U.S. patent application number 11/708232 was filed with the patent office on 2007-07-05 for advanced glycation end-product intermediaries and post-amadori inhibition.
This patent application is currently assigned to University of Kansas Medical Center. Invention is credited to Aaron Ashley Booth, Billy G. Hudson, Raja Gabriel Khalifah, Parvin Todd.
Application Number | 20070155801 11/708232 |
Document ID | / |
Family ID | 25518160 |
Filed Date | 2007-07-05 |
United States Patent
Application |
20070155801 |
Kind Code |
A1 |
Hudson; Billy G. ; et
al. |
July 5, 2007 |
Advanced glycation end-product intermediaries and post-amadori
inhibition
Abstract
The instant invention provides compositions and methods for
modeling post-Amadori AGE formation and the identification and
characterization of effective inhibitors of post-Amadori AGE
formation, and such identified inhibitor compositions.
Inventors: |
Hudson; Billy G.; (Omaha,
AR) ; Todd; Parvin; (US) ; Khalifah; Raja
Gabriel; (Cary, NC) ; Booth; Aaron Ashley;
(Prairie Village, KS) |
Correspondence
Address: |
MCDONNELL BOEHNEN HULBERT & BERGHOFF LLP
300 S. WACKER DRIVE
32ND FLOOR
CHICAGO
IL
60606
US
|
Assignee: |
University of Kansas Medical
Center
|
Family ID: |
25518160 |
Appl. No.: |
11/708232 |
Filed: |
February 20, 2007 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10214540 |
Aug 8, 2002 |
|
|
|
11708232 |
Feb 20, 2007 |
|
|
|
09723770 |
Nov 28, 2000 |
6472411 |
|
|
10214540 |
Aug 8, 2002 |
|
|
|
08971285 |
Nov 17, 1997 |
6228858 |
|
|
09723770 |
Nov 28, 2000 |
|
|
|
08711555 |
Sep 10, 1996 |
5985857 |
|
|
08971285 |
Nov 17, 1997 |
|
|
|
Current U.S.
Class: |
514/349 ;
514/350; 546/290; 546/298 |
Current CPC
Class: |
A61K 31/44 20130101;
C07K 5/06052 20130101; C07C 215/50 20130101; A61K 38/06 20130101;
A61K 31/675 20130101; A61P 43/00 20180101; A61K 31/506 20130101;
C07D 213/66 20130101; A61K 31/04 20130101; A61K 38/07 20130101;
G01N 2400/02 20130101; A61K 31/4415 20130101; G01N 33/6842
20130101; A61K 38/05 20130101; C07K 5/06034 20130101; A61K 31/435
20130101; A61K 31/00 20130101; C07K 5/0812 20130101; A61P 13/12
20180101; G01N 33/68 20130101; G01N 2500/04 20130101 |
Class at
Publication: |
514/349 ;
514/350; 546/290; 546/298 |
International
Class: |
A61K 31/4415 20060101
A61K031/4415; C07D 213/78 20060101 C07D213/78 |
Goverment Interests
STATEMENT OF GOVERNMENT RIGHTS
[0002] Some of the work disclosed has been supported in part by NIH
Grant DK 43507, therefore, the United States Government may have
certain rights in the invention.
Claims
1. A compound of the general formula: ##STR10## wherein R.sub.1 is
CH.sub.2NH.sub.2, CH.sub.2SH, COOH, CH.sub.2CH.sub.2NH.sub.2,
CH.sub.2CH.sub.2SH, or CH.sub.2COOH; R.sub.2 and R.sub.6 is H, OH,
SH, NH.sub.2, C 1-6 alkyl, alkoxy or alkene; R.sub.4 and R.sub.5
are H, C 1-6 alkyl, alkoxy or alkene; Y is N or C, such that when Y
is N R.sub.3 is nothing, and when Y is C, R.sub.3 is NO.sub.2 or
another electron withdrawing group, and salts thereof, wherein said
compound is not pyridoxamine.
2. A pharmaceutical composition comprising a compound of claim 1,
or salt thereof in a suitable carrier.
3. A method for inhibiting post-Amadori AGE formation comprising
administering an effective post-Amadori AGE inhibiting amount of a
compound of claim 1.
4. A method for treating a patient with AGE related pathology
comprising administering an effective therapeutic amount of a
compound of claim 1.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/214,540 filed Aug. 6, 2002, which is a
continuation of U.S. patent application Ser. No. 09/723,770 filed
Nov. 28, 2000, now U.S. Pat. No. 6,472,411, which is a continuation
of U.S. patent application Ser. No. 08/971,285 filed Nov. 17, 1997,
now U.S. Pat. No. 6,228,858, which is a continuation-in-part of
U.S. patent application Ser. No. 08/711,555, filed Sep. 10, 1996,
now U.S. Pat. No. 5,985,857, which claims priority to U.S.
Provisional Application for Patent Ser. No. 60/003,268, filed Sep.
12, 1995, the contents of each of which are hereby incorporated by
reference in their entirety.
BACKGROUND OF THE INVENTION
[0003] The instant invention is in the field of Advanced Glycation
End-products (AGEs), their formation, detection, identification,
inhibition, and inhibitors thereof.
Protein Aging and Advanced Glycosylation End-products
[0004] Nonenzymatic glycation by glucose and other reducing sugars
is an important post-translational modification of proteins that
has been increasingly implicated in diverse pathologies.
Irreversible nonenzymatic glycation and crosslinking through a
slow, glucose-induced process may mediate many of the complications
associated with diabetes. Chronic hyperglycemia associated with
diabetes can cause chronic tissue damage which can lead to
complications such as retinopathy, nephropathy, and atherosclerotic
disease. (Cohen and Ziyadeh, 1996, J. Amer. Soc. Nephrol.
7:183-190). It has been shown that the resulting chronic tissue
damage associated with long-term diabetes mellitus arise in part
from in situ immune complex formation by accumulated
immunoglobulins and/or antigens bound to long-lived structural
proteins that have undergone Advanced Glycosylation End-product
(AGE) formation, via non-enzymatic glycosylation (Brownlee et al.,
1983, J. Exp. Med. 158:1739-1744). The primary protein target is
thought to be extra-cellular matrix associated collagen.
Nonenzymatic glycation of proteins, lipids, and nucleic acids may
play an important role in the natural processes of aging. Recently
protein glycation has been associated with .beta.-amyloid deposits
and formation of neurofibrillary tangles in Alzheimer disease, and
possibly other neurodegenerative diseases involving amyloidosis
(Colaco and Harrington, 1994, NeuroReport 5: 859-861). Glycated
proteins have also been shown to be toxic, antigenic, and capable
of triggering cellular injury responses after uptake by specific
cellular receptors (see for example, Vlassara, Bucala &
Striker, 1994, Lab. Invest. 70:138-151; Vlassara et al., 1994,
PNAS(USA) 91:11704-11708; Daniels & Hauser, 1992, Diabetes
41:1415-1421; Brownlee, 1994, Diabetes 43:836-841; Cohen et al.,
1994, Kidney Int. 45:1673-1679; Brett et al., 1993, Am. J. Path.
143:1699-1712; and Yan et al., 1994, PNAS(USA) 91:7787-7791).
[0005] The appearance of brown pigments during the cooking of food
is a universally recognized phenomenon, the chemistry of which was
first described by Maillard in 1912, and which has subsequently led
to research into the concept of protein aging. It is known that
stored and heat-treated foods undergo nonenzymatic browning that is
characterized by crosslinked proteins which decreases their
bioavailibility. It was found that this Maillard reaction occurred
in vivo as well, when it was found that a glucose was attached via
an Amadori rearrangement to the amino-terminal of the .alpha.-chain
of hemoglobin.
[0006] The instant disclosure teaches previously unknown, and
unpredicted mechanism of formation of post-Amadori advanced
glycation end products (Maillard products; AGEs) and methods for
identifying and characterizing effective inhibitors of post-Amadori
AGE formation. The instant disclosure demonstrates the unique
isolation and kinetic characterization of a reactive protein
intermediate competent in forming post-Amadori AGEs, and for the
first time teaching methods which allow for the specific
elucidation and rapid quantitative kinetic study of "late" stages
of the protein glycation reaction.
[0007] In contrast to such "late" AGE formation, the "early" steps
of the glycation reaction have been relatively well characterized
and identified for several proteins (Harding, 1985, Adv. Protein
Chem. 37:248-334; Monnier & Baynes eds., 1989, The Maillard
Reaction in Aging, Diabetes, and Nutrition (Alan R. Liss, New
York); Finot et al., 1990, eds. The Maillard Reaction in Food
Processing, Human Nutrition and Physiology (Birkhauser Verlag,
Basel)). Glycation reactions are known to be initiated by
reversible Schiff-base (aldimine or ketimine) addition reactions
with lysine side-chain .epsilon.-amino and terminal .alpha.-amino
groups, followed by essentially irreversible Amadori rearrangements
to yield ketoamine products e.g. 1-amino-1-deoxy-ketoses from the
reaction of aldoses (Baynes et al., 1989, in The Maillard Reaction
in Aging, Diabetes, and Nutrition, ed. Monnier and Baynes, (Alan R.
Liss, New York, pp 43-67). Typically, sugars initially react in
their open-chain (not the predominant pyranose and furanose
structures) aldehydo or keto forms with lysine side chain
.epsilon.-amino and terminal .alpha.-amino groups through
reversible Schiff base condensation (Scheme I). The resulting
aldimine or ketimine products then undergo Amadori rearrangements
to give ketoamine Amadori products, i.e. 1-amino-1-deoxy-ketoses
from the reaction of aldoses (Means & Chang, 1982, Diabetes 31,
Suppl. 3:1-4; Harding, 1985, Adv. Protein Chem. 37:248-334). These
Amadori products then undergo, over a period of weeks and months,
slow and irreversible Maillard "browning" reactions, forming
fluorescent and other products via rearrangement, dehydration,
oxidative fragmentation, and cross-linking reactions. These
post-Amadori reactions, (slow Maillard "browning" reactions), lead
to poorly characterized Advanced Glycation End-products (AGEs).
[0008] As with Amadori and other glycation intermediaries, free
glucose itself can undergo oxidative reactions that lead to the
production of peroxide and highly reactive fragments like the
dicarbonyls glyoxal and glycoaldehyde. Thus the elucidation of the
mechanism of formation of a variety of AGEs has been extremely
complex since most in vitro studies have been carried out at
extremely high sugar concentrations.
[0009] In contrast to the relatively well characterized formation
of these "early" products, there has been a clear lack of
understanding of the mechanisms of forming the "late" Maillard
products produced in post-Amadori reactions, because of their
heterogeneity, long reaction times, and complexity. The lack of
detailed information about the chemistry of the "late" Maillard
reaction stimulated research to identify fluorescent AGE
chromophores derived from the reaction of glucose with amino groups
of polypeptides. One such chromophore,
2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole (FFI) was identified
after nonenzymatic browning of bovine serum albumin and polylysine
with glucose, and postulated to be representative of the
chromophore present in the intact polypeptides. (Pongor et al.,
1984, PNAS(USA) 81:2684-2688). Later studies established FFI to be
an artifact formed during acid hydrolysis for analysis.
[0010] A series of U.S. Patents have issued in the area of
inhibition of protein glycosylation and cross-linking of protein
sugar amines based upon the premise that the mechanism of such
glycosylation and cross-linking occurs via saturated glycosylation
and subsequent cross-linking of protein sugar amines via a single
basic, and repeating reaction. These patents include U.S. Pat. Nos.
4,665,192; 5,017,696; 4,758,853; 4,908,446; 4,983,604; 5,140,048;
5,130,337; 5,262,152; 5,130,324; 5,272,165; 5,221,683; 5,258,381;
5,106,877; 5,128,360; 5,100,919; 5,254,593; 5,137,916; 5,272,176;
5,175,192; 5,218,001; 5,238,963; 5,358,960; 5,318,982; and
5,334,617. (All U.S. Patents cited are hereby incorporated by
reference in their entirety).
[0011] The focus of these U.S. Patents, are a method for inhibition
of AGE formation focused on the carbonyl moiety of the early
glycosylation Amadori product, and in particular the most effective
inhibition demonstrated teaches the use of exogenously administered
aminoguanidine. The effectiveness of aminoguanidine as an inhibitor
of AGE formation is currently being tested in clinical trials.
[0012] Inhibition of AGE formation has utility in the areas of, for
example, food spoilage, animal protein aging, and personal hygiene
such as combating the browning of teeth. Some notable, though
quantitatively minor, advanced glycation end-products are
pentosidine and N.epsilon.-carboxymethyllysine (Sell and Monnier,
1989, J. Biol. Chem. 264:21597-21602; Ahmed et al., 1986, J. Biol.
Chem. 261:4889-4894).
[0013] The Amadori intermediary product and subsequent post-Amadori
AGE formation, as taught by the instant invention, is not fully
inhibited by reaction with aminoguanidine. Thus, the formation of
post-Amadori AGEs as taught by the instant disclosure occurs via an
important and unique reaction pathway that has not been previously
shown, or even previously been possible to demonstrate in
isolation. It is a highly desirable goal to have an efficient and
effective method for identifying and characterizing effective
post-Amadori AGE inhibitors of this "late" reaction. By providing
efficient screening methods and model systems, combinatorial
chemistry can be employed to screen candidate compounds
effectively, and thereby greatly reducing time, cost, and effort in
the eventual validation of inhibitor compounds. It would be very
useful to have in vivo methods for modeling and studying the
effects of post-Amadori AGE formation which would then allow for
the efficient characterization of effective inhibitors.
[0014] Inhibitory compounds that are biodegradable and/or naturally
metabolized are more desirable for use as therapeutics than highly
reactive compounds which may have toxic side effects, such as
aminoguanidine.
SUMMARY OF THE INVENTION
[0015] In accordance with the present invention, a stable
post-Amadori advanced glycation end-product (AGE) precursor has
been identified which can then be used to rapidly complete the
post-Amadori conversion into post-Amadori AGEs. This stable product
is a presumed sugar saturated Amadori/Schiff base product produced
by the further reaction of the early stage protein/sugar Amadori
product with more sugar. In a preferred embodiment, this
post-Amadori/Schiff base intermediary has been generated by the
reaction of target protein with ribose sugar.
[0016] The instant invention provides for a method of generating
stable protein-sugar AGE formation intermediary precursors via a
novel method of high sugar inhibition. In a preferred embodiment
the sugar used is ribose.
[0017] The instant invention provides for a method for identifying
an effective inhibitor of the formation of late Maillard products
comprising: generating stable protein-sugar post-Amadori advanced
glycation end-product intermediates by incubating a protein with
sugar at a sufficient concentration and for sufficient length of
time to generate stable post-Amadori AGE intermediates; contacting
said stable protein-sugar post-Amadori advanced glycation
end-product intermediates with an inhibitor candidate; identifying
effective inhibition by monitoring the formation of post-Amadori
AGEs after release of the stable protein-sugar post-Amadori
advanced glycation end-product intermediates from sugar induced
equilibrium. Appropriate sugars include, and are not limited to
ribose, lyxose, xylose, and arabinose. It is believed that certain
conditions will also allow for use of glucose and other sugars. In
a preferred embodiment the sugar used is ribose.
[0018] The instant invention teaches that an effective inhibitor of
post-Amadori AGE formation via "late" reactions can be identified
and characterized by the ability to inhibit the formation of
post-Amadori AGE endproducts in an assay comprising; generating
stable protein-sugar post-Amadori advanced glycation end-product
intermediates by incubating a protein with sugar at a sufficient
concentration and for sufficient length of time to generate stable
post-Amadori AGE intermediates; contacting said stable
protein-sugar post-Amadori advanced glycation end-product
intermediates with an inhibitor candidate; identifying effective
inhibition by monitoring the formation of post-Amadori AGEs after
release of the stable protein-sugar post-Amadori advanced glycation
end-product intermediates from sugar induced equilibrium. In a
preferred embodiment the assay uses ribose.
[0019] Thus the methods of the instant invention allow for the
rapid screening of candidate post-Amadori AGE formation inhibitors
for effectiveness, greatly reducing the cost and amount of work
required for the development of effective small molecule inhibitors
of post-Amadori AGE formation. The instant invention teaches that
effective inhibitors of post-Amadori "late" reactions of AGE
formation include derivatives of vitamin B.sub.6 and vitamin
B.sub.1, in the preferred embodiment the specific species being
pyridoxamine, pyridoxamine-5'-phosphate, and thiamine
pyrophosphate.
[0020] The instant invention teaches new methods for rapidly
inducing diabetes like pathologies in rats comprising administering
ribose to the subject animal. Further provided for is the use of
identified inhibitors pyridoxamine, pyridoxamine-5'-phosphate, and
thiamine pyrophosphate in vivo to inhibit post-Amadori AGE induced
pathologies.
[0021] The present invention encompasses compounds for use in the
inhibition of AGE formation and post-Amadori AGE pathologies, and
pharmaceutical compositions containing such compounds of the
general formula: ##STR1## wherein R.sub.1 is CH.sub.2NH.sub.2,
CH.sub.2SH, COOH, CH.sub.2CH.sub.2NH.sub.2, CH.sub.2CH.sub.2SH, or
CH.sub.2COOH; [0022] R.sub.2 is OH, SH or NH.sub.2; [0023] Y is N
or C, such that when Y is N R.sub.3 is nothing, and when Y is C,
R.sub.3 is NO.sub.2 or another electron withdrawing group; and
salts thereof.
[0024] The present invention also encompasses compounds of the
general formula ##STR2## wherein R.sub.1 is CH.sub.2NH.sub.2,
CH.sub.2SH, COOH, CH.sub.2CH.sub.2NH.sub.2, CH.sub.2CH.sub.2SH, or
CH.sub.2COOH; [0025] R.sub.2 is OH, SH or NH.sub.2; [0026] Y is N
or C, such that when Y is N R.sub.3 is nothing, and when Y is C,
R.sub.3 is NO.sub.2 or another electron withdrawing group; [0027]
R.sub.4 is H, or C 1-6 alkyl; [0028] R.sub.5 and R.sub.6 are H, C
1-6 alkyl; and salts thereof.
[0029] In a preferred embodiment at least one of R.sub.4, R.sub.5
and R.sub.6 are H. The present invention also encompasses compounds
wherein R.sub.4 and R.sub.5 are H, C 1-6 alkyl, alkoxy or alkene.
In keeping with the present invention, it is also encompassed that
R.sub.2 and R.sub.6 can be H, OH, SH, NH.sub.2, C 1-6 alkyl, alkoxy
or alkene. It is also envisioned that R.sub.4, R.sub.5 and R.sub.6
can be larger functional groups, such as and not limited to aryl,
heteroaryl, and cycloalkyl alkoxy groups.
[0030] In addition, the instant invention also envisions compounds
of the formulas ##STR3##
[0031] The compounds of the present invention can embody one or
more electron withdrawing groups, such as and not limited to
--NH.sub.2, --NHR, --NR.sub.2, --OH, --OCH.sub.3, --OCR, and
--NH--COCH.sub.3 where R is C 1-6 alkyl.
[0032] The instant invention encompasses pharmaceutical
compositions which comprise one or more of the compounds of the
present invention, or salts thereof, in a suitable carrier. The
instant invention encompasses methods for administering
pharmaceuticals of the present invention for therapeutic
intervention of pathologies which are related to AGE formation in
vivo. In one preferred embodiment of the present invention the AGE
related pathology to be treated is related to diabetic
nephropathy.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIG. 1 is a series of graphs depicting the effect of vitamin
B.sub.6 derivatives on AGE formation in bovine serum albumin (BSA).
FIG. 1A Pyridoxamine (PM); FIG. 1B pyridoxal phosphate (PLP); FIG.
1C pyridoxal (PL); FIG. 1D pyridoxine (PN).
[0034] FIG. 2 is a series of graphs depicting the effect of vitamin
B.sub.1 derivatives and aminoguanidine (AG) on AGE formation in
bovine serum albumin. FIG. 2A Thiamine pyrophosphate (TPP); FIG. 2B
thiamine monophosphate (TP); FIG. 2C thiamine (T); FIG. 2D
aminoguanidine (AG).
[0035] FIG. 3 is a series of graphs depicting the effect of vitamin
B.sub.6 derivatives on AGE formation in human methemoglobin (Hb).
FIG. 3A Pyridoxamine (PM); FIG. 3B pyridoxal phosphate (PLP); FIG.
3C pyridoxal (PL); FIG. 3D pyridoxine (PN).
[0036] FIG. 4 is a series of graphs depicting the effect of vitamin
B.sub.1 derivatives and aminoguanidine (AG) on AGE formation in
human methemoglobin. FIG. 2A Thiamine pyrophosphate (TPP); FIG. 2B
thiamine monophosphate (TP); FIG. 2C thiamine (T); FIG. 2D
aminoguanidine (AG).
[0037] FIG. 5 is a bar graph comparison of the inhibition of the
glycation of ribonuclease A by thiamine pyrophosphate (TPP),
pyridoxamine (PM) and aminoguanidine (AG).
[0038] FIG. 6A is a graph of the kinetics of glycation of RNase A
(10 mg/mL) by ribose as monitored by ELISA. FIG. 6B is a graph
showing the dependence of reciprocal half-times on ribose
concentration at pH 7.5.
[0039] FIG. 7 are two graphs showing a comparison of uninterrupted
and interrupted glycation of RNase by glucose (7B) and ribose (7A),
as detected by ELISA.
[0040] FIG. 8 are two graphs showing kinetics of pentosidine
fluorescence (arbitrary units) increase during uninterrupted and
interrupted ribose glycation of RNase. FIG. 8A Uninterrupted
glycation in the presence of 0.05 M ribose. FIG. 8B Interrupted
glycation after 8 and 24 hours of incubation.
[0041] FIG. 9 is a graph which shows the kinetics of reactive
intermediate buildup.
[0042] FIG. 10 are graphs of Post-Amadori inhibition of AGE
formation by ribose. FIG. 10A graphs data where aliquots were
diluted into inhibitor containing buffers at time 0. FIG. 10B
graphs data where samples were interrupted at 24 h, and then
diluted into inhibitor containing buffers.
[0043] FIG. 11 is a graph showing dependence of the initial rate of
formation of antigenic AGE on pH following interruption of
glycation.
[0044] FIG. 12 are two graphs showing the effect of pH jump on
ELISA detected AGE formation after interrupted glycation.
Interrupted samples left 12 days at 37.degree. C. in pH 5.0 buffer
produced substantial AGEs (33%; FIG. 12B) when pH was changed to
7.5, as compared to the normal control sample not exposed to low pH
(FIG. 12A).
[0045] FIG. 13 is a series of graphs depicting the effect of
vitamin B.sub.6 derivatives on AGE formation during uninterrupted
glycation of ribonuclease A (RNase A) by ribose. FIG. 13A
Pyridoxamine (PM); FIG. 13B pyridoxal-5'-phosphate (PLP); FIG. 13C
pyridoxal (PL); FIG. 13D pyridoxine (PN).
[0046] FIG. 14 is a series of graphs depicting the effect of
vitamin B.sub.1 derivatives and aminoguanidine (AG) on AGE
formation during uninterrupted glycation of ribonuclease A (RNase
A) by ribose. FIG. 14A Thiamine pyrophosphate (TPP); FIG. 14B
thiamine monophosphate (TP); FIG. 14C thiamine (T); FIG. 14D
aminoguanidine (AG).
[0047] FIG. 15 is a series of graphs depicting the effect of
vitamin B.sub.6 derivatives on AGE formation during uninterrupted
glycation of bovine serum albumin (BSA) by ribose. FIG. 15A
Pyridoxamine (PM); FIG. 15B pyridoxal-5'-phosphate (PLP); FIG. 15C
pyridoxal (PL); FIG. 15D pyridoxine (PN).
[0048] FIG. 16 is a series of graphs depicting the effect of
vitamin B.sub.1 derivatives and aminoguanidine (AG) on AGE
formation during uninterrupted glycation of bovine serum albumin
(BSA) by ribose. FIG. 16A Thiamine pyrophosphate (TPP); FIG. 16B
thiamine monophosphate (TP); FIG. 16C thiamine (T); FIG. 16D
aminoguanidine (AG).
[0049] FIG. 17 is a series of graphs depicting the effect of
vitamin B.sub.6 derivatives on AGE formation during uninterrupted
glycation of human methemoglobin (Hb) by ribose. FIG. 17A
Pyridoxamine (PM); FIG. 17B pyridoxal-5'-phosphate (PLP); FIG. 17C
pyridoxal (PL); FIG. 17D pyridoxine (PN).
[0050] FIG. 18 is a series of graphs depicting the effect of
vitamin B.sub.6 derivatives on post-Amadori AGE formation after
interrupted glycation by ribose. FIG. 18A BSA and Pyridoxamine
(PM); FIG. 18B BSA and pyridoxal-5'-phosphate (PLP); FIG. 18C BSA
and pyridoxal (PL); FIG. 18D RNase and pyridoxamine (PM).
[0051] FIG. 19 are graphs depicting the effect of thiamine
pyrophosphate on post-Amadori AGE formation after interrupted
glycation by ribose. FIG. 19A RNase, FIG. 19B BSA.
[0052] FIG. 20 are graphs depicting the effect of aminoguanidine on
post-Amadori AGE formation after interrupted glycation by ribose.
FIG. 20A RNase, FIG. 20B BSA.
[0053] FIG. 21 is a graph depicting the effect of
N.sup..alpha.-acetyl-L-lysine on post-Amadori AGE formation after
interrupted glycation by ribose.
[0054] FIG. 22 are bar graphs showing a comparison of post-Amadori
inhibition of AGE formation by thiamine pyrophosphate (TPP),
pyridoxamine (PM) and aminoguanidine (AG) after interrupted
glycation of RNase (FIG. 22A) and BSA (FIG. 22B) by ribose.
[0055] FIG. 23 is a bar graph showing the effects of Ribose
treatment in vivo alone on rat tail-cuff blood pressure. Treatment
was with 0.05 M, 0.30 M, and 1 M Ribose (R) injected for 1, 2 or 8
Days (D).
[0056] FIG. 24 is a bar graph showing the effects of Ribose
treatment in vivo alone on rat creatinine clearance (Clearance per
100 g Body Weight). Treatment was with 0.05 M, 0.30 M, and 1 M
Ribose (R) injected for 1, 2 or 8 Days (D).
[0057] FIG. 25 is a bar graph showing the effects of Ribose
treatment in vivo alone on rat Albuminuria (Albumin effusion rate).
Treatment was with 0.30 M, and 1 M Ribose (R) injected for 1, 2 or
8 Days (D).
[0058] FIG. 26 is a bar graph showing the effects of inhibitor
treatment in vivo, with or without ribose, on rat tail-cuff blood
pressure. Treatment groups were: 25 mg/1000 g body weight
aminoguanidine (AG); 25 or 250 mg/1000 g body weight Pyridoxamine
(P); 250 mg/1000 g body weight Thiamine pyrophosphate (T), or with
1 M Ribose (R).
[0059] FIG. 27 is a bar graph showing the effects of inhibitor
treatment in vivo, with or without ribose, on rat creatinine
clearance (Clearance per 100 g body weight). Treatment groups were:
25 mg/1000 g body weight aminoguanidine (AG); 25 or 250 mg/1000 g
body weight Pyridoxamine (P); 250 mg/1000 g body weight Thiamine
pyrophosphate (T), or with 1 M Ribose (R).
[0060] FIG. 28 is a bar graph showing the effects of inhibitor
treatment in vivo without ribose, and ribose alone on rat
Albuminuria (Albumin effusion rate). Treatment groups were: 25
mg/1000 g body weight aminoguanidine (AG); 250 mg/1000 g body
weight Pyridoxamine (P); 250 mg/1000 g body weight Thiamine
pyrophosphate (T), or treatment with 1 M Ribose (R) for 8 days (D).
Control group had no treatment.
[0061] FIG. 29 is a bar graph showing the effects of inhibitor
treatment in vivo, with 1 M ribose , on rat Albuminuria (Albumin
effusion rate). Treatment groups were: 25 mg/1000 g body weight
aminoguanidine (AG); 25 and 250 mg/1000 g body weight Pyridoxamine
(P); 250 mg/1000 g body weight Thiamine pyrophosphate (T), or
treatment with 1 M Ribose (R) for 8 days (D) alone. Control group
had no treatment.
[0062] FIG. 30A depicts Scheme 1 showing a diagram of AGE formation
from protein. FIG. 30B depicts Scheme 2, a chemical structure of
aminoguanidine. FIG. 30C depicts Scheme 3, chemical structures for
thiamine, thiamine-5'-phosphate, and thiamine pyrophosphate. FIG.
30D depicts Scheme 4, chemical structures of pyridoxine,
pyridoxamine, pyridoxal-5'-phosphate, and pyridoxal. FIG. 30E
depicts Scheme 5, kinetics representation of AGE formation. FIG.
30F depicts Scheme 6, kinetics representation of AGE formation and
intermediate formation.
[0063] FIG. 31A and 31B shows a 125 MHz C-13 NMR Resonance spectrum
of Riobonuclease Amadori Intermediate prepared by 24 HR reaction
with 99% [2-C13]Ribose.
[0064] FIG. 32A is a set of graphs which show AGE intermediary
formation using the pentoses Xylose, Lyxose, Arabinose and Ribose.
The graphs illustrate dependence of post-Amadori AGE formation on
time of pre-incubation with 0.5M pentose sugar. RNase mixed with
0,5M pentose for the indicated times, then assayed 7 days after
removal of the pentose by dilution.
[0065] FIG. 32B is a graph which shows the inhibition of AGE
formation by pyridoxamine (PM) and pyridoxamine-5'-phosphate (PMP).
The graph illustrates the effect of PM and PMP on post-Amadori AGE
formation on Bovine Serum Albumin (BSA) modified by interrupted
glycation with 0.5M ribose.
[0066] FIG. 33 is a graph showing the results of glomeruli staining
at pH 2.5 with Alcian blue.
[0067] FIG. 34 is a graph showing the results of glomeruli staining
at pH 1.0 with Alcian blue.
[0068] FIG. 35 is a graph showing the results of immunofluroescent
glomeruli staining for RSA.
[0069] FIG. 36 is a graph showing the results of immunofluroescent
glomeruli staining for Heparan Sulfate Proteoglycan Core
protein.
[0070] FIG. 37 is a graph showing the results of immunofluroescent
glomeruli staining for Heparan Sulfate Proteoglycan side-chain.
[0071] FIG. 38 is a graph showing the results of analysis of
glomeruli sections for average glomerular volume.
DETAILED DESCRIPTION
Animal Models for Protein Aging
[0072] Alloxan induced diabetic Lewis rats have been used as a
model for protein aging to demonstrate the in vivo effectiveness of
inhibitors of AGE formation. The correlation being demonstrated is
between inhibition of late diabetes related pathology and effective
inhibition of AGE formation (Brownlee, Cerami, and Vlassara, 1988,
New Eng. J. Med. 318(20):1315-1321). Streptozotocin induction of
diabetes in Lewis rats, New Zealand White rabbits with induced
diabetes, and genetically diabetic BB/Worcester rats have also been
utilized, as described in, for example, U.S. Pat. No. 5,334,617
(incorporated by reference). A major problem with these model
systems is the long time period required to demonstrate AGE related
injury, and thus to test compounds for AGE inhibition. For example,
16 weeks of treatment was required for the rat studies described in
U.S. Pat. No. 5,334,617, and 12 weeks for the rabbit studies. Thus
it would be highly desirable and useful to have a model system for
AGE related diabetic pathology that will manifest in a shorter time
period, allowing for more efficient and expeditious determination
of AGE related injury and the effectiveness of inhibitors of
post-Amadori AGE formation.
Antibodies to AGEs
[0073] An important tool for studying AGE formation is the use of
polyclonal and monoclonal antibodies that are specific for AGEs
elicited by the reaction of several sugars with a variety of target
proteins. The antibodies are screened for resultant specificity for
AGEs that is independent of the nature of the protein component of
the AGE (Nakayama et al., 1989, Biochem. Biophys. Res. Comm. 162:
740-745; Nakayama et al., 1991, J. Immunol. Methods 140: 119-125;
Horiuchi et al., 1991, J. Biol. Chem. 266: 7329-7332; Araki et al.,
1992, J. Biol. Chem. 267: 10211-10214; Makita et al., 1992, J.
Biol. Chem. 267: 5133-5138). Such antibodies have been used to
monitor AGE formation in vivo and in vitro.
Thiamine--Vitamin B.sub.1
[0074] The first member of the Vitamin B complex to be identified,
thiamine is practically devoid of pharmacodynamic actions when
given in usual therapeutic doses; and even large doses were not
known to have any effects. Thiamine pyrophosphate is the
physiologically active form of thiamine, and it functions mainly in
carbohydrate metabolism as a coenzyme in the decarboxylation of
.alpha.-keto acids. Tablets of thiamine hydrochloride are available
in amounts ranging from 5 to 500 mg each. Thiamine hydrochloride
injection solutions are available which contain 100 to 200
mg/ml.
[0075] For treating thiamine deficiency, intravenous doses of as
high as 100 mg/L of parenteral fluid are commonly used, with the
typical dose of 50 to 100 mg being administered. GI absorption of
thiamine is believed to be limited to 8 to 15 mg per day, but may
be exceed by oral administration in divided doses with food.
[0076] Repeated administration of glucose may precipitate thiamine
deficiency in under nourished patients, and this has been noted
during the correction of hyperglycemia.
[0077] Surprisingly, the instant invention has found, as shown by
in vitro testing, that administration of thiamine pyrophosphate at
levels above what is normally found in the human body or
administered for dietary therapy, is an effective inhibitor of
post-Amadori antigenic AGE formation, and that this inhibition is
more complete than that possible by the administration of
aminoguanidine.
Pyridoxine--Vitamin B.sub.6
[0078] Vitamin B.sub.6 is typically available in the form of
pyridoxine hydrochloride in over-the-counter preparations available
from many sources. For example Beach pharmaceuticals Beelith
Tablets contain 25 mg of pyridoxine hydrochloride that is
equivalent to 20 mg of B.sub.6, other preparations include Marlyn
Heath Care Marlyn Formula 50 which contain 1 mg of pyridoxine HCl
and Marlyn Formula 50 Mega Forte which contains 6 mg of pyridoxine
HCl, Wyeth-Ayerst Stuart Prenatal.RTM. tablets which contain 2.6 mg
pyridoxine HCl, J&J-Merck Corp. Stuart Formula.RTM. tablets
contain 2 mg of pyridoxine HCl, and the CIBA Consumer Sunkist
Children's chewable multivitamins which contain 1.05 mg of
pyridoxine HCl, 150% of the U.S. RDA for children 2 to 4 years of
age, and 53% of the U.S. RDA for children over 4 years of age and
adults. (Physician's Desk Reference for nonprescription drugs, 14th
edition (Medical Economics Data Production Co., Montvale, N.J.,
1993).
[0079] There are three related forms of pyridoxine, which differ in
the nature of the substitution on the carbon atom in position 4 of
the pyridine nucleus: pyridoxine is a primary alcohol, pyridoxal is
the corresponding aldehyde, and pyridoxamine contains an
aminomethyl group at this position. Each of these three forms can
be utilized by mammals after conversion by the liver into
pyridoxal-5'-phosphate, the active form of the vitamin. It has long
been believed that these three forms are equivalent in biological
properties, and have been treated as equivalent forms of vitamin
B.sub.6 by the art. The Council on Pharmacy and Chemistry has
assigned the name pyridoxine to the vitamin.
[0080] The most active antimetabolite to pyridoxine is
4-deoxypyridoxine, for which the antimetabolite activity has been
attributed to the formation in vivo of
4-deoxypyridoxine-5-phosphate, a competitive inhibitor of several
pyridoxal phosphate-dependent enzymes. The pharmacological actions
of pyridoxine are limited, as it elicits no outstanding
pharmacodynamic actions after either oral or intravenous
administration, and it has low acute toxicity, being water soluble.
It has been suggested that neurotoxicity may develop after
prolonged ingestion of as little as 200 mg of pyridoxine per day.
Physiologically, as a coenzyme, pyridoxine phosphate is involved in
several metabolic transformations of amino acids including
decarboxylation, transamination, and racemization, as well as in
enzymatic steps in the metabolism of sulfur-containing and
hydroxy-amino acids. In the case of transamination, pyridoxal
phosphate is aminated to pyridoxamine phosphate by the donor amino
acid, and the bound pyridoxamine phosphate is then deaminated to
pyridoxal phosphate by the acceptor .alpha.-keto acid. Thus vitamin
B complex is known to be a necessary dietary supplement involved in
specific breakdown of amino acids. For a general review of the
vitamin B complex see The Pharmacological Basis of Therapeutics,
8th edition, ed. Gilman, Rall, Nies, and Taylor (Pergamon Press,
New York, 1990, pp. 1293-4; pp. 1523-1540).
[0081] Surprisingly, the instant invention has discovered that
effective dosages of the metabolically transitory pyridoxal amine
form of vitamin B.sub.6 (pyridoxamine), at levels above what is
normally found in the human body, is an effective inhibitor of
post-Amadori antigenic AGE formation, and that this inhibition may
be more complete than that possible by the administration of
aminoguanidine.
Formation of Stable Amadori/Schiff Base Intermediary
[0082] The typical study of the reaction of a protein with glucose
to form AGEs has been by ELISA using antibodies directed towards
antigenic AGEs, and the detection of the production of an
acid-stable fluorescent AGE, pentosidine, by HPLC following acid
hydrolysis. Glycation of target proteins (i.e. BSA or RNase A) with
glucose and ribose were compared by monitoring ELISA reactivity of
polyclonal rabbit anti-Glucose-AGE-RNase and anti-Glucose-AGE-BSA
antibodies. The antigen was generated by reacting 1 M glucose with
RNase for 60 days and BSA for 90 days. The antibodies (R618 and
R479) were screened and showed reactivity with only AGEs and not
the protein, except for the carrier immunogen BSA.
EXAMPLE 1
Thiamine Pyrophosphate and Pyridoxamine Inhibit the Formation of
Antigenic Advanced Glycation End-Products from Glucose: Comparison
with Aminoguanidine
[0083] Some B.sub.6 vitamers, especially pyridoxal phosphate (PLP),
have been previously proposed to act as "competitive inhibitors" of
early glycation, since as aldehydes they themselves can form Schiff
bases adducts with protein amino groups (Khatami et al., 1988, Life
Sciences 43:1725-1731) and thus limit the amount of amines
available for glucose attachment. However, effectiveness in
limiting initial sugar attachment is not a predictor of inhibition
of the conversion of any Amadori products formed to AGEs. The
instant invention describes inhibitors of "late" glycation
reactions as indicated by their effects on the in vitro formation
of antigenic AGEs (Booth et al., 1996, Biochem. Biophys. Res. Com.
220:113-119).
Chemicals
[0084] Bovine pancreatic ribonuclease A (RNase) was
chromatographically pure, aggregate-free grade from Worthington
Biochemicals. Bovine Serum albumin (BSA; fraction V, fatty-acid
free), human methemoglobin (Hb), D-glucose, pyridoxine, pyridoxal,
pyridoxal 5'phosphate, pyridoxamine, thiamine, thiamine
monophosphate, thiamine pyrophosphate, and goat alkaline
phosphatase-conjugated anti-rabbit IgG were all from Sigma
Chemicals. Aminoguanidine hydrochloride was purchased from Aldrich
Chemicals.
Uninterrupted Glycation with Glucose
[0085] Bovine serum albumin, ribonuclease A, and human hemoglobin
were incubated with glucose at 37.degree. C. in 0.4 M sodium
phosphate buffer of pH 7.5 containing 0.02% sodium azide. The
protein, glucose (at 1.0 M), and prospective inhibitors (at 0.5, 3,
15 and 50 mM) were introduced into the incubation mixture
simultaneously. Solutions were kept in the dark in capped tubes.
Aliquots were taken and immediately frozen until analyzed by ELISA
at the conclusion of the reaction. The incubations were for 3 weeks
(Hb) or 6 weeks (RNase, BSA).
Preparation of Polyclonal Antibodies to AGE Proteins
[0086] Immunogen preparation followed earlier protocols (Nakayama
et al., 1989, Biochem. Biophys. Res. Comm. 162:740-745; Horiuchi et
al., 1991, J. Biol. Chem. 266:7329-7332; Makita et al., 1992, J.
Biol. Chem. 267:5133-5138). Briefly, immunogen was prepared by
glycation of BSA (R479 antibodies) or RNase (R618 antibodies) at
1.6 g protein in 15 ml for 60-90 days using 1.5 M glucose in 0.4 M
sodium phosphate buffer of pH 7.5 containing 0.05% sodium azide at
pH 7.4 and 37.degree. C. New Zealand white rabbit males of 8-12
weeks were immunized by subcutaneous administration of a 1 ml
solution containing 1 mg/ml of glycated protein in Freund's
adjuvant. The primary injection used the complete adjuvant and
three boosters were made at three week intervals with Freund's
incomplete adjuvant. Rabbits were bled three weeks after the last
booster. The serum was collected by centrifugation of clotted whole
blood. The antibodies are AGE-specific, being unreactive with
either native proteins (except for the carrier) or with Amadori
intermediates. The polyclonal anti-AGE antibodies have proven to be
a sensitive and valuable analytical tool for the study of "late"
AGE formation in vitro and in vivo. The nature of the dominant
antigenic AGE epitope or hapten remains in doubt, although recently
it has been proposed that the protein glycoxidation product
carboxymethyl lysine (CmL) may be a dominant antigen of some
antibodies (Reddy et al., 1995, Biochem. 34:10872-10878). Earlier
studies have failed to reveal ELISA reactivity with model CmL
compounds (Makita et al., 1992, J. Biol. Chem. 267:5133-5138).
ELISA Detection of AGE Products
[0087] The general method of Engvall (1981, Methods Enzymol.
70:419-439) was used to perform the ELISA. Typically, glycated
protein samples were diluted to approximately 1.5 ug/ml in 0.1 M
sodium carbonate buffer of pH 9.5 to 9.7. The protein was coated
overnight at room temperature onto 96-well polystyrene plates by
pippetting 200 ul of the protein solution in each well (0.3
ug/well). After coating, the protein was washed from the wells with
a saline solution containing 0.05% Tween-20. The wells were then
blocked with 200 ul of 1% casein in carbonate buffer for 2 h at
37.degree. C. followed by washing. Rabbit anti-AGE antibodies were
diluted at a titer of about 1:350 in incubation buffer, and
incubated for 1 h at 37.degree. C., followed by washing. In order
to minimize background readings, antibodies R479 used to detect
glycated RNase were raised against glycated BSA, and antibodies
R618 used to detect glycated BSA and glycated Hb were raised
against glycated RNase. An alkaline phosphatase-conjugated antibody
to rabbit IgG was then added as the secondary antibody at a titer
of 1:2000 or 1:2500 (depending on lot) and incubated for 1 h at
37.degree. C., followed by washing. The p-nitrophenylphosphate
substrate solution was then added (200 ul/well) to the plates, with
the absorbance of the released p-nitrophenolate being monitored at
410 nm with a Dynatech MR 4000 microplate reader.
[0088] Controls containing unmodified protein were routinely
included, and their readings were subtracted, the corrections
usually being negligible. The validity of the use of the ELISA
method in quantitatively studying the kinetics of AGE formation
depends on the linearity of the assay (Kemeny & Challacombe,
1988, ELISA and Other Solid Phase Immunoassays, John Wiley &
Sons, Chichester, U.K.). Control experiments were carried out, for
example, demonstrating that the linear range for RNase is below a
coating concentration of about 0.2-0.3 ug/well.
Results
[0089] FIG. 1A-D are graphs which show the effect of vitamin
B.sub.6 derivatives on post-Amadori AGE formation in bovine serum
albumin glycated with glucose. BSA (10 mg/ml) was incubated with
1.0 M glucose in the presence and absence of the various indicated
derivative in 0.4 M sodium phosphate buffer of pH 7.5 at 37.degree.
C. for 6 weeks. Aliquots were assayed by ELISA using R618 anti-AGE
antibodies. Concentrations of the inhibitors were 3, 15 and 50 mM.
Inhibitors used in FIGS. (1A) Pyridoxamine (PM); (1B) pyridoxal
phosphate (PLP); (1C) pyridoxal (PL); (1D) pyridoxine (PN).
[0090] FIG. 1 (control curves) demonstrates that reaction of BSA
with 1.0 M glucose is slow and incomplete after 40 days, even at
the high sugar concentration used to accelerate the reaction. The
simultaneous inclusion of different concentrations of various
B.sub.6 vitamers markedly affects the formation of antigenic AGEs.
(FIG. 1A-D) Pyridoxamine and pyridoxal phosphate strongly
suppressed antigenic AGE formation at even the lowest
concentrations tested, while pyridoxal was effective above 15 mM.
Pyridoxine was slightly effective at the highest concentrations
tested.
[0091] FIG. 2A-D are graphs which show the effect of vitamin
B.sub.1 derivatives and aminoguanidine (AG) on AGE formation in
bovine serum albumin. BSA (10 mg/ml) was incubated with 1.0 M
glucose in the presence and absence of the various indicated
derivative in 0.4 M sodium phosphate buffer of pH 7.5 at 37.degree.
C. for 6 weeks. Aliquots were assayed by ELISA using R618 anti-AGE
antibodies. Concentrations of the inhibitors were 3, 15 and 50 mM.
Inhibitors used in FIGS. (2A) Thiamine pyrophosphate (TPP); (2B)
thiamine monophosphate (TP); (2C) thiamine (T); (2D) aminoguanidine
(AG).
[0092] Of the various B.sub.1 vitamers similarly tested (FIG.
2A-D), thiamine pyrophosphate was effective at all concentrations
tested (FIG. 2C), whereas thiamine and thiamine monophosphate were
not inhibitory. Most significantly it is remarkable to note the
decrease in the final levels of AGEs formed observed with thiamine
pyrophosphate, pyridoxal phosphate and pyridoxamine. Aminoguanidine
(FIG. 2D) produced some inhibition of AGE formation in BSA, but
less so than the above compounds. Similar studies were carried out
with human methemaglobin and bovine ribonuclease A.
[0093] FIG. 3A-D are graphs which show the effect of vitamin
B.sub.6 derivatives on AGE formation in human methemoglobin. Hb (1
mg/ml) was incubated with 1.0 M glucose in the presence and absence
of the various indicated derivative in 0.4 M sodium phosphate
buffer of pH 7.5 at 37.degree. C. for 3 weeks. Aliquots were
assayed by ELISA using R618 anti-AGE antibodies. Concentrations of
the inhibitors were 0.5, 3, 15 and 50 mM. Inhibitors used in FIGS.
(3A) Pyridoxamine (PM); (3B) pyridoxal phosphate (PLP); (3C)
pyridoxal (PL); (3D) pyridoxine (PN).
[0094] It had been previously reported that Hb of a diabetic
patient contains a component that binds to anti-AGE antibodies, and
it was proposed that this glycated Hb (termed Hb-AGE, not to be
confused with Hb.sub.Alc) could be useful in measuring long-term
exposure to glucose. The in vitro incubation of Hb with glucose
produces antigenic AGEs at an apparently faster rate than observed
with BSA. Nevertheless, the different B.sub.6 (FIG. 3A-D) and
B.sub.1 (FIG. 4A-C) vitamers exhibited the same inhibition trends
in Hb, with pyridoxamine and thiamine pyrophosphate being the most
effective inhibitors in each of their respective families.
Significantly, in Hb, aminoguanidine only inhibited the rate of AGE
formation, and not the final levels of AGE formed (FIG. 4D).
[0095] With RNase the rate of antigenic AGE formation by glucose
was intermediate between that of Hb and BSA, but the extent of
inhibition within each vitamer series was maintained. Again
pyridoxamine and thiamine pyrophosphate were more effective that
aminoguanidine (FIG. 5).
[0096] FIG. 4A-D are graphs which show the effect of vitamin
B.sub.1 derivatives and aminoguanidine (AG) on AGE formation in
human methemoglobin. Hb (1 mg/ml) was incubated with 1.0 M glucose
in the presence and absence of the various indicated derivative in
0.4 M sodium phosphate buffer of pH 7.5 at 37.degree. C. for 3
weeks. Aliquots were assayed by ELISA using R618 anti-AGE
antibodies. Concentrations of the inhibitors were 0.5, 3, 15 and 50
mM. Inhibitors used in FIGS. (4A) Thiamine pyrophosphate (TPP);
(4B) thiamine monophosphate (TP); (4C) thiamine (T); (4D)
aminoguanidine (AG).
[0097] FIG. 5 is a bar graph which shows a comparison of the
inhibition of the glycation of ribonuclease A by thiamine
pyrophosphate (TPP), pyridoxamine (PM) and aminoguanidine (AG).
RNase (1 mg/ml) was incubated with 1.0 M glucose (glc) in the
presence and absence of the various indicated derivative in 0.4 M
sodium phosphate buffer of pH 7.5 at 37.degree. C. for 6 weeks.
Aliquots were assayed by ELISA using R479 anti-AGE antibodies. The
indicated percent inhibition was computed from ELISA readings in
the absence and presence of the inhibitors at the 6 week time
point. Concentrations of the inhibitors were 0.5, 3, 15 and 50
mM.
Discussion
[0098] These results demonstrate that certain derivatives of
B.sub.1 and B.sub.6 vitamins are capable of inhibiting "late" AGE
formation. Some of these vitamers successfully inhibited the final
levels of AGE produced, in contrast to aminoguanidine, suggesting
that they have greater interactions with Amadori or post-Amadori
precursors to antigenic AGEs. The Amadori and post-Amadori
intermediates represent a crucial juncture where the "classical"
pathway of nonenzymatic glycation begins to become essentially
irreversible (Scheme I). In earlier inhibition studies "glycation"
was usually measured either as Schiff base formed (after reduction
with labeled cyanoborohydride) or as Amadori product formed (after
acid precipitation using labeled sugar). Such assays do not yield
information on inhibition of post-Amadori conversion steps to
"late" AGE products, since such steps lead to no change in the
amount of labeled sugar that is attached to the proteins. Other
"glycation" assays have relied on the sugar-induced increase of
non-specific protein fluorescence, but this can also be induced by
dicarbonyl oxidative fragments of free sugar, such as glycoaldehyde
or glyoxal (Hunt et al., 1988, Biochem. 256:205-212), independently
of Amadori product formation.
[0099] In the case of pyridoxal (PL) and pyridoxal phosphate (PLP),
the data support the simple mechanism of inhibition involving
competitive Schiff-base condensation of these aldehydes with
protein amino groups at glycation sites. Due to internal hemiacetal
formation in pyridoxal but not pyridoxal phosphate, stronger
inhibition of AGE formation by PLP is expected by this competitive
mechanism. This indeed is observed in the data (FIG. 1B, 1C, FIG.
3B, 3C). The inhibition by pyridoxamine is necessarily different,
as pyridoxamine lacks an aldehyde group. However, pyridoxamine is a
candidate amine potentially capable of forming a Schiff-base
linkage with the carbonyls of open-chain sugars, with dicarbonyl
fragments, with Amadori products, or with post-Amadori
intermediates. The mechanism of inhibition of B.sub.1 compounds is
not obvious. All the forms contain an amino functionality, so that
the marked efficiency of only the pyrophosphate form suggests an
important requirement for a strong negative charge.
[0100] A significant unexpected observation is that the extent of
inhibition by aminoguanidine, and some of the other compounds, is
considerably less at late stages of the reaction, than during the
early initial phase. This suggests a different mechanism of action
than that of pyridoxamine and thiamine pyrophosphate, suggesting
that the therapeutic potential of these compounds will be enhanced
by co-administration with aminoguanidine.
EXAMPLE 2
Kinetics of Non-enzymatic Glycation: Paradoxical Inhibition by
Ribose and Facile Isolation of Protein Intermediate for Rapid
Post-Amadori AGE Formation
[0101] While high concentrations of glucose are used to cause the
non-enzymatic glycation of proteins, paradoxically, it was found
that ribose at high concentrations is inhibitory to post-Amadori
AGE formation in ribonuclease by acting on the post-Amadori "late"
stages of the glycation reaction. This unexpectedly inhibitory
effect suggests that the "early" reactive intermediates, presumably
Amadori products, can be accumulated with little formation of
"late" post-Amadori AGE products (AGEs; Maillard products).
Investigation into this phenomenon has demonstrated: (1) ability to
define conditions for the kinetic isolation of Amadori (or
post-Amadori) glycated intermediate(s); (2) the ability study the
fast kinetics of buildup of such an intermediate; (3) the ability
to study the surprisingly rapid kinetics of conversion of such
intermediates to AGE products in the absence of free or reversibly
bound sugar; (4) the ability to use these intermediates to study
and characterize inhibition of post-Amadori steps of AGE formation
thus providing a novel system to investigate the mechanism of
reaction and the efficacy of potential agents that could block AGE
formation; and (5) with this knowledge it is also further possible
to use .sup.13C NMR to study the reactive intermediates and monitor
their conversion to various candidate AGEs (Khalifah et al., 1996,
Biochemistry 35(15):4645-4654).
Chemicals and Materials As in Example 1 above.
Preparation of polyclonal antibodies to AGEs As in Example 1
above.
ELISA detection of AGE products As in Example 1 above.
Amino Acid Analysis
[0102] Amino acid analyses were carried out at the Biotechnology
Support Facility of the Kansas University Medical Center. Analyses
were performed after hydrolysis of glycated protein (reduced with
sodium cyanoborohydride) with 6 N HCl at 110.degree. C. for 18-24
h. Phenyl isothiocyanate was used for derivatization, and PTH
derivatives were analyzed by reverse-phase HPLC on an Applied
Biosystems amino acid analyzer (420A derivatizer, 130A separation
system, 920A data analysis system).
Pentosidine Reverse-Phase HPLC Analysis
[0103] Pentosidine production in RNase was quantitated by HPLC
(Sell & Monnier, 1989, J. Biol. Chem. 264:21597-21602; Odetti
et al., 1992, Diabetes 41:153-159). Ribose-modified protein samples
were hydrolyzed in 6 N HCl for 18 h at 100.degree. C. and then
dried in a Speed Vac. The samples were then redissolved, and
aliquots were taken into 0.1% trifluoroacetic acid and analyzed by
HPLC on a Shimadzu system using a Vydac C-18 column equilibrated
with 0.1% TFA. A gradient of 0-6% acetonitrile (0.1% in TFA) was
run in 30 min at a flow rate of about 1 ml/min. Pentosidine was
detected by 335 nm excitation/385 nm emission fluorescence, and its
elution time was determined by running a synthesized standard. Due
to the extremely small levels of pentosidine expected (Grandhee
& Monnier, 1991, J. Biol. Chem. 266:11649-11653; Dyer et al.,
1991, J. Biol. Chem. 266:11654-11660), no attempt was made to
quantitate the absolute concentrations. Only relative
concentrations were determined from peak areas.
Glycation Modifications
[0104] Modification with ribose or glucose was generally done at
37.degree. C. in 0.4 M phosphate buffer of pH 7.5 containing 0.02%
sodium azide. The high buffer concentration was always used with
0.5 M ribose modifications. The solutions were kept in capped tubes
and opened only to remove timed aliquots that were immediately
frozen for later carrying out the various analyses. "Interrupted
glycation" experiments were carried out by first incubating protein
with the ribose at 37.degree. C. for 8 or 24 h, followed by
immediate and extensive dialysis against frequent cold buffer
changes at 4.degree. C. The samples were then reincubated by
quickly warming to 37.degree. C. in the absence of external ribose.
Aliquots were taken and frozen at various intervals for later
analysis. Due to the low molecular weight of RNase, protein
concentrations were remeasured after dialysis even when low
molecular weight cut-off dialysis tubing was used. An alternative
procedure was also devised (see below) in which interruption was
achieved by simple 100-fold dilution from reaction mixtures
containing 0.5 M ribose. Protein concentrations were estimated from
UV spectra. The difference in molar extinction between the peak
(278 nm) and trough (250 nm) was used for RNase concentration
determinations in order to compensate for the general increase in
UV absorbance that accompanies glycation. Time-dependent
UV-difference spectral studies were carried out to characterize the
glycation contributions of the UV spectrum.
Data Analysis and Numerical Simulations of Kinetics
[0105] Kinetic data were routinely fit to monoexponential or
biexponential functions using nonlinear least-squares methods. The
kinetic mechanisms of Schemes 5-6 have been examined by numerical
simulations of the differential equations of the reaction. Both
simulations and fitting to observed kinetics data were carried out
with the SCIENTIST 2.0 software package (Micromath, Inc.).
Determination of apparent half-times (FIG. 6B) from kinetic data
fit to two-exponential functions (FIG. 6A) was carried out with the
"solve" function of MathCAD 4.0 software (MathSoft, Inc.).
RESULTS
Comparison of Glycation by Glucose and Ribose
[0106] The reaction of RNase A with ribose and glucose has been
followed primarily with ELISA assays, using R479 rabbit
AGE-specific antibodies developed against glucose-modified BSA. To
a lesser extent, the production of pentosidine, the only known
acid-stable fluorescent AGE, was quantiated by HPLC following acid
hydrolysis. Preliminary studies using 0.05 M ribose at 37.degree.
C. showed that the rate of antigenic AGE formation appears to be
modestly increased (roughly 2-3 fold as measured by the apparent
half-time) as the pH is increased from 5.0 to 7.5, with an apparent
small induction period at the beginning of the kinetics in all
cases. The glycation of RNase with 0.05 M ribose at pH 7.5
(half-time near 6.5 days) appears to be almost an order of
magnitude faster than that of glycation with 1.0 M glucose
(half-time in excess of 30 days; see FIG. 7B, solid line). The
latter kinetics also displayed a small induction period but
incomplete leveling off even after 60 days, making it difficult to
estimate a true half-time.
[0107] When the dependence of the kinetics on ribose concentration
was examined at pH 7.5, a most unexpected result was obtained. The
rate of reaction initially increased with increasing ribose
concentration, but at concentrations above 0.15 M the rate of
reaction leveled off and then significantly decreased (FIG. 6A). A
plot of the dependence of the reciprocal half-time on the
concentration of ribose (FIG. 6B) shows that high ribose
concentrations are paradoxically inhibitory to post-Amadori
antigenic AGE formation. This unusual but consistent effect was
found to be independent of changes in the concentration of either
buffer (2-fold) or RNase (10-fold), and it was not changed by
affinity purification of the R479 antibody on a column of
immobilized AGE-RNase. It is also not due to effects of ribose on
the ELISA assay itself. The measured inhibitory effect by ribose on
post-Amadori AGE formation is not likely due to ribose interference
with antibody recognition of the AGE antigenic sites on protein in
the ELISA assay. Prior to the first contact with the primary
anti-AGE antibody on the ELISA plates, glycated protein has been
diluted over 1000-fold, washed extensively with Tween-20 after
adsorption, and blocked with a 1% casein coating followed by
further washing with Tween-20.
Kinetics of Formation of Post-Amadori Antigenic AGEs by
"Interrupted Glycation"
[0108] In view of the small induction period seen, an attempt was
made to determine whether there was some accumulation during the
reaction, of an early precursor such as an Amadori intermediate,
capable of producing the ELISA-detectable post-Amadori antigenic
AGEs. RNase was glycated at pH 7.5 and 37.degree. C. with a high
ribose concentration of 0.5 M, and the reaction was interrupted
after 24 h by immediate cooling to 4.degree. C. and dialysis
against several changes of cold buffer over a period of 24 h to
remove free and reversibly bound (Schiff base) ribose. Such a
ribose-free sample was then rapidly warmed to 37.degree. C. without
re-adding any ribose, and was sampled for post-Amadori AGE
formation over several days. The AGE antigen production of this 24
h "interrupted glycation" sample is shown by the dashed line and
open triangles in FIG. 7A, the time spent in the cold dialysis is
not included. An uninterrupted control (solid line and filled
circles) is also shown for comparison. Dramatically different
kinetics of post-Amadori antigenic AGE formation are evident in the
two samples. The kinetics of AGE antigen production of the
ribose-free interrupted sample now show (1) monoexponential
kinetics with no induction period, (2) a greatly enhanced rate of
antigenic AGE formation, with remarkable half-times of the order of
10 h, and (3) production of levels of antigen comparable to those
seen in long incubations in the continued presence of ribose (see
FIG. 6A). Equally significant, the data also demonstrate that
negligible AGE antigen was formed during the cold dialysis period,
as shown by the small difference between the open triangle and
filled circle points at time 1 day in FIG. 7A. Very little, if any,
AGE was formed by the "interruption" procedure itself. These
observations show that a fully competent isolatable intermediate or
precursor to antigenic AGE has been generated during the 24 h
contact with ribose prior to the removal of the free and reversibly
bound sugar.
[0109] Samples interrupted after only 8 h produced a final amount
of AGE antigen that was about 72% of the 24 h interrupted sample.
Samples of RNase glycated with only 0.05 M ribose and interrupted
at 8 h by cold dialysis and reincubation at 37.degree. C. revealed
less than 5% production of ELISA-reactive antigen after 9 days.
Interruption at 24 h, however, produced a rapid rise of ELISA
antigen (similar to FIG. 7A) to a level roughly 50% of that
produced in the uninterrupted presence of 0.05 M ribose.
[0110] The same general interruption effects were also seen with
other proteins (BSA and Hemoglobin). Except for a somewhat
different absolute value of the rate constants, and the amount of
antigenic AGEs formed during the 24 h 0.5 M ribose incubation, the
same dramatic increase in the rate of AGE antigen formation was
observed after removal of 0.5 M ribose.
[0111] Glycation is much slower with glucose than with ribose (note
the difference in time scales between FIG. 7A and FIG. 7B).
However, unlike the case with ribose, interruption after 3 days of
glycation by 1.0 M glucose produced negligible buildup of precursor
to ELISA-reactive AGE antigens (FIG. 7B, dashed curve).
Kinetics of Pentosidine Formation
[0112] The samples subjected to ELISA testing were also assayed for
the production of pentosidine, an acid-stable AGE. The content of
pentosidine was measured for the same RNase samples analyzed for
antibody reactivity by ELISA. Glycation by ribose in 0.4 M
phosphate buffer at pH 7.5 produced pentosidine in RNase A that was
quantitated by fluroescence after acid hydrolysis. FIG. 8A shows
that under uninterrupted conditions, 0.05 M ribose produces a
progressive increase in pentosidine. However, when glycation is
carried out under "interrupted" conditions using 0.5 M ribose, a
dramatic increase in the rate of pentosidine formation is seen
immediately after removal of excess ribose (FIG. 8B), which is
similar to, but slightly more rapid than, the kinetics of the
appearance of antigenic AGEs (FIG. 7A). A greater amount of
pentosidine was also produced with 24 h interruption as compared
with 8 h. Reproducible differences between the kinetics of
formation of pentosidine and antigenic AGEs can also be noted. A
significant amount of pentosidine is formed during the 24 h
incubation and also during the cold dialysis, resulting in a jump
of the dashed vertical line in FIG. 8B. Our observations thus
demonstrate that a pentosidine precursor accumulates during ribose
glycation that can rapidly produce pentosidine after ribose removal
(cf. Odetti et al., 1992, Diabetes 41:153-159).
Rate of Buildup of the Reactive Intermediate(s)
[0113] The "interrupted glycation" experiments described above
demonstrate that a precursor or precursors to both post-Amadori
antigenic AGEs and pentosidine can be accumulated during glycation
with ribose. The kinetics of formation of this intermediate can be
independently followed and quantitated by a variation of the
experiments described above. The amount of intermediate generated
in RNase at different contact times with ribose can be assayed by
the maximal extent to which it can produce antigenic AGE after
interruption. At variable times after initiating glycation, the
free and reversibly-bound ribose is removed by dialysis in the cold
or by rapid dilution (see below). Sufficient time (5 days, which
represents several half-lives according to FIG. 7A) is then allowed
after warming to 37.degree. C. for maximal development of
post-Amadori antigenic AGEs. The ELISA readings 5 days after each
interruption point, representing maximal AGE development, would
then be proportional to the intermediate concentration present at
the time of interruption.
[0114] FIG. 9 shows such an experiment where the kinetics of
intermediate buildup are measured for RNase A in the presence of
0.5 M ribose (solid symbols and curve). For comparison, the amount
of AGE present before ribose removal at each interruption point is
also shown (open symbols and dashed lines). As expected (cf. FIG.
7A), little AGE is formed prior to removal (or dilution) of ribose,
so that ELISA readings after the 5 day secondary incubation periods
are mostly a measure of AGE formed after ribose removal. The
results in FIG. 9 show that the rate of buildup of intermediate in
0.5 M ribose is exponential and very fast, with a half-time of
about 3.3 h. This is about 3-fold more rapid than the observed rate
of conversion of the intermediate to antigenic AGEs after
interruption (open symbols and dashed curve FIG. 7A).
[0115] In these experiments the removal of ribose at each
interruption time was achieved by 100-fold dilution, and not by
dialysis. Simple dilution reduced the concentration of ribose from
0.05 M to 0.005 M. It was independently determined (FIG. 6A) that
little AGE is produced in this time scale with the residual 5 mM
ribose. This dilution approach was primarily dictated by the need
for quantitative point-to-point accuracy. Such accuracy would not
have been achieved by the dialysis procedure that would be carried
out independently for each sample at each interruption point. Our
results show that dilution was equivalent to dialysis.
[0116] A separate control experiment (see FIG. 10 below)
demonstrated that the instantaneous 100-fold dilution gave nearly
identical results to the dialysis procedure. These control
experiments demonstrate that the reversible ribose-protein binding
(Schiff base) equilibrium is quite rapid on this time scale. This
is consistent with data of Bunn and Higgins (1981, Science 213:
222-224) that indicated that the half-time of Schiff base formation
with 0.5 M ribose should be on the order of a few minutes. The
100-fold rapid dilution method to reduce ribose is a valid method
where quantitative accuracy is essential and cannot be achieved by
multiple dialysis of many samples.
Direct Inhibition of Post-Amadori AGE Formation from the
Intermediate by Ribose and Glucose
[0117] The increase in the rate of AGE formation after interruption
and sugar dilution suggests, but does not prove, that high
concentrations of ribose are inhibiting the reaction at or beyond
the first "stable" intermediate, presumably the Amadori derivative
(boxed in Scheme I). A test of this was then carried out by
studying the effect of directly adding ribose, on the post-Amadori
reaction. RNase was first incubated for 24 h in 0.5 M ribose in
order to prepare the intermediate. Two protocols were then carried
out to measure possible inhibition of the post-Amadori formation of
antigenic AGEs by different concentrations of ribose. In the first
experiment, the 24 h ribated sample was simply diluted 100-fold
into solutions containing varying final concentrations of ribose
ranging from 0.005 M to 0.505 M (FIG. 10A). The rate and extent of
AGE formation are clearly seen to be diminished by increasing
ribose concentrations. Significantly, up to the highest
concentration of 0.5 M ribose, the kinetics appear exponential and
do not show the induction period that occurs with uninterrupted
glycation (FIGS. 6A and 7A) in high ribose concentrations.
[0118] A second experiment (FIG. 10B) was also conducted in which
the 24 h interrupted sample was extensively dialyzed in the cold to
release free and reversibly bound ribose as well as any inhibitory
products that may have formed during the 24 h incubation with
ribose. Following this, aliquots were diluted 100-fold into varying
concentrations of freshly made ribose, and the formation of
antigenic AGE products was monitored as above. There results were
nearly identical to the experiment of FIG. 10A where the dialysis
step was omitted. In both cases, the rate and extent of AGE
formation were diminished by increasing concentrations of ribose,
and the kinetics appeared exponential with no induction period.
[0119] The question of whether glucose or other sugars can also
inhibit the formation of AGEs from the reactive intermediate
obtained by interrupted glycation in 0.5 M ribose was also
investigated. The effects of glucose at concentrations of 1.0-2.0 M
were tested (data not shown). Glucose was clearly not as inhibitory
as ribose. When the 24 h ribose interrupted sample was diluted
100-fold into these glucose solutions, the amount of antigenic AGE
formed was diminished by about 30%, but there was little decrease
in the apparent rate constant. Again, the kinetics appeared
exponential.
Effect of pH on Post-Amadori Kinetics of AGE Formation
[0120] The interrupted glycation method was used to investigate the
pH dependence of the post-Amadori kinetics of AGE formation from
the reactive intermediate. In these experiments, RNase A was first
reacted for 24 h with 0.5 M ribose at pH 7.5 to generate the
reactive intermediate. The kinetics of the decay of the
intermediate to AGEs were then measured by ELISA. FIG. 11 shows
that an extremely wide pH range of 5.0-9.5 was achievable when the
kinetics were measured by initial rates. A remarkable bell-shaped
dependence was observed, showing that the kinetics of antigenic
AGEs formation are decreased at both acidic and alkaline pH ranges,
with an optimum near pH 8.
[0121] A single "pH jump" experiment was also carried out on the pH
5.0 sample studied above which had the slowest rate of antigenic
AGE formation. After 12 days at 37.degree. C. in pH 5.0 buffer, the
pH was adjusted quickly to 7.5, and antigenic AGE formation was
monitored by ELISA. Within experimental error, the sample showed
identical kinetics (same first order rate constant) of AGE
formation to interrupted glycation samples that had been studied
directly at pH 7.5 (FIG. 12). In this experiment, the relative
amounts of antigenic AGE could not be directly compared on the same
ELISA plate, but the pH-jumped sample appeared to have formed
substantial though somehow diminished levels of antigenic AGEs.
These results demonstrate that intermediate can be prepared free of
AGE and stored at pH 5 for later studies of conversion to AGEs.
Inhibition of Post-Amadori AGE formation by Aminoguanidine
[0122] The efficacy of aminoguanidine was tested by this
interrupted glycation method, i.e., by testing its effect on
post-Amadori formation of antigenic AGEs after removal of excess
and reversibly bound ribose. FIG. 20A demonstrates that
aminoguanidine has modest effects on blocking the formation of
antigenic AGEs in RNase under these conditions, with little
inhibition below 50 mM. Approximately 50% inhibition is achieved
only at or above 100-250 mM. Note again that in these experiments,
the protein was exposed to aminoguanidine only after interruption
and removal of free and reversibly bound ribose. Comparable results
were also obtained with the interrupted glycation of BSA (FIG.
20B).
Amino Acid Analysis of Interrupted Glycation Samples
[0123] Amino acid analysis was carried out on RNase after 24 h
contact with 0.5 M ribose (undialyzed), after extensive dialysis of
the 24 h glycated sample, and after 5 days of incubation of the
latter sample at 37.degree. C. These determinations were made after
sodium cyanoborohydride reduction, which reduces Schiff base
present on lysines or the terminal amino group. All three samples,
normalized to alanine (12 residues), showed the same residual
lysine content (4.0.+-.0.5 out of the original 10 in RNase). This
indicates that after 24 h contact with 0.5 M ribose, most of the
formed Schiff base adducts had been converted to Amadori or
subsequent products. No arginine or histidine residues were lost by
modification.
Discussion
[0124] The use of rapidly reacting ribose and the discovery of its
reversible inhibition of post-Amadori steps have permitted the
dissection and determination of the kinetics of different steps of
protein glycation in RNase. Most previous kinetic studies of
protein "glycation" have actually been restricted to the "early"
steps of Schiff base formation and subsequent Amadori
rearrangement. Some kinetic studies have been carried out starting
with synthesized fructosylamines, i.e. small model Amadori
compounds of glucose (Smith and Thornalley, 1992, Eur. J. Biochem.
210:729-739, and references cited therein), but such studies, with
few exceptions, have hitherto not been possible with proteins. One
notable exception is the demonstration by Monnier (Odetti et al.,
1992, supra) that BSA partially glycated with ribose can rapidly
produce pentosidine after ribose removal. The greater reactivity of
ribose has also proven a distinct advantage in quantitatively
defining the time course of AGE formation. It is noted that glucose
and ribose are both capable of producing similar AGE products, such
as pentosidine (Grandhee & Monnier, 1991, supra; Dyer et al.
1991, supra), and some .sup.13C NMR model compound work has been
done with ADP-ribose (Cervantes-Laurean et al., 1993, Biochemistry
32:1528-1534). The present work shows that antigenic AGE products
of ribose fully cross-react with anti-AGE antibodies directed
against glucose-modified proteins, suggesting that ribose and
glucose produce similar antigenic AGEs. The primary kinetic
differences observed between these two sugars are probably due to
relative differences in the rate constants of steps leading to
post-Amadori AGE formation, rather than in the mechanism.
[0125] The results presented reveal a marked and paradoxical
inhibition of overall AGE formation by high concentrations of
ribose (FIG. 6) that has not been anticipated by earlier studies.
This inhibition is rapidly reversible in the sense that it is
removed by dialysis of initially modified protein (FIG. 7A) or by
simple 100-fold dilution (as used in FIG. 11). The experiments of
FIG. 10 demonstrate that it is not due to the accumulation of
dialyzable inhibitory intermediates during the initial glycation,
since dialysis of 24 h modified protein followed by addition of
different concentrations of fresh ribose induces the same
inhibition. The data of FIG. 10A, B show that the inhibition occurs
by reversible and rapid interaction of ribose with protein
intermediate containing reactive Amadori products. The inhibition
is unlikely to apply to the early step of formation of Amadori
product due to the rapid rate of formation of the presumed Amadori
intermediate that was determined in the experiment of FIG. 9. The
identification of the reactive intermediate as an Amadori product
is well supported by the amino acid analysis carried out (after
sodium cyanoborohydrate reduction) before and after dialysis at the
24 h interruption point. The unchanged residual lysine content
indicates that any dischageable Schiff bases have already been
irreversibly converted (presumably by Amadori rearrangement) by the
24 h time.
[0126] The secondary ribose suppression of "late" but not "early"
glycation steps significantly enhances the accumulation of a
fully-competent reactive Amadori intermediate containing little
AGE. Its isolation by the interruption procedure is of importance
for kinetic and structural studies, since it allows one to make
studies in the absence of free or Schiff base bound sugar and their
attendant reactions and complications. For example, the
post-Amadori conversion rates to antigenic AGE and pentosidine AGE
products have been measured (FIG. 7A, open symbols, and FIG. 8B),
and demonstrated to be much faster (t 1/2.about.10 h) than
reflected in the overall kinetics under uninterrupted conditions
(FIG. 6A and FIG. 8A). The rapid formation of pentosidine that was
measured appears consistent with an earlier interrupted ribose
experiment on BSA by Odetti et al. (1992, supra). Since ribose and
derivatives such as ADP-ribose are normal metabolites, the very
high rates of AGE formation seen here suggest that they should be
considered more seriously as sources of potential glycation in
various cellular compartments (Cervantes-Laurean et al., 1993,
supra), even though their concentrations are well below those of
the less reactive glucose.
[0127] Another new application of the isolation of intermediate is
in studying the pH dependence of this complex reaction. The unusual
bell-shaped pH profile seen for the post-Amadori AGE formation
(FIG. 11) is in striking contrast to the mild pH dependence of the
overall reaction. The latter kinetics reflect a composite effect of
pH on all steps in the reaction, including Schiff base and Amadori
product formation, each of which may have a unique pH dependence.
This complexity is especially well illustrated by studies of
hemoglobin glycation (Lowery et al., 1985, J. Biol. Chem.
260:11611-11618). The bell-shaped pH profile suggests, but does not
prove, the involvement of two ionizing groups. If true, the data
may imply the participation of a second amino group, such as from a
neighboring lysine, in the formation of dominant antigenic AGEs.
The observed pH profile and the pH-jump observations described
suggest that a useful route to isolating and maintaining the
reactive intermediate would be by the rapid lowering of the pH to
near 5.0 after 24 h interruption.
[0128] The kinetic studies provide new insights into the mechanisms
of action of aminoguanidine (guanylhydrazine), an AGE inhibitor
proposed by Cerami and co-workers to combine with Amadori
intermediates (Brownlee et al., 1986, supra). This proposed
pharmacological agent is now in Phase III clinical trials for
possible therapeutic effects in treating diabetes (Vlassara et al.,
1994, supra). However, its mechanism of AGE inhibition is likely to
be quite complex, since it is multifunctional. As a nucelophilic
hydrazine, it can reversibly add to active carbonyls, including
aldehydo carbonyls of open-chain glucose and ribose (Khatami et
al., 1988, Life Sci. 43:1725-1731; Hirsch et al., 1995, Carbohyd.
Res. 267:17-25), as well as keto carbonyls of Amadori compounds. It
is also a guanidinium compound that can scavange highly reactive
dicarbonyl glycation intermediates such as glyoxal and glucosones
(Chen & Cerami, 1993, J. Carbohyd. Chem. 12:731-742; Hirsch et
al., 1992, Carbohyd. Res. 232:125-130; Ou & Wolff, 1993,
Biochem. Pharmacol. 46:1139-1144). The interrupted glycation method
allowed examination of aminoguanidine efficacy on only post-Amadori
steps of AGE formation. Equally important, it allowed studies in
the absence of free sugar or dicarbonyl-reactive fragments from
free sugar (Wolff & Dean, 1987, Biochem. J. 245:243-250;
Wells-Knecht et al., 1995, Biochemistry 34:3702-3709) that can
combine with aminoguanidine. The results of FIG. 20 demonstrate
that aminoguanidine has, at best, only a modest effect on
post-Amadori AGE formation reactions, achieving 50% inhibition at
concentrations above 100-250 mM. The efficacy of aminoguanidine
thus predominantly arises either from inhibiting early steps of
glycation (Schiff base formation) or from scavenging highly
reactive dicarbonyls generated during glycation. Contrary to the
original claims, it does not appear to inhibit AGE formation by
complexing the Amadori intermediate.
[0129] The use of interrupted glycation is not limited for kinetic
studies. Interrupted glycation can simplify structural studies of
glycated proteins and identifying unknown AGEs using techniques
such as .sup.13C NMR that has been used to detect Amadori adducts
of RNase (Neglia et al., 1983, J. Biol. Chem. 258:14279-14283;
1985, J. Biol. Chem. 260:5406-5410). The combined use of structural
and kinetic approaches should also be of special interest. For
example, although the identity of the dominant antigenic AGEs
reacting with the polyclonal antibodies remains uncertain,
candidate AGEs, such as the recently proposed (carboxymethyl)lysine
(Reddy et al., 1995, Biochemistry 34:10872-10878; cf. Makita et
al., 1992, J. Biol. Chem. 267:5133-5138) should display the same
kinetics of formation from the reactive intermediate that we have
observed. The availability of the interrupted kinetics approach
will also help to determine the importance of the Amadori pathway
to the formation of this AGE. Similarly, monitoring of the
interrupted glycation reaction by techniques such as .sup.13C NMR
should identify resonances of other candidate antigenic AGEs as
being those displaying similar kinetics of appearance. Table I
lists the .sup.13C NMR peaks of the Amadori intermediate of RNase
prepared by reaction with C-2 enriched ribose. The downfield peak
near 205 ppm is probably due to the carbonyl of the Amadori
product. In all cases, the ability to remove excess free and Schiff
base sugars through interrupted glycation will considerably
simplify isolation, identification, and structural
characterization.
[0130] Table I lists the peaks that were assigned to the
Post-Amadori Intermediate due to their invariant or decreasing
intensity with time. Peak positions are listed in ppm downfield
from TMS. TABLE-US-00001 TABLE I 125 MHz C-13 NMR Resonances of
Ribonuclease Amadori Intermediate Prepared by 24 HR Reaction with
99% [2-C13]Ribose 216.5 ppm 108.5 ppm 211.7 105.9 208 103.9 103 172
95.8 165 163.9 73.65 162.1 70.2 69.7
[0131] Ribonuclease A was reacted for 24 hr with 0.5 M ribose 99%
enriched at C-2, following which excess and Schiff base bound
ribose was removed by extensive dialysis in the cold. The sample
was then warmed back to 37.degree. C. immediately before taking a 2
hr NMR scan. The signals from RNase reacted with natural abundance
ribose under identical conditions were then subtracted from the NMR
spectrum. Thus all peaks shown are due to enriched C-13 that
originated at the C-2 position. Some of the peaks arise from
degradation products of the intermediate, and these can be
identified by the increase in the peak intensity over time. FIG. 31
shows the NMR spectrum obtained.
EXAMPLE 3
In Vitro Inhibition of the Formation of Antigenic Advanced
Glycation End-Products (AGES) by Derivatives of Vitamins B.sub.1
and B.sub.6 and Aminoguanidine. Inhibition of Post-Amadori Kinetics
Differs from that of Overall Glycation
[0132] The interrupted glycation method for following post-Amadori
kinetics of AGE formation allows for the rapid quantitative study
of "late" stages of the glycation reaction. Importantly, this
method allows for inhibition studies that are free of pathways of
AGE formation which arise from glycoxidative products of free sugar
or Schiff base (Namiki pathway) as illustrated in Scheme I. Thus
the interrupted glycation method allows for the rapid and unique
identification and characterization of inhibitors of "late" stages
of glycation which lead to antigenic AGE formation.
[0133] Among the vitamin B.sub.1 and B.sub.6 derivatives examined,
pyridoxamine and thiamine pyrophosphate are unique inhibitors of
the post-Amadori pathway of AGE formation. Importantly, it was
found that efficacy of inhibition of overall glycation of protein,
in the presence of high concentrations of sugar, is not predictive
of the ability to inhibit the post-Amadori steps of AGE formation
where free sugar is removed. Thus while pyridoxamine, thiamine
pyrophosphate and aminoguanidine are potent inhibitors of AGE
formation in the overall glycation of protein by glucose,
aminoguanidine differs from the other two in that it is not an
effective inhibitor of post-Amadori AGE formation. Aminoguanidine
markedly slows the initial rate of AGE formation by ribose under
uninterrupted conditions, but has no effect on the final levels of
antigenic AGEs produced. Examination of different proteins (RNase,
BSA and hemoglobin), confirmed that the inhibition results are
generally non-specific as to the protein used, even though there
are individual variations in the rates of AGE formation and
inhibition.
Chemicals and Materials As in Example 1 above.
Preparation of polyclonal antibodies to AGEs As in Example 1
above.
ELISA detection of AGE products As in Example 1 above.
Uninterrupted Ribose Glycation Assays
[0134] Bovine serum albumin, ribonuclease A, and human hemoglobin
were incubated with ribose at 37.degree. C. in 0.4 M sodium
phosphate buffer of pH 7.5 containing 0.02% sodium azide. The
protein (10 mg/ml or 1 mg/ml), 0.05 M ribose, and prospective
inhibitors (at 0.5, 3, 15 and 50 mM) were introduced into the
incubation mixture simultaneously. Solutions were kept in the dark
in capped tubes. Aliquots were taken and immediately frozen until
analyzed by ELISA at the conclusion of the reaction. The
incubations were for 3 weeks (Hb) or 6 weeks (RNase, BSA).
Glycation reactions were monitored for constant pH throughout the
duration of the experiments.
Interrupted (Post-Amadori) Ribose Glycation Assays
[0135] Glycation was first carried out by incubating protein (10
mg/ml) with 0.5 M ribose at 37.degree. C. in 0.4 M phosphate buffer
at pH 7.5 containing 0.2% sodium azide for 24 h in the absence of
inhibitors. Glycation was then interrupted to remove excess and
reversibly bound (Schiff base) sugar by extensive dialysis against
frequent cold buffer changes at 4.degree. C. The glycated
intermediate samples containing maximal amount of Amadori product
and little AGE (depending on protein) were then quickly warmed to
37.degree. C. without re-addition of ribose. This initiated
conversion of Amadori intermediates to AGE products in the absence
or presence of various concentrations (typically 3, 15 and 50 mM)
of prospective inhibitors. Aliquots were taken and frozen at
various intervals for later analysis. The solutions were kept in
capped tubes and opened only to remove timed aliquots that were
immediately frozen for later carrying out the various analyses.
Numerical Analysis of Kinetics Data
[0136] Kinetics data (time progress curves) was routinely fit to
mono- or bi-exponential functions using non-linear least squares
methods utilizing either SCIENTIST 2.0 (MicroMath, Inc.) or ORIGIN
(Microcal, Inc.) software that permit user-defined functions and
control of parameters to iterate on. Standard deviations of the
parameters of the fitted functions (initial and final ordinate
values and rate constants) were returned as measures of the
precision of the fits. Apparent half-times for bi-exponential
kinetics fits were determined with the "solve" function of MathCad
software (MathSoft, Inc.).
Results
Inhibition by Vitamin B.sub.6 Derivatives of the Overall Kinetics
of AGE Formation from Ribose.
[0137] The inhibitory effects of vitamin B.sub.1 and B.sub.6
derivatives on the kinetics of antigenic AGE formation were
evaluated by polyclonal antibodies specific for AGEs. Initial
inhibition studies were carried out on the glycation of bovine
ribonuclease A (RNase) in the continuous presence of 0.05 M ribose,
which is the concentration of ribose where the rate of AGE
formation is near maximal. FIG. 13 (control curves, filled
rectangles) demonstrates that the formation of antigenic AGEs on
RNase when incubated with 0.05 M ribose is relatively rapid, with a
half-time of approximately 6 days under these conditions.
Pyridoxal-5'-phosphate (FIG. 13B) and pyridoxal (FIG. 13C)
significantly inhibited the rate of AGE formation on RNase at
concentrations of 50 mM and 15 mM. Surprisingly, pyridoxine, the
alcohol form of vitamin B.sub.6, also moderately inhibited AGE
formation on RNase (FIG. 13D). Of the B.sub.6 derivatives examined,
pyridoxamine at 50 mM was the best inhibitor of the "final" levels
of AGE formed on RNase over the 6-week time period monitored (FIG.
13A).
Inhibition by Vitamin B.sub.1 Derivatives of the Overall Kinetics
of AGE Formation from Ribose.
[0138] All of the B.sub.1 vitamers inhibited antigenic AGE
formation on RNase at high concentrations, but the inhibition
appeared more complex than for the B.sub.6 derivatives (FIG.
14A-C). In the case of thiamine pyrophosphate as the inhibitor
(FIG. 14A), both the rate of AGE formation and the final levels of
AGE produced at the plateau appeared diminished. In the case of
thiamine phosphate as the inhibitor (FIG. 14B), and thiamine (FIG.
14C), there appeared to be little effect on the rate of AGE
formation, but a substantial decrease in the final level of AGE
formed in the presence of the highest concentration of inhibitor.
In general, thiamine pyrophosphate demonstrated greater inhibition
than the other two compounds, at the lower concentrations
examined.
Inhibition by Aminoguanidine of the Overall Kinetics of AGE
Formation from Ribose
[0139] Inhibition of AGE formation by aminoguanidine (FIG. 14D) was
distinctly different from that seen in the B.sub.1 and B.sub.6
experiments. Increasing concentration of aminoguanidine decreased
the rate of AGE formation on RNase, but did not reduce the final
level of AGE formed. The final level of AGE formed after the
6-weeks was nearly identical to that of the control for all tested
concentrations of aminoguanidine.
Inhibition of the Overall Kinetics of AGE Formation in Serum
Albumin and Hemoglobin from Ribose
[0140] Comparative studies were carried out with BSA and human
methemoglobin (Hb) to determine whether the observed inhibition was
protein-specific. The different derivatives of vitamin B.sub.6
(FIG. 15A-D) and vitamin B.sub.1 (FIG. 16A-C) exhibited similar
inhibition trends when incubated with BSA as with RNase,
pyridoxamine and thiamine pyrophosphate being the most effective
inhibitors or each family. Pyridoxine failed to inhibit AGE
formation on BSA (FIG. 15D). Pyridoxal phosphate and pyridoxal
(FIG. 15B-C) mostly inhibited the rate of AGE formation, but not
the final levels of AGE formed. Pyridoxamine (FIG. 15A) exhibited
some inhibition at lower concentrations, and at the highest
concentration tested appeared to inhibit the final levels of AGE
formed more effectively than any of the other B.sub.6 derivatives.
In the case of B.sub.1 derivatives, the overall extent of
inhibition of AGE formation with BSA (FIG. 16A-C), was less than
that observed with RNase (FIG. 14A-C). Higher concentrations of
thiamine and thiamine pyrophosphate inhibited the final levels of
AGEs formed, without greatly affecting the rate of AGE formation
(FIG. 16C). Aminoguanidine again displayed the same inhibition
effects with BSA as seen with RNase (FIG. 16D), appearing to slow
the rate of AGE formation without significantly affecting the final
levels of AGE formed.
[0141] The kinetics of AGE formation was also examined using Hb in
the presence of the B.sub.6 and B.sub.1 vitamin derivatives, and
aminoguanidine. The apparent absolute rates of AGE formation were
significantly higher with Hb than with either RNase or BSA.
However, the tested inhibitors showed essentially similar behavior.
The effects of the vitamin B.sub.6 derivatives are shown in FIG.
17. Pyridoxamine showed the greatest inhibition at concentrations
of 3 mM and above (FIG. 17A), and was most effective when compared
to pyridoxal phosphate (FIG. 17B), pyridoxal (FIG. 17C), and
pyridoxine (FIG. 17D). In the case of the B.sub.1 vitamin
derivatives (data not shown), the inhibitory effects were more
similar to the BSA inhibition trends than to RNase. The inhibition
was only modest at the highest concentrations tested (50 mM), being
nearly 30-50% for all three B.sub.1 derivatives. The primary
manifestation of inhibition was in the reduction of the final
levels of AGE formed.
Inhibition by Vitamin B.sub.6 Derivatives of the Kinetics of
Post-Amadori Ribose AGE Formation
[0142] Using the interrupted glycation model to assay for
inhibition of the "late" post-Amadori AGE formation, kinetics were
examined by incubating isolated Amadori intermediates of either
RNase or BSA at 37.degree. C. in the absence of free or reversibly
bound ribose. Ribose sugar that was initially used to prepare the
intermediates was removed by cold dialysis after an initial
glycation reaction period of 24 h. After AGE formation is allowed
to resume, formation of AGE is quite rapid (half-times of about 10
h) in the absence of any inhibitors. FIG. 18 shows the effects of
pyridoxamine (FIG. 18A), pyridoxal phosphate (FIG. 18B), and
pyridoxal (FIG. 18C) on the post-Amadori kinetics of BSA.
Pyridoxine did not produce any inhibition (data not shown). Similar
experiments were carried out on RNase. Pyridoxamine caused nearly
complete inhibition of AGE formation with RNase at 15 mM and 50 mM
(FIG. 18D). Pyridoxal did not show any significant inhibition at 15
mM (the highest tested), but pyridoxal phosphate showed significant
inhibition at 15 mM. Pyridoxal phosphate is known to be able to
affinity label the active site of RNase (Raetz and Auld, 1972,
Biochemistry 11:2229-2236).
[0143] With BSA, unlike RNase, a significant amount of antigenic
AGE formed during the 24 h initial incubation with BSA (25-30%), as
evidenced by the higher ELISA readings after removal of ribose at
time zero for FIGS. 18A-C. For both BSA and RNase, the inhibition,
when seen, appears to manifest as a decrease in the final levels of
AGE formed rather than as a decrease in the rate of formation of
AGE.
Inhibition by Vitamin B.sub.1 Derivatives of the Kinetics of
Post-Amadori Ribose AGE Formation
[0144] Thiamine pyrophosphate inhibited AGE formation more
effectively than the other B.sub.1 derivatives with both RNase and
BSA (FIG. 19). Thiamine showed no effect, while thiamine phosphate
showed some intermediate effect. As with the B.sub.6 assays, the
post-Amadori inhibition was most apparently manifested as a
decrease in the final levels of AGE formed.
Effects of Aminoguanidine and N.sup..alpha.-acetyl-L-lysine on the
Kinetics of Post-Amadori Ribose AGE Formation
[0145] FIG. 20 shows the results of testing aminoguanidine for
inhibition of post-Amadori AGE formation kinetics with both BSA and
RNase. At 50 mM, inhibition was about 20% in the case of BSA (FIG.
20B), and less than 15% with RNase (FIG. 20A). The possibility of
inhibition by simple amino-containing functionalities was also
tested using N.sup..alpha.-acetyl-L-lysine (FIG. 21), which
contains only a free N.epsilon.-amino group.
N.sup..alpha.-acetyl-L-lysine at up to 50 mM failed to exhibit any
significant inhibition of AGE formation.
Discussion
[0146] Numerous studies have demonstrated that aminoguanidine is an
apparently potent inhibitor of many manifestations of nonenzymatic
glycation (Brownlee et al., 1986; Brownlee, 1992,1994, 1995). The
inhibitory effects of aminoguanidine on various phenomena that are
induced by reducing sugars are widely considered as proof of the
involvement of glycation in many such phenomena. Aminoguanidine has
recently entered into a second round of Phase III clinical trials
(as pimagedine) for ameliorating the complications of diabetes
thought to be caused by glycation of connective tissue proteins due
to high levels of sugar.
[0147] Data from the kinetic study of uninterrupted "slow" AGE
formation with RNase induced by glucose (Example 1) confirmed that
aminoguanidine is an effective inhibitor, and further identified a
number of derivatives of vitamins B.sub.1 and B.sub.6 as equally or
slightly more effective inhibitors. However, the inhibition by
aminoguanidine unexpectedly appeared to diminish in effect at the
later stages of the AGE formation reaction. Due to the slowness of
the glycation of protein with glucose, this surprising observation
could not be fully examined. Furthermore, it has been suggested
that there may be questions about the long-term stability of
aminoguanidine (Ou and Wolff, 1993, supra).
[0148] Analysis using the much more rapid glycation by ribose
allowed for the entire time-course of AGE formation to be
completely observed and quantitated during uninterrupted glycation
of protein. The use of interrupted glycation uniquely allowed
further isolation and examination of only post-Amadori antigenic
AGE formation in the absence of free and reversibly bound (Schiff
base) ribose. Comparison of the data from these two approaches with
the earlier glucose glycation kinetics has provided novel insights
into the mechanisms and effectiveness of various inhibitors. FIG.
22 are bar graphs which depict summarized comparative data of
percent inhibition at defined time points using various
concentrations of inhibitor. FIG. 22A graphs the data for
inhibition after interrupted glycation of RNase AGE formation in
ribose. FIG. 22B graphs the data for inhibition after interrupted
glycation of BSA AGE formation by ribose.
[0149] The overall results unambiguously demonstrate that
aminoguanidine slows the rate of antigenic AGE formation in the
presence of sugar but has little effect on the final amount of
post-Amadori AGE formed. Thus observations limited to only the
initial "early" stages of AGE formation which indicate efficacy as
an inhibitor may in fact be misleading as to the true efficacy of
inhibition of post-Amadori AGE formation. Thus the ability to
observe a full-course of reaction using ribose and interrupted
glycation gives a more complete picture of the efficacy of
inhibition of post-Amadori AGE formation.
EXAMPLE 4
Animal Model & Testing of in Vivo Effects of AGE
Formation/Inhibitors
[0150] Hyperglycemia can be rapidly induced (within one or two
days) in rats by administration of streptozocin (aka.
streptozotocin, STZ) or alloxan. This has become a common model for
diabetes melitus. However, these rats manifest nephropathy only
after many months of hyperglycemia, and usually just prior to death
from end-stage renal disease (ESRD). It is believed that this
pathology is caused by the irreversible glucose chemical
modification of long-lived proteins such as collagen of the
basement membrane. STZ-diabetic rats show albuminuria very late
after induction of hyperglycemia, at about 40 weeks usually only
just prior to death.
[0151] Because of the dramatic rapid effects of ribose demonstrated
in vitro in the examples above, it was undertaken to examine the
effects of ribose administration to rats, and the possible
induction of AGEs by the rapid ribose glycation. From this study, a
rat model for accelerated ribose induced pathology has been
developed.
Effects of Very Short-term Ribose Administration in Vivo
Phase I Protocol
[0152] Two groups of six rats each were given in one day either:
[0153] a. 300 mM ribose (two intraperitoneal infusions 6-8 hours
apart, each 5% of body weight as ml); or [0154] b. 50 mM ribose
(one infusion)
[0155] Rats were then kept for 4 days with no further ribose
administration, at which time they were sacrificed and the
following physiological measurements were determined: (i) initial
and final body weight; (ii) final stage kidney weight; (iii)
initial and final tail-cuff blood pressure; (iv) creatinine
clearance per 100 g body weight.
[0156] Albumin filtration rates were not measured, since no rapid
changes were initially anticipated. Past experience with
STZ-diabetic rats shows that albuminuria develops very late
(perhaps 40 weeks) after the induction of hyperglycemia and just
before animals expire.
Renal Physiology Results
[0157] a. Final body weight and final single kidney weight was same
for low and high ribose treatment groups.
[0158] b. Tail-cuff blood pressure increased from 66.+-.4 to
83.+-.3 to rats treated with low ribose (1.times.50 mM), and from
66.+-.4 to 106.+-.5 for rats treated with high ribose (2.times.300
mM). These results are shown in the bar graph of FIG. 23.
[0159] c. Creatinine clearance, as cc per 100 g body weight, was
decreased (normal range expected about 1.0-1.2) in a dose-dependent
fashion to 0.87.+-.0.15 for the low ribose group, and decreased
still further 30% to 0.62.+-.0.13 for the high ribose group. These
results are shown in the bar graph of FIG. 24.
Phase I Conclusion
[0160] A single day's ribose treatment caused a dose-dependent
hypertension and a dose-dependent decrease in glomerular clearance
function manifest 4 days later. These are significant metabolic
changes of diabetes seen only much later in STZ-diabetic rats.
These phenomenon can be hypothesized to be due to ribose
irreversible chemical modification (glycation) of protein in
vivo.
Effect of Exposure to Higher Ribose Concentrations for Longer
Time
Phase II Protocol
[0161] Groups of rats (3-6) were intraperitoneally given 0.3 M "low
ribose dose" (LR) or 1.0 M "high ribose dose" (HR) by twice-daily
injections for either (i) 1 day, (ii) a "short-term" (S) of 4 days,
or (iii) a "long-term" (L) of 8 days. Additionally, these
concentrations of ribose were included in drinking water.
Renal Physiology Results
[0162] a. Tail-cuff blood pressure increased in all groups of
ribose-treated rats, confirming Phase I results. (FIG. 23).
[0163] b. Creatinine clearance decreased in all groups in a ribose
dose-dependent and time-dependent manner (FIG. 24).
[0164] c. Albumin Effusion Rate (AER) increased significantly in a
ribose-dependent manner at 1-day and 4-day exposures. However, it
showed some recovery at 8 day relative to 4 day in the high-dose
group but not in the low-dose group. These results are shown in the
bar graph of FIG. 25.
[0165] d. Creatinine clearance per 100 g body weight decreased for
both low- and high-ribose groups to about the same extent in a
time-dependent manner (FIG. 24).
Phase II Conclusion
[0166] Exposure to ribose for as little as 4 days leads to
hypertension and renal dysfunction, as manifest by both decreased
creatinine clearance and increased albumin filtration. These
changes are typical of diabetes and are seen at much later times in
STZ-diabetic rats.
Intervention by Two New Therapeutic Compounds and
Aminoguanidine
Phase III Protocol
[0167] Sixty rats were randomized into 9 different groups,
including those exposed to 1 M ribose for 8 days in the presence
and absence of aminoguanidine, pyridoxamine, and thiamine
pyrophosphate as follows:
Control Groups:
[0168] (i) no treatment; [0169] (ii) high dose (250 mg/kg body
weight) of pyridoxamine ("compound-P"); [0170] (iii) high dose (250
mg/kg body weight of thiamine pyrophosphate ("compound-T" or
"TPP"); and [0171] (iv) low dose (25 mg/kg body weight) of
aminoguanidine ("AG"). Test Groups: [0172] (i) only 1 M
ribose-saline (2.times.9 cc daily IP for 8 days); [0173] (ii)
ribose plus low dose ("LP") of pyridoxamine (25 mg/kg body weight
injected as 0.5 ml with 9 cc ribose); [0174] (iii) ribose plus high
dose ("HP") of pyridoxamine (250 mg/kg body weight injected as 0.5
ml with 9 cc ribose); [0175] (iv) ribose plus high dose ("HT") of
thiamine pyrophosphate (250 mg/kg body weight injected as 0.5 ml
with 9 cc ribose); and [0176] (v) ribose plus low dose of amino
guanidine (25 mg/kg body weight injected as 0.5 ml with 9 cc
ribose).
[0177] Unlike Phase II, no ribose was administered in drinking
water. Intervention compounds were pre-administered for one day
prior to introducing them with ribose.
Renal Physiology Results
[0178] a. Blood pressure was very slightly increased by the three
compounds alone (control group); ribose-elevated BP was not
ameliorated by the co-administration of compounds. These results
are shown in the bar graph of FIG. 26.
[0179] b. Creatinine clearance in controls was unchanged, except
for TPP which diminished it.
[0180] c. Creatinine clearance was normalized when ribose was
co-administerd with low dose (25 mg/kg) of either aminoguanidine or
pyridoxamine. These results are shown in the bar graph of FIG.
27.
[0181] d. High concentrations (250 mg/kg) of pyridoxamine and TPP
showed only partial protection against the ribose-induced decrease
in creatinine clearance (FIG. 27).
[0182] e. Albumin effusion rate (AER) was elevated by ribose, as
well as by high dose of pyridoxamine and TPP, and low dose of
aminoguanidine in the absence of ribose. These results are shown in
the bar graph of FIG. 28.
[0183] f. Albumin effusion rate was restored to normal by the
co-administration of low dose of both aminoguanidine and
pyridoxamine. These results are shown in the bar graph of FIG.
29.
Phase III Conclusions
[0184] As measured by two indicies of renal function, pyridoxamine
and aminoguanidine, both at 25 mg/kg, were apparently effective,
and equally so, in preventing the ribose-induced decrease in
creatinine clearance and ribose-induced mild increase in
albuminuria.
[0185] (i) Thiamine pyrophosphate was not tested at 25 mg/kg; (ii)
thiamine pyrophosphate and pyridoxamine at 250 mg/kg were partially
effective in preventing creatinine clearance decreases but possibly
not in preventing mild proteinuria; (iii) at these very high
concentrations and in the absence of ribose, thiamine pyrophosphate
alone produced a decrease in creatinine clearance, and both
produced mild increases in albuminuria.
Summary
Renal Function and Diabetes
[0186] Persistent hyperglycemia in diabetes mellitus leads to
diabetic nephropathy in perhaps one third of human patients.
Clinically, diabetic nephropathy is defined by the presence of:
[0187] 1. decrease in renal function (impaired glomerular
clearance)
[0188] 2. an increase in urinary protein (impaired filtration)
[0189] 3. the simultaneous presence of hypertension
[0190] Renal function depends on blood flow (not measured) and the
glomerular clearance, which can be measured by either inulin
clearance (not measured) or creatinine clearance. Glomerular
permeability is measured by albumin filtration rate, but this
parameter is quite variable. It is also a log-distribution
function: a hundred-fold increase in albumin excretion represents
only a two-fold decrease in filtration capacity.
Ribose Diabetic Rat Model
[0191] By the above criteria, ribose appears to very rapidly induce
manifestations of diabetic nephropathy, as reflected in
hypertension, creatinine clearance and albuminuria, even though the
latter is not large. In the established STZ diabetic rat,
hyperglycemia is rapidly established in 1-2 days, but clinical
manifestations of diabetic nephropathy arise very late, perhaps as
much as 40 weeks for albuminuria. In general, albuminuria is highly
variable from day to day and from animal to animal, although unlike
humans, most STZ rats do eventually develop nephropathy.
Intervention by Compounds
[0192] Using the ribose-treated animals, pyridoxamine at 25 mg/kg
body weight appears to effectively prevent two of the three
manifestations usually attributed to diabetes, namely the
impairment of creatinine clearance and albumin filtration. It did
so as effectively as aminoguanidine. The effectiveness of thiamine
pyrophosphate was not manifest, but it should be emphasized that
this may be due to its use at elevated concentrations of 250 mg/kg
body weight. Pyridoxamine would have appeared much less effective
if only the results at 250 mg/kg body weight are considered.
Effect of Compounds Alone
[0193] Overall, the rats appeared to tolerate the compounds well.
Kidney weights were not remarkable and little hypertension
developed. The physiological effects of the compounds were only
tested at 250 mg/kg. Thiamine pyrophosphate, but not pyridoxamine,
appeared to decrease creatinine clearance at this concentration.
Both appeared to slightly increase albuminuria, but these
measurements were perhaps the least reliable.
Human Administration
[0194] A typical adult human being of average size weighs between
66-77 Kg. Typically, diabetic patients may tend to be overweight
and can be over 112 Kg. The Recommended dietary allowances for an
adult male of between 66-77 Kg, as revised in 1989, called for 1.5
mg per day of thiamine, and 2.0 mg per day of Vitamin B.sub.6
(Merck Manual of Diagnosis and Therapy, 16th edition (Merck &
Co., Rathaway, N.J., 1992) pp 938-939).
[0195] Based upon the rat model approach, a range of doses for
administration of pyridoxamine or thiamine pyrophosphate that is
predicted to be effective for inhibiting post-Amadori AGE formation
and thus inhibiting related pathologies would fall in the range of
1 mg/100 g body weight to 200 mg/100 g body weight. The appropriate
range when co-administered with aminoguanidine will be similar.
Calculated for an average adult of 75 Kg, the range (at for example
1 mg/l Kg body weight) can be approximately 75 mg to upwards of 150
g (at for example 2 g/l Kg body weight). This will naturally vary
according to the particular patient.
EXAMPLE 5
Inhibition of Advanced Glycation End-Product (AGE) Formation by
Pyridoxamine-5'-Phosphate (PMP)
[0196] ##STR4##
[0197] Current data (FIG. 32B) utilizing the interrupted glycation
assay as described above has demonstrated that AGE formation is
inhibited by administration of Pyridoxamine-5'-Phosphate (PMP) as
compared to PM.
[0198] The instant invention teaches pharmaceutical compositions
comprising PMP, or salts thereof, in suitable pharmaceutical
carriers for treatment of AGE related disorders.
[0199] Thus the instant invention further teaches a method for
inhibiting post-Amadori AGE formation comprising administering an
effective post-Amadori AGE inhibiting amount of
pyridoxamine-5'-Phosphate. Also encompassed is a method of
inhibiting protein cross-linking by the administration of an
effective post-Amadori AGE inhibiting amount of
pyridoxamine-5'-Phosphate.
EXAMPLE 6
In Vivo Inhibition of the Formation of Advanced Glycation
End-Products (AGEs) by Derivatives of Vitamin B.sub.6 and
Aminoguanidine. Inhibition of Diabetic Nephropathy
[0200] The interrupted glycation method, as described in the
examples above, allows for the rapid generation of stable
well-defined protein Amadori intermediates from ribose and other
pentose sugars for use in in vivo studies.
[0201] The effects of 25 mg/kg/day pyridoxamine (PM) and
aminoguanidine (AG) on renal pathology induced by injecting
Sprague-Dawley rats daily with 50 mg/kg/day of ribose-glycated
Amadori-rat serum albumin (RSA), AGE-RSA, and unmodified RSA for 6
weeks. Hyperfiltration (increased creatinine clearance) was
transiently seen with rats receiving Amadori-RSA and AGE-RSA,
regardless of the presence of PM and AG.
[0202] Individuals from each group receiving Amadori-RSA and
AGE-RSA exhibited microalbuminuria, but none was seen if PM was
co-administered. Immunostaining with anti-RSA revealed glomerular
staining in rats treated with AGE-RSA and with Amadori-RSA; and
this staining was decreased by treatment with PM but not by AG
treatment. A decrease in glomerular sulfated glycosaminoglycans
(Alcian blue pH 1.0 stain) was also found in rats treated with
glycated (Amadori and AGE) RSA. This appears to be due to reduced
heparan sulfate proteoglycans (HSPG), as evidenced by diminished
staining with mAb JM-403 that is specific for HSPG side-chain.
These HSPG changes were ameliorated by treatment with PM, but not
by AG treatment.
[0203] Thus we conclude that pyridoxamine can prevent both
diabetic-like glomerular loss of heparan sulfate and glomerular
deposition of glycated albumin in SD rats chronically treated with
ribose-glycated albumin.
Materials and Methods
Chemicals
[0204] Rat serum albumin (RSA) (fraction V, essentially fatty
acid-free 0.005%; A2018), D-ribose, pyridoxamine, and goat alkaline
phosphatase-conjugated anti-rabbit IgG were all from Sigma
Chemicals. Aminoguanidine hydrochloride was purchased from Aldrich
Chemicals.
Preparation of Ribated RSA
[0205] Rat serum albumin was passed down an Affi-Gel Blue column
(Bio-Rad), a heparin-Sepharose CL-6B column (Pharmacia) and an
endotoxin-binding affinity column (Detoxigel, Pierce Scientific) to
remove any possible contaminants. The purified rat serum albumin
(RSA) was then dialyzed in 0.2 M phosphate buffer (pH 7.5). A
portion of the RSA (20 mg/ml) was then incubated with 0.5 M ribose
for 12 hours at 37.degree. C. in the dark. After the 12 hour
incubation the reaction mixture was dialyzed in cold 0.2 M sodium
phosphate buffer over a 36 hour period at 4.degree. C. (this
dialysis removes not only the free ribose, but also the Schiff-base
intermediaries). At this stage of the glycation process, the
ribated protein is classified as Amadori-RSA and is negative for
antigenic AGEs, as determined by antibodies reactive with AGE
protein (as described previously; R618, antigen:glucose modified
AGE-Rnase). The ribated protein is then divided into portions that
will be injected either as: a)Amadori-RSA, b)NaBH.sub.4-reduced
Amadori-RSA, c)AGE-RSA.
[0206] The ribated protein to be injected as Amadori-RSA is simply
dialyzed against cold PBS at 4.degree. C. for 24 hours. A portion
of the Amadori-RSA in 0.2 M sodium phosphate is reduced with
NaBH.sub.4 to form NaBH.sub.4-reduced Amadori-RSA. Briefly,
aliquots were reduced by adding 5 uL of NaBH.sub.4 stock solution
(100 mg/ml in 0.1 M NaOH) per mg of protein, incubated for 1 hour
at 37.degree. C., treated with HCl to discharge excess NaBH.sub.4,
and then dialyzed extensively in cold PBS at 4.degree. C. for 36
hours. The AGE-RSA was formed by reincubating the Amadori-RSA in
the absence of sugar for 3 days. The mixture was then dialyzed
against cold PBS at 4.degree. C. for 24 hours. All solutions were
filtered (22 um filter) sterilized and monitored for endotoxins by
a limulus amoebocyte lysate assay (E-Toxate, Sigma Chemical) and
contained <0.2 ng/ml before being frozen (-70.degree. C.) down
into individual aliquots until it was time for injection.
Animal Studies
[0207] Male Sprague-Dawley rats (Sasco, 100 g) were used. After a 1
week adaptation period, rats were placed in metabolic cages to
obtain a 24 hour urine specimen for 2 days before administration of
injections. Rats were then divided into experimental and control
groups and given tail vein injections with either saline,
unmodified RSA (50 mg/kg), Amadori-RSA (50 mg/kg),
NaBH.sub.4-reduced Amadori-RSA (50 mg/kg), or AGE-RSA (50
mg/kg).
[0208] Rats injected with Amadori-RSA and AGE-RSA were then either
left untreated, or futher treated by the administration of either
aminoguanidine (AG; 25 mg/kg), pyridoxamine (PM; 25 mg/kg), or a
combination of AG and PM (10 mg/kg each) through the drinking
water. Body weight and water intake of the rats were monitored
weekly in order to adjust dosages. At the conclusion of the
experimental study the rats were placed in metabolic cages to
obtain 24 hour urine specimen for 2 days prior to sacrificing the
animals.
[0209] Total protein in the urine samples was determined by Bio-Rad
assay. Albumin in urine was determined by competitive ELISA using
rabbit anti-rat serum albumin (Cappell) as primary antibody (
1/2000) and goat anti-rabbit IgG (Sigma Chemical) as a secondary
antibody ( 1/2000). Urine was tested with Multistix 8 SG (Miles
Laboratories) for glucose, ketone, specific gravity, blook, pH,
protein, nitrite, and leukocytes. Nothing remarkable was detected
other than some protein.
[0210] Creatinine measurements were performed with a Beckman
creatinine analyzer II. Blood samples were collected by heart
puncture before termination and were used in the determination of
creatinine clearance, blood glucose (glucose oxidase, Sigma
chemical), fructosamine (nitroblue tetrazolium, Sigma chemical),
and glycated Hb (columns, Pierce chemicals). Aorta, heart, both
kidneys and the rat tail were visually inspected and then quickley
removed after perfusing with saline through the right ventricle of
the heart. One kidney, aorta, rat tail, and the lower 2/3 of the
heart were snap-frozen and then permanently stored at -70.degree.
C. The other kidney was sectioned by removing both ends (cortex) to
be snap-frozen, with the remaining portions of the kidney being
sectioned into thirds with two portions being placed into neutral
buffered formalin and the remaining third minced and placed in 2.5%
glutaraldehyde/2% paraformaldehyde.
Light Microscopy
[0211] After perfusion with saline, kidney sections were fixed in
ice-cold 10% neutral buffered formalin. Paraffin-embedded tissue
sections from all rat groups (n=4 per group) were processed for
staining with Harris' alum hematoxylin and eosin (H&E), perodic
acid/Schiff reagent (PAS), and alcian blue (pH 1.0 and pH 2.5)
stains for histological examination. The alcian blue sections were
scored by two investigators in a blinded fashion.
Electron Microscopy
[0212] Tissues were fixed in 2.5% glutaraldehyde/2%
paraformaldehyde (0.1 M sodium cacodylate, pH 7.4), post-fixed for
1 hour in buffered osmium tetroxide (1.0%), prestained in 0.5%
uranyl acetate for 1 hour and embedded in Effapoxy resin. Ultrathin
sections were examined by electron microscopy.
Immunofluorescence
[0213] Parrafin-embedded sections were deparaffinized and then
blocked with 10% goat serum in PBS for 30 min at room temperature.
The sections were then incubated for 2 hour at 37.degree. C. with
primary antibody, either affinity purified polyclonal rabbit
anti-AGE antibody, or a polyclonal sheep anti-rat serum albumin
antibody (Cappell). The sections were then rinsed for 30 min with
PBS and incubated with secondary antibody, either affinity purified
FITC-goat anti-rabbit IgG (H+L) double stain grade (Zymed) or a
Rhodamine-rabbit anti-sheep IgG (whole) (Cappell) for 1 hour at
37.degree. C. The sections were then rinsed for 30 min with PBS in
the dark, mounted in aqueous mounting media for immunocytochemistry
(Biomeda), and cover slipped. Sections were scored in a blinded
fashion. Kidney sections were evaluated by the number and intensity
of glomerular staining in 5 regions around the periphery of the
kidney. Scores were normalized for the immunofluorescent score per
100 glomeruli with a scoring system of 0-3.
Preparation of Polyclonal Antibodies to AGE-Proteins
[0214] Immunogen was prepared by glycation of BSA (R479 antibodies)
or Rnase (R618 antibodies) at 1.6 g protein in 15 ml for 60-90 days
using 1.5 M glucose in 0.4 M phosphate containing 0.05% sodium
azide at pH 7.4 and 37.degree. C. New Zealand white rabbit males of
8-12 weeks were immunized by subcutaneous administration of a 1 ml
solution containing 1 mg/ml of glycated protein in Freund's
adjuvant. The primary injection used the complete adjuvant and
three boosters were made at three week intervals with Freund's
incomplete adjuvant. The rabbits were bled three weeks after the
last booster. The serum was collected by centrifugation of clotted
whole blood. The antibodies are AGE-specific, being unreactive with
either native proteins or with Amadori intermediates.
ELISA Detection of AGE Products
[0215] The general method of Engvall (21) was used to perform the
ELISA. Glycated protein samples were diluted to approximately 1.5
ug/ml in 0.1 M sodium carbonate buffer of pH 9.5 to 9.7. The
protein was coated overnight at room temperature onto a 96-well
polystyrene plate by pippetting 200 ul of protein solution into
each well (about 0.3 ug/well). After coating, the excess protein
was washed from the wells with a saline solution containing 0.05%
Tween-20. The wells were then blocked with 200 ul of 1% casein in
carbonate buffer for 2 hours at 37.degree. C. followed by washing.
Rabbit anti-AGE antibodies were diluted at a titer of 1:350 in
incubation buffer and incubated for 1 hour at 37.degree. C.,
followed by washing. In order to minimize background readings,
antibody R618 used to detect glycated RSA was generated by
immunization against glycated Rnase. An alkaline
phosphatase-conjugated antibody to rabbit IgG was then added as the
secondary antibody at a titer of 1:2000 and incubated for 1 hour at
37.degree. C., followed by washing. The p-nitrophenolate being
monitored at 410 nm with a Dynatech MR4000 microplate reader.
Results
[0216] The rats in this study survived the treatments and showed no
outward signs of any gross pathology. Some of the rats showed some
small weight changes and tail scabbing.
[0217] Initial screening of kidney sections with PAS and H&E
stains did not reveal any obvious changes, and some EM sections did
not reveal any gross changes in the glomerular basement membrane
(GBM). However, upon Alcian Blue staining, striking differences
were discovered. Alcian blue staining is directed towards
negatively charged groups in tissues and can be made selective via
changes in the pH of staining. At pH 1.0 Alcian blue is selective
for mucopolysaccharides, and at pH 2.5 detects glucoronic groups.
Thus negative charges are detected depending upon the pH of the
stain.
[0218] At pH 2.5 Alcian blue staining showed that Amadori-RSA
(p<0.05) and AGE-RSA (p<0.01) induced increased staining for
acidic glycosaminoglycans (GAG) over control levels (FIG. 33). For
both AGE-RSA and Amadori-RSA, treatment with pyridoxamine (PM)
prevented the increase in staining (p<0.05 as compared with
controls). In contrast, treatment with aminoguanidine (AG) or
combined PM and AG at 10 mg/kg each, did not prevent the
increase.
[0219] At pH 1.0 Alcian blue staining was significantly decreased
by AGE-RSA (p<0.001) (FIG. 34). However, no significant
difference was seen with Amadori-RSA. Due to faint staining,
treatment with PM, AG and combined could not be quantitated.
[0220] Immunofluorescent glomerular staining for RSA showed
elevated staining with Amadori-RSA and AGE-RSA injected animals
(FIG. 35). Significant reduction of this effect was seen in the
rats treated with PM, and not with AG or combined AG & PM.
[0221] Immunofluorescent glomerular staining for Heparan Sulfate
Proteoglycan Core protein showed slightly reduced staining with
Amadori-RSA and AGE-RSA injected animals but were not statistically
significant(FIG. 36). A reduction of this effect was seen in the
rats treated with PM, and not with AG or combined AG & PM.
However, immunofluorescent glomerular staining for Heparan Sulfate
Proteoglycan side-chain showed highly reduced staining with
Amadori-RSA and AGE-RSA injected animals (FIG. 37). A significant
reduction of this effect was seen in the rats treated with PM, and
not with AG or combined AG & PM.
[0222] Analysis of average glomerular volume by blinded scoring
showed that Amadori-RSA and AGE-RSA caused significant increase in
average glomeruli volume (FIG. 38). A significant reduction of this
effect was seen with treatment of the rats with PM. No effect was
seen with treatment with AG or combined AG and PM at 10 mg/kg
each.
EXAMPLE 7
AGE Inhibitor Compounds
[0223] The present invention encompasses compounds, and
pharmaceutical compositions containing compounds having the general
formula: ##STR5## wherein R.sub.1 is CH.sub.2NH.sub.2, CH.sub.2SH,
COOH, CH.sub.2CH.sub.2NH.sub.2, CH.sub.2CH.sub.2SH, or
CH.sub.2COOH; [0224] R.sub.2 is OH, SH or NH.sub.2; [0225] Y is N
or C, such that when Y is N R.sub.3 is nothing, and when Y is C,
R.sub.3 is NO.sub.2 or another electron withdrawing group; and
salts thereof.
[0226] The present invention also encompasses compounds of the
general formula ##STR6## wherein R.sub.1 is CH.sub.2NH.sub.2,
CH.sub.2SH, COOH, CH.sub.2CH.sub.2NH.sub.2, CH.sub.2CH.sub.2SH, or
CH.sub.2COOH; [0227] R.sub.2 is OH, SH or NH.sub.2; [0228] Y is N
or C, such that when Y is N R.sub.3 is nothing, and when Y is C,
R.sub.3 is NO.sub.2 or another electron withdrawing group; [0229]
R.sub.4 is H, or C 1-6 alkyl; [0230] R.sub.5 and R.sub.6 are H, C
1-6 alkyl, alkoxy or alkane; and salts thereof.
[0231] In addition, the instant invention also envisions compounds
of the formulas ##STR7##
[0232] The compounds of the present invention can embody one or
more electron withdrawing groups, such as and not limited to
--NH.sub.2, --NHR', --NR'.sub.2, --OH, --OCH.sub.3, --OCR', and
--NH--COCH.sub.3 where R' is C 1-6 alkyl.
[0233] In a preferred embodiment at least one of R.sub.4, R.sub.5
and R.sub.6 are H. The present invention also encompasses compounds
wherein R.sub.4 and R.sub.5 are H, C 1-6 alkyl, alkoxy or alkene.
In keeping with the present invention, it is also encompassed that
R.sub.2 and R.sub.6 can be H, OH, SH, NH.sub.2, C 1-6 alkyl, alkoxy
or alkene. It is also envisioned that R.sub.4, R.sub.5 and R.sub.6
can be larger functional groups, such as and not limited to
phosphate, aryl, heteroaryl, and cycloalkyl alkoxy groups.
[0234] As used herein, the term "aryl" refers to aromatic
carbocyclic groups having a single ring (e.g., phenyl), multiple
rings (e.g., biphenyl), or multiple condensed rings in which at
least one is aromatic, (e.g., 1,2,3,4-tetrahydronaphthyl, naphthyl,
anthryl, or phenanthryl), which can optionally be substituted with
e.g., halogen, lower alkyl, lower alkylthio, trifluoromethyl, lower
acyloxy, aryl, and heteroaryl.
[0235] A preferred aryl group is phenyl optionally substituted with
up to five groups selected independently from halogen, cyano,
hydroxy, straight or branched chain lower alkyl having 1-6 carbon
atoms or cycloalkyl having 3-7 carbon atoms, amino, mono or
dialkylamino where each alkyl is independently straight or branched
chain lower alkyl having 1-6 carbon atoms or cycloalkyl having 3-7
carbon atoms, straight or branched chain lower alkoxy having 1-6
carbon atoms, cycloalkyl alkoxy having 3-7 carbon atoms, or
NR1COR.sup.2, COR.sup.2, CONR.sup.1R.sup.2 or CO.sub.2R.sup.2 where
R.sup.1 and R.sup.2 are the same or different and represent
hydrogen or straight or branched chain lower alkyl having 1-6
carbon atoms or cycloalkyl having 3-7 carbon atoms
[0236] By heteroaryl is meant aromatic ring systems having at least
one and up to four hetero atoms selected from the group consisting
of nitrogen, oxygen and sulfur. Examples of heteroaryl groups are
pyridyl, pyrimidinyl, pyrrolyl, pyrazolyl, pyrazinyl, pyridazinyl,
oxazolyl, napthyridinyl, isoxazolyl, phthalazinyl, furanyl,
quinolinyl, isoquinolinyl, thiazolyl, and thienyl, which can
optionally be substituted with, e.g., halogen, lower alkyl, lower
alkoxy, lower alkylthio, trifluoromethyl, lower acyloxy, aryl,
heteroaryl, and hydroxy.
[0237] The aryl and heteroaryl groups herein are systems
characterized by 4n+2.cndot. electrons, where n is an integer.
[0238] In addition to those mentioned above, other examples of the
aryl and heteroaryl groups encompassed within the invention are the
following: ##STR8##
[0239] As noted above, each of these groups can optionally be mono-
or polysubstituted with groups selected independently from, for
example, halogen, lower alkyl, lower alkoxy, lower alkylthio,
trifluoromethyl, lower acyloxy, aryl, heteroaryl, and hydroxy.
[0240] Still other examples of various aryl and heteroaryl groups
are shown in Chart D of published International Application Wo
93/17025 (hereby incorporated by reference).
[0241] As used herein "cycloalkyl alkoxy" refers to groups of the
formula ##STR9## where a is an integer of from 2 to 6; R' and R''
independently represent hydrogen or alkyl; and b is an integer of
from 1 to 6.
[0242] By "alkyl" and "lower alkyl" in the present invention is
meant straight or branched chain alkyl groups having 1-12 carbon
atoms, such as, for example, methyl, ethyl, propyl, isopropyl,
n-butyl, sec-butyl, tert-butyl, pentyl, 2-pentyl, isopentyl,
neopentyl, hexyl, 2-hexyl, 3-hexyl, and 3-methylpentyl. Unless
indicated otherwise, the alkyl group substituents herein are
optionally substituted with at least one group independently
selected from hydroxy, mono- or dialkyl amino, phenyl or
pyridyl.
[0243] By "alkyl" and "lower alkyl" in the present invention is
meant straight or branched chain alkyl groups having from 1-12
carbon atoms, such as, for example, methyl, ethyl, propyl,
isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, 2-pentyl,
isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl, and 3-methylpentyl.
Unless indicated otherwise, the alkyl group substituents herein are
optionally substituted with at least one group independently
selected from hydroxy, mono- or dialkyl amino, phenyl or
pyridyl.
[0244] By "alkoxy" and "lower alkoxy" in the present invention is
meant straight or branched chain alkoxy groups having 1-6 carbon
atoms, such as, for example, methoxy, ethoxy, propoxy, isopropoxy,
n-butoxy, sec-butoxy, tert-butoxy, pentoxy, 2-pentyl, isopentoxy,
neopentoxy, hexoxy, 2-hexoxy, 3-hexoxy, and 3-methylpentoxy.
[0245] By "alkene" and "lower alkene" in the present invention is
meant straight and branched chain alkene groups having 1-6 carbon
atoms, such as, for example, ethlene, propylene, 1-butene,
1-pentene, 1-hexene, cis and trans 2-butene or 2-pentene,
isobutylene, 3-methyl-1-butene, 2-methyl-2-butene, and
2,3-dimethyl-2-butene.
[0246] By "salts thereof" in the present invention is meant
compounds of the present invention as salts and metal complexes
with said compounds, such as with, and not limited to, Al,Zn, Mn,
Cu, and Fe.
[0247] One of ordinary skill in the art will be able to make
compounds of the present invention using standard methods and
techniques.
[0248] The instant invention encompasses pharmaceutical
compositions which comprise one or more of the compounds of the
present invention, or salts thereof, in a suitable carrier. The
instant invention encompasses methods for administering
pharmaceuticals of the present invention for therapeutic
intervention of pathologies which are related to AGE formation in
vivo. In one preferred embodiment of the present invention the AGE
related pathology to be treated is related to diabetic
nephropathy.
[0249] The compounds of the invention may be formulated as a
solution of lyophilized powders for paraenteral administration.
Powders may be reconstituted by addition of a suitable diluent or
other pharmaceutically acceptable carrier prior to use. The liquid
formulation is generally a buffered, isotonic, aqueous solution.
Examples of suitable diluents are normal isotonic saline solution,
standard 5% dextrose in water or in buffered sodium of ammonium
acetate solution. Such formulation is especially suitable for
paraenteral administration, but may also be used for oral
administration. It may be desirable to add excipients such as
polyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia,
polyethylene glycol, mannitol, sodium choride or sodium
citrate.
[0250] Alternatively, the compounds of the present invention may be
encapsulated, tableted or prepared in an emulsion (oil-in-water or
water-in-oil) syrup for oral administration. Pharmaceutically
acceptable solids or liquid carriers, which are generally known in
the pharmaceutical formulary arts, may be added to enhance or
stabilize the composition, or to facilitate preparation of the
composition. Solid carriers include starch (corn or potato),
lactose, calcium sulfate dihydrate, terra alba, croscarmellose
sodium, magnesium stearate or stearic acid, talc, pectin, acacia,
agar, gelatin, or colloidal silicon dioxide. Liquid carriers
include syrup, peanut oil, olive oil, saline and water. The carrier
may also include a sustained release material such as glyceryl
monostearate or glyceryl distearate, alone or with a wax. The
amount of solid carrier varies but, preferably, will be between
about 1 mg to about 1 g per dosage unit.
[0251] The instant invention may be embodied in other forms or
carried out in other ways without departing from the spirit or
essential characteristics thereof. The present disclosure and
enumerated examples are therefore to be considered as in all
respects illustrative and not restrictive, the scope of the
invention being indicated by the appended claims, and all
equivalency are intended to be embraced therein. One of ordinary
skill in the art would be able to recognize equivalent embodiments
of the instant invention, and be able to practice such embodiments
using the teaching of the instant disclosure and only routine
experimentation.
* * * * *