Integrative Medicine and Experimental Pharmacogenomic Therapy in a Child with Niemann-Pick Disease (NPD), Type A

Slide 1: Integrative Medicine and Experimental Pharmacogenomic Therapy in a Child with Niemann-Pick Disease (NPD), Type A Sylvestre Quevedo, MD Osher Center for Integrative Medicine University of California School of Medicine San Francisco, California, USA Center for Integrative Medicine San Jose, California, USA Sophia’s Garden Foundation Scientific Advisory Board Palo Alto, California, USA Collaborator: Richard Ellson, Chair, Scientific Advisory Board, Sophia’s Garden Foundation

Slide 2: Incidences of LSD

Slide 3: Background of NPD Types A and B NPD— two forms of same disease caused by • mutations in gene for acid sphingomyelinase (ASM) Results in reduced ASM activity in cellular lysosomes leading to • sphingomyelin storage (lysosomal storage) NPD Type A • * 0-5% ASM activity * Niemann (Germany) 1914; more severe disease, neurologic involvement; life expectancy is 2 to 3 years NPD Type B • * 2-10% ASM activity, ASM greater substrate effect; less lysosomal storage * Pick (Germany) 1927; Less severe, can live into adulthood Types C and D NPD— two forms of distinct disease caused by • abnormalities in cholesterol-metabolizing gene

Slide 4: Characteristics of NPD Type A Child Neurology © 2000 Menkes, M.D., John H. & Sarnat, M.D., Harvey B.

Slide 5: Genetics of NPD Type A Autosomal Recessive The gene for ASM located on human chromosome 11 * Occurs in Ashkenazi Jews at rate of 1 in 40,000 * 90% of Ashkenazi Jews with Type A NPD have "common" * mutations occurring in ASM genes: "L302P," "fsP330," and "R496L" “Wildcard” or unique family mutations account for other 10% * Some patients have both common and wildcard mutations such as * the patient at hand

Slide 6: Biochemistry of Sphingomyelin

Slide 7: Therapeutic Approaches •Enzyme Replacement Therapy NPD A—Doesn’t cross blood-brain barrier; may be used in * tandem with gene therapy. * NPD B—In preclinical studies •Small Molecule Pharmacogenomics—Stop Codon or “nonsense” mutation * * Pharmacological Chaperone —“missense” mutation •Gene Therapy Gene transplantation using viral vectors (Adeno Associated * Virus) * Stem Cell

Slide 8: Case Review 35 month female with NPD Type A common missense mutation (R496L) * rare nonsense (stop codon) mutation (R441X) * Both in ASM gene * With approval Stanford Ethics Committee * Intranasal gentamicin used to promote read through of stop * codon and production of full length ASM Based on report of gentamicin-induced read through of CF * stop codon (NEJM 10/9/2003) Problem: gentamicin minimally crosses blood brain barrier *

Slide 9: Diagnosis of NPD Type A

Slide 10: Mode of Action of Aminoglycosides in Altering Translational Fidelity • During bacterial protein synthesis • Translation initiated with 30S ribosomal subunit binding to mRNA sequence at start codon AUG • tRNA with complementary anticodon (UAC) • tRNA carries the aa f-methionine (fMet); forms H-bonds with the AUG start codon • Forms initiation complex for translation

Slide 11: Mode of Action of Aminoglycosides in Altering Translational Fidelity • Next - 50S ribosomal subunit joins complex • Aminoglycoside antibiotics (e.g. streptomycin, gentamicin) • Bind to the 30S subunit of bacterial ribosomes • Block the attachment of the 50S subunit to the initiation complex.

Slide 12: Mammalian Read through Patient’s R441X mutation Context 1 Context 2 Most 1 UGAC UGAC Read- tetranucleotide sequence (UGAA) is through 2 UAGU UAGU amenable to read through 3 UAGC UGAA 4 UGAA UGAG 5 UAGA UAGC UGAA context ranked near the top of 6 . . 12 tetratnucleotide stop sequences 7 . . 8 . . 9 . . Similar rank to CF mutations (UGAG, 10 . . UAGA) Least 11 . . Read- 12 UAAU UAAU through

Slide 13: Progress to Date with Patient Gentamicin start May 23, 2003 Intranasal administration. Under 0.1mg/kg through August. Ramped up dose to 1.5 mg/kg through September to combat rising cholesterol and triglycerides. Cholesterol Triglycerides Average of 3 tests prior to 273 383 Gentamicin treatment (pre 5/23/03) Peak during ramp up (9/16/03) 274 808 Most recent (3/9/04) 187 284

Slide 14: Oxazolidinone for Read Through of Stop Codons

Slide 15: Oxazolidinone for Read Through of Stop Codons • Oxazolidinones - new class of synthetic antibiotics • Bind to 50 S ribosomal subunit • Causes translational inaccuracy in vivo: (1) Frameshifting, (2) Read through of stop codons, (3) aa misincorporation • Linezolid - an oxazolidinone - recently released for resistant gram positive bacterial infections (VRE) • Safe, used in children, good oral absorption

Slide 16: Linezolid Trial as Stop Codon Read Through Promoter • Linezolid preferable to gentamicin Better read through of stop codon * Penetration of blood brain barrier * Good oral absorption * • Started on 4/6/04 at 5mg/kg every 8 hrs orally

Slide 17: Example of Integrative Medicine: Sophia’s Healing Circle

Slide 18: Example of Integrative Medicine: Sophia’s Healing Circle Allopathic Medicine Nutrition Traditional Medicine Family & Community Spiritual/ Naturopathy Psychological Support Physical/ Medical Energy Research Therapies

Slide 19: Acknowledgements Research Team Other Contributors • • Richard Ellson Greg Enns, M.D. • • Richard Moss, M.D. Karen Herzog • Lucile Packard Children’s • Lucy Hu, O.M.D., LAc Hospital at Stanford Ethics • Sylvestre Quevedo, M.D. Committee • Richard Sachs • Ed Schuchman, PhD. • Greg Stewart, PhD.

Slide 20: For More Information Center for Integrative Medicine (CIM) www.cimsj.com International Center for Types A and B Niemann-Pick Disease www.mssm.edu/niemann-pick Osher Center for Integrative Medicine University of California School of Medicine www.ucsf.edu/ocim Sophia’s Garden Foundation www.sophiasgarden.org