Disorders of Cholesterol Homeostasis Niemann-Pick disease, type C • Autosomal recessive, progressive, lethal, neurodegenerative disorder due to mutation of either

NPC1

or

NPC2

• Endolysosomal storage of unesterified cholesterol and lipids • Incidence: 1/100,000-120,000 • No FDA approved therapies

Niemann-Pick Disease, type C1

• Variable phenotype and age of onset – Classical Disease: Late-infantile and juvenile (60-70%) – Infantile (20%) – Adult • Late-infantile and juvenile (classical) – Transient neonatal jaundice – Splenomegaly – Neurological symptoms (insidious onset) • Vertical supranuclear ophthalmoplegia • Cerebellar ataxia and dysfunction • Cognitive impairment and dementia • Gelastic cataplexy • Seizures

Niemann-Pick Disease, type C1 • Therapeutic trial issues – Rare Disease – Heterogeneous phenotype • Variable age of onset • Variable symptom complex – Clinical progression occurs over years – Goal: Stabilization or delay of neurological disease – Optimal treatment may be prior to the onset of neurological signs and symptoms – Significant diagnostic delay – Lack of defined and accepted outcome measures

Niemann-Pick disease, type C1 • Natural History Trial • 78 patients enrolled since August 2006 – Age range: 3 months to 54 years (median 10 years) – NPC1 Neurological Severity

40 20

(range 0-50, median 14) – Miglustat therapy (42%)

0 10 20 30 40 50 Age (years)

• Goals: – Identify clinical or biochemical markers that can be used as an outcome measure in a therapeutic trial – Identify a biochemical marker that can be used for diagnostic testing or screening

Niemann-Pick Disease, type C1

Mean Diffusivity Volume

Niemann-Pick Disease, type C

Biomarker Development (Assembling a Tool Box)

Niemann-Pick disease, type C1 • Biomarkers – Insight into pathology – Diagnostic/screening test – Tools to guide therapeutic trials • Biomarker identification – Candidate proteins/lipids – Multi-analyte profiling – Expression analysis – Proteomics

Oxysterols Blood-based diagnostic test

Niemann-Pick Disease, type C

Filipin Staining • Filipin Staining – Fluorescent antibiotic that binds unesterified cholesterol – Specialized testing – Requires a skin biopsy-invasive – Variable staining • 80-85% “ classical ” • Molecular Testing – Expensive and requires a high index of suspicion – ~80% sensitive • 4-5 year diagnostic delay Control NPC Vanier and Millat (2003) Clin Genet. 64: 269-281 Dan Ory (personal communication)

NPC1: Clinical Science • Oxysterols – Hypothesis: In NPC1 the unique combination of increased oxidative stress and intracellular accumulation of unesterified cholesterol will result in increased nonenzymatic oxysterols.

Intracellular Cholesterol Accumulation in NPC1

10

Decreased Serum Antioxidant Capacity in NPC1 Subjects

8 6 4

n=40 p<0.0001

2

n=40

0

Control NPC Fu et al (2010) MGM, 101:214

Niemann-Pick Disease, type C1 Plasma Oxysterols 3β,5α,6β-cholestane-triol 7-ketocholesterol

3β,5α,6β-cholestane-triol 7-ketocholesterol

Porter et al. (2010) STM, 2: 56ra81

Niemann-Pick Disease, type C1

Triol (24.5 ng/ml) Sensitivity 97% Specificity 100%

Porter et al. (2010) STM, 2: 56ra81 Jiang et al. (2011) JLR, 52:1435

Niemann-Pick Disease, type C1 CSF Protein Biomarkers

Niemann-Pick Disease, type C1

+/+

Calbindin D 20 15 10 5 0 p<0.001

Control

-/-

NPC1 20 15 p=0.28

10 5 0 Control Untreated Treated

Niemann-Pick Disease, type C

20 Serial Calbindin pre/post miglustat 15 10 5 0 0 10 Months 20 30 A B Calbindin Percent Change +/- miglustat 30 20 10 0 -10 -20 -30 -40 -50

n=16,15,5

un tr ea ted tr ea ted pr e po st Miglustat C D Untreated Treated Pre/post

p<0.05

B/A D/C C/B

Niemann-Pick disease, type C1 • Fatty Acid Binding Protein 3 (FABP3)

62 59

Npc1

+/+ 100 80 60 40 20 0

CSF

p<0.0001

Controls NPC 62

Npc1

-/ 59 -20 -40 -60 -80 40 20 0

untreated p<0.0001

p=0.05

treated pre/post miglustat

Niemann-Pick disease, type C1 • Macrophage Inflammatory Protein 1-alpha (Mip1α,

CCL3

) – Proinflammatory cytokine – Activated microglia

30

CSF

p<0.0001

20 10 0 Control NPC Control 4.8

±

NPC 14.7

±

1.9 pg/ml 4.2 30 25 20 15 10 5 0 0 10 20 30 40 50 Severity Score Ranking 60

Confidential

CSF Biomarkers

• CSF Biomarkers – Lipid-based • 3β,5α,6β-cholestane-triol and cholesterol esters – Protein-based • Markers of inflammation – MIP-1α, IL1a, IL3, IL4, IL12p40, IL13, IL15, and MMP2 • Markers of neuronal injury – Aβ(42), phosphorylated tau, fatty acid binding protein 3 (FABP3), and calbindin-D • Markers of oxidative stress – glutathione S-transferase alpha (GSTα) and superoxide dismutase 1 (SOD1)

2-Hydroxypropyl-β-cyclodextrin

NPC1 HPβCD Trial

• 2-Hydroxypropyl-β-cyclodextrin (HPβCD) – Cyclic oligosaccharide with a hydrophobic core – Pharmaceutical excipient • Drug repurposing

NPC1 HPβCD Trial

•

Npc1

mouse model Survival Cerebellar pathology: 49-days

Npc1 +/+ Npc1 -/ Npc1 -/ +

CD Ramirez et al. (2010) Ped. Res. 68: 309 Liu et al. (2009) PNAS 106: 2377

NPC1 HPβCD Trial

•

Npc1

cat model – 24 week

Npc1

mutant cats (HPβCD, miglustat, untreated) Personal Communication: Charles Vite

NPC1 HPβCD Trial

• HPβCD Therapeutic trial – Pilot project for TRND/NCATS • Goal: Improve drug development for rare and neglected diseases – HPβCD does not cross the blood-brain barrier – Dose limiting toxicity: Ototoxicity • Cat, dog, and mouse

NPC1 HPβCD Trial

Lancet (1963) 282: 983-984

Risks Bleeding/strokes Infection Seizure

NPC1 HPβCD Trial

• Phase 1 trial – Goal: Establish a safe and biochemically effective dose – Cohort dose-escalation • Safety • Pharmacokinetics – Quantitative and validated assay for HPβCD • Pharmacodynamics (Biochemical efficacy ) – Quantitative and validated assays for 24-hydroxycholesterol • Pathological efficacy – CSF protein and lipid biomarkers

NPC1 HPβCD Trial

• Pharmacodynamic response • Plasma 24(S)-hydroxycholesterol Neuron

Cholesterol Synthesis ApoE NPC1 HPβCD CYP46 24(S)HC CSF 24(S)HC Blood

NPC1 HPβCD Trial

• Pharmacodynamic response • Plasma 24(S)-hydroxycholesterol Neuron

Cholesterol Synthesis ApoE NPC1 HPβCD CYP46 24(S)HC CSF 24(S)HC Blood

NPC1 HPβCD Trial

Saline

CSF and Plasma Sampling (0 to 72 hours)

HPβCD

CSF and Plasma Sampling (0 to 72 hours)

• Baseline standard deviation of the saline area under the curve (AUC) estimated by resampling (bootstrapping) • Biochemical response defined as: – In an individual patient the difference between the drug and saline AUC greater than 2.3 x SD is considered a positive response – Positive response observed in at least 2 out of 3 patients

NPC1 HPβCD Trial

• 50 mg HPβCD – No drug related adverse events or reactions • • 2/3 patients had a biochemical response – AUC 8-72 • Patient 1 + 3.1 x sd • Patient 2 + 0.8 x sd • Patient 3 + 4.1 x sd

35 30

P. acnes

– Infection/Colonization

25 0 12 24 36 48 Time (hours) Saline 50 mg 60 72

Niemann-Pick disease, type C1 • ICV versus lumbar IT • Dose escalation response • Repetitive dosing response: Attenuation • Response of other biomarkers • Safety-Ototoxicity

Cohort 1 50 mg Dose

Cohort Dose-escalation

Not Tolerated Not Tolerated Tolerated No Response STOP Cohort 3 Tolerated No Response Cohort 1/2 200 mg Dose Cohort 1/2/3 300 mg Cohort 3/4 400 mg Maintain 200 mg Tolerated Biochemical Response Cohort 1/2 Maintain 300 mg Maintain 400 mg

Niemann-Pick disease, type C1 • No significant response at 50 and 200 mg IT • Positive response in 2/3 subjects at 300 mg IT – Response similar to 50 mg ICV – Grade 1 Ototoxicity in 2 subjects (siblings) • 400 mg cohort completed this week

Niemann-Pick disease, type C1 • Clinical efficacy trial • Multicenter: Funding and IRB complexity • Multinational: Funding, IRB and regulatory complexity • NeuroNext Network – Concept proposal accepted • Clinical outcome measures – Ataxia (Cerebellar Function) • Gross motor • Fine motor – Swallowing 26 US Sites Common IRB Common Trial Agreement

NPC1 HPβCD Trial

• HPβCD selected as a TRND clinical candidate in February 2011 • PreIND meetings with FDA in October and December 2011 • Juvenile dog toxicity study, formulation, and device compatibility studies performed in 2012 • IRB approval July 2012 and IND filing November 2012 • First ICV infusion February 4, 2013 • First IT infusion September 28. 2013

NPC1 TRND Team

NICHD Nicole Yanjanin Aiyi Liu (DESPR) Roopa Shankar NHGRI Bill Pavan NINDS Russell Lonser John Heiss TRND/NCATS Chris Austin John McKew Nuria Carrillo Liz Ottinger Washington University School of Medicine Daniel Ory University of Pennsylvania Charles Vite Albert Einstein Collage of Medicine Steven Walkley Johnson and Johnson Steven Silber Mark Kao Marcus Brewster Juan Marugan Pramod Terse Xin Xu Wei Zheng Clinical Center RRD International Charles Finn Frank Hurley Joy Vanderwal Carmen Brewer Beth Solomon Naomi O ’ Grady Kelly King SAIC/Leidos Jon Stocker Molly Buehn Leah Giambarresi Patrick Frenchick Sandra Morseth Kimberly Lilly

Acknowledgements

SMD, PDEGEN, NICHD Nicole Yanjanin Lee Ann Knight Roopa Shankar Chris Wassif Celine Cluzeau Stephanie Cologna Ryan Lee Cynthia Toth Jiang Xiao-Sheng Ian Williams Antony Cougnoux ORD/CC Bench to Bedside Awards Ara Parseghian Medical Research Foundation National Niemann Pick Disease Foundation SOAR-NPC Dana ’ s Angels Research Trust

Niemann-Pick disease, type C1

Niemann-Pick disease, type C1

Hydroxypropyl-β-Cyclodextrin

Mouse Cat Mouse Cat 0.5 g 30.0

Human 1400.0

Newborn 375.0

Niemann-Pick Disease, type C1

Vertical supranuclear ophthalmoplegia Differential -Niemann-Pick Disease, type C1 -Progressive Supranuclear Palsy -Midbrain lesions

Niemann-Pick disease, type C1 Attenuation of the 24OHC response with repetitive dosing