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Non-Physiological Amino Acid (NPAA) Therapy in Pah enu2 Mice Relevance to PKU Therapy Mike Gibson, Professor and Head Section of Clinical Pharmacology Washington State University (WSU) Spokane College of Pharmacy, Spokane WA Disclosures: None

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Rationale and Goals To Selectively Lower Brain Phenylalanine To Selectively Lower Brain Phenylalanine NPAAs targeting L and A systems (BBB and gut) Maintain Other LNAAs at or Near Normal Levels Adjuvant Therapy, Target Cognitive Improvements Adjuvant Therapy, Target Cognitive Improvements Mammalian System L Transporters Specific for LNAAs TransporterExpression Amino AcidsTransported LAT-1 Li (fetal), BM, Br, Pl, Te L-I-V-F-Y-W-M-H LAT-2 Je, Ile, Ki, Pl, Br, Te, Sp L-F-W-T-N-I-C-S-Y-V-Q LAT-3 Pa, Li (fetal, adult), SM L-I-V-F LAT-4 Pl, Ki, Leuc L-F-I-M

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Non-Physiological Amino Acids A=Norleucine B=2-Aminoisobutyrate C=N-Methyl-2- aminoisobutyrate D=2-aminonorbornane- 2-carboxylic acid Only NL previously used in a mammalian system K m values of LNAAs for LAT(s) may lead to NPAA concentrations that selectively lower Phe while minimally altering other LNAAs

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Brain Amino Acid Transport Systems ~ Amino Acid Specificity ~ Amino Acid Specificity Considerable Overlap Considerable Overlap Glutamine (Q): Numerous Glutamine (Q): Numerous Na Dependent/Independent Na Dependent/Independent A ~ Smaller Amino Acids A ~ Smaller Amino Acids L ~ Larger Amino Acids L ~ Larger Amino Acids

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Approach Dietary Administration-Clinical Relevance Dietary Administration-Clinical Relevance Control Nitrogen Load with Casein Control Nitrogen Load with Casein Monitor Brain LNAAs and Monoamines Monitor Brain LNAAs and Monoamines Monitor Behavior/Movement in Future Blood Chemistries (Safety) Blood Chemistries (Safety) Assess Effects on Nitrogen Load Develop Methods to Quantify NPAAs Develop Methods to Quantify NPAAs

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LNAA Metabolic Roles

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Pilot Studies-Effect on Phe/Tyr Phenylalanine Tyrosine

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Results for Monoamines SerotoninDopamine

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Additional LNAA Outcomes Tryptophan Total Branched Chain AAs

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Effects on 1-Methyl Transfer MethionineSAMe

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Conclusions Proof-of-Principle: Feasibility Proof-of-Principle: Feasibility Phe Reduction with NL, NB and MAIB Other LNAA Effects: Lower Levels of NL and NB Movement Effects of 3-5% NL Prominent First Use of These in an Murine PKU Model MAIB is a Selective A System Inhibitor MAIB is a Selective A System Inhibitor Can Clearly Reduce Phe, However Not Previously Documented More Benign Effects than 5% NL, 0.5% NB

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Goals in Future Studies Different Concentrations of NPAAs Different Concentrations of NPAAs Combinatorial Administration (BBB-Gut) Combinatorial Administration (BBB-Gut) NPAA Characterization in High/Low Protein NPAA Characterization in High/Low Protein Address Variability in Some LNAA Levels Address Variability in Some LNAA Levels Evaluate Method of Sacrifice Evaluate Method of Sacrifice Characterize Behavior during NPAA Intervention Characterize Behavior during NPAA Intervention

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Acknowledgements Colleagues/Funding Alliance Support Alliance Support Kara Vogel Kara Vogel Brandi Wasek Brandi Wasek Erland Arning Erland Arning Terry Bottiglieri Terry Bottiglieri R01 HD 58553 R01 HD 58553 U54 DK 83916 U54 DK 83916