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A Novel Anti-Cancer Strategy Through Biological Metal Overloading in Cancer Cells via pH-sensitive Metalo-organic Nanoparticles Jinhyuk Fred Chung, Ph. D., CEO NanoMedicine Research Co. April 18 th , 2008 The 5 th Nanoforum Jeju, Korea

Challenges & Limitations in Anti-cancer Medicines: Seesaw Dillemma Toxicity / Efficacy Selectivity Cytotoxin Enzyme Inhibitor Which side to leverage?

◈ Theory – A lesson from nature 1: Mol Med. 2008 Mar-Apr;14(3-4):98-108. Links Iron-mediated inhibition of mitochondrial manganese uptake mediates mitochondrial Benign biological metals can dysfunction in a mouse model of KILL UPON hemochromatosis. OVERLOADING! Jouihan HA, Cobine PA, Cooksey RC, Hoagland EA, Boudina S, Abel ED, Winge DR, McClain DA. Departments of Medicine and Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah, USA and.

“Calcium & Iron Bomb” Strategy *Source of the depiction unknown

◈ OFeCa-1: The Anti-Cancer Particle • OFeCa-1 • pH-sensitive organo-metallic nanoparticles with iron & calcium as the main therapeutic components. • Degrades near neutral pH, releasing metals. Transmission Electron Microscope (TEM) Image taken near pH 2 ICP-MS metal analysis of 11% w/w OFeCa-1 solution (same concentration solution as those used for cell & animal experiments)

◈ OFeCa-1’s Degradation via pH Mass Distribution at pH 6.4 • Dynamic light scattering results 120 on OFeCa-1 at varying pH, 100 %Mass(Per max peak) pH 6.4 80 showing pH-dependent 60 40 degradation of OFeCa-1 near 20 physiological ranges (~pH 7). 0 0.4 0.62 0.97 1.5 2.33 3.62 5.61 8.72 13.5 21 32.7 50.7 78.8 122 190 295 459 712 1110 Diameter • Horizontal axis denote Mass Distribution at pH 7 120 nanoparticle sizes in diameter, pH 7.0 100 %Mass(Per Max Peak) graphs denote mass distribution, 80 60 normalized against the norm. 40 20 • This degradation property allows 0 0.4 0.62 0.97 1.5 2.33 3.62 5.61 8.72 13.5 21 32.7 50.7 78.8 122 190 295 459 712 1110 OFeCa-1 to “unload” bound Diameter (nm) Mass Distribution at pH 7.4 metals that, in turn, kill target 120 pH 7.4 cancer mass 100 %Mass(Per Max Peak) 80 60 40 20 0 0.4 0.62 0.97 1.5 2.33 3.62 5.61 8.72 13.5 21 32.7 50.7 78.8 122 190 295 459 712 1110 Diameter (nm)

◈ Human Cancer Cell Screening Note this occurance of 4.500 24hr Treated 48 72 “critical dosage”, at which 4.000 3.500 point cancer suddenly 3.000 5000 per well disappear altogether 2.500 Cancer Survival 2.000 1.500 1.000 Hep2 (larynx cancer) 0.500 0.000 0 5 10 20 30 40 50 60 0 5 10 20 30 40 50 60 60 60 0 5 24hrs 48hrs 72hrs 24 48 72 4.500 4.000 3.500 3.000 5000 per well 2.500 2.000 1.500 1.000 293T (kidney cancer) 0.500 0.000 0 5 10 20 30 40 50 60 0 5 10 20 30 40 50 60 0 5 10 20 30 40 50 60 24hrs 48hrs 72hrs *Cell counting kit-8 (Dojindo, Tokyo) was used to study cell viability. OFeCa-1 Added (uL/mL Media)

Continued 3.000 24hr 48hr 72hr 2.500 2.000 5000 per well 1.500 1.000 Ramos (Burkitt's lymphoma ) 0.500 0.000 0 5 10 20 30 40 50 60 0 5 10 20 30 40 50 60 0 5 10 20 30 40 50 60 24hrs 48hrs 72hrs 5.000 4.500 4.000 3.500 3.000 2.500 2.000 1.500 T98g (brain cancer) 1.000 0.500 0.000 0 5 10 20 30 40 50 60 0 5 10 20 30 40 50 60 0 5 10 20 30 40 50 60 24hrs 48hrs 72hrs 0.900 0.800 0.700 0.600 ell 0.500 er w 5000 p 0.400 0.300 H460 (lung cancer) 0.200 0.100 0.000 0 5 10 20 30 40 50 60 0 5 10 20 30 40 50 60 0 5 10 20 30 40 50 60 24hrs 48hrs 72hrs

◈ Toxicity Testing against HSC HSC survival (A.U.) • Adult stem cells such as hair cells & hematopoetic stem cells 1. 6 1. 4 (HSC: blood stem cell) are 1. 2 easily killed by conventional 1 anti-cancer drugs, leading to hair 0. 8 loss & bone marrow suppression 0. 6 0. 4 during cancer treatment. 0. 2 0 • OFeCa-1, on the other hand, c 1 2. 5 5 10 20 40 displays no signs of adverse 11% OFeCa-1 in Media (uL/mL) effect on mouse HSC’s survival .

Efficacy against Mouse Cancer Cell Effect of OFeCa-1 on B16F10 • OFeCa-1 shows Melanoma & Normal Stromal Cells no toxicity # of Initial 120 against non- Melanoma Cells % Cell Survival against Control Group cancerous 10^ 5 100 normal mouse 2.5*10^ 4 stromal cells. 80 • Within same 1.25*10^ 4 range, OFeCa-1 60 0.6*10^ 4 shows powerful anti-cancer 0.3*10^ 4 40 activity against mouse 0.15*10^ 4 20 melanoma Norm al cell B16F10. 0 40 20 5 1 11% w/w OFeCa-1 Added (uL/mL media)

◈ Mouse Model Bearing Metastatic Lung Cancer Effect of sustained administration of OFeCa-1 on A intravanous metastatic mouse model using B16/F10. OFeCa-1 was passively administered as water-substitute by diluting 11 % OFeCa-1 ten-fold with dietary water. Quick survey B showed that the mice consumed roughly 2~3 mL of the diluted OFeCa-1 daily. (A) B16/F10 control. (B) B16/F10 mice fed 50 Melanoma Colony Count C with 1/10 diluted OFeCa-1. (C) Quantified B16/F10 colonies 40 found in the lungs of test subjects. P10 refers to passively 30 fed 1/10 diluted 11% w/w OFeCa-1 against water. 20 Experiment terminated before the 10 extinction of melanoma colonies in the 0 treatment group for visual B16/F10 B16/F10 + P10 confirmation of successful metastasis Control Treatment

◈ 2W Acute Toxicity Test using SD rats

◈ Replenishment Effect: More Effectiveness • The graph left shows clear evidence of improvement in Cancer Cell Survival After 72hr Treatment anti-cancer efficacy of OFeCa-1 upon daily 5 replacement of the media 4.5 containing OFeCa-1. 4 • This data indicates that frequent use of OFeCa-1 is 3.5 likely to be recommended in 3 the future. Kept Media Abs 2.5 Fresh Media • Considering the data thus far, 2 greater dose and more 1.5 frequent intake of OFeCa-1 1 is likely to be highly recommended, increasing 0.5 the demand for OFeCa-1 for 0 extra assurance in cancer 0 1 10 20 30 40 treatment. OFeCa-1 Added (uL/mL media) *B16F10 mouse melanoma as model. Media of appropriate condition were replaced daily for “fresh media” group.

OFeCa-1: Ideal Anti-Cancer Agent • Ultra-low toxicity* • Composed only of biological metals & organic nanoparticle backbone that degrades into non-toxic biological metabolites • Displays effectiveness against multiple aggressive cancer cell lines (both human & mouse origins) • Designed to work in multiple modes, increasing effectiveness & reducing chances of resistance development • Outlook of good efficacy against Lung cancer • Significantly more effective upon frequent & prolonged use • Expected to be compatible with conventional chemotherapeutic agents. • And many more ideal behaviors & properties that qualify OFeCa-1 as an ideal candidate for future broad-spectrum cancer treatments. *Based on toxicity tests against cell-based tests & acute toxicity test using SD rats