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

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

Incidences of LSD

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

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

Biochemistry of Sphingomyelin

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

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

Diagnosis of NPD Type A

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

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.

Mammalian Read through

Patient’s R441X mutation tetranucleotide sequence (UGAA) is amenable to read through

UGAA context ranked near the top of 12 tetratnucleotide stop sequences

Similar rank to CF mutations (UGAG, UAGA)

Context 2 UGAC UAGU UGAA UGAG UAGC . . . . . . UAAU Context 1 UGAC UAGU UAGC UGAA UAGA . . . . . . UAAU Most Read- through Least Read- through 1 2 3 4 5 6 7 8 9 10 11 12

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.

808 274 Peak during ramp up (9/16/03) 284 187 Most recent (3/9/04) 383 273 Average of 3 tests prior to Gentamicin treatment (pre 5/23/03) Triglycerides Cholesterol

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

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

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Acknowledgements