U.S. patent application number 11/419714 was filed with the patent office on 2006-11-09 for methods of selecting compounds for modulation of bladder function.
This patent application is currently assigned to Wyeth. Invention is credited to Thomas M. Argentieri, Jeffrey H. Sheldon.
Application Number | 20060252104 11/419714 |
Document ID | / |
Family ID | 26933980 |
Filed Date | 2006-11-09 |
United States Patent
Application |
20060252104 |
Kind Code |
A1 |
Argentieri; Thomas M. ; et
al. |
November 9, 2006 |
Methods of selecting compounds for modulation of bladder
function
Abstract
The present invention involves an assay system and method of
selecting compounds useful in the treatment of bladder instability
and related bladder conditions through the activation of KCNQ
potassium channels in the bladder smooth muscle.
Inventors: |
Argentieri; Thomas M.;
(Yardley, PA) ; Sheldon; Jeffrey H.; (Trappe,
PA) |
Correspondence
Address: |
DARBY & DARBY P.C.
P.O. BOX 5257
NEW YORK
NY
10150-5257
US
|
Assignee: |
Wyeth
Madison
NJ
07940
|
Family ID: |
26933980 |
Appl. No.: |
11/419714 |
Filed: |
May 22, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10399489 |
Apr 17, 2003 |
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PCT/US01/32371 |
Oct 17, 2001 |
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11419714 |
May 22, 2006 |
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60241078 |
Oct 17, 2000 |
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60281428 |
Apr 4, 2001 |
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Current U.S.
Class: |
435/7.2 ;
435/369; 435/455 |
Current CPC
Class: |
A61P 43/00 20180101;
G01N 2800/341 20130101; C12N 2510/00 20130101; A61P 13/08 20180101;
A61P 13/02 20180101; G01N 33/6872 20130101; A61K 31/405 20130101;
C12N 5/0686 20130101; A61K 31/00 20130101; A61K 31/24 20130101;
G01N 2500/10 20130101; C12N 2503/02 20130101; A61P 13/00 20180101;
A61P 13/10 20180101 |
Class at
Publication: |
435/007.2 ;
435/369; 435/455 |
International
Class: |
G01N 33/567 20060101
G01N033/567; C12N 5/08 20060101 C12N005/08; G01N 33/53 20060101
G01N033/53 |
Claims
1. A method of selecting compounds for the treatment, prevention,
or inhibition of urge urinary incontinence, bladder instability,
abnormal bladder contractility, hyperactive bladder, hyperreflexive
bladder, neurogenic bladder, or bladder-related sensory urgency
comprising: (a) expressing a target KCNQ protein in a host cell;
and (b) detecting activation of said target KCNQ protein.
2. The method of claim 1, wherein the host cell is an animal
cell.
3. The method of claim 1, wherein the host cell is mammalian.
4. The method of claim 1, wherein the host cell is human.
5. The method of claim 1, wherein the host cell is human embryonic
kidney.
6. The method of claim 1, wherein the host cell is HEK293 or a COS
cell.
7. The method of claim 1, wherein the membrane potential is
measured using electrophysiological techniques or fluorescence
techniques.
8. The method of claim 1, wherein compounds are selected that
exhibit at least 2 times greater activity on a target KCNQ protein
in bladder smooth muscle than on other target KCNQ proteins in
tissue other than bladder smooth muscle.
9. The method of claim 1, wherein compounds are selected that
exhibit at least 10 times greater activity on a target KCNQ protein
in bladder smooth muscle than on other target KCNQ proteins in
tissue other than bladder smooth muscle.
10. The method of claim 1, wherein compounds are selected that
exhibit at least 100 times greater activity on a target KCNQ
protein in bladder smooth muscle than on other target KCNQ proteins
in tissue other than bladder smooth muscle.
11. The method of claim 1, wherein compounds are selected that
exhibit substantially no effect on other target KCNQ proteins in
tissue other than bladder smooth muscle.
12. The method of claim 1, wherein compounds are selected that
exhibit at least 2 times greater activity on a target KCNQ protein
in bladder smooth muscle than on non-target KCNQ proteins.
13. The method of claim 1, wherein compounds are selected that
exhibit at least 10 times greater activity on a target KCNQ protein
in bladder smooth muscle than on non-target KCNQ proteins.
14. The method of claim 1, wherein compounds are selected that
exhibit at least 100 times greater activity on a target KCNQ
protein in bladder smooth muscle than on non-target KCNQ
proteins.
15. The method of claim 1, wherein compounds are selected that
exhibit substantially no effect on non-target KCNQ proteins.
16. The method of claim 1, wherein compounds are selected that
exhibit at least 2 times greater activity on a target KCNQ protein
in bladder smooth muscle than on proteins which form potassium
channels other than KCNQ proteins.
17. The method of claim 1, wherein compounds are selected that
exhibit at least 10 times greater activity on a target KCNQ protein
in bladder smooth muscle than on proteins which form potassium
channels other than KCNQ proteins.
18. The method of claim 1, wherein compounds are selected that
exhibit at least 100 times greater activity on a target KCNQ
protein in bladder smooth muscle than on proteins which form
potassium channels other than KCNQ proteins.
19. The method of claim 1, wherein compounds are selected that
exhibit substantially no effect on proteins which form potassium
channels other than KCNQ proteins.
20. A method of selecting compounds for the treatment, prevention,
or inhibition of urge urinary incontinence, bladder instability,
abnormal bladder contractility, hyperactive bladder, hyperreflexive
bladder, neurogenic bladder, or bladder-related sensory urgency
comprising: (a) expressing a target KCNQ channel in a host cell;
and (b) detecting activation of said target KCNQ channel.
21. The method of claim 20, wherein the host cell is an animal
cell.
22. The method of claim 20, wherein the host cell is mammalian.
23. The method of claim 20, wherein the host cell is human.
24. The method of claim 20, wherein the host cell is human
embryonic kidney.
25. The method of claim 20, wherein the host cell is HEK293 or a
COS cell.
26. The method of claim 20, wherein the membrane potential is
measured using electrophysiological techniques or fluorescence
techniques.
27. The method of claim 20, wherein compounds are selected that
exhibit at least 2 times greater activity on a target KCNQ protein
in bladder smooth muscle than on other target KCNQ proteins in
tissue other than bladder smooth muscle.
28. The method of claim 20, wherein compounds are selected that
exhibit at least 10 times greater activity on a target KCNQ protein
in bladder smooth muscle than on other target KCNQ proteins in
tissue other than bladder smooth muscle.
29. The method of claim 20, wherein compounds are selected that
exhibit at least 100 times greater activity on a target KCNQ
protein in bladder smooth muscle than on other target KCNQ proteins
in tissue other than bladder smooth muscle.
30. The method of claim 20, wherein compounds are selected that
exhibit substantially no effect on other target KCNQ proteins in
tissue other than bladder smooth muscle.
31. The method of claim 20, wherein compounds are selected that
exhibit at least 2 times greater activity on a target KCNQ protein
in bladder smooth muscle than on non-target KCNQ proteins.
32. The method of claim 20, wherein compounds are selected that
exhibit at least 10 times greater activity on a target KCNQ protein
in bladder smooth muscle than on non-target KCNQ proteins.
33. The method of claim 20, wherein compounds are selected that
exhibit at least 100 times greater activity on a target KCNQ
protein in bladder smooth muscle than on non-target KCNQ
proteins.
34. The method of claim 20, wherein compounds are selected that
exhibit substantially no effect on non-target KCNQ proteins.
35. The method of claim 20, wherein compounds are selected that
exhibit at least 2 times greater activity on a target KCNQ protein
in bladder smooth muscle than on proteins which form potassium
channels other than KCNQ proteins.
36. The method of claim 20, wherein compounds are selected that
exhibit at least 10 times greater activity on a target KCNQ protein
in bladder smooth muscle than on proteins which form potassium
channels other than KCNQ proteins.
37. The method of claim 20, wherein compounds are selected that
exhibit at least 100 times greater activity on a target KCNQ
protein in bladder smooth muscle than on proteins which form
potassium channels other than KCNQ proteins.
38. The method of claim 20, wherein compounds are selected that
exhibit substantially no effect on proteins which form potassium
channels other than KCNQ proteins.
39. A method of selecting compounds for the treatment, prevention,
or inhibition of urge urinary incontinence, bladder instability,
abnormal bladder contractility, hyperactive bladder, hyperreflexive
bladder, neurogenic bladder, or bladder-related sensory urgency
comprising: (a) recombinantly expressing a target KCNQ channel in a
host cell; (b) measuring the membrane potential of the host cell in
the presence or absence of the a substance; (c) selecting those
compounds whose presence causes hyperpolarization of the host
cell.
40. The method of claim 39, wherein the host cell is an animal
cell.
41. The method of claim 39, wherein the host cell is mammalian.
42. The method of claim 39, wherein the host cell is human.
43. The method of claim 39, wherein the host cell is human
embryonic kidney.
44. The method of claim 39, wherein the host cell is HEK293 or a
COS cell.
45. The method of claim 39, wherein the membrane potential is
measured using electrophysiological techniques or fluorescence
techniques.
46. The method of claim 39, wherein compounds are selected that
exhibit at least 2 times greater activity on a target KCNQ protein
in bladder smooth muscle than on other target KCNQ proteins in
tissue other than bladder smooth muscle.
47. The method of claim 39, wherein compounds are selected that
exhibit at least 10 times greater activity on a target KCNQ protein
in bladder smooth muscle than on other target KCNQ proteins in
tissue other than bladder smooth muscle.
48. The method of claim 39, wherein compounds are selected that
exhibit at least 100 times greater activity on a target KCNQ
protein in bladder smooth muscle than on other target KCNQ proteins
in tissue other than bladder smooth muscle.
49. The method of claim 39, wherein compounds are selected that
exhibit substantially no effect on other target KCNQ proteins in
tissue other than bladder smooth muscle.
50. The method of claim 39, wherein compounds are selected that
exhibit at least 2 times greater activity on a target KCNQ protein
in bladder smooth muscle than on non-target KCNQ proteins.
51. The method of claim 39, wherein compounds are selected that
exhibit at least 10 times greater activity on a target KCNQ protein
in bladder smooth muscle than on non-target KCNQ proteins.
52. The method of claim 39, wherein compounds are selected that
exhibit at least 100 times greater activity on a target KCNQ
protein in bladder smooth muscle than on non-target KCNQ
proteins.
53. The method of claim 39, wherein compounds are selected that
exhibit substantially no effect on non-target KCNQ proteins.
54. The method of claim 39, wherein compounds are selected that
exhibit at least 2 times greater activity on a target KCNQ protein
in bladder smooth muscle than on proteins which form potassium
channels other than KCNQ proteins.
55. The method of claim 39, wherein compounds are selected that
exhibit at least 10 times greater activity on a target KCNQ protein
in bladder smooth muscle than on proteins which form potassium
channels other than KCNQ proteins.
56. The method of claim 39, wherein compounds are selected that
exhibit at least 100 times greater activity on a target KCNQ
protein in bladder smooth muscle than on proteins which form
potassium channels other than KCNQ proteins.
57. The method of claim 39, wherein compounds are selected that
exhibit substantially no effect on proteins which form potassium
channels other than KCNQ proteins.
58. A method of selecting a compound comprising: (a) selecting
compounds that do not cross the blood-brain barrier; (b) testing
those compounds for the ability to modulate a target KCNQ protein
in bladder smooth muscle, and; (c) selecting those compounds that
show at least a greater ability to modulate a target KCNQ protein
in bladder smooth muscle than on other target KCNQ proteins in
tissue other than bladder smooth muscle.
59. The method of claim 58, wherein the compound selected shows at
least a 2 times greater ability to modulate a target KCNQ protein
in bladder smooth muscle than on other target KCNQ proteins in
tissue other than bladder smooth muscle.
60. The method of claim 58, wherein the compound selected shows at
least a 10 times greater ability to modulate a target KCNQ protein
in bladder smooth muscle than on other target KCNQ proteins in
tissue other than bladder smooth muscle.
61. The method of claim 58, wherein the compound selected shows at
least a 100 times greater ability to modulate a target KCNQ protein
in bladder smooth muscle than on other target KCNQ proteins in
tissue other than bladder smooth muscle.
62. The method of claim 58, wherein the compound selected shows
substantially no effect on other target KCNQ proteins in tissue
other than bladder smooth muscle.
63. A method of selecting a compound comprising: (a) selecting
compounds that do not cross the blood-brain barrier; (b) testing
those compounds for the ability to modulate a target KCNQ protein
in bladder smooth muscle, and; (c) selecting those compounds that
show a greater ability to modulate a target KCNQ protein in bladder
smooth muscle than on non-target KCNQ proteins.
64. The method of claim 63, wherein the compound selected shows at
least a 2 times greater ability to modulate a target KCNQ protein
in bladder smooth muscle than on non-target KCNQ proteins.
65. The method of claim 63, wherein the compound selected shows at
least a 10 times greater ability to modulate a target KCNQ protein
in bladder smooth muscle than on non-target KCNQ proteins.
66. The method of claim 63, wherein the compound selected shows at
least a 100 times greater ability to modulate a target KCNQ protein
in bladder smooth muscle than on non-target KCNQ proteins.
67. The method of claim 63, wherein the compound selected shows
substantially no effect on non-target KCNQ proteins.
68. A method of selecting a compound comprising: (a) selecting
compounds that do not cross the blood-brain barrier; (b) testing
those compounds for the ability to modulate a target KCNQ protein
in bladder smooth muscle, and; (c) selecting those compounds that
show a greater ability to modulate a target KCNQ protein in bladder
smooth muscle than proteins which form potassium channels other
than KCNQ proteins.
69. The method of claim 68, wherein the compound selected shows at
least a 2 times greater ability to modulate a target KCNQ protein
than proteins which form potassium channels other than KCNQ
proteins.
70. The method of claim 68, wherein the compound selected shows at
least a 10 times greater ability to modulate a target KCNQ protein
than proteins which form potassium channels other than KCNQ
proteins.
71. The method of claim 68, wherein the compound selected shows at
least a 100 times greater ability to modulate a target KCNQ protein
than proteins which form potassium channels other than KCNQ
proteins.
72. The method of claim 68, wherein the compound selected shows
substantially no effect on proteins which form potassium channels
other than KCNQ proteins.
73. A method of selectively activating a target KCNQ protein in
bladder smooth muscle of an animal for the treatment of bladder
instability, comprising administering a therapeutically effective
amount of a compound, which selectively activates a target KCNQ
protein in bladder smooth muscle, to an animal.
74. The method of claim 73, wherein the compound activates a target
KCNQ protein in bladder smooth muscle at least 2 times greater than
other target KCNQ proteins in tissue other than bladder smooth
muscle.
75. The method of claim 73, wherein the compound activates a target
KCNQ protein in bladder smooth muscle at least 10 times greater
than other target KCNQ proteins in tissue other than bladder smooth
muscle.
76. The method of claim 73, wherein the compound activates a target
KCNQ protein in bladder smooth muscle at least 100 times greater
than other target KCNQ proteins in tissue other than bladder smooth
muscle.
77. The method of claim 73, wherein the compound activates a target
KCNQ protein in bladder smooth muscle and shows substantially no
effect on other target KCNQ proteins in tissue other than bladder
smooth muscle.
78. The method of claim 73, wherein the compound activates a target
KCNQ protein in bladder smooth muscle at least 2 times greater than
on non-target KCNQ proteins.
79. The method of claim 73, wherein the compound activates a target
KCNQ protein in bladder smooth muscle at least 10 times greater
than on non-target KCNQ proteins.
80. The method of claim 73, wherein the compound activates a target
KCNQ protein in bladder smooth muscle at least 100 times greater
than on non-target KCNQ proteins.
81. The method of claim 73, wherein the compound activates a target
KCNQ protein in bladder smooth muscle and shows substantially no
effect on non-target KCNQ proteins.
82. The method of claim 73, wherein the compound activates a target
KCNQ protein in bladder smooth muscle at least 2 times greater than
proteins which form potassium channels other than KCNQ
proteins.
83. The method of claim 73, wherein the compound activates a target
KCNQ protein in bladder smooth muscle at least 10 times greater
than proteins which form potassium channels other than KCNQ
proteins.
84. The method of claim 73, wherein the compound activates a target
KCNQ protein in bladder smooth muscle at least 100 times greater
than proteins which form potassium channels other than KCNQ
proteins.
85. The method of claim 73, wherein the compound activates a target
KCNQ protein in bladder smooth muscle and shows substantially no
effect on proteins which form potassium channels other than KCNQ
proteins.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the fields of
pharmaceutical chemistry and urology and to a method of selecting
compounds useful in the treatment of urologic conditions and to
methods of treatment of said urologic conditions and, more
particularly, to an assay system and method of selecting compounds
useful in the treatment of bladder instability and related bladder
conditions through the activation of KCNQ potassium channels in the
bladder smooth muscle.
[0002] The invention also relates to a method of treatment for
bladder instability by activating KCNQ potassium channels in the
bladder smooth muscle. This invention also relates to novel methods
for modulating bladder tissues utilizing compounds, which modulate
the KCNQ family of potassium or M channels, particularly compounds
which open or agonize the channels. The methods of this invention
include the treatment, prevention, inhibition and amelioration of
urge urinary incontinence also known as bladder instability,
neurogenic bladder, voiding dysfunction, hyperactive bladder,
hyperreflexic bladder, or detrusor overactivity. The methods of
this invention also include the prevention and treatment of mixed
stress and urge urinary incontinence, including that associated
with secondary conditions such as prostate hypertrophy.
BACKGROUND OF THE INVENTION
[0003] Transmembrane currents play a fundamental role in the
activation and functioning of excitable tissues. In urinary bladder
smooth muscle, depolarization, excitation-contraction, and
repolarization are dependent upon the activation of transmembrane
currents through voltage dependent ion channels. The current
underlying repolarization in detrusor smooth muscle is carried
through several ion channels, virtually all of which utilize
potassium as the charge carrier. These include a transient,
4-aminopyridine sensitive current (Fujii K, Foster C D, Brading A F
and Parekh A B. Potassium channel blockers and the effects of
cromakalim on the smooth muscle of the guinea-pig bladder. Br J
Pharmacol 99: 779-785, 1990), a delayed rectifier (Klockner, U. and
Isenberg, G. Calcium currents of cesium loaded isolated smooth
muscle cells (urinary bladder of the guinea pig). Pflugers Arch
405: 340-348, 1985), an ATP-dependent current (Bonev A D and Nelson
M T. ATP-sensitive potassium channels in smooth muscle cells from
guinea pig urinary bladder. Am J Physiol 264(Cell Physiol 33):
C1190-C1200, 1993; Trivedi S, Stetz S L, Potter-Lee L, McConville
M, Li J H, Empfield J, Ohnmacht C J, Russell K, Brown F J, Trainor
D A et al. K-channel opening activity of ZD6169 and its analogs:
effect on 86Rb efflux and 3H-P1075 binding in bladder smooth
muscle. Pharmacol 50: 388-397, 1994) and a charybdotoxin-sensitive
current consistent with the large-conductance, calcium-dependent
potassium current (BKCa) (Zografos P, Li J H and Kau S T.
Comparison of the in vitro effects of K.sup.+ channel modulators on
detrusor and portal vein strips from guinea pigs. Pharmacol 45:
216-230, 1992). Several of these channels have been the target of
compounds and drugs aimed at modulating the physiology and
functioning of smooth muscle and other tissues (Edwards, G. and
Weston, A H.: Pharmacology of the potassium channel openers.
Cardiovasc Drugs and Ther 9: 185-193, 1995).
[0004] It has been suggested (Foster D C and Brading A F. The
effect of potassium channel antagonists on the BRL 34915 activated
potassium channel in guinea-pig bladder. Br J Pharmacol 92: 751,
1987) that a potassium channel opener (KCO) may be useful in the
treatment of detrusor hyperactivity. An increase in potassium
channel permeability would hyperpolarize the cell, bring the
membrane potential further from the threshold for activation of
calcium channels and reduce excitability (Brading A F. Ion channels
and control of contractile activity in urinary bladder smooth
muscle. Jap J Pharmacol 58 Suppl 2: 120P-127P, 1992). A number of
potassium channel openers have shown activity in isolated tissues
(Fujii et al., 1990; Malmgren A, Andersson K E, Andersson P O,
Fovaeus M and Sjogren C. Effects of cromakalim (BRL 34915) and
pinacidil on normal and hypertrophied rat detrusor in vitro. J Urol
143: 828-834, 1990; Grant T L and Zuzack J S. Effects of K.sup.+
channel blockers and cromakalim (BRL 34915) on the mechanical
activity of guinea pig detrusor smooth muscle. J Pharmacol Exp
Thera 269(3): 1158-1164, 1991) and efficacy in both experimental
(Foster and Brading, 1987; Malmgren A, Andersson K E, Sjogren C and
Andersson P O. Effects of pinacidil and cromakalim (BRL 34915) on
bladder function in rats with detrusor instability. J Urol 142:
1134-1138, 1989; Wojdan A, Freeden C, Woods M, Norton W. Warga D,
Spinelli W, Colatsky T, Antane M, Antane S, Butera J and Argentieri
T M. Comparison of the potassium channel openers ZD6169, celikalim
and WAY-133537 on isolated bladder tissue and in vivo bladder
instability in the rat. J Pharmacol Exp Therap 289: 1410-1418,
1999) and clinical bladder instability (Nurse et al., 1991).
However, because these compounds also activate channels in vascular
smooth muscle (causing vasodilation), the clinical utility has been
severely limited by hemodynamic side effects including hypotension
and tachycardia.
[0005] It has been stated previously that retigabine
(N-[2-amino-4-(4-fluorobenzylamino)-phenyl]carbamic acid ethyl
ester) activates a member of the KCNQ family of potassium channel
in the bladder which is most likely KCNQ2/3 and/or KCNQ3/5.
(Wickenden A. D., Yu, W., Zou, A., Jegla, T., & Wagoner, P. K.
Retigabine, a novel anti-convulsant, enhances activation of
KCNQ2/Q3 potassium channels. Molec Pharmacol 58: 591-600 (2000);
Wickenden, A. D., Zou, A., Wagoner, P. K., & Jela, T.
Characterization of the KCNQ5/Q3 potassium channels expressed in
mammalian cells. Br J Pharmacol 132: 381-384 (2001); Rundfeldt, C.,
Netzer, R. The novel anticonvulsant retigabine activates M-currents
in Chinese hamster ovary-cells tranfected with human KCNQ2/3
subunits. Neuroscience Letters 282: 73-76 (2000); Main, M. J.,
Cryan, J. E., Dupere, J. R. B., Cox, B., Clare, J. J. &
Burbidge, S. A. Modulation of KCNQ2/3 potassium channels by the
novel anticonvulsant retigabine. Molec Pharm 58: 253-262 (2000)).
The result is an inhibition of bladder smooth muscle contractility.
In addition, recent data provides evidence for the existence of the
KCNQ4 channel in human bladder smooth muscle. Current knowledge of
KCNQ4 suggests that it may form a functional ion channel on its own
(Sogaard S, Ljungstrom T, Perersen K A, Olesen S P, Jensen, B S.
KCNQ4 channels expressed in mammalian cells: functional
characteristics and pharmacology. Am J Physiol 280: C859-C866,
2001), or that it may combine with KCNQ3 (Kubisch C. Schroeder B C.
Friedrich T. Lutjohann B. El-Amraoui A. Marlin S. Petit C. Jentsch
T J. KCNQ4, a novel potassium channel expressed in sensory outer
hair cells, is mutated in dominant deafness. Cell 96(3):437-446,
1999). It is likely therefore, that retigabine's effects on bladder
smooth muscle include activation of the KCNQ4 channel in addition
to the channels formed by KCNQ2/3 and KCNQ3/5. Activation of this
channel will hyperpolarize the bladder smooth muscle cells and, in
doing so, relax the bladder. Since these KCNQ channels are not
present in the cardiovascular system, retigabine and other
molecules that activate these channels should be useful in the
treatment of bladder instability without hemodynamic
compromise.
[0006] M-currents have been shown to play an important functional
role as determinants of cell excitability. Recent evidence
indicates that the KCNQ potassium channel subunit form the
molecular basis for M-current activity in a variety of tissues.
From their initial report in peripheral sympathetic neurons the
gene family has evolved to contain at least five major sub-units
designated KCNQ1 though KCNQ5 (see reviews in Rogowski, M. A.
KCNQ2/KCNQ3 K.sup.+ channels and the molecular pathogenesis of
epilepsy: implications for therapy. TINS 23: 393-398, (2000);
Jentsch, T. J. Neuronal KCNQ potassium channels: physiology and
role in disease, Nature Rev, (2000)). These sub-units have been
shown to co-assemble to form both heteromeric and homomeric
functional ion channels. Recent reports indicate that both KCNQ2
and KCNQ5 can co-assemble with KCNQ3 (Tinel, N., Lauritzen, I.,
Chouabe, C., Lazdunski, M., Borsotto, M. The KCNQ2 potassium
channel: splice variants, functional and developmental expression.
Brain localization and comparison with KCNQ3. FEBS Letters. 438:
171-176 (1998); Yang, W., P., Levesque, P., C., Little, W., A.,
Conder, M., L., Ramakrishnan, P., Neubauer, M., G., Blanar, M., A.
Functional expression of two KvLQT1-related potassium channels
responsible for an inherited idiopathic epilepsy. J Biological
Chemistry. 273:19419-19423 (1998); Wang, H. S., Pan, Z., Shi, W.,
Brown, B. S., Wymore, R. S., Cohen, I. S., Dixon, J. E. &
McKinnon, D. KCNQ2 and KCNQ3 potassium channel subunits: molecular
correlets of the M-channel. Science 282: 1890-1893, (1998); Lerche,
C., Scherer, C. R., Seebohm, G., Derst, C., Wei, A. D., Busch, A.
E., Steinmeyer, K. J Biologic Chem (2000); Schroeder, B., C.,
Hechenberger, M., Weinreich, F., Kubisch, C., Jentsch, T., J.
KCNQ5, a novel potassium channel broadly expressed in brain,
mediates M-type currents. [Journal Article] J Biological Chemistry.
275: 24089-24095 (2000)) to form a functional M-channel activatable
by retigabine (Wickenden A. D., Yu, W., Zou, A., Jegla, T., &
Wagoner, P. K. Retigabine, a novel anti-convulsant, enhances
activation of KCNQ2/Q3 potassium channels. Molec Pharmacol 58:
591-600 (2000); Wickenden, A. D., Zou, A., Wagoner, P. K., &
Jela, T. Characterization of the KCNQ5/Q3 potassium channels
expressed in mammalian cells. Br J Pharmacol 132: 381-384 (2001);
Rundfeldt, C., Netzer, R. The novel anticonvulsant retigabine
activates M-currents in Chinese hamster ovary-cells transfected
with human KCNQ213 subunits. Neuroscience Letters 282: 73-76
(2000); Main, M. J., Cryan, J. E., Dupere, J. R. B., Cox, B.,
Clare, J. J. & Burbidge, S. A. Modulation of KCNQ2/3 potassium
channels by the novel anticonvulsant retigabine. Molec Pharm 58:
253-262 (2000)) and blocked by either acetylcholine (Adams, P., R.,
Brown, D., A., Constanti, A. M-currents and other potassium
currents in bullfrog sympathetic neurones. J Physiology 330:
537-72(1982); Brown, D., A., Adams, P., R. Muscarinic suppression
of a novel voltage-sensitive K+ current in a vertebrate neurone.
Nature 283: 673-676(1980); Shapiro, M., S., Roche, J., P., Kaftan,
E., J., Cruzblanca, H., Mackie, K., Hille, B. Reconstitution of
muscarinic modulation of the KCNQ2/KCNQ3 K(+) channels that
underlie the neuronal M current. J Neuroscience 20: 1710-1721
(2000)) linopirdine or XE-991
(10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone (Aiken, S. P.,
Lamp, B. J. Murphy, P. A. & Brown B. S. Reduction of spike
frequency adaptation and blockade of M-current in rat CA1 pyramidal
neurons by linopirdine (DuP 996) a neurotransmitter release
enhancer. Br J Pharm 115: 1163-1168, (1995); Zaczek R. Chorvat R J.
Saye J A. Pierdomenico M E. Maciag C M. Logue A R. Fisher B N.
Rominger D H. Earl R A. Two new potent neurotransmitter release
enhancers, 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone and
10,10-bis(2-fluoro-4-pyridinylmethyl)-9(10H)-anthracenone:
comparison to linopirdine. J Pharmacology & Exp Therap 285:
724-730 (1998). The parasympathetic neurotransmitter acetylcholine
(Ach) is known to produce several physiological responses in
bladder smooth muscle. The net result of Ach exposure is a
contraction of the smooth muscle mainly through the mobilization of
transmembrane and intracellular calcium stores (Hashitani H.
Bramich N J. Hirst G D. Mechanisms of excitatory neuromuscular
transmission in the guinea-pig urinary bladder. Journal of
Physiology 524: 565-579 (2000)). The role that Ach plays in
modulating the cell transmembrane potential, however, is more
complex. Pathways for both hyperpolarization and depolarization are
present with muscarinic stimulation of bladder smooth muscle.
Hyperpolarization may be associated with a mechanism that involves
calcium sparks and activation of calcium-dependent potassium
currents (Herrera G M. Heppner T J. Nelson M T. Voltage dependence
of the coupling of Ca(2+) sparks to BK(Ca) channels in urinary
bladder smooth muscle. American Journal of Physiology-Cell
Physiology 280: C481-490 (2001)).
[0007] Furthermore, there is a need to develop methods of selecting
compounds useful in the treatment bladder instability and related
urologic or bladder conditions. The present invention meets this
need and includes methods of treatment of bladder instability and
related urologic and bladder conditions.
SUMMARY OF THE INVENTION
[0008] The present invention provides a method of selecting
compounds for the treatment of bladder instability comprising,
expressing a target KCNQ protein in a host cell and detecting
activation of said target KCNQ protein. In one embodiment, a target
KCNQ protein is expressed or overexpressed naturally in a host cell
or host line. In another embodiment of the present invention, a
target KCNQ protein is expressed recombinantly in a host cell.
[0009] The present invention provides a method of selecting
compounds for the treatment of bladder instability comprising,
expressing a target KCNQ protein in a host cell and detecting
activation of said target KCNQ protein, wherein said detection is
performed by measuring the membrane potential of the host cell in
the presence or absence of a substance; and selecting those
compounds whose presence causes a change in membrane potential of
the host cell.
[0010] The present invention provides a method of selecting
compounds for the treatment of bladder instability comprising,
expressing a target KCNQ protein in a host cell and detecting
activation of said target KCNQ protein, wherein said detection is
performed by fluorescence techniques with the host cell in the
presence or absence of a substance; and selecting those compounds
whose presence causes a hyperpolarization of said host cell as
evidenced by the presence of fluorescence.
[0011] The invention also provides for this method, wherein the
compounds selected exhibit the following characteristics: at least
2 times greater activity with respect to target KCNQ proteins in
bladder smooth muscle compared with KCNQ proteins in other tissue;
at least 2 times greater activity with respect to target KCNQ
proteins in bladder smooth muscle compared with non-target KCNQ
proteins; or at least 2 times greater activity with respect to
target KCNQ proteins in bladder smooth muscle compared with other
potassium channels. For the various embodiments of this invention,
one may also select compounds that do not cross the blood brain
barrier. Some compounds, which leak across the blood brain barrier,
may be used as long as they exhibit no undesirable side effects.
The latter is not preferred.
[0012] The present invention also provides a method of selecting a
compound comprising, selecting compounds that do not cross the
blood brain barrier; testing those compounds for the ability to
activate a target KCNQ protein in bladder smooth muscle; selecting
those compounds which show a greater ability to activate a target
KCNQ protein in bladder smooth muscle when compared with activation
of target KCNQ proteins in other tissue; activation of non-target
KCNQ proteins, or activation of other potassium channels.
[0013] The present invention also provides a method of treatment of
bladder instability by selectively activating target KCNQ channels
in bladder smooth muscle, comprising administering a compound to an
animal, wherein said compound selectively activates a target KCNQ
protein in bladder smooth muscle.
[0014] This invention comprises methods for modulating urinary
bladder tissues in a mammal, particularly including uses thereof
for maintaining urinary bladder control, the methods comprising
administering to a mammal in need thereof a pharmaceutically
effective amount of a compound which acts as an agonist or opener
of the KCNQ family of potassium channels, including the KCNQ1,
KCNQ2, KCNQ3, KCNQ4, and KCNQ5 potassium channels, alone or in
combination. A particular embodiment of this invention includes use
in the methods described herein of one or more agonists or openers
of KCNQ2/3 potassium channels. Another series of methods of this
invention comprises use of one or more agonists or openers of
KCNQ3/5 potassium channels. Yet another series of methods of this
invention comprises use of one or more agonists or openers of KCNQ4
potassium channels.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1. is a graph depicting Retigabine concentration
response curve for inhibition of isolated rat bladder strip
contractions. Closed circled represent data from preparations
contracted with 20 mM KCl; IC.sub.50 was 1.4.+-.0.1 .mu.M. Open
circles represent data from preparations contracted with 60 mM KCl;
the IC.sub.50 was 21.8.+-.0.8 .mu.M. This profile is consistent
with a potassium channel opening mechanism where higher
concentrations of KCl diminish the driving force for potassium and
inhibit the potency of potassium channel openers
[0016] FIG. 2. A graph of KCNQ gene expression levels in (a) human
urinary bladders and in (b) rat urinary bladders measured as KCNQ
mRNA/GADPH mRNA. In FIG. 2a, quantitative reverse transcriptase
polymerase chain reaction (rtPCR) was performed on RNA isolated
from rat bladder smooth muscle. Message for KCNQ1, KCNQ3 and KCNQ5
was seen. No message for KCNQ2 or KCNQ4 was present. In FIG. 2b,
rtPCR performed on RNA isolated from cultured human bladder smooth
muscle cell. Message was seen for KCNQ3 and KCNQ5
[0017] FIG. 3. a. A graph of a current clamp tracing from an
isolated rat bladder smooth muscle cell. Resting membrane potential
was -40 mV prior to exposure to 10 .mu.M retigabine. Retigabine
hyperpolarized the cell by approximately 10 mV. This
hyperpolarization was reversed by the addition of 10 .mu.M XE-991.
b. A graph of a current clamp tracing from an isolated rat bladder
smooth muscle cell showing a retigabine-induced hyperpolarization
followed by a depolarization by 100 nM Ach. c. A graph of
current-voltage relationship for outward current before (control)
and after retigabine. d. Three graphs of voltage clamp tracings
from isolated human bladder smooth muscle cell. Retigabine
increased an outward current that was partially reversed by 10
.mu.M XE-991.
[0018] FIG. 4. a. depicts cystometrograms from four rats. Bladder
infusate contained 0.25% acetic acid to induce spontaneous
contractions and shorten the micturition interval. Micturition was
completely blocked within minutes of dosing retigabine (10 mg/kg,
i.p.). FIG. 4b. depicts 2 cystometrograms showing spontaneous
contractions during bladder filling. Retigabine (0.1 mg/kg) (first
cystometogram) significantly reduced the frequency of spontaneous
contractions in comparison to the control.
DEFINITIONS
[0019] A KCNQ subunit is a KCNQ whole protein that forms part of a
potassium channel known as the .alpha.-subunit.
[0020] A KCNQ channel is a potassium channel tetramer composed of
at least one KCNQ subunit type (i.e., KCNQ1, KCNQ2, KCNQ3, KCNQ4,
or KCNQ5).
[0021] A KCNQ protein is any protein of the KCNQ family, proteins
predominantly involved in M-channel or potassium channel
regulation. These proteins include, but are not limited to the
following types: KCNQ1 (SEQ ID NO:1), KCNQ2 (SEQ ID NO:2), KCNQ3
(SEQ ID NO:3), KCNQ4 (SEQ ID NO:4), KCNQ5 (SEQ ID NO:5), KCNQ2/3,
and KCNQ3/5, or any combination thereof.
[0022] A "target KCNQ protein" is a KCNQ protein occurring in the
bladder smooth muscle. Preferably, it is a protein which appears at
greater concentrations in the bladder smooth muscle than in other
tissue. By way of example and in no way intended to limit, a
"target KCNQ protein" is KCNQ3/5 or KCNQ4.
[0023] A "non-target KCNQ protein" is a KCNQ protein not occurring
in bladder smooth muscle.
[0024] "Other potassium channels" are all potassium channels not
composed of at least one KCNQ subunit.
DETAILED DESCRIPTION OF THE INVENTION
[0025] The present invention provides a method of selecting
compounds for the treatment of bladder instability comprising,
expressing a target KCNQ protein in a host cell and detecting
activation of said target KCNQ protein. In one embodiment, a target
KCNQ protein is expressed or overexpressed naturally in a host cell
or host line. In another embodiment of the present invention, a
target KCNQ protein is expressed recombinantly in a host cell.
[0026] The present invention provides a method of selecting
compounds for the treatment of bladder instability comprising,
expressing a target KCNQ protein in a host cell and detecting
activation of said target KCNQ protein, wherein said detection is
performed by measuring the membrane potential of the host cell in
the presence or absence of a substance; and selecting those
compounds whose presence causes a change in membrane potential of
the host cell.
[0027] The present invention provides a method of selecting
compounds for the treatment of bladder instability comprising,
expressing a target KCNQ protein in a host cell and detecting
activation of said target KCNQ protein, wherein said detection is
performed by fluorescence techniques with the host cell in the
presence or absence of a substance; and selecting those compounds
whose presence causes a hyperpolarization of said host cell as
evidenced by the presence of fluorescence.
[0028] In one embodiment, the host cell is an animal cell. In a
further embodiment, the host cell is mammalian. In a further
embodiment, the host cell is human. In a further embodiment, the
host cell is human kidney. In a further embodiment, the host cell
is human embryonic kidney. In a further embodiment, the host cell
is HEK293. In an alternative embodiment, the host cell is COS.
[0029] In the present invention, compound selection is based upon
detection of target KCNQ protein or KCNQ channel activation
measured by various conventional means, including
electrophysiological techniques (i.e., current clamping and voltage
clamping). In one embodiment, membrane potential is measured using
fluorescence methods. In another embodiment, membrane current is
measured using voltage clamp methods. In yet another embodiment,
membrane voltage is measured using current clamp techniques.
[0030] In one embodiment, substances are further selected based
upon their ability to cause greater activation in target KCNQ
proteins in the bladder smooth muscle than in target KCNQ proteins
in other tissue. Preferably, substances are selected which cause at
least 2 times greater activity in target KCNQ proteins in the
bladder smooth muscle than in target KCNQ proteins in other tissue.
More preferably, substances are selected which cause at least 10
times greater activity in target KCNQ proteins in bladder smooth
muscle than in target KCNQ proteins in other tissue. Alternatively,
substances are selected which cause at least 20, 30, 40, 50, 60,
70, 80, or 90 times greater activity in target KCNQ proteins in
bladder smooth muscle than in target KCNQ proteins in other tissue.
Most preferably, substances are selected which cause at least 100
times greater activity in target KCNQ proteins in bladder smooth
muscle than in target KCNQ proteins in other tissue.
[0031] In another embodiment, substances are further selected based
upon their ability to cause greater activation in target KCNQ
proteins in bladder smooth muscle than on non-target KCNQ proteins.
Preferably, substances are selected which cause at least 2 times
greater activity in target KCNQ proteins in bladder smooth muscle
than in non-target KCNQ proteins. More preferably, substances are
selected which cause at least 10 times greater activity in target
KCNQ proteins in the bladder smooth muscle than in non-target KCNQ
proteins. Alternatively, substances are selected which cause at
least 20, 30, 40, 50, 60, 70, 80, or 90 times greater activity in
target KCNQ proteins in the bladder smooth muscle than in
non-target KCNQ proteins. Most preferably, substances are selected
which cause at least 100 times greater activity in target KCNQ
proteins in bladder smooth muscle than on non-target KCNQ
proteins.
[0032] In another embodiment, substances are further selected based
upon their ability to cause greater activation in target KCNQ
proteins than in other potassium channels. Preferably, substances
are selected which cause at least 2 times greater activity in
target KCNQ proteins in bladder smooth muscle than in other
potassium channels. More preferably, substances are selected which
cause at least 10 times greater activity in target KCNQ proteins in
bladder smooth muscle than in other potassium channels.
Alternatively, substances are selected which cause at least 20, 30,
40, 50, 60, 70, 80, or 90 times greater activity in target KCNQ
proteins than in other potassium channels. Most preferably,
substances are selected which cause 100 times greater activity in
target KCNQ proteins than in other potassium channels.
[0033] For the various embodiments of the present invention, any
one of a variety of compounds selected through the as a result of
an increase in activity of a target KCNQ protein or channel is
further analyzed by in vivo or in vitro analysis to detect
correlation with an improvement in treatment of bladder instability
or a variety of related bladder conditions as described herein.
[0034] The present invention also provides for a method of
selecting a compound, comprising selecting those compounds, which
substantially do not cross the blood-brain barrier or which leak
across the blood brain barrier without causing undesirable side
effects; testing those compounds for the ability to modulate a
target KCNQ protein in bladder smooth muscle; and selecting those
compounds that show a greater ability to activate a target KCNQ
protein in bladder smooth muscle than to activate target KCNQ
proteins in other tissue.
[0035] Known methods for predicting blood brain barrier penetration
include computational methods using mathematical tools, cell
culture methods using endothelial cell cultures from animal origin,
high performance liquid chromotography (HPLC) using immobilized
artificial membrane columns, measurement of surface activity using
critical micelle concentration methodology, microdialysis
techniques involving sampling tissue from the brain of a living
animal for external HPLC analysis, the use of postmortem human
brain capillaries, and in vivo animal studies. (Clark, D. E.;
Pickett, S. Computational Methods for the Prediction of
`Drug-likeness`. Drug Discovery Today 5(2): 49-58, 2000; Eddy, E.
P.; Maleef, B. E., Hart, T. K., Smith, P. L. In Vitro Models to
Predict Blood-Brain Barrier Permeability. Adv Drug Delivery Rev 23:
185-1981, 1997; Gumbleton, M. and Kenneth L. Audus. Progress and
Limitations in the Use of In Vitro Cell Cultures to Serve as a
Permeability Screen for the Blood-Brain Barrier. J Pharm Sci 90:
1681-1698, 2001).
[0036] The present invention also provides for a method of
treatment for bladder instability comprising administering to an
animal, preferably a mammal or a human, a compound that selectively
activates a target KCNQ protein in bladder smooth muscle.
[0037] The methods of this invention are useful for inducing,
assisting or maintaining desirable bladder control in a mammal
experiencing or susceptible to bladder instability or urinary
incontinence. These methods include prevention, treatment or
inhibition of bladder-related urinary conditions and bladder
instability, including nocturnal enuresis, nocturia, voiding
dysfunction and urinary incontinence. Also treatable or preventable
with the methods of this invention is bladder instability secondary
to prostate hypertrophy. The compounds described herein are also
useful in promoting the temporary delay of urination whenever
desirable. The compounds of this invention may also be utilized to
stabilize the bladder and treat or prevent incontinence, including
urge urinary incontinence or a combination of urge and stress
incontinence in a mammal, which may also be referred to as mixed
urge and stress incontinence. These methods include assistance in
preventing or treating urinary incontinence associated with
secondary conditions such as prostate hypertrophy.
[0038] These methods may be utilized to allow a recipient to
control the urgency and frequency of urination. The methods of this
invention include the treatment, prevention, inhibition and
amelioration of urge urinary incontinence also known as bladder
instability, neurogenic bladder, voiding dysfunction, hyperactive
bladder, detrusor overactivity, detrusor hyper-reflexia or
uninhibited bladder.
[0039] As described above, methods of this invention include
treatments, prevention, inhibition or amelioration of hyperactive
or unstable bladder, neurogenic bladder or hyperreflexic bladder.
These uses include, but are not limited to, those for bladder
activities and instabilities in which the urinary urgency is
associated with prostatitis, prostatic hypertrophy, interstitial
cystitis, urinary tract infections or vaginitis. The methods of
this invention may also be used to assist in inhibition or
correction of the conditions of Frequency-Urgency Syndrome.
[0040] The methods of this invention may also be used to treat,
prevent, inhibit, or limit the urinary incontinence, urinary
instability or urinary urgency associated with or resulting from
administrations of other medications.
[0041] The methods of this invention are useful for inducing or
assisting in urinary bladder control or preventing or treating the
maladies described herein in humans in need of such relief,
including adult and pediatric uses. However, they may also be
utilized for veterinary applications, particularly including canine
and feline bladder control methods. If desired, the methods herein
may also be used with farm animals, such as ovine, bovine, porcine
and equine breeds.
[0042] The applications may utilize conventional oral, rectal,
parenteral or intravenous delivery methods as conventionally
utilized in veterinary practice. Most preferable in most instance
for home use with companion animals are oral tablets or capsules or
neat compound or powdered or granular pharmaceutical formulations
which may be mixed with chewable or liquid veterinary formulations
or food materials or liquids acceptable to the animal in
question.
[0043] As used herein, the terms "pharmaceutically effective
amount" or "therapeutically effective amount" mean the total amount
of each active component of the pharmaceutical composition or
method that is sufficient to show a meaningful patient benefit
i.e., treatment, prevention or amelioration of urinary incontinence
or the excessive or undesirable urge to urinate, or a decrease in
the frequency of incidence of urinary incontinence. When applied to
an individual active ingredient, administered alone, the term
refers to that ingredient alone. When applied to a combination, the
term refers to combined amounts of the active ingredients that
result in the therapeutic effect, whether administered in
combination, serially or simultaneously.
[0044] The examples described below are for illustrative purposes,
and the present invention is not meant to be limited to these
examples.
[0045] In one embodiment, the present invention involves a high
throughput screening of compounds and selection of lead compounds
by indirectly measuring the membrane potential of transfected cells
as described in more detail below.
EXAMPLE 1
[0046] High Throughput Screening
[0047] Mammalian (e.g., HEK-293, COS) cells are transfected (either
stable or transient) with cDNA or cRNA for KCNQ potassium channels.
These cells express and incorporate functional KCNQ channels in
their membrane that modulate membrane potential. Several methods
are available to monitor transmembrane potential. One such method
employs the use of a Fluorescence Imaging Plate Reader (FLIPR), and
voltage-sensitive fluorescent dyes. Transfected cells are grown on
the well bottoms e.g., of a 96 or 384 well plate. The instrument
has the capability of pipetting into and recording fluorescent
signals from all wells simultaneously. In this way, large numbers
of compounds can be tested rapidly for their ability to modulate
the KCNQ-dependent transmembrane potential. Compounds, which cause
hyperpolarization of the transfected cell membrane, are further
analyzed through various techniques. In one example, the
fluorescence imaging plate reader (FLIPR) uses bis-oxonol (DiBAC4)
as the voltage sensitive fluorescent probe.
[0048] In one embodiment, following, high throughput screening,
secondary analysis of compounds selected via FLIPR is performed in
order to obtain functional data for lead compounds. In particular,
secondary analysis is performed as described more fully below.
EXAMPLE 2
In Vitro (Activity) Assays--Secondary Analysis
[0049] Isolated Rat Bladder Strip
[0050] Male Sprague-Dawley rats (200-400 grams) are euthanized by
CO.sub.2 inhalation and exsanguination. Their urinary bladders are
rapidly removed and placed in 37.degree. C. physiological salt
solution (PSS) that contained the following (mM): NaCl (118.4), KCl
(5), CaCl.sub.2 (2.5), MgSO.sub.4 (1.2), KH.sub.2PO.sub.4 (1.2),
NaHCO.sub.3 (24.9) and D-glucose (11.1) gassed with
O.sub.2/CO.sub.2 (95%/5%) to achieve a pH of 7.4. The dome of the
bladder is isolated from the trigon region and this tissue is then
cut into 4-5 mm wide by 10 mm long strips. One end is secured to
the bottom of a water jacketed tissue bath and the other to a Grass
isometric force transducer (Grass Instruments, Quincy, Mass.).
Tissues are pretensioned (0.25 to 0.5 grams), and after 30 minutes
of equilibration are contracted with an additional 15 mM KCl (total
of 20 mM) and again allowed to equilibrate until the preparations
are contracting steadily. Any of a variety of compounds are
administered directly into the tissue baths as sequential
concentrations. Transducer signals are digitized (12 bit
resolution) and analyzed on-line using a 586-based computer and
custom software. The area under the contraction curve (AUC) is used
as a measure of contractility since the spontaneous bladder
contractions are irregular in amplitude and frequency. A 5-minute
AUC value is taken 30 minutes after administration of each compound
concentration to the tissue bath.
[0051] Isolation of Rat Detrusor Cells
[0052] Rat detrusor cells are isolated in a manner previously
described for guinea-pig detrusor (Sheldon J H, Norton N W and
Argentieri T M (1997) Inhibition of guinea pig detrusor contraction
by NS-1619 is associated with activation of BKCa and inhibition of
calcium currents. J Pharmacol Exp Thera 283(3): 1193-1200). Male
Sprague-Dawley rats (Charles River, Wilmington, Mass.; 200-400
grams) are euthanized by CO.sub.2 inhalation and exsanguination.
Their urinary bladders are rapidly removed and placed in 37.degree.
C. physiological solution with the following composition (mM): Na
glutamate (80.0), NaCl (54.7), KCl (5.0), NaHCO.sub.3 (25.0),
MgCl.sub.2.2H.sub.2O (2.5), D-glucose (11.8) and CaCl.sub.2 (0.2)
gassed with O.sub.2--CO.sub.2, 95%/5% for a final pH of 7.4. The
dome of the bladder is isolated from the trigone region and the
mucosa is removed. This tissue is then cut into 2-3 mm wide strips
and placed into fresh buffer for 1 hour. Tissues are then
transferred into 10 ml of an isolation buffer containing the above
composition plus collagenase type VIII (1.0 mg/ml) and pronase
(0.25 mg/ml). After 10 minutes the isolation buffer is replaced
with fresh isolation buffer for an additional 10 min. The tissue is
then washed 3 times in fresh collagenase and pronase free solution
and stored at room temperature until studied. Cells for study are
prepared by triturating 1-2 pieces of detrusor tissue in 2 ml of
fresh isolation buffer for 5 minutes with a polished Pasteur
pipette, (tip diameter .about.1.5 mm) attached to a modified
Harvard Respirator pump (Harvard Apparatus, Southnatic, Mass.) at a
rate of 20.times./min. with an approximate volume of 5 ml. Cells
are then placed on a microscope stage in a temperature regulated
tissue bath at 32.5.degree. C. and continually superfused with
PSS.
[0053] Cell Electrophysiology
[0054] Single cell recordings are performed with a List-Medical
EPC-7 patch clamp amplifier (Adams & List Assoc., Westbury,
N.Y.). Pipette electrodes had tip resistances of 2-4 M.OMEGA. and
are filled with the following composition (mM): KCl (126.0),
MgCl.sub.2.6H.sub.2O (4.5), ATP Mg salt (4.0), GTP tris salt (0.3),
creatine PO.sub.4 (14.0), D-glucose (9.0), EGTA (9.0), HEPES (9.0).
The pH is adjusted to 7.4 with KOH. Signals are acquired (3 kHz
high frequency cut-off, 12 bit resolution) using a 586-based
personal computer.
[0055] To validate, changes in membrane potential observed
indirectly via FLIPR, voltage and current clamp analysis are
performed as described.
EXAMPLE 3
[0056] Current Clamp Recordings
[0057] Cell resting membrane potential (RMP) is measured in current
clamp using the above mentioned instrumentation and pipette
solutions. For these experiments nystatin is also added to the
pipette solution (100 .mu.g/ml) to allow recording through
utilization of the perforated patch technique (Korn, et al, 1991).
After stable access is achieved, RMP is recorded for a 5 minute
control period followed by 5 minute of drug application (0.3 and
1.0 .mu.M). After this time, various antagonists (linopirdine,
XE-991) are added to the perfusate and RMP is recorded for an
additional 5 minutes.
[0058] Voltage Clamp Recordings
[0059] Whole cell recordings are made using broken patch access.
Currents are evoked using either voltage steps (Vh=-50; Vt=-60 to
40 mV) or voltage ramps (-60 to 40 mV at 3.3 mV/sec.). The exact
voltage clamp protocols are well known in the art. After stability
is achieved control currents are recorded. Next, test compound is
added to the superfusate. Currents are recorded for 5 to 10 minutes
or until compound effects reach steady state. This is followed
either by washout or addition of antagonists (linopirdine, XE-991)
to the superfusate.
[0060] To further verify that changes in membrane potential and
membrane current are occurring as a result of activation of KCNQ
potassium channels, a Xenopus oocyte assay is performed as
follows:
EXAMPLE 4
[0061] Xenopus Oocyte Assay
[0062] Xenopus laevis are used because their ovaries always contain
oocytes at different stages (stages V and VI are considered mature
and used for expression purposes). These oocytes have very limited
number of endogenous ion channels and receptors and can express
"foreign" mRNAs easily. Therefore, in modern electrophysiology and
cellular and molecular biology, expression of mRNAs in Xenopus
oocytes has become a good tool for examining the properties of
receptors and ion channels from mammalian (including human)
tissues. Frogs are anesthetized in 0.3 tricaine methanesulphonate
(MS222) for at least 45 min. A lateral incision (<1 cm) is made
through the epidermis and the muscle fascia. The distal lobe of the
ovary is pulled out using blunt, atraumatic forceps and cut. Each
layer of the wound is closed separately using 4-O black
monofilament nylon and FS-2 cutting needles.
[0063] After removal, oocytes are cleaned and separated by
incubating with enzyme solutions. Eggs are then injected with
message for KCNQ subunits. After several days the channel proteins
are expressed in the oocyte membrane. Trans membrane currents and
voltage can be measured using standard two microelectrode recording
techniques.
[0064] Additional known, available, or conventional techniques are
applied to selected compounds to obtain functional in vitro or in
vivo data.
EXAMPLE 5
In Vivo (Efficacy) Assays
[0065] A) Hyperreflexic Bladders
[0066] Micturition frequency is enhanced by the stimulation of
sensory afferents using a dilute acetic acid solution in the
cystometric infusate as previously described by Birder and de Groat
(Birder L A. de Groat W C. Increased c-fos expression in spinal
neurons after irritation of the lower urinary tract in the rat. J
Neuroscience 12:4878-89, (1992)). Briefly, female Sprague-Dawley
rats (190-210 g) are anesthetized with urethane (1 g/kg/10 mL,
i.p.; 1 g/kg/10 mL, s.q.). The trachea is cannulated with PE205 to
ensure a patent airway. The external jugular is cannulated with
PE50 tubing for administration of compound. The bladder is exposed
through a midline incision, and an angiocatheter (24 g, teflon), is
heat flared at the end and inserted into the dome of the bladder
and secured with 4-0 silk. The bladder is flushed with normal
saline and allowed to equilibrate for 1 hour before cystometry is
performed. Using a "T" connector, the bladder catheter is connected
to a Statham pressure transducer (Model P23Db) and to a Harvard
infusion pump. Cystometric recordings are monitored on a Grass
polygraph while infusing the bladder with saline containing 0.25%
acetic acid at a rate of 2.4 mL/hr. for one hour. Next, compound is
administered intravenously and the cystometry monitored for an
additional 2 hours. The following cystometric parameters are
recorded: micturition interval, micturition amplitude, micturition
threshold pressure, bladder capacity, bladder compliance and the
number of spontaneous bladder contractions (SBC) during the filling
phase. The control period is taken as the 30 minute time period of
acetic acid saline perfusion before dosing. Compound effect is
analyzed 15 minutes after dosing, for 105 minutes.
[0067] B (i) Hypertrophied Bladders
[0068] The method for producing hypertrophied, unstable bladders
was modified from that reported by Malmgren, et al. (Malmgren A.
Sjogren C. Uvelius B. Mattiasson A. Andersson K E. Andersson P O.
Cystometrical evaluation of bladder instability in rats with
infravesical outflow obstruction. J Urology 137:1291-4, (1987)) and
reported by Wojdan, et al. (Wojdan A. Freeden C. Woods M. Oshiro G.
Spinelli W. Colatsky T J. Sheldon J H. Norton N W. Warga D. Antane
M M. Antane S A. Butera J A. Argentieri T M. Comparison of the
potassium channel openers, WAY-133537, ZD6169, and celikalim on
isolated bladder tissue and in vivo bladder instability in rat. J
Pharmacol Exp Therap 289:1410-1418, (1999)). Briefly, female
Sprague-Dawley rats (190-210 g) are used. Animals are anesthetized
with isoflurane. Once the animals are anesthetized, the bladder and
urethra are exposed through a midline incision and a 4-0 silk
ligature is tied around the proximal urethra in the presence of a
stainless steel rod (1 mm diameter). When the rod is then removed,
a calibrated partial occlusion of the urethra results. The
abdominal muscle is closed using 3-0 silk and the skin is closed
with surgical staples. Each rat receives 150,000 units of bicilin
C--R, i.m. During the following 6-9 weeks, bladder hypertrophy and
instability results from the partial outlet obstruction.
[0069] (ii) Catheter Implantation
[0070] Using the above animals, after approximately 6-9 weeks of
resulting hypertrophy as described above, the animals are
re-anesthetized with isoflurane, the ligature is removed from the
proximal urethra, and a flared catheter (PE60) is placed in the
dome of the bladder; secured with a suture. The catheter is
exteriorized under the skin through an opening in the back of the
neck. The abdominal incision is sutured, and the free end of the
catheter sealed. Following surgery, animals are given a second dose
of bicilin C--R (150,000 units/rat, i.m.).
[0071] (iii) Cystometric Evaluation
[0072] Two days after catheter implantation, animals are placed in
a metabolic cage, and the bladder catheter is attached (using a "T"
connector) to both a Statham pressure transducer (Model P23Db) and
to a Harvard infusion pump. Urine volume is monitored with a
plastic beaker attached to a force displacement transducer (Grass
FTO3). The cystometric evaluation of bladder function is started by
infusing the bladder with saline (10-20 mL/hr depending upon the
degree of hypertrophy). The following cystometric parameters are
recorded: number of spontaneous bladder contractions (SBC) during
the filling phase, micturition amplitude and micturition volume.
Cystometric recordings are made on a Grass polygraph and included
at least 2 micturition intervals or 20 minutes. Next, the rats are
rested for a two-hour period then orally gavaged with the test
compound. A second cystometry is performed approximately 60 minutes
after administration of test compound. A separate group of vehicle
(saline) treated animals with hypertrophied bladders serve as
time/vehicle controls.
[0073] Use of Retigabine and Other Experiments Establishing KCNQ as
a Target
[0074] In this report, we show that retigabine can relax isolated
KCl or carbachol-contracted rat bladder strips, and this relaxation
can be reversed by either linopirdine or XE-991. Using quantitative
rtPCR we have identified the expression of KCNQ1, 3 and 5 in the
rat urinary bladder and KCNQ3 and 5 in cultured human bladder
smooth muscle cells. The highest levels of expression were seen for
KCNQ5>KCNQ1>KCNQ3 in the rat and KCNQ5>KCNQ3 in human
cells. M-current activity was demonstrated by the presence of a
retigabine-induced increase in repolarizing current in isolated rat
and human bladder smooth muscle cells. The retigabine-dependent
current and hyperpolarization was reversed by the addition of
either linopirdine or XE-991, or acetylcholine to the tissue bath.
Finally, bladder cystometry revealed that retigabine could inhibit
spontaneous bladder contractions and micturition in a rat
neurogenic bladder model in a dose-dependent manner.
[0075] At present KCNQ derived M-current channels have mainly been
identified in neuronal, cardiac (Barhanin, J., Lesage, F.,
Guillemare, E., Fink, M., Lazdunski, M., Romey, G. K(V)LQT1 and lsK
(minK) proteins associate to form the I(Ks) cardiac potassium
current. Nature 384: 78-80 (1996); Sanguinetti, M., C., Curran, M.,
E., Zou, A., Shen, J., Spector, P., S., Atkinson, D., L., Keating,
M., T. Coassembly of K(V)LQT1 and mink (IsK) proteins to form
cardiac I(Ks) potassium channel. Nature 384: 80-83 (1996)) and
skeletal muscle (Schroeder, B., C., Hechenberger, M., Weinreich,
F., Kubisch, C., Jentsch, T., J. KCNQ5, a novel potassium channel
broadly expressed in brain, mediates M-type currents. J Biological
Chemistry 275: 24089-24095 (2000)) tissue. A number of syndromes
have been associated with defects in these proteins including: long
QT syndrome and cardiac arrhythmias (KCNQ1; Sanguinetti et al),
benign familial neonatal convulsions (KCNQ2 and KCNQ3 (Biervert,
C., Schroeder, B., C., Kubisch, C., Berkovic, S., F., Propping, P.,
Jentsch, T., J., Steinlein, O., K. A potassium channel mutation in
neonatal human epilepsy. Science 279: 403-406 (1998); Singh, N.,
A., Charlier, C., Stauffer, D., DuPont, B., R., Leach, R., J.,
Melis, R., Ronen, G., M., Bjerre, I., Quattlebaum, T., Murphy, J.,
V., McHarg, M., L., Gagnon, D., Rosales, T., O., Peiffer, A.,
Anderson, V., E., Leppert, M. A novel potassium channel gene,
KCNQ2, is mutated in an inherited epilepsy of newborns. Nature
Genetics 18: 25-29 (1998); Charlier, C., Singh, N., A., Ryan, S.,
G., Lewis, T., B., Reus, B., E., Leach, R., J., Leppert, M. A pore
mutation in a novel KQT-like potassium channel gene in an
idiopathic epilepsy family. Nature Genetics 18: 53-55 (1998)) and
nonsyndromic autosomal dominant deafness (KCNQ4 (Kubisch, C.,
Schroeder, B., C., Friedrich, T., Lutjohann, B., El-Amraoui, A.,
Marlin, S., Petit, C., Jentsch T., J., KCNQ4, a novel potassium
channel expressed in sensory outer hair cells, is mutated in
dominant deafness. Cell 96: 437-446 (1999); Coucke, P., J., Van
Hauwe, P., Kelley, P., M., Kunst, H., Schatteman, I., Van Velzen,
D., Meyers, J., Ensink, R., J., Verstreken, M., Declau, F., Marres,
H., Kastury, K., Bhasin, S., McGuirt, W., T., Smith, R., J.,
Cremers, C., W., Van de Heyning, P., Willems, P., J., Smith, S.,
D., Van Camp, G. Mutations in the KCNQ4 gene are responsible for
autosomal dominant deafness in four DFNA2 families. Human Molecular
Genetics 8:1321-1328 (1999)). To date, however, there have been no
reports of evidence for KCNQ currents in other tissue types,
including bladder smooth muscle. The data presented here provide
molecular and physiological evidence for the existence of
KCNQ-based M currents that contribute to membrane potential and
functioning of urinary bladder smooth muscle.
[0076] We isolated rat bladder strips from male Sprague-Dawley rats
as previously described, (Wojdan, A., Freeden, C., Woods, M.,
Oshiro, G., Spinelli, W. et al., J Pharmacol Exp Therap 289:
1410-1418 (1999)) and precontracted them with 20 mM KCl. The KCNQ
channel agonist retigabine, was added to the tissue bath in
increasing concentrations, and area under the contraction curve was
analyzed. Retigabine inhibited the spontaneous contractions in a
concentration-dependent manner with an IC.sub.50=1.4.+-.0.1 .mu.M
(n=4; FIG. 1). The effects of drug were not reversed by the
ATP-sensitive K.sup.+ channel blocker, glyburide (10 .mu.M), but
were antagonized 94.8.+-.17.5% by 10 .mu.M of the M-current
inhibitor, linopirdine or the selective KCNQ channel blocker
XE-991.
[0077] In another study, isolated rat bladder strips were
precontracted with 60 mM KCl (FIG. 1). The IC.sub.50 for inhibition
of contraction under this condition was significantly greater
(21.8.+-.0.8 .mu.M; n=4), than the IC.sub.50 obtained with 20 mM
KCl (p<0.05).
[0078] In a third set of bladder strips contracted with the
muscarinic agonist carbachol (200 nM), retigabine produced a
concentration-dependent inhibition of contraction with an IC.sub.50
of 3.5.+-.0.9 nM (n=14). The difference in IC.sub.50s between 20
and 60 mM KCl depolarizations are consistent with a potassium
channel opening mechanism. The inability of the ATP-dependent
K.sup.+ channel antagonist glyburide, and the ability of
linopirdine or XE-991 to antagonize the effects of retigabine
suggests that the bladder smooth muscle contractility is inhibited
via activation of a KCNQ channel.
[0079] We next probed the KCNQ subunit composition in rat bladder
using quantitative reverse transcriptase polymerase chain reaction
(rtPCR). Data (rtPCR) are presented as percent RNA/GAPDH RNA level.
The highest level of expression was seen with the KCNQ5 gene
(0.2.+-.0.1 ng KCNQ5 mRNA/GAPDH mRNA). KNCQ1 showed levels of
0.07.+-.0.1 ng mRNA/GAPDH mRNA, while KCNQ3 was calculated at
0.01.+-.0.01 ng mRNA/GAPDH mRNA. No signals were seen for either
the KCNQ2 or KCNQ4 gene (FIG. 2b). The KCNQ subunit composition in
cultured human bladder smooth muscle cells was KCNQ5 (0.07.+-.0.06
01 ng mRNA/GAPDH mRNA) and KCNQ3
(1.5.times.10.sup.-3.+-.0.1.times.10.sup.-3 ng mRNA/GAPDH mRNA.
There was no evidence for expression of KCNQ1, KCNQ2 or KCNQ4 in
these cells.
[0080] Current data suggest that the KCNQ2 and KCNQ3 subunits form
a heteromultimeric channel that can be agonized by retigabine.
KCNQ3 and KCNQ5 also appear to form a functional ion channel
similarly sensitive to retigabine. KCNQ4 may form a heteromultimer
with KCNQ3 or a homermeric ion channel that can be activated by
retigabine (Schroder, R. L., Jespersen, T., Christophersen, P.,
Strobaek, D., Jensen, B. et al. KCNQ4 channel activation by
BMS-204352 and retigabine. Neuropharmacol 40: 888-898 (2001)). The
above data provides molecular evidence for the expression of KCNQ
mRNA in rat and human bladder smooth muscle. Activation of the ion
channels formed by this message would be consistent with the
retigabine-induced relaxation of bladder smooth muscle.
[0081] Cellular electrophysiological studies were conducted using
both voltage and current clamp techniques (Hammill, O., P., Marty,
A., Neher, E., Sakmann, B. & Sigworth, F., J.: Improved
patch-clamp techniques for high-resolution current recordings from
cells and cell-free membrane patches. Pflugers Arch 391: 85-100
(1981)) from Sprague-Dawley rat (200-400 grams) bladders as
previously described, (Wojdan, A., Freeden, C., Woods, M., Oshiro,
G., Spinelli, W. et al., J. Pharmacol Exp Therap 289: 1410-1418
(1999)) and from a human bladder, primary cell culture (Colnetics,
San Diego, Calif.). In rat cells, the average, control resting
membrane potential (RMP) was -29.0.+-.4.5 mV. After exposure to 10
.mu.M retigabine, there was a significant (p<0.5, n=3)
hyperpolarization to -43.0.+-.3.5 mV. Hyperpolarization was
completely reversed by washout or the addition of 10 .mu.M XE-991
(FIG. 3a). Interestingly, XE-991 depolarized the cell below its
initial RMP suggesting that KCNQ currents are a determinant of
normal membrane potential. The retigabine-induced hyperpolarization
could also be antagonized by the application of 100 nM Ach (FIG.
3b). Voltage clamp studies revealed a retigabine induced increase
in outward current at test potentials between -50 and 80 mV (FIG.
3b,c; n=5). Increases in outward current were not sensitive to 100
nM iberiotoxin (Galvez, A. et al.: Purification and
characterization of a unique, potent, peptidyl probe for the high
conductance calcium-activated potassium channel from venom of the
scorpion buthus tamulus. J Biol Chem 265: 11083-11090 (1990))
(IbTx), but were partially reversed by linopirdine (50 .mu.M) or
XE-991 (10 .mu.M) (data not shown). Cultured human bladder smooth
muscle cells were more depolarized with resting membrane potentials
of -8.0.+-.2.8 mV. Exposure to retigabine hyperpolarized these
cells by 11.+-.1.1 mV (n=3) and increased outward currents (FIG.
3d). These changes could be partially reversed by XE-991.
[0082] These data demonstrate the existence of an outward current
in rat and human bladder smooth muscle that can be activated by the
KCNQ channel opener retigabine. Activation of this current was
associated with a hyperpolarization that was blocked by Ach and the
KCNQ channel blockers linopirdine and XE-991. It can be concluded
that the retigabine-dependent outward current in bladder smooth
muscle is electrophysiologically and pharmacologically consistent
with that reported for the M-current in other tissues.
[0083] Rat bladder micturition frequency (enhanced by infusate
containing 0.25% acetic acid (Birder, L. A., & deGroat, W., C.
Increased c-fos expression in spinal neurons after irritation of
the lower urinary tract in the rat. J Neurosc 12: 4878-4889
(1992))) was inhibited by retigabine in a dose-dependent (0.1-10
mg/kg, i.p.) manner. At 10 mg/kg, micturition was blocked in 100%
of animals dosed; the population ED.sub.50 was 1-2 mg/kg (n=5-8).
Micturition block lasted for up to 90 minutes (FIG. 4a).
Cystometrograms showed a high degree of spontaneous contractions
that were sensitive to doses of retigabine that did not completely
bock micturition. There was a 43.5.+-.14.1% inhibition (p<0.05)
of spontaneous contractions at 1 mg/kg, i.p. (FIG. 4b).
[0084] Summary
[0085] M-currents have been shown to play an important functional
role in a variety of tissues. The gene family contains at least
five major sub-units--KCNQ1 though KCNQ5. These sub-units have been
shown to co-assemble to form functional heteromeric and homomeric
ion channels. The only previous evidence of M-currents in smooth
muscle has been that reported in toad gastric smooth muscle (Sims,
S., T., Singer J., J., & Walsh, J., V. Antagonistic
adrenergic-muscarinic regulation of M current in smooth muscle
cells. Science 239: 190-193 (1988). Our data provide physiological
and pharmacological support for an M-current in rat and human
bladder smooth muscle, and provides molecular evidence suggesting
that the KCNQ potassium channel underlies this current. We have
shown that the KCNQ channel agonist, retigabine, can relax
precontracted, isolated rat bladder strips. The fact that the
addition of glyburide did not antagonize the relaxation suggests
that retigabine does not work via activation of the ATP-dependent
potassium channel. Using quantitative rtPCR we have identified the
expression of mRNA for KCNQ1, 3 and 5 in the rat and KCNQ3 and 5 in
human urinary bladder. Electrophysiological assessment revealed a
retigabine-induced outward current and hyperpolarization that was
antagonized by the M-current blocker linopirdine and KCNQ channel
antagonist XE-991. These data provide evidence for a KCNQ mediated
M-current that appears to be an important determinant of urinary
bladder smooth muscle excitability. We believe that this channel
may represent a novel molecular target for the treatment of bladder
hyperactivity associated with urge urinary incontinence.
[0086] Methods
[0087] Rat bladder strips were isolated and prepared as previously
described (Wojdan, A., Freeden, C., Woods, M., Oshiro, G.,
Spinelli, W. et al., J Pharmacol Exp Therap 289: 1410-1418 (1999)).
Preparations were contracted with either 20 or 60 mM KCl, or 200 nM
carbachol. A five minute area under the contraction curve was
acquired 20 minutes after addition of each concentration of
retigabine using a 12 bit D/A and a 586 based personal computer
running custom software. Message for KCNQ subunits was probed using
quantitative rtPCR on an ABI PRISM.RTM. 7700 Sequence Detection
System (Taqman). Forward and reverse primers and Taqman probes were
designed using published RNA sequences for human KCNQ1-5 and rat
KCNQ1-4 listed within the NCBI data base. Since no rat sequence for
KCNQ5 were currently published, probes and primers were designed by
BLAST analysis of rat Expressed Sequence Tags (EST) using the known
mouse KNCQ5 sequence. From homologous EST sequences, a contiguous
sequence of 384 base pairs was constructed. Probes and primers were
designed against this sequence. BLAST analysis of our rat KCNQ5
probes and primer were selective for mouse and human KCNQ5
sequences. PCR products were confirmed by gel electrophoresis (data
not shown). Cells for electrophysiology were prepared from rat
bladder smooth muscle as previously described (Wojdan et al.,
1999). Human bladder smooth muscle cells were obtained from
Clonetics, San Diego, Calif. Cells were removed from culture with
trypsin and added directly into the recording chamber. All
recordings were made at 32.degree. C. and acquired on a 586 based
personal computer using pCLAMP (Axon Instruments) software. Current
clamp recordings were performed with nystatin access as previously
described (Wojdan et al.). Rat bladder cystometry was performed as
previously described (Woods, M., Carson, N., Norton N., W., Sheldon
J., H., & Argentieri T., M. Efficacy of the beta 3-adrenergic
receptor agonist CL-316243 on experimental bladder hyperreflexia
and detrusor instability in the rat. Journal of Urology
166:1142-1147 (2001)). Tracings were acquired and analyzed off-line
using a PowerLab ML795 (16 bit A/D) data acquisition system.
[0088] Publications cited herein above are incorporated by
reference.
Sequence CWU 1
1
5 1 2924 DNA Homo sapiens 1 gcagcttcca tggcctgggg ctgtgagagg
cccgggaagg cactgtcttt gcgcctgcac 60 atgtgtgtgt ctggagtgta
ggatggcact ggtgccgggc ctgggcttcc tcgagcgtcc 120 caccggctgg
aagttgtaga cgcggccctg gacgtgggtg cgcgccaaca ccgggcggcg 180
cgtgctgtag atggagacgc gcgggtctag gctcaccggc ggccagggcc gcgtctacaa
240 cttcctcgag cgtcccaccg gctggaaatg cttcgtttac cacttcgccg
tcttcctcat 300 cgtcctggtc tgcctcatct tcagcgtgct gtccaccatc
gagcagtatg ccgccctggc 360 cacggggact ctcttctgga tggagatcgt
gctggtggtg ttcttcggga cggagtacgt 420 ggtccgcctc tggtccgccg
gctgccgcag caagtacgtg ggcctctggg ggcggctgcg 480 ctttgcccgg
aagcccattt ccatcatcga cctcatcgtg gtcgtggcct ccatggtggt 540
cctctgcgtg ggctccaagg ggcaggtgtt tgccacgtcg gccatcaggg gcatccgctt
600 cctgcagatc ctgaggatgc tacacgtcga ccgccaggga ggcacctgga
ggctcctggg 660 ctccgtggtc ttcatccacc gccaggagct gataaccacc
ctgtacatcg gcttcctggg 720 cctcatcttc tcctcgtact ttgtgtacct
ggctgagaag gacgcggtga acgagtcagg 780 ccgcgtggag ttcggcagct
acgcagatgc gctgtggtgg ggggtggtca cagtcaccac 840 catcggctat
ggggacaagg tgccccagac gtgggtcggg aagaccatcg cctcctgctt 900
ctctgtcttt gccatctcct tctttgcgct cccagcgggg attcttggct cggggtttgc
960 cctgaaggtg cagcagaagc agaggcagaa gcacttcaac cggcagatcc
cggcggcagc 1020 ctcactcatt cagaccgcat ggaggtgcta tgctgccgag
aaccccgact cctccacctg 1080 gaagatctac atccggaagg ccccccggag
ccacactctg ctgtcaccca gccccaaacc 1140 caagaagtct gtggtggtaa
agaaaaaaaa gttcaagctg gacaaagaca atggggtgac 1200 tcctggagag
aagatgctca cagtccccca tatcacgtgc gaccccccag aagagcggcg 1260
gctggaccac ttctctgtcg acggctatga cagttctgta aggaagagcc caacactgct
1320 ggaagtgagc atgccccatt tcatgagaac caacagcttc gccgaggacc
tggacctgga 1380 aggggagact ctgctgacac ccatcaccca catctcacag
ctgcgggaac accatcgggc 1440 caccattaag gtcattcgac gcatgcagta
ctttgtggcc aagaagaaat tccagcaagc 1500 gcggaagcct tacgatgtgc
gggacgtcat tgagcagtac tcgcagggcc acctcaacct 1560 catggtgcgc
atcaaggagc tgcagaggag gctggaccag tccattggga agccctcact 1620
gttcatctcc gtctcagaaa agagcaagga tcgcggcagc aacacgatcg gcgcccgcct
1680 gaaccgagta gaagacaagg tgacgcagct ggaccagagg ctggcactca
tcaccgacat 1740 gcttcaccag ctgctctcct tgcacggtgg cagcaccccc
ggcagcggcg gcccccccag 1800 agagggcggg gcccacatca cccagccctg
cggcagtggc ggctccgtcg accctgagct 1860 cttcctgccc agcaacaccc
tgcccaccta cgagcagctg accgtgccca ggaggggccc 1920 cgatgagggg
tcctgaggag gggatggggc tgggggatgg gcctgagtga gaggggaggc 1980
caagagtggc cccacctggc cctctctgaa ggaggccacc tcctaaaagg cccagagaga
2040 agagccccac tctcagaggc cccaataccc catggaccat gctgtctggc
acagcctgca 2100 cttgggggct cagcaaggcc acctcttcct ggccggtgtg
ggggccccgt ctcaggtctg 2160 agttgttacc ccaagcgccc tggcccccac
atggtgatgt tgacatcact ggcatggtgg 2220 ttgggaccca gtggcagggc
acagggcctg gcccatgtat ggccaggaag tagcacaggc 2280 tgagtgcagg
cccaccctgc ttggcccagg gggcttcctg aggggagaca gagcaacccc 2340
tggaccccag cctcaaatcc aggaccctgc caggcacagg cagggcagga ccagcccacg
2400 ctgactacag ggccaccggc aataaaagcc caggagccca tttggagggc
ctgggcctgg 2460 ctccctcact ctcaggaaat gctgacccat gggcaggaga
ctgtggagac tgctcctgag 2520 cccccagctt ccagcaggag ggacagtctc
accatttccc cagggcacgt ggttgagtgg 2580 ggggaacgcc cacttccctg
ggttagactg ccagctcttc ctagctggag aggagccctg 2640 cctctccgcc
cctgagccca ctgtgcgtgg ggctcccgcc tccaacccct cgcccagtcc 2700
cagcagccag ccaaacacac agaaggggac tgccacctcc ccttgccagc tgctgagccg
2760 cagagaagtg acggttccta cacaggacag gggttccttc tgggcattac
atcgcataga 2820 aatcaataat ttgtggtgat ttggatctgt gttttaatga
gtttcacagt gtgattttga 2880 ttattaattg tgcaagcttt tcctaataaa
cgtggagaat caca 2924 2 2750 DNA Homo sapiens 2 ccccgctgag
cctgagcccg acccggggcg cctcccgcca ggcaccatgg tgcagaagtc 60
gcgcaacggc ggcgtatacc ccggcccgag cggggagaag aagctgaagg tgggcttcgt
120 ggggctggac cccggcgcgc ccgactccac ccgggacggg gcgctgctga
tcgccggctc 180 cgaggccccc aagcgcggca gcatcctcag caaacctcgc
gcgggcggcg cgggcgccgg 240 gaagcccccc aagcgcaacg ccttctaccg
caagctgcag aatttcctct acaacgtgct 300 ggagcggccg cgcggctggg
cgttcatcta ccacgcctac gtgttcctcc tggttttctc 360 ctgcctcgtg
ctgtctgtgt tttccaccat caaggagtat gagaagagct cggagggggc 420
cctctacatc ctggaaatcg tgactatcgt ggtgtttggc gtggagtact tcgtgcggat
480 ctgggccgca ggctgctgct gccggtaccg tggctggagg gggcggctca
agtttgcccg 540 gaaaccgttc tgtgtgattg acatcatggt gctcatcgcc
tccattgcgg tgctggccgc 600 cggctcccag ggcaacgtct ttgccacatc
tgcgctccgg agcctgcgct tcctgcagat 660 tctgcggatg atccgcatgg
accggcgggg aggcacctgg aagctgctgg gctctgtggt 720 ctatgcccac
agcaaggagc tggtcactgc ctggtacatc ggcttccttt gtctcatcct 780
ggcctcgttc ctggtgtact tggcagagaa aggggagaac gaccactttg acacctacgc
840 ggatgcactc tggtggggcc tgatcacgct gaccaccatt ggctacgggg
acaagtaccc 900 ccagacctgg aacggcaggc tccttgcggc aaccttcacc
ctcatcggtg tctccttctt 960 cgcgctgcct gcaggcatct tggggtctgg
gtttgccctg aaggttcagg agcaacacag 1020 gcagaagcac tttgagaaga
ggcggaaccc ggcagcaggc ctgatccagt cggcctggag 1080 attctacgcc
accaacctct cgcgcacaga cctgcactcc acgtggcagt actacgagcg 1140
aacggtcacc gtgcccatgt acagttcgca aactcaaacc tacggggcct ccagacttat
1200 ccccccgctg aaccagctgg agctgctgag gaacctcaag agtaaatctg
gactcgcttt 1260 caggaaggac cccccgccgg agccgtctcc aagcccccga
ggcgtggccg ccaaggggaa 1320 ggggtccccg caggcccaga ctgtgaggcg
gtcacccagc gccgaccaga gcctcgagga 1380 cagccccagc aaggtgccca
agagctggag cttcggggac cgcagccggg cacgccaggc 1440 tttccgcatc
aagggtgccg cgtcacggca gaactcagaa gcaagcctcc ccggagagga 1500
cattgtggat gacaagagct gcccctgcga gtttgtgacc gaggacctga ccccgggcct
1560 caaagtcagc atcagagccg tgtgtgtcat gcggttcctg gtgtccaagc
ggaagttcaa 1620 ggagagcctg cggccctacg acgtgatgga cgtcatcgag
cagtactcag ccggccacct 1680 ggacatgctg tcccgaatta agagcctgca
gtccagagtg gaccagatcg tggggcgggg 1740 cccagcgatc acggacaagg
accgcaccaa gggcccggcc gaggcggagc tgcccgagga 1800 ccccagcatg
atgggacggc tcgggaaggt ggagaagcag gtcttgtcca tggagaagaa 1860
gctggacttc ctggtgaata tctacatgca gcggatgggc atccccccga cagagaccga
1920 ggcctacttt ggggccaaag agccggagcc ggcgccgccg taccacagcc
cggaagacag 1980 ccgggagcat gtcgacaggc acggctgcat tgtcaagatc
gtgcgctcca gcagctccac 2040 gggccaggag aacttctcgg cgcccccggc
cgcgccccct gtccagtgtc cgccctccac 2100 ctcctggcag ccacagagcc
acccgcgcca gggccacggc acctcccccg tgggggacca 2160 cggctccctg
gtgcgcatcc cgccgccgcc tgcccacgag cggtcgctgt ccgcctacgg 2220
cgggggcaac cgcgccagca tggagttcct gcggcaggag gacaccccgg gctgcaggcc
2280 ccccgagggg aacctgcggg acagcgacac gtccatctcc atcccgtccg
tggaccacga 2340 ggagctggag cgttccttca gcggcttcag catctcccag
tccaaggaga acctggatgc 2400 tctcaacagc tgctacgcgg ccgtggcgcc
ttgtgccaaa gtcaggccct acattgcgga 2460 gggagagtca gacactgact
ccgacctctg taccccgtgc gggcccccgc catgctcggc 2520 caccggcgag
ggtccctttg gtgacgtggg ctgggccggg cccaggaagt gaggcggcgc 2580
tgggccagtg gacccgcccg cggccctcct cagcacggtg cctccgaggt tttgaggcgg
2640 gaaccctctg gggccctttt cttacagtaa ctgggtgtgg cgggaagggt
gggccctgga 2700 ggggcccatg tgggctgaag gatgggggct cctggcagtg
accttttaca 2750 3 3005 DNA Homo sapiens Unsure (1)..(5) N is
unknown, but could be A, T, G, or C. 3 nnnnngaccc cctgaacccc
ctgcctggcc tcccctgccc cccaggggcc cgcctttgcc 60 tgcttttggg
ggggggtggg gaggggcgcg cggatcatgg cattggagtt cccgggcttg 120
cagccgccgc cgccgcctcg ttcacgcacc ccgagcgccc cttcttccca gagcagcagc
180 ggagaaggcg aagcgttctt cgggggcgag gcagatgggg ctcaaggcgc
gcagggcggc 240 gggggcggct ggcggcggcg gcgacggggg cggcggaggc
ggcggggcgg ctaacccagc 300 cggaggggac gcggcggcgg ccggcgacga
ggagcggaaa gtggggctgg cgcccggcga 360 cgtggagcaa gtcaccttgg
cgctcggggc cggagccgac aaagacggga ccctgctgct 420 ggagggcggc
ggccgcgacg aggggcagcg gaggaccccg cagggcatcg ggctcctggc 480
caagaccccg ctgagccgcc cagtcaagag aaacaacgcc aagtaccggc gcatccaaac
540 tttgatctac gacgccctgg agagaccgcg gggctgggcg ctgctttacc
acgcgttggt 600 gttcctgatt gtcctggggt gcttgattct ggctgtcctg
accacattca aggagtatga 660 gactgtctcg ggagactggc ttctgttact
ggagacattt gctattttca tctttggagc 720 cgagtttgct ttgaggatct
gggctgctgg atgttgctgc cgatacaaag gctggcgggg 780 ccgactgaag
tttgccagga agcccctgtg catgttggac atctttgtgc tgattgcctc 840
tgtgccagtg gttgctgtgg gaaaccaagg caatgttctg gccacctccc tgcgaagcct
900 gcgcttcctg cagatcctgc gcatgctgcg gatggaccgg agaggtggca
cctggaagct 960 tctgggctca gccatctgtg cccacagcaa agaactcatc
acggcctggt acatcggttt 1020 cctgacactc atcctttctt catttcttgt
ctacctggtt gagaaagacg tcccagaggt 1080 ggatgcacaa ggagaggaga
tgaaagagga gtttgagacc tatgcagatg ccctgtggtg 1140 gggcctgatc
acactggcca ccattggcta tggagacaag acacccaaaa cgtgggaagg 1200
ccgtctgatt gccgccacct tttccttaat tggcgtctcc ttttttgccc ttccagcggg
1260 catcctgggg tccgggctgg ccctcaaggt gcaggagcaa caccgtcaga
agcactttga 1320 gaaaaggagg aagccagctg ctgagctcat tcaggctgcc
tggaggtatt atgctaccaa 1380 ccccaacagg attgacctgg tggcgacatg
gagattttat gaatcagtcg tctcttttcc 1440 tttcttcagg caagtggggn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1500 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnatt 1560
ttgtagccaa aagctgggtc tcttggatcg ggttcgcctt tctaatcctc gtggtagcaa
1620 tactaaagga aagctattta cccctctgaa tgtagatgcc atagaagaaa
gtccttctaa 1680 agaaccaaag cctgttggct taaacaataa agagcgtttc
cgcacggcct tccgcatgaa 1740 agcctacgct ttctggcaga gttctgaaga
tgccgggaca ggtgacccca tggcggaaga 1800 caggggctat gggaatgact
tccccatcga agacatgatc cccaccctga aggccgccat 1860 ccgagccgtc
agaattctac aattccgtct ctataaaaaa aaattcaagg agactttgag 1920
gccttacgat gtgaaggatg tgattgagca gtattctgcc gggcatctcg acatgctttc
1980 caggataaag taccttcaga cgagaataga tatgattttc acccctggac
ctccctccac 2040 gccaaaacac aagaagtctc agaaagggtc agcattcacc
ttcccatccc agcaatctcc 2100 caggaatgaa ccatatgtag ccagaccatc
cacatcagaa atcgaagacc aaagcatgat 2160 ggggaagttt gtaaaagttg
aaagacaggt tcaggacatg gggaagaagc tggacttcct 2220 cgtggatatg
cacatgcaac acatggaacg gttgcaggtg caggtcacgg agtattaccc 2280
aaccaagggc acctcctcgc cagctgaagc agagaagaag gaggacaaca ggtattccga
2340 tttgaaaacc atcatctgca actattctga gacaggcccc ccggaaccac
cctacagctt 2400 ccaccaggtg accattgaca aagtcagccc ctatgggttt
tttgcacatg accctgtgaa 2460 cctgccccga gggggaccca gttctggaaa
ggttcaggca actcctcctt cctcagcaac 2520 aacgtatgtg gagaggccca
cggtcctgcc tatcttgact cttctcgact cccgagtgag 2580 ctgccactcc
caggctgacc tgcagggccc ctactcggac cgaatctccc cccggcagag 2640
acgtagcatc acgcgagaca gtgacacacc tctgtccctg atgtcggtca accacgagga
2700 gctggagagg tctccaagtg gcttcagcat ctcccaggac agagatgatt
atgtgttcgg 2760 ccccaatggg gggtcgagct ggatgaggga gaagcggtac
ctcgccgagg gtgagacgga 2820 cacagacacg gaccccttca cgcccagcgg
ctccatgcct ctgtcgtcca caggggatgg 2880 gatttctgat tcagtatgga
ccccttccaa taagcccatt taaaagaggt cactggctga 2940 cccctccttg
taatgtagac agactttgta tagttcactt actcttacac ccgacgctta 3000 ccagc
3005 4 2335 DNA Homo sapiens 4 agccatgcgt ctctgagcgc cccgagcgcg
cccccgcccc ggaccgtgcc cgggccccgg 60 cgcccccagc ccggcgccgc
ccatggccga ggcccccccg cgccgcctcg gcctgggtcc 120 cccgcccggg
gacgcccccc gcgcggagct agtggcgctc acggccgtgc agagcgaaca 180
gggcgaggcg ggcgggggcg gctccccgcg ccgcctcggc ctcctgggca gccccctgcc
240 gccgggcgcg cccctccctg ggccgggctc cggctcgggc tccgcctgcg
gccagcgctc 300 ctcggccgcg cacaagcgct accgccgcct gcagaactgg
gtctacaacg tgctggagcg 360 gccccgcggc tgggccttcg tctaccacgt
cttcatattt ttgctggtct tcagctgcct 420 ggtgctgtct gtgctgtcca
ctatccagga gcaccaggaa cttgccaacg agtgtctcct 480 catcttggaa
ttcgtgatga tcgtggtttt cggcttggag tacatcgtcc gggtctggtc 540
cgccggatgc tgctgccgct accgaggatg gcagggtcgc ttccgctttg ccagaaagcc
600 cttctgtgtc atcgacttca tcgtgttcgt ggcctcggtg gccgtcatcg
ccgcgggtac 660 ccagggcaac atcttcgcca cgtccgcgct gcgcagcatg
cgcttcctgc agatcctgcg 720 catggtgcgc atggaccgcc gcggcggcac
ctggaagctg ctgggctcag tggtctacgc 780 gcatagcaag gagctgatca
ccgcctggta catcgggttc ctggtgctca tcttcgcctc 840 cttcctggtc
tacctggccg agaaggacgc caactccgac ttctcctcct acgccgactc 900
gctctggtgg gggacgatta cattgacaac catcggctat ggtgacaaga caccgcacac
960 atggctgggc agggtcctgg ctgctggctt cgccttactg ggcatctctt
tctttgccct 1020 gcctgccggc atcctaggct ccggctttgc cctgaaggtc
caggagcagc accggcagaa 1080 gcacttcgag aagcggagga tgccggcagc
caacctcatc caggctgcct ggcgcctgta 1140 ctccaccgat atgagccggg
cctacctgac agccacctgg tactactatg acagtatcct 1200 cccatccttc
agagagctgg ccctcttgtt tgagcacgtg caacgggccc gcaatggggg 1260
cctacggccc ctggaggtgc ggcgggcgcc ggtacccgac ggagcaccct cccgttaccc
1320 gcccgttgcc acctgccacc ggccgggcag cacctccttc tgccctgggg
aaagcagccg 1380 gatgggcatc aaagaccgca tccgcatggg cagctcccag
cggcggacgg gtccttccaa 1440 gcagcagctg gcacctccaa caatgcccac
ctccccaagc agcgagcagg tgggtgaggc 1500 caccagcccc accaaggtgc
aaaagagctg gagcttcaat gaccgcaccc gcttccgggc 1560 atctctgaga
ctcaaacccc gcacctctgc tgaggatgcc ccctcagagg aagtagcaga 1620
ggagaagagc taccagtgtg agctcacggt ggacgacatc atgcctgctg tgaagacagt
1680 catccgctcc atcaggattc tcaagttcct ggtggccaaa aggaaattca
aggagacact 1740 gcgaccgtac gacgtgaagg acgtcattga gcagtactca
gcaggccacc tggacatgct 1800 gggccggatc aagagcctgc aaactcgggt
ggaccaaatt gtgggtcggg ggcccgggga 1860 caggaaggcc cgggagaagg
gcgacaaggg gccctccgac gcggaggtgg tggatgaaat 1920 cagcatgatg
ggacgcgtgg tcaaggtgga gaagcaggtg cagtccatcg agcacaagct 1980
ggacctgctg ttgggcttct attcgcgctg cctgcgctct ggcacctcgg ccagcctggg
2040 cgccgtgcaa gtgccgctgt tcgaccccga catcacctcc gactaccaca
gccctgtgga 2100 ccacgaggac atctccgtct ccgcacagac gctcagcatc
tcccgctcgg tcagcaccaa 2160 catggactga gggacttctc agaggcaggg
cagcacacgg ccagccccgc ggcctggcgc 2220 tccgactgcc ctctgaggcc
tccggactcc tctcgtactt gaactcactc cctcacgggg 2280 agagagacca
cacgcagtat tgagctgcct gagtgggcgt ggtacctgct gtggg 2335 5 3074 DNA
Homo sapiens 5 gggcgccccg tcggccgccg gcttcctcct tgaaacccgc
cggcgcacat gaggccgctg 60 cccccgccgc aggcgctggc ggccccctcg
cggtgcccgt ggtgatgcca tgccccgcca 120 ccacgcggga ggagaggagg
gcggcgccgc cgggctctgg gtgaagagcg gcgcagcggc 180 ggcggcggcg
ggcggggggc gcttgggcag cggcatgaag gatgtggagt cgggccgggg 240
cagggtgctg ctgaactcgg cagccgccag gggcgacggc ctgctactgc tgggcacccg
300 cgcggccacg cttggtggcg gcggcggtgg cctgagggag agccgccggg
gcaagcaggg 360 ggcccggatg agcctgctgg gaagccgcct ctcttacacg
agtagccaga gctgccggcg 420 caacgtcaag taccggcggg tgcagaacta
cctgtacaac gtgctggaga gaccccgcgg 480 ctgggcgttc atctaccacg
ctttcgtttt cctccttgtc tttggttgct tgattttgtc 540 agtgttttct
accatccctg agcacacaaa attggcctca agttgcctct tgatcctgga 600
gttcgtgatg attgtcgtct ttggtttgga gttcatcatt cgaatctggt ctgcgggttg
660 ctgttgtcga tatagaggat ggcaaggaag actgaggttt gctcgaaagc
ccttctgtgt 720 tatagatacc attgttctta tcgcttcaat agcagttgtt
tctgcaaaaa ctcagggtaa 780 tatttttgcc acgtctgcac tcagaagtct
ccgtttccta cagatcctcc gcatggtgcg 840 catggaccga aggggaggca
cttggaaatt actgggttca gtggtttatg ctcacagcaa 900 ggaattaatc
acagcttggt acataggatt tttggttctt attttttcgt ctttccttgt 960
ctatctggtg gaaaaggatg ccaataaaga gttttctaca tatgcagatg ctctctggtg
1020 gggcacaatt acattgacaa ctattggcta tggagacaaa actcccctaa
cttggctggg 1080 aagattgctt tctgcaggct ttgcactcct tggcatttct
ttctttgcac ttcctgccgg 1140 cattcttggc tcaggttttg cattaaaagt
acaagaacaa caccgccaga aacactttga 1200 gaaaagaagg aacccagctg
ccaacctcat tcagtgtgtt tggcgtagtt acgcagctga 1260 tgagaaatct
gtttccattg caacctggaa gccacacttg aaggccttgc acacctgcag 1320
ccctaccaag aaagaacaag gggaagcatc aagcagtcag aagctaagtt ttaaggagcg
1380 agtgcgcatg gctagcccca ggggccagag tattaagagc cgacaagcct
cagtaggtga 1440 caggaggtcc ccaagcaccg acatcacagc cgagggcagt
cccaccaaag tgcagaagag 1500 ctggagcttc aacgaccgaa cccgcttccg
gccctcgctg cgcctcaaaa gttctcagcc 1560 aaaaccagtg atagatgctg
acacagccct tggcactgat gatgtatatg atgaaaaagg 1620 atgccagtgt
gatgtatcag tggaagacct caccccacca cttaaaactg tcattcgagc 1680
tatcagaatt atgaaatttc atgttgcaaa acggaagttt aaggaaacat tacgtccata
1740 tgatgtaaaa gatgtcattg aacaatattc tgctggtcat ctggacatgt
tgtgtagaat 1800 taaaagcctt caaacacgtg ttgatcaaat tcttggaaaa
gggcaaatca catcagataa 1860 gaagagccga gagaaaataa cagcagaaca
tgagaccaca gacgatctca gtatgctcgg 1920 tcgggtggtc aaggttgaaa
aacaggtaca gtccatagaa tccaagctgg actgcctact 1980 agacatctat
caacaggtcc ttcggaaagg ctctgcctca gccctcgctt tggcttcatt 2040
ccagatccca ccttttgaat gtgaacagac atctgactat caaagccctg tggatagcaa
2100 agatctttcg ggttccgcac aaaacagtgg ctgcttatcc agatcaacta
gtgccaacat 2160 ctcgagaggc ctgcagttca ttctgacgcc aaatgagttc
agtgcccaga ctttctacgc 2220 gcttagccct actatgcaca gtcaagcaac
acaggtgcca attagtcaaa gcgatggctc 2280 agcagtggca gccaccaaca
ccattgcaaa ccaaataaat acggcaccca agccagcagc 2340 cccaacaact
ttacagatcc cacctcctct cccagccatc aagcatctgc ccaggccaga 2400
aactctgcac cctaaccctg caggcttaca ggaaagcatt tctgacgtca ccacctgcct
2460 tgttgcctcc aaggaaaatg ttcaggttgc acagtcaaat ctcaccaagg
accgttctat 2520 gaggaaaagc tttgacatgg gaggagaaac tctgttgtct
gtctgtccca tggtgccgaa 2580 ggacttgggc aaatctttgt ctgtgcaaaa
cctgatcagg tcgaccgagg aactgaatat 2640 acaactttca gggagtgagt
caagtggctc cagaggcagc caagattttt accccaaatg 2700 gagggaatcc
aaattgttta taactgatga agaggtgggt cccgaagaga cagagacaga 2760
cacttttgat gccgcaccgc agcctgccag ggaagctgcc tttgcatcag actctctaag
2820 gactggaagg tcacgatcat ctcagagcat ttgtaaggca ggagaaagta
cagatgccct 2880 cagcttgcct catgtcaaac tgaaataagt tcttcatttt
ctttccaggc atagcagttc 2940 tttagccata catatcattg catgaactat
ttcgaaagcc cttctaaaaa gttgaaattg 3000 caagaatcgg gaagaacatg
aaaggcagtt tataagcccg ttacctttta attgcatgaa 3060 aatgcatgtt tagg
3074
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