U.S. patent application number 11/351655 was filed with the patent office on 2007-02-01 for preventing and reversing the formation of advance glycosylation endproducts.
Invention is credited to Anthony Cerami, John J. Egan, San-Bao Hwang, Peter C. Ulrich, Sara Vasan, Dilip R. Wagle.
Application Number | 20070025926 11/351655 |
Document ID | / |
Family ID | 27409228 |
Filed Date | 2007-02-01 |
United States Patent
Application |
20070025926 |
Kind Code |
A1 |
Cerami; Anthony ; et
al. |
February 1, 2007 |
Preventing and reversing the formation of advance glycosylation
endproducts
Abstract
The present invention relates to compositions and methods for
inhibiting and reversing nonenzymatic cross-linking (protein
aging). Accordingly, compositions are disclosed which comprise an
agent capable of inhibiting the formation of advanced glycosylation
endproducts of target proteins, and which additionally reverse
pre-formed crosslinks in the advanced glycosylation endproducts by
cleaving alpha-dicarbonyl-based protein crosslinks present in the
advanced glycosylation endproducts. Certain useful agents are
thiazolium salts. The method comprises contacting the target
protein with the composition. Both industrial and therapeutic
applications for the invention are envisioned, as food spoilage and
animal protein aging can be treated. A novel immunoassay for
detection of the reversal of the nonenzymatic crosslinking is also
disclosed.
Inventors: |
Cerami; Anthony; (Shelter
Island, NY) ; Ulrich; Peter C.; (Old Tappan, NJ)
; Wagle; Dilip R.; (Valley Cottage, NY) ; Hwang;
San-Bao; (Sudbury, MA) ; Vasan; Sara;
(Yonkers, NY) ; Egan; John J.; (Mountain Lakes,
NJ) |
Correspondence
Address: |
MINTZ, LEVIN, COHN, FERRIS, GLOVSKY;AND POPEO, P.C.
ONE FINANCIAL CENTER
BOSTON
MA
02111
US
|
Family ID: |
27409228 |
Appl. No.: |
11/351655 |
Filed: |
February 9, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10418398 |
Apr 18, 2003 |
7022719 |
|
|
11351655 |
Feb 9, 2006 |
|
|
|
10174883 |
Jun 19, 2002 |
|
|
|
10418398 |
Apr 18, 2003 |
|
|
|
09470482 |
Dec 22, 1999 |
6440749 |
|
|
10174883 |
Jun 19, 2002 |
|
|
|
08971878 |
Nov 19, 1997 |
6007865 |
|
|
09470482 |
Dec 22, 1999 |
|
|
|
08588249 |
Jan 18, 1996 |
5853703 |
|
|
08971878 |
Nov 19, 1997 |
|
|
|
08473184 |
Jun 7, 1995 |
|
|
|
08588249 |
Jan 18, 1996 |
|
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08375155 |
Jan 18, 1995 |
5656261 |
|
|
08473184 |
Jun 7, 1995 |
|
|
|
Current U.S.
Class: |
424/49 ; 514/365;
548/204 |
Current CPC
Class: |
G01N 33/6842 20130101;
C07D 277/22 20130101; A61P 7/00 20180101; A61P 19/02 20180101; A61P
3/10 20180101; A61Q 19/00 20130101; A61Q 19/08 20130101; A61K
31/428 20130101; G01N 33/68 20130101; G01N 2500/02 20130101; A61P
27/02 20180101; A61Q 11/00 20130101; A61P 13/02 20180101; G01N
33/6854 20130101; C07D 277/40 20130101; A61P 3/08 20180101; A61K
31/426 20130101; A61P 15/00 20180101; A61K 8/49 20130101; A61K
31/425 20130101 |
Class at
Publication: |
424/049 ;
514/365; 548/204 |
International
Class: |
A61K 31/426 20070101
A61K031/426; A61K 8/49 20070101 A61K008/49; C07D 277/30 20060101
C07D277/30 |
Claims
1. A compound of Formula (I), ##STR22## wherein R.sup.1 and R.sup.2
are methyl; Z is hydrogen; Y is a group of the formula: ##STR23##
wherein R is phenyl; and X is halide.
2. A compound according to claim 1, wherein said compound is
3-(2-phenyl-2-oxoethyl)-4,5-dimethylthiazolium chloride.
3. A method for treating diabetes or treating or ameliorating
adverse sequelae of diabetes in an animal, said method comprising
administering to the animal a diabetes treating or adverse sequelae
of diabetes treating or ameliorating effective amount of a
pharmaceutical composition, said pharmaceutical composition
comprising the compound of claim 1 and a pharmaceutically
acceptable carrier therefor.
4. A method of treating or ameliorating the discoloration of teeth,
said method comprising administering a teeth discoloration
inhibiting effective amount of a pharmaceutical composition, said
pharmaceutical composition comprising the compound of claim 1 and a
pharmaceutically acceptable carrier therefor.
5. A method for treating or ameliorating kidney damage in an
animal, said method comprising administering a kidney treating or
ameliorating effective amount of a pharmaceutical composition, said
pharmaceutical composition comprising the compound of claim 1 and a
pharmaceutically acceptable carrier therefor.
6. A method for treating or ameliorating damage to blood
vasculature in an animal, said method comprising administering a
blood vasculature damage treating or ameliorating effective amount
of a pharmaceutical composition, said pharmaceutical composition
comprising the compound of claim 1 and a pharmaceutically
acceptable carrier therefor.
7. A method for treating or ameliorating the elasticity or reducing
wrinkles of the skin of an animal, said method comprising
administering a skin elasticity or wrinkle reducing effective
amount of a pharmaceutical composition, said pharmaceutical
composition comprising the compound of claim 1 and a
pharmaceutically acceptable carrier therefor.
8. A method for treating or ameliorating hypertension in an animal,
said method comprising administering a hypertension treating or
ameliorating effective amount of a pharmaceutical composition, said
pharmaceutical composition comprising the compound of claim 1 and a
pharmaceutically acceptable carrier therefor.
9. A method for treating or ameliorating retinopathy in an animal,
said method comprising administering a retinopathy treating or
ameliorating effective amount of a pharmaceutical composition, said
pharmaceutical composition comprising the compound of claim 1 and a
pharmaceutically acceptable carrier therefor.
10. A method for treating or ameliorating damage to lens proteins
in an animal, said method comprising administering a lens damage
treating effective amount of a pharmaceutical composition, said
pharmaceutical composition comprising the compound of claim 1 and a
pharmaceutically acceptable carrier therefor.
11. A method for treating or ameliorating peripheral neuropathy in
an animal, said method comprising administering a peripheral
neuropathy treating or ameliorating effective amount of a
pharmaceutical composition, said pharmaceutical composition
comprising the compound of claim 1 and a pharmaceutically
acceptable carrier therefor.
12. A method for treating or ameliorating cataracts in an animal,
said method comprising administering a cataract treating or
ameliorating effective amount of a pharmaceutical composition, said
pharmaceutical composition comprising the compound of claim 1 and a
pharmaceutically acceptable carrier therefor.
13. A method for treating or ameliorating damage to a tissue caused
by contact with elevated levels of reducing sugars in an animal,
said method comprising administering a peripheral neuropathy
treating or ameliorating effective amount of a pharmaceutical
composition, said pharmaceutical composition comprising the
compound of claim 1 and a pharmaceutically acceptable carrier
therefor.
14. A method for treating or ameliorating stroke in an animal, said
method comprising administering a stroke treating or ameliorating
effective amount of a pharmaceutical composition, said
pharmaceutical composition comprising the compound of claim 1 and a
pharmaceutically acceptable carrier therefor.
15. A method for treating or ameliorating osteoarthritis in an
animal, said method comprising administering an osteoarthritis
treating or ameliorating effective amount of a pharmaceutical
composition, said pharmaceutical composition comprising the
compound of claim 1 and a pharmaceutically acceptable carrier
therefor.
16. A method for increasing red blood cell deformability in an
animal, said method comprising administering a red blood cell
deformability increasing effective amount of a pharmaceutical
composition, said pharmaceutical composition comprising the
compound of claim 1 and a pharmaceutically acceptable carrier
therefor.
Description
[0001] This patent application is a Continuation of U.S. Ser. No.
10/418,398, filed Apr. 18, 2003, which is a Continuation of U.S.
Ser. No. 10/174,883, filed Jun. 19, 2002, which is a Divisional of
U.S. Ser. No. 09/470,482, filed Dec. 22, 1999, now U.S. Pat. No.
6,440,749, which is a Divisional of U.S. application Ser. No.
08/971,878, now U.S. Pat. No. 6,007,865, which is a Divisional of
U.S. application Ser. No. 08/588,249, now U.S. Pat. No. 5,853,703,
which is a Continuation-in-part of U.S. application Ser. No.
08/473,184, now abandoned, which is a Continuation-in-part of U.S.
application Ser. No. 08/375,155, now U.S. Pat. No. 5,656,261. The
contents of these applications are each incorporated herein by
reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] The present invention relates generally to the aging of
proteins resulting from their reaction with glucose and other
reducing sugars, and more particularly to the inhibition of the
reaction of nonenzymatically glycosylated proteins and the breaking
of cross-linked formed subsequent to formation of advanced
glycosylation (glycation) endproducts.
[0003] The reaction between glucose and proteins has been known for
some time. Its earliest manifestation was in the appearance of
brown pigments during the cooking of food, which was identified by
Maillard in 1912, who observed that glucose or other reducing
sugars react with amino acids to form adducts that undergo a series
of dehydrations and rearrangements to form stable brown pigments.
Further studies have suggested that stored and heat treated foods
undergo nonenzymatic browning as a result of the reaction between
glucose and the polypeptide chain, and that the proteins are
resultantly cross-linked and correspondingly exhibit decreased
bioavailability.
[0004] This reaction between reducing sugars and food proteins was
found to have its parallel in vivo. Thus, the nonenzymatic reaction
between glucose and the free amino groups on proteins to form a
stable, 1-deoxyketosyl adduct, known as the Amadori product, has
been shown to occur with hemoglobin, wherein a rearrangement of the
amino terminal of the beta-chain of hemoglobin by reaction with
glucose, forms the adduct known as hemoglobin A1c. The reaction has
also been found to occur with a variety of other body proteins,
such as lens crystallins, collagen and nerve proteins. See Bucala
et al., "Advanced Glycosylation; Chemistry, Biology, and
Implications for Diabetes and Aging" in Advances in Pharmacology,
Vol. 2, pp. 1-34, Academic Press (1992).
[0005] Moreover, brown pigments with spectral and fluorescent
properties similar to those of late-stage Maillard products have
also been observed in vivo in association with several long-lived
proteins, such as lens proteins and collagen from aged individuals.
An age-related linear increase in pigment was observed in human
dura collagen between the ages of 20 to 90 years. Interestingly,
the aging of collagen can be mimicked in vitro by the cross-linking
induced by glucose; and the capture of other proteins and the
formation of adducts by collagen, also noted, is theorized to occur
by a cross-linking reaction, and is believed to account for the
observed accumulation of albumin and antibodies in kidney basement
membrane.
[0006] In U.S. Pat. No. 4,758,583, a method and associated agents
were disclosed that served to inhibit the formation of advanced
glycosylation endproducts by reacting with an early glycosylation
product that results from the original reaction between the target
protein and glucose. Accordingly, inhibition was postulated to take
place as the reaction between the inhibitor and the early
glycosylation product appeared to interrupt the subsequent reaction
of the glycosylated protein with additional protein material to
form the cross-linked late-stage product. One of the agents
identified as an inhibitor was amino guanidine, and the results of
further testing have borne out its efficacy in this regard.
[0007] While the success that has been achieved with aminoguanidine
and similar compounds is promising, a need continues to exist to
identify and develop additional inhibitors that broaden the
availability and perhaps the scope of this potential activity and
its diagnostic and therapeutic utility. A further need exists to
find agents which not only inhibit this reaction and its
consequences, but agents capable of breaking the cross-links formed
as a result of pre-existing advanced glycosylation endproducts,
thereby reversing the resultant effects thereof.
SUMMARY OF THE INVENTION
[0008] In accordance with the present invention, a method and
compositions are disclosed for the inhibition of formation of
advanced glycosylation of proteins (protein aging) and for breaking
the cross-links that form between advanced glycosylation
(glycation) endproducts (AGEs) or between AGEs and other proteins.
Advanced glycosylation (glycation) endproducts and cross-linking
caused by other reactive sugars present in vivo or in foodstuffs,
including ribose, galactose and fructose would also be prevented
and reversed by the methods and compositions of the present
invention.
[0009] In particular, the compositions comprise agents for
inhibiting the formation of and reversing the pre-formed advanced
glycosylation (glycation) endproducts and breaking the subsequent
cross-links. While not wishing to be bound by any theory, it is
believed that the breaking of the pre-formed advanced glycosylation
(glycation) endproducts and cross-links is a result of the cleavage
of a dicarbonyl-based protein crosslinks present in the advanced
glycosylation endproducts. The methods and compositions of this
invention are thus directed to agents which, by their ability to
effect such cleavage, can be utilized to break the pre-formed
advanced glycosylation endproduct and cross-link, and the resultant
deleterious effects thereof, both in vitro and in vivo.
[0010] Certain of the agents useful in the present invention are
members of the class of compounds known as thiazoliums.
[0011] The agents comprise thiazolium compounds having the
following structural formula: ##STR1## wherein R.sup.1 and R.sup.2
are independently selected from the group consisting of hydrogen,
hydroxy (lower) alkyl, acetoxy (lower) alkyl, lower alkyl, lower
alkenyl, or R.sup.1 and R.sup.2 together with their ring carbons
may be an aromatic fused ring, optionally substituted by one or
more amino, halo or alkylenedioxy groups; [0012] Z is hydrogen or
an amino group; [0013] Y is amino, a group of the formula ##STR2##
wherein R is a lower alkyl, alkoxy, hydroxy, amino or an aryl
group, said aryl group optionally substituted by one or more lower
alkyl, lower alkoxy, halo, dialkylamino, hydroxy, nitro or
alkylenedioxy groups; [0014] a group of the formula: --CH.sub.2R'
[0015] wherein R' is hydrogen, or a lower alkyl, lower alkenyl, or
aryl group; or a group of the formula: ##STR3## [0016] wherein R''
is hydrogen and R'' is a lower alkyl group, optionally substituted
by an aryl group, or an aryl group, said aryl group optionally
substituted by one or more lower alkyl, halo, or alkoxylcarbonyl
groups; or R'' and R''' are both lower alkyl groups; [0017] X is a
halide, tosylate, methanesulfonate or mesitylenesulfonate ion; and
mixtures thereof, and a carrier therefor.
[0018] The compounds, and their compositions, utilized in this
invention appear to react with an early glycosylation product
thereby preventing the same from later forming the advanced
glycosylation end products which lead to cross-links, and thereby,
to molecular or protein aging and other adverse molecular
consequences. Additionally, they react with already formed advanced
glycosylation end products to reduce the amount of such
products.
[0019] The present invention also relates to a method for
inhibiting protein aging and other adverse molecular consequences
by contacting the initially glycosylated molecules at the stage of
the early glycosylation product with a quantity of one or more of
the agents of the present invention, or a composition containing
the same, and to a method for breaking the already formed advanced
glycosylation end products to reduce the amount of such products by
cleaving the .alpha.-dicarbonyl-based crosslinks present in the
advanced glycosylation endproducts. In the instance where the
present method has industrial application, one or more of the
agents may be applied to the proteins in question, for instance,
either by introduction into a mixture of the same in the instance
of a protein extract, or by application or introduction into
foodstuffs susceptible to advanced glycation and crosslinking, all
to prevent premature aging and spoilage of the particular
foodstuffs, and to reverse the effects of already formed advanced
glycosylation end products.
[0020] The ability to inhibit the formation of advanced
glycosylation endproducts, and to reverse the already formed
advanced glycosylation products in the body carries with it
significant implications in all applications where advanced
glycation. and concomitant molecular crosslinking is a serious
detriment. Thus, in the area of food technology, for instance, the
retardation of food spoilage would confer an obvious economic and
social benefit by making certain foods of marginal stability less
perishable and therefore more available for consumers. Spoilage
would be reduced as would the expense of inspection, removal, and
replacement, and the extended availability of the foods could aid
in stabilizing their price in the marketplace. Similarly, in other
industrial applications where the perishability of proteins is a
problem, the admixture of the agents of the present invention in
compositions containing such proteins would facilitate the extended
useful life of the same. Presently used food preservatives and
discoloration preventatives such as sulfur dioxide, known to cause
toxicity including allergy and asthma in animals, can be replaced
with compounds such as those described herein.
[0021] The present method has particular therapeutic application as
the Maillard process acutely affects several of the significant
protein masses in the body, among them collagen, elastin, lens
proteins, and the kidney glomerular basement membranes. These
proteins deteriorate both with age (hence the application of the
term "protein aging") and as a consequence of diabetes.
Accordingly, the ability to either retard or substantially inhibit
the formation of advanced glycosylation endproducts, and to reduce
the amount of cross-links formed between advanced glycosylation
endproducts and other proteins in the body carries the promise for
treatment of the complications of diabetes and aging for instance,
and thereby improving the quality and, perhaps, duration of animal
and human life.
[0022] The present agents are also useful in the area of personal
appearance and hygiene, as they prevent, and reverse, the staining
of teeth by cationic anti-microbial agents with anti-plaque
properties, such as chlorhexidine.
[0023] The invention additionally comprises a novel analytic method
for the determination of the "breaking" or reversal of the
formation of non-enzymatic endproducts. In this connection, the
invention further extends to the identification and use of a novel
cross-link structure which is believed to represent a significant
number of the molecular crosslinks that form in vitro and in vivo
as a consequence of advanced glycation. More particularly, the
cross-link structure includes a sugar-derived a-dicarbonyl segment
or moiety, such as a diketone, that is capable of cleavage by a
dinucleophilic, thiazolium-like compound. Specifically, the
cross-link structure may be according to the formula: ##STR4##
where A and B independently, are sites of attachment to the
nucleophilic atom of a biomolecule.
[0024] Accordingly, it is a principal object of the present
invention to provide a method for inhibiting the formation of
advanced glycosylation endproducts and extensive cross-linking of
molecules, and a method of breaking the cross-links formed from
pre-existing advanced glycosylation endproducts, that occur as a
consequence of the reaction of susceptible molecules such as
proteins with glucose and other reactive sugars, by correspondingly
inhibiting the formation of advanced glycosylation endproducts, and
breaking the advanced glycosylation mediated cross-linking that has
previously occurred.
[0025] It is a further object of the present invention to provide a
method as aforesaid which is characterized by a reaction with an
initially glycosylated protein identified as an early glycosylation
product.
[0026] It is a further object of the present invention to provide a
method as aforesaid which prevents the rearrangement and
cross-linking of the said early glycosylation products to form the
said advanced glycosylation endproducts.
[0027] It is a yet further object of the present invention to
provide agents capable of participating in the reaction with the
said early glycosylation products in the method as aforesaid.
[0028] It is a yet further object of the present invention to
provide agents which break or reverse the advanced glycosylation
endproducts formed as a consequence of the aforesaid advanced
glycosylation reaction sequence by cleaving the a-dicarbonyl-based
protein crosslinks present in the advanced glycosylation
endproducts.
[0029] It is a still further object of the present invention to
provide therapeutic methods of treating the adverse consequences of
molecular or protein aging by resort to the aforesaid method and
agents.
[0030] It is a still further object of the present invention to
provide a method of inhibiting, and reversing, the discoloration of
teeth by resort to the aforesaid method and agents.
[0031] It is a still further object of the present invention to
provide compositions, including pharmaceutical compositions, all
incorporating the agents of the present invention.
[0032] It is still further object of the present invention to
provide novel compounds, as well as processes for their
preparation, for use in the methods and compositions of the present
invention.
[0033] It is a still further object of the present invention to
provide novel assays which can be utilized to detect compounds
having the ability to "break" or reverse the formation of
non-enzymatic glycosylation endproducts, and their subsequent
cross-links.
[0034] It is a yet further object of the present invention to
provide a cross-link structure that is capable of cleavage by the
agents that break or reverse the formation of advanced
glycosylation endproducts as set forth herein, and the antibodies
specific to said cross-link structure, and the diagnostic and
therapeutic uses thereof.
[0035] Other objects and advantages will become apparent to those
skilled in the art from a consideration of the ensuing description
which proceeds with reference to the following illustrative
drawings.
BRIEF DESCRIPTION OF THE DRAWING FIGURES
[0036] FIG. 1 is an SDS-PAGE gel showing the CNBr Peptide Maps of
Tail Tendon Collagen from Normal and diabetic rates following
incubation with 3-(2-phenyl-2-oxoethyl)thiazolium bromide
(designated as ALT-766) of the present invention.
[0037] FIG. 2 is an SDS-PAGE gel showing the physical evidence for
the breaking of cross-linked AGE-BSA by
3-(2-phenyl-2-oxoethyl)thiazolium bromide, a compound of the
present invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0038] In accordance with the present invention, agents,
compositions including pharmaceutical compositions containing said
agents and associated methods have been developed which are
believed to inhibit the formation of advanced glycosylation
endproducts in a number of target molecules, including particularly
proteins, existing in both animals and plant material, and to
reverse the already formed advanced glycosylation endproducts. In
particular, the invention relates to a composition which may
contain one or more agents comprising compounds having the ability
to effect cleavage of .alpha.-dicarbonyl-based molecular crosslinks
present in the advanced glycosylation endproducts. Useful agents,
for instance, comprise compounds having the structural formula:
##STR5## [0039] wherein R.sup.1 and R.sup.2 are independently
selected from the group consisting of hydrogen,
hydroxy(lower)alkyl, acetoxy(lower)alkyl, lower alkyl, lower
alkenyl, or R.sup.1 and R.sup.2 together with their ring carbons
may be an aromatic fused ring, optionally substituted by one or
more amino, halo or alkylenedioxy groups; [0040] Z is hydrogen or
art amino group; [0041] Y is amino, a group of the formula ##STR6##
[0042] wherein R is a lower alkyl, alkoxy, hydroxy, amino or an
aryl group, said aryl group optionally substituted by one or more
lower alkyl, lower alkoxy, halo, dialkylamino, hydroxy, nitro or
alkylenedioxy groups; [0043] a group of the formula --CH.sub.2R'
[0044] wherein R' is hydrogen, or a lower alkyl, lower alkynyl, or
aryl group; [0045] or a group of the formula: ##STR7## [0046]
wherein R'' is hydrogen and R''' is a lower alkyl group, optionally
substituted by an aryl group, or an aryl group, said aryl group
optionally substituted by one or more lower alkyl, halo, or
alkoxylcarbonyl groups; or R'' and R''' are both lower alkyl
groups; X is a halide, tosylate, methanesulfonate or
mesitylenesulfonate ion; and mixtures thereof, and a carrier
therefor.
[0047] The lower alkyl groups referred to above contain 1-6 carbon
atoms and include methyl, ethyl, propyl, butyl, pentyl, hexyl, and
the corresponding branched-chain isomers thereof. The lower alkynyl
groups contain from 2 to 6 carbon atoms. Similarly, the lower
alkoxy groups contain from 1 to 6 carbon atoms, and include
methoxy, ethoxy, propoxy, butoxy, pentoxy, and hexoxy, and the
corresponding branched-chain isomers thereof. These groups are
optionally substituted by one or more halo, hydroxy, amino or lower
alkylamino groups.
[0048] The lower acyloxy(lower)alkyl groups encompassed by the
above formula include those wherein the acyloxy portion contain
from 2 to 6 carbon atoms and the lower alkyl portion contains from
1 to 6 carbon atoms. Typical acyloxy portions are those such as
acetoxy or ethanoyloxy, propanoyloxy, butanoyloxy, pentanoyloxy,
hexanoyloxy, and the corresponding branched chain isomers thereof.
Typical lower alkyl portions are as described hereinabove.
[0049] The aryl groups encompassed by the above formula are those
containing 6-10 carbon atoms, such as naphthyl, phenyl and lower
alkyl substituted-phenyl, e.g., tolyl and xylyl, and are optionally
substituted by 1-2 halo, hydroxy, lower alkoxy or di (lower)
alkylamino groups. Preferred aryl groups are phenyl, methoxyphenyl
and 4-bromophenyl groups.
[0050] The halo atoms in the above formula may be fluoro, chloro,
bromo or iodo.
[0051] For the purposes of this invention, the compounds of formula
(I) are formed as biologically and pharmaceutically acceptable
salts. Useful salt forms are the halides, particularly the bromide
and chloride, tosylate, methanesulfonate, and mesitylenesulfonate
salts. Other related salts can be formed using similarly non-toxic,
and biologically and pharmaceutically acceptable anions.
[0052] Of the compounds encompassed by Formula I, certain
substituents are preferred. For instance, the compounds wherein
R.sup.1 or R.sup.2 are lower alkyl groups are preferred. Also
highly preferred are the compounds wherein Y is an amino group, a
2-amino-2-oxoethyl group, a 2-phenyl-2-oxoethyl or a 2-(substituted
phenyl)-2 -oxoethyl group.
[0053] Representative compounds of the present invention are:
[0054] 3-aminothiazolium mesitylenesulfonate; [0055]
3-amino-4,5-dimethylaminothiazolium mesitylenesulfonate; [0056]
2,3-diaminothiazoliniurn mesitylenesulfonate; [0057]
3-(2-methoxy-2-oxoethyl)-thiazolium bromide; [0058]
3-(2-methoxy-2-oxoethyl)-4,5-dimethyithiazolium bromide; [0059]
3-(2-methoxy-2-oxoethyl)-4-methylthiazolium bromide; [0060]
3-(2-phenyl-2-oxoethyl)-4-methyithiazolium bromide; [0061]
3-(2-phenyl-2-oxoethyl)-4,5-dimethylthiazolium bromide; [0062]
3-amino-4-methylthiazolium mesitylenesulfonate; [0063]
3-(2-methoxy-2-oxoethyl)-5-methylthiazolium bromide; [0064]
3-(3-(2-phenyl-2-oxoethyl)-5-methyithiazolium bromide; [0065]
3-[2-(4'-bromophenyl)-2-oxoethyl]thiazolium bromide; [0066]
3-[2-(4'-bromophenyl)-2-oxoethyl]-4-methylth'iazolium bromide;
[0067] 3-[2-(4'-bromophenylDhenyl)-2-oxoethyl]-5-methylthiazolium
bromide; [0068]
3-[2-(4'bromophenyl)-2-oxoethyl)-4,5-dimethyithiazolium bromide;
[0069]
3-(2-methoxy-2-oxoethyl)-4-methyl-5-(2-hydroxyethyl)thiazolium
bromide; [0070]
3-(2-phenyl-2-oxoethyl)-4-methyl-5-(2-hydroxyethyl)thiazolium
bromide; [0071]
3-[2-(4'-bromophenyl)-2-oxoethyl]-4-methyl-5-(2-hydroxyethyl)thia-
zolium bromide; [0072] 3,4-dimethyl-5-(2-hydroxyethyl)thiazolium
iodide; [0073] 3-ethyl-5-(2-hydroxyethyl)-4-methylthiazolium
bromide; [0074] 3-benzyl-5-(2-hydroxyethyl)-4-methylthiazolium
chloride; [0075] 3-(2-methoxy-2-oxoethyl)benzothiazolium bromide;
[0076] 3-(2-phenyl-2-oxoethyl)benzothiazolium bromide; [0077]
3-[2-(4'bromophenyl)-2-oxoethyl]benzothiazolium bromide; [0078]
3-(carboxymethyl)benzothiazolium bromide; [0079]
2,3-(diamino)benzothiazolium mesitylenesulfonate; [0080]
3-(2-amino-2-oxoethyl)thiazolium bromide; [0081]
3-(2-amino-2-oxoethyl)-4-methylthiazolium bromide; [0082]
3-(2-amino-2-oxoethyl)-5-methyithiazolium bromide; [0083]
3-(2-amino-2-oxoethyl)4,5-dimethylthiazolium bromide; [0084]
3-(2-amino-2-oxoethyl)benzothiazolium bromide; [0085]
3-(2-amino-2-oxoethyl)4-methyl-5-(2-hydroxyethyl)thiazolium
bromide; [0086] 3-amino-5-(2-hydroxyethyl)-4-methyithiazolium
mesitylenesulfonate; [0087] 3-(2-methyl-2-oxoethyl)thiazolium
chloride; [0088] 3-amino-4-methyl-5-(2-acetoxyethyl)thiazolium
mesitylenesulfonate; [0089] 3-(2-phenyl-2-oxoethyl)thiazolium
bromide; [0090]
3-(2-methoxy-2-oxoethyl)-4-methyl-5-(2-acetoxyethyl)thiazolium
bromide; [0091]
3-(2-amino-2-oxoethyl)-4-methyl-5-(2-acetoxyethyl)thiazolium
bromide; [0092] 2-amino-3-(2-methoxy-2-oxoethyl)thiazolium bromide;
[0093] 2-amino-3-(2-methoxy-2-oxoethyl)benzothiazolium bromide;
[0094] 2-amino-3-(2-amino-2-oxoethyl)thiazolium bromide; [0095]
2-amino-3-(2-amino-2-oxoethyl)benzothiazolium bromide; [0096]
3-[2-(4'-methoxyphenyl)-2-oxoethyl)-thiazolinium bromide; [0097]
3-[2-(2',4'-dimethoxyphenyl)-2-oxoethyl]-thiazolinium bromide;
[0098] 3-[2-(4'-fluorophenyl)-2-oxoethyl]-thiazolinium bromide;
[0099] 3-[2-(2',4'-difluorophenyl)-2-oxoethyl]-thiazolinium
bromide; [0100]
3-[2-(4'-diethylaminophenyl)-2-oxoethyl]-thiazolinium bromide;
[0101] 3-propargyl-thiazolinium bromide; [0102]
3-propargyl-4-methylthiazolinium bromide; [0103]
3-propargyl-5-methyithiazolinium bromide; [0104]
3-propargyl-4,5-dimethylthiazolinium bromide; [0105]
3-propargyl-4-methyl-5-(2-hydroxyethyl)-thiazolinium bromide;
[0106] 3-(2-(3'-methoxyphenyl)-2-oxoethyl)-thiazolium bromide;
[0107] 3-(2-(3'-methoxy
phenyl)-2-oxoethyl)-4methyl-5-(2'-hydroxyethyl)-thiazolium bromide;
[0108] 3-(2-(3'-methoxyphenyl)-2-oxoethyl)-benzothiazolium bromide;
[0109] 2,3-diamino-4-chlorobenzothiazolium mesitylenesulfonate;
[0110] 2,3-diamino-4-methyl-thiazolium mesitylenesulfonate; [0111]
3-amino-4-methyl-5-vinyl-thiazolium mesitylenesulfonate; [0112]
2,3-diamino-6-chlorobenzothiazolium mesitylenesulfonate; [0113]
2,6-diamino-benzothiazole dihydrochloride; [0114]
2,6-diamino-3[2-(4'-methoxyphenyl)-2-oxoethyl)benzothiazolium
bromide; [0115]
2,6-diamino-3[2-(3'-methoxyphenyl)-2-oxoethyl)benzothiazolium
bromide; [0116]
2,6-diamino-3[2-(4'-diethylaminophenyl)-2-oxoethyl]benzothiazolium
bromide; [0117]
2,6-diamino-3(2-(4'-bromophenyl)-2-oxoethyl]benzothiazolium
bromide; [0118]
2,6-diamino-3(2-(2-phenyl-2-oxoethyl)benzothiazolium bromide;
[0119] 2,6-diamino-3[2-(4'-fluorophenyl-2-oxoethyl)benzothiazolium
bromide; [0120] 3-acetamido-4-methyl-5-thiazolyl-ethyl acetate
mesitylenesulfonate; [0121] 2,3-diamino-5-methylthiazolium
mesitylenesulfonate; [0122]
3-[2-(2'-naphtyl)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-thiazolium
bromide; [0123]
3-[2-(3',5'-di-ter-butyl-4'-hydroxyphenyl)-2-oxoethyl]-4-methyl-5-(2'-hyd-
roxyethyl-thiazolium bromide; [0124]
3-[2-(2',6'-dichlorophenethylamino)-2-oxoethyl]-4-methyl-5-(2'-hydroxyeth-
yl)-thiazolium-bromide; [0125]
3-[2-dibutylamino-2-oxoethyl)-4-methyl-5-(2'-hydroxyethyl)-thiazolium
bromide; [0126]
3-[2-4'-carbethoxyanilino)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-thiaz-
olium bromide; [0127]
3-[2-(2',6'-diisopropylanilino)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)--
thiazolium bromide; [0128]
3-amino-4-methyl-5-(2-(2',6'-dichlorobenzyloxy)ethyl]-thiazolium
mesitylenesulfonate; [0129]
3-[2-(4'-carbmethoxy-3'-hydroxyanilino)-2-oxoethyl)-4-methyl-5-(2'-hydrox-
yethyl)-thiazolium bromide; [0130]
2,3-diamino-4,5-dimethyithiazolium mesitylenesulfonate; [0131]
2,3-diamino-4-methyl-5-hydroxyethyl-thiazolium mesitylene
sulfonate; [0132] 2,3-diamino-5-(3',4'-trimethylenedioxy
phenyl)-thiazolium mesitylene sulfonate; [0133]
3-[2-(1',4'-benzodioxan-6-yl)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-th-
iazolium bromide; [0134]
3-[2-(3',4'-trimethylenedioxyphenyl)-2-oxoethyl]-4-methyl-5-(2'-hydroxyet-
hyl)-thiazolium bromide; [0135]
3-(2-[1',4-benzodioxan-6-yl)-2-oxoethyl)-thiazolium bromide; [0136]
3-[2-(3',4'-trimethylenedioxyphenyl)-2-oxoethyl]-thiazolium
bromide; [0137]
3-[2-(3',5'-di-tert-butyl-4'-hydroxyphenyl)-2-oxoethyl]-thiazoliu-
m bromide; [0138]
3-[2-(3',5'-di-tert-butyl-4'-hydroxyphenyl)-2-oxoethyl]-4-methyl-thiazoli-
um bromide; [0139]
3-[2-(3',5'-di-tert-butyl-4'-hydroxyphenyl)-2-oxoethyl)-5-methyl-thiazoli-
um bromide; [0140]
3-[2-(3',5'-di-tert-butyl-4'-hydroxyphenyl)-2-oxoethyl]-4,5-dimethyl-thia-
zolium bromide; [0141]
3-[2-(3',5'-di-tert-butyl-4'-hydroxyphenyl)-2-oxoethyl]-benzothiazolium
bromide; [0142]
1-methyl-3-(2-(3',5'-di-tert-butyl-4'-hydroxyphenyl)-2-oxoethyl]-imidazol-
ium bromide; [0143]
3-[2-(4'-n-pentylphenyl)-2-oxoethyl]-thiazolinium bromide; [0144]
3-[2-(4'-n-pentylphenyl)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-thiazol-
inium bromide; [0145]
3-[2-4'-diethylaminophenyl)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-thia-
zolinium bromide; [0146]
3-(2-phenyl-2-oxoethyl)-4-methyl-5-vinyl-thiazolium bromide; [0147]
3-[2-(3',5'-tert-butyl-4'-hydroxyphenyl)-2-oxoethyl)-4-methyl-5-vinyl-thi-
azolium bromide; [0148] 3-(2-tert-butyl-2-oxoethyl)-thiazolium
bromide; [0149]
3-(2-tert-butyl-2-oxoethyl)-4-methyl-5-(2'-hydroxyethyl)-thiazoli-
um bromide; [0150]
3-(3'-methoxybenzyl)-4-methyl-5-(2'-hydroxyethyl)-thiazolium
chloride; [0151]
3-(2',6'-dichlorobenzyl)-4-methyl-5-(2'-hydroxyethyl)-thiazolium
chloride; [0152]
3-(2'-nitrobenzyl)-4-methyl-5-(2'-hydroxyethyl)-thiazolium bromide;
[0153] 3[2-(4'-chiorophenyl)-2-oxoethyl]-thiazolium bromide; [0154]
3[2-(4'-chlorophenyl)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-thiazolium
bromide; and [0155]
3[2-(4'-methoxyphenyl)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-thiazoliu-
m bromide.
[0156] Certain of the compounds represented by Formula I are novel
compounds which represent a further embodiment of the present
invention. These compounds are represented by the formula ##STR8##
wherein R.sup.1 and R.sup.2 are independently selected from the
group consisting of hydrogen, hydroxy(lower)alkyl,
acetoxy(lower)alkyl, lower alkyl, lower alkenyl, or R.sup.1 and
R.sup.2 together with their ring carbons may be an aromatic fused
ring, optionally substituted by one or more amino, halo or
alkylenedioxy groups; [0157] Z is hydrogen or an amino group;
[0158] Y is amino, a group of the formula ##STR9## [0159] wherein R
is a lower alkyl, alkoxy, hydroxy, amino or an aryl group, said
aryl group optionally substituted by one or more lower alkyl, lower
alkoxy, halo, dialkylamino, hydroxy, nitro or alkylenedioxy groups;
[0160] a group of the formula --CH.sub.2R' [0161] wherein R' is
hydrogen, or a `lower alkyl, lower alkynyl, or aryl group; [0162]
or a group of the formula ##STR10## [0163] wherein R'' is hydrogen
and R''' is a lower alkyl group, optionally substituted by an aryl
group, or an aryl group, said aryl group optionally substituted by
one or more lower alkyl, halo, or alkoxylcarbonyl groups; or R''
and R'' are both lower alkyl groups; with the proviso that at least
one of Y and Z is an amino group, and the further proviso that when
Y is amino and R.sup.2 and Z are both hydrogen, then R.sup.1 is
other than a lower alkyl group; and X is a halide, tosylate,
methanesulfonate or mesitylenesulfonate ion.
[0164] Other novel compounds are those of the formula ##STR11##
wherein R.sup.1 and R.sup.2 are independently selected from the
group consisting of, hydroxy (lower) alkyl, acetoxy(lower)alkyl,
lower acyloxy(lower)alkyl, lower alkyl, or R.sup.1 and R.sup.2
together with their ring carbons may be an aromatic fused ring;
[0165] Z is hydrogen or an amino group; [0166] Y is an
alkynylmethyl group, or a group of the formula ##STR12## wherein
R'' is hydrogen and R'' is a lower alkyl group, optionally
substituted by an aryl group, or an aryl group, said aryl group
optionally substituted by one or more lower alkyl, halo, or
alkoxylcarbonyl groups; or R'' and R''' are both lower alkyl
groups; and
[0167] X is a halide, tosylate, methanesulfonate or
mesitylenesulfonate ion. The above compounds are capable of
inhibiting the formation of advanced glycosylation endproducts on
target molecules, including, for instance, proteins, as well as
being capable of breaking or reversing already formed advanced
glycosylation endproducts on such proteins. The cross-linking of
protein by formation of advanced glycosylation endproducts
contributes to the entrapment of other proteins and results in the
development in vivo of conditions such as reduced elasticity and
wrinkling of the skin, certain kidney diseases, atherosclerosis,
osteoarthritis and the like. Similarly, plant material that
undergoes nonenzymatic browning deteriorates and, in the case of
foodstuffs, become spoiled or toughened and, consequently,
inedible, unpalatable or non-nutritious. Thus, the compounds
employed in accordance with this invention inhibit this late-stage
Maillard effect and intervene in the deleterious changes described
above, and reduce the level of the advanced glycosylation
endproducts already present in the protein material.
[0168] The rationale of the present invention is to use agents
which block, as well as reverse, the post-glycosylation step, e.g.,
the formation of fluorescent chromophores and cross-links, the
presence of which is associated with, and leads to adverse sequelae
of diabetes and aging. An ideal agent would prevent the formation
of such chromophores and of cross-links between protein strands and
trapping of proteins onto other proteins, such as occurs in
arteries and in the kidney, and reverse the level of such
cross-link formation already present.
[0169] The chemical nature of the early glycosylation products with
which the compounds of the present invention are believed to react
may vary, and accordingly the term "early glycosylation product(s)"
as used herein is intended to include any and all such variations
within its scope. For example, early glycosylation products with
carbonyl moieties that are involved in the formation of advanced
glycosylation endproducts, and that may be blocked by reaction.
with the compounds of the present invention, have been postulated.
In one embodiment, it is envisioned that the early glycosylation
product may comprise the reactive carbonyl moieties of Amadori
products or their further condensation, dehydration and/or
rearrangement products, which may condense to form advanced
glycosylation endproducts. In another scenario, reactive carbonyl
compounds, containing one or more carbonyl moieties (such as
glycolaldehyde, glyceraldehyde or 3-deoxyglucosone) may form from
the cleavage of Amadori or other early glycosylation endproducts,
and by subsequent reactions with an amine or Amadori product, may
form carbonyl containing advanced glycosylation products such as
alkylformyl-glycosylpyrroles.
[0170] Several investigators have studied the mechanism of advanced
glycosylation product formation. In vitro studies by Eble et al.,
(1983), "Nonenzymatic Glucosylation and Glucose-dependent
Cross-linking of Protein", J. Biol. Chem., 258:9406-9412, concerned
the cross-linking of glycosylated protein with nonglycosylated
protein in the absence of glucose. Eble et al. sought to elucidate
the mechanism of the Maillard reaction and accordingly conducted
controlled initial glycosylation of RNase as a model system, which
was then examined under `varying conditions. In one aspect, the
glycosylated protein material was isolated and placed in a
glucose-free environment and thereby observed to determine the
extent of cross-linking.
[0171] Eble et al. thereby observed that cross-linking continued to
occur not only with the glycosylated protein but with
non-glycosylated proteins as well. One of the observations noted by
Eble et al. was that the reaction between glycosylated protein and
the protein material appeared to occur at the location on the amino
acid side chain of the protein. Confirmatory experimentation
conducted by Eble et al. in this connection demonstrated that free
lysine would compete with the lysine on RNase for the binding of
glycosylated protein. Thus, it might be inferred from these data
that lysine may serve as an inhibitor of advanced glycosylation;
however, this conclusion and the underlying observations leading to
it should be taken in the relatively limited context of the model
system prepared and examined by Eble et al. Clearly, Eble et al.
does not appreciate, nor is there a suggestion therein, of the
discoveries that underlie the present invention, with respect to
the inhibition of advanced glycosylation of proteins both in vitro
and in vivo.
[0172] The experiments of Eble et al. do not suggest the reactive
cleavage product mechanism or any other mechanism in the in vivo
formation of advanced glycosylation endproducts in which glucose is
always present. In fact, other investigators support this mechanism
to explain the formation of advanced glycosylated endproducts in
vivo (see for example, Hayase et al, J. Biol. Chem., 263:3758-3764
(1989); Sell and Monnier, J. Biol. Chem., 264:21597-21602 (1989);
Oimomi et al., Agric. Biol. Chem., 53(6):1727-1728 (1989); and
Diabetes Research and Clinical Practice, 6:311-313 (1989).
Accordingly, the use of lysine as an inhibitor in the Eble et al.
model system has no bearing upon the utility of the compounds of
the present invention in the `inhibition of advanced glycosylated
endproducts formation` in the presence of glucose in vivo, and the
amelioration of complications of diabetes and aging.
[0173] While not wishing to be bound by any particular theory as to
the mechanism by which the compounds of the instant invention
reverse already formed advanced glycosylation endproducts, studies
have been structured to elucidate a possible mechanism. Earlier
studies examining the fate of the Amadori product (AP) in vivo have
identified one likely route that could lead to the formation of
covalent, glucose-derived protein crosslinks. This pathway proceeds
by dehydration of the AP via successive beta-eliminations as shown
in the Scheme A below. Thus, loss of the 4-hydroxyl of the AP (1)
gives a 1,4-dideoxy-1-alkylamino-2,3-hexodiulose (AP-dione) (2). An
AP-dione with the structure of an amino-1,4-dideoxyosone has been
isolated by trapping model APs with the AGE-inhibitor
aminoguanidine. Subsequent elimination of the 5-hydroxyl gives a
1,4,5-trideoxy-1-alkylamino-2,3-hexulos-4-ene (AP-ene-dione) (3),
which has been isolated as a triacetyl derivative of its 1,2-enol
form. Amadori-diones, particularly the AP-ene-dione, would be
expected to be highly reactive toward protein cross linking
reactions by serving as targets for the addition of the amine (Lys,
His)-, or sulfhydryl (Cys)-based nucleophiles that exist in
proteins, thereby producing stable cross links of the form (4).
##STR13##
[0174] Note that the linear AP-ene-dione of (3) and the stable 20
cross-link of, (4) may cyclize to form either 5- or 6-member lactol
rings, although only the 6-member cyclic variant is shown in Scheme
A set forth above.
[0175] The possibility that a major pathway of glucose-derived
cross link formation proceeds through an AP-ene-dione intermediate
was investigated by experiments designed to test the occurrence of
this pathway in vivo as well as to effect the specific cleavage of
the resultant .alpha.-dicarbonyl-based protein crosslinks. The
thiazolium compounds of the instant invention are thus believed to
act as novel "bidentate" nucleophiles, particularly designed to
effect a carbon-carbon breaking reaction between the two carbonyls
of the cross link, as shown in Scheme B below under physiological
conditions. This scheme shows the reaction of a prototypic
.alpha.-dione cleaving agent of the formula I,N-phenacylthiazolium
bromide, with an AP-ene-dione derived cross link. ##STR14##
[0176] A further experiment to `elucidate this reaction involves
the reaction of a compound of the formula I,N-phenacyithiazolium
bromide, with 1-phenyl-1,2-propanedione to produce the predicted
fission product, benzoic acid. The reaction between
N-phenacylthiazolium bromide and 1-phenyl-i,2-propanedione was
rapid and readily proceeded, confirming this possible
mechanism.
[0177] Once early, glucose-derived addition products form on
proteins, further reactions can ensue to effect a covalent,
protein-protein crosslinking reaction. In this regard, a compound
of the formula I,N-phenacylthiazolium bromide, was allowed to react
with the AGE-crosslinks that form when AGE-modified BSA (AGE-BSA)
is allowed to react with unmodified, native collagen. This resulted
in a concentration-dependent release of BSA from the pre-formed
AGE-mediated complexes. Again, this study confirmed that a
significant portion of the AGE-crosslinks that form under
experimental conditions consist of an .alpha.-diketone or related
structure that is susceptible to cleavage by the advantageous
bidentate-type molecules of the compounds of formula I under
physiological conditions.
[0178] To confirm that, the same situation occurs in vivo, isolated
collagen from the tail tendons of rats which had been diabetic for
32 weeks were treated with a compound of the formula
I,N-phenacylthiazolium bromide, prior to cyanogen bromide digestion
and gel electrophoresis analysis. The subsequent electrophoresis
revealed that the treated collagen was indistinguishable from
untreated, non-diabetic (control) collagen, in marked contrast to
the AGE-modified, highly cross linked, digestion-resistant collagen
that is typically isolated from diabetic animals.
[0179] The present invention likewise relates to methods for
inhibiting the formation of advanced glycosylation endproducts, and
reversing the level of already formed advanced glycosylation
endproducts, which comprise contacting the target molecules with a
composition of the present invention. In the instance where the
target proteins are contained in foodstuffs, whether of plant or
animal origin, these foodstuffs could have applied to them by
various conventional means a composition containing the present
agents.
[0180] In the food industry, sulfites were found years ago to
inhibit the Maillard reaction and are commonly used in processed
and stored foods. Recently, however, sulfites in food have been
implicated in severe and even fatal reactions in asthmatics. As a
consequence, the sulfite treatment of fresh fruits and vegetables
has been banned.
[0181] The mechanism for the allergic reaction is not known.
Accordingly, the present compositions and agents offer a nontoxic
alternative to sulfites in the treatment of foods in this
manner.
[0182] As is apparent from a discussion of the environment of the
present invention, the present methods and compositions hold the
promise for arresting, and to some extent reversing, the aging of
key proteins both in animals and plants, and concomitantly,
conferring both economic and medical benefits as a result thereof.
In the instance of foodstuffs, the administration of the present
composition holds the promise for retarding food spoilage thereby
making foodstuffs of increased shelf life and greater availability
to consumers. Replacement of currently-used preservatives, such as
sulfur dioxide known to cause allergies and asthma in humans, with
nontoxic, biocompatible compounds is a further advantage of the
present invention.
[0183] The therapeutic implications of the present invention relate
to the arrest, and to some extent, the reversal of the aging
process which has, as indicated earlier, been identified and
exemplified in the aging of key proteins by advanced glycosylation
and cross-linking. Thus, body proteins, and particularly structural
body proteins, such as collagen, elastin, lens proteins, nerve
proteins, kidney glomerular basement membranes and other
extravascular matrix components would all benefit in their
longevity and operation from the practice of the present invention.
The present invention thus reduces the incidence of pathologies
involving the entrapment of proteins by cross-linked target
proteins, such as retinopathy, cataracts, diabetic kidney disease,
glomerulosclerosis, peripheral vascular disease, arteriosclerosis
obliterans, peripheral neuropathy, stroke, hypertension,
atherosclerosis, osteoarthritis, periarticular rigidity, loss of
elasticity and wrinkling of skin, stiffening of joints,
glomerulonephritis, etc. Likewise, all of these conditions are in
evidence and tend to occur at an accelerated rate in patients
afflicted with diabetes mellitus as a consequence of this
hyperglycemia. Thus, the present therapeutic method is relevant to
treatment of these and related conditions in patients either of
advanced age or those suffering from one of the mentioned
pathologies.
[0184] Molecular cross-linking through advanced glycosylation
product formation can decrease solubility of structural proteins
such as collagen in vessel walls and can also trap serum proteins,
such as lipoproteins to the collagen. Also, this may result in
increased permeability of the endothelium and consequently covalent
trapping of extravasated plasma proteins in subendothelial matrix,
and reduction in susceptibility of both plasma and matrix proteins
to physiologic degradation by enzymes. For these reasons, the
progressive occlusion of diabetic vessels induced by chronic
hyperglycemia has been hypothesized to result from excessive
formation of sugar-derived and particularly, glucose-derived
cross-links. Such diabetic, microvascular changes and microvascular
occlusion can be effectively prevented and reversed by chemical
inhibition and reversal of the advanced glycosylation product
formation utilizing a composition and the methods of the present
invention.
[0185] Studies indicate that the development of chronic diabetic
damage in target organs is primarily linked to hyperglycemia so
that tight metabolic control would delay or even prevent end-organ
damage. See Nicholls et al., Lab. Invest., 60, No. 4, p. 486
(1989), which discusses the effects of islet isografting and
aminoguanidine in murine diabetic nephropathy. These studies
further evidence that aminoguanidine diminishes aortic wall protein
cross-linking in diabetic rats and confirm earlier studies by
Brownlee et al., Science, 232:1629-1632 (1986) to this additional
target organ of complication of diabetes. Also, an additional study
showed the reduction of immunoglobulin trapping in the kidney by
aminoguanidine (Brownlee et al., Diabetes, 35(1):42A (1986)).
[0186] Further evidence in the streptozotocin-diabetic rat model
that aminoguanidine administration intervenes in the development of
diabetic nephropathy was presented by Brownlee et al., 1988, supra,
with regard to morphologic changes in the kidney which are
hallmarks of diabetic renal disease. These investigators reported
that the increased glomerular basement membrane thickness, a major
structural abnormality characteristic of diabetic renal disease,
was prevented with aminoguanidine.
[0187] Taken together, these data strongly suggest that inhibition
and reversal of the formation of advanced glycosylation end
products (AGEs), by the teaching of the present invention, may
prevent, as well as to some extent reverse late, as well as early,
structural lesions due to diabetes, as well as changes during aging
caused by the formation of AGEs.
[0188] Diabetes-induced changes in the deformability of red blood
cells, leading to more rigid cell membranes, is another
manifestation of cross-linking and aminoguanidine has been shown to
prevent it in vivo. In such studies, New Zealand White rabbits,
with induced, long-term diabetes are used to study the effects of a
test compound on red blood cell (RBC) deformability (df). The test
compound is administered at a rate of 100 mg/kg by oral gavages to
diabetic rabbits.
[0189] A further consequence of diabetes is the
hyperglycemia-induced matrix bone differentiation resulting in
decreased bone formation usually associated with chronic diabetes.
In animal models, diabetes reduces matrix induced bone
differentiation by 70%.
[0190] In the instance where the compositions of the present
invention are utilized for in vivo or therapeutic purposes, it may
be noted that the compounds or agents used therein are
biocompatible. Pharmaceutical compositions may be prepared with a
therapeutically effective quantity of the agents or compounds of
the present invention and may include a pharmaceutically acceptable
carrier, selected from known materials utilized for this purpose.
Such compositions may be prepared in a variety of forms, depending
on the method of administration. Also, various pharmaceutically
acceptable addition salts of the compounds of Formula I may be
utilized.
[0191] A liquid form would be utilized in the instance where
administration is by intravenous, intramuscular or intraperitoneal
injection. When appropriate, solid dosage forms such as tablets,
capsules, or liquid dosage formulations such as solutions and
suspensions, etc., may be prepared for oral administration. For
topical or dermal application to the skin or eye, a solution, a
lotion or ointment may be formulated with the agent in a suitable
vehicle such as water, ethanol, propylene glycol, perhaps including
a carrier to aid in penetration into the skin or eye. For example,
a topical preparation could include up to about 10% of the compound
of Formula I. Other suitable forms for administration to other body
tissues are also contemplated.
[0192] In the instance where the present method has therapeutic
application, the animal host intended for treatment may have
administered to it a quantity of one or more of the agents, in a
suitable pharmaceutical form. Administration may be accomplished by
known techniques, such as oral, topical and parenteral techniques
such as intradermal, subcutaneous, intravenous or intraperitoneal
injection, as well as by other conventional means. Administration
of the agents may take place over an extended period of time at a
dosage level of, for example, up to about 30 mg/kg.
[0193] As noted earlier, the invention also extends to a method of
inhibiting and reversing the discoloration of teeth resulting from
nonenzymatic browning in the oral cavity which comprises
administration to a subject in need of such therapy an amount
effective to inhibit and reverse the formation of advanced
glycosylation endproducts of a composition comprising an agent of
structural Formula I.
[0194] The nonenzymatic browning reaction which occurs in the oral
cavity results in the discoloration of teeth. Presently used
anti-plaque agents accelerate this nonenzymatic browning reaction
and further the staining of the teeth. Recently, a class of
cationic antimicrobial agents with remarkable anti-plaque
properties have been formulated in oral rinses for regular use to
kill bacteria in the mouth. These agents, the cationic antiseptics,
include such agents as alexidine, cetyl pyridinium chloride,
chlorhexidine gluconate, hexetidine, and benzalkonium chloride.
[0195] Tooth staining by chlorhexidine and other anti-plaque agents
apparently results from the enhancement of the Maillard reaction.
Nordbo, J. Dent. Res., 58:1429 (1979) reported that chlorhexidine
and benzalkonium chloride catalyze browning reactions in vitro.
Chlorhexidine added to mixtures containing a sugar derivative and a
source of amino groups underwent increased color formation,
attributed to the Maillard reaction. It is also known that use of
chlorhexidine results in an increased dental pellicle. Nordbo
proposed that chlorhexidine resulted in tooth staining in two ways:
first, by increasing formation of pellicle which contains more
amino groups, and secondly, by catalysis of the Maillard reaction
leading to colored products.
[0196] In accordance with this method, the compounds of Formula I
are formulated into compositions adapted for use in the oral
cavity. Particularly suitable formulations are oral rinses and
toothpastes incorporating the active agent.
[0197] In the practice of this invention, conventional formulating
techniques are utilized with nontoxic, pharmaceutically acceptable
carriers typically utilized in the amounts and combinations that
are well-known for the formulation of such oral rinses and
toothpastes.
[0198] The agent of Formula I is formulated in compositions in an
amount effective to inhibit and reverse the formation of advanced
glycosylation endproducts. This amount will, of course, vary with
the particular agent being utilized and the particular dosage form,
but typically is in the range of 0.01% to 1.0%, by weight, of the
particular formulation.
[0199] The compounds encompassed by Formula I are conveniently
prepared by chemical syntheses well-known in the art. Certain of
the compounds encompassed by Formula I are well-known compounds
readily available from chemical supply houses and/or are prepared
by synthetic methods specifically published therefor. For instance,
3,4-dimethyl-5-(2-hydroxyethyl) thiazolium iodide;
3-ethyl-5-(2-hydroxyethyl)-4-methylthiazolium bromide;
3-benzyl-5-(2-hydroxyethyl)-4-methylthiazolium chloride; and
3-(carboxymethyl)benzothiazolium bromide are obtainable from
Compounds described in the chemical and patent literature or
directly prepared by methods described therein and encompassed by
Formula I are those such as
3-(2-phenyl-2-oxoethyl)-4-methylthiazolium bromide and
3-benzyl-5-(2-hydroxyethyl)-4-methyl thiazolium chloride [Potts et
al., J. Org. Chem., 41:187-191(1976)].
[0200] Certain of the compounds of formula (I) are novel compounds,
not heretofore known in the art. These compounds are those
represented by the formula Ia ##STR15## wherein R.sup.1 and R.sup.2
are independently selected from the group consisting of hydrogen,
hydroxy (lower)alkyl, acetoxy(lower)alkyl, lower alkyl, lower
alkenyl, or R.sup.1 and R.sup.2 together with their ring carbons
may be an aromatic fused ring, optionally substituted by one or
more amino, halo or alkylenedioxy groups;
[0201] Z is hydrogen or an amino group;
[0202] Y is amino, a group of the formula ##STR16## wherein R is a
lower alkyl, alkoxy, hydroxy, amino or an aryl group, said aryl
group optionally substituted by one or more lower alkyl, lower
alkoxy, halo, dialkylamino, hydroxy, nitro or alkylenedioxy groups;
[0203] a group of the formula --CH.sub.2R' wherein R' is hydrogen,
or a lower alkyl, lower alkynyl, or aryl, group; [0204] or a group
of the formula ##STR17## wherein R'' is hydrogen and R'' is a lower
alkyl group, optionally substituted by an aryl group, or an aryl
group, said aryl group optionally substituted by one or more lower
alkyl, halo, or alkoxylcarbonyl groups; or R'' and R''' are both
lower alkyl groups; with the proviso that at least one of Y and Z
is an amino group, and the further proviso that when Y is amino and
R.sub.2 and Z are both hydrogen, then R.sub.1 is other than a lower
alkyl group; and
[0205] X is a halide, tosylate, methanesulfonate or
methanesulfonate ion.
[0206] Other novel compounds are those of formula I wherein Y is a
lower alkynylmethyl group or a group of the formula ##STR18##
wherein R'' is hydrogen and R'' is a lower alkyl group, optionally
substituted by an aryl group, or an aryl group, said aryl group
optionally substituted by one or more lower alkyl, halo, or
alkoxylcarbonyl groups; or R'' and R'' are both lower alkyl
groups.
[0207] The compounds of formula I wherein Y is a group of the
formula wherein R is a lower alkyl, alkoxy, hydroxy, amino or aryl
group; ##STR19##
[0208] Wherein R is lower alkyl, alkoxy, hydroxy, amino or aryl
group; [0209] or a group of the formula --CH.sub.2R' wherein R' is
hydrogen, or a lower alkyl, lower alkynyl or aryl group;
[0210] X is a halide, tosylate, methanesulfonate or
mesitylenesulfonate ion;
[0211] can be prepared according to the methods described in Potts
et al., J. Org. Chem., 41:187 (1976); and Potts et al., J. Org.
Chem., 42:1648 (1977), or as shown in Scheme I below. ##STR20##
wherein R.sup.1, R.sup.2, Z, and R are as hereinabove defined, and
X is a halogen atom.
[0212] In reaction Scheme I, the appropriate substituted thiazole
compound of formula II wherein R.sup.1, R.sup.2 and Z are as
hereinbefore defined, is reacted with the appropriate halo compound
of. formula III wherein R and X are as hereinbefore defined, to
afford the desired compound of formula I wherein R.sup.1, R.sup.2,
Z, R and X are as hereinbefore defined.
[0213] Typically, this reaction is conducted at reflux temperatures
for times of about 1-3 hours.
[0214] Typically, a polar solvent such as ethanol is utilized for
the conduct of the reaction.
[0215] The compounds of formula I wherein Y is an amino group can
be prepared according to the methods described in Tamura et al.,
Synthesis, 1 (1977), or as shown below in Scheme II. ##STR21##
wherein R.sup.1, R.sup.2 and Z are as defined hereinabove.
[0216] In the reaction shown in Scheme II, typically conducted in
an anhydrous polar solvent at room temperatures, typical reaction
temperatures range from room temperature to reflux, and typical
times vary from 1 to about 4 hours. This reaction affords the
mesitylene sulfonate, which can then be optionally converted to
other thiazolium salts by typical exchange reactions.
[0217] The present invention also involves a novel sandwich enzyme
immunoassay used to ascertain the ability of test compounds to
"break" or reverse already formed advanced glycosylation
endproducts by detecting the breaking of AGE (Advanced
glycosylation endproduct) moieties from AGE-crosslinked protein.
This assay comprises:
[0218] a) incubation of AGE-modified bovine serum albumin (AGE BSA)
on collagen-coated wells of microtiter plates for a period of 2-6
hours' at a temperature of 37.degree. C.;
[0219] b) washing of the wells with PBS-Tween;
[0220] c) application of the test compounds to the washed wells of
step b;
[0221] d) incubation of the test compounds applied to the washed
wells for an additional 12-24 hours at a temperature of about
37.degree. C.; and
[0222] e) detection of the AGE-breaking using an antibody raised
against AGE-ribonuclease or cross-link breaking with an antibody
against BSA.
[0223] The following examples are illustrative of the
invention.
EXAMPLE 1
3-(2-Methoxy-2-oxoethyl)-thiazolium bromide
[0224] Thiazole, (850 mg, 10 mmol), methyl bromoacetate (1.52, 10
mmol) and absolute ethanol (50 ml) were refluxed for 2 hours. On
cooling, the salt separated and was recrystallized from absolute
ethanol to give the title compound (1.59 g), m.p. 189-190.degree.
C. (dec).
EXAMPLE 2
3-Amino-4,5-dimethylthiazoliurn mesitylenesulfonate
[0225] An ice cold solution of the 4,5-dimethyl thiazole (2.26 g,
20 mmol) in dry dichloromethane (15 ml) was treated dropwise with a
solution of o-mesitylenesulfonylhydroxylamine (4.3 g, 20 mmol) in
dry dichloromethane (15 ml). After stirring for 2 hours at room
temperature, anhydrous ether (10 ml) was added. On cooling,
colorless needles of the title product,
3-amino-4,5-dimethyl-thiazolium mesitylenesulfonate, separated
(3.48 g), m.p. 165-168.degree. C.
EXAMPLE 3
[0226] Using the procedures described above in Examples 1 and 2,
the following compounds are prepared. [0227] 423 3-amino-thiazolium
mesitylenesulfonate, m.p. 102-104.degree. C.
[0228] 427 2,3-diarmino-thiazolium mesitylenesulfonate, m.p.
173-175.degree. C. (dec). [0229] 670
3-(2-methoxy-2-oxoethyl)-4,5-dimethylthiazolium bromide, m.p.
184-185.degree. C. (dec). [0230] 709
3-(2-methoxy-2-oxoethyl)-4-methylthiazolium bromide, m.p.
149-151.degree. C. (dec). [0231] 710
3-(2-phenyl-2-oxoethyl)-4-methylthiazolium bromide, m.p.
218-220.degree. C. (dec). [0232] 711
3-(2-phenyl-2-oxoethyl)-4,5-dimethylthiazolium bromide, m.p.
212-213.degree. C. (dec). [0233] 717 3-amino-4-methyl-thiazolium
mesitylene sulfonate, m.p. 143-144.degree. C. [0234] 719
3-(2-methoxy-2-oxoethyl)-5-methyl-thiazolium bromide, m.p.
193-194.degree. C. (dec). [0235] 720
3-(2-phenyl-2-oxoethyl)-5-methyl-thiazolium bromide, m.p.
193-194.degree. C. [0236] 721
3-(2-(4.sup.1-bromophenyl]-2-oxoethyl)-thiazolium bromide, m.p.
269-270.degree. C. (dec). [0237] 722
3-(2-[4.sup.1-bromophenyl)-2-oxoethyl)-4-methyl-thiazolium bromide,
m.p. 248-249.degree. C. (dec). [0238] 723
3-(2-(4.sup.1-bromophenyl]-2-oxoethyl)-5-methyl-thiazolium bromide,
m.p. 216-217.degree. C. [0239] 724
3-(2-(4-bromophenyl]-2-oxoethyl)-4,5-dimethylthiazolium bromide,
m.p. 223-224.degree. C. (dec). [0240] 725
3-(2-methoxy-2-oxoethyl)-4-methyl-5-(2-hydroxyethyl)-thiazolium
bromide, m.p. 137-138.degree. C. [0241] 726
3-(2-phenyl-2-oxoethyl)-4-methyl-5-(2-hydroxyethyl)-thiazolium
bromide, m.p. 180-181.degree. C. [0242] 727
3-(2-(41-bromophenyl]-2-oxoethyl)-4-methyl-5-(2-hydroxyethyl)thiazolium
bromide, m.p. 251-252.degree. C. (dec). [0243] 728
3,4-dimethyl-5-(2-hydroxyethyl)-thiazolium iodide, m.p.
85-87.degree. C. [0244] 729 3-ethyl-5-(2-hydroxyethyl)-4-methyl
thiazolium bromide, m.p. 84-85.degree. C. [0245] 730
3-benzyl-5-(2-hydroxyethyl)-4-methyl thiazolium chloride, m.p.
144-146.degree. C. [0246] 731
3-(2-methoxy-2-oxoethyl)-benzothiazolium bromide, m.p.
144-145.degree. C. (dec). [0247] 732
3-(2-phenyl-2-oxoethyl)-benzothiazolium bromide, m.p. [0248] 733
240-241.degree. C. (dec). [0249] 734
3-(2-(41-bromophenyl)-2-oxoethyl)-benzothiazolium bromide, m.p.
261-262.degree. C. (dec). [0250] 734
3-(carboxymethyl)-benzothiazolium bromide m.p. 250.degree. C.
(dec). [0251] 735 2,3-diaminio-benzothiazolium mesitylenesulfonate,
m.p. 212-214.degree. C. (dec). [0252] 738
3-(2-amino-2-oxoethyl)-thiazolium bromide, m.p. 205-206.degree. C.
[0253] 739 3-(2-amino-2-oxoethyl)-4-methyl-thiazolium bromide, m.p.
220-222.degree. C. [0254] 740
3-(2-amino-2-oxoethyl)-5-methyl-thiazolium bromide, m.p.
179-180.degree. C. [0255] 741
3-(2-amino-2-oxoethyl)-4,5-dimethyl-thiazolium bromide, m.p.
147-148.degree. C. [0256] 742
3-(2-amino-2-oxoethyl)-benzothiazolium bromide, m.p.
222-223.degree. C. [0257] 743
3-(2-amino-2-oxoethyl)-4-methyl-5-(2-hydroxyethyl)thiazolium
bromide, m.p. 182-183.degree. C. [0258] 744 3-amino-5
-(2-hydroxyethyl)-4-methyl-thiazolium mesitylenesulfonate, m.p.
94-95.degree. C. (dec). [0259] 755
3-(2-methyl-2-oxoethyl)thiazolium chloride, m.p. 178 -179.degree.
C. [0260] 763 3-amino-4-methyl-5-(2-acetoxyethyl)thiazolium
mesitylenesulfonate, m.p. 118-120.degree. C. [0261] 766
3-(2-phenyl-2-oxoethyl)thiazolium bromide, m.p. 217-218.degree. C.
[0262] 769
3-(2-methoxy-2-oxoethyl)-4-methyl-5-(2-acetoxyethyl)thiazolium
bromide, m.p. 217-218.degree. C. [0263] 770
3-(2-amino-2-oxoethyl)-4-methyl-5-(2-acetoxyethyl)thiazolium
bromide, m.p. 233-234.degree. C. [0264] 771
2-amino-3-(2-methoxy-2-oxoethyl)thiazolium bromide, m.p.
191-192.degree. C. [0265] 772
2-amino-3-(2-methoxy-2-oxoethyl)benzothiazolium bromide, m.p.
236-237.degree. C. [0266] 773
2-amino-3-(2-amino-2-oxoethyl)thiazolium bromide, m.p.
209-210.degree. C. [0267] 774 2-amino-3
-(2-amino-2-oxoethyl)benzothiazolium bromide, m.p. 234-235.degree.
C. [0268] 798 3-(2-(4'-methoxyphenyl)-2-oxoethy].]-thiazolinium
bromide, m.p. 248-249.degree. C. (dec.); [0269] 799
3-(2-(2',4'-dimethoxyphenyl)-2-oxoethyl]-thiazolinium bromide, m.p.
214-216.degree. C. (dec.); 35 800
3-(2-(4'-fluorophenyl-2-oxoethyl]-thiazolinium bromide, m.p.
209-210.degree. C. (dec.); [0270] 801
3-(2-(2',4'-difluorophenyl)-2-oxoeethyl)-thiazolinium bromide, m.p.
226-228.degree. C. (dec.); [0271] 802
3-(2-(4'-diethylaminophenyl)-2-oxoethyl]-thiazolinium bromide, m.p.
233-235.degree. C. (dec.); [0272] 803 3-propargyl-thiazolium
bromide, m.p. 64-66.degree. C.; [0273] 804 3-Propargyl-4-methyl
thiazolium bromide, m.p. 213-215.degree. C.; [0274] 805
3-Propargyl-5-methyl thiazolium bromide, m.p. 127-129.degree. C.;
[0275] 806 3-Propargyl-4,5-dimethyl thiazolium bromide, m.p.
198-200.degree. C.; [0276] 807
3-Propargyl-4-methyl-5-(2-hydroxyethyl)-thiazolium bromide, m.p.
132-134.degree. C.; [0277] 824
3-(2-(3'-methoxyphenyl]-2-oxoethyl)-thiazolium bromide, m.p.
224-225.degree. C.; [0278] 825
3-(2-[3'-methoxyphenyl]-2-oxoethyl)-4 methyl
5-(2'-hydroxyethyl)-thiazolium bromide. m.p. 164-165.degree. C.;
[0279] 826 3-(2-[3'-methoxyphenyl]-2-oxoethyl)-benzothiazolium
bromide, m.p. 215-217.degree. C.; [0280] 836
2,3-diamino-4-chlorobenzothiazolium mesitylenesulfonate, m.p.
228-230.degree. C.; [0281] 847 2,3-diamino-4-methyl-thiazolium
mesitylene sulfonate, m.p. 204-205.degree. C.; [0282] 848
3-amino-4-methyl-5-vinyl-thiazolium mesitylene sulfonate, m.p.
145-147.degree. C.; [0283] 858 2,3-diamino-6-chlorobenzothiazolium
mesitylenesulfonate, m.p. 244-246.degree. C.; [0284] 862
2,6-diamino-benzothiazole dihydrochloride, m.p. 318-320.degree. C.
(dec.); [0285] 876
2,6-diamino-3(2-(4'-methoxyphenyl)-2-oxoethyl]benzothiazolium
bromide, m.p. 243-245.degree. C. (dec.); [0286] 877
2,6-diamino-3(2-(3'-methoxyphenyl)-2'-oxoethyl]benzothiazolium
bromide, m.p. 217-218.degree. C. (dec.); [0287] 878
2,6-diamino-3(2-(4'-diethylaminophenyl)-2-oxoethyl)benzothiazolium
bromide, m.p. 223-225.degree. C. (dec.); [0288] 887 2,6
-diamino-3(2-(4'-bromophenyl)-2-oxoethyl]benzothiazolium bromide,
m.p. 258-259.degree. C. (dec.); [0289] 888
2,6-diamino-3(2-(2-phenyl-2-oxoethyl)benzothiazolium bromide, m.p.
208-210.degree. C. (dec.); [0290] 889
2,6-diamino-3(2-(4'-fluorophenyl-2-oxoethyl]benzothiazolium
bromide, m.p. 251-252.degree. C. (dec.); [0291] 897
3-acetamido-4-methyl-5-thiazolyl-ethyl acetate mesitylenesulfonate,
m.p. syrup material; [0292] 913 2,3-diamino-5-methylthiazolium
mesitylenesulfonate, m.p. 149-152.degree. C.; [0293] 924
3-(2-(2'-naphthyl)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-thiazolium
bromide, m.p. 219-220.degree. C.; [0294] 925
3-(2-(3',5'-Di-tert-butyl-4'-hydroxyphenyl)-2-15
oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-thiazolium bromide, m.p.
206-207.degree. C.; [0295] 928
3-[2-(2',6'-Dichlorophenethylamino)-2-oxoethyl]-4-methyl-5-(2'-hydroxyeth-
yl) thiazolium bromide, m.p. 193-195.degree. C.; [0296] 929
3-(2-Dibutylamino-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-thiazolium
bromide, m.p. 78-80.degree. C.; [0297] 930
3-(2-4'-carbethoxyanilino)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-thiaz-
olium bromide, m.p. 204-206.degree. C.; [0298] 931
3-(2-(2',6'-Diisopropylanilino)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)--
thiazolium bromide, m.p. 166-168.degree. C.; [0299] 932
3-amino-4-methyl-5-(2(2',6'-dichlorobenzyloxy)ethyl]-thiazolium
mesitylenesulfonate, 30 m.p. 164-166.degree. C.; [0300] 935
3-(2-(4'-carbmethoxy-3'-hydroxyanilino)-2-oxoethyl]-4-methyl-5-(2'-hydrox-
yethyl)-thiazolium bromide, m.p. 222-223.degree. C.; [0301] 938 2,
3-Diamino-4, 5-dimethyl thiazolium mesitylene sulfonate, m.p.
166-168.degree. C.; [0302] 939
2,3-Diamino-4-methyl-5-hydroxyethyl-thiazolium mesitylene
sulfonate, m.p. 132-134.degree. C.; [0303] 940
2,3-Diamino-5-(3',4'-trimethylenedioxy phenyl)thiazolium mesitylene
sulfonate, m.p. 224-226.degree. C.; [0304] 941
3(2-(1',4'-benzodioxan-6-yl)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-
-thiazolium bromide, m.p. 196-198.degree. C.; [0305] 942
3-(2-(3',4'-trimethylenedioxyphenyl)-2-oxoethyl]-4-methyl-5-(2'-hydroxyet-
hyl) thiazolium bromide, m.p. 164-166.degree. C.; [0306] 943
3-(2-(1',4'-benzodioxan-6-yl]-2-oxoethyl)-thiazolium bromide, m.p.
238-239.degree. C.; [0307] 944
3-(2-(3',4'-trimethylenedioxyphenyl)-2-oxoethyl]-thiazolium
bromide, m.p. 246-248.degree. C. (dec.); [0308] 948
3-[2-(3',5''-di-tert-butyl-4'-hydroxyphenyl)-2-oxoethyl]-thiazolium
bromide, m.p. [0309] 949
3-[2-(3',5'-di-tert-butyl-4'-hydroxyphenyl)-2-oxoethyl]-4-methyl-thiazoli-
um bromide, m.p. 226-228.degree. C. (dec.); [0310] 950,
3-[2-(3',5'-di-tert-butyl-4'-hydroxyphenyl)-2-oxoethyl)-5-methyl-thiazoli-
um bromide, m.p. 210-211.degree. C.; [0311] 951
3-(2-(3',5'-di-tert-butyl-4'-hydroxypheny].)-2-oxoethyl]-4,5-dimethyl-thi-
azolium bromide, m.p. 243-244.degree. C. (dec.); [0312] 952
3-(2-(3',5'-di-tert-butyl-4'-hydroxyphenyl)-2-oxoethyl]-benzothiazolium
bromide, m.p. 239-294.degree. C. (dec.); [0313] 953
1-methyl-3-(2-(3',5'-di-tert-butyl-4'-hydroxyphenyl)-2-oxoethyl]-imidazol-
ium bromide, m.p. 148-150.degree. C.; [0314] 954
3-[2-(4'-n-pentylphenyl)-2-oxoethyl]-thiazolinium bromide, m.p.
218-220.degree. C. (dec.); [0315] 955
3-(2-(4'-n-pentylphenyl)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-thiazol-
inium, m.p. 178-180.degree. C. (dec.); [0316] 956
3-(2-4'-diethylaminophenyl)-2-oxoethyl]-4-qj.
methyl-5-(2'-hydroxyethyl)-thiazolinium bromide, m.p.
184-186.degree. C. (dec.); [0317] 957
3-(2-phenyl-2-oxoethyl)-4-methyl-5-vinyl-thiazolium bromide, m.p.
176-177.degree. C.; [0318] 958
3-(2-(3',5'-tert-butyl-4'-hydroxyphenyl)-2-oxoethyl)-4-methyl-5-vinyl-thi-
azolium bromide, m.p. 208-209.degree. C.; [0319] 959
3-(2-tert-butyl-2-oxoethyl)-thiazolium bromide, m.p.
211-212.degree. C.; [0320] 960
3-(2-tert-butyl-2-oxoethyl)-4-methyl-5-(2'-hydroxyethyl)-thiazolium
bromide, m.p. 186-187.degree. C.; [0321] 961
3-(3'-methoxybenzyl)-4-methyl-5-(2'-hydroxyethyl)-thiazolium
chloride, m:p. 135-136.degree. C.; [0322] 962
3-(2',6'-dichlorobenzyl)-4-methyl-5-(2'-15 hydroxyethyl)-thiazolium
chloride, m.p. 192-194.degree. C.; [0323] 963
3-(2'-nitrobenzyl)-4-methyl-5-(2'-hydroxyethyl)-thiazolium bromide,
m.p. 215-216.degree. C.; [0324] 964 3
(2-(4'-chlorophenyl)-2-oxoethyl]-thiazolium bromide, m.p.
239-241.degree. C. (dec.); [0325] 965 3
(2-(4'-chlorophenyl)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-thiazolium
bromide, m.p. 240-251.degree. C. (dec.); and [0326] 966
3(2-(4'-methoxyphenyl)-2-oxoethyl]-4-methyl-5-(2'hydroxyethyl)-thiazolium
bromide, m.p. 229-231.degree. C. 25 (dec.).
EXAMPLE 4
[0327] TABLE-US-00001 Mg/tablet Compound of Formula I 50 Starch 50
Mannitol 75 Magnesium stearate 2 Stearic acid 5
[0328] The compound, a portion of the starch and the lactose are
combined and wet granulated with starch paste. The wet granulation
is placed on trays and allowed to dry overnight at a temperature of
45.degree. C. The dried granulation is comminuted in a comminutor
to a particle size of approximately 20 mesh. Magnesium stearate,
stearic acid and the balance of the starch are added and the entire
mix blended prior to compression on a suitable tablet press. The
tablets are compressed at a weight of 232 mg. using a 11/32'' punch
with a hardness of 4 kg. These tablets will disintegrate within a
half hour according to the method described in USP XVI.
EXAMPLE 5
[0329] TABLE-US-00002 Lotion mg/g Compound of Formula I 1.0 Ethyl
alcohol 400.0 Polyethylene glycol 400 300.0 Hydroxypropyl cellulose
5.0 Propylene glycol to make 1.0 g
EXAMPLE 6
[0330] TABLE-US-00003 Oral Rinse Compound of Formula I: 1.4%
Chlorhexidine gluconate 0.12% Ethanol 11.6% Sodium saccharin 0.15%
FD&C Blue No. 1 0.001% Peppermint Oil 0.5% Glycerine 10.0%
Tween 60 0.3% Water to 100%
EXAMPLE 7
[0331] TABLE-US-00004 Toothpaste Compound of Formula I: 5.5%
Sorbitol, 70% in water 25% Sodium saccharin 0.15% Sodium lauryl
sulfate 1.75% Carbopol 934, 6% dispersion in 15% Oil of Spearmint
1.0% Sodium hydroxide, 50% in water 0.76% Dibasic calcium phosphate
dihydrate 45% Water to 100%
EXAMPLE 8
Cross-Linking Inhibition Assay
[0332] The following method was used to evaluate the ability of the
compounds of the present invention to inhibit the cross-linking of
glycated bovine serum albumin (AGE-BSA) to the rat tail tendon
collagen-coated 96-well plate.
[0333] The AGE-BSA was prepared by incubating BSA at a
concentration of 200 mg per ml with 200 mM glucose in 0.4M sodium
phosphate buffer, pH 7.4 at 37.degree. C. for 12 weeks. The
glycated ESA was then extensively dialyzed against phosphate buffer
solution (PBS) for 48 hours with additional 5 times buffer
exchanges. The rat tail tendon collagen coated plate was blocked
first with 300 ml of superbloc blocking buffer (Pierce #37515X) for
one hour. The blocking solution was removed from the wells by,
washing the plate twice with PBS-'Tween 20 solution (0.05% Tween
20) using a NUNq-multiprobe or Dynatech ELISA-plate washer.
Cross-linking of AGE-BSA (1 to 10 mg per well depending on the
batch of AGE-BSA) to rat tail tendon collagen coated plate was
performed with and without the testing `compound dissolved in PBS
buffer at pH 7.4 at the desired concentrations by the, addition of
50 .mu.l each of the AGE-BSA diluted in PBS or in the solution of
test compound at 37.degree. C. for 4 hours. Unbrowned BSA in PBS
buffer with or without testing compound were added to the separate
wells as the blanks. The un-cross-linked AGE-BSA was then removed
by washing the wells three times with PBS-Tween buffer. The amount
of AGE-BSA cross-linked to the tail tendon collagen-coated plate
was then quantitated using a polyclonal antibody raised against
AGE-RNase. After a one-hour incubation period, AGE antibody was
removed by washing 4 times with PBS-Tween.
[0334] The bound AGE antibody was then detected with the addition
of horseradish peroxidase-conjugated secondary antibody--goat
anti-rabbit immunoglobulin and incubation for 30 minutes. The
substrate of 2,2-azino-di(3-ethylbenzthiazoline sulfonic acid)
(ABTS chromogen) (Zymed #00-2011) was added. The reaction was
allowed for an additional 15 minutes and the absorbance was read at
410 nm in a Dynatech plate reader.
[0335] The % inhibition of each test compound was calculated as 15
follows. % inhibition= {(Optical density (without compound)-optical
density (with compound)]/optical density (without
compound)).times.100%
[0336] The IC.sub.50 values or the inhibition at various
concentrations by test compounds is as follows: TABLE-US-00005
Relative Cross-link Test Compound IC.sub.50 Inhibition Inhibition
Data (mM) (at 10 mM) 3-amino-4,5-dimethylaminothiazolium
mesitylenesulfonate 2.8 2,3-diaminothiazolinium mesitylenesulfonate
>.10 27% 3-(2-methoxy-2-oxoethyl)-thiazolium bromide 0.25
3-(2-methoxy-2-oxoethyl)-4,5-dimethylthiazolium bromide 0.48
3-(2-methoxy-2-oxoethyl)-4-methylthiazolium bromide 58%
3-(2-phenyl-2-oxoethyl)-4-methylthiazolium bromide 5.6
3-(2-phenyl-2-oxoethyl)-4,5-dimethylthiazolium bromide 37%
3-amino-4-methylthiazolium mesitylenesulfonate 46%
3-(2-methoxy-2-oxoethyl)-5-methylthiazolium bromide 3.2
3-(3-(2-phenyl-2-oxoethyl)-5-methylthiazolium bromide 12.6
3-[2-(4'-bromophenyl)-2-oxoethyl]-4-methylthiazolium 37% bromide
3-[2-(4' bromophenyl)-2-oxoethyl]-4,5- 2.92 dimethylthiazolium
3-(2-methoxy-2-oxoethyl)-4-methyl-5-(2-hydroxyethyl) 38% thiazolium
bromide 3-(2-phenyl-2-oxoethyl)-4-methyl-5-(2-hydroxyethyl) >10
36% thiazolium bromide
3-[2-(4'-bromophenyl)-2-oxoethyl]-4-methyl-5-(2- 2.95 hydroxyethyl)
thiazolium bromide 3-(2-methoxy-2-oxoethyl) benzothiazolium bromide
>10 35% 3-(carboxymethyl) benzothiazolium bromide 16%
2,3-(diamino) benzothiazolium mesitylenesulfonate 0.0749
3-(2-amino-2-oxoethyl) thiazolium bromide 0.53
3-(2-amino-2-oxoethyl)-4-methylthiazolium bromide 0.7
3-(2-amino-2-oxoethyl)-5-methylthiazolium bromide 0.0289
3-(2-amino-2-oxoethyl) 4,5-dimethylthiazolium bromide 9.9
3-(2-amino-2-oxoethyl) benzothiazolium bromide 0.02
3-(2-amino-2-oxoethyl) 4-methyl-5-(2-hydroxyethyl) 1.42 thiazolium
bromide 3-amino-5-(2-hydroxyethyl)-4-methylthiazolium 3.6 .times.
10.sup.5 mesitylenesulfonate 3-(2-phenyl-2-oxoethyl) thiazolium
bromide 11.1 34% 3-(2-[3'-methoxyphenyl]-2-oxoethyl-thiazolium
bromide 29% 2,3-diamino-4-chlorobenzothiazolium mesitylenesulfonate
33% 2,3-diamino-4-methyl-thiazolium mesitylenesulfonate 40%
3-amino-4-methyl-5-vinyl-thiazolium mesitylenesulfonate 11.3
2,3-diamino-6-chlorobenzothiazolium mesitylenesulfonate 23.2 (2 mm)
2,6-diamino-3[2-(4'-methoxyphenyl)-2-oxoethyl] benzothiazolium
bromide 2,6-diamino-3[2-(4'-bromophenyl)-2-oxoethyl]
benzothiazolium bromide
2,6-diamino-3[2-(4'-fluorophenyl-2-oxoethyl] benzothiazolium
bromide 2,3-diamino-5-methylthiazolium mesitylenesulfonate
3-[2-(2'-naphthyl)-2-oxoethyl]-4-methyl-5-(2'-hydroxy 61%
ethyl)-thiazolium bromide
3-[2-Dibutylamino-2-oxoethyl]-4-methyl-5-(2'- 0.8% (10 mm)
hydroxyethyl)-thiazolium bromide
3-[2-4'-carbethoxyanilino)-2-oxoethyl]-4-methyl-5-(2'- 8.8% (1 mm)
hydroxyethyl)-thiazolium bromide
3-[2-(2',6'-Diisopropylanilino)-2-oxoethyl]-4-methyl-5- 19%
(2'-hydroxyethyl)-thiazolium bromide
3-amino-4-methyl-5-[2(2',6'-dichlorobenzyloxy) ethyl]- 26.5% (3 mm)
thiazolium mesitylenesulfonate
3-[2-(4'-carbmethoxy-3'-hydroxyanilino)-2-oxoethyl]-4- 1.76
methyl-5-(2'-hydroxyethyl)-thiazolium bromide
2,3-Diamino-4,5-dimdethyl thiazolium mesitylene sulfonate 39%
2,3-Diamino-4-methyl-5-hydroxyethyl-thiazolium mesitylene 18%
sulfonate 2,3-Diamino-5-(3',4'-trimethylenedioxy phenyl)-thiazolium
40% @ 3 mM mesitylene sulfonate
3[2-(1',4'-benzodioxan-6-yl)-2-oxoethyl]-4-methyl-5-(2'- 13%
hydroxyethyl)-thiazolium bromide
3-[2-(3',4'-trimethylenedioxyphenyl)-2-oxoethyl]- 4.4 thiazolium
bromide 3-[2-(3',4'-trimethylenedioxyphenyl)-2-oxoethyl]- 45%
thiazolium bromide
3-[2-(3',5'-di-tert-butyl-4'-hydroxyphenyl)-2-oxoethyl]-4- 24% @
0.3 mM methyl-thiazolium bromide
3-[2-(3',5'-di-tert-butyl-4'-hydroxyphenyl)-2-oxoethyl]-5- 0.78 69%
@ 1 mM methyl-thiazolium bromide
3-[2-(3',5'-di-tert-butyl-4'-hydroxyphenyl)-2-oxoethyl]- 0.16
4,5-dimethyl-thiazolium bromide
1-methyl-3-[2-(3',5'-di-tert-butyl-4'-hydroxyphenyl)-2- 4.5
oxoethyl]-imidazolium bromide
3-[2-(4'-n-pentylphenyl)-2-oxoethyl]-thiazolinium bromide ND
3-[2-(4'-n-pentylphenyl)-2-oxoethyl]-4-methyl-5-(2'- 1.53 52% @ 3
mM hydroxyethyl)-thiazolinium bromide
3-[2-4'-diethylaminophenyl)-2-oxoethyl]-4-methyl-5-(2'- 2.8
hydroxyethyl)-thiazolinium bromide
3-(2-phenyl-2-oxoethyl)-4-methyl-5-vinyl-thiazolium ND bromide
3-[2-(3',5'-tert-butyl-4'-hydroxyphenyl)-2-oxoethyl)-4- ND
methyl-5-vinyl-thiazolium bromide
[0337] The above experiments suggest that this type of drug therapy
may have benefit in reducing the pathology associated with the
advanced glycosylation of proteins and the formation of cross-links
between proteins and other macromolecules. Drug therapy may be used
to prevent the increased trapping and cross-linking of proteins
that occurs in diabetes and aging which leads to sequelae such as
retinal damage, and extravascularly, damage to tendons, ligaments
and other joints. This therapy might retard atherosclerosis and
connective tissue changes that occur with diabetes and aging. Both
topical, oral, and parenteral routes of administration to provide
therapy locally and systemically are contemplated.
EXAMPLE 9
Cross-Link Breaking Assay
[0338] In order to ascertain the ability of the compounds of the
instant invention to "break" or reverse already formed advanced
glycosylation endproducts, a novel sandwich enzyme immunoassay was
developed which detects breaking of AGE (Advanced glycosylation
endproduct) moieties from AGE-crosslinked protein. The assay
utilizes collagen-coated 96 well microtiter plates that are
obtained commercially. AGE-modified protein (AGE-BSA), prepared,
for instance, as in Example 8, above, is incubated on the
collagen-coated wells for four hours, is washed off the wells with
PBS-Tween and solutions of the test compounds are added. Following
an incubation period of 16 hours (37.degree. C.)
cross-link-breaking is detected using an antibody raised against
AGE-ribonuclease or with an antibody against BSA. Positive results
in this assay indicate compounds that are capable of reducing the
amount of AGE-BSA previously crosslinked to the collagen by
breaking the crosslinks and allowing the liberated material to be
flushed away in subsequent washing steps. Details of the assay are
as follows:
[0339] Materials
[0340] Immunochemicals and Chemicals
[0341] Bovine Serum Albumin (Type V), (BSA) Calbiochem
[0342] Dextrose
[0343] Superbloc, Pierce, Inc.
[0344] Rabbit anti-Bovine Serum Albumin
[0345] Horseradish Peroxidase (HRP)-Goat-anti-rabbit), Zymed
[0346] HRP substrate buffer, Zymed
[0347] ABTS chromogen, Zymed
[0348] Phosphate Buffer Saline
[0349] Tween 20, Sigma
[0350] Equipment
[0351] ELISA Plate Washer, Dynatech
[0352] ELISA Plate Reader, Dynatech
[0353] Precision Water Bath
[0354] Corning digital pH meter
[0355] Glassware and Plasticware
[0356] Finneppette Multichannel Pipettor, Baxter
[0357] Eppendorf pipettes, Baxter
[0358] Eppendorf repeater pipette, Baxter
[0359] Pipettor tips for Finneppetter, Baxter
[0360] Pipettor tips for Eppendorf, Baxter
[0361] Glass test tubes, 13.times.100 mm; Baxter
[0362] Mylar Sealing Tape for 96 well plates, Corning
[0363] Biocoat Cellware Rat Tail Collagen Type-1 coated 96-well
plates, Collaborative Biomedical Products.
[0364] Methods
[0365] Preparation of Solutions and Buffers
[0366] 1. AGE-BSA stock solutions were prepared as follows. Sodium
phosphate buffer (0.4 M) was prepared by dissolving 6 grams of
monobasic sodium phosphate in 100 ml of distilled water, 7 grams of
dibasic sodium phosphate (0.4 M) in 100 ml of distilled water and
adjusting the pH of the dibasic solution to 7.4 with the monobasic
solution. Sodium azide (0.02 grams) was added per 100 ml volume to
inhibit bacterial growth. The BSA solution was prepared as follows:
400 mg of Type V BSA (bovine serum albumin) was added for each ml
of sodium phosphate buffer (above). A 400 mM glucose solution was
prepared by dissolving 7.2 grams of dextrose in 100 ml of sodium
phosphate buffer (above). The BSA and glucose solutions were mixed
1:1 and incubated at 37.degree. C. for 12 weeks. The pH of the
incubation mixture was monitored weekly and adjusted to pH 7.4 if
necessary. After 12 weeks, the AGE-BSA solution was dialyzed
against PBS for 48 hours with four buffer changes, each at a 1:500
ratio of solution to dialysis buffer. Protein concentration was
determined by the micro-Lowry method. The AGE-BSA stock solution
was aliquoted and stored at -20.degree. C. Dilute solutions of
AGE-BSA were unstable when stored at -20.degree. C.
[0367] 2. Working solutions for crosslinking and breaking studies
were prepared as follows. Test compounds were dissolved in PBS and
the pH was adjusted to pH 7.4 if necessary. AGE-BSA stock solution
was diluted in PBS to measure maximum crosslinking and in the
inhibitor solution for testing inhibitory activity of compounds.
The concentration of AGE-BSA necessary to achieve the optimum
sensitivity was determined by initial titration of each lot of
AGE-BSA.
[0368] 3. Wash buffer ("PBS-Tween") was, prepared as follows. PBS
was prepared by dissolving the following salts in one liter of
distilled water: NaC1, 8 grams; KC1, 0.2 gram, KH.sub.2PO.sub.4.
1.15 grams; NaN.sub.3, 0.2 gram. Tween-20 was added to a final
concentration of 0.05% (vol/vol).
[0369] 4. Substrates for detection of secondary antibody binding
were prepared by diluting the HRP substrate buffer 1:10 in
distilled water and mixing with ABTS chromogen 1:50 just prior to
use.
[0370] Assay Procedures
[0371] 1. Biocoat plates were blocked with 300 .mu.l of
"Superbloc". Plates were blocked for one hour at room temperature
and were washed with PBS-Tween three times with the Dynatech
platewasher before addition of test reagents.
[0372] 2. Each experiment was set up in the following manner. The
first three wells of the Biocoat plate were used for the reagent
blank. Fifty microliters of solutions AGE-BSA were added to test
wells in triplicate and only PBS in blank wells. The plate was
incubated at 37.degree. C. for four hours and washed with PBS-Tween
three times. Fifty microliters of PBS was added to the control
wells and 50 .mu.l of the test "AGE Cross-link breaker" compound
was added to the test wells and blank. The plate was incubated
overnight (approximately 16 hours) with the test "AGE Cross-link
breaker" compound, followed by washing in PBS before addition of
primary antibody (below).
[0373] 3. Each lot of primary antibody, either anti-BSA or
anti-RNase, was tested for optimum binding capacity in this assay
by preparing serial dilutions (1:500 to 1:2000) and plating 50
.mu.l of each dilution in the wells of Biocoat plates. Optimum
primary antibody was determined from saturation kinetics. Fifty
microliters of primary antibody of appropriate dilution, determined
by initial titration, was added and incubated for one hour at room
temperature. The plate was then washed with PBS-Tween.
[0374] 4. Plates were incubated with the secondary antibody, HRP
(Goat-anti-rabbit), which was diluted 1:4000 in PBS and used as the
final secondary antibody. The incubation was performed at room
temperature for thirty minutes.
[0375] 5. Detection of maximum crosslinking and breaking of AGE
crosslinking was performed as follows. HRP substrate (100 ul) was
added to each well of the plate and was incubated at 37.degree. C.
for fifteen minutes. Readings were taken in the Dynatech
ELISA-plate reader. The sample filter was set to "1" and the
reference filter `was set to "5".
[0376] Standard Operating Procedure
[0377] Preliminary Steps
[0378] 1. Titrate each new lot of AGE-BSA preparation as described
in Table 4 and determine the optimum AGE-BSA concentration for the
ELISA assay from saturation kinetics.
[0379] 2. At the beginning of the day, flush the plate washer head
with hot water, rinse with distilled water and 50% ethanol. Fill
the buffer reservoir of the plate washer with PBS-Tween (0.05%) and
purge the system three times before use.
[0380] 3. Prepare an assay template for setting up the experiment
as described under "Assay Setup", #2, below.
[0381] Assay Setup
[0382] 1. Warm Superbloc reagent to 37.degree. C. Add 300 .mu.l of
Superbloc to each well of the Biocoat plate and let stand for sixty
minutes at 37.degree. C. Wash the wells three times with PBS-Tween
(0.05%). Turn the plate 180 degrees and repeat this wash cycle.
[0383] 2. Dilute the AGE-BSA in PBS so that 50 .mu.l of the diluted
sample will contain the amount of AGE-BSA necessary for minimum
crosslinking and inhibition by pimagedine (aminoguanidine), as
determined by initial titration described above. Prepare negative
controls by dissolving non-browned BSA in PBS at the same
concentration as the AGE-BSA. Add 50 .mu.l of AGE-BSA or BSA to
each well which correspond to the "AGE-BSA" and "BSA" labels on the
template.
[0384] 3. Dissolve the test compounds in PBS at 30 mM concentration
for preliminary evaluation. The pH must be checked and adjusted to
7.4 when necessary. Pretreat the collagen-coated plates with
AGE-BSA to obtain maximum crosslinking. Prepare negative controls
for inhibition experiments by dissolving BSA in the inhibition
solution at the same protein concentration as that used for
AGE-BSA. Add 50 .mu.l of AGE-BSA or BSA in the inhibitor solutions
to the wells which correspond to "ALT#+AGE-BSA and to "ALT# blank",
respectively, on the template. Incubate the plate at 37.degree. C.
for four hours. Following covalent binding of AGE-BSA to the
plates, wash the plates with PBS-Tween in preparation of the
detection reaction (below).
[0385] 4. Binding of primary antibody to the Biocoat plates is
carried out as follows. At the end of the four hour incubation, the
wells are washed with PBS-Tween. Appropriate dilutions (as
determined by initial titration) of the rabbit-anti-AGE-RNase or
rabbit-anti-BSA antibodies were prepared in PBS, and 50 .mu.l is
added to each well and the plate is allowed to stand at room
temperature for sixty minutes.
[0386] 5. Secondary antibody binding wells are washed with
PBS-Tween and 50 microliters HRP (Horseradish Periodase) (Goat
anti-rabbit serum) diluted to 1-4000 in PBS and is added to each
well. The plate is allowed to stand at room temperature for 30
minutes.
[0387] 6. Color development was carried out as follows. Plates are
washed as in Step 4 above. Dilute the HRP-substrate buffer 1:10 in
water. Add 200 .mu.l of ABTS solution, mix well and add 100 .mu.l
of this reagent to each well. Incubate the plate at 37.degree. C.
for 15 minutes. Read the optical density at 410 run with the sample
filter set to "1" and the reference filter set to "5" on the
Dynatech ELISA plate reader. Calculate the percent inhibition by
the compound as described above. Compounds which are found to
reduce the amount of immunoreactivity are considered to be
therapeutically useful insofar as they reverse and reduce the
levels of advanced glycosylation endproducts. TABLE-US-00006
IC.sub.50 (mM) Anti- Breaking Anti- AGE/Anti- AGE/Anti-BSA (at Test
Compound BSA mM) 3-aminothiazolium mesitylenesulfonate 0.005/3.0
71%/67% (30) 3-amino-4,5dimenthylaminothizaolium 63%/44% (10)
mesitylenesulfonate 2,3-diminothiazolinium mesitylenesulfonate
0.28/0.18 79%/90% (10) 3-(2-methoxy-2-oxoethyl)-thiazolium bromide
38%/41% (30) 3-(2-methoxy-2-oxoethyl)-4,5-dimethylthiazolium
63%/47% (30) bromide 3-(2-methoxy-2-oxoethyl)-4-methylthiazolium
bromide 54%/51% (30)
3-(2-phenyl-2-oxoethyl)-4-methylthizaoliumbromide 0.23/0.30 68%/66%
(30) 3-(2-phenyl-2-oxoethyl)-4,5-dimethylthizaolium bromide 56%/ND
(30) 3-amino-4-methylthiazolium mesithylenesulfonate 55%/ND (30)
3-(2-methoxy-2-oxoethyl)-5-methylthiazolium bromide 72%/27% (30)
3-[2-(4'-bromophenyl)-2-oxoethyl) thiazolium bromide 76%/25% (30)
3-(2-phenyl-2-oxoethyl)-4-methyl-5-(2- 14.3/112.0 67%/13% (30)
hyroxyethyl)thiazolium bromide
3-benzyl-5-(2-hydroxyethyl)-4-methylthiazolium chloride 0.42/0.55
65%/61% (30) 3-(2-methoxy-2-oxoethyl)benzothiazolium bromide
1.20/25.9 66%/37% (30) 3-(carboxymethyl) benzothiazolium bromide
63.7%/17.9% (30) 2,3-(diamino) benzothiazolium mesithylenesulfonate
87%/54% (30) 3-(2-amino-2-oxoethyl)-4-methylthiazolium bromide
4.70/38.6 89%/44% (30) 3-(2-amino-2-oxoethyl)4,5-dimethylthiazolium
bromide 61%/16% (30) 3-(2-amino-2-oxoethyl) benzothiazolium bromide
0.4/0.52 77%/65% 3-(2-amino-2-oxoethyl) 4-methyl-5-(2- 0.012/0.120
65%/57% hydroxyethyl)thiazolium bromide
3-amino-5-(2-hydroxyethyl)-4-methylthiazoium 0.18/0.50 76%/48%
mesitylenesulfonate 3-(2-methyl-2-oxoethyl) thiazolium chloride
0.83/0.75 56%/93% 3-(2-phenyl-2-oxoethyl) thiazolium bromide
0.020/0.014 73%/98% 3-(2-[3'-methoxyphenyl]-2-oxoethyl)-thiazolium
bromide 22%/44% (10) 2,3-diamino-4-chorobenzothiazolium
mesitylenesulfonate 21%/26 (10) 2,3-diamino-4-methyl-thiazolium
mesithylenesulfonate 25%/30% (10)
3-amino-4-methyl-5-vinyl-thiazolium mesitylenesulfonate ND/2.0
51%/74% (10) 2,3-diamino-6-chlorobenzothiazolium 25%/51 (10)
mesithylenesulfonate 2,6-diamino-3[2-(4'-methoxyphenyl)-2-oxoethyl]
29%/35% (10) benzothiazolium bromide 2,6-diamino-3
(2-(4'-bromophenyl)-2-oxoethyl)] 27%/44% (10) benzothiazolium
bromide 2,6-diamino-3 [2-(4'-fluorophenyl-2-oxoethyl] 24%/40% (10)
benzothiazolium bromide 2,3-diamino-5-methylthizaolium
mesitylenesulfonate 14%/17% (10)
3-[2-(2'-naphthyl)-2-oxoethyl]-4-methyl-5-(2'- 52%/61% (10)
hydroxyethyl)-thiazolium bromide
3-[Dibutylamino-2-oxoethyl]-4-methyl-5-(1'- 25%/38% (10)
hydroxyethyl)-thiazolium bromide
3-[2-4'-carbethoxyanilino)-2-oxoethyl]-4-methyl-5-(2'- 48%-57% (10)
hydroxtethyl)-thiazolium bromide
3-[2-(2',6'-Diisopropylanilino)-2-oxoethyl]-4-methyl-5- 31%/48%
(10) (2'-dhyroxyethyl)-thiazolium bromide
3-amino-4-methyl-5-[2(2',6'-dichlorobenzyloxy)ethyl]- 31%/54% (10)
thiazolium mesitylenesulfonate
3-[2-(4'-carbmethoxy-3'-hydroxyanilino)-2-oxoethyl]-4- 24%/18% (10)
methyl-5-(2'-hydroxyethyl)-thiazolium bromide
2,3-Diamino-4,5-dimethyl thiazolium mesitylene 24%/23% (10)
sulfonate 2,3-Diamino-4-methyl-5-hydrozyethyl-thiazolium 20%/18%
(10) mesitylene sulfonate 2,3-Diamino-5-(3',4'-trimethylenedioxy
phenyl)- 13%/42% (1) thiazolium mesitylene sulfonate
3[2-(1',4'-benzodioxan-6-yl)-2-oxoethyl]-4-methyl-5-(2'- 11%/21%
(3) hydroxyethyl)-thizaolium bromide
3-[2-(3',4'-trimethylenedioxyphenyl)-2-oxoethyl]- 17%/18% (10)
thiazolium bromide
3-[2-(3',5'-di-tert-butyl-4'-hydroxyphenyl)-2-oxoethyl]-4- 14%/2%
(0.3) methyl-thiazolium bromide
3-[2-(3',5'-di-tert-butyl-4'-hydroxyphenyl)-2-oxoethyl]-5- 3/0/74
65%/69% (1) methyl-thiazolium bromide
3-[2-(3',5'-di-tert-butyl-4'-hydroxyphenyl)-2-oxoethyl]-5- 48%/49%
(10) methyl-thiazolium bromide
1-methyl-3-[2[(3',5'-di-tert-butyl-4'-hydroxyphenyl)-2- 56%/38%
(10) oxoethyl]-imidazolium bromide
3-(2-phenyl-2-oxoethyl)-4-methyl-5-vinyl-thiazolium ND/0.1 62%/82%
(1) bromide 3-[2-(3',5'-tert-butyl-4'-hydroxyphenyl)-2-oxoethyl)-4-
ND/0/60% 32%/50% (0.3) methyl-5-vinyl-thiazolium bromide
3-(2-tert-butyl-2-oxoethyl)-thiazolium bromide 28%/37% (10)
3-(3'-methoxybenzyl)-4-methyl-5-(2'-hydroxyethyl)- 4%/19% (10)
thiazolium chloride
3-(3'-methoxybenzyl)-4-methyl-5-(2'-hydroxyethyl)- 14%/25% (10)
thiazolium chloride
3-(2',6'-dichlorobenzyl)-4-methyl-5-(2'-hydroxyethyl)- 6%/27% (10)
thiazolium chloride
3-(2'-nithrobenzyl)-4-methyl-5-(2'-hydroxyethyl)- 11%/13% (10)
thiazolium bromide
EXAMPLE 10
[0388] To ascertain the ability of the compounds of the invention
to decrease the amount of IgG crosslinked to circulating red blood
cells in streptozotocin-induced diabetic rats, was measured by the
following assay. The test compounds are administered to the test
animals either orally or intraperitoneally, and the blood samples
are collected are tested at various times, e.g. 4, 7 or 19 days,
after administration to assess efficacy.
Protocol for RBC-IgG Assay
[0389] A. Preparation of Red Blood Cells
[0390] Blood is collected from the rats in heparinized tubes and
spun at 2000.times.g for 10 minutes, and the plasma carefully
removed. Then, about 5 ml of PBS per ml blood is added, gently
mixed, and then spun again. The supernatant is then removed by
aspiration. The wash is then repeated two more times. Then, 0.2 to
0.3 ml of packed RBC is withdrawn from the bottom of the tube,
using a pipette, and added to the PBS to make a 1 to 10 dilution.
This dilution is then further diluted 1 to 25 and 1 to 50 in
PBS.
[0391] B. Assay Set Up. [0392] 1. Warm Superbloc to 37.degree. C.
[0393] 2. Take a plate of Multiscreen-HA, 0.45 u. Cellulose ester
membrane-sealed 96 well plate (Millipore MARAS45). [0394] 3. Wet
the wells with 100 .mu.l of PBS. [0395] 4. Add 300 .mu.l of
superblock to each well and incubate at 37.degree. C. for one hour.
[0396] 5. Place the plate on the Millititer Vacuum holder, turn on
the vacuum and press the plate down once for tight hold. The
liquids in the wells will be suctioned off. Wash the wells with 300
.mu.l of PBS-Tween 0.05%. [0397] 6. Turn off the vacuum and add 100
.mu.l of PBS to each well. [0398] 7. Gently vortex the RBC samples
and pipette 50 .mu.l to the wells, as per the protocol sheet. Leave
first three wells for reagent blanks. Leave another three wells for
antibody blank. [0399] 8. Suction-off the liquid as above and wash
the RBCs twice with PBS. [0400] 9. Dilute AP(Rb-anti-rat) (Sigma
A-6066), 1 to 25000 in PBS. [0401] 10. Add 50 .mu.l to the wells
and let stand at room temp. for two hours. [0402] 11. Suction-off
the liquid as above and wash the RBCs twice with PBS. [0403] 12.
Add pNPP substrate (1 mg/mi in DEA buffer). 100 .mu.l per well.
[0404] 13. Let the color develop for two hours at 37.degree. C.
[0405] 14. Place a 96 well coming micrometer plate in the vacuum
chamber. [0406] 15. Place the sample plate on the vacuum manifold.
Make sure the bottom of the plate is completely dry. [0407] 16.
Apply vacuum for about 5 minutes. Add 100 .mu.l of PBS to all wells
and apply vacuum again for 5 minutes. Gently lift the plate and
make sure that no liquid drops are hanging at the bottom of the
plate. If necessary apply vacuum for few more minutes. Read OD of
the solution collected in the Corning plate on Dynatech Plate
reader Sample filter 1 and Ref. filter 4. [0408] 17. Calculate
percent breaking: 100* (OD410 control-OD410 treated)/OD410
control.
[0409] Percent Inhibition in animals dosed orally at a rate of 10
mg/kg body weight are as listed below: TABLE-US-00007
3-amino-4-methyl-5-vinyl-thiazolium 11- .+-. 1 @ 19 days
mesitylenesulfonate 3-[2-(2'-naphthyl)-2-oxoethyl)-4- 40 .+-. 24 @
19 days methyl-5-(2'-hydroxyethyl)-thiazolium bromide
3-[2-(3',5'-di-tert-butyl-4'-hydroxyphenyl)- 65 .+-. 15 @ 19 days
2-oxoethyl]-5-methyl-thiazolium bromide
3-(2-phenyl-2-oxoethyl)-4-methyl-5- 58 .+-. 21 .RTM. 19 days
vinyl-thiazolium bromide
[0410] The extensive degree of reversal of crosslinking observed in
these studies underscores two important conclusions by Applicants.
First, a large percentage of cross-links formed in vivo are
susceptible to attack and cleavage by the dinucleophilic,
thiazolium-based compounds of the present invention, and thus, by
inference, that these cross-links comprise an .alpha.-diketone
segment consistent with the model shown in Schemes A and B. Second,
the crosslink-breaking agents of the present invention can act
catalytically, in the sense that a single, dinucleophilic
thiazolium-based molecule of the present invention can attack and
cause the cleavage of more than one glycation cross-link.
EXAMPLE 11
[0411] This example describes the preparation of CNBr peptide maps
of rat laid tendon collagen from normal and diabetic animals
following treatment with a compound of formula I, i.e.,
3-(2-phenyl-2-oxoethyl)thiazolium bromide (ALT 766). Collagen
fibers (5 mg) from streptozotocin diabetic rats and age-matched
control animals hydrated in land PBS at 60.degree. C. for one hour,
the soluble collagen was removed and the pellets were washed
several times with PBS then treated with
3-(2-phenyl-2-oxoethyl)thiazolium bromide at a concentration of 30
mM for 16 hours. Following incubation, the pellets were
centrifuged, washed, and treated with CNBr (40 mg/ml in formic acid
at 30.degree. C. for 48 hours. The CNBr digests were lyophilized
repeatedly to remove CNBr and acid and then subjected to SDS-PAGE
(20% acrylamide) under reducing conditions (Lanes 1, 2 and 9, MWS;
lane 3, 4 and 5, tail tendon collagen from non-diabetic animals
with 3 and 5 treated with 3-(2-phenyl-2-oxoethyl)thiazolium
bromide, 4 was treated with PBS; lanes 6, 7 and 8, collagen from
diabetic animals with 6 and 8 treated with 3-(2-phenyl-2-oxoethyl)
thiazolium bromide, 7 was treated with PBS). The gels which result
are as shown in FIG. 1.
EXAMPLE 12
[0412] Preparation of AGE-BSA and Crosslinked-AGE-BSA:
[0413] Prepare the following solutions.
[0414] 1. Buffer: 0.4 M sodium phosphate pH 7.4. TABLE-US-00008
NaH.sub.2PO.sub.4 6 g/100 ml NaH.sub.2PO.sub.4 7 g/100 ml
pH of the monobasic sodium phosphate was adjusted to 7.4 with the
dibasic 0.02 sodium azide was added per 100 ml of the buffer.
[0415] 2. BSA Solution
[0416] BSA: Calbiochem Type V; 400 mg/ml in the buffer 1. Total
volume prepared 50 g/125 ml. Filtered through a 0.45 u filter into
a sterile one liter Corning flask.
[0417] 3. Glucose solution. 400 uM
[0418] Glucose: 400 mM 9 g/125 ml of buffer. Filtered through a
0.45 u filter into one liter Coming sterile flask.
[0419] Reaction Setup:
[0420] BSA and glucose solutions (100 ml each) were mixed in the
one liter Coming sterile flask, screw-capped tight and incubated at
56.degree. C. without shaking. The bottle was opened once a week to
remove aliquots for testing. Reaction was continued for 9 weeks
when AGE-BSA polymer formation was observed.
[0421] Breaking the Polymer:
[0422] Pieces of AGE-BSA gel was washed with PBS until no more
protein was leached in the supernatant, blotted dry with paper
towels. About 50 mg of the washed gel was incubated either with PBS
or 10 mm 3-(2-phenyl-2-oxoethyl)thiazolium bromide (ALT 766)
overnight at 37.degree. C. The supernatants were analyzed by
SDS-PAGE and stained with coommassie blue. The resulting gels are
shown in FIG. 2.
EXAMPLE 13
[0423] To further study the ability of AGE crosslink-inhibiting and
reversing agents of the present invention to prevent the
discoloration of protein on a surface, such as that which occurs on
the tooth surface, the following surface browning experiment is
performed. As a substitute for a pellicle-covered tooth surface,
unexposed and developed photographic paper is used to provide a
fixed protein (gelatin, i.e., collagen) surface on a paper backing.
Five millimeter circles are punched and immersed for one week at
50.degree. C. in a solution of 100 mM glucose-6-phosphate in a 0.5
M phosphate buffer, pH 7.4, containing 3 mM sodium azide.
Glucose-6-phosphate is a sugar capable of participating in
nonenzymatic browning at a more rapid rate than glucose. In
addition to the glucose-6-phosphate, chlorhexidine and/or a
compound of Formula I are included. After incubation, the
gelatin/paper disks are rinsed with water, observed for brown
color, and photographed.
[0424] Incubation of the disks in glucose-6-phosphate alone shows
slight brown color versus disks soaked in buffer alone. Inclusion
of chlorhexidine (in the form of PERIDEX.RTM. at a final
concentration of 0.04% chlorhexidine) shows significant browning.
Addition of a compound of Formula I to the chlorhexidine completely
inhibits browning of the gelatin, as does inclusion of a compound
of Formula I in the absence of chlorhexidine.
[0425] The slight brown color formed by the action of
glucose-6-phosphate on the gelatin surface alone and its prevention
by a compound of Formula I demonstrates the utility of the present
invention in preventing nonenzymatic browning of tooth surfaces.
The enhanced browning in the presence of chlorhexidine and its
prevention with a compound of Formula I demonstrates the utility of
the present invention in preventing the anti-plaque agent-enhanced
nonenzymatic browning which occurs with chlorhexidine.
EXAMPLE 14
[0426] As a demonstration of the general utility of compounds of
the present invention to break undesired crosslinks in medically
relevant biomolecules, Applicants conducted the following
experiment with the amyloid peptide of Alzheimer's disease. This 14
kDalton peptide comprises a main constituent of the large,
plaque-like aggregates which form within the brain parenchyma of
Alzheimer's disease patients. The gradual accumulation of such
amyloid plaques, together with other abnormal features such as
perivascular amyloid and neurofibrillary tangles, is thought to
account for certain of the neurotoxic and other pathogenic
processes of this dementia, which is invariably fatal and presently
incurable. The Alzheimer's amyloid peptide is known to accumulate
AGE modifications in vivo, and upon exposure to physiologically
relevant concentrations of glucose, in vivo, which glycation
enhances the formation of insoluble aggregates of the peptide,
reminiscent of Alzheimer's amyloid plaques.
[0427] AGE-.beta.-peptide was prepared by incubating an aliquot of
the soluble .beta.-amyloid peptide, synthetically prepared and
corresponding in sequence to the 13-amyloid peptide found in the
plaques, typical of Alzheimer's disease, in a neutral buffered
glucose solution for three months, generally as described above for
the preparation of AGE-BSA except that .beta.-peptide was
substituted for BSA as the glycation substrate. The
AGE-.beta.-peptide, glycated and cross-linked after this prolonged
exposure to glucose in vivo, was separated from low molecular
weight reactants by size exclusion chromatography (e.g. over a
PFD-10 column), and iodinated by standard methods to give
.sup.125I-AGE-.beta.-peptide as the desired radiolabeled reagent
useful to test or screen compounds for molecular AGE-breaking
activity according to the following procedure. Aliquots of
1.sup.25I-AGE-.beta.-peptide were incubated with or without added
test compounds of the present invention, at predetermined
concentrations (e.g., k 10 mM Compound 766) for a predetermined
tine (e.g. overnight), after which a sample of the incubation
mixture was prepared for denaturing gel electrophoresis (SDS-PAGE)
and analyzed to determine apparent molecular weight according to
well-known procedures. Autoradiograms exposed on the resulting
electrophoresis gels were scanned into a digital radiographic
imaging and analysis system which was used to record radioactivity
as a function of apparent molecular weight (electrophoretic
mobility in SDS-containing buffer). Inspection of the results of
this experiment showed that if .sup.125I-AGE-.beta.-peptide were
not exposed to an "AGE-breaker" compound of the present invention,
it eluted a high molecular weight (>40 kDalton) band, suggesting
that its glycation was accompanied by aggregation and the formation
of stable covalent cross-links. If, however,
.sup.125I-AGE-.beta.-peptide was first incubated in a solution of
an AGE crosslink-bearing agent of the present invention, the
.sup.125I-AGE-.beta.-peptide was significantly disaggregated as
shown by the appearance of low molecular weight (>18 kDalton)
iodinated material in the final radiogram. This experiment suggests
not only that dinucleophilic thiazolium-like agents of the present
invention can be used to hydrolyze covalent AGE-mediated crosslinks
between protein strands, but also that such inhibition and reversal
of AGEs can reverse the adverse molecular consequences of AGE
accumulation on a protein relevant to human disease.
EXAMPLE 15
[0428] The cross-link structure and related compounds of the
present invention also find utility as antigens or haptens, to
elicit antibodies specifically directed thereto. Such antibodies,
likewise of the present invention, are useful in turn to identify
AAA structures of the present invention. By constructing
immunoassays employing anti-cross-link structure antibodies of the
present invention, for instance, the degree to which proteins are
modified by such cross-links can be measured. As discussed above,
and depending on the half-life of the protein so modified,
immunochemical measurement of the cross-link epitopes on a protein
sample, such as hemoglobin, provides an index of recent
AGE-formation. Likewise, immunochemical detection of cross-link
epitopes on circulating and/or tissue proteins can be used to
monitor the course of therapy with agents of the present invention,
which agents are directed toward inhibition of, and breaking of
advanced glycation.
[0429] Cross-link-modified BSA for use as an immunogen can be
prepared by coupling a cross-link structure with bovine serum
albumin (BSA) using any of a number of well-known divalent coupling
reagents such as a carbodiimide like EDC. Various other haptens,
antigens, and conjugated immunogens corresponding to the cross-link
structures of the present invention, including without limitation
those described specifically herein, can conveniently be prepared,
either by isolation from incubation mixtures or by direct synthetic
approaches. This cross-structure may then be used as an immunogen
to raise a variety of antibodies which recognize specific epitopes
or molecular features thereof.
[0430] In a preferred embodiment, the cross-link structure itself
is considered a hapten, which is correspondingly coupled to any of
several preferred carrier proteins, including for instance keyhole
limpet hemocyanin (KLH), thyroglobulin, and most preferred, bovine
serum albumin (BSA), using a divalent coupling reagents such as
EDC, according to protocols widely circulated in the art.
[0431] The cross-link structure, whether alone or coupled to a
carrier protein, may be employed in any well-recognized
immunization protocol to generate antibodies and related
immunological reagents that are useful in a number of applications
owing to the specificity of the antibodies for molecular features
of the cross-link structure.
[0432] Following a preferred protocol, any of several animal
species may be immunized to produce polyclonal antisera directed
against the cross-link structure-protein conjugate, including for
instance mice, rats, hamsters, goats, rabbits, and chickens. The
first of three of the aforesaid animal species are particularly
desired choices for the subsequent production of hybridomas
secreting hapten-specific monoclonal antibodies. The production of
said hybridomas from spleen cells of immunized animals may
conveniently be accomplished by any of several protocols popularly
practiced in the art, and which describe conditions suitable for
immortalization of immunized spleen cells by fusion with an
appropriate cell line, e.g. a myeloma cell line. Said protocols for
producing hybridomas also provide methods for selecting and cloning
immune splenocyte/myeloma cell hybridomas and for identifying
hybridomas clones that stably secrete antibodies directed against
the desired epitope(s). Animal species such as rabbit and goat are
more commonly employed for the generation of polyclonal antisera,
but regardless of whether polyclonal antisera or monoclonal
antibodies are desired ultimately, the hapten-modified carrier
protein typically is initially administered in conjunction with an
adjuvant such as Complete Freund's Adjuvant. Immunizations may be
administered by any of several routes, typically intraperitoneal,
intramuscular or intradermal; certain routes are preferred in the
art according to the species to be immunized and the type of
antibody ultimately to be produced. Subsequently, booster
immunizations are generally administered in conjunction with an
adjuvant such as alum or Incomplete Freund's Adjuvant. Booster
immunizations are administered at intervals after the initial
immunization; generally one month is a suitable interval, with
blood samples taken between one and two weeks after each booster
immunization. Alternatively, a variety of so-called
hyperimmunization schedules, which generally feature booster
immunizations spaced closer together in time, are sometimes
employed in an effort to produce anti-hapten antibodies
preferentially over anti-carrier protein antibodies.
[0433] The antibody titers in post-boost blood samples can be
compared for hapten-specific immune titer in any of several
convenient formats including, for instance, Ouchterlony diffusion
gels and direct ELISA protocols. In a typical direct ELISA, a
defined antigen is immobilized onto the assay well surface,
typically in a 96-well or microtiter plate format, followed by a
series of incubations separated by rinses of the assay well surface
to remove unbound binding partners. By way of non-limiting example,
the wells of an assay plate may receive a dilute, buffered aqueous
solution of the hapten/carrier conjugate, preferably wherein the
carrier protein differs from that used to immunize the
antibody-producing animal to be tested; e.g. serum from AAA/KLH
conjugate-immunized animal might be tested against assays wells
decorated with immobilized AAA/BSA conjugate. Alternatively, the
assay surface may be decorated by incubation with the hapten alone.
Generally, the surface of the assay wells is then exposed to a
solution of an irrelevant protein, such as casein, .to block
unoccupied sites on the plastic surfaces. After rinsing with a
neutral buffered solution that typically contains salts and a
detergent to minimize non-specific interactions, the well is then
contacted with one of a serial dilution of the serum prepared from
the blood sample of interest (the primary antiserum). After rinsing
again, the extent of test antibodies immobilized Onto the assay
wells by interaction with the desired hapten or hapten/carrier
conjugate can be estimated by incubation with a commercially
available enzyme-antibody conjugate, wherein the antibody portion
of this secondary conjugate is directed against the species used to
produce the primary antiserum; e.g. if the primary antiserum was
raised in rabbits, a commercial preparation of anti-rabbit
antibodies raised in goat and conjugated to one of several enzymes,
such as horseradish peroxidase, can be used as the secondary
antibody. Following procedures specified by the manufacturer, the
amount of this secondary antibody can then be estimated
quantitatively by the activity of the associated conjugate enzyme
in a colorimetric assay. Many related ELISA or radioimmunometric
protocols, such as competitive ELISAs or sandwich ELISAs, all of
which are well know in the art, may optionally be substituted, to
identify the desired antisera of high titer; that is, the
particular antisera which give a true positive result at high
dilution (e.g. greater than 1/1000 and more preferably greater than
1/10,000).
[0434] Similar immunometric protocols can be used to estimate the
titer of antibodies in culture supernatants from hybridomas
prepared from spleen cells of immunized animals. In so
characterizing antisera or hybridoma supernatants, it is desirable
to employ a variety of control incubations, e.g. with different
carrier proteins, related but structurally distinct haptens or
antigens, and omitting various reagents in the immunometric
procedure in order to minimize non-specific signals in the assay
and to identify reliable determinations of antibody specificity and
titer from false positive and false negative results. The types of
control incubations to use in this regard are well known. Also, the
same general immunometric protocols subsequently may be employed
with the antisera identified by the above procedures to be of high
titer and to be directed against specific structural determinants
in the cross-link structures on biological samples, foodstuffs or
other comestibles, or other amine-bearing substances and
biomolecules of interest. Such latter applications of the desired
anti-aldehyde-modified Amadori product antibodies, whether
polyclonal or monoclonal, together with instructions and optionally
with other useful reagents and diluents, including, without
limitation, a set of molecular standards of the cross-link
structure, may be provided in kit form for the convenience of the
operator.
[0435] This 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 is 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 changes which come within the meaning and range of equivalency
are intended to be embraced therein.
* * * * *