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

• 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

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

(range 0-50, median 14)

– Miglustat therapy (42%)

• 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

0 10 20 30 40 50

20

40

Sev

erit

y S

core

Age (years)

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– 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

Filipin Staining

Control NPCVanier 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.

TE

AC

(m

M)

Control NPC0

2

4

6

8

10

n=40

n=40

p<0.0001

Fu et al (2010) MGM, 101:214

Intracellular CholesterolAccumulation in NPC1

Decreased Serum Antioxidant Capacity in NPC1 Subjects

Niemann-Pick Disease, type C1

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

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

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

Niemann-Pick Disease, type C1

Porter et al. (2010) STM, 2: 56ra81Jiang et al. (2011) JLR, 52:1435

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

Niemann-Pick Disease, type C1

CSF Protein Biomarkers

Niemann-Pick Disease, type C1

+/+

-/- Control NPC10

5

10

15

20

Cal

bind

in n

g/m

L

p<0.001

Calbindin D

Control Untreated Treated0

5

10

15

20C

alb

ind

in (

ng

/mL

)p=0.28

Niemann-Pick Disease, type CCalbindin Percent Change

+/- miglustat

untreat

ed

treat

ed

pre-p

ost-50

-40

-30

-20

-10

0

10

20

30

n=16,15,5

p<0.05

Per

cen

t C

han

ge

A B C D

Miglustat

Untreated B/ATreated D/CPre/post C/B

Serial Calbindin pre/post miglustat

0 10 20 300

5

10

15

20

Months

Cal

bin

din

ng

/mL

Niemann-Pick disease, type C1

• Fatty Acid Binding Protein 3 (FABP3)

Controls NPC0

20

40

60

80

100

FA

BP

3 (n

g/m

l)

-80

-60

-40

-20

0

20

40

untreated

treated

pre/postmiglustat

Per

cen

t ch

ang

eF

AB

P3

p=0.05

p<0.0001

6259

6259

Npc1+/+ Npc1-/-

p<0.0001

CSF

Niemann-Pick disease, type C1

• Macrophage Inflammatory Protein 1-alpha (Mip1α, CCL3)

– Proinflammatory cytokine

– Activated microglia

Control NPC0

10

20

30

Mip

1 (

pg

/mL

)

p<0.0001

Control 4.8 ± 1.9 pg/mlNPC 14.7 ± 4.2

0 10 20 30 40 50 600

5

10

15

20

25

30

Severity Score Ranking

Mip

1alp

ha

(pg/

mL

)CSF

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

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

Npc1+/+ Npc1-/- Npc1-/- + CD

Cerebellar pathology: 49-daysSurvival

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

ApoE

CholesterolSynthesis

CYP46

24(S)HC

Blood24(S)HC

CSF

Neuron

HPβCD

NPC1

NPC1 HPβCD Trial

ApoE

CholesterolSynthesis

24(S)HC

Blood24(S)HC

CSF

Neuron

HPβCD

NPC1

CYP46

• Pharmacodynamic response• Plasma 24(S)-hydroxycholesterol

NPC1 HPβCD Trial

CSF and Plasma Sampling(0 to 72 hours)

Saline

CSF and Plasma Sampling(0 to 72 hours)

HPβCD

• 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– AUC8-72

• Patient 1 + 3.1 x sd

• Patient 2 + 0.8 x sd

• Patient 3 + 4.1 x sd

• P. acnes– Infection/Colonization

0 12 24 36 48 60 72

25

30

35Saline

50 mg

Time (hours)

Pla

sm

a 2

4-O

H C

ho

les

tero

l(n

g/m

l)

Niemann-Pick disease, type C1

• ICV versus lumbar IT

• Dose escalation response

• Repetitive dosing response: Attenuation

• Response of other biomarkers

• Safety-Ototoxicity

Cohort Dose-escalation

Cohort 150 mg Dose

ToleratedBiochemical Response

ToleratedNo Response

Cohort 3/4400 mg

Not Tolerated

Cohort 1/2200 mg Dose

Cohort 1/2/3300 mg

ToleratedNo Response

Maintain200 mg

ToleratedBiochemical Response

Maintain300 mg

ToleratedBiochemical Response

Maintain400 mg

Not Tolerated

Cohort 1/2

STOP

Cohort 3

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

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 CougnouxORD/CC Bench to Bedside Awards

Ara Parseghian Medical Research FoundationNational Niemann Pick Disease Foundation

SOAR-NPCDana’s Angels Research Trust

Niemann-Pick disease, type C1

Niemann-Pick disease, type C1

Mouse

Cat

Hydroxypropyl-β-Cyclodextrin

Mouse 0.5 gCat 30.0Human 1400.0Newborn 375.0

Vertical supranuclear ophthalmoplegia

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

Niemann-Pick Disease, type C1

Niemann-Pick disease, type C1

Attenuation of the 24OHCresponse with repetitive dosing