WO2009023207A1 - Composés de thiazolium pour le traitement des complications gastro-intestinales - Google Patents

Composés de thiazolium pour le traitement des complications gastro-intestinales Download PDF

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WO2009023207A1
WO2009023207A1 PCT/US2008/009660 US2008009660W WO2009023207A1 WO 2009023207 A1 WO2009023207 A1 WO 2009023207A1 US 2008009660 W US2008009660 W US 2008009660W WO 2009023207 A1 WO2009023207 A1 WO 2009023207A1
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oxoethyl
bromide
thiazolium
group
methyl
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PCT/US2008/009660
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English (en)
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Raj Kumar
Pankaj Jay Pasricha
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Synvista Therapeutics, Inc.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/426—1,3-Thiazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/428—Thiazoles condensed with carbocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system

Definitions

  • 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.
  • gastrointestinal complications e.g., complications related to a reduction of neuronal nitric oxide synthase (nNOS) expression and/or diabeties.
  • nNOS neuronal nitric oxide synthase
  • nNOS neuronal nitric oxide synthase
  • NO nitric oxide
  • 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.
  • AGEs advanced glycation end products
  • N-carboxymethyl-lysine, pentosidine and methylglyoxal derivatives are classical examples for AGEs.
  • 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).
  • RAGE advanced glycation end products
  • 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.
  • 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.
  • aminoguanidine also has a variety of other effects including acting as an antioxidant and a cytoprotective agent by increasing total sulphydryl (SH) content.
  • SH total sulphydryl
  • Giardino I et al. Diabetes 1998;47:1114-20; Mustafa A, et al. Comp Biochem Physiol C Toxicol Pharmacol 2002;132:391-7.
  • 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.
  • 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.
  • AGE signaling is involved in the suppression of enteric nNOS in diabetes.
  • 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.
  • 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,
  • R 1 and R 2 are selected from the group consisting of hydrogen, hydroxy (lower) alkyl, acetoxy (lower) alkyl, lower alkyl, lower alkenyl; or R 1 and R 2 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:
  • 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:
  • R' is hydrogen, or a lower alkyl, lower alkenyl, or aryl group; or a group of the formula:
  • 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.
  • 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.
  • compositions of the invention are 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.
  • the compositions comprise compounds for inhibiting the formation of and reversing the pre-formed advanced glycosylation (glycation) endproducts and breaking the subsequent cross-links.
  • 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.
  • AGEs advanced glycation end-products
  • nNOS intestinal neuronal nitric oxide synthase
  • 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:
  • R 1 is selected from the group consisting of hydrogen, hydroxy (lower) alkyl, acetoxy (lower) alkyl, lower alkyl, lower alkenyl;
  • R 2 is selected from the group consisting of hydrogen, hydroxy (lower) alkyl, acetoxy (lower) alkyl, lower alkyl, lower alkenyl; or R 1 and R 2 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:
  • 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: -CH 2 R' wherein R' is hydrogen, or a lower alkyl, lower alkenyl, or aryl group; or a group of the formula:
  • 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 R 1 and R 2 are lower alkyl, Z is hydrogen, Y is a group of the formula
  • 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.
  • 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.
  • 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.
  • the cross-link structure includes a sugar-derived ⁇ -dicarbonyl segment or moiety, such as a diketone, that is capable of cleavage by a dinucleophilic, thiazolium-like compound.
  • the cross-link structure may be according to the formula:
  • a and B independently, are sites of attachment to the nucleophilic atom of a biomolecule.
  • gastrointestinal complications e.g., that arise from a reduction in nNOS expression such as diabetes-related complications
  • the compound of the invention is administered prophylatically or therapeutically.
  • 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.
  • 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 ⁇ -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.
  • 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.
  • the invention relates to a composition which may contain one or more compounds having the ability to effect cleavage of ⁇ -dicarbonyl-based molecular crosslinks present in the advanced glycosylation endproducts.
  • 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:
  • R 1 and R 2 are independently selected from the group consisting of hydrogen, hydroxy(lower)alkyl, acetoxy(lower)alkyl, lower alkyl, lower alkenyl, or
  • R 1 and R 2 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
  • Il -CH 2 C-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 -CH 2 R' wherein R' is hydrogen, or a lower alkyl, lower alkynyl, or aryl group; or a group of the formula:
  • 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.
  • 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.
  • lower alkynyl means that the group contains from 2, 3, 4,
  • 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.
  • 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.
  • aryl groups encompassed by the formulae of the invention are those containing
  • aryl groups are phenyl, methoxyphenyl and 4- bromophenyl groups.
  • halo atoms in the formulae of the invention may be fluoro, chloro, bromo or iodo.
  • 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 R 1 and R are lower alkyl, Z is hydrogen, Y is a group of the formula
  • 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.
  • 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.
  • treating 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.
  • 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.
  • R 1 or R 2 are lower alkyl groups are preferred.
  • 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 ;
  • R 1 is independently selected from the group consisting of hydrogen, hydroxy(lower)alkyl, acetoxy(lower)alkyl, lower alkyl, lower alkenyl;
  • R 2 is independently selected from the group consisting of hydrogen, hydroxy(lower)alkyl, acetoxy(lower)alkyl, lower alkyl, lower alkenyl, or R 1 and R 2 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
  • 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
  • R' is hydrogen, or a 'lower alkyl, lower alkynyl, or aryl group; or a group of the formula
  • R" is hydrogen and R'" is a lower alkyl group, optionally substituted by an aryl group, or an aryl group, said aryl group optionally substituted by one or more lower alkyl, halo, or alkoxylcarbonyl groups; or R" and R" are both lower alkyl groups; with the proviso that at least one of Y and Z is an amino group, and the further proviso that when Y is amino and R 2 and Z are both hydrogen, then R 1 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
  • 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.
  • 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.
  • R 1 is independently selected from the group consisting of , hydroxy (lower) alkyl, acetoxy(lower)alkyl, lower acyloxy(lower)alkyl, lower alkyl
  • R 2 is independently selected from the group consisting of , hydroxy (lower) alkyl, acetoxy(lower)alkyl, lower acyloxy(lower)alkyl, lower alkyl, or R 1 and R 2 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
  • 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):
  • R 1 and R 2 are methyl; Z is hydrogen; Y is a group of the formula:
  • 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.
  • 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.
  • 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.
  • 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.
  • carbonyl moieties such as glycolaldehyde, glyceraldehyde or 3-deoxyglucosone
  • 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.
  • 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.
  • 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.
  • 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).
  • 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 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.
  • 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.
  • 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.
  • solid dosage forms such as tablets, capsules, or liquid dosage formulations such as solutions and suspensions, etc.
  • 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.
  • 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.
  • 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. Or
  • R 1 and R 2 are independently selected from the group consisting of hydrogen, hydroxy (lower) alkyl, acetoxy(lower)alkyl, lower alkyl, lower alkenyl, or R 1 and R 2 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
  • 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 -CH 2 R' wherein R' is hydrogen, or a lower alkyl, lower alkynyl, or aryl, group; or a group of the formula
  • R" is hydrogen and R" is a lower alkyl group, optionally substituted by an aryl group, or an aryl group, said aryl group optionally substituted by one or more lower alkyl, halo, or alkoxylcarbonyl groups; or R" and R'" are both lower alkyl groups; with the proviso that at least one of Y and Z is an amino group, and the further proviso that when Y is amino and R 2 and Z are both hydrogen, then Ri is other than a lower alkyl group; and
  • X is a halide, tosylate, methanesulfonate or methanesulfonate ion.
  • 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.
  • R is a group of the formula wherein R is a lower alkyl, alkoxy, hydroxy, amino or aryl group;
  • R is lower alkyl, alkoxy, hydroxy, amino or aryl group; or a group of the formula -CH 2 R' 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.
  • reaction Scheme I the appropriate substituted thiazole compound of formula II wherein R 1 , R 2 and Z are as hereinbefore defined, is reacted with the appropriate halo compound of. formula III wherein R and X are as hereinbefore defined, to afford the desired compound of the invention e.g., formula I wherein R 1 , R 2 , Z, R and X are as hereinbefore defined.
  • this reaction is conducted at reflux temperatures for times of about 1 -3 hours.
  • 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.
  • R 1 , R 2 and Z are as defined hereinabove.
  • 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.
  • AGE Advanced glycosylation endproduct
  • This assay comprises: a) incubation of AGE-modif ⁇ ed bovine serum albumin (AGE BSA) on collagen-coated wells of microtiter plates for a period of 2-6 hours' at a temperature of 37 0 C ; 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 0 C; and e) detection of the AGE-breaking using an antibody raised against AGE- ribonuclease or cross-link breaking with an antibody against BSA.
  • AGE BSA AGE-modif ⁇ ed bovine serum albumin
  • 965 3 (2- (4' -chlorophenyl) -2-oxoethyl] -4-methyl-5-(2'-hydroxyethyl)-thiazolium bromide, m.p. 240-251 0 C (dec); and 966 3 (2- (4' -methoxyphenyl) -2-oxoethyl] -4-methyl-5- (2' hydroxyethyl)-thiazolium bromide, m.p. 229-231°C 25 (dec).
  • 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.
  • 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.
  • AGE-BSA glycated bovine serum albumin
  • 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.
  • PBS phosphate buffer solution
  • 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 ⁇ l each of the AGE-BSA diluted in PBS or in the solution of test compound at 37 0 C for 4 hours. Unbrowned BSA in PBS buffer with or without testing compound were added to the separate wells as the blanks.
  • the un-cross-linked AGE-BSA was then removed by washing the wells three times with PBS-Tween buffer.
  • the amount of AGE-BSA cross- linked to the tail tendon collagen-coated plate was then quantitated using a polyclonal antibody raised against AGE-RNase. After a one-hour incubation period, AGE antibody was removed by washing 4 times with PBS-Tween.
  • 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.
  • IC50 values or the inhibition at various concentrations by test compounds is as follows:
  • 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.
  • AGE-BSA AGE-modified protein
  • HRP Horseradish Peroxidase
  • AGE-BSA stock solutions were prepared as follows. Sodium phosphate buffer (0.4%)
  • 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 0 C for 12 weeks.
  • the pH of the incubation mixture was monitored weekly and adjusted to pH 7.4 if necessary.
  • 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 0 C. Dilute solutions of AGE-BSA were unstable when stored at -20 0 C.
  • 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, KH 2 PO 4 . 1.15 grams; NaN 3 , 0.2 gram. Tween-20 was added to a final concentration of 0.05% (vol/vol).
  • 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 Setup 1. Warm Superbloc reagent to 37 0 C. Add 300 ⁇ of Superbloc to each well of the Biocoat plate and let stand for sixty minutes at 37 0 C. Wash the wells three times with PBS-Tween (0.05%). Turn the plate 180 degrees and repeat this wash cycle.
  • 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 ⁇ is added to each well and the plate is allowed to stand at room temperature for sixty minutes.
  • Test Compound ICsn Breaking Anti- Anti- AGE/Anti-BSA fat
  • test compounds of the invention 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.
  • 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.
  • 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.
  • 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.
  • BSA Calbiochem Type V; 400 mg/ml in the buffer 1. Total volume prepared 50g/125ml.
  • Glucose 400 mM 9g/125ml of buffer. Filtered through a 0.45u filter into one liter Corning sterile flask.
  • 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 0 C. 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.
  • 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 0 C in a solution of 100 mM glucose-6-phosphate in a 0.5 M phosphate buffer, pH 7.4, containing 3 mM sodium azide.
  • Glucose-6-phosphate is a sugar capable of participating in nonenzymatic browning at a more rapid rate than glucose.
  • 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.
  • amyloid peptide of Alzheimer's disease 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- ⁇ -peptide was prepared by incubating an aliquot of the soluble ⁇ -amyloid peptide, synthetically prepared and corresponding in sequence to the ⁇ -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 ⁇ -peptide was substituted for BSA as the glycation substrate.
  • Aliquots of 125 I- AGE- ⁇ -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.
  • 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.
  • 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.
  • immunochemical measurement of the cross-link epitopes on a protein sample such as hemoglobin, provides an index of recent AGE-formation.
  • 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.
  • BSA bovine serum albumin
  • Various other haptens, antigens, and conjugated immunogens corresponding to the cross-link structures of the present invention 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.
  • 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.
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • EDC divalent coupling reagents
  • the cross-link structure 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.
  • 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.
  • 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.
  • 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-specif ⁇ c immune titer in any of several convenient formats including, for instance, Ouchterlony diffusion gels and direct ELISA protocols.
  • 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.
  • 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.
  • 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.
  • the assay surface may be decorated by incubation with the hapten alone.
  • 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.
  • 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).
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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
  • 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
  • polyclonal anti-AGE antibody AGE102; 10 ⁇ g/ml; Biologo, Kronshagen, Germany
  • AGE102 polyclonal anti-AGE antibody
  • carbonate buffer pH 9.6
  • 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 ⁇ g/ml; Biologo) for 2 h at room temperature followed by anti-mouse IgG-HRP (1 : 7500; BioRad) for 1 h at room temperature.
  • monoclonal anti-AGE antibody clone 6Dl 2; 0.5 ⁇ g/ml; Biologo
  • 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 -80 0 C 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).
  • Samples 60 ⁇ g 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 0 C.
  • blocking buffer 5% non-fat dry milk in TBST
  • mouse monoclonal anti-RAGE antibody e.g., Chemicon international, Temecula, CA, USA
  • HRP horseradish peroxidase
  • Ig immunoglobulin G antibody
  • Bio-Rad horseradish peroxidase
  • the immunoreactive bands were visualized using enhanced chemiluminescence (e.g., ECL kit; Amersham, Buckinghamshire, UK).
  • ECL kit horseradish peroxidase
  • Amersham Buckinghamshire, UK
  • the membranes were exposed to X-ray films and subsequently stripped and re-probed with mouse monoclonal ⁇ - 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).
  • 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). ⁇ -III tubulin was measured as a reference gene.
  • sequence-specific primers and probes were designed using primer express software 2.0 (Applied Biosystems) - for rat nNOS: forward - 5 I -ACGGACCCGACCTCAGAGA-3 1 (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 ⁇ -i ⁇ tubulin: forward - 5'-GGGCCTTTGGACACCTATTCA-S' (SEQ ID NO.
  • Polymerase chain reaction conditions were 50 0 C for 2 min, 95 0 C for 10 min, followed by 40 cycles of 95 0 C for 15 s, 55 0 C for 15 s and 60 for 1 min.
  • Data were normalized to ⁇ -III tubulin and relative quantification of gene expression was performed using the 2 [ ⁇ -C (T)] or 2-(DDCt) relative quantification method (to the difference on normalized number of cycles to threshold).
  • 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.
  • X-100 100 ⁇ mol 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).
  • PMSF phenylmethylsulphonylfluoride
  • samples 300 ⁇ g 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).
  • 4X SDS loading buffer 0.25 mol Tris-HCl (pH 6.8), 8% SDS, 40% glycerol, 2.5% DTT, 0.05% bromophenol blue
  • 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 0 C.
  • blocking buffer 5% non-fat dry milk in TBS containing 0.1% Tween 20; TBST
  • mouse monoclonal anti-nNOS antibody e.g., BD Transduction Laboratories, San Jose, CA, USA
  • 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.
  • HRP horseradish peroxidase
  • Ig immunoglobulin G antibody
  • Bio-Rad horseradish peroxidase
  • the immunoreactive bands were visualized using enhanced chemiluminescence (e.g., ECL kit; Amersham, Buckinghamshire, UK).
  • ECL kit enhanced chemiluminescence
  • the membranes were exposed to X-ray films and subsequently stripped and re-probed with mouse monoclonal ⁇ - 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,
  • 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 ⁇ -tubulin. Control rats received no treatment (Group 1); diabetes induced rats received STZ (Group 2) and STZ + aminoguanidine
  • 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.
  • 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.
  • 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.
  • nNOS neuronal nitric oxide synthase
  • 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.

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Abstract

La présente invention concerne un procédé de traitement des complications gastro-intestinales, par exemple des complications liées à une réduction de l'expression de nNOS. Ledit procédé utilise des composés et des compositions de la présente invention.
PCT/US2008/009660 2007-08-13 2008-08-13 Composés de thiazolium pour le traitement des complications gastro-intestinales WO2009023207A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021125905A1 (fr) * 2019-12-20 2021-06-24 가천대학교 산학협력단 Nouveau dérivé thiazole et son utilisation

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