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Allomed Pharmaceuticals

D-Chiro-Inositol and its Role in Diabetes and Insulin Resistance Joseph Larner. Allomed Pharmaceuticals. The Search for an Insulin Mediator. Two Enzyme Bioassays Initial Mediator Hypothesis and Search Inositol Glycans Isolated from Rat Liver Scale up to Beef Liver

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Allomed Pharmaceuticals

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  1. D-Chiro-Inositol and its Role in Diabetes and Insulin Resistance Joseph Larner Allomed Pharmaceuticals

  2. The Search for an Insulin Mediator • Two Enzyme Bioassays • Initial Mediator Hypothesis and Search • Inositol Glycans Isolated from Rat Liver • Scale up to Beef Liver • Isolate and Determine Novel Structure of Pinitol Galactosamine Pseudodisaccharide Mn++ Chelate • Bioactivity of Synthetic Pseudodisaccharide • Theoretical Docking and Allosteric Mechanism of Action of Pseudodisaccharide to Activate PP2C • Pathophysiology of Inositol Imbalance and Relation to Insulin Resistance • Epimerization Defect • Repletion with D-Chiro-Inositol • Future New Possible Therapeutic Agents: • Dibutyryl D-Chiro-Inositol • Pseudodisaccharide • Modified Pseudodisaccharide

  3. PDH (% Maximum Activity)

  4. TLC chromatogram of 2 purified inositol glycans from rat liver. TLC plate developed in isopropanol:pyridine:acetic acid:H2O (8:8:1:4) sprayed with ninhydrin. Lanes 1 and 4, galactosamine standards; Lane 5, PDH phosphatase stimulator from control rat; Lane 6, PDH phosphatase stimulator from insulin treated rat; Lane 2, cAMP-kinase inhibitor from control rat; Lane 3, cAMP-kinase inhibitor from insulin treated rat.

  5. 1 2 3 4 TLC analysis of silica column fractions from beef liver. Aliquots of fractions 1,2,3 eluted from silica column, stained with ninhydrin together with a galactosamine standard are shown. Solvent used contained pyridine: isopropanol:acetic acid:water (8:8:1:4). Lane 1 – galactosamine standard; lanes 2,3,4 fractions 1,2,3 eluated from the silica column. Note the higher Rf peach staining spots in lanes 2,3 most prominent in lane 3 separated from the heavier blue ninhydrin staining peptide material in lane 4, fraction 3. Allomed Pharmaceuticals

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  10. Allosteric site Catalytic site

  11. Pseudodisaccharide

  12. Asp 243 Pseudodisaccharide Phosphorylated peptide substrate

  13. ANALYSIS OF INS2 BINDING INTERACTIONS Using FlexX to find the highest affinity binding conformation, Sybyl was then used to analyze the hydrogen bonding of this conformation of INS2 to PP2C. As we expected, INS2 has strong hydrogen bonding interactions with Asp 243 from two points in the molecule. It was also found that INS2 had a similar interaction with Asp 163. The site mutagenesis data at 163 showing 50% reduced activity upon the removal of this residue helps to support our binding hypothesis for INS2.

  14. SITE POINT MUTAGENESIS OF PP2C Mutations in catalytic site Mutations in allosteric site

  15. Pathophysiology of Inositol Imbalance and Relation to Insulin Resistance

  16. Control Subjects Type II Diabetic Subjects

  17. Urinary Excretion of Inositols (µmol/day ± SEM) UVA Pima Indian Subjects

  18. Urinary Inositol Excretion (nmol/day) Wistar Rats GK rats

  19. Myo-Inositol to Chiro-Inositol Epimerization Defect

  20. •Conversion of Myo-Inositol to Chiro-Inositol is defective in diabetes.

  21. Future New Possible Therapeutic Agents

  22. Suppression by D-Chiro-Inositol and Allo-1 of ROS concentrations in Bovine Aortic Endothelial Cells Incubated in the presence of 30 mM Glucose 250 200 150 * OS (nmoles/mL) ** * 100 * * * * R 50 0 20 µM Chiro 50 µM Chiro 20 µM Allo-1 50 µM Allo-1 100 µM Chiro 100 µM Allo-1 5 mM Glucose 30 mM Glucose

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