Patent Application
20220378814
The current obesity epidemic presents a major challenge to global health management. In the United States, the prevalence of obesity in adults was 42.4% in 2017-2018 [1]. The estimate of healthcare cost attributable to obesity was $190 billion in 2013, or approximately 21% of US healthcare expenditure [2]. The World Health Organization estimated that approximately 650 million adults were obese worldwide in 2016. Obesity is an established risk factor for the development of type 2 diabetes mellitus and chronic inflammatory diseases, such as dyslipidemia, non-alcoholic fatty liver disease, hypertension, coronary heart disease, stroke, rheumatoid arthritis, and certain cancers [3]. Weight loss is clearly the most obvious strategy for the prevention of obesity and associated diseases. However, achieving weight loss by means of lifestyle intervention has proven to be challenging for many patients to maintain in the long term. Alternative intervention strategies include pharmacotherapy to restrict caloric intake for weight management or glycemic control for diabetes management [4]. Two prevalent pharmacotherapy types are being pursued: those developed for glycemic control that might lead to weight gain, and those developed for weight management that improve glycemic control. Approved anti-obesity and antidiabetic drugs by the United States Food and Drug Administration have proven to be moderately beneficial although they are often associated with adverse side effects, which limit their long-term usage [5]. Clearly, there is still an unmet demand for an effective and safe intervention that is capable of long-term management of obesity and associated medical conditions.
The present disclosure relates to novel compositions of phytonutrients and methods of treating obesity by administering these compositions to subjects in need thereof. The compositions described herein are rationally designed compositions of phytonutrients that interfere with fat cell differentiation, a process commonly known as “adipogenesis”, to prevent weight gain and improve glycemic control. Phytonutrients are natural compounds in plants and mushrooms that have beneficial effects on human health. Phytonutrients have been proven to have anti-obesity effects, such as appetite reduction, modulation of lipid absorption and metabolism, enhancement of insulin sensitivity, thermogenesis and changes to the gut microbiota [6]. Consumption of phytonutrients is generally considered as a safe, widely available and inexpensive approach to prevent obesity and associated conditions.
The compositions include formulation of rationally combined phytonutrients for obesity prevention. The phytonutrients are rationally combined based on their complementary effects on the expression level of six adipogenic biomarker proteins. The formulation of rationally combined phytonutrients exhibit unique anti-adipogenic properties that are distinct from those of individual phytonutrients. In addition, the formulation of rationally combined phytonutrients is more potent and longer lasting than those of individual phytonutrients for the suppression of adipogenesis in cell cultures. The formulations of rationally combined phytonutrients disclosed herein may prevent weight gain, improve glucose tolerance, reduce blood triglyceride and LDL cholesterol, reduce liver steatosis and visceral adiposity, and/or reduce the level of inflammatory cytokines and chemokines in a subject's blood. Collectively, the formulation of rationally combined phytonutrients described herein may prevent weight gain, improve glycemic control, reduce blood lipid level, suppress liver steatosis, and/or reduce systemic inflammation, thus, lowering the risks of developing obesity-associated disease.
In one aspect, the compositions disclosed herein may include any combination of phytonutrients C1, C2, C3, C4, C5, C6, C7, C8, and C9. For example, the combination of phytonutrients may be (1) C1, C2, C3, and C5; (2) C1, C2, C3, and C7; (3) C1, C3, C4, and C5; or C1, C3, C4, and C7. In a particular embodiment, the composition may include all nine phytonutrients C1, C2, C3, C4, C5, C6, C7, C8, and C9.
The ratio of individual components may vary, for example in an embodiment comprising all nine phytonutrients, C1 may account for about 18% (e.g., about 15%, 16%, 17%, 18%, 19%, 20%, 21%) of the formulation, C2 may account for about 10% (e.g., about 7%, 8%, 9%, 10%, 11%, 12%, 13%) of the formulation, C3 may account for about 18% (e.g., about 15%, 16%, 17%, 18%, 19%, 20%, 21%) of the formulation, C4 may account for about 9% (e.g., about 6%, 7%, 8%, 9%, 10%, 11%, 12%) of the formulation, C5 may account for about 9% (e.g., about 6%, 7%, 8%, 9%, 10%, 11%, 12%) of the formulation, C6 may account for about 9% (e.g., about 6%, 7%, 8%, 9%, 10%, 11%, 12%) of the formulation, C7 may account for about 9% of the formulation, C8 may account for about 9% (e.g., about 6%, 7%, 8%, 9%, 10%, 11%, 12%) of the formulation, and C9 may account for about 9% (e.g., about 6%, 7%, 8%, 9%, 10%, 11%, 12%) of the formulation. In some formulations, the ratio of C1:C2:C3:C4:C5:C6:C7:C8:C9 is about 2:1:2:1:1:1:1:1:1.
In another aspect, the invention relates to methods of treating or preventing obesity comprising administering a therapeutically effective amount of the compositions disclosed herein.
The compositions disclosed herein may improve glycemic control, reduce blood lipid level, suppress liver steatosis, reduce systemic inflammation, and/or lower the risk of developing obesity-associated disease.
Using advanced proteomic methods, we screened hundreds of phytonutrients for their anti-adipogenic properties and identified nine phytonutrients (C1-C9) that exhibit complementary effects. We combined these nine phytonutrients into a formulation called F1 to synergize their anti-adipogenic effects. Similar to the beneficial synergistic interactions among multiple ingredients in botanical extracts, we anticipate that the interactions of nine phytonutrients provide synergistic multitargeted effects and neutralize the adverse side effects of individual phytonutrients. In cultures of human primary preadipocytes, we demonstrated that F1 was a much more potent and longer-lasting inhibitor of adipogenesis than individual phytonutrients. In an animal model of diet-induced obesity, we showed that F1 was effective at preventing weight gain, improving glucose tolerance, suppressing liver steatosis, and reducing visceral adiposity, blood lipids and systemic inflammation. Our experimental approaches, methods, conditions, and supporting data are presented in the following sections.
Obesity is characterized by increased adipose tissue mass via hypertrophy, an increase in size of existing fat cells or adipocytes, or hyperplasia, the formation of new adipocytes from precursor cells or preadipocytes [7]. Adipogenesis is the process by which preadipocytes cells commit to the adipogenic lineage, express adipogenic genes and proteins, accumulate intracellular lipid storage and become fully differentiated adipocytes. Our approach toward obesity prevention was to identify phytonutrients that interfere with the expression of six following adipogenic biomarker proteins: PPARγ, SREBP1c, FASN, PLIN1, FABP4 and β-catenin. The function and expected expression level of these biomarker proteins during adipogenesis are listed in Table 1.
Briefly, preadipocytes grown to 2 days post-confluence were induced for adipogenesis via the addition of complete differentiation media for 6 days. On day 7, complete differentiation media were replaced with maintenance media and differentiation was allowed to continue until day 14. Phytonutrients were supplemented to the complete differentiation media to screen for their anti-adipogenic effects. Out of hundreds of phytonutrients screened, nine phytonutrients (C1-C9) were selected that had complementary effects on the expression level of six adipogenic biomarker proteins aforementioned. The effects of C1-C9 on the expression level of six biomarker proteins during adipogenesis are presented in
The identities, chemical structures of C1-C9 and their half-maximal effective concentrations (EC50) for the suppression of adipogenesis are listed in Table 3.
Table legend: d0: preadipocytes in growth media; d6: differentiating adipocytes on the sixth day post-differentiation induced by complete differentiation media; C1-C9: differentiating adipocytes on the sixth day post-differentiation induced by complete differentiation media supplemented with individual C1-C9 phytonutrients; F1: differentiating adipocytes on the sixth day post-differentiation induced by complete differentiation media supplemented with F1; −: low expression level; +: high expression level.
Based on the complementary effects of C1-C9 on the expression level of six biomarker proteins, we aimed to synergize their anti-adipogenic properties via rational combinations. The three must-have objectives for the rationally designed formulations were: (1) suppression of the expression of lipogenic genes, de novo fatty acid biosynthesis, formation of lipid droplets, and fatty acids transport via negative regulation of PPARγ, FASN, PLIN1, and FABP4 expression, respectively; (2) activation of glycolysis via positive regulation of SREBP1c expression; and (3) preservation of cell-cell adhesion via inhibition of β-catenin degradation. The rationally designed formulations aimed to maintain the capability for glucose uptake and utilization of differentiating adipocytes while suppressing their capability for de novo fatty acid biosynthesis, lipid droplet formation, fatty acid transport, and morphological transformation from spindle to round shape.
Based on the complementary effects of phytonutrients on six protein biomarkers summarized in Table 2, the following four possible combinations should theoretically meet the proposed design objectives: (1) C2 & C5, (2) C2 & C7, (3) C4 & C5, and (4) C4 & C7. The combinations of two phytonutrients were inadequate for prolonged suppression of adipogenesis (
Next, the number of phytonutrients in the combinations was increased and reported substantial improvement in both anti-adipogenic potency and duration with the addition of both C1 and C3 compounds to the existing combinations of two phytonutrients. The combinations of four phytonutrients included the following: (1) C1, C2, C3, & C5; (2) C1, C2, C3, & C7; (3) C1, C3, C4, & C5; and (4) C1, C3, C4, & C7. These combinations of four phytonutrients were able to suppress cytoplasmic lipid droplet accumulation by more than 90% during the first six days of differentiation and up to 50% on the 14th day post-differentiation (
The combination of all nine phytonutrients (C1-C9) was the most effective for the suppression of adipogenesis. The formulation comprising all nine phytonutrients was named F1. Formulation F1 met all of the must-have design objectives including negative regulation of the expression of PPARγ, FASN, PLIN1, and FABP4; positive regulation the expression of SREBP1c; and preservation of the expression level of β-catenin in differentiating adipocytes (
Most importantly, F1 was a highly effective inhibitor of adipogenesis (
The therapeutic effects of F1 for obesity prevention were evaluated using a DIO mouse model (
At 16th week, glucose tolerance tests were performed for all mice after 16 hours of overnight fasting (
At 17th week, blood and tissue samples were terminally collected from all mice in this study. Blood samples were sent to IDEXX Analytics (West Sacramento, Calif.) for measurement of triglyceride, total cholesterol, high-density lipoprotein (HDL) cholesterol, and low-density lipoprotein (LDL) cholesterol. The average triglyceride levels were 76 mg/dL, 123 mg/dL, and 93 mg/dL for mice fed with a lean diet, a high-fat diet, and a high-fat diet supplemented with F1, respectively (
Terminally collected liver and visceral adipose tissues were sent to IHC World (Woodstock, Md.) for hematoxylin & eosin (H&E) histology preparation. H&E histology revealed a complete absence of any lipid droplet accumulation in liver tissues of mice fed with a lean diet, severe lipid droplet accumulation in liver tissues of mice fed with a high-fat diet, and substantially reduced level of lipid droplet accumulation in liver tissues of mice fed with a high-fat diet supplemented with F1 compared to those of mice fed with a high-fat diet alone (
Obesity is associated with systemic low-grade chronic inflammation that increases the risks for the development of metabolic disease. Using a membrane-based immunoassay to measure an array of inflammatory chemokines and cytokines, substantial increases in the presence of chemokines (CCL3, CCL4, CXCL2, and RANTES) and cytokines (IL-1F2, IL-1F3, IL-2, IL-12p70, IL16, IL17, IL23, and IL27) were reported in the blood samples of mice fed with a high-fat diet compared to those of mice fed with a lean diet (
In summary, described herein is a formulation of rationally combined phytonutrients for obesity prevention. The phytonutrients were rationally combined based on their complementary effects on the expression level of six adipogenic biomarker proteins. The formulation of rationally combined phytonutrients had unique anti-adipogenic properties that were distinct from those of individual phytonutrients. In addition, the formulation of rationally combined phytonutrients was more potent and longer lasting than those of individual phytonutrients for the suppression of adipogenesis in cell cultures. Furthermore, in a DIO animal model, dietary supplementation with the formulation of rationally combined phytonutrients prevented weight gain, improved glucose tolerance, reduced blood triglyceride and LDL cholesterol, reduced liver steatosis and visceral adiposity, and reduced the level of inflammatory cytokines and chemokines in the blood. Collectively, the formulation of rationally combined phytonutrients described herein is capable of preventing weight gain, improving glycemic control, reducing blood lipid level, suppressing liver steatosis, and reducing systemic inflammation, thus, lowering the risks of developing obesity-associated disease.
Primary human preadipocytes were isolated from subcutaneous adipose tissues of a single donor who was undergoing elective surgery. Preadipocytes were cultured and differentiated into adipocytes using a previously published protocol [8]. Briefly, preadipocytes were grown to confluence in growth media comprising Minimum Essential Medium a supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. At 2 days post-confluence, growth media were aspirated off the culture dishes and complete differentiation media were added. Complete differentiation media comprise DMEM/F12 with 18.5 mM glucose, HEPES (15 mM), NaHCO3 (25 mM), 100 units/ml penicillin, 100 μg/ml streptomycin, d-biotin (33 μM), pantothenate (17 μM), dexamethasone (100 nM), insulin (100 nM), rosiglitazone (1 μM), IBMX (0.5 mM), triiodothyronine (T3, 2 nM), and transferrin (10 μg/ml). On day three post-differentiation, complete differentiation media were replenished. On day seventh post-differentiation, complete differentiation media were replaced with maintenance media. Maintenance media comprise DMEM/F12, 100 units/ml penicillin, 100 μg/ml streptomycin, HEPES (15 mM), NaHCO3 (25 mM), d-biotin, pantothenate, insulin (10 nM), and dexamethasone (10 nM). Maintenance media were replenished on days tenth post-differentiation. Complete differentiation of preadipocytes into adipocytes were achieved on day fourteenth post-differentiation.
Three cell cultures were generated for the screening of each phytonutrient: a cell culture of preadipocytes at 2 days post-confluence (d0), a cell culture of differentiating adipocytes at 6 days post-differentiation (d6), and cell culture of differentiating adipocytes at 6 days post-differentiation in the present of C1-C9 or F1. Phytonutrients were added to the complete differentiation media on day 0 and day 3 post-differentiation. The concentrations of C1-C9 and F1 are listed in Table 3.
Approximately one million cells were incubated on ice for 10 minutes with 60 μl of lysis buffer (cat. no. 040-764, ProteinSimple, Santa Clara, Calif., USA), sonicated 4 times for 5 seconds each, mixed by rotation for 2 hours at 4° C., and centrifuged at 12,000 rpm in an Eppendorf 5430R microfuge for 20 minutes at 4° C. The supernatant was collected as the cell lysate. The total protein concentration in the cell lysate was determined with a Bradford protein assay and adjusted to a final concentration of 0.3 μg/μl with separation gradients (cat. no. Premix G2, pH 5-8, ProteinSimple, Santa Clara, Calif.) for charge-based cIEF immunoassays or to 0.4 μg/μl with denaturing buffers (cat. no. PS-ST01EZ or PS-ST03EZ, ProteinSimple) for size-based Western immunoassays.
Cell lysates in denaturing buffers were denatured at 95° C. for 5 minutes, and then transferred to assay plates (cat. no. SM-W004 or SM-W008, ProteinSimple) preloaded with blocking reagents, wash buffer, primary and secondary antibodies, and chemiluminescent substrates. Sized-based protein separation and detection in capillaries were performed using the default protocols of the Jess system (ProteinSimple). β-Actin and HSP60 were used as loading controls. All capillary Western immunoassays were performed in triplicate for each protein, and duplicate experiments were performed for each treatment condition, producing six repeated measurements per protein. Expression levels of PPARγ, SREBP1c, FASN, PLIN1, and β-catenin were detected with capillary Western immunoassays.
Cell lysates in separation gradients were loaded into 384-well assay plates (cat. no. 040-663, ProteinSimple) preloaded with primary and secondary antibodies and chemiluminescent substrates. Charge-based protein separation and detection in individual capillaries were performed using the default protocols of the NanoPro 1000 system (ProteinSimple). Hsp70 was used as the loading control. All cIEF immunoassays were performed in triplicate for each protein, and duplicate experiments were performed for each treatment condition, producing six repeated measurements per protein. Expression level of FABP4 was detected with capillary isoelectric focusing immunoassays.
The antibodies used to measure protein expression levels are listed in Table 4.
C57BL/6J mice (male, ˜10 weeks old, Jackson Lab, Bar Harbor, Me.) were divided into three groups: a group of 40 mice fed with a lean diet, a group of 40 mice fed with a high-fat diet, and a group comprising mice fed with a high-fat diet supplemented with F1. The lean diet (cat. no. TD7001, Teklad Diets, Madison, Wis.) comprised protein (25.2% by weight), carbohydrate (39.5% by weight), fat (4.4% by weight), and others (30.9% by weight, ash, fibers, others). The lean diet has 3 kcal/g, with 34% of kcal from protein, 53% of kcal from carbohydrate, and 13% of kcal from fat. The high-fat diet (cat. no. TD88137, Teklad Diets) comprised protein (17.3% by weight), carbohydrate (48.5% by weight), fat (21.2% by weight), and others (13% by weight, ash, fibers, others). The high fat diet has 4.5 g/kcal, with 15.2% of kcal from protein, 42.7% of kcal from carbohydrate, and 42% of kcal from fat. F1 was supplemented at 0.1% by weight leading to an approximately daily dose of 200 mg/kg for mice, or approximately 16 mg/kg of human equivalent dose. Mice groups were placed on their respective diets in the form of ground pellets for 17 weeks. Glucose tolerance tests using standard protocols were performed at 16th week. Terminal tissue and blood samples collection were performed at 17th week. Collected liver and visceral adipose tissues were sent to IHC WORLD (Woodstock, Md.) for histopathology analysis. Collected blood samples were sent to IDEXX Analytics (West Sacramento, Calif.) for measurement of triglyceride, cholesterol, HDL, and LDL. The Proteome Profiler Mouse Cytokine Array Kits (cat. no. ARY006, R&D Systems, Minneapolis, Minn.) were used to measure inflammatory cytokines in collected blood samples. All animal studies were performed in conformity with the Public Health Service Policy on Humane Care and Use of Laboratory Animals and with the approval of the Animal Care and Use Committee at Roseman University of Health Sciences.
The present application claims the benefit of U.S. Provisional Patent Application 63/186,993, filed May 11, 2021.
| Number | Date | Country | |
|---|---|---|---|
| 63186993 | May 2021 | US |
