U.S. patent application number 10/721351 was filed with the patent office on 2004-06-03 for method for kidney disease treatment by drug intervention.
This patent application is currently assigned to MONASH UNIVERSITY. Invention is credited to Comper, Wayne D..
Application Number | 20040106155 10/721351 |
Document ID | / |
Family ID | 25645955 |
Filed Date | 2004-06-03 |
United States Patent
Application |
20040106155 |
Kind Code |
A1 |
Comper, Wayne D. |
June 3, 2004 |
Method for kidney disease treatment by drug intervention
Abstract
A method is disclosed for diagnosing early stage of a disease in
which an intact protein found in urine is an indicator of the
disease, followed by early drug intervention to prevent and treat
the disease are also disclosed. The drug treatment involves the use
of a lysosome activating compound.
Inventors: |
Comper, Wayne D.; (Victoria,
AU) |
Correspondence
Address: |
McDermott, Will & Emery
600 13th Street, N.W.
Washington
DC
20005-3096
US
|
Assignee: |
MONASH UNIVERSITY
Victoria
AU
|
Family ID: |
25645955 |
Appl. No.: |
10/721351 |
Filed: |
November 26, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10721351 |
Nov 26, 2003 |
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09893346 |
Jun 28, 2001 |
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09893346 |
Jun 28, 2001 |
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09415217 |
Oct 12, 1999 |
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6447989 |
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Current U.S.
Class: |
435/7.1 |
Current CPC
Class: |
A61P 9/10 20180101; A61P
29/00 20180101; A61P 35/00 20180101; A61P 9/12 20180101; A61P 13/12
20180101; A61P 1/18 20180101; A61P 31/18 20180101; A61P 17/00
20180101; A61P 3/10 20180101; G01N 33/5091 20130101; A61P 7/00
20180101; A61P 17/06 20180101; A61P 11/00 20180101; A61P 21/00
20180101; A61P 9/00 20180101; Y10S 436/811 20130101; G01N 33/50
20130101; A61P 25/30 20180101; G01N 33/68 20130101 |
Class at
Publication: |
435/007.1 |
International
Class: |
G01N 033/53 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 21, 1998 |
AU |
PP7843 |
Dec 20, 1999 |
WO |
PCT/IB99/02029 |
Claims
What is claimed is:
1. A method for treating a person with a renal disease and/or renal
complications of a disease, comprising: (a) administering a
treatment agent to a person in need thereof; (b) obtaining a sample
of body fluid from the person; and (c) assaying for a protein in
the sample, wherein either presence of or lack of presence of the
protein in the urine or decreasing amount of the protein over time
in the urine indicates that the treatment agent is therapeutically
effective for the renal disease and/or renal complications of a
disease.
2. The method according to claim 1, wherein the renal disease
and/or renal complications of the disease is selected from the
group consisting of nephropathy, diabetes insipidus, diabetes type
I, diabetes II, renal disease (glomerulonephritis, bacterial and
viral glomerulonephritides, IgA nephropathy and Henoch-Schonlein
Purpura, membranoproliferative glomerulonephritis, membranous
nephropathy, Sjogren's syndrome, nephrotic syndrome (minimal change
disease, focal glomerulosclerosis and related disorders), acute
renal failure, acute tubulointerstitial nephritis, pyelonephritis,
GU tract inflammatory disease, Pre-clampsia, renal graft rejection,
leprosy, reflux nephropathy, nephrolithiasis), genetic renal
disease (medullary cystic, medullar sponge, polycystic kidney
disease (autosomal dominant polycystic kidney disease, autosomal
recessive polycystic kidney disease, tuborous sclerosis), von
Hippel-Lindau disease, familial thin-glomerular basement membrane
disease, collagen III glomerulopathy, fibronectin glomerulopathy,
Alport's syndrome, Fabry's disease, Nail-Patella Syndrome,
congenital urologic anomalies), monoclonal gammopathies (multiple
myeloma, amyloidosis and related disorders), febrile illness
(familial Mediterranean fever, HIV infection -AIDS), inflammatory
disease (systemic vasculitides (polyarteritis nodosa, Wegener's
granulomatosis, polyarteritis, necrotizing and crescentic
glomerulonephritis), polymyositis-dermatomyositis, pancreatitis,
rheumatoid arthritis, systemic lupus erythematosus, gout), blood
disorders (sickle cell disease, thrombotic thrombocytopenia
purpura, hemolytic-uremic syndrome, acute corticol necrosis, renal
thromboembolism), trauma and surgery (extensive injury, burns,
abdominal and vascular surgery, induction of anesthesia), drugs
(penicillamine, steroids) and drug abuse, malignant disease
(epithelial (lung, breast), adenocarcinoma (renal), melanoma,
lymphoreticular, multiple myeloma), circulatory disease (myocardial
infarction, cardiac failure, peripheral vascular disease,
hypertension, coronary heart disease, non-atherosclerotic
cardiovascular disease, atherosclerotic cardiovascular disease),
skin disease (psoriasis, systemic sclerosis), respiratory disease
(COPD, obstructive sleep apnoea, hypoia at high altitude) and
endocrine disease (acromegaly, diabetes mellitus, diabetes
insipidus).
3. The method according to claim 1, wherein the treatment agent is
a lysosome-activating compound.
4. The method according to claim 3, wherein the lysosome-activating
compound is selected from the group consisting of ACE inhibitors,
anti-glycation agents, anticancer compounds, antiproliferation
compounds, and compounds that neutralize TGF-beta.
5. The method according to claim 3, wherein the lysosome-activating
compound is selected from the group consisting of ramipril,
aminoguanidine, paracetamol, vitamin A (retinoic acid), retinol
derivatives, and anti-TGF beta antibodies.
6. The method according to claim 1, wherein the sample of body
fluid is a urine, blood or laboratory sample.
7. The method according to claim 1, wherein the protein comprises
albumin, globulin (.alpha.-globulin(.alpha..sub.1-globulin,
.alpha..sub.2-globulin),.beta.-globulin,.gamma.-globulin),
euglobulin, pseudoglobulin I and II, fibrinogen, .alpha..sub.1 acid
glycoprotein (orosomucoid), .alpha..sub.1 glycoprotein,
.alpha..sub.1 lipoprotein, ceruloplasmin, .alpha..sub.2 19S
glycoprotein, .beta..sub.1 transferrin, .beta..sub.1 lipoprotein,
immunoglobulins A, E, G, and M, horseradish peroxidase, lactate
dehydrogenase, glucose oxidase, myoglobin, lysozyme, protein
hormone, growth hormone, insulin, or parathyroid hormone.
8. The method according to claim 1, wherein the assaying for a
protein in the sample comprises a method selected from the group
consisting of: (a) assaying for albumin by a conventional method;
and (b) assaying for intact modified albumin.
9. The method according to claim 8, wherein the conventional method
comprises a method selected from the group consisting of: (a) an
antibody method, and (b) a non-antibody method comprising loading
the sample on a chromatography, electrophoresis or sedimentation
apparatus to test for native or intact modified albumin.
10. The method according to claim 9, wherein the albumin is
detected by an antibody that is specific for both unmodified and
modified forms of the protein.
11. The method according to claim 9, wherein the albumin is
detected by an antibody that is specific for the modified
protein.
12. The method according to claim 9, wherein the albumin is
detected by an antibody that is attached to an enzymatic,
radioactive, fluorescent or chemiluminescent label, wherein the
detecting step comprises radioimmunoassay, immunoradiometric assay,
fluorescent immunoassay, enzyme linked immunoassay, or protein A
immunoassay.
13. The method according to claim 1, wherein the assaying for a
protein in the sample comprises the steps of: (i) detecting native
albumin amount by conventional antibody assay: (ii) detecting
intact modified albumin by a non-antibody method; and (iii) adding
the values obtained in (i) and (ii) to obtain an accurate reading
of total albumin content in the sample.
14. The method according to claim 13, wherein the non-antibody
method comprises loading the sample on a chromatography,
electrophoresis or sedimentation apparatus to test for native or
intact modified albumin.
15. The method according to claim 1, wherein the assaying for a
protein in the sample is a non-antibody method comprising detecting
a sum of native protein and intact modified protein in a
sample.
16. The method according to claim 1, wherein the assaying for a
protein in the sample is by a method selected from the group
consisting of partition chromatography, adsorption chromatography,
paper chromatography, thin-layer chromatography, gas-liquid
chromatography, gel chromatography, ion-exchange chromatography,
affinity chromatography, or hydrophobic interaction chromatography,
moving-boundary electrophoresis, zone electrophoresis, or
isoelectric focusing.
17. The method according to claim 1, wherein the assaying for a
protein in the sample is by hydrophobic interaction chromatography
carried out in a high pressure liquid chromatography (HPLC)
apparatus.
18. The method according to claim 1, wherein the assaying for a
protein in the sample is by detecting albumin in the sample with
specific albumin dyes.
19. The method according to claim 1, wherein an early stage of the
disease is diagnosed when modified albumin is present in the sample
in increasing amounts over time.
20. A method for identifying a treatment agent for renal disease
and/or renal complications of a disease, comprising: (a)
administering to a person in need thereof an agent that is
suspected of being able to treat the disease; (b) obtaining a urine
sample from the person; and (c) assaying for a protein in the
sample, wherein either presence of or lack of presence of the
protein in the urine or decreasing amount of the protein over time
in the urine indicates that the agent is a treatment agent for the
renal disease and/or renal complications of a disease.
21. The method of claim 19, wherein the for a protein in the sample
comprises assaying for a modified form of albumin in the sample,
wherein either presence of or a lack of presence of the modified
form of the protein in the sample or decreasing amount of the
modified form of the protein over time in the urine indicates that
the agent is a treatment agent for the renal disease and/or renal
complications of a disease.
Description
CLAIM OF PRIORITY
[0001] This application is a continuation-in-part of and claims
priority to parent application Ser. No. ______, filed Jun. 22,
2001, which is a continuation-in-part of and claims priority to
Ser. No. 09/415,217, filed Oct. 12, 1999.
FIELD OF THE INVENTION
[0002] The present invention relates to methods of detecting an
early stage of renal disease and/or renal complications of a
disease. The invention also relates to preventing and treating the
disease.
BACKGROUND OF THE INVENTION
[0003] The appearance of excess protein such as albumin in the
urine is indicative of kidney disease. Diabetic nephropathy is such
a disease. By the time the excess albumin is detected, kidney
disease has progressed, possibly to a stage where it is
irreversible and treatment has little effect. Therefore it is an
object of the invention to provide a test that is more sensitive
than the currently known radioimmunoassay to detect such a disease
as early as possible so that the disease can be either prevented or
a treatment protocol commenced early on in the disease.
[0004] Specific proteinuria, and in particular, albuminuria (micro-
and macro-), is a marker of disease including renal disease
(glomerulonephritis, bacterial and viral glomerulonephritides, IgA
nephropathy and Henoch-Schonlein Purpura, membranoproliferative
glomerulonephritis, membranous nephropathy, Sjogren's syndrome,
diabetic nephropathy, nephrotic syndrome (minimal change disease,
focal glomerulosclerosis and related disorders), acute renal
failure, acute tubulointerstitial nephritis, pyelonephritis, GU
tract inflammatory disease, Pre-clampsia, renal graft rejection,
leprosy, reflux nephropathy, nephrolithiasis), genetic renal
disease (medullary cystic, medullar sponge, polycystic kidney
disease (autosomal dominant polycystic kidney disease, autosomal
recessive polycystic kidney disease, tuborous sclerosis), von
Hippel-Lindau disease, familial thin-glomerular basement membrane
disease, collagen III glomerulopathy, fibronectin glomerulopathy,
Alport's syndrome, Fabry's disease, Nail-Patella Syndrome,
congenital urologic anomalies), monoclonal gammopathies (multiple
myeloma, amyloidosis and related disrders), febrile illness
(familial Mediterannean fever, HIV infection -AIDS), inflammatory
disease (systemic vasculitides (polyarteritis nodosa, Wegener's
granulomatosis, polyarteritis, necrotizing and crescentic
glomerulonephritis), polymyositis-dermatomyositis, pancreatitis,
rheumatoid arthritis, systemic lupus erythematosus, gout), blood
disorders (sickle cell disease, thrombotic thrombocytopenia
purpura, hemolytic-uremic syndrome, acute corticol necrosis, renal
thromboembolism), trauma and surgery (extensive injury, burns,
abdominal and vascular surgery, induction of anesthesia), drugs
(penicillamine, steroids) and drug abuse, malignant disease
(epithelial (lung, breast), adenocarcinoma (renal), melanoma,
lymphoreticular, multiple myeloma), circulatory disease (myocardial
infarction, cardiac failure, peripheral vascular disease,
hypertension, coronary heart disease, non-atherosclerotic
cardiovascular disease, atherosclerotic cardiovascular disease),
skin disease (psoriasis, systemic sclerosis), respiratory disease
(COPD, obstructive sleep apnoea, hypoia at high altitude) and
endocrine disease (acromegaly, diabetes mellitus, and diabetes
insipidus).
[0005] Kidney disease may result from bacterial infection,
allergies, congenital defects, stones, antibiotics,
immunosuppressives, antineoplastics, nonsteroidal anti-inflammatory
drugs, analgesics, heavy metals, tumors, chemicals.
[0006] The applicant has found that proteins, including albumin,
are normally excreted as a mixture of native protein and fragments
that are specifically produced during renal passage. Osicka T. M.
et al., Nephrology 2:199-212 (1996). Proteins are heavily degraded
during renal passage by post-glomerular (basement membrane) cells,
which may include tubular cells. Lysosomes in renal tubular cells
may be responsible for the breakdown of proteins excreted during
renal passage (see, FIG. 1). The breakdown products are excreted
into the tubular lumen. In normal individuals, most of the albumin
in the urine is fragmented.
SUMMARY OF THE INVENTION
[0007] The invention provides for the use of a lysosome-activating
compound in reactivating lysosomes or processes that direct
substrates to the lysosome or products away from the lysosome. When
lysosome activity or intracellular processes directing substrates
to lysosomes is reduced as in kidney disease, more of the high
molecular weight, and substantially full length filtered protein
including albumin appears in the urine. The applicant has found
that conventional drugs, the ACE inhibitor ramipril and the
anti-glycation agent aminoguanidine, are able to prevent changes in
the renal lysosomal processing of protein.
[0008] The invention is directed to a method for treating a person
suffering from a disease in which a diseased state is indicated by
specific proteinuria, by administering a therapeutically effective
amount of the treatment agent, obtained according to the above
method, to a person in need thereof. Preferably, the treatment
agent is a lysosome-activating compound.
[0009] In one aspect of the invention, there is provided a method
of preventing or treating kidney disease, the method including
administering an effective amount of lysosome-activating compound
to a subject in need thereof.
[0010] In another aspect of the present invention, there is
provided a composition comprising a lysosome-activating compound
and a carrier.
[0011] In yet another aspect of the present invention, there is
provided a method of screening a multiplicity of compounds to
identify a compound capable of activating or processes that direct
substrates to the lysosome or products away from the lysosome, said
method including the steps of:
[0012] (a) exposing said compound to a lysosome and assaying said
compound for the ability to activate a lysosome wherein said
lysosome when activated has a changed activity;
[0013] (b) assaying for the ability to restore a cellular process
to substantially normal levels in kidney tissue, wherein said
kidney has a low lysosome activity; and/or
[0014] (c) assaying for the ability to restore tissue turnover to
substantially normal levels in kidney tissue, wherein said kidney
tissue has low lysosome activity.
[0015] Although not limited to any particular disease, according to
the method of the invention, the disease sought to be treated
includes nephropathy, diabetes insipidus, diabetes type I, diabetes
II, renal disease (glomerulonephritis, bacterial and viral
glomerulonephritides, IgA nephropathy and Henoch-Schonlein Purpura,
membranoproliferative glomerulonephritis, membranous nephropathy,
Sjogren's syndrome, nephrotic syndrome (minimal change disease,
focal glomerulosclerosis and related disorders), acute renal
failure, acute tubulointerstitial nephritis, pyelonephritis, GU
tract inflammatory disease, Pre-clampsia, renal graft rejection,
leprosy, reflux nephropathy, nephrolithiasis), genetic renal
disease (medullary cystic, medullar sponge, polycystic kidney
disease (autosomal dominant polycystic kidney disease, autosomal
recessive polycystic kidney disease, tuborous sclerosis), von
Hippel-Lindau disease, familial thin-glomerular basement membrane
disease, collagen III glomerulopathy, fibronectin glomerulopathy,
Alport's syndrome, Fabry's disease, Nail-Patella Syndrome,
congenital urologic anomalies), monoclonal gammopathies (multiple
myeloma, amyloidosis and related disorders), febrile illness
(familial Mediterranean fever, HIV infection--AIDS), inflamrnatory
disease (systemic vasculitides (polyarteritis nodosa, Wegener's
granulomatosis, polyarteritis, necrotizing and crescentic
glomerulonephritis), polymyositis-dermatomyosi- tis, pancreatitis,
rheumatoid arthritis, systemic lupus erythematosus, gout), blood
disorders (sickle cell disease, thrombotic thrombocytopenia
purpura, hemolytic-uremic syndrome, acute cortical necrosis, renal
thromboembolism), trauma and surgery (extensive injury, burns,
abdominal and vascular surgery, induction of anesthesia), drugs
(penicillamine, steroids) and drug abuse, malignant disease
(epithelial (lung, breast), adenocarcinoma (renal), melanoma,
lymphoreticular, multiple myeloma), circulatory disease (myocardial
infarction, cardiac failure, peripheral vascular disease,
hypertension, coronary heart disease, non-atherosclerotic
cardiovascular disease, atherosclerotic cardiovascular disease),
skin disease (psoriasis, systemic sclerosis), respiratory disease
(COPD, obstructive sleep apnoea, hypoia at high altitude) and
endocrine disease (acromegaly, diabetes mellitus, diabetes
insipidus).
[0016] The invention can be used in conjunction with a method of
diagnosing early stage of renal disease and/or renal complications
of a disease, comprising:
[0017] (a) separating all of the proteins in a urine sample;
and
[0018] (b) detecting a modified form of a protein in the sample,
wherein detection of the modified protein is indicative of an early
stage of the renal disease and/or renal complications of a
disease.
[0019] The invention can be used for determining a treatment agent
(such as a lysosome-activating compound) for renal disease and/or
renal complications of a disease, comprising:
[0020] (a) administering to a person in need thereof an agent that
is suspected of being able to treat the disease;
[0021] (b) obtaining a urine sample from the person; and
[0022] (c) assaying for a modified form of the protein in the
sample, wherein either presence or lack of presence of the modified
form of the protein in the urine or decreasing amount of the
modified form of the protein over time indicates that the agent is
a treatment agent for the renal disease and/or renal complications
of a disease.
[0023] These and other objects of the invention will be more fully
understood from the following description of the invention, the
referenced drawings attached hereto and the claims appended
hereto.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1 illustrates the progress of filtered intact albumin
into tubular cells and breakdown of albumin to provide excreted
albumin fragments.
[0025] FIG. 2 (2a and 2b) illustrate a representative profile
of(.sup.3H) HSA in (a) urine and (b) plasma collected from normal,
healthy volunteers by size exclusion chromatography. Urine contains
mostly fragmented albumin. And plasma contains mostly intact
albumin.
[0026] FIG. 3 illustrates urine from normal, healthy volunteer
showing a fragmented albumin peak, but no intact albumin peak from
size exclusion chromatography.
[0027] FIG. 4 illustrates urine from a diabetic patient showing
both intact and fragmented albumin peaks from size exclusion
chromatography.
[0028] FIG. 5 illustrates a HPLC profile of albumin alone.
[0029] FIG. 6 illustrates the HPLC profile of plasma from normal,
healthy volunteer showing albumin peaks.
[0030] FIG. 7 shows the HPLC profile of urine from normal, healthy
volunteer with fragmented products of albumin but no intact albumin
peak.
[0031] FIG. 8 shows the HPLC profile of a urine sample from a
normoalbuminuric diabetic patient showing albumin breakdown
products and a small-modified albumin peak at approximately 39-44
minutes retention time.
[0032] FIG. 9 shows the HPLC profile of urine from a
normoalbuminuric diabetic patient showing signs of kidney failure
and the presence of the characteristic spiked albumin peak at
approximately 39-44 minutes retention time.
[0033] FIG. 10 illustrates a HPLC profile of a normoalbuminuric
diabetic patient showing signs of kidney failure and the presence
of the characteristic spiked modified albumin peak at approximately
39-44 minutes retention time.
[0034] FIG. 11 illustrates a HPLC of a macroalbuminuric diabetic
patient showing high levels of the normal albumin as well as the
characteristic spiked appearance at approximately 39-44 minutes
retention time.
[0035] FIG. 12 illustrates a longitudinal study of a patient in
which the modified protein was detected at a time prior to onset of
diabetic nephropathy, indicating predisposition to diabetic
nephropathy, and the delay in treatment caused by relying on
conventional RIA methods.
[0036] FIG. 13 illustrates a longitudinal study of a patient in
which the modified protein was detected at a time prior to onset of
diabetic nephropathy, indicating predisposition to diabetic
nephropathy, and the delay in treatment caused by relying on
conventional RIA methods.
[0037] FIG. 14 illustrates a longitudinal study of a patient in
which the modified protein was detected at a time prior to onset of
diabetic nephropathy, indicating predisposition to diabetic
nephropathy, and the delay in treatment caused by relying on
conventional RIA methods.
[0038] FIG. 15 shows the HPLC chromatogram used as a criterion of
purity of the modified albumin of Example 4.
[0039] FIG. 16 illustrates (.sup.3H)HSA in urine collected from a
microalbuminuric type I diabetic patient by size exclusion
chromatography. Urine contains mostly fragmented albumin. Plasma
contains only intact albumin. Conventional RIA (human albumin
ordinate) measures only intact albumin and not fragments.
[0040] FIG. 17 illustrates the size exclusion profiles of
(.sup.3H)HSA in urine from four different patients with varying
albumin excretion rate (AER) as measured by conventional RIA. As
intact albuminuria increases the amount of intact albumin in the
urine increases and the relative amount of fragments decreases. The
total excretion represents the excretion of intact albumin plus
albumin derived fragments.
DETAILED DESCRIPTION OF THE INVENTION
[0041] The applicant has discovered that when proteins, including
major plasma proteins such as albumin and immunoglobulin, are
filtered by the kidney they are subsequently degraded by cells in
the kidney prior to the material being excreted. It is likely that
filtered proteins are taken up by tubular cells. Tubular cells lie
beyond the kidney filter and come in direct contact with the
primary filtrate. When proteins are internalized by the tubular
cells, they are directed towards the lysosomes, where they are
partially degraded to various size fragments, and then regurgitated
to outside the cell. These regurgitated fragments, of which there
may be at least 60 different fragments generated from any one
particular type of protein, are then excreted into the urine.
[0042] The applicant has discovered that in renal disease
fragmentation of proteins is inhibited. This means that
substantially full-length filtered proteins will be excreted in a
person suffering from renal disease. This transition from
fragmentation to inhibition of fragmentation of excreted proteins
is a basis for the development of new drugs and diagnostic assays.
For example, initial changes that occur with the onset of renal
complications in diabetes are associated with a change in the
fragmentation profile of excreted albumin. This leads to an
apparent microalbuminuria, which is synonymous with the development
of diabetic nephropathy. It is likely that this is due to an
inhibition in the lysosomal activity of tubular cells in diabetes.
Thus, drugs can be formulated to turn on lysosomal activity in
diabetes where renal complications are occurring. The drugs may
also be useful in other renal diseases where lysosomal activities
are affected, or in diabetes without renal complications in
situations where lysosomal activity is turned off in non-renal
tissues. Such drugs include antiproliferative drugs, such as
anticancer drugs or antibodies to neutralize TGF-beta.
[0043] The invention is also directed to a method of determining a
treatment agent for renal disease and/or renal complications of a
disease, comprising: (a) administering to a person an agent that is
suspected of being able to treat the disease; (b) obtaining a urine
sample from the person; and (c) assaying for the protein in the
sample, wherein decreasing amount of the protein over time
indicates that the agent is a treatment agent for the disease. The
treatment agent may be a lysosome-activating agent that may act
directly or indirectly to activate lysosome, and thereby cause the
lysosome to digest post-glomerular filtered proteins, which is a
sign of a healthy kidney.
[0044] The process of trafficking of proteins to the lysosomes
plays a role in the mechanism of albuminuria in diabetes. An
intracellular molecule that is involved in trafficking is protein
kinase C (PKC). It is contemplated that a drug or agent can be
formulated that activates lysosomal trafficking or inhibits
PKC.
[0045] Accordingly, in one aspect of the present invention, there
is provided a lysosome-activating compound for use in reactivating
lysosomes or processes that direct substrates to the lysosome or
products away from the lysosome.
[0046] In another aspect of the present invention, there is
provided a composition comprising a lysosome-activating compound
and a carrier.
[0047] In yet another aspect of the invention there is provided a
method of preventing or treating kidney disease, said method
including administering an effective amount of a
lysosome-activating compound to a subject.
[0048] In yet another aspect of the present invention, there is
provided a method of screening a multiplicity of compounds to
identify a compound capable of activating lysosomes or processes
that direct substrates to the lysosome or products away from the
lysosome, said method including the steps of: (a) exposing said
compound to a lysosome and assaying said compound for the ability
to activate a lysosome wherein said lysosome when activated has a
changed activity; (b) assaying for the ability to restore a
cellular process to substantially normal levels in kidney tissue,
wherein said kidney tissue has a low lysosome activity; and/or (c)
assaying for the ability to restore tissue turnover to
substantially normal levels in kidney tissue, wherein said kidney
tissue has low lysosome activity.
[0049] Lysosomes are associated with the breakdown of proteins,
particularly albumin, in the kidney (see, FIG. 1). In cases of
microalbuminuria, substantial amounts of albumin escape lysosomal
breakdown possibly due to a deactivated lysosome. Restoration of
lysosomal breakdown restores the balance in the kidney of cellular
processes and tissue turnover.
[0050] A lysosome-activating compound is a compound that acts
directly or indirectly on the lysosome. By acting indirectly, the
compound may act on a component that influences the activity of the
lysosome. Nevertheless, the outcome results in an activation of the
lysosome, thereby providing enhanced protein breakdown.
[0051] In another aspect of the present invention, there is
provided a composition comprising a lysosome-activating compound
and a carrier.
[0052] The composition may be a physiologically acceptable or
pharmaceutically acceptable composition. However, it will be a
composition that allows for stable storage of the
lysosome-activating compound. Where the composition is a
pharmaceutically acceptable composition, it may be suitable for use
in a method of preventing or treating kidney disease.
[0053] In yet another aspect of the invention there is provided a
method of preventing or treating kidney disease, said method
including administering an effective amount of a
lysosome-activating compound to a subject.
[0054] As described above, the lysosome-activating compound may act
by reactivating the lysosome so that cellular processes and tissue
turnover are restored fully or in part, thereby resulting in the
kidney being restored partially or fully. In any case,
administering a lysosome-activating compound to an animal having
kidney disease may restore lysosome activity fully or in part.
[0055] Methods of administering may be oral or parenteral. Oral may
include administering with tablets, capsules, powders, syrups, etc.
Parenteral administration may include intravenous, intramuscular,
subcutaneous or intraperitoneal routes. Dosages for administration
of the compounds of the invention may be calculated by those of
skill in the art (see, Goodman & Gilman, The Pharmacological
Basis of Therapeutics, 8th Ed. (Pergamon Press, NY, 1990); and The
Merck Index, 11th Ed. (Merck and Co., Inc., Rahway, N.J. 1989);
both incorporated herein by reference). Preparation of aqueous
solutions, liposomes, emulsion and suspensions are known to those
of ordinary skill in the art (see, Remington's Pharmaceutical
Sciences, 18th Ed. (Mack Publishing Co., Easton, Pa., 1990);
incorporated herein by reference).
[0056] The changed activity of the lysosome is preferably a change
that enhances the activity of the lysosome so that albumin
breakdown is improved. The ability to not only activate lysosome
but also improve cellular processes and/or tissue turnover is a
characteristic of the most desirable lysosome-activating compound.
Preferably, it is desired to use the lysosome-activating compound
to restore kidney function.
[0057] DEFINITIONS
[0058] "Fragmented protein or fragment albumin" includes
post-glomerular breakdown products after chemical, enzymatic or
physical breakdown that occurs during renal passage. These
components have a reduced size and/or may have changed
hydrophobicity.
[0059] "Intact albumin, modified albumin, or modified form of
albumin" as used herein means a compound having similar size and
structural characteristics to native albumin, wherein the amino
acid sequence is substantially the same as the native albumin. It
is preferably a filtered intact protein. It elutes at or near the
same position as native albumin on high-pressure liquid
chromatography (HPLC) (FIG. 5). However, the structure has been
modified biochemically either by minor enzyme mediated modification
or addition to its basic structure and/or physically through a
change in its three dimensional structure so that it escapes
detection by conventionally used anti-albumin antibodies.
Biochemical modification may be made by enzymes such as endo- or
exo- peptidases. The 3D structure of albumin may have been altered
in some way. Ligands may have bound to the albumin, or it may be
any combination of these. The modified albumin detected in the
method of the invention is not detectable by current and
conventional radioimmunoassays using available antibodies and is
not a fragment.
[0060] Conventional anti-albumin antibodies can be purchased from
any purveyor of immunochemicals. For example, monoclonal antibody
catalog numbers A6684 (clone no. HSA-11), and A2672 (clone no.
HSA-9), as well as liquid whole serum, lyophilized fractionates,
liquid IgG fraction, and the monoclonal antibodies in liquid
ascites fluids form, can be obtained from Sigma, St. Louis, Mo., as
found in the Immunochemicals section at pages 1151-1152 in the 1994
Sigma--Biochemicals Organic Compounds for Research and Diagnostic
Reagents catalog.
[0061] As used herein, intact/modified albumin includes albumin
that is substantially full-length, fragmented, chemically modified,
or physically modified. As used herein, intact/modified albumin is
meant to indicate albumin that is less than, equal to, or greater
in molecular weight than the full-length albumin, and elutes at or
near the native albumin position in a separation medium, such as
chromatography, preferably HPLC, and most preferably hydrophobicity
HPLC. As used herein, fragmented albumin is meant to refer to the
fragment of albumin that is not detected by conventional
anti-albumin antibody, and its presence is detected in diagnosing
an early stage of renal disease and/or renal complications of a
disease. The detection of the presence of intact/modified albumin
is an indication of a predisposition to renal disease.
[0062] "Intact protein, modified protein or modified form of a
protein" as used herein includes those forms of substantially
full-length protein which are undetectable by conventional
radioimmunoassay. The protein includes, but is not limited to,
albumins, globulins (.alpha.-globulin(.alpha..sub.1-globulin,
.alpha..sub.2-globulin),.beta.-- globulins,.gamma.-globulins),
euglobulins, pseudoglobulin I and II, fibrinogen, .alpha..sub.1,
acid glycoprotein (orosomucoid), .alpha..sub.1 glycoprotein,
.alpha..sub.1 lipoproteins, ceruloplasmin, .alpha..sub.2 19S
glycoprotein, .beta..sub.1 transferrin, .beta..sub.1 lipoprotein,
immunoglobulins A, E ,G, and M, protein hormones including growth
hormone, insulin, parathyroid hormone and other proteins including
horseradish peroxidase, lactate dehydrogenase, glucose oxidase,
myoglobin, and lysozyme.
[0063] "Kidney disease" as used herein includes any malfunction of
the kidney. Kidney disease may be identified by the presence of
intact or modified albumin in the urine. Preferably, an early
diagnosis of the kidney disease may be made by detecting the
presence of modified protein in the urine, or an increase in the
modified protein in the urine over time.
[0064] "Low lysosome activity" as used herein is compared against
normal levels of lysosome activity and/or lysosome machinery that
traffics protein to the lysosome in a normal individual. The
activity is insufficient for the lysosome to fragment proteins so
that intact protein is excreted at a greater amount than at
normally low levels.
[0065] "Lysosome-activating compound" as used herein refers to a
compound that is beneficial to reactivation of the lysosome. The
compound may work directly or indirectly on the lysosome resulting
in activation of lysosomal function. These compounds may be
selected from the group including, but not limited to, anticancer
compounds, antiproliferation compounds, paracetamol, insulin
vitamin A (retinoic acid) or derivatives of retinol, or compounds,
including antibodies, to neutralize TGF-beta.
[0066] Osicka T. M. et al., Diabetologia 44: 230-236 (2001)
examined the time course for the diabetes-related changes in renal
lysosomal processing and to determine whether these changes can be
prevented by aminoguanidine or ramipril treatment. The percentage
desulphation of intravenously injected tritium labelled dextran
sulphate ([.sup.3H]DSO.sub.4) in the urine, as determined by
ion-exchange chromatography, was used as a marker of lysosomal
sulphatase activity. The amount of totally desulphated
[.sup.3H]DSO.sub.4 was significantly reduced in diabetic rats. The
significant decrease in the amount of totally desulphated
([.sup.3H]DSO.sub.4) in the urine was not affected by drug
treatment. However, the increase in totally sulphated
([.sup.3H]DSO.sub.4) in the urine collected from diabetic rats
compared with that of control rats was normalised by treatment with
both aminoguanidine or ramipril. These results indicate that the
diabetes-induced changes in renal lysosomal processing may be one
of the initial events in the development of diabetic nephropathy.
Aminoguanidine and ramipril, which have been previously thought to
have different mechanism of action, seem to prevent similar
diabetes-induced changes in lysosomal processing either through
their similar effects on enzyme activity within the lysosome or
through their similar effects on the trafficking of molecules to
and from the lysosome.
[0067] "Macroalbuminuria" is a condition where an individual
excretes greater than 200 .mu.g albumin/min in the urine as
measured by conventional radioimmunoassay (RIA).
[0068] "Microalbuminuria" is a condition where an individual
excretes at least 20 .mu.g albumin/min in the urine as measured by
conventional radioimmunoassay (RIA). RIA measures down to 15.6
ng/ml and is able to measure albumin in urine of normal subjects
who have clearance of less than 6 .mu.g/min. However, when albumin
excretion exceeds 20 .mu.g/min, treatment of the kidney disease is
limited and full recovery is difficult from this point.
[0069] "Microalbuminuric" as used herein is a condition when
albumin is detected in the urine at an excretion rate of at least
20 .mu.g/min as measured by conventional RIA.
[0070] As used herein, "native" and "unmodified" are used
interchangeably to describe a protein that is naturally found in an
organism, preferably a human, which has not been modified by the
filtering process of the renal glomeruli.
[0071] "Normal individual" as used herein is an individual who does
not have a disease in which intact protein found in urine is an
indicator of the disease. Preferably, the disease is kidney
disease.
[0072] "Normal levels of lysosome activity" are levels of lysosome
activity found in undiseased kidney of a normal individual.
[0073] "Normoalbuminuric" as used herein means a condition where
albumin is excreted in the urine and is not detectable by RIA, or
less than 20 .mu.g/min (as measured by RIA) is excreted.
[0074] "Propensity for a disease" as used herein means that a
disease may result in an individual as judged by a determination of
the presence and excretion rate of a modified protein such as
modified albumin.
[0075] "Proteinuria" as used herein is the existence of protein in
the urine, usually in the form of albumin, a protein that is
soluble in water and can be coagulated by heat. Related to this,
"specific proteinuria" refers to the existence of a particular
protein in the urine.
[0076] "Radioimmunoassay" as used herein is a method for detection
and measurement of substances using radioactively labeled specific
antibodies or antigens.
[0077] "Reactivation of the lysosome" as used herein includes an
activation of lysosome activity preferably so that breakdown of
proteins, particularly albumin, is increased compared with an
inactivated state of the lysosome.
[0078] "Restore" as used herein means to restore in full or in part
so that the component being restored has an improved function
compared with its previous function.
[0079] The "sum of intact and intact modified protein" as used
herein refers to the total amount of intact protein, and intact
modified protein present in a biological sample.
[0080] "Total protein" as used herein refers to a particular
filtered protein present in native, unmodified, modified or
fragmented form that is excreted in urine. It includes protein that
is not detected by conventional radioimmunoassay or conventional
methods, which are currently available to detect the protein.
Preferably the protein is albumin.
[0081] According to the present invention, the diseases to be
treated include, but are not limited to renal disease
(glomerulonephritis, bacterial and viral glomerulonephritides, IgA
nephropathy and Henoch-Schonlein Purpura, membranoproliferative
glomerulonephritis, membranous nephropathy, Sjogren's syndrome,
diabetic nephropathy, nephrotic syndrome (minimal change disease,
focal glomerulosclerosis and related disorders), acute renal
failure, acute tubulointerstitial nephritis, pyelonephritis, GU
tract inflammatory disease, pre-clampsia, renal graft rejection,
leprosy, reflux nephropathy, nephrolithiasis), genetic renal
disease (medullary cystic, medullar sponge, polycystic kidney
disease (autosomal dominant polycystic kidney disease, autosomal
recessive polycystic kidney disease, tuborous sclerosis), von
Hippel-Lindau disease, familial thin-glomerular basement membrane
disease, collagen III glomerulopathy, fibronectin glomerulopathy,
Alport's syndrome, Fabry's disease, Nail-Patella Syndrome,
congenital urologic anomalies), monoclonal gammopathies (multiple
myeloma, amyloidosis and related disrders), febrile illness
(familial Mediterannean fever, HIV infection -AIDS), inflammatory
disease (systemic vasculitides (polyarteritis nodosa, Wegener's
granulomatosis, polyarteritis, necrotizing and crescentic
glomerulonephritis), polymyositis-dermatomyositis, pancreatitis,
rheumatoid arthritis, systemic lupus erythematosus, gout), blood
disorders (sickle cell disease, thrombotic thrombocytopenia
purpura, hemolytic-uremic syndrome, acute corticol necrosis, renal
thromboembolism), trauma and surgery (extensive injury, burns,
abdominal and vascular surgery, induction of anaesthesia), drugs
(penicillamine, steroids) and drug abuse, malignant disease
(epithelial (lung, breast), adenocarcinoma (renal), melanoma,
lymphoreticular, multiple myeloma), circulatory disease (myocardial
infarction, cardiac failure, peripheral vascular disease,
hypertension, coronary heart disease, non-atherosclerotic
cardiovascular disease, atherosclerotic cardiovascular disease),
skin disease (psoriasis, systemic sclerosis), respiratory disease
(COPD, obstructive sleep apnoea, hypoia at high altitude) and
endocrine disease (acromegaly, diabetes mellitus, and diabetes
insipidus).
[0082] Kidney disease may result from bacterial infection,
allergies, congenital defects, stones, antibiotics,
immunosuppressives, antineoplastics, nonsteroidal antiinflammatory
drugs, analgesics, heavy metals, tumors, chemicals.
[0083] In one aspect of the invention, there is provided a method
for determining a propensity for or early diagnosis of renal
disease and/or renal complications of a disease. The method
includes determining a change in the albumin content in a urine
sample. The disease may be a kidney disease, although not
necessarily limited to a kidney disease.
[0084] In the method of the invention, albumin is used herein only
as an example of a protein to be detected in urine. When the
albumin in a patient is analyzed by conventional RIA, it is
expected that a normoalbuminuric patient or normal individual would
have albumin in the urine in the range of 3-10 .mu.g/min in young
people and greater in older people. However, normoalbuminuric
patients also show levels of albumin in the urine if measured by
HPLC. Applicant has found that these levels may be in the order of
5 .mu.g/min. As kidney disease progresses, the level of
intact/modified albumin will increase to microalbuminuria levels in
the order of 20 to 200 .mu.g/min as determined by RIA. This will be
much higher when determined by HPLC or a method that determines the
sum of intact albumin and intact modified albumin. By monitoring
the increase in intact/modified albumin, early signs of kidney
disease may be detected. However, these levels are not detectable
by the methods currently available such as radioimmunoassay using
antibodies currently commercially in use, possibly for the reason
that antibodies detect certain epitopes. If the albumin is modified
in any way as described above, the epitope may be destroyed thereby
leaving the modified albumin undetectable,
[0085] A patient suspected of having diabetic kidney disease will
not show signs of kidney degeneration until well after 10 to 15
years when albumin is detected by currently available methods such
as RIA methods. Urinary excretion rates of at least 20 .mu.g/min
may be detected by RIA when an individual enters a microalbuminuric
state. Again, by observing the excretion of modified albumin, a
change in the kidney and possibly onset of a kidney disease may be
detected.
[0086] A normoalbuminuric subject, or normoalbuminuric diabetic
patient may continue to have a low albumin excretion rate of less
than 20 .mu.g/min as determined by RIA, for many years. The
presence of albumin in the urine is a sign that functions of the
kidney may be impaired. Once this level begins to change, treatment
may be initiated.
[0087] In a normal individual a small amount of albumin is
detectable in the urine. Total filtered albumin appears mainly as
fragmented albumin in urine. Some albumin may be detected in
normoalbuminuric individuals. However, the excretion rate of
albumin in urine in a normoalbuminuric individual may be as low as
5 .mu.g/min. This level is generally detectable by RIA.
[0088] The modified protein of the invention can be detected by a
variety of methods that are well-known in the art, including, but
not limited to chromatography, electrophoresis and sedimentation,
or a combination of these, which are described in Karger B L,
Hancock W S (eds.) High Resolution Separation and Analysis of
biological Macromolecules. Part A Fundamentals in Methods in
Enzymology, Vol. 270, 1996, Academic Press, San Diego, Calif., USA;
Karger B L, Hancock W S (eds.) High Resolution Separation and
Analysis of biological Macromolecules. Part B Applications in
Methods in Enzymology, Vol. 271, 1996, Acadermic Press, San Diego,
Calif., USA; or Harding S E, Rowe, A J, Horton J C (eds.)
Analytical Ultracentrifugation in Biochemistry and Polymer Science.
1992, Royal Soc. Chemistry, Cambridge, UK, which references are
incorporated herein by reference in their entirety.
[0089] The electrophoresis method includes, but is not limited to,
moving-boundary electrophoresis, zone electrophoresis, and
isoelectric focusing.
[0090] The chromatography method includes, but is not limited to,
partition chromatography, adsorption chromatography, paper
chromatography, thin-layer chromatography, gas-liquid
chromatography, gel chromatography, ion-exchange chromatography,
affinity chromatography, and hydrophobic interaction
chromatography. Preferably, the method is a sizing gel
chromatography and hydrophobic interaction chromatography. More
preferably, the method is hydrophobic interaction chromatography
using a HPLC column.
[0091] The modified protein can also be detected by the use of
specific albumin dyes. Such methods are described by Pegoraro et
al., American Journal of Kidney Diseases 35(4): 739-744 (April
2000), the entire disclosure of which is hereby incorporated by
reference. The modified albumin, as well as the whole albumin, is
detectable by this dye method to provide the sum of modified
albumin and whole or intact albumin. This detection method may be
used with or without an initial separation of the albumin
components from urine. Such dyes normally do not detect fragments
<10,000 in molecular weight, but will detect the modified
albumin.
[0092] In this dye method of detection, a dye such as Albumin Blue
580 is used. Such dyes are naturally non-fluorescent, but fluoresce
on binding to intact albumin as well as the modified albumin, but
do not bind to globulins. Therefore, globulins do not interfere
with the assay so that measurements can be made in unfractionated
urine.
[0093] Applicant has found that among diabetics, a normoalbuminuric
diabetic patient has almost undetectable levels of modified or
fragments of albumin when analyzed by conventional RIA. They appear
to be normal. However, when the urine is tested by HPLC, the levels
of modified albumin are much greater than found in a normal
individual. This difference in albumin may be attributed to the
inability of conventional RIA's to adequately detect all albumin
(total albumin) in intact or modified forms. Thus, HPLC is
preferred for generating a fragmentation profile. A fragmentation
profile on HPLC is characterized by a series of peaks representing
a number of species of albumin as fragments or in intact or
modified forms.
[0094] In a preferred aspect of the present invention, the method
of determining a propensity for or early diagnosis of a kidney
disease in a subject is determined before the subject becomes
microalbuminuric.
[0095] Measuring albumin content in a sample by an HPLC method of
the present invention may provide different results from its
measurement by conventional RIA. In the HPLC technique, a low level
of albumin is observed in normal individuals. When the level of
modified albumin begins to be detected and its level increases, and
progresses toward microalbuminuria then a patient can be determined
to have a propensity for kidney disease.
[0096] In a normal individual, the HPLC generated fragmentation
profile is characterized by the absence of a peak in a region where
full-length native albumin elutes. Instead, multiple fragmented
albumin is detectable. A pure protein product (unmodified) produces
essentially a single peak. For example, using a hydrophobicity
HPLC, albumin was observed to elute in the range of 39-44 minutes
(FIG. 5). Thus, a normal individual would provide a distinct
fragmentation profile indicative of an absence of kidney disease or
no propensity for a kidney disease. However, as kidney disease
progresses, an increasing amount of modified albumin first, and
then native form later are detectable. The fragmentation profile
begins to change and more products in the region of full-length
albumin manifests as additional spikes or an enlarged peak
indicative of more intact/modified albumin in the urine.
[0097] In a HPLC generated fragmentation profile of a urine sample,
the modified albumin may appear in a region where native albumin
elutes but may be manifest as multiple peaks indicating the
presence of multiple forms of modified albumin.
[0098] In a further preferred embodiment, the propensity for kidney
disease may be measured by determining the presence of or
identifying at least one species of modified albumin. This may be
determined or identified by the presence of a specific peak on a
HPLC profile, preferably the peak is within the range of position
that corresponds to the elution position of the native albumin.
[0099] A HPLC column for detecting modified albumin or unmodified
albumin may be a hydrophobicity column, such as Zorbax 300 SB-CB
(4.6 mm.times.150 mm). A 50 .mu.l sample loop may be used. Elution
solvents suitable for HPLC in detecting albumin and its breakdown
products may include standard elution solvents such as acetonitrile
solvents. Preferably a buffer of water/1% trifluoro acetic acid
(TFA) followed by a buffer of 60% acetonitrile/0.09% TFA may be
used. A gradient of 0 to 100% of a 60% acetonitrile/0.09% TFA has
been found to be suitable.
[0100] Suitable HPLC conditions for a hydrophobicity column may be
as follows:
[0101] Solvent A H.sub.2O, 1% trifluoro acetic acid
[0102] Solvent B 60% acetonitrile, 0.09% TFA
[0103] Solvent A2 99.96>00.00:49.58 min Pressure 9.014Mpascalls
(.about.1100 psi)
[0104] Solvent B2 0.04>100.0:49.58 min Pressure
7.154Mpascalls
[0105] The wavelength used in HPLC may be approximately 214 nm.
[0106] Modified albumin may elute between 39-44 minutes (FIG. 5).
Albumin fragments may elute much earlier, mainly at less than 20
minutes.
[0107] The method for determining the propensity for kidney disease
is applicable to any individual. Kidney disease may be caused by a
number of factors including bacterial infection, allergic,
congenital defects, stones, tumors, chemicals or from diabetes.
Preferably, the method is applicable for determining a propensity
for kidney disease in diabetic patients that may progress to a
kidney disease. Preferably, the individual is a normoalbuminuric
diabetic. However, normal individuals may be monitored for
propensity for the disease by determining increased levels of
intact or modified albumin in the urine.
[0108] The method of the invention can be carried out using
non-antibody separation procedures as described above. However,
antibody specific for modified protein may also be used to detect
the presence of the modified protein.
[0109] The antibody to the modified protein may be obtained using
the following method. The procedure is described specifically for
albumin by way of example only, and can be readily applied to
antibody production against any other protein in the urine. The
method seeks to determine which modified albumin molecule is the
most sensitive marker to identify diabetic patients, for example,
who will progress to kidney complications.
[0110] The modified albumin is characterized by carrying out a
quantitative separation of the modified albumin molecules, such as
by preparative HPLC. The modified proteins are analyzed for ligand
binding, such as glycation. Subsequently, amino acid sequence of
the individual modified protein is determined, preferably by mass
spectrometry using methods described in Karger B L, Hancock W S
(eds.) High Resolution Separation and Analysis of biological
Macromolecules. Part A Fundamentals in Methods in Enzymology, Vol.
270, 1996, Academic Press, San Diego, Calif., USA; or Karger B L,
Hancock W S (eds.) High Resolution Separation and Analysis of
biological Macromolecules. Part B Applications in Methods in
Enzymology, Vol. 271, 1996, Academic Press, San Diego, Calif., USA,
for example, which references are incorporated herein by reference
in their entirety. In a preferred embodiment, there may be about 3
to 4 modified albumin species.
[0111] The method of generating antibody against the modified
albumin seeks to develop a diagnostic immunoassay for the modified
albumin that predicts those diabetic patients, for example, that
progress to kidney complications. To accomplish this, sufficient
quantities of modified albumin is prepared by HPLC. Antibodies are
made by sequential injection of the modified albumin in an animal
such as a rabbit, to generate good titer, and the antibodies are
isolated using conventional techniques using methods described in
Goding J W, Monoclonal Antibodies: Principles and Practice.
Production and Application of Monoclonal Antibodies in Cell
Biology, Biochemistry and Immunology, 2nd Edition 1986, Academic
Press, London, UK; or Johnstone A, Thorpe R, Immunochemistry in
Practice, 3rd edition 1996, Blackwell Science Ltd, Oxford, UK, for
example, which references are incorporated herein by reference in
their entirety. The obtained antibodies may be polyclonal
antibodies or monoclonal antibodies.
[0112] Preferably, at least one species of a modified albumin is
isolated and identified for use in determining a propensity for
kidney disease. The isolated species may be used to generate
antibodies for use in immunoassays. The antibodies may be tagged
with an enzymatic, radioactive, fluorescent or chemiluminescent
label. The detection method may include, but is not limited to
radioimmunoassay, immunoradiometric assay, fluorescent immunoassay,
enzyme linked immunoassay, and protein A immunoassay. The assays
may be carried out in the manner described in Goding J W,
Monoclonal Antibodies: Principles and Practice. Production and
Application of Monoclonal Antibodies in Cell Biology, Biochemistry
and Immunology. 2nd Edition 1986, Academic Press, London, UK;
Johnstone A, Thorpe R, Immunochemistry in Practice, 3rd edition
1996, Blackwell Science Ltd, Oxford, UK; or Price C P, Newman D J
(eds.) Principles and Practice of Immunoassay, 2nd Edition, 1997
Stockton Press, New York, N.Y., USA, for example, which references
are incorporated herein by reference in their entirety.
[0113] It is an object of this invention to provide an article of
matter or a kit for rapidly and accurately determining the presence
or absence of modified protein such as modified albumin, in a
sample quantitatively or non-quantitatively as desired. Each
component of the kit(s).may be individually packaged in its own
suitable container. The individual container may also be labeled in
a manner, which identifies the contents. Moreover, the individually
packaged components may be placed in a larger container capable of
holding all desired components. Associated with the kit may be
instructions, which explain how to use the kit. These instructions
may be written on or attached to the kit.
[0114] The invention is also directed to a method of determining a
treatment agent for renal disease and/or renal complications of a
disease, comprising:
[0115] (a) administering to a person an agent that is suspected of
being able to treat the disease;
[0116] (b) obtaining a urine sample from the person; and
[0117] (c) assaying for the modified form of the protein in the
sample, wherein either the presence of or lack of presence of a
modified form of the protein in the urine or decreasing amount of
the modified form of the protein over time indicates that the agent
is a treatment agent for the disease. The treatment agent may be a
lysosome activating agent that may act directly or indirectly to
activate lysosome, and thereby cause the lysosome to digest
post-glomerular filtered proteins, which is a sign of a healthy
kidney.
[0118] The process of trafficking of proteins to the lysosomes
plays a role in the mechanism of albuminuria in diabetes. An
intracellular molecule that is involved in trafficking is protein
kinase C (PKC). It is contemplated that a drug or agent can be
formulated that will activate lysosomal trafficking or inhibit
PKC.
[0119] Accordingly, in one aspect of the present invention, there
is provided a lysosome-activating compound for use in reactivating
lysosomes or processes that direct substrates to the lysosome or
products away from the lysosome.
[0120] In another aspect of the present invention, there is
provided a composition comprising a lysosome-activating compound
and a carrier.
[0121] In yet another aspect of the invention there is provided a
method of preventing or treating kidney disease, said method
including administering an effective amount of a
lysosome-activating compound to a subject.
[0122] In yet another aspect of the present invention, there is
provided a method of screening a multiplicity of compounds to
identify a compound capable of activating lysosomes or processes
that direct substrates to the lysosome or products away from the
lysosome, said method including the steps of:
[0123] (a) exposing said compound to a lysosome and assaying said
compound for the ability to activate a lysosome wherein said
lysosome when activated has a changed activity;
[0124] (b) assaying for the ability to restore a cellular process
to substantially normal levels in kidney tissue, wherein said
kidney tissue has a low lysosome activity; and/or
[0125] (c) assaying for the ability to restore tissue turnover to
substantially normal levels in kidney tissue, wherein said kidney
tissue has low lysosome activity.
[0126] Lysosomes may be associated with the breakdown of proteins,
particularly albumin, in the kidney. In cases of microalbuminuria,
substantial amounts of albumin escape lysosomal breakdown possibly
due to a deactivated lysosome. Restoration of lysosomal breakdown
may restore the balance in the kidney of cellular processes and
tissue turnover.
[0127] A lysosome-activating compound may be a compound that acts
directly or indirectly on the lysosome. By acting indirectly, the
compound may act on a component, which influences the activity of
the lysosome. Nevertheless, the outcome results in an activation of
the lysosome, thereby providing enhanced protein breakdown.
[0128] In another aspect of the present invention, there is
provided a composition comprising a lysosome-activating compound
and a carrier.
[0129] The composition may be a physiologically acceptable or
pharmaceutically acceptable composition. However, it will be a
composition which allows for stable storage of the lysosome
activating compound. Where the composition is a pharmaceutically
acceptable composition, it may be suitable for use in a method of
preventing or treating kidney disease.
[0130] In yet another aspect of the invention there is provided a
method of preventing or treating kidney disease, said method
including administering an effective amount of a
lysosome-activating compound to a subject.
[0131] As described above, the lysosome-activating compound may act
by reactivating the lysosome so that cellular processes and tissue
turnover are restored fully or in part, thereby resulting in the
kidney being restored partially or fully. In any case,
administering a lysosome activating compound to an animal having
kidney disease may restore lysosome activity fully or in part.
[0132] Methods of administering may be oral or parenteral. Oral may
include administering with tablets, capsules, powders, syrups, etc.
Parenteral administration may include intravenous, intramuscular,
subcutaneous or intraperitoneal routes.
[0133] The changed activity of the lysosome is preferably a change
which enhances the activity of the lysosome so that albumin
breakdown is improved. The ability to not only activate lysosome
but also improve cellular processes and/or tissue turnover is a
characteristic of the most desirable lysosome activating compound.
Preferably, it is desired to use the lysosome activating compound
to restore kidney function.
[0134] In another aspect of the present invention there is provided
a method for preventing kidney disease in a subject, said method
including:
[0135] (a) measuring the amount of intact and modified intact
albumin content in a urine sample;
[0136] (b) determining a change in the amount of intact albumin in
the urine that has been modified so as to be not detectable by
conventional RIA methods wherein the change is indicative of a
propensity for kidney disease; and
[0137] (c) treating the animal for a kidney disease when a change
is determined.
[0138] The following examples are offered by way of illustration of
the present invention, and not by way of limitation.
EXAMPLES
Example 1
Size Exclusion Chromatography of Human Serum Albumin (HSA)
[0139] Normal, healthy volunteers were used to provide urine for
analyzing the distribution of albumin in their urine.
[0140] .sup.3H[HSA] (Human Serum Albumin) was injected into healthy
volunteers and urine and plasma were collected and analyzed by size
exclusion chromatography using a G-100 column. The column was
eluted with PBS (pH=7.4) at 20 ml/hr at 4.degree. C. The void
volume (V.sub.0) of the column was determined with blue dextran
T2000 and the total volume with tritiated water.
[0141] Tritium radioactivity was determined in 1 ml aqueous samples
with 3 ml scintillant and measured on a Wallac 1410 liquid
scintillation counter (Wallac Turku, Finland).
[0142] FIG. 2 illustrates the distribution of albumin in urine and
in plasma.
Example 2
Albumin Excretion in a Normal, Healthy Volunteer and Diabetic
Patient
[0143] .sup.3H[HSA] as used in Example 1 was injected into a
normal, healthy volunteer and a diabetic patient. Samples of urine
were collected and .sup.3H[HSA] was determined as in Example 1.
[0144] The normal, healthy volunteer (FIG. 3) shows the excretion
of fragments of albumin on a size exclusion chromatography as
performed in Example 1.
[0145] The diabetic patient (FIG. 4) shows the presence of
substantially full-length and fragmented albumin on size exclusion
chromatography. However, excretion rates of albumin detectable by
these methods were in the order of 5 .mu.g/min (control) and 1457
.mu.g/min (diabetic).
Example 3
Determination of Intact Albumin, and Intact/Modified Albumin on
HPLC.
[0146] Urine samples were collected from normal, healthy volunteer,
normoalbuminuric diabetic patients and from macroalbuminuric
patients. Urine was collected midstream in 50 ml urine specimen
containers. The urine was frozen until further use. Prior to HPLC
analysis the urine was centrifuged at 5000 g.
[0147] Samples were analyzed on HPLC using a hydrophobicity column
Zorbax 300 SB-CB (4.6 mm.times.150 mm). A 50 .mu.l sample loop was
used.
[0148] Samples were eluted from the columns using the following
conditions.
[0149] Solvent A H.sub.2O, 1% trifluoro acetic acid
[0150] Solvent B 60% acetonitrile, 0.09% TFA
[0151] Solvent A2 99.96>00.00:49.58 min Pressure 9.014Mpascalls
(.about.1100 psi)
[0152] Solvent B2 0.04>100.0:49.58 min Pressure
7.154Mpascalls
[0153] A wavelength of 214 nm was used.
Example 4
Purification of Modified Albumin for Antibody Production by
Standard Techniques
[0154] Urine from microalbuminuric patient which had an intact
albumin concentration of 43.5 mg/L as determined by turbitimer
(involving conventional immunochemical assay) was initially
filtered through a 30 kDa membrane to separate the modified albumin
from low molecular weight (<30,000) protein fragments in urine.
The material that was retained by the filter gave a yield of intact
albumin of 27.4 mg/L as determined by turbitimer assay. This
retained material was then subjected to size exclusion
chromotography on Sephadex G100. The material collected was the
peak fraction that coelutes with intact albumin. This material gave
a yield of 15.2 ml/L of albumin as determined by the turbitimer
method. This material was then subjected to affinity chromatography
on an intact,albumin antibody column. This column will only bind
albumin that has conventional epitopes. The yield of material that
eluted from the column was <6 mg/L (lowest sensitivity of the
turbitimer). This is expected as the immunoreactive albumin would
have bound to the affinity column. The eluate was then subject to
reverse phase HPLC chromatography(as described above) to determine
the amount of immuno-unreactive albumin in the sample. A 1452 unit
area corresponding to 30.91 mg/L of purified modified albumin was
noted as shown in FIG. 15. This purified modified albumin can then
be used for antibody production by standard means.
[0155] Results
[0156] FIG. 5 illustrates a HPLC profile of albumin alone.
Essentially a single peak which elutes at approximately 39-44
minutes retention time was obtained.
[0157] FIG. 6 illustrates a HPLC profile of plasma showing a
distinct albumin peak at approximately 39-44 minutes as well as
other peaks corresponding to other plasma proteins.
[0158] FIG. 7 illustrates a HPLC profile of a normal, healthy
volunteer showing no albumin peak in the urine sample. This
individual breaks down the albumin excreted into the urine possibly
via an active lysosome. Substantial fragmented products were
evident showing prominence of some species, particularly of a
species at approximately less than 14.5 minutes retention time.
[0159] When urine from a normoalbuminuric diabetic patient (with an
albumin excretion rate of 8.07 .mu.g/min, as measured by RIA) is
analyzed (FIG. 8), small amounts of modified albumin eluting at
approximately 39-44 minutes retention time is evident. Whereas
conventional test indicates the presence of <6 mg/l of albumin
in the urine sample, the method of the invention showed that the
true albumin content in the urine sample was 26.7 mg/l. Treatment
for the disease should have begun on this individual. Albumin
by-products or fragmented albumin is present as in the normal,
healthy volunteer.
[0160] Another urine sample from normoalbuminuric diabetic patient
(with albumin excretion rate of 17.04 .mu.g/min) was analyzed (FIG.
9). RIA tests show albumin excreted in the urine for this patient.
However, on HPLC (FIG. 9) an albumin or modified albumin peak is
evident at approximately 39-44 minutes retention time. Whereas
conventional test indicates the presence of <6 mg/l of albumin
in the urine sample, the method of the invention showed that the
true albumin content in the urine sample was 81.3 mg/l. Treatment
for the disease should have begun on this individual. This peak
begins to show a multiple peaked appearance. A smaller peak
corresponding to intact albumin shows that modified albumin may
represent the peak at 39-44 minutes. The presence of this albumin
peak compared with the profile of a normal, healthy volunteer
having no albumin peak shows a change in the detectable levels of
the amount of intact/modified albumin. This may signal a propensity
for a kidney disease.
[0161] A further urine sample from a normoalbuminuric diabetic
patient (with an albumin excretion rate of 4.37 .mu.g/min) was
analyzed, and the HPLC profile is illustrated in FIG. 10. Again,
modified albumin was detected at approximately 39-44 minutes
retention time showing multiple peaks. This patient again did
register normal albumin by RIA. Whereas conventional test indicates
the presence of <6 mg/l of albumin in the urine sample, the
method of the invention showed that the true albumin content in the
urine sample was 491 mg/l. Treatment for the disease should have
begun on this individual. It is clear that modified albumin
assessment is necessary to identify these changes. This patient
would be determined to have a propensity for kidney disease. As
kidney disease progresses, the modified albumin peak will continue
to increase.
[0162] This is shown in FIG. 11 where a urine sample of a
macroalbuminuric patient was analyzed. A quite significant albumin
peak at approximately 39-44 minutes retention time showing multiple
peaks was evident. The patient's albumin content was 1796 mg/l.
Treatment for this individual is in progress.
[0163] The method of the invention results in early detection of a
propensity for a renal disease as illustrated by the longitudinal
studies in FIGS. 12-14. FIGS. 12-14 show situations in which the
ACE inhibitor treatment for diabetes was begun later than it should
have had the modified albumin detection method of the invention
been used. Detecting modified protein using the method according to
the invention is a more effective method for predicting the onset
of a renal disease than using conventional RIA.
Example 5
(.sup.3H)HSA in Urine Collected from a Microalbuminuric Type I
Diabetic
[0164] FIG. 16 illustrates (.sup.3H)HSA in urine collected from a
microalbuminuric type I diabetic patient by size exclusion
chromatography. Urine contains mostly fragmented albumin. Plasma
contains only intact albumin. Conventional RIA (human albumin
ordinate) measures only intact albumin and not fragments.
Example 4
Size Exclusion Profiles of (.sup.3H)HSA in Patient Urine
[0165] When lysosome activity or intracellular processes directing
substrates to lysosomes is reduced as in kidney disease, more of
the high molecular weight, and substantially full length filtered
protein including albumin appears in the urine. FIG. 17 shows that
in renal disease in type I diabetic patients the severity of the
disease as measured by albuminuria is associated with the increased
presence of intact albumin in urine.
[0166] FIG. 17 illustrates the size exclusion profiles
of(.sup.3H)HSA in urine from four different patients with varying
albumin excretion rate (AER) as measured by conventional RIA. As
intact albuminuria increases the amount of intact albumin in the
urine increases and the relative amount of fragments decreases. The
total excretion represents the excretion of intact albumin plus
albumin derived fragments.
[0167] All of the references cited herein are incorporated by
reference in their entirety.
[0168] Finally, it is to be understood that various other
modifications and/or alterations may be made without departing from
the spirit of the present invention as outlined herein.
* * * * *