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Phylum Chordata Cephalochordata (“lancelets”) Urochordata (“tunicates) Agnatha (jawless fish, e.g. lampreys) Placodermi (“jawfish”) -EXTINCT Chondrichthyes (sharks and rays) Osteichthyes (“bony fish”) Amphibia Vertebrates Reptilia Aves (birds) Mammalia Phylum Chordata Cephalochordata (“lancelets”) Urochordata (“tunicates) Agnatha (jawless fish, e.g. lampreys) Placodermi (“jawfish”) -EXTINCT Chondrichthyes (sharks and rays) Osteichthyes (“bony fish”) Amphibia Vertebrates Reptilia Aves (birds) Mammalia Marine Mammals Marine Fish Class Chondrichthyes (“cartilaginous fish”) Class Osteichthyes (“bony fish”) Bioactive Compounds from Osteichthyes: Tetrodotoxin OHO O O +H2N N H Fugu spp. (“Pufferfish”) …and others. N H OH HO OH Tetrodotoxin (TTX) Voltage-Gated Sodium Channels + + + + + + + + ++-- - - - - - K+ Na+ Voltage-Gated Ion Channels Membrane Potential (mV) Action Potential Action Potential + 50 0 Depolarizing Phase Repolarizing Phase - 50 Threshold Resting Potential - 70 Time (milliseconds) Voltage-Gated Sodium Channels + + + + + + + + - - - ++ -- - - - Action Potential K+ Na+ TTX Inhibits Depolarization (Action Potential) in Neurons TTX Cell-Specific 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 Bioactive Compounds from Chondrichthyes: Squalamine Squalus acanthias (“Dogfish Shark”) Stomach Tissue (400 g) Extract: 60% CH3CN/1% TFA Extract Lipids: “Folch Method” 2:1 CHCl3/MeOH, Salt Wash Aqueous Phase Gel Filtration (Bio-Gel P-30) C18 HPLC SCX HPLC (Ion Exchange) C4 HPLC FAB-MS, 1H- and 13C-NMR, 2-D NMR (NOESY, HMQC) Moore et al. (1993) Proc. Natl. Acad. Sci., 90: 1354-8. OSO2 H3 C CH3 CH3 CH3 CH3 H +H3 N N H2+ N H2+ OH H Squalamine MW 628.4739 Moore et al. (1993) Proc. Natl. Acad. Sci., 90: 1354-8. Condensation of Squalene and Spermidine H2 N N H NH2 Spermidine Squalene OSO2 H3 C CH3 CH3 CH3 CH3 H +H3 N N H2+ N H2+ OH H Tissue Distribution of Squalamine Tissue Stomach Liver Gallbladder Spleen Testes Gills Intestine Amount (µg/g tissue) 1 4-7 4-7 2 2 0.5 0.02 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 210 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: 136-44. Squalamine Inhibits H+ Efflux in NHE3-Transformed Cells Akhter et al. (1999) Am. J. Physiol., 276: 136-44. Squalamine Inhibition is DoseDependent Akhter et al. (1999) Am. J. Physiol., 276: 136-44. Squalamine Does NOT Inhibit H+ Efflux in NHE1- or NHE2Transformed Cells Akhter et al. (1999) Am. J. Physiol., 276: 136-44. Squalamine Inhibition of NHE3Transformed Cells Requires –COOH Terminal (76 Amino Acids) Akhter et al. (1999) Am. J. Physiol., 276: 136-44. Squalamine Inhibits Na+/H+ Exchange in Rabbit Illeal Brush Border Vesicles 22Na+ Uptake Akhter et al. (1999) Am. J. Physiol., 276: 136-44. 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: 1344-54. Squalamine Inhibits VEGF-Induced Phosphorylation of MAPK Squalamine Inhibition of NHE3Transformed Cells Requires –COOH Terminal (76 Amino Acids) Pre-Clinical Trials with Squalamine Xenograft Mice: Lung, Breast, Ovarian and Prostate Cancer Allograft Rats: Brain and Breast Cancer Inhibit Tumor Growth and Angiogenesis Phase I/IIa Clinical Trials with Squalamine Lung and Ovarian Cancer MTD = 384 mg/m2/day; 120-h Continuous Infusion Co-Administration with Other Drugs Co-Administration of Squalamine Control Cisplatin + Squalamine Cisplatin + Squalamine Schiller and Bittner (1999) Clin. Cancer Res., 5: 4287-94 Squalamine and Macular Degeneration Squalamine Lactate (MSI-1256F) EVIZON™ (Genaera® Pharmaceuticals)
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