METHODS AND COMPOSITIONS FOR INDUCING BROWN ADIPOGENESIS
20230190689 · 2023-06-22
Inventors
Cpc classification
A61K31/4174
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
International classification
A61K31/4174
HUMAN NECESSITIES
Abstract
This disclosure features compositions, methods, and kits for the treatment of metabolic disorders such as diabetes and obesity.
Claims
1-19. (canceled)
20. A pharmaceutical composition comprising bezafibrate as a first active ingredient, a second active ingredient selected from the group consisting of oxaprozin, zaltoprofen and ozagrel, and a pharmaceutically acceptable carrier.
21. The pharmaceutical composition of claim 20, comprising bezafibrate and oxaprozin.
22. The pharmaceutical composition of claim 21, comprising: (a) a therapeutically effective amount of bezafibrate ranging from about 25% to about 75% of the clinically approved dosage of BEZALIP® SR (bezafibrate sustained release); and (b) a therapeutically effective amount of oxaprozin ranging from about 25% to about 100% of the clinically approved dosage of DAYPRO® (oxaprozin).
23. The pharmaceutical composition of claim 22, wherein the therapeutically effective amount of bezafibrate ranges from about 100 mg to about 300 mg, and wherein the therapeutically effective amount of oxaprozin ranges from about 300 mg to about 1200 mg.
24. The pharmaceutical composition of claim 21, comprising: (a) a therapeutically effective amount of bezafibrate ranging from about 25% to about 100% of the clinically approved dosage of BEZALIP® SR; and (b) a therapeutically effective amount of oxaprozin ranging from about 25% to about 75% of the clinically approved dosage of DAYPRO®.
25. The pharmaceutical composition of claim 24, wherein the therapeutically effective amount of bezafibrate ranges from about 100 mg to about 400 mg or about 5 mg to about 500 mg, and wherein the therapeutically effective amount of oxaprozin ranges from about 300 mg to about 900 mg or about 5 mg to about 500 mg.
26. The pharmaceutical composition of claim 20, comprising bezafibrate and zaltoprofen.
27. The pharmaceutical composition of claim 20, comprising bezafibrate and ozagrel.
28. The pharmaceutical composition of claim 20, wherein said first and second active ingredients are provided in therapeutically effective amounts that, when administered to a patient, are sufficient to treat or reduce obesity.
29. The pharmaceutical composition of claim 20, wherein said first and second active ingredients are provided in therapeutically effective amounts that, when administered to a patient, are sufficient to treat or reduce type II diabetes.
30. The pharmaceutical composition of claim 20, wherein said first and second active ingredients are provided in therapeutically effective amounts capable of inducing the expression of UCP1, FABP4 (aP2), PPARγ2, mtTFA, PGC-1α, and/or COX IV in BAT progenitor cells in human skeletal muscle, in vitro, in vivo, or both.
31. The pharmaceutical composition of claim 20, wherein said composition has one or more biological activities selected from the group consisting of: (a) an increase in thermogenesis in brown adipose tissue and/or skeletal muscle tissue; (b) an increase in insulin sensitivity of skeletal muscle, white adipose tissue, or liver; (c) an increase in glucose tolerance; (d) an increase in basal respiration, maximal respiration rate, or uncoupled respiration; (e) an increase in metabolic rate; (f) a decrease in hepatosteatosis; (g) a decrease in body weight; (h) a decrease in body fat mass; (i) a decrease in plasma leptin levels; (j) a decrease in glycemia; (k) a decrease in plasma insulin levels; and (l) a decrease in insulin resistance; or a combination thereof.
32. A method of modulating a metabolic response in a subject comprising administering a composition of claim 20 to a subject in need thereof.
33. A method of treating a metabolic disorder in a subject comprising administering a composition of claim 20 to a subject in need thereof.
34. The method of claim 33, wherein the metabolic disorder is one or more of obesity, overweight, type II diabetes, insulin resistance, hyperinsulinemia, hyperglycemia, pre-diabetes, hypertension, hyperlipidemia, hepatosteatosis, fatty liver, non-alcoholic fatty liver disease, hyperuricemia, polycystic ovarian syndrome, acanthosis nigricans, hyperphagia, endocrine abnormalities, triglyceride storage disease, Bardet-Biedl syndrome, Laurence-Moon syndrome, Prader-Willi syndrome, neurodegenerative diseases, and Alzheimer's disease.
35. A method of promoting brown adipogenesis in a subject in need thereof, the method comprising administering to the subject the pharmaceutical composition of claim 20.
36. The method of claim 35, further comprising modulating a metabolic response in the subject and/or treating a metabolic disorder in the subject.
37. The method of claim 36, wherein the metabolic disorder is one or more of obesity, overweight, type II diabetes, insulin resistance, hyperinsulinemia, hyperglycemia, pre-diabetes, hypertension, hyperlipidemia, hepatosteatosis, fatty liver, non-alcoholic fatty liver disease, hyperuricemia, polycystic ovarian syndrome, acanthosis nigricans, hyperphagia, endocrine abnormalities, triglyceride storage disease, Bardet-Biedl syndrome, Laurence-Moon syndrome, Prader-Willi syndrome, neurodegenerative diseases, and Alzheimer's disease.
38. The method of claim 35, wherein the pharmaceutical composition comprises a therapeutically effective amount of bezafibrate that ranges from about 100 mg to about 400 mg, about 100 mg to about 300 mg, or about 5 mg to about 500 mg, and a therapeutically effective amount of oxaprozin that ranges from about 300 mg to about 900 mg, about 300 mg to about 1200 mg, or about 5 mg to about 500 mg.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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[0073] a. Adipocyte scores as determined by light microscopy of multilocular lipid-containing cells (on a per-well basis) at the end of the differentiation of the cells, approximately 6 to 12 days following the end of compound treatment and switch into MDM media. Concentrations are in μM. [0074] b. Expression of PPARγ2 mRNA (on a per-well basis) at the end of the differentiation of the cells, approximately 6 to 12 days following the end of compound treatment and switch into MDM media. Concentrations are in μM. Y axis units are percent of vehicle-treated cells. [0075] c. Expression of UCP1 mRNA (on a per-well basis) at the end of the differentiation of the cells, approximately 6 to 12 days following the end of compound treatment and switch into MDM media. Concentrations are in μM.
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DETAILED DESCRIPTION
[0160] PCT International Patent Application Nos. PCT/US2009/003217 and PCT/US2012/064366 previously identified the presence of cells in various tissues that are capable of differentiating into brown adipocytes. A population of such cells, referred to as BAT progenitor cells, are found to be present in skeletal muscle. PCT International Patent Application No. PCT/US2015/017392 provided assays that allow identification of agents (e.g., compounds, proteins, biologicals, and the like) that induce the expression of the UCP1 gene, promote the differentiation of BAT progenitor cells into brown adipocytes in vitro, promote the differentiation of BAT progenitor cells to brown adipocytes in vivo, or combinations of these activities.
[0161] Provided herein, in some embodiments, is a combination of two or more effective brown adipocyte recruiting agents that can have additive or synergistic effects, and thereby provide greater efficacy than a single agent alone, or reduce the toxicity associated with a single agent by permitting the use of lower doses, and thereby provide for superior product candidates for treating metabolic diseases such as obesity, type 2 diabetes, insulin-resistance, dyslipidemia, and the like. For example, the combination of rosiglitazone and BMP7 results in greater in vitro PPARγ2 and UCP1 expression that either agent alone. Multi-agent combinations, including two-compound combinations that show additive or synergistic activity on metabolic parameters of importance in metabolic disorders were identified by testing candidate brown adipocyte recruiting agents in an animal model of obesity and type 2 diabetes (DIO mice).
[0162] The present disclosure provides combinations of agents that promote the differentiation of BAT progenitor cells to brown adipocytes, both in vitro and in vivo. Combinations of effective brown adipocyte recruiters can provide greater efficacy than either compound alone. The combination of rosiglitazone and BMP7 results in greater PPARγ2 and UCP1 expression that either alone.
[0163] Accordingly, in some embodiments compositions containing the following can be used to promote the differentiation of BAT progenitor cells into brown adipocytes and/or induce the expression of UCP1, FABP4 (aP2), PPARγ2, mtTFA, PGC-1α, and/or COX IV in BAT progenitor cells in vitro, in vivo, or both: a cyclo-oxygenase inhibitor such as zaltoprofen or oxaprosin, an inhibitor of thromboxane synthetase such as ozagrel, or a pan-PPAR (α, δ, γ) ligand like bezafibrate, in compositions such as bezafibrate in combination with oxaprozin or bezafibrate in combination with zaltoprofen or bezafibrate in combination with ozagrel.
[0164] In still other embodiments, additional agents or combinations thereof that can be used to promote the differentiation of BAT progenitor cells into brown adipocytes and/or induce the expression of UCP1 include rosiglitazone or pioglitazone with oxaprozin or with zaltoprofen or ozagrel; Bezafibrate in combination with diflunisal or probenecid or tianeptine or glimepiride; Zaltoprofen in combination with glimepiride or probenecid; Probenecid in combination with tianeptine or ozagrel or diflunisal; or Tianeptine in combination with glimepiride.
[0165] In some embodiments, treatment of a subject, including a human subject, with a composition shown here results in an increase in the production of UCP1 mRNA or protein in the subject's skeletal muscle. For example, treatment of subjects with rosiglitazone can, in some embodiments, induce the appearance or differentiation of brown adipocytes in skeletal muscle, enhance expression of the UCP1 gene in existing brown adipocytes in or near skeletal muscle (between myofibers, at the surface of and/or adjacent to skeletal muscle tissue), or both. In some embodiments the appearance or differentiation of brown adipocytes in skeletal muscle can be induced in a subject suffering from a metabolic disease. The brown adipocytes can provide a glucose sink with high mitochondrial and cellular respiration and fatty acid oxidation rates, dissipating energy as heat (uncoupled oxidative phosphorylation). The subject metabolic rate can be enhanced, and a decrease in body weight can be induced. Induction of the appearance or differentiation of brown adipocytes can also yield improvements in insulin sensitivity, blood glucose homeostasis and cardiovascular disease risk factors. Brown adipocytes may further secrete factors that contribute to reaching a healthy energy balance and low body fat levels, increased insulin sensitivity and improved blood glucose homeostasis or cardiovascular health.
[0166] Accordingly, in some embodiments the agents disclosed herein, or combinations thereof, can be used for treatment of a subject, including a human subject. In some aspects, these agents may promote the differentiation of BAT progenitor cells into brown adipocytes. In other aspects these agents may induce the expression of UCP1, FABP4 (aP2), PPARγ2, mtTFA, PGC-1α, and/or COX IV in BAT progenitor cells in vitro, in vivo, or both.
[0167] In some aspects the treated metabolic disease may be obesity, overweight, type II diabetes, insulin resistance, hyperinsulinemia, hyperglycemia, pre-diabetes, hypertension, hyperlipidemia, hepatosteatosis, fatty liver, non-alcoholic fatty liver disease, hyperuricemia, polycystic ovarian syndrome, acanthosis nigricans, hyperphagia, endocrine abnormalities, triglyceride storage disease, Bardet-Biedl syndrome, Laurence-Moon syndrome, Prader-Willi syndrome, neurodegenerative diseases, and Alzheimer's disease.
[0168] In other embodiments, compositions may be used to activate isolated BAT progenitor cells that are then used for treatment of a subject, including a human subject.
[0169] The assay previously disclosed by PCT International Patent Application No. PCT/US2015/017392 can be used to identify additive or synergistic effects of 2 or more compounds that promote the differentiation of BAT progenitor cells into brown adipocytes. However, as some compounds individually induce almost complete (100%, or close to 100%) brown adipocyte differentiation, it can be challenging to detect significant differences between 2-compound combinations And individual compounds. In vivo studies in mice with obesity, insulin resistance, and impaired glucose tolerance were therefore used to complement the cell culture assays to identify 2-compound combinations producing additive or synergistic effects on any of the metabolic parameters of interest: body weight, body fat content, adipose depot mass, plasma leptin concentration, blood glucose concentration, plasma glucose concentration, plasma insulin concentration, HOMA-IR (Homeostatic Model Assessment of Insulin Resistance), glucose tolerance, or plasma triglyceride concentration.
[0170] The combinations tested in animals contain compounds having different known or suspected molecular mechanisms of action. For example, bezafibrate, a fibrate used to treat dyslipidemia, acts through a different molecular target (PPARs) and pathway than oxaprozin. Oxaprozin is an NSAID acting as an inhibitor of cyclooxygenase-1 and −2 (prostaglandin G/H synthase-1 and −2). However, it is likely that oxaprozin acts as described on body weight, body fat, etc., through a molecular target other than cyclooxygenase.
[0171] We have discovered that certain bezafibrate-containing compound combinations have in vitro and in vivo activities that suggest that these combinations may be useful for treating a patient that has been diagnosed with or is at risk of having a metabolic disorder. In the case of obesity and diabetes, for example, such administration may reduce the levels of body weight/fat and/or blood glucose.
[0172] In one example, we propose that the administration of bezafibrate and oxaprozin to a patient having a metabolic disorder such as obesity or diabetes within 14 days of each other will treat, prevent, or reduce the metabolic disorder.
[0173] In another example, the administration of bezafibrate and zaltoprofen to the patient within 14 days of each other will also treat, prevent, or reduce the metabolic disorder.
[0174] In another example, the administration of bezafibrate and ozagrel to the patient within 14 days of each other will also treat, prevent, or reduce the metabolic disorder.
[0175] The two agents are desirably administered within 10 days of each other, more desirably within seven days of each other, and even more desirably within twenty-four hours of each other, one hour of each other, or even simultaneously (i.e., concomitantly). If desired, either one of the two agents may be administered in low dosage.
[0176] In view of this discovery, the aforementioned combinations of drugs can be used in a variety of compositions, methods, and kits, as described herein.
[0177] By “treating, reducing, or preventing a metabolic disorder” is meant ameliorating such a condition before or after it has occurred. As compared with an equivalent untreated control, such reduction or degree of prevention is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90°/%, 95%, or 100% as measured by any standard technique.
[0178] A patient who is being treated for a metabolic disorder is one who a medical practitioner has diagnosed as having such a condition. Diagnosis may be performed by any suitable means, such as those described herein. A patient in whom the development of diabetes or obesity is being prevented may or may not have received such a diagnosis. One in the art will understand that patients of the disclosure may have been subjected to standard tests or may have been identified, without examination, as one at high risk due to the presence of one or more risk factors, such as family history, obesity, particular ethnicity (e.g., African Americans and Hispanic Americans), gestational diabetes or delivering a baby that weighs more than nine pounds, hypertension, having a pathological condition predisposing to obesity or diabetes, high blood levels of triglycerides, high blood levels of cholesterol, presence of molecular markers (e.g., presence of autoantibodies), and age (over 45 years of age). An individual is considered obese when their weight is 20% (25% in women) or more over the maximum weight desirable for their height. An adult who is more than 100 pounds overweight, is considered to be morbidly obese. Obesity is also defined as a body mass index (BMI) over 30 kg/m2.
[0179] By “a metabolic disorder” is meant any pathological condition resulting from an alteration in a patient's metabolism. Such disorders include those resulting from an alteration in glucose homeostasis resulting, for example, in hyperglycemia. According to this disclosure, an alteration in glucose levels is typically an increase in glucose levels by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100/relative to such levels in a healthy individual. Metabolic disorders include obesity and diabetes (e.g., diabetes type I, diabetes type II, MODY, and gestational diabetes), dyslipidemia, and endocrine deficiencies of aging.
[0180] By “reducing glucose levels” is meant reducing the level of glucose by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 1000% relative to an untreated control. Desirably, glucose levels are reduced to normoglycemic levels, i.e., between 150 to 60 mg/dL, between 140 to 70 mg/dL, between 130 to 70 mg/dL, between 125 to 80 mg/dL, and preferably between 120 to 80 mg/dL.
[0181] By “patient” is meant any animal (e.g., a human), including horses, dogs, cats, pigs, goats, rabbits, hamsters, monkeys, guinea pigs, rats, mice, lizards, Snakes, sheep, cattle, fish, and birds.
[0182] By “an amount sufficient” is meant the amount of a compound, alone or in combination with another therapeutic regimen, required to treat, prevent, or reduce a metabolic disorder such as diabetes in a clinically relevant manner. A sufficient amount of an active compound used to practice the present disclosure for therapeutic treatment of metabolic disorders varies depending upon the manner of administration, the age, body weight, and general health of the mammal or patient. Ultimately, the prescribers will decide the appropriate amount and dosage regimen. Additionally, an effective amount may be an amount of compound in the combination of the disclosure that is safe and efficacious in the treatment of a patient having a metabolic disorder such as diabetes over each agent alone as determined and approved by a regulatory authority (such as the U.S. Food and Drug Administration).
[0183] By “more effective” is meant that a treatment exhibits greater efficacy, or is less toxic, safer, more convenient, or less expensive than another treatment with which it is being compared. Efficacy may be measured by a skilled practitioner using any standard method that is appropriate for a given indication.
[0184] Compounds useful in the disclosure include those described herein in any of their pharmaceutically acceptable forms, including isomers such as diastereomers and enantiomers, salts, esters, solvates, and polymorphs thereof, as well as racemic mixtures and pure isomers of the compounds described herein.
[0185] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Methods and materials are described herein for use in the present disclosure; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
Diagnosis of Metabolic Disorders
[0186] The methods and compositions of the present disclosure are useful for treating any patient that has been diagnosed with or is at risk of having a metabolic disorder, such as obesity or diabetes. A patient in whom the development of a metabolic disorder (e.g., obesity or diabetes) is being prevented may or may not have received such a diagnosis. One in the art will understand that patients of the disclosure may have been subjected to standard tests or may have been identified, without examination, as one at high risk due to the presence of one or more risk factors.
[0187] An individual is considered obese when their Body Mass Index is 30 kg/M.sup.2 or higher. An adult with a BMI>40 is considered to be morbidly obese.
[0188] Diagnosis of other metabolic disorders may be performed using any standard method known in the art, such as those described herein. Methods for diagnosing diabetes are described, for example, in U.S. Pat. No. 6,537,806, hereby incorporated by reference. Diabetes may be diagnosed and monitored using, for example, urine tests (urinalysis) that measure glucose and ketone levels (products of the breakdown of fat); tests that measure the levels of glucose in blood; glucose tolerance tests; and assays that detect molecular markers characteristic of a metabolic disorder in a biological sample (e.g., blood, serum, or urine) collected from the mammal (e.g., measurements of Hemoglobin A1c (Hb A1c) levels in the case of diabetes).
[0189] Patients may be diagnosed as being at risk or as having diabetes if a random plasma glucose test (taken at any time of the day) indicates a value of 200 mg/dL or more, if a fasting plasma glucose test indicates a value of 126 mg/dL or more (after 8 hours), or if an oral glucose tolerance test (OGTT) indicates a plasma glucose value of 200 mg/dL or more in a blood sample taken two hours after a person has consumed a drink containing 75 grams of glucose dissolved in water. The OGTT measures plasma glucose at timed intervals over a 3-hour period. Desirably, the level of plasma glucose in a diabetic patient that has been treated according to the disclosure ranges between 160 to 60 mg/dL, between 150 to 70 mg/dL, between 140 to 70 mg/dL, between 135 to 80 mg/dL, and preferably between 120 to 80 mg/dL.
[0190] Optionally, a hemoglobin A1c (Hb A1c) test, which assesses the average blood glucose levels during the previous two and three months, may be employed. A person without diabetes typically has an HbA1c value that ranges between 4% and 6%. For every 1% increase in Hb A1c, blood glucose level increases by approximately 30 mg/dL and the risk of complications increases. Preferably, the Hb A1c value of a patient being treated according to the present disclosure is reduced to less than 9%, less than 7%, less than 6%, and most preferably to around 5%. Thus, the Hb A1c levels of the patient being treated are preferably lowered by 10%, 20%, 30%, 40%, 50%, or more relative to such levels prior to treatment.
[0191] Gestational diabetes is typically diagnosed based on plasma glucose values measured during the OGTT. Since glucose levels are normally lower during pregnancy, the threshold values for the diagnosis of diabetes in pregnancy are lower than in the same person prior to pregnancy. If a woman has two plasma glucose readings that meet or exceed any of the following numbers, she has gestational diabetes: a fasting plasma glucose level of 95 mg/dL, a 1-hour level of 180 mg/dL, a 2-hour level of 155 mg/dL, or a 3-hour level of 140 mg/dL.
[0192] The use of any of the above tests or any other tests known in the art may be used to monitor the efficacy of the present treatment. Since the measurements of hemoglobin A1c (HbA1c) levels is an indication of average blood glucose during the previous two to three months, this test may be used to monitor a patient's response to diabetes treatment.
Bezafibrate (2-(4-{2-[(4-chlorobenzoyl)amino]ethyl}phenoxy)−2-methylpropanoic acid) has the following structure:
##STR00001##
[0193] Oxaprozin (3-(4,5-Diphenyloxazol-2-yl)propionic acid) has the following structure:
##STR00002##
[0194] Zaltoprofen (2-(6-Oxo-5H-benzo[b][1]benzothiepin-3-yl)propanoic acid) has the following structure:
##STR00003##
[0195] Ozagrel ((2E)−3-{4-[(1H-imidazol-1-yl)methyl]phenyl}prop-2-enoic acid) has the following structure:
##STR00004##
EXAMPLES
[0196] Aspects of the present teachings may be further understood in light of the following examples, which should not be construed as limiting the scope of the present teachings in any way.
Example 1: Screening of Potential Modulators of Human UCP1 mRNA by Quantification Using TaqMan Real-Time PCR
[0197] CD34+ cells can be used as a tool to identify agents (small molecule compounds, proteins, biologicals, and the like) that induce the differentiation of these cells into brown adipocytes or modulate the expression of UCP1. For example, an RT-PCR based approach can be used to measure UCP1 mRNA levels which may be affected by certain agents.
[0198] This allows the identification of agents that can enhance the differentiation of CD34+ cells into brown adipocytes and/or the expression of UCP1 by enhancing the transcription of the UCP1 gene and/or by stabilizing the UCP1 transcript.
[0199] For example, a PPARγ ligand like rosiglitazone can be used to promote the differentiation of CD34+ progenitor cells into brown adipocytes (
[0200] A robust method, previously disclosed, was used for detection of CD34+ cell differentiation into brown adipocytes by simultaneously quantifying mRNA species corresponding to the brown adipocyte marker UCP1, the adipocyte marker PPARγ2, and the “housekeeping” gene cyclophilin A which was used as the internal control.
[0201] This method permits analysis of a large number of samples to identify agents that enhance the differentiation of CD34+ cells into brown adipocytes. When differentiated into brown adipocytes, CD34+ cells express much higher levels of UCP1 and PPARγ2 mRNA for a given level of cyclophilin A. UCP1 and PPARγ2 mRNA levels normalized to cyclophilin A mRNA levels give an indication of the level of differentiation of the CD34+ cells into brown adipocytes, independent of the total number of cells in the sample.
[0202] Quantification of UCP1, PPARγ2 and cyclophilin A mRNA by multiplexed TaqMan real-time PCR was thus used to quantify differentiation of the CD34+ cells into brown adipocytes.
[0203] Applicants hypothesized that some previously approved drugs may show activity in recruiting brown adipocytes (inducing the differentiation of brown adipocyte progenitor cells into brown adipocytes), and could thus be of benefit to treat obesity and diabetes. Furthermore, two agents recruiting brown adipocytes through distinct molecular mechanisms could produce effects on brown adipocyte recruitment greater than the individual agents, enhancing in vivo efficacy on parameters of metabolic health.
[0204] Materials and Methods
[0205] Screening for Brown Adipocyte Recruiters:
[0206] UCP1 is the key protein in brown adipocytes responsible for uncoupled respiration and is also highly specific for brown adipocyte differentiation. It is expressed exclusively by fully differentiated brown adipocytes and not by the CD34+ progenitor cells. We used a real-time semi-quantitative RT PCR-based assay for detection of UCP1 expression, in order to screen for brown adipocyte recruiters. The assay is multiplexed for the simultaneous detection of UCP1 (brown fat specific), PPARγ2 (adipocyte-specific and confirmatory for brown differentiation/maturation in the cells as the CD34+ cells do not become white, only brown), and cyclophilin A, for message normalization to cell number. The assay has the added advantage of allowing light microscopic visualization of the distinct morphological changes accompanying differentiation into brown adipocytes. This serves as further confirmation of differentiation. The system responds as predicted to several positive controls (including PPARγ activators like rosiglitazone, pioglitazone, and ciglitazone, and the protein BMP7) with appropriate dose-responsive behavior.
[0207] Combinations of compounds can exhibit combinatorial effects. We have shown that with CD34+ cells rosiglitazone and BMP7 both robustly increase the recruitment of brown adipocytes. Given that they may be acting via different mechanisms, they were tested together at their most effective individual concentrations to determine whether they could promote additional recruitment beyond that seen with either compound. In fact, the two together are considerably more effective than either alone (
[0208] Cell Culture
[0209] Cells were seeded at 10,000 per cm.sup.2 (48-well tissue culture, Chemglass #CLS-3500-048), cultured until confluency (1-4 days) at 37° C. in Endothelial cell growth medium-2 (EGM2) (BulletKit growth medium, Lonza #CC-3162) and until differentiation (6-12 more days). After 1-4 days in EGM2 media the cells were incubated with test agents (or positive controls) for 2-3 days (from day −3 or −2 to day 0). Rosiglitazone (1 μM) and rhBMP7 (6.3 nM) were used as reference agents (positive controls). Then (on day 0) the culture media (containing the test agents or positive controls) was removed, and minimal adipogenic medium (MDM) was added and the cells were left to differentiate of 6-12 days. The MDM media is a modification of the adipogenic media described by Rodriguez et al. [21], and contains: DMEM/Ham's F-12 50/50 Mix (3.151 g/1, 17.5 mM D-glucose, 3.651 g/l L-glutamine) (Cellgro #10-090-CV), 5 μg/ml (0.86 μM) insulin, 1 μM dexamethasone, 100 μM 3-isobutyl-1-methylxanthine, 0.2 nM 3,3′,5-triiodo-L-thyronine, 10 μg/ml (127 nM) transferrin, and 1% penicillin-streptomycin. Adipocyte scores were determined by light microscopy and defined as the number of cells, on a per well basis, that contain multilocular lipid droplets, at the end of the differentiation of the cells, approximately 6 to 12 days following the end of compound treatment and switch into MDM media.
[0210] Quantfication of UCP1 and PPARγ2 mRNA by quantitative reverse transcription, real-time PCR
[0211] Total RNA was prepared from cells using PureLink RNA Isolation Kit (Invitrogen #12183-016). Alternatively, cells were simply lyzed by freezing (−80° C.). First strand cDNA were synthesized using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, Calif.) and random primers.
[0212] Quantitative real-time PCR was performed using an Applied Biosystems StepOnePlus™ instrument, TaqMan Gene Expression Master Mix (Applied Biosystems #4369016), and custom TaqMan gene expression probes and primers for human uncoupling protein-1 “UCP1” (GenBank NM_021833) and for human peptidylprolyl isomerase A “cyclophilin A” (GenBank NM_021130). Custom TaqMan Gene Expression reagents were also developed for simultaneous measurement of peroxisome proliferator-activated receptor gamma, transcript variant 2 (PPARγ2) (GenBank NM_015869) in a multiplexed fashion (with UCP1 and cyclophilin A): UCP1 FAM-MGB probe: TCA AGG GGT TGG TAC CTT CC (SEQ ID NO: 1), sense primer: CAC TAA CGA AGG ACC AAC GG (SEQ ID NO: 2), and antisense primer: TTC CAG GAT CCA AGT CGC AA (SEQ ID NO: 3). Cyclophilin A NED-MGB probe: ACT GCC AAG ACT GAG TGG TT (SEQ ID NO: 4), sense primer: CAA ATG CTG GAC CCA ACA CA (SEQ ID NO: 5), and antisense primer: TCA CTT TGC CAA ACA CCA CA (SEQ ID NO: 6). PPARγ2 VIC-MGB probe: TCA CAA GAA ATG ACC ATG GTT G (SEQ ID NO: 7), sense primer: AGC GAT TCC TTC ACT GAT ACA C (SEQ ID NO: 8), and antisense primer: CCA GAA TGG CAT CTC TGT GT (SEQ ID NO: 9).
[0213] Cyclophilin A was used as a control to account for any variations due to the efficiency of reverse transcription. Arbitrary units were determined by normalizing target mRNA levels to cyclophilin A mRNA levels (based on Cts).
[0214] Pictures for Cell Morphology
[0215] Pictures of cells were taken using a hand-held digital camera (Nikon Coolpix 950) and inverted microscope (Nikon TMS) used for cell culture observations; images were optimized using Paint.net version 4.0 functions for Auto-Level Brightness and Contrast.
[0216] Results
[0217] We incubated CD34+ brown adipocyte progenitors with compounds (at 10 μM) contained in a collection of 1018 FDA approved drugs (FDA Approved Drug Screening Library collection, Selleckchem, Houston, Tex.). Compounds that were found to be active in the first screen (at 10 μM) were retested at concentrations 1 nM to 50 μM for dose-dependency confirmation.
[0218] Using this method the following agents were identified or confirmed to promote the differentiation of brown adipocyte progenitor cells into brown adipocytes, as indicated by expression of UCP1 and PPARγ2, in vitro: an analog of prostaglandin E1 (PGE1) such as Alprostadil, a pan-PPAR (α, δ, γ) ligand like benzofibrate (bezafibrate), a cyclooxygenase inhibitor such as Diflunisal, Zaltoprofen, Indomethacin, Acemethacin, Diclofenac, Mefenamic acid, Niflumic acid, Meclofenamate, or Oxaprozin, an inhibitor of thromboxane synthetase such as Ozagrel, a sulfonylurea such as Glimepiride or Gliquidone, or Probenecid, Tianeptine, Epalrestat (
[0219] These agents (with the possible exception of bezafibrate and indomethacin) would not have been expected to induce brown adipocyte progenitor cell differentiation, and their biological activity as brown adipocyte-recruiting agents in vitro could not have been predicted based on their respective known molecular targets or approved indications.
[0220] Except where otherwise indicated, all organic and inorganic chemicals of analytical or molecular biology grade were purchased from Cayman Chemical (including rosiglitazone, #71742), R&D Systems (including recombinant human BMP7 (rhBMP7), 100 μg/ml, 6.3 μM, #354-BP-010), Sigma Chemical Co. (St Louis, Mich.), Life Technologies (Grand Island, N.Y.). The FDA Approved Drug Screening Library collection of 1018 FDA approved drugs was purchased from Selleck Chemicals/Selleckchem (Houston, Tex.).
[0221] Using this method the following agents were identified or confirmed to promote the differentiation of BAT progenitor cells into brown adipocytes and/or induce the expression of UCP1, FABP4 (aP2), PPARγ2, mtTFA, PGC-1α, and/or COX IV in BAT progenitor cells in vitro, in vivo, or both: an analog of prostaglandin E1 (PGE1) such as Alprostadil, a pan-PPAR (α, δ, γ) ligand like benzofibrate (bezafibrate), a cyclooxygenase inhibitor such as Diflunisal, Zaltoprofen, Indomethacin, Acemethacin, Diclofenac, Mefenamic acid, Niflumic acid, Meclofenamate, or Oxaprozin, an inhibitor of thromboxane synthetase such as Ozagrel, a sulfonylurea such as Glimepiride or Gliquidone, or Probenecid, Tianeptine, Epalrestat.
Example 2: Quantification of UCP1 Protein by Fluorescence Immunohistochemistry (IHC)
[0222] Differentiation of brown adipocyte progenitors into brown adipocytes can be detected through quantification of UCP1 protein by immunohistochemistry
[0223] Culturing and differentiation of CD34+ cells into brown adipocytes were performed using adipogenic differentiation medium lacking (Minimal Differentiation Medium, MDM) or containing 1 μM rosiglitazone (Reference Differentiation Medium, RDM). After 15 days of differentiation cells were fixed with 4% Paraformaldehyde PBS pH 7.4, and incubated with a UCP1 antibody (Abcam ab23841) and Alexafluor 488 goat anti-rabbit antibody to quantify relative UCP1 levels (green) according to standard protocols. Prior to fixation of cells, nuclei were labeled with 5 μM DAPI (blue) for 10 minutes. Each treatment condition was evaluated in triplicate in a 96-well plate corresponding to approximately 360-480 cells for each data point in total. The InCell 1000 Developer Toolbox software was used to develop an automated cell detection script to measure UCP1 signal intensity, using the nuclei and cytoplasm detection algorithms. As a readout, total intensity of UCP1 signal within the cell was used, normalized to cell number.
[0224] In some embodiments, agents or combinations thereof that were identified using this technique include Famotidine, Tiapride hydrochloride, Guanfacine hydrochloride, Reserpine, Minoxidil, Spiperone, Diflunisal, Syrosingopine, Probenecid, Metformin, Thiethylperazine, Colchicine, and Felodipine.
Example 3: Detection of Brown Adipocyte Differentiation Using BODIPY
[0225] BODIPY fluorescent dye-labeled neutral lipids become incorporated in cytoplasmic lipid droplets allowing analysis of cellular fatty acid uptake and adipocyte differentiation by fluorescent cellular imaging. Cells are incubated with C1-BODIPY® 500/510 C12 (Molecular Probes #D-3823) for 3 to 6 hours before imaging on a microplate-based high-throughput, high-content, brightfield and fluorescence cellular imager and analyzer (Cyntellect Celigo® or GE Healthcare IN Cell Analyzer).
Example 4: The Combination of Bezafibrate and Oxaprozin Induces Body Weight Loss, an Increase in Insulin Sensitivity, and Improvement in Blood Glucose Homeostasis in a Mouse Model of Obesity and Diabetes
[0226] An agent that promotes the differentiation of brown adipocyte progenitor cells into brown adipocytes, i.e., an agent that recruits brown adipocytes or brown adipose tissue in vivo could be expected to cause improvement of any of the following parameters of metabolic health in obese individuals or animals: decreases in body weight, body fat content, plasma levels of leptin, glucose and insulin, and index of insulin resistance HOMA-IR (HOMA-IR=(plasma insulin [microIU/ml] x plasma glucose [mM])/22.5).
[0227] We investigated the effects of agents that were found to recruit brown adipocytes in vitro on parameters of metabolic health in obese, pre-diabetic mice. In order to uncover possible combinatorial effects between several compounds, we also investigated the effects of combinations of two agents that recruit brown adipocytes in vitro and are known, or believed to, affect different molecular targets and intracellular signaling pathways.
[0228] Materials and Methods
[0229] Animal Studies
[0230] Obesity and insulin resistance, an early stage in the development of type 2 diabetes (or pre-diabetes), was induced in C57Bl/6 mice by feeding the mice with a high fat diet (Research diet, Cat #D12492, 60/a fat kcal) for 12 weeks starting at 6 weeks of age. The mice were maintained at 22-23° C. with abundant bedding material, maintaining the animals in an environmental temperature close to their thermoneutrality, starting 2 weeks prior to the dosing period and for the full dosing period with a 12 h/12 h light/dark cycle.
[0231] Mice were dosed once per day by oral gavage (200 μl per mouse), first for 3 days with the vehicle (PBS+0.5% CMC+0.1% Tween-80) as acclimation for running into the study, then with either vehicle alone or testing material dissolved in the vehicle for 28 to 55 days (4 to 8 weeks).
[0232] Body weight was recorded every 3 days, body composition (fat and lean mass with EchoMRI) was assessed at the end of the study (University of Cincinnati Mouse Metabolic Phenotyping Center), blood glucose level (measured with a glucometer) was monitored 3-5 days before (baseline) and at the end of the dosing period.
[0233] At the end of the dosing period, animals were fasted for 6 hours and euthanized by CO.sub.2, blood was collected, and plasma was isolated and saved at −20° C. Plasma glucose, insulin and leptin levels were assessed 3-5 days before (baseline) and at the end of the dosing period (mice were fasted for 6 hours before all plasma collection) (University of Cincinnati Mouse Metabolic Phenotyping Center). Insulin sensitivity was determined using the homeostasis model assessment of insulin resistance (HOMA-IR) [38]. HOMA-IR=(plasma insulin [microIU/ml] x plasma glucose [mM])/22.5.
[0234] The mice were maintained on a high fat diet (60% calories from fat) throughout the study. An overnight fast was performed the last night of the study in preparation for glucose tolerance testing.
[0235] Animal Study with Indirect Calorimetry (Bezafibrate (60)+Oxaprozin (50))
[0236] Diet-induced obesity mice: C57Bl/6 males mice were fed with a high fat diet (Research diet, Cat #D12492, 60% fat kcal) for 12 weeks starting at 6 weeks of age. The mice were maintained at 22-23° C. with abundant bedding material, maintaining the animals in an environmental temperature close to their thermoneutrality, starting 2 weeks prior to the dosing period and for the full dosing period with a 12 h/12 h light/dark cycle.
[0237] Mice were dosed once per day by oral gavage (200 μl per mouse) with either vehicle alone (PBS+0.5% CMC+0.1% Tween-80) or the combination of bezafibrate at 60 mg/kg+oxaprozin at 50 mg/kg dissolved in the vehicle for 24 days.
[0238] Body weight was recorded daily, body composition (fat and lean mass with EchoMRI) was assessed at baseline (before dosing) and at the end of the dosing period (University of Michigan Mouse Metabolic Phenotyping Center).
[0239] At the end of the dosing period, animals were fasted for 6 hours and euthanized by CO.sub.2, blood was collected, and plasma was isolated and saved at −20° C. Plasma glucose, insulin and leptin levels were assessed at baseline and at the end of the dosing period (mice were fasted for 6 hours before all plasma collection) (University of Michigan Mouse Metabolic Phenotyping Center).
[0240] After 24 days of dosing the mice were transferred to calorimetry chambers (1 mouse per cage) and maintained 3 days at 30° C., a temperature close to mouse thermoneutrality. During the 3 days in the calorimetry chambers (TSE Systems Phenomaster) the following parameters were measured: energy expenditure by indirect calorimetry (using oxygen consumption rate (VO2), carbon dioxide production (VCO2), respiratory quotient (RQ)), food intake, locomotor activity, ambulatory activity and distance traveled (beam breaks).
[0241] The first day in the calorimetric chamber was used as acclimation period (parameters measured were not used in the data analysis). The values obtained during the second day (over 24 h) in the calorimetric chamber were used as non-stimulated/resting values. On the third day in the calorimetric chamber we measured whole-body non-shivering thermogenesis of the mice, a measure of whole-body brown adipose tissue capacity [39] using a single injection of 1 mg/kg norepinephrine.
[0242] Statistical Analysis
[0243] Data from cell culture studies are expressed as mean f SEM. Significances were evaluated using the paired or unpaired Student's t-test using online GraphPad QuickCalcs t test calculator (GraphPad Software, San Diego, Calif.). Significances were set at p<00.05.
[0244] Data from in vivo mouse studies are presented as mean f SEM. Significances were evaluated using the paired or unpaired Student's t-test, 2-way ANOVA or 1-way ANOVA with Bonferroni's or Dunnett's multiple comparison test using GraphPad Prism version 7 or 8 (GraphPad Software, San Diego, Calif.). Significances were set at p<0.05, with *: p<0.05 vs. Vehicle, **: p<0.01 vs. Vehicle, ***: p<0.001 vs. Vehicle.
[0245] Results
[0246] In the first study reported here we tested the effects of bezafibrate (60 mg/kg), oxaprozin (50 mg/kg) and the combination of bezafibrate (60)+oxaprozin (50) in DIO mice over 56 days of dosing on parameters of metabolic health.
[0247] These data were generated using bezafibrate at approximately half of the recommended dosing level in humans being treated for hyperlipidemia, allometrically scaled for mice according to current FDA guidance. Oxaprozin was used at approximately one-quarter of the scaled recommended human dose in our animal studies.
[0248] Treatment of DIO mice with bezafibrate (60 mg/kg) or with oxaprozin (50 mg/kg) over 56 days, compared to treatment with vehicle, induced significant decreases in body weight (
[0249] In addition, we found that the combination of bezafibrate (60 mg/kg)+oxaprozin (50 mg/kg) caused further decreases in all these metabolic parameters. In fact, the weight loss inducing effect of the combination (
[0250] We found that bezafibrate+oxaprozin produced highly significant reduction in body weight in DIO mice over 56 days (p=2.6×10-10). The observed effect was synergistic (greater than the sum of the effects of the 2 individual agents vs vehicle (p=0.043). P-values for all figures from this 56 day study of bezafibrate (60)+oxaprozin were determined based on two-sided Z-tests with standard errors estimated via the bootstrap method. To assess synergistic effects, we tested whether the percent change from baseline with the combination is higher than the sum of the percent changes from baseline of the 2 individual drugs. A log-transformation was used for Z-tests on group differences for all end of study measurements. These data were generated using bezafibrate at approximately half of the recommended dosing level in humans being treated for hyperlipidemia, allometrically scaled for mice according to current FDA guidance. Oxaprozin was used at approximately one-quarter of the scaled recommended human dose in our animal studies.
[0251] Bezafibrate is known to lower plasma levels of triglycerides and LDL cholesterol through activation of PPARα in the liver, and is used to treat dyslipidemia and increased cardiovascular risk. Oxaprozin is a non-steroid agent anti-inflammatory (NSAID) agent that inhibits cyclooxygenases 1 and 2, and is used to treat rheumatoid arthritis.
[0252] Based on this, the synergistic effects of the combination of bezafibrate and oxaprozin on body weight, body fat content and plasma leptin levels could not have been anticipated based on the known effects of these individual agents. It is possible that the known molecular targets of these two agents mediate the brown adipocyte-recruiting (and metabolic) effects of these agents. Alternatively, it is possible that one or both of these agents affect brown adipocyte recruitment and metabolic health through other molecular targets.
[0253] Using this general method, the following, comprising combinations of certain existing drugs, were identified or confirmed to induce body weight loss, an increase in insulin sensitivity, and improvement in blood glucose homeostasis in a mouse model of obesity and diabetes: Bezafibrate in combination with oxaprozin or zaltoprofen or ozagrel or diflunisal or probenecid or tianeptine or glimepiride, Rosiglitazone or Pioglitazone in combination with oxaprozin or zaltoprofen or ozagrel, Zaltoprofen in combination with glimepiride or probenecid, Probenecid in combination with tianeptine or ozagrel or diflunisal, and Tianeptine in combination with glimepiride.
[0254] In a second study the effects of the combination of bezafibrate (60 mg/kg)+oxaprozin (50 mg/kg) was investigated in DIO mice over 24 days of dosing on parameters of metabolic health and energy expenditure (metabolic rate) at rest and after maximal sympathetic stimulation with norepinephrine. The aim of this study was to assess the energy expenditure and whole-body thermogenic capacity of the mice after 24 days of dosing with bezafibrate (60)+oxaprozin (50). The purpose was to assess whether the mice treated with bezafibrate (60)+oxaprozin (50) showed evidence of increased brown adipose mass (and capacity) compared to the mice given the vehicle.
[0255] Treatment of DIO mice with the combination of bezafibrate (60 mg/kg)+oxaprozin (50 mg/kg) over 24 days, compared to treatment with vehicle, induced significant decreases in body weight (
[0256] In addition, energy expenditure assessment by indirect calorimetry showed that the mice treated with the combination of bezafibrate (60)+oxaprozin (50) had significantly higher energy expenditure (over 24 h, unstimulated,
[0257] Furthermore, the thermogenic (energy expenditure) response to the norepinephrine injection was significantly higher in the mice treated with the combination of bezafibrate (60)+oxaprozin (50) vs. the mice treated with vehicle (
[0258] There was no significant difference between the groups of mice in food intake, locomotor activity, ambulatory activity and distance traveled.
[0259] These data clearly demonstrate that, in DIO mice, the combination of bezafibrate (60)+oxaprozin (50) induces a recruitment, an increase, in thermogenic brown/brite/beige adipose tissue, which results in enhanced energy expenditure (metabolic rate) and an improvement in parameters of metabolic health (decreases in body weight, fat mass, plasma leptin, glucose and insulin levels, and index of insulin resistance HOMA-IR).
[0260] The section headings and subheadings used in this specification are for organizational purposes only and are not to be construed as limiting the subject matter described in any way. Further, while the present teachings are described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents as will be appreciated by those of skill in the art.
Other Embodiments
[0261] Various aspects of the present disclosure may be used alone, in combination, or in a variety of arrangements not specifically discussed in the embodiments described in the foregoing and is therefore not limited in its application to the details and arrangement of components set forth in the foregoing description or illustrated in the drawings. For example, aspects described in one embodiment may be combined in any manner with aspects described in other embodiments.
[0262] Use of ordinal terms such as “first,” “second,” “third,” etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements.
[0263] Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” or “having,” “containing,” “involving,” and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.
[0264] The present disclosure provides among other things novel compositions capable of recruiting brown adipocytes in vitro and in vivo. While specific embodiments of the subject disclosure have been discussed, the above specification is illustrative and not restrictive. Many variations of the disclosure will become apparent to those skilled in the art upon review of this specification. The full scope of the disclosure should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.
Incorporation by Reference
[0265] All publications, patents and patent applications referenced in this specification are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent or patent application were specifically indicated to be so incorporated by reference.
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