U.S. patent application number 11/995943 was filed with the patent office on 2010-05-13 for compositions and methods for treatment and prevention of hyperuricemia related health consequneces.
Invention is credited to Richard J. Johnson, Takahiko Nakagawa.
Application Number | 20100120796 11/995943 |
Document ID | / |
Family ID | 35786704 |
Filed Date | 2010-05-13 |
United States Patent
Application |
20100120796 |
Kind Code |
A1 |
Johnson; Richard J. ; et
al. |
May 13, 2010 |
COMPOSITIONS AND METHODS FOR TREATMENT AND PREVENTION OF
HYPERURICEMIA RELATED HEALTH CONSEQUNECES
Abstract
Disclosed herein are methods of delaying the onset or treating
diabetes that comprises administering a uric acid lowering agent.
The inventors have made the remarkable discovery that elevated uric
acid levels are not a corollary to insulin resistance, but rather a
primary mediator of insulin resistance. Specifically exemplified
are methods that involve administering to a patient susceptible to
development of diabetes a composition comprising a uric acid
lowering agent in a regimen that maintains serum uric acid levels
below at least 5.5 mg/dl, or below at least 5.2 mg/dl.
Inventors: |
Johnson; Richard J.;
(Gainesville, FL) ; Nakagawa; Takahiko;
(Gainesville, FL) |
Correspondence
Address: |
Beusse Wolter Sanks Mora & Maire
390 N. ORANGE AVENUE, SUITE 2500
ORLANDO
FL
32801
US
|
Family ID: |
35786704 |
Appl. No.: |
11/995943 |
Filed: |
May 31, 2006 |
PCT Filed: |
May 31, 2006 |
PCT NO: |
PCT/US06/20998 |
371 Date: |
January 17, 2008 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
PCT/US05/25910 |
Jul 21, 2005 |
|
|
|
11995943 |
|
|
|
|
60589921 |
Jul 21, 2004 |
|
|
|
Current U.S.
Class: |
514/262.1 ;
514/365 |
Current CPC
Class: |
A61K 31/4439 20130101;
A61P 13/02 20180101; A61K 31/426 20130101; A61K 31/519
20130101 |
Class at
Publication: |
514/262.1 ;
514/365 |
International
Class: |
A61K 31/519 20060101
A61K031/519; A61K 31/425 20060101 A61K031/425 |
Claims
1. A method of preventing or delaying the onset of insulin
resistance in a patient comprising: determining said patient's
average serum uric acid level; and administering to said patient a
composition comprising UALA according to a regimen effective to
maintain said patient's average serum uric acid level at or below
5.5 mg/dl.
2. The method of claim 1, further comprising determining said
patient's average serum uric acid occurs prior to said
administering.
3. The method of claim 1, wherein said composition is administered
over the course of at least one week.
4. The method of claim 1, wherein said composition is administered
over the course of at least 2 weeks.
5. The method of claim 1, wherein said composition is administered
over the course of at least 4 weeks.
6. The method of claim 1, wherein said composition is administered
according to a regimen to maintain the average serum uric acid
level equal to or below 5.5 mg/dl for at least 2 weeks.
7. The method of claim 6, wherein regimen is designed to maintain
the average serum uric acid level equal to or below 5.5 mg/dl for
at least 4 weeks.
8. The method of claim 6, wherein regimen is designed to maintain
the average serum uric acid level equal to or below 5.5 mg/dl for
at least 8 weeks.
9. The method of claim 6, wherein regimen is designed to maintain
the average serum uric acid level equal to or below 5.5 mg/dl for
at least 24 weeks.
10. The method of claim 6, wherein regimen is designed to maintain
the average serum uric acid level equal to or below 5.5 mg/dl for
at least 2 years.
11. The method of claim 1, wherein said composition is administered
according to a regimen to maintain average serum uric acid levels
between about 3.5 mg/dl to about 5.5 mg/dl for at least 12
weeks.
12. The method of claim 11, wherein said composition is
administered according to a regimen to maintain average serum uric
acid levels between about 3.5 mg/dl to about 5.5 mg/dl for at least
1 year.
13. A method of preventing, delaying the onset of, or treating
insulin resistance of a patient comprising: determining said
patient's average serum uric acid level; and administering to said
patient a composition comprising UALA according to a regimen
effective to maintain said patient's average serum uric acid level
between about 4.0 mg/dl and 5.5 mg/dl for at least 4 weeks.
14. The method of claim 13, wherein said administering occurs
according to a regimen effective to maintain said patient's average
serum uric acid level between about 4.0 mg/dl and 5.5 mg/dl for at
least 12 weeks.
15. The method of claim 13, wherein said administering occurs
according to a regimen effective to maintain said patient's average
serum uric acid level between about 4.0 mg/dl and 5.5 mg/dl for at
least 36 weeks.
16. A composition comprising UALA and at least one antioxidant.
17. A combination therapy comprising the administration
concomitantly, simultaneously or sequentially, of therapeutically
effective amounts of a combination of UALA and antioxidant.
18. The combination therapy of the claim 17, wherein UALA is
administered according to a dosage to lower a patient's average
serum uric acid level below 5.5 mg/dl.
19. The combination therapy of claim 17 wherein UALA is
administered according to a regimen effective to maintain said
patient's average serum uric acid level between about 4.0 mg/dl and
5.5 mg/dl for at least 2 weeks.
20. A method of reducing the risk of developing, delaying the onset
of, or treating metabolic syndrome in a patient in need thereof
comprising administering to said patient a composition comprising
UALA according to a regimen effective to maintain said patient's
average serum uric acid level below 5.5 mg/dl for at least 12
weeks.
21. The method of claim 20, wherein said patient is determined to
have asymptomatic hyperuricemia.
22. A method of reducing the risk of developing, delaying the onset
of, or treating insulin resistance in patient in need thereof
comprising administering to said patient a composition comprising
UALA according to a regimen effective to maintain said patient's
average serum uric acid level between about 4.0 mg/dl and 5.5 mg/dl
for at least 12 weeks.
23. The method of claim 22, wherein said patient is determined to
have asymptomatic hyperuricemia.
24. A method of reducing the risk of developing, delaying the onset
of, or treating obesity associated with metabolic syndrome in a
patient in need thereof comprising administering to said patient a
composition comprising UALA according to a regimen effective to
maintain said patient's average serum uric acid level below 5.5
mg/dl for at least 12 weeks.
25. A method of reducing the risk of developing
hyperuricemia-induced health consequences in a patient experiencing
asymptomatic hyperuricemia with a uric acid level higher than 5.5
mg/dl, said method comprising administering to said patient a
composition comprising UALA according to a regimen effective to
maintain said patient's average serum uric acid level at or below
5.5 mg/dl for at least 12 weeks.
26. The method of claim 25, wherein said administering occurs
according to a regimen effective to maintain said patient's average
serum uric acid level between about 4.0 mg/dl and 5.5 mg/dl for at
least 52 weeks.
27. The method of claim 25, wherein said hyperuricemia-induced
health consequences are insulin resistance, obesity,
hypertriglyceridemia, nonalcoholic fatty liver disease, metabolic
syndrome or diabetic nephropathy.
28. A method of delaying the onset of or reducing the risk of
developing diabetic nephropathy of a patient with diabetes and a
uric acid level higher than 5.5 mg/dl, said method comprising
administering to said patient a composition comprising UALA
according to a regimen effective to maintain said patient's average
serum uric acid level below 5.5 mg/dl for at least 12 weeks.
29. The method of claim 27, wherein said UALA is allopurinol or
febuxostat, or both.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation in part of International
Application No. PCT/US05/25910; filed Jul. 21, 2005. This
application also claims benefit of the Jul. 21, 2004, filing date
of U.S. provisional patent application No. 60/589,921.
BACKGROUND OF THE INVENTION
[0002] Diabetes mellitus is characterized by a broad array of
physiologic and anatomic abnormalities, for example, altered
glucose disposition, hypertension, retinopathy, abnormal platelet
activity, aberrations involving large, medium and small sized
vessels, and other problems encountered in diabetic patients.
Diabetes is classified into two categories: primary and secondary.
Primary diabetes includes: 1) Insulin-dependent diabetes mellitus
(IDDM, Type 1), 2) Non-insulin-dependent diabetes mellitus (NIDDM),
Type 2) including a) Nonobese NIDDM, b) Obese NIDDM and c)
Maturity-onset diabetes of the young. Primary diabetes implies that
no associated disease is present, while in the secondary diabetes
some other identifiable condition causes or allows a diabetic
syndrome to develop, for example, 1) Pancreatic disease, 2)
Hormonal abnormalities, 3) Drug or chemical induced, 4) Insulin
receptor abnormalities, 5) Genetic syndromes and 6) Others.
[0003] Insulin dependence in this classification is not equivalent
to insulin therapy, but means that the patient is at risk for
ketoacidosis in the absence of insulin. It has been suggested that
the terms insulin-dependent and non-insulin-dependent describe
physiologic states (ketoacidosis-prone and ketoacidosis-resistant,
respectively), while the terms Type 1 and Type 2 refer to
pathogenetic mechanisms (immune-mediated and non-immune-mediated,
respectively). Using this classification, three major forms of
primary diabetes are recognized: (1) type 1 insulin-dependent
diabetes, (2) type 1 non-insulin-dependent diabetes, and (3) type 2
non-insulin-dependent diabetes.
[0004] Secondary forms of diabetes encompass a host of conditions
such as pancreatic disease, hormonal abnormalities, genetic
syndromes, and others.
[0005] Insulin-dependent diabetes mellitus often develops in
childhood or adolescence while the onset of NIDDM generally occurs
in middle or late life. Patients with NIDDM are usually overweight
and constitute 90 to 95 percent of all diabetics. IDDM results from
the destruction of beta cells by an autoimmune process that may be
precipitated by a viral infection. NIDDM is characterized by a
gradual decline in beta cell function and varying degrees of
peripheral resistance to insulin. The annual incidence of IDDM
ranges from 10 cases per 100,000 persons for nonwhite males to 16
cases per 100,000 persons for white males. LaPorte, R. E. et al.,
1981, Diabetes 30: 279. The prevalence of NIDDM increases with age,
especially after age 45 and is higher among blacks than whites and
certain populations such as Asian Indians living in South Africa
and England. Matter, H. M. et al., 1985, Br. Med. J. 291: 1081.
Gestational diabetes occurs in 2.4 percent of all pregnancies in
the United States annually. Freinkel, N. et al., 1985, N. Engl. J.
Med. 313: 96. Pregnancy is also a state of insulin resistance. This
insulin resistance is exacerbated in gestational diabetes which may
predispose patients to the various hypertensive syndromes of
pregnancy associated with Type 2 NIDDM. Bardicef, M. et al., 1995,
Am. J. Gynecol. 172: 1009-1013.
[0006] Current therapies for IDDM include insulin therapy, and for
NIDDM will include dietary modification in a patient who is
overweight and hypoglycemic agents, e.g., tolbutamide,
chlorpropamide, acetohexamide, tolazamide, glipizide and glyburide,
all of which act by stimulating the release of insulin from the
beta cells. Also, thiazolidone drugs like rosiglitazone are being
used to treat insulin resistance.
[0007] Insulin resistance and hyperuricemia are considered a part
of the `metabolic syndrome` or `syndrome X` of obesity, insulin
resistance, hypertriglyceridemia and hyperuricemia, which underlies
the pathogenesis of type II diabetes. Insulin resistance is an
impaired metabolic response to our body's own insulin so that
active muscle cells cannot take up glucose as easily as they
should. The condition can exist unrecognized and metabolic damage
can occur before a full blown Type 2 diabetes is finally diagnosed.
Insulin resistant diabetics are 2-5 times more likely to die from
heart attack or stroke than are non diabetics. Currently metabolic
syndrome is epidemic both in the United States and throughout the
world, resulting in exponential increases in health care cost and
causing great morbidity and mortality due to the increased risk for
cardiovascular and renal disease in this population. Most studies
suggest that the epidemic is due to the adaptation of `Westernized
diet` this diet is also known to increase our risk for gout
(Johnson R J, Rideout B: Uric acid and diet: insights into the
Epidemic of Cardiovascular Disease. N Engl J Med (editorial) 2004;
350:1071-1074).
[0008] It has widely been assumed that the rise in serum uric acid
associated with insulin resistance is due to the effect of insulin
to increase urate reabsorption in the renal tubule, and hence it
had been assumed that the hyperuricemia associated with insulin
resistance does not have a causal role in the syndrome.
SUMMARY OF THE INVENTION
[0009] The inventors have made the remarkable discovery that
elevated levels of uric acid is a primary mediator of insulin
resistance. The subject invention provides a new approach to
combating the epidemic of the metabolic syndrome. In one
embodiment, the subject invention provides an approach to
preventing and/or treating one or more metabolic syndrome related
characteristics.
[0010] In a specific embodiment, the subject invention pertains to
methods of administering a uric acid lowering agent (UALA) to a
patient susceptible to developing insulin resistance or suffering
from insulin resistance. As part of the medical treatment, serum
samples may be obtained and tested so that serum uric acid levels
may be monitored in conjunction with the administration of the
UALA.
[0011] In another embodiment, the subject invention provides an
approach to preventing and/or treating metabolic syndrome related
obesity. In a specific embodiment, the subject invention pertains
to methods of administering a uric acid lowering agent (UALA) to a
patient susceptible to developing or suffering from metabolic
syndrome related obesity.
[0012] In another embodiment, the subject invention provides an
approach to reducing the risk of developing, delaying the onset of
and/or treating nonalcoholic fatty liver disease.
[0013] In another embodiment, the subject invention provides an
approach to reducing the risk of developing, delaying the onset of
and/or diabetic nephropathy.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 is a graph showing the relationship of serum uric
acid and serum nitrites at 1 and 7 Days of hyperuricemic induced
rats. Serum was analyzed for uric acid concentration and
nitrites/nitrates (NO.sub.X) by chemiluminescence method.
[0015] FIG. 2 represents a graph that shows the linear correlation
of serum uric acid and serum nitrites.
[0016] FIG. 3. Survival rate of diabetic eNOS KO mice at 5 months.
NonDM; non diabetic mice. DMIns; diabetes with insulin
treatment.
[0017] FIG. 4. Histology in glomeruli from C57BL6 and eNOS KO mice.
(A-K; X 1000, L; X 2000) (A) glomerulus in non-diabetic C57BL6 mice
at 3 months. (B) diabetic C57BL6 mice at 3 months, (C) non-diabetic
eNOS KO mice at 3 months. (D) diabetic eNOS KO mice at 3 months.
Mesangiolysis can be observed in glomerulus. (E) Glomerular
microaneurysm in diabetic eNOS KO mice at 3 months. (F) Nodular
glomerular expansion in diabetic eNOS KO mice at 5 months. (G)
Nodular lesion with acellular PAS-positive material in diabetic
eNOS KO mice at 3 months. (H) Mesangiolysis Diffuse
glomerulosclerosis with fibrillar mesagial matrix in diabetic eNOS
KO mice at 3 months. (I) Arterioler hyalinosis (arrow) associated
with glomerular mesangiolysis in diabetic eNOS KO mice at 5 months.
(J) Nodular glomerulosclerosis (arrow) in diabetic eNOS KO mice at
5 months. (K) Nodular glomerulosclerosis on PAM staining in serious
section of (J) in diabetic eNOS KO mice at 5 months. (L) (X 2000)
Hyalinosis (arrow) at vascular pole of glomerulus in diabetic eNOS
KO mice at 5 months. Size Bar; 10 .mu.m
[0018] FIG. 5. Correlation between blood glucose and renal injury
at 3 months. (A) Mesangial expansion in C57BL6 and eNOS KO mice.
(B) Mesangiolysis in C57BL6 and eNOS KO mice
[0019] FIG. 6. Arteriolar lesion in diabetic eNOS KO mice at 3
months. (A) Inner lumen size in afferent arteriole. a; p<0.01
vs. C57BL6, b; p<0.05 vs. non-DM in eNOS KO mice. (B) Inner
lumen of afferent arteriole in diabetic eNOS KO mice. Glomerulus
with mesangiolysis is associated with dilated arteriole. (C) Wall
area of afferent arteriole. (D) Wall area of arteriole in diabetic
eNOS KO mice (E) Immunohistochemistry for Smooth muscle actin (SMA)
in afferent arteriole in non-DM C57BL6 (X1000). Brown color
indicates SMA staining. (F) Immunohistochemistry for Smooth muscle
actin (SMA) (Brown color) in afferent arteriole in DM eNOS KO
(X1000). (G) PAS staining in non-DM eNOS KO mice (X200). (H)
Immunohistochemistry for SMA (Brown color) in non-DM eNOS KO mic
(X400).
[0020] FIG. 7. Endothelial cell proliferation in diabetic eNOS KO
mice. (A) PAS staining of injured glomerulus in diabetic eNOS KO
mice at 3 months (X 630). (B) Immunostaining for CD34. Brown color
indicates CD34 staining as a marker of endothelial cell. Blue color
indicates counter staining for nucleus with hematoxyline. Loss of
endothelial cell is observed in injured glomerulus in diabetic eNOS
KO mice at 3 months. (C) Immunohistochemistry for CD34 in non-DM
C57BL6 (X200). (D) CD34 in Diabetic eNOS KO kidney (X200). Some
glomeruli show strong immunoreactivity (block arrow) whereas some
show less endothelial staining (white arrow). (C) Immunostaining
for Thrombomodulin (TM) (Brown color) in non-DM C57BL6 (X200). TM
is primarily expressed in peritubular capillary. (D) TM in diabetic
eNOS KO (X200). (E) Double staining for TM (Bjoran Purple) (white
arrow) and Ki67 (Dark brown). Double staining can indicate
proliferating endothelial cell (black arrow). (F) Proliferating
endothelial cell detected (black arrow) by double staining of
glomerular capillary for TM (white arrow) and Ki67 (Dark
brown).
[0021] FIG. 8. (A) Quantification of CD34 in cortex. (B)
Quantification of Thrombomoduline staining. (C) Cell number with
double staining for TM and Ki67 in renal cortex per 100
.mu.m.sup.2. (H) Real time PCR for VEGF mRNA expression in whole
kidney at 3 month. a; p<0.05 vs. non-DM and DM Ins in C57BL6. b;
p<0.05 vs. non-DM in C57BL6. c; p<0.05 vs. DMIns in eNOSKO.
d; p<0.05 vs. nonDM and DMIns in eNOS KO. e; p<0.05 vs. DM in
C57BL6 nonDM; non-diabetes, DM; diabetes, DMIns; diabetes+Insulin
treatment.
[0022] FIG. 9. Effects of allopurinol treatment for hypuricemia on
the metabolic parameters in Fructose-fed Rats. Fructose-fe (Fr) are
weeks and this is prevented by allopurinol (AP; 150 m p<0.01 vs.
con<0.05 vs. Fr.) (B) Fructose reduced urinary excretion of uric
acid at 9 weeks and this is prevented by allopurinol. (*p<0.01
vs. Fr; #p<0.05 vs. control.) (C) Hypertension develops in
fructose-fed rats, which is significantly reduced with allopurinol
(#p<0.01 vs. control, and Fr) (D) Serum triglycerides are
increased in fructose-fed rats, and this completely prevented by
allopurinol (#p<0.01 vs. control, and Fr+AP). (E) The serum
triglyceride level correlates directly with the serum uric acid.
Data are mean.+-.SD.
[0023] FIG. 10. Effect of allopurinol treatment on glucose
metabolism in Fructose-fed rats. (A) Glucose tolerance test at 10
weeks. Similar blood glucose levels were observed in all groups.
(B) Plasma insulin levels following the glucose tolerance test.
Fructose ingestion was associated with fasting and postprandial
hyperinsulinemia. Allopurinol (AP; 150 mg/L) prevented basal
hyperinsulinemia and significantly reduced postprandial
hyperinsulinemia. (*p<0.01 vs. control; #p<0.05 vs. Fr.) (C)
Insulin sensitivity index (ISI). Insulin sensitivity was reduced
with fructose diet and improved by allopurinol. All data are
means.+-.SD. Statistical analysis among three groups were analyzed
by ANOVA with Bonferoni correction in Figure B. (*p<0.01 vs.
control; #p<0.05 vs. Fr.). Comparison was done between Fr and
Fr+AP using unpaired t test in Figure C.
[0024] FIG. 11. Blocking of hyperuricemia in fructose-fed rats with
allopurinol prevents features of the metabolic syndrome. (A)
Allopurinol (AP; 150 mg/L) prevented the rise in uric acid in
fructose-fed rats. (#, p<0.05 vs con, Fr+AP) (B) Allopurinol
treatment was associated with significantly lower fasting insulin
levels compared to fructose-fed rats at 8 weeks. (C) Allopurinol
also prevented the increase in BW induced with fructose.
Statistical analysis among three groups was analyzed by ANOVA with
Bonferoni correction.
[0025] FIG. 12. Uric Acid Inhibits Acetylcholine-Mediate
Vasodilation in Rat Aortic Artery Segments. Acetylcholine (5
.mu.M)-induced vasorelaxation was assessed in the presence of
various concentration of uric acid for 10 min after stable
construction by U-46619 (0.5 .mu.M). n=4, *p<0.01 vs. control,
#p<0.05 vs. 0.7 mg/dl, ##p<0.01 vs. 0.7 mg/dl.
DETAILED DESCRIPTION OF THE INVENTION
[0026] The pharmaceutical compositions provided herein contain
therapeutically effective amounts of one or more agents to lower
uric acid that are useful in the treatment or prevention of insulin
resistance. The inventors have discovered that hyperuricemia plays
a critical role in causing insulin resistance.
[0027] The term "uric acid lowering agent" or UALA refers to
substances known to lower serum uric acid levels in mammals.
Typically, the UALA may limit serum uric acid levels by at least
about 0.2 mg/dl. UALAs include, but are not limited to, xanthine
oxidase inhibitors such as allopurinol, hydroxyakalone, TEI-6720,
carprofen, febuxostat, and y-700; uricosurics such as benziodarone,
benzbromarone, probenecid; uricase derivatives such as Rasburicase
and Pegylated uricase; gene based therapies such as uricase
overexpression or blockade of URAT-1; a supplement of the unease
protein which might be delivered as a conjugate with polyethylene
glycol or another delivery system; and a urate transport channel
inhibitor.
[0028] The compounds are preferably formulated into suitable
pharmaceutical preparations such as solutions, suspensions,
tablets, dispersible tablets, pills, capsules, powders, sustained
release formulations or elixirs, for oral administration or in
sterile solutions or suspensions for parenteral administration, as
well as transdermal patch preparation and dry powder inhalers.
Typically the compounds described above are formulated into
pharmaceutical compositions using techniques and procedures well
known in the art (see, e.g., Ansel Introduction to Pharmaceutical
Dosage Forms, Fourth Edition 1985, 126).
[0029] In the compositions, effective concentrations of one or more
compounds or pharmaceutically acceptable derivatives is (are) mixed
with a suitable pharmaceutical carrier or vehicle. The compounds
may be derivatized as the corresponding salts, esters, enol ethers
or esters, acids, bases, solvates, hydrates or prodrugs prior to
formulation, as described above. The concentrations of the
compounds in the compositions are effective for delivery of an
amount, upon administration, that reduces serum uric acid levels at
least 0.5 mg/dl to be equal to or less than 5.5 mg/dl. In a most
preferred embodiment, effective amount is such as to lower serum
uric acid levels to less than or equal to 5.5 mg/dl and more than
or equal to 3.5 mg/dl. Preferably still, the effective amount is
such as to lower serum uric acid levels to less than or equal to
5.2 mg/dl and more than or equal to 4.0 mg/dl. It is known that
uric acid acts as antioxidant in the body. Epidemiological studies
performed by the inventor have uncovered that the positive effects
of avoiding insulin resistance are achieved by lowering serum uric
acid levels to at least 5.5 mg/dl. However, the positive effects
are largely negated as serum uric acid levels fall below 4.0 mg/dl.
At levels below 4.0 mg/dl, the loss of antioxidant activity of uric
acid may actually predispose to an increased incidence of
cardiovascular disease and mortality. The UALA may be administered
concomitantly or sequentially with one or more known antioxidants,
such as, but not limited to, vitamin C, alpha-lipoic acid, Vitamin
E, beta carotene, selenium, zinc, carnosine, green tea, soy and
isoflavones, tempol, etc. Such combination may be beneficial
regardless of uric acid levels, but may be particularly helpful if
dosages of UALA are administered that lower the uric acid below 4.5
mg/dl.
[0030] Typically, the compositions are formulated for single dosage
administration. To formulate a composition, the weight fraction of
compound is dissolved, suspended, dispersed or otherwise mixed in a
selected vehicle at an effective concentration such that the
treated condition is relieved or ameliorated. Pharmaceutical
carriers or vehicles suitable for administration of the compounds
provided herein include any such carriers known to those skilled in
the art to be suitable for the particular mode of
administration.
[0031] The term "average serum uric acid level(s)" as used herein
refers to an average of two or more uric acid readings obtained
from a patient. The two or more uric acid readings may be taken
within hours of each other. Preferably, the two or more readings
are obtained at least a week from each other.
[0032] The term "regimen" as used herein refers to an
administration of two or more dosages sequentially spaced in time
so as to maintain average serum uric acid levels at a predetermined
level. The space in time is preferably 3 or more hours. The regimen
may be based on empirically determined optimal dosages. Naturally,
it goes without saying that the administration of UALA according to
a regimen `so as to maintain (or effective to maintain) average
serum uric acid levels` at a predetermined level is understood to
mean that readings from a patient are not necessarily obtained, but
rather that the regimen is designed to be effective to maintain
serum uric acid levels at a desired average level over a period of
time whether or not such average is actually determined for a given
patient.
[0033] Asymptomatic hyperuricemia refers to the state of
hyperuricemia without clinical gout, renal stones or tophi.
Hyperuricemia is traditionally considered to pertain to serum uric
acid levels 7.0 mg/dL and higher, but as is noted herein, for
purposes of embodiments of the present invention, hyperuricemia is
considered to pertain to serum uric acid levels higher than 5.5
mg/dL. Conventional wisdom dictates that asymptomatic hyperuricemia
is benign and should not medically be treated (Harris et al., 1999
Feb. 15; 59(4):925-34). The inventors have elucidated that chronic
hyperuricemia can promote the onset of the metabolic syndrome,
diabetic nephropathy, and non-alcoholic fatty liver disease and
that lowering and maintaining levels of uric acid to 5.5 and below
can reduce the onset of such health issues.
[0034] In addition, the compounds may be formulated as the sole
pharmaceutically active ingredient in the composition or may be
combined with other active ingredients. Liposomal suspensions,
including tissue-targeted liposomes, particularly tumor-targeted
liposomes, may also be suitable as pharmaceutically acceptable
carriers. These may be prepared according to methods known to those
skilled in the art. For example, liposome formulations may be
prepared as described in U.S. Pat. No. 4,522,811.
[0035] The active compound is included in the pharmaceutically
acceptable carrier in an amount sufficient to exert a
therapeutically useful effect in the absence of undesirable side
effects on the patient treated. The therapeutically effective
concentration may be determined empirically by testing the
compounds in known in vitro and in vivo systems (see, e.g.,
Rosenthal et al. (1996) Antimicrob. Agents Chemother.
40(7):1600-1603; Dominguez et al. (1997) J. Med. Chem.
40:2726-2732; Clark et al. (1994) Molec. Biochem. Parasitol.
17:129; Ring et al. (1993) Proc. Natl. Acad. Sci. USA 90:3583-3587;
Engel et al. (1998) J. Exp. Med. 188(4):725-734; Li et al. (1995)
J. Med. Chem. 38:5031) and then extrapolated therefrom for dosages
for humans.
[0036] The concentration of active compound in the pharmaceutical
composition will depend on absorption, inactivation and excretion
rates of the active compound, the physicochemical characteristics
of the compound, the dosage schedule, and amount administered as
well as other factors known to those of skill in the art. For
example, the amount that is delivered is sufficient to lower uric
acid concentrations at least 0.5 mg/dl to achieve 5.5 mg/dl or
lower serum uric acid levels.
[0037] Typically a therapeutically effective dosage should produce
a serum concentration of active ingredient of from about 0.1 ng/ml
to about 50-100 .mu.g/ml. The pharmaceutical compositions typically
should provide a dosage of from about 0.001 mg to about 2000 mg of
compound per kilo-gram of body weight per day. Pharmaceutical
dosage unit forms are prepared to provide from about 1 mg to about
1000 mg and preferably from about 10 to about 500 mg of the
essential active ingredient or a combination of essential
ingredients per dosage unit form.
[0038] The active ingredient may be administered at once, or may be
divided into a number of smaller doses to be administered at
intervals of time. It is understood that the precise dosage and
duration of treatment is a function of the disease being treated
and may be determined empirically using known testing protocols or
by extrapolation from in vivo or in vitro test data. It is to be
noted that concentrations and dosage values may also vary with the
severity of the condition to be alleviated. It is to be further
understood that for any particular subject, specific dosage
regimens should be adjusted over time according to the individual
need and the professional judgment of the person administering or
supervising the administration of the compositions, and that the
concentration ranges set forth herein are exemplary only and are
not intended to limit the scope or practice of the claimed
compositions.
[0039] Preferred pharmaceutically acceptable derivatives include
acids, bases, enol ethers and esters, salts, esters, hydrates,
solvates and prodrug forms. The derivative is selected such that
its pharmacokinetic properties are superior to the corresponding
neutral compound.
[0040] Thus, effective concentrations or amounts of one or more of
the compounds described herein or pharmaceutically acceptable
derivatives thereof are mixed with a suitable pharmaceutical
carrier or vehicle for systemic, topical or local administration to
form pharmaceutical compositions. Compounds are included in an
amount effective for reducing uric acid at or below 5.5 mg/dl. The
concentration of active compound in the composition will depend on
absorption, inactivation, excretion rates of the active compound,
the dosage schedule, amount administered, particular formulation as
well as other factors known to those of skill in the art.
[0041] The compositions are intended to be administered by a
suitable route, including orally, parenterally, rectally, topically
and locally. For oral administration, capsules and tablets are
presently preferred. The compositions are in liquid, semi-liquid or
solid form and are formulated in a manner suitable for each route
of administration. Preferred modes of administration include
parenteral and oral modes of administration. Oral administration is
presently most preferred.
[0042] Solutions or suspensions used for parenteral, intradermal,
subcutaneous, or topical application can include any of the
following components: a sterile diluent, such as water for
injection, saline solution, fixed oil, polyethylene glycol,
glycerine, propylene glycol or other synthetic solvent;
antimicrobial agents, such as benzyl alcohol and methyl parabens;
antioxidants, such as ascorbic acid and sodium bisulfite; chelating
agents, such as ethylenediaminetetraacetic acid (EDTA); buffers,
such as acetates, citrates and phosphates; and agents for the
adjustment of tonicity such as sodium chloride or dextrose.
Parenteral preparations can be enclosed in ampules, disposable
syringes or single or multiple dose vials made of glass, plastic or
other suitable material.
[0043] In instances in which the compounds exhibit insufficient
solubility, methods for solubilizing compounds may be used. Such
methods are known to those of skill in this art, and include, but
are not limited to, using cosolvents, such as dimethylsulfoxide
(DMSO), using surfactants, such as TWEEN.RTM., or dissolution in
aqueous sodium bicarbonate. Derivatives of the compounds, such as
prodrugs of the compounds may also be used in formulating effective
pharmaceutical compositions.
[0044] Upon mixing or addition of the compound(s), the resulting
mixture may be a solution, suspension, emulsion or the like. The
form of the resulting mixture depends upon a number of factors,
including the intended mode of administration and the solubility of
the compound in the selected carrier or vehicle. The effective
concentration is sufficient for ameliorating the symptoms of the
disease, disorder or condition treated and may be empirically
determined.
[0045] The pharmaceutical compositions are provided for
administration to humans and animals in unit dosage forms, such as
tablets, capsules, pills, powders, granules, sterile parenteral
solutions or suspensions, and oral solutions or suspensions, and
oil-water emulsions containing suitable quantities of the compounds
or pharmaceutically acceptable derivatives thereof. The
pharmaceutically therapeutically active compounds and derivatives
thereof are typically formulated and administered in unit-dosage
forms or multiple-dosage forms. Unit-dose forms as used herein
refers to physically discrete units suitable for human and animal
subjects and packaged individually as is known in the art. Each
unit-dose contains a predetermined quantity of the therapeutically
active compound sufficient to produce the desired therapeutic
effect, in association with the required pharmaceutical carrier,
vehicle or diluent. Examples of unit-dose forms include ampoules
and syringes and individually packaged tablets or capsules.
Unit-dose forms may be administered in fractions or multiples
thereof. A multiple-dose form is a plurality of identical
unit-dosage forms packaged in a single container to be administered
in segregated unit-dose form. Examples of multiple-dose forms
include vials, bottles of tablets or capsules or bottles of pints
or gallons. Hence, multiple dose form is a multiple of unit-doses
which are not segregated in packaging.
[0046] The composition can contain along with the active
ingredient: a diluent such as lactose, sucrose, dicalcium
phosphate, or carboxymethylcellulose; a lubricant, such as
magnesium stearate, calcium stearate and talc; and a binder such as
starch, natural gums, such as gum acaciagelatin, glucose, molasses,
polvinylpyrrolidine, celluloses and derivatives thereof, povidone,
crospovidones and other such binders known to those of skill in the
art. Liquid pharmaceutically administrable compositions can, for
example, be prepared by dissolving, dispersing, or otherwise mixing
an active compound as defined above and optional pharmaceutical
adjuvants in a carrier, such as, for example, water, saline,
aqueous dextrose, glycerol, glycols, ethanol, and the like, to
thereby form a solution or suspension. If desired, the
pharmaceutical composition to be administered may also contain
minor amounts of nontoxic auxiliary substances such as wetting
agents, emulsifying agents, or solubilizing agents, pH buffering
agents and the like, for example, acetate, sodium citrate,
cyclodextrine derivatives, sorbitan monolaurate, triethanolamine
sodium acetate, triethanolamine oleate, and other such agents.
Actual methods of preparing such dosage forms are known, or will be
apparent, to those skilled in this art; for example, see
Remington's Pharmaceutical Sciences, Mack Publishing Company,
Easton, Pa., 15th Edition, 1975. The composition or formulation to
be administered will, in any event, contain a quantity of the
active compound in an amount sufficient to alleviate the symptoms
of the treated subject.
[0047] Dosage forms or compositions containing active ingredient in
the range of 0.005% to 100% with the balance made up from non-toxic
carrier may be prepared. For oral administration, a
pharmaceutically acceptable non-toxic composition is formed by the
incorporation of any of the normally employed excipients, such as,
for example pharmaceutical grades of mannitol, lactose, starch,
magnesium stearate, talcum, cellulose derivatives, sodium
crosscarmellose, glucose, sucrose, magnesium carbonate or sodium
saccharin. Such compositions include solutions, suspensions,
tablets, capsules, powders and sustained release formulations, such
as, but not limited to, implants and microencapsulated delivery
systems, and biodegradable, biocompatible polymers, such as
collagen, ethylene vinyl acetate, polyanhydrides, polyglycolic
acid, polyorthoesters, polylactic acid and others. Methods for
preparation of these compositions are known to those skilled in the
art. The contemplated compositions may contain 0.001%-100% active
ingredient, preferably 0.1-85%, typically 75-95%.
[0048] The active compounds or pharmaceutically acceptable
derivatives may be prepared with carriers that protect the compound
against rapid elimination from the body, such as time release
formulations or coatings.
[0049] 1. Compositions for Oral Administration
[0050] Oral pharmaceutical dosage forms are either solid, gel or
liquid. The solid dosage forms are tablets, capsules, granules, and
bulk powders. Types of oral tablets include compressed, chewable
lozenges and tablets which may be enteric-coated, sugar-coated or
film-coated. Capsules may be hard or soft gelatin capsules, while
granules and powders may be provided in non-effervescent or
effervescent form with the combination of other ingredients known
to those skilled in the art.
[0051] In certain embodiments, the formulations are solid dosage
forms, preferably capsules or tablets. The tablets, pills,
capsules, troches and the like can contain any of the following
ingredients, or compounds of a similar nature: a binder, a diluent;
a disintegrating agent; a lubricant; a glidant; a sweetening agent;
and a flavoring agent.
[0052] Examples of binders include microcrystalline cellulose, gum
tragacanth, glucose solution, acacia mucilage, gelatin solution,
sucrose and starch paste. Lubricants include talc, starch,
magnesium or calcium stearate, lycopodium and stearic acid.
Diluents include, for example, lactose, sucrose, starch, kaolin,
salt, mannitol and dicalcium phosphate. Glidants include, but are
not limited to, colloidal silicon dioxide. Disintegrating agents
include crosscarmellose sodium, sodium starch glycolate, alginic
acid, corn starch, potato starch, bentonite, methylcellulose, agar
and carboxymethylcellulose. Coloring agents include, for example,
any of the approved certified water soluble FD and C dyes, mixtures
thereof; and water insoluble FD and C dyes suspended on alumina
hydrate. Sweetening agents include sucrose, lactose, mannitol and
artificial sweetening agents such as saccharin, and any number of
spray dried flavors. Flavoring agents include natural flavors
extracted from plants such as fruits and synthetic blends of
compounds which produce a pleasant sensation, such as, but not
limited to peppermint and methyl salicylate. Wetting agents include
propylene glycol monostearate, sorbitan monooleate, diethylene
glycol monolaurate and polyoxyethylene laural ether.
Emetic-coatings include fatty acids, fats, waxes, shellac,
ammoniated shellac and cellulose acetate phthalates. Film coatings
include hydroxyethylcellulose, sodium carboxymethylcellulose,
polyethylene glycol 4000 and cellulose acetate phthalate.
[0053] If oral administration is desired, the compound could be
provided in a composition that protects it from the acidic
environment of the stomach. For example, the composition can be
formulated in an enteric coating that maintains its integrity in
the stomach and releases the active compound in the intestine. The
composition may also be formulated in combination with an antacid
or other such ingredient.
[0054] When the dosage unit form is a capsule, it can contain, in
addition to material of the above type, a liquid carrier such as a
fatty oil. In addition, dosage unit forms can contain various other
materials which modify the physical form of the dosage unit, for
example, coatings of sugar and other enteric agents. The compounds
can also be administered as a component of an elixir, suspension,
syrup, wafer, sprinkle, chewing gum or the like. A syrup may
contain, in addition to the active compounds, sucrose as a
sweetening agent and certain preservatives, dyes and colorings and
flavors.
[0055] The active materials can also be mixed with other active
materials which do not impair the desired action, or with materials
that supplement the desired action, such as antacids, H2 blockers,
and diuretics. The active ingredient is a compound or
pharmaceutically acceptable derivative thereof as described herein.
Higher concentrations, up to about 98% by weight of the active
ingredient may be included.
[0056] Pharmaceutically acceptable carriers included in tablets are
binders, lubricants, diluents, disintegrating agents, coloring
agents, flavoring agents, and wetting agents. Enteric-coated
tablets, because of the enteric-coating, resist the action of
stomach acid and dissolve or disintegrate in the neutral or
alkaline intestines. Sugar-coated tablets are compressed tablets to
which different layers of pharmaceutically acceptable substances
are applied. Film-coated tablets are compressed tablets which have
been coated with a polymer or other suitable coating. Multiple
compressed tablets are compressed tablets made by more than one
compression cycle utilizing the pharmaceutically acceptable
substances previously mentioned. Coloring agents may also be used
in the above dosage forms. Flavoring and sweetening agents are used
in compressed tablets, sugar-coated, multiple compressed and
chewable tablets. Flavoring and sweetening agents are especially
useful in the formation of chewable tablets and lozenges.
[0057] Liquid oral dosage forms include aqueous solutions,
emulsions, suspensions, solutions and/or suspensions reconstituted
from non-effervescent granules and effervescent preparations
reconstituted from effervescent granules. Aqueous solutions
include, for example, elixirs and syrups. Emulsions are either
oil-in-water or water-in-oil.
[0058] Elixirs are clear, sweetened, hydroalcoholic preparations.
Pharmaceutically acceptable carriers used in elixirs include
solvents. Syrups are concentrated aqueous solutions of a sugar, for
example, sucrose, and may contain a preservative. An emulsion is a
two-phase system in which one liquid is dispersed in the form of
small globules throughout another liquid. Pharmaceutically
acceptable carriers used in emulsions are non-aqueous liquids,
emulsifying agents and preservatives. Suspensions use
pharmaceutically acceptable suspending agents and preservatives.
Pharmaceutically acceptable substances used in non-effervescent
granules, to be reconstituted into a liquid oral dosage form,
include diluents, sweeteners and wetting agents. Pharmaceutically
acceptable substances used in effervescent granules, to be
reconstituted into a liquid oral dosage form, include organic acids
and a source of carbon dioxide. Coloring and flavoring agents are
used in all of the above dosage forms.
[0059] Solvents include glycerin, sorbitol, ethyl alcohol and
syrup. Examples of preservatives include glycerin, methyl and
propylparaben, benzoic add, sodium benzoate and alcohol. Examples
of non-aqueous liquids utilized in emulsions include mineral oil
and cottonseed oil. Examples of emulsifying agents include gelatin,
acacia, tragacanth, bentonite, and surfactants such as
polyoxyethylene sorbitan monooleate. Suspending agents include
sodium carboxymethylcellulose, pectin, tragacanth, Veegum and
acacia. Diluents include lactose and sucrose. Sweetening agents
include sucrose, syrups, glycerin and artificial sweetening agents
such as saccharin. Wetting agents include propylene glycol
monostearate, sorbitan monooleate, diethylene glycol monolaurate
and polyoxyethylene lauryl ether. Organic adds include citric and
tartaric acid. Sources of carbon dioxide include sodium bicarbonate
and sodium carbonate. Coloring agents include any of the approved
certified water soluble FD and C dyes, and mixtures thereof.
Flavoring agents include natural flavors extracted from plants such
fruits, and synthetic blends of compounds which produce a pleasant
taste sensation.
[0060] For a solid dosage form, the solution or suspension, in for
example propylene carbonate, vegetable oils or triglycerides, is
preferably encapsulated in a gelatin capsule. Such solutions, and
the preparation and encapsulation thereof, are disclosed in U.S.
Pat. Nos. 4,328,245; 4,409,239; and 4,410,545. For a liquid dosage
form, the solution, e.g., for example, in a polyethylene glycol,
may be diluted with a sufficient quantity of a pharmaceutically
acceptable liquid carrier, e.g., water, to be easily measured for
administration.
[0061] Alternatively, liquid or semi-solid oral formulations may be
prepared by dissolving or dispersing the active compound or salt in
vegetable oils, glycols, triglycerides, propylene glycol esters
(e.g., propylene carbonate) and other such carriers, and
encapsulating these solutions or suspensions in hard or soft
gelatin capsule shells. Other useful formulations include those set
forth in U.S. Pat. No. Re 28,819 and U.S. Pat. No. 4,358,603.
[0062] In all embodiments, tablets and capsules formulations may be
coated as known by those of skill in the art in order to modify or
sustain dissolution of the active ingredient. Thus, for example,
they may be coated with a conventional enterically digestible
coating, such as phenylsalicylate, waxes and cellulose acetate
phthalate.
[0063] 2. Injectables, Solutions and Emulsions
[0064] Parenteral administration, generally characterized by
injection, either subcutaneously, intramuscularly or intravenously
is also contemplated herein. Injectables can be prepared in
conventional forms, either as liquid solutions or suspensions,
solid forms suitable for solution or suspension in liquid prior to
injection, or as emulsions. Suitable excipients are, for example,
water, saline, dextrose, glycerol or ethanol. In addition, if
desired, the pharmaceutical compositions to be administered may
also contain minor amounts of non-toxic auxiliary substances such
as wetting or emulsifying agents, pH buffering agents, stabilizers,
solubility enhancers, and other such agents, such as for example,
sodium acetate, sorbitan monolaurate, triethanolamine oleate and
cyclodextrins. Implantation of a slow-release or sustained-release
system, such that a constant level of dosage is maintained (see,
e.g., U.S. Pat. No. 3,710,795) is also contemplated herein. The
percentage of active compound contained in such parenteral
compositions is highly dependent on the specific nature thereof, as
well as the activity of the compound and the needs of the
subject.
[0065] Parenteral administration of the compositions includes
intravenous, subcutaneous and intramuscular administrations.
Preparations for parenteral administration include sterile
solutions ready for injection, sterile dry soluble products, such
as lyophilized powders, ready to be combined with a solvent just
prior to use, including hypodermic tablets, sterile suspensions
ready for injection, sterile dry insoluble products ready to be
combined with a vehicle just prior to use and sterile emulsions.
The solutions may be either aqueous or nonaqueous.
[0066] If administered intravenously, suitable carriers include
physiological saline or phosphate buffered saline (PBS), and
solutions containing thickening and solubilizing agents, such as
glucose, polyethylene glycol, and polypropylene glycol and mixtures
thereof.
[0067] Pharmaceutically acceptable carriers used in parenteral
preparations include aqueous vehicles, nonaqueous vehicles,
antimicrobial agents, isotonic agents, buffers, antioxidants, local
anesthetics, suspending and dispersing agents, emulsifying agents,
sequestering or chelating agents and other pharmaceutically
acceptable substances.
[0068] Examples of aqueous vehicles include Sodium Chloride
Injection, Ringers Injection, Isotonic Dextrose Injection, Sterile
Water Injection, Dextrose and Lactated Ringers Injection.
Nonaqueous parenteral vehicles include fixed oils of vegetable
origin, cottonseed oil, corn oil, sesame oil and peanut oil.
Antimicrobial agents in bacteriostatic or fungistatic
concentrations must be added to parenteral preparations packaged in
multiple-dose containers which include phenols or cresols,
mercurials, benzyl alcohol, chlorobutanol, methyl and propyl
p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and
benzethonium chloride. Isotonic agents include sodium chloride and
dextrose. Buffers include phosphate and citrate. Antioxidants
include sodium bisulfate. Local anesthetics include procaine
hydrochloride. Suspending and dispersing agents include sodium
carboxymethylcelluose, hydroxypropyl methylcellulose and
polyvinylpyrrolidone. Emulsifying agents include Polysorbate 80
(TWEEN.RTM. 80). A sequestering or chelating agent of metal ions
include EDTA. Pharmaceutical carriers also include ethyl alcohol,
polyethylene glycol and propylene glycol for water miscible
vehicles and sodium hydroxide, hydrochloric acid, citric acid or
lactic acid for pH adjustment.
[0069] The concentration of the pharmaceutically active compound is
adjusted so that an injection provides an effective amount to
produce the desired pharmacological effect. The exact dose depends
on the age, weight and condition of the patient or animal as is
known in the art.
[0070] The unit-dose parenteral preparations are packaged in an
ampoule, a vial or a syringe with a needle. All preparations for
parenteral administration must be sterile, as is known and
practiced in the art.
[0071] Illustratively, intravenous or intraarterial infusion of a
sterile aqueous solution containing an active compound is an
effective mode of administration. Another embodiment is a sterile
aqueous or oily solution or suspension containing an active
material injected as necessary to produce the desired
pharmacological effect.
[0072] Injectables are designed for local and systemic
administration. Typically a therapeutically effective dosage is
formulated to contain a concentration of at least about 0.1% w/w up
to about 90% w/w or more, preferably more than 1% w/w of the active
compound to the treated tissue(s). The active ingredient may be
administered at once, or may be divided into a number of smaller
doses to be administered at intervals of time. It is understood
that the precise dosage and duration of treatment is a function of
the tissue being treated and may be determined empirically using
known testing protocols or by extrapolation from in vivo or in
vitro test data. It is to be noted that concentrations and dosage
values may also vary with the age of the individual treated. It is
to be further understood that for any particular subject, specific
dosage regimens should be adjusted over time according to the
individual need and the professional judgment of the person
administering or supervising the administration of the
formulations, and that the concentration ranges set forth herein
are exemplary only and are not intended to limit the scope or
practice of the claimed formulations.
[0073] The compound may be suspended in micronized or other
suitable form or may be derivatized to produce a more soluble
active product or to produce a prodrug. The form of the resulting
mixture depends upon a number of factors, including the intended
mode of administration and the solubility of the compound in the
selected carrier or vehicle. The effective concentration is
sufficient for ameliorating the symptoms of the condition and may
be empirically determined.
[0074] 3. Lyophilized Powders
[0075] Of interest herein are also lyophilized powders, which can
be reconstituted for administration as solutions, emulsions and
other mixtures. They may also be reconstituted and formulated as
solids or gels.
[0076] The sterile, lyophilized powder is prepared by dissolving a
compound of formula I in a suitable solvent. The solvent may
contain an excipient which improves the stability or other
pharmacological component of the powder or reconstituted solution,
prepared from the powder. Excipients that may be used include, but
are not limited to, dextrose, sorbital, fructose, corn syrup,
xylitol, glycerin, glucose, sucrose or other suitable agent. The
solvent may also contain a buffer, such as citrate, sodium or
potassium phosphate or other such buffer known to those of skill in
the art at, typically, about neutral pH. Subsequent sterile
filtration of the solution followed by lyophilization under
standard conditions known to those of skill in the art provides the
desired formulation. Generally, the resulting solution will be
apportioned into vials for lyophilization. Each vial will contain a
single dosage (10-1000 mg, preferably 100-500 mg) or multiple
dosages of the compound. The lyophilized powder can be stored under
appropriate conditions, such as at about 4.degree. C. to room
temperature.
[0077] Reconstitution of this lyophilized powder with water for
injection provides a formulation for use in parenteral
administration. For reconstitution, about 1-50 mg, preferably 5-35
mg, more preferably about 9-30 mg of lyophilized powder, is added
per mL of sterile water or other suitable carrier. The precise
amount depends upon the selected compound. Such amount can be
empirically determined.
[0078] 4. Topical Administration
[0079] Topical mixtures are prepared as described for the local and
systemic administration. The resulting mixture may be a solution,
suspension, emulsions or the like and are formulated as creams,
gels, ointments, emulsions, solutions, elixirs, lotions,
suspensions, tinctures, pastes, foams, aerosols, irrigations,
sprays, suppositories, bandages, dermal patches or any other
formulations suitable for topical administration.
[0080] The compounds or pharmaceutically acceptable derivatives
thereof may be formulated as aerosols for topical application, such
as by inhalation (see, e.g., U.S. Pat. Nos. 4,044,126, 4,414,209,
and 4,364,923, which describe aerosols for delivery of a steroid
useful for treatment inflammatory diseases, particularly asthma).
These formulations for administration to the respiratory tract can
be in the form of an aerosol or solution for a nebulizer, or as a
microfine powder for insufflation, alone or in combination with an
inert carrier such as lactose. In such a case, the particles of the
formulation will typically have diameters of less than 50 microns,
preferably less than 10 microns.
[0081] The compounds may be formulated for local or topical
application, such as for topical application to the skin and mucous
membranes, such as in the eye, in the form of gels, creams, and
lotions and for application to the eye or for intracisternal or
intraspinal application. Topical administration is contemplated for
transdermal delivery and also for administration to the eyes or
mucosa, or for inhalation therapies. Nasal solutions of the active
compound alone or in combination with other pharmaceutically
acceptable excipients can also be administered.
[0082] These solutions, particularly those intended for ophthalmic
use, may be formulated as 0.01%-10% isotonic solutions, pH about
5-7, with appropriate salts.
[0083] 5. Compositions for Other Routes of Administration
[0084] Other routes of administration, such as transdermal patches
and rectal administration are also contemplated herein.
[0085] For example, pharmaceutical dosage forms for rectal
administration are rectal suppositories, capsules and tablets for
systemic effect. Rectal suppositories are used herein mean solid
bodies for insertion into the rectum which melt or soften at body
temperature releasing one or more pharmacologically or
therapeutically active ingredients. Pharmaceutically acceptable
substances utilized in rectal suppositories are bases or vehicles
and agents to raise the melting point. Examples of bases include
cocoa butter (theobroma oil), glycerin-gelatin, carbowax
(polyoxyethylene glycol) and appropriate mixtures of mono-, di- and
triglycerides of fatty acids. Combinations of the various bases may
be used. Agents to raise the melting point of suppositories include
spermaceti and wax. Rectal suppositories may be prepared either by
the compressed method or by molding. The typical weight of a rectal
suppository is about 2 to 3 gm.
[0086] Tablets and capsules for rectal administration are
manufactured using the same pharmaceutically acceptable substance
and by the same methods as for formulations for oral
administration.
[0087] 6. Articles of Manufacture
[0088] The compounds or pharmaceutically acceptable derivatives may
be packaged as articles of manufacture containing packaging
material, a compound or pharmaceutically acceptable derivative
thereof provided herein, which is effective for reducing serum uric
levels.
[0089] The articles of manufacture provided herein contain
packaging materials. Packaging materials for use in packaging
pharmaceutical products are well known to those of skill in the
art. See, e.g., U.S. Pat. Nos. 5,323,907, 5,052,558 and 5,033,352.
Examples of pharmaceutical packaging materials include, but are not
limited to, blister packs, bottles, tubes, inhalers, pumps, bags,
vials, containers, syringes, bottles, and any packaging material
suitable for a selected formulation and intended mode of
administration and treatment. A wide array of formulations of the
compounds and compositions provided herein are contemplated for
treatment and prevention of insulin resistance.
[0090] It has recently been reported that raised uric acid levels
do not impair endothelial function in humans. Waring et al., Heart
2004, 90:155-159. The inventors believe that this report does not
fully reveal the effects of raised uric acid levels in the blood.
Waring et al reported that the infusion of uric acid into the
forearm vein of humans does not impair endothelial function as
measured by brachial artery reactivity. However, the authors
examined the effect immediately after infusion of uric acid, and it
remains possible that the effect on NO production is delayed.
Indeed, with experimental hyperuricemia, hypertension does not
develop until several weeks after the uric acid is raised. Contrary
to the Waring et al. report, the inventors believe that uric acid
does indeed impair endothelial dysfunction and as a result NO
production is impaired.
Example 1
Hyperuricemia Induces Endothelial Dysfunction by Inhibiting the
Production of NO in Rats
Methods
[0091] Male Sprague-Dawley rats were housed in standard conditions
and fed normal diets. Hyperuricemia was induced with an uricase
inhibitor, oxonic acid (OA; 750 mg/kg/day), by gavage, with control
rats receiving vehicle. Allopurinol (AP) was used to block
hyperuricemia by placing AP in the drinking water (150 mg/L). Rats
were divided into four groups: (1) Control, (2) AP only, (3) OA
only, and (4) OA+AP. Systolic blood pressure was measured using a
tail-cuff sphygmomanometer. The amount of drinking water consumed
and changes in body weight were noted. Rats were sacrificed at one
and seven days. Serum was analyzed for uric acid concentration and
nitrites/nitrates (NO.sub.X) by chemiluminescence method.
(Prabhakar S S: Inhibition of mesangial iNOS by reduced
extracellular pH is associated with uncoupling of NADPH oxidation.
Kidney Int 61:2015-2024, 2002). Statistical analysis between
subgroups was performed using ANOVA.
Results
[0092] There was no difference in the amount of water consumed and
the change in body weight between the three groups over seven days.
OA induced a mild hyperuricemia at both 1 day (1.7.+-.0.7 vs.
0.8.+-.0.4 mg/dL in OA vs. Control, p<0.05) and 7 days
(1.8.+-.0.4 vs. 0.9.+-.0.7 mg/dL in OA vs. Control, p<0.05). AP
only had a mild and non-significant effect on serum uric acid
concentrations at day 1 (1.52.+-.0.3 mg/dL, p=NS), but effectively
reversed the hyperuricemia at 7 days (0.3.+-.0.2 mg/dL,
p<0.001). Serum nitrites and nitrates (NO.sub.X) were reduced by
40-50% in hyperuricemic rats at both 1 day (15.6.+-.0.4 vs.
22.6.+-.1.0 .mu.mol/L in OA vs. Control, p<0.001) and 7 days
(14.6.+-.1.1 vs. 27.5.+-.1.3 .mu.mol/L in OA vs. Control,
p<0.001). This decrease in NO.sub.X was improved slightly by AP
at 1 day (17.4.+-.0.8 .mu.mol/L, p<0.001) and reversed
completely at 7 days (25.0.+-.0.8 .mu.mol/L, p<0.001). (FIG. 1.)
There was also a direct linear correlation between serum UA and
NO.sub.X (FIG. 2). Rats treated with AP alone did not show a
significant change in either serum UA or NO.sub.X concentration.
Rats treated with OA also showed a trend toward higher systolic
blood pressure at 7 days (178.+-.18 vs. 158.+-.16 vs. 147.+-.11 mm
Hg in OA vs. Control vs. OA/AP, p=NS).
Conclusions
[0093] Most mammals have the enzyme uricase that degrades uric acid
to allantoin with the generation of oxidants. In humans, uricase is
mutated resulting in higher uric acid levels. Rats administered an
uricase inhibitor (oxonic acid) develop mild hyperuricemia,
hypertension, and vascular disease that is mediated by activation
of the renin-angiotensin system, a loss of macula densa NO
synthase, and the development of microvascular disease (Mazzali M,
Hughes J, Kim Y G, Jefferson J A, Kang D H, Gordon K L, Lan H Y,
Kivlighn S, Johnson R J: Elevated uric acid increases blood
pressure in the rat by a novel crystal-independent mechanism.
Hypertension 38:1101-1106, 2001). In this study, it was
demonstrated that hyperuricemic rats have a fall in serum nitrites
(a reflection of NO production) that is reversed by allopurinol.
Furthermore, there was a direct linear correlation between serum
uric acid and serum nitric oxide. The induction of hyperuricemia
also showed a trend towards increased systolic blood pressure. This
data shows that hyperuricemia leads to endothelial dysfunction in
the rat. As discussed briefly above, this is a contrary conclusion
to that was earlier reported by Waring et al which concluded that
the infusion of uric acid into humans does not impair endothelial
function (Waring W S, Adwani S H, Breukels O, Webb D J, Maxwell S
R: Hyperuricaemia does not impair cardiovascular function in
healthy adults. Heart 90:155-159, 2004). However, these studies did
not measure nitric oxide levels nor mention effects of sustained
hyperuricemia on endothelial-dependent vasodilatation.
[0094] Without being held to any specific mechanism, the inventors
believe that raised serum uric acid levels ultimately lead to
insulin resistance mediated by impairment of endothelial function
and inhibition of NO production. As support for this mechanistic
theory, the inventors cite to Cook et al., Swiss Med Wkly, 2003,
133:360-363, which shows that knock-out mice harboring a genetic
defect for endothelial nitric oxide synthase develop many of the
abnormalities associated with the metabolic syndrome. Accordingly,
it is the inventors' position that insulin resistance, and other
metabolic related characteristics, results from raised serum uric
acid levels, likely caused by the high sugar, fructose-generating
western diet, which results in endothelial dysfunction and
inhibition of NO production, and ultimately to insulin resistance.
Thus, controlling a person's average serum uric acid levels by
administration of UALA will have the dramatic affect of delaying
the onset of the characteristics of the metabolic syndrome, namely
insulin resistance, obesity and hypertriglyceridemia.
[0095] According to another embodiment, the subject invention
pertains to a method of determining the uric acid increasing load
per mass of food. The method may comprise the administration of a
quantity of a food item and determination of the affect of such
administration on the uric acid levels of such food. Thus, one or
more food items are tested and the information is used to generate
a uric acid increasing index (or `UA index`). WO-A 2005040752 and
U.S. Patent Pub No. 2004043106 are incorporated by reference, which
describes methodology for establishing glycemic loads of foods. The
teachings of such publication may be easily adaptable to producing
correlating types of information relating to Uric Acid generating
loads of foods, including fluids.
Example 2
Metabolic Syndrome Characteristics are Treated by Normalizing Uric
Acid Levels Methods
In Vivo Studies.
[0096] Treatment of fructose-induced hyperuricemia with
allopurinol: Male Sprague-Dawley rats (150-200 g) were housed in
standard conditions and fed control (n=7) or 60% fructose diet
(Harlan, Madison, Wis., n=14) for 10 weeks. "Control diet" contains
46% carbohydrate, which is mainly composed of starch whereas the
fructose diet contained 60% fructose as the carbohydrate. The
caloric content of these diets are 3.1 kcal/g and 3.6 kcal/g,
respectively. At 4 weeks, blood sample were obtained at 11 am in
the morning after 4 h fasting. Half of the fructose-fed rats were
administered allopurinol (AP, 150 mg/L in the drinking water)
(Sigma, St. Louis, Mo.) for an additional 6 weeks to lower serum
uric acid. Fresh drinking water containing allopurinol was replaced
every 2 days. Rats were divided into 3 groups: Control; Fructose
(Fr); and Fr+AP. At 10 weeks an oral glucose tolerance test was
performed, in which rats were fasted overnight (16 hours), and then
administered 1.5 g/kg OGTT (50% glucose solution) by gavage. Blood
was sampled at 0, 30, 60, 120 min for blood glucose and serum
insulin measurement. Rats were then sacrificed.
[0097] Prevention of fructose-induced hyperuricemia with
allopurinol: To assess the effect of preventing hyperuricemia
during the period of the study, allopurinol was initiated on the
day when fructose diet was given (from week 0 to Week 8). Three
groups (control, Fr, and Fr+AP; n=8 each) were designed for this
prevention study. Body weight was measured every 2 weeks. Food
consumption was measured for 3 days at 8 weeks.
[0098] The effect of lowering of uric acid by either allopurinol or
Benzbromarone (BZ) on body weight and food consumption: In this
experiment, the effect of BZ, a uricosuric agent (150 mg/L in the
drinking water) (Sigma, St. Louis, Mo.), was also examined to
confirm the effect of lowering of uric acid on body weight and food
intake. Fresh drinking water containing Benzbromarone was replaced
every 2 days. Three groups (control, AP, and BZ; n=8 each) were
studied. All groups were fed with "Control diet" for 8 weeks. Body
weight and the consumption of food were measured weekly for 8
weeks.
[0099] Comparison between 60% dextrose and 60% fructose on the
development of metabolic syndrome and the effect of lowering uric
acid with Benzbromarone: Rats were pair-fed with 60% dextrose diet
or 60% fructose diet for 4 weeks, which are isocaloric. Since
Experiment II showed that each rat normally eats 25-30 g/day, the
inventors administered 25 g of diet to each rat every day. At 4
weeks, total food intake per animal was calculated from the food
left over. Total food intake is the subtraction of the left-over
food from total administered food (1425 g/rat/28 days). In addition
to the above two groups, a third group of fructose fed rats were
administered BZ. Body weight was measured weekly. At 4 weeks, after
5 h fasting, insulin, triglyceride and uric acid were measured. All
protocols were approved by the Animal Care Committee of the
University of Florida.
[0100] Measurements: Systolic blood pressure was assessed as the
mean value of 3 consecutive measurements obtained in the morning
using a tail-cuff sphygmomanometer (Visitech BP2000, Visitech
Systems, Inc., Apex, N.C.). All animals were preconditioned for
blood pressure measurements 1 wk before each experiment. Serum uric
acid was measured by uricase method. Blood glucose was measured
with the ONE TOUCH system (Johnson&Johnson, Milpitas, Calif.).
Rat insulin was measured by ELISA (Crystal Chem. Inc., Chicago,
Ill.). Insulin sensitivity index was calculated using the formula
of Matsuda and DeFronzo (10,000/square root of [fasting glucose X
fasting linsulin] X [mean glucose X mean insulin during OGTT]),
which is highly correlated (r=0.73, p<0.0001) with rate of
whole-body glucose disposal during the euglycemic insulin clamp
(Matsuda M and DeFronzo R A, Insulin sensitivity indices obtained
from oral glucose tolerance testing: comparison with the euglycemic
insulin clamp, Diabetes Care 22: 1462-1470, 1999). Serum lipids
were measured with an autoanalyzer (VETAce, Alfa Wassermann Inc,
West Caldwell, N.J.) or Triglyceride-SL assay kit (Diagnostic
chemicals Limited, Charlottetown, PE, Canada).
[0101] Vasorelaxation of rat Aortic Artery (AA) segments: Rat AA
segments (1-0.5 mm diameter.times.3-4 mm length) were isolated from
the 2- to 3-month-old rats, AA segments were suspended in
individual organ chambers (Radnoti Four-Unit Tissue Bath System)
with 5 ml in Earl's solution, oxygenated with 95% O2 and 5% CO2 at
37.degree. C. After 1hr equilibration of resting force of 1.5 g,
vascular smooth muscle cell or endothelium integrity in this AA
segment was confirmed by monitoring 0.5 .mu.M U-46619 (a
thromboxane A2 mimetic, sigma)-mediated AA contraction or
acetylcholine (5 .mu.M)-mediated vasodilation, respectively. After
washing several times, the segments were incubated with various
concentration of uric acid (0-15 mg/dl) in organ bath chamber for
30 min. Stable construction was induced by 0.5 .mu.M U-46619 for 10
min prior to acetylcholine-induced vasorelaxation. The vascular
tensions were continuously monitored with an isometric force
transducer (Harvard Apparatus, Holliston, Mass.). To standardize
the data, U-46619-induced stable increase in vascular tone was set
as 100%.
[0102] Statistical analysis. All values presented are expressed as
mean.+-.SD and analyzed by one-way analysis of variance (ANOVA) or
by unpaired Student's t test. Significance was defined as
p<0.05.
Results
[0103] In Vivo Study
[0104] Serum uric acid levels, systolic blood pressure, and fasting
insulin levels were elevated in fructose-fed rats compared to rats
fed a control diet at 4 weeks (Table 1). In addition, the body
weight of fructose-fed rats tended to increase compared to rats fed
a normal diet (Table 1). These data demonstrate that fructose
feeding induces early features of the metabolic syndrome in
rats.
[0105] In order to examine the role of uric acid in this model,
half of the fructose-fed rats were treated with allopurinol (a
xanthine oxidase inhibitor) for 6 additional weeks. This treatment
was effective at lowering uric acid, whereas the fructose-fed rats
that did not receive treatment continued to be hyperuricemic (FIG.
9A). In addition, the inventors examined the urinary excretion of
uric acid in these animals to clarify the mechanisms of
hyperuricemia in fructose-fed rats. As shown in FIG. 9B,
fructose-fed rats had lower urinary excretion of uric acid.
Interestingly, allopurinol prevented the reduced excretion of uric
acid in fructose-fed rats.
[0106] Fructose-fed rats treated with allopurinol showed an
improvement in the metabolic syndrome. Allopurinol significantly
reduced systolic blood pressure in fructose-fed rats (FIG. 9C),
although pressures remained higher than that observed in control
rats. Fructose-fed rats also developed marked hypertriglyceridemia
that was abolished by allopurinol treatment (FIG. 9D). The
reduction in serum uric acid correlated directly with the decrease
in triglyceride levels (FIG. 9E). Fructose-fed rats also showed an
increase in body weight compared to controls. Allopurinol prevented
the increase in body weight although this did not reach
significance (522.+-.57 g in Fr vs. 470.+-.28 g in control, and
474.+-.37 g in Fr+AP, p=NS).
[0107] While no groups developed fasting or postprandial
hyperglycemia (FIG. 10A), fructose-fed rats developed fasting
hyperinsulinemia that was reversed with allopurinol (FIG. 10B).
Postprandial hyperinsulinemia also occurred in fructose-fed rats
administered an oral glucose tolerance test, and this was partially
but significantly, lower in allopurinol-treated rats (FIG. 10),
resulting in improved insulin sensitivity (FIG. 10C).
[0108] The inventors also examined the effectiveness of allopurinol
in preventing as opposed to treating rats with fructose-induced
metabolic syndrome. Allopurinol was given simultaneously with the
fructose diet from the starting point to avoid fructose-induced
hyperuricemia. As shown in FIG. 11A, the elevation of uric acid by
fructose diet was prevented over the 6 week period in fructose-fed
rats. Allopurinol treated rats had significantly lower fasting
insulin levels compared to fructose-fed rats (FIG. 11B) and the
development of hypertriglyceridemia was completely prevented (FIG.
11D). In addition, while fructose-fed rats gained weight compared
to control rats (456.+-.24 vs. 414.+-.24 g, final weights in Fr vs.
control, p<0.01), allopurinol treated rats had lower weight gain
(final weight 426.+-.26 g, p<0.05 vs. Fructose-fed rats). At 8
weeks, total food intake over 3 days in fructose-fed rats was
slightly higher (92.+-.2 g) compared to that of the
Fructose+Allopurinol group (88.+-.4 g), although this did not reach
statistical significance. The observation that administration of
allopurinol to fructose fed rats prevented obesity led to
additional studies to ensure that allopurinol did not have specific
effects on food intake or body weight. To address this possibility,
allopurinol or benzbromarone (a uricosuric) was administered to
rats on control diets for 8 weeks. A third group received control
diet alone. Total food consumption at 8 weeks and final body weight
were not different among the three groups (Table 2).
[0109] Finally, the inventors compared the effects of 60% Dextrose
diet and 60% Fructose diet on the development of metabolic
syndrome. In this experiment food intake was controlled so that
each group received the same intake of calories and had the same
weight gain. Nevertheless, only the fructose fed rats developed
hyperuricemia, hypertriglyceridemia, and hyperinsulinemia (Table
3). Importantly, these effects observed in fructose fed rats were
significantly improved by lowering uric acid levels with the
uricosuric agent, benzbromarone (Table 3).
[0110] In Vitro Studies
[0111] Endothelial dysfunction is common in metabolic syndrome. It
is known that impaired nitric oxide response to insulin may be a
mechanism for the development of insulin resistance (Shinozaki K,
Kashiwagi A, Nishio Y, Okamura T, Yoshida Y, MasadaToda N, and
Kikkawa R, Abnormal biopterin metabolism is a major cause of
impaired endothelium-dependent relaxation through nitric
oxide/O2-imbalance in insulin-resistant rat aorta, Diabetes 48:
2437-2445, 1999). Previously, uric acid has been shown to potently
reduce NO levels in cultured bovine endothelial cells (Khosla U M,
Zharikov S, Finch J L, Nakagawa T, Roncal C, Mu W, Krotova, Block E
R, Prabhakar S, and Johnson R J, Hyperuricemia induces endothelial
dysfunction, Kidney Int 67: 1739-1742, 2005). To further examine
this relationship, the inventors examined the acute effect of uric
acid on acetylcholine-induced vasodilation of rat aortic artery
rings. As shown in FIG. 12, uric acid dose-dependently blocked the
vasorelaxation of aortic arterial rings in response to
acetylcholine.
Example 3
Treatment or Delaying the Progression of Diabetic Nephropathy
[0112] The inventors hypothesize that an uncoupling of VEGF with
endothelial NO might contribute to the vascular complications
observed in diabetes. Indeed, the inventors were able to
demonstrate that uncoupling of VEGF with endothelial NO could
stimulate an excessive endothelial cell proliferation under high
glucose conditions. To test their hypothesis in an in vivo model of
diabetes, the inventors utilized eNOS KO mice which are incapable
of endogenously producing endothelial cell NO. The inventors
performed experiments to determine if diabetic mice lacking
endothelial NO synthase might be predisposed to diabetic
nephropathy.
Methods
[0113] Experimental Animals
[0114] Experiments were performed following protocol approval by
the Animal Care and Use Committee of the University of Florida
(IACUC). C57Bl/6J mice (C57BL6) and C57BL/6J-Nos3tm1Unc (eNOS KO
mice) (Jackson Laboratory, Bar Harbor, Me.) aged 8 weeks were
rendered diabetic with intraperitoneal injections of streptozotocin
(STZ) (100 mg/kg/day for 2 consecutive days) freshly dissolved in
0.1M citrate buffer (pH 4.5). Development of diabetes (defined by
blood glucose greater than 250 mg/dL) was verified 1 week after the
first STZ injection with ONE TOUCH system (Johnson & Johnson,
Milpitas, Calif.). For blood sugar control, a single insulin pellet
(Linshin Canada Inc, Ontario, Canada) was implanted subcutaneously
for 5 months. Blood glucose was monitored every 2 weeks and if the
fasting blood glucose was >200 mg/dl, an additional insulin
pellet was inserted. Mice were housed in the animal care facility
with 12-hour light/dark cycle and allowed free access to food and
water. Body weight was recorded monthly. At 3 and 5 months, mice
were euthanized for histological analysis. A total of 6 groups were
examined with 10 mice for each group at starting points.
Non-diabetic (non-DM), diabetic (DM), and diabetic mice (C57BL6 and
eNOS KO) with insulin treatment (DMIns) were examined. Systolic
blood pressure was assessed as the mean value of 5-10 consecutive
measurements obtained in the morning using a tail-cuff
sphygmomanometer (Visitech BP2000, Visitech Systems, Inc., Apex,
N.C.). BUN was measured by BUN assay (Diagnostic chemicals limited,
PE, Canada). Urine in bladder was obtained for urinary albumin
excretion at sacrifice. Albumin-to-Creatinine ratio was measured
with Albuwell M (Exocell Inc., Philadelphia, Pa.) and Liquid
Creatinine Assay (Bioquant, San Diego, Calif.), respectively.
[0115] Renal Histology
[0116] Kidneys were fixed in Fekete's fixative (mixture of ethanol,
distilled water, 37% formalin and glacial acetic acid), and
embedded in paraffin. 2-.mu.m sections were stained with the
periodic acid-Schiff reagent (PAS) or the periodic acid-methenamine
silver (PAM) and counterstained with hematoxylin. Indirect
immunoperoxidase staining was performed using antibodies to the
endothelial antigen, thrombomodulin (TM) (Yuzawa Y, Brentjens J R,
Brett J, Caldwell P R, Esposito C, Fukatsu A, Godman G, Stern D,
Andres G: Antibody-mediated redistribution and shedding of
endothelial antigens in the rabbit. J Immunol 150: 5633-5646, 1993)
or CD34 (BD Pharmingen, San Jose, Calif.) (Fina L, Molgaard H V,
Robertson D, Bradley N J, Monaghan P, Delia D, Sutherland D R,
Baker M A, Greaves M F: Expression of the CD34 gene in vascular
endothelial cells. Blood 75: 2417-2426, 1990), and to vascular
smooth muscle cells with anti-smooth muscle actin (Abeam,
Cambridge, Mass.). To detect endothelial cell proliferation, double
immunostaining was performed with an antibody to the proliferating
cell nuclear antigen, Ki67 (Abeam, Cambridge, Mass.) and
thrombomodulin. Color was developed using DAB as a chromogen. In
double staining, Bjoran Purple (BioCare Medical, Concord, Calif.)
was used for thrombomodulin.
[0117] Quantification of Morphology
[0118] All quantifications were performed in a blinded fashion.
Using coronal sections of the kidney, all glomeruli (100-200 of
glomeruli per each animal) were examined. Glomerular mesangial
expansion, mesangiolysis, and nodular lesions were evaluated. The
percentage of mesangiolysis was calculated as the number of
glomeruli with mesangiolysis divided by that of total glomeruli.
Arteriolar morphology was assessed by indirect peroxidase
immunostaining for alpha-smooth muscle actin. Only vessels which
were adjacent to glomeruli in the outer cortex and possessed
flattened endothelial cells were selected for arterioles as
previously described (Mazzali M, Kanellis J, Han L, Feng L, Xia Y
Y, Chen Q, Kang D H, Gordon K L, Watanabe S, Nakagawa T, Lan H Y,
Johnson R J: Hyperuricemia induces a primary renal arteriolopathy
in rats by a blood pressure-independent mechanism. Am J Physiol
Renal Physiol 282: F991-997, 2002). Afferent arteriolar wall
thickness was measured by computer image analysis. For each
arteriole, the outline of the vessel and its internal lumen
(excluding the endothelium) were generated by using computer
analysis to calculate the total wall area (outline-inline) in a
minimum of 12 arterioles. Vessels that were cross-sectioned or not
sectioned transversally, providing an asymmetrical wall, were
excluded from the present study. Proliferating endothelial cells
were identified by double staining with Ki67 and TM or CD34.
[0119] Real Time PCB
[0120] To quantify mRNA expression for VEGF, real time PCR was
performed as described previously (Nakagawa T, Lan H Y, Zhu H J,
Kang D H, Schreiner G F, Johnson R J: Differential regulation of
VEGF by TGF-beta and hypoxia in rat proximal tubular cells. Am J
Physiol Renal Physiol 287: F658-664, 2004). Briefly, after 1 .mu.g
of total RNA was converted to cDNA with Platinum PCR supermix
(Biorad), PCR was performed with mouse VEGF or GAPDH primers mixed
with SYBR Green JumpStat Taq ReadyMix (Sigma) using a DNA Engine
OPTICON (MJ Research, Waltham, Mass.) as follows: 94.degree. C. for
5 min, then 35 cycles of denaturation at 94.degree. C. for 30 sec,
annealing at 61.degree. C. for 1 min and extension at 72.degree. C.
for 90 sec. The sizes of amplicons were 111 by (mouse VEGF)
(Emanueli C, Salis M B, Van Linthout S, Meloni M, Desortes E,
Silvestre J S, Clergue M, Figueroa C D, Gadau S, Condorelli G,
Madeddu P: Akt/protein kinase B and endothelial nitric oxide
synthase mediate muscular neovascularization induced by tissue
kallikrein gene transfer. Circulation 110: 1638-1644, 2004).
Reaction specificity was confirmed by electrophoretic analysis of
products in 2% agarose gel prior to real-time RT-PCR and bands of
expected size were detected. Ratios to GAPDH mRNA were calculated
for each sample and expressed as mean.+-.SD.
[0121] Statistical Analysis
[0122] All values presented are expressed as mean.+-.SD. The
unpaired Student's t-test was used to determine statistical
difference between two experimental groups. Significance was
defined as p<0.05.
Results
[0123] General parameters. The induction of type 1 diabetes by
streptozotocin resulted in equivalent hyperglycemia in C57BL6 and
eNOS KO mice when measured at 3 and 5 months, as shown in Table 4.
However, loss of body weight was more severe in diabetic eNOS KO
mice compared to diabetic C57BL6 mice. Systolic blood pressure was
higher in non-diabetic eNOS KO mice at 3 months but fell to lower
levels that wildtype controls at 5 months. Indeed, blood pressure
was unmeasurable in 2 out of 6 diabetic eNOS KO mice at 5 months
whereas 2 other diabetic eNOS KO mice demonstrated low blood
pressures of 92 and 104 mmHg, respectively. Survival of diabetic
eNOS knockout mice was also lower at 5 months compared to diabetic
wild type mice (FIG. 3).
[0124] Insulin treatment was associated with significant
improvements in blood glucose levels in both wildtype and eNOS
knockout mice. Interestingly, insulin treatment significantly
improved blood sugar, blood pressure, and survival in eNOS knockout
mice (Table 4 and FIG. 3). Elevated blood pressure at 3 months was
improved with insulin treatment in eNOS knockout mice, while lower
blood pressures at 5 months was also largely prevented by insulin
treatment.
[0125] Renal function and gross morphology. Diabetes-induced renal
hypertrophy was more pronounced in eNOS KO mice (Table 4). Diabetic
wildtype and eNOS knockout mice demonstrate higher urinary albumin
excretion as well as high BUN levels at 3 months. However, urinary
albumin excretion and BUN levels were higher in eNOS knockout mice
compared to diabetic wildtype mice at 5 months (Table 4). The
administration of insulin at doses that resulted in normalization
of blood sugar prevented the development of renal hypertrophy,
proteinuria and renal dysfunction both in the wildtype and eNOS
knockout mice (Table 4).
[0126] Glomerular Histology
[0127] Both C57BL6 and eNOS KO diabetic mice developed mesangial
expansion but it was more prominent in the eNOS knockout mice (FIG.
4A, B and Table 5). As shown in FIG. 5, blood glucose levels
correlated with mesangial expansion both in C57BL6 mice and eNOS KO
mice. Interestingly, glomeruli in eNOS KO were more susceptible to
blood glucose than wild type mice in terms of development of
mesangial expansion (FIG. 5A). Most importantly, at 3 months there
were striking findings in diabetic eNOS KO mice, in which
mesangiolysis (FIG. 4D) and glomerular microaneurysms (FIG. 4E)
developed. Furthermore, Kimmelstiel-Wilson-like nodular lesions
were observed in occasional glomeruli at both 3 and 5 months. These
nodular lesions were composed of nodular mesangial expansion (FIG.
4F), acellular PAS-positive material (FIG. 4G), and dense fibrillar
mesangial matrix (FIG. 4H). Nodular glomerulosclerosis was
demonstrated by serial section of glomeruli with PAS and PAM
staining (FIGS. 4J and K). Hyalinosis of arterioles (FIG. 4I) or of
the vascular pole of the glomerulus (FIG. 4L) were also observed in
diabetic eNOS KO mice. Interestingly the presence of significant
arteriolar disease in individual glomeruli were often associated
with glomerular mesangiolysis (FIG. 4I). Mesangiolysis also
correlated with blood glucose levels in diabetic eNOS KO mice (FIG.
5B). In addition, non-diabetic eNOS KO mice rarely developed
mesangiolysis at 5 months (Table 6). Interestingly, insulin
treatment blocked the development of mesangial expansion,
mesangiolysis, and the development of the nodular lesions at 3 and
5 months (Table 5, 6).
[0128] Renal Arteriolar Histology
[0129] The inventors have previously demonstrated in other models
that the development of preglomerular arteriolar disease results in
altered autoregulation and can predispose kidneys to progression
(Johnson R J, Feig D I, Herrera-Acosta J, Kang D H: Resurrection of
uric acid as a causal risk factor in essential hypertension.
Hypertension 45: 18-20, 2005). The inventors have also shown that
preglomerular arteriolar disease occurs with blockade of NO
synthesis with L-NAME (Quiroz Y, Pons H, Gordon K L, Rincon J,
Chavez M, Parra G, Herrera-Acosta J, Gomez-Garre D, Largo R, Egido
J, Johnson R J, Rodriguez-Iturbe B: Mycophenolate mofetil prevents
salt-sensitive hypertension resulting from nitric oxide synthesis
inhibition. Am J Physiol Renal Physiol 281: F38-47, 2001). The
inventors therefore examined the morphology of the afferent
arteriole in both diabetic and nondiabetic mice. As shown in FIG.
5, the lumen of arterioles of eNOS KO mice were larger than that
observed in C57BL6 mice. In animals with diabetes there was a
further increase in the inner lumen size in eNOS KO mice compared
to non-diabetic C57BL6 mice (FIG. 6A, 6E, 6F). This increase was
blocked by insulin treatment (FIG. 6A). On the other hand, the
total vascular smooth muscle wall area was not different in these
mice (FIG. 6C). Interestingly, glomeruli with mesangiolysis were
significantly associated with dilated arterioles (FIG. 6B) as well
as an increase in vascular smooth muscle wall area (FIG. 6D)
compared to those glomeruli without mesangiolysis.
[0130] eNOS KO mice also demonstrated rare focal areas of tubular
atrophy with condensed, hypoplastic glomeruli (FIG. 6G). In these
areas, the arterioles were severely constricted or occluded (FIG.
6H).
[0131] Angiogenesis (Endothelial Cell Proliferation)
[0132] VEGF mRNA expression was increased in diabetic C57BL6 and
eNOS KO mice (FIG. 8D). Importantly, insulin treatment blocked this
up-regulation of VEGF, demonstrating a key role for glucose in
regulating VEGF regardless of the status of the endothelial NO
system.
[0133] Endothelial morphology was assessed by immunostaining for
CD34 (Fina L, Molgaard H V, Robertson D, Bradley N J, Monaghan P,
Delia D, Sutherland D R, Baker M A, Greaves M F: Expression of the
CD34 gene in vascular endothelial cells. Blood 75: 2417-2426, 1990)
and thrombomodulin (Yuzawa Y, Brentjens J R, Brett J, Caldwell P R,
Esposito C, Fukatsu A, Godman G, Stern D, Andres G:
Antibody-mediated redistribution and shedding of endothelial
antigens in the rabbit. J Immunol 150: 5633-5646, 1993). Both
diabetic eNOS knockout and wildtype mice showed a generalized
increase in endothelial cells in the cortex as noted by
immunostaining for either CD34 or thrombomodulin (FIGS. 7 and 8),
and this was associated with enhanced endothelial cell
proliferation, as noted by double staining with Ki67 and
thrombomodulin or CD34 (FIG. 7G, 7H). Both endothelial
proliferation and endothelial immunostaining were increased in eNOS
diabetic knockout compared to diabetic wildtype mice. Insulin
treatment also largely corrected the increase in endothelial cell
proliferation and number.
[0134] In contrast, focal loss of endothelial cell staining was
occasionally observed, particularly in glomeruli displaying
mesangiolysis (FIGS. 7A and 7B).
Discussion
[0135] In this study, the inventors present a mouse model of
diabetic kidney disease that closely resembles human diabetic
nephropathy. Diabetic mice lacking the eNOS gene demonstrated
classic features of diabetic nephropathy with intrarenal vascular
disease, mesangial expansion with mesangiolysis and occasional
microaneurysm formation, and with the development of mesangial
nodular (Kimmelsteil-Wilson) lesions. These changes could be
largely prevented by insulin. Collectively, the data strongly
suggests that a relative deficiency in endothelial NO levels may be
one of the long-sought risk factors that is critical for the
increased susceptibility for nephropathy in subjects with
diabetes.
[0136] Without being bound to any particular theory, the inventors
believe that one potential mechanism by which eNOS knockout mice
may be more susceptible to diabetic nephropathy is due to the
dysregulation of the VEGF-NO axis. Normally VEGF acts on
endothelial cells largely via stimulation of eNOS. However, in the
setting where endothelial NO levels are low, an increase in VEGF
expression is associated with a marked NO-independent endothelial
proliferative response. The inventors have found that elevated
glucose can cause this uncoupling in vitro.
[0137] Consistent with the uncoupling hypothesis was our
observation that endothelial cell staining and proliferation were
increased in diabetic eNOS knockout mice compared to diabetic
control mice. Importantly, the increased expression of VEGF was
blocked in both groups of mice with insulin treatment. This
demonstrates that the regulation of VEGF expression appears to be
primarily dependent on glucose levels as opposed to endothelial NO
levels in this model. In addition, the observation that endothelial
staining was greater in eNOS knockout mice compared to wild type
mice regardless of presence of diabetes suggests the importance of
the uncoupling hypothesis in augmenting the endothelial
proliferative response (Nakagawa T, Sato W, Sautin Y Y, Glushakova
O, Croker B, Atkinson M A, Tisher C C, Johnson R J: Uncoupling of
vascular endothelial growth factor with nitric oxide as a mechanism
for diabetic vasculopathy. J Am Soc Nephrol 17: 736-745, 2006). In
contrast, Murohara et al have reported that eNOS KO mice exhibited
impaired angiogenesis in the hindlimb ischemic model (Murohara T,
Asahara T, Silver M, Bauters C, Masuda H, Kalka C, Kearney M, Chen
D, Symes J F, Fishman M C, Huang P L, Isner J M: Nitric oxide
synthase modulates angiogenesis in response to tissue ischemia. J
Clin Invest 101: 2567-2578, 1998). In their model, however, the
ischemic insult failed to increase VEGF expression whereas in our
model the primary stimulus appeared to be hyperglycemia.
[0138] An interesting finding was that the presence of
mesangiolysis was associated with loss of glomerular endothelial
cells whereas most other glomeruli showed an endothelial
proliferative response in diabetic eNOS KO mice. This heterogeneity
of endothelial response could be associated with the heterogeneity
of mesangial cell proliferation. Indeed, it has been demonstrated
that a glomerulus simultaneously exhibits mesangial proliferation
and mesangiolysis in human diabetic nephropathy (Stout L C, Kumar
S, Whorton E B: Focal mesangiolysis and the pathogenesis of the
Kimmelstiel-Wilson nodule. Hum Pathol 24: 77-89, 1993). Furthermore
it is also compatible with the evidence that anti-Thy1-induced
mesangiolysis in rat is associated with loss of both mesangial and
endothelial cells followed by both mesangial and glomerular
endothelial cell proliferation (Iruela-Arispe L, Gordon K, Hugo C,
Duijvestijn A M, Claffey K P, Reilly M, Couser W G, Alpers C E,
Johnson R J: Participation of glomerular endothelial cells in the
capillary repair of glomerulonephritis. Am J Pathol 147: 1715-1727,
1995).
[0139] It is also possible that the deletion of eNOS gene could
have altered local endothelial viability and thereby predisposed
glomeruli to mesangiolysis (Table 6). However, the fact that the
mesangiolysis was largely prevented by insulin treatment suggests
that elevated glucose (and/or AGEs) are also likely important
factors. However, a high glucose cannot be the sole factor since
diabetic C57BL6 mice did not develop mesangiolysis. However, a high
glucose could additionally impair endothelial function, and thereby
accelerate the development of glomerular injury.
[0140] The inventors believe that this new murine model of diabetic
nephropathy may be relevant to human diabetic disease. In addition
to the similar histological findings, human diabetic nephropathy is
also strongly associated with endothelial dysfunction due to the
effects of glucose and AGEs, but also because of the frequent
elevations in uric acid (Bo S, Cavallo-Perin P, Gentile L, Repetti
E, Pagano G: Hypouricemia and hyperuricemia in type 2 diabetes: two
different phenotypes. Eur J Clin Invest 31: 318-321, 2001.), CRP
(Tan K C, Chow W S, Tam S C, Ai V H, Lam C H, Lam K S: Atorvastatin
lowers C-reactive protein and improves endothelium-dependent
vasodilation in type 2 diabetes mellitus. J Clin Endocrinol Metab
87: 563-568, 2002), oxidative stress (Beckman J A, Goldline A B,
Gordon M B, Garrett L A, Kearley J F, Jr., Creager M A: Oral
antioxidant therapy improves endothelial function in Type 1 but not
Type 2 diabetes mellitus. Am J Physiol Heart Circ Physiol 285:
H2392-2398, 2003) and asymmetric dimethylarginine (ADMA) (Fard A,
Tuck C H, Donis J A, Sciacca R, Di Tullio M R, Wu H D, Bryant T A,
Chen N T, Torres-Tamayo M, Ramasamy R, Berglund L, Ginsberg H N,
Homma S, Cannon P J: Acute elevations of plasma asymmetric
dimethylarginine and impaired endothelial function in response to a
high-fat meal in patients with type 2 diabetes. Arterioscler Thromb
Vasc Biol 20: 2039-2044, 2000; Tarnow L, Hovind P, Teerlink T,
Stehouwer C D, Parving H H: Elevated plasma asymmetric
dimethylarginine as a marker of cardiovascular morbidity in early
diabetic nephropathy in type 1 diabetes. Diabetes Care 27: 765-769,
2004) all which are known to reduce endothelial NO bioavailability
(Landmesser U, Harrison D G, Drexler H: Oxidant stress-a major
cause of reduced endothelial nitric oxide availability in
cardiovascular disease. Eur J Can Pharmacol: 1-7, 2005). Since uric
acid, CRP, and ADMA tend to be higher in humans than rodents, this
could provide a potential explanation why rodents are much less
likely to develop classic diabetic renal disease. It is well known
that only 30-40% of subjects with type I diabetes will develop
significant nephropathy. Based on the findings in this study, the
inventors propose that it is the level of endothelial NO that may
be one of the critical determinants for whether diabetic subjects
are at risk for developing nephropathy. By extension, normalizing
uric acid levels in diabetic patients may delay the onset or
prevent diabetic nephropathy.
Example 4
Treatment of Prevention of Fatty Liver Syndrome
[0141] Non-alcoholic fatty liver disease (NAFLD), a more recently
appreciated component of the Metabolic Syndrome with a more than
30-fold relative risk in obese individuals, is believed to be the
most prevalent form of liver disease worldwide. Fatty liver
syndrome is dramatically increased in patients with metabolic
syndrome. Liver steatosis associated with obesity results from
increased plasma free fatty acids uptake, enhanced rate of de novo
fatty acid synthesis, and/or dysregulation of intracellular lipid
partitioning in which fatty acid oxidation is impaired and its
esterification enhanced (Fromenty B, et al. Diabetes Metab.
2004,30:121; Festi D, et al. Obes. Rev. 2004, 5:27). As discussed
above, the inventors show that fructose enriched diet induces the
metabolic syndrome (hyperinsulinemia, hypertriglyceridemia,
hyperuricemia and weight gain) in rats at 8 weeks. Further, the
inventors realize that fructose is known to cause fatty liver. The
inventors demonstrate that administration the uric acid lowering
agent, allopurinol, reduces the exhibition of these
characteristics. Metabolic syndrome produces an increase
triglyceride plasma levels, which likely reflect intracellular
triglyceride stores that are responsible for the fatty liver. By
logical extension, the inventors assert that reducing the onset of
the metabolic syndrome will reduce the onset of fatty liver.
[0142] The disclosures of the cited patent documents, publications
and references are incorporated herein in their entirety to the
extent not, inconsistent with the teachings herein. It should be
understood that the examples and embodiments described herein are
for illustrative purposes only and that various modifications or
changes in light thereof will be suggested to persons skilled in
the art and are to be included within the spirit and purview of
this application and the scope of the appended claims
* * * * *