U.S. patent application number 12/360501 was filed with the patent office on 2009-05-21 for treatment for cardiovascular disease.
Invention is credited to Richard J. Johnson, Salah Kivlighn, Marilda Mazzali.
Application Number | 20090130078 12/360501 |
Document ID | / |
Family ID | 22800556 |
Filed Date | 2009-05-21 |
United States Patent
Application |
20090130078 |
Kind Code |
A1 |
Kivlighn; Salah ; et
al. |
May 21, 2009 |
Treatment for Cardiovascular Disease
Abstract
This invention relates to a method for treating and preventing
hypertension by administering a therapeutically effective amount of
an agent capable of reducing uric acid levels in a patient in need
of such treatment. Additionally, the scope of the invention
includes a method of treating coronary heart disease by
administering a therapeutically effective amount of an agent
capable of reducing uric acid levels in a patient in need of such
treatment.
Inventors: |
Kivlighn; Salah;
(Doylestown, PA) ; Johnson; Richard J.; (Bellaire,
TX) ; Mazzali; Marilda; (Houston, TX) |
Correspondence
Address: |
McDERMOTT, WILL & EMERY
600 13th Street, N.W.
Washington
DC
20005-3096
US
|
Family ID: |
22800556 |
Appl. No.: |
12/360501 |
Filed: |
January 27, 2009 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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11696330 |
Apr 4, 2007 |
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12360501 |
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09892505 |
Jun 28, 2001 |
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11696330 |
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60214825 |
Jun 28, 2000 |
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Current U.S.
Class: |
424/94.1 ;
514/44R |
Current CPC
Class: |
A61K 31/343 20130101;
A61K 31/4152 20130101; A61P 9/12 20180101; A61K 31/513 20130101;
A61K 31/195 20130101; A61K 31/403 20130101; A61K 45/06 20130101;
A61K 31/381 20130101; A61P 43/00 20180101; A61K 47/60 20170801;
A61P 9/10 20180101; A61K 31/4178 20130101; A61K 38/13 20130101;
A61P 25/08 20180101; A61K 31/505 20130101; A61P 19/06 20180101;
A61K 31/519 20130101; A61K 31/401 20130101; A61K 31/423 20130101;
A61K 31/405 20130101; A61K 31/403 20130101; A61K 2300/00 20130101;
A61K 31/513 20130101; A61K 2300/00 20130101; A61K 31/519 20130101;
A61K 2300/00 20130101; A61K 38/13 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
424/94.1 ;
514/44 |
International
Class: |
A61K 38/43 20060101
A61K038/43; A61K 31/7088 20060101 A61K031/7088 |
Goverment Interests
[0002] The U.S. Government has a paid-up license in this invention
and a right in limited circumstances to require the patent owner to
license others on reasonable terms as provided for by the terms of
National Institute of Health Grant No. DK 47659.
Claims
1. A method of treating hypertension comprising administering a
therapeutically effective amount of an agent, or pharmaceutically
acceptable salt thereof, capable of reducing uric acid levels in a
patient in need of such treatment.
2. A method of preventing hypertension comprising administering a
therapeutically effective amount of an agent, or pharmaceutically
acceptable salt thereof, capable of reducing uric acid levels in a
patient in need of such treatment.
3. A method of treating coronary heart disease comprising
administering a therapeutically effective amount of an agent, or
pharmaceutically acceptable salt thereof, capable of reducing uric
acid levels in a patient in need of such treatment.
4. A method of treating and preventing eclampsia comprising
administering a therapeutically effective amount of an agent, or
pharmaceutically acceptable salt thereof, capable of reducing uric
acid levels in a patient in need of such treatment.
5. An agent capable of reducing uric acid levels selected from the
group consisting of: gene therapy, a xanthine oxidase inhibitor; a
uricosuric agent; supplements of the uricase protein and a urate
channel inhibitor, or pharmaceutically acceptable salts, or
combinations therefrom.
6. The agent capable of reducing uric acid levels, as recited in
claim 5, which is gene therapy that targets the overexpression of
uricase.
7. The agent capable of reducing uric acid levels, as recited in
claim 5, which is a xanthine oxidase inhibitor selected from the
group consisting of: allopurinol, and carprofen, or
pharmaceutically acceptable salt thereof.
8. The agent capable of reducing uric acid levels, as recited in
claim 5, which is a uricosuric agent selected from the group
consisting of: losartan, benzbromarone, benziodarone, probenecid,
sulfinpyrazone, ethebencid, orotic acid, ticrynafen and
zoxazolamine, or pharmaceutically acceptable salt thereof.
9. The agent capable of reducing uric acid levels, as recited in
claim 5, which is a supplement of uricase protein that is delivered
as a conjugate with polyethylene glycol or an alternate delivery
system.
10. A pharmaceutical composition comprising a renin angiotensin
system (RAS) inhibitor, or pharmaceutically acceptable salt thereof
and the agent, or pharmaceutically acceptable salt thereof, capable
of reducing uric acid levels as recited in claim 5, and a
pharmaceutical carrier.
11. A combination therapy comprising the administration,
concomitantly, simultaneously or sequentially, of therapeutically
effective amounts of a RAS inhibitor, or pharmaceutically
acceptable salt thereof, and the agent, or pharmaceutically
acceptable salt thereof, capable of reducing uric acid levels as
recited in claim 5.
12. The pharmaceutical composition levels as recited in claim 10,
further comprising a diuretic, or pharmaceutically acceptable salt
thereof.
13. A combination therapy comprising the administration,
concomitantly, simultaneously or sequentially, of therapeutically
effective amounts of a combination of a RAS inhibitor, or
pharmaceutically acceptable salt thereof with a diuretic, or
pharmaceutically acceptable salt thereof and the agent, or
pharmaceutically acceptable salt thereof, capable of reducing uric
acid levels as recited in claim 5.
Description
[0001] This application claims priority from co-pending provisional
application Ser. No. 60/214,825 filed on Jun. 28, 2000.
BACKGROUND OF THE INVENTION
[0003] Uric acid is a purine metabolite that in most animals is
degraded by the hepatic enzyme uricase to allantoin. However,
several mutations of the gene for this enzyme occurred during early
primate development with the consequence that man and other
primates have relatively higher levels of serum uric acid [Wu, X.,
Muzny, D. M., Lee, C. C., and Caskey, C. T., Two independent
mutational events resulted in the loss of urate oxidase during
hominoid evolution. J. Mol. Evol. 34:78-84 (1992)]. The adaptive
benefit of this deletion is not known nor has the modern day
consequences of these mutations been fully understood. It has been
hypothesized that the loss of uricase provided a protective benefit
to prehistoric man who was known to have a very low sodium diet
[Eaton, S. B., Konner, and M., Paleolithic nutrition: A
consideration of its nature and current implications. N Engl J Med
312: 283-289 (1985)] but in modern times these mutations resulted
in the development of hypertension and other cardiovascular
diseases. In most subjects, the loss of uricase appears to be of no
significance, but for the 10 to 15 percent of the general
population with the highest uric acid levels (>6.0 mg/dl in
women and >6.5 mg/dl in men), there is an increased risk for the
development of hypertension, atherosclerosis, and other
cardiovascular diseases. Additionally 25 to 50% of hypertensive
individuals have elevated serum uric acid, based upon the current
standards 7 mg/dl [Cannon, P. J., Stason, W. B., Demartini, F. E.,
Sommers, S. C., and Laragh, J. H., Hyperuricemia in primary and
renal hypertension. N Engl J Med 275:457-464 (1966]. This invention
demonstrates for the first time mechanistic evidence that uric acid
is directly related to the development of increased blood
pressure.
[0004] An association between an elevated uric acid and an
increased risk for cardiovascular disease was originally suggested
by Haig in the late 1800s. Haig postulated that uric acid crystals
might precipitate in the circulation and occlude the
microvasculature [Haig, A., On uric acid and arterial tension. Br
Med J 1:288-291 (1889)], thereby assuming that the damaging effects
of uric acid were related to the formation of uric acid crystals
and not to the soluble form of uric acid. Recent epidemiological
studies have reported that an elevated uric acid confers an
increased risk for the development of hypertension [Selby, J. V.,
Friedman, G. D., and Quesenberry, C. P., Precursors of essential
hypertension: pulmonary function, heart rate, uric acid, serum
cholesterol, and other serum chemistries. Am J Epidemiol
131:1017-27 (1990); Jossa, F., et al. Serum uric acid and
hypertension: the Olivetti heart study. J Hum Hypertens 8:677-681
(1994); and Goldstein, H. S., and Manowitz, P., Relationship
between serum uric acid and blood pressure in adolescents. Annals
Hum Biol 20:423-431 (1993)], ischemic heart disease [Fang, J., and
Alderman, M. H. Serum uric acid and cardiovascular mortality. The
NHANES I Epidemiologic Follow-up Study, 1971-1992 JAMA
283:2404-2410 (2000); Bengtsson, C., Lapidus, L., Stendahl, C., and
Waldenstrom, J., Hyperuricemia and risk of cardiovasular disease
and overall death. Acta Med Scand 224:549-55 (1988); and Alderman,
M. H., Cohen, H., Madhavan, S., Kivlighn, S. Serum uric acid and
cardiovascular events in successfully treated hypertensive
patients. Hypertension 34:144-150 (1999).], and stroke [Lehto, S.,
Niskanen, L., Ronnemaa, T., and Laakso, M., Serum uric acid is a
strong predictor of stroke in patients with non-insulin dependent
diabetes mellitus. Stroke 29:635-639 (1998)]. In the Worksite study
an increase of 1 mg/dl of uric acid conferred the same
cardiovascular risk as an increase of 10 mm Hg in systolic blood
pressure or 20 mg/dl of cholesterol [Alderman, M. H., Cohen, H.,
Madhavan, S., and Kivlighn, S., Serum uric acid and cardiovascular
events in successfully treated hypertensive patients. Hypertension
34:144-150 (1999).]. Several studies have also reported that the
increased mortality associated with diuretic use can be attributed
to the increase in uric acid induced by these agents [Franse, L.
V., Pahor, M., and Barli, M. D., Serum uric acid, it's change with
diuretic use and risk of cardiovascular events in the Systolic
Hypertension in the Elderly Program (SHEP). American Society of
Hypertension Annual Meeting, May 1999, New York.]. Others have
shown that an increased uric acid confers increased risk for
cardiovascular mortality, especially in women [Fang, J., and
Alderman, M. H., Serum uric acid and cardiovascular mortality. The
NHANES I Epidemiologic Follow-up Study, 1971-1992 JAMA
283:2404-2410 (2000); Bengtsson, C., Lapidus, L., Stendahl, C., and
Waldenstrom, J., Hyperuricemia and risk of cardiovasular disease
and overall death. Acta Med Scand 224:549-55 (1988); and Persky, V.
W., et al. Uric acid: A risk factor for coronary heart disease?
Circulation 59:969-979 (1979)]. Despite the clinical and
epidemiological evidence, some authorities do not consider an
elevated uric acid to be a true cardiovascular risk factor
[Vaccarino, V., and Krumholz, H. M., Risk factors for
cardiovascular disease: One down, many more to evaluate. Ann Int
Med 131:62-63 (1999); and Wannamethee, S. G., Is serum uric acid a
risk factor for coronary heart disease? J Hum Hypertens 13:153-156
(1999)]. This is because many patients with an elevated uric acid
have other well-established risk factors for cardiovascular
disease, such as hypertension, renal disease, obesity,
dyslipidemia, and insulin resistance [Barlow, K. A., Hyperlipidemia
in primary gout. Metabolism 17:289-299 (1968) and Grahame, R., and
Stott, J. T., Clinical survey of 354 patients with gout. Ann Rheum
Dis 29:461-468 (1970)]. Whereas some studies have found that an
elevated uric acid level is an independent risk factor after
controlling for the contribution of these other risk factors by
multivariate analyses [Fang, J., and Alderman, M. H., Serum uric
acid and cardiovascular mortality. The NHANES I Epidemiologic
Follow-up Study, 1971-1992 JAMA 283:2404-2410 (2000); Bengtsson,
C., Lapidus, L., Stendahl, C., and Waldenstrom, J. Hyperuricemia
and risk of cardiovasular disease and overall death. Acta Med Scand
224:549-55 (1988); and Persky, V. W., et al. Uric acid: A risk
factor for coronary heart disease? Circulation 59:969-979 (1979)],
other studies including the recent Framingham analysis could not
[Culleton, B. F., Larson, M. G., Kannel, W. B., and Levy, D., Serum
uric acid and risk for cardiovascular disease and death: The
Framingham Study. Ann Intern Med 131:7-13 (1999); Klein, R., et al.
Serum uric acid: its relationship to coronary heart disease risk
factors and cardiovascular disease. Evans County, Ga. Arch Int Med
132:401-410 (1973); and Yano, K., Reed, D. M., and McGee, D. L.,
Ten year incidence of coronary heart disease in the Honolulu Heart
Program: relationship to biologic and lifestyle characteristics. Am
J Epidemiol 119:653-666 (1984).]. The lack of a mechanistic pathway
by which uric acid can cause cardiovascular disease, coupled with
the inconclusive clinical and epidemiological data, have left this
issue unresolved. In considering this controversy, it is important
to note that no animal model existed to study the effects of a
mildly elevated uric acid.
[0005] Cyclosporine (CSA) was introduced in the 1980's as an
immunosuppressant, and quickly become a first line treatment in
organ transplantation as well as in other immunologically mediated
diseases [Bennett, W. M., De Mattos, A., Meyer, M. M., Andoh, T.
F., and Barry, J. M., Chronic cyclosporine nephropathy. The
Achille's heel of immunossupressive therapy. Kidney Int 1996;
50:1089.]. Cyclosporine has numerous side effects, of which two of
the most important are nephrotoxicity [Myers, B. D. and Newton, L.,
Cyclosporine induced chronic nephropathy: an obbliterative
microvascular renal injury. J Am Soc Nephrol 1991; 2: S45, and
Chapman, J. R., Harding, N. G. L., Griffiths, D., and Morris, P.
J., Reversibility of cyclosporine nephrotoxicity after three months
treatment. Lancet 1985; 1:128.] and hyperuricemia [Gores, P. F.,
Fryd, D. S., Sutherland D. E. R., Najarian, J. S., and Simmons, R.
L., Hyperuricemia after renal transplantation. Am J Surg 1988; 156:
397.]. As many as 50% of patients taking CSA develop hyperuricemia
[Kahl, L. E., Thompson, M. E., and Griffith, B. P., Gout in the
heart transplant recipient: Physiological puzzle and therapeutic
challenge. Am J Med 1989; 87: 289, Najarian, J. S., Fryd, D. S.,
and Stransd, M., A single institution, randomized, prospective
trial of cyclosporine versus azathioprine-antilymphocyte globulin
for immunossupression in renal allograft recipients. Ann Surg 1985;
201:142 and Sutherland, D. E. R., Fryd, D. S., and Strand, M. H.,
Minnesota randomized prospective trial of cyclosporine versus
azathioprine-antilymphocyte globulin for immunossupression in renal
allograft recipients. Am J Kidney Dis 1985; 5:318.] and 9 to 10%
develop gout [West, C., Carpenter, B. J., and Hakala, T. R., The
incidence of gout in renal transplant recipients. Am J Kidney Dis
1987; 10: 369.]. The hyperuricemia from CSA is thought to result
from both a decrease in GFR [Zurcher, R. M., Bock, H. A., and
Thiel, G., Hyperuricemia in cyclosporine treated patients: A GFR
related effect. Nephrol Dial Transplant 1996; 11:153.], as well as
an increase in net tubular urate reabsorption [Laine, J., and
Hohnberg, C., Mechanisms of hyperuricemia in cyclosporine-treated
renal transplanted children. Nephron 1996; 74: 318, and Marcen, R.,
Gallego, N., Orofino, L. et al., Impairment of tubular secretion of
urate in renal transplant patients on cyclosporine. Nephron 1995;
70: 307.]. The most important complication of CSA is
nephrotoxicity, which is characterized histologically by striped
interstitial fibrosis, tubular atrophy and arteriolar hyalinosis
[Bennett, W. M., De Mattos, A., Meyer, M. M., Andoh, T. F., and
Barry, J. M., Chronic cyclosporine nephropathy. The Achille's heel
of immunossupressive therapy. Kidney Int 1996; 50:1089, Myers, B.,
Cyclosporine nephrotoxicity. Kidney Int 1986; 30:964, and Bennett,
W. M., Burdmann, E. A., Andoh, T. F., Houghton, D. C., Lindsley,
J., and Elzinga, L. W., Nephrotoxicity of immunossupressive drugs.
Nephrol Dial Transplant 1994; 9:141.]. The pathogenesis of CSA
nephropathy is multifactorial but likely involves afferent
arteriolar vasoconstriction with activation of the renin
angiotensin pathway and inhibition of nitric oxide (NO) production
[Bennett, W. M., Burdmann, E. A., Andoh, T. F., Houghton, D. C.,
Lindsley, J., and Elzinga, L. W.: Nephrotoxicity of
immunossupressive drugs. Nephrol Dial Transplant 1994; 9:141,
Burdmann, E. A., Andoh, T. F., Nast, C. C., et al., Prevention of
experimental cyclosporine induced interstitial fibrosis by losartan
and enalapril. Am J Physiol 1995; 269: F491, and Pichler, R.,
Franceschini, N., Young, B. A. et al., Pathogenesis of cyclosporine
nephropathy. Roles of angiotensin II and osteopontin. J Am Soc
Nephrol 1995; 6: 1186.].
[0006] The possibility that cyclosporine induced hyperuricemia may
have a role in either mediating or exacerbating cyclosporine
nephropathy has not previously been considered. However, it is
known that hyperuricemia is also associated with reduced renal
blood flow and increased renal vascular resistance [Hoyer, P. F.,
Lee, I. K., Oemar, B. S., Krohn, H. P., Offner, G., and Brodhel,
J., Renal handling of uric acid under cyclosporine A treatment.
Pediatr Nephrol 1988; 2:18, and Messerli, F. H., Frolich, E. D.,
Drelinski, C. R., Suarez, D. H., and Aristimuno, G. G., Serum uric
acid in essential hypertension: an indicator of renal vascular
involvement. Ann Int Med 1980; 93:817.] and those patients with
long-standing gout may develop chronic tubulointerstitial disease
[Beck, L. H., Requiem for gouty nephropathy. Kidney Int 30:280-287,
1986, Emmerson, B. T., and Row, P. G., An evaluation of the
pathogenesis of the gout kidney. Kidney Int. 1975; 8:65, and
Johnson, R. J., Kivlighn, S. D., Kim, Y. G., Suga, S., and Fogo, A.
B., Reapprasial of the pathogenesis and consequences of
hyperuricemia in hypertension, cardiovascular disease and renal
disease. Am J Kidney Dis 1999; 33: 225.]. Controversy has existed,
however, over whether hyperuricemia is the cause or consequence of
renal vasoconstriction and tubulointerstitial lesions [Nickeleit,
V., and Mihatsh, M. J., Uric acid nephropathy and end-stage renal
disease. Review of a non-disease. Nephrol Dial Transplant 1997; 12:
1832, and Yu, T., Berger, L., Dorph, D. J., and Smith, H., Renal
function in gout: V-Fators influencing the renal hemodynamics. Am J
Med 1979: 67:766.].
[0007] A recent report suggested that allopurinol, an inhibitor of
uric acid production, could protect the kidney from CSA
nephrotoxicity [Assis, S. M., Monteiro, J. L., and Seguro, A. C.,
L-arginine and allopurinol protect against cyclosporine
nephrotoxicity. Transplantation 1997; 63(8): 1070.]. Thus the
hypothesis that hyperuricemia might exacerbate cyclosporine
nephropathy was tested. As rodents normally do not become
hyperuricemic because they have the hepatic enzyme uricase, which
degrades uric acid to allantoin [Becker, B. F., Towards the
physiological function of uric acid. Free Rad Biol Med 1993;
14:615, and Waisman, J., Bluestone, R. and Klinemberg, J. R., A
preliminary report of nephropathy in hyperuricemic rats. Lab Invest
1974; 30:716.], rats with cyclosporine nephropathy, in the presence
and absence of the uricase inhibitor, oxonic acid were compared.
This invention demonstrates that hyperuricemia exacerbates CSA
nephropathy through a crystal independent mechanism.
[0008] Hyperuricemia, defined as serum uric acid levels >7.0
mg/dl in man and >6.0 mg/dl in women, is a common metabolic
abnormality that is observed in 4 to 6% of the population
(Wyngaarden J. B. and Kelley W. N., Epidemiology of hyperuricemia
and gout. In Gout and Hyperuricemia, Grune and Stratton, New York,
1976, pp 21-37). The major risks classically attributed to
hyperuricemia have been the risk of developing gout and/or uric
acid renal stones. Patients with longstanding hyperuricemia and/or
gout are also at risk for developing chronic renal disease (Talbot,
J. H., and Terplan, K. L., The kidney in gout. Medicine 39:405-467,
1960, and Gonick, H. C., Rubini, M. D., Gleason, I. O., and
Sommers, S. C., The renal lesion in gout. Ann Int Med 62:667-74,
1965). Several large studies have documented that between 30 and
60% of patients with gout will develop renal insufficiency and up
to 10% will develop end stage renal disease (Talbot, J. H., and
Terplan, K. L., The kidney in gout. Medicine 39:405-467, 1960;
Gonick, H. C., Rubini, M. D., Gleason, I. O., and Sommers, S. C.,
The renal lesion in gout. Ann Int Med 62:667-74, 1965; Yu, T.,
Berger, L., Dorph, D. J., and Smith, H., Renal function in gout:
V-Factors influencing the renal hemodynamics. Am J Med 67:766-71,
1979; and Berger, L., and Yu, T., Renal Function in Gout: IV. An
Analysis of 524 Gouty Subjects Including long-term follow-up
studies. Am J Med 59:605-613, 1975). Renal structural changes are
even more common than the functional abnormalities (Greenbaum, D.,
and Ross, J. H., Renal biopsy in gout., Brit Med J 1:1502-1504,
1961), and in one study renal disease was observed in 287 of 290
patients with gout (Talbot, J. H., and Terplan, K. L., The kidney
in gout. Medicine 39:405-467, 1960). The renal disease, which has
been termed `gouty nephropathy`, is characterized by chronic
tubulointerstitial fibrosis, often with arteriolosclerosis and
glomerular sclerosis (Talbot, J. H., and Terplan, K. L., The kidney
in gout. Medicine 39:405-467, 1960). In addition, many biopsies
show focal deposits of urate crystals, particularly in the outer
medulla (Talbot, J. H., and Terplan, K. L., The kidney in gout.
Medicine 39:405-467, 1960; Gonick, H. C., Rubini, M. D., Gleason,
I. O., and Sommers, S. C., The renal lesion in gout. Ann Int Med
62:667-74, 1965; and Cannon, P. J., Stason, W. B., Dematini, F. E.,
Sommers, S. C., and Laragh, J. H., Hyperuricemia in Primary and
Renal Hypertension. New Engl J Med 275:457-464, 1966).
[0009] However, investigators have challenged if `gouty
nephropathy` truly exists (Beck, L. H., Requiem for gouty
nephropathy. Kidney Int 30:280-287, 1986, and Nickeleit, V. and
Mihatsh, M. J., Uric acid nephropathy and end-stage renal disease.
Review of a non-disease. Nephrol Dial Transplant 12: 1832-38,
1997). Some studies have suggested that the renal functional
changes could be attributed to co-existing hypertension or the
consequence of aging (Yu, T., Berger, L., Dorph, D. J., and Smith,
H., Renal function in gout: V-Factors influencing the renal
hemodynamics. Am J Med 67:766-71, 1979, and Yu, T. and Berger, L.,
Impaired Renal Function in Gout: Its Association with Hypertensive
Vascular Disease and Intrinsic Renal Disease. Am J Med 72:95-100,
1982). Others have noted the apparent discrepancy between the focal
nature of the urate deposits and the diffuse interstitial disease
((Beck, L. H., Requiem for gouty nephropathy. Kidney Int
30:280-287, 1986, and Nickeleit, V. and Mihatsh, M. J., Uric acid
nephropathy and end-stage renal disease. Review of a non-disease.
Nephrol Dial Transplant 12: 1832-38, 1997). Furthermore, the effect
of uric acid lowering agents on improving renal function in
patients with gout has been variable, with both positive
(Perez-Ruiz, F., Calabozo, M., Fernandez-Lopez, M. J.,
Herrero-Beites, A., Ruiz-Lucea, E., Garcia-Erasukin, G., Duruelo,
J., and Alonso-Ruiz, A., Treatment of chronic gout in patients with
renal function impairment. An open, randomized actively controlled
study. J Clin Rheumatol 1999; 5:49-55, and Perez-Ruiz F,
Alonso-Ruiz A, Calabozo M, Herrero-Beites A, Garcia-Erauskin G, and
Ruiz-Lucca E., Efficacy of allopurinol and benzbromarone for the
control of hyperuricemia. A pathogenic approach to the treatment of
primary chronic gout. Ann Rheum Dis 1998; 57:545-549.) and negative
(Fessel, W. J., Renal Outcomes of Gout and Hyperuricemia. Am J Med
67:74-82, 1979, and Rosenfeld, J. B., Effect of long-term
allopurinol administration on serial GFR in normotensive and
hypertensive hyperuricemic subjects. Adv Exp Med Biol 41B:581-596,
1974) studies reported.
[0010] A novel pathway has been demonstrated where uric acid, a
purine metabolite present in the blood, actually causes
hypertension and renal disease. It is known that markedly elevated
uric acid can crystallize in the tubules of the kidney and cause
kidney failure. The invention disclosed herein is that mildly
elevated uric acid levels can also cause renal disease and
hypertension. Furthermore, it has been shown that this action is
mediated in part by activation of the renin-angiotensin system in
the kidney and by the inhibition of nitric oxide synthases (NOS)
within the kidney.
SUMMARY OF THE INVENTION
[0011] This invention relates to a method for treating and
preventing hypertension by administering a therapeutically
effective amount of an agent capable of reducing uric acid levels
in a patient in need of such treatment. Additionally, the scope of
the invention includes a method of treating coronary heart disease
by administering a therapeutically effective amount of an agent
capable of reducing uric acid levels in a patient in need of such
treatment. The agent, or pharmaceutically acceptable salt thereof,
capable of reducing uric acid levels is selected from the group
consisting of gene therapy, a xanthine oxidase inhibitor, a
uricosuric agent, supplements of the uricase protein and a urate
channel inhibitor or combinations thereof. Also within the scope of
the invention is a pharmaceutical composition, comprising a renin
angiotensin system (RAS) inhibitor, or pharmaceutically acceptable
salt thereof and the agent, or pharmaceutically acceptable salt
thereof capable of reducing uric acid levels, and a pharmaceutical
carrier, or a combination therapy comprising the concomitant,
simultaneous or sequential administration of the RAS inhibitor, or
pharmaceutically acceptable salt thereof, and the agent, or
pharmaceutically acceptable salt thereof, capable of reducing uric
acid levels.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] The file of this patent contains at least one drawing
executed in color. Copies of this patent with color drawing(s) will
be provided by the Patent and Trademark Office upon request and
payment of the necessary fee.
[0013] FIG. 1--A Model of Mild Hyperuricemia in Rats. Rats treated
with oxonic acid (2%) develop mild hyperuricemia compared to
controls on a normal salt diet (FIG. 1A). Light microscopy (PAS,
50.times.) at 7 weeks is normal (FIG. 1B) and no urate crystals are
present by DeGalantha (50.times.) stain of 100% ethanol-fixed
tissue (FIG. 1C). For comparison, we have included a DeGalantha
stain in rats with acute urate nephropathy showing intratubular
urate crystals (FIG. 1D). [Key: .box-solid., oxonic acid; .DELTA.,
control]
[0014] FIG. 2--Hyperuricemia Elevates Blood Pressure in Rats. Rats
placed on oxonic acid (2%) develop a modest elevation of blood
pressure after 3 weeks on a normal sodium (0.26% NaCl) diet.
*p<0.05 versus control. [Key: .box-solid., oxonic acid; .DELTA.,
control]
[0015] FIG. 3--Hyperuricemia Maintains and Elevates Blood Pressure
in Rats on a Low Salt diet. Control rats placed on a mild salt
restriction (0.125% NaCl diet) have a fall in blood pressure after
several weeks; this is prevented in the presence of oxonic acid
(2%). Rats placed on oxonic acid and low salt diet that are also
administered allopurinol do not show the increase in blood
pressure. FIG. 3A shows the blood pressures, and FIG. 3B shows the
uric acid levels. [Key: .box-solid., oxonic acid; .DELTA., control;
.largecircle., allopurinol+oxonic acid]*p<0.05 versus control,
.dagger.p<0.05 vs. oxonic acid alone.
[0016] FIG. 4--Hyperuricemia Correlates with Blood Pressure. Shown
are the serum uric acid levels in individual rats on a low salt
diet (closed circles), low salt+oxonic acid (open circles) and on a
low salt diet+oxonic acid+allopurinol (triangles) at 7 weeks with
the corresponding systolic blood pressures. A strong correlation is
present (r=0.7, n=52, p<0.001).
[0017] FIG. 5--Effect of Allopurinol Intervention or Oxonic Acid
Withdrawal on Blood Pressure in Hyperuricemic Rats. Rats were
placed on low salt diet plus oxonic acid (2%) for 7 weeks and then
matched on basis of uric acid level and blood pressure into 3
groups (Oxonic acid plus low salt diet (OA/LSD); Withdrawal of
oxonic acid but continuation of the LSD (OA withdrawal); and the
addition of allopurinol (150 mg/L drinking water) with continuation
of the OA/LSD diet (+allopurinol). FIG. 5A shows the blood
pressures, and FIG. 5B shows the uric acid levels. [Key:
.box-solid., oxonic acid; .DELTA., oxonic acid withdrawal;
.largecircle., allopurinol+oxonic acid].
[0018] FIG. 6--Renal Fibrosis Develops in Hyperuricemic Rats. Rats
treated with oxonic acid (2%) and a low salt diet for 11 weeks
develop significant striped interstitial fibrosis, as shown by
immunostaining for interstitial type III collagen (FIG. 6 A).
Control rats on a low salt diet do not develop any evidence of
interstitial disease (FIG. 6B). Renal fibrosis was less in oxonic
acid-treated rats in which allopurinol was administered for 4 weeks
prior to sacrifice (FIG. 6C).
[0019] FIG. 7--Renin Correlates with Serum Uric Acid in Rats on a
Low Salt Diet. There was a direct correlation between renin
(measured as the % of glomeruli with juxtaglomerular renin
staining) and serum uric acid levels in rats on a low sodium diet
(r=0.7, n=18, p=0.0006). [Key: .largecircle., oxonic acid; , low
salt diet control; .DELTA., allopurinol+oxonic acid].
[0020] FIG. 8--The Elevated BP in Hyperuricemic Rats is Prevented
by Treatment with an ACE inhibitor or with L-Arginine. Rats placed
on a low salt diet with oxonic acid develop an elevated blood
pressure (OA/LSD), which is prevented if enalapril (1 mg/kg/d) or
L-Arginine (1%) is added to the drinking water. *p<0.05 versus
OA/LSD. [Key: .box-solid., oxonic acid; .diamond-solid.,
L-arginine; , enalapril].
[0021] FIG. 9--Hyperuricemia exacerbates chronic cyclosporine
nephropathy. Cyclosporine alone results in classic chronic
tubulointerstitial disease [A], which is worse in rats that are
also hyperuricemic [B](striped fibrosis indicated by arrows).
Similarly, Cyclosporine-Oxonic Acid rats show greater osteopontin
expression [D], macrophage infiltration [F] and type HI collagen
deposition [H], compared to respective controls [C,E,G].
(Magnification: hematoxilin-eosin .times.50, OPN .times.25, ED-1
.times.100, type III collagen .times.50).
[0022] FIG. 10--Effect of Hyperuricemia alone and in combination
with cyclosporine on renal interstitium. Group 3, cyclosporine
treated rats (CSA, gray columns) presented increased interstitial
fibrosis score [A], arteriolar hyalinosis [B], osteopontin
expression [C], macrophage infiltration [D] and type III collagen
deposition [E], compared to Group 1, vehicle treated rats (VEH,
white columns). These findings are greatest in group 4, rats
treated with both cyclosporine and oxonic acid (CSA-OA group, black
columns). (*p<0.05 compared to VEH, **p<0.05 compared to VEH
and CSA).
DETAILED DESCRIPTION OF THE INVENTION
[0023] This invention relates to a method of treating hypertension
comprising administering a therapeutically effective amount of an
agent capable of reducing uric acid levels in a patient in need of
such treatment. A reduction in uric acid levels would reduce the
risk of hypertension, coronary heart disease, renal dysfunction,
cardiovascular morbidity and mortality. Current standards for
elevated uric acid levels are 7 mg/dl. However, patients with uric
acid levels of 10 mg/dl are a high risk for the above-noted
cardiovascular conditions, between 6 and 10 mg/dl are at an
increased risk for the above-noted cardiovascular conditions, or a
reduced risk with uric acid levels of >4 and <6 mg/dl.
[0024] A method of preventing hypertension comprising administering
a therapeutically effective amount of an agent capable of reducing
uric acid levels in a patient in need of such treatment.
[0025] A method of treating coronary heart disease comprising
administering a therapeutically effective amount of an agent
capable of reducing uric acid levels in a patient in need of such
treatment.
[0026] A method of treating and preventing eclampsia comprising
administering a therapeutically effective amount of an agent
capable of reducing uric acid levels in a patient in need of such
treatment.
[0027] An agent capable of reducing uric acid levels by about 0.2
mg/dl. The agent capable of reducing uric acid levels, which is
selected from the group consisting of: gene therapy, a xanthine
oxidase inhibitor; a uricosuric agent; supplements of the uricase
protein and a urate channel inhibitor, or combinations of these
agents. Specific examples of agents that are capable of reducing
uric acid levels include but are not limited to: [0028] a gene
therapy such as one that targets the overexpression of uricase, the
enzyme responsible for the breakdown of uric acid to allantoin;
[0029] a xanthine oxidase inhibitor, such as allopurinol, and
carprofen; [0030] a uricosuric agent, which is defined as an
inhibitor of the organic anion transport channels and/or voltage
sensitive transport channels acting in the kidney, such agents
include but are not limited to: losartan, benzbromaraone,
benziodarone, probenecid, sulfinpyrazone ethebencid, orotic acid,
ticrynafen and zoxazolamine; [0031] a supplement of the uricase
protein which might be delivered as a conjugate with polyethylene
glycol or another delivery system; and [0032] a urate channel
inhibitor, is a means for interfering with the uric acid transport
mechanism by blocking the influx of uric acid into cells.
[0033] A pharmaceutical composition comprising a renin angiotensin
system (RAS) inhibitor or a pharmaceutically acceptable salt
thereof and the agent capable of reducing uric acid levels or a
pharmaceutically acceptable salt thereof as recited above and a
pharmaceutical carrier. The renin angiotensin system inhibitor,
such as an angiotensin converting enzyme inhibitor, an angiotensin
II antagonist and a renin inhibitor. Representative RAS inhibitors
include: captopril, cilazapril, enalapril, fosinopril, lisinopril,
quinapril, ramapril, zofenopril, candesartan cilexetil, eprosartan,
irbesartan, losartan, tasosartan, tehnisartan, and valsartan, or
pharmaceutically acceptable salts thereof. Also within the scope of
this invention is the combination of an agent capable of reducing
uric acid levels with a combination RAS inhibitor with a diuretic,
such as hydrochlorothiazide, furosemide, etc. Specific examples,
include but are not limited to the above RAS inhibitors with
hydrochlorothiazide.
[0034] A combination therapy comprising the administration,
concomitantly, simultaneously or sequentially, of therapeutically
effective amounts of a RAS inhibitor and the agent capable of
reducing uric acid levels as recited above. Also within the scope
of this invention is the combination therapy as recited above that
includes an agent capable of reducing uric acid levels with a
combination RAS inhibitor with a diuretic, such as
hydrochlorothiazide, furosemide, etc. Specific examples, include
but are not limited to the above RAS inhibitors with
hydrochlorothiazide.
[0035] Also within the scope of the invention is a pharmaceutical
composition comprising an agent which stimulates nitric oxide
production via endothelial and/or neuronal nitric oxide synthase or
a pharmaceutically acceptable salt thereof and the agent capable of
reducing uric acid levels or a pharmaceutically acceptable salt
thereof as recited above and a pharmaceutical carrier. An agent
which stimulates nitric oxide production via endothelial and/or
neuronal nitric oxide including, but not limited to L-Arginine,
nitrates and nitrate-mimetics and gene therapy, such as one that
targets the overexpression endothelial and/or neuronal nitric oxide
synthase.
[0036] The studies were performed on rats in which the serum uric
acid was raised to a mild (1.5 to 2.0-fold) degree, using an enzyme
inhibitor of uricase, an enzyme involved in the degradation of uric
acid. Rats made mildly hyperuricemic developed significant
hypertension within a few weeks, and this was associated with
stimulation of renin (documented by renin staining in the kidney)
and by a fall in neuronal NOS in the macula densa (a tubular
segment in the kidney involved in regulation of renal blood flow)
and of endothelial cell NOS. Associated with these findings was the
development of renal fibrosis with increased collagen deposition
and macrophage infiltration. These changes could be prevented by
lowering the uric acid with allopurinol.
[0037] The studies provide a mechanism for the long-observed
association of uric acid with hypertension, cardiovascular disease
and renal disease, and for the first time provides direct
experimental evidence that uric acid is causal rather than simply a
marker for associated cardiovascular risk factors. It thus provides
the first direct rationale for lowering uric acid as a means for
not only preventing the development of hypertension but also for
its treatment--a substantial finding given that 25% of the worlds
population will become hypertensive. It is also relevant to a
number of other diseases, including eclampsia (a disease afflicting
pregnant women associated with hypertension, renal disease and an
elevated uric acid but in which the latter was thought only to be a
marker), to cyclosporine nephropathy (one of the complications of
transplantation in which hypertension, renal disease and an
elevated uric acid are central features), to progressive renal
disease, and even to aging associated hypertension and renal
disease. The observation that blacks have higher uric acid levels
also provides a mechanism to explain the reason they are more
susceptible to hypertension.
[0038] The studies show that increasing the uric acid level in the
rat will cause hypertension and renal disease, and that lowering it
will lower the blood pressure and prevent the development of renal
disease. So far we have used pharmacologic agents for this
purpose--such as the use of allopurinol, losartan, or benziodarone.
However, there are numerous other possible ways to lower uric
acid--these could include repleting humans with uricase (the enzyme
that degrades uric acid to allantoin). Unlike most mammals, man had
a series of mutations of the uricase gene early in his
evolution--we have hypothesized that during this period, when man
was on a very low salt diet, that the increase in uric acid would
have helped maintain blood pressure under those conditions. Indeed,
this was confirmed by our experimental studies in the rat. However,
it may be now prudent to replace uricase in man as a means for
preventing the development of hypertension--this could be done by
gene therapy or by supplying the uricase protein, such as by
conjugation with polyethylene glycol or other method. Future
therapies might also be directed at blocking the influx of uric
acid into cells by interfering with the uric acid transport
mechanism.
[0039] The instant invention provides direct evidence that mild
hyperuricemia in rats induces hypertension, as well as subtle renal
injury and fibrosis, through a crystal-independent mechanism
mediated by activation of the renin angiotensin system and
downregulation of neuronal nitric oxide synthase in the macula
densa. This observation may explain why hyperuricemia has been
found to predict the development of hypertension [Selby, J. V.,
Friedman, G. D., and Quesenberry, C. P., Precursors of essential
hypertension: pulmonary function, heart rate, uric acid, serum
cholesterol, and other serum chemistries. Am J Epidemiol
131:1017-27 (1990), Jossa, F., et al., Serum uric acid and
hypertension: the Olivetti heart study. J Hum Hypertens 8:677-681
(1994), and Goldstein, H. S., and Manowitz, P., Relationship
between serum uric acid and blood pressure in adolescents. Annals
Hum Biol 20:423-431 (1993)], and may also be relevant to the 25 to
50% of the hypertensive population who are found to be
hyperuricemic at presentation [Cannon, P. J., Stason, W. B.,
Demartini, F. E., Sommers, S. C., and Laragh, J. H., Hyperuricemia
in primary and renal hypertension. N Engl J Med 275:457-464
(1966)]. These studies may also provide a mechanism to explain how
hyperuricemia can thwart the beneficial effects of diuretics on
overall cardiovascular mortality [Franse, L. V., Pahor, M., and
Barli, M. D., Serum uric acid, it's change with diuretic use and
risk of cardiovascular events in the Systolic Hypertension in the
Elderly Program (SHEP). American Society of Hypertension Annual
Meeting, May 1999, New York.]. Furthermore, the finding that
hyperuricemia can induce renal fibrosis may provide a mechanism for
the development of `gouty nephropathy` [Talbott, J. H., and
Terplan, K. L., The kidney in gout Medicine 39:405-50, 1960, and
Gonick, H. C., Rubini, M. E., Gleason, I. O., and Sommers, S. C.,
The renal lesion in gout Annals Int Med 62:667-674, 1968], as it
has been hard to attribute the diffuse injury to urate crystal
deposition alone [Beck, L. H., Requiem for gouty nephropathy Kidney
Int 30:280-287 (1986), and Nickeleit, V., and Mihatscli, M. J.,
Uric acid nephropathy and end-stage renal disease-Review of a
nondisease. Nephrol Dial Transpl 12:1832-1838 (1997)]. It also
suggests a true pathogenic role for uric acid in familial
hyperuricemic nephropathy, an inherited disorder in which
hyperuricemia, renal vasoconstriction, hypertension and
interstitial renal disease develop [McBride, M. B., Simmonds, H.
A., Moro, F. Familial renal disease or familial juvenile
hyperuricaemic nephropathy? J Inher Metab Dis 20:351-353 (1997)].
The documentation that an elevated uric acid causes hypertension
also helps resolve the clinical and epidemiological controversies
surrounding the role of uric acid in cardiovascular disease, as
multivariate analyses would not be expected to show uric acid to be
an independent risk factor when controlled for variables to which
it is causally linked [Johnson, R. J., and Tuttle, K., Much ado
about nothing, or much to do about something: The continuing
controversy on the role of uric acid in cardiovascular disease.
Hypertension 35:E10-E10 (2000)].
[0040] While the data suggests that an elevated uric acid can
increase blood pressure and induce renal disease through a
mechanism that involves activation of the renin angiotensin system
and inhibition of neuronal nitric oxide synthase, it is important
to recognize that there may be additional mechanisms by which uric
acid contributes to cardiovascular disease. Indeed, there are other
studies have shown that uric acid remains an independent
cardiovascular risk factor even after controlling for hypertension
and renal disease [Fang, J., and Alderman, M. H., Serum uric acid
and cardiovascular mortality. The NHANES I Epidemiologic Follow-up
Study, 1971-1992 JAMA 283:2404-2410 (2000), Bengtsson, C., Lapidus,
L., Stendahl, C., and Waldenstrom, J., Hyperuricemia and risk of
cardiovasular disease and overall death. Acta Med Scand 224:549-55
(1988), and Alderman, M. H., Cohen, H., Madhavan, S., and Kivlighn,
S., Serum uric acid and cardiovascular events in successfully
treated hypertensive patients. Hypertension 34:144-150 (1999)].
[0041] Finally, the observation that inhibition of uricase can
prevent the fall of blood pressure under low salt conditions
provides a mechanism to explain why the mutations of the uricase
gene, giving rise to an elevated uric acid, were preferentially
conserved during early primate development. Indeed, studies had
suggested that humans were on a very low sodium diet (20-40
mmol/day of sodium) for the great majority (99.8%) of the last 3.5
million years, and it is only in the last several thousand years
that man has been on the modern day, high salt diet [Eaton, S. B.,
and Konner, M., Paleolithic nutrition: A consideration of its
nature and current implications. N Engl J Med 312: 283-289 (1985)].
It is also of interest that studies of primitive societies have
documented a low prevalence of hypertension and cardiovascular
disease [Young, D. B., Lin, H., and McCabe, R. D., Potassium's
cardioprotective mechanisms. Am J Physiol 268:R825-R837 (1995), and
Tobian, L. Salt and hypertension. Lessons from animal models that
relate to human hypertension. Hypertension 17[suppl I]:I52-I58
(1991)], suggesting that the current `epidemic` of cardiovascular
disease and hypertension may be a consequence of modern society.
While this mutation may have benefited early humans, it is
hypothesized that in modern societies it plays a critical role in
the pathogenesis of hypertension and cardiovascular disease.
[0042] A major complication of chronic cyclosporine treatment is
CSA nephropathy [Myers, B. D., Newton, L., Cyclosporine induced
chronic nephropathy: an obbliterative microvascular renal injury. J
Am Soc Nephrol 1991; 2: S45, and Myers, B., Cyclosporine
nephrotoxicity, Kidney Int 1986; 30:964.], which is characterized
by arteriolar hyalinosis and tubulointerstitial disease. The
pathogenesis is considered to be secondary to intense renal
vasoconstriction induced by angiotensin II and other vasoactive
substances [Bennett, W. M., De Mattos, A., Meyer, M. M., Andoh, T.
F., and Barry, J. M., Chronic cyclosporine nephropathy. The
Achille's heel of immunossupressive therapy. Kidney Int 1996;
50:1089, Myers, B., Cyclosporine nephrotoxicity. Kidney Int 1986;
30:964, and Bennett, W. M., Burdmann, E. A., Andoh, T. F.,
Houghton, D. C., Lindsley, J., and Elzinga, L. W., Nephrotoxicity
of immunossupressive drugs. Nephrol Dial Transplant 1994; 9:141,
and Burdmann, E. A., Andoh, T. F., and Nast, C. C., et al.,
Prevention of experimental cyclosporine induced interstitial
fibrosis by losartan and enalapril. Am J Physiol 1995; 269:
F491.].
[0043] Cyclosporine use is also associated with the development of
hyperuricemia, secondary to a decrease in uric acid excretion
[Hoyer, P. F., Lee, I. K., Oemar, B. S., Krohn, H. P., Offner, G.,
and Brodhel, J., Renal handling of uric acid under cyclosporine A
treatment. Pediatr Nephrol 1988; 2:18, Cohen, S. L., Boner, G.,
Rosenfeld, J. B., et al., The mechanism of hyperuricaemia in
cyclosporine-treated renal transplant recipients. Transplant Proc
1987; 19:1829, and Noordzij, T. C., Leunissen, K. L. M., and Van
Hoff, J. P., Renal handling of urate and the incidence of gouty
arthritis during cyclosporine and diuretic use. Transplantation
1991; 52(1): 64.]. While the risk of hyperuricemia in patients on
CSA has generally been considered only to be gout [West, C.,
Carpenter, B. J., and Hakala, T. R., The incidence of gout in renal
transplant recipients. Am J Kidney Dis 1987; 10: 369.], it is of
interest that there has been a longstanding controversy on the role
of hyperuricemia in mediating tubulointerstitial diseases. Numerous
studies have documented that patients with gout have a high
prevalence of tubulointerstitial disease ("gouty nephropathy")
[Beck, L. H., Requiem for gouty nephropathy. Kidney Int 30:280-287,
1986, Emmerson, B. T., and Row, P. G., An evaluation of the
pathogenesis of the gout kidney. Kidney Int. 1975; 8:65, Gonick, H.
C., Rubini, M. D., Gleason, I. O., and Sommers, S. C. The renal
lesion in gout. Ann Int Med 1965; 62:667, and Talbot, J. H., and
Terplan, K. L., The kidney in gout. Medicine 1960; 39:405. Steele
TH: Hyperuricemic nephropathies. Nephron 1999; 81 (1 suppl 1): 45.]
and these patients also have evidence for intense renal
vasoconstriction [Messerli, F. H., Frolich, E. D., Drelinski, G.
R., Suarez, D. H., and Mistimuno, G. G., Serum uric acid in
essential hypertension: an indicator of renal vascular involvement.
Ann Int Med 1980; 93:817.]. However, it has remained controversial
as to whether the hyperuricemia per se contributes to the renal
disease or whether the renal disease results from other associated
risk factors such as hypertension [Nickeleit, V., and Mihatsh, M.
J., Uric acid nephropathy and end-stage renal disease. Review of a
non-disease. Nephrol Dial Transplant 1997; 12: 1832.]. In this
study we have addressed the role of uric acid in a model of CSA
nephropathy in rats and examined the hypothesis that hyperuricemia
may significantly augment cyclosporine mediated renal injury.
[0044] The first finding was that CSA, independent of oxonic acid,
was associated with an increase in serum uric acid with a tendency
for a reduction in fractional urate excretion. In rats receiving
CSA and oxonic acid, the serum urate levels were higher but the
fractional urate excretion remained low to normal. These findings
are similar to those observed in humans [Hoyer, P. F., Lee, I. K.,
Oemar, B. S., Krohn, H. P., Offner, G., and Brodhel, J., Renal
handling of uric acid under cyclosporine A treatment. Pediatr
Nephrol 1988; 2:18, and Cohen, S. L., Boner, G., Rosenfeld, J. B.,
et al., The mechanism of hyperuricaemia in cyclosporine-treated
renal transplant recipients. Transplant Proc 1987; 19:1829.] and
document the clinical relevance of this model.
[0045] The second important finding of this study was that
hyperuricemia significantly exacerbated the tubulointerstitial
disease and arteriolar hyalinosis induced by cyclosporine.
Parameters analyzed included osteopontin, which is a sensitive
marker of tubulointerstitial injury, interstitial and glomerular
macrophage accumulation and interstitial deposition of type III
collagen. Interestingly, all of these parameters were significantly
worse in rats treated with CSA and OA compared to rats treated with
CSA alone.
[0046] The mechanism by which hyperuricemia exacerbates renal
disease is of intense interest. An important finding in our study
is that it does not involve intrarenal crystal deposition.
Utilizing different stains for uric acid we were unable to identify
crystals in these lesions. Furthermore, the pattern of tissue
injury was more consistent with an "ischemic" pattern [Duncan, H.,
and Dixon, A. S., Gout, familial hyperuricemia and renal disease. Q
J Med 1960; 29: 127.] as opposed to an "obstructive" pattern as
seen with crystal induced intratubular deposition [Waisman, J.,
Mwasi, L. M., Bluestone, R., and Klinemberg, J. R., Acute
hyperuricemic nephropathy in rats. An electron microscopy study. Am
J Pathol 1975; 81(2): 367, and Tykarski, A., Evaluation of renal
handling of uric acid in essential hypertension: hyperuricemia
related to decreased urate secretion. Nephron 1991; 59:364.]. In
addition the ability of CSA to reduce the fractional excretion of
urate resulted in urinary levels lower than that associated with
the acute urate nephropathy model, in which urinary urate excretion
is typically increased [Bluestone, J., Waisman, J., and Klinemberg,
J. R., Chronic experimental hyperuricemia nephropathy. Biochemical
and morphological characterization. Lab Invest 1975; 33(3):
273.].
[0047] Therefore, it is our contention that hyperuricemia may
augment renal injury in this model by potentiating CSA-mediated
renal vasoconstriction. Furthermore, gout is associated with both
tubulointerstitial disease and renal vasoconstriction. It is of
interest that familial hyperuricemic nephropathy is characterized
by reduced fractional urate excretion, renal vasoconstriction and
tubulointerstitial disease in which intrarenal urate crystal
deposition is often absent [Mateos, F. A., and Puig, J. C., Renal
hemodynamics in familial nephropathy associated with hyperuricemia.
Adv Exp Med Biol 1991; 309: 301, and Simmonds, H. A., Warren, D.
J., Cameron, J. S., Potter, C. F., and Farebrother, D. A., Familial
gout and renal failure in young women. Clin Nephrol 1980;
14:176.].
[0048] The presence of hyperuricemia in rats with CSA-induced
nephropathy is associated with significantly worse
tubulointerstitial renal injury, but does not involve intrarenal
crystal deposition. This finding has significant implications not
only in our understanding of the pathogenesis of CSA nephropathy,
but also in the role of hyperuricemia in the progression of renal
disease.
[0049] The following examples illustrate this method of
treatment/prevention, and as such are not to be considered as
limiting the invention set forth in the claims appended hereto.
EXAMPLES
Experimental Design
[0050] All studies utilized adult male Sprague-Dawley rats
(Simonsen Laboratories, Gilroy Calif.) (200-250 g).
Example 1
[0051] Rats were placed on a normal salt (NaCl 0.26%) diet with or
without 2% oxonic acid (Ziegler Bros, Gardners, Pa.) added to the
diet and rats were sacrificed at week 7.
[0052] Systolic blood pressure was measured by tail cuff
sphyngomanometer using an automated system with photoelectric
sensor (IITC, Life Science) that has been shown to closely
correlate with intra-arterial blood pressure measurements [Fischer
E, Schnermann, J., Briggs, J. P, Kriz, W. Ronco, P. M., Bachmann,
S. Ontogeny of NO synthase and renin in juxtaglomerular apparatus
of rat kidneys. Am J Physiol 268:F1164-76, 1995].
Example 2
[0053] Rats were placed on a low sodium diet (NaCl, 0.125%) with or
without 2% oxonic acid for 7 weeks. A third group were administered
allopurinol in the drinking water (150 mg/L) with weekly
adjustments of the dose depending on the uric acid level.
[0054] Systolic blood pressure was measured by tail cuff
sphyngomanometer using an automated system with photoelectric
sensor (IITC, Life Science) that has been shown to closely
correlate with intra-arterial blood pressure measurements [Fischer
E, Schnermann, J., Briggs, J. P, Kriz, W. Ronco, P. M., and
Bachmann, S., Ontogeny of NO synthase and renin in juxtaglomerular
apparatus of rat kidneys. Am J Physiol 268:F1164-76, 1995].
Example 3
[0055] Rats were placed on the low sodium diet with oxonic acid for
7 weeks, and then were matched based on uric acid level and blood
pressure in various groups to either receive allopurinol, have the
oxonic acid withdrawn from the diet, or continue the oxonic
acid/low salt diet. A control group of six rats were placed on the
low sodium diet alone for 11 weeks. All of these rats were
sacrificed at week 11.
[0056] Systolic blood pressure was measured by tail cuff
sphyngomanometer using an automated system with photoelectric
sensor (IITC, Life Science) that has been shown to closely
correlate with intra-arterial blood pressure measurements [Fischer,
E., Schnermann, J., Briggs, J. P, Kriz, W., Ronco, P. M., and
Bachmann, S., Ontogeny of NO synthase and renin in juxtaglomerular
apparatus of rat kidneys. Am J Physiol 268:F1164-76, 1995].
Example 4
[0057] Rats were placed on either low salt diet, low salt diet with
2% oxonic acid, low salt diet with oxonic acid and enalapril (1
mg/kg/d in drinking water), or low salt diet with oxonic acid and
L-Arginine (1% in drinking water).
[0058] Systolic blood pressure was measured by tail cuff
sphyngomanometer using an automated system with photoelectric
sensor (IITC, Life Science) that has been shown to closely
correlate with intra-arterial blood pressure measurements [Fischer,
E., Schnermann, J., Briggs, J. P., Kriz, W., Ronco, P. M., and
Bachmann, S., Ontogeny of NO synthase and renin in juxtaglomerular
apparatus of rat kidneys. Am J Physiol 268:F1164-76, 1995].
Functional Data:
[0059] Serum and urine uric acid were measured by a carbonate
phosphotungstate method [Henry, R. J., Sobel, C., and Kim, J., A
modified carbonate phosphotungstate method for the determination of
uric acid and comparison with the spectophotometric uricase method.
Am J Clin Pathol 1957; 28:152.]. Serum blood urea nitrogen was
measured by a standard kit (Sigma, St Louis, Mo.).
Renal Immunohistochemistry:
[0060] Renal biopsies were fixed in Methyl-Carnoy's, 10% formalin
or 100% ethanol and embedded in paraffin. The presence of uric acid
crystals was evaluated by staining 4-.mu.m ethanol-fixed sections
with de Galantha and modified Von Kossa stains. Kidney tissue from
rats with acute uric acid nephropathy, induced with oxonic acid and
uric acid administration was used as a positive control [Stavric,
B., Johnson, W. J., and Grice, H. C., Uric acid nephropathy: An
experimental model, Proc. Soc. Exp. Biol. Med. 130: 512-16
(1969).]. Light microscopy was performed in 4-.mu.m sections of
Methyl-Carnoy's fixed tissue stained with periodic acid Schiff
(PAS) reagent or with hematoxylin and eosin.
[0061] Methyl-Carnoy's fixed tissue sections were analyzed by
indirect immunoperoxidase [Lombardi, D., Gordon, K. L., Polinsky,
P., Suga, S., Schwartz, S. M., and Johnson, R. J. Salt sensitive
hypertension develops after transient exposure to angiotensin II.
Hypertension 33:1013-1019, 1999] staining with the following
primary antibodies: OP199, a goat polyclonal antibody against
osteopontin (OPN) (gift of C. Giachelli, Univ of WA, Seattle);
ED-1, a monoclonal antibody to rat monocytes and macrophages
(Serotec); goat antihuman type III collagen (Southern Biotechnology
Associates Inc, Birmingham Ala.); and anti-renin, a mouse antibody
to human renin (Sanofi Recherche, Montpellier, France). Sections
were incubated with a secondary antibody followed by horseradish
peroxidase-conjugated avidin D (Vector Laboratories, Burlingame,
Calif.), diaminobenzidine (Sigma) with or without nickel chloride
as a chromogen, and counsterstained with methyl green.
[0062] NOS1 was detected on formalin fixed tissue sections with a
rabbit anti-rat neuronal nitric oxide synthase (Transduction
Laboratories, Lexington, Ky.), followed by a biotinylated secondary
antibody, diaminobenzidine with nickel chloride and counterstained
with nuclear fast red.
Quantification of Morphologic Data:
[0063] AU quantification was performed blinded. The tubular
expression of osteopontin (OPN), which is a sensitive marker of
tubulointerstitial injury, was calculated as the percent of renal
cortex occupied by OPN-positive tubules [Lombardi, D., Gordon, K.
L., Polinsky, P., Suga, S., Schwartz, S. M., and Johnson, R. J.
Salt sensitive hypertension develops after transient exposure to
angiotensin II. Hypertension 33:1013-1019, 1999]. Utilizing
computer-assisted image analysis software (Optimas V6.2, Media
Cybernetics, Silver Springs, Md.) and digitized images, the percent
of area occupied by OPN positive tubules per 4 mm.sup.2 field at a
magnification of 50.times. was measured and the mean percent area
calculated for each biopsy. The interstitial deposition of collagen
type III was calculated as the % of renal cortex occupied by
collagen III, noted by immunostaining, by computer image analysis.
The mean number of interstitial macrophages (ED-1+ cells) in each
biopsy was calculated in a blinded manner by counting the total
number of positive interstitial cortical cells in 20 sequentially
selected 0.25 mm.sup.2 grids at 200.times. magnification. Renin
expression was quantified by the number of glomeruli with positive
staining for juxtaglomerular renin using a minimum of 100 glomeruli
in each biopsy as previously described [Eng, E., et al., Renal
proliferation and phenotypic changes in rats with two-kidney,
one-clip Goldblatt hypertension. Am J Hypertens 7:177-185 (1994)];
this has been shown previously to correlate with intrarenal renin
content [Eng, E., et al., Renal proliferation and phenotypic
changes in rats with two-kidney, one-clip Goldblatt hypertension.
Am J Hypertens 7:177-185 (1994)]. NOS1 was quantified by a blinded
counting of the number of positive macula densa cells staining with
anti-NOS1 antibody using a minimum of 100 glomeruli per biopsy
[Eng, E., et al., Renal proliferation and phenotypic changes in
rats with two-kidney, one-clip Goldblatt hypertension. Am J
Hypertens 7:177-185 (1994)]. Previous studies have shown that the
number of NOS1 cells correlates with intrarenal NOS1 activity
[Fischer, E., Schnermann, J., Briggs, J. P, Kriz, W., Ronco, P. M.,
and Bachmann, S., Ontogeny of NO synthase and renin in
juxtaglomerular apparatus of rat kidneys. Am J Physiol
268:F1164-76, 1995].
Statistical Analysis
[0064] All values are expressed as mean.+-.standard error, unless
otherwise stated. Statistical significance (p<0.05) was
evaluated by ANOVA and unpaired Student's t test with appropriate
correction for multiple comparisons.
An Animal Model of Mild Hyperuricemia
[0065] An animal model of mild hyperuricemia was developed using
the rat. Several previous groups had reported that hyperuricemia
could be induced in rats by feeding them oxonic acid, which is a
uricase inhibitor [Stavric, B., Johnson, W. J., and Grice, H. C.,
Uric acid nephropathy: An experimental model, Proc Soc Exp Biol Med
130:512-16 (1969)]. In most studies uric acid supplements were also
added to the diet. Unfortunately, this model usually results in a
six to ten-fold increase in serum uric acid levels with marked
uricosuria, resulting in acute renal failure from obstruction of
the renal tubules with urate crystals [Stavric, B., Johnson, W. J.,
and Grice, H. C., Uric acid nephropathy: An experimental model,
Proc Soc Exp Biol Med 130:512-16 (1969)]. Likewise, targeted
deletion of the uricase gene in mice also results in marked
hyperuricemia, intrarenal urate crystal deposition, and renal
failure [Bradley, A., and Caskey, C. T., Hyperuricemia and urate
nephropathy in urate oxidase deficient mice. Proc Natl Acad Sci USA
91:742-746 (1994)]. While these latter models mimic the acute urate
nephropathy syndrome observed in occasional patients with cancers
following chemotherapy (`tumor lysis syndrome`) [Robinson, R. R.,
and Yarger, W. E., Acute uric acid nephropathy Arch Int Med
17:839-840 (1977)], they are inappropriate models for the mild
hyperuricemia observed in patients with cardiovascular disease.
[0066] Hyperuricemia was induced in rats by feeding 2% oxonic acid
in the diet, resulting in a mild increase in the serum uric acid
level. Although urinary uric acid increased two-fold, it was not at
a level sufficient to cause intrarenal crystal deposition (FIG. 1).
Routine light microscopy of the kidney revealed normal histology at
7 weeks, and special stains for uric acid crystals were negative
(FIG. 1). Rats administered the oxonic diet also appeared
completely healthy, although the body weight at the end of the
study (7 weeks) was slightly lower in the hyperuricemic animals
(367.6.+-.17 vs. 394.+-.18 g body weight, oxonic acid diet vs.
control, p<0.05).
Hyperuricemia Induces Blood Pressure Elevation
[0067] A remarkable finding was that rats with hyperuricemia
developed increased blood pressure within 4 weeks after commencing
the diet (FIG. 2). Systolic blood pressures averaged 10 to 30 mm Hg
higher in the hyperuricemic rats compared to controls. The
observation that an elevated uric acid induced an increase in blood
pressure, suggests that it might act to help maintain blood
pressure in conditions associated with a low salt intake, such as
occurred during early hominoid evolution [Eaton, S. B., and Konner,
M., Paleolithic nutrition: A consideration of its nature and
current implications. N Engl J Med 312: 283-289 (1985)]. As shown
in FIG. 3, control rats placed on a modest sodium restricted diet
had a fall in blood pressure within 3 weeks. In contrast,
hyperuricemic rats on a low salt diet showed a significant increase
in blood pressure resulting in 30 to 40 mmHg differences between
groups. Blood pressures showed a direct correlation with uric acid
levels in both experiments (n=52, r=0.7, p<0.0001 for the low
salt study; n=12, r=0.7, p<0.0001 for the normal salt study)
(FIG. 4). An increase of 0.5 mg/dl in uric acid resulted in an
increase in systolic blood pressure of 20 mm Hg (FIG. 4). At uric
acid levels of 2 mg/dl or higher (corresponding to a 50% increase
in uric acid over baseline) blood pressures were in the
hypertensive range (systolic blood pressure>140 mm Hg).
Interestingly, rats on an oxonic acid diet that did not develop
hyperuricemia did not have elevated blood pressures (FIG. 4).
[0068] To document that the elevation in blood pressure was due to
the hyperuricemia and not a nonspecific effect of the oxonic acid,
hyperuricemic rats were co-administered the xanthine oxidase
inhibitor, allopurinol, with the oxonic acid. Allopurinol
administered from the initiation of the oxonic acid diet prevented
the development of hyperuricemia and hypertension (FIGS. 3A and B).
Furthermore, in hypertensive, hyperuricemic rats, either withdrawal
of the oxonic acid or adding allopurinol also resulted in a
reduction in the blood pressure in association with a fall in serum
uric acid values (FIGS. 5A and B).
Mild Hyperuricemia Causes Renal Fibrosis
[0069] In an attempt to understand the mechanism for the
hypertensive effect of hyperuricemia, we carefully examined the
kidneys of the hyperuricemic and control animals. At 7 weeks both
routine light microscopy (FIG. 1) and blood urea nitrogen levels
were normal in the hyperuricemic rats. However, special
immunohistochemical stains showed a striped pattern of early
interstitial fibrosis, with increased deposition of interstitial
collagen, macrophage accumulation, and with tubular expression of
osteopontin, which is a sensitive marker of tubular injury
[Lombardi, D., Gordon, K. L., Polinsky, P., Suga, S., Schwartz, S.
M., and Johnson, R. J., Salt sensitive hypertension develops after
transient exposure to angiotensin II. Hypertension 33:1013-1019,
1999]. The administration of allopurinol from the time the diet was
initiated prevented the development of the fibrotic changes (Table
1).
[0070] A second study was also performed in which allopurinol was
added or oxonic acid withdrawn at 7 weeks and then the rats were
followed for an additional 4 weeks before they were sacrificed
(FIG. 5). In this study the hyperuricemic rats showed more
pronounced renal fibrosis and a statistical increase in blood urea
nitrogen (Table 1) (FIG. 6). Rats in which the hyperuricemia was
treated by either the addition of allopurinol or by the withdrawal
of oxonic acid showed significantly less renal fibrosis and lower
blood urea nitrogen levels (Table 1).
TABLE-US-00001 TABLE 1 Hyperuricemic Rats Develop Renal Injury Type
III ED-1 OPN BUN collagen (%) (cells/mm2) (% increase) (mg/dl)
Example 2: Renal Findings at 7 weeks after Oxonic Acid (OA) in
presence/absence of allopurinol (AP) Control 5.4 .+-. 0.3 18.3 .+-.
1.3 0.9 .+-. 0.06 14.4 .+-. 1.2 (LSD) OA + LSD 8.8 .+-. 1.5* 27.2
.+-. 1.9* 1.8 .+-. 0.06* 23.2 .+-. 2.3 OA/LSD + 6.2 .+-. 0.6 20.5
.+-. 0.6 1.3 .+-. 0.15* 15.2 .+-. 2.3 Allopurinol Example 3. Effect
of Oxonic Acid (OA) withdrawal or addition of Allopurinol (AP) at 7
weeks on Renal Findings at 11 weeks. Control 7.2 .+-. 0.6 27.8 .+-.
1.4 0.7 .+-. 0.08 16.6 .+-. 0.9 (LSD) OA + LSD 13.9 .+-. 0.6* 36.5
.+-. 1.4* 1.33 .+-. 0.14* 24.1 .+-. 1.7* OA 8.9 .+-. 33.0 .+-. 1.6*
1.08 .+-. 0.06* 18.8 .+-. 0.8 withdrawal 1.6* OA/LSD + 9.6 .+-.
25.2 .+-. 2.6 0.97 .+-. 0.03* 17.7 .+-. 0.5 Allopurinol 0.3*
Abbreviations: BUN, blood urea nitrogen; ED-1, macrophages; LSD,
low NaCl diet (0.125%); OA, oxonic acid; TI, tubulointerstitial. *p
< .05 vs. control. 24.1 .+-.1.7* p < 0.05 vs LSD/OA.
Hyperuricemia Activates the Renin Angiotensin System and Inhibits
Intrarenal Neuronal Nitric Oxide Synthase
[0071] The `striped` fibrotic pattern of renal injury is
characteristic of chronic vasoconstriction and/or ischemia, which
is of interest given Messerli's observation that hyperuricemia in
man is associated with renal vasoconstriction [Messerli, F. H.,
Frohlich, E. D., Dreslinski, G. R., Suarez, D. H., and Aristimuno,
G. G., Serum uric acid in essential hypertension: An indicator of
renal vascular involvement. Ann Int Med 93:817-821, 1980.]. The
renal expression of two important vasoactive mediators were
examined in these rats (Table 2). The percentage of glomeruli with
juxtaglomerular renin staining was markedly increased in the
hyperuricemic animals, a finding that correlates with increased
renal renin content [Eng, E., et al., Renal proliferation and
phenotypic changes in rats with two-kidney, one-clip Goldblatt
hypertension. Am J Hypertens 7:177-185 (1994)]. There was also a
direct correlation of serum uric acid levels with the percentage of
renin-positive glomeruli, both in the studies using a low salt diet
(r=0.7, n=18, p=0.0006, FIG. 7) and in the study using a normal
salt diet (r=0.6, n=12, p=0.05). Interestingly, Saito et al., have
previously reported that uric acid levels correlate with plasma
renin activity in patients with essential hypertension [Saito, I.,
et. al. Serum uric acid and the renin-angiotensin system in
hypertension. J Am Geriatrics Soc 26:241-2471976.].
[0072] The effect of hyperuricemia on neuronal nitric oxide
synthase (NOS1) expression in the macula densa, which is involved
in regulating afferent arteriolar tone and tubuloglomerular
feedback were also examined. As shown in Table 2, the number of
neuronal nitric oxide synthase (NOS1) positive cells in the macula
densa was decreased in hyperuricemic rats. This is particularly
relevant, as chronic inhibition of NOS1 has been reported to
elevate blood pressure in rats [Ollerstam, A. Pittner, J., Persson
E. G., and Thorup, C., Increased blood pressure in rats after
long-term inhibition of the neuronal isoform of nitric oxide
synthase. J Clin Invest 99:2212-2218, 1997.]. As with renin, the
decrease in NOS-1 positive cells was largely prevented by
allopurinol treatment (Table 2).
[0073] To further document a role for these mediators, we
administered enalapril, an angiotensin converting enzyme inhibitor,
or L-Arginine, which is a substrate for nitric oxide production, to
the hyperuricemic rats from the outset. As shown in FIG. 8,
hyperuricemic control rats have an approximately 20 mm Hg increase
in systolic blood pressure over the 7 week dietary period.
L-Arginine treatment largely prevented this increase.
Enalapril-treated hyperuricemic rats had the lowest systolic blood
pressures. At 7 weeks, the systolic blood pressures in the
L-Arginine and enalapril groups averaged 25 mm Hg lower than the
hyperuricemic controls (p<0.05). This suggests that the
hypertension and renal disease induced by hyperuricemia are
dependent on both angiotensin II and file nitric oxide system.
TABLE-US-00002 TABLE 2 Hyperuricemia Induces Changes in Vasoactive
Mediators NOS-1 Renin (positive cells/100 (% positive JGA)
glomeruli) Example 2. Renal Findings at 7 weeks after Oxonic Acid
(OA) in presence/absence of Allopurinol (AP) Control (LSD) 39.6
.+-. 2.0 147.2 .+-. 12.4 OA + LSD 60.9 .+-. 1.5* 80.4 .+-. 4.3*
OA/LSD + Allopurinol 44.1 .+-. 1.6 97.4 .+-. 5.6* Example 3. Effect
of Oxonic Acid (OA) withdrawal or addition of Allopurinol (AP) at 7
weeks on Renal Findings at 11 weeks. Control (LSD) 41.0 .+-. 1.9
104.4 .+-. 11.5 OA + LSD 58.4 .+-. 0.8* 65.6 .+-. 7.1* OA
withdrawal 50.5 .+-. 2.1* 83.1 .+-. 10.8 OA/LSD + Allopurinol 44.2
.+-. 1.3 98.6 .+-. 5.1 Abbreviations: AP, allopurinol; glom,
glomeruli; LSD, low NaCl diet (0.125%); OA, oxonic acid; MD, macula
densa. *p < .05 vs. control. p < 0.05 vs LSD/OA.
Example 5
Animals
[0074] Studies were conducted in 20 adult male Sprague-Dawley rats
(Simmonsen Laboratories, Gilroy, Calif., USA) weighing 200 to 250
grams. All rats were fed a low salt diet (0.125% NaCl) (Zeigler
Bros, Gardners, Pa.), with water ad libitum. The use of low salt
diet has been shown to accelerate the development of CSA
nephropathy [13,26]. In order to induce hyperuricemia, oxonic acid
2% was added to low salt diet. Because rats have uricase, an
hepatic enzyme which degrades uric acid to allantoin, the blockade
of this enzyme by oxonic acid is necessary.
Experimental Design
[0075] After one week on a low salt diet, weight-matched rats were
randomly divided into four groups: [0076] Group 1 (Vehicle (VH);
n=6): These rats received a daily subcutaneous (SC) injection of
olive oil, for 7 weeks. [0077] Group 2 (Oxonic acid plus vehicle
(OA); n=4): these rats received a daily SC injection of olive oil,
1 mg/kg, and a supplement of 2% oxonic acid in their chow, for 7
weeks. [0078] Group 3 (Cyclosporine (CSA); n=6): these rats
received a daily injection of cyclosporine 15 mg/kg, for 7 weeks.
[0079] Group 4 (Cyclosporine plus oxonic acid (CSA-OA); n=4): these
rats received a daily injection of cyclosporine 15 mg/kg, and a
supplement of 2% oxonic acid in their chow for 7 weeks.
[0080] After 7 weeks, rats were placed in individual metabolic
cages for 24-hour urine collection. The following day, rats were
anesthetized with xylazine and ketamine, serum was collected for
creatinine and uric acid measurements, and both kidneys were
obtained for histology evaluation. Biopsies were fixed in 10%
formalin, 100% ethanol or Methyl Carnoy's.
Functional Data
[0081] Serum and urine creatinine were measured by a standard
picric acid method (Sigma Diagnostics creatinine kit, St. Louis,
Mo.). Serum and urine uric acid were measured by a modified
carbonate-phosphotungstate method [Henry, R. J., Sobel, C., and
Kim, J., A modified carbonate phosphotungstate method for the
determination of uric acid and comparison with the
spectophotometric uricase method. Am J Clin Pathol 1957; 28:152.].
Fractional uric acid excretion and creatinine clearance were
calculated by standard formulas. Cyclosporine levels were measured
by high performance liquid chromatography (HPLC) of whole
blood.
Drugs
[0082] Cyclosporine (Novartis) was diluted in olive oil to a final
concentration of 15 mg/ml and injected SC in a dose of 15 mg/kg of
body weight.
Renal Morphology and Immunohistochemistry
[0083] Methyl Carnoy's fixed tissue was processed and paraffin
embedded, and 4 .mu.m sections were stained with PAS reagents and
hematoxilin-eosin. Alcohol-fixed tissue was processed and paraffin
embedded, and 4 .mu.m sections were stained for uric acid crystals
by de Galantha's and Von Kossa stains. The positive control was
kidney tissue from a rat with acute uric acid nephropathy, induced
with oxonic acid and uric acid administration [Waisman, J.,
Bluestone, R., and Klinemberg, J. R., A preliminary report of
nephropathy in hyperuricemic rats. Lab Invest 1974; 30:716,
Bluestone, J., Waisman, J., Klinemberg, J. R., Chronic experimental
hyperuricemia nephropathy. Biochemical and morphological
characterization. Lab Invest 1975; 33(3): 273, and Waisman, J.,
Mwasi, L. M., Bluestone, R., and Klinemberg, J. R., Acute
hyperuricemic nephropathy in rats. An electron microscopy study. Am
J Pathol 1975; 81(2): 367.].
[0084] Methyl-Carnoy's fixed tissue sections were analyzed by
indirect immunoperoxidase with primary antibodies against
osteopontin (OP199, gift of C. Giachelli, University of Washington,
Seattle, Wash.), monocytes and macrophages, ED-1, Serotec, Oxford,
UK) and collagen type III (Southern Biotechnology Associates Inc,
Birmingham, Ala., USA).
Quantification of Morphologic Data
[0085] Interstitial fibrosis was scored semi quantitatively on
biopsies stained with PAS and hematoxilin-eosin, using the
following scoring system: zero=normal interstitium and tubules,
1=mild fibrosis with minimal thickening between the tubules,
2=moderate fibrosis with moderate interstitial thickening between
the tubules, 3=severe fibrosis with severe interstitial thickening
between the tubules.
[0086] The tubular expression of osteopontin (OPN), which is a
sensitive marker of tubulointerstitial injury [Giachelli, C. M.,
Pichler, R., and Lombardi, D., Osteopontin expression in
angiotensin II-induced tubulointerstitial nephritis. Kidney Int
1994; 45: 515, and Thomas, S. E., Lombardi, D., Giachelli, C.,
Bohle, A., and Johnson, R. J., Osteopontin expression,
tubulointerstitial disease and essential hypertension. Am J
Hypertens 1998; 11:954.], was calculated as the percentage (%) of
renal cortex occupied by OPN-positive tubules [Johnson, R. J.,
Alpers, C. E., Yoshimura, A., et al., Renal injury from angiotensin
II mediated hypertension. Hypertension 1992; 19: 464.], utilizing
computer-assisted image analysis software (Optimas V6.2, Media
Cybernetics, Silver Systems Md.) and digitized images. The % area
occupied by OPN positive tubules per 4 mm.sup.2 field at 50.times.
was measured and the mean % area calculated for each biopsy. The
same method was used to quantify the interstitial expression of
collagen III.
[0087] The mean number of interstitial macrophages (ED-1+
cells/mm.sup.2) in each biopsy was calculated in a blinded manner
by counting the total number of positive cells in 20 sequentially
selected 0.25 mm.sup.2 grids at 200.times. magnification. The
number of macrophages per glomerular cross section (utilizing a
minimum of 100 glomeruli per biopsy) was also determined.
Statistical Analysis
[0088] All values are expressed as mean.+-.SD, unless otherwise
stated. The differences between groups were compared with unpaired
Student's t tests.
TABLE-US-00003 TABLE 3 Body weight, renal function and uric acid
levels at 7 weeks of study Group 1 Group 2 Group 3 Group 4 Weight
(grams) 422.2 .+-. 31.2 421.2 .+-. 28.8 .sup. 357.2 .+-.
28.8.sup.a,b .sup. 351.2 .+-. 10.7.sup.a,b Serum Uric Acid (mg/dl)
1.62 .+-. 0.31 .sup. 4.08 .+-. 1.22.sup.a .sup. 3.15 .+-.
0.85.sup.a 5.90 .+-. 1.55.sup.a,b,c Urinary Uric Acid (mg/day) 2.19
.+-. 0.66 .sup. 5.06 .+-. 2.81.sup.a 3.05 .+-. 1.00 .sup. 4.74 .+-.
3.22.sup.a Urate/creatinine (urine) 0.20 .+-. 0.08 0.35 .+-. 0.27
0.18 .+-. 0.09 0.23 .+-. 0.13 FE urate 0.12 .+-. 0.06 0.11 .+-.
0.14 0.07 .+-. 0.04 0.06 .+-. 0.04 Serum Creatinine (mg/dl) 0.94
.+-. 0.24 0.88 .+-. 0.19 .sup. 1.35 .+-. 0.52.sup.b .sup. 1.42 .+-.
0.32.sup.b Creatnine Clearance (ml/min) 1.39 .+-. 0.73 1.94 .+-.
0.73 .sup. 0.96 .+-. 0.43.sup.b .sup. 0.79 .+-. 0.29.sup.b
Cyclosporine (ng/dl) 4560.0 .+-. 602.0 4765.0 .+-. 486.0 .sup.ap
< 0.05 compared to Group 1; .sup.bp < 0.05 compared to Group
2; .sup.cp < 0.05 compared to Group 3. FE urate = fractional
excretion of uric acid
Uric Acid
[0089] Serum uric acid in control rats on a low salt diet was
1.6.+-.0.3 mg/dl (Table 3). In rats receiving CSA alone, the uric
acid levels were increased almost 2-fold, and were similar to the
levels in vehicle rats in which uricase was blocked by oxonic acid
(OA). Serum uric acid levels were highest in rats treated with CSA
and oxonic acid (CSA-OA). (Table 3).
[0090] Urinary uric acid excretion was increased in rats fed oxonic
acid (OA alone and CSA-OA groups). CSA treated rats (CSA alone and
CSA-OA) had urate/creatinine ratios comparable to normal controls
and the fractional urate excretion tended to be lower than either
vehicle or OA alone groups (p=0.06). (Table 3).
Cyclosporine Levels
[0091] CSA was measured by HPLC in whole blood at 7 weeks. No
difference in CSA levels was observed between CSA and CSA-OA rats.
(Table 3).
Renal Function
[0092] Glomerular filtration rate (GFR) evaluated by serum
creatinine and creatinine clearance, were reduced in both. CSA and
CSA-OA groups, but no statistical difference was observed between
CSA and CSA-OA groups (Table 3).
Histological Analysis
Tubulointerstitial and Micro Vascular Changes
[0093] Rats treated with CSA for 7 weeks displayed classic
histological findings of chronic CSA nephropathy, with arteriolar
hyalinosis, tubular dilatation and atrophy in a stripped pattern
extending from medulla to cortex (FIG. 9A). Similar histological
findings were observed in CSA-OA rats, except that the changes were
more severe, including arteriolar hyalinosis (61.8% vs 44.8%, CSA
vs. CSA-OA, p<0.05) (FIG. 9B). In contrast, no significant
tubulointerstitial changes were noted by light microscopy of PAS
stained sections from VEH or OA alone rat kidneys.
Osteopontin Expression
[0094] Osteopontin is a macrophage-adhesive protein that is
expressed by tubules in CSA nephropathy and has been shown to
correlate with the macrophage infiltration, tubulointerstitial
fibrosis and renal function [Pichler, R., Franceschini N., Young,
B. A. et al., Pathogenesis of cyclosporine nephropathy. Roles of
angiotensin II and osteopontin. J Am Soc Nephrol 1995; 6: 1186.].
Whereas minimal osteopontin is expressed in normal (VEH) control
rats, a significant increase was observed in rats treated with CSA
(FIG. 9C). The highest expression was observed in CSA-OA treated
rats (FIGS. 9D and 10).
Macrophage Accumulation
[0095] The marked increase in OPN expression in CSA and CSA-OA
treated rats was associated with accumulation of ED-1+ macrophages
in the interstitium (FIG. 9E). Similar to the findings of OPN,
CSA-OA had a greater number of macrophages than CSA alone
(395.6.+-.92.5 vs. 271.9.+-.43.4 ED-1+ cells/mm.sup.2, p<0.05).
In addition, CSA treated rats exhibited a mild glomerular
macrophage accumulation, which was more pronounced in CSA-OA
treated animals (1.8.+-.0.5 vs. 3.5.+-.1.7 ED-1+ cells/glomerular
cross section, p<0.05) (FIGS. 9E and 10).
Type III Collagen Deposition
[0096] In normal kidney, type III collagen was minimally present in
renal cortex, with slight accumulation around interlobular arteries
and veins. In rats on CSA, type III collagen was increased in both
the cortex and subcapsular area, and displayed a striped
interstitial pattern similar to that observed by routine light
microscopy (FIG. 9G). This general pattern was more severe in
CSA-OA treated rats (FIGS. 9H and 10).
Crystal Deposition
[0097] In order to determine if the increased tubulointerstial
injury observed in CSA-OA was associated with intrarenal crystal
deposition, alcohol-fixed tissue was stained for uric acid crystals
using both the De Galantha and modified Von Kossa stain. Whereas
crystals could easily be identified in positive control tissue from
rats with acute urate nephropathy induced by a combination of
oxonic acid and uric acid, no crystals were present in any of the
experimental groups.
Example 6
Uric Acid Excretion Activity (Excerpted from U.S. Pat. No.
5,260,322, Columns 41-42)
[0098] Twenty-four (24) male adults (25 to 48 years old, 161 cm to
187 cm tall, weighing 48 kg to 85 kg) were divided into 4 groups, 6
per group. Compound No. 9 [(COZAAR (losartan potassium)] was orally
administered under hunger in the form of capsules in Example 2, in
a definite dose (25 mg, 50 mg, 100 mg or 200 mg) per person, by
varying the dose in each group. Further in order to examine
influence of diet on uric acid excretion increasing activity of
Compound No. 9, the capsule of Example 2 containing 100 mg of
Compound No. 9 was orally administered at 2 weeks after the test
under hunger was completed. Concentration of uric acid in urine and
blood was determined by the uricase-POD method at every definite
period of time after the administration. The results are shown in
Tables 11 through 14.
[0099] As is clear from Tables 11 through 14, the concentration of
uric acid in serum decreased in 4 hours after medication
dose-dependently. However, a tendency that the uric acid
concentration was recovered to the concentration level prior to
medication was noted 24 hours after. On the other hand, when
medicated after meals, the concentration of uric acid in serum was
kept as it decreased even 24 hours after.
[0100] The uric acid concentration in urine dose-dependently
increased from 0 to 4 hours by administering Compound No. 9 in
doses of 25 mg, 50 mg and 100 mg per person. In the dose of 200 mg,
however, the uric acid concentration in urine did not increase
dose-dependently but was kept almost constant. On the other hand,
when medicated after meals, the uric acid concentration in urine
increased in 0 to 8 hours.
[0101] The foregoing results reveal that the non-peptide type
compounds having an angiotensin II receptor-antagonizing activity
in accordance with the present concentration in blood and
increasing excretion of uric acid into urine. Accordingly, the
non-peptide type compounds having an angiotensin II
receptor-antagonizing activity in accordance with the present
invention are useful as drugs for the prevention or treatment of
hyperuricemia.
TABLE-US-00004 TABLE 11 Change of uric acid concentration in serum
with passage of time when administered in hunger Dose (mg/man)
Concentration of Uric Acid (mg/dl) Time (hr) 25 50 100 200 0 (when
administered) 5.2 .+-. 0.5 6.1 .+-. 1.4 5.9 .+-. 0.9 5.6 .+-. 0.7 4
4.8 .+-. 0.6 5.3 .+-. 1.3 4.6 .+-. 0.7 4.3 .+-. 0.9 24 4.6 .+-. 0.6
5.6 .+-. 1.4 5.2 .+-. 0.8 5.0 .+-. 0.9
TABLE-US-00005 TABLE 12 Change in uric acid concentration in serum
with passage of time after meal Dose 100 mg/man Time (hr)
Concentration of Uric Acid (mg/hr) 0 (when administered) 5.8 .+-.
1.1 4 4.9 .+-. 1.0 24 4.7 .+-. 0.9
TABLE-US-00006 TABLE 13 Change of uric acid excretion in urine with
passage of time when administered in hunger Dose (mg/man)
Concentration of Uric Acid (mg/hr) Time (hr) 25 50 100 200 0-4 43.0
.+-. 24.5 52.8 .+-. 4.3 81.2 .+-. 15.7 78.7 .+-. 15.3 4-8 32.4 .+-.
14.7 42.8 .+-. 8.5 36.4 .+-. 7.7 25.4 .+-. 6.6 8-12 28.7 .+-. 13.6
39.1 .+-. 4.4 30.1 .+-. 6.8 19.6 .+-. 5.2 12-24 19.7 .+-. 9.9 22.2
.+-. 3.8 19.2 .+-. 4.2 13.4 .+-. 2.3 24-40 33.2 .+-. 21.9 26.6 .+-.
5.4 28.0 .+-. 7.2 21.0 .+-. 3.0
TABLE-US-00007 TABLE 14 Change in uric acid excretion in urine with
passage of time after meal Dose 100 mg/man Time (hr) Concentration
of Uric Acid (mg/hr) 0-4 75.9 .+-. 19.0 4-8 59.0 .+-. 3.8 8-12 31.8
.+-. 4.5 12-24 19.7 .+-. 2.5 24-40 29.5 .+-. 4.1
* * * * *