US 20070077310A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2007/0077310 A1 Zemel et al. (43) Pub. Date: Apr. 5, 2007
(54) METHODS OF REDUCING THE Publication Classification PRODUCTION OF REACTIVE OXYGEN SPECIES AND METHODS OF SCREENING (51) Int. Cl. ORDENTIFYING COMPOUNDS AND CI2O I/26 (2006.01) COMPOSITIONS THAT REDUCE THE A 6LX 3L/355 (2006.01) PRODUCTION OF REACTIVE OXYGEN A6IR 33/42 (2006.01) SPECIES A6IR 33/10 (2006.01) (75) Inventors: Michael B. Zemel, Knoxville, TN (52) U.S. Cl...... 424/602; 424/687: 514/355; (US); Xiaocun Sun, Knoxville, TN 514/458; 435/25 (US) Correspondence Address: SALWANCHIK LLOYD & SALWANCHK (57) ABSTRACT A PROFESSIONAL ASSOCATION PO BOX 142950 The Subject application provides methods of identifying GAINESVILLE, FL 32614-2950 (US) compounds, combinations of compounds, compositions, and/or combinations of compositions that are Suitable for (73) Assignee: University of Tennessee Research reducing the production of reactive oxygen species (ROS) in Foundation, Knoxville, TN an individual with the proviso that said compound, combi Appl. No.: 11/542,703 nation of compounds, composition, or combination of com (21) positions is not a dietary material containing calcium or (22) Filed: Oct. 3, 2006 dietary calcium. Also provided in the Subject application are methods of treating diseases or disorders associated with Related U.S. Application Data ROS production and methods of reducing ROS production in an individual with the proviso that said compound, (60) Provisional application No. 60/723,042, filed on Oct. combination of compounds, composition, or combination of 3, 2005. Provisional application No. 60/787,819, filed compositions is not a dietary material containing calcium or on Mar. 31, 2006. dietary calcium. Patent Application Publication Apr. 5, 2007 Sheet 1 of 38 US 2007/007731.0 A1
Z. A *(p<0.015) 250 se (p<0.01) Owild-type mice 200 Transgenic mices
150 100 O A1 50
O Baseline After Diet
*(p<0.001)
a 4 *(-0.01<0.01) Owild-type mice cos Transgenic mice- gld 3 - s O 2 A. aS 1 O Baseline After Diet
FIG. 2 Patent Application Publication Apr. 5, 2007 Sheet 2 of 38 US 2007/007731.0 A1
12 - * (p P O.04) - BodyE. Weightfat weight
Basal diet High calcium diet
FIG. 3
180 150 120
Basal diet High calcium diet
FIG. 4 Patent Application Publication Apr. 5, 2007 Sheet 3 of 38 US 2007/007731.0 A1
46 2 600 (p<0.001)(p * (p<0.012) a 2.5 O Viceral fat Subcutaneous fat isr 2 m-- o 1.5 A. A <6 1 Z. 0.5 O Basal diet High calcium diet FIG. 6 Patent Application Publication Apr. 5, 2007 Sheet 4 of 38 US 2007/007731.0 A1 (p<0.001) O Viceral fat Subcutaneous fat 2 O O Basal diet High calcium diet FIG. 7 * (p<0.001) # (p<0.001) Viceral fat Subcutaneous fat Basal diet High calcium diet FIG. 8 Patent Application Publication Apr. 5, 2007 Sheet 5 of 38 US 2007/007731.0 A1 Basal diet High calcium diet FIG. 10 Patent Application Publication Apr. 5, 2007 Sheet 6 of 38 US 2007/007731.0 A1 Viceral fat Subcutaneous fat Basal diet High calcium diet FIG. 11 D Control 600 attocopherol 500 4 OO 300 200 100 O -- -- GDP Nifedipine Patent Application Publication Apr. 5, 2007 Sheet 7 of 38 US 2007/0077310 A1 80 O Control or it tocopherol 6 O - 4 0 20 O Control H.O, Ho. Ho, GDP Nifedipine FIG. 13 D Wild type l UCP2 transfected 0.8 .S. 5 0.6 d d is 0.4 r s2 0.2 9 S. > O Control H2O, -- -- GDP Nifedipine FIG. 14 Patent Application Publication Apr. 5, 2007 Sheet 8 of 38 US 2007/007731.0 A1 600 is 450 E s 300 es S. 150 O Control H2O2 -- otocopherol 280Ž O) S560 s s 40 C A3 20 O Control Glucose Glucose Glucose Glucose -- -- 1,25D GDP Nifedipine FIG. 16 Patent Application Publication Apr. 5, 2007 Sheet 9 of 38 US 2007/007731.0 A1 300 S 200 *cy 100 9. O Control Glucose Glucose -- dittocopherol FIG. 17 UCP2 transfected 2 O Wild-type 1 6 1 2 Control Glucose Glucose Glucose Glucose ------Nifedipine 1, 25D GDP FIG. 18 Patent Application Publication Apr. 5, 2007 Sheet 10 of 38 US 2007/0077310 A1 1 8 DUCP2 transfected 12 Control Glucose Glucose Glucose Glucose ------Nifedipine 1, 25D GDP FIG. 19 Control Glucose Glucose Glucose Glucose -H -H -H 125D GDP Nifedipine FIG. 20 Patent Application Publication Apr. 5, 2007 Sheet 11 of 38 US 2007/0077310 A1 3 & t 2 . 3. 1 O Control Glucose Glucose Glucose Glucose ------Nifedipine 1, 25D GDP FIG 21 Subcutaneous fat Visceral fat FIG.22A Patent Application Publication Apr. 5, 2007 Sheet 12 of 38 US 2007/0077310 A1 Basal diet High-Ca diet Basal diet High-Ca diet Subcutaneous fat Visceralfat FIG. 22B Basal diet High-Cadiet -N-1 Visceral fat FIG. 23A Patent Application Publication Apr. 5, 2007 Sheet 13 of 38 US 2007/007731.0 A1 1.6 b 3 1.2 s 0.8 O . t O Basal diet High-Ca diet N-N-1 Visceral fat 2.5 2 b 5 àis ,1 a O Basal diet High-Cadiet Y-N-1 Soleus muscle FIG. 23C Patent Application Publication Apr. 5, 2007 Sheet 14 of 38 US 2007/007731.0 A1 ; Control 1, 25 (OHDD, Nifedipine 1, 25(OH).D. -- Nifedipine FIG. 24A Control 1, 25(OH)D. Nifedipine 1, 25 (OH). D. re Nifedipine FIG. 24B Basal diet High-Ca Diet FIG. 24C Patent Application Publication Apr. 5, 2007 Sheet 15 of 38 US 2007/007731.0 A1 0.3 O C tr ol l, 2 5( O H), D N ifedipine 1,25(OH),D, r Nifedipine FIG. 25A Control l, 25(OH),D, Nifedipine 1,25(OH),D, -- Nifedipine FIG. 2SB Patent Application Publication Apr. 5, 2007 Sheet 16 of 38 US 2007/007731.0 A1 i Control l, 25(OH), D, Nifedipine l, 25(OH),D, -- Nifedipine FIG. 25C O Control 1, 25OH).D. Nifedipine 1,25OHDDs -- Nifedipine FIG. 25D Patent Application Publication Apr. 5, 2007 Sheet 17 of 38 US 2007/007731.0 A1 1.2 0.9 3 0.6 S NaC s H 0.3 O Control attocopherol HO, HO, -- attocopherol C y 9 e S. es Control attocopherol HO HO, attocopherol FIG. 26B Patent Application Publication Apr. 5, 2007 Sheet 18 of 38 US 2007/007,731.0 A1 1. 0.8 0.6 CO S '? 0.4 s 0.2 O Control attocopherol HO HO -H otocopherol (A) so l d Control Calcitriol Calcitriol Nifedipine -- Nifedipine FIG. 27A Patent Application Publication Apr. 5, 2007 Sheet 19 of 38 US 2007/007731.0 A1 0.6 So 0.4 s'? | | | || 8" | | | | | | | | O ... . . Control Calcitriol Calcitriol Nifedipine Nifedipine FIG. 27B Control Calcitriol Calcitriol Nifedipine FIG. 28A Patent Application Publication Apr. 5, 2007 Sheet 20 of 38 US 2007/007731.0 A1 Control Calcitriol Calcitriol Nifedipine FIG. 28B OControl Co-Culture Control Calcitriol Calcitriol Calcitriol He s Nifedipine DNP FIG. 29 Patent Application Publication Apr. 5, 2007 Sheet 21 of 38 US 2007/007731.0 A1 Control 3 Co-Culture 8 2 n 2 O Control Calcitriol Calcitriol Calcitrio H Nifedipine DNP FIG. 30 Patent Application Publication Apr. 5, 2007 Sheet 22 of 38 US 2007/007731.0 A1 Control on 3 Co-Culture OO S 2 1 O Control Calcitriol Calcitriol Calcitriol -- H. Nifedipine DNP FIG. 31 1 2 3 4 5 6 7 8 910 Control FIG. 32A Patent Application Publication Apr. 5, 2007 Sheet 23 of 38 US 2007/007731.0 A1 -.' . . v , . , -, real-e-r-e- Calcitrio-teated FIG. 32B 1 2 M-CSF (NFS) fy IL-12 Schematic diagram of antibody array FIG. 32C Patent Application Publication Apr. 5, 2007 Sheet 24 of 38 US 2007/007731.0 A1 8C: s It Wis s b & S as c y NW s 3.c s (S 3. S Q1 & s W sa c C. S S Sto co to Sr S 2 V Asued Patent Application Publication Apr. 5, 2007 Sheet 25 of 38 US 2007/007731.0 A1 1 2 3 4 5 S 7 8 9 10 E Control FIG. 33A Calcitrio-treated FIG. 33B Patent Application Publication Apr. 5, 2007 Sheet 26 of 38 US 2007/007731.0 A1 Schematic diagram of antibody array FIG. 33C Patent Application Publication Apr. 5, 2007 Sheet 27 of 38 US 2007/007731.0 A1 8CS3 | 8 W It QoisS s 'f & S 3. s N W s & s é (Sf NS S W 8 as g c g Patent Application Publication Apr. 5, 2007 Sheet 28 of 38 US 2007/007731.0 A1 Control Co-Culture Control Calcitrio Calcitrio Calcitrio se Nifedipine DNP . FIG. 34 O Control Co-Culture Control Calcitriol Calcitrio Calcitriol s an Nifedipine DNP FIG. 35 Patent Application Publication Apr. 5, 2007 Sheet 29 of 38 US 2007/007731.0 A1 O Control Co-Culture 4 O - Control Calcitrio Calcitrio Calcitrio as o Nifedipine DNP FIG. 36 P = 0.012 basal vs. calcium. p = 0.285 basal vs. milk p = 0.005 calcium vs. milk. p = 0.018 Basal High-Ca Milk FIG. 37 Patent Application Publication Apr. 5, 2007 Sheet 30 of 38 US 2007/007731.0 A1 P = 0.002 basal vs. calcium p = 0.038 basal vs. milk. p = 0.001 calcium vs. milk. p = 0.018 i 24 H P = 0.05 basal vs. calcium: p = 0.290 basal vs. milk: p = 0.060 calcium vs. milk. p = 0.20 O 1 0. O 8 0. O 6 0.02 Patent Application Publication Apr. 5, 2007 Sheet 31 of 38 US 2007/007731.0 A1 P = 0.001 basal vs. calcium. p = 0.178 basal vs. milk p < 0.001 calcium vs. milk. p = 0.002 o E O) CD 3. 5 .2 - Basal High-Ca Milk P & O.001 Plasma 1, 25 D basal vs. calcium. p = 0.002 basal vs. milk p < 0.001 50 calcium vs. milk. p = 0.059 O) Ot LD (N Basal High-Ca Milk FIG. 41 Patent Application Publication Apr. 5, 2007 Sheet 32 of 38 US 2007/007731.0 A1 P = 0.001 basal vs. calcium. p = 0.002 basal vs. milk. p = 0.001 calcium vs. milk. p = 0.030 SOO 500 400 3OO 2OO 1 OO Adipose (perirenal fat) P & O.OO1 gene expression basal vs. calcium p < 0.001 basal vs. milk. p < 0.001 Calcium vs. milk. p = 0.058 | Basal High-Ca Milk FIG. 43 Patent Application Publication Apr. 5, 2007 Sheet 33 of 38 US 2007/007731.0 A1 P & O.OO1 Plasma MDA basal VS. Calcium. p = 0.001 basal vs. milk. p < 0.001 calcium vs. milk. p = 0.039 400 . 200 100 | | | | | | Basal High-Ca Milk FIG. 44 Adipose (perirenal fat) If s P & O.OO1 gene expression basal vs. Calcium. p = 0.001 basal vs. milk. p < 0.001 calcium vs. milk. p = 0.078 Patent Application Publication Apr. 5, 2007 Sheet 34 of 38 US 2007/007731.0 A1 Adipose (perirenal fat) P = 0.002 basal vs. calcium. p = 0.006 gene expression basal vs. milk p = 0.001 calcium vs. milk. p = 0.217 Adipose (perirenal fat) P 0001 basal vs. calcium p = 0.003 gene expression basal vs. milk" p < 0.001 calcium vs. milk. p = 0.183 Patent Application Publication Apr. 5, 2007 Sheet 35 of 38 US 2007/007731.0 A1 Adipose cvtokine release P-0130 p yt S Basal vs. Calcium: 0.118 Basal vs. milk. O.059 Calcium vS. milk. 0.725 0.01 O.008 O.OO6 O.OO4 O.OO2 Basal High-Ca Milk FIG. 48 Adipose4. cytokinep release pBasal = 0.008 VS. Calcium: 0.033 BaSal VS. milk. 0.003 Calcium V.S. Milk. O.122 1.6 E 92 9. 1.2 Oe g S 0.8 9. O 0.4 Basal High-Ca Milk FIG. 49 Patent Application Publication Apr. 5, 2007 Sheet 36 of 38 US 2007/007731.0 A1 g p = 0.001 Adipose (perirenal fat) basal VS. Calcium: 0 = 0.007 gene expression basal vs. milk. p40.001 calcium vs. milk. p = 0.073 Basal High-Ca Milk FIG. 50 P & 0.001 basal vs. Calcium. p = 0.001 basal vs. milk. p < 0.001 3 calcium vs. milk. p = 0.068 Basal High-Ca Milk FIG. 51 Patent Application Publication Apr. 5, 2007 Sheet 37 of 38 US 2007/007731.0 A1 P & O.OO1 basal vs. calcium. p < 0.001 basal vs. milk p < 0.001 calcium vs. milk p = 0.07 2 () OO y 1 o O Basal High-Ca Milk FIG. 52 900 a 720 2 O 540 S 360 ". % 180 3 s leucine -- is es Nifedipine as ca Adiponectin a- s as ar sis 1- s Calcitrio - as a as a a 1 Patent Application Publication Apr. 5, 2007 Sheet 38 of 38 US 2007/007731.0 A1 Leucine Nifedipine 1 - - - Adiponectin - Calcitriol FIG. 54 ,+ Calcitriol FIG. 55 US 2007/00773 10 A1 Apr. 5, 2007 METHODS OF REDUCING THE PRODUCTION OF 0007 FIG. 3: Effect of dietary calcium on body weight REACTIVE OXYGEN SPECIES AND METHODS and fat pads weight in aP2-agouti transgenic mice. Values OF SCREENING OR DENTIFYING COMPOUNDS are presented as meant-SEM, n=10. AND COMPOSITIONS THAT REDUCE THE 0008 FIG. 4: Effect of dietary calcium on fasting blood PRODUCTION OF REACTIVE OXYGEN SPECIES glucose in aP2-agouti transgenic mice. Values are presented CROSS-REFERENCE TO RELATED as mean-SEM, n=10. * indicates significant difference from APPLICATIONS the basal diet, p<0.05. 0001) This application claims the benefit of U.S. Provi 0009 FIG. 5: Effect of dietary calcium on adipose intra sional Application Ser. No. 60/723,042, filed Oct. 3, 2005 cellular ROS production in aP2-agouti transgenic mice. and U.S. Provisional Application Ser. No. 60/787,819, filed Values are presented as meant-SEM, n=10. Mar. 31, 2006. 0010 FIG. 6: Effect of dietary calcium on adipose NADPH oxidase expression in aP2-agouti transgenic mice. BACKGROUND OF THE INVENTION Values are presented as meant-SEM, n=10. 0002 Reactive oxygen species (ROS) production is 0011 FIG. 7: Effect of dietary calcium on adipose intra increased in obesity and diabetes (Furukaw et al., 2004: cellular calcium (Cali) in aP2-agouti transgenic mice. Atabek et al., 2004; Lin et al., 2005; Sonta et al., 2004). It Values are presented as meant-SEM, n=10. has been postulated that hyperglycemia and hyperlipidemia, key clinical manifestations of obesity and diabetes, may 0012 FIG. 8: Effect of dietary calcium on adipose UCP2 promote ROS production through multiple pathways expression in aP2-agouti transgenic mice. Values are pre (Inoguchi et al., 2000; Shangari et al., 2004; Chung et al., sented as meantSEM, n=10. 2003). ROS are also associated with a variety of diseases or 0013 FIG. 9: Effect of dietary calcium on soleus muscle disorders. For example, ROS are associated with cataracts, UCP3 expression in aP2-agouti transgenic mice. Values are heart disease, cancer, male infertility, aging, and various presented as meant-SEM, n=10. * indicates significant dif neurodegenerative diseases such as Alzheimer's disease, ference from the basal diet, p<0.05. amyotrophic lateral Sclerosis, Parkinson's disease, multiple Sclerosis and aging. 0014 FIG.10: Effect of dietary calcium on soleus muscle NADPH oxidase in aP2-agouti transgenic mice. Values are 0003 Previous studies from have demonstrated an anti presented as meantSEM, n=10. * indicates significant dif obesity effect of dietary calcium, with increasing dietary ference from the basal diet, p<0.05. calcium inhibiting lipogenesis, Stimulating lipolysis and thermogenesis and increasing adipocyte apoptosis (Zemel, 0.015 FIG. 11: Effect of dietary calcium on adipose 2005a). These effects are mediated by suppression of 1C. 113-HSD expression in aP2-agouti transgenic mice. Values 25-(OH),D,-induced stimulation of Ca" influx and sup are presented as meant-SEM, n=10. pression of adipose UCP2 gene expression (Shi et al., 2001; 0016 FIG. 12: Effect of HO, on DNA synthesis in Shi et al., 2002). Further, ROS production is modulated by cultured 3T3-L1 adipocytes. Adipocytes were treated with mitochondrial uncoupling status and cytosol calcium signal either HO (100 nmol/L) or C-tocopherol (1 umol/L), com ing, and that 1C, 25(OH),D, regulates ROS production in bined with or without GDP (100 umol/L) or nifedipine (10 cultured murine and human adipocytes (Sun et al., 2005). umol/L) for 48 hours. Data are expressed as meantSE (n=6). Different letters above the bars indicate a significant differ BRIEF DESCRIPTION OF THE INVENTION ence at level of p-0.05. 0004 The subject application provides methods of iden 0017 FIG. 13: ROS production in cultured 3T3-L1 adi tifying compounds, combinations of compounds, composi pocytes. Adipocytes were treated with either HO (100 tions, and/or combinations of compositions that are Suitable nmol/L) or C-tocopherol (1 umol/L), combined with or for reducing the production of reactive oxygen species without GDP (100 umol/L) or nifedipine (10 umol/L) for 48 (ROS) in an individual with the proviso that said compound, hours. Data are expressed as mean+SE (n=6). Different combination of compounds, composition, or combination of letters above the bars indicate a significant difference at level compositions is not a dietary material containing calcium or of p-0.05. dietary calcium. Also provided in the Subject application are methods of treating diseases or disorders associated with 0018 FIG. 14: Mitochondrial potential in cultured wild ROS production and methods of reducing ROS production type 3T3-L1 adipocytes and UCP2 transfected 3T3-L1 adi in an individual with the proviso that said compound, pocytes. Adipocytes were treated with either HO (100 combination of compounds, composition, or combination of nmol/L) or C-tocopherol (1 umol/L), combined with or compositions is not a dietary material containing calcium or without GDP (100 umol/L) or nifedipine (10 umol/L) for 48 dietary calcium. hours. Data are expressed as meantSE (n=6). 0.019 FIG. 15: Intracellular calcium (Cali)in cultured BRIEF DESCRIPTION OF THE FIGURES 3T3-L1 adipocytes. Adipocytes were treated with either 0005 FIG. 1: Adipose intracellular ROS production in HO, (100 nmol/L) or H.O. (100 nmol/L) plus C-tocopherol wild-type and aP2-agouti transgenic mice. Values are pre (1 umol/L) for 4 hours. Data are expressed as meaniSE sented as meantSEM, n=6. (n=6). Different letters above the bars indicate a significant 0006 FIG. 2: Adipose NADPH oxidase expression in difference at level of p<0.05. wild-type and aP2-agouti transgenic mice. Values are pre 0020 FIG. 16: ROS production in cultured 3T3-L1 adi sented as meantSEM, n=6. pocytes. Adipocytes were treated with either glucose (30 US 2007/00773 10 A1 Apr. 5, 2007 mmol/L) or glucose (30 mmol/L) plus nifedipine (10 umol/ FIG. 24C illustrates plasma 1C, 25-(OH)-D in aP2-agouti L), or glucose (30 mmol/L) plus GDP, or glucose (30 transgenic mice fed low calcium (basal) or high calcium mmol/L) plus 1C, 25-(OH)D for 48 hours. Data are diets. Values are presented as meant-SEM, n=10. Means expressed as mean+SE (n=6). Different letters above the with different letter differ, p=0.005. bars indicate a significant difference at level of p-0.05. 0029 FIG. 25A shows IL-6 expression, FIG. 25B shows 0021 FIG. 17: Cati in cultured 3T3-L1 adipocytes. IL-8 expression, FIG. 25C shows IL-15 expression and FIG. Adipocytes were treated with either glucose (30 mmol/L) or 25D shows adiponectin expression in differentiated Zen-bio glucose (30 mmol/L) plus C-tocopherol (1 Limol/L) for 4 human adipocytes. Adipocytes were treatment with 10 hours. Data are expressed as meaniSE (n=6). Different nmol/L 1C, 25-(OH)-D, 10 umol/L nifepipine, and 10 letters above the bars indicate a significant difference at level nmol/L 1C, 25-(OH)2-D plus 10 umol/L nifepipine respec of p-0.05. tively for 48 h. Data are normalized to 18s expression. 0022 FIG. 18: Expression ratio of NADPH oxidase to Values are presented as meant SEM, n=6. Means with 18s in cultured 3T3-L1 adipocytes. Adipocytes were treated different letter differ, p<0.005. with either glucose (30 mmol/L) or glucose (30 mmol/L ) 0030 FIG. 26A shows Adiponectin expression and FIG. plus nifedipine (10 umol/L), glucose (30 mmol/L) plus GDP. 26B shows IL-15 expression in differentiated 3T3-L1 adi or glucose (30 mmol/L) plus 1C, 25-(OH), D for 48 hours. pocytes. Adipocytes were treatment with 100 nmol/L H.O. Data are expressed as meaniSE (n=6). Different letters 1 umol/L Cittocopherol, and 100 nmol/L H.O. 1 umol/L above the bars indicate a significant difference at level of Cittocopherol respectively for 48 h. Data are normalized to p-0.05. 18s expression. Values are presented as meant SEM, n=6. Means with different letter differ, p<0.05. FIG. 26C: There 0023 FIG. 19: Expression ratio of UCP2 to 18s in was no direct effect of ROS on IL-15 expression; however, cultured 3T3-L1 adipocytes. Adipocytes were treated with addition of Cittocopherol markedly increased IL-15 by 2.2- either glucose (30 mmol/L) or glucose (30 mmol/L ) plus nifedipine (10 umol/L), glucose (30 mmol/L) plus GDP, or fold as compared to HO-treated cells (P=0.043). glucose (30 mmol/L) plus 1C, 25-(OH),D, for 48 hours. 0031 FIG. 27 demonstrates that calcitriol increased MIF Data are expressed as meaniSE (n=6). Different letters (FIG. 27A) and CD14 (FIG. 27B) expression in human above the bars indicate a significant difference at level of adipocytes, and addition of a calcium channel antagonist p-0.05. (nifedipine) reversed this effect, indicating a role of intrac ellular calcium in mediating this effect. 0024 FIG. 20: DNA synthesis in cultured 3T3-L1 adi pocytes. Adipocytes were treated with either glucose (30 0032 FIG. 28 demonstrates that calcitriol increased MIF mmol/L) or glucose (30 mmol/L) plus nifedipine (10 umol/ (FIG. 28A) and CD14 (FIG. 28B) expression in mouse L), glucose (30 mmol/L) plus GDP, or glucose (30 mmol/L) (3T3-L1) adipocytes and the addition of a calcium channel plus 1C, 25-(OH)D for 48 hours. Data are expressed as antagonist (nifedipine) reversed this effect. meantSE (n=6). Different letters above the bars indicate a 0033 FIGS. 29, 30 and 31 show that calcitriol markedly significant difference at level of p-0.05. stimulate inflammatory cytokines M-CSF (FIG. 29), MIP 0025 FIG. 21: Expression ratio of cyclin A to 18s in (FIG. 30) and IL-6 (FIG. 31) expression in 3T3-L1 adipo cultured 3T3-L1 adipocytes. Adipocytes were treated with cytes, and co-culture with RAW264 macrophages enhance either glucose (30 mmol/L) or glucose (30 mmol/L) plus this effect, indicating a potential role of adipocytes in nifedipine (10 umol/L), glucose (30 mmol/L) plus GDP, or regulation of local resident macrophages activity and that glucose (30 mmol/L) plus 1C, 25-(OH), D for 48 hours. calcitriol regulates this effect via a calcium and mitochon Data are expressed as meaniSE (n=6). Different letters drial uncoupling-dependent mechanism. Main effects of above the bars indicate a significant difference at level of chemical treatment and culture status were significant p-0.05. (p<0.02). 0026 FIG. 22A shows adipose tissue TNFC. expression 0034 FIGS. 32A-D illustrate the effect of calcitriol on ratio and FIG. 22B shows IL-6 expression ratio in aP2 mouse cytokine protein production. Calcitriol markedly agouti transgenic mice. Data are normalized to 18s expres increases production of several cytokines in 3T3-L1 adipo sion. Values are presented as meant-SEM, n=6. Means with cytes, as indicated in the schematic diagram. different letter differ, p<0.001. 0035 FIGS. 33A-D demonstrate that the effect of cal 0027 FIG. 23A shows adipose tissue IL-15 expression, citriol on mouse cytokine protein production in a co-culture FIG. 23B shows Adipose adiponectin expression and FIG. system. Calcitriol markedly increased cytokine production 23C shows Muscle IL-15 expression in aP2-agouti trans in a 3T3-L1 adipocytes-RAW264 macrophage co-culture, as genic mice. Data are normalized to 18s expression. Values indicated in the schematic diagram. are presented as meant SEM, n=6. Means with different 0.036 FIG. 34: MCP-1 expression in 3T3-L1 adipocytes. letter differ, p<0.03. 0037 FIGS. 35-36: Calcitriol stimulates expression of 0028 FIG. 24A shows TNFC. expression and FIG. 24B TNFC. and IL-6. Calcitriol stimulated TNFC. expression by shows IL-6 expression in differentiated 3T3-L1 adipocytes. 91% (FIG. 35) and IL-6 by 796% (FIG. 36) in RAW 264 Adipocytes were treatment with 10 nmol/L 1C, 25-(OH)- macrophages cultured alone. These effects were blocked by D, 10umol/L nifepipine, and 10 nmol/L 1C, 25-(OH)-D- adding nifedipine or DNP. Co-culture of macrophages with plus 10 umol/L nifepipine respectively for 48 h. Data are differentiated 3T3-L1 adipocytes markedly augmented normalized to 18s expression. Values are presented as TNFC. (FIG. 35) and IL-6 (FIG. 36) expression in macroph meaniSEM, n=6. Means with different letter differ, p<0.02. ages, and these effects were further enhanced by calcitriol. US 2007/00773 10 A1 Apr. 5, 2007 0038 FIG. 37: The high calcium diet was without effect 0050 FIG. 54: C2C12 myotubes were treated with or on body weight, but the milk diet did induce a significant without leucine (2.5 mM), nifedipine (10), adiponectin (70 decrease in total body weight. ng/ml) or/and calcitriol (10 nM) for 48 hours. IL-15 release in the medium was determined using ELISA. Data are 0039 FIG. 38: Both the calcium and the milk diets corrected for DNA content. Values are presented as caused significant decreases in body fat, with the milk diet meaniSEM, n=6. Means with different letter differ with eliciting a significantly greater effect. p-0.05. 0040 FIG. 39: The milk group had significantly greater skeletal muscle mass than the calcium group (p=0.02) and a 0051 FIG. 55: C2C12 myotubes were treated with or tendency towards greater skeletal muscle mass than the without leucine (2.5 mM), nifedipine (10), adiponectin(70 basal group (p=0.06). ng/ml) or/and calcitriol (10 nM) for 48 hours. IL-6 release in the medium was determined using ELISA. Data are 0041 FIG. 40: Liver weight was slightly, but signifi corrected for DNA content. Values are presented as cantly, reduced by the milk diet. meaniSEM, n=6. Means with different letter differ with 0.042 FIG. 41: The high calcium diet caused a reduction p-0.05. in plasma 1.25-(OH)-D (calcitriol) (p=0.002), and there was a trend (p=0.059) towards a further decrease in plasma DETAILED DESCRIPTION OF THE calcitriol on the high milk diet. INVENTION 0.043 FIG. 42: Adipose tissue reactive oxygen species 0052 The subject application provides a method of (ROS) production was significantly reduced by the high screening compounds or compositions suitable for reducing calcium diet (p=0.002) and further reduced by the milk diet the production of reactive oxygen species (ROS) compris (p=0.03). ing: a) contacting one or more adipocyte cell(s) with a candidate compound, combination of candidate compounds, 0044 FIG. 43: The high calcium diet caused a significant candidate composition, or combination of candidate com reduction in adipose tissue NADPH oxidase (Nox; one of positions with the proviso that said compound, combination the sources of intracellular ROS) expression (p=0.001) and of compounds, composition, or combination of composi there was a strong trend (p=0.056) towards a further sup tions is not a dietary material containing calcium or dietary pression of NOX on the milk diet. calcium; b) measuring the intracellular concentrations of 004.5 FIG. 44: Plasma MDA was significantly decreased calcium in said adipocyte cell(s), wherein a decrease of by both the calcium and milk diets (p=0.001), with a intracellular calcium concentration in said adipocyte cell(s) significantly greater effect of the milk diet (p=0.039). is indicative of a compound or composition Suitable for use in reducing the production of ROS. Cells suitable for these 0046 FIGS. 45-49: The high calcium diet resulted in screening methods include 3T3-L1 adipocytes (ATCC, Suppression of inflammatory markers and an upregulation of Manassas, Va.) and human adipocytes (Zen Bio, Inc., anti-inflammatory markers, and the milk diet exerted a Research Triangle, N.C.). These cells can be maintained in greater effect than the high calcium diet. Adipose tissue culture as described in Example 2. expression of TNF-C. (FIG. 45), IL-6 (FIG. 46) and MCP (FIG. 47) were all significantly suppressed by the high 0053 Another screening method provided by the subject calcium diet. Expression of each of these inflammatory application provides a method of identifying or screening cytokines was lower on the milk diet than on the high compounds or compositions suitable for reducing the pro calcium diet, but this difference was only statistically evi duction of reactive oxygen species (ROS) comprising: a) dent as a trend for TNF-C. (p=0.076). The calcium and milk administering a candidate compound, combination of can diets caused significant reductions in the release of inflam didate compounds, candidate composition, or combination matory cytokines (TNF-C, FIG. 48; IL6, FIG. 49) from of candidate compositions to at least one Subject with the adipose tissue. There was trend towards a greater effect of proviso that said compound, combination of compounds, the milk vs. calcium diet, but this difference was not composition, or combination of compositions is not a dietary statistically significant. material containing calcium or dietary calcium; b) measur ing intracellular concentrations of calcium in cells of said at 0047 FIGS. 50-51: The high calcium and milk diets least one Subject, wherein a decrease of intracellular calcium increased adiponectin expression (p=0.001; FIG. 50) and concentration in said cells of said at least one test Subject as IL-15 expression (p=0.001; FIG. 51), and there was a trend compared to the intracellular concentrations of calcium in for a further increase on the milk diet vs. high calcium diet the cells of at least one control subject is indicative of a (p=0.073 for adiponectin; p=0.068 for IL-15). compound, composition, combination of compounds or 0.048 FIG. 52: There was a marked increase in skeletal combination of compositions suitable for use in reducing the muscle IL-15 expression on the high calcium diet (p<0.001). production of ROS in a subject. In some embodiments of the Il-15 expression was further increased on the milk diet invention, intracellular concentrations of Ca" are measured (p=0.07). in adipocyte cells (e.g., visceral adipocytes or cutaneous adipocytes). Other embodiments allow for the administra 0049 FIG. 53: Fatty acid oxidation was determined via tion of a candidate compound, combination of candidate palmitate oxidation. C2C12 myotubes were treated with or compounds, candidate composition, or combination of can without leucine (2.5 mM), nifedipine (10 u), adiponectin (70 didate compositions to at least one test Subject orally with ng/ml) and/or calcitriol (10 nM) for 48 hours. Data are the proviso that said candidate compound, combination of corrected for DNA content. Values are presented as candidate compounds, candidate composition, or combina meaniSEM, n=6. Means with different letter differ with tion of candidate compositions is not being administered to p-0.05. the subjects orally as a component of the diet of the subject. US 2007/00773 10 A1 Apr. 5, 2007 0054 As used herein, the term “subject” or “individual' tration of a compound, composition, combination of com includes mammals. Non-limiting examples of mammals pounds, or combination of compositions that decrease intra include transgenic mice (such as aP2-agouti transgenic cellular calcium levels to an individual in need of such mice) or human test Subjects. Other mammals include, and treatment in amounts sufficient to decrease intracellular are not limited to, apes, chimpanzees, orangutans, monkeys; levels of calcium in the cells of the individual, decrease domesticated animals (pets) such as dogs, cats, guinea pigs, TNF-O, CD14, MIP, MIF, M-CSF, MCP-1, G-CSF or IL-6 hamsters, mice, rats, rabbits, and ferrets; domesticated farm expression (or any combination of the aforementioned animals such as cows, buffalo, bison, horses, donkey, Swine, cytokines) in the individual, and increase the expression of sheep, and goats; or exotic animals typically found in Zoos, IL-15, adiponectin, or both IL-15 or adiponectin in the Such as bear, lions, tigers, panthers, elephants, hippopota individual with the proviso that the compound, composition, mus, rhinoceros, giraffes, antelopes, sloth, gazelles, Zebras, combination of compounds, or combination of compositions wildebeests, prairie dogs, koala bears, kangaroo, pandas, is not dietary calcium or dietary material that contains giant pandas, hyena, seals, sea lions, and elephant seals. calcium. 0.055 “Dietary material containing dietary calcium' is 0058 Also provided are methods of increasing the in defined herein as any item normally consumed in the diet of vitro expression of MIF, M-CSF, MIP, IL-6, IL-10, IL-4, a human or mammal. Non-limiting examples of such dietary IL-13, MIG, IL-5, VEGF, CD14, G-CSF, TNF-O, RANTES, materials are salmon, beans, tofu, spinach, turnip greens, and/or MIP-1C. comprising contacting a composition com kale, broccoli, waffles, pancakes, pizza, milk, yogurt, prising a carrier and calcitriol (1, 25-(OH)-D) with a cheeses, cottage cheese, ice cream, frozen yogurt, calcium culture of cells. The cells are cultured in the presences of this fortified nutrient supplements, calcium fortified vitamin composition and MIF, M-CSF, MIP, IL-6, IL-10, IL-4, Supplements, or liquids Supplemented with calcium. Spe IL-13, MIG, IL-5, VEGF, CD14, G-CSF, TNF-O, RANTES, cifically excluded from Such a definition are those compo and/or MIP-C. can be recovered for the cell culture according sitions that would be prescribed by a physician or veteri to methods known in the art. In some embodiments, the cells narian for the treatment of a disease or condition. Also can be derived from adipose tissue (adipocytes); skeletal specifically excluded from the definition of “dietary cal muscle cells (or commercially available skeletal cell lines); cium' or "dietary material containing dietary calcium' are or human or murine adipocyte cell lines (e.g., 3T3-L1 cells). compounds found in compound libraries (such as chemical In some aspects of the invention, the culture of cells com compound libraries or peptide libraries) and compositions prises a co-culture system that includes macrophage (e.g., comprising such compounds or peptides. Also excluded see Example 4). from the definition of “dietary material containing dietary calcium is any source of calcium that does not form a part 0059. The subject invention also provides methods of of the diet of a mammal or human. Pharmaceutical compo increasing the production of IL-15 and/or adiponectin com sitions prescribed by a physician or veterinarian that contain prising contacting a composition comprising a carrier and calcium (or physiologically acceptable salts of calcium) via calcium with a culture of cells. The cells are cultured in the intravenous, intraarterial, oral, buccal, topical, transdermal, presences of this composition and IL-15 and/or adiponectin rectal, intramuscular, Subcutaneous, intraosseous, transmu can be recovered for the cell culture according to methods cosal, or intraperitoneal routes of administration are not known in the art. In some embodiments, the cells can be construed to a "dietary material containing dietary calcium’ cells derived from adipose tissue (adipocytes); skeletal or as "dietary calcium'. One or more physiologically accept muscle cells (or commercially available skeletal cell lines); able salt(s) of calcium include, and are not limited to, or human or murine adipocyte cell lines (e.g., 3T3-L1 cells). calcium phosphates, calcium carbonate, calcium chloride, 0060 ROS associated diseases include, and are not lim calcium sulfate, calcium tartrate, calcium magnesium car ited to, cataracts, diabetes, Alzheimer's disease, heart dis bonate, calcium metasilicate, calcium malate, secondary ease, inflammation, cancer, male infertility, amyotrophic calcium orthophosphate, calcium citrate, or calcium hydrox lateral Sclerosis, Parkinson's disease, and multiple Sclerosis ide. and aging. Thus, the Subject application provides methods of 0056. The subject application also provides methods of treating cataracts, Alzheimer's disease, heart disease, cancer, treating diseases associated with reactive oxygen species male infertility, amyotrophic lateral sclerosis, Parkinson's (ROS) comprising the administration of a compound, com disease, and multiple Sclerosis and aging that comprises the position, combination of compounds, or combination of administration of compounds, compositions, combinations compositions that decrease intracellular calcium levels to an of compounds or combinations of compositions in amounts individual in need of Such treatment in amounts sufficient to sufficient to decrease the intracellular levels of calcium in an decrease the intracellular concentrations of calcium in the individual. cells of the individual with the proviso that the compound, 0061 The subject application also provides methods of composition, combination of compounds, or combination of treating cancer-associated ROS disease comprising the compositions is not dietary calcium or dietary material that administration of a composition comprising an appropriate contains calcium. In some embodiments, the methods of therapeutic agent and calcium (or physiologically acceptable treating diseases associated with ROS also include a step salts of calcium) in an amount Sufficient to reduce the that comprises the diagnosis or identification of an indi production of ROS. In the context of this aspect of the vidual as having a disease or disorder associated with ROS invention, the phrase “appropriate therapeutic agent' or suffering from elevated ROS levels. includes, and is not limited to, alkylating agents (e.g., 0057 The subject application also provides methods of cyclophosphamide, ifosfamide), antibiotics which affect altering the expression of cytokines in an individual (or the nucleic acids (e.g., doxorubicin, bleomycin), platinum com cytokine profile of an individual) comprising the adminis pounds (e.g., cisplatin), mitotic inhibitors (e.g., Vincristine), US 2007/00773 10 A1 Apr. 5, 2007 antimetabolites (e.g., 5-fluorouracil), camptothecin deriva expression in said cell(s) is indicative of a compound or tives (e.g., topotecan), biological response modifiers (e.g., composition Suitable for use in reducing the production interferon or monoclonal antibodies), and hormonetherapies of ROS; (e.g., tamoxifen). Additional non-limiting examples of 0071 iv) UCP3 expression in said one or more cell(s), “appropriate therapeutic agents' are identified in Table 1 of wherein an increase in UCP3 expression in said cell(s) this application as are the indications (types of cancer) that is indicative of a compound or composition Suitable for can be treated with a particular therapeutic agent. use in reducing the production of ROS; 0062) The terms “administer”, “administered”, “admin isters' and “administering are defined as the providing a 0072 v) NADPH oxidase expression in said one or candidate compound, combination of candidate compounds, more cell(s), wherein a decrease in NADPH oxidase candidate composition, or combination of candidate com expression in said cell(s) is indicative of a compound or positions to a Subject via intravenous, intraarterial, oral, composition Suitable for use in reducing the production buccal, topical, transdermal, rectal, intramuscular, Subcuta of ROS; neous, intraosseous, transmucosal, or intraperitoneal routes 0073 vi) 113-HSD expression in said one or more of administration. In certain embodiments of the subject cell(s), wherein a decrease in the expression of 11 B application, oral routes of administering a candidate com HSD in said cell(s) is indicative of a compound or pound, combination of candidate compounds, candidate composition Suitable for use in reducing the production composition, or combination of candidate compositions to a of ROS; Subject are specifically excluded. 0074 vii) TNF-ct, CD14, MIF, M-CSF, MIP, MCP-1, 0063. The term “physiologically acceptable salts' of cal G-CSF or IL-6 expression in said one or more cell(s), cium include, and are not limited to, calcium phosphate, wherein a decrease in the expression of TNF-C, CD14, calcium carbonate, calcium chloride, calcium sulfate, cal MIF (macrophage inhibitory factor), MIP (macrophage cium tartrate, calcium magnesium carbonate, calcium meta inhibitory protein), M-CSF (macrophage colony stimu silicate, calcium malate, secondary calcium orthophosphate, lating factor), G-CSF (granulocyte colony Stimulating calcium citrate, or calcium hydroxide. In certain aspect of factor) or IL-6 in said cell(s) is indicative of a com the invention, amounts of calcium that are administered in pound or composition Suitable for use in reducing the combination with appropriate therapeutic agents for the production of ROS; or treatment of cancer provide at least 400 to 2000 mg, 900 to 1500 mg, 1000 to 1400, 1100 to 1300 mg, or 1200 to 1300 0075 viii) IL-15 or adiponectin expression in said one mg of calcium per day. Alternatively, X.YZ mg (or about or more cell(s), wherein an increase in the expression X.YZ mg or at least X.YZ mg) of calcium per day are of IL-15 or adiponectin in said cell(s) is indicative of a provided to the subject wherein X is any integer selected compound or composition Suitable for use in reducing from 400 to 2000, Y is an integer selected from 0, 1, 2, 3, 4, the production of ROS; 5, 6, 7, 8, or 9, and Z is an integer selected from 0, 1, 2, 3, 0076 B) The embodiment as set forth in A, wherein said 4, 5, 6, 7, 8, or 9. one or more cell(s) is a adipocyte or an adipocyte cell line; 0064. As set forth herein, the subject application also 0.077 C) An embodiment as set forth in A or B, wherein provides the following non-limiting aspects of the invention: the one or more cell(s) is afare human adipocyte(s) or a 0065 A) An in vitro method of screening compounds or murine adipocyte; compositions suitable for reducing the production of reac 0078 D) An embodiment as set forth in A, B or C, tive oxygen species (ROS) comprising: wherein the one or more cell(s) are an adipocyte cell line; 0.066 a) contacting one or more cell(s) with a candidate 0079 E) An embodiment as set forth in A, B, C or D, compound, combination of candidate compounds, candidate wherein the one or more cell(s) are a human adipocyte cell composition, or combination of candidate compositions with line; the proviso that said candidate compound, combination of candidate compounds, candidate composition, or combina 0080 F) An embodiment as set forth in A, B, C or D, tion of candidate compositions is not dietary material con wherein the one or more cell(s) are a murine adipocyte cell taining calcium or dietary calcium; and line; 0067 b) measuring one or more of the following param 0081 G) An embodiment as set forth in A, B or C. wherein the one or more cell(s) are a murine or human eters: adipocyte; 0068 i) intracellular concentrations of calcium in said one or more cell(s), wherein a decrease of intracellular 0082 H) An embodiment as set forth in G, wherein the calcium concentration in said cell(s) is indicative of a murine or human adipocytes are obtained from visceral, or compound or composition Suitable for use in reducing Subcutaneous, or both visceral and Subcutaneous fat tissue; the production of ROS; 0083) I) An embodiment as set forth in A, B, C, D, E, F, 0069 ii) UCP2 expression in said one or more cell(s), G, or H, wherein the cell(s) are obtained from a transgenic wherein an increase in UCP2 expression in said cell(s) mouse; is indicative of a compound or composition Suitable for 0084 J) An embodiment as set forth in I, wherein the use in reducing the production of ROS; transgenic mouse is an aP2-agouti transgenic mouse; 0070) iii) NADPH oxidase expression in said one or 0085 K) An embodiment as set forth in A, B, C, D, E, F more cell(s), wherein a decrease in NADPH oxidase or G, wherein the cell(s) 3T3-L1 adipocytes; US 2007/00773 10 A1 Apr. 5, 2007 0.086 L) A method of identifying or screening com bination of compounds or combination of compositions pounds or compositions Suitable for reducing the production suitable for use in reducing the production of ROS in a of reactive oxygen species (ROS) comprising: Subject; 0087 a) administering a candidate compound, combina 0094 vi) 113-HSD expression in visceral adipocyte tion of candidate compounds, candidate composition, or tissue or cells of said at least one test Subject and at least combination of candidate compositions to at least one test one control subject, wherein a decrease of 113-HSD Subject with the proviso that said candidate compound, expression in the visceral adipocyte tissue or cells of a combination of candidate compounds, candidate composi test subject as compared to the 113-HSD expression in tion, or combination of candidate compositions, if adminis the visceral adipocyte tissue or cells of at least one tered orally to said test Subject, is not being administered to control Subject is indicative of a compound, composi said at least one test Subject orally as a component of the diet tion, combination of compounds or combination of of said at least one test Subject or as dietary calcium to said compositions suitable for use in reducing the produc test Subject (i.e., (said candidate compound, combination of tion of ROS in a subject; candidate compounds, candidate composition, or combina tion of candidate compositions, if administered orally to said 0.095 vii) TNF-ct, CD14, MIF, MIP, M-CSF, MCP-1, test Subject, is not a dietary material containing calcium or G-CSF or IL-6 expression in said one or more cell(s), dietary calcium); and wherein a decrease in the expression of TNF-C, CD14, MIF, MIP, M-CSF, G-CSF or IL-6 in said cell(s) is 0088 b) measuring one or more of the following param indicative of a compound or composition Suitable for eters: use in reducing the production of ROS; or 0089 i) intracellular calcium concentrations in cells of 0096 viii) IL-15 or adiponectin expression in said one said at least one test Subject and at least one control or more cell(s), wherein an increase in the expression Subject, wherein a decrease of intracellular calcium of IL-15 or adiponectin in said cell(s) is indicative of a concentration in the cells of a test Subject as compared compound or composition Suitable for use in reducing to the intracellular concentrations of calcium in the the production of ROS; cells of at least one control subject is indicative of a compound, composition, combination of compounds or 0097 M) An embodiment as set forth in L(b)(i)-(iii), combination of compositions suitable for use in reduc (vii), or (viii), wherein the cells are adipocyte cells obtained ing the production of ROS in a subject; from at least one test subject and at least one control subject; 0090 ii) UCP2 expression in cells of said at least one 0.098 N) An embodiment as set forth in M, wherein the test Subject and at least one control Subject, wherein an cells are cutaneous adipocyte cells obtained from at least one increase of UCP2 expression in the cells of a test test Subject and at least one control Subject; subject as compared to the UCP2 expression in the cells of at least one control Subject is indicative of a com 0099 O) An embodiment as set forth in M, wherein the pound, composition, combination of compounds or intracellular concentration of calcium is measured in vis combination of compositions suitable for use in reduc ceral adipocyte cells obtained from at least one test Subject ing the production of ROS in a subject; and at least one control Subject; 0.091 iii) NADPH oxidase expression in cells of said at 0100 P) An embodiment as set forth in M, wherein the least one test Subject and at least one control Subject, intracellular concentration of calcium is measured in cuta wherein a decrease of NADPH oxidase expression in neous, or visceral, or both cutaneous and visceral adipocyte the cells of a test subject as compared to the NADPH cells obtained from at least one test Subject and at least one oxidase expression in the cells of at least one control control Subject; Subject is indicative of a compound, composition, com 0101 Q) An embodiment as set forth in L, M, N, O or P. bination of compounds or combination of compositions wherein the test Subject and control Subject are human; suitable for use in reducing the production of ROS in a Subject; 0102 R) An embodiment as set forth in L, M, N, O or P. wherein the test Subject and control Subject are murine; 0092) iv) UCP3 expression in skeletal muscle cells of said at least one test Subject and at least one control 0.103 S) An embodiment as set forth in R, wherein the subject, wherein an increase in UCP3 expression in the test Subject and control Subject are transgenic mice; skeletal muscle cells of a test Subject as compared to 0.104 T) An embodiment as set forth in S, wherein the UCP3 expression in the skeletal muscle cells of at least test Subject and control Subject are aP2-agouti transgenic one control Subject is indicative of a compound, com mice; position, combination of compounds or combination of compositions Suitable for use in reducing the produc 0105 U) An embodiment as set forth in L, M, N, O, P, Q, R, S or T, wherein: 1) a candidate compound, combination tion of ROS in a subject; of candidate compounds, candidate composition, or combi 0093 v) NADPH oxidase expression in skeletal nation of candidate compositions is administered to a subject muscle cells of said at least one test Subject and at least via intravenous, intraarterial, oral, buccal, topical, transder one control subject, wherein a decrease of NADPH mal, rectal, intramuscular, Subcutaneous, intraosseous, oxidase expression in the skeletal muscle cells of a test transmucosal, or intraperitoneal routes of administration or subject as compared to the NADPH oxidase expression wherein said candidate compound, combination of candidate in the skeletal muscle cells of at least one control compounds, candidate composition or combination of can Subject is indicative of a compound, composition, com didate compositions is administered to at least one test US 2007/00773 10 A1 Apr. 5, 2007 subject orally with the proviso that said candidate com 0114 DD) An embodiment as set forth in CC, wherein pound, combination of candidate compounds, candidate the one or more physiologically acceptable salt(s) of calcium composition, or combination of candidate compositions is include, and are not limited to, calcium phosphates, calcium not being administered to at least one test Subject orally as carbonate, calcium chloride, calcium Sulfate, calcium tar a component of the diet of said test subject; or 2) wherein trate, calcium magnesium carbonate, calcium metasilicate, said candidate compound, combination of candidate com calcium malate, secondary calcium orthophosphate, calcium pounds, candidate composition, or combination of candidate citrate, or calcium hydroxide; compositions is administered to at least one test Subject 0115 EE) An embodiment as set forth in W, X, Y, Z, AA, orally as a component of the Subject’s diet (i.e., as dietary BB, CC or DD wherein a dosage of 400 to 2000 mg of calcium); calcium are administered to a subject per day; 0106 V). An embodiment as set forth in L, M, N, O, P, Q, R, S or T, wherein a candidate compound, combination of 0116 FF) An embodiment as set forth in W, X, Y, Z, AA, candidate compounds, candidate composition, or combina BB, CC or DD wherein a dosage of X.YZ mg of calcium is tion of candidate compositions is administered to a subject administered to a subject per day, wherein X is any integer parenterally (e.g., via intravenous, intraarterial, buccal, topi from 400 to 2000, Y is 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9, and Z is cal, transdermal, rectal, intramuscular, Subcutaneous, 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9: intraosseous, transmucosal, or intraperitoneal routes of 0117 GG) An embodiment as set forth in W, X, Y, Z, AA, administration); BB, CC, DD or EE wherein a dosage of 900 to 1500 mg of 0107 W) A method of treating diseases associated with calcium are administered to a subject per day; reactive oxygen species (ROS) comprising the administra 0118) HH) An embodiment as set forth in W, X, Y, Z, AA, tion of a compound, composition, combination of com BB, CC, DD or EE wherein a dosage of 1000 to 1400 mg of pounds, or combination of compositions that decrease intra calcium are administered to a subject per day; cellular calcium levels to an individual in need of such treatment in amounts sufficient to decrease the intracellular 0119) II) An embodiment as set forth in W, X, Y, Z, AA, concentrations of calcium in the cells of the individual with BB, CC, DD or EE wherein a dosage of 1100 to 1300 mg of the proviso that the compound, composition, combination of calcium are administered to a subject per day; compounds, or combination of compositions is not dietary 0120 JJ) An embodiment as set forth in W, X, Y, Z, AA, calcium or dietary material that contains calcium; BB, CC, DD or EE wherein a dosage of 1200 to 1300 mg of 0108) X) An embodiment as set forth in W, wherein the calcium are administered to a subject per day; method includes a step that comprises the diagnosis or 012.1 KK) An embodiment as set forth in W. Z. AA, BB, identification of an individual as having a disease or disorder CC, DD, EE, FF, GG, HH, II or JJ, wherein the therapeutic associated with ROS or diagnosing or identifying an indi agent or therapeutic agents and calcium (or physiologically vidual having elevated ROS levels (e.g., measuring the acceptable salts of calcium) are administered as a single levels of ROS and comparing the measured levels against a composition; standard or collection of control Subjects); 0.122 LL) An embodiment as set forth in Z. AA, BB, CC, 0109) Y) An embodiment as set forth in W or X, wherein DD, EE, FF, GG, HH, II or JJ, wherein the therapeutic agent the ROS associated diseases include, and are not limited to, or therapeutic agents and calcium (or physiologically cataracts, diabetes, Alzheimer's disease, heart disease, can acceptable salts of calcium) are administered as separate or cer, male infertility, inflammation, amyotrophic lateral scle different compositions; rosis, Parkinson's disease, and multiple Sclerosis and aging; 0123 MM) An embodiment as set forth in X, Y or LL. 0110 Z) A method of treating cancer-associated ROS wherein the separate or different compositions are adminis disease or disorders comprising the administration of one or tered simultaneously, sequentially or contemporaneously; more composition comprising an appropriate therapeutic agent and calcium (or physiologically acceptable salts of 0.124 NN) An embodiment as set forth in KK, LL or calcium) in an amount Sufficient to reduce the production of MM, wherein the compositions are administered at multiple ROS; times during the day; 0111 AA) An embodiment as set forth in Z, wherein the 0.125 OO) An embodiment as set forth in X, Y, Z or KK, “appropriate therapeutic agent includes, and is not limited wherein the composition is administered once per day; to, alkylating agents (e.g., cyclophosphamide, ifosfamide), 0126 PP)An embodiment as set forth in W, X, Y, Z, AA, antibiotics which affect nucleic acids (e.g., doxorubicin, BB, CC, DD, EE, FF, GG, HH, II, JJ, KK, LL., MM, NN or bleomycin), platinum compounds (e.g., cisplatin), mitotic OO, wherein said administration is parenteral; inhibitors (e.g., Vincristine), antimetabolites (e.g., 5-fluorou racil), camptothecin derivatives (e.g., topotecan), biological 0.127 QQ) A method of reducing ROS production in a diabetic individual comprising the administration of one or response modifiers (e.g., interferon or this application; more composition comprising an appropriate therapeutic 0112 BB) An embodiment as set forth in Z or AA, agent and calcium (or physiologically acceptable salts of wherein the type of cancer to be treated is identified as an calcium) in an amount Sufficient to reduce the production of indication in Table 1: ROS; 0113 CC) An embodiment as set forth in W, X, Y, Z, AA 0.128 RR). An embodiment as set forth in QQ, wherein or BB, wherein one or more physiologically acceptable one or more physiologically acceptable salt(s) of calcium are salt(s) of calcium is administered in the composition; present in at least one of said one or more composition; US 2007/00773 10 A1 Apr. 5, 2007 0129. SS) An embodiment as set forth in RR, wherein the 0.143 GGG) An embodiment as set forth in EEE, wherein one or more physiologically acceptable salt(s) of calcium said appropriate therapeutic agent is selected from those set include, and are not limited to, calcium phosphates, calcium forth in Table 2 or any combination of the therapeutic agents carbonate, calcium chloride, calcium Sulfate, calcium tar set forth therein; trate, calcium magnesium carbonate, calcium metasilicate, calcium malate, secondary calcium orthophosphate, calcium 0.144 HHH) An embodiment as set forth in any of QQ. RR, SS, TT, UU, VV, WW, XX, YY, ZZ, AAA, BBB, CCC, citrate, or calcium hydroxide; DDD, EEE, FFF, or GGG, wherein said diabetic individual 0130 TT) An embodiment as set forth in QQ, RR, or SS has a Type II diabetes (non-insulin dependent diabetes wherein a dosage of 400 to 2000 mg of calcium are mellitus NIDDM)); administered to a subject per day; 0145 III) A composition comprising one or more thera 0131 UU) An embodiment as set forth in QQ, RR, SS, or peutic agent selected from Tables 2 or 3 in combination with TT wherein a dosage of X.YZ mg of calcium are adminis calcium or one or more physiological salts of calcium; tered to a subject per day, wherein X is any integer from 400 0146) JJJ) An embodiment as set forth in III, wherein said to 2000, Y is O, 1, 2, 3, 4, 5, 6, 7, 8 or 9, and Z is O, 1, 2, composition contains between 1 and 2000 mg of calcium or 3, 4, 5, 6, 7, 8 or 9: one or more physiologically acceptable salts thereof; 0132) VV) An embodiment as set forth in QQ, RR, SS, 0147 KKK) An embodiment as set forth in III or JJJ, TT or UU wherein a dosage of 900 to 1500 mg of calcium wherein said composition contains X.YZ mg of calcium or are administered to a Subject per day; physiological salts of calcium, wherein X is any integer 0.133 WW) An embodiment as set forth in QQ, RR, SS, between 1 to 2000 (inclusive of 1 and 2000), Y is 0, 1, 2, 3, TT, UU or VV wherein a dosage of 1000 to 1400 mg of 4, 5, 6, 7, 8 or 9, and Z is 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9: calcium are administered to a Subject per day; 0.148 LLL) An embodiment as set forth in III, JJJ, or 0134) XX). An embodiment as set forth in QQ, RR, SS, KKK, wherein said composition contains between 900 and TT, UU, VV or WW wherein a dosage of 1100 to 1300 mg 1500 mg of calcium or one or more physiological salts of of calcium are administered to a subject per day; calcium; 0149 MMM) An embodiment as set forth in III, JJJ, 0135) YY) An embodiment as set forth in QQ, RR, SS, KKK, or LLL, wherein said composition contains between TT, UU, VV, WW or XX wherein a dosage of 1200 to 1300 1000 and 1400 mg of calcium or one or more physiological mg of calcium are administered to a subject per day; salts of calcium; 0.136 ZZ) An embodiment as set forth in QQ, RR, SS, 0150. NNN) An embodiment as set forth in III, JJJ, KKK, TT, UU, VV, WW, XX or YY, wherein the therapeutic agent LLL, or MMM, wherein said composition contains between or therapeutic agents and calcium (or physiologically 1100 and 1300 mg of calcium or one or more physiological acceptable salts of calcium) are administered as a single composition; salts of calcium; 0151 OOO) An embodiment as set forth in III, JJJ, KKK, 0137 AAA) An embodiment as set forth in QQ, RR, SS, LLL, MMM or NNN, wherein said composition contains TT, UU, VV, WW, XX or YY, wherein the therapeutic agent between 1200 and 1300 mg of calcium or one or more or therapeutic agents and calcium (or physiologically physiological salts of calcium; acceptable salts of calcium) are administered as separate or different compositions; 0152 PPP)An embodiment as set forth in III, JJJ, KKK, LLL, MMM, NNN or OOO, wherein said one or more 0138 BBB) An embodiment as set forth in QQ, RR, SS, physiological salts of calcium include, and are not limited to, TT, UU, VV, WW, XX, YY or AAA, wherein the separate or calcium phosphates, calcium carbonate, calcium chloride, different compositions are administered simultaneously, calcium sulfate, calcium tartrate, calcium magnesium car sequentially or contemporaneously; bonate, calcium metasilicate, calcium malate, secondary 0139 CCC) An embodiment as set forth in QQ, RR, SS, calcium orthophosphate, calcium citrate, or calcium hydrox TT, UU, VV, WW, XX, YY, ZZ, AAA or BBB, wherein the ide; compositions are administered at multiple times during the 0.153 QQQ) A method of altering the expression of day; cytokines in an individual (or the cytokine profile of an individual) comprising the administration of a compound, 0140. DDD) An embodiment as set forth in QQ, RR, SS, composition, combination of compounds, or combination of TT, UU, VV, WW, XX, YY, ZZ, AAA, or BBB, wherein the compositions that decrease intracellular calcium levels to an composition is administered once per day; individual in need of Such treatment in amounts sufficient to 0141 EEE) An embodiment as set forth in QQ, RR, SS, decrease intracellular levels of calcium in the cells of the TT, UU, VV, WW, XX, YY, ZZ, AAA, BBB, CCC, or DDD, individual, decrease TNF-O, CD14, MIP, MIF, M-CSF, wherein said administration is parenteral; G-CSF or IL-6 expression (or any combination of the aforementioned cytokines) in the individual, and increase 0142 FFF) An embodiment as set forth in any of QQ. the expression of IL-15, adiponectin, or both IL-15 or RR, SS, TT, UU, VV, WW, XX, YY, ZZ, AAA, BBB, CCC, adiponectin in the individual with the proviso that the or DDD, wherein said appropriate therapeutic agent is compound, composition, combination of compounds, or selected from those set forth in Table 3 or any combination combination of compositions is not dietary calcium or of the therapeutic agents set forth therein; dietary material that contains calcium; US 2007/00773 10 A1 Apr. 5, 2007 0154 RRR) An embodiment as set forth in QQQ. 0168 Levels of NADPH oxidase, UCP2, UCP3, cyclin wherein one or more physiologically acceptable salt(s) of A, 11 B-HSD, TNF-ct, CD14, MIF, MIP, M-CSF, G-CSF, calcium are present in at least one of said one or more IL-6, IL-15, adiponectin and/or intracellular levels of cal composition; cium can be measured according to methods well-known in the art or as set forth in the following examples. By way of 0155 SSS) An embodiment as set forth in RRR, wherein non-limiting examples, relative levels of expressions of the one or more physiologically acceptable salt(s) of calcium NADPH oxidase, UCP2, UCP3, cyclin A, 11B-HSD, TNF include, and are not limited to, calcium phosphates, calcium C, CD14, MIF, MIP, M-CSF, G-CSF, IL-6, IL-15, and/or carbonate, calcium chloride, calcium Sulfate, calcium tar adiponectin can be determined by: 1) nuclear run-on assay, trate, calcium magnesium carbonate, calcium metasilicate, 2) slot blot assay, 3) Northern blot assay (Alwine et al., calcium malate, secondary calcium orthophosphate, calcium 1977), 4) magnetic particle separation, 5) nucleic acid or citrate, or calcium hydroxide; DNA chips, 6) reverse Northern blot assay, 7) dot blot assay, 0156 TTT). An embodiment as set forth in QQQ, RRR, or 8) in situ hybridization, 9) RNase protection assay (Melton SSS wherein a dosage of 400 to 2000 mg of calcium are et al., 1984, and as described in the 1998 catalog of Ambion, administered to a subject per day; Inc., Austin,Tex.), 10) ligase chain reaction, 11) polymerase chain reaction (PCR), 12) reverse transcriptase (RT)-PCR 0157 UUU) An embodiment as set forth in QQQ, RRR, (Berchtold et al., 1989), 13) differential display RT-PCR SSS, or TTT wherein a dosage of X.YZ mg of calcium are (DDRT-PCR) or other suitable combinations of techniques administered to a subject per day, wherein X is any integer and assays. Labels Suitable for use in these detection meth from 400 to 2000, Y is O, 1, 2, 3, 4, 5, 6, 7, 8 or 9, and Z odologies include, and are not limited to 1) radioactive is 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9: labels, 2) enzyme labels, 3) chemiluminescent labels, 4) fluorescent labels, 5) magnetic labels, or other suitable 0158 VVV) An embodiment as set forth in QQQ, RRR, labels, including those set forth below. These methodologies SSS, TTT or UUU wherein a dosage of 900 to 1500 mg of and labels are well known in the art and widely available to calcium are administered to a Subject per day; the skilled artisan. Likewise, methods of incorporating 0159 WWW) An embodiment as set forth in QQQ, RRR, labels into the nucleic acids are also well known to the SSS, TTT, UUU or VVV wherein a dosage of 1000 to 1400 skilled artisan. mg of calcium are administered to a subject per day; 0169. Alternatively, the expression of NADPH oxidase, 0160 XXX) An embodiment as set forth in QQQQQ. UCP2, UCP3, cyclin A, 11B-HSD, TNF-ct, CD 14, MIF, RRR, SSS, TTT, UUU, VVV or WWW wherein a dosage of MIP, M-CSF, G-CSF, IL-6, IL-15, and/or adiponectin can be 1100 to 1300 mg of calcium are administered to a subject per measured at the polypeptide level by using labeled antibod day; ies that specifically bind to the polypeptides in immunoas says such as commercially available protein arrayS/assays, 0161 YYY) An embodiment as set forth in QQQ, RRR, ELISA assays, RIA assays, Western blots or immunohis SSS, TTT, UUU, VVV, WWW or XXX wherein a dosage of tochemical assays. Reagents for Such detection and/or quan 1200 to 1300 mg of calcium are administered to a subject per tification assays can be obtained from commercial sources or day; made by the skilled artisan according to methods well known in the art. 0162 ZZZ) An embodiment as set forth in QQQ, RRR, SSS, TTT, UUU, VVV, WWW, XXX or YYY, wherein the EXAMPLE 1. calcium (or physiologically acceptable salts of calcium) is administered in a single composition; In Vivo Studies 0163 AAAA) An embodiment as set forth in QQQ, RRR, Animals and Diets: SSS, TTT, UUU, VVV, WWW, XXX or YYY, the calcium 0170 A. Animal Pilot Study (or physiologically acceptable salts of calcium) is adminis tered as separate or different compositions; 0171 Six-week old male aP2-agouti transgenic mice and wild-type male littermates (n=12/group) from our colony 0164 BBBB) An embodiment as set forth in QQQ, RRR, were utilized. Six mice randomly selected from each group SSS, TTT, UUU, VVV, WWW, XXX, YYY or AAAA, were sacrificed to provide baseline data and the remaining 6 wherein the separate or different compositions are adminis mice in each group were put on a modified AIN 93 G diet tered simultaneously, sequentially or contemporaneously; (Reeves 1997) with sucrose as the sole carbohydrate source and providing 64% of energy, and fat increased to 25% of 0165 CCCC) An embodiment as set forth in QQQ, RRR, energy with lard as previously described (Zemel et al., 2000; SSS, TTT, UUU, VVV, WWW, XXX, YYY, AAAA or Sun et al., 2004). Mice were studied for 9 days, during which BBBB, wherein the compositions are administered at mul food intake and spillage were measured daily and body tiple times during the day; weight, fasting blood glucose, food consumption assessed 0166 DDDD) An embodiment as set forth in QQQ, RRR, weekly. At the conclusion of the study, all mice were killed SSS, TTT, UUU, VVV, WWW, XXX, YYY, AAAA or under isofluorane anesthesia and fat pads were immediately BBBB, wherein the composition is administered once per excised, weighed and used for further study, as described day; or below. 0167 EEEE) An embodiment as set forth in QQQ, RRR, 0172 B. Diet Study SSS, TTT, UUU, VVV, WWW, XXX, YYY, AAAA, BBBB, 0173 At 6 wk of age, 20 male aP2-agouti transgenic mice CCCC, or DDDD, wherein said administration is parenteral. from our colony were randomly divided into two groups (10 US 2007/00773 10 A1 Apr. 5, 2007 mice/group) and fed a modified AIN 93 G diet with subop diluted in the range of 1.5625-25 ng and used to establish a timal calcium (calcium carbonate, 0.4%) or high calcium standard curve; total RNAs for unknown samples were also (calcium carbonate, 1.2%) respectively, with Sucrose as the diluted in this range. Reactions of quantitative RT-PCR for sole carbohydrate source and providing 64% of energy, and standards and unknown samples were also performed fat increased to 25% of energy with lard. Mice were studied according to the instructions of Smart Cycler System (Cep for three weeks, during which food intake and spillage were heid, Sunnyvale, Calif.) and TaqMan Real Time PCR Core measured daily and body weight, fasting blood glucose, food Kit (Applied Biosystems, Branchburg, N.J.). The mRNA consumption assessed weekly. At the conclusion of the quantitation for each sample was further normalized using study, all mice were killed under isofluorane anesthesia and the corresponding 18s quantitation (Sun et al., 2004c). blood collected via cardiac puncture; fat pads and soleus muscle were immediately excised, weighed and used for Determination of Intracellular ROS Generation further study, as described below. 0.178 Adipose tissue digestion and adipocytes prepara tion were prepared as described in Cali measurement. 0.174. This study was approved from an ethical standpoint Intracellular ROS generation was assessed using 6-carboxy by the Institutional Care and Use Committee of The Uni 2,7'-dichlorodihydrofluorescein diacetate (H2-DCFDA) as versity of Tennessee. described previously (Manea et al., 2004). Cells were loaded Measurement of Adipocyte Intracellular Ca"(Cali) with H2-DCFDA (2 umol/L) 30 min before the end of the 0175 Adipose tissue was first washed several times with incubation period (48 h). After washing twice with PBS, Hank's Balanced Salt Solution (HBSS), minced into small cells were scraped and disrupted by Sonication on ice (20s). pieces, and digested with 0.8 mg/ml type I collagenase in a Fluorescence (emission 543 nm or 527 nm) and DNA shaking water bath at 37° C. for 30 min. Adipocytes were content were then measured as described previously. The then filtered through sterile 500-um nylon mesh and cultured intensity of fluorescence was expressed as arbitary units per in Dulbecco's Modified Eagle's Medium (DMEM) supple ng DNA. mented with 1% fetal bovine serum (FBS). Cells were Statistical Analysis. cultured in Suspension and maintained in a thin layer at the top of culture media for 2 h for cell recovery. Cali in 0.179 Data were evaluated for statistical significance by isolated mouse adipocytes was measured by using a fura-2 analysis of variance (ANOVA), and significantly different dual wavelength fluorescence imaging system. Prior to group means were then separated by the least significant Cali measurement, adipocytes were pre-incubated in difference test by using SPSS (SPSS Inc, Chicago, Ill.). All serum-free medium for 2 hand rinsed with HBSS containing data presented are expressed as meant-SEM. the following components (in mmol/L): NaCl 138, CaCl Results 1.8, MgSO 0.8, NaHPO, 0.9, NaHCO, 4, glucose 5, glutamine 6, Hepes 20, and bovin serum albumin 1%. 0180 Our previous work indicated that aP2-agouti trans Adipocytes were loaded with fura-2 acetoxymethyl ester genic mice are a useful model for diet-induced obesity in a (fura-2 AM) (10 umol/L) in the same buffer in dark for 1 h genetically susceptible human population, as they are non at 37° C. Adipocytes were rinsed with HBSS three times to obese on standard diets but develop mild to moderate remove extracellular dye and then post-incubated at room obesity, hyperglycemia and insulin resistance when fed high temperature for an additional 30 min to permit complete sucrose and/or high fat diets (Zemel et al., 2000; Sun et al., hydrolysis of cytoplasmic fura-2 AM. A thin layer of adi 2004). Given the role of obesity and diabetes in oxidative pocytes was plated in 35 mm dishes with glass cover slips stress, we first investigated whether aP2-agouti transgenic mice are also a suitable model for the study of diet-induced (P35G-0-14-C, Mattek Corporation, Ashland, Mass.). The oxidative stress. Transgenic mice exhibited significantly dishes with dye-loaded cells were mounted on the stage of greater baseline ROS production compared with wild-type Nikon TMS-F fluorescence inverted microscope with a controls prior to the feeding period, and the consumption of Cohu 4915 CCD camera. Fluorescent images were captured the obesity-promoting diet significantly increased adipose alternatively at excitation wavelength of 340 nm and 380 nm tissue ROS production only in aP2-agouti transgenic mice with an emission wavelength of 520 nm. Catli was cal (FIG. 1). This effect was also associated with increased culated by using a ratio equation as described previously NADPH oxidase expression in adipose tissue of aP2-agouti (Zemel, 2003). transgenic mice prior to and following consumption of the Total RNA Extraction. obesity-promoting diet (FIG. 2). 0176). A total cellular RNA isolation kit (Ambion, Austin, 0181 Based on the suitability of this model, we utilized Tex.) was used to extract total RNA from cells according to aP2 transgenic mice as the animal to investigate the effect of manufacturers instruction. dietary calcium in regulation of diet-induced oxidative stress in a three-week obesity induction period on high Sucrose/ Quantitative Real Time PCR high fat diets with either low calcium (0.4% from 0177 Adipocyte 18s, UCP2, NADPH oxidase and 11 B CaCO)(basal diet) or high calcium (1.2% from HSD, and muscle UCP3 and NADPH oxidase were quan CaCO) (high calcium diet) content. Although feeding high titatively measured using a Smart Cycler Real Time PCR fat/high sucrose diets ad libitum for 3 weeks induced weight System (Cepheid, Sunnyvale, Calif.) with a TaqMan 1000 and fat gain in all animals, mice on the high calcium diet Core Reagent Kit (Applied Biosystems, Branchburg, N.J.). gained only 50% of the body weight (p=0.04) and fat The primers and probe sets were obtained from Applied (p<0.001) as mice on the basal diet (FIG. 3). The high Biosystems TaqMan R. Assays-on-DemandTM Gene Expres calcium diet also suppressed diet-induced hyperglycemic sion primers and probe set collection according to manu and reduced fasting blood glucose by 15% compared to mice facture's instruction. Pooled adipocyte total RNA was serial on basal diet (p=0.003) (FIG. 4). The high calcium diet US 2007/00773 10 A1 Apr. 5, 2007 significantly reduced adipose intracellular ROS production stress, dietary calcium may play a role in modulating diet by 64% and 18% (p<0.001) in visceral and subcutaneous induced oxidative stress. Data from the present study dem adipose tissue respectively (FIG. 5). Consistent with this, the onstrate that dietary calcium decreased diet-induced ROS high calcium diet also inhibited adipose tissue NADPH production. Our previous data demonstrate that 1C.. oxidase expression, by 49% (p=0.012) in visceral adipose 25(OH),D, stimulates Ca" signaling and suppresses UCP2 tissue and by 63% (p=0.05) in subcutaneous adipose tissue, expression on human and murine adipocytes (Shi et al., respectively, compared to mice on the basal diet (FIG. 6), 2002: indicating that dietary calcium may inhibit oxidative stress by Suppressing cytosolic enzymatic ROS production. More 0.184 Sun et al., 2004) and suppresses UCP3 expression over, adipocyte intracellular calcium (Cali) levels, which in skeletal muscle (Sun et al., 2004); accordingly, dietary were previously demonstrated to favor adipocyte ROS pro calcium suppression of ROS production is likely due to duction, were markedly Suppressed in mice on the high Suppression of circulating 1C... 25(OH)D levels and result calcium diet by 73%-80% (p<0.001) versus mice on the ant reductions in Ca" signaling and increases in UCP2 and basal diet (FIG. 7), suggesting a role of Cali in regulation UCP3 expression. Furthermore, dietary calcium also of oxidative stress by dietary calcium. Consistent with our appeared to regulate cytosol enzymatic ROS production by previous study, the high calcium diet also induced 367% and inhibiting NADPH oxidase expression, which also contrib 191% increases in adipose UCP2 expression (p<0.001) in utes to cellular ROS production. visceral and Subcutaneous adipose tissue respectively, com 0185. The interaction between ROS and calcium have pared to mice on the basal diet (FIG. 8). Moreover, the been intensively investigated (Toescu 2004; Ermak et al., pattern of UCP3 expression and indices of ROS production 2002; Miwa et al., 2003; Brookes 2005). Calcium signaling in skeletal muscle was consistent with these findings. UCP3 is essential for production of ROS, and elevated intracellular expression was 22% higher (p=0.006) (FIG.9) and NADPH calcium (Cali) activates ROS-generating enzymes, such oxidase expression was 36% lower (p=0.001) (FIG. 10) in as NADPH-oxidase and myeloperoxidase, as well as the soleus muscle of mice on the high calcium diet compared to formation of free radicals by the mitochondrial respiratory mice on the low calcium diet, Suggesting that increases in chain (Gordeeva et al., 2003). Interestingly, increased ROS UCP2 and UCP3 expression in adipose tissue and muscle, production also stimulates Cali by activating calcium respectively, of animals on high calcium diets may contrib channels on both the plasma membrane and endoplasmic ute to reduced ROS levels. reticulum (ER) (Volk et al., 1997). Thus, there is a bi 0182 We have recently shown that lo, 25(OH),D, pro directional interaction wherein ROS cellular calcium motes cortisol production by stimulating 113-HSD expres homeostasis and calcium-dependent physiological processes sion in cultured human adipocytes (Morris et al., 2005). while manipulation of calcium signaling may also regulate However, the effect of modulation of 1C, 25(OH),D, via cellular ROS production. Consistent with this concept, the dietary calcium on this gene expression in vivo had not been present data show that suppression Cali by high dietary investigated. Data from the present study demonstrates that calcium was associated with amelioration of ROS produc the high calcium diet suppressed 113-HSD expression in tion in adipose tissue. visceral adipose tissue by 39% (p=0.034) compared to mice 0186 Respiration is associated with production of ROS, on the basal diet (FIG. 11). Interestingly, 113-HSD expres and mitochondria produce a large fraction of the total ROS sion in visceral fat was markedly higher than Subcutaneous made in cells (Brand et al., 2004). Mild uncoupling of fat in mice on basal low calcium group (p=0.034) whereas respiration diminishes mitochondrial ROS formation by no difference was observed between the fat depots in mice dissipating mitochondrial proton gradient and potential on the high calcium diet. (Miwa et al., 2003). Korshunov et al. has demonstrated that Discussion slight increase of the H backflux (to the matrix), which diminishes Alp, results in a Substantial decrease in mito 0183 Previous data from our laboratory demonstrate that chondrial ROS formation (Korshunov et al., 1997). Accord dietary calcium exerts an anti-obesity effect via a 10. ingly, the H backflow induced by uncoupling via UCPs 25-(OH)-D-mediated mechanism (Zemel, 2005a). We would be expected to down-regulate ROS production. Mild have reported that 1 C, 25-(OH)-D plays a direct role in the activation of UCPs may therefore play a role in the antioxi modulation adipocyte Ca" signaling, resulting in an dant defense system and it is reasonable to propose that increased lipogenesis and decreased lipolysis (Xue et al., dietary calcium induced Suppression of 1C, 25-(OH)2D, 1998; Xue et al., 2000). In addition, 1C..., 25-(OH)-D also which has been demonstrated to inhibit UCP2 expression plays a role in regulating human adipocyte UCP2 expres (Shi et al., 2002), may inhibit ROS production. Indeed, in Sion, Suggesting that the Suppression of 1C, 25-(OH)-D- the present study, we have shown that high dietary calcium and the resulting up-regulation of UCP2 may contribute to up-regulated both UCP2 expression in adipose tissue and increased rates of energy utilization (Shi et al., 2001; Shi et UCP3 expression in skeletal muscle, and these findings were al., 2002). Accordingly, the suppression of 1C, 25-(OH)-D- associated decreased ROS production, indicating a role of by increasing dietary calcium attenuates adipocyte triglyc mitochondrial uncoupling in regulation of oxidative stress. eride accumulation and caused a net reduction in fat mass in both mice and humans in the absence of caloric restriction 0187 We also compared the ROS production between (Zemel et al., 2000), a marked augmentation of body weight Subcutaneous and visceral adipose tissue. Consistent with and fat loss during energy restriction in both mice and our previous data (Zemel, 2005a; Zemel et al., 2005a), humans (Zemel et al., 2000; Zemel, 2004), and a reduction animals on the basal low calcium diet showed markedly in the rate of weight and fat regain following energy higher visceral fat gain than Subcutaneous fat versus mice on restriction in mice (Sun et al., 2004a). Given that obesity and the high calcium diet (data not shown) and exhibited strik related disorders are associated with increased oxidative ingly enhanced ROS production and NADPH oxidase US 2007/00773 10 A1 Apr. 5, 2007 expression in visceral fat versus Subcutaneous fat. Con tiation before conducting chemical treatment. Chemicals versely, high dietary calcium ameliorated visceral fat gain were freshly diluted in adipocyte medium before treatment. and mice on the high calcium diet showed no significantly Cells were washed with fresh adipocyte medium, re-fed with greater ROS production in visceral fat versus subcutaneous medium containing the different treatments, and incubated at fat. These results therefore indicated that higher visceral fat 37° C. in 5% CO, in air before analysis. Cell viability was predisposes to enhanced ROS production. Accordingly, we measured via trypan blue exclusion. further evaluated the involvement of glucocorticoid by mea suring 11B hydroxysteroid dehydrogenase (113-HSD) 0.190 Human preadipocytes used in this study were Sup expression, the key enzyme responsible for converting glu plied by Zen-Bio (Research Triangle, N.C.). Preadipocytes cocorticoid into its active form (Agarwal 2003). We dem were inoculated in DMEM/Ham's F-10 medium (DMEM onstrated that 113-HSD expression in visceral fat was mark F10) (1:1, vol/vol) containing 10% FBS, 15 mmol/L edly higher than Subcutaneous fat in mice on basal low HEPES, and antibiotics at a density of 30,000 cells/cm. calcium group whereas no difference was observed between Confluent monolayers of preadipocytes were induced to the fat depots in mice on the high calcium diet. We also differentiate with a standard differentiation medium consist found the high calcium diet suppressed 113-HSD expression ing of DMEM-F10 (1:1, vol/vol) medium supplemented in visceral adipose tissue compared to mice on the low with 15 mmol/L HEPES, 3% FBS, 33 umol/L biotin, 17 calcium diet. These findings demonstrated that dietary cal umol/L pantothenate, 100 nmol/L insulin, 0.25 umol/L cium exerts greater effect on inhibition of visceral fat gain methylisobutylxanthine (MIX), 1 umol/L dexamethasone, 1 via Suppressing formation of active glucocoticoid and thus umol/L BRL49653, and antibiotics. Preadipocytes were explained the markedly decreased visceral fat gain in mice maintained in this differentiation medium for 3 days and on the high calcium diet than mice on the low calcium diet. Subsequently cultured in adipocyte medium in which Therefore, the enhanced ROS production observed in vis BRL49653 and MIX were omitted. Cultures were refed ceral fat compare to Subcutaneous fat in response to the high every 2-3 days. fat/high Sucrose diet only in mice on low calcium diet UCP2 Transfection Suggested that Suppression of ROS production by dietary 0191 UCP2 full-length cDNAs was amplified by RT calcium may be mediated, at least in part, by the regulation PCR using mRNAs isolated from mouse white adipose of glucocorticoid associated fat distribution. We recently tissues. The PCR primers for this amplification are shown as reported in vitro observation that 1C, 25 (OH), D directly follows: UCP2 forward, 5'-GCTAGCATGGTTG regulates adipocyte 113-HSD 1 expression and local cortisol GTTTCAAG-3 (SEQ ID NO: 1), reverse, 5'-GCTAGCT levels in cultured human adipocytes (Morris et al., 2005), CAGAAAGGTGAATC-3' (SEQID NO: 2). The PCR prod and data from this study provides the first in vivo evidence ucts were then subcloned into pcDNA4/His expression that dietary calcium may contribute to the preferential loss vectors. The linearized constructs were transfected into of visceral adiposity and obesity associated oxidative stress 3T3-L1 preadipocytes using lipofectamine plus standard by regulating adipose tissue 113-HSD expression and glu protocol (Invitrogen, Carlsbad, Calif.). After 48 hrs of cocorticoid production. transfection, cells were split and cultured in selective 0188 In conclusion, these data support a role for dietary medium containing 400 ug/ml Zeocin for the selection of calcium in the regulation of diet- and obesity-induced oxi resistant colonies. Cells were fed with selective medium dative stress. Potential mechanisms include increases in every 3 days until resistant colonies could be identified. UCP2 and UCP3 expression, suppression of Cali, and/or These resistant foci were picked, expanded, tested for inhibition of NADPH oxidase and 113-HSD gene expres expression, and frozen for future experiments. Sion. These data also Support our previous observation that Determination of Mitochondrial Membrane Potential dietary calcium inhibits obesity, with partially selective effects on visceral adipose tissue, and leads to significant 0.192 Mitochondrial membrane potential was analyzed changes in adipose tissue metabolism, including accelerated fluorometrically with a lipophilic cationic dye JC-1 (5.5".6, adipose tissue deposition and reduced ROS production. 6'-tetrachloro-1,1,3,3'-tetraethylbenzimidazol carbocyanine iodide) using a mitochondrial potential detection kit (Bio EXAMPLE 2 carta, San Diego, Calif.). Mitochondrial potential was deter mined as the ratio of red fluorescence (excitation 550 nm, 1.25-Dihyrdoxyvitamin D Modulation of Reactive emission 600 nm) and green fluorescence (excitation 485 Oxigen Species Production and Cell Proliferation in nm, emission 535 nm) using a fluorescence microplate Human and Murine Adipocytes reader. 0189 3T3-L1 preadipocytes were incubated at a density Measurement of Intracellular Ca"(Ca'i) of 8000 cells/cm (10 cm dish) and grown in Dulbecco's 0193 Cali in adipocytes was measured using a fura-2 modified Eagle's medium (DMEM) containing 10% FBS dual-wavelength fluorescence imaging system. Cells were and antibiotics (adipocyte medium) at 37° C. in 5% CO in plated in 35-mm dishes (P35G-0-14-C, Mattek). Prior to air. Confluent preadipocytes were induced to differentiate Cali measurement, cells were put in serum-free medium with a standard differentiation medium consisting of overnight and rinsed with HEPES balanced salt solution DMEM-F10 (1:1, vol/vol) medium supplemented with 1% (HBSS) containing the following components (in mmol/L): FBS, 1 uM dexamethasone, IBMX (0.5 mM) and antibiotics 138 NaCl, 1.8 CaCl, 0.8 MgSO, 0.9 NaHPO, 4 (1% Penicillin-Streptomycin). Preadipocytes were main NaHCO, 5 glucose, 6 glutamine, 20 HEPES, and 1% and tained in this differentiation medium for 3 days and subse 1% bovine serum albumin. Cells were loaded with fura-2 quently cultured in adipocyte medium. Cultures were re-fed acetoxymethyl ester (fura-2 AM) (10 umol/L) in the same every 2-3 days to allow 90% of cells to reach full differen buffer for 2 hat 37° C. in a dark incubator with 5% CO. To US 2007/00773 10 A1 Apr. 5, 2007 remove extracellular dye, cells were rinsed with HBSS three DNA content were then measured as described previously. times and then post-incubated at room temperature for an The intensity of fluorescence was expressed as arbitary units additional 1 h for complete hydrolysis of cytoplasmic fura-2 per ng DNA. AM. The dishes with dye-loaded cells were mounted on the stage of Nikon TMS-F fluorescence inverted microscope Statistical Analysis with a Cohu model 4915 charge-coupled device (CCD) 0198 All data are expressed as meant SEM. Data were camera. Fluorescent images were captured alternatively at evaluated for statistical significance by analysis of variance excitation wavelengths of 340 and 380 nm with an emission (ANOVA), and significantly different group means were wavelength of 520 nm. After establishment of a stable then separated by the least significant difference test by baseline, the responses to 1C, 25-(OH)-D was determined. using SPSS (SPSS Inc, Chicago, Ill.). Cali was calculated using a ratio equation as described previously. Each analysis evaluated responses of 5 repre Results sentative whole cells. Images were analyzed with InCyt Im2 0199 Our first aim was to examine whether ROS have version 4.62 imaging software (Intracellular Imaging, Cin effect on adipocyte proliferation. The data presented in FIG. cinnati, Ohio). Images were calibrated using a fura-2 cal 12 indicate that this is indeed the case. Treatment of 3T3-L1 cium imaging calibration kit (Molecular Probes, Eugene, adipocytes with H2O, increased the total DNA of cultured Oreg.) to create a calibration curve in solution, and cellular cells by 39% (p<0.001), while addition of antioxidant C.-to calibration was accomplished using digitonin (25 umol/L) copherol completely blocked this effect. The effect of ROS and pH 8.7 Tris-EGTA (100 mmol/L) to measure maximal on adipocyte proliferation appears to be regulated by mito and minimal Cali levels respectively. chondrial uncoupling and intracellular calcium homeostasis. Total RNA Extraction Addition of mitochondrial uncoupling inhibitor GDP aug mented the stimulation of cell proliferation by HO, by 0194 A total cellular RNA isolation kit (Ambion, Austin, 183% (p<0.005) while calcium channel antagonist nife Tex.) was used to extract total RNA from cells according to dipine had the opposite effect and suppressed HO induced manufacturers instruction. cell DNA synthesis (p<0.05). Since inhibiting mitochondrial uncoupling and increasing Cali have been demonstrated Quantitative Real Time PCR to contribute to increased ROS production, GDP may 0.195 Adipocyte 18s, cyclin A, NADPH oxidase, and increases DNA synthesis by increasing ROS production UCP2 were quantitatively measured using a Smart Cycler while nifedipine exerts the opposite effect via suppression of Real Time PCR System (Cepheid, Sunnyvale, Calif.) with a ROS production. Consistent with this, FIG. 13 shows that TaqMan 1000 Core Reagent Kit (Applied Biosystems, addition of GDP increased ROS production by 24% Branchburg, N.J.). The primers and probe sets were ordered (p<0.01) compare H.O. treatment alone while nifedipine from Applied Biosystems TaqMan(R) Assays-on-DemandTM inhibited HO, induced ROS production by 25% (p<0.003). Gene Expression primers and probe set collection according FIGS. 12 and 13 also demonstrate that addition of antioxi to manufacture's instruction. Pooled adipocyte total RNA dant C.-tocopherol inhibited both ROS production and DNA was serial-diluted in the range of 1.5625-25 ng and used to synthesis in all groups. These results suggest that ROS establish a standard curve; total RNAs for unknown samples stimulated cell proliferation in cultured adipocytes and that were also diluted in this range. Reactions of quantitative this effect can be regulated by mitochondrial uncoupling RT-PCR for standards and unknown samples were also status and intracellular homeostasis. Similar results were performed according to the instructions of Smart Cycler also observed in human adipocytes (data not shown). System (Cepheid, Sunnyvale, Calif.) and TaqMan RealTime 0200. To further investigate the interaction between ROS PCR Core Kit (Applied Biosystems, Branchburg, N.J.). The and mitochondrial uncoupling status, we measured mito mRNA quantitation for each sample was further normalized chondrial potential in both wild-type 3T3-L1 adipocytes and using the corresponding 18s quantitation. UCP2 transfected 3T3-L1 adipocytes. FIG. 14 demonstrates that HO, increased mitochondrial potential by 72% and that Assessment of Cell Proliferation addition of GDP augmented this effect by 10%, indicating 0196) Cells were plated in DMEM with different treat that ROS production inhibits mitochondrial uncoupling. ment in duplicate in 96-well plates. After 48 h, a CyOUANT Nifedipine Suppressed the H2O induced increase in mito Cell Proliferation Kit (Molecular Probes, Eugene, Oreg.) chondrial potential and this result confirms that calcium was used following the manufacturer's protocol. a micro channel antagonist inhibits ROS production. UCP2 trans plate fluorometer (Packard Instrument Company, Inc., fection increased mitochondrial potential and Suppressed the Downers Grove, Ill.) was used to measure CyOUANT effect of HO on mitochondrial uncoupling, indicating that fluorescence. Cell viability was determined by Trypan blue ROS production is regulated, in part by mitochondrial exclusion examination. potential and UCP2. Determination of Intracellular ROS Generation 0201 FIG. 15 demonstrates that ROS has a direct role in regulation of intracellular calcium homeostasis in 3T3-L1 0197) Intracellular ROS generation was assessed using adipocytes. HO, induced a 5-fold increase in Cali 6-carboxy-2,7'-dichlorodihydrofluorescein diacetate (H2 (p<0.001) and this effect was reversed by addition of anti DCFDA) as described previously (Manea et al., 2004). Cells oxidant C-tocopherol. Since Suppression of intracellular were loaded with H2-DCFDA (2 umol/L) 30 minute before calcium influx by nifedipine decreased ROS production as the end of the incubation period (48 h). After washing twice described in FIG. 13, this result suggests a positive feedback with PBS, cells were scraped and disrupted by sonication on interaction between ROS production and intracellular cal ice (20s). Fluorescence (emission 543 nm or 527 nm) and cium homeostasis: ROS stimulate Cali and elevated US 2007/00773 10 A1 Apr. 5, 2007 Cali also favors ROS production. Similar results were proliferation in cultured adipocytes. This effect can be observed in Zen-Bio human adipocytes (data not shown). augmented by a mitochondrial uncoupling inhibitor and 0202 Hyperglycemia is one of the most common clinical Suppressed by a calcium channel antagonist, indicating that signs in obesity and diabetes, which has been demonstrated mitochondrial potential and intracellular calcium homeosta to be associated with increased ROS production. Accord sis may play a role in regulation of ROS induced cell ingly, we next investigated the effect and mechanism of high proliferation. 1C, 25-(OH)D, which has been demon glucose level on ROS production and consequent adipocyte strated to stimulate Cali and to inhibit UCP2 expression, proliferation. As shown in FIG. 16, high glucose treatment stimulates ROS production and cell proliferation in adipo increased ROS production significantly (p<0.05) and this cytes. High glucose also exerts stimulatory effect on ROS effect was partially reversed by addition of nifedipine. production and this effect can be augmented by addition of Addition of GDP further stimulated ROS production com 1C, 25-(OH)2D, Suggesting that 1C, 25-(OH)D may pared to glucose alone. Notably, treatment of adipocytes involved in regulation of ROS production in adipocytes. with 1C, 25-(OH)D, which was previously found to sup These results indicate that strategies to Suppress 1C.. press mitochondrial uncoupling and to increase Cali in 25-(OH).D. levels, such as increasing dietary calcium, may adipocytes, resulted in greater stimulation of ROS produc reduce oxidative stress and thereby inhibit ROS-induced tion than either glucose alone or glucose plus GDP (p<0.05), stimulation of adipocyte proliferation. Suggesting that 1C, 25-(OH)D stimulates ROS production by both inhibition of mitochondrial uncoupling and stimu 0205 Elevated oxidative stress has been reported in both lation of Cali. Glucose also increased Cali by 3-fold humans and animal models of obesity (Sonta et al., 2004: (p<0.001) (FIG. 17) and this effect was partially blocked by Atabek et al., 2004), Suggesting that ROS may play a critical addition of C-tocopherol, indicating that stimulation of role in the mechanisms underlying proliferative responses. Cali by high glucose is partially attributable to ROS This concept is supported by evidence that both HO and production. Consistent with this, FIG. 18 shows that high Superoxide anion induce mitogenesis and cell proliferation glucose also increased expression of NADPH oxidase in several mammmalian cell types (Burdon 1995). Further (p<0.001), a key enzyme in ROS production, in both wild more, reduction of oxidants via Supplementation with anti type and UCP2 transfected 3T3-L1 adipocytes, but UCP2 oxidants inhibits cell proliferation in vitro (Khan et al., overexpression attenuated this effect. These results suggest 2004; Simeone et al., 2004). Although the mechanisms for that high glucose may increase ROS production by Stimu the involvement of oxidative stress in the induction of cell lating NADPH oxidase expression. Addition of 1 O. proliferation are not known, it has been demonstrated that 25-(OH) D, stimulated NADPH oxidase expression while ROS and other free radicals influence the expression of nifedipine suppressed its expression. Although GDP has number of genes and transduction pathways involved in cell been shown to increases ROS production, we found GDP growth and proliferation. The most significant effects of suppressed NADPH oxidase expression, indicating that oxidant on signaling pathways have been observed in the regulation of ROS production by GDP is not via up mitogen-activated protein (MAP) kinase/AP1, and it has regulation of ROS-generating enzyme gene expression. FIG. been suggested that ROS can activate MAP kinases and 19 provides further evidence for the role of UCP2 in the thereby transcription factors activator protein-1 (AP-1) regulation high glucose induced ROS production. High (Chang et al., 2001), a collection of dimeric basic region glucose inhibits UCP2 expression in both wild type and leucine Zipper proteins which activates cyclin-dependent UCP2 transfected adipocytes, indicating that high glucose kinase and entry into cell division cycle (Kouzarides et al., stimulates ROS production by regulating mitochondrial 1989). Furthermore, the elevation of cytosolic calcium level uncoupling status. induced by ROS results in activation of protein kinase C (PKC) required for expression of positive regulators of cell 0203 FIG. 20 demonstrates that stimulation of ROS proliferation Such as c-fos and c-jun (Lin 2004, Amstad et production by high glucose is associated with increased al., 1992; Hollander et al., 1989). ROS have also been DNA synthesis. High glucose alone significantly increased implicated as a second messenger involved in activation of DNA synthesis (p<0.03) and this effect was by augmented NF-kB (Song et al., 2004), whose expression has been by addition of GDP or 1C, 25-(OH)2D. In contrast, inhi shown to stimulate cell proliferation via tumor necrosis bition of ROS production by nifedipine decreased glucose factor (TNF) and interleukin-1 (IL-1) (Giri et al., 1998). The induced DNA synthesis (p<0.05). To further investigate the effect of ROS on NF-kB activation is further supported by effect of high glucose on adipocyte proliferation, we also studies which demonstrated that expression NF-kB can be observed the expression of cyclin A (FIG. 21). Consistent suppressed by antioxidants (Nomura et al., 2000; Schulze with the DNA synthesis data, high glucose stimulated cyclin Osthoff et al., 1997). In addition, ROS can modify DNA A expression by 3-fold (p<0.001), and GDP and 1C. methylation and cause oxidative DNA damage, which result 25-(OH),D, augmented this effect while nifedipine sup in decreased methylation patterns (Weitzman et al., 1994) pressed its expression. These data Suggest high glucose and consequently contribute to an overall aberrant gene stimulates adipocyte proliferation and this effect may be at expression. ROS may also attribute to the inhibition of least partially mediated by its stimulation of ROS produc cell-to-cell communication and this effect can result in tion. decreased regulation of homeostatic growth control of nor Discussion mal Surrounding cells and lead to clonal expansion (Cerutti et al., 1994; Upham et al., 1997). Despite these mechanisms 0204 Obesity and diabetes are associated with increased proposed to explain the stimulatory effect on cell prolifera oxidative stress, and ROS may play a role in regulation of tion, limited Studies have been conducted on adipocytes. In adipocyte proliferation. In the present study, we demon present study, we demonstrated that low concentrations of strated that a low concentration of HO stimulates cell ROS promote cell proliferation in cultured human and US 2007/00773 10 A1 Apr. 5, 2007 murine adipocytes. However, further investigation for the and this effect can by Suppressed by mitochondrial uncou underlying molecular mechanisms is required. pling and stimulated by elevation of intracellular calcium. 0206. The yield of ROS can be efficiently modulated by 1C, 25-(OH)D increases ROS production by inhibiting mitochondrial uncoupling. Korshunov et al. has demon UCP2 expression and increasing Cali and consequently strated that slight increase of the H backflux (to the matrix), favors adipocyte proliferation. Accordingly, the present data which diminishes Alp, results in a Substantial decrease of suggest that suppression 1C, 25-(OH)D by increasing mitochondrial ROS formation (Korshunov et al., 1997). dietary calcium may reduce 1C, 25-(OH)D mediated ROS Accordingly, the H backflow from UCP-induced uncou production and limit ROS induced adipocyte proliferation, pling would be expected to down-regulate ROS production. resulting in reduced adiposity. In addition, calcium can active ROS-generating enzymes 0208. This work demonstrated a direct effect of oxidative directly and activation of calcium dependent PKC favors stress on adipocyte proliferation in white adipose tissue and assembly of the active NADPH-oxidase complex (Gordeeva this observation may have important implications in under et al., 2003), indicating that Cali may be another key standing the adipose mass changes observed under oxidative player in regulation of ROS production. Accordingly, it is stress. However, cell proliferation was only evaluated by reasonable to propose that 1C, 25-(OH). D., which has been DNA content and cyclin expression level. Further, various demonstrated both to inhibit mitochondrial uncoupling and sources of ROS production may play different roles in to stimulate Cali in adipocytes, would stimulate ROS regulation of cell signaling in cell cycle and cell metabolism. production and may consequently be involved in the regu Although we demonstrated both mitochondrial ROS pro lation of adipocyte proliferation. Indeed, in the present duction and cellular enzymatic ROS production are associ study, we have shown that addition of 1C, 25-(OH). D. ated with adipocyte proliferation, the contribution of each augmented high glucose-induced ROS production and adi Source needs further investigation. pocyte proliferation. This effect was further enhanced by a mitochondrial uncoupling inhibitor and Suppressed by cal EXAMPLE 3 cium channel antagonism, indicating that 1 C, 25-(OH)D. Calcium and 1.25-(OH)-D Regulation of stimulates ROS production by increasing Cali and by Adipokine Expression in Murine and Human inhibiting mitochondrial uncoupling. Furthermore, previous Adipocytes and aP2-Agouti Transgenic Mice studies Suggest that 1C, 25-(OH)D may act as an prooxi dant in various cell types (Koren et al., 2001) and treatment Materials and Methods with 1C, 25-(OH)2Ds inhibited the expression of the major Animals and Diets constituents of the cellular defense system against ROS (Banakar et al., 2004). 0209 At 6 wk of age, 20 male aP2-agouti transgenic mice 0207 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 subop lipid and energy metabolism via both genomic and non timal calcium (0.4% from calcium carbonate) or high cal genomic pathways (Zemel, 2004; Shi et al., 2001; Shi et al., cium (1.2% from calcium carbonate) respectively. Sucrose 2002). We have reported that 1C, 25-(OH)-D plays a direct was the sole carbohydrate source, providing 64% of energy, role in the modulation adipocyte Ca" signaling, resulting in and fat was increased to 25% of energy with lard. Mice were an increased lipogenesis and decreased lipolysis (Shi et al., studied for three weeks, during which food intake and 2001). In addition, 1C.., 25-(OH)-D, also plays a role in spillage were measured daily and body weight, fasting blood regulating human adipocyte UCP2 mRNA and protein lev glucose, food consumption assessed weekly. At the conclu els, indicating that the Suppression of 1C, 25-(OH)-D and sion of the study, all mice were killed under isofluorane the resulting up-regulation of UCP2 may contribute to anesthesia and blood collected via cardiac puncture; visceral increased rates of lipid oxidation (Shi et al., 2002). In fat pads (perirenal and abdominal), Subcutaneous fat pads addition, we also demonstrate that physiological doses of (Subscapular) and soleus muscle were immediately excised, 1C, 25-(OH)-D inhibit apoptosis in differentiated human weighed and used for further study, as described below. and 3T3-L1 adipocytes (Sun et al., 2004b), and that the 0210. This study was approved from an ethical standpoint Suppression of 1C, 25-(OH)2-D in vivo by increasing by the Institutional Care and Use Committee of The Uni dietary calcium stimulates adipocyte apoptosis in aP2 trans versity of Tennessee. genic mice (Sun et al., 2004b), Suggesting that the stimula tion of adipocyte apoptosis contributes to the observed Cell Culture reduction in adipose tissue mass after administration of high 0211 3T3-L1 pre-adipocytes were incubated at a density calcium diets (Shi et al., 2002). Accordingly, the Suppression of 8000 cells/cm (10 cm dish) and grown in Dulbecco's of 1C, 25-(OH)-D by increasing dietary calcium attenuates modified Eagle's medium (DMEM) containing 10% FBS adipocyte triglyceride accumulation and caused a net reduc and antibiotics (adipocyte medium) at 37°C. in 5% CO in tion in fat mass in both mice and humans in the absence of air. Confluent pre-adipocytes were induced to differentiate caloric restriction (Zemel et al., 2000), a marked augmen with a standard differentiation medium consisting of tation of body weight and fat loss during energy restriction DMEM-F10 (1:1, vol/vol) medium supplemented with 1% in both mice and humans (Zemel et al., 2000; Zemel et al., fetal bovine serum (FBS), 1 uM dexamethasone, isobutyl 2004), and a reduction in the rate of weight and fat regain methylxanthine (IBMX) (0.5 mM) and antibiotics (1% Peni following energy restriction in mice (Sun et al., 2004). Data cillin-Streptomycin). Pre-adipocytes were maintained in this from present study provide further evidence to support the differentiation medium for 3 days and subsequently cultured role of 1C, 25-(OH)D in favoring energy storage and fat in adipocyte medium. Cultures were re-fed every 2-3 days to mass expansion by stimulating ROS production and adipo allow 90% cells to reach fully differentiation before con cyte proliferation. ROS stimulates adipocyte proliferation ducting chemical treatment. US 2007/00773 10 A1 Apr. 5, 2007 0212 Human pre-adipocytes used in this study were Statistical Analysis supplied by Zen-Bio (Research Triangle, N.C.). Preadipo 0217. Data were evaluated for statistical significance by cytes were inoculated in DMEM/Ham's F-10 medium analysis of variance (ANOVA) or t-test, and significantly (DMEM-F10) (1:1, vol/vol) containing 10% FBS, 15 different group means were then separated by the least mmol/L 4-2-hydroxyethyl-1-piperazineethanesulfonic acid significant difference test by using SPSS (SPSS Inc, Chi (HEPES), and antibiotics at a density of 30,000 cells/cm. cago, Ill.). All data presented are expressed as meantSEM. The cells are isolated from the stromal vascular fraction of human Subcutaneous adipose tissue and differentiated in Results vitro as follows: Confluent monolayers of pre-adipocytes 0218. Dietary calcium regulates inflammatory cytokine were induced to differentiate with a standard differentiation production in adipose tissue and skeletal muscle. Feeding medium consisting of DMEM-F10 (1:1, vol/vol) medium the high calcium ad libitum for 3 weeks significantly supplemented with 15 mmol/L HEPES, 3% FBS, 33 umol/L decreased weight and fat gain (Table 4) and Suppressed biotin, 17 umol/L pantothenate, 100 nmol/L insulin, 0.25 TNFC. gene expression by 64% in visceral, but not subcu umol/L methylisobutylxanthine, 1 umol/L dexamethasone, 1 taneous, fat compared with mice on low calcium basal diet umol/L BRL49653, and antibiotics. Preadipocytes were (FIG. 22A)(p<0.001). Similarly, IL-6 expression was maintained in this differentiation medium for 3 days and decreased by 51% in visceral fat of mice on the high calcium Subsequently cultured in adipocyte medium in which diet versus mice on the low calcium basal diet (FIG. 22B) BRL49653 and MIX were omitted. Cultures were re-fed (p<0.001) and this effect was absent in subcutaneous fat. In every 2-3 days till fully differentiated. contrast, dietary calcium up-regulated IL-15 expression in visceral fat, with a 52% increases in mice on high calcium 0213 Cells were incubated in serum free medium over diet compared with animals on low calcium diet (FIG. 23A) night before chemical treatment. Chemicals were freshly (p=0.001). Adiponectin expression was similarly elevated in diluted in adipocyte medium before treatment. Cells were visceral fat of mice on the high calcium diet versus mice on washed with fresh adipocyte medium, re-fed with medium low calcium diet (FIG. 23B) (p=0.025). The high calcium containing the different treatments (control, 10 nmol/L 1C. diet also induced a 2-fold increase in IL-15 expression in 25-(OH)-D, 10 umol/L nifedipine, 10 nmol/L 1C, soleus muscle compared with mice on low calcium diet 25-(OH)-D, plus 10 umol/L nifepipine, 100 nmol/L H.O. (FIG. 23C) (p=0.01). 1 umol/L Cittocopherol, or 100 nmol/L H.O. plus 1 umol/L 0219 Intracellular calcium and 1C, 25-(OH)-D regu Cittocopherol) and incubated at 37° C. in 5% CO, for 48 h. lates cytokine production in cultured murine and human in air before analysis. Cell viability was measured via trypan adipocytes. 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 0214) 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 manufacturers instruction. larly, 1C.., 25-(OH)-D markedly increased IL-6 expression in 3T3-L1 adipocyte and this effect was reversed by addition Plasma lo, 25-(OH)-D, Assay of nifedipine (p=0.016) (FIG. 24B). Similar results were observed in human adipocytes (data not shown). These data 0215 A1C, 25-(OH)-D-vitamin D ELISA kit was used suggested that 1C, 25-(OH)-D3 stimulated cytokine pro to measure plasma 1 C, 25-(OH)-D content according to duction by increasing intracellular calcium influx. The high the manufacturers instructions (Alpco Diagnostics, calcium diet suppressed plasma 1C, 25-(OH)-D (FIG. Windham, N.H.). 24C). Quantitative Real Time PCR 0220 Similar results were also observed in differentiated 0216 Adipocyte and muscle 18s, TNFO, IL-6, IL-8, human adipocytes; 1 C, 25-(OH)2-D stimulated IL-6 and IL-15 and adiponectin were quantitatively measured using a IL-8 expression by 53% and 49% respectively (FIG. 25A, smart cycler real-time PCR system (Cepheid, Sunnyvale, p=0.004) (FIG. 25B, p<0.001), and the addition of nife Calif.) with a TaqMan 1000 Core Reagent Kit (Applied dipine blocked this effect. However, we found no effect of Biosystems, Branchburg, N.J.). The primers and probe sets 1C, 25-(OH)-D, or nifedipine on IL-15 (FIG. 25C, were obtained from Applied Biosystems TaqMan(R) Assays p=0.473) or adiponectin expression (FIG. 25D, p=0.377) in on-DemandTM Gene Expression primers and probe set col the human adipocytes. lection and utilized according to manufactures instructions. 0221) Reactive oxygen species exerted direct impact on Pooled adipocyte total RNA was serial-diluted in the range cytokine production in cultured adipocytes. The direct role of 1.5625-25 ng and used to establish a standard curve; and of ROS in regulation of adipose cytokine production was total RNA for the unknown samples were also diluted in this investigated in differentiated 3T3-L1 adipocytes. FIG. 26A range. Reactions of quantitative RT-PCR for standards and shows that hydrogen peroxides increased IL-6 expression by unknown samples were also performed according to the 167% (p<0.001) and that this effect was attenuated by the instructions of Smart Cycler System (Cepheid, Sunnyvale, addition of anti-oxidant Cittocopherol (p=0.016), indicating Calif.) and TaqMan Real Time PCR Core Kit (Applied that ROS exerted a direct role in stimulation of inflammatory Biosystems, Branchburg, N.J.). The mRNA quantitation for cytokine production. Clittocopherol also increased adiponec each sample was further normalized using the corresponding tin production (p=0.002), although ROS (hydrogen perox 18s quantitation. ide) was without significant effect (p=0.06) (FIG. 26B). US 2007/00773 10 A1 Apr. 5, 2007 Similarly, there was no direct effect of ROS on IL-15 lating IL-6, with visceral fat contributing markedly more expression; however, addition of Cittocopherol markedly IL-6 compared with subcutaneous fat (Fried et al., 1998: increased IL-15 by 2.2-fold as compared to HO-treated Fain et al., 2004). Expression of TNFC. is increased in cells (P=0.043) (FIG. 26C), providing further evidence that inflammatory conditions such as obesity and cachexia and oxidative stress is involved in adipocyte cytokine production considered a likely mediator of insulin resistance associated with visceral adiposity (Hotamisligil et al., 1994: Ofei et al., Discussion 1996). Consistent with this, diet-induced obesity in present 0222 Previous data from our laboratory demonstrate that study resulted in increased expression of TNFC. and IL-6 in dietary calcium exerts an anti-obesity effect and Suppresses visceral fat, and dietary calcium attenuated these effects. obesity associated oxidative stress via a 1C, 25-(OH)-D- 0224 IL-15 is highly expressed in skeletal muscle, where mediated mechanism (Zemel, 2005b, Zemel, 2004). We it exerts anabolic effects (Busquets et al., 2005). IL-15 have demonstrated that 1C, 25-(OH)-D plays a direct role administration reduces muscle protein degradation and in the modulation of adipocyte Ca" signaling, resulting in inhibits skeletal muscle wasting in degenerative conditions an increased lipogenesis and decreased lipolysis (Shi et al., such as cachexia (Carbo et al., 2000a). Interestingly, IL-15 2001). In addition, 1C, 25-(OH)-D is also involved in exerts the opposite effect in adipose tissue; administration of regulation of metabolic efficiency by modulating adipocyte IL-15 reduced fat deposition without altering food intake UCP2 expression (Shi et al., 2003). Accordingly, the Sup and Suppressed fat gain in growing rats (Carbo et al., 2000b. pression of 1C..., 25-(OH)-D by increasing dietary calcium Carbo et al., 2001). IL-15 also stimulates adiponectin secre attenuates adipocyte triglyceride accumulation and causes a tion in cultured 3T3-L1 adipocytes (Quinn et al., 2005), net reduction in fat mass in both mice and humans in the indicating a role for IL-15 in regulating adipocyte metabo absence of caloric restriction (Zemel et al., 2000; Zemel et lism. These observations Suggest that IL-15 might be al., 2005b), a marked augmentation of body weight and fat involved in a muscle-fat endocrine axis and regulate energy loss during energy restriction in both mice and humans utilization between the two tissues (Argiles et al., 2005). We (Zemel et al., 2000; Thompson et al., 2005; Zemel et al., previously found calcium-rich diets to Suppress fat gain and 2004; Zemel et al., 2005a), and a reduction in the rate of accelerate fat loss while protecting muscle mass in diet weight and fat regain following energy restriction in mice induced obesity and during energy restriction, indicating (Sun et al., 2004a). Given that obesity and related disorders that dietary calcium may similarly regulate energy partition are associated with low grade systemic inflammation (Lee et ing in a tissue selective manner. In the present study, we al., 2005), it is possible that dietary calcium may also play provide the first in vivo evidence that dietary calcium a role in modulating adipose tissue cytokine production. up-regulates IL-15 expression in visceral adipose tissue and Data from the present study demonstrate that dietary cal skeletal muscle, and stimulates adiponectin expression in cium decreased production of pro-inflammatory factors such visceral adipose tissue, skeletal muscle and stimulates adi as TNFC. and IL-6 and increased anti-inflammatory mol ponectin expression in visceral adipose tissue in aP2 agouti ecules such as IL-15 and adiponectin in visceral fat. We also transgenic mice. This suggests that dietary calcium may also found that 1C, 25-(OH)-D, stimulated TNFO, IL-6 and IL-8 regulate energy metabolism, in part, by modulating these production in cultured human and murine adipocytes and cytokines in both adipose tissue and skeletal muscle, thereby that this effect was completely blocked by a calcium channel favoring elevated energy expenditure in adipose tissue and antagonist, Suggesting that dietary calcium Suppresses preserving energy storage in skeletal muscle. However, we inflammation factor production in adipocyte and that 10. found no effect of 1C, 25-(OH)-D on IL-15 expression in 25-(OH)-D-induced Ca" influx may be a key mediator of human adipocytes. Since these human adipocytes were this effect. FIGS. 22-23 demonstrate that dietary calcium originally developed from Subcutaneous fat, these results decreased expression of pro-inflammatory factors (TNFC. further support our in vivo observations of dietary calcium and IL-6) and increased anti-inflammatory molecules (IL-15 regulation of adipocyte cytokine production in a depot and adiponectin) in visceral adipose tissue and that dietary specific manner, although we do not have data from human calcium up-regulates expression of IL-15 in both visceral visceral adipocytes for comparison. adipose tissue and skeletal muscle, and stimulates adiponec 0225. We have recently shown that 1C, 25-(OH)-D, tin expression in visceral adipose tissue in aP2 agouti stimulated ROS production in cultured adipocytes and that transgenic mice. This suggests that dietary calcium is Suppression of 1C, 25-(OH)2-D via dietary calcium also involved in regulation of energy metabolism by modulating attenuates adipose oxidative stress (Sun et al., 2006), Sug endocrine function of both adipose tissue and skeletal gesting a potential connection between oxidative tress and muscle, resulting in a pattern which favors reduced energy production of inflammatory factors. The present data dem storage in adipose tissue and elevated protein synthesis and onstrate that hydrogen peroxide stimulates adipocyte IL-6 energy expenditure in skeletal muscle. expression and Cittocopherol inhibits this effect. Although 0223 Obesity is associated with increased expression of hydrogen peroxide showed no direct effect on the expression inflammatory markers (Valle et al., 2005), while weight loss of anti-inflammatory factors adiponectin and IL-15, addition results in decreased expression and secretion of pro-inflam of Ottocopherol markedly elevated the expression of both, matory components in obese individuals (Clement et al., Suggesting a direct role of oxidative stress in regulating 2004). Accordingly, modulation of the adipose tissue mass inflammation. Indeed, previous studies have demonstrated appears to result in corresponding modulation of cytokine that oxidative stress was augmented in adiposity, with ROS production. TNFC. and IL-6 are two intensively studied elevated in blood and tissue in various animal model of cytokines in obesity and have been consistently found to be obesity (Suzuki et al., 2003: Furukawa et al., 2004), while increased in the white adipose tissue of obese subjects markers of systemic oxidative stress were inversely related (Cottam et al., 2004). Previous studies suggest that white to plasma adiponectin in human Subjects (Furukawa et al., adipose tissue contributes a considerable portion of circu 2004: Soares et al., 2005). Moreover, addition of oxidants US 2007/00773 10 A1 Apr. 5, 2007 Suppressed expression of adiponectin and increased expres survival, in differentiated human adipocytes. Calcitriol sion of IL-6, MCP-1 and PAI-1 (Soares et al., 2005). These markedly increased MIF and CD 14 expression by 59%(p= results indicate that a local increase in oxidative stress in 0.001) and 33% (p=0.008). respectively, while calcium accumulated fat causes dysregulated production of adipo channel antagonism with nifedipine completely reversed cytokines. The role of adiposity in up-regulation of oxidative these effects, indicating that calcitriol stimulates MIF and stress and inflammation has been investigated intensively. CD14 expression via a calcium-dependent mechanism. Fat accumulation stimulates NADPH oxidase expression in Similar results were also found in cultured 3T3-L1 adipo white adipose tissue (Sun et al., 2004d; Inoguchi et al., cytes; in addition, calcitriol also up-regulated M-CSF, MIP. 2000). Further, NOX4, an isoform of NADPH oxidase, is MCP-1 (monocyte chemoattractant protein-1) and IL-6 expressed in adipocytes, but not in macrophage (Mahadev et expression in 3T3-L1 adipocyte and stimulated tumor necro al., 2004; Sorescu et al., 2002). Xu et al. (2003) and sis factor-O. (TNF-C.) and IL-6 expression in RAW264 Weisberg et al. (2003) also reported that ROS stimulated macrophage cultured alone and this effect was blocked by macrophages infiltration of obese adipose tissue via ROS either a calcium channel antagonist (nifedipine) or a mito induced MCP-1 production and stimulated local NADPH chondrial uncoupler (DNP). Moreover, co-culture of 3T3-L1 oxidase expression and ROS production, indicating that both adipocytes with RAW 264 macrophages significantly adipocytes and macrophages contribute to elevated oxida increased the expression and production of multiple inflam tive stress in obesity. matory cytokines in response to calcitriol in both cell types. These data Suggest that calcitriol may regulate macrophage 0226 Notably, the anti-inflammatory effect of dietary activity by modulating adipocyte production of factors asso calcium is greater in visceral versus Subcutaneous fat. We ciated with macrophage function. These data also provide have previously observed similar pattern in adipocyte ROS additional explanation for our recent observations that Sup production (Sun et al., 2006), in that ROS production and pression of calcitriol by dietary calcium decreases obesity NADPH oxidase expression were markedly higher in vis associated oxidative stress and inflammation ceral fat versus Subcutaneous fat, Suggesting that there may be an association between oxidative stress and inflammation Materials and Methods in diet-induced obesity. Indeed, it was postulated that because visceral fat is more sensitive to lipolytic stimuli than 0228 Cell culture: Human preadipocytes used in this adipose tissue stored at other sites, turnover of triacylglyc study were supplied by Zen-Bio (Research Triangle, N.C.). erols and release of fatty acids into the portal circulation are Preadipocytes were inoculated in DMEM/Ham's F-10 increased (Wajchenberg, 2000). Free fatty acids, in addition, medium (DMEM-F10) (1:1, vol/vol) containing 10% FBS, can stimulate ROS production by stimulating NADPH oxi 15 mmol/L HEPES, and antibiotics at a density of 30,000 dase expression and activation (Soares et al., 2005). Accord cells/cm2. Confluent monolayers of preadipocytes were ingly, obesity associated with oxidative stress and inflam induced to differentiate with a standard differentiation mation may occur in a depot specific manner in adipose medium consisting of DMEM-F10 (1:1, vol/vol) medium tissue, with significant higher ROS and inflammatory cytok supplemented with 15 mmol/L HEPES, 3% FBS, 33 umol/L ines produced in visceral fat versus Subcutaneous fat (Li et biotin, 17 umol/L pantothenate, 100 nmol/L insulin, 0.25 al., 2003). In Summary, the present study demonstrates that umol/L methylisobutylxanthine, 1 umol/L dexamethasone, 1 dietary calcium Suppresses obesity associated inflammatory umol/L BRL49653, and antibiotics. Preadipocytes were status by modulating pro-inflammatory and anti-inflamma maintained in this differentiation medium for 3 days and tory factor expression, providing the evidence for the first Subsequently cultured in adipocyte medium in which time that increasing dietary calcium may contribute to BRL49653 and MIX were omitted. Cultures were re-fed Suppression of obesity associated inflammation. every 2-3 days. 0229 RAW264 macrophages and 3T3-L1 preadipocytes EXAMPLE 4 (American Type Culture Collection) were incubated at a density of 8000 cells/cm2 (10 cm2 dish) and grown in Calcium-Dependent Regulation of Macrophage Dulbecco's modified Eagle's medium (DMEM) containing Inhibitory Factor and CD14 Expression by 10% FBS and antibiotics (adipocyte medium) at 37° C. in Calcitriol in Human Adipocytes 5% CO2 in air. Confluent 3T3-L1 preadipocytes were induced to differentiate with a standard differentiation 0227 Obesity increases oxidative stress and inflamma medium consisting of DMEM-F10 (1:1, vol/vol) medium tory cytokine production in adipose tissue, and our recent supplemented with 1% FBS, 1 uM dexamethasone, IBMX data demonstrate that dietary calcium attenuates obesity (0.5 mM) and antibiotics (1% Penicillin-Streptomycin). induced oxidative stress and inflammation. This effect may Preadipocytes were maintained in this differentiation be explained by dietary calcium inhibition of calcitriol, medium for 3 days and Subsequently cultured in adipocyte which we have shown to stimulate reactive oxygen species medium. Cultures were re-fed every 2-3 days to allow 90% and inflammatory cytokine production in cultured adipo cells to reach fully differentiation for 3T3-L1 adipocytes or cytes. However, adipose tissue includes both endothelial grow to a confluence for RAW 264 before conducting cells and leukocytes as well as adipocytes; these appear to chemical treatment. Cells were treated with or without contribute to a low-grade inflammatory state in obesity. Accordingly, the interaction between adipocytes and leuko calcitriol (10 nmol/L), GDP (100 umol/L) and/or nifedipine cytes may play an important role in the local modulation of (10 umol/L) for 48 hours, as indicated in each figure. inflammation. Consequently, we investigated calcitriol 0230 Cells were washed with fresh adipocyte medium, modulation of the expression of macrophage inhibitory re-fed with medium containing the indicated treatments, and factor (MIF) and macrophage surface specific protein CD14, incubated at 37°C. in 5% CO for 48 hours before analysis. two key factors in regulating macrophage function and Cell viability was measured via trypan blue exclusion. US 2007/00773 10 A1 Apr. 5, 2007 Cell Culture Chicago, Ill.). The co-culture experiments were analyzed via 0231 Human adipocytes (Zen-Bio, Inc.), 3T3-L1 adipo two-way (treatment X culture condition) ANOVA. cytes, RAW264 macrophages were obtained and co-cultured Results and Discussion by using transwell inserts with 0.4 um porous membranes 0236 Obesity is characterized by increased oxidative and (Coming) to separate adipocytes and macrophages. All data inflammatory stress. Adipose tissue is a significant Source of are expressed as meant SEM. Data were evaluated for reactive oxygen species (ROS) and expresses and secretes a statistical significance by analysis of one-way or two-way wide variety of pro-inflammatory components in obese variance (ANOVA: means with different letter differ, individuals, such as TNF-C. and IL-6. Notably, the adipose p-0.05). tissue is not only composed of adipocytes but also contains Total RNA Extraction: a stromal vascular fraction that includes blood cells, endot helial cells and macrophages. Although adipocytes directly 0232 A total cellular RNA isolation kit (Ambion, Austin, generate inflammatory mediators, adipose tissue-derived Tex.) was used to extract total RNA from cells according to cytokines also originate Substantially from non-fat cells, manufacturers instruction. The concentration and purity of among which infiltrated macrophages appear to play a the isolated RNA was measured spectrophotometrically and prominent role. Infiltration and differentiation of adipose the integrity of RNA sample was analyzed by BioAnalyzer tissue-resident macrophages are under the local control of (Agilent 2100, Agilent Tenchnologies). chemokines, many of which are produced by adipocytes. Quantitative Real Time PCR: Accordingly, the cross-talk between adipocytes and mac 0233 Adipocyte and muscle 18s, CD14, TNFC, MIP. rophages may be a key factor in mediating inflammatory and M-CSF, IL-6 and MCP-1 were quantitatively measured oxidative changes in obesity. using a Smart Cycler Real Time PCR System (Cepheid, 0237 FIG. 27 demonstrates that calcitriol increased MIF Sunnyvale, Calif.) with a TaqMan 1000 Core Reagent Kit (FIG. 27A) and CD14 (FIG. 27B) expression in human (Applied Biosystems, Branchburg, N.J.). The primers and adipocytes by 59% and 33% respectively, and addition of a probe sets were obtained from Applied Biosystems Taq calcium channel antagonist (nifedipine) reversed this effect, Man(R) Assays-on-DemandTM Gene Expression primers and indicating a role of intracellular calcium in mediating this probe set collection according to manufactures instruction. effect. FIG. 28, consistent with FIG. 27, demonstrates that Pooled adipocyte total RNA was serial-diluted in the range calcitriol increased MIF expression by 50% (FIG. 28A) and of 1.5625-25 ng and used to establish a standard curve; total CD14 expression by 45% (FIG. 28B) in mouse (3T3-L1) RNAs for unknown samples were also diluted in this range. adipocytes and the addition of a calcium channel antagonist Reactions of quantitative RT-PCR for standards and (nifedipine) reversed this effect. FIGS. 29, 30 and 31 show unknown samples were also performed according to the that calcitriol markedly stimulate inflammatory cytokines instructions of Smart Cycler System (Cepheid, Sunnyvale, M-CSF (FIG. 29), MIP (FIG. 30), IL-6 (FIG.31) and MCP-1 Calif.) and TaqMan Real Time PCR Core Kit (Applied (FIG. 34) expression in 3T3-L1 adipocytes, and co-culture Biosystems, Branchburg, N.J.). The mRNA quantitation for with RAW264 macrophages enhance this effect, indicating each sample was further normalized using the corresponding a potential role of adipocytes in regulation of local resident 18s quantitation. macrophages activity and that calcitriol may regulate mac Cytokine Antibody Array: rophage activity by modulating adipocyte production of factors associated with macrophage function. Main effects 0234. A TansSignal?TM mouse cytokine antibody array kit of chemical treatment and culture status were significant (Panomics, Fremont, Calif.) was used to detect cytokine (p<0.02). protein released in culture medium according to the manu 0238 A cytokine antibody array was used to further facture's instruction. Briefly, membranes immobilized with investigate the effects of calcitriol on release of major capture antibodies specific to particular cytokine proteins inflammatory cytokines from adipocytes. These protein data was incubated with 1X blocking buffer for 2 hours and then Support the gene expression observations, as calcitriol up blocking buffer was washed three times using washing regulated production of multiple inflammatory cytokine buffer. Then, membranes were incubated in samples for 2 proteins in differentiated 3T3-L1 adipocytes cultured alone hours to allow cytokine protein in the culture medium to (FIG. 32); these include TNFO, IL-6, IL-2, Granulocyte/ bind to the capture antibody on the membrane. At the end of Macrophage-Colony Stimulating Factor (GM-CSF), Inter the incubation, unbound protein was washed away using feron-inducible protein-10 (IP-10), IL-4, IL-13, macrophage washing buffer. The membranes were then incubated with induced gene (MIG), regulated upon T cell activation biotin-conjugated antibody mix which binds to a second expressed secreted (RANTES), IL-5, macrophage inflam epitope on the protein. The membrane was then washed and matory protein 1C.. (MIP-1C) and vascular endothelial incubated with strepavidin-HRP to visualize the antibody growth factor (VEGF). Co-culture of 3T3-L1 adipocytes protein complexes on the array to determine which cytok with macrophages significantly up-regulated production of ines are present in the sample via chemiluminescent signal cytokines such as interferon Y (IFNY), TNFC, G-CSF and which was detected using X-ray film. MIP-1C. compared with 3T3-L1 cultured alone (FIG. 33), Statistical Analysis: and calcitriol further stimulated inflammatory cytokine pro 0235. Each treatment was replicated with n=6, and data duction (FIG. 33). are expressed as meant SEM. Data were evaluated for 0239 Calcitriol also markedly stimulated TNFC. expres statistical significance by analysis of variance (ANOVA) and sion by 91% (FIG. 35) and IL-6 by 796% (FIG. 36) in RAW significantly different group means were then separated by 264 macrophages cultured alone and these effects were the least significant difference test by using SPSS (SPSS Inc. blocked by adding nifedipine or DNP. Co-culture of mac US 2007/00773 10 A1 Apr. 5, 2007 20 rophages with differentiated 3T3-L1 adipocytes markedly calcium was derived from the milk and the remainder was augmented TNFC. (FIG. 35) and IL-6 (FIG. 36) expression added as CaCO. Food intake and spillage was monitored in macrophages, and these effects were further enhanced by daily and body weight and blood glucose was measured calcitriol. weekly. Following three weeks of feeding, all animals from each group were killed for determination of the following 0240 Data from this study demonstrate that calcitriol outcome measurements: plasma insulin, MDA calcitriol and stimulates production of adipokines associated with mac cytokine (IL-6, MCP IL-15, adiponectin and TNFC.; adipose rophage function and increases inflammatory cytokine Tissue:IL-6, MCP IL-15, adiponectin, TNF-C. and NADPH expression in both macrophages and adipocytes; these oxidase expression, tissue release of adipokines, ROS pro include CD14, MIF, M-CSF, MIP, TNFO, IL-6 and MCP-1 in adipocytes, and TNFC. and IL-6 in macrophages. Consis duction; muscle: real-time PCR of NADPH oxidase, IL-6 tent with this, the cytokine protein array identified multiple and IL-15; Tissue release of cytokines, ROS production. additional inflammatory cytokines which were up-regulated 0244 Results: Body weight and composition: A three by calcitriol in adipocytes. Moreover, calcitriol also regu week study duration was utilized in order to avoid major lated cross-talk between macrophages and adipocytes, as calcium- and milk-induced alterations in adiposity, as adi shown by augmentation of expression and production of posity-induced oxidative stress could cause a degree of inflammatory cytokines from adipocytes and macrophages confounding. Nonetheless, there were modest, but statisti in coculture versus individual culture. These effects were cally significant diet-induced changes in body weight and attenuated by either calcium channel antagonism or mito composition. The high calcium diet was without effect on chondrial uncoupling, indicating that the pro-inflammatory body weight, but the milk diet did induce a significant effect of calcitriol are mediated by calcitriol-induced stimu decrease in total body weight (FIG. 37). In contrast, both the lation of Ca2+ signaling and attenuation of mitochondrial calcium and the milk diets caused significant decreases in uncoupling. body fat, with the milk diet eliciting a significantly greater 0241 These data demonstrate that calcitriol regulates effect (FIG. 38). both adipocyte and macrophage production of inflammatory 0245 Skeletal muscle weight (soleus+gastrocnemius) factors via calcium-dependent and mitochondrial uncou exhibited overall differences (p=0.05) among the dietary pling-dependent mechanisms and that these effects are groups. The milk group had significantly greater skeletal amplified with co-culture of both cell types. These data muscle mass than the calcium group (p=0.02) and a ten further suggest that strategies for reducing circulating cal dency towards greater skeletal muscle mass than the basal citriol levels, such as increasing dietary calcium, may regu group (p=0.06) (FIG. 39). Liver weight was slightly, but lating adipocyte macrophage interaction and thereby attenu significantly, reduced by the milk diet (FIG. 40). ate local inflammation in adipose tissue. 0246 Circulating calcitriol: The high calcium diet caused EXAMPLE 5 a reduction in plasma 1.25-(OH)-D (calcitriol) (p=0.002), and there was a trend (p=0.059) towards a further decrease in plasma calcitriol on the high milk diet (FIG. 41). The Dietary Calcium and Dairy Modulation of reason for the difference between the calcium and milk diets Oxidative and Inflammatory Stress in Mice in Suppressing calcitriol is not clear, as they contain the same 0242 Obesity is associated with subclinical chronic levels of dietary calcium. inflammation which contributes to obesity-associated co 0247 Reactive Oxygen Species and Oxidative Stress: morbidities. Calcitriol (1, 25-(OH)-D) regulates adipocyte Adipose tissue reactive oxygen species (ROS) production lipid metabolism, while dietary calcium inhibits obesity by was significantly reduced by the high calcium diet suppression of calcitriol. We have recently shown this (p=0.002), consistent with our previous data, and further anti-obesity effect to be associated with decreased oxidative reduced by the milk diet (p=0.03) (FIG. 42). Consistent with and inflammatory stress in adipose tissue in vivo. However, this, the high calcium diet caused a significant reduction in dairy contains additional bioactive compounds which mark adipose tissue NADPH oxidase (Nox; one of the sources of edly enhance its anti-obesity activity and which we propose intracellular ROS) expression (p=0.001) and there was a will also enhance its ability to Suppress oxidative and inflammatory stress. Accordingly, the objective of this study strong trend (p=0.056) towards a further suppression of was to determine the effects of dietary calcium and dairy on NOX on the milk diet (FIG. 43). oxidative and inflammatory stress in a mouse model (alP2 0248. These changes were reflected in significant agouti transgenic mice) that we have previously demon decreases in systemic lipid peroxidation, as demonstrated by strated to be highly predictive of the effects of calcium and significant decreases in plasma malonaldehyde (MDA). dairy on adiposity in humans and have recently established Plasma MDA was significantly decreased by both the cal as a model for the study of oxidative stress. cium and milk diets (p=0.001), with a significantly greater 0243 Study: Six-week old aP2-agouti transgenic mice effect of the milk diet (p=0.039) (FIG. 44). were fed a modified AIN 93-G diet with Sucrose as the sole 0249 Inflammatory Stress: In general, the high calcium carbohydrate source (64% of energy), and fat increased to diet resulted in Suppression of inflammatory markers and an 25% of energy with lard. A total of 30 animals will be upregulation of anti-inflammatory markers, and the milk diet studied for three weeks (n=10/group), as follows: Control exerted a greater effect than the high calcium diet. Adipose (low Ca) suboptimal calcium (0.4%); High Ca with 1.2% tissue expression of TNFC. (FIG. 45), IL-6 (FIG. 46) and calcium in the form of CaCOs; High Dairy: 50% of the MCP (FIG. 47) were all significantly suppressed by the high protein was replaced by nonfat dry milk and dietary calcium calcium diet. Expression of each of these inflammatory will be increased to 1.2%. Approximately 4 of the additional cytokines was lower on the milk diet than on the high US 2007/00773 10 A1 Apr. 5, 2007 calcium diet, but this difference was only statistically evi by suppression of calcitriol, while the additional effect of dent as a trend for TNF-C. (p=0.076). dairy is mediated by additional bioactive components; these include the high concentration of leucine, a key factor in the 0250 Consistent with these data, the calcium and milk regulation of muscle protein turnover. These data Suggest diets caused significant reductions in the release of inflam that dietary calcium provided with leucine may regulate matory cytokines (TNF-C, FIG. 48; IL6, FIG. 49) from energy partitioning in a tissue selective manner and regulate adipose tissue. There was trend towards a greater effect of energy metabolism by modulating endocrine function of the milk vs. calcium diet, but this difference was not both adipose tissue and skeletal muscle, favoring elevated statistically significant. energy expenditure in adipose tissue and promoting protein 0251 There was a corresponding up-regulation of adi synthesis in skeletal muscle. However, the effect of leucine pose tissue anti-inflammatory cytokine expression on the and calcium, in regulating this process is unclear. Accord high calcium diets. The high calcium and milk diets ingly, present study was designed to investigate the effect of increased adiponectin expression (p=0.001; FIG. 50) and leucine, calcitriol and calcium on energy metabolism in IL-15 expression (p=0.001; FIG. 51), and there was a trend murine adipocytes and muscle cells. Leucine induced a 41% for a further increase on the milk diet vs. high calcium diet increase in fatty acid oxidation in C2C12 muscle cells (p=0.073 for adiponectin; p=0.068 for IL-15). (p<0.001) and decreased fatty acid synthase gene expression by 66% (p<0.001) in 3T3-L1 adipocytes. Calcitriol 0252 Similarly, there was a marked increase in skeletal decreased muscle cell fatty acid oxidation by 17% (p<0.05) muscle IL-15 expression on the high calcium diet (p<0.001), and increased adipocyte FAS gene expression by 3-fold with a further increase on the milk diet (p=0.07: FIG. 52). (p<0.05). These effects were partially reversed by either 0253) These data clearly demonstrate that dietary calcium leucine or calcium channel antagonist nifedipine. Incubation Suppresses both adipose tissue and systemic oxidative stress, of muscle cells with 48-h adipocyte conditioned medium and that dairy (milk) exerts a significantly greater effect. It decreased fatty acid oxidation by 56% (p<0.001), and leu may be argued that the reduced adipose tissue mass may cine and/or nifedipine conditioned adipocyte attenuated this have contributed to the decrease in oxidative stress on the effect in muscle cells. These data Suggest that leucine and high milk diet. However, this is unlikely, as the decrease in nifedipine promote energy partitioning from adipocytes to adiposity was quite modest compared to the decrease in muscle cells, resulting in decreased energy storage in adi oxidative stress. Moreover, the decrease in adipose tissue pocytes and increasing fatty acid utilization in muscle. ROS production and Nox expression are normalized to 0255 Although adiponectin has been reported to increase reflect decreases per adipocyte as well as total systemic fatty acid oxidation in both mice and humans, the role of this decreases. Accordingly, these decreases in oxidative stress adipokine in mediating the effects of leucine and calcium on appear to be direct effects of the high calcium and high dairy energy metabolism in skeletal muscle and adipocytes is yet diets. Data from this study also demonstrate a marked unclear. Consistent with previous studies, the present data reduction in adipose tissue-derived inflammatory cytokines demonstrate that adiponectin markedly increased fatty acid on the high calcium diets, with a strong trend towards further oxidation in C2C12 myotubes (FIG. 53). Further, adiponec Suppression of inflammatory cytokines on the milk vs. high tin restored fatty acid oxidation Suppressed by calcitriol in calcium diet. Moreover, anti-inflammatory cytokine expres the present of leucine. Comparable effects of leucine, cal sion is significantly up-regulated on the high calcium diet, citriol and adiponectin were found in myotubes co-cultured with further improvements evident on the milk vs. calcium with adipocytes; however, the presence of adipocytes mark diet. Although there are additional analyses to be completed, edly suppressed fatty acid oxidation. This was due to these data indicate a marked shift in the ratio of anti secreted factor(s), as a comparable Suppression resulted inflammatory to inflammatory cytokines on high calcium from exposure of the myotubes to adipocyte conditioned diets, with further improvements in this ratio when milk is medium (data not shown). used as the calcium source. Thus, data from this pilot study strongly suggest that dietary calcium Suppresses oxidative 0256 The new data also demonstrate that adiponectin and inflammatory stress, consistent with our previous data, regulates IL-15 and IL-6 release by myotubes in response to and that other components of milk enhance this effect to calcitriol, leucine and nefedipine. Adiponectin significantly produce greater control of both oxidative and inflammatory increased IL-15 release, and partially reversed the inhibitory effects of calcitriol (FIG. 54). Leucine was without effect on StreSS. IL-15 release, while nifedipine alone promoted IL-15 EXAMPLE 6 release but adiponectin exerted no addition effect. Adiponec tin also increases IL-15 release in muscle cells treated with Leucine and Calcium Modulation of both leucine and nifedipine and this effect was not attenuated Adipocyte-Skeletal Muscle Energy Partitioning by addition of calcitriol, indicating the effect of calcitriol is mediated, at lease in part by calcium signaling. Comparable 0254 The adipose tissue-skeletal muscle endocrine axis effects were found in myotubes co-cultured with adipocytes. may play a potential role in regulating metabolic energy Interestingly, the presence of adipocytes decreased IL-15 partitioning. We have previous shown that dietary calcium release under basal conditions but increased IL-15 release in exhibits an inhibitory effect on obesity and that dairy prod with the presence of leucine and/or nifedipine. These data ucts exert a greater effect on adiposity compared to Supple Suggest that leucine and nifedipine regulate muscle-adipo mental or fortified sources of calcium. While both calcium cyte cross-talk by modulating production of cytokines which and dairy accelerate adipose tissue loss, dairy exerts a affect energy partitioning between adipose tissue and skel substantially greater effect and exerts a protective effect on etal muscle. Similar effects of adiponectin, calcitriol, leucine lean tissue during hypocaloric diets. We have shown that and nifedipine were observed in the regulation of IL-6 dietary calcium modulation of adiposity is mediated, in part, release in C2C12 myotubes (FIG. 55). US 2007/00773 10 A1 Apr. 5, 2007 22 0257 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 regu muscle via a fat-muscle endocrine axis may play a potential lation, and that IL-15 and IL-6 may serve as skeletal role in regulating overall metabolic energy partitioning, and muscle-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 Pharmaceuticals S8CO8. 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 and/or 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 C8Ce anastrozole Arimidex Treatment of advanced breast cancer AstraZeneca in postmenopausal women with Pharmaceuticals disease progression following tamoxifen therapy. anastrozole Arimidex For first-line treatment of AstraZeneca postmenopausal women with hormone Pharmaceuticals receptor positive or hormone receptor unknown locally advanced or metastatic breast cancer. arsenic trioxide Trisenox Second line treatment of relapsed or Cell 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 capsules Targretin For the treatment by oral capsule of Ligand cutaneous manifestations of cutaneous Pharmaceuticals T-cell lymphoma in patients who are refractory to at least one prior systemic therapy. bexarotenegel Targretin For the topical treatment of cutaneous Ligand manifestations of cutaneous T-cell Pharmaceuticals lymphoma in patients who are US 2007/00773 10 A1 Apr. 5, 2007 23 TABLE 1-continued Drug Name Trade Name Indication Manufacturer 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 intravenous Busulfex Use in combination with Orphan 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 Pharmaceuticals Implant recurrent glioblastoma multiforme who 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 tumors whoc have already received appropriate Surgical andfor radiotherapeutic procedures. An US 2007/00773 10 A1 Apr. 5, 2007 24 TABLE 1-continued Drug Name Trade Name Indication Manufacturer established combinati on therapy consists of Platinol, Blenoxane and Velbam. cisplatin Platinol Metastatic ovarian tumors —in Bristol-Myers established combinati on therapy with Squibb other approved chemotherapeutic agents: Ovarian-in es ablished combination therapy with other approved chemothera peutic agents in patients with metasta ic ovarian tumors who have already rec eived appropriate Surgical and/or radiot herapeutic procedures. An establ ished 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/or radiotherapy. cladribine Leustatin, 2 Treatment of active hairy cell leukemia. R.W. Johnson CdA Pharmaceutical 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 liposomal DepoCyt Accel. Approv. (clinical benefit not Skye established) Intrathecal therapy of Pharmaceuticals lymphomatous meningitis dacarbazine DTIC-Dome Bayer dactinomycin, Cosmegen Merck actinomycin D dactinomycin, Cosmegan Merck actinomycin D Darbepoetin alfa Aranesp Treatment of anemia associated with Amgen, Inc. chronic renal failure. Darbepoetin alfa Aranesp Aranesp is indicated for the treatment Amgen, Inc. 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 erythroid Bedford Labs daunomycin of adults/remission 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 diftitox Ontak Accel. Approv. (clinical benefit not Seragen, Inc. established) treatment of patients with persistent or recurrent cutaneous T cell lymphoma whose malignant cells express the CD25 component of the IL-2 receptor dexrazoxane Zinecard Accel. Approv. (clinical benefit Pharmacia & subsequently established) Prevention Upjohn of cardiomyopathy associated with Company doxorubicin administration US 2007/00773 10 A1 Apr. 5, 2007 25 TABLE 1-continued Drug Name Trade Name Indication Manufacturer 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 benefi Aventis subsequently established) Treatment Pharmaceutical of patients with locally advanced or 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 Pharmaceutical progressed during anthracycline-based treatment or relapsed during anthracycline-based adjuvant therapy. docetaxel Taxotere For locally advanced or metastatic Aventis non-Small cell lung cancer after failure Pharmaceutical of prior platinum-based chemotherapy. docetaxel Taxotere Aventis Pharmaceutical docetaxel Taxotere in combination with cisplatin for the Aventis treatment of patients with Pharmaceutical unresectable, locally advanced or 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 liposomal Doxi Accel. Approv. (clinical benefit not Sequus established) Treatment of AIDS-related Pharmaceuticals, Kaposi's sarcoma in patients with Inc. disease that has progressed on prior combination chemotherapy or in patients who are intolerant to Such therapy. doxorubicin liposomal Doxi Accel. Approv. (clinical benefit not Sequus established) Treatment of metastatic Pharmaceuticals, carcinoma of the ovary in patient with Inc. disease that is refractory to both paclitaxel and platinum based regimens DROMOSTANOLONE DROMOSTANOLONE Eli Lilly PROPIONATE DROMOSTANOLONE MASTERONE SYNTEX PROPIONATE INJECTION Eliott's B Solution Eliott's B Diluent for the intrathecal Orphan 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. Epoetin alfa epogen EPOGENB is indicated for the Amgen, Inc. reatment of anemia related to therapy with zidovudine in HIV-infected patients. EPOGENB is indicated to elevate or maintain the red blood cell level (as manifested by the hematocrit or hemoglobin determinations) and to US 2007/00773 10 A1 Apr. 5, 2007 26 TABLE 1-continued Drug Name Trade Name Indication Manufacturer decrease the need for transfusions in these patients. EPOGEND is not indicated for the treatment of anemia in HIV-infected patients due to other factors 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 of anemia associated with CRF, including patients on dialysis (ESRD) and patients not on dialysis. estramustine Emcyt palliation of prostate cancer Pharmacia & Upjohn Company etoposide phosphate Etopophos Management of refractory testicular Bristol-Myers tumors, in combination with other Squibb approved chemotherapeutic agents. etoposide phosphate Etopophos Management of Small cell lung cancer, Bristol-Myers first-line, in combination with other Squibb approved chemotherapeutic agents. etoposide phosphate Etopophos Management of refractory testicular Bristol-Myers tumors and Small cell lung cancer. Squibb etoposide, VP-16 Vepesid Refractory testicular tumors-in Bristol-Myers 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, VP-16 WePesid in combination with other approved Bristol-Myers chemotherapeutic agents as first line Squibb treatment in patients with Small cell ung cancer. etoposide, VP-16 Vepesid in combination with other approved Bristol-Myers 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. 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. US 2007/00773 10 A1 Apr. 5, 2007 27 TABLE 1-continued Drug Name Trade Name Indication Manufacturer 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, 5-FU Adrucil prolong Survival in combination with ICN Puerto 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 acetate Zoladex Palliative treatment of advanced breast AstraZeneca Implant cancer in pre- and perimenopausal Pharmaceuticals WOCl. goserelin acetate Zoladex AstraZeneca Pharmaceuticals hydroxyurea Hydrea Bristol-Myers Squibb hydroxyurea Hydrea Decrease need for transfusions in Bristol-Myers sickle cell anemia Squibb Ibritumomab Tuxetan Zevalin Accel. Approv. (clinical benefit not IDEC established) treatment of patients with Pharmaceuticals relapsed or refractory low-grade, Corp follicular, or transformed B-cell non Hodgkin’s lymphoma, including patients with Rituximab refractory follicular non-Hodgkin's lymphoma. 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. US 2007/00773 10 A1 Apr. 5, 2007 28 TABLE 1-continued Drug Name Trade Name Indication Manufacturer ifosfamide IFEX Third line chemotherapy of germ cell Bristol-Myers testicular cancer when used in Squibb combination with certain other approved antineoplastic agents. imatinib meSylate Gleevec Accel. Approv. (clinical benefit not Novartis established) Initial therapy of chronic myelogenous leukemia imatinib meSylate Gleevec Accel. Approv. (clinical benefit not Novartis established) metastatic or unresectable malignant gastrointestinal Stromal tumors imatinib meSylate Gleevec Accel. Approv. (clinical benefit not Novartis established) Initial treatment of newly diagnosed Ph-- chronic myelogenous leukemia (CML). Interferon alfa-2a. Roferon-A Hoffmann-La Roche Inc. Interferon alfa-2b Intron A Interferon alfa-2b, recombinant for Schering 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 alfa-2b Intron A interferon alfa-2b, recombinant for Schering 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 alfa-2b Intron A interferon alfa-2b, recombinant for Schering 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 alfa-2b Intron A interferon alfa-2b, recombinant for Schering 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-product exposure and/or are HCV antibody positive. Interferon alfa-2b Intron A interferon alfa-2b, recombinant for Schering 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 alfa-2b Intron A Interferon alfa-2b, recombinant for Schering Injection is indicated for the treatment Corp of patients 18 years of age or older with hairy cell leukemia. Interferon alfa-2b Intron A Interferon alfa-2b, recombinant for Schering Injection 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 INTRON A therapy is greater in patients who are without systemic symptoms, who have limited lymphadenopathy and who have a relatively intact immune system as indicated by total CD4 count. Interferon alfa-2b Intron A Schering Corp Interferon alfa-2b Intron A Schering Corp Interferon alfa-2b Intron A Intron A Schering Corp irinotecan Camptosar Accel. Approv. (clinical benefit Pharmacia & subsequently established) Treatment Upjohn US 2007/00773 10 A1 Apr. 5, 2007 29 TABLE 1-continued Drug Name Trade Name indication Manufacturer 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 herapy. irinotecan Camptosar For first line treatment in combination Pharmacia & with 5-FU leucovorin of metastatic Upjohn carcinoma of the colon or rectum. Company letrozole Femara Treatment of advanced breast cancer Novartis in postmenopausal women. letrozole Femara First-line treatment of postmenopausal Novartis women with hormone receptor positive or hormone receptor unknown locally advanced or metastatic breast cancer. letrozole Femara Novartis leucovorin Wellcovorin, Leucovorin calcium is indicated fro use Immunex Leucovorin in combination with 5-fluorouraci to Corporation prolong Survival in the palliative treatment of patients with advanced colorectal cancer. leucovorin Leucovorin leX Corporation leucovorin Leucovorin leX Corporation leucovorin Leucovorin leX Corporation leucovorin Leucovorin In combination with fluorouraci to Lederle prolong Survival in the palliative Laboratories treatment of patients with advanced colorectal cancer. levamisole Ergamisol Adjuvant treatment in combination with 8SScil 5-fluorouracil after Surgical resection in Research patients with Dukes' Stage C colon Foundation C8CC. lomustine, CCNU CeeBU meclorethamine, Mustargen nitrogen mustard megestrol acetate Megace melphalan, L-PAM Alkeran melphalan, L-PAM Alkeran Systemic administration for palliative treatment of patients with multiple myeloma for whom oral therapy is not appropriate. mercaptopurine, 6-MP Purimethol Glaxo 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 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 US 2007/00773 10 A1 Apr. 5, 2007 30 TABLE 1-continued Drug Name Trade Name Indication Manufacturer 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 Pharmaceuticals, line or Subsequent systemic 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 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 for use in combination with cisplatin, Bristol-Myers for the first-line treatment of non-small Squibb cell lung cancer in patients who are not US 2007/00773 10 A1 Apr. 5, 2007 31 TABLE 1-continued Drug Name Trade Name Indication Manufacturer 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 conjunction 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 of febrile neutropenia. pentostatin Nipent Single agent treatment for adult Parke-Davis patients with alpha interferon refractory Pharmaceutical hairy cell leukemia Co. pentostatin Nipent Single-agent treatment for untreated Parke-Davis hairy cell leukemia patients with active Pharmaceutical disease as defined by clinically Co. significant anemia, neutropenia, thrombocytopenia, or disease-related symptoms. (Supplement for front-line therapy.) pipobroman Vercyte Abbott Labs plicamycin, Mithracin Pfizer Labs mithramycin porfimer Sodium Photofrin For use in photodynamic therapy QLT Photo (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. porfimer Sodium Photofrin For use in photodynamic therapy for QLT Photo treatment of microinvasive herapeutics endobronchial nonsmall cell lung IlC. cancer in patients for whom Surgery and radiotherapy are not indicated. porfimer Sodium Photofrin For use in photodynamic therapy QLT Photo (PDT) for reduction of obstruction and herapeutics palliation of symptoms in patients with IlC. 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, IlC. lymphoma, and Solid tumor malignancies who are receiving anti cancer therapy expected to result in tumor lysis and Subsequent elevation of plasma uric acid. Rituximab Rituxan Genentech, Inc. Sargramostim Prokine Immunex Corp streptozocin Zanosar Antineoplastic agent. Pharmacia & Upjohn Company US 2007/00773 10 A1 Apr. 5, 2007 32 TABLE 1-continued Drug Name Trade Name Indication Manufacturer talc Sclerosol For the prevention of the recurrence of Bryan malignant pleural effusion in symptomatic patients. tamoxifen Novadex AstraZeneca Pharmaceuticals tamoxifen Novadex As a single agent to delay breast AstraZeneca cancer recurrence following total Pharmaceuticals mastectomy and axillary dissection in postmenopausal women with breast cancer (T1-3, N1, MO) amoxifen Novadex For use in premenopausal women with AstraZeneca metastatic breast cancer as an Pharmaceuticals alternative to oophorectomy or ovarian irradiation amoxifen Novadex For use in women with axillary node AstraZeneca negative breast cancer adjuvant Pharmaceuticals herapy. amoxifen Novadex Metastatic breast cancer in men. traZeneca armaceuticals amoxifen Novadex Equal bioavailability of a 20 mg traZeneca Nolvadex tablet taken once a day to a armaceuticals 0 mg Nolvadex tablet taken twice a day. amoxifen Novadex o reduce the incidence of breast AstraZeneca cancer in women at high risk for breast Pharmaceuticals C8CC amoxifen Novadex in women with DCIS, following breast AstraZeneca Surgery and radiation, Nolvadex is Pharmaceuticals indicated to reduce the risk of invasive 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, VM-26 Vulmon In combination with other approved Bristol-Myers 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, 6-TG Thioguanine Glaxo SmithKline hiotepa Thioplex leX Corporation hiotepa Thioplex leX 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 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. US 2007/00773 10 A1 Apr. 5, 2007 33 TABLE 1-continued Drug Name Trade Name Indication Manufacturer 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, ATRA Vesanoid Induction of remission in patients with Roche acute promyelocytic leukemia (APL) who are refractory to or unable to tolerate anthracycline based cytotoxic chemotherapeutic regimens. Uracil Mustard Uracil Mustard Roberts Labs 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. zoledronate Zometa the treatment of patients with multiple Novartis myeloma and patients with US 2007/00773 10 A1 Apr. 5, 2007 34 TABLE 1-continued Drug Name Trade Name Indication Manufacturer documented bone metastases from solid tumors, in conjunction with standard antineoplastic therapy. Prostate ancer should have progressed after treatment with at least one hormonal therapy 0258 0259 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 Nowolin 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 Biguanide Metformin Glucophage Bristol Myers Novolin L (Novo Nordisk) Squibb Intermediate- and Humulin 50:50 Metformin (long Glucophage XR Bristol Myers short-acting mixtures Humulin 70.30 lasting) Squibb Humalog Mix 75/25 Metformin with Glucowance Bristol Myers Humalog Mix 50/50 glyburide Squibb RosiglitaZone Avandia SmithKline (Eli Lilly) Olesolidine Beecham (now Nowolin 70:30 (GlitaZone) GlaxoSmithKline) Novolog Mix 70/30 PioglitaZone ActOS Takeda (Novo Nordisk) Pharmaceuticals Long-acting Ultralente Alpha-Glucose Acarbose Precose Bayer (Eli Lilly) Inhibitor Miglitol Glyset Pharmacia and Lantus (glargine) Upjohn (Aventis) 0260) 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-Ca diet High-Ca diet Low-Ca diet High-Ca diet p value Body weight (g)' 25.28 O.39 24.47 O.47 32.96 - 0.95 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 - 0.17 O.94 - 0.11* P = 0.015 Visceral fat (g) NA NA 2.48 O.19 1.31 - 0.11* 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-Ca diet. ‘Subscapular fat pad Sum of perirenal and abdominal fat pads US 2007/00773 10 A1 Apr. 5, 2007 REFERENCES 0274 Cerutti, P., Ghosh, R., Oya, Y., Amstad, P. (1994) 0261) Agarwal, A. K. (2003) “Cortisol metabolism and “The role of the cellular antioxidant defense in oxidant visceral obesity: role of 11beta-hydroxysteroid dehydro carcinogenesis’Environ Health Perspect 102:123-9. genase type I enzyme and reduced co-factor NADPH'En 0275 Chang, L., Karin, M. 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SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 2 <210> SEQ ID NO 1 <211& LENGTH: 21 US 2007/00773 10 A1 Apr. 5, 2007 -continued &212> TYPE DNA <213> ORGANISM: Artificial sequence &220s FEATURE <223> OTHER INFORMATION: Forward PCR primer for UCP2 <400 SEQUENCE: 1 gctago atgg ttggtttcaa g 21 <210> SEQ ID NO 2 <211& LENGTH 21 &212> TYPE DNA <213> ORGANISM: Artificial sequence &220s FEATURE <223> OTHER INFORMATION: Reverse PCR primer for UCP2 <400 SEQUENCE: 2 gctagotcag aaaggtgaat c 21 We claim: said cell(s) is indicative of a compound or compo 1. An in vitro method of Screening compounds or com sition Suitable for use in reducing the production of positions Suitable for reducing the production of reactive ROS; oxygen species (ROS) comprising: vii) TNF-ct, CD14, MIF, M-CSF, MIP, MCP-1, G-CSF a) contacting one or more cell(s) with a candidate com or IL-6 expression in said one or more cell(s), pound, combination of candidate compounds, candi wherein a decrease in the expression of TNF-C. date composition, or combination of candidate compo CD14, MIF (macrophage inhibitory factor), MIP sitions with the proviso that said candidate compound, (macrophage inhibitory protein), M-CSF (macroph combination of candidate compounds, candidate com age colony stimulating factor), MCP-1 (monocyte position, or combination of candidate compositions is chemoattractant protein-1), G-CSF (granulocyte not dietary material containing calcium or dietary cal colony Stimulating factor) or IL-6 in said cell(s) is cium; and indicative of a compound or composition Suitable for use in reducing the production of ROS; or b) measuring one or more of the following parameters: viii) IL-15 or adiponectin expression in said one or i) intracellular concentrations of calcium in said one or more cell(s), wherein an increase in the expression of more cell(s), wherein a decrease of intracellular IL-15 or adiponectin in said cell(s) is indicative of a calcium concentration in said cell(s) is indicative of compound or composition Suitable for use in reduc a compound or composition Suitable for use in ing the production of ROS. reducing the production of ROS; 2. The method according to claim 1, wherein said one or more cell(s) is a adipocyte or an adipocyte cell line. ii) UCP2 expression in said one or more cell(s), 3. The method according to claim 2, wherein said adipo wherein an increase in UCP2 expression in said cyte or adipocyte cell line is human(s) or a murine. cell(s) is indicative of a compound or composition 4. A method of identifying or screening compounds or suitable for use in reducing the production of ROS; compositions suitable for reducing the production of reac iii) NADPH oxidase expression in said one or more tive oxygen species (ROS) comprising: cell(s), wherein a decrease in NADPH oxidase a) administering a candidate compound, combination of expression in said cell(s) is indicative of a compound candidate compounds, candidate composition, or com or composition Suitable for use in reducing the bination of candidate compositions to at least one test production of ROS; Subject with the proviso that said candidate compound, combination of candidate compounds, candidate com iv) UCP3 expression in said one or more cell(s), position, or combination of candidate compositions, wherein an increase in UCP3 expression in said when administered orally to said test Subject, is not cell(s) is indicative of a compound or composition being administered to said at least one test Subject suitable for use in reducing the production of ROS; orally as a component of the diet of said at least one test v) NADPH oxidase expression in said one or more Subject or as dietary calcium to said test Subject; and cell(s), wherein a decrease in NADPH oxidase b) measuring one or more of the following parameters: expression in said cell(s) is indicative of a compound or composition Suitable for use in reducing the i) intracellular calcium concentrations in cells of said at production of ROS; least one test Subject and at least one control Subject, wherein a decrease of intracellular calcium concen vi) 113-HSD expression in said one or more cell(s), tration in the cells of a test Subject as compared to the wherein a decrease in the expression of 113-HSD in intracellular concentrations of calcium in the cells of US 2007/00773 10 A1 Apr. 5, 2007 39 at least one control Subject is indicative of a com 6. The method according to claim 4, wherein a candidate pound, composition, combination of compounds or compound, combination of candidate compounds, candidate combination of compositions Suitable for use in composition, or combination of candidate compositions is reducing the production of ROS in a subject; administered to a subject via intravenous, intraarterial, buc ii) UCP2 expression in cells of said at least one test cal, topical, transdermal, rectal, intramuscular, Subcutane Subject and at least one control Subject, wherein an ous, intraosseous, transmucosal, or intraperitoneal routes of increase of UCP2 expression in the cells of a test administration. subject as compared to the UCP2 expression in the 7. The method according to claim 5, wherein said candi cells of at least one control subject is indicative of a date compound, combination of candidate compounds, can compound, composition, combination of compounds didate composition, or combination of candidate composi or combination of compositions suitable for use in tions is administered to at least one test subject orally with reducing the production of ROS in a subject; the proviso that said candidate compound, combination of iii) NADPH oxidase expression in cells of said at least candidate compounds, candidate composition, or combina one test Subject and at least one control Subject, tion of candidate compositions is not being administered to wherein a decrease of NADPH oxidase expression in said at least one test Subject orally as a component of the diet the cells of a test subject as compared to the NADPH of said at least one test Subject or as dietary calcium to said oxidase expression in the cells of at least one control test Subject. Subject is indicative of a compound, composition, 8. A method of treating diseases associated with reactive combination of compounds or combination of com oxygen species (ROS) comprising the administration of a positions Suitable for use in reducing the production compound, composition, combination of compounds, or of ROS in a subject; combination of compositions that increase intracellular cal iv) UCP3 expression in skeletal muscle cells of said at cium levels to an individual in need of such treatment in least one test Subject and at least one control Subject, amounts sufficient to increase the intracellular concentra wherein an increase in UCP3 expression in the tions of calcium in the cells of the individual with the skeletal muscle cells of a test Subject as compared to proviso that said compound, combination of compounds, UCP3 expression in the skeletal muscle cells of at composition, or combination of compositions is not a dietary least one control Subject is indicative of a compound, material containing calcium or dietary calcium. composition, combination of compounds or combi 9. The method according to claim 8, further comprising nation of compositions Suitable for use in reducing the step of diagnosing or identifying an individual as having the production of ROS in a subject; a disease or disorder associated with ROS or diagnosing or v) NADPH oxidase expression in skeletal muscle cells identifying an individual having elevated ROS levels com of said at least one test Subject and at least one prising measuring the levels of ROS and comparing the control subject, wherein a decrease of NADPH oxi measured levels against a standard or collection of ROS dase expression in the skeletal muscle cells of a test levels from control subjects. subject as compared to the NADPH oxidase expres 10. The method according to claim 8, wherein the ROS sion in the skeletal muscle cells of at least one associated disease or disorder is cataracts, diabetes, Alzhe control Subject is indicative of a compound, compo imer's disease, heart disease, cancer, male infertility, inflam sition, combination of compounds or combination of mation, amyotrophic lateral Sclerosis, Parkinson's disease, compositions suitable for use in reducing the pro multiple Sclerosis or aging. duction of ROS in a subject; 11. The method according to claim 9, wherein the ROS vi) 113-HSD expression in visceral adipocyte tissue or associated disease or disorder is cataracts, diabetes, Alzhe cells of said at least one test Subject and at least one imer's disease, heart disease, cancer, male infertility, inflam control subject, wherein a decrease of 113-HSD mation, amyotrophic lateral Sclerosis, Parkinson's disease, expression in the visceral adipocyte tissue or cells of multiple Sclerosis or aging. a test subject as compared to the 113-HSD expres 12. The method according to claim 8, wherein said sion in the visceral adipocyte tissue or cells of at least ROS-associated disease or disorder is cancer-associated one control Subject is indicative of a compound, ROS disease or disorders and said method comprises the composition, combination of compounds or combi administration of one or more composition comprising cal nation of compositions Suitable for use in reducing cium, or physiologically acceptable salts of calcium, and a the production of ROS in a subject; therapeutic agent selected from alkylating agents, antibiotics which affect nucleic acids, platinum compounds, mitotic vii) TNF-ct, CD14, MIF, MIP, M-CSF, MCP-1, G-CSF or inhibitors, antimetabolites, camptothecin derivatives, bio IL-6 expression in said one or more cell(s), wherein a logical response modifiers, hormone therapies or any of the decrease in the expression of TNF-C, CD14, MIF, MIP, therapeutic agents is identified in Table 1. M-CSF, MCP-1, G-CSF or IL-6 in said cell(s) is 13. The method according to claim 12, wherein the indicative of a compound or composition Suitable for therapeutic agent or therapeutic agents and calcium, or use in reducing the production of ROS; or physiologically acceptable salts of calcium, are administered viii) IL-15 or adiponectin expression in said one or as a single composition. more cell(s), wherein an increase in the expression of 14. The method according to claim 12, wherein the IL-15 or adiponectin in said cell(s) is indicative of a therapeutic agent or therapeutic agents and calcium, or compound or composition Suitable for use in reduc physiologically acceptable salts of calcium, are administered ing the production of ROS. as separate or different compositions. US 2007/00773 10 A1 Apr. 5, 2007 40 15. The method according to claim 14, wherein the calcium sulfate, calcium tartrate, calcium magnesium car separate or different compositions are administered simul bonate, calcium metasilicate, calcium malate, secondary taneously, sequentially or contemporaneously. calcium orthophosphate, calcium citrate, or calcium hydrox 16. A method of reducing ROS production in a diabetic ide. individual comprising the administration of one or more 21. A method of increasing the in vitro expression of MIF, composition comprising one or more therapeutic agent as set M-CSF, MIP, IL-6, IL-10, IL-4, IL-13, MIG, IL-5, VEGF, forth in Table 2 or Table 3 and calcium, or physiologically CD14, G-CSF, TNF-C, RANTES, or MIP-1C. comprising acceptable salts of calcium, in an amount Sufficient to reduce contacting a composition comprising a carrier and calcitriol the production of ROS in said individual. (1, 25-(OH)-D) with a adipocytes, skeletal muscle cells, 17. A method of altering the expression of cytokines in an skeletal muscle cell lines, human adipocyte cell lines, individual or the cytokine profile of an individual compris murine adipocyte cell lines or transformed host cells com ing the administration of a compound, composition, com prising MIF, M-CSF, MIP, IL-6, IL-10, IL-4, IL-13, MIG, bination of compounds, or combination of compositions that IL-5, VEGF, CD14, G-CSF, TNF-O, RANTES, or MIP-1C. decrease intracellular calcium levels to an individual in need genes and culturing said cells under conditions that allow for of Such treatment in amounts sufficient to decrease intrac the production of MIF, M-CSF, MIP, IL-6, IL-10, IL-4, ellular levels of calcium in the cells of the individual, IL-13, MIG, IL-5, VEGF, CD14, G-CSF, TNF-O, RANTES, decrease TNF-O, CD14, MIP, MIF, M-CSF, G-CSF or IL-6 or MIP-1C. expression, or any combination thereof, in the individual, 22. The method according to claim 21, further comprising and increase the expression of IL-15. adiponectin, or both the recovery of MIF, M-CSF, MIP, IL-6, IL-10, IL-4, IL-13, IL-15 or adiponectin in the individual with the proviso that MIG, IL-5, VEGF, CD14, G-CSF, TNF-O, RANTES, or said compound, combination of compounds, composition, or MIP-1 O. combination of compositions is not a dietary material con 23. A method of increasing fatty acid oxidation in muscle taining calcium or dietary calcium. cells or decreasing the expression of fatty acid synthase in 18. A composition comprising one or more therapeutic adipocytes comprising the administration of a composition agent selected from Tables 1 or 2 or 3 in combination with comprising leucine to a Subject in an amount Sufficient to calcium or one or more physiological salts of calcium. increase fatty acid oxidation in said muscle cells or decreas 19. The composition according to claim 18, wherein said ing the expression of fatty acid synthase in adipocytes. composition contains between: 1 and 2000 mg; 900 and 1500 mg: 1000 and 1400 mg. 1200 and 1300 mg: 1100 and 24. The method according to claim 23, wherein about 1 to 1300 mg. or 1200 and 1300 mg of calcium or one or more 10 grams of leucine is administered to said individual. physiologically acceptable salts thereof. 25. The method according to claim 24, wherein said 20. The composition according to claim 18, wherein said leucine is administered orally. one or more physiological salts of calcium are selected from calcium phosphates, calcium carbonate, calcium chloride,