1. 1 Mitigating Effects of Oxidation in Aging and Diseases M. Shchepinov, R.J. Molinari *, V. Shmanai, C.Clarke, A. Manning-Bog March 5, 2010 ASENT Pipeline Session * Presentor

2. Background & Hypothesis Many CNS disease etiologies have recently been associated with oxidation damage of mitochondrial membranes, Such oxidation occurs at a very few, weak-link chemical bonds in polyunsaturated fatty acids, Isotope effect can stabilize target bonds in well-understood ways, with little, if any, toxicity. Supplementing isotope-‘fortified’ components into essential fatty acids will increase resistance to oxidation, and may mitigate disease.

3. 3 Oxidation by ROS and Simplest Isotope Protection ROS abstract hydrogens from PUFAs Some specific stabilization schema.

4. 4 Preliminary Data:. D-4 Linolenic Acid Co-enzyme Q10 deficient yeast oxidative stress models. Complete replacement of dietary PUFA with d-PUFA, Treatment of MPTP mice with dietary PUFAs.

5. 5 Yeast Co-Q10 minus Oxidative Stress Model Linolenic Acid vs. D-4 Linolenic Acid * Clarke Lab, UCLA

6. MPTP Mouse Study  Primary goal: Does dietary D-PUFA protect nigrostriatal dopamine from MPTP-induced depletion?  Secondary goals: -Do dietary D-PUFAs incorporate into the brain ? -Are they toxic ? -Does dietary D-PUFA decrease nigrostriatal lipid hydroperoxide formation (HNE) due to MPTP challenge ?

7.  Dietary PUFA dosing: Fat-free chow (5-6g/mouse) supplemented with saturated fats, oleic acid, and H-PUFA or D-PUFA (30 mg linolenic and 30 mg linoleic coated on pellets) for 6 days + 6 days post MPTP  Toxicant challenge: Single injection of MPTP (40 mg/kg) or saline vehicle, i.p. Cohorts (4) were: H-PUFA-saline; H-PUFA-MPTP; D- PUFA-saline; D-PUFA-MPTP  Readouts: Striatal dopamine levels 4-HNE immunohistochemistry/nigral stereology Remaining brain for total deuterium levels Study Design:

8. * p = 0.0077 Nigrostriatal dopamine levels Striatal DA (ng/mg protein) n=3 n=4 Manning-Bog Lab, Stanford Research Institute

9.  Brain tissues in treated, saline samples were highly enriched in deuterium.  No observed mortality or side effects from dietary dosing, normal weight gain in all mice except as noted below.  MPTP produced marked dopamine depletion of >75% with abnormally high mortality in the untreated, MPTP cohort (3/7) vs. 1/7 in D-PUFA cohort.  Study Notations: Less than 1 in 7 were removed due to dissected protein amounts in excess of two std. deviations (n=2), or failure to thrive (n=1) Additional Observations

10. 10 Further Strategies Focus on diseases in which oxidation products of PUFAs (e.g. 4-HNE) are directly implicated (PD, AMD, ALS, ALZ, CHF) Expand POC efficacy and toxicity of PUFA reinforcement in predictive cellular models & mammals Retrotope supplies materials/expertise for trials, work done by disease model experts Triage and accelerate disease programs with new funding / partners when POC established Develop early regulatory Tox Approach

11. Acknowledgments InvestigatorInstitutionWork Co-PI: Mikhail Shchepinov, CSO Robert Molinari *, CEO Retrotope, Inc.All studies Co-PI: Amy Manning-Bog RA, Vivian Chou Advisor: J. Wm. Langston Stanford Research Institute/ The Parkinson’s Institute Preclinical in vivo MPTP modeling studies Collaborator: Catherine Clarke UCLA Department of Chemistry Co-Q10 Yeast Studies Collaborator: Vadim Shmanai Belarussian Academy of Sciences, Institute for Physical Organic Chemistry Chemical Synthesis of deuterated PUFAs Contractor: Eric Pollock Univ. of Arkansas Stable Isotopes Laboratory Mass Spec for deuterated PUFA incorporation This work was funded by the Michael J. Fox Foundation for Parkinson’s Research