the resistance to low pH discovered for
polypyrimidine oligonucleoides; in certain embodiments such a
composition is adapted for delivery to an acidic in vivo site,
e.g., oral delivery or vaginal delivery.
[0102] In particular embodiments of compositions and formulations
for oral administration containing such polypyrimidine
oligonucleotides, the composition or formulation is prepared in the
form of a powder, granules, microparticulates, nanoparticulates,
suspensions or solutions in water or non-aqueous media, emulsion
(e.g., microemulsion), capsule, gel capsule, sachet, tablet, or
minitablet. In certain embodiments, thickeners, flavoring agents,
diluents, emulsifiers, dispersing aids or binders may be included.
In some embodiments, the oral formulations are those in which
oligonucleotides of the invention are administered in conjunction
with one or more penetration enhancers surfactants and/or
chelators, e.g. and without restriction, fatty acids and/or esters
or salts thereof (for example, arachidonic acid, undecanoic acid,
oleic acid, lauric acid, caprylic acid, capric acid, myristic acid,
palmitic acid, stearic acid, linoleic acid, linolenic acid,
dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,
1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or
a monoglyceride, a diglyceride or a pharmaceutically acceptable
salt thereof (e.g. sodium), bile acids and/or salts thereof (for
example, chenodeoxycholic acid (CDCA) and ursodeoxychenedeoxycholic
acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid,
glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic
acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate,
sodium glycodihydrofusidate). Some embodiments include a
combination of penetration enhancers, for example, fatty
acids/salts in combination with bile acids/salts such as the sodium
salt of lauric acid, capric acid and UDCA. Further exemplary
penetration enhancers include polyoxyethylene-9-lauryl ether,
polyoxyethylene-20-cetyl ether.
[0103] In particular embodiments in which the oligonucleotides of
the invention are prepared in granular form (including sprayed
dried particles) or complexed to form micro or nanoparticles, a
complexing agent(s) is used that is selected, without restriction,
from poly-amino acids; polyimines; polyacrytates;
polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates;
cationized gelatins, albumins, starches, acrylates,
polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates;
DEAE-derivatized polyimines, pollulans, celluloses, and starches,
or more specifically selected from chitosan, N-trimethytchitosan,
poly-L-lysine, polyhistidine, polyorithine, polyspermines,
protamine, polyvinylpyridine, polythiodiethylamino-methylethylene
P(TDAE), polyaminostyrene (e.g. p-amino),
poly(methylcyanoacrylate), poly(ethylcyanoacrylate),
poly(butylcyanoacrylatc), poly(isobutylcyanoacrylate),
poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,
DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,
polyhexylacrylate, poly(D,L-lactic acid),
poly(DL-lactic-co-glycolic acid (PLGA), alginate, and
polyethyleneglycol (PEG).
[0104] In particular embodiments, the composition is adapted for
vaginal administration. In such embodiments, the composition may be
prepared, without limitation, in the form of tablets, a solution, a
cream, a gel, a suppository.
[0105] In particular embodiments, the composition is adapted for
topical administration.
[0106] As used herein, the terms "polypyrimidine oligonucleotide"
or "pyrimidine oligonucleotide" means an oligonucleotide that
contains greater than 50% pyrimidine nucleotides.
[0107] As used in relation to in vivo administration of the present
oligonucleotides and compositions, the term "acidic site" means a
site that has a pH of less than 7. Examples include the stomach (pH
generally 1-2), the vagina (pH generally 4-5 but may be lower), and
to a lesser degree, the skin (pH generally 4-6).
[0108] As used herein, the phrase "adapted for oral delivery" and
like terms indicate that the composition is sufficiently resistant
to acidic pH to allow oral administration without a clinically
excessive loss of activity, e.g., an excessive first pass loss due
to stomach acidity of less than 50% (or is indicated, less than
40%, 30%, 20%, 10%, or 5%).
[0109] As used herein, the phrase "adapted for vaginal
administration" and like terms indicate that the composition is
prepared such that when appropriately administered, the composition
will not degrade to a clinically unacceptable extent (e.g., less
than 50%, 40%, 30%, 20%, 10%, or 5% over a specified time for
retention) and will remain substantially in the vagina (excluding
material that is absorbed) for at least 1 hour (or if indicated,
for at least 2 hr, 4 hr, 8 hr, 12 hr, 1 day, or 2 days). Such
retention may be due to any of a number of different factors or
combinations of factors, for example, due to physical form or
adhesive properties, and the like.
[0110] As used herein in connection with antiviral oligonucleotides
and formulations, and the like, in reference to a particular virus
or group of viruses the term "targeted" indicates that the
oligonucleotide is selected to inhibit that virus or group of
viruses. As used in connection with a particular tissue or cell
type, the term indicates that the oligonucleotide, formulation, or
delivery system is selected such that the oligonucleotide is
preferentially present and/or preferentially exhibits an antiviral
effect in or proximal to the particular tissue or cell type.
[0111] As used herein, the term "delivery system" refers to a
component or components that, when combined with an oligonucleotide
(e.g., an antisense oligo, siRNA, or oligonucleotide as described
herein), increases the amount of the oligonucleotide that contacts
the intended location in vivo, and/or extends the duration of its
presence at the target, e.g., by at least 20, 50, or 100%, or even
more as compared to the amount and/or duration in the absence of
the delivery system, and/or prevents or reduces interactions that
cause side effects.
[0112] As used herein in connection with antiviral agents and other
drugs or test compounds, the term "small molecule" means that the
molecular weight of the molecule is 1500 daltons or less. In some
cases, the molecular weight is 1000, 800, 600, 500, or 400 daltons
or less.
[0113] In another aspect, the invention provides a kit that
includes at least one antiviral oligonucleotide, antiviral
oligonucleotide mixture, antiviral oligonucleotide formulation, or
antiviral pharmaceutical composition that includes such
oligonucleotide, oligonucleotide mixture, or oligonucleotide
formulation in a labeled package, where the antiviral activity of
the oligonucleotide occurs principally by a sequence independent
e.g., non-sequence complementary, mode of action and the label on
the package indicates that the antiviral oligonucleotide can be
used against at least one virus.
[0114] In particular embodiments the kit includes a pharmaceutical
composition that includes at least one antiviral oligonucletide as
described herein. In one embodiment, the kit contains a mixture of
at least two different antiviral oligonucleotides. In one
embodiment, the antiviral oligonucleotide is adapted for in vivo
use in an animal and/or the label indicates that the
oligonucleotide or composition is acceptable and/or approved for
use in an animal; the animal is a mammal, such as human, or a
non-human mammal such as bovine, porcine, a ruminant, ovine, or
equine; the animal is a non-human animal; the animal is a bird, the
kit is approved by a regulatory agency such as the U.S. Food and
Drug Administration or equivalent agency for use in an animal,
e.g., a human.
[0115] In another aspect, the invention provides a method for
selecting an antiviral oligonucleotide, e.g, a non-sequence
complementary antiviral oligonucleotide, for use as an antiviral
agent. The method involves synthesizing a plurality of different
random oligonucleotides, testing the oligonucleotides for activity
in inhibiting the ability of a virus to produce infectious virions,
and selecting an oligonucleotide having a pharmaceutically
acceptable level of activity for use as an antiviral agent.
[0116] In particular embodiments, the different random
oligonucleotides comprises randomers of different lengths; the
random oligonucleotides can have different sequences or can have
sequence in common, such as the sequence of the shortest oligos of
the plurality; and/or the different random oligonucleotides
comprise a plurality of oligonucleotides comprising a randomer
segment at least 5 nucleotides in length or the different random
oligonucleotides include a plurality of randomers of different
lengths. Other oligonucleotides, e.g., as described herein for
antiviral oligonucleotides, can be tested in a particular
system.
[0117] In yet another aspect, the invention provides a method for
the prophylaxis or treatment of a viral infection in a subject by
administering to a subject in need of such treatment a
therapeutically effective amount of at least one pharmacologically
acceptable oligonucleotide as described herein, e.g., a
non-sequence complementary oligonucleotide at least 6 nucleotides
in length, or an antiviral pharmaceutical composition or
formulation or mixture containing such oligonucleotide(s). In
particular embodiments, the virus can be any of those listed herein
as suitable for inhibition using the present invention; the
infection is related to a disease or condition indicated herein as
related to a viral infection; the subject is a type of subject as
indicated herein, e.g., human, non-human animal, non-human mammal,
bird, plant, and the like; the treatment is for a viral disease or
disease with a viral etiology, e.g., a disease as indicated in the
Background section herein.
[0118] In yet another aspect, the invention provides a method for
the prophylaxis or treatment of a viral infection in an acidic
environnement in a subject, comprising administering to a subject
in need of such a treatment a therapeutically effective amount of
at least one pharmacologically acceptable antiviral pharmaceutical
composition of the invention, said composition being adapted for
administration to an acidic in vivo site.
[0119] In particular embodiments, an antiviral oligonucleotide (or
oligonucleotide formulation or pharmaceutical composition) as
described herein is administered; administration is a method as
described herein; a delivery system or method as described herein
is used; the viral infection is of a DNA virus or an RNA virus; the
virus is a parvoviridae, papovaviridae, adenoviridae,
herpesviridae, poxviridae, hepadnaviridae, or papillomaviridae; the
virus is a arenaviridae, bunyaviridae, calciviridae, coronaviridae,
filoviridae, flaviridae, orthomyxoviridae, paramyxoviridae,
picornaviridae, reoviridae, rhabdoviridae, retroviridae, or
togaviridae; the herpesviridae virus is EBV, HSV-1, HSV-2, CMV,
VZV, HHV-6, HHV-7, or HHV-8; the virus is HIV-1 or HIV-2; the virus
is respiratory syncytical virus (RSV); the virus is parainfluenza-3
virus; the virus is an influenza virus, e.g., influenza A; the
virus is HBV; the virus is smallpox virus or vaccinia virus; the
virus is a coronavirus; the virus is SARS virus; the virus is West
Nile Virus; the virus is a hantavirus; the virus is a parainfluenza
virus; the virus is coxsackievirus; the virus is rhinovirus; the
virus is yellow fever virus; the virus is dengue virus; the virus
is hepatitis C virus; the virus is Ebola virus; the virus is
Marburg virus; the virus is Lassa fever virus; the virus is
Varicella Zoster Virus; the virus is Epstein Barr Virus; the virus
is Human Herpesvirus 6A or 6B; the virus is HBV; the virus is
parainfluenza virus; the virus is human metapneumovirus; the virus
is Rift Valley fever virus; the virus is Crimean Congo Hemorrhagic
Fever virus; the virus is Western Equine Encephalitis virus.
[0120] In particular embodiments, the oligonucleotide is a
polypyrimidine oligonucleotide (or a formulation or pharmaceutical
composition containing such polypyrimidine oligonucleotide), which
may be adapted for oral or vaginal administration, e.g., as
described herein.
[0121] Similarly, in a related aspect, the invention provides a
method for the prophylactic treatment of cancer caused by
oncoviruses in a human or animal by administering to a human or
animal in need of such treatment, a pharmacologically acceptable,
therapeutically effective amount of at least one random
oligonucleotide of at least 6 nucleotides in length (or another
length as described herein), or a formulation or pharmaceutical
composition containing such oligonucleotide. In one embodiment, a
mixture of oligonucleotides of the invention.
[0122] In particular embodiments, the oligonucleotide(s) is as
described herein for the present invention, e.g., having a length
as described herein; a method of administration as described herein
is used; a delivery system as described herein is used.
[0123] The term "therapeutically effective amount" refers to an
amount that is sufficient to effect a therapeutically or
prophylactically significant reduction in production of infectious
virus particles when administered to a typical subject of the
intended type. In aspects involving administration of an antiviral
oligonucleotide to a subject, typically the oligonucleotide,
formulation, or composition should be administered in a
therapeutically effective amount.
[0124] In certain embodiments involving oligonucleotide
formulations, pharmaceutical compositions, and/or treatment and
prophylactic methods described herein, the oligonucleotide(s)
having a sequence independent mode of action is not associated with
a transfection agent; the oligonucleotide(s) having a sequence
independent mode of action is not encapsulated in liposomes and/or
non-liposomal lipid particles. In certain embodiments, the
oligonucleotide(s) having a sequence independent mode of action is
in a pharmaceutical composition or is administered in conjunction
with (concurrently or sequentially) an antiviral oligonucleotide
that acts principally by a sequence dependent mode of action, e.g.,
antisense oligonucleotide or siRNA, where the oligonucleotide(s)
having a sequence dependent mode of action can be associated with a
transfection agent and/or encapsulated in liposomes and/or
non-liposomal lipid particles.
[0125] In another aspect, the discovery that sequence independent,
e.g., non-sequence complementary, interactions produce effective
antiviral activity provides a method of screening to identify a
compound that alters binding of an oligonucleotide to a viral
component, such as one or more viral proteins (e.g., extracted or
purified from a viral culture of infected host organisms, or
produced by recombinant methods). For example, the method can
involve determining whether a test compound reduces the binding of
oligonucleotide to one or more viral components.
[0126] As used herein, the term "screening" refers to assaying a
plurality of compounds to determine if they possess a desired
property. The plurality of compounds can, for example, be at least
10, 100, 1000, 10,000 or more test compounds.
[0127] In particular embodiments, any of a variety of assay formats
and detection methods can be used to identify such alteration in
binding, e.g., by contacting the oligonucleotide with the viral
component(s) in the presence and absence of a compound(s) to be
screened (e.g., in separate reactions) and determining whether a
difference occurs in binding of the oligo the viral component(s) in
the presence of the compound compared to the absence of the
compound. The presence of such a difference is indicative that the
compound alters the binding of the random oligonucleotide to the
viral component. Alternatively, a competitive displacement can be
used, such that oligonucleotide is bound to the viral component and
displacement by added test compound is determined, or conversely
test compound is bound and displacement by added oligonucleotide is
determined.
[0128] In particular embodiments, the oligonucleotide is as
described herein for antiviral oligonucleotides; the
oligonucleotide is at least 6, 8, 10, 15, 20, 25, 29, 30, 32, 34,
36, 38, 40, 46, 50, 60, 70, 80, 90, 100, 110, or 120 nucleotides in
length or at least another length specified herein for the
antiviral oligonucleotides, or is in a range defined by taking any
two of the preceding values as inclusive endpoints of the range;
the test compound(s) is a small molecule; the test compound has a
molecular weight of less than 400, 500, 600, 800, 1000, 1500, 2000,
2500, or 3000 daltons, or is in a range defined by taking any two
of the preceding values as inclusive endpoints of the range; the
viral extract or component is from a virus as listed herein; at
least 100, 1000, 10,000, 20,000, 50,000, or 100,000 compounds are
screened; the oligonucleotide has an in vitro IC.sub.50 of equal to
or less than 10, 5, 2, 1, 0.500, 0.200, 0.100, 0.075, 0.05, 0.045,
0.04, 0.035, 0.03, 0.025, 0.02, 0.015, or 0.01 .mu.M.
[0129] The present invention further provides oligonucleotides
described in Table 21.
[0130] The present invention further provides an antiviral
oligonucleotide as set forth in any one of REP 1001, REP 2001, REP
3007, REP 2004, REP 2005, REP 2006, REP 2007, REP 2008, REP 2017,
REP 2018, REP 2020, REP 2021, REP 2024, REP 2036, A20, G20, C20,
REP 2029, REP 2031, REP 2030, REP 2033, REP 2055, REP 2056, REP
2057, REP 2060 and REP 2107.
[0131] As used herein, the term "viral component" refers to a
product encoded by a virus or produced by infected host cells as a
consequence of the viral infection. Such components can include
proteins as well as other biomolecules. Such viral components, can,
for example, be obtained from viral cultures, infected host
organisms, e.g., animals and plants, or can be produced from viral
sequences in recombinant systems (prokaryotes and eukaryotes), as
well synthetic proteins having amino acid sequences corresponding
to viral encoded proteins. The term "viral culture extract" refers
to an extract from cells infected by a virus that will include
virus-specific products. Similarly, a "viral protein" refers to a
virus-specific protein, usually encoded by a virus, but can also be
encoded at least in part by host sequences as a consequence of the
viral infection.
[0132] In a related aspect, the invention provides an antiviral
compound identified by the preceding method, e.g., a novel
antiviral compound.
[0133] In a further aspect, the invention provides a method for
purifying oligonucleotides binding to at least one viral component
from a pool of oligonucleotides by contacting the pool with at
least one viral component, e.g., bound to a stationary phase
medium, and collecting oligonucleotides that bind to the viral
component(s). Generally, the collecting involves displacing the
oligonucleotides from the viral component(s). The method can also
involve sequencing and/or testing antiviral activity of collected
oligonucleotides (i.e., oligonucleotides that bound to viral
protein).
[0134] In particular embodiments, the bound oligonucleotides of the
pool are displaced from the stationary phase medium by any
appropriate method, e.g., using an ionic displacer, and displaced
oligonucleotides are collected. Typically for the various methods
of displacement, the displacement can be performed in increasing
stringent manner (e.g., with an increasing concentration of
displacing agent, such as a salt concentration, so that there is a
stepped or continuous gradient), such that oligonucleotides are
displaced generally in order of increased binding affinity. In many
cases, a low stringency wash will be performed to remove weakly
bound oligonucleotides, and one or more fractions will be collected
containing displaced, tighter binding oligonucleotides. In some
cases, it will be desired to select fractions that contain very
tightly binding oligonucleotides (e.g., oligonucleotides in
fractions resulting from displacement by the more stringent
displacement conditions) for further use.
[0135] Similarly, the invention provides a method for enriching
oligonucleotides from a pool of oligonucleotides binding to at
least one viral component, by contacting the pool with one or more
viral proteins, and amplifying oligonucleotides bound to the viral
proteins to provide an enriched oligonucleotide pool. The
contacting and amplifying can be performed in multiple rounds,
e.g., at least 1, 2, 3, 4, 5, 10, or more additional times using
the enriched oligonucleotide pool from the preceding round as the
pool of oligonucleotides for the next round. The method can also
involve sequencing and testing antiviral activity of
oligonucleotides in the enriched oligonucleotide pool following one
or more rounds of contacting and amplifying.
[0136] The method can involve displacing oligonucleotides from the
viral component (e.g., viral protein bound to a solid phase medium)
with any of a variety of techniques, such as those described above,
e.g., using a displacement agent. As indicated above, it can be
advantageous to select the tighter binding oligonucleotides for
further use, e.g., in further rounds of binding and amplifying. The
method can further involve selecting one or more enriched
oligonucleotides, e.g., high affinity oligonucleotides, for further
use. In particular embodiments, the selection can include
eliminating oligonucleotides that have sequences complementary to
host genomic sequences (e.g., human) for a particular virus of
interest. Such elimination can involve comparing the
oligonucleotide sequence(s) with sequences from the particular host
in a sequence database(s), e.g., using a sequence alignment program
(e.g., a BLAST search), and eliminating those oligonucleotides that
have sequences identical or with a particular level of identity to
a host sequence. Eliminating such host complementary sequences
and/or selecting one or more oligonucleotides that are not
complementary to host sequences can also be done for the other
aspects of the present invention.
[0137] In the preceding methods for identifying, purifying, or
enriching oligonucleotides, the oligonucleotides can be of types as
described herein. The above methods are advantageous for
identifying, purifying or enriching high affinity oligonucleotides,
e.g., from an oligonucleotide randomer preparation.
[0138] In a related aspect, the invention concerns an antiviral
oligonucleotide preparation that includes one or more
oligonucleotides identified using a method of any of the preceding
methods for identifying, obtaining, or purifying antiviral
oligonucleotides from an initial oligonucleotide pool, where the
oligonucleotides in the oligonucleotide preparation exhibit higher
mean binding affinity with one or more viral proteins than the mean
binding affinity of oligonucletides in the initial oligonucleotide
pool.
[0139] In particular embodiments, the mean binding affinity of the
oligonucleotides is at least two-fold, 3-fold, 5-fold, 10-fold,
20-fold, 50-fold, or 100-fold greater than the mean binding
affinity of oligonucleotides in the initial oligonucleotide pool,
or even more; the median of binding affinity is at least two-fold,
3-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold greater
relative to the median of the binding affinity of the initial oligo
pool, where median refers to the middle value.
[0140] In yet another aspect, the invention provides an antiviral
polymer mix that includes at least one antiviral oligonucleotide
and at least one non-nucleotide antiviral polymer. In particular
embodiments, the oligonucleotide is as described herein for
antiviral oligonucleotides and/or the antiviral polymer is as
described herein or otherwise known in the art or subsequently
identified.
[0141] In yet another aspect, the invention provides an
oligonucleotide randomer, where the randomer is at least 6
nucleotides in length. In particular embodiments the randomer has a
length as specified above for antiviral oligonucleotides; the
randomer includes at least one phosphorothioate linkage, the
randomer includes at least one phosphorodithioate linkage or other
modification as listed herein; the randomer oligonucleotides
include at least one non-randomer segment (such as a segment
complementary to a selected virus nucleic acid sequence), which can
have a length as specified above for oligonucleotides; the randomer
is in a preparation or pool of preparations containing at least 5,
10, 15, 20, 50, 100, 200, 500, or 700 micromol, 1, 5, 7, 10, 20,
50, 100, 200, 500, or 700 mmol, or 1 mole of randomer, or a range
defined by taking any two different values from the preceding as
inclusive end points, or is synthesized at one of the listed scales
or scale ranges.
[0142] Likewise, the invention provides a method for preparing
antiviral randomers, by synthesizing at least one randomer, e.g., a
randomer as described above.
[0143] As indicated above, for any aspect involving a viral
infection or risk of viral infection or targeting to a particular
virus, in particular embodiments the virus is as listed above.
[0144] The expression "human and animal viruses" is intended to
include, without limitation, DNA and RNA viruses in general. DNA
viruses include, for example, parvoviridae, papovaviridae,
adenoviridae, herpesviridae, poxviridae, hepadnaviridae, and
papillomaviridae. RNA viruses include, for example, arenaviridae,
bunyaviridae, calciviridae, coronaviridae, filoviridae, flaviridae,
orthomyxoviridae, paramyxoviridae, picornaviridae, reoviridae,
rhabdoviridae, retroviridae, or togaviridae.
[0145] In connection with modifying characteristics of an
oligonucleotide by linking or conjugating with another molecule or
moiety, the modifications in the characteristics are evaluated
relative to the same oligonucleotide without the linked or
conjugated molecule or moiety.
[0146] Additional embodiments will be apparent from the Detailed
Description and from the claims.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0147] The present invention is concerned with the identification
and use of antiviral oligonucleotides that act by a sequence
independent mechanism, and includes the discovery that for many
viruses, the antiviral activity is greater for larger
oligonucleotides, and is typically optimal for oligonucleotides
that are 40 nucleotides or more in length.
[0148] In accordance with the present invention there is provided
an oligonucleotide comprising at least one modified
internucleotidic linkage, wherein said oligonucleotide has an
antiviral activity against a target virus wherein said activity
operates predominantly by a sequence independent mode of
action.
[0149] In accordance with the present invention, there is provided
an oligonucleotide, having at least 50% of its nucleotides in said
oligonucleotide modified at the 2'-position of the ribose moiety
and having at least 50% of its internucleotidic linkages modified,
wherein said oligonucleotide has an antiviral activity against a
target virus, said activity operating predominantly by a sequence
independent mode of action. In one embodiment, 50%, 80%
respectively. In one embodiment, 80%, 80% respectively. In one
embodiment, 90%, 90% respectively. In one embodiment, 100%, 100%
respectively.
[0150] Lengths & Not Self-Complementary
[0151] The present invention further provides an oligonucleotide
having at least 15 nucleotides in length. In one embodiment, at
least 20 nucleotides in length. In one embodiment, at least 25
nucleotides in length. In one embodiment, at least 30 nucleotides
in length. In one embodiment, at least 35 nucleotides in length. In
one embodiment, at least 40 nucleotides in length. In one
embodiment, at least 45 nucleotides in length. In one embodiment,
at least 50 nucleotides in length. In one embodiment, at least 60
nucleotides in length. In one embodiment, at least 80 nucleotides
in length.
[0152] The present invention further provides an oligonucleotide
having 20-30 nucleotides in length. In one embodiment, 30-40
nucleotides in length.in one embodiment, 40-50 nucleotides in
length. In one embodiment, 50-60 nucleotides in length. In one
embodiment, 60-70 nucleotides in length. In one embodiment, 70-80
nucleotides in length.
[0153] The present invention further provides an oligonucleotide
which is free from self-complementary sequences of greater than 5
contiguous nucleotides. In one embodiment, greater than 10
contiguous nucleotides. In one embodiment, greater than 20
contiguous nucleotides.
[0154] The present invention further provides an oligonucleotide
which is free of catalytic activity.
Random
[0155] The present invention further provides an oligonucleotide
having an antiviral activity against a target virus, and the
sequence of said oligonucleotide not being complementary to any
equal length portion of the genomic sequence of said target
virus.
[0156] The present invention further provides an oligonucleotide,
wherein said oligonucleotide is not complementary to any equal
length portion of the genomic sequence of a human pathogenic
virus.
[0157] The present invention further provides an oligonucleotide,
wherein said oligonucleotide is not complementary to any equal
length portion of the genomic sequence of a human pathogenic virus
sequenced as of Jan. 1st, 2005.
[0158] The present invention further provides an oligonucleotide
which is not complementary to any equal length portion of the
genomic sequence of a human.
[0159] The present invention further provides an oligonucleotide
which is not complementary to any equal length portion of the
genomic sequence of one or more animals selected from the group
consisting of cattle, horse, swine, sheep, bird, dog, cat and
fish.
RNA and Other Chain Moiety Limitations
[0160] The present invention further provides an oligonucleotide
wherein at least 30% of the nucleotides are ribonucleotides. In one
embodiment, at least 50% of the nucleotides are ribonucleotides. In
one embodiment, at least 70% of the nucleotides are
ribonucleotides. In one embodiment, at least 80% of the nucleotides
are ribonucleotides. In one embodiment, at least 90% of the
nucleotides are ribonucleotides. In one embodiment, all of the
nucleotides are ribonucleotides.
[0161] The present invention further provides an oligonucleotide
comprising 1-4 non-nucleotide chain moieties.
Randomer
[0162] The present invention further provides an oligonucleotide
comprising at least 10 contiguous nucleotides of randomer sequence.
In one embodiment, at least 20 nucleotides of randomer sequence. In
one embodiment, at least 30 nucleotides of randomer sequence. In
one embodiment, at least 40 nucleotides of randomer sequence.
[0163] The present invention further provides an oligonucleotide
wherein said oligonucleotide is randomer oligonucleotide.
Homopolymer
[0164] The present invention further provides an oligonucleotide
comprising a homopolymer sequence of at least 10 contiguous A
nucleotides. In one embodiment, at least 10 contiguous T
nucleotides In one embodiment, at least 10 contiguous U
nucleotides. In one embodiment, at least 10 contiguous C
nucleotides. In one embodiment, at least 10 contiguous G
nucleotides. In one embodiment, at least 10 contiguous I nucleotide
analogs.
Heterodimers
[0165] The present invention further provides an oligonucleotide
comprising a polyAT sequence at least 10 nucleotides in length. In
one embodiment, a polyAC sequence at least 10 nucleotides in
length. In one embodiment, a polyAG sequence at least 10
nucleotides in length. In one embodiment, a polyAU sequence at
least 10 nucleotides in length. In one embodiment, a polyAI
sequence at least 10 nucleotides in length. In one embodiment, a
polyGC sequence at least 10 nucleotides in length. In one
embodiment, a polyGT sequence at least 10 nucleotides in length. In
one embodiment, a polyGU sequence at least 10 nucleotides in
length. In one embodiment, a polyGI sequence at least 10
nucleotides in length. In one embodiment, a polyCT sequence at
least 10 nucleotides in length. In one embodiment, a polyCU
sequence at least 10 nucleotides in length. In one embodiment, a
polyCI sequence at least 10 nucleotides in length. In one
embodiment, a polyTI sequence at least 10 nucleotides in
length.
Modified Linkages, Including ps and ps2
[0166] The present invention further provides an oligonucleotide,
wherein the modified linkages are selected from the group
consisting of phosphorothioate linkages, phosphorodithioate
linkages, and boranophosphate linkages.
[0167] The present invention further provides an oligonucleotide
wherein at least 50% of the internucleotidic linkages are modified
linkages. In one embodiment, wherein at least 80% of the
internucleotidic linkages are modified linkages. In one embodiment,
wherein at least 90% of the internucleotidic linkages are modified
linkages. In one embodiment, wherein all of the internucleotidic
linkages are modified linkages.
[0168] The present invention further provides an oligonucleotide,
wherein at least 50% of the internucleotidic linkages are
phosphorothioate linkages. In one embodiment, wherein at least 80%
of the internucleotidic linkages are phosphorothioate linkages. In
one embodiment, wherein at least 90% of the internucleotidic
linkages are phosphorothioate linkages. In one embodiment, wherein
all of the internucleotidic linkages are phosphorothioate
linkages.
[0169] The present invention further provides an oligonucleotide,
wherein at least 50% of the internucleotidic linkages are
phosphorodithioate linkages. In one embodiment, wherein at least
80% of the internucleotidic linkages are phosphorodithioate
linkages. In one embodiment, wherein all of the internucleotidic
linkages are phosphorodithioate linkages.
2'-Modifications, Combinations with Modified Linkages
[0170] The present invention further provides an oligonucleotide,
wherein said oligonucleotide comprises at least one phosphodiester
linkage. In one embodiment, wherein said oligonucleotide comprises
at least 10% phosphodiester linkages. In one embodiment, wherein
said oligonucleotide comprises at least 20% phosphodiester
linkages.
[0171] In one embodiment, wherein at least 50% of the nucleotides
in said oligonucleotide are modified at the 2'-position of the
ribose moiety. In one embodiment, wherein at least 60% of the
nucleotides in said oligonucleotide are modified at the 2'-position
of the ribose moiety. In one embodiment, wherein at least 70% of
the nucleotides in said oligonucleotide are modified at the
2'-position of the ribose moiety. In one embodiment, wherein at
least 80% of the nucleotides in said oligonucleotide are modified
at the 2'-position of the ribose moiety. In one embodiment, wherein
at least 90% of the nucleotides in said oligonucleotide are
modified at the 2'-position of the ribose moiety. In one
embodiment, wherein 100% of the nucleotides in said oligonucleotide
are modified at the 2'-position of the ribose moiety.
[0172] The present invention further provides an oligonucleotide,
wherein at least 50% of the internucleotidic linkages are modified
and at least 50% of the nucleotides in said oligonucleotide are
modified at the 2'-position of the ribose moiety. In one
embodiment, wherein at least 60% of the internucleotidic linkages
are modified and at least 60% of the nucleotides in said
oligonucleotide are modified at the 2'-position of the ribose
moiety. In one embodiment, wherein at least 70% of the
internucleotidic linkages are modified and at least 70% of the
nucleotides in said oligonucleotide are modified at the 2'-position
of the ribose moiety. In one embodiment, wherein at least 80% of
the internucleotidic linkages are modified and at least 80% of the
nucleotides in said oligonucleotide are modified at the 2'-position
of the ribose moiety. In one embodiment, wherein all of the
internucleotidic linkages are modified and all of the nucleotides
in said oligonucleotide are modified at the 2'-position of the
ribose moiety.
[0173] The present invention further provides an oligonucleotide,
wherein at least 15% of the nucleotides in said oligonucleotide
comprise 2'-OMe moieties at the 2'-position of the ribose moiety.
In one embodiment, wherein at least 20% of the nucleotides in said
oligonucleotide comprise 2'-OMe moieties at the 2'-position of the
ribose moiety. In one embodiment, wherein at least 30% of the
nucleotides in said oligonucleotide comprise 2'-OMe moieties at the
2'-position of the ribose moiety. In one embodiment, wherein at
least 50% of the nucleotides in said oligonucleotide comprise
2'-OMe moieties at the 2'-position of the ribose moiety. In one
embodiment, wherein at least 60% of the nucleotides in said
oligonucleotide comprise 2'-OMe moieties at the 2'-position of the
ribose moiety. In one embodiment, wherein at least 70% of the
nucleotides in said oligonucleotide comprise 2'-OMe moieties at the
2'-position of the ribose moiety. In one embodiment, wherein at
least 80% of the nucleotides in said oligonucleotide comprise
2'-OMe moieties at the 2'-position of the ribose moiety. In one
embodiment, wherein at least 90% of the nucleotides in said
oligonucleotide comprise 2'-OMe moieties at the 2'-position of the
ribose moiety. In one embodiment, wherein all of the nucleotides in
said oligonucleotide comprise 2'-OMe moieties at the 2'-position of
the ribose moiety.
[0174] Misc. Characteristics
[0175] The present invention further provides an oligonucleotide,
wherein said oligonucleotide is a concatemer consisting of two or
more oligonucleotide sequences joined by a linker.
[0176] The present invention further provides an oligonucleotide,
wherein said oligonucleotide is linked or conjugated at one or more
nucleotide residues, to a molecule modifying the characteristics of
the oligonucleotide to obtain one or more characteristics selected
from the group consisting of higher stability, lower serum
interaction, higher cellular uptake, higher viral protein
interaction, an improved ability to be formulated for delivery, a
detectable signal, higher antiviral activity, better
pharmacokinetic properties, specific tissue distribution, lower
toxicity.
[0177] The present invention further provides an oligonucleotide,
wherein said oligonucleotide is double stranded.
[0178] The present invention further provides an oligonucleotide,
wherein said oligonucleotide is double or single stranded and
comprises at least one base which is capable of hybridizing via
non-watson-crick interactions.
[0179] The present invention further provides an oligonucleotide,
wherein said oligonucleotide comprises a portion complementary to a
viral mRNA.
[0180] The present invention further provides an oligonucleotide,
wherein said oligonucleotide binds to one or more viral
components.
[0181] The present invention further provides an oligonucleotide,
wherein said oligonucleotide interacts with one or more host
components, wherein said interaction results in inhibition of viral
activity or production.
[0182] The present invention further provides an oligonucleotide,
wherein at least a portion of the sequence of said oligonucleotide
is derived from a viral genome.
[0183] The present invention further provides an oligonucleotide,
wherein at least a portion of the sequence of said oligonucleotide
is derived from a viral genome and has an antiviral activity that
is predominantly a non-sequence complementary mode of action.
[0184] The present invention further provides an oligonucleotide,
wherein at least a portion of the sequence of said oligonucleotide
is derived from a viral packaging sequence or other viral sequence
involved in an aptameric interaction.
[0185] The present invention further provides an oligonucleotide,
wherein at least a portion of the sequence of said oligonucleotide
is involved in an aptameric interaction with a viral component or a
host component or both.
Activity Levels
[0186] The present invention further provides an oligonucleotide,
wherein said oligonucleotide has an IC.sub.50 for a target virus of
0.10 .mu.m or less. In one embodiment, wherein said oligonucleotide
has an IC.sub.50 for a target virus of 0.05 .mu.m or less. In one
embodiment, wherein said oligonucleotide has an IC.sub.50 for a
target virus of 0.025 .mu.m or less. In one embodiment, wherein
said oligonucleotide has an IC.sub.50 for a target virus of 0.015
.mu.m or less.
Target Viruses
[0187] The present invention further provides an oligonucleotide,
wherein said oligonucleotide targets a DNA virus. In one
embodiment, an RNA virus. In one embodiment, a member of the
herpesviridae. In one embodiment, HSV-1. In one embodiment, HSV-2.
In one embodiment, CMV. In one embodiment, a member of the
hepadnaviridae In one embodiment, HBV. In one embodiment, a member
of the parvoviridae. In one embodiment, a member of the poxviridae.
In one embodiment, a member of the papillomaviridae. In one
embodiment, a member of the adenoviridae In one embodiment, a
member of the retroviridae In one embodiment, HIV-1. In one
embodiment, HIV-2 In one embodiment, a member of the
paramyxoviridae. In one embodiment, RSV. In one embodiment,
parainfluenza virus. In one embodiment, a member of the
bunyaviridae. In one embodiment, hantavirus In one embodiment, a
member of the picornaviridae In one embodiment, coxsackievirus. In
one embodiment, rhinovirus. In one embodiment, a member of the
flaviviridae In one embodiment, yellow fever virus In one
embodiment, dengue virus. In one embodiment, West Nile virus In one
embodiment, hepatitis C virus. In one embodiment, a member of the
filoviridae. In one embodiment, Ebola virus In one embodiment,
Marburg virus In one embodiment, a member of the orthomyxoviridae.
In one embodiment, influenza virus. In one embodiment, a member of
the togaviridae. In one embodiment, a member of the coronaviridae.
In one embodiment, a member of the reoviridae. In one embodiment, a
member of the rhabdoviridae. In one embodiment, a member of the
arenaviridae. In one embodiment, a member of the calciviridae. In
one embodiment, Varicella Zoster Virus. In one embodiment, Epstein
Barr Virus. In one embodiment, Herpesvirus 6A or 6B. In one
embodiment, a member of hepadnaviridae. In one embodiment, human
metapneumovirus. In one embodiment, Rift Valley fever virus. In one
embodiment, Crimean Congo Hemorrhagic Fever virus. In one
embodiment, Western Equine Encephalitis virus. In one embodiment,
lassa fever virus.
Oligonucleotide
[0188] The present invention further provides an oligonucleotide
comprising at least 20 linked nucleotides, wherein at least 80% of
the linkages are modified; and at least 80% of the nucleotides
comprise 2'-modifications of the ribose sugar moiety. In one
embodiment, this oligonucleotide has an antiviral activity.
[0189] In one embodiment, wherein at least 90% of the
internucleotidic linkages are modified In one embodiment, wherein
all of the internucleotidic linkages are modified. In one
embodiment, wherein at least 90% of the nucleotides comprise
2'-modifications of the ribose sugar. In one embodiment, wherein
all of the nucleotides comprise 2'-modifications of the ribose
sugar.
[0190] The present invention further provides an oligonucleotide,
wherein said 2'-modifications are 2'-OMe substitutions. In one
embodiment, wherein at least 90% of the nucleotides comprise 2'-OMe
substitutions. In one embodiment, wherein all of the nucleotides
comprise 2'-OMe substitutions.
[0191] The present invention further provides an oligonucleotide,
wherein said 2'-modifications are 2'-methoxyethoxy substitutions.
In one embodiment, at least 15% of the nucleotides comprise
2'-methoxyethoxy substitutions. In one embodiment, at least 50% of
the nucleotides comprise 2'-methoxyethoxy substitutions. In one
embodiment, at least 90% of the nucleotides comprise
2'-methoxyethoxy substitutions. In one embodiment, all of the
nucleotides comprise 2'-methoxyethoxy substitutions.
[0192] The present invention further provides an oligonucleotide,
wherein said oligonucleotide is at least 40 nucleotides in length.
In one embodiment, at least 50 nucleotides in length. In one
embodiment, at least 60 nucleotides in length. In one embodiment,
at least 80 nucleotides in length
[0193] The present invention further provides an oligonucleotide,
wherein said oligonucleotide is 30-40 nucleotides in length. In one
embodiment, 40-50 nucleotides in length. In one embodiment, 50-60
nucleotides in length. In one embodiment, 60-70 nucleotides in
length. In one embodiment, 70-80 nucleotides in length.
[0194] The present invention further provides an oligonucleotide,
wherein said oligonucleotide is free from self-complementary
sequences of greater than 5 contiguous nucleotides. In one
embodiment, greater than 10 contiguous nucleotides. In one
embodiment, greater than 20 contiguous nucleotides.
[0195] The present invention further provides an oligonucleotide,
wherein said oligonucleotide is free of catalytic activity.
Chain Moiety Limitations
[0196] The present invention further provides an oligonucleotide,
further comprising 1-4 non-nucleotide chain moieties.
Mixtures
[0197] The present invention further provides an oligonucleotide
mixture, comprising a mixture of at least two different antiviral
oligonucleotides of the invention. In one embodiment, at least ten
different antiviral oligonucleotides. In one embodiment, at least
100 different antiviral oligonucleotides. In one embodiment, at
least 1000 different antiviral oligonucleotides. In one embodiment,
at least 106 different antiviral oligonucleotides.
[0198] The present invention further provides a mixture, wherein a
plurality of said different oligonucleotides are at least 10
nucleotides in length. In one embodiment, at least 20 nucleotides
in length. In one embodiment, at least 30 nucleotides in length. In
one embodiment, at least 40 nucleotides in length. In one
embodiment, at least 50 nucleotides in length. In one embodiment,
at least 60 nucleotides in length. In one embodiment, at least 70
nucleotides in length. In one embodiment, at least 80 nucleotides
in length. In one embodiment, at least 120 nucleotides in
length.
Pharmaceutical Compositions
[0199] The present invention further provides an antiviral
pharmaceutical composition comprising a therapeutically effective
amount of at least one pharmacologically acceptable, antiviral
oligonucleotide, polypyrimidine or oligonucleotide mixture, wherein
the antiviral activity of said oligonucleotide or the
oligonucleotides in said mixture occurs principally by a sequence
independent mode of action; and a pharmaceutically acceptable
carrier.
[0200] The present invention further provides an antiviral
pharmaceutical composition, adapted for the treatment, control, or
prevention of a disease with a viral etiology.
[0201] The present invention further provides an antiviral
pharmaceutical composition, adapted for the treatment, control or
prevention of a prion disease.
[0202] The present invention further provides an antiviral
pharmaceutical composition, adapted for delivery by a mode selected
from the group consisting of intraocular, oral ingestion,
enterally, inhalation, cutaneous injection, subcutaneous injection,
intramuscular injection, intraperitoneal injection, intrathecal
injection, intratrachael injection, and intravenous injection.
[0203] The present invention further provides an antiviral
pharmaceutical composition, wherein said composition further
comprises a delivery system. In one embodiment, said delivery
system targets specific cells or specific tissues. In one
embodiment, said composition further comprises at least one other
antiviral drug in combination. In one embodiment, said composition
further comprises a non-nucleotide antiviral polymer in
combination. In one embodiment, said composition further comprises
an antiviral antisense oligonucleotide in combination. In one
embodiment, said comoposition further comprises an antiviral
RNAi-inducing oligonucleotide. In one embodiment, said antiviral
RNAi-inducing oligonucleotide is an siRNA.
[0204] The present invention further provides an antiviral
pharmaceutical composition, wherein said composition has an
IC.sub.50 for a target virus of 0.10 .mu.M or less. In one
embodiment, an IC.sub.50 for a target virus of 0.05 .mu.M or less.
In one embodiment, an IC.sub.50 for a target virus of 0.025 .mu.M
or less. In one embodiment, an IC.sub.50 for a target virus of
0.015 .mu.M or less.
[0205] Kits
[0206] The present invention further provides a kit comprising at
least one antiviral oligonucleotide, mixture, or antiviral
pharmaceutical composition in a labeled package, wherein the
antiviral activity of said oligonucleotide occurs principally by a
non-sequence complementary mode of action and the label on said
package indicates that said antiviral oligonucleotide can be used
against at least one virus.
[0207] The present invention further provides a kit, wherein said
kit contains a mixture of at least two different antiviral
oligonucleotides.
[0208] The present invention further provides a kit approved by a
regulatory agency for use in humans.
[0209] The present invention further provides a kit approved by a
regulatory agency for use in at least one non-human animal. In one
embodiment, said non-human animal is a primate In one embodiment,
said non-human animal is a feline In one embodiment, said non-human
animal is a bovine. In one embodiment, said non-human animal is an
ovine. In one embodiment, said non-human animal is a canine In one
embodiment, said non-human animal is a porcine. In one embodiment,
said non-human animal is an equine.
Method of Treatment
[0210] The present invention further provides a method for the
prophylaxis or treatment of a viral infection in a subject,
comprising administering to a subject in need of such a treatment a
therapeutically effective amount of at least one pharmacologically
acceptable oligonucleotide of the invention.
[0211] The present invention further provides use of at least one
oligonucleotide according to the invention, or pharmaceutical
composition according to the invention in the manufacture of a
medicament for the prophylaxis or treatment of a viral infection in
a subject.
[0212] In one embodiment, said subject is a human. In one
embodiment, said subject is a non-human animal. In one embodiment,
said non-human animal is a primate. In one embodiment, said
non-human animal is a feline. In one embodiment, said non-human
animal is a bovine. In one embodiment, said non-human animal is an
ovine. In one embodiment, said non-human animal is a canine. In one
embodiment, said non-human animal is a porcine. In one embodiment,
said non-human animal is an equine. In one embodiment, said subject
is a plant.
[0213] The present invention further provides use of at least one
oligonucleotide according to the invention, or pharmaceutical
composition according to the invention in the manufacture of a
medicament for the prophylactic treatment of cancer caused by
oncoviruses in a human or a non-human animal.
[0214] In one embodiment, said oligonucleotide is administered to a
human. In one embodiment, said oligonucleotide is administered to a
non-human animal. In one embodiment, said non-human animal is a
primate. In one embodiment, said non-human animal is a feline. In
one embodiment, said non-human animal is a bovine. In one
embodiment, said non-human animal is an ovine. In one embodiment,
said non-human animal is a canine. In one embodiment, said
non-human animal is a porcine. In one embodiment, said non-human
animal is an equine.
Polypyrimidine Oligo-Related
[0215] The present invention further provides an oligonucleotide
comprising at least 50% of pyrimidine residues. In one embodiment,
at least 80%. In one embodiment, at least 90%. In one embodiment,
only pyrimidine residues.
[0216] The present invention further provides an oligonucleotide
wherein the pyrimidine residues are cytosine residues. In one
embodiment, thymine residues. In one embodiment, cytosine or
thymine residues.
[0217] The present invention further provides an antiviral
pharmaceutical composition comprising a therapeutically effective
amount of at least one pharmacologically acceptable, polypyrimidine
oligonucleotide or polypyrimidine oligonucleotide mixture, wherein
the antiviral activity of said oligonucleotide or the
oligonucleotides in said mixture occurs principally by a sequence
independent mode of action; and a pharmaceutically acceptable
carrier. In one embodiment, said oligonucleotide comprises at least
one modified internucleotidic linkage.
[0218] In one embodiment, said composition is adapted for
administration to an acidic in vivo site.
[0219] In one embodiment, said composition further comprises a
penetration enhancer.
[0220] In one embodiment, said composition further comprises a
surfactant.
[0221] In one embodiment, said composition is in the form of a
powder.
[0222] In one embodiment, said composition is in the form of
granules.
[0223] In one embodiment, said composition is in the form of
microparticulates.
[0224] In one embodiment, said composition is in the form of
nanoparticulates.
[0225] In one embodiment, said composition is in the form of a
suspension or solution.
[0226] In one embodiment, said composition is in the form of an
emulsion.
[0227] In one embodiment, said composition is adapted for oral
administration.
[0228] In one embodiment, wherein said composition is adapted for
vaginal administration.
[0229] In one embodiment, said composition comprises at least one
polyC oligonucleotide.
[0230] In one embodiment, said composition comprises at least one
polyT oligonucleotide.
[0231] In one embodiment, said composition comprises at least one
polyCT oligonucleotide.
[0232] In one embodiment, said composition is approved for
administration to a human.
[0233] In one embodiment, said composition is approved for
administration to a mammal.
[0234] In one embodiment, said composition is approved for
administration to a non-mammal animal.
[0235] The present invention further provides use of a
pharmaceutical composition adapted for administration to an acidic
in vivo site, wherein said composition contains at least one
pharmacologically acceptable polypyrimidine oligonucleotide in the
manufacture of a medicament for the prophylaxis or treatment of a
viral infection in a subject.
[0236] In one embodiment, said subject is a human. In one
embodiment, said subject is a mammal. In one embodiment, said
subject is a non-mammal animal.
[0237] As described in the Background, a number of antisense
oligonucleotides (ONs) have been tested for antiviral activity.
However, such antisense ONs are sequence-specific, and typically
are about 16-20 nucleotides in length.
[0238] As demonstrated by the results in Examples 1 and 2, the
antiviral effect of random PS-ONs is not sequence specific.
Considering the volumes and concentrations of PS-ONs used in those
tests, it is almost theoretically impossible that a particular
random sequence is present at more than 1 copy in the mixture. This
means than there can be no antisense effect in these PS-ON
randomers. In the latter example, should the antiviral effect be
caused by the sequence-specificity of the PS-ONs, such effect would
thus have to be caused by only one molecule, a result that does not
appear possible. For example, for an ON randomer 40 bases in
length, any particular sequence in the population would
theoretically represent only 1/4.sup.40 or 4.1.times.10.sup.-41 of
the total fraction. Given that 1 mole=6.022.times.10.sup.23
molecules, and the fact that our largest synthesis is currently
done at the 15 micromole scale, all possible sequences will not be
present and also, each sequence is present most probably as only
one copy. Of course, one skilled in the art applying the teaching
of the present invention could also use ONs that have sequences of
such sequence specific ONs, but utilize the sequence independent
activity discovered in the present invention. Accordingly, the
present invention is not to be restricted to non-sequence
complementary ONs, but disclaims what has been disclosed in the
prior art regarding sequence-specific antisense and RNAi (e.g.,
siRNA) ONs for treating viral infections.
[0239] For applicable viruses (including, for example, those for
which data is described herein), as the size of the randomer
increases, so does its antiviral potency for lengths up to and even
exceeding 40 nucleotides. It should be pointed out that due to
limitations in current phosphoramidite-based oligonucleotide
synthesis, the larger PS-ONs (e.g., 80- and 120-mers) have a
significant contamination of fragments smaller than the desired
size. The weaker effects (on a per base basis) seen with larger
oligos (80 and 120 bp) may reflect the lower concentration of
full-length randomers in these populations and may also reflect a
decreased availability at the appropriate site. It may be possible
to achieve much larger increases in antiviral activity if larger
randomers (>40 bases) of reasonable purity (e.g., at least 75%
full length) are synthesized or purified, and/or if the delivery of
any of these ONs is facilitated by a delivery system, e.g., a
delivery system providing targeting or sustained release.
[0240] In the present invention, randomers (or other antiviral
oligonucleotides as described herein) may block viral replication
by several mechanisms, including but not limited to the following:
1. preventing the adsorption or receptor interaction of virions,
thus preventing infection, 2. doping the virus assembly or the
packaging of viral genomes into capsids (competing with viral DNA
or RNA for packaging), resulting in defective virions, 3.
disrupting and or preventing the formation of capsids during
packaging or the interaction of capsid proteins with other
structural proteins, resulting in inhibition of viral release or
causing the release of defective virions, 4. binding to key viral
components and preventing or reducing their activity, 5. binding to
key host components required for viral proliferation.
[0241] Without being limited on the mechanism by which the present
viral inhibition is achieved, as indicated above there are several
possible mechanisms that could explain and/or predict the
inhibitory properties of ONs against viral replication. The first
of these is that the general aptameric effect of ONs is allowing
for their attachment, either to proteins on the cell surface or to
viral proteins, preventing viral adsorption and fusion. The size
threshold for effect may be a result of a certain cumulative charge
required for interaction.
[0242] A second possible mechanism is that ONs may function within
the cell by preventing packaging and/or assembly of the virus. ONs
above a certain size threshold may compete or interfere with the
normal capsid/nucleic acid interaction, preventing the packaging of
a functional viral genome inside new viruses. Alternatively, ONs
may prevent the formation of a normal capsid, which could prevent
normal viral budding, alter viral stability, or prevent proper
virion disassembly upon internalization.
[0243] While the mechanism of action is not yet entirely clear,
assay results demonstrate that the present ONs can exhibit greater
efficacy in viral inhibition compared to the clinical correlates,
acyclovir, gancyclovir, Ribavirin, and protease inhibitors. ONs in
accordance with the present invention could thus be used for
treating or preventing viral infection. The viral infections
treated could be those caused by human, animal, and plant
viruses
[0244] Chemical modification of oligonucleotides can advantageously
be used to enhance the stability and/or activity of the present
antiviral oligonucleotides. Methoxylation and other modifications
at the 2'-position of the ribose on RNA have been shown to render
RNA stable to nucleases, to minimize the protein binding observed
with phosphorothioated nucleic acids and to increase the melting
temperature of these oligos with their target sequences. While 2'-O
methylation and other 2'-modifications are currently used to
improve the characteristics of antisense oligonucleotides,
oligonucleotides with such modfications do not elicit RNase H
activity when present on every ribose, making completely
2'-modified oligonucleotides poorer candidates for antisense
activity. This has resulted in the use of 2'-O methyl and other 2'
modification "gapmers" which contain 2' modifications only at the
extremities of the oligonucleotide, thus retaining the ability of
the oligo to activate RNase H. To our knowledge, there is no report
of a non-sequence specific antiviral oligonucleotide with
phosphorothioate linkages and ribonucleotides such as 2'-O-methyl
or other 2' modification on each ribose sugar in the
oligonucleotide.
[0245] As described herein, we had found that the 40 base PS-ON
randomer is a potent inhibitor of several different viruses. We
suggest the non-limiting hypothesis that the thioated backbone
imparts an increased hydrophobic character to the ON randomer,
which may allow it to interact with hydrophobic domains in viral
fusion proteins. These hydrophobic domains are believed to be
essential for the membrane fusion activity of many different
viruses including HSV, HIV, influenza, RSV, and Ebola. In the case
of HIV, such hydrophobic domain has been used as a target for the
development of fusion inhibitors.
[0246] Thus, the incorporation of phosphorothioate linkages and
ribonucleotide modifications, including 2'-O-methyl and other 2'
sugar modifications, into oligonucleotides of this invention, is
useful for improving characteristics of non-sequence specific
antiviral oligonucleotides. Results demonstrate that modification
at the '2-position of each ribose of PS-ONs does not significantly
alter their antiviral activity, but that such modification reduces
the general interaction of the PS-ONs with serum proteins and
renders them significantly more resistant to low pH. These
properties promise to increase the pharmacokinetic performance and
reduce the toxic side effects normally seen with PS-ONs. For
example, their pH resistance make them more suitable for oral
delivery. Also their lowered interaction with serum proteins
promises to improve their pharmacokinetic behaviour without
affecting their antiviral activity. Thus, oligonucleotides having
each linkage phosphorothioated and each ribonucleotide modified at
the 2'-position of the ribose, e.g., 2'-O-methyl modifications,
have antiviral activity but do not trigger RNase H activity, a
property desirable for traditional antisense oligonucleotide but
completely dispensable for the activity described in this present
invention. Results also demonstrate that modifications at the
'2-position of each ribose of PS-ONs renders the ON more resistant
to nucleases in comparison with a PS-ON comprising the same
modifications but only at both ends (gapmer). Gapmers are
preferentially used in the antisense technology. Nuclease
resistance of PS-ONs including modifications at the '2-position of
each ribose should display beneficial properties, such as improved
pharmakokinetics and/or oral availability.
[0247] In addition, while PS-ONs that include modifications at the
2'-position of each ribose show desirable characteristics, PS-ONs
with substantial numbers of modifications at the 2'-position of
riboses would also display desirable characteristic, e.g.,
modification at at least 50% of the riboses and more preferably 80%
or even more.
[0248] As described above, the activity of the present
oligonucleotides does not target any nucleic acid by hybridization
since randomers, for example, have no antisense activity. Thus, we
believe that the oligonucleotides target proteins. Since the
addition of 2'-O-methyl ribose modifications to phosphorothioate
oligonucleotides lowers the protein binding activity (Kandimalla et
al., 1998, Bioorganic Med Chem Lett. 8:2103-2108; Mou et al., 2002,
Nucleic Acids Res. 30:749-758), it would be expected that these
modifications would lower antiviral activity. Unexpectedly, we
found that addition of 2'-O-methyl ribose modifications to
phosphorothioate oligonucleotides does not affect the antiviral
activity.
[0249] Assay results for a number of different oligonucleotides are
described herein. Unless otherwise indicated, the tested
oligonucleotides have 2'-H moieties (2'-deoxy) and are thus ODNs.
However, the sequence independent activities of the present
invention are not limited to oligonucleotides with such 2'-H
moieties, but is also present for oligos containing nucleotides
having 2'-OH moieties as well as other 2'-modifications, for
example, 2'-O-methyl and 2'-fluoro.
[0250] The description herein utilizes a number of abbreviations,
including the following:
[0251] Selected Abbreviations
[0252] ON: Oligonucleotide
[0253] ODN: Oligodeoxynucleotide
[0254] PS: Phosphorothioate
[0255] PS2: Phosphorodithioate
[0256] PRA: Plaque reduction assay
[0257] PFU: Plaque forming unit
[0258] INF A: Influenza A virus
[0259] HIV: Human immunodeficiency virus (includes both HIV-1 and
HIV-2 if not specified)
[0260] HSV: Herpes simplex virus (includes both HSV-2 and HSV-3 if
not specified)
[0261] RSV: Respiratory syncytial virus
[0262] COX: Coxsackievirus
[0263] DHBV: Duck hepatitis B virus
[0264] Broad Spectrum Antiviral Activity
[0265] According to the conclusions discussed above and the data
reported herein, it appeared that random ONs and ON randomers could
have broad-spectrum antiviral activity with viruses where assembly
and/or packaging and/or encapsidation of the viral genome is a
required step in replication. Therefore to test this hypothesis,
several PS-ON randomers of different sizes were selected to be
tested in cellular models of various viral Infections. A number of
such tests are described herein in the Examples, including tests
with CMV, HIV-1, RSV, Coxsackie virus B2, DHBV, Hantavirus,
Parainfluenza virus, and Vaccinia virus, as well as the tests on
HSV-1 and HSV-2 described in Examples 1 and 2.
[0266] Conclusions on Broad Spectrum Antiviral Activity
[0267] The efficacy studies with different viruses demonstrate that
random ONs and randomers display inhibitory properties against a
variety of different viruses. Moreover, these studies support the
conclusion that larger randomers display greater efficacy for viral
inhibition than smaller randomers. This suggests a common size
and/or charge dependent mechanism for the random ONs or ON
randomers activity in all encapsidating viruses.
[0268] While HSV and CMV are both double-stranded DNA viruses of
the herpesviridae family, HIV is a RNA virus from the retroviridae,
and RSV a RNA virus from the paramyxoviridae. Given the fact that
ON randomers can inhibit viral function in a variety of different
viruses, without being limited to the mechanisms listed, as
discussed above the following mechanisms are reasonable: A) ONs/ON
randomers are inhibiting viral infection via an aptameric effect,
preventing viral fusion with the plasma membrane; and/or B) ONs/ON
randomers are preventing or doping the assembly of virions or the
packaging of viral DNA within capsids resulting in defective
virions; and/or C) ONs/ON randomers are interfering with host
proteins or components required in the assembly and/or packaging
and/or gene expression of the virus.
[0269] Requirement for Antiviral Activity
[0270] Since a randomized DNA sequence seems to be sufficient for
viral inhibition, it was interesting to see if antiviral activity
could be maintained in the absence of the phosphorothioate
modification and also if the efficacy was augmented by either
choosing a random sequence or a specific sequence found in the
viral genome.
[0271] Accordingly, DNA and RNA modifications were investigated
with respect to their effect on the antiviral efficacy of the
randomers. Since randomers work via a sequence independent, e.g.,
non-sequence complementary, mechanism, these experiments were
designed to test the slight changes in nucleic acid conformation
and charge distribution on antiviral efficacy.
[0272] To test if ONs with different nucleotide/nucleoside
modifications could inhibit HSV-1, REP 2024, 2026, 2059, and 2060
were tested in the HSV-1 PRA as described in the Examples. REP 2024
(a PS-ON with a 2-0-Methyl modification to the ribose on 4 bases at
both termini of the ON), REP 2026 (a PO-ON with methylphosphonate
modifications to the linkages between the 4 bases at both termini
of the ON), REP 2059 (RNA PS-ON randomer 20 bases in length), and
REP 2060 (RNA PS-ON randomer 30 bases in length) showed anti-HSV-1
activity. The assay was conducted as a plaque reduction assay in
VERO cells using HSV-1 (strain KOS). The PS-ONs were tested in
increasing concentrations. IC.sub.50 values calculated from linear
regressions were 0.14, 3.41, 1.36, and 0.80 respectively.
[0273] In the latter example, should the antiviral effect be caused
only by the ONs consisting of DNA phosphorothioate backbone, such
effect would thus be caused by only one molecule. But other
backbones and modifications gave positive antiviral activity. Of
course, one skilled in the art applying the teaching of the present
invention could also use different chemistry ONs. A modification of
the ON, such as, but not limited to, a phosphorothioate
modification, appears to be beneficial for antiviral activity. This
is most likely due to the needed charge of ONs and/or the
requirement for stabilization of DNA both in the media and
intracellularly, and it may also be due to the chirality of the
PS-ONs.
[0274] Compound REP 2026 showed an antiviral activity while having
a central portion comprising unmodified PO-nucleotides and 4
methylphosphonate linkages at both termini protecting from
degradation. This indicates that PO-ONs can be used as antivirals
while protected from degradation. This protection can be achieved
by modifying nucleotides at termini and/or by using a suitable
delivery system as described later.
[0275] In general, the sequence composition of the DNA used has
little effect on the overall efficacy, whether randomer, random
sequence or a specific HSV-1 sequence. However, at intermediate
lengths, HSV-1 sequence was almost 3.times. more potent than a
random sequence. This data suggests that while specific antisense
functionality exists for specific HSV sequences, sequence
independent mechanism (the non-antisense mechanism) elucidated
herein may represent the predominant part of this activity. Indeed,
as the ON grows to 40 bases, essentially all of the antiviral
activity can be attributed to a sequence independent (e.g.,
non-antisense) effect.
[0276] Lower Toxicity of Randomer
[0277] One goal of using an ON randomer is to lower the toxicity.
It is known that different sequences may trigger different
responses in the animal, such as general toxicity, interaction with
serum proteins, and interaction with immune system (Monteith et al
(1998) Toxicol Sci 46:365-375). The mixture of ONs may thus
decrease toxic effects because the level of any particular sequence
will be very low, so that no significant interaction due to
sequence or nucleotide composition is likely.
[0278] Pharmaceutical Compositions
[0279] The ONs of the invention may be in the form of a therapeutic
composition or formulation useful for treating (or prophylaxis of)
viral diseases, which can be approved by a regulatory agency for
use in humans or in non-human animals, and/or against a particular
virus or group of viruses. These ONs may be used as part of a
pharmaceutical composition when combined with a physiologically
and/or pharmaceutically acceptable carrier. The characteristics of
the carrier may depend on the route of administration. The
pharmaceutical composition of the invention may also contain other
active factors and/or agents which enhance activity.
[0280] Administration of the ONs of the invention used in the
pharmaceutical composition or formulation or to practice the method
of treating an animal can be carried out in a variety of
conventional ways, such as intraocular, oral ingestion, enterally,
inhalation, or cutaneous, subcutaneous, intramuscular,
intraperitoneal, intrathecal, intratracheal, or intravenous
injection.
[0281] The pharmaceutical composition or oligonucleotide
formulation of the invention may further contain other
chemotherapeutic drugs for the treatment of viral diseases, such
as, without limitation, Rifampin, Ribavirin, Pleconaryl, Cidofovir,
Acyclovir, Pencyclovir, Gancyclovir, Valacyclovir, Famciclovir,
Foscarnet, Vidarabine, Amantadine, Zanamivir, Oseltamivir,
Resquimod, antiproteases, pegylated interferon (Pegasys.TM.) anti
HIV proteases (e.g. lopinivir, saquinivir, amprenavir, HIV fusion
inhibitors, nucleotide HIV RT inhibitors (e.g., AZT, Lamivudine,
Abacavir), non-nucleotide HIV RT inhibitors, Doconosol,
Interferons, Butylated Hydroxytoluene (BHT) and Hypericin. Such
additional factors and/or agents may be included in the
pharmaceutical composition, for example, to produce a synergistic
effect with the ONs of the invention.
[0282] The pharmaceutical composition or oligonucleotide
formulation of the invention may further contain a polymer, such
as, without restriction, polyanionic agents, sulfated
polysaccharides, heparin, dextran sulfate, pentosan polysulfate,
polyvinylalcool sulfate, acemannan, polyhydroxycarboxylates,
cellulose sulfate, polymers containing sulfonated benzene or
naphthalene rings and naphthalene sulfonate polymers, acetyl
phthaloyl cellulose, poly-L-lysine, sodium caprate, cationic
amphiphiles, cholic acid. Polymers are known to affect the entry of
virions in cells by, in some cases, binding or adsorbing to the
virion itself. This characteristic of antiviral polymers can be
useful in competing with ONs for the binding, or adsorption to the
virion, the result being an increased intracellular activity of the
ONs compared to its extracellular activity.
[0283] Exemplary Lipid Encapsulation and Delivery
[0284] Although PS-ONs (as well as oligonucleotides with other
modified linkages) are more resistant to endogenous nucleases than
natural phosphodiesters, they are not completely stable and are
slowly degraded in blood and tissues. A limitation in the clinical
application of PS oligonucleotide drugs is their propensity to
activate complement on i.v. administration. In general, liposomes
and other delivery systems enhance the therapeutic index of drugs,
including ONs, by reducing drug toxicity, increasing residency time
in the plasma, and delivering more active drug to disease tissue by
extravasation of the carriers through hyperpermeable vasculature.
Moreover in the case of PS-ON, lipid encapsulation prevents the
interaction with potential protein-binding sites while in
circulation (Klimuk et al. (2000) J Pharmacol Exp Ther
292:480-488).
[0285] According to our results described herein, an approach is to
use a delivery system such as, but without restriction, lipophilic
molecules, polar lipids, liposomes, monolayers, bilayers, vesicles,
programmable fusogenic vesicles, micelles, cyclodextrins, PEG,
iontophoresis, powder injection, and nanoparticles (such as PIBCA,
PIHCA, PHCA, gelatine, PEG-PLA) for the delivery of ONs described
herein and/or antisense and siRNA oligonucleotides. Use of such
delivery systems can, without limitation, provide one or more of
the following benefits: lower the toxicity of the active compound
in animals and humans, lower the IC.sub.50, increase the duration
of action from the standpoint of drug delivery, and protect the
oligonucleotides from non-specific binding with serum proteins.
[0286] Thus, we have shown that the antiviral activity of PS-ON
randomers increases with increasing size. Moreover this activity is
correlated with increased affinity for viral proteins (in a viral
lysate). Since it is well known in the art that the
phosphorothioate modification increases the affinity of protein-DNA
interaction, we tested the ability of increasingly larger PS-ON
randomers to bind to fetal bovine serum (FBS) using the same
FP-based assay used for measuring interaction with viral lysates.
In this assay, 250 ug of non-heat inactivated FBS was complexed
with a fluorescently labeled 20 base PS-ON randomer, under
conditions where the binding (mP value) was saturated. Unlabelled
PS-ON randomers of increasing size (REP 2003, REP 2004, REP 2006
and REP 2007) were used to compete the interaction of FBS with the
labeled bait. The results of this test clearly show that as the
size of the PS-ON randomer increases, so does its affinity for FBS.
This result suggests that the most highly active anti-viral PS-ONs
will also be the ones to bind with the highest affinity to
proteins.
[0287] However, it is known in the art that one of the main
therapeutic problems for phosphorothioate antisense
oligonucleotides is their side effects due mainly to an increased
interaction with proteins (specifically with serum proteins) as
described by Kandimalla and co-workers (Kandimalla et al. (1998)
Bioorg. Med. Chem. Lett. 8:2103-2108). Therefore, in some cases it
may be beneficial to use a suitable delivery system capable of
delivering antiviral ONs to the site of action while preventing
their interaction with serum proteins. In addition, it may be
beneficial to use suitable delivery systems for combination use of
the present sequence independent ONs with other types of ant-viral
ONs such as antisense oligonucleotides and siRNAs.
[0288] To demonstrate certain effects of a delivery system, we
tested two different delivery technologies which are liposomal
based; Cytofectin and DOTAP. We measured the protection of REP2006
from serum protein interactions by DOTAP and Cytofectin in our in
vitro FP-based interaction assay. Unencapsulated REP 2006 was able
to compete bound fluorescent oligo from serum but when REP 2006 was
encapsulated with either DOTAP or Cytofectin it was no longer able
to compete for serum binding. These data suggest that encapsulation
protects oligos from serum interaction and will result in better
pharmacokinetic behaviour with fewer side effects.
[0289] We also measured the delivery of the PS-ON randomer REP 2006
(encapsulated with either Cytofectin or DOTAP) into 293A cells in
the presence of high concentrations of serum (50%) by measuring the
intracellular concentration of labeled REP 2006 by fluorometry.
These results show that such delivery agents increase the
intracellular concentration of REP 2006, and also that, in the case
of DOTAP, the levels of intracellular REP 2006 after 24 hours were
markedly increased. Finally, we measured the protection of REP2006
from serum protein interactions by DOTAP and cytofectin in our in
vitro FP-based interaction assay. Unencapsulated REP 2006 was able
to compete bound fluorescent oligo from serum but when REP 2006 was
encapsulated with either DOTAP or cytofectin it was no longer able
to compete for serum binding. These data suggest that encapsulation
protects oligos from serum interaction and will result in better
pharmacokinetic behaviour with fewer side effects.
[0290] Similarly demonstrating the effect of lipid encapsulation of
oligonucleotides, we monitored the uptake of an additional PS-ON
randomer by exposing cultured cells to fluorescently labeled
randomers and then examined the fluorescence intensity in lysed
cells after two rounds of washing. The cellular uptake of cells
exposed to 250 nM REP 2004-FL was tested with no delivery and after
encapsulation in one of the following lipid based delivery systems;
Lipofectamine.TM. (Invitrogen), Polyfect.TM. (Qiagen) and
Oligofectamine.TM. (Invitrogen). After 4 hours, cells were washed
twice with PBS and lysed using MPER lysis reagent (PROMEGA). The
relative fluorescence yield from equivalent numbers of exposed
cells with and without lipid system was detected. We observed that
in the presence of all three agents tested, there was a significant
increase in the intracellular PS-ON concentration compared to no
delivery.
[0291] In keeping with the test results, the use of a delivery
system can serve to protect oligonucleotides from serum
interactions, reducing side effects and increasing tissue
distribution and/or can significantly increase the intracellular
delivery of ONs.
[0292] Another potential benefit in using a delivery system is to
protect the ONs from interactions, such as adsorption, with
infective virions in order to prevent amplification of viral
infection through different mechanisms such as increased cellular
penetration of virions.
[0293] Another approach is to accomplish cell specific delivery by
associating the delivery system with a molecule(s) that will
increase affinity with specific cells, such molecules being without
restriction antibodies, receptor ligands, vitamins, hormones and
peptides.
[0294] Additional options for delivery systems are provided
below.
[0295] Linked ON
[0296] In certain embodiments, ONs of the invention are modified in
a number of ways without compromising their ability to inhibit
viral replication. For example, the ONs are linked or conjugated,
at one or more of their nucleotide residues, to another moiety.
Thus, modification of the oligonucleotides of the invention can
involve chemically linking to the oligonucleotide one or more
moieties or conjugates which enhance the activity, cellular
distribution, increase transfer across cellular membranes
specifically or not, or protecting against degradation or
excretion, or providing other advantageous characteristics. Such
advantageous characteristics can, for example, include lower serum
interaction, higher viral-protein interaction, the ability to be
formulated for delivery, a detectable signal, improved
pharmacokinetic properties, and lower toxicity. Such conjugate
groups can be covalently bound to functional groups such as primary
or secondary hydroxyl groups. For example, conjugate moieties can
include a steroid molecule, a non-aromatic lipophilic molecule, a
peptide, cholesterol, bis-cholesterol, an antibody, PEG, a protein,
a water soluble vitamin, a lipid soluble vitamin, another ON, or
any other molecule improving the activity and/or bioavailability of
ONs.
[0297] In greater detail, exemplary conjugate groups of the
invention can include intercalators, reporter molecules,
polyamines, polyamides, polyethylene glycols, polyethers, SATE,
t-butyl-SATE, groups that enhance the pharmacodynamic properties of
oligomers, and groups that enhance the pharmacokinetic properties
of oligomers. Typical conjugate groups include cholesterols,
lipids, phospholipids, biotin, phenazine, folate, phenanthridine,
anthraquinone, acridine, fluoresceins, rhodamines, coumarins,
fluorescent nucleobases, and dyes.
[0298] Groups that enhance the pharmacodynamic properties, in the
context of this invention, include groups that enhance oligomer
resistance to degradation and/or protect against serum interaction.
Groups that enhance the pharmacokinetic properties, in the context
of this invention, include groups that improve oligomer uptake,
distribution, metabolism or excretion. Exemplary conjugate groups
are described in International Patent Application PCT/US92/09196,
filed Oct. 23, 1992, which is incorporated herein by reference in
its entirety.
[0299] Conjugate moieties can include but are not limited to lipid
moieties such as a cholesterol moiety (Letsinger et al., Proc.
Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan
et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether,
e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci.,
1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let.,
1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl.
Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g.,
dodecandiol or undecyl residues (Saison-Behmoaras et at., EMBO J.,
1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259,
327-330; Svinarchuk et at., Biochimie, 1993, 75, 49-54), a
phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium
1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et at.,
Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids
Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol
chain (Manoharan et at., Nucleosides & Nucleotides, 1995, 14,
969-973), or adamantane acetic acid (Manoharan et at, Tetrahedron
Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et at.,
Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine
or hexylaminocarbonyl-oxycholesterol moiety (Crooke et al., J.
Pharmacol Exp. Ther., 1996, 277, 923-937.
[0300] The present oligonucleotides may also be conjugated to
active drug substances, for example without limitation, aspirin,
warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen,
ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine,
2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a
benzothiadiazide, chlorothiazide, a diazepine, indomethicin, a
barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an
antibacterial or an antibiotic.
[0301] Exemplary U.S. patents that describe the preparation of
exemplary oligonucleotide conjugates include, for example, U.S.
Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313;
5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584;
5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439;
5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779;
4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013;
5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136;
5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873;
5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475;
5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481;
5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and
5,688,941, each of which is incorporated by reference herein in its
entirety.
[0302] Another approach is to prepare antiviral ONs as lipophilic
pro-oligonucleotides by modification with enzymatically cleavable
charge neutralizing adducts such as s-acetylthio-ethyl or
s-pivasloylthio-ethyl (Vives et al., 1999, Nucl Acids Res 27:
4071-4076). Such modifications have been shown to increase the
uptake of ONs into cells, and therefore are beneficial for ONs that
are active intracellularly.
[0303] Design of Non-Specific ONs
[0304] In another approach, an antiviral ON demonstrating low,
preferably the lowest possible, homology with the human (or other
subject organism) genome is designed. The goal is to obtain an ON
that will show the lowest toxicity due to interactions with human
or animal genome sequence(s) and mRNAs. The first step is to
produce the desired length sequence of the ON, e.g., by aligning
nucleotides A, C, G, T in a random fashion, manually or, more
commonly, using a computer program. The second step is to compare
the ON sequence with a library of human sequences such as GenBank
and/or the Ensemble Human Genome Database. The sequence generation
and comparison can be performed repetitively, if desired, to
identify a sequence or sequences having a desired low homology
level with the subject genome. Desirably, the ON sequence is at the
lowest homology possible with the entire genome, while also
preferably minimizing self interaction.
[0305] Non-Specific ONs with Antisense Activity
[0306] In another approach, an antiviral non-specific sequence
portion(s) is/are coupled with an antisense sequence portion(s) to
increase the activity of the final ON. The non-specific portion of
the ONs is described in the present invention. The antisense
portion is complementary to a viral mRNA.
[0307] Non-Specific ONs with a G-Rich Motif Activity
[0308] In another approach, an antiviral non-specific sequence
portion(s) is/are coupled with a motif portion(s) to improve the
activity of the final ON. The non-specific portion of the ON is
described in the present invention. The motif portion can, as
non-limiting examples, include, CpG, Gquartet, and/or CG that are
described in the literature as stimulators of the immune system.
Agrawal et al. (2001) Curr. Cancer Drug Targets 3:197-209.
[0309] Non-Watson-Crick ONs
[0310] Another approach is to use an ON composed of one type or
more of non-Watson-Crick nucleotides/nucleosides. Such ONs can
mimic PS-ONs with some of the following characteristics similar to
PS-ONs: a) the total charge; b) the space between the units; c) the
length of the chain; d) a net dipole with accumulation of negative
charge on one side; e) the ability to bind to proteins; f) the
ability to bind viral proteins, g) the ability to penetrate cells,
h) an acceptable therapeutic index, i) an antiviral activity. The
ON has a preferred phosphorothioate backbone but is not limited to
it. Examples of non-Watson-Crick nucleotides/nucleosides are
described in Kool, 2002, Acc. Chem. Res. 35:936-943; and Takeshita
et al., (1987) J. Biol. Chem. 262:10171-10179 where ONs containing
synthetic abasic sites are described.
[0311] Antiviral Polymer
[0312] Another approach is to use a polymer mimicking the activity
of phosphorothioate ONs. As described in the literature, several
anionic polymers were shown to have antiviral inhibitory activity.
These polymers belong to several classes: (1) sulfate esters of
polysaccharides (dextrin and dextran sulfates; cellulose sulfate);
(2) polymers containing sulfonated benzene or naphthalene rings and
naphthalene sulfonate polymers; (3) polycarboxylates (acrylic acid
polymers); and acetyl phthaloyl cellulose (Neurath et al. (2002)
BMC Infect Dis 2:27); and (4) abasic oligonucleotides (Takeshita et
al., 1987, J. Biol. Chem. 262:10171-10179). Other examples of
non-nucleotide antiviral polymers are described in the literature.
The polymers described herein mimic PS-ONs described in this
invention and have the following characteristics similar to PS-ONs:
a) the length of the chain; b) a net dipole with accumulation of
negative charge on one side; c) the ability to bind to proteins; d)
the ability to bind viral protein, e) an acceptable therapeutic
index, f) an antiviral activity. In order to mimic the effect of a
PS-ON, the antiviral polymer may preferably be a polyanion
displaying similar space between its units as compared to a PS-ON.
It may also have the ability to penetrate cells alone or in
combination with a delivery system.
[0313] Antiviral Activity of Double-Stranded PS-ONs
[0314] A random sequence (REP 2017) and its complement (either PS
modified or unmodified) are fluorescently labeled as described
elsewhere and tested for their ability to bind to purified HSV-1
and HIV-1 proteins by fluorescence polarization as described in the
present invention. Hybridization was verified by acrylamide gel
electrophoresis. Unmodified REP 2017 (2017U), either single (ss) or
double stranded (ds), had no binding activity in either HSV-1 or
HIV-1 lysates. However, PS modified REP 2017, either single
stranded or double stranded, was capable of HSV-1 and HIV-1
interaction.
[0315] According to our results described herein, an approach is to
use double stranded ONs as effective antiviral agents.
Preferentially such ONs have a phosphorothioate backbone but may
also have other and/or additional modifications which increase
antiviral activity and/or stability and/or delivery characteristics
as described herein for single stranded ONs.
[0316] In Vitro Assay for Drug Discovery
[0317] An in vitro assay is developed based on fluorescence
polarization to measure the ability of PS-ONs to bind to viral
components, e.g., viral proteins. When a protein (or another
interactor) binds to the fluorescently labeled bait, the three
dimensional tumbling of the bait in solution is retarded. The
retardation of this tumbling is measured by an inherent increase in
the polarization of excited light from the labeled bait. Therefore,
increased polarization (reported as a dimensionless measure [mP])
is correlated with increased binding.
[0318] One methodology is to use as bait a PS-ON randomer labeled
at the 3' end with FITC using an inflexible linker
(3'-(6-Fluorescein) CPG). This PS-ON randomer is diluted to 2 nM in
assay buffer (10 mM Tris, pH7.2, 80 mM NaCl, 10 mM EDTA, 100 mM
b-mercaptoethanol and 1% tween 20). This oligo is then mixed with
an appropriate interactor. In this case, we use lysates of sucrose
gradient purified HSV-1 (strain MacIntyre), HIV-1 (strain Mn) or
RSV (strain A2) suspended in 0.5M KCl and 0.5% Triton X-100 (HSV-1
and HIV-1) or 10 mM Tris, pH7.5, 150 mM NaCl, 1 mM EDTA and 0.1%
Triton X-100 (RSV). Following bait interaction, the complexes are
challenged with various unlabelled PS-ONs to assess their ability
to displace the bait from its complex.
[0319] In a preliminary test with three baits of different sizes; 6
(REP 2032-FL), 10 (REP 2003-FL) and 20 bases (REP 2004-FL), the
baits were tested for their ability to interact with HSV-1, HIV-1,
and RSV lysates. Viral lysate binding to baits of different sizes
was determined by fluorescence polarization. In the presence of any
of the viral lysates the degree of binding was dependent on the
size of the bait used, with 2004-FL displaying the largest shift in
mP (binding) in the presence of viral lysate. We note that this is
similar to the size dependent antiviral efficacy of PS-ON
randomers. This bait was then used to assess the ability of PS-ONs
of different sizes to compete the interaction of the bait with the
lysate.
[0320] The interaction of REP 2004-FL with HSV-1, HIV-1, and RSV
lysates was challenged with PS-ONs of increasing size.
Determination of affinity of PS-ON randomers for the viral lysates
was detected by fluorescence polarization. Using REP 2004-FL as the
bait, complex formation with HSV-1 lysate, HIV-1 lysate, or RSV
lysate was challenged with increasing concentrations of REP 2003,
REP 2004, REP 2006 or REP 2007. For each viral lysate tested, we
note that REP 2003 is unable to compete the bait away from the
lysate. The bait interaction was very strong as revealed by the
relatively weak competition elicited by the REP 2004 (unlabeled
bait) competitor. However, it was observed that as the size of the
competitor PS-ON increased above 20 bases, its ability to displace
the bait became more robust. This indicates an increased affinity
to protein components in the viral lysate as the PS-ON randomer
size increases. This phenomenon mirrors the increased antiviral
activity of larger PS-ON randomers against HSV-1, HSV-2, CMV, HIV-1
and RSV.
[0321] The similarity between the efficacy in bait competition and
antiviral activity of PS-ON randomers indicates that this assay
paradigm is a good predictor of antiviral activity. This assay is
robust, easy to perform and very stable, making it a very good
candidate for a high throughput screen to identify novel antiviral
molecules based not on specific target identification but on their
ability to interact with one or more components, e.g., viral
proteins.
[0322] While the exemplary method described herein utilizes
fluorescence polarization to measure interaction with the viral
lysate, numerous techniques are known in the art for monitoring
protein interactions, and can be used in the present methods. These
include without restriction surface plasmon resonance, fluorescence
resonance energy transfer (FRET), enzyme linked immunosorbent assay
(ELSIA), gel electrophoresis (to measure mobility shift),
isothermal titration and differential scanning microcalorimetry and
column chromatography. These other different techniques can be
applied to measure the interaction of ONs with a viral lysate or
component, and thus can be useful in screening for compounds which
have anti-viral activity.
[0323] The method described herein is used to screen for novel
compounds from any desired source, for example, from a library
synthesized by combinatorial chemistry or isolated by purification
of natural substances. It can be used to a) determine appropriate
size, modifications, and backbones of novel ONs; b) test novel
molecules including novel polymers; predict a particular virus'
susceptibility to novel ONs or novel compounds; or d) determine the
appropriate suite of compounds to maximally inhibit a particular
virus.
[0324] The increased lysate affinity with larger sized PS-ON
randomers suggests that the antiviral mechanism of action of PS-ON
randomers is based on an interaction with one or more viral protein
components which prevents either the infection or correct
replication of virions. It also suggests that this interaction is
charge (size) dependent and not dependent on sequence. As these
PS-ON randomers have a size dependent activity across multiple
viruses spanning several different families, we suggest that PS-ON
randomers interfere with common, charge dependent protein-protein
interactions, protein-DNA/RNA interactions, and/or other
molecule-molecule interactions. These interactions can include (but
are not limited to): [0325] The interaction between individual
capsid subunits during capsid formation. [0326] The interaction
between the capsid/nucleocapsid protein and the viral genome.
[0327] The interaction between the capsid and glycoproteins during
budding. [0328] The interaction between glycoproteins and their
receptors during infection. [0329] The interaction between other
key viral components involved in viral replication.
[0330] These multiple, simultaneous inhibitions of protein-protein
interactions represent a novel mechanism for antiviral
inhibition.
[0331] Effect of PS-ON Sequence Composition on Viral Lysate
Interaction
[0332] We monitored the ability of PS-ONs of different sequences to
interact with several viral lysates. In each case, a 20-mer PS-ON
is labeled at the 3' end with FITC as previously described herein.
The PS-ONs tested consisted of A20, T20, G20, C20, AC10, AG10,
TC10, TG10, REP 2004 and REP 2017. Each of these sequences is
diluted to 4nM in assay buffer and incubated in the presence of lug
of HSV-1, HIV-1 or RSV lysate. Interaction is measured by
fluorescence polarization.
[0333] The profile of interaction with all sequences tested is
similar in all viral lysates, indicating that the nature of the
binding interaction is very similar. The ability of 20-mer PS-ONs
of different sequence compositions (A20, C20, G20, T20, AC10, AG10,
TC10, TG10, REP2004, REP2017) to bind to viral lysates was measured
by fluorescence polarization. PS-ONs 3' labeled with FITC were
incubated in the presence of lug of HSV-1, HIV-1 or RSV lysates.
Within each lysate, the PS-ONs of uniform composition (A20, G20,
T20, C20) were the weakest interactors with A20 being the weakest
interactor of these by a significant margin. For the rest of the
PS-ONs tested, all of them displayed a similar, strong interaction
with the exception of TG10, which consistently displayed the
strongest interaction in each lysate. The binding profiles for
these PS-ONs is similar in all three viral lysates.
[0334] Target Identification for PS-ON Randomers in HIV-I
[0335] The ability of PS-ON randomers to bind to purified HIV-1
proteins was tested by fluorescence polarization as described in
example 9. Increasing quantities of purified HIV-1 p24 or purified
HIV-1 gp41 were reacted with REP 2004-FL. We note that for both
these proteins, there is a protein concentration dependent shift in
fluorescence polarization, indicating an interaction with both
these proteins.
[0336] The ability of a range of sizes of PS-ON randomers to bind
to these proteins was also tested using fluorescent versions of REP
2032, REP 2003, REP 2004, REP 2006 and REP 2007. We observed that
for p24, there is no size dependent interaction with p24, however;
we did see an increase in gp41 binding in PS-ON randomers larger
than 20 bases versus those less than 20 bases. This suggests when
PS-ON randomer length increases above 20 bases, multiple copies of
gp41 can bind to individual randomers, increasing their
polarization.
[0337] This is a significant observation as it demonstrates the
potential of larger ONs to sequester structural proteins during
viral synthesis and limit their availability for the formation of
new virions.
[0338] High Affinity Oligonucleotides
[0339] Another approach is a method to enrich or purify antiviral
ON(s) having a higher affinity for viral components, such as viral
proteins, than the average affinity of the ONs in a starting pool
of ONs. The method will thus provide one or more non-sequence
complementary ON(s) that will exhibit increased affinity to one or
more viral components, e.g., having a three-dimensional shape
contributing to such elevated binding affinity. The rationale is
that while ON(s) will act as linear molecules in binding with viral
components, they can also fold into a 3-dimensional shape that can
enhance the interaction with such viral components. Without being
limited to the specific technique, high affinity ONs can be
purified or enriched in the following ways.
[0340] One method for purifying a high affinity ON, or a plurality
of high affinity ONs, involves using a stationary phase medium with
bound viral protein(s) as an affinity matrix to bind ONs, which can
then be eluted under increasingly stringent conditions (e.g.,
increasing concentration of salt or other chaotropic agent, and/or
increasing temperture and/or changes in pH). Such a method can, for
example, be carried out by: [0341] (a) loading a pool of ONs onto
an exchange column having a viral protein or several viral proteins
or a viral lysate bound to a stationary phase; [0342] (b)
displacing (eluting) bound ONs from the column, e.g., by using a
displacer solution such as an increasing salt solution; [0343] (c)
collecting fractions of eluted ONs at different salt concentration;
[0344] (d) cloning and sequencing eluted ONs from different
fractions, more preferably from a fraction(s) at high salt
concentration, such that the ONs eluted at the high salt
concentration have a greater binding affinity with the viral
protein(s); and [0345] (e) Testing the activity of sequenced ON(s)
in assays such binding and/or viral inhibiton assay, e.g., a
fluorescence polarization-binding assay as decribed herein and/or
in a cellular viral inhibition assay and/or in an animal viral
inhibition assay.
[0346] In a second example, a method derived and modified from the
SELEX methodology (Morris et al (1998) Biochemistry 95:2902-2907)
can be used for purifying the high affinity ON. One implementation
of such a method can be performed as: [0347] (a) providing a
starting ON pool material, for example, a collection of synthetic
random ONs containing a high number of sequences, e.g., one hundred
trillion (10.sup.14) to ten quadrillion (10.sup.16) different
sequences. Each ON molecule contains a segment of random sequence
flanked by primer-binding sequences at each end to facilitate
polymerase chain reaction (PCR). Because the nucleotide sequences
of essentially all of the molecules are unique, an enormous number
of structures are sampled in the population. These structures
determine each molecule's biochemical properties, such as the
ability to bind a given viral target molecule; [0348] (b)
contacting ONs with a viral protein or several viral proteins or a
viral lysate; [0349] (c) selecting ONs that bind to viral
protein(s), using a partition technique(s) that can partition bound
and unbound ONs, such as native gel shifts and nitrocellulose
filtration. Either of these methods physically separates the bound
species from the unbound species, allowing preferential recovery of
those sequences that bind best. Also, to select ON (s) that bind to
a small protein, it is desirable to attach the target to a solid
support and use that support as an affinity purification matrix.
Those molecules that are not bound get washed off and the bound
ones are eluted with free target, again physically separating bound
and unbound species; [0350] (d) amplifying the eluted binding
ON(s), e.g., by using PCR using primers hybridizing with both
flanking sequences of ONs; [0351] (e) steps (b) (c) and (d) can be
performed multiple times (i.e., multiple cycles or rounds of
enrichment and amplification) in order to preferentially recover
ONs that display the highest binding affinity to viral protein(s).
After several cycles of enrichment and amplification, the
population is dominated by sequences that display the desired
biochemical property; [0352] (f) cloning and sequencing one or more
ONs selected from from an enrichment cycle, e.g., the last such
cycle; and [0353] (g) testing the binding and/or activity of
sequenced ON(s) in assays, e.g., in a fluorescence polarization
binding assay as decribed herein and/or in a cellular viral
inhibition assay and/or in an animal viral inhibition assay.
[0354] Another approach is to apply a modification of a split
synthesis methodology to create one-bead one-PS-ON and one-bead
one-PS2-ON libraries as described in Yang et al (2002) Nucl. Acids
Res. 30(e132):1-8. Binding and selection of specific beads to viral
proteins can be done. Sequencing both the nucleic acid bases and
the positions of any thioate/dithioate linkages can be carried out
by using a PCR-based identification tag of the selected beads. This
approach can allow for the rapid and convenient identification of
PS-ONs or PS2-ONs that bind to viral proteins and that exhibit
potent antiviral properties.
[0355] Once the specific sequences that bind to the viral proteins
with high affinity are determined (e.g., by amplification and
sequencing of individual sequences), one or more such high affinity
sequences can be selected and synthesized (e.g., by either chemical
or enzymatic synthesis) to provide a preparation of high affinity
ON(s), which can be modified to improve their activity, including
improving their pharmacokinetic properties. Such high affinity ONs
can be used in the present invention.
[0356] Prion Diseases
[0357] Another approach is used in an alternative embodiment of the
present invention for the treatment, the control of the
progression, or the prevention of prion disease. This fatal
neurodegenerative disease is infectious and can affect both humans
and animals. Structural changes in the cellular prion protein, PrPC
to its scrapie isoform, PrPSC, are considered to be the obligatory
step in the occurrence and propagation of the prion disease.
Amyloid polymers are associated with neuropathology of the prion
disease.
[0358] The incubation of a prion protein fragment and double
stranded nucleic acid results in the formation of amyloid fibres
(Nandi et al (2002), J Mol Biol 322: 153-161). ONs having affinity
to proteins such as phosphorothioates are used to compete or
inhibit the interaction of double stranded nucleic acid with the
PrPC and consequently stop the formation of the amyloid polymers.
Such ONs of different sizes and different compositions can be used
in an appropriate delivery form to treat patients suffering from
prion diseases or for prophylaxis in high risk situations. Such
interfering ONs can be identified by measuring folding changes of
amyloid polymerase as described by Nandi et al. (supra) in the
presence of test ONs.
[0359] Putative Viral Etiologies
[0360] Another approach is used in another embodiment of the
present invention for the treatment or prevention of diseases or
conditions with putative viral etiologies as described without
limitation in the following examples. Viruses are putative causal
agents in diseases and conditions that are not related to a primary
viral infection. For example, arthritis is associated with HCV
(Olivieri et al. (2003) Rheum Dis Clin North Am 29:111-122),
Parvovirus B19, HIV, HSV, CMV, EBV, and VZV (Stahl et al. (2000)
Clin Rheumatol 19:281-286). Other viruses have also been identified
as playing a role in different diseases. For example, influenza A
in Parkinson's disease (Takahashi et al. (1999), Jpn J Infect Dis
52:89-98), Coronavirus, EBV and other viruses in Multiple Sclerosis
(Talbot et al (2001) Curr Top Microbiol Immunol 253:247-71); EBV,
CMV and HSV-6 in Chronic Fatigue Syndrome (Lerner et al. (2002)
Drugs Today 38:549-561); and paramyxoviruses in asthma (Walter et
al (2002) J Clin Invest 110:165-175) and in Paget's disease; and
HBV, HSV, and influrenza in Guillain-Barre Syndrome.
[0361] Because of these etiologies, inhibition of the relevant
virus using the present invention can delay, slow, or prevent
development of the corresponding disease or condition, or at least
some symptoms of that disease.
[0362] Oligonucleotide Modifications and Synthesis
[0363] As indicated in the Summary above, modified oligonucleotides
are useful in this invention. Such modified oligonucleotides
include, for example, oligonucleotides containing modified
backbones or non-natural internucleoside linkages. Oligonucleotides
having modified backbones include those that retain a phosphorus
atom in the backbone and those that do not have a phosphorus atom
in the backbone.
[0364] Such modified oligonucleotide backbones include, for
example, phosphorothioates, chiral phosphorothioates,
phosphorodithioates, phosphotriesters, aminoalkylphosphotri-esters,
methyl and other alkyl phosphonates including 3'-alkylene
phosphonates, 5'-alkylene phosphonates and chiral phosphonates,
phosphinates, phosphoramidates including 3'-amino phosphoramidate
and aminoalkylphosphoramidates, thionophosphoramidates,
thionoalkylphosphonates, thionoalkylphosphotriesters,
selenophosphates, carboranyl phosphate and boranophosphates having
normal 3'-5' linkages, 2'-5' linked analogs of these, and those
having inverted polarity wherein one or more internucleotide
linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage.
Oligonucleotides having inverted polarity typically include a
single 3' to 3' linkage at the 3'-most internucleotide linkage i.e.
a single inverted nucleoside residue which may be abasic (the
nucleobase is missing or has a hydroxyl group in place thereof).
Various salts, mixed salts and free acid forms are also
included.
[0365] Preparation of oligonucleotides with phosphorus-containing
linkages as indicated above are described, for example, in U.S.
Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;
5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;
5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;
5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;
5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697
and 5,625,050, each of which is incorporated by reference herein in
its entirety.
[0366] Some exemplary modified oligonucleotide backbones that do
not include a phosphodiester linkage have backbones that are formed
by short chain alkyl or cycloalkyl internucleoside linkages, mixed
heteroatom and alkyl or cycloalkyl internucleoside linkages, or one
or more short chain heteroatomic or heterocyclic internucleoside
linkages. These include those having morpholino linkages (formed in
part from the sugar portion of a nucleoside); siloxane backbones;
sulfide, sulfoxide and sulfone backbones; formacetyl and
thioformacetyl backbones; methylene formacetyl and thioformacetyl
backbones; riboacetyl backbones; alkene containing backbones;
sulfamate backbones; methyleneimino and methylenehydrazino
backbones; sulfonate and sulfonamide backbones; amide backbones;
and others having mixed N, 0, S and CH.sub.2 component parts.
Particularly advantageous are backbone linkages that include one or
more charged moieties. Examples of U.S. patents describing the
preparation of the preceding oligonucleotides include U.S. Pat.
Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141;
5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677;
5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240;
5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070;
5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and
5,677,439, each of which is incorporated by reference herein in its
entirety.
[0367] Modified oligonucleotides may also contain one or more
substituted sugar moieties. For example, such oligonucleotides can
include one of the following 2'-modifications: OH; F; O--, S--, or
N-alkyl; O--, S--, or N-alkenyl; O--, S-- or N-- alkynyl; or
O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be
substituted or unsubstituted C.sub.1 to C.sub.10 alkyl or C.sub.2
to C.sub.10 alkenyl and alkynyl, or 2'-O--(O-carboran-1-yl)methyl.
Particular examples are O[(CH.sub.2).sub.nO].sub.mCH.sub.3,
O(CH.sub.2).about.OCH.sub.3, O(CH.sub.2).sub.nNH.sub.2,
O(CH.sub.2).sub.nCH.sub.3, O(CH.sub.2).sub.nONH.sub.2, and
O(CH.sub.2).sub.nON[(CH.sub.2).sub.nCH.sub.3)].sub.2, where n and m
are from 1 to 10. Other exemplary oligonucleotides include one of
the following 2'-modifications: C.sub.1 to C.sub.10 lower alkyl,
substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl,
O-alkaryl or O-aralkyl, SH, SCH.sub.3, OCN, Cl, Br, CN,
CF.sub.3.OCF.sub.3, SOCH.sub.3, SO.sub.2CH.sub.3, ONO.sub.2,
NO.sub.2, N.sub.3, NH.sub.2, heterocycloalkyl, heterocycloalkaryl,
aminoalkylamino, polyalkylamino, substituted silyl, a reporter
group, an intercalator, a group for improving the pharmacokinetic
properties of an oligonucleotide, or a group for improving the
pharmacodynamic properties of an oligonucleotide. Examples include
2'-methoxyethoxy (2'-O--CH.sub.2CH.sub.2OCH.sub.3, also known as
2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta,
1995, 78, 486-504) i.e., an alkoxyalkoxy group;
2'-dimethy-laminooxyethoxy, i.e., a
O(CH.sub.2).sub.2ON(CH.sub.3).sub.2 group, also known as 2'-DMAOE;
and 2'-dimethylaminoethoxyethoxy (also known as
2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e.,
2'-O--CH.sub.2--O--CH.sub.2--N(CH.sub.2).sub.2.
[0368] Other modifications include Locked Nucleic Acids (LNAs) in
which the 2'-hydroxyl group is linked to the 3' or 4' carbon atom
of the sugar ring thereby forming a bicyclic sugar moiety. The
linkage can be a methelyne (--CH.sub.2--).about. group bridging the
2' oxygen atom and the 4' carbon atom wherein n is 1 or 2. LNAs and
preparation thereof are described in WO 98/39352 and WO 99/14226,
which are incorporated herein by reference in their entireties.
[0369] Other modifications include sulfur-nitrogen bridge
modifications, such as locked nucleic acid as described in Orum et
al. (2001) Curr. Opin. Mol. Ther. 3:239-243.
[0370] Other modifications include 2'-methoxy (2'-O--CH.sub.3),
2'-methoxyethyl (2-O--CH.sub.2--CH.sub.3 ), 2'-ethyl, 2'-ethoxy,
2'-aminopropoxy (2'-OCH.sub.2CH.sub.2CH.sub.2NH.sub.2), 2'-allyl
(2'-CH.sub.2--CH.dbd.CH.sub.2), 2'-O-allyl
(2'-O--CH.sub.2--CH.dbd.CH.sub.2) and 2'-fluoro (2'-F).
[0371] The 2'-modification may be in the arabino (up) position or
ribo (down) position. Similar modifications may also be made at
other positions on the oligonucleotide, particularly the 3'
position of the sugar on the 3' terminal nucleotide or in 2'-5'
linked oligonucleotides and the 5' position of the 5' terminal
nucleotide. Oligonucleotides may also have sugar mimetics such as
cyclobutyl moieties in place of the pentofuranosyl sugar. Exemplary
U.S. patents describing the preparation of such modified sugar
structures include, for example, U.S. Pat. Nos. 4,981,957;
5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;
5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;
5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633;
5,792,747; and 5,700,920, each of which is incorporated by
reference herein in its entirety.
[0372] Still other modifications include an ON concatemer
consisting of multiple oligonucleotide sequences joined by a
linker(s). The linker may, for example, consist of modified
nucleotides or non-nucleotide units. In some embodiments, the
linker provides flexibility to the ON concatemer. Use of such ON
concatemers can provide a facile method to synthesize a final
molecule, by joining smaller oligonucleotide building blocks to
obtain the desired length. For example, a 12 carbon linker (C12
phosphoramidite) can be used to join two or more ON concatemers and
provide length, stability, and flexibility.
[0373] As used herein, "unmodified" or "natural" bases
(nucleobases) include the purine bases adenine (A) and guanine (G),
and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
Oligonucleotides may also include base modifications or
substitutions. Modified bases include other synthetic and
naturally-occurring bases such as 5-methylcytosine (5-me-C),
5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,
6-methyl and other alkyl derivatives of adenine and guanine,
2-propyl and other alkyl derivatives of adenine and guanine,
2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and
cytosine, 5-propynyl(-C.ident.C--CH.sub.3) uracil and cytosine and
other alkynyl derivatives of pyrimidine bases, 6-azo uracil,
cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo,
8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted
adenines and guanines, 5-halo particularly 5-bromo,
5-trifluoromethyl and other 5-substituted uracils and cytosines,
7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine,
8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine
and 3-deazaguanine and 3-deazaadenine. Additional modified bases
include tricyclic pyrimidines such as phenoxazine
cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one),
phenothiazine
cytidine(1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps
such as a substituted phenoxazine cytidine (e.g.
9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one),
carbazole cytidine(2H-pyrimido[4,5-b]indol-2-one), pyridoindole
cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one).
Modified bases may also include those in which the purine or
pyrimidine base is replaced with other heterocycles, for example
7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.
Further nucleobases include those described in U.S. Pat. No.
3,687,808, those disclosed in The Concise Encyclopedia Of Polymer
Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John
Wiley & Sons, 1990, those disclosed by Englisch et al.,
Angewandte Chemie, International Edition, 1991, 30, 613, and those
disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and
Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC
Press, 1993.
[0374] Another modification includes phosphorodithioate linkages.
Knowing that phosphorodithioate ONs (PS2-ONs) and PS-ONs have a
similar binding affinity to proteins (Tonkinson et al. (1994)
Antisense Res. Dev. 4 :269-278)(Cheng et al. (1997) J. Mol. Recogn.
10:101-107) and knowing that a possible mechanism of action of ONs
is binding to viral proteins, it could be desirable to include
phosphorodithioate linkages on the antiviral ONs described in this
invention.
[0375] Another approach to modify ONs is to produce stereodefined
or stereo-enriched ONs as described in Yu at al (2000) Bioorg. Med.
Chem. 8:275-284 and in Inagawa et al. (2002) FEBS Lett. 25:48-52.
ONs prepared by conventional methods consist of a mixture of
diastereomers by virtue of the asymmetry around the phosphorus atom
involved in the internucleotide linkage. This may affect the
stability of the binding between ONs and viral components such as
viral proteins. Previous data showed that protein binding is
significantly stereo-dependent (Yu et al.). Thus, using
stereodefined or stereo-enriched ONs could improve their protein
binding properties and improve their antiviral efficacy.
[0376] The incorporation of modifications such as those described
above can be utilized in many different incorporation patterns and
levels. That is, a particular modification need not be included at
each nucleotide or linkage in an oligonucleotide, and different
modifications can be utilized in combination in a single
oligonucleotide, or even in a single nucleotide.
[0377] As examples and in accordance with the description above,
modified oligonucleotides containing phosphorothioate or dithioate
linkages may also contain one or more substituted sugar moieties
particularly modifications at the sugar moieties including, without
restriction, 2'-ethyl, 2'-ethoxy, 2'-methoxy, 2'-aminopropoxy,
2'-allyl, 2'-fluoro, 2'-pentyl, 2'-propyl,
2'-dimethylaminooxyethoxy, and 2'-dimethylaminoethoxyethoxy. The
2'-modification may be in the arabino (up) position or ribo (down)
position. A preferred 2'-arabino modification is 2'-fluoro. Similar
modifications may also be made at other positions on the
oligonucleotide, particularly the 3' position of the sugar on the
3' terminal nucleotide or in 2'-5' linked oligonucleotides and the
5' position of 5' terminal nucleotide. Oligonucleotides may also
have sugar mimetics such as cyclobutyl moieties in place of the
pentofuranosyl sugar. Moreover ONs may have a structure of or
comprise a portion consisting of glycol nucleic acid (GNA) with an
acyclic propylene glycol phosphodiester backbone (Zhang L, et al
(2005) J. Am. Chem. Soc. 127(12):4174-5). Such GNA may comprise
phosphorothioate linkages and may comprise only pyrimidine
bases.
[0378] Oligonucleotide Synthesis
[0379] The present oligonucleotides can by synthesized using
methods known in the art. For example, unsubstituted and
substituted phosphodiester (P.dbd.O) oligonucleotides can be
synthesized on an automated DNA synthesizer (e.g., Applied
Biosystems model 380B) using standard phosphoramidite chemistry
with oxidation by iodine. Phosphorothioates (P.dbd.S) can be
synthesized as for the phosphodiester oligonucleotides except the
standard oxidation bottle can be replaced by 0.2 M solution of
311-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the
step-wise thioation of the phosphite linkages. The thioation wait
step can be increased to 68 sec, followed by the capping step.
After cleavage from the CPG column and deblocking in concentrated
ammonium hydroxide at 55.degree. C. (18 h), the oligonucleotides
can be purified by precipitating twice with 2.5 volumes of ethanol
from a 0.5 M NaCl solution.
[0380] Phosphinate oligonucleotides can be prepared as described in
U.S. Pat. No. 5,508,270; alkyl phosphonate oligonucleotides can be
prepared as described in U.S. Pat. No. 4,469,863;
3'-Deoxy-3'-methylene phosphonate oligonucleotides can be prepared
as described in U.S. Pat. Nos. 5,610,289 and 5,625,050;
phosphoramidite oligonucleotides can be prepared as described in
U.S. Pat. No. 5,256,775 and U.S. Pat. No. 5,366,878;
alkylphosphonothioate oligonucleotides can be prepared as described
in published PCT applications PCT/US94/00902 and PCT/US93/06976
(published as WO 94/17093 and WO 94/02499, respectively);
3'-Deoxy-3'-amino phosphoramidate oligonucleotides can be prepared
as described in U.S. Pat. No. 5,476,925; Phosphotriester
oligonucleotides can be prepared as described in U.S. Pat. No.
5,023,243; boranophosphate oligonucleotides can be prepared as
described in U.S. Pat. Nos. 5,130,302 and 5,177,198;
methylenemethylimino linked oligonucleotides, also identified as
MMI linked oligonucleotides, methylenedimethyl-hydrazo linked
oligonucleotides, also identified as MDII linked oligonucleotides,
and methylenecarbonylamino linked oligonucleotides, also identified
as amide-3 linked oligonucleotides, and methyleneaminocarbonyl
linked oligo-nucleotides, also identified as amide-4 linked
oligonucleo-sides, as well as mixed backbone compounds having, for
instance, alternating MMI and P.dbd.O or P.dbd.S linkages can be
prepared as described in U.S. Pat. Nos. 5,378, 825, 5,386,023,
5,489,677, 5,602,240 and 5,610,289; formacetal and thioformacetal
linked oligonucleotides can be prepared as described in U.S. Pat.
Nos. 5,264,562 and 5,264,564; and ethylene oxide linked
oligonucleotides can be prepared as described in U.S. Pat. No.
5,223,618. Each of the cited patents and patent applications is
incorporated by reference herein in its entirety.
[0381] Oligonucleotide Formulations and Pharmaceutical
Compositions
[0382] The present oligonucleotides can be prepared in an
oligonucleotide formulation or pharmaceutical composition. Thus,
the present oligonucleotides may also be admixed, encapsulated,
conjugated or otherwise associated with other molecules, molecule
structures or mixtures of compounds, as for example, liposomes,
receptor targeted molecules, oral, rectal, topical or other
formulations, for assisting in uptake, distribution and/or
absorption. Exemplary U.S. patents that describe the preparation of
such uptake, distribution and/or absorption assisting formulations
include, for example, U.S. Pat. Nos. 5,108,921; 5,354,844;
5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020;
5,591,721; 4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804;
5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978;
5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152;
5,556,948; 5,580,575; and 5,595,756, each of which is incorporated
herein by reference in its entirety.
[0383] The oligonucleotides, formulations, and compositions of the
invention include any pharmaceutically acceptable salts, esters, or
salts of such esters, or any other compound which, upon
administration to an animal including a human, is capable of
providing (directly or indirectly) the biologically active
metabolite or residue thereof. Accordingly, for example, the
disclosure is also drawn to prodrugs and pharmaceutically
acceptable salts of the compounds of the invention,
pharmaceutically acceptable salts of such prodrugs, and other
bioequivalents.
[0384] The term "prodrug" indicates a therapeutic agent that is
prepared in an inactive form that is converted to an active form
(i.e., drug) within the body or cells thereof by the action of
endogenous enzymes or other chemicals and/or conditions. In
particular embodiments, prodrug versions of the present
oligonucleotides are prepared as SATE
[(S-acetyl-2-thioethyl)phosphate] derivatives according to the
methods disclosed in Gosselin et al., WO 93/24510 and in Imbach et
al., WO 94/26764 and U.S. Pat. No. 5,770,713, which are hereby
incorporated by reference in their entireties.
[0385] The term "pharmaceutically acceptable salts" refers to
physiologically and pharmaceutically acceptable salts of the
present compounds: i.e., salts that retain the desired biological
activity of the parent compound and do not impart undesired
toxicological effects thereto. Many such pharmaceutically
acceptable salts are known and can be used in the present
invention.
[0386] For oligonucleotides, useful examples of pharmaceutically
acceptable salts include but are not limited to salts formed with
cations such as sodium, potassium, ammonium, magnesium, calcium,
polyamines such as spermine and spermidine, etc.; acid addition
salts formed with inorganic acids, for example hydrochloric acid,
hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and
the like; salts formed with organic acids such as, for example,
acetic acid, oxalic acid, tartaric acid, succinic acid, maleic
acid, fumaric acid, gluconic acid, citric acid, malic acid,
ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic
acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic
acid, p-toluenesulfonic acid, naphthalenedisulfonic acid,
polygalacturonic acid, and the like; and salts formed from
elemental anions such as chlorine, bromine, and iodine.
[0387] The present invention also includes pharmaceutical
compositions and formulations which contain the antiviral
oligonucleotides of the invention. Such pharmaceutical compositions
may be administered in a number of ways depending upon whether
local or systemic treatment is desired and upon the area to be
treated. For example, administration may be topical (including
ophthalmic and to mucous membranes including vaginal and rectal
delivery); pulmonary, e.g., by inhalation or insufflation of
powders or aerosols, including by nebulizer; intratracheal;
intranasal; epidermal and transdermal; oral; or parenteral.
Parenteral administration includes intravenous, intraarterial,
subcutaneous, intraperitoneal or intramuscular injection or
infusion; or intracranial, e.g., intrathecal or intraventricular,
administration.
[0388] Pharmaceutical compositions and formulations for topical
administration may include transdermal patches, ointments, lotions,
creams, gels, drops, suppositories, sprays, liquids and powders.
Conventional pharmaceutical carriers, aqueous, powder or oily
bases, thickeners and the like may be necessary or desirable.
Coated condoms, gloves and the like may also be useful. Preferred
topical formulations include those in which the oligonucleotides of
the invention are in admixture with a topical delivery agent such
as lipids, liposomes, fatty acids, fatty acid esters, steroids,
chelating agents and surfactants. Preferred lipids and liposomes
include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine,
dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl
choline) negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and
cationic (e.g. dioleoyltetramethylaminopropyl DOTAP and
dioleoylphosphatidyl ethanolamine DOTMA). Oligonucleotides may be
encapsulated within liposomes or may form complexes thereto, in
particular to cationic liposomes. Alternatively, oligonucleotides
may be complexed to lipids, in particular to cationic lipids.
Preferred fatty acids and esters include but are not limited
arachidonic acid, oleic acid, eicosanoic acid, laurie acid,
caprylic acid, capric acid, myristic acid, palmitic acid, stearic
acid, linoleic acid, linolenic acid, dicaprate, tricaprate,
monoolein, dilaurin, glyceryl 1-monocaprate,
1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or
a C.sub.1-10 alkyl ester (e.g. isopropylmyristate IPM),
monoglyceride, diglyceride or pharmaceutically acceptable salt
thereof.
[0389] Compositions and formulations for oral administration
include powders or granules, microparticulates, nanoparticulates,
suspensions or solutions in water or non-aqueous media, capsules,
gel capsules, sachets, tablets or minitablets. Thickeners,
flavoring agents, diluents, emulsifiers, dispersing aids or binders
may be desirable. Preferred oral formulations are those in which
oligonucleotides of the invention are administered in conjunction
with one or more penetration enhancers surfactants and chelators.
Exemplary surfactants include fatty acids and/or esters or salts
thereof, bile acids and/or salts thereof. Exemplary bile
acids/salts include chenodeoxycholic acid (CDCA) and
ursodeoxychenedeoxycholic acid (UDCA), cholic acid, dehydrocholic
acid, deoxycholic acid, glucholic acid, glycholic acid,
glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid,
sodium tauro-24,25-dihydro-fusidate, sodium glycodihydrofusidate.
Exemplary fatty acids include arachidonic acid, undecanoic acid,
oleic acid, lauric acid, caprylic acid, capric acid, myristic acid,
palmitic acid, stearic acid, linoleic acid, linolenic acid,
dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,
1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or
a monoglyceride, a diglyceride or a pharmaceutically acceptable
salt thereof (e.g. sodium). Also preferred are combinations of
penetration enhancers, for example, fatty acids/salts in
combination with bile acids/salts. A particularly preferred
combination is the sodium salt of lauric acid, capric acid and
UDCA. Further exemplary penetration enhancers include
polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.
Oligonucleotides of the invention may be delivered orally in
granular form including sprayed dried particles, or complexed to
form micro or nanoparticles. Oligonucleotide complexing agents
include poly-amino acids; polyimines; polyacrytates;
polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates;
cationized gelatins, albumins, starches, acrylates,
polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates;
DEAE-derivatized polyimines, pollulans, celluloses, and starches.
Particularly advantageous complexing agents include chitosan,
N-trimethytchitosan, poly-L-lysine, polyhistidine, polyorithine,
polyspermines, protamine, polyvinylpyridine,
polythiodiethylamino-methylethylene P(TDAE), polyaminostyrene (e.g.
p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),
poly(butylcyanoacrylatc), poly(isobutylcyanoacrylate),
poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,
DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,
polyhexylacrylate, poly(D,L-lactic acid),
poly(DL-lactic-co-glycolic acid (PLGA), alginate, and
polyethyleneglycol (PEG).
[0390] Compositions for vaginal delivery can be in various forms,
including for example, a gel, cream, tablet, pill, capsule,
suppository, film, or any other pharmaceutically acceptable form
that adheres to the mucosa and does not wash away easily. A large
variety of different formulations for vaginal delivery are further
described in the art, for example in U.S. Pat. Nos. 4,615,697 and
6,699,494, which are incorporated herein by reference in their
entireties.
[0391] Additionally, additives (such as those described in the U.S.
Pat. No. 4,615,697 patent) may be combined in the formulation for
maximum or desired efficacy of the delivery system or for the
comfort of the patient. Such additives include, for example,
lubricants, plasticizing agents, preservatives, gel formers, tablet
formers, pill formers, suppository formers, film formers, cream
formers, disintegrating agents, coatings, binders, vehicles,
coloring agents, taste and/or odor controlling agents, humectants,
viscosity controlling agents, pH-adjusting agents, and similar
agents.
[0392] In certain embodiments, a composition can include a
cross-linked polycarboxylic acid polymer formulation, generally
described in U.S. Pat. No. 4,615,697. In general, in such
embodiments at least eighty percent of the monomers of the polymer
in such a formulation should contain at least one carboxyl
functionality. The cross-linking agent should be present at such an
amount as to provide enough bioadhesion to allow the system to
remain attached to the target epithelial surfaces for a sufficient
time to allow the desired dosing to take place.
[0393] For vaginal administration, such a formulation remains
attached to the epithelial surfaces for a period of at least about
twenty-four to forty-eight hours. Such results may be measured
clinically over various periods of time, by testing samples from
the vagina for pH reduction due to the continued presence of the
polymer. This preferred level of bioadhesion is usually attained
when the cross-linking agent is present at about 0.1 to 6.0 weight
percent of the polymer, with about 1.0 to 2.0 weight percent being
most preferred, as long as the appropriate level of bioadhesion
results. Bioadhesion can also be measured by commercially available
surface tensiometers utilized to measure adhesive strength.
[0394] The polymer formulation can be adjusted to control the
release rate by varying the amount of cross-linking agent in the
polymer. Suitable cross-linking agents include divinyl glycol,
divinylbenzene, N,N-diallylacrylamide, 3,4-dihydroxy-1,5-hexadiene,
2,5-dimethyl-1,5-hexadiene and similar agents.
[0395] A preferred polymer for use in such a formulation is
Polycarbophil, U.S.P., which is commercially available from B. F.
Goodrich Speciality Polymers of Cleveland, Ohio under the trade
name NOVEON.RTM.-AA1. The United States Pharmacopeia, 1995 edition,
United States Pharmacopeial Convention, Inc., Rockville, Md., at
pages 1240-41, indicates that polycarbophil is a polyacrylic acid,
cross-linked with divinyl glycol.
[0396] Other useful bioadhesive polymers that may be used in such a
drug delivery system formulation are mentioned in the U.S. Pat. No.
4,615,697 patent. For example, these include polyacrylic acid
polymers cross-linked with, for example,
3,4-dihydroxy-1,5-hexadiene, and polymethacrylic acid polymers
cross-linked with, for example, divinyl benzene. Typically, these
polymers would not be used in their salt form, because this would
decrease their bioadhesive capability. Such bioadhesive polymers
may be prepared by conventional free radical polymerization
techniques utilizing initiators such as benzoyl peroxide,
azobisisobutyronitrile, and the like. Exemplary preparations of
useful bioadhesives are provided in the U.S. Pat. No. 4,615,697
patent.
[0397] Compositions and formulations for parenteral, intrathecal or
intraventricular administration may include sterile aqueous
solutions which may also contain buffers, diluents and other
suitable additives such as, but not limited to, penetration
enhancers, carrier compounds and other pharmaceutically acceptable
carriers or excipients.
[0398] Pharmaceutical compositions of the present invention
include, but are not limited to, solutions, emulsions, and
liposome-containing formulations. These compositions may be
generated from a variety of components that include, but are not
limited to, preformed liquids, self-emulsifying solids and
self-emulsifying semisolids.
[0399] The pharmaceutical formulations of the present invention,
which may conveniently be presented in unit dosage form, may be
prepared according to conventional techniques well known in the
pharmaceutical industry. Such techniques include the step of
bringing into association the active ingredients with the
pharmaceutical carrier(s) or excipient(s). In general the
formulations are prepared by uniformly and intimately bringing into
association the active ingredients with liquid carriers or finely
divided solid carriers or both, and then, if necessary, shaking the
product.
[0400] The compositions of the present invention may be formulated
into any of many possible dosage forms such as, but not limited to,
tablets, capsules, gel capsules, liquid syrups, soft gels,
suppositories, and enemas. The compositions of the present
invention may also be formulated as suspensions in aqueous,
non-aqueous or mixed media. Aqueous suspensions may further contain
substances which increase the viscosity of the suspension
including, for example, sodium carboxymethylcellulose, sorbitol
and/or dextran. The suspension may also contain stabilizers.
[0401] In one embodiment of the present invention the
pharmaceutical compositions may be formulated and used as foams.
Pharmaceutical foams include formulations such as, but not limited
to, emulsions, microemulsions, creams, jellies and liposomes. While
basically similar in nature these formulations vary in the
components and the consistency of the final product. The
preparation of such compositions and formulations is generally
known to those skilled in the pharmaceutical and formulation arts
and may be applied to the formulation of the compositions of the
present invention.
[0402] Emulsions
[0403] The formulations and compositions of the present invention
may be prepared and formulated as emulsions. Emulsions are
typically heterogenous systems of one liquid dispersed in another
in the form of droplets usually exceeding 0.1 .mu.m in diameter.
(Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and
Banker (lids.), 1988, Marcel Dekker, Inc., New York, N.Y., volume
1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman,
Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York,
N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms,
Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New
York, N.Y., volume 2, p. 335; Higuchi et at., in Remington's
Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p.
301). Emulsions are often biphasic systems comprising of two
immiscible liquid phases intimately mixed and dispersed with each
other. In general, emulsions may be either water-in-oil (w/o) or of
the oil-in-water (o/w) variety. When an aqueous phase is finely
divided into and dispersed as minute droplets into a bulk oily
phase the resulting composition is called a water-in-oil (w/o)
emulsion. Alternatively, when an oily phase is finely divided into
and dispersed as minute droplets into a bulk aqueous phase the
resulting composition is called an oil-in-water (o/w) emulsion.
Emulsions may contain additional components in addition to the
dispersed phases and the active drug which may be present as a
solution in either the aqueous phase, oily phase or itself as a
separate phase. Pharmaceutical excipients such as emulsifiers,
stabilizers, dyes, and anti-oxidants may also be present in
emulsions as needed. Pharmaceutical emulsions may also be multiple
emulsions that are comprised of more than two phases such as, for
example, in the case of oil-in-water-in-oil (o/w/o) and
water-in-oil-in-water (w/o/w) emulsions. Such complex formulations
often provide certain advantages that simple binary emulsions do
not. Multiple emulsions in which individual oil droplets of an o/w
emulsion enclose small water droplets constitute a w/o/w emulsion.
Likewise a system of oil droplets enclosed in globules of water
stabilized in an oily continuous provides an o/w/o emulsion.
[0404] Emulsions are characterized by little or no thermodynamic
stability. Often, the dispersed or discontinuous phase of the
emulsion is well dispersed into the external or continuous phase
and maintained in this form through the means of emulsifiers or the
viscosity of the formulation. Either of the phases of the emulsion
may be a semisolid or a solid, as is the case of emulsion-style
ointment bases and creams. Other means of stabilizing emulsions
entail the use of emulsifiers that may be incorporated into either
phase of the emulsion. Emulsifiers may broadly be classified into
four categories: synthetic surfactants, naturally occurring
emulsifiers, absorption bases, and finely dispersed solids (Idson,
in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker
(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.
199).
[0405] Synthetic surfactants, also known as surface active agents,
have found wide applicability in the formulation of emulsions and
have been reviewed in the literature (Rieger, in Pharmaceutical
Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel
Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in
Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.),
Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199).
Surfactants are typically amphiphilic and comprise a hydrophilic
and a hydrophobic portion. The ratio of the hydrophilic to the
hydrophobic nature of the surfactant has been termed the
hydrophile/lipophile balance (HLB) and is a valuable tool in
categorizing and selecting surfactants in the preparation of
formulations. Surfactants may be classified into different classes
based on the nature of the hydrophilic group: non-ionic, anionic,
cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms,
Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New
York, N.Y., volume 1, p. 285).
[0406] Naturally occurring emulsifiers used in emulsion
formulations include lanolin, beeswax, phosphatides, lecithin and
acacia. Absorption bases possess hydrophilic properties such that
they can soak up water to form w/o emulsions yet retain their
semisolid consistencies, such as anhydrous lanolin and hydrophilic
petrolatum. Finely divided solids have also been used as good
emulsifiers especially in combination with surfactants and in
viscous preparations. These include polar inorganic solids, such as
heavy metal hydroxides, nonswelling clays such as bentonite,
attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum
silicate and colloidal magnesium aluminum silicate, pigments and
nonpolar solids such as carbon or glyceryl tristearate.
[0407] A large variety of non-emulsifying materials are also
included in emulsion formulations and contribute to the properties
of emulsions. These include fats, oils, waxes, fatty acids, fatty
alcohols, fatty esters, humectants, hydrophilic colloids,
preservatives and antioxidants (Block, in Pharmaceutical Dosage
Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,
Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical
Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel
Dekker, Inc., New York, N.Y., volume 1, p. 199).
[0408] Hydrophilic colloids or hydrocolloids include naturally
occurring gums and synthetic polymers such as polysaccharides (for
example, acacia, agar, alginic acid, carrageenan, guar gum, karaya
gum, and tragacanth), cellulose derivatives (for example,
carboxymethylcellulose and carboxypropylcellulose), and synthetic
polymers (for example, carbomers, cellulose ethers, and
carboxyvinyl polymers). These disperse or swell in water to form
colloidal solutions that stabilize emulsions by forming strong
inter-facial films around the dispersed-phase droplets and by
increasing the viscosity of the external phase.
[0409] Since emulsions often contain a number of ingredients such
as carbohydrates, proteins, sterols and phosphatides that may
readily support the growth of microbes, these formulations often
incorporate preservatives. Commonly used preservatives included in
emulsion formulations include methyl paraben, propyl paraben,
quaternary ammonium salts, benzalkonium chloride, esters of
p-hydroxybenzoic acid, and boric acid, Antioxidants are also
commonly added to emulsion formulations to prevent deterioration of
the formulation. Antioxidants used may be free radical scavengers
such as tocopherols, alkyl gallates, butylated hydroxyanisole,
butylated hydroxytoluene, or reducing agents such as ascorbic acid
and sodium metabisulfite, and antioxidant synergists such as citric
acid, tartaric acid, and lecithin.
[0410] The application of emulsion formulations via dermatological,
oral and parenteral routes and methods for their manufacture have
been reviewed in the literature (Idson, in Pharmaceutical Dosage
Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,
Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for
oral delivery have been very widely used because of reasons of ease
of formulation, efficacy from an absorption and bioavailabiity
standpoint. (Rosoff, in Pharmaceutical Dosage Forms, Lieberman,
Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York,
N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms,
Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New
York, N.Y., volume 1, p. 199). Mineral-oil base laxatives,
oil-soluble vitamins and high fat nutritive preparations are among
the materials that have commonly been administered orally as o/w
emulsions.
[0411] In one embodiment of the present invention, the compositions
of oligonucleotides are formulated as microemulsions. A
microemulsion may be defined as a system of water, oil and
amphiphile which is a single optically isotropic and
thermodynamically stable liquid solution (Rosoff, in Pharmaceutical
Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel
Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically
micro-emulsions are systems that are prepared by first dispersing
an oil in an aqueous surfactant solution and then adding a
sufficient amount of a fourth component, generally an intermediate
chain-length alcohol to form a transparent system. Therefore,
microemulsions have also been described as thermodynamically
stable, isotropically clear dispersions of two immiscible liquids
that are stabilized by interfacial films of surface-active
molecules (Leung and Shah, in: Controlled Release of Drugs:
Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH
Publishers, New York, pages 185-215). Microemulsions commonly are
prepared via a combination of three to five components that include
oil, water, surfactant, cosurfactant and electrolyte. Whether the
microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w)
type is dependent on the properties of the oil and surfactant used
and on the structure and geometric packing of the polar heads and
hydrocarbon tails of the surfactant molecules (Schott, in
Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,
Pa., 1985, p. 271).
[0412] The phenomenological approach utilizing phase diagrams has
been extensively studied and has yielded a comprehensive knowledge,
to one skilled in the art, of how to formulate microemulsions
(Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and
Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,
p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger
and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,
volume 1, p. 335). Compared to conventional emulsions,
microemulsions offer the advantage of solubilizing water-insoluble
drugs in a formulation of thermodynamically stable droplets that
are formed spontaneously.
[0413] Surfactants used in the preparation of microemulsions
include, but are not limited to, ionic surfactants, non-ionic
surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol
fatty acid esters, tetraglycerol monolaurate (ML31O), tetraglycerol
monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol
pentaoleate (PO500), decaglycerol monocaprate (MCA750),
decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750),
decaglycerol decaoleate (DA0750), alone or in combination with
cosurfactants. The cosurfactant, usually a short-chain alcohol such
as ethanol, 1-propanol, and 1-butanol, serves to increase the
interfacial fluidity by penetrating into the surfactant film and
consequently creating a disordered film because of the void space
generated among surfactant molecules. Microemulsions may, however,
be prepared without the use of cosurfactants and alcohol-free
self-emulsifying microemulsion systems are known in the art. The
aqueous phase may typically be, but is not limited to, water, an
aqueous solution of the drug, glycerol, PEG300, PEG400,
polyglycerols, propylene glycols, and derivatives of ethylene
glycol. The oil phase may include, but is not limited to, materials
such as Captex 300, Captex 355, Capmul MCM, fatty acid esters,
medium chain (C8-C12) mono, di, and tri-glycerides,
polyoxyethylated glyceryl fatty acid esters, fatty alcohols,
polyglycolized glycerides, saturated polyglycolized C8-C10
glycerides, vegetable oils and silicone oil.
[0414] Microemulsions are particularly of interest from the
standpoint of drug solubilization and the enhanced absorption of
drugs. Lipid based microemulsions (both o/w and w/o) have been
proposed to enhance the oral bioavailability of drugs, including
peptides (Constantinides et al., Pharmaceutical Research, 1994, 11,
1385-1390; Ritschet, Met/i. Find. Exp. Clin. PharmacoL, 1993, 13,
205). Micro-emulsions afford advantages of improved drug
solubilization, protection of drug from enzymatic hydrolysis,
possible enhancement of drug absorption due to surfactant-induced
alterations in membrane fluidity and permeability, ease of
preparation, ease of oral administration over solid dosage forms,
improved clinical potency, and decreased toxicity (Constantinides
et at., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J.
Pharm. Set, 1996, 85, 138-143). Often microemulsions may form
spontaneously when their components are brought together at ambient
temperature. This may be particularly advantageous when formulating
thermolabile drugs, peptides or oligonucleotides. Microemulsions
have also been effective in the transdermal delivery of active
components in both cosmetic and pharmaceutical applications. It is
expected that the microemulsion compositions and formulations of
the present invention will facilitate the increased systemic
absorption of oligonucteotides and nucleic acids from the
gastrointestinal tract, as well as improve the local cellular
uptake of oligonucleotides and nucleic acids within the
gastrointestinal tract, vagina, buccal cavity and other areas of
administration.
[0415] Microemulsions of the present invention may also contain
additional components and additives such as sorbitan monostearate
(Grill 3), Labrasol, and penetration enhancers to improve the
properties of the formulation and to enhance the absorption of the
oligonucleotides and nucleic acids of the present invention.
Penetration enhancers used in the microemulsions of the present
invention may be classified as belonging to one of five broad
categories--surfactants, fatty acids, bile salts, chelating agents,
and non-chelating non-surfactants (Lee et al., Critical Reviews in
Therapeutic Drug Carrier Systems, 1991, p. 92).
[0416] Liposomes
[0417] There are many organized surfactant structures besides
microemulsions that have been studied and used for the formulation
of drugs. These include monolayers, micelles, bilayers and
vesicles. Vesicles offer specificity and extended duration of
action for drug delivery. Thus, as used herein, the term "liposome"
refers to a vesicle composed of amphiphilic lipids arranged in a
spherical bilayer or bilayers, i.e., liposomes are unilamellar or
multilamellar vesicles which have a membrane formed from a
lipophilic material and an aqueous interior. The aqueous portion
typically contains the composition to be delivered. In order to
cross intact mammalian skin, lipid vesicles must pass through a
series of fine pores, each with a diameter less than 50 nm, under
the influence of a suitable transdermal gradient. Therefore, it is
desirable to use a liposome which is highly deformable and able to
pass through such fine pores. Additional factors for liposomes
include the lipid surface charge, and the aqueous volume of the
liposomes.
[0418] Further advantages of liposomes include; liposomes obtained
from natural phospholipids are biocompatible and biodegradable;
liposomes can incorporate a wide range of water and lipid soluble
drugs; liposomes can protect encapsulated drugs in their internal
compartments from metabolism and degradation (Rosoff, in
Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.),
1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245).
[0419] For topical administration, there is evidence that liposomes
present several advantages over other formulations. Such advantages
include reduced side-effects related to high systemic absorption of
the administered drug, increased accumulation of the administered
drug at the desired target, and the ability to administer a wide
variety of drugs, both hydrophilic and hydrophobic, into the skin.
Compounds including analgesics, antibodies, hormones and
high-molecular weight DNAs have been administered to the skin,
generally resulting in targeting of the upper epidermis.
[0420] Liposomes fall into two broad classes. Cationic liposomes
are positively charged liposomes which interact with the negatively
charged DNA molecules to form a stable complex. The positively
charged DNA/liposome complex binds to the negatively charged cell
surface and is internalized in an endosome. Due to the acidic pH
within the endosome, the liposomes are ruptured, releasing their
contents into the cell cytoplasm (Wang et at., Biochem. Biophys.
Res. Commun., 1987, 147, 980-985).
[0421] Liposomes which are pH-sensitive or negatively-charged,
entrap DNA rather than complex with it. Since both the DNA and the
lipid are similarly charged, repulsion rather than complex
formation occurs. The DNA is thus entrapped in the aqueous interior
of these liposomes. pH-sensitive liposomes have been used, for
example, to deliver DNA encoding the thymidine kinase gene to cell
monolayers in culture (Zhou et al., Journal of Controlled Release,
1992, 19, 269-274).
[0422] One major type of liposomal composition includes
phospholipids other than naturally-derived phosphatidylcholine.
Neutral liposome compositions, for example, can be formed from
dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl
phosphatidylcholine (DPPC). Anionic liposome compositions generally
are formed from dimyristoyl phosphatidylglycerol, while anionic
fusogenic liposomes are formed primarily from dioleoyl
phosphatidylethanolamine (DOPE). Another type of liposomal
composition is formed from phosphatidylcholine (PC) such as, for
example, soybean PC, and egg PC. Another type is formed from
mixtures of phospholipid and/or phosphatidylcholine and/or
cholesterol.
[0423] Several studies have assessed the topical delivery of
liposomal drug formulations to the skin. Application of liposomes
containing interferon to guinea pig skin resulted in a reduction of
skin herpes sores while delivery of interferon via other means
(e.g. as a solution or as an emulsion) were ineffective (Weiner et
at., Journal of Drug Targeting, 1992, 2, 405-410). Further, an
additional study tested the efficacy of interferon administered as
part of a liposomal formulation to the administration of interferon
using an aqueous system, and concluded that the liposomal
formulation was superior to aqueous administration (du Plessis et
al., Antiviral Research, 1992, 18, 259-265).
[0424] Non-ionic liposomal systems have also been examined to
determine their utility in the delivery of drugs to the skin, in
particular systems comprising non-ionic surfactant and cholesterol.
Non-ionic liposomal formulations comprising Novasone.TM. I
(glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether)
and Novasome.TM. II (glyceryl
distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used
to deliver cyclosporin-A into the dermis of mouse skin. Results
indicated that such non-ionic liposomal systems were effective in
facilitating the deposition of cyclosporin-A into different layers
of the skin (Hu et at. S.T.P. Pharma. Sci., 1994, 4, 6, 466).
[0425] Liposomes also include "sterically stabilized" liposomes, a
term which, as used herein, refers to liposomes comprising one or
more specialized lipids that, when incorporated into liposomes,
result in enhanced circulation lifetimes relative to liposomes
lacking such specialized lipids. Examples of sterically stabilized
liposomes are those in which part of the vesicle-forming lipid
portion of the liposome include one or more glycolipids, such as
monosialoganglioside G.sub.M1, or is derivatized with one or more
hydrophilic polymers, such as a polyethylene glycol (PEG) moiety.
Without being bound by any particular theory, it is believed that
for sterically stabilized liposomes containing gangliosides,
sphingomyelin, or PEG-derivatized lipids, the increase in
circulation half-life of these sterically stabilized liposomes is
due to a reduced uptake into cells of the reticuloendothelial
system (RES) (Allen et at., FEBS Lett., 1987, 223, 42; Wu et al.,
Cancer Research, 1993, 53, 3765).
[0426] Various liposomes that include one or more glycolipids have
been reported in Papahadjopoulos et al., Ann. N.Y. Acad. Sci.,
1987, 507, 64 (monosiatoganglioside G.sub.M1, galactocerebroside
sulfate and phosphatidylinositol); Gabizon et at., Proc. Natl.
Acad. Sci. USA., 1988, 85, 6949; Allen et al., U.S. Pat. No.
4,837,028 and International Application Publication WO 88/04924
(sphingomyelin and the ganglioside G.sub.M1 or a galactocerebroside
sulfate ester); Webb et al., U.S. Pat. No. 5,543,152
(sphingomyelin); Lim et al., WO 97/13499
(1,2-sn-dimyristoylphosphatidylcholine).
[0427] Liposomes that include lipids derivatized with one or more
hydrophilic polymers, and methods of preparation are described, for
example, in Sunamoto et al., Bull. Chem. Soc. Jpn., 1980, 53, 2778
(a nonionic detergent, 2C.sub.1215G, that contains a PEG moiety);
Illum et al., FEBS Lett, 1984, 167, 79 (hydrophilic coating of
polystyrene particles with polymeric glycols); Sears, U.S. Pat.
Nos. 4,426,330 and 4,534,899 (synthetic phospholipids modified by
the attachment of carboxylic groups of polyalkylene glycols (e.g.,
PEG)); Klibanov et al., FEBS Lett., 1990, 268, 235
(phosphatidylethanolamine (PE) derivatized with PEG or PEG
stearate); Blume et al., Biochimica et Biophysica Acta, 1990, 1029,
91 (PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the
combination of distearoylphosphatidylethanolamine (DSPE) and PEG);
Fisher, European Patent No. EP 0 445 131 B1 and WO 90/04384
(covalently bound PEG moieties on liposome external surface);
Woodle et al., U.S. Pat. Nos. 5,013,556 and 5,356,633, and Martin
et al., U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496
813 B1 (liposome compositions containing 1-20 mole percent of PE
derivatized with PEG); Martin et al., WO 91/05545 and U.S. Pat. No.
5,225,212 and in Zalipsky et al., WO 94/20073 (liposomes containing
a number of other lipid-polymer conjugates); Choi et al., WO
96/10391 (liposomes that include PEG-modified ceramide lipids);
Miyazaki et al., U.S. Pat. No. 5,540,935, and Tagawa et al., U.S.
Pat. No. 5,556,948 (PEG-containing liposomes that can be further
derivatized with functional moieties on their surfaces).
[0428] Liposomes that include nucleic acids have been described,
for example, in Thierry et al., WO 96/40062 (methods for
encapsulating high molecular weight nucleic acids in liposomes);
Tagawa et al., U.S. Pat. No. 5,264,221 (protein-bonded liposomes
containing RNA); Rahman et al., U.S. Pat. No. 5,665,710 (methods of
encapsulating oligodeoxynucleotides in liposomes); Love et al., WO
97/04787 (liposomes that include antisense oligonucleotides).
[0429] Another type of liposome, transfersomes are highly
deformable lipid aggregates which are attractive for drug delivery
vehicles. (Cevc et al., 1998, Biochim Biophys Acta.
1368(2):201-15.) Transfersomes may be described as lipid droplets
which are so highly deformable that they can penetrate through
pores which are smaller than the droplet. Transfersomes are
adaptable to the environment in which they are used, for example,
they are shape adaptive, self-repairing, frequently reach their
targets without fragmenting, and often self-loading. Transfersomes
can be made, for example, by adding surface edge-activators,
usually surfactants, to a standard liposomal composition.
[0430] Surfactants
[0431] Surfactants are widely used in formulations such as
emulsions (including microemulsions) and liposomes. The most common
way of classifying and ranking the properties of the many different
types of surfactants, both natural and synthetic, is by the use of
the hydrophile/lipophile balance (HLB). The nature of the
hydrophilic group (also known as the "head") provides the most
useful means for categorizing the different surfactants used in
formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel
Dekker, Inc., New York, N.Y., 1988, p. 285).
[0432] If the surfactant molecule is not ionized, it is classified
as a nonionic surfactant. Nonionic surfactants are widely used in
pharmaceutical and cosmetic products and are usable over a wide
range of pH values, and with typical HLB values from 2 to about 18
depending on structure. Nonionic surfactants include nonionic
esters such as ethylene glycol esters, propylene glycol esters,
glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose
esters, and ethoxylated esters; and nonionic alkanolamides and
ethers such as fatty alcohol ethoxylates, propoxylated alcohols,
and ethoxylated/propoxylated block polymers are also included in
this class. The polyoxyethylene surfactants are the most commonly
used members of the nonionic surfactant class.
[0433] Surfactant molecules that carry a negative charge when
dissolved or dispersed in water are classified as anionic. Anionic
surfactants include carboxylates such as soaps, acyl lactylates,
acyl amides of amino acids, esters of sulfuric acid such as alkyl
sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl
benzene sulfonates, acyl isothionates, acyl laurates and
sulfosuccinates, and phosphates. The alkyl sulfates and soaps are
the most commonly used anionic surfactants.
[0434] Surfactant molecules that carry a positive charge when
dissolved or dispersed in water are classified as cationic.
Cationic surfactants include quaternary ammonium salts and
ethoxylated amines, with the quaternary ammonium salts used most
often.
[0435] Surfactant molecules that can carry either a positive or
negative charge are classified as amphoteric. Amphoteric
surfactants include acrylic acid derivatives, substituted
alkylamides, N-alkylbetaines and phosphatides.
[0436] The use of surfactants in drug products, formulations and in
emulsions has been reviewed in Rieger, in Pharmaceutical Dosage
Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).
[0437] Penetration Enhancers
[0438] In some embodiments, penetration enhancers are used in or
with a composition to increase the delivery of nucleic acids,
particularly oligonucleotides, to the skin or across mucous
membranes of animals. Most drugs are present in solution in both
ionized and nonionized forms. However, usually only lipid soluble
or lipophilic drugs readily cross cell membranes. It has been
discovered that even non-lipophilic drugs may cross cell membranes
if the membrane to be crossed is treated with a penetration
enhancer. In addition to aiding the diffusion of non-lipophilic
drugs across cell membranes, penetration enhancers also enhance the
permeability of lipophilic drugs.
[0439] Penetration enhancers may be classified as belonging to one
of five broad categories, i.e., surfactants, fatty acids, bile
salts, chelating agents, and non-chelating nonsurfactants (Lee et
al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.
92). Each of these classes of penetration enhancers is described
below in greater detail.
[0440] Surfactants: In connection with the present invention,
surfactants (or "surface-active agents") are chemical entities
which, when dissolved in an aqueous solution, reduce the surface
tension of the solution or the interfacial tension between the
aqueous solution and another liquid, with the result that
absorption of oligonucleotides through the mucosa is enhanced.
These penetration enhancers include, for example, sodium lauryl
sulfate, polyoxyethylene-9-lauryl ether and
polyoxyethylene-20-cetyl ether) (Lee et at., Critical Reviews in
Therapeutic Drug Carrier Systems, 1991, p. 92); and
perfluorochemical emulsions, such as FC-43. Takahashi et al., J.
Pharm. Pharmacol., 1988, 40, 252), each of which is incorporated
herein by reference in its entirety.
[0441] Fatty acids: Various fatty acids and their derivatives which
act as penetration enhancers include, for example, oleic acid,
lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic
acid, stearic acid, linoleic acid, linolenic acid, dicaprate,
tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin,
caprylic acid, arachidonic acid, glycerol 1-monocaprate,
1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines,
C.sub.1-10 alkyl esters thereof (e.g., methyl, isopropyl and
t-butyl), and mono- and diglycerides thereof (i.e., oleate,
laurate, caprate, myristate, palmitate, stearate, linoleate, etc.)
(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems,
1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug
Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm.
Pharmacol., 1992, 44, 651-654), each of which is incorporated
herein by reference in its entirety.
[0442] Bile salts: The physiological role of bile includes the
facilitation of dispersion and absorption of lipids and fat-soluble
vitamins (Brunton, Chapter 38 in: Goodman & Gilman's The
Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al.
Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural
bile salts, and their synthetic derivatives, act as penetration
enhancers. Thus the term "bile salts" includes any of the naturally
occurring components of bile as well as any of their synthetic
derivatives. The bile salts of the invention include, for example,
cholic acid (or its pharmaceutically acceptable sodium salt, sodium
cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic
acid (sodium deoxycholate), glucholic acid (sodium glucholate),
glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium
glycodeoxycholate), taurocholic acid (sodium taurocholate),
taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic
acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA),
sodium tauro-24,25-dihydro-fusidate (STDHF), sodium
glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee
et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991,
page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical
Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa.,
1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic
Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm.
Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990,
79, 579-583).
[0443] Chelating Agents: In the present context, chelating agents
can be regarded as compounds that remove metallic ions from
solution by forming complexes therewith, with the result that
absorption of oligonucleotides through the mucosa is enhanced. With
regards to their use as penetration enhancers in the present
invention, chelating agents have the added advantage of also
serving as DNase inhibitors, as most characterized DNA nucleases
require a divalent metal ion for catalysis and are thus inhibited
by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339).
Without limitation, chelating agents include disodium
ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g.,
sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl
derivatives of collagen, laureth-9 and N-amino acyl derivatives of
beta-diketones(enamines) (Lee et al., Critical Reviews in
Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi,
Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7,
1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).
[0444] Non-chelating non-surfactants: As used herein, non-chelating
non-surfactant penetration enhancing compounds are compounds that
do not demonstrate significant chelating agent or surfactant
activity, but still enhance absorption of oligonucleotides through
the alimentary mucosa (Muranishi, Critical Reviews in Therapeutic
Drug Carrier Systems, 1990, 7, 1-33). Examples of such penetration
enhancers include unsaturated cyclic ureas, 1-alkyl- and
1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical
Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and
nonsteroidal anti-inflammatory agents such as diclofenac sodium,
indomethacin and phenylbutazone (Yamashita et al, J. Pharm.
Pharmacol., 1987, 39, 621-626).
[0445] Agents that enhance uptake of oligonucleotides at the
cellular level may also be added to the pharmaceutical and other
compositions and formulations of the present invention. For
example, cationic lipids, such as lipofectin (Junichi et al, U.S.
Pat. No. 5,705,188), cationic glycerol derivatives, and
polycationic molecules, such as polylysine (Lollo et al., PCT
Application WO 97/30731), are also known to enhance the cellular
uptake of oligonucleotides.
[0446] Other agents may be utilized to enhance the penetration of
the administered nucleic acids, including glycols such as ethylene
glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and
terpenes such as limonene and menthone.
[0447] Carriers
[0448] Certain compositions of the present invention also
incorporate carrier compounds in the formulation. As used herein,
"carrier compound" or "carrier" can refer to a nucleic acid, or
analog thereof, which is inert (i.e., does not possess biological
activity per se) but is recognized as a nucleic acid by in vivo
processes that reduce the bioavailability of a nucleic acid having
biological activity by, for example, degrading the biologically
active nucleic acid or promoting its removal from circulation. The
coadministration of a nucleic acid and a carrier compound, often
with an excess of the latter substance, can result in a substantial
reduction of the amount of nucleic acid recovered in the liver,
kidney or other extracirculatory reservoirs. For example, the
recovery of a partially phosphorothioate oligonucleotide in hepatic
tissue can be reduced when it is coadministered with polyinosinic
acid, dextran sulfate, polycytidic acid or
4-acetamido-4'isothiocyano-stilbene-2,2-disulfonic acid (Miyao et
al., Antisense Res. Dev., 1995,5, 115-121; Takakura et al.,
Antisense & NucL Acid Drug Dev., 1996, 6, 177-183), each of
which is incorporated herein by reference in its entirety.
[0449] Excipients
[0450] In contrast to a carrier compound, a "pharmaceutical
carrier" or "excipient" is a pharmaceutically acceptable solvent,
suspending agent or any other pharmacologically inert vehicle for
delivering one or more nucleic acids to an animal, and is typically
liquid or solid. A pharmaceutical carrier is generally selected to
provide for the desired bulk, consistency, etc., when combined with
a nucleic acid and the other components of a given pharmaceutical
composition, in view of the intended administration mode. Typical
pharmaceutical carriers include, but are not limited to, binding
agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or
hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and
other sugars, microcrystalline cellulose, pectin, gelatin, calcium
sulfate, ethyl cellulose, polyacrylates or calcium hydrogen
phosphate, etc.); lubricants (e.g., magnesium stearate, talc,
silica, colloidal silicon dioxide, stearic acid, metallic
stearates, hydrogenated vegetable oils, corn starch, polyethylene
glycols, sodium benzoate, sodium acetate, etc.); disintegrants
(e.g., starch, sodium starch glycotate, etc.); and wetting agents
(e.g., sodium lauryl sulphate, etc.).
[0451] Pharmaceutically acceptable organic or inorganic excipients
suitable for non-parenteral administration which do not
deleteriously react with nucleic acids can also be used to
formulate the compositions of the present invention. Suitable
pharmaceutically acceptable carriers include, but are not limited
to, water, salt solutions, alcohols, polyethylene glycols, gelatin,
lactose, amylose, magnesium stearate, talc, silicic acid, viscous
paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the
like.
[0452] Formulations for topical administration of nucleic acids may
include sterile and non-sterile aqueous solutions, non-aqueous
solutions in common solvents such as alcohols, or solutions of the
nucleic acids in liquid or solid oil bases. The solutions may also
contain buffers, diluents and other suitable additives.
Pharmaceutically acceptable organic or inorganic excipients
suitable for non-parenteral administration which do not
deleteriously react with nucleic acids can be used.
[0453] Other Pharmaceutical Composition Components
[0454] The present compositions may additionally contain other
components conventionally found in pharmaceutical compositions, at
their art-established usage levels. Thus, for example, the
compositions may contain additional, compatible,
pharmaceutically-active materials such as, for example,
antipruritics, astringents, local anesthetics or anti-inflammatory
agents, or may contain additional materials useful in physically
formulating various dosage forms of the compositions of the present
invention, such as dyes, flavoring agents, preservatives,
antioxidants, opacifiers, thickening agents and stabilizers.
However, such materials, when added, should not unduly interfere
with the biological activities of the components of the
compositions of the present invention. The formulations can be
sterilized and, if desired, mixed with auxiliary agents, e.g.,
lubricants, preservatives, stabilizers, wetting agents,
emulsifiers, salts for influencing osmotic pressure, buffers,
colorings, flavorings and/or aromatic substances and the like which
do not deleteriously interact with the nucleic acid(s) of the
formulation.
[0455] Aqueous suspensions may contain substances which increase
the viscosity of the suspension including, for example, sodium
carboxymethylcellulose, sorbitol and/or dextran, and/or
stabilizers.
[0456] Certain embodiments of the invention provide pharmaceutical
compositions containing (a) one or more antiviral oligonucleotides
and (b) one or more other chemotherapeutic agents which function by
a different mechanism. Examples of such chemotherapeutic agents
include but are not limited to daunorubicin, daunomycin,
dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin,
bleomycin, mafosfamide, ifosfamide, cytosine arabinoside,
bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D,
mithramycin, prednisone, hydroxyprogesterone, testosterone,
tamoxifen, dacarbazine, procarbazine, hexamethylmelamine,
pentamethytmetamine, mitoxantrone, amsacrine, chlorambucil,
methylcyclohexylnitrosurea, nitrogen mustards, melphalan,
cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,
5-azacytidine, hydroxyurea, deoxycoformycin,
4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),
5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine,
taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate,
irinotecan, topotecan, gemcitabine, teniposide, cisplatin, and
diethylstilbestrol (DES). See, generally, The Merck Manual of
Diagnosis and Therapy, 15th Ed. 1987, pp. 1206-1228, Berkow et al.,
eds., Rahway, N.J. When used with the compounds of the invention,
such chemotherapeutic agents may be used individually (e.g., 5-FU
and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide
for a period of time followed by MTX and oligonucleotide), or in
combination with one or more other such chemotherapeutic agents
(e.g., 5-EU, MTX and oligonucleotide, or 5-FU, radiotherapy and
oligonucleotide). Anti-inflammatory drugs, including but not
limited to nonsteroidal anti-inflammatory drugs and
corticosteroids, and antiviral drugs, including but not limited to
Ribavirin, cidofovir, vidarabine, acyclovir and ganciclovir, may
also be combined in compositions of the invention. See, generally,
The Merck Manual of Diagnosis and Therapy, 15th Ed., Berkow et al.,
eds., 1987, Rahway, N.J., pages 2499-2506 and 46-49, respectively).
Other non-oligonucleotide chemotherapeutic agents are also within
the scope of this invention. Two or more combined compounds may be
used together or sequentially.
EXAMPLES
Example 1
Herpes Simplex Virus
[0457] Herpes simplex virus (HSV) affects a significant proportion
of the human population. It was found in the present invention that
random ONs or ON randomers inhibited the infectivity of viruses
such as HSV. Using cellular HSV replication assays in VERO cells
(susceptible to HSV-1 (strain KOS) and HSV-2 (strain MS2)
infection) it was found that a single stranded PS-ON complementary
to the HSV origin of replication inhibited replication of HSV-1 and
HSV-2. Surprisingly, control PS-ONs complementary to human (343
ARS) and plasmid (pBR322/pUC) origins also inhibited viral
infectivity. Experiments with random sequence PS-ONs and PS-ON
randomers demonstrated that inhibition of viral infection increased
with increasing ON size. These data show that ONs are potent
antiviral agents useful for therapeutic treatment of viral
infection.
[0458] The inventors have theorized that a potential mechanism for
blocking the spread of viruses such as HHVs was to prevent the
replication of its DNA. With this in mind, phosphorothioate
oligonucleotides (ONs) complementary to the origin of replication
of HSV1 and HSV2 were introduced into infected cells. These ONs
would cause DNA triplex formation at the viral origin of
replication, blocking the association of necessary trans-acting
factors and viral DNA replication. Surprising results are presented
herein of these experiments which show that, in an experimental
paradigm, the potency of ONs in inhibiting viral infection
increases as their size (length) increases.
[0459] Inhibition of HSV-1
[0460] The ability of PS-ONs to inhibit HSV-1 is measured in a
plaque reduction assay (PRA). Immortalized African Green Monkey
kidney (VERO) cells are cultured at 37.degree. C. and 5% CO.sub.2
in MEM (minimal essential medium) plus 10% fetal calf serum
supplemented with gentamycin, vancomycin and amphoterecin B. Cells
are seeded in 12 well plates at a density which yields a confluent
monolayer of cells after 4 days of growth. Upon reaching
confluency, the media is changed to contain only 5% serum plus
supplements as described above and cells are then exposed to HSV-1
(strain KOS, approximately 40-60 PFU total) in the presence of the
test compound for 90 minutes. After viral exposure, the media is
replaced with new "overlay" media containing 5% serum, 1% human
immunoglobulins, supplements as described above and the test
compound. Plaque counting is performed 3-4 days post infection
following formalin fixation and cresyl violet staining of infected
cultures.
[0461] All ONs (except where noted otherwise) were synthesized at
the University of Calgary Core DNA Services lab. ONs (see table 21)
are prepared on a 1 or 15 micromol synthesis scale, deprotected and
desalted on a 50 cm Sephadex G-25 column. The resulting ONs are
analyzed by UV shadowing gel electrophoresis and are determined to
contain .about.95% of the full length, n-1 and n-2 oligo and up to
5% of shorter oligo species (these are assumed to have random
deletions). For random oligo synthesis, adenine, guanosine,
cytosine and thymidine amidites are mixed together in equimolar
quantities to maximize the randomness of incorporation at each
position of the ONs during synthesis.
[0462] To test if PS-ONs could inhibit HSV-1, REP 1001, 2001 and
3007 are tested in the HSV-1 PRA. It would have been expected that
only REP 2001 will show any activity as this PS-ON is directed
against the origin of replication in HSV (the other two are
directed against replication origins in humans and plasmids).
However all three PS-ONs showed anti-HSV-1 activity. The testing
was carried out in a plaque reduction assay conducted in VERO cells
using HSV-1 (strain KOS). Infected cells were treated with
increasing concentrations of REP 1001, REP 2001, or REP 3007.
IC.sub.50 values calculated from linear regressions of the assay
results were 2.76, 0.77, and 5.33 micromolar respectively.
Moreover, the potentcy of the anti-HSV-1 effect was found to be
dependent on the size of the oligo.
[0463] To confirm the size dependence and relative sequence
independence of PS-ONs on anti-HSV-1 activity, we tested PS-ONs
that vary in size (REP 2002, 2003, 2004, 2005 and 2006) along with
the antiviral drug Acyclovir. These PS-ONs are rendered inert with
respect to sequence specific effects by synthesizing each base as a
"wobble" (N) so that each PS-ON actually represents a population of
different random sequences with the same size; these PS-ONs are
termed "randomers". Plaque reduction assay was conducted in VERO
cells using HSV-1 (strain KOS). Infected cells are treated with
increasing concentrations of REP 2001, REP 2002 or REP 3003, REP
2004, REP 2005, REP 2006, and Acyclovir. IC.sub.50 values were
calculated from linear regressions of assay data. The relationship
between PS-ON size and IC.sub.50 against HSV-1 was determined by
plotting the IC.sub.50 values against the specific size of each
PS-ON tested which showed anti-HSV-1 activity. The IC.sub.50 for
Acyclovir was used as a reference to a clinical correlate. We found
that oligos 10 bases or lower have no detectable anti-HSV-1
activity but as the size of the PS-ON increases above 10 bases, the
potency also increases (IC.sub.50 decreases). We also noted that
PS-ONs greater than 20 bases had IC.sub.50 values significantly
lower than a clinically accepted anti-HSV-1 drug, acyclovir.
[0464] To better define the effective size range for PS-ON
anti-HSV-1 activity, we tested PS-ON randomers covering a broader
range of sizes from 10 to 120 bases. Plaque reduction assay was
conducted in VERO cells using HSV-1 (strain KOS). A broad range of
PS-ON randomer sizes were tested in increasing concentrations; REP
2003, REP 2009, REP 2010, REP 2011, REP 2012, REP 2004, REP 2006,
REP 2007, and REP 2008. IC.sub.50 values were calculated from
linear regressions. We discovered that oligos 12 bases and larger
have detectable anti-HSV-1 activity and that the efficacy against
HSV-1 also increases with increased PS-ON randomer length up to at
least 120 bases. However, the increases in efficacy per base
increase in size are smaller in PS-ON randomers greater than 40
bases.
[0465] To compare the efficacy of non-PS-ON randomers, a random
sequence PS-ON and a HSV-1 specific sequence PS-ON, we tested these
three types of modifications in ONs 10, 20 and 40 bases in size.
Plaque reduction assay was conducted in VERO cells using HSV-1
(strain KOS). Unmodified ONs, PS-ONs with a random sequence, and
PS-ONs targeting the start codon of HSV-1 IE110 were tested in
increasing concentrations. The ONs were REP 2013, REP 2014, REP
2015, REP 2016, REP 2017, REP 2018, REP 2019, REP 2020, and REP
2021. IC.sub.50 values were calculated from linear regressions. In
this system, unmodified ON randomers have no detectable anti-HSV-1
activity at tested sizes. Both random sequence and specific HSV-1
sequence PS-ONs show size dependent anti-HSV-1 activity (no
activity is observed at 10 bases for either of these modifications.
A comparison of random sequence, specific HSV-1 sequence and
randomer PS-ONs showed that for PS-ONs 20 bases in length, there is
an enhancement of anti-HSV-1 activity with the specific HSV-1
sequence but that at 40 bases in length, all modifications, whether
randomer, random sequence or specific HSV-1 sequence were equally
efficacious against HSV-1.
[0466] To the best of our knowledge, this is the first time
IC.sub.50s for HSV-1 as low as 0.059 .mu.M and 0.043 .mu.M are
reported for PS-ONs.
Example 2
Inhibition of HSV-2
[0467] The ability of PS-ONs to inhibit HSV-2 is measured by PRA.
Immortalized African Green Monkey kidney (VERO) cells are cultured
at 37.degree. C. and 5% CO.sub.2 in MEM plus 10% fetal calf serum
supplemented with gentamycin, vancomycin and amphoterecin B. Cells
are seeded in 12 well plates at a density which yields a confluent
monolayer of cells after 4 days of growth. Upon reaching
confluency, the media is changed to contain only 5% serum plus
supplements as described above and cells are then exposed to HSV-2
(strain MS2, approximately 40-60 PFU total) in the presence of the
test compound for 90 minutes. After viral exposure, the media is
replaced with new "overlay" media containing 5% serum, 1% human
immunoglobulins, supplements as described above and the test
compound. Plaque counting is performed 3-4 days post infection
following formalin fixation and cresyl violet staining of infected
cultures.
[0468] To test if PS-ONs could inhibit HSV-2, REP 1001, 2001 and
3007 are tested in the HSV-2 PRA. Plaque reduction assay was
conducted in human fibroblast cells using HSV-2 (strain MS2), with
infected cells treated with increasing concentrations of REP 1001,
REP 2001, or REP 3007. IC.sub.50 values were calculated from linear
regressions. If the inhibitory activity were due to an antisense or
other sequence complementary mechanism, it would be expected that
only REP 2001 would show any activity as this PS-ON is directed
against the origin of replication in HSV-1/2 (the other two are
directed against replication origins in humans and plasmids
respectively). However all three PS-ONs showed anti-HSV-2 activity.
Moreover, the potency of the anti-HSV-2 effect is dependent on the
size of the PS-ON and independent of the sequence.
[0469] To confirm the size dependence and sequence independence of
PS-ONs on anti-HSV-2 activity, we tested PS-ONs that vary in size
(REP 2001, 2002, 2003, 2004, 2005 and 2006). These PS-ONs are
rendered inert with respect to sequence specific effects by
synthesizing each base as a "wobble" (N) so that each PS-ON
actually represents a population of different random sequences with
the same size, these PS-ONs are termed "randomers". When these
PS-ONs are tested in the HSV-2 PRA, we find that PS-ONs 10 bases or
lower had no detectable anti-HSV-2 activity but as the size of the
PS-ON increases above 10 bases, the potency also increases
(IC.sub.50 decreases). We also noted that PS-ONs greater than 20
bases had IC.sub.50 values significantly lower than a clinically
accepted anti-HSV-2 drug, acyclovir.TM..
[0470] To the best of our knowledge, this is the first time an
IC.sub.50 for HSV-2 as low as 0.012 .mu.M has been reported for a
PS-ON.
[0471] To determine if non-specific sequence composition has an
effect on ON antiviral activity, several PS-ONs of equivalent size
but differing in their sequence composition were tested for
anti-HSV1 activity in the HSV-1 PRA. The PS-ONs tested were REP
2006 (N20), REP 2028 (G40), REP 2029 (A40), REP 2030 (T40) and REP
2031 (C40). The IC.sub.50 values generated from the HSV-1 PRA show
that REP 2006 (N40) was the most active of all sequences tested
while REP 2029 (A40) was the least active. We also note that, all
the other PS-ONs were significantly less active than N40 with their
rank in terms of efficacy being
N40>C40>T40>A40>>G40.
[0472] We also tested the efficacy of different PS ONs having
varying sequence composition with two different nucleotides. The
PS-ON randomer (REP 2006) was significantly more efficacious
against HSV-1 than AC20 (REP 2055), TC20 (REP 2056) or AG20 (REP
2057) with their efficacies ranked as follows:
N40>AG>AC>TC. This data suggests that although the
anti-viral effect is non-sequence complementary, certain
non-specific sequence compositions (ie C40 and N40) have more
potent anti-viral activity. We suggest that this phenomenon can be
explained by the fact that, while retaining intrinsic protein
binding ability, sequences like C40, A40, T40 and G40 bind fewer
viral proteins with high affinity, probably due to some restrictive
tertiary structure formed in these sequences. On the other hand,
due to the random nature of N40, it retains its ability to bind
with high affinity to a broad range of anti-viral proteins which
contributes to its robust anti-viral activity.
Example 3
Inhibition of CMV
[0473] The ability of PS-ONs to inhibit CMV is measured in a plaque
reduction assay (PRA). This assay is identical to the assay used
for testing anti-HSV-1 and anti-HSV-2 except that CMV (strain
AD169) is used as the viral innoculum and human fibroblasts were
used as cellular host.
[0474] To test the size dependence and sequence independence of
PS-ONs on anti-CMV activity, we tested PS-ON randomers that vary in
size. Plaque reduction assay was conducted in VERO cells using CMV
(strain AD169). Infected cells were treated with increasing
concentrations of REP 2004 (a) or REP 2006 (b). IC.sub.50 values
were calculated from linear regressions, and relationship between
PS-ON size and IC.sub.50 against CMV was determined by plotting
IC.sub.50 values against the specific size of each PS-ON tested.
When these PS-ONs are tested in the CMV PRA, we find that as the
size of the PS-ON increases, the potency also increases (IC.sub.50
decreases).
[0475] To more clearly elucidate the effective size range for PS-ON
anti-CMV activity, we tested PS-ON randomers covering a broader
range of sizes from 10 to 80 bases. We also included several
clinically accepted small molecule CMV treatments (Gancyclovir,
Foscarnet and Cidofovir) as well as 2 versions of a marketed
antisense treatment for CMV retinitis, (Vitravene.TM.; commercially
available and synthesized by the University of Calgary). Plaque
reduction assay was conducted in VERO cells using CMV (strain
AD169). Three clinical CMV therapies were tested: Gancyclovir,
Foscarnet, and Cidofovir. A broad range of PS-ON randomer sizes
were also tested in increasing concentrations; REP 2003, REP 2004,
REP 2006, and REP 2007. Finally, REP 2036 (Vitravene) was tested as
synthesized in house and as commercially available. IC.sub.50
values were calculated from linear regressions. We discovered that
while increased PS-ON randomer size leads to increased efficacy,
this effect saturates at about 40 bases. Moreover, the 20, 40 and
80 base PS-ON randomers are all significantly more efficacious than
any of the small molecule treatments tested. In addition, 40 and 80
base PS-ON randomers are more efficacious than Vitravene.TM..
[0476] To the best of our knowledge, this is the first time an
IC.sub.50 for CMV as low as 0.067 .mu.M has been reported for a
PS-ON.
Example 4
Inhibition of HIV-1
[0477] The ability of PS-ON randomers to inhibit HIV-1 is measured
by two different assays:
[0478] Cytopathic Effect (CPE)
[0479] Cytopathic effect is monitored using MTT dye to report the
extent of cellular metabolism. Immortalized human lymphocyte (MT4)
cells are cultured at 37.degree. C. and 5% CO.sub.2 in MEM plus 10%
fetal calf serum supplemented with antibiotics. Cells are seeded in
96 well plates in media containing the appropriate test compound
and incubated for 2 hours. After preincubation with the test
compound, HIV-1 (strain NL 4-3) was added to the wells (0.0002
TCID.sub.50/cell). After 6 days of additional incubation, CPE is
monitored by MTT conversion. Cytotoxicity is measured by incubating
the drugs for 6 days in the absence of viral inoculation. For
transformation of MTT absorbance values into % survival, the
absorbance of uninfected, untreated cells is set to 100% and the
absorbance of infected, untreated cells is set to 0%.
[0480] Replication Assay (RA)
[0481] The ability of HIV to replicate is monitored in immortalized
human embryonic kidney (293A) cells. These cells are cotransfected
with two plasmids. One plasmid contains a recombinant wild type
HIV-1 genome (NL 4-3) having its env gene disrupted by a luciferase
expression cassette (identified as strain CNDO), the other plasmid
contains the env gene from murine leukemia virus (MLV). These two
plasmids provide all the protein factors in trans to produce a
mature chimeric virus having all the components from HIV-1 except
the protein products provided in trans from the MLV env gene.
Virions produced from these cells are infectious and replicative
but cannot produce another generation of infectious virions because
they will lack the env gene products.
[0482] 24 hours after transfection, these cells are trypsinized and
plated in 96 well plates. After the cells have adhered, the media
is washed and replaced with media containing the test compound.
Virus production is allowed to proceed for an additional 24 hours.
The supernatant is then harvested and used to reinfect naive 293A
cells. Naive cells that are infected are identified by the
luciferase gene product. The number of luciferase positive cells is
a measure of the extent of replication and/or infection by the
recombinant HIV-1. This assay is also adapted to test the
resistance to many clinically accepted anti-HIV-1 drugs by using a
HIV-1 genome with several point mutations known to induce
resistance to several different classes of anti-HIV drugs.
Percentage inhibition is set to 100% for no detectable luciferase
positive cells and 0% for the number of positive cells in infected,
untreated controls.
[0483] To test the size dependence and sequence independence of
PS-ONs on anti-HIV-1 activity, we tested PS-ON randomers that vary
in size. CPE assay was conducted in MT4 cells using HIV-1 (strain
NL4-3). Infected cells were treated with increasing concentrations
of REP 2004 or REP 2006. IC.sub.50 values were calculated from
linear regressions. Cytotoxicity profiles in uninfected MT4 cells
were determined for REP 2004 and REP 2006. We found that as the
size of the PS-ON increases, the potency also increases (IC.sub.50
decreases). We also noted that the PS-ON randomers exhibited no
significant toxicity to the host cells in this assay.
[0484] To the best of our knowledge, this is the first time an
IC.sub.50 for HIV-1 as low as 0.011 .mu.M has been reported for a
PS-ON.
[0485] To more clearly elucidate the effective size range for PS-ON
anti-HIV-1 activity, we tested more PS-ON randomers covering a
broader range of sizes from 10 to 80 bases by RA using wild-type
HIV-I (recombinant NL 4-3 (CNDO)). Replication assay was conducted
in 293A cells using recombinant wild type HIV-1NL4-3 (strain CNDO).
In addition, we tested four protease inhibitors currently used in
the clinic (aprenavir, indinavir, lopinavir and saquinavir).
Infected cells were treated with increasing concentrations of
Amprenavir, Indinavir, Lopinavir, Saquinavir, REP 2003, REP 2004,
REP 2006, and REP 2007. We discovered that PS-ON randomers 10 bases
and larger have anti-HIV-1 activity and that the efficacy against
HIV-1 also increases with increased PS-ON randomer length but is
saturated at about 40 bases. Moreover, the 40 and 80 base PS-ON
randomers were almost equivalent in efficacy with the 4 clinical
controls.
[0486] To the best of our knowledge, this is the first time an
IC.sub.50 for HIV-1 as low as 0.014 .mu.M has been reported for a
PS-ON.
[0487] To test the ability of PS-ON randomers to inhibit a drug
resistant strain of HIV, we duplicated the above test using the
recombinant MDRC4 strain of HIV-1. This recombinant strain exhibits
significant resistance to at least 16 different clinically accepted
drugs from all classes: nucleotide RT inhibitors, non-nucleotide RT
inhibitors and protease inhibitors. We found that all the PS-ON
randomers tested perform as well against the resistant strain as
they do against the wild type strain. However, three of the four
protease inhibitors show a reduction in their efficacy against the
mutant strain, such that the 40 and 80 base PS-ON randomers were
more potent against this resistant strain than these drugs.
Example 5
Inhibition of RSV
[0488] The ability of PS-ON randomers to inhibit RSV is measured by
monitoring CPE with alamar blue (an indirect measure of cellular
metabolism). Human larynx carcinoma (Hep2) cells are cultured at
37.degree. C. and 5% CO.sub.2 in MEM plus 5% fetal calf serum.
Cells are seeded in 96 well plates at a density which yields a
confluent monolayer of cells after 5-6 days of growth. The day
after plating, cells were infected with RSV (strain A2,
10.sup.8.2TCID.sub.50/ml) in the presence of the test compound in a
reduced volume for 2 hours. Following inoculation, the media was
changed and was supplemented with test compound. 6 days after
infection, CPE was monitored by measuring the fluorescent
conversion of alamar blue. Toxicity of test compounds in Hep2 cells
was monitored by treating uninfected cells for 7 days and measuring
alamar blue conversion in these cells. The alamar blue readings in
uninfected, untreated cells were set to 100% survival and the
readings in infected, untreated cells were set to 0% survival.
[0489] To confirm the size dependence and sequence independence of
PS-ONs on anti-RSV activity, we tested PS-ON randomers that vary in
size. In addition, we tested the clinically accepted treatment for
RSV infection, Ribavirin (Virazole.TM.). CPE assay was conducted in
Hep2 cells using RSV (strain A2). Infected cells are treated with
increasing concentrations of REP 2004, REP 2006, REP 2007, or
Ribavirin. IC.sub.50 values were calculated from linear regressions
are reported in each graph. Cytotoxicity profiles in uninfected
Hep2 cells were determined for REP 2004, REP 2006, REP 2007, or
Ribavirin. We found that as the size of the PS-ON randomer
increases, the potency also increases but saturates at about 40
bases in size. We also noted that 20, 40 and 80 base PS-ON
randomers had IC.sub.50 values significantly lower than a
clinically accepted anti-RSV drug, Ribavirin. PS-ON randomers
exhibited no toxicity in Hep2 cells while Ribavirin was
significantly toxic (therapeutic index=2.08).
[0490] To the best of our knowledge, this is the first time an
IC.sub.50 for RSV-1 as low as 0.015 .mu.M has been reported for a
PS-ON.
Example 6
Inhibition of Coxsackie Virus B2
[0491] The ability of PS-ON randomers to inhibit COX B2 is measured
monitoring CPE with alamar blue (an indirect measure of cellular
metabolism). Rhesus monkey kidney (LLC-MK2) cells are cultured at
37.degree. C. and 5% CO.sub.2 in MEM plus 5% fetal calf serum.
Cells are seeded in 96 well plates at a density which yields a
confluent monolayer of cells after 5-6 days of growth. The day
after plating, cells were infected with COX B2 (strain Ohio-1,
10.sup.7.8TCID.sub.50ml) in the presence of the test compound in a
reduced volume for 2 hours. Following inoculation, the media was
changed and was supplemented with test compound. 6 days after
infection, CPE was monitored by measuring the fluorescent
conversion of alamar blue. Toxicity of test compounds in LLC-MK2
cells was monitored by treating uninfected cells for 7 days and
measuring alamar blue conversion in these cells. The alamar blue
readings in uninfected, untreated cells were set to 100% survival
and the readings in infected, untreated cells were set to 0%
survival.
[0492] We tested the anti-COX B2 activity of REP 2006 in the COX B2
CPE assay. The CPE assay was conducted in LLC-MK2 cells using
Coxsackievirus B2 (strain Ohio-1). Infected cells were treated with
increasing concentrations of REP 2006. The cytotoxicity profile for
REP 2006 in LLC-MK2 cells was determined. We found that, while
exhibiting some slight toxicity in LLC-MK2 cells, this PS-ON
randomer was able to partially rescue infected LLC-MK2 cells from
COX B2 infection.
Example 7
Inhibition of Vaccinia Virus
[0493] We used the vaccinia infection model as an indicator of the
efficacy of our compounds against poxviruses, including smallpox
virus. The ability of PS-ON randomers to inhibit Vaccinia is
measured by monitoring CPE with alamar blue (an indirect measure of
cellular metabolism). Vero cells are cultured at 37.degree. C. and
5% CO.sub.2 in MEM plus 5% fetal calf serum. Cells are seeded in 96
well plates at a density which yields a confluent monolayer of
cells after 5-6 days of growth. The day after plating, cells were
infected with Vaccinia (10.sup.7.9TCID.sub.50/ml) in the presence
of the test compound in a reduced volume for 2 hours. Following
inoculation, the media was changed and was supplemented with test
compound (all at 10 .mu.M, except for Cidofovir which was used at
50 .mu.M). Five days after infection, the supernatants were
harvested. The viral load in the supernatant was determined by
reinfection of VERO cells with supernatant diluted 1:100 and the
monitoring of CPE 7 days after reinfection by measuring the
fluorescent conversion of alamar blue.
[0494] We tested PS-ON randomers that vary in size (REP 2004, 2006
and 2007). In addition, we tested a known effective treatment for
Vaccinia infection, Cidofovir (Vistide.TM.). Indirect determination
of viral load in infected supernatants from vaccinia infected VERO
cells was determined by measuring the CPE induced by these
supernatants in naive cells. REP 2004, 2006 and 2007 were tested at
10 .mu.M while Cidofovir was tested at 50 .mu.M. When tested in the
Vacinnia CPE assay, we found that treatment with REP 2004, 2006 and
2007 all displayed antiviral activity (ie. resulted in supernatants
which showed a decreased CPE upon reinfection) but that this
activity was weaker than that seen for Cidofovir.
Example 8
Inhibition of DHBV, Parainfluenza-3 Virus, and Hanta Virus
[0495] Because DHBV, Parainfluenza-3 virus and Hanta virus do not
readily generate measurable plaques or CPE, we tested the efficacy
of REP 2006 in these viruses using a fluorescence focus forming
unit (FFFU) detection. In this assay, REP 2006 (at a final
concentration of 10 .mu.M) is mixed with the virus which is then
adsorbed onto the cells. After adsorption, infected cells are
allowed to incubate for an additional 7-14 days at which point they
are fixed in methanol. Regions of viral replication are detected by
immunofluorescence microscopy against the appropriate viral
antigen. For each of the three viruses tested, the specific
experimental conditions and results are described in Table 1 below:
TABLE-US-00001 TABLE 1 Inhibition of DHBV, Parainfluenza-3 virus,
and Hanta virus. FFFU count Antibody for FFFU FFFU count (10 .mu.M
REP Virus Cellular Host detection (no drug) 2006) DHBV (HBV Primary
duck Mouse anti-DHBV 163 +/- 38.5 0 surrogate) hepatocytes IgG
Parainfluenza-3 LLC-MK2 cells Mouse anti-PI3 IgG 288 +/- 126 0
Hanta Virus VERO E6 cells Mouse anti- 232.3 +/- 38.17 0 (Strain
SinNombre Prospect Hill) nucleoprotein IgG
[0496] This initial data shows that at 10 .mu.M, REP 2006 is
effective in inhibiting DHBV, Parainfluenza-2 and Hanta Virus. We
anticipate that given the robust response in the preliminary test
that IC.sub.50 values will be lower. These data support the
efficacy of PS-ON randomers for the treatment of human infections
of Hanta Virus and Hepatitis B (closely related to DHBV) as well as
providing a rationale for the immediate treatment of pediatric
bronchiolitis caused by RSV and Parainfluenza-3, which may not
require differential diagnosis for treatment to begin.
Example 9
Currently Non-Responsive Viruses
[0497] To date we have not observed a detectable anti-viral
efficacy with PS-ON randomers (up to 10 .mu.M) without using a
delivery system, a drug combination, or a chemical modification in
the following viral systems described in Table 2: TABLE-US-00002
TABLE 2 Viral Systems Assay Virus Strain Cellular Host paradigm
Corona virus MHV2 (mouse) NCTC-1496 cells Plaque (SARS surrogate)
MHV-A59 (mouse) DBT cells reduction HCoV-OC43 HRT-18 cells (human)
BVDV (HCV NA BT cells CPE by surrogate) alamar blue Rhinovirus HGP
HeLa cells CPE by alamar blue Adenovirus Human Ad5 293A cells
Plaque reduction
[0498] Under the current testing procedures, we did not demonstrate
activity. However, the lack of demonstrated antiviral activity may
be due to limitations of the particular assays used. Additional
testing is underway to demonstrate efficacious results with these
viruses.
[0499] Since our evidence indicates that the charge characteristics
of a PS-ON are important for the inhibition of viruses from several
different families, we expect that this charge dependent mechanism
for the inhibition of viral activity has the potential to inhibit
the activity of all encapsidating viruses. The corollary to this is
that the lack of detected anti-viral efficacy against those viruses
listed in Example 9 suggests that the interaction between the PS-ON
and the structural proteins of these viruses may not strong be
enough to prevent the interaction of viral proteins during the
replication of these viruses. In this case, one way of achieving
efficacy against these viruses is to alter the charge
characteristics of the DNA or anti-viral polymer (e.g.,
substituting phosphorodithioate for phosphorothioate linkages in
DNA) so their affinity for viral proteins is increased.
Example 10
Inhibition of Influenza A
[0500] The ability of PS-ONs to inhibit the influenza virus (INF) A
is measured in a plaque reduction assay (PRA). Immortalized Canine
kidney (MDCK) cells are cultured at 37.degree. C. and 5 CO.sub.2 in
MEM plus 10% fetal calf serum supplemented with gentamycin,
vancomycin and amphoterecin B. Cells are seeded in 6 well plates at
a density which yields a confluent monolayer of cells after 6 days
of growth. Upon reaching confluency, the media is changed to
contain only supplements as described above and cells are then
exposed to INF A (strain H3N2, approximately 35-70 PFU total) in
the presence of the test compound for 60 minutes. After viral
exposure, the media is replaced with new media containing drug
only. 24 hours after infection, media is again replaced with
overlay media containing 4% albumin, 0.025% DEAE dextran, 2 mg/ml
TPCK-treated trypsin and 0.8% seaplaque agarose, supplements as
described above and no test compound. Plaque counting is performed
2-3 days post infection following formalin fixation and cresyl
violet staining of infected cultures.
[0501] We tested the anti INF A activity of a variety of PS-ON
randomers in the INF A PRA assay. We found that only REP 2006
showed any measurable antiviral activity but that this activity was
significant (see following table 3). TABLE-US-00003 TABLE 3
Activity of PS-ON randomers against INF A (H3N2). Randomer
IC.sub.50 (.mu.M) REP 2003 >10 REP 2004 >10 REP 2006
.about.3
[0502] Since only the largest randomer seemed to have any activity
and we know that the activity of randomers in many other viruses
was size dependent, we tested the antiviral activity of a larger
size distribution of randomers using a broader dilution range. We
discovered that as for other viruses we had tested, the anti-INF A
activity of randomers became more potent as their length increased
but that no significant increase in activity was seen for randomers
above 40 bases in length. TABLE-US-00004 TABLE 4 Size dependent
anti-INF A activity of PS-ON randomers. Randomer IC.sub.50 (.mu.M)
REP 2032 >50 REP 2003 >50 REP 2004 .about.25 REP 2005
.about.6.25 REP 2006 .about.1.25 REP 2007 .about.0.625
[0503] To determine the mechanism of action of REP 2006 we
attempted to determine the effect of adding REP 2006 (at IC99
concentration) at various times before, during and after infection.
In this experiment, we observed that even 5 hours (300 min) after
infection, adding REP 2006 resulted in a complete inhibition of INF
A activity (see following table). These results indicate that at
least a significant portion of the action of REP 2006 against
influenza occurs post infection. Since PS-ON randomers do not
readily enter the cell, PS-ON randomers may also interfere with
viral budding from the host cell. TABLE-US-00005 TABLE 5 Time of
addition of REP 2006 versus effect on INF A activity. Time of REP
2006 mixing with virus relative to Infectivity infection (min) (%)
no drug (ctl) 100 -30 0 -5 0 0 0 5 0 30 0 60 0 90 0 120 0 180 0 240
0 300 0
Example 11
Tests for Determining if an Oligonucleotide Acts Predominantly by a
Sequence Independent Mode of Action
[0504] We have shown herein that the antiviral activity of the
present ONs occurs by a sequence-independent mode of action. Of
course a person skilled in the art could prepare sequence-specific
ONs, for example an antisense ON targeting a mRNA of a particular
virus and incorporating all phosphorothioate and 2' O-methyl
modifications. However such an ON would have benefited from the ON
modifications we have described herein and the fact that we have
demonstrated herein that the activity of such a modified ON is
sequence independent. Thus, an ON shall be considered to have
sequence-independent activity if it meets the criteria of any one
of the 5 tests outlined below, i.e., if a substantial part of its
function is due to a sequence-independent activity. The ONs used in
the following tests can be prepared following the general
methodology described in example 12 for the synthesis of
PS-ONs.
Test #1--Effect of Partial Degeneracy of a Candidate ON on its
Antiviral Efficacy
[0505] This test serves to measure the antiviral activity of a
candidate ON sequence when part of its sequence is made degenerate.
If the degenerate version of the candidate ON having the same
chemistry retains its activity as described below, is it deemed to
have sequence-independent activity. Candidate ONs will be made
degenerate according to the following rule: [0506] L=the number of
bases in the candidate ON [0507] X=the number of bases on each end
of the oligo to be made degenerate (but having the same chemistry
as the candidate ON) [0508] If L is even, then X=integer (L/4)
[0509] If L is odd, then X=integer ((L+1)/4) [0510] X must be equal
to or greater than 4
[0511] If the candidate ON is claimed to have an anti-viral
activity against a member of the herpesviridae, retroviridae, or
paramyxoviridae families, the IC.sub.50 generation will be
performed using the assay described herein for that viral family
preferably using the viral strains indicated. If the candidate ON
is claimed to have an anti-viral activity against a member of a
particular virus family not mentioned above, then the IC.sub.50
values shall be generated by a test of antiviral efficacy accepted
by the pharmaceutical industry. IC.sub.50 values shall be generated
using a minimum of seven concentrations of compound, with three or
more points in the linear range of the dose response curve. Using
these tests, the IC.sub.50 of the candidate ON shall be compared to
its degenerate counterpart. If the IC.sub.50 of the partially
degenerate ON is less than 5-fold greater than the original
candidate ON (based on minimum triplicate measurements, standard
deviation not to exceed 15% of mean) then the ON shall be deemed to
act predominantly by a sequence independent mode of action.
Test #2--Comparison of Antiviral Activity of a Candidate ON with an
ON Randomer.
[0512] This test serves to compare the anti-viral efficacy of a
candidate ON with the antiviral efficacy of a randomer ON of
equivalent size and chemistry in the same virus.
[0513] If the candidate ON is claimed to have an anti-viral
activity against a member of the herpesviridae, retroviridae, or
paramyxoviridae families, the IC.sub.50 generation will be
performed using the assay described herein for that viral family
preferably using the viral strains indicated. If the candidate ON
is claimed to have an anti-viral activity against a member of a
particular virus family not mentioned above, then the IC.sub.50
values shall be generated by a test of antiviral efficacy accepted
by the pharmaceutical industry. IC.sub.50 values shall be generated
using a minimum of seven concentrations of compound, with three or
more points in the linear range of the dose response curve. Using
this test, the IC.sub.50 of the candidate ON shall be compared to
an ON randomer of equivalent size and chemistry. If the IC.sub.50
of the ON randomer is less than 5-fold greater than the candidate
ON (based on minimum triplicate measurements, standard deviation
not to exceed 15% of mean) then the candidate ON shall be deemed to
act predominantly by a sequence independent mode of action.
Test #3--Comparison of Antiviral Activity of a Candidate ON in Two
Non-Homologous Viruses from the Same Viral Family
[0514] This test serves to compare the efficacy of a candidate ON
against a target virus whose genome is homologous to the candidate
ON with the efficacy of the candidate ON against a second virus
whose genome has no homology to that candidate ON but is in the
same viral family. For example, if a candidate ON is reported to
have activity against HSV, its activity against HSV will be
compared to its activity against CMV or VZV etc. The comparison of
the relative activities of the candidate ON in the target virus and
the second virus is accomplished by using the activities of an ON
randomer of the same length and chemistry in both viruses to
normalize the IC.sub.50 values for the candidate ON obtained in the
two viruses.
[0515] Thus, if the candidate ON is claimed to have an anti-viral
activity against a certain virus, then the IC.sub.50 generation
will be determined in this virus using one of the assays described
herein for the herpesviridae, retroviridae, or paramyxoviridae
families, or other assays known in the art. Similarly, IC.sub.50
generation will be performed for the candidate ON against a second
virus using one of the assays as described herein or an assay
accepted by the industry for a virus whose genome has no homology
to the sequence of the candidate ON but is from the same viral
family. IC.sub.50 generation is also performed for a randomer of
equivalent size and chemistry against each of the viruses. The
IC.sub.50 of the ON randomer against the two viruses are used to
normalize the IC.sub.50 values for the candidate ON against the two
viruses as follows: [0516] An equivalent algebraic transformation
is applied to the IC.sub.50 of the candidate ON and the ON randomer
in the first (homologous) virus such that the IC.sub.50 of the
randomer is now 1. [0517] An equivalent algebraic transformation is
applied to the IC.sub.50 of the candidate ON and the ON randomer in
the second (non-homologous) virus such that the IC.sub.50 of the
randomer is now 1. [0518] The fold difference in the IC.sub.50s for
the candidate ON in the homologus versus the non-homologous virus
is calculated by dividing the transformed IC.sub.50 of the
candidate ON in the non-homologous virus by the transformed
IC.sub.50 of the candidate ON in the homologous virus.
[0519] The candidate ON shall be deemed to act predominantly by a
sequence independent mode of action if the fold difference in
IC.sub.50 between the two viruses is less than 5.
Test #4: Antiviral Activity of a Candidate ON in a Different Viral
Family
[0520] This test serves to determine if a candidate ON has a
drug-like activity in a virus where the sequence of the candidate
ON is not homologous to any portion of the viral genome and the
virus is from a different family. Thus the candidate ON shall be
tested using one of the assays described herein for the
herpesviridae, retroviridae or paramyxoviridae such that the
sequence of the candidate ON tested is not homologous to any
portion of the genome of the virus to be used. An IC.sub.50 value
shall be generated using a minimum of seven concentrations of the
candidate ON, with three or more points in the linear range. If the
resulting dose response curve indicates a drug-like activity (which
can typically be seen as a decay or sigmoidal curve, having reduced
anti-viral efficacy with decreasing concentrations of candidate ON)
and the IC.sub.50 generated from the curve is less than 10 .mu.M,
the candidate ON shall be deemed to have a drug-like activity. If
the candidate ON is deemed to have a drug-like activity in a virus
from a different family for which the candidate ON is not
complementary and thus can have no sequence dependent antisense
activity, it shall be considered to act predominantly by a sequence
independent mode of action.
Test #5. Extracellular Antiviral Activity of a Candidate ON
[0521] Our current results indicate that the sequence-independent
antiviral activity of ONs occurs outside the cell. The state of the
art in ON technology teaches that, since ONs are not readily cell
permeable, they must be delivered across the cell membrane by an
appropriate carrier to have antisense activity. Thus, the antiviral
activity of antisense ONs by definition is dependent on delivery
inside cells for activity. If a particular sequence-specific
candidate ON has in vitro antiviral activity when used naked (and
therefore having poor intracellular penetration), it must benefit
from the sequence-independent properties of ONs described in this
invention.
[0522] If the sequence-specific candidate ON is complementary to a
portion of the genome of HSV-1, HIV-1 or RSV, then the presence of
a sequence-independent antiviral activity of the candidate ON shall
be determined in the appropriate assay described below. If the
candidate ON is complementary to a virus which is not HSV-1, HIV-1
or RSV, then the antiviral activity of the candidate ON shall be
determined using an assay accepted by the pharmaceutical
industry.
[0523] Using the appropriate assay, the antiviral activity of the
naked candidate ON shall be compared to that of the encapsulated
(for transfection) candidate ON (using identical candidate ON
concentrations in both naked and encapsulated conditions). The
activity shall be measured by a dose response curve with not less
than 7 concentrations, at least 3 of which fall in the linear range
which includes the 50% inhibition of viral activity. The IC.sub.50
(the concentration which reduces viral activity by 50%) shall be
calculated by linear regression of the linear range of the dose
response curve as defined above. If the IC.sub.50 of the naked
candidate ON is less than 5-fold greater than that of the
encapsulated candidate ON, then the activity of the candidate ON
shall be deemed to act predominantly by a sequence independent mode
of action.
Thresholds Used in These Tests
[0524] The purpose of these tests are to determine by a reasonable
analysis, if ONs benefit from or utilize the sequence-independent
antiviral properties of ONs which we have described herein and is
acting with sequence-independent activity. Of course anyone skilled
in the art will realize that, given the inherent variability of all
testing methodologies, especially antiviral testing methods, a
determination of differences in antiviral activity between two
compounds may not be reliably concluded if the threshold is set at
a 2 or 3 fold difference between the activities of said compounds.
This is due to the fact that variations from experiment to
experiment with the same compound in these assays can yield
IC.sub.50s which vary in this range. Thus, to be reasonably certain
that real differences between the activities of two compounds (e.g.
two ONs) exist, we set a threshold of at least a 5-fold difference
between the IC.sub.50s of said compounds. This threshold ensures
the reliability of the assessment of the above mentioned tests.
[0525] The thresholds described in tests 1 to 3 and 5 above are the
default thresholds. If specifically indicated, other thresholds can
be used in the comparison tests 1 to 3 and 5 described above. Thus
for example, if specifically indicated, the threshold for
determining whether an ON is acting with sequence-independent
activity can be any of 10-fold, 8-fold, 6-fold, 5-fold, 4-fold,
3-fold, 2-fold, 1.5-fold, or equal. The threshold described in test
4 above is also a default threshold. If specifically indicated, the
threshold for determining whether an ON has sequence-independent
activity in test 4 can be an IC.sub.50 of less than 10 .mu.M, 5
.mu.M, 1 .mu.M, 0.8 .mu.M, 0.6 .mu.M, 0.5 .mu.M, 0.4 .mu.M, 0.3
.mu.M, 0.2 .mu.M or 0.1 .mu.M.
[0526] Similarly, though the default is that satisfying any one of
the above 5 tests is sufficient, if specifically indicated, the ON
can be required to satisfy any two (e.g., tests 1 & 2, 1 &
3, 1 & 4, 1 & 5, 2 & 3, 2& 4, 2 & 5, 3 & 4,
and 3 & 5), any three (e.g., tests 1 & 2 & 3,1 & 2
& 4, 1,& 2 & 5, 1 & 3 & 4, 1 & 3 & 5, 2
& 3 & 4, and 2 & 4 & 5), any 4 of the tests (e.g.,
1 & 2 & 3 & 4, 1 & 2 & 3 & & 5, and 2
& 3 & 4 & 5) at a default threshold, or if specifically
indicated, at another threshold(s) as indicated above.
Example 12
Methodologies
[0527] The following methods are provided for application in the
tests described in example 11.
Oligonucleotide Synthesis
[0528] The present oligonucleotides can by synthesized using
methods known in the art. For example, unsubstituted and
substituted phosphodiester (P.dbd.O) oligonucleotides can be
synthesized on an automated DNA synthesizer (e.g., Applied
Biosystems model 380B or Akta Oligopilot 100) using standard
phosphoramidite chemistry with oxidation by iodine.
Phosphorothioates (P.dbd.S) can be synthesized as for the
phosphodiester oligonucleotides except the standard oxidation
bottle can be replaced by 0.2 M solution of
311-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the
step-wise thioation of the phosphite linkages. The thioation wait
step can be increased to 68 sec, followed by the capping step.
After cleavage from the support column and deblocking in
concentrated ammonium hydroxide at 55.degree. C. (18 h), the
oligonucleotides can be purified by precipitating twice with 2.5
volumes of ethanol from a 0.5 M NaCl solution.
Antiviral Assay for Herpesviridae
[0529] A plaque reduction assay for herpesviridae is performed as
follows:
[0530] For HSV-1 or HSV-2, VERO cells (ATCC# CCL-81) are grown to
confluence in 12 well tissue culture plates (NUNC or equivalent) at
37.degree. C. and 5% CO.sub.2 in the presence of MEM supplemented
with 10% heat inactivated fetal calf serum and gentamycin,
vancomycin and amphoterecin B. Upon reaching confluency, the media
is changed to contain 5% fetal calf serum and antibiotics as
detailed above supplemented with either HSV-1 (strain KOS, 40-60
PFU total) or HSV-2 (strain MS2, 40-60 PFU total). Viral adsorbtion
proceeds for 90 minutes, after which cells are washed and replaced
with new "overlay" media containing 5% fetal calf serum and 1%
human immunoglobins. Three to four days after adsorbtion, cells are
fixed by formalin and plaques are counted following formalin
fixation and cresyl violet staining.
[0531] For CMV, human fibroblasts are grown as specified for VERO
cells in the HSV-1/2 assay. Media components and adsorbtion/overlay
procedures are identical with the following exceptions: [0532] 1.
CMV (strain AD169, 40-60 PFU total) is used to infect cells during
the adsorbtion. [0533] 2. In the overlay media, 1% human
immunoglobins are replaced by 4% sea-plaque agarose.
[0534] For other herpesviridae, testing is to be conducted in the
plaque assay described above using an appropriate cellular host and
40-60 PFU of virus during the adsorbtion.
[0535] This test is only valid if identifiable plaques are present
in the absence of compound at the end of the test.
[0536] In this test, IC.sub.50 is the concentration at which 50% of
the plaques are present compared to the untreated control.
[0537] Compound to be tested is present during the adsorption and
in the overlay.
Antiviral Assay for Retroviridae
[0538] Assaying for the retroviridae HIV-1 is performed by
detection of total p24 in the supernatant of HIV-1 infected cells
by ELISA is performed as follows:
[0539] PBMCs were isolated from fresh human blood obtained from
screened donors, seronegative for HIV and HBV. Peripheral blood
cells were pelleted/washed 2-3 times by low speed centrifugation
and resuspension in PBS to remove contaminating platelets. The
washed blood cells were then diluted 1:1 with Dulbecco's phosphate
buffered saline (PBS) and layered over 14 mL of Lymphocyte
Separation Medium (LSM; celigro.RTM. by Mediatech, Inc.; density
1.078.+-.0.002 g/ml; Cat.# 85-072-CL) in a 50 mL centrifuge tube
and centrifuged for 30 minutes at 600.times.g. Banded PBMCs were
gently aspirated from the resulting interface and subsequently
washed 2.times. with PBS by low speed centrifugation. After the
final wash, cells were counted by trypan blue exclusion and
resuspended at 1.times.10.sup.7 cells/mL in RPMI 1640 supplemented
with 15% Fetal Bovine Serum (FBS), 2 mM L-glutamine, 4 .mu.g/mL
PHA-P. The cells were allowed to incubate for 48-72 hours at
37.degree. C. After incubation, PBMCs were centrifuged and
resuspended in RPMI 1640 with 15% FBS, 2 mM L-glutamine, 100 U/mL
penicillin, 100 .mu.g/mL streptomycin, 10 .mu.g/mL gentamycin, and
20 U/mL recombinant human IL-2. PBMCs were maintained in this
medium at a concentration of 1-2.times.10.sup.6 cells/mL with
biweekly medium changes until used in the assay protocol. Monocytes
were depleted from the culture as the result of adherence to the
tissue culture flask.
[0540] For the standard PBMC assay, PHA-P stimulated cells from at
least two normal donors were pooled, diluted in fresh medium to a
final concentration of 1.times.10.sup.6 cells/mL, and plated in the
interior wells of a 96 well round bottom microplate at 50
.mu.L/well (5.times.10.sup.4 cells/well). Test drug dilutions were
prepared at a 2.times. concentration in microtiter tubes and 100
.mu.L of each concentration was placed in appropriate wells in a
standard format. After a 2-hr preincubation period (cells+drug), 50
.mu.L of a predetermined dilution of virus stock was placed in each
test well (final MOI 0.1). Wells with cells and virus alone were
used for virus control. Separate plates were prepared identically
without virus for drug cytotoxicity studies using an MTS assay
system (described below). The PBMC cultures were maintained for
seven days following infection, at which time cell-free supernate
samples were collected and assayed for reverse transcriptase
activity as described below.
[0541] P24 ELISA kits were purchased from Coulter Electronics. The
assay is performed according to the manufacturer's instructions.
Control curves are generated in each assay to accurately quantify
the amount of p24 antigen in each sample. Data are obtained by
spectrophotometric analysis at 450 nm using a Molecular Devices
Vmax plate reader. Final concentrations are calculated from the
optical density values
[0542] This test is only valid if there is an accumulation of p24
in the tissue culture supernatant in the infected, untreated
cells.
[0543] In this test, IC.sub.50 is the concentration at which the
amount of p24 detectable is 50% of the p24 present in the untreated
control.
[0544] Compound to be tested is present during the adsorption and
in the media after adsorption.
Antiviral Assay for Paramyxoviridae
[0545] For RSV, a measurement of CPE is performed as follows:
[0546] Hep2 cells were plated in 96 well plates and allowed to grow
overnight in MEM plus 5% fetal calf serum at 37.degree. C. and 5%
CO.sub.2. The next day, cells are infected with RSV (strain A2,
10.sup.8.2 TCID50/ml in 100 ul/well) by adsorbtion for 2 hours.
Following adsorbtion, media is changed and after 7 days growth, CPE
is measured by conversion of Alamar Blue dye to its fluorescent
adduct by living cells.
[0547] This test is only valid if CPE measurement (as measured by
Alamar Blue conversion) in infected cells in the absence of
compound is 10% of the conversion measured in uninfected cells.
[0548] For purposes of IC.sub.50 comparison, 100% CPE is set at the
conversion level seen in infected cells and 0% CPE is set at the
conversion seen in uninfected cells. Therefore IC.sub.50 is the
concentration of compound which generates 50% CPE.
[0549] Compound to be tested is present during the adsorption and
in the media after adsorption.
Example 13
2'-O Methylated Phosphorothioated Randomers Exhibit Potent
Antiviral Activity with Increased pH Resistance and Lower Serum
Protein Binding
[0550] We show herein that PS-ON randomers do not act via a
sequence specific mechanism (i.e. their activity does not require
them to bind to nucleic acid and their activity is not due to a
sequence specific aptameric effect). We further show in this
example the effect of oligonucleotides combining unmodified
linkages, phosphorothiate linkages, 2'-O methyl modified riboses
and unmodified ribonucleotides on a 40 base randomer with respect
to their antiviral activity, serum protein interaction and chemical
stability.
[0551] All randomers were prepared using standard solid phase,
batch synthesis at the University of Calgary Core DNA Services lab
on a 1 or 15 mol synthesis scale, deprotected and desalted on a 50
cm Sephadex G-25 column.
[0552] For antiviral activity testing in influenza A (INF A),
immortalized Canine kidney (MDCK) cells are cultured at 37.degree.
C. and 5% CO.sub.2 in MEM plus 10% fetal calf serum supplemented
with gentamycin, vancomycin and amphoterecin B. Cells are seeded in
6 well plates at a density which yields a confluent monolayer of
cells after 6 days of growth. Upon reaching confluency, the media
is changed to contain only supplements as described above and cells
are then exposed to INF A (strain H3N2, approximately 35-70 PFU
total) for 60 minutes. After viral exposure, the media is replaced
with new media containing drug only. Plaque counting is performed
2-3 days post infection following formalin fixation and cresyl
violet staining of infected cultures.
[0553] For antiviral testing in HSV, immortalized African Green
Monkey kidney (VERO) cells are cultured at 37.degree. C. and 5%
CO.sub.2 in MEM plus 10% fetal calf serum supplemented with
gentamycin, vancomycin and amphoterecin B. Cells are seeded in 12
well plates at a density which yields a confluent monolayer of
cells after 4 days of growth. Upon reaching confluency, the media
is changed to contain only 5% serum plus supplements as described
above and cells are then exposed to HSV-1 (strain KOS,
approximately 40-60 PFU total) in the presence of the test compound
for 90 minutes. After viral exposure, the media is replaced with
new "overlay" media containing 5% serum, 1% human immunoglobulins,
supplements as described above and the test compound. Plaque
counting is performed 3-4 days post infection following formalin
fixation and cresyl violet staining of infected cultures.
[0554] To determine serum protein interaction, a phosphorothioate
randomer labeled at the 3' end with FITC (the bait) is diluted to 2
nM in assay buffer (10 mM Tris, pH7.2, 80 mM NaCl, 10 mM EDTA, 100
mM b-mercaptoethanol and 1% tween 20). This oligo is then mixed
with the appropriate amount of non heat-inactivated FBS. Following
randomer-FBS interaction, the complexes are challenged with various
unlabelled randomers to assess their ability to displace the bait
from its complex. Displaced bait is measured by fluorescence
polarization. The displacement curve was used to determine Kd.
[0555] pH resistance was determined by incubation of randomers in
PBS adjusted to the appropriate pH with HCl. 24 hours after
incubation, samples were neutralized with 1M TRIS, pH 7.4 and run
on denaturing acryalmide gels and visualized following EtBr
staining.
[0556] For these experiments, we compared the behaviours of
different modified randomers: REP 2006, REP 2024, REP 2107, REP
2086 and REP 2060 (see Table 6 in this example). The antiviral
activities of these randomers were tested for antiviral activity in
HSV and influenza A by plaque reduction assay (see Table 7 in this
example). In these two viruses, REP 2006, 2024 and 2107 had similar
and potent anti-viral activity, REP 2060 showed significant
anti-HSV activity and REP 2086 had no detectable antiviral activity
in either HSV-1 or influenza A under these assay conditions.
TABLE-US-00006 TABLE 6 Randomer description Randomer Description (N
= A, G, T/U or C) REP 2006 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
REP 2024 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN REP 2107
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN REP 2086
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN REP 2060
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNN N = unmodified deoxyribonucleotide,
unmodified linkage N = unmodified deoxyribonucleotide,
phosphorothiate linkage N = 2'-O methyl modified ribose, unmodified
linkage N = 2'-O methyl modified ribose + phosphorothioate linkage
N = unmodified ribonucleotide + phosphorothioate linkage
[0557] TABLE-US-00007 TABLE 7 Antiviral activity of various
randomers in HSV and influenza A Randomer IC.sub.50 (.mu.M) (HSV)
IC.sub.50 (.mu.M) (influenza A) REP 2006 0.1 .about.2 REP 2024 0.14
.about.1.5 REP 2107 0.085 .about.1 REP 2086 No activity no activity
REP 2060 0.85 Not tested
[0558] The relative affinity of these randomers for serum proteins
was determined as described above. The results of these experiments
showed that REP 2107 has a lower affinity to serum proteins than
REP 2006 or REP 2024 (see Table 8 in this example) and that there
was no interaction detected between REP 2086 and serum proteins.
Moreover, at saturation of competition, REP 2107 was less effective
at displacing bound bait than REP 2006 or REP 2024 (see Table 9 in
this example). TABLE-US-00008 TABLE 8 Serum protein affinity of
various randomers. Randomer Kd (nM) (FBS) 2006 13 2024 13 2107 27
2086 no binding
[0559] TABLE-US-00009 TABLE 9 Displacement of bait randomer at
saturation. Randomer % displaced bait 2006 75 2024 80 2107 60 2086
no displacement
[0560] Finally, we tested the pH stability of these randomers in
the range of pH 1 to pH 7 over 24 hours of incubation at 37.degree.
C. While REP 2006 and REP 2024 showed significant degredation at pH
3 and complete degredation at pH 2.5, REP 2107, 2086 and 2060 were
completely stable at pH 1 after 24 h of incubation.
[0561] These results duplicate our previous findings that the
phosphorothioation of ON randomers is highly beneficial for their
antiviral activity. We further demonstrate here that the
incorporation of 2'-O methyl modifications in PS-ON randomers does
not affect the antiviral activity of these molecules, even when
every ribose in the PS-ON randomer contains a 2'-O-methyl
modification. Moreover, the fully 2'-O-methylated, fully
phosphorothioated randomer (REP 2107) has a weaker interaction with
serum proteins and shows a significantly increased resistance to
low pH induced hydrolysis
Example 14
PS-ONs Act by a Predominantly Extracellular Mode of Action
[0562] Prior art has taught that the use of delivery agents to
increase the intracellular concentrations of PS-ONs would be
beneficial to their activity. We demonstrate here that the
antiviral activity of PS-ONs acts predominantly outside the cell
and therefore would not receive a major benefit from the
transfection enhancement of an intracellular delivery agent.
[0563] In this example, we use a PS-ON made of deoxyribonucleotides
(DNA) without other modifications such as ribonucleotides (RNA) or
2'-O-methyl modification. It is safe to consider that this data
will apply to PS-ON bearing additional modifications because it is
known is the art that these molecules do not penetrate cells in
vitro easily without the aide of a delivery system or a tranfection
agent, especially in cases of antisense antivity.
[0564] For the determination of cellular delivery, HeLa cells were
cultured under standard conditions and then incubated with
fluorescently labelled REP 2006 (FL-REP2006, a 3' fluorescein
isothiocynate conjugated 40 base PS-ON randomer), either naked or
encapsulated with a delivery agent (in this case DOTAP
[1,2-Dioleoyl-3-Trimethylammonium-Propane], a cationic lipid).
After various times of incubation, cells were thoroughly washed
with PSB to remove any non-internalized ON and the cells were
subsequently lysed. The level of intracellular ON in the cell
lysate was determined using a fluorescence plate reader.
[0565] The determination of antiviral efficacy with naked, DOTAP
and PEI (polyethylene imine) encapsulated REP 2006 in HSV-1 and
influenza was determined as described above.
[0566] The determination of the time of action of REP 2006 during
the infectious cycle of HSV-1 was determined as described above,
but adding REP 2006 at various times before, during and after
infection. In HIV-1, this was determined by adding REP 2006 to
HIV-LTR-beta-gal HeLa cells at various time before, during and
after infection. HIV-1 infection was monitored by a colourmetric
assay of beta-gal production using absorbance spectroscopy.
[0567] We first determined that DOTAP and PEI could deliver
fluorescent REP 2006 inside cells (see table 10). This data showed
that both DOTAP and PEI were capable of delivering FL-2006 (and by
inference REP 2006) inside cells. TABLE-US-00010 TABLE 10
Intracellular concentration of FL-REP 2006 with and without
delivery (pmol/cell) Incubation FL-REP 2006 + time FL-REP2006 DOTAP
FL-REP2006 + PEI 1 hour 6 .times. 10.sup.-5 5 .times. 10.sup.-4 6
.times. 10.sup.-5 6 hours 6 .times. 10.sup.-5 9 .times. 10.sup.-4 3
.times. 10.sup.-4 24 hours 6 .times. 10.sup.-5 1.7 .times.
10.sup.-3 7 .times. 10.sup.-4
[0568] We then determined the activity of encapsulated (DOTAP or
PEI) REP2006 in HSV-1 influenza A (see Table 11 and 12 in this
example) These results showed that encapsulated REP 2006 had no
detectable antiviral activity in both HSV-1 and influenza.
TABLE-US-00011 TABLE 11 Activity of encalsulated REP 2006 in HSV-1
(IC.sub.50, .mu.M) REP2006 REP2006 + DOTAP REP 2006 + PEI 0.074 No
activity No activity
[0569] TABLE-US-00012 TABLE 12 Activity of PEI encapsulated REP
2006 in influenza A (% inhibition of plaque formation) ON
concentration (.mu.M) REP 2006 REP 2006 + PEI 0 0 0 0.625 0 0 0.125
50 0 2.5 75 0 5 100 0 10 100 0
[0570] Finally, a time of addition study in HSV-1 and HIV-1 was
performed where REP 2006 was added at various times before, during
and after the infection. These results showed that in both viruses,
REP 2006 was most effective when present before or during the
infection, indicating that it was a fusion/entry inhibitor in HSV-1
and HIV-1.
[0571] These results demonstrate that the antiviral activity or REP
2006 and PS-ONs bearing additional modifications such as, but
without restriction, ribonucleotides (RNA) or 2'-O-methyl, occurs
principally outside the cell.
Example 15
REP 2107 Exhibits Superior Nuclease Resistance
[0572] 40 mer randomers of various chemistries were assessed for
their ability to resist degredation by various nucleases for 4
hours at 37.degree. C. (see Table 13 in this example). While most
chemistries exhibited resistance to more than one nuclease, only
REP 2107 was resistant to all four nucleases tested. It is
important to note that REP 2024 (which has 2'-O methyl
modifications at the 4 riboses at each end of the molecule) showed
the same resistance profile as its parent molecule REP 2006, being
sensitive to S1 nuclease degradation while 2107 (fully 2'-O methyl
modified) was resistant to this enzyme. These results suggest that
REP 2107 will be the most effective of the tested oligonucleotides
in resisting degredation by nucleases in the blood. TABLE-US-00013
TABLE 13 Resistance to various nucleases by different randomer
chemistries. Sensitive (S) or Resistant (R) after 4 h incubation at
37.degree. C. Phosphodiesterase S1 Nuclease Bal 31 Exonuclease I II
(Fermentas (NEB (NEB Randomer (Sigma P9041) #EN0321) M0213S)
M0293S) REP2015 R S S S REP2107 R R R R REP2006 R S R R REP2086 R R
S R REP2024 R S R R
Example 16
Phosphorothioated Polypyrimidine ONs Exhibit Acid and Nuclease
Resistance
[0573] To determine the extent of ONs acid resistance of ONs,
various 40 base ONs having different chemistries and/or sequences
are incubated in PBS buffered to different pH values for 24 hours
at 37.degree. C. The degradation of these ONs was assessed by
urea-polyacryamide gel electrophoresis (see table 14).
[0574] The results of these studies show that randomer ONs
(containing both pyrimidine and purine nucleotides) are resistant
to acidic pH only when they were fully 2'-O-methylated. Our data
indicated that even partially 2'-O-methylated ONs (gapmers, REP
2024) do not display any significant increase in acid resistance
compared to fully phosphorothioated ONs. Even fully
phosphorothioated randomers show no increased pH resistance
compared to unmodified ONs. In contrast, we noted that the
phosphorothioated 40mer ONs containing only the pyrimidine
nucleotides cytosine (polyC, REP 2031) or thymidine (polyT, REP
2030) or the polyTC heteropolymer (REP 2056) had equivalent acid
resistance compared to the fully 2'-O-methylated randomers whether
phosphorothioated (REP 2107) or not (REP 2086). Contrary to the
results for the polypyrimidine oligonucleotides, phosphorothioated
oligonucleotides containing only the purine nucleotide adenosine
(polyA, REP 2029) or any adenosine or guanosine nucleotides (REP
2033, 2055, 2057) showed no greater acid resistance compared to
unmodified DNA.
[0575] These results are significant because the preferred way
described in the prior art to achieve greater acid resistance
compared to phosphorothioated ONs was to add 2'-O-methyl
modifications (or other 2'-ribose modifications) or other
modifications. The present data demonstrates that the 2'-O-methyl
ribose modification or other 2'-ribose modifications are not
required if the ON is a polypyrimidine (i.e. contains only
pyrimidine nucleotides [e.g. homopolymers of cytosine or thymidine
or a heteropolymer of cytosines and thymidines]) to achieve pH and
nuclease resistance. The presence of purines (A or G) even in the
presence of pyrimidines, can render ONs acid labile. TABLE-US-00014
TABLE 14 Acid stability of various 40 mer ONs stability to various
pHs after 24 h at 37.degree. C. pH ON name sequence modification pH
1 pH 2 2.5 pH 3 pH 4 pH 5 pH 7 REP 2015 randomer none - - -/+ + +++
+++ +++ REP 2006 randomer PS - - -/+ + +++ +++ +++ REP 2086
randomer 2'OMe +++ +++ +++ +++ +++ +++ +++ REP 2107 randomer PS,
2'OMe +++ +++ +++ +++ +++ +++ +++ REP 2024 randomer PS, 2'OMe - -
-/+ + +++ +++ +++ gapmer REP 2031 polyC PS +++ +++ +++ +++ +++ +++
+++ REP 2030 polyT PS +++ +++ +++ +++ +++ +++ +++ REP 2029 polyA PS
- - - - ++ +++ +++ REP 2033 polyTG PS - - - - ++ +++ +++ REP 2055
polyAC PS - - - - ++ +++ +++ REP 2056 polyTC PS +++ +++ +++ +++ +++
+++ +++ REP 2057 polyAG PS - - - - ++ +++ +++ PII =
phosphodiesterase II, S1 = S1 nuclease, Exo1 = Exonuclease 1, PS =
all linkages phosphorothioated, 2'OMe = all riboses are 2'O
methylated. +++ = no degradation, ++ = less than 5-% degradation,
-/+ = more than 90% degradation, - = completely degraded
[0576] To determine the extent of ON nucleotide composition and
modifications on nuclease resistance, various 40 base ONs having
different nucleotide compositions and modifications were incubated
in the presence of various endo and exonucleases for 4 hours at
37.degree. C. The degradation of these ONs was assessed by
urea-polyacryamide gel electrophoresis.
[0577] The results of these studies showed that randomer ONs were
resistant to all four enzymes tested (phosphodiesterase II [Sigma],
S1 nuclease [Fermentas], Bal31 [New England Biolabs] and
exonuclease 1 [New England Biolabs]) only when they were fully
phosphorothioated and fully 2'-O -methylated (see table 15).
Omission of any of these modifications in randomers resulted in
increased sensitivity to one or more of the nucleases tested. We
noted that the fully phosphorothioated, partially 2'-O -methylated
randomer (REP 2024) was equivalent in nuclease resistance to REP
2006, indicated that 2'-O-methylation may be required on each
nucleotide of a phosphorothioated ON to achieve the optimal
nuclease resistance. However, we noted that the phosphorothioated
40mer polypyrimidine poly cytosine (poly C, REP 2031) had
equivalent nuclease resistance compared to the fully
phosphorothioated, fully 2'O methylated randomer (REP 2107).
[0578] These results are significant because the prior art teaches
that the preferred way to enhance nuclease resistance of
phosphorothioated ONs is to add 2'-O-methyl modifications, other
2'-ribose modifications, or other modifications. This new data
demonstrates that the 2'-O-methyl modification or other 2'-ribose
modifications or any other modifications are not required to
enhance nuclease resistance if the ON is fully phosphorothioated
and consists of a homopolymer of pyrimidines. TABLE-US-00015 TABLE
15 Nuclease resistance of various 40 mer ONs Nuclease resistance ON
after 4 h at 37.degree. C. name sequence modification PII S1 Bal 31
Exo 1 REP randomer none - - - - 2015 REP randomer PS +++ - ++++
++++ 2006 REP randomer 2'OMe ++++ ++++ - ++++ 2086 REP randomer PS,
2'OMe ++++ ++++ ++++ ++++ 2107 REP randomer PS, 2'OMe ++++ - ++++
++++ 2024 gapmer REP polyC PS ++++ ++++ ++++ ++++ 2031 REP2029 Poly
A PS - - ++++ ++++ REP2030 Poly T PS - - ++++ ++++ REP2033 Poly TG
PS + - ++++ ++++ REP2055 Poly AC PS + - ++++ ++++ REP2056 Poly TC
PS + - ++++ ++++ REP2057 Poly AG PS ++ - ++++ ++++ PII =
phosphodiesterase II, S1 = S1 nuclease, Exo1 = Exonuclease 1, PS =
all linkages phosphorothioated, 2'OMe = all riboses are 2'O
methylated. - = complete degredation, ++++ = no degredation,. PS =
phosphorothioate, 2'OMe = 2'-O-methyl modification of the
ribose.
These results demonstrate that phosphorothioated ONs containing
only pyrimidine nucleotides, including cytosine and/or thymidine
and/or other pyrimidines are resistant to low pH and
phosphorothioated ONs containing only cytosine nucleotides exhibit
superior nuclease resistance, two important characteristics for
oral administration of an antiviral ON. Thus, high pyrimidine
nucleotide content of an antiviral ON is advantageous to provide
resistance to low pH resistance and high cytosine content is
advantagaeous to provide improved nuclease resistance. For example,
in certain embodiments, the pyrimidine content of such an
oligonucleotide is more than 50%, more than 60%, or more than 70%,
or more than 80%, or more than 90%, or 100%. Furthermore, these
results show the potential of a method of treatment using oral
administration of a therapeutically effective amount of at least
one pharmacologically acceptable ON composed of pyrimidine
nucleotides. These results also show the potential of ONs
containing high levels of pyrimidine nucleotides as a component of
an antiviral ON formulation.
Example 17
Sequence Independent Broad Spectrum Activity of ONs In Vivo
[0579] We show here that a 40 base sequence-independent PS-ON
randomer has potent antiviral activity in six different animal
models of viral infection (see table 16). The 40 base PS-ON
randomer was introduced to animals by multiple routes of
administration including subcutaneous, intraperitoneal and aerosol
(inhalation). These data strongly support the therapeutic potential
of sequence independent ONs as broad spectrum antivirals.
TABLE-US-00016 TABLE 16 PS-ON randomers have potent broad spectrum
in vivo antiviral activity Virus (strain) Reduction in viral titer
(organ) (Animal/mode of infection) relative to placebo (route)
p-value Ebola Zaire (Mayinga) 100% survival (n = 6) ND (mouse/IP,
lethal model) (intraperitoneal) Influenza A/HK/68 3.8 log.sub.10
(lung) <0.001 (Mouse/IN) (aerosol) MCMV (Smith) 2.67 log.sub.10
(spleen) <0.0001 (Mouse/IV) (subcutaneous) 1.67 log.sub.10
(spleen) 0.012 (intraperitoneal) HSV-2 .about.70% of animals
protected from ND (Mouse/vaginal gel) HSV-2 transmission Friend's
Leukemia Virus 68%(Reduction of infected 0.0084 (Mouse/IV)
splenocytes) (subcutaneous) Respiratory Syncytial Virus 1.1
log.sub.10 (lung) <0.01 (Long) (Cotton rat/IN) (aerosol) ND =
not determined
Example 18
Oligonucleotides have Antiviral Activity in a Broad Spectrum of
Viruses
[0580] We show here that a 40 PS-ON randomer has antiviral activity
in vitro against 13 viral families (see table 17). TABLE-US-00017
TABLE 17 PS-ON randomers have broad spectrum in vitro antiviral
activity Activity Family Virus (Strain) (IC.sub.50, .mu.M) Assay
Used Herpesviridae HSV-1 (KOS) 0.06 Plaque reduction HSV-1 0.2
Inhibition of CPE HSV-2 (MS2) 0.1 Plaque reduction HSV-2 0.02
Inhibition of CPE CMV (AD169) 0.13 Plaque reduction Human CMV 0.02
Inhibition of CPE VZV <0.02 Inhibition of CPE Retroviridae HIV-1
.about.0.1 p24 ELISA (multiple clinical isolates) (in human PBMCs)
HIV-1 (NL4-3) 0.011 Inhibition of CPE HIV/MLV Chimera 0.014
fluorescence-based infection assay Hepadnaviridae HBV 0.007
detection of virions in the supernatant Paramyxoviridae RSV (A2)
0.019 inhibition of CPE Parainfluenza-3 0.125 plaque reduction
Coronaviridae SARS (Toronto-2) 100 Inhibition of CPE Filoviridae
Ebola Zaire (Mayinga) 0.1 FACS analysis of infected cells Marburg
(Muskoke) IC99 < 1 fluorescent plaque reduction Arenaviridae
Lassa Fever (Josiah) IC99 < 1 reduction of virus in supernatant
Bunyaviridae Hantavirus (Prospect Hill) IC99 < 10 fluorescent
plaque reduction Orthopoxviridae vaccinia (WR) .about.1.5 plaque
reduction vaccinia .about.0.5 plaque reduction ectromelia
(mousepox) .about.1.5 plaque reduction Flaviviridae West Nile 3.02
inhibition of CPE (NY-99) Yellow Fever 3.47 inhibition of CPE
Dengue .about.10 plaque reduction (Den-4) Togaviridae Western
Equine Encephalitis 0.12 inhibition of CPE Rhabdoviridae Rabies
(ERA) IC99 < 1 fluorescent plaque reduction Orthomyxoviridae
Influenza A .about.1 plaque reduction
Example 19
In Vivo and In Vitro Anti-Influenza Activity of PS-ONs
[0581] In order to further assess the anti-influenza activity of
ONs, REP 2006 was tested against different strains of influenza
using a hemagluttination assay. REP 2006 displayed a broad spectrum
anti-influenza activity as shown in Table 18. TABLE-US-00018 TABLE
18 Broad spectrum antiviral activity of a REP 2006 against multiple
strains of influenza. Trial 1 IC.sub.50 Trial 2 IC.sub.50 Influenza
strain (mM) (mM) A/New Caledonia (H1N1) 0.014 0.055 A/Taiwan (H1N1)
0.014 0.055 B/Panama 0.038 0.055 B/Singapore 0.038 0.055 A/PR8
(H1N1) 0.055 0.015 A/HK/68 (H3N2) 0.008 0.0017 A/WSN (H1N1) 0.038
Not tested
[0582] In order to asses the potential of ONs as drugs for the
treatment of influenza, REP 2006 was tested in a mouse model of
influenza infection. REP 2006 was prepared at two concentrations in
water for injection and aerosolized by nebulization where the
outlet was connected to an Anderson cascade chamber. 20 g Balb/c
mice were exposed daily to aerosolized randomer 1 for 30 minutes
using 10 ml of REP 2006 at various concentrations in an aerosol
chamber. Mice were intranasally infected with .about.100TCID of
influenza A (H3N2, A/Hong Kong/68) and after 4 days of infection,
animals were sacrificed and lung viral titers were determined by
hemagluttination assay. REP 2006 demonstrated a potent
anti-influenza activity in vivo as shown in Table 19.
TABLE-US-00019 TABLE 19 In vivo efficacy of the REP2006 against
influenza A. Dose Viral titer ANOVA mg/ml - SDA (log10/g lung) Non-
Treatment mg/kg - IP/SC Regimen (days) Mean St. dev. Parametric
parametric Influenza A/HK/68 (n = 5) in Balb/c mice dH.sub.2O 0
(SDA) -1, 0, 1, 2 6.6 0.9 NA NA ribavirin 180 (IP) -1, 0, 1, 2 3.3
0.6 <0.001 NS REP 2006 10 (SDA) -1, 0, 1, 2 2.8 0.9 <0.001 NS
REP 2006 100 (SDA) -1, 0, 1, 2 <2.3 0.0 <0.001 <0.01
Influenza A/HK/68 (n = 5) in Balb/c mice dH.sub.2O 0 (SDA) -1, 0,
1, 2 5.8 0.4 NA NA REP 2006 10 (SDA) -1, 0, 1, 2 3.9 0.4 <0.001
NS 2 .times. 10 (SDA)** -1, 0, 1, 2 3.3 0.5 <0.001 <0.01 2
.times. 100 (SDA)** 1, 2 1.1 1.5 <0.001 NS 20 (IP) 1, 2 3.2 0.4
<0.001 NS 20 (SC) 1, 2 3.9 0.2 <0.001 NS SDA = small droplet
aerosol, IP = intraperitoneal, SC = subcutaneous) **indicates two
daily doses given 12 hours apart.
Example 20
Phosphorothioated Polypyrimidine ON Exhibits Improved Antiviral
Activity in Acidic Environment In Vivo
[0583] In order to assess the resistance of polypyrimidine ONs to
low pH and their capacity to be active drugs at lower pH in vivo,
REP 2031 (PS polyC) was tested in a HSV-2 vaginal mouse model.
Groups of Female Swiss Webster were administered a 0.1 ml
suspension containing 3 mg of medroxyprogesterone acetate by
subcutaneous injection 7 and 1 days prior to viral challenge, to
increase susceptibility to vaginal HSV-2 infection. The vaginal
vault was swabbed twice, first with a moistened type 1 calcium
alginate-tipped swab and then with a dry swab. Animals were treated
with 15 .mu.l of either the candidate solution or a placebo control
using a positive displacement pipetter. Five minutes later, animals
were inoculated by instillation of 15 .mu.l of a suspension
containing 10.sup.4 pfu of HSV-2, strain 186. Vaginal swabs samples
were collected from all animals on day 2 after inoculation and
stored frozen (-80.degree. C.) until assayed for the presence of
virus by culture. Mice were evaluated daily up to day 21 after
inoculation, for evidence of symptomatic infection that can include
hair loss and erythema around the perineum, chronic urinary
incontinence, hind-limb paralysis, and mortality. Animals that did
not develop symptoms were defined as infected if virus was isolated
from vaginal swab samples collected on day 2 after inoculation.
Results showed (Table 20) that polypyrimidine REP 2031 had an
antiviral activity in an acidic environment, such as the vagina in
this example or the stomach. TABLE-US-00020 TABLE 20 Vaginal
efficacy of ONs against HSV-2 Virus (strain) Route of Reduction in
viral (Animal/ administration titre (organ) mode of and relative to
infection) Compound dosing regimen placebo (log.sub.10) HSV-2 (186)
REP 2006 Single prophylactic 8/12 animals (Swiss Webster
(PS-randomer) topical application protected from mice/vaginal) to
vagina transmission (100 mg/ml gel) (0/12 in untreated animals) REP
2031 12/12 animals (PS-poly C) protected from transmission
[0584] All patents and other references cited in the specification
are indicative of the level of skill of those skilled in the art to
which the invention pertains, and are incorporated by reference in
their entireties, including any tables and figures, to the same
extent as if each reference had been incorporated by reference in
its entirety individually.
[0585] One skilled in the art would readily appreciate that the
present invention is well adapted to obtain the ends and advantages
mentioned, as well as those inherent therein. The methods,
variances, and compositions described herein as presently
representative of preferred embodiments are exemplary and are not
intended as limitations on the scope of the invention. Changes
therein and other uses will occur to those skilled in the art,
which are encompassed within the spirit of the invention, are
defined by the scope of the claims.
[0586] It will be readily apparent to one skilled in the art that
varying substitutions and modifications may be made to the
invention disclosed herein without departing from the scope and
spirit of the invention. For example, variations can be made to
synthesis conditions and compositions of the oligonucleotides.
Thus, such additional embodiments are within the scope of the
present invention and the following claims.
[0587] The invention illustratively described herein suitably may
be practiced in the absence of any element or elements, limitation
or limitations which is not specifically disclosed herein. Thus,
for example, in each instance herein any of the terms "comprising",
"consisting essentially of" and "consisting of" may be replaced
with either of the other two terms. The terms and expressions which
have been employed are used as terms of description and not of
limitation, and there is no intention that in the use of such terms
and expressions of excluding any equivalents of the features shown
and described or portions thereof, but it is recognized that
various modifications are possible within the scope of the
invention claimed. Thus, it should be understood that although the
present invention has been specifically disclosed by preferred
embodiments and optional features, modification and variation of
the concepts herein disclosed may be resorted to by those skilled
in the art, and that such modifications and variations are
considered to be within the scope of this invention as defined by
the appended claims.
[0588] In addition, where features or aspects of the invention are
described in terms of Markush groups or other grouping of
alternatives, those skilled in the art will recognize that the
invention is also thereby described in terms of any individual
member or subgroup of members of the Markush group or other
group.
[0589] Also, unless indicated to the contrary, where various
numberical values are provided for embodiments, additional
embodiments are described by taking any 2 different values as the
endpoints of a range. Such ranges are also within the scope of the
described invention.
[0590] Thus, additional embodiments are within the scope of the
invention and within the following claims.
Sequence CWU 1
1
26 1 20 DNA Artificial Sequence REP 1001 20mer from human
autonomously replicating sequence 1 ttgataaata gtactaggac 20 2 22
DNA Artificial Sequence REP 2001 - 22mer from HSV-1 origin of
replication 2 gaagcgttcg cacttcgtcc ca 22 3 16 DNA Artificial
Sequence REP 3007 - 16 mer from pUC19/pBR322 origin of replication
3 cttgcggtat tcggaa 16 4 10 DNA Artificial Sequence REP 2016 - 10
mer random sequence 4 tccgaagacg 10 5 20 DNA Artificial Sequence
REP 2017 - 20mer random sequence 5 acacctccga agacgataac 20 6 40
DNA Artificial Sequence REP 2018 - 40mer random sequence 6
ctacagacat acacctccga agacgataac actagacata 40 7 10 DNA Artificial
Sequence REP 2019 - 10mer centered around start codon of HSV-1
IE110 protein 7 cccccatgga 10 8 20 DNA Artificial Sequence REP 2020
- 20mer centered around start codon of HSV-1 IE110 protein 8
tacgaccccc atggagcccc 20 9 40 DNA Artificial Sequence REP 2021 -
40mer centered around start codon of HSV-1 IE110 protein 9
tccagccgca tacgaccccc atggagcccc gccccggagc 40 10 21 DNA Artificial
Sequence REP 2036 - 21mer commercially marketed antisense against
CMV 10 gcgtttgctc ttcttcttgc g 21 11 20 DNA Artificial Sequence A20
oligomer 11 aaaaaaaaaa aaaaaaaaaa 20 12 20 DNA Artificial Sequence
G20 oligomer 12 gggggggggg gggggggggg 20 13 20 DNA Artificial
Sequence C20 oligomer 13 cccccccccc cccccccccc 20 14 20 DNA
Artificial Sequence T20 oligomer 14 tttttttttt tttttttttt 20 15 20
DNA Artificial Sequence AC10 oligomer 15 acacacacac acacacacac 20
16 20 DNA Artificial Sequence AG10 oligomer 16 agagagagag
agagagagag 20 17 20 DNA Artificial Sequence TC10 oligomer 17
tctctctctc tctctctctc 20 18 20 DNA Artificial Sequence TG10
oligomer 18 tgtgtgtgtg tgtgtgtgtg 20 19 40 DNA Artificial Sequence
REP 2029 oligomer 19 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 40
20 40 DNA Artificial Sequence REP 2028 oligomer 20 gggggggggg
gggggggggg gggggggggg gggggggggg 40 21 40 DNA Artificial Sequence
REP 2031 oligomer 21 cccccccccc cccccccccc cccccccccc cccccccccc 40
22 40 DNA Artificial Sequence REP 2030 oligomer 22 tttttttttt
tttttttttt tttttttttt tttttttttt 40 23 40 DNA Artificial Sequence
REP 2055 oligomer 23 acacacacac acacacacac acacacacac acacacacac 40
24 40 DNA Artificial Sequence REP 2056 oligomer 24 tctctctctc
tctctctctc tctctctctc tctctctctc 40 25 40 DNA Artificial Sequence
REP 2057 oligomer 25 agagagagag agagagagag agagagagag agagagagag 40
26 40 DNA Artificial Sequence Rep 2033 oligomer 26 tgtgtgtgtg
tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 40
* * * * *