U.S. patent application number 11/834314 was filed with the patent office on 2007-11-22 for methods for alleviating deleterious effects of 3-deoxyglucosone.
Invention is credited to Truman R. Brown, Francis Kappler.
Application Number | 20070270335 11/834314 |
Document ID | / |
Family ID | 37187699 |
Filed Date | 2007-11-22 |
United States Patent
Application |
20070270335 |
Kind Code |
A1 |
Brown; Truman R. ; et
al. |
November 22, 2007 |
METHODS FOR ALLEVIATING DELETERIOUS EFFECTS OF 3-DEOXYGLUCOSONE
Abstract
Disclosed is a class of compounds which inhibit the enzymatic
conversion of fructose-lysine into fructose-lysine-3-phosphate in
an ATP dependent reaction in a newly discovered metabolic pathway.
According to the normal functioning on this pathway,
fructose-lysine-3-phosphate (FL3P) is broken down to form free
lysine, inorganic phosphate and 3-deoxyglucosone (3DG), the latter
being a reactive protein modifying agent. 3DG can be detoxified by
reduction to 3-deoxyfructose (3DF), or it can react with endogenous
proteins to form advanced glycation end-product modified proteins
(AGE-proteins) Also disclosed are therapeutic methods of using such
inhibitors to alleviate deleterious effects of 3DG.
Inventors: |
Brown; Truman R.; (Cornwall
on Hudson, NY) ; Kappler; Francis; (Philadelphia,
PA) |
Correspondence
Address: |
DANN, DORFMAN, HERRELL & SKILLMAN
1601 MARKET STREET
SUITE 2400
PHILADELPHIA
PA
19103-2307
US
|
Family ID: |
37187699 |
Appl. No.: |
11/834314 |
Filed: |
August 6, 2007 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11478497 |
Jun 29, 2006 |
|
|
|
11834314 |
Aug 6, 2007 |
|
|
|
09974323 |
Oct 10, 2001 |
7071298 |
|
|
11478497 |
Jun 29, 2006 |
|
|
|
09182114 |
Oct 28, 1998 |
|
|
|
09974323 |
Oct 10, 2001 |
|
|
|
09095953 |
Jun 11, 1998 |
|
|
|
09182114 |
Oct 28, 1998 |
|
|
|
PCT/US98/02192 |
Feb 5, 1998 |
|
|
|
09095953 |
Jun 11, 1998 |
|
|
|
08794433 |
Feb 5, 1997 |
6004958 |
|
|
PCT/US98/02192 |
Feb 5, 1998 |
|
|
|
Current U.S.
Class: |
530/322 ;
514/6.8; 530/300; 530/350 |
Current CPC
Class: |
A61K 31/197 20130101;
A61P 3/10 20180101; A61K 31/7008 20130101; G01N 33/66 20130101;
A61K 31/5375 20130101; A61K 31/00 20130101; A61K 31/198 20130101;
A61K 31/13 20130101; C07H 19/20 20130101; C07H 1/00 20130101; A61K
31/275 20130101 |
Class at
Publication: |
514/008 ;
514/002; 530/300; 530/322; 530/350 |
International
Class: |
A61K 38/16 20060101
A61K038/16; A61K 38/00 20060101 A61K038/00; A61P 3/10 20060101
A61P003/10; C07K 14/00 20060101 C07K014/00; C07K 9/00 20060101
C07K009/00 |
Goverment Interests
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY-SPONSORED
RESEARCH AND DEVELOPMENT
[0002] Pursuant to 35 U.S.C. .sctn.202(c), it is hereby
acknowledged that the U.S. Government has certain rights in the
invention described herein, which was made in part with funds from
the National Institutes of Health (Grant Nos. DK44050, DK50317,
DK50364, and DK55079).
Claims
1. A compound having the structural formula: ##STR11## wherein X is
--NR'--, --S(O)--, --S(O).sub.2--, or --O--, R' being selected from
the group consisting of H, and linear or branched chain alkyl group
(C.sub.1-C.sub.4) and an unsubstituted or substituted aryl group
(C.sub.6-C.sub.10) or aralkyl group (C.sub.7-C.sub.10); R is a
substituent selected from the group consisting of H, an amino acid
residue, a polyaminoacid residue, a peptide chain, a linear or
branched chain aliphatic group (C.sub.3-C.sub.8), which is
unsubstituted or substituted with at least one nitrogen or
oxygen-containing substituent, a linear or branched chain aliphatic
group (C.sub.1-C.sub.8), which is unsubstituted or substituted with
at least one nitrogen or oxygen-containing substituent and
interrupted by at least one --O--, --NH--, or --NR''-- moiety, R''
being linear or branched chain alkyl (C.sub.1-C.sub.6) and an
unsubstituted or substituted aryl group (C.sub.6-C.sub.10) or
aralkyl group (C.sub.7-C.sub.10), with the proviso that when X
represents --NR'--, R and R', together with the nitrogen atom to
which they are attached, may also represent a substituted or
unsubstituted heterocyclic ring having from 5 to 7 ring atoms, with
at least one of nitrogen and oxygen being the only heteroatoms in
said ring, said aryl group (C.sub.6-C.sub.10) or aralkyl group
(C.sub.7-C.sub.10), and said heterocyclic ring substituents being
selected from the group consisting of H, alkyl (C.sub.1-C.sub.6),
halogen, CF.sub.3, CN and --O-alkyl (C.sub.1-C.sub.6); R.sub.1 is a
polyol moiety having 1 to 4 linear carbon atoms, Y is a
hydroxymethylene moiety --CHOH--; Z is selected from the group
consisting of --H, --O-alkyl (C.sub.1-C.sub.6), -halogen,
--CF.sub.3, --CN, --COOH and --SO.sub.3H.sub.2, and optionally
--OH; or its pharmaceutically acceptable salt or its stereoisomer,
except that X--R in the above formula does not represent hydroxyl
or thiol.
2. A compound according to claim 1, selected from the group
consisting of sorbitol-lysine, mannitol-lysine, and
galactitol-lysine.
3. The compound according to claim 1,
3-O-methyl-sorbitol-lysine.
4. A method of treating glycogen storage diseases, including
Fanconi's syndrome, in a patient in need thereof by administering a
therapeutically effective amount of a compound of the formula
##STR12## wherein X is --NR'--, --S(O)--, --S(O).sub.2--, or --O--,
R' being selected from the group consisting of H, and linear or
branched chain alkyl group (C.sub.1-C.sub.4) and an unsubstituted
or substituted aryl group (C.sub.6-C.sub.10) or aralkyl group
(C.sub.7-C.sub.10); R is a substituent selected from the group
consisting of H, an amino acid residue, a polyaminoacid residue, a
peptide chain, a linear or branched chain aliphatic group
(C.sub.3-C.sub.8), which is unsubstituted or substituted with at
least one nitrogen or oxygen-containing substituent, a linear or
branched chain aliphatic group (C.sub.1-C.sub.8), which is
unsubstituted or substituted with at least one nitrogen- or
oxygen-containing substituent and interrupted by at least one
--O--, --NH--, or --NR''-- moiety, R'' being linear or branched
chain alkyl (C.sub.1-C.sub.6) and an unsubstituted or substituted
aryl group (C.sub.6-C.sub.10) or aralkyl group (C.sub.7-C.sub.10),
with the proviso that when X represents --NR'--, R and R', together
with the nitrogen atom to which they are attached, may also
represent a substituted or unsubstituted heterocyclic ring having
from 5 to 7 ring atoms, with at least one of nitrogen and oxygen
being the only heteroatoms in said ring, said aryl group
(C.sub.6-C.sub.10) or aralkyl group (C.sub.7-C.sub.10), and said
heterocyclic ring substituents being selected from the group
consisting of H, alkyl (C.sub.1-C.sub.6), halogen, CF.sub.3, CN and
--O-alkyl (C.sub.1-C.sub.6); R.sub.1 is a polyol moiety having 1 to
4 linear carbon atoms, Y is a hydroxymethylene moiety --CHOH--; Z
is selected from the group consisting of --H, --O-alkyl
(C.sub.1-C.sub.6), -halogen, --CF.sub.3, --CN, --COOH and
--SO.sub.3H.sub.2, and optionally --OH; or its pharmaceutically
acceptable salt or its stereoisomer, except that X--R in the above
formula does not represent hydroxyl or thiol.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation-in-part of U.S.
patent application Ser. No. 09/182,114, filed Oct. 28, 1998, which
is a continuation-in-part of U.S. application Ser. No. 09/095,953,
filed Jun. 11, 1998, now abandoned, which is a continuation-in-part
of International Application No. PCT/US98/02192, filed Feb. 5,
1998, which is a continuation-in-part of U.S. application Ser. No.
08/794,433, filed Feb. 5, 1997, now U.S. Pat. No. 6,004,958. The
entire disclosure of each of the aforesaid is incorporated by
reference herein.
BACKGROUND OF THE INVENTION
[0003] The present invention relates to therapeutic agents and
their use for the treatment of diabetes, and in particular for
preventing, reducing or delaying the onset of diabetic
complications and other disorders of related etiology, such as
glycogen storage diseases, including Fanconi's syndrome. More
particularly, the present invention relates to a class of enzyme
inhibitors which inhibit the enzymatic conversion of fructose
lysine (FL) to fructose-lysine-3-phosphate (FL3P), which is
believed to be an important step in the biochemical mechanism
leading to diabetic complications. This invention also relates to a
method of assessing a diabetic patient's risk of experiencing
diabetic complications, as well as a method of determining the
efficacy of therapeutic intervention in preventing, reducing or
delaying the onset of diabetic complications.
[0004] There are four particularly serious complications of
diabetes, namely, diabetic nephropathy or kidney disease; diabetic
retinopathy which causes blindness due to destruction of the
retina; diabetic neuropathy involving the loss of peripheral nerve
function; and circulatory problems due to capillary damage. Both
retinopathy and nephropathy are thought to be subsets of the
general circulatory problems associated with this disease state.
The role of microvascular dysfunction in late stage diabetes has
been recently summarized (Tooke, Diabetes, 44: 721 (1995)).
Throughout this disclosure, the terms "diabetes-associated
pathologic conditions" and synonymous terms are meant to include
the various well-known retinopathic, neuropathic, nephropathic,
macroangiopathic, as well as other complications of diabetes and
diseases of related etiology, including glycogen storage
diseases.
[0005] The similarities between the pathologies arising from
diabetes and those resulting from aging have been extensively
reported. Studies have shown that many diabetes-associated
pathologic conditions are clinically very similar to the
pathologies normally associated with aging. It has been shown, for
example, that in diabetes arteries and joints prematurely stiffen,
lung elasticity and vital capacity prematurely decrease. Moreover,
atherosclerosis, myocardial infarction and strokes occur more
frequently in diabetics than in age-matched non-diabetic
individuals. Diabetics are also more susceptible to infections, and
are more likely to have hypertension, accelerated bone loss,
osteoarthritis ard impaired T-cell function at a younger age than
non-diabetics.
[0006] The similarities between diabetes-associated pathologic
conditions and aging would appear to suggest a common mechanistic
rationale. A variety of mechanisms have been proposed as a common
biochemical basis for both diabetes-associated pathologic
conditions and aging. The hypothesis most strongly supported by
data from human subjects is premised on a non-enzymatic
glycosylation mechanism. This hypothesis states that the aging
process and diabetes-associated pathologic conditions, such as
those described above, are caused, at least in part, by protein
modification and cross-linking by glucose and glucose-derived
metabolites via the Maillard reaction (Monnier et al., Proc. Natl.
Acad. Sci. USA, 81: 583 (1984) and Lee et al., Biochem. Biophys.
Res. Comm., 123: 888 (1984)). The modified proteins resulting from
such glycosylation reactions are referred to herein as advanced
glycation end product-modified proteins (AGE-proteins). It is
widely accepted that 3-deoxyglucosone (3DG) is a key intermediate
in the multi-step reaction sequence leading to AGE-protein
formation. 3DG is a glucose-derived metabolite that can react with
proteins leading to the cross-linking of both intracellular and
extracellular proteins, such as collagen and basement
membranes.
[0007] In the case of diabetic complications, the reactions that
lead to AGE-proteins are thought to be kinetically accelerated by
the chronic hyperglycemia associated with this disease. Evidence
supporting this mechanism includes data showing that long-lived
proteins such as collagen and lens crystallins from diabetic
subjects contain a significantly greater AGE-protein content than
do those from age-matched normal controls. Thus, the unusual
incidence of cataracts in diabetics at a relatively early age is
explainable by the increased rate of modification and cross-linking
of lens crystalline. Similarly, the early onset of joint and
arterial stiffening, as well as loss of lung capacity observed in
diabetics is explained by the increased rate of modification and
cross-linking of collagen, the key structural protein. Because
these proteins are long-lived, the consequences of modification
tend to be cumulative.
[0008] Another factor demonstrating cause and effect relationship
between diabetic complications and hyperglycemia is hyperglycemic
memory. One particularly striking example of this phenomenon is the
development of severe retinopathy in dogs that were initially
diabetic, then treated to restore normal blood glucose levels.
Although the dog eyes were histologically normal at the time of the
treatment, over time diabetic retinopathy developed in these
animals in spite of the normalized glucose concentrations (Engerman
et al., Diabetes, 36: 808 (1987)). Thus, the underlying damage to
the eyes irreversibly occurred during the period of early
hyperglycemia, before clinical symptoms were evident.
[0009] Diabetic humans and animals have been shown to have higher
than normal concentrations of early and late sugar modified
AGE-proteins. In fact, the increase in AGE-proteins is greater than
the increase in blood glucose levels. The concentration of
AGE-proteins can be estimated by fluorescence measurement, as some
percentage of sugar molecules rearrange to produce protein-bound
fluorescent molecules.
[0010] The pathogenic role of AGE-proteins is not limited to
diabetes. Protein glycation has been implicated in Alzheimer's
disease (Harrington et al., Nature, 370: 247 (1994)). Increased
protein fluorescence is also seen with aging. Indeed, some theories
trace the aging process to a combination of oxidative damage and
sugar-induced protein modification. Thus, a therapy that reduces
AGE-protein formation may also be useful in treating other
etiologically-similar human disease states, and perhaps slow the
aging process.
[0011] It has generally been assumed that the formation of
AGE-proteins begins with the reaction of a protein amino group and
a sugar, primarily glucose. One typical literature citation states
"The initial adduct formed by glycation of .epsilon.-amino groups
of lysine residues is the Amadori compound, fructoselysine.
Glycation is an initial step in a complex series of reactions,
known collectively as the Maillard or browning reaction, which
ultimately leads to the formation of crosslinked, precipitated,
oxidized, brown and fluorescent proteins". K. J. Knecht et al.,
Archives of Biochem. Biophys., 294: 130 (1992).
[0012] The formation of AGE-proteins from sugars is a multi-step
process, involving early, reversible reactions with sugars to
produce fructose-lysine containing proteins. These modified
proteins then continue to react to produce irreversibly modified
AGE-proteins. It is clear that AGE-proteins are not identical to
proteins containing glycated-lysine residues, as antibodies raised
against AGE-proteins do not react with fructose-lysine. It is also
clear that AGE-proteins exist as multiple chemical species; however
few have been identified. The chemical species
.epsilon.-Amino-(carboxymethyl)lysine has been identified as one
important final AGE-protein structure in recent studies (Reddy et
al., Biochem., 34: 10872 (1995) and Ikeda et al., Biochemistry, 35:
8075 (1996)). This study failed to chemically identify another
AGE-protein epitope that made up approximately 50% of the modified
sites. A method of studying the kinetics of AGE-protein formation
from ribose has recently been developed (Khalifah et al.,
Biochemistry, 35: 4645 (1996)). However, this study suggests that
ribose may play an important physiological role in AGE-protein
formation, supporting the relatively broad definitions of
glycated-lysines and fructose-lysine provided below.
[0013] Other references point out the distinction between proteins
containing glycated lysine residues and AGE proteins, "Equilibrium
levels of Schiff-base and Amadori products are reached in hours and
weeks, respectively. The reversible, equilibrium nature of early
glycosylation products is important, because it means that the
total amount of such products, even on very long-lived proteins,
reaches a steady-state plateau within a short period of time. Since
these early glycosylation products do not continue to accumulate on
collagen and other stable tissue proteins over years in chronic
diabetes, it is not surprising that their concentration does not
correlate with either the presence or the severity of diabetic
retinopathy . . . Some of the early glycosylation products on
collagen and other long-lived proteins of the vessel walls do not
dissociate, however. Instead, they undergo a slow, complex series
of chemical rearrangements to form irreversible advanced
glycosylation end products. M. Brownlee et al., New England Journal
of Medicine, 318: 1315 (1988). The only route for production of
these modified proteins which is described in the scientific
literature involves an initial reaction between proteins and sugar
molecules.
[0014] Numerous references point out that the formation of
AGE-proteins occurs through a multi-step pathway and that
3-deoxyglucosone (3-DG) is a key intermediate in this pathway. M.
Brownlee, Diabetes, 43: 836 (1994); M. Brownlee, Diabetes Care, 15:
1835 (1992); T. Niwa et al., Nephron, 69: 438 (1995); W. L. Dills,
Jr., Am. J. Clin. Nutr., 58: S779 (1993); H. Yamadat et al., J.
Biol. Chem., 269: 20275 (1994); N. Igaki et al., Clin. Chem., 36:
631 (1990). The generally accepted pathway for formation of 3DG
from the reaction of sugars and proteins is illustrated in FIG. 1.
As can be seen in FIG. 1, a sugar (glucose) molecule initially
forms a Schiff base with a protein-lysine amino group (I). The
resulting Schiff base then rearranges to produce fructose-lysine
modified proteins (II). The reactions leading up to (II) are freely
reversible. (II) can rearrange to produce 3DG and free protein
lysine. Subsequent reaction between 3DG and protein is the first
irreversible step in AGE-protein formation.
[0015] Insofar as is known, it has never been reported that 3DG can
be produced by alternative pathways, or indeed, that the major
source of 3-DG is from an enzyme catalyzed metabolic pathway,
rather than from the uncatalyzed reactions shown in FIG. 1.
[0016] Diabetic patients have significantly more 3DG in serum than
do non-diabetic patients (12.78.+-.2.49 .mu.M versus 1.94.+-.0.17
.mu.M). (Toshimitsu Niwa et al., Nephron, 69: 438 (1995)).
Nonetheless, this toxic compound is found in normal healthy
individuals. Thus, it is not surprising that the body has developed
a detoxification pathway for this molecule. One of these reactions
is catalyzed by aldehyde reductase which detoxifies 3DG by reducing
it to 3-deoxyfructose (3DF) which is efficiently excreted in urine
(Takahashi et al., Biochem, 34: 1433 (1995)). Another
detoxification reaction oxidizes 3DG to 3-deoxy-2-ketogluconic acid
(DGA) by oxoaldehyde dehydrogenase (Fujii et al., Biochem. Biophys.
Res. Comm., 210: 852 (1995)).
[0017] Results of studies to date show that the efficiency of at
least one of these enzymes, aldehyde reductase, is adversely
affected in diabetes. When isolated from normal rat liver, a
fraction of this enzyme is partially glycated on lysines 67, 84 and
140 and has a low catalytic efficiency when compared with the
normal, unmodified enzyme (Takahaski et al., Biochem., 34: 1433
(1995)). Since diabetic patients have higher ratios of glycated
proteins than normoglycemic individuals they are likely to have
both higher levels of 3DG and a reduced ability to detoxify this
reactive molecule by reduction to 3DF.
[0018] The mechanism of aldehyde reductase has been studied. These
studies determined that this important detoxification enzyme is
inhibited by aldose reductase inhibitors (ARIs) (Barski et al.,
Biochem., 34: 11264 (1995)). ARIs are currently under clinical
investigation for their potential to reduce diabetic complications.
These compounds, as a class, have shown some effect on short term
diabetic complications. However, they lack clinical effect on long
term diabetic complications and they worsen kidney function in rats
fed a high protein diet. As will appear hereinbelow, this finding
is consistent with the newly discovered metabolic pathway for
lysine recovery underlying the present invention. A high protein
diet will increase the consumption of fructose-lysine, which
undergoes conversion into 3DG by the kidney lysine recovery
pathway. The detoxification of the resulting 3DG by reduction to
3DF will be inhibited by ARIs therapy, which consequently leads to
an increase in kidney damage, as compared to rats not receiving
ARIs. This is because inhibition of the aldose reductase by the
ARIs would reduce availability of aldose reductase for reducing 3DG
and 3DF.
[0019] The role of 3-DG in contributing to human disease has been
previously investigated as will be appreciated from a review of the
patents summarized below.
[0020] U.S. Pat. No. 5,476,849 to Ulrich et al. describes a method
of inhibiting the formation of AGE-proteins using amino-benzoic
acids and derivatives. These compounds presumably act by reacting
with 3-DG and removing it from the system before it can react with
proteins to begin the irreversible steps of AGE-protein
formation.
[0021] U.S. Pat. Nos. 4,798,583 and 5,128,360 to Cerami et al.
describes the use of aminoguanidine to prevent AGE-protein
formation and diabetes-induced arterial wall protein cross-linking.
Aminoguanidine was shown to react with an early glycosylation
product. This early product is 3DG, as defined herein. These
patents do not contemplate the possibility of inhibiting the
formation of 3-DG. They focus exclusively on complexing this toxic
molecule.
[0022] U.S. Pat. No. 5,468,777 to France et al. describes methods
and agents for preventing the staining of teeth caused by the
non-enzymatic browning of proteins in the oral cavity. Cysteine and
cysteine derivatives are described as particularly useful in this
application.
[0023] U.S. Pat. No. 5,358,960 to Ulrich et al. describe a method
for inhibiting AGE-protein formation using aminosubstituted
imidazoles. These compounds were shown to react with an early
glycosylation product (3DG). No mention is made in this patent that
a metabolic source of 3DG may exist. This patent envisions that 3DG
is made exclusively as an intermediate in the non-enzymatic
browning of proteins.
[0024] U.S. Pat. No. 5,334,617 to Ulrich et al. describes amino
acids useful as inhibitors of AGE-protein formation. Lysine and
other bifunctional amino acids are described as particularly useful
in this regard. These amino acids are described as reacting with
the early glycosylation product from the reaction of glucose and
proteins. It appears that the early glycosylation product described
in this patent is 3DG.
[0025] U.S. Pat. No. 5,318,982 to Ulrich et al. describes the
inhibition of AGE-protein formation using as the inhibitory agent
1,2,4-triazoles. The inhibitors described in this patent contain
diamino-substituents that are positioned to react with and complex
3DG. The patent describes these compounds as reacting with an early
glycosylation product (3DG as defined herein).
[0026] U.S. Pat. No. 5,272,165 to Ulrich et al. describes the use
of 2-alkylidene-aminoguanidines as inhibitors of AGE-protein
formation. The inhibitors described in this patent are said to be
highly reactive with 3DG. No mention is made of inhibiting the
metabolic formation of 3DG in this patent.
[0027] U.S. Pat. No. 5,262,152 to Ulrich et al. describes the use
of amidrazones and derivatives to inhibit AGE-protein formation.
The compounds described in this patent are .alpha.-effect amines.
W. P. Jencks, 3rd ed., McGraw Hill, New York. Compounds of this
category are known to react with dicarbonyl compounds, e.g.
3DG.
[0028] U.S. Pat. No. 5,258,381 to Ulrich et al. describes the use
of 2-substituted-2-imidazolines to inhibit AGE-protein formation.
The compounds described in this patent contain adjacent amino
groups that can readily react with 3DG.
[0029] U.S. Pat. No. 5,243,071 to Ulrich et al. describes the use
of 2-alkylidene-aminoguanidies to inhibit AGE-protein formation.
The compounds described in this patent are highly reactive with 3DG
and function by complexing this reactive, toxic molecule.
[0030] U.S. Pat. No. 5,221,683 to Ulrich et al. describes the use
of diaminopyridine compounds to inhibit AGE-protein formation. The
diaminopyridine compounds described as particularly useful will
react with 3DG to form a stable, six-member ring containing
complex.
[0031] U.S. Pat. No. 5,130,337 to Ulrich et al. describes the use
of amidrazones and derivatives to inhibit AGE-protein formation.
The inhibitors described in this patent are a-effect amines which,
as is know in the art, will rapidly react with 3DG and form stable
complexes.
[0032] U.S. Pat. No. 5,130,324 to Ulrich et al. describes the use
of 2-alkylidene-aminoguanidines to inhibit AGE-protein formation.
The compounds described in this patent function by reacting with
the early glycosylation product resulting from the reaction of
glucose with proteins (3DG).
[0033] U.S. Pat. No. 5,114,943 by Ulrich et al. describes the use
of amino-substituted pyrimidines to inhibit AGE-protein formation.
The compounds described in this patent are said to rapidly react
with and detoxify 3DG.
[0034] None of the above-mentioned patents suggest inhibition of
the metabolic formation of 3DG as a means of therapeutic
intervention to prevent diabetic complications. Indeed, none of
these patents even suggest the involvement of an enzymatic pathway
in the production of 3DG.
[0035] U.S. Pat. No. 5,108,930 to Ulrich et al. describes a method
for detecting the levels of minoguanidine in biological samples.
This assay is described as having potential utility in determining
kidney function by measuring the aminoguanidine elimination time.
The principal utility intended for the assay method described in
this patent is in the measurement of tissue levels of
aminoguanidine, so that doses sufficient to inhibit AGE-protein
formation can be maintained in animal and human studies. No mention
is made in this patent of using urine 3DG, 3DF or DGA ratios to
determine diabetics at risk for complications.
[0036] U.S. Pat. No. 5,231,031 to Szwergold et al. describes a
method for assessing the risk of diabetic-associated pathologic
conditions and determining the efficacy of therapies for these
complications. This patent describes the measurement of two
phosphorylated compounds in erythrocytes of diabetic patients.
These two compounds were not chemically identified in this patent.
However, neither compound is 3DG or 3DF, whose levels are measured
in urine in the diagnostic embodiment of the present invention.
[0037] Methods for monitoring metabolic control in diabetic
patients by measurement of glycosylation end-products are known.
The concentration of glycosylated hemoglobin is known to reflect
mean blood glucose concentration during the preceding several
weeks. U.S. Pat. No. 4,371,374, issued to A. Cerami et al.,
describes a method for monitoring glucose levels by quantitation of
the degradation products of glycosylated proteins, more
specifically non-enzymatically glycosylated amino acids and
peptides, in urine. This method purports to utilize the affinity of
alkaline boronic acids for forming specific complexes with the
coplanar cis-diol groups found in glycosylation end-products to
separate and quantitate such end-products.
[0038] U.S. Pat. No. 4,761,368 issued to A. Cerami describes the
isolation and purification of a chromophore present in browned
polypeptides, e.g., bovine serum albumin and poly-L-lysine. The
chromophore, 2-(2-furoyl)-4(5)-2(furoyl)-1H-imidazole (FFI) is a
conjugated heterocycle derived from the condensation of two
molecules of glucose with two lysine-derived amino groups. This
patent further describes the use of FFI in a method for measuring
"aging" (the degree of advanced glycosylation) in a protein sample
wherein the sample "age" is determined by measuring the amount of
the above-described chromophore in the sample and then comparing
this measurement to a standard (a protein sample having an amount
of FFI which has been correlated to the "age" of the sample).
[0039] Without wishing to be bound by any theory, it is believed
that the present invention may be used to treat any glycogen
storage disease. Glycogen storage diseases (glycogenoses or GSDS)
are hereditary disorders in which a patient is missing one or more
of the enzymes that interconvert sugar and glycogen. The GSDs that
are presently known are classified as Types 0 to VII, depending on
the identity of the missing enzyme or enzymes, and are also known
by common names including von Gierke's disease, Pompe's disease,
Forbes' disease, Andersen's disease, McArdle's disease, Hers'
disease, and Tarui's disease. Fanconi's syndrome is also believed
to be a glycogen storage disease, and, as such, amenable to
treatment with compounds of the present invention.
[0040] There is a long-standing, unfilled need in existing
treatment regimens of diabetic patients for effective means to
identify those at risk of developing diabetes-associated pathologic
conditions, to prevent, reduce or delay the onset of such
conditions by therapeutic intervention and to determine the benefit
of such therapeutic intervention. A parallel need exists in the
treatment regimens of patients affected with glycogen storage
diseases, including Fanconi's syndrome.
SUMMARY OF THE INVENTION
[0041] The present invention arose from the discovery of a
metabolic pathway that involves the enzyme-mediated conversion of
FL to FL3P and produces relatively high concentrations of
3-deoxyglucosone (3DG) in organs affected by diabetes. Subsequent
research into the biochemical function of this newly discovered
pathway tends to indicate that it has an important role in the
etiology of diabetic kidney disease. It is also suspected that this
pathway contributes to the development of the various known
diabetes-associated pathologic conditions.
[0042] This discovery has found practical application in the
present invention which, in one aspect, provides a class of
compounds which have enzyme inhibitory activity and are effective
to inhibit the enzymatic conversion of fructose-lysine to
fructose-lysine-3-phosphate. The relevant enzyme inhibitory
activity of the compounds of the present invention is readily
determinable by assay. The assay method comprises providing an
aqueous solution of fructose-lysine, adenosine triphosphate (ATP),
a source of fructose-lysine-3-phosphate kinase and a compound of
the present invention in an amount sufficient to demonstrate
inhibitory activity, subjecting the resulting solution to
conditions promoting the formation of fructose-lysine-3-phosphate
and adenosine diphosphate as products of the interaction of the
above-mentioned kinase, fructose-lysine and adenosine triphosphate,
and measuring the production of at least one of such products, the
compounds of the present invention reducing the amount of such
products, as compared to an aqueous solution of the same relative
amounts of fructose-lysine, adenosine triphosphate and source of
fructose-lysine-3-phosphate kinase, without the addition of a
compound of the present invention. The assay method just described
is also within the scope of the present invention.
[0043] According to another aspect, the present invention provides
a pharmaceutical preparation for preventing, reducing or delaying
the onset of diabetic complications in a diabetic patient,
comprising, as an active agent, a compound of the invention, as
described above, and a pharmaceutically acceptable vehicle.
[0044] According to a further aspect of the present invention,
there is provided a method for preventing, reducing or delaying the
onset of diabetic complications in a patient at risk of developing
same, which method comprises administering to the patient a
compound of the present invention in an amount effective to inhibit
the enzymatic conversion of fructose-lysine to
fructose-lysine-3-phosphate. This same method may be used for the
prevention or treatment of other etiologically-similar disease
states, as will be further described hereinbelow.
[0045] According to still another aspect, the present invention
provides a method for assessing a diabetic patient's risk of
experiencing a diabetes-associated pathologic condition. This
method comprises administering to the patient a source of
glycated-lysine residues in an amount providing a predetermined
dose of the glycated-lysine residues, and measuring the ratio of
3-deoxyglucosone to 3-deoxyfructose in a biological sample obtained
from the patient, with reference to the ratio of 3-deoxyglucosone
to 3-deoxyfructose in a normal subject, i.e., a non-diabetic
subject or one having no clinical symptoms of diabetes. The higher
ratio of 3-deoxyglucosone to 3-deoxyfructose in the diabetic
patient sample, in comparison to that of the asymptomatic subject
is indicative that the diabetic patient is at higher risk of
experiencing a diabetes-associated pathologic condition.
[0046] The present invention also provides a method for assessing
the efficacy of therapeutic intervention in preventing diabetic
complications. The method involves measuring the concentration of
3-deoxyglucosone, 3-deoxyfructose and fructose-lysine in biological
samples obtained from a diabetic patient, both before and after
initiation of the therapeutic intervention. The sum of the
3-deoxyglucosone and 3-deoxyfructose concentrations are then
compared to the concentration of fructose-lysine in the samples. A
decrease in the sum of 3-deoxyglucosone and 3-deoxyfructose
concentrations relative to the fructose-lysine concentration in the
biological sample taken after initiation of therapeutic
intervention, as compared to the same concentrations measured in
the biological sample taken before initiation of the therapeutic
intervention, is indicative of the efficacy of the therapeutic
intervention.
[0047] As yet another aspect of the present invention, there is
provided a method for apprising a diabetic person of the potential
of a food product to contribute to the development of a
diabetic-associated pathologic condition. This method involves
measuring the content of glycated-lysine residues in the food
product and providing this information to diabetic patients, e.g.,
on the package of the food product or in a publication intended for
use by diabetics. In research leading up to the present invention,
it has been discovered that elevated levels of 3DF in biological
samples, e.g., urine, are associated with a significant risk of
developing diabetic complications. Thus, a method has been provided
for assessing a diabetic patient's risk of experiencing a
diabetes-associated pathologic condition based on measurement of
the 3DF present in a biological sample of a diabetic patient with
reference to one or more predetermined baseline levels of 3DF as an
indicator of the likelihood that the patient will develop diabetic
complications, or not.
[0048] Other related research led to the discovery of a method of
reducing susceptibility to carcinoma in a patient associated with
the intake of glycated proteins. The method comprises the
administration of a pharmaceutical composition which contains an
active compound having inhibitory activity for the enzymatic
conversion of fructose-lysine to fructose-lysine-3-phosphate. Also
embodied in the present invention is a method of preventing,
reducing, or delaying the onset of carcinoma caused by the
formation of AGE-proteins. The method comprises administering a
therapeutic amount of an agent that inhibits production of
3-deoxyglucosone.
[0049] As a means to further assess the molecular mechanism of
malignant transformation associated with administration of a diet
containing glycated proteins, a method for inducing carcinoma in a
susceptible test animal has been discovered which comprises feeding
the animal with a glycated protein diet for a sufficient time
period, such that 3-deoxyglucosone is elevated in biological fluids
at least three fold. Such animals would be assessed relative to
untreated control animals.
[0050] A method of screening for substances which affect the
development of carcinoma has also been discovered. Carcinoma will
be induced in test animals via feeding of glycated protein diet
such that 3DG levels are elevated at least 3 fold in biological
fluids. The animals are then divided into two groups, one of which
will receive the compound to be assessed, while the other group
serves as a negative control. After a suitable time period, both
groups of animals will be sacrificed and the presence and/or
absence of carcinoma in both groups assessed.
[0051] Finally, another method for screening for substances which
prevent, reduce or delay the onset of carcinoma comprises the steps
of feeding susceptible test animals a glycated protein diet in an
amount and for a time sufficient to maintain 3-deoxyglucosone (3DG)
content of a biological fluid elevated at least 3-fold relative to
the 3DG content of a biological fluid from a similar susceptible
test animal fed a diet substantially free of the glycated protein.
A test substance will then be administered to one portion of the
test animals but not to the other portion. The animals will then be
sacrificed and tissue sections compared from each such portion of
susceptible test animals to assess the effects of the test
substance.
BRIEF DESCRIPTION OF DRAWINGS
[0052] FIG. 1 shows the initial step involved in the multi-step
reaction leading to irreversibly-modified AGE-proteins.
[0053] FIG. 2 illustrates the reactions involved in the lysine
recovery pathway.
[0054] FIG. 3 is a graphical representation of a urinary profile
showing the variation over time of 3DF, 3DG and FL from a single
individual fed 2 g. of FL and followed for 24 hours.
[0055] FIG. 4 is a graphical representation of urinary excretion
over time of 3DF from seven volunteers fed 2 g. of
fructoselysine.
[0056] FIG. 5 shows a graphical comparison of 3DF and
N-acetyl-.beta.-glucosaminidase (NAG) between a group of control
animals and an experimental group maintained on a feed containing
0.3% glycated protein.
[0057] FIG. 6 is a graph showing the linear relationship between
3DF and 3DG levels in urine of rats fed either a control diet or
one enriched in glycated protein.
[0058] FIGS. 7A and 7B are graphical representations of fasting
levels of 3DG in the urine of normals and diabetic patients plotted
against the fasting level of 3DF.
DETAILED DESCRIPTION OF THE INVENTION
[0059] The following definitions are provided to facilitate
understanding of the present invention, as described in further
detail hereinbelow:
[0060] 1. Glycated-Lysine Residues--The expression "glycated lysine
residues", as used herein, refers to the modified lysine residue of
a stable adduct produced by the reaction of a reducing sugar and a
lysine-containing protein.
[0061] The majority of protein lysine residues are located on the
surface of proteins as expected for a positively charged amino
acid. Thus, lysine residues on proteins which come in contact with
serum, or other biological fluids, can freely react with sugar
molecules in solution. This reaction occurs in multiple stages. The
initial stage involves the formation of a Schiff base between the
lysine free amino group and the sugar keto-group. This initial
product then undergoes the Amadori rearrangement, to produce a
stable ketoamine compound.
[0062] This series of reactions can occur with various sugars. When
the sugar involved is glucose, the initial Schiff base product will
involve imine formation between the aldehyde moiety on C-1 of the
glucose and the lysine .epsilon.-amino group. The Amadori
rearrangement will result in formation of lysine coupled to the C-1
carbon of fructose, 1-deoxy-1-(.epsilon.-aminolysine)-fructose,
herein referred to as fructose-lysine or FL.
[0063] Similar reactions will occur with other aldose sugars, for
example galactose and ribose (Dills, Am. J. Clin. Nutr., 58: S779
(1993)). For the purpose of the present invention, the early
products of the reaction of any reducing sugar and the e-amino
residue of protein lysine are included within the meaning of
glycated-lysine residue, regardless of the exact structure of the
modifying sugar molecule.
[0064] Also, the terms glycated-lysine residue, glycated protein
and glycosylated protein or lysine residue are used interchangeably
herein, which is consistent with current usage in scientific
journals where such expressions are often used interchangeably.
[0065] 2. Fructose-lysine--The term "fructose-lysine" (FL) is used
herein to signify any glycated-lysine, whether incorporated in a
protein/peptide or released from a protein/peptide by proteolytic
digestion. This term is specifically not limited to the chemical
structure commonly referred to as fructose-lysine, which is
reported to form from the reaction of protein lysine residues and
glucose. As noted above, lysine amino groups can react with a wide
variety of sugars. Indeed, one report indicates that glucose is the
least reactive sugar out of a group of sixteen (16) different
sugars tested (Bunn et al., Science, 213: 222 (1981)). Thus,
tagatose-lysine formed from galactose and lysine, analogously to
glucose is included wherever the term fructose-lysine is mentioned
in this description, as is the condensation product of all other
sugars, whether naturally-occurring or not. It will be understood
from the description herein that the reaction between
protein-lysine residues and sugars involves multiple reaction
steps. The final steps in this reaction sequence involve the
crosslinking of proteins and the production of multimeric species,
known as AGE-proteins, some of which are fluorescent. Proteolytic
digestion of such modified proteins does not yield lysine
covalently linked to a sugar molecule. Thus, these species are not
included within the meaning of "fructose-lysine", as that term is
used herein.
[0066] 3. Fructose-lysine-3-phosphate--This compound is formed by
the enzymatic transfer of a high energy phosphate group from ATP to
FL. The term fructose-lysine-3-phosphate (FL3P), as used herein, is
meant to include all phosphorylated fructose-lysine moieties that
can be enzymatically formed whether free or protein-bound.
[0067] 4. Fructose-lysine-3-phosphate kinase--This term refers to
one or more proteins which can enzymatically convert FL to FL3P, as
defined above, when additionally supplied with a source of high
energy phosphate.
[0068] 5. 3-Deoxyglucosone-3-Deoxyglucosone (3DG) is the
1,2-dicarbonyl-3-deoxysugar (also known as 3-deoxyhexylosone) which
is formed upon breakdown of FL3P to yield free lysine and inorganic
phosphate. For purposes of the present description, the term
3-deoxyglucosone is intended to include all possible dicarbonyl
sugars which are formed upon breakdown of FL3P, having the broad
definition of FL3P stated above.
[0069] 6. FL3P Lysine Recovery Pathway--A lysine recovery pathway
exists in human kidney, and possibly other tissues, which
regenerates unmodified lysine as a free amino acid or incorporated
in a polypeptide chain. As will be further explained below, this
pathway is an important factor contributing to the complications of
diabetes.
[0070] 7. AGE-Proteins--The term "AGE-proteins" (Advanced Glycation
End-product modified proteins) has been used in scientific
journals, and is used herein, to refer to the final product of the
reaction between sugars and proteins (Brownlee, Diabetes Care, 15:
1835 (1992) and Niwa et al., Nephron, 69: 438 (1995)). It is clear
that the reaction, for example, between protein lysine residues and
glucose does not stop with the formation of fructose-lysine. FL can
undergo multiple dehydration and rearrangement reactions to produce
non-enzymatic 3DG, which reacts again with free amino groups,
leading to cross-linking and browning of the protein involved.
Indeed, there is reasonable evidence that 3DG, as defined
hereinabove, is a central intermediate in this modification
reaction.
[0071] 8. "Glycated Diet"--As used herein, this expression refers
to any given diet in which a percentage of normal protein is
replaced with glycated protein. The expression "glycated diet" and
"glycated protein diet" are used interchangeably herein.
[0072] At least some, and possibly all, of the complications of
diabetes are due to the covalent modification of proteins by
glucose and other reactive sugars. M. Brownlee, Diabetes, 43: 836
(1994). As noted above, diabetic humans and animals have been shown
to have higher concentrations of sugar modified proteins than
normal. In fact, the increase in diabetes-associated AGE-proteins
is greater than the increase in blood glucose levels.
[0073] Previously, it had been generally accepted that the origin
of 3DG in vivo was from the decomposition of proteins containing
glycated lysine residues. It had also been commonly believed that
these glycated-lysines could not be used as an amino acid source.
As will appear hereinbelow, this previous belief was incorrect.
[0074] 9. "Susceptible test animal"--As used herein this expression
refers a strain of laboratory animals which, due to the presence of
certain genetic mutations, have a higher propensity towards
malignant transformation and tumor formation. Unless otherwise
specified, the Eker rat which has a mutation in the tuberous
sclerous gene (Tsc-2) was utilized in the studies described herein.
One of ordinary skill in the art is aware of a variety of other
laboratory rat or mouse strains with increased propensity for tumor
formation. The phrase "similar susceptible test animal" refers to
animals of a comparable genetic background which are used as
control, untreated animals.
[0075] As mentioned above, the present invention evolved from the
discovery of a previously unknown metabolic pathway which produces
3DG in an enzyme-catalyzed reaction. This enzymatic pathway is
capable of enzymatic inhibition, thereby reducing the production of
toxic 3DG.
[0076] During the course of a series of studies on diabetic
kidneys, examination of .sup.31P NMR spectra from perchloric acid
extracts of kidneys from streptozotoxin induced diabetic rats
revealed an unusual new peak in the NMR spectrum. Previous studies
by the present inventors had demonstrated the presence of
fructose-3-phosphate in rat lens and human erythrocytes (A.
Petersen et al., Biochem. J., 284: 363-366 (1992); Lal et al.,
Arch. Biochem. Biophys., 318: 191 (1995); Szwergold et al.,
Science, 247: 451 (1990) and Lal et al., Investigative opthalmology
and Visual Science, 36(5): 969 (1995)). Earlier studies had
identified other unusual phosphorylated sugars in rat lens
(Szwergold et al., Diabetes, 44: 810 (1995) and Kappler et al.,
Metabolism, 44: 1527 (1995)). Thus it was reasonable to assume that
this newly identified peak was another phosphorylated sugar.
Further extensive laboratory investigation revealed that this new
compound was not a simple sugar, but rather fructose-lysine
phosphorylated on the 3 position of the fructose component.
[0077] This identification was confirmed in two ways. Authentic
fructose-lysine-3-phosphate (FL3P) was synthesized by the procedure
disclosed in Example 2, below, and shown to co-resonate in the
.sup.31P NMR spectrum with the peak in diabetic rat kidneys.
Synthetic fructose-lysine was also injected into non-diabetic rats.
These rats showed a substantial increase in the levels of FL3P in
their kidneys following this injection.
[0078] Two experiments were conducted to demonstrate that FL3P is
derived directly from FL in an enzyme catalyzed reaction.
Fructose-lysine labeled with deuterium at the C3 position of the
fructose moiety was synthesized and injected into rats. Three hours
after injection, the kidneys of these rats were removed and
extracted with perchloric acid. NMR spectroscopy revealed that the
FL3P material isolated from these rats contained the deuterium
label at the C3 position of the fructose moiety. In addition, rat
kidney homogenates demonstrate the ability to produce FL3P in a
reaction requiring both ATP and fructose-lysine. This
last-mentioned experiment confirms the presence of a specific FL3P
kinase, as no FL3P is formed when only fructoselysine and ATP are
incubated together under physiological conditions. Further
experiments which involved the fractionation of kidney cortex have
demonstrated that this kinase activity is not distributed uniformly
in the kidney but is concentrated in the proximal tubular region,
which is one of the earliest anatomical sites to demonstrate damage
in human and animal diabetic kidneys.
[0079] FL3P is not stable in aqueous solution. It rapidly degrades
to form 3DG, lysine and inorganic phosphate. This reaction also
occurs in vivo. It is not currently know if the degradation of FL3P
in vivo is a spontaneous or enzyme catalyzed reaction. It is
strongly suspected, however, that enzymatic catalysis is involved,
as the production of 3DG from fructose-lysine occurs very rapidly
in intact kidney.
[0080] The reaction steps in the FL3P lysine recovery pathway are
presented in FIG. 2. In the first step, fructose-lysine and ATP
react to form fructose-lysine-3-phosphate (FL3P) and ADP in a
reaction catalyzed by FL3P kinase. Phosphorylation occurs on the
3-position of the fructose moiety, leading to destabilization of
the fructoselysine molecule. The resulting FL3P then decomposes to
form 3-deoxyglucosone (3DG), inorganic phosphate, and unmodified,
free, reusable lysine, which is available for utilization in
protein synthesis. Aldehyde reductase detoxifies 3DG by reduction
to 3-deoxyfructose (3DF), which is excreted in urine.
[0081] Although FIG. 2 illustrates this pathway using the most
prevalent glycated-lysine, fructose-lysine, it will be readily
apparent to those skilled in the art that a wide variety of similar
molecules can flux through this pathway. Indeed, as will be
explained in further detail below, the substrate selectivity of the
FL3P lysine recovery pathway is quite broad, warranting the broad
definition of the terms given above.
[0082] Additional experiments have shown that the lysine recovery
pathway is found in a wide variety of animal species, including
sheep, pig, dog, rabbit, cow, mice and chicken. This pathway is
also present in humans. The ubiquitous presence of the FL3P lysine
recovery pathway can be understood, given that lysine is an
essential amino acid which is present in relatively low
concentrations in most foods. In addition, an appreciable
percentage of the lysine residues in food will exist in the
glycated form and the proportion of this modified lysine will
increase when the food is cooked. Since these glycated lysine
residues can not be utilized for protein synthesis, a recovery
pathway for lysine is of great utility and affords a selective
advantage to organisms which possess it.
[0083] Diabetes has two effects on the lysine recovery pathway.
Blood proteins contain higher concentrations of glycated-lysines
when isolated from diabetics than from non-diabetic-individuals.
Thus, diabetics are subject to greater flux through the lysine
recovery pathway than non-diabetics. Additionally, from preliminary
observations on the ratios of 3DG and 3DF in the urine of diabetics
and normals, diabetics appear to have a reduced ability to detoxify
3DG that is produced via this pathway. These two factors combine to
produce higher urinary concentrations of 3DG in diabetics (See FIG.
7; also Lal et al., Arch. Biochem. and Biophys., 342(1): 254-60
(1997).
[0084] The agents involved in the lysine recovery pathway have been
identified in other tissues besides kidney, specifically red blood
cells, lens, and peripheral nerve tissues. All of these tissues are
affected by the complications of diabetes. The location in red
blood cells correlates with the microvascular complications of
diabetes, e.g., diabetic retinopathy, the kidney location
correlates with diabetic nephropathy, while the location in
peripheral nerve correlates with diabetic peripheral neuropathy.
These agents are also found in pancreas. Experiments are in
progress to determine the presence of these agents in skin. If
found to be present, it is believed that their deleterious effects
may be ameliorated by a topical treatment using the inhibitory
compounds of the invention in a suitable vehicle to prevent
collagen crosslinking, and thereby improve skin elasticity.
[0085] Experiments have been conducted that tend to prove that
humans produce both 3DG and 3DF from orally ingested proteins
containing glycated-lysine residues. These experiments, which are
described in detail below, convincingly demonstrate that the lysine
recovery pathway exists in humans. These experiments also shed
light on a puzzling phenomenon, namely, that some diabetics develop
diabetic complications, while others, even those in poor glycemic
control, do not develop such complications. The reason for this
phenomenon is apparent from the data presented herein. Diabetics
have a differing ability to detoxify 3DG. A subset of the diabetic
population appears to have relatively higher aldehyde reductase
activities than does the majority of diabetics. Consequently, these
individuals are capable of handling the increased flux through the
lysine recovery pathway by efficiently detoxifying the higher than
normal level of 3DG. Others with impaired capacity are less able to
detoxify their elevated 3DG levels, and consequently are at higher
risk of developing diabetic complications.
[0086] As will be described in more detail below, it has been
experimentally demonstrated that stimulation of the lysine recovery
pathway can occur through the use of a glycated protein diet. As
was the case with FL above, elevation of FL3P, 3DG and 3DF was
observed in test animals that were fed the glycated protein
diet.
[0087] The enzyme inhibitor compounds of the invention block the
lysine recovery pathway, preventing formation of toxic 3DG from
FL3P.
[0088] Described below is a set of extensive criteria that a
suitable enzyme inhibitor should display for use in the practice of
this invention, as well as certain tests for determining if any
putative inhibitor meets these criteria. Candidate kinase
inhibitors for use in accordance with this invention may be natural
products isolated from plants or microorganisms. Alternatively,
they may be synthetic molecules derived from the rational knowledge
of the enzymatic reaction and its mechanism. Inhibitors may also be
synthesized by combinatorial methods. Combinatorial libraries may
be generated from a random starting point. Furthermore,
combinatorial methods can be utilized to generate a wide variety of
compounds related to previously identified inhibitors of the target
FL3P kinase.
[0089] Regardless of the source of the putative inhibitor,
compounds that do not meet all of the criteria listed below are not
considered useful therapeutic agents capable of inhibiting the
lysine recovery pathway and thereby preventing, reducing or
delaying the onset of diabetic complications or disorders of
related etiology.
[0090] 1. The inhibitor should be a small molecule and readily
taken up by cells. In order to meet this criteria, the inhibitor
must have a molecular weight of less than 2,000 and more ideally
approximately 1,000 daltons or less.
[0091] 2. The inhibitor must show competitive, noncompetitive,
irreversible or suicide inhibition of the FL3P kinase. If the
inhibitor is a competitive or noncompetitive inhibitor, the
inhibition constant, Ki, must be less than about 1 mM. Ideally, it
must be less than 100 .mu.M and more ideally, about 40 .mu.M or
less. If the inhibitor shows suicide or other irreversible
inhibition, this requirement for inhibition constant is rendered
moot.
[0092] 3. The inhibitor must be both soluble in aqueous solution
and stable in aqueous solution at physiological pH. The requirement
for solubility is met only if the inhibitor, or a salt of the
inhibitor, is soluble in physiological saline or serum at a
concentration equal to or greater than 10 .mu.M. This stability
requirement is met only if a solution of inhibitor dissolved in
physiological saline at 37.degree. C. retains greater than 50% of
its activity after incubation for one hour. Ideally, the inhibitor
must retain greater than 50% activity upon incubation for one day
or more.
[0093] 4. The Inhibitor Must Show Acceptable pharmacokinetics. That
is, it must remain at a therapeutically effective concentration for
at least one hour following administration of the agent. Ideally,
it should maintain effective concentration for at least eight
hours. More ideally, once per day dosing should be all that is
necessary in order to maintain a therapeutic concentration of the
inhibitor. This requirement does not mean that the inhibitor must
be able to establish a therapeutic concentration after the first
dose. Numerous examples of successful pharmaceuticals exist where
medical efficacy is seen only upon prolonged dosing. The criterion
does mean that, once an efficacious concentration is reached, this
concentration should be able to be maintained for greater than one
hour following the last administration of medication. A test for
therapeutic efficacy is described herein.
[0094] 5. The inhibitor must be non-toxic. This criteria requires
that the inhibitor not demonstrate human toxicity when administered
at the therapeutic dose. Ideally, toxicity should not be evident
when the inhibitor is present at blood and/or target tissue levels
of twice that needed for therapeutic effect. More ideally, there
should be no appreciable toxicity at levels 6 or more times the
therapeutic range. Diabetic complications can only be prevented by
long term inhibitor treatment. Therefore, the requirement for
non-toxicity must include both acute toxicity and chronic toxicity
that may become evident over extended, long term use. Toxicity of
candidate molecules can be readily assessed using well established
animal studies. Human toxicity is assessed in stage one clinical
trials.
[0095] Included among the compounds useful in the practice of this
invention are those of the formula: ##STR1##
[0096] wherein X is --NR'--, --S(O)--, --S(O).sub.2--, or --O--, R'
being selected from the group consisting of H, and linear or
branched chain alkyl group (C.sub.1-C.sub.4) and an unsubstituted
or substituted aryl group (C.sub.6-C.sub.10) or aralkyl group
(C.sub.7-C.sub.10); R is a substituent selected from the group
consisting of H, an amino acid residue, a polyaminoacid residue, a
peptide chain, a linear or branched chain aliphatic group
(C.sub.1-C.sub.8), which is unsubstituted or substituted with at
least one nitrogen- or oxygen-containing substituent, a linear or
branched chain aliphatic group (C.sub.1-C.sub.8), which is
unsubstituted or substituted with at least one nitrogen- or
oxygen-containing substituent and interrupted by at least one
--O--, --NH--, or --NR''-- moiety, R'' being linear or branched
chain alkyl group (C.sub.1-C.sub.6) and an unsubstituted or
substituted aryl group (C.sub.6-C.sub.10) or aralkyl group
(C.sub.7-C.sub.10), with the proviso that when X represents
--NR'--, R and R', together with the nitrogen atom to which they
are attached, may also represent a substituted or unsubstituted
heterocyclic ring having from 5 to 7 ring atoms, with at least one
of nitrogen and oxygen being the only heteroatoms in said ring,
said aryl group (C.sub.6-C.sub.10) or aralkyl group
(C.sub.7-C.sub.10) and said heterocyclic ring substituents being
selected from the group consisting of H, alkyl (C.sub.1-C.sub.6),
halogen, CF.sub.3CN, NO.sub.2 and --O-alkyl (C.sub.1-C.sub.6);
R.sub.1 is a polyol moiety having 1 to 4 linear carbon atoms, Y is
a hydroxymethylene moiety --CHOH--; Z is selected from the group
consisting of --H, --O-alkyl (C.sub.1-C.sub.6), -halogen
--CF.sub.3, --CN, --COOH, and --SO.sub.3H.sub.2, and optionally
--OH; and the isomers and pharmaceutically acceptable salts of said
compound, except that X--R in the above formula does not represent
hydroxyl or thiol.
[0097] Illustrative examples of nitrogen- or oxygen-containing "R"
substituents include those derived from
.gamma.-amino-.alpha.-hydroxy butyric acid
(--(CH.sub.2).sub.2--CHOH--COOH), 1,2,4 triaminobutane
(--(CH.sub.2).sub.2--CHNH.sub.2--CH.sub.2NH.sub.3),
3,6-diamino-5-hydroxyheptanoic acid
(--CH.sub.2--CH(OH)--CH.sub.2--CH(NH.sub.2)--CH.sub.2--COOH), and
the like.
[0098] The structure of formula I has asymetric centers and may
occur as racemates, racemic mixtures and various stereoisomers, all
of such isomeric forms being within the scope of this invention, as
well as mixtures thereof.
[0099] Although certain of the compounds having the structure of
formula I, above, were previously known, others are believed to be
novel and as such are within the scope of the present invention, as
is the use of all of the compounds of formula I for inhibiting the
enzyme-catalyzed production of 3DG in vivo.
[0100] Inhibitors of the above formula may be prepared by reacting
the appropriate sugar, e.g., glucose, galactose, mannose, ribose,
xylose, or the like, with an amino- or hydroxyl-substituted
reactant of the type described herein in the presence of an agent,
such as NaBH.sub.3CN, that selectively reduces the Schiff-base
intermediate to an amine, thereby producing an inhibitor having an
alcohol moiety (i.e., Y.ident.CH(--OH)--). The reactive moiety of
an amino acid reactant, when used, may be the amine group on the
alpha-carbon, or the amine group or hydroxyl group on the acid side
chain. Suitable amino acids encompass the essential amino acids.
Specific examples include without limitation, glycine, alanine,
valine, leucine, isoleucine, serine, threonine, methionine,
aspartic acid, phenylalanine, tyrosine, histidine and tryptophan.
Other suitable reactants are from the broader class of
aminocarboxylic acid, for example, pyroglutamic acid, beta-alanine,
gamma-aminobutyric acid, epsilon-amino caproic acid and the like.
N-acyl derivatives of the above-mentioned amino acids, such as
formyl lysine, may also be used if desired.
[0101] Other appropriate reactants include, without limitation,
unsubstituted or substituted aryl (C.sub.6-C.sub.10) compounds,
wherein the substituent may be alkyl (C.sub.1-C.sub.3), alkoxy,
carboxy, nitro or halogen groups, unsubstituted or substituted
alkanes, wherein the substituent may be at least one alkoxy group;
or unsubstituted or substituted nitrogen-containing heterocyclic
compounds, wherein the substituents may be alkyl (C.sub.1-C.sub.3),
aryl (C.sub.6-C.sub.10), alkoxy, carboxy, nitro or halogen groups.
Illustrative examples of the last-mentioned group of reactants
include m-methyl-, p-methyl-, m-methoxy-, o-methoxy- and
m-nitro-aminobenzenes, o- and p-aminobenzoic acids; n-propylamine,
n-butylamine, 3-methoxypropylamine; morpholine and piperidine.
[0102] Representative inhibitor compounds having the above formula
are set forth in the attached Table A. Examples of known compounds
that may be used as inhibitors in practicing this invention
include, without limitation, meglumine, sorbitol lysine and
mannitol lysine. A preferred inhibitor is 3-O-methyl
sorbitollysine.
[0103] It appears that the locus of uptake of the inhibitors in
vivo is the kidney, as demonstrated by the data in Example 16,
below. TABLE-US-00001 TABLE A Compound Name X R R.sub.1 Y Z a
3-O-methyl sorbitollysine --N--H ##STR2## ##STR3## ##STR4##
--O--CH.sub.3 galactitol lysine '' '' '' ##STR5## --OH 3-deoxy
sorbitol lysine '' '' '' '' --H 3-deoxy-3-fluoro- xylitol lysine ''
'' ##STR6## '' --F 3-deoxy-3-cyano sorbitol lysine '' '' ##STR7##
'' --C.ident.N b 3-deoxy-sedoheptitol spermine --N--CH.sub.3
##STR8## ##STR9## ##STR10## H a -lysine residue b -spermine
residue
[0104] The inhibitor compounds described herein can form
pharmaceutically acceptable salts with various inorganic or organic
acids or bases. Suitable bases include, e.g., alkali metal salts,
alkaline earth metal salts, ammonium, substituted ammonium and
other amine salts. Suitable acids include, e.g., hydrochloric acid,
hydrobromic acid and methanesulfonic acid.
[0105] The pharmaceutically acceptable salts of the compounds of
formula I can be prepared following procedures which are familiar
to those skilled in the art.
[0106] The ability of a compound to inhibit the FL3P kinase can be
determined using a wide variety of kinase activity assays. One
useful assay involves incubating the potential inhibitor with
fructose-lysine and ATP in the presence of kidney homogenate or
other enzyme source.
[0107] A solution of the assay components is prepared, which
typically contains 1 millimole or less of the inhibitor compound of
this invention, an amount of fructose lysine (FL) in the range of
1-10 millimoles, an amount of ATP in the range of 0.1-10 millimoles
and an amount of the enzyme source which is sufficient to convert
FL to fructose lysine-3-phosphate. The incubation should be
conducted within a pH range of 4.5 to 9.5 and ideally at neutral or
near neutral pH. The incubation should be carried out at a
temperature that is compatible with enzyme activity, between
4.degree. and 40.degree. C. Ideally, the incubation is carried out
at physiological temperature. After incubation, the reaction is
stopped by acid precipitation of the protein and the production of
FL3P measured by .sup.31P-NMR spectroscopy. FL3P production will be
reduced or eliminated in samples containing an inhibitor compound
when compared to control samples that are free of inhibitor.
[0108] Other assays have utility for the rapid determination of
enzyme inhibition. One such assay involves the use of
fructose-lysine and .gamma.-labelled .sup.32P or .sup.33P-ATP.
Since FL3P does not bind to Dow-1 but ATP and most other phosphates
do, it is possible to separate the product FL3P from the remaining
reaction mixture by passing the assay solution through a column of
Dow-1 resin after a predetermined reaction time, typically 10
minutes. The resultant solution is added to a container of
scintillation liquid, e.g., Ecoscint A, and counted to determine
the amount of radioactivity produced.
[0109] As it is difficult to obtain large quantities of human
tissue, it is preferable to use a recombinant version of the kinase
that is cloned into an expression system, such as E. Coli. The
cloned kinase can be readily obtained from the "shotgun" cloning of
tissue specific cDNA libraries. Such libraries are readily
available from commercial sources. For example they may be obtained
from Clontech, Palo Alto, Calif. The shotgun cloning envisioned may
be performed using the lambda cloning system commercially available
from Stratagen, located in San Diego, Calif. This cloning kit
contains detailed instructions for its use.
[0110] The pharmaceutical preparations of the present invention
comprise one or more of the compounds described above, as the
active ingredient, in combination with a pharmaceutically
acceptable carrier medium or auxiliary agent.
[0111] These ingredients may be prepared in various forms for
administration, including both liquids and solids. Thus, the
preparation may be in the form of tablets, caplets, pills or
dragees, or can be filled in suitable containers, such as capsules,
or, in the case of suspensions, filled into bottles. As used
herein, "pharmaceutically acceptable carrier medium" includes any
and all solvents, diluents, or other liquid vehicle, dispersion or
suspension aids, surface active agents, isotonic agents, thickening
or emulsifying agents, preservatives, solid binders, lubricants and
the like, as suited to the particular dosage form desired.
Representative examples of suitable carrier media include gelatine,
lactose, starch, magnesium stearate, talc, vegetable and animal
fats and oils, gum, polyalkylene glycol, or the like. Remington's
Pharmaceutical Sciences, Fifteenth Edition, E. W. Martin (Mack
Publishing Co., Easton, Pa. 1975) discloses various carriers used
in formulating pharmaceutical compositions and known techniques for
the preparation thereof. Except insofar as any conventional carrier
medium is incompatible with the enzyme inhibitors of the invention,
such as by producing any undesirable biological effect or otherwise
interacting in a deleterious manner with any other component(s) of
the pharmaceutical preparation, its use is contemplated to be
within the scope of this invention.
[0112] In the pharmaceutical preparations of the invention, the
active agent(s) may be present in an amount of at least 5% and
generally not more than 98% by weight, based on the total weight of
the preparation, including carrier medium and/or auxiliary
agent(s), if any. Preferably, the proportion of active agent varies
between 65%-95% by weight of the composition.
[0113] Preferred supplementary active agents are compounds that
bind to 3DG in vivo. This class of compounds includes, without
limitation, aminoguanidine, amino benzoic acid and derivatives
thereof, cysteine and derivatives thereof, amino-substituted
imidazoles, 1,2-disubstituted benzimidazoles, substituted
1,2,4-triazoles, diaminopyridine and derivatives thereof,
amino-substituted pyrimidines, aminoalcohols, diamines and the
like. Anti-hypertensive drugs, including particularly the
angiotensin-converting enzyme (ACE) inhibitors, may also be
included as supplementary active agents in the pharmaceutical
preparations of this invention.
[0114] Auxiliary agents, such as compounds that will protect the
active compound from acid destruction in the stomach or facilitate
the absorption of the active compound into the bloodstream can also
be incorporated into the pharmaceutical preparation, if necessary
or desirable. Such auxiliary agents may include, for example,
complexing agents such as borate or other salts which partially
offset the acid conditions in the stomach, and the like. Absorption
can be increased by delivering the active compound as the salt of a
fatty acid (in those cases where the active compound contains one
or more basic functional groups).
[0115] The compounds of the invention, along with any supplementary
active ingredient(s) may be administered, using any amount and any
route of administration effective for inhibiting the FL3P lysine
recovery pathway. Thus, the expression "therapeutically effective
amount", as used herein, refers to a nontoxic but sufficient amount
of the enzyme inhibitor to provide the desired therapy to
counteract diabetic complications or to inhibit the metabolic
production of 3DG for other medical reasons, such as reducing the
effects of aging or other human disease states where AGE-Protein
formation has a causative role. The exact amount required may vary,
depending on the species, age, and general condition of the
patient, the nature of the complications, the particular enzyme
inhibitor and its mode of administration, and the like.
[0116] The compounds of the invention are preferably formulated in
dosage form for ease of administration and uniformity of dosage.
Dosage unit form as used herein refers to a physically discrete
unit of enzyme inhibitor appropriate for the patient to be treated.
Each dosage should contain the quantity of active material
calculated to produce the desired therapeutic effect either as
such, or in association with the selected pharmaceutical carrier
medium. Typically, the compounds of the invention will be
administered in dosage units containing from about 1 mg to about
2,500 mg of the compound, by weight of the preparation, with a
range of about 5 mg to about 250 mg being preferred.
[0117] The compounds of the invention may be administered orally,
parenterally, such as by intramuscular injection, intraperitoneal
injection, intravenous infusion or the like, depending on the
nature of the diabetic complication being treated. The compounds of
the invention may be administered orally or parenterally at dosage
levels of about 0.7 .mu.g to about 20 mg and preferably from about
30 .mu.g to about 3.5 mg/kg, of patient body weight per day, one or
more times a day, to obtain the desired therapeutic effect.
[0118] Orally active enzyme inhibitors are particularly preferred,
provided the oral dose is capable of generating blood and/or target
tissue levels of the inhibitor that are therapeutically active.
Those skilled in the art can readily measure the levels of a small
molecule inhibitor in deproteinized samples of blood, kidney and
other target tissues. The concentration of inhibitor in these
samples can be compared with the predetermined inhibitory constant.
Tissue levels that are far below the inhibitory constant suggest a
lack of therapeutic activity. In the case of irreversible
inhibitors, this lack can be confirmed or refuted by assay of the
FL3P kinase levels in the respective tissue. In all cases,
therapeutic activity can be assessed by feeding the human or animal
subject a food rich in glycated lysine residues or fructose-lysine
and measuring the amount of 3DG and 3DF in their urine, both before
and after feeding. Subjects that have therapeutically active
inhibitor in their systems will experience decreased secretion of
both 3DG and 3DF and increased urinary secretion of fructose-lysine
when compared to levels secreted by these same subjects prior to
inhibitor therapy as will be described in further detail
hereinbelow.
[0119] The compounds of the invention will typically be
administered once per day or up to 4-5 times per day, depending
upon the exact inhibitor chosen. While a dosing schedule of
once-a-day is preferred, diabetic patients are accustomed to paying
close attention to their disease state, and so will readily accept
more frequent dosing schedules if required, so as to deliver the
above-mentioned daily dosage. However, the exact regimen for
administration of the compounds and compositions described herein
will necessarily be dependent on the needs of the individual
patient being treated, the type of treatment administered and the
judgment of the attending physician. As used herein, the term
"patient" includes both humans and animals.
[0120] The inhibitor compounds described herein are useful in
counteracting diabetic complications, especially diabetic
nephropathy which affects greater than forty percent of diabetics
and is the primary cause of end stage renal disease requiring
dialysis and transplantation. In addition, these inhibitors may be
used for the prevention or treatment of other pathological
conditions attributable to the formation of AGE-proteins, such as
hypertension, stroke, neurodegenerative disorders, e.g., senile
dementia of the Alzheimers type, circulatory disease, glycogen
storage diseases including Fanconi's syndrome, atherosclerosis,
osteoarthritis, cataracts and the general debilitating effects of
aging.
[0121] Preliminary experiments have shown that serious adverse
health effects result from stimulation of the lysine recovery
pathway through long-term consumption of glycated proteins. As was
the case with FL, elevation of FL3P, 3DG and 3DF was observed in
test animals that were fed a glycated protein diet. See Table B.
After eight months of such a diet clear evidence of kidney
pathology, resembling that found in diabetic kidneys, was seen in
the animals on the glycated protein diet, as described further in
Example 10, below. Transient elevation of 3DG and 3DF levels were
also observed in the urine of human volunteers who ate a small
amount of the glycated protein. TABLE-US-00002 TABLE B % Glycated
FL3P conc. 3DG/3DF concs Protein (nM-in Kidney) (.mu.M-in plasma) 0
97 1.4/0.05 1 295 -- 2.5 605 -- 5 937 -- 10 1066 3.6/0.12 20 1259
5.2/0.14 30 1267 6.2/0.28
[0122] Since stimulation of the newly discovered lysine recovery
pathway leads to substantial increases in systemic 3DG levels, an
investigation was carried out to determine whether a glycated diet
would cause significant effects on pregnancy. The results obtained
so far suggest there is a very strong effect due to this pathway,
as will appear in the examples that follow.
[0123] Furthermore, it is well known that in susceptible strains of
rats and mice the diets on which the animals are maintained in
early life (following weaning), can have a marked effect on the
incidence of type 1 diabetes, with the incidence ranging from 10%
to 90%. Considerable effort has been put into investigating this
phenomenon over the last 10 years. See, for example, Diabetes,
46(4): 589-98 (1997) and Diabetes Metab. Rev., 12(4): 341-59
(1996), and references cited therein. An investigation has been
undertaken by certain of the present inventors with respect to two
diets which are at the extremes for induction of diabetes. AIN-93
(Dyets, Inc.) causes the least incidence of diabetes and produces
the lowest ratio of urinary 3DF/creatinine (1.0) yet observed.
Purina 500 induces the highest incidence of diabetes and produces a
2.5 fold increase in the 3DF/creatinine ratio. Since FL3P, 3DG and
3DF were observed in the pancreas of rats, it is likely that
fructoselysine kinase and the metabolites of this metabolic pathway
are involved in the development of Type I diabetes. Animals which
are susceptible to this type of diabetes (a useful model of insulin
dependent or Type I diabetes in humans) have an abnormal immune
system which makes them sensitive to an unknown antigen which
develops in the .beta.-cells of the pancreas, resulting in an
autoimmune attack by the animal's own immune system on its
.beta.-cells. This results in their subsequent destruction, thereby
depriving the animal of the ability to make insulin. It is well
known that 3DG reacting with proteins can make new antigenic sites.
Thus, the source of the antigenic properties of the various diets
appears to be the 3DG created by the decomposition of
fructoselysine-3-phosphate in the pancreas.
[0124] Also, because 3DG is known to interact with amines
generally, it may be able to interact with DNA and show mutagenic
and carcinogenic potential, as well as crosslink proteins.
[0125] The discovery of the FL3P lysine recovery pathway makes it
practical, for the first time, to differentiate the diabetic
population and to determine which subset of the population is
likely to develop to diabetic complications. This determination can
be conveniently carried out on a biological fluid of the test
subject, such as urine, blood fractions (particularly plasma or
serum), lymph fluid, interstitial fluid or the like.
[0126] After an overnight fast, a human subject is fed a food
source containing a relatively high concentration of
glycated-lysine residues. By way of example, this food can be in
the form of a casein/sugar "cookie", such as described in Example
5, below, or some other suitable source of glycated-lysines or
synthetic fructose-lysine. When proteins containing glycated-lysine
residues are utilized, the content of glycated-lysine should be
preferably between 0.02 and 10% of total protein amino acid, or
more preferably between about 0.2 and 0.4%. The total amount of
glycated-lysine residues in the oral dose should be about 0.3
grams. Preferably, a urine sample is collected before consumption
of the glycated-lysine source, then at one, three and five hours,
or such other appropriate times as may be warranted by the
individual clinical situation.
[0127] The 3DG and 3DF levels in these urine samples are measured
and the ratios of these metabolites calculated. The particular
methodology utilized in this measurement is not essential to the
practice of this invention. The GC method described in Example 5,
below, may be utilized, if desired. Alternatively, calorimetric or
immunological assay methods can be used, as will be apparent to
those skilled in the art.
[0128] It is clear that the major risk factor faced by diabetics is
glycemic control, as was clearly demonstrated by the recently
completed Diabetes Control and Complications Trial. However, the
incidence of diabetic complications cannot be explained solely by
blood sugar levels; a fairly wide scatter is seen when the
incidence of diabetic complications is compared to historical blood
sugar levels.
[0129] One method for determining that subset of the diabetic
population which is most at risk for developing diabetic
complications is a particularly significant aspect of the present
invention. This method involves the measurement of FL, 3DG and 3DF
levels before and, optimally, after ingesting a source of glycated
lysine.
[0130] For example, normal subjects have a fasted 3DG to 3DF ratio
in urine of about 0.025, whereas diabetics have higher ratios,
which may be up to five fold higher, or more. This is borne out by
the data in FIG. 7, which shows that normoglycemics have a 3DG/3DF
ratio of 0.025 (1/39.77) with quite tight scatter around this
value, whereas diabetics have a more than 2 fold higher average
ratio (average 0.069) with much more scatter around the
average.
[0131] As demonstrated herein, diabetics have increased production
of 3DG. Therefore, resistance to diabetic complicatiors requires
highly efficient removal of this toxic metabolite. The ratio of 3DG
to 3DF, calculated by the method described herein, allows one to
assess the efficiency of the 3DG detoxification pathways. Those
individuals with low ratio will be generally resistant to
developing diabetic complications. Individuals with higher ratios,
including ratios contained within the normal range, are more at
risk, while individuals with elevated ratios above the normal range
are particularly at risk for developing these complications.
[0132] Recent measurements of fructoselysine (FL) in the plasma and
urine of four different rat strains have demonstrated considerable
variability in the manner in which their respective kidneys process
FL in blood. In two of the four strains (Long Evans, Brown Norway)
virtually all of the FL filtered by the kidney appeared in the
urine based upon ratios of this compound and its metabolites with
creatinine. With the other two strains (Sprague Dawley, Fischer)
10-20% of the FL in the plasma appeared in the urine, based on
comparisons with creatinine filtration. These measurements strongly
suggest a major variability in FL processing in the mammalian
kidney. Given what is known about the functional equivalence of
rodent and human kidneys, it is reasonable to assume a similar
variation in FL processing will exist among humans. Since FL is the
primary input to the fructoselysine recovery pathway, the entire
pathway is likely to be substantially stimulated in those humans in
whom a large amount of FL is absorbed from the ultrafiltrate,
leading to the high local levels of 3-deoxyglucosone (3DG) in the
kidney, as well as systemically throughout the body. This
observation may serve as the basis of a diagnostic test in which
the comparison of a sample of plasma or serum contemporaneously
obtained with a urine sample would determine the flux of FL into
the kidney, and the fraction of that flux which appears in the
urine. Those individuals in whom this ratio is substantially lower
than one (1) would then be at risk for developing a variety of
kidney pathologies including, but not limited to, diabetic
nephropathy, kidney failure in old age and kidney carcinoma.
[0133] Therapeutic efficacy of the kinase inhibitors of the
invention can be easily and safely determined using a test of the
lysine recovery pathway. The test protocol is identical to the one
presented immediately above, with the exception that urinary
fructoselysine levels are measured in addition to urinary 3DG and
3DF levels. It is useful to conduct this test both before and after
initiating FL3P kinase inhibitor therapy. The urine levels of 3DG
and 3DF are summed at each time point and compared to the levels of
fructose-lysine measured in the same sample.
[0134] The peak levels of 3DG and 3DF found in urine following
ingestion of food rich in glycated-lysine residues are derived from
the activity of the lysine recovery pathway. The ratio of the
concentration of these metabolites to unreacted fructose-lysine
(which is a normal component of human urine) reflects the activity
of this pathway. Inhibition of the lysine recovery pathway will
cause a decrease in the amount of 3DG and 3DF excreted, and an
increase in the excreted levels of fructose-lysine. Thus,
therapeutic efficacy of a kinase inhibitor can be quantitated by
measuring the decrease of the (3DG+3DF)/fructose-lysine ratio
following initiation of therapy. It is noteworthy that urine volume
or metabolite concentrations are not a factor in interpreting this
assay, as only a ratio of metabolites is considered.
[0135] It will be appreciated from the foregoing disclosure that
orally digested food containing high concentrations of
glycated-lysine residues will lead to the production of kidney and
serum 3DG. It is reasonable to caution individuals at risk for
kidney disease, for example diabetics, to avoid food with these
high concentrations. Concentrations of glycated-lysine residues can
be measured using a wide variety of methods. One such measurement
method is described in Example 4, below. However, any suitable
measurement methodology that accurately determines the levels of
glycated-lysine residues can be substituted in place of the assay
method exemplified below. Examples of assay methods specifically
contemplated include but are not limited to calorimetric and
immunological methods.
[0136] Regardless of the method of measurement employed, it is
within the scope of the present invention to determine the content
of glycated-lysine residues in prepared foods and to apprise
individuals at risk for developing kidney dysfunction of these
determinations, so that such individuals may refrain from ingesting
foods high in glycated-lysine content.
[0137] The following examples are provided to describe the
invention in further detail. These examples are provided for
illustrative purposes only, and should in no way be construed as
limiting the invention. All temperatures given in the examples are
in degrees centigrade unless otherwise indicated.
EXAMPLE 1
Isolation and Identification of FL3P
[0138] A .sup.31P NMR analysis of a perchloric acid extract of
diabetic rat kidneys showed a new sugar monophosphate resonance at
6.24 ppm which is not observed in non-kidney tissue and is present
at greatly reduced levels in non-diabetic kidney. The compound
responsible for the observed resonance was isolated by
chromatography of the extract on a microcrytalline cellulose column
using 1-butanol-acetic acid-water (5:2:3) as eluent. The structure
was determined by proton 2D COSY to be fructose-lysine 3-phosphate.
This was later confirmed by injecting animals with FL, prepared as
previously described (Finot and Mauson, Helv. Chim. Acta, 52: 1488
(1969)), and showing direct phosphorylation to FL3P. Using FL
specifically deuterated in position-3 confirmed the position of the
phosphate at carbon-3. This was performed by analyzing the .sup.31P
NMR spectra both coupled and decoupled. The normal P--O--C--H
coupling produces a doublet in FL3P with a J value of 10.3 Hz,
whereas P--O--C-D has no coupling and produces a singlet both
coupled and decoupled, as was found for 3-deuterated FL3P. A unique
property of FL3P is that when treated with sodium borohydride it is
converted into two new resonances at 5.85 and 5.95 ppm, which
correspond to mannitol and sorbitol-lysine 3-phosphates.
EXAMPLE 2
Synthesis of FL3P
[0139] 1 mmol of dibenzyl-glucose 3-phosphate and 0.25 mmol of
.alpha.-carbobenzoxy-lysine was refluxed in 50 ml of MeOH for 3
hours. The solution was diluted with 100 ml water and
chromatographed on a Dow-50 column (2.5.times.20 cm) in the
pyridinium form and eluted first with water (200 ml) and then with
600 ml buffer (0.1M pyridine and 0.3M acetic acid). The target
compound eluted at the end of the water wash and the beginning of
the buffer wash. Removal of the cbz and benzyl blocking groups with
5% Pd/C at 20 psi of hydrogen gave FL3P in 6% yield.
EXAMPLE 3
Enzymatic Production of FL3P from FL and ATP and Assay for
Screening Inhibitors
[0140] Initially .sup.31P NMR was used to demonstrate kinase
activity in the kidney cortex. A 3 g. sample of fresh pig kidney
cortex was homogenized in 9 ml. of 50 mM Tris HCl containing 150 mM
KCl, 5 mM DTT, 15 mM MgCl.sub.21 pH 7.5. This was centrifuged at
10,000 g for 30 minutes, and then the supernate centrifuged at
100,000 g for 60 minutes. Ammonium sulfate was added to 60%
saturation. After 1 hour at 4.degree. the precipitate was collected
by centrifugation and dissolved in 5 ml. of original buffer. A 2 ml
aliquot of this solution was incubated with 10 mM ATP and 10 mM of
FL (prepared as in Example 1, above) for 2 hours at 370. The
reaction was quenched with 300 uL of perchloric acid, centrifuged
to remove protein, and desalted on a column of Sephadex G 10
(5.times.10 cm). .sup.31P NMR analysis of the reaction mixture
detected formation of FL3P.
[0141] Based on the proof of kinase activity thus obtained, a
radioactive assay was developed. This assay was designed to take
advantage of the lack of binding to Dow-1 anion exchange resin by
FL3P. This characteristic of FL3P was discovered during efforts to
isolate it. Since most phosphates bind to this resin, it was
suspected that the bulk of all compounds that react with ATP as
well as any excess ATP would be bound, leaving FL3P in solution.
The first step was to determine the amount of resin required to
remove the ATP in the assay. This was accomplished by pipetting the
mixture into a suspension of 200 mg. of Dow-1 in 0.9 ml H.sub.2O,
vortexing and centrifuging to pack the resin. From this 0.8 ml. of
supernate was pipetted onto 200 mg. of fresh dry resin, vortexed
and centrifuged. A 0.5 ml volume of supernate was pipetted into 10
ml of Ecoscint A and counted. Residual counts were 85 cpm. This
procedure was used for the assay. The precipitate from 60% ammonium
sulfate precipitation of the crude cortex homogenate was
redissolved in the homogenate buffer at 40. The assay contains 10
mM y.sup.33P-ATP (40,000 cpm), 10 mM FL, 150 mM KCl, 15 mM
MgCl.sub.2, 5 mM DTT in 0.1 ml of 50 mM TrisHCl, pH 7.5. The
relationship between rates of FL3P production and enzyme
concentration was determined using triplicate determinations with
1, 2 and 4 mg of protein for 30 minutes at 370. Blanks run
concurrently without FL were subtracted and the data recorded. The
observed activity corresponds to an approximate FL3P synthesis rate
of 20 nmols/hr./mg. protein.
EXAMPLE 4
Inhibition of the Formation of 3-Deoxyglucosone by Meglumine and
Various Polyollysines
[0142] a. General Polyollysine Synthesis.
[0143] The sugar (11 mmoles), .alpha.-carbobenzoxy-lysine (10
mmols) and NaBH.sub.3CN (15 mmoles) were dissolved in 50 ml of
MeOH--H.sub.2O (3:2) and stirred at 25.degree. for 18 hours. The
solution was treated with an excess of Dow-50 (H) ion exchange
resin to decompose excess NaBH.sub.3CN. This mixture (liquid plus
resin) was transferred onto a Dow-50 (H) column (2.5.times.15 cm)
and washed well with water to remove excess sugar and boric acid.
The carbobenzoxy-polyollysine was eluted with 5% NH.sub.4 OH. The
residue obtained upon evaporation was dissolved in water-methanol
(9:1) and reduced with hydrogen gas (20 psi) using a 10% palladium
on charcoal catalyst. Filtration and evaporation yields the
polyollysine.
[0144] B. Experimental Protocol for Reduction of Urinary and Plasma
3-Deoxyglucosone by Sorbitollysine, Mannitollysine and
Galactitollysine.
[0145] Urine was collected from six rats for three hours. A plasma
sample was also obtained. The animals were then given 10 .mu.mols
of either sorbitollysine, mannitollysine, or galactitollysine by
intraperitoneal injection. Urine was collected for another three
hours, and a plasma sample obtained at the end of the three
hours.
[0146] 3-deoxyglucosone was measured in these samples, as described
in Example 5, below, and variable volumes were normalized to
creatinine. The average reduction of urinary 3-deoxyglucosone was
50% by sorbitollysine, 35% by mannitollysine and 35% by
galactitollysine. Plasma 3-deoxyglucosone was reduced 40% by
sorbitollysine, 58% by mannitolysine and 50% by
galactitollysine.
[0147] c. Use of Meglumine to Reduce Urinary 3-Deoxyglucosone.
[0148] Three rats were treated as in b), immediately above, except
meglumine (100 .mu.mols) was injected intraperitoneally instead of
the above-mentioned lysine derivatives. Three hours after the
injection the average 3-deoxyglucosone concentrations in the urine
were decreased 42%.
EXAMPLE 5
Elevation of Urinary FL, 3DG and 3DF in Humans Following Ingestion
of Glycated Protein
[0149] a. Preparation of glycated protein containing food product:
260 g. of casein, 120 g. of glucose and 720 ml. of water were mixed
to give a homogeneous mixture. This mixture was transferred to a
metal plate and cooked at 650 for 68 hours. The resulting cake was
then pulverized to a coarse powder.
[0150] This powder contained 60% protein as determined by the
Kjeldahl procedure.
[0151] b. Measurement of glycated lysine content: 1 g of the powder
prepared as in step a., above, was hydrolyzed by refluxing with 6N
HCl for 20 hours. The resulting solution was adjusted to pH 1.8
with NaOH solution and diluted to 100 ml. The fructoselysine
content was measured on an amino acid analyzer as furosine, the
product obtained from acid hydrolysis of fructoselysine. In this
way, it was determined that the cake contained 5.5% (w/w)
fructoselysine.
[0152] c. Experimental protocol: Volunteers spent two days on a
fructoselysine-free diet and then consumed 22.5 g of the food
product prepared as described herein, thus effectively receiving a
2 g. dose of fructoselysine. Urine was collected at 2 hour
intervals for 14 hours and a final collection was made at 24
hours.
[0153] d. Measurement of FL, 3DG and 3DF in urine: FL was measured
by HPLC with a Waters 996 diode Array using a Waters C18 Free Amino
Acid column at 460 and a gradient elution system of
acetonitrile-methyl alcohol-water (45:15:40) into
acetonitrile-sodium acetate-water (6:2:92) at 1 ml./min.
Quantitation employed an internal standard of meglumine.
[0154] 3DF was measured by HPLC after deionization of the sample.
Analyses were performed on a Dionex DX-500 HPLC system employing a
PA1 column (Dionex) and eluting with 32 mM sodium hydroxide at 1
ml./min. Quantitation was performed from standard curves obtained
daily with synthetic 3DF.
[0155] 3DG was measured by GC-MS after deionization of the sample.
3DG was derivatized with a 10-fold excess of diaminonaphthalene in
PBS. Ethyl acetate extraction gave a salt free fraction which was
converted to the trimethyl silyl ethers with Tri-Sil (Pierce).
Analysis was performed on a Hewlett-Packard 5890 selected ion
monitoring GC-MS system. GC was performed on a fused silica
capillary column (DB-5,25 mx.25 mm) using the following temperature
program: injector port 250.degree., initial column temperature
150.degree. which is held for 1 minute, then increased to
290.degree. at 16.degree./minute and held for 15 minutes.
Quantitation of 3DG employed selected ion monitoring using an
internal standard of U-13C-3DG.
[0156] The graph shown in FIG. 3 represents production of FL, 3DF
and 3DG in the urine of one volunteer after consuming the glycated
protein. The rapid appearance of all three metabolites is clearly
evident. Both 3DF and 3DG show a slight elevation even after
twenty-four hours.
[0157] The graph shown in FIG. 4 represents the formation of 3DF in
each of the members of a seven person test group. A similar pattern
was seen in all cases. As appears in FIG. 4, 3DF excretion peaks
about 4 hours after the FL bolus and a slight elevation of 3DF is
noticeable even 24 h after the bolus.
EXAMPLE 6
Feeding Experiment
[0158] N-acetyl-.beta.-glucosaminidase (NAGase) is an enzyme
excreted into the urine in elevated concentration in diabetics. It
is thought to be an early marker of tubular damage, but the
pathogenesis of increased NAGase in urine is not well understood.
The increased urinary output of NAGase in diabetics has been
proposed to be due to activation of lysosomes in proximal tubules
induced by diabetes with an increased output into the urine rather
than destruction of cells.
[0159] The results obtained in this example show that in all
comparisons 3DF and NAGase levels are elevated in the experimental
group relative to the control. Thus, animals fed glycated protein
excrete excess NAGase into their urine, similar to results obtained
with diabetics. There is an approximate 50% increase in NAGase
output compared with control animals. These animals also have a
five-fold increase in urine 3DF compared with controls. Urinary 3DF
correlates extremely well with 3DG, as can be seen in FIGS. 5 and
6. Both compounds appear to be removed from the plasma at the
glomerular filtration rate, with no reabsorption.
EXAMPLE 7
SDS Gel of Kidney Proteins
[0160] Two rats were injected daily with 5 .mu.mols. of either FL
or mannitol (used as a control) for 5 days. The animals were
sacrificed and the kidneys removed and dissected into the cortex
and medulla. Tissues were homogenized in 5 volumes of 50 mM TrisHCl
containing 150 mM KCl, 15 mM MgCl.sub.2 and 5 mM DTT, pH 7.5.
Cellular debris was removed by centrifugation at 10,000 g for 15
minutes, and the supernate was then centrifuged at 150,000 g for 70
minutes. The soluble proteins were analyzed by SDS PAGE on 12%
polyacrylamide gels as well as on 4-15 and 10-20% gradient gels. In
all cases, lower molecular weight bands were missing or visually
reduced from the kidney extract of the animal injected with FL when
compared with the animal injected with mannitol.
EXAMPLE 8
Synthesis of 3-O-Methylfructose Lysine
[0161] A suspension of 19.4 g (0.1 mol) of anhydrous 3-O-methyl
glucose and 1 g of sodium bisulfite in 30 ml of methanol and 15 ml
of glycerol was refluxed for 30 minutes, followed by the addition
of 0.035 mol of .alpha.-carbobenzoxy-lysine and 4 ml of acetic
acid. This solution was refluxed for 3 hours. The solution was
treated with 1 volume of water and chromatographed on a Dowex-50
column (4.times.50 cm) in the pyridinium form, and eluted first
with water and then with pyridinium acetate. Fractions containing
the pure material were combined and evaporated. The resulting
material was dissolved in 50 ml of water-methanol (9:1) and reduced
with hydrogen gas (20 psi) using a 10% palladium on charcoal
catalyst. Filtration and evaporation gave
3-O-methyl-fructoselysine.
[0162] Other specific compounds having the structure of formula
(I), above, may be made e.g. by glycation of a selected nitrogen-
or oxygen-containing starting material, which maybe an amino acid,
polyaminoacid, peptide or the like, with a glycating agent, such as
fructose, which may be chemically modified, if desired, according
to procedures well know to those skilled in the art.
EXAMPLE 9
Additional Assay for FL3P Kinase Activity
[0163] a. Preparation of Stock Solutions:
[0164] An assay buffer solution was prepared which was 100 mM HEPES
pH 8.0, 10 mM ATP, 2 mM MgCl.sub.2, 5 mM DTT, 0.5 mM PMSF. A
fructosyl-spermine stock solution was prepared which was 2 mM
fructosyl-spermine Hcl. A spermine control solution was prepared
which was 2 mM spermine Hcl.
[0165] b. Synthesis of Fructosyl-spermine:
[0166] Synthesis of fructosyl-spermine was performed by an
adaptation of a known procedure (J. Hodge and B. Fisher, Methods
Carbohydr. Chem., 2: 99-107 (1963)). A mixture of spermine (500
mg), glucose (500 mg) and sodium pyrosulfite (80 mg) was prepared
in a molar ratio of 8:4:1 (spermine:glucose:pyrosulfite) in 50 ml
of methanol-water (1:1) and refluxed for 12 hours. The product was
diluted to 200 ml with water and loaded onto a DOW-50 column
(5.times.90 cm). The unreacted glucose was removed by 2 column
volumes of water and the product and unreacted spermine were
removed with 0.1 M NH.sub.4OH. Pooled peak fractions of the product
were lyophilized and concentration of fructosyl-spermine was
determined by measuring the integral of the C-2 fructosyl peak in a
quantitative .sup.13C NMR spectrum of the product (NMR data
collected with a 450 pulse, a 10 second relaxation delay and
without NOE decoupling).
[0167] c. Assay of Kinase for Purification:
[0168] An incubation mixture was prepared including 10 .mu.l of the
enzyme preparation, 10 .mu.l of assay buffer, 1.0 .mu.Ci of
.sup.33P ATP, 10 .mu.l of fructosyl-spermine stock solution and 70
.mu.l of water and incubated at 37.degree. C. for 1 hour. At the
end of the incubation 90 .mu.l (2.times.45 .mu.l) of the sample is
spotted onto two 2.5 cm diameter cellulose phosphate disks (Whatman
P-81) and allowed to dry. The disks were washed extensively with
water. After drying, the disks were placed in scintillation vials
and counted.
[0169] Each enzyme fraction was assayed in duplicate with an
appropriate spermine control.
EXAMPLE 10
Kidney Pathology Observed in Test Animals on Glycated Protein
Diet
[0170] Three rats were maintained on a glycated protein diet (20%
total protein; 3% glycated) for 8 months and compared to 9 rats of
the same age maintained on a control diet. The primary finding was
a substantial increase in damaged glomeruli in the animals on the
glycated diet. Typical lesions observed in these animals were
segmental sclerosis of the glomerular tuft with adhesion to
Bowman's capsule, tubular metaplasia of the parietal epithelium and
intestitial fibrosis. All three of the animals on the glycated
protein diet, and only one of the animals on the control diet
showed more than 13% damaged glomeruli. The probablity of this
happening by chance is less than 2%. In addition to the pathology
observed in the glomeruli, a number of hylinated casts within
tubules were observed. More of these were found in animals on the
glycated diet, although these were not quantitated. Increased
levels of NAGase were also observed in the animals on the glycated
diet.
[0171] From the results of this experiment, the glycated diet
appeared to cause the test animals to develop a series of
histological lesions similar to those seen in the diabetic
kidney.
EXAMPLE 11
Effects of Glycated Diets on Pregnancy
[0172] In a preliminary experiment, 5 mice pairs were placed on a
glycated diet (18% total protein; 3% glycated) and bred six times
over a period of 7 months. The resulting six pregnancies produced
the following live pups; 17, 23, 13, 0, 3 and 0. In view of this
sharp drop in live pups after the third breeding, two cohorts of
ten pairs each were put on either a glycated diet (13% total
protein; 3% glycated) or a control diet (13% total protein; 0%
glycated). Thus far, the two groups of pups have been bred four
times obtaining similar results in both groups. The first pregnancy
produced 49/20 (glycated/control) pups; the second, 18/41; the
third 37/27; and the fourth 20/33. The fifth pregnancy is currently
underway. The mice pairs have been tested for hyperglycemia. The
blood glucose levels are 120 and 112 mg/dl in the experimental and
control groups, respectively.
[0173] Preliminary measurements of the 3DF levels in the mice urine
indicate, as expected, a substantial elevation (approximately 5-10
fold) of the systemic 3DF when on the glycated diet described
herein.
EXAMPLE 12
Carcinogenic Effects of Fructoselysine Pathway
[0174] To investigate the carcinogenic potential of metabolites
formed in the fructoselysine pathway, experiments have been
conducted on a strain of rats with a high susceptibility to kidney
carcinomas. Four rats were put on a glycated protein diet and three
rats on a control diet. After ten weeks on the diet, the animals
were sacrificed and their kidneys examined. In all four animals on
the diet, kidney carcinomas of size greater than 1 mm were found,
whereas no lesions this large were found in the control animals.
The probability of this happening by chance is less than 2%. The
data show that the elevated 3DG levels caused by the excess
fructoselysine coming from the glycated protein in the animals diet
found in the kidney tubular cells (known to be the cell of origin
of most kidney carcinomas) can interact with the cellular DNA
leading to a variety of mutogenic and ultimately carcinogenic
events. The possibility exists that this process is important in
the development of human cancers in the kidney and elsewhere.
EXAMPLE 13
Dietary Effects of Glycated Protein Diet on Renal Cell Carcinoma in
Susceptible Rats
[0175] In addition experiments assessing the relationship between a
glycated protein diet and renal cell carcinoma, twenty-eight rats
with a mutation making them susceptible to the development of
kidney carcinoma were divided into two cohorts. One cohort was fed
a glycated protein diet: the other cohort was on a control diet.
The glycated protein diet consisted of a standard nutritious diet
to which 3% glycated protein had been added. The glycated protein
was made by mixing together casein and glucose (2:1), adding water
(2.times. the weight of the dried material), and baking the mixture
at 60.degree. for 72 hours. The control was prepared in the same
way except that no water was used and the casein and glucose were
not mixed prior to baking. Rats were placed on the diets
immediately following weaning at three weeks of age and maintained
on the diets ad libitum for the next 16 weeks. The animals were
then sacrificed, the kidneys fixed and hemotoxylin and eosin
sections were made. These were examined for lesions by a trained
pathologist. Four types of lesions were identified. These included:
cysts, very small collections of tumor-like cells, typically less
than 10 cells; small tumors, 0.5 mm or less, and tumors greater
than 0.5 mm. For every type, more lesions were observed in the
animals on the glycated diet than on the control diet as shown in
the following table. TABLE-US-00003 CYSTS .ltoreq.10 CELLS
.ltoreq.0.5 mm >0.5 mm TOTAL CONTROL 2 9 9 3 23 GLYCATED 9 21 32
6 68
[0176] To summarize the results, the average number of lesions per
kidney section was computed for each diet. These were 0.82.+-.0.74
and 2.43.+-.2.33 in the control and glycated diet, respectively.
The likelihood of this happening by chance is about 2 in
100,000.
[0177] These results provide strong support for the premise that
the effects of the lysine recovery pathway, the discovery of which
underlies the present invention, extend to causing mutations, and
thus produce a carcinogenic effect as well. These results provide a
basis for the development of therapeutic methods and agents to
inhibit this pathway in order to reduce cancer in the kidney as
well as in other organs where this pathway may have similar
effects.
EXAMPLE 14
Urinary Excretion of 3-Deoxy-Fructose is Indicative of Progression
to Microalbuminuria in Patients with Type I Diabetes
[0178] As set forth hereinabove, serum levels of the glycation
intermediate, three deoxy-glucosone (3DG) and its reductive
detoxification product, three deoxy-fructose (3DF), are elevated in
diabetes. The relationship between baseline levels of these
compounds and subsequent progression of microalbuminura (MA) has
been examined in a group of 39 individuals from a prospective
cohort of patients at the Joslin Diabetes Center with
insulin-dependent diabetes mellitus (IDDM) and microalbuminuria
(based on multiple measurements during the two years of baseline
starting between 1990-1993) and not on ACE inhibitors.
[0179] Baseline levels of 3DF and 3DG in random spot urines were
measured by HPLC and GC-MS. Individuals that progressed to either a
higher level of MA or proteinuria in the next four years (n=24) had
significantly higher baseline levels of log 3DF/urinary creatinine
ratios compared to non-progressors (n=15) (p=0.02). Baseline levels
determined in this study were approximately 0.24 .mu.mole/mg of
creatinine in the progressors vs. approximately 0.18 .mu.mole/mg of
creatinine ratios in the non-progressors. Baseline 3DG/urine
creatinine ratios did not differ between the groups. Adjustment of
the baseline level of HgA.sub.Ic (the major fraction of
glycoslyated hemoglobin) did not substantially alter these
findings. These results provide additional evidence of the
association between urinary 3DF and progression of kidney
complications on diabetes.
[0180] A. Quantification of 3-Deoxyfructose
[0181] Samples were processed by passing a 0.3 mL aliquot of the
test sample through an ion-exchange column containing 0.15 mL of AG
1-X8 and 0.15 mL of AG 50W-X8 resins. The columns were then washed
twice with 0.3 mL deionized water, aspirated to remove free liquid
and filtered through a 0.45 mm Millipore filter.
[0182] Injections (50 .mu.L) of the treated samples were analyzed
using a Dionex DX 500 chromatography system. A carbopac PA1
anion-exchange column was employed with an eluant consisting of 16%
sodium hydroxide (200 mM) and 84% deionized water. 3DF was detected
electrochemically using a pulsed amperometric detector. Standard
3DF solutions spanning the anticipated 3DF concentrations were run
both before and after each unknown sample.
[0183] B. Measurement of Urine Creatinine
[0184] Urine creatinine concentrations were determined by the
end-point colormetric method (Sigma Diagnostic kit 555-A) modified
for use with a plate reader. Creatinine concentrations were
assessed to normalize urine volumes for measuring metabolite levels
present therein.
[0185] C. Measurement of Albumin in the Urine
[0186] To assess albumin levels in the urine of the test subjects,
spot urines were collected and immunoephelometry performed on a BN
100 apparatus with the N-albumin kit (Behring). Anti-albumin
antibodies are commercially available. Albumin levels in urine may
be assessed by any suitable assay including but not limited to
ELISA assays, radioimmunoassays, Western and dot blotting.
[0187] Based on the data obtained in the study of the Joslin
Diabetes Center patients, it appears that elevated levels of
urinary 3DF are associated with progression to microalbuminuria in
diabetes. This observation provides a new diagnostic parameter for
assessing the likelihood of progression to serious kidney
complications in patients afflicted with diabetes.
EXAMPLE 15
3-O-Methyl Sorbitollysine Lowers Systemic Levels of 3DG in Normal
and Diabetic Rats
[0188] A cohort of twelve diabetic rats was divided into two groups
of six. The first group received saline-only injections, and the
second received injections of 3-O-methyl sorbitollysine in saline
solution. The same procedure was conducted on a cohort of twelve
non-diabetic rats. As summarized in Table C, within one week, the
3-O-methyl sorbitollysine treatment significantly reduced the
plasma 3DG levels as compared to the respective saline controls in
both diabetic and non-diabetic rats. TABLE-US-00004 TABLE C
3-O-Methyl sorbitollysine reduces plasma 3DG levels in diabetic and
non-diabetic rats. Diabetic Rats Non-diabetic Rats Plasma, Day 8
Plasma, Day 8 Control (n = 6) 0.94 .+-. 0.28 .mu.M 0.23 .+-. 0.07
.mu.M 3-O-methyl 0.44 .+-. 0.10 .mu.M 0.13 .+-. 0.02 .mu.M
Sorbitollysine (n = 6) Percent 53% 43% reduction t-test P = 0.0006
P = 0.0024
[0189] The ability of 3-O-methyl sorbitollysine to reduce systemic
3DG levels suggests that diabetic complications other than
nephropathy (e.g., retinopathy and stiffening of the aorta) may
also be controllable by Amadorase inhibitor therapy.
EXAMPLE 16
Locus of 3-O-Methyl Sorbitollysine Uptake In Vivo is the Kidney
[0190] Six rats were injected intraperitoneally with 13.5 mmoles
(4.4 mg) of 3-O-methyl sorbitollysine. The rats' urine was
collected for 3 hours, after which the rats were sacrificed. The
tissues to be analyzed were removed and freeze clamped in liquid
nitrogen. Perchloric acid extracts of the tissues were used for
metabolite analysis. The tissues examined were taken from the
brain, heart, muscle, sciatic nerve, spleen, pancreas, liver and
kidney. Plasma and urine were also analyzed.
[0191] The only tissue extract found to contain 3-O-methyl
sorbitollysine was that of the kidney. The urine also contained
3-O-methyl sorbitollysine, but plasma did not. The percentage of
the injected dose recovered from urine and kidney varied between 39
and 96%, as shown in Table D, below. TABLE-US-00005 TABLE D nmols
nmols nmols total % 3OMeSL* 3OMeSL 3OMeSL 3OMeSL 3OMeSL Rat #
Injected in urine in kidneys recovered recovered 2084 13500 2940
10071 13011 96.4 2085 13500 1675 6582 8257 61.2 2086 13500 1778
5373 7151 53.0 2087 13500 2360 4833 7193 53.3 2088 13500 4200 8155
12355 91.5 2089 13500 1355 3880 5235 38.8 *3-O-methyl
sorbitollysine
[0192] While certain embodiments of the present invention have been
described and/or exemplified above, various other embodiments will
be apparent to those skilled in the art from the foregoing
disclosure. The present invention is, therefore, not limited to the
particular embodiments described and/or exemplified, but is capable
of consideration variation and modification without departure from
the scope of the appended claims.
* * * * *