Cephalochordata(“lancelets”)

Urochordata(“tunicates)

Agnatha(jawless fish, e.g. lampreys)

Placodermi (“jawfish”) -EXTINCTChondrichthyes(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”) -EXTINCTChondrichthyes(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.

NH

NH

OO

O-

HO

HO OH

OH

+H2N

Tetrodotoxin (TTX)

Bioactive Compounds from Osteichthyes:Bioactive Compounds from Osteichthyes:Tetrodotoxin Tetrodotoxin

Voltage-Gated Sodium Channels

+ + - - - - - - - -+ + + + + + + +

Na+

K+

Voltage-Gated Ion Channels

- 70

0

- 50

+ 50

Action Potential

Resting Potential

Threshold

Mem

bran

e P

oten

tial (

mV

)

Time (milliseconds)

Action Potential

DepolarizingPhase

RepolarizingPhase

- - - ++ -- - - - -+ + + + + + + +

Na+

K+

ActionPotential

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 Bioactive Compounds from ChondrichthyesChondrichthyes

Other Myths/Misconceptions?“Sharks Don’t Have Immune Systems,

But Don’t Get Infections”

““Dietary Supplements” from SharksDietary Supplements” from Sharks

Shark Oil or “Snake Oil?”Shark Oil or “Snake Oil?”

Shark Liver Oil

Ingredients

71.9% Petrolatum14% Mineral Oil

3% Shark Liver Oil0.25% Phenylephrine HCl

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)

Bioactive Compounds from Bioactive Compounds from Chondrichthyes: Chondrichthyes: SqualamineSqualamine

Moore et al. (1993) Proc. Natl. Acad. Sci., 90: 1354-8.

Moore et al. (1993) Proc. Natl. Acad. Sci., 90: 1354-8.

SqualamineMW 628.4739

OSO2-

CH3

CH3

H3C

NH2+

NH2+

+H3N

CH3

CH3

OH

H

H

NH2NH

H2N

OSO2-

CH3

CH3

H3C

NH2+

NH2+

+H3N

CH3

CH3

OH

H

H

Condensation of Squalene and Condensation of Squalene and SpermidineSpermidine

Spermidine Squalene

Amount Tissue (µg/g tissue)

Stomach 1Liver 4-7Gallbladder 4-7Spleen 2Testes 2Gills 0.5Intestine 0.02

Tissue Distribution of SqualamineTissue Distribution of Squalamine

Squalamine Inhibits Na+/H+ Squalamine Inhibits Na+/H+ Exchanger (NHEs)Exchanger (NHEs)

Brush Border Cells

Squalamine Inhibits Na+/H+ Squalamine Inhibits Na+/H+ Exchanger (NHEs)Exchanger (NHEs)

PS120 Fibroblasts(No NHE)

NHE1, 2 and 3 cDNA(Transformed)

40 mM NH4Cl for 2-10 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 Squalamine Inhibits H+ Efflux in NHE3-Transformed CellsNHE3-Transformed Cells

Akhter et al. (1999) Am. J. Physiol., 276: 136-44.

Squalamine Inhibition is Dose-Squalamine Inhibition is Dose-DependentDependent

Akhter et al. (1999) Am. J. Physiol., 276: 136-44.

Squalamine Does NOT Inhibit H+ Squalamine Does NOT Inhibit H+ Efflux in NHE1- or NHE2-Efflux in NHE1- or NHE2-

Transformed CellsTransformed Cells

Akhter et al. (1999) Am. J. Physiol., 276: 136-44.

Squalamine Inhibition of NHE3-Squalamine Inhibition of NHE3-Transformed Cells Requires –COOH Transformed Cells Requires –COOH

Terminal (76 Amino Acids)Terminal (76 Amino Acids)

Akhter et al. (1999) Am. J. Physiol., 276: 136-44.

Squalamine Inhibits Na+/H+ Squalamine Inhibits Na+/H+ Exchange in Rabbit Illeal Brush Exchange in Rabbit Illeal Brush

Border VesiclesBorder Vesicles

Akhter et al. (1999) Am. J. Physiol., 276: 136-44.

22Na+ Uptake

Squalamine Inhibits Tumor Squalamine Inhibits Tumor AngiogenesisAngiogenesis

Tumors Recruit Vascular Endothelial Tumors Recruit Vascular Endothelial Cells in AngiogenesisCells in Angiogenesis

Vascular Endothelial Growth Factor Vascular Endothelial Growth Factor (VEGF) Stimulates Vessel Formation(VEGF) Stimulates Vessel Formation

VEGF Acts Via Mitogen-Associated VEGF Acts Via Mitogen-Associated Protein Kinase (MAPK)Protein Kinase (MAPK)

MAPK Phosphorylation is pH MAPK Phosphorylation is pH DependentDependent

Stathopoulou et al. (2006) J. Exp. Biol., 209: 1344-54.

Squalamine Inhibits VEGF-Induced Squalamine Inhibits VEGF-Induced Phosphorylation of MAPKPhosphorylation of MAPK

Squalamine Inhibition of NHE3-Squalamine Inhibition of NHE3-Transformed Cells Requires –COOH Transformed Cells Requires –COOH

Terminal (76 Amino Acids)Terminal (76 Amino Acids)

Pre-Clinical Trials with SqualaminePre-Clinical Trials with Squalamine

Phase I/IIa Clinical Trials with Phase I/IIa Clinical Trials with SqualamineSqualamine

Xenograft Mice: Lung, Breast, Ovarian and Prostate CancerAllograft Rats: Brain and Breast CancerInhibit Tumor Growth and Angiogenesis

Lung and Ovarian CancerMTD = 384 mg/m2/day; 120-h Continuous InfusionCo-Administration with Other Drugs

Control

Cisplatin + Squalamine

Cisplatin + Squalamine

Schiller and Bittner (1999) Clin. Cancer Res., 5: 4287-94

Co-Administration of SqualamineCo-Administration of Squalamine

Squalamine and Macular DegenerationSqualamine and Macular Degeneration

Squalamine Lactate Squalamine Lactate (MSI-1256F)(MSI-1256F)

EVIZON™EVIZON™(Genaera® (Genaera®

Pharmaceuticals)Pharmaceuticals)