U.S. patent application number 10/980002 was filed with the patent office on 2005-09-01 for modulation of sglt2 expression.
Invention is credited to Freier, Susan M., Leedom, Thomas A., Monia, Brett P., Siwkowski, Andrew M., Wancewicz, Edward, Watts, Lynnetta.
Application Number | 20050191653 10/980002 |
Document ID | / |
Family ID | 34556290 |
Filed Date | 2005-09-01 |
United States Patent
Application |
20050191653 |
Kind Code |
A1 |
Freier, Susan M. ; et
al. |
September 1, 2005 |
Modulation of SGLT2 expression
Abstract
Compounds, compositions and methods are provided for modulating
the expression of SGLT2. The compositions comprise
oligonucleotides, targeted to nucleic acid encoding SGLT2. Methods
of using these compounds for modulation of SGLT2 expression and for
diagnosis and treatment of diseases and conditions associated with
expression of SGLT2 are provided.
Inventors: |
Freier, Susan M.; (San
Diego, CA) ; Wancewicz, Edward; (Poway, CA) ;
Monia, Brett P.; (Encinitas, CA) ; Siwkowski, Andrew
M.; (Carlsbad, CA) ; Watts, Lynnetta;
(Carlsbad, CA) ; Leedom, Thomas A.; (Fallbrook,
CA) |
Correspondence
Address: |
COZEN O'CONNOR, P.C.
1900 MARKET STREET
PHILADELPHIA
PA
19103-3508
US
|
Family ID: |
34556290 |
Appl. No.: |
10/980002 |
Filed: |
November 2, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60517334 |
Nov 3, 2003 |
|
|
|
Current U.S.
Class: |
435/6.11 ;
514/44A; 536/23.1 |
Current CPC
Class: |
C12N 2500/40 20130101;
C12N 2310/321 20130101; A61P 3/00 20180101; A61P 3/10 20180101;
C07H 21/02 20130101; C12N 2310/11 20130101; A61P 9/12 20180101;
A61P 3/04 20180101; C07H 21/04 20130101; C12N 15/1138 20130101;
A61P 9/00 20180101; C12Q 2600/158 20130101; C12N 2310/315 20130101;
A61P 35/00 20180101; C12N 2320/32 20130101; C12N 15/1136 20130101;
C12N 2310/341 20130101; C12N 2310/321 20130101; C12N 15/111
20130101; C12N 2310/3527 20130101; C12N 2310/3525 20130101; C12N
2310/3341 20130101; A61P 3/06 20180101; A61P 43/00 20180101; A61P
3/08 20180101; A61K 31/70 20130101; C12N 2310/321 20130101; C12Q
1/6886 20130101; C12N 2310/346 20130101 |
Class at
Publication: |
435/006 ;
514/044; 536/023.1 |
International
Class: |
C12Q 001/68; C07H
021/02; A61K 048/00 |
Claims
What is claimed is:
1. An oligomeric compound 8 to 80 nucleobases in length targeted to
a nucleic acid molecule encoding SGLT2, wherein the compound is at
least 70% complementary to the nucleic acid molecule encoding
SGLT2, and wherein the compound inhibits the expression of SGLT2
mRNA by at least 10%.
2. The compound of claim 1 comprising 10 to 50 nucleobases in
length.
3. The compound of claim 2 comprising 13 to 30 nucleobases in
length.
4. The compound of claim 3 comprising 15 to 25 nucleobases in
length.
5. The compound of claim 4 comprising 18 to 22 nucleobases in
length.
6. The compound of claim 1 comprising an oligonucleotide.
7. The compound of claim 6 comprising a DNA oligonucleotide.
8. The compound of claim 6 comprising an RNA oligonucleotide.
9. The compound of claim 6 comprising a chimeric
oligonucleotide.
10. The compound of claim 6 wherein at least a portion of the
compound hybridizes with RNA to form an oligonucleotide-RNA
duplex.
11. The compound of claim 1 comprising at least 80% complementarity
with the nucleic acid molecule encoding SGLT2.
12. The compound of claim 1 comprising at least 90% complementarity
with the nucleic acid molecule encoding SGLT2.
13. The compound of claim 1 comprising at least 95% complementarity
with the nucleic acid molecule encoding SGLT2.
14. The compound of claim 1 comprising at least 99% complementarity
with the nucleic acid molecule encoding SGLT2.
15. The compound of claim 1 comprising at least one modified
internucleoside linkage, sugar moiety, or nucleobase.
16. The compound of claim 1 comprising at least one
2'-O-methoxyethyl sugar moiety.
17. The compound of claim 1 comprising at least one
phosphorothioate internucleoside linkage.
18. The compound of claim 1 wherein at least one cytosine is a
5-methylcytosine.
19. A method of inhibiting the expression of SGLT2 in a cell or
tissue comprising contacting the cell or tissue with the compound
of claim 1 so that expression of SGLT2 is inhibited.
20. A method of screening for a modulator of SGLT2 comprising:
contacting a target segment of a nucleic acid molecule encoding
SGLT2 with one or more candidate modulators of SGLT2; and
identifying one or more modulators of SGLT2 expression which
modulate the expression of SGLT2.
21. The method of claim 20 wherein the modulator of SGLT2
expression comprises an oligonucleotide, an antisense
oligonucleotide, a DNA oligonucleotide, an RNA oligonucleotide, an
RNA oligonucleotide having at least a portion of said RNA
oligonucleotide capable of hybridizing with RNA to form an
oligonucleotide-RNA duplex, or a chimeric oligonucleotide.
22. A method for identifying a disease state comprising identifying
the presence of SGLT2 in a sample using at least one primer
comprising SEQ ID NO: 5 or SEQ ID NO: 6, or a probe comprising SEQ
ID NO: 7.
23. A kit or assay device comprising the compound of claim 1.
24. A method of treating an animal having a disease or condition
associated with SGLT2 comprising administering to the animal a
therapeutically or prophylactically effective amount of the
compound of claim 1 so that expression of SGLT2 is inhibited.
25. The method of claim 24 wherein the disease or condition is a
hyperproliferative or metabolic disorder.
26. The compound of claim 1 comprising at least an 8-nucleobase
portion of SEQ ID NO: 19, 20, 21, 22, 23, 26, 27, 28, 29, 32, 33,
34, 35, 37, 38, 39, 40, 41, 43, 47, 48, 49, 50, 51, 52, 53, 54, 55,
56, 57, 58, 59, 61, 62, 63, 64, 66, 67, 68, 69, 70, 73, 74, 77, 79,
80, 82, 85, 88, 90, 91, 92, 93, 94, or 95.
27. The compound of claim 26 wherein the compound comprises SEQ ID
NO: 19, 20, 21, 22, 23, 26, 27, 28, 29, 32, 33, 34, 35, 37, 38, 39,
40, 41, 43, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61,
62, 63, 64, 66, 67, 68, 69, 70, 73, 74, 77, 79, 80, 82, 85, 88, 90,
91, 92, 93, 94, or 95.
28. The compound of claim 1 wherein the compound comprises at least
an 8-nucleobase portion of SEQ ID NO: 99, 105, 107, 109, 110, 117,
121, 124, 125, 126, 130, 131, 132, 136, 138, 139, 142, 147, 148,
150, 153, 155, 162, 173, or 175.
29. The compound of claim 28 wherein the compound comprises SEQ ID
NO: 99, 105, 107, 109, 110, 117, 121, 124, 125, 126, 130, 131, 132,
136, 138, 139, 142, 147, 148, 150, 153, 155, 162, 173, or 175.
30. The compound of claim 1 wherein the compound comprises an
antisense nucleic acid molecule that is specifically hybridizable
with a 5'-untranslated region of a nucleic acid molecule encoding
SGLT2.
31. The compound of claim 1 wherein the compound comprises an
antisense nucleic acid molecule that is specifically hybridizable
with a start region of a nucleic acid molecule encoding SGLT2.
32. The compound of claim 1 wherein the compound comprises an
antisense nucleic acid molecule that is specifically hybridizable
with a coding region of a nucleic acid molecule encoding SGLT2.
33. The compound of claim 1 wherein the antisense compound
comprises an antisense nucleic acid molecule that is specifically
hybridizable with a stop region of a nucleic acid molecule encoding
SGLT2.
34. The compound of claim 1 wherein the antisense compound
comprises an antisense nucleic acid molecule that is specifically
hybridizable with a 3'-untranslated region of a nucleic acid
molecule encoding SGLT2.
35. The compound of claim 1 which comprises a first region
consisting of at least 5 contiguous 2'-deoxy.nucleosides flanked by
a second region and a third region, wherein each of the second and
third regions, independently, comprises at least one
2'-O-methoxyethyl nucleoside, and wherein the internucleoside
linkages of the first region are phosphorothioate linkages and the
internucleoside linkages of the second and third regions are
phosphodiester linkages.
36. A method of inhibiting the expression of SGLT2 in a kidney cell
or kidney tissue comprising contacting the kidney cell or kidney
tissue with the compound of claim 35.
37. A method of enhancing inhibition of expression of SGLT2 in a
kidney cell or kidney tissue comprising contacting the kidney cell
or kidney tissue with the compound of claim 35 so that expression
of SGLT2 is inhibited.
38. The method of claim 37 wherein the compound comprises SEQ ID
NO: 106, 255, or 256.
39. A method of preventing or delaying the onset of a disease or
condition in an animal comprising administering to the animal an
effective amount of the compound of claim 35 so that expression of
SGLT2 is inhibited, wherein the disease or condition is associated
with expression of SGLT2 in the kidney.
40. The method of claim 39 wherein the compound comprises SEQ ID
NO: 106, 255, or 256.
41. A method of preventing or delaying the onset of type 2 diabetes
in an animal comprising administering to the animal the compound of
claim 35 so that expression of SGLT2 is inhibited.
42. The method of claim 41 wherein said animal is a primate or a
rodent.
43. The method of claim 41 wherein the compound comprises SEQ ID
NO: 106, 255, or 256.
44. A method of preventing or delaying the onset of an increase in
blood glucose level in an animal comprising administering to the
animal the compound of claim 35 so that expression of SGLT2 is
inhibited.
45. The method of claim 44 wherein the animal is a primate or a
rodent.
46. The method of claim 44 wherein the blood glucose level is
plasma glucose level or serum glucose level.
47. The method of claim 44 wherein the animal is a diabetic
animal.
48. The method of claim 44 wherein the animal is insulin-resistant
as compared to a normal animal.
49. The method of claim 44 wherein the compound comprises SEQ ID
NO: 106, 255, or 256.
50. A method of decreasing blood glucose level in an animal
comprising administering to the animal the compound of claim 35 so
that expression of SGLT2 is inhibited.
51. The method of claim 50 wherein the animal is a primate or a
rodent.
52. The method of claim 50 wherein the blood glucose level is
plasma glucose level or serum glucose level.
53. The method of claim 50 wherein the animal is a diabetic
animal.
54. The method of claim 50 wherein the animal is insulin-resistant
as compared to a normal animal.
55. The method of claim 50 wherein the compound comprises SEQ ID
NO: 106, 255, or 256.
56. A method of enhancing inhibition of expression of SGLT2 in a
kidney cell or kidney tissue comprising contacting the cell or
tissue with an antisense compound comprising a first central region
comprising at least 5 contiguous 2'-deoxy nucleosides flanked by a
second 5' region and a third 3' region, wherein each of the second
and third regions, independently, comprises at least one
2'-O-methoxyethyl nucleoside, and wherein the internucleoside
linkages of the first region are phosphorothioate linkages and the
internucleoside linkages of the second and third regions are
phosphodiester linkages except one or both of the extreme 5'
linkage and the extreme 3' linkage are phosphorothioate linkages,
so that expression of the RNA target is inhibited.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority to U.S.
provisional patent application Ser. No. 60/517,334 filed Nov. 3,
2003, and the benefit of priority to U.S. application Ser. No.
10/946,498 filed Sep. 21, 2004, each of which is incorporated
herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention provides compositions and methods for
modulating the expression of SGLT2. In particular, this invention
relates to antisense compounds, particularly oligonucleotide
compounds, which, in some embodiments, hybridize with nucleic acid
molecules encoding SGLT2. Such compounds are shown herein to
modulate the expression of SGLT2.
BACKGROUND OF THE INVENTION
[0003] A fundamental component of energy metabolism is glucose
transport. The transport of glucose across cell membranes is
essential to metabolic processes, including the maintenance of a
relatively constant blood glucose concentration and the delivery of
glucose to peripheral tissues for storage and utilization. As cell
membranes are essentially impermeable to glucose, the movement of
glucose across membranes must be accomplished by protein
transporters (Brown, J. Inherit. Metab. Dis., 2000, 23,
237-246).
[0004] Mediated glucose transport occurs in two forms, secondary
active transport and facilitated transport. In cells where glucose
is rapidly metabolized, the concentration gradient across the
plasma membrane is used to drive facilitated transport, and an
active mechanism is not required. Secondary active transport of
glucose enables cells to transport glucose against a concentration
gradient. This mechanism involves cotransport of glucose and sodium
ions across the apical surface of the cells and the energy is
provided by the sodium gradient maintained by the sodium/potassium
ATPase in the basolateral membrane. Efflux of glucose from the
cells into the circulation is then mediated by a facilitative
transporter (Brown, J. Inherit. Metab. Dis., 2000, 23, 237-246;
Wright, Am. J. Physiol. Renal Physiol., 2001, 280, F10-18).
[0005] Secondary active transport of glucose operates in the
mucosal cells of the intestine and the proximal tubular cells of
the kidney and functions to ensure efficient uptake of dietary
glucose and minimal urinary loss. Plasma glucose is normally
filtered in the kidney in the glomerulus and actively reabsorbed in
the proximal tubule. Glucose is essentially completely reabsorbed
from the urine in the proximal tubule of the kidney through the
action of the sodium-glucose cotransporters (SGLTs) located in the
brush border membrane (BBM). Comparison of the glucose transport
properties of proximal tubule BBM vesicles prepared from the outer
cortex and the outer medulla of rabbit kidney revealed the presence
of two distinct sodium-coupled D-glucose transport systems. The
outer cortex preparation exhibited a low-affinity/high-capacity
activity (K.sub.m=6 mM), whereas the outer medulla displayed a
high-affinity/low-capacity activity (K.sub.m=0.35 mM) (Turner and
Moran, Am. J. Physiol. Endocrinol. Metab., 1982, 242, F406-414;
Wright, Am. J. Physiol. Renal Physiol., 2001, 280, F10-18). Further
characterization of the renal outer cortical BBM transport system
revealed a glucose to sodium coupling ratio of 1:1, whereas the
ratio is 2:1 in vesicles isolated from the outer medullary tissue
(Turner and Moran, J. Membr. Biol., 1982, 67, 73-80).
[0006] Isolation of nucleic acid molecules encoding SGLTs confirmed
the presence of multiple transport systems. A cDNA encoding human
SGLT2 (also known as solute carrier family 2, member 5,
Na-dependent glucose cotransporter 2 or SLC2A5) was identified in a
screen for sodium cotransporter-like sequences in a cDNA library
prepared from human kidney (Kanai et al., J. Clin. Invest., 1994,
93, 397-404; Wells et al., Am. J. Physiol. Endocrinol. Metab.,
1992, 263, F459-465). Human SGLT2 localizes to chromosome 16p11.2
(Wells et al., Genomics, 1993, 17, 787-789). Subsequent
investigations of human SGLT2 revealed that has functional
properties characteristic of a low-affinity, sodium-dependent
glucose cotransporter.
[0007] Studies of human SGLT2 injected into Xenopus oocytes
demonstrated that this protein mediates sodium-dependent transport
of D-glucose and .alpha.-methyl-D-glucopyranoside (.alpha.-MeGlc; a
glucose analog) with a K.sub.m value of 1.6 mM for .alpha.-MeGlc
and a sodium to glucose coupling ratio of 1:1 (Kanai et al., J.
Clin. Invest., 1994, 93, 397-404; You et al., J. Biol. Chem., 1995,
270, 29365-29371). This transport activity was suppressed by
phlorizin, a plant glycoside that binds to the glucose site but is
not transported and thus inhibits SGLTs (You et al., J. Biol.
Chem., 1995, 270, 29365-29371). These findings indicated that SGLT2
is responsible for the low-affinity transport observed in BBM
vesicle preparations from rabbit kidney outer cortex.
[0008] The tissue distribution of SGLT2 further suggested that this
cotransporter is the kidney low-affinity glucose transporter.
Northern blotting revealed that human SGLT2 is primarily expressed
in kidney, and in situ hybridization of a human SGLT2 probe to rat
kidney tissue demonstrated that SGLT2 is expressed in the proximal
tubule S1 segments in the outer cortex (Kanai et al., J. Clin.
Invest., 1994, 93, 397-404; Wells et al., Am. J. Physiol.
Endocrinol. Metab., 1992, 263, F459-465). This localization pattern
distinguishes SGLT2 from SGLT1, the high-affinity/low-capacity
sodium/glucose transporter that is expressed in the proximal tubule
S3 segments of the outer medulla, where it is appropriately
positioned to reabsorb the remainder of filtered glucose not
reabsorbed by SGLT2 in the proximal tubule S1 segments.
[0009] Rat SGLT2, like human SGLT2, is strongly expressed in
proximal S1 segments and this expression is developmentally
regulated, with expression appearing on embryonic day 17, gradually
increasing until day 19 and subsequently decreasing between day 19
and birth. Interestingly, rat SGLT2 mRNA is 2.6 kb before birth and
2.2 kb after birth, suggesting the presence of a different splice
variant in embryonic kidney compared to the adult (You et al., J.
Biol. Chem., 1995, 270, 29365-29371).
[0010] The transport properties of rat SGLT2, i.e K.sub.m of 3.0 mM
and sodium to glucose coupling of 1:1, are also characteristic of a
kidney cortical low-affinity transport system. Hybrid depletion
studies in which rat kidney superficial cortex mRNA was mixed with
an antisense oligonucleotide corresponding to the 5' portion of the
rat SGLT2 coding region completely suppressed the uptake of
.alpha.-MeGlc in Xenopus oocytes into which the
mRNA/oligonucleotide mix was injected. An antisense oligonucleotide
targeted to SGLT1 had no effect on the uptake of .alpha.-MeGlc.
These data demonstrate that the .alpha.-MeGlc uptake was entirely
due to the expression of rat SGLT2 and support the proposal that
SGLT2 is the major kidney cortical low affinity glucose transporter
(You et al., J. Biol. Chem., 1995, 270, 29365-29371).
[0011] A second low-affinity SGLT, named SAAT-pSGLT2, was isolated
from porcine kidney cells and was initially proposed to be the main
low-affinity glucose transporter. However, further studies have
revealed that the molecular characteristics of SAAT-pSGLT2 differ
from those of SGLT2 and consequently SAAT-pSGLT2 has been renamed
SGLT3 (Kong et al., J. Biol. Chem., 1993, 268, 1509-1512; Mackenzie
et al., J. Biol. Chem., 1996, 271, 32678-32683; Mackenzie et al.,
J. Biol. Chem., 1994, 269, 22488-22491; You et al., J. Biol. Chem.,
1995, 270, 29365-29371). Whether SGLT3 contributes to glucose
reabsorption in a physiologically relevant manner is unclear.
[0012] The importance of SGLT2 function was demonstrated in
hepatocyte nuclear factor 1 .alpha. (HNF1.alpha.)-deficient
animals, which are diabetic and also suffer from a renal Fanconi
syndrome characterized by urinary glucose loss. HNF1.alpha. is a
transcriptional activator expressed in liver, kidney, pancreas and
intestine. The renal defect in these mice is due to an 80-90%
reduction in SGLT2 expression. Thus, HNF1.alpha. is one gene
product that controls SGLT2 expression, which is essential to
proper glucose reabsorption in vivo (Pontoglio et al., EMBO Rep.,
2000, 1, 359-365).
[0013] Reduction of SGLT2 mRNA was also observed upon exposure of
mouse kidney cortical cells to cadmium, along with inhibition of
sodium-dependent uptake of the glucose analog .alpha.-MeGlc.
Interestingly, while both SGLT1 and SGLT2 mRNA were decreased in
mouse kidney cortical cells exposed to cadmium, SGLT3 mRNA was
upregulated, suggesting that individual SGLT species are not
regulated in a similar manner (Tabatabai et al., Toxicol. Appl.
Pharmacol., 2001, 177, 163-173). Changes in glucose or sodium
filtrated rate also modulate the expression of sodium-glucose
transporter mRNA. Diabetic rats with glycosuria and rats fed a high
sodium diet exhibited increased SGLT2 expression in the renal
proximal tubule. The finding that SGLT1 levels in these rats were
not altered to the same extent as SGLT2 levels further supports the
hypothesis that the cotransporters are differentially regulated
(Vestri et al., J. Membr. Biol., 2001, 182, 105-112).
[0014] Although studies of SGLT function and localization in
multiple mammalian species, including rat, mouse, pig, rabbit and
dog, indicated that SGLT2 is the low-affinity renal SGLT, the
identity of the human SGLT responsible for glucose reabsorption
across the brush border of the human proximal tubule remained
unclear. The lack of information describing SGLT protein
localization in renal brush border further hindered the
identification of the human low-affinity SGLT. Molecular genetic
analysis of SGLT1 and SGLT2 indicated that a genetic alteration in
the SGLT2 gene is a likely cause of renal glycosuria, a condition
characterized by elevated excretion of glucose in the urine
(Hediger et al., Klin. Wochenschr., 1989, 67, 843-846). Direct
evidence of SGLT function in the reabsorption of glucose came from
analysis of the SGLT2 gene in a patient with congenital isolated
renal glucosuria. Sequence analysis revealed a homozygous nonsense
mutation in exon 11 of the SGLT2 gene leading to the formation of a
truncated protein which is predicted to lack cotransport function
(van den Heuvel et al., Hum. Genet., 2002, 111, 544-547).
[0015] Whereas SGLT2 deficiency leads to inhibited reabsorption of
glucose, SGLT2 elevation potentially allows for increased glucose
uptake and is observed in metastatic lesions of lung cancer.
Quantitation of SGLT2 gene expression revealed no significant
difference between normal lung tissue and primary lung cancer.
However, the metatstatic lesions of both the liver and lymph node
exhibited significantly higher expression of SGLT2 (Ishikawa et
al., Jpn. J. Cancer Res., 2001, 92, 874-879). This finding is
significant in light of evidence that different clinical tumors
show significantly increased glucose uptake in vivo compared to
normal tissue. Such a change in metabolism confers an advantage to
tumor cells which allows them to survive and invade. Furthermore,
glucose uptake correlates with tumor aggressiveness and prognosis
(Dang and Semenza, Trends Biochem. Sci., 1999, 24, 68-72).
[0016] Diabetes is a disorder characterized by hyperglycemia due to
deficient insulin action. Chronic hyperglycemia is a major risk
factor for diabetes-associated complications, including heart
disease, retinopathy, nephropathy and neuropathy. As the kidneys
play a major role in the regulation of plasma glucose levels, renal
glucose transporters are becoming attractive drug targets (Wright,
Am. J. Physiol. Renal Physiol., 2001, 280, F10-18). Synthetic
agents that are derived from phlorizin, a specific inhibitor of
sodium/glucose transporters, have been designed and include T-1095,
and its metabolically active form T-1095A (Tsujihara et al., J.
Med. Chem., 1999, 42, 5311-5324). Phlorizin, T-1095 and T-1095A all
inhibited sodium-dependent glucose uptake in brush border membranes
prepared from normal and diabetic rat kidney, rat small intestine,
mouse kidney and dog kidney, as well as in Xenopus oocytes injected
with human SGLT mRNA (Oku et al., Diabetes, 1999, 48, 1794-1800;
Oku et al., Eur. J. Pharmacol., 2000, 391, 183-192). These agents
have been tested as antidiabetic compounds in laboratory animals
with genetic and streptozotocin-induced diabetes. In these models,
administration of these compounds inhibited renal SGLT activity,
increased urinary glucose excretion and improved glucose tolerance,
hyperglycemia and hypoinsulemia (Arakawa et al., Br. J. Pharmacol.,
2001, 132, 578-586; Oku et al., Diabetes, 1999, 48, 1794-1800; Oku
et al., Eur. J. Pharmacol., 2000, 391, 183-192). Prolonged
treatment of db/db mice with T-1095 yielded similar results and
also almost completely suppressed the increase of urinary albumin
and improved renal glomeruli pathology, indicating a beneficial
influence on renal disfunction and a protective effect against
nephropathy, respectively (Arakawa et al., Br. J. Pharmacol., 2001,
132, 578-586). Diabetic nephropathy is the most common cause of
end-stage renal disease that develops in many patients with
diabetes. In Zucker diabetic fatty rats, long-term treatment with
T-1095 lowered both fed and fasting glucose levels to near normal
ranges. Also observed were recovered hepatic glucose production and
glucose utilization rates without a significant improvement in
skeletal muscle glucose utilization rate, indicating that
hyperglycemia contributes to insulin resistance in hepatic and
adipose tissue in this rat model of diabetes. These results further
suggest that glucotoxicity, which results from long-term
hyperglycemia, induces tissue-dependent insulin resistance in
diabetic patients (Nawano et al., Am. J. Physiol. Endocrinol.
Metab., 2000, 278, E535-543).
[0017] Other SGLT2 inhibiting compounds are known in the art, such
as the c-aryl glucosides disclosed in U.S. Pat. No. 6,414,126,
which are inhibitors of sodium dependent glucose transporters found
in the intestine and kidney and are proposed to treat diabetes,
hyperglycemia and related diseases when used alone or in
combination with other antidiabetic agents (Ellsworth et al.,
2002).
[0018] The US pre-grant publication 20030055019 discloses isolated
mutant proteins selected from a group which includes SGLT2, the
corresponding nucleic acid molecules encoding said mutant proteins,
isolated antisense derivatives of the nucleic acid sequences
encoding said mutant proteins, as well as methods of delivering
said antisense nucleic acid derivatives to treat or prevent
hypertension, diabetes, insulin sensitivity, obesity, dyslipidemia
and stroke. This application also discloses the antisense molecules
may be DNA or RNA or a chimeric mixture, single-stranded or
double-stranded or may comprise a ribozyme or catalytic RNA
(Shimkets, 2003).
[0019] The European Patent Applications EP 1 293 569 and EP 1 308
459 disclose a polynucleotide comprising a protein-coding region of
the nucleotide sequence of any one of a group of sequences which
includes a nucleic acid sequence encoding human SGLT2, an
oligonucleotide comprising at least 15 nucleotides complementary to
the nucleotide sequence or to a complementary strand thereof and an
antisense polynucleotide against the disclosed polynucleotide or a
part thereof. These applications disclose the use of said antisense
polynucleotides for suppressing the expression of a polypeptide of
the invention and for gene therapy (Isogai et al., 2003, ; Isogai
et al., 2003).
[0020] Although phlorizin and its derivatives are potent inhibitors
of sodium-glucose cotransporters, these agents do not specifically
inhibit a single species of SGLT, thus all SGLTs in all tissues are
affected. Thus, there remains a need for therapeutic compounds that
targets specific SGLT species. Antisense technology is an effective
means for reducing the expression of specific gene products and may
therefore prove to be uniquely useful in a number of therapeutic,
diagnostic and research applications for the modulation of SGLT2
expression.
[0021] The present invention provides compounds and methods for
modulating SGLT2 expression.
SUMMARY OF THE INVENTION
[0022] The present invention is directed to oligomeric compounds,
especially nucleic acid and nucleic acid-like oligomers, such as
antisense compounds, which are targeted to a nucleic acid encoding
SGLT2, and which modulate the expression of SGLT2. Pharmaceutical
and other compositions comprising the compounds of the invention
are also provided. Further provided are methods of screening for
modulators of SGLT2 and methods of modulating the expression of
SGLT2 in cells, tissues or animals comprising contacting the cells,
tissues or animals with one or more of the compounds or
compositions of the invention. Further provided are diagnostic
methods for identifying a disease state by identifying the presence
of SGLT2 in a sample using one or more of the compounds of the
invention. Methods of treating an animal, particularly a human,
suspected of having or being prone to a disease or condition
associated with expression of SGLT2 are also set forth herein. Such
methods comprise administering a therapeutically or
prophylactically effective amount of one or more of the compounds
or compositions of the invention to the person, who may be in need
of treatment.
[0023] Also provided are methods of enhancing inhibition of
expression of preselected cellular RNA targets in kidney cells and
kidney tissue using compounds, such as antisense compounds, of the
invention. Further provided are methods of preventing or delaying
the onset of a disease or condition in an animal, wherein the
disease or condition is associated with expression of a preselected
cellular RNA target expressed in the kidney, particularly SGLT2.
Methods of lowering blood glucose levels in an animal and methods
of delaying or preventing the onset of type 2 diabetes also are set
forth herein. Such methods comprise administering a therapeutically
or prophylactically effective amount of one or more of the
compounds of the invention to the animal, which may be in need of
treatment. Provided herein are methods of enhancing inhibition of
expression of SGLT2 in kidney cells or kidney tissues, comprising
contacting the cells or tissues with one or more of the compounds
of the invention, such as antisense compounds.
DETAILED DESCRIPTION OF THE INVENTION
[0024] The present invention employs oligomeric compounds,
preferably oligonucleotides and similar species, such as antisense
compounds, for use in modulating the function or effect of nucleic
acid molecules encoding SGLT2. This is accomplished by providing
oligomeric compounds, such as oligonucleotides, which specifically
hybridize with one or more nucleic acid molecules encoding
SGLT2.
[0025] In one embodiment, the oligomeric compounds of the invention
are chimeric oligonucleotides ("gapmers"), composed of a central
"gap" region consisting of 2'-deoxynucleotides, which is flanked on
both sides (5' and 3' directions) by "wings" composed of
2'-methoxyethyl (2'-MOE) nucleotides. In some embodiments, the
internucleoside (backbone) linkages are phosphorothioate (P.dbd.S)
throughout the oligonucleotide. In some embodiments, one or more
cytidine residues are 5-methylcytidines.
[0026] In another embodiment, the oligomeric compounds of the
invention are chimeric oligonucleotides having mixed
phosphorothioate and phosphodiester backbones, referred to herein
as "mixed backbone compounds." The mixed backbone compounds of the
invention can have a central "gap" region consisting of at least 5
contiguous 2'-deoxy nucleosides flanked by two "wing" regions
consisting of at least one 2'-O-methoxyethyl nucleoside in each
region. The internucleoside linkages of the mixed backbone
compounds can be phosphorothioate linkages in the central "gap"
region and phosphodiester linkages in the two "wing" regions. In
another embodiment, mixed backbone compounds have phosphodiester
linkages in the "wing" regions except for one phosphodiester
linkage at one or both of the extreme 5' and 3' ends of the
oligonucleotide.
[0027] It is shown herein that mixed backbone compounds are
efficiently delivered to the kidney and treatment with the mixed
backbone compounds results in efficient modulation of target gene
expression in the kidney without liver or kidney toxicity. It is
further shown herein that treatment with mixed backbone compounds
in animal models of type 2 diabetes reduces blood glucose levels in
diabetic animals.
[0028] As used herein, the terms "target nucleic acid" and "nucleic
acid molecule encoding SGLT2" have been used for convenience to
encompass DNA encoding SGLT2, RNA (including pre-mRNA and mRNA or
portions thereof) transcribed from such DNA, and also cDNA derived
from such RNA. The hybridization of a compound of this invention
with its target nucleic acid is generally referred to as
"antisense." Consequently, one mechanism believed to be included in
the practice of some embodiments of the invention is referred to
herein as "antisense inhibition." Such antisense inhibition is
typically based upon hydrogen bonding-based hybridization of
oligonucleotide strands or segments such that at least one strand
or segment is cleaved, degraded, or otherwise rendered inoperable.
In this regard, specific nucleic acid molecules and their functions
can be targeted for such antisense inhibition.
[0029] The functions of DNA to be interfered with can include
replication and transcription. Replication and transcription, for
example, can be from an endogenous cellular template, a vector, a
plasmid construct or otherwise. The functions of RNA to be
interfered with can include functions such as, for example,
translocation of the RNA to a site of protein translation,
translocation of the RNA to sites within the cell which are distant
from the site of RNA synthesis, translation of protein from the
RNA, splicing of the RNA to yield one or more RNA species, and
catalytic activity or complex formation involving the RNA which may
be engaged in or facilitated by the RNA. One result of such
interference with target nucleic acid function is modulation of the
expression of SGLT2. In the context of the present invention,
"modulation" and "modulation of expression" mean either an increase
(stimulation) or a decrease (inhibition) in the amount or levels of
a nucleic acid molecule encoding the gene, e.g., DNA or RNA.
Inhibition is often the desired form of modulation of expression
and mRNA is often a desired target nucleic acid.
[0030] In the context of this invention, "hybridization" means the
pairing of complementary strands of oligomeric compounds. In the
present invention, one mechanism of pairing involves hydrogen
bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen
hydrogen bonding, between complementary nucleoside or nucleotide
bases (nucleobases) of the strands of oligomeric compounds. For
example, adenine and thymine are complementary nucleobases which
pair through the formation of hydrogen bonds. Hybridization can
occur under varying circumstances.
[0031] An oligomeric compound, such as an antisense compound, is
specifically hybridizable when binding of the compound to the
target nucleic acid interferes with the normal function of the
target nucleic acid to cause a loss of activity, and there is a
sufficient degree of complementarity to avoid non-specific binding
of the antisense compound to non-target nucleic acid sequences
under conditions in which specific binding is desired, i.e., under
physiological conditions in the case of in vivo assays or
therapeutic treatment, and under conditions in which assays are
performed in the case of in vitro assays.
[0032] In the present invention the phrase "stringent hybridization
conditions" or "stringent conditions" refers to conditions under
which a compound of the invention will hybridize to its target
sequence, but to a minimal number of other sequences. Stringent
conditions are sequence-dependent and will be different in
different circumstances and in the context of this invention,
"stringent conditions" under which oligomeric compounds hybridize
to a target sequence are determined by the nature and composition
of the oligomeric compounds and the assays in which they are being
investigated.
[0033] "Complementary," as used herein, refers to the capacity for
precise pairing between two nucleobases of an oligomeric compound.
For example, if a nucleobase at a certain position of an
oligonucleotide (an oligomeric compound), is capable of hydrogen
bonding with a nucleobase at a certain position of a target nucleic
acid, said target nucleic acid being a DNA, RNA, or oligonucleotide
molecule, then the position of hydrogen bonding between the
oligonucleotide and the target nucleic acid is considered to be a
complementary position. The oligonucleotide and the further DNA,
RNA, or oligonucleotide molecule are complementary to each other
when a sufficient number of complementary positions in each
molecule are occupied by nucleobases which can hydrogen bond with
each other. Thus, "specifically hybridizable" and "complementary"
are terms which are used to indicate a sufficient degree of precise
pairing or complementarity over a sufficient number of nucleobases
such that stable and specific binding occurs between the
oligonucleotide and a target nucleic acid.
[0034] It is understood in the art that the sequence of an
oligomeric compound need not be 100% complementary to that of its
target nucleic acid to be specifically hybridizable. Moreover, an
oligomeric compound may hybridize over one or more segments such
that intervening or adjacent segments are not involved in the
hybridization event (e.g., a loop structure or hairpin structure).
The antisense compounds of the present invention can comprise at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
at least 95%, or at least 99% sequence complementarity to the
target region within the target nucleic acid sequence to which they
are targeted. For example, an antisense compound in which 18 of 20
nucleobases of the antisense compound are complementary to a target
region, and would therefore specifically hybridize, would represent
90 percent complementarity. In this example, the remaining
noncomplementary nucleobases may be clustered or interspersed with
complementary nucleobases and need not be contiguous to each other
or to complementary nucleobases. As such, an antisense compound
which is 18 nucleobases in length having 4 (four) noncomplementary
nucleobases which are flanked by two regions of complete
complementarity with the target nucleic acid would have 77.8%
overall complementarity with the target nucleic acid and would thus
fall within the scope of the present invention. Percent
complementarity of an antisense compound with a region of a target
nucleic acid can be determined routinely using BLAST programs
(basic local alignment search tools) and PowerBLAST programs known
in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403-410;
Zhang and Madden, Genome Res., 1997, 7, 649-656).
[0035] Percent homology, sequence identity or complementarity, can
be determined by, for example, the Gap program (Wisconsin Sequence
Analysis Package, Version 8 for Unix, Genetics Computer Group,
University Research Park, Madison Wis.), using default settings,
which uses the algorithm of Smith and Waterman (Adv. Appl. Math.,
1981, 2, 482-489). In some embodiments, homology, sequence identity
or complementarity, between the oligomeric and target is from about
50% to about 60%, from about 60% to about 70%, from about 70% to
about 80%, from about 80% to about 90%, about 90%, about 92%, about
94%, about 95%, about 96%, about 97%, about 98%, about 99%, or
about 100%. As used herein, the term "about" means .+-.5% of the
value modified.
[0036] According to the present invention, oligomeric compounds,
such as antisense compounds, include antisense oligomeric
compounds, antisense oligonucleotides, ribozymes, external guide
sequence (EGS) oligonucleotides, alternate splicers, and other
oligomeric compounds which hybridize to at least a portion of the
target nucleic acid. As such, these compounds may be introduced in
the form of single-stranded, double-stranded, circular or hairpin
oligomeric compounds and may contain structural elements such as
internal or terminal bulges or loops. Once introduced to a system,
the compounds of the invention may elicit the action of one or more
enzymes or structural proteins to effect modification of the target
nucleic acid.
[0037] One non-limiting example of such an enzyme is RNAse H, a
cellular endonuclease which cleaves the RNA strand of an RNA:DNA
duplex. It is known in the art that single-stranded antisense
compounds which are "DNA-like" elicit RNAse H. Activation of RNase
H, therefore, results in cleavage of the RNA target, thereby
greatly enhancing the efficiency of oligonucleotide-mediated
inhibition of gene expression. Similar roles have been postulated
for other ribonucleases such as those in the RNase III and
ribonuclease L family of enzymes.
[0038] While one form of antisense compound is a single-stranded
antisense oligonucleotide, in many species the introduction of
double-stranded structures, such as double-stranded RNA (dsRNA)
molecules, has been shown to induce potent and specific
antisense-mediated reduction of the function of a gene or its
associated gene products. This phenomenon occurs in both plants and
animals and is believed to have an evolutionary connection to viral
defense and transposon silencing.
[0039] The first evidence that dsRNA could lead to gene silencing
in animals came in 1995 from work in the nematode, Caenorhabditis
elegans (Guo and Kempheus, Cell, 1995, 81, 611-620). Montgomery et
al. have shown that the primary interference effects of dsRNA are
posttranscriptional (Montgomery et al., Proc. Natl. Acad. Sci. USA,
1998, 95, 15502-15507). The posttranscriptional antisense mechanism
defined in Caenorhabditis elegans resulting from exposure to
double-stranded RNA (dsRNA) has since been designated RNA
interference (RNAi). This term has been generalized to mean
antisense-mediated gene silencing involving the introduction of
dsRNA leading to the sequence-specific reduction of endogenous
targeted mRNA levels (Fire et al., Nature, 1998, 391, 806-811).
Recently, it has been shown that it is, in fact, the
single-stranded RNA oligomers of antisense polarity of the dsRNAs
which are the potent inducers of RNAi (Tijsterman et al., Science,
2002, 295, 694-697).
[0040] The oligomeric compounds of the present invention also
include modified compounds in which a different base is present at
one or more of the nucleotide positions in the compound. For
example, if the first nucleotide is an adenosine, modified
compounds may be produced which contain thymidine, guanosine or
cytidine at this position. This may be done at any of the positions
of the antisense compound. These compounds are then tested using
the methods described herein to determine their ability to inhibit
expression of SGLT2 mRNA.
[0041] In the context of this invention, the term "oligomeric
compound" refers to a polymer or oligomer comprising a plurality of
monomeric units. In the context of this invention, the term
"oligonucleotide" refers to an oligomer or polymer of ribonucleic
acid (RNA) or deoxyribonucleic acid (DNA) or mimetics, chimeras,
analogs and homologs thereof. This term includes oligonucleotides
composed of naturally occurring nucleobases, sugars and covalent
internucleoside (backbone) linkages as well as oligonucleotides
having non-naturally occurring portions which function similarly.
Such modified or substituted oligonucleotides are often desired
over native forms because of desirable properties such as, for
example, enhanced cellular uptake, enhanced affinity for a target
nucleic acid and increased stability in the presence of
nucleases.
[0042] While oligonucleotides are one form of the antisense
compounds of this invention, the present invention comprehends
other families of antisense compounds as well, including but not
limited to oligonucleotide analogs and mimetics such as those
described herein.
[0043] The antisense compounds in accordance with this invention
can comprise from about 8 to about 80 nucleobases (i.e. from about
8 to about 80 linked nucleosides). One of ordinary skill in the art
will appreciate that the invention embodies compounds of 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,
62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,
79, or 80 nucleobases in length, or any range therewithin.
[0044] In one embodiment, the antisense compounds of the invention
are 10 to 50 nucleobases in length. One having ordinary skill in
the art will appreciate that this embodies compounds of 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 or 50 nucleobases in length, or any range
therewithin.
[0045] In another embodiment, the antisense compounds of the
invention are 13 to 30 nucleobases in length. One having ordinary
skill in the art will appreciate that this embodies compounds of
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29
or 30 nucleobases in length, or any range therewithin.
[0046] In another embodiment, the antisense compounds of the
invention are 15 to 25 nucleobases in length. One having ordinary
skill in the art will appreciate that this embodies compounds of
15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleobases in length,
or any range therewithin.
[0047] In another embodiment, the antisense compounds of the
invention are 18 to 22 nucleobases in length. One having ordinary
skill in the art will appreciate that this embodies compounds of
18, 19, 20, 21 or 22 nucleobases in length, or any range
therewithin.
[0048] Particularly suitable compounds are oligonucleotides from
about 10 to about 50 nucleobases, from about 13 to about 30
nucleobases, from about 15 to about 25, and from about 18 to about
22 nucleobases.
[0049] Antisense compounds 8 to 80 nucleobases in length comprising
a stretch of at least eight (8) consecutive nucleobases selected
from within the illustrative antisense compounds are considered to
be suitable antisense compounds as well.
[0050] Exemplary antisense compounds include oligonucleotide
sequences that comprise at least the 8 consecutive nucleobases from
the 5'-terminus of one of the illustrative antisense compounds (the
remaining nucleobases being a consecutive stretch of the same
oligonucleotide beginning immediately upstream of the 5'-terminus
of the antisense compound which is specifically hybridizable to the
target nucleic acid and continuing until the oligonucleotide
contains about 8 to about 80 nucleobases). Similarly suitable
antisense compounds are represented by oligonucleotide sequences
that comprise at least the 8 consecutive nucleobases from the
3'-terminus of one of the illustrative antisense compounds (the
remaining nucleobases being a consecutive stretch of the same
oligonucleotide beginning immediately downstream of the 3'-terminus
of the antisense compound which is specifically hybridizable to the
target nucleic acid and continuing until the oligonucleotide
contains about 8 to about 80 nucleobases). It is also understood
that antisense compounds may be represented by oligonucleotide
sequences that comprise at least 8 consecutive nucleobases from an
internal portion of the sequence of an illustrative antisense
compound, and may extend in either or both directions until the
oligonucleotide contains about 8 to about 80 nucleobases.
[0051] One having skill in the art armed with the antisense
compounds illustrated herein will be able, without undue
experimentation, to identify additional antisense compounds.
[0052] "Targeting" an antisense compound to a particular nucleic
acid molecule, in the context of this invention, can be a multistep
process. The process usually begins with the identification of a
target nucleic acid whose function is to be modulated. This target
nucleic acid may be, for example, a cellular gene (or mRNA
transcribed from the gene) whose expression is associated with a
particular disorder or disease state, or a nucleic acid molecule
from an infectious agent. In the present invention, the target
nucleic acid encodes SGLT2.
[0053] The targeting process usually also includes determination of
at least one target region, segment, or site within the target
nucleic acid for the antisense interaction to occur such that the
desired effect, e.g., modulation of expression, will result. Within
the context of the present invention, the term "region" is defined
as a portion of the target nucleic acid having at least one
identifiable structure, function, or characteristic. Within regions
of target nucleic acids are segments. "Segments" are defined as
smaller or sub-portions of regions within a target nucleic acid.
"Sites," as used in the present invention, are defined as positions
within a target nucleic acid.
[0054] Since, as is known in the art, the translation initiation
codon is typically 5'-AUG (in transcribed mRNA molecules; 5'-ATG in
the corresponding DNA molecule), the translation initiation codon
is also referred to as the "AUG codon," the "start codon" or the
"AUG start codon." A minority of genes have a translation
initiation codon having the RNA sequence 5'-GUG, 5'-UUG or 5'-CUG,
and 5'-AUA, 5'-ACG and 5'-CUG have been shown to function in vivo.
Thus, the terms "translation initiation codon" and "start codon"
can encompass many codon sequences, even though the initiator amino
acid in each instance is typically methionine (in eukaryotes) or
formylmethionine (in prokaryotes). It is also known in the art that
eukaryotic and prokaryotic genes may have two or more alternative
start codons, any one of which may be preferentially utilized for
translation initiation in a particular cell type or tissue, or
under a particular set of conditions. In the context of the
invention, "start codon" and "translation initiation codon" refer
to the codon or codons that are used in vivo to initiate
translation of an mRNA transcribed from a gene encoding SGLT2,
regardless of the sequence(s) of such codons. It is also known in
the art that a translation termination codon (or "stop codon") of a
gene may have one of three sequences, i.e., 5'-UAA, 5'-UAG and
5'-UGA (the corresponding DNA sequences are 5'-TAA, 5'-TAG and
5'-TGA, respectively).
[0055] The terms "start codon region" and "translation initiation
codon region" refer to a portion of such an mRNA or gene that
encompasses from about 25 to about 50 contiguous nucleotides in
either direction (i.e., 5' or 3') from a translation initiation
codon. Similarly, the terms "stop codon region" and "translation
termination codon region" refer to a portion of such an mRNA or
gene that encompasses from about 25 to about 50 contiguous
nucleotides in either direction (i.e., 5' or 3') from a translation
termination codon. Consequently, the "start codon region" (or
"translation initiation codon region") and the "stop codon region"
(or "translation termination codon region") are all regions which
may be targeted effectively with the antisense compounds of the
present invention.
[0056] The open reading frame (ORF) or "coding region," which is
known in the art to refer to the region between the translation
initiation codon and the translation termination codon, is also a
region which may be targeted effectively. Within the context of the
present invention, a suitable region is the intragenic region
encompassing the translation initiation or termination codon of the
open reading frame (ORF) of a gene.
[0057] Other target regions include the 5' untranslated region
(5'UTR), known in the art to refer to the portion of an mRNA in the
5' direction from the translation initiation codon, and thus
including nucleotides between the 5' cap site and the translation
initiation codon of an mRNA (or corresponding nucleotides on the
gene), and the 3' untranslated region (3'UTR), known in the art to
refer to the portion of an mRNA in the 3' direction from the
translation termination codon, and thus including nucleotides
between the translation termination codon and 3' end of an mRNA (or
corresponding nucleotides on the gene). The 5' cap site of an mRNA
comprises an N7-methylated guanosine residue joined to the 5'-most
residue of the mRNA via a 5'-5' triphosphate linkage. The 5' cap
region of an mRNA is considered to include the 5' cap structure
itself as well as the first 50 nucleotides adjacent to the cap
site. It is also suitable to target the 5' cap region.
[0058] Although some eukaryotic mRNA transcripts are directly
translated, many contain one or more regions, known as "introns,"
which are excised from a transcript before it is translated. The
remaining (and therefore translated) regions are known as "exons"
and are spliced together to form a continuous mRNA sequence.
Targeting splice sites, i.e., intron-exon junctions or exon-intron
junctions, may also be particularly useful in situations where
aberrant splicing is implicated in disease, or where an
overproduction of a particular splice product is implicated in
disease. Aberrant fusion junctions due to rearrangements or
deletions are also suitable target sites. mRNA transcripts produced
via the process of splicing of two (or more) mRNAs from different
gene sources are known as "fusion transcripts." It is also known
that introns can be effectively targeted using antisense compounds
targeted to, for example, DNA or pre-mRNA.
[0059] It is also known in the art that alternative RNA transcripts
can be produced from the same genomic region of DNA. These
alternative transcripts are generally known as "variants." More
specifically, "pre-mRNA variants" are transcripts produced from the
same genomic DNA that differ from other transcripts produced from
the same genomic DNA in either their start or stop position and
contain both intronic and exonic sequence.
[0060] Upon excision of one or more exon or intron regions, or
portions thereof during splicing, pre-mRNA variants produce smaller
"mRNA variants." Consequently, mRNA variants are processed pre-mRNA
variants and each unique pre-mRNA variant must always produce a
unique mRNA variant as a result of splicing. These mRNA variants
are also known as "alternative splice variants." If no splicing of
the pre-mRNA variant occurs then the pre-mRNA variant is identical
to the mRNA variant.
[0061] It is also known in the art that variants can be produced
through the use of alternative signals to start or stop
transcription and that pre-mRNAs and mRNAs can possess more that
one start codon or stop codon. Variants that originate from a
pre-mRNA or mRNA that use alternative start codons are known as
"alternative start variants" of that pre-mRNA or mRNA. Those
transcripts that use an alternative stop codon are known as
"alternative stop variants" of that pre-mRNA or mRNA. One specific
type of alternative stop variant is the "polyA variant" in which
the multiple transcripts produced result from the alternative
selection of one of the "polyA stop signals" by the transcription
machinery, thereby producing transcripts that terminate at unique
polyA sites. Within the context of the invention, the types of
variants described herein are also suitable target nucleic
acids.
[0062] The locations on the target nucleic acid to which the
antisense compounds hybridize are hereinbelow referred to as
"suitable target segments." As used herein the term "suitable
target segment" is defined as at least an 8-nucleobase portion of a
target region to which an active antisense compound is targeted.
While not wishing to be bound by theory, it is presently believed
that these target segments represent portions of the target nucleic
acid which are accessible for hybridization.
[0063] While the specific sequences of certain preferred target
segments are set forth herein, one of skill in the art will
recognize that these serve to illustrate and describe particular
embodiments within the scope of the present invention. Additional
target segments may be identified by one having ordinary skill.
[0064] Target segments 8-80 nucleobases in length comprising a
stretch of at least eight (8) consecutive nucleobases selected from
within the illustrative target segments are considered to be
suitable for targeting as well.
[0065] Target segments can include DNA or RNA sequences that
comprise at least the 8 consecutive nucleobases from the
5'-terminus of one of the illustrative target segments (the
remaining nucleobases being a consecutive stretch of the same DNA
or RNA beginning immediately upstream of the 5'-terminus of the
target segment and continuing until the DNA or RNA contains about 8
to about 80 nucleobases). Target segments are also represented by
DNA or RNA sequences that comprise at least the 8 consecutive
nucleobases from the 3'-terminus of one of the illustrative target
segments (the remaining nucleobases being a consecutive stretch of
the same DNA or RNA beginning immediately downstream of the
3'-terminus of the target segment and continuing until the DNA or
RNA contains about 8 to about 80 nucleobases). It is also
understood that antisense target segments may be represented by DNA
or RNA sequences that comprise at least 8 consecutive nucleobases
from an internal portion of the sequence of an illustrative target
segment, and may extend in either or both directions until the
oligonucleotide contains about 8 to about 80 nucleobases. One
having skill in the art armed with the target segments illustrated
herein will be able, without undue experimentation, to identify
further target segments.
[0066] Once one or more target regions, segments or sites have been
identified, antisense compounds are chosen which are sufficiently
complementary to the target, i.e., hybridize sufficiently well and
with sufficient specificity, to give the desired effect.
[0067] The oligomeric antisense compounds may also be targeted to
regions of the target nucleobase sequence (e.g., such as those
disclosed in Example 16) comprising nucleobases 1-80, 81-160,
161-240, 241-320, 321-400, 401-480, 481-560, 561-640, 641-720,
721-800, 801-880, 881-960, 961-1040, 1041-1120, 1121-1200,
1201-1280, 1281-1360, 1361-1440, 1441-1520, 1521-1600, 1601-1680,
1681-1760, 1761-1840, 1841-1920, 1921-2000, 2001-2080, 2081-2160,
2161-2240, 2241-2273, or any combination thereof.
[0068] In a further embodiment, the "suitable target segments"
identified herein may be employed in a screen for additional
compounds that modulate the expression of SGLT2. "Modulators" are
those compounds that decrease or increase the expression of a
nucleic acid molecule encoding SGLT2 and which comprise at least an
8-nucleobase portion which is complementary to a target segment.
The screening method comprises the steps of contacting a target
segment of a nucleic acid molecule encoding SGLT2 with one or more
candidate modulators, and selecting for one or more candidate
modulators which decrease or increase the expression of a nucleic
acid molecule encoding SGLT2. Once it is shown that the candidate
modulator or modulators are capable of modulating (e.g. either
decreasing or increasing) the expression of a nucleic acid molecule
encoding SGLT2, the modulator may then be employed in further
investigative studies of the function of SGLT2, or for use as a
research, diagnostic, or therapeutic agent in accordance with the
present invention.
[0069] The target segments of the present invention may be also be
combined with their respective complementary antisense compounds of
the present invention to form stabilized double-stranded (duplexed)
oligonucleotides.
[0070] Such double stranded oligonucleotide moieties have been
shown in the art to modulate target expression and regulate
translation as well as RNA processsing via an antisense mechanism.
Moreover, the double-stranded moieties may be subject to chemical
modifications (Fire et al., Nature, 1998, 391, 806-811; Timmons and
Fire, Nature 1998, 395, 854; Timmons et al., Gene, 2001, 263,
103-112; Tabara et al., Science, 1998, 282, 430-431; Montgomery et
al., Proc. Natl. Acad. Sci. USA, 1998, 95, 15502-15507; Tuschl et
al., Genes Dev., 1999, 13, 3191-3197; Elbashir et al., Nature,
2001, 411, 494-498; Elbashir et al., Genes Dev. 2001, 15, 188-200).
For example, such double-stranded moieties have been shown to
inhibit the target by the classical hybridization of antisense
strand of the duplex to the target, thereby triggering enzymatic
degradation of the target (Tijsterman et al., Science, 2002, 295,
694-697).
[0071] The antisense compounds of the present invention can also be
applied in the areas of drug discovery and target validation. The
present invention comprehends the use of the compounds and target
segments identified herein in drug discovery efforts to elucidate
relationships that exist between SGLT2 and a disease state,
phenotype, or condition. These methods include detecting or
modulating SGLT2 comprising contacting a sample, tissue, cell, or
organism with the compounds of the present invention, measuring the
nucleic acid or protein level of SGLT2 and/or a related phenotypic
or chemical endpoint at some time after treatment, and optionally
comparing the measured value to a non-treated sample or sample
treated with a further compound of the invention. These methods can
also be performed in parallel or in combination with other
experiments to determine the function of unknown genes for the
process of target validation or to determine the validity of a
particular gene product as a target for treatment or prevention of
a particular disease, condition, or phenotype.
[0072] The antisense compounds of the present invention can be
utilized for diagnostics, therapeutics, prophylaxis and as research
reagents and kits. Furthermore, antisense oligonucleotides, which
are able to inhibit gene expression with exquisite specificity, are
often used by those of ordinary skill to elucidate the function of
particular genes or to distinguish between functions of various
members of a biological pathway.
[0073] For use in kits and diagnostics, the compounds of the
present invention, either alone or in combination with other
compounds or therapeutics, can be used as tools in differential
and/or combinatorial analyses to elucidate expression patterns of a
portion or the entire complement of genes expressed within cells
and tissues.
[0074] As one nonlimiting example, expression patterns within cells
or tissues treated with one or more antisense compounds are
compared to control cells or tissues not treated with antisense
compounds and the patterns produced are analyzed for differential
levels of gene expression as they pertain, for example, to disease
association, signaling pathway, cellular localization, expression
level, size, structure or function of the genes examined. These
analyses can be performed on stimulated or unstimulated cells and
in the presence or absence of other compounds which affect
expression patterns.
[0075] Examples of methods of gene expression analysis known in the
art include DNA arrays or microarrays (Brazma and Vilo, FEBS Lett.,
2000, 480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE
(serial analysis of gene expression)(Madden, et al., Drug Discov.
Today, 2000, 5, 415-425), READS (restriction enzyme amplification
of digested cDNAs) (Prashar and Weissman, Methods Enzymol., 1999,
303, 258-72), TOGA (total gene expression analysis) (Sutcliffe, et
al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 1976-81), protein
arrays and proteomics (Celis, et al., FEBS Lett., 2000, 480, 2-16;
Jungblut, et al., Electrophoresis, 1999, 20, 2100-10), expressed
sequence tag (EST) sequencing (Celis, et al., FEBS Lett., 2000,
480, 2-16; Larsson, et al., J. Biotechnol., 2000, 80, 143-57),
subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal.
Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41,
203-208), subtractive cloning, differential display (DD) (Jurecic
and Belmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative
genomic hybridization (Carulli, et al., J. Cell Biochem. Suppl.,
1998, 31, 286-96), FISH (fluorescent in situ hybridization)
techniques (Going and Gusterson, Eur. J. Cancer, 1999, 35,
1895-904) and mass spectrometry methods (To, Comb. Chem. High
Throughput Screen, 2000, 3, 235-41).
[0076] The antisense compounds of the invention are useful for
research and diagnostics, because these compounds hybridize to
nucleic acids encoding SGLT2. For example, oligonucleotides that
are shown to hybridize with such efficiency and under such
conditions as disclosed herein as to be effective SGLT2 inhibitors
will also be effective primers or probes under conditions favoring
gene amplification or detection, respectively. These primers and
probes are useful in methods requiring the specific detection of
nucleic acid molecules encoding SGLT2 and in the amplification of
said nucleic acid molecules for detection or for use in further
studies of SGLT2. Hybridization of the antisense oligonucleotides,
particularly the primers and probes, of the invention with a
nucleic acid encoding SGLT2 can be detected by means known in the
art. Such means may include conjugation of an enzyme to the
oligonucleotide, radiolabelling of the oligonucleotide or any other
suitable detection means. Kits using such detection means for
detecting the level of SGLT2 in a sample may also be prepared.
[0077] The specificity and sensitivity of antisense is also
harnessed by those of skill in the art for therapeutic uses.
Antisense compounds have been employed as therapeutic moieties in
the treatment of disease states in animals, including humans.
Antisense oligonucleotide drugs, including ribozymes, have been
safely and effectively administered to humans and numerous clinical
trials are presently underway. It is thus established that
antisense compounds can be useful therapeutic modalities that can
be configured to be useful in treatment regimes for the treatment
of cells, tissues and animals, especially humans.
[0078] For therapeutics, an animal, such as a human, suspected of
having a disease or disorder which can be treated by modulating the
expression of SGLT2 is treated by administering antisense compounds
in accordance with this invention. For example, in one non-limiting
embodiment, the methods comprise the step of administering to the
animal a therapeutically effective amount of a SGLT2 inhibitor. The
animal may or may not have already been identifies as being in need
of treatment. That is, the animal may or may not have been
diagnosed with a particular disease or disorder. The SGLT2
inhibitors of the present invention effectively inhibit the
activity of the SGLT2 protein or inhibit the expression of the
SGLT2 protein. In some embodiments, the activity or expression of
SGLT2 in an animal or cell is inhibited by at least about 10%, by
at least about 20%, by at least about 30%, by at least about 40%,
by at least about 50%, by at least about 60%, by at least about
70%, by at least about 75%, by at least about 80%, by at least
about 85%, by at least about 90%, by at least about 95%, by at
least about 97%, by at least about 99%, or by 100%.
[0079] For example, the reduction of the expression of SGLT2 may be
measured in serum, adipose tissue, liver or any other body fluid,
tissue or organ of the animal. The cells contained within the
fluids, tissues or organs being analyzed can contain a nucleic acid
molecule encoding SGLT2 protein and/or the SGLT2 protein
itself.
[0080] The antisense compounds of the invention can be utilized in
pharmaceutical compositions by adding an effective amount of a
compound to a suitable pharmaceutically acceptable diluent or
carrier. Use of the compounds and methods of the invention may also
be useful prophylactically.
[0081] As is known in the art, a nucleoside is a base-sugar
combination. The base portion of the nucleoside is normally a
heterocyclic base sometimes referred to as a "nucleobase" or simply
a "base." The two most common classes of such heterocyclic bases
are the purines and the pyrimidines. Nucleotides are nucleosides
that further include a phosphate group covalently linked to the
sugar portion of the nucleoside. For those nucleosides that include
a pentofuranosyl sugar, the phosphate group can be linked to either
the 2', 3' or 5' hydroxyl moiety of the sugar. In forming
oligonucleotides, the phosphate groups covalently link adjacent
nucleosides to one another to form a linear polymeric compound. In
turn, the respective ends of this linear polymeric compound can be
further joined to form a circular compound, however, linear
compounds are generally desired. In addition, linear compounds may
have internal nucleobase complementarity and may therefore fold in
a manner as to produce a fully or partially double-stranded
compound. Within oligonucleotides, the phosphate groups are
commonly referred to as forming the internucleoside backbone of the
oligonucleotide. The normal linkage or backbone of RNA and DNA is a
3' to 5' phosphodiester linkage.
[0082] Modified Internucleoside Linkages (Backbones)
[0083] Specific examples of antisense compounds useful in this
invention include oligonucleotides containing modified backbones or
non-natural internucleoside linkages. As defined in this
specification, oligonucleotides having modified backbones include
those that retain a phosphorus atom in the backbone and those that
do not have a phosphorus atom in the backbone. For the purposes of
this specification, and as sometimes referenced in the art,
modified oligonucleotides that do not have a phosphorus atom in
their internucleoside backbone can also be considered to be
oligonucleosides.
[0084] Modified oligonucleotide backbones containing a phosphorus
atom therein include, for example, phosphorothioates, chiral
phosphorothioates, phosphorodithioates, phosphotriesters,
aminoalkylphosphotriaminoalkylphosphotriesters, methyl and other
alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene
phosphonates and chiral phosphonates, phosphinates,
phosphoramidates including 3'-amino phosphoramidate and
aminoalkylphosphoramidates, thionophosphoramidates,
thionoalkylphosphonates, thionoalkylphosphotriest- ers,
selenophosphates and boranophosphates having normal 3'-5' linkages,
2'-5' linked analogs of these, and those having inverted polarity
wherein one or more internucleotide linkages is a 3' to 3', 5' to
5' or 2' to 2' linkage. Oligonucleotides having inverted polarity
can comprise a single 3' to 3' linkage at the 3'-most
internucleotide linkage i.e. a single inverted nucleoside residue
which may be abasic (the nucleobase is missing or has a hydroxyl
group in place thereof). Various salts, mixed salts and free acid
forms are also included.
[0085] Representative U.S. patents that teach the preparation of
the above phosphorus-containing linkages include, but are not
limited to, U.S. Pat. No. 3,687,808; 4,469,863; 4,476,301;
5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302;
5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233;
5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111;
5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899;
5,721,218; 5,672,697 and 5,625,050.
[0086] Modified oligonucleotide backbones that do not include a
phosphorus atom therein can have backbones that are formed by short
chain alkyl or cycloalkyl internucleoside linkages, mixed
heteroatom and alkyl or cycloalkyl internucleoside linkages, or one
or more short chain heteroatomic or heterocyclic internucleoside
linkages. These include those having morpholino linkages (formed in
part from the sugar portion of a nucleoside); siloxane backbones;
sulfide, sulfoxide and sulfone backbones; formacetyl and
thioformacetyl backbones; methylene formacetyl and thioformacetyl
backbones; riboacetyl backbones; alkene containing backbones;
sulfamate backbones; methyleneimino and methylenehydrazino
backbones; sulfonate and sulfonamide backbones; amide backbones;
and others having mixed N, O, S and CH.sub.2 component parts.
[0087] Representative U.S. patents that teach the preparation of
the above oligonucleosides include, but are not limited to, U.S.
Pat. No. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141;
5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677;
5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240;
5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070;
5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and
5,677,439.
[0088] Modified Sugar and Internucleoside Linkages-Mimetics
[0089] In other antisense compounds, e.g., oligonucleotide
mimetics, both the sugar and the internucleoside linkage (i.e. the
backbone), of the nucleotide units are replaced with novel groups.
The nucleobase units are maintained for hybridization with an
appropriate target nucleic acid. One such compound, an
oligonucleotide mimetic that has been shown to have excellent
hybridization properties, is referred to as a peptide nucleic acid
(PNA). In PNA compounds, the sugar-backbone of an oligonucleotide
is replaced with an amide containing backbone, in particular an
aminoethylglycine backbone. The nucleobases are retained and are
bound directly or indirectly to aza nitrogen atoms of the amide
portion of the backbone. Representative U.S. patents that teach the
preparation of PNA compounds include, but are not limited to, U.S.
Pat. No. 5,539,082; 5,714,331; and 5,719,262. Further teaching of
PNA compounds can be found in Nielsen et al., Science, 1991, 254,
1497-1500.
[0090] Some embodiments of the invention include oligonucleotides
with phosphorothioate backbones and oligonucleosides with
heteroatom backbones, and in particular
--CH.sub.2--NH--O--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--O--CH.sub.2-- (known as a methylene
(methylimino) or MMI backbone),
--CH.sub.2--O--N(CH.sub.3)--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--CH.sub.2-- and
--O--N(CH.sub.3)--CH.sub.2--CH.sub.2-- (wherein the native
phosphodiester backbone is represented as --O--P--O--CH.sub.2--) of
the above referenced U.S. Pat. No. 5,489,677, and the amide
backbones of the above referenced U.S. Pat. No. 5,602,240. Also
suitable are oligonucleotides having morpholino backbone structures
of the above-referenced U.S. Pat. No. 5,034,506.
[0091] Modified Sugars
[0092] Modified antisense compounds may also contain one or more
substituted sugar moieties. Antisense compounds, such as antisense
oligonucleotides, can comprise one of the following at the 2'
position: OH; F; O--, S--, or N-alkyl; O--, S--, or N-alkenyl; O--,
S-- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl
and alkynyl may be substituted or unsubstituted C.sub.1 to C.sub.10
alkyl or C.sub.2 to C.sub.10 alkenyl and alkynyl. Also suitable are
O((CH.sub.2).sub.nO).sub.- mCH.sub.3, O(CH.sub.2).sub.nOCH.sub.3,
O(CH.sub.2).sub.nNH.sub.2, O(CH.sub.2).sub.nCH.sub.3,
O(CH.sub.2).sub.nONH.sub.2, and
O(CH.sub.2).sub.nON((CH.sub.2).sub.nCH.sub.3).sub.2, where n and m
are from 1 to about 10. Other oligonucleotides can comprise one of
the following at the 2' position: C.sub.1 to C.sub.10 lower alkyl,
substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl,
O-alkaryl or O-aralkyl, SH, SCH.sub.3, OCN, Cl, Br, CN, CF.sub.3,
OCF.sub.3, SOCH.sub.3, SO.sub.2CH.sub.3, ONO.sub.2, NO.sub.2,
N.sub.3, NH.sub.2, heterocycloalkyl, heterocycloalkaryl,
aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving
group, a reporter group, an intercalator, a group for improving the
pharmacokinetic properties of an oligonucleotide, or a group for
improving the pharmacodynamic properties of an oligonucleotide, and
other substituents having similar properties. One modification
includes 2'-methoxyethoxy (2'-O--CH.sub.2CH.sub.2OCH.sub- .3, also
known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv.
Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. Another
modification includes 2'-dimethylaminooxyethoxy, i.e., a
O(CH.sub.2).sub.2ON(CH.sub.3).sub.2 group, also known as 2'-DMAOE,
as described in examples hereinbelow, and
2'-dimethylaminoethoxyethoxy (also known in the art as
2'-O-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), i.e.,
2'-O--CH.sub.2--O--CH.sub.2--N(CH.sub.3).sub.2, also described in
examples hereinbelow.
[0093] Other modifications include 2'-methoxy (2'-O--CH.sub.3),
2'-aminopropoxy (2'-OCH.sub.2CH.sub.2CH.sub.2NH.sub.2), 2'-allyl
(2'-CH.sub.2--CH.dbd.CH.sub.2), 2'-O-allyl
(2'-O--CH.sub.2--CH.dbd.CH.sub- .2) and 2'-fluoro (2'-F). The
2'-modification may be in the arabino (up) position or ribo (down)
position. A preferred 2'-arabino modification is 2'-F. Similar
modifications may also be made at other positions on the
oligonucleotide, particularly the 3' position of the sugar on the
3' terminal nucleotide or in 2'-5' linked oligonucleotides and the
5' position of 5' terminal nucleotide. Antisense compounds may also
have sugar mimetics such as cyclobutyl moieties in place of the
pentofuranosyl sugar. Representative U.S. patents that teach the
preparation of such modified sugar structures include, but are not
limited to, U.S. Pat. No. 4,981,957; 5,118,800; 5,319,080;
5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134;
5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053;
5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and
5,700,920.
[0094] Another modification of the sugar includes Locked Nucleic
Acids (LNAs) in which the 2'-hydroxyl group is linked to the 3' or
4' carbon atom of the sugar ring, thereby forming a bicyclic sugar
moiety. The linkage can be a methylene (--CH.sub.2--).sub.n group
bridging the 2' oxygen atom and the 4' carbon atom wherein n is 1
or 2. LNAs and preparation thereof are described in WO 98/39352 and
WO 99/14226.
[0095] Natural and Modified Nucleobases
[0096] Antisense compounds may also include nucleobase (often
referred to in the art as heterocyclic base or simply as "base")
modifications or substitutions. As used herein, "unmodified" or
"natural" nucleobases include the purine bases adenine (A) and
guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and
uracil (U). Modified nucleobases include other synthetic and
natural nucleobases such as 5-methylcytosine (5-me-C),
5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,
6-methyl and other alkyl derivatives of adenine and guanine,
2-propyl and other alkyl derivatives of adenine and guanine,
2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and
cytosine, 5-propynyl (--C.ident.C--CH.sub.3) uracil and cytosine
and other alkynyl derivatives of pyrimidine bases, 6-azo uracil,
cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil,
8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other
8-substituted adenines and guanines, 5-halo particularly 5-bromo,
5-trifluoromethyl and other 5-substituted uracils and cytosines,
7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine,
8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine
and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases
include tricyclic pyrimidines such as phenoxazine
cytidine(1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one),
phenothiazine cytidine
(1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps such as a
substituted phenoxazine cytidine (e.g.
9-(2-aminoethoxy)-H-pyrimido(- 5,4-b)(1,4)benzoxazin-2(3H)-one),
carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindole
cytidine (H-pyrido(3',2':4,5)pyrrolo(2,3-d)pyrimidin-2-one).
Modified 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, pages 858-859, Kroschwitz, J. I., ed. John
Wiley & Sons, 1990, those disclosed by Englisch et al.,
Angewandte Chemie, International Edition, 1991, 30, 613, and those
disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and
Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC
Press, 1993. Certain of these nucleobases are particularly useful
for increasing the binding affinity of the compounds of the
invention. These include 5-substituted pyrimidines,
6-azapyrimidines and N-2, N-6 and O-6 substituted purines,
including 2-aminopropyladenine, 5-propynyluracil and
5-propynylcytosine. 5-methylcytosine substitutions have been shown
to increase nucleic acid duplex stability by 0.6-1.2.degree. C. and
are presently suitable base substitutions, even more particularly
when combined with 2'-O-methoxyethyl sugar modifications.
[0097] Representative U.S. patents that teach the preparation of
certain of the above noted modified nucleobases as well as other
modified nucleobases include, but are not limited to, the above
noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 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; 5,830,653; 5,763,588;
6,005,096; and 5,681,941, and 5,750,692.
[0098] Conjugates
[0099] Another modification of the antisense compounds of the
invention involves chemically linking to the antisense compound one
or more moieties or conjugates which enhance the activity, cellular
distribution or cellular uptake of the oligonucleotide. These
moieties or conjugates can include conjugate groups covalently
bound to functional groups such as primary or secondary hydroxyl
groups. Conjugate groups of the invention include, but are not
limited to, intercalators, reporter molecules, polyamines,
polyamides, polyethylene glycols, polyethers, groups that enhance
the pharmacodynamic properties of oligomers, and groups that
enhance the pharmacokinetic properties of oligomers. Typical
conjugate groups include, but are not limited to, cholesterols,
lipids, phospholipids, biotin, phenazine, folate, phenanthridine,
anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and
dyes. Groups that enhance the pharmacodynamic properties, in the
context of this invention, include groups that improve uptake,
enhance resistance to degradation, and/or strengthen
sequence-specific hybridization with the target nucleic acid.
Groups that enhance the pharmacokinetic properties, in the context
of this invention, include groups that improve uptake,
distribution, metabolism or excretion of the compounds of the
present invention. Representative conjugate groups are disclosed in
International Patent Application PCT/US92/09196, filed Oct. 23,
1992, and U.S. Pat. No. 6,287,860. Conjugate moieties include, but
are not limited to, lipid moieties such as a cholesterol moiety,
cholic acid, a thioether, e.g., hexyl-S-tritylthiol, a
thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl
residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or
triethylammonium 1,2-di-O-hexadecyl-rac-glyc- ero-3-H-phosphonate,
a polyamine or a polyethylene glycol chain, or adamantane acetic
acid, a palmityl moiety, or an octadecylamine or
hexylamino-carbonyl-oxycholesterol moiety. Antisense compounds of
the invention may also be conjugated to active drug substances, for
example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen,
fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen,
dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid,
folinic acid, a benzothiadiazide, chlorothiazide, a diazepine,
indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an
antidiabetic, an antibacterial or an antibiotic.
Oligonucleotide-drug conjugates and their preparation are described
in U.S. patent application Ser. No. 09/334,130 (filed Jun. 15,
1999).
[0100] Representative U.S. patents that teach the preparation of
such oligonucleotide conjugates include, but are not limited to,
U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465;
5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731;
5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603;
5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025;
4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582;
4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963;
5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250;
5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463;
5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142;
5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928
and 5,688,941.
[0101] Chimeric Compounds
[0102] It is not necessary for all positions in a given compound to
be uniformly modified, and in fact more than one of the
aforementioned modifications may be incorporated in a single
compound or even at a single nucleoside within an
oligonucleotide.
[0103] The present invention also includes antisense compounds
which are chimeric compounds. "Chimeric" antisense compounds or
"chimeras," in the context of this invention, are antisense
compounds, particularly oligonucleotides, which contain two or more
chemically distinct regions, each made up of at least one monomer
unit, i.e., a nucleotide in the case of an oligonucleotide
compound. Chimeric antisense oligonucleotides are thus a form of
antisense compound. These oligonucleotides typically contain at
least one region wherein the oligonucleotide is modified so as to
confer upon the oligonucleotide increased resistance to nuclease
degradation, increased cellular uptake, increased stability and/or
increased binding affinity for the target nucleic acid. An
additional region of the oligonucleotide may serve as a substrate
for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way
of example, RNAse H is a cellular endonuclease which cleaves the
RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore,
results in cleavage of the RNA target, thereby greatly enhancing
the efficiency of oligonucleotide-mediated inhibition of gene
expression. The cleavage of RNA:RNA hybrids can, in like fashion,
be accomplished through the actions of endoribonucleases, such as
RNAseL which cleaves both cellular and viral RNA. Cleavage of the
RNA target can be routinely detected by gel electrophoresis and, if
necessary, associated nucleic acid hybridization techniques known
in the art.
[0104] Chimeric antisense compounds of the invention may be formed
as composite structures of two or more oligonucleotides, modified
oligonucleotides, oligonucleosides and/or oligonucleotide mimetics
as described above. Such compounds have also been referred to in
the art as hybrids or gapmers. Representative U.S. patents that
teach the preparation of such hybrid structures include, but are
not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007;
5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065;
5,652,355; 5,652,356; and 5,700,922.
[0105] The compounds of the invention may also be admixed,
encapsulated, conjugated or otherwise associated with other
molecules, molecule structures or mixtures of compounds, as for
example, liposomes, receptor-targeted molecules, oral, rectal,
topical or other formulations, for assisting in uptake,
distribution and/or absorption. Representative U.S. patents that
teach the preparation of such uptake, distribution and/or
absorption-assisting formulations include, but are not limited to,
U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127;
5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330;
4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221;
5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854;
5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575;
and 5,595,756.
[0106] The antisense compounds of the invention encompass any
pharmaceutically acceptable salts, esters, or salts of such esters,
or any other compound which, upon administration to an animal,
including a human, is capable of providing (directly or indirectly)
the biologically active metabolite or residue thereof.
[0107] The term "pharmaceutically acceptable salts" refers to
physiologically and pharmaceutically acceptable salts of the
compounds of the invention: i.e., salts that retain the desired
biological activity of the parent compound and do not impart
undesired toxicological effects thereto. For oligonucleotides,
examples of pharmaceutically acceptable salts and their uses are
further described in U.S. Pat. No. 6,287,860. Potassium and sodium
salts are typical salts.
[0108] The present invention also includes pharmaceutical
compositions and formulations which include the antisense compounds
of the invention. The pharmaceutical compositions of the present
invention may be administered in a number of ways depending upon
whether local or systemic treatment is desired and upon the area to
be treated. Administration may be topical (including ophthalmic and
to mucous membranes including vaginal and rectal delivery),
pulmonary, e.g., by inhalation or insufflation of powders or
aerosols, including by nebulizer; intratracheal, intranasal,
epidermal and transdermal), oral or parenteral. Parenteral
administration includes intravenous, intraarterial, subcutaneous,
intraperitoneal or intramuscular injection or infusion; or
intracranial, e.g., intrathecal or intraventricular,
administration. Oligonucleotides with at least one
2'-O-methoxyethyl modification are believed to be particularly
useful for oral administration. Pharmaceutical compositions and
formulations for topical administration may include transdermal
patches, ointments, lotions, creams, gels, drops, suppositories,
sprays, liquids and powders. Conventional pharmaceutical carriers,
aqueous, powder or oily bases, thickeners and the like may be
necessary or desirable. Coated condoms, gloves and the like may
also be useful.
[0109] The pharmaceutical formulations of the present invention,
which may conveniently be presented in unit dosage form, may be
prepared according to conventional techniques well known in the
pharmaceutical industry. Such techniques include the step of
bringing into association the active ingredients with the
pharmaceutical carrier(s) or excipient(s). In general, the
formulations are prepared by uniformly and intimately bringing into
association the active ingredients with liquid carriers or finely
divided solid carriers or both, and then, if necessary, shaping the
product.
[0110] The compositions of the present invention may be formulated
into any of many possible dosage forms such as, but not limited to,
tablets, capsules, gel capsules, liquid syrups, soft gels,
suppositories, and enemas. The compositions of the present
invention may also be formulated as suspensions in aqueous,
non-aqueous or mixed media. Aqueous suspensions may further contain
substances which increase the viscosity of the suspension
including, for example, sodium carboxymethylcellulose, sorbitol
and/or dextran. The suspension may also contain stabilizers.
[0111] Pharmaceutical compositions of the present invention
include, but are not limited to, solutions, emulsions, foams and
liposome-containing formulations. The pharmaceutical compositions
and formulations of the present invention may comprise one or more
penetration enhancers, carriers, excipients or other active or
inactive ingredients.
[0112] Emulsions are typically heterogenous systems of one liquid
dispersed in another in the form of droplets usually exceeding 0.1
.mu.m in diameter. Emulsions may contain additional components in
addition to the dispersed phases, and the active drug which may be
present as a solution in either the aqueous phase, oily phase or
itself as a separate phase. Microemulsions are included as an
embodiment of the present invention. Emulsions and their uses are
well known in the art and are further described in U.S. Pat. No.
6,287,860.
[0113] Formulations of the present invention include liposomal
formulations. As used in the present invention, the term "liposome"
means a vesicle composed of amphiphilic lipids arranged in a
spherical bilayer or bilayers. Liposomes are unilamellar or
multilamellar vesicles which have a membrane formed from a
lipophilic material and an aqueous interior that contains the
composition to be delivered. Cationic liposomes are positively
charged liposomes which are believed to interact with negatively
charged DNA molecules to form a stable complex. Liposomes that are
pH-sensitive or negatively-charged are believed to entrap DNA
rather than complex with it. Both cationic and noncationic
liposomes have been used to deliver DNA to cells.
[0114] Liposomes also include "sterically stabilized" liposomes, a
term which, as used herein, refers to liposomes comprising one or
more specialized lipids that, when incorporated into liposomes,
result in enhanced circulation lifetimes relative to liposomes
lacking such specialized lipids. Examples of sterically stabilized
liposomes are those in which part of the vesicle-forming lipid
portion of the liposome comprises one or more glycolipids or is
derivatized with one or more hydrophilic polymers, such as a
polyethylene glycol (PEG) moiety. Liposomes and their uses are
further described in U.S. Pat. No. 6,287,860.
[0115] The pharmaceutical formulations and compositions of the
present invention may also include surfactants. The use of
surfactants in drug products, formulations and in emulsions is well
known in the art. Surfactants and their uses are further described
in U.S. Pat. No. 6,287,860.
[0116] In one embodiment, the present invention employs various
penetration enhancers to effect the efficient delivery of nucleic
acids, particularly oligonucleotides. In addition to aiding the
diffusion of non-lipophilic drugs across cell membranes,
penetration enhancers also enhance the permeability of lipophilic
drugs. Penetration enhancers may be classified as belonging to one
of five broad categories, i.e., surfactants, fatty acids, bile
salts, chelating agents, and non-chelating non-surfactants.
Penetration enhancers and their uses are further described in U.S.
Pat. No. 6,287,860.
[0117] One of skill in the art will recognize that formulations are
routinely designed according to their intended use, i.e. route of
administration.
[0118] Formulations for topical administration include those in
which the oligonucleotides of the invention are in admixture with a
topical delivery agent such as lipids, liposomes, fatty acids,
fatty acid esters, steroids, chelating agents and surfactants.
Lipids and liposomes include neutral (e.g. dioleoylphosphatidyl
DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC,
distearolyphosphatidyl choline) negative (e.g.
dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.
dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl
ethanolamine DOTMA).
[0119] For topical or other administration, oligonucleotides of the
invention may be encapsulated within liposomes or may form
complexes thereto, in particular to cationic liposomes.
Alternatively, oligonucleotides may be complexed to lipids, in
particular to cationic lipids. Fatty acids and esters,
pharmaceutically acceptable salts thereof, and their uses are
further described in U.S. Pat. No. 6,287,860. Topical formulations
are described in detail in U.S. patent application Ser. No.
09/315,298 filed on May 20, 1999.
[0120] Compositions and formulations for oral administration
include powders or granules, microparticulates, nanoparticulates,
suspensions or solutions in water or non-aqueous media, capsules,
gel capsules, sachets, tablets or minitablets. Thickeners,
flavoring agents, diluents, emulsifiers, dispersing aids or binders
may be desirable. Suitable oral formulations are those in which
oligonucleotides of the invention are administered in conjunction
with one or more penetration enhancers surfactants and chelators.
Surfactants include fatty acids and/or esters or salts thereof,
bile acids and/or salts thereof. Bile acids/salts and fatty acids
and their uses are further described in U.S. Pat. No. 6,287,860.
Also suitable are combinations of penetration enhancers, for
example, fatty acids/salts in combination with bile acids/salts. A
particularly suitable combination is the sodium salt of lauric
acid, capric acid and UDCA. Further penetration enhancers include
polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.
Oligonucleotides of the invention may be delivered orally, in
granular form including sprayed dried particles, or complexed to
form micro or nanoparticles. Oligonucleotide complexing agents and
their uses are further described in U.S. Pat. No. 6,287,860. Oral
formulations for oligonucleotides and their preparation are
described in detail in U.S. applications Ser. No. 09/108,673 (filed
Jul. 1, 1998), Ser. No. 09/315,298 (filed May 20, 1999) and Ser.
No. 10/071,822, filed Feb. 8, 2002 and published as U.S.
Application No. 2003-0027780.
[0121] Compositions and formulations for parenteral, intrathecal or
intraventricular administration may include sterile aqueous
solutions which may also contain buffers, diluents and other
suitable additives such as, but not limited to, penetration
enhancers, carrier compounds and other pharmaceutically acceptable
carriers or excipients.
[0122] Certain embodiments of the invention provide pharmaceutical
compositions containing one or more oligomeric compounds and one or
more other chemotherapeutic agents which function by a
non-antisense mechanism. Examples of such chemotherapeutic agents
include, but are not limited to, cancer chemotherapeutic drugs such
as daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin,
idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide,
cytosine arabinoside, bis-chloroethylnitrosurea, busulfan,
mitomycin C, actinomycin D, mithramycin, prednisone,
hydroxyprogesterone, testosterone, tamoxifen, dacarbazine,
procarbazine, hexamethylmelamine, pentamethylmelamine,
mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea,
nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine,
6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea,
deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil
(5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX),
colchicine, taxol, vincristine, vinblastine, etoposide (VP-16),
trimetrexate, irinotecan, topotecan, gemcitabine, teniposide,
cisplatin and diethylstilbestrol (DES). When used with the
compounds of the invention, such chemotherapeutic agents may be
used individually (e.g., 5-FU and oligonucleotide), sequentially
(e.g., 5-FU and oligonucleotide for a period of time followed by
MTX and oligonucleotide), or in combination with one or more other
such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide,
or 5-FU, radiotherapy and oligonucleotide). Anti-inflammatory drugs
including, but not limited to, nonsteroidal anti-inflammatory drugs
and corticosteroids, and antiviral drugs including, but not limited
to, ribivirin, vidarabine, acyclovir and ganciclovir, may also be
combined in compositions of the invention. Combinations of
antisense compounds and other non-antisense drugs are also within
the scope of this invention. Two or more combined compounds may be
used together or sequentially.
[0123] In another related embodiment, compositions of the invention
may contain one or more antisense compounds, particularly
oligonucleotides, targeted to a first nucleic acid and one or more
additional antisense compounds targeted to a second nucleic acid
target. Alternatively, compositions of the invention may contain
two or more antisense compounds targeted to different regions of
the same nucleic acid target. Numerous examples of antisense
compounds are known in the art. Two or more combined compounds may
be used together or sequentially.
[0124] The formulation of therapeutic compositions and their
subsequent administration (dosing) is believed to be within the
skill of those in the art. Dosing is dependent on severity and
responsiveness of the disease state to be treated, with the course
of treatment lasting from several days to several months, or until
a cure is effected or a diminution of the disease state is
achieved. Optimal dosing schedules can be calculated from
measurements of drug accumulation in the body of the patient.
Persons of ordinary skill can easily determine optimum dosages,
dosing methodologies and repetition rates. Optimum dosages may vary
depending on the relative potency of individual oligonucleotides,
and can generally be estimated based on EC.sub.50s found to be
effective in in vitro and in vivo animal models. In general, dosage
is from 0.01 .mu.g to 100 g per kg of body weight, from 0.1 .mu.g
to 10 g per kg of body weight, from 1 .mu.g to 1 g per kg of body
weight, from 10 .mu.g to 100 mg per kg of body weight, from 100
.mu.g to 10 mg per kg of body weight, or from 100 .mu.g to 1 mg per
kg of body weight, and may be given once or more daily, weekly,
monthly or yearly, or even once every 2 to 20 years. Persons of
ordinary skill in the art can easily estimate repetition rates for
dosing based on measured residence times and concentrations of the
drug in bodily fluids or tissues. Following successful treatment,
it may be desirable to have the patient undergo maintenance therapy
to prevent the recurrence of the disease state, wherein the
oligonucleotide is administered in maintenance doses, ranging from
0.0001 .mu.g to 100 g per kg of body weight, once or more daily, to
once every 20 years.
[0125] In order that the invention disclosed herein may be more
efficiently understood, examples are provided below. It should be
understood that these examples are for illustrative purposes only
and are not to be construed as limiting the invention in any
manner. Throughout these examples, molecular cloning reactions, and
other standard recombinant DNA techniques, were carried out
according to methods described in Maniatis et al., Molecular
Cloning--A Laboratory Manual, 2nd ed., Cold Spring Harbor Press
(1989), using commercially available reagents, except where
otherwise noted.
EXAMPLES
Example 1
Synthesis of Nucleoside Phosphoramidites
[0126] The following compounds, including amidites and their
intermediates were prepared as described in U.S. Pat. No. 6,426,220
and published PCT WO 02/36743; 5'-O-Dimethoxytrityl-thymidine
intermediate for 5-methyl dC amidite,
5'-O-Dimethoxytrityl-2'-deoxy-5-methylcytidine intermediate for
5-methyl-dC amidite,
5'-O-Dimethoxytrityl-2'-deoxy-N4-benzoyl-5-methylcyt- idine
penultimate intermediate for 5-methyl dC amidite,
(5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-deoxy-N.sup.4-benzoyl-5-methylcy-
tidin-3'-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite
(5-methyl dC amidite), 2'-Fluorodeoxyadenosine,
2'-Fluorodeoxyguanosine, 2'-Fluorouridine, 2'-Fluorodeoxycytidine,
2'-O-(2-Methoxyethyl) modified amidites,
2'-O-(2-methoxyethyl)-5-methyluridine intermediate,
5'-O-DMT-2'-O-(2-methoxyethyl)-5-methyluridine penultimate
intermediate,
(5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-O-(2-methoxyethyl)-5-methyluridi-
n-3'-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE T
amidite),
5'-O-Dimethoxytrityl-2'-O-(2-methoxyethyl)-5-methylcytidine
intermediate,
5'-O-dimethoxytrityl-2'-0-(2-methoxyethyl)-N.sup.4-benzoyl-5-methyl-cytid-
ine penultimate intermediate,
(5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-O-(-
2-methoxyethyl)-N.sup.4-benzoyl-5-methylcytidin-3'-O-yl)-2-cyanoethyl-N,N--
diisopropylphosphoramidite (MOE 5-Me--C amidite),
(5'-O-(4,4'-Dimethoxytri-
phenylmethyl)-2'-O-(2-methoxyethyl)-N.sup.6-benzoyladenosin-3'-O-yl)-2-cya-
noethyl-N,N-diisopropylphosphoramidite (MOE A amdite),
(5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-O-(2-methoxyethyl)-N.sup.4-isobu-
tyrylguanosin-3'-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite
(MOE G amidite), 2'-O-(Aminooxyethyl) nucleoside amidites and
2'-O-(dimethylaminooxyethyl) nucleoside amidites,
2'-(Dimethylaminooxyeth- oxy) nucleoside amidites,
5'-O-tert-Butyldiphenylsilyl-O.sup.2-2'-anhydro-- 5-methyluridine,
5'-O-tert-Butyldiphenylsilyl-2'-O-(2-hydroxyethyl)-5-meth-
yluridine,
2'-O-((2-phthalimidoxy)ethyl)-5'-t-butyldiphenylsilyl-5-methylu-
ridine,
5'-O-tert-butyldiphenylsilyl-2'-O-((2-formadoximinooxy)ethyl)-5-me-
thyluridine, 5'-O-tert-Butyldiphenylsilyl-2'-O-(N,N
dimethylaminooxyethyl)-5-methyluridine,
2'-O-(dimethylaminooxyethyl)-5-me- thyluridine,
5'-O-DMT-2'-O-(dimethylaminooxyethyl)-5-methyluridine,
5'-O-DMT-2'-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3'-((2-cyanoe-
thyl)-N,N-diisopropylphosphoramidite), 2'-(Aminooxyethoxy)
nucleoside amidites,
N2-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-ethylacetyl)-5'-O-(-
4,4'-dimethoxytrityl)guanosine-3'-((2-cyanoethyl)-N,N-diisopropylphosphora-
midite), 2'-dimethylaminoethoxyethoxy (2'-DMAEOE) nucleoside
amidites, 2'-O-(2(2-N,N-dimethylaminoethoxy)ethyl)-5-methyl
uridine,
5'-O-dimethoxytrityl-2'-O-(2(2-N,N-dimethylaminoethoxy)-ethyl))-5-methyl
uridine and
5'-O-Dimethoxytrityl-2'-O-(2(2-N,N-dimethylaminoethoxy)-ethyl-
))-5-methyl
uridine-3'-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite.
Example 2
Oligonucleotide and Oligonucleoside Synthesis
[0127] The antisense compounds used in accordance with this
invention may be conveniently and routinely made through the
well-known technique of solid phase synthesis. Equipment for such
synthesis is sold by several vendors including, for example,
Applied Biosystems (Foster City, Calif.). Any other means for such
synthesis known in the art may additionally or alternatively be
employed. It is well known to use similar techniques to prepare
oligonucleotides such as the phosphorothioates and alkylated
derivatives.
[0128] Oligonucleotides:
[0129] Unsubstituted and substituted phosphodiester (P.dbd.O)
oligonucleotides are synthesized on an automated DNA synthesizer
(Applied Biosystems model 394) using standard phosphoramidite
chemistry with oxidation by iodine.
[0130] Phosphorothioates (P.dbd.S) are synthesized similar to
phosphodiester oligonucleotides with the following exceptions:
thiation was effected by utilizing a 10% w/v solution of
3,H-1,2-benzodithiole-3-o- ne 1,1 -dioxide in acetonitrile for the
oxidation of the phosphite linkages. The thiation reaction step
time was increased to 180 sec and preceded by the normal capping
step. After cleavage from the CPG column and deblocking in
concentrated ammonium hydroxide at 55.degree. C. (12-16 hr), the
oligonucleotides were recovered by precipitating with >3 volumes
of ethanol from a 1 M NH.sub.4OAc solution. Phosphinate
oligonucleotides are prepared as described in U.S. Pat. No.
5,508,270.
[0131] Alkyl phosphonate oligonucleotides are prepared as described
in U.S. Pat. No. 4,469,863.
[0132] 3'-Deoxy-3'-methylene phosphonate oligonucleotides are
prepared as described in U.S. Pat. Nos. 5,610,289 or 5,625,050.
[0133] Phosphoramidite oligonucleotides are prepared as described
in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878.
[0134] Alkylphosphonothioate oligonucleotides are prepared as
described in published PCT applications PCT/US94/00902 and
PCT/US93/06976 (published as WO 94/17093 and WO 94/02499,
respectively).
[0135] 3'-Deoxy-3'-amino phosphoramidate oligonucleotides are
prepared as described in U.S. Pat. No. 5,476,925.
[0136] Phosphotriester oligonucleotides are prepared as described
in U.S. Pat. No. 5,023,243.
[0137] Borano phosphate oligonucleotides are prepared as described
in U.S. Pat. Nos. 5,130,302 and 5,177,198.
[0138] Oligonucleosides:
[0139] Methylenemethylimino linked oligonucleosides, also
identified as MMI linked oligonucleosides, methylenedimethylhydrazo
linked oligonucleosides, also identified as MDH linked
oligonucleosides, and methylenecarbonylamino linked
oligonucleosides, also identified as amide-3 linked
oligonucleosides, and methyleneaminocarbonyl linked
oligo-nucleosides, also identified as amide-4 linked
oligonucleosides, as well as mixed backbone compounds having, for
instance, alternating MMI and P.dbd.O or P.dbd.S linkages are
prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023,
5,489,677, 5,602,240 and 5,610,289.
[0140] Formacetal and thioformacetal linked oligonucleosides are
prepared as described in U.S. Pat. Nos. 5,264,562 and
5,264,564.
[0141] Ethylene oxide linked oligonucleosides are prepared as
described in U.S. Pat. No. 5,223,618.
Example 3
RNA Synthesis
[0142] In general, RNA synthesis chemistry is based on the
selective incorporation of various protecting groups at strategic
intermediary reactions. Although one of ordinary skill in the art
will understand the use of protecting groups in organic synthesis,
a useful class of protecting groups includes silyl ethers. In
particular bulky silyl ethers are used to protect the 5'-hydroxyl
in combination with an acid-labile orthoester protecting group on
the 2'-hydroxyl. This set of protecting groups is then used with
standard solid-phase synthesis technology. It is important to
lastly remove the acid labile orthoester protecting group after all
other synthetic steps. Moreover, the early use of the silyl
protecting groups during synthesis ensures facile removal when
desired, without undesired deprotection of 2' hydroxyl.
[0143] Following this procedure for the sequential protection of
the 5'-hydroxyl in combination with protection of the 2'-hydroxyl
by protecting groups that are differentially removed and are
differentially chemically labile, RNA oligonucleotides were
synthesized.
[0144] RNA oligonucleotides are synthesized in a stepwise fashion.
Each nucleotide is added sequentially (3'- to 5'-direction) to a
solid support-bound oligonucleotide. The first nucleoside at the
3'-end of the chain is covalently attached to a solid support. The
nucleotide precursor, a ribonucleoside phosphoramidite, and
activator are added, coupling the second base onto the 5'-end of
the first nucleoside. The support is washed and any unreacted
5'-hydroxyl groups are capped with acetic anhydride to yield
5'-acetyl moieties. The linkage is then oxidized to the more stable
and ultimately desired P(V) linkage. At the end of the nucleotide
addition cycle, the 5'-silyl group is cleaved with fluoride. The
cycle is repeated for each subsequent nucleotide.
[0145] Following synthesis, the methyl protecting groups on the
phosphates are cleaved in 30 minutes utilizing 1 M
disodium-2-carbamoyl-2-cyanoethyl- ene-1,1-dithiolate trihydrate
(S.sub.2Na.sub.2) in DMF. The deprotection solution is washed from
the solid support-bound oligonucleotide using water. The support is
then treated with 40% methylamine in water for 10 minutes at
55.degree. C. This releases the RNA oligonucleotides into solution,
deprotects the exocyclic amines, and modifies the 2'-groups. The
oligonucleotides can be analyzed by anion exchange HPLC at this
stage.
[0146] The 2'-orthoester groups are the last protecting groups to
be removed. The ethylene glycol monoacetate orthoester protecting
group developed by Dharmacon Research, Inc. (Lafayette, Colo.), is
one example of a useful orthoester protecting group which, has the
following important properties. It is stable to the conditions of
nucleoside phosphoramidite synthesis and oligonucleotide synthesis.
However, after oligonucleotide synthesis the oligonucleotide is
treated with methylamine which not only cleaves the oligonucleotide
from the solid support but also removes the acetyl groups from the
orthoesters. The resulting 2-ethyl-hydroxyl substituents on the
orthoester are less electron withdrawing than the acetylated
precursor. As a result, the modified orthoester becomes more labile
to acid-catalyzed hydrolysis. Specifically, the rate of cleavage is
approximately 10 times faster after the acetyl groups are removed.
Therefore, this orthoester possesses sufficient stability in order
to be compatible with oligonucleotide synthesis and yet, when
subsequently modified, permits deprotection to be carried out under
relatively mild aqueous conditions compatible with the final RNA
oligonucleotide product.
[0147] Additionally, methods of RNA synthesis are well known in the
art (Scaringe, S. A. Ph.D. Thesis, University of Colorado, 1996;
Scaringe, S. A., et al., J. Am. Chem. Soc., 1998, 120, 11820-11821;
Matteucci, M. D. and Caruthers, M. H. J. Am. Chem. Soc., 1981, 103,
3185-3191; Beaucage, S. L. and Caruthers, M. H. Tetrahedron Lett.,
1981, 22, 1859-1862; Dahl, B. J., et al., Acta Chem. Scand,. 1990,
44, 639-641; Reddy, M. P., et al., Tetrahedrom Lett., 1994, 25,
4311-4314; Wincott, F. et al., Nucleic Acids Res., 1995, 23,
2677-2684; Griffin, B. E., et al., Tetrahedron, 1967, 23,
2301-2313; Griffin, B. E., et al., Tetrahedron, 1967, 23,
2315-2331).
[0148] RNA antisense compounds (RNA oligonucleotides) of the
present invention can be synthesized by the methods herein or
purchased from Dharmacon Research, Inc (Lafayette, Colo.). Once
synthesized, complementary RNA antisense compounds can then be
annealed by methods known in the art to form double stranded
(duplexed) antisense compounds. For example, duplexes can be formed
by combining 30 .mu.l of each of the complementary strands of RNA
oligonucleotides (50 uM RNA oligonucleotide solution) and 15 .mu.l
of 5.times. annealing buffer (100 mM potassium acetate, 30 mM
HEPES-KOH pH 7.4, 2 mM magnesium acetate) followed by heating for 1
minute at 90.degree. C., then 1 hour at 37.degree. C. The resulting
duplexed antisense compounds can be used in kits, assays, screens,
or other methods to investigate the role of a target nucleic acid,
or for diagnostic or therapeutic purposes.
Example 4
Synthesis of Chimeric Compounds
[0149] Chimeric oligonucleotides, oligonucleosides or mixed
oligonucleotides/oligonucleosides of the invention can be of
several different types. These include a first type wherein the
"gap" segment of linked nucleosides is positioned between 5' and 3'
"wing" segments of linked nucleosides and a second "open end" type
wherein the "gap" segment is located at either the 3' or the 5'
terminus of the oligomeric compound. Oligonucleotides of the first
type are also known in the art as "gapmers" or gapped
oligonucleotides. Oligonucleotides of the second type are also
known in the art as "hemimers" or "wingmers".
(2'-O--Me)-(2'-deoxy)-(2'O--Me) Chimeric Phosphorothioate
Oligonucleotides
[0150] Chimeric oligonucleotides having 2'-O-alkyl phosphorothioate
and 2'-deoxy phosphorothioate oligonucleotide segments are
synthesized using an Applied Biosystems automated DNA synthesizer
Model 394, as above. Oligonucleotides are synthesized using the
automated synthesizer and
2'-deoxy-5'-dimethoxytrityl-3'-O-phosphoramidite for the DNA
portion and 5'-dimethoxytrityl-2'-O-methyl-3'-O-phosphoramidite for
5' and 3' wings. The standard synthesis cycle is modified by
incorporating coupling steps with increased reaction times for the
5'-dimethoxytrityl-2'-O-methyl-3'-O- -phosphoramidite. The fully
protected oligonucleotide is cleaved from the support and
deprotected in concentrated ammonia (NH.sub.4OH) for 12-16 hr at
55.degree. C. The deprotected oligo is then recovered by an
appropriate method (precipitation, column chromatography, volume
reduced in vacuo and analyzed spetrophotometrically for yield and
for purity by capillary electrophoresis and by mass
spectrometry.
(2 '-O-(2-Methoxyethyl))-(2'-deoxy)-(2'-O-(Methoxyethyl)) Chimeric
Phosphorothioate Oligonucleotides
[0151] (2'-O-(2-methoxyethyl))-(2'-deoxy)-(2'-O-(methoxyethyl))
chimeric phosphorothioate oligonucleotides were prepared as per the
procedure above for the 2'-O-methyl chimeric oligonucleotide, with
the substitution of 2'-O-(methoxyethyl) amidites for the
2'-O-methyl amidites.
(2 '-O-(2-Methoxyethyl)Phosphodiester)-(2 '-deoxy
Phosphorothioate)-(2'-O-- (2-Methoxyethyl) Phosphodiester) Chimeric
Oligonucleotides
[0152] (2'-O-(2-methoxyethyl phosphodiester)-(2'-deoxy
phosphorothioate)-(2'-O-(methoxyethyl) phosphodiester) chimeric
oligonucleotides are prepared as per the above procedure for the
2'-O-methyl chimeric oligonucleotide with the substitution of
2'-O-(methoxyethyl) amidites for the 2'-O-methyl amidites,
oxidation with iodine to generate the phosphodiester
internucleotide linkages within the wing portions of the chimeric
structures and sulfurization utilizing 3,H-1,2 benzodithiole-3-one
1,1 dioxide (Beaucage Reagent) to generate the phosphorothioate
internucleotide linkages for the center gap.
[0153] Other chimeric oligonucleotides, chimeric oligonucleosides
and mixed chimeric oligonucleotides/oligonucleosides are
synthesized according to U.S. Pat. No. 5,623,065, herein
incorporated by reference.
Example 5
Design and Screening of Duplexed Antisense Compounds Targeting
SGLT2
[0154] In accordance with the present invention, a series of
nucleic acid duplexes comprising the antisense compounds of the
present invention and their complements can be designed to target
SGLT2. The nucleobase sequence of the antisense strand of the
duplex comprises at least an 8-nucleobase portion of an
oligonucleotide in Table 1. The ends of the strands may be modified
by the addition of one or more natural or modified nucleobases to
form an overhang. The sense strand of the dsRNA is then designed
and synthesized as the complement of the antisense strand and may
also contain modifications or additions to either terminus. For
example, in one embodiment, both strands of the dsRNA duplex would
be complementary over the central nucleobases, each having
overhangs at one or both termini.
[0155] For example, a duplex comprising an antisense strand having
the sequence CGAGAGGCGGACGGGACCG (SEQ ID NO: 268) and having a
two-nucleobase overhang of deoxythymidine(dT) would have the
following structure:
1 cgagaggcggacgggaccgTT Antisense (SEQ ID NO:269)
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline. Strand
TTgctctccgcctgccctggc Comple- (SEQ ID NO:270) ment
[0156] In another embodiment, a duplex comprising an antisense
strand having the same sequence CGAGAGGCGGACGGGACCG (SEQ ID NO:
268) may be prepared with blunt ends (no single stranded overhang)
as shown:
2 cgagaggcggacgggaccg Antisense .vertline..vertline..vertl-
ine..vertline..vertline..vertline..vertline..vertline..vertline..vertline.-
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ver-
tline..vertline. Strand gctctccgcctgccctggc Complement (SEQ ID
NO:271)
[0157] RNA strands of the duplex can be synthesized by methods
disclosed herein or purchased from Dharmacon Research Inc.,
(Lafayette, Colo.). Once synthesized, the complementary strands are
annealed. The single strands are aliquoted and diluted to a
concentration of 50 .mu.M. Once diluted, 30 .mu.L of each strand is
combined with 15 .mu.L of a 5.times. solution of annealing buffer.
The final concentration of said buffer is 100 mM potassium acetate,
30 mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate. The final
volume is 75 .mu.L. This solution is incubated for 1 minute at
90.degree. C. and then centrifuged for 15 seconds. The tube is
allowed to sit for 1 hour at 37.degree. C. at which time the dsRNA
duplexes are used in experimentation. The final concentration of
the dsRNA duplex is 20 .mu.M. This solution can be stored frozen
(-20.degree. C.) and freeze-thawed up to 5 times.
[0158] Once prepared, the duplexed antisense compounds are
evaluated for their ability to modulate SGLT2 expression.
[0159] When cells reached 80% confluency, they are treated with
duplexed antisense compounds of the invention. For cells grown in
96-well plates, wells are washed once with 200 .mu.L OPTI-MEM-1
reduced-serum medium (Gibco BRL) and then treated with 130 .mu.L of
OPTI-MEM-1 containing 12 .mu.g/mL LIPOFECTIN (Gibco BRL) and the
desired duplex antisense compound at a final concentration of 200
mM. After 5 hours of treatment, the medium is replaced with fresh
medium. Cells are harvested 16 hours after treatment, at which time
RNA is isolated and target reduction measured by RT-PCR.
Example 6
Oligonucleotide Isolation
[0160] After cleavage from the controlled pore glass solid support
and deblocking in concentrated ammonium hydroxide at 55.degree. C.
for 12-16 hours, the oligonucleotides or oligonucleosides are
recovered by precipitation out of 1 M NH.sub.4OAc with >3
volumes of ethanol. Synthesized oligonucleotides were analyzed by
electrospray mass spectroscopy (molecular weight determination) and
by capillary gel electrophoresis and judged to be at least 70% full
length material. The relative amounts of phosphorothioate and
phosphodiester linkages obtained in the synthesis was determined by
the ratio of correct molecular weight relative to the -16 amu
product (.+-.32 .+-.48). For some studies oligonucleotides were
purified by HPLC, as described by Chiang et al., J. Biol. Chem.
1991, 266, 18162-18171. Results obtained with HPLC-purified
material were similar to those obtained with non-HPLC purified
material.
Example 7
Oligonucleotide Synthesis--96 Well Plate Format
[0161] Oligonucleotides were synthesized via solid phase P(III)
phosphoramidite chemistry on an automated synthesizer capable of
assembling 96 sequences simultaneously in a 96-well format.
Phosphodiester internucleotide linkages were afforded by oxidation
with aqueous iodine. Phosphorothioate internucleotide linkages were
generated by sulfurization utilizing 3,H-1,2 benzodithiole-3-one
1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile. Standard
base-protected beta-cyanoethyl-diiso-propyl phosphoramidites were
purchased from commercial vendors (e.g. PE-Applied Biosystems,
Foster City, Calif., or Pharmacia, Piscataway, N.J.). Non-standard
nucleosides are synthesized as per standard or patented methods.
They are utilized as base protected beta-cyanoethyldiisopropyl
phosphoramidites.
[0162] Oligonucleotides were cleaved from support and deprotected
with concentrated NH.sub.4OH at elevated temperature (55-60.degree.
C.) for 12-16 hours and the released product then dried in vacuo.
The dried product was then re-suspended in sterile water to afford
a master plate from which all analytical and test plate samples are
then diluted utilizing robotic pipettors.
Example 8
Oligonucleotide Analysis--96-Well Plate Format
[0163] The concentration of oligonucleotide in each well was
assessed by dilution of samples and ]UV absorption spectroscopy.
The full-length integrity of the individual products was evaluated
by capillary electrophoresis (CE) in either the 96-well format
(Beckman P/ACE.TM. MDQ) or, for individually prepared samples, on a
commercial CE apparatus (e.g., Beckman P/ACE.TM. 5000, ABI 270).
Base and backbone composition was confirmed by mass analysis of the
compounds utilizing electrospray-mass spectroscopy. All assay test
plates were diluted from the master plate using single and
multi-channel robotic pipettors. Plates were judged to be
acceptable if at least 85% of the compounds on the plate were at
least 85% full length.
Example 9
Cell Culture and Oligonucleotide Treatment
[0164] The effect of antisense compounds on target nucleic acid
expression can be tested in any of a variety of cell types provided
that the target nucleic acid is present at measurable levels. This
can be routinely determined using, for example, PCR or Northern
blot analysis. The following cell types are provided for
illustrative purposes, but other cell types can be routinely used,
provided that the target is expressed in the cell type chosen. This
can be readily determined by methods routine in the art, for
example Northern blot analysis, ribonuclease protection assays, or
RT-PCR.
[0165] T-24 Cells:
[0166] The human transitional cell bladder carcinoma cell line T-24
was obtained from the American Type Culture Collection (ATCC)
(Nanassas, Va.). T-24 cells were routinely cultured in complete
McCoy's 5A basal media (Invitrogen Corporation, Carlsbad, Calif.)
supplemented with 10% fetal calf serum (Invitrogen Corporation,
Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin
100 micrograms per mL (Invitrogen Corporation, Carlsbad, Calif.).
Cells were routinely passaged by trypsinization and dilution when
they reached 90% confluence. Cells were seeded into 96-well plates
(Falcon-Primaria #353872) at a density of 7000 cells/well for use
in RT-PCR analysis.
[0167] A549 Cells:
[0168] The human lung carcinoma cell line A549 was obtained from
the American Type Culture Collection (ATCC) (Manassas, Va.). A549
cells were routinely cultured in DMEM basal media (Invitrogen
Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf
serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100
units per mL, and streptomycin 100 micrograms per mL (Invitrogen
Corporation, Carlsbad, Calif.). Cells were routinely passaged by
trypsinization and dilution when they reached 90% confluence.
[0169] NHDF Cells:
[0170] Human neonatal dermal fibroblast (NHDF) were obtained from
the Clonetics Corporation (Walkersville, Md.). NHDFs were routinely
maintained in Fibroblast Growth Medium (Clonetics Corporation,
Walkersville, Md.) supplemented as recommended by the supplier.
Cells were maintained for up to 10 passages as recommended by the
supplier.
[0171] HEK Cells:
[0172] Human embryonic keratinocytes (HEK) were obtained from the
Clonetics Corporation (Walkersville, Md.). HEKs were routinely
maintained in Keratinocyte Growth Medium (Clonetics Corporation,
Walkersville, Md.) formulated as recommended by the supplier. Cells
were routinely maintained for up to 10 passages as recommended by
the supplier.
[0173] HK-2 Cells:
[0174] HK-2 (human kidney 2) is a proximal tubular cell (PTC) line
derived from normal kidney cells immortalized by transduction with
human papilloma virus 16 (HPV-16) E6/E7 genes (CRL-2190, American
Type Culture Collection, Manassus, Va.). HK-2 cells were routinely
cultured in Keratinocyte-Serum Free Medium (17005-042, Invitrogen
Corporation, Carlsbad, Calif.) which includes 5 ng/ml recombinant
epidermal growth factor and 0.05 mg/ml bovine pituitary extract.
Cells were routinely passaged by trypsinization and split at a
ratio of 1:4 when they reached 70-80% confluence. One day prior to
transfection, cells were seeded into 96-well plates
(Falcon-Primaria #353872, BD Biosciences, Bedford, Mass.) at a
density of 10,000 cells/well.
[0175] b. END Cells:
[0176] The mouse brain endothelial cell line b.END was obtained
from Dr. Werner Risau at the Max Plank Instititute (Bad Nauheim,
Germany). b.END cells were routinely cultured in DMEM, high glucose
(Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10%
fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.).
Cells were routinely passaged by trypsinization and dilution when
they reached 90% confluence. Cells were seeded into 96-well plates
(Falcon-Primaria #3872) at a density of 3000 cells/well for use in
RT-PCR analysis.
[0177] Treatment with Antisense Compounds:
[0178] When cells reached 65-75% confluency, they were treated with
oligonucleotide. For cells grown in 96-well plates, wells were
washed once with 100 .mu.L OPTI-MEM.TM.-1 reduced-serum medium
(Invitrogen Corporation, Carlsbad, Calif.) and then treated with
130 .mu.L of OPTI-MEM.TM.-1 containing 3.75 .mu.g/mL LIPOFECTIN.TM.
(Invitrogen Corporation, Carlsbad, Calif.) and the desired
concentration of oligonucleotide. Cells are treated and data are
obtained in triplicate. After 4-7 hours of treatment at 37.degree.
C., the medium was replaced with fresh medium. Cells were harvested
16-24 hours after oligonucleotide treatment.
[0179] The concentration of oligonucleotide used varies from cell
line to cell line. To determine the optimal oligonucleotide
concentration for a particular cell line, the cells are treated
with a positive control oligonucleotide at a range of
concentrations. For human cells the positive control
oligonucleotide is selected from either ISIS 13920
(TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1) which is targeted to human
H-ras, or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 2) which is
targeted to human Jun-N-terminal kinase-2 (JNK2). Both controls are
2'-O-methoxyethyl gapmers (2'-O-methoxyethyls shown in bold) with a
phosphorothioate backbone. For mouse or rat cells the positive
control oligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID
NO: 3, a 2'-O-methoxyethyl gapmer (2'-O-methoxyethyls shown in
bold) with a phosphorothioate backbone which is targeted to both
mouse and rat c-raf. The concentration of positive control
oligonucleotide that results in 80% inhibition of c-H-ras (for ISIS
13920), JNK2 (for ISIS 18078) or c-raf (for ISIS 15770) mRNA is
then utilized as the screening concentration for new
oligonucleotides in subsequent experiments for that cell line. If
80% inhibition is not achieved, the lowest concentration of
positive control oligonucleotide that results in 60% inhibition of
c-H-ras, JNK2 or c-raf mRNA is then utilized as the oligonucleotide
screening concentration in subsequent experiments for that cell
line. If 60% inhibition is not achieved, that particular cell line
is deemed as unsuitable for oligonucleotide transfection
experiments. The concentrations of antisense oligonucleotides used
herein are from 50 nM to 300 nM.
[0180] For Northern blotting or other analysis, cells may be seeded
onto 100 mm or other standard tissue culture plates and treated
similarly, using appropriate volumes of medium and
oligonucleotide.
Example 10
Analysis of Oligonucleotide Inhibition of SGLT2 Expression
[0181] Antisense modulation of SGLT2 expression can be assayed in a
variety of ways known in the art. For example, SGLT2 mRNA levels
can be quantitated by, e.g., Northern blot analysis, competitive
polymerase chain reaction (PCR), or real-time PCR (RT-PCR).
Real-time quantitative PCR is presently preferred. RNA analysis can
be performed on total cellular RNA or poly(A)+ mRNA. The preferred
method of RNA analysis of the present invention is the use of total
cellular RNA as described in other examples herein. Methods of RNA
isolation are well known in the art. Northern blot analysis is also
routine in the art. Real-time quantitative (PCR) can be
conveniently accomplished using the commercially available ABI
PRISM.TM. 7600, 7700, or 7900 Sequence Detection System, available
from PE-Applied Biosystems, Foster City, Calif. and used according
to manufacturer's instructions.
[0182] Protein levels of SGLT2 can be quantitated in a variety of
ways well known in the art, such as immunoprecipitation, Western
blot analysis (immunoblotting), enzyme-linked immunosorbent assay
(ELISA) or fluorescence-activated cell sorting (FACS). Antibodies
directed to SGLT2 can be identified and obtained from a variety of
sources, such as the MSRS catalog of antibodies (Aerie Corporation,
Birmingham, Mich.), or can be prepared via conventional monoclonal
or polyclonal antibody generation methods well known in the
art.
Example 11
Design of Phenotypic Assays for the Use of SGLT2 Inhibitors
[0183] Phenotypic Assays
[0184] Once SGLT2 inhibitors have been identified by the methods
disclosed herein, the compounds are further investigated in one or
more phenotypic assays, each having measurable endpoints predictive
of efficacy in the treatment of a particular disease state or
condition. Phenotypic assays, kits and reagents for their use are
well known to those skilled in the art and are herein used to
investigate the role and/or association of SGLT2 in health and
disease. Representative phenotypic assays, which can be purchased
from any one of several commercial vendors, include those for
determining cell viability, cytotoxicity, proliferation or cell
survival (Molecular Probes, Eugene, Oreg.; PerkinElmer, Boston,
Mass.), protein-based assays including enzymatic assays (Panvera,
LLC, Madison, Wis.; BD Biosciences, Franklin Lakes, N.J.; Oncogene
Research Products, San Diego, Calif.), cell regulation, signal
transduction, inflammation, oxidative processes and apoptosis
(Assay Designs Inc., Ann Arbor, Mich.), triglyceride accumulation
(Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tube
formation assays, cytokine and hormone assays and metabolic assays
(Chemicon International Inc., Temecula, Calif.; Amersham
Biosciences, Piscataway, N.J.).
[0185] In one non-limiting example, cells determined to be
appropriate for a particular phenotypic assay (i.e., MCF-7 cells
selected for breast cancer studies; adipocytes for obesity studies)
are treated with SGLT2 inhibitors identified from the in vitro
studies as well as control compounds at optimal concentrations
which are determined by the methods described above. At the end of
the treatment period, treated and untreated cells are analyzed by
one or more methods specific for the assay to determine phenotypic
outcomes and endpoints.
[0186] Phenotypic endpoints include changes in cell morphology over
time or treatment dose as well as changes in levels of cellular
components such as proteins, lipids, nucleic acids, hormones,
saccharides or metals. Measurements of cellular status which
include pH, stage of the cell cycle, intake or excretion of
biological indicators by the cell, are also endpoints of
interest.
[0187] Analysis of the genotype of the cell (measurement of the
expression of one or more of the genes of the cell) after treatment
is also used as an indicator of the efficacy or potency of the
SGLT2 inhibitors. Hallmark genes, or those genes suspected to be
associated with a specific disease state, condition, or phenotype,
are measured in both treated and untreated cells.
Example 12
RNA Isolation
[0188] Poly(A)+ mRNA Isolation
[0189] Poly(A)+ mRNA was isolated according to Miura et al., (Clin.
Chem., 1996, 42, 1758-1764). Other methods for poly(A)+ mRNA
isolation are routine in the art. Briefly, for cells grown on
96-well plates, growth medium was removed from the cells and each
well was washed with 200 .mu.L cold PBS. 60 .mu.L lysis buffer (10
mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM
vanadyl-ribonucleoside complex) was added to each well, the plate
was gently agitated and then incubated at room temperature for five
minutes. 55 .mu.L of lysate was transferred to Oligo d(T) coated
96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated
for 60 minutes at room temperature, washed 3 times with 200 .mu.L
of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl).
After the final wash, the plate was blotted on paper towels to
remove excess wash buffer and then air-dried for 5 minutes. 60
.mu.L of elution buffer (5 mM Tris-HCl pH 7.6), preheated to
70.degree. C., was added to each well, the plate was incubated on a
90.degree. C. hot plate for 5 minutes, and the eluate was then
transferred to a fresh 96-well plate.
[0190] Cells grown on 100 mm or other standard plates may be
treated similarly, using appropriate volumes of all solutions.
[0191] Total RNA Isolation
[0192] Total RNA was isolated using an RNEASY 96.TM. kit and
buffers purchased from Qiagen Inc. (Valencia, Calif.) following the
manufacturer's recommended procedures. Briefly, for cells grown on
96-well plates, growth medium was removed from the cells and each
well was washed with 200 .mu.L cold PBS. 150 .mu.L Buffer RLT was
added to each well and the plate vigorously agitated for 20
seconds. 150 .mu.L of 70% ethanol was then added to each well and
the contents mixed by pipetting three times up and down. The
samples were then transferred to the RNEASY 96.TM. well plate
attached to a QIAVAC.TM. manifold fitted with a waste collection
tray and attached to a vacuum source. Vacuum was applied for 1
minute. 500 .mu.L of Buffer RW1 was added to each well of the
RNEASY 96.TM. plate and incubated for 15 minutes and the vacuum was
again applied for 1 minute. An additional 500 .mu.L of Buffer RW1
was added to each well of the RNEASY 96.TM. plate and the vacuum
was applied for 2 minutes. 1 mL of Buffer RPE was then added to
each well of the RNEASY 96.TM. plate and the vacuum applied for a
period of 90 seconds. The Buffer RPE wash was then repeated and the
vacuum was applied for an additional 3 minutes. The plate was then
removed from the QIAVAC.TM. manifold and blotted dry on paper
towels. The plate was then re-attached to the QIAVAC.TM. manifold
fitted with a collection tube rack containing 1.2 mL collection
tubes. RNA was then eluted by pipetting 140 .mu.L of RNAse free
water into each well, incubating 1 minute, and then applying the
vacuum for 3 minutes.
[0193] The repetitive pipetting and elution steps may be automated
using a QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.).
Essentially, after lysing of the cells on the culture plate, the
plate is transferred to the robot deck where the pipetting, DNase
treatment and elution steps are carried out.
Example 13
Real-Time Quantitative PCR Analysis of SGLT2 mRNA Levels
[0194] Quantitation of SGLT2 mRNA levels was accomplished by
real-time quantitative PCR using the ABI PRISM.TM. 7600, 7700, or
7900 Sequence Detection System (PE-Applied Biosystems, Foster City,
Calif.) according to manufacturer's instructions. This is a
closed-tube, non-gel-based, fluorescence detection system which
allows high-throughput quantitation of polymerase chain reaction
(PCR) products in real-time. As opposed to standard PCR in which
amplification products are quantitated after the PCR is completed,
products in real-time quantitative PCR are quantitated as they
accumulate. This is accomplished by including in the PCR reaction
an oligonucleotide probe that anneals specifically between the
forward and reverse PCR primers, and contains two fluorescent dyes.
A reporter dye (e.g., FAM or JOE, obtained from either PE-Applied
Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda,
Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is
attached to the 5' end of the probe and a quencher dye (e.g.,
TAMRA, obtained from either PE-Applied Biosystems, Foster City,
Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA
Technologies Inc., Coralville, Iowa) is attached to the 3' end of
the probe. When the probe and dyes are intact, reporter dye
emission is quenched by the proximity of the 3' quencher dye.
During amplification, annealing of the probe to the target sequence
creates a substrate that can be cleaved by the 5'-exonuclease
activity of Taq polymerase. During the extension phase of the PCR
amplification cycle, cleavage of the probe by Taq polymerase
releases the reporter dye from the remainder of the probe (and
hence from the quencher moiety) and a sequence-specific fluorescent
signal is generated. With each cycle, additional reporter dye
molecules are cleaved from their respective probes, and the
fluorescence intensity is monitored at regular intervals by laser
optics built into the ABI PRISM.TM. Sequence Detection System. In
each assay, a series of parallel reactions containing serial
dilutions of mRNA from untreated control samples generates a
standard curve that is used to quantitate the percent inhibition
after antisense oligonucleotide treatment of test samples.
[0195] Prior to quantitative PCR analysis, primer-probe sets
specific to the target gene being measured are evaluated for their
ability to be "multiplexed" with a GAPDH amplification reaction. In
multiplexing, both the target gene and the internal standard gene
GAPDH are amplified concurrently in a single sample. In this
analysis, mRNA isolated from untreated cells is serially diluted.
Each dilution is amplified in the presence of primer-probe sets
specific for GAPDH only, target gene only ("single-plexing"), or
both (multiplexing). Following PCR amplification, standard curves
of GAPDH and target mRNA signal as a function of dilution are
generated from both the single-plexed and multiplexed samples. If
both the slope and correlation coefficient of the GAPDH and target
signals generated from the multiplexed samples fall within 10% of
their corresponding values generated from the single-plexed
samples, the primer-probe set specific for that target is deemed
multiplexable. Other methods of PCR are also known in the art.
[0196] PCR reagents were obtained from Invitrogen Corporation,
(Carlsbad, Calif.). RT-PCR reactions were carried out by adding 20
.mu.L PCR cocktail (2.5.times. PCR buffer minus MgCl.sub.2, 6.6 mM
MgCl.sub.2, 375 .mu.M each of dATP, dCTP, dCTP and dGTP, 375 nM
each of forward primer and reverse primer, 125 nM of probe, 4 Units
RNAse inhibitor, 1.25 Units PLATINUM.RTM. Taq, 5 Units MuLV reverse
transcriptase, and 2.5.times. ROX dye) to 96-well plates containing
30 .mu.L total RNA solution (20-200 ng). The RT reaction was
carried out by incubation for 30 minutes at 48.degree. C. Following
a 10 minute incubation at 95.degree. C. to activate the
PLATINUM.RTM. Taq, 40 cycles of a two-step PCR protocol were
carried out: 95.degree. C. for 15 seconds (denaturation) followed
by 60.degree. C. for 1.5 minutes (annealing/extension).
[0197] Gene target quantities obtained by real time RT-PCR are
normalized using either the expression level of GAPDH, a gene whose
expression is constant, or by quantifying total RNA using
RiboGreen.TM. (Molecular Probes, Inc. Eugene, Oreg.). GAPDH
expression is quantified by real time RT-PCR, by being run
simultaneously with the target, multiplexing, or separately. Total
RNA is quantified using RiboGreen.TM. RNA quantification reagent
(Molecular Probes, Inc. Eugene, Oreg.). Methods of RNA
quantification by RiboGreen.TM. are taught in Jones, L. J., et al,
(Analytical Biochemistry, 1998, 265, 368-374).
[0198] In this assay, 170 .mu.L of RiboGreen.TM. working reagent
(RiboGreen.TM. reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA,
pH 7.5) is pipetted into a 96-well plate containing 30 .mu.L
purified, cellular RNA. The plate is read in a CytoFluor 4000 (PE
Applied Biosystems) with excitation at 485 nm and emission at 530
nm.
[0199] Probes and primers to human SGLT2 were designed to hybridize
to a human SGLT2 sequence, using published sequence information
(GenBank accession number NM.sub.--003041.1, incorporated herein as
SEQ ID NO: 4). For human SGLT2 the PCR primers were:
3 forward primer: TCGGCGTGCCCAGCT (SEQ ID NO: 5) reverse primer:
AGAACAGCACAATGGCGAAGT (SEQ ID NO: 6) and the PCR probe was:
FAM-TCCTCTGCGGCGTGCACTACCTC- -TAMRA (SEQ ID NO: 7)
[0200] where FAM is the fluorescent dye and TAMRA is the quencher
dye. For human GAPDH the PCR primers were:
4 forward primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 8) reverse
primer: GAAGATGGTGATGGGATTTC (SEQ ID NO: 9) and the PCR probe was:
5' JOE-CAAGCTTCCCGTTCTCAGCC- (SEQ ID NO: 10) TAMRA 3'
[0201] where JOE is the fluorescent reporter dye and TAMRA is the
quencher dye.
[0202] Probes and primers to mouse SGLT2 were designed to hybridize
to a mouse SGLT2 sequence, using published sequence information
(the concatenation of the sequences with the GenBank accession
numbers: AJ292928, AW106808, AI789450, AW046901, the complement of
AI647605, the complement of AW107250, and the complement of
AI788744, incorporated herein as SEQ ID NO: 11). For mouse SGLT2
the PCR primers were:
5 forward primer: TGTTGGACCCTCACAAAGAGTAAG (SEQ ID NO: 12) reverse
primer: GCTGTATTCTTGCCCTGTTCCT (SEQ ID NO: 13) and the PCR probe
was: FAM-TTCTGGGATCCACTCCAAGCTGCTCA- (SEQ ID NO: 14) TAMRA
[0203] where FAM is the fluorescent reporter dye and TAMRA is the
quencher dye. For mouse GAPDH the PCR primers were:
6 forward primer: GGCAAATTCAACGGCACAGT (SEQ ID NO: 15) reverse
primer: GGGTCTCGCTCCTGGAAGAT (SEQ ID NO: 16) and the PCR probe was:
5' JOE- (SEQ ID NO: 17) AAGGCCGAGAATGGGAAGCTTGTCATC- TAMRA 3'
[0204] where JOE is the fluorescent reporter dye and TAMRA is the
quencher dye.
Example 14
Northern Blot Analysis of SGLT2 mRNA Levels
[0205] Eighteen hours after antisense treatment, cell monolayers
were washed twice with cold PBS and lysed in 1 mL RNAZOL.TM.
(TEL-TEST "B" Inc., Friendswood, Tex.). Total RNA was prepared
following manufacturer's recommended protocols. Twenty micrograms
of total RNA was fractionated by electrophoresis through 1.2%
agarose gels containing 1.1% formaldehyde using a MOPS buffer
system (AMRESCO, Inc. Solon, Ohio). RNA was transferred from the
gel to HYBOND.TM.-N+ nylon membranes (Amersham Pharmacia Biotech,
Piscataway, N.J.) by overnight capillary transfer using a
Northern/Southern Transfer buffer system (TEL-TEST "B" Inc.,
Friendswood, Tex.). RNA transfer was confirmed by UV visualization.
Membranes were fixed by UV cross-linking using a STRATALINKER.TM.
UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then
probed using QUICKHYB.TM. hybridization solution (Stratagene, La
Jolla, Calif.) using manufacturer's recommendations for stringent
conditions.
[0206] To detect human SGLT2, a human SGLT2 specific probe was
prepared by PCR using the forward primer TCGGCGTGCCCAGCT (SEQ ID
NO: 5) and the reverse primer AGAACAGCACAATGGCGAAGT (SEQ ID NO: 6).
To normalize for variations in loading and transfer efficiency
membranes were stripped and probed for human
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech,
Palo Alto, Calif.).
[0207] To detect mouse SGLT2, a mouse SGLT2 specific probe was
prepared by PCR using the forward primer TGTTGGACCCTCACAAAGAGTAAG
(SEQ ID NO: 12) and the reverse primer GCTGTATTCTTGCCCTGTTCCT (SEQ
ID NO: 13). To normalize for variations in loading and transfer
efficiency membranes were stripped and probed for mouse
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech,
Palo Alto, Calif.).
[0208] Hybridized membranes were visualized and quantitated using a
PHOSPHORIMAGER.TM. and IMAGEQUANT.TM. Software V3.3 (Molecular
Dynamics, Sunnyvale, Calif.). Data was normalized to GAPDH levels
in untreated controls.
Example 15
Antisense Inhibition of Human SGLT2 Expression by Chimeric
Phosphorothioate Oligonucleotides Having 2'-MOE Wings and a Deoxy
Gap
[0209] In accordance with the present invention, a series of
antisense compounds was designed to target different regions of the
human SGLT2 RNA, using published sequences (GenBank accession
number NM.sub.--003041.1, incorporated herein as SEQ ID NO: 4). The
compounds are shown in Table 1. "Target site" indicates the first
(5'-most) nucleotide number on the particular target sequence to
which the compound binds. All compounds in Table 1 are chimeric
oligonucleotides ("gapmers") 20 nucleotides in length, composed of
a central "gap" region consisting of ten 2'-deoxynucleotides, which
is flanked on both sides (5' and 3' directions) by five-nucleotide
"wings". The wings are composed of 2'-methoxyethyl
(2'-MOE)nucleotides. The internucleoside (backbone) linkages are
phosphorothioate (P.dbd.S) throughout the oligonucleotide. All
cytidine residues are 5-methylcytidines. The compounds were
analyzed for their effect on human SGLT2 mRNA levels by
quantitative real-time PCR as described in other examples herein.
HK-2 cells were treated with 500 nM of antisense oligonucleotide
mixed with 15 .mu.g/mL LIPOFECTIN. Data are averages from three
experiments in which HK-2 cells were treated with the antisense
oligonucleotides of the present invention. If present, "N.D."
indicates "no data".
7TABLE 1 Inhibition of human SGLT2 mRNA levels by chimeric
phosphorothioate oligonucleotides having 2'-MOE wings and a deoxy
gap TARGET SEQ ID TARGET % SEQ ID ISIS # REGION NO SITE SEQUENCE
INHIB NO 337873 Start Codon 4 1 tctccccaggatctgccccc 17 18 337874
Start Codon 4 15 gtgtgctcctccattctccc 41 19 337875 Coding 4 42
cccatctctggtgccgagcc 33 20 337876 Coding 4 70 aggattgtcaatcagggcct
49 21 337877 Coding 4 95 atgcagcaatgactaggatg 45 22 337878 Coding 4
124 caagccaacgccaatgacca 54 23 337879 Coding 4 150
cctctgttggttctgcacat 26 24 337880 Coding 4 182 tgcgtcctgccaggaagtag
25 25 337881 Coding 4 204 ccaaccggccaccacaccat 45 26 337882 Coding
4 262 agtccctgccaggcccacaa 43 27 337883 Coding 4 291
ccagcaacagccaagccact 37 28 337884 Coding 4 354 aggtacacgggtgcaaacag
48 29 337885 Coding 4 384 tactgtggcatcgtgatgac 28 30 337886 Coding
4 426 aggtagaggcggatgcggcg 24 31 337887 Coding 4 442
aagggagagcacagacaggt 50 32 337888 Coding 4 474 tccactgagatcttggtgaa
41 33 337889 Coding 4 501 tggatgaatacagctccgga 49 34 337890 Coding
4 529 ggcatagatgttccagccca 36 35 337891 Coding 4 560
tcatggtgatgcccagaagc 23 36 337892 Coding 4 577 tcctgtcaccgtgtaaatca
39 37 337893 Coding 4 600 gtgtacatcagcgcggccag 33 38 337894 Coding
4 624 atgacgaaggtctgtaccgt 41 39 337895 Coding 4 651
cccatgaggatgcaggcgcc 55 40 337896 Coding 4 694 gtcgaagagacccgaatacc
30 41 337897 Coding 4 716 aagtcgctgctcccaggtat 0 42 337898 Coding 4
772 tcgatagcagaagctggaga 47 43 337899 Coding 4 849
agtccgaggagcagcgcggg 5 44 337900 Coding 4 884 ggtcgctgcaccagtaccag
29 45 337901 Coding 4 909 gccaggcagcgctgcacgat 22 46 337902 Coding
4 944 tgcagcccgccttgatgtgg 67 47 337903 Coding 4 954
ccacacaggatgcagcccgc 37 48 337904 Coding 4 991 catgaccatgagaaacatgg
43 49 337905 Coding 4 1006 gctgatcatgcctggcatga 54 50 337906 Coding
4 1033 cgccacctcgtctgggtaca 45 51 337907 Coding 4 1051
cacctcaggcaccacgcacg 54 52 337908 Coding 4 1073
ccgtgccgcacacgcgcctg 34 53 337909 Coding 4 1100
ggtaggcgatgttggagcag 30 54 337910 Coding 4 1122
atgagcttcacgacgagccg 48 55 337911 Coding 4 1151
ccagcatgagtccgcgcaga 50 56 337912 Coding 4 1180
cgaggacatgagcgcggcca 71 57 337913 Coding 4 1211
gcgtgctgctgctgttgaag 37 58 337914 Coding 4 1232
tgtagatgtccatggtgaag 39 59 337915 Coding 4 1272
agcagcagctcgcggtcgcc 21 60 337916 Coding 4 1292
ccacccagagccgtcccacc 47 61 337917 Coding 4 1319
aggccaccgacactaccacg 38 62 337918 Coding 4 1360
gaagagctgcccgccctgtg 38 63 337919 Coding 4 1372
ctggatgtaatcgaagagct 45 64 337920 Coding 4 1415
cgaagacggcggacacgggc 3 65 337921 Coding 4 1433 gcacgaagagcgccagcacg
32 66 337922 Coding 4 1453 gccctgctcattaacgcgcg 34 67 337923 Coding
4 1479 aggcccccgatgagtcccca 48 68 337924 Coding 4 1497
cgtgccaggcccatcagcag 37 69 337925 Coding 4 1526
ccgagccgaaggagaactcg 47 70 337926 Coding 4 1544
agggctgcacacagctgccc 0 71 337927 Coding 4 1570 gccgcagaggaaagctgggc
15 72 337928 Coding 4 1595 caatggcgaagtagaggtag 37 73 337929 Coding
4 1615 gccagagcagaagaacagca 41 74 337930 Coding 4 1641
cacagggagaccgtgagggt 11 75 337931 Coding 4 1677
aggcggtggaggtgctttct 29 76 337932 Coding 4 1706
cctccttgctatgccggaga 47 77 337933 Coding 4 1729
atcagcatccaggtcctccc 0 78 337934 Coding 4 1763 cattctgtacagggagtgag
50 79 337935 Coding 4 1788 atctccatggcactctctgg 58 80 337936 Coding
4 1835 gcaggcactggcggaagagg 29 81 337937 Coding 4 1861
acctctgctcattccacaaa 56 82 337938 Coding 4 1881
ggcggaggactgcccacccc 22 83 337939 Coding 4 1917
cgcctggctgctgccgctgc 11 84 337940 Coding 4 1939
gtcctcgctgatgtcctcca 40 85 337941 Coding 4 1972
ggcattgaggttgaccacac 2 86 337942 Coding 4 2003 agaggaacacggccactgcc
8 87 337943 Coding 4 2014 atagaagccccagaggaaca 39 88 337944 Stop
Codon 4 2025 tggtcttaggcatagaagcc 28 89 337945 3'UTR 4 2048
tggcttatggtgtccaacgc 35 90 337946 3'UTR 4 2072 tcacccccacttcctgtgag
42 91 337947 3'UTR 4 2120 tctcaccccactgccccttc 38 92 337948 3'UTR 4
2158 caggcagaggaaggccggga 38 93 337949 3'UTR 4 2197
cctcatgggaagtgactgcc 37 94 337950 3'UTR 4 2230 ttccttagggcaactgcagc
34 95
[0210] As shown in Table 1, SEQ ID NOs 19, 20, 21, 22, 23, 26, 27,
28, 29, 32, 33, 34, 35, 37, 38, 39, 40, 41, 43, 47, 48, 49, 50, 51,
52, 53, 54, 55, 56, 57, 58, 59, 61, 62, 63, 64, 66, 67, 68, 69, 70,
73, 74, 77, 79, 80, 82, 85, 88, 90, 91, 92, 93, 94 and 95
demonstrated at least 30% inhibition of human SGLT2 expression in
this assay. The target regions to which these sequences are
complementary are herein referred to as "suitable target segments"
and are therefor suitable for targeting by compounds of the present
invention. These target segments are shown in Table 3. These
sequences are shown to contain thymine (T) but one of skill in the
art will appreciate that thymine (T) is generally replaced by
uracil (U) in RNA sequences. The sequences represent the reverse
complement of the suitable antisense compounds shown in Table 1,
"Target site" indicates the first (5'-most) nucleotide number on
the particular target nucleic acid to which the oligonucleotide
binds. Also shown in Table 3 is the species in which each of the
suitable target segments was found.
Example 16
Antisense Inhibition of Mouse SGLT2 Expression by Chimeric
Phoshorothioate Oligonucleotides Having 2'-MOE Wings and a Deoxy
Gap
[0211] In accordance with the present invention, a second series of
antisense compounds was designed to target different regions of the
mouse SGLT2 RNA, using published sequences (the concatenation of
the sequences with the GenBank accession numbers: AJ292928,
AW106808, AI789450, AW046901, the complement of AI647605, the
complement of AW107250, and the complement of AI788744,
incorporated herein as SEQ ID NO: 11; GenBank accession number
AJ292928. 1, incorporated herein as SEQ ID NO: 96; and GenBank
accession number AW045170.1, incorporated herein as SEQ ID NO: 97).
The compounds are shown in Table 2. "Target site" indicates the
first (5'-most) nucleotide number on the particular target nucleic
acid to which the compound binds. All compounds in Table 2 are
chimeric oligonucleotides ("gapmers") 20 nucleotides in length,
composed of a central "gap" region consisting of ten
2'-deoxynucleotides, which is flanked on both sides (5' and 3'
directions) by five-nucleotide "wings". The wings are composed of
2'-methoxyethyl (2'-MOE) nucleotides. The internucleoside
(backbone) linkages are phosphorothioate (P.dbd.S) throughout the
oligonucleotide. All cytidine residues are 5-methylcytidines. The
compounds were analyzed for their effect on mouse SGLT2 mRNA levels
by quantitative real-time PCR as described in other examples
herein. Data are averages from three experiments in which b.END
cells were treated with 100 nM of the antisense oligonucleotides of
the present invention. The positive control for each datapoint is
identified in the table by sequence ID number. If present, "N.D."
indicates "no data".
8TABLE 2 Inhibition of mouse SGLT2 mRNA levels by chimeric
phosphorothioate oligonucleotides having 2'-MOE wings and a deoxy
gap TARGET CONTROL SEQ ID TARGET % SEQ ID SEQ ID ISIS # REGION NO
SITE SEQUENCE INHIB NO NO 145725 Coding 11 27 tgctccccaagttcagagcc
16 98 1 145726 Coding 11 39 atcaggaccttctgctcccc 30 99 1 145727
Coding 11 50 caggattatcaatcaggacc 15 100 1 145728 Coding 11 62
ccagaatgtcagcaggatta 9 101 1 145729 Coding 11 93
ccaatgaccagcaggaaata 15 102 1 145730 Coding 11 117
ctgaacatagaccacaagcc 0 103 1 145731 Coding 11 127
tctattggttctgaacatag 9 104 1 145732 Coding 11 138
ccaactgtgcctctattggt 43 105 1 145733 Coding 11 148
gaagtagccaccaactgtgc 16 106 1 145734 Coding 11 189
gaggctccaaccggccacca 43 107 1 145735 Coding 11 213
ctgccgatgttgctggcgaa 2 108 1 145736 Coding 11 230
ggcccacaaaatgaccgctg 44 109 1 145737 Coding 11 261
gccaagccacttgctgcacc 29 110 1 145738 Coding 11 294
acgaagagcgcattccactc 7 111 1 145739 Coding 11 299
gcaccacgaagagcgcattc 0 112 1 145740 Coding 11 375
cgcttgcggaggtactgagg 0 113 1 145741 Coding 11 420
agcgagagcacggacaggta 0 114 1 145742 Coding 11 462
gagaacatatccaccgagat 5 115 1 145743 Coding 11 490
cagggcctgttgaatgaata 0 116 1 145744 Coding 11 550
cacagtataaatcatggtga 35 117 1 145745 Coding 11 581
ctgtgtacatcagtgccgcc 18 118 1 145746 Coding 11 592
ctgcacagtgtctgtgtaca 7 119 1 145747 Coding 11 605
gaatgacgaaggtctgcaca 14 120 1 145748 Coding 11 616
ggccccggcaagaatgacga 25 121 1 145749 Coding 11 659
agtacccgcccacttcatgg 0 122 1 145750 Coding 11 706
acccgtcagtgaagtcattg 18 123 1 145751 Coding 11 784
gtcacgcagcaggtgatagg 24 124 1 145752 Coding 11 795
cctgtcacagggtcacgcag 40 125 1 145753 Coding 11 840
gagacaatggtaagccccag 20 126 1 145754 Coding 11 902
tcagattctttccagccagg 12 127 1 145755 Coding 11 912
ttgatgtgagtcagattctt 13 128 1 145756 Coding 11 998
ggtagagaatgcggctgatc 8 129 1 145757 Coding 11 1039
ccgcttacacacctcaggta 32 130 1 145758 Coding 11 1050
gtgccacacacccgcttaca 39 131 1 145759 Coding 11 1068
ttagagcagcccacctcagt 28 132 1 145760 Coding 11 1081
tgggtaggcgatgttagagc 15 133 1 145761 Coding 11 1113
agaccattgggcatgagctt 2 134 1 145762 Coding 11 1128
agcatgagtccgcgcagacc 0 135 1 145763 Coding 11 1142
ccagcatgactgccagcatg 22 136 1 145764 Coding 11 1177
gttaaagatggatgccagag 0 137 1 145765 Coding 11 1246
cagctccttatcacctgcac 55 138 1 145766 Coding 11 1320
gctgcctgcaccactggcag 44 139 1 145767 Coding 11 1393
aaagaccgcagacacttgag 0 140 1 145768 Coding 11 1403
gtgcaagcacaaagaccgca 6 141 1 145769 Coding 11 1475
gagctaggcccatcagcagg 55 142 1 145770 Coding 11 1485
ggtatgagacgagctaggcc 0 143 1 145771 Coding 11 1496
agaagaactcgggtatgaga 0 144 1 145772 Coding 11 1524
gagggtcgcacacagctgcc 8 145 1 145773 Coding 11 1563
tagaggtagtgtacccgaca 0 146 1 145774 Coding 11 1682
ccttgctgtgccggagactg 40 147 1 145775 Coding 11 1707
tcagcatccaggtcctcccg 46 148 1 145776 Coding 11 1722
ggaccttctaactcatcagc 2 149 1 145777 Coding 11 1765
cattgcacattcctggcccc 23 150 1 145778 Coding 11 1839
ttgctcatcccacagaacca 15 151 1 145779 Coding 11 1851
cctgacccactcttgctcat 1 152 1 145780 Coding 11 1881
gccacctcctcggtagtggg 21 153 1 145781 Coding 11 1909
gatgtcctccagccgcctgg 0 154 1 145782 Coding 11 1921
gggatcctcactgatgtcct 25 155 1 145783 Coding 11 1953
agggcattgaggttgactac 11 156 1 145784 Coding 11 1992
tagaagccccagaggaacac 0 157 1 145785 3'UTR 11 2164
aatcaaatggactggacccc 0 158 1 145786 3'UTR 11 2174
agtgacaaccaatcaaatgg 10 159 1 145787 3'UTR 11 2186
catcttgtgggaagtgacaa 14 160 1 145788 3'UTR 11 2199
accaattggccatcatcttg 0 161 1 145789 3'UTR 11 2237
ggagggcagttttatttttg 20 162 1 145790 exon:intron 96 2123
caatgtctcacccacaagcc 4 163 1 145791 intron 96 2239
ctaaatctaggtttctccct 11 164 1 145792 intron 96 2291
ttttgcacaatccagaaggt 9 165 1 145793 intron 96 2407
gaccttaaatataggctgct 0 166 1 145794 intron 96 2477
aacccaggccctaatcctag 4 167 1 145795 intron 96 2551
aggctgaagattaaccagcc 8 168 1 145796 intron 96 2595
ttggacttccttagcttcct 9 169 1 145797 exon:intron 96 2647
gaacatagactgggaaacag 0 170 1 145798 intron 96 2797
gaggctccaacctgggtggc 12 171 1 145799 intron 97 133
tccagcaaatgaacctgtgt 0 172 1 145800 intron 97 284
cacagcggaagtgcctgggc 21 173 1 145801 intron 97 316
tgtcctagtcctcacaccca 12 174 1 145802 intron 97 338
gggacagcatcctgagcagg 25 175 1
[0212] As shown in Table 2, SEQ ID NOs 99, 105, 107, 109, 110, 117,
121, 124, 125, 126, 130, 131, 132, 136, 138, 139, 142, 147, 148,
150, 153, 155, 162, 173 and 175 demonstrated at least 20%
inhibition of mouse SGLT2 expression in this experiment. Also
suitable are SEQ ID NOs 105, 119 and 135. The target regions to
which these sequences are complementary are herein referred to as
"suitable target segments" and are therefore suitable for targeting
by compounds of the present invention. These target segments are
shown in Table 3. These sequences are shown to contain thymine (T)
but one of skill in the art will appreciate that thymine (T) is
generally replaced by uracil (U) in RNA sequences. The sequences
represent the reverse complement of the preferred antisense
compounds shown in Tables 1 and 2. "Target site" indicates the
first (5'-most) nucleotide number on the particular target nucleic
acid to which the oligonucleotide binds. Also shown in Table 3 is
the species in which each of the suitable target segments was
found.
9TABLE 3 Sequence and position of preferred target segments
identified in human and mouse SGLT2. TARGET REV SITE SEQ ID TARGET
COMP OF ACTIVE SEQ ID ID NO SITE SEQUENCE SEQ ID IN NO 253571 4 15
gggagaatggaggagcacac 19 H. sapiens 176 253572 4 42
ggctcggcaccagagatggg 20 H. sapiens 177 253573 4 70
aggccctgattgacaatcct 21 H. sapiens 178 253574 4 95
catcctagtcattgctgcat 22 H. sapiens 179 253575 4 124
tggtcattggcgttggcttg 23 H. sapiens 180 253578 4 204
atggtgtggtggccggttgg 26 H. sapiens 181 253579 4 262
ttgtgggcctggcagggact 27 H. sapiens 182 253580 4 291
agtggcttggctgttgctgg 28 H. sapiens 183 253581 4 354
ctgtttgcacccgtgtacct 29 H. sapiens 184 253584 4 442
acctgtctgtgctctccctt 32 H. sapiens 185 253585 4 474
ttcaccaagatctcagtgga 33 H. sapiens 186 253586 4 501
tccggagctgtattcatcca 34 H. sapiens 187 253587 4 529
tgggctggaacatctatgcc 35 H. sapiens 188 253589 4 577
tgatttacacggtgacagga 37 H. sapiens 189 253590 4 600
ctggccgcgctgatgtacac 38 H. sapiens 190 253591 4 624
acggtacagaccttcgtcat 39 H. sapiens 191 253592 4 651
ggcgcctgcatcctcatggg 40 H. sapiens 192 253593 4 694
ggtattcgggtctcttcgac 41 H. sapiens 193 253595 4 772
tctccagcttctgctatcga 43 H. sapiens 194 253599 4 944
ccacatcaaggcgggctgca 47 H. sapiens 195 253600 4 954
gcgggctgcatcctgtgtgg 48 H. sapiens 196 253601 4 991
ccatgtttctcatggtcatg 49 H. sapiens 197 253602 4 1006
tcatgccaggcatgatcagc 50 H. sapiens 198 253603 4 1033
tgtacccagacgaggtggcg 51 H. sapiens 199 253604 4 1051
cgtgcgtggtgcctgaggtg 52 H. sapiens 200 253605 4 1073
caggcgcgtgtgcggcacgg 53 H. sapiens 201 253606 4 1100
ctgctccaacatcgcctacc 54 H. sapiens 202 253607 4 1122
cggctcgtcgtgaagctcat 55 H. sapiens 203 253608 4 1151
tctgcgcggactcatgctgg 56 H. sapiens 204 253609 4 1180
tggccgcgctcatgtcctcg 57 H. sapiens 205 253610 4 1211
cttcaacagcagcagcacgc 58 H. sapiens 206 253611 4 1232
cttcaccatggacatctaca 59 H. sapiens 207 253613 4 1292
ggtgggacggctctgggtgg 61 H. sapiens 208 253614 4 1319
cgtggtagtgtcggtggcct 62 H. sapiens 209 253615 4 1360
cacagggcgggcagctcttc 63 H. sapiens 210 253616 4 1372
agctcttcgattacatccag 64 H. sapiens 211 253618 4 1433
cgtgctggcgctcttcgtgc 66 H. sapiens 212 253619 4 1453
cgcgcgttaatgagcagggc 67 H. sapiens 213 253620 4 1479
tggggactcatcgggggcct 68 H. sapiens 214 253621 4 1497
ctgctgatgggcctggcacg 69 H. sapiens 215 253622 4 1526
cgagttctccttcggctcgg 70 H. sapiens 216 253625 4 1595
ctacctctacttcgccattg 73 H. sapiens 217 253626 4 1615
tgctgttcttctgctctggc 74 H. sapiens 218 253629 4 1706
tctccggcatagcaaggagg 77 H. sapiens 219 253631 4 1763
ctcactccctgtacagaatg 79 H. sapiens 220 253632 4 1788
ccagagagtgccatggagat 80 H. sapiens 221 253634 4 1861
tttgtggaatgagcagaggt 82 H. sapiens 222 253637 4 1939
tggaggacatcagcgaggac 85 H. sapiens 223 253640 4 2014
tgttcctctggggcttctat 88 H. sapiens 224 253642 4 2048
gcgttggacaccataagcca 90 H. sapiens 225 253643 4 2072
ctcacaggaagtgggggtga 91 H. sapiens 226 253644 4 2120
gaaggggcagtggggtgaga 92 H. sapiens 227 253645 4 2158
tcccggccttcctctgcctg 93 H. sapiens 228 253646 4 2197
ggcagtcacttcccatgagg 94 H. sapiens 229 253647 4 2230
gctgcagttgccctaaggaa 95 H. sapiens 230 58683 11 39
ggggagcagaaggtcctgat 99 M. musculus 231 58689 11 138
accaatagaggcacagttgg 105 M. musculus 232 58691 11 189
tggtggccggttggagcctc 107 M. musculus 233 58693 11 230
cagcggtcattttgtgggcc 109 M. musculus 234 58694 11 261
ggtgcagcaagtggcttggc 110 M. musculus 235 58701 11 550
tcaccatgatttatactgtg 117 M. musculus 236 58705 11 616
tcgtcattcttgccggggcc 121 M. musculus 237 58708 11 784
cctatcacctgctgcgtgac 124 M. musculus 238 58709 11 795
ctgcgtgaccctgtgacagg 125 M. musculus 239 58710 11 840
ctggggcttaccattgtctc 126 M. musculus 240 58714 11 1039
tacctgaggtgtgtaagcgg 130 M. musculus 241 58715 11 1050
tgtaagcgggtgtgtggcac 131 M. musculus 242 58716 11 1068
actgaggtgggctgctctaa 132 M. musculus 243 58720 11 1142
catgctggcagtcatgctgg 136 M. musculus 244 58722 11 1246
gtgcaggtgataaggagctg 138 M. musculus 245 58723 11 1320
ctgccagtggtgcaggcagc 139 M. musculus 246 58726 11 1475
cctgctgatgggcctagctc 142 M. musculus 247 58731 11 1682
cagtctccggcacagcaagg 147 M. musculus 248 58732 11 1707
cgggaggacctggatgctga 148 M. musculus 249 58734 11 1765
ggggccaggaatgtgcaatg 150 M. musculus 250 58737 11 1881
cccactaccgaggaggtggc 153 M. musculus 251 58739 11 1921
aggacatcagtgaggatccc 155 M. musculus 252 58746 11 2237
caaaaataaaactgccctcc 162 M. musculus 253 58757 97 284
gcccaggcacttccgctgtg 173 M. musculus 254 58759 97 338
cctgctcaggatgctgtccc 175 M. musculus 255
[0213] As these "suitable target segments" have been found by
experimentation to be open to, and accessible for, hybridization
with the antisense compounds of the present invention, one of skill
in the art will recognize or be able to ascertain, using no more
than routine experimentation, further embodiments of the invention
that encompass other compounds that specifically hybridize to these
suitable target segments and consequently inhibit the expression of
SGLT2.
[0214] According to the present invention, antisense compounds
include antisense oligomeric compounds, antisense oligonucteotides,
ribozymes, external guide sequence (EGS) oligonucleotides,
alternate splicers, and other short oligomeric compounds which
hybridize to at least a portion of the target nucleic acid.
Example 17
Western Blot Analysis of SGLT2 Protein Levels
[0215] Western blot analysis (immunoblot analysis) is carried out
using standard methods. Cells are harvested 16-20 h after
oligonucleotide treatment, washed once with PBS, suspended in
Laemmli buffer (100 .mu.l/well), boiled for 5 minutes and loaded on
a 16% SDS-PAGE gel. Gels are run for 1.5 hours at 150 V, and
transferred to membrane for western blotting. Appropriate primary
antibody directed to SGLT2 is used, with a radiolabeled or
fluorescently labeled secondary antibody directed against the
primary antibody species. Bands are visualized using a
PHOSPHORIMAGER.TM. (Molecular Dynamics, Sunnyvale Calif.).
Example 18
Design of Chemically Modified Antisense Compounds Targeting
SGLT2
[0216] A series of chemically modified antisense compounds were
designed using the sequence of ISIS 145733 (SEQ ID NO: 106), ISIS
145742 (SEQ ID NO: 265) or ISIS 145746 (SEQ ID NO: 266).
Modifications were made to the internucleoside linkages such that
the oligonucleotides consisted of either full phosphorothioate
backbones or mixed phosphorothioate and phosphodiester backbones
(mixed backbone compounds). Modified antisense compounds also
contained sugar moiety substitutions at the 2' position, comprising
a 2'-methoxyethyl (2'-MOE) or a 2'-0-dimethylaminoethoxyethyl
(2'-DMAEOE). Further modifications included nucleobase
substitutions, wherein the unmodified cytosine nucleobase was used
in place of the modified 5-methylcytosine at one position in the
antisense compound. The compounds are shown in Table 4.
[0217] ISIS 145733 (SEQ ID NO: 106), ISIS 145742 (SEQ ID NO: 265)
and ISIS 145746 (SEQ ID NO: 266) are chimeric oligonucleotides
having 2'-MOE wings and a deoxy gap with phosphorothioate linkages
throughout the oligonucleotide. ISIS 257016 (SEQ ID NO: 106), ISIS
341699 (SEQ ID NO: 265) and ISIS 351642 (SEQ ID NO: 266) are
chimeric oligonucleotides having 2'-MOE wings and a deoxy gap, with
phosphodiester linkages in the wings and phosphorothioate linkages
in the gap. ISIS 351641 (SEQ ID NO: 106), ISIS 360886 (SEQ ID NO:
106) and ISIS 360887 (SEQ ID NO: 106) are chimeric oligonucleotides
having 2'-MOE wings and a deoxy gap, with phosphorothioate linkages
in the gap and phosphodiester linkages in the wings, except for one
phosphorothioate linkage in the wing(s) at either the extreme 5'
end (ISIS 360886), the extreme 3' end (ISIS 360887) or both of the
extreme 5' and 3' ends (ISIS 351641).
[0218] ISIS 323294 (SEQ ID NO: 106) consists of 2'-MOE nucleotides
at positions 1, 2, 3, 4, 17 and 19, 2'-DMAEOE nucleotides at
positions 5, 16, 18 and 20 and 2'-deoxynucleotides at positions 6
through 15, with phosphorothioate linkages throughout the
oligonucleotide. ISIS 323295 (SEQ ID NO: 106) consists of 2'-MOE
nucleotides at positions 1, 2, 3, 4, 17 and 19, 2'-DMAEOE
nucleotides at positions 5, 16, 18 and 20 and 2'-deoxynucleotides
at positions 6 through 15, wherein the first and last 4
internucleoside linkages are phosphodiester and the central
internucleoside linkages are phosphorothioate.
[0219] The nucleotides in the 3' most positions in ISIS 251017 and
257018 are cytosine residues (indicated by an asterisk in Table 4).
All other cytosine residues of the oligonucleotides listed above
are 5-methylcytosines. The compounds are shown in Table 4.
Phosphodiester (P.dbd.O) internucleoside linkages are indicated by
an "o" between nucleotide positions. Phosphorothioate (P.dbd.S)
internucleoside linkages are indicated by an "s" between nucleotide
positions. 2'-MOE nucleotides are underscored and 2'-DMAEOE
nucleotides are emboldened. All compounds in Table 4 target the
coding region of murine SGLT2 (provided herein as SEQ ID NO:
11).
10TABLE 4 Chemical modifications of antisense compounds targeting
SGLT2 SEQ ID ISIS # Sequence NO 145733
GsAsAsGsTsAsGsCsCsAsCsCsAsAsCsTs- GsTsGsC 106 257016
GoAoAoGoTsAsGsCsCsAsCsCsAsAsCsToGoToGo- C 106 257017
GsAsAsGsTsAsGsCsCsAsCsCsAsAsCsTsGsTsGsC* 106 257018
GoAoAoGoTsAsGsCsCsAsCsCsAsAsCsToGoToGoC* 106 145742
GsAsGsAsAsCsAsTsAsTsCsCsAsCsCsGsAsGsAsT 265 341699
GoAoGoAoAsCsAsTsAsTsCsCsAsCsCsGoAoGoAoT 265 145746
CsTsGsCsAsCsAsGsTsGsTsCsTsGsTsGsTsAsCsA 266 351642
CoToGoCoAsCsAsGsTsGsTsCsTsGsTsGoToAoCoA 266 351641
GsAoAoGoTsAsGsCsCsAsCsCsAsAsCsToGoToGsC 106 360886
GsAoAoGoTsAsGsCsCsAsCsCsAsAsCsToGoToGoC 106 360887
GoAoAoGoTsAsGsCsCsAsCsCsAsAsCsToGoToGsC 106 323294
GsAsAsGsTsAsGsCsCsAsCsCsAsAsCsTsGsTsGsC 106 323295
GoAoAoGoTsAsGsCsCsAsCsCsAsAsCsToGoToGoC 106
Example 19
Effects of Antisense Inhibition of SGLT2 in Mice: Comparison of
Various Chemistries
[0220] In accordance with the present invention, SGLT2 antisense
compounds described in Example 18 were investigated for their
activity in vivo. ISIS 29837 (TCGATCTCCTTTTATGCCCG, SEQ ID NO: 256)
served as a control compound and is a chimeric oligonucleotide
("gapmer") 20 nucleotides in length, composed of a central "gap"
region consisting of ten 2'-deoxynucleotides, which is flanked on
both sides (5' and 3' directions) by five-nucleotide "wings". The
wings are composed of 2'-methoxyethyl (2'-MOE) nucleotides. The
internucleoside (backbone) linkages are phosphorothioate (P.dbd.S)
throughout the oligonucleotide. All cytidine residues are
5-methylcytidines.
[0221] Male 6-week old Balb/c mice (Charles River Laboratories,
Wilmington, Mass.) were given intraperitoneal injections of ISIS
145733, ISIS 257016, ISIS 323294, ISIS 323295 or ISIS 29837 at a
dose of 25 mg/kg twice per week for two weeks. Saline-injected
animals also served as a control. Each treatment group contained
four animals. The mice were sacrificed 2 days following
administration of the fourth and final dose of oligonucleotide or
saline.
[0222] Mice were evaluated for SGLT2 levels in kidney. Target
levels were determined by quantitative real-time PCR as described
by other examples herein. PCR results were normalized to the
ubiquitously expressed mouse cyclophilin A gene.
[0223] Probes and primers to mouse SGLT2 were designed to hybridize
to a mouse SGLT2 sequence, using published sequence information
(incorporated herein as SEQ ID NO: 11). For mouse SGLT2 the PCR
primers were:
11 (SEQ ID NO: 257) forward primer: CTCGTCTCATACCCGAGTTCTTC- T (SEQ
ID NO: 258) reverse primer: AATGATGGCGAAATAGAGGTAGTGTAC and the PCR
probe was: (SEQ ID NO: 259) FAM-TGCGACCCTCAGCGTGCCC-TAMRA
[0224] where FAM is the fluorescent dye and TAMRA is the quencher
dye. For mouse cyclophilin A the PCR primers were:
12 (SEQ ID NO: 260) forward primer: TCGCCGCTTGCTGCA (SEQ ID NO:
261) reverse primer: ATCGGCCGTGATGTCGA and the PCR probe was: (SEQ
ID NO: 262) 5' JOE-CCATGGTCAACCCCACCGTGTTC-3'
[0225] where JOE is the fluorescent reporter dye and TAMRA is the
quencher dye.
[0226] The data are expressed as percent change ("-" indicates a
decrease) relative to saline treated animals and are shown in Table
5.
13TABLE 5 Antisense inhibition of SGLT2 mRNA expression in vivo by
25 mg/kg doses of antisense compounds % change in SGLT2 expression
relative to saline ISIS ISIS ISIS ISIS ISIS 145733 257016 323294
323295 29837 -44 -82 -40 -31 -23
[0227] These data illustrate that antisense compounds of different
chemistries inhibit the expression of SGLT2 mRNA in mouse
kidney.
[0228] Mice were further evaluated for total body weight, liver
weight and spleen weight. Significant changes in spleen, liver or
body weight can indicate that a particular compound causes toxic
effects. The data are expressed as percent change ("+" indicates an
increase, "-" indicates a decrease) relative to saline control. The
results are presented in Table 6.
14TABLE 6 Effects of antisense compounds on total body weight,
liver weight and spleen weight in mice Weight as % change relative
to saline control 145733 257016 323294 323295 29837 Total Body 0 0
-1 -3 0 Liver +1 +1 +9 +4 +12 Spleen +4 +1 +19 +8 +1
[0229] All changes were within the margin of error of the
experiment. No significant changes in body weight were observed
during the treatment or at study termination. No significant
changes in liver or spleen weights were observed.
[0230] Toxic effects of compounds administered in vivo can also be
assessed by measuring the levels of enzymes and proteins associated
with disease or injury of the liver or kidney. Elevations in the
levels of the serum transaminases aspartate aminotransferase (AST)
and alanine aminotransferase (ALT) are often indicators of liver
disease or injury. Serum total bilirubin is an indicator of liver
and biliary function, and albumin and blood urea nitrogen (BUN) are
indicators of renal function. Glucose and triglyceride levels are
sometimes altered due to toxicity of a treatment. Serum glucose
also depends in part upon the activity of SGLT2.
[0231] In accordance with the present invention, the levels of ALT,
AST, total bilirubin, albumin, BUN, glucose and triglyceride were
measured in mice treated with the compounds of the invention. Serum
was analyzed by LabCorp Testing Facility (San Diego, Calif.). The
results are expressed as units measured and are shown in Table
7.
15TABLE 7 Effects of antisense compounds targeting SGLT2 on liver
and kidney function in mice Serum Normal Treatment and units
measured indicator Range Saline 145733 257016 323294 323295 29837
BUN mg/dL 15-40 27 29 33 29 30 30 Albumin 2.5-4.0 3 3 3 3 3 3 g/dL
Bilirubin mg/dL 0.1-1.0 0.1 0.1 0.1 0.1 0.1 0.1 AST 30-300 124 83
129 174 89 114 IU/L ALT 30-200 33 26 47 61 32 31 IU/L Triglycerides
25-100* 179 154 157 160 209 198 mg/dL Glucose 80-150* 242 270 222
284 271 235 mg/dL *Triglyceride and glucose levels are routinely
higher in the Balb/c strain of mice than in other strains of
mice.
[0232] The levels of routine clinical indicators of liver and
kidney injury and disease are within normal ranges and are not
significantly changed relative to saline-treated animals,
demonstrating that the compounds of the invention do not
significantly affect renal or hepatic function. Triglyceride and
glucose levels, while outside the normal range for most mice as is
common in the Balb/c strain, are not significantly elevated
relative to saline-treated animals.
[0233] Mice injected with ISIS 145733, 257016, 323294 and 323295
were also evaluated histologically following routine procedures.
Liver, spleen, kidney, intestine, pancreas, lung, skin, heart and
muscle samples were procured, fixed in 10% neutral-buffered
formalin and processed for staining with hematoxylin and eosin, to
visualize nuclei and cytoplasm, or with the anti-oligonucleotide
IgG1 antibody 2E1-B5 (Berkeley Antibody Company, Berkeley, Calif.)
to assess oligonucleotide staining patterns. Hematoxylin and eosin
staining in most tissues exhibited no significant difference
between saline- and oligonucleotide-treated animals. Heart sections
from animals treated with 323294 and 323295 showed a high amount of
inflammation relative to hearts from saline-treated mice. 2E1-B5
antibody was recognized using an isospecific anti-IgG2 horse-radish
peroxidase-conjugated secondary antibody (Zymed, San Francisco,
Calif.) and immunostaining was developed with 3,3'-diaminobenzidene
(DAKO, Carpenteria, Calif.). 2E1-B5 staining was performed in
duplicate and showed that none of the chemistries significantly
stained the liver, while staining was observed in the kidney
proximal tubules.
[0234] The results illustrated in this example demonstrate that
antisense compounds of different chemistries are delivered to the
kidney, reduce SGLT2 expression in vivo, and that treatment with
these compounds does not result in liver or kidney toxicity.
Example 20
Effects of Antisense Compounds on SGLT2 mRNA Expression in vivo:
Dose Response Study Comparing Mixed Backbone and Full
Phosphorothioate Backbones
[0235] ISIS 145733 and ISIS 257016 were selected for a dose
response study in mice. Male 8-week old Balb/c mice (Charles River
Laboratories, Wilmington, Mass.) were given intraperitoneal
injections of either ISIS 145733 or ISIS 257016 at doses of 6.25,
12.5, 25 and 50 mg/kg twice per week for two weeks. Saline-injected
animals served as controls. A total of 4 animals were injected per
group. The mice were sacrificed 2 days following administration of
the fourth and final dose of oligonucleotide or saline.
[0236] Mice were evaluated for SGLT2 levels in kidney. Target
levels were determined by quantitative real-time PCR as described
by other examples herein. PCR results were normalized to
cyclophilin as described in Example 19. The data are expressed as
percent change ("+" indicates an increase, "-" indicates a
decrease) relative to saline treated animals and are illustrated in
Table 8.
16TABLE 8 Antisense inhibition of SGLT2 mRNA expression in vivo by
antisense compounds with varying chemistries % change in SGLT2
expression relative to saline Dose of oligonucleotide ISIS ISIS
mg/kg 145733 257016 6.25 -3 -58 12.5 -7 -68 25 -37 -68 50 -34
-77
[0237] These results illustrate that the compounds of the
invention, both full phosphorothioate and mixed backbone
oligonucleotides, inhibit the expression of SGLT2 in vivo in a
dose-dependent manner.
[0238] The levels of SGLT2 expression were also evaluated by
Northern blot analysis of both pooled and individual RNA samples,
to validate the target reduction observed by real-time PCR. Total
RNA was prepared from procured tissues of sacrificed mice by
homogenization in GITC buffer (Invitrogen, Carlsbad, Calif.)
containing 2-mercaptoethanol (Sigma-Aldrich, St. Louis, Mo.)
following manufacturer's recommended protocols followed by
ultracentrifugation through a CsCl cushion. Twenty micrograms of
total RNA was fractionated by electrophoresis through 1.2% agarose
gels containing 1.1% formaldehyde using a MOPS buffer system
(AMRESCO, Inc. Solon, Ohio). RNA was transferred from the gel to
HYBOND.TM.-N+ nylon membranes (Amersham Pharmacia Biotech,
Piscataway, N.J.) by overnight capillary transfer. RNA transfer was
confirmed by UV visualization. Membranes were fixed by UV
cross-linking using a STRATALINKER.TM. UV Crosslinker 2400
(Stratagene, Inc, La Jolla, Calif.) and then probed using
RapidHYB.TM. hybridization solution (Amersham Pharmacia Biotech,
Piscataway, N.J.) using manufacturer's recommendations for
stringent conditions.
[0239] To detect mouse SGLT2, a mouse SGLT2 specific template was
prepared by PCR using the forward primer
5'-ATGGAGCAACACGTAGAGGCAGGCT-3' (SEQ ID NO: 263) and the reverse
primer 5'-GAGTGCCGCCAGCCCTCCTGTCACA-3' (SEQ ID NO: 264) and gel
purified. The probe was prepared by asymmetric PCR with the
purified template and the reverse primer incorporating .sup.32p CTP
to label the probe. Following hybridization blots were exposed
overnight to phosphorimager screens (Molecular Dynamics, Amersham)
and quantitated. To normalize for variations in loading and
transfer efficiency membranes were stripped and probed for mouse
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech,
Palo Alto, Calif.).
[0240] For pooled sample analysis, equal amounts of RNA isolated
from the kidneys of mice in the same treatment was combined for a
total of 20 .mu.g, and the pooled sample was subjected to Northern
blot analysis. The results of the pooled sample analysis are shown
in Table 9 and are normalized to saline controls ("+" indicates an
increase, "-" indicates a decrease).
17TABLE 9 Northern Analysis of SGLT2 message in pooled kidney RNA
samples % change in SGLT2 expression Dose of relative to saline
oligonucleotide ISIS ISIS mg/kg 145733 257016 6.25 +21 -57 12.5 +7
-50 25 -35 -75 50 -35 -82
[0241] These results demonstrate that, as determined by Northern
blot analysis of pooled samples, ISIS 257016 inhibits SGLT2
expression inhibits SGLT2 expression at all doses of antisense
compound in a dose-dependent manner, where as ISIS 145733 inhibits
SLGT2 expression at the two highest doses of antisense
compound.
[0242] Target levels in kidney RNA samples from individual mice
were also measured by Northern blot analysis. Equal amounts of RNA
were individually subjected to Northern blot analysis to determine
the level of SGLT2. Target level measurements for each treatment
group were then averaged. The results are shown in Table 10 and are
normalized to saline controls ("-" indicates a decrease).
18TABLE 10 Northern analysis of SGLT2 message in individually
measured RNA samples % change in SGLT2 expression Dose of relative
to saline oligonucleotide ISIS ISIS mg/kg 145733 257016 6.25 -34
-66 12.5 -38 -68 25 -39 -74 50 -59 -82
[0243] Treated mice were further evaluated at the end of the
treatment period for total body, liver and spleen weight. The data
are expressed as percent change ("+" indicates an increase, "-"
indicates a decrease) relative to saline control. The results are
presented in Table 11
19TABLE 11 Effects of antisense compounds on total body weight,
liver weight and spleen weight in mice % Change relative to
saline-treated ISIS ISIS Dose of 145733 257016 oligonucleotide
Total Total mg/kg Body Liver Spleen Body Liver Spleen 6.25 -4 -10
-12 -1 -3 +1 12.5 -6 -2 -7 -3 -13 -9 25 1 -1 +10 1 -8 +8 50 -1 +6
+10 -3 -9 +12
[0244] These data demonstrate that no significant changes in total
body, liver or spleen weights are observed following treatment with
ISIS 145733 or ISIS 257016 at 4 different doses. No changes in
total body weight were observed during the treatment period, or at
study termination.
[0245] In addition to the indicators of toxicity listed in Example
19, creatinine levels are also used to evaluate renal function. In
accordance with the present invention, the levels of ALT, AST,
total bilirubin, creatinine, BUN, glucose and triglyceride were
measured in mice treated with the compounds of the invention. Serum
was analyzed by LabCorp Testing Facility (San Diego, Calif.). The
results are expressed as units measured and are shown in Table
12.
20TABLE 12 Effects of antisense compounds targeting SGLT2 on liver
and kidney function in mice Units measured per treatment and dose
Serum Normal 145733 indicator Range Saline 25 mg/kg 145733 50 mg/kg
257016 25 mg/kg 257016 50 mg/kg BUN 15-40 24 24 25 26 26 mg/dL
Creatinine 0.0-1.0 0.1 0.1 0.1 0.125 0.1 mg/L Bilirubin mg/dL
0.1-1.0 0.125 0.1 0.1 0.1 0.1 AST 30-300 77 65 96 133 141 IU/L ALT
30-200 24 18 22 34 35 IU/L Triglycerides 25-100* 165 169 230 130
111 mg/dL Glucose 80-150* 236 280 256 244 248 mg/dL *Triglyceride
and glucose levels are routinely higher in the Balb/c strain of
mice than in other strains of mice.
[0246] The AST levels in animals treated with 25 mg/kg of ISIS
145733 are slightly below the normal range, as is the ALT level for
saline treated mice. Otherwise, the levels of routine clinical
indicators of liver and kidney injury and disease are within normal
ranges and are not significantly changed relative to saline-treated
animals, demonstrating that the compounds of the invention do not
significantly affect renal or hepatic function. Triglyceride and
glucose levels, while outside the normal range as is common in the
Balb/c strain, are not significantly elevated relative to
saline-treated animals.
[0247] Mice injected with ISIS 145733 and 257016 at doses from 6.25
to 50 mg/kg were also evaluated histologically following routine
procedures. Liver and kidney samples were procured, fixed in 10%
neutral-buffered formalin and processed for staining with
hematoxylin and eosin, to visualize nuclei and cytoplasm, or with
the anti-oligonucleotide IgG1 antibody 2E1-B5 (Berkeley Antibody
Company, Berkeley, Calif.) to assess oligonucleotide staining
patterns. Hematoxylin and eosin staining exhibited no significant
difference between saline- and oligonucleotide-treated animals.
2E1-B5 antibody was recognized using an isospecific anti-IgG2
horseradish peroxidase-conjugated secondary antibody (Zymed, San
Francisco, Calif.) and immunostaining was developed with
3,3'-diaminobenzidene (DAKO, Carpenteria, Calif.). 2E1 staining
showed no detectable oligonucleotide in the liver, while staining
was observed in the kidney proximal tubules. Staining intensity
lessened concomitantly with a decrease in oligonucleotide dose.
[0248] The results illustrated in this example demonstrate that
antisense compounds of different chemistries are delivered to the
kidney, reduce SGLT2 expression in vivo in a dose-dependent manner,
and that treatment with these compounds does not result in liver or
kidney toxicity.
Example 21
Effects of Antisense Compounds on SGLT2 mRNA Expression in vivo: an
Additional Dose Response Study Comparing Mixed Backbone and Full
Phosphorothioate Backbones
[0249] ISIS 145733 and ISIS 257016 were selected for a dose
response study in mice using two identical and two lower doses with
respect to the doses used in Example 20.
[0250] Male 8-week old Balb/c mice (Charles River Laboratories,
Wilmington, Mass.) were given intraperitoneal injections of ISIS
145733 or ISIS 257016 at doses of 1, 5, 25 or 50 mg/kg twice per
week for two weeks. Saline-injected animals served as a control. In
addition, as a specificity control, the same doses of SGLT2
antisense oligomeric compounds do not significantly inhibit
expression of SGLT1 mRNA in kidney cells. Each treatment group
contained 4 mice. The mice were sacrificed 2 days following
administration of the fourth and final dose of oligonucleotide or
saline.
[0251] Mice were evaluated for SGLT2 levels in kidney and liver.
Target levels were determined by quantitative real-time PCR as
described by other examples herein. PCR results were normalized to
cyclophilin. The data are expressed as percent change relative to
saline treated animals ("+" indicates an increase, "-" indicates a
decrease) and are illustrated in Table 13.
21TABLE 13 Antisense inhibition of SGLT2 mRNA expression in vivo by
antisense compounds with varying chemistries % change in SGLT2
expression relative to saline Dose of Kidney Liver oligonucleotide
ISIS ISIS ISIS ISIS mg/kg 145733 257016 145733 257016 1 +2 -46 -19
+13 5 -15 -64 -39 +1 25 -34 -74 -21 -5 50 -40 -76 -59 -12
[0252] These results illustrate that the compounds of the
invention, both full phosphorothioate and mixed backbone
oligonucleotides, can inhibit the expression of kidney SGLT2 in a
dose-dependent manner. Greater inhibition is observed in kidneys
from mice treated with ISIS 257016, a mixed backbone antisense
compound. SGLT2 is not highly expressed in liver, therefore target
levels are low before treatment and therefore more difficult to
accurately measure. While ISIS 145733 and ISIS 257016 also lowered
liver SGLT2 expression, with 145733 having a greater effect in
liver than the mixed backbone ISIS 257016.
[0253] Treated mice were further evaluated for liver and spleen
weight. The data are expressed as percent change ("+" indicates an
increase, "-" indicates a decrease) relative to saline control. The
results are presented in Table 14.
22TABLE 14 Effects of antisense compounds on total body weight,
liver weight and spleen weight in mice % change in body, liver and
spleen weight ISIS ISIS Dose of 145733 257016 oligonucleotide Total
Total mg/kg Body Liver Spleen Body Liver Spleen 1 0 -6 +10 -2 -8
+13 5 +3 +1 +10 -3 -9 +5 25 -1 +2 -4 +2 +2 +12 50 -1 +13 +35 -2 -6
+15
[0254] No significant change was observed in total body weight at
timepoints throughout or at the termination of the study.
Treatments of 25 mg/kg ISIS 145733 and 50 mg/kg 257016 resulted in
a decrease and increase in liver weight, respectively, however,
these changes are within the margin of error for the data and are
therefore not significant.
[0255] In addition to the other serum markers described herein,
cholesterol levels can be used as a measure of toxicity. In
accordance with the present invention, the levels of ALT, AST,
total bilirubin, albumin, creatinine, BUN, triglyceride,
cholesterol and glucose were measured in mice treated with the
compounds of the invention. Plasma samples were analyzed using the
Olympus AU400e automated chemistry analyzer (Olympus America,
Irving, Tex.). The results are expressed as units measured are
shown for ISIS 145733 in Table 15 and for ISIS 257016 in Table
16.
23TABLE 15 Effects of the full phosphorothioate antisense compound
ISIS 145733 on indicators of liver and kidney function Units
measured per Normal dose of ISIS 145733 Serum indicator Range
Saline 1 mg/kg 5 mg/kg 25 mg/kg 50 mg/kg BUN 15-40 27 31 31 30 25
mg/dL Creatinine 0.0-1.0 0.2 0.2 0.2 0.2 0.2 mg/L Bilirubin mg/dL
0.1-1.0 0.3 0.2 0.1 0.3 0.1 AST 30-300 92 91 45 133 56 IU/L ALT
30-200 35 27 26 37 31 IU/L Albumin 2.5-4.0 3 3 3 3 3 g/dL
Triglycerides mg/dL 25-100* 136 188 183 153 224 Cholesterol 70-125
122 116 117 120 132 mg/dL Glucose 80-150* 208 202 173 170 161
mg/dL
[0256]
24TABLE 16 Effects of the mixed backbone antisense compound ISIS
257016 on indicators of liver and kidney function Units measured
per Normal dose of ISIS 257016 Serum indicator Range Saline 1 mg/kg
5 mg/kg 25 mg/kg 50 mg/kg BUN 15-40 27 23 29 25 28 mg/dL Creatinine
0.0-1.0 0.2 0.2 0.2 0.2 0.2 mg/L Bilirubin mg/dL 0.1-1.0 0.3 0.2
0.2 0.2 0.2 AST 30-300 92 74 73 99 138 IU/L ALT 30-200 35 34 34 46
48 IU/L Albumin 2.5-4.0 3 3 3 3 3 g/dL Triglycerides mg/dL 25-100*
136 271 233 225 136 Cholesterol 70-125 122 116 124 144 137 mg/dL
Glucose 80-150* 208 180 178 154 182 mg/dL *Triglyceride and glucose
levels are routinely higher in the Balb/c strain of mice than in
other strains of mice.
[0257] The levels of routine clinical indicators of liver and
kidney injury and disease are within normal ranges and are not
significantly changed relative to saline-treated animals,
demonstrating that the compounds of the invention do not
significantly affect renal or hepatic function. Triglyceride and
glucose levels, while outside the normal range as is common in the
Balb/c strain, are not significantly elevated relative to
saline-treated animals.
[0258] Mice injected ISIS 145733 and 257016 at 1-50 mg/kg were also
evaluated histologically following routine procedures. Liver and
kidney samples were procured, fixed in 10% neutral-buffered
formalin and processed for staining with hematoxylin and eosin, to
visualize nuclei and cytoplasm, or with the anti-oligonucleotide
IgG1 antibody 2E1-B5 (Berkeley Antibody Company, Berkeley, Calif.)
to assess oligonucleotide staining patterns. Hematoxylin and eosin
staining in most tissues exhibited no significant difference
between saline- and 145733-treated animals, with the exception of
slight inflammatory cell infiltration in the liver tissue. Livers
from mice treated with ISIS 257016 showed evidence of nuclear
degradation and mitosis at 50 mg/kg and slight mitosis at 25 mg/kg.
Kidneys from ISIS 257016 exhibited no significant differences
compared to saline-treated kidneys. 2E1-B5 antibody was recognized
using an isospecific anti-IgG2 horse-radish peroxidase-conjugated
secondary antibody (Zymed, San Francisco, Calif.) and
immunostaining was developed with 3,3'-diaminobenzidene (DAKO,
Carpenteria, Calif.). Staining with the 2E1 antibody showed weak
staining in liver and kidneys from animals treated with ISIS
145733, whereas staining was strong in liver and kidney from
animals treated with ISIS 257016. Kidney 2E1 staining appears in a
punctate pattern.
Example 22
Dose Response Study Comparing Mixed Backbone and Full
Phosphorothioate Backbones: a Second SGLT2 Antisense Sequence
[0259] A second mixed backbone SGLT2 oligonucleotide, ISIS 341699
(SEQ ID NO: 265), and control phosphorothioate SGLT2
oligonucleotide, ISIS 145742 (SEQ ID NO: 265), were selected for a
dose response study in mice. For comparison, ISIS 257016 (mixed
backbone; SEQ ID NO: 106) also was included in this study.
[0260] Male 8-week old Balb/c mice (Charles River Laboratories,
Wilmington, Mass.) were given intraperitoneal injections of ISIS
341699, ISIS 145742 or ISIS 257016 twice per week for two weeks
with the doses shown in Table 17. Saline-injected animals served as
controls. Each treatment group contained 4 mice. The mice were
sacrificed 2 days following administration of the fourth and final
dose of oligonucleotide or saline.
[0261] Mice were evaluated for SGLT2 levels in kidney. Target
levels were determined by quantitative real-time PCR as described
by other examples herein. PCR results were normalized to
cyclophilin. The data are expressed as percent change relative to
saline treated animals ("+" indicates an increase, "-" indicates a
decrease) and are illustrated in Table 17.
25TABLE 17 Antisense inhibition of SGLT2 mRNA expression in vivo by
mixed backbone and full phosphorothioate oligonucleotides
(expressed as percent change in SGLT2 mRNA expression relative to
saline) Dose of oligonucleotide ISIS ISIS ISIS mg/kg 145742 341699
257016 0.2 -- -- -18.9 1 -- -1.8 -50.5 5 -0.6 -10.9 -56.7 25 -24.9
-23.9 -- 50 -32.6 -- --
[0262] These results illustrate that the compounds of the
invention, both full phosphorothioate and mixed backbone
oligonucleotides, can inhibit the expression of kidney SGLT2 in a
dose-dependent manner. However, lower doses of the mixed backbone
compound are required to inhibit SGLT2 expression in kidneys from
treated mice.
[0263] Treated mice were further evaluated for liver and spleen
weight. The data are expressed as percent change in body or organ
weight ("+" indicates an increase, "-" indicates a decrease). The
results are presented in Table 18 and Table 19.
26TABLE 18 Effects of antisense compounds on total body weight of
mice (expressed as percent change in body weight) Dose of
oligonucleotide ISIS ISIS ISIS mg/kg 145742 341699 257016 0.2 -- --
+7.9 1 -- +5.7 +5.8 5 +5.0 +5.8 +3.2 25 +2.0 +2.5 -- 50 +7.2 --
--
[0264]
27TABLE 19 Effects of antisense compounds on liver weight and
spleen weight of mice (expressed as percent change in organ weight)
Dose of Liver Spleen oligonucleotide ISIS ISIS ISIS ISIS ISIS ISIS
mg/kg 145742 341699 257016 145742 341699 257016 0.2 -- -- -6.0 --
-- -4.7 1 -- +2.3 +14.9 -- -4.2 +1.4 5 +7.1 +2.2 +7.0 +10.6 -2.8
-7.6 25 +7.2 +5.8 -- +0.8 -0.2 -- 50 +12.1 -- -- +9.4 -- --
[0265] No significant change was observed in total body weight,
liver weight or spleen weight at timepoints throughout or at the
termination of the study.
[0266] Levels of BUN, creatinine, AST, ALT, albumin, triglycerides,
cholesterol and glucose were measured in mice treated with the
compounds of the invention. Plasma samples were analyzed using the
Olympus AU400e automated chemistry analyzer (Olympus America,
Irving, Tex.). The results, expressed as units measured, are shown
for ISIS 145742 in Table 20, ISIS 341699 in Table 21 and ISIS
257016 in Table 22.
28TABLE 20 Effect of the full phosphorothioate antisense compound
ISIS 145742 on indicators of liver and kidney function Units
measured per Serum Normal dose of ISIS 145742 indicator Range
Saline 5 mg/kg 25 mg/kg 50 mg/kg BUN 15-40 20 21.3 25.5 20.8 mg/dL
Creatinine 0.0-1.0 0.1 0.2 0.2 0.2 mg/L AST 30-300 113 75.3 83.5
145.3 IU/L ALT 30-200 35.5 29.8 40.3 47.5 IU/L Albumin 2.5-4.0 3.0
3.0 2.9 2.9 g/dL Triglycerides 25-100* 223.8 176.5 192 176.8 mg/dL
Cholesterol 70-125 129 119.5 119.5 113.5 mg/dL Glucose 80-150*
176.5 196.5 192 194.8 mg/dL
[0267]
29TABLE 21 Effect of mixed backbone antisense compound ISIS 341699
on indicators of liver and kidney function Units measured per
Normal dose of ISIS 341699 Serum indicator Range Saline 1 mg/kg 5
mg/kg 25 mg/kg BUN 15-40 20 20 21.8 22 mg/dL Creatinine 0.0-1.0 0.1
0.2 0.2 0.2 mg/L AST 30-300 113 78.2 119 64.8 IU/L ALT 30-200 35.5
36.2 37.3 33.0 IU/L Albumin 2.5-4.0 3.0 3.3 3.1 3.2 g/dL
Triglycerides 25-100* 223.8 206.4 186.8 183.5 mg/dL Cholesterol
70-125 129 135 124 120.8 mg/dL Glucose 80-150* 176.5 203.2 171.5
197 mg/dL
[0268]
30TABLE 22 Effect of mixed backbone antisense compound ISIS 257016
on indicators of liver and kidney function Units measured per
Normal dose of ISIS 257016 Serum indicator Range Saline 0.2 mg/kg 1
mg/kg 5 mg/kg BUN 15-40 20 21.8 26.3 20.5 mg/dL Creatinine 0.0-1.0
0.1 0.2 0.2 0.2 mg/L AST 30-300 113 123.8 85.3 69.5 IU/L ALT 30-200
35.5 36.8 44 43 IU/L Albumin 2.5-4.0 3.0 3.1 3.4 3.1 g/dL
Triglycerides 25-100* 223.8 138.8 268.3 212.8 mg/dL Cholesterol
70-125 129 128 152 135.3 mg/dL Glucose 80-150* 176.5 208.8 212.3
164.5 mg/dL *Triglyceride and glucose levels are routinely higher
in the Balb/c strain of mice than in other strains of mice.
[0269] In some oligonucleotide-treated animals cholesterol levels
were above the normal range; however, this elevation is not
significant since saline-treated animals also exhibited cholesterol
above the normal range. The levels of the remaining routine
clinical indicators of liver and kidney injury and disease are
within normal ranges and are not significantly changed relative to
saline-treated animals, demonstrating that the compounds of the
invention do not significantly affect renal or hepatic function.
Triglyceride and glucose levels, while outside the normal range as
is common in the Balb/c strain, are not significantly elevated
relative to saline-treated animals.
[0270] Mice injected with ISIS 145742, ISIS 341699 and ISIS 257016
at 0.2-50 mg/kg were also evaluated histologically following
routine procedures. Liver and kidney samples were procured, fixed
in 10% neutral-buffered formalin and processed for staining with
hematoxylin and eosin or with the anti-oligonucleotide IgGI
antibody 2E1-B5, as described in other examples herein. Hematoxylin
and eosin staining in both liver and kidney tissues exhibited no
significant difference between saline- and antisense
oligonucleotide-treated animals. Staining with the 2E1 antibody
showed high background in sinusoidal tissues of liver from the
saline-injected animals, therefore making it difficult to interpret
positive staining in the oligonucleotide-treated livers. Kidney
samples from saline-injected animals and animals treated with 0.2
mg/kg ISIS 257016 showed no positive oligonucleotide staining;
however, the remainder of the oligonucleotide-treated animals
demonstrated high levels of staining in the proximal tubules, which
increased with dose.
[0271] The results illustrated in this example demonstrate that
antisense compounds of different chemistries are delivered to the
kidney, reduce SGLT2 expression in vivo in a dose-dependent manner,
and that treatment with these compounds does not result in liver or
kidney toxicity. The results further demonstrate that mixed
backbone compounds ISIS 341699 and ISIS 257016 are particularly
effective at reducing target mRNA levels in the kidney.
Example 23
Dose Response Study Comparing Mixed Backbone and Full
Phosphorothioate Backbones: a Third SGLT2 Antisense Sequence
[0272] A third mixed backbone SGLT2 oligonucleotide, ISIS 351642
(SEQ ID NO: 266), and control phosphorothioate SGLT2
oligonucleotide, ISIS 145746 (SEQ ID NO: 266), were selected for a
dose response study in mice.
[0273] Male 7-week old Balb/c mice (Charles River Laboratories,
Wilmington, Mass.) were given intraperitoneal injections of ISIS
145746 or ISIS 351642 twice per week for two weeks with the doses
shown in Table 23. Saline-injected animals served as controls. Each
treatment group contained 4 mice. The mice were sacrificed 2 days
following administration of the fourth and final dose of
oligonucleotide or saline.
[0274] Mice were evaluated for SGLT2 levels in kidney. Target
levels were determined by quantitative real-time PCR as described
by other examples herein. PCR results were normalized to
cyclophilin. The data are expressed as percent change relative to
saline treated animals ("+" indicates an increase, "-" indicates a
decrease) and are illustrated in Table 23.
31TABLE 23 Antisense inhibition of SGLT2 mRNA expression in vivo by
mixed backbone and full phosphorothioate oligonucleotides
(expressed as percent change in SGLT2 mRNA expression relative to
saline) Dose of oligonucleotide ISIS ISIS mg/kg 145746 351642 1 --
-26.7 5 -5.8 -35.1 25 -10.5 -44.3 50 -35.6 -31.8
[0275] These results illustrate that the compounds of the
invention, both full phosphorothioate and mixed backbone
oligonucleotides, can inhibit the expression of kidney SGLT2 in a
dose-dependent manner. At doses of 5 and 25 mg/kg, greater
inhibition is observed in kidneys from mice treated with ISIS
351462, suggesting the mixed backbone antisense compound is a more
efficient inhibitor of target mRNA expression in the kidney.
[0276] Treated mice were further evaluated for body weight, liver
weight and spleen weight. The data are expressed as percent change
in body or organ weight ("+" indicates an increase, "-" indicates a
decrease). The results are presented in Table 24.
32TABLE 24 Effects of antisense compounds on total body weight,
liver weight and spleen weight of mice Percent change in weight
ISIS ISIS Dose of 145746 351642 oligonucleotide Total Total mg/kg
Body Liver Spleen Body Liver Spleen 1 -- -- -- +6.9 -8.2 +0.8 5
+3.6 -5.7 +6.5 +4.6 -0.6 -7.9 25 +5.4 -2.0 +3.7 +4.7 -10.6 +1.1 50
+12.1 -8.4 +10.0 +7.4 -3.0 +1.3
[0277] No significant change was observed in total body weight,
liver weight or spleen weight at timepoints throughout or at the
termination of the study.
[0278] Levels of BUN, creatinine, AST, ALT, albumin, triglycerides,
cholesterol and glucose were measured in mice treated with the
compounds of the invention. Plasma samples were analyzed using the
Olympus AU400e automated chemistry analyzer (Olympus America,
Irving, Tex.). The results, expressed as units measured, are shown
for ISIS 145746 in Table 25 and ISIS 351642 in Table 26.
33TABLE 25 Effect of the full phosphorothioate antisense compound
ISIS 145746 on indicators of liver and kidney function Units
measured per dose of ISIS 145746 Serum Normal Sa- 1 5 25 50
indicator Range line mg/kg mg/kg mg/kg mg/kg Creatinine 0.0-1.0 0.1
-- 0.2 0.2 0.1 mg/L AST 30-300 129 -- 60 84 155 IU/L ALT 30-200 30
-- 28 26 77 IU/L Albumin 2.5-4.0 2.8 -- 2.9 2.8 2.9 g/dL
Triglycerides 25-100* 298 -- 268 259 236 mg/dL Cholesterol 70-125
116 -- 118 108 106 mg/dL Glucose 80-150* 163 -- 162 181 179
mg/dL
[0279]
34TABLE 26 Effect of mixed backbone antisense compound ISIS 351642
on indicators of liver and kidney function Units measured per dose
of ISIS 351642 Serum Normal Sa- 1 5 25 50 indicator Range line
mg/kg mg/kg mg/kg mg/kg Creatinine 0.0-1.0 0.1 0.1 0.1 0.2 0.2 mg/L
AST 30-300 129 132 75 131 160 IU/L ALT 30-200 30 31 28 29 31 IU/L
Albumin 2.5-4.0 2.8 2.9 3.0 2.7 2.8 g/dL Triglycerides 25-100* 298
238 287 240 233 mg/dL Cholesterol 70-125 116 117 122 106 113 mg/dL
Glucose 80-150* 163 195 175 164 171 mg/dL *Triglyceride and glucose
levels are routinely higher in the Balb/c strain of mice than in
other strains of mice.
[0280] The levels of routine clinical indicators of liver and
kidney injury and disease are within normal ranges and are not
significantly changed relative to saline-treated animals,
demonstrating that the compounds of the invention do not
significantly affect renal or hepatic function. Triglyceride and
glucose levels, while outside the normal range as is common in the
Balb/c strain, are not significantly elevated relative to
saline-treated animals.
[0281] The results illustrated in this example demonstrate that
antisense compounds of different chemistries are delivered to the
kidney, reduce SGLT2 expression in vivo in a dose-dependent manner,
and that treatment with these compounds does not result in liver or
kidney toxicity. The results further suggest that mixed backbone
compound ISIS 351642 is more effective than full phosphorothioate
oligonucleotides at reducing target mRNA levels in the kidney,
particularly at low doses.
Example 24
Comparison of a Standard Mixed Backbone Compound and a Mixed
Backbone Compound with Phosphorothioate Linkages at the Extreme 5'
and 3' Ends: a Single Dose Study
[0282] In accordance with the present invention, ISIS 257016 (SEQ
ID NO: 106) and ISIS 351641 (SEQ ID NO: 106) were analyzed for
their ability to inhibit SGLT2 expression in vivo. ISIS 257016 is a
standard mixed backbone compound having 2'-MOE wings and a deoxy
gap, with phosphodiester linkages in the wings and phosphorothioate
linkages in the gap. ISIS 351641 differs from the standard mixed
backbone compounds by having one phosphorothioate linkage at each
of the extreme 5' and 3' ends of the wings.
[0283] Male 8-week old Balb/c mice (Charles River Laboratories,
Wilmington, Mass.) were given a single intraperitoneal injection of
ISIS 257016 or ISIS 351641 at a dose of 1, 5, 25 or 50 mg/kg.
Saline-injected animals served as controls. Each treatment group
contained 4 mice. The mice were sacrificed 2 days following
administration of the single dose of oligonucleotide or saline.
[0284] Mice were evaluated for SGLT2 levels in kidney. Target
levels were determined by quantitative real-time PCR as described
by other examples herein. PCR results were normalized to
cyclophilin. The data are expressed as percent change relative to
saline treated animals ("+" indicates an increase, "-" indicates a
decrease) and are illustrated in Table 27.
35TABLE 27 Antisense inhibition of SGLT2 mRNA expression in vivo by
mixed backbone oligonucleotides (expressed as percent change in
SGLT2 mRNA expression relative to saline) Dose of oligonucleotide
ISIS ISIS mg/kg 257016 351641 1 -21.5 -14.0 5 -26.4 -19.3 25 -24.2
-12.5 50 -36.3 -22.0
[0285] These results illustrate that mixed backbone compounds of
the invention, with either complete phosphodiester linkages in the
wings, or with the extreme 5' and 3 ' ends substituted with
phosphorothioate linkages, inhibit the expression of kidney SGLT2
in a dose-dependent manner. However, greater inhibition is observed
in kidneys from mice treated with ISIS 257016, which contains all
phosphodiester linkages in the wings.
[0286] Treated mice were further evaluated for body weight and
liver and spleen weight. The data are expressed as percent change
in body or organ weight ("+" indicates an increase, "-" indicates a
decrease). The results are presented in Table 28.
36TABLE 28 Effects of antisense compounds on total body weight,
liver weight and spleen weight of mice Percent change in weight
ISIS ISIS Dose of 257016 351641 oligonucleotide Total Total mg/kg
Body Liver Spleen Body Liver spleen 1 -0.9 +1.2 -1.6 +2.8 +3.0 -0.1
5 -5.1 +5.4 +20.1 +4.0 +2.1 +9.7 25 -1.1 +3.5 +3.8 -0.7 +9.3 +5.9
50 -2.5 -2.3 +7.8 +0.9 -0.7 +10.2
[0287] No significant change was observed in total body weight,
liver weight or spleen weight at timepoints throughout or at the
termination of the study.
[0288] Levels of creatinine, AST, ALT, albumin, triglycerides,
cholesterol and glucose were measured in mice treated with the
compounds of the invention. Plasma samples were analyzed using the
Olympus AU400e automated chemistry analyzer (Olympus America,
Irving, Tex.). The results, expressed as units measured, are shown
for ISIS 257016 in Table 29 and for ISIS 351641 in Table 30.
37TABLE 29 Effect of mixed backbone antisense compound ISIS 257016
on indicators of liver and kidney function Units measured per dose
of ISIS 257016 Serum Normal Sa- 1 5 25 50 indicator Range line
mg/kg mg/kg mg/kg mg/kg Creatinine 0.0-1.0 0.0 0.0 0.0 0.0 0.2 mg/L
AST 30-300 141 62 77 89 88 IU/L ALT 30-200 30 29 28 27 33 IU/L
Albumin 2.5-4.0 2.9 2.8 2.8 3.0 2.9 g/dL Triglycerides 25-100* 213
253 255 347 245 mg/dL Cholesterol 70-125 118 111 116 125 120 mg/dL
Glucose 80-150* 155 186 172 174 169 mg/dL
[0289]
38TABLE 30 Effect of mixed backbone antisense compound ISIS 351641
on indicators of liver and kidney function Units measured per dose
of ISIS 351641 Serum Normal Sa- 1 5 25 50 indicator Range line
mg/kg mg/kg mg/kg mg/kg Creatinine 0.0-1.0 0.0 0.2 0.1 0.1 0.2 mg/L
AST 30-300 141 75 117 68 98 IU/L ALT 30-200 30 25 33 30 27 IU/L
Albumin 2.5-4.0 2.9 2.9 2.9 2.9 2.9 g/dL Triglycerides 25-100* 213
271 280 296 271 mg/dL Cholesterol 70-125 118 120 126 112 117 mg/dL
Glucose 80-150* 155 162 171 189 175 mg/dL *Triglyceride and glucose
levels are routinely higher in the Balb/c strain of mice than in
other strains of mice.
[0290] The levels of routine clinical indicators of liver and
kidney injury and disease are within normal ranges and are not
significantly changed relative to saline-treated animals,
demonstrating that the compounds of the invention do not
significantly affect renal or hepatic function. Triglyceride and
glucose levels, while outside the normal range as is common in the
Balb/c strain, are not significantly elevated relative to
saline-treated animals.
[0291] The results illustrated in this example demonstrate that
mixed backbone compounds of varying chemistries are delivered to
the kidney, reduce SGLT2 expression in vivo, and that treatment
with these compounds does not result in liver or kidney toxicity.
The results further indicate that mixed backbone compounds with
wings composed completely of phosphodiester linkages are more
efficient inhibitors of target mRNA.
Example 25
Effects of Modified Antisense Compounds on SGLT2 mRNA Expression in
vivo: Two and Three Dose Protocols
[0292] In accordance with the present invention, mixed backbone
compound ISIS 257016 (SEQ ID NO; 106) was analyzed for its ability
to inhibit SGLT2 expression in vivo when administered in either two
or three doses. ISIS 353003 (CCTTCCCTGAAGGTTCCTCC; SEQ ID NO: 267),
a mixed backbone oligonucleotide which targets human PTP1B, was
used as a control.
[0293] Male 8-week old Balb/c mice (Charles River Laboratories,
Wilmington, Mass.) were given two or three intraperitoneal
injections of ISIS 257016 or ISIS 353003 at three day intervals.
ISIS 257016 was administered at doses of 1, 5 or 25 mg/kg and ISIS
353003 was administered at a dose of 25 mg/kg. Saline-injected
animals served as controls. Each treatment group contained 4 mice.
The mice were sacrificed 2 days following administration of the
final dose of oligonucleotide or saline.
[0294] Mice were evaluated for SGLT2 levels in kidney. Target
levels were determined by quantitative real-time PCR as described
in other examples herein. PCR results were normalized to
cyclophilin. The data are expressed as percent change relative to
saline treated animals ("+" indicates an increase, "-" indicates a
decrease) and are illustrated in Table 31.
39TABLE 31 Antisense inhibition of SGLT2 mRNA expression in vivo by
two doses or three doses of mixed backbone oligonucleotides
(expressed as percent change in SGLT2 mRNA expression relative to
saline control) Oligonucleotide Three (dose in mg/kg) Two Doses
Doses ISIS 257016 (1 mg/kg) -43.2 -39.1 ISIS 257016 (5 mg/kg) -39.7
-42.9 ISIS 257016 (25 mg/kg) -53.8 -65.5 ISIS 353003 (25 mg/kg)
-8.0 -6.9
[0295] These results illustrate that the mixed backbone compounds
of the invention efficiently inhibit the expression of kidney SGLT2
in a dose-dependent manner. Furthermore, inhibition increases with
the number of doses administered.
[0296] Treated mice were further evaluated for body weight, kidney
weight, liver weight and spleen weight. The data are expressed as
percent change in body or organ weight ("+" indicates an increase,
"-" indicates a decrease). The results are presented in Table 32
and Table 33.
40TABLE 32 Effects of antisense compounds on total body weight of
mice (expressed as percent change in body weight) Oligonucleotide
Two Three (dose in mg/kg) Doses Doses ISIS 257016 (1 mg/kg) -1.1 0
ISIS 257016 (5 mg/kg) +1.3 +0.8 ISIS 257016 (25 mg/kg) +0.1 +1.3
ISIS 353003 (25 mg/kg) -0.8 +0.8
[0297]
41TABLE 33 Effects of antisense compounds on total kidney weight,
liver weight and spleen weight of mice Percent change in weight
Oligonucleotide Two Doses Three Doses (dose in mg/kg) Kidney Liver
Spleen Kidney Liver Spleen ISIS 257016 -0.5 -2.2 -4.3 -5.6 -3.8
-5.9 (1 mg/kg) ISIS 257016 -5.4 +2.5 +7.4 -6.6 -7.1 -9.0 (5 mg/kg)
ISIS 257016 -7.9 -1.1 +4.2 -8.6 -8.8 -1.2 (25 mg/kg) ISIS 353003
-5.5 +1.2 -2.7 -0.2 -4.0 +6.5 (25 mg/kg)
[0298] No significant change was observed in total body weight,
kidney weight, liver weight or spleen weight at timepoints
throughout or at the termination of the study.
[0299] Levels of BUN, creatinine, bilirubin, AST, ALT, albumin,
triglycerides, cholesterol and glucose were measured in mice
treated with the compounds of the invention. Plasma samples were
analyzed using the Olympus AU400e automated chemistry analyzer
(Olympus America, Irving, Tex.). The results, expressed as units
measured, are shown for the two dose protocol in Table 34 and for
the three dose protocol in Table 35.
42TABLE 34 Effect of mixed backbone antisense compound ISIS 257016
administered according to the two dose protocol on indicators of
liver and kidney function Units measured per dose of ISIS 257016
Serum Normal Sa- 1 5 25 ISIS indicator Range line mg/kg mg/kg mg/kg
353003 BUN 15-40 32 34 29 25 28 mg/dL Creatinine 0.0-1.0 0.1 0.1
0.2 0.1 0.1 mg/L Bilirubin 0.1-1.0 0.1 0.1 0.1 0.1 0.1 mg/dL AST
30-300 54 119 156 116 154 IU/L ALT 30-200 27 36 45 30 36 IU/L
Albumin 2.5-4.0 2.7 3.2 3.1 3.0 2.8 g/dL Triglycerides 25-100* 221
263 234 264 278 mg/dL Cholesterol 70-125 113 118 117 125 125 mg/dL
Glucose 80-150* 170 157 177 163 152 mg/dL
[0300]
43TABLE 35 Effect of mixed backbone antisense compound ISIS 257016
administered according to the three dose protocol on indicators of
liver and kidney function Units measured per dose of ISIS 257016
Serum Normal Sa- 1 5 25 ISIS indicator Range line mg/kg mg/kg mg/kg
353003 BUN 15-40 30 32 30 27 27 mg/dL Creatinine 0.0-1.0 0.1 0.1
0.2 0.1 0.1 mg/L Bilirubin 0.1-1.0 0.1 0.1 0.1 0.1 0.1 mg/dL AST
30-300 126 83 81 59 57 IU/L ALT 30-200 35 30 57 27 24 IU/L Albumin
2.5-4.0 3.0 2.8 2.8 2.7 2.8 g/dL Triglycerides 25-100* 223 236 202
153 188 mg/dL Cholesterol 70-125 112 113 114 116 106 mg/dL Glucose
80-150* 152 169 161 181 192 mg/dL *Triglyceride and glucose levels
are routinely higher in the Balb/c strain of mice than in other
strains of mice.
[0301] The levels of routine clinical indicators of liver and
kidney injury and disease are within normal ranges and are not
significantly changed relative to saline-treated animals,
demonstrating that the compounds of the invention do not
significantly affect renal or hepatic function. Triglyceride and
glucose levels, while outside the normal range as is common in the
Balb/c strain, are not significantly elevated relative to
saline-treated animals.
[0302] Mice injected with ISIS 257016 and control animals were also
evaluated histologically following routine procedures. Liver and
kidney samples were procured, fixed in 10% neutral-buffered
formalin and processed for staining with hematoxylin and eosin.
Hematoxylin and eosin staining exhibited no significant difference
between saline- and oligonucleotide-treated animals. All tissue
samples exhibited normal kidney and liver morphology.
[0303] The results illustrated in this example demonstrate that
mixed backbone compounds are delivered to the kidney, reduce SGLT2
expression in vivo, and that treatment with these compounds does
not result in liver or kidney toxicity. The results further
indicate that inhibition of target mRNA expression in the kidney
increases with the number of doses administered.
Example 26
Effects of Mixed Backbone Antisense Compounds on SGLT2 mRNA
Expression in vivo: Two to Five Day Consecutive Daily Dosing
Protocols
[0304] In accordance with the present invention, mixed backbone
compound ISIS 257016 (SEQ ID NO: 106) was analyzed for its ability
to inhibit SGLT2 expression in vivo when administered in two to
five doses (consecutive daily doses). ISIS 353003 (SEQ ID NO: 267),
a mixed backbone oligonucleotide which targets human PTP IB, was
used as a control.
[0305] Male 9-week old Balb/c mice (Charles River Laboratories,
Wilmington, Mass.) were given two, three, four or five
intraperitoneal injections of ISIS 257016 or ISIS 353003 once a day
for the treatment period. ISIS 257016 was administered at doses of
2.5 or 25 mg/kg and ISIS 353003 was administered at a dose of 25
mg/kg. Saline-injected animals served as controls. Each treatment
group contained 4 mice. The mice were sacrificed 2 days following
administration of the final dose of oligonucleotide or saline.
[0306] Mice were evaluated for SGLT2 levels in kidney. Target
levels were determined by quantitative real-time PCR as described
in other examples herein. PCR results were normalized to
cyclophilin. The data are expressed as percent change relative to
saline treated animals ("+" indicates an increase, "-" indicates a
decrease) and are illustrated in Table 36.
44TABLE 36 Antisense inhibition of SGLT2 mRNA expression in vivo by
mixed backbone oligonucleotide (expressed as percent change in
SGLT2 mRNA expression relative to saline control) Oligonucleotide
Three Four Five (dose in mg/kg) Two Doses Doses Doses Doses ISIS
257016 (2.5 mg/kg) -14.2 -35.4 -25.3 -42.0 ISIS 257016 (25 mg/kg)
-12.5 -32.9 -39.1 -68.9 ISIS 353003 (25 mg/kg) -4.5 -9.6 +0.5
-11.3
[0307] These results illustrate that the mixed backbone compounds
of the invention efficiently inhibit the expression of kidney SGLT2
and inhibition increases with the number of doses administered.
[0308] Treated mice were further evaluated for body weight, kidney
weight, liver weight and spleen weight. The data are expressed as
percent change in body or organ weight ("+" indicates an increase,
"-" indicates a decrease). The results are presented in Tables
37-40.
45TABLE 37 Effects of antisense compounds on total body weight of
mice (expressed as percent change in body weight) Oligonucleotide
Two Three Four Five (dose in mg/kg) Doses Doses Doses Doses ISIS
257016 (2.5 mg/kg) +2.7 +2.7 +3.2 +1.5 ISIS 257016 (25 mg/kg) +2.0
+2.0 +3.1 -0.7 ISIS 353003 (25 mg/kg) +0.6 +0.8 +2.5 +1.3
[0309]
46TABLE 38 Effects of antisense compounds on total kidney weight
(expressed as percent change in kidney weight) Oligonucleotide Two
Three Four Five (dose in mg/kg) Doses Doses Doses Doses ISIS 257016
(2.5 mg/kg) +8.2 -1.4 +8.9 +1.5 ISIS 257016 (25 mg/kg) +11.5 +3.6
+2.7 -7.7 ISIS 353003 (25 mg/kg) +5.3 -3.6 +4.9 +7.1
[0310]
47TABLE 39 Effects of antisense compounds on total liver weight
(expressed as percent change in liver weight) Oligonucleotide Two
Three Four Five (dose in mg/kg) Doses Doses Doses Doses ISIS 257016
(2.5 mg/kg) +9.2 +7.5 +4.8 +4.8 ISIS 257016 (25 mg/kg) +11.8 +5.2
+0.6 -8.0 ISIS 353003 (25 mg/kg) +7.4 -3.4 +12.9 +9.5
[0311]
48TABLE 40 Effects of antisense compounds on total spleen weight
(expressed as percent change in spleen weight) Oligonucleotide Two
Three Four Five (dose in mg/kg) Doses Doses Doses Doses ISIS 257016
(2.5 mg/kg) +22.2 +10.1 +15.3 +10.7 ISIS 257016 (25 mg/kg) +13.3
+5.1 +6.7 +4.5 ISIS 353003 (25 mg/kg +7.3 +1.4 +19.8 +8.6
[0312] No significant change was observed in total body weight,
kidney weight, liver weight or spleen weight at timepoints
throughout or at the termination of the study.
[0313] Levels of creatinine, AST, ALT, albumin, triglycerides,
cholesterol and glucose were measured in mice treated with the
compounds of the invention. Plasma samples were analyzed using the
Olympus AU400e automated chemistry analyzer (Olympus America,
Irving, Tex.). The results, expressed as units measured, are shown
in Tables 41-44.
49TABLE 41 Effect of mixed backbone antisense compound ISIS 257016
administered as two consecutive daily doses on indicators of liver
and kidney function Units measured per dose of oligonucleotide ISIS
ISIS ISIS Serum Normal 257016 257016 353003 indicator Range Saline
2.5 mg/kg 25 mg/kg 25 mg/kg Creatinine 0.0-1.0 0.2 0.1 0.1 0.2 mg/L
AST 30-300 160 132 75 131 IU/L ALT 30-200 31 31 28 29 IU/L Albumin
2.5-4.0 2.8 2.9 3.0 2.7 g/dL Triglycerides 25-100* 233 238 287 240
mg/dL Cholesterol 70-125 113 117 122 106 mg/dL Glucose 80-150* 171
195 175 164 mg/dL
[0314]
50TABLE 42 Effect of mixed backbone antisense compound ISIS 257016
administered as three consecutive daily doses on indicators of
liver and kidney function Units measured per dose of
oligonucleotide ISIS ISIS ISIS Serum Normal 257016 257016 353003
indicator Range Saline 2.5 mg/kg 25 mg/kg 25 mg/kg Creatinine
0.0-1.0 0.1 0.2 0.2 0.1 mg/L AST 30-300 199 60 84 155 IU/L ALT
30-200 29 28 26 77 IU/L Albumin 2.5-4.0 2.8 2.9 2.8 2.9 g/dL
Triglycerides 25-100* 289 268 259 236 mg/dL Cholesterol 70-125 111
118 108 106 mg/dL Glucose 80-150* 204 162 181 179 mg/dL
[0315]
51TABLE 43 Effect of mixed backbone antisense compound ISIS 257016
administered as four consecutive daily doses on indicators of liver
and kidney function Units measured per dose of oligonucleotide ISIS
ISIS ISIS Serum Normal 257016 257016 353003 indicator Range Saline
2.5 mg/kg 25 mg/kg 25 mg/kg Creatinine 0.0-1.0 0.1 0.1 0.1 0.2 mg/L
AST 30-300 199 92 120 144 IU/L ALT 30-200 29 30 30 36 IU/L Albumin
2.5-4.0 2.8 3.0 2.8 3.0 g/dL Triglycerides 25-100* 289 252 269 294
mg/dL Cholesterol 70-125 111 126 115 120 mg/dL Glucose 80-150* 204
173 198 192 mg/dL
[0316]
52TABLE 44 Effect of mixed backbone antisense compound ISIS 257016
administered as five consecutive daily doses on indicators of liver
and kidney function Units measured per dose of oligonucleotide ISIS
ISIS ISIS Serum Normal 257016 257016 353003 indicator Range Saline
2.5 mg/kg 25 mg/kg 25 mg/kg Creatinine 0.0-1.0 0.1 0.1 0.1 0.1 mg/L
AST 30-300 129 121 125 97 IU/L ALT 30-200 30 30 33 29 IU/L Albumin
2.5-4.0 2.8 2.9 2.8 2.9 g/dL Triglycerides 25-100* 298 298 285 277
mg/dL Cholesterol 70-125 116 126 122 126 mg/dL Glucose 80-150* 163
177 204 185 mg/dL *Triglyceride and glucose levels are routinely
higher in the Balb/c strain of mice than in other strains of
mice.
[0317] The levels of routine clinical indicators of liver and
kidney injury and disease are within normal ranges and are not
significantly changed relative to saline-treated animals,
demonstrating that the compounds of the invention do not
significantly affect renal or hepatic function. Triglyceride and
glucose levels, while outside the normal range as is common in the
Balb/c strain, are not significantly elevated relative to
saline-treated animals.
[0318] The results illustrated in this example demonstrate that
mixed backbone compounds are delivered to the kidney, reduce SGLT2
expression in vivo, and that treatment with these compounds does
not result in liver or kidney toxicity. The results further
indicate that inhibition of target mRNA expression in the kidney
increases with the number of doses administered.
Example 27
Comparison of a Standard Mixed Backbone Compound and Mixed Backbone
Compounds with Phosphorothioate Linkages at Either or Both of the
Extreme 5' and 3' Ends: a Four Dose Protocol
[0319] In accordance with the present invention, ISIS 257016 (SEQ
ID NO: 106), ISIS 351641 (SEQ ID NO: 106), ISIS 360886 (SEQ ID NO:
106) and ISIS 360887 (SEQ ID NO: 106) were analyzed for their
ability to inhibit SGLT2 expression in vivo. ISIS 257016 is a
standard mixed backbone compound having 2'-MOE wings and a deoxy
gap, with phosphodiester linkages in the wings and phosphorothioate
linkages in the gap. ISIS 351641 differs from the standard mixed
backbone compounds by having one phosphorothioate linkage at each
of the extreme 5' and 3' ends of the wings. ISIS 360886 and ISIS
360887 are mixed backbone compounds with one phosphorothioate
linkage at the extreme 5' end or extreme 3' end, respectively.
[0320] Male 7-week old Balb/c mice (Charles River Laboratories,
Wilmington, Mass.) were given intraperitoneal injections of ISIS
257016, ISIS 351641, ISIS 360886 or ISIS 360887 twice a week for
two weeks at doses of 1.56, 6.25 or 25 mg/kg. Saline-injected
animals served as controls. Each treatment group contained 4 mice.
The mice were sacrificed 2 days following administration of the
final dose of oligonucleotide or saline.
[0321] Mice were evaluated for SGLT2 levels in kidney. Target
levels were determined by quantitative real-time PCR as described
in other examples herein. PCR results were normalized to
cyclophilin. The data are expressed as percent change relative to
saline treated animals ("+" indicates an increase, "-" indicates a
decrease) and are illustrated in Table 45.
53TABLE 45 Antisense inhibition of SGLT2 mRNA expression in vivo by
mixed backbone oligonucleotides (expressed as percent change in
SGLT2 mRNA expression relative to saline) Dose of oligonucleotide
ISIS ISIS ISIS ISIS mg/kg 257016 351641 360886 360887 1.56 -39.1
-4.2 -12.7 -9.7 6.25 -52.8 -4.87 -19.7 -7.3 25 -57.8 -11.0 -29.0
-4.9
[0322] These results illustrate that mixed backbone compounds of
the invention, with either complete phosphodiester linkages in the
wings, or with the extreme 5' and 3' ends substituted with
phosphorothioate linkages, can inhibit the expression of kidney
SGLT2 in a dose-dependent manner. With the exception of ISIS
360887, inhibition of target mRNA was dose-dependent. Although all
mixed backbone compounds inhibited SGLT2 expression, greater
inhibition is observed in kidneys from mice treated with ISIS
257016, which is a mixed backbone compound that contains all
phosphodiester linkages in the wings.
[0323] Treated mice were further evaluated for body weight, kidney
weight, liver weight and spleen weight. The data are expressed as
percent change in body or organ weight ("+" indicates an increase,
"-" indicates a decrease). The results are presented in Table
46.
54TABLE 46 Effects of antisense compounds on total body weight,
kidney weight, liver weight and spleen weight of mice (expressed as
percent change in weight) Dose Body Kidney Liver Spleen
Oligonucleotide mg/kg weight weight weight weight ISIS 257016 1.56
+11.6 -3.5 -4.2 -2.4 ISIS 257016 6.25 +7.9 -3.0 +3.8 -1.3 ISIS
257016 25 +11.7 -4.1 +1.4 +8.9 ISIS 351641 1.56 +7.9 -0.9 -5.4 +9.4
ISIS 351641 6.25 +11.1 +1.3 -2.2 +13.4 ISIS 351641 25 +7.4 -2.1
-0.5 -1.4 ISIS 360886 1.56 +7.6 -1.0 -13.7 -5.0 ISIS 360886 6.25
+8.9 -3.7 -16.6 +1.2 ISIS 360886 25 +11.1 -5.5 -11.6 +0.8 ISIS
360887 1.56 +8.5 +1.0 -10.4 -0.4 ISIS 360887 6.25 +7.5 -1.8 -8.4
+1.1 ISIS 360887 25 +9.8 +2.2 -9.0 +11.8
[0324] No significant change was observed in total body weight,
liver weight or spleen weight at timepoints throughout or at the
termination of the study.
[0325] Levels of BUN, creatinine, bilirubin, AST, ALT, albumin,
triglycerides, cholesterol and glucose were measured in mice
treated with the compounds of the invention. Plasma samples were
analyzed using the Olympus AU400e automated chemistry analyzer
(Olympus America, Irving, Tex.). The results, expressed as units
measured, are shown in Tables 47-50.
55TABLE 47 Effect of mixed backbone antisense compound ISIS 257016
on indicators of liver and kidney function Units measured per dose
of ISIS 257016 Serum Normal 1.56 6.25 25 indicator Range Saline
mg/kg mg/kg mg/kg BUN 15-40 23 21 26 22 mg/dL Creatinine 0.0-1.0
0.2 0.2 0.2 0.2 mg/L Bilirubin mg/dL 0.1-1.0 0.2 0.2 0.2 0.1 AST
30-300 75 61 83 71 IU/L ALT 30-200 30 30 33 39 IU/L Albumin 2.5-4.0
2.8 2.9 2.9 2.7 g/dL Triglycerides 25-100* 208 210 243 150 mg/dL
Cholesterol 70-125 116 125 130 135 mg/dL Glucose 80-150* 207 184
184 215 mg/dL
[0326]
56TABLE 48 Effect of mixed backbone antisense compound ISIS 351641
on indicators of liver and kidney function Units measured per dose
of ISIS 351641 Normal 1.56 6.25 25 Serum indicator Range Saline
mg/kg mg/kg mg/kg BUN 15-40 23 23 25 22 mg/dL Creatinine 0.0-1.0
0.2 0.2 0.2 0.2 mg/L Bilirubin mg/dL 0.1-1.0 0.2 0.1 0.2 0.1 AST
30-300 75 61 67 54 IU/L ALT 30-200 30 32 31 30 IU/L Albumin 2.5-4.0
2.8 2.7 2.7 2.8 g/dL Triglycerides 25-100* 208 169 176 185 mg/dL
Cholesterol 70-125 116 110 115 107 mg/dL Glucose 80-150* 207 205
199 208 mg/dL
[0327]
57TABLE 49 Effect of mixed backbone antisense compound ISIS 360886
on indicators of liver and kidney function Units measured per dose
of ISIS 360886 Normal 1.56 6.25 25 Serum indicator Range Saline
mg/kg mg/kg mg/kg BUN 15-40 23 21 23 24 mg/dL Creatinine 0.0-1.0
0.2 0.1 0.2 0.2 mg/L Bilirubin mg/dL 0.1-1.0 0.2 0.2 0.2 0.1 AST
30-300 75 56 77 73 IU/L ALT 30-200 30 26 27 28 IU/L Albumin 2.5-4.0
2.8 2.7 2.7 2.7 g/dL Triglycerides 25-100* 208 164 181 169 mg/dL
Cholesterol 70-125 116 105 108 108 mg/dL Glucose 80-150* 207 189
202 200 mg/dL
[0328]
58TABLE 50 Effect of mixed backbone antisense compound ISIS 360887
on indicators of liver and kidney function Units measured per dose
of ISIS 360887 Normal 1.56 6.25 25 Serum indicator Range Saline
mg/kg mg/kg mg/kg BUN 15-40 23 23 22 23 mg/dL Creatinine 0.0-1.0
0.2 0.2 0.2 0.2 mg/L Bilirubin mg/dL 0.1-1.0 0.2 0.2 0.1 0.2 AST
30-300 75 142 83 108 IU/L ALT 30-200 30 40 39 34 IU/L Albumin
2.5-4.0 2.8 2.7 2.7 2.7 g/dL Triglycerides 25-100* 208 136 157 200
mg/dL Cholesterol 70-125 116 109 107 110 mg/dL Glucose 80-150* 207
199 201 187 mg/dL *Triglyceride and glucose levels are routinely
higher in the Balb/c strain of mice than in other strains of
mice.
[0329] Cholesterol levels of mice treated with either 6.25 or 25
mg/kg were slightly elevated; however, these levels are not
significantly greater than the cholesterol levels observed in
saline-treated control animals. Otherwise, the levels of routine
clinical indicators of liver and kidney injury and disease are
within normal ranges and are not significantly changed relative to
saline-treated animals, demonstrating that the compounds of the
invention do not significantly affect renal or hepatic function.
Triglyceride and glucose levels, while outside the normal range as
is common in the Balb/c strain, are not significantly elevated
relative to saline-treated animals.
[0330] Saline- and oligonucleotide-injected animals also were
evaluated histologically following routine procedures. Liver and
kidney samples were procured, fixed in 10% neutral-buffered
formalin and processed for staining with hematoxylin and eosin.
Hematoxylin and eosin staining exhibited no significant difference
between control and oligonucleotide-treated animals.
[0331] The results illustrated in this example demonstrate that
mixed backbone compounds are delivered to the kidney, reduce SGLT2
expression in vivo, and that treatment with these compounds does
not result in liver or kidney toxicity. The results further
indicate that mixed backbone compounds with complete phosphodiester
linkages in the wings are more effective modulators of target mRNA
expression in the kidney than mixed backbone compounds with a
phosphorothioate linkage at one or both of the extreme 5' and 3'
ends.
Example 28
Comparison of a Standard Mixed Backbone Compound and Mixed Backbone
Compounds with Phosphorothioate Linkages at Either or Both of the
Extreme 5' and 3' Ends: an Eight Dose Protocol
[0332] A second study of SGLT2 antisense oligonucleotides ISIS
257016, ISIS 351641, ISIS 360886 and ISIS 360887 was undertaken in
which mice received eight doses over a four week period. As
described previously, ISIS 257016 is a standard mixed backbone
compound having 2'-MOE wings and a deoxy gap, with phosphodiester
linkages in the wings and phosphorothioate linkages in the gap.
ISIS 351641 differs from the standard mixed backbone compounds by
having one phosphorothioate linkage at each of the extreme 5' and
3' ends of the wings. ISIS 360886 and ISIS 360887 are mixed
backbone compounds with one phosphorothioate linkage at the extreme
5' end and extreme 3' end, respectively.
[0333] Male 8-week old Balb/c mice (Charles River Laboratories,
Wilmington, Mass.) were given intraperitoneal injections of ISIS
257016, ISIS 351641, ISIS 360886 or ISIS 360887 twice a week for
four weeks at doses of 1, 5 or 25 mg/kg. Saline-injected animals
served as controls. Each treatment group contained 4 mice. The mice
were sacrificed 2 days following administration of the final dose
of oligonucleotide or saline.
[0334] Mice were evaluated for SGLT2 levels in kidney. Target
levels were determined by quantitative real-time PCR as described
by other examples herein. PCR results were normalized to
cyclophilin. The data are expressed as percent change relative to
saline treated animals ("+" indicates an increase, "-" indicates a
decrease) and are illustrated in Table 51.
59TABLE 51 Antisense inhibition of SGLT2 mRNA expression in vivo by
mixed backbone oligonucleotides (expressed as percent change in
SGLT2 mRNA expression relative to saline) Dose of oligonucleotide
ISIS ISIS ISIS ISIS mg/kg 257016 351641 360886 360887 1 -53 -14 -24
-23 5 -64 -23 -30 -26 25 -68 -37 -50 -40
[0335] These results illustrate that mixed backbone compounds of
the invention, with either complete phosphodiester linkages in the
wings, or with the extreme 5' and 3' ends substituted with
phosphorothioate linkages, can inhibit the expression of kidney
SGLT2 in a dose-dependent manner. However, greater inhibition is
observed in kidneys from mice treated with ISIS 257016, which
contains all phosphodiester linkages in the wings.
[0336] Treated mice were further evaluated for body weight and
liver and spleen weight. The data are expressed as percent change
in body or organ weight ("+" indicates an increase, "-" indicates a
decrease). The results are presented in Table 52.
60TABLE 52 Effects of antisense compounds on total body weight,
liver weight and spleen weight of mice (expressed as percent change
in weight) Dose Body Liver Spleen Oligonucleotide mg/kg weight
weight weight ISIS 257016 1 +11.8 -6.9 -10.1 ISIS 257016 5 +8.4
-4.3 +4.4 ISIS 257016 25 +5.4 -2.1 +12.5 ISIS 351641 1 +12.3 -2.8
-2.9 ISIS 351641 5 +9.2 -8.7 -5.5 ISIS 351641 25 +9.4 -0.8 +3.3
ISIS 360886 1 +9.2 -5.2 -4.5 ISIS 360886 5 +10.3 -2.7 +15.1 ISIS
360886 25 +9.4 -2.1 -11.4 ISIS 360887 1 +10.0 -7.0 -1.5 ISIS 360887
5 +12.6 -3.2 +4.0 ISIS 360887 25 +11.8 -7.6 +14.7
[0337] No significant change was observed in total body weight,
liver weight or spleen weight at timepoints throughout or at the
termination of the study.
[0338] Levels of BUN, creatinine, bilirubin, AST, ALT, albumin,
triglycerides, cholesterol and glucose were measured in mice
treated with the compounds of the invention. Plasma samples were
analyzed using the Olympus AU400e automated chemistry analyzer
(Olympus America, Irving, Tex.). The results, expressed as units
measured, are shown in Tables 53-56.
61TABLE 53 Effect of mixed backbone antisense compound ISIS 257016
on indicators of liver and kidney function Units measured per
Normal dose of ISIS 257016 Serum indicator Range Saline 1 mg/kg 5
mg/kg 25 mg/kg BUN 15-40 27 31 29 23 mg/dL Creatinine 0.0-1.0 0.2
0.2 0.2 0.2 mg/L Bilirubin mg/dL 0.1-1.0 0.2 0.2 0.2 0.2 AST 30-300
60 58 82 119 IU/L ALT 30-200 22 27 35 66 IU/L Albumin 2.5-4.0 2.7
2.8 2.7 2.6 g/dL Triglycerides 25-100* 178 263 187 99 mg/dL
Cholesterol 70-125 123 142 138 162 mg/dL Glucose 80-150* 193 201
201 185 mg/dL
[0339]
62TABLE 54 Effect of mixed backbone antisense compound ISIS 351641
on indicators of liver and kidney function Units measured per
Normal dose of ISIS 351641 Serum indicator Range Saline 1 mg/kg 5
mg/kg 25 mg/kg BUN 15-40 27 27 26 28 mg/dL Creatinine 0.0-1.0 0.2
0.2 0.2 0.2 mg/L Bilirubin 0.1-1.0 0.2 0.1 0 0.1 mg/dL AST 30-300
60 48 49 50 IU/L ALT 30-200 22 23 23 20 IU/L Albumin 2.5-4.0 2.7
2.8 2.8 2.7 g/dL Triglycerides 25-100* 178 165 197 222 mg/dL
Cholesterol 70-125 123 118 120 118 mg/dL Glucose 80-150* 193 192
200 197 mg/dL
[0340]
63TABLE 55 Effect of mixed backbone antisense compound ISIS 360886
on indicators of liver and kidney function Units measured per
Normal dose of ISIS 360886 Serum indicator Range Saline 1 mg/kg 5
mg/kg 25 mg/kg BUN 15-40 27 27 26 27 mg/dL Creatinine 0.0-1.0 0.2
0.2 0.2 0.2 mg/L Bilirubin 0.1-1.0 0.2 0 0.1 0.1 mg/dL AST 30-300
60 52 71 90 IU/L ALT 30-200 22 23 23 29 IU/L Albumin 2.5-4.0 2.7
2.8 2.8 2.8 g/dL Triglycerides 25-100* 178 230 250 227 mg/dL
Cholesterol 70-125 123 122 129 133 mg/dL Glucose 80-150* 193 187
182 185 mg/dL
[0341]
64TABLE 56 Effect of mixed backbone antisense compound ISIS 360887
on indicators of liver and kidney function Units measured per
Normal dose of ISIS 360887 Serum indicator Range Saline 1 mg/kg 5
mg/kg 25 mg/kg BUN 15-40 27 25 24 23 mg/dL Creatinine 0.0-1.0 0.2
0.2 0.2 0.1 mg/L Bilirubin 0.1-1.0 0.2 0.2 0.2 0.2 mg/dL AST 30-300
60 60 44 92 IU/L ALT 30-200 22 24 22 31 IU/L Albumin 2.5-4.0 2.7
2.7 2.5 2.7 g/dL Triglycerides 25-100* 178 240 262 171 mg/dL
Cholesterol 70-125 123 121 129 134 mg/dL Glucose 80-150* 193 189
186 181 mg/dL *Triglyceride and glucose levels are routinely higher
in the Balb/c strain of mice than in other strains of mice.
[0342] The levels of routine clinical indicators of liver and
kidney injury and disease are within normal ranges and are not
significantly changed relative to saline-treated animals,
demonstrating that the compounds of the invention do not
significantly affect renal or hepatic function. Triglyceride and
glucose levels, while outside the normal range as is common in the
Balb/c strain, are not significantly elevated relative to
saline-treated animals.
[0343] The results illustrated in this example demonstrate that
mixed backbone compounds are delivered to the kidney, reduce SGLT2
expression in vivo, and that treatment with these compounds does
not result in liver or kidney toxicity. Furthermore, the eight dose
protocol resulted in greater inhibition of target mRNA levels in
the kidney than observed for the four dose protocol shown in
Example 22.
Example 29
Antisense Inhibition of SGLT2 in a Murine Model of Type 2 Diabetes:
Comparison of Full Phosphorothioate and Mixed Backbone
Oligonucleotides
[0344] The Animal Models of Diabetic Complications Consortium
(AMDCC) has developed protocols for the induction of diabetes in a
number of animal models. The genetic C57BLKS/J
Lep.sup.db/Lep.sup.db model has been approved by the AMDCC as an
appropriate model system for studies of diabetic nephropathy
associated with type 2 diabetes.
[0345] Leptin is a hormone produced by fat that regulates appetite.
Deficiencies in this hormone in both humans and non-human animals
lead to obesity. Lep.sup.db/Lep.sup.db mice have a mutation in the
leptin receptor gene which results in obesity and hyperglycemia. As
such, these mice are a useful model for the investigation of
obesity and diabetes and treatments designed to treat these
conditions. In accordance with the present invention, oligomeric
compounds of the present invention were tested in the
Lep.sup.db/Lep.sup.db model of type 2 diabetes.
[0346] Male Lep.sup.db/Lep.sup.db (db/db) mice were given
intraperitoneal injections of either ISIS 257016 (SEQ ID NO: 106),
which has a mixed backbone, or ISIS 145733 (SEQ ID NO: 106), which
has a phosphorothioate backbone, twice a week for four weeks at
doses of 12.5, 25 or 37.5 mg/kg. Saline-injected animals served as
controls. Each treatment group contained 6 mice. The mice were
sacrificed 2 days following administration of the final dose of
oligonucleotide or saline.
[0347] Mice were evaluated for SGLT2 levels in kidney. Target
levels were determined by quantitative real-time PCR as described
by other examples herein. PCR results were normalized to
cyclophilin. The data are expressed as percent change relative to
saline treated animals ("+" indicates an increase, "-" indicates a
decrease) and are illustrated in Table 57.
65TABLE 57 Antisense inhibition of SGLT2 mRNA expression in db/db
mice (expressed as percent change in SGLT2 mRNA expression relative
to saline) Dose of oligonucleotide ISIS ISIS mg/kg 145733 257016
12.5 -48 -72 25 -71 -72 37.5 -64 -72
[0348] These results illustrate that both mixed backbone compound
ISIS 257016 and full phosphorothioate compound ISIS 145733
effectively inhibit the expression of kidney SGLT2. However,
greater inhibition is observed in kidneys from mice treated with
ISIS 257016, particularly at the lowest dose of 12.5 mg/kg.
[0349] Treated mice were further evaluated for body weight and
liver and spleen weight. The data are expressed as weight in grams.
The results are presented in Table 58.
66TABLE 58 Effects of antisense compounds on total body weight,
liver weight and spleen weight of db/db mice (in grams) Dose Body
Kidney Liver Spleen Oligonucleotide mg/kg weight weight weight
weight Saline -- 35 0.32 1.5 0.09 ISIS 145733 12.5 34 0.32 1.9 0.12
ISIS 145733 25 37 0.37 2.1 0.15 ISIS 145733 37.5 38 0.35 2.3 0.14
ISIS 257016 12.5 34 0.31 1.6 0.09 ISIS 257016 25 36 0.31 1.7 0.08
ISIS 257016 37.5 34 0.35 1.8 0.11
[0350] No significant change was observed in total body weight,
liver weight or spleen weight at timepoints throughout or at the
termination of the study.
[0351] Levels of AST, ALT, triglycerides, cholesterol and glucose
were measured in mice treated with the compounds of the invention.
Plasma samples were analyzed using the Olympus AU400e automated
chemistry analyzer (Olympus America, Irving, Tex.). The results,
expressed as units measured, are shown in Table 59 and Table
60.
67TABLE 59 Effect of full phosphorothioate backbone compound ISIS
145733 on indicators of toxicity Units measured per dose of ISIS
145733 Normal 12.5 mg/ 25 mg/ 37.5 mg/ Serum indicater Range Saline
kg kg kg AST 30-300 61 72 80 93 IU/L ALT 30-200 63 87 101 120 IU/L
Triglycerides 25-100* 245 216 243 204 mg/dL Cholesterol 70-125* 182
196 211 224 mg/dL Glucose 80-150* 611 452 391 351 mg/dL
[0352]
68TABLE 60 Effect of mixed backbone antisense compound ISIS 257016
on indicators of toxicity Units measured per dose of ISIS 257016
Normal 12.5 mg/ 25 mg/ 37.5 mg/ Serum indicater Range Saline kg kg
kg AST 30-300 61 120 144 175 IU/L ALT 30-200 63 123 142 154 IU/L
Triglycerides 25-100* 245 167 188 183 mg/dL Cholesterol 70-125* 182
248 264 265 mg/dL Glucose 80-150* 611 281 320 326 mg/dL
*Triglyceride, cholesterol and glucose levels are routinely higher
in the Lep.sup.db/Lep.sup.db strain of mice than in other strains
of mice.
[0353] The levels of routine clinical indicators of liver injury
and disease are within normal ranges and are not significantly
changed relative to saline-treated animals, demonstrating that the
compounds of the invention do not significantly affect hepatic
function. Given the genetic defect of the Lep.sup.db/Lep.sup.db
mice and the diabetic phenotype exhibited by these mice, it is
expected that triglyceride, cholesterol and glucose levels will
exceed the normal range. Importantly, treatment with either of the
SGLT2 antisense compounds resulted in a significant decrease in
blood glucose levels, with ISIS 257016, the mixed backbone
compound, achieving greater levels of target mRNA inhibition.
Treatment with ISIS 257016 also resulted in a significant decrease
in serum triglyceride levels.
[0354] The results illustrated in this example demonstrate that
mixed backbone compounds are effectively delivered to the kidney,
reduce SGLT2 expression in vivo, and that treatment with these
compounds does not result in liver or other toxicity. Furthermore,
these results indicate that mixed backbone compounds targeted to
SGLT2 efficiently decrease blood glucose levels and serum
triglyceride levels in a mouse model of type 2 diabetes.
Example 30
Antisense Inhibition of SGLT2 in a Murine Model of Type 2 Diabetes:
Low Dose Comparison of Full Phosphorothioate and Mixed Backbone
Oligonucleotides
[0355] Since treatment with ISIS 257016 resulted in significant
reduction in SGLT2 expression levels even at the lowest dose of
12.5 mg/kg, a second dose-response study was conducted using a
lower dose range of 1.56, 3.12 and 6.25 mg/kg. Male
Lep.sup.db/Lep.sup.db mice were given intraperitoneal injections of
either mixed backbone compound ISIS 257016 or full phosphorothioate
compound ISIS 145733 twice a week for four weeks at doses of 1.56,
3.12 or 6.25 mg/kg. Saline-injected animals served as controls.
Each treatment group contained 4 mice. The mice were sacrificed 2
days following administration of the final dose of oligonucleotide
or saline.
[0356] Mice were evaluated for SGLT2 levels in kidney. Target
levels were determined by quantitative real-time PCR as described
by other examples herein. PCR results were normalized to
cyclophilin. The data are expressed as percent change relative to
saline treated animals ("+" indicates an increase, "-" indicates a
decrease) and are illustrated in Table 61.
69TABLE 61 Antisense inhibition of SGLT2 mRNA expression in db/db
mice (expressed as percent change in SGLT2 mRNA expression relative
to saline) Dose of oligonucleotide ISIS ISIS mg/kg 145733 257016
1.56 -13 -75 3.12 -14 -83 6.25 -12 -80
[0357] These results illustrate that mixed backbone compound ISIS
257016 is a more effective inhibitor of SGLT2 mRNA expression in
the kidney, particularly at low doses of oligonucleotide.
[0358] Levels of glucose were measured in mice treated with the
compounds of the invention. Plasma samples were analyzed using the
Olympus AU400e automated chemistry analyzer (Olympus America,
Irving, Tex.). The data are expressed as percent change relative to
saline treated animals ("+" indicates an increase, "-" indicates a
decrease) and are illustrated in Table 62.
70TABLE 62 Blood glucose levels in db/db mice treated with SGLT2
antisense compounds (expressed as percent change in blood glucose
relative to saline) Dose of oligonucleotide ISIS ISIS mg/kg 145733
257016 1.56 -5 -41 3.12 -7 -37 6.25 -14 -40
[0359] The results demonstrate that treatment with mixed backbone
compound ISIS 257016 results in a significant decrease in blood
glucose levels and that mixed backbone compounds are more effective
at lowering blood glucose levels than full phosphorothioate
antisense compounds.
[0360] Antisense inhibition of SGLT2 by ISIS 257016 was further
evaluated using a dose range of 0.39, 0.78 and 1.56 mg/kg. As
described above, male Lep.sup.db/Lep.sup.db mice were given
intraperitoneal injections of mixed backbone compound ISIS 257016
twice a week for four weeks. Saline-injected animals served as
controls. Each treatment group contained 4 mice. The mice were
sacrificed 2 days following administration of the final dose of
oligonucleotide or saline.
[0361] Mice were evaluated for SGLT2 levels in kidney. Target
levels were determined by quantitative real-time PCR as described
by other examples herein. PCR results were normalized to
cyclophilin. Blood glucose levels also were determined. Plasma
samples were analyzed using the Olympus AU400e automated chemistry
analyzer (Olympus America, Irving, Tex.). The data are expressed as
percent change relative to saline treated animals ("+" indicates an
increase, "-' indicates a decrease) and are illustrated in Table
63.
71TABLE 63 Antisense inhibition of SGLT2 mRNA expression and blood
glucose levels in db/db mice (expressed as percent change in SGLT2
mRNA expression or blood glucose levels relative to saline) Dose of
ISIS 257016 SGLT2 Blood mg/kg mRNA glucose 0.39 -66 -16 0.78 -68
-21 1.56 -82 -21
[0362] These results further demonstrate the effectiveness of mixed
backbone compounds at inhibiting SGLT2 expression in the kidney and
lowering blood glucose levels when administered at very low doses
of oligonucleotide.
[0363] Mice treated with the compounds of the invention also were
evaluated for liver and kidney toxicity, organ and body weights and
tissue histology. These studies demonstrated no significant level
of toxicity or change in body or organ weight, indicating that
mixed backbone compounds are effective in vivo without toxicity to
the animal.
[0364] The results illustrated in this example demonstrate that
mixed backbone compounds are effectively delivered to the kidney,
reduce SGLT2 expression in vivo, and that treatment with these
compounds lowers blood glucose levels in diabetic animals.
Example 31
Antisense Inhibition of SGLT2 in a Murine Model of Obesity and
Diabetes Using Mixed Backbone Compounds
[0365] Leptin is a hormone produced by fat that regulates appetite.
Deficiencies in this hormone in both humans and non-human animals
leads to obesity. C57B1/6J-Lep ob/ob mice have a mutation in the
leptin gene which results in obesity and hyperglycemia. As such,
these mice are a useful model for the investigation of obesity and
diabetes and treatments designed to treat these conditions. In
accordance with the present invention, the oligomeric compounds of
the invention were tested in the ob/ob model of obesity and
diabetes.
[0366] Male C57B1/6J-Lep ob/ob mice (Jackson Laboratory, Bar
Harbor, Me.) were subcutaneously injected with ISIS 257016 (SEQ ID
NO: 106) at a dose of 25 mg/kg two times per week for 4 weeks.
Saline-injected animals served as controls. Each treatment group
contained 4 mice. The mice were sacrificed 2 days following
administration of the final dose of oligonucleotide or saline.
[0367] Mice were evaluated for SGLT2 levels in kidney. Target
levels were determined by quantitative real-time PCR as described
by other examples herein. PCR results were normalized to
cyclophilin. Blood glucose levels also were determined. Plasma
samples were analyzed using the Olympus AU400e automated chemistry
analyzer (Olympus America, Irving, Tex.). The data are expressed as
percent change relative to saline treated animals ("+" indicates an
increase, "-" indicates a decrease) and are illustrated in Table
64.
72TABLE 64 Antisense inhibition of SGLT2 mRNA expression and blood
glucose levels in ob/ob mice (expressed as percent change in SGLT2
mRNA expression or blood glucose levels relative to saline) Dose of
oligonucleotide SGLT2 Blood mg/kg mRNA glucose 25 -83 -39
[0368] The results demonstrate that treatment with a mixed backbone
SGLT2 antisense compound results in a significant decrease in SGLT2
mRNA expression in the kidney of diabetic mice. Importantly, blood
glucose levels also are significantly decreased in treated
animals.
Example 32
Comparison of Mixed Backbone Compounds 16 to 20 Nucleobases in
Length
[0369] In accordance with the present invention, mixed backbone
compounds with less than 20 nucleobases were evaluated for their
ability to inhibit SGLT2 expression in the kidney. Four compounds
were synthesized based on the sequence of ISIS 257016 (SEQ ID NO:
106). ISIS 366847, ISIS 366848, ISIS 366849 and ISIS 366850 are
comprised of the 5'-most 19, 18, 17 and 16 nucleobases,
respectively, of ISIS 257016 (see Table 65). ISIS 257016 has 2'-MOE
wings of five nucleobases each and a deoxy gap of 10 nucleobases.
ISIS 366847, ISIS 366848, ISIS 366849 and ISIS 366850 have a 10
nucleobases gap, a five nucleobase 2'-MOE wing at the 5' end, but
contain a shortened 3' wing of 1 to 4 nucleobases.
73TABLE 65 Antisense compounds 16 to 20 nucleobases in length ISIS
# SEQUENCE SEQ ID NO: 257016 GAAGTAGCCACCAACTGTGC 106 366847
GAAGTAGCCACCAACTGTG 366848 GAAGTAGCCACCAACTGT 366849
GAAGTAGCCACCAACTG 366850 GAAGTAGCCACCAACT
[0370] Male 6-week old Balb/c mice (Charles River Laboratories,
Wilmington, Mass.) were given intraperitoneal injections of ISIS
257016, ISIS 366847, ISIS 366848, ISIS 366849 or ISIS 366850 twice
a week for two weeks at doses of 0.14, 0.7 or 3.5 micromoles per
kilogram (EM/kg). Saline-injected animals served as controls. Each
treatment group contained 4 mice. The mice were sacrificed 2 days
following administration of the final dose of oligonucleotide or
saline.
[0371] Mice were evaluated for SGLT2 levels in kidney. Target
levels were determined by quantitative real-time PCR as described
in other examples herein. PCR results were normalized to
cyclophilin. The data are expressed as percent change relative to
saline treated animals ("+" indicates an increase, "-" indicates a
decrease) and are illustrated in Table 66.
74TABLE 66 Antisense inhibition of SGLT2 mRNA expression in vivo by
mixed backbone oligonucleotides (expressed as percent change
relative to saline control) Dose of oligonucleotide ISIS ISIS ISIS
ISIS ISIS .mu.M/kg 257016 366847 366848 366849 366850 0.14 -53 -55
-58 -57 -49 0.7 -56 -63 -59 -61 -57 3.5 -70 -64 -72 -69 -69
[0372] These results illustrate that mixed backbone compounds of
the invention, containing 16 to 20 nucleobases, are effective
inhibitors of SGLT2 expression in the kidney.
[0373] Treated mice were further evaluated for body weight, kidney
weight, liver weight and spleen weight. The data are expressed as
percent change in body or organ weight ("+" indicates an increase,
"-" indicates a decrease). The results are presented in Table
67.
75TABLE 67 Effects of antisense compounds on total body weight,
kidney weight, liver weight and spleen weight of mice (expressed as
percent change in weight) Dose Body Kidney Liver Spleen
Oligonucleotide .mu.M/kg weight weight weight weight ISIS 257016
0.14 +9.0 -4.5 -6.1 -8.3 ISIS 257016 0.7 +11.1 -5.3 +4.1 -3.7 ISIS
257016 3.5 +10.2 -3.6 +3.7 +11.9 ISIS 366847 0.14 +15.0 -0.5 +0.2
-6.9 ISIS 366847 0.7 +12.7 +1.2 +6.8 -4.9 ISIS 366847 3.5 +10.3
+3.6 +3.8 +2.9 ISIS 366848 0.17 +8.5 -7.1 -7.9 -2.4 ISIS 366848 0.7
+7.7 +6.4 +5.9 +3.8 ISIS 366848 3.5 +10.8 +3.0 +4.6 +9.3 ISIS
366849 0.14 +6.9 -3.3 -2.6 -7.2 ISIS 366849 0.7 +7.4 +0.1 -4.3 -2.2
ISIS 366849 3.5 +8.4 -2.9 -5.2 -3.9 ISIS 366850 0.14 +11.1 -3.8
-4.6 +2.0 ISIS 366850 0.7 +4.8 -0.8 -1.7 +0.9 ISIS 366850 3.5 11.2
-6.0 +4.5 +9.8
[0374] No significant change was observed in total body weight,
liver weight or spleen weight at timepoints throughout or at the
termination of the study.
[0375] Levels of BUN, creatinine, bilirubin, AST, ALT, albumin,
triglycerides, cholesterol and glucose were measured in mice
treated with the compounds of the invention. Plasma samples were
analyzed using the Olympus AU400e automated chemistry analyzer
(Olympus America, Irving, Tex.). The results, expressed as units
measured, are shown in Tables 68-72.
76TABLE 68 Effect of mixed backbone antisense compound ISIS 257016
on indicators of liver and kidney function Units measured per Serum
Normal dose of ISIS 257016 indicator Range Saline 0.14 .mu.M/kg 0.7
.mu.M/kg 3.5 .mu.M/kg BUN 15-40 31 32 32 31 mg/dL Creatinine
0.0-1.0 0.2 0.2 0.2 0.2 mg/L Bilirubin 0.1-1.0 0.2 0.1 0.1 0.1
mg/dL AST 30-300 82 68 85 117 IU/L ALT 30-200 22 24 26 32 IU/L
Albumin 2.5-4.0 3.0 3.2 3.1 3.1 g/dL Triglycerides 25-100* 225 266
308 225 mg/dL Cholesterol 70-125 123 128 128 147 mg/dL Glucose
80-150* 181 195 187 183 mg/dL
[0376]
77TABLE 69 Effect of mixed backbone antisense compound ISIS 366847
on indicators of liver and kidney function Units measured per Serum
Normal dose of ISIS 366847 indicator Range Saline 0.14 .mu.M/kg 0.7
.mu.M/kg 3.5 .mu.M/kg BUN 15-40 31 29 32 29 mg/dL Creatinine
0.0-1.0 0.2 0.2 0.2 0.2 mg/L Bilirubin 0.1-1.0 0.2 0.1 0 0.1 mg/dL
AST 30-300 82 53 69 131 IU/L ALT 30-200 22 23 28 50 IU/L Albumin
2.5-4.0 3.0 3.1 3.2 3.0 g/dL Triglycerides 25-100* 225 289 308 184
mg/dL Cholesterol 70-125 123 122 132 145 mg/dL Glucose 80-150* 181
173 193 181 mg/dL
[0377]
78TABLE 70 Effect of mixed backbone antisense compound ISIS 366848
on indicators of liver and kidney function Units measured per dose
of ISIS 366848 Serum Normal 0.14 .mu.M/ 0.7 .mu.M/ 3.5 .mu.M/
indicator Range Saline kg kg kg BUN 15-40 31 31 29 32 mg/dL
Creatinine 0.0-1.0 0.2 0.2 0.2 0.2 mg/L Bilirubin 0.1-1.0 0.2 0.1
0.1 0.1 mg/dL AST 30-300 82 82 105 123 IU/L ALT 30-200 22 23 34 46
IU/L Albumin 2.5-4.0 3.0 3.1 3.1 3.0 g/dL Triglycerides 25-100* 225
320 374 246 mg/dL Cholesterol 70-125 123 132 142 147 mg/dL Glucose
80-150* 181 200 187 190 mg/dL
[0378]
79TABLE 71 Effect of mixed backbone antisense compound ISIS 366849
on indicators of liver and kidney function Units measured per dose
of ISIS 366849 Serum Normal 0.14 .mu.M/ 0.7 .mu.M/ 3.5 .mu.M/
indicator Range Saline kg kg kg BUN 15-40 31 25 30 33 mg/dL
Creatinine 0.0-1.0 0.2 0.2 0.2 0.2 mg/L Bilirubin 0.1-1.0 0.2 0.1
0.1 0.1 mg/dL AST 30-300 82 98 90 92 IU/L ALT 30-200 22 26 24 33
IU/L Albumin 2.5-4.0 3.0 3.0 3.0 3.0 g/dL Triglycerides 25-100* 225
354 308 240 mg/dL Cholesterol 70-125 123 133 129 150 mg/dL Glucose
80-150* 181 170 173 192 mg/dL
[0379]
80TABLE 72 Effect of mixed backbone antisense compound ISIS 366850
on indicators of liver and kidney function Units measured per dose
of ISIS 366850 Serum Normal 0.14 .mu.M/ 0.7 .mu.M/ 3.5 .mu.M/
indicator Range Saline kg kg kg BUN 15-40 31 26 25 23 mg/dL
Creatinine 0.0-1.0 0.2 0.2 0.2 0.2 mg/L Bilirubin 0.1-1.0 0.2 0.1
0.1 0 mg/dL AST 30-300 82 83 69 108 IU/L ALT 30-200 22 21 27 38
IU/L Albumin 2.5-4.0 3.0 3.0 3.0 3.0 g/dL Triglycerides 25-100* 225
320 380 271 mg/dL Cholesterol 70-125 123 127 131 164 mg/dL Glucose
80-150* 181 192 187 179 mg/dL *Triglyceride and glucose levels are
routinely higher in the Balb/c strain of mice than in other strains
of mice.
[0380] Some oligonucleotide treated animals exhibited elevated
levels of cholesterol; however, saline control animals also
demonstrated cholelsterol levels at the high end of the normal
range. Thus, the slightly elevated cholesterol levels do not appear
to be significant. Otherwise, the levels of routine clinical
indicators of liver and kidney injury and disease are within normal
ranges and are not significantly changed relative to saline-treated
animals, demonstrating that the compounds of the invention do not
significantly affect renal or hepatic function. Triglyceride and
glucose levels, while outside the normal range as is common in the
Balb/c strain, are not significantly elevated relative to
saline-treated animals.
[0381] The results illustrated in this example demonstrate that
mixed backbone compounds of 16 to 20 nucleobases are delivered to
the kidney, reduce SGLT2 expression in vivo, and that treatment
with these compounds does not result in liver or kidney
toxicity.
Example 33
Antisense Inhibition of SGLT2 in Sprague Dawley Rats
[0382] In accordance with the present invention, 7-week old Sprague
Dawley rats (purchased from Charles River Labs, Wilmington, Mass.)
were treated with SGLT2 mixed backbone compound ISIS 257016 (SEQ ID
NO: 106) or SGLT2 full phosphorothioate compound ISIS 145733 (SEQ
ID NO: 106). Rats were injected i.p. twice a week for three weeks
with 10 mg/kg of oligonucleotide. Saline-injected animals served as
controls. The rats were sacrificed 2 days following administration
of the final dose of oligonucleotide or saline.
[0383] Rats were evaluated for SGLT2 levels in kidney. Target
levels were determined by quantitative real-time PCR as described
in other examples herein. PCR results were normalized to
cyclophilin. The data are expressed as percent change relative to
saline treated animals ("+" indicates an increase, "-" indicates a
decrease) and are illustrated in Table 73.
81TABLE 73 Antisense inhibition of SGLT2 mRNA expression in Sprague
Dawley rats (expressed as percent change in SGLT2 mRNA expression
relative to saline) % Change Treatment in mRNA Saline 0 ISIS 257016
-83.9 ISIS 145733 -38.5
[0384] These results illustrate that both full phosphorothioate and
mixed backbone compounds inhibit SGLT2 expression in the kidney of
rats. However, the mixed backbone compound is a more effective
inhibitor of SGLT2.
[0385] Treated rats were further evaluated for body weight, kidney
weight, liver weight and spleen weight. For body weight, the data
are expressed as percent change in body weight ("+" indicates an
increase, "-" indicates a decrease). For organ weights, the results
are expressed as percent of saline control normalized to body
weight. The results are presented in Table 74 and Table 75.
82TABLE 74 Effects of antisense compounds on total body weight of
rats (expressed as percent change in weight) Body Treatment weight
Saline +60.7 ISIS 257016 +58.4 ISIS 145733 +57.1
[0386]
83TABLE 75 Effects of antisense compounds on total kidney weight,
liver weight and spleen weight of rats (expressed as percent of
saline control normalized to body weight) Kidney Liver Spleen
Treatment weight weight weight ISIS 257016 99.3 93.4 105.8 ISIS
145733 107.2 105.2 123.4
[0387] No significant change was observed in total body weight,
kidney weight, liver weight or spleen weight at timepoints
throughout or at the termination of the study.
[0388] Levels of BUN, creatinine, bilirubin, AST, ALT, albumin,
triglycerides, cholesterol and glucose were measured in rats
treated with the compounds of the invention. Plasma samples were
analyzed using the Olympus AU400e automated chemistry analyzer
(Olympus America, Irving, Tex.). The results, expressed as units
measured, are shown in Table 76.
84TABLE 76 Effect of mixed backbone antisense compound ISIS 257016
and full phosphorothioate compound ISIS 145733 on indicators of
liver and kidney function (expressed as units measured) ISIS ISIS
Serum Indicator Saline 257016 145733 BUN 19 19 17 mg/dL Creatinine
0.3 0.4 0.2 mg/L Bilirubin mg/dL 0.1 0.1 0.1 AST 157 105 105 IU/L
ALT 65 44 36 IU/L Albumin 3.7 3.8 3.6 g/dL Triglycerides 42 47 53
mg/dL Cholesterol 68 66 54 mg/dL Glucose 189 173 180 mg/dL
[0389] The levels of routine clinical indicators of liver and
kidney injury are not significantly changed relative to
saline-treated animals, demonstrating that the compounds of the
invention do not significantly affect renal or hepatic function in
rats.
[0390] The results illustrated in this example demonstrate that
both full phosphorothioate and mixed backbone compounds are
delivered to the kidney, reduce SGLT2 expression in vivo, and that
treatment with these compounds does not result in liver or kidney
toxicity. The results further indicate that mixed backbone
compounds are more effective inhibitors of SGLT2 expression in
vivo.
[0391] Various modifications of the invention, in addition to those
described herein, will be apparent to those skilled in the art from
the foregoing description. Such modifications are also intended to
fall within the scope of the appended claims. Each reference
(including, but not limited to, journal articles, U.S. and non-U.S.
patents, patent application publications, international patent
application publications, gene bank accession numbers, and the
like) cited in the present application is incorporated herein by
reference in its entirety.
Sequence CWU 0
0
* * * * *