US 20110251 158A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2011/0251158 A1 KEPLEY et al. (43) Pub. Date: Oct. 13, 2011

(54) FULLERENE THERAPES FOR (60) Provisional application No. 61/033,309, filed on Mar. NFLAMMATION AND INHIBITION OF 3, 2008, provisional application No. 61/033,336, filed BUILD-UP OF ARTERIAL PLAQUE on Mar. 3, 2008. Publication Classification (75) Inventors: Christopher L. KEPLEY, Ringgold, VA (US); Robert P. (51) Int. Cl. LENK, Danville, VA (US); Darren A63/685 (2006.01) K. MACFARLAND, Danville, VA C07C 69/76 (2006.01) (US); Zhiguo ZHOU, A63/492 (2006.01) Winston-Salem, NC (US) C07D 249/04 (2006.01) A6IP 9/02 (2006.01) (73) Assignee: Luna Innovations Incorporated, C07D 40/4 (2006.01) Roanoke, VA (US) C07F 9/10 (2006.01) A6II 3/66 (2006.01) A6IP 9/00 (2006.01) (21) Appl. No.: 12/884,076 A6II 3L/235 (2006.01) A63L/455 (2006.01) Filed: Sep. 16, 2010 (22) (52) U.S. Cl...... 514/78: 514/510; 560/80: 514/359; Related U.S. Application Data 548/255; 514/332:546/263:554/80; 514/107 (57) ABSTRACT (63) Continuation-in-part of application No. 12/921,049, filed on Sep. 3, 2010, filed as application No. PCT/ Described herein are methods for treating inflammatory dis US2009/001333 on Mar. 3, 2009, Continuation-in orders or for inhibiting the build-up of arterial plaque. The part of application No. 12/921,083, filed on Dec. 16, methods comprise administering to a Subject in need thereof 2010, filed as application No. PCT/US2009/001335 atherapeutically effective amount of a synthetically modified on Mar. 3, 2009. fullerene.

Patent Application Publication Oct. 13, 2011 Sheet 1 of 37 US 2011/0251158A1

FIGURE 1 Patent Application Publication Oct. 13, 2011 Sheet 2 of 37 US 2011/0251158A1

Co-tetrainositol C60-ethanolamine

FIGURE 2 Patent Application Publication Oct. 13, 2011 Sheet 3 of 37 US 2011/0251158A1

FIGURE 3 Patent Application Publication Oct. 13, 2011 Sheet 4 of 37 US 2011/0251158A1

7 (TGA) 5 (ALM)

FIGURE 4 Patent Application Publication Oct. 13, 2011 Sheet 5 of 37 US 2011/0251158A1

A Clinical Indices

120 Ankle thickness 100

80

60

40

20

-20

-0- 200 ng/100 ul LNWOO42 -O- 2000 ng/100 ul LNW0042 -v- 200 ng/100 ul LNW0048 was a 2000 ng/100 ul LNW0048 -- 200 mg/100 ulPBS FIGURE 5 Patent Application Publication Oct. 13, 2011 Sheet 6 of 37 US 2011/0251158A1

:

FIGURE 6 Patent Application Publication Oct. 13, 2011 Sheet 7 of 37 US 2011/0251158A1

Walues represent mast ceilgeries upregulated by at east 10% by FCeR stimulation that were inhibited >20% by inos (sorted most inhibited to least inhibited in column D) symbol % increase (-) to (+) % inhibition by inositol LECT1 1024.5% 99.08% PTBP2 1538.29% 98.69% FIGURE 7 ABCG5 4480.00% 97.39% NIT1. 1913.64% 95.15% W.L 84.3% 88.13% MARVELD3 270.87% 84.86% CFH R5 34.99% 76.92% MER3 23.03% 72.22% DH3B 40.70% 69.26% LMX1B 35.8% 67.49% XYLT2 38.93% . . . . 65.09% ANTXRL 43.68% 64.64% KYNU 74.87% 64.52% FAM22 29.86% 62.28% C12Orf52 36.53% 62.04% CA7 42.42% 60.63% CHST1. 33.91% 59.95% C20orf144, C20orf134 w 66.44% 59.92% VIBLAC1 359.97% 59.87% CDCA3 83.10% 59.76% OTUB1 62.69% 59.22% EIF1AD 30.98% 58.92% GNB2 29.14% 58.46% EF6 17.89% 58.35% RP5-981O7.3 473.55% 58.35% WASH1. 97.42% 58.22% RAD51L, 92.07% 58.17% C3orf62 A1.37% 58.07% SEPN1 20.10% 57.79% PHLDA1 35.31% 57.77% ADAD2 14.51% 57.47% SMOC2 18.70% 57.19% ACO98823.3 67.54% 57.10% RP11-318K123 . 10.62% 56.91% ASTN). 45.29% 56.41% PARP15 27.69% 55.63% RP11-1919.4 29.43% 55.51% ANKRD39,ANKRD23 61.60% 55.38% POU3F3 73.70% 55.28% FAT3 68.38% 55.27% C2OOrf102 20.76% 55.27% FGL2 6499.13% 54.84% RHO 92.42% 54.78% NOG 78.26% 54.62% Patent Application Publication Oct. 13, 2011 Sheet 8 of 37 US 2011/0251158A1

PLA2G16 34.38% 54.56% GNG4 62.55% 54.53% RP13-143G15.3 78.91% 54.53% FAM177B 41.68% 54.29% EFHD1 19.83% 53.96% FGURE 7 TCF4 67.42% 53.95% ACO92296.2-1. 43.21% 53.85% GPRC5C 34.63% 53.85% IF6 55.53% 53.77% AC146944.1-4AC146944.1-5, 25.81% 53.64% TMEM161A 33.21% 53.48% EIF2B5, FAM131A 165.68% 53.47% C9orf110 64.61% 53.00% MAFA 31.22% 52.94% RPPLY1. 16.62% 52.85% FGF22 18.24% 52.73% ACOO61588 50.22% 52.71% TRAF6 204.92% 52.67% MYBPC2SPIB 10.13% 52.27% AC116353.7 20.07% 52.24% AC135.457.2-3 54,47% 52.22% KREMEN1. 25.36% 51.87% PLAGL1. 33O46% 51.77% EIF4EBP2 99.24% 51.72% CPB1 45.44% 51.71% TNFRSF14 67.26% 51.70% C7orfA7 32.34% 51.68% CYP27C1 135.47% 51.64% COO.7 38.84% 51.60% AJAP1 39.30% 51.52% GFRA4 32.56% 51.50% ACOA3479.1. 186.02% 5.41% DPM3 10.38% 51.28% LSSAPOO1468.1 16.76% 50.97% AC38O281-3 37.18% 50.78% OR12 64.87% 50.75% ATP1A4 51.41% 50.65% PAPPA 359.84% 50.63% MAF 16.55% 50.46% AMPD2 67.64% 50.45% LRRC51. 140.25% 50.37% NKG7 40.70% 50.36% USE. 24.11% 50.24% TNRC6B 21.65% 50.24% CALML3 27.07% 50.18% ABCB11 32.38% 50.12% ETS1 25.62% 50.10%

Patent Application Publication Oct. 13, 2011 Sheet 10 of 37 US 2011/0251158A1

HSD11B2 26.67% 45.44% SLC8A1 89.40% 45.13% C22Orf13 18.45% 45.05% APOOO354.4 49.26% 44.37% CPNE7 13.59% 44.79% IOCA 19.76% 447.9% FGURE 7 UTP23 18.42% 44.72% NTF3 15.58% 44.61% UBE2C 27.93% 44.53% RAB5C 29.92% 44.40% TNRSF14 1125.00% 44.39% CLC 80.17% 44.23% Corf143 177.35% 44.22% HSD4. 21.0% 44.18% MOBP 32.53% 44.16% SLT1. 27.55% 44.12% CD-233OK9.3 47.14% 44.08% SLC46A3 585.71% 44.05% SYNPO 20.17% 44.00% LRRCAO 36.19% 43.88% CLIC1, CLIC1, CLIC1, CLIC1, CLIC1 25.62% 43.76% PK3R3 62.54% 43.73% KRTAP20-2 67.04% 43.68% AC105345.3 91.52% 43.61% DPM2 73.59% 43.61% GPR371. 46.15% 43.61% ZFAND2A 36.18% 43.56% ACO84018.1 72.53% 43.38% RPUSD1 53.63% 43.36% LRRC56 32.15% 43.31% TSSK3 12.34% 43.28% SPRED3 20.52% 43.22% MURC 81.85% 43.21% PPARGCB 18.39% 43.11% YSK4 74.96% 43.03% HPAC009087.4-2 17.83% 42.99% RP11-343.24. 108.98% 42.69% DO1 36.59% 42.65% EPHB4 30.18% 42.59% NAT8 161.43% 42.54% ARTS-1 19.17% 42.52% DG1. 45.95% 42.52% SLC4A2 31.63% 42.49% KF2A 195.88% 42.39% RP1-212P92 10,04% 42.33% LRB3 25.55% 42.28% KREMEN2 37.37% 42.0% Patent Application Publication Oct. 13, 2011 Sheet 11 of 37 US 2011/0251158A1

CYP4F3LPAC140481.7AC132. 45.34% 42.09% SP140 46.87% 42.02% TMEM30A,COX7A2 24.85% 41.99% SGECP16 47.57% 41.96% PNCK 85.10% 41.91% ANKRD45 133.16% 41.88% a six KRTAP3-3 34.80% 41.83% FIGURE 7 ADRA1B 117.59% 41.62% TBX20 47.30% 41.55% KK10 76.71% 41.55% GUDP2 68.24% 41.54% HS3ST1. 52.04% 41.50% PDIA3P, PDIA3 14.75% 41.36% NDUFV1 16.57% 41.36% C20Orf)1 1.93% 41.22% KIAA1467 78.86% 41.19% AFG31 55.17% 41.17% TNRC6C 16,02% 41.12% ATP7B 54.89% 41.05% RAB36 62.31% 40.98% UBE22 33.89% 40.96% RX4 1767.86% 40.92% ACO254496-1. 48.79% 40.84% WBSCR17 16.59% 40.82% RUNX1. 11.65% 40.77% RAD54L 41.66% 40.73% ADAMSA 103.68% 40.73% NGN4Y 125.23% 40.60% ZNF587 155.56% 40.58% AMAC1, POLR2A,AMAC1L3 34.30% 40.51% NEK6 113.93% A-0.45% FBXL18 61.09% 40.38% SLC38A10 19.51% 40.34% CFH 3O7.64% 40.26% HS6ST3 37.89% 40.24% RPL3 42.63% 40.24% PAOR5 497.59% 40.20% ACO10536.8-2 34.93% A.O.19% SMCY 38.54% 40.14% NDUFA4 70.48% 40.06% HC1. 34.51% 39.97% KBTBD3 68.43% 39.95% A4GNT 19.32% 39.84% TNFAP1 196.99% 39.67% ART3 20.56% 39.64% UP2 62.19% 39.63% HNRNPAB 57.60% 39.36% Patent Application Publication Oct. 13, 2011 Sheet 12 of 37 US 2011/025.1158A1

ANKRD18B 19.25% 39.32% ACO93323.3 69.74% 39.32% OR1OJ8P 25.51% 39.30% CTNND2 13.59% 39.23% PSMB2 155.99% 39, 10% EDEM2 39.01% 39.10% FIGURE 7 DNASE 47.80% 39.05% AGXT22 65.83% 39.03% GPR26 27.50% 38.98% NEUROG2 64.12% 38.91% GPR172A A.O.17% 38.75% L17B 23.51% 38.73% SSTR5 63.31% 38.67% CCN 46.57% 38.59% ZNF497 33.48% 38.54% PA2G2C 31.22% 38.53% CTA 23.35% 38.52% GNA15 10.27% 38.48% BCR 22.86% 38.41% UBXN6 69.04% 38.40% PXN 29.11% 38.38% UPB1 167.67% 38.37% GPR62 22.30% 38.37% THRBAC112217.2 22.00% 38.31% ARF1. 28.31% 38.27% C1orf106 213.44% 38.25% SFN 47.69% 38.24% HSD17B2 51.03% 38.16% AC136632.3-2 10.30% 38.13% KAAO319 46.11% 38.10% ULBP3 10.28% 38.07% C11orf63 37.76% 37.99% FOXA2 69.27% 37.95% DSCR4 53.02% 37.94% CYP2R1. 18.87% 37.86% CDADC1. 36.55% 37.81% GLB1, TMPPE 327.70% 37.72% ARG LU1 122.90% 37.70% RP11-178A10.1 116.53% 37.62% SC4MOL 211.86% 37.56% C11orfA2 53.52% 37.56% NPBWR2 62.66% 37.48% NGFRAP1 35.76% 37.47% FRM D3 58.04% 37.46% CCRL2 22.29% 37.45% CECAF 49.12% 37.39% PHF19 62.46% 37.37% Patent Application Publication Oct. 13, 2011 Sheet 13 of 37 US 2011/0251158A1

ACOO6328.8 24.87% 37.36% PPFBP1 24.72% 37.31% TMEM99 126.74% 37.30% EFTUD2 30.32% 37.27% BRD1. 45.08% 37.18% WT1A 88.96% 37.18% FIGURE 7 ACOO7750.5 93.52% 37.15% TRA2A 247.37% 37.05% OBSCN 53.85% 36.94% MRPS21. 10.04% 36.88% SWL 26.05% 36.85% CGN 40.21% 36.84% PH 34.27% 36.83% MAP1LC3A 58.58% 36.70% GPR176 65.83% 36.68% SLC10A3 91.89% 36.68% THOC4 78.89% 36.65% ELOVL5 477.24% 36.63% FCGBP 17.53% 36.60% RP11-122G1.85 12.85% 36.56% CALU 476.10% 36.50% CADM4 22.81% 36.48% GPHA2 40.53% 36.24% ADCY6 37.95% 36.20% MYOSB 26.98% 36.15% PHLDB3 40.72% 36.14% GOT1. 37.86% 36.08% C21Orf 05 60.06% 36.01% SERTAD1 40.60% 35.96% OR3A3 62.55% 35.87% MPDH2 22.91% 35.82% C2Oorf58 22.62% 35.75% GPR19 114.22% 35.70% WPS4A 15.48% 35.68% COX6B1. 39.27% 35.57% TCTEX1D1 193.70% 35,55% NCKIPSD 20.34% 35.49% PRAMEF1 168,00% 35.45% NUPL1 843.58% 35.42% MMP24. 56.10% 35.41% C17Orf65 48,03% 35.39% RP11-332 P22. 173.00% 35.38% PDCD4. 144.41% 35.34% CWF19L2 58.81% 35.28% RP11-4C20.3 51.45% 35.25% LEP1 38.03% 35.24% MFF 115.35% 35.23% Patent Application Publication Oct. 13, 2011 Sheet 14 of 37 US 2011/0251158A1

MEX3D 24.45% 35.19% C150f27 11.64% 35.09% ACCN4 10,33% 35.08% PAD1 53.84% 35.00% FIGURE 7 FGF11 91.54% 34.98% NRARP 2171.84% 34.96% TCERG1. 100.80% 34.93% ERCC6 129.30% 34.92% ANKRD58 55.90% 34.89% ZBP. 19.13% 34.89% RP11-307L3.2ACO69236.27 162.49% 34.79% DPT 15.74% 34.79% LMTK3 22.51% 34.72% NFKB 12.18% 34.72% ZER1 30.26% 34.66% DNM1P33, DNM1P24. 43.94% 34.62% C2OOf3 62.19% 34.57% PLK2 78.86% 34.48% MAN2B2 11.38% 34.36% NAV2 59.25% 34.27% HLA-DRB1 HLA-DRB1, HLA-DRE 13,09% 34.24% FA2H 32.76% 34.22% AC020922.9 121.38% 34.17% APOOO344.1. 14.34% 34.06% CALN 116.87% 34.01% COL17A1 101.67% 33.98% ADAM10 116.41% 33.96% PXDN 151.01% 33.96% ACTBL2, PLEKHB2, POTEEA26C 112.99% 33.90% C19 Orf36 15.92% 33.88% SLC25A2 10.50% 33.86% KHL 20 78.09% 33.84% PRKAR1B 66.37% 33.80% DMRT3 10.45% 33.74% RPS27 72.67% 33.71% SFRS9 35.36% 33.71% ZNF806,ZNF285B 56.24% 33.50% CLSPN 15.99% 33.48% C14orf142 457.74% 33.46% NRF1 61.56% 33.44% DUSP18 60.12% 33.38% CCDC4.6 92.01% 33.28% SLC6A11 60.86% 33.08% PLEC1 24.56% 33.05% DCTN5 112.18% 33.04% PRSS36 334.82% 32.97% C2Orf27,CR382.287,7-1, RP 11-7 35.98% 32.86% Patent Application Publication Oct. 13, 2011 Sheet 15 of 37 US 2011/0251158A1

C21orf74 31.13% 32.85% TBC1D2B 33.68% 32.79% LDR1 89.25% 32.74% FIGURE 7 REB2 366.37% 32.72% 34.95% 32.70% COL5A1 41.61% 32.69% RP11-107.14.1 36.33% 32.63% ADAMS6 65.26% 32,61% WDR68, KCNH6 52.36% 32.60% MCRS1 47.02% 32.41% CAMK1 15.02% 32.39% 1CAM 19.78% 32.09% ALG5 33.91% 31.98% AC105233.12-3, AF228730.8-1 50.13% 31.97% CD82 82,51% 31.87% SLC8A2 27.01% 31.85% ACOO4836.2 34.75% 31,78% KLFG1. 253.40% 31.75% SIM2 28.08% 31.70% ACO93162,3,.SNRPE 179.29% 31.69% NRL 25.83% 31.68% AHSA2 21.48% 31.68% AC145098, 2-2 13.21% 31.66% CLDN14 66.59% 31.64% GRD1 287.58% 31.60% PRDM7 13.44% 31.58% GATAD2A 176.25% 31.54% FBRSL1. 46.94% 31.51% RGS10 24,06% 31.47% NKX2-1. 26.65% 31.44% SNIP1 117.59% 31.40% MFRP 87.05% 31.36% WDFY2 61.34% 31.32% FPR1 21.41% 31.30% DNAH2 71.62% 31.27% C1Orf89 106.10% 31.25% FARSA 33.12% 31.22% TAX1BP3 25.02% 31.19% PTPNM2 276.70% 31.07% RP4-758, 18.6 44,24% 3.07% RP11-216L13.14, LCN8 13.74% 31.01% MTMR15 133.72% 31.01% CFLP2 CFL1 18,00% 30.99% RBPSUH 41.96% 30.98% L2 70,76% 30.90% ACOO6050.3,ACOO6050.2 57.97% 30.80% ACO15849.2 53.75% 30.80% Patent Application Publication Oct. 13, 2011 Sheet 16 of 37 US 2011/0251158A1

TOMM40 27.5% 30.79% ABCC13 49.59% 30.75% TRIB3 106.60% 3O.75% FMO1 13.48% 30.74% PGAM5 61.61% 30.73% FGURE 7 AOX1 82.24% 30.72% ACO84398.25-1 73.33% 30.71% HTR6 52.62% 30.70% RP1-43E13.2 233.39% 30.68% GABRA4 51.98% 30.65% C3orf.3 87.08% 30.65% ACSM1 13.75% 30.60% C12Orf72 45.8% 30.49% RP-66N13.3 76.26% 30.47% TSPAN31. 66.50% 30.44% AL139020.5 54.88% 30.39% UBB 65.68% 30.37% FAM13B2 54.70% 30.36% OTUD7A 69.27% 30.25% MYH9 48.06% 30.21% ZNF598 69.58% 30.19% DEFB126 34.24% 30.19% NANOS1. 1834.2% 30.18% CAMKV 58.49% 30.17% CYP4F12 35.92% 30.16% GSF11. 10.32% 30.15% - SLT3 30.52% 30.06% MSRB2 44.24% 29.99% ANK3 29.46% 29.97% BFSP2 20.25% 29.94% HM13 242.86% 29.91% HRA 12.69% 29.86% NELL2 2O7.56% 29.71% CCN 161.64% 29.70% PUS7 112.1.1% 29.60% ABLIM2 24.55% 29.59% TMEM179 117.42% 29.51% SLC35E4 66.33% 29.48% GRIFIN 85.53% 29.46% SCN8A 83.92% 29.40% CSDC2 35.01% 29.36% C9 Of 127 21.76% 29.17% FAM45B, FAM45A 83.31% 29.16% GPR110 51.33% 29.16% ZNF621 26.02% 29.15% GR6 75.62% 29.11% DNAJC6 58.53% 29.06% Patent Application Publication Oct. 13, 2011 Sheet 17 of 37 US 2011/0251158A1

RM58 38.19% 29.03% SCNN1G 312.29% 29.01% ZNF398 26.19% 28.90% AP005435.2-1APOO6587.1-2, 52.25% 28.86% ACOO7551.3 14.89% 28.75% FIGURE 7 TTC9 100.35% 28.75% DDX50 16.52% 28.73% C12Orf67 140.39% 28.73% TUBB3 11.52% 28.71% FNDC3A 743.05% 28.71% GAS3 25.59% 28.68% RBBP7 58.99% 28.62% MAN 33.72% 28.58% NUBP1 70.88% 28.57% C19Orf15 62.40% 28.54% SEMA4D 430.04% 28.52% RAI2 90.35% 28.48% PPP5C 29.53% 28.40% ZNF37A 29.15% 28.40% DG3 32,05% 28.40% GLRXP,GLRX 160.55% 28.31% WNT1 OA 17.91% 28.30% PRKAR1B 20.59% 28.27% SCD 320.95% 28.20% TM2D3 32.83% 28.19% RASD2 62.05% 28.11% ANKRD13B 22.55% 28.11% XOH 62.36% 28.03% SLC25A21. 52.10% 27.98% RP5-1061.H2O.3 11.26% 27,96% CF 92.11% 27.79% CHRNA6 43.36% 27.78% CTSZ 46.24% 27.78% C19 Orf73 16.92% 27.76% CRYBB2 91.22% 27.74% COX7A2 2O2.34% 27.74% NMT1 19.91% 27.68% FBXO15 80.49% 27.52% RG1. 45.09% 27.49% OSTF1 117.17% 27.46% C16orf2 17.13% 27.44% SECISBP2 111.81% 27.35% RP11-172F4.2,TINP1 88.71% 27.32% C14orf68 45.53% 27.27% MMP2 184.16% 27.26% RP11-389K14.3 220.65% 27.23% PYCR1 75.00% 27.21% Patent Application Publication Oct. 13, 2011 Sheet 18 of 37 US 2011/0251158A1

EWISL 39.74% 27.20% C12Orf33 223.53% 27.13% C4OrfA2 33.33% 27.12% CSE1 99.60% 27.05% PMF1 14.13% 27.00% C22orf29,GNB1L. 68.70% 26.99% RP11-25OJ16.1 45.49% 26.92% FIGURE 7 FAM104B 32.79% 26.92% ARSG 38.72% 26.91% ACOO507711 23.74% 26.89% CYTH2 56.13% 26.78% PSD3 257.27% 26.73% CHKA 31.8% 26.72% LRTM2 26.84% 26.72% SLC38A10 41.55% 26.71% NCRNAOO118 43.23% 26.67% RP4-662A9.2 12.55% 26.65% SMCR8 299.58% 26.60% LRBA 209.02% 26.59% FAM69A 36.29% 26.56% OAZ3 18.25% 26.54% TM9SF3 76.87% 26.54% GALT 31.46% 26.45% CACNG6 37.76% 26.44% HYDIN - . 66.22% 26.42% KNG1. 93.66% 26.40% EVX2 43.31% 26.37% COLEC 1 41.89% 26.34% APOOO345.1 30.44% 26.31% PDX1 112.99% 26.22% HAO2 - 16.37% 26.15% SH3BP2 76.90% 26.12% FI6 319.76% 26.10% CENPV 86.09% 26.09% C1orf212 76.49% 26.08% ACP2 35.39% 26.06% PCBP3 85.71% 25.99% HTR3E 169.76% 25.99% ITGB1. 46.28% 25.92% PLP2 147.23% 25.92% P2RY6 12.23% 25.92% PREPL 168.17% 25.89% TEX264 A8.06% 25.88% CA13 45.08% 25.87% MFAP2 64.76% 25.84% CHRNE 97.35% 25.83% C4OrfA. 81.35% 25.80% Patent Application Publication Oct. 13, 2011 Sheet 19 of 37 US 2011/0251158A1

HDAC4 46, 12% 25.73% SLC17A4 62.31% 25.70% MCHR1 12,05% 25.69% SAMD9 38.56% 25.68% ZNF644 56.17% 25.67% TPM2 19.92% 25.61% AC124944.3 57.63% 25.61% FGURE 7 ACOO3O75.4 11.99% 25.59% LEFTY1 71.43% 25.58% GPHBP1 106.90% 25.52% OLFM2 45.18% 25.50% RP11-239.3 27.05% 25.48% SRPK2. 56.39% 25.47% TMEM126B 210.37% 25.40% FAM53C 55.88% 25.38% STAT3 483.13% 25.31% DACT3 14.38% 25.30% SLC35C1 15.35% 25.18% HGD 31.93% 25.14% UBE2N 52.50% 25.13% CYP2A7 28.65% 25.07% GUCY2F 28.90% 25.07% NAPB 110.92% 25.05% GGYP1 69.12% 25.05% ARAP1 23.31% 25.03% ACRV1 21.45% 24.94% FAM129C 20.03% 24.90% KF1C 84.56% 24.88% PPB 79.79% 24.87% TYMP 40.94% 24.85% SIGLEC14 34.13% 24.80% TPK1 24.39% 24.78% CACHD1 157.89% 24,77% STBD1. 171.36% 24.74% CLASP1 22.70% 24.73% NPR3 32.47% 24.71% ZMYND15 35.13% 24.70% DPP6 15.94% 24.61% ENOX2 393.90% 24.59% ETNK1 20.16% 24.49% ZNF320,7NF28,ZNF816A 52.66% 24.44% PARP1 20.28% 24.41% AT2 42.83% 24.37% GFM2 91.53% 24.32% TAAR5 34.59% 24.30% COPS6 38.10% 24.24% REPS2 92.22% 24.21% Patent Application Publication Oct. 13, 2011 Sheet 20 of 37 US 2011/0251158A1

FNBP1 372.48% 24.5% SPINT2 105.74% 24.07% NIPA1 112.55% 24.07% LVK2 68.93% 24.01% C4Orf32 704.78% 23.97%. , N DUFA11. 131.59% 23.96% FIGURE 7 TMEM225 78.50% 23.93% HLA-DRA, HLA-DRA, HLA-DRA, 62.96% 23.87% TSSK6 60.50% 23.83% C2Orf50 28.56% 23.82% TMEM101. 22.75% 23.72% GDPD4. 23.98% 23.66% RTN3 32.37% 23.64% ZNF649,ZNF577 22.48% 23.63% L22RA2 233.67% 23.61% SH3TC1 16.81% 23.61% ANFA89 96.35% 23.61% WDR40A 49.36% 23.58% MCRS1. 14.30% 23.52% LENG9 89.53% 23.40% LRP3 30.93% 23.33% ACO21,054.28 106.26% 23.31% ACOO5077.11 25.27% 23.27% C6orf168 97.26% 23.26% SOX7 23.47% 23.25% C7orf51 16.62% 23.21% TMCO1 25.57% 23.18% APOOO/69.5-1. 10.92% 23.17% RAB31 47.87% 23.15% C12OrfA2 34.87% 23.13% RP4-604K51. 1332.50% 23.04% MPEG1 99.17% 22.98% FPR3 181.40% 22.93% COX5B 19.74% 22.86% POLC1 18.78% 22.80% PEF1. 27.20% 22.76% SDP2 127.93% 22.70% ETV3 169.58% 22.67% RHOV 22.10% 22.67% SUVO3 50.41% 22.62% SLC25A28 14.34% 22.60% UBOX5, FASTKD5 93.08% 22.60% HPK1. 128.41% 22.56% ZMAT3 115.35% 22.54% CD34 42.15% 22.53% TCP111 26.01% 22.52% C7orf33 216.57% 22.51% Patent Application Publication Oct. 13, 2011 Sheet 21 of 37 US 2011/0251158A1

AL031846,2ASCC2, ASCC2 14.10% 22.48% CA5B 66.09% 22.45% GSF3 50.29% 22.43% CALR3, MED26 55.71% 22.34% SLC27A 108.68% 22.27% SCRN1. 116.34% 22.26% OTUD7A, 31.80% 22.22% FIGURE 7 RP11-552E20.3 113.87% 22.19% DNAJB3AC114812.5 54.90% 22.17% UBE2I 45.52% 22.2% EPHA1O 55.69% 22.05% C9orf73 45.83% 21.97% ELOW. 24.82% 21.95% C21orf29, KRTAP10-2 68.40% 21.94% CHD9 27.91% 21.91% FAM98A 93.78% 21.90% RP11-44.5H22.4 17.21% 21.87% FAM103A1, RP3-427A4.2,RPS6 46,07% 21.85% GPC2 26.75% 21.85% SUMF 31.90% 21.81% ZK1. 88.3% 21.80% 3MBTL4 53.93% 21.77% MAN2C1 13.67% 21.77% NX1. 90.55% 21.76% HNRNPUL1 51.62% 21.69% MSX2 50.79% 21.55% 12.47% 21.54% TFR2 16.86% 21.53% 17.55% 21.51% 26.65% 21.45% EFNB3 10.36% 2.44% MVD2 11.98% 21.40% ZNF324. 16.07% 21.38% SPMBT2 81.49% 21.37% HTATIP2 125.16% 21.35% HHAT 61.92% 21.35% 30.89% 21.33% PTPR 65.32% 21.32% SETD5 34.39% 21.25% PHTF2 36.72% 21.21% TMEM45A 313.26% 21.10% TGIT 16.32% 21.04% ANF238 19.54% 20.98% HBP1 193.98% 20.95% SLC22A4 206.49% 20.94% GSTK1 27.53% 20.94% MOGAT3 38.02% 20.89% Patent Application Publication Oct. 13, 2011 Sheet 22 of 37 US 2011/025.1158A1

KRTCAP2 26,39% 20.85% DKK3 28.91% 20.84% AP1 24.02% 20.81% ACTL7A 28.25% 20.77% MLX 19.73% 20.77% UBR4 48.50% 20.76% CASZ1. 10.16% 20.72% FGURE 7 PBX2AGER, PBX2,AGER, PBX2, 26.58% 20.70% CAPZA 40.59% 20.69% ZNFA45 13.72% 20.66% CDC42EP1 33.74% 20.66% BRC5 91.26% 20,62% 7SK 170.56% 20.50% SERPINA11 107.32% 20.47% MFAP3 1343.53% 20.46% RC8B 60.68% 20.43% CYP46A1 68.91% 20.40% ASTL 12.14% 20.38% RAD1 90.98% 20.38% CP111 218.57% 20.36% PFN1, RP4-56OB9.2 60.28% 20.30% ND1 39.38% 20.23% KBKAP 55.02% 20.23% H2AFX 66.84% 20.22% CUGBP1 116.60% 2O.22% TM2C 34.82% 20.20% WPS35 21.30% 20.20% MUC5AC, MUC5B 45.49% 20.16% FANCE 43.30% 20.14% LCE1C,LCE2B, LCE1F 35.11% 20.14% NEO1 98.96% 20.13% DHRS11 165.45% 20.12% SORBS1 457.37% 20.11% STOM 63.27% 20.10% AC1304542 46.15% 20.10% KLC3 113.21% 20.06% RP4-617 C6.1 37.65% 20.00% MAP2K5 120.26% 20.00% Patent Application Publication Oct. 13, 2011 Sheet 23 of 37 US 2011/0251158A1

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FULLERENE THERAPES FOR 0006 Prolonged inflammation, known as chronic inflam NFLAMMATION AND INHIBITION OF mation, leads to a progressive shift in the type of cells which BUILD-UP OF ARTERIAL PLAQUE are present at the site of inflammation and is characterized by simultaneous destruction and healing of the tissue from the CROSS-REFERENCE TO RELATED inflammatory process. Chronic inflammation is a pathologi APPLICATIONS cal condition characterized by concurrent active inflamma tion, tissue destruction, and attempts at repair. Chronic 0001. This application is a continuation-in-part of U.S. inflammation is not characterized by the classic signs of acute application Ser. No. 12/921,049, filed on Sep. 3, 2010, which inflammation listed above. Instead, chronically inflamed tis in turn is a national stage of PCT/US2009/001333 filed on sue is characterized by the infiltration of mononuclear Mar. 3, 2009, published as WO 2009/114088, which in turn immune cells (monocytes, macrophages, lymphocytes, and claims priority of U.S. Provisional Application No. 61/033, plasma cells), tissue destruction, and attempts at healing, 309, filed on Mar. 3, 2008; and of U.S. application Ser. No. which include angiogenesis and fibrosis. Tissue mast cells 12/921,083, filed on Sep. 3, 2010, which in turn is a national contain many of the mediators that are released that mediate stage of PCT/US2009/001335 filed on Mar. 3, 2009, pub this influx of inflammatory cells. Endogenous causes include lished as WO 2009/114090, which claims priority of U.S. persistent acute inflammation. Exogenous causes are varied Provisional Application No. 61/033,336, filed on Mar. 3, and include bacterial infection, prolonged exposure to chemi 2008, the entire content of each of which is incorporated cal agents such as silica, or autoimmune reactions such as herein by reference. rheumatoid arthritis. 0007 Cells of the immune system use a signal cascade to BACKGROUND mount an escalating response to a real or perceived insult. The 0002 Various embodiments described herein relate to the inflammatory response becomes pathogenic when the signal use of fullerenes to treat inflammatory disorders and to inhibit cascade is invoked inappropriately. For example, autoim the build-up of arterial plaque in human. mune diseases are the consequence of the immune system 0003 Inflammation is the complex biological response of mounting a response against antigens which are intrinsic. tissues to harmful stimuli. Such as pathogens, damaged cells, Many anti-inflammatory agents function by inhibiting the or irritants. However, inflammation which runs unchecked signal cascade. Such as by blocking intracellular or intercel can lead to a host of disorders, such as inflammatory arthritis, lular effectors. Glucocorticoids, for example, mimic the natu rheumatoid arthritis, allergic disease (hay fever), and athero ral immune suppressant, cortisol, to block at the tran Sclerosis. Scription level, and cylco-oxygenase inhibitors are Small 0004 Mast cells (MC) are granule-rich tissue cells that molecules that bind to and inhibit enzymes that process an significantly contribute to a wide range of diseases through internal signal molecule in cells. the release of noxious mediators. Peripheral blood basophils 0008 Cardiovascular disease is a major health risk (PBB) are similar to MC in that they are the only leukocytes throughout the industrialized world. Atherosclerosis, the that contain pre-stored histamine within their granules. The most prevalent of cardiovascular diseases, is the principal high affinity IgE receptor, FceRI, is one of many ways in cause of heart attack, stroke, and gangrene of the extremities, which MC/PBB can be activated for mediator release. Cross and thereby the principal cause of death in the United States. linking IgE-primed FceRI leads to the release of various Atherosclerosis is a complex disease involving many cell pre-formed and newly generated mediators which can cause types and molecular factors (for a detailed review, see Ross, allergic disease. Ryan, J. J., Kashyap, M., Bailey, D., Nature 362:801-809 (1993)). Kennedy, S. Speiran, K., Brenzovich, J., Barnstein, B., Osk 0009 Atherosclerosis is a disease characterized by the eritzian, C., and Gomez, G. Crit Rev. Immunol. 27, 15-32 deposition offatty Substances, primarily , and Sub (2007). Moreover, MC are suspected to play a role in other sequent fibrosis in the inner layer (intima) of an artery, result inflammatory disorders such as arthritis and cardiovascular ing in plaque deposition on the inner Surface of the arterial disease through non-FceRI mediated mechanisms. Kovanen, wall and degenerative changes within it. The ubiquitous arte P. T. Immunol. Rev. 217, 105-122 (2007). Basophils, also rial fatty plaque is the earliest lesion of atherosclerosis and is established effector cells in allergic disease, have recently a grossly flat, -rich atheroma consisting of macrophages been implicated as playing a major role in adaptive and innate (white blood cells) and smooth muscle fibers. The fibrous immunity. Alter, S.C. and Schwartz, L. B. Biochim. Biophys. plaque of the various forms of advanced atherosclerosis has Acta 991, 426-430 (1989). increased intimal Smooth muscle cells Surrounded by a con 0005 Inflammation can be classified as either acute or nective tissue matrix and variable amounts of intracellular chronic. Acute inflammation is the initial response of the and extracellular lipid. At the luminal surface of the artery, a body to harmful stimuli and is achieved by the increased dense fibrous cap of Smooth muscle or connective tissue movement of plasma and leukocytes from the blood into the usually covers this plaque or lesion. Beneath the fibrous cap, injured tissues. A cascade of biochemical events propagates the lesions are highly cellular consisting of macrophages, and matures the inflammatory response, involving the local other leukocytes and smooth muscle cells. Deep in this cell vascular system, the immune system, and various cells within rich region may be areas of cholesterol crystals, necrotic the injured tissue. Acute inflammation is a short-term process debris and calcification. which is characterized by the classic signs of inflammation— 0010. If allowed to progress, the disease can cause narrow Swelling, redness, pain, heat, and loss of function—due to the ing and obstruction of the lumen of the artery, diminished or infiltration of the tissues by plasma and leukocytes. It occurs occluded blood flow and, consequently, ischemia or infarc as long as the injurious stimulus is present and ceases once the tion of the predominantly affected organ or anatomical part stimulus has been removed, broken down, or walled off by Such as the brain, heart, intestine or extremities. The result can scarring (fibrosis). be significant loss of function, loss of cellular Substance, US 2011/025 1158 A1 Oct. 13, 2011 emergency medical and/or Surgical procedures, and signifi tor that readily recognizes oxidatively modified Lp(a). Mar cant disability or death. Alternatively, the arterial wall can be covina & Morrisett, Current Opinion. In , 6:136 severely weakened by the infiltration of the muscular layer 145 (1995). with the lipid (cholesterol), inflammatory white blood cells, (0015 Cholesterol levels below 200 mg/dl are considered connective tissue and calcium, resulting in Soft and/or brittle "desirable.” A Scandinavian study showed that reduction of areas which can become segmentally dilated (aneurysmal) cholesterol reduced mortality associated with coronary artery and rupture or crack leading to organ, limb or even life disease (CAD) by 42% over six year period and reduced threatening hemorrhage. overall mortality by 30%. J. Hardman & L. Lipman, Good 0011. Once the disease has progressed to the stage of man & Gilman's The Pharmacological Basis Of Therapeu significant persistent symptoms and compromised function, tics (9th ed. 1996) (hereinafter “J. Hardman'). Researchers the next treatment step has conventionally been artery bypass have shown that a 1-mmol/L increase in levels grafting to repair and/or replace the damaged artery. While produces a 76% increase in cardiovascular disease risk in coronary artery bypass has become one of the more common women and a 31% increase in men. Austin, American Journal major cardiovascular Surgical procedures in the United of Cardiology, 83 (9B):13F-16F (1999). Even inpatients with States, Surgery clearly is not the Solution to the pathologic established disease, lowering of LDL cholesterol to between process. Moreover, there is a significant risk of morbidity and 2 and 2.5 mmol/L retards its progression and may even lead to mortality associated with Surgery that many patients are regression. Illingsworth, Drugs, 41 (20): 151-160 (1991). reluctant to accept. Indeed, the autogenous veins or arteries 0016. It is recommended that persons with elevated cho used to bypass the disease-impaired arteries undergo athero lesterol concentrations above 240 mg/dL (6.2 mM/L) receive Sclerosis changes postoperatively generally at a faster rate treatment and that those with borderline values between 200 than the original, affected arteries. The Coronary-Artery Sur 239 mg/dL (5.2 to 6.2 mM/L) be further evaluated according gery Study (CASS) sponsored by the National Heart, Lung to the presence of risk factors for coronary artery disease and Blood Institute (NHLBI) concluded that certain subsets including the sex of the patient, post-menopausal status, a low of patients do not gain any overall statistical benefit from plasma concentration of high-density lipoprotien cholesterol bypass Surgery in comparison to other medical treatments. (HDL) cholesterol (below 35 mg/dL 0.9 mM/L), positive Carraciolo, Circulation, 91 (9): 2335-44 (1995). family history, Smoking, hypertension and diabetes mellitus. 0012. As an alternative to coronary bypass Surgery, certain Expert Panel on Detection, Evaluation, and Treatment of medications and procedures are used to treat the results of High Blood Cholesterol in Adults, J. Am. Medical A., 269 atherosclerosis. These treatments include chelation with eth (23):3015-3023 (1993). Other factors include obesity, hyper ylene diamine tetra-acetic acid (EDTA) and percutaneous triglyceridemia, sedentary lifestyle, Steroid use...beta.-adren transluminal coronary angioplasty (PTCA). EDTA treat ergic blocking agents, some diuretics and genetic factors. ments, however, are still experimental, unproved and poten Frohlich & Pritchard, Clinical Biochemistry, 22:417-433 tially as harmful as they are beneficial. PTCA treatments are (1989). invasive, of limited application and Success and occasionally (0017. By the 1980's, it was recognized that HDL levels manifest lethal complications. Highly experimental intra-ar could be more important in predicting atherosclerotic disease terial laser beam plaque vaporization has limited application than LDL and that HDL may prevent the development of and requires an open operative approach to affected vessels. CAD. Id. Factors such as Smoking, obesity, hypertriglyceri 0013. It is now well established that vascular blockage and demia, genetic factors and lack of exercise are major causes of cardiovascular disorders including myocardial infarction, reduced serum HDL. HDL cholesterol move coronary heart disease, hypertension and hypotension, cere excess cholesterol from the extrahepatic organs to the liver for brovascular disorders including stroke, cerebral thrombosis excretion. Dietschy, Am. J. Clinical Nutrition, 65:1581 S-9S and memory loss due to stroke; peripheral vascular disease (1997). There is evidence that virtually every body tissue is and intestinal infarction are caused by blockage of arteries capable of at least some cholesterol synthesis from the pre and arterioles by atherosclerotic plaque. The production of cursor acetyl-coenzyme A (CoA). Every day, HDL carries atherosclerotic plaque formation is multi-factorial in its pro back to the liver an amount of cholesterol equal to the amount duction. Hypercholesterolemia, especially elevated levels of synthesized and taken up as LDL by all extrahepatic organs low-density cholesterol (LDL) is an important except endocrine glands. There is a second LDL transport risk factor for atherosclerosis and arteriosclerosis and asso process that is receptor independent. Id. Removal of free ciated diseases. cholesterol from arterial wall cells may be an important 0014 Lipoproteins are spherical particles with the lipo mechanism by which HDL plays an anti-atherogenic role. J. philic and cholesteryl esters in the hydrophobic Hardman, Supra, at 878. core, and the amphiphilic , phospholipids and free cho 0018. The earliest recognized gross lesion in atherogen lesterol on the surface with . When the esis is the fatty streak, characterized by an accumulation of amount of cholesterol entering the body increases, the pools cells loaded with cholesteryl esters (“foam cells) just ofsterol within liver cells expands and the receptors that clear beneath the vascular endothelium. The LDL receptor in the LDL from the blood down-regulate, thus increasing LDL arteries gives rise to foam cells and fatty streaks, the earliest levels in the blood. When cholesterol intake is constant, some lesion in atherosclerosis, but there is also a receptor-indepen long-chain Saturated fatty acids further Suppress the hepatic dent mechanism for their formation. This has been demon LDL receptor whereas several unsaturated fatty acids have strated by the development of lesions rich in macrophage the opposite effect. Lipoprotein (a) Lp(a) has emerged as a derived foam cells, even in patients and animals deficient in plasma lipoprotein linked to both diseases of the coronary LDL receptors, and the failure to produce foam cells from arteries, the carotid and the cerebral arteries. It is structurally normal monocytes and monocyte derived macrophages incu related to LDL and possesses one molecule of bated with LDL. This led researchers to explore the possibil Boo per particle. Macrophages express the scavenger recep ity of a post-secretory modification of LDL before it is taken US 2011/025 1158 A1 Oct. 13, 2011

up into foam cells by a new, specific receptor: the "scavenger 0-13-120198-0 (2004); Mitchelet. al., Inorg. Chem., 40:2751 receptor.” Steinberg, New Eng.J. Medicine, 320(14): 915-924 (2001); Sano, Nature (London), 414: 506 (2001); Shvarts (1989). burg, Phys. Chem. 103: 5275 (1999); and Li et al., Chem. 0019. At any given level of hypercholesterolemia there is Phys. Lett. 335: 524 (2001). considerable variation in clinical disease. Postsecretory 0024. In general, fullerenes are hydrophobic and sparingly modifications in the structure of lipoproteins appear to affect their atherogenic potential. Steinberg, supra, at 915. It is not soluble in many solvents. See, e.g., Braun et al., Fullerenes, only the elevated levels of LDL cholesterol that are important, Nanotubes and Carbon Nanostructures, 15: 311-314 (2007). but also its oxidation that leads to atherosclerosis. For this However, a variety of procedures for functionalizing reason, antioxidants are believed to reduce the risk of athero fullerenes are known in the art, and some of the derivative Sclerotic disease. Mortensen, Molecular Aspects of Medicine, fullerenes are water soluble. See, e.g., U.S. Pat. No. 5,648, 18:s137-s144, (Supp. 1997). Peroxidation of polyunsaturated 243 to Chiang; U.S. Patent Application Publication Nos. fatty acids in the LDL lipids is the common initiating factor of 2008/0004345 and 2004/0044062; Jensen et al., Bioorganic the changes and the cytotoxicity of oxidized LDL has been & Medicinal Chemistry, 4:767-79 (1996); Da Ros et al., proven by several research groups and may lead to the denu Croatica Chemica Acta CCACAA 74:743-55 (2001); Wilson, dation of the benign fatty-streak lesion into the atheromatous Perspectives in Fullerene Nanotechnology, Osawa, ed., (Klu plaque. Steinberg, Supra, at 918. wer Academic Publishers, Dorcrecht, Netherlands, 2000); 0020 Researchers believe that the oxidation of LDL Syrensky, et al., Kopf Carrier #63, (David Kopf Instruments within the arterial wall itself is most important. Ocana, New Tujunga, California, September 2006); Y. L. Lai and L. Y. Eng. J. Medicine, 321 (17): 1196-1197 (1989). Auto-antibod Chiang, J. Autonomic Pharmacol., 17:229 (1997); Schinazi et ies to MDL-LDL were seen at significantly higher titers in al., Proc. Electrochem. Soc.,97:10, (1997); Laiet al., World.J. men with atherosclerosis than in normal controls, and in a Surg., 24:450 (2000); Jin et al., J. Neuroscience Res., 62:600 greater proportion of Smokers, those with higher LDL cho (2000); Huanget al., Free Radical Biol. Med., 30:643 (2001): lesterol, and those with higher serum levels of copper in the Chietal. Perspectives of Fullerene Nanotechnology, pp 165 case group. Salonen, 339 LANCET883-887 (1992). 183, E. Osawa ed., (Kluwer Academic Publisher, Great Brit 0021 Researchers also have studied the effects of incuba ain, 2002): Dugan et al., P.N.A.S. 94:9434-39 (1997); Dugan tion of LDL with macrophages and found that in that envi et al., Parkinsonism & Related Disorders 7:243-46 (2001); ronment LDL is oxidized and recognized and taken up by the Quick et al., Neurobiol of Aging (electronic publication acetyl LDL or scavenger receptor in the same cell. Alpha 2006); Kato et al., Chem & Biodiv, 2:1232-1241 (2005): tocopherol, butylated hydroxytoluene (BHT) and Probucol Georgakilas etal, Proc. Nat. Acad. Sci. 99:5075-5080 (2002). block this process. Parthasarathy, Arteriosclerosis, 6(5):505 0025 Incorporation of fullerenes into lipid vesicles has 10 (1986). Treatment with Probucol, a potent anti-oxidant, also been studied (see, e.g., Bensasson et al., Journal of significantly lowered the rate of development of fatty streak Physical Chemistry, 98:3492-3500 (1994); Hirsch et al., lesions in hyperlipidemic rabbits, although the plasma cho Angewandte Chemie International Edition, 39:1845-1848 lesterol level was not lower than in lovastatin-treated animals. (1999); U.S. Pat. No. 7,070,810; Felder, et al., Helv. Chim. Carew, Schwenke & Steinberg, PNAS USA, 84:7725-7729 Acta, 85:288-319 (2002). (1987). Similar results have been demonstrated in cultures of 0026 Fullerenes can also be modified at their surface to LDL with endothelial cells. Steinbrecher, PNAS, 81:3883 present specific biologically active groups, such as lectins or 3887 (1984). Monocytes and neutrophils, when incubated antibodies. See, e.g., U.S. Patent Application Publication No. with LDL, oxidize LDL and render it toxic. Cathcart, Morel 2005/0043787; U.S. Pat. No. 5,310,669. Certain chemically & Chisolm, J. Leukocyte Biology, 38:341-350 (1985). modified fullerenes are commercially available. See, e.g., 0022 Fullerene molecules are a family of carbon allot Bucky USA, Houston, Tex. and American Dye Source, Inc., ropes that comprise closed cages of generally 60 to 200 car Quebec, Canada. bon atoms and may also include chemical moieties attached 0027 Fullerenes and derivatives of fullerenes have been to the exterior or incorporated within the cage. Fullerenes can proposed as free radical scavengers. See, e.g., Haddon, J. Am. be in the form of a hollow sphere, ellipsoid, or tube. The most Chem. Soc. 112:3389 (1990); U.S. Pat. No. 5,648,243 to common fullerene to date is the Co. Buckminsterfullerene Chiang, U.S. Patent Application Publication No. 2003/ (IUPAC name (Co-Ih)5.6 fullerene). Another fairly com 0162837 by Dugan; U.S. Pat. No. 7,163,956 to Wilson; Kep mon buckminsterfullerene is Co., but fullerenes with 72, 76, ley, J. Immunol. 179:665 (2007). 84 and even up to 100 carbon atoms are commonly obtained. Fullerene molecules can contain as few as 20 or more than 500 carbonatoms. Fullerenes may enclose one or more atoms SUMMARY Such as metal atoms, or other Small chemical groups, inside the carbon cage; Such fullerenes are sometimes called 0028. Described herein are methods for treating inflam endohedral fullerenes. Fullerenes may also be modified or matory disorders, comprising administering to a subject in derivatized to include chemical functional groups attached to need thereofatherapeutically effective amount of a syntheti the Surface of the carbon cage. cally modified fullerene. 0023 Structural variations include nonclosed-cage struc 0029. Also disclosed herein are methods for stabilizing tures, heterofullerenes, derivatives formed by substitution of mast cells to prevent mast cell-driven disease Such as asthma, hydrofullerenes, the fusion of organic rings or ring systems to arthritis, and allergy, and methods for inhibiting the build-up the fullerene cage, chiral fullerenes, buckyball clusters, nano of arterial plaque in an individual. tubes, megatubes, polymers, nano'onions.” linked "ball-and 0030. According to various embodiments, disclosed chain dimers, and fullerene rings. See, e.g., Miessler and herein are methods for treating inflammatory disorders or for Tarr, Inorg. Chem. 3, Pearson Education International. ISBN inhibiting the build-up of arterial plaque in an individual, US 2011/025 1158 A1 Oct. 13, 2011

comprising administering to the Subject in need thereof a ence to “the dosage' includes reference to one or more therapeutically effective amount of fullerenes. dosages and equivalents thereof known to those skilled in the art, and so forth. BRIEF DESCRIPTION OF THE DRAWINGS Inflammatory Disorders 0031 FIGS. 1 to 4 illustrate non-limiting examples of 0054 The term “inflammatory disorder” or “inflamma synthetically modified fullerenes. tory disease' is used to refer to abnormalities associated with 0032 FIG. 5 illustrates the attenuation of arthritis in inflammation, and comprises a large group of disorders. An fullerene-treated mice. FIG. 5A shows clinical indices, and inflammatory disorder can be associated with acute inflam FIG. 5B shows ankle thickness. mation and/or chronic inflammation. Examples of inflamma 0033 FIG. 6 illustrates the serum levels of TNF-C. at day tory disorders include, without limitation, autoimmune dis 14 in control and mice treated with fullerene derivatives. eases, inflammatory arthritis, rheumatoid arthritis, 0034 FIG. 7 shows data of affected genes by fullerene osteoarthritis, gouty arthritis, shoulder tendonitis or bursitis, derivative preincubation. polymyalgia rheumatica, inflammatory lung disease, asthma, 0035 FIG. 8 shows inhibition of MC degranulation by type 1 diabetes melitis, multiple Sclerosis, Systemic lupus fullerene derivatives. erthematosus, psoriasis, chronic prostatitis, glomerulone 0036 FIG.9 shows inhibition of MC cytokine production phritis, mast cell-mediated type 1 hypersenitivity, hypersen by fullerene derivatives. sitivity reactions (such as type 2 and type 3 hyperSensitivity), 0037 FIGS. 10A and 10B show concentration (dose)-de inflammatory bowel diseases (such as ulcerative colitis and pendent inhibition of histamine release and IL-13 release. Crohn's disease), pelvic inflammatory disease, reperfusion 0038 FIG. 11 shows inhibition of anti-FceRI-dependent injury, transplant rejection, vasculitis, allergic reactions, increases in intracellular calcium and ROS levels by fullerene inflammatory myopathies (such as dermatomyositis, poly derivatives. myositis, and inclusion body myositis), and leukocyte defects 0039 FIG. 12 shows FceRI-mediated activation of early (such as Chediak-Higashi syndrome and chronic granuloma tous disease). signaling molecules is inhibited by fullerene derivatives. 0055 Inflammatory arthritis comprises a condition where 0040 FIG. 13 shows microarray analysis of FceRI acti arthritis is present because of localized joint inflammation. vated genes affected by TGA. Rheumatoid arthritis, generally considered a type of inflam 0041 FIG. 14 shows microarray analysis of FceRI acti matory arthritis, involves many joints all of which are dam vated genes affected by InoS. aged to some degree by inflammation and it’s sequelae. In 0042 FIG. 15 shows that fullerene derivatives blunt MC certain embodiments, the inflammatory disorder described driven anaphylaxis in vivo. FIG. 15A: Average change of herein is an inflammatory arthritis, including but not limited temperature with TGA injection; FIG. 15B: Average change to rheumatoid arthritis and mast cells mediated inflammatory of temperature with Inos injection; FIG. 15C: serum hista arthritis. mine production. 0056 Allergic diseases are the result of B cell-produced, 0043 FIG. 16 shows effects on lipid uptake by fullerene specific IgE antibody to common, normally innocuous anti derivatives. gens. In simplistic terms, mast cells and drive the initial, 0044 FIG. 17 shows effects on foam cell formation by allergen-inducing reaction through the production of IL-4, fullerene derivatives. and other TH2-specific cytokines which result in IgE sensi 0045 FIG. 18 shows inhibition of clumping of activated tization. Re-exposure to the allergen triggers an allergic monocytes by fullerene derivatives. response through the release of inflammatory mediators from 0046 FIG. 19 shows inhibition of induction of foam cell mast cells and basophils. The IgE produced binds to FceRI on formation by fullerene derivatives. mast cells and basophils and the release of pre-allergic media 0047 FIG. 20 illustrates an exemplary synthesis scheme tors is induced when 2 or more IgE molecules are crosslinked for producing Compound 5. with allergen. Mast cells and basophils are unique in driving 0048 FIG. 21 illustrates an exemplary synthesis scheme this process as they are the only cells that express IgE-binding for producing Czo-tetraglycolic acid, compound 7. FceRI receptors that control the release of histamine (pre stored only in mast cells and basophils) when the IgE encoun 0049 FIG. 22 illustrates an exemplary synthesis scheme ters allergen. Indeed, most allergy medications are aimed at for producing Czo-tetrainositol, compound 10. neutralizing (anti-histamines, H1-receptor blockers) or pre 0050 FIG. 23 illustrates an exemplary synthesis scheme venting (“Omalizumab’’) mast cells and basophils FceRI for producing Co TEG acid (TTA), compound 12. responses. 0051 FIG. 24 illustrates an exemplary synthesis scheme 0057 Mast cells and basophils also mediate other disease for producing Co with a phenyl propionic acid group as one processes. Mice genetically engineered to not express mast of its hydrophilic groups. cells fail to develop asthma-like pulmonary disease when sensitized with less-aggressive immunization protocols and DETAILED DESCRIPTION challenged with aerosolized allergen. 0.058 Mast cells also are involved in other disease pro 0052. In accordance with this detailed description, the fol cesses. Mast cells have traditionally been established partici lowing definitions apply. pants in allergic disease and in protection against extracellu 0053 As used herein, the singular forms “a”, “an', and lar parasites. However, research over the past several years “the include plural referents unless the context clearly dic has revealed that the role of mast cells is not limited to tates otherwise. Thus, for example, reference to “com IgE-mediated immune responses. Mast cells play a critical pounds includes a plurality of Such compounds and refer role in the pathogenesis of synovitis in a murine model of US 2011/025 1158 A1 Oct. 13, 2011

rheumatoid arthritis (RA). The synovium of patients with RA the frequent occurrence ofatherosclerotic plaques in regions is chronically inflamed and characterized by an expanded of the circulatory system where turbulent blood flow occurs, population of MC, as in the mouse model. Mast cells are Such as branch points and irregular structures. markedly increased in number and can make up 5% or more 0063. The first observable event in the formation of an of the expanded population of total synovial cells. The num atherosclerotic plaque occurs when blood-borne monocytes ber of accumulated MC differs substantially from patient to adhere to the vascular endothelial layer and transmigrate patient, in general varying directly with the intensity of joint through to the Sub-endothelial space. Adjacent endothelial inflammation. Mast cell mediators (histamine and tryptase) cells at the same time produce oxidized LDL. These oxidized are also present at higher concentrations in the synovial fluid LDLs are then absorbed in large amounts by the monocytes of inflamed human joints. through Scavenger receptors expressed on their Surfaces. In 0059 MC degranulation has long been associated with contrast to the regulated pathway by which native LDL arthritis in several animal models, but a critical functional role (nLDL) is absorbed by nLDL specific receptors, the scaven in the disease was established in the K/BxN mouse model. ger pathway of uptake is not regulated by the monocytes. This arthritis model closely mimics human RA via symmetric Oxidation of LDL into oxidized LDL results in the loss of the joint involvement, chronicity, a distal-to-proximal gradient of recognition of the apo B component by cellular LDL recep joint involvement, and histological features including syn tors, and in the preferential uptake of oxidized LDL by mac ovial infiltrates, pannus, and erosions of cartilage and bone. rophage'scavenger receptors. The enhanced endocytosis of Mice deficient in mast cells are highly resistant to arthritis, oxidized LDL by vascular wall macrophages transforms whereas reconstitution with normal mast cells restores the them into lipid-laden foam cells that characterize early ath wild-type phenotype. Furthermore, degranulation of mast erosclerotic lesions. cells in the synovium is the first event observed histologically, 0064. The lipid-filled monocytes are called foam cells, and occurring within 1-2 hours of administration of K/BxN are the major constituent of the fatty streak. Interactions serum. Thus, mast cells are a normal cell population within between foam cells and the endothelial and SMCs which the human synovium and have a critical role in the pathogen Surround them lead to a state of chronic local inflammation esis of inflammatory arthritis. which can eventually lead to smooth muscle cell proliferation 0060 Mast cells also mediate multiple sclerosis. Experi and migration, and the formation of a fibrous plaque. Such mental allergic encephalomyelitis (EAE) is a rodent model of plaques occlude the blood vessel concerned and thus restrict human multiple sclerosis (MS) characterized by inflamma the flow of blood, resulting in ischemia. tion in the central nervous system (CNS). Like the human 0065 Ischemia is a condition characterized by a lack of disease, EAE is associated with an early breach of the blood oxygen Supply in tissues of organs due to inadequate perfu brain barrier, focal perivascular mononuclear cell infiltrates, Sion. Such inadequate perfusion can have number of natural and demyelination leading to paralysis of the extremities. causes, including atherosclerotic or restenotic lesions, ane While CD4-positive T cells have been implicated, the under mia, or stroke, to name a few. Many medical interventions, lying cause of increased vascular permeability that facilitates such as the interruption of the flow of blood during bypass the entry of T cells into the CNS is unknown. Surgery, for example, also lead to ischemia. In addition to 0061 Mast cell contribution to the pathogenesis of MS has Sometimes being caused by diseased cardiovascular tissue, been hypothesized based on their presence in CNS plaques of ischemia may sometimes affect cardiovascular tissue, such as MS patients and the correlation between the number, distri in ischemic heart disease. Ischemia may occur in any organ; bution, or MC markers and MS or EAE pathology. Further however, that is suffering a lack of oxygen Supply. evidence for past cells involvement in EAE/MS came from 0066. The most common cause of ischemia in the heart is studies using mast cells-deficient mice. The mast cells-defi atherosclerotic disease of epicardial coronary arteries. By cient W/Wv mice exhibited significantly reduced disease reducing the lumen of these vessels, atherosclerosis causes an incidence, delayed disease onset, and decreased mean clinical absolute decrease in myocardial perfusion in the basal state or scores when compared with their wild-type congenic litter limits appropriate increases in perfusion when the demand for mates. No differences were observed in the T and B cell flow is augmented. Coronary blood flow can also be limited responses between the two groups and reconstitution of the by arterial thrombi, spasm, and, rarely, coronary emboli, as mast cells population in W/Wv mice restores induction of well as by ostial narrowing due to luetic aortitis. Congenital early and severe disease to wild-type levels. These data pro abnormalities, such as anomalous origin of the left anterior vide a new mechanism for immune destruction in EAE and descending coronary artery from the pulmonary artery, may indicate that mast cells may be sentinels of neurologic inflam cause myocardial ischemia and infarction in infancy, but this mation. cause is very rare in adults. Myocardial ischemia can also occur if myocardial oxygen demands are abnormally Build-Up of Arterial Plaque increased, as in severe ventricular hypertrophy due to hyper 0062 “Arterial plaque’ or “atherosclerosis' as used herein tension or aortic stenosis. The latter can be present with are interchangeable. In normal circumstances, the build-up of angina that is indistinguishable from that caused by coronary arterial plaque is a protective response to stresses on the atherosclerosis. A reduction in the oxygen-carrying capacity endothelium and smooth muscle cells (SMCs) of the wall of of the blood, as in extremely severe anemia or in the presence the artery. In response to such stresses, atherosclerosis con of carboxy-hemoglobin, is a rare cause of myocardial sists of the formation of fibrofatty and fibrous lesions or ischemia. Not infrequently, two or more causes of ischemia plaques, preceded and accompanied by inflammation. The will coexist, Such as an increase in oxygen demand due to left advanced lesions of atherosclerosis may occlude the artery Ventricular hypertrophy and a reduction in oxygen Supply concerned, and result from an excessive inflammatory-fibro secondary to coronary atherosclerosis. See, for example, U.S. proliferative response to numerous different forms of insult. Pat. No. 6,492,126 for additional information regarding ath For example, shear stresses are thought to be responsible for erosclerosis and ischemia. US 2011/025 1158 A1 Oct. 13, 2011

0067 Free radical "scavengers' such as vitamins A, E, C, aqueous solution and do not significantly precipitate. Water and selenium are believed to react with oxidized LDLs and soluble fullerenes are known in the art as described above, render them incapable of oxidation. The inhibitory action of and can be synthesized for example by attaching one or more these antioxidants thus inhibits the formation of oxidized hydrophilic chemical groups to the Surface of the carbon LDL, thereby lowering the levels of arterial plaque deposits in cage. Suitable hydrophilic chemical groups include niacin blood vessels. See, for example, U.S. Pat. No. 6,326,031 for group, hydroxyl or polyhydroxyl groups and N-eth additional background regarding LDL, O-LDL, HDL, and ylpolyamino groups. Non-limiting examples of water soluble arterial plaque. fullerenes include Co(OH), Co(NH-CH CH), and 0068. Fullerenes effectively block the immune cascade Czo-tetraglycolate. Many other examples of water-soluble that follows subcutaneous injection of phorbol myristate fullerenes can involve the addition of one or more charged (PMA). Without wishing to be bound by theory, it is believed groups such as phosphates, Sulfates, ammonium, carboxy that a mechanism of action of this blockade may involve free lates, or other charged groups; or hydrophilic, such as radical Scavenging. Membrane trafficking and permeability hydroxyl and polyhydroxyl groups; and carbohydrates, pep may be contributing to the biological response. tides, , nucleotides and DNA. 0069 Peripheral blood monocytes, when placed in a tissue 0073. In another embodiment, chemical groups such as culture dish will adhere and become macrophages. However amphiphilic or lipophilic groups can be attached to the carbon they do not normally ingest LDL added to the culture cage instead of or in combination with hydrophilic chemical medium. Chemical modification of LDL, e.g., by oxidation, groups. will stimulate macrophages to take up LDL. Another tech (0074 “Fullerene.” “fullerene compound” or “fullerene nique for stimulating human peripheral monocytes to ingest derivative' as used herein refers to certain synthetically modi LDL is to incubate the macrophages with PMA, as shown by fied fullerene molecules as described herein, including Syn Kruth et al., J Biol Chem, 277:34573 (2002). thetically modified fullerenes of the formula Z. F. Y. 0070. Without wishing to be bound by theory, it is believed The fullerenes comprise closed cages of 60 to 200 carbon that a compound that blocks PMA inflammatory response in atoms which may also include chemical moieties attached to skin could also block the same pathway in foam cells. Thus, the exterior and/or incorporated within the cage. a proposed intracellular mechanism for controlling the uptake 0075 Certain synthetically modified fullerene molecules of LDL is to usefullerenes to block the inflammatory mecha are described in copending U.S. patent application Ser. No. nism in foam cells and thereby preventing these cells from 12/073,230, U.S. Patent Application Publication No. 2008 accumulating lipids. 0213324-A1, filed Mar. 3, 2008, entitled “AMPHIPHILIC OR LIPOPHILIC POLAR FUNCTIONALIZED Fullerenes FULLERENES AND THEIR USES, the entire disclosure of (0071 “Fullerene' or “fullerene molecule' generally which is incorporated by reference herein. refers to any member of the fullerene family of carbon cage 0076. The synthetically modified fullerene molecules as molecules. Fullerenes are generally carbon structures formed described in the copending application include fullerenes that offive and six membered rings arranged so that the rings form have an aspect ratio z1, with an equatorial band and two a closed geodesic sphere or spheroid held together by a com opposing poles, and comprise an adduct at one or both poles. bination of single and double carbon:carbon covalent bonds. 0077. In one embodiment, the synthetically modified The fullerenes in this disclosure can be defined by the for fullerene has the formula mula: C whereins is greater than or equal to 30, such as from Z-F Y, about 30 to about 200 or from about 30 to about 100. For example, the fullerenes include Co. Co. and similar mol (0078 wherein F is a fullerene of formula C, or X(aC, the ecules that range in molecular weight from Coup to Cs Coo. fullerene having two opposing poles and an equatorial region; and larger Such molecules, with shapes ranging from sphe (0079 C represents a fullerene cage having p carbon roidal to ellipsoidal, elongated and other shapes, and includ atoms, and X(a)C represents such a fullerene cage having a ing not only single-walled but also multi-walled cages con chemical group X within the cage. sisting of stacked or parallel layers. The fullerenes may be 0080 Z and Y are positioned near respective opposite unmodified or underivatized. Alternatively, the fullerenes poles of C: may enclose one or more atoms such as metal atoms, or other I0081 m is an integer of from 1 to 5 and Z is a hydrophilic, Small chemical groups, inside the carbon cage; Such lipophilic, or amphiphilic chemical moiety; fullerenes are sometimes called endohedral fullerenes. I0082 n is an integer of from 1 to 5 and Y is a hydrophilic Fullerenes, as used herein, also include structures with chemical moiety; chemical functional groups attached to the Surface of the I0083 p is an even number between 60 and 200; and carbon cage. The functional groups can be covalently bound I0084 X, if present, represents one or more metal atoms to the carbon cage via opening carbon:carbon double bonds. within the fullerene (F), optionally in the form of a trinitride Fullerenes also include other structural variants, derivatives, of formula G, H, -N in which G and Hare metal atoms. and/or modified or functionalized fullerenes as described In one embodiment, at least one of G and H represents a rare herein and/or as known in the art. The fullerenes can be earth element, a group IIIB element in the periodic table of synthetic or naturally-occurring. Synthetic fullerene mol elements or the like. Examples of suitable rare earth elements ecules can be prepared in a laboratory by known methods and group IIIB elements may include, but are not limited to, (see, e.g., U.S. Pat. No. 5,177.248 and Krätschmer et al., scandium (Sc), erbium (Er), holmium (Ho), yttrium (Y), lan Chem. Phys. Lett., 170, 167-170 (1990)) or can be purchased thanum (La), gadolinium (Gd), thulium (Tm), dysprosium commercially. (Dy), terbium (Tb) and ytterbium (Yb). 0072. In one embodiment, the fullerenes are water soluble, I0085. In exemplary variations, p is an even number meaning the fullerenes distribute more or less uniformly in an between 60 and 120, with p=60-96 being preferred, and p=60 US 2011/025 1158 A1 Oct. 13, 2011

or p=70 being more preferred. The synthetically modified 0091. In another embodiment, the synthetically modified fullerene can be arranged wherein each chemical moiety Z is fullerene molecule has the formula Z(C7)Y wherein: Y is a composed of formula AB in which A is a hydrophilic, lipo hydrophilic moiety covalently connected to C, optionally philic or amphiphilic chemical moiety, r is an integer of from through a linking group, at or near a pole thereof. Z is a 1 to 4, and B is a chemical linker connecting A to the fullerene, hydrophilic moiety covalently connected to C, optionally and each chemical moiety Y is composed of formula DE, in through a linking group, at or near apole opposite to Y, and Y which E is a hydrophilic chemical moiety, V is an integer of is capable of anchoring the synthetic fullerene molecule to a from 1 to 4, and D is a chemical linker connecting the hydro lipid membrane. philic chemical moiety E to the fullerene. 0092. In another embodiment, the synthetically modified fullerene molecule has the formula Z. F. Y., wherein: 0.086. In certain embodiments, Band/or E contain at least (0093. F is a fullerene of formula C, having 60-200, pref one—C(O)C)— moiety. In certain embodiments, one or more erably 60 or 70, carbon atoms: A contains, at a free end thereof, a -(CH2)CH or 0094 m is an integer of from 1 to 5 such that each Z is a —(OCH2CH), OCH moiety. q is an integer of from 3 to 25, group AB in which r is an integer of from 1 to 4, S is an preferably from 4 to 20, and more preferably from 5 to 17. w integer of from 1 to 4, and A is one or more hydrophilic or is an integer of from 1 to 12, preferably from 1 to 9, and more polar group bonded to the fullerene through one or more preferably from 1 to 6. In certain embodiments, A and/or D linker B; contain at least one niacin moiety at a free end thereof. In a 0.095 n is an integer of from 1 to 5 and each Y is a group preferred embodiment, the chemical moiety Y contains two DE in which t is an integer of from 1 to 4, V is an integer of niacin moieties. In another preferred embodiment, the chemi from 1 to 4 and E is one or more hydrophilic group bonded to cal moieties Y and Z each contains two niacin moieties. In a the fullerene through one or more linker D; and, further embodiment, Y has formula DE, wherein each E 0096 XandYare positioned at or near opposite poles of F. contains niacin moiety at the end thereof, and Z has formula 0097. In certain embodiments, the synthetically modified AB, wherein each A contains niacin moiety at the end fullerene has a geometrical configuration capable of causing thereof. the fullerene molecule to locate within phospholipid bilayers 0087. The synthetically modified fullerene can be a pro of a cell Such that a radical scavenging Zone near the equato late ellipsoid shaped fullerene having a major axis Such that rial band of the fullerene is situated within or in close prox said poles are located at opposing ends of the major axis of the imity to the phospholipid bilayer. prolate ellipsoid fullerene. Alternatively, the fullerene can be I0098. A plurality of such synthetically modified fullerene spheroid with opposing poles defined by an axis through molecules can be uniformly dispersed in phospholipids, Such opposing carbon rings. ZandY can configured such that when as in liposomes. The amphipathic fullerene molecules the molecule is contacted with a lipid bilayer in an aqueous described herein do not generally form vesicles by them medium, the equatorial region of F is selectively located selves, but require membrane-forming phospholipids in mole within or in close proximity to the phospholipid bilayer. The ratios greater than 1:1 (lipid:fullerene adduct) to form molecule can be configured so that in an extended configura vesicles. tion has an aspect ratio of about 2.1 to 15, and a diameter less 0099. In exemplary embodiments, the fullerene comprises than about 2 nm. Such configurations are preferred configu any one or more of compounds shown in FIGS. 1-4. rations for incorporation of the molecules into lipid bilayers. 0.100 Suitable fullerenes are also described in the follow 0088. In another embodiment, the synthetically modified ing co-pending U.S. application Ser. No. 12/921, 106, filed fullerene molecule has the formula Z(C)Y wherein: p is an Sep. 3, 2010, which is a national stage of PCT Application even number between 60 and 200, preferably p=60 or 70;Y is No. PCT/US2009/001334, filed on Mar. 3, 2009, published as a hydrophilic moiety covalently connected to C optionally WO 2009/114089, entitled “USING FULLERENES TO through a linking group, at or near a pole thereof. Z is a ENHANCE AND STIMULATE HAIR GROWTH: U.S. hydrophilic, lipophilic or amphiphilic moiety covalently con application Ser. No. 12/921,072, filed Sep. 3, 2010, which is nected to C, optionally through a linking group, at or near a a national stage of PCT/US2009/001332, filed on Mar. 3, pole opposite to Y, and Y is capable of anchoring the synthetic 2009, published as WO 2009/114087, entitled “METHOD fullerene molecule to a lipid membrane. FOR TREATING PRURITUS BY ADMINISTERING 0089. In another embodiment, the synthetically modified FULLERENES:” U.S. application Ser. No. 12/921,143, filed fullerene molecule has the formula Z(C)Y wherein: p is an Sep. 3, 2010, which is a national stage of PCT/US2009/ even number between 60 and 200, preferably p=60 or 70;Y is 001329, filed on Mar. 3, 2009, published as WO 2009/ a hydrophilic moiety covalently connected to C, optionally 114084, entitled “METHOD FOR TREATING WOUNDS through a linking group, at or near a pole thereof. Z is a BY ADMINISTERING FULLERENES:” and Sarah K. hydrophilic moiety covalently connected to C, optionally Norton, Anthony Dellinger, Zhiguo Zhou, Robert Lenk, Dar through a linking group, at or near apole opposite to Y; and Y ren MacFarland, Bechy Vonakis, Daniel Conrad, and Chris is capable of anchoring the synthetic fullerene molecule to a topher L. Kepley. A new class of human mast cell and periph lipid membrane. eral blood basophil stabilizers that differentially control 0090. In another embodiment, the synthetically modified allergic mediator release, Clin. Transl Sci. 2010 August: 3(4): fullerene molecule has the formula Z(C)Y: wherein Y is a 158-169, the entire disclosure of each of which are incorpo hydrophilic moiety covalently connected to Co. optionally rated herein by reference. through a linking group, at or near a pole thereof. Z is a 0101 "Pharmaceutically acceptable salt” refers to phar hydrophilic, lipophilic or amphiphilic moiety covalently con maceutically acceptable salts of fullerenes which salts are nected to Co., optionally through a linking group, at or near a derived from a variety of organic and inorganic counter ions pole opposite to Y, and Y is capable of anchoring the synthetic well known in the art and include, by way of example only, fullerene molecule to a lipid membrane. Sodium, potassium, calcium, magnesium, ammonium, tet US 2011/025 1158 A1 Oct. 13, 2011

raalkylammonium, and the like; and when the molecule con of the drug. Further modes of administration include buccal, tains a basic functionality, salts of organic or inorganic acids, Sublingual, vaginal, Subcutaneous, intramuscular, or intrad Such as hydrochloride, hydrobromide, tartrate, mesylate, ermal administration. acetate, maleate, oxalate and the like. 0105 Modes of administration can include delivery via a 0102) The terms “inhibiting, “treating,” or “treatment” sustained release and/or controlled release drug delivery for and the like are used herein to generally mean obtaining a mulation and/or device. “Sustained release' refers to release of a drug or an active metabolite thereof into the systemic desired pharmacological and physiological effect, and refer circulation over a prolonged period of time relative to that to complete elimination as well as to any clinically or quan achieved by oral administration of a conventional formulation titatively measurable reduction in the condition for which the of the drug. “Controlled release' is a Zero order release; that subject is being treated. “Treatment' is an intervention per is, the drug releases over time irrespective of concentration. formed with the intention of preventing the development or Single, multiple, continuous or intermittent administration altering the pathology or symptoms of a disorder. Accord can be effected. ingly, “treatment” refers to both therapeutic treatment and 0106. In one embodiment, a composition comprising prophylactic or preventative measures. “Treatment may also fullerenes is administered orally to a subject having an be specified as palliative care. More specifically, the inflammatory arthritis Such as rheumatoid arthritis. In another fullerenes described herein are used to treat a subject with an embodiment, a composition comprising fullerenes is injected inflammatory disorder or to inhibit the build-up of arterial directly into an affected joint of a Subject having an inflam plaque in a Subject. These fullerenes are provided in a thera matory arthritis Such as rheumatoid arthritis. In yet another peutically effective amount to: prevent the disorder (i.e., embodiment, a composition comprising fullerenes is admin inhibit the onset or occurrence of the disorder and/or cause the istered via a topical formulation applied to the skin proximal clinical symptoms of the disorder not to develop in a mammal to an affected joint of a Subject having an inflammatory arthri that may be exposed to or predisposed to the disorder but does tis Such as rheumatoid arthritis. not yet experience or display symptoms of the disorder): 0107. In some embodiments, a pharmaceutical composi inhibit the disorder (i.e., arrest or reduce the development of tion or formulation comprising plaque targeted fullerenes is the disorder or its clinical symptoms); or relieve the disorder administered orally to a subject in whom the inhibition of the (i.e., cause regression of the disorder or its clinical Symp build-up of arterial plaque is desired. These fullerenes are toms). Subjects in need of treatment include those already substantially absorbed in the intestine and become incorpo with one or more inflammatory disorder as well as those in rated into LDL particles in the liversuch that atherapeutically which one or more inflammatory disorder is to be prevented, effective amount of fullerenes is delivered to the foam cells and all subjects in whom the inhibition of the build-up of and the fullerenes block further accumulation of LDL into arterial plaque is desired. plaque. 0103) A “subject in need thereof refers to any subject or 0108. In another embodiment, a composition comprising individual who could benefit from the method of treatment fullerenes is injected directly into the vasculature of a subject described herein. In certain embodiments, a subject in need in whom the inhibition of the build-up of arterial plaque is thereof is a subject predisposed for the development of one or desired, such that a therapeutically effective amount of more inflammatory disorders; a subject having one or more fullerenes are absorbed by arterial plaque to block further inflammatory disorders but not exhibiting any clinical Symp accumulation of LDL into arterial plaque. In yet another toms; or a Subject having one or more inflammatory disorders embodiment, a composition comprising cholesterol modified and Suffering from the symptoms of the one or more iron fullerenes is administered directly to vasculature wherein inflammatory disorders. The “subject in need thereof refers such cholesterol modified fullerenes form micelles which to a vertebrate. Such as a mammal. Mammals include, but are partition into LDL particles within the vasculature such that a not limited to, humans, other primates, rodents (i.e., mice, therapeutically effective amount of the fullerenes is absorbed rats, and hamsters), farm animals, sport animals and pets. In by arterial plaque to block further accumulation of LDL into one embodiment, the Subject is a mammal such as a human. In arterial plaque. Targeting of foam cells in arterial plaque is certain embodiments, the methods find use in experimental accomplished through the attachment of groups (i.e., choles animals, in veterinary application, and/or in the development terol derivatives) which home to cholesterol receptors on the of animal models for disease. foam cells. 0104. As used herein, the term “administering or “intro 0109 “Optional” or “optionally” means that the subse ducing a fullerene to a Subject means providing the fullerene quently described event or circumstance may, but need not, to a subject. Methods of administering fullerenes to subjects occur, and that the description includes instances where the include any of a number of convenient means including, but event or circumstance occurs and instances in which it does not limited to, systemic administration (e.g. intravenous not injection, intraparenteral injection, inhalation, transdermal 0110. As used herein, “pharmaceutical composition' and delivery, oral delivery, nasal delivery, rectal delivery, etc.) “pharmaceutical formulation' are interchangeable. and/or local administration (e.g. direct injection into a target 0111. A “therapeutically effective amount’ or “pharma tissue, delivery into a tissue via cannula, delivery into a target ceutically effective amount’ means the amount of a fullerene tissue by implantation of a time-release material, or delivery that, when administered to a subject for treating an inflam through the skin via a topical composition Such as a cream, matory disorder, or to a subject in whom the inhibition of the lotion, or the like), delivery into a tissue by a pump, etc., build-up of arterial plaque is desired, is sufficient to effect intraosseously, in the cerebrospinal fluid, or the like. “Orally such treatment for the disorder or desired inhibition of the delivery refers to administration in an oral form, such as in a build-up of arterial plaque. Thus, a “therapeutically effective pharmaceutically acceptable carrier and/or diluent. Oral amount' is an amount indicated for treatment while not delivery includes ingestion of the drug as well as oral gavage exceeding an amount which may cause significant adverse US 2011/025 1158 A1 Oct. 13, 2011 effects. The “therapeutically effective amount” will vary compositions comprising the fullerenes as described herein in depending on the type of fullerene to be administered, and combination with other agents suitable for the treatment of will also be determined by physical and physiological factors inflammatory disorders or for the inhibition of the build-up of such as the disorder and its severity or the degree of inhibition arterial plaque. of the build-up arterial plaque desired, and the age, body 0117 The fullerenes may be formulated into a variety of weight, and/or clinical history of the subject to be treated. compositions (i.e., formulations or preparations). These com Methods for evaluating the effectiveness of therapeutic treat positions may comprise any component that is suitable for the ments are known to those of skill in the art. intended purpose. Such as conventional physiologically 0112 Doses to be administered are variable according to acceptable delivery vehicles, diluents and excipients includ the treatment period, frequency of administration, the host, ing isotonising agents, pH regulators, solvents, solubilizers, and the nature and severity of the disorder. The dose can be dyes, gelling agents and thickeners and buffers and combina determined by one of skill in the art without an undue amount tions thereof. Pharmaceutical formulations suitable for use of experimentation. The fullerenes are administered in dos with the instant fullerenes can be found, for instance, in age concentrations Sufficient to ensure the release of a Suffi Remington's Pharmaceutical Sciences. Physiologically cient dosage unit into the patient to provide the desired treat acceptable carriers are carriers that are nontoxic at the dos ment of the inflammatory disorder or the desired level of ages and concentrations employed. Pharmaceutical formula inhibition of the build-up of arterial plaque. The actual dosage tions herein comprise pharmaceutical excipients or carriers administered will be determined by physical and physiologi capable of directing the fullerenes to the area where the sub cal factors such as age, body weight, severity of condition, ject in need thereof is a subject in whom the inhibition of the and/or clinical history of the patient. In some embodiments, build-up of arterial plaque is desired. Suitable excipients for the active ingredients may be administered to achieve thera use with fullerenes include water, Saline, dextrose, glycerol peutic or prophylactic blood concentrations, such as in vivo and the like. plasma concentrations of the fullerenes of from about 0.01 to 0118. In various embodiments, the fullerenes are admin about 10,000 ng/cc, such as from about 0.01 to about 1,000 istered to a subject in need thereof in the form of pharmaceu ng/cc. “Therapeutic or prophylactic blood concentrations' tical compositions or formulations. These pharmaceutical refers to systemic exposure to a sufficient concentration of a compositions or formulations comprise fullerenes and can drug or an active metabolite thereof over a sufficient period of also include one or more pharmaceutically acceptable carri time to effect disease therapy or to prevent the onset or reduce ers or excipients. The excipient is typically one suitable for the severity of a disease in the treated animal. administration to human subjects or other mammals. In mak 0113 For example, the methods described herein may use ing the compositions of this disclosure, the active ingredient compositions to provide from about 0.01 to about 100 mg/kg (i.e., fullerenes) is usually mixed with an excipient, and/or body weight/day of the fullerenes, from about 0.01 to about diluted by an excipient. When the excipient serves as a dilu 10 mg/kg body weight/day of the fullerenes, or about 30 ent, it can be a Solid, semi-solid, or liquid material, which acts mg/kg body weight/day of the fullerenes. It will be under as a vehicle, carrier or medium for the active ingredient. For stood, however, that dosage levels that deviate from the additional information regarding Suitable methods and for ranges provided may also be suitable in the treatment of a mulations for use in the present disclosure are found in REM given disorder. INGTON'S PHARMACEUTICAL SCIENCES, Mace Pub 0114. The fullerenes may be in any form suitable for lishing Company, Philadelphia, Pa., 17th ed. (1985). administration. Such administrable forms include tablets, 0119. According to one embodiment, the fullerenes may buffered tablets, pills, capsules, enteric-coated capsules, dra be administered alone, or in combination with any other gees, cachets, powders, granules, aerosols, liposomes, Sup medicament. Thus, the formulation may comprise fullerenes positories, creams, lotions, ointments, skin patches, parenter in combination with another active ingredient, such as a drug, als, lozenges, oral liquids such as Suspensions, Solutions and in the same formulation. When administered in combination, emulsions (oil-in-water or water-in-oil), ophthalmic liquids the fullerenes may be administered in the same formulation as and injectable liquids, or Sustained- and/or controlled release other compounds as shown, or in a separate formulation. forms thereof. The desired dose may be provided in several When administered in combination, the fullerenes may be increments at regular intervals throughout the day, by con administered prior to, following, or concurrently with the tinuous infusion, or by Sustained and/or controlled release other compounds and/or compositions. formulations, or may be presented as a bolus, electuary or I0120 In certain embodiments, the pharmaceutical com paste. positions or formulations described herein have a viscosity at 0115 “Practical dosage regimen” refers to a schedule of 20° C. of from about 5 cps to about 50000 cps, such as from drug administration that is practical for a patient to comply about 500 cps to about 40000 cps, or about 5000 cps to about with. For human patients, a practical dosage regimen for an 30000 cps. orally administered drug is likely to be an aggregate dose of I0121 Preparation of dry formulations that are reconsti less than 10 g/day. tuted immediately before use also is contemplated. The 0116. In one embodiment, a pharmaceutical composition preparation of dry or lyophilized formulations can be effected or formulation comprising the fullerenes is prepared by in a known manner, conveniently from the Solutions of the admixture with one or more pharmaceutically acceptable car invention. The dry formulations of this invention are also riers and/or excipients. Other active ingredients and/or addi storable. By conventional techniques, a solution can be tives may be added, if desired, to maximize fullerene preser evaporated to dryness under mild conditions, especially after Vation, to optimize a particular method of delivery, or to the addition of solvents for azeotropic removal of water, optimize the desired effects in the subject to be treated. In typically a mixture of toluene and ethanol. The residue is addition, according to certain embodiments, the pharmaceu thereafter conveniently dried, e.g., for Some hours in a drying tical composition or formulation includes use of combination OVC. US 2011/025 1158 A1 Oct. 13, 2011

0122) The fullerene-containing preparations described given as the sum of observed inflammation (per paw): 0 no above may be administered systemically or locally and may evidence of inflammation; 1 =subtle inflammation (metatarsal be used alone or as components of mixtures. In one embodi phalanges joints, individual phalanx, or localized edema); ment the administration is local. The route of administration 2=easily identified swelling but localized to either dorsal or for the fullerenes may be intravenous, oral, or by use of an Ventral Surface of paw; and 3 Swelling on all aspects of paw. implant. Maximum score=12. In FIG.SB, the sum of the measurement 0123. Additional routes of administration are subcutane in ankle Swelling at each day was given. The * indicates ous, intramuscular, or intraperitoneal injections of the significant differences observed on that day in fullerene com fullerenes in conventional or convenient forms. pared to non-fullerene-treated mice. 0.124 Generally, the pharmaceutical compositions or for I0129. In FIG. 5, LNW0042 refers to compound 5 (see, mulations described herein can be administered as a pharma e.g., FIG. 4), and LNW0048 refers to compound 7 (see, e.g., ceutical or nutritional formulation. These compositions or FIG. 4). formulations can be administered orally, intravenously, or as 0.130 Tumor necrosis factor-alpha (TNF-C.) is a major a Suppository. mediator of inflammatory arthritis. Several clinical trials have 0125 “Pharmaceutically acceptable carrier” or “diluent” shown that TNF-C. blocking agents, such as etanercept (co means a carrier that is useful in preparing a pharmaceutical marketed by Amgen and Wyeth under the trade name composition that is generally safe, neither biologically nor ENBREL(R): infliximab (marketed under the trade name otherwise undesirable, not toxic or otherwise unacceptable REMICADE(R) by Centocor); and adalimumab (marketed as commensurate with a reasonable benefit/risk ratio, compat HUMIRAR) by Abbott Laboratories, Illinois, U.S.A.), sig ible with other ingredients of the formulation, and includes a nificantly reduce the morbidity associated with inflammatory carrier that is acceptable for Veterinary use as well as human arthritis. As seen in FIG. 6, TNF-C. in the serum at day 14 was pharmaceutical use. "A pharmaceutically acceptable carrier' significantly inhibited in the mice treated with fullerene as used in the specification and claims includes both one and derivatives. These results demonstrate that fullerene deriva more than one such carrier. tives can inhibit inflammatory arthritis, possibly through the 0126. A “pharmaceutically acceptable carrier includes inhibition of TNF-C. any and all solvents, dispersion media, coatings, antibacterial I0131 Prior to injection, 5 was incorporated into liposomes and anti-fungal agents, isotonic and absorption delaying with egg phosphatidylcholine (PC) at a ratio of 1:2. 7 was agents, and the like, compatible with pharmaceutical admin dissolved in PBS buffer at pH 7.4. istration of a composition comprising fullerenes. Examples 0.132. The mouse arthritis model is characterized by the of such carriers or diluents include, but are not limited to, development of disease with many of the features of rheuma water, Saline, Ringer's solutions and dextrose solution. The toid arthritis in humans. As seen in FIGS. 5 and 6, different Volume of the pharmaceutical composition is based on the fullerene formulations inhibited inflammatory arthritis. After intended mode of administration and the safe volume for the arthritogenic serum transfer, PBS-treated mice exhibited individual patient, as determined by a medical professional. typical clinical arthritis as determined using clinical indices 0127. The selection of carrier also depends on the intended and quantitative ankle Swelling measurements. In contrast, mode of administration. Fullerenes of the present invention mice treated with fullerene derivatives demonstrated a sig may be administered by any of a number of convenient means nificant inhibition in both the clinical indices as well as ankle including, but not limited to systemic administration (e.g., Swelling measurements. Inflammation was significantly intravenous injection, intraparenteral injection, inhalation, inhibited (p<0.04) by 5 from day 6 to 14. At day 14, concen transdermal delivery, oral delivery, nasal delivery, rectal trations of 200 ng/100 ul or 2000 ng/100 ul both had p<0. delivery, etc.) and/or local administration (e.g., direct injec 0001. In addition, at 200 ng/100 ul 7, inflammation was tion into a target tissue, delivery into a tissue via cannula, significantly inhibited from days 6 to 12 (p<0.03) and 2000 delivery into a target tissue by implantation of a time-release ng/100 ul 7, inflammation was significantly inhibited from material, or delivery through the skin via a topical composi days 10 to 14 (p<0.02). tion Such as a cream, lotion, or the like), delivery into a tissue by a pump, etc., orally, parenterally, intraosseously, in the Inhibition of FceRI-Mediated MC/PBB Activation cerebrospinal fluid, or the like. Further modes of administra 0.133 Fullerene derivatives were tested in different con tion include buccal, Sublingual, vaginal, Subcutaneous, intra centrations for their ability to inhibit FceRI-mediated muscular, or intradermal administration. MC/PBB activation. Early activation events (ROS, Ca", and phosphorylation of signaling molecules) and later events Fullerene Derivatives in a Murine Arthritis Model (gene expression by microarray and quantification of those 0128. To induce disease, C57/B6 (5 mice/cage/group) FceRI-activated signaling molecules most affected by mice were injected intraperitoneally (IP) on Days 1 and 3 with fullerene derivatives pre-treatment) were examined. In vivo 100 ul of arthritogenic serum. Fullerene derivatives (200 inhibition of MC-dependent anaphylaxis was also analyzed. 2000 ng/100 ul phosphate buffered saline (PBS)) were See, e.g., Sarah K. Norton, Anthony Dellinger, Zhiguo Zhou, injected IP on Day 0, 2, and every 2nd day thereafter. As a Robert Lenk, Darren MacFarland, Bechy Vonakis, Daniel control 100 ul of PBS without serum was injected in the Conrad, and Christopher L. Kepley. A new class of human control group. Swelling in each ankle was measured along mast cell and peripheral blood basophil stabilizers that dif with the clinical indices as described (Lee, D M. Science ferentially control allergic mediator release, Clin. Transl Sci. 2002, Sep. 6; 297(5587):1689-92). Measurements were per 2010 August: 3(4): 158-169. formed every second day by personnel blinded to the identity of the injections. After 14 days mice were sacrificed and ankle Toxicity of Fullerene Derivatives sections removed for histology and serum obtained for cytok I0134) Fullerene derivatives were tested for cell toxicity by ine analysis. Error bars, SEM. In FIG.5A, clinical index was incubation with increasing concentrations up to 100 ug/ml US 2011/025 1158 A1 Oct. 13, 2011

and viability counts taken on days three, six, and nine. No cell debris. Proteins were separated on 8% or 10% NuPage toxicity was observed with any of fullerene derivatives com Tris-Bisgels using Licor running buffer. Western blotting was pared to control cells (not shown). performed using Licor blocking buffer and IR800 and IR700 anti-rabbit F(ab) secondary Abs (1:1000). Primary Abs were MC/PBB Fullerene Derivative Culture and FceRI-Mediated from Cell Signaling Technologies or Santa Cruz, unless oth Activation erwise noted. Band intensities were captured using the Odys sey Imaging System and bands quantified by measuring the 0135 MC are a component of the inflammatory response. number of pixels in each band using a box drawn for the same As such, cultures of human Mast Cells can be used for screen area of measurement for each separate blot. The band inten ing the activity of different fullerene derivatives to evaluate sity was then normalized for loading by dividing the number their potential activity in animal models. In this example, of pixels in each band with the housekeeping band intensity human skin tissue was received from the Cooperative Human (B-actin) performed on the same blot. Tissue Network and MC purified and cultured as described in Kepley, C. L. Int. Arch. Allergy Immunol. 138, 29-39 (2005). The MC were cultured in media containing stem cell factor Calcium and Reactive Oxygen Species (ROS) Measurement which is removed from the culture 24 hours prior to experi I0139 MC were pre-treated with or without fullerene mentation. derivatives as above, washed with Tyrodes buffer supple 0136 PBB were obtained from two sources: donors mented with BSA, and incubated with Fura-2 AM (2 uM) for recruited under an IRB-approved protocol after informed 30 minutes at 37° C. Cells were washed, stimulated with consent or from leukopheresis packs obtained from the Johns anti-FceRI, and calcium flux measured in real time on a Hopkins Hemapheresis Center. PBB were purified to 2.99% PerkinElmer LS55 Spectrofluorometer. For ROS production, purity as described in Vonakis, B.M., Gibbons SJr, Sora, R., cells were exposed to fullerene derivatives as above. After Langdon, J. M., and MacDonald, S.M.J. Allergy Clin. Immu washing, cells were re-suspended in X-Vivo medium contain mol. 108, 822-831 (2001), and Miura, K., Saini, S. S., ing 5uM dichlorodihydrofluorescein (DCF) at 37° C. for 30 Gauvreau, G., and MacGlashan, D. W., Jr. J. Immunol. 167, minutes, washed, and activation-induced changes in mean 2282-2291 (2001). Purified PBB were incubated overnight fluorescence was measured with excitation at 502 nM and (20 hours) with a fullerene derivative (5 lug/ml) or vehicle emission at 523 nM for 15 minutes (19). The data is presented control and a minimal (non-stimulatory) concentration of as fluorescence intensity of the 523 nM emission over time. IL-3 (2 pg/ml) to prevent apoptosis. The next day, cells were All experiments were performed in triplicate and degranula washed and aliquoted for the histamine release assay by treat tion was measured in parallel. Separate experiments were ment with 0.1 mg/ml of goat polyclonal anti-IgE, buffer alone performed to ensure that the fullerene derivatives do not inter (spontaneous release) or perchloric acid (total histamine fere with indicator dye binding (not shown). determination). Histamine was quantified in cell free Super natants using automated fluorimetry in duplicate. In a second Gene Microarray Studies and Validation Using Western Blot set of experiments the two fullerene derivatives (at 5 g/ml) ting or Flow Cytometry were incubated with PBB for 20 hours, washed cells stimu lated with 15 ng/ml anti-IgE for 18 hours in duplicate and I0140 Mast cells (1x107 cell/condition; each condition Supernatants collected for quantification of IL-13 by in-house performed in triplicate) were incubated with or without ELISA. The optimal doses of anti-IgE are chosen for activa fullerene derivatives as above and incubated with or without tion of PBB. anti-FceRI antibodies for 10 minutes, supernatants were 0.137 For activation, MC were suspended in fresh medium removed (to remove pre-formed mediators), and fresh warm (without cytokines) and incubated for 16 hours with or with medium containing anti-FceRI antibodies (14 ml) added for 2 out fullerene derivatives at 37°C. in a 6% CO incubator. The hours. Cells were centrifuged, the Supernatant and the pellet 16 hour time point was chosen based on preliminary experi immediately frozen and microarray performed using the ments demonstrating this was optimal for inhibition of Human Whole Genome OneArrayTM gene expression profil mediator release (not shown) and uptake within FceRI cells. ing service (Phalanx Biotech Group). Separate samples, The next morning, cells were washed and stimulated with assayed in parallel were lysed and expression ana anti-FceRIAbs (3B4; 1 g/ml) for 30 minutes (B-hexosamini lyzed by Western blotting or flow cytometry (CD45) as dase) or overnight (GM-CSF) at 37°C. in a 6% CO, incubator described in Kepley, C. L., Pfeiffer, J., Wilson, B. W., and mediator release measured as described in Zhao, W., Schwartz, L. B., and Oliver, J. M. J. Leukocyte Biol. 64. Kepley, C. L., Morel, P.A., Okumoto, L. M., Fukuoka, Y., and 474-483 (1998). Schwartz, L. B. J Immunol. 177, 694-701 (2006). All MC 0141 For microarray RNA was isolated using the Ambion mediator release studies were performed in triplicate. Message AmpaRNAkit; all samples passed the internal qual ity control checks. For hybridization each sample was run in Western Blotting and Phospho-Signaling Quantification triplicate. Optical density was measured by NanoDrop ND-1000. The ratio of absorbance at 260 nm and 280 nm 0138 Cell lysate preparation and Western blotting were provides an estimate of RNA purity. Samples were found to performed using a protocol optimized for extracting phos have ratios between 1.8 and 2.2 indicating highly pure pho-proteins from human MC. Tkaczyk, C., Metcalfe, D.D., samples. The reactive amino group of 5-(3-aminoallyl)-UTP/ and Gilfillan, A. M. J. Immunol. Methods 268, 239-243 5-(3-aminoallyl)-dUTP was used to conjugate the purified (2002). Following activation, cells were lysed directly in boil aRNA/cDNA with the NHS-CyDye. Labeling efficiency was ing denaturing sample buffer consisting of tris-buffered calculated by the concentration of CyDye and aRNA/cDNA saline with triton-X-100 (0.5%) and protease and phos which was above 10. For hybridization 10 ug Cy5-labeled phatase inhibitors. The cell Suspension was then passed aRNA was utilized by the Phalanx Hybridization Protocol through a 20-gauge needle, boiled, and centrifuged to remove Array Version HOA 4.3. US 2011/025 1158 A1 Oct. 13, 2011

0142 Pearson correlation tables (R values) for each tech described in Booth, G., Newham, P., Barlow, R., Raines, S., nical repeat were calculated from raw log 2 intensity (R) and Zheng, B., and Han, S. Autoimmunity 41, 512-521 (2008). normalized log 2 intensity (N) values and compared to each Data is presented as an average of 4 (Untreated) or 4 (Treated) other. Only probes with P value (detected) less than 0.05 were micetStandard Deviation. included in the calculation. This analysis showed good cor relation between platforms when filtered stringently for fold Fullerene Derivatives Reduce Degranulation and Cytokine change and loosely for significance (p-value). Greater than Production from MC after Anti-FceRI Activation 95% of the samples had R values of 0.8 or greater indicating 0147 Cells were cultured with fixed concentrations of a strong correlation between the two parameters. fullerene derivative (10 ug/ml: FIGS. 8A and 9A) or different 0143. In order to examine the genes most affected by concentrations (FIGS. 8B and 9B) for 16 h, washed and fullerene derivative preincubation the resulting values were stimulated for 30 min (FIGS. 8A and 8B) or 24 hours (FIGS. sorted and filtered the following way. First, the mean (SD) of 9A and 9B) with optimal concentrations of Canti-FceRIAbs the six values from each condition for each of the 30,970 (3B4; 1 ug/ml). Cells were centrifuged and B-hexosaminidase genes was calculated. Second, only normalized value inten release and GMCSF levels determined by ELISA. sities of >100 were included so that follow up detection of 0.148. In FIGS. 8A and 9A, data shown are means-SE of protein levels would be more likely to be successful. Third, triplicate samples that is representative of at least four sepa these data were further truncated to include only normalized rate experiments with separate MC cultures. All values shown data in which the average FceRI activation (without pre treatment) significantly (P<0.05) increased at least 10% com demonstrated a significant (P<0.05) inhibition of at least pared to non-FceRI resting cells. Fourth, the genes with >10% >10% inhibition compared to non-treated samples. In FIG. upregulation by FceRI stimulation alone were compared to 8B, Czo (OH)2 (black square), Czo-tetraphosphate (grey those samples pretreated with each fullerene derivative and diamond), Czo-tetrapyridine (black X), Co-niacin (grey only those showing at least >20% inhibition or >20% upregu circle), Czo (PC) (grey triangle), and CCC (black +). In lation (non-treated+anti-FceRI compared to treated+anti FIG. 9B, Czo-tetrapyridine (black diamond), Czo-tetraphos FceRI) were examined. Data are presented as the average phate (black *). Co-tetrasulfonate (grey triangle), Co-etha percentage downregulated withfullerene derivative treatment nolamine (black X), and CCC (grey circle). Fullerene deriva tives with no effect (approximately 76% of those tested) are derived from the following equation: not shown. I0149 Further, Co (PC). Co-tetrainositol and Co-tet raglucolate (TGA) showed >10% inhibition of histamine (Non-treated -- FiceRI activation) - release when screened at 5 g/mL, compared to the relevant (FD-treated -- FoeRI activation) vehicle control (PBS or 10% DMSO in PBS) (Table 1). In Non-treated -- Fosr activation addition, these fullerene derivatives showed significant inhi bition of FceRI-induced IL-13 release (optimal concentra 0144 Downregulation observed at the gene level was veri tions of anti-IgE was 15 ng/ml) (Table 2). fied at the protein level for several representative molecules using Western blotting or flow cytometry. A complete list of TABLE 1 those genes downregulated or upregulated by each fullerene Inhibition of Histamine Release derivative (>20%) are shown in FIG. 7. Mean 96 Mouse Models of Anaphylaxis and Assessment of Toxicity Vehicle Inhibition control + FD- P value of 0145 MC-dependent anaphylaxis and treatments are Fullerene anti-IgE anti-IgE (n); Histamine described in Ryan, J. J., Bateman, H. R. Stover, A., Gomez, Derivative (meant SEM) (meant SEM) T-test Release G., Norton, S. K., Zhao, W., Schwartz, L. B., Lenk, R., and C7o-(PC)4 46.25.0 42.04.7 0.098 (6) 9.18 Kepley, C. L. J Immunol. 179, 665-672 (2007). Female C7o- SO.89.2 42.19.7 0.007 (7) 22.1 C57BL/6J mice (Jackson Laboratory, Bar Harbor, Me.) aged Tetrainositol 10-12 weeks were injected i.p. with 50 Lug IgE-DNP. Two C7o-TGA 454 - 6.7 39.42.3 0.001 (9) 15.8 hours later mice were injected i.p. with TGA, Inos (100 ng/200 ul in PBS), or 200 ul PBS alone as a vehicle control. After 16 hours mice were challenged i.p. with 100 ug DNP TABLE 2 BSA (Sigma-Aldrich) in 100 ul PBS. Body temperature mea surements were recorded with a digital rectal thermometer Inhibition of IL-13 Secretion every 10 minutes for a total of 50 minutes. Peripheral blood Vehicle Mean 96 was harvested by cardiac puncture 50 minutes after antigen control + FD- Inhibition challenge and serum histamine measurements determined by anti-IgE anti-IgE P value of ELISA. Fullerene (meant SEM, (meant SEM, (n); IL-13 0146 To examine potential toxicity, alanine aminotran?fer Derivative ng/ml) ng/ml) T-test Secretion ease (ALT) and aspartate aminotransferase (AST) were mea C7o-(PC)4 101.1 - 20.3 37.6 20.7 0.047 (3) 62.7 Sured in serum. These enzymes leak out into the general C7o- 1773 - 61.0 6.O. O.8 0.05 (3) 90.9 circulation when the liver is injured. Mice were treated with Tetrainositol or without 1000 ug/100 ul (50 times more than used for the in C7o-TGA 1974 + 46.2 79.3 + 20.9 0.029 (4) 52.4 vivo study) of TGA or Inos by tail vein injection and i.p. After two and 14 days mice were sacrificed and blood, obtained by (O150 FIGS. 10A and 10B show concentration (dose)-de cardiac puncture. ALT and AST activity were measured as pendent inhibition of histamine release and IL-13 release. As US 2011/025 1158 A1 Oct. 13, 2011

shown in FIG. 10A, the maximal inhibition of anti-IgE-in 0151. Following Tables 3-6 show the results obtained with duced histamine release was observed at the 50 mg/ml dose two fullerene derivatives: TGA and Inos. TGA was observed (Czo (PC), 25.19.5%: Czo-tetrainositol=23+16.3%; and to be one of the most efficient inhibitors and significantly Co-TGA=37.8+16.25%: meant-SEM, n=2-3). As shown in FIG. 10B, the basophils were more sensitive to inhibition of reduced both degranulation and cytokine production in MC anti-IgE stimulated IL-13 secretion with maximal inhibition and PBB. However, Inos significantly inhibited degranulation detected at 5 lug/ml for Czo (PC) (64.8%), 5 lug/ml for and cytokine production in PBB (Tables 5 and 6) but signifi Czo-tetrainositol (52.5%); and 0.5 g/ml for C7-TGA (37. cantly inhibited only cytokine production in MC. degranula 4%); mean, n=2). tion was not affected in MC.

TABLE 3 Mean percent degranulation (SD) in TGA treated and untreated MC Mast cells

no XL XL TGA XLF * % inh IC50 (ug/ml)

Exp 1 3.4 76.7 39.1 48.9 Exp 2 3.2 81.1 SO.1 38.2 Exp 3 5.5 74.5 53.4 28.3 Exp 4 4 77.9 40.1 48.5 Exp 5 8.1 78.5 52.7 32.8 Average 4.8 (+2.0) 77.7 (+2.4) 47.1 (+6.9) 39.3 (9.2) 10.9 (+3.5) f: TGA Treatment (a) 10 g/ml * P value = 0.0001 Mean percent GMCSF-cytokine production (SD) in TGA treated and untreated MC Mast cells

no XL XL TGA XLF: % inh IC50 (ug/ml)

Exp 1 SO4 211.4 69.5 67.8 Exp 2 1354 893.8 121.9 84.9 Exp 3 91.6 309.5 104.5 66.2 Exp 4 65.4 271.4 74.1 72.7 Exp 5 1054 741 43.4 95.2 Average 89.6 (+33.4) 485.4 (+309.8) 82.7 (+30.8) 77.4 (+12.4) 3.3 (+0.6) f: TGA Treatment (a) 10 g/ml * P value = 0.02

TABLE 4 Mean percent degranulation (SD) in Inos treated and untreated MC Mast cells

no XL XL Inos + XLF % inh IC50 (ug/ml) Exp 1 3.4 76.7 78.1 1.9 Exp 2 3.2 81.1 80.3 1 Exp 3 5.5 74.5 70.2 5.8 Exp 4 4 77.9 75.5 3.1 Exp 5 8.1 78.5 91.6 16.8 Average 4.8 (+2.0) 77.7 (+2.4) 79.1 (+7.9) 3.3 (+2.4) fInos Treatment (a) 10 g/ml *P value = 0.358 Mean percent GMCSF-cytokine production (SD) in Inos treated and untreated MC Mast cells

no XL XL Inos + XLTF % inh IC50 (ug/ml)

Exp 1 SO4 211.4 11.9 94.4 Exp 2 1354 893.8 20.4 97.7 Exp 3 91.6 309.5 23.5 92.4 Exp 4 65.4 271.4 63.1 76.8 Exp 5 1054 741 43.4 95.2 Average 89.6 (+33.4) 485.4 (+309.8) 32.5 (+20.6) 91.3 (+8.3) 1.9 (+1.1) Inos Treatment (a) 10 g/ml P value = 0.006 US 2011/025 1158 A1 Oct. 13, 2011 14

0152 IC50 values was calculated by setting the fullerene TABLE 5 derivative dose that resulted in maximum inhibition to 100% and then extrapolating the dose at which 50% inhibition was Mean percent degranulation (SD) in TGA treated and untreated PBB seen. The majority of the compounds tested to date had no Peripheral Blood Basophils significant effect on FceRI mediator release. These data dem onstrate selective inhibition of FceRI mediator release from IC50 primary human MC/PBB using nano-engineered fullerene no XL XL TGA-XLF % inh (Ig/ml) derivatives which depends on the moieties added to the car Exp 1 7.3 35 31 11.4 bon cage. Exp 2 22.2 37 34 8.1 0153. Unlike TGA, Inos does not interfere with degranu Exp 3 16.8 36 24 33.3 lation in MC but is highly effective at blocking cytokine Exp 4 8.1 43 40 6.9 Exp 5 9.5 21 17 19 production in MC, while it does inhibit degranulation in PBB. Exp 6 17.8 62.5 51 18.4 This suggests that the InoS interacts and/or indirectly inhibits Exp 7 9.2 64 63 1.56 a signaling molecule found in human MC FceRI signaling Exp 8 11 83 78 6 and not in PBB. Exp 9 19.2 27.5 17 38.1 0154 Mechanistically, the variations in how Inos and Average 13.5 45.4 39.4 15.8 9.9 TGA differentially inhibit intercellular FceRI responses (+5.5) (+20.1) (+7.0) (+4.2) (+2.9) between MC and PBB may be explained in how they affect f TGA Treatment (a) 5 g/ml Lyn. In mouse Lyn-f- basophils, FceRI-mediated degranu 'P value = 0.001 Mean percent IL-13-cytokine production(SD) in TGA lation is inhibited. Charles, N., Watford, W.T., Ramos, H. L., treated and untreated PBB Hellman, L., Oettgen, H. C., Gomez, G., Ryan, J. J. O'Shea, Peripheral Blood Basophils J. J., and Rivera, J. Immunity. 30, 533-543 (2009). However, Lyn-/- BMMCs can have the opposite phenotype with IC50 degranulation being upregulated or inhibited depending on no XL XL TGA + XLF % inh (Ig/ml) the Lyn/Fyn ratio. Parravicini, V., Gadina, M., Kovarova, M., Exp 1 42.8 25.2 119.5 S2.6 Odom, S., Gonzalez-Espinosa, C. Furumoto, Y., Saitoh, S., Exp 2 12.6 297.5 35 88.2 Samelson, L.E., O'Shea, J.J., and Rivera, J. Nat. Immunol. 3, Exp 3 16.4 102.5 52.5 48.8 741-748 (2002); and Yamashita, Y. Charles, N. Furumoto.Y., Exp 4 39.4 137.5 110 2O Odom, S., Yamashita, T., Gilfillan, A. M., Constant, S., Average 27.8 1974 79.3 S2.4 10.6 Bower, M. A., Ryan, J. J., and Rivera, J. J. Immunol. 179, (+924) (+924) (+41.8) (+28.0) (+3.4) 740–743 (2007). In RBL cells degranulation can be inhibited f TGA Treatment (a) 5 g/ml while TNF-C. secretion is unaffected by overexpressing Lyn. 'P value = 0.029 Vonakis, B. M., Gibbons, S.P., Jr., Rotte, M.J., Brothers, E. A., Kim, S. C., Chichester, K., and MacDonald, S. M. J. Immunol. 175, 4543-4554 (2005). TABLE 6 0155 There is also precedence demonstrating that intrac Mean percent degranulation (SD) in Inos ellular FceRI-signaling pathways diverge Subsequent to acti treated and untreated PBB vation in MC and PBB. Concentrations of FceRI cross-link Peripheral Blood Basophils ing agents leading to optimal cytokine production are IC50 consistently lower than concentrations needed for optimal no XL XL Inos + XLF * % inh (Lig/ml) degranulation. MacGlashan, D., Jr. Immunology Series 57. 273–299 (1992). The release of pre-formed mediators through Exp 1 7.3 35 23 34.2 FceRI-mediated degranulation follows the activation of PKC Exp 2 16.8 36 21 41.6 Exp 3 12.6 41 36 12.1 and calcium mobilization, cytokine and chemokine produc Exp 4 10.4 19 11 42.1 tion requires activation of MAP kinases p38 and JNK, Exp 5 12.9 62.5 60 4 whereas lipid mediator production follows the activation of Exp 6 12.5 86 68 20.9 ERK 1/2 pathway. Gilfillan, A. M. and Tkaczyk, C. Nat. Rev. Exp 7 11.4 76 76 O Immunol. 6, 218-230(2006). Average 12 SO.8 42.1 22.1 17.8 0156. A central control point that possibly mediates (+2.9) (+24.5) (+25.7) (+17.5) (+3.2) FceRI-signals leading to cytokine production and mediator f: Inos Treatment (a) 5 g/ml release occurs at LAT, the phospho-activation of this signal * P value = 0.007 Mean percent IL-13-cytokine production (SD) in Inos ing molecule was affected significantly (see below). Rivera, J. treated and untreated PBB and Gilfillan, A. M.J. Allergy Clin. Immunol. 117, 1214-1225 Peripheral Blood Basophils (2006). 0157. In mice, tumor necrosis factor-associated factor 6 L IC50 (TRAF6) is specifically required for cytokine generating no XL X Inos + XLF * % inh (Lig/ml) FceRI-signals of NF-kB, p38 MAP kinase and JNKyet is not Exp 1 42.8 252 7 97.2 required for proximal signaling and Subsequent degranula Exp 2 16.4 102.5 5 95.1 tion. Yang, Y.J., Chen, W., Carrigan, S.O., Chen, W.M., Roth, Exp 3 1.2 51.8 10.1 8O.S Average 29.6 177.3 6.0 90.9 2.6 K., Akiyama, T., Inoue, J., Marshall, J.S., Berman, J. N., and (+18.7) (+105.7) (+1.4) (+9.0) (+1.2) Lin, T. J. J. Biol. Chem. 283,32110-32118 (2008). f: Inos Treatment (a) 5 g/ml Mechanisms of MC-FceRI Inhibition * P value = 0.05 0158 To understand how TGA and Inos differentially influence FceRI-dependent mediator release, those events US 2011/025 1158 A1 Oct. 13, 2011

that occur immediately (within 30 minutes) upon FceRI acti separate experiments. The two gels were run side-by-side, so vation were first examined. The activation of MC and PBB that each time point could be examined, and are shown next to FceRI leading to degranulation is calcium dependent and each other. induces elevated cellular levels of ROS. Swindle, E. J. and 0164. As seen in FIG. 12, several signaling intermediates Metcalfe, D. D. Immunol. Rev. 217:186-205., 186-205 that were phosphorylated by FceRI activation were inhibited (2007). It is hypothesized that the underlying mechanism of by pre-incubation with both fullerene derivatives including inhibition involved the blocking of FceRI-mediated calcium phosphorylation of extracellular signal regulated kinases 1/2 and ROS responses based on previous studies with mixed (ERK 1/2), p38-mitogen-activated protein kinase (p38 MAPK), linker of activated T cells (LAT), AKT, phosphoi isomer fullerene derivatives. Ryan, J. J., Bateman, H. R. nositide 3-kinase (PI3-K), and SRC. For example, fullerene Stover, A., Gomez, G., Norton, S. K., Zhao, W., Schwartz, L. derivative inhibition of MC signaling was time dependent as B., Lenk, R., and Kepley, C. L. J. Immunol. 179, 665-672 pre-incubation with TGA inhibited AKT phosphorylation (2007). 10%, 36%, and 43% at 3, 5 and, 10 minutes while Inos 0159. The responses of MC treated with TGA or Inos were inhibited AKT phosphorylation 18%, 48%, and 42% at 3, 5 compared. and, 10 minutes. Both fullerene derivatives dramatically (0160 Cells were incubated with or without fullerene reduced LAT phosphorylation at later time points with 36%, derivative (10 g/mL) overnight. The next day cells were 72%, and 69% for TGA and 33%, 79%, and 70% for Inos at challenged with anti-FceRI (3B4; 1 ug/mL) and calcium 3, 5 and, 10 minutes, respectively. PI3-kinase, a kinase stores release was determined by the 340/380 nm ratio (FIGS. strongly associated with calcium flux, was dramatically 11A and 11B) and ROS measured by DCF detection at 523 inhibited by both compounds with TGA reducing phospho nm (FIGS. 11C and 11D). FIGS. 11A and 11C contain cells rylation by 15%, 35%, and 50%, while Inos reduced phos incubated with the TGA fullerene derivative, and FIGS. 11B phorylation by 8%, 40%, and 56% at 3, 5, and 10 minutes, and 11D contain cells incubated with the Inos fullerene respectively. There were also variations in the phosphoryla derivative (10 g/mL). The dark gray Squares are the unchal tion of other signaling intermediates examined at several time lenged negative control, dark thick Solid black line is the points. Little change was observed with a phospho-Lyn Ab to anti-FceRI challenged positive control cells, and the two light the negative regulatory tyrosine; a kinase with positive and gray lines (hashed and double line) are the anti-FceRI chal negative signaling roles in MC. Rivera, J., Fierro, N. A., lenged cells pretreated with respective fullerene derivative. Olivera, A., and Suzuki, R. Adv. Immunol. 98, 85-120 (2008). Results are representative of at least three separate experi Thus, molecules previously implicated in the release of cal mentS. cium stores and ROS production in response to FceRI aggre gation were inhibited by certain fullerene derivatives. How 0161 The increase in FceRI-induced intracellular calcium ever, differences in how TGA and Inos inhibit FceRI mediator stores release (FIG. 11A) and ROS (FIG. 11B) upon FceRI release were not due to differential phosphorylation of the cross-linking was inhibited with TGA. However, Inos pre signaling molecules examined. incubation did not affect calcium (FIG. 11C) or ROS (FIG. 0.165. It was shown for the first time that fullerene deriva 11D) levels. Gene microarray data further suggests that TGA tives can inhibit the phosphorylation of signaling intermedi and Inos differentially influence FceRI-associated signaling ates involved with calcium and ROS generation. The Inos and molecules involved in calcium stores release and oxidative TGA both reduced the phosphorylation of several intermedi stress. Thus, TGA and Inos differentially affect ROS and ates; especially LAT and PI3-K. These results are consistent calcium responses induced by FceRI activation. with other findings showing that LAT is critical for calcium mobilization and MC from LAT-deficient mice have inhibited Fullerene Derivatives Block Early FceRI-Activated Signal FceRI-mediated degranulation. Gilfillan, A.M. and Tkaczyk, ing Molecules C. Nat. Rev. Immunol. 6, 218-230 (2006). Similarly, PI3-K is 0162 To further investigate the early signaling events in critical for functional responses in MC as the PI3-Kinhibitors FceRI-dependent mediator release that are affected by TGA wortmannin and LY294.002 inhibit antigen-induced calcium or Inos, Western blotting analysis using phospho-specific mobilization, degranulation and cytokine production by antibodies were performed. The phosphorylation of signaling murine and human MC. Gilfillan, A.M. and Rivera, J. Immu intermediates is an important early step in FceRI-induced mol. Rev. 228, 149-169 (2009). However, the activation of mediator release. Gilfillan, A.M. and Rivera, J. Immunol. Rev. these signaling molecules is dependent on prior activation of 228, 149-169 (2009). Lyn. In this study, the early (10 minute) phosphorylation of 0163 MC were pretreated with Inos or TGA (10 ug/mL) Lyn was not influenced by either fullerene derivative. overnight at 37°C./6% CO. The next day washed cells were Gene Microarray Analysis of TGA and Inos-Treated MC activated with or without anti-FceRI (1 lug/mL) for the indi cated times and lysed using protocols described above. Two (0166 Given that optimal inhibition of MC or PBB secre separate 10% Tris-Glycine gels were used for sodium dode tion was observed after overnight pre-incubation with cyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) fullerene derivative, it was hypothesized that a transcriptional and Western blotting performed with the indicated phospho mechanism of action was involved. rylation-specific (i.e., phosph-Lyn) antibodies. To ensure 0.167 Microarray analysis was used to obtain a broad equal loading, an antiactin antibody was used in parallel. The overview of those FceRI-associated signaling molecules intensity of each band was detected and quantified using an influenced by TGA and Inos pre-incubation following FceRI Odyssey imaging system. Band intensities are presented as a activation at later (2 hours) times. Of the approximately ratio compared to antiactin band intensities probed in the 31,000 genes examined the level of expression of 1,771 same lane and presented for each time point in the colored increased at least 10% in MCs following cross linking of graphs on the right side. Results are representative of three FceRI. In cells pretreated with TGA 2,603 of those were US 2011/025 1158 A1 Oct. 13, 2011

decreased at least 20% compared to non TGA treated cells binding protein localized in the endoplasmic reticulum (ER) and 515 demonstrated greater than 50% inhibition. involved in protein folding and sorting, a disintegrin and 0168 FIG. 13A represents several of those genes that were metalloproteinase (ADAM10), SOX9, tumor necrosis factor upregulated after 2 hours of FceRI activation and signifi alpha-induced protein 1 (TNFAIP1) an immediate-early cantly inhibited by TGA pre-incubation. The gene microarray response gene of endothelium induced by TNF-C., and data was verified at the protein level using Western blotting. SAMD9 involved in the regulation of TNF-C. signaling. For example, the gene for the tyrosine kinase Lyn was upregu Pomerantz, J. L. and Baltimore, D. EMBO.J. 18, 6694-6704 lated 293% upon FceRI stimulation. However, gene expres (1999); Boulares, A.H., Zoltoski, A.J., Sherif, Z.A., Jolly, P. sion levels were reduced by 51% at 2 hours which resulted in Massaro, D., and Smulson, M. E. Am. J Respir: Cell Mol. Biol. a 92% inhibition at the protein level observed at 6 hours (FIG. 28, 322-329 (2003); Sarma, V., Wolf, F. W., Marks, R. M., 13). E74-like factor 2 (ELF-2), which transcriptionally regu Shows, T. B., and Dixit, V. M. J. Immunol. 148, 3302-3312 lates Lyn, was also downregulated 47% suggesting Lyn is (1992); Topaz, O., Indelman, M., Chefetz, I., Geiger, D., controlled at the transcription/translation level and protein Metzker, A., Altschuler, Y., Choder, M., Bercovich, D., Uitto, level after two hours of FceRI aggregation but not after 10 J., Bergman, R., Richard, G., and Sprecher, E. Am. J. Hum. minutes of activation. Conversely, Fyn, which was not inhib Genet. 79, 759-764 (2006); and Chefetz, I., Ben, A. D., ited at the genetic level (not shown) had no change in protein Browning, S., Skorecki, K., Adir, N., Thomas, M. G., levels when pre-incubated with TGA (FIG.3-insert). Several Kogleck, L., Topaz, O., Indelman, M., Uitto, J., Richard, G., other molecules including ADAM10, MAP2K, BTK, and Bradman, N., and Sprecher, E.J. Invest Dermatol. 128, 1423 Syk, were inhibited at the gene level which resulted in lower 1429 (2008). protein levels. The ability to down regulate multiple compo (0175 Second, these were the first data to demonstrate that nents of a signaling pathway is novel and may provide a fullerene derivatives can influence gene expression. Given platform for engineering potent anti-inflammatory com that current dogma Suggest their biological activity depends pounds. These results indicate the ability to block both the solely on their anti-oxidant properties these results suggest activation as well as the expression of distinct signaling mol fullerene derivative effects are not entirely due to ROS scav ecules within the FceRI pathway. enging capabilities. 0169. There was a dramatic difference in the FceRI genes 0176 Third, it proves that fullerenes as a class cannot be affected by Inos compared to TGA. In cells preincubated with considered to behave the same in vitro, in situ, and in vivo. this fullerene derivative, only 741 genes were decreased at Two Co-based derivatives with very similar molecular least 20% compared to non fullerene derivative treated cells weights had practically no common effects in the microarray and 92 demonstrated greater than 50% inhibition. Instead, studies. This suggests that the biological effects of fullerene there was a dramatic increase in expression of genes associ derivatives critically depends on the side chains added to the ated with inhibitory signaling. core carbon cage. (0170 The Inos fullerene derivative had 6 times more 0177. It was observed that fullerenes are potent ROS scav (1847) genes upregulated compare to TGA (FIG. 7). engers and there is evidence that ROS is involved in FceRI (0171 MC were treated and analyzed as in FIG. 13. Given signaling. Further, these test data demonstrate that selective the large size of CD45 (220 kDa) and difficulty in gel-to inhibition of FceRI mediator release from primary human membrane transfer FACS analysis was used to monitor MC/PBB using nano-engineered fullerene derivatives upregulation: mouse IgG Isotype control (black line), depends on the moieties added to the fullerene carbon cage. untreated FITC labeled CD45 (gray filled), and Inos treated Moreover, fullerene derivatives have been shown to affect Fluorescein isothiocyanate (FITC) labeled CD45 (red filled) both phosphorylation of signaling molecules as well gene (FIG. 14). expression. Furthermore, these results show that appropriate 0172 Unlike with TGA, Inos induced the upregulation of fullerene derivatives may be effective treatments for diseases over 20 protein tyrosine phosphatases (PTP) and dual speci that are influenced by MC activation and may represent a new ficity phosphatases (DUSP). The upregulation of several of way to control MC responses before they occur. these inhibitory molecules is shown in FIG. 14. For example, (0178 Complete inhibition of FceRI MC/PBB mediator DUSP1 was upregulated 126% at the gene level at 2 hours and release was not observed with TGA yet it was sufficient to 99% at 6 hours at the protein level (FIG. 14-insert). There improve disease outcomes. Previous studies showed that MC fore, the inhibition of cytokine production by Inos (opposed targeting and complete inhibition of FceRI mediator release to TGA) was due in part to increasing protein expression of in vitro is not necessary for in vivo efficacy. PTP. Since TGA and Inos may be differentially affecting two (0179 To explore the in vivo effects of fullerene derivatives groups of enzymes (kinases vs. PTP), the fullerene derivative on MC responses, the degranulation/cytokine-blocking TGA may be recognizing a common regulatory mechanism or and the cytokine blocking InoS on MC-induced anaphylaxis motif in the signaling molecule. were tested. DNP-IgE-sensitized animals injected i.p. with (0173 These results revealed that TGA profoundly DNP-BSA demonstrated a characteristic drop in core body reduced the FceRI-induced activation of over 1000 genes temperature resulting from MC-driven anaphylactic shock. which was selectively verified at the protein level. Several 0180 Mice (5 per group) were sensitized with 50 ug DNP important discoveries were made from these experiments. IgE in 100 uL PBS. Two hours later the mice were injected i.p. 0.174 First, many upregulated genes were identified that with PBS alone or 100 ng of fullerene derivative that inhibits were previously not associated with MC/PBB FceRI signal degranulation and cytokine production, e.g., TGA (FIG.15A) ing. These include tank binding kinase 1 (TBK-1) involved in or cytokine production only, e.g., Inos (FIG. 15B) in 100 uL mediating NFKB activation, Poly ADP-ribose polymerase 1 PBS. The following day rectal temperatures were recorded (PARP 1) previously shown to be a target for the development before mice were challenged i.p. with 100 ug of DNP-BSA in of new therapeutic strategies in the treatment of lung disor 100 uL PBS. Temperatures were recorded every 10 minutes derS Such as asthma, calumenin (CALU) which is a calcium (up to 50 minutes) following challenge with DNP-BSA. US 2011/025 1158 A1 Oct. 13, 2011 17

Increase in antigen-induced serum histamine release is Cell Culture blunted by fullerene derivative (FIG. 15C). Blood was col 0185. The human monocytic cell line U937 was obtained lected from control (black bars), degranulation inhibiting from the American Type Culture Collection (Manassas, Va., fullerene derivatives (light gray bars) or cytokine inhibiting USA). Cells are maintained in RPMI 1640 media enriched fullerene derivatives (dark gray bars) at 50 minutes and his with 10% heat-inactivated FBS, 2 mM L-Glutamine, 10 mM tamine content determined by ELISA. HEPES buffer, 0.1 mM non-essential Amino Acids, 1% anti 0181. When mice were injected with TGA before FceRI biotic/antimycotic, and 50 uM beta mercaptoethanol at 37° challenge there was a significant reduction in the anaphylac tic-induced drop in core body temperature and behavioral C., 6% CO. responses that accompany anaphylactic shock (FIG. 15A). Foam Cell Differentiation However, Inos had no effect on MC-induced anaphylaxis 0186 LDL from human plasma was oxidized as described (FIG. 15B). This suggests that the Inos interacts and/or indi by Kuzuya M. Yamada K. Hayashi T, et al. Oxidation of rectly inhibits a signaling molecule found in human MC low-density lipoprotein by copper and iron in phosphate FceRI signaling and not in PBB. As expected, serum hista buffer. Biochim. Biophys. Acta 1991; 1084:198-201. To mine levels were significantly lower in animals treated with induce foam cell formation, the U937 monocytic cells were the degranulation/cytokine inhibitor compared to controls seeded at 10° cells/mL in 24 well plates prior to experimental (FIG.15C). There was no significant increase in serum activ treatments. For differentiation into macrophage cells, the ity of ALT and AST between the untreated and fullerene U937 cells were treated with 0.7 ug/mL phorbol myristilic treated mice injected with fullerene derivative concentrations acid (PMA) and incubated for 24 hours at 37° C., 6% CO. 50 fold higher than that needed for in vivo efficacy (Table 7: Oxidized-LDL (10 ug/mL) was added to the PMA-differen tail vein or i.p. routes gave the same result). The injections tiated macrophage cells and incubated for 48 hours at 37°C., were well tolerated and no change in behavior or body 6% CO. weights was noted. These experiments demonstrate that the efficacy of fullerene derivatives in vivo depends on how the Determination of Foam Cell Formation Using Oil Red-O carbon cage is derivatized and Suggest they can be engineered 0187 Cells were fixed with 4% paraformaldehyde and Oil at the nanoscale level to perform specific cellular functions. Red-O (ORO) staining was conducted for foam cell differen

TABLE 7 No liver toxicity is detected following FD iniection Day 2 Day 14 Inositol Inositol Treated TGA Treated Untreated Treated TGA Treated Untreated in Activity in Activity in Activity in Activity in Activity in Activity Aspartate 3 27.6 3 8.5 3 29.S 3 33.5 3 29.8 3 51.1 aminotransferase (AST) (+2.1) (+2.3) (+9.2) (+4.2) (9.9) (+4.1) Alanine 3 26.4 3 29.2 3 64.3 3 44.2 3 36.7 3 47.0 aminotransferase (ALT) (+2.0) (+2.5) (+11.5) (+1.5) (+6.6) (+2.9) n = Number of Mice evaulated in Duplicates Untreated = Normal Mice with PBS injection Treated = Tail Vein injection of 100 ul of Co-inositol or Co-itetragylcolate

0182. As discussed herein, no toxicity was observed using tiated macrophages. Fixed and stained cell preparations were up to 100 g/ml of fullerene derivatives well above the cytocentrifuged and washed in deionized HO for five min concentrations in which efficacy was observed in vitro and in utes and then viewed under the microscope for detection of vivo. It is noted that thoroughly purified fullerene derivatives ORO stain as described in Koopman R. Schaart G. Hesselink were employed in the studies, limiting the likelihood of con MK. Optimisation of oil red Ostaining permits combination founding results due to sample impurities. with immunofluorescence and automated quantification of 0183 In separate experiments the in vivo administration lipids. Histochem. Cell Biol. 2001; 116:63-68. (i.v. daily; 200 ng/three weeks) of TGA to mice showed no notable differences in behavior and there were no abnormali Flow Cytometry ties observed upon gross pathological examination. No mutagenic potential (using the Ames test) was observed (not 0188 Cells were recovered by centrifugation at 800xg at shown). No adverse reactions were observed in the anaphy 4°C., washed with PBS/1% BSA, and blocked for 30 min at laxis model when fullerene derivatives were injected and no 4°C. with a 1/500 dilution of normal human serum. The cells liver damage was noted at the concentrations sufficient for in were washed and incubated with either FITC-labelled anti vivo efficacy. Taken together, the derivatives described herein CD11b (10 ug/mL) or FITC-labelled Isotype Control (10 are not cytotoxic to several cell lines tested and appear to have ug/mL) for one hour at 4°C. After three washes, cells were no acute in vivo cytotoxic effects. resuspended in 400 ul of PBS. The mean intensity of fluores 0184 Cultured monocytes will adhere to tissue culture cence was determined for at least 10,000 cells using a FAC flasks and differentiate into macrophages. Such cultured mac Scan flow cytometer (BDBiosciences). All experiments were rophages can be studied as a model system for foam cells. performed in duplicates. US 2011/025 1158 A1 Oct. 13, 2011

Fullerene Effects on TNF-C. Release from Macrophage Foam cellular clumping as part thereof, equal numbers of U937 Cells monocytes were separated into three different groups. One (0189 In order to observe the TNF-C. release from foam group of cells was treated with 0.7 ug/mL PMA for 24 hours cells, duplicate samples of cells (107 cells/ml) were treated while another was pre-treated with 5 lug/mL ALM for 24 with or without fullerenes (5.0 ug/mL) for 24 hours. The next hours before receiving PMA. The control group did not day cells were challenged with or without 0.7 ug/mL of PMA receive PMA or ALM. After the appropriate time had elapsed, and varying concentrations of OX-LDL (0.5 ug/mL, 1.0 the PMA-treated cells showed significant clumping (FIG. ug/mL, 5.0 ug/mL, 8.0 g/mL and 10.0 g/mL) as described 17). However, the ALM-treated cells showed no significant above. Controls samples were treated with 5.0 lug/mL ALM clumping. only and 0.7 g/mL PMA only. Cells were placed in a 37°C. 0194 To test whether the fullerene derivatives inhibit the incubator and samples were taken at 12 and 24 hours. TNF-C. clumping of activated monocytes through Mac 1 inhibition, levels were measured as previously described in Kepley C L. cells were treated as above and examined for the upregulation Antigen-induced reduction in mast cell and basophil func of Mac 1 using FACs analysis. As seen in FIG. 18, the same tional responses due to reduced Syk protein levels. Int. Arch. fullerene treatment conditions that prevented cell clumping Allergy Immunol. 2005: 138:29-39. When the assay was com also prevented the upregulation of Mac1. Thus, fullerenes pleted, the plate was read on a Biotek ELX800 plate reader and prevent activation-induced monocytic cell-cell adhesion pos the resulting data was analyzed. sibly through the inhibition of Mac 1. Effects of ALM on U937 Monocyte Cell Viability Preincubation of Monocytes Prevents TNF-C.-Induced Foam Cell Formation 0190. The effects of fullerenes on cell viability was exam ined in parallel with Vitamin C, a potent anti-oxidant (Table (0195 To test whether fullerenes inhibited the induction of 8): foam cell formation in part through the inhibition of TNF-C. the effects ALM had on TNF-C. release from activated mono TABLE 8 cytes were examined. As shown in FIG. 19, cells treated for 24 hours with 0.7 ug/mL of PMA and any concentration up to 90 Viability 8 g/mL of OX-LDL released a significantly higher amount of TNF-C. than untreated monocytes. However, when cells were Day 3 Day 6 Day 9 pretreated with 5.0 ug/mL of ALM, there was a statistically Untreated 98.8% O.2O 93.9% O.81 69.9% O.90 significant decrease in the amount of TNF-C. released ranging ALM (67 uM) 98.2% O.6O 91.1% 0.33 67.0% 1.03 ALM (6.7 uM) 99.4% O.56 90.9% O.O7 66.5% 0.03 from 58% to 77% inhibition. Similar statistically significant ALM (0.67 uM) 98.3% O.S2 92.6% 2.31 67.2% O.82 inhibition of release was seen with ALM when levels were Vitamin C (67 uM) 98.2% O.67 90.9% O.O7 60.7% O.28 monitored at 12 hours (data not shown). Thus, ALM may Vitamin C (6.7 uM) 97.4% 1.86 91.2% 0.33 61.8% 1.10 inhibit foam cell formation through reductions in TNF-C. Vitamin C (0.67 uM) 98.8% OO6 93.8% 1.09 60.9% 1.13 levels. 0196. The results showed that cells pretreated with the 0191 As seen in above Table 8, monocyte cells incubated fullerenes had a significant decrease in total ORO staining in with Vitamin C or ALM did not have toxic effects on the the cell as opposed to those that were untreated. Current viability of serum-starved cell. No significant differences in treatment for atherosclerosis is the management of lipid accu cell viability was observed using up to 100 ug/mL ALM mulation and several trials have demonstrated reduced car compared to control cells at days six and nine. Similar results diovascular events and mortality with lipid-lowering therapy. were obtained with the monocytic cell line THP-1 and mono Ashen MD, Blumenthal RS. Clinical practice. Low HDL cytes derived from whole blood (data not shown). cholesterol levels. N. Engl. J. Med. 353:1252-1260 (2005). It is assumed that the reduction in lipids results in the reduction Fullerenes Prevent Lipid Accumulation in Macrophage Foam in foam cells that line arteriole walls. Thus, fullerenes may be Cells a new approach for lowering a patient’s lipid burden and Subsequent plaque accumulation. 0.192 Differentiated monocytes were used to observe the 0197) There are several studies which support the impor effects that ALM would have onlipid uptake. Untreated U937 tance of the monocyte to macrophage differentiation in the monocytes showed very little ORO staining (FIG. 16A). initiation and progression of atherosclerosis. Davis S C, Analysis of lipid uptake in cells incubated with PMA and Ricotti C, Cazzaniga A, Welsh E, Eaglstein WH, Mertz PM, various concentrations of OX-LDL showed a significant Microscopic and physiologic evidence for biofilm-associated amount of accumulation in the cytoplasms of cells as seen in wound colonization in vivo. Wound. Repair Regen. 16:23-29 FIG. 16B. However, cells pre-incubated with ALM prior to (2008). One of the first events of atherogenesis is invasion of the addition of PMA and OX-LDL had significantly less stain the arterial wall by monocyte derived macrophages. Mono ing than those not receiving ALM, indicating less lipid accu cytes are further induced toward foam cell formation through mulation (FIG. 16C). Dose response studies demonstrated the induction of cellular adhesion molecules that mediate that 5ug/mL ALM for 24 hours was optimal and this concen their adhesion to vessel walls. Galkina E. Ley K. Vascular tration was used in all Subsequent experiments (data not adhesion molecules in atherosclerosis. Arterioscler: Thromb. shown). Vasc. Biol. 27:2292-2301 (2007). While there are several Fullerenes Inhibit Foam Cell Adhesion Via Mac 1 Down steps involved in leukocyte trafficking into vascular tissues Regulation ICAM-1 is particularly involved in atherosclerosis through the regulation of monocyte recruitment (which express Mac 1: 0193 To determine if fullerene derivatives affect the foam a ligand for ICAM) into atherosclerosis-prone areas. In ath cell formation process where activated monocytes initiate erosclerosis OX-LDL induces endothelial ICAM-1 upregula US 2011/025 1158 A1 Oct. 13, 2011 tion which would provide more opportunities for interactions fullerenes and suggest they may represent a novel therapeutic with monocytic Mac1. Several other studies in humans and candidate for the treatment of atherosclerosis. mice Suggest that ICAM-1-Mac 1 interactions participates in 0201 The present disclosure relates to use of any one or the initial adhesion of monocytes/macrophages onto vessels. more of the fullerenes described herein for the treatment of an Martineau L, Davis S. C., Controlling methicillin resistant inflammatory disease or for inhibiting the build-up of arterial StaphylocCocus aureus and Pseudomonas aeruginosa wound plaque. The present disclosure also relates to the use of any infections with a novel biomaterial. J. Invest Surg. 20:217 one or more of the fullerenes described herein for manufac ture of a medicament, particularly the manufacture of a medi 227 (2007). The studies here demonstrate that fullerenes cament for treating inflammatory disease or for inhibiting the reduce cellular adhesion in monocytic cells through reduction build-up of arterial plaque. of CD11 b expression. These results suggest fullerenes may 0202 The publications discussed herein are provided block the initial steps involved in atherosclerosis—the adhe solely for their disclosure prior to the filing date of the present sion of monocytes to blood vessels—by preventing the application. Nothing herein is to be construed as an admission upregulation of adhesion receptors on activated monocytes. that the present disclosure is not entitled to antedate such 0198 Inflammation plays a vital role in all phases of ath publication by virtue of prior disclosure. Further, the dates of erosclerosis. Hansson G. K. Robertson A K, Soderberg-Nau publication provided may be different from the actual publi cler C., Inflammation and atherosclerosis. Annu. Rev. Pathol. cation dates, which may need to be independently confirmed. 1:297-329 (2006). This inflammation is a result of the stimu All publications, patents, patent applications and other refer lation of monocyte and macrophage cells: both of which ences cited herein are hereby incorporated by reference. release pro-inflammatory cytokines: IL-1B, IL-6, and TNF-C. 0203 While the disclosure has been described in detail which has been shown to have a profound influence on the with reference to certain embodiments thereof, it will be exacerbation of atherosclerosis. These cytokines are often apparent to one skilled in the art that various changes can be observed in theatheroscleroticlesions. Studies examining the made, and equivalents employed, without departing from the suppression of TNF-C. (mostly from rheumatoid arthritis tri Scope of the disclosure. In addition, the following examples als) suggest that anti-TNF-C. therapy seems to be, at least in are illustrative of the methods described herein and should not part, associated with concomitant reduction of the risk of be considered as limiting the foregoing disclosure in any way. cardiovascular events. Avouac J. Allanore Y., Cardiovascular risk in rheumatoid arthritis: effects of anti-TNF drugs. EXAMPLES Expert. Opin. Pharmacother: 9:1121-1128 (2008). Appli Example 1 cants findings show that with varying concentrations of fullerenes the production of TNF-C. could be reduced in some Preparation of Amphiphilic Fullerene Derivative cases as much as 72%. These differences of TNF-C. release Depicted in FIG. 20 may be important in providing a new way to prevent athero Sclerosis. Compound 5; Labeled LnW0042 in FIG. 6 0199. Several studies have demonstrated that the induc Step 1. Synthesis of Didodecylmalonate (Compound 1 in tion of foam cell formation in atherosclerosis is mediated in FIG. 20) part through oxidative stress Suggesting anti-oxidant therapy may be beneficial for preventing this process. Iuliano L., The 0204 10 mmole dodecyl alcohol was dissolved in 30 mL oxidant stress hypothesis of atherogenesis. Lipids 36 Suppl: dry dichloromethane (DCM), to which 12 mmole triethy S41-S44 (2001). For example the mechanism for lipid accu lamine (TEA) was added and stirred under nitrogen atmo mulation in monocyte cells is governed by the CD36 and SRA sphere. Then, 5 mmole malonyl chloride was dissolved in 1 receptors. Furthermore, oxidative stress increases CD36 ml dry DCM, and dropwise added to the above solution expression. Fuhrman B, Volkova N. Aviram M., Oxidative within a period of 10 minutes. Upon completion of the addi stress increases the expression of the CD36 scavenger recep tion of malonyl chloride, the reaction mixture was stirred for torand the cellular uptake of oxidized low-density lipoprotein a few hours and monitored by TLC. When reaction was com in macrophages from atherosclerotic mice: protective role of pleted, the reaction mixture was washed with brine twice and antioxidants and of paraoxonase. Atherosclerosis 161:307 the combined organic phase was dried over MgSO, filtered, 316 (2002). Previous reports further support the hypothesis and concentrated to 1-2 mL. Flash column with silica gel was and have proposed that membrane expression of CD36 used to purify the products with DCM as the solvents. Yield: involves redox signaling pathway via NADPH oxidase acti 85%. Vation and the administration of antioxidants leads to a reduc tion in CD36 expression in monocytes derived from humans. Step 2. Synthesis of Czo Monoadduct (Compound 2 Given that fullerenes are extremely potent anti-oxidants (Wil in FIG. 20) son SR, Schuster DI, Nuber B, Meier M, Prato M, Taylor R. 0205 840 mg (1.0 mmole) of Czo was dissolved in 50 mL Fullerenes. Chemistry, Physics, and Technology. K. Kadish, o-xylene and sonicated for 3 minutes, and then 400 mL tolu and R. Ruoff, eds, John Wiley & Sons, NY, 2000), it is ene was added. Next, 1.0 mmole of the malonate 1 was added tempting to speculate that the derivatives described here may and the whole mixture was stirred, to which 1.0 mmole of be exerting their inhibitory effects through the inhibition of iodine (MW-254 Da) was added. After stirred for 10 minutes, oxidative stress. a 20 mL toluene solution of DBU (2.5 mmole, MW=151 Da, 0200. The results show that fullerenes exert their anti 1,8-diazabicyclo5.4.0]undec-7-ene) was added to the mix atherogenic effects by inhibiting the formation of foam cell ture overa period of 15 minutes, and stirred for two hour. TLC formation and adhesion through the reduction of inflamma monitored the reaction progress with 3:7 toluene/hexanes. tory cytokine release and adhesion molecule membrane Upon completion of the reaction, the product was concen expression. These results further extend the utilization of trated to 10 mL (not to dryness) and 30 mL hexanes were US 2011/025 1158 A1 Oct. 13, 2011 20 added to dilute. Next, the mixture was loaded to the top of a which was rotavaped and vacuum pumped for overnight for silica gel column for purification. A mixture of solvents of 3:7 NMR and MALDI-MS. Yield: 70%. toluene? hexanes was used to elute unreacted Co (first band), and then the product (second band), which was then Step 3. Synthesis of Czo Tetraglycolic Acid rotavaped and then pumped under vacuum for overnight (Compound 7) before NMR and MALDI-MS. Yield: 60%. 0209. The tert-butyl ester precursor 6 was dissolved in DCM at 20 mg/mL, and equal volume of TFA was added, and Step 3. Synthesis of Compound 5 stirred overnight.TFA was removed by evaporation and water washing. The DCM layer was then dried to give pure TGA 0206 127.8 mg (0.1 mmole) of Czo monoadduct 2 product. Yield: quantitative. (MW=1278) was dissolved in 60 mL toluene. Next, 0.1 mmole of the malonate 4 (see detailed synthesis in Example Example 3 2) was added and the whole mixture was stirred, to which 0.1 mmole of iodine (MW-254 Da) was added. After stirred for Preparation of Czo-Tetrainositol 15 minutes, a 10 mL toluene solution of DBU (0.25 mmol. Compound 10 in FIG. 22 MW=151 Da) was added to the mixture over a period of 10 Step 1. Synthesis of Protected Inositol Malonate minutes, and stirred for two hour. TLC monitored the reaction (Compound 8 in FIG. 22) progress with DCM or toluene? EA (98:2). When complete, the mixture was concentrated to 10 mL (not to dryness), and 0210 10 mmole 1.2:5,6-bis-O-(1-methylethylene)-3-me loaded to the top of a silica gel column for purification. thyl-1D-chiro-inositol was dissolved in 50 mL dry DCM, to Toluene was used first to elute unreacted Czo monoadduct which 12 mmole TEA was added and stirred under nitrogen. (first band), and then the product (second band), which was Then, 5 mmole malonyl chloride was dissolved in 1 ml dry then rotavaped and then pumped under vacuum for overnight DCM, and dropwise added to the above solution within a before NMR and MALDI-MS. Yield: 60%. The obtained the period of 10 minutes. Upon completion of the addition, the tert-butyl ester of ALM was dissolved in DCM and TFA (v:v reaction mixture was stirred for 6 hours. When completed, the 3:1) at 20 mg/mL and stirred at RT for 6 hours. Solvents were reaction mixture was washed with brine twice and the com evaporated and dried under vacuum for overnight to quanti bined organic phase was dried over MgSO filtered, and tatively yield the final product ALM, which was characterized concentrated to 1-2 mL. Flash column with silica gel was by MALDI-MS and NMR. used to purify the products with 20% EA in DCM as the solvents. The product was colorless viscous liquid. Yield: Example 2 55%. Proton and carbon NMR confirmed the structure. Preparation of Czo-Tetraglycolic Acid Depicted in Step 2. Synthesis of Czo-Tetrainositol-Acetal FIG 21 Protected (Compound 9 in FIG.22) TGA or Compound 7: Labeled “LnW0048 in FIG. 0211 84 mg (0.1 mmole) of Co was dissolved in 60 mL 5 toluene. Next, 0.2 mmole of the malonate 8 was added and the mixture was stirred, to which 0.2 mmole of iodine was added. Step 1. Synthesis of di(tert-butylacetoxy)malonate After stirred for 15 minutes, a 10 mL toluene solution of DBU (Compound 4 in FIG. 21) (0.50 mmol) was added to the mixture over a period of 10 0207 To a solution of malonic acid (40.0 mmol. 4.16 g) in minutes, and stirred for 6 hours. When it was complete, the dioxane was added 11.1 mL TEA (80.0 mmol). The mixture reaction mixture was concentrated to 10 mL and loaded to the was stirred for 30 minutes, and then 11.8 mL (80.0 mmol) of top of a silica gel column for purification. DCM was used first tert-butyl bromoacetate in 12 mL dioxane was added and to elute unreacted Co and its monoadduct and then solvent stirred over weekend. TLC monitored the reaction progress was changed to DCM/EA to elute the product, which was until completion. The precipitate was filtered and washed rotavaped and pumped under vacuum for overnight before with ether. The filtrate was then washed with brine twice, NMR and MALDI-MS. dried over MgSO, and concentrated for NMR analysis. Step 3. Synthesis of Co-Tetrainositol (Compound 10 Yield: 75%. in FIG. 22) Step 2. Synthesis of Czo Tetraglycolic Acid 0212 50 mg of the obtained octakis-acetal protected pre Tert-Butyl Ester (Compound 6 in FIG. 21) cursor compound 9 was dissolved in 20 mL 4.0M hydrochlo ride solution in dioxane. A few drops of water was added and 0208 840 mg (1.0 mmole) of Czo was dissolved in 50 mL stirred for 30 hours. Complete deprotection was achieved. o-xylene and sonicated for 3 minutes, and then 200 mL tolu Solvent were removed completely and dried under vacuum ene was added. Next, 2.0 mmole of the malonate 4 was added overnight to yield pure final product, with NMR and MALDI and the whole mixture was stirred, to which 2.0 mmole of MS data confirmed. iodine was added. After stirred for 10 minutes, a 20 mL toluene solution of DBU (5.0 mmole, MW=151 Da, 1.8- Example 4 diazabicyclo5.4.0]undec-7-ene) was added to the mixture Preparation of Co-TEG Acid over a period of 15 minutes, and stirred for 8 hours. TLC was Compound 12 or TTAFIG. 23 used to monitor the reaction progress. When complete, it was concentrated to 40 mL and loaded onto the top of a silica gel Step 1. Synthesis of Di(Tert-Butyl TEG Propionate) column for purification. Toluene was used to remove unre Malonate 11 acted Czo and then DCM was to remove the monoadduct. 0213 10 mmole tert-butyl 12-hydroxy-4,7,10-trioxa Finally a mixture of EA and DCM was to elute the product, dodecanoate was dissolved in 50 mL dry DCM, to which 12 US 2011/025 1158 A1 Oct. 13, 2011

mmole TEA was added and stirred under nitrogen. Then, 5 extracted with ethyl acetate and water. The ethyl acetate layer mmole malonyl chloride was dissolved in 1 ml dry DCM, and was further purified by column chromatography to yield the dropwise added to the above solution within a period of 10 triazole product. minutes. Upon completion of the addition, the reaction mix ture was stirred for 4 hours. When completed, the reaction Step 4. Preparation of bis(tert-butyl hydroxy mixture was washed with brine twice and the combined trioxadodecanoate (Compound 17) organic phase was dried over MgSO4, filtered, and concen 0218 bis(tert-Butyl 12-hydroxy-4,7,10-trioxadode trated to 1-2 mL. Flash column with silica gel was used to canoate)malonate was synthesized by reaction with I, DBU. purify the products with DCM/EA as the solvents. Yield: and Co in Xylene, followed by column chromotography puri 85%. Proton and carbon NMR confirmed the structure. fication. Step 2. Synthesis of Czo-TEG Acid 12 Step 5. Preparation of Triazole Propionic Acid 0214 84 mg (0.1 mmole) of Czo was dissolved in 60 mL Malonamide (Compound 18) toluene. Next, 0.2 mmole of the malonate 11 was added and 0219 Using the C7o mono-adduct from above, I, DBU, the mixture was stirred, to which 0.2 mmole of iodine was and the triazole-containing malonamide, a second adduct added. After stirred for 15 minutes, a 10 mL toluene solution were added to the C7o cage. After 20 minutes of reaction of DBU (0.50 mmol) was added to the mixture over a period under nitrogen at room temperature, the reaction was poured of 10 minutes, and stirred for 6 hours. When it was complete, onto a silica column and purified by chromatography. the reaction mixture was concentrated to 10 mL and loaded to the top of a silica gel column for purification. DCM was used Step 6. Synthesis of Compound 19 first to elute unreacted Czo and its monoadduct and then solvent was changed to DCM/EA to elute the product, which 0220 Finally, deprotection of the t-butyl esters was was rotavaped and pumped under vacuum for overnight achieved by reaction with trifluoroacetic acid in CHCl (1:1) before NMR and MALDI-MS. Yield: 70%. The obtained overnight. Volatiles were removed under reduced pressure to tert-butyl ester of Czo TEG acid was dissolved in DCM and yield the product, 19. TFA (v:v 3:1) at 20 mg/mL and stirred at RT for 8 hours. Solvents were evaporated and dried under vacuum for over Example 6 night to quantitatively yield the final product C7 TEG acid, Preparation of Czo-Tetraniacin which was characterized by MALDI-MS and NMR. Step 1. Synthesis of Czo-Tetrabromide Example 5 0221) Co. (84 mg) was dissolved in anhydrous toluene (50 mL) and the mixture was Sonicated for 2 minutes, to which Preparation of Phenylpropionic Acid-Triazole-Mixed 2-bromoethyl malonate (63.2 mg) and iodine (50.8 mg) was Malonateamide-C7-TEG-COOH added and stirred for 5 minutes under argon. A toluene solu Compound 19 (FIG. 24) tion of DBU (75.5 mg in 10 mL anhydrous and deoxygenated toluene) was added dropwise. The reaction mixture was Step 1. Synthesis of 2-(4-azidomethylphenyl)-propi stirred for additional 5 hours before it was washed with brine. onic acid (compound 14 in FIG. 24) The toluene solution was dried, concentrated and subjected to silica gel column for purification of the co-tetrabromide 0215. To a solution of 2-(4-bromomethylphenyl)-propri using toluene as the eluant. The final compound was dried in onic acid (1.5 g) in 1,4-dioxane was added NaNs (5 g) and vacuum and characterized by MALDI and NMR. 15-crown-5 (100 mg). The mixture was heated to 80° C. for 16 h, then cooled. The solvent was removed under reduced Step 2. Synthesis of Co-Tetraniacin pressure, and the residue chromatographed on silica to yield 2-(4-azidomethylphenyl)-proprionic acid as an off white 0222 Czo-tetrabromide (50 mg) was reacted with exces solid. sive niacin (2 g) (MW-123.1 g/mol, m.p. 235°C.) at 240°C. for 6 hours in a pre-dried flask under argon protection. After Step 2. Preparaton of Acetylene Malonate the reaction was done, the mixture was cooled to room tem (Compound 15) perature and dissolved in a dilute bicarbonate basic water (pH=9.0). The mixture was filtered to remove undissolved 0216) To a separate solution of ethoxy malonyl chloride materials, and the filtrate was thoroughly dialyzed with a was added triethylamine and propargylamine in equal molar 1000 MWCO membrane to remove unreacted niacin. equivalents. The mixture was stirred 20 minutes and purified 0223) The dialyzed products were further purified by crys by column chromatography. tallization. In brief, the dialysate containing the products was adjusted to neutral pH and then concentrated and isopropyl Step 3. Preparation of Triazole Propionic Acid alcohol was slowly added until precipitation occurs. After the (Compound 16) resulting mixture was placed in fridge for 10 hours, the pre cipitates were collected and dried in a vacuum oven. Alterna 0217. A mixture of the acetylene malonate (1 equivalent), tively, Czo-tetraniacin can be synthesized by reacting Co 2-(4-azidomethylphenyl)-propionic acid (1 equivalent), tetrabromide (50 mg) with ethyl pyridine-3-carboxylate CuSO (5 mol%), triethylamine (5 equivalents), and ascorbic (ethyl niacin, 5 mL) at 150° C. for overnight. After cooled to acid (50 mol %) was stirred in 1:1 THF: water overnight room temperature, precipitates were collected and washed following a procedure widely used in the literature. Solvents with cold ethanol. The precipitates were then dried and were removed under reduced pressure and the residue was hydrolyzed in basic water to remove the four ethyl groups to US 2011/025 1158 A1 Oct. 13, 2011 22 afford the Co-tetraniacin. The final product was character dried. Yield is 75%. The product was characterized by ized by NMR, IR and UV-Vis. MALDI and NMR (d6-DMSO as solvent). Example 7 Example 8 Preparation of Czo—(OH)2 Preparation of Czo-(DMAE) 0224 0226

TMSO TMSO N,N-dimethyl amino 2) Acetic acid ethanol He

B.1N-O

HO

le N1 on

HOS-1a n-1No Co-(DMAE)

0227 Czo-tetrabromide (10 mg) was dissolved in deoxy genated DMF (5 mL) and deoxygenated N,N-dimethylami 0225. A solution of NHS (23 mg) and DCC (41 mg) in dry noethanol (10 mL) was added. The mixture was completely DMF was added to a solution of TGA (28 mg) in dry DMF (5 deoxygenated with argon for 30 minutes and reacted 28 hours mL), and stirred for 8 hours. TLC shows complete conversion at 80° C. After the reaction was complete, the mixture was to NHS esters. TMS-protected TRIS in large excess was cooled and dialyzed against DI water with MWCO of 1000 to added to the above mixture and stirred overnight. All solvents remove any non-fullerene materials. The dialyzed product were evaporated under vacuo and the residue was reconsti was dried and characterized by NMR, UV-Vis and MALDI. tuted in ethyl ether and filtered. This step was repeated 3 times UV-Vis shows the characteristic absorption peaks of Co until all byproducts were removed. The ether solution was bisadduct in the range of 400-550 nm, showing no reaction dried and redissolved in a mixture of THF/acetic acid/water occurred between the C7o fullerene cage and tertiary amines and stirred overnight. THF and acetic acid were rotavaped in under the employed conditions. Quantitative yield was vacuo, and the product was collected, washed with THF and obtained. The product has moderate solubility in buffer. US 2011/025 1158 A1 Oct. 13, 2011

Example 9 Preparation of Co (PC) 0228

P-CI 1) O)M O 2) Bromine 3) TMA He

0229. To a solution of Co (OH) (0.7 mmol) which was rotavapored to dryness. The residue was then reconstituted in made reacting Co with bis(2-hydroxylethyl)malonate (2 1:1 DCM and isopropanol. 40% trimethylamine (17 mmol) solution (1 mL) was added and stirred for 2 hours at 0°C. The equivalents) under typical Bingel conditions followed by mixture was stirred at RT for another 12 hours and concen chromatographic purification, in THF (20 mL) cooled with trated. The residue was chromatographed on silica gel with salt-ice bath was added DIPEA (0.5 mL, 2.8 mmol) and DCM/EA/CH-OH as the solvent system. The product was ethylene chlorophosphite (0.2 mL, 2.1 mmol). The reaction eluted as the third band after unreacted and intermediate mixture was stirred for 90 minutes and then neat bromine (0.1 fullerene compounds were removed. NMR shows singlet mL, 2.1 mmol) was added. After 30 min, water (5 mL) was peak at 3.1-3.2 ppm corresponding to the three methyl groups added and stirred with the temperature slowly rising to RT on the quaternary amine as well as methylene protons adja after salt-ice bath was removed. Then DCM (20 mL) was cent to the malonate ester and phosphate esters in the region added and phase separated. The organic phase was of 4.1-4.5 ppm. US 2011/025 1158 A1 Oct. 13, 2011 24

Example 10 Preparation of Co-Tetrasulfonate 0230

C70-tetraDMABM

C70-tetrasulfonate US 2011/025 1158 A1 Oct. 13, 2011 25

0231. Czo-tetral DMABM was made by reacting Czo with N,N-dimethylaminobutyl malonate (DMABM) (2 equiva -continued lents), iodine (2 equiv.) and DBU (Diaza(1,3)bicyclo5.4.0 undecane) (5 equiv.) in toluene under typical Bingel condi tions and purified with neutral alumina column. Co tetraDMABM (0.1 mmol) was subsequently refluxed with excessive 1,3-propane Sulfone (0.8 mmol) overnight in a mix ture of DMF and water. Excessive sulfone was destroyed by adding more TEA and stirring for 2 hours at RT. The final material were purified by dialysis with 1000 MWCO regen erated cellulose tubes against PBS solution, to remove all Small molecule reactants or impurities. The product shows characteristic UV-V is absorption peaks of Czobisadducts, and NMR spectrum conforms to the Zwitterionic structure. Example 11 Preparation of Czo-Tetrapyridine 0232

C70-tetrapyridine

0233 Czo-tetrapyridine was synthesized by reacting Czo tetrabromide (50mg) with neat pyridine (5 mL) at 70° C. for 21 hours in a pressure tube reactor, and the tube remained Pyridine tightly closed during the reaction period. After the reaction Her Heating was complete, the majority of fullerene materials precipitate onto the bottom of the tube. The mixture was centrifuged to remove pyridine solution with light reddish colors, and the precipitates were washed 3 times with toluene and ether respectively, and then dried in vacuo for 6 hours. The product was readily dissolved in both DI water and phosphate buff ered saline with solubility of >2 mg/mL. NMR spectrum in DO revealed the presence of aromatic protons of pyridine with low-field shifts of 0.2-0.6 ppm due to the quaternization of the pyridine nitrogen atom. Co-tetrabromide Example 12 Preparation of Co-Glu 0234

O "Sr." in-1-X O O

1) Co., DBU, I ><------>< Her2) TFA, DCM O O US 2011/025 1158 A1 Oct. 13, 2011 26

-continued 1) O

H

Boc /N o-(-

O OH DCC, NHS Ho 2) HCl, dioxane

C70-tetraamine NH2 HN

OH HO

OH HO

HN

0235 Czo-tetraamine building block was synthesized by Example 13 reacting Czo with Boc-aminoethyl malonate (2 equivalents), Preparation of Co-Tetraphosphate iodine (2 equiv.), and DBU (5 equiv.) in toluene at RT for 7 0236 hours. The product was purified on silica gel column with DCM/EA as the eluant, followed by acidic removal of the Boc groups in a mixture of 20% TFA in DCM for 4 hours at RT to quantitatively yield the TFA salt of the C-tetraamine com pound, which (0.1 mmol, neutralized with DIPEA) was sub sequently added to and reacted with a premade solution of Boc-Glu-OBut (0.5 mmol), DCC (0.5 mmol and NHS (0.5 Phosphoryl mmol) in anhydrous THF. The mixture was stirred overnight chloride and the desired product was purified by silica gel column - THF - - using DCA/EA/MeOH as the solvent. The product was char acterized by MALDI-MS and NMR. It was then re-dissolved in a 4.0M HCl dioxane solution to remove the Boc and tert butyl protecting groups to generate compound Co-Glu. NMR of the final compound confirmed the complete removal of all Boc and butyl groups. This method can be used to synthesize a number of Czo amino acid derivatives starting with different partially protected natural amino acids which C70-tetraamine can be obtained from Advanced Chemtech Inc. US 2011/025 1158 A1 Oct. 13, 2011 27

1. A method for treating an inflammatory disease, compris -continued ing administering to a subject in need thereof a therapeuti cally effective amount of a synthetically modified fullerene of the formula

Z-F Y, wherein F is a fullerene of formula C, or X(aC, the fullerene having two opposing poles and an equatorial region; C, represents a fullerene cage having p carbon atoms, and X(aC, represents such a fullerene cage having a chemi cal group X within the cage; ZandY are positionednearrespective opposite poles of C: m is an integer of from 1 to 5 and Z is a hydrophilic, lipophilic, or amphiphilic moiety; n is an integer of from 1 to 5 and Y is a hydrophilic moiety; p is an even number between 60 and 200; and X, if present, represents one or more metal atoms within the fullerene (F), optionally in the form of a trinitride of formula G, H, -N in which G and H are metal atOmS. C70-tetraphosphate 2. The method of claim 1, wherein p is an even number between 60 and 96. 3. The method of claim 2, wherein p is 60 or 70. 4. The method of claim 1, wherein said synthetically modi fied fullerene is a prolate ellipsoid shaped fullerene having a major axis such that said poles are located at opposing ends of the major axis of the prolate ellipsoid fullerene. 5. The method of claim 1, wherein said synthetically modi 0237) TFA salt of the C-tetramine compound (0.1 mmol) fied fullerene is spheroid with opposing poles defined by an was dissolved in anhydrous TFA, and neutralized with axis through opposing carbon rings. DIPEA (diisopropylethylamine) (0.4 mmol). The in situ gen 6. A method for treating an inflammatory disease, compris erated amine groups were reacted with phosphorous oxychlo ing administering to a subject in need thereof a therapeuti ride (phosphoryl chloride) (50 mmol) for 4 hours at RT under cally effective amount of a synthetically modified fullerene of argon. After the reaction was completed, the reaction mixture the formula was cooled with a salt/ice bath and water was dropwise added to neutralize all unreacted phosphorous chloride and convert the remaining P Cl bonds in the product to P OH. The wherein p is an even number between 60 and 200; Y is a hydrophilic moiety covalently connected to C, option final product was dissolved in basic carbonate solution and ally through a linking group, at or near a pole thereof; dialyzed with MWCO 1000 for 8 hours to remove phosphoric and Z is a hydrophilic, lipophilic, or amphiphilic moiety acid salts. The final product can be dissolved in slightly basic covalently connected to C, optionally through a linking aqueous solution and it remained dissolved when the pH was group, at or near a pole opposite to said Y. adjusted to 7.4. NMR shows the conversion of amines to 7. The method of claim 6, wherein C is Co. phosphamide. 8. The method of claim 6, wherein Z comprises at least one 0238 While various embodiments have been particularly -(CH2)AH, or (OCH2CH2), OCH moiety, wherein q is shown and described herein, it will be understood by those an integer of from 5 to 17 and w is an integer of from 1 to 6. skilled in the art that various changes inform and details may 9. The method of claim 6, wherein: (a) Z comprises at least be made therein without departing from the spirit and scope of one niacin moiety at a free end thereof; (b) Z comprises at least one —C(O)O— moiety; (c) Y comprises at least one these embodiments as further defined by the appended niacin moiety at a free end thereof; or (d)Y comprises at least claims. one —C(O)O— moiety. 0239) All of the above-mentioned references are herein 10. The method of claim 6, wherein: (a) Z comprises two incorporated by reference in their entirety to the same extent niacin moieties at two free ends thereof; (b) Z comprises two as if each individual reference was specifically and individu —C(O)O— moieties; (c)Y comprises two niacin moieties at ally indicated to be incorporated herein by reference in its two free ends thereof; or (d)Y comprises at least two —C(O) entirety. O— moieties. US 2011/025 1158 A1 Oct. 13, 2011 28

11. The method of claim 6, wherein the synthetically modi fied fullerene is selected from the group consisting of

HO

OH

OH OH

HO

HO

OH Co-(OH)12

Co-(DMAE) US 2011/025 1158 A1 Oct. 13, 2011 29

-continued COOH

COOH

Co-Niacin

US 2011/025 1158 A1 Oct. 13, 2011 30

-continued

Co-tetrasulfonate

C70-tetrapyridine US 2011/025 1158 A1 Oct. 13, 2011 31

-continued

OH US 2011/025 1158 A1 Oct. 13, 2011 32

-continued

ALM

US 2011/025 1158 A1 Oct. 13, 2011 33

-continued

C70-tetraphosphate

US 2011/025 1158 A1 Oct. 13, 2011 34

-continued OH OH HOtti,

and

C70-tetrainositol

- QC -

HO / N N \ OH C60-ethanolamine

12. The method of claim 6, wherein the inflammatory C, represents a fullerene cage having p carbon atoms, and disease is inflammatory arthritis or an allergic disease. X(a)C represents such a fullerene cage having a chemi 13. A method of inhibiting build-up of arterial plaque, cal group X within the cage; comprising administering a therapeutically effective amount of one or more fullerenes to a subject in need thereof. ZandY are positionednear respective opposite poles of C: 14. The method of claim 13, wherein said fullerenes inhibit m is an integer of from 1 to 5 and Z is a hydrophilic, accumulation of LDL in foam cells of the subject. lipophilic, or amphiphilic moiety; 15. The method of claim 13, wherein said fullerenes are n is an integer of from 1 to 5 and Y is a hydrophilic moiety; delivered directly to the foam cells of the subject. p is an even number between 60 and 200; and 16. The method of claim 13, wherein said subject is a X, if present, represents one or more metal atoms within the human. fullerene (F), optionally in the form of a trinitride of 17. The method of claim 13, wherein at least one of said one formula G, H, -N in which G and H are metal or more fullerenes is a synthetically modified fullerene of the atOmS. formula 18. The method of claim 17, wherein p is 60 or 70. Z-F Y, 19. The method of claim 17, wherein at least one of said one wherein F is a fullerene of formula C or X(aC, the or more fullerenes is a synthetically modified fullerene of the fullerene having two opposing poles and an equatorial formula region; US 2011/025 1158 A1 Oct. 13, 2011

wherein p is an even number between 60 and 200; Y is a 22. The method of claim 19, wherein: (a) Z comprises at hydrophilic moiety covalently connected to C, option least one niacin moiety at a free end thereof; (b) Z comprises ally through a linking group, at or near a pole thereof, at least one —C(O)O— moiety; (c)Y comprises at least one and wherein Z is a hydrophilic, lipophilic, or niacin moiety at a free end thereof; or (d)Y comprises at least amphiphilic moiety covalently connected to C, option one —C(O)O— moiety. ally through a linking group, at or near apole opposite to 23. The method of claim 19, wherein: (a) Z comprises two said Y. niacin moieties at two free ends thereof; (b) Z comprises two 20. The method of claim 19, wherein C is Co. —C(O)O— moieties; (c)Y comprises two niacin moieties at 21. The method of claim 19, wherein Z comprises at least two free ends thereof; or (d)Y comprises at least two —C(O) one-(CH2)CH or (OCH2CH2), OCH moiety, wherein O— moieties. q is an integer of from 5 to 17 and w is an integer of from 1 to 24. The method of claim 19, wherein the synthetically 6. modified fullerene is selected from the group consisting of

HO

OH

OH OH

HO

HO

OH

OH

Co-(DMAE) US 2011/025 1158 A1 Oct. 13, 2011 36

-continued

COOH

COOH

Co-Niacin

US 2011/025 1158 A1 Oct. 13, 2011 37

-continued

Cio-tetrasulfonate

Co-tetrapyridine US 2011/025 1158 A1 Oct. 13, 2011 38

-continued

OH US 2011/025 1158 A1 Oct. 13, 2011 39

-continued

ALM

US 2011/025 1158 A1 Oct. 13, 2011 40

-continued

C70-tetraphosphate

US 2011/025 1158 A1 Oct. 13, 2011 41

-continued

OH OH HOit.

and

HO HO Co-tetrainositol

HO

OH

NH

HN NH

C60-ethanolamine

25. A synthetically modified fullerene of the formula ZandY are positionednear respective opposite poles of C: m is an integer of from 1 to 5 and Z is a hydrophilic, Z—F Y, lipophilic, or amphiphilic moiety; wherein F is a fullerene of formula C, or X(aC, the n is an integer of from 1 to 5 and Y is a hydrophilic moiety; fullerene having two opposing poles and an equatorial p is an even number between 60 and 200; and region; X, if present, represents one or more metal atoms within the C, represents a fullerene cage having p carbon atoms, and fullerene (F), optionally in the form of a trinitride of X(a)C represents such a fullerene cage having a chemi formula G, H, -N in which G and H are metal cal group X within the cage. atoms, US 2011/025 1158 A1 Oct. 13, 2011 42

wherein: (a)Z comprises at least one niacin moiety at a free —C(O)O— moieties; (c)Y comprises two niacin moieties at end thereof; (b) Z comprises at least one —C(O)O— two free ends thereof; or (d)Y comprises at least two —C(O) moiety; (c)Y comprises at least one niacin moiety at a O— moieties. free end thereof; or (d)Y comprises at least one —C(O) O— moiety. 26. The method of claim 25, wherein: (a) Z comprises two 27. A compound selected from the group consisting of is niacin moieties at two free ends thereof; (b) Z comprises two selected from the group consisting of

HO

OH

OH OH

HO

HO US 2011/025 1158 A1 Oct. 13, 2011 43

-continued

COOH

HOOC

COOH

Co-Niacin

US 2011/025 1158 A1 Oct. 13, 2011 44

-continued

Cio-tetrasulfonate

Co-tetrapyridine US 2011/025 1158 A1 Oct. 13, 2011 45

-continued

OH

and