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Mitigating Effects of Oxidation in Mitigating Effects of Oxidation in Aging and DiseasesAging and Diseases

M. Shchepinov, R.J. Molinari *, V. Shmanai, M. Shchepinov, R.J. Molinari *, V. Shmanai, C.Clarke, A. Manning-Bog C.Clarke, A. Manning-Bog

March 5, 2010March 5, 2010ASENT Pipeline SessionASENT Pipeline Session

* Presentor

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.

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Oxidation by ROS and Simplest Isotope Protection

ROS abstract hydrogens from PUFAs

Some specific stabilization schema.

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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.

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Yeast Co-Q10 minus Oxidative Stress ModelLinolenic Acid vs. D-4 Linolenic Acid *

Clarke Lab, UCLA

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 ?

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 stereologyRemaining brain for total deuterium levels

Study Design:

* p = 0.0077

Nigrostriatal dopamine levels

Str

iata

l DA

(n

g/m

g p

rote

in)

n=3 n=3 n=4 n=4

Manning-Bog Lab, Stanford Research Institute

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

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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

Acknowledgments Investigator Institution Work

Co-PI: Mikhail Shchepinov, CSORobert Molinari *, CEO

Retrotope, Inc. All studies

Co-PI: Amy Manning-BogRA, Vivian Chou

Advisor: J. Wm. Langston

Stanford Research Institute/The Parkinson’s Institute

Preclinical in vivo MPTP modeling studies

Collaborator:Catherine Clarke

UCLADepartment 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 ArkansasStable Isotopes Laboratory

Mass Spec for deuterated PUFA incorporation

This work was funded by the Michael J. Fox Foundation for Parkinson’s Research