U.S. patent application number 16/203156 was filed with the patent office on 2019-10-17 for antisense oligonucleotide compositions.
This patent application is currently assigned to Ionis Pharmaceuticals, Inc.. The applicant listed for this patent is Ionis Pharmaceuticals, Inc.. Invention is credited to Bret Coldren, Andrew Dibble, Marc Lim, Lloyd Tillman.
Application Number | 20190316123 16/203156 |
Document ID | / |
Family ID | 49584478 |
Filed Date | 2019-10-17 |
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United States Patent
Application |
20190316123 |
Kind Code |
A1 |
Dibble; Andrew ; et
al. |
October 17, 2019 |
ANTISENSE OLIGONUCLEOTIDE COMPOSITIONS
Abstract
The present disclosure provides compositions comprising an
antisense oligonucleotide and one or more excipients that modulates
viscosity, turbidity or both viscosity and turbidity. In certain
embodiments, compositions comprising an antisense oligonucleotide
and one or more excipients having low viscosity are provided. In
certain embodiments, compositions comprising an antisense
oligonucleotide and one or more excipients having low turbidity are
provided. In certain embodiments, pharmaceutical compositions
comprising an antisense oligonucleotide and one or more excipients
having low viscosity and turbidity are provided.
Inventors: |
Dibble; Andrew; (Vista,
CA) ; Tillman; Lloyd; (Encinitas, CA) ;
Coldren; Bret; (Vista, CA) ; Lim; Marc;
(Fullerton, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ionis Pharmaceuticals, Inc. |
Carlsbad |
CA |
US |
|
|
Assignee: |
Ionis Pharmaceuticals, Inc.
Carlsbad
CA
|
Family ID: |
49584478 |
Appl. No.: |
16/203156 |
Filed: |
November 28, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15293613 |
Oct 14, 2016 |
10174318 |
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16203156 |
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14401761 |
Nov 17, 2014 |
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PCT/US2013/041701 |
May 17, 2013 |
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15293613 |
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61648556 |
May 17, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2320/50 20130101;
C12N 15/111 20130101; C12N 2310/11 20130101; C12N 15/113 20130101;
C12N 2310/321 20130101; C12N 2320/52 20130101; C12N 2310/3231
20130101; C12N 2320/51 20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113; C12N 15/11 20060101 C12N015/11 |
Claims
1.-275. (canceled)
276. An aqueous solution comprising: an antisense oligonucleotide;
and at least one excipient that modulates viscosity, turbidity or
both viscosity and turbidity; wherein the dynamic viscosity of the
solution is less than 100 cP at 25.degree. C.; and wherein the
antisense oligonucleotide is present at a concentration of 250
mg/ml to 500 mg/ml.
277. The solution of claim 276, having a dynamic viscosity of less
than 90 cP at 25.degree. C.
278. The solution of claim 276, having a dynamic viscosity of less
than 80 cP at 25.degree. C.
279. The solution of claim 276, having a dynamic viscosity of less
than 70 cP at 25.degree. C.
280. The solution of claim 276, having a dynamic viscosity of less
than 60 cP at 25.degree. C.
281. The solution of claim 276, having a dynamic viscosity of less
than 50 cP at 25.degree. C.
282. The solution of claim 276, having a dynamic viscosity of less
than 40 cP at 25.degree. C.
283. The solution of claim 276, having a dynamic viscosity of less
than 30 cP at 25.degree. C.
284. The solution of claim 276, having a dynamic viscosity of less
than 20 cP at 25.degree. C.
285. The solution of claim 276, having a dynamic viscosity of less
than 10 cP at 25.degree. C.
286. The solution of claim 276, having a turbidity of in the range
of 0 to 4000 NTU.
287. The solution of claim 276, having a turbidity of in the range
of 0 to 3000 NTU.
288. The solution of claim 276, having a turbidity of in the range
of 0 to 2000 NTU.
289. The solution of claim 276, having a turbidity of in the range
of 0 to 1000 NTU.
290. The solution of claim 276, having a turbidity of in the range
of 0 to 100 NTU.
291. The solution of claim 276, having a turbidity of in the range
of 0 to 90 NTU.
292. The solution of claim 276, having a turbidity of the solution
is in the range of 0 to 80 NTU.
293. The solution of claim 276, having a turbidity in the range of
0 to 70 NTU.
294. The solution of claim 276, having a turbidity in the range of
0 to 60 NTU.
295. The solution of claim 276, having a turbidity in the range of
0 to 50 NTU.
Description
SEQUENCE LISTING
[0001] The present application is being filed along with a Sequence
Listing in electronic format. The Sequence Listing is provided as a
file entitled CORE0105USC2SEQ.txt, created Nov. 28, 2018, which is
8 Kb in size. The information in the electronic format of the
sequence listing is incorporated herein by reference in its
entirety.
BACKGROUND OF THE INVENTION
[0002] Antisense compounds have been used to modulate target
nucleic acids. Antisense compounds comprising a variety of chemical
modifications and motifs have been reported. In certain instances,
such compounds are useful as research tools, diagnostic reagents,
and as therapeutic agents. Certain DNA-like oligomeric compounds
have been shown to reduce protein expression. Certain RNA-like
compounds are known to inhibit protein expression in cells. Such
RNA-like compounds function, at least in part, through the
RNA-inducing silencing complex (RISC). RNA-like compounds may be
single-stranded or double-stranded. Antisense compounds have also
been shown to alter processing of pre-mRNA and to modulate
non-coding RNA molecules. In certain instances antisense compounds
have been shown to modulate protein expression by binding to a
target messenger RNA (mRNA) encoding the protein. In certain
instances, such binding of an antisense compound to its target mRNA
results in cleavage of the mRNA. Antisense compounds that modulate
processing of a pre-mRNA have also been reported. Such antisense
compounds alter splicing, interfere with polyadenlyation or prevent
formation of the 5'-cap of a pre-mRNA. Compositions and methods
that increase productive uptake of antisense compounds in cells are
desired. Compositions and methods that facilitate the manufacture,
storage, administration, and delivery of antisense compounds are
also desired.
SUMMARY OF THE INVENTION
[0003] The present disclosure provides compositions and methods
that facilitate the manufacture, storage, administration, and
delivery of antisense oligonucleotide solutions. For example, in
certain embodiments, the present disclosure provides a number of
excipients that reduce the viscosity of an antisense
oligonucleotide solution. In certain embodiments, the present
disclosure provides a number of excipients that reduce the
turbidity of an antisense oligonucleotide solution. In certain
embodiments, the present disclosure provides a number of excipients
that reduce both the turbidity and the viscosity of an antisense
oligonucleotide solution. In certain embodiments, the present
disclosure provides a number of excipients that modulate viscosity,
turbidity or both viscosity and turbidity of antisense
oligonucleotide solutions wherein the antisense oligonucleotide is
present in the solution at a high concentration. In certain
embodiments, the present disclosure provides a number of excipients
that reduce both the turbidity and the viscosity of an antisense
oligonucleotide solution and wherein the excipient also modulates
the osmolality of the antisense oligonucleotide solution. In
certain embodiments, the present disclosure provides a number of
excipients that modulate viscosity, turbidity, or osmolality or
viscosity and turbidity and osmolality of an antisense
oligonucleotide solution and wherein the antisense oligonucleotide
is present in the solution at a high concentration.
[0004] The present disclosure provides the following non-limiting
numbered embodiments:
Embodiment 1
[0005] An aqueous solution comprising: [0006] an antisense
oligonucleotide; and [0007] at least one excipient that modulates
viscosity, turbidity or both viscosity and turbidity; [0008]
wherein the dynamic viscosity of the solution is less than 100 cP
at 25.degree. C.
Embodiment 2
[0009] The solution of embodiment 1, having a dynamic viscosity of
less than 90 cP at 25.degree. C.
Embodiment 3
[0010] The solution of embodiment 1, having a dynamic viscosity of
less than 80 cP at 25.degree. C.
Embodiment 4
[0011] The solution of embodiment 1, having a dynamic viscosity of
less than 70 cP at 25.degree. C.
Embodiment 5
[0012] The solution of embodiment 1, having a dynamic viscosity of
less than 60 cP at 25.degree. C.
Embodiment 6
[0013] The solution of embodiment 1, having a dynamic viscosity of
less than 50 cP at 25.degree. C.
Embodiment 7
[0014] The solution of embodiment 1, having a dynamic viscosity of
less than 40 cP at 25.degree. C.
Embodiment 8
[0015] The solution of embodiment 1, having a dynamic viscosity of
less than 30 cP at 25.degree. C.
Embodiment 9
[0016] The solution of embodiment 1, having a dynamic viscosity of
less than 20 cP at 25.degree. C.
Embodiment 10
[0017] The solution of embodiment 1, having a dynamic viscosity of
less than 10 cP at 25.degree. C.
Embodiment 11
[0018] The solution of any of embodiments 1-10, having a turbidity
of in the range of 0 to 4000 NTU.
Embodiment 12
[0019] The solution of any of embodiments 1-10, having a turbidity
of in the range of 0 to 3000 NTU.
Embodiment 13
[0020] The solution of any of embodiments 1-10, having a turbidity
of in the range of 0 to 2000 NTU.
Embodiment 14
[0021] The solution of any of embodiments 1-10, having a turbidity
of in the range of 0 to 1000 NTU.
Embodiment 15
[0022] The solution of any of embodiments 1-10, having a turbidity
of in the range of 0 to 100 NTU.
Embodiment 16
[0023] The solution of any of embodiments 1-10, having a turbidity
of in the range of 0 to 90 NTU.
Embodiment 17
[0024] The solution of any of embodiments 1-10, having a turbidity
of the solution is in the range of 0 to 80 NTU.
Embodiment 18
[0025] The solution of any of embodiments 1-10, having a turbidity
in the range of 0 to 70 NTU.
Embodiment 19
[0026] The solution of any of embodiments 1-10, having a turbidity
in the range of 0 to 60 NTU.
Embodiment 20
[0027] The solution of any of embodiments 1-10, having a turbidity
in the range of 0 to 50 NTU.
Embodiment 21
[0028] The solution of any of embodiments 1-10, having a turbidity
in the range of 0 to 40 NTU.
Embodiment 22
[0029] The solution of any of embodiments 1-10, having a turbidity
in the range of 0 to 30 NTU.
Embodiment 23
[0030] The solution of any of embodiments 1-10, having a turbidity
in the range of 0 to 25 NTU.
Embodiment 24
[0031] The solution of any of embodiments 1-10, having a turbidity
in the range of 0 to 20 NTU.
Embodiment 25
[0032] The solution of any of embodiments 1-10, having a turbidity
in the range of 0 to 15 NTU.
Embodiment 26
[0033] The solution of any of embodiments 1-10, having a turbidity
in the range of 0 to 10 NTU.
Embodiment 27
[0034] The solution of any of embodiments 1-10, having a turbidity
in the range of 0 to 5 NTU.
Embodiment 28
[0035] The solution of any of embodiments 1 to 27, wherein the
antisense oligonucleotide is present at a concentration of 1 mg/ml
to 500 mg/ml.
Embodiment 29
[0036] The solution of any of embodiments 1 to 27, wherein the
antisense oligonucleotide is present at a concentration of 1 to 400
mg/mL.
Embodiment 30
[0037] The solution of any of embodiments 1 to 27, wherein the
antisense oligonucleotide is present at a concentration of 1 to 300
mg/mL.
Embodiment 31
[0038] The solution of any of embodiments 1 to 27, wherein the
antisense oligonucleotide is present at a concentration of 1 to 280
mg/mL.
Embodiment 32
[0039] The solution of any of embodiments 1 to 27, wherein the
antisense oligonucleotide is present at a concentration of 1 to 260
mg/mL.
Embodiment 33
[0040] The solution of any of embodiments 1 to 27, wherein the
antisense oligonucleotide is present at a concentration of 1 to 240
mg/mL.
Embodiment 34
[0041] The solution of any of embodiments 1 to 27, wherein the
antisense oligonucleotide is present at a concentration of 1 to 220
mg/mL.
Embodiment 35
[0042] The solution of any of embodiments 1 to 27, wherein the
antisense oligonucleotide is present at a concentration of 1 to 200
mg/mL.
Embodiment 36
[0043] The solution of any of embodiments 1 to 27, wherein the
antisense oligonucleotide is present at a concentration of 100 to
220 mg/mL.
Embodiment 37
[0044] The solution of any of embodiments 1 to 27, wherein the
antisense oligonucleotide is present at a concentration of 120 to
220 mg/mL.
Embodiment 38
[0045] The solution of any of embodiments 1 to 27, wherein the
antisense oligonucleotide is present at a concentration of 140 to
220 mg/mL.
Embodiment 39
[0046] The solution of any of embodiments 1 to 27, wherein the
antisense oligonucleotide is present at a concentration of 160 to
220 mg/mL.
Embodiment 40
[0047] The solution of any of embodiments 1 to 27, wherein the
antisense oligonucleotide is present at a concentration of 180 to
220 mg/mL.
Embodiment 41
[0048] The solution of any of embodiments 1 to 27, wherein the
antisense oligonucleotide is present at a concentration of 130 to
170 mg/mL.
Embodiment 42
[0049] The solution of any of embodiments 1 to 27, wherein the
antisense oligonucleotide is present at a concentration of 135 to
165 mg/mL.
Embodiment 43
[0050] The solution of any of embodiments 1 to 27, wherein the
antisense oligonucleotide is present at a concentration of 140 to
160 mg/mL.
Embodiment 44
[0051] The solution of any of embodiments 1 to 27, wherein the
antisense oligonucleotide is present at a concentration of 145 to
155 mg/mL.
Embodiment 45
[0052] The solution of any of embodiments 1 to 27, wherein the
antisense oligonucleotide is present at a concentration of 150
mg/mL.
Embodiment 46
[0053] The solution of any of embodiments 1 to 27, wherein the
antisense oligonucleotide is present at a concentration of 50
mg/mL.
Embodiment 47
[0054] The solution of any of embodiments 1 to 27, wherein the
antisense oligonucleotide is present at a concentration of 40
mg/mL.
Embodiment 48
[0055] The solution of any of embodiments 1 to 27, wherein the
antisense oligonucleotide is present at a concentration of 60
mg/mL.
Embodiment 49
[0056] The solution of any of embodiments 1 to 48, wherein at least
one of the at least one excipient that modulates viscosity,
turbidity or both viscosity and turbidity comprises an aromatic
compound.
Embodiment 50
[0057] The solution of any of embodiments 1 to 48, wherein at least
one of the at least one excipient that modulates viscosity,
turbidity or both viscosity and turbidity is an aromatic
compound.
Embodiment 51
[0058] The solution of any of embodiments 1 to 50, wherein at least
one of the at least one excipient that modulates viscosity,
turbidity or both viscosity and turbidity is a heterocyclic
compound.
Embodiment 52
[0059] The solution of any of embodiments 1 to 51, wherein at least
one of the at least one excipient that modulates viscosity,
turbidity or both viscosity and turbidity is a heterocyclic
aromatic compound.
Embodiment 53
[0060] The solution of any of embodiments 1 to 50, wherein at least
one of the at least one excipient that modulates viscosity,
turbidity or both viscosity and turbidity comprises a heterocyclic
compound.
Embodiment 54
[0061] The solution of embodiment 51 or 52, wherein the heteroatom
of the heterocyclic compound is nitrogen.
Embodiment 55
[0062] The solution of embodiment 51 or 52, wherein the heteroatom
of the heterocyclic compound is oxygen.
Embodiment 56
[0063] The solution of embodiment 51 or 52, wherein the heteroatom
of the heterocyclic compound is not oxygen.
Embodiment 57
[0064] The solution of embodiment 51 or 52, wherein the heteroatom
of the heterocyclic compound is sulfur.
Embodiment 58
[0065] The solution of any of embodiments 51 to 57, wherein
heterocyclic compound has more than one heteroatom.
Embodiment 59
[0066] The solution of any of embodiments 51 to 57, wherein
heterocyclic compound has two or more heteroatoms.
Embodiment 60
[0067] The solution of any of embodiments 51 to 59, wherein
heterocyclic compound comprises a monocyclic ring having two or
more heteroatoms.
Embodiment 61
[0068] The solution of any of embodiments 51 to 59, wherein
heterocyclic compound is a heterocyclic aromatic compound that
comprises a monocyclic ring having two or more heteroatoms.
Embodiment 62
[0069] The solution of any of embodiments 60 to 61 where the
heteroatoms are different.
Embodiment 63
[0070] The solution of any of embodiments 60 to 61 where the
heteroatoms are the same.
Embodiment 64
[0071] The solution of embodiment 60 or 63, wherein the heteroatom
of the heterocyclic compound is nitrogen.
Embodiment 65
[0072] The solution of embodiment 60 or 63, wherein the heteroatom
of the heterocyclic compound is oxygen.
Embodiment 66
[0073] The solution of embodiment 60 or 63, wherein the heteroatom
of the heterocyclic compound is not oxygen.
Embodiment 67
[0074] The solution of embodiment 60 or 63, wherein the heteroatom
of the heterocyclic compound is sulfur.
Embodiment 68
[0075] The solution of any of embodiments 1 to 52, wherein at least
one of the at least one excipient that modulates viscosity,
turbidity or both viscosity and turbidity is a pyridine.
Embodiment 69
[0076] The solution of any of embodiments 1 to 52, wherein at least
one of the at least one excipient that modulates viscosity,
turbidity or both viscosity and turbidity is a diazine.
Embodiment 70
[0077] The solution of embodiment 69, wherein the diazine is
1,2-diazine.
Embodiment 71
[0078] The solution of embodiment 69, wherein the diazine is
1,3-diazine.
Embodiment 72
[0079] The solution of embodiment 69, wherein the diazine is
1,4-diazine.
Embodiment 73
[0080] The solution of any of embodiments 1 to 52, wherein at least
one of the at least one excipient that modulates viscosity,
turbidity or both viscosity and turbidity is an imidazole.
Embodiment 74
[0081] The solution of any of embodiments 1 to 52, wherein at least
one of the at least one excipient that modulates viscosity,
turbidity or both viscosity and turbidity is a tetrazine.
Embodiment 75
[0082] The solution of any of embodiments 1 to 52, wherein at least
one of the at least one excipient that modulates viscosity,
turbidity or both viscosity and turbidity is a hexazine.
Embodiment 76
[0083] The solution of embodiment 53, wherein the heterocyclic
compound comprises a heterocyclic amine.
Embodiment 77
[0084] The solution of any of embodiments 1 to 69, wherein the
excipient is a hydrogen bond donor.
Embodiment 78
[0085] The solution of any of embodiments 1 to 69, wherein the
excipient is a hydrogen bond acceptor.
Embodiment 79
[0086] The solution of any of embodiments 1 to 78, wherein at least
one of the at least one excipient that modulates viscosity,
turbidity or both viscosity and turbidity is selected from the
group consisting of Adenine, Benzyl Alcohol, m-Cresol, Cytidine,
Cytidine Monophosphate, Cytosine, Dextran, Guanine Monophosphate,
D-Mannitol, Methylparaben, Nicotinamide, Phenol, 2-Phenoxyethanol,
L-Phenylalanine, Pyridoxine, Sodium Chloride, Thymine, tryptophan,
L-Tryptophan, L-Tyrosine, Ascorbic Acid, Benzamide, o-Benzenediol,
Benzenehexol, L-Histidine, Hydroxypyridine, Indole,
D-Mannitol+Phenol mixture, D-Mannitol+Pyridine mixture, 2H-Pyran,
Pyrazinamide, Pyridine, Pyrimidine, 2-Pyrone, Riboflavin, Thiamine,
Tryptamine, ethanol, (2-Hydroxypropyl)-.beta.-cyclodextrin, Niacin,
Polyethylene Glycol 600, Polyethylene Glycol 4600, Propylene
Glycol, Pyridoxine, Sucrose, Thymidine, Tween 80, Uridine, Thymine,
caffeine, acridine orange, ethidium bromide, propidium iodide,
cyanine dyes such as PicoGreen.RTM., thiamine hydrochloride,
ethylene diamine tetraacetic acid, 1,2-dihydroxybenzene,
1,2-dihydroxybenzene (catechol), D-Mannitol+phenol mixture,
D-Mannitol+niacinamide mixture, calcium folinate,
L-phenylalanine+pyridoxine mixture, pyridoxine+benzyl alcohol
mixture, pyridoxine HCl+benzyl alcohol mixture, niacin sodium salt,
dextran 1500, and pyridoxine HCl+phenylalanine mixture.
Embodiment 80
[0087] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is Adenine.
Embodiment 81
[0088] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is Benzyl Alcohol.
Embodiment 82
[0089] The solution of embodiment 79, wherein at least one of the
one or more excipient is m-Cresol.
Embodiment 83
[0090] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is Cytidine.
Embodiment 84
[0091] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is Cytidine Monophosphate.
Embodiment 85
[0092] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is Cytosine.
Embodiment 86
[0093] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is Dextran.
Embodiment 87
[0094] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is Guanine Monophosphate.
Embodiment 88
[0095] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is D-Mannitol.
Embodiment 89
[0096] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is Methylparaben.
Embodiment 90
[0097] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is Nicotinamide.
Embodiment 91
[0098] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is Phenol.
Embodiment 92
[0099] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is 2-Phenoxyethanol.
Embodiment 93
[0100] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is L-Phenylalanine.
Embodiment 94
[0101] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is Pyridoxine.
Embodiment 95
[0102] The solution of embodiment 79, wherein at least one of at
least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is Sodium Chloride.
Embodiment 96
[0103] The solution of embodiment 79, wherein at least one of at
least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is Thymine.
Embodiment 97
[0104] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is L-Tryptophan.
Embodiment 98
[0105] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is tryptophan.
Embodiment 99
[0106] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is L-Tyrosine.
Embodiment 100
[0107] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is Ascorbic Acid.
Embodiment 101
[0108] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is Benzamide.
Embodiment 102
[0109] The solution of embodiment 79, wherein at least one of the
one or more excipient is o-Benzenediol.
Embodiment 103
[0110] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is Benzenehexol.
Embodiment 104
[0111] The solution of embodiment 79, wherein at least one of the
one or more excipient is L-Histidine.
Embodiment 105
[0112] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is a hydroxypyridine.
Embodiment 106
[0113] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is an indole.
Embodiment 107
[0114] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is D-Mannitol.
Embodiment 108
[0115] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is a D-Mannitol and phenol mixture.
Embodiment 109
[0116] The solution of embodiment 79, wherein at least one of at
least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is a D-Mannitol and pyridine mixture.
Embodiment 110
[0117] The solution of embodiment 79, wherein at least one of at
least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is 2H-Pyran.
Embodiment 111
[0118] The solution of embodiment 79, wherein at least one of at
least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is a pyrazinamide.
Embodiment 112
[0119] The solution of embodiment 79, wherein at least one of at
least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is a pyridine.
Embodiment 113
[0120] The solution of embodiment 79, wherein at least one of at
least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is a pyrimidine.
Embodiment 114
[0121] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is 2-Pyrone.
Embodiment 115
[0122] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is riboflavin.
Embodiment 116
[0123] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is thiamine.
Embodiment 117
[0124] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is tryptamine.
Embodiment 118
[0125] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is ethanol.
Embodiment 119
[0126] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is (2-Hydroxypropyl)-o-cyclodextrin.
Embodiment 120
[0127] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is Niacin.
Embodiment 121
[0128] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is Polyethylene Glycol 790.
Embodiment 122
[0129] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is Polyethylene Glycol 4790.
Embodiment 123
[0130] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is Propylene Glycol.
Embodiment 124
[0131] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is Pyridoxine.
Embodiment 125
[0132] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is Sucrose.
Embodiment 126
[0133] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is Thymidine.
Embodiment 127
[0134] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is Tween 80.
Embodiment 128
[0135] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is Uridine.
Embodiment 129
[0136] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is Thymine.
Embodiment 130
[0137] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is caffeine.
Embodiment 131
[0138] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is acridine orange.
Embodiment 132
[0139] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is ethidium bromide.
Embodiment 133
[0140] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is propidium iodide.
Embodiment 134
[0141] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is a cyanine dye.
Embodiment 135
[0142] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is PicoGreen.RTM..
Embodiment 136
[0143] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is 1,2-dihydroxybenzene (catechol).
Embodiment 137
[0144] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is D-Mannitol+phenol mixture.
Embodiment 138
[0145] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is D-Mannitol+niacinamide mixture.
Embodiment 139
[0146] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is calcium folinate.
Embodiment 140
[0147] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is L-phenylalanine+pyridoxine mixture.
Embodiment 141
[0148] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is pyridoxine+benzyl alcohol mixture.
Embodiment 142
[0149] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is pyridoxine HCl+benzyl alcohol
mixture.
Embodiment 143
[0150] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is pyridoxine HCl+phenylalanine
mixture.
Embodiment 144
[0151] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is D-Mannitol (0.5% w/v)+phenol (0.5% w/v)
mixture.
Embodiment 145
[0152] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is D-Mannitol (0.5% w/v)+niacinamide (0.5%
w/v) mixture.
Embodiment 146
[0153] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is D-Mannitol (0.5% w/v)+pyridine (0.5%
w/v) mixture.
Embodiment 147
[0154] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is L-phenylalanine (1.0% w/v)+pyroxidine
(0.5% w/v) mixture.
Embodiment 148
[0155] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is pyroxidine (0.5% w/v)+benzyl alcohol
(0.5% w/v) mixture.
Embodiment 149
[0156] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is pyroxidine HCl (0.5% w/v)+benzyl alcohol
(0.5% w/v) mixture.
Embodiment 150
[0157] The solution of embodiment 79, wherein at least one of the
at least one excipient that modulates viscosity, turbidity or both
viscosity and turbidity is pyroxidine HCl (0.5% w/v)+phenylalanine
(1.0% w/v) mixture.
Embodiment 151
[0158] The solution of any of embodiments 1 to 150, wherein the
solution does not comprise cyclodextrin.
Embodiment 152
[0159] The solution of any of embodiments 1 to 150, wherein the
solution does not comprise mannitol.
Embodiment 153
[0160] The solution of any of embodiments 1 to 152, wherein at
least one of the at least one excipient that modulates viscosity,
turbidity or both viscosity and turbidity is an antimicrobial.
Embodiment 154
[0161] The solution of any of embodiments 1 to 153, wherein at
least one of the at least one excipient that modulates viscosity,
turbidity or both viscosity and turbidity is not an
antimicrobial.
Embodiment 155
[0162] The solution of any of embodiments 1 to 152 or 154, wherein
the solution does not comprise any excipient that is an
antimicrobial.
Embodiment 156
[0163] The solution of any of embodiments 1 to 155, wherein at
least one of the at least one excipient that modulates viscosity,
turbidity or both viscosity and turbidity increases the osmolarity
of the solution.
Embodiment 157
[0164] The solution of any of embodiments 1 to 156, wherein at
least one of the at least one excipient that modulates viscosity,
turbidity or both viscosity and turbidity increases the pH of the
solution.
Embodiment 158
[0165] The solution of any of embodiments 1 to 156, wherein at
least one of the at least one excipient that modulates viscosity,
turbidity or both viscosity and turbidity decreases the pH of the
solution.
Embodiment 159
[0166] The solution of any of embodiments 1 to 158, wherein at
least one of the at least one excipient that modulates viscosity,
turbidity or both viscosity and turbidity buffers the pH of the
solution.
Embodiment 160
[0167] The solution of any of embodiments 1 to 159, wherein the
solution comprises only one excipient that modulates viscosity,
turbidity or both viscosity and turbidity.
Embodiment 161
[0168] The solution of any of embodiments 1 to 160, wherein the
solution comprises only one excipient.
Embodiment 162
[0169] The solution of any of embodiments 1 to 160, wherein the
solution comprises at least two excipients.
Embodiment 163
[0170] The solution of any of embodiments 1 to 160, wherein the
solution comprises at least three excipients.
Embodiment 164
[0171] The solution of any of embodiments 1 to 160, wherein the
solution comprises at least four excipients.
Embodiment 165
[0172] The solution of any of embodiments 1 to 160, wherein the
solution comprises at least two excipients that modulate viscosity,
turbidity or both.
Embodiment 166
[0173] The solution of any of embodiments 1 to 160, wherein the
solution comprises at least three excipients that modulate
viscosity, turbidity or both.
Embodiment 167
[0174] The solution of any of embodiments 1 to 160, wherein the
solution comprises at least four excipients that modulate
viscosity, turbidity or both.
Embodiment 168
[0175] The solution of any of embodiments 1 to 167, wherein the
total amount of excipient in the solution is in the range of 0.1 to
7 parts by weight.
Embodiment 169
[0176] The solution of any of embodiments 1 to 167, wherein the
total amount of excipient in the solution is in the range of 0.3 to
7 parts by weight.
Embodiment 170
[0177] The solution of any of embodiments 1 to 167, wherein the
total amount of excipient in the solution is in the range of 0.5 to
5 parts by weight.
Embodiment 171
[0178] The solution of any of embodiments 1 to 167, wherein the
total amount of excipient in the solution is in the range of 1 to 5
parts by weight.
Embodiment 172
[0179] The solution of any of embodiments 1 to 167, wherein the
total amount of excipient in the solution is in the range of 2 to 5
parts by weight.
Embodiment 173
[0180] The solution of any of embodiments 1 to 167, wherein the
total amount of excipient in the solution is in the range of 3 to 5
parts by weight.
Embodiment 174
[0181] The solution of any of embodiments 1 to 167, wherein the
total amount of excipient in the solution is in the range of 4 to 5
parts by weight.
Embodiment 175
[0182] The solution of any of embodiments 1 to 167, wherein the
total amount of excipient in the solution is in the range of 4.5 to
5.5 parts by weight.
Embodiment 176
[0183] The solution of any of embodiments 1 to 167, wherein the
total amount of excipient that modulates viscosity, turbidity or
both in the solution is in the range of 0.1 to 7 parts by
weight.
Embodiment 177
[0184] The solution of any of embodiments 1 to 167, wherein the
total amount of excipient that modulates viscosity, turbidity or
both in the solution is in the range of 0.3 to 7 parts by
weight.
Embodiment 178
[0185] The solution of any of embodiments 1 to 167, wherein the
total amount of excipient that modulates viscosity, turbidity or
both in the solution is in the range of 0.5 to 5 parts by
weight.
Embodiment 179
[0186] The solution of any of embodiments 1 to 167, wherein the
total amount of excipient that modulates viscosity, turbidity or
both in the solution is in the range of 1 to 5 parts by weight.
Embodiment 180
[0187] The solution of any of embodiments 1 to 167, wherein the
total amount of excipient that modulates viscosity, turbidity or
both in the solution is in the range of 2 to 5 parts by weight.
Embodiment 181
[0188] The solution of any of embodiments 1 to 167, wherein the
total amount of excipient that modulates viscosity, turbidity or
both in the solution is in the range of 3 to 5 parts by weight.
Embodiment 182
[0189] The solution of any of embodiments 1 to 167, wherein the
total amount of excipient that modulates viscosity, turbidity or
both in the solution is in the range of 4 to 5 parts by weight.
Embodiment 183
[0190] The solution of any of embodiments 1 to 167, wherein the
total amount of excipient that modulates viscosity, turbidity or
both in the solution is in the range of 4.5 to 5.5 parts by
weight.
Embodiment 184
[0191] The solution of any of embodiments 1 to 183, wherein the
antisense oligonucleotide comprises at least one modified
nucleoside.
Embodiment 185
[0192] The solution of embodiment 184, wherein at least one
modified nucleoside comprises a modified sugar moiety.
Embodiment 186
[0193] The solution of embodiment 185, wherein at least one
modified sugar moiety is a 2'-substituted sugar moiety.
Embodiment 187
[0194] The solution of embodiment 186, wherein the 2'-substitutent
of at least one 2'-substituted sugar moiety is selected from among:
2'-OMe, 2'-F, and 2'-MOE.
Embodiment 188
[0195] The solution of embodiment 187, wherein the 2'-substituent
of at least one 2'-substituted sugar moiety is a 2'-MOE.
Embodiment 189
[0196] The solution of any of embodiments 1 to 188, wherein at
least one modified sugar moiety is a bicyclic sugar moiety.
Embodiment 190
[0197] The solution of embodiment 189, wherein at least one
bicyclic sugar moiety is LNA or cEt.
Embodiment 191
[0198] The solution of any of embodiments 1 to 191, wherein at
least one sugar moiety is a sugar surrogate.
Embodiment 192
[0199] The solution of embodiment 191, wherein at least one sugar
surrogate is a morpholino.
Embodiment 193
[0200] The solution of embodiment 191, wherein at least one sugar
surrogate is a modified morpholino.
Embodiment 194
[0201] The solution of any of embodiments 1 to 193, wherein the
antisense oligonucleotide comprises at least 5 modified
nucleosides, each independently comprising a modified sugar
moiety.
Embodiment 195
[0202] The solution of any of embodiments 1 to 193, wherein the
antisense oligonucleotide comprises at least 10 modified
nucleosides, each independently comprising a modified sugar
moiety.
Embodiment 196
[0203] The solution of any of embodiments 1 to 193, wherein the
antisense oligonucleotide comprises at least 15 modified
nucleosides, each independently comprising a modified sugar
moiety.
Embodiment 197
[0204] The solution of any of embodiments 1 to 196, wherein each
nucleoside of the antisense oligonucleotide is a modified
nucleoside, each independently comprising a modified sugar moiety
Embodiment 198. The solution of any of any of embodiments 1 to 197,
wherein the antisense oligonucleotide comprises at least two
modified nucleosides comprising modified sugar moieties that are
the same as one another.
Embodiment 199
[0205] The solution of any of embodiments 1 to 198, wherein the
antisense oligonucleotide comprises at least two modified
nucleosides comprising modified sugar moieties that are different
from one another.
Embodiment 200
[0206] The solution of any of embodiments 1 to 199, wherein the
antisense oligonucleotide comprises a modified region of at least 5
contiguous modified nucleosides.
Embodiment 201
[0207] The solution of any of embodiments 1 to 200, wherein the
antisense oligonucleotide comprises a modified region of at least
10 contiguous modified nucleosides.
Embodiment 202
[0208] The solution of any of embodiments 1 to 201, wherein the
antisense oligonucleotide comprises a modified region of at least
15 contiguous modified nucleosides.
Embodiment 203
[0209] The solution of any of embodiments 1 to 202, wherein the
antisense oligonucleotide comprises a modified region of at least
20 contiguous modified nucleosides.
Embodiment 204
[0210] The solution of any of embodiments 197 to 203, wherein each
modified nucleoside of the modified region has a modified sugar
moiety independently selected from among: 2'-F, 2'-OMe, 2'-MOE,
cEt, LNA, morpholino, and modified morpholino.
Embodiment 205
[0211] The solution of any of embodiments 197 to 203, wherein the
modified nucleosides of the modified region each comprise the same
modification as one another.
Embodiment 206
[0212] The solution of embodiment 205, wherein the modified
nucleosides of the modified region each comprise the same
2'-substituted sugar moiety.
Embodiment 207
[0213] The solution of embodiment 206, wherein the 2'-substituted
sugar moiety of the modified nucleosides of the region of modified
nucleosides is selected from 2'-F, 2'-OMe, and 2'-MOE.
Embodiment 208
[0214] The solution of embodiment 207, wherein the 2'-substituted
sugar moiety of the modified nucleosides of the region of modified
nucleosides is 2'-MOE.
Embodiment 209
[0215] The solution of embodiment 205, wherein the modified
nucleosides of the region of modified nucleosides each comprise the
same bicyclic sugar moiety.
Embodiment 210
[0216] The solution of embodiment 209, wherein the bicyclic sugar
moiety of the modified nucleosides of the region of modified
nucleosides is selected from LNA and cEt.
Embodiment 211
[0217] The solution of embodiment 205, wherein the modified
nucleosides of the region of modified nucleosides each comprises a
sugar surrogate.
Embodiment 212
[0218] The solution of embodiment 211, wherein the sugar surrogate
of the modified nucleosides of the region of modified nucleosides
is a morpholino.
Embodiment 213
[0219] The solution of embodiment 211, wherein the sugar surrogate
of the modified nucleosides of the region of modified nucleosides
is a modified morpholino.
Embodiment 214
[0220] The solution of any of embodiments 1 to 213, wherein the
modified nucleotide comprises no more than 4 contiguous naturally
occurring nucleosides.
Embodiment 215
[0221] The solution of any of embodiments 1 to 214, wherein each
nucleoside of the antisense oligonucleotide is a modified
nucleoside.
Embodiment 216
[0222] The solution of embodiment 215 wherein each modified
nucleoside comprises a modified sugar moiety.
Embodiment 217
[0223] The solution of embodiment 216, wherein the modified
nucleosides of the antisense oligonucleotide comprise the same
modification as one another.
Embodiment 218
[0224] The solution of embodiment 217, wherein the modified
nucleosides of the antisense oligonucleotide each comprise the same
2'-substituted sugar moiety.
Embodiment 219
[0225] The solution of embodiment 218, wherein the 2'-substituted
sugar moiety of the antisense oligonucleotide is selected from
2'-F, 2'-OMe, and 2'-MOE.
Embodiment 220
[0226] The solution of embodiment 219, wherein the 2'-substituted
sugar moiety of the antisense oligonucleotide is 2'-MOE.
Embodiment 221
[0227] The solution of embodiment 217, wherein the modified
nucleosides of the antisense oligonucleotide each comprise the same
bicyclic sugar moiety.
Embodiment 222
[0228] The solution of embodiment 221, wherein the bicyclic sugar
moiety of the antisense oligonucleotide is selected from LNA and
cEt.
Embodiment 223
[0229] The solution of embodiment 217, wherein the modified
nucleosides of the antisense oligonucleotide each comprises a sugar
surrogate.
Embodiment 224
[0230] The solution of embodiment 223, wherein the sugar surrogate
of the antisense oligonucleotide is a morpholino.
Embodiment 225
[0231] The solution of embodiment 223, wherein the sugar surrogate
of the antisense oligonucleotide is a modified morpholino.
Embodiment 226
[0232] The solution of any of embodiments 1 to 225, wherein the
antisense oligonucleotide comprises at least one modified
internucleoside linkage.
Embodiment 227
[0233] The solution of embodiment 226, wherein each internucleoside
linkage is a modified internucleoside linkage.
Embodiment 228
[0234] The solution of embodiment 226 or 227, comprising at least
one phosphorothioate internucleoside linkage.
Embodiment 229
[0235] The solution of embodiment 227, wherein each internucleoside
linkage is a modified internucleoside linkage and wherein each
internucleoside linkage comprises the same modification.
Embodiment 230
[0236] The solution of embodiment 229, wherein each internucleoside
linkage is a phosphorothioate internucleoside linkage.
Embodiment 231
[0237] The solution of any of embodiments 1 to 230, comprising at
least one conjugate.
Embodiment 232
[0238] The solution of any of embodiments 1 to 231, wherein the
antisense oligonucleotide consists of a single-strand antisense
oligonucleotide.
Embodiment 233
[0239] The solution of any of embodiments 1 to 232, wherein the
antisense oligonucleotide consists of a double-strand antisense
oligonucleotide.
Embodiment 234
[0240] The solution of any of embodiments 1 to 233, wherein the
antisense oligonucleotide is selected from the group consisting of
ISIS 426115, ISIS 104838, ISIS 416858, ISIS 420915, ISIS 494372,
ISIS 487660, ISIS 404173, ISIS 449884, ISIS 501861, ISIS 540175,
ISIS 396443, ISIS 463588, ISIS 301012, ISIS 329993, ISIS 304801,
ISIS 426115, ISIS 481464, ISIS 183750, and ISIS 333611.
Embodiment 235
[0241] The solution of any of embodiments 1 to 234, wherein the
antisense oligonucleotide has a dynamic viscosity of more than 60
cP at 25.degree. C. when mixed in water or saline solution in the
absence of an excipient that modulates viscosity, turbidity or both
viscosity and turbidity.
Embodiment 236
[0242] The solution of any of embodiments 1 to 234, wherein the
antisense oligonucleotide has a dynamic viscosity of more than 55
cP at 25.degree. C. when mixed in water or saline solution and in
the absence of an excipient that modulates viscosity, turbidity or
both viscosity and turbidity.
Embodiment 237
[0243] The solution of any of embodiments 1 to 234, wherein the
antisense oligonucleotide has a dynamic viscosity of more than 50
cP at 25.degree. C. when mixed in water or saline solution and in
the absence of an excipient that modulates viscosity, turbidity or
both viscosity and turbidity.
Embodiment 238
[0244] The solution of any of embodiments 1 to 234, wherein the
antisense oligonucleotide has a dynamic viscosity of more than 45
cP at 25.degree. C. when mixed in water or saline solution and in
the absence of an excipient that modulates viscosity, turbidity or
both viscosity and turbidity.
Embodiment 239
[0245] The solution of any of embodiments 1 to 234, wherein the
antisense oligonucleotide has a dynamic viscosity of more than 40
cP at 25.degree. C. when mixed in water or saline solution and in
the absence of an excipient that modulates viscosity, turbidity or
both viscosity and turbidity.
Embodiment 240
[0246] The solution of any of embodiments 1 to 234, wherein the
antisense oligonucleotide has a turbidity of more than 100 NTU when
mixed in water or saline solution and in the absence of an
excipient that modulates viscosity, turbidity or both viscosity and
turbidity.
Embodiment 241
[0247] The solution of any of embodiments 1 to 234, wherein the
antisense oligonucleotide has a turbidity of more than 70 NTU when
mixed in water or saline solution and in the absence of an
excipient that modulates viscosity, turbidity or both viscosity and
turbidity.
Embodiment 242
[0248] The solution of any of embodiments 1 to 234, wherein the
antisense oligonucleotide has a turbidity of more than 50 NTU when
mixed in water or saline solution and in the absence of an
excipient that modulates viscosity, turbidity or both viscosity and
turbidity.
Embodiment 243
[0249] The solution of any of embodiments 1 to 234, wherein the
antisense oligonucleotide has a turbidity of more than 25 NTU when
mixed in water or saline solution and in the absence of an
excipient that modulates viscosity, turbidity or both viscosity and
turbidity.
Embodiment 244
[0250] The solution of any of embodiments 1 to 234, wherein the
antisense oligonucleotide has a turbidity of more than 20 NTU when
mixed in water or saline solution and in the absence of an
excipient that modulates viscosity, turbidity or both viscosity and
turbidity.
Embodiment 245
[0251] A method of reducing the turbidity of an antisense
oligonucleotide solution, said method comprising adding an
effective amount of an excipient that modulates viscosity,
turbidity or both viscosity and turbidity to the antisense
oligonucleotide solution.
Embodiment 246
[0252] The method of embodiment 245, wherein the excipient that
modulates viscosity, turbidity or both viscosity and turbidity is
an excipient according to any of embodiments 49-133.
Embodiment 247
[0253] The method of embodiment 245 or 246, wherein the antisense
compound is an antisense compound according to any of embodiments
184-234.
Embodiment 248
[0254] The method of any of embodiments 245-247, wherein the
turbidity is reduced by 0-1000 NTU.
Embodiment 249
[0255] The method of any of embodiments 245-247, wherein the
turbidity is reduced by 0-500 NTU.
Embodiment 250
[0256] The method of any of embodiments 245-247, wherein the
turbidity is reduced by 0-100 NTU.
Embodiment 251
[0257] The method of any of embodiments 245-247, wherein the
turbidity is reduced by 0-50 NTU.
Embodiment 252
[0258] The method of any of embodiments 245-247, wherein the
turbidity is reduced by 0-30 NTU.
Embodiment 253
[0259] The method of any of embodiments 245-247, wherein the
turbidity is reduced by 0-20 NTU.
Embodiment 254
[0260] The method of any of embodiments 245-247, wherein the
turbidity is reduced by 0-10 NTU.
Embodiment 255
[0261] A method of reducing the viscosity of an antisense
oligonucleotide solution, said method comprising adding an
effective amount of an excipient that modulates viscosity,
turbidity or both viscosity and turbidity to the antisense
oligonucleotide solution.
Embodiment 256
[0262] The method of embodiment 255, wherein the excipient that
modulates viscosity, turbidity or both viscosity and turbidity is
an excipient according to any of embodiments 49-155.
Embodiment 257
[0263] The method of embodiment 255 or 256, wherein the antisense
compound is an antisense compound according to any of embodiments
184-234.
Embodiment 258
[0264] The method of any of embodiments 255-257, wherein the
viscosity is reduced by 1-100 cP at 25.degree. C.
Embodiment 259
[0265] The method of any of embodiments 255-257, wherein the
viscosity is reduced by 1-50 cP at 25.degree. C.
Embodiment 260
[0266] The method of any of embodiments 255-257, wherein the
viscosity is reduced by 1-40 cP at 25.degree. C.
Embodiment 261
[0267] The method of any of embodiments 255-257, wherein the
viscosity is reduced by 1-30 cP at 25.degree. C.
Embodiment 262
[0268] The method of any of embodiments 255-257, wherein the
viscosity is reduced by 1-20 cP at 25.degree. C.
Embodiment 263
[0269] The method of any of embodiments 255-257, wherein the
viscosity is reduced by 1-10 cP at 25.degree. C.
Embodiment 264
[0270] The method of any of embodiments 255-257, wherein the
viscosity is reduced by 1-5 cP at 25.degree. C.
Embodiment 265
[0271] A method of reducing both the viscosity and the turbidity of
an antisense oligonucleotide solution, said method comprising
adding an effective amount of an excipient that modulates
viscosity, turbidity or both to the antisense oligonucleotide
solution.
Embodiment 266
[0272] The method of embodiment 265, wherein the excipient that
modulates viscosity, turbidity or both viscosity and turbidity is
an excipient according to any of embodiments 49-155.
Embodiment 267
[0273] The method of embodiment 245 or 246, wherein the antisense
compound is an antisense compound according to any of embodiments
184-234.
Embodiment 268
[0274] A method of selecting a pharmaceutically acceptable
antisense oligonucleotide from among many antisense
oligonucleotides, said method comprising: [0275] measuring the
dynamic viscosity of an antisense oligonucleotide solution, wherein
the solution comprises between 100 mg/mL and 250 mg/mL of the
antisense oligonucleotide; [0276] measuring the turbidity of the
antisense oligonucleotide solution; and [0277] selecting an
antisense oligonucleotide that has a dynamic viscosity below 40 cP
and a turbidity below 30 NTU.
Embodiment 269
[0278] A method of reducing the turbidity of an antisense
oligonucleotide solution, said method comprising adding an
effective amount of an excipient that modulates viscosity,
turbidity or both viscosity and turbidity to the antisense
oligonucleotide solution.
Embodiment 270
[0279] The method of embodiment 269, wherein the excipient that
modulates viscosity, turbidity or both viscosity and turbidity is
an excipient according to any preceeding embodiment.
Embodiment 271
[0280] A method of reducing the viscosity of an antisense
oligonucleotide solution, said method comprising adding an
effective amount of an excipient that modulates viscosity,
turbidity or both viscosity and turbidity to the antisense
oligonucleotide solution.
Embodiment 272
[0281] The method of embodiment 271, wherein the excipient that
modulates viscosity, turbidity or both viscosity and turbidity is
an excipient according to any preceeding embodiment.
Embodiment 273
[0282] A method of reducing both the viscosity and the turbidity of
an antisense oligonucleotide solution, said method comprising
adding an effective amount of an excipient that modulates
viscosity, turbidity or both to the antisense oligonucleotide
solution.
Embodiment 274
[0283] The method of embodiment 273, wherein the excipient that
modulates viscosity, turbidity or both viscosity and turbidity is
an excipient according to any preceeding embodiment.
Embodiment 275
[0284] A method of increasing the viscosity, turbidity, or both the
viscosity and turbidity of an antisense oligonucleotide solution,
comprising reducing or removing the amount of an excipient that
modulates viscosity, turbidity or both from the antisense
oligonucleotide solution.
BRIEF DESCRIPTION OF THE FIGURES
[0285] FIG. 1 illustrates a standard curve conversion for 13-mm
sample tubes for 0-1000 NTU. CF for the tubes is shown to be the
slope of the plot, which is 1.8148.
[0286] FIG. 2 illustrates the turbidity and temperature profiles of
220 mg/mL ISIS NO. 426115 over time. The turbidity reaches 20 NTU,
which is approximately the lower limit of visible turbidity, at
around 50 minutes.
[0287] FIG. 3 illustrates the turbidity plotted over (approximate)
temperature profile of 220 mg/mL ISIS NO.
[0288] FIG. 4 illustrates the effect of NTU of Formazin reference
suspensions on visible turbidity. FIG. 4A These standards are 0
(visually clear), 20, 100, 1000, and 4000 (visually turbid) NTU,
respectively. FIG. 4B These standards shown in 2 mL, 13 mm vials
are 20, 10, and 0 (WFI control) NTU respectively.
DETAILED DESCRIPTION OF THE INVENTION
[0289] Unless specific definitions are provided, the nomenclature
used in connection with, and the procedures and techniques of,
analytical chemistry, synthetic organic chemistry, and medicinal
and pharmaceutical chemistry described herein are those well known
and commonly used in the art. Standard techniques may be used for
chemical synthesis, and chemical analysis. Certain such techniques
and procedures may be found for example in "Carbohydrate
Modifications in Antisense Research" Edited by Sangvi and Cook,
American Chemical Society, Washington D.C., 1994; "Remington's
Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa.,
21.degree. edition, 2005; and "Antisense Drug Technology,
Principles, Strategies, and Applications" Edited by Stanley T.
Crooke, CRC Press, Boca Raton, Fla.; and Sambrook et al.,
"Molecular Cloning, A laboratory Manual," 2.sup.nd Edition, Cold
Spring Harbor Laboratory Press, 1989, which are hereby incorporated
by reference for any purpose. Where permitted, all patents,
applications, published applications and other publications and
other data referred to throughout in the disclosure are
incorporated by reference herein in their entirety.
[0290] Unless otherwise indicated, the following terms have the
following meanings:
[0291] As used herein, "aqueous solution" means a solution
comprising one or more solutes in water. In certain embodiments,
the water is sterile water. In certain embodiments, an aqueous
solution is saline.
[0292] As used herein, "antisense oligonucleotide solution" means
an aqueous solution comprising one or more antisense
oligonucleotides. In certain embodiments, an antisense
oligonucleotide solution comprises an aqueous solution having only
one antisense oligonucleotide. In certain embodiments, an antisense
oligonucleotide solution comprises an aqueous solution having more
than one antisense oligonucleotide. In certain embodiments, an
antisense oligonucleotide solution comprises an aqueous solution
wherein an antisense oligonucleotide is present at a concentration
between 0.1 and 500 mg/mL.
[0293] As used herein, "excipient" means any compound or
composition other than water or an antisense oligonucleotide.
[0294] As used herein, "excipient that modulates viscosity,
turbidity or both viscosity and turbidity" or "excipient that
modulates viscosity, turbidity or both" means an excipient, the
presence of which increases or decreases the viscosity and/or
turbidity of an antisense oligonucleotide solution, compared to the
viscosity or turbidity of the antisense oligonucleotide solution at
the same concentration and temperature in the absence of the
excipient.
[0295] As used herein, "aromatic compound" refers to a mono- or
polycyclic carbocyclic ring system having one or more aromatic
rings. Preferred aromatic ring systems have from about 5 to about
20 carbon atoms in one or more rings. Aromatic groups as used
herein may optionally include further substituent groups.
[0296] The term "heterocyclic compound" as used herein, refers to a
mono-, or poly-cyclic ring system that includes at least one
heteroatom and is unsaturated, partially saturated, or fully
saturated, thereby including heteroaryl groups and heterocyclic
aromatic compounds. Heterocyclic compound is also meant to include
fused ring systems wherein one or more of the fused rings contain
at least one heteroatom and the other rings can contain one or more
heteroatoms or optionally contain no heteroatoms. A heterocyclic
compound typically includes at least one atom selected from sulfur,
nitrogen or oxygen. In certain embodiments, a heterocyclic compound
may include one or more rings, wherein each ring has one or more
heteroatoms. In certain embodiments, a heterocyclic compound
includes a monocyclic ring system with one or more heteroatoms. In
certain embodiments, a heterocyclic compound includes a monocyclic
ring system with two or more heteroatoms. Examples of heterocyclic
compounds include, but are not limited to, [1,3]dioxolane,
pyrrolidine, pyrazoline, pyrazolidine, imidazoline, imidazolidine,
piperidine, piperazine, oxazolidine, isoxazolidine, morpholine,
thiazolidine, isothiazolidine, quinoxaline, pyridazinone,
tetrahydrofuran and the like. Heterocyclic compounds as used herein
may optionally include further substituent groups.
[0297] As used herein, "heterocyclic aromatic compound" means any
compound comprising a mono- or poly-cyclic aromatic ring system or
fused ring system wherein at least one of the rings is aromatic and
includes one or more heteroatoms. Heterocyclic aromatic compounds
also include fused ring systems, including systems where one or
more of the fused rings contain no heteroatoms. Heterocyclic
aromatic compounds typically include one ring atom selected from
sulfur, nitrogen or oxygen. Examples of heterocyclic aromatic
compounds groups include without limitation, pyridine, pyrazine,
pyrimidine, pyrrole, pyrazole, imidazole, thiazole, oxazole,
isoxazole, thiadiazole, oxadiazole, thiophene, furan, quinoline,
isoquinoline, benzimidazole, benzooxazole, quinoxaline and the
like. Heterocyclic aromatic compounds can be attached to a parent
molecule directly or through a linking moiety such as an aliphatic
group or hetero atom. Heterocyclic aromatic compounds as used
herein may optionally include further substituent groups.
[0298] As used herein, "nucleoside" means a compound comprising a
nucleobase moiety and a sugar moiety. Nucleosides include, but are
not limited to, naturally occurring nucleosides (as found in DNA
and RNA) and modified nucleosides. Nucleosides may be linked to a
phosphate moiety.
[0299] As used herein, "chemical modification" means a chemical
difference in a compound when compared to a naturally occurring
counterpart. In reference to an oligonucleotide, chemical
modification does not include differences only in nucleobase
sequence. Chemical modifications of oligonucleotides include
nucleoside modifications (including sugar moiety modifications and
nucleobase modifications) and internucleoside linkage
modifications.
[0300] As used herein, "furanosyl" means a structure comprising a
5-membered ring comprising four carbon atoms and one oxygen
atom.
[0301] As used herein, "naturally occurring sugar moiety" means a
ribofuranosyl as found in naturally occurring RNA or a
deoxyribofuranosyl as found in naturally occurring DNA.
[0302] As used herein, "sugar moiety" means a naturally occurring
sugar moiety or a modified sugar moiety of a nucleoside.
[0303] As used herein, "modified sugar moiety" means a substituted
sugar moiety, a bicyclic or tricyclic sugar moiety, or a sugar
surrogate.
[0304] As used herein, "substituted sugar moiety" means a furanosyl
comprising at least one substituent group that differs from that of
a naturally occurring sugar moiety. Substituted sugar moieties
include, but are not limited to furanosyls comprising substituents
at the 2'-position, the 3'-position, the 5'-position and/or the
4'-position.
[0305] As used herein, "2'-substituted sugar moiety" means a
furanosyl comprising a substituent at the 2'-position other than H
or OH. Unless otherwise indicated, a 2'-substituted sugar moiety is
not a bicyclic sugar moiety (i.e., the 2'-substituent of a
2'-substituted sugar moiety does not form a bridge to another atom
of the furanosyl ring.
[0306] As used herein, "MOE" means
--OCH.sub.2CH.sub.2OCH.sub.3.
[0307] As used herein, "bicyclic sugar moiety" means a modified
sugar moiety comprising a 4 to 7 membered ring (including but not
limited to a furanosyl) comprising a bridge connecting two atoms of
the 4 to 7 membered ring to form a second ring, resulting in a
bicyclic structure. In certain embodiments, the 4 to 7 membered
ring is a sugar ring. In certain embodiments the 4 to 7 membered
ring is a furanosyl. In certain such embodiments, the bridge
connects the 2'-carbon and the 4'-carbon of the furanosyl.
[0308] As used herein the term "sugar surrogate" means a structure
that does not comprise a furanosyl and that is capable of replacing
the naturally occurring sugar moiety of a nucleoside, such that the
resulting nucleoside is capable of (1) incorporation into an
oligonucleotide and (2) hybridization to a complementary
nucleoside. Such structures include rings comprising a different
number of atoms than furanosyl (e.g., 4, 6, or 7-membered rings);
replacement of the oxygen of a furanosyl with a non-oxygen atom
(e.g., carbon, sulfur, or nitrogen); or both a change in the number
of atoms and a replacement of the oxygen. Such structures may also
comprise substitutions corresponding to those described for
substituted sugar moieties (e.g., 6-membered carbocyclic bicyclic
sugar surrogates optionally comprising additional substituents).
Sugar surrogates also include more complex sugar replacements
(e.g., the non-ring systems of peptide nucleic acid). Sugar
surrogates include without limitation morpholino, modified
morpholinos, cyclohexenyls and cyclohexitols.
[0309] As used herein, "nucleotide" means a nucleoside further
comprising a phosphate linking group. As used herein, "linked
nucleosides" may or may not be linked by phosphate linkages and
thus includes, but is not limited to "linked nucleotides." As used
herein, "linked nucleosides" are nucleosides that are connected in
a continuous sequence (i.e. no additional nucleosides are present
between those that are linked).
[0310] As used herein, "nucleobase" means a group of atoms that can
be linked to a sugar moiety to create a nucleoside that is capable
of incorporation into an oligonucleotide, and wherein the group of
atoms is capable of bonding with a complementary naturally
occurring nucleobase of another oligonucleotide or nucleic acid.
Nucleobases may be naturally occurring or may be modified.
[0311] As used herein, "heterocyclic base" or "heterocyclic
nucleobase" means a nucleobase comprising a heterocyclic
structure.
[0312] As used herein the terms, "unmodified nucleobase" or
"naturally occurring nucleobase" means the naturally occurring
heterocyclic nucleobases of RNA or DNA: the purine bases adenine
(A) and guanine (G), and the pyrimidine bases thymine (T), cytosine
(C) (including 5-methyl C), and uracil (U).
[0313] As used herein, "modified nucleobase" means any nucleobase
that is not a naturally occurring nucleobase.
[0314] As used herein, "modified nucleoside" means a nucleoside
comprising at least one chemical modification compared to naturally
occurring RNA or DNA nucleosides. Modified nucleosides comprise a
modified sugar moiety and/or a modified nucleobase.
[0315] As used herein, "bicyclic nucleoside" or "BNA" means a
nucleoside comprising a bicyclic sugar moiety.
[0316] As used herein, "constrained ethyl nucleoside" or "cEt"
means a nucleoside comprising a bicyclic sugar moiety comprising a
4'-CH(CH.sub.3)--O-2'bridge.
[0317] As used herein, "locked nucleic acid nucleoside" or "LNA"
means a nucleoside comprising a bicyclic sugar moiety comprising a
4'-CH.sub.2--O-2'bridge.
[0318] As used herein, "2'-substituted nucleoside" means a
nucleoside comprising a substituent at the 2'-position other than H
or OH. Unless otherwise indicated, a 2'-substituted nucleoside is
not a bicyclic nucleoside.
[0319] As used herein, "2'-deoxynucleoside" means a nucleoside
comprising 2'-H furanosyl sugar moiety, as found in naturally
occurring deoxyribonucleosides (DNA). In certain embodiments, a
2'-deoxynucleoside may comprise a modified nucleobase or may
comprise an RNA nucleobase (e.g., uracil).
[0320] As used herein, "oligonucleotide" means a compound
comprising a plurality of linked nucleosides. In certain
embodiments, an oligonucleotide comprises one or more unmodified
ribonucleosides (RNA) and/or unmodified deoxyribonucleosides (DNA)
and/or one or more modified nucleosides.
[0321] As used herein "oligonucleoside" means an oligonucleotide in
which none of the internucleoside linkages contains a phosphorus
atom. As used herein, oligonucleotides include
oligonucleosides.
[0322] As used herein, "modified oligonucleotide" means an
oligonucleotide comprising at least one modified nucleoside and/or
at least one modified internucleoside linkage.
[0323] As used herein "internucleoside linkage" means a covalent
linkage between adjacent nucleosides in an oligonucleotide.
[0324] As used herein "naturally occurring internucleoside linkage"
means a 3' to 5' phosphodiester linkage.
[0325] As used herein, "modified internucleoside linkage" means any
internucleoside linkage other than a naturally occurring
internucleoside linkage.
[0326] As used herein, "oligomeric compound" means a polymeric
structure comprising two or more sub-structures. In certain
embodiments, an oligomeric compound comprises an oligonucleotide.
In certain embodiments, an oligomeric compound comprises one or
more conjugate groups and/or terminal groups. In certain
embodiments, an oligomeric compound consists of an
oligonucleotide.
[0327] As used herein, "terminal group" means one or more atom
attached to either, or both, the 3' end or the 5' end of an
oligonucleotide. In certain embodiments a terminal group is a
conjugate group. In certain embodiments, a terminal group comprises
one or more terminal group nucleosides.
[0328] As used herein, "conjugate" means an atom or group of atoms
bound to an oligonucleotide or oligomeric compound. In general,
conjugate groups modify one or more properties of the compound to
which they are attached, including, but not limited to
pharmacodynamic, pharmacokinetic, binding, absorption, cellular
distribution, cellular uptake, charge and/or clearance
properties.
[0329] As used herein, "conjugate linking group" means any atom or
group of atoms used to attach a conjugate to an oligonucleotide or
oligomeric compound.
[0330] As used herein, "antisense compound" means a compound
comprising or consisting of an oligonucleotide at least a portion
of which is complementary to a target nucleic acid to which it is
capable of hybridizing, resulting in at least one antisense
activity.
[0331] As used herein, "antisense activity" means any detectable
and/or measurable change attributable to the hybridization of an
antisense compound to its target nucleic acid.
[0332] As used herein, "detecting" or "measuring" means that a test
or assay for detecting or measuring is performed. Such detection
and/or measuring may result in a value of zero. Thus, if a test for
detection or measuring results in a finding of no activity
(activity of zero), the step of detecting or measuring the activity
has nevertheless been performed.
[0333] As used herein, "detectable and/or measurable activity"
means a statistically significant activity that is not zero.
[0334] As used herein, "essentially unchanged" means little or no
change in a particular parameter, particularly relative to another
parameter which changes much more. In certain embodiments, a
parameter is essentially unchanged when it changes less than 5%. In
certain embodiments, a parameter is essentially unchanged if it
changes less than two-fold while another parameter changes at least
ten-fold. For example, in certain embodiments, an antisense
activity is a change in the amount of a target nucleic acid. In
certain such embodiments, the amount of a non-target nucleic acid
is essentially unchanged if it changes much less than the target
nucleic acid does, but the change need not be zero.
[0335] As used herein, "expression" means the process by which a
gene ultimately results in a protein. Expression includes, but is
not limited to, transcription, post-transcriptional modification
(e.g., splicing, polyadenlyation, addition of 5'-cap), and
translation.
[0336] As used herein, "target nucleic acid" means a nucleic acid
molecule to which an antisense compound hybridizes.
[0337] As used herein, "mRNA" means an RNA molecule that encodes a
protein.
[0338] As used herein, "pre-mRNA" means an RNA transcript that has
not been fully processed into mRNA. Pre-RNA includes one or more
intron.
[0339] As used herein, "transcript" means an RNA molecule
transcribed from DNA. Transcripts include, but are not limited to
mRNA, pre-mRNA, and partially processed RNA.
[0340] As used herein, "targeting" or "targeted to" means the
association of an antisense compound to a particular target nucleic
acid molecule or a particular region of a target nucleic acid
molecule. An antisense compound targets a target nucleic acid if it
is sufficiently complementary to the target nucleic acid to allow
hybridization under physiological conditions.
[0341] As used herein, "nucleobase complementarity" or
"complementarity" when in reference to nucleobases means a
nucleobase that is capable of base pairing with another nucleobase.
For example, in DNA, adenine (A) is complementary to thymine (T).
For example, in RNA, adenine (A) is complementary to uracil (U). In
certain embodiments, complementary nucleobase means a nucleobase of
an antisense compound that is capable of base pairing with a
nucleobase of its target nucleic acid. For example, if a nucleobase
at a certain position of an antisense compound is capable of
hydrogen bonding with a nucleobase at a certain position of a
target nucleic acid, then the position of hydrogen bonding between
the oligonucleotide and the target nucleic acid is considered to be
complementary at that nucleobase pair. Nucleobases comprising
certain modifications may maintain the ability to pair with a
counterpart nucleobase and thus, are still capable of nucleobase
complementarity.
[0342] As used herein, "non-complementary" in reference to
nucleobases means a pair of nucleobases that do not form hydrogen
bonds with one another.
[0343] As used herein, "complementary" in reference to oligomeric
compounds (e.g., linked nucleosides, oligonucleotides, or nucleic
acids) means the capacity of such oligomeric compounds or regions
thereof to hybridize to another oligomeric compound or region
thereof through nucleobase complementarity under stringent
conditions. Complementary oligomeric compounds need not have
nucleobase complementarity at each nucleoside. Rather, some
mismatches are tolerated. In certain embodiments, complementary
oligomeric compounds or regions are complementary at 70% of the
nucleobases (70% complementary). In certain embodiments,
complementary oligomeric compounds or regions are 80%
complementary. In certain embodiments, complementary oligomeric
compounds or regions are 90% complementary. In certain embodiments,
complementary oligomeric compounds or regions are 95%
complementary. In certain embodiments, complementary oligomeric
compounds or regions are 100% complementary.
[0344] As used herein, "hybridization" means the pairing of
complementary oligomeric compounds (e.g., an antisense compound and
its target nucleic acid). While not limited to a particular
mechanism, the most common mechanism of pairing involves hydrogen
bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen
hydrogen bonding, between complementary nucleobases.
[0345] As used herein, "specifically hybridizes" means the ability
of an oligomeric compound to hybridize to one nucleic acid site
with greater affinity than it hybridizes to another nucleic acid
site. In certain embodiments, an antisense oligonucleotide
specifically hybridizes to more than one target site.
[0346] As used herein, "percent complementarity" means the
percentage of nucleobases of an oligomeric compound that are
complementary to an equal-length portion of a target nucleic acid.
Percent complementarity is calculated by dividing the number of
nucleobases of the oligomeric compound that are complementary to
nucleobases at corresponding positions in the target nucleic acid
by the total length of the oligomeric compound.
[0347] As used herein, "percent identity" means the number of
nucleobases in a first nucleic acid that are the same type
(independent of chemical modification) as nucleobases at
corresponding positions in a second nucleic acid, divided by the
total number of nucleobases in the first nucleic acid.
[0348] As used herein, "modulation" means a change of amount or
quality of a molecule, function, or activity when compared to the
amount or quality of a molecule, function, or activity prior to
modulation. For example, modulation includes the change, either an
increase (stimulation or induction) or a decrease (inhibition or
reduction) in gene expression. As a further example, modulation of
expression can include a change in splice site selection of
pre-mRNA processing, resulting in a change in the absolute or
relative amount of a particular splice-variant compared to the
amount in the absence of modulation.
[0349] As used herein, "motif" means a pattern of chemical
modifications in an oligomeric compound or a region thereof. Motifs
may be defined by modifications at certain nucleosides and/or at
certain linking groups of an oligomeric compound.
[0350] As used herein, "nucleoside motif" means a pattern of
nucleoside modifications in an oligomeric compound or a region
thereof. The linkages of such an oligomeric compound may be
modified or unmodified. Unless otherwise indicated, motifs herein
describing only nucleosides are intended to be nucleoside motifs.
Thus, in such instances, the linkages are not limited.
[0351] As used herein, "sugar motif" means a pattern of sugar
modifications in an oligomeric compound or a region thereof.
[0352] As used herein, "linkage motif" means a pattern of linkage
modifications in an oligomeric compound or region thereof. The
nucleosides of such an oligomeric compound may be modified or
unmodified. Unless otherwise indicated, motifs herein describing
only linkages are intended to be linkage motifs. Thus, in such
instances, the nucleosides are not limited.
[0353] As used herein, "nucleobase modification motif" means a
pattern of modifications to nucleobases along an oligonucleotide.
Unless otherwise indicated, a nucleobase modification motif is
independent of the nucleobase sequence.
[0354] As used herein, "sequence motif" means a pattern of
nucleobases arranged along an oligonucleotide or portion thereof.
Unless otherwise indicated, a sequence motif is independent of
chemical modifications and thus may have any combination of
chemical modifications, including no chemical modifications.
[0355] As used herein, "type of modification" in reference to a
nucleoside or a nucleoside of a "type" means the chemical
modification of a nucleoside and includes modified and unmodified
nucleosides. Accordingly, unless otherwise indicated, a "nucleoside
having a modification of a first type" may be an unmodified
nucleoside.
[0356] As used herein, "differently modified" mean chemical
modifications or chemical substituents that are different from one
another, including absence of modifications. Thus, for example, a
MOE nucleoside and an unmodified DNA nucleoside are "differently
modified," even though the DNA nucleoside is unmodified. Likewise,
DNA and RNA are "differently modified," even though both are
naturally-occurring unmodified nucleosides. Nucleosides that are
the same but for comprising different nucleobases are not
differently modified. For example, a nucleoside comprising a 2'-OMe
modified sugar and an unmodified adenine nucleobase and a
nucleoside comprising a 2'-OMe modified sugar and an unmodified
thymine nucleobase are not differently modified.
[0357] As used herein, "the same type of modifications" refers to
modifications that are the same as one another, including absence
of modifications. Thus, for example, two unmodified DNA nucleoside
have "the same type of modification," even though the DNA
nucleoside is unmodified. Such nucleosides having the same type
modification may comprise different nucleobases.
[0358] As used herein, "pharmaceutically acceptable carrier or
diluent" means any substance suitable for use in administering to
an animal. In certain embodiments, a pharmaceutically acceptable
carrier or diluent is sterile saline. In certain embodiments, such
sterile saline is pharmaceutical grade saline.
[0359] As used herein, "substituent" and "substituent group," means
an atom or group that replaces the atom or group of a named parent
compound. For example a substituent of a modified nucleoside is any
atom or group that differs from the atom or group found in a
naturally occurring nucleoside (e.g., a modified 2'-substituent is
any atom or group at the 2'-position of a nucleoside other than H
or OH). Substituent groups can be protected or unprotected. In
certain embodiments, compounds of the present invention have
substituents at one or at more than one position of the parent
compound. Substituents may also be further substituted with other
substituent groups and may be attached directly or via a linking
group such as an alkyl or hydrocarbyl group to a parent
compound.
[0360] Likewise, as used herein, "substituent" in reference to a
chemical functional group means an atom or group of atoms differs
from the atom or a group of atoms normally present in the named
functional group. In certain embodiments, a substituent replaces a
hydrogen atom of the functional group (e.g., in certain
embodiments, the substituent of a substituted methyl group is an
atom or group other than hydrogen which replaces one of the
hydrogen atoms of an unsubstituted methyl group). Unless otherwise
indicated, groups amenable for use as substituents include without
limitation, halogen, hydroxyl, alkyl, alkenyl, alkynyl, acyl
(--C(O)R.sub.aa), carboxyl (--C(O)O--R.sub.aa), aliphatic groups,
alicyclic groups, alkoxy, substituted oxy (--O--R.sub.aa), aryl,
aralkyl, heterocyclic radical, heteroaryl, heteroarylalkyl, amino
(--N(R.sub.bb)(R.sub.cc)), imino (.dbd.NR.sub.bb), amido
(--C(O)N(R.sub.bb)(R.sub.cc) or --N(R.sub.bb)C(O)R.sub.aa), azido
(--N.sub.3), nitro (--NO.sub.2), cyano (--CN), carbamido
(--OC(O)N(R.sub.bb)(R.sub.cc) or --N(R.sub.bb)C(O)OR.sub.aa),
ureido (--N(R.sub.bb)C(O)N(R.sub.bb)(R.sub.cc)), thioureido
(--N(R.sub.bb)C(S)N(R.sub.bb)--(R.sub.cc)), guanidinyl
(--N(R.sub.bb)C(.dbd.NR.sub.bb)N(R.sub.bb)(R.sub.cc)), amidinyl
(--C(.dbd.NR.sub.bb)N(R.sub.bb)(R.sub.cc) or
--N(R.sub.bb)C(.dbd.NR.sub.bb)(R.sub.aa)), thiol (--SR.sub.bb),
sulfinyl (--S(O)R.sub.bb), sulfonyl (--S(O).sub.2R.sub.bb) and
sulfonamidyl (--S(O).sub.2N(R.sub.bb)(R.sub.cc) or
--N(R.sub.bb)S--(O).sub.2R.sub.bb). Wherein each R.sub.aa, R.sub.bb
and R.sub.cc is, independently, H, an optionally linked chemical
functional group or a further substituent group with a preferred
list including without limitation, alkyl, alkenyl, alkynyl,
aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl, alicyclic,
heterocyclic and heteroarylalkyl. Selected substituents within the
compounds described herein are present to a recursive degree.
[0361] As used herein, "alkyl," as used herein, means a saturated
straight or branched hydrocarbon radical containing up to twenty
four carbon atoms. Examples of alkyl groups include without
limitation, methyl, ethyl, propyl, butyl, isopropyl, n-hexyl,
octyl, decyl, dodecyl and the like. Alkyl groups typically include
from 1 to about 24 carbon atoms, more typically from 1 to about 12
carbon atoms (C.sub.1-C.sub.12 alkyl) with from 1 to about 6 carbon
atoms being more preferred.
[0362] As used herein, "alkenyl," means a straight or branched
hydrocarbon chain radical containing up to twenty four carbon atoms
and having at least one carbon-carbon double bond. Examples of
alkenyl groups include without limitation, ethenyl, propenyl,
butenyl, 1-methyl-2-buten-1-yl, dienes such as 1,3-butadiene and
the like. Alkenyl groups typically include from 2 to about 24
carbon atoms, more typically from 2 to about 12 carbon atoms with
from 2 to about 6 carbon atoms being more preferred. Alkenyl groups
as used herein may optionally include one or more further
substituent groups.
[0363] As used herein, "alkynyl," means a straight or branched
hydrocarbon radical containing up to twenty four carbon atoms and
having at least one carbon-carbon triple bond. Examples of alkynyl
groups include, without limitation, ethynyl, 1-propynyl, 1-butynyl,
and the like. Alkynyl groups typically include from 2 to about 24
carbon atoms, more typically from 2 to about 12 carbon atoms with
from 2 to about 6 carbon atoms being more preferred. Alkynyl groups
as used herein may optionally include one or more further
substituent groups.
[0364] As used herein, "acyl," means a radical formed by removal of
a hydroxyl group from an organic acid and has the general Formula
--C(O)--X where X is typically aliphatic, alicyclic or aromatic.
Examples include aliphatic carbonyls, aromatic carbonyls, aliphatic
sulfonyls, aromatic sulfinyls, aliphatic sulfinyls, aromatic
phosphates, aliphatic phosphates and the like. Acyl groups as used
herein may optionally include further substituent groups.
[0365] As used herein, "alicyclic" means a cyclic ring system
wherein the ring is aliphatic. The ring system can comprise one or
more rings wherein at least one ring is aliphatic. Preferred
alicyclics include rings having from about 5 to about 9 carbon
atoms in the ring. Alicyclic as used herein may optionally include
further substituent groups.
[0366] As used herein, "aliphatic" means a straight or branched
hydrocarbon radical containing up to twenty four carbon atoms
wherein the saturation between any two carbon atoms is a single,
double or triple bond. An aliphatic group preferably contains from
1 to about 24 carbon atoms, more typically from 1 to about 12
carbon atoms with from 1 to about 6 carbon atoms being more
preferred. The straight or branched chain of an aliphatic group may
be interrupted with one or more heteroatoms that include nitrogen,
oxygen, sulfur and phosphorus. Such aliphatic groups interrupted by
heteroatoms include without limitation, polyalkoxys, such as
polyalkylene glycols, polyamines, and polyimines. Aliphatic groups
as used herein may optionally include further substituent
groups.
[0367] As used herein, "alkoxy" means a radical formed between an
alkyl group and an oxygen atom wherein the oxygen atom is used to
attach the alkoxy group to a parent molecule. Examples of alkoxy
groups include without limitation, methoxy, ethoxy, propoxy,
isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy,
neopentoxy, n-hexoxy and the like. Alkoxy groups as used herein may
optionally include further substituent groups.
[0368] As used herein, "aminoalkyl" means an amino substituted
C.sub.1-C.sub.12 alkyl radical. The alkyl portion of the radical
forms a covalent bond with a parent molecule. The amino group can
be located at any position and the aminoalkyl group can be
substituted with a further substituent group at the alkyl and/or
amino portions.
[0369] As used herein, "aralkyl" and "arylalkyl" mean an aromatic
group that is covalently linked to a C.sub.1-C.sub.12 alkyl
radical. The alkyl radical portion of the resulting aralkyl (or
arylalkyl) group forms a covalent bond with a parent molecule.
Examples include without limitation, benzyl, phenethyl and the
like. Aralkyl groups as used herein may optionally include further
substituent groups attached to the alkyl, the aryl or both groups
that form the radical group.
[0370] As used herein, "aryl" and mean a mono- or polycyclic
carbocyclic ring system radicals having one or more aromatic rings.
Examples of aryl groups include without limitation, phenyl,
naphthyl, tetrahydronaphthyl, indanyl, idenyl and the like.
Preferred aryl ring systems have from about 5 to about 20 carbon
atoms in one or more rings. Aryl groups as used herein may
optionally include further substituent groups.
[0371] As used herein, "halo" and "halogen," mean an atom selected
from fluorine, chlorine, bromine and iodine.
[0372] As used herein, "heteroaryl," mean a radical comprising a
mono- or poly-cyclic aromatic ring, ring system or fused ring
system wherein at least one of the rings is aromatic and includes
one or more heteroatoms. Heteroaryl is also meant to include fused
ring systems including systems where one or more of the fused rings
contain no heteroatoms. Heteroaryl groups typically include one
ring atom selected from sulfur, nitrogen or oxygen. Examples of
heteroaryl groups include without limitation, pyridinyl, pyrazinyl,
pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl,
isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl,
quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl,
quinoxalinyl and the like. Heteroaryl radicals can be attached to a
parent molecule directly or through a linking moiety such as an
aliphatic group or hetero atom. Heteroaryl groups as used herein
may optionally include further substituent groups.
Oligomeric Compounds
[0373] In certain embodiments, the present invention provides
oligomeric compounds. In certain embodiments, such oligomeric
compounds comprise oligonucleotides optionally comprising one or
more conjugate and/or terminal groups. In certain embodiments, an
oligomeric compound consists of an oligonucleotide. In certain
embodiments, oligonucleotides comprise one or more chemical
modifications. Such chemical modifications include modifications
one or more nucleoside (including modifications to the sugar moiety
and/or the nucleobase) and/or modifications to one or more
internucleoside linkage.
[0374] Certain Sugar Moieties
[0375] In certain embodiments, oligomeric compounds of the
invention comprise one or more modified nucleosides comprising a
modified sugar moiety. Such oligomeric compounds comprising one or
more sugar-modified nucleosides may have desirable properties, such
as enhanced nuclease stability or increased binding affinity with a
target nucleic acid relative to oligomeric compounds comprising
only nucleosides comprising naturally occurring sugar moieties. In
certain embodiments, modified sugar moieties are substituted sugar
moieties. In certain embodiments, modified sugar moieties are
bicyclic or tricyclic sugar moieties. In certain embodiments,
modified sugar moieties are sugar surrogates. Such sugar surrogates
may comprise one or more substitutions corresponding to those of
substituted sugar moieties.
[0376] In certain embodiments, modified sugar moieties are
substituted sugar moieties comprising one or more substituent,
including but not limited to substituents at the 2' and/or 5'
positions. Examples of sugar substituents suitable for the
2'-position, include, but are not limited to: 2'-F, 2'-OCH.sub.3
("OMe" or "O-methyl"), and 2'-O(CH.sub.2).sub.2OCH.sub.3 ("MOE").
In certain embodiments, sugar substituents at the 2' position is
selected from allyl, amino, azido, thio, O-allyl,
O--C.sub.1-C.sub.10 alkyl, O--C.sub.1-C.sub.10 substituted alkyl;
O--C.sub.1-C.sub.10 alkoxy; O-- C.sub.1-C.sub.10 substituted
alkoxy, OCF.sub.3, O(CH.sub.2).sub.2SCH.sub.3,
O(CH.sub.2).sub.2--O--N(Rm)(Rn), and
O--CH.sub.2--C(.dbd.O)--N(Rm)(Rn), where each Rm and Rn is,
independently, H or substituted or unsubstituted C.sub.1-C.sub.10
alkyl. Examples of sugar substituents at the 5'-position, include,
but are not limited to: 5'-methyl (R or S); 5'-vinyl, and
5'-methoxy. In certain embodiments, substituted sugars comprise
more than one non-bridging sugar substituent, for example,
2'-F-5'-methyl sugar moieties (see, e.g., PCT International
Application WO 2008/101157, for additional 5', 2'-bis substituted
sugar moieties and nucleosides).
[0377] Nucleosides comprising 2'-substituted sugar moieties are
referred to as 2'-substituted nucleosides. In certain embodiments,
a 2'-substituted nucleoside comprises a 2'-substituent group
selected from halo, allyl, amino, azido, O--C.sub.1-C.sub.10
alkoxy; O--C.sub.1-C.sub.10 substituted alkoxy, SH, CN, OCN,
CF.sub.3, OCF.sub.3, O-alkyl, S-alkyl, N(R.sub.m)-alkyl; O-alkenyl,
S-alkenyl, or N(R.sub.m)-alkenyl; O-alkynyl, S-alkynyl,
N(R.sub.m)-alkynyl; O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl,
O-alkaryl, O-aralkyl, O(CH.sub.2).sub.2SCH.sub.3,
O--(CH.sub.2).sub.2--O--N(R.sub.m)(R.sub.n) or
O--CH.sub.2--C(.dbd.O)--N(R.sub.m)(R.sub.n), where each R.sub.m and
R.sub.n is, independently, H, an amino protecting group or
substituted or unsubstituted C.sub.1-C.sub.10 alkyl. These
2'-substituent groups can be further substituted with one or more
substituent groups independently selected from hydroxyl, amino,
alkoxy, carboxy, benzyl, phenyl, nitro (NO.sub.2), thiol,
thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and
alkynyl.
[0378] In certain embodiments, a 2'-substituted nucleoside
comprises a 2'-substituent group selected from F, NH.sub.2,
N.sub.3, OCF.sub.3, O--CH.sub.3, O(CH.sub.2).sub.3NH.sub.2,
CH.sub.2--CH.dbd.CH.sub.2, O--CH.sub.2--CH.dbd.CH.sub.2,
OCH.sub.2CH.sub.2OCH.sub.3, O(CH.sub.2).sub.2SCH.sub.3,
O--(CH.sub.2).sub.2--O--N(R.sub.m)(R.sub.n),
O(CH.sub.2).sub.2O(CH.sub.2).sub.2N(CH.sub.3).sub.2, and
N-substituted acetamide
(O--CH.sub.2--C(.dbd.O)--N(R.sub.m)(R.sub.n) where each R.sub.m and
R.sub.n is, independently, H, an amino protecting group or
substituted or unsubstituted C.sub.1-C.sub.10 alkyl.
[0379] In certain embodiments, a 2'-substituted nucleoside
comprises a sugar moiety comprising a 2'-substituent group selected
from F, OCF.sub.3, O--CH.sub.3, OCH.sub.2CH.sub.2OCH.sub.3,
O(CH.sub.2).sub.2SCH.sub.3,
O--(CH.sub.2).sub.2--O--N(CH.sub.3).sub.2,
--O(CH.sub.2).sub.2O(CH.sub.2).sub.2N(CH.sub.3).sub.2, and
O--CH.sub.2--C(.dbd.O)--N(H)CH.sub.3.
[0380] In certain embodiments, a 2'-substituted nucleoside
comprises a sugar moiety comprising a 2'-substituent group selected
from F, O--CH.sub.3, and OCH.sub.2CH.sub.2OCH.sub.3.
[0381] Certain modified sugar moieties comprise a bridging sugar
substituent that forms a second ring resulting in a bicyclic sugar
moiety. In certain such embodiments, the bicyclic sugar moiety
comprises a bridge between the 4' and the 2' furanose ring atoms.
Examples of such 4' to 2' sugar substituents, include, but are not
limited to: --[C(R.sub.a)(R.sub.b)].sub.n--,
--[C(R.sub.a)(R.sub.b)].sub.n--O--, --C(R.sub.aR.sub.b)--N(R)--O--
or, --C(R.sub.aR.sub.b)--O--N(R)--; 4'- CH.sub.2-2',
4'-(CH.sub.2).sub.2-2', 4'-(CH.sub.2).sub.3-2'; 4'-(CH.sub.2)--O-2'
(LNA); 4'-(CH.sub.2)--S-2'; 4'-(CH.sub.2).sub.2--O-2' (ENA);
4'-CH(CH.sub.3)--O-2' (cEt) and 4'-CH(CH.sub.2OCH.sub.3)--O-2', and
analogs thereof (see, e.g., U.S. Pat. No. 7,399,845, issued on Jul.
15, 2008); 4'-C(CH.sub.3)(CH.sub.3)--O-2' and analogs thereof,
(see. e.g., WO2009/006478, published Jan. 8, 2009);
4'-CH.sub.2--N(OCH.sub.3)-2' and analogs thereof (see, e.g.,
WO2008/150729, published Dec. 11, 2008);
4'-CH.sub.2--O--N(CH.sub.3)-2' (see, e.g., US2004/0171570,
published Sep. 2, 2004); 4'-CH.sub.2--O--N(R)-2', and
4'-CH.sub.2--N(R)--O-2'-, wherein each R is, independently, H, a
protecting group, or C.sub.1-C.sub.12 alkyl;
4'-CH.sub.2--N(R)--O-2', wherein R is H, C.sub.1-C.sub.12 alkyl, or
a protecting group (see, U.S. Pat. No. 7,427,672, issued on Sep.
23, 2008); 4'-CH.sub.2--C(H)(CH.sub.3)-2' (see, e.g.,
Chattopadhyaya, et al., J. Org. Chem., 2009, 74, 118-134); and
4'-CH.sub.2--C(.dbd.CH.sub.2)-2' and analogs thereof (see,
published PCT International Application WO 2008/154401, published
on Dec. 8, 2008).
[0382] In certain embodiments, such 4' to 2' bridges independently
comprise from 1 to 4 linked groups independently selected from
--[C(R.sub.a)(R.sub.b)].sub.n--, --C(R.sub.a).dbd.C(R.sub.b)--,
--C(R.sub.a).dbd.N--, --C(.dbd.NR.sub.a)--, --C(.dbd.O)--,
--C(.dbd.S)--, --O--, --Si(R.sub.a).sub.2--, --S(.dbd.O).sub.x--,
and --N(R.sub.a)--;
[0383] wherein:
[0384] x is 0, 1, or 2;
[0385] n is 1, 2, 3, or 4;
[0386] each R.sub.a and R.sub.b is, independently, H, a protecting
group, hydroxyl, C.sub.1-C.sub.12 alkyl, substituted
C.sub.1-C.sub.12 alkyl, C.sub.2-C.sub.12 alkenyl, substituted
C.sub.2-C.sub.12 alkenyl, C.sub.2-C.sub.12 alkynyl, substituted
C.sub.2-C.sub.12 alkynyl, C.sub.5-C.sub.20 aryl, substituted
C.sub.5-C.sub.20 aryl, heterocycle radical, substituted heterocycle
radical, heteroaryl, substituted heteroaryl, C.sub.5-C.sub.7
alicyclic radical, substituted C.sub.5-C.sub.7 alicyclic radical,
halogen, OJ.sub.1, NJ.sub.1J.sub.2, SJ.sub.1, N.sub.3, COOJ.sub.1,
acyl (C(.dbd.O)--H), substituted acyl, CN, sulfonyl
(S(.dbd.O).sub.2-J.sub.1), or sulfoxyl (S(.dbd.O)-J.sub.1); and
[0387] each J.sub.1 and J.sub.2 is, independently, H,
C.sub.1-C.sub.12 alkyl, substituted C.sub.1-C.sub.12 alkyl,
C.sub.2-C.sub.12 alkenyl, substituted C.sub.2-C.sub.12 alkenyl,
C.sub.2-C.sub.12 alkynyl, substituted C.sub.2-C.sub.12 alkynyl,
C.sub.5-C.sub.20 aryl, substituted C.sub.5-C.sub.20 aryl, acyl
(C(.dbd.O)--H), substituted acyl, a heterocycle radical, a
substituted heterocycle radical, C.sub.1-C.sub.12 aminoalkyl,
substituted C.sub.1-C.sub.12 aminoalkyl, or a protecting group.
[0388] Nucleosides comprising bicyclic sugar moieties are referred
to as bicyclic nucleosides or BNAs. Bicyclic nucleosides include,
but are not limited to, (A) .alpha.-L-Methyleneoxy
(4'-CH.sub.2--O-2') BNA, (B) .beta.-D-Methyleneoxy
(4'-CH.sub.2--O-2') BNA (also referred to as locked nucleic acid or
LNA), (C) Ethyleneoxy (4'-(CH.sub.2).sub.2--O-2') BNA, (D) Aminooxy
(4'-CH.sub.2--O--N(R)-2') BNA, (E) Oxyamino
(4'-CH.sub.2--N(R)--O-2') BNA, (F) Methyl(methyleneoxy)
(4'-CH(CH.sub.3)--O-2') BNA (also referred to as constrained ethyl
or cEt), (G) methylene-thio (4'-CH.sub.2--S-2') BNA, (H)
methylene-amino (4'-CH.sub.2--N(R)-2') BNA, (I) methyl carbocyclic
(4'-CH.sub.2--CH(CH.sub.3)-2') BNA, and (J) propylene carbocyclic
(4'-(CH.sub.2).sub.3-2') BNA as depicted below.
##STR00001## ##STR00002##
wherein Bx is a nucleobase moiety and R is, independently, H, a
protecting group, or C.sub.1-C.sub.12 alkyl.
[0389] Additional bicyclic sugar moieties are known in the art, for
example: Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et
al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc.
Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638; Kumar et al., Bioorg.
Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem.,
1998, 63, 10035-10039; Srivastava et al., J. Am. Chem. Soc.,
129(26) 8362-8379 (Jul. 4, 2007); Elayadi et al., Curr. Opinion
Invens. Drugs. 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001,
8, 1-7; Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243;
U.S. Pat. Nos. 7,053,207, 6,268,490, 6,770,748, 6,794,499,
7,034,133, 6,525,191, 6,670,461, and 7,399,845; WO 2004/106356, WO
1994/14226, WO 2005/021570, and WO 2007/134181; U.S. Patent
Publication Nos. US2004/0171570, US2007/0287831, and
US2008/0039618; U.S. patent Ser. Nos. 12/129,154, 60/989,574,
61/026,995, 61/026,998, 61/056,564, 61/086,231, 61/097,787, and
61/099,844; and PCT International Applications Nos.
PCT/US2008/064591, PCT/US2008/066154, and PCT/US2008/068922.
[0390] In certain embodiments, bicyclic sugar moieties and
nucleosides incorporating such bicyclic sugar moieties are further
defined by isomeric configuration. For example, a nucleoside
comprising a 4'-2' methylene-oxy bridge, may be in the .alpha.-L
configuration or in the .beta.-D configuration. Previously,
.alpha.-L-methyleneoxy (4'-CH.sub.2--O-2') bicyclic nucleosides
have been incorporated into antisense oligonucleotides that showed
antisense activity (Frieden et al., Nucleic Acids Research. 2003,
21, 6365-6372).
[0391] In certain embodiments, substituted sugar moieties comprise
one or more non-bridging sugar substituent and one or more bridging
sugar substituent (e.g., 5'-substituted and 4'-2' bridged sugars).
(see, PCT International Application WO 2007/134181, published on
Nov. 22, 2007, wherein LNA is substituted with, for example, a
5'-methyl or a 5'-vinyl group).
[0392] In certain embodiments, modified sugar moieties are sugar
surrogates. In certain such embodiments, the oxygen atom of the
naturally occurring sugar is substituted, e.g., with a sulfur,
carbon or nitrogen atom. In certain such embodiments, such modified
sugar moiety also comprises bridging and/or non-bridging
substituents as described above. For example, certain sugar
surrogates comprise a 4'-sulfur atom and a substitution at the
2'-position (see, e.g., published U.S. Patent Application
US2005/0130923, published on Jun. 16, 2005) and/or the 5' position.
By way of additional example, carbocyclic bicyclic nucleosides
having a 4'-2' bridge have been described (see, e.g., Freier et
al., Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et
al., J. Org. Chem., 2006, 71, 7731-7740).
[0393] In certain embodiments, sugar surrogates comprise rings
having other than 5-atoms. For example, in certain embodiments, a
sugar surrogate comprises a six-membered tetrahydropyran. Such
tetrahydropyrans may be further modified or substituted.
Nucleosides comprising such modified tetrahydropyrans include, but
are not limited to, hexitol nucleic acid (HNA), anitol nucleic acid
(ANA), manitol nucleic acid (MNA) (see Leumann, C J. Bioorg. &
Med. Chem. (2002) 10:841-854), fluoro HNA (F-HNA), and those
compounds having Formula VII:
##STR00003##
wherein independently for each of said at least one tetrahydropyran
nucleoside analog of Formula VII:
[0394] Bx is a nucleobase moiety;
[0395] T.sub.3 and T.sub.4 are each, independently, an
internucleoside linking group linking the tetrahydropyran
nucleoside analog to the antisense compound or one of T.sub.3 and
T.sub.4 is an internucleoside linking group linking the
tetrahydropyran nucleoside analog to the antisense compound and the
other of T.sub.3 and T.sub.4 is H, a hydroxyl protecting group, a
linked conjugate group, or a 5' or 3'-terminal group;
q.sub.1, q.sub.2, q.sub.3, q.sub.4, q.sub.5, q.sub.6 and q.sub.7
are each, independently, H, C.sub.1-C.sub.6 alkyl, substituted
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, substituted
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, or substituted
C.sub.2-C.sub.6 alkynyl; and
[0396] each of R.sub.1 and R.sub.2 is independently selected from
among: hydrogen, halogen, substituted or unsubstituted alkoxy,
NJ.sub.1J.sub.2, SJ.sub.1, N.sub.3, OC(.dbd.X)J.sub.1,
OC(.dbd.X)NJ.sub.1J.sub.2, NJ.sub.3C(.dbd.X)NJ.sub.1J.sub.2, and
CN, wherein X is O, S or NJ.sub.1, and each J.sub.1, J.sub.2, and
J.sub.3 is, independently, H or C.sub.1-C.sub.6 alkyl.
[0397] In certain embodiments, the modified THP nucleosides of
Formula VII are provided wherein q.sub.1, q.sub.2, q.sub.3,
q.sub.4, q.sub.5, q.sub.6 and q.sub.7 are each H. In certain
embodiments, at least one of q.sub.1, q.sub.2, q.sub.3, q.sub.4,
q.sub.5, q.sub.6 and q.sub.7 is other than H. In certain
embodiments, at least one of q.sub.1, q.sub.2, q.sub.3, q.sub.4,
q.sub.5, q.sub.6 and q.sub.7 is methyl. In certain embodiments, THP
nucleosides of Formula VII are provided wherein one of R.sub.1 and
R.sub.2 is F. In certain embodiments, R.sub.1 is fluoro and R.sub.2
is H, R.sub.1 is methoxy and R.sub.2 is H, and R.sub.1 is
methoxyethoxy and R.sub.2 is H.
[0398] Many other bicyclic and tricyclic sugar and sugar surrogate
ring systems are known in the art that can be used to modify
nucleosides (see, e.g., review article: Leumann, J. C, Bioorganic
& Medicinal Chemistry, 2002, 10, 841-854).
[0399] In certain embodiments, sugar surrogates comprise rings
having more than 5 atoms and more than one heteroatom. For example
nucleosides comprising morpholino sugar moieties and their use in
oligomeric compounds has been reported (see for example: Braasch et
al., Biochemistry, 2002, 41, 4503-4510; and U.S. Pat. Nos.
5,698,685; 5,166,315; 5,185,444; and 5,034,506). As used here, the
term "morpholino" means a sugar surrogate having the following
structure:
##STR00004##
In certain embodiments, morpholinos may be modified, for example by
adding or altering various substituent groups from the above
morpholino structure. Such sugar surrogates are referred to herein
as "modified morpholinos."
[0400] Combinations of modifications are also provided without
limitation, such as 2'-F-5'-methyl substituted nucleosides (see PCT
International Application WO 2008/101157 Published on Aug. 21, 2008
for other disclosed 5', 2'-bis substituted nucleosides) and
replacement of the ribosyl ring oxygen atom with S and further
substitution at the 2'-position (see published U.S. Patent
Application US2005-0130923, published on Jun. 16, 2005) or
alternatively 5'-substitution of a bicyclic nucleic acid (see PCT
International Application WO 2007/134181, published on Nov. 22,
2007 wherein a 4'-CH.sub.2--O-2' bicyclic nucleoside is further
substituted at the 5' position with a 5'-methyl or a 5'-vinyl
group). The synthesis and preparation of carbocyclic bicyclic
nucleosides along with their oligomerization and biochemical
studies have also been described (see, e.g., Srivastava et al., J.
Am. Chem. Soc. 2007, 129(26), 8362-8379).
[0401] Certain Nucleobases
[0402] In certain embodiments, nucleosides of the present invention
comprise one or more unmodified nucleobases. In certain
embodiments, nucleosides of the present invention comprise one or
more modified nucleobases.
[0403] In certain embodiments, modified nucleobases are selected
from: universal bases, hydrophobic bases, promiscuous bases,
size-expanded bases, and fluorinated bases as defined herein.
5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6
substituted purines, including 2-aminopropyladenine,
5-propynyluracil; 5-propynylcytosine; 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, 3-deazaguanine and
3-deazaadenine, universal bases, hydrophobic bases, promiscuous
bases, size-expanded bases, and fluorinated bases as defined
herein. Further modified nucleobases include tricyclic pyrimidines
such as phenoxazine cytidine([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 nucleobases 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 disclosed in U.S. Pat. No.
3,687,808, those disclosed in The Concise Encyclopedia Of Polymer
Science And Engineering, Kroschwitz, J. I., Ed., John Wiley &
Sons, 1990, 858-859; 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,
Crooke, S. T. and Lebleu, B., Eds., CRC Press, 1993, 273-288.
[0404] Representative United States patents that teach the
preparation of certain of the above noted modified nucleobases as
well as other modified nucleobases include without limitation, U.S.
Pat. Nos. 3,687,808; 4,845,205; 5,130,302; 5,134,066; 5,175,273;
5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177;
5,525,711; 5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617;
5,645,985; 5,681,941; 5,750,692; 5,763,588; 5,830,653 and
6,005,096, certain of which are commonly owned with the instant
application, and each of which is herein incorporated by reference
in its entirety.
[0405] Certain Internucleoside Linkages
[0406] In certain embodiments, the present invention provides
oligomeric compounds comprising linked nucleosides. In such
embodiments, nucleosides may be linked together using any
internucleoside linkage. The two main classes of internucleoside
linking groups are defined by the presence or absence of a
phosphorus atom. Representative phosphorus containing
internucleoside linkages include, but are not limited to,
phosphodiesters (P.dbd.O), phosphotriesters, methylphosphonates,
phosphoramidate, and phosphorothioates (P.dbd.S). Representative
non-phosphorus containing internucleoside linking groups include,
but are not limited to, methylenemethylimino
(--CH.sub.2--N(CH.sub.3)--O--CH.sub.2--), thiodiester
(--O--C(O)--S--), thionocarbamate (--O--C(O)(NH)--S--); siloxane
(--O--Si(H).sub.2--O--); and N,N'-dimethylhydrazine
(--CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--). Modified linkages,
compared to natural phosphodiester linkages, can be used to alter,
typically increase, nuclease resistance of the oligomeric compound.
In certain embodiments, internucleoside linkages having a chiral
atom can be prepared as a racemic mixture, or as separate
enantiomers. Representative chiral linkages include, but are not
limited to, alkylphosphonates and phosphorothioates. Methods of
preparation of phosphorous-containing and
non-phosphorous-containing internucleoside linkages are well known
to those skilled in the art.
[0407] The oligonucleotides described herein contain one or more
asymmetric centers and thus give rise to enantiomers,
diastereomers, and other stereoisomeric configurations that may be
defined, in terms of absolute stereochemistry, as (R) or (S),
.alpha. or .beta. such as for sugar anomers, or as (D) or (L) such
as for amino acids etc. Included in the antisense compounds
provided herein are all such possible isomers, as well as their
racemic and optically pure forms.
[0408] Neutral internucleoside linkages include without limitation,
phosphotriesters, methylphosphonates, MMI
(3'-CH.sub.2--N(CH.sub.3)--O-5'), amide-3
(3'-CH.sub.2--C(.dbd.O)--N(H)-5'), amide-4
(3'-CH.sub.2--N(H)--C(.dbd.O)-5'), formacetal
(3'-O--CH.sub.2--O-5'), and thioformacetal (3'-S--CH.sub.2--O-5').
Further neutral internucleoside linkages include nonionic linkages
comprising siloxane (dialkylsiloxane), carboxylate ester,
carboxamide, sulfide, sulfonate ester and amides (See for example:
Carbohydrate Modifications in Antisense Research; Y. S. Sanghvi and
P. D. Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4,
40-65). Further neutral internucleoside linkages include nonionic
linkages comprising mixed N, O, S and CH.sub.2 component parts.
[0409] Certain Motifs
[0410] In certain embodiments, the present invention provides
oligomeric compounds comprising oligonucleotides. In certain
embodiments, such oligonucleotides comprise one or more chemical
modification. In certain embodiments, chemically modified
oligonucleotides comprise one or more modified nucleosides. In
certain embodiments, chemically modified oligonucleotides comprise
one or more modified nucleosides comprising modified sugars. In
certain embodiments, chemically modified oligonucleotides comprise
one or more modified nucleosides comprising one or more modified
nucleobases. In certain embodiments, chemically modified
oligonucleotides comprise one or more modified internucleoside
linkages. In certain embodiments, the chemically modifications
(sugar modifications, nucleobase modifications, and/or linkage
modifications) define a pattern or motif. In certain embodiments,
the patterns of chemical modifications of sugar moieties,
internucleoside linkages, and nucleobases are each independent of
one another. Thus, an oligonucleotide may be described by its sugar
modification motif, internucleoside linkage motif and/or nucleobase
modification motif (as used herein, nucleobase modification motif
describes the chemical modifications to the nucleobases independent
of the sequence of nucleobases).
[0411] Certain Sugar Motifs
[0412] In certain embodiments, oligonucleotides comprise one or
more type of modified sugar moieties and/or naturally occurring
sugar moieties arranged along an oligonucleotide or region thereof
in a defined pattern or sugar modification motif. Such motifs may
include any of the sugar modifications discussed herein and/or
other known sugar modifications.
[0413] In certain embodiments, the oligonucleotides comprise or
consist of a region having a gapmer sugar modification motif, which
comprises two external regions or "wings" and an internal region or
"gap." The three regions of a gapmer motif (the 5'-wing, the gap,
and the 3'-wing) form a contiguous sequence of nucleosides wherein
at least some of the sugar moieties of the nucleosides of each of
the wings differ from at least some of the sugar moieties of the
nucleosides of the gap. Specifically, at least the sugar moieties
of the nucleosides of each wing that are closest to the gap (the
3'-most nucleoside of the 5'-wing and the 5'-most nucleoside of the
3'-wing) differ from the sugar moiety of the neighboring gap
nucleosides, thus defining the boundary between the wings and the
gap. In certain embodiments, the sugar moieties within the gap are
the same as one another. In certain embodiments, the gap includes
one or more nucleoside having a sugar moiety that differs from the
sugar moiety of one or more other nucleosides of the gap. In
certain embodiments, the sugar modification motifs of the two wings
are the same as one another (symmetric gapmer). In certain
embodiments, the sugar modification motifs of the 5'-wing differs
from the sugar modification motif of the 3'-wing (asymmetric
gapmer). In certain embodiments, oligonucleotides comprise 2'-MOE
modified nucleosides in the wings and 2'-F modified nucleosides in
the gap.
[0414] In certain embodiments, oligonucleotides are fully modified.
In certain such embodiments, oligonucleotides are uniformly
modified. In certain embodiments, oligonucleotides are uniform
2'-MOE. In certain embodiments, oligonucleotides are uniform 2'-F.
In certain embodiments, oligonucleotides are uniform morpholino. In
certain embodiments, oligonucleotides are uniform BNA. In certain
embodiments, oligonucleotides are uniform LNA. In certain
embodiments, oligonucleotides are uniform cEt.
[0415] In certain embodiments, oligonucleotides comprise a
uniformly modified region and additional nucleosides that are
unmodified or differently modified. In certain embodiments, the
uniformly modified region is at least 5, 10, 15, or 20 nucleosides
in length. In certain embodiments, the uniform region is a 2'-MOE
region. In certain embodiments, the uniform region is a 2'-F
region. In certain embodiments, the uniform region is a morpholino
region. In certain embodiments, the uniform region is a BNA region.
In certain embodiments, the uniform region is a LNA region. In
certain embodiments, the uniform region is a cEt region.
[0416] In certain embodiments, the oligonucleotide does not
comprise more than 4 contiguous unmodified 2'-deoxynucleosides. In
certain circumstances, antisense oligonucleotides comprising more
than 4 contiguous 2'-deoxynucleosides activate RNase H, resulting
in cleavage of the target RNA. In certain embodiments, such
cleavage is avoided by not having more than 4 contiguous
2'-deoxynucleosides, for example, where alteration of splicing and
not cleavage of a target RNA is desired.
[0417] Certain Internucleoside Linkage Motifs
[0418] In certain embodiments, oligonucleotides comprise modified
internucleoside linkages arranged along the oligonucleotide or
region thereof in a defined pattern or modified internucleoside
linkage motif. In certain embodiments, internucleoside linkages are
arranged in a gapped motif, as described above for sugar
modification motif. In such embodiments, the internucleoside
linkages in each of two wing regions are different from the
internucleoside linkages in the gap region. In certain embodiments
the internucleoside linkages in the wings are phosphodiester and
the internucleoside linkages in the gap are phosphorothioate. The
sugar modification motif is independently selected, so such
oligonucleotides having a gapped internucleoside linkage motif may
or may not have a gapped sugar modification motif and if it does
have a gapped sugar motif, the wing and gap lengths may or may not
be the same.
[0419] In certain embodiments, oligonucleotides comprise a region
having an alternating internucleoside linkage motif. In certain
embodiments, oligonucleotides of the present invention comprise a
region of uniformly modified internucleoside linkages. In certain
such embodiments, the oligonucleotide comprises a region that is
uniformly linked by phosphorothioate internucleoside linkages. In
certain embodiments, the oligonucleotide is uniformly linked by
phosphorothioate. In certain embodiments, each internucleoside
linkage of the oligonucleotide is selected from phosphodiester and
phosphorothioate. In certain embodiments, each internucleoside
linkage of the oligonucleotide is selected from phosphodiester and
phosphorothioate and at least one internucleoside linkage is
phosphorothioate.
[0420] In certain embodiments, the oligonucleotide comprises at
least 6 phosphorothioate internucleoside linkages. In certain
embodiments, the oligonucleotide comprises at least 8
phosphorothioate internucleoside linkages. In certain embodiments,
the oligonucleotide comprises at least 10 phosphorothioate
internucleoside linkages. In certain embodiments, the
oligonucleotide comprises at least one block of at least 6
consecutive phosphorothioate internucleoside linkages. In certain
embodiments, the oligonucleotide comprises at least one block of at
least 8 consecutive phosphorothioate internucleoside linkages. In
certain embodiments, the oligonucleotide comprises at least one
block of at least 10 consecutive phosphorothioate internucleoside
linkages. In certain embodiments, the oligonucleotide comprises at
least block of at least one 12 consecutive phosphorothioate
internucleoside linkages. In certain such embodiments, at least one
such block is located at the 3' end of the oligonucleotide. In
certain such embodiments, at least one such block is located within
3 nucleosides of the 3' end of the oligonucleotide.
[0421] Certain Nucleobase Modification Motifs
[0422] In certain embodiments, oligonucleotides comprise chemical
modifications to nucleobases arranged along the oligonucleotide or
region thereof in a defined pattern or nucleobases modification
motif. In certain such embodiments, nucleobase modifications are
arranged in a gapped motif. In certain embodiments, nucleobase
modifications are arranged in an alternating motif. In certain
embodiments, each nucleobase is modified. In certain embodiments,
none of the nucleobases is chemically modified.
[0423] In certain embodiments, oligonucleotides comprise a block of
modified nucleobases. In certain such embodiments, the block is at
the 3'-end of the oligonucleotide. In certain embodiments the block
is within 3 nucleotides of the 3'-end of the oligonucleotide. In
certain such embodiments, the block is at the 5'-end of the
oligonucleotide. In certain embodiments the block is within 3
nucleotides of the 5'-end of the oligonucleotide.
[0424] In certain embodiments, nucleobase modifications are a
function of the natural base at a particular position of an
oligonucleotide. For example, in certain embodiments each purine or
each pyrimidine in an oligonucleotide is modified. In certain
embodiments, each adenine is modified. In certain embodiments, each
guanine is modified. In certain embodiments, each thymine is
modified. In certain embodiments, each cytosine is modified. In
certain embodiments, each uracil is modified.
[0425] In certain embodiments, some, all, or none of the cytosine
moieties in an oligonucleotide are 5-methyl cytosine moieties.
Herein, 5-methyl cytosine is not a "modified nucleobase."
Accordingly, unless otherwise indicated, unmodified nucleobases
include both cytosine residues having a 5-methyl and those lacking
a 5 methyl. In certain embodiments, the methylation state of all or
some cytosine nucleobases is specified.
[0426] Certain Overall Lengths
[0427] In certain embodiments, the present invention provides
oligomeric compounds including oligonucleotides of any of a variety
of ranges of lengths. In certain embodiments, the invention
provides oligomeric compounds or oligonucleotides consisting of X
to Y linked nucleosides, where X represents the fewest number of
nucleosides in the range and Y represents the largest number of
nucleosides in the range. In certain such embodiments, X and Y are
each independently selected from 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and
50; provided that X.ltoreq.Y. For example, in certain embodiments,
the invention provides oligomeric compounds which comprise
oligonucleotides consisting of 8 to 9, 8 to 10, 8 to 11, 8 to 12, 8
to 13, 8 to 14, 8 to 15, 8 to 16, 8 to 17, 8 to 18, 8 to 19, 8 to
20, 8 to 21, 8 to 22, 8 to 23, 8 to 24, 8 to 25, 8 to 26, 8 to 27,
8 to 28, 8 to 29, 8 to 30, 9 to 10, 9 to 11, 9 to 12, 9 to 13, 9 to
14, 9 to 15, 9 to 16, 9 to 17, 9 to 18, 9 to 19, 9 to 20, 9 to 21,
9 to 22, 9 to 23, 9 to 24, 9 to 25, 9 to 26, 9 to 27, 9 to 28, 9 to
29, 9 to 30, 10 to 11, 10 to 12, 10 to 13, 10 to 14, 10 to 15, 10
to 16, 10 to 17, 10 to 18, 10 to 19, 10 to 20, 10 to 21, 10 to 22,
10 to 23, 10 to 24, 10 to 25, 10 to 26, 10 to 27, 10 to 28, 10 to
29, 10 to 30, 11 to 12, 11 to 13, 11 to 14, 11 to 15, 11 to 16, 11
to 17, 11 to 18, 11 to 19, 11 to 20, 11 to 21, 11 to 22, 11 to 23,
11 to 24, 11 to 25, 11 to 26, 11 to 27, 11 to 28, 11 to 29, 11 to
30, 12 to 13, 12 to 14, 12 to 15, 12 to 16, 12 to 17, 12 to 18, 12
to 19, 12 to 20, 12 to 21, 12 to 22, 12 to 23, 12 to 24, 12 to 25,
12 to 26, 12 to 27, 12 to 28, 12 to 29, 12 to 30, 13 to 14, 13 to
15, 13 to 16, 13 to 17, 13 to 18, 13 to 19, 13 to 20, 13 to 21, 13
to 22, 13 to 23, 13 to 24, 13 to 25, 13 to 26, 13 to 27, 13 to 28,
13 to 29, 13 to 30, 14 to 15, 14 to 16, 14 to 17, 14 to 18, 14 to
19, 14 to 20, 14 to 21, 14 to 22, 14 to 23, 14 to 24, 14 to 25, 14
to 26, 14 to 27, 14 to 28, 14 to 29, 14 to 30, 15 to 16, 15 to 17,
15 to 18, 15 to 19, 15 to 20, 15 to 21, 15 to 22, 15 to 23, 15 to
24, 15 to 25, 15 to 26, 15 to 27, 15 to 28, 15 to 29, 15 to 30, 16
to 17, 16 to 18, 16 to 19, 16 to 20, 16 to 21, 16 to 22, 16 to 23,
16 to 24, 16 to 25, 16 to 26, 16 to 27, 16 to 28, 16 to 29, 16 to
30, 17 to 18, 17 to 19, 17 to 20, 17 to 21, 17 to 22, 17 to 23, 17
to 24, 17 to 25, 17 to 26, 17 to 27, 17 to 28, 17 to 29, 17 to 30,
18 to 19, 18 to 20, 18 to 21, 18 to 22, 18 to 23, 18 to 24, 18 to
25, 18 to 26, 18 to 27, 18 to 28, 18 to 29, 18 to 30, 19 to 20, 19
to 21, 19 to 22, 19 to 23, 19 to 24, 19 to 25, 19 to 26, 19 to 29,
19 to 28, 19 to 29, 19 to 30, 20 to 21, 20 to 22, 20 to 23, 20 to
24, 20 to 25, 20 to 26, 20 to 27, 20 to 28, 20 to 29, 20 to 30, 21
to 22, 21 to 23, 21 to 24, 21 to 25, 21 to 26, 21 to 27, 21 to 28,
21 to 29, 21 to 30, 22 to 23, 22 to 24, 22 to 25, 22 to 26, 22 to
27, 22 to 28, 22 to 29, 22 to 30, 23 to 24, 23 to 25, 23 to 26, 23
to 27, 23 to 28, 23 to 29, 23 to 30, 24 to 25, 24 to 26, 24 to 27,
24 to 28, 24 to 29, 24 to 30, 25 to 26, 25 to 27, 25 to 28, 25 to
29, 25 to 30, 26 to 27, 26 to 28, 26 to 29, 26 to 30, 27 to 28, 27
to 29, 27 to 30, 28 to 29, 28 to 30, or 29 to 30 linked
nucleosides. In embodiments where the number of nucleosides of an
oligomeric compound or oligonucleotide is limited, whether to a
range or to a specific number, the oligomeric compound or
oligonucleotide may, nonetheless further comprise additional other
substituents. For example, an oligonucleotide comprising 8-30
nucleosides excludes oligonucleotides having 31 nucleosides, but,
unless otherwise indicated, such an oligonucleotide may further
comprise, for example one or more conjugates, terminal groups, or
other substituents. In certain embodiments, a gapmer
oligonucleotide has any of the above lengths.
[0428] One of skill in the art will appreciate that certain lengths
may not be possible for certain motifs. For example: a gapmer
having a 5'-wing region consisting of four nucleotides, a gap
consisting of at least six nucleotides, and a 3'-wing region
consisting of three nucleotides cannot have an overall length less
than 13 nucleotides. Thus, one would understand that the lower
length limit is 13 and that the limit of 10 in "10-20" has no
effect in that embodiment.
[0429] Further, where an oligonucleotide is described by an overall
length range and by regions having specified lengths, and where the
sum of specified lengths of the regions is less than the upper
limit of the overall length range, the oligonucleotide may have
additional nucleosides, beyond those of the specified regions,
provided that the total number of nucleosides does not exceed the
upper limit of the overall length range. For example, an
oligonucleotide consisting of 20-25 linked nucleosides comprising a
5'-wing consisting of 5 linked nucleosides; a 3'-wing consisting of
5 linked nucleosides and a central gap consisting of 10 linked
nucleosides (5+5+10=20) may have up to 5 nucleosides that are not
part of the 5'-wing, the 3'-wing, or the gap (before reaching the
overall length limitation of 25). Such additional nucleosides may
be 5' of the 5'-wing and/or 3' of the 3' wing.
[0430] Certain Oligonucleotides
[0431] In certain embodiments, oligonucleotides of the present
invention are characterized by their sugar motif, internucleoside
linkage motif, nucleobase modification motif and overall length. In
certain embodiments, such parameters are each independent of one
another. Thus, each internucleoside linkage of an oligonucleotide
having a gapmer sugar motif may be modified or unmodified and may
or may not follow the gapmer modification pattern of the sugar
modifications. Thus, the internucleoside linkages within the wing
regions of a sugar-gapmer may be the same or different from one
another and may be the same or different from the internucleoside
linkages of the gap region. Likewise, such sugar-gapmer
oligonucleotides may comprise one or more modified nucleobase
independent of the gapmer pattern of the sugar modifications.
Herein if a description of an oligonucleotide or oligomeric
compound is silent with respect to one or more parameter, such
parameter is not limited. Thus, an oligomeric compound described
only as having a gapmer sugar motif without further description may
have any length, internucleoside linkage motif, and nucleobase
modification motif. Unless otherwise indicated, all chemical
modifications are independent of nucleobase sequence.
[0432] Certain Conjugate Groups
[0433] In certain embodiments, oligomeric compounds are modified by
attachment of one or more conjugate groups. In general, conjugate
groups modify one or more properties of the attached oligomeric
compound including but not limited to pharmacodynamics,
pharmacokinetics, stability, binding, absorption, cellular
distribution, cellular uptake, charge and clearance. Conjugate
groups are routinely used in the chemical arts and are linked
directly or via an optional conjugate linking moiety or conjugate
linking group to a parent compound such as an oligomeric compound,
such as an oligonucleotide. Conjugate groups includes without
limitation, intercalators, reporter molecules, polyamines,
polyamides, polyethylene glycols, thioethers, polyethers,
cholesterols, thiocholesterols, cholic acid moieties, folate,
lipids, phospholipids, biotin, phenazine, phenanthridine,
anthraquinone, adamantane, acridine, fluoresceins, rhodamines,
coumarins and dyes. Certain conjugate groups have been described
previously, for example: 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., do-decan-diol or undecyl residues (Saison-Behmoaras et al.,
EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990,
259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a
phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium
1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,
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 al., Nucleosides & Nucleotides, 1995, 14,
969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron
Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al.,
Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine
or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J.
Pharmacol. Exp. Ther., 1996, 277, 923-937).
[0434] In certain embodiments, a conjugate group comprises an
active drug substance, for example, aspirin, warfarin,
phenylbutazone, ibuprofen, suprofen, fen-bufen, ketoprofen,
(S)-(+)-pranoprofen, carprofen, dansylsarcosine,
2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a
benzothiadiazide, chlorothiazide, a diazepine, indo-methicin, a
barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an
antibacterial or an antibiotic.
[0435] In certain embodiments, conjugate groups are directly
attached to oligonucleotides in oligomeric compounds. In certain
embodiments, conjugate groups are attached to oligonucleotides by a
conjugate linking group. In certain such embodiments, conjugate
linking groups, including, but not limited to, bifunctional linking
moieties such as those known in the art are amenable to the
compounds provided herein. Conjugate linking groups are useful for
attachment of conjugate groups, such as chemical stabilizing
groups, functional groups, reporter groups and other groups to
selective sites in a parent compound such as for example an
oligomeric compound. In general a bifunctional linking moiety
comprises a hydrocarbyl moiety having two functional groups. One of
the functional groups is selected to bind to a parent molecule or
compound of interest and the other is selected to bind essentially
any selected group such as chemical functional group or a conjugate
group. In some embodiments, the conjugate linker comprises a chain
structure or an oligomer of repeating units such as ethylene glycol
or amino acid units. Examples of functional groups that are
routinely used in a bifunctional linking moiety include, but are
not limited to, electrophiles for reacting with nucleophilic groups
and nucleophiles for reacting with electrophilic groups. In some
embodiments, bifunctional linking moieties include amino, hydroxyl,
carboxylic acid, thiol, unsaturations (e.g., double or triple
bonds), and the like.
[0436] Some nonlimiting examples of conjugate linking moieties
include pyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO),
succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC)
and 6-aminohexanoic acid (AHEX or AHA). Other linking groups
include, but are not limited to, substituted C.sub.1-C.sub.10
alkyl, substituted or unsubstituted C.sub.2-C.sub.10 alkenyl or
substituted or unsubstituted C.sub.2-C.sub.10 alkynyl, wherein a
nonlimiting list of preferred substituent groups includes hydroxyl,
amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy,
halogen, alkyl, aryl, alkenyl and alkynyl.
[0437] Conjugate groups may be attached to either or both ends of
an oligonucleotide (terminal conjugate groups) and/or at any
internal position.
[0438] In certain embodiments, conjugate groups are at the 3'-end
of an oligonucleotide of an oligomeric compound. In certain
embodiments, conjugate groups are near the 3'-end. In certain
embodiments, conjugates are attached at the 3'end of an oligomeric
compound, but before one or more terminal group nucleosides. In
certain embodiments, conjugate groups are placed within a terminal
group.
[0439] In certain embodiments, the present invention provides
oligomeric compounds. In certain embodiments, oligomeric compounds
comprise an oligonucleotide. In certain embodiments, an oligomeric
compound comprises an oligonucleotide and one or more conjugate
and/or terminal groups. Such conjugate and/or terminal groups may
be added to oligonucleotides having any of the chemical motifs
discussed above. Thus, for example, an oligomeric compound
comprising an oligonucleotide having region of alternating
nucleosides may comprise a terminal group.
Antisense Compounds
[0440] In certain embodiments, oligomeric compounds of the present
invention are antisense compounds.
[0441] Such antisense compounds are capable of hybridizing to a
target nucleic acid, resulting in at least one antisense activity.
In certain embodiments, antisense compounds specifically hybridize
to one or more target nucleic acid. In certain embodiments, a
specifically hybridizing antisense compound has a nucleobase
sequence comprising a region having sufficient complementarity to a
target nucleic acid to allow hybridization and result in antisense
activity and insufficient complementarity to any non-target so as
to avoid non-specific hybridization to any non-target nucleic acid
sequences under conditions in which specific hybridization is
desired (e.g., under physiological conditions for in vivo or
therapeutic uses, and under conditions in which assays are
performed in the case of in vitro assays).
[0442] In certain embodiments, the present invention provides
antisense compounds comprising oligonucleotides that are fully
complementary to the target nucleic acid over the entire length of
the oligonucleotide. In certain embodiments, oligonucleotides are
99% complementary to the target nucleic acid. In certain
embodiments, oligonucleotides are 95% complementary to the target
nucleic acid. In certain embodiments, such oligonucleotides are 90%
complementary to the target nucleic acid.
[0443] In certain embodiments, such oligonucleotides are 85%
complementary to the target nucleic acid. In certain embodiments,
such oligonucleotides are 80% complementary to the target nucleic
acid. In certain embodiments, an antisense compound comprises a
region that is fully complementary to a target nucleic acid and is
at least 80% complementary to the target nucleic acid over the
entire length of the oligonucleotide. In certain such embodiments,
the region of full complementarity is from 6 to 14 nucleobases in
length.
[0444] TABLE 1 below provides certain non-limiting examples of
antisense compounds and their targets:
TABLE-US-00001 TABLE 1 Antisense Compounds SEQ ID Target ISIS No
Indication Sequence Chemistry NO Factor XI 416858 Clotting
ACGGCATTGGTGCACAGTTT 5-10-5 MOE 1 disorder TTR 420915 Amyloidosis
TCTTGGTTACATGAAATCCC 5-10-5 MOE 2 Apo(a) 494372 CAD
TGCTCCGTTGGTGCTTGTTC 5-10-5 MOE 3 Alpha1- 487660 Liver disease
CCAGCTCAACCCTTCTTTAA 5-10-5 MOE 4 antitrypsin PTP-1B 404173
Diabetes AATGGTTTATTCCATGGCCA 5-10-5 MOE 5 GCGR 449884 Diabetes
GGTTCCCGAGGTGCCCA 3-10-4 MOE 6 DGAT2 501861 NASH
TCACAGAATTATCAGCAGTA 5-10-5 MOE 7 Factor VII 540175 Cancer-
GGACACCCACGCCCCC 3-10-3 8 associated cEt/MOE thrombosis SMN 396443
SMA TCACTTTCATAATGCTGG Uniform 9 MOE FGFR4 463588 Obesity
GCACACTCAGCAGGACCCCC 5-10-5 MOE 10 apoB-100 301012 High
GCCTCAGTCTGCTTCGCACC 5-10-5 MOE 11 Cholesterol CRP 329993 CAD
AGCATAGTTAACGAGCTCCC 5-10-5 MOE 12 ApoC-III 304801 High
AGCTTCTTGTCCAGCTTTAT 5-10-5 MOE 13 triglycerides GCCR 426115
Diabetes GCAGCCATGGTGATCAGGAG 5-10-5 MOE 14 STAT3 Cancer 481464
CTATTTGGATGTCAGC 3-10-3 (S)-cEt 15 eIF-4E 183750 Cancer
TGTCATATTCCTGGATCCTT 5-10-5 MOE 16 SOD1 333611 ALS
CCGTCGCCCTTCAGCACGCA 5-10-5 MOE 17 GHR 227452 Acromegaly
TCAGGGCATTCTTTCCATTC 5-10-5 MOE 18 Clusterin 112989 Cancer
CAGCAGCAGAGTCTTCATCAT 4-13-4 MOE 19 Hsp27 306053 Cancer
GGGACGCGGCGCTCGGTCAT 4-12-4 MOE 20 CMV 2922 Retinitis
GCGTTTGCTCTTCTTCTTGCG Uniform 21 deoxy ICAM-1 2302 Ulcerative
GCCCAAGCTGGCATCCGTCA Uniform 22 colitis deoxy VLA-4 107248 Multiple
CTGAGTCTGTTTTCCATTCT 3-9-8 MOE 23 sclerosis CTGF 412294 Fibrosis
GTTTGACATGGCACAATGTT 2-13-5 MOE 24 c-raf kinase 13650 Ocular
disease TCCCGCCTGTGACATGCATT 6-8-6 MOE 25
Certain Target Nucleic Acids and Mechanisms
[0445] In certain embodiments, antisense compounds comprise or
consist of an oligonucleotide comprising a region that is
complementary to a target nucleic acid. In certain embodiments, the
target nucleic acid is an endogenous RNA molecule. In certain
embodiments, the target nucleic acid is a pre-mRNA. In certain
embodiments, an antisense oligonucleotide modulates splicing of a
pre-mRNA.
Certain Properties of Antisense Compounds
[0446] Viscosity
[0447] In certain embodiments, antisense oligonucleotide solutions
possess varying degrees of viscosity, and the viscosity of
antisense oligonucleotide solutions depends on many factors. Some
factors include, but are not limited to, length, nucleobase
sequence, nucleobase modifications, nucleobase modification motif,
and/or sugar modification of the antisense oligonucleotide. In
certain embodiments, the viscosity of an antisense oligonucleotide
solution may be difficult to predict. In certain embodiments an
antisense oligonucleotide solution may exhibit a relatively high
viscosity, while an antisense oligonucleotide solution comprising a
similar antisense oligonucleotide (partially homologous sequence,
similar modifications, etc.) may exhibit a relatively low
viscosity. In certain embodiments it may be desirable to lower the
viscosity of a given antisense oligonucleotide solution, for
example, in certain embodiments it may be desirable to lower the
viscosity of a given antisense oligonucleotide solution so that
higher amounts of an antisense oligonucleotide may be present in a
given volume of solution.
[0448] In certain embodiments it may be desirable to increase the
viscosity of a given antisense oligonucleotide solution. In certain
embodiments, viscosity may be increased or decreased depending on a
variety of factors, e.g. concentration, volume of solute,
temperature, and/or pH. In certain embodiments, the desirable
viscosity varies. In certain embodiments it may be desirable to
increase or decrease viscosity, depending on the particular route
of delivery or application a given antisense oligonucleotide
solution.
[0449] In certain embodiments, viscosity is often a
concentration-limiting factor. While not wishing to be bound by
theory, in certain embodiments, as the concentration of an
antisense oligonucleotide is increased, interactions between
antisense oligonucleotide molecules likewise increase. In certain
embodiments, certain antisense oligonucleotides may interact to
form aggregates which may increase viscosity and/or turbidity.
Certain antisense oligonucleotides may interact to form antisense
oligonucleotide polymers, which may increase viscosity and/or
turbidity. In certain embodiments, as the concentration of an
antisense oligonucleotide in solution increases, the viscosity of
the antisense oligonucleotide solution also increases. In certain
embodiments, it is undesirable to have an antisense oligonucleotide
solution having a high viscosity. For example, if the viscosity of
an antisense oligonucleotide solution is too high, then it may
become difficult to manufacture an antisense oligonucleotide
solution including but not limited to an antisense oligonucleotide
solution drug product. As another example, if the viscosity of an
antisense oligonucleotide solution is too high, filtration time
during manufacturing may increase, adding time and cost to the
manufacturing process. As another example, if the viscosity of an
antisense oligonucleotide solution is too high, it may become more
difficult to pre-fill syringes with accurate and precise amounts of
the antisense oligonucleotide solution. As another example, if the
viscosity of an antisense oligonucleotide solution is too high, it
may become more difficult to administer to an animal, human, or
patient. As another example, if the viscosity of an antisense
oligonucleotide solution is too high, drug clearance from the
subcutaneous injection site may be slowed. As another example, if
the viscosity of an antisense oligonucleotide solution is too high,
a larger gauge needle may be required to effectively administer
doses of the antisense oligonucleotide solution, which may
exacerbate discomfort upon injection. In certain embodiments, it is
therefore desirable to have an antisense oligonucleotide solution
having both a high concentration of antisense oligonucleotide and
low viscosity.
[0450] In certain embodiments, if the viscosity of an antisense
oligonucleotide solution is too high, then the concentration of the
antisense oligonucleotide in solution may be reduced to reduce the
viscosity of the antisense oligonucleotide solution to a desirable
level. The reduction of the concentration of an antisense
oligonucleotide in the antisense oligonucleotide solution may be
undesirable for several reasons. For example, as the concentration
of an antisense oligonucleotide in an antisense oligonucleotide
solution is decreased, animals, humans, and/or patients must
receive a larger volume of antisense oligonucleotide solution to
receive the desired amount of an antisense oligonucleotide. In
certain embodiments, it is therefore desirable to reduce the
viscosity of an antisense oligonucleotide solution without reducing
the concentration of the antisense oligonucleotide within the
antisense oligonucleotide solution. In certain embodiments, it is
therefore desirable to have an antisense oligonucleotide solution
having both a high concentration of antisense oligonucleotide and
low viscosity.
[0451] In certain embodiments, certain antisense oligonucleotide
solutions having desirable concentrations of antisense
oligonucleotides have undesirably high viscosities. In certain
embodiments, the addition of one or more excipients that modulate
viscosity, turbidity or both may reduce the viscosity of a certain
antisense oligonucleotide solution to a desirable level.
[0452] In certain embodiments described herein, it is recognized
that viscosity of an oligonucleotide solution may vary with
temperature. In certain embodiments, the viscosity may be expressed
as a range of cP units relative to a concentration of
oligonucleotide in solution. For example, in certain embodiments,
the viscosity of the antisense oligonucleotide solution is less
than 40 cP when the concentration of the antisense oligonucleotide
is between 40 to 60 mg/mL. In certain such embodiments, the
viscosity of the solution and the concentration of the
oligonucleotide represent an approximate measurement of viscosity
and approximate concentration of oligonucleotide at a given
temperature or range of temperatures. For example, in certain
embodiments described herein, the temperature of the solution for
which viscosity is measured is about 25 degrees Celsius.
[0453] In certain embodiments, the addition of one or more
excipients that modulate viscosity, turbidity or both to the
antisense oligonucleotide solution reduces the viscosity of the
antisense oligonucleotide solution to less than 40 cP when the
concentration of the antisense oligonucleotide is between 40 to 60
mg/mL. In certain embodiments, the addition of one or more
excipients that modulate viscosity, turbidity or both to the
antisense oligonucleotide solution reduces the viscosity of the
antisense oligonucleotide solution to less than 40 cP when the
concentration of the antisense oligonucleotide is between 45 to 55
mg/mL. In certain embodiments, the addition of one or more
excipients that modulate viscosity, turbidity or both to the
antisense oligonucleotide solution reduces the viscosity of the
antisense oligonucleotide solution to less than 40 cP when the
concentration of the antisense oligonucleotide is between 80 to 120
mg/mL. In certain embodiments, the addition of one or more
excipients that modulate viscosity, turbidity or both to the
antisense oligonucleotide solution reduces the viscosity of the
antisense oligonucleotide solution to less than 40 cP when the
concentration of the antisense oligonucleotide is between 90 to 110
mg/mL. In certain embodiments, the addition of one or more
excipients that modulate viscosity, turbidity or both to the
antisense oligonucleotide solution reduces the viscosity of the
antisense oligonucleotide solution to less than 40 cP when the
concentration of the antisense oligonucleotide is between 140 to
160 mg/mL. In certain embodiments, the addition of one or more
excipients that modulate viscosity, turbidity or both to the
antisense oligonucleotide solution reduces the viscosity of the
antisense oligonucleotide solution to less than 40 cP when the
concentration of the antisense oligonucleotide is between 165 to
185 mg/mL. In certain embodiments, the addition of one or more
excipients that modulate viscosity, turbidity or both to the
antisense oligonucleotide solution reduces the viscosity of the
antisense oligonucleotide solution to less than 40 cP when the
concentration of the antisense oligonucleotide is between 180 to
220 mg/mL. In certain embodiments, the addition of one or more
excipients that modulate viscosity, turbidity or both to the
antisense oligonucleotide solution reduces the viscosity of the
antisense oligonucleotide solution to less than 40 cP when the
concentration of the antisense oligonucleotide is between 190 to
210 mg/mL. In certain embodiments, the addition of one or more
excipients that modulate viscosity, turbidity or both to the
antisense oligonucleotide solution reduces the viscosity of the
antisense oligonucleotide solution to less than 40 cP when the
concentration of the antisense oligonucleotide is between 210 to
230 mg/mL. In certain embodiments, the addition of one or more
excipients that modulate viscosity, turbidity or both to the
antisense oligonucleotide solution reduces the viscosity of the
antisense oligonucleotide solution to less than 40 cP when the
concentration of the antisense oligonucleotide is between 230 to
260 mg/mL. In certain embodiments, the addition of one or more
excipients that modulate viscosity, turbidity or both to the
antisense oligonucleotide solution reduces the viscosity of the
antisense oligonucleotide solution to less than 40 cP when the
concentration of the antisense oligonucleotide is between 245 to
255 mg/mL. In certain embodiments, the addition of one or more
excipients that modulate viscosity, turbidity or both to the
antisense oligonucleotide solution reduces the viscosity of the
antisense oligonucleotide solution to less than 40 cP when the
concentration of the antisense oligonucleotide is between 260 to
300 mg/mL. In certain embodiments, the addition of one or more
excipients that modulate viscosity, turbidity or both to the
antisense oligonucleotide solution reduces the viscosity of the
antisense oligonucleotide solution to less than 40 cP when the
concentration of the antisense oligonucleotide is between 300 to
400 mg/mL.
[0454] In certain embodiments, the addition of one or more
excipients that modulate viscosity, turbidity or both to the
antisense oligonucleotide solution reduces the viscosity of the
antisense oligonucleotide solution to less than 40 cP when the
antisense oligonucleotide has one or more modified sugars having
2'-MOE modifications. In certain embodiments, the addition of one
or more excipients that modulate viscosity, turbidity or both to
the antisense oligonucleotide solution reduces the viscosity of the
antisense oligonucleotide solution to less than 40 cP when the
antisense oligonucleotide has one or more modified sugars having
2'-OMe modifications. In certain embodiments, the addition of one
or more excipients that modulate viscosity, turbidity or both to
the antisense oligonucleotide solution reduces the viscosity of the
antisense oligonucleotide solution to less than 40 cP when the
antisense oligonucleotide has one or more modified sugars having
2'-F modifications. In certain embodiments, the addition of one or
more excipients that modulate viscosity, turbidity or both to the
antisense oligonucleotide solution reduces the viscosity of the
antisense oligonucleotide solution to less than 40 cP when the
antisense oligonucleotide has one or more modified sugars having
LNA modifications. In certain embodiments, the addition of one or
more excipients that modulate viscosity, turbidity or both to the
antisense oligonucleotide solution reduces the viscosity of the
antisense oligonucleotide solution to less than 40 cP when the
antisense oligonucleotide has one or more modified sugars having
cEt modifications.
[0455] Turbidity
[0456] While not wishing to be bound by theory, the presence of
turbidity in antisense oligonucleotide solutions is associated with
the formation of antisense oligonucleotide strand aggregates. In
certain embodiments, the presence of turbidity in antisense
oligonucleotide solutions makes the antisense oligonucleotide
solution appear white and cloudy. In certain embodiments, the
presence of turbidity in antisense oligonucleotide solutions makes
the antisense oligonucleotide solution appear to contain small
particles. In certain embodiments, it is desirable to have
antisense oligonucleotide solutions that have low turbidity. In
certain embodiments, it is desirable to have antisense
oligonucleotide solutions that appear clear and particle-free when
viewed by the naked eye. In certain embodiments, it is desirable to
have antisense oligonucleotide solutions that have turbidity below
20 NTU.
[0457] In certain embodiments, turbidity may be induced via a
freeze-thaw method, wherein an antisense oligonucleotide solution
is frozen and then rapidly thawed, as described herein. In certain
embodiments, the freeze-thaw method induces turbidity wherein a
given antisense oligonucleotide solution would not normally
demonstrate turbidity, or wherein a given antisense oligonucleotide
solution would demonstrate turbidity after a long period of time.
In certain embodiments turbidity represents an aesthetic problem,
but does not affect the efficacy or safety of a given antisense
oligonucleotide solution.
[0458] In certain embodiments, certain antisense oligonucleotide
solutions having desirable concentrations of antisense
oligonucleotides have undesirably high turbidity. In certain
embodiments, certain antisense oligonucleotide solutions having
desirable concentrations of antisense oligonucleotides have
undesirably high turbidity wherein the antisense oligonucleotide
solution has a cloudy or milky white appearance. In certain
embodiments, certain antisense oligonucleotide solutions having
desirable concentrations of antisense oligonucleotides have
undesirably high turbidity wherein the antisense oligonucleotide
solution appears to have small amounts of particulate interspersed
throughout the antisense oligonucleotide solution. In certain
embodiments, the addition of one or more excipients that modulate
viscosity, turbidity or both may reduce the turbidity of a certain
antisense oligonucleotide solution to a desirable level. In certain
embodiments, the addition of one or more excipients that modulate
viscosity, turbidity or both may reduce the turbidity of a certain
antisense oligonucleotide solution from having a cloudy appearance
to a having a clear appearance to the naked eye. In certain
embodiments, the addition of one or more excipients that modulate
viscosity, turbidity or both may reduce the turbidity of a certain
antisense oligonucleotide solution from having a milky white
appearance to a having a clear appearance to the naked eye. In
certain embodiments, the addition of one or more excipients that
modulate viscosity, turbidity or both may reduce the turbidity of a
certain antisense oligonucleotide solution from visible particles
to a having a clear particle-free appearance to the naked eye.
[0459] In certain embodiments, the addition of one or more
excipients that modulate viscosity, turbidity or both to the
antisense oligonucleotide solution reduces the turbidity of the
antisense oligonucleotide solution to less than 20 NTU when the
concentration of the antisense oligonucleotide is between 40 to 60
mg/mL. In certain embodiments, the addition of one or more
excipients that modulate viscosity, turbidity or both to the
antisense oligonucleotide solution reduces the turbidity of the
antisense oligonucleotide solution to less than 20 NTU when the
concentration of the antisense oligonucleotide is between 45 to 55
mg/mL. In certain embodiments, the addition of one or more
excipients that modulate viscosity, turbidity or both to the
antisense oligonucleotide solution reduces the turbidity of the
antisense oligonucleotide solution to less than 20 NTU when the
concentration of the antisense oligonucleotide is between 80 to 120
mg/mL. In certain embodiments, the addition of one or more
excipients that modulate viscosity, turbidity or both to the
antisense oligonucleotide solution reduces the turbidity of the
antisense oligonucleotide solution to less than 20 NTU when the
concentration of the antisense oligonucleotide is between 90 to 110
mg/mL. In certain embodiments, the addition of one or more
excipients that modulate viscosity, turbidity or both to the
antisense oligonucleotide solution reduces the turbidity of the
antisense oligonucleotide solution to less than 20 NTU when the
concentration of the antisense oligonucleotide is between 140 to
160 mg/mL. In certain embodiments, the addition of one or more
excipients that modulate viscosity, turbidity or both to the
antisense oligonucleotide solution reduces the turbidity of the
antisense oligonucleotide solution to less than 20 NTU when the
concentration of the antisense oligonucleotide is between 165 to
185 mg/mL. In certain embodiments, the addition of one or more
excipients that modulate viscosity, turbidity or both to the
antisense oligonucleotide solution reduces the turbidity of the
antisense oligonucleotide solution to less than 20 NTU when the
concentration of the antisense oligonucleotide is between 180 to
220 mg/mL. In certain embodiments, the addition of one or more
excipients that modulate viscosity, turbidity or both to the
antisense oligonucleotide solution reduces the turbidity of the
antisense oligonucleotide solution to less than 20 NTU when the
concentration of the antisense oligonucleotide is between 190 to
210 mg/mL. In certain embodiments, the addition of one or more
excipients that modulate viscosity, turbidity or both to the
antisense oligonucleotide solution reduces the turbidity of the
antisense oligonucleotide solution to less than 20 NTU when the
concentration of the antisense oligonucleotide is between 210 to
230 mg/mL. In certain embodiments, the addition of one or more
excipients that modulate viscosity, turbidity or both to the
antisense oligonucleotide solution reduces the turbidity of the
antisense oligonucleotide solution to less than 20 NTU when the
concentration of the antisense oligonucleotide is between 230 to
260 mg/mL. In certain embodiments, the addition of one or more
excipients that modulate viscosity, turbidity or both to the
antisense oligonucleotide solution reduces the turbidity of the
antisense oligonucleotide solution to less than 20 NTU when the
concentration of the antisense oligonucleotide is between 245 to
255 mg/mL. In certain embodiments, the addition of one or more
excipients that modulate viscosity, turbidity or both to the
antisense oligonucleotide solution reduces the turbidity of the
antisense oligonucleotide solution to less than 20 NTU when the
concentration of the antisense oligonucleotide is between 260 to
300 mg/mL. In certain embodiments, the addition of one or more
excipients that modulate viscosity, turbidity or both to the
antisense oligonucleotide solution reduces the turbidity of the
antisense oligonucleotide solution to less than 20 NTU when the
concentration of the antisense oligonucleotide is between 300 to
400 mg/mL.
[0460] In certain embodiments, the addition of one or more
excipients that modulate viscosity, turbidity or both to the
antisense oligonucleotide solution reduces the turbidity of the
antisense oligonucleotide solution to less than 20 NTU when the
antisense oligonucleotide has one or more modified sugars having
2'-MOE modifications. In certain embodiments, the addition of one
or more excipients that modulate viscosity, turbidity or both to
the antisense oligonucleotide solution reduces the turbidity of the
antisense oligonucleotide solution to less than 20 NTU when the
antisense oligonucleotide has one or more modified sugars having
2'-OMe modifications. In certain embodiments, the addition of one
or more excipients that modulate viscosity, turbidity or both to
the antisense oligonucleotide solution reduces the turbidity of the
antisense oligonucleotide solution to less than 20 NTU when the
antisense oligonucleotide has one or more modified sugars having
2'-F modifications. In certain embodiments, the addition of one or
more excipients that modulate viscosity, turbidity or both to the
antisense oligonucleotide solution reduces the turbidity of the
antisense oligonucleotide solution to less than 20 NTU when the
antisense oligonucleotide has one or more modified sugars having
LNA modifications. In certain embodiments, the addition of one or
more excipients that modulate viscosity, turbidity or both to the
antisense oligonucleotide solution reduces the turbidity of the
antisense oligonucleotide solution to less than 20 NTU when the
antisense oligonucleotide has one or more modified sugars having
cEt modifications.
[0461] Osmolarity
[0462] While not wishing to be bound by theory, antisense
oligonucleotide solutions possess varying concentrations of
osmolarity, and the osmolarity of antisense oligonucleotide
solutions depends on many factors. Some factors include but are not
limited to, length, nucleobase sequence, nucleobase modifications,
nucleobase modification motif, and/or sugar modification of the
antisense oligonucleotide. In certain embodiments, the osmolarity
of an antisense oligonucleotide solution may be difficult to
predict. In certain embodiments an antisense oligonucleotide
solution may exhibit a relatively high concentrations of
osmolarity, while a similar antisense oligonucleotide solution may
exhibit a relatively low concentrations of osmolarity.
[0463] In certain embodiments, certain antisense oligonucleotide
solutions having desirable concentrations of antisense
oligonucleotides have undesirably low osmolarity, and are
hypotonic. In certain embodiments, it is undesirable to have an
antisense oligonucleotide solution having low osmolarity, and
hypotonicity. For example, if the osmolarity of an antisense
oligonucleotide solution is too low, then an animal, human, or
patient may experience pain at the injection site. In certain
embodiments, it is therefore desirable to increase the osmolarity
of an antisense oligonucleotide solution. In certain embodiments,
it is therefore desirable to have the antisense oligonucleotide
solution become isotonic.
[0464] In certain embodiments, the addition of one or more
excipients that modulate viscosity, turbidity or both to the
antisense oligonucleotide solution increases the osmolarity of the
antisense oligonucleotide solution. In certain embodiments, the
addition of one or more excipients that modulate viscosity,
turbidity or both to the antisense oligonucleotide solution
increases the osmolarity of the antisense oligonucleotide solution
wherein the antisense oligonucleotide solution becomes
isotonic.
[0465] PH
[0466] While not wishing to be bound by theory, antisense
oligonucleotide solutions possess varying pH levels, and the pH
levels of antisense oligonucleotide solutions depends on many
factors. Some factors include, but are not limited to length,
nucleobase sequence, nucleobase modifications, nucleobase
modification motif, and/or sugar modification of the antisense
oligonucleotide. In certain embodiments, the pH of an antisense
oligonucleotide solution may be difficult to predict. In certain
embodiments an antisense oligonucleotide solution may exhibit a
relatively high pH, while an antisense oligonucleotide solution of
a similar antisense oligonucleotide may exhibit a relatively low
pH.
[0467] In certain embodiments, the addition of one or more
excipients that modulate viscosity, turbidity or both to the
antisense oligonucleotide solution alters the pH of the antisense
oligonucleotide solution. In certain embodiments, the addition of
one or more excipients that modulate viscosity, turbidity or both
to the antisense oligonucleotide solution increases pH of the
antisense oligonucleotide solution. In certain embodiments, the
addition of one or more excipients that modulate viscosity,
turbidity or both to the antisense oligonucleotide solution
decreases pH of the antisense oligonucleotide solution. In certain
embodiments, the addition of one or more excipients that modulate
viscosity, turbidity or both to the antisense oligonucleotide
solution buffers pH of the antisense oligonucleotide solution. In
certain embodiments, the addition of one or more excipients that
modulate viscosity, turbidity or both to the antisense
oligonucleotide solution buffers the pH of the antisense
oligonucleotide solution around a pH of 8.0. (e.g., 7.8 to 8.2) In
certain embodiments, the addition of one or more excipients that
modulate viscosity, turbidity or both to the antisense
oligonucleotide solution buffers the pH of the antisense
oligonucleotide solution around a pH of 7.4. (e.g., 7.2-7.6) In
certain embodiments, the addition of one or more excipients that
modulate viscosity, turbidity or both to the antisense
oligonucleotide solution buffers the pH of the antisense
oligonucleotide solution around a pH of 7.0. (e.g. 6.8-7.2).
[0468] Antimicrobial
[0469] In certain embodiments it is desirable to include an
antimicrobial agent to an antisense oligonucleotide solution to
facilitate storage or delivery. In certain embodiments it is not
necessary to include an antimicrobial agent to an antisense
oligonucleotide solution to facilitate storage or delivery of the
antisense oligonucleotide solution. In certain embodiments an
antimicrobial agent is added to an antisense oligonucleotide
solution. In certain embodiments no antimicrobial agents are added
to an antisense oligonucleotide solution. In certain embodiments
antisense oligonucleotide solutions are prepared without
antimicrobial agents. In certain embodiments one or more excipients
that modulate viscosity, turbidity or both may also serve as an
antimicrobial agent or preservative. In certain embodiments one or
more excipients that modulate viscosity, turbidity or both may not
have any antimicrobial or preservative properties.
Certain Pharmaceutical Compositions
[0470] In certain embodiments, the present invention provides
pharmaceutical compositions comprising one or more antisense
compound. In certain embodiments, such pharmaceutical composition
comprises a suitable pharmaceutically acceptable diluent or
carrier. In certain embodiments, a pharmaceutical composition
comprises a sterile saline solution and one or more antisense
compound. In certain embodiments, such pharmaceutical composition
consists of a sterile saline solution and one or more antisense
compound. In certain embodiments, the sterile saline is
pharmaceutical grade saline. In certain embodiments, a
pharmaceutical composition comprises one or more antisense compound
and sterile water. In certain embodiments, a pharmaceutical
composition consists of one or more antisense compound and sterile
water. In certain embodiments, the sterile saline is pharmaceutical
grade water. In certain embodiments, a pharmaceutical composition
comprises one or more antisense compound and phosphate-buffered
saline (PBS). In certain embodiments, a pharmaceutical composition
consists of one or more antisense compound and sterile
phosphate-buffered saline (PBS). In certain embodiments, the
sterile saline is pharmaceutical grade PBS.
[0471] In certain embodiments, antisense compounds may be admixed
with pharmaceutically acceptable active and/or inert substances for
the preparation of pharmaceutical compositions or formulations.
Compositions and methods for the formulation of pharmaceutical
compositions depend on a number of criteria, including, but not
limited to, route of administration, extent of disease, or dose to
be administered.
[0472] Pharmaceutical compositions comprising antisense compounds
encompass any pharmaceutically acceptable salts, esters, or salts
of such esters. In certain embodiments, pharmaceutical compositions
comprising antisense compounds comprise one or more oligonucleotide
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 pharmaceutically acceptable salts of
antisense compounds, prodrugs, pharmaceutically acceptable salts of
such prodrugs, and other bioequivalents. Suitable pharmaceutically
acceptable salts include, but are not limited to, sodium and
potassium salts.
[0473] A prodrug can include the incorporation of additional
nucleosides at one or both ends of an oligomeric compound which are
cleaved by endogenous nucleases within the body, to form the active
antisense oligomeric compound.
[0474] Lipid moieties have been used in nucleic acid therapies in a
variety of methods. In certain such methods, the nucleic acid is
introduced into preformed liposomes or lipoplexes made of mixtures
of cationic lipids and neutral lipids. In certain methods, DNA
complexes with mono- or poly-cationic lipids are formed without the
presence of a neutral lipid. In certain embodiments, a lipid moiety
is selected to increase distribution of a pharmaceutical agent to a
particular cell or tissue. In certain embodiments, a lipid moiety
is selected to increase distribution of a pharmaceutical agent to
fat tissue. In certain embodiments, a lipid moiety is selected to
increase distribution of a pharmaceutical agent to muscle
tissue.
[0475] In certain embodiments, a pharmaceutical composition
provided herein comprises a delivery system. Examples of delivery
systems include, but are not limited to, liposomes and emulsions.
Certain delivery systems are useful for preparing certain
pharmaceutical compositions including those comprising hydrophobic
compounds. In certain embodiments, certain organic solvents such as
dimethylsulfoxide are used.
[0476] In certain embodiments, a pharmaceutical composition
provided herein comprises one or more tissue-specific delivery
molecules designed to deliver the one or more pharmaceutical agents
of the present invention to specific tissues or cell types. For
example, in certain embodiments, pharmaceutical compositions
include liposomes coated with a tissue-specific antibody.
[0477] In certain embodiments, a pharmaceutical composition
provided herein comprises a co-solvent system. Certain of such
co-solvent systems comprise, for example, benzyl alcohol, a
nonpolar surfactant, a water-miscible organic polymer, and an
aqueous phase. In certain embodiments, such co-solvent systems are
used for hydrophobic compounds. A non-limiting example of such a
co-solvent system is the VPD co-solvent system, which is a solution
of absolute ethanol comprising 3% w/v benzyl alcohol, 8% w/v of the
nonpolar surfactant Polysorbate 80.TM. and 65% w/v polyethylene
glycol 300. The proportions of such co-solvent systems may be
varied considerably without significantly altering their solubility
and toxicity characteristics. Furthermore, the identity of
co-solvent components may be varied: for example, other surfactants
may be used instead of Polysorbate 80.TM.; the fraction size of
polyethylene glycol may be varied; other biocompatible polymers may
replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other
sugars or polysaccharides may substitute for dextrose.
[0478] In certain embodiments, a pharmaceutical composition
provided herein is prepared for oral administration. In certain
embodiments, pharmaceutical compositions are prepared for buccal
administration.
[0479] In certain embodiments, a pharmaceutical composition is
prepared for administration by injection (e.g., intravenous,
subcutaneous, intramuscular, etc.). In certain of such embodiments,
a pharmaceutical composition comprises a carrier and is formulated
in aqueous solution, such as water or physiologically compatible
buffers such as Hanks's solution, Ringer's solution, or
physiological saline buffer. In certain embodiments, other
ingredients are included (e.g., ingredients that aid in solubility
or serve as preservatives). In certain embodiments, injectable
suspensions are prepared using appropriate liquid carriers,
suspending agents and the like. Certain pharmaceutical compositions
for injection are presented in unit dosage form, e.g., in ampoules
or in multi-dose containers. Certain pharmaceutical compositions
for injection are suspensions, solutions or emulsions in oily or
aqueous vehicles, and may contain formulatory agents such as
suspending, stabilizing and/or dispersing agents. Certain solvents
suitable for use in pharmaceutical compositions for injection
include, but are not limited to, lipophilic solvents and fatty
oils, such as sesame oil, synthetic fatty acid esters, such as
ethyl oleate or triglycerides, and liposomes. Aqueous injection
suspensions may contain substances that increase the viscosity of
the suspension, such as sodium carboxymethyl cellulose, sorbitol,
or dextran. Optionally, such suspensions may also contain suitable
stabilizers or agents that increase the solubility of the
pharmaceutical agents to allow for the preparation of highly
concentrated solutions.
[0480] In certain embodiments, a pharmaceutical composition is
prepared for transmucosal administration. In certain of such
embodiments penetrants appropriate to the barrier to be permeated
are used in the formulation. Such penetrants are generally known in
the art.
[0481] In certain embodiments, a pharmaceutical composition
provided herein comprises an oligonucleotide in a therapeutically
effective amount. In certain embodiments, the therapeutically
effective amount is sufficient to prevent, alleviate or ameliorate
symptoms of a disease or to prolong the survival of the subject
being treated. Determination of a therapeutically effective amount
is well within the capability of those skilled in the art.
[0482] In certain embodiments, one or more modified oligonucleotide
provided herein is formulated as a prodrug. In certain embodiments,
upon in vivo administration, a prodrug is chemically converted to
the biologically, pharmaceutically or therapeutically more active
form of an oligonucleotide. In certain embodiments, prodrugs are
useful because they are easier to administer than the corresponding
active form. For example, in certain instances, a prodrug may be
more bioavailable (e.g., through oral administration) than is the
corresponding active form. In certain instances, a prodrug may have
improved solubility compared to the corresponding active form. In
certain embodiments, prodrugs are less water soluble than the
corresponding active form. In certain instances, such prodrugs
possess superior transmittal across cell membranes, where water
solubility is detrimental to mobility. In certain embodiments, a
prodrug is an ester. In certain such embodiments, the ester is
metabolically hydrolyzed to carboxylic acid upon administration. In
certain instances the carboxylic acid containing compound is the
corresponding active form. In certain embodiments, a prodrug
comprises a short peptide (polyaminoacid) bound to an acid group.
In certain of such embodiments, the peptide is cleaved upon
administration to form the corresponding active form.
[0483] In certain embodiments, the present invention provides
compositions and methods for reducing the amount or activity of a
target nucleic acid in a cell. In certain embodiments, the cell is
in an animal. In certain embodiments, the animal is a mammal. In
certain embodiments, the animal is a rodent. In certain
embodiments, the animal is a primate. In certain embodiments, the
animal is a non-human primate. In certain embodiments, the animal
is a human.
[0484] In certain embodiments, the present invention provides
methods of administering a pharmaceutical composition comprising an
oligomeric compound of the present invention to an animal. Suitable
administration routes include, but are not limited to, oral,
rectal, transmucosal, intestinal, enteral, topical, suppository,
through inhalation, intrathecal, intracerebroventricular,
intraperitoneal, intranasal, intraocular, intratumoral, and
parenteral (e.g., intravenous, intramuscular, intramedullary, and
subcutaneous). In certain embodiments, pharmaceutical intrathecals
are administered to achieve local rather than systemic exposures.
For example, pharmaceutical compositions may be injected directly
in the area of desired effect (e.g., into the eyes, ears).
Certain Excipients
[0485] As reported in scientific or patent literature, some
oligonucleotide compounds form aggregates when in solution and
aggregation may causes undesired viscosity, turbidity, or both. In
certain embodiments, oligonucleotide solutions having high
viscosity are undesirable because it complicates delivery through a
syringe. For example, oligonucleotide solutions having high
viscosity may only effectively get delivered using a high gauge
needle which may cause excess pain or discomfort to a patient. In
certain embodiments, oligonucleotide solutions having high
turbidity are undesirable because the oligonucleotide solution may
appear to have one or more impurities present.
[0486] In certain embodiments, oligonucleotide solutions having
additives can mitigate only turbidity or only viscosity (see US
20110098343). In certain embodiments, this disclosure provides that
both turbidity and viscosity can be comitigated by a single
excipient, for example, L-tryptophan, pyridoxine, L-Phenylalanine,
and nicotinamide. In certain embodiments, additional excipients
that have potential to mitigate both turbidity and viscosity
include but are not limited to thymine, adenine, riboflavin,
thiamine, and tryptamine. In certain embodiments, effective
excipients have heterocyclic character and/or an ability to act as
a hydrogen bond donor and/or a hydrogen bond acceptor. In certain
embodiments, excipients with nonaromatic rings may not be effective
for viscosity reduction.
[0487] In certain embodiments, certain properties of effective
excipients, e.g. (i) aromatic homocyclicity or heterocyclicity and
(ii) the ability to act as a hydrogen bond donor and/or acceptor,
may be consolidated into one excipient or compound. In certain
embodiments, two or more excipients may together possess one or
more properties of effective excipients. For example, in certain
embodiments, a first excipient may be an aromatic homocyclic or
heterocyclic compound but may not have the ability to act as a
hydrogen bond donor and/or acceptor. In certain such embodiments, a
second excipient may have the ability to act as a hydrogen bond
donor and/or acceptor, but may not be an aromatic homocyclic or
heterocyclic compound. In certain such embodiments, the first
excipient may act as a aromatic homocyclic or heterocyclic compound
and a second excipient may act as a hydrogen bond donor and/or
acceptor and the combination of the first and second excipient may
produce an effective excipient for mitigating turbidity, viscosity,
or both. In certain embodiments, a mixture of two or more
excipients, the sum of which contain (i) aromatic homocyclicity or
heterocyclicity and (ii) the ability to act as a hydrogen bond
donor and/or acceptor, are effective in mitigating both turbidity
and viscosity.
[0488] In certain embodiments, antisense oligonucleotide
compositions provided herein comprise one or more modified
antisense oligonucleotides and one or more excipients. Any suitable
excipient known to those having skill in the art may be used. For
example, suitable excipients may be found in, for example "Handbook
of Pharmaceutical Excipients," American Pharmaceutical Association
Publications, Washington D.C., 6.sup.th Edition, 2009; which is
hereby incorporated herein by reference in its entirety. In certain
such embodiments, excipients are selected from salt solutions,
alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium
stearate, talc, silicic acid, viscous paraffin,
hydroxymethylcellulose and polyvinylpyrrolidone.
[0489] In certain embodiments, excipients are selected from
Adenine, Benzyl Alcohol, m-Cresol, Cytidine, Cytidine
Monophosphate, Cytosine, Dextran, Guanine Monophosphate,
D-Mannitol, Methylparaben, Nicotinamide, Phenol, 2-Phenoxyethanol,
L-Phenylalanine, Pyridoxine, Sodium Chloride, Thymine, tryptophan,
L-Tryptophan, L-Tyrosine, Ascorbic Acid, Benzamide, o-Benzenediol,
Benzenehexol, L-Histidine, Hydroxypyridine, Indole,
D-Mannitol+Phenol mixture, D-Mannitol+Pyridine mixture, 2H-Pyran,
Pyrazinamide, Pyridine, Pyrimidine, 2-Pyrone, Riboflavin, Thiamine,
Tryptamine, ethanol, (2-Hydroxypropyl)-o-cyclodextrin, Niacin,
Polyethylene Glycol 600, Polyethylene Glycol 4600, Propylene
Glycol, Pyridoxine, Sucrose, Thymidine, Tween 80, Uridine, Thymine,
caffeine, acridine orange, ethidium bromide, propidium iodide,
cyanine dyes such as PicoGreen.RTM., thiamine hydrochloride,
ethylene diamine tetraacetic acid, and 1,2-dihydroxybenzene.
[0490] In certain embodiments, excipients comprise heterocyclic
molecules. In certain embodiments, excipients comprise heterocyclic
amines. In certain embodiments, excipients comprise aromatic
molecules or molecules having one or more aromatic ring. In certain
embodiments, excipients comprise heterocyclic molecules wherein the
heteroatom is oxygen. In certain embodiments, excipients comprise
heterocyclic molecules wherein the heteroatom is nitrogen. In
certain embodiments, excipients comprise heterocyclic molecules
wherein the heteroatom is sulfur.
[0491] In certain embodiments, the excipient lowers the viscosity
of an antisense oligonucleotide composition. In certain
embodiments, the excipient lowers the turbidity of an antisense
oligonucleotide composition. In certain embodiments, the excipient
increases the osmolarity of an antisense oligonucleotide
composition. In certain embodiments, the excipient decreases the
osmolarity of an antisense oligonucleotide composition. In certain
embodiments, the excipient increases the pH of an antisense
oligonucleotide composition. In certain embodiments, the excipient
decreases the pH of an antisense oligonucleotide composition. In
certain embodiments, the excipient buffers the pH of an antisense
oligonucleotide composition. In certain embodiments, the excipient
lowers the turbidity and the viscosity of an antisense
oligonucleotide composition. In certain embodiments, the excipient
lowers the turbidity and the viscosity of an antisense
oligonucleotide composition and also increases the osmolarity of
the antisense composition. In certain embodiments, the excipient
lowers the turbidity and the viscosity of an antisense
oligonucleotide composition and also decreases the osmolarity of
the antisense composition.
[0492] In certain embodiments, a mixture of two or more excipients
lowers the viscosity of an antisense oligonucleotide composition.
In certain embodiments, a mixture of two or more excipients lowers
the turbidity of an antisense oligonucleotide composition. In
certain embodiments, a mixture of two or more excipients increases
the osmolarity of an antisense oligonucleotide composition. In
certain embodiments, a mixture of two or more excipients decreases
the osmolarity of an antisense oligonucleotide composition. In
certain embodiments, a mixture of two or more excipients increases
the pH of an antisense oligonucleotide composition. In certain
embodiments, a mixture of two or more excipients decreases the pH
of an antisense oligonucleotide composition. In certain
embodiments, a mixture of two or more excipients buffers the pH of
an antisense oligonucleotide composition. In certain embodiments, a
mixture of two or more excipients lowers the turbidity and the
viscosity of an antisense oligonucleotide composition. In certain
embodiments, a mixture of two or more excipients lowers the
turbidity and the viscosity of an antisense oligonucleotide
composition and also increases the osmolarity of the antisense
composition. In certain embodiments, a mixture of two or more
excipients lowers the turbidity and the viscosity of an antisense
oligonucleotide composition and also decreases the osmolarity of
the antisense composition.
[0493] In certain embodiments, one or more excipients may effective
reduce the viscosity, turbidity, or both the viscosity and
turbidity of a solution of one or more modified antisense
oligonucleotides. In certain embodiments, one or more excipients
may effective reduce the viscosity, turbidity, or both the
viscosity and turbidity of a solution of one or more modified
antisense oligonucleotides but this excipient may, for example,
increase the osmolality of the solution to an undesirable amount.
In certain embodiments,
[0494] In certain embodiments, salts such as NaCl, KCl, LiC,
MgCl.sub.2, or CaCl.sub.2 may used to modulate the osmolality of an
oligonucleotide in solution, but may also induce the turbidity of
certain oligonucleotides. In certain such embodiments, it may
therefore be desirable to use an excipient selected from among
L-Tryptophan, Niacinamide, L-Phenylalanine, and L-Histidine to
modulate the osmolality of an oligonucleotide solution and to also
modulate the viscosity and/or turbidity of the oligonucleotide
solution. In certain such embodiments, it may therefore be
desirable to use an excipient selected from among L-Tryptophan,
Niacinamide, L-Phenylalanine, and L-Histidine to increase the
osmolality of an oligonucleotide solution and to also decrease the
viscosity of the oligonucleotide solution. In certain such
embodiments, it may therefore be desirable to use an excipient
selected from among L-Tryptophan, Niacinamide, L-Phenylalanine, and
L-Histidine to increase the osmolality of an oligonucleotide
solution and to also decrease the turbidity of the oligonucleotide
solution. In certain such embodiments, it may therefore be
desirable to use an excipient selected from among L-Tryptophan,
Niacinamide, L-Phenylalanine, and L-Histidine to increase the
osmolality of an oligonucleotide solution and to also decrease the
viscosity and decrease the turbidity of the oligonucleotide
solution.
NONLIMITING DISCLOSURE AND INCORPORATION BY REFERENCE
[0495] While certain compounds, compositions and methods described
herein have been described with specificity in accordance with
certain embodiments, the following examples serve only to
illustrate the compounds described herein and are not intended to
limit the same. Each of the references, GenBank accession numbers,
and the like recited in the present application is incorporated
herein by reference in its entirety.
[0496] Although the sequence listing accompanying this filing
identifies each sequence as either "RNA" or "DNA" as required, in
reality, those sequences may be modified with any combination of
chemical modifications. One of skill in the art will readily
appreciate that such designation as "RNA" or "DNA" to describe
modified oligonucleotides is, in certain instances, arbitrary. For
example, an oligonucleotide comprising a nucleoside comprising a
2'-OH sugar moiety and a thymine base could be described as a DNA
having a modified sugar (2'-OH for the natural 2'-H of DNA) or as
an RNA having a modified base (thymine (methylated uracil) for
natural uracil of RNA).
[0497] Accordingly, nucleic acid sequences provided herein,
including, but not limited to those in the sequence listing, are
intended to encompass nucleic acids containing any combination of
natural or modified RNA and/or DNA, including, but not limited to
such nucleic acids having modified nucleobases. By way of further
example and without limitation, an oligomeric compound having the
nucleobase sequence "ATCGATCG" encompasses any oligomeric compounds
having such nucleobase sequence, whether modified or unmodified,
including, but not limited to, such compounds comprising RNA bases,
such as those having sequence "AUCGAUCG" and those having some DNA
bases and some RNA bases such as "AUCGATCG" and oligomeric
compounds having other modified or naturally occurring bases, such
as "AT.sup.meCGAUCG," wherein .sup.meC indicates a cytosine base
comprising a methyl group at the 5-position.
EXAMPLES
[0498] The following examples illustrate certain embodiments of the
present invention and are not limiting. Moreover, where specific
embodiments are provided, the inventors have contemplated generic
application of those specific embodiments. For example, disclosure
of an oligonucleotide having a particular motif provides reasonable
support for additional oligonucleotides having the same or similar
motif. And, for example, where a particular high-affinity
modification appears at a particular position, other high-affinity
modifications at the same position are considered suitable, unless
otherwise indicated.
Example 1
General Method for Evaluating Turbidity, and Viscosity
[0499] The general method for measuring turbidity is as follows.
Turbidity qualitative assessment is performed by visually
inspecting control and sample vials that have frozen at -20.degree.
C. and subsequently thawed at 5.degree. C. Their appearance is
noted before the samples equilibrate to room temperature. These
results are often documented with photographs. Turbidity
quantitative measurement is performed on a Hach 2100 AN Laboratory
Turbidimeter and is used to test turbidity of 3.2-3.4 mL of
solution filled in a 13-mm glass tube. Typically the solution has
received freeze-thaw treatment similar to the one described above
to induce turbidity formation prior to measurement. The instrument
is standardized with Formazin reference suspensions over a range of
Nephelometric Turbidity Units (NTUs) that brackets the turbidity of
the samples being tested.
[0500] Viscosity qualitative assessment is performed by visually
inspecting the flow of a sample solution in a container and
comparing it to a control. In this case, sample viscosities were
noted as increased or decreased only when an obvious difference was
noted (e.g., if at 5.degree. C. the control sample appeared to
mimic honey in viscosity, the viscosity of excipient samples
appearing as free-flowing liquids are noted as "decreased", while
excipient samples appearing as solid gel are noted as "increased").
Quantitative viscosity measurement comprises of two forms, dynamic
and kinematic. The form relating to antisense oligonucleotide (ASO)
drug product characterization is dynamic viscosity. Two instruments
that are currently used in the Pharmaceutical Development (PD)
laboratory to measure dynamic viscosity are RheoSense m-VROC
viscometer and Malvern Instruments Bohlin CVO 100 rheometer.
Further information about the instruments can be obtained from the
manufacturer's websites (rheosense.com and malvern.com/CVO).
RheoSense m-VROC System
[0501] The RheoSense m-VROC utilizes a microelectromechanical
systems (MEMS) chip which consists of three silicone pressure
sensor arrays embedded lengthwise along the center of a rectangular
channel. The liquid sample is loaded into a syringe pump, which
dispenses the sample into the microfluidic chip at a specified flow
rate. The pressure drop over distance of a flowing test liquid is
measured, and it is expected to be linear for Newtonian fluids if a
fully developed flow is ensured within the channel. The shear
stress and viscosity are calculated according to fluid dynamic
principles.
[0502] There are four classifications of chips available for the
RheoSense m-VROC, labeled A, B, C, and D. They vary consecutively
in size, with the A chip having the smallest inner channel
dimensions that enables precise measurements of low viscosity
samples, while the D chip is built for high viscosity samples.
Within each chip category there are also three channel depths: 20,
50, and 100 .mu.m, indicated as the "02", "05", and "10" series.
The maximum recommended sample viscosities for use with each chip
are shown in Table 2.
TABLE-US-00002 TABLE 2 Recommended upper viscosity limits for each
Rheosense m-VROC chip Chip Maximum Recommended Viscosity (cP) A 100
B 400 C 3,000 D 10,000
Malvern Instruments Bohlin CVO 100 System
[0503] The Bohlin CVO 100 operates via a bob-and-cup mechanism,
whereby the torque required to accelerate a bob in a cup filled
with sample fluid to a specified angular speed is measured and
converted mathematically to viscosity. A single interface is used
for all sample types evaluated in, with the bob and cup being
removable for sample loading and cleaning. The instrument
specifications are shown in Table 3.
TABLE-US-00003 TABLE 3 Instrument Specifications for Bohlin CVO 100
System Method Shear ramp method with a spinning bob rheometer and
no Description inertial corrections Cell 1 mL Mooney-Ewart Gap 75
microns Temperature Isothermal, typically at 25.degree. C.,
15.degree. C., or 5.degree. C. Standard and 1.2 mL Sample Volume
Standards 10, 50, and 100 cP polydimethylsiloxane viscosity
reference fluids Model Fit Newtonian
Example 1a
General Method for Evaluating Osmolality
[0504] Osmolality is measured using Wescor VAPRO 5600 Vapor
Pressure Osmometer. A small paper disc is loaded onto the sampling
area, and 10 uL of test solution is dispensed into the disc.
Calibration is performed using 100, 290, and 1000 mOsm/kg
standards. Further information about this instrument can be
obtained from the manufacturer's website
(wescor.com/biomedical/osmometer/vapro5600.html).
Wescor VAPRO 5600 Vapor Pressure Osmometer
[0505] VAPRO 5600 osmometer measures a test solution's dew point
temperature depression, which is related to its vapor pressure.
Vapor pressure is a colligative property of a solution which
linearly correlates to the concentration of particles dissolved in
the solvent (i.e. osmolality). The particle's size, density,
configuration, or electrical charge has no bearing on a colligative
property. Increasing osmolality decreases the solution vapor
pressure.
[0506] In the VAPRO system, an internal thermocouple hygrometer
joins with the sample holder when the sample has been loaded to
form a small chamber enclosing the sample disc. After the chamber
temperature has equilibrated, the thermocouple is cooled, thus
inducing dew formation on the thermocouple surface. The
thermocouple is then heated until it reaches the dew point. The
difference between ambient temperature and the dew point
temperature is the dew point temperature depression. The instrument
processes this result and provides a reading in mmol/kg, equaling
mOsm/kg. (Note: mOsm/kg refers to the concentration of dissociated
molecules in solution. Therefore, 1 mmol/L NaCl yields 2 mmol/L of
dissociated ions; i.e. Na.sup.+ and Cl.sup.-, which equals 2
mOsm/L. Applying the density conversion of 1 kg/L yields 2
mOsm/kg). The instrument specifications are shown in Table 3a.
TABLE-US-00004 TABLE 3a Instrument Specifications for Wescor VAPRO
5600 Sample Volume 10 .mu.L Measurement Range 20-300 mmol/kg
Measurement Time 90 seconds Resolution 1 mmol/kg Calibration
Opti-mole .TM. osmolality standards
Example 2
General Method Used to Screen Excipients for the Mitigation of
Turbidity and Viscosity
Turbidity Screening
[0507] The screening of the excipients for mitigating turbidity is
performed by adding 0.1 to 5% (w/v) of the excipient at pH 7-8 to a
solution of ASO in water at a concentration of 200 mg/mL to 250
mg/mL. After the solution is prepared, a 1 mL sample is aliquoted
and filled into a 2-mL clean glass vial, stoppered, sealed, frozen
at -20.degree. C., and thawed at 5.degree. C. The sample is then
evaluated for turbidity with the turbidimeter or more commonly by
visual inspection. The results from visual inspection is converted
to a score from 0 to 3 with 0 being visually clear (i.e., <20
NTU); 1 being less turbid than a control but not clear; 2
approximately the same turbidity as a control; and 3 being more
turbid than a control. A solution of ASO in water is used as the
control.
[0508] The excipients investigated for mitigation of turbidity are
independently selected from but are not limited to adenine, benzyl
alcohol, m-cresol, cytidine, cytidine monophosphate, cytosine,
dextran, guanine monophosphate, D-mannitol, methylparaben,
nicotinamide, phenol, 2-phenoxyethanol, L-phenylalanine,
pyridoxine, sodium chloride, thymine, L-tryptophan, L-tyrosine,
ascorbic acid, benzamide, o-benzenediol, benzenehexol, caffeine,
L-histidine, hydroxypyridine, indole, 2H-pyran, pyrazinamide,
pyridine, pyrimidine, 2-pyrone, riboflavin, thiamine, thiamine
hydrochloride, 1,2-dihydroxybenzene (catechol), ethylene diamine
tetraacetic acid (EDTA), tryptamine, calcium folinate, sodium
folinate (vitamin B9), D-mannitol and phenol mixture, D-mannitol
and pyridine mixture, D-mannitol and niacinamide (nicotinamide)
mixture, L-phenylalanine and pyridoxine mixture, pyridoxine and
benzyl alcohol mixture, L-phenylalanine and L-histidine mixture;
any mixture combination of L-phenylalanine, L-tryptophan,
L-histidine, and niacinamide; and dinucleotides or trinucleotides
or other shorter oligonucleotide probes; and nucleic acid stains
such as acridine orange, ethidium bromide, propidium iodide, and
cyanine dyes such as PicoGreen.RTM..
Viscosity Screening
[0509] A concentrated stock solution of ASO in water is diluted
using solid excipient or a concentrated stock solution of
excipient. A typical dilution is from a concentration of 250 to 200
mg/mL of ASO at pH 8. Viscosity would either be visually noted, or
measured using the RheoSense m-VROC viscometer or Malvern
Instruments Bohlin CVO 100 rheometer as described in Example 1. The
measured results for viscosity are then obtained and normalized to
the results when the diluent is only water with no excipient
present.
[0510] The excipients investigated for mitigation of viscosity are
independently selected from but are not limited to benzyl alcohol,
m-cresol, cytidine, cytosine, dextran, ethanol,
(2-Hydroxypropyl)-.beta.-cyclodextrin, D-mannitol, niacin,
nicotinamide, polyethylene glycol 600 (PEG.sub.600), polyethylene
glycol 4600 (PEG.sub.4600), propylene glycol, pyridoxine, sodium
chloride, sucrose, thymidine, L-tryptophan, uridine, adenine,
thymine, Tween 80, cytidine monophosphate, guanine monophosphate,
methylparaben, phenol, 2-phenoxyethanol, L-phenylalanine,
L-tyrosine, ascorbic acid, benzamide, o-benzenediol, benzenehexol,
caffeine, L-histidine, hydroxypyridine, indole, 2H-pyran,
pyrazinamide, pyridine, pyrimidine, 2-pyrone, riboflavin, thiamine,
tryptamine, thiamine hydrochloride, 1,2-dihydroxybenzene
(catechol), ethylene diamine tetraacetic acid (EDTA), calcium
folinate, sodium folinate (vitamin B9), D-mannitol and phenol
mixture, D-mannitol and pyridine mixture, D-mannitol and phenol
mixture, D-mannitol and pyridine mixture, D-mannitol and
niacinamide (nicotinamide) mixture, L-phenylalanine and pyridoxine
mixture, pyridoxine and benzyl alcohol mixture, L-phenylalanine and
L-histidine mixture; any mixture combination of L-phenylalanine,
L-tryptophan, L-histidine, and niacinamide; and dinucleotides or
trinucleotides or other shorter oligonucleotide probes; and nucleic
acid stains such as acridine orange, ethidium bromide, propidium
iodide, and cyanine dyes such as PicoGreen.RTM..
[0511] Note:
[0512] Not all the aforementioned excipients are viable for
formulation. Some are used in the screening simply for mechanistic
studies.
Example 3
Turbidity and Viscosity Evaluation for ISIS NO. 426115
[0513] Antisense oligonucleotide Isis No. 426115 was selected for
turbidity and viscosity evaluation. The ASO and its motif are
described in Table 4. The internucleoside linkages throughout each
modified oligonucleotide are phosphorothioate linkages (P.dbd.S).
Nucleosides without a subscript are
.beta.-D-2'-deoxyribonucleosides. Nucleosides followed by a
subscript "e" indicate 2'-O-methoxyethyl (MOE) modified
nucleosides. An "N" indicates a U, T, C, .sup.meC, G or A
nucleoside.
TABLE-US-00005 TABLE 4 Antisense Oligonucleotide Isis No. 426115
Selected for Turbidity and Viscosity Evaluation Isis No.
Composition (5' to 3') Motif SEQ ID No. 426115
N.sub.eN.sub.eN.sub.eN.sub.eN.sub.eNNNNNNNNNNN.sub.eN.sub.eN.sub.eN-
.sub.eN.sub.e 5-10-5 14
Turbidity Evaluation for ISIS NO. 426115
[0514] Several excipients in Example 2 were selected and screened
for their effect in mitigating the turbidity of ISIS NO. 426115.
The turbidity experiment was performed in the same manner as
described in Example 2. To an aqueous solution of ISIS NO. 426115
at a concentration of 220 mg/mL, the excipient was added at the
percentage (%, w/v) as indicated in Table 5. The solution was
frozen at -20.degree. C., thawed to 5.degree. C. and subjected to
turbidity evaluation. Turbidity was analyzed and compared to a
control by visual inspection using a scoring format of 0 to 3 with
0 being visually clear; 1 being less turbid than a control but not
clear; 2 approximately the same turbidity as a control; and 3 being
more turbid than a control. A solution of ISIS NO. 426115 at a
concentration of 220 mg/mL at pH 7-8 in only water was used as the
control. The results are presented in Table 5.
[0515] As illustrated, several excipients when used individually or
in combination with other excipients at various concentrations
demonstrated desirable reduction in turbidity with a score of 1 or
lower as compared to the control.
TABLE-US-00006 TABLE 5 Effect of various excipients on turbidity
for ISIS NO. 426115 at 220 mg/mL at 5.degree. C. Excipient
Turbidity Excipient concentration (%, w/v) (visual inspection)
Adenine 0.5 0 Ascorbic acid 0.5 2 Benzyl alcohol 0.5 2 1.5 0
Caffeine 0.5 0 1.5 0 m-Cresol 0.3 3 Cytidine 0.01 2 0.2 0.5 5.0 0
Cytidine Monophosphate 0.5 2 Cytosine 0.005 2 0.2 2 0.5 0 1.0
Dextran 0.5 2 1,2-Dihydroxybenzene 0.5 3 (catechol) Ethylene
diamine 0.2 2 tetraacetic acid (EDTA) 1.0 3 Guanine Monophosphate
0.5 2 L-Histidine 1.5 0 2.0 0 Hydroxypyridine or 2 0.5 1 Pyridone
1.0 0 D-Mannitol (0.5) + Phenol 1.0 (total) 2 (0.5) mixture
D-Mannitol (0.5) + 1.0 (total) 1 Pyridine (0.5) mixture D-Mannitol
(0.5) + 1.0 (total) 1 Niacinamide (0.5) mixture D-Mannitol 2.0 2
5.0 10 15 Methylparaben 0.2 3 0.5 2 1.5* 0 *excipient saturated and
precipitated Nicotinamide or 0.5 2 Niacinamide 1.0 1 1.5 0 2.0 2.5
3.0 5.0 Phenol 0.5 3 2-Phenoxyethanol 0.5 3 L-Phenylalanine 0.5 1
1.5 0 Pyrazinamide 0.5 1 1.0 0 Pyridine 0.5 2 Pyridoxine 1.0 2 1.5
Sodium Chloride 0.5 2 Thiamine HCl 1 0 Thymine 0.5 0 L-Tryptophan
0.1 2 0.2 0.3 0.4 1 0.5 0 1.5 0 L-Tyrosine 0.04 2 Calcium Folinate
0.5 2 L-Histidine 0.5 2 1.5 1 2 0 Indole 0.1 2 2H-Pyran 0.5 2
Pyrimidine 0.5 2 2-Pyrone 0.5 1 Riboflavin (Vitamin B2) 0.025 2
D-Mannitol (0.5) + 1 (total) 1 Nicotinamide/Niacinamide (0.5)
mixture L-phenylalanine (1) + 1.5 (total) 0 Pyridoxine (0.5)
Pyridoxine (0.5) + Benzyl 1 (total) 0 alcohol (0.5)
Viscosity Evaluation for ISIS NO. 426115
[0516] Several excipients in Example 2 were selected and screened
for their effect in mitigating the viscosity of ISIS NO. 426115.
The viscosity experiment was performed in the same manner as
described in Example 2. A concentrated stock solution of ASO in
water was diluted using solid excipient or a concentrated stock
solution of excipient. The dilution was at a concentration of 220
mg/mL of ISIS NO. 426115 at pH 7-8. Viscosity at 25.degree. C. and
5 OC was measured using the RheoSense m-VROC viscometer as
described in Example 1. The results for viscosity were obtained and
normalized to the results of the control when the diluent was only
water. Normalized viscosity was calculated by dividing the
viscosity of the excipient present vs the viscosity of the control.
The results are presented in Table 6.
[0517] As illustrated, at 25.degree. C. or 5 OC, several excipients
when used alone or in combination with other excepients at various
concentrations demonstrated desirable reduction in the viscosity
with a normalized viscosity below 1.00.
TABLE-US-00007 TABLE 6 Effect of various excipients on viscosity of
ISIS NO. 426115 at 220 mg/mL at 25.degree. C. and 5.degree. C.
Excipient Viscosity Normalized Conc. (cP) Viscosity* Excipient (%,
w/v) 25.degree. C. 5.degree. C. 25.degree. C. 5.degree. C. None
(Control) 0 79 28,500 1.00 1.00 L-Tryptophan 0.5 73 12,000 0.9 0.4
1.5 64 7,758 0.8 0.3 Nicotinamide 1.5 50 3,000 0.6 0.1
(Niacinamide) Phenylalanine 1.5 41 1,604 0.5 0.1 Thiamine HCl 1 95
79,000 1.2 2.8 Pyridoxine HCl (0.5%) + 1 106 19,000 1.3 0.7 benzyl
alcohol (0.5%) Pyridoxine HCl (0.5%) + 1.5 46 2,539 0.6 0.1
phenylalanine (1%) Caffeine 1.5 148 35,000 1.9 1.2 Histidine 0.5 73
NT 0.9 NT 1.5 63 NT 0.8 NT 2 47 4,759 0.6 0.2 Pyrazinamide 1 59 NT
0.7 NT Methylparaben 1.5 120 NT 1.5 NT *Normalized to ISIS NO.
426115 at concentration of 220 mg/mL in water NT = Not Tested
[0518] As reported in scientific or patent literature, some
additives can mitigate only turbidity or only viscosity (see US
20110098343). Unlike the literature, results from Tables 4 and 5
demonstrated that both turbidity and viscosity can be comitigated
by a single excipient such as L-tryptophan, benzyl alcohol,
L-histidine, L-phenylalanine, and nicotinamide. In certain
embodiments, additional excipients that have potential to mitigate
both turbidity and viscosity based on our current study include but
are not limited to thymine, adenine, riboflavin, thiamine, and
tryptamine since, in certain embodiments, several effective
excipients have aromatic character (with heterocylicity enhancing
their turbidity mitigation property) and an ability to act as a
hydrogen bond donor and/or a hydrogen bond acceptor.
Osmolality Measurement
[0519] The osmolality of some samples from the screenings above was
measured and compared to the control using the procedure
illustrated in Example 1a. The results are presented in Table 6a,
below.
[0520] The molar increase in solution osmolality compared to the
control was expected to equal the molar amount of excipient added,
since no ionic dissociation was expected. For example, adding 73.4
mmol/kg of L-Tryptophan (which equals to 73.4 mOsm/kg assuming the
density is 1 kg/L) to a control solution of 431 mOsm/kg, the
expected increase in osmolality of the solution would be 73.4
mOsm/kg (which equals to 504.4 mOsm/kg total solution osmolality).
As illustrated in Table 6a, both L-tryptophan and niacinamide
showed an increase in osmolality less than expected at 26 mOsm/kg,
and 66 mOsm/kg rather than at 73.4 mOsm/kg and 122.8 mOsm/kg,
respectively compared to the control. Phenylalanine showed an
increase in osmolality as expected, while histidine showed an
increase in osmolality more than expected at 119 mOsm/kg.
TABLE-US-00008 TABLE 6a Effect of various excipients on osmolality
for ISIS 426115 at 220 mg/mL Expected Measured Excipient Excipient
Expected Measured Osmolality Osmolality Conc. Conc. Osmolality
Osmolality Increase Increase Excipient (%, w/v) (mmol/kg)*
(mOsm/kg) (mOsm/kg) (mOsm/kg) (mOsm/kg) Control 0 0.0 431 431 0 0
L-Tryptophan 1.5 73.4 504.4 457 73.4 26 Niacinamide 1.5 122.8 553.8
497 122.8 66 L-Phenylalanine 1.5 90.8 521.8 522 90.8 91 L-Histidine
1.5 96.7 527.7 550 96.7 119 *assuming density = 1 kg/L
Example 4
Evaluation of Properties of Effective Turbidity and Viscosity
Comitigator Excipients for ISIS NO. 426115
[0521] The properties of excipients which can effectively
comitigate turbidity and viscosity of ISIS NO. 426115 were
investigated. Several excipients in Example 2 were selected and
screened using the same method as described previously. The
properties that were examined included, but not limited to
aromaticity, homocyclic vs. heterocyclic aromatic rings, and the
number of hydrogen bond donor and/or acceptor.
[0522] The ASO samples were prepared in the same manner as
described in Example 3. To an aqueous solution of ISIS NO. 426115
at a concentration of 220 mg/mL, the excipient was added at the
percentage (%, w/v) as indicated in Table 7. The solution was
frozen at -20.degree. C., thawed to 5.degree. C. and subjected to
turbidity and viscosity evaluation. Turbidity and viscosity were
analyzed and compared to a control by visual inspection. For
turbidity evaluation, a scoring format of 0 to 3 was employed with
0 being visually clear; 1 being less turbid than a control but not
clear; 2 approximately the same turbidity as a control; and 3 being
more turbid than a control. A solution of ISIS NO. 426115 at a
concentration of 220 mg/mL at pH 8 in only water was used as the
control. The results are presented in Table 7.
[0523] As illustrated, ascorbic acid reduced turbidity but not
viscosity compared to the control, while L-phenylalanine was able
to reduce both turbidity and viscosity. Similarly, pyrazinamide was
effective for co-mitigation at 0.5% (w/v). Benzamide was effective
for mitigating turbidity but not for viscosity at 2% (w/v), while
nicotinamide was able to co-mitigate turbidity and viscosity at the
same concentration. Phenol was unable to mitigate turbidity and
viscosity, while hydroxypyridine was able to co-mitigate. Thus, in
certain embodiments, heterocyclicity with increasing number of
non-carbon substituents such as nitrogen and oxygen, seem to
improve turbidity reduction.
[0524] Both phenol and catechol were ineffective in reducing
turbidity at 0.5% (w/v). Phenol appeared to increase viscosity
while catechol had no effect. Pyridine was ineffective for both
turbidity and viscosity reduction, while hydroxypyridine was
effective for co-mitigation. These results suggest that excipients
with more hydrogen bond donors and/or acceptors can be effective at
turbidity and viscosity co-mitigation.
[0525] In certain embodiments, some excipients effective at
decreasing turbidity and/or viscosity have heterocyclic or
homocyclic aromatic character. In certain embodiments, some
excipients effective at decreasing turbidity and/or viscosity are
heterocyclic and nonaromatic, for example, ascorbic acid as shown
in Table 7.
[0526] The results from Table 7 suggest that in certain
embodiments, excipients that have heterocyclic aromatic or
nonaromatic character and contain hydrogen-bond donor(s) and/or
hydrogen-bond acceptor(s) mitigate both turbidity and
viscosity.
[0527] In certain embodiments, the properties given in this example
are consistent with single excipients that are effective at
mitigating both turbidity and viscosity, for example, compounds
that resemble nucleobases, and heterocyclic compounds that possess
both hydrogen bond donors and acceptors. In certain embodiments,
aromatic ring character appears to provide a benefit based on the
planar nature that facilitates: (i) positioning hydrogen bonds at a
low energy state and (ii) interference of base stacking.
TABLE-US-00009 TABLE 7 Effect of excipients in mitigating turbidity
and viscosity of ISIS NO. 426115 Viscosity Compared to Turbidity
Control Concentration (visual (visual Chemical Structure Excipient
(%, w/v) inspection) inspection) ##STR00005## Phenol 0.5 3
Increased ##STR00006## Catechol (1,2- Dihydroxybenzene) 0.5 3 No
notable difference ##STR00007## Hydroxypyridine 0.5 1 Decreased
##STR00008## Benzamide 2 3 Decreased ##STR00009## Nicotinamide 0.5
2 2 0 No notable difference Decreased ##STR00010## Pyrazinamide 0.5
1 Decreased ##STR00011## Pyridine 0.5 2 No notable difference
##STR00012## Ascorbic acid 0.5 0 No notable difference ##STR00013##
Phenylalanine 0.5 1 Slightly decreased
Example 5
Effect of Combining Singly Ineffective Excipients for Co-Mitigation
of ISIS NO. 426115
[0528] The effect of combining excipients which are ineffective by
themselves at mitigating both turbidity and viscosity of ISIS NO.
426115 was investigated. Several excipients in Example 2 were
selected and screened using the same method as described
previously. The ASO samples were prepared in the same manner as
described in Example 3. To an aqueous solution of ISIS NO. 426115
at a concentration of 220 mg/mL at pH 8 the excipient mixture was
added at the percentage (%, w/v) indicated in Table 8. The solution
was frozen at -20.degree. C., thawed to 5.degree. C. and subjected
to turbidity and viscosity evaluation. Turbidity and viscosity were
analyzed and compared to a control by visual inspection. For
turbidity evaluation, a scoring format of 0 to 3 was employed with
0 being visually clear; 1 being less turbid than a control but not
clear; 2 approximately the same turbidity as a control; and 3 being
more turbid than a control. A solution of ISIS NO. 426115 at a
concentration of 220 mg/mL at pH 8 in only water was used as the
control. The results are presented in Table 8.
[0529] In certain embodiments, the chemical properties listed
previously namely (i) aromatic homocyclicity or heterocyclicity and
(ii) the ability to act as a hydrogen bond donor and/or acceptor do
not have to be consolidated into one excipient or compound. In
certain embodiments, it was discovered from our finding that a
mixture of two excipients, which satisfies the two properties as a
whole, can also be effective in mitigating both turbidity and
viscosity. In certain embodiments, this was demonstrated by the
mixture of 0.5% (w/v) mannitol and 0.5% (w/v) pyridine. Mannitol is
a saturated linear carbon chain with hydroxyl groups, therefore
possessing both hydrogen bond donors and acceptors; whereas
pyridine is a heterocyclic aromatic compound lacking a hydrogen
bond donor but has a hydrogen bond acceptor from its nitrogen atom.
This mixture visually reduced both turbidity and viscosity compared
to the control. In contrast, mannitol and pyridine by itself are
ineffective at mitigating either turbidity or viscosity. In support
of our observation for chemical properties of the excipients, it
was found that mannitol and phenol mixture lacking a heterocyclic
ring showed no significant mitigation in turbidity (Table 5).
[0530] The results from Table 8 suggest that in certain
embodiments, other mixtures may potentially have such synergistic
effects for both turbidity and viscosity mitigation as demonstrated
by D-mannitol and pyridine mixture. Such mixtures include, but are
not limited to, dextran and pyrimidine mixture or ascorbic acid and
phenanthroline mixture.
TABLE-US-00010 TABLE 8 Effect of excipient mixtures in mitigating
turbidity and viscosity of ISIS NO. 426115 Viscosity Compared to
Turbidity Control Concentration (visual (visual Chemical Structure
Excipient (%, w/v) inspection) inspection) ##STR00014## Pyridine
0.5 2 No notable difference ##STR00015## D-Mannitol 2 5 10 15 2 No
notable difference See boxes above D-Mannitol + 0.5 + 0.5 1
Decreased Pyridine
Example 6
Effect of Temperature and Time Dependency on ISIS NO. 426115
Turbidity
[0531] The effect of temperature and time dependency on the
turbidity profile of freeze-thawed ISIS NO. 426115 at 220 mg/mL as
it warmed up from 5.degree. C. to room temperature was evaluated.
The experiment was performed in the following manner.
Materials
[0532] The materials used to carry out the experiment included a
solution of ISIS NO. 426115 in water at a concentration of 220
mg/mL and 150 mg/mL; a Hach 2100AN Nephelometer; a StablCal
Calibration Set for 2100AN Nephelometer, a StablCal Formazin
Turbidity Standard at 1000 NTU; a VWR Precision 0.01.degree.
thermometer and Gerresheimer 13.times.100 mm glass culture
tubes.
Method
[0533] A 4 mL sample of ISIS NO. 426115 at concentration of 220
mg/mL or 150 mg/mL was pipetted into a sample tube, capped and
parafilmed at the cap joint. Both samples were allowed to freeze in
a -20.degree. C. storage and then moved to a 2-8.degree. C. storage
to thaw.
Turbidity Measurement
[0534] The turbidimeter was calibrated using Formazin turbidity
calibration set. The 220 mg/mL sample from 2-8.degree. C. storage
was removed and allowed to stand at room temperature. The
temperature of the sample was monitored until it reached
approximately 13.degree. C. or until condensation does not recur
upon wiping of the tube surface and the temperature was recorded.
The tube was then inserted in the nephelometer using a tube adaptor
and turbidity measurement was taken every 2 minutes until turbidity
value does not change by more than 0.1 NTU.
Temperature Profile
[0535] The 150 mg/mL sample from 2-8.degree. C. storage was removed
and allowed to stand at room temperature. The sample tube was
inserted into the nephelometer (which does not need to be turned
on). Condensation was not accounted for since it did not
significantly affect the temperature profile. A temperature probe
was inserted into the sample tube and the temperature was recorded
over time.
[0536] Note:
[0537] The temperature profiles of 150 mg/mL and 220 mg/mL samples
of ISIS NO. 426115 has been shown to be identical in previous
studies.
Sample Tube Turbidity Correction Factor (CF)
[0538] Since the 13-mm sample tubes used differ from the
nephelometer's recommended set, a different path length of light
applies for taking the sample turbidity. As such a CF was required
to convert the readings into accurate NTU (Nephelometric Turbidity
Units) values.
[0539] Formazin turbidity standards were volumetrically prepared by
serial dilution of 1000 NTU standard using sterile water for
injection. The standard values are 0, 100, 250, 500, 750 or 1000
NTU. The standard solutions were filled into 13-mm sample tubes and
turbidity was measured. The standard values were plotted over
measured turbidity values and a linear trend over the data points
were fitted to obtain the slope. The slope is the CF for 13-mm
sample tubes. The CF was used to convert measured values from 220
mg/mL ISIS NO. 426115 sample readings into actual NTU values.
Data Analysis and Results
[0540] The temperature and turbidity measurements over time were
tabulated. The temperature profile was plotted by designating time
zero to be at the same starting temperature as the turbidity
sample. Overlaid the turbidity and temperature profiles over time
and matched up the turbidity value with a temperature value along
the time profile to generate an approximate turbidity vs
temperature profile (FIG. 1).
[0541] The results showed that the CF for the tubes is the slope of
the plot which is 1.8148 (FIG. 1). A decrease in turbidity appeared
to lag behind the increase in temperature as shown in FIGS. 2 and
3. Turbidity remained at approximately 1800 NTU for the first 10
minutes when the temperature had increased by about 5 degrees. The
solution temperature reached room temperature after approximately
30 minutes, however visually the turbidity did not completely
dissipate until approximately 50 minutes, when it reached 20 NTU.
FIG. 4 shows that 20 NTU is the approximate lower limit of visible
turbidity in a solution.
[0542] In certain embodiments, this observation is indicative of a
certain amount of thermal energy being required to break up the
turbid species, which has been hypothesized to be self associated
oligonucleotides. In addition, in certain embodiments, as shown in
FIG. 3, there is an apparent melting temperature existing at
approximately 19.degree. C. At temperatures below that, the turbid
species persist, and at temperatures above that they
dissociate.
Example 7
Viscosity Evaluation for ISIS NO. 104838
[0543] Antisense oligonucleotide Isis No. 104838 was selected for
viscosity evaluation. The ASO and its motif are described in Table
9. The internucleoside linkages throughout each modified
oligonucleotide are phosphorothioate linkages (P.dbd.S).
Nucleosides without a subscript are
.beta.-D-2'-deoxyribonucleosides. Nucleosides followed by a
subscript "e" indicate 2'-O-methoxyethyl (MOE) modified
nucleosides. An "N" indicates a U, T, C, .sup.meC, G or A
nucleoside.
[0544] Several excipients in Example 2 were selected and screened
for their effect in mitigating the viscosity of ISIS NO. 104838.
The viscosity experiment was performed in the same manner as
described in Example 2. A concentrated stock solution of ASO in
water was diluted using solid excipient or a concentrated stock
solution of excipient. The dilution was at a concentration of 200
mg/mL of ISIS NO. 104838 at pH 8. Viscosity at 25.degree. C. was
measured using the Malvern Instruments Bohlin CVO 100 rheometer as
described in Example 1. The results for viscosity were obtained and
normalized to the results of the control when the diluent was only
water. The range of the control viscosity (105.1-113.6 cP) provided
in Table 10 was generated from various independent studies and
therefore, normalized viscosity was calculated based on the control
value obtained from the same study. The results are shown
below.
[0545] As illustrated, several excipients at various concentrations
demonstrated a desirable reduction in viscosity with a normalized
viscosity of below 1.00 as compared to the control.
TABLE-US-00011 TABLE 9 Antisense Oligonucleotide Isis No. 104838
Selected for Viscosity Evaluation Isis SEQ ID No. Composition (5'
to 3') Motif No. 104838
N.sub.eN.sub.eN.sub.eN.sub.eN.sub.eNNNNNNNNNNN.sub.eN.sub.eN.sub.eN-
.sub.eN.sub.e 5405 26
TABLE-US-00012 TABLE 10 Effect of various excipients on viscosity
of ISIS NO. 104838 at 200 mg/mL at 25.degree. C. Excipient
Viscosity Normalized Excipient Conc. (%, w/v) (cP) Viscosity* None
(control) 0 105.1-113.6 1.00 PEG.sub.4600 5 194.8 1.85
(2-Hydroxypropyl)-.beta.- 5 178.2 1.60 cyclodextrin PEG.sub.600 5
147.2 1.40 Dextran 1500 5 130.9 1.18 D-Mannitol 5 115.5 1.10
Sucrose 5 122.1 1.10 Niacin (Vitamin B.sub.3) 5 123.8 1.09 sodium
salt Tween 80 0.5 115.1 1.04 Thymidine 5 94.75 0.86 Uridine 5 89.05
0.81 Cytosine 1 81.89 0.74 L-Tryptophan 1.1 82.69 0.73 Benzyl
alcohol 0.9 82.09 0.72 3.8 44.08 0.39 Cytidine 5 67.64 0.61
m-Cresol 2 62.40 0.55 Nicotinamide/Niacinamide 5 42.46 0.38
*Normalized to ISIS NO. 104838 concentration of 200 mg/mL in
water
Example 8
Combining Singly Effective Excipients for Enhanced Turbidity and
Viscosity Co-Mitigation of ISIS NO. 426115
[0546] The effect of combining effective excipients was evaluated
for co-mitigation of turbidity and viscosity of ISIS NO. 426115
beyond what each excipient could perform individually. Tryptophan,
niacinamide (nicotinamide), L-phenylalanine and L-histidine were
selected for a factorial mixture design-of-experiment (DOE) study,
where the total of all excipient mixture combinations equal 60 mM.
The ratios are presented in Table 11, below.
[0547] Stock solutions of 220 mg/mL ISIS NO. 426115 containing 60
mM single excipients were prepared by adding solid ASO and
excipient powders, diluting with water, and then pH-adjusting to
target pH 7-8 as necessary. Subsequently, the stock solutions were
combined in relevant ratios to total 1 mL solutions, and filled
into 2-mL clean glass vials which were stoppered and sealed, to
produce all 24 samples. Control solution of 220 mg/mL ISIS NO.
426115 without excipient was prepared separately using similar
method. These samples were frozen and thawed in two cycles, by
first freezing at -20.degree. C. and thawing at 5.degree. C., and
then the process was repeated. Turbidity was analyzed after the two
freeze-thaw cycles and compared to the control by visual
inspection. A scoring format of 0 to 2 was employed with 0 being
visually clear; 0.5 being very slightly turbid, 1 being slightly
turbid; and 2 being as turbid as the control. Viscosity was
measured at 25.degree. C. using the Rheosense m-VROC system as
described in Example 1. The results for viscosity were obtained and
normalized to the results of the control when the diluent was only
water. The results presented in Table 11 were then entered into a
DOE software (Design-Expert.RTM. 8) to determine statistical
significance of the data and render a response surface showing the
most ideal mixture for viscosity reduction.
[0548] As illustrated, all combinations of excipients at the ratios
tested mitigated turbidity with the score of 1 or lower and also
mitigated viscosity to less than 65% of the control. Design-Expert
ANOVA analysis determined that the results possess adequate
signal-to-noise ratio, and could be used to generate statistically
significant models. Additionally, a viscosity response surface
generated by the software showed that 50/50 combination of
L-phenylalanine and L-histidine proved most effective at lowering
viscosity.
TABLE-US-00013 TABLE 11 Effect of excipient mixtures in mitigating
turbidity and viscosity of ISIS NO. 426115 at 25.degree. C.
Nicotinamide Turbidity Sample L-tryptophan (Niacinamide)
L-phenylalanine L-histidine (visual Viscosity Normalized Type (mM)
(mM) (mM) (mM) inspection) (cP) Viscosity Control 0 0 0 0 2 66.841*
1.0 Test 60 0 0 0 0 38.855 0.58 Samples 0 60 0 0 0 35.254 0.53 0 0
60 0 0 34.296 0.51 0 0 0 60 1 34.863 0.52 30 30 0 0 0 36 0.54 30 0
30 0 0 33.306 0.50 30 0 0 30 0 38.378 0.57 0 30 30 0 0 35.216 0.53
0 30 0 30 0.5 35.61 0.53 0 0 30 30 0.5 29.147 0.44 20 20 20 0 0
35.863 0.54 20 20 0 20 0 36.114 0.54 20 0 20 20 0 32.279 0.48 0 20
20 20 0.5 32.829 0.49 15 15 15 15 0 34.739 0.52 38 8 8 8 0 37.625
0.56 8 38 8 8 0 34.778 0.52 8 8 38 8 0 26.026 0.39 8 8 8 38 0.5
35.13 0.53 60 0 0 0 0 41.662 0.62 0 60 0 0 0 36.625 0.55 0 0 60 0 0
34.082 0.51 0 0 0 60 1 35.56 0.53 30 30 0 0 0 37.494 0.56 *Measured
by taking the average from three independent studies
Example 10
Turbidity Evaluation for ISIS 442245 in the Presence of Osmolality
Adjusters
[0549] Another ASO sequence, ISIS 442245 was used for turbidity
evaluation. ISIS 442245 and its motif are described in Table 12.
The internucleoside linkages throughout each modified
oligonucleotide are phosphorothioate linkages (P.dbd.S).
Nucleosides without a subscript are D-D-2'-deoxyribonucleosides.
Nucleosides followed by a subscript "g" indicate 3'-F-HNA modified
nucleosides. An "N" indicates a U, T, C, .sup.meC, G or A
nucleoside.
[0550] Osmolality adjusters for ISIS 442245 formulated at low ASO
concentration can comprise salts such as NaCl, or sugars such as
mannitol, or other substances. The effect of adding 0.6% (w/v) NaCl
to 55 mg/mL ISIS 442245 was compared to adding 0.4% (w/v) mannitol.
A buffered aqueous solution of ISIS 442245 at a concentration of 55
mg/mL was prepared by adding solid drug substance powder to 5 mM
phosphate buffer (pH 7-8). To this stock solution, either 0.4%
(w/v) mannitol or 0.6% NaCl (w/v) was added. A control solution was
also prepared without osmolality adjusters.
[0551] The solutions were frozen at -20.degree. C., thawed to
5.degree. C. Turbidity was evaluated by visual inspection and noted
as either "clear" or "turbid". The results are presented in Table
13, below.
[0552] As illustrated, only ISIS 442245 with NaCl solution became
turbid after freeze-thaw. The solution containing mannitol remained
clear, which suggests that mannitol or other sugars such as
glucose, sucrose, or fructose, may likely be suitable for use as
osmolality adjusters replacing NaCl for turbidity mitigation. Table
12
TABLE-US-00014 TABLE 12 Antisense Oligonucleotide Isis 442245
Selected for Turbidity Evaluation Isis SEQ ID No. Composition (5'
to 3') Motif No. 442245 N.sub.gN.sub.gNNNNNNNN.sub.gN.sub.g 2-10-2
27
TABLE-US-00015 TABLE 13 Turbidity Evaluation of ISIS 442245 in the
Presence or Absence of Osmolality Adjusters Samples (all buffered
with Osmolality Turbidity (visual 5 mM phosphate buffer, pH 7-8)
(mOsm/kg) inspection) Control: 55 mg/mL 442245 274 Clear 55 mg/mL
442245 + 0.4% mannitol 505 Clear 55 mg/mL 442245 + 0.6% NaCl 488
Turbid
Example 11
[0553] Turbidity Evaluation for ISIS 442245 with Excipients
[0554] Excipients such as tryptophan, niacinamide (nicotinamide)
and phenylalanine were selected and screened for their effect in
mitigating the turbidity of ISIS 442245 formulated with 5 mM
phosphate buffer (pH 7-8) and 0.6% NaCl. The turbidity experiment
was performed in the same manner as described in Example 2. To an
aqueous solution of ISIS 442245 at a concentration of 55 mg/mL the
excipients were added at the percentage (%, w/v) indicated in Table
14. L-Tryptophan and L-Phenylalanine concentrations tested were
limited by their solubility. The solutions were frozen at
-20.degree. C., thawed to 5.degree. C. and subjected to turbidity
evaluation. Turbidity was analyzed and compared to a control by
visual inspection using a scoring format of 0 to 3 with 0 being
visually clear; 1 being less turbid than a control but not clear; 2
approximately the same turbidity as a control; and 3 being more
turbid than a control. A solution of ISIS 442245 at a concentration
of 55 mg/mL with 5 mM phosphate buffer (pH 7-8) and 0.6% NaCl is
used as the control. The results are presented in Table 14.
[0555] As illustrated, the control appeared to be a turbid gel at
5.degree. C. L-phenylalanine was shown to be effective in
co-mitigating turbidity and viscosity at approximately 200 mM when
the solution was stored for several days in 5.degree. C. Upon
freeze-thaw, the saturated L-phenylalanine precipitated and
therefore caused the solution to become more turbid than the
control. L-Tryptophan was also effective at turbidity mitigation at
50 mM after freeze-thaw, and would likely be a successful
co-mitigator if it had been more soluble. Further, niacinamide was
shown to co-mitigate turbidity and viscosity successfully at 400 mM
while remaining stable after freeze-thaw. The results demonstrate
that these excipients can be effective at mitigating turbidity
and/or viscosity and can be used as co-mitigators for
oligonucleotide sequences other than the ones exemplified
herein.
TABLE-US-00016 TABLE 14 Effect of various excipients on turbidity
and viscosity of ISIS NO. 442245 at 55 mg/mL formulated with 5 mM
buffer pH 7-8 and 0.6% NaCl Turbidity after Turbidity after
Viscosity Excipient Excipient 3 days at 5.degree. C. freeze-thaw at
5.degree. C. Viscosity Conc. Conc. (visual (visual (visual at
5.degree. C. Excipient (% w/v) (mM) inspection) inspection)
inspection) (cP) Control 0 0 2 2 N/A See note** Niacinamide 4.9 400
0 0 Decreased 5 L-Tryptophan 1.0 50 2 1 No notable See note**
difference L-Phenylalanine 3.3* 200* 0 3 Decreased 31 2.5 150 0 2
Decreased NT *Due to excipient saturation, some excipients
precipitated at this concentration **Viscosity traces were erratic,
showing some maximum values above 100 cP and minima around 0 cP.
This is likely due to inhomogeneous sample gelling. NT = not tested
Sequence CWU 1
1
27120DNAArtificial SequenceSynthetic oligonucleotide 1acggcattgg
tgcacagttt 20220DNAArtificial SequenceSynthetic oligonucleotide
2tcttggttac atgaaatccc 20320DNAArtificial SequenceSynthetic
oligonucleotide 3tgctccgttg gtgcttgttc 20420DNAArtificial
SequenceSynthetic oligonucleotide 4ccagctcaac ccttctttaa
20520DNAArtificial SequenceSynthetic oligonucleotide 5aatggtttat
tccatggcca 20617DNAArtificial SequenceSynthetic oligonucleotide
6ggttcccgag gtgccca 17720DNAArtificial SequenceSynthetic
oligonucleotide 7tcacagaatt atcagcagta 20816DNAArtificial
SequenceSynthetic oligonucleotide 8ggacacccac gccccc
16918DNAArtificial SequenceSynthetic oligonucleotide 9tcactttcat
aatgctgg 181020DNAArtificial SequenceSynthetic oligonucleotide
10gcacactcag caggaccccc 201120DNAArtificial SequenceSynthetic
oligonucleotide 11gcctcagtct gcttcgcacc 201220DNAArtificial
SequenceSynthetic oligonucleotide 12agcatagtta acgagctccc
201320DNAArtificial SequenceSynthetic oligonucleotide 13agcttcttgt
ccagctttat 201420DNAArtificial SequenceSynthetic oligonucleotide
14gcagccatgg tgatcaggag 201516DNAArtificial SequenceSynthetic
oligonucleotide 15ctatttggat gtcagc 161620DNAArtificial
SequenceSynthetic oligonucleotide 16tgtcatattc ctggatcctt
201720DNAArtificial SequenceSynthetic oligonucleotide 17ccgtcgccct
tcagcacgca 201820DNAArtificial SequenceSynthetic oligonucleotide
18tcagggcatt ctttccattc 201921DNAArtificial SequenceSynthetic
oligonucleotide 19cagcagcaga gtcttcatca t 212020DNAArtificial
SequenceSynthetic oligonucleotide 20gggacgcggc gctcggtcat
202121DNAArtificial SequenceSynthetic oligonucleotide 21gcgtttgctc
ttcttcttgc g 212220DNAArtificial SequenceSynthetic oligonucleotide
22gcccaagctg gcatccgtca 202320DNAArtificial SequenceSynthetic
oligonucleotide 23ctgagtctgt tttccattct 202420DNAArtificial
SequenceSynthetic oligonucleotide 24gtttgacatg gcacaatgtt
202520DNAArtificial SequenceSynthetic oligonucleotide 25tcccgcctgt
gacatgcatt 202620DNAArtificial SequenceSynthetic
oligonucleotidemisc_feature(1)..(20)n = A,T,C or G 26nnnnnnnnnn
nnnnnnnnnn 202714DNAArtificial SequenceSynthetic
oligonucleotidemisc_feature(1)..(14)n = A,T,C or G 27nnnnnnnnnn
nnnn 14
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