(12) Patent Application Publication (10) Pub. No.: US 2011/0038948 A1 Zemel Et Al

US 2011 0038948A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2011/0038948 A1 Zemel et al. (43) Pub. Date: Feb. 17, 2011

(54) METHODS OF REDUCING THE (60) Provisional application No. 60/723,042, filed on Oct. PRODUCTION OF REACTIVE OXYGEN 3, 2005, provisional application No. 60/787,819, filed SPECIES AND METHODS OF SCREENING on Mar. 31, 2006. ORDENTIFYING COMPOUNDS AND Publication Classification COMPOSITIONS THAT REDUCE THE PRODUCTION OF REACTIVE OXYGEN (51) Int. Cl. SPECIES A633/42 (2006.01) A2.3L I/304 (2006.01) (75) Inventors: Michael B. Zemel, Knoxville, TN GOIN 33/53 (2006.01) (US); Xiaocun Sun, Knoxville, TN A633/10 (2006.01) (US) A633/14 (2006.01) A633/00 (2006.01) Correspondence Address: A6IP39/00 (2006.01) SALWANCHIK LLOYD & SALWANCHIK (52) U.S. Cl...... 424/602: 426/2: 435/7.21; 424/715; A PROFESSIONAL ASSOCATION 424/678; 424/722 PO Box 142950 GAINESVILLE, FL 32614 (US) (57) ABSTRACT The Subject application provides methods of identifying com (73) Assignee: UNIVERSITY OF TENNESSEE pounds, combinations of compounds, compositions, and/or RESEARCH FOUNDATION, combinations of compositions that are suitable for reducing KNOXVILLE, TN (US) the production of reactive oxygen species (ROS) in an indi vidual with the proviso that said compound, combination of (21) Appl. No.: 12/893,003 compounds, composition, or combination of compositions is not a dietary material containing calcium or dietary calcium. Filed: Sep. 29, 2010 Also provided in the subject application are methods of treat (22) ing diseases or disorders associated with ROS production and methods of reducing ROS production in an individual with Related U.S. Application Data the proviso that said compound, combination of compounds, (62) Division of application No. 1 1/542,703, filed on Oct. composition, or combination of compositions is not a dietary 3, 2006. material containing calcium or dietary calcium.

2 * (p<0.015) 250 . * (b<0.01 *(p<0.01) O wild-type mice Transgenic mice

Baseline After Diet Patent Application Publication Feb. 17, 2011 Sheet 1 of 38 US 2011/0038948 A1

2 *(p<0.015) O wild-type mice Transgenic mice

Baseline After Diet

FIG. 1

*(p<0.001)

wild-type mice Transgenic mice

Baseline After Diet

FIG. 2 Patent Application Publication Feb. 17, 2011 Sheet 2 of 38 US 2011/0038948 A1

12------k (n=0.04 Body Weight aa (p ) l Body fat weight s 9 ------ms------E. dS 6– - - - it (p<0.001) . . . . .

3 -

ol------Basal diet High calcium diet

Fs so 5 g 3 E C

Basal diet High calcium diet

FIG. 4 Patent Application Publication Feb. 17, 2011 Sheet 3 of 38 US 2011/0038948 A1

(p<0.001) 6 O O ------... w --- Ali in Viceral fat Subcutaneous fat

4 O O ---

Basal diet High calcium diet

FIG. 5

* (p<0.012 - p - --- ) ----- O Viceral fat Subcutaneous fat

Basal diet High calcium diet

FIG. 6 Patent Application Publication Feb. 17, 2011 Sheet 4 of 38 US 2011/0038948 A1

* (p<0.001) O Viceral fat Subcutaneous fat

Basal diet High calcium diet

* (p<0.001) # (p<0.001) 3 ------rom-W-m------

32 Viceral fat Subcutaneous fat isA. 2 - - O P

1 ---

Basal diet High calcium diet

FIG. 8 Patent Application Publication Feb. 17, 2011 Sheet 5 of 38 US 2011/0038948 A1

Basal diet High calcium diet

FIG 10 Patent Application Publication Feb. 17, 2011 Sheet 6 of 38 US 2011/0038948 A1

- - O Viceral fat --- Subcutaneous fat

Basal diet High calcium diet

FIG 11

600 D attocopherolControl

HO, -- -- GDP Nifedipine Patent Application Publication Feb. 17, 2011 Sheet 7 of 38 US 2011/0038948 A1

80 Control 2 ot tocopherol A 60 C

S b .9

40 d c r 5. C ?y

O ------Control H.O, HO, GDP Nifedipine FIG. 13

O Wild type UCP2 transfected

0.8 -

0.6

0.4

(). L. L. L. Control H2O, HO H2O, -- -- GDP Nifedipine

FIG. 14 Patent Application Publication Feb. 17, 2011 Sheet 8 of 38 US 2011/0038948 A1

600 a 450 si is 300

SO. C 150

Control HO -- dittocopherol

< 280 O) C .9 60 d 3 b a 40 al a. C 3.?y 20

O |- unit — ------Control Glucose Glucose Glucose Glucose - -- 1,25D GDP Nifedipine

FIG. 16 Patent Application Publication Feb. 17, 2011 Sheet 9 of 38 US 2011/0038948 A1

300

Control Glucose Glucose -- at tocopherol

FIG. 1 7

DUCP2 transfected 2 O c Wild-type 1. 6

1 2 b

4 al d

Ot - Control Glucose Glucose Glucose Glucose ------Nifedipine 1,25D GDP

FIG. 18 Patent Application Publication Feb. 17, 2011 Sheet 10 of 38 US 2011/0038948 A1

8 DUCP2 transfected wild-type 12 6 . Control Glucose Glucose Glucose Glucose ------Ni?edipine 1, 25D GDP

FIG. 19

600

4 5 O C

3 O O d 150 - 5

Control Glucose Glucose Glucose Glucose -- -- 1,25D GDP Nifedipine FIG. 20 Patent Application Publication Feb. 17, 2011 Sheet 11 of 38 US 2011/0038948 A1

3 C

3 s. 2 < : d S 3, 1 b

Control Glucose Glucose Glucose Glucose ------Nifedipine 1, 25D GDP

2 s

asal diet High-Cadiet Basal diet High-Cadiet

Subcutaneous fat Wisceral fat FIG.22A Patent Application Publication Feb. 17, 2011 Sheet 12 of 38 US 2011/0038948 A1

Basal diet High-Cadiet Basal diet High-Cadet

Subcutaneous fat Wiscerafat FIG. 22B

Basal diet High-Cadiet ------Visceral fat FIG. 23A Patent Application Publication Feb. 17, 2011 Sheet 13 of 38 US 2011/0038948A1

Basal diet High-Ca diet

----- N- --- Visceral fat FIG. 23B

Basal diet High-Ca diet --- Soleus muscle FIG. 23C Patent Application Publication Feb. 17, 2011 Sheet 14 of 38 US 2011/0038948 A1

: s z

Control 1, 25 (OH). D. Nifedipine 1, 25 (OH)D -- Nife dipine FIG. 24A

Cotrol 1, 25(OH). D. Nifedipine 1, 25 (OH) D.

Nifedipine FIG. 24B

Basal diet High-Ca Diet

FIG. 24C Patent Application Publication Feb. 17, 2011 Sheet 15 of 38 US 2011/0038948 A1

D. Nifedipine 1,25(OH),D, -- Nifedipine FIG. 25A

Control 1,25(OH),D, Nifedipine 1,25(OH),D, -- Nifedipine FIG. 25B Patent Application Publication Feb. 17, 2011 Sheet 16 of 38 US 2011/0038948A1

: O.8 .4.

Control l, 25(OH),D, Nifedipine 1,25(OH),D, -- Nifedipine FIG. 25C

O Control 1, 25OH).D. Nifedipine 1,25OH),D -- Ni?ed pine FIG. 25D Patent Application Publication Feb. 17, 2011 Sheet 17 of 38 US 2011/0038948 A1

Control attocopherol HO, HO, -- uttocopherol FIG. 26A

Control attocopher ol HO, H, O,

attocophero FIG. 26B Patent Application Publication Feb. 17, 2011 Sheet 18 of 38 US 2011/0038948 A1

A. an f al

Control attocopherol HO, HO

-- attocopherol FIG. 26C

() o l >

Control Calcitriol Calcitriol Nifedipine -- Nifedipine FIG. 27A Patent Application Publication Feb. 17, 2011 Sheet 19 of 38 US 2011/0038948A1

O.6 () S2 0.4 N O30.2

Control Calcitrio Calcitriol Nifedipine Nifedipine

FIG. 27B

3 ... ------

O ------Control Calcitrio Calcitriol -- Nifedipine

FIG. 28A Patent Application Publication Feb. 17, 2011 Sheet 20 of 38 US 2011/0038948 A1

2 c) to 1.5 N O5 O Control Calcitrio Calcitriol Nifedipine

FIG. 28B

OControl

Co-Culture

Control Calcitriol Calcitriol Calcitriol -- - Nifedipine DNP FIG. 29 Patent Application Publication Feb. 17, 2011 Sheet 21 of 38 US 2011/0038948 A1

Control Calcitrio Calcitriol Calcitrio r -- Nifedipine DNP

FIG. 30 Patent Application Publication Feb. 17, 2011 Sheet 22 of 38 US 2011/0038948 A1

4. -- --- k Control 3 Co-Culture (f) OO

is 2 - a 1 -

O - ---

Control Calcitrio Calcitrio Calcitrio -- Nifedipine DNP

FIG. 31

2 3 4 5 6 8 9 O

Control FIG. 32A Patent Application Publication Feb. 17, 2011 Sheet 23 of 38 US 2011/0038948A1

1 2 3 4 5 6 78 910

Calcitrio-treated FIG. 32B

Schematic diagram of antibody array FIG. 32C Patent Application Publication Feb. 17, 2011 Sheet 24 of 38 US 2011/0038948A1

4. Basa 12 - Cacierie

FIG. 32D Patent Application Publication Feb. 17, 2011 Sheet 25 of 38 US 2011/0038948 A1

1 2 3 4 5 6 78 910 A. B C D E

Control G. 33A

B C D E

Calcitriol-treated FIG. 33B Patent Application Publication Feb. 17, 2011 Sheet 26 of 38 US 2011/0038948 A1

* Incs NF C GM-CSFP-10

Schematic diagram of antibody array

FIG. 33C Patent Application Publication Feb. 17, 2011 Sheet 27 of 38 US 2011/0038948A1

o Basa

120 acilig 1. 8

FIG. 33D Patent Application Publication Feb. 17, 2011 Sheet 28 of 38 US 2011/0038948 A1

i: Coltrol CQ-Cite

Control Calcitrio Calcitrio Calcitriot - Nifedipine DNP

FIG. 34

D Control 8

Co-Culture

Control Calcitrio Calcitrio Calcitrio Nifedipine DNP FIG. 35 Patent Application Publication Feb. 17, 2011 Sheet 29 of 38 US 2011/0038948A1

Control Co-Culture

Control Calcitriol Calcitriol Calcitriol an Nifedipine DNP FIG. 36

P - OO12 basal vs. calcium p = 0.285 basal vs. milk. p = 0.005

Calcium w8. milk. p = 0.018 Patent Application Publication Feb. 17, 2011 Sheet 30 of 38 US 2011/0038948 A1

P = OOO2 basaws. Calcium. p = 0.038 basal wS. milk. p = 0.001 calcium vs. milk. p = 0.018 i 24

P - O.O.5. basal vs. calcium p = 0.290 basal vs. milk. p = 0.060

Calcium wS. milk p = 0.20 Patent Application Publication Feb. 17, 2011 Sheet 31 of 38 US 2011/0038948 A1

P - OOO1 basal vs. Calcium. p = 0.178 basal vs. milk p < 0.001 Calcium vs. milk. p = 0.002

o E g S. g will

Basal High-Ca Mik FIG. 40

Plasma 1, 25 D P & OOO basal vs. calcium. p = 0.002 basal vs. milk p < 0.001

calcium vs. milk. p = 0.059 Patent Application Publication Feb. 17, 2011 Sheet 32 of 38 US 2011/0038948 A1

P = OOO1 basal vs. calcium p = 0.002 basal vs. milk p = 0.001 calcium vs. milk. p = 0.030

Basal High-Ca Milk FIG. 42

Adipose (perirenal fat) P & OOO gene expression basal vs. Calcium: p < 9001 basaws milk p < 0.001

calcium vs. milk. p = 0.058 Patent Application Publication Feb. 17, 2011 Sheet 33 of 38 US 2011/0038948 A1

P & OOO1 Plasma MDA basal vs. Calcium p = 0.001 basal vs. milk. p < 0.001 calcium vs. milk p = 0.039

400

300

O * OO | | | | | | Basal High-Ca Milk FIG. 44

Adipose (perirenal fat) NFOOO1 gene expression basal vs. calcium. p = 0.001 basal vs. milk p < 0.001

calcium vs. milk. p = 0.078 Patent Application Publication Feb. 17, 2011 Sheet 34 of 38 US 2011/0038948 A1

Adipose (perirenal fat) 92 EC OOO6 gene expressionWirrae ei basalaSal vs.VS. milk.CaFCum. p = p0.001 = U.UU

calcium vs. milk. p = 0.217

Basal High-Ca Miik FIG. 46

Adipose (perirenal fat) P 0001 basal WS. Calcium p = 0.003 gene expression basal vs. milk p < 0.001 r calcium vs. milk. p = 0.183

gi1 d

*...w: Patent Application Publication Feb. 17, 2011 Sheet 35 of 38 US 2011/0038948 A1

Adipose cytokine release 0.130. Basaws. Calcium 0.118 Basal WS. milk. O.O.59 Calcium wS. milk. O.25 OO1 S OOO8

Basal High-Ca Mik FIG. 48

Adipose cytokine release p = 0.008 Basaws. Calcium. O.033 BasayS. milk. OOO3 Calcium WS, Mik. O.122

Patent Application Publication Feb. 17, 2011 Sheet 36 of 38 US 2011/0038948 A1

Adipose:- - - - (perirenal- - - - fat)Y pbasal = 0.001 WS. Calcium. O - O.OOT gene expression basal vs. milk. p-0.001 E.calcium vS. milk. p = 0.073

O123A. Basal High-Ca Milk FIG. 5 O

P & OOO1 basal vs. calcium p = 0.001 basal vs. milk. p < 0.001

calcium vs. milk p = 0.068 Patent Application Publication Feb. 17, 2011 Sheet 37 of 38 US 2011/0038948A1

P & OOO basal vs. calcium p < 0.001 basal wS. milk p < 0.001 calcium vs. milk. p = 0.07

Basal High-Ca Milk FIG. 52

k 2. f

et Cie ------

hifedipine ------

Adiponectin ------a------

Cacific ------s: e. as - Patent Application Publication Feb. 17, 2011 Sheet 38 of 38 US 2011/0038948A1

3. 2 s E 5 s S. s

leucire ------Nifedipine - - - - . . . Adiponectin ------

Calcitrio -- - - ... -- --

E s E. t s

Leucire Mifedipine -- - - - Adiponectin ------Calcitrio - US 2011/0038948 A1 Feb. 17, 2011

METHODS OF REDUCING THE 0006 FIG. 2: Adipose NADPH oxidase expression in PRODUCTION OF REACTIVE OXYGEN wild-type and aP2-agouti transgenic mice. Values are pre SPECIES AND METHODS OF SCREENING sented as meant SEM, n=6. ORDENTIFYING COMPOUNDS AND 0007 FIG. 3: Effect of dietary calcium on body weight COMPOSITIONS THAT REDUCE THE and fat pads weight in aP2-agouti transgenic mice. Values are PRODUCTION OF REACTIVE OXYGEN presented as meant SEM, n=10. SPECIES 0008 FIG. 4: Effect of dietary calcium on fasting blood glucose in aP2-agouti transgenic mice. Values are presented CROSS-REFERENCE TO RELATED as meaniSEM, n=10. * indicates significant difference from APPLICATIONS the basal diet, p<0.05. 0009 FIG. 5: Effect of dietary calcium on adipose intrac 0001. This application is a divisional of U.S. Ser. No. ellular ROS production in aP2-agouti transgenic mice. Values 1 1/542,703, filed Oct. 3, 2006 which claims the benefit of are presented as meant-SEM, n=10. U.S. Provisional Application Ser. No. 60/723,042, filed Oct. (0010 FIG. 6: Effect of dietary calcium on adipose 3, 2005 and U.S. Provisional Application Ser. No. 60/787, NADPH oxidase expression in aP2-agouti transgenic mice. 819, filed Mar. 31, 2006. Values are presented as mean-SEM, n=10. 0011 FIG. 7: Effect of dietary calcium on adipose intrac BACKGROUND OF THE INVENTION ellular calcium (ICati) in aP2-agouti transgenic mice. Val 0002 Reactive oxygen species (ROS) production is ues are presented as meaniSEM, n=10. increased in obesity and diabetes (Furukaw et al., 2004: (0012 FIG. 8: Effect of dietary calcium on adipose UCP2 Atabek et al., 2004; Lin et al., 2005; Sonta et al., 2004). It has expression in aP2-agouti transgenic mice. Values are pre been postulated that hyperglycemia and hyperlipidemia, key sented as meant SEM, n=10. clinical manifestations of obesity and diabetes, may promote (0013 FIG. 9: Effect of dietary calcium on soleus muscle ROS production through multiple pathways (Inoguchi et al., UCP3 expression in aP2-agouti transgenic mice. Values are 2000; Shangarietal., 2004; Chung et al., 2003). ROS are also presented as meant-SEM, n=10. * indicates significant differ associated with a variety of diseases or disorders. For ence from the basal diet, p<0.05. example, ROS are associated with cataracts, heart disease, 0014 FIG.10: Effect of dietary calcium on soleus muscle cancer, male infertility, aging, and various neurodegenerative NADPH oxidase in aP2-agouti transgenic mice. Values are diseases such as Alzheimer's disease, amyotrophic lateral presented as meant-SEM, n=10. * indicates significant differ Sclerosis, Parkinson's disease, multiple Sclerosis and aging. ence from the basal diet, p<0.05. 0003 Previous studies from have demonstrated an anti (0015 FIG. 11: Effect of dietary calcium on adipose 11 B obesity effect of dietary calcium, with increasing dietary cal HSD expression in aP2-agouti transgenic mice. Values are cium inhibiting lipogenesis, Stimulating lipolysis and ther presented as meant SEM, n=10. mogenesis and increasing adipocyte apoptosis (Zemel, (0016 FIG. 12: Effect of HO, on DNA synthesis in cul 2005a). These effects are mediated by suppression of 1C. tured 3T3-L1 adipocytes. Adipocytes were treated with either 25-(OH),D,-induced stimulation of Ca" influx and suppres HO, (100 nmol/L) or C-tocopherol (1 umol/L), combined sion of adipose UCP2 gene expression (Shi et al., 2001; Shiet with or without GDP (100 umol/L) or nifedipine (10 umol/L) al., 2002). Further, ROS production is modulated by mito for 48 hours. Data are expressed as mean+SE (n-6). Different chondrial uncoupling status and cytosol calcium signaling, letters above the bars indicate a significant difference at level and that 25(OH),D, regulates ROS production in cultured of p-0.05. murine and human adipocytes (Sun et al., 2005). (0017 FIG. 13: ROS production in cultured 3T3-L1 adipo cytes. Adipocytes were treated with either HO (100 nmol/L) BRIEF DESCRIPTION OF THE INVENTION or C-tocopherol (1 umol/L), combined with or without GDP 0004. The subject application provides methods of identi (100LLmol/L) or nifedipine (10 umol/L) for 48 hours. Data are fying compounds, combinations of compounds, composi expressed as meant-SE (n-6). Different letters above the bars tions, and/or combinations of compositions that are Suitable indicate a significant difference at level of p-0.05. for reducing the production of reactive oxygen species (ROS) (0018 FIG. 14: Mitochondrial potential in cultured wild in an individual with the proviso that said compound, com type 3T3-L1 adipocytes and UCP2 transfected 3T3-L1 adi bination of compounds, composition, or combination of com pocytes. Adipocytes were treated with either HO (100 positions is not a dietary material containing calcium or nmol/L) or C-tocopherol (1 umol/L), combined with or with dietary calcium. Also provided in the Subject application are out GDP (100 umol/L) or nifedipine (10 umol/L) for 48 methods of treating diseases or disorders associated with hours. Data are expressed as meaniSE (n=6). ROS production and methods of reducing ROS production in 0019 FIG. 15: Intracellular calcium (Cali) in cultured an individual with the proviso that said compound, combina 3T3-L1 adipocytes. Adipocytes were treated with either tion of compounds, composition, or combination of compo H2O (100 nmol/L) or HO (100 nmol/L) plus C.-tocopherol sitions is not a dietary material containing calcium or dietary (1 umol/L) for 4 hours. Data are expressed as mean+SE (n-6). calcium. Different letters above the bars indicate a significant differ ence at level of p<0.05. BRIEF DESCRIPTION OF THE FIGURES (0020 FIG. 16: ROS production in cultured 3T3-L1 adipo cytes. Adipocytes were treated with either glucose (30 mmol/ 0005 FIG. 1: Adipose intracellular ROS production in L) or glucose (30 mmol/L) plus nifedipine (10 umol/L), or wild-type and aP2-agouti transgenic mice. Values are pre glucose (30 mmol/L) plus GDP, or glucose (30 mmol/L) plus sented as meantSEM, n=6. 1C, 25-(OH)D for 48 hours. Data are expressed as US 2011/0038948 A1 Feb. 17, 2011

meantSE (n-6). Different letters above the bars indicate a tively for 48 h. Data are normalized to 18s expression. Values significant difference at level of p-0.05. are presented as mean-SEM, n=6. Means with different letter 0021 FIG. 17: Cati in cultured 3T3-L1 adipocytes. differ, p<0.005. Adipocytes were treated with either glucose (30 mmol/L) or 0030 FIG. 26A shows Adiponectin expression and FIG. glucose (30 mmol/L) plus C-tocopherol (1 Limol/L) for 4 26B shows IL-15 expression in differentiated 3T3-L1 adipo hours. Data are expressed as meant-SE (n=6). Different letters cytes. Adipocytes were treatment with 100 nmol/L H.O. 1 above the bars indicate a significant difference at level of p<0.05. umol/L Cittocopherol, and 100 nmol/L H.O. 1 umol/L 0022 FIG. 18: Expression ratio of NADPH oxidase to 18s Cittocopherol respectively for 48 h. Data are normalized to in cultured 3T3-L1 adipocytes. Adipocytes were treated with 18s expression. Values are presented as meant-SEM, n=6. either glucose (30 mmol/L) or glucose (30 mmol/L) plus Means with different letter differ, p<0.05. FIG. 26C: There nifedipine (10 umol/L), glucose (30 mmol/L) plus GDP, or was no direct effect of ROS on. IL-15 expression; however, glucose (30 mmol/L) plus 1, 25-(OH)2D for 48 hours. Data addition of Cittocopherol markedly increased IL-15 by 2.2- are expressed as meaniSE (n-6). Different letters above the fold as compared to HO-treated cells (P=0.043). bars indicate a significant difference at level of p-0.05. 0031 FIG. 27 demonstrates that calcitriol increased MIF 0023 FIG. 19: Expression ratio of UCP2 to 18s in cultured (FIG. 27A) and CD14 (FIG. 27B) expression in human adi 3T3-L1 adipocytes. Adipocytes were treated with either glu pocytes, and addition of a calcium channel antagonist (nife cose (30 mmol/L) or glucose (30 mmol/L) plus nifedipine (10 dipine) reversed this effect, indicating a role of intracellular umol/L), glucose (30 mmol/L) plus GDP, or glucose (30 calcium in mediating this effect. mmol/L) plus 1C, 25-(OH)D for 48 hours. Data are 0032 FIG. 28 demonstrates that calcitriol increased MIF expressed as meant-SE (n-6). Different letters above the bars indicate a significant difference at level of p-0.05. (FIG.28A) and CD14 (FIG. 28B) expression in mouse (3T3 0024 FIG. 20: DNA synthesis in cultured 3T3-L1 adipo L1) adipocytes and the addition of a calcium channel antago cytes. Adipocytes were treated with either glucose (30 mmol/ nist (nifedipine) reversed this effect. L) or glucose (30 mmol/L) plus nifedipine (10 umol/L), glu 0033 FIGS. 29, 30 and 31 show that calcitriol markedly cose (30 mmol/L) plus GDP, or glucose (30 mmol/L) plus 1C. stimulate inflammatory cytokines M-CSF (FIG. 29), MIP 25-(OH)D for 48 hours. Data are expressed as meant-SE (FIG. 30) and IL-6 (FIG. 31) expression in 3T3-L1 adipo (n-6). Different letters above the bars indicate a significant cytes, and co-culture with RAW264 macrophages enhance difference at level of p-0.05. this effect, indicating a potential role of adipocytes in regu 0025 FIG. 21: Expression ratio of cyclin A to 18s in cul lation of local resident macrophages activity and that cal tured 3T3-L1 adipocytes. Adipocytes were treated with either citriol regulates this effect via a calcium and mitochondrial glucose (30 mmol/L) or glucose (30 mmol/L) plus nifedipine uncoupling-dependent mechanism. Main effects of chemical (10 umol/L), glucose (30 mmol/L) plus GDP, or glucose (30 treatment and culture status were significant (p<0.02). mmol/L) plus 1C, 25-(OH)D for 48 hours. Data are 0034 FIGS. 32A-D illustrate the effect of calcitriol on expressed as meant-SE (n-6). Different letters above the bars mouse cytokine protein production. Calcitriol markedly indicate a significant difference at level of p-0.05. increases production of several cytokines in 3T3-L1 adipo 0026 FIG. 22A shows adipose tissue TNFC. expression cytes, as indicated in the schematic diagram. ratio and FIG.22B shows IL-6 expression ratio in aP2-agouti 0035 FIGS. 33 A-D demonstrate that the effect of cal transgenic mice. Data are normalized to 18s expression. Val citriol on mouse cytokine protein production in a co-culture ues are presented as mean-SEM, n=6. Means with different system. Calcitriol markedly increased cytokine production in letter differ, p<0.001. a 3T3-L1 adipocytes-RAW264 macrophage co-culture, as 0027 FIG. 23A shows adipose tissue IL-15 expression, indicated in the schematic diagram. FIG. 23B shows Adipose adiponectin expression and FIG. 0036 FIG. 34: MCP-1 expression in 3T3-L1 adipocytes. 23C shows Muscle IL-15 expression in aP2-agoutitransgenic 0037 FIGS. 35-36: Calcitriol stimulates expression of mice. Data are normalized to 18s expression. Values are pre TNFC. and IL-6. Calcitriol stimulated TNFC. expression by sented as meant SEM, n=6. Means with different letter differ, 91% (FIG. 35) and IL-6 by 796% (FIG. 36) in RAW 264 p<0.03. macrophages cultured alone. These effects were blocked by 0028 FIG. 24A shows TNFC. expression and FIG. 24B adding nifedipine or DNP. Co-culture of macrophages with shows IL-6 expression in differentiated 3T3-L1 adipocytes. differentiated 3T3-L1 adipocytes markedly augmented Adipocytes were treatment with 10 nmol/L 1C, 25-(OH)-D, TNFC. (FIG. 35) and IL-6 (FIG. 36) expression in macroph 10 umol/L nifepipine, and 10 nmol/L 1C, 25-(OH)-D plus ages, and these effects were further enhanced by calcitriol. 10 umol/L nifepipine respectively for 48 h. Data are normal ized to 18s expression. Values are presented as mean-SEM, 0038 FIG.37: The high calcium diet was without effect on n=6. Means with different letter differ, p<0.02. FIG. 24C body weight, but the milk diet did induce a significant illustrates plasma 1 C, 25-(OH)-D in aP2-agouti transgenic decrease in total body weight. mice fed low calcium (basal) or high calcium diets. Values are 0039 FIG.38: Both the calcium and the milk diets caused presented as meant SEM, n=10. Means with different letter significant decreases in body fat, with the milk diet eliciting a differ, p=0.005. significantly greater effect. 0029 FIG. 25A shows IL-6 expression, FIG. 25B shows 0040 FIG. 39: The milk group had significantly greater IL-8 expression, FIG. 25C shows IL-15 expression and FIG. skeletal muscle mass than the calcium group (p=0.02) and a 25D shows adiponectin expression in differentiated Zen-bio tendency towards greater skeletal muscle mass than the basal human adipocytes. Adipocytes were treatment with 10 group (p=0.06). nmol/L 1C, 25-(OH)-D, 10 umol/L nifepipine, and 10 0041 FIG. 40: Liver weight was slightly, but significantly, nmol/L 1C, 25-(OH)-D plus 10 Limol/L nifepipine respec reduced by the milk diet. US 2011/0038948 A1 Feb. 17, 2011

0042 FIG.41: The high calcium diet caused a reduction in composition, or combination of candidate compositions with plasma 1.25-(OH)-D (calcitriol) (p=0.002), and there was a the proviso that said compound, combination of compounds, trend (p=0.059) towards a further decrease in plasma cal composition, or combination of compositions is not a dietary citriol on the high milk diet. material containing calcium or dietary calcium; b) measuring 0043 FIG. 42: Adipose tissue reactive oxygen species the intracellular concentrations of calcium in said adipocyte (ROS) production was significantly reduced by the high cal cell(s), wherein a decrease of intracellular calcium concen cium diet (p=0.002) and further reduced by the milk diet tration in said adipocyte cell(s) is indicative of a compound or (p=0.03). composition Suitable for use in reducing the production of 0044 FIG. 43: The high calcium diet caused a significant ROS. Cells suitable for these screening methods include 3T3 reduction in adipose tissue NADPH oxidase (Nox; one of the L1 adipocytes (ATCC, Manassas, Va.) and human adipocytes sources of intracellular ROS) expression (p=0.001) and there (Zen Bio, Inc., Research Triangle, N.C.). These cells can be was a strong trend (p=0.056) towards a further suppression of maintained in culture as described in Example 2. NOX on the milk diet. 0053 Another screening method provided by the subject 0045 FIG. 44: Plasma MDA was significantly decreased application provides a method of identifying or screening by both the calcium and milk diets (p=0.001), with a signifi compounds or compositions suitable for reducing the produc cantly greater effect of the milk diet (p=0.039). tion of reactive oxygen species (ROS) comprising: a) admin 0046 FIGS. 45-49: The high calcium diet resulted in sup istering a candidate compound, combination of candidate pression of inflammatory markers and an upregulation of compounds, candidate composition, or combination of can anti-inflammatory markers, and the milk diet exerted a didate compositions to at least one subject with the proviso greater effect than the high calcium diet. Adipose tissue that said compound, combination of compounds, composi expression of TNF-C. (FIG. 45), IL-6 (FIG. 46) and MCP tion, or combination of compositions is not a dietary material (FIG. 47) were all significantly suppressed by the high cal containing calcium or dietary calcium; b) measuring intrac cium diet. Expression of each of these inflammatory cytok ellular concentrations of calcium in cells of said at least one ines was lower on the milk diet than on the high calcium diet, Subject, wherein a decrease of intracellular calcium concen but this difference was only statistically evident as a trend for tration in said cells of said at least one test Subject as com TNF-C. (p=0.076). The calcium and milk diets caused signifi pared to the intracellular concentrations of calcium in the cant reductions in the release of inflammatory cytokines cells of at least one control Subject is indicative of a com (TNF-C, FIG. 48; IL6, FIG. 49) from adipose tissue. There pound, composition, combination of compounds or combi was trend towards a greater effect of the milk vs. calcium diet, nation of compositions suitable for use in reducing the pro but this difference was not statistically significant. duction of ROS in a subject. In some embodiments of the 0047 FIGS. 50-51: The high calcium and milk diets invention, intracellular concentrations of Ca" are measured increased adiponectin expression (p=0.001; FIG. 50) and in adipocyte cells (e.g., visceral adipocytes or cutaneous adi IL-15 expression (p=0.001; FIG. 51), and there was a trend pocytes). Other embodiments allow for the administration of for a further increase on the milk diet vs. high calcium diet a candidate compound, combination of candidate com (p=0.073 for adiponectin: p=0.068 for IL-15). pounds, candidate composition, or combination of candidate 0.048 FIG. 52: There was a marked increase in skeletal compositions to at least one test Subject orally with the pro muscle IL-15 expression on the high calcium diet (p<0.001). Viso that said candidate compound, combination of candidate IL-15 expression was further increased on the milk diet (p=0. compounds, candidate composition, or combination of can 07). didate compositions is not being administered to the Subjects 0049 FIG. 53: Fatty acid oxidation was determined via orally as a component of the diet of the subject. palmitate oxidation. C2C12 myotubes were treated with or 0054 As used herein, the term “subject” or “individual' without leucine (2.5 mM), nifedipine (10), adiponectin (70 includes mammals. Non-limiting examples of mammals ng/ml) and/or calcitriol (10 nM) for 48 hours. Data are cor include transgenic mice (Such as aP2-agouti transgenic mice) rected for DNA content. Values are presented as mean-SEM, or human test Subjects. Other mammals include, and are not n=6. Means with different letter differ with p<0.05. limited to, apes, chimpanzees, orangutans, monkeys; domes 0050 FIG. 54: C2C12 myotubes were treated with or ticated animals (pets) such as dogs, cats, guinea pigs, ham without leucine (2.5 mM), nifedipine (10), adiponectin (70 sters, mice, rats, rabbits, and ferrets; domesticated farm ani ng/ml) or/and calcitriol (10 nM) for 48 hours. IL-15 release in mals such as cows, buffalo, bison, horses, donkey, Swine, the medium was determined using ELISA. Data are corrected sheep, and goats; or exotic animals typically found in Zoos, for DNA content. Values are presented as mean+SEM, n=6. Such as bear, lions, tigers, panthers, elephants, hippopotamus, Means with different letter differ with p-0.05. rhinoceros, giraffes, antelopes, sloth, gazelles, Zebras, wilde 0051 FIG. 55: C2C12 myotubes were treated with or beests, prairie dogs, koala bears, kangaroo, pandas, giant without leucine (2.5 mM), nifedipine (10), adiponectin (70 pandas, hyena, seals, sea lions, and elephant seals. ng/ml) or/and calcitriol (10 nM) for 48 hours. IL-6 release in 0055 “Dietary material containing dietary calcium is the medium was determined using ELISA. Data are corrected defined herein as any item normally consumed in the diet of a for DNA content. Values are presented as mean+SEM, n=6. human or mammal. Non-limiting examples of Such dietary Means with different letter differ with p-0.05. materials are salmon, beans, tofu, spinach, turnip greens, DETAILED DESCRIPTION OF THE INVENTION kale, broccoli, waffles, pancakes, pizza, milk, yogurt, cheeses, cottage cheese, ice cream, frozen yogurt, calcium 0052. The subject application provides a method of fortified nutrient supplements, calcium fortified vitamin screening compounds or compositions suitable for reducing Supplements, or liquids Supplemented with calcium. Specifi the production of reactive oxygen species (ROS) comprising: cally excluded from Such a definition are those compositions a) contacting one or more adipocyte cell(s) with a candidate that would be prescribed by a physician or veterinarian for the compound, combination of candidate compounds, candidate treatment of a disease or condition. Also specifically excluded US 2011/0038948 A1 Feb. 17, 2011

from the definition of "dietary calcium' or "dietary material the invention, the culture of cells comprises a co-culture containing dietary calcium' are compounds found in com system that includes macrophage (e.g., see Example 4). pound libraries (such as chemical compound libraries or pep 0059. The subject invention also provides methods of tide libraries) and compositions comprising Such compounds increasing the production of IL-15 and/or adiponectin com or peptides. Also excluded from the definition of “dietary prising contacting a composition comprising a carrier and material containing dietary calcium' is any source of calcium calcium with a culture of cells. The cells are cultured in the that does not form a part of the diet of a mammal or human. presences of this composition and IL-15 and/or adiponectin Pharmaceutical compositions prescribed by a physician or can be recovered for the cell culture according to methods Veterinarian that contain calcium (or physiologically accept known in the art. In some embodiments, the cells can be cells able salts of calcium) via intravenous, intraarterial, oral, buc derived from adipose tissue (adipocytes); skeletal muscle cal, topical, transdermal, rectal, intramuscular, Subcutaneous, cells (or commercially available skeletal cell lines); or human intraosseous, transmucosal, or intraperitoneal routes of or murine adipocyte cell lines (e.g., 3T3-L1 cells). 0060 ROS associated diseases include, and are not limited administration are not construed to a "dietary material con to, cataracts, diabetes, Alzheimer's disease, heart disease, taining dietary calcium' or as "dietary calcium'. One or more inflammation, cancer, male infertility, amyotrophic lateral physiologically acceptable salt(s) of calcium include, and are Sclerosis, Parkinson's disease, and multiple Sclerosis and not limited to, calcium phosphates, calcium carbonate, cal aging. Thus, the Subject application provides methods of cium chloride, calcium sulfate, calcium tartrate, calcium treating cataracts, Alzheimer's disease, heart disease, cancer, magnesium carbonate, calcium metasilicate, calcium malate, male infertility, amyotrophic lateral sclerosis, Parkinson's secondary calcium orthophosphate, calcium citrate, or cal disease, and multiple Sclerosis and aging that comprises the cium hydroxide. administration of compounds, compositions, combinations 0056. The subject application also provides methods of of compounds or combinations of compositions in amounts treating diseases associated with reactive oxygen species sufficient to decrease the intracellular levels of calcium in an (ROS) comprising the administration of a compound, com individual. position, combination of compounds, or combination of com 0061 The subject application also provides methods of positions that decrease intracellular calcium levels to an indi treating cancer-associated ROS disease comprising the vidual in need of Such treatment in amounts sufficient to administration of a composition comprising an appropriate decrease the intracellular concentrations of calcium in the therapeutic agent and calcium (or physiologically acceptable cells of the individual with the proviso that the compound, salts of calcium) in an amount sufficient to reduce the pro composition, combination of compounds, or combination of duction of ROS. In the context of this aspect of the invention, compositions is not dietary calcium or dietary material that the phrase “appropriate therapeutic agent' includes, and is contains calcium. In some embodiments, the methods of not limited to, alkylating agents (e.g., cyclophosphamide, treating diseases associated with ROS also include a step that ifosfamide), antibiotics which affect nucleic acids (e.g., comprises the diagnosis or identification of an individual as doxorubicin, bleomycin), platinum compounds (e.g., cispl having a disease or disorder associated with ROS or suffering atin), mitotic inhibitors (e.g., Vincristine), antimetabolites from elevated ROS levels. (e.g., 5-fluorouracil), camptothecin derivatives (e.g., topote 0057 The subject application also provides methods of can), biological response modifiers (e.g., interferon or mono altering the expression of cytokines in an individual (or the clonal antibodies), and hormone therapies (e.g., tamoxifen). cytokine profile of an individual) comprising the administra Additional non-limiting examples of “appropriate therapeu tion of a compound, composition, combination of com tic agents' are identified in Table 1 of this application as are pounds, or combination of compositions that decrease intra the indications (types of cancer) that can be treated with a cellular calcium levels to an individual in need of such particular therapeutic agent. treatment in amounts sufficient to decrease intracellular lev 0062. The terms “administer”, “administered”, “adminis els of calcium in the cells of the individual, decrease TNF-C., ters' and “administering are defined as the providing a can CD14, MIP, MIF, M-CSF, MCP-1, G-CSF or IL-6 expression didate compound, combination of candidate compounds, (or any combination of the aforementioned cytokines) in the candidate composition, or combination of candidate compo individual, and increase the expression of IL-15. adiponectin, sitions to a Subject via intravenous, intraarterial, oral, buccal, or both IL-15 or adiponectin in the individual with the proviso topical, transdermal, rectal, intramuscular, Subcutaneous, that the compound, composition, combination of compounds, intraosseous, transmucosal, or intraperitoneal routes of or combination of compositions is not dietary calcium or administration. In certain embodiments of the Subject appli dietary material that contains calcium. cation, oral routes of administering a candidate compound, 0058 Also provided are methods of increasing the in vitro combination of candidate compounds, candidate composi expression of MIF, M-CSF, MIP, IL-6, IL-10, IL-4, IL-13, tion, or combination of candidate compositions to a subject MIG, IL-5, VEGF, CD14, G-CSF, TNF-O, RANTES, and/or are specifically excluded. MIP-1C. comprising contacting a composition comprising a 0063. The term “physiologically acceptable salts' of cal carrier and calcitriol (1.25-(OH)-D) with a culture of cells. cium include, and are not limited to, calcium phosphate, The cells are cultured in the presences of this composition and calcium carbonate, calcium chloride, calcium sulfate, cal MIF, M-CSF, MIP, IL-6, IL-10, IL-4, IL-13, MIG, IL-5, cium tartrate, calcium magnesium carbonate, calcium meta VEGF, CD14, G-CSF, TNF-O, RANTES, and/or MIP-1C. can silicate, calcium malate, secondary calcium orthophosphate, be recovered for the cell culture according to methods known calcium citrate, or calcium hydroxide. In certain aspect of the in theart. In some embodiments, the cells can be derived from invention, amounts of calcium that are administered in com adipose tissue (adipocytes); skeletal muscle cells (or com bination with appropriate therapeutic agents for the treatment mercially available skeletal cell lines); or human or murine of cancer provide at least 400 to 2000 mg,900 to 1500 mg. adipocyte cell lines (e.g., 3T3-L1 cells). In some aspects of 1000 to 1400, 1100 to 1300 mg, or 1200 to 1300 mg of US 2011/0038948 A1 Feb. 17, 2011

calcium per day. Alternatively, X.YZ mg (or about X.YZ mg (0077 C) An embodiment as set forth in A or B, wherein or at least X.YZ mg) of calcium per day are provided to the the one or more cell(s) is afare human adipocyte(s) or a subject wherein X is any integer selected from 400 to 2000,Y murine adipocyte, is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9, and Z 0078 D) An embodiment as set forth in A, B or C, wherein is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9. the one or more cell(s) are an adipocyte cell line; 0064. As set forth herein, the subject application also pro (0079 E) An embodiment as set forth in A, B, C or D, vides the following non-limiting aspects of the invention: wherein the one or more cell(s) are a human adipocyte cell 0065 A) An in vitro method of screening compounds or line; compositions suitable for reducing the production of reactive 0080 F) An embodiment as set forth in A, B, C or D, oxygen species (ROS) comprising: wherein the one or more cell(s) are a murine adipocyte cell 0066 a) contacting one or more cell(s) with a candidate line; compound, combination of candidate compounds, candidate I0081 G) An embodiment as set forth in A, B or C, wherein composition, or combination of candidate compositions with the one or more cell(s) are a murine or human adipocyte; the proviso that said candidate compound, combination of I0082 H) An embodiment as set forth in G, wherein the candidate compounds, candidate composition, or combina murine or human adipocytes are obtained from visceral, or tion of candidate compositions is not dietary material con Subcutaneous, or both visceral and Subcutaneous fat tissue; taining calcium or dietary calcium; and I0083) I) An embodiment as set forth in A, B, C, D, E, F, G, 0067 b) measuring one or more of the following param or H, wherein the cell(s) are obtained from a transgenic eters: mouse; 0068 i) intracellular concentrations of calcium in said I0084 J) An embodiment as set forth in I, wherein the one or more cell(s), wherein a decrease of intracellular transgenic mouse is an aP2-agouti transgenic mouse; calcium concentration in said cell(s) is indicative of a I0085 K) An embodiment as set forth in A, B, C, D, E, F or compound or composition Suitable for use in reducing G, wherein the cell(s) 3T3-L1 adipocytes; the production of ROS; I0086 L) A method of identifying or screening compounds 0069 ii) UCP2 expression in said one or more cell(s), or compositions suitable for reducing the production of reac wherein an increase in UCP2 expression in said cell(s) is tive oxygen species (ROS) comprising: indicative of a compound or composition Suitable for use I0087 a) administering a candidate compound, combina in reducing the production of ROS; tion of candidate compounds, candidate composition, or 0070) iii) NADPH oxidase expression in said one or combination of candidate compositions to at least one test more cell(s), wherein a decrease in NADPH oxidase Subject with the proviso that said candidate compound, com expression in said cell(s) is indicative of a compound or bination of candidate compounds, candidate composition, or composition Suitable for use in reducing the production combination of candidate compositions, if administered of ROS: orally to said test Subject, is not being administered to said at least one test Subject orally as a component of the diet of said 0071 iv) UCP3 expression in said one or more cell(s), at least one test Subject or as dietary calcium to said test wherein an increase in UCP3 expression in said cell(s) is Subject (i.e., (said candidate compound, combination of can indicative of a compound or composition Suitable for use didate compounds, candidate composition, or combination of in reducing the production of ROS; candidate compositions, if administered orally to said test 0072 v) NADPH oxidase expression in said one or Subject, is not a dietary material containing calcium or dietary more cell(s), wherein a decrease in NADPH oxidase calcium); and expression in said cell(s) is indicative of a compound or I0088 b) measuring one or more of the following param composition Suitable for use in reducing the production eters: of ROS: 0089 i) intracellular calcium concentrations in cells of 0073 vi) 11 B-HSD expression in said one or more said at least one test Subject and at least one control cell(s), wherein a decrease in the expression of 11 Subject, wherein a decrease of intracellular calcium con B-HSD in said cell(s) is indicative of a compound or centration in the cells of a test Subject as compared to the composition Suitable for use in reducing the production intracellular concentrations of calcium in the cells of at of ROS: least one control Subject is indicative of a compound, 0074 vii) TNF-ct, CD14, MIF, M-CSF, MIP, MCP-1, composition, combination of compounds or combina G-CSF or IL-6 expression in said one or more cell(s), tion of compositions suitable for use in reducing the wherein a decrease in the expression of TNF-C, CD14, production of ROS in a subject; MIF (macrophage inhibitory factor), MIP (macrophage 0090 ii) UCP2 expression in cells of said at least one inhibitory protein), M-CSF (macrophage colony stimu test Subject and at least one control Subject, wherein an lating factor), G-CSF (granulocyte colony Stimulating increase of UCP2 expression in the cells of a test subject factor) or IL-6 in said cell(s) is indicative of a compound as compared to the UCP2 expression in the cells of at or composition Suitable for use in reducing the produc least one control Subject is indicative of a compound, tion of ROS; or composition, combination of compounds or combina 0075 viii) IL-15 or adiponectin expression in said one tion of compositions suitable for use in reducing the or more cell(s), wherein an increase in the expression of production of ROS in a subject; IL-15 or adiponectin in said cell(s) is indicative of a (0.091 iii) NADPH oxidase expression in cells of said at compound or composition Suitable for use in reducing least one test Subject and at least one control Subject, the production of ROS; wherein a decrease of NADPH oxidase expression in the 0076 B) The embodiment as set forth in A, wherein said cells of a test subject as compared to the NADPH oxi one or more cell(s) is a adipocyte or an adipocyte cell line; dase expression in the cells of at least one control Subject US 2011/0038948 A1 Feb. 17, 2011

is indicative of a compound, composition, combination 0104 T) An embodiment as set forth in S, wherein the test of compounds or combination of compositions Suitable Subject and control Subject are aP2-agouti transgenic mice; for use in reducing the production of ROS in a subject; 0105 U) An embodiment as set forth in L, M, N, O, P, Q, 0092 iv) UCP3 expression in skeletal muscle cells of R, S or T, wherein: 1) a candidate compound, combination of said at least one test Subject and at least one control candidate compounds, candidate composition, or combina subject, wherein an increase in UCP3 expression in the tion of candidate compositions is administered to a subject skeletal muscle cells of a test Subject as compared to via intravenous, intraarterial, oral, buccal, topical, transder UCP3 expression in the skeletal muscle cells of at least mal, rectal, intramuscular, Subcutaneous, intraosseous, trans one control Subject is indicative of a compound, compo mucosal, or intraperitoneal routes of administration or sition, combination of compounds or combination of wherein said candidate compound, combination of candidate compositions suitable for use in reducing the production compounds, candidate composition or combination of candi of ROS in a subject; date compositions is administered to at least one test Subject (0093 v) NADPH oxidase expression in skeletal muscle orally with the proviso that said candidate compound, com cells of said at least one test Subject and at least one bination of candidate compounds, candidate composition, or control subject, wherein a decrease of NADPH oxidase combination of candidate compositions is not being admin expression in the skeletal muscle cells of a test Subject as istered to at least one test Subjectorally as a component of the compared to the NADPH oxidase expression in the skel diet of said test Subject; or 2) wherein said candidate com etal muscle cells of at least one control Subject is indica pound, combination of candidate compounds, candidate tive of a compound, composition, combination of com composition, or combination of candidate compositions is pounds or combination of compositions suitable for use administered to at least one test Subjectorally as a component in reducing the production of ROS in a subject; of the Subject's diet (i.e., as dietary calcium); 0094 vi) 11 B-HSD expression in visceral adipocyte 0106 V). An embodiment as set forth in L, M, N, O, P, Q, tissue or cells of said at least one test Subject and at least R, S or T, wherein a candidate compound, combination of one control subject, wherein a decrease of 11 B-HSD candidate compounds, candidate composition, or combina expression in the visceral adipocyte tissue or cells of a tion of candidate compositions is administered to a subject test subject as compared to the 11 B-HSD expression in parenterally (e.g., via intravenous, intraarterial, buccal, topi the visceral adipocyte tissue or cells of at least one cal, transdermal, rectal, intramuscular, Subcutaneous, control Subject is indicative of a compound, composi intraosseous, transmucosal, or intraperitoneal routes of tion, combination of compounds or combination of administration); compositions suitable for use in reducing the production 0107 W) A method of treating diseases associated with of ROS in a subject; reactive oxygen species (ROS) comprising the administration (0.095 vii) TNF-ct, CD14, MIF, MIP, M-CSF, MCP-1, of a compound, composition, combination of compounds, or G-CSF or IL-6 expression in said one or more cell(s), combination of compositions that decrease intracellular cal wherein a decrease in the expression of TNF-C, CD14, cium levels to an individual in need of such treatment in MIF, MIP, M-CSF, G-CSF or IL-6 in said cell(s) is amounts Sufficient to decrease the intracellular concentra indicative of a compound or composition Suitable for use tions of calcium in the cells of the individual with the proviso in reducing the production of ROS; or that the compound, composition, combination of compounds, 0096 viii) IL-15 or adiponectin expression in said one or combination of compositions is not dietary calcium or or more cell(s), wherein an increase in the expression of dietary material that contains calcium; IL-15 or adiponectin in said cell(s) is indicative of a 0108) X) An embodiment as set forth in W, wherein the compound or composition Suitable for use in reducing method includes a step that comprises the diagnosis or iden the production of ROS; tification of an individual as having a disease or disorder associated with ROS or diagnosing or identifying an indi 0097 M) An embodiment as set forth in L(b)(i)-(iii), (vii), vidual having elevated ROS levels (e.g., measuring the levels or (viii), wherein the cells are adipocyte cells obtained from at of ROS and comparing the measured levels against a standard least one test Subject and at least one control Subject; or collection of control subjects); 0098 N) An embodiment as set forth in M, wherein the 0109 Y) An embodiment as set forth in W or X, wherein cells are cutaneous adipocyte cells obtained from at least one the ROS associated diseases include, and are not limited to, test Subject and at least one control Subject; cataracts, diabetes, Alzheimer's disease, heart disease, can 0099 O) An embodiment as set forth in M, wherein the cer, male infertility, inflammation, amyotrophic lateral scle intracellular concentration of calcium is measured in visceral rosis, Parkinson's disease, and multiple Sclerosis and aging; adipocyte cells obtained from at least one test Subject and at 0110 Z) A method of treating cancer-associated ROS dis least one control Subject; ease or disorders comprising the administration of one or 0100 P) An embodiment as set forth in M, wherein the more composition comprising an appropriate therapeutic intracellular concentration of calcium is measured in cutane agent and calcium (or physiologically acceptable salts of ous, or visceral, or both cutaneous and visceral adipocyte calcium) in an amount Sufficient to reduce the production of cells obtained from at least one test Subject and at least one ROS; control Subject; 0111 AA) An embodiment as set forth in Z, wherein the 0101 Q) An embodiment as set forth in L, M, N, O or P. “appropriate therapeutic agent includes, and is not limited wherein the test Subject and control Subject are human; to, alkylating agents (e.g., cyclophosphamide, ifosfamide), 0102 R) An embodiment as set forth in L, M, N, O or P. antibiotics which affect nucleic acids (e.g., doxorubicin, wherein the test Subject and control Subject are murine; bleomycin), platinum compounds (e.g., cisplatin), mitotic 0103 S) An embodiment as set forth in R, wherein the test inhibitors (e.g., Vincristine), antimetabolites (e.g., 5-fluorou Subject and control Subject are transgenic mice; racil), camptothecin derivatives (e.g., topotecan), biological US 2011/0038948 A1 Feb. 17, 2011

response modifiers (e.g., interferon or monoclonal antibod I0128 RR). An embodiment as set forth in QQ, wherein one ies), and hormone therapies (e.g., tamoxifen) or is identified or more physiologically acceptable salt(s) of calcium are in Table 1 of this application; present in at least one of said one or more composition; 0112 BB) An embodiment as set forthin Zor AA, wherein I0129. SS) An embodiment as set forth in RR, wherein the the type of cancer to be treated is identified as an indication in one or more physiologically acceptable salt(s) of calcium Table 1: include, and are not limited to, calcium phosphates, calcium 0113 CC) An embodiment as set forth in W, X,Y,Z, AA or carbonate, calcium chloride, calcium Sulfate, calcium tar BB, wherein one or more physiologically acceptable salt(s) of trate, calcium magnesium carbonate, calcium metasilicate, calcium is administered in the composition; calcium malate, secondary calcium orthophosphate, calcium 0114 DD) An embodiment as set forth in CC, wherein the citrate, or calcium hydroxide; one or more physiologically acceptable salt(s) of calcium I0130 TT) An embodiment as set forth in QQ, RR, or SS include, and are not limited to, calcium phosphates, calcium wherein a dosage of 400 to 2000 mg of calcium are adminis carbonate, calcium chloride, calcium Sulfate, calcium tar tered to a Subject per day; trate, calcium magnesium carbonate, calcium metasilicate, I0131 UU) An embodiment as set forth in QQ, RR, SS, or calcium malate, secondary calcium orthophosphate, calcium TT wherein a dosage of X.YZ mg of calcium are administered citrate, or calcium hydroxide; to a subject per day, wherein X is any integer from 400 to 0115 EE) An embodiment as set forth in W, X, Y, Z, AA, 2000, Y is 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9, and Z is 0, 1, 2, 3, 4, 5, BB, CC or DD whereina dosage of 400 to 2000 mg of calcium 6, 7, 8 or 9: are administered to a subject per day; (0132) VV) An embodiment as set forth in QQ, RR, SS, TT 0116 FF) An embodiment as set forth in W, X, Y, Z, AA, or UU wherein a dosage of 900 to 1500 mg of calcium are BB, CC or DD wherein a dosage of X.YZ mg of calcium is administered to a subject per day; administered to a Subject per day, wherein X is any integer (0.133 WW) An embodiment as set forth in QQ, RR, SS, from 400 to 2000, Y is 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9, and Z is 0, TT, UU or VV wherein a dosage of 1000 to 1400 mg of 1, 2, 3, 4, 5, 6, 7, 8 or 9: calcium are administered to a Subject per day; 0117 GG) An embodiment as set forth in W, X, Y, Z, AA, I0134) XX) An embodiment as set forthin QQ, RR, SS, TT, BB, CC, DD or EE wherein a dosage of 900 to 1500 mg of UU, VV or WW wherein a dosage of 1100 to 1300 mg of calcium are administered to a subject per day; calcium are administered to a Subject per day; 0118 HH) An embodiment as set forth in W, X, Y, Z, AA, I0135 YY) An embodiment as set forthin QQ, RR, SS, TT, BB, CC, DD or EE wherein a dosage of 1000 to 1400 mg of UU,VV, WW or XX wherein a dosage of 1200 to 1300 mg of calcium are administered to a subject per day; calcium are administered to a Subject per day; 0119 II) An embodiment as set forth in W, X, Y, Z, AA, (0.136 ZZ) An embodiment as set forth in QQ, RR, SS, TT, BB, CC, DD or EE wherein a dosage of 1100 to 1300 mg of UU, VV, WW, XX or YY, wherein the therapeutic agent or calcium are administered to a subject per day; therapeutic agents and calcium (or physiologically accept 0120 JJ) An embodiment as set forth in W, X, Y, Z, AA, able salts of calcium) are administered as a single composi BB, CC, DD or EE wherein a dosage of 1200 to 1300 mg of tion; calcium are administered to a subject per day; 0.137 AAA) An embodiment as set forth in QQ, RR, SS, 0121 KK) An embodiment as set forth in W. Z. AA, BB, TT, UU,VV, WW, XXorYY, wherein the therapeutic agentor CC, DD, EE, FF, GG, HH, II or JJ, wherein the therapeutic therapeutic agents and calcium (or physiologically accept agent or therapeutic agents and calcium (or physiologically able salts of calcium) are administered as separate or different acceptable salts of calcium) are administered as a single com compositions; position; I0138 BBB) An embodiment as set forth in QQ, RR, SS, 0122 LL) An embodiment as set forth in Z. AA, BB, CC, TT, UU, VV, WW, XX, YY or AAA, wherein the separate or DD, EE, FF, GG, HH, II or JJ, wherein the therapeutic agent different compositions are administered simultaneously, ortherapeutic agents and calcium (or physiologically accept sequentially or contemporaneously; able salts of calcium) are administered as separate or different I0139 CCC) An embodiment as set forth in QQ, RR, SS, compositions; TT, UU, VV, WW, XX, YY, ZZ, AAA or BBB, wherein the (0123 MM) An embodiment as set forth in X, Y or LL. compositions are administered at multiple times during the wherein the separate or different compositions are adminis day; tered simultaneously, sequentially or contemporaneously; 0140. DDD) An embodiment as set forth in QQ, RR, SS, (0.124 NN) An embodiment as set forth in KK, LL or MM, TT, UU, VV, WW, XX,YY, ZZ, AAA, or BBB, wherein the wherein the compositions are administered at multiple times composition is administered once per day; during the day; 0141 EEE) An embodiment as set forth in QQ, RR, SS, 0125 OO) An embodiment as set forth in X, Y, Z or KK, TT, UU, VV, WW, XX,YY, ZZ, AAA, BBB, CCC, or DDD, wherein the composition is administered once per day; wherein said administration is parenteral; 0126 PP)An embodiment as set forth in W, X, Y, Z, AA, 0.142 FFF) An embodiment as set forth in any of QQ, RR, BB, CC, DD, EE, FF, GG, HH, II, JJ, KK, LL., MM, NN or SS, TT, UU, VV, WW, XX, YY, ZZ, AAA, BBB, CCC, or OO, wherein said administration is parenteral; DDD, wherein said appropriate therapeutic agent is selected 0127 QQ) A method of reducing ROS production in a from those set forth in Table 3 or any combination of the diabetic individual comprising the administration of one or therapeutic agents set forth therein; more composition comprising an appropriate therapeutic 0.143 GGG) An embodiment as set forth in EEE, wherein agent and calcium (or physiologically acceptable salts of said appropriate therapeutic agent is selected from those set calcium) in an amount Sufficient to reduce the production of forth in Table 2 or any combination of the therapeutic agents ROS: set forth therein; US 2011/0038948 A1 Feb. 17, 2011

0144 HHH) An embodiment as set forth in any of QQ, 0156 TTT). An embodiment as set forth in QQQ, RRR, or RR, SS, TT, UU, VV, WW, XX, YY. ZZ, AAA, BBB, CCC, SSS wherein a dosage of 400 to 2000 mg of calcium are DDD, EEE, FFF, or GGG, wherein said diabetic individual administered to a subject per day; has a Type II diabetes (non-insulin dependent diabetes mel (O157 UUU) An embodiment as set forth in QQQ, RRR, litus (NIDDM): SSS, or TTT wherein a dosage of X.YZ mg of calcium are 0145 III) A composition comprising one or more thera administered to a Subject per day, wherein X is any integer peutic agent selected from Tables 2 or 3 in combination with from 400 to 2000, Y is 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9, and Z is 0, calcium or one or more physiological salts of calcium; 1, 2, 3, 4, 5, 6, 7, 8 or 9: 0146) JJJ) An embodiment as set forth in III, wherein said 0158 VVV) An embodiment as set forth in QQQ, RRR, composition contains between 1 and 2000 mg of calcium or SSS, TTT or UUU wherein a dosage of 900 to 1500 mg of one or more physiologically acceptable salts thereof; calcium are administered to a Subject per day; 0147 KKK) An embodiment as set forth in III or JJJ, 0159 WWW) An embodiment as set forth in QQQ, RRR, wherein said composition contains X.YZ mg of calcium or SSS, TTT, UUU or VVV wherein a dosage of 1000 to 1400 physiological salts of calcium, wherein X is any integer mg of calcium are administered to a Subject per day; between 1 to 2000 (inclusive of 1 and 2000), Y is 0, 1, 2, 3, 4, 0160 XXX) An embodiment as set forth in QQQQQ, 5, 6, 7, 8 or 9, and Z is 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9: RRR, SSS, TTT, UUU, VVV or WWW wherein a dosage of 0148 LLL) An embodiment as set forth in III, JJJ, or 1100 to 1300 mg of calcium are administered to a subject per KKK, wherein said composition contains between 900 and day; 1500 mg of calcium or one or more physiological salts of (0161 YYY) An embodiment as set forth in QQQ, RRR, calcium; SSS, TTT, UUU, VVV, WWW or XXX wherein a dosage of 0149 MMM) An embodiment as set forthin III, JJJ, KKK, 1200 to 1300 mg of calcium are administered to a subject per or LLL, wherein said composition contains between 1000 and day; 1400 mg of calcium or one or more physiological salts of 0162 ZZZ) An embodiment as set forth in QQQ, RRR, SSS, TTT, UUU, VVV, WWW, XXX or YYY, wherein the calcium; calcium (or physiologically acceptable salts of calcium) is 0150. NNN) An embodiment as set forth in III, JJJ, KKK, administered in a single composition; LLL, or MMM, wherein said composition contains between 0163 AAAA) An embodiment as set forth in QQQ, RRR, 1100 and 1300 mg of calcium or one or more physiological SSS, TTT, UUU, VVV, WWW, XXX orYYY, the calcium (or salts of calcium; physiologically acceptable salts of calcium) is administered 0151 OOO) An embodiment as set forth in III, JJJ, KKK, as separate or different compositions; LLL, MMM or NNN, wherein said composition contains (0164 BBBB) An embodiment as set forth in QQQ, RRR, between 1200 and 1300 mg of calcium or one or more physi SSS, TTT, UUU, VVV, WWW, XXX, YYY or AAAA, ological salts of calcium; wherein the separate or different compositions are adminis 0152 PPP) An embodiment as set forth in III, JJJ, KKK, tered simultaneously, sequentially or contemporaneously; LLL, MMM, NNN or OOO, wherein said one or more physi (0165 CCCC) An embodiment as set forth in QQQ, RRR, ological salts of calcium include, and are not limited to, SSS, TTT, UUU, VVV, WWW, XXX, YYY, AAAA or calcium phosphates, calcium carbonate, calcium chloride, BBBB, wherein the compositions are administered at mul calcium sulfate, calcium tartrate, calcium magnesium car tiple times during the day; bonate, calcium metasilicate, calcium malate, secondary cal (0166 DDDD) An embodiment as set forth in QQQ, RRR, cium orthophosphate, calcium citrate, or calcium hydroxide; SSS, TTT, UUU, VVV, WWW, XXX, YYY, AAAA or 0153 QQQ) A method of altering the expression of cytok BBBB, wherein the composition is administered once per ines in an individual (or the cytokine profile of an individual) day; or comprising the administration of a compound, composition, (0167 EEEE) An embodiment as set forth in QQQ, RRR, combination of compounds, or combination of compositions SSS, TTT, UUU, VVV, WWW, XXX,YYY, AAAA, BBBB, that decrease intracellular calcium levels to an individual in CCCC, or DDDD, wherein said administration is parenteral. need of Such treatment in amounts Sufficient to decrease (0168 Levels of NADPH oxidase, UCP2, UCP3, cyclin A, intracellular levels of calcium in the cells of the individual, 11 B-HSD, TNF-O, CD14, MIF, MIP, M-CSF, G-CSF, IL-6, decrease TNF-O, CD14, MIP, MIF, M-CSF, G-CSF or IL-6 IL-15, adiponectin and/or intracellular levels of calcium can expression (or any combination of the aforementioned cytok be measured according to methods well-known in the art or as ines) in the individual, and increase the expression of IL-15. set forth in the following examples. By way of non-limiting adiponectin, or both IL-15 or adiponectin in the individual examples, relative levels of expressions of NADPH oxidase, with the proviso that the compound, composition, combina UCP2, UCP3, cyclin A, 11 B-HSD, TNF-ct, CD14, MIF, MIP tion of compounds, or combination of compositions is not M-CSF, G-CSF, IL-6, IL-15, and/or adiponectin can be deter dietary calcium or dietary material that contains calcium; mined by: 1) nuclear run-on assay, 2) slot blot assay, 3) 0154 RRR) An embodiment as set forth in QQQ, wherein Northern blotassay (Alwine et al., 1977), 4) magnetic particle one or more physiologically acceptable salt(s) of calcium are separation, 5) nucleic acid or DNA chips, 6) reverse Northern present in at least one of said one or more composition; blotassay, 7) dot blotassay, 8) in situ hybridization, 9) RNase (O155 SSS) An embodiment as set forth in RRR, wherein protection assay (Melton et al., 1984, and as described in the the one or more physiologically acceptable salt(s) of calcium 1998 catalog of Ambion, Inc., Austin, Tex.), 10) ligase chain include, and are not limited to, calcium phosphates, calcium reaction, 11) polymerase chain reaction (PCR), 12) reverse carbonate, calcium chloride, calcium Sulfate, calcium tar transcriptase (RT)-PCR (Berchtold et al., 1989), 13) differ trate, calcium magnesium carbonate, calcium metasilicate, ential display RT-PCR (DDRT-PCR) or other suitable com calcium malate, secondary calcium orthophosphate, calcium binations oftechniques and assays. Labels suitable for use in citrate, or calcium hydroxide; these detection methodologies include, and are not limited to US 2011/0038948 A1 Feb. 17, 2011

1) radioactive labels, 2) enzyme labels, 3) chemiluminescent tured in Suspension and maintained in a thin layer at the top of labels, 4) fluorescent labels, 5) magnetic labels, or other suit culture media for 2 h for cell recovery. Cali in isolated able labels, including those set forth below. These method mouse adipocytes was measured by using afura-2 dual wave ologies and labels are well known in the art and widely length fluorescence imaging system. Prior to Cali mea available to the skilled artisan. Likewise, methods of incor Surement, adipocytes were pre-incubated in serum-free porating labels into the nucleic acids are also well known to medium for 2 hand rinsed with HBSS containing the follow the skilled artisan. ing components (in mmol/L); NaCl 138, CaCl2 1.8, MgSO (0169. Alternatively, the expression of NADPH oxidase, 0.8, NaH2PO 0.9, NaHCO 4, glucose 5, glutamine 6, Hepes UCP2, UCP3, cyclin A, 11 B-HSD, TNF-ct, CD14, MIF, MIP 20, and bovine serum albumin 1%. Adipocytes were loaded M-CSF, G-CSF, IL-6, IL-15, and/or adiponectin can be mea with fura-2 acetoxymethyl ester (fura-2 AM) (10 umol/L) in sured at the polypeptide level by using labeled antibodies that the same buffer in dark for 1 h at 37° C. Adipocytes were specifically bind to the polypeptides in immunoassays such as rinsed with HBSS three times to remove extracellular dye and commercially available protein arrayS/assays, ELISA assays, then post-incubated at room temperature for an additional 30 RIA assays, Western blots or immunohistochemical assays. minto permit complete hydrolysis of cytoplasmic fura-2AM. Reagents for Such detection and/or quantification assays can A thin layer of adipocytes was plated in 35 mm dishes with be obtained from commercial sources or made by the skilled glass cover slips (P35G-0-14-C, Mattek Corporation, Ash artisan according to methods well known in the art. land, Mass.). The dishes with dye-loaded cells were mounted on the stage of Nikon TMS-F fluorescence inverted micro Example 1 scope with a Cohu 4915 CCD camera. Fluorescent images In Vivo Studies were captured alternatively at excitation wavelength of 340 nm and 380 nm with an emission wavelength of 520 nm. Animals and Diets Cali was calculated by using a ratio equation as described (0170 A. Animal Pilot Study previously (Zemel, 2003). 0171 Six-week old male aP2-agouti transgenic mice and Total RNA Extraction. wild-type male littermates (n=12/group) from our colony were utilized. Six mice randomly selected from each group 0176 A total cellular RNA isolation kit (Ambion, Austin, were sacrificed to provide baseline data and the remaining 6 Tex.) was used to extract total RNA from cells according to mice in each group were put on a modified AIN 93 G diet manufacturer's instruction. (Reeves 1997) with sucrose as the sole carbohydrate source Quantitative RealTime PCR and providing 64% of energy, and fat increased to 25% of (0177 Adipocyte 18s, UCP2, NADPH oxidase and 11 B energy with lard as previously described (Zemel et al., 2000; HSD, and muscle UCP3 and NADPH oxidase were quanti Sun et al., 2004). Mice were studied for 9 days, during which tatively measured using a Smart Cycler RealTime PCR Sys food intake and spillage were measured daily and body tem (Cepheid, Sunnyvale, Calif.) with a TaqMan 1000 Core weight, fasting blood glucose, food consumption assessed Reagent Kit (Applied Biosystems, Branchburg, N.J.). The weekly. At the conclusion of the study, all mice were killed primers and probe sets were obtained from Applied Biosys under isofluorane anesthesia and fat pads were immediately tems TaqMan(R) Assays-on-DemandTM Gene Expression excised, weighed and used for further study, as described primers and probe set collection according to manufacture's below. instruction. Pooled adipocyte total RNA was serial-diluted in 0172 B. Diet Study the range of 1.5625-25 ng and used to establish a standard 0173 At 6 wk of age, 20 male aP2-agouti transgenic mice curve; total RNAs for unknown samples were also diluted in from our colony were randomly divided into two groups (10 this range. Reactions of quantitative RT-PCR for standards mice/group) and fed a modified AIN 93 G diet with subopti and unknown samples were also performed according to the mal calcium (calcium carbonate, 0.4%) or high calcium (cal instructions of Smart Cycler System (Cepheid, Sunnyvale, cium carbonate, 1.2%) respectively, with Sucrose as the sole Calif.) and TaqMan Real Time PCR Core Kit (Applied Bio carbohydrate source and providing 64% of energy, and fat systems, Branchburg, N.J.). The mRNA quantitation for each increased to 25% of energy with lard. Mice were studied for three weeks, during which food intake and spillage were sample was further normalized using the corresponding 18S measured daily and body weight, fasting blood glucose, food quantitation (Sun et al., 2004c). consumption assessed weekly. At the conclusion of the study, Determination of Intracellular ROS Generation all mice were killed under isofluorane anesthesia and blood 0.178 Adipose tissue digestion and adipocytes preparation collected via cardiac puncture; fat pads and soleus muscle were prepared as described in Cali measurement. Intrac were immediately excised, weighed and used for further ellular ROS generation was assessed using 6-carboxy-2,7- study, as described below. dichlorodihydrofluorescein diacetate (H2-DCFDA) as 0.174. This study was approved from an ethical standpoint described previously (Manea et al., 2004). Cells were loaded by the Institutional Care and Use Committee of The Univer with H2-DCFDA (2 umol/L) 30 min before the end of the sity of Tennessee. incubation period (48 h). After washing twice with PBS, cells Measurement of Adipocyte Intracellular Ca"(Cal.) were scraped and disrupted by sonication on ice (20s). Fluo 0175 Adipose tissue was first washed several times with rescence (emission 543 nm or 527 nm) and DNA content Hank's Balanced Salt Solution (HBSS), minced into small were then measured as described previously. The intensity of pieces, and digested with 0.8 mg/ml type I collagenase in a fluorescence was expressed as arbitrary units per ng DNA. shaking water bath at 37°C. for 30 min. Adipocytes were then filtered through sterile 500-um nylon mesh and cultured in Statistical Analysis. Dulbecco's Modified Eagle's Medium (DMEM) supple 0179 Data were evaluated for statistical significance by mented with 1% fetal bovine serum (FBS). Cells were cul analysis of variance (ANOVA), and significantly different US 2011/0038948 A1 Feb. 17, 2011

group means were then separated by the least significant 0182 We have recently shown that 1.O. 25(OH)2D pro difference test by using SPSS (SPSS Inc, Chicago, Ill.). All motes cortisol production by stimulating 113-HSD expres data presented are expressed as meant-SEM. sion in cultured human adipocytes (Morris et al., 2005). How ever, the effect of modulation of 1C, 25(OH),D via dietary Results calcium on this gene expression in Vivo had not been inves 0180. Our previous work indicated that aP2-agouti trans tigated. Data from the present study demonstrates that the genic mice are a useful model for diet-induced obesity in a high calcium diet suppressed 113-HSD expression in visceral genetically Susceptible human population, as they are non adipose tissue by 39% (p=0.034) compared to mice on the obese on standard diets but develop mild to moderate obesity, basal diet (FIG. 11). Interestingly, 11B-HSD expression in hyperglycemia and insulin resistance when fed high Sucrose visceral fat was markedly higher than Subcutaneous fat in and/or high fat diets (Zemel et al., 2000; Sun et al., 2004). mice on basal low calcium group (p=0.034) whereas no dif Given the role of obesity and diabetes in oxidative stress, we ference was observed between the fat depots in mice on the first investigated whether aP2-agouti transgenic mice are also high calcium diet. a suitable model for the study of diet-induced oxidative stress. Transgenic mice exhibited significantly greater baseline ROS Discussion production compared with wild-type controls prior to the 0183 Previous data from our laboratory demonstrate that feeding period, and the consumption of the obesity-promot dietary calcium exerts an anti-obesity effect via a 1 C, 25 ing diet significantly increased adipose tissue ROS produc (OH)-D-mediated mechanism (Zemel, 2005a). We have tion only in aP2-agouti transgenic mice (FIG. 1). This effect reported that 1C, 25-(OH)-D plays a direct role in the modu was also associated with increased NADPH oxidase expres lation adipocyte Ca" signaling, resulting in an increased sion in adipose tissue of aP2-agouti transgenic mice prior to lipogenesis and decreased lipolysis (Xue et al., 1998; Xue et and following consumption of the obesity-promoting diet al., 2000). In addition, 1C, 25-(OH)-D, also plays a role in (FIG. 2). regulating human adipocyte UCP2 expression, Suggesting 0181 Based on the suitability of this model, we utilized that the suppression of 1C, 25-(OH)-D and the resulting aP2 transgenic mice as the animal to investigate the effect of up-regulation of UCP2 may contribute to increased rates of dietary calcium in regulation of diet-induced oxidative stress energy utilization (Shi et al., 2001; Shi et al., 2002). Accord in a three-week obesity induction period on high Sucrose/high ingly, the suppression of 1C, 25-(OH)2-D by increasing fat diets with either low calcium (0.4% from CaCO) (basal dietary calcium attenuates adipocyte triglyceride accumula diet) or high calcium (1.2% from CaCO) (high calcium diet) tion and caused a net reduction in fat mass in both mice and content. Although feeding high fat/high Sucrose diets ad libi humans in the absence of caloric restriction (Zemel et al., tum for 3 weeks induced weight and fat gain in all animals, 2000), a marked augmentation of body weight and fat loss mice on the high calcium diet gained only 50% of the body during energy restriction in both mice and humans (Zemel et weight (p=0.04) and fat (p<0.001) as mice on the basal diet al., 2000; Zemel, 2004), and a reduction in the rate of weight (FIG. 3). The high calcium diet also suppressed diet-induced and fat regain following energy restriction in mice (Sun et al., hyperglycemic and reduced fasting blood glucose by 15% 2004a). Given that obesity and related disorders are associ compared to mice on basal diet (p=0.003) (FIG. 4). The high ated with increased oxidative stress, dietary calcium may play calcium diet significantly reduced adipose intracellular ROS a role in modulating diet-induced oxidative stress. Data from production by 64% and 18% (p<0.001) in visceral and sub the present study demonstrate that dietary calcium decreased cutaneous adipose tissue respectively (FIG. 5). Consistent diet-induced ROS production. Our previous data demonstrate with this, the high calcium diet also inhibited adipose tissue that 1C,25(OH)2D, stimulates Ca' signaling and suppresses NADPH oxidase expression, by 49% (p=0.012) in visceral UCP2 expression on human and murine adipocytes (Shietal. adipose tissue and by 63% (p=0.05) in subcutaneous adipose 2002; Sun et al., 2004) and suppresses UCP3 expression in tissue, respectively, compared to mice on the basal diet (FIG. skeletal muscle (Sun et al., 2004); accordingly, dietary cal 6), indicating that dietary calcium may inhibit oxidative stress cium Suppression of ROS production is likely due to Suppres by Suppressing cytosolic enzymatic ROS production. More sion of circulating 1C... 25(OH)2D levels and resultant reduc over, adipocyte intracellular calcium (Cali) levels, which tions in Ca" signaling and increases in UCP2 and UCP3 were previously demonstrated to favor adipocyte ROS pro expression. Furthermore, dietary calcium also appeared to duction, were markedly Suppressed in mice on the high cal regulate cytosol enzymatic ROS production by inhibiting cium diet by 73%-80% (p<0.001) versus mice on the basal NADPH oxidase expression, which also contributes to cellu diet (FIG. 7), suggesting a role of Cali in regulation of lar ROS production. oxidative stress by dietary calcium. Consistent with our pre 0.184 The interaction between ROS and calcium have vious study, the high calcium diet also induced 367% and been intensively investigated (Toescu 2004; Ermak et al., 191% increases in adipose UCP2 expression (p<0.001) in 2002; Miwa et al., 2003; Brookes 2005). Calcium signaling is visceral and Subcutaneous adipose tissue respectively, com essential for production of ROS, and elevated intracellular pared to mice on the basal diet (FIG. 8). Moreover, the pattern calcium (Cali) activates ROS-generating enzymes, such as of UCP3 expression and indices of ROS production in skel NADPH-oxidase and myeloperoxidase, as well as the forma etal muscle was consistent with these findings. UCP3 expres tion of free radicals by the mitochondrial respiratory chain sion was 22% higher (p=0.006) (FIG.9) and NADPH oxidase (Gordeeva et al., 2003). Interestingly, increased ROS produc expression was 36% lower (p=0.001) (FIG. 10) in soleus tion also stimulates Cali by activating calcium channels on muscle of mice on the high calcium diet compared to mice on both the plasma membrane and endoplasmic reticulum (ER) the low calcium diet, Suggesting that increases in UCP2 and (Volk et al., 1997). Thus, there is a bi-directional interaction UCP3 expression in adipose tissue and muscle, respectively, wherein ROS cellular calcium homeostasis and calcium-de of animals on high calcium diets may contribute to reduced pendent physiological processes while manipulation of cal ROS levels. cium signaling may also regulate cellular ROS production. US 2011/0038948 A1 Feb. 17, 2011

Consistent with this concept, the present data show that Sup visceral adiposity and obesity associated oxidative stress by pression Cati by high dietary calcium was associated with regulating adipose tissue 113-HSD expression and glucocor amelioration of ROS production in adipose tissue. ticoid production. 0185. Respiration is associated with production of ROS, 0187. In conclusion, these data support a role for dietary calcium in the regulation of diet- and obesity-induced oxida and mitochondria produce a large fraction of the total ROS tive stress. Potential mechanisms include increases in UCP2 made in cells (Brand et al., 2004). Mild uncoupling of respi and UCP3 expression, suppression of Cali, and/or inhibi ration diminishes mitochondrial ROS formation by dissipat tion of NADPH oxidase and 113-HSD gene expression. ing mitochondrial proton gradient and potential (Miwa et al., These data also support our previous observation that dietary 2003). Korshunov et al. has demonstrated that slight increase calcium inhibits obesity, with partially selective effects on of the H backflux (to the matrix), which diminishes Alp, visceral adipose tissue, and leads to significant changes in results in a substantial decrease in mitochondrial ROS forma adipose tissue metabolism, including accelerated adipose tis tion (Korshunov et al., 1997). Accordingly, the H backflow sue deposition and reduced ROS production. induced by uncoupling via UCPs would be expected to down regulate ROS production. Mild activation of UCPs may there Example 2 fore play a role in the antioxidant defense system and it is reasonable to propose that dietary calcium induced Suppres 1.25-Dihyrdoxyvitamin D Modulation of Reactive sion of 1C, 25-(OH),D, which has been demonstrated to Oxygen Species Production and Cell Proliferation in inhibit UCP2 expression (Shi et al., 2002), may inhibit ROS Human and Murine Adipocytes production. Indeed, in the present study, we have shown that 0188 3T3-L1 preadipocytes were incubated at a density high dietary calcium up-regulated both UCP2 expression in of 8000 cells/cm (10 cm dish) and grown in Dulbecco's adipose tissue and UCP3 expression in skeletal muscle, and modified Eagle's medium (DMEM) containing 10% FBS and these findings were associated decreased ROS production, antibiotics (adipocyte medium) at 37°C. in 5% CO, in air. indicating a role of mitochondrial uncoupling in regulation of Confluent preadipocytes were induced to differentiate with a oxidative stress. standard differentiation medium consisting of DMEM-F10 (1:1, vol/vol) medium supplemented with 1% FBS, 1 uM 0186 We also compared the ROS production between dexamethasone, IBMX (0.5 mM) and antibiotics (1% Peni Subcutaneous and visceral adipose tissue. Consistent with our cillin-Streptomycin). Preadipocytes were maintained in this previous data (Zemel, 2005a; Zemel et al., 2005a), animals on differentiation medium for 3 days and subsequently cultured the basal low calcium diet showed markedly higher visceral in adipocyte medium. Cultures were re-fed every 2-3 days to fat gain than Subcutaneous fat versus mice on the high cal allow 90% of cells to reach full differentiation before con cium diet (data not shown) and exhibited Strikingly enhanced ducting chemical treatment. Chemicals were freshly diluted ROS production and NADPH oxidase expression in visceral in adipocyte medium before treatment. Cells were washed fat versus Subcutaneous fat. Conversely, high dietary calcium with fresh adipocyte medium, re-fed with medium containing ameliorated visceral fat gain and mice on the high calcium the different treatments, and incubated at 37°C. in 5% CO, in diet showed no significantly greater ROS production in vis air before analysis. Cell viability was measured via trypan ceral fat versus subcutaneous fat. These results therefore indi blue exclusion. cated that higher visceral fat predisposes to enhanced ROS 0189 Human preadipocytes used in this study were sup production. Accordingly, we further evaluated the involve plied by Zen-Bio (Research Triangle, N.C.). Preadipocytes ment of glucocorticoid by measuring 11B hydroxysteroid were inoculated in DMEM/Ham's F-10 medium (DMEM dehydrogenase (113-HSD) expression, the key enzyme F10) (1:1, vol/vol) containing 10% FBS, 15 mmol/L HEPES, responsible for converting glucocorticoid into its active form and antibiotics at a density of 30,000 cells/cm. Confluent (Agarwal 2003). We demonstrated that 113-HSD expression monolayers of preadipocytes were induced to differentiate in visceral fat was markedly higher than Subcutaneous fat in with a standard differentiation medium consisting of DMEM mice on basal low calcium group whereas no difference was F10 (1:1, vol/vol) medium supplemented with 15 mmol/L observed between the fat depots in mice on the high calcium HEPES, 3% FBS, 33 umol/L biotin, 17 umol/L pantothenate, diet. We also found the high calcium diet suppressed 11B 100 nmol/L insulin, 0.25 umol/L methylisobutylxanthine HSD expression in visceral adipose tissue compared to mice (MIX), 1 umol/L dexamethasone, 1 umol/L BRL49653, and on the low calcium diet. These findings demonstrated that antibiotics. Preadipocytes were maintained in this differen dietary calcium exerts greater effect on inhibition of visceral tiation medium for 3 days and Subsequently cultured in adi fat gain via Suppressing formation of active glucocoticoid and pocyte medium in which BRL49653 and MIX were omitted. thus explained the markedly decreased visceral fat gain in Cultures were refed every 2-3 days. mice on the high calcium diet than mice on the low calcium diet. Therefore, the enhanced ROS production observed in UCP2 Transfection visceral fat compare to Subcutaneous fat in response to the (0190. UCP2 full-length cDNAs was amplified by RT-PCR high fat/high Sucrose diet only in mice on low calcium diet using mRNAS isolated from mouse white adipose tissues. Suggested that Suppression of ROS production by dietary The PCR primers for this amplification are shown as follows: calcium may be mediated, at least in part, by the regulation of UCP2 forward, 5'-GCTAGCATGGTTGGTTTCAAG-3' glucocorticoid associated fat distribution. We recently (SEQ ID NO: 1), reverse, 5'-GCTAGCTCAGAAAGGT reported in vitro observation that 1C, 25 (OH), D, directly GAATC-3' (SEQ ID NO: 2). The PCR products were then regulates adipocyte 113-HSD 1 expression and local cortisol subcloned into pcDNA4/His expression vectors. The linear levels in cultured human adipocytes (Morris et al., 2005), and ized constructs were transfected into 3T3-L1 preadipocytes data from this study provides the first in vivo evidence that using lipofectamine plus standard protocol (Invitrogen, dietary calcium may contribute to the preferential loss of Carlsbad, Calif.). After 48 hrs of transfection, cells were split US 2011/0038948 A1 Feb. 17, 2011

and cultured in selective medium containing 400 g/ml Zeo establish a standard curve; total RNAs for unknown samples cin for the selection of resistant colonies. Cells were fed with were also diluted in this range. Reactions of quantitative selective medium every 3 days until resistant colonies could RT-PCR for standards and unknown samples were also per be identified. These resistant foci were picked, expanded, formed according to the instructions of Smart Cycler System tested for expression, and frozen for future experiments. (Cepheid, Sunnyvale, Calif.) and TaqMan Real Time PCR Core Kit (Applied Biosystems, Branchburg, N.J.). The Determination of Mitochondrial Membrane Potential mRNA quantitation for each sample was further normalized 0191 Mitochondrial membrane potential was analyzed using the corresponding 18s quantitation. fluorometrically with a lipophilic cationic dye JC-1 (5.5".6, 6'-tetrachloro-1,1,3,3'-tetraethylbenzimidazol carbocyanine Assessment of Cell Proliferation iodide) using a mitochondrial potential detection kit (Bio (0195 Cells were plated in DMEM with different treat carta, San Diego, Calif.). Mitochondrial potential was deter ment in duplicate in 96-well plates. After 48 h, a CyOUANT mined as the ratio of red fluorescence (excitation 550 nm, Cell Proliferation Kit (Molecular Probes, Eugene, Oreg.) was emission 600 nm) and green fluorescence (excitation 485 nm, used following the manufacturer's protocol. a microplate emission 535 nm) using a fluorescence microplate reader. fluorometer (Packard Instrument Company, Inc., Downers Grove, Ill.) was used to measure CyOUANT fluorescence. Measurement of Intracellular Ca"(ICati) Cell viability was determined by Trypan blue exclusion (0192 Cali in adipocytes was measured using a fura-2 examination. dual-wavelength fluorescence imaging system. Cells were plated in 35-mm dishes (P35G-0-14-C, Mattek). Prior to Determination of Intracellular ROS Generation Cali measurement, cells were put in serum-free medium 0.196 Intracellular ROS generation was assessed using overnight and rinsed with HEPES balanced salt solution 6-carboxy-2,7'-dichlorodihydrofluorescein diacetate (H2 (HBSS) containing the following components (in mmol/L): DCFDA) as described previously (Manea et al., 2004). Cells 138 NaCl, 1.8 CaCl, 0.8 MgSO, 0.9 NaH2PO4 NaHCO, were loaded with H2-DCFDA (2 umol/L) 30 minute before 5 glucose, 6 glutamine, 20 HEPES, and 1% bovine serum the end of the incubation period (48 h). After washing twice albumin. Cells were loaded with fura-2 acetoxymethyl ester with PBS, cells were scraped and disrupted by sonication on (fura-2 AM) (10 umol/L) in the same buffer for 2 hat 37° C. ice (20s). Fluorescence (emission 543 nm or 527 nm) and in a dark incubator with 5% CO. To remove extracellular DNA content were then measured as described previously. dye, cells were rinsed with HBSS three times and then post The intensity of fluorescence was expressed as arbitrary units incubated at room temperature for an additional 1 h for com per ng DNA. plete hydrolysis of cytoplasmic fura-2 AM. The dishes with dye-loaded cells were mounted on the stage of Nikon TMS-F Statistical Analysis fluorescence inverted microscope with a Cohu model 4915 0.197 All data are expressed as meaniSEM. Data were charge-coupled device (CCD) camera. Fluorescent images evaluated for statistical significance by analysis of variance were captured alternatively at excitation wavelengths of 340 (ANOVA), and significantly different group means were then and 380 nm with an emission wavelength of 520 nm. After separated by the least significant difference test by using establishment of a stable baseline, the responses to 1C, 25 (OH)-D was determined. Cai was calculated using a SPSS (SPSS Inc, Chicago, Ill.). ratio equation as described previously. Each analysis evalu Results ated responses of 5 representative whole cells. Images were analyzed with InCyt Im2 version 4.62 imaging Software (In 0198 Our first aim was to examine whether ROS have tracellular Imaging, Cincinnati, Ohio). Images were cali effect on adipocyte proliferation. The data presented in FIG. brated using a fura-2 calcium imaging calibration kit (Mo 12 indicate that this is indeed the case. Treatment of 3T3-L1 lecular Probes, Eugene, Oreg.) to create a calibration curve in adipocytes with HO, increased the total DNA of cultured Solution, and cellular calibration was accomplished using cells by 39% (p<0.001), while addition of antioxidant C-to digitonin (25umol/L) and pH 8.7 Tris-EGTA (100 mmol/L) copherol completely blocked this effect. The effect of ROS on to measure maximal and minimal Cali levels respectively. adipocyte proliferation appears to be regulated by mitochon drial uncoupling and intracellular calcium homeostasis. Total RNA Extraction Addition of mitochondrial uncoupling inhibitor GDP aug mented the stimulation of cell proliferation by HO by 183% 0193 A total cellular RNA isolation kit (Ambion, Austin, (p<0.005) while calcium channel antagonist nifedipine had Tex.) was used to extract total RNA from cells according to the opposite effect and suppressed HO induced cell DNA manufacturer's instruction. synthesis (p<0.05). Since inhibiting mitochondrial uncou pling and increasing Cali have been demonstrated to con Quantitative RealTime PCR tribute to increased ROS production, GDP may increases 0194 Adipocyte 18s, cyclin A, NADPH oxidase, and DNA synthesis by increasing ROS production while nife UCP2 were quantitatively measured using a Smart Cycler dipine exerts the opposite effect via suppression of ROS Real Time PCR System (Cepheid, Sunnyvale, Calif.) with a production. Consistent with this, FIG. 13 shows that addition TaqMan 1000 Core Reagent Kit (Applied Biosystems, of GDP increased ROS production by 24% (p<0.01) compare Branchburg, N.J.). The primers and probe sets were ordered HO, treatment alone while nifedipine inhibited H.O. from Applied Biosystems TaqMan(R) Assays-on-DemandTM induced ROS production by 25% (p<0.003). FIGS. 12 and 13 Gene Expression primers and probe set collection according also demonstrate that addition of antioxidant C-tocopherol to manufacture's instruction. Pooled adipocyte total RNA inhibited both ROS production and DNA synthesis in all was serial-diluted in the range of 1.5625-25 ng and used to groups. These results suggest that ROS stimulated cell pro US 2011/0038948 A1 Feb. 17, 2011

liferation in cultured adipocytes and that this effect can be adipocytes, indicating that high glucose stimulates ROS pro regulated by mitochondrial uncoupling status and intracellu duction by regulating mitochondrial uncoupling status. lar homeostasis. Similar results were also observed in human 0202 FIG. 20 demonstrates that stimulation of ROS pro adipocytes (data not shown). duction by high glucose is associated with increased DNA (0199 To further investigate the interaction between ROS synthesis. High glucose alone significantly increased DNA and mitochondrial uncoupling status, we measured mito synthesis (p<0.03) and this effect was by augmented by addi chondrial potential in both wild-type 3T3-L1 adipocytes and tion of GDP or 1C, 25-(OH). D. In contrast, inhibition of UCP2 transfected 3T3-L1 adipocytes. FIG. 14 demonstrates ROS production by nifedipine decreased glucose induced that H2O, increased mitochondrial potential by 72% and that DNA synthesis (p<0.05). To further investigate the effect of addition of GDP augmented this effect by 10%, indicating high glucose on adipocyte proliferation, we also observed the that ROS production inhibits mitochondrial uncoupling. expression of cyclin A (FIG. 21). Consistent with the DNA Nifedipine suppressed the HO induced increase in mito synthesis data, high glucose stimulated cyclin A expression chondrial potential and this result confirms that calcium chan by 3-fold (p<0.001), and GDP and 1C, 25-(OH)D aug nel antagonist inhibits ROS production. UCP2 transfection mented this effect while nifedipine suppressed its expression. increased mitochondrial potential and Suppressed the effect These data Suggest high glucose stimulates adipocyte prolif of H2O, on mitochondrial uncoupling, indicating that ROS eration and this effect may be at least partially mediated by its production is regulated, in part by mitochondrial potential stimulation of ROS production. and UCP2. 0200 FIG. 15 demonstrates that ROS has a direct role in Discussion regulation of intracellular calcium homeostasis in 3T3-L1 0203 Obesity and diabetes are associated with increased adipocytes. HO, induced a 5-fold increase in Cali (p<0. oxidative stress, and ROS may play a role in regulation of 001) and this effect was reversed by addition of antioxidant adipocyte proliferation. In the present study, we demon C-tocopherol. Since Suppression of intracellular calcium strated that a low concentration of H2O, stimulates cell pro influx by nifedipine decreased ROS production as described liferation in cultured adipocytes. This effect can be aug in FIG. 13, this result suggests a positive feedback interaction mented by a mitochondrial uncoupling inhibitor and between ROS production and intracellular calcium homeo Suppressed by a calcium channel antagonist, indicating that stasis: ROS stimulate Cati and elevated Cai also favors mitochondrial potential and intracellular calcium homeosta ROS production. Similar results were observed in Zen-Bio sis may play a role in regulation of ROS induced cell prolif human adipocytes (data not shown). eration. 1C, 25-(OH)D, which has been demonstrated to 0201 Hyperglycemia is one of the most common clinical stimulate Cati and to inhibit UCP2 expression, stimulates signs in obesity and diabetes, which has been demonstrated to ROS production and cell proliferation in adipocytes. High be associated with increased ROS production. Accordingly, glucose also exerts stimulatory effect on ROS production and we next investigated the effect and mechanism of high glu this effect can be augmented by addition of 1C, 25-(OH)D. cose level on ROS production and consequent adipocyte pro suggesting that 1C, 25-(OH)D may involved in regulation liferation. As shown in FIG. 16, high glucose treatment of ROS production in adipocytes. These results indicate that increased ROS production significantly (p<0.05) and this strategies to Suppress 1C, 25-(OH)2D levels. Such as increas effect was partially reversed by addition of nifedipine. Addi ing dietary calcium, may reduce oxidative stress and thereby tion of GDP further stimulated ROS production compared to inhibit ROS-induced stimulation of adipocyte proliferation. glucose alone. Notably, treatment of adipocytes with 1C.. 0204 Elevated oxidative stress has been reported in both 25-(OH)D, which was previously found to suppress mito humans and animal models of obesity (Sonta et al., 2004: chondrial uncoupling and to increase (Cai in adipocytes, Atabek et al., 2004), Suggesting that ROS may play a critical resulted in greater stimulation of ROS production than either role in the mechanisms underlying proliferative responses. glucose alone or glucose plus GDP (p<0.05), Suggesting that This concept is supported by evidence that both HO and 1C, 25-(OH),D, stimulates ROS production by both inhibi Superoxide anion induce mitogenesis and cell proliferation in tion of mitochondrial uncoupling and stimulation of Cali. several mammalian cell types (Burdon 1995). Furthermore, Glucose also increased Caliby 3-fold (p<0.001) (FIG. 17) reduction of oxidants via Supplementation with antioxidants and this effect was partially blocked by addition of C-toco inhibits cell proliferation in vitro (Khan et al., 2004; Simeone pherol, indicating that stimulation of Cali by high glucose et al., 2004). Although the mechanisms for the involvement of is partially attributable to ROS production. Consistent with oxidative stress in the induction of cell proliferation are not this, FIG. 18 shows that high glucose also increased expres known, it has been demonstrated that ROS and other free sion of NADPH oxidase (p<0.001), a key enzyme in ROS radicals influence the expression of number of genes and production, in both wild-type and UCP2 transfected 3T3-L1 transduction pathways involved in cell growth and prolifera adipocytes, but UCP2 overexpression attenuated this effect. tion. The most significant effects of oxidant on signaling These results suggest that high glucose may increase ROS pathways have been observed in the mitogen-activated pro production by stimulating NADPH oxidase expression. Addi tein (MAP) kinase/AP1, and it has been suggested that ROS tion of 1C, 25-(OH)D stimulated NADPH oxidase expres can activate MAP kinases and thereby transcription factors sion while nifedipine Suppressed its expression. Although activator protein-1 (AP-1) (Changet al., 2001), a collection of GDP has been shown to increases ROS production, we found dimeric basic region-leucine Zipper proteins which activates GDP suppressed NADPH oxidase expression, indicating that cyclin-dependent kinase and entry into cell division cycle regulation of ROS production by GDP is not via up-regulation (Kouzarides et al., 1989). Furthermore, the elevation of cyto of ROS-generating enzyme gene expression. FIG. 19 pro solic calcium level induced by ROS results in activation of vides further evidence for the role of UCP2 in the regulation protein kinase C (PKC) required for expression of positive high glucose induced ROS production. High glucose inhibits regulators of cell proliferation Such as c-fos and c-jun (Lin UCP2 expression in both wild type and UCP2 transfected 2004; Amstad et al., 1992; Hollander et al., 1989). ROS have US 2011/0038948 A1 Feb. 17, 2011 also been implicated as a second messenger involved in acti cytes (Sun et al., 2004b), and that the suppression of 1C. vation of NF-kB (Song et al., 2004), whose expression has 25-(OH)2-D in vivo by increasing dietary calcium stimulates been shown to stimulate cell proliferation via tumor necrosis adipocyte apoptosis in aP2 transgenic mice (Sun et al., factor (TNF) and interleukin-1 (IL-1) (Giri et al., 1998). The 2004b), Suggesting that the stimulation of adipocyte apopto effect of ROS on NF-kB activation is further supported by sis contributes to the observed reduction in adipose tissue studies which demonstrated that expression NF-kB can be mass after administration of high calcium diets (Shi et al., suppressed by antioxidants (Nomura et al., 2000; Schulze 2002). Accordingly, the suppression of 1C, 25-(OH)-D by Osthoff et al., 1997). In addition, ROS can modify DNA increasing dietary calcium attenuates adipocyte triglyceride methylation and cause oxidative DNA damage, which result accumulation and caused a net reduction in fat mass in both in decreased methylation patterns (Weitzman et al., 1994) and mice and humans in the absence of caloric restriction (Zemel consequently contribute to an overall aberrant gene expres et al., 2000), a marked augmentation of body weight and fat sion. ROS may also attribute to the inhibition of cell-to-cell loss during energy restriction in both mice and humans (Ze communication and this effect can result in decreased regu melet al., 2000; Zemelet al., 2004), and a reduction in the rate lation of homeostatic growth control of normal Surrounding of weight and fat regain following energy restriction in mice cells and lead to clonal expansion (Cerutti et al., 1994: Upham (Sun et al., 2004). Data from present study provide further et al., 1997). Despite these mechanisms proposed to explain evidence to support the role of 1C, 25-(OH),D. in favoring the stimulatory effect on cell proliferation, limited studies energy storage and fat mass expansion by stimulating ROS have been conducted on adipocytes. In present study, we production and adipocyte proliferation. ROS stimulates adi demonstrated that low concentrations of ROS promote cell pocyte proliferation and this effect can by Suppressed by proliferation in cultured human and murine adipocytes. How mitochondrial uncoupling and stimulated by elevation of ever, further investigation for the underlying molecular intracellular calcium. 1C, 25-(OH)D increases ROS pro mechanisms is required. duction by inhibiting UCP2 expression and increasing 0205 The yield of ROS can be efficiently modulated by Cali and consequently favors adipocyte proliferation. mitochondrial uncoupling. Korshunov et al. has demon Accordingly, the present data Suggest that Suppression 1C.. strated that slight increase of the H backflux (to the matrix), 25-(OH)D by increasing dietary calcium may reduce 1C.. which diminishes Alp, results in a Substantial decrease of 25-(OH)D mediated ROS production and limit ROS mitochondrial ROS formation (Korshunov et al., 1997). induced adipocyte proliferation, resulting in reduced adipos Accordingly, the H backflow from UCP-induced uncoupling ity. would be expected to down-regulate ROS production. In 0207. This work demonstrated a direct effect of oxidative addition, calcium can active ROS-generating enzymes stress on adipocyte proliferation in white adipose tissue and directly and activation of calcium dependent PKC favors this observation may have important implications in under assembly of the active NADPH-oxidase complex (Gordeeva standing the adipose mass changes observed under oxidative et al., 2003), indicating that Caimay be another key player stress. However, cell proliferation was only evaluated by in regulation of ROS production. Accordingly, it is reasonable DNA content and cyclin expression level. Further, various to propose that 1 C, 25-(OH)D, which has been demon sources of ROS production may play different roles in regu strated both to inhibit mitochondrial uncoupling and to stimu lation of cell signaling in cell cycle and cell metabolism. late Cali in adipocytes, would stimulate ROS production Although we demonstrated both mitochondrial ROS produc and may consequently be involved in the regulation of adi tion and cellular enzymatic ROS production are associated pocyte proliferation. Indeed, in the present study, we have with adipocyte proliferation, the contribution of each source shown that addition of 1C, 25-(OH)D augmented high glu needs further investigation. cose-induced ROS production and adipocyte proliferation. This effect was further enhanced by a mitochondrial uncou Example 3 pling inhibitor and Suppressed by calcium channel antago Calcium and 1, 25-(OH)-D Regulation of Adipok nism, indicating that 1C, 25-(OH)D stimulates ROS pro ine Expression in Murine and Human Adipocytes duction by increasing Cali and by inhibiting mitochondrial and aP2-agouti Transgenic Mice uncoupling. Furthermore, previous studies suggest that 1C.. 25-(OH)2D may act as an prooxidant in various cell types Materials and Methods (Koren et al., 2001) and treatment with 1C, 25-(OH),D, inhibited the expression of the major constituents of the cel Animals and Diets lular defense system against ROS (Banakar et al., 2004). 0208. At 6 wk of age, 20 male aP2-agouti transgenic mice 0206 Previous data from our laboratory have demon from our colony were randomly divided into two groups (10 strated that 10, 25-(OH)-D appears to modulate adipocyte mice/group) and fed a modified AIN 93 G diet with subopti lipid and energy metabolism via both genomic and non-ge mal calcium (0.4% from calcium carbonate) or high calcium nomic pathways (Zemel, 2004; Shi et al., 2001; Shi et al., (1.2% from calcium carbonate) respectively. Sucrose was the 2002). We have reported that 1C, 25-(OH)-D plays a direct sole carbohydrate source, providing 64% of energy, and fat role in the modulation adipocyte Ca" signaling, resulting in was increased to 25% of energy with lard. Mice were studied an increased lipogenesis and decreased lipolysis (Shi et al., for three weeks, during which food intake and spillage were 2001). In addition, 1C.., 25-(OH)-D, also plays a role in measured daily and body weight, fasting blood glucose, food regulating human adipocyte UCP2 mRNA and protein levels, consumption assessed weekly. At the conclusion of the study, indicating that the suppression of 1C, 25-(OH)-D and the all mice were killed under isofluorane anesthesia and blood resulting up-regulation of UCP2 may contribute to increased collected via cardiac puncture; visceral fat pads (perirenal rates of lipid oxidation (Shi et al., 2002). In addition, we also and abdominal), Subcutaneous fat pads (subscapular) and demonstrate that physiological doses of 1C, 25-(OH)-D- soleus muscle were immediately excised, weighed and used inhibit apoptosis in differentiated human and 3T3-L1 adipo for further study, as described below. US 2011/0038948 A1 Feb. 17, 2011

0209. This study was approved from an ethical standpoint cycler real-time PCR system (Cepheid, Sunnyvale, Calif.) by the Institutional Care and Use Committee of The Univer with a TaqMan 1000 Core Reagent Kit (Applied Biosystems, sity of Tennessee. Branchburg, N.J.). The primers and probe sets were obtained from Applied Biosystems TaqMan(R) Assays-on-DemandTM Cell Culture Gene Expression primers and probe set collection and ulti 0210 3T3-L1 pre-adipocytes were incubated at a density lized according to manufacture's instructions. Pooled adipo of 8000 cells/cm (10 cm dish) and grown in Dulbecco's cyte total RNA was serial-diluted in the range of 1.5625-25 ng modified Eagle's medium (DMEM) containing 10% FBS and and used to establish a standard curve; and total RNA for the antibiotics (adipocyte medium) at 37°C. in 5% CO in air. unknown samples were also diluted in this range. Reactions Confluent pre-adipocytes were induced to differentiate with a of quantitative RT-PCR for standards and unknown samples standard differentiation medium consisting of DMEM-F10 were also performed according to the instructions of Smart (1:1, vol/vol) medium supplemented with 1% fetal bovine Cycler System (Cepheid, Sunnyvale, Calif.) and TaqMan serum (FBS), 1 uM dexamethasone, isobutylmethylxanthine Real Time PCR Core Kit (Applied Biosystems, Branchburg, (IBMX) (0.5 mM) and antibiotics (1% Penicillin-Streptomy N.J.). The mRNA quantitation for each sample was further cin). Pre-adipocytes were maintained in this differentiation normalized using the corresponding 18s quantitation. medium for 3 days and Subsequently cultured in adipocyte medium. Cultures were re-fed every 2-3 days to allow 90% Statistical Analysis cells to reach fully differentiation before conducting chemi cal treatment. 0216 Data were evaluated for statistical significance by 0211 Human pre-adipocytes used in this study were sup analysis of variance (ANOVA) or t-test, and significantly plied by Zen-Bio (Research Triangle, N.C.). Preadipocytes different group means were then separated by the least sig were inoculated in DMEM/Ham's F-10 medium (DMEM nificant difference test by using SPSS (SPSS Inc, Chicago, F10) (1:1, vol/vol) containing 10% FBS, 15 mmol/L 4-2- Ill.). All data presented are expressed as mean-SEM. hydroxyethyl-1-piperazineethanesulfonic acid (HEPES), and antibiotics at a density of 30,000 cells/cm. The cells are Results isolated from the Stromal vascular fraction of human Subcu taneous adipose tissue and differentiated in vitro as follows: 0217 Dietary calcium regulates inflammatory cytokine Confluent monolayers of pre-adipocytes were induced to dif production in adipose tissue and skeletal muscle. Feeding the ferentiate with a standard differentiation medium consisting high calcium ad libitum for 3 weeks significantly decreased of DMEM-F10 (1:1, vol/vol) medium supplemented with 15 weight and fat gain (Table 4) and Suppressed TNFC. gene mmol/L HEPES, 3% FBS, 33 umol/L biotin, 17 umol/L pan expression by 64% in visceral, but not subcutaneous, fat tothenate, 100 nmol/L insulin, 0.25umol/L methylisobutylx compared with mice on low calcium basal diet (FIG. 22A) anthine, 1 umol/L dexamethasone, 1 umol/L BRL49653, and (p<0.001). Similarly, IL-6 expression was decreased by 51% antibiotics. Preadipocytes were maintained in this differen in visceral fat of mice on the high calcium diet versus mice on tiation medium for 3 days and Subsequently cultured in adi the low calcium basal diet (FIG. 22B) (p<0.001) and this pocyte medium in which BRL49653 and MIX were omitted. effect was absent in Subcutaneous fat. In contrast, dietary Cultures were re-fed every 2-3 days till fully differentiated. calcium up-regulated IL-15 expression in visceral fat, with a 0212. Cells were incubated in serum free medium over 52% increases in mice on high calcium diet compared with night before chemical treatment. Chemicals were freshly animals on low calcium diet (FIG. 23A) (p=0.001). Adi diluted in adipocyte medium before treatment. Cells were ponectin expression was similarly elevated in visceral fat of washed with fresh adipocyte medium, re-fed with medium mice on the high calcium diet versus mice on low calcium diet containing the different treatments (control, 10 nmol/L 1C. (FIG. 23B) (p=0.025). The high calcium diet also induced a 25-(OH)-D, 10umol/L nifedipine, 10 nmol/L 1C, 25-(OH) 2-fold increase in IL-15 expression in soleus muscle com 2-D plus 10 umol/L nifepipine, 100 nmol/L H.O. 1 umol/L pared with mice on low calcium diet (FIG. 23C) (p=0.01). Cittocopherol, or 100 nmol/L H.O. plus 1 umol/L 0218 Intracellular calcium and 1C, 25-(OH)-D regu Cittocopherol) and incubated at 37°C. in 5% CO for 48 h in lates cytokine production in cultured murine and human adi air before analysis. Cell viability was measured via trypan pocytes. We investigated the role of 1C, 25-(OH)-D and blue exclusion. calcium in regulation of adipokine production in vitro. FIG. 24A shows that 1C, 25-(OH)-D stimulated TNFC. expres Total RNA Extraction sion by 135% in 3T3-L1 adipocyte and addition of calcium 0213 A total cellular RNA isolation kit (Ambion, Austin, channel antagonist nifedipine completely blocked this effect Tex.) was used to extract total RNA from cells according to (p<0.001), while nifedipine alone exerted no effect. Simi manufacturer's instruction. larly, 1C.., 25-(OH)-D markedly increased IL-6 expression in 3T3-L1 adipocyte and this effect was reversed by addition Plasma 1C, 25-(OH)-D Assay of nifedipine (p=0.016) (FIG. 24B). Similar results were observed in human adipocytes (data not shown). These data 0214. A 1C, 25-(OH)-D-vitamin DELISA kit was used suggested that 1C... 25-(OH)-D stimulated cytokine produc to measure plasma 1 C, 25-(OH)2-D content according to the tion by increasing intracellular calcium influx. The high cal manufacturer's instructions (Alpco Diagnostics, Windham, cium diet suppressed plasma 1C... 25(OH)-D (FIG.24C). N.H.). 0219. Similar results were also observed in differentiated human adipocytes; 1 C, 25-(OH)2-D stimulated IL-6 and Quantitative RealTime PCR IL-8 expression by 53% and 49% respectively (FIG. 25A, 0215 Adipocyte and muscle 18s, TNFO, IL-6, IL-8, IL-15 p=0.004) (FIG. 25B, p<0.001), and the addition ofnifedipine and adiponectin were quantitatively measured using a Smart blocked this effect. However, we found no effect of 1C, 25 US 2011/0038948 A1 Feb. 17, 2011

(OH)-D, or nifedipine on IL-15 (FIG. 25C, p=0.473) or favors reduced energy storage in adipose tissue and elevated adiponectin expression (FIG. 25D, p=0.377) in the human protein synthesis and energy expenditure in skeletal muscle. adipocytes. 0222 Obesity is associated with increased expression of 0220 Reactive oxygen species exerted direct impact on inflammatory markers (Valle et al., 2005), while weight loss cytokine production in cultured adipocytes. The direct role of results in decreased expression and secretion of pro-inflam ROS in regulation of adipose cytokine production was inves matory components in obese individuals (Clement et al., tigated in differentiated 3T3-L1 adipocytes. FIG. 26A shows 2004). Accordingly, modulation of the adipose tissue mass that hydrogen peroxides increased IL-6 expression by 1.67% appears to result in corresponding modulation of cytokine (p<0.001) and that this effect was attenuated by the addition production. TNFC. and IL-6 are two intensively studied cytok of anti-oxidant Cittocopherol (p=0.016), indicating that ROS ines in obesity and have been consistently found to be exerted a direct role in stimulation of inflammatory cytokine increased in the white adipose tissue of obese subjects (Cot production. Clittocopherol also increased adiponectin produc tam et al., 2004). Previous studies suggest that white adipose tion (p=0.002), although ROS (hydrogen peroxide) was with tissue contributes a considerable portion of circulating IL-6, out significant effect (p=0.06) (FIG. 26B). Similarly, there with visceral fat contributing markedly more IL-6 compared was no direct effect of ROS on IL-15 expression; however, with subcutaneous fat (Fried et al., 1998: Fain et al., 2004). addition of Cittocopherol markedly increased IL-15 by 2.2- Expression of TNFC. is increased in inflammatory conditions foldas compared to HO-treated cells (P=0.043) (FIG.26C), Such as obesity and cachexia and considered a likely mediator providing further evidence that oxidative stress is involved in of insulin resistance associated with visceral adiposity (Hota adipocyte cytokine production misligil et al., 1994: Ofei et al., 1996). Consistent with this, diet-induced obesity in present study resulted in increased Discussion expression of TNFC. and IL-6 in visceral fat, and dietary 0221 Previous data from our laboratory demonstrate that calcium attenuated these effects. dietary calcium exerts an anti-obesity effect and Suppresses 0223 IL-15 is highly expressed in skeletal muscle, where obesity associated oxidative stress via a 1C, 25-(OH)-D- it exerts anabolic effects (Busquets et al., 2005). IL-15 admin mediated mechanism (Zemel, 2005b, Zemel, 2004). We have istration reduces muscle protein degradation and inhibits demonstrated that 1C, 25-(OH)-D plays a direct role in the skeletal muscle wasting in degenerative conditions such as modulation of adipocyte Ca" signaling, resulting in an cachexia (Carbo et al., 2000a). Interestingly, IL-15 exerts the increased lipogenesis and decreased lipolysis (Shi et al., opposite effect in adipose tissue; administration of IL-15 2001). In addition, lo, 25-(OH)-D is also involved in regu reduced fat deposition without altering food intake and Sup lation of metabolic efficiency by modulating adipocyte UCP2 pressed fat gain in growing rats (Carbo et al., 2000b, Carbo et expression (Shi et al., 2003). Accordingly, the suppression of al., 2001). IL-15 also stimulates adiponectin secretion in cul 1C, 25-(OH)-D by increasing dietary calcium attenuates tured 3T3-L1 adipocytes (Quinn et al., 2005), indicating a adipocyte triglyceride accumulation and causes a net reduc role for IL-15 in regulating adipocyte metabolism. These tion in fat mass in both mice and humans in the absence of observations suggest that IL-15 might be involved in a caloric restriction (Zemel et al., 2000; Zemel et al., 2005b), a muscle-fat endocrine axis and regulate energy utilization marked augmentation of body weight and fat loss during between the two tissues (Argiles et al., 2005). We previously energy restriction in both mice and humans (Zemel et al., found calcium-rich diets to suppress fat gain and accelerate 2000; Thompson et al., 2005; Zemelet al., 2004; Zemelet al., fat loss while protecting muscle mass in diet-induced obesity 2005a), and a reduction in the rate of weight and fat regain and during energy restriction, indicating that dietary calcium followingenergy restriction in mice (Sun et al., 2004a). Given may similarly regulate energy partitioning in a tissue selec that obesity and related disorders are associated with low tive manner. In the present study, we provide the first in vivo grade systemic inflammation (Lee et al., 2005), it is possible evidence that dietary calcium up-regulates IL-15 expression that dietary calcium may also play a role in modulating adi in visceral adipose tissue and skeletal muscle, and stimulates pose tissue cytokine production. Data from the present study adiponectin expression in visceral adipose tissue, skeletal demonstrate that dietary calcium decreased production of muscle and stimulates adiponectin expression in visceral adi pro-inflammatory factors such as TNFC. and IL-6 and pose tissue in aP2 agouti transgenic mice. This suggests that increased anti-inflammatory molecules such as IL-15 and dietary calcium may also regulate energy metabolism, in part, adiponectin in visceral fat. We also found that 1C, 25-(OH) by modulating these cytokines in both adipose tissue and 2-D stimulated TNFO, IL-6 and IL-8 production in cultured skeletal muscle, thereby favoring elevated energy expendi human and murine adipocytes and that this effect was com ture in adipose tissue and preserving energy storage in skel pletely blocked by a calcium channel antagonist, Suggesting etal muscle. However, we found no effect of 1C, 25-(OH)-D- that dietary calcium Suppresses inflammation factor produc on IL-15 expression in human adipocytes. Since these human tion in adipocyte and that 1C, 25-(OH)-D-induced Ca" adipocytes were originally developed from Subcutaneous fat, influx may be a key mediator of this effect. FIGS. 22-23 these results further support our in vivo observations of demonstrate that dietary calcium decreased expression of dietary calcium regulation of adipocyte cytokine production pro-inflammatory factors (TNFC. and IL-6) and increased in a depot specific manner, although we do not have data from anti-inflammatory molecules (IL-15 and adiponectin) in vis human visceral adipocytes for comparison. ceral adipose tissue and that dietary calcium up-regulates 0224. We have recently shown that 1C, 25-(OH)-D, expression of IL-15 in both visceral adipose tissue and skel stimulated ROS production in cultured adipocytes and that etal muscle, and stimulates adiponectin expression in visceral Suppression of 1C, 25-(OH)-D via dietary calcium also adipose tissue in aP2 agouti transgenic mice. This Suggests attenuates adipose oxidative stress (Sun et al., 2006), Suggest that dietary calcium is involved in regulation of energy ing a potential connection between oxidative tress and pro metabolism by modulating endocrine function of both adi duction of inflammatory factors. The present data demon pose tissue and skeletal muscle, resulting in a pattern which strate that hydrogen peroxide stimulates adipocyte IL-6 US 2011/0038948 A1 Feb. 17, 2011 expression and Cittocopherol inhibits this effect. Although inflammatory cytokine production in cultured adipocytes. hydrogen peroxide showed no direct effect on the expression However, adipose tissue includes both endothelial cells and of anti-inflammatory factors adiponectin and IL-15, addition leukocytes as well as adipocytes; these appear to contribute to of Ottocopherol markedly elevated the expression of both, a low-grade inflammatory state in obesity. Accordingly, the Suggesting a direct role of oxidative stress in regulating interaction between adipocytes and leukocytes may play an inflammation. Indeed, previous studies have demonstrated important role in the local modulation of inflammation. Con that oxidative stress was augmented in adiposity, with ROS sequently, we investigated calcitriol modulation of the elevated in blood and tissue in various animal model of obe expression of macrophage inhibitory factor (MIF) and mac sity (Suzuki et al., 2003: Furukawa et al., 2004), while mark rophage surface specific protein CD14, two key factors in ers of systemic oxidative stress were inversely related to regulating macrophage function and Survival, in differenti plasma adiponectin inhuman Subjects (Furukawa et al., 2004; ated human adipocytes. Calcitriol markedly increased MIF Soares et al., 2005). Moreover, addition of oxidants sup and CD14 expression by 59% (p=0.001) and 33% (p=0.008). pressed expression of adiponectin and increased expression respectively, while calcium channel antagonism with nife of IL-6, MCP-1 and PAI-1 (Soares et al., 2005). These results dipine completely reversed these effects, indicating that cal indicate that a local increase in oxidative stress in accumu citriol stimulates MIF and CD14 expression via a calcium lated fat causes dysregulated production of adipocytokines. dependent mechanism. Similar results were also found in The role of adiposity in up-regulation of oxidative stress and cultured 3T3-L1 adipocytes; in addition, calcitriol also up inflammation has been investigated intensively. Fat accumu regulated M-CSF, MIP, MCP-1 (monocyte chemoattractant lation stimulates NADPH oxidase expression in white adi protein-1) and IL-6 expression in 3T3-L1 adipocyte and pose tissue (Sun et al., 2004d; Inoguchi et al., 2000). Further, stimulated tumor necrosis factor-O. (TNF-C.) and IL-6 expres NOX4, an isoform of NADPH oxidase, is expressed in adi sion in RAW264 macrophage cultured alone and this effect pocytes, but not in macrophage (Mahadev et al., 2004; was blocked by either a calcium channel antagonist (nife Sorescu et al., 2002). Xu et al. (2003) and Weisberg et al. dipine) or a mitochondrial uncoupler (DNP). Moreover, co (2003) also reported that ROS stimulated macrophages infil culture of 3T3-L1 adipocytes with RAW264 macrophages tration of obese adipose tissue via ROS induced MCP-1 pro significantly increased the expression and production of mul duction and stimulated local NADPH oxidase expression and tiple inflammatory cytokines in response to calcitriol in both ROS production, indicating that both adipocytes and mac cell types. These data Suggest that calcitriol may regulate rophages contribute to elevated oxidative stress in obesity. macrophage activity by modulating adipocyte production of 0225. Notably, the anti-inflammatory effect of dietary cal factors associated with macrophage function. These data also cium is greater in visceral versus Subcutaneous fat. We have provide additional explanation for our recent observations previously observed similar pattern in adipocyte ROS pro that Suppression of calcitriol by dietary calcium decreases duction (Sun et al., 2006), in that ROS production and obesity associated oxidative stress and inflammation NADPH oxidase expression were markedly higher in visceral fat versus Subcutaneous fat, Suggesting that there may be an Materials and Methods association between oxidative stress and inflammation in 0227 Cell culture: Human preadipocytes used in this diet-induced obesity. Indeed, it was postulated that because study were supplied by Zen-Bio (Research Triangle, N.C.). visceral fat is more sensitive to lipolytic stimuli than adipose Preadipocytes were inoculated in DMEM/Ham's F-10 tissue stored at other sites, turnover of triacylglycerols and medium (DMEM-F10) (1:1, vol/vol) containing 10% FBS, release of fatty acids into the portal circulation are increased 15 mmol/L HEPES, and antibiotics at a density of 30,000 (Wajchenberg, 2000). Free fatty acids, in addition, can stimu cells/cm2. Confluent monolayers of preadipocytes were late ROS production by stimulating NADPH oxidase expres induced to differentiate with a standard differentiation sion and activation (Soares et al., 2005). Accordingly, obesity medium consisting of DMEM-F10 (1:1, vol/vol) medium associated with oxidative stress and inflammation may occur supplemented with 15 mmol/L HEPES, 3% FBS, 33 umol/L in a depot specific manner in adipose tissue, with significant biotin, 17 umol/L pantothenate, 100 nmol/L insulin, 0.25 higher ROS and inflammatory cytokines produced in visceral umol/L methylisobutylxanthine, 1 umol/L dexamethasone, 1 fat versus subcutaneous fat (Liet al., 2003). In summary, the umol/L BRL49653, and antibiotics. Preadipocytes were present study demonstrates that dietary calcium Suppresses maintained in this differentiation medium for 3 days and obesity associated inflammatory status by modulating pro Subsequently cultured in adipocyte medium in which inflammatory and anti-inflammatory factor expression, pro BRL49653 and MIX were omitted. Cultures were re-fed viding the evidence for the first time that increasing dietary every 2-3 days. calcium may contribute to Suppression of obesity associated 0228 RAW264 macrophages and 3T3-L1 preadipocytes inflammation. (AmericanType Culture Collection) were incubated at a den sity of 8000 cells/cm2 (10 cm2 dish) and grown in Dulbecco's Example 4 modified Eagle's medium (DMEM) containing 10% FBS and Calcium-Dependent Regulation of Macrophage antibiotics (adipocyte medium) at 37°C. in 5% CO2 in air. Inhibitory Factor and CD14 Expression by Calcitriol Confluent 3T3-L1 preadipocytes were induced to differenti in Human Adipocytes ate with a standard differentiation medium consisting of DMEM-F10 (1:1, vol/vol) medium supplemented with 1% 0226 Obesity increases oxidative stress and inflammatory FBS, 1 uM dexamethasone, IBMX (0.5 mM) and antibiotics cytokine production in adipose tissue, and our recent data (1% Penicillin-Streptomycin). Preadipocytes were main demonstrate that dietary calcium attenuates obesity-induced tained in this differentiation medium for 3 days and subse oxidative stress and inflammation. This effect may be quently cultured in adipocyte medium. Cultures were re-fed explained by dietary calcium inhibition of calcitriol, which every 2-3 days to allow 90% cells to reach fully differentia we have shown to stimulate reactive oxygen species and tion for 3T3-L1 adipocytes or grow to a confluence for RAW US 2011/0038948 A1 Feb. 17, 2011

264 before conducting chemical treatment. Cells were treated on the array to determine which cytokines are present in the with or without calcitriol (10 nmol/L), GDP (100 umol/L) sample via chemiluminescent signal which was detected and/or nifedipine (10 umol/L) for 48 hours, as indicated in using X-ray film. each figure. 0229 Cells were washed with fresh adipocyte medium, Statistical Analysis: re-fed with medium containing the indicated treatments, and 0234 Each treatment was replicated with n=6, and data incubated at 37°C. in 5% CO for 48 hours before analysis. are expressed as meant-SEM. Data were evaluated for statis Cell viability was measured via trypan blue exclusion. tical significance by analysis of variance (ANOVA) and sig nificantly different group means were then separated by the Cell Culture least significant difference test by using SPSS (SPSS Inc. Chicago, Ill.). The co-culture experiments were analyzed via 0230 Human adipocytes (Zen-Bio, Inc.), 3T3-L1 adipo cytes, RAW264 macrophages were obtained and co-cultured two-way (treatmentxculture condition) ANOVA. by using transwell inserts with 0.4 um porous membranes (Corning) to separate adipocytes and macrophages. All data Results and Discussion are expressed as meant-SEM. Data were evaluated for statis 0235. Obesity is characterized by increased oxidative and tical significance by analysis of one-way or two-way variance inflammatory stress. Adipose tissue is a significant Source of (ANOVA: means with different letter differ, p<0.05). reactive oxygen species (ROS) and expresses and secretes a wide variety of pro-inflammatory components in obese indi Total RNA Extraction: viduals, such as TNF-C. and IL-6. Notably, the adipose tissue is not only composed of adipocytes but also contains a stro 0231. A total cellular RNA isolation kit (Ambion, Austin, mal vascular fraction that includes blood cells, endothelial Tex.) was used to extract total RNA from cells according to cells and macrophages. Although adipocytes directly gener manufacturer's instruction. The concentration and purity of ate inflammatory mediators, adipose tissue-derived cytokines the isolated RNA was measured spectrophotometrically and also originate Substantially from non-fat cells, among which the integrity of RNA sample was analyzed by BioAnalyzer infiltrated macrophages appear to play a prominent role. Infil (Agilent 2100, Agilent Technologies). tration and differentiation of adipose tissue-resident mac rophages are under the local control of chemokines, many of Quantitative RealTime PCR: which are produced by adipocytes. Accordingly, the cross 0232 Adipocyte and muscle 18s, CD14, TNFO, MIP, talk between adipocytes and macrophages may be a key fac M-CSF, IL-6 and MCP-1 were quantitatively measured using tor in mediating inflammatory and oxidative changes in obe a Smart Cycler RealTime PCRSystem (Cepheid, Sunnyvale, S1ty. Calif.) with a TaqMan 1000 Core Reagent Kit (Applied Bio 0236 FIG. 27 demonstrates that calcitriol increased MIF systems, Branchburg, N.J.). The primers and probe sets were (FIG. 27A) and CD14 (FIG. 27B) expression in human adi obtained from Applied Biosystems TaqMan R. Assays-on-De pocytes by 59% and 33% respectively, and addition of a mandTM Gene Expression primers and probe set collection calcium channel antagonist (nifedipine) reversed this effect, according to manufacture's instruction. Pooled adipocyte indicating a role of intracellular calcium in mediating this total RNA was serial-diluted in the range of 1.5625-25ng and effect. FIG. 28, consistent with FIG. 27, demonstrates that used to establish a standard curve; total RNAs for unknown calcitriol increased MIF expression by 50% (FIG. 28A) and samples were also diluted in this range. Reactions of quanti CD14 expression by 45% (FIG. 28B) in mouse (3T3-L1) tative RT-PCR for standards and unknown samples were also adipocytes and the addition of a calcium channel antagonist performed according to the instructions of Smart Cycler Sys (nifedipine) reversed this effect. FIGS. 29, 30 and 31 show tem (Cepheid, Sunnyvale, Calif.) and TaqMan Real Time that calcitriol markedly stimulate inflammatory cytokines PCR Core Kit (Applied Biosystems, Branchburg, N.J.). The M-CSF (FIG. 29), MIP (FIG. 30), IL-6 (FIG.31) and MCP-1 mRNA quantitation for each sample was further normalized (FIG. 34) expression in 3T3-L1 adipocytes, and co-culture using the corresponding 18s quantitation. with RAW264 macrophages enhance this effect, indicating a potential role of adipocytes in regulation of local resident macrophages activity and that calcitriol may regulate mac Cytokine Antibody Array: rophage activity by modulating adipocyte production of fac 0233 A TansSignalTM mouse cytokine antibody array kit tors associated with macrophage function. Main effects of (Panomics, Fremont, Calif.) was used to detect cytokine pro chemical treatment and culture status were significant (p<0. tein released in culture medium according to the manufac 02). ture's instruction. Briefly, membranes immobilized with cap 0237. A cytokine antibody array was used to further inves ture antibodies specific to particular cytokine proteins was tigate the effects of calcitriol on release of major inflamma incubated with 1x blocking buffer for 2 hours and then block tory cytokines from adipocytes. These protein data Support ing buffer was washed three times using washing buffer. the gene expression observations, as calcitriol up-regulated Then, membranes were incubated in samples for 2 hours to production of multiple inflammatory cytokine proteins in allow cytokine protein in the culture medium to bind to the differentiated 3T3-L1 adipocytes cultured alone (FIG. 32); capture antibody on the membrane. At the end of the incuba these include TNFO, IL-6, IL-2, Granulocyte/Macrophage tion, unbound protein was washed away using washing Colony Stimulating Factor (GM-CSF), Interferon-inducible buffer. The membranes were then incubated with biotin-con protein-10 (IP-10), IL-4, IL-13, macrophage induced gene jugated antibody mix which binds to a second epitope on the (MIG), regulated upon T cell activation expressed secreted protein. The membrane was then washed and incubated with (RANTES), IL-5, macrophage inflammatory protein 1C. strepavidin-HRP to visualize the antibody-protein complexes (MIP-1C...) and vascular endothelial growth factor (VEGF). US 2011/0038948 A1 Feb. 17, 2011

Co-culture of 3T3-L1 adipocytes with macrophages signifi 25% of energy with lard. A total of 30 animals will be studied cantly up-regulated production of cytokines such as inter for three weeks (n=10/group), as follows: Control (low Ca) feron Y (IFNY), TNFC, G-CSF and MIP-1C. compared with suboptimal calcium (0.4%); High Ca with 1.2% calcium in 3T3-L1 cultured alone (FIG.33), and calcitriol further stimu the form of CaCO: High Dairy: 50% of the protein was lated inflammatory cytokine production (FIG. 33). replaced by nonfat dry milk and dietary calcium will be 0238 Calcitriol also markedly stimulated TNFC. expres increased to 1.2% Approximately of the additional calcium sion by 91% (FIG. 35) and IL-6 by 796% (FIG. 36) in RAW was derived from the milk and the remainder was added as 264 macrophages cultured alone and these effects were CaCO. Food intake and spillage was monitored daily and blocked by adding nifedipine or DNP. Co-culture of mac body weight and blood glucose was measured weekly. Fol rophages with differentiated 3T3-L1 adipocytes markedly lowing three weeks of feeding, all animals from each group augmented TNFC. (FIG.35) and IL-6 (FIG. 36) expression in were killed for determination of the following outcome mea macrophages, and these effects were further enhanced by Surements: plasma insulin, MDA calcitriol and cytokine (IL calcitriol. 6, MCP, IL-15, adiponectin and TNF-C.; adipose Tissue:IL-6, 0239 Data from this study demonstrate that calcitriol MCP, IL-15, adiponectin, TNF-C. and NADPH oxidase stimulates production of adipokines associated with mac expression, tissue release of adipokines, ROS production; rophage function and increases inflammatory cytokine muscle: real-time PCR of NADPH oxidase, IL-6 and IL-15; expression in both macrophages and adipocytes; these Tissue release of cytokines, ROS production. include CD14, MIF, M-CSF, MIP, TNFO, IL-6 and MCP-1 in 0243 Results: Body weight and composition: A three adipocytes, and TNFC. and IL-6 in macrophages. Consistent week study duration was utilized in order to avoid major with this, the cytokine protein array identified multiple addi calcium- and milk-induced alterations in adiposity, as adipos tional inflammatory cytokines which were up-regulated by ity-induced oxidative stress could cause a degree of con calcitriol in adipocytes. Moreover, calcitriol also regulated founding. Nonetheless, there were modest, but statistically cross-talk between macrophages and adipocytes, as shown by significant diet-induced changes in body weight and compo augmentation of expression and production of inflammatory sition. The high calcium diet was without effect on body cytokines from adipocytes and macrophages in coculture ver weight, but the milk diet did induce a significant decrease in sus individual culture. These effects were attenuated by either total body weight (FIG.37). In contrast, both the calcium and calcium channel antagonism or mitochondrial uncoupling, the milk diets caused significant decreases in body fat, with indicating that the pro-inflammatory effect of calcitriol are the milk diet eliciting a significantly greater effect (FIG. 38). mediated by calcitriol-induced stimulation of Cal signaling 0244 Skeletal muscle weight (soleus+gastrocnemius) and attenuation of mitochondrial uncoupling. exhibited overall differences (p=0.05) among the dietary 0240. These data demonstrate that calcitriol regulates both groups. The milk group had significantly greater skeletal adipocyte and macrophage production of inflammatory fac muscle mass than the calcium group (p=0.02) and a tendency tors via calcium-dependent and mitochondrial uncoupling towards greater skeletal muscle mass than the basal group dependent mechanisms and that these effects are amplified (p=0.06) (FIG. 39). Liver weight was slightly, but signifi with co-culture of both cell types. These data further suggest cantly, reduced by the milk diet (FIG. 40). that strategies for reducing circulating calcitriol levels, such 0245 Circulating calcitriol: The high calcium diet caused as increasing dietary calcium, may regulating adipocyte mac a reduction in plasma 1.25-(OH)-D (calcitriol) (p=0.002), rophage interaction and thereby attenuate local inflammation and there was a trend (p=0.059) towards a further decrease in in adipose tissue. plasma calcitriol on the high milk diet (FIG. 41). The reason for the difference between the calcium and milk diets in Example 5 Suppressing calcitriol is not clear, as they contain the same levels of dietary calcium. Dietary Calcium and Dairy Modulation of Oxidative 0246 Reactive Oxygen Species and Oxidative Stress: and Inflammatory Stress in Mice Adipose tissue reactive oxygen species (ROS) production 0241 Obesity is associated with subclinical chronic was significantly reduced by the high calcium diet (p=0.002), inflammation which contributes to obesity-associated consistent with our previous data, and further reduced by the co-morbidities. Calcitriol (1.25-(OH)2-D) regulates adipo milk diet (p=0.03) (FIG. 42). Consistent with this, the high cyte lipid metabolism, while dietary calcium inhibits obesity calcium diet caused a significant reduction in adipose tissue by suppression of calcitriol. We have recently shown this NADPH oxidase (Nox; one of the sources of intracellular anti-obesity effect to be associated with decreased oxidative ROS) expression (p=0.001) and there was a strong trend and inflammatory stress in adipose tissue in vivo. However, (p=0.056) towards a further suppression of NOX on the milk dairy contains additional bioactive compounds which mark diet (FIG. 43). edly enhance its anti-obesity activity and which we propose 0247 These changes were reflected in significant will also enhance its ability to Suppress oxidative and inflam decreases in systemic lipid peroxidation, as demonstrated by matory stress. Accordingly, the objective of this study was to significant decreases in plasma malonaldehyde (MDA). determine the effects of dietary calcium and dairy on oxida Plasma MDA was significantly decreased by both the calcium tive and inflammatory stress in a mouse model (aP2-agouti and milk diets (p=0.001), with a significantly greater effect of transgenic mice) that we have previously demonstrated to be the milk diet (p=0.039) (FIG. 44). highly predictive of the effects of calcium and dairy on adi 0248 Inflammatory Stress: In general, the high calcium posity in humans and have recently established as a model for diet resulted in Suppression of inflammatory markers and an the study of oxidative stress. upregulation of anti-inflammatory markers, and the milk diet 0242 Study: Six-week old aP2-agouti transgenic mice exerted a greater effect than the high calcium diet. Adipose were fed a modified AIN 93-G diet with Sucrose as the sole tissue expression of TNF-C. (FIG. 45), IL-6 (FIG. 46) and carbohydrate source (64% of energy), and fat increased to MCP (FIG. 47) were all significantly suppressed by the high US 2011/0038948 A1 Feb. 17, 2011 20 calcium diet. Expression of each of these inflammatory by additional bioactive components; these include the high cytokines was lower on the milk diet than on the high calcium concentration of leucine, a key factor in the regulation of diet, but this difference was only statistically evident as a muscle protein turnover. These data Suggest that dietary cal trend for TNF-C. (p=0.076). cium provided with leucine may regulate energy partitioning 0249 Consistent with these data, the calcium and milk in a tissue selective manner and regulate energy metabolism diets caused significant reductions in the release of inflam by modulating endocrine function of both adipose tissue and matory cytokines (TNF-C, FIG. 48; IL6, FIG. 49) from adi skeletal muscle, favoring elevated energy expenditure in adi pose tissue. There was trend towards a greater effect of the pose tissue and promoting protein synthesis in skeletal milk vs. calcium diet, but this difference was not statistically muscle. However, the effect of leucine and calcium, in regu significant. lating this process is unclear. Accordingly, present study was 0250. There was a corresponding up-regulation of adipose tissue anti-inflammatory cytokine expression on the high cal designed to investigate the effect of leucine, calcitriol and cium diets. The high calcium and milk diets increased adi calcium on energy metabolism in murine adipocytes and ponectin expression (p=0.001; FIG.50) and IL-15 expression muscle cells. Leucine induced a 41% increase in fatty acid (p=0.001; FIG. 51), and there was a trend for a further oxidation in C2C12 muscle cells (p<0.001) and decreased increase on the milk diet vs. high calcium diet (p=0.073 for fatty acid synthase gene expression by 66% (p<0.001) in adiponectin; p=0.068 for IL-15). 3T3-L1 adipocytes. Calcitriol decreased muscle cell fatty 0251 Similarly, there was a marked increase in skeletal acid oxidation by 17% (p<0.05) and increased adipocyte FAS muscle IL-15 expression on the high calcium diet (p<0.001), gene expression by 3-fold (p<0.05). These effects were par with a further increase on the milk diet (p=0.07: FIG. 52). tially reversed by either leucine or calcium channel antagonist 0252. These data clearly demonstrate that dietary calcium nifedipine. Incubation of muscle cells with 48-h adipocyte Suppresses both adipose tissue and systemic oxidative stress, conditioned medium decreased fatty acid oxidation by 56% and that dairy (milk) exerts a significantly greater effect. It (p<0.001), and leucine and/or nifedipine conditioned adipo may be argued that the reduced adipose tissue mass may have cyte attenuated this effect in muscle cells. These data Suggest contributed to the decrease in oxidative stress on the high milk that leucine and nifedipine promote energy partitioning from diet. However, this is unlikely, as the decrease in adiposity adipocytes to muscle cells, resulting in decreased energy was quite modest compared to the decrease in oxidative storage in adipocytes and increasing fatty acid utilization in stress. Moreover, the decrease in adipose tissue ROS produc muscle. tion and Nox expression are normalized to reflect decreases 0254 Although adiponectin has been reported to increase per adipocyte as well as total systemic decreases. Accord fatty acid oxidation in both mice and humans, the role of this ingly, these decreases in oxidative stress appear to be direct adipokine in mediating the effects of leucine and calcium on effects of the high calcium and high dairy diets. Data from this energy metabolism in skeletal muscle and adipocytes is yet study also demonstrate a marked reduction in adipose tissue unclear. Consistent with previous studies, the present data derived inflammatory cytokines on the high calcium diets, demonstrate that adiponectin markedly increased fatty acid with a strong trend towards further Suppression of inflamma oxidation in C2C12 myotubes (FIG.53). Further, adiponectin tory cytokines on the milk vs. high calcium diet. Moreover, restored fatty acid oxidation suppressed by calcitriol in the anti-inflammatory cytokine expression is significantly up present of leucine. Comparable effects of leucine, calcitriol regulated on the high calcium diet, with further improve and adiponectin were found in myotubes co-cultured with ments evident on the milk vs. calcium diet. Although there are adipocytes; however, the presence of adipocytes markedly additional analyses to be completed, these data indicate a Suppressed fatty acid oxidation. This was due to secreted marked shift in the ratio of anti-inflammatory to inflamma factor(s), as a comparable Suppression resulted from expo tory cytokines on high calcium diets, with further improve Sure of the myotubes to adipocyte conditioned medium (data ments in this ratio when milk is used as the calcium source. not shown). Thus, data from this pilot study strongly suggest that dietary 0255. The new data also demonstrate that adiponectin calcium Suppresses oxidative and inflammatory stress, con regulates IL-15 and IL-6 release by myotubes in response to sistent with our previous data, and that other components of calcitriol, leucine and nifedipine. Adiponectin significantly milk enhance this effect to produce greater control of both increased IL-15 release, and partially reversed the inhibitory oxidative and inflammatory stress. effects of calcitriol (FIG. 54). Leucine was without effect on IL-15 release, while nifedipine alone promoted IL-15 release Example 6 but adiponectin exerted no addition effect. Adiponectin also Leucine and Calcium Modulation of Adipocyte-Skel increases IL-15 release in muscle cells treated with both etal Muscle Energy Partitioning leucine and nifedipine and this effect was not attenuated by addition of calcitriol, indicating the effect of calcitriol is 0253) The adipose tissue-skeletal muscle endocrine axis mediated, at least in part by calcium signaling. Comparable may play a potential role in regulating metabolic energy effects were found in myotubes co-cultured with adipocytes. partitioning. We have previous shown that dietary calcium Interestingly, the presence of adipocytes decreased IL-15 exhibits an inhibitory effection obesity and that dairy products release under basal conditions but increased IL-15 release in exert a greater effect on adiposity compared to Supplemental with the presence of leucine and/or nifedipine. These data or fortified sources of calcium. While both calcium and dairy Suggest that leucine and nifedipine regulate muscle-adipo accelerate adipose tissue loss, dairy exerts a Substantially cyte cross-talk by modulating production of cytokines which greater effect and exerts a protective effect on lean tissue affect energy partitioning between adipose tissue and skeletal during hypocaloric diets. We have shown that dietary calcium muscle. Similar effects of adiponectin, calcitriol, leucine and modulation of adiposity is mediated, in part, by Suppression nifedipine were observed in the regulation of IL-6 release in of calcitriol, while the additional effect of dairy is mediated C2C12 myotubes (FIG. 55). US 2011/0038948 A1 Feb. 17, 2011 21

0256 These data provide further supporting evidence for calcium, calcitriol and leucine modulate this process. These our proposal that the interaction between adipose tissue and data also suggest that adiponectin is involved in this regula muscle via a fat-muscle endocrine axis may play a potential tion, and that IL-15 and IL-6 may serve as skeletal muscle role in regulating overall metabolic energy partitioning, and derived messengers in this cross-talk.

TABLE 1.

Drug Name Trade Name Indication Manufacturer Aldesleukin Proleukin Chiron Corp Alemtuzumab Campath Accel. Approv. (clinical benefit not Millennium established) Campath is indicated for and ILEX the treatment of B-cell chronic Partners, LP lymphocytic leukemia (B-CLL) in patients who have been treated with alkylating agents and who have failed fludarabine therapy. alitretinoin Panretin Topical treatment of cutaneous lesions Ligand in patients with AIDS-related Kaposi's Pharma S80O8. ceuticals allopurinol Zyloprim Patients with leukemia, lymphoma and Glaxo solid tumor malignancies who are SmithKline receiving cancer therapy which causes elevations of serum and urinary uric acid levels and who cannot tolerate oral therapy. altretamine Hexalen Single agent palliative treatment of US patients with persistent or recurrent Bioscience ovarian cancer following first-line therapy with a cisplatin andfor alkylating agent based combination. amifostine Ethyol To reduce the cumulative renal toxicity US associated with repeated Bioscience administration of cisplatin in patients with advanced ovarian cancer amifostine Ethyol Accel. Approv. (clinical benefit not US established) Reduction of platinum Bioscience toxicity in non-Small cell lung cancer amifostine Ethyol To reduce post-radiation xerostomia US for head and neck cancer where the Bioscience radiation port includes a Substantial portion of the parotid glands. anastrozole Arimidex Accel. Approv. (clinical benefit not AstraZeneca established) for the adjuvant treatment of postmenopausal women with hormone receptor positive early breast

anastrozole Arimidex Treatment of advanced breast cancer AstraZeneca in postmenopausal women with Pharma disease progression following ceuticals tamoxifen therapy. anastrozole Arimidex For first-line treatment of AstraZeneca postmenopausal women with hormone Pharma receptor positive or hormone receptor ceuticals unknown locally advanced or metastatic breast cancer. arsenic Trisenox Second line treatment of relapsed or Cell trioxide refractory APL following ATRA plus an Therapeutic anthracycline. Asparaginase Elspar ELSPAR is indicated in the therapy of Merck & Co., patients with acute lymphocytic Inc. leukemia. This agent is useful primarily in combination with other chemotherapeutic agents in the induction of remissions of the disease in pediatric patients. BCG Live TICE BCG Organon Teknika Corp bexaroteine Targretin For the treatment by oral capsule of Ligand capsules cutaneous manifestations of cutaneous Pharma T-cell lymphoma in patients who are ceuticals refractory to at least one prior systemic therapy. US 2011/0038948 A1 Feb. 17, 2011 22

TABLE 1-continued Drug Name Trade Name Indication Manufacturer bexaroteine Targretin For the topical treatment of cutaneous Ligand gel manifestations of cutaneous T-cell Pharma lymphoma in patients who are ceuticals refractory to at least one prior systemic therapy. bleomycin Blenoxane Bristol-Myers Squibb bleomycin Blenoxane Sclerosing agent for the treatment of Bristol-Myers malignant pleural effusion (MPE) and Squibb prevention of recurrent pleural effusions. busulfan Busulfex Use in combination with Orphan intravenous cyclophoshamide as conditioning Medical, Inc. regimen prior to allogeneic hematopoietic progenitor cell transplantation for chronic myelogenous leukemia. busulfan oral Myleran Chronic Myelogenous Leukemia Glaxo palliative therapy SmithKline calusterone Methosarb Pharmacia & Upjohn Company capecitabine Xeloda Accel. Approv. (clinical benefit Roche Subsequently established) Treatment of metastatic breast cancer resistant to both paclitaxel and an anthracycline containing chemotherapy regimen or resistant to paclitaxel and for whom urther anthracycline therapy may be contraindicated, e.g., patients who have received cumulative doses of 400 mg/m2 of doxorubicin or doxorubicin equivalents capecitabine Xeloda initial therapy of patients with Roche metastatic colorectal carcinoma when treatment with fluoropyrimidine therapy alone is preferred. Combination chemotherapy has shown a Survival benefit compared to 5-FU/LV alone. A survival benefit over 5 FU/LV has not been demonstrated with Xeloda monotherapy. capecitabine Xeloda Treatment in combination with Roche docetaxel of patients with metastatic breast cancer after failure of prior anthracycline containing chemotherapy carboplatin Paraplatin Palliative treatment of patients with Bristol-Myers ovarian carcinoma recurrent after prior Squibb chemotherapy, including patients who have been previously treated with cisplatin. carboplatin Paraplatin initial chemotherapy of advanced Bristol-Myers ovarian carcinoma in combination with Squibb other approved chemotherapeutic agents. carmustine BCNU, BiCNU Bristol-Myers Squibb carmustine with Gliadel Wafer For use in addition to Surgery to Guilford Polifeprosan 20 prolong Survival in patients with Pharma Implant recurrent glioblastoma multiforme who ceuticals Inc. qualify for Surgery. celecoxib Celebrex Accel. Approv. (clinical benefit not Searle established) Reduction of polyp number in patients with the rare genetic disorder of familial adenomatous polyposis. chlorambucil Leukeran Chronic Lymphocytic Leukemia Glaxo palliative therapy SmithKline chlorambucil Leukeran Glaxo SmithKline cisplatin Platinol Metastatic testicular-in established Bristol-Myers combination therapy with other Squibb approved chemotherapeutic agents in patients with metastatic testicular US 2011/0038948 A1 Feb. 17, 2011 23

TABLE 1-continued Drug Name Trade Name Indication Manufacturer tumors who have alre ady received appropriate Surgical andfor radiotherapeutic procedures. An established combination therapy consists of Platinol, Blenoxane and Velbam. cisplatin Platinol Metastatic ovarian tumors - in Bristol-Myers established combination therapy with Squibb other approved chemotherapeutic agents: Ovarian-ines ablished combination therapy with other approved chemothera peutic agents in patients with metasta ic ovarian tumors who have already received appropriate Surgical and/or radio herapeutic procedures. An established combination consists of Platinol and Adriamycin. Platinol, as a single agent, is indicated as secon airy therapy in patients with metasta ic ovarian tumors refractory to standar chemotherapy who have not previously received Platinol therapy. cisplatin Platinol as a single agent for patients with Bristol-Myers transitional cell blad er cancer which is Squibb no longer amenable to local treatments Such as Surgery and/o r radiotherapy. cladribine Leustatin, 2 Treatment of active hairy cell leukemia. R. W. Johnson CdA Pharma ceutical Research institute cyclophosphamide Cytoxan, Bristol-Myers Neosar Squibb cyclophosphamide Cytoxan Bristol-Myers Injection Squibb cyclophosphamide Cytoxan Bristol-Myers Injection Squibb cyclophosphamide Cytoxan Bristol-Myers Tablet Squibb cytarabine Cytosar-U Pharmacia & Upjohn Company cytarabine DepoCyt Accel. Approv. (clinical benefit not Skye liposomal established) Intrathec altherapy of Pharma lymphomatous meningitis ceuticals dacarbazine DTIC-Dome Bayer dactinomycin, Cosmegen Merck actinomycin D dactinomycin, Cosmegan Merck actinomycin D Darbepoetin Aranesp Treatment of anemia associated with Amgen, Inc. alfa chronic renal failure. Darbepoetin Aranesp Aranesp is indicated for the treatment Amgen, Inc. alfa of anemia in patients with non-myeloid malignancies where anemia is due to the effect of concomitantly administered chemotherapy. daunorubicin DanuoXome First line cytotoxic therapy for Nexstar, Inc. liposomal advanced, HIV related Kaposi's daunorubicin, Daunorubicin Leukemia myelogenous/monocytic Bedford Labs daunomycin erythroid of adultsfremission induction in acute lymphocytic leukemia of children and adults. daunorubicin, Cerubidine In combination with approved Wyeth Ayerst daunomycin anticancer drugs for induction of remission in adult ALL. Denileukin Ontak Accel. Approv. (clinical benefit not Seragen, Inc. diftitox established) treatment of patients with persistent or recurrent cutaneous T cell lymphoma whose malignant cells express the CD25 component of the IL-2 receptor US 2011/0038948 A1 Feb. 17, 2011 24

TABLE 1-continued

Drug Name Trade Name Indication Manufacturer dexrazoxane Zinecard Accel. Approv. (clinical benefit Pharmacia & Subsequently established) Prevention Upjohn of cardiomyopathy associated with Company doxorubicin administration dexrazoxane Zinecard reducing the incidence and severity of Pharmacia & cardiomyopathy associated with Upjohn doxorubicin administration in women Company with metastatic breast cancer who have received a cumulative doxorubicin dose of 300 mg/m2 and who will continue to receive doxorubicin therapy to maintain tumor control. It is not recommended for use with the initiation of doxorubicin therapy. docetaxel Taxotere Accel. Approv. (clinical benefit Aventis Subsequently established) Treatment Pharma of patients with locally advanced or ceutical metastatic breast cancer who have progressed during anthracycline-based therapy or have relapsed during anthracycline-based adjuvant therapy. docetaxel Taxotere For the treatment of locally advanced Aventis or metastatic breast cancer which has Pharma progressed during anthracycline-based ceutical treatment or relapsed during anthracycline-based adjuvant therapy. docetaxel Taxotere For locally advanced or metastatic Aventis non-Small cell lung cancer after failure Pharma of prior platinum-based chemotherapy. ceutical docetaxel Taxotere Aventis Pharma ceutical docetaxel Taxotere in combination with cisplatin for the Aventis treatment of patients with Pharma unresectable, locally advanced or ceutical metastatic non-Small cell lung cancer who have not previously received chemotherapy for this condition. doxorubicin Adriamycin, Pharmacia & Rubex Upjohn Company doxorubicin Adriamycin Antibiotic, antitumor agent. Pharmacia & PFS Injection Upjohn intravenous Company injection doxorubicin Doxi Accel. Approv. (clinical benefit not Sequus liposomal established) Treatment of AIDS-related Pharma Kaposi's sarcoma in patients with ceuticals, Inc. disease that has progressed on prior combination chemotherapy or in patients who are intolerant to Such therapy. doxorubicin Doxi Accel. Approv. (clinical benefit not Sequus liposomal established) Treatment of metastatic Pharma carcinoma of the ovary in patient with ceuticals, Inc. disease that is refractory to both paclitaxel and platinum based regimens DROMOSTANOLONE, DROMO Eli Lilly PROPIONATE STANOLONE DROMOSTANOLONE MASTERONE SYNTEX PROPIONATE INJECTION Eliott's B Eliott's B Diluent for the intrathecal Orphan Solution Solution administration of methotrexate Sodium Medical, Inc. and cytarabine for the prevention or treatment of meningeal leukemia or lymphocytic lymphoma. epirubicin Ellence A component of adjuvant therapy in Pharmacia & patients with evidence of axillary node Upjohn tumor involvement following resection Company of primary breast cancer. US 2011/0038948 A1 Feb. 17, 2011 25

TABLE 1-continued Drug Name Trade Name indication Manufacturer Epoetin alfa epogen EPOGENB is indicated for the Amgen, Inc. treatment of anemia related to therapy with zidovudine in HIV-infected patients. EPOGENB is indicated to elevate or maintain the red blood cell evel (as manifested by the hematocrit or hemoglobin determinations) and to decrease the need for transfusions in hese patients. EPOGEND is not indicated for the treatment of anemia in HIV-infected patients due to other actors such as iron or folate deficiencies, hemolysis or gastrointestinal bleeding, which should be managed appropriately. Epoetin alfa epogen EPOGENB is indicated for the Amgen, Inc. treatment of anemic patients (hemoglobin >10 to <13 g/dL) Scheduled to undergo elective, noncardiac, nonvascular Surgery to reduce the need for allogeneic blood transfusions. Epoetin alfa epogen EPOGENB is indicated for the Amgen, Inc. treatment of anemia in patients with non-myeloid malignancies where anemia is due to the effect of concomitantly administered chemotherapy. EPOGEND is indicated to decrease the need for transfusions in patients who will be receiving concomitant chemotherapy for a minimum of 2 months. EPOGENB is not indicated for the treatment of anemia in cancer patients due to other actors such as iron or folate deficiencies, hemolysis or gastrointestinal bleeding, which should be managed appropriately. Epoetin alfa epogen EPOGEN is indicated for the treatment Amgen, Inch ofanemia associated with CRF, including patients on dialysis (ESRD) and patients not on dialysis. estramustine Emcyt palliation of prostate cancer Pharmacia & Upjohn Company etoposide Etopophos Management of refractory testicular Bristol-Myers phosphate tumors, in combination with other Squibb approved chemotherapeutic agents. etoposide Etopophos Management of Small cell lung cancer, Bristol-Myers phosphate first-line, in combination with other Squibb approved chemotherapeutic agents. etoposide Etopophos Management of refractory testicular Bristol-Myers phosphate tumors and Small cell lung cancer. Squibb etoposide, Vepesid Refractory testicular tumors-in Bristol-Myers VP-16 combination therapy with other Squibb approved chemotherapeutic agents in patients with refractory testicular tumors who have already received appropriate Surgical, chemotherapeutic and radiotherapeutic therapy. etoposide, WePesid in combination with other approved Bristol-Myers VP-16 chemotherapeutic agents as first line Squibb treatment in patients with Small cell ung cancer. etoposide, Vepesid in combination with other approved Bristol-Myers VP-16 chemotherapeutic agents as first line Squibb treatment in patients with Small cell ung cancer. exeneStane Aromasin Treatment of advance breast cancer in Pharmacia & postmenopausal women whose Upjohn disease has progressed following Company amoxifen therapy. US 2011/0038948 A1 Feb. 17, 2011 26

TABLE 1-continued Drug Name Trade Name Indication Manufacturer Filgrastim Neupogen Amgen, Inc. Filgrastim Neupogen NEUPOGEN is indicated to reduce the Amgen, Inc. duration of neutropenia and neutropenia-related clinical sequelae, eg, febrile neutropenia, in patients with nonmyeloid malignancies undergoing myeloablative chemotherapy followed by marrow transplantation. Filgrastim Neupogen NEUPOGEN is indicated to decrease Amgen, Inc. the incidence of infection, as manifested by febrile neutropenia, in patients with nonmyeloid malignancies receiving myelosuppressive anticancer drugs associated with a significant incidence of severe neutropenia with fever. Filgrastim Neupogen NEUPOGEN is indicated for reducing Amgen, Inc. the time to neutrophil recovery and the duration of fever, following induction or consolidation hemotherapy treatment of adults with AML. floxuridine FUDR Roche (intraarterial) fludarabine Fludara Palliative treatment of patients with B- Berlex cell lymphocytic leukemia (CLL) who Laboratories have not responded or have Inc. progressed during treatment with at east one standard alkylating agent containing regimen. fluorouracil, Adrucil prolong Survival in combination with ICN Puerto 5-FU eucovorin Rico fulvestrant Faslodex he treatment of hormone receptor- IPR positive metastatic breast cancer in postmenopausal women with disease progression following antiestrogen herapy gemcitabine Gemzar Treatment of patients with locally Eli Lilly advanced (nonresectable stage II or II) or metastatic (stage IV) adenocarcinoma of the pancreas. indicated for first-line treatment and for patients previously treated with a 5 fluorouracil-containing regimen. gemcitabine Gemzar For use in combination with cisplatin Eli Lilly or the first-line treatment of patients with inoperable, locally advanced (Stage IIIA or IIIB) or metastatic (Stage V) non-Small cell lung cancer. gemtuzumab Mylotarg Accel. Approv. (clinical benefit not Wyeth Ayerst OZogamicin established) Treatment of CD33 positive acute myeloid leukemia in patients in first relapse who are 60 years of age or older and who are not considered candidates for cytotoxic chemotherapy. goserelin Zoladex Palliative treatment of advanced breast AstraZeneca acetate Implant cancer in pre- and perimenopausal Pharma WOleil. ceuticals goserelin Zoladex AstraZeneca acetate Pharma ceuticals hydroxyurea Hydrea Bristol-Myers Squibb hydroxyurea Hydrea Decrease need for transfusions in Bristol-Myers sickle cell anemia Squibb Ibritumomab Zevalin Accel. Approv. (clinical benefit not IDEC Tiuxetan established) treatment of patients with Pharma relapsed or refractory low-grade, ceuticals Corp follicular, or transformed B-cell non Hodgkin's lymphoma, including patients with Rituximab refractory follicular non-Hodgkin's lymphoma. US 2011/0038948 A1 Feb. 17, 2011 27

TABLE 1-continued

Drug Name Trade Name Indication Manufacturer idarubicin Idamycin For use in combination with other Adria approved antileukemic drugs for the Laboratories treatment of acute myeloid leukemia (AML) in adults. idarubicin Idamycin In combination with other approved Pharmacia & antileukemic drugs for the treatment of Upjohn acute non-lymphocytic leukemia in Company adults. ifosfamide IFEX Third line chemotherapy of germ cell Bristol-Myers testicular cancer when used in Squibb combination with certain other approved antineoplastic agents. imatinib Gleevec Accel. Approv. (clinical benefit not Novartis meSylate established) Initial therapy of chronic myelogenous leukemia imatinib Gleevec Accel. Approv. (clinical benefit not Novartis meSylate established) metastatic or unresectable malignant gastrointestinal stromal tumors imatinib Gleevec Accel. Approv. (clinical benefit not Novartis meSylate established) Initial treatment of newly diagnosed Ph-- chronic myelogenous leukemia (CML). Interferon Roferon-A Hoffmann-La alfa-2a. Roche Inc. Interferon Intron A Interferon alfa-2b, recombinant for Schering alfa-2b injection is indicated as adjuvant to Corp Surgical treatment in patients 18 years of age or older with malignant melanoma who are free of disease but at high risk for systemic recurrence within 56 days of surgery. Interferon Intron A interferon alfa-2b, recombinant for Schering alfa-2b njection is indicated for the initial Corp treatment of clinically aggressive ollicular Non-Hodgkin's Lymphoma in conjunction with anthracycline containing combination chemotherapy in patients 18 years of age or older. Interferon Intron A interferon alfa-2b, recombinant for Schering alfa-2b njection is indicated for intralesional Corp treatment of selected patients 18 years of age or older with condylomata acuminata involving external Surfaces of the genital and perianal areas. Interferon Intron A interferon alfa-2b, recombinant for Schering alfa-2b njection is indicated for the treatment Corp of chronic hepatitis C in patients 18 years of age or older with compensated liver disease who have a history of blood or blood-produc exposure and/or are HCV antibody positive. Interferon Intron A interferon alfa-2b, recombinant for Schering alfa-2b njection is indicated for the treatment Corp of chronic hepatitis B in patients 18 years of age or older with compensated liver disease and HBV replication. Interferon Intron A interferon alfa-2b, recombinant for Schering alfa-2b njection is indicated for the treatment Corp of patients 18 years of age or older with hairy cell leukemia. Interferon Intron A interferon alfa-2b, recombinant for Schering alfa-2b njection is indicated for the treatment Corp of selected patients 18 years of age or older with AIDS-Related Kaposi's Sarcoma. The likelihood of response to NTRONA therapy is greater in patients who are without systemic symptoms, who have limited ymphadenopathy and who have a relatively intact immune system as indicated by total CD4 count. US 2011/0038948 A1 Feb. 17, 2011 28

TABLE 1-continued Drug Name Trade Name Indication Manufacturer Interferon Intron A Schering alfa-2b Corp Interferon Intron A Schering alfa-2b Corp Interferon Intron A Schering alfa-2b Intron A Corp irinotecan Camptosar Accel. Approv. (clinical benefit Pharmacia & Subsequently established) Treatment Upjohn of patients with metastatic carcinoma Company of the colon or rectum whose disease has recurred or progressed following 5-FU-based therapy. irinotecan Camptosar Follow up of treatment of metastatic Pharmacia & carcinoma of the colon or rectum Upjohn whose disease has recurred or Company progressed following 5-FU-based therapy. irinotecan Camptosar For first line treatment in combination Pharmacia & with 5-FU leucovorin of metastatic Upjohn carcinoma of the colon or rectum. Company etrozole Femara Treatment of advanced breast cancer Novartis in postmenopausal women. etrozole Femara First-line treatment of postmenopausal Novartis women with hormone receptor positive or hormone receptor unknown locally advanced or metastatic breast cancer. etrozole Femara Novartis eucovorin Wellcovorin, Leucovorin calcium is indicated fro use illeX Leucovorin in combination with 5-fluorouraci to Corporation prolong Survival in the palliative treatment of patients with advanced colorectal cancer. eucovorin Leucovorin illeX Corporation eucovorin Leucovorin illeX Corporation eucovorin Leucovorin illeX Corporation eucovorin Leucovorin In combination with fluorouraci to Lederle prolong Survival in the palliative Laboratories treatment of patients with advanced colorectal cancer. evamisole Ergamisol Adjuvant treatment in combination with 5-fluorouracil after Surgical resection in patients with Dukes' Stage C colon C8CC. omustine, CeeBU CCNU meclorethamine, Mustargen nitrogen mustard megestrol Megace Bristol-Myers acetate Squibb melphalan, Alkeran Glaxo L-PAM SmithKline melphalan, Alkeran Systemic administration for palliative Glaxo L-PAM treatment of patients with multiple SmithKline myeloma for whom oral therapy is not appropriate. mercaptopurine, Purimethol Glaxo 6-MP SmithKline le:S3 MeSnex Prevention of ifosfamide-induced Asta Medica hemorrhagic cystitis methotrexate Methotrexate Lederle Laboratories methotrexate Methotrexate Lederle Laboratories methotrexate Methotrexate Lederle Laboratories methotrexate Methotrexate Lederle Laboratories methotrexate Methotrexate OSteoSarcoma Lederle Laboratories methotrexate Methotrexate Lederle Laboratories US 2011/0038948 A1 Feb. 17, 2011 29

TABLE 1-continued

Drug Name Trade Name Indication Manufacturer methoXSalen Uvadex For the use of UWADEX with the UWAR Therakos Photopheresis System in the palliative treatment of the skin manifestations of cutaneous T-cell lymphoma (CTCL) that is unresponsive to other forms of treatment. mitomycin C Mutamycin Bristol-Myers Squibb mitomycin C MitoZytrex therapy of disseminated Supergen adenocarcinoma of the stomach or pancreas in proven combinations with other approved chemotherapeutic agents and as palliative treatment when other modalities have failed. mitotane Lysodren Bristol-Myers Squibb mitoxantrone Nowantrone For use in combination with Immunex corticosteroids as initial chemotherapy Corporation for the treatment of patients with pain related to advanced hormone refractory prostate cancer. mitoxantrone Nowantrone For use with other approved drugs in Lederle the initial therapy for acute Laboratories nonlymphocytic leukemia (ANLL) in adults. nandrolone Durabolin-50 Organon phenpropionate Nofetumomab Verluma Boehringer Ingelheim Pharma KG (formerly Dr. Karl Thomae GmbH) Oprelvekin Neumega Genetics Institute, Inc. Oprelvekin Neumega Genetics Institute, Inc. Oprelvekin Neumega Neumega is indicated for the Genetics prevention of severe thrombocytopenia Institute, Inc. and the reduction of the need for platelet transfusions following myelosuppressive chemotherapy in adult patients with nonmyeloid malignancies who are at high risk of Severe thrombocytopenia. oxaliplatin Eloxatin Accel. Approv. (clinical benefit not Sanofi established) in combination with Synthelabo infusional 5-FU/LV, is indicated for the treatment of patients with metastatic carcinoma of the colon or rectum whose disease has recurred or progressed during or within 6 months of completion of first line therapy with the combination of bolus 5-FU/LV and irinotecan. paclitaxel Paxene treatment of advanced AIDS-related Baker Norton Kaposi's sarcoma after failure of first Pharma line or Subsequent systemic ceuticals, Inc. chemotherapy paclitaxel Taxol Treatment of patients with metastatic Bristol-Myers carcinoma of the ovary after failure of Squibb first-line or Subsequent chemotherapy. paclitaxel Taxol Treatment of breast cancer after failure Bristol-Myers of combination chemotherapy for Squibb metastatic disease or relapse within 6 months of adjuvant chemotherapy. Prior therapy should have included an anthracycline unless clinically contraindicated. paclitaxel Taxol New dosing regimen for patients who Bristol-Myers have failed initial or Subsequent Squibb chemotherapy for metastatic carcinoma of the ovary US 2011/0038948 A1 Feb. 17, 2011 30

TABLE 1-continued

Drug Name Trade Name indication Manufacturer paclitaxel Taxol second line therapy for AIDS related Bristol-Myers Kaposi's sarcoma. Squibb paclitaxel Taxol For first-line therapy for the treatment Bristol-Myers of advanced carcinoma of the ovary in Squibb combination with cisplatin. paclitaxel Taxol or use in combination with cisplatin, Bristol-Myers or the first-line treatment of non-small Squibb cell lung cancer in patients who are not candidates for potentially curative Surgery and/or radiation therapy. paclitaxel Taxol For the adjuvant treatment of node Bristol-Myers positive breast cancer administered Squibb sequentially to standard doxorubicin containing combination therapy. paclitaxel Taxol First line ovarian cancer with 3 hour Bristol-Myers infusion. Squibb pamidronate Aredia Treatment of osteolytic bone Novartis metastases of breast cancer in coniunction with standard antineoplastic therapy. pegademase Adagen Enzyme replacement therapy for Enzon (Pegademase patients with severe combined Bovine) immunodeficiency asa result of adenosine deaminase deficiency. Pegaspargase Oncaspar Enzon, Inc. Pegfilgrastim Neulasta Neulasta is indicated to decrease the Amgen, Inc. incidence of infection, as manifested by febrile neutropenia, in patients with non-myeloid malignancies receiving myelosuppressive anti-cancer drugs associated with a clinically significant incidence offebrile neutropenia. pentostatin Nipent Single agent treatment for adult Parke-Davis patients with alpha interferon refractory Pharma hairy cell leukemia. ceutical Co. pentostatin Nipent Single-agent treatment for untreated Parke-Davis hairy cell leukemia patients with active Pharma disease as defined by clinically ceutical Co. significant anemia, neutropenia, thrombocytopenia, or disease-related symptoms. (Supplement for front-line therapy.) pipobroman Vercyte Abbott Labs plicamycin, Mithracin Pfizer Labs mithramycin portimer Photofrin For use in photodynamic therapy QLT Photo Sodium (PDT) for palliation of patients with therapeutics completely obstructing esophageal Inc. cancer, or patients with partially obstructing esophageal cancer who cannot be satisfactorily treated with ND-YAG laser therapy. portimer Photofrin For use in photodynamic therapy for QLT Photo Sodium treatment of microinvasive therapeutics endobronchial nonsmall cell lung Inc. cancer in patients for whom Surgery and radiotherapy are not indicated. portimer Photofrin For use in photodynamic therapy QLT Photo Sodium (PDT) for reduction of obstruction and therapeutics palliation of symptoms in patients with Inc. completely or partially obstructing endobroncial nonsmall cell lung cancer (NSCLC). procarbazine Matulane Sigma Tau Pharms quinacrine Atabrine Abbott Labs Rasburicase Elitek ELITEK is indicated for the initial Sanofi management of plasma uric acid levels Synthelabo, in pediatric patients with leukemia, Inc. lymphoma, and solid tumor malignancies who are receiving anti cancer therapy expected to result in tumor lysis and Subsequent elevation of plasma uric acid. US 2011/0038948 A1 Feb. 17, 2011 31

TABLE 1-continued Drug Name Trade Name Indication Manufacturer Rituximab Rituxan Genentech, Inc. Sargramostim Prokine Immunex Corp streptozocin Zanosar Antineoplastic agent. Pharmacia & Upjohn Company talc Sclerosol For the prevention of the recurrence of Bryan malignant pleural effusion in symptomatic patients. tamoxifen Novadex AstraZeneca Pharma ceuticals tamoxifen Novadex As a single agent to delay breast AstraZeneca cancer recurrence following total Pharma mastectomy and axillary dissection in ceuticals postmenopausal women with breast cancer (T1-3, N1, MO) amoxifen Novadex For use in premenopausal women with AstraZeneca metastatic breast cancer as an Pharma alternative to oophorectomy or ovarian ceuticals irradiation amoxifen Novadex For use in women with axillary node- AstraZeneca negative breast cancer adjuvant Pharma herapy. ceuticals amoxifen Novadex Metastatic breast cancer in men. AstraZeneca Pharma ceuticals amoxifen Novadex Equal bioavailability of a 20 mg AstraZeneca Nolvadex tablet taken once a day to a Pharma Omg Nolvadex tablet taken twice a ceuticals day. amoxifen Novadex o reduce the incidence of breast AstraZeneca cancer in women at high risk for breast Pharma C8Ce ceuticals amoxifen Novadex in women with DCIS, following breast AstraZeneca Surgery and radiation, Nolvadex is Pharma indicated to reduce the risk of invasive ceuticals breast cancer. emozolomide Temodar Accel. Approv. (clinical benefit not Schering established) Treatment of adult patients with refractory anaplastic astrocytoma, i.e., patients at first relapse with disease progression on a nitrosourea and procarbazine containing regimen eniposide, Vulmon In combination with other approved Bristol-Myers VM-26 anticancer agents for induction therapy Squibb in patients with refractory childhood acute lymphoblastic leukemia (all). estolactone Teslac Bristol-Myers Squibb estolactone Teslac Bristol-Myers Squibb hioguanine, Thioguanine Glaxo 6-TG SmithKline hiotepa Thioplex illeX Corporation hiotepa Thioplex illeX Corporation hiotepa Thioplex Lederle Laboratories opotecan Hycamtin Treatment of patients with metastatic Glaxo carcinoma of the ovary after failure of SmithKline initial or Subsequent chemotherapy. opotecan Hycamtin Treatment of Small cell lung cancer Glaxo sensitive disease after failure of first- SmithKline line chemotherapy. In clinical studies Submitted to Support approval, sensitive disease was defined as disease responding to chemotherapy but Subsequently progressing at least US 2011/0038948 A1 Feb. 17, 2011 32

TABLE 1-continued

Drug Name Trade Name Indication Manufacturer 60 days (in the phase 3 study) or at least 90 days (in the phase 2 studies) after chemotherapy toremifene Fareston Treatment of advanced breast cancer Orion Corp. in postmenopausal women. Tositumomab Bexxar Accel. Approv. (clinical benefit not Corixa established) Treatment of patients with Corporation CD20 positive, follicular, non-Hodgkin's ymphoma, with and without transformation, whose disease is refractory to Rituximab and has relapsed following chemotherapy Trastuzumab Herceptin HERCEPTIN as a single agent is Genentech, indicated for the treatment of patients Inc. with metastatic breast cancer whose tumors overexpress the HER2 protein and who have received one or more chemotherapy regimens for their metastatic disease. Trastuzumab Herceptin Herceptin in combination with Genentech, paclitaxel is indicated for treatment of Inc. patients with metastatic breast cancer whose tumors overexpress the HER-2 protein and had not received chemotherapy for their metastatic disease Trastuzumab Herceptin Genentech, Inc. Trastuzumab Herceptin Genentech, Inc. Trastuzumab Herceptin Genentech, Inc. tretinoin, Vesanoid Induction of remission in patients with Roche ATRA acute promyelocytic leukemia (APL) who are refractory to or unable to tolerate anthracycline based cytotoxic chemotherapeutic regimens. Uracil Uracil Mustard Roberts Labs Mustard Capsules walrubicin Valstar For intravesical therapy of BCG- Anthra --> refractory carcinoma in situ (CIS) of Medeva the urinary bladder in patients for whom immediate cystectomy would be associated with unacceptable morbidity or mortality. vinblastine Welban Eli Lilly Vincristine Oncovin Eli Lilly Vincristine Oncovin Eli Lilly Vincristine Oncovin Eli Lilly Vincristine Oncovin Eli Lilly Vincristine Oncovin Eli Lilly Vincristine Oncovin Eli Lilly Vincristine Oncovin Eli Lilly vinorelbine Navelbine Single agent or in combination with Glaxo cisplatin for the first-line treatment of SmithKline ambulatory patients with unresectable, advanced non-Small cell lung cancer (NSCLC). vinorelbine Navelbine Navelbine is indicated as a single Glaxo agent or in combination with cisplatin SmithKline for the first-line treatment of ambulatory patients with unreseactable, advanced non-Small cell lung cancer (NSCLC). In patients with Stage IV NSCLC, Navelbine is indicated as a single agent or in combination with cisplatin. In Stage III NSCLC, Navelbine is indicated in combination with cisplatin. US 2011/0038948 A1 Feb. 17, 2011 33

TABLE 1-continued Drug Name Trade Name Indication Manufacturer zoledronate Zometa the treatment of patients with multiple Novartis myeloma and patients with documented bone metastases from Solid tumors, in conjunction with standard antineoplastic therapy. Prostate cancer should have progressed after treatment with at least one hormonal therapy

TABLE 2 TABLE 3

Injectable therapeutic agents for diabetes. Oral therapeutic agents for the treatment of diabetes.

Type of Insulin Example Category Generic Name Brand Name Manufacturer

-- Sulfonylurea Chlorpropamide Diabinese Pfizer Rapid-acting Humalog (lispro) (Eli Lilly) Glipizide Glucotrol Pfizer NovoLog (aspart) (Novo Nordisk) Glyburide DiaBeta Aventis, Short-acting (Regular) Humulin R (Eli Lilly) Micronase? Pharmacia Novolin RNovo Nordisk Glynase and Upjohn Intermediate-acting (NPH) Humulin N (Eli Lilly) Glimepiride Amaryl Aventis Novolin N (Novo Nordisk) Meglitinide Repaglinide Prandin Novo Nordisk Humulin L (Eli Lilly) Nateglinide Nateglinide Starlix Novartis Novolin L (Novo Nordisk) Biguanide Metformin Glucophage Bristol Myers Intermediate- and Humulin 50:50 Squibb short-acting mixtures Humulin 7030 Metformin (long Glucophage Bristol Myers Humalog Mix 75/25 lasting) XR Squibb Humalog Mix 50/50 Metformin with Glucovance Bristol Myers (Eli Lilly) glyburide Squibb Nowolin 70:30 Thiazolidinedione RosiglitaZone Avandia SmithKline Novolog Mix 70/30 (GlitaZone) Beecham (now GlaxoSmithKline) (Novo Nordisk) PioglitaZone Actos Takeda Long-acting Ultralente Pharmaceuticals (Eli Lilly) Alpha-Glucose Acarbose Precose Bayer Lantus (glargine) Inhibitor Miglitol Glyset Pharmacia and (Aventis) Upjohn

TABLE 4

Body weight and fat pad weights at baseline and 3-week after diet treatment in aP2-agouti transgenic mice fed low and high calcium diets'.

Baseline 3-week after

Low-Cadiet High-Ca diet Low-Cadiet High-Ca diet p value

Body weight (g)' 25.28 0.39 24.47 O.47 32.96 O.95 28.56 - O.S7* P = 0.023 Body fat (g) NA NA 4.47 0.37 2.44 0.23* P = 0.007 Subcutaneous fat (g) NA NA 1.76 O.17 O.94 0.11* P = 0.015 Visceral fat (g) NA NA 2.48 O.19 1.31 O.11* P = 0.004

Values are means + SD, n = 10, p-values indicate significant level between animals on the basal diet and those on the high-Cadiet, *Subscapular fat Sum of perirenal and abdominal fatpads US 2011/0038948 A1 Feb. 17, 2011 34

REFERENCES 0272 Chung, S. S., Ho, E. C., Lam, K. S., Chung, S. K. (2003) “Contribution of polyol pathway to diabetes-in 0257 Agarwal, A. K. (2003) “Cortisol metabolism and duced oxidative stress'JAm Soc Nephrol 14:S233-S236. visceral obesity: role of 11beta-hydroxysteroid dehydro 0273 Clement, K., Viguerie, N., Poitou, C., Carette, C., genase type I enzyme and reduced co-factor NADPH Pelloux, V., Curat, C. A., Sicard, A., Rome, S., Benis, A., Endocr Res. 29:411-418. Zucker, J. D., Vidal, H., Laville, M., Barsh, Basdevant, A., 0258 Alwine, J. C. et al. (1977) “Method for detection of Stich, V., Cancello, R., Langin, D. (2004) “Weight loss specific RNAS in agarose gels by transfer to diazobenzy regulates inflammation-related genes in white adipose tis loxymethyl-paper and hybridization with DNA probes’ sue of obese subjects' FASEBJ 18:1657-1669. Proc. Natl. Acad. Sci. USA 74:5350-5354. 0274 Cottam, D. R. S. G. Mattar, E. Barinas-Mitchell, G. 0259 Amstad, P. A., Krupitza, G., Cerutti, P. A. (1992) Eid, L. Kuller and D. E. Kelley et al. (2004) “The chronic “Mechanism of c-fos induction by active oxygen Cancer inflammatory hypothesis for the morbidity associated with ReS. 52:3952-6O. morbid obesity: implications and effects of weight loss” 0260 Argiles, J. M., Lopez-Soriano, J., Almendro, V., Obes Surg 14:589-600. Busquets, S., Lopez-Soriano, F. J. (2005) “Cross-talk (0275 Ermak, G., Davies, K. J. (2002) “Calcium and oxi between skeletal muscle and adipose tissue: a link with dative stress: from cell signaling to cell death' Mol Immu obesity' Med Res Rev. 25:49-65. nol 38:713-721. 0261 Atabek, M. E., Vatansev, H., Erkul, I. (2004) “Oxi 0276 Fain, J. N., A. K. Madan, M. L. Hiler, P. Cheema and dative stress in childhood obesity' J Pediatr Endocrinol S. W. Bahouth (2004) “Comparison of the release of adi Metab 17:1063-1068. pokines by adipose tissue, adipose tissue matrix, and adi 0262 Banakar, M.C., Paramasivan, S.K., Chattopadhyay, pocytes from visceral and Subcutaneous abdominal adi M. B., Datta, S., Chakraborty, P. Chatterjee, M., Kannan, pose tissues of obese humans' Endocrinology 145:2273 K., Thygarajan, E. (2004) “1 alpha, 25-dihydroxyvitamin 82. D3 prevents DNA damage and restores antioxidant 0277. Furukawa, S., Fujita, T., Shimabukuro, M., Iwaki, enzymes in rathepatocarcinogenesis induced by diethylni M. Yamada, Y., Nakajima, Y., Nakayama, O., Makishima, trosamine and promoted by Phenobarbital World J. Gas M. Matsuda, M., Shimomura, I. (2004) “Increased oxida troenterol 10:1268-75. tive stress in obesity and its impact on metabolic Syn 0263 Berchtold, M. W. et al. (1989) “A simple method for drome' J Clin Invest. 114:1752-1761. direct cloning and sequencing cDNA by the use of a single (0278 Fried, S. K. D. A. Bunkin and A. S. Greenberg specific oligonucleotide and oligo(dT) in a polymerase (1998) “Omental and subcutaneous adipose tissues of chain reaction (PCR) Nuc. Acids. Res. 17:453. obese subjects release interleukin-6: depot difference and 0264 Brand, M. D., Buckingham, J. A., Esteves, T. C., regulation by glucocorticoid’ J Clin Endocrinol Metab Green, K., Lambert, A.J., Miwa, S., Murphy, M.P., Pakay 83:847-50. J. L., Talbot, D. A., Echtay, K. S. (2004) “Mitochondrial (0279 Giri D. K. Aggarwal, B. B. (1998) “Constitutive Superoxide and aging: uncoupling-protein activity and activation of NF-kappaB causes resistance to apoptosis in superoxide production’ Biochem Soc Symp 71: 203-213. human cutaneous T cell lymphoma HuT-78 cells. Auto 0265 Brookes, P. S. (2005)“Mitochondrial H(+) leak and crine role of tumor necrosis factor and reactive oxygen ROS generation: an odd couple’ Free Radic Biol Med intermediates' J Biol. Chen. 273:14008-14. 38:12-23. 0280 Gordeeva, A. V., Zvyagilskaya, R. A., Labas, Y.A. 0266 Busquets, S., Figueras, M. T., Meijsing, S., Carbo, (2003) “Cross-talk between reactive oxygen species and N., Quinn, L. S., Almendro, V., Argiles, J. M., Lopez calcium in living cells' Biochemistry (Mosc). 68: 1077 Soriano, F. J. (2005) “Interleukin-15 decreases proteolysis 1080. in skeletal muscle: a direct effect' IntJ Mol. Med. 16:471 (0281 Hollander, M. C., Fornace, A. J. Jr. (1989) “Induc 6 tion of fos RNA by DNA-damaging agents' Cancer Res. 0267 Carbo, N., Lopez-Soriano, J., Costelli, P., Busquets, 49:1687-92. S., Alvarez, B., Baccino, F. M., Quinn, L. S., Lopez-Sori 0282 Hotamisligil, G.S., D. L. Murray, L. N. Choy and B. ano, F. J., Argiles, J. M. (2000a) “Interleukin-15 antago M. Spiegelman (1994)“Tumor necrosis factor alpha inhib nizes muscle protein waste in tumour-bearing rats' Br J its signaling from the insulin receptor Proc Natl AcadSci Cancer 83:526-531. USA 91.4854-8. 0268 Carbó, N., J. Lopez-Soriano, P. Costelli, S. 0283 Inoguchi, T., Li, P. Umeda, F.Yu. H.Y., Kakimoto, Busquets, B. Alvarez and F. M. Baccino et al. (2000b) M., Imamura, M., Aoki, T., Etoh, T., Hashimoto, T., “Interleukin-15 antagonizes muscle protein waste in Naruse, M., Sano, H., Utsumi, H., Nawata, H. (2000) tumour-bearing rats' Br Cancer 83:526-531. “High glucose level and free fatty acid stimulate reactive 0269 Carbó, N.J. Lopez-Soriano, P. Costelli, B. Alvarez, oxygen species production through protein kinase C-de S. Busquets and F. M. Baccino et al. (2001) “Interleukin-15 pendent activation of NAD(P)H oxidase in cultured vascu mediates reciprocal regulation of adipose tissue and lar cells’ Diabetes. 49:1939-1945. muscle mass: a potential role in body weight control Bio 0284. Khan, N., Sultana, S. (2004) “Induction of renal chim Biophy's Acta 1526:17-24. oxidative stress and cell proliferation response by ferric (0270. Cerutti, P., Ghosh, R., Oya, Y., Amstad, P. (1994) nitrilotriacetate (Fe-NTA): diminution by soy isoflavones' “The role of the cellular antioxidant defense in oxidant Chen Biol Interact. 149:23-35. carcinogenesis' Environ Health Perspect 102:123-9. 0285 Koren, R., Hadari-Naor, I., Zuck, E., Rotem, C., (0271 Chang, L., Karin, M. (2001) “Mammalian MAP Liberman, U.A., Ravid, A. (2001) “Vitamin D is a prooxi kinase signalling cascades' Nature 410:37-40. dant in breast cancer cells' Cancer Res. 61:1439-44. US 2011/0038948 A1 Feb. 17, 2011 35

0286 Korshunov, S. S., Skulachev, V. P. Starkov, A. A. (0301 Schulze-Osthoff, K., Bauer, M. K. Vogt, M., Wes (1997) “High protonic potential actuates a mechanism of selborg, S. (1997) “Oxidative stress and signal transduc production of reactive oxygen species in mitochondria' tion Int J. Vitam Nutr Res. 67:336-42. FEBS Left. 416: 15-18. (0302 Shangari, N., O'Brien, P. J. (2004) “The cytotoxic (0287 Kouzarides, T., Ziff, E. (1989) “Leucine zippers of mechanism of glyoxal involves oxidative stress’ Biochem fos, jun and GCN4 dictate dimerization specificity and Pharmacol. 68:1433-1442. thereby control DNA binding Nature. 340:568-71. 0303 Shi, H., Norman, A. W., Okamura, W. H., Sen, A., 0288 Lee, Y. H. Pratley, R. E. (2005)“The evolving role of Zemel, M. B. (2001) “1alpha,25-Dihydroxyvitamin D3 inflammation in obesity and the metabolic syndrome'. Curr modulates human adipocyte metabolism via nongenomic Diab Rep. 5:70-75. action FASEBI. 15:2751-2753. 0289 Li, Y. Bujo, H., Takahashi, K., Shibasaki, M., Zhu, 0304 Shi, H., Norman, A. W., Okamura, W. H., Sen, A., Y.Yoshida, Y., Otsuka, Y., Hashimoto, N., Saito, Y. (2003) Zemel, M.B. (2002) “1C.25-dihydroxyvitamin D. inhibits 'Visceral fat: higher responsiveness of fat mass and gene uncoupling protein 2 expression in human adipocytes' expression to calorie restriction than Subcutaneous fat” FASEB 16:1808-1810. Exp Biol Med (Maywood) 228:1118-1123. (0305 Simeone, A. M., Tari, A. M. (2004) “How retinoids 0290 Lin, J. K. (2004) “Suppression of protein kinase C regulate breast cancer cell proliferation and apoptosis' and nuclear oncogene expression as possible action Cell Mol Life Sci. 61:1475-84. mechanisms of cancer chemoprevention by Curcumin' 0306 Soares, A. F., Guichardant, M., Cozzone, D., Ber Arch Pharm Res. 27:683-92. noud-Hubac, N., Bouzaidi-Tiali, N., Lagarde, M., Geloen, 0291 Lin, T. K., Chen, S. D., Wang, P.W., Wei, Y. H., Lee, A. (2005) “Effects of oxidative stress on adiponectin secre C. F., Chen, T. L., Chuang, Y.C., Tan, T.Y., Chang, K.C., tion and lactate production in 3T3-L1 adipocytes' Free Liou, C. W. (2005) “Increased Oxidative Damage with Radic Biol Med. 38:882-889. Altered Antioxidative Status in Type 2 Diabetic Patients (0307 Song, Y. S., Rosenfeld, M. E. (2004) “Methionine Harboring the 16189 T to C Variant of Mitochondrial induced hyperhomocysteinemia promotes Superoxide DNA Ann NYAcad. Sci. 1042:64-69. anion generation and NFkappaB activation in peritoneal 0292. Mahadev, K., Motoshima, H. Wu. X., Ruddy, J. M., macrophages of C57BL/6 mice' J Med Food. 7:229-34. Arnold, R. S., Cheng, G., Lambeth, J. D., Goldstein, B.J. 0308 Sonta, T., Inoguchi, T., Tsubouchi, H., Sekiguchi, (2004)“The NAD(P)H oxidase homolog Nox4 modulates N. Kobayashi, K., Matsumoto, S., Utsumi, H., Nawata, H. insulin-stimulated generation of H2O2 and plays an inte (2004) “Evidence for contribution of vascular NAD(P)H gral role in insulin signal transduction” Mol Cell Biol. oxidase to increased oxidative stress in animal models of 24:1844-54. diabetes and obesity' Free Radic Biol Med. 37: 115-123. 0309 Sorescu, D., Weiss, D., Lassegue, B., Clempus, R. 0293. Manea, A., Constantinescu, E., Popov, D., Raicu, M. E., Szocs, K., Sorescu, G. P. Valppu, L., Quinn, M. T., (2004) “Changes in oxidative balance in rat pericytes Lambeth, J. D., Vega, J. D., Taylor, W. R. Griendling, K. K. exposed to diabetic conditions' J Cell Mol Med. 8:117 (2002)“Superoxide production and expression of nox fam 126. ily proteins in human atherosclerosis' Circulation 105: 0294 Melton, D. A. et al. (1984) “Efficient in vitro syn 1429-1435. thesis of biologically active RNA and RNA hybridization 0310 Sun, X. C., Morris, K., Zemel, M. B. (2005) “1, probes form plasmids containing a bacteriophage SP6 pro 25(OH)2D3 and reactive oxygen species interactively moter Nuc. Acids Res. 12:7035-7056. stimulate angiotensinogen expression in differentiated 0295 Morris, K. L., Zemel, M. B. (2005)“1,25-dihydrox 3T3-L1 adipocytes' FASEB.J. 19:A70 (abstract). yvitamin D3 modulation of adipocyte glucocorticoid func 0311 Sun, X. C., Zemel, M. B. (2004a) “Calcium and tion Obes Res. 13:670-677. Dairy Products Inhibit Weight and Fat Regain during Ad 0296 Miwa, S., Brand, M. D. (2003) “Mitochondrial Libitum Consumption Following Energy Restriction in matrix reactive oxygen species production is very sensitive Ap2-Agouti Transgenic Mice'J Nutr. 134:3054-3060. to mild uncoupling Biochem Soc Trans. 31: 1300-1. 0312 Sun, X. Zemel, M. B. (2004b) “Role of uncoupling 0297 Nomura, M., Ma, W., Chen, N., Bode, A. M., Dong, protein 2 (UCP2) expression and 1C.25-dihydroxyvitamin Z. (2000) “Inhibition of 12-O-tetradecanoylphorbol-13 D. in modulating adipocyte apoptosis” FASEB.J. 18:1430 acetate-induced NF-kappaB activation by tea polyphenols, 1432. (-)-epigallocatechin gallate and theaflavins' Carcinogen 0313 Sun, X. C., Zemel, M. B. (2004c) “Dual effect of eSiS. 21:1885-90. 1-alpha,25-dihydroxyvitamin D-3 on adipocyte apoptosis 99 0298. Ofei, F. S. Hurel, J. Newkirk, M. Sopwith and R. FASEB.J. 18:A49 (abstract). Taylor (1996) “Effects of an engineered human anti-TNF 0314 Sun, X. C., Zemel, M. B. (2004d) “Reactive oxygen alpha antibody (CDP571) on insulin sensitivity and glyce species stimulate cell proliferation and down-regulate mic control in patients with NIDDM Diabetes 45:881 UCP2 expression in 3T3-L1 adipocytes' Obesity Research 885. 11:A37(abstr). 0299 Quinn, L. S., Strait-Bodey, L., Anderson, B. G., 0315 Sun, X. C., Zemel, M. B (2006) “Dietary calcium Argiles, J. M., Havel, P. J. (2005) “Interleukin-15 stimu regulates ROS production in aP2-agouti transgenic mice lates adiponectin secretion by 3T3-L1 adipocytes: Evi on high fat/high sucrose diets’ IntJ Obesity, in press. dence for a skeletal muscle-to-fat signaling pathway Cell 0316 Suzuki, K., Ito, Y., Ochiai, J., Kusuhara, Y., Hash Biol Int. 29:449-57 imoto, S., Tokudome, S. Kojima, M., Wakai, K., 0300 Reeves, P. G. (1997) “Components of the AIN-93 Toyoshima, H., Tamakoshi, K., Watanabe. Y., Hayakawa, diets as improvements in the AIN-76A diet J Nutr. 127: N., Maruta, M., Watanabe, M., Kato, K., Ohta, Y., Tama 838 S-841S. koshi, A.; JACC Study Group (2003) “Relationship US 2011/0038948 A1 Feb. 17, 2011 36

between obesity and serum markers of oxidative stress and 0325 Weisberg, S. P. McCann, D., Desai, M., Rosen inflammation in Japanese Asian Pac J Cancer Prev. baum, M., Leibel, R. L., Ferrante, A. W.Jr. (2003)“Obesity 4:259-266. is associated with macrophage accumulation in adipose 0317 Thompson, W. G., Rostad Holdman, N., Janzow, D. tissue” J. Clin Invest. 112:1796-808. J., Slezak, J. M., Morris, K. L., Zemel, M.B. (2005)“Effect 0326 Weitzman, S.A., Turk, P. W., Milkowski, D. H., of energy-reduced diets high in dairy products and fiber on Kozlowski, K. (1994) “Free radical adducts induce alter weight loss in obese adults' Obes Res. 2005:13:1344-53. ations in DNA cytosine methylation” Proc Natl Acad Sci 0318 Valle, M., Martos, R., Gascon, F., Canete, R. Zafra, USA. 91:1261-4. M. A., Morales, R. (2005) “Low-grade systemic inflam 0327 Zemel, M. B., Shi, H., Greer, B., Dirienzo, D., mation, hypoadiponectinemia and a high concentration of Zemel, P. C. (2000) “Regulation of adiposity by dietary leptin are present in very young obese children, and corre calcium FASEB.J. 14:1132-1138. late with metabolic syndrome’ Diabetes Metab. 31:55-62. 0328. Zemel, M. B. (2003) “Effects of mitochondrial 0319 Volk, T., Hensel, M., Kox, W.J. (1997) “Transient uncoupling on adipocyte intracellular Ca2+ and lipid Ca2+ changes in endothelial cells induced by low doses of metabolism'J Nutr Biochem. 14:219-226. reactive oxygen species: role of hydrogen peroxide' Mol 0329 Zemel, M. B. (2004) “Role of calcium and dairy Cell Biochen. 171:11-21. products in energy partitioning and weight management’ 0320 Upham, B. L., Kang, K. S., Cho, H.Y., Trosko, J. E. (1997) “Hydrogen peroxide inhibits gap junctional inter An I Clin Nutr 79:907 S-912S. cellular communication in glutathione Sufficient but not 0330 Zemel, M. B., Thompson, W., Milstead, A., Morris, glutathione deficient cells' Carcinogenesis 18:37-42. K., Campbell P. (2004) “Calcium and dairy acceleration of 0321 Xu, H., Barnes, G. T., Yang, Q. Tan, G., Yang, D., weight and fat loss during energy restriction in obese Chou, C.J., Sole, J., Nichols, A., Ross, J. S., Tartaglia, L. adults' Obes Res. 12:582-90. A., Chen, H. (2003) “Chronic inflammation in fat plays a 0331 Zemel, M. B. (2005a) “Calcium and dairy modula crucial role in the development of obesity-related insulin tion of obesity risk” Obes Res. 13(1):192-193. resistance J Clin Invest. 112:1821-1830. 0332 Zemel, M. B. (2005b) “The role of dairy foods in 0322 Xue, B., Zemel, M. B. (2000) “Relationship weight management J. Am Coll Nutr. 24(6 Suppl):537S between human adipose tissue agouti and fatty acid syn 546S thase (FAS)”J Nutr. 130:2478-2481. 0333 Zemel, M.B., Richards, J., Mathis, S., Milstead, A., 0323 Xue, B., Moustaid, N., Wilkison, W. O., Zemel, M. Gebhardt, L., Silva, E. (2005a) “Dairy augmentation of B. (1998) “The agouti gene product inhibits lipolysis in total and central fat loss in obese subjects' IntJ Obes Relat human adipocytes via a Ca"-dependent mechanism” Metab Disord. 29:391-397. FASEBI. 12:1391-1396. 0334 Zemel, M. B., Richards, J., Milstead, A., Campbell, 0324 Wajchenberg, B. L. (2000) “Subcutaneous and vis P. (2005b) “Effects of calcium and dairy on body compo ceral adipose tissue: their relation to the metabolic syn sition and weight loss in African-American adults' Obes drome Endocr Rev. 21:697-738. ReS. 2005:13:1218-25.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 2

<21 Oc SEO ID NO 1 <211 LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <22 Os FEATURE; <223> OTHER INFORMATION: Forward PCR primer for UCP2

<4 OOs SEQUENCE: 1

gctagoatgg ttggitttcaa g 21

SEO ID NO 2 LENGTH: 21 TYPE: DNA ORGANISM: Artificial sequence FEATURE; OTHER INFORMATION: Reverse PCR primer for UCP2

<4 OOs SEQUENCE: 2

gctagotcag aaaggtgaat c 21 US 2011/0038948 A1 Feb. 17, 2011 37

We claim: bination of candidate compositions to at least one test 1. An in vitro method of screening compounds or compo subject with the proviso that said candidate compound, sitions suitable for reducing the production of reactive oxy combination of candidate compounds, candidate com gen species (ROS) comprising: position, or combination of candidate compositions, a) contacting one or more cell(s) with a candidate com when administered orally to said test subject, is not pound, combination of candidate compounds, candidate being administered to said at least one test subject orally composition, or combination of candidate compositions as a component of the diet of said at least one test Subject with the proviso that said candidate compound, combi or as dietary calcium to said test subject; and nation of candidate compounds, candidate composition, b) measuring one or more of the following parameters: or combination of candidate compositions is not dietary i) intracellular calcium concentrations in cells of said at material containing calcium or dietary calcium; and least one test subject and at least one control Subject. b) measuring one or more of the following parameters: wherein a decrease of intracellular calcium concen i) intracellular concentrations of calcium in said one or tration in the cells of a test subject as compared to the more cell(s), wherein a decrease of intracellular cal intracellular concentrations of calcium in the cells of cium concentration in said cell(s) is indicative of a at least one control subject is indicative of a com compound or composition suitable for use in reducing pound, composition, combination of compounds or the production of ROS; combination of compositions suitable for use in ii) UCP2 expression in said one or more cell(s), wherein reducing the production of ROS in a subject; an increase in UCP2 expression in said cell(s) is ii) UCP2 expression in cells of said at least one test indicative of a compound or composition suitable for subject and at least one control subject, wherein an use in reducing the production of ROS; increase of UCP2 expression in the cells of a test iii) NADPH oxidase expression in said one or more subject as compared to the UCP2 expression in the cell(s), wherein a decrease in NADPH oxidase cells of at least one control subject is indicative of a expression in said cell(s) is indicative of a compound compound, composition, combination of compounds or composition suitable for use in reducing the pro or combination of compositions suitable for use in duction of ROS; reducing the production of ROS in a subject; iv) UCP3 expression in said one or more cell(s), wherein iii) NADPH oxidase expression in cells of said at least an increase in UCP3 expression in said cell(s) is one test subject and at least one control Subject, indicative of a compound or composition suitable for wherein a decrease of NADPH oxidase expression in use in reducing the production of ROS; the cells of a test subject as compared to the NADPH v) NADPH oxidase expression in said one or more cell oxidase expression in the cells of at least one control (s), whereina decrease in NADPH oxidase expression subject is indicative of a compound, composition, in said cell(s) is indicative of a compound or compo combination of compounds or combination of com sition suitable for use in reducing the production of positions suitable for use in reducing the production ROS: of ROS in a subject; vi) 11 B-HSD expression in said one or more cell(s), iv) UCP3 expression in skeletal muscle cells of said at wherein a decrease in the expression of 11 B-HSD in least one test subject and at least one control subject, said cell(s) is indicative of a compound or composi wherein an increase in UCP3 expression in the skel tion suitable for use in reducing the production of etal muscle cells of a test subject as compared to ROS; UCP3 expression in the skeletal muscle cells of at vii) TNF-ct, CD14, MIF, M-CSF, MIP, MCP-1, G-CSF least one control subject is indicative of a compound, or IL-6 expression in said one or more cell(s), wherein composition, combination of compounds or combi a decrease in the expression of TNF-C., CD14, MIF nation of compositions suitable for use in reducing the (macrophage inhibitory factor), MIP (macrophage production of ROS in a subject; inhibitory protein), M-CSF (macrophage colony v) NADPH oxidase expression in skeletal muscle cells stimulating factor), MCP-1 (monocyte chemoattrac of said at least one test subject and at least one control tant protein-1), G-CSF (granulocyte colony stimulat subject, wherein a decrease of NADPH oxidase ing factor) or IL-6 in said cell(s) is indicative of a expression in the skeletal muscle cells of a test subject compound or composition suitable for use in reducing as compared to the NADPH oxidase expression in the the production of ROS; or skeletal muscle cells of at least one control subject is viii) IL-15 or adiponectin expression in said one or more indicative of a compound, composition, combination cell(s), wherein an increase in the expression of IL-15 of compounds or combination of compositions Suit or adiponectin in said cell(s) is indicative of a com able for use in reducing the production of ROS in a pound or composition suitable for use in reducing the subject; production of ROS. vi) 11 B-HSD expression in visceral adipocyte tissue or 2. The method according to claim 1, wherein said one or cells of said at least one test subject and at least one more cell(s) is a adipocyte or an adipocyte cell line. control subject, wherein a decrease of 11 B-HSD 3. The method according to claim 2, wherein said adipo expression in the visceral adipocyte tissue or cells of cyte or adipocyte cell line is human(s) or a murine. a test subject as compared to the 11 f-HSD expression 4. A method of identifying or screening compounds or in the visceral adipocyte tissue or cells of at least one compositions suitable for reducing the production of reactive control subject is indicative of a compound, compo oxygen species (ROS) comprising: sition, combination of compounds or combination of a) administering a candidate compound, combination of compositions suitable for use in reducing the produc candidate compounds, candidate composition, or com tion of ROS in a subject; US 2011/0038948 A1 Feb. 17, 2011 38

vii) TNF-ct, CD14, MIF, MIP, M-CSF, MCP-1, G-CSF 12. The method according to claim 11, wherein the thera or IL-6 expression in said one or more cell(s), wherein peutic agent ortherapeutic agents and calcium, or physiologi a decrease in the expression of TNF-C, CD14, MIF, cally acceptable salts of calcium, are administered as a single MIP, M-CSF, MCP-1, G-CSF or IL-6 in said cell(s) is composition. indicative of a compound or composition Suitable for 13. The method according to claim 12, wherein the thera use in reducing the production of ROS; or peutic agent ortherapeutic agents and calcium, or physiologi viii) IL-15 or adiponectin expression in said one or more cally acceptable salts of calcium, are administered as separate cell(s), wherein an increase in the expression of IL-15 or different compositions. or adiponectin in said cell(s) is indicative of a com 14. The method according to claim 13, wherein the sepa pound or composition Suitable for use in reducing the rate or different compositions are administered simulta production of ROS. neously, sequentially or contemporaneously. 5. The method according to claim 4, wherein a candidate 15. A method of reducing ROS production in a diabetic compound, combination of candidate compounds, candidate individual comprising the administration of one or more com composition, or combination of candidate compositions is position comprising one or more therapeutic agent as set forth administered to a subject via intravenous, intraarterial, buc in Table 2 or Table 3 and calcium, or physiologically accept cal, topical, transdermal, rectal, intramuscular, Subcutaneous, able salts of calcium, in an amount Sufficient to reduce the intraosseous, transmucosal, or intraperitoneal routes of production of ROS in said individual. administration. 16. A method of altering the expression of cytokines in an 6. The method according to claim 5, wherein said candidate individual or the cytokine profile of an individual comprising compound, combination of candidate compounds, candidate the administration of a compound, composition, combination composition, or combination of candidate compositions is of compounds, or combination of compositions that decrease administered to at least one test subjectorally with the proviso intracellular calcium levels to an individual in need of such that said candidate compound, combination of candidate treatment in amounts Sufficient to decrease intracellular lev compounds, candidate composition, or combination of can els of calcium in the cells of the individual, decrease TNF-C., didate compositions is not being administered to said at least CD14, MIP, MIF, M-CSF, G-CSF or IL-6 expression, or any one test Subject orally as a component of the diet of said at combination thereof, in the individual, and increase the least one test Subject or as dietary calcium to said test Subject. expression of IL-15, adiponectin, or both IL-15 or adiponec 7. A method of treating diseases associated with reactive tin in the individual with the proviso that said compound, oxygen species (ROS) comprising the administration of a combination of compounds, composition, or combination of compound, composition, combination of compounds, or compositions is not a dietary material containing calcium or combination of compositions that increase intracellular cal cium levels to an individual in need of such treatment in dietary calcium. amounts sufficient to increase the intracellular concentrations 17. A composition comprising one or more therapeutic of calcium in the cells of the individual with the proviso that agent selected from Tables 1 or 2 or 3 in combination with said compound, combination of compounds, composition, or calcium or one or more physiological salts of calcium. combination of compositions is not a dietary material con 18. The composition according to claim 17, wherein said taining calcium or dietary calcium. composition contains between: 1 and 2000 mg:900 and 1500 8. The method according to claim 7, further comprising the mg: 1000 and 1400 mg: 1200 and 1300 mg: 1100 and 1300 step of diagnosing or identifying an individual as having a mg; or 1200 and 1300 mg of calcium or one or more physi disease or disorder associated with ROS or diagnosing or ologically acceptable salts thereof. identifying an individual having elevated ROS levels com 19. The composition according to claim 17, wherein said prising measuring the levels of ROS and comparing the mea one or more physiological salts of calcium are selected from sured levels against a standard or collection of ROS levels calcium phosphates, calcium carbonate, calcium chloride, from control Subjects. calcium sulfate, calcium tartrate, calcium magnesium car 9. The method according to claim 7, wherein the ROS bonate, calcium metasilicate, calcium malate, secondary cal associated disease or disorder is cataracts, diabetes, Alzhe cium orthophosphate, calcium citrate, or calcium hydroxide. imer's disease, heart disease, cancer, male infertility, inflam 20. A method of increasing the in vitro expression of MIF, mation, amyotrophic lateral Sclerosis, Parkinson's disease, M-CSF, MIP, IL-6, IL-10, IL-4, IL-13, MIG, IL-5, VEGF, multiple Sclerosis or aging. CD14, G-CSF, TNF-C, RANTES, or MIP-1C. comprising 10. The method according to claim 8, wherein the ROS contacting a composition comprising a carrier and calcitriol associated disease or disorder is cataracts, diabetes, Alzhe (1, 25-(OH)-D) with a adipocytes, skeletal muscle cells, imer's disease, heart disease, cancer, male infertility, inflam skeletal muscle cell lines, human adipocyte cell lines, murine mation, amyotrophic lateral Sclerosis, Parkinson's disease, adipocyte cell lines or transformed host cells comprising multiple Sclerosis or aging. MIF, M-CSF, MIP, IL-6, IL-10, IL-4, IL-13, MIG, IL-5, 11. The method according to claim 7, wherein said ROS VEGF, CD14, G-CSF, TNF-ct, RANTES, or MIP-1C genes associated disease or disorder is cancer-associated ROS dis and culturing said cells under conditions that allow for the ease or disorders and said method comprises the administra production of MIF, M-CSF, MIP, IL-6, IL-10, IL-4, IL-13, tion of one or more composition comprising calcium, or MIG, IL-5, VEGF, CD14, G-CSF, TNF-C, RANTES, or MIP physiologically acceptable salts of calcium, and a therapeutic 1C. agent selected from alkylating agents, antibiotics which 21. The method according to claim 20, further comprising affect nucleic acids, platinum compounds, mitotic inhibitors, the recovery of MIF, M-CSF, MIP, IL-6, IL-10, IL-4, IL-13, antimetabolites, camptothecin derivatives, biological MIG, IL-5, VEGF, CD14, G-CSF, RANTES, or MIP-1C. response modifiers, hormone therapies or any of the thera peutic agents is identified in Table 1. c c c c c