Cephalochordata (“lancelets”) Urochordata (“tunicates) Agnatha (jawless fish, e.g. lampreys) Placodermi (“jawfish”) -EXTINCT Chondrichthyes (sharks and rays) Osteichthyes (“bony fish”) Amphibia Reptilia Aves (birds) Mammalia Phylum Chordata Vertebrates

Cephalochordata (“lancelets”) Urochordata (“tunicates) Agnatha (jawless fish, e.g. lampreys) Placodermi (“jawfish”) -EXTINCT Chondrichthyes (sharks and rays) Osteichthyes (“bony fish”) Amphibia Reptilia Aves (birds) Mammalia Phylum Chordata Vertebrates

Marine Mammals

Marine Fish Class Chondrichthyes (“cartilaginous fish”) Class Osteichthyes (“bony fish”)

Fugu spp. (“Pufferfish”) …and others. Tetrodotoxin (TTX) Bioactive Compounds from Osteichthyes: Tetrodotoxin

Voltage-Gated Sodium Channels Na + K+K+ Voltage-Gated Ion Channels

Action Potential Resting Potential Threshold Membrane Potential (mV) Time (milliseconds) Action Potential Depolarizing Phase Repolarizing Phase

Na + K+K+ Action Potential Voltage-Gated Sodium Channels

TTX Inhibits Depolarization (Action Potential) in Neurons Cell-Specific TTX

TTX Binds and Blocks Voltage-Gated Sodium Channels

Bioactive Compounds from Chondrichthyes Other Myths/Misconceptions? “Sharks Don’t Have Immune Systems, But Don’t Get Infections”

“Dietary Supplements” from Sharks

Shark Oil or “Snake Oil?”

Shark Liver Oil Ingredients 71.9% Petrolatum 14% Mineral Oil 3% Shark Liver Oil 0.25% Phenylephrine HCl

Squalus acanthias (“Dogfish Shark”) Stomach Tissue (400 g) Extract: 60% CH 3 CN/1% TFA Extract Lipids: “Folch Method” 2:1 CHCl 3 /MeOH, Salt Wash Aqueous Phase Gel Filtration (Bio-Gel P-30) C18 HPLC SCX HPLC (Ion Exchange) C4 HPLC FAB-MS, 1 H- and 13 C-NMR, 2-D NMR (NOESY, HMQC) Bioactive Compounds from Chondrichthyes: Squalamine Moore et al. (1993) Proc. Natl. Acad. Sci., 90:

Squalamine MW

Condensation of Squalene and Spermidine Spermidine Squalene

Amount Tissue(µg/g tissue) Stomach1 Liver4-7 Gallbladder4-7 Spleen2 Testes2 Gills0.5 Intestine0.02 Tissue Distribution of Squalamine

Squalamine Inhibits Na+/H+ Exchanger (NHEs) Brush Border Cells

Squalamine Inhibits Na+/H+ Exchanger (NHEs) PS120 Fibroblasts (No NHE) NHE1, 2 and 3 cDNA (Transformed) 40 mM NH4Cl for min.; Perfuse with Medium (Acidification) 2’, 7’-bis(2-carboxyethyl)- 5(6)-carboxyfluorescein (BCECF)-AM (Intracellular pH Indicator) Akhter et al. (1999) Am. J. Physiol., 276:

Squalamine Inhibits H+ Efflux in NHE3-Transformed Cells Akhter et al. (1999) Am. J. Physiol., 276:

Squalamine Inhibition is Dose- Dependent Akhter et al. (1999) Am. J. Physiol., 276:

Squalamine Does NOT Inhibit H+ Efflux in NHE1- or NHE2- Transformed Cells Akhter et al. (1999) Am. J. Physiol., 276:

Squalamine Inhibition of NHE3- Transformed Cells Requires –COOH Terminal (76 Amino Acids) Akhter et al. (1999) Am. J. Physiol., 276:

Squalamine Inhibits Na+/H+ Exchange in Rabbit Illeal Brush Border Vesicles Akhter et al. (1999) Am. J. Physiol., 276: Na + Uptake

Squalamine Inhibits Tumor Angiogenesis

Tumors Recruit Vascular Endothelial Cells in Angiogenesis

Vascular Endothelial Growth Factor (VEGF) Stimulates Vessel Formation

VEGF Acts Via Mitogen-Associated Protein Kinase (MAPK)

MAPK Phosphorylation is pH Dependent Stathopoulou et al. (2006) J. Exp. Biol., 209:

Squalamine Inhibits VEGF-Induced Phosphorylation of MAPK

Squalamine Inhibition of NHE3- Transformed Cells Requires –COOH Terminal (76 Amino Acids)

Pre-Clinical Trials with Squalamine Phase I/IIa Clinical Trials with Squalamine Xenograft Mice: Lung, Breast, Ovarian and Prostate Cancer Allograft Rats: Brain and Breast Cancer Inhibit Tumor Growth and Angiogenesis Lung and Ovarian Cancer MTD = 384 mg/m2/day; 120-h Continuous Infusion Co-Administration with Other Drugs

Control Cisplatin + Squalamine Cisplatin + Squalamine Schiller and Bittner (1999) Clin. Cancer Res., 5: Co-Administration of Squalamine

Squalamine and Macular Degeneration Squalamine Lactate (MSI-1256F) EVIZON™ (Genaera® Pharmaceuticals)