U.S. patent application number 10/694641 was filed with the patent office on 2004-05-13 for inhibitors of epoxide hydrolases for the treatment of hypertension.
This patent application is currently assigned to THE REGENTS OF THE UNIVERSITY OF CALIFORNIA. Invention is credited to Hammock, Bruce D., Kroetz, Deanna L., Morisseau, Christophe, Zeldin, Darryl C..
Application Number | 20040092487 10/694641 |
Document ID | / |
Family ID | 26942082 |
Filed Date | 2004-05-13 |
United States Patent
Application |
20040092487 |
Kind Code |
A1 |
Kroetz, Deanna L. ; et
al. |
May 13, 2004 |
Inhibitors of epoxide hydrolases for the treatment of
hypertension
Abstract
The invention provides compounds that inhibit epoxide hydrolase
in therapeutic applications for treating hypertension. A preferred
class of compounds for practicing the invention have the structure
shown by Formula 1 1 wherein Z is oxygen or sulfur, W is carbon
phosphorous or sulfur, X and Y is each independently nitrogen,
oxygen, or sulfur, and X can further be carbon, at least one of
R.sub.1-R.sub.4 is hydrogen, R.sub.2 is hydrogen when X is nitrogen
but is not present when X is sulfur or oxygen, R.sub.4 is hydrogen
when Y is nitrogen but is not present when Y is sulfur or oxygen,
R.sub.1 and R.sub.3 is each independently C.sub.1-C.sub.20
substituted or unsubstituted alkyl, cycloalkyl, aryl, acyl, or
heterocyclic.
Inventors: |
Kroetz, Deanna L.; (San
Francisco, CA) ; Zeldin, Darryl C.; (Chapel Hill,
NC) ; Hammock, Bruce D.; (Davis, CA) ;
Morisseau, Christophe; (Sacramento, CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
THE REGENTS OF THE UNIVERSITY OF
CALIFORNIA
Oakland
CA
THE GOVERNMENT OF THE U.S., as represented by the Sec. of the
Dept. of Health and Human Services
Rockville
MD
|
Family ID: |
26942082 |
Appl. No.: |
10/694641 |
Filed: |
October 27, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10694641 |
Oct 27, 2003 |
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10328495 |
Dec 23, 2002 |
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6693130 |
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10328495 |
Dec 23, 2002 |
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09721261 |
Nov 21, 2000 |
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6531506 |
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09721261 |
Nov 21, 2000 |
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09252148 |
Feb 18, 1999 |
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6150415 |
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Current U.S.
Class: |
514/114 ;
514/44R; 514/475; 514/478; 514/580; 514/588 |
Current CPC
Class: |
A61K 31/336 20130101;
A61K 31/325 20130101; A61P 9/12 20180101; A61K 31/00 20130101 |
Class at
Publication: |
514/114 ;
514/475; 514/044; 514/478; 514/580; 514/588 |
International
Class: |
A61K 048/00; A61K
031/66; A61K 031/335; A61K 031/325; A61K 031/17 |
Goverment Interests
[0002] This invention was made with Government support under Grant
Nos. HL53994, ES02710, and ES04699 awarded by the National
Institutes of Health.
Claims
What is claimed is:
1. A method of treating hypertension in a patient, the method
comprising administering to the patient a therapeutically effective
amount of a nucleic acid which inhibits epoxide hydrolase (EH) gene
expression.
2. A method of claim 1, wherein the nucleic acid is complementary
to a portion of a human gene encoding EH.
3. A method of claim 1, wherein the nucleic acid is DNA.
4. A method of claim 1, wherein the nucleic acid is RNA.
5. A method of claim 1, wherein the nucleic acid is modified to
increase resistance to nucleases.
6. A method of delivering a reactive functionality to epoxide
hydrolase, said method comprising contacting said epoxide hydrolase
with a compound selected from the group consisting of Formula I and
of Formula II, wherein (a) compounds of Formula I have the
structure 137wherein Z is oxygen or sulfur, W is carbon phosphorous
or sulfur, X and Y is each independently nitrogen, oxygen, or
sulfur, and X can further be carbon, at least one of
R.sub.1-R.sub.4 is hydrogen, R.sub.2 is hydrogen when X is nitrogen
but is not present when X is sulfur or oxygen, R.sub.4 is hydrogen
when Y is nitrogen but is not present when Y is sulfur or oxygen,
R.sub.1 and R.sub.3 are each independently a substituted or
unsubstituted alkyl, haloalkyl, cycloalkyl, aryl, acyl, or
heterocyclic, and (b) compounds of Formula II have the structure
138wherein R is alkyl or aryl, the compound is trans-across the
epoxide ring, OX is a carbonyl (.dbd.O) or hydroxy group (OH) and
R' is a H, alkyl or aryl group, and further wherein said compound
of Formula I or Formula II is derivatized with a reactive
functionality.
7. A method of claim 6, wherein the reactive functionality is an
alkylating agent or a Michael acceptor
8. A method of claim 7, wherein the alkylating agent is a
halogen.
9. A method of claim 7, wherein the alkylating agent is an
epoxide.
10. A method of claim 6, wherein said compound of Formula I or
Formula II is derivatized with a Michael acceptor.
11. A method of claim 6, wherein said compound is a compound of
Formula I.
12. A method of claim 6, wherein said compound is a compound of
Formula II.
13. A method of detecting epoxide hydrolase, said method comprising
contacting said epoxide hydrolase with a compound selected from the
group consisting of Formula I and of Formula II, wherein (a)
compounds of Formula I have the structure 139wherein Z is oxygen or
sulfur, W is carbon phosphorous or sulfur, X and Y is each
independently nitrogen, oxygen, or sulfur, and X can further be
carbon, at least one of R.sub.1-R.sub.4 is hydrogen, R.sub.2 is
hydrogen when X is nitrogen but is not present when X is sulfur or
oxygen, R.sub.4 is hydrogen when Y is nitrogen but is not present
when Y is sulfur or oxygen, R.sub.1 and R.sub.3 are each
independently a substituted or unsubstituted alkyl, haloalkyl,
cycloalkyl, aryl, acyl, or heterocyclic, and (b) compounds of
Formula II have the structure 140wherein R is alkyl or aryl, the
compound is trans-across the epoxide ring, OX is a carbonyl
(.dbd.O) or hydroxy group (OH) and R' is a H, alkyl or aryl group,
and further wherein said compound of Formula I or Formula II is
derivatized with a fluorescent or affinity label.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 10/328,495, filed Dec. 23, 2002, which is a continuation of
U.S. application Ser. No. 09/721,261, now U.S. Pat. No. 6,531,506,
which is a continuation in part of U.S. application Ser. No
09/252,148, filed Feb. 18, 1999, now U.S. Pat. No. 6,150,415. The
disclosures of all of these applications are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention generally relates to methods of
treating hypertension using inhibitors of epoxide hydrolases.
Preferred inhibitors include compounds, such as ureas, amides, and
carbamates that can interact with the enzyme catalytic site and
mimic transient intermediates. Other useful inhibitors include
glycodiols and chalcone oxides which can interact with the enzyme
as irreversible inhibitors.
[0005] 2. Background of the Invention
[0006] Hypertension is the most common risk factor for
cardiovascular disease, the leading cause of death in many
developed countries. Essential hypertension, the most common form
of hypertension, is usually defined as high blood pressure in which
secondary causes such as renovascular disease, renal failure,
pheochromocytoma, aldosteronism, or other causes are not present
(for a discussion of the definition and etiology of essential
hypertension see, Carretero and Oparil Circulation 101:329-335
(2000) and Carretero, O. A. and S. Oparil. Circulation 101:446-453
(2000)
[0007] A combination of genetic and environmental factors
contribute to the development of hypertension and its successful
treatment is limited by a relatively small number of therapeutic
targets for blood pressure regulation. Renal cytochrome P450 (CYP)
eicosanoids have potent effects on vascular tone and tubular ion
and water transport and have been implicated in the control of
blood pressure (Makita et al. FASEB J 10:1456-1463 (1996)). The
major products of CYP-catalyzed arachidonic acid metabolism are
regio- and stereoisomeric epoxyeicosatrienoic acids (EETs) and
20-hydroxyeicosatetraenoic acid (20-HETE). 20-HETE produces potent
vasoconstriction by inhibition of the opening of a
large-conductance, calcium-activated potassium channel leading to
arteriole vascular smooth muscle depolarization (Zou et al. Am J.
Physiol. 270:R228-237 (1996)). In contrast, the EETs have
vasodilatory properties associated with an increased open-state
probability of a calcium-activated potassium channel and
hyperpolarization of the vascular smooth muscle and are recognized
as putative endothelial derived hyperpolarizing factors (Campbell
et al. Cir. Res. 78:415-423 (1996)). Hydrolysis of the EETs to the
corresponding dihydroxyeicosatrienoic acids (DHETs) is catalyzed
largely by soluble epoxide hydrolase (sEH) (Zeldin et al. J. Biol.
Chem. 268:6402-64-07 (1993)).
[0008] Recent studies have indicated that renal CYP-mediated
20-HETE and EET formation are altered in genetic rat models of
hypertension and that modulation of these enzyme activities is
associated with corresponding changes in blood pressure (Omata et
al. Am J Physiol 262:F8-16 (1992); Makita et al. J Clin Invest
94:2414-2420 (1994); Kroetz et al. Mol Pharmacol 52:362-372 (1997);
Su, P. et al., Am J Physiol 275, R426-438 (1998)). Modulation of
the CYP pathways of arachidonic acid metabolism as a means to
regulate eicosanoid levels is limited by multiple isoforms
contributing to a single reaction and the general lack of
selectivity of most characterized inhibitors and inducers.
Similarly, modulating EET levels by regulation of their hydrolysis
to the less active diols has not been considered in light of
concerns that EETs are involved in many physiological processes.
(Campbell, Trends Pharmacol Sci 21:125-7 (2000)).
SUMMARY OF THE INVENTION
[0009] The present invention provides methods of treating
hypertension by administering to a patient a therapeutically
effective amount of an inhibitor of epoxide hydrolase. A preferred
class of compounds for practice in accordance with the invention
has the structure shown by Formula 1.
Formula 1
[0010] 2
[0011] wherein Z is oxygen or sulfur, W is carbon phosphorous or
sulfur, X and Y is each independently nitrogen, oxygen, or sulfur,
and X can further be carbon, at least one of R.sub.1-R.sub.4 is
hydrogen, R.sub.2 is hydrogen when X is nitrogen but is not present
when X is sulfur or oxygen, R.sub.4 is hydrogen when Y is nitrogen
but is not present when Y is sulfur or oxygen, R.sub.1 and R.sub.3
are each independently a substituted or unsubstituted alkyl,
haloalkyl, cycloalkyl, aryl, acyl, or heterocyclic.
[0012] Preferred compounds of the invention have an IC.sub.50
(inhibition potency or, by definition, the concentration of
inhibitor which reduces enzyme activity by 50%) of less than about
500 .mu.M. Exemplary compounds of the invention are listed in Table
1. The Table shows inhibition of recombinant mouse sEH (MsEH) and
Human sEH (HsEH). The enzyme concentrations were 0.13 and 0.26
micromolar respectively
1TABLE 1 Inhibition of MsEH (0.13 .mu.M) and HsEH (0.26 .mu.M)
Mouse sEH Human sEH Structure inhibitors nb IC.sub.50 (.mu.M)*
IC.sub.50 (.mu.M)* 3 72 0.11 .+-. 0.01 0.48 .+-. 0.01 4 248 0.33
.+-. 0.05 2.9 .+-. 0.6 5 42 0.06 .+-. 0.01 0.13 .+-. 0.01 6 224
0.99 .+-. 0.02 0.32 .+-. 0.08 7 225 0.84 .+-. 0.10 1.05 .+-. 0.03 8
276 1.1 .+-. 0.1 0.34 .+-. 0.02 9 277 0.12 .+-. 0.01 0.22 .+-. 0.02
10 460 0.10 .+-. 0.02 0.18 .+-. 0.01 11 110 0.21 .+-. 0.01 0.35
.+-. 0.01 12 259 0.45 .+-. 0.09 0.10 .+-. 0.02 13 260 0.06 .+-.
0.01 0.10 .+-. 0.01 14 261 0.08 .+-. 0.01 0.12 .+-. 0.01 15 262
0.05 .+-. 0.01 0.10 .+-. 0.02 16 263 3.0 .+-. 0.3 0.33 .+-. 0.06 17
255 0.48 .+-. 0.07 2.88 .+-. 0.04 18 514 0.27 .+-. 0.01 19 515 0.19
.+-. 0.05 20 187 0.05 .+-. 0.01 0.42 .+-. 0.03 21 374 0.25 .+-.
0.01 2.03 .+-. 0.07 22 381 0.06 .+-. 0.01 0.68 .+-. 0.03 23 0.25
.+-. 0.02 0.47 .+-. 0.01 24 189 0.80 .+-. 0.03 1.0 .+-. 0.2 25 375
0.18 .+-. 0.01 0.11 .+-. 0.01 26 157 0.85 .+-. 0.01 1.43 .+-. 0.03
27 143 1.0 .+-. 0.1 0.57 .+-. 0.01 28 178 0.31 .+-. 0.01 0.25 .+-.
0.01 29 380 0.73 .+-. 0.03 0.68 .+-. 0.03 30 125 0.09 .+-. 0.01
0.72 .+-. 0.02 31 183 1.06 .+-. 0.07 5.9 .+-. 0.3 32 175 0.24 .+-.
0.01 33 181 0.52 .+-. 0.02 1.71 .+-. 0.23 34 168 0.06 .+-. 0.01
0.12 .+-. 0.01 35 151 2.29 .+-. 0.03 0.58 .+-. 0.01 36 170 0.12
.+-. 0.01 0.18 .+-. 0.01 37 429 0.38 .+-. 0.04 1.7 .+-. 0.4 38 153
0.06 .+-. 0.01 0.10 .+-. 0.01 39 148 0.21 .+-. 0.01 0.61 .+-. 0.02
40 172 0.12 .+-. 0.01 0.30 .+-. 0.01 41 556 0.20 .+-. 0.02 0.74
.+-. 0.07 42 478 0.05 .+-. 0.01 0.26 .+-. 0.02 43 562 0.5 .+-. 0.1
15 .+-. 3 44 531 0.14 .+-. 0.02 0.64 .+-. 0.03 45 504 0.8 .+-. 0.1
23 .+-. 4 46 479 0.60 .+-. 0.06 5.0 .+-. 0.1 47 103 0.12 .+-. 0.01
2.2 .+-. 0.1 48 347 0.07 .+-. 0.01 3.10 .+-. 0.07 49 124 0.05 .+-.
0.01 0.14 .+-. 0.01 50 509 0.06 .+-. 0.01 0.92 .+-. 0.08 51 286
0.11 .+-. 0.03 0.07 .+-. 0.02 52 344 0.05 .+-. 0.01 2.50 .+-. 0.08
53 508 0.05 .+-. 0.01 0.10 .+-. 0.01 54 473 0.05 .+-. 0.01 0.10
.+-. 0.01 55 297 0.05 .+-. 0.01 0.10 .+-. 0.01 56 425 0.05 .+-.
0.01 0.10 .+-. 0.01 57 354 0.05 .+-. 0.01 0.10 .+-. 0.01 58 477
0.11 .+-. 0.01 0.24 .+-. 0.01 59 23 0.10 .+-. 0.01 1.69 .+-. 0.05
60 0.09 .+-. 0.01 0.16 .+-. 0.01 61 538 0.05 .+-. 0.01 0.10 .+-.
0.01 62 551 0.05 .+-. 0.01 0.10 .+-. 0.01 63 57 0.06 .+-. 0.01 0.16
.+-. 0.01 64 360 0.05 .+-. 0.01 0.10 .+-. 0.01 65 359 0.05 .+-.
0.01 0.10 .+-. 0.01 66 461 0.42 .+-. 0.01 0.55 .+-. 0.02 67 533
0.05 .+-. 0.1 0.10 .+-. 0.01 68 463 0.90 .+-. 0.07 8.3 .+-. 0.4 69
377 0.7 .+-. 0.1 17.8 .+-. 0.7 70 428 0.05 .+-. 0.1 0.10 .+-. 0.01
71 22 0.05 .+-. 0.1 0.10 .+-.0.01 72 58 0.05 .+-. 0.01 0.09 .+-.
0.01 73 119 0.05 .+-. 0.01 0.10 .+-. 0.01 74 543 0.05 .+-. 0.01
0.10 .+-. 0.01 75 192 0.05 .+-. 0.01 0.10 .+-. 0.01 76 427 0.05
.+-. 0.01 0.10 .+-. 0.01 77 358 0.05 .+-. 0.01 0.18 .+-. 0.04 78 21
0.76 .+-. 0.02 1.39 .+-. 0.02 79 435 0.05 .+-. 0.01 0.18 .+-. 0.01
80 270 0.05 .+-. 0.01 1.9 .+-. 0.1 81 544 0.05 .+-. 0.01 0.10 .+-.
0.01 82 545 0.05 .+-. 0.01 3.7 .+-. 0.3 83 437 0.05 .+-. 0.01 0.10
.+-. 0.01 84 176 0.06 .+-. 0.01 0.53 .+-. 0.03 85 36 0.06 .+-. 0.01
0.16 .+-. 0.02 86 104 0.04 .+-. 0.01 0.29 .+-. 0.01 87 105 0.05
.+-. 0.01 0.58 .+-. 0.03 88 100 0.07 .+-. 0.01 0.15 .+-. 0.01 89
188 0.73 .+-. 0.08 2.50 .+-. 0.03 90 434 0.05 .+-. 0.01 0.10 .+-.
0.01 91 59 0.85 .+-. 0.02 0.48 .+-. 0.01 92 559 0.08 .+-. 0.01 0.14
.+-. 0.01 93 169 0.06 .+-. 0.01 0.13 .+-. 0.01 94 140 0.05 .+-.
0.01 0.10 .+-. 0.01 95 108 0.13 .+-. 0.01 0.17 .+-. 0.01 96 67 0.71
.+-. 0.04 0.23 .+-. 0.01 97 384 0.05 .+-. 0.01 1.0 .+-. 0.2 98 343
0.05 .+-. 0.01 0.10 .+-. 0.01 99 118 0.06 .+-. 0.01 0.10 .+-. 0.01
100 126 0.06 .+-. 0.01 0.27 .+-. 0.02 101 66 0.09 .+-. 0.01 0.07
.+-. 0.01 102 180 0.06 .+-. 0.01 0.10 .+-. 0.01 103 75 0.06 .+-.
0.01 0.23 .+-. 0.01 104 501 0.05 .+-. 0.01 0.16 .+-. 0.01 105 60
0.78 .+-. 0.02 0.43 .+-. 0.02 106 20 0.19 .+-. 0.02 0.40 .+-. 0.05
107 193 0.05 .+-. 0.01 0.19 .+-. 0.01 108 361 0.07 .+-. 0.02 0.20
.+-. 0.02 109 44 0.07 .+-. 0.01 0.19 .+-. 0.01 110 179 0.06 .+-.
0.01 0.10 .+-. 0.01 111 65 0.17 .+-. 0.01 0.11 .+-. 0.01 112 53
0.07 .+-. 0.01 0.12 .+-. 0.01 113 385 0.17 .+-. 0.02 2.2 .+-. 0.1
114 379 0.05 .+-. 0.01 1.5 .+-. 0.1 115 362 0.05 .+-. 0.01 1.5 .+-.
0.1 116 38 0.17 .+-. 0.01 0.36 .+-. 0.02 117 341 2.3 .+-. 0.3 4.3
.+-. 0.4 118 128 0.79 .+-. 0.08 11.1 .+-. 0.8 119 411 0.05 .+-.
0.01 0.10 .+-. 0.01 120 412 0.05 .+-. 0.01 0.10 .+-. 0.01 121 413
0.05 .+-. 0.01 0.10 .+-. 0.01 122 438 0.05 .+-. 0.01 0.10 .+-. 0.01
123 430 0.21 .+-. 0.02 0.55 .+-. 0.03 124 470 0.59 .+-. 0.08 7.6
.+-. 0.1 125 471 0.25 .+-. 0.03 2.2 .+-. 0.1 126 159 0.59 .+-. 0.03
3.40 .+-. 0.04 127 156 0.20 .+-. 0.01 0.48 .+-. 0.01 128 287 0.09
.+-. 0.01 0.10 .+-. 0.01 129 167 0.39 .+-. 0.02 3.77 .+-. 0.03 130
0.36 .+-. 0.02 0.12 .+-. 0.01 131 299 0.7 0.1 0.26 0.02 132 253
0.10 .+-. 0.02 0.28 .+-. 0.01 133 283 0.7 .+-. 0.2 1.12 .+-. 0.03
134 257 0.05 .+-. 0.01 0.10 .+-. 0.04
[0013] A second preferred class of compounds for practice in
accordance with the invention has the structure shown by Formula
2,
Formula 2
[0014] 135
[0015] wherein R is alkyl or aryl, the compound is trans-across the
epoxide ring, OX is a carbonyl (.dbd.O) or hydroxy group (OH) and
R' is a H, alkyl or aryl group. The preparation of these compounds
is described in U.S. Pat. No. 5,955,496 and in WO98/06261.
[0016] Exemplary compounds are shown in Table 2, below.
2TABLE 2 Inhibition of MsEH (0.13 .mu.M) and HsEH (0.26 .mu.M) 136
Mouse sEH IC.sub.50 (.mu.M)* Human sEH IC.sub.50 (.mu.M)* R R' X--Y
Abs. Conf. C.sub.6H.sub.5 C.sub.6H.sub.5 C.dbd.O .+-. 2.9 .+-. 0.3
0.3 .+-. 0.1 C.sub.6H.sub.5 C.sub.6H.sub.5 CH--OH .+-. 12.6 .+-.
0.9 22 .+-. 2 C.sub.6H.sub.5 C.sub.6H.sub.5 C.dbd.NOH .+-. 3.5 .+-.
0.5 0.29 .+-. 0.01 C.sub.6H.sub.5 C.sub.6H.sub.5 S.dbd.O .+-. 2.3
.+-. 0.4 0.31 .+-. 0.02 C.sub.6H.sub.5 C.sub.6H.sub.5 CH--OCH.sub.3
.+-. 103 .+-. 5 34 .+-. 1 4-F--C.sub.6H.sub.4 C.sub.6H.sub.5
C.dbd.O .+-. 1.3 .+-. 0.3 0.3 .+-. 0.1 4-F--C.sub.6H.sub.4
C.sub.6H.sub.5 CH--OH .+-. 72 .+-. 16 18 .+-. 2
4-C.sub.6H.sub.5--C.sub.6H.sub.4 C.sub.6H.sub.5 C.dbd.O .+-. 0.14
.+-. 0.01 0.20 .+-. 0.01 4-C.sub.6H.sub.5--C.sub.6H.sub.4
C.sub.6H.sub.5 CH--OH .+-. 0.51 .+-. 0.04 0.72 .+-. 0.03
4-C.sub.6H.sub.5--C.sub.6H.sub.4 C.sub.6H.sub.5 C.dbd.NOH .+-. 42
.+-. 3 35 .+-. 1 4-C.sub.6H.sub.5--C.sub.6H.sub.4 C.sub.6H.sub.5
S.dbd.O .+-. 73 .+-. 5 70 .+-. 3 4-C.sub.6H.sub.5--C.sub.6H.sub.4
C.sub.6H.sub.5 CH--OCH.sub.3 .+-. 0.48 .+-. 0.05 1.36 .+-. 0.07
C.sub.10H.sub.7 C.sub.6H.sub.5 C.dbd.O .+-. 0.49 .+-. 0.02 0.85
.+-. 0.06 4-C.sub.6H.sub.5--C.sub.6H.sub.4
4-CH.sub.3--C.sub.6H.sub.4 C.dbd.O .+-. 0.10 .+-. 0.01 0.19 .+-.
0.03 4-C.sub.6H.sub.5--C.sub.6H.sub.4 4-CH.sub.3--C.sub.6H.sub.4
CH--OH .+-. 0.09 .+-. 0.01 0.15 .+-. 0.02
4-NO.sub.2--C.sub.6H.sub.4 CH.sub.3 C.dbd.O .+-. 163 .+-. 11 269
.+-. 5 4-NO.sub.2--C.sub.6H.sub.4 CH.sub.3 CH--OH .+-. 6.5 .+-. 0.2
39 .+-. 1 C.sub.6H.sub.5 H CH--OH R,R 1100 .+-. 23 C.sub.6H.sub.5 H
CH--OH S,S 2400 .+-. 46 4-NO.sub.2--C.sub.6H.sub- .4 H CH--OH .+-.
5 .+-. 1 4-NO.sub.2--C.sub.6H.sub.4 H CH--OH R,R 1200 .+-. 25
4-NO.sub.2--C.sub.6H.sub.4 H CH--OH S,S 1.6 .+-. 0.6 Abs. Conf.:
Absolute configuration
[0017] The enzymes of interest for this invention typically are
able to distinguish enantiomers. Thus, in choosing an inhibitor for
use for an application in accordance with the invention it is
preferred to screen different optical isomers of the inhibitor with
the selected enzyme by routine assays so as to choose a better
optical isomer, if appropriate, for the particular application. The
pharmacophores described here can be used to deliver a reactive
functionality to the catalytic site. These could include alkylating
agents such as halogens or epoxides or Michael acceptors which will
react with thiols and amines. These reactive functionalities also
can be used to deliver fluorescent or affinity labels to the enzyme
active site for enzyme detection.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIGS. 1 and 2 show that renal microsomal DHET formation is
increased in the SHR relative to the WKY and this is due to
increased renal EET hydrolysis. The NADPH-dependent formation of
11,12-DHET (FIG. 1A), 8,9-DHET (FIG. 1B) and 14,15-DHET (FIG. 1C)
was measured in incubations of WKY (.largecircle.) and SHR
(.smallcircle.) renal microsomes with [.sup.14C]-arachidonic acid
(FIG. 1) or [.sup.14C]-regioisomeric EETs (FIG. 2). Values are the
mean.+-.SE from three to six animals of a given age and strain
(FIG. 1) or means of two separate animals (FIG. 2). Significant
differences between WKY and SHR samples at a given age are
indicated (p<0.05). The hydrolysis of all of the major EETs was
increased in the SHR kidney.
[0019] FIG. 3 shows that sEH expression is increased in the SHR
kidney relative to the WKY kidney and that DHET urinary excretion
is also increased in the SHR. (A) Microsomal proteins from WKY (W)
and SHR (S) renal cortex were separated on a 8% SDS-polyacrylamide
gel, transferred to nitrocellulose, and blotted with antisera
against rat mEH (top panel) or mouse sEH (bottom panel). The age of
the rats is indicated on the top of the blot. (B) Microsomal (top
panel) and cytosolic (bottom panel) proteins from WKY (W) and SHR
(S) cortex, outer medulla and liver were separated and transferred
as described above and blotted with antisera against mouse sEH. A
renal cortex sample from a Sprague-Dawley (SD) rat and a purified
recombinant sEH protein sample are also included on the blot.
Immunoreactive proteins were detected by chemiluminescence. The
blots are representative of the results from three to six
animals/experimental group. (C) Urine was collected for 24 hr from
untreated WKY rats (solid bars) and SHR (hatched bars). DHETs were
extracted from urine and quantified by GC-MS as described in the
Methods section. The values shown are the means.+-.SE of four
animals/strain. Significant differences between WKY and SHR are
indicated (p<0.0005).
[0020] FIG. 4 shows that DCU is a potent and selective inhibitor of
sEH and has antihypertensive effects in the SHR. (A) The formation
of [1-.sup.14C]11,12- (A), 8,9- (B), and 14,15-DHET (C) from
[1-.sup.14C]EETs (50 .mu.M) was measured in SHR renal S9 fractions
in the presence of increasing concentrations of DCU. The values
shown are the average of two samples/concentration, expressed as %
of control. The difference between the individual values was 7-33%.
Control formation rates were 7193 pmol/min/mg protein for
14,15-DHET, 538 pmol/min/mg protein for 11,12-DHET, and 595
pmol/min/mg protein for 8,9-DHET. DCU was a potent and selective
inhibitor of EET hydrolysis in vitro. (B) Urine was collected for
24 hr following treatment of SHR with vehicle (solid bars) or DCU
(hatched bars) daily for 3 days. EETs and DHETs were extracted from
urine and quantified by GC-MS as described in the Methods section.
The values shown are the means.+-.SE of four animals/strain.
Significant differences between vehicle- and DCU-treated SHR are
indicated (p<0.05). DCU was a potent inhibitor of 14,15-EET
hydrolysis in vivo. (C) Male SHR rats were treated with a single 3
mg/kg dose of DCU (.smallcircle.) or vehicle (.largecircle.).
Systolic blood pressure was measured with a photoelectric tail cuff
for up to 96 hr after the dose. The values shown are the mean.+-.SE
from DCU- and vehicle-treated rats (n=5/group). Baseline systolic
blood pressure was 143.+-.3 mm Hg in the SHRs. (D) Male WKY rats
were treated with a single 3 mg/kg dose of DCU (.smallcircle.) or
vehicle (.largecircle.). Systolic blood pressure was measured with
a photoelectric tail cuff for up to 96 hr after the dose. The
values shown are the mean.+-.SE from DCU- and vehicle-treated rats
(n=5/group). Baseline systolic blood pressure was and 118.+-.2 mm
Hg in the WKY rats. Blood pressure decreased an average of 22 mm Hg
in the DCU-treated SHRs 6 hr after the dose (p<0.01) and was
unaffected by DCU in the WKY rats.
[0021] FIG. 5 shows that a structurally related urea inhibitor of
sEH also has antihypertensive effects in the SHR. Male SHRs were
treated with a single dose of vehicle (.smallcircle.) or
N-cyclohexyl-N'-dodecylurea (.smallcircle.) (equimolar to 3 mg/kg
DCU). Systolic blood pressure was measured with a photoelectric
tail cuff for 24 hr after the dose. The values shown are the
mean.+-.SE from inhibitor- and vehicle-treated rats (n=5/group).
Baseline systolic blood pressures were 135.+-.5 mm Hg in the
N-cyclohexyl-N'-dodecylurea group. Blood pressure decreased an
average of 12 mm Hg in the N-cyclohexyl-N'-dodecylurea-treated SHRs
6 hr after the dose (p<0.01).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0022] The present invention is based on the discovery that epoxide
hydrolase activity is associated with hypertension. The
epoxyeicosatrienoic acids (EETs) are regarded as antihypertensive
eicosanoids due to their potent effects on renal vascular tone and
sodium and water transport in the renal tubule. As shown here, EET
activity is regulated by hydrolysis to the corresponding
dihydroxyeicosatrienoic acids by epoxide hydrolase. Inhibition of
EET hydrolysis in vivo with a potent and selective soluble epoxide
hydrolase inhibitor leads to a decrease in blood pressure. Thus,
the present invention provides a new therapeutic approach for the
control of blood pressure.
Abbreviations and Definitions
[0023] The abbreviations used herein have their conventional
meaning within the chemical and biological arts.
[0024] The term "alkyl," by itself or as part of another
substituent, means, unless otherwise stated, a straight or branched
chain, or cyclic hydrocarbon radical, or combination thereof, which
may be fully saturated, mono- or polyunsaturated and can include
di- and multivalent radicals, having the number of carbon atoms
designated (i.e. C.sub.1-C.sub.10 means one to ten carbons).
Examples of saturated hydrocarbon radicals include groups such as
methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl,
sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl,
homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl,
n-octyl, and the like. An unsaturated alkyl group is one having one
or more double bonds or triple bonds. Examples of unsaturated alkyl
groups include vinyl, 2-propenyl, crotyl, 2-isopentenyl,
2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1-
and 3-propynyl, 3-butynyl, and the higher homologs and isomers.
[0025] The term "alkylene" by itself or as part of another
substituent means a divalent radical derived from an alkane, as
exemplified by --CH.sub.2CH.sub.2CH.sub.2CH.sub.2--, and further
includes those groups described below as "heteroalkylene."
Typically, an alkyl (or alkylene) group will have from 1 to 24
carbon atoms, with those groups having 10 or fewer carbon atoms
being preferred in the present invention. A "lower alkyl" or "lower
alkylene" is a shorter chain alkyl or alkylene group, generally
having eight or fewer carbon atoms.
[0026] The terms "cycloalkyl" and "heterocycloalkyl", by themselves
or in combination with other terms, represent, unless otherwise
stated, cyclic versions of "alkyl" and "heteroalkyl", respectively.
Additionally, for heterocycloalkyl, a heteroatom can occupy the
position at which the heterocycle is attached to the remainder of
the molecule. Examples of cycloalkyl include cyclopentyl,
cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the
like. Examples of heterocycloalkyl include
1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,
3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,
tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,
1-piperazinyl, 2-piperazinyl, and the like.
[0027] The term "aryl" means, unless otherwise stated, a
polyunsaturated, typically aromatic, hydrocarbon substituent which
can be a single ring or multiple rings (up to three rings) which
are fused together or linked covalently. The term "heteroaryl"
refers to aryl groups (or rings) that contain from zero to four
heteroatoms selected from N, O, and S, wherein the nitrogen and
sulfur atoms are optionally oxidized, and the nitrogen atom(s) are
optionally quaternized. A heteroaryl group can be attached to the
remainder of the molecule through a heteroatom. Non-limiting
examples of aryl and heteroaryl groups include phenyl, 1-naphthyl,
2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl,
3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl,
4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl,
4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl,
2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl,
4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl,
2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl,
2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl.
Substituents for each of the above noted aryl and heteroaryl ring
systems are selected from the group of acceptable substituents
described below.
[0028] For brevity, the term "aryl" when used in combination with
other terms (e.g., aryloxy, arylthioxy, arylalkyl) includes both
aryl and heteroaryl rings as defined above. Thus, the term
"arylalkyl" is meant to include those radicals in which an aryl
group is attached to an alkyl group (e.g., benzyl, phenethyl,
pyridylmethyl and the like) including those alkyl groups in which a
carbon atom (e.g., a methylene group) has been replaced by, for
example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl,
3-(1-naphthyloxy)propyl, and the like).
[0029] Each of the above terms (e.g., "alkyl," "heteroalkyl,"
"aryl" and "heteroaryl") are meant to include both substituted and
unsubstituted forms of the indicated radical. Preferred
substituents for each type of radical are provided below.
[0030] Substituents for the alkyl and heteroalkyl radicals
(including those groups often referred to as alkylene, alkenyl,
heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl,
heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be a
variety of groups selected from: --OR', .dbd.O, .dbd.NR',
.dbd.N--OR', --NR'R", --SR', -halogen, --SiR'R"R'", --OC(O)R',
--C(O)R', --CO.sub.2R', --CONR'R", --OC(O)NR'R", --NR"C(O)R',
--NR'--C(O)NR"R'", --NR"C(O).sub.2R', --NH--C(NH.sub.2).dbd.NH,
--NR'C(NH.sub.2).dbd.NH, --NH--C(NH.sub.2).dbd.NR', --S(O)R',
--S(O).sub.2R', --S(O).sub.2NR'R", --CN and --NO.sub.2 in a number
ranging from zero to (2m'+1), where m' is the total number of
carbon atoms in such radical. R', R" and R'" each independently
refer to hydrogen, unsubstituted. (C.sub.1-C.sub.8)alkyl and
heteroalkyl, unsubstituted aryl, aryl substituted with 1-3
halogens, unsubstituted alkyl, alkoxy or thioalkoxy groups, or
aryl-(C.sub.1-C.sub.4)alkyl groups. When R' and R" are attached to
the same nitrogen atom, they can be combined with the nitrogen atom
to form a 5-, 6-, or 7-membered ring. For example, --NR'R" is meant
to include 1-pyrrolidinyl and 4-morpholinyl. From the above
discussion of substituents, one of skill in the art will understand
that the term "alkyl" is meant to include groups such as haloalkyl
(e.g., --CF.sub.3 and --CH.sub.2CF.sub.3) and acyl (e.g.,
--C(O)CH.sub.3, --C(O)CF.sub.3, --C(O)CH.sub.2OCH.sub.3, and the
like).
[0031] Similarly, substituents for the aryl and heteroaryl groups
are varied and are selected from: -halogen, --OR', --OC(O)R',
--NR'R", --SR', --R', --CN, --NO.sub.2, --CO.sub.2R', --CONR'R",
--C(O)R', --OC(O)NR'R", --NR"C(O)R', --NR"C(O).sub.2R',
--NR'--C(O)NR"R'", --NH--C(NH.sub.2).dbd.NH, --NR'C(NH.sub.2)'NH,
--NH--C(NH.sub.2).dbd.NR', --S(O)R', --S(O).sub.2R',
--S(O).sub.2NR'R", --N.sub.3, --CH(Ph).sub.2,
perfluoro(C.sub.1-C.sub.4)alkoxy, and
perfluoro(C.sub.1-C.sub.4)alkyl, in a number ranging from zero to
the total number of open valences on the aromatic ring system; and
where R', R" and R'" are independently selected from hydrogen,
(C.sub.1-C.sub.8)alkyl and heteroalkyl, unsubstituted aryl and
heteroaryl, (unsubstituted aryl)-(C.sub.1-C.sub.4)alkyl, and
(unsubstituted aryl)oxy-(C.sub.1-C.sub.4)alkyl.
[0032] Two of the substituents on adjacent atoms of the aryl or
heteroaryl ring may optionally be replaced with a substituent of
the formula -T-C(O)--(CH.sub.2).sub.q--U--, wherein T and U are
independently --NH--, --O--, --CH.sub.2-- or a single bond, and q
is an integer of from 0 to 2. Alternatively, two of the
substituents on adjacent atoms of the aryl or heteroaryl ring may
optionally be replaced with a substituent of the formula
-A-(CH.sub.2).sub.r--B--, wherein A and B are independently
--CH.sub.2--, --O--, --NH--, --S--, --S(O)--, --S(O).sub.2--,
--S(O).sub.2NR'-- or a single bond, and r is an integer of from 1
to 3. One of the single bonds of the new ring so formed may
optionally be replaced with a double bond. Alternatively, two of
the substituents on adjacent atoms of the aryl or heteroaryl ring
may optionally be replaced with a substituent of the formula
--(CH.sub.2).sub.s--X--(CH.sub.2).sub.t- --, where s and t are
independently integers of from 0 to 3, and X is --O--, --NR'--,
--S--, --S(O)--, --S(O).sub.2--, or --S(O).sub.2NR'--. The
substituent R' in --NR'-- and --S(O).sub.2NR'-- is selected from
hydrogen or unsubstituted (C.sub.1-C.sub.6)alkyl.
[0033] The terms "halo" or "halogen," by themselves or as part of
another substituent, mean, unless otherwise stated, a fluorine,
chlorine, bromine, or iodine atom. Additionally, terms such as
"haloalkyl," are meant to include monohaloalkyl and polyhaloalkyl.
For example, the term "halo(C.sub.1-C.sub.4)alkyl" is mean to
include trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl,
3-bromopropyl, and the like.
[0034] As used herein, the term "heteroatom" is meant to include
oxygen (O), nitrogen (N), Boron (B), phosphorous (P) and sulfur
(S).
[0035] The term "pharmaceutically acceptable salts" is meant to
include salts of the active compounds which are prepared with
relatively nontoxic acids or bases, depending on the particular
substituents found on the compounds described herein. When
compounds of the present invention contain relatively acidic
functionalities, base addition salts can be obtained by contacting
the neutral form of such compounds with a sufficient amount of the
desired base, either neat or in a suitable inert solvent. Examples
of pharmaceutically acceptable base addition salts include sodium,
potassium, calcium, ammonium, organic amino, or magnesium salt, or
a similar salt. When compounds of the present invention contain
relatively basic functionalities, acid addition salts can be
obtained by contacting the neutral form of such compounds with a
sufficient amount of the desired acid, either neat or in a suitable
inert solvent. Examples of pharmaceutically acceptable acid
addition salts include those derived from inorganic acids like
hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic,
phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,
monohydrogensulfuric, hydriodic, or phosphorous acids and the like,
as well as the salts derived from relatively nontoxic organic acids
like acetic, propionic, isobutyric, maleic, malonic, benzoic,
succinic, suberic, fumaric, lactic, mandelic, phthalic,
benzenesulfonic, p-tolylsulfonic, citric, tartaric,
methanesulfonic, and the like. Also included are salts of amino
acids such as arginate and the like, and salts of organic acids
like glucuronic or galactunoric acids and the like (see, for
example, Berge, S. M., et al, "Pharmaceutical Salts", Journal of
Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds
of the present invention contain both basic and acidic
functionalities that allow the compounds to be converted into
either base or acid addition salts.
[0036] The neutral forms of the compounds may be regenerated by
contacting the salt with a base or acid and isolating the parent
compound in the conventional manner. The parent form of the
compound differs from the various salt forms in certain physical
properties, such as solubility in polar solvents, but otherwise the
salts are equivalent to the parent form of the compound for the
purposes of the present invention.
[0037] In addition to salt forms, the present invention provides
compounds which are in a prodrug form. Prodrugs of the compounds
described herein are those compounds that readily undergo chemical
changes under physiological conditions to provide the compounds of
the present invention. Additionally, prodrugs can be converted to
the compounds of the present invention by chemical or biochemical
methods in an ex vivo environment. For example, prodrugs can be
slowly converted to the compounds of the present invention when
placed in a transdermal patch reservoir with a suitable enzyme or
chemical reagent.
[0038] Certain compounds of the present invention can exist in
unsolvated forms as well as solvated forms, including hydrated
forms. In general, the solvated forms are equivalent to unsolvated
forms and are intended to be encompassed within the scope of the
present invention. Certain compounds of the present invention may
exist in multiple crystalline or amorphous forms. In general, all
physical forms are equivalent for the uses contemplated by the
present invention and are intended to be within the scope of the
present invention.
[0039] Certain compounds of the present invention possess
asymmetric carbon atoms (optical centers) or double bonds; the
racemates, diastereomers, geometric isomers and individual isomers
are all intended to be encompassed within the scope of the present
invention.
Inhibitors of Epoxide Hydrolases
[0040] As noted above, a preferred class of inhibitors of the
invention are compounds shown by Formulas 1 and 2, above. Means for
preparing such compounds and assaying desired compounds for the
ability to inhibit epoxide hydrolases is described in the parent
application, U.S. Ser. No. 09/252,148. Compounds of Formula 2 are
described in Pat. No. 5,955,496 and in WO98/06261.
[0041] In addition to the compounds in Formula 1 which interact
with the enzyme in a reversible fashion based on the inhibitor
mimicking an enzyme-substrate transition state or reaction
intermediate, one can have compounds that are irreversible
inhibitors of the enzyme. The active structures such as those in
the Tables or Formula 1 can direct the inhibitor to the enzyme
where a reactive functionality in the enzyme catalytic site can
form a covalent bond with the inhibitor. One group of molecules
which could interact like this would have a leaving group such as a
halogen or tosylate which could be attacked in an S.sub.N2 manner
with a lysine or histidine. Alternatively, the reactive
functionality could be an epoxide or Michael acceptor such as a
.alpha./.beta.-unsatura- ted ester, aldehyde, ketone, ester, or
nitrile.
[0042] Further, in addition to the Formula 1 compounds, active
derivatives can be designed for practicing the invention. For
example, dicyclohexyl thio urea can be oxidized to
dicyclohexylcarbodiimide which, with enzyme or aqueous acid
(physiological saline), will form an active dicyclohexylurea.
Alternatively, the acidic protons on carbamates or ureas can be
replaced with a variety of substituents which, upon oxidation,
hydrolysis or attack by a nucleophile such as glutathione, will
yield the corresponding parent structure. These materials are known
as prodrugs or protoxins (Gilman et al., The Pharmacological Basis
of Therapeutics, 7.sup.th Edition, MacMillan Publishing Company,
New York, p. 16 (1985)) Esters, for example, are common prodrugs
which are released to give the corresponding alcohols and acids
enzymatically (Yoshigae et al., Chirality, 9:661-666 (1997)). The
prodrugs can be chiral for greater specificity. These derivatives
have been extensively used in medicinal and agricultural chemistry
to alter the pharmacological properties of the compounds such as
enhancing water solubility, improving formulation chemistry,
altering tissue targeting, altering volume of distribution, and
altering penetration. They also have been used to alter toxicology
profiles.
[0043] There are many prodrugs possible, but replacement of one or
both of the two active hydrogens in the ureas described here or the
single active hydrogen present in carbamates is particularly
attractive. Such derivatives have been extensively described by
Fukuto and associates. These derivatives have been extensively
described and are commonly used in agricultural and medicinal
chemistry to alter the pharmacological properties of the compounds.
(Black et al., Journal of Agricultural and Food Chemistry,
21(5):747-751 (1973); Fahmy et al, Journal of Agricultural and Food
Chemistry, 26(3):550-556 (1978); Jojima et al., Journal of
Agricultural and Food Chemistry, 31(3):613-620 (1983); and Fahmy et
al., Journal of Agricultural and Food Chemistry, 29(3):567-572
(1981).)
[0044] Such active proinhibitor derivatives are within the scope of
the present invention, and the just-cited references are
incorporated herein by reference. Without being bound by theory, it
is believed that suitable inhibitors of the invention mimic the
enzyme transition state so that there is a stable interaction with
the enzyme catalytic site. The inhibitors appear to form hydrogen
bonds with the nucleophilic carboxylic acid and a polarizing
tyrosine of the catalytic site.
[0045] Where the modified activity of the complexed epoxide
hydrolase is enzyme inhibition, then particularly preferred
compound embodiments have an IC.sub.50 (inhibition potency or, by
definition, the concentration of inhibitor which reduces enzyme
activity by 50%) of less than about 500 .mu.M.
[0046] Although the preferred inhibitors of the invention
specifically inhibit the activity of sEH, some inhibitors of the
invention can be used to inhibit the activity of microsomal epoxide
hydrolase (mEH). The micosomal enzyme play a significant role in
the metabolism of xenobiotics such as polyaromatic toxicants.
Additionally, polymorphism studies have underlined a potential role
of this enzyme in relation to several diseases, such as emphysema,
spontaneous abortion and several forms of cancer. Inhibition of
recombinant rat and human mEH can be obtained using primary ureas,
amides, and amines. For example, elaidamide, has a K.sub.i of 70 nM
for recombinant rat mEH. This compound interacts with the enzyme
forming a non-covalent complex, and blocks substrate turnover
through an apparent mix of competitive and non-competitive
inhibition kinetics. Furthermore, in insect cell culture expressing
rat mEH, elaidamide enhances the toxicity effects of
epoxide-containing xenobiotics.
Assays for Epoxide Hydrolase Activity
[0047] The invention also provide methods for assaying for epoxide
hydrolase activity as diagnostic assay to identify individuals at
increased risk for hypertension and/or those that would benefit
from the therapeutic methods of the invention. Any of a number of
standard assays for determining epoxide hydrolase activity can be
used. For example, suitable assays are described in Gill, et al.,
Anal Biochem 131, 273-282 (1983); and Borhan, et al., Analytical
Biochemistry 231, 188-200 (1995)). Suitable in vitro assays are
described in Zeldin et al. J Biol. Chem. 268:6402-6407 (1993).
Suitable in vivo assays are described in Zeldin et al. Arch Biochem
Biophys 330:87-96 (1996). Assays for epoxide hydrolase using both
putative natural substrates and surrogate substrates have been
reviewed (see, Hammock, et al. In: Methods in Enzymology, Volume
III, Steroids and Isoprenoids, Part B, (Law, J. H. and H. C.
Rilling, eds. 1985), Academic Press, Orlando, Fla., pp. 303-311 and
Wixtrom et al., In: Biochemical Pharmacology and Toxicology, Vol.
1: Methodological Aspects of Drug Metabolizing Enzymes, (Zakim, D.
and D. A. Vessey, eds. 1985), John Wiley & Sons, Inc., New
York, pp. 1-93. Several spectral based assays exist based on the
reactivity or tendency of the resulting diol product to hydrogen
bond (see, e.g., Wixtrom, and Hammock. Anal. Biochem. 174:291-299
(1985) and Dietze, et al. Anal. Biochem. 216:176-187 (1994)).
[0048] The enzyme also can be detected based on the binding of
specific ligands to the catalytic site which either immobilize the
enzyme or label it with a probe such as luciferase, green
fluorescent protein or other reagent. For the data in this
disclosure the enzyme was assayed by its hydration of EETs, its
hydrolysis of an epoxide to give a colored product as described by
Dietze et al. (1994) or its hydrolysis of a radioactive surrogate
substrate (Borhan et al., 1995)
[0049] The assays of the invention are carried out using an
appropriate sample from the patient. Typically, such a sample is a
blood sample.
Other Means of Inhibiting EH Activity
[0050] Other means of inhibiting EH activity or gene expression can
also be used. For example, a nucleic acid molecule complementary to
at least a portion of the human EH gene can be used to,inhibit EH
gene expression. Means for inhibiting gene expression using, for
example, antisense molecules, ribozymes, and the like are well
known to those of skill in the art. The nucleic acid molecule can
be a DNA probe, a riboprobe, a peptide nucleic acid probe, a
phosphorothioate probe, or a 2'-O methyl probe.
[0051] Generally, to assure specific hybridization, the antisense
sequence is substantially complementary to the target sequence. In
certain embodiments, the antisense sequence is exactly
complementary to the target sequence. The antisense polynucleotides
may also include, however, nucleotide substitutions, additions,
deletions, transitions, transpositions, or modifications, or other
nucleic acid sequences or non-nucleic acid moieties so long as
specific binding to the relevant target sequence corresponding to
the EH gene is retained as a functional property of the
polynucleotide. As one embodiment of the antisense molecules form a
triple helix-containing, or "triplex" nucleic acid. Triple helix
formation results in inhibition of gene expression by, for example,
preventing transcription of the target gene (see, e.g., Cheng et
al., 1988, J. Biol. Chem. 263:15110; Ferrin and Camerini-Otero,
1991, Science 354:1494; Ramdas et al., 1989, J. Biol. Chem.
264:17395; Strobel et al., 1991, Science 254:1639; and Rigas et
al., 1986, Proc. Natl. Acad. Sci. U.S.A. 83:9591)
[0052] In another embodiment, ribozymes can be used (see, e.g.,
Cech, 1995, Biotechnology 13:323; and Edgington, 1992,
Biotechnology 10:256 and Hu et al., PCT Publication WO
94/03596).
[0053] The antisense nucleic acids (DNA, RNA, modified, analogues,
and the like) can be made using any suitable method for producing a
nucleic acid, such as the chemical synthesis and recombinant
methods disclosed herein and known to one of skill in the art. In
one embodiment, for example, antisense RNA molecules of the
invention may be prepared by de novo chemical synthesis or by
cloning. For example, an antisense RNA can be made by inserting
(ligating) an EH gene sequence in reverse orientation operably
linked to a promoter in a vector (e.g., plasmid). Provided that the
promoter and, preferably termination and polyadenylation signals,
are properly positioned, the strand of the inserted sequence
corresponding to the noncoding strand will be transcribed and act
as an antisense oligonucleotide of the invention.
[0054] It will be appreciated that the oligonucleotides can be made
using nonstandard bases (e.g., other than adenine, cytidine,
guanine, thymine, and uridine) or nonstandard backbone structures
to provides desirable properties (e.g., increased
nuclease-resistance, tighter-binding, stability or a desired
T.sub.m). Techniques for rendering oligonucleotides
nuclease-resistant include those described in PCT Publication WO
94/12633. A wide variety of useful modified oligonucleotides may be
produced, including oligonucleotides having a peptide-nucleic acid
(PNA) backbone (Nielsen et al., 1991, Science 254:1497) or
incorporating 2'-O-methyl ribonucleotides, phosphorothioate
nucleotides, methyl phosphonate nucleotides, phosphotriester
nucleotides, phosphorothioate nucleotides, phosphoramidates.
[0055] Proteins have been described that have the ability to
translocate desired nucleic acids across a cell membrane.
Typically, such proteins have amphiphilic or hydrophobic
subsequences that have the ability to act as membrane-translocating
carriers. For example, homeodomain proteins have the ability to
translocate across cell membranes. The shortest internalizable
peptide of a homeodomain protein, Antennapedia, was found to be the
third helix of the protein, from amino acid position 43 to 58 (see,
e.g., Prochiantz, 1996, Current Opinion in Neurobiology 6:629-634.
Another subsequence, the h (hydrophobic) domain of signal peptides,
was found to have similar cell membrane translocation
characteristics (see, e.g., Lin et al., 1995, J. Biol. Chem.
270:14255-14258). Such subsequences can be used to translocate
oligonucleotides across a cell membrane. Oligonucleotides can be
conveniently derivatized with such sequences. For example, a linker
can be used to link the oligonucleotides and the translocation
sequence. Any suitable linker can be used, e.g., a peptide linker
or any other suitable chemical linker.
Therapeutic Administration
[0056] The compounds of the present invention can be prepared and
administered in a wide variety of oral, parenteral and topical
dosage forms. Thus, the compounds of the present invention can be
administered by injection, that is, intravenously, intramuscularly,
intracutaneously, subcutaneously, intraduodenally, or
intraperitoneally. Also, the compounds described herein can be
administered by inhalation, for example, intranasally.
Additionally, the compounds of the present invention can be
administered transdermally. Accordingly, the present invention also
provides pharmaceutical compositions comprising a pharmaceutically
acceptable carrier or excipient and either a compound of the
invention or a pharmaceutically acceptable salt of the
compound.
[0057] For preparing pharmaceutical compositions from the compounds
of the present invention, pharmaceutically acceptable carriers can
be either solid or liquid. Solid form preparations include powders,
tablets, pills, capsules, cachets, suppositories, and dispersible
granules. A solid carrier can be one or more substances which may
also act as diluents, flavoring agents, binders, preservatives,
tablet disintegrating agents, or an encapsulating material.
[0058] In powders, the carrier is a finely divided solid which is
in a mixture with the finely *o divided active component. In
tablets, the active component is mixed with the carrier having the
necessary binding properties in suitable proportions and compacted
in the shape and size desired.
[0059] The powders and tablets preferably contain from 5% or 10% to
70% of the active compound. Suitable carriers are magnesium
carbonate, magnesium stearate, talc, sugar, lactose, pectin,
dextrin, starch, gelatin, tragacanth, methylcellulose, sodium
carboxymethylcellulose, a low melting wax, cocoa butter, and the
like. The term "preparation" is intended to include the formulation
of the active compound with encapsulating material as a carrier
providing a capsule in which the active component with or without
other carriers, is surrounded by a carrier, which is thus in
association with it. Similarly, cachets and lozenges are included.
Tablets, powders, capsules, pills, cachets, and lozenges can be
used as solid dosage forms suitable for oral administration.
[0060] For preparing suppositories, a low melting wax, such as a
mixture of fatty acid glycerides or cocoa butter, is first melted
and the active component is dispersed homogeneously therein, as by
stirring. The molten homogeneous mixture is then poured into
convenient sized molds, allowed to cool, and thereby to
solidify.
[0061] Liquid form preparations include solutions, suspensions, and
emulsions, for example, water or water/propylene glycol solutions.
For parenteral injection, liquid preparations can be formulated in
solution in aqueous polyethylene glycol solution.
[0062] Aqueous solutions suitable for oral use can be prepared by
dissolving the active component in water and adding suitable
colorants, flavors, stabilizers, and thickening agents as desired.
Aqueous suspensions suitable for oral use can be made by dispersing
the finely divided active component in water with viscous material,
such as natural or synthetic gums, resins, methylcellulose, sodium
carboxymethylcellulose, and other well-known suspending agents.
[0063] Also included are solid form preparations which are intended
to be converted, shortly before use, to liquid form preparations
for oral administration. Such liquid forms include solutions,
suspensions, and emulsions. These preparations may contain, in
addition to the active component, colorants, flavors, stabilizers,
buffers, artificial and natural sweeteners, dispersants,
thickeners, solubilizing agents, and the like.
[0064] The pharmaceutical preparation is preferably in unit dosage
form. In such form the preparation is subdivided into unit doses
containing appropriate quantities of the active component. The unit
dosage form can be a packaged preparation, the package containing
discrete quantities of preparation, such as packeted tablets,
capsules, and powders in vials or ampoules. Also, the unit dosage
form can be a capsule, tablet, cachet, or lozenge itself; or it can
be the appropriate number of any of these in packaged form.
[0065] The term "unit dosage form", as used in the specification,
refers to physically discrete units suitable as unitary dosages for
human subjects and animals, each unit containing a predetermined
quantity of active material calculated to produce the desired
pharmaceutical effect in association with the required
pharmaceutical diluent, carrier or vehicle. The specifications for
the novel unit dosage forms of this invention are dictated by and
directly dependent on (a) the unique characteristics of the active
material and the particular effect to be achieved and (b) the
limitations inherent in the art of compounding such an active
material for use in humans and animals, as disclosed in detail in
this specification, these being features of the present
invention.
[0066] A therapeutically effective amount of the compounds of the
invention is employed in treatment. The dosage of the specific
compound for treatment depends on many factors that are well known
to those skilled in the art. They include for example, the route of
administration and the potency of the particular compound. An
exemplary dose is from about 0.001 .mu.M/kg to about 100 mg/kg body
weight of the mammal. Without further elaboration, it is believed
that one skilled in the art can, using the preceding description,
practice the present invention to its fullest extent. The following
detailed examples describe how to prepare the various compounds
and/or perform the various processes of the invention and are to be
construed as merely illustrative, and not limitations of the
preceding disclosure in any way whatsoever. Those skilled in the
art will promptly recognize appropriate variations from the
procedures both as to reactants and as to reaction conditions and
techniques.
EXAMPLE
[0067] This example shows that inhibitors of epoxide hydrolase are
effective in decreasing blood pressure in mammals.
Methods
[0068] Animals. Male SHR and WKY rats 3-13 wks of age were
purchased from Charles River Laboratories (Wilmington, Mass.) and
housed in a controlled environment with 12 hr light/dark cycles and
fed standard laboratory chow for .quadrature. 3 days before
euthanasia. All animal use was approved by the University of
California San Francisco Committee on Animal Research and followed
the National Institutes of Health guidenlines for the care and use
of laboratory animals. For isolation of kidney subcellular
fractions, rats were anesthetized with methoxyflurane, the
abdominal cavities were opened, and the kidneys were perfused with
ice-cold saline. Perfused kidneys were rapidly removed, the cortex
and medulla dissected out and immersed in liquid nitrogen. All
tissue was stored at -80.degree. C. until preparation of
microsomes. In some cases WKY and SHR rats were housed in metabolic
cages for up to three days and urine was collected over
triphenylphosphine in 24 hr intervals. The urine volume was noted
and aliquots were stored at -80.degree. C. prior to extraction and
quantitation of DHETs and EETs. For the sEH inhibition studies,
groups of 8 wk old male SHRs and WKY rats were treated daily for
1-4 days with a 3 mg/kg i.p. dose of N,N'-dicyclohexylurea (DCU) in
a 1.5:1 mixture of corn oil and DMSO. Systolic blood pressure was
measured at room temperature by a photoelectric tail cuff system
(Model 179, IITC, Inc., Woodland Hills, Calif.) for up to four days
following the dose of inhibitor. Blood pressures are reported as
the average of three separate readings over a 30 min period. Urine
was collected for 24 hr immediately following a dose of DCU or
vehicle for quantification of DHET and EET excretion. Similar
inhibition studies were carried out with equimolar doses of
N-cyclohexyl-N'-dodecylurea, N-cyclohexyl-N'-ethyl urea and
dodecylamine.
[0069] Renal microsomal arachidonic acid metabolism. Microsomes,
cytosol, and S9 fractions were prepared from the renal cortex or
outer medulla samples from a single animal as described previously
(Kroetz, D. L. et al. Mol Pharmacol 52, 362-372 (1997), Su, P. et
al. Am J Physiol 275, R426-438 (1998)). Reaction conditions for the
in vitro determination of arachidonic acid epoxygenase activity,
metabolite extraction and HPLC analysis were described in detail
elsewhere (Su, P. et al. Am J Physiol 275, R426-438 (1998)).
[0070] Western immunoblotting. Renal and hepatic microsomes and
cytosol (4 to 10 .quadrature.g) were separated on a 8% sodium
dodecyl sulfate-polyacrylamide gel and transferred to
nitrocellulose in 25 mM Tris/192 mM glycine/20% methanol using a
semidry transfer system (BioRad, Hercules, Calif.). The primary
antibody used in these studies was a rabbit anti-mouse sEH antisera
(Silva, M. H. et al. Comp. Biochem Physiol B: Comp Biochem 87,
95-102 (1987)). Western blots were incubated with a 1:2000 fold
dilution of the primary antibody followed by a 1:2000-fold dilution
of horseradish peroxidase-conjugated goat anti-rabbit IgG.
Immunoreactive proteins were visualized using an ECL detection kit
(Amersham Life Science, Arlington Heights, Ill.).
[0071] EET hydrolysis. Racemic [1-14C]EETs were synthesized and
purified according to published methods from [1-14C]arachidonic
acid (56-57 .mu.Ci/.mu.mole) by nonselective epoxidation (Falck, J.
R. et al., Meth Enzymol 187, 357-364 (1990)). Hydrolysis of
[1-14C]EETs was measured in WKY and SHR renal S9 fractions at
37.degree. C. as described previously (Zeldin, D. C. et al. J Biol
Chem 268, 6402-6407 (1993)). The reaction mixture consisted of 50
.quadrature.M EET (0.045-0.09 .quadrature.Ci) and 1 mg/ml S9
protein (0.5 mg/ml SHR S9 protein for 14,15-EET hydrolysis) in 150
mM KCl, 10 mM MgCl2, 50 mM potassium phosphate buffer pH 7.4.
Reactions were carried out for 40 min (10 min for 14,15-EET
hydrolysis in SHR samples) and the reaction products were extracted
into ethyl acetate, evaporated under a blanket of nitrogen and
detected by reverse phase HPLC with radiometric detection as
described for the arachidonic acid incubations.
[0072] DHET urinary excretion. Urinary creatinine concentrations
were measured by the Medical Center Clinical Laboratories at the
University of California San Francisco. Methods used to quantify
endogenous EETs and DHETs present in rat urine were similar to
those described by Capdevila et al. (Capdevila, J. H. et al. J Biol
Chem 267, 21720-21726 (1992)). DHET and [1-14C]DHET internal
standards were prepared by chemical hydration of EETs and
[1-14C]EETs as described (Zeldin, D. C. et al. J Biol Chem 268,
6402-6407 (1993)). All synthetic EETs and DHETs were purified by
reverse-phase HPLC. EET quantifications were made by GC/MS analysis
of their pentafluorobenzyl (PFB) esters with selected ion
monitoring at m/z 319 (loss of PFB from endogenous EET-PFB) and m/z
321 (loss of PFB from [1-14C]EET-PFB internal standard). The
EET-PFB/[1-14C]EET-PFB ratios were calculated from the integrated
values of the corresponding ion current intensities.
Quantifications of DHETs were made from GC/MS analysis of their PFB
esters, trimethylsilyl (TMS) ethers with selected ion monitoring at
m/z 481 (loss of PFB from endogenous DHET-PFB-TMS) and m/z 483
(loss of PFB from [1-14C]DHET-PFB-TMS internal standard). The
DHET-PFB-TMS/[1-14C]DHET-PFB-- TMS ratios were calculated from the
integrated values of the corresponding ion current intensities.
Data were normalized for kidney function by expressing per mg
creatinine. Control studies demonstrated that under the conditions
used, artifactual EET or DHET formation was negligible.
[0073] Other enzyme assays. Activities of microsomal and soluble EH
were determined in liver and kidney samples according to previously
published protocols (Gill, S. S. et al., Anal Biochem 131, 273-282
(1983); Borhan, B., et al., Anal Biochem 231, 188-200 (1995)).
Inhibition of recombinant soluble EH by DCU was described recently
(Morisseau, C., et al. Proc. Natl. Acad. Sci. USA 96, 8849-8854,
(1999)). Epoxide hydrolase activities are reported as the transdiol
formation rates.
[0074] Statistics. Statistical significance of differences between
mean values was evaluated by a one-way analysis of variance or a
Student's t-test. Significance was set at a p value of
<0.05.
Results
[0075] The spontaneously hypertensive rat (SHR) is a well accepted
experimental model of essential hypertension and was used in the
present study to characterize the contribution of EET hydrolysis to
the elevated blood pressure in these animals. Renal transplantation
studies support a role for the kidneys in the development of
hypertension in the SHR and altered renal function is essential for
the development and maintenance of elevated blood pressure
(Bianchi, G. et al., Clin Sci Mol Med 47, 435-448 (1974); Cowley,
A. W. et al., JAMA 275, 1581-1589 (1996)). Arachidonic acid
metabolism was measured in renal cortical microsomes of SHR and WKY
rats and a dramatic increase in DHET formation was observed in the
SHR relative to the WKY samples (FIG. 1). The formation of 11,12-
and 8,9-DHET was measurable in both strains and was 2- to 8-fold
higher in the SHR relative to the WKY rat throughout their
development (FIGS. 1A and 1B). Interestingly, 14,15-DHET formation
was readily detected in the 3-13 wk old SHR kidneys but could not
be measured in the majority of the WKY samples (FIG. 1C). In the
several instances where 14,15-DHET formation was detectable in the
WKY kidneys it was never greater than 17% of the corresponding
value in SHR. Calculation of the percentage of EETs that were
converted to the corresponding DHETs revealed a large discrepancy
between the WKY and SHR strains. In the WKY renal microsomes
32.+-.3.1% of the EETs were converted to DHETs, while the DHET
recovery was 66.+-.2.1% in the SHR renal microsomes
(p<0.00001).
[0076] A dramatic increase in DHET formation in incubations of
arachidonic acid with SHR renal cortical microsomes relative to the
WKY samples (FIG. 1) led us to hypothesize that EET hydrolysis may
be altered in this experimental model of hypertension and that
epoxide hydrolase activity may be an important determinant of blood
pressure regulation. Two major EH isoforms, a microsomal (mEH) and
soluble (sEH) form are expressed in most tissues and species
(Vogel-Bindel, U. et al., Eur J Biochem 126, 425-431 (1982); Kaur,
S. et al., Drug Metab Disp 13, 711-715 (1985)). EET hydrolysis
rates in cytosol and microsomes and the regioisomeric product
distribution in urine relative to recombinant EH proteins are
consistent with the majority of EET hydrolysis being catalyzed by
sEH (Zeldin, D. C. et al., J Biol Chem 268, 6402-6407, (1993)).
Direct hydrolysis of the regioisomeric EETs was measured in S9
fractions (containing both the soluble and microsomal forms of EH)
from WKY and SHR renal cortex. There was measurable hydrolysis of
8,9-, 11,12- and 14,15-EET in S9 fractions from both WKY and SHR
kidneys (FIG. 2), with a significant increase in hydrolysis in the
SHR relative to the WKY. For example, 8,9- and 11,12-EET hydrolysis
rates were 5- to 15-fold higher in the SHR compared to the WKY and
14,15-EET hydrolysis was as much as 54-fold higher in the SHR.
These data also showed a distinct preference of sEH for the
14,15-EET regioisomer. In the SHR kidney hydrolysis of 14,15-EET
was 10-fold higher than that of 8,9- and 11,12-EET.
[0077] To investigate the possibility that altered EH expression is
responsible for the differences in EET hydrolysis in the SHR and
WKY rat renal microsomes and S9 fractions, we measured EH protein
levels in these samples. mEH was abundantly expressed in the renal
microsomes at relatively constant levels throughout development and
there was no evidence of altered expression of mEH in the SHR
kidney (FIG. 3A). The soluble EH isoform was also easily detected
in SHR cortical microsomes but not in the corresponding WKY samples
(FIG. 3A). Quantitation of the immunoreactive protein bands
indicated that levels of sEH protein in the SHR microsomes were 6-
to 90-fold higher than the corresponding levels in the WKY
microsomes. The high levels of expression of sEH in the SHR
microsomes were limited to the renal cortex (FIG. 3B).
Immunodetectable sEH was barely detectable in SHR outer medulla and
liver microsomes by Western blot. Relatively high levels of sEH
were detected in SHR cortex, outer medulla and liver cytosol (FIG.
3B). In the WKY rats, the level of sEH protein was uniformly low in
both microsomes and cytosol from the kidney and liver. Importantly,
sEH protein in the normotensive Sprague-Dawley rat kidney was also
barely detectable. Increased sEH expression in SHR vs. WKY rats
provides an explanation for the increased EET hydrolysis in the SHR
kidney and the absence or very low levels of 14,15-EET hydrolysis,
the preferred sEH substrate (Zeldin, D. C., et al., J Biol Chem
268, 6402-6407 (1993)), in the WKY kidney.
[0078] Increased sEH activity in the SHR kidney was independently
confirmed using the sEH substrate trans-1,3-diphenylpropene oxide
(tDPPO). There was a 26-fold increase in tDPPO hydrolysis in the
SHR cortical cytosol relative to that of the WKY rat cortical
cytosol (Table 2). The corresponding difference in the microsomal
fraction was 32-fold. Hydrolysis of tDPPO was also significantly
higher in SHR vs. WKY rat liver microsomes and cytosol. Consistent
with the Western blots, sEH activity was easily detectable in the
SHR microsomes and very low in the WKY cytosol and microsomes from
kidney. In contrast, mEH activity, as measured by cSO hydrolysis,
was similar in WKY and SHR cortex and liver (Table 2).
[0079] Urinary excretion of DHETs was measured to evaluate whether
increased sEH expression and EET hydrolysis in the SHR was also
apparent in vivo. Urine was collected in untreated 4 and 8 wk old
SHR and WKY rats and their DHET excretion rates are shown in FIG.
3C. The excretion rates were similar for the 4 and 8 wk animals and
the reported numbers are averages from all samples of a given
strain. The excretion of 14,15-DHET was 2.6-fold higher in the SHR
relative to the WKY rat, consistent with the increased EET
hydrolysis and sEH expression in SHR kidney. In contrast, the 8,9-
and 11,12-DHET urinary excretion in the SHR and WKY rats were
comparable.
[0080] A tight binding sEH specific inhibitor, dicyclohexylurea
(DCU) (Morisseau, C. et al., Proc Natl Acad Sci USA 96, 8849-8854
(1999)), was used to reduce sEH activity in vivo and to determine
the effect of decreased EET hydrolysis on blood pressure.
Inhibition of EET hydrolysis by DCU was confirmed in incubations of
renal S9 fractions with the regioisomeric EETs (FIG. 4A). A
dose-dependent inhibition of EET hydrolysis by DCU was apparent for
all three regioisomers. DCU had the most significant effect on the
hydrolysis of 8,9-EET, inhibiting this reaction with an IC50 of
0.086.+-.0.014 .quadrature.M. The corresponding IC50 values for
inhibition of 11,12- and 14,15-EET hydrolysis were 0.54.+-.0.08
.quadrature.M and 0.45.+-.0.16 .quadrature.M, respectively. At
concentrations up to 25 .quadrature.M, DCU had no effect on CYP
epoxygenase or .quadrature.-hydroxylase activity and previous
studies from our laboratory have shown that DCU does not inhibit
mEH (Morisseau, C. et al., Proc Natl Acad Sci USA 96, 8849-8854
(1999)). The potent inhibition of sEH by DCU was confirmed with
purified recombinant rat sEH. DCU inhibited sEH-catalyzed tDPPO
hydrolysis with a Ki of 34 nM. This is comparable to the Ki values
for DCU with human (30 nM) and murine (26 nM) sEH (Morisseau, C. et
al., Proc Natl Acad Sci USA 96, 8849-8854 (1999)).
[0081] DCU was administered to eight wk old SHRs daily for four
days and urinary DHET excretion was measured during the 24 hr
period immediately following the third dose. The dose of DCU was
based on in vitro estimates of inhibitory potency and previous
studies in the mouse (Morisseau, C. et al., Proc Natl Acad Sci USA
96, 8849-8854 (1999). In the DCU-treated rats there was a
significant 65% decrease in 14,15-DHET urinary excretion and a
corresponding 30% increase in 14,15-EET urinary excretion relative
to vehicle-treated controls (FIG. 4B), consistent with DCU-mediated
inhibition of sEH in vivo. The excretion of total
epoxygenase-derived products (EETs and DHETs) was decreased from
2020 pg/mg creatinine in the vehicle-treated animals to 1237 pg/mg
creatinine in the DCU-treated rats (p<0.05). This inhibition of
14,15-DHET excretion was accompanied by a significant decrease in
blood pressure measured in conscious animals three to five hr after
the fourth dose. Systolic blood pressure decreased from 128.+-.5 mm
Hg in the vehicle-treated rats to 102.+-.5 mm Hg (p<0.01) in the
DCU-treated animals.
[0082] A study of the time course of the effect of a single dose of
DCU (3 mg/kg) demonstrated that the antihypertensive effect in the
SHR was acute (FIG. 4C). Blood pressure was decreased 22.+-.4 mm Hg
6 hr after DCU treatment (p<0.01) and returned to baseline
levels by 24 hr after the dose. Importantly, there was no effect of
DCU on blood pressure in the WKY (FIG. 4D). This is consistent with
the very low levels of sEH protein in the WKY kidney. Several
additional structurally related inhibitors were also studied in the
SHR. N-cyclohexyl-N'-dodecylurea is a sEH inhibitor with similar
potency to DCU (IC50 with mouse sEH=0.05.+-.0.01 compared to
0.09.+-.0.01 .quadrature.M for DCU; unpublished data, C. Morisseau
and B. Hammock, 2000). A single dose of N-cyclohexyl-N'-dodecyl-
urea significantly decreased systolic blood pressure 12.+-.2 mm Hg
6 hr after the dose, and similar to DCU, blood pressure returned to
normal by 24 hours after the dose (FIG. 5). The
N-cyclohexyl-N'-ethylurea analog is a weak sEH inhibitor (IC50 with
mouse sEH=51.7.+-.0.7 .quadrature.M; unpublished data, C. Morisseau
and B. Hammock, 2000) and had no effect on blood pressure in the
SHR. Likewise, the selective mEH inhibitor dodecylamine also had no
effect on blood pressure. Collectively, these data suggest that the
effect of DCU and N-cyclohexyl-N'-dodecylurea on blood pressure is
related to their ability to inhibit sEH and EET hydrolysis in
vivo.
Discussion
[0083] The EET eicosanoids are recognized as important mediators of
vascular tone and renal tubular sodium and water transport (Makita,
K. et al., FASEB J 10, 1456-1463 (1996)). These data provide
substantial evidence in support of the protective role of the EETs.
The potential protective effects of increased EET formation in the
SHR kidney are attenuated by an even greater increase in FET
hydrolysis. Increased expression of sEH in the SHR kidney results
in increased EET hydrolysis in vitro and in vivo and therefore
lower levels of the antihypertensive EETs. In contrast, inhibition
of EET hydrolysis in vivo is associated with elevated EET levels
and a reduction in blood pressure. Importantly, this provides a
common link between the pathophysiological regulation of blood
pressure in rats and humans (Catella, F. et al., Proc Natl Acad Sci
USA 87, 5893-5897 (1990)). The 8,9-DHET regioisomer is easily
detected in the urine of healthy women while excretion of 14,15-
and 11,12-DHET is minimal in this population. For all three isomers
DHET excretion is increased during a normal pregnancy and during
pregnancy-induced hypertension 14,15- and 11,12-DHET excretion
increased even further. This was most dramatic for 14,15-EET, the
preferred substrate for sEH (Zeldin, D. G. et al., J Biol Chem 268,
6402-6407 (1993)). The excretion of 14,15-DHET increased from a
median of 85 pg/mg of creatinine during normal pregnancy to 2781
pg/mg of creatinine in women with pregnancy-induced hypertension.
Pharmacokinetic evidence is consistent with a renal origin of the
urinary DHETs (Catella, F. et al., Proc Natl Acad Sci USA 87,
5893-5897 (1990)) so altered EET and DHET levels could potentially,
affect tubular ion transport and/or renal vascular tone. The
present results in the SHR support the possibility that sEH
expression is altered in women with pregnancy induced
hypertension.
[0084] Inhibition of EET hydrolysis is a new therapeutic approach
to regulating renal eicosanoid formation. Recently, inhibition of
arachidonic acid c-hydroxylase activity with a mechanism-based CYP
inhibitor has been shown to effectively lower blood pressure in the
SHR (Su, P. et al., Am J Physiol 275, R426-438 (1998)). The
approach of inhibition of EET hydrolysis in the present study
produces a significantly greater decrease in blood pressure than
the CYP inhibition strategy. The possibility exists of a
synergistic effect of CYP4A inhibition resulting in decreased
levels of the prohypertensive 20-HETE eicosanoid and sEH inhibition
leading to increased levels of the antihypertensive EETs. Parallel
inhibition of related enzymes is a limitation of CYP epoxygenase
and .omega.-hydroxylase inhibition but is of little concern with
sEH inhibition. These findings make it of interest to fully
characterize the impact of sEH inhibition on renal tubular ion
transport, vascular tone and blood pressure. The possibility of
similar changes in sEH activity in human hypertensive populations
is compelling. Identification of individuals with elevated sEH
activity may prove useful in designing the most effective
antihypertensive therapy.
[0085] It is to be understood that while the invention has been
described above in conjunction with preferred specific embodiments,
the description and examples are intended to illustrate and not
limit the scope of the invention, which is defined by the scope of
the appended claims. All publications, sequences referred to in
GenBank accession numbers, patents, and patent applications cited
herein are hereby incorporated by reference for all purposes.
* * * * *