WO2009023207A1 - Thiazolium compounds for treating gastrointestinal complications - Google Patents

Abstract

The present invention relates to a method of treating gastrointestinal complications e.g., complications related to a reduction of nNOS expression, and more particularly gastroparesis and diabetic enteropathy, using thiazolium derivatives.

Description

THIAZOLIUM COMPOUNDS FOR TREATING GASTROINTESTINAL COMPLICATIONS

RELATED APPLICATIONS

This application claims priority to, and the benefit of, U.S. Provisional Application No. 60/964,609, filed on August 13, 2007, and U.S. Provisional Application No.

61/004,798, filed on November 29, 2007. The contents of each of these applications is incorporated herein by reference in their entirety.

FIELD OF THE INVENTION The present invention relates to methods of treating gastrointestinal complications e.g., complications related to a reduction of neuronal nitric oxide synthase (nNOS) expression and/or diabeties, using the thiazolium compounds and compositions of the invention.

BACKGROUND OF THE INVENTION

Patients with diabetes mellitus commonly experience gastric and intestinal dysfunction. The pathogenesis of these complications remains poorly understood, although the degree of hyperglycemia appears to be an important determinant in their incidence and severity. Bytzer P, et al., Am J Gastroenterol 2002;97.604-11. At a molecular level, much attention has recently been devoted to the potential role of altered nitrergic signaling in the enteric nervous system. Nerves throughout the luminal gastrointestinal tract express neuronal nitric oxide synthase (nNOS), which generates nitric oxide (NO), a key neurotransmitter in the regulation of gastrointestinal motility. Russo A, et al. , Gut 1999;44:72-6. Impairment of NO production appears to be important in the pathogenesis of gastroparesis as evidenced by mice with genetic deletion of nNOS. Further, several investigators have shown that experimental diabetes in rodents can result in reduced nitrergic signaling from mechanisms that may potentially include reduced nNOS expression as well as loss of the functionally active dimer form of nNOS. Gangula PR, et al., Am J Physiol Gastrointest Liver Physiol 2007;292:725-33. By contrast to gastroparesis, diabetic intestinal dysfunction has received little attention, even though it is relatively common. The term diabetic enteropathy is often used to explain disturbances in bowel function such as chronic diarrhea which occurs in 15% or more of diabetic patients as reported in large prospective population based studied. Bytzer P, et al. , Arch Intern Med 2001 ;161 : 1989-96. A role for decreased nNOS expression has been suggested by experimental studies in animals as well as by isolated human reports. Nakao K, et al, J Physiol 1998;507:549-60; Martinez-Cuesta MA, et al, Br J Pharmacol 1995;114:919-24; Watkins CC, et al, J Clin Invest 2000;106:373-84; He CL, et al, Gastroenterology 2001;121:427-34. It has also been shown that myenteric nNOS expression in the intestine, unlike the stomach, is not dependent on vagal innervation. Nakao K, et al., J Physiol 1998;507:549- 60. It therefore remains unknown how diabetes induces changes in nNOS expression. Potential mediators of this effect include advanced glycation end products (AGEs), which are a heterogeneous group of molecules formed by nonenzymatic attachment of reducing sugars to the amino groups of various proteins through a series of complex intermediary reactions including Schiff bases and amadori products. N-carboxymethyl-lysine, pentosidine and methylglyoxal derivatives are classical examples for AGEs. Although some AGE formation occurs even in physiological states, this process is significantly accelerated in uncontrolled diabetes due to the abundant availability of glucose. Although glycation itself can lead to structural and functional changes in the target protein, perhaps a more important consequence may be the ability of the conjugate to activate the receptor for advanced glycation end products (RAGE), a member of the immunoglobulin superfamily of cell surface molecules, capable of recognizing not only AGE but a variety of other ligands including fibrillar amyloid, amphoterin and SlOO/calgranulins (including EN-RAGE). Morbini P, et al, Mod Pathol 2006;19: 1437-45; Bierhaus A, Curr Opin Investig Drugs 2006;7:985-91.

Serum and tissue levels of both AGEs, as well as other potential ligands for RAGE are elevated in diabetes, both in the serum and within tissues and have been linked to many other complications of diabetes mellitus including those affecting the blood vessels, kidneys, nerves and retina. Singh R, et al. Advanced glycation end-products: a review. Diabetologia 2001 ;44: 129-46. RAGE is also expressed by myenteric neurons in the intestine and that its activation in vitro can suppress nNOS expression an NO release. Korenaga K, et al, Neurogastroenterol Motil 2006;18:392-400. AGE-RAGE signaling is also important in the modulation of intestinal nNOS expression in an in vivo model of diabetes.

In U.S. Patent 4,758,583, a method and compounds 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 compounds identified as an inhibitor was aminoguanidine (AG), and the results of further testing have borne out its efficacy in this regard. Aminoguanidine, an older compound, has been shown to prevent AGE formation in hyperglycemic states and effectively treat experimental diabetic neuropathy, retinopathy and nephropathy. Thornalley PJ. Arch Biochem Biophys 2003;419:31-40. Aminoguanidine prevents the AGEs formation possibly through the trapping of reactive carbonyl groups in glycating agents such as methylglyoxal, glyoxal, and 3-deoxyglucosone. Thornalley PJ, Biochem Pharmacol 2000;60:55-65.

However, aminoguanidine also has a variety of other effects including acting as an antioxidant and a cytoprotective agent by increasing total sulphydryl (SH) content. Giardino I, et al. Diabetes 1998;47:1114-20; Mustafa A, et al. Comp Biochem Physiol C Toxicol Pharmacol 2002;132:391-7. Further, aminoguanidine inhibits aldose reductase and can chelate metal ions. Kumari K, Biochem Pharmacol 1991 ; 41 : 1527-8. Price DL, et al., J Biol Chem 2001;276:48967-72.

Perhaps most importantly, at concentrations required for AGE prevention, aminoguanidine inhibits all isoforms of NOS, with iNOS being much more sensitive than nNOS and eNOS. Alderton WK, Biochem J2001;357:593-615; Jianmongkol S, J Biol Chem 2000; 275: 13370-6.

The results of clinical trials with aminoguanidine in diabetic states have been equivocal. Because of its relative lack of specificity and some concern about adverse effects, attention has shifted to newer compounds such as the compounds of the invention e.g., thiazoliums. The compounds of the invention are a class of novel cross-link breakers which have been shown to cleave preformed AGEs and in the diabetic milieu to reduce AGE accumulation. Vasan S, Nature 1996;382:275-8; Cooper ME, et al., Diabetologia 2000;43:660-4.

Late administration of the compounds of the invention reduces the serum AGEs to control levels. In agreement with previous reports, these results confirm the reduction in AGEs level in serum after administration of compounds of the invention. Cellek S, et al., Diabetologia 2004;47:331-9; Usta MF, et al., J Sex Med 2006;3:242-50.

In contrast to the results with aminoguanidine, restoration of nNOS expression is observed with compounds of the invention of the protein level and not the mRNA. Aminoguanidine prevents the depletion in the retinal nNOS-expressing neurons, implying that the AGE specific effect may be more important for this effect. Roufail E, et al, Diabetologia 1998;41 : 1419-25.

A recent study investigated the effects of aminoguanidine on diabetes-induced changes in small intestinal myenteric neurons. Cellek S, et al, Diabetologia 2004;47:331- 9. These findings are notable for several reasons. First, contrast to most of the literature, the authors reported that after 8 weeks of streptozotocin-inducted (STZ-induced) diabetes, a significant increase in nNOS activity was observed. This was accompanied by a significant thickening of nNOS-positive fibers was seen in both the myenteric plexus and within the muscle layer. Aminoguanidine reversed the increase in nNOS activity and the changes in intramuscular nitrergic nerve morphology (but not in the myenteric plexus). Although a similar effect was seen on nerves expressing VTP, there was no effect of aminoguanidine on diabetic changes sympathetic nerves. Secondly, aminoguanidine at very high doses (1.8 g/1) did not affect myenteric nNOS activity in control rats. Shotton HR, Auton Neurosci 2007;132: 16-26.

The non-specific nature of aminoguanidine and the results of the experiments with compounds of the invention indicate that AGE signaling is involved in the suppression of enteric nNOS in diabetes. As specific anti-AGE compounds, the differential effects of the compounds of the invention on nNOS mRNA and protein expression are significance for two reasons. First, suppression of nNOS gene transcription in diabetes may be due to factors other than activation of the AGE-RAGE pathways, whereas the latter may be more important in post-transcriptional or post-translational modifications.

Treatment directed against AGEs is useful for the treatment and/or prevention of gastrointestinal complications. Countering the AGE-RAGE signaling pathway is a target for related gastrointestinal complications, particularly those that arise from a reduction in nNOS expression. Gastrointesinal complications include complications related to diabetes such as diabetic intestinal dysfunction, diabetic enteropathy and diabetic gastroparesis, as well as other forms such as postsurgical and medication-related gastroparesis. A need exists to identify and develop compounds that broaden the availability and scope of this potential activity and its therapeutic utility. A further need exists to find compounds which not only inhibit AGE formation and its consequences, but also compounds 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

The present invention provides a method of treating, or ameloriating (lessening or reducing) a symptom of, a gastrointestinal disorder or condition in a patient in need thereof, comprising administering a pharmaceutical composition comprising a compound of Formula I, or a pharmaceutically acceptable salt of the compound of Formula I,

wherein:

R¹ and R² are selected from the group consisting of hydrogen, hydroxy (lower) alkyl, acetoxy (lower) alkyl, lower alkyl, lower alkenyl; or R¹ and R² together with their ring carbons may be an aromatic fused ring, optionally substituted by one or more amino, halo or alkylenedioxy groups;

Z is hydrogen or an amino group;

Y is amino, a group of the formula:

O Il

-CH₂C-R, 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; a group of the formula:

-CH₂R' wherein R' is hydrogen, or a lower alkyl, lower alkenyl, or aryl group; or a group of the formula:

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

X is a pharmaceutically acceptable anion, and a pharmaceutically acceptable carrier, thereby treating or preventing said gastrointestinal disorder or condition. The compound of Formula I can include where Rl and R2 are independently lower alkyl. The lower alkyl can be methyl. The compound of Formula I can include where Z is hydrogen and where R is an aryl group. The pharmaceutically acceptable anion can be halide.

Preferably, the compound of Formula I is 3-(2-phenyl-2-oxoethyl)-4,5- dimethylthiazolium. More preferably, the compound of Formula I is 3-(2-phenyl-2- oxoethyl)-4,5-dimethylthiazolium chloride or 3-(2-phenyl-2-oxoethyl)-4,5- dimethylthiazolium bromide.

The gastrointestinal disorder or condition can be a gastrointestinal complication related to diabetes or related to a decrease in nNOS expression. More preferably, related to a decrease in nNOS expression in the gastrointestinal system or tract, such as the duodenum.

The gastrointestinal disorder or condition can be gastroparesis. The gastroparesis can be diabetic gastroparesis, postsurgical gastroparesis or medication-related gastroparesis.

The gastrointestinal disorder or condition can also be diabetes-induced delayed gastric emptying, diabetic intestinal dysfunction, diabetic enteropathy or gastric and intestinal dysfunction.

The subject or patient treated by the methods of the invention is an animal, preferably a mammal, more preferably a human. The following properties or applications of these methods will essentially be described for humans although they may also be applied to non-human mammals, e.g., apes, monkeys, dogs, mice, etc. The patient can have diabetes (e.g., suffering from or diagnosed with diabetes). The diabetes can be diabetes mellitus. The patient can be hyperglycemic.

The patient can have decreased intestinal neuronal nitric oxide synthase (nNOS) protein expression. The decrease in intestinal nNOS expression can be diabetes induced. The administration of said pharmaceutical composition increases the intestinal protein expression of nNOS. The protein expression of nNOS can be increased in the duodenal myenteric plexus or in the myenteric ganglia in the duodenum (or both) of said patient.

In accordance with the present invention, a method for the treatment of gastrointestinal complications using compositions of the invention is disclosed. Gastrointesinal complications include those related to diabetes such as diabetic gastxoparesis, and other less common forms of gastroparesis such as postsurgical and medication-related. In particular, the compositions comprise compounds 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 method and compositions of this invention are thus directed to compounds 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.

Further, it is known that advanced glycation end-products (AGEs), can inhibit the expression of intestinal neuronal nitric oxide synthase (nNOS) in vitro acting via their receptor RAGE. This effect may be important in experimental diabetes in vivo. The generation of AGEs in diabetes results in a loss of intestinal nNOS expression and may be responsible for enteric dysfunction in diabetes. Treatment directed against AGEs may be useful for the treatment or prevention of gastrointestinal complications e.g., complications that arise from the reduction of nNOS expression such as diabetes-related complications.

The invention includes a method for treating or preventing gastroparesis in an mammal, where the method comprises administering to the mammal an effective amount of a compound of the invention. The invention further includes a method for treating or preventing gastroparesis in an mammal, where the method comprises administering to the mammal an effective amount of a pharmaceutical composition, said pharmaceutical composition comprising the compound of claim 1 and a pharmaceutically acceptable carrier therefor. Certain of the compounds useful in the present invention are members of the class of compounds known as thiazoliums.

The invention comprises thiazolium compounds having the following structural formula:

(I) wherein R¹ is selected from the group consisting of hydrogen, hydroxy (lower) alkyl, acetoxy (lower) alkyl, lower alkyl, lower alkenyl;

R² is selected from the group consisting of hydrogen, hydroxy (lower) alkyl, acetoxy (lower) alkyl, lower alkyl, lower alkenyl; or R¹ and R² together with their ring carbons may be an aromatic fused ring, optionally substituted by one or more amino, halo or alkylenedioxy groups;

Z is hydrogen or an amino group;

Y is amino, a group of the formula:

O Il -CH₂C-R, 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; a group of the formula: -CH₂R' wherein R' is hydrogen, or a lower alkyl, lower alkenyl, or aryl group; or a group of the formula:

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.

The preferred thiazolium compound of the instant invention comprises the structure of Formula I, wherein R¹ and R² are lower alkyl, Z is hydrogen, Y is a group of the formula

O Il

-CH₂C-R wherein R is an aryl group and X is halide. In more preferred embodiments, the compound of the invention is 3-(2-phenyl-2-oxoethyl)-4,5-dimethylthiazolium chloride or N-phenacyl-4,5-dimethylthiazolium chloride, also referred to as ALT-711 or algebrium chloride herein. In other embodiments, the compound of the invention is 3-(2-phenyl-2- oxoethyl)-4,5-dimethylthiazolium bromide or N-phenacyl-4,5-dimethylthiazolium bromide, also referred to as DMPTB or PMTB.

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. The invention additionally comprises an analytic method for identifying compounds for the treatment or prevention of gastrointestinal complications e.g., complications that arise from a reduction of nNOS expressions such as complications of diabetes, where method determines 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 α-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:

where A and B independently, are sites of attachment to the nucleophilic atom of a biomolecule.

Accordingly, it is a principal object of the present invention to provide a method for the treatment or prevention of gastrointestinal complications e.g., that arise from a reduction in nNOS expression such as diabetes-related complications, where the formation of advanced glycosylation endproducts and extensive cross-linking of molecules is inhibited, and 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, are broken and the breaking of the advanced glycosylation mediated cross-linking has previously occurred. It is a further object of the present invention to provide a method for the treatment or prevention of gastrointestinal complications e.g., complications that arise from a reduction of nNOS expression such as diabetes-related complications which are characterized by a reaction with an initially glycosylated protein identified as an early glycosylation product. It is a further object of the present invention to provide a method the treatment or prevention of gastrointestinal complications e.g., complications that arise from a reduction of nNOS expression such as diabetes-related complications which prevents the rearrangement and cross-linking of the said early glycosylation products to form the said advanced glycosylation endproducts. It is a yet further object of the present invention to provide compounds capable of participating in the reaction with the said early glycosylation products in the method the treatment or prevention of gastrointestinal complications e.g., complications that arise from a reduction of nNOS expression such as diabetes-related complications e.g., gastroparesis. It is a yet further object of the present invention to provide compounds which break or reverse the advanced glycosylation endproducts formed as a consequence of the aforesaid advanced glycosylation reaction sequence by cleaving the α-dicarbonyl-based protein crosslinks present in the advanced glycosylation endproducts.

It is a further objective of the present invention to provide a method for the treatment or prevention of gastrointestinal complications, where the accumulation of AGEs within the small intestine, accompanied by the reduction in myenteric nNOS expression is reversed by a compound of the invention. The compound of the invention is administered prophylatically or therapeutically.

It is a still further object of the present invention to provide compositions, including pharmaceutical compositions, all incorporating the compounds of the present invention. It is still further object of the present invention to provide compounds, as well as processes for their preparation, for use in the method and compositions of the present invention.

It is a still further object of the present invention to provide 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 in order to identify compounds that are useful for the treatment or prevention of gastrointestinal complications e.g., complications that arise from a reduction of nNOS expression such as diabetes-related complications. It is a yet further object of the present invention to provide a cross-link structure that is capable of cleavage by the compounds 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. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE IA shows the body weight of control and experimental rats. FIGURE IB shows blood glucose concentration of control and experimental rats. FIGURE 2 shows the effect of aminoguanidine and ALT-711 on AGEs accumulation.

FIGURE 3 shows expression of RAGE in duodenum of control and experimental rats.

FIGURE 4 shows the effect of aminoguanidine and ALT-711 on nNOS mRNA expression. FIGURES 5 A and 5B show the effect of aminoguanidine and ALT-711 on nNOS protein expression in duodenum of control and experimental rats. Figure 5 A is a representative Western blot showing nNos immunoreactive bands relevant to 155kDa. Figure 5B shows band intensities that were measured by densitometry and graphed as a proportion of γ-tubulin. FIGURE 6A shows immunohistochemical localization of nNOS in the duodenal myenteric plexus (arrows) of control and experimental rats. Figure 6B shows quantification of nNOS positive cells. FIGURE 7 shows CNBr peptide maps of rat laid tendon collagen from normal and diabetic animals following treatment with a test compound of the invention.

FIGURE 8 shows the break up of crosslinked- AGE-BS A by a test compound of the invention.

DETAILED DESCRIPTION

In accordance with the present invention, compounds, compositions including pharmaceutical compositions containing said compounds 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 compounds having the ability to effect cleavage of α-dicarbonyl-based molecular crosslinks present in the advanced glycosylation endproducts. In particular, the invention relates to compositions that can reverse the accumulation of AGEs and reduction of nNOS expression which occurs in diabetes. Useful compounds, for instance, comprise compounds having the structural formula:

wherein R¹ and R² are independently selected from the group consisting of hydrogen, hydroxy(lower)alkyl, acetoxy(lower)alkyl, lower alkyl, lower alkenyl, or

R¹ and R² together with their ring carbons may be an aromatic fused ring, optionally substituted by one or more amino, halo or alkylenedioxy groups;

Z is hydrogen or an amino group;

Y is amino, a group of the formula

O

Il -CH₂C-R 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; a group of the formula -CH₂R' wherein R' is hydrogen, or a lower alkyl, lower alkynyl, or aryl group; or a group of the formula:

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.

The term "lower alkyl" means that the group contains 1, 2, 3, 4, 5, or 6 carbon atoms and includes methyl, ethyl, propyl, butyl, pentyl, hexyl, and the corresponding branched- chain isomers thereof. The term "lower alkynyl" means that the group contains from 2, 3, 4,

5, or 6 carbon atoms. Similarly, the term "lower alkoxy" means that the group contains from 1, 2, 3, 4, 5, or 6 carbon atoms, and includes 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.

The term "lower acyloxy(lower)alkyl" means that the acyloxy portion contain from 2, 3, 4, 5, or 6 carbon atoms and the lower alkyl portion contains from 1, 2, 3, 4, 5, or 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.

The aryl groups encompassed by the formulae of the invention are those containing

6, 7, 8, 9, or 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.

The halo atoms in the formulae of the invention may be fluoro, chloro, bromo or iodo. For the purposes of this invention, the compounds of the invention 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.

The preferred thiazolium compound of the instant invention comprises the structure of Formula I, wherein R¹ and R are lower alkyl, Z is hydrogen, Y is a group of the formula

O

Il -CH2C— R wherein R is an aryl group and X is halide. In more preferred embodiments, the compound of the invention is 3-(2-phenyl-2-oxoethyl)-4,5-dimethylthiazolium chloride or N-phenacyl-4,5-dimethylthiazolium chloride, also referred to as ALT-711 or algebrium chloride herein. In other embodiments, the compound of the invention is 3-(2-phenyl-2- oxoethyl)-4,5-dimethylthiazolium bromide or N-phenacyl-4,5-dimethylthiazolium bromide, also referred to as DMPTB or PMTB.

As used herein, "treating" or "treatment" includes any effect e.g., lessening, reducing, modulating, or eliminating, that results in the improvement of the condition, disease, disorder, etc. "Treating" or "treatment" of a disease state means the treatment of a disease-state in a mammal, particularly in a human, and include: (a) inhibiting an existing disease-state, i.e., arresting its development or its clinical symptoms; and/or (c) relieving the disease-state, i.e., causing regression of the disease state.

As used herein, "preventing" means causing the clinical symptoms of the disease state not to develop i.e., inhibiting the onset of disease, in a subject that may be exposed to or predisposed to the disease state, but does not yet experience or display symptoms of the disease state.

Gastroparesis is the term used to describe a significant delay in the emptying of solids and liquids from the stomach. Such a delay in gastric emptying might asymptomatic but can also be associated with nausea, vomiting, bloating, dyspepsia, early satiety and pain. The most common forms of gastroparesis are diabetic and idiopathic, other less common forms include postsurgical and medical-related gastroparesis. See also, Vittal, H., et al. Mechanism of Disease: the Pathological Basis of Gastroparesis-a Review of Experimental and Clinical Studies. Gastroenterology & Hepatology 2007; Vol. 4, pages 1-12, the contents of which are incorporated herein.

Of the compounds encompassed by Formula I, certain substituents are preferred. For instance, the compounds wherein R¹ or R² 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. Representative compounds of the present invention are: 3 -aminothiazolium mesitylenesulfonate ;

3-amino-4, 5-dimethylaminothiazolium mesitylenesulfonate;

2,3-diaminothiazolinium mesitylenesulfonate;

3-(2-methoxy-2-oxoethyl)-thiazolium bromide; 3-(2-methoxy-2-oxoethyl)-4,5-dimethylthiazolium bromide;

3-(2-methoxy-2-oxoethyl)-4-methylthiazolium bromide;

3-(2-phenyl-2-oxoethyl)-4-methylthiazolium bromide;

3-(2-phenyl-2-oxoethyl)-4,5-dimethylthiazolium bromide;

3-(2-phenyl-2-oxoethyl)-4,5-dimethylthiazolium chloride; 3-amino-4-methylthiazolium mesitylenesulfonate;

3-(2-methoxy-2-oxoethyl)-5-methylthiazolium bromide;

3-(3-(2-phenyl-2-oxoethyl)-5-methylthiazolium bromide;

3-[2-(4'-bromophenyl)-2-oxoethyl] thiazolium bromide;

3-[2-(4'-bromophenyl) -2-oxoethyl]-4-methylth'iazolium bromide; 3-[2-(4'-bromophenylDhenyl)-2-oxoethyl] -5-methylthiazolium bromide;

3-[2- (4 'bromophenyl)-2-oxoethyl) -4, 5-dimethylthiazolium bromide;

3-(2-methoxy-2-oxoethyl)-4-methyl-5-(2-hydroxyethyl) thiazolium bromide;

3-(2-phenyl-2-oxoethyl)-4-methyl-5- (2-hydroxyethyl) thiazolium bromide;

3-[2-(4'-bromophenyl)-2-oxoethyl] -4-methyl-5- (2-hydroxyethyl) thiazolium bromide; 3, 4-dimethyl-5-(2-hydroxyethyl) thiazolium iodide;

3 -ethyl-5-(2-hydroxyethyl)-4-methylthiazolium bromide;

3 -benzyl-5-(2-hydroxyethyl)-4-methylthiazolium chloride;

3-(2-methoxy-2-oxoethyl) benzothiazolium bromide;

3-(2-phenyl-2-oxoethyl) benzothiazolium bromide; 3-[2-(4'bromophenyl)-2-oxoethyl] benzothiazolium bromide;

3-(carboxymethyl) benzothiazolium bromide;

2,3-(diamino) benzothiazolium mesitylenesulfonate;

3-(2-amino-2-oxoethyl) thiazolium bromide;

3-(2-amino-2-oxoethyl)-4-methylthiazolium bromide; 3-(2-amino-2-oxoethyl) -5-methylthiazolium bromide;

3-(2-amino-2-oxoethyl) 4, 5-dimethylthiazolium bromide;

3-(2-amino-2-oxoethyl) benzothiazolium bromide;

3-(2-amino-2-oxoethyl) 4-methyl-5- (2-hydroxyethyl) thiazolium bromide;

3-amino-5-(2-hydroxyethyl)-4-methylthiazolium mesitylenesulfonate; 3-(2-methyl-2-oxoethyl) thiazolium chloride;

3-amino-4-methyl-5-(2-acetoxyethyl) thiazolium mesitylenesulfonate;

3-(2-phenyl-2-oxoethyl) thiazolium bromide;

3-(2-methoxy-2-oxoethyl)-4-methyl-5-(2-acetoxyethyl) thiazolium bromide; 3-(2-amino-2-oxoethyl) -4-methyl-5-(2-acetoxyethyl) thiazolium bromide;

2-amino-3- (2-methoxy-2-oxoethyl) thiazolium bromide;

2-amino-3- (2-methoxy-2-oxoethyl) benzothiazolium bromide;

2-amino-3-(2-amino-2-oxoethyl) thiazolium bromide;

2-amino-3- (2-amino-2-oxoethyl) benzothiazolium bromide; 3-[2-(4'-methoxyphenyl)-2-oxoethyl)-thiazolinium bromide;

3-[2-(2',4'-dimethoxyphenyl)-2-oxoethyl] -thiazolinium bromide;

3-[2-(4'-fluorophenyl)-2-oxoethyl]-thiazolinium bromide;

3-[2-(2',4'-difluorophenyl)-2-oxoethyl]-thiazolinium bromide;

3-[2-(4'-diethylaminophenyl)-2-oxoethyl]-thiazolinium bromide; 3-propargyl-thiazolinium bromide;

3 -propargyl-4-methylthiazolinium bromide;

3-propargyl-5-methylthiazolinium bromide;

3-propargyl-4,5-dimethylthiazolinium bromide;

3-propargyl-4-methyl-5-(2-hydroxyethyl)-thiazolinium bromide; 3-(2-(3'-methoxyphenyl) -2-oxoethyl)-thiazolium bromide;

3-(2-(3'-methoxy phenyl)-2-oxoethyl)-4 methyl-5-(2'-hydroxyethyl)-thiazolium bromide;

3-(2-(3 '-methoxyphenyl)-2-oxoethyl)-benzothiazolium bromide;

2,3-diamino-4-chlorobenzothiazolium mesitylenesulfonate;

2,3-diamino-4-methyl-thiazolium mesitylenesulfonate; 3-amino-4-methyl-5-vinyl-thiazolium mesitylenesulfonate;

2,3-diamino-6-chlorobenzothiazolium mesitylenesulfonate;

2,6-diamino-benzothiazole dihydrochloride;

2,6-diamino-3 [2-(4'-methoxyphenyl)-2-oxoethyl) benzothiazolium bromide;

2,6-diamino-3 [2-(3'-methoxyphenyl)-2-oxoethyl) benzothiazolium bromide; 2,6-diamino-3 [2-(4'-diethylaminophenyl)-2-oxoethyl] benzothiazolium bromide;

2,6-diamino-3 (2-(4'-bromophenyl)-2-oxoethyl] benzothiazolium bromide;

2,6-diamino-3 (2- (2-phenyl-2-oxoethyl) benzothiazolium bromide;

2,6-diamino-3 [2- (4' -fluorophenyl-2-oxoethyl) benzothiazolium bromide;

3-acetamido-4-methyl-5-thiazolyl-ethyl acetate mesitylenesulfonate; 2,3-diamino-5-methylthiazolium mesitylenesulfonate;

3-[2-(2'-naphtyl) -2-oxoethyl] -4-methyl-5-(2'-hydroxyethyl)-thiazolium bromide;

3-[2-(3',5'-di-ter-butyl-4'-hydroxyphenyl)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl- thiazolium bromide; 3-[2-(2',6'-dichlorophenethylamino)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl) - thiazolium-bromide;

3-[2-dibutylamino-2-oxoethyl) -4-methyl-5- (2'-hydroxyethyl) -thiazolium bromide;

3-[2-4' -carbethoxyanilino) -2-oxoethyl] -4-methyl-5- (2'-hydroxyethyl) -thiazolium bromide; 3-[2-(2',6'-diisopropylanilino) -2-oxoethyl] -4-methyl-5-(2'-hydroxyethyl) -thiazolium bromide;

3-amino-4-methyl-5- (2- (2' ,6' -dichlorobenzyloxy) ethyl]-thiazolium mesitylenesulfonate;

3-[2-(4'-carbmethoxy- 3' -hydroxyanilino) -2 -oxoethyl) -4-methyl-5- (2' -hydroxyethyl)- thiazolium bromide; 2,3-diamino-4, 5-dimethylthiazolium mesitylenesulfonate;

2,3-diamino-4-methyl-5-hydroxyethyl-thiazolium mesitylene sulfonate;

2,3-diamino-5- (3' ,4' -trimethylenedioxy phenyl) -thiazolium mesitylene sulfonate;

3-[2-(l', 4' -benzodioxan-6-yl) -2-oxoethyl] -4-methyl-5- (2'-hydroxyethyl) -thiazolium bromide; 3-[2-(3',4'-trimethylenedioxyphenyl)-2-oxoethyl] -4-methyl-5-(2'-hydroxyethyl)- thiazolium bromide;

3-(2-[F,4-benzodioxan-6-yl) -2-oxoethyl) -thiazolium bromide;

3-[2-(3',4' -trimethylenedioxyphenyl) -2-oxoethyl] -thiazolium bromide;

3-[2-(3', 5'-di-tert-butyl-4' -hydroxyphenyl) -2-oxoethyl] -thiazolium bromide; 3-[2- (3', 5'-di-tert-butyl-4' -hydroxyphenyl) -2-oxoethyl] -4-methyl-thiazolium bromide;

3-[2- (3', 5'-di-tert-butyl-4' -hydroxyphenyl) -2-oxoethyl) -5-methyl-thiazolium bromide;

3-[2-(3', 5'-di-tert-butyl-4' -hydroxyphenyl)-2-oxoethyl] -4, 5-dimethyl-thiazolium bromide;

3-[2- (3', 5' -di-tert-butyl-4' -hydroxyphenyl)-2-oxoethyl]-benzothiazolium bromide; l-methyl-3- (2- (3', 5 '-di-tert-butyl-4' -hydroxyphenyl) -2-oxoethyl] -imidazolium bromide;

3-[2-(4'-n-pentylphenyl) -2-oxoethyl] -thiazolinium bromide;

3-[2- (4'-n-pentylphenyl) -2-oxoethyl] -4-methyl-5-(2'-hydroxyethyl) -thiazolinium bromide; 3-[2-4' -diethylaminophenyl) -2-oxoethyl] -4-methyl-5-(2'-hydroxyethyl) -thiazolinium bromide;

3-(2-phenyl-2-oxoethyl)-4-methyl-5 -vinyl-thiazolium bromide;

3-[2-(3' ,5' -tert-butyl-4'-hydroxyphenyl) -2-oxoethyl) -4-methyl-5 -vinyl-thiazolium bromide;

3-(2-tert-butyl-2-oxoethyl) -thiazolium bromide;

3-(2-tert-butyl-2-oxoethyl) -4-methyl-5- (2' -hydroxyethyl) -thiazolium bromide;

3-(3'-methoxybenzyl) -4-methyl-5- (2' -hydroxyethyl) -thiazolium chloride;

3-(2',6'-dichlorobenzyl) -4-methyl-5- (2' -hydroxyethyl) -thiazolium chloride; 3-(2'-nitrobenzyl)-4-methyl - 5- (T - hydroxyethyl) -thiazolium bromide;

3 [2-(4'-chiorophenyl) -2-oxoethyl] -thiazolium bromide;

3[2-(4'-chlorophenyl) -2-oxoethyl] -4-methyl-5- (2 '-hydroxyethyl) -thiazolium bromide; and

3[2-(4'-methoxyphenyl) -2-oxoethyl] -4-methyl-5- (2 '-hydroxyethyl) -thiazolium bromide. Compounds of the invention fixrther include compounds represented by the formula

Ia:

wherein R¹ is independently selected from the group consisting of hydrogen, hydroxy(lower)alkyl, acetoxy(lower)alkyl, lower alkyl, lower alkenyl;

R² is independently selected from the group consisting of hydrogen, hydroxy(lower)alkyl, acetoxy(lower)alkyl, lower alkyl, lower alkenyl, or R¹ and R² together with their ring carbons may be an aromatic fused ring, optionally substituted by one or more amino, halo or alkylenedioxy groups; Z is hydrogen or an amino group;

Y is amino, a group of the formula

O

Il

-CH₂C-R 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; a group of the formula

-CH₂R' wherein R' is hydrogen, or a 'lower alkyl, lower alkynyl, or aryl group; or a group of the formula

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 R² and Z are both hydrogen, then R¹ is other than a lower alkyl group;' and X is a halide, tosylate, methanesulfonate or mesitylenesulfonate ion.

The preferred thiazolium compound of the instant invention comprises the structure of Formula I, wherein R and R are lower alkyl, Z is hydrogen, Y is a group of the formula

O

Il

-CH₂C-R wherein R is an aryl group and X is halide. In more preferred embodiments, the compound of the invention is 3-(2-phenyl-2-oxoethyl)-4,5-dimethylthiazolium chloride or N-phenacyl-4,5-dimethylthiazolium chloride, also referred to as ALT-711 or algebrium chloride herein. In other embodiments, the compound of the invention is 3-(2-phenyl-2- oxoethyl)-4,5-dimethylthiazolium bromide or N-phenacyl-4,5-dimethylthiazolium bromide, also referred to as DMPTB or PMTB.

Other compounds of the invention are those of the formula Ib:

wherein R¹ is independently selected from the group consisting of , hydroxy (lower) alkyl, acetoxy(lower)alkyl, lower acyloxy(lower)alkyl, lower alkyl; R² is independently selected from the group consisting of , hydroxy (lower) alkyl, acetoxy(lower)alkyl, lower acyloxy(lower)alkyl, lower alkyl, or R¹ and R² together with their ring carbons may be an aromatic fused ring;

Z is hydrogen or an amino group; Y is an alkynylmethyl group, or a group of the formula

-CH₂C-N

R"¹ 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 X is a halide, tosylate, methanesulfonate or mesitylenesulfonate ion. Other compounds of the invention are those of formula (Ic):

wherein R¹ and R² are methyl; Z is hydrogen; Y is a group of the formula:

O

Il

-CH₂C-R, wherein R is phenyl; and X is halide.

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 compounds employed in accordance with this invention inhibit this late-stage Maillard effect and reduce the level of the advanced glycosylation endproducts already present in the protein material.

The rationale of the present invention is to use compounds 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. 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.

Several investigators have studied the mechanism of advanced glycosylation product formation. In vitro studies by EbIe 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. EbIe 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.

EbIe 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 EbIe 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 EbIe 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 EbIe et al. Clearly, EbIe 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.

The experiments of EbIe 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 EbIe 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.

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-l- 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-l- 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).

X-[Protein]

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.

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 α-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 α-dione cleaving agent of the formula I, N-phenacylthiazolium bromide, with an AP-ene-dione derived cross link.

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 l-phenyl-i,2-propanedione was rapid and readily proceeded, confirming this possible mechanism.

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-modifϊed 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 α-diketone or related structure that is susceptible to cleavage by the advantageous bidentate-type molecules of the compounds of formula I under physiological conditions. 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.

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. 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. The therapeutic implications of the present invention relate to the a method of treating or preventing gastrointestinal complications e.g. diabetes-related complications. The present invention relates to a method of treating or preventing complications that arise from a reduction in nNOS expression. The present invention relates to a method of treating or preventing gastroparesis. 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 used therein are biocompatible. Pharmaceutical compositions may be prepared with a therapeutically effective quantity of the 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 the invention may be utilized.

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 the invention. Other suitable forms for administration to other body tissues are also contemplated.

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 compounds, 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 compounds may take place over an extended period of time at a dosage level of, for example, up to about 30 mg/kg. The compound of the invention is formulated in compositions in an amount effective to inhibit and reverse the formation of advanced glycosylation endproducts. The compound of the invention is formulated in compositions in an amount effective to inhibit the expression of intestinal neuronal nitric oxide synthase nNOS. 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.

The compounds encompassed by the invention are conveniently prepared by chemical syntheses well-known in the art. Certain of the compounds encompassed by the invention 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 the present invention 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., 7. Org. Chem., 41 :187-191 (1976)].

Certain of the compounds of the invention are novel compounds, not heretofore known in the art. These compounds are those represented by the formula Ia

wherein R¹ and R² are independently selected from the group consisting of hydrogen, hydroxy (lower) alkyl, acetoxy(lower)alkyl, lower alkyl, lower alkenyl, or R¹ and R² together with their ring carbons may be an aromatic fused ring, optionally substituted by one or more amino, halo or alkylenedioxy groups; Z is hydrogen or an amino group;

Y is amino, a group of the formula

O

Il

-CH₂C-R 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; a group of the formula -CH₂R' wherein R' is hydrogen, or a lower alkyl, lower alkynyl, or aryl, group; or a group of the formula

⁰ /"

-CH₂C-N R"' 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 R₂ and Z are both hydrogen, then Ri is other than a lower alkyl group; and

X is a halide, tosylate, methanesulfonate or methanesulfonate ion.

Other novel compounds are those of formula I wherein Y is a lower alkynylmethyl group or a group of the formula

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. 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;

O

Il

-CH₂C-R Wherein R is lower alkyl, alkoxy, hydroxy, amino or aryl group; or a group of the formula -CH₂R' wherein R' is hydrogen, or a lower alkyl, lower alkynyl or aryl group; X is a halide, tosylate, methanesulfonate or mesitylenesulfonate ion; 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. Scheme I

Scheme I

wherein R¹, R², Z, and R are as hereinabove defined, and X is a halogen atom. In reaction Scheme I, the appropriate substituted thiazole compound of formula II wherein R¹, R² 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 the invention e.g., formula I wherein R¹, R², Z, R and X are as hereinbefore defined. Typically, this reaction is conducted at reflux temperatures for times of about 1 -3 hours. Typically, a polar solvent such as ethanol is utilized for the conduct of the reaction. 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.

SCHEME II

JP

wherein R¹, R² and Z are as defined hereinabove.

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. 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: a) incubation of AGE-modifϊed bovine serum albumin (AGE BSA) on collagen-coated wells of microtiter plates for a period of 2-6 hours' at a temperature of 37 ⁰C ; b) washing of the wells with PBS-Tween; c) application of the test compounds to the washed wells of step b; d) incubation of the test compounds applied to the washed wells for an additional 12-24 hours at a temperature of about 37 ⁰C; and e) detection of the AGE-breaking using an antibody raised against AGE- ribonuclease or cross-link breaking with an antibody against BSA.

While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

The present invention is further illustrated by the following examples that should not be construed as limiting in any way.

EXAMPLES EXAMPLE 1

3- (2-Methoxy-2-oxoethyl)-thiazolium bromide

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 ⁰C (dec).

EXAMPLE 2

3-Amino-4.5-dimethylthiazoliurn mesitylenesulfonate

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°C.

EXAMPLE 3 Using the procedures described above in Examples 1 and 2, the following compounds are prepared.

423 3-amino-thiazolium mesitylenesulfonate, m.p. 102-104 ⁰C. 427 2,3-diarmino-thiazolium mesitylenesulfonate, m.p. 173-175 ⁰C (dec). 670 3- (2-methoxy-2-oxoethyl) -4,5-dimethylthiazolium bromide, m.p. 184- 185 ⁰C (dec).

709 3- (2-methoxy-2-oxoethyl) -4-methylthiazolium bromide, m.p. 149-151 ⁰C (dec).

710 3- (2-phenyl-2-oxoethyl) -4-methylthiazolium bromide, m.p. 218-220 ⁰C (dec).

711 3- (2-phenyl-2-oxoethyl) -4,5-dimethylthiazolium bromide, m.p. 212-213°C (dec). 717 3-amino-4-methyl-thiazolium mesitylene sulfonate, m.p. 143-144 ⁰C.

719 3-(2-methoxy-2-oxoethyl)-5-methyl-thiazolium bromide, m.p. 193- 194 ⁰C (dec).

720 3-(2-phenyl-2-oxoethyl)-5-methyl-thiazolium bromide, m.p. 193- 194 ⁰C.

721 3- (2-(4¹-bromophenyl]-2-oxoethyl)-thiazolium bromide, m.p. 269-270 ⁰C (dec).

722 3- (2-[4'-bromophenyl)-2-oxoethyl)-4-methyl-thiazolium bromide, m.p. 248-249 ⁰C (dec).

723 3-(2-(4¹-bromophenyl]-2-oxoethyl)-5-methyl-thiazolium bromide, m.p. 216-217 ⁰C.

724 3- (2- (4-bromophenyl] -2-oxoethyl)-4, 5-dimethylthiazolium bromide, m.p. 223- 224 ⁰C (dec).

725 3-(2-methoxy-2-oxoethyl)-4-methyl-5-(2-hydroxyethyl)-thiazolium bromide, m.p. 137-138°C.

726 3-(2-phenyl-2 -oxoethyl)-4-methyl- 5- (2 -hydroxyethyl)- thiazolium bromide, m.p. 180-181⁰C.

727 3-(2-(41 -bromophenyl]-2-oxoethyl)-4-methyl-5-(2-hydroxyethyl)thiazolium bromide, m.p. 251-252°C (dec). 728 3,4-dimethyl-5- (2-hydroxyethyl) -thiazolium iodide, m.p. 85-87⁰C.

729 3-ethyl-5- (2-hydroxyethyl) -4-methyl thiazolium bromide, m.p. 84-85°C.

730 3-benzyl-5- (2-hydroxyethyl) -4-methyl thiazolium chloride, m.p. 144-146°C.

731 3-(2-methoxy-2-oxoethyl) -benzothiazolium bromide, m.p. 144-145°C (dec).

732 3-(2-phenyl-2-oxoethyl) -benzothiazolium bromide, m.p. 733 240-241⁰C (dec).

734 3-(2-(41-bromophenyl) -2-oxoethyl) -benzothiazolium bromide, m.p. 261-262°C (dec).

734 3-(carboxymethyl) -benzothiazolium bromide m.p. 250⁰C (dec).

735 2,3-diaminio-benzothiazolium mesitylenesulfonate, m.p. 212-214°C (dec). 738 3-(2-amino-2-oxoethyl) -thiazolium bromide, m.p. 205-206⁰C.

739 3- (2-amino-2-oxoethyl) -4-methyl-thiazolium bromide, m.p. 220-222⁰C.

740 3- (2-amino-2-oxoethyl) -5-methyl-thiazolium bromide, m.p. 179-180⁰C.

741 3-(2-amino-2-oxoethyl)-4,5-dimethyl-thiazolium bromide, m.p. 147-148°C. 742 3-(2-amino-2-oxoethyl) -benzothiazolium bromide, m.p. 222-223°C.

743 3-(2-amino-2-oxoethyl)-4-methyl-5-(2-hydroxyethyl) thiazolium bromide, m.p. 182- 183°C.

744 3-amino-5 - (2-hydroxyethyl) -4 -methyl-thiazolium mesitylenesulfonate, m.p. 94- 95°C (dec). 755 3-(2-methyl-2-oxoethyl) thiazolium chloride, m.p. 178 -179°C.

763 3-amino-4-methyl-5- (2-acetoxyethyl) thiazolium mesitylenesulfonate, m.p. 118-

120⁰C. 766 3-(2-phenyl-2-oxoethyl) thiazolium bromide, m.p. 217-218°C.

769 3-(2-methoxy-2-oxoethyl)-4-methyl-5-(2- acetoxyethyl) thiazolium bromide, m.p. 217-218°C.

770 3-(2-amino-2-oxoethyl)-4-methyl-5-(2~ acetoxyethyl)thiazolium bromide, m.p. 233-234°C.

771 2-amino-3- (2-methoxy-2-oxoethyl) thiazolium bromide, m.p. 191-192°C.

772 2-amino-3- (2-methoxy-2-oxoethyl) benzothiazolium bromide, m.p. 236-237°C. 773 2-amino-3- (2-amino-2-oxoethyl) thiazolium bromide, m.p.209-210⁰C.

774 2-amino-3 - (2-amino-2-oxoethyl) benzothiazolium bromide, m.p. 234-235°C.

798 3-(2-(4'-methoxyphenyl) -2-oxoethy].] -thiazolinium bromide, m.p. 248-249°C (dec);

799 3- (2-(2', 4'-dimethoxyphenyl) -2~oxoethyl] - thiazolinium bromide, m.p. 214- 216°C (dec); 35 800 3- (2- (4' -fluorophenyl-2-oxoethyl] - thiazolinium bromide, m.p. 209-210⁰C (dec);

801 3-(2-(2', 4'-difluorophenyl) -2-oxoeethyl) - thiazolinium bromide, m.p. 226-228°C (dec);

802 3- (2- (4' -diethylaminophenyl) -2-oxoethyl] - thiazolinium bromide, m.p. 233- 235°C (dec);

803 3-propargyl-thiazolium bromide, m.p. 64-66°C;

804 3-Propargyl-4-methyl thiazolium bromide, m.p. 213-215°C;

805 3-Propargyl-5-methyl thiazolium bromide, m.p. 127-129° C;

806 3-Propargyl-4, 5-dimethyl thiazolium bromide, m.p. 198-200⁰C; 807 3-Propargyl-4-methyl-5- (2-hydroxyethyl) -thiazolium bromide, m.p. 132- 134°C;

824 3- (2- (3' -methoxyphenyl] -2-oxoethyl) -thiazolium bromide, m.p. 224-225°C;

825 3- (2- [3' -methoxyphenyl] -2-oxoethyl) -4 methyl 5- (2' -hydroxyethyl) -thiazolium bromide, m.p. 164-165°C; 826 3- (2- [3' -methoxyphenyl] -2-oxoethyl) - benzothiazolium bromide, m.p. 215-

217°C; 836 2, 3-diamino-4-chlorobenzothiazolium mesitylenesulfonate, m.p. 228-230⁰C;

847 2, 3-diamino-4-methyl-thiazolium mesitylene sulfonate, m.p. 204-205⁰C;

848 3-amino-4-methyl-5-vinyl-thiazolium mesitylene sulfonate, m.p. 145-147°C; 858 2, 3-diamino-6-chlorobenzothiazolium mesitylenesulfonate, m.p. 244-246°C;

862 2, 6-diamino-benzothiazole dihydrochloride, m.p. 318-320°C (dec);

876 2,6 -diamino-3 (2- (4' -methoxyphenyl) -2-oxoethyl] benzothiazolium bromide, m.p. 243-245°C (dec);

877 2,6 -diamino-3 (2- (3' -methoxyphenyl) -2-'oxoethyl] benzothiazolium bromide, m.p. 217-218°C (dec);

878 2,6 -diamino-3 (2- (4' -diethylaminophenyl) -2-oxoethyl) benzothiazolium bromide, m.p. 223-225°C (dec);

887 2,6 -diamino-3 (2- (4' -bromophenyl) -2 -oxoethyl] benzothiazolium bromide, m.p.

258-259°C (dec); 888 2,6-diamino-3(2-(2-phenyl-2-oxoethyl) benzothiazolium bromide, m.p. 208-210⁰C

(dec); 889 2, 6-diamino-3 (2- (4' -fluorophenyl-2 -oxoethyl] benzothiazolium bromide, m.p.

251-252°C (dec);

897 3-acetamido-4-methyl-5-thiazolyl-ethyl acetate mesitylenesulfonate, m.p. syrup material;

913 2, 3-diamino-5-methylthiazolium mesitylenesulfonate, m.p. 149-152°C;

924 3-(2-(2'-naphthyl)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-thiazolium bromide, m.p. 219-220⁰C;

925 3- (2- (3' , 5' -Di-tert-butyl-4' -hydroxyphenyl) -2-15 oxoethyl] -4-methyl-5- (2' - hydroxyethyl) -thiazolium bromide, m.p. 206-207⁰C;

928 3- [2- (2', 6' -Dichlorophenethylamino) -2-oxoethyl] -4-methyl-5- (2' - hydroxyethyl) -thiazolium bromide, m.p. 193-195°C;

929 3-(2-Dibutylamino-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl) -thiazolium bromide, m.p. 78-80⁰C; 930 3- (2-4' -carbethoxyanilino) -2-oxoethyl] -4- methyl-5- (2' -hydroxyethyl) - thiazolium bromide, m.p. 204-206°C;

931 3-(2-(2' ,6'-Diisopropylanilino)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl) - thiazolium bromide, m.p. 166-168°C; 932 3-amino-4-methyl-5-(2(2' ,6'-dichlorobenzyloxy) ethyl] -thiazolium mesitylenesulfonate, 30 m.p. 164-166°C; 935 3- (2- (4' -carbmethoxy-3' -hydroxyanilino) -2-oxoethyl] -4-methyl-5- (2' - hydroxyethyl) -thiazolium bromide, m.p. 222-223°C;

938 2, 3-Diamino-4, 5-dimethyl thiazolium mesitylene sulfonate, m.p. 166-168°C; 939 2,3-Diamino-4-methyl-5-hydroxyethyl-thiazolium mesitylene sulfonate, m.p. 132- 134°C;

940 2, 3-Diamino-5- (3' ,4' -trimethylenedioxy phenyl) thiazolium mesitylene sulfonate, m.p. 224-226°C;

941 3 (2- (1 ' , 4' -benzodioxan-6-yl) -2-oxoethyl] -4-methyl-5- (2' -hydroxyethyl) - thiazolium bromide, m.p. 196- 198°C;

942 3- (2- (3' , 4' -trimethylenedioxyphenyl) -2-oxoethyl] -4-methyl-5- (2' - hydroxyethyl) -thiazolium bromide, m.p. 164-166°C;

943 3- (2- (1 ' , 4 -benzodioxan-6-yl] — 2-oxoethyl) - thiazolium bromide, m.p. 238- 239°C; 944 3- (2- (3' , 4' -trimethylenedioxyphenyl) -2- oxoethyl]-thiazolium bromide, m.p. 246-248°C (dec);

948 3-[2-(3',5"-di-tert-butyl-4'-hydroxyphenyl)-2- oxoethyl] -thiazolium bromide, m.p.

949 3-[2-(3',5'~di-tert-butyl-4'-hydroxyphenyl)-2- oxoethyl] -4-methyl-thiazolium bromide, m.p. 226-228°C (dec); 950 , 3-[2-(3',5'-di-tert-butyl-4'-hydroxyphenyl)-2- oxoethyl) -5-methyl-thiazolium bromide, m.p. 210-211° C;

951 3-(2-(3',5'-di-tert-butyl-4'-hydroxypheny].)-2- oxoethyl] -4, 5-dimethyl- thiazolium bromide, m.p. 243-244°C (dec);

952 3-(2-(3',5'-di-tert-butyl-4'-hydroxyphenyl)-2- oxoethyl] -benzothiazolium bromide, m.p. 239- 294°C (dec);

953 l-methyl-3-(2-(3',5'-di-tert-butyl-4'-hydroxyphenyl) -2-oxoethyl] -imidazolium bromide, m.p. 148-150°C;

954 3- [2- (4' -n-pentylphenyl) -2-oxoethyl] - thiazolinium bromide, m.p. 218-22O⁰C (dec); 955 3- (2- (4' -n-pentylphenyl) -2 -oxoethyl] -4-methyl -5-(2' -hydroxyethyl) - thiazolinium, m.p. 178-180⁰C (dec);

956 3- (2-4' -diethylaminophenyl) -2-oxoethyl] -4-qj. methyl-5- (2' -hydroxyethyl) - thiazolinium bromide, m.p. 184-186°C (dec); 957 3-(2-phenyl-2-oxoethyl)-4-methyl-5-vinyl-thiazolium bromide, m.p. 176-177°C;

958 3- (2- (3' , 5' -tert-butyl-4' -hydroxyphenyl) -2- oxoethyl) -4-methyl-5-vinyl- thiazolium bromide, m.p. 208-209° C;

959 3- (2-tert-butyl-2-oxoethyl) -thiazolium bromide, m.p. 211 -212°C;

960 3-(2-tert-butyl-2-oxoethyl)-4-methyl-5-(2'-hydroxyethyl) -thiazolium bromide, m.p. 186-187°C;

961 3-(3'-methoxybenzyl)-4-methyl-5-(2'-hydroxyethyl) -thiazolium chloride, m.p. 135- 136°C;

962 3- (2' , 6' -dichlorobenzyl) -4-methyl-5- (2' -15 hydroxyethyl)- thiazolium chloride, m.p. 192-194°C; 963 3-(2'-nitrobenzyl)-4-methyl-5-(2'- hydroxyethyl) - thiazolium bromide, m.p. 215- 216°C;

964 3 (2- (4' -chlorophenyl) -2-oxoethyl] -thiazolium bromide, m.p. 239-241⁰C (dec);

965 3 (2- (4' -chlorophenyl) -2-oxoethyl] -4-methyl-5-(2'-hydroxyethyl)-thiazolium bromide, m.p. 240-251⁰C (dec); and 966 3 (2- (4' -methoxyphenyl) -2-oxoethyl] -4-methyl-5- (2' hydroxyethyl)-thiazolium bromide, m.p. 229-231°C 25 (dec).

EXAMPLE 4

Mg/tablet

Compound of the invention 50 Starch 50

Mannitol 75

Magnesium stearate 2

Stearic acid 5

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°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

Lotion mg/g

Compound of the invention 1.0 Ethyl alcohol 400.0 Polyethylene glycol 400 300.0 Hydroxypropyl cellulose 5.0 Propylene glycol to make 1.0 g

EXAMPLE 6

Oral Rinse

Compound of the invention: 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

Toothpaste

Compound of the invention: 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

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. 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°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 μl each of the AGE-BSA diluted in PBS or in the solution of test compound at 37 ⁰C 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.

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 ran in a Dynatech plate reader.

The % inhibition of each test compound was calculated as 15 follows.

% inhibition =

{(Optical density (without compound) - optical density (with compound)]/optical density

(without compound)) x 100%

The IC50 values or the inhibition at various concentrations by test compounds is as follows:

Test Compound ICsn Relative Cross-

(mM) link Inhibition fat 10 mM) Inhibition Data

3-amino-4, 5-dimethylaminothiazolium 2.8 mesitylenesulfonate

2, 3-diaminothiazolinium mesitylenesulfonate >.10 27%

3- (2-methoxy-2-oxoethyl) -thiazolium bromide 0.25

3- (2-methoxy-2-oxoethyl) -4, 5-dimethylthiazolium 0.48 bromide

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 37% bromide

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 bromide 37%

3- [2- (4' bromophenyl) -2-oxoethyl] -4, 5- 2.92 dimethylthiazolium

3- (2-methoxy-2-oxoethyl) -4-methyl-5- (2-hydroxyethyl) thiazolium bromide 38%

3- (2-phenyl-2-oxoethyl) -4-methyl-5- (2-hydroxyethyl) thiazolium bromide >10 36% 3- [2- (4' -bromophenyl) -2-oxoethyl] -4-methyl-5- (2- hydroxyethyl) thiazolium bromide 2.95

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) thiazolium bromide 1.42

3-amino-5- (2-hydroxyethyl) -4-methylthiazolium 3.6 x 10⁵ mesitylenesulfonate

3- (2-phenyl-2-oxoethyl) thiazolium bromide 11.1 34%

3- (2- [3' - methoxyphenyl] -2-oxoethyl -thiazolium 29% bromide

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-ethyl) -thiazolium bromide 61%

3- [2-Dibutylamino-2-oxoethyl] -4-methyl-5- (2' - hydroxyethyl) -thiazolium bromide 0.8% ( 10mm) 3- [2-4'-carbethoxyanilino) -2-oxoethyl] -4-methyl-5- (2'

-hydroxyethyl) -thiazolium bromide 8.8% (1 mm)

3- [2- (2', 6' - Diisopropylanilino) -2-oxoethyl] -4-methyl-

5- (2' -hydroxyethyl) -thiazolium bromide 19%

3-amino-4-methyl-5- [2(2', 6' -dichlorobenzyloxy) ethyl]

-thiazolium mesitylenesulfonate 26.5% (3mm)

3- [2- (4' -carbmethoxy-3' -hydroxyanilino) -2-oxoethyl] -

4-methyl-5- (2' -hydroxyethyl) -thiazolium bromide 1.76

2,3-Diamino-4,5-dimdethyl thiazolium mesitylene 39% sulfonate

2,3-Diamino-4-methyl-5-hydroxyethyl-thiazolium mesitylene sulfonate 18%

2,3-Diamino-5- (3',4' -trimethylenedioxy phenyl) - thiazolium mesitylene sulfonate 40% @ 3mM

3[2- (l',4' -benzodioxan-6-yl) -2-oxoethyl] -4-methyl-5-

(2' -hydroxyethyl) -thiazolium bromide 13%

3- [2- (3 ',4' -trimethylenedioxyphenyl) -2-oxoethyl] - thiazolium bromide 4.4

3- [2- (3',4' - trimethylenedioxyphenyl) -2-oxoethyl] - thiazolium bromide 45%

3- [2- (3',5'-di-tert-butyl-4'-hydroxyphenyl) -2-oxoethyl]

-4-methyl-thiazolium bromide 24% @ 0.3mM

3- [2- (3',5'-di-tert-butyl-4'-hydroxyphenyl) -2-oxoethyl]

-5-methyl-thiazolium bromide 0.78 69% @ ImM

3- [2- (3',5'-di-tert-butyl-4'-hydroxyphenyl) -2-oxoethyl]

-4,5-dimethyl-thiazolium bromide 0.16 l-methyl-3- [2- (3',5' -di-tert-butyl-4'-hydroxyphenyl) -2- oxoethyl] -imidazolium bromide 4.5

3- [2- (4'-n-pentylphenyl) -2-oxoethyl] -thiazolinium ND bromide

3- [2- (4'-n-pentylphenyl) -2-oxoethyl] -4-methyl-5- (2' - hydroxyethyl) -thiazolinium bromide 1.53 52% @ 3mM 3- [2-4' -diethylaminophenyl) -2-oxoethyl] -4-methyl-5- (2' -hydroxyethyl) -thiazolinium bromide 2.8

3- (2-phenyl-2-oxoethyl) -4-methyl-5-vinyl-thiazolium bromide ND

3- [2- (3 ',5' -tert-butyl-4' -hydroxyphenyl) -2-oxoethyl) - 4-methyl-5-vinyl-thiazolium bromide ND

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

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 ⁰C) 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: MATERIALS

Immunochemicals and Chemicals Bovine Serum Albumin (Type V), (BSA) Calbiochem Dextrose

Superbloc, Pierce, Inc.

Rabbit anti-Bovine Serum Albumin

Horseradish Peroxidase (HRP) -Goat-anti-rabbit), Zymed HRP substrate buffer, Zymed

ABTS chromogen, Zymed

Phosphate Buffer Saline

Tween 20, Sigma

Equipment ELISA Plate Washer, Dynatech

ELISA Plate Reader, Dynatech

Precision Water Bath

Corning digital pH meter

Glassware and Plasticware Finneppette Multichannel Pipettor, Baxter

Eppendorf pipettes, Baxter

Eppendorf repeater pipette, Baxter

Pipettor tips for Finneppetter, Baxter

Pipettor tips for Eppendorf, Baxter Glass test tubes, 13x100 mm; Baxter

Mylar Sealing Tape for 96 well plates, Corning

Biocoat Cellware Rat Tail Collagen Type-1 coated 96-well plates, Collaborative Biomedical

Products.

METHODS Preparation of solutions and buffers

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 ⁰C 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 ⁰C. Dilute solutions of AGE-BSA were unstable when stored at -20 ⁰C.

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.

3. Wash buffer ("PBS-Tween") was, prepared as follows. PBS was prepared by dissolving the following salts in one liter of distilled water: NaCl, 8 grams; KCl, 0.2 gram, KH₂PO₄. 1.15 grams; NaN₃, 0.2 gram. Tween-20 was added to a final concentration of 0.05% (vol/vol).

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.

Assay procedures 1. Biocoat plates were blocked with 300 μ\ 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.

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 ⁰C for four hours and washed with PBS-Tween three times. Fifty microliters of PBS was added to the control wells and 50 μ\ 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).

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 μ\ 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.

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.

5. Detection of maximum crosslinking and breaking of AGE crosslinking was performed as follows. HRP substrate (10OuI) was added to each well of the plate and was incubated at 37 ⁰C 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". STANDARD OPERATING PROCEDURE Preliminary Steps

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.

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.

3. Prepare an assay template for setting up the experiment as described under "Assay Setup", #2, below.

Assay Setup 1. Warm Superbloc reagent to 37 ⁰C. Add 300 μ\ of Superbloc to each well of the Biocoat plate and let stand for sixty minutes at 37 ⁰C. Wash the wells three times with PBS-Tween (0.05%). Turn the plate 180 degrees and repeat this wash cycle.

2. Dilute the AGE-BSA in PBS so that 50 μ\ 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 μ\ of AGE-BSA or BSA to each well which correspond to the "AGE-BSA" and "BSA" labels on the template.

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 μ\ of AGE-BSA or BSA in the inhibitor solutions to the wells which correspond to "test compound + AGE-BSA and to "test compound blank", respectively, on the template. Incubate the plate at 37 ⁰C 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).

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 μ\ is added to each well and the plate is allowed to stand at room temperature for sixty minutes.

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.

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 μ\ of ABTS solution, mix well and add 100 μ\ of this reagent to each well. Incubate the plate at 37°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.

Test Compound ICsn (mM) Breaking Anti- Anti- AGE/Anti-BSA fat

AGE/Anti- mM) BSA

3- aminothiazolium mesitylenesulfonate 0.005/3.0 71%/67% (30)

3-amino-4,5dimenthylaminothiazolium 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 54%/51% (30) bromide

3-(2-phenyl-2-oxoethyl)-4-methylthiazoliumbromide 0.23/0.30 68%/66% (30)

3-(2-phenyl-2-oxoethyl)-4, 5-dimethylthiazolium 56%/ND (30) bromide

3-amino-4-methylthiazolium mesithylenesulfonate 55%/ND (30)

3-(2-methoxy-2-oxoethyl)-5-methylthiazolium 72%/27% (30) bromide

3-[2-(4'-bromophenyl)-2-oxoethyl) thiazolium 76%/25% (30) bromide

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 0.42/0.55 65%/61% (30) chloride

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 61%/16% (30) bromide

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 22%/44% (10) bromide

2, 3-diamino-4-chorobenzothiazolium 21%/26 (10) mesitylenesulfonate

2, 3-diamino-4-methyl-thiazolium 25%/30% (10) mesithylenesulfonate 3-amino-4-methyl-5-vinyl-thiazolium ND/2.0 51%/74% (10) mesitylenesulfonate

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-methylthiazolium 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-(l '- 25%/38% (10) hydroxyethyl)-thiazolium bromide

3 - [2-4 ' -carbethoxyanilino)-2-oxoethyl] -4-methyl-5- 48%-57% (10)

(2 ' -hydroxtethyl)-thiazolium bromide

3-[2-(2', 6'-Diisopropylanilino) -2-oxoethyl]-4- 31%/48% (10) methyl-5-(2'-dhyroxyethyl)-thiazolium bromide

3-amino-4-methyl-5-[2(2',6'- 31%/54% (10) dichlorobenzyloxy)ethyl] -thiazolium mesitylenesulfonate

3 - [2-(4 ' -carbmethoxy-3 ' -hydroxyanilino)-2-oxoethyl] - 24%/18% (10)

4-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- 11%/21% (3)

(2 ' -hydroxyethyl)-thiazolium bromide 3-[2-(3 ' ,4 ' -trimethylenedioxyphenyl)-2-oxoethyl]- 17%/18% (10) thiazolium bromide

3-[2-(3',5'-di-tert-butyl-4'-hydroxyphenyl)-2- 14%/2% (0.3) oxoethyl] -4-methyl-thiazolium bromide

3-[2-(3',5'-di-tert-butyl-4'-hydroxyphenyl)-2- 3/0/74 65%/69% (1) oxoethyl] -5 -methyl-thiazolium bromide

3-[2-(3',5'-di-tert-butyl-4'-hydroxyphenyl)-2- 48%/49% (10) oxoethyl]-5-methyl-thiazolium bromide

1 -methyl-3-[2 [(3 ',5 ' -di-tert-butyl-4 '-hydroxyphenyl)- 56%/38% (10)

2-oxoethyl]-imidazolium bromide

3-(2-phenyl-2-oxoethyl)-4-methyl-5-vinyl-thiazolium ND/0.1 62%/82% (l) bromide

3-[2-(3',5'-tert-butyl-4'-hydroxyphenyl)-2-oxoethyl)- ND/0/60% 32%/50% (0.3)

4-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'- 6%/27% (10) hydroxyethyl)-thiazolium chloride

3-(2'-nithrobenzyl)-4-methyl-5-(2'-hydroxyethyl)- l l%/13% (10) thiazolium bromide

EXAMPLE 10

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 A. Preparation of Red Blood Cells

Blood is collected from the rats in heparinized tubes and spun at 2000 x 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.

B. Assay set up. 1. Warm Superbloc to 37° C.

2. Take a plate of Multiscreen-HA, 0.45u. Cellulose ester membrane-sealed 96 well plate (Millipore MARAS45).

3. Wet the wells with 100 μl of PBS.

4. Add 300 μl of superblock to each well and incubate at 37 ⁰C. for one hour. 5. Place the plate on the Milliliter 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 μl of PBS-Tween 0.05%.

6. Turn off the vacuum and add 100 μl of PBS to each well.

7. Gently vortex the RBC samples and pipette 50 μl to the wells, as per the protocol sheet. Leave first three wells for reagent blanks. Leave another three wells for antibody blank.

8. Suction-off the liquid as above and wash the RBCs twice with PBS.

9. Dilute AP(Rb-anti-rat) (Sigma A-6066), 1 to 25000 in PBS.

10. Add 50 μl to the wells and let stand at room temp, for two hours. 11. Suction-off the liquid as above and wash the RBCs twice with PBS.

12. Add pNPP substrate ( 1 mg/mi in DEA buffer). 100 μl per well.

13. Let the color develop for two hours at 37 ⁰C.

14. Place a 96 well corning micrometer plate in the vacuum chamber.

15. Place the sample plate on the vacuum manifold. Make sure the bottom of the plate is completely dry.

16. Apply vacuum for about 5 minutes. Add 100 μl 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.

17. Calculate percent breaking: 100* (OD410 control-OD410 treated) /OD410 control. Percent Inhibition in animals dosed orally at a rate of 10 mg/kg body weight are as listed below:

3 -amino-4-methyl-5 -vinyl-thiazolium mesitylenesulfonate 11- ± 1 @ 19 days

3- [2- (2' -naphthyl) -2-oxoethyl) A- methyl-5- (2' -hydroxyethyl) -thiazolium bromide 40 ± 24 @ 19 days

3- [2- (3' , 5' -di-tert-butyl-4' -hydroxy- phenyl) -2-oxoethyl] -5-methyl-thiazolium bromide 65 ± 15 @ 19 days

3- (2-phenyl-2-oxoethyl) -4-methyl-5- vinyl-thiazolium bromide 58 ± 21 ® 19 days

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 α- diketone segment consistent with the model shown in Schemes A and B. Second, the crosslink-breaking compounds 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 I l

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 the invention, i.e., 3-(2-phenyl-2-oxoethyl) thiazolium bromide. Collagen fibers (5mg) from streptozotocin diabetic rats and age-matched control animals hydrated in land PBS at 60 °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 3OmM for 16 hours. Following incubation, the pellets were centrifuged, washed, and treated with CNBr (40mg/ml in formic acid at 30 ⁰C 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 Figure 7.

EXAMPLE 12

Preparation of AGE-BSA and crosslinked- AGE-BSA:

Prepare the following solutions. 1. Buffer: 0.4 M sodium phosphate pH 7.4.

NaH₂PO₄ :6g/100ml

NaH₂PO₄ :7g/100ml 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. 2. BSA Solution

BSA: Calbiochem Type V; 400 mg/ml in the buffer 1. Total volume prepared 50g/125ml.

Filtered through a 0.45u filter into a sterile one liter Corning flask.

3. Glucose solution. 40OuM

Glucose: 400 mM 9g/125ml of buffer. Filtered through a 0.45u filter into one liter Corning sterile flask.

Reaction setup:

BSA and glucose solutions (100 ml each) were mixed in the one liter Corning sterile flask, screw-capped tight and incubated at 56 ⁰C. 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.

Breaking the polymer:

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 overnight at 37 ⁰C. The supernatants were analyzed by SDS-PAGE and stained with coommassie blue. The resulting gels are shown in Figure 8.

EXAMPLE 13 To further study the ability of AGE crosslink-inhibiting and reversing compounds 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 ⁰C 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 the invention are included. After incubation, the gelatin/paper disks are rinsed with water, observed for brown color, and photographed. 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^® at a final concentration of 0.04% chlorhexidine) shows significant browning. Addition of a compound of the invention to the chlorhexidine completely inhibits browning of the gelatin, as does inclusion of a compound of the invention in the absence of chlorhexidine. The slight brown color formed by the action of glucose-6-phosphate on the gelatin surface alone and its prevention by a compound of the invention 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 the invention demonstrates the utility of the present invention in preventing the anti-plaque agent-enhanced nonenzymatic browning which occurs with chlorhexidine.

EXAMPLE 14

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. AGE-β-peptide was prepared by incubating an aliquot of the soluble β-amyloid peptide, synthetically prepared and corresponding in sequence to the β-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 β-peptide was substituted for BSA as the glycation substrate. The AGE-β-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 ¹²⁵I- AGE-β-peptide as the desired radiolabeled reagent useful to test or screen compounds for molecular AGE-breaking activity according to the following procedure. Aliquots of ¹²⁵I- AGE-β-peptide were incubated with or without added test compounds of the present invention, at predetermined concentrations (e.g., k 1OmM 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 ¹²⁵I- AGE-β-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, ¹²⁵I- AGE-β-peptide was first incubated in a solution of an AGE crosslink-bearing agent of the present invention, the ¹²⁵I- AGE-β-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 compounds 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 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 compounds of the present invention, which compounds are directed toward inhibition of, and breaking of advanced glycation.

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. 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. 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.

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.

The antibody titers in post-boost blood samples can be compared for hapten-specifϊc 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).

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.

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.

The following sections below entitled Animals, Experimental Design, Experimental Diabetes, and Results apply to Examples 16-21: ANIMALS Eight weeks old male Sprague Dawley rats were housed in pairs in cages with wire mesh bases into controlled environment at a constant room temperature (22°C), humidity, and 12 hour light-darkness cycles. Rats were fed standard chow, and water was given ad libitum. Experiments were approved by the Institutional Animal Care Committee at the University of Texas Medical Branch. EXPERIMENTAL DESIGN

Rats were divided into four groups of six animals each. Group I rats were maintained as healthy controls. Diabetes was induced in Groups II, III and IV as described below. Rats in group II were maintained as disease controls. Diabetic rats in Group in received aminoguanidine (lg/1 daily, a dose similar to that shown to be previously effective in diabetic peripheral neuropathy* in drinking water from day 2 of induction to the end of the experimental period, whereas those in Group FV received ALT-711 (3mg/kg daily) by th intraperitoneal injection beginning at the 6 week of induction through the entire experimental period. *Cameron NE, Cotter MA , Dines K , et al. Effects of aminoguanidine on peripheral nerve function and polyol pathway metabolites in streptozotocin-diabetic rats. Diabetologia 1992;35:946-50. EXPERIMENTAL DIABETES

A single intraperitoneal injection of 55 mg/kg STZ dissolved in freshly prepared 50 mmol/1 citrate buffer (pH 4.0) immediately before administration was given to the rats. Blood concentrations of glucose were measured 48 hours later (day 0) in blood obtained from the cut tip of the tail and measured by a glucometer (USA). Only rats with a blood glucose concentration above 200mg/dl were included in diabetic groups. Diabetic rats also received minimal doses (4u) of insulin every alternative day to maintain body mass and improve survival during the experimental period. Animals were sacrificed 12 weeks after induction of diabetes. Blood was centrifuged to obtain serum for measuring AGEs.

RESULTS

Values are expressed as mean (SE). Where necessary, multiple comparisons between groups were performed using ANOVA and unless specifically indicated otherwise, a post-test analysis was done using Dunnett's method for comparing all three diabetic groups to healthy controls (group I).

EXAMPLE 16 Body weight and blood glucose concentrations were subsequently measured every two weeks in overnight fasted animals through out the period. Data are shown in Fig. IA and Fig. IB. Control rats received no treatment (Group 1); diabetes induced rats received STZ (Group 2) and STZ + aminoguanidine (Group 3) or STZ + ALT-711 (Group 4). Data are expressed as mean (SE) for six rats in each group; AG=aminoguanidine; ALT=ALT- 711; STZ = streptozotocin.

During the experimental period mean body weight was significantly decreased in all the diabetic rats whereas control rats gained in body weight. Mean blood glucose concentrations were higher in all the diabetic induced animals than in the control animals. Administration of low dose insulin (4U) did not affect the blood glucose concentration of diabetic induced animals during the experimental period. Further, treatment with aminoguanidine or ALT-711 did not show any significant effect on body weight or blood glucose levels.

EXAMPLE 17 The effect of the compounds of the invention on AGEs accumulation was determined as follows using an enzyme linked immunosorbent assay (ELISA) for AGEs. Wells (96-well ELISA plate, FALCON, Franklin Lakes, NJ USA) were coated with polyclonal anti-AGE antibody (AGE102; 10 μg/ml; Biologo, Kronshagen, Germany) in 50 mmol/1 carbonate buffer (pH 9.6) overnight at room temperature as previously described in Cellek S, Qu W , Schmidt AM , et al. Synergistic action of advanced glycation end products and endogenous nitric oxide leads to neuronal apoptosis in vitro: a new insight into selective nitrergic neuropathy in diabetes. Diabetologia 2004;47:331-9. The wells were then washed with PBS containing 0.05% Tween 20 and blocked at room temperature with PBS containing 0.25% BSA. After washing, the wells were incubated with the standards (AGE-BSA as described below; diluted 1:10 -1 : 100,000) or samples (rat serum diluted in PBS 1:10-1:10,000) at room temperature for 3 h. After washing, the wells were incubated with monoclonal anti-AGE antibody (clone 6Dl 2; 0.5 μg/ml; Biologo) for 2 h at room temperature followed by anti-mouse IgG-HRP (1 : 7500; BioRad) for 1 h at room temperature. The wells were washed again and developed with peroxidase substrate (Alpha diagnostic, San Antonio, TX, USA) for 20 min. After adding the stopping solution (Alpha diagnostic, San Antonio, TX, USA) the yellow color was read at 450 nm. The absorbance obtained using AGE-BSA was used as positive control. BSA-AGEs were produced by incubating BSA (50 mg/ml) was incubated with 1 mol/1 glucose in PBS in sterile conditions at 37°C for 12 weeks. Excess unbound glucose was then removed using dialysis against a high volume of PBS then stored at -80⁰C until needed.

Figure 2 shows the effect of aminoguanidine and ALT-711 on AGEs accumulation. Serum AGE level as a percentage of the control values. Control rats received no treatment (Group 1); diabetes induced rats received STZ (Group 2) and STZ + aminoguanidine (Group 3) or STZ + ALT-711 (Group 4). Data from 3 independent experiments are expressed as mean (SE) of three rats in each group.* p < 0.001 significantly different from control (Group 1).

An evaluation of serum AGE levels in control and experimental animals and found significant differences among the four groups (P<0.001 by ANOVA), with elevations in the diabetic group that were not seen in either treatment group as compared with controls.

EXAMPLE 18

The expression of RAGE in duodenum of control and rats treated with compounds of the invention using Western immunoblotting analysis using mouse monoclonal anti- RAGE antibody.

Duodenum of all experimental animals were homogenized in ice-cold lysis buffer containing 125mM Tris (pH 7.8) with H₃PO₄WiIh 1OmM CDTA, 1OmM DTT, 50% Triton

X-IOO, 100 μmol proteinase cocktail inhibitor, 1 mM phenylmethylsulphonylfluoride (PMSF). After centrifugation (for 2 min, at 4°C, 12000 X g) the supernatants were collected and protein content was determined (e.g., BCA Protein Assay Kit, Pierce, Rockford, IL, USA).

For RAGE analysis, Samples (60 μg) were subjected to 10% SDS-PAGE e.g., with Mini-PROTEAN II xi system (Bio-Rad) and transferred to PVDF membranes, and incubated in blocking buffer (5% non-fat dry milk in TBST) for 1 h at room temperature and probed with mouse monoclonal anti-RAGE antibody (e.g., Chemicon international, Temecula, CA, USA) at a dilution of 1: 200 in blocking buffer overnight at 4 ⁰C. After washing in TBST, the blots were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin (Ig) G antibody (e.g., Bio-Rad) at a dilution of 1 : 1000 in TBST containing 2.5% non-fat dry milk for 1 h at room temperature. The immunoreactive bands were visualized using enhanced chemiluminescence (e.g., ECL kit; Amersham, Buckinghamshire, UK). The membranes were exposed to X-ray films and subsequently stripped and re-probed with mouse monoclonal γ- tubulin antibody (1 : 2000; e.g., Sigma, Saint Louis, MO, USA). The intensities of bands were quantified using Alpha digidoc software (e.g., San Leandro, CA, USA).

Figure 3 shows the expression of RAGE in duodenum of control and experimental rats. Control rats received no treatment (Group 1); diabetes induced rats received STZ (Group 2) and STZ + aminoguanidine (Group 3) or STZ + ALT-711 (Group 4). A representative Western blot showing RAGE immunoreactive bands; AG=aminoguanidine; ALT=ALT-711.

Western immunoblotting analysis revealed immunoreactive bands of protein extracts at approximately 48 kDa (Fig 3). There was no significant change observed between immunoreactive bands between the groups.

EXAMPLE 19

The effect of compounds of the invention on nNOS mRNA expression was determined using real time RT-PCR.

Total RNA from the duodenum was extracted by using Trizol reagent (Invitrogen, Carlsbad, CA, USA) phenol-chloroform extraction method. First-strand cDNA was then generated using TaqMan Reverse Transcription Regents (Applied Biosystems, Foster City, CA, USA). Quantitative real-time polymerase chain reaction (PCR) was carried out using the ABI Prism 5700 Sequence Detector with TaqMan Universal PCR Master Mix Kit (Applied Biosystems). β-III tubulin was measured as a reference gene. The following sequence-specific primers and probes were designed using primer express software 2.0 (Applied Biosystems) - for rat nNOS: forward - 5^I-ACGGACCCGACCTCAGAGA-3¹ (SEQ ID NO. 1); reverse - 5'-CGAGGCCGAACACTGAGAAC-S' (SEQ ID NO. 2) and probe: 5^I-6FAM-AAGTACTGGACCCCTGGCCAATGTGA-TAMRA-3^I (SEQ ID NO. 3); for β-iπ tubulin: forward - 5'-GGGCCTTTGGACACCTATTCA-S' (SEQ ID NO.

4); reverse - 5'-GCCCTTTGGCCCAGTTGT-S' (SEQ ID NO. 5) and probe: 5 '-όFAM-CCTGACAACTTTATCTTCGGTCAGAGTGGTG-TAMRA-S ' (SEQ ID NO. 6).

Polymerase chain reaction conditions were 50 ⁰C for 2 min, 95 ⁰C for 10 min, followed by 40 cycles of 95 ⁰C for 15 s, 55 ⁰C for 15 s and 60 for 1 min. Data were normalized to β-III tubulin and relative quantification of gene expression was performed using the 2 [ΔΔ-C (T)] or 2-(DDCt) relative quantification method (to the difference on normalized number of cycles to threshold).

Figure 4 shows control rats received no treatment (Group 1); diabetes induced rats received STZ (Group 2) and STZ + aminoguanidine (Group 3) or STZ + ALT-711 (Group 4). Data were normalized by β-Iϋ tubulin and expressed as percentage of control rats, using comparative CT method. Data are expressed as mean (SE) of three rats in each group; * p = 0.003 as significantly different when compared with control.

Significant differences in mRNA were seen (P=0.003 by ANOVA), with nNOS mRNA decreasing to 56% of control levels in diabetic rats (Fig 4), an effect that was prevented by aminoguanidine but not by ALT-711 (P >0.05 when the values in the diabetic group were compared to the ALT-711 group, using ar Newman-Keuls multiple comparison test).

EXAMPLE 20 The effect on nNOS protein expression in duodenum in control and rats treated with compounds of the invention was determined using Western blotting.

Duodenum of all experimental animals were homogenized in ice-cold lysis buffer containing 125mM Tris (pH 7.8) with H PO with 1OmM CDTA, 1OmM DTT, 50% Triton

X-100, 100 μmol proteinase cocktail inhibitor, 1 mM phenylmethylsulphonylfluoride (PMSF). After centrifugation (for 2 min, at 4°C, 12000 X g) the supernatants were collected and protein content was determined (e.g., BCA Protein Assay Kit, Pierce, Rockford, IL, USA). For nNOS protein analysis, samples (300 μg protein) were diluted in 4X SDS loading buffer [0.25 mol Tris-HCl (pH 6.8), 8% SDS, 40% glycerol, 2.5% DTT, 0.05% bromophenol blue], boiled for 5 min, and subjected to 8% SDS-polyacrylamide gel electrophoresis (PAGE) e.g., with PROTEAN II xi system (Bio-Rad, Richmond, CA, USA). After electrophoresis, proteins were transferred to PVDF membranes and were incubated in blocking buffer (5% non-fat dry milk in TBS containing 0.1% Tween 20; TBST) for 1 h at room temperature and probed with mouse monoclonal anti-nNOS antibody (e.g., BD Transduction Laboratories, San Jose, CA, USA) at a dilution of 1 : 1000 in blocking buffer overnight at 4 ⁰C. After washing in TBST, the blots were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin (Ig) G antibody (Bio-Rad) at a dilution of 1 : 2000 in TBST containing 2.5% non-fat dry milk for 1 h at room temperature. The immunoreactive bands were visualized using enhanced chemiluminescence (e.g., ECL kit; Amersham, Buckinghamshire, UK). The membranes were exposed to X-ray films and subsequently stripped and re-probed with mouse monoclonal γ- tubulin antibody (1:2000; e.g., Sigma, Saint Louis, MO, USA). The intensities of bands were quantified using Alpha digidoc software (e.g., San Leandro, CA, USA).

Figure 5A shows a representative Western blot showing nNOS immunoreactive bands relevant to 155 kDa. Diabetes induced nNOS suppression was reversed by treatment with aminoguanidine and ALT-711. Figure 5B shows band intensities that were measured by densitometry and graphed as a proportion of γ-tubulin. Control rats received no treatment (Group 1); diabetes induced rats received STZ (Group 2) and STZ + aminoguanidine

(Group 3) or STZ + ALT-711 (Group 4). Data are expressed as the percentage of control and represent mean (SE) of three rats in each group; * p = 0.001 significantly different from control (Groupl); AG=aminoguanidine; ALT=ALT-711.

Western immunoblotting of the duodenal tissue homogenate using anti-nNOS antibody revealed a band at 155 kDa (Fig 5) in control and all experimental groups. nNOS protein expressions differed significantly (P=0.001 by ANOVA) with a markedly lower value in the diabetic group, an effect not seen in either treatment group (which did not differ from controls).

EXAMPLE 21 Immunohistochemistry was used to determine the localization of nNOS in the duodenal myenteric plexus (arrows) of control and rats treated with compounds of the invention.

Whole duodenum with adherent myenteric plexus were fixed in Zamboni's fixative for 10 min at room temperature. The Zamboni-fixed tissue samples were later dehydrated and embedded in paraffin wax at 55 °C according to a previously described method. Six- micrometer-thick sections were cut on a microtome and placed in a water bath at 48 ⁰C. Thereafter, sections were transferred onto prewashed microscopic slides, which were dried in an oven at 55°C for 30 min to enhance attachment of sections. The sections were then de- paraffinized in xylene and processed for immunohistochemistry. After 30 min incubation in the blocking reagent, the appropriate dilution of primary mouse monoclonal anti-nNOS antibody (e.g., BD Transduction Laboratories, San Jose, CA, USA) and negative control reagents were applied. The sections were incubated in primary antibodies for 60 min at room temperature. The slides were then washed and incubated for 30 min with prediluted biotinylated anti-mouse IgG. After washing in TBST containing Tween 20 the sections were incubated with streptavidin- HRP conjugate for 20 min followed by washing with TBST. The peroxidase activity was visualized by incubating the specimens for 3 min in TBS solution containing 3,3-diaminobenzidine tetrahydrochloride and 0.03% hydrogen peroxide. The slides were later washed, counter-stained with haematoxylin for 30 sec, and dehydrated before mounting. The antiserum to nNOS was used at 1 :7500 dilution. The specificity of the antibody was confirmed by processing tissue samples in the absence of anti-nNOS serum. The number of nNOS positive cells in each myenteric ganglion was counted from 5 different microscopic fields for each group of rats.

Figure 6A shows immunohistochemical localization of nNOS in the duodenal myenteric plexus in control and rats treated with a compound of the invention. Note the markedly diminished staining in the diabetic group. Figure 6B shows quantification of nNOS positive cells. Control rats received no treatment (Group 1); diabetes induced rats received STZ (Group 2) and STZ + aminoguanidine (Group 3) or STZ + ALT-711 (Group 4).The number of nNOS positive cells against the total number of cells was counted from 5 different microscopic fields for each group of rats and graphed in percentage. The percentage of nNOS positive cells was significantly reduced in diabetes rats and was reversed back with aminoguanidine and ALT-711 treatment. Data from five different locations are shown as mean (SE) for each group; * p < 0.001 as significantly different when compared with control (Group 1). Fig 6A shows myenteric ganglia in the duodenum of rats in the various experimental groups. Although nNOS expression was observed in the myenteric plexus of all experimental groups, the number of nNOS positive cells per ganglia was significantly different amongst the groups (P< 0.001), being reduced by nearly half in untreated diabetic rats compared with healthy controls, an effect that was reversed with treatment by either drug.

EXAMPLE 22

The role of advanced glycation end products in diabetes-induced delayed gastric emptying was also investigated. As described herein, the loss of neuronal nitric oxide synthase (nNOS) expression in the myenteric plexus is important in the pathogenesis of diabetic gastroparesis and enteropathy. As further described herein, AGEs play a significant role in the suppression of duodenal nNOS in the diabetic rats. Thus, two drugs: 1) aminoguanidine and 2) ALT-711, were used to determine whether AGEs affect gastric emptying by altering nNOS expression in a diabetic rat model. Male Sprague-Dawley rats were randomly divided into age-matched controls, streptozotocin (STZ)-induced diabetic rats (55 mg/kg; intraperitoneal injection), STZ induced diabetic rats treated with aminoguanidine (1 gm/L in drinking water) at the induction of diabetes, and STZ induced diabetic rats treated with ALT-711 (3 mg/kg/day, intraperitoneally) beginning at week 6 of the 12 week study. 12 weeks after induction of diabetes, rats were sacrificed, and nNOS protein expression analyzed by immunohistochemistry and Western blots. The level of solid gastric emptying was also studied in these animals.

Figure 9 shows the effect of aminoguanidine and ALT-711 on AGEs accumulation. Serum AGE level as a percentage of the control values. Control rats received no treatment; diabetes induced rats received STZ and STZ + aminoguanidine (AG) or STZ + ALT-711 (ALT). Data from 3 independent experiments are expressed as mean (SE) of three rats in each group.* p < 0.001 significantly different from control. Figures 10 and 11 show the protein expression of nNOS in plyorous tissue and the percentage of nitric oxide released from plyorous tissue, respectively. Data in these figures are expressed as mean (SE) of three rats in each group; * p < 0.05 as significantly different when compared with control; AG=aminoguanidine; ALT= ALT-711. Figure 12 shows the percentage of gastric emptying. Data are expressed as mean (SE) of four rats in each group; * p < 0.05 as significantly different when compared with control; AG=aminoguanidine; ALT= ALT-711. The results show that diabetes resulted in a significant elevation in serum levels of

AGEs (p<0.05), accompanied by a significant increase of nNOS protein expression (p<0.05) in pylorus, and delayed gastric emptying (p<0.05) as compared with healthy controls. These effects were attenuated by the treatment of aminoguanidine or ALT-711. These results indicate that increased serum AGEs level and impaired nNOS expression can play a crucial role in delayed gastric emptying, and aminoguanidine and ALT-711 can be useful therapeutic agents for diabetes induced gastrointestinal complications.

Claims

We Claim:

1. A method of treating, or ameloriating a symptom of, a gastrointestinal disorder or condition in a patient in need thereof, comprising administering a pharmaceutical composition comprising a compound of Formula I, or a pharmaceutically acceptable salt of the compound of Formula I,

wherein:

R¹ and R² are selected from the group consisting of hydrogen, hydroxy (lower) alkyl, acetoxy (lower) alkyl, lower alkyl, lower alkenyl; or R and R together with their ring carbons may be an aromatic fused ring, optionally substituted by one or more amino, halo or alkylenedioxy groups;

Z is hydrogen or an amino group;

Y is amino, a group of the formula:

O Il

-CH₂C-R₅ 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; a group of the formula:

-CH₂R' wherein R' is hydrogen, or a lower alkyl, lower alkenyl, or aryl group; or a group of the formula:

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

X is a pharmaceutically acceptable anion, and a pharmaceutically acceptable carrier, thereby treating or preventing said gastrointestinal disorder or condition.

2. The method of claim 1 , wherein said gastrointestinal disorder or condition is a gastrointestinal complication related to diabetes.

3. The method of claim 1 , wherein said gastrointestinal disorder or condition is gastroparesis.

4. The method of claim 3, wherein said gastroparesis is diabetic gastroparesis, postsurgical gastroparesis or medication-related gastroparesis.

5. The method of claim 1, wherein said gastrointestinal disorder or condition is diabetes-induced delayed gastric emptying.

6. The method of claim 1, wherein said gastrointestinal disorder or condition is diabetic intestinal dysfunction.

7. The method of claim 1 , wherein said gastrointestinal disorder or condition is diabetic enteropathy.

8. The method of claim 1, wherein said gastrointestinal disorder or condition is gastric and intestinal dysfunction.

9. The method of claim 1 , wherein Rl and R2 are independently lower alkyl.

10. The method of claim 1 , wherein Z is hydrogen.

11. The method of claim 1 , wherein R is an aryl group.

12. The method of claim 1 , wherein the compound of Formula I is 3-(2-phenyl-2- oxoethyl)-4,5-dimethylthiazolium.

13. The method of claim 1, wherein the compound of Formula I is 3-(2-phenyl-2- oxoethyl)-4,5-dimethylthiazolium chloride.

14. The method of claim 1, wherein the compound of Formula I is 3-(2-phenyl-2- oxoethyl)-4,5-dimethylthiazolium bromide.

15. The method of claim 1 , wherein said patient has diabetes.

16. The method of claim 1, wherein said patient has decreased intestinal neuronal nitric oxide synthase (nNOS) protein expression.

17. The method of claim 16, wherein the decrease in intestinal nNOS expression is diabetes induced.

18. The method of claim 1 , wherein the administration of said pharmaceutical composition increases the intestinal protein expression of nitric oxide synthase (nNOS)

19. The method of claim 18, wherein the protein expression of nitric oxide synthase (nNOS) is increased in the duodenal myenteric plexus of said patient.

20. The method of claim 18, wherein the protein expression of nitric oxide synthase (nNOS) is increased in the myenteric ganglia in the duodenum of said patient.