US 2011 01 65096A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2011/0165096 A1 Liu et al. (43) Pub. Date: Jul. 7, 2011

(54) FICUS EXTRACTS HAVING ANGIOGENESIS Publication Classification HIBITING ACTIVITY AND METHODS OF (51) Int. Cl. SOLATING AND USING THE SAME A6IR 9/12 (2006.01) A636/00 (2006.01) CI2O 1/02 (2006.01) (75) Inventors: Zhijun Liu, Baton Rouge, LA A6IP35/00 (2006.01) (US); Eugene A. Woltering, A6IP3L/00 (2006.01) Kenner, LA (US) A6IP3L/04 (2006.01) A6IP37/00 (2006.01) A6IP27/02 (2006.01) (73) Assignee: ULLMAN MEDICAL, INC., A6IP 9/02 (2006.01) Silver Spring, MD (US) A6IP3/10 (2006.01) A6IP 7/02 (2006.01) A6IP 7/06 (2006.01) A6IP3/04 (2006.01) (21) Appl. No.: 13/058,957 A6IP4/00 (2006.01) A6IP 9/12 (2006.01) A6IP 9/00 (2006.01) (22) PCT Filed: Aug. 14, 2009 A6IPI/00 (2006.01) (52) U.S. Cl...... 424/45; 424/725; 435/29 (86). PCT No.: PCT/USO9/53860 (57) ABSTRACT The present invention is directed to methods for extracting S371 (c)(1), and isolating extracts having angiogenesis inhibiting activity (2), (4) Date: Mar. 17, 2011 from a latex-containing portion of a Ficus variant, pharma ceutical and nutraceutical compositions comprising the extracts, methods of administering the extracts to treat angio Related U.S. Application Data genesis-dependent diseases and to reduce or inhibit neovessel growth in a Subject in need thereof, and the use of the extracts (60) Provisional application No. 61/088.924, filed on Aug. in the manufacture of a composition for reducing or inhibiting 14, 2008. neovessel growth. Patent Application Publication Jul. 7, 2011 Sheet 1 of 23 US 2011/O165096 A1

100

FIG. 1 Patent Application Publication Jul. 7, 2011 Sheet 2 of 23 US 2011/O165096 A1

Discard Solids S/S 22

225 M liquid Extract

24

are - P -- a Organic Fraction (F) Y242

Aqueous Extract (F) \ 43

Diluted Aqueous Extract

Column Fractionation

Eluted with DE HO (Fo) 27

Eluted with EtOH:HO (Fass) \)72

FIG. 2 Patent Application Publication Jul. 7, 2011 Sheet 3 of 23 US 2011/O165096 A1

300

312

31

FIG. 3A FIG. 3B Patent Application Publication Jul. 7, 2011 Sheet 4 of 23 US 2011/O165096 A1 4OO

Mean Angiogenic Index = 1+1+1+1 = 4 FIG. 4A

Mean Angiogenic Index = 4+4+4+4 = 16 FIG. 4B Patent Application Publication Jul. 7, 2011 Sheet 5 of 23 US 2011/O165096 A1

Mean Angiogenic Index (Ficus carica L., variety kadota, day 14) (series 1A) DExcluding Zeros DIncluding Zeros

d s 3 3 38 g 3 E 3 E st S S SS2 S. S. C. S. C. S. w CN o S CN o S (NE (NE da o da C) 8 C N). C C N C C N1 N1 a E a E as CD go 8 cc E S C8 d). g 3- it c. it c. d d Treatment FIG. 5A Angiogenesis Initiation (%) 80 (Ficus carica L., variety kadota, day 14) (series 1A)

70 – 60 - 9. 50 –

S. 30 —

is 20 ------

10 – O 9 £ 35, 35 S.F S.F D - - s s - N N CN E CN E cN SC) cN EC. CC N4 W- N1 cy a N1 Na N1 O CD o o O) Co Oc C. v . C. c. 3-es ifC S ifC S H- O H- C C O Treatment FIG. 5B Patent Application Publication Jul. 7, 2011 Sheet 6 of 23 US 2011/O165096 A1

Mean Angiogenic Index 11 (Ficus carica L., variety kadota, day 14) (series 1B)

------DIncluding(IDExcluding Zeros Zeros

g 3 - S - 3 3 S 3 s 58 C w CN o S CN o S C E N CN c es CD go 8 cc E o O3 d g 3. in c. in c. O O Treatment F.G. 6A Angiogenesis initiation (%) 70 ------(Ficus carica L., variety kadota, day 14) (series 1B).

60 - Initiati

to 50 40 30 - S. 20 - 10 O –

s- St3 E- S3 2 - S.3 SS. SR3 5 SESS SS38 8 CC N2 R S S2 C. S & N1 N2 E S2 es CD go 8 cc odd O3 g . o st- it c ic O C Treatment F.G. 6B Patent Application Publication Jul. 7, 2011 Sheet 7 of 23 US 2011/O165096 A1

Mean Angiogenic Index (Ficus carica L., variety kadota, day 14) (series 1C) DExcluding Zeros DIncluding Zeros

5

C CC CC cd CO - CO - 9 "as 9 - 9 - - & E SE O E O E C c 8 CN go S CN go S S S cN 9) cN 9) 3 N. C. S & C S N) Y S. E S. E as go E go E is .95 g St. CD in c. - CS C C Treatments

Angiogenesis Initiation (%) (Ficus carica L., variety kadota, day 14) (series 1C) Initiation

9 C5 COS SCO SE SE (Ogo -8 COgo -8 E < e SS E S 3 S 2 S 2 SS, SS 3 S. S N. C. raS. Na Q S.9 S.N) SSY. N2 E & E St.ea CD go g g os 23cs g g g Treatment FIG. 7B Patent Application Publication Jul. 7, 2011 Sheet 8 of 23 US 2011/O165096 A1

Mean Angiogenic Index (Ficus carica L., variety kadota, day 14) (series 1D) 3 |Excluding Zeros DIncluding Zeros 2

1

O d s InS S SE L.SE SE SE t E 9 W- Scy O Gs O Gs O S. O S. w- & S & SS Sg Sg N. O. N to 8 a. a S. g. a g a N2 E Na E as (5 E () E (p & D8 (p (p 2 3t - C - O C C Treatments FIG. 8A Angiogenesis initiation (%) 30 (Ficus carica L., variety kadota, day 14) (series 1D)

25 - Initiation

20------15 - d 10 al

O s g g E gE SE SE SE S SS, SS SS SS SS SS O - cN S CN S E SE Na E & E C k N. W. M cy) - cy) - cy 1. CD isOS isC 9. it9 C.c isop C it 2 itS2' Ye' C P Treatments FI G Patent Application Publication Jul. 7, 2011 Sheet 9 of 23 US 2011/O165096 A1

Mean Angiogenic Index (Ficus carica L., variety kadota, day 14) (series 1E) T Excluding Zeros DIncluding Zeros

234.5671 O C C CC CC C - CC - 9 C C as E as 8 S E S E w-C - &92 - Sc. &92 38cyd SgC is SgC is oCN Sto oCN Sto 8--a CDCC YgDo goa 2 gE gasa opY 8 S^p. S N.p S2 3t - - C C C C Treatments FIG. 9A Angiogenesis initiation (%) 7 O (Ficus carica L., variety kadota, day 14) (series 1E) 1234.56 O O s SSto E SScd E cdSS cdSS S.CO -S S.CO -S OOOOOO K- C cod C cod S. S. S as o St. o St. 3 < a - Yoo N1 Sa E Sa E --- CD CDS (DS 9 Sv 9 S 9 9 ar' - CS- - CS Co - C Treatments FIG. 9B Patent Application Publication Jul. 7, 2011 Sheet 10 of 23 US 2011/O165096 A1

Mean Angiogenic Index (Ficus carica L., variety gold, day 14) (series 2A) (IDExcluding Zeros O Including Zeros

C CC CO O CO CO - (O - 9E "as &3 - S- &3 38- SSN 9 SSN 2 &SS ds &SS S. OO E 8 CD r 2 CD 2 s CD S CD CD E CD E as is E E SC 92 3. CD c. c. C C Treatments FIG. 10A Angiogenesis Initiation (%) 70 (Ficus carica L., variety gold, day 14) (series 2A)

60 -

50 - 9. S 40 E 30 9 20 10 -

O o g8 g8 SS SE SE SE w K- c) O O) O C S.N s. SOn as coc asto CCN - OS E2 D E NDg E o E S 8E Y . C2E 1. s pa pa ics in c. C ics Ya-Y CD - c. - c. Treatments FIG. 10B Patent Application Publication Jul. 7, 2011 Sheet 11 of 23 US 2011/O165096 A1

Mean Angiogenic Index

Patent Application Publication Jul. 7, 2011 Sheet 12 of 23 US 2011/O165096 A1

Mean Angiogenic Index 7 (Ficus carica L., variety gold, day 14) (series 2C) |Excluding Zeros DIncluding Zeros

c s 8 8 - - - sts v- CDcD 3S S c 3C S dCD3E S, 93ECD S, OSSS OSS) O CDk S2us c 8s is2 c. 89 LS2 SO 89 LS 8 asN E on5 E E S S CN CN Treatments FIG. 12A Angiogenesis Initiation (%) 40 - (Ficus carica L., variety gold, day 14) (Series 2C)

o 3 SE SE g E SE SE SE E - S2 S 2 se S 5.5, 55. 3 is CD S. O O CD2 c. CD(2 v . CD2. c. 1. 2 C. 9 c. CD - C - O Y- - c. - c.5 O L. C. - - Treatments FIG. 12B Patent Application Publication Jul. 7, 2011 Sheet 13 of 23 US 2011/O165096 A1

Mean Angiogenic Index 10 ------(Ficus carica L., variety magnolia, day 14) (series 3A) |Excluding Zeros DIncluding Zeros

cE s > 3d > 38 E SEe- as8- > S SE > 3 SE o C (DS, CDS, CD se S CD so 9s 92 9. CD S S. L. S. 8 LS 8 S. S. -- C C CN CN Ye- CN CN Treatments FIG. 13A Angiogenesis initiation (%) 70 - (Ficus carica L., variety magnolia, day 14) (Series 3A)

60 50 9.

40 ------D ------

30 - 9.is 20 - 10 –

O 9 5d Scd S cdS S SCO -S SCO S S(O -E SE(O - 8E aCw &> 2- & co2 &> 2 &2 2 &> 82 SB> 8 3.es CD CD 3s (D3C. 9LL &C 9LL C 9 9 Treatments FIG. 13B Patent Application Publication Jul. 7, 2011 Sheet 14 of 23 US 2011/O165096 A1

Mean Angiogenic Index (Ficus carica L., variety magnolia, day 14) (series 3B) IDExcluding Zeros DIncluding Zeros

1 O c s s 8 as - 8- s s C St -s > Scd E > SC SE CD 38c S, CD SEc. 5 CD SC28 OS28 9 O CD ig E if g E L S 8 is E L S. S 2. C Cd (N N Y- cy CN Treatments FIG. 14A Angiogenesis Initiation (%) 1OO (Ficus carica L., variety magnolia, day 14) (series 3B)

90 – 80 -

7O ------C

is 60 ------E 50 - s 40 – 30 a 20

10 – ------O s cd E co E CC C (O - (O - 9E 5- c\ S c\ S cy S2 N S2 (\ CN 3 a. e - co > 2 > 8 s a- (D (DS (DS CD e CD & CD CD 2 t ii i u c ill d is to Treatments FIG. 14B Patent Application Publication Jul. 7, 2011 Sheet 15 of 23 US 2011/O165096 A1

Mean Angiogenic Index (Ficus carica L., variety magnolia, day 14) (series 3C) IDExcluding Zeros DIncluding Zeros

o is is 2 SE3 SS 8SE > S S. s3.S 9.O CD osg g osg g g (2is g 8 (2g2LS 8 5.CN 2 2S2CN -- O C CN CN Ye- CN N Treatments FIG. 15A Angiogenesis Initiation (%)

1OO - (Ficus carica L., variety magnolia, day 14) (series 3C)

90 ------80 70 - 9. is 60

E 50 Hir ------E 40 - 30 - 20 10 - O s C to F C - CO - O - CO - SC s 5 (N S.a On S. S S.2 S S.2 CN EC. SSCN C 8 35 a w > co > 2 > 8 is 8 es (DS CD3 CD C. CD C. CD CD 2 t c. C. LL C C d o Treatments FIG. 15B Patent Application Publication Jul. 7, 2011 Sheet 16 of 23 US 2011/O165096 A1

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18 b. 82a 185 s S. 814a ty 1814b J

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FIG. 18 Patent Application Publication Jul. 7, 2011 Sheet 19 of 23 US 2011/O165096 A1

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FIG. 19 Patent Application Publication Jul. 7, 2011 Sheet 20 of 23 US 2011/O165096 A1

Patent Application Publication Jul. 7, 2011 Sheet 21 of 23 US 2011/O165096 A1

OVERALL ANGIOGENICEFFECT FIG O Control A Fig 1 mg/ml — Linear (Control) — Linear (Fig 1 mg/ml) y = 0.3241x + 1.5817 y = 0.0768x - 0.1018 10 R 2-= 0.9462 R 2= 0.1459

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FIG. 21 Patent Application Publication Jul. 7, 2011 Sheet 22 of 23 US 2011/O165096 A1

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FICUS EXTRACTS HAVING ANGIOGENESIS during reproduction, e.g., menstrual cycle and pregnancy). In IHIBITING ACTIVITY AND METHODS OF contrast, the process of angiogenesis, the proliferation and ISOLATING AND USING THE SAME development of new blood vessels, often occurs in wound healing and in pathological processes, e.g., tumor growth. CROSS-REFERENCE TO RELATED Angiogenesis is a complex process involving many stages, APPLICATIONS including extracellular matrix remodeling, endothelial cell 0001. This Non-Provisional Patent Application claims the migration and proliferation, capillary differentiation, and benefit of U.S. Provisional Patent Application No. 61/088, anastomosis. All detectable solid tumors (tumors over 2 mm 924, filed on Aug. 14, 2008, and which is hereby incorporated in diameter, a size reflecting the limit of simple diffusion to by reference in its entirety. supply cells with oxygen and nutrients or to remove wastes) exploit angiogenesis to supply the needed blood to prolifer FIELD OF THE INVENTION ating tumor cells. Studies have demonstrated that the level of vascularization in a tumor is strongly associated with 0002 The present invention is directed to methods for metastasis in melanoma, breast, and lung carcinomas. See R. extracting and isolating extracts having angiogenesis inhib Bicknell, “Vascular targeting and the inhibition of angiogen iting activity from the latex-containing portion of the Ficus esis.” Annals of Oncology, vol. 5, pp. 45-50 (1994). genus, pharmaceutical dosage forms and nutraceutical com 0006 Angiogenesis inhibitors have been suggested to positions comprising the Ficus extracts, and methods of intervene into neoplastic processes. See G. Gasparini, “The administering the Ficus extracts to treat angiogenesis-depen rationale and future potential of angiogenesis inhibitors in dent diseases and inhibit or reduce neovessel growth in Sub neoplasia.” Drugs, vol. 58, pp. 17-38 (1999). The inhibitory jects in need thereof. agents block angiogenesis, thereby causing tumor regression in various types of neoplasia. Known therapeutic candidates DESCRIPTION OF RELATED ART include naturally occurring angiogenic inhibitors (e.g., 0003. The search for effective and less toxic treatments for angiostatin, endostatin, platelet factor-4), specific inhibitors cancer and cancer cell proliferation is an ongoing area of of endothelial cell growth (e.g., TNP-470, thalidomide, inter intense research. One promising area of research is the devel leukin-12), agents that neutralize angiogenic molecules (e.g., opment of anti-tumor agents that inhibit tumor angiogenesis, antibodies to fibroblast growth factor or vascular endothelial a necessary step for tumors and their metastases to grow growth factor), Suramin and its analogs, tecogalan, agents that beyond a microscopic size. Angiogenesis, the process of neutralize receptors for angiogenic factors, agents that inter blood vessel growth, occurs not only in the development, fere with vascular basement membrane and extracellular reproduction and repair of new tissue, but also in tumor for matrix (e.g., metalloprotease inhibitors, angiostatic steroids). mation in cancers and a variety of other non-neoplastic dis and anti-adhesion molecules (e.g., antibodies such as anti eases such as autoimmune disorders, age-related macular integrin alpha V beta 3). See L. Rosen, 'Antiangiogenic strat degeneration and atherosclerosis. Such "pathological' angio egies and agents in clinical trials. Oncologist, Vol. 5. Supple genesis can persist for years, and therefore active agents that ment 1, pp. 20-27 (2000). target angiogenesis inhibition provide the possibility for low 0007 Pathogenic angiogenesis occurs when improper dose, local treatment that can both stop the growth of existing control of angiogenesis causes either excessive or insufficient tumors and inhibit future tumor growth. blood vessel growth. Excessive blood vessel proliferation in 0004 Mechanisms of angiogenesis inhibition typically cancer-related conditions favors tumor growth and develop focus on inhibition of vascular endothelial growth factor ment of distant metastases. In other diseases, it is the root (VEGF) secretion and/or VEGF binding at its receptor cause of tissue injury, including blindness associated with (VEGFR2) and its binding to neuropilin on endothelial cells. proliferative retinopathies, skin disorders such as psoriasis, Known angiogenesis inhibiting active agents include Beva and rheumatoid arthritis. Diseases that have been associated cizumab (AVAsTINR; Genentech, Inc., South San Francisco, with neovascularization include, for example, Crohn's dis Calif.), Bortezomib (VELCADER); Millennium Pharmaceu ease, diabetic retinopathy, macular degeneration, obesity, ticals, Inc., Cambridge, Mass.), Erlotinib (TARCEVAR: OSI corneal neovascularization, malignant tumor growth beyond Pharmaceuticals, Inc., Melville, N.Y.), Pegaptainib (MACU 2 mm diameter, benign tumors, benign functional endocrine GENR: OSI Pharmaceuticals, Inc.), Ranibizumab (LucFN tumors, hemangioma, arterial/venous malformations, sickle Tis(R; Genentech, Inc.), Sorafenib (NEXAVAR(R); Bayer cell anemia, sarcoidosis, syphilis, pseudoxanthoma elasti Aktiengesellschaft, Leverkusen-Bayerwerk, Germany), cum, Paget’s disease, vein occlusion in the eye, infections of Lenalidomide (REVLIMIDR); Celgene Corp., Summit, N.J.), the retina, primary hyperparathyroidism, secondary hyper and Sunitinib (SUTENTR); Pfizer, Inc., New York, N.Y.). parathyroidism, tertiary hyperparathyroidism, artery occlu However, for the most part these active agents are biological sion, carotid obstructive disease, chronic uveitis/vitritis, molecules (i.e., polypeptides) that must be prepared using mycobacterial infections, Lyme disease, systemic lupus costly biotechnology manufacturing and purification pro erythematosis, psoriasis, retinopathy of prematurity, Eales cesses. The high cost and difficulty in manufacturing angio disease. Behcet’s disease, infections causing retinitis or chor genesis inhibiting agents could be greatly reduced by identi oiditis, presumed ocular histoplasmosis. Best's disease, myo fying natural products having angiogenesis inhibiting activity pia, optic pits, Stargardt’s disease, pars planitis, chronic reti that can be readily isolated from a plant source. nal detachment, hyperviscosity syndrome, toxoplasmosis, 0005. In adults, two types of blood vessels can potentially trauma, rheumatoid arthritis, and post-laser laser complica be found. The normal blood vessel is a resting, quiescent, tions. Other angiogenic-related diseases may include, for fully-developed vessel. A second form, a proliferating or example, diseases associated with rubeosis (neovasculariza developing blood vessel, occurs rarely during the normal tion of the angle), and diseases caused by abnormal prolifera human life cycle (occurring only in early development and tion of fibrovascular or fibrous tissue, including all forms of US 2011/01 65096 A1 Jul. 7, 2011 proliferative vitreoretinopathy. Any disease having a known a critical size, the immunological system is no longer able to angiogenic counterpart could potentially be treated with an effectively destroy the tumor; i.e., there is a negative correla anti-angiogenic factor, e.g., psoriasis. See D. Creamer et al., tion between tumor size and immune competence. See A. K. “Overexpression of the angiogenic factor platelet-derived Eerola et al., “Tumour infiltrating lymphocytes in relation to endothelial cell growth factor/thymidine phosphorylase in tumour angiogenesis, apoptosis.” Lung Cancer, Vol. 26, pp. psoriatic epidermis. Br. J. Dermatol., vol. 137, pp. 851-855 73-83 (1999); and F. A. Wenger et al., “Tumor size and (1997). lymph-node status in pancreatic carcinoma is there a cor 0008 Angiogenesis is a prominent contributor to solid relation to the preoperative immune function?” Langenbecks tumor growth and the formation of distant metastases. Several Archives of Surgery, vol. 384, pp. 473–478 (1999). Early experimental studies have concluded that primary tumor adjuvant use of an effective anti-angiogenic agent to preclude growth, tumor invasiveness, and metastasis all require development of tumor metastases beyond 1 to 2 mm may neovascularization. The process of tumor growth and allow more effective tumor attack and control by the body’s metastasis is complex, involving interactions among trans immunological mechanisms. In addition, prolonged adjuvant formed neoplastic cells, resident tissue cells (e.g., fibroblasts, use of a non-toxic angiogenic inhibitor may prevent tumor macrophages, and endothelial cells), and recruited circulating dissemination by blocking the growth of vessels required for cells (e.g., platelets, neutrophils, monocytes, and lympho the transport of tumor cells that would form metastatic foci. cytes). A possible mechanism for the maintenance of tumor 0012 Angiogenesis has also been implicated in obesity. growth is an imbalance, or disregulation, of stimulatory and Several mice strains, both young and aged animals, used as inhibitory growth factors in and around the tumor. Disregu obesity models treated with anti-angiogenic agents lost lation of multiple systems allows the perpetuation of tumor weight. See M. A. Rupnicket al., Adipose tissue mass can be growth and eventual metastasis. Angiogenesis is one of many regulated through the vasculature.” PNAS, vol.99, pp. 10730 systems that is disregulated in tumor growth. In the past it has 10735 (2002). This same study also found that adipose tissue been difficult to distinguish between disregulation of angio mass was reduced by the anti-angiogenic compounds. genesis and disregulation of other systems affecting a devel 0013 Angiogenesis has also been implicated in psoriasis. oping tumor. Another complicating factor is that aggressive Psoriasis is a chronic inflammatory skin disease characterized human melanomas mimic vasculogenesis by producing chan by exaggerated keratinocyte proliferation. A current para nels of patterned networks of interconnected loops of extra digm indicates that psoriasis is driven by T cell-mediated cellular matrix, in which red blood cells, but not endothelial immune responses targeting keratinocytes. However, psoria cells, are detected. See A. J. Maniotis et al., “Vascular channel sis cannot be explained solely on the basis of T-cell activation, formation by human melanoma cells in vivo and in vitro: and it is likely that intrinsic alterations in epidermal kerati Vasculogenic mimicry. Am. J. Pathol. Vol. 155, pp. 739-52 nocytes play a very relevant role in disease expression. In (1999). These channels may facilitate perfusion of tumors, particular, keratinocytes may be important in initiating, Sus independent of perfusion from angiogenesis. taining, and amplifying the inflammatory responses by 0009. A tumor cannot expand beyond approximately 2 expressing molecules involved in T-cell recruitment, reten mm in diameter without a blood Supply to provide nutrients tion, and activation. Keratinocytes are also a relevant Source and remove cellular wastes. Tumors in which angiogenesis is of growth factors for angiogenesis. In fact, a hallmark of important include Solid tumors, and benign tumors including psoriatic skin is the Substantial transformation of the local acoustic neuroma, neurofibroma, trachoma, and pyogenic microvascular system, showing dilatation and tortuosity of granulomas. Inhibiting angiogenesis could halt the growth capillaries, increased permeability, and high endothelial and potentially lead to regression of these tumors. Angiogenic Venule formation. factors have been reported as being associated with several 0014. In psoriasis, an abundance of blood vessels is Solid tumors, including rhabdomyosarcoma, retinoblastoma, present in the papillary dermis, showing microvascular Ewing's sarcoma, neuroblastoma, and osteosarcoma. changes such as pronounced dilatation and tortuosity. Expan 0010 Angiogenesis has also been associated with some sion of the microvascular dermal plexus is believed to be non-Solid tumors, including blood-born tumors such as leu mediated by angiogenesis, which is an active vasoprolifera kemias, various acute or chronic neoplastic diseases of the tive process. It has been suggested that VEGF, which has been bone marrow marked by unrestrained proliferation of white shown to be dramatically elevated in human psoriatic skin, blood cells, usually accompanied by anemia, impaired blood might play a causative role in the vascular changes seen in this clotting, and enlargement of the lymph nodes, liver, and disease and also in epidermal and inflammatory alterations. spleen. It is believed that angiogenesis may play a role in the Recently, vascular endothelial growth factor receptors (VEG abnormalities in the bone marrow that give rise to leukemias FRs, including VEGFR-1, VEGFR-2 and VEGFR-3) were and multiple myelomas. found to be expressed in normal human epidermis and asso 0011 Anti-angiogenic factors inhibit tumor growth ciated with proliferation and migration of keratinocytes. beyond 2 mm in diameter by inhibiting the angiogenic Overexpression of VEGFR-1, VEGFR-2 and VEGFR-3 in response and thus inhibiting blood vessel growth to the tumor. psoriatic epidermis was demonstrated both at mRNA and Althoughangiogenesis in a tumor may beginatan early stage, protein levels in vitro, suggesting that VEGFRs are overex a tumor requires a blood Supply to grow much beyond about pressed in lesional psoriatic epidermal keratinocytes. 2 mm in diameter. Up to 2 mm diameter, tumors can Survive 0015 New anti-angiogenic factors are needed, in particu by obtaining nutrients and oxygen by simple diffusion. Most lar, compounds that not only inhibit new angiogenic growth, anti-angiogenic factors are not cytotoxic, i.e., capable of kill but also that degrade existing capillary networks. Very few ing the tumor cells directly. Small tumors of a size about 1 anti-angiogenic factors have been reported to diminish exist mm can be effectively inhibited and destroyed by factors, ing capillary networks. either endogenous or exogenous, that stimulate the immune 0016. The latex (i.e., milksap, resin, or sap) of many spe system. It is generally accepted that once a tumor has reached cies of the genus Ficus exhibit strong pharmacological effects US 2011/01 65096 A1 Jul. 7, 2011

in man and animal models, including antitumor and/or cyto of a Ficus variant with a polar solvent to provide a liquid toxic activity. For example, alkaloid compounds exhibiting extract; washing the liquid extract with an organic solvent to tumor necrotic effects in mice were isolated from Ficus provide an aqueous extract; and fractionating the aqueous carica latex by filtration followed by dialysis (see, e.g., S. B. extract to provide a Ficus extract having angiogenesis inhib Ullman et al., Exp. Med. Sur. 3:11 (1945)). The latex was iting activity. shown to also contain a water-soluble non-dialyzable fraction 0022. The present invention is also directed to a process that exhibited strong cytotoxic effects, which was hypoth for obtaining a Ficus extract having angiogenesis inhibiting esized to contain a proteolytic enzyme. Enzymatic cytotoxic activity in a human or animal, the process comprising: pre activity has been observed in extracts from both Ficus carica paring an aqueous extract from a latex-containing portion of and Ficus glabrata (see, e.g., U.S. Pat. Nos. 2,163,643 and a Ficus variant, fractionating the aqueous extract; and select 2.995,493, and U.S. Pat. No. 1,616,291, respectively). ing as the Ficus extract having angiogenesis inhibiting activ 0017 More recently, researchers have characterized a ity in a human or animal, a Substantially protein-free fraction broad range of lipophilic extracts from the latex, seeds, roots, containing one or more compounds having a molecular and bark of Ficus species using, for example, an organic weight of about 200 Da to about 2,000 Da wherein the Ficus solvent extraction method. For example, I3-sitosterols extract exhibits angiogenesis inhibiting activity. extracted from Ficus carica latex have been investigated for 0023. In some embodiments, angiogenesis inhibiting activity involving Suppression of cancer cell proliferation activity is exhibited as an inhibition or a reduction of neoves (see, e.g., S. Rubnov et al., J. Nat. Prod. 64:993 (2001)), as sel growth in an in vitro model Such as, but not limited to, a well as mitigating oxidative damage in response to ferric tumor fragment, a placental sample, and the like. In some nitrilotriacetate (see, e.g., N. Khan and S. Sultana, Life Sci embodiments, angiogenesis inhibiting activity is exhibited as ences 77:1194 (2005)). Other lipophilic compounds present an inhibition or a reduction of neovessel growth in a human or in Ficus latex that have been characterized by gas chroma animal Subject in need thereof. tography/mass spectrometry (“GC/MS) include campes 0024. In some embodiments, the process further com terol, Stigmasterol, fucosterol, and fatty acids such as myristic prises macerating the latex-containing portion of the Ficus acid, palmitic acid, Stearic acid, oleic acid, linoleic acid, and variant. linolenic acid (see, e.g., W. S. Jeong and P. A. Lachance, J. 0025. In some embodiments, fractionating comprises Food Sci. 66:278 (2001)). Additionally, cytotoxic triterpenes applying the aqueous extract to an adsorbent, and eluting a have been extracted from the aerial roots of Ficus microcaoa Ficus extract having angiogenesis inhibiting activity from the (see, e.g., Y.-M. Chiang et al., Phytochemistiy 66:495 (2005)). adsorbent with an eluting solvent. In some embodiments, an Similar triterpenes such as betulinic acid have been investi eluting solvent comprises an organic solvent. In some gated for regulating hair growth (see, e.g., U.S. Pat. Nos. embodiments, an eluting solvent comprises about 95% etha 6,124.362 and 6,482,857 B1). nol and about 5% water. In some embodiments, an adsorbent 0018. The observed activity of Ficus extracts is highly is porous and has an average pore size of about 20 nm or less. dependent on the extraction and purification procedures. For 0026. In some embodiments, a process further comprises example, 2,5-dihydroxytetrahydro-2-furancarboxylic acids adding a Ficus extract having angiogenesis inhibiting activity have been extracted from Ficus elastica and shown to have to an alcoholic Solution to provide a precipitate having angio anticancer, apoptosis-inducing activity (see, e.g., U.S. Pat. genesis inhibiting activity. In some embodiments, a Ficus No. 6,133,238). Methanol extraction followed by silica gel extract is added to an alcoholic Solutionina dropwise manner. chromatography has been demonstrated as useful for extract 0027. In some embodiments, a process further comprises ing agents from Ficus benghalensis bark having anti-tumor treating a Ficus extract having angiogenesis inhibiting activ activity (see, e.g., U.S. Pat. No. 6,660,309 B2). Additionally, ity with activated carbon. flavonoids and polyphenols extracted from Ficus citrifblia, 0028. In some embodiments, a process further comprises administered to patients undergoing chemotherapy, have removing at least a portion of a polar solvent from a liquid been demonstrated to exhibit improved modulation of cancer extract. In some embodiments, a polar solvent is a C-Co cell resistance to chemotherapeutic agents (see, e.g., P-N. compound comprising a heteroatom selected from: N, O, P, S, Simon et al., Anticancer Res. 21:1023 (2001)). Anti-cancer and combinations thereof. In some embodiments, a polar extracts from a broad spectrum of herbs and fruits including Solvent is a solvent selected from: a C-Co alcohol, a C-Co Ficus racemosa have also been extracted using freon gas (see, ether, a C-C aldehyde, a C-Coketone, a C-C carboxy e.g., U.S. Pat. No. 6,780,441 B2). lic acid, a C-Co ester, a Ca-Co amine, a C-Cs amide, and 0019 Substituted aurone derivatives having antifungal combinations thereof. In some embodiments, a polar solvent activity have been extracted from the seeds of Ficus religiosa has a boiling point of about 200° C. or less. (see, e.g., U.S. Pat. No. 6,307,070 B1). Anthocyanin and 0029. In some embodiments, a process further comprises other flavonoid derivatives extracted from fig fruit have also filtering a liquid extract. In some embodiments, washing fur been shown to have anti-inflammatory COX-2 inhibiting ther comprises dissolving a liquid extract in water. In some activity (see, e.g., U.S. Pat. Nos. 6,818,234 B1 and 7,192,611 embodiments, a process further comprises freeze-drying one B2). or more of the extracts. 0020. However, extracts from Ficus variants such as Ficus 0030 The present invention is also directed to a Ficus carica, or any other Ficus species, have not been previously extract prepared by the above processes. shown to exhibit angiogenesis inhibiting activity. 0031. In some embodiments, a latex-containing portion of a Ficus variant comprises at least a portion of a fruit of a Ficus BRIEF SUMMARY OF THE INVENTION variant. In some embodiments, a latex-containing portion of a 0021. The present invention is directed to a process for Ficus variant comprises a Ficus carica fruit. isolating a Ficus extract having angiogenesis inhibiting activ 0032. In some embodiments, a Ficus extract has angiogen ity, the process comprising: mixing a latex-containing portion esis inhibiting activity in the micromolar range. In some US 2011/01 65096 A1 Jul. 7, 2011 embodiments, a Ficus extract is Substantially lacking cyto ing material having a particle size of about 3 um to about a toxic activity. In some embodiments, a Ficus extract is Sub pore size of about 100 A, and a carbon loading of about 19%. stantially free from one or more of shikimic acid, fumaric 0038. The present invention is also directed to a method of acid, Syringin, chlorogenic acid, catechin, coumaric acid, inhibiting or reducing neovessel growth in a humanoranimal, psoralen, and bergapten. the method comprising administering a composition com 0033. In some embodiments, a Ficus extract of the present prising a Ficus extract. Preferably, the Ficus extract com invention has a chromatograph having peaks at about 4.9 prises rutin and exhibits angiogenesis inhibiting activity to a minutes, about 6.0 minutes, about 21.3 minutes, about 22.5 human or animal in need thereof. minutes, about 35.0 minutes, and about 49.7 minutes, when 0039. The present invention is also directed to a method of Subjected to HPLC using a Cs reverse phase column having inhibiting or reducing neoVessel growth associated with an an internal diameter of about 4.6 mm and a length of about angiogenesis-dependent disease in a human or animal in need 250 mm; wherein a first eluent is acetonitrile and a second thereof, the method comprising: administering to the Subject eluent is water containing about 0.3% phosphoric acid and a composition comprising a Ficus extract having angiogen about 2.5% acetonitrile, the concentration of the first eluent esis inhibiting activity. Preferably, the Ficus extract is isolated and second eluent is 100% by volume, and from 0 minutes to by a process of the present invention. The present invention is about 20 minutes the first eluent increases linearly from 0% to also directed to a method of treating an angiogenesis-depen about 10% by volume, from about 20 minutes to about 50 dent disease in a Subject in need thereof, the method compris minutes the first eluent increases linearly from about 10% to ing: administering to the Subject a composition comprising a about 20% by volume, from about 50 minutes to about 65 Ficus extract having angiogenesis inhibiting activity, wherein minutes the first eluent increases linearly from about 20% to the Ficus extract is isolated by a process of the present inven about 40% by volume, and from about 65 minutes to about 80 tion. minutes the first eluent increases linearly from about 40% to 0040. The present invention is also directed to the use of a about 60% by volume; and wherein the column temperature is Ficus extract comprising rutin, wherein the Ficus extract has about 25°C.; the injection volume is about 10 uL: the flow angiogenesis inhibiting activity, in the manufacture of a com rate is about 1 mL/minute; from 0 minutes to about 80 min position for reducing or inhibiting neovessel growth. Prefer utes the pressure increases linearly from about 1,000 psi to ably, the Ficus extract is isolated by a process of the present about 3,000 psi; and detection is at about 254 nm. invention. The neovessel growth may be associated with an 0034. In some embodiments, a Ficus extract of the present angiogenesis-dependent disease. invention further comprises rutin, wherein the rutin appears 0041 Angiogenesis-dependent diseases suitable for treat as a peak in the chromatograph having a retention time of ment by a method of a present invention or inhibiting or about 51 minutes. reducing neoVessel growth associated with the diseases 0035. In some embodiments, the Ficus extract of the include, but are not limited to: cancer, infectious diseases, present invention has a HPLC chromatograph that further including mycobacterial infections, infections of the retina, comprises a chromatograph peak of at least one of about 3.0 presumed ocular histoplasmosis, and infections causing minutes, about 3.2 minutes, about 4.9 minutes, about 6.0 retinitis or choroiditis; autoimmune disorders; benign minutes, about 8.6 minutes, about 15.3 minutes, about 19.6 tumors, e.g., haemangiomas (including infantile haemangio minutes, about 27.7 minutes, about 28.7 minutes, about 29.3 mas, capillary haemangiomas, and cavernous haemangio minutes, about 30.4 minutes, about 33.1 minutes, about 34.0 mas), functional endocrine tumors, acoustic neuromas, neu minutes, about 37.0 minutes, about 44.1 minutes, about 45.7 rofibromas, trachomas, and pyogenic granulomas; minutes, about 46.3 minutes, about 48.2 minutes, about 53.0 atherosclerosis and atherosclerotic plaques: ocular angio minutes, about 57.3 minutes, and combinations thereof. genic diseases, e.g., diabetic retinopathy, retinopathy of pre 0036. In some embodiments, a Ficus extract of the present maturity, macular degeneration, corneal graft rejection, invention has a chromatograph substantially in accordance neovascular glaucoma, retrolental fibroplasia, rubeosis, ret with FIG. 16 when subjected to HPLC using a Cs reverse inoblastoma, persistent hyperplastic vitreous syndrome, cho phase column having an internal diameter of about 4.6 mm roidal or corneal neovascularization, Venous occlusion, uvei and a length of about 250 mm; wherein a first eluent is tis, vitritis, Eales disease, Behcet’s disease, proliferative acetonitrile and a second eluent is water containing about vitreoretinopathy, ocularischemic syndrome, and pterygium; 0.3% phosphoric acid and about 2.5% acetonitrile, the con myopia; optic pits; Best disease; Stargardt's macular dystro centration of the first eluent and second eluent is 100% by phy; pars planitis; chronic retinal detachment; hyperviscosity Volume, and from 0 minutes to about 20 minutes the first syndrome; rheumatoid arthritis; psoriasis; warts; allergic der eluent increases linearly from 0% to about 10% by volume, matitis; blistering disease; Karposi sarcoma; delayed wound from about 20 minutes to about 50 minutes the first eluent healing; endometriosis; uterine bleeding; ovarian cysts; ova increases linearly from about 10A to about 20% by volume, rian hyperstimulation; vasculogenesis; granulations; hyper from about 50 minutes to about 65 minutes the first eluent trophic Scars (keloids); nonunion fractures; Scleroderma; tra increases linearly from about 20% to about 40% by volume, choma; vascular adhesions; vascular malformations; and from about 65 minutes to about 80 minutes the first eluent DiGeorge syndrome; transplantarteriopathy; restenosis; obe increases linearly from about 40% to about 60% by volume: sity; myocardial angiogenesis; coronary collaterals; cerebral and wherein the column temperature is about 25° C.; the collaterals; arteriovenous malformations; ischemic limb injection volume is about 10 uI: the flow rate is about 1 angiogenesis; primary pulmonary hypertension; pulmonary mL/minute; from 0 minutes to about 80 minutes the pressure edema; asthma, nasal polyps; inflammatory bowel disease; increases linearly from about 1,000 psi to about 3,000 psi; and periodontal disease; ascites; peritoneal adhesions; Osler-We detection is at about 254 nm. ber-Rendu syndrome (hereditary hemorrhagic telangiecta 0037. In some embodiments, a Cs reverse phase column sia); plaque neovascularization; telangiectasia; hemophiliac Suitable for use with the present invention comprises a pack joints; synovitis; osteomyelitis; osteophyte formation; US 2011/01 65096 A1 Jul. 7, 2011 angiofibroma; fibromuscular dysplasia; wound granulation; 0049. In some embodiments, a composition of the present Crohn's disease; peptic ulcer, malignant tumor growth invention further comprises a pharmaceutically or nutraceu beyond 2 mm in diameter, sickle cell anemia; Sarcoidosis: tically acceptable carrier. syphilis; pseudoxanthoma elasticum, Paget’s disease; pri 0050. In some embodiments, a Ficus extract of the present mary hyperparathyroidism; secondary hyperparathyroidism; invention is formulated into a pharmaceutical dosage form. tertiary hyperparathyroidism; arterial occlusion; carotid Suitable pharmaceutical dosage forms include, but are not obstructive disease; Lyme disease; systemic lupus erythema limited to, a tablet, a caplet, a pellet, a capsule, a gelcap, a tosis; toxoplasmosis; trauma; Wegner's granulomatosis: troche, a lozenge, a syrup, a gel, an ointment, an emulsion, a post-laser complications; and combinations thereof. patch, a solution, a dispersion, a mist, an aerosol, and com binations thereof. 0042. The present invention is also directed to a method of 0051. In some embodiments, a Ficus extract of the present analytically determining and characterizing a Ficus extract invention is formulated into a nutraceutical composition. In having angiogenesis inhibiting activity, the method compris Some embodiments, the nutraceutical composition is formu ing providing a Ficus extract by a process of the present lated into a food preparation. Suitable food preparations invention, and analyzing the Ficus extract having angiogen include, but are not limited to, a food bar, a beverage, a liquid, esis inhibiting activity by applying the Ficus extract to a a solution, a Suspension, a food gel, a food Supplement, a HPLC column, eluting a component of the Ficus extract from powder, and a syrup. Additional Suitable nutraceutical com the HPLC column, and detecting the component of the Ficus positions include, but are not limited to a tablet, a capsule, a extract as the component elutes from the HPLC column. softgel, a gelcap, a liquid, a powder, a solution, a Suspension, 0043. In some embodiments, the analyzing provides that and a syrup. the Ficus extract comprises rutin, having a retention time of 0052. In some embodiments, the Ficus extract of the about 51.0 minutes. In some embodiments, the analyzing present invention comprises rutin and the Ficus extract is provides that the Ficus extract is substantially free from one substantially free from one or more of: shikimic acid, fumaric or more of shikimic acid, fumaric acid, Syringin, chlorogenic acid, Syringin, chlorogenic acid, catechin, coumaric acid, acid, catechin, coumaric acid, psoralen, and bergapten. psoralen, and bergapten. 0053. Further embodiments, features, and advantages of 0044. In some embodiments, the process further com the present inventions, as well as the structure and operation prises macerating the latex-containing portion of a Ficus Vari of the various embodiments of the present invention, are ant, wherein the latex-containing portion of the Ficus variant described in detail below with reference to the accompanying includes a fruit of a Ficus variant. drawings. 0045. In some embodiments, the fractionating comprises applying the aqueous extract to an adsorbent and eluting the BRIEF DESCRIPTION OF THE DRAWINGS extract having angiogenesis inhibiting activity from the adsorbent with an eluting solvent. 0054 For a further understanding of the nature, objects, 0046. In some embodiments, the analyzing comprises and advantages of the present invention, reference should be eluting with a first eluent of acetonitrile and a second eluent of had to the following detailed description, read in conjunction water containing about 0.3% phosphoric acid and about 2.5% with the following drawings, wherein like reference numerals acetonitrile, wherein the concentration of the first eluent and denote like elements. second eluent is 100% by volume, and wherein from 0 min 0055 FIGS. 1 and 2 provide schematic representations of utes to about 20 minutes of the eluting the first eluent processes of the present invention for extracting and isolating increases linearly from 0% to about 10% by volume, from an angiogenesis inhibiting extract from a Ficus fruit. about 20 minutes to about 50 minutes of the eluting the first 0056 FIGS. 3A-3B and 4A-4B provide schematic repre eluent increases linearly from about 10% to about 20% by sentations of an angiogenesis inhibition screening process of Volume, from about 50 minutes to about 65 minutes of the the present invention. eluting the first eluent increases linearly from about 20% to 0057 FIGS. 5A-5B; 6A-6B; 7A-7B; 8A-8B; and 9A-9B about 40% by volume, and from about 65 minutes to about 80 provide graphic representations of the in vitro angiogenesis minutes of the eluting the first eluent increases linearly from inhibiting activity of extracts from Ficus carica L., variety about 40% to about 60% by volume; and wherein the column kadota, at concentrations of 0.001 mg/mL, 0.003 mg/mL, temperature is about 25°C.; the injection volume is about 10 0.01 mg/mL, 0.03 mg/mL, 0.1 mg/mL and 0.3 mg/mL, as LL; the flow rate is about 1 mL/minute; and from 0 minutes to tested by a screening method of the present invention. about 80 minutes the pressure increases linearly from about 0.058 FIGS. 10A-10B; 11A-11B; and 12A-12B provide 1,000 psi to about 3,000 psi. graphic representations of the in vitro angiogenesis inhibiting 0047. The present invention is also directed to a method of activity of extracts from Ficus carica L., variety gold, at screening a Ficus extract having angiogenesis inhibiting concentrations of 0.001 mg/mL, 0.003 mg/mL, 0.01 mg/mL, activity, the method comprising isolating a Ficus extract hav 0.03 mg/mL, 0.1 mg/mL and 0.3 mg/mL, as tested by a ing angiogenesis inhibiting activity by a process of the screening method of the present invention. present invention, applying the Ficus extract having angio 0059 FIGS. 13 A-13B; 14A-14B; and 15A-15B provide genesis inhibiting activity to a tumor, and measuring neoves graphic representations of the in vitro angiogenesis inhibiting sel growth from the tumor. activity of extracts from Ficus carica L., variety magnolia, at 0048. The present invention is also directed to a pharma concentrations of 0.001 mg/mL, 0.003 mg/mL, 0.01 mg/mL, ceutical composition or a nutraceutical composition compris 0.03 mg/mL, 0.1 mg/mL and 0.3 mg/mL, as tested by a ing a Ficus extract comprising rutin, wherein the Ficus extract screening method of the present invention. exhibits angiogenesis inhibiting activity, and the Ficus extract 0060 FIG. 16 provides a graphical representation of a is isolated by a process of the present invention. high-performance liquid chromatograph of a Ficus extract US 2011/01 65096 A1 Jul. 7, 2011 having angiogenesis inhibiting activity that was isolated from 0070 FIG. 1 provides a generalized schematic represen a Ficus variant according to the present invention. tation, 100, of an extraction process of the present invention. 0061 FIGS. 17, 18 and 19 provide graphical representa The latcx-containing portion of the Ficus variant, 101, is tions of high-performance liquid chromatographs of various placed inapolar organic solvent, and then separated, 110, into Ficus carica L. raw materials, extracts and other process a solid fraction, 111, and a liquid fraction, 112. The liquid products of the present invention. fraction, 112, is further separated into four major fractions. 0062 FIGS. 20A, 20B, and 20O show the result of treat An organic fraction (F), 121, is isolated by washing the ment with a Ficus extract on psoriasis in a male human Sub liquid fraction with an organic solvent, 120. The remaining ject. aqueous fraction (F), 122, is then separated into two or more 0063 FIG. 21 shows the result of treatment with a Ficus fractions via column chromatography, 130, utilizing, for extract on angiogenesis in carcinoid tumors. example, an adsorbent capable of separating species based 0.064 FIG.22 shows the results of treatment with various upon relative molecular size. An aqueous eluate fraction (F- compounds, including a Ficus extract, on angiogenesis in o), 131, is eluted from the adsorbent using deionized water. fragments from a neuroendocrine lymph node. The adsorbent is then washed with a solvent that includes an 0065 FIG. 23 shows the results of treatment with various organic (e.g., a species comprising at least one carbon atom, compounds, including a Ficus extract, on angiogenesis in Such as an alcohol) to provide an organic eluate fraction fragments from a sarcoma. (F-9s), 132. The Ficus extract of the present invention is present in the organic eluate fraction (Fos), 132. DETAILED DESCRIPTION OF THE INVENTION 0071. In some embodiments, the organic eluate fraction 0066. Before the subject invention is further described, it (Fos), 132, is further purified. Referring to FIG. 1, the is to be understood that the invention is not limited to the organic eluate fraction (Fos), 132, is precipitated, 140, particular embodiments of the invention described below, as from alcohol to form a solid precipitate (Fost), 141. In variations of the particular embodiments may be made and Some embodiments, precipitation, 140, can increase the still fall within the scope of the appended claims. It is also to purity and the potency of the angiogenesis inhibiting activity be understood that the terminology employed is for the pur of the extract by about 20%, about 30%, about 40%, or about pose of describing particular embodiments, and is not 50%. intended to be limiting. Instead, the scope of the present 0072 Referring to FIG. 1, either of the organic eluate invention will be established by the appended claims. fraction (F-9s), 132, or the solid precipitate (Fose), 141, 0067. In this specification and the appended claims, the can also be optionally further purified by treating, 150 or 151, singular forms “a,” “an,” and “the include plural reference with activated carbon to provide a fraction retaining angio unless the context clearly dictates otherwise. Unless defined genesis inhibiting activity (Fos), 152. In some embodi otherwise, all technical and scientific terms used herein have ments, the resulting fraction (Fos), 152, can be light the same meaning as commonly understood to one of ordi green, beige, light yellow, white, and/or clear in color. Thus, nary skill in the art to which this invention belongs. in Some embodiments, the treating with activated carbon 0068. The present invention is directed to a process for removes intensely colored compounds (i.e., compounds hav isolating a natural product having angiogenesis inhibiting ing an electronic absorption in the visible region of the spec activity from a latex-containing portion of a Ficus variant. As trum) from the extract. In some embodiments, an extract of used herein, a “Ficus variant” refers to a species of Ficus and the present invention is treated with activated carbon in a varieties thereof, including both wild and engineered Ficus weight ratio of about 100:1, about 10:1, about 5:1, about 2:1, species and varieties thereof. In some embodiments, a Ficus about 1:1, about 1:2, about 1:5 or about 1:10 (extract:acti variant is from a variety of Ficus carica L.). However, there vated carbon, by weight). In some embodiments, the treating are hundreds of species of Ficus and hundreds of varieties of with activated carbon improves the purity and/or potency of Ficus carica that are suitable for use with the present inven the Ficus extract by about 20%, about 50%, about 75%, about tion. See, e.g., I. Condit, “Fig Varieties: A Monograph.J.Agr. 100%, about 150%, or about 200%. Sci. 23:323 (1955) and A. Salhi-Hannachi et al., Hereditas 0073. The process of the present invention comprises mix 143:15 (2006), which disclose many Ficus variants, and ing a latex-containing portion of a Ficus variant (e.g., a fruit) which are incorporated herein by reference in their entirety. In with a polar solvent to form a mixture comprising a liquid Some embodiments, a Ficus fruit is from a Ficus carica L. extract and Solids. In some embodiments, the latex-contain variety Such as, but not limited to, gold, magnolia, louisiana, ing portion can be macerated, mashed, chopped, diced, or kadota, and combinations thereof. otherwise prepared to form a pulp or slurry prior to mixing 0069. Latex or resin is present in the fruit of the Ficus in with a polar solvent. The mixing is not particularly limited by Small quantities. Even Smaller quantities of latex can be the polar solvent that is used or by the duration of the mixing. present in the cellulosic matter of a Ficus variant. The present 0074 Polar solvents suitable for use with the present invention provides a straightforward process for isolating an invention include, but are not limited to, a C-C compound extract from a Ficus variant that eliminates the painstaking having at least one heteroatom selected from: N, O, P, S, and collection of fig latex from individual fruits. Latex (i.e., resin combinations thereof. In some embodiments, the polar Sol or sap) concentration in the Ficus fruit is typically highest vent includes at least one of water, a C-Co alcohol, a C-Co when the fruit is premature. Thus, in some embodiments, the ether, C-C aldehyde, a C-C ketone, a C-C carboxylic present invention is directed to isolating an extract from a acid, a C-Co ester, a C-Co amine, a C1-C5 amide, and latex-containing portion of a Ficus variant comprising a pre combinations thereof. In some embodiments, the polar Sol mature Ficus fruit, and in particular premature fruits of Ficus vent comprises a polar, protic solvent (e.g., methanol). In carica. Depending on the variety and the growing region, Some embodiments, the polar solvent comprises a polar, apro premature fruit from Ficus species in the Northern hemi tic solvent (e.g., acetone). Polar solvents suitable for use with sphere can typically be harvested between February and July. the present invention include, but are not limited to, methanol, US 2011/01 65096 A1 Jul. 7, 2011

ethanol, n-propanol, iso-propanol, a butanol, a pentanol, removed from the liquid extract so long as the amount of the acetone, methyethylketone, ethylacetate, acetonitrile, tet polar solvent remaining in the liquid extract does not nega rahydrofuran, dimethylformamide, dimethylsulfoxide, tively affect the partition of components during the washing water, and combinations thereof. with an organic solvent. 0075. In some embodiments, the polar solvent has a I0082. The method for removing the polar solvent is not dielectric constant of about 5 or greater, about 10 or greater, particularly limited, and can include solvent evaporation at a about 15 or greater, about 20 or greater, about 25 or greater, reduced pressure (e.g., Subatmospheric pressure) and/or an about 30 or greater, or about 40 or greater. elevated temperature (e.g., above about 25° C.). A Ficus 0076. In some embodiments, the polar solvent has a boil extract of the present invention is generally stable at elevated ing point of about 200° C. or less, about 175°C. or less, about temperatures for short periods of time (e.g., at a temperature 150° C. or less, about 125° C. or less, or about 100° C. or less. of about 100° C. or greater for a duration of about 30 minutes 0077. The concentration or amount of a polar solvent used or less, or about 15 minutes or less). However, the present to extract materials from a latex-containing portion of a Ficus invention also includes the removal of the polar solvent (and variant can be varied. Generally, the ratio of a polar solvent to other process steps) being conducted under controlled tem a latex-containing portion of a Ficus variant (weight to perature conditions such as, but not limited to, about 120° C. weight) is the amount of a polar solvent Sufficient to extract or less, about 100° C. or less, about 80°C. or less, about 60° about 75% or more, about 85% or more, about 90% or more, C. or less, or about 40° C. or less. about 95% or more, about 97% or more, or about 99% or more 0083. In some embodiments, it can be difficult to com of a material having angiogenesis inhibiting activity. For pletely remove a polar solvent from a liquid extract by stan example, further processing of a latex-containing portion of a dard solvent removal procedures such as evaporation. In some Ficus variant with an additional polar solvent after an initial embodiments, processes such as co-evaporation, lyophiliza extraction would provide about 25% or less, about 15% or tion, and the like can be used to completely remove the polar less, about 10% or less, about 5% or less, about 3% or less, or solvent from a liquid fraction to form a dry powder, dry pellet, about 1% or less of a Ficus extract having angiogenesis inhib dry granulate, paste, and the like. iting activity in addition to that extracted by an initial extrac I0084. A liquid extract is then washed with an organic tion with a polar solvent. In some embodiments, the ratio of Solvent. In some embodiments, washing comprises suspend polar solvent to fruit is about 100:1 to about 1:100, or about ing a solid form of a liquid extract (e.g., a powder, pellet, 10:1 to about 1:10 by weight. granulate, etc.) in an organic solvent for a period of time 0078. In some embodiments, a latex-containing portion of followed by filtering the suspension, or alternatively, placing a Ficus variant is contacted with a polar solvent for about 15 a solid form of a liquid extract in a filter system and rinsing minutes or more, about 30 minutes or more, about 1 hour or with an organic solvent. In some embodiments, the liquid more, about 4 hours or more, about 8 hours or more, about 16 extract or a solid form of the liquid extract is taken up or hours or more, about 24 hours or more, about 48 hours or dissolved in water (e.g., deionized water) and then washed more, or about 72 hours or more. In some embodiments, the with an organic solvent. In some embodiments, the washing efficiency of an initial extraction with a polar solvent can be includes at least one washing with an organic solvent. More limited by contacting for too short a time interval. The result than one washing step can be performed, for example, two or ing liquid extract, or a solid resulting therefrom, is stable more washings, three or more washings, or four or more under ambient conditions for an extended period of time, for Washings. example, for at least about 1 month, at least about 6 months, I0085. The washing is not particularly limited by the at least about 1 year, or at least about 2 years. organic solvent. In some embodiments, an organic solvent 007.9 Temperature can also be controlled during the con Suitable forwashing a liquid extract is at least partially immis tacting. In some embodiments, a latex-containing portion of a cible with water. As used herein, an organic solvent at least Ficus variant is contacted with a polar solvent at a tempera partially immiscible with water refers to an organic Solvent ture of about -25° C. to about 200° C., about 0°C. to about that forms at least a partial bilayer with deionized water at 150° C., or about 25°C. to about 100° C. about 20°C. In some embodiments, an organic solvent Suit 0080. In some embodiments, the process of the present able for washing a liquid extract has a solubility in deionized invention comprises isolating a liquid extract from the mix water at about 20°C. of about 100 g/L or less, about 80 g/L or ture comprising the liquid extract and solids. Suitable means less, about 50 g/L or less, about 25 g/L or less, about 15 g/L or for isolating the liquid extract include those known in the art less, about 10 g/L or less, about 5 g/L or less, about 2 g/L or of organic synthesis and include, but are not limited to, grav less, about 1 g/L or less, or about 0.5 g/L or less. Organic ity filtration, Suction and/or vacuum filtration, centrifuging, solvents suitable for washing the liquid extract include, but settling and decanting, and the like. In some embodiments, are not limited to, ethyl acetate, chloroform, methylene chlo the isolating comprises filtering a liquid extract through a ride, hexane, benzene, toluene, petroleum ether, diethyl ether, porous membrane, sponge, Zeolite, paper, or the like having a and the like, and combinations thereof. pore size of about 100 um or less, about 50 um or less, about I0086 Washing the liquid extract with an organic solvent 20um or less, about 10um or less, about 5um or less, or about provides an organic fraction (F) and an aqueous extract 1 um or less. (F). Not being bound by any particular theory, washing the 0081. A liquid extract comprises an aqueous solution that liquid extract with an organic solventis effective in extracting includes water present in the latex-containing portion of a from the liquid extract compounds present in a liquid extract Ficus variant (e.g., water present in a fruit, stem, or leaf) in Such as, but not limited to, waxes, lipids, chloroplasts and addition to a polar solvent used for the extracting. In some other light harvesting chromophores, sitosterol, campsterol, embodiments the process comprises removing at least a por Stigmasterol, fucosterol, fatty acids (e.g., myristic acid, palm tion of the polar solvent from the liquid extract. It is not itic acid, Stearic acid, oleic acid, linoleic acid, and linolenic crucial that the polar solvent be removed or completely acid), triterpene compounds, lupeol, quercetin, and other US 2011/01 65096 A1 Jul. 7, 2011

lipophilic compounds as disclosed by W. S. Jeong and P. A. binations thereof. In some embodiments, an eluting solvent Lachance, J. Food Chem. 66:278 (2001), and by S. Rubnov et comprises an organic and water, e.g., about 95% ethanol and al., J. Nat. Prod. 64:993 (2001), which is incorporated by about 5% water. herein in its entirety. 0094. In some embodiments, an organic is present in an 0087. The process of the present invention comprises frac eluting solvent in a concentration of about 1% to about 99%, tionating the aqueous extract. Fractionating can be performed about 2% to about 98%, about 5% to about 95%, about 10% by processes such as, but not limited to: column chromatog to about 90%, about 25% to about 75%, about 40% to about raphy, preparative high performance liquid chromatography 60%, about 10% to about 99%, about 25% to about 99%, (“HPLC), reduced pressure distillation, and combinations about 40% to about 99%, about 60% to about 99%, about 75% thereof. to about 99%, about 85% to about 99%, about 10% to about 95%, about 25% to about 95%, about 40% to about 95%, 0088. In some embodiments, the fractionating comprises about 60% to about 95%, about 75% to about 95%, or about applying the aqueous extract to an adsorbent and isolating a 85% to about 95%, by volume of the eluting solvent. Ficus extract having angiogenesis inhibiting activity by col 0095. In some embodiments, a first eluting solvent is umn chromatography. In some embodiments, the aqueous water, and a second eluting solvent comprises water and an extract can be purified using a chromatographic separation organic. system comprising an adsorbent. In some embodiments, a chromatographic separation system further comprises a 0096. In some embodiments, the aqueous extract is applied to an adsorbent and an eluting solvent of water is material in addition to an adsorbent, such as, but not limited applied to elute from the adsorbent an aqueous eluate fraction to, a porous membrane, an ion exchange resin, a silica gel, a (Fo). Not being bound by any particular theory, an aqueous reverse phase silica gel, or any resin, polymer, colloid, and the eluate fraction eluted from the adsorbent having an aqueous like Suitable for performing a separation based upon a extract applied thereto can comprise protcolytic enzymes molecular property Such as, but not limited to, polarity, size, and/or active moieties having cytotoxic activity, and the like functional group, and combinations thereof. (e.g., water soluble compounds having a molecular weight of 0089. In some embodiments, the aqueous extract is further about 1,000 to about 10,000 Daltons or greater). diluted with water prior to application to an adsorbent. 0097. In some embodiments, the aqueous extract is 0090. In some embodiments, an adsorbent is porous. In applied to an adsorbent and an eluting solvent comprising an Some embodiments, a porous adsorbent has a pore size of organic is applied to elute from the adsorbent an organic about 20 nm or less, about 15 nm or less, about 10 nm or less, eluate fraction (Fos). Not being bound by any particular about 8 nm or less, about 6 nm or less, about 5 nm or less, theory, an eluting solvent comprising an organic is effective about 4 nm or less, about 3 nm or less, about 2 nm or less, or for eluting from an adsorbent having an aqueous extract about 1 nm or less. In some embodiments, a porous adsorbent applied thereto an organic eluate fraction (i.e., Fos) com has a pore size of about 0.6nm to about 20 nm, about 0.8 nm prising an extract having angiogenesis inhibiting activity. to about 15 nm, about 1 nm to about 10 nm, about 1.5 nm to 0098. In some embodiments, after fractionating the Ficus about 8 nm, about 2 nm, about 4 nm, about 6 nm, or about 8 extract having angiogencsis inhibiting activity can be further purified by a process comprising at least one of filtering 0091 Adsorbents suitable for use with the present inven through activated carbon, precipitation into a solution, and tion include, but are not limited to, cross-linked styrene combinations thereof. divinylbenzene resins (e.g., DOWEX(R) OPTIPORE(R) Resins, 0099. The present invention is also directed to a product The Dow Chemical Co., Midland, Mich. and AMBERLITE(R) prepared by the process of the present invention. In some XAD4, XAD16, XAD1180, and XAD 1600, Rohm and Haas embodiments, the Ficus extract of the present invention is Co., Philadelphia, Pa.); highly cross-linked, aliphatic, orphe Substantially free of cyclotoxic compounds. In some embodi nol-formaldehyde condensate polymers (e.g., AMBERLITE(R) ments, the Ficus extract of the present invention substantially XAD7HP and XAD761, Rohm and Haas Co.); carbonaceous lacks cytotoxic activity. As used herein, “substantially lacks resins (e.g., AMBERSORB(R) 563 and 572, Rohm and Haas cytotoxic activity” refers to extracts that are not appreciably Co.); granular activated carbon (e.g., FILTRASORBR 300 and cytotoxic under in vitro or in vivo testing and/or administer 400, Calgon Carbon Corp., Pittsburgh, Pa.); and combina ing conditions. In some embodiments, “substantially lacks tions thereof. cytotoxic activity” refers to extracts lacking cytotoxic activity 0092 An eluting solvent is applied to an adsorbent loaded as described in S. B. Ullman eted. Exp. Med. Sur. 3:11 (1945) with the aqueous extract to elute fractions from the adsorbent. and S. B. Ullman eted. Exp. Med. Sur:/0:287 (1952), both of In some embodiments, an eluting solvent is an aqueous eluent which are incorporated herein by reference in their entirety. comprising water. In some embodiments, an eluting solventis 0100. In some embodiments, the Ficus extract of the deionized (e.g., deionized water). Alternatively, the tonicity present invention is Substantially free of anti-inflammatory of an eluting solvent can be increased by including one or compounds, and does not exhibit Substantial anti-inflamma more ions, salts, and the like to an eluting solvent. tory activity. In some embodiments, an extract of the present 0093. In some embodiments, an eluting solvent comprises invention is Substantially free of terpenoid compounds. an “organic.” which as used herein refers to a liquid, Solid, or 0101. In some embodiments, a Ficus extract of the present gas that includes at least one carbon atom in its molecular invention is substantially free from one or more of shikimic structure. Organics Suitable for use as eluting solvents acid, fumaric acid, Syringin, chlorogenic acid, catechin, cou include, but are not limited to, methanol, ethanol, propanol, maric acid, psoralen, and bergapten. As used herein, 'Sub acetone, carbon dioxide, methylethyl ketone, acetonitrile, stantially free from refers to a Ficus extract that does not butyronitrile, carbon dioxide, ethyl acetate, tetrahydrofuran, contain an appreciable quantity of a compound. In some di-iso-propylether, ammonia, triethylamine, N,N-dimethyl embodiments, substantially free from refers to a Ficus extract formamide, N,N-dimethylacetamide, and the like, and com comprising about 5% or less, about 2% or less, about 1% or US 2011/01 65096 A1 Jul. 7, 2011

less, about 0.5% or less, about 0.2% or less, about 0.1% or packing material having a particle size of about 3 um to about less, about 0.05% or less, or about 0.01% or less of a specified 5um, a pore size of about 100 A, and a carbon loading of compound. about 19%. Additional column packing materials suitable for 0102) A Ficus extract of the present invention can be char use with the present invention include are known to persons of acterized by analytical methods such as, but not limited to, ordinary skill in the art, and have, e.g., a particle size of about spectroscopic methods such as, but not limited to, ultraviolet 2 um, to about 10 Lim, about 3.5um, about 5 and about 7um; visible spectroscopy (“UV-Vis'), infrared spectroscopy a pore size of about 50 A to about 500 A, about 100 A, about (“IR”), and the like; mass-spectrometry (“MS) methods 200 A, and about 300 A; and a carbon load of about 2% to such as, but not limited to, time-of-flight MS; quadrupole MS; about 20%, about 2.8%, about 8.5%, about 12%, about 15%, electrospray MS. Fourier-transform MS, Matrix-Assisted and about 17%. Laser Desorption/Ionization ("MALDI), and the like; chro 0106. In some embodiments, a Ficus extract of the present matographic methods such as, but not limited to, gas-chro invention has a HPLC chromatograph, obtained under the matography ("GC), liquid chromatograph ("LC), high-per conditions described herein, that comprises at least one peak formance liquid chromatography (“HPLC), and the like; and of about 3.4 minutes, about 4.8 minutes, about 18.3 minutes, combinations thereof (e.g., GC/MS, LC/MS, HPLC/UV-Vis, about 18.6 minutes, about 19.6 minutes, about 21.7 minutes, and the like), and other analytical methods known to persons about 22.4 minutes, about 23.2 minutes, about 24.2 minutes, of ordinary skill in the art. about 25.5 minutes, about 26.3 minutes, about 27.1 minutes, 0103) Not being bound by any particular theory, a Ficus about 27.4 minutes, about 28.3 minutes, about 29.0 minutes, extract of the present invention comprises one or more com about 31.0 minutes, about 32.2 minutes, about 34.0 minutes, pounds (i.e., active moieties) having a molecular weight of about 35.7 minutes, about 39.6 minutes, about 41.5 minutes, about 2,000 Daltons (“Da') or less, about 1,500 Da or less, about 42.2 minutes, about 47.1 minutes, about 50.2 minutes, about 1,200 Da or less, about 1,000 Da or less, about 900 Da about 50.8 minutes, and combinations thereof. or less, about 800 Da or less, about 700 Da or less, about 600 0107. In some embodiments, a Ficus extract of the present Da or less, or about 500 Da or less. In some embodiments, a invention has a chromatograph substantially in accordance Ficus extract of the present invention comprises one or more with FIG. 16 (as described herein in Example 9) when sub compounds having a molecular weight of about 200 Da to jected to HPLC using a Cs reverse phase column having an about 2,000 Da, about 250 Da to about 1,500 Da, about 300 internal diameter of about 4.6 mm and a length of about 250 Dato about 1,000 Da, about 400 Dato about 2,000 Da, about mm under the conditions provided above. 600 Da to about 2,000 Da, about 800 Da to about 2,000 Da, 0108. In some embodiments, “substantially free from can about 1,200 Da to about 2,000 Da, about 500 Da to about further be related to a HPLC chromatograph of a Ficus extract 1,500 Da, about 200 Da to about 900 Da, about 300 Da to in which the chromatograph lacks peaks corresponding to a about 900 Da, about 400 Da to about 900 Da, about 200 Dato specified compound. For example, a Ficus extract of the about 800 Da, about 300 Da to about 800 Da, about 400 Dato present invention has a HPLC chromatograph, obtained about 800 Da, about 300 Da, about 350 Da, about 400 Da, under the conditions described herein, that is substantially about 450 Da, about 500 Da, about 600 Da, about 650 Da, free from one or more peaks corresponding to shikimic acid about 750 Da, or about 950 Da. (having a retention time of about 3.6 minutes), fumaric acid 0104. In some embodiments, a Ficus extract of the present (having a retention time of about 7.7 minutes), Syringin (hav invention can be characterized by HPLC, and has a chromato ing a retention time of about 31.6 minutes) chlorogenic acid graph that exhibits peaks characteristic of the Ficus extract. (having a retention time of about 33.3 minutes), catechin For example, in some embodiments a Ficus extract having (having a retention time of about 33.3 minutes), coumaric angiogenesis inhibiting activity has a chromatograph having acid (having a retention time of about 46.2 minutes), psoralen peaks at about 4.9 minutes, about 6.0 minutes, about 21.3 (having a retention time of about 74.1 minutes), and ber minutes, about 22.5 minutes, about 35.0 minutes, and about gapten (having a retention time of about 79.0 minutes). 49.7 minutes, when subjected to analytical HPLC when sub jected to HPLC using a Cs reverse phase column having an Methods of Inhibiting or Reducing Neovessel Growth and internal diameter of about 4.6 mm and a length of about 250 Methods of Treating mm, wherein a first eluent is acetonitrile and a second eluent is water containing about 0.3% phosphoric acid and about 0109. In some embodiments, the present invention is 2.5% acetonitrile, the concentration of the first eluent and directed to a method of treating an angiogenesis dependent second eluent is 100% by volume, and from 0 minutes to disease in a Subject in need thereof, the method comprising: about 20 minutes the first eluent increases linearly from 0% to administering to a subject suffering from an angiogenesis about 10% by volume, from about 20 minutes to about 50 dependent disease a pharmaceutical composition comprising minutes the first eluent increases linearly from about 10% to a Ficus extract having angiogenesis inhibiting activity, about 20% by volume, from about 50 minutes to about 65 wherein the Ficus extract is isolated from a latex-containing minutes the first eluent increases linearly from about 20% to portion of a Ficus variant by a process of the present inven about 40% by volume, and from about 65 minutes to about 80 tion. minutes the first eluent increases linearly from about 40% to 0110. Non-limiting examples of angiogenesis dependent about 60% by volume; and wherein the column temperature is diseases and disorders suitable for treatment with an extract about 25°C.; the injection volume is about 10 uL: the flow of the present invention include, but are not limited to: a rate is about 1 mL/minute; from 0 minutes to about 80 min cancer, an age-related macular degenerative disorder, athero utes the pressure increases linearly from about 1,000 psi to Sclerosis, a psoriasis, a haemangioma (e.g., an infantile hae about 3,000 psi; and detection is at about 254 nm. mangioma, a capillary haemangioma, or a cavernous hae 0105. A non-limiting example of a Cs reverse phase col mangioma), a peptic ulcer, an ocular neovascularization, a umn Suitable for use with the present invention comprises a rheumatoid arthritis, and combinations thereof. US 2011/01 65096 A1 Jul. 7, 2011

0111. As used herein, the terms “treat,” “treating, and complications or adverse reactions occur. Appropriate thera “treatment” refer to both therapeutic treatment and prophy peutically effective amounts can also be determined readily lactic or preventative measures, wherein the object is to pre by routine experimentation using, for example, animal mod vent, inhibit, reverse or slow down (lessen) an undesired els. In any event, the effectiveness of treatment can be deter physiological condition, disorder or disease, or obtain a ben mined by monitoring the extent of angiogenic inhibition or eficial or desired clinical result. For purposes of this inven remission by methods well known to those in the field. In tion, beneficial or desired clinical results include, but are not Some embodiments, a therapeutically effective amount of a limited to, alleviation of symptoms; diminishing the extent of Ficus extract of the present invention comprises about 0.01 a condition, disorder or disease; stabilization (i.e., not wors micrograms per kilogram (ug/kg) to about 20 mg/kg of body ening) of a state of a condition, disorder or disease; delay of weight of a subject per day. In some embodiments, an extract the onset or progression of a condition, disorder or disease; is administered to a subject in a dosage of about 0.02 g/kg to amelioration of a condition, disorder or disease state; and about 10 mg/kg, about 0.05 ug/kg to about 5 mg/kg, about remission (whether partial or total) or enhancement or 0.08 ug/kg to about 1 mg/kg, about 0.1 g/kg to about 500 improvement of a condition, disorder or disease, whether ug/kg, about 0.5ug/kg to about 200 g/kg, about 1 g/kg to detectable or undetectable. Treatment also includes, but is not about 100 ug/kg, about 2 ug/kg to about 50 ug/kg, or about 5 limited to, eliciting a clinically significant response (e.g., a ug/kg to about 30 g/kg of body weight of a subject per day. cellular response) without excessive levels of side effects. For 0.115. In some embodiments, a Ficus extract of the present example, treating can include decreasing the time required for invention has angiogenesis inhibiting activity in the micro a patient to recover from an angiogenesis dependent disease, molar range. As used herein, “the micromolar range” refers to decreasing, eliminating, reducing, or disabling an angiogen an extract of the present invention that exhibits angiogenesis esis dependent disease, or alleviating or decreasing the sever inhibiting activity when administered to a Subject in need ity of symptoms associated with aanangiogenesis dependent thereof in an amount of about 1 umol to about 999 umol daily disease. Treatment also includes prolonging Survival as com dosage. pared to expected Survival if not receiving treatment. 0116. In some embodiments, a therapeutically effective 0112 The term “subject,” as used herein, refers to any amount of an angiogenesis inhibiting active agent, or of an mammal, including humans and non-humans, such as, but not extract comprising an angiogenesis inhibiting active agent, limited to, domestic and farm animals, Zoo animals, sports can be about 10 ug to about 100 mg per day. In some embodi animals, and pets. ments, a minimum therapeutically effective amount of an 0113. As used herein, “angiogenesis inhibiting activity” angiogenesis inhibiting active agent, or of an extract compris refers to the ability to delay, retard, slow, decrease, diminish, ing an angiogenesis inhibiting active agent, is about 10 ug. or otherwise inhibit neovessel growth. Any assay or testing about 20 Jug, about 50 ug, about 100 ug, about 200 ug, about protocol known to a person of ordinary skill in the art that is 500 ug, about 1 mg, about 2 mg, about 5 mg, about 10 mg. Suitable for quantifying an inhibition of neovessel growth can about 15 mg, about 20 mg. or about 25 mg per day. In some be utilized. In some embodiments, an inhibition of neovessel embodiments, a maximum therapeutically effective amount growth (i.e., an angiogenesis inhibiting activity) can be deter of an angiogenesis inhibiting active agent, or of an extract mined by an in vitro test protocol, an in vivo test protocol, an comprising an angiogenesis inhibiting active agent, is about animal model, an animal clinical trial, a human clinical trial, 100 mg, about 90 mg, about 80 mg, about 70 mg, about 60 mg. or a combination thereof. In some embodiments, the angio about 50 mg, about 40 mg, about 35 mg, about 30 mg. or genesis inhibiting activity of a Ficus extract of the present about 25 mg per day. invention can be quantified using the in vitro testing protocols 0117. A Ficus extract of the present invention can be and procedures described in the Examples herein. administered to a Subject in need thereof systemically (e.g., 0114. The term “therapeutically effective amount’ as used intravenously or orally) or locally, (e.g., injected, applied herein refers to an amount of a Ficus extract sufficient to topically, and the like). Conditions affecting specific organs either inhibit angiogenesis or to degrade existing capillary Such as macular degeneration and the like can be more ame networks. The term therefore includes, for example, an nable to local administration of a therapeutic agent because a amount of a Ficus extract sufficient to prevent the growth of high local concentration of the therapeutic agent can be angiogenic vessels found in diseases of tumor growth, dia achieved, which can also, for example, avoid possible side betic retinopathy, psoriasis, retinopathy of prematurity, and effects that can arise due to systemic administration of a preferably to reduce by at least 50%, and more preferably to therapeutic agent. reduce by at least 90%, the amount of angiogensis. The dos 0118. In some embodiments, a method of treating an age ranges for the administration of a Ficus extract are those angiogenesis dependent condition further comprises admin that produce the desired effect. The precise therapeutic dos istering a Ficus extract of the present invention with an age of a Ficus extract of the present invention that is thera another active agent either in the same dosage form or in peutically effective can vary between Subjects (e.g., due to separate and/or divided dosage forms. age, body weight, sex, condition of the Subject, the nature and 0119 The present invention is also directed to methods of severity of the disorder or disease to be treated, and the like). reducing or inhibiting neovessel growth in a human or mam Thus, in some embodiments a therapeutically effective mal, the method comprising administering a composition amount of a Ficus extract of the present invention is not comprising an angiogenesis inhibiting Ficus extract from a specified in advance of administering the Ficus extract to a latex-containing portion of a Ficus variant to a human or Subject in need thereof. In some embodiments, a therapeuti animal in need thereof. cally effective amount of a Ficus extract of the present inven I0120 In some embodiments, the neovessel growth that is tion and can be determined by a caregiver, for example, by a inhibited and/or reduced can be associated with an angiogen physician using, for example, dose titration. The dosage can esis-dependent disease. Such as those listed herein. Thus, in be adjusted by the individual physician in the event that any Some embodiments the present invention is directed to a US 2011/01 65096 A1 Jul. 7, 2011

method of inhibiting or reducing neovessel growth associated istration, which is a product taken by mouth that contains a with an angiogenesis-dependent disease in a humanoranimal "dietary ingredient'. Such as, but not limited to, a vitamin, a in need thereof, the method comprising: administering to the mineral, an herb or other botanical, an amino acid, and Sub Subject a composition comprising a Ficus extract having stances such as an enzyme, an organ tissue, a glandular, a angiogenesis inhibiting activity, wherein the extract is iso metabolite, or an extract or concentrate thereof. lated from a latex-containing portion of a Ficus variant by a process of the present invention. I0128. Non-limiting forms of nutraceutical compositions 0121 Inhibition and/or reduction of neovessel growth can of the present invention include: a tablet, a capsule, a softgel, be quantified using statistical methods known to persons of a gelcap, a liquid, a powder, a solution, a tincture, a Suspen ordinary skill in the art, and as described herein. In some Sion, a syrup, or other forms known to persons of skill in the embodiments, an inhibition or reduction of neoVessel growth art. A nutraceutical composition can also be in the form of a comprises the inhibition of new blood vessel growth in a food. Such as, but not limited to, a food bar, a beverage, a food anatomical region of interest, Such as, but not limited to, the gel, a food Supplement, a powder, a syrup, and combinations eye (e.g., a Subconjunctival region of an eye, and the like), the thereof. mouth, the tongue, the esophagus, the stomach, the intestine, I0129. In some embodiments, a Ficus extract of the present the colon, the rectum, the skin (e.g., the cutaneous, Subcuta invention is administered as a pharmaceutical composition to neous, and/or endometrial regions of the skin), the liver, the a Subject (e.g., a human or animal) in need thereof. As used pancreas, the kidney(s), the bladder, the prostate gland, the herein, a “pharmaceutical composition” refers to a pharma thyroid gland, the lungs, the breast, the ovary(ies), the tes ceutically acceptable composition comprising a Ficus extract ticles, the brain, or a tumor associated therewith, and combi of the present invention. As used herein, "pharmaceutically nations thereof. acceptable' refers to those compounds, materials, composi 0122. In some embodiments, angiogenesis inhibiting tions, and/or dosage forms which arc, within the scope of activity is exhibited as an inhibition or a reduction of neoves Sound medical judgment, Suitable for contact with the tissues sel growth in an in vitro model Such as, but not limited to, a of human beings and animals without excessive toxicity, irri tumor fragment, a placental sample, and the like. In some tation, allergic response, or other problem complications embodiments, angiogenesis inhibiting activity is exhibited as commensurate with a reasonable benefit/risk ratio. In some an inhibition or a reduction of neovessel growth in a human or embodiments, the term “pharmaceutically acceptable' refers animal Subject in need thereof. to excipients that have been approved by a regulatory agency 0123. In some embodiments, administering a Ficus extract of the Federal or a state government or listed in the U.S. of the present invention to a subject inhibits neovessel growth Pharmacopeia or other generally recognized international in a tissue of a human or animal body, or a tumor associated pharmacopeia for use in animals, and more particularly in therewith, by about 10% or more, about 20% or more, about humans. One of skill in the art will recognize that pharma 30% or more, about 50% or more, about 75% or more, about ceutically acceptable excipients can be used in the present 80% or more, about 85% or more, about 90% or more, or invention including those listed in The Handbook of Pharma about 95% or more compared to a subject having a similar ceutical Excipients, 5th Ed., The Pharmaceutical Press and condition that is not treated with the Ficus extract. American Pharmacists Association, London, UK and Wash 0.124. In some embodiments, a Ficus extract of the present ington, D.C. (2006) and Remington. The Science and Prac invention inhibits neovessel growth in a tissue of a human or tice of Pharmacy, Lippincott Williams & Wilkins, 21st Ed. animal body, a tumor associated therewith, or an in vitro (2005), which are incorporated herein by reference in their tissue sample taken therefrom, by about 10% or more, about entirety. 20% or more, about 30% or more, about 50% or more, about 0.130. When a Ficus extract of the present invention is 75% or more, about 80% or more, about 85% or more, about employed as an angiogenesis inhibiting agent in a human or 90% or more, or about 95% or more compared to a tissue or animal, the Ficus extract can be administered alone or in tumor that is not treated with the Ficus extract. combination with pharmacologically acceptable carriers, the proportion of which is determined by the solubility and Compositions Comprising the Ficus Extracts chemical nature of the extract or the composition, chosen 0.125 A Ficus extract of the present invention can be route of administration and standard pharmacological or administered to a subject (e.g., a human or animal) in need nutraceutical practice. thereof in a variety of forms such as, but not limited to, a I0131. In some embodiments, a Ficus extract of the present nutraceutical composition and a pharmaceutical composition invention is present in a nutraceutical orpharmaceutical com (i.e., a pharmaceutically acceptable dosage form). position in a concentration of about 0.5% to about 99%, about 0126. As used herein, a “nutraceutical composition” refers 1% to about 98%, about 5% to about 95%, about 10% to about to any substance that may be considered a food or part of a 90%, about 20% to about 85%, about 50% to about 80%, or food and provides medical or health benefits, including the about 60% by weight of the nutraceutical or pharmaceutical prevention and treatment of disease. In some embodiments, a composition. nutraceutical composition is intended to Supplement the diet 0.132. In some embodiments, a nutraceutical composition and contains at least one or more of the following ingredients: or pharmaceutical composition provides a unit dosage to a a vitamin; a mineral; an herb: a botanical; a fruit; a vegetable; Subject (e.g., a human or animal) in need thereof. As used an amino acid; or a concentrate, metabolite, constituent, or herein, a “unit dosage” refers to an amount of a Ficus extract extract of any of the previously mentioned ingredients; and of the present invention that is sufficient for once-daily, twice combinations thereof. daily, thrice daily, four-times daily dosing, each dose of which 0127. In some embodiments, a nutraceutical composition is administered as a single unit. Representative unit dosage of the present invention can be administered as a "dietary forms of the present invention include, but are not limited to, supplement, as defined by the U.S. Food and Drug Admin a tablet, a capsule, a packet, a lozenge, and the like. US 2011/01 65096 A1 Jul. 7, 2011

0.133 Pharmaceutically acceptable dosage forms of the greater than about 15% after about 1 hour and at least about present invention include, but are not limited to, oral, 50% complete within about 12 hours; not greater than about parenteral, transmucosal, transdermal, Sublingual, intraocu 25% complete after about 1 hour and at least about 50% lar, and pulmonary dosage forms comprising an extract from complete within about 8 hours, or not greater than about 35% a Ficus variant, in particular varieties of Ficus carica L. For complete after about 1 hour and at least about 50% complete example, Suitable pharmaceutically acceptable dosage forms within about 4 hours. include, but are not limited to, tablets, caplets, pellets, cap 0.138. In some embodiments, dissolution of a Ficus extract Sules, shearform matrices, gelcaps, troches, lozenges, syrups, from a dosage form of the present invention can be related to gels, ointments, emulsions, patches, Solutions, dispersions, pharmacokinetic parameters and/or the in Vivo concentration mists, aerosols, and the like that contain a therapeutically of an angiogenesis inhibiting active agent and/or its metabo effective amount of a Ficus extract of the present invention. lite present in the Ficus extract. The in vivo concentration of Parenteral infusions include intramuscular, intravenous, an active agent and its metabolites, as well as pharmacoki intraarterial, or intraperitoneal administration. netic parameters can be determined by sampling the blood 0134) For example, a Ficus extract of the present invention plasma of a Subject after administration of the pharmaceutical can be orally administered in the form of a tablet, a capsule, a dosage forms of the present invention. Pharmacokinetic lozenge, a syrup, a mist, an aerosol, a troche, a strip, a sus parameters that can be measured include, but are not limited pension, a solution, and the like containing excipients such as, to ln(CA), AUC, AUC.M. ln(AUCs) and IntraCV. but not limited to, starch, milk, Sugar, certain types of clay, coloring and flavoring agents, as well as optional dispersants, Therapeutic Packages solubilizers, and the like. A Ficus extract can be injected 0.139. The present invention is also directed to a therapeu parenterally in the form of a sterile solution containing other tic package comprising a Ficus extract enclosed within a Solutes; for example, saline or glucose Sufficient to make a container. The container is Suitable for maintaining the sta Solution isotonic. bility of the Ficus extract for an extended period of time 0135. As used herein, an “excipient” refers to a pharma during storage, shipping, and administration of the Ficus ceutically acceptable Substance, or mixture of Substances, eXtract. that can be used to give desirable physical characteristics to a 0140. As used herein, a “package” refers to a container, pharmaceutical dosage form comprising a Ficus extract of the vessel, and the like Suitable for stably containing, e.g., a present invention. Pharmaceutically acceptable excipients for pharmaceutical or nutraceutical composition comprising a use with the dosage forms include, but are not limited to, Ficus extract of the present invention. A package can com diluents, binders, disintegrants, solubilizers, extended release prise a wrapper, a box, a bottle (i.e., a child safety bottle, a polymers, lubricants, preservatives, inorganic excipients, punctureable bottle, and the like), a Syringe, a peelable plastic glidants, taste-masking excipients, flavorants, flavor enhanc container, and the like, and combinations thereof. ers, and Sweeteners. 0.141. The therapeutic packages of the present invention 0.136 Dosages comprising the therapeutic Ficus extracts can also include a second container for storing the compo of the present invention can vary with the form of adminis nents (i.e., the container of unit dosages, printed matter, etc.) tration and the particular compound chosen. Furthermore, the of the therapeutic packages. The second container can be, for dosage forms comprising the Ficus extracts can vary with the example, a bag, box, envelope or any other container that particular Subject under treatment. For example, treatment would be suitable for use with the present invention. In some can be initiated with Small dosages Substantially less than the cases, it can be desirable to have a small second container optimum dose of a Ficus extract. Thereafter, the dosage is which can be placed in a subject's luggage, pocketbook, increased by Small increments until an optimum effect under briefcase or pocket. the circumstances is reached. In general, a Ficus extract of the 0.142 Packages comprising a nutraceutical composition present invention is administered at a concentration level that of the present invention further include information such as a will generally afford effective results without causing any label that clearly identifies the nutraceutical composition as harmful or deleterious side effects. not being a conventional food or a suitable sole item of a meal 0.137 Oral dosage forms containing a Ficus extract of the or diet. Information accompanying the package further com present invention can be designed to undergo efficient disso prises language representing a nutraceutical compositions as lution either immediately upon administration of the dosage a “food” (i.e., not a “drug or pharmaceutical), as well as form, as a function of time after administration, or as a func identifying any additional Supplement, extract, and the like tion of location within the gastrointestinal tract. As used present in a nutraceutical composition. herein, “dissolution” refers to the process by which a Ficus 0143. In some embodiments, a therapeutic package of the extract dissolves into solution from a dosage form of the present invention further comprises a label directing admin present invention. The dissolution rate of an angiogenesis istering a dosage form comprising a Ficus extract to a human inhibiting active agent can be measured using, for example, a or animal Subject (e.g., a human or animal) in need thereof. USPType I or Type II dissolution apparatus. In some embodi 0144. In some embodiments, a label can comprise printed ments, dissolution of a Ficus extract from a dosage form can matter. “Printed matter can be, for example, one of a book, be measured using, for example, a USP Type I or Type II booklet, brochure or leaflet. Possible formats include, but are Apparatus containing 0.1 NHCl with a paddle speed of 25 not limited to, a bullet point list, a list of frequently asked rpm. In some embodiments, dissolution of a Ficus extract questions (FAQ) or a chart. Additionally, the information to from an immediate release dosage form is at least about 75% be imparted can be illustrated in non-textual terms using complete within about 30 minutes, at least about 80% com pictures, graphics or other symbols. For example, printed plete within about 30 minutes, or at least about 90% complete matter can be in a form prescribed by a governmental agency within about 30 minutes. In some embodiments, dissolution regulating the manufacture, use or sale of pharmaceuticals or of a Ficus extract from an extended release dosage form is not biological products, which notice reflects approval by the US 2011/01 65096 A1 Jul. 7, 2011

agency of the manufacture, sale, or use for human adminis Solvent at reduced pressure using a rotary evaporator to yield tration in treating a subject (e.g., a human or animal) in need a concentrated liquid extract (approximately 25 L), which thereof with a dosage form of the present invention. The was then lyophilized using a freeze drier to yield a paste printed matter can also contain information on the dangers (approximately 1.84 kg), 231. The ratio of dry fruit, 201, to associated with taking an angiogenesis inhibiting active paste, 231, was about 5:1 by weight. The paste, 231, was then agents and/or extracts in excessive amounts, for excessive suspended in deionized water (25 L), and washed with an durations, or in any manner other than that which is recom organic solvent (ethyl acetate:deionized water, 1:1 V/v), 240. mended or approved. In some embodiments, printed matter The washing was conducted three times, and after each wash can be accompanied by a pre-recorded media device and/or a ing the organic solvent layer was removed, and the organic planner. Solvent was evaporated at reduced pressure to yield an 0145 A“pre-recorded media device' can be, for example, organic fraction, (F, approximately 90 g in powder form), a visual media device, such as a videotape cassette, a DVD 242. The aqueous layer (containing a small amount of ethyl (digital video disk), filmstrip, 35 mm movie or any other acetate due to overlapping layers) was concentrated in a visual media device. Alternately, a pre-recorded media device rotary evaporator to completely remove the water and any can be an interactive software application, such as a CD-ROM organic solvent, and then optionally lyophilized to yield an (compact disk-read only memory) or floppy disk. Alternately, aqueous extract (F. 1.75 kg in paste form), 243. Alterna a pre-recorded media device can be an audio media device, tively, the liquid extract can be washed directly with an Such as a record, audiocassette or audio compact disk. The organic solvent, 241, so long as the organic solvent used for information contained on a pre-recorded media device can the washing is at least partially immiscible with the liquid describe the use of a pharmaceutical dosage form of the extract. The concentrated aqueous extract, 243, was further present invention for treating a condition in a Subject (e.g., a purified by diluting with deionized water, 250, to provide a human or animal) in need thereof. A pre-recorded media diluted aqueous extract (having a volume of about 20 L), 251, device can also contain information on the dangers associated which was then applied, 260, to a column (8" diameterx48" with taking an angiogenesis inhibiting active agent in exces length) packed with an adsorbent (15 kg of DOWEX(R) OPTI sive amounts, for excessive durations, or in any manner other PORER 493, The Dow Chemical Co., Midland, Mich.). The than recommended or approved. diluted aqueous extract was passed through the column, 270, 0146 A “planner can be, for example, a weekly, a to yield an eluate (approximately 20 L) and the column was monthly, a multi-monthly, a yearly, or a multi-yearly planner. then washed with additional deionized water (5 L). The The planner can be used as a diary to monitor dosage deionized water eluates were combined to yield a purified amounts, to keep track of dosages administered, or to prepare aqueous fraction (Fo). The purified aqueous fraction was for future events wherein taking a regularly administered concentrated using a rotary evaporator and lyophilized to pharmaceutical dosage form of the present invention can be yield a paste product (1.4 kg), 271. An eluting solvent com difficult. Alternately, the planner can be a calendar which will prising an organic (ethanol: water, 95:5 V/V, 20 L) was then provide a means to monitor when a dosage has been taken and applied to the adsorbent, and eluted from the column to yield when it has not been taken. This type of planner will be an extract that exhibited angiogenesis inhibiting activity (F- particularly useful for Subjects or care providers (e.g., physi 95), 272. The extract was concentrated under reduced pressure cians, nurses, veterinarians, and the like) having unusual and then lyophilized to yield a dry powder (350 g). The ratio schedules for administering medication to themselves (e.g., of the Ficus extract having angiogenesis inhibiting activity non-daily self-administration regimens) or their subjects. (Fos), 272, to the dry portion of the fresh Ficus fruit, 201, Additionally, the planner can be useful for the elderly, chil was approximately 1:27, and the ratio of the Ficus extract dren, or other subject group who can administer medication to having angiogenesis inhibiting activity (Fos), 272, to the themselves and may become forgetful, or when multiple total weight of the fresh fruit, 201, was approximately 1:151. doses must be administered in a single 24 hour period. One skilled in the art will appreciate the variety of planning tools Example 2 that would be appropriate for use with the present invention. 0147 The following examples of extraction, isolation and 0149 An extract exhibiting angiogenesis inhibiting activ screening conditions and parameters arc given for the purpose ity was isolated from Ficus carica L., variety gold, by the of illustrating the present invention and shall not be construed procedure outlined in Example 1. as being limitations on the scope or spirit of the invention. Example 3 Example 1 0150. An extract exhibiting angiogenesis inhibiting activ 0148 An extract exhibiting angiogenesis inhibiting activ ity was isolated from Ficus carica L., variety magnolia, by the ity was isolated from Ficus carica L., variety kadota. FIG. 2 procedure outlined in Example 1. provides a schematic outline of the isolation and purification process, 200. Raw fruit (fresh), 201, of Ficus carica L., vari Example 4 ety kadota fruit (53 kg), containing approximately 82% of 0151. An extract exhibiting angiogenesis inhibiting activ water and 18% solids (or approximately 9.5 kg dry weight) ity was isolated from Ficus carica L., variety louisiana, by the was macerated, 210, using a fruit chopper and mixed with a procedure outlined in Example 1. polar solvent (methanol, 130 L) for 72 hours at room tem perature, to form a liquid-Solid mixture, 211, having liquid Example 5 and solid portions. The mixture was filtered, 220, using What man #4 filter paper and the Solid portion, 221, was discarded. 0152 The Ficus extracts isolated by the procedure out The liquid extract (approximately 230 L), 222, was then lined in Examples 1-3 were screened for angiogenesis inhib concentrated and lyophilized, 230, by first removing the polar iting activity by the following protocols. US 2011/01 65096 A1 Jul. 7, 2011

0153 Discarded portions of fresh human placenta were score of each of the four quadrants, 411,412,413 and 414, is obtained anonymously with the approval of the Louisiana 4, giving a total mean AI of 16 (4+4+4+4–16). Thus, the State University Health Sciences Center's Institutional neovessel structure in FIG. 4A resulted in an AI four times Review Board (New Orleans, La.). Tissues were obtained and less than the neovessel structure in FIG. 4B. These AI data transported to the laboratory in a saline-soaked gauze pad. correlate well with more objective measures of vessel growth, Dissected placental veins were trimmed of placental tissue Such as vessel length or vessel Surface area as determined by and adventitia, and then opened longitudinally to produce a digital image analysis. At the completion of the experiment, flat film having a full thickness of venous tissue. selected wells were fixed in 10% neutral buffered formalin for 0154) A schematic representation of the angiogenesis hematoxylin and eosin staining or for immunohistochemical assay is depicted in FIGS. 3A and 3B. Discs were prepared evaluation for factor VIII. from the placental veins using a sterile 0.2 mm diameter skin 0158 Referring to FIGS.5A, 6A, 7A, 8A, 9A, 10A, 11A, punch. Referring to FIG. 3A, the placental discs were allo 12A, 13A, 14A and 15A, the mean AI was calculated twice cated to wells in a 96-well plate (Corning Inc., Corning, for each set of experiments. In the first calculation, “exclud N.Y.), 300, in a pattern designed to minimize inclusion of one ing Zeros.' quadrants exhibiting no neovessel growth (AI-0) section of the placenta into a specific treatment group. All were excluded from mean AI score. Thus, the “excluding wells, 301, were preloaded with thrombin solution (0.05IU in Zeros' data includes the Angiogenic Index scores from only 1.0 uL/well) (Sigma Chemical Co., St. Louis, Mo.). Wells those quadrants that exhibited neovessel growth. In the sec were allowed to evaporate to dryness. ond calculation, “including Zeros.' neovessel growth in all 0155. A fibrin-thrombin clot was created in each well by quadrants is included in the mean AI score. The AI “including covering the discs with 100 of a clot-forming medium com Zeros' is a parameter that evaluates both initiation and prising human fibrinogen (3 mg/mL) and 6-aminocaproic growth, while the AI “excluding Zeros' evaluates pure angio acid (5.0%) (M-199, Sigma) in HPVAM medium. The genic growth. HPVAM medium comprised Medium 199 and an antibiotic/ 0159. Second, the ability of the preformed blood vessels to antimycotic solution comprising 100 U penicillin, 100 U invade into the fibrin-thrombin clot was assessed over time, streptomycin Sulfate, and 0.25 ug amphotericin/mL (Gibco and the percentage of placental discs that developed invasion BRL, Gaithersburg, Md.). This mixture was placed in a (% I) was calculated using the formula: humidified incubator and allowed to clot at 37° C. in a 5% CO, 95% air environment. A nutrient medium (100 uL) containing the HPVAM supplemented with 20% bovine # of wells exhibiting invasion 100 = % serum (Gibco BRL) was added to the tissue-containing clot. # of wells placed X 2 Drug-treated wells contained the nutrient medium Supple mented with a Ficus extract of the present invention or a control compound. Each well's total volume was 200 uL. An (0160. The data for % I is provided in FIGS. 5B, 6B, 7B, enlargement of an individual well is schematically repre 8B,9B, 10B, 11B, 12B, 13B, 14B and 15B, respectively. sented in FIG.3B. At the center of the well, 311, the placental discs, 312, is shown with its surrounding neovessel structure. Example 6 0156 Placental discs (30 for each dosage regimen) were 0.161 The effect on the angiogenic response of an extract treated with a nutrient medium or a Ficus extract-containing from Ficus carica L., variety kadota, as prepared in Example medium starting on the first day in culture, followed by obser 1, was evaluated by the procedure of Example 5. The protocol Vation of the development of an angiogenic response for 14 described in Example 5 was repeated five times using the days. Representative in vitro concentrations of the Ficus extracts from five different batches of fruit. The angiogenesis extracts present in medium were about 0.001 mg/mL, 0.003 inhibiting data for each of the five batches of fruit from Ficus mg/mL, 0.01 mg/mL, 0.03 mg/mL, 0.1 mg/mL and 0.3 carica L., variety kadota, is provided in FIGS. 5A-5B, mg/mL. An additional tissue sample was incubated with gal 6A-6B, 7A-7B, 8A-8B and 9A-9B, respectively. lic acid (1 mM or 3 mM) as a standard. 0157 Wells were assessed for microcapillary growth on Example 7 day 14 using an inverted phase microscope. Two different assay criteria were evaluated. First, the degree of angiogenic 0162 The effect on the angiogenic response of an extract response was assessed using a semi-quantitative visual rating from Ficus carica L., variety gold, as prepared in Example 1, scale, a schematic representation of which is provided in was evaluated by the procedure of Example 5. The protocol FIGS. 4A and 4B. Referring to FIG. 4A, tissue discs, 400, described in Example 5 was repeated five times using the were visually divided into four quadrants, 401, 402,403 and extracts from five different batches of fruit. The angiogenesis 404. At the center of each quadrant was the placental disc with inhibiting data for each of the three batches of fruit from Ficus its neovascular structure, 405, penetrating into the domains of carica L., variety gold, is provided in FIGS. 10A-10B, 11A each quadrant. Each quadrant was given a numeric score from 11B and 12A-12B, respectively. 0 to 4 based on neovessel length, density, and percentage of the quadrants circumference involved with the angiogenic Example 8 response. Numeric results from the four quadrants were Summed and expressed as an Angiogcnic Index (AI’, 0-16). 0163 The effect on the angiogenic response of an extract In FIG. 4A, the quantitative score of each of the four quad from Ficus carica L., variety magnolia, as prepared in rants, 401, 402,403 and 404, is 1, giving a total mean AI of 4 Example 1, was evaluated by the procedure of Example 5. (1+1+1+1=4). Referring to FIG. 4B, after treatment, the tis The protocol described in Example 5 was repeated five times Sue disc, 410, was again measured to determine the growth of using the extracts from five different batches of fruit. The the neovessel structure, 415. In FIG. 4B, the quantitative angiogenesis inhibiting data for each of the three batches of US 2011/01 65096 A1 Jul. 7, 2011 fruit from Ficus carica L., variety magnolia, is provided in genesis inhibiting activity, rutin can serve as a chemical FIGS. 13 A-13B, 14A-14B and 15A-15B, respectively. marker for the angiogenesis inhibiting Ficus extract of the present invention. Example 9 Example 10 0164. The extract obtained from Ficus carica L., variety 0.167 Compounds present in various extracts and samples kadota (isolated by the process described in Example 1), that isolated by the process of the present invention were charac exhibited angiogenesis inhibiting activity was characterized terized using HPLC. A reference solution (10 mL) was pre by High-Performance Liquid Chromatography (“HPLC) pared by mixing ten compounds that were previously char using a WATERS 600E HPLC system (Waters Corp., Mil acterized as present in the latex-containing portion (e.g., fruit) ford, Mass.), equipped with a 717 auto sampler and a 2996 of Ficus carica L. Variants: psoralen (32 mg/mL), catechin UV-Vis photodiode array detector (having a detector range of (39 mg/mL), rutin (68 mg/mL), Syringin (38 mg/mL), ber 200 nm-400 nm) and using a Cs reverse phase column (250 gapten (16 mg/mL), chlorogenic acid (39 mg/mL), fumaric mm lengthx4.6 mm internal diameter WATERS SYMMETRYR) acid (90 mg/mL), shikimic acid (61 mg/mL), quinic acid (90 Cs column) in combination with a YMCCs guard column mg/mL) and coumaric acid (56 mg/mL) in methanol. The (7.5 mm lengthx4.6 mm internal diameter). The reverse phase resulting solution was filtered through a membrane (pore column contained a spherical packing material having a par size=0.2 mm) and characterized by HPLC by the method ticle size of 3.5um and about 5um, havingapore size of about described in Example 9. 100 A, and having a carbon load of about 19%. The reverse 0168 FIG. 17 provides a graphical representation of the phase column was also end-capped. The eluent was a first chromatograph, 1710, obtained from the reference solution. Referring to FIG. 17, a chart, 1700, shows the HPLC chro eluent, which consisted of HPLC-grade acetonitrile, and a matograph, 1710, (absorbance at 254 nm vs. retention time) second eluent, which consisted of HPLC-grade water con of the reference solution. Peaks in the chromatograph corre taining 2.5% by volume HPLC-grade acetonitrile and 0.3% sponding to Substances having an absorption at 254 nm were by volume phosphoric acid. The concentration of the first observed for quinic acid (1711a, having retention time of eluent and second eluent was provided a total eluent concen about 3.0 minutes); shikimic acid (1711b, having a retention tration of 100% by volume. From 0 minutes to 20 minutes and time of about 3.6 minutes); fumaric acid (1712, having a 59 seconds the first eluent increased linearly from 0% to 10% retention time of about 7.7 minutes); syringin (1713, having a by volume of the total eluent, from 21 minutes to 50 minutes retention time of about 31.6 minutes); chlorogenic acid and 59 seconds the first eluent increased linearly from 10% to (1714a, having a retention time of about 33.3 minutes); cat 20% by volume of the total eluent, from 51 minutes to 65 echin (1714b, having a retention time of about 33.3 minutes); minutes and 59 seconds the first eluent increased linearly coumaric acid (1715, having a retention time of about 46.2 from 20% to 40% by volume of the total eluent, and from 66 minutes); rutin (1716, having a retention time of about 51.3 minutes to 80 minutes and 59 seconds the first eluent minutes); psoralen (1717, having a retention time of about increased linearly from 41% to 60% by volume of the total 74.1 minutes); and bergapten (1718, having a retention time eluent. The column temperature was maintained at 25°C. The of about 79.0 minutes); respectively. injection volume was 10 uL. The flow rate was 1 mL/minute. The pressure of the column increased linearly from 0 minutes Example 11 to about 80 minutes, from a starting pressure of 1,000 psi to a 0169. The HPLC chromatograph for the reference solu final pressure of 3,000 psi. The photodiode array detected tion was compared with several other process products of the peak intensity at 254 nm, and run-to-run variability for peak present invention. Dried fruit of Ficus carica L., variety retention times was about 0.2 minutes. kadota was taken up in absolute methanol and Sonicated for 0.165 Referring to FIG. 16, plot 1610 is a chromatograph 60 minutes to provide a sonicated fraction, F. The soni of the angiogenesis inhibiting extract. Plot 1610 displayed a cated fraction, F, was characterized by HPLC as described series of peaks in the chromatograph having retention times in Example 9, the results of which are depicted in FIG. 17 as of about 4.9 minutes (1611), about 6.0 minutes (1612), about chromatograph 1620. 21.3 minutes (1613), about 22.5 minutes (1614), about 35.0 (0170 Referring to FIG. 17, chromatograph 1720 displays minutes (1615), and about 49.7 minutes (1616). Also multiple peaks having a retention time of about 2-8 minutes, observed were peaks having retention times of about 3.0 1721, as well as two closely spaced peaks having a retention minutes (1617), about 3.2 minutes (1618), about 4.9 minutes time of about 50 minutes, 1726 and 1729. Peak 1726 corre (1619), about 6.0 minutes (1620), about 8.6 minutes (1621), sponds to rutin, present in the chromatograph of the reference about 15.3 minutes (1622), about 17.0 minutes (1623), about solution, 1710, a peak 1716, whereas peak 1729 lacks a 18.0 minutes (1624), about 19.6 minutes (1625), about 27.7 corresponding peak in the reference chromatograph, 1710. minutes (1626), about 28.7 minutes (1627), about 29.3 min utes (1628), about 30.4 minutes (1629), about 33.1 minutes Example 12 (1630), about 34.0 minutes (1631), about 37.0 minutes (1632), about 44.1 minutes (1633), about 45.7 minutes 0171 Whole fresh figs, Ficus carica L., variety kadota, (1634), about 46.3 minutes (1635), about 48.2 minutes were placed in methanol (1:10 w/v fresh fruit:methanol) and (1636), about 53.0 minutes (1637), and about 57.3 minutes allowed to incubate for 72 hours at room temperature. The (1638). resulting Supernatant was decanted from the Solid portion and 0166 Also observed in the chromatograph was a peak, then filtered (filter pore size=20 um). The filtered solution 1640, having a retention time of about 51.0 minutes and was then concentrated using a rotary evaporator. The result corresponding to rutin. While rutin does not exhibit angio ing crude solid fraction, F, was dissolved in water and char US 2011/01 65096 A1 Jul. 7, 2011 acterized by HPLC using the procedure described Example 9. (0179 Referring to FIG. 18, plot 1820 is a chromatograph The chromatograph of the dissolved solid is displayed in FIG. of the organic eluate fraction (F9s) isolated from Ficus 17. carica L., variety kadota (as described in Example 1). Plot (0172 Referring to FIG. 17, the chromatograph, 1730, of 1820 displayed a series of peaks, 1821, having retention times the dissolved solid, displays a series of peaks having retention of about 2-8 minutes, a series of peaks, 1822, having retention times of about 2-8 minutes, 1731, including peak 1732, which times of about 15-40 minutes, as well as peak 1826, which corresponds to fumaric acid. Chromatograph 1730 also dis corresponds to rutin. Also observed were peaks 1823 and plays a pair of peaks having a retention time of about 50 1824, which do not correspond to any peaks in the chromato minutes, 1736 and 1739, the former, 1736, which corresponds graph of the reference solution, 1810. to rutin, the latter, 1739, lacking a corresponding peak in the 0180 Referring to FIG. 18, plot 1830 is a chromatograph reference chromatograph, 1710. of the organic eluate fraction (F9s) isolated from Ficus carica L., variety magnolia (as described in Example 3). Example 13 Chromatograph 1830 displayed peaks similar to those observed for in the chromatograph of the organic eluate frac 0173 Fractions resulting from isolation of extracts from tion isolated from fruit of the Ficus carica L. kadota variety, the latex-containing portion of Ficus carica L., variety plot 1820. Specifically also observed in plot 1830 were a kadota, as described in Example 1 were further characterized series of peaks, 1831, having retention times of about 2-8 by HPLC according to the procedure of Example 9, the chro minutes, a series of peaks, 1832, having retention times of matographs of which are displayed in FIG. 17. Referring to about 15-40 minutes, and peak 1836, corresponding to rutin. FIG. 17, the following chromatographs are displayed: plot Also observed in plot 1830 were peaks 1833 and 1834, which 1740 is a chromatograph of the organic fraction, 241 (F) in do not correspond to any peaks in the chromatograph of the FIG. 1; plot 1750 is a chromatograph of the aqueous fraction, 242 (F) in FIG. 1; plot 1760 is a chromatograph of the water reference solution, 1810. eluate fraction, 271 (Fo) in FIG. 1; and plot 1770 is a chromatograph of the organic eluate fraction, 272 (F-9s) in Example 15 FIG 1. (0174 Referring to FIG. 17, plot 1740 is a chromatograph 0181. The water-soluble portion of latex from Ficus of the organic fraction (F), and displays a series of peaks, carica L. (37% by weight of the raw fruit) was isolated by the 1741, having retention times of about 2-8 minutes, as well as procedure of Example 1 to provide a water-soluble fraction a peak 1744, corresponding to chlorogenic acid or catechin; a (F). The aqueous fraction, F. was screened for angiogen peak 1745, corresponding to coumaric acid; and a peak 1746, esis inhibiting activity by the procedure described in Example corresponding to rutin. The chromatograph, 1740, also dis 5. The aqueous fraction, F,ag was a moderate inhibitor of plays peaks 1748 and 1749 that lack corresponding peaks in angiogenesis. the reference solution chromatograph, 1710. (0182. The aqueous fraction, F, was also characterized by (0175 Referring to FIG. 17, plot 1750 is a chromatograph HPLC using the procedure described in Example 9. Referring of the aqueous fraction (F), and displays a series of peaks to FIG. 18, plot 1840 is a chromatograph of the water-soluble similar to the chromatograph, 1720, of the sonicated fraction, fraction F, which displays peaks 1843 (having a retention (F), isolated by the procedure of Example 10. time of about 34.0 minutes) and 1844 (having a retention time (0176 Referring to FIG. 17, plot 1760 is a chromatograph of about 49.6 minutes). Peaks having similar retention times of the water eluate fraction (Fo), and displayed peaks 1761, were also observed in plots 1820 and 1830, while these peaks having retention times of about 2-25 minutes. were not observed in the chromatograph of the reference (0177 Referring to FIG. 17, plot 1760 is a chromatograph Solution. The compounds corresponding to peaks 1843 and of the organic eluate fraction (F-9s) displayed peaks 1771, 1844 therefore likely contribute to the angiogenesis inhibit having retention times of about 2-8 minutes, a series of peaks, ing activity of the latex extract. 1773, having retention times of about 15-40 minutes, as well a peak 1776, corresponding to rutin, and a peak 1779, corre Example 16 sponding to an unidentified compound. The latter peak, 1779, is also present in chromatographs 1720, 1730, 1740, 1750 and (0183) The organic eluate fraction, Fos, isolated from the 1760. fruit of Ficus carica L., variety kadota, as described in Example 1 (i.e., fraction 132 in FIG.1) was further purified by Example 14 precipitation in ethanol. The solid extract (about 1 g) was added to ethanol (95% ethanol, industrial grade, 20 mL), the (0178 FIG. 18 provides a comparison of the HPLC chro Suspension was Sonicated for 30 minutes at room tempera matograph of the reference solution with several additional ture. The resulting Supernatant was filtered using Whatman process products of the present invention. Referring to FIG. #4 paper (having a pore size of about 20 um). The liquid was 18, a chart, 1800, is provided that includes plot 1810, the then dried using a rotary evaporator, and freeze-dried. The chromatograph of the reference solution (absorbance at 254 resulting Solid was dark green in color. This procedure nm vs. retention time; also displayed in FIG. 17 as plot 1710). removed approximately 18% by weight of the material Peaks in the chromatograph, 1810, corresponding to sub present in F-9s, the resulting fraction, Fose, having a stances having an absorption at 254 nm were observed for purity about 1.2 times higher than the starting material. quinic acid (1811a); shikimic acid (1811b); fumaric acid (0184 The precipitate fraction, Fose, was characterized (1812); syringin (1813); chlorogenic acid (1814a); catechin by HPLC using the procedure described in Example 9. Refer (1814b); coumaric acid (1815); rutin (1816); psoralen (1817); ring to FIG. 18, plot 1850 is a chromatograph of Fose, andbergapten (1818); respectively. which displays peaks 1851 having retention times of about US 2011/01 65096 A1 Jul. 7, 2011

2-8 minutes, and peaks 1853 and 1854, which are also present plaques. A 1% Ficus extract isolated by the procedures out in other fractions that exhibited angiogenesis inhibiting activ lined in Example 1 (organic eluate fraction (F-9s) isolated 1ty. from Ficus carica L., variety Kadota—see, e.g., plot 1920 Example 17 of FIG. 19) was prepared in a sterile cream base for applica (0185. The precipitate fraction, Fose, isolated in tion to affected dermal lesions on a twice-daily basis. FIG. Example 16 was further purified by treating the Ficus extract 20A shows the pre-treatment appearance of the psoriatic with activated carbon. The extract (5 g) was dissolved in plaques on the left forearm of a male subject. This subject had ethanol (95% ethanol, industrial grade, 100 mL) and mixed been afflicted with psoriasis for many years, and was refrac with active carbon (5 g, Sigma Chemical, St. Louis, Mo.). The tory to treatment with commonly-prescribed antipsoriatic resulting mixture was Sonicated for 30 minutes at room tem medications. FIG.20B shows the same forearm 39 days later, perature, then filtered using Whatman #4 paper. The resulting following twice-daily application of the Ficus extract, which Solution was dried using a rotary evaporator and then freeze produced marked reduction of psoriatic scales. FIG. 20O dried to yield a solid extract, Fosc, having a mass that was shows the same forearm after an additional 37 days (76 days about 47% of the mass of the starting material. Thus, the after treatment was initiated), showing dramatic reduction of treating with activated carbon removed about 53% by weight the psoriatic lesions. After the photograph of FIG. 20O was of the extract material. The purified extract was light-green in taken, treatment with Ficus extract was discontinued to esti color. mate the time to relapse. Psoriatic lesions did not recur until 0186. The activated carbon fraction, Fos, was charac about 4 months following discontinuation of treatment. terized by HPLC using the procedure described in Example 9. Referring to FIG. 18, plot 1860 is a chromatograph of Fos, Example 19 which displays peaks 1861 having retention times of about 2-8 minutes, as well as numerous low-intensity peaks 1862, 0190. Fibrin clots were prepared in 96-well plates (Corn having retention times of about 15-50 minutes, which are also ing Inc., Corning, N.Y.). Each well was preloaded with a present in other fractions that exhibited angiogenesis inhib human thrombin solution (0.05 IU/mL.; in 2.0 uL/well), iting activity. (Sigma Chemical Company, St Louis, Mo.). A pre-coagula 0187. The HPLC characterization of the angiogenesis tion solution was prepared by dissolving fibrinogen (0.3%: inhibiting fractions indicates that about eight out of the ten Sigma) and 6-aminocaproic acid (0.5%; Sigma) in serum compounds present in the reference Solution are not present in free growth medium consisting of medium 199 (Mediatech, an angiogenesis inhibiting extract of the present invention. Herndon Va.) Supplemented with an antibiotic-antifungal Two compounds that were present in several of the extracts solution (Gibco/Invitrogen, Grand Island, N.Y.; 100 Units/ isolated by the procedures described herein contained mL of Sodium penicillin G, 100 ug/mL of streptomycin Sul fumaric acid and rutin. Fumaric acid was present in the crude fate, and 25ug of amphotericin B) and buffered to a pH of 7.6. solid fraction (Fs, Example 11), the organic fraction (F), 0191 Tumor specimens were placed in chilled serum the aqueous fraction (F), and the aqueous eluate fraction containing growth media (medium 199 with 20% fetal bovine (Fo), but not the organic eluate fraction (F-9s) that exhib serum (FBS), Gibco/Invitrogen) and kept under refrigeration ited angiogenesis inhibiting activity. Thus, the active fraction until they were processed. The specimens were processed did not contain fumaric acid, and this compound is therefore within 24 hours of Surgical harvesting. Minced tumor frag not responsible for the angiogenesis inhibiting activity of the ments approximately 2 mm in diameter and 1 mm thick were extract of the present invention. Rutin was present in the crude prepared and rapidly embedded within the fibrin clots. Each solid fraction (Fs, Example 11), the organic fraction (F), tumor fragment was placed in the center of a thrombin-treated the aqueous fraction (F), and the organic eluate fraction well. The pre-coagulation solution (0.1 mL) was layered over (F-9s) that exhibited angiogenesis inhibiting activity, but the tumor fragments. Clot formation took place within 30 to not the aqueous eluate fraction (Fo). Therefore, in addition 60 minutes at 37°C. in a humidified environment using 6% to the angiogenesis inhibiting fraction, rutin is also present in CO. 94% air. The tumor-containing clot was then Supple several other fractions that did not exhibit angiogenesis inhib mented with 100 uL of a nutrient growth medium (medium iting activity. 199 supplemented with antibiotics, antimycotic, and 20% 0188 FIG. 19 provides a chart, 1900, comparing plots FBS; control medium) or 100 uL of nutrient growth medium 1920 and 1940, which are HPLC chromatographs of the containing a drug. The drugs were present at the concentra organic eluate fraction (F-9s) isolated from Ficus carica L., tions shown in FIGS. 21, 22, and 23. Total well volume was variety kadota (as described in Example 1) and the water 200 uL. Nutrient or drug treatments were added on day 0 and soluble fraction, F, respectively. (Also shown in FIG. 18 as replenished on day 7. The cultures were maintained for 14 chromatographs 1820 and 1840, respectively.) As discussed days. above, both of these extracts exhibited angiogenesis inhibit 0.192 The visual evaluation of all wells was performed ing activity. Peaks common to both samples are highlighted using an inverted phase-contrast microscope. Tumor frag by vertical dashed lines (------). The plots show that peaks ments were graded using two criteria: initiation of angiogenic 1941, having retention times of about 4-8 minutes; peaks response percent initiation, (%. 1) and the degree of angio 1922, having retention times of about 30 minutes; peak 1923, genic neovessel development, angiogenic index (AI). Ini having a retention time of about 35 minutes; peak 1924, tiation of an angiogenic response was defined as the develop having a retention time of about 37 minutes; and peaks 1925 ment of three or more sprouts around the periphery of the and 1926, having retention times of about 50 minutes are disk, visible at 10x magnification. Percent initiation (%I) is a present in both samples. ratio calculated from the number of wells that developed an angiogenic response divided by the total number of wells Example 18 prepared (x100). For the angiogenic index (AI), each well (0189 FIGS. 20A, 20B, and 20O illustrate the results containing a tumor fragment was visually divided into four obtained from topical application of Ficus extract to psoriatic quadrants and each quadrant was rated on a 0 to 4 scale for the US 2011/01 65096 A1 Jul. 7, 2011 amount (length, density, and percentage of the circumference tine 54 nM. Valproic Acid 1 mM, Sweet LeafTea 0.5 mg/mL, involved in the angiogenic response) of angiogenic growth. A Black Raspberry 1 mg/mL, and Fig (Ficus) extract 1 mg/mL. total score (sum of the four quadrants) of 0 to 16 was created Controls included media alone (Control), and Gallic Acid (1 for each well. The angiogenic index of individual wells was mM; total inhibition). In this specimen, the angiogenic then averaged in one of two manners: 1) average of wells that response of the control (untreated) was significant (angio had an AI of 1 or greater (AI growth), 2) average of all wells genic index of 9.2) while the angiogenic response with drug prepared (AI overall angiogenic response). The first growth treatment varied. Fig extract completely inhibited angiogen based calculation includes only wells that initiated an angio esis in this specimen. genic response and excludes wells that did not sprout neoves sels; it is considered a quantification of pure neovessel 0.196 FIG. 23 shows the effects of various drugs on sar growth. The second calculation averages all wells regardless coma. Fragments from a sarcoma were embedded in fibrin of whether or not an angiogenic response was observed. clots. The fragments were treated with various drugs, 30 wells Because this value includes all wells, it incorporates both the per treatment group. On day 14, fragments were examined degree of new vessel initiation as well as the degree of neoves microscopically and scored for an angiogenic response (an sel growth thus yielding a score that reflects the overall angio giogenic index=0-16). Data presented are the Mean angio genic response. genic response +2 S.E.M. for each drug treatment (n=30). 0193 Previous experience with culture techniques in a Treatments included PTK787 20 uM, Gleevec 2.5uM, SOM human placental vein-based model and a variety of tumor 23010 nM, Interferon C. 0.25 IU/mL, CP 673451, Avastin 168 based models showed an excellent correlation between uM, pothilone B (Epo) 10 nM, RAD001 10 nM, 2 Methox observer scores and more objective ratings, such as vessel yestradiol 1 uM, Taxol 10 nM, Colchicine 100 uM, Vincris length (mm) or a total vessel Surface area (mm) determined tine 54 nM. Valproic Acid 1 mM, Sweet LeafTea 0.5 mg/mL, by digital image analysis. In addition, comparisons of Black Raspberry 1 mg/mL, and Fig (Ficus) extract 1 mg/mL. observer scores generated by multiple graders have a high Controls included media alone (Control), and Gallic Acid (1 degree of correlation. The visual rating system was utilized mM; total inhibition). In this specimen, the angiogenic because neovessel growth could be more rapidly evaluated by response of the control (untreated) was low (angiogenic index visual inspection by a trained grader than with digital image of 1.3) and the angiogenic response with drug treatment var analysis. ied. Fig extract completely inhibited angiogenesis in this 0194 FIG. 21 shows the overall effect of treatment with specimen. Ficus extract on angiogenesis in carcinoid tumors. The data reflect the effect on both initiation and neovessel growth (overall effect; all wells plated were included in the analysis) CONCLUSION in individual specimens. Data were sorted in ascending order based on the control response value (filled circles) and the 0197) These examples illustrate possible embodiments of corresponding drug-treated value is indicated (filled tri the present invention. While various embodiments of the angles). AS FIG. 21 shows, Ficus extract at 1 mg/mL is a present invention have been described above, it should be highly effective inhibitor of angiogenesis in human carcinoid understood that they have been presented by way of example tumors compared to responses obtained in untreated control only, and not limitation. It will be apparent to persons skilled samples from the same specimen. In FIG. 21, a theoretical in the relevant art that various changes in form and detail can efficacy was estimated using the slopes of the control and be made therein without departing from the spirit and scope of drug-treated specimens; trendlines and their associated equa the invention. Thus, the breadth and scope of the present tions are shown. Values on the X-axis (individual tumor speci invention should not be limited by any of the above-described mens) were ordered from the lowest to highest control exemplary embodiments, but should be defined only in accor response for eachangiogenic parameter; the drug-treated val dance with the following claims and their equivalents. All the ues were then paired with the control value. The slopes, various embodiments or options described herein can becom obtained from the trendline equations, provide a numerical bined in any and all variations. estimate of angiogenic potential (control) or response (drug 0198 All references cited in this specification are herein treated) for the tumor sample population evaluated in this incorporated by reference as though each reference was spe series. The sloperatio (drug/control) relates the drug effect, cifically and individually indicated to be incorporated by be it stimulation or inhibition, to the normal control angio reference. The citation of any reference is for its disclosure genic response for this tumor sample. The theoretic efficacy prior to the filing date and should not be construed as an was calculated as (1-drug slope/control slope)x100. A admission that the present invention is not entitled to antedate higher value Suggests greater antiangiogenic efficacy. such reference by virtue of prior invention. 0.195 FIG.22 shows the effects of various drugs on neu (0199. It will be understood that each of the elements roendocrine lymph node metastasis. Fragments from a neu described above, or two or more together may also find a roendocrine lymph node metastasis were embedded in fibrin useful application in other types of methods differing from clots. The fragments were treated with various drugs, 30 wells the type described above. Without further analysis, the fore per treatment group. On day 14, fragments were examined going will so fully reveal the gist of the present invention that microscopically and scored for an angiogenic response (an others can, by applying current knowledge, readily adapt it giogenic index 0-16). Data presented are the Mean angio for various applications without omitting features that, from genic response +2 S.E.M. for each drug treatment (n=30). the standpoint of prior art, fairly constitute essential charac Treatments included PTK787 20 uM, Gleevec 2.5 LM, SOM teristics of the generic or specific aspects of this invention set 23010 nM, Interferon C. 0.25 IU/mL, CP 673451, Avastin 168 forth in the appended claims. The foregoing embodiments are uM, Epothilone B (Epo) 10 nM, RAD001 10 nM, 2 Methox presented by way of example only; the scope of the present yestradiol 1 uM, Taxol 10 nM, Colchicine 100 uM, Vincris invention is to be limited only by the following claims. US 2011/01 65096 A1 Jul. 7, 2011

What is claimed is: a) wherein a first eluent is acetonitrile and a second eluent 1. A process for isolating a Ficus extract having angiogen is water containing about 0.3% phosphoric acid and esis inhibiting activity, the process comprising: about 2.5% acetonitrile, the concentration of the first a) mixing a latex-containing portion of a Ficus variant with eluent and second eluent is 100% by volume, and from 0 a polar solvent to provide a liquid extract; minutes to about 20 minutes the first eluent increases b) washing the liquid extract with an organic solvent to linearly from 0% to about 10% by volume, from about provide an aqueous extract; and 20 minutes to about 50 minutes the first eluent increases c) fractionating the aqueous extract to provide a Ficus linearly from about 10% to about 20% by volume, from extract having angiogenesis inhibiting activity. about 50 minutes to about 65 minutes the first eluent 2. The process of claim 1, wherein the latex-containing increases linearly from about 20% to about 40% by portion of the Ficus variant comprises at least a portion of a Volume, and from about 65 minutes to about 80 minutes fruit of the Ficus variant. the first eluent increases linearly from about 40% to 3. The process of claim 1, further comprising macerating about 60% by volume; and the latex-containing portion of the Ficus variant. b) wherein the column temperature is about 25° C.; the 4. The process of claim 1, wherein the fractionating com injection volume is about 10 uL: the flow rate is about 1 prises applying the aqueous extract to an adsorbent, and elut mL/minute; from 0 minutes to about 80 minutes the ing the Ficus extract having angiogenesis inhibiting activity pressure increases linearly from about 1,000 psi to about from the adsorbent with an eluting solvent. 3,000 psi; and detection is at about 254 nm. 5. The process of claim 4, wherein the adsorbent is porous 21. The Ficus extract of claim 20, wherein the Cs reverse and has an average pore size of about 20 nm or less. phase column comprises a packing material having a particle 6. The process of claim 4, wherein the eluting solvent size of about 3 um to about 5um, a pore size of about 100 A. comprises an organic. and a carbon loading of about 19%. 7. The process of claim 4, wherein the eluting solvent 22. A Ficus extract prepared by the process of claim 1, comprises about 95% ethanol and about 5% water. having a chromatograph Substantially in accordance with 8. The process of claim 1, further comprising adding the FIG. 16 when subjected to HPLC using a Cs reverse phase Ficus extract having angiogenesis inhibiting activity to an column having an internal diameter of about 4.6 mm and a alcoholic Solution to provide a precipitate having angiogen length of about 250 mm: esis inhibiting activity. a) wherein a first eluent is acetonitrile and a second eluent 9. The process of claim 1, further comprising treating the is water containing 0.3% phosphoric acid and 2.5% Ficus extract having angiogenesis inhibiting activity with acetonitrile, the concentration of the first eluent and activated carbon. second eluent is 100% by volume, and from 0 minutes to 10. The process of claim 1, wherein the latex-containing about 20 minutes the first eluent increases linearly from portion of the Ficus variant comprises a Ficus carica fruit. 0% to about 10% by volume, from about 20 minutes to 11. The process of claim 1, wherein the polar solvent is a about 50 minutes the first eluent increases linearly from C-C compound comprising a heteroatom selected from: N. about 10% to about 20% by volume, from about 50 O, P, S, and combinations thereof. minutes to about 65 minutes the first eluent increases 12. The process of claim 1, wherein the polar solvent is a linearly from about 20% to about 40% by volume, and Solvent selected from: a C-C alcohol, a C-Co ether, from about 65 minutes to about 80 minutes the first Cs-Co aldehyde, a C-Coketone, a C-Co carboxylic acid, eluent increases linearly from about 40% to about 60% a C-Co ester, a C-Co amine, a C-Cs amide, and combi by volume; and nations thereof. b) wherein the column temperature is about 25° C.; the 13. The process of claim 1, wherein the polar solvent has a injection volume is about 10 uL: the flow rate is about 1 boiling point of about 200° C. or less. mL/minute; from 0 minutes to about 80 minutes the 14. The process of claim 1, further comprising filtering the pressure increases linearly from about 1,000 psi to about liquid extract. 3,000 psi; and detection is at about 254 nm. 15. The process of claim 1, further comprising removing at 23. The Ficus extract of claim 22, wherein the Cs reverse least a portion of the polar solvent from the liquid extract. phase column comprises a packing material having a particle 16. The process of claim 1, wherein the washing further size of about 3 um to about 5um, a pore size of about 100 A. comprises dissolving the liquid extract in water. and a carbon loading of about 19%. 17. The process of claim 1, further comprising freeze 24. A process for obtaining a Ficus extract having angio drying one or more of the extracts. genesis inhibiting activity in a human or animal, the process 18. A Ficus extract prepared by the process of claim 1, comprising: wherein the Ficus extract is Substantially lacking cytotoxic a) preparing an aqueous extract from a latex-containing activity. portion of a Ficus variant; fractionating the aqueous 19. A Ficus extract prepared by the process of claim 1, extract; and wherein the Ficus extract is substantially free from one or b) selecting as the Ficus extract having angiogenesis inhib more of shikimic acid, fumaric acid, Syringin, chlorogenic iting activity in a human or animal, a Substantially pro acid, catechin, coumaric acid, psoralen, and bergapten. tein-free fraction containing one or more compounds 20. A Ficus extract prepared by the process of claim 1, having a molecular weight of about 200 Da to about having a chromatograph having peaks at about 4.9 minutes, 2,000 Da, wherein the Ficus extract exhibits angiogen about 6.0 minutes, about 21.3 minutes, about 22.5 minutes, esis inhibiting activity. about 35.0 minutes, and about 49.7 minutes, when subjected 25. The process of claim 24, wherein the angiogenesis to HPLC using a Cs reverse phase column having an internal inhibiting activity is exhibited as an inhibition or a reduction diameter of about 4.6 mm and a length of about 250 mm: of neovessel growth in an in vitro model. US 2011/01 65096 A1 Jul. 7, 2011 20

26. The process of claim 24, wherein the angiogenesis size of about 3 um to about 5um, a pore size of about 100 A. inhibiting activity is exhibited as an inhibition or a reduction and a carbon loading of about 19%. of neoVessel growth in a human or animal Subject in need 33. Use of a Ficus extract comprising rutin, wherein the thereof. Ficus extract has angiogenesis inhibiting activity, in the 27. The process of claim 24, wherein the Ficus extract has manufacture of a medicament for reducing or inhibiting angiogenesis inhibiting activity in the micromolar range. neovessel growth. 28. The process of claim 24, wherein the Ficus extract is 34. The method of claim 33, wherein the Ficus extract is substantially free from one or more of: shikimic acid, fumaric substantially free from one or more of: shikimic acid, fumaric acid, Syringin, chlorogenic acid, catechin, coumaric acid, acid, Syringin, chlorogenic acid, catechin, coumaric acid, psoralen, and bergapten. psoralen, and bergapten. 29. A Ficus extract obtained by the process of claim 24, 35. The method of claim 33, wherein the medicament is a having a chromatograph having peaks at about 4.9 minutes, nutraceutical composition. about 6.0 minutes, about 21.3 minutes, about 22.5 minutes, 36. The method of claim 35, wherein the nutraceutical about 35.0 minutes, and about 49.7 minutes, when subjected composition is a food preparation selected from: a food bar, a to HPLC using a C reverse phase column having an internal beverage, a food gel, a food Supplement, a powder, and a diameter of about 4.6 mm and a length of about 250 mm: syrup. a) wherein a first eluent is acetonitrile and a second eluent 37. The method of claim 35, wherein the nutraceutical is water containing about 0.3% phosphoric acid and composition is formulated into a form selected from: a tablet, about 2.5% acetonitrile, the concentration of the first a capsule, a softgel, a gelcap, a liquid, a powder, a Solution, a eluent and second eluent is 100% by volume, and from 0 Suspension, a syrup, and combinations thereof. minutes to about 20 minutes the first eluent increases 38. The method of claim 33, wherein the medicament is a linearly from 0% to about 10% by volume, from about pharmaceutical composition formulated into a pharmaceuti 20 minutes to about 50 minutes the first eluent increases cal dosage form. linearly from about 10% to about 20% by volume, from 39. The method of claim 38, wherein the pharmaceutical about 50 minutes to about 65 minutes the first eluent dosage form is a tablet, a caplet, a pellet, a capsule, a gelcap. increases linearly from about 20% to about 40% by a troche, a lozenge, a syrup, a gel, an ointment, an emulsion, Volume, and from about 65 minutes to about 80 minutes a patch, a solution, a dispersion, a mist, or an aerosol. the first eluent increases linearly from about 40% to 40. The method of claim 33, wherein the neovessel growth about 60% by volume; and is associated with a disease. b) wherein the column temperature is about 25° C.; the 41. The method of claim 40, wherein the disease is selected injection volume is about 10 uL: the flow rate is about 1 from the group consisting of cancer, infectious diseases, mL/minute; from 0 minutes to about 80 minutes the including mycobacterial infections, infections of the retina, pressure increases linearly from about 1,000 psi to about presumed ocular histoplasmosis, and infections causing 3,000 psi; and detection is at about 254 nm. retinitis or choroiditis; autoimmune disorders; benign 30. The Ficus extract of claim 29, wherein the Cs reverse tumors, e.g., haemangiomas (including infantile haemangio phase column comprises a packing material having a particle mas, capillary haemangiomas, and cavernous haemangio size of about 3 um to about 5um, a pore size of about 100 A. mas), functional endocrine tumors, acoustic neuromas, neu and a carbon loading of about 19%. rofibromas, trachomas, and pyogenic granulomas; atherosclerosis and atherosclerotic plaques: ocular angio 31. A Ficus extract obtained by the process of claim 24, genic diseases, e.g., diabetic retinopathy, retinopathy of pre having a chromatograph Substantially in accordance with maturity, macular degeneration, corneal graft rejection, FIG. 16 when subjected to HPLC using a Cs reverse phase neovascular glaucoma, retrolental fibroplasia, rubeosis, ret column having an internal diameter of about 4.6 mm and a inoblastoma, persistent hyperplastic vitreous syndrome, cho length of about 250 mm: roidal or corneal neovascularization, Venous occlusion, uvei a) wherein a first eluent is acetonitrile and a second eluent tis, vitritis, Eales disease, Behcet’s disease, proliferative is water containing about 0.3% phosphoric acid and vitreoretinopathy, ocularischemic syndrome, and pterygium; about 2.5% acetonitrile, the concentration of the first myopia; optic pits; Best disease; Stargardt's macular dystro eluent and second eluent is 100% by volume, and from 0 phy; pars planitis; chronic retinal detachment; hyperviscosity minutes to about 20 minutes the first eluent increases syndrome; rheumatoid arthritis; psoriasis; warts; allergic der linearly from 0% to about 10% by volume, from about matitis; blistering disease; Karposi sarcoma; delayed wound 20 minutes to about 50 minutes the first eluent increases healing; endometriosis; uterine bleeding; ovarian cysts; ova linearly from about 10% to about 20% by volume, from rian hyperstimulation; vasculogenesis; granulations; hyper about 50 minutes to about 65 minutes the first eluent trophic Scars (keloids); nonunion fractures; Scleroderma; tra increases linearly from about 20% to about 40% by choma; vascular adhesions; vascular malformations; Volume, and from about 65 minutes to about 80 minutes DiGeorge syndrome; transplantarteriopathy; restenosis; obe the first eluent increases linearly from about 40% to sity; myocardial angiogenesis; coronary collaterals; cerebral about 60% by volume; and collaterals; arteriovenous malformations; ischemic limb b) wherein the column temperature is about 25° C.; the angiogenesis; primary pulmonary hypertension; pulmonary injection volume is about 10 uL: the flow rate is about 1 edema; asthma, nasal polyps; inflammatory bowel disease; mL/minute; from 0 minutes to about 80 minutes the periodontal disease; ascites; peritoneal adhesions; Osler-We pressure increases linearly from about 1,000 psi to about ber-Rendu syndrome (hereditary hemorrhagic telangiecta 3,000 psi; and detection is at about 254 nm. sia); plaque neovascularization; telangiectasia; hemophiliac 32. The Ficus extract of claim 31, wherein the Cs reverse joints; synovitis; osteomyelitis; osteophyte formation; phase column comprises a packing material having a particle angiofibroma; fibromuscular dysplasia; wound granulation; US 2011/01 65096 A1 Jul. 7, 2011

Crohn's disease; peptic ulcer, malignant tumor growth pressure increases linearly from about 1,000 psi to about beyond 2 mm in diameter, sickle cell anemia; Sarcoidosis: 3,000 psi; and detection is at about 254 nm. syphilis; pseudoxanthoma elasticum, Paget’s disease; pri 48. The Ficus extract of claim 46, further comprising rutin, mary hyperparathyroidism; secondary hyperparathyroidism; wherein the rutin appears as a peak in the chromatograph tertiary hyperparathyroidism; arterial occlusion; carotid obstructive disease; Lyme disease; systemic lupus erythema having a retention time of about 51.0 minutes. tosis; toxoplasmosis; trauma; Wegner's granulomatosis: 49. The Ficus extract of claim 46, wherein the Cs reverse post-laser complications; and combinations thereof. phase column comprises a packing material having a particle 42. The method of claim 41, wherein the disease is psoria size of about 3 um to about 5um, a pore size of about 100 A. S1S. and a carbon loading of about 19%. 43. The method of claim 41 wherein the disease is cancer. 50. The Ficus extract of claim 46, wherein the HPLC 44. A method of Screening a Ficus extract having angio chromatograph further comprises a chromatograph peak of at genesis inhibiting activity, the method comprising: least one of about 3.0 minutes, about 3.2 minutes, about 4.9 a) isolating a Ficus extract having angiogenesis inhibiting minutes, about 6.0 minutes, about 8.6 minutes, about 15.3 activity by a process comprising: mixing a latex-con minutes, about 19.6 minutes, about 27.7 minutes, about 28.7 taining portion of a Ficus variant with a polar solvent to minutes, about 29.3 minutes, about 30.4 minutes, about 33.1 provide a liquid extract; minutes, about 34.0 minutes, about 37.0 minutes, about 44.1 b) washing the liquid extract with an organic solvent to minutes, about 45.7 minutes, about 46.3 minutes, about 48.2 provide an aqueous extract; and fractionating the aque minutes, about 53.0 minutes, about 57.3 minutes, and com ous extract to provide an extract having angiogenesis binations thereof. inhibiting activity; 51. The Ficus extract of claim 46, wherein the HPLC c) applying the Ficus extract having angiogenesis inhibit chromatograph is substantially free from a chromatograph ing activity to a tumor; and measuring neovessel growth peak selected from: about 7.7 minutes, about 31.6 minutes, from the tumor. about 74.1 minutes, about 79.0 minutes, and combinations 45. A composition comprising a Ficus extract and rutin, thereof. wherein the Ficus extract exhibits angiogenesis inhibiting activity, and wherein the composition is Substantially free 52. A Ficus extract having angiogenesis inhibiting activity from quinic acid. and having a chromatograph Substantially in accordance with 46. The composition of claim 44, wherein the composition FIG. 16 when subjected to HPLC using a Cs reverse phase is substantially free from at least one of shikimic acid, column having an internal diameter of about 4.6 mm and a fumaric acid, Syringin, chlorogenic acid, catechin, coumaric length of about 250 mm: acid, psoralen, and bergapten. a) wherein a first eluent is acetonitrile and a second eluent 47. A Ficus extract having angiogenesis inhibiting activity is water containing about 0.3% phosphoric acid and and having a chromatograph having peaks at about 4.9 min about 2.5% acetonitrile, the concentration of the first utes, about 6.0 minutes, about 21.3 minutes, about 22.5 min eluent and second eluent is 100% by volume, and from 0 utes, about 35.0 minutes, and about 49.7 minutes, when sub minutes to about 20 minutes the first eluent increases jected to HPLC using a Cs reverse phase column having an linearly from 0% to about 10% by volume, from about internal diameter of about 4.6 mm and a length of about 250 20 minutes to about 50 minutes the first eluent increases mm, linearly from about 10% to about 20% by volume, from a) wherein a first eluent is acetonitrile and a second eluent about 50 minutes to about 65 minutes the first eluent is water containing about 0.3% phosphoric acid and increases linearly from about 20% to about 40% by about 2.5% acetonitrile, the concentration of the first Volume, and from about 65 minutes to about 80 minutes eluent and second eluent is 100% by volume, and from 0 the first eluent increases linearly from about 40% to minutes to about 20 minutes the first eluent increases about 60% by volume; and linearly from 0% to about 10% by volume, from about b) wherein the column temperature is about 25° C.; the 20 minutes to about 50 minutes the first eluent increases injection volume is about 10 uL: the flow rate is about 1 linearly from about 10% to about 20% by volume, from mL/minute; from 0 minutes to about 80 minutes the about 50 minutes to about 65 minutes the first eluent pressure increases linearly from about 1,000 psi to about increases linearly from about 20% to about 40% by 3,000 psi; and detection is at about 254 nm. Volume, and from about 65 minutes to about 80 minutes 53. The Ficus extract of claim 51, wherein the Cs reverse the first eluent increases linearly from about 40% to phase column comprises a packing material having a particle about 60% by volume; and size of about 3 um to about 5um, a pore size of about 100 A. b) wherein the column temperature is about 25° C.; the and a carbon loading of about 19%. injection volume is about 10 uL: the flow rate is about 1 mL/minute; from 0 minutes to about 80 minutes the c c c c c