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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)