Compositions for targeting receptor for advanced glycation end products (RAGE) in chronic inflammatory conditions

Abstract

This invention discloses a composition and method comprising bis(demethoxycurcumin) enriched in at least 20% w / w for inhibiting receptor for advanced glycation end products (RAGE) expression in subjects with chronic inflammatory conditions. The composition further comprises β-amyrin palmitate (BAP). This invention also discloses the use of the above composition in controlling chronic inflammatory conditions in subjects.

Description

[0001] Cross-reference to related applications

[0002] This is a PCT application claiming priority to U.S. Provisional Application No. 63126920, filed on December 17, 2020, the contents of which are incorporated herein by reference. Invention Field

[0003] This invention generally relates to compositions and methods for use in subjects suffering from chronic inflammatory conditions, comprising compositions containing enriched bis(demethoxycurcumin) (BDMC) present at not less than 20% w / w to inhibit receptor for complete glycation end products (RAGE) expression. The compositions further comprise β-amyrin palmitate (BAP). This invention also includes the therapeutic control of chronic inflammatory conditions in subjects using the above-described compositions. Background Technology

[0004] Non-enzymatic oxidation and glycation of proteins lead to the formation of advanced glycation end products (AGEs). Advanced renal failure, atherosclerosis, diabetes, and aging are some of the conditions that contribute to AGEs. They are also increased in inflammatory diseases such as systemic lupus erythematosus, rheumatoid arthritis, osteoarthritis, and dialysis-related complications, indicating their intrinsic link to chronic inflammatory diseases. These chronic inflammatory conditions can increase the risk of accelerating atherosclerosis and its complications. Conversely, hypoxia and ischemia-reperfusion injury are rapid contributors to AGEs and can further increase complications of inflammatory diseases. Hyperglycemia or diabetes, leading to elevated blood glucose levels, is a condition that promotes AGEs. The mechanisms underlying glucose-induced inflammation and other related pathological disturbances in cells, which are yet to be fully elucidated, remain to be determined. Furthermore, existing hypotheses suggest that AGE deposition in tissues leads to organ failure (Lin et al., Curcumin inhibits gene expression of receptor for advanced glycation end-products (RAGE) in hepatic stellate cells in vitro by elevating PPARγ activity and attenuating oxidative stress, British Journal of Pharmacology 2212-2227 (2012)). AGEs are a variety of macromolecules, including carboxymethyl lysine (CML), carboxyethyl lysine (CEL), pentosine, glucosepane, glyoxal lysine dimer, and glycolate lysine amide, which are formed through non-enzymatic processes of protein and lipid glycosylation. AGEs induce their cellular effects by interacting with their receptors. The receptor for advanced glycation end products (RAGE) is a ubiquitous transmembrane receptor that binds to a range of endogenous ligands. The interaction between AGE and RAGE initiates a complex intracellular signaling cascade, leading to the production of reactive oxygen species (ROS), immune inflammatory effects, cell proliferation or apoptosis, and the upregulation of RAGE itself. Several studies have found correlations between RAGE activity and pathological conditions such as cancer, diabetes, cardiovascular disease, and neurodegeneration.Although AGE can be benign and nonresponsive, several studies have found correlations between RAGE activity and pathological conditions such as cancer, diabetes, cardiovascular disease, and neurodegeneration. It may be a contributing factor to complications of chronic diseases such as type II diabetes, cardiovascular disease, Alzheimer's disease, cancer, peripheral neuropathy, sensory loss, and blindness (Rehman et al., Effect of non-enzymatic glycosylation in the epigenetics of cancer, Semin Cancer Biol. Dec 2: S1044-579X(20)30257(2020); (Laura et al., The AGE-RAGE Axis: Implications for Age-Associated Arterial Diseases, Frontiers in Genetics, 8, 1-10 (2017). The mechanism of action caused by AGE-RAGE binding leads to stimulation of NADPH oxidase, increasing the production of reactive oxygen species (ROS), thereby regulating the expression of tumor necrosis factor (TNF-α), the transcription factor nuclear factor-κB (NF-κB), the release of cytokines, inflammatory expression, and the activation of cell signal transduction. ROS generated during RAGE activation is the source of protein oxidation to form protein carbonyl substances. In the "primary protein carbonylation" reaction, the direct oxidation of the side chains of lysine, arginine, proline, and threonine residues, among other amino acids, produces DNPH-detectable protein products called reactive carbonyl substances (RCS) (Suzuki et al., Protein carbonylation. Antioxidant Redox). Signal. 2010; 12(3):323-325. Conversely, the reactive carbonyl compounds of carbohydrates bind to the amino groups of proteins, lipids, or nucleic acids to form Schiff bases, which rearrange to Amadori products. In a series of slow reactions, the Amadori reaction, Schiff base reaction, and Maillard reaction ultimately form AGEs. Amadori compounds can further degrade into various late-stage glycosylation end products, including reactive α-dicarbonyl compounds, and release ROS such as superoxide anions and hydrogen peroxide.Oxidative decomposition of Schiff bases and Amadori adducts, as well as the formation of α-dicarbonyl compounds during glucose autoxidation, can lead to the oxidative deamination of Lys via a Strecker-type reaction, resulting in the formation of 3-deoxyglucose (3DG) and methylglyoxal (MG) (Ros et al., Protein Carbonylation (Principles, Analysis, and Biological Implications) Diversity of Protein Carbonylation Pathways., 48–8 (2017)). α-Dicarbonyl compounds are also produced in vivo during lipid peroxidation, glucose autoxidation, or glucose metabolism. Glyoxal (GO) and methylglyoxal (MGO) react with lysine and arginine residues in proteins to form AGEs, such as carboxymethyl lysine (CML), carboxyethyl lysine (CEL), and arginine pyrimidine (ArgP). Therefore, AGE formation, protein carbonyl compounds, their interaction with RAGEs, and downstream oxidative stress and inflammation are closely linked. Increased levels of RCS and AGE homeostasis lead to carbonyl stress that interferes with normal metabolism. Reactive carbonyl groups (RCS) are ubiquitous compounds with relatively long half-lives and stability, especially compared to reactive oxygen species (ROS). Their low molecular weight, uncharged structure, and relatively high stability allow them to cross biological membranes, diffuse through peripheral circulation, and even cross the blood-brain barrier. Glycosylated RCS derivatives such as methylglyoxal, glyoxal, 3-deoxyfructose, glucone, and 3-deoxyglucone are approximately 20,000 times more reactive than reduced carbohydrates. Alzheimer's disease (AD), rheumatoid arthritis, diabetes, sepsis, chronic renal failure, and acute respiratory distress syndrome are some of the conditions caused by increased protein carbonylation (Isabella et al., Protein carbonyl groups as biomarkers of oxidative stress, 329(1), 23-38 (2003)). The formation of AGEs is accelerated under conditions of hyperglycemia, oxidative stress, aging, and inflammation (Laura et al., The AGE-RAGE Axis: Implications for Age-Associated Arterial Diseases, Frontiers in Genetics, 8, 1-10 (2017)).Although AGEs can be benign and unresponsive, they can be a contributing factor to complications of chronic diseases such as type 2 diabetes, cardiovascular disease, Alzheimer's disease, cancer, peripheral neuropathy, sensory loss, and blindness (Rehman et al., Effect of non-enzymatic glycosylation in the epigenetics of cancer, Semin Cancer Biol. Dec 2:S1044-579X(20)30257(2020)). Reducing glycosylation, RAGE expression, and protein carbonylation could ultimately reduce oxidative and carbonyl stress, which may be beneficial for chronic inflammatory conditions. RAGEs, as potential biomarkers for several diseases, are attractive targets for developing inhibitors to control these conditions. Altered circulating levels of RAGEs have been identified in patients with diabetic complications, cardiovascular disease, and Alzheimer's disease. RAGEs have been investigated as potential targets for the treatment of cancer, cardiovascular disease, and neurodegeneration. (Salvatore et al., Targeting the Receptor for Advanced Glycation End products (RAGE): A Medicinal Chemistry Perspective, 60(17), 7213-7232(2017)).

[0005] Numerous studies have demonstrated the role of RAGE in the formation of atherosclerotic lesions and its promotion of pro-inflammatory pathways, suggesting that diabetes mediates atherosclerosis. In these studies, diabetes-related AGE is increased in diabetic RAGE. - / - / apoE - / -It was significantly reduced in mice, accompanied by an inflammatory response with macrophage accumulation, decreased expression of cytokines and chemokines (Paavonen et al., Receptor for Advanced Glycation End Products (RAGE) Deficiency Attenuates the Development of Atherosclerosis in Diabetes, Diabetes, 57, 2461-2469 (2008)). This is also supported by numerous studies showing that neutralizing antibodies or soluble RAGE inhibit RAGE activation (Park et al., Suppression of accelerated diabetic atherosclerosis by the soluble receptor for advanced glycation end products. Nat Med 4, 1025–1031 (1998); Bucciarelli et al., RAGE blockade stabilizes established atherosclerosis in diabeticapolipoprotein E-null mice. Circulation 106, 2827–2835 (2002); Sakaguchi et al., Central role of RAGE-dependent neointimal expansion in arterial restenosis. J Clin Invest 111, 959–972 (2003)).

[0006] The effects of curcumin on RAGE signaling, inhibition of AGE accumulation, and attenuation of RAGE expression in experimental diabetic rats have been reported (Lin et al., Curcumin inhibits gene expression of receptor for advanced glycation end products (RAGE) in hepatic stellate cells in vitro by elevating PPARγ activity and attenuating oxidative stress, British Journal of Pharmacology 166, 2212-2227 (2012); Yu et al., Curcumin Alleviates Diabetic Cardiomyopathy in Experimental Diabetic Rats, PLOS One 7(12)1-11). However, commercially available curcumin contains three types of curcuminoids: 72-77% curcumin, 14-18% dimethoxycurcumin, and 3-5% didemethoxycurcumin. Furthermore, the high proportion of curcumin makes it hydrophobic, thus affecting bioavailability and absorption (Pushpakumari et al., Enhancing the Absorption of Curcuminoids from Formulated Turmeric Extracts, 6(6)2468-2476(2015)). The biological properties of curcumin, bis(demethoxy)curcumin, and demethoxycurcumin vary in different disease symptoms. In recent years, bis(demethoxy)curcumin and demethoxycurcumin have attracted much attention due to their similar and superior efficacy in controlling certain disease symptoms (Majeed et al., Reductive Metabolites of Curcuminoids, Nutriscience Publishers LLC, 2019). The pharmacological challenges associated with targeting RAGE involve not only controlling the gene expression of inflammatory genes, but also controlling NF-κB activation, which induces RAGE expression in the feedforward loop (Armando et al., Inhibition of RAGE AxisSignalling: A Pharmacological Challenge, Current Drug Targets, 20, 340-346, (2019)).Promising results have been achieved with blocking peptides and antibodies against RAGE, but their use is limited due to their limitations as therapeutic compounds (Arumugam T et al., S100P-derived RAGE antagonisticpeptide reduces tumor growth and metastasis. Clin Cancer Res. 18(16):4356-64 (2012); Kokkola et al., Successful treatment of collagen-induced arthritis in mice and rats by targeting extracellular high mobility group box chromosomalprotein 1 activity. Arthritis Rheum 48(7):2052-8 (2003)). In addition, several small molecules, such as TTP488, azeliragon, pioglitazone, and PPARγ agonists, also block RAGE signaling (Salvatore et al., Targeting the Receptor for Advanced Glycation Endproducts (RAGE): A Medicinal Chemistry Perspective, 60(17), 7213-7232 (2017); Burstein et al., Effect of TTP488 in patients with mild to moderate Alzheimer's disease. BMC Neurol. 14, 12 (2014); Burstein et al., Development of azeliragon, an oral small molecule antagonist of the receptor for advanced glycation endproducts, for the potential slowing of loss of cognition in mild Alzheimer's disease, J Prev Alzheimers Dis 5(2):149-54 (2018)). Given the complexities associated with targeting RAGE and the very limited list of compounds available for clinical trials, new approaches to targeting RAGE are needed, especially those that are safe and have low toxicity.

[0007] Purpose of the invention

[0008] The primary objective of this invention is to disclose a composition and method for inhibiting RAGE expression in subjects suffering from chronic inflammatory conditions, using a composition comprising enriched bis(demethoxycurcumin) (BDMC) present at not less than 20% w / w. The composition further comprises β-amyrin palmitate (BAP).

[0009] In yet another principal objective of the present invention, compositions and methods for controlling RAGE in subjects suffering from chronic inflammatory conditions are disclosed using compositions comprising enriched bis(demethoxycurcumin) (BDMC) present in a concentration of not less than 20% w / w. The compositions further comprise β-amyrin palmitate (BAP). Summary of the Invention

[0010] This invention broadly addresses the aforementioned problems mentioned in the background art by encompassing methods and compositions for inhibiting RAGE expression in subjects suffering from chronic inflammatory conditions using a composition comprising enriched bis(demethoxycurcumin) (BDMC) present at not less than 20% w / w. The composition further comprises β-amyrin palmitate (BAP).

[0011] A first aspect of the invention relates to a composition for use in subjects suffering from chronic inflammatory conditions to inhibit RAGE expression using a composition comprising enriched bis(demethoxycurcumin) (BDMC) present at not less than 20% w / w. The composition further comprises β-amyrin palmitate (BAP).

[0012] In yet another aspect of the invention, a composition is included for controlling chronic inflammatory conditions in subjects using a composition comprising bis(demethoxycurcumin) enriched in at least 20% w / w. The composition further comprises β-amyrin palmitate (BAP).

[0013] In another aspect of the invention, a method is covered for inhibiting RAGE expression in subjects suffering from chronic inflammatory conditions using a composition comprising bis(demethoxycurcumin) enriched in at least 20% w / w. The composition further comprises β-amyrin palmitate (BAP).

[0014] In another aspect of the invention, a method for treating a chronic inflammatory condition in a subject is included, wherein the subject is administered a composition comprising bis(demethoxycurcumin) enriched in at least 20% w / w. The composition further comprises β-amyrin palmitate (BAP).

[0015] The broader scope of the invention will become apparent from the following detailed description. However, it should be understood that while the following detailed description and specific embodiments point to preferred embodiments of the invention, they should not be construed as limiting the invention, and various changes and modifications can be made within the scope of those skilled in the art, such as altering the concentration range of the sample used, curcumin derivatives / analogs, BAP, experimental conditions, and the selection of mammals, all of which are within the spirit and scope of the invention. Attached Figure Description

[0016] Figure 1 The effects of AC3, BAP, and their combinations on RAGE expression in the pancreas were shown. *P<0.05.

[0017] Figure 2 The effects of AC3, BAP, and their combinations on anti-glycosylation in the pancreas were shown. *P<0.05. Detailed Implementation

[0018] Selected definition

[0019] Unless otherwise stated, all terms used in this application have their common meanings known in the prior art. Several other specific definitions used in this invention are explained below, and they apply throughout this specification. Unless otherwise stated, the claims provide for a broader range of definitions.

[0020] In this application, any reference to a sample refers to one or a combination of the following agents that produce the disclosed therapeutic effects. The agents include, for example, an enriched BDMC composition, which refers to a curcuminoid composition containing at least 20% w / w BDMC. More specifically, AC3 is the preferred curcuminoid used in this invention, and any reference to curcuminoid refers to AC3, which is 20-50% w / w bis(demethoxy)curcumin, 10-25% w / w bis(demethoxy)curcumin, and 30-50% w / w curcumin; BAP refers to β-amyrin palmitate. Any reference to the C3 complex refers to 75-81% curcumin, 15-19% bis(demethoxy)curcumin, and 2.2-6.5% bis(demethoxy)curcumin. Again, curcuminoid refers to BDMC, DMC, or AC3, depending on the disclosed example.

[0021] Therapeutic management refers to the effective improvement of the condition disclosed in this invention. Any reference to control in this specification refers to the diabetes control, untreated control, and metformin control covered in the experiments and examples.

[0022] This invention generally covers methods and compositions for inhibiting RAGE expression in subjects suffering from chronic inflammatory conditions using a composition comprising enriched bis(demethoxycurcumin) (BDMC) present in at least 20% w / w. The invention also covers compositions for therapeutically controlling chronic inflammatory conditions in subjects using a composition comprising enriched bis(demethoxycurcumin) (BDMC) present in at least 20% w / w. Furthermore, it covers methods for treating chronic inflammatory conditions in subjects by administering to said subjects a composition comprising enriched bis(demethoxycurcumin) (BDMC) present in at least 20% w / w. The composition comprises 20-50% w / w BDMC, 10-25% w / w demethoxycurcumin (DMC), and 30-50% w / w curcumin, and the total curcuminoids in the composition are in the range of 20-95% w / w. The composition further comprises β-amyrin palmitate (BAP). In this respect, the subjects are mammals.

[0023] In a preferred embodiment, the present invention discloses a composition for inhibiting RAGE expression in subjects suffering from chronic inflammatory conditions, wherein the composition comprises enriched bis(demethoxy)curcumin (BDMC) present in a concentration of not less than 20% w / w. In another aspect of this embodiment, the composition comprises 20-50% w / w BDMC, 10-25% w / w demethoxycurcumin (DMC), and 30-50% w / w curcumin, and the total curcuminoids in the composition are in the range of 20-95% w / w. In a related aspect of this embodiment, the composition further comprises β-amyrin palmitate (BAP). In this related aspect, the subject is a mammal.

[0024] In another preferred embodiment of the invention, a composition for therapeutically controlling chronic inflammatory conditions in a subject is disclosed, wherein the composition comprises enriched bis(demethoxy)curcumin (BDMC) present in a concentration of not less than 20% w / w. In another aspect of this embodiment, the composition comprises 20-50% w / w BDMC, 10-25% w / w demethoxycurcumin (DMC), and 30-50% w / w curcumin, and the total curcuminoids in the composition are in the range of 20-95% w / w. In a related aspect of this embodiment, the composition further comprises β-amyrin palmitate (BAP). In this related aspect, the subject is a mammal.

[0025] In another preferred embodiment of the invention, a method for inhibiting RAGE expression in a subject suffering from a chronic inflammatory condition is disclosed, comprising: (a) identifying the subject suffering from the chronic inflammatory condition; and (b) administering to the subject a composition comprising enriched bis(demethoxy)curcumin (BDMC) present at a concentration of not less than 20% w / w. In another aspect of this embodiment, the composition comprises 20-50% w / w BDMC, 10-25% w / w demethoxy)curcumin (DMC), and 30-50% w / w curcumin, and the total curcuminoids in the composition are in the range of 20-95% w / w. In a related aspect of this embodiment, the composition further comprises β-amyrin palmitate (BAP). In a related aspect, the subject is a mammal.

[0026] In another preferred embodiment of the invention, a method for treating a chronic inflammatory condition in a subject is disclosed, comprising: (a) identifying the subject suffering from the chronic inflammatory condition; and (b) administering to the subject a composition comprising enriched bis(demethoxy)curcumin (BDMC) present at a concentration of not less than 20% w / w. In another aspect of this embodiment, the composition comprises 20-50% w / w BDMC, 10-25% w / w demethoxy)curcumin (DMC), and 30-50% w / w curcumin, and the total curcuminoids in the composition are in the range of 20-95% w / w. In a related aspect of this embodiment, the composition further comprises β-amyrin palmitate (BAP). In a related aspect, the subject is a mammal.

[0027] In a related embodiment of the invention, inhibition of RAGE expression in subjects with chronic inflammatory conditions is induced by reducing the expression of inflammatory markers, decreasing oxidative stress, and slowing glycosylation levels. A further aspect of this embodiment is that RAGE inhibition is induced by curcuminoids, BAP, or combinations thereof selected from the range of 1 μg / mL to 10 μg / mL, or preferably from the range of 2 μg / mL to 8 μg / mL, or preferably from 4 μg / mL to 6 μg / mL (Example 1, Tables 1-3). In another related aspect of this embodiment, slowing RAGE expression is induced by a decrease in RAGE expression caused by curcuminoids, BAP, or combinations thereof. RAGE expression is increased in diabetic rats. BAP has no effect on pancreatic RAGE expression, and AC3 reduced it by 30.3%. The combination reduced expression by 44.6% and 76.7%, respectively, while metformin effectively reduced it by 6.9% (…). Figure 1(Example 3). In a relevant aspect of this embodiment of the invention, the reduction in expression of inflammatory markers (TNF-α, IL-6, IL-1β) was induced by treatment with curcuminoids, BAP, or a combination thereof. The combination showed a 20-50 fold reduction compared to diabetic controls, and the effect was more significant when using 100 mg / kg curcuminoids and 200 μg / kg BAP compared to treatment alone (Table 6, Example 3). In another aspect of this embodiment of the invention, oxidative stress was reduced in subjects, and this was induced by curcuminoids, BAP, or a combination thereof. The combination showed a better effect of a 3-4 fold reduction compared to a 2-fold reduction in BAP relative to diabetic controls (Table 7, Example 4). In another aspect of this embodiment of the invention, the reduction in glycosylation levels was induced by treatment with curcuminoids, BAP, or a combination thereof. When curcuminoids or BAP were used alone, the effect of protein carbonylation was inhibited by 6-10 fold, while the combination provided a 20-fold change relative to hyperglycemic controls ( Figure 2 (Example 5). In this respect, the subjects were mammals.

[0028] In relevant embodiments of the invention, the therapeutic effect in the subjects is achieved through treatment with curcumin and BAP in the range of 50 μg / kg to 100 mg / kg. More preferably, the curcumin concentration is 1-100 mg / kg, or more preferably, 50-100 mg / kg, or most preferably, 100 mg / kg. BAP is selected from the range of 50 μg / kg to 200 μg / kg, or more preferably, 50 μg / kg, or most preferably, 50 μg / kg or 200 μg / kg. The combination of curcumin and BAP is selected from the range of 50 μg / kg to 100 mg / kg, or more preferably, 50 μg / kg or 200 μg / kg of BAP and 100 mg / kg of curcumin. In relevant embodiments of the invention, the chronic inflammatory condition is selected from the group consisting of: type II diabetes, cardiovascular disease, Alzheimer's disease, cancer, peripheral neuropathy, sensory loss, and blindness. In related aspects, the subjects are mammals.

[0029] In another related embodiment of the invention, the composition further comprises stabilizers, bioavailability enhancers and antioxidants, pharmaceutically, nutritionally or cosmetically acceptable excipients and enhancers, and is suitably formulated for oral administration in the form of tablets, capsules, syrups, gummies, powders, suspensions, emulsions, chewables, confectionery or edibles (Example 7). Proposing suitable administration formulations is entirely within the scope of those skilled in the art.

[0030] In another embodiment of the invention, it is disclosed that the use of AC3, C3 or curcuminoid compositions alone inhibits DPP4 (dipeptidyl peptidase 4), α-glucosidase and anti-glycation (Tables 8-11).

[0031] Based on the foregoing disclosure and teachings, other modifications and variations of the invention will be apparent to those skilled in the art. Therefore, although only certain embodiments of the invention have been specifically described herein, it will be apparent that many modifications can be made thereto without departing from the spirit and scope of the invention.

[0032] Example

[0033] Example 1: Anti-glycation - Measures for in vitro prevention of advanced glycation end products (AGEs)

[0034] Glycosylation is a non-enzymatic glycosylation reaction involving the amino group of a protein, lipid, or nucleic acid with a glycosyl or ketone group, leading to the formation of advanced glycation end products (AGEs) (Yamagishi et al., Pathologic role of dietary advanced glycation end products in cardiometabolic disorders, and therapeutic intervention, Nutrition, 32(2), 157-65 (2016)). The reactive carbonyl derivative of a sugar binds to the amino group of a protein, lipid, or nucleic acid to produce a Schiff base, which rearranges into an Amadori product. In a series of slow reactions, the Amadori reaction, the Schiff base reaction, and the Maillard reaction ultimately form AGEs. Although glycosylation is slow in vivo, the glycosylated products have a durable effect. The efficacy of the test substance in preventing AGE formation was evaluated in vitro.

[0035] Anti-glycosylation activity was evaluated as previously described (Sero et al., Tuning a 96-Well Microtiter Plate Fluorescence-Based Assay to Identify AGE Inhibitors in Crude Plant Extracts). Briefly, 10 μL of different sample concentrations were added to 40 μL of 25 mg / ml bovine serum albumin and 50 μL of 150 mg / ml D-ribose in 96-well black microplates. Buffered D-ribose served as a control. Plates containing the mixture were incubated at 37°C for 24 hours. Late glycosylation products were detected by measuring fluorescence intensity at excitation / emission (Ex / Em) wavelengths of 390 / 460 nm using a BMG FLUOstar Optima microplate reader. AGE formation (a non-enzymatic reaction between protein (BSA) and sugar (ribose)) was inhibited by AC3 in a concentration-related manner (Table 2). BAP was a poor inhibitor of AGE formation (Table 1). The combination of AC3 and BAP synergistically increased the inhibition of glycosylation in vitro (Table 3).

[0036] Tables 1 & 2: Concentration-related inhibition of BAP and AC3

[0037]

[0038] Table 3: Concentration-related inhibition of AC3-BAP combination

[0039]

[0040]

[0041] Example 2: Inhibition of physiological AGE and RAGE, taking diabetes as an example.

[0042] To investigate the effects of the interaction between AGE and RAGE and their pathological consequences, diet-induced diabetes was used as a model.

[0043] Wistar rats were fed a high-fat, fructose-containing diet (HFFD) of 150 g to induce type 2 diabetes (T2D). HFFD induced the development of diabetes associated with long-term metabolic disorders, including fasting hyperglycemia, pre- and postprandial hyperinsulinemia, insulin resistance, glucose intolerance, and dyslipidemia. HFFD-treated animals exhibited T2D-related complications such as hepatic steatosis with fibrosis, inflammation, hyperleptinemia, and endothelial dysfunction.

[0044] Rats were co-administered with AC3 (100 mg / kg), BAP (200 μg / kg), BAP+AC3 (200 μg + 100 mg / kg), BAP+AC3 (50 μg + 100 mg / kg), and 100 mg / kg metformin as a positive control, along with HFFD, for 90 days (Table 4). Organs were harvested at the end of the experiment to assess the effects of the supplements on RAGE expression, oxidative stress, and inflammation.

[0045] Table 4: Animals in the study group (rats)

[0046]

[0047]

[0048] Example 3: RAGE expression in the pancreas

[0049] DNA was extracted from pancreatic samples using the trizol method. Pancreatic tissue was homogenized in liquid nitrogen, followed by trizol extraction and removal of any trace DNA using DNase. Oligo dT primers and Superscript III reverse transcriptase (cDNA synthesis kit, Invitrogen) were used. TM First-strand cRNA was prepared from the RNA sample. Using a Light cycler 96, following the manufacturer's instructions (Light... FastStart DNAMaster SYBR Green I (Roche) was used for quantitative real-time PCR (qRT-PCR) with SYBR Green I fluorescent dye. Primers used for analysis are provided in Table 5. The β-actin gene was used as a housekeeping gene. Gene expression of the target gene in each test sample was determined by relative quantification using the comparative Ct (ΔΔCt) method.

[0050] RAGE expression was increased in diabetic rats. BAP had no effect on pancreatic RAGE expression, while AC3 reduced it by 30.3%. The combination of these drugs reduced expression by 44.6% and 76.7%, respectively, while metformin effectively reduced it by 6.9%. Figure 1 ).

[0051] Table 5: List of primers used for marker expression

[0052]

[0053] Table 6: Expression Levels of Biomarkers

[0054]

[0055] Compared with the control, diabetic rats showed increased expression of inflammatory cytokines. 200 μg / kg BAP was not effective in reducing cytokine expression in the pancreas, with AC3 showing the lowest activity. The combination was highly effective in reducing the expression levels of inflammatory cytokines (Table 6) (p<0.05).

[0056] Example 4: Estimation of Oxidative Stress

[0057] Oxidative stress levels in tissues were assessed using 20,70-dichlorofluorescein diacetate (DCFDA), a fluorescent dye that measures the activity of hydroxyl, peroxy, and other reactive oxygen species (ROS). Briefly, aliquots (10 μL) of tissue homogenate were mixed with 150 μL of an ethanol solution of DCFDA to a final concentration of 10 mM. After incubation at room temperature in the dark for 30 minutes, fluorescence was measured at excitation and emission wavelengths of 488 and 520 nm. Higher fluorescence indicated higher oxidative stress. Oxidative stress was reduced in subjects through curcuminoids, BAP, or combinations thereof. This combination showed a 3-4 fold reduction compared to a 2-fold reduction in BAP relative to diabetic controls (Table 7).

[0058] Table 7: Relative fluorescence intensity measured under oxidative stress

[0059]

[0060]

[0061] Example 5: Protein carbonylation and AGE-protein carbonylation in the pancreas

[0062] Protein carbonylation is defined as the introduction of reactive carbonyl moieties, such as aldehydes, ketones, or lactams, into proteins via oxidative stress-related reactions. Therefore, the term "carbonyl stress" has been suggested to describe the abnormal accumulation of reactive carbonyl substances due to their production or disruption of cellular metabolism. Compared to other oxidative modifications, protein carbonylates exhibit unique stability, can circulate in the bloodstream for longer periods, and have a wide range of downstream functional consequences. Chronic diseases such as diabetes, lung disease, kidney failure, and Alzheimer's disease are some consequences of carbonylated proteins. In addition to AGEs, hyperglycemia can increase protein carbonylation. In diabetes, increased levels of reactive oxygen species (ROS) combined with hyperglycemia lead to the formation of intermediates containing reactive carbonyl groups, such as glyoxal and methylglyoxal (MG) derived from glucose oxidation. Therefore, reducing protein carbonyl compounds is being sought as a novel mechanism for controlling chronic diseases.

[0063] Assay of the fluorescence of protein carbonyl compounds (PCs) using NBDH (7-hydrazino-4-nitrobenzo-2,1,3-oxadiazole).

[0064] This assay is based on the hydrazone formation reaction of NBDH with carbonyl compounds to form a highly fluorescent product (Vidal et al., 2014). All protein-containing or biological samples were diluted 2-fold in PBS. 100 μL of the diluted protein sample was placed in a black 96-well microplate. 100 μL of NBDH solution (200 μM NBDH in PBS (pH 7.4) containing 1 M HCl) was added, and the plate was incubated at 37°C with gentle shaking for 20 min. Fluorescence was measured at 560 nm and excited at 480 nm. When curcuminoids or BAP were used alone, the effect of protein carbonylation was inhibited by 6 to 10-fold, while this combination provided a 20-fold change relative to the hyperglycemic control. Figure 2 The combination of AC3 and BAP showed promising results compared to treatment alone.

[0065] Example 6: Activity against DPP4, α-glucosidase, and glycosylation

[0066] By inhibiting DPP4 enzyme (Table 8), α-glucosidase (Table 9), and the anti-glycation and DPP4 effects of curcuminoids alone, the C3 complex, and the AC3 complex, the didemethoxycurcumin (AC3) combination showed dose-related control of hyperglycemia. The AC3 complex was a better anti-glycation inhibitor than curcuminoids alone (Table 10), and curcumin was as effective against DPP4 as AC3 (Table 11).

[0067] Table 8: Inhibition of DPP4

[0068]

[0069] Table 9: Inhibition of α-glucosidase activity

[0070]

[0071] Table 10: Anti-glycation activity of curcuminoids at 24 and 72 hours

[0072]

[0073]

[0074] Table 11: Anti-DPP4 activity of curcuminoids

[0075]

[0076] Example 7: Formulations containing AC3 and β-amyrin palmitate

[0077] The composition is formulated with pharmaceutically / nutritionalally acceptable excipients, adjuvants, diluents, stabilizers, dispersible gums, bioavailability enhancers, or carriers, and administered orally in the form of tablets, capsules, syrups, gummies, powders, suspensions, emulsions, chewables, confectionery, or edibles.

[0078] In related aspects, piperine was selected as the bioavailability enhancer. The group consisting of quercetin, garlic extract, ginger extract, and naringin. In another related aspect, the stabilizer is selected from the group consisting of rosmarinic acid, butylated hydroxyanisole, butylated hydroxytoluene, sodium metabisulfite, propyl gallate, cysteine, ascorbic acid, and tocopherol. In another related aspect, the dispersible gum is selected from the group consisting of agar, alginate, carrageenan, gum arabic, guar gum, locust bean gum, konjac gum, xanthan gum, and pectin.

[0079] Table 12-16 provides illustrative examples of nutritional preparations containing didemethoxycurcumin.

[0080] Table 12: Tablets

[0081]

[0082] Table 13: Capsules

[0083]

[0084] Table 14: Powders

[0085]

[0086] Table 15: Gluten Candy Formulations

[0087]

[0088] Table 16: Confectionery Preparations

[0089]

[0090] The above formulations are merely illustrative examples, and any formulation containing the above active ingredients and intended for the stated purpose is considered equivalent.

[0091] Based on the foregoing disclosure and teachings, other modifications and variations of the invention will be apparent to those skilled in the art. Therefore, while only certain embodiments of the invention have been specifically described herein, it will be apparent that many modifications can be made thereto without departing from the spirit and scope of the invention, and the invention is interpreted only in conjunction with the appended claims. sequence list <110> Sami-Sabinsa Group Limited <120> Compositions for targeting the receptor for advanced glycation end products (RAGE) in chronic inflammatory conditions <130> BDMC-RAGE <160> 10 <170> PatentIn version 3.5 <210> 1 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Forward primers for RAGE <400> 1 acagaaaccg gtgatgaagg 20 <210> 2 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Reverse primers for RAGE <400> 2 ctctcctcga gtctgggttg 20 <210> 3 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Forward primers targeting β-actin <400> 3 cccgcgagta caaccttct 19 <210> 4 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Reverse primer targeting β-actin <400> 4 cgtcatccat ggcgaact 18 <210> 5 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Forward primers targeting TNFα <400> 5 actgaacttc ggggtgattg 20 <210> 6 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Reverse primers targeting TNFα <400> 6 gcttggtggt ttgctacgac 20 <210> 7 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Forward primers targeting IL6 <400> 7 ctctccgcaa gagacttcca g 21 <210> 8 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Reverse primer targeting IL6 <400> 8 ttctgacagt gcatcatcgc t 21 <210> 9 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Forward primers targeting IL1β <400> 9 caccttcttt tccttcatct ttg 23 <210> 10 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Reverse primers targeting IL1β <400> 10 gtcgttgctt gtctctcctt gta 23

Claims

1. Use of a composition of AC3 and β-amyrin palmitate (BAP) in the preparation of a medicament for the therapeutic control of diabetes in subjects, wherein AC3 is a curcuminoid composition comprising 20-50% w / w bis(demethoxy)curcumin (BDMC), 10-25% w / w demethoxy)curcumin (DMC), and 30-50% w / w curcumin.

2. The use as described in claim 1, wherein the total curcumin in AC3 is in the range of 20-95% w / w.

3. The use as described in claim 1, wherein the diabetes is controlled by inhibiting RAGE, slowing RAGE expression, reducing inflammatory marker expression, reducing oxidative stress, and slowing down glycation levels.

4. The use as claimed in claim 3, wherein the inhibition of RAGE is caused by a combination of curcuminoids and BAP selected from the range of 1 µg / mL to 10 µg / mL.

5. The use as described in claim 3, wherein the reduction in RAGE expression is caused by treatment with a combination of curcuminoids and BAP selected from the range of 50 µg / kg to 100 mg / kg, resulting in a decrease in RAGE expression levels.

6. The use as claimed in claim 3, wherein the inflammatory marker is selected from the group consisting of TNF-α, IL-6 and IL-1β, and wherein the decrease in expression of the inflammatory marker is caused by treatment with a combination of curcuminoids and BAP selected from the range of 50 µg / kg to 100 mg / kg.

7. The use as claimed in claim 3, wherein the oxidative stress is reduced in the subject by treatment with a combination of curcuminoids and BAP selected from the range of 50 µg / kg to 100 mg / kg.

8. The use as described in claim 3, wherein the reduction in glycosylation levels is induced by a combination of curcuminoids and BAP selected from the range of 50 µg / kg to 100 mg / kg.

9. The use as claimed in claim 1, wherein the composition further comprises stabilizers, bioavailability enhancers and antioxidants, pharmaceutically or nutritionally or cosmeceutically acceptable excipients and enhancers, and is administered orally in the form of tablets, capsules, syrups, powders, suspensions, emulsions, chewables, confectionery or other edible forms.

10. The use as claimed in claim 1, wherein the composition further comprises a stabilizer, a bioavailability enhancer and an antioxidant, a pharmaceutically or nutritionally or cosmeceutically acceptable excipient and enhancer, and is administered orally in the form of a gummy candy.

11. The use as claimed in claim 1, wherein the subject is a mammal.

12. A composition comprising AC3 and β-amyrin palmitate (BAP), wherein the composition is used to therapeutically control diabetes in a subject, wherein AC3 is a curcuminoid composition comprising 20-50% w / w bis(demethoxy)curcumin (BDMC), 10-25% w / w demethoxy)curcumin (DMC), and 30-50% w / w curcumin.

13. The composition of claim 12, wherein the total curcuminoids in AC3 are in the range of 20-95% w / w.

14. The composition of claim 12, wherein the diabetes is controlled by inhibiting RAGE, slowing RAGE expression, reducing inflammatory marker expression, reducing oxidative stress, and slowing down glycation levels.

15. The composition of claim 14, wherein the inhibition of RAGE is caused by a combination of curcuminoids and BAP selected from the range of 1 µg / mL to 10 µg / mL.

16. The composition of claim 14, wherein the reduction in RAGE expression is caused by treatment with a combination of curcuminoids and BAP selected from the range of 50 µg / kg to 100 mg / kg, resulting in a decrease in RAGE expression levels.

17. The composition of claim 14, wherein the inflammatory marker is selected from the group consisting of TNF-α, IL-6 and IL-1β, and wherein the reduction in expression of the inflammatory marker is caused by treatment with a combination of curcuminoids and BAP selected from the range of 50 µg / kg to 100 mg / kg.

18. The composition of claim 14, wherein the oxidative stress was reduced in the subject by treatment with a combination of curcumin and BAP selected from the range of 50 µg / kg to 100 mg / kg.

19. The composition of claim 14, wherein the reduction in glycosylation levels is caused by a combination of curcuminoids and BAP selected from the range of 50 µg / kg to 100 mg / kg.

20. The composition of claim 12, wherein the composition further comprises stabilizers, bioavailability enhancers and antioxidants, pharmaceutically or nutritionally or cosmeceutically acceptable excipients and enhancers, and is administered orally in the form of tablets, capsules, syrups, powders, suspensions, emulsions, chewables, confectionery or other edible forms.

21. The composition of claim 12, wherein the composition further comprises a stabilizer, a bioavailability enhancer and an antioxidant, pharmaceutically or nutritionally or cosmeceutically acceptable excipients and enhancers, and is administered orally in gummy form.

22. The composition of claim 12, wherein the subject is a mammal.

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