Composition for targeting the advanced glycation end product receptor (RAGE) in chronic inflammatory states

Abstract

The present invention discloses compositions and methods comprising enriched bisdemethoxycurcumin (BDMC) present at 20% by weight or greater for use in inhibiting receptor for advanced glycation end products (RAGE) expression in a subject with a chronic inflammatory condition. The composition further comprises beta-amyrin palmitate (BAP). The present invention also discloses the use of the composition in managing a chronic inflammatory condition in a subject.

Description

Technical Field

[0001] Cross - reference to related applications This application is a PCT application claiming priority to U.S. Provisional Patent Application No. 63126920, filed on December 17, 2020, the content of which is incorporated herein by reference.

[0002] The present invention generally relates to compositions and methods for use in inhibiting the expression of receptor for advanced glycation end products (RAGE) in subjects with chronic inflammatory conditions using a composition comprising concentrated bisdemethoxycurcumin (BDMC) present at 20% by mass or more. The composition further comprises β - amylin palmitate (BAP). The present invention also includes the step of therapeutically managing chronic inflammatory conditions in a subject using the above composition.

Background Art

[0003] Non-enzymatic oxidation and glycation of proteins lead to the formation of advanced glycation end products (AGEs). Progressive renal failure, atherosclerosis, diabetes, and aging are some of the conditions that promote AGE formation. AGEs are increased in inflammatory conditions such as systemic lupus erythematosus, rheumatoid arthritis, osteoarthritis, and dialysis-related complications, suggesting a specific link to chronic inflammatory diseases. These chronic inflammatory conditions can accelerate atherosclerosis and increase the risk of its complications. Conversely, hypoxia and ischemic-reperfusion injury can rapidly generate AGEs and further increase complications of inflammatory diseases. Hyperglycemia or diabetes is a condition that leads to elevated blood glucose levels in the bloodstream, promoting the formation of advanced glycation end products (AGEs). Cells develop glucose-induced inflammation and other related pathological damage when excessively exposed to glucose, although the mechanisms are not yet fully established. Furthermore, existing hypotheses suggest that AGE deposition in tissues can lead 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 diverse macromolecules, including carboxymethyllysine (CML), carboxyethyllysine (CEL), pentosidine, glucospan, glyoxallysine dimer, and glycolic acid lysine amide, formed by non-enzymatic glycation processes of proteins and lipids. AGEs induce cellular effects by interacting with receptors. The receptor for advanced glycation end products (RAGE) is a ubiquitous transmembrane receptor that binds to various endogenous ligands. The interaction between AGEs and RAGE initiates a complex intracellular signaling cascade, leading to the upregulation of RAGE itself, as well as the production of reactive oxygen species (ROS), immunoinflammatory effects, cell proliferation, or apoptosis.Several studies have found correlations between RAGE activity and pathological conditions such as cancer, diabetes, cardiovascular disease, and neurodegeneration. While AGEs can be benign and unresponsive, some studies have found correlations between RAGE activity and pathological conditions such as cancer, diabetes, cardiovascular disease, and neurodegeneration, suggesting that AGEs may be a cause of complications in chronic diseases such as type II diabetes mellitus, cardiovascular disease, Alzheimer's disease, cancer, peripheral neuropathy, loss of sensation, 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 resulting from 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), cytokine release, inflammatory expression, and activation of cellular signaling. The ROS generated during RAGE activation are a source of protein oxidation, forming protein carbonyl species. In the "primary protein carbonylation" reaction, among other amino acids, the direct oxidation of the side chains of lysine, arginine, proline, and threonine residues produces DNPH-detectable protein products called reactive carbonyl species (RCS) (Suzuki et al. Protein carbonylation. Antioxid Redox Signal). (2010;12(3):323-325). Conversely, the reactive carbonyl of sugars combines with the amino group of a protein, lipid, or nucleic acid to produce a Schiff base, which then rearranges the Amadori product. In a series of slow reactions, the Amadori reaction, the Schiff base reaction, and the Maillard reaction ultimately form an AGE.Amadori compounds can be further broken down into different advanced glycation end products containing reactive α-dicarbonyls, releasing ROS such as superoxide anions and hydrogen peroxide. α-dicarbonyls formed by the oxidative breakdown of Schiff bases and Amadori adducts, as well as during glucose autooxidation, can lead to oxidative deamination of Lys via Strecker-type reactions, resulting in the formation of 3-deoxyglucosone (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 autooxidation, or glucose metabolism. The reaction of glyoxal (GO) and methylglyoxal (MGO) with lysine and arginine residues in proteins leads to the formation of AGEs such as carboxymethyllysine (CML), carboxyethyllysine (CEL), and argpyrimidine (ArgP). Therefore, AGEs, protein carbonyl formation, and their interaction with RAGE, as well as downstream oxidative stress and inflammation, are closely related. Increased steady-state levels of RCS and AGEs lead to carbonyl stress and disrupt normal metabolism. RCS are ubiquitous compounds with relatively high half-lives and stability, especially compared to reactive oxygen species (ROS). Due to their low molecular bulk, uncharged structure, and relatively high stability, RCS can traverse biological membranes, diffuse through peripheral circulation, and cross the blood-brain barrier. Glycated derivatives such as methylglyoxal, glyoxal, 3-deoxyfructose, glucosone, and 3-deoxyglucosone are approximately 20,000 times more reactive than reduced carbohydrates. Alzheimer's disease (AD), rheumatoid arthritis, diabetes mellitus, sepsis, chronic renal failure, and respiratory distress syndrome are some of the conditions associated with increased protein carbonylation (Isabella et al. Protein carbonyl groups as biomarkers of oxidative stress, 329(1), 23-38 (2003)).AGE formation is accelerated under 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)). While AGEs can be benign and non-reactive, they can contribute to complications in chronic diseases such as type II diabetes mellitus, cardiovascular disease, Alzheimer's disease, cancer, peripheral neuropathy, loss of sensation, 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 glycation, RAGE expression, and protein carbonylation can ultimately reduce oxidative and carbonyl stress and may be beneficial in chronic inflammatory states. RAGE is an attractive target for the development of inhibitors to manage several diseases, serving as a potential biomarker for these conditions. Altered circulating levels of RAGE have been identified in patients with diabetic complications, cardiovascular disease, and Alzheimer's disease. RAGE has also been investigated as a potential target for therapies for 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)).

[0004] Numerous studies link the role of RAGE in the formation of atherosclerotic lesions and promote pro-inflammatory pathways, suggesting diabetes-mediated atherosclerosis. - / - / apoE - / -In mice, increased AGEs linked to diabetes were significantly reduced, along with inflammatory responses characterized by decreased macrophage accumulation and reduced cytokine and chemokine expression (Paavonen et al. Receptor for Advanced Glycation End Products (RAGE) Deficiency Attenuates the Development of Atherosclerosis in Diabetes, Diabetes, 57, 2461-2469 (2008)). This has also been supported by numerous studies demonstrating inhibition of RAGE activation using neutralizing antibodies or soluble RAGE (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 diabetic apolipoprotein 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)).

[0005] The role of curcumin in attenuating the effects of RAGE signaling, inhibiting AGE accumulation, and influencing RAGE expression has been reported in experimentally diabetic rats (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 curcuminoids: 72-77% curcumin, 14-18% dimethoxycurcumin, and 3-5% bisdemethoxycurcumin. Furthermore, fractions larger than curcumin make it hydrophobic, thus affecting its 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, bisdemethoxycurcumin, and demethoxycurcumin vary depending on the disease state, and recently, bisdemethoxycurcumin and demethoxycurcumin have attracted considerable attention due to their similar and superior efficacy to curcumin in managing certain disease symptoms (Majeed et al., Reductive Metabolites of Curcuminoids, Nutriscience Publishers LLC, 2019).The pharmacological challenges associated with targeting RAGE involve not only regulating the gene expression of inflammatory genes in the feedforward loop, but also regulating NF-κB activation that induces RAGE expression (Armando et al. Inhibition of RAGE Axis Signalling: A Pharmacological Challenge, Current Drug Targets, 20, 340-346, (2019)). While promising results were obtained with blockading peptides and antibodies produced against RAGE, their use faced limitations due to constraints as therapeutic compounds (Arumugam T et al. S100P-derived RAGE antagonistic peptide 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 chromosomal protein 1 activity. Arthritis Rheum 48(7): 2052-8 (2003)).In addition, several small molecules, including TTP488, azeliragon, pioglitazone, and PPARγ agonists, also block RAGE signaling (Salvatore et al. Targeting the Receptor for Advanced Glycation End products (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 end products, for the potential slowing of loss of cognition in mild Alzheimer's disease, J Prev Alzheimers Dis 5(2): 149-54 (2018)). Given the complexity associated with targeting RAGE and the very limited list of compounds available in clinical trials, there is a need for novel methods of targeting RAGE, particularly safe and less toxic ones.

[0006] Object of the invention A primary object of the present invention is to disclose compositions and methods for use in inhibiting RAGE expression in subjects with chronic inflammatory conditions using compositions containing concentrated bisdemethoxycurcumin (BDMC) present at a concentration of 20% by mass or more. The compositions further comprise β-amylin palmitic acid (BAP). In another primary object of the present invention, compositions and methods for therapeutically managing RAGE in subjects with chronic inflammatory conditions are disclosed, using compositions comprising concentrated bisdemethoxycurcumin (BDMC) present at a concentration of 20% by mass or more. The compositions further comprise β-amylin palmitic acid (BAP). [Overview of the project]

[0007] The present invention broadly solves the aforementioned problems described in the background by encompassing methods and compositions for use in inhibiting RAGE expression in subjects with chronic inflammatory conditions using compositions containing concentrated bisdemethoxycurcumin (BDMC) present at 20% by mass or more. The composition further comprises β-amylin palmitic acid (BAP). A first aspect of the present invention relates to a composition for use in inhibiting RAGE expression in subjects with a chronic inflammatory state using a composition comprising concentrated bisdemethoxycurcumin (BDMC) present at a concentration of 20% by mass or more. The composition further comprises β-amylin palmitic acid (BAP). In yet another aspect of the present invention, the present invention includes a composition for use in therapeutically managing a chronic inflammatory condition in a subject using a composition comprising concentrated bisdemethoxycurcumin (BDMC) present in an amount of 20% by mass or more. The composition further comprises β-amylin palmitic acid (BAP). Another embodiment of the present invention includes a method for inhibiting RAGE expression in subjects with a chronic inflammatory state using a composition comprising concentrated bisdemethoxycurcumin (BDMC) present at a concentration of 20% by mass or more. The composition further comprises β-amyrin palmitic acid (BAP). Another aspect of the present invention comprises a method for treating a chronic inflammatory condition in a subject by administering a composition comprising concentrated bisdemethoxycurcumin (BDMC) present in an amount of 20% by mass or more to the subject. The composition further comprises β-amylin palmitic acid (BAP). The broader applications of the present invention will become apparent from the detailed description below. However, it should be understood that the following detailed description and specific examples illustrate preferred embodiments of the present invention, but should not be construed as limitations on the invention, and various changes and modifications, such as changes in the concentration range of the sample used, curcuminoid derivatives / analogs, BAP, experimental conditions, and selection of mammals, are within the scope of the articulate and well within the spirit and scope of the invention as described in this detailed description. [Brief explanation of the drawing]

[0008] [Figure 1] This figure shows the effects of AC3, BAP, and their combined use on RAGE expression in the pancreas. *P<0.05. [Figure 2] This figure shows the effects of AC3, BAP, and their combined use on anti-glycation in the pancreas. *P<0.05. [Modes for carrying out the invention]

[0009] Selected definition Unless otherwise specified, all terms used in this application have their ordinary meanings known in the prior art. A few other specific definitions used in this invention are described below and apply throughout this specification. Unless otherwise specified, claims provide broader definitions.

[0010] In this application, any reference to a sample refers to one or a combination of the following agents that produce the disclosed therapeutic effect. The agent includes a concentrated BDMC composition, which refers to a curcuminoid composition containing at least 20% by mass of BDMC. More specifically, AC3 is a preferred curcuminoid used in the present invention, and any reference to a curcuminoid refers to AC3, which consists of 20-50% by mass of bisdemethoxycurcumin, 10-25% by mass of demethoxycurcumin, and 30-50% by mass of curcumin, and BAP refers to β-amylin palmitic acid. Any reference to a C3 complex consists of 75-81% curcumin, 15-19% demethoxycurcumin, and 2.2-6.5% bisdemethoxycurcumin. Also, curcuminoid refers to any of BDMC, DMC, or AC3, as disclosed in the examples. Therapeutic management or control means a state that effectively improves the condition disclosed in this invention. Any reference to controls in this specification refers to diabetic controls, untreated controls, and metformin controls for the experiments and examples included.

[0011] The present invention generally encompasses methods and compositions for use in inhibiting RAGE expression in subjects with chronic inflammatory conditions using compositions containing concentrated bisdemethoxycurcumin (BDMC) present at 20% by mass or more. The present invention also encompasses compositions for use in therapeutically managing chronic inflammatory conditions in subjects using compositions containing concentrated bisdemethoxycurcumin (BDMC) present at 20% by mass or more. Furthermore, the present invention encompasses a method for treating chronic inflammatory conditions in a subject by administering a composition containing concentrated bisdemethoxycurcumin (BDMC) present at 20% by mass or more to the subject, wherein the composition contains 20-50% by mass of BDMC, 10-25% by mass of demethoxycurcumin (DMC), and 30-50% by mass of curcumin, and the total curcuminoid content in the composition is in the range of 20-95% by mass. The composition further comprises β-amylin palmitic acid (BAP). In relevant embodiments, the subject is a mammal.

[0012] In its most preferred embodiment, the present invention discloses a composition for use in inhibiting RAGE expression in subjects with a chronic inflammatory state, the composition comprising concentrated bisdemethoxycurcumin (BDMC) present at 20% by mass or more. In another embodiment of this embodiment, the composition comprises 20-50% by mass of BDMC, 10-25% by mass of demethoxycurcumin (DMC), and 30-50% by mass of curcumin, the total curcuminoids in the composition being in the range of 20-95% by mass. In a related embodiment of this embodiment, the composition further comprises β-amylin palmitic acid (BAP). In a related embodiment, the subject is a mammal.

[0013] In another most preferred embodiment of the present invention, the present invention discloses a composition for use in therapeutically managing a chronic inflammatory condition in a subject, the composition comprising concentrated bisdemethoxycurcumin (BDMC) present at 20% by mass or more. In another embodiment of this embodiment, the composition comprises 20-50% by mass of BDMC, 10-25% by mass of demethoxycurcumin (DMC), and 30-50% by mass of curcumin, the total curcuminoids in the composition being in the range of 20-95% by mass. In a related embodiment of this embodiment, the composition further comprises β-amylin palmitic acid (BAP). In a related embodiment, the subject is a mammal. In a most preferred alternative embodiment of the present invention, the present invention discloses a method for inhibiting RAGE expression in a subject with a chronic inflammatory state, comprising the steps of (a) identifying a subject with a chronic inflammatory state and (b) administering to the subject a composition comprising concentrated bisdemethoxycurcumin (BDMC) present at 20% by mass or more. In another embodiment of this embodiment, the composition comprises 20-50% by mass of BDMC, 10-25% by mass of demethoxycurcumin (DMC), and 30-50% by mass of curcumin, wherein the total curcuminoids in the composition are in the range of 20-95% by mass. In a related embodiment of this embodiment, the composition further comprises β-amylin palmitic acid (BAP). In a related embodiment, the subject is a mammal.

[0014] In a most preferred yet another embodiment of the present invention, the present invention discloses a method for treating a chronic inflammatory condition in a subject, comprising the steps of (a) identifying a subject with a chronic inflammatory condition; and (b) administering to the subject a composition comprising concentrated bisdemethoxycurcumin (BDMC) present at 20% by mass or more. In another embodiment of this embodiment, the composition comprises 20-50% by mass of BDMC, 10-25% by mass of demethoxycurcumin (DMC), and 30-50% by mass of curcumin, wherein the total curcuminoids in the composition are in the range of 20-95% by mass. In a related embodiment of this embodiment, the composition further comprises β-amylin palmitic acid (BAP). In a related embodiment, the subject is a mammal.

[0015] In related embodiments of the present invention, inhibition of RAGE expression in subjects with a chronic inflammatory state is achieved by reducing the expression of inflammatory markers, reducing oxidative stress, and mitigating glycation levels. In a further embodiment of this embodiment, inhibition of RAGE is achieved by curcuminoids, BAP, or a combination thereof selected from the range of 1 to 10 μg / mL, preferably 2 to 8 μg / mL, preferably 4 to 6 μg / mL (Example 1, Tables 1 to 3). In another related embodiment of this embodiment, reduction of RAGE expression is achieved by curcuminoids, BAP, or a combination thereof resulting in a decrease in RAGE expression. RAGE expression was increased in diabetic rats. BAP had no effect on pancreatic RAGE expression, but AC3 reduced expression by 30.3%. Combinations reduced expression by 44.6% and 76.7%, respectively. Metformin was effective in reducing expression by 6.9% (Figure 1, Example 3). In a related embodiment of this invention, a reduction in the expression of inflammatory markers (TNF-α, IL-6, IL-1β) was achieved by treatment with curcuminoids, BAP, or a combination thereof. The combination showed a reduction of 1 / 20 to 1 / 50 compared to diabetic controls, and the effect was even more pronounced when 100 mg / kg curcuminoids and 200 μg / kg BAP were used compared to the individual treatments (Table 5, Example 3). In another embodiment of this invention, oxidative stress was reduced in the subjects by curcuminoids, BAP, or a combination thereof. Compared to BAP, which showed a reduction of 1 / 2 compared to diabetic controls, the effect of the combination was superior, showing a reduction of 1 / 3 to 1 / 4 (Table 6, Example 4). In another embodiment of this invention, a reduction in glycan levels was achieved by treatment with curcuminoids, BAP, or a combination thereof. The effect of protein carbonylation was inhibited to 1 / 6 to 1 / 10 of the hyperglycemic control when curcuminoids or BAP were used individually, and the combination resulted in a 1 / 20 change (Figure 2, Example 5). In the relevant embodiments, the subjects are mammals.

[0016] In related embodiments of the present invention, the therapeutic effect in the subject is obtained by treatment with curcuminoids in the range of 50 μg / kg to 100 mg / kg, and BAP. More preferably, 1 to 100 mg / kg, more preferably 50 to 100 mg / kg, and most preferably 100 mg / kg of curcuminoids. BAP is selected from the range of 50 to 200 μg / kg, more preferably 50 μg / kg, and most preferably either 50 μg / kg or 200 μg / kg. The combination of curcuminoids and BAP is selected from BAP in the range of 50 μg / kg to 100 mg / kg, more preferably either 50 μg / kg or 200 μg / kg, and 100 mg / kg of curcuminoids. In related embodiments of the present invention, the chronic inflammatory state is selected from the group consisting of type II diabetes mellitus, cardiovascular disease, Alzheimer's disease, cancer, peripheral neuropathy, loss of sensation, and blindness. In related embodiments, the subject is a mammal.

[0017] In another related embodiment of the present invention, the composition further comprises stabilizers, bioavailable enhancers, and antioxidants, pharmaceutically or nutritionally or cosmetically acceptable excipients and enhancers, and is appropriately formulated for oral administration in the form of tablets, capsules, syrups, gummies, powders, suspensions, emulsions, chewables, candies or edible products (Example 7). It is well within the capabilities of those skilled in the art to devise formulations suitable for administration. Another embodiment of the present invention discloses the use of AC3, C3, or individual curcuminoid compositions to inhibit DPP4 (dipeptidyl peptidase 4), α-glucosidase, and anti-glycation (Tables 7-10). Other modifications and changes to the present invention will be apparent to those skilled in the art from the foregoing disclosure and teachings. Thus, although only specific embodiments of the present invention are described herein, it will be apparent that numerous modifications may be made without departing from the spirit and scope of the invention. [Examples]

[0018] (Example 1) Anti-glycation - Strategies to prevent advanced glycation end products in vitro Glycation is a non-enzymatic glycosylation reaction involving amino groups of proteins, lipids, or nucleic acids and aldehyde or keto groups of sugars, 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 group of the sugar binds to the amino group of the protein, lipid, or nucleic acid to form a Schiff base, which then rearranges the Amadori product. In a series of slow reactions, the Amadori reaction, Schiff base formation, and Maillard reaction ultimately lead to the formation of AGEs. While glycation is slow in vivo, the effects of glycated products are persistent. The effects of test substances that prevent AGE formation were evaluated in vitro.

[0019] As previously described, anti-glycation activity was evaluated (Sero et al. Tuning a 96-Well Microtiter Plate Fluorescence-Based Assay to Identify AGE Inhibitors in Crude Plant Extracts). Briefly, 10 μL of various sample concentrations were added to 40 μL of 25 mg / mL bovine serum albumin and 50 μL of 150 mg / mL D-ribose in a 96-well black microplate. D-ribose and buffer were used as controls. Plates containing the mixtures were incubated at 37°C for 24 hours. Glycation products were detected by measuring fluorescence intensity at 390 / 460 nm Ex / Em using a BMG FLUOstar Optima microplate reader. AGE formation [non-enzymatic reaction between protein (BSA) and sugar (ribose)] was inhibited by AC3 in a concentration-dependent manner (Table 1). BAP was a weak inhibitor of AGE formation (Table 1). The combined use of AC3 and BAP may synergistically enhance the inhibition of glycation in vitro (Table 2).

[0020]

Table 1

Table 2

[0021] (Example 2) Inhibition of AGE and RAGE in a physiological state using an example of diabetes. To study the interaction effect of AGE and RAGE and its pathological consequences, diet-induced diabetes was used as a model. Wistar rats (150 g) were fed a high-fat and fructose diet (HFFD) to induce type 2 diabetes (T2D). HFFD induces the onset of diabetes associated with long-term metabolic disorders including fasting hyperglycemia, hyperinsulinemia before and after meals, insulin resistance, impaired glucose tolerance, and dyslipidemia. Animals on HFFD exhibit complications associated with T2D such as hepatic steatosis with fibrosis, inflammation, hyperleptinemia, and endothelial cell dysfunction.

[0022] Rats were co-administered 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 with HFFD for 90 days (Table 3). Organs were harvested at the end of the experiment to evaluate the effect of the agents on RAGE expression, oxidative stress, and inflammation.

Table 3

[0023] (Example 3) RAGE expression in the pancreas DNA was extracted from pancreatic samples using the trizol method. Pancreatic tissue was homogenized in liquid nitrogen, followed by trizol extraction and DNAse treatment to remove any trace amounts of DNA. First-strand cDNA was prepared from RNA samples using oligo dT primers and Superscript III reverse transcriptase [cDNA synthesis kit, Invitrogen®]. Quantitative real-time PCR (qRT-PCR) was performed using a Light Cycler 96 according to the manufacturer's instructions, with SYBR Green I fluorescent dye [Light Cycler® FastStart DNA Master SYBR Green I, Roche]. The primers used for the analysis are provided in Table 4. The beta-actin gene was used as a housekeeping gene. Gene expression of target genes in each test sample was determined by relative quantification using the comparative Ct (ΔΔCt) method.

[0024] RAGE expression was increased in diabetic rats. BAP had no effect on pancreatic RAGE expression, but AC3 reduced expression by 30.3%. Combination therapy reduced expression by 44.6% and 76.7%, respectively. Metformin was effective in reducing expression by 6.9% (Figure 1).

[0025] [Table 4] [Table 5]

[0026] Inflammatory cytokine expression was increased in diabetic rats compared to controls. BAP at 200 ug / kg was not effective in reducing cytokine expression in the pancreas, while AC3 showed minimal activity. Their combined use was highly effective in reducing inflammatory cytokine expression levels (Table 5) (p<0.05).

[0027] (Example 4) Assessment of oxidative stress The level of oxidative stress in tissues was estimated by using 2',7'-dichlorofluorescein diacetate (DCFDA) fluorescent dye to measure hydroxyl, peroxyl, and other reactive oxygen species (ROS) activity. Briefly, an aliquot (10 μL) of tissue homogenate was mixed with 150 μL of DCFDA ethanol solution to a final concentration of 10 mM. After incubation in the dark at room temperature 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 the subjects and was achieved by curcuminoids, BAP, or a combination thereof. Compared to a 1 / 2 reduction in diabetic controls with BAP, the effect of the combination was superior, with a 1 / 3 to 1 / 4 reduction (Table 6).

[0028] [Table 6]

[0029] (Example 5) Protein carbonylation and AGE-protein carbonylation in the pancreas 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” suggests a description of the abnormal accumulation of reactive carbonyl species due to their production or interference with cellular metabolism. Compared to other oxidative modifications, protein carbonyls possess inherent stability, can circulate in the blood for longer periods, and have a wide range of downstream functional consequences. Chronic diseases such as diabetes, lung diseases, renal failure, and Alzheimer's disease are some of the consequences of carbonylated proteins. Apart from AGEs, hyperglycemia can increase protein carbonylation. In diabetes, increased reactive oxygen species (ROS) levels accompanied by hyperglycemia lead to the formation of reactive carbonyl-containing intermediates such as glyoxal and methylglyoxal (MG), derived from glucose oxidation. Therefore, reducing protein carbonyl compounds is being pursued as a novel mechanism for managing chronic diseases.

[0030] Fluorescent quantitative assay of protein carbonyls (PC) using NBDH (7-hydrazino-4-nitrobenzo-2,1,3-oxadiazole) assay. This assay is based on the reaction of NBDH with carbonyl via hydrazone formation, resulting in the formation of a highly fluorescent product (Vidal et la., 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 with 1 M HCl (pH 7.4)] was added, and the plate was incubated at 37°C for 20 minutes with gentle shaking. Fluorescence was excited at 480 nm and measured at 560 nm. The effect of protein carbonylation was inhibited to 1 / 6 to 1 / 10 of the hyperglycemic control when curcuminoids or BAP were used individually, and the combined use resulted in a 1 / 20 change (Figure 2). The combined use of AC3 and BAP showed a greater effect than the individual treatments.

[0031] (Example 6) DPP4, α-glucosidase, and activity in glycation Bisdemethoxycurcumin (AC3) compositions exhibit hyperglycemia control through their inhibitory effects on DPP4 enzyme (Table 7) and α-glucosidase enzyme (Table 8). Individual curcuminoids, the C3 complex, and the AC3 complex exert anti-glycation and anti-DPP4 effects in a dose-dependent manner. The AC3 complex is a superior inhibitor of glycation compared to individual curcuminoids (Table 9), and curcumin was as effective against DPP4 as AC3 (Table 10).

[0032] [Table 7] [Table 8] [Table 9] [Table 10]

[0033] (Example 7) A preparation containing AC3 and β-amylin palmitic acid The composition is formulated with pharmaceutically / nutritionally acceptable excipients, adjuvants, diluents, stabilizers, dispersible rubbers, bioavailable enhancers, or carriers, and is administered orally in the form of tablets, capsules, syrups, gummies, powders, suspensions, emulsions, chewables, candies, or edibles.

[0034] In a related embodiment, the bioavailability enhancer is selected from the group consisting of piperine [BioPerine®], quercetin, garlic extract, ginger extract, and naringin. In another related embodiment, 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 yet another related embodiment, the dispersible gum is selected from the group consisting of agar, alginate, carrageenan, acacia gum, guar gum, locust bean gum, konjac gum, xanthan gum, and pectin.

[0035] Tables 11 to 15 provide examples of functional food formulations containing bisdemethoxycurcumin. [Table 11] [Table 12] [Table 13] [Table 14] [Table 15] The above formulations are merely examples, and any formulation containing the above active ingredients intended for the aforementioned purposes will be considered equivalent.

[0036] Other modifications and changes to the present invention will be apparent to those skilled in the art from the foregoing disclosure and teaching. Thus, although only specific embodiments of the present invention are described herein, it is clear that numerous modifications may be made without departing from the spirit and scope of the invention and should be interpreted only in conjunction with the appended claims. Another aspect of the present invention may be as follows: [1] A composition for use in inhibiting the expression of advanced glycation end product receptor (RAGE) in subjects with a chronic inflammatory state, comprising concentrated bisdemethoxycurcumin (BDMC) present at a concentration of 20% by mass or more. [2] The composition for use according to [1], wherein the composition comprises 20-50% by mass of BDMC, 10-25% by mass of demethoxycurcumin (DMC), and 30-50% by mass of curcumin, and the total amount of curcuminoids in the composition is in the range of 20-95% by mass. [3] The composition for use according to [1], wherein the composition further comprises β-amylin palmitic acid (BAP). [4] The composition for use according to [1], wherein inhibition of RAGE expression results in a reduction in the expression of inflammatory markers, a reduction in oxidative stress, and a reduction in glycation levels. [5] The composition for use according to [1], wherein the inhibition of RAGE expression is obtained by a curcuminoid selected from 1 to 10 μg / mL, BAP, or a combination thereof. [6] The composition for use described in [1] above, wherein inhibition of RAGE expression is obtained by treatment with a curcuminoid, BAP, or a combination thereof selected from a range of 50 μg / kg to 100 mg / kg, resulting in a decrease in the RAGE expression level. [7] The composition for use according to [4], wherein the inflammatory marker is selected from the group consisting of TNF-α, IL-6, and IL-1β, and the reduction in the expression of the inflammatory marker is obtained by treatment with a curcuminoid selected from 50 μg / kg to 100 mg / kg, BAP, or a combination thereof. [8] The composition for use described in [4], wherein the oxidative stress is reduced in the subject by treatment with a curcuminoid selected from 50 μg / kg to 100 mg / kg, BAP, or a combination thereof. [9] The composition for use described in [4] above, wherein the reduction of glycan levels is achieved by treatment with curcuminoids selected from 50 μg / kg to 100 mg / kg, BAP, or a combination thereof.

[10] The composition for use according to [1] above, wherein the chronic inflammatory state is selected from the group consisting of type II diabetes mellitus, cardiovascular disease, Alzheimer's disease, cancer, peripheral neuropathy, loss of sensation and blindness.

[11] The composition for use described in [1], wherein the subject is a mammal.

[12] The composition for use as described in [1], further comprising a stabilizer, a bioavailable enhancer, and an antioxidant, a pharmaceutically or nutritionally or cosmetically acceptable excipient and enhancer, and administered orally in the form of a tablet, capsule, syrup, gummy, powder, suspension, emulsion, chewable, candy or food product.

[13] A composition for use in the therapeutic management of a chronic inflammatory state in a subject, wherein the composition contains concentrated bisdemethoxycurcumin (BDMC) present in an amount of 20% by mass or more.

[14] The composition for use according to

[13] , wherein the composition comprises 20-50% by mass of BDMC, 10-25% by mass of demethoxycurcumin (DMC), and 30-50% by mass of curcumin, and the total amount of curcuminoids in the composition is in the range of 20-95% by mass.

[15] The composition for use according to

[13] , wherein the composition further comprises β-amyrin palmitic acid (BAP).

[16] The composition for use according to

[13] , wherein the therapeutic management of a chronic inflammatory state is obtained by inhibiting RAGE, mitigating RAGE expression, reducing the expression of inflammatory markers, reducing oxidative stress, and mitigating glycation levels.

[17] The composition for use according to

[16] , wherein the inhibition of RAGE is obtained by a curcuminoid selected from 1 to 10 μg / mL, BAP, or a combination thereof.

[18] The composition for use described in

[16] , wherein the reduction of RAGE expression is obtained by treatment with a curcuminoid, BAP, or a combination thereof selected from the range of 50 μg / kg to 100 mg / kg, resulting in a decrease in the RAGE expression level.

[19] The composition for use according to

[16] , wherein the inflammatory marker is selected from the group consisting of TNF-α, IL-6, and IL-1β, and the reduction in the expression of the inflammatory marker is obtained by treatment with a curcuminoid selected from 50 μg / kg to 100 mg / kg, BAP, or a combination thereof.

[20] The composition for use according to

[16] , wherein the oxidative stress is reduced in the subject by treatment with a curcuminoid selected from 50 μg / kg to 100 mg / kg, BAP, or a combination thereof.

[21] The composition for use described in

[16] , wherein the reduction of glycan levels is achieved by treatment with curcuminoids selected from 50 μg / kg to 100 mg / kg, BAP, or a combination thereof.

[22] The composition for use according to

[16] , wherein the chronic inflammatory state is selected from the group consisting of type II diabetes mellitus, cardiovascular disease, Alzheimer's disease, cancer, peripheral neuropathy, loss of sensation and blindness.

[23] The composition for use as described in

[13] , further comprising a stabilizer, a bioavailable enhancer, and an antioxidant, a pharmaceutically or nutritionally or cosmetically acceptable excipient and enhancer, and administered orally in the form of a tablet, capsule, syrup, gummy, powder, suspension, emulsion, chewable, candy or food product.

[24] The composition for use described in

[13] , wherein the subject is a mammal.

Claims

1. A composition for use in inhibiting the expression of advanced glycation end product receptors (RAGEs) in subjects with chronic inflammatory conditions, wherein the composition comprises 20 to 95% by mass of a curcuminoid composition and β-amylin palmitic acid (BAP), and the composition comprises 20% by mass or more of BDMC, wherein the curcuminoid composition comprises 20 to 50% by mass of BDMC, 10 to 25% by mass of demethoxycurcumin (DMC), and 30 to 50% by mass of curcumin, relative to the total mass of the curcuminoid composition.

2. A composition for use in the therapeutic management of a chronic inflammatory state in a subject, wherein the composition comprises 20 to 95% by mass of a curcuminoid composition and β-amylin palmitic acid (BAP), and the composition comprises 20% by mass or more of BDMC, wherein the curcuminoid composition comprises 20 to 50% by mass of BDMC, 10 to 25% by mass of demethoxycurcumin (DMC), and 30 to 50% by mass of curcumin, relative to the total mass of the curcuminoid composition.

3. A composition for use according to claim 1 or 2, wherein a chronic inflammatory state is controlled by inhibiting RAGE, mitigating RAGE expression, reducing the expression of inflammatory markers, reducing oxidative stress, and mitigating glycation levels.

4. The composition for use according to claim 3, wherein the inhibition of RAGE is obtained by using the composition in a range of 1 to 10 μg / mL.

5. The composition for use according to claim 3, wherein the reduction of RAGE expression is obtained by treatment with the composition in a range of 50 μg / kg to 100 mg / kg that results in a decrease in the RAGE expression level.

6. The composition for use according to claim 3, wherein the inflammatory marker is selected from the group consisting of TNF-α, IL-6, and IL-1β, and the reduction in the expression of the inflammatory marker is obtained by treatment with the composition in the range of 50 μg / kg to 100 mg / kg.

7. The composition for use according to claim 3, wherein the oxidative stress is reduced in the subject by treatment with the composition in the range of 50 μg / kg to 100 mg / kg.

8. The composition for use according to claim 3, wherein the reduction of glycan levels is achieved by treatment with the composition in the range of 50 μg / kg to 100 mg / kg.

9. The composition for use according to claim 3, wherein the chronic inflammatory state is selected from the group consisting of type II diabetes mellitus, cardiovascular disease, Alzheimer's disease, cancer, peripheral neuropathy, loss of sensation, and blindness.

10. The composition for use according to claim 1 or 2, further comprising a stabilizer, a bioavailable enhancer, and an antioxidant, a pharmaceutically or nutritionally or cosmetically acceptable excipient and enhancer, and administered orally in the form of a tablet, capsule, syrup, gummy, powder, suspension, emulsion, chewable, candy or food product.

11. The composition for use according to claim 1 or 2, wherein the subject is a mammal.

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