2-Hydroxypropyl-β-cyclodextrin is the active component in a triple combination formulation for treatment of Niemann-Pick C1 disease

Article abstract of DOI:10.1016/j.bbalip.2019.04.011

Niemann-Pick type C1 (NPC1) disease is a fatal neurovisceral disease for which there are no FDA approved treatments, though cyclodextrin (HPβCD) slows disease progression in preclinical models and in an early phase clinical trial. Our goal was to evaluate the mechanism of action of a previously described combination-therapy, Triple Combination Formulation (TCF) – comprised of the histone deacetylase inhibitor (HDACi) vorinostat/HPβCD/PEG – shown to prolong survival in Npc1 mice. In these studies, TCF's benefit was attributed to enhanced vorinostat pharmacokinetics (PK). Here, we show that TCF reduced lipid storage, extended lifespan, and preserved neurological function in Npc1 mice. Unexpectedly, substitution of an inactive analog for vorinostat in TCF revealed similar efficacy. We demonstrate that the efficacy of TCF was attributable to enhanced HPβCD PK and independent of NPC1 protein expression. We conclude that although HDACi effectively reduce cholesterol storage in NPC1-deficient cells, HDACi are ineffective in vivo in Npc1 mice.

Full text of DOI:10.1016/j.bbalip.2019.04.011

Accepted Manuscript
2
-Hydroxypropyl-β-cyclodextrin is the active component in a
triple combination formulation for treatment of Niemann-Pick C1
disease
Jessica Davidson, Elizabeth Molitor, Samantha Moores, Sarah
E. Gale, Kanagaraj Subramanian, Xuntian Jiang, Rohini Sidhu,
Pamela Kell, Jesse Zhang, Hideji Fujiwara, Cristin Davidson,
Paul Helquist, Bruce J. Melancon, Michael Grigalunas, Gang Liu,
Farbod Salahi, Olaf Wiest, Xin Xu, Forbes D. Porter, Nina H.
Pipalia, Dana L. Cruz, Edward B. Holson, Jean E. Schaffer,
Steven U. Walkley, Frederick R. Maxfield, Daniel S. Ory
PII:
S1388-1981(19)30064-2
DOI:
https://doi.org/10.1016/j.bbalip.2019.04.011
BBAMCB 58451
Reference:
To appear in:
BBA - Molecular and Cell Biology of Lipids
Received date:
Revised date:
Accepted date:
3 January 2019
25 April 2019
26 April 2019
Please cite this article as: J. Davidson, E. Molitor, S. Moores, et al., 2-Hydroxypropyl-β-
cyclodextrin is the active component in a triple combination formulation for treatment of
Niemann-Pick C1 disease, BBA - Molecular and Cell Biology of Lipids, https://doi.org/
1
0.1016/j.bbalip.2019.04.011
This is a PDF file of an unedited manuscript that has been accepted for publication. As
a service to our customers we are providing this early version of the manuscript. The
manuscript will undergo copyediting, typesetting, and review of the resulting proof before
it is published in its final form. Please note that during the production process errors may
be discovered which could affect the content, and all legal disclaimers that apply to the
journal pertain.

ACCEPTED MANUSCRIPT
1
2
-hydroxypropyl--cyclodextrin is the active component in a triple combination formulation for
treatment of Niemann-Pick C1 Disease
1
1
1
1
Authors: Jessica Davidson , Elizabeth Molitor , Samantha Moores , Sarah E. Gale , Kanagaraj
1
1
1
1
1
1
Subramanian , Xuntian Jiang , Rohini Sidhu , Pamela Kell , Jesse Zhang , Hideji Fujiwara , Cristin
2
3
3
3
3
3
3
Davidson , Paul Helquist , Bruce J. Melancon , Michael Grigalunas, Gang Liu, Farbod Salahi, Olaf Wiest ,
4
5
6
6
7
1
Xin Xu , Forbes D. Porter , Nina H. Pipalia , Dana L. Cruz , Edward B. Holson , Jean E. Schaffer , Steven U.
2
6
1*
Walkley , Frederick R. Maxfield , Daniel S. Ory
Affiliations:
1
Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA
2
Dominick P. Purpura Department of Neuroscience, Rose F. Kennedy Intellectual and Developmental
Disabilities Research Center, Albert Einstein College of Medicine, 1410 Pelham Parkway South, Bronx, NY
10461, USA
3
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556 5670, USA
Division of Preclinical Innovation, National Center for Advancing Translational Sciences (NCATS), 9800
4
Medical Center Dr., National Institutes of Health, Rockville, MD 20850, USA
5
Section on Molecular Dysmorphology, Eunice Kennedy Shriver National Institute of Child Health and Human
Development, NIH, DHHS, Bethesda, MD 20892, USA
6
Department of Biochemistry, Weill Cornell Medical College, New York, NY 10065, USA
7
KDac Therapeutics, Cambridge, MA 02139, USA
*
To whom correspondence should be addressed: dory@wustl.edu
One Sentence Summary: HDACi are ineffective in treating NPC1 disease

ACCEPTED MANUSCRIPT
2
ABSTRACT
Niemann-Pick type C1 (NPC1) disease is a fatal neurovisceral disease for which there are no FDA approved
treatments, though cyclodextrin (HPβCD) slows disease progression in preclinical models and in an early
phase clinical trial. Our goal was to evaluate the mechanism of action of a previously described combination-
therapy, Triple Combination Formulation (TCF) – comprised of the histone deacetylase inhibitor (HDACi)
vorinostat/HPβCD/PEG – shown to prolong survival in Npc1 mice. In these studies, TCF’s benefit was
attributed to enhanced vorinostat pharmacokinetics (PK). Here, we show that TCF reduced lipid storage,
extended lifespan, and preserved neurological function in Npc1mice. Unexpectedly, substitution of an inactive
analog for vorinostat in TCF revealed similar efficacy. We demonstrate that the efficacy of TCF was attributable
to enhanced HPβCD PK and independent of NPC1 protein expression. We conclude that although HDACi
effectively reduce cholesterol storage in NPC1-deficient cells, HDACi are ineffective in vivo in Npc1mice.
1. INTRODUCTION
Niemann-Pick Type C (NPC) is a rare, autosomal recessive disease characterized by a defect in cholesterol
trafficking with subsequent lysosomal lipid accumulation. The clinical presentation of NPC disease is variable.
Age of onset ranges from perinatal to adulthood and initial manifestations may be visceral, neurologic, or
psychiatric. Patients who survive past the perinatal stage will develop a progressive neurological disease that
consists of cerebellar ataxia, dysarthria, dysphagia, and dementia, and ultimately leads to premature death [1].
NPC disease is caused by mutations in the NPC1 and NPC2 genes. NPC1 is a large transmembrane
glycoprotein and NPC2 is a small, soluble protein; both proteins reside in lysosomes and facilitate cholesterol
transport from that compartment [2-5]. Defects in the NPC1 gene are responsible for 95% of the disease, with
over 400 mutations having been identified (https://medgen.medizin.uni-tuebingen.de/NPC-db2/index.php). The
I1061T
most prevalent NPC1 mutation, NPC1
, represents approximately 15-20% of all disease alleles [6, 7].
I1061T
Previous studies have shown that the NPC1
protein is misfolded and targeted to endoplasmic reticulum
(
ER)-associated degradation (ERAD) through the proteasome [8]. The use of chemical chaperones or protein
I1061T
overexpression has been shown to partially correct the NPC1
phenotype by allowing a portion of the
I1061T
mutant protein to evade ERAD. NPC1
protein that escapes this checkpoint is then targeted to lysosomes
where it is functional with respect to cholesterol trafficking [8].
There are currently no FDA approved treatments for NPC disease. Miglustat, an inhibitor of glycosphingolipid
synthesis that has shown limited efficacy in treatment of NPC disease, is approved in over 40 countries,
though not in the US [9]. The most promising therapeutic under evaluation is 2-hydroxypropyl-β-cyclodextrin
(
HPCD), a cholesterol-binding compound. In preclinical models, treatment with HPCD causes a reduction in
unesterified cholesterol and glycolipid storage, prolonged survival, and a delay in clinical onset [10-12]. In a
Phase 1/2a trial HPCD was effective in slowing neurodegenerative disease progression in NPC1 patients and
is currently being evaluated in a late stage clinical trial [13]. Because cyclodextrin is largely excluded by the
blood brain barrier, optimal treatment requires direct central nervous system (CNS) delivery [11, 14-16].

ACCEPTED MANUSCRIPT
3
A potentially more tractable therapeutic approach is the use of CNS-penetrant, small molecule proteostatic
regulators, such as histone deacetylase (HDAC) inhibitors [17]. Through posttranslational modification of
histones, transcription factors, and chaperones, HDACs can modify gene transcription and expression. HDAC
inhibitors (HDACi) have been evaluated in a broad range of neurodegenerative disorders such as Alzheimer,
Parkinson, and Huntington disease [18]. In previous studies, HDACi, including suberoylanilide hydroxamic acid
(
also known as vorinostat or SAHA), were found to be effective in reducing cholesterol storage in murine
I1061T
embryonic fibroblasts and human fibroblasts expressing the NPC1
protein [19-22]. While the mechanism
I1061T
of the correction is not well understood, treatment with the HDACi increased NPC1
expression through
stabilization of the mutant protein, presumably through alteration of the proteostatic environment [21].
Intriguingly, when treated with HDACi, ~85% of all the NPC1 missense mutants tested exhibited reduced
cholesterol storage, suggesting that proteostatic regulation may be relevant to large majority of NPC1 patients
[
21]. The potential proteostatic benefit of vorinostat is currently being evaluated in NPC1 patients in a Phase
/2a trial [23].
1
Although HDACi appear to be effective in vitro, these findings have proved difficult to translate to in vivo
nmf164
models. Treatment of Npc1
mice, which harbor a D1005G point mutation, with 150mg/kg vorinostat in a
PEG/DMSO vehicle led to normalization of expression of lipid homeostatic genes and limited improvement in
liver pathology and function [24]. However, NPC1 mutant protein expression and lipid accumulation remained
unchanged [24]. Moreover, vorinostat did not show efficacy with respect to disease progression, life span, or
weight loss, which is unsurprising in light of its poor blood brain barrier penetration [25]. In an effort to
circumvent this limitation, Alam and colleagues proposed a triple combination formulation (TCF), in which both
nmf164
vorinostat and HPCD were complexed in a PEG/DMSO vehicle [26, 27]. Treating Npc1
mice with the
TCF led to an almost two-fold increase in lifespan relative to mice treated with HPCD alone. They reported
greater exposure of the plasma and brain to vorinostat with the TCF, attributing the benefit to improved drug
pharmacokinetics (PK) [26, 27]. Although the formulation appears to offer an attractive approach to improving
HDACi efficacy in vivo, the results are difficult to interpret due to the confounding use of HPCD in the
formulation.
I1061T/I1061T
In the present study, we evaluate the effects of the TCF formulation in the Npc1
mouse model, which
nih
-/-
express the prevalent I1061T point mutation, and in BALB/c Npc1 (Npc1 ) mice. Using additional controls,
such as an inactive analog of vorinostat and HPCD dissolved in the PEG/DMSO vehicle, we show that TCF
efficacy results from enhanced HPCD PK rather than enhanced HDACi activity. Our findings of lack of
efficacy for the HDACi in vivo should prompt reevaluation of the application of these drugs for treatment of
NPC1 and other diseases.

ACCEPTED MANUSCRIPT
4
2. MATERIALS AND METHODS
2.1. Study Design
The study objectives were to evaluate the Triple Combination Formulation (TCF) [26] as a therapeutic for
Niemann-Pick C and determine its mechanism of action in a murine model. For the 6-week treatment regimen
tm(I1061T)Dso
and survival studies, Npc1
mice [22] were randomly allocated to one of ten different treatment
groups: vehicle, HPCD in vehicle (CD/Vehicle), HPCD in water (CD/H O), HPCD in water delivered 3x
2
weekly (CD/H O (3x/wk)), vorinostat, the inactive analog of vorinostat (IVA), TCF with vorinostat (TCF Active),
2
TCF with inactive vorinostat (TCF/IVA), and TCF with CI-994 (TCF/CI994). A parallel 6-week study was
nih
performed in BALB/c Npc1 mice to determine dependency on NPC1 mutant protein expression. This study
included three of the treatment groups: vehicle, CD/H O, and TCF.
2
2.2. Animals
tm(I1061T)Dso
I1061T
nih
-/-
I1061T
For treatment studies, Npc1
(NPC1
) and BALB/c Npc1 (NPC1 ) mice were used. NPC1
-/-
mice harbor the clinically relevant p.I1061T mutation in the Npc1 gene [22]. Npc1 carries a null mutation gene
due to a retroposon insertion in the NPC1 locus [28, 29]. Breeding colonies of the NPC1 mouse lines are
maintained at Washington University. C57BL/6J mice obtained from Jackson Laboratories (Bar Harbor, ME,
USA) were used for PK analysis and as wild type controls. Mice were kept in a temperature and humidity-
controlled animal facility and were given access to standard chow and water ad libitum. Experimental
procedures were approved by the Washington University Animal Studies Committee and were conducted in
accordance with the USDA Animal Welfare Act and the Public Health Service Policy for the Humane Care and
Use of Laboratory Animals.
2
.3. Materials
All chemicals including powdered 2-hydroxypropyl-β-cyclodextrin (H107), DMSO, polyethylene glycol
PEG400), methoxypolyethylene glycol 350 (MeO-PEG-350) ammonium fluoride and citric acid were
(
purchased from Sigma-Aldrich (St. Louis, MO, USA). Vorinostat (suberanilohydroxamic acid), inactive
vorinostat (suberanilic acid), d5-vorinostat, and d5-inactive vorinostat were synthesized in-house initially in the
Helquist lab and later by the Notre Dame Chemical Synthesis Drug Discovery Facility following published
procedures [30]. The LC-MS grade methanol, isopropanol, and acetonitrile were purchased from Honeywell
(
Morristown, NJ).
2
.4. Synthesis of inactive vorinostat
Octane-1,8-dioic acid was purchased from Ark Pharma (Libertyville, IL). A solution of octane-1,8-dioic acid
5.00 g, 28.7 mmol) in acetic anhydride (10 mL) was heated at reflux for 1 h. The mixture was cooled to 25°C
(
and concentrated under vacuum. The remaining yellow solid was recrystallized from acetonitrile to afford the
cyclic anhydride (1.75 g; 20%) as a white solid, which was used in the next step. The anhydride was dissolved

ACCEPTED MANUSCRIPT
5
in anhydrous THF (50 mL), and aniline (1.03 g, 1.01 mL; 11.1mmol) was added to the solution at 25 °C. After
being stirred for 30 min, the mixture became cloudy white. Water (20 mL) was added, and the resulting white
solid was isolated by filtration. Recrystallization from water gave 8-oxo-8-(phenylamino)octanoic acid (0.92 g,
1
3
2
1
2
3%) as a white powder. H NMR (400 MHz, CH OD) δ 7.54 (d, 2H), 7.27 (t, 2H), 7.06 (m, 1H) 2.30 (m, 2H),
3
13
.27 (m, 2H), 1.61 (m, 4H), 1.39 (m, 4H). C NMR (126 MHz, CH OD) δ 176.43, 173.45, 138.72, 128.58,
3
20.08, 36.72, 33.67, 28.82, 28.75, 25.59, 24.75. HRMS m/z calculated for C H NO (M+1) 250.1438; found
14
20
3
1
13
50.1457. (lit.[30] H NMR, C NMR).
2.5. Dose response of fibroblasts and bone marrow-derived macrophages
I1061T
As previously described [21], human NPC1
fibroblasts were grown in Modified Eagle Medium (MEM)
supplemented with 10% FBS. For drug treatment, cells were maintained in MEM supplemented with 5% FBS.
These cells were seeded in 384-well plates at 450 cells/well in growth medium on day one. After overnight
incubation, test compounds were added at 40 nM to 10 μM final concentration. DMSO was used as a control
treatment in each plate. After 72 hours, the plate was washed with PBS three times, fixed with 1.5% PFA, and
stained with 50 μg/ml filipin and the nuclear stain Draq5. Measurements were made from four wells for each
condition in each experiment, and the experiment was repeated three times. Images were acquired on an
Micro
ImageXpress
automatic fluorescence microscope, at four sites per well and analyzed to obtain the LSO
compartment ratio, which is a measure of filipin labeling of stored cholesterol [19, 20]. A high LSO ratio is
associated with high levels of cholesterol in late endosomes/lysosomes. The LSO compartment ratio for each
concentration was normalized to the DMSO treated controls. The identical procedure was used for bone
I1061T/I1061T
marrow derived macrophages, which were obtained from the femurs of Npc1
mice.
2.6. Drug preparations and regimen
Vorinostat formulations and TCF active were prepared as described [26]. The inactive formulations and
TCF/CI994 were prepared in the same manner using the inactive analog of vorinostat and CI-994, respectively.
HPCD was dissolved in either the 45% PEG/5% DMSO vehicle or water, as specified. Final compositions of
all formulations are summarized in Table 1, and were prepared fresh for each use and filtered prior to delivery.
In HPCD-containing formulations, final concentration was 2000 mg/kg, except in the CD/H O (3x/wk), in which
2
the final concentration was 4000 mg/kg. Treatments began at four weeks of age and were administered weekly
for the duration of the six-week study, with the exception of CD/H O (3x/wk), in which the treatment was
2
delivered three times per week. Animal body weight was determined before each injection. All treatments were
administered via intraperitoneal injection at a dose of 10 µL/g body weight. For survival studies, mice were
euthanized when they showed clinical signs of end-stage disease as previously described [10].
2.7. Gait analysis
Gait analysis was performed using the Noldus CatWalk XT 10.6 (Noldus Information Technology, Wageningen,
Netherlands). CatWalk XT is a highly sensitive tool to assess gait and locomotion. Gait analysis was performed

ACCEPTED MANUSCRIPT
6
once per week, prior to compound injection. The following data were analyzed: standing on diagonal, coupling,
and stride length. Standing on diagonal describes the relative duration of simultaneous contact with the glass
plate of all combinations of paws. Coupling, a measure of inter-paw coordination, is the temporal relationship
between the placement of a pair paws relative to the duration of a step cycle. Stride length is the distance
between two successive placements of the same paw.
2.8. Tissue collection
Mice were euthanized according to established procedures one-hour post final treatment. Euthanasia was
performed via ketamine/xylazine cocktail and mice were perfused with 0.9% saline. Plasma was collected via
retroorbital bleed. Liver and brain tissue were resected with one lobe of liver, half of the cortex, and the whole
cerebellum fixed in 4% paraformaldehyde for tissue histology. All remaining liver and the remaining half of the
cortex were flash frozen in liquid nitrogen and stored at -80C until use.
2.9. Pharmacokinetics
Five-week-old male C57BL/6J (000664) mice were purchased from Jackson Laboratories (Bar Harbor, ME,
USA). Mice were enrolled into seven treatment groups with five harvest time points per group and n=4 mice
per harvest time point. Across all treatment groups, a dose of 10 µL/g of body weight was used and all drug
compounds were delivered via a single intraperitoneal injection. Tissue harvests occurred at 0.25, 1, 2, 4, or 7
hours post injection as indicated above.
For the quantification of vorinostat and IVA, each liquid chromatography-tandem mass spectrometry
(
LC-MS/MS) run included eight calibration standards in duplicate, one blank, one blank without IS, and study
samples. The internal standards (IS) for plasma and liver (600/600/30,000 ng/mL d5-vorinostat/d5-suberanilic
acid/HP-d6-β-CD) and for brain (100/100/5000 ng/mL d5-vorinostat/d5-suberanilic acid/HP-d6-β-CD) were
prepared by dilution of d5-vorinostat, d5-suberanilic acid, and HP-d6-β-CD stock solutions with methanol-water
(
1:1). The PEG-400 IS spiking solutions for liver (2 g/mL MeO-PEG-350) and for brain (1 g/mL MeO-PEG-
50) were prepared by dilution of MeO-PEG-350 stock solution with acetonitrile. Brain samples were
3
homogenized in 4 volumes of Milli Q water and liver samples in 0.04 M of citric acid in water for analysis of
vorinostat/suberanilic acid/HPCD. For PEG400, brain and liver samples were homogenized in 4 volumes of
Milli Q water.
LC-MS/MS analysis was conducted on a Shimadzu (Columbia, MD) Prominence UFLC system coupled
with an Applied Biosystems/MDS Sciex (Ontario, Canada) 4000QTRAP mass spectrometer using positive
multiple reaction monitoring (MRM). Separation of vorinostat and suberanilic acid was carried out using a
Waters (Milford, MA) Xselect HSS C18 analytical column (3 × 50 mm, 3.5 µm) connected to a Phenomenex
(
Torrance, CA) SecurityGuard C18 guard column (4 × 3 mm) at a flow rate of 0.6 mL/min. The mobile phase
consisted of 5 mM ammonium fluoride in water (solvent A), and 0.1% formic acid in methanol-acetonitrile (4:1)
solvent B). The step gradient was as follows: 0–0.1 min, 50% solvent B; 0.1–1.5 min, 50% to 100% solvent B;
.5–2.0 min, 100% solvent B; 2.0–2.1 min, 100% to 50% solvent B; 2.1–3.5 min 50% solvent B. The HPLC
(
1

ACCEPTED MANUSCRIPT
7
eluate was directed into the mass spectrometer for data acquisition within the 1.6-min time window (0.8 – 2.4
min) in which vorinostat and suberanilic acid were eluted; elsewhere, eluate was sent to waste to minimize
source contamination. The total run-time was 3.5 min. The injection volume was 5, 10, 20 µL for plasma, liver,
and brain samples, respectively. The ESI source temperature was 600 °C; the ESI needle was 2000 V; the
declustering potential was 50 V; the entrance potential was 10 V; and the collision cell exit potential was 10 V
for vorinostat, suberanilic acid, d5-vorinostat, and d5-suberanilic acid. The collision and curtain gas were set at
medium and 20, respectively. Both desolvation gas and nebulizing gas were set at 55 L/min and 35 L/min,
respectively. The collision energies were 17, 32, 17, and 32 eV for vorinostat, suberanilic acid, d5-vorinostat,
and d5-suberanilic acid, respectively. MRM mass transitions m/z 265.2→172.1, 250.2→94.1, 270.2→172.1,
and m/z 255.2→94.1 were used for vorinostat, suberanilic acid, d5-vorinostat, and d5-suberanilic acid,
respectively, with a dwell time of 50 ms for each mass transition. The mass spectrometer was operated at unit
mass resolution for both the first and third quadruple.
Sample preparation, instrumentation, LC-MS/MS conditions, and quantification of cyclodextrin by
tandem mass spectrometry were performed as previously reported [31]. Data were acquired and analyzed by
Analyst software (version 1.5.2).
For analysis of PEG400, the mobile phase consisted of 5 mM ammonium acetate in water (solvent A),
methanol (solvent B), and isopropanol (solvent C). Separation of PEG-400 and MeO-PEG-350 was carried out
using solvent A and B on a Waters (Milford, MA) Xbridge C18 analytical column (3 × 50 mm, 3.5 μm)
connected to a Phenomenex (Torrance, CA) SecurityGuard C18 guard column (4 × 3 mm) at a flow rate of 0.6
mL/min. The step gradient for solvents A and B was as follows: 0 – 3.0 min, 20% to 50% solvent B; 3.0–3.1
min, 50% to 20% solvent B; 3.1–6.5 min 20% solvent B. The solvent A and B eluate was controlled by valve 1
and directed into the mass spectrometer for data acquisition within the 1.6-min time window (1.2 – 3.0 min) in
which PEG-400 and MeO-PEG-350 were eluted; elsewhere, eluate was sent to waste to minimize source
contamination. Solvent C is controlled with valve 2 and used to back flush column after PEG-400 and MeO-
PEG-350 were eluted at 0.6 mL/min for 1.5 min (3.0 – 4.5 min). The total run-time was 6.5 min. The injection
volume was 5 μL for liver and brain samples. The ESI source temperature was 650°C; the ESI needle was
5000 V; the declustering potentials was 35 and 80 V for PEG-400 and MeO-PEG-350, respectively; the
entrance potential was 10 V for both PEG-400 and MeO-PEG-350; and the collision cell exit potential was 10 V
for both PEG-400 and MeO-PEG-350. The collision and curtain gas were set at medium and 20, respectively.
Both desolvation gas and nebulizing gas were set at 65 L/min and 55 L/min, respectively. The collision
energies were 30 eV for both PEG-400 and MeO-PEG-350. The multiple reaction monitoring (MRM) mass
transitions m/z 344.1→133.1, and m/z 314.1→103.1 were used for PEG-400 and MeO-PEG-350, respectively,
with a dwell time of 50 ms for each mass transition. The mass spectrometer was operated at unit mass
resolution for both the first and third quadruple.
Pharmacokinetic parameters were calculated with the mean concentrations of 3-4 mice at each time
point using the non-compartmental method (NCA; Model 200) of the pharmacokinetic software Phoenix

ACCEPTED MANUSCRIPT
8
WinNonlin (Certara, St. Louis, MO). The area under the concentration-time curve (AUC) was calculated using
the linear trapezoidal method.
2
.10. Measurement of histone acetylation
Tissues were homogenized and sonicated in TNEN buffer (1% NP-40, 0.25mM EDTA, 50mM Tris pH 8,
50mM sodium chloride) with 1x protease complete inhibitors and 1mM phenylmethylsulfonyl fluoride. The
1
lysate was centrifuged for 10 min at 10,000 × g at 4°C. Supernatant was collected and protein quantified using
a BCA Protein Assay kit (Pierce). Equal amounts of protein were loaded and separated by NuPage 4-12% Bis-
Tris gel (Invitrogen) under reducing conditions with 1x MES running buffer (Invitrogen). Proteins were
transferred to nitrocellulose using the iBlot 2 dry transfer system (Invitrogen) and probed with rabbit anti-human
histone H3 polyclonal antibody at a 1:10,000 dilution (Abcam) and rabbit anti-human acetyl-histone H3
polyclonal antibody at a 1:1000 dilution (Millipore). A rabbit anti-human Hsp90 (Enzo) was used as a protein
loading control. Peroxidase-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories) and
chemiluminescence were used for detection.
2.11. Measurement of NPC1 protein expression
For quantification of NPC1 protein expression in liver, tissues were homogenized and sonicated in HEPES
buffer (50mM Tris pH 8, 150mM sodium chloride, 5mM EDTA, 1% Triton X-100, 0.1%SDS) with 1x protease
complete inhibitors and 1mM phenylmethylsulfonyl fluoride. The lysate was centrifuged for 10 min at 10,000
×
g at 4°C. Supernatant was collected and protein quantified using a BCA Protein Assay kit (Pierce). Sample
protein was incubated overnight at 4°C with 1.8μg of a rabbit polyclonal antibody raised against mouse NPC1
amino acids 1261–1278, CTTYERYRGTERERLLNF) and produced in-house [32]. Immunoprecipitation was
(
performed using Dynabeads Protein A (Thermo Fisher) and protein was eluted using 2x Laemmli sample
buffer (Bio-Rad) with 5% 2-mercaptoethanol. Samples were separated by NuPage 3-8% Tris-Acetate gel
(
Invitrogen) under reducing conditions with 1x Tris-Acetate running buffer (Invitrogen). Proteins were
transferred to nitrocellulose using the iBlot 2 dry transfer system (Invitrogen) and probed with aforementioned
mouse-NPC1 antibody at a concentration of 1:1000. Protein detection was performed as described above.
For quantification of NPC1 protein expression in brain, membrane proteins in tissues were isolated
using a membrane protein extraction kit, Mem-PER Plus (Thermo Scientific), and quantified using a BCA
Protein Assay kit (Pierce). Samples were separated by NuPage 3-8% Tris-Acetate gel (Invitrogen) under
reducing conditions with 1x Tris-Acetate running buffer (Invitrogen). Proteins were transferred to nitrocellulose
using the iBlot 2 dry transfer system (Invitrogen). NPC1 expression was analyzed using the rabbit polyclonal
antibody raised against human NPC1 C-terminus described above. Detection of p63 by a rabbit polyclonal
antibody [33] was used as a loading control. Their respective concentrations were 1:1000 and 1:5000.
Peroxidase-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories) and
chemiluminescence were used for detection.

ACCEPTED MANUSCRIPT
9
2
.12. Lipidomic analysis
The tissue samples were homogenized with 8-fold excess volume of PBS buffer using an Omni Bead Ruptor
4 (Omni International, Inc.). 50 µL of the tissue homogenate was extracted with 200 µL of methanol
2
containing internal standards. For sterol analysis, d7-cholestane-3,5,6-triol, d7-7-ketocholesterol, d7-24(S)-
hydroxycholesterol, and d7-cholesterol were used as the internal standards. All sterols as well as their
deuterated standards were derivatized with N, N-dimethylglycine. For sphingolipid measurements, 17:0
glycosylceramide, d3-16:0 lactosyl-ceramide, d7-sphingosine, d7-sphingosine-1-phosphate, N-omega-CD3-
octadecanoyl monosialoganglioside GM3, and N-heptadecanoylceramide trihexoside were added as the
internal standards. Lipid analysis was performed by LC-MS/MS as previously described [22, 34].
2.13. Histopathological analysis
Liver and brain tissue samples fixed in 4% paraformaldehyde and stored cold in 0.1 M phosphate buffer were
sectioned at 35 microns on a Vibratome and subjected to filipin staining (liver and brain) and to GM2
immunohistochemical staining (brain only). Staining procedures followed published methods [10, 35]. Briefly,
for filipin labeling, prepared sections were incubated with PBS containing 0.005% filipin complex (Sigma
F9765) from a 25 mg/mL solubilized stock in DMSO (Sigma D128-500), or with an equivalent concentration of
DMSO only to serve as a control. The sections were mounted on Gold Seal fluorescent antibody slides (Fisher
12-568-20) with Prolong Antifade Kit (Molecular Probes P7481) and circular coverslips (Fisher 12-545-81). For
GM2 staining, prepared sections were incubated overnight at 4°C in diluent with primary antibody to GM2
ganglioside (produced in house) or with nonspecific mouse IgM (Sigma) as control. On day 2, the sections
were incubated with the secondary antibody (Texas Red-conjugated goat anti-mouse IgM), and mounted as
previously described for filipin labeling. Prepared slides were imaged on a Leica fluorescence microscope.
Blinded analysis by two independent reviewers was carried out on sections that were coded
independently by a third party. A scale of 0 – 5 was used to evaluate staining intensity as an indicator of
relative storage of cholesterol and GM2 ganglioside (0 = no storage, 5 = extensive storage present). In liver,
cholesterol storage was assessed based on staining intensity and the distribution of filipin staining in
hepatocytes and liver macrophages (Kupffer cells). The identity of the latter were established by evaluation of
CD68 stained cells in liver sections which reveal the typical distribution of Kupffer cells vs. hepatocytes. In
brain, assessments were carried on for both GM2 ganglioside and cholesterol, focusing on upper (layers 1-3)
and lower (4-6) dorsal neocortex. Brain sections were matched so that analysis occurred just above the dorsal
placement of the hippocampus at -1.6-2.0 from the bregma (based on Franklin and Paxinos, The Mouse Brain
rd
in Stereotaxic Coordinates, 3 edition). Following completion of scoring, the third party code was broken and
scoring results of all sections were averaged for each tissue area and statistical analyses performed using an
ANOVA (significance deemed p<0.05). If warranted, Dunnett’s test was employed to determine significant
differences between vehicle and treatment groups.
2.14. HDAC inhibitor treatment of bone marrow-derived macrophages

ACCEPTED MANUSCRIPT
10
I1061T
Bone marrow was isolated from Npc1
mice that were housed in a pathogen-free environment at Albert
Einstein College of Medicine and used in accordance with protocols approved by the Institutional Animal Care
I1061T
and Utilization Committees. Sterilized femurs and tibias from Npc1
mice were flushed in bone marrow
(
BM) media (DMEM with L-Glutamine, 4.5 g/L glucose, sodium pyruvate and 1% penicillin/streptomycin,
supplemented with 10% heat-inactivated fetal calf serum (FCS) and 20% L-cell conditioned medium). Cells
were differentiated for seven days by culturing in BM media in a humidified atmosphere (5% CO ) at 37°C.
2
For vorinostat treatment and filipin staining, differentiated BMMs were plated in 384 well plates in BM
media. On the following day vorinostat was added at concentrations from 0.004 µM to 10 µM in 4-6 wells per
dose, and the cells were incubated in control medium or vorinostat for 48h or 72h. To load the BMMs with
lipoprotein, the cells were supplemented for the final 24 hours with 20 µg/ml acetylated low density lipoprotein
(
AcLDL). At the end of the treatment cells were washed with phosphate buffered saline (PBS), fixed with 2%
para-formaldehyde (PFA) in PBS and stained with filipin and Draq5. Automated microscopy and data analysis
was performed as described in section 2.5.
3. RESULTS
3.1. Characterization of an inactive vorinostat control
We reasoned that if HPCD was the active component in the TCF, then a formulation containing the
PEG/DMSO vehicle, but lacking HDACi activity, would show similar efficacy in an NPC1 mouse model. We
selected suberoylanilide acid, an inactive analog and synthetic precursor of suberoylanilide hydroxamic acid
(
vorinostat)[30] for inclusion in TCF (Fig. 1A) to minimize perturbation to the formulation. In contrast to
1061T
vorinostat, which reduces lysosomal cholesterol in human fibroblasts with homozygous NPC1
mutations
(
Fig. 1B), suberoylanilide acid (inactive vorinostat, IVA), had no effect on cholesterol storage. To demonstrate
that IVA lacks HDACi activity in vivo, the inactive compound along with a vorinostat control were injected into
mice – both alone and as a component of TCF – and liver acetylation of histone H3 was assessed one hour
post injection, a method similarly used by Alam et al [26]. Levels of both acetylated and total histone 3 were
determined by western blot, and their ratio used to determine acetylation status. Increased histone 3
acetylation was observed in treatment groups containing an active HDACi: vorinostat alone, TCF containing
vorinostat (TCF active), and TCF containing CI-994 (TCF/CI-994) [36], a more CNS-penetrant HDACi (Fig.
1C). No increase in acetylation was observed in treatment groups not containing an active HDACi, including
groups treated with IVA and TCF/IVA. Together, these findings support use of suberoylanilide acid as an
inactive analog for examination of the TCF mechanism.

ACCEPTED MANUSCRIPT
11
1
1
0
0
.5
.0
.5
.0
1.0
IVA
Vorinostat
#
*
0
.8
Suberoylanilide Hydroxamic Acid
vorinostat)
0.6
(
0
0
.4
.2
0.0
0
.04
0.12
1.1
3.3
10
t
le
C
O
le
A
e
4
A
a
i
v
t
I99
hic
2
hic
t
I
V
c
a
/IV
s
/
H
o
F
concentration (mM)
e
/ve
F/C
D
rin
C
v
Suberoylanilide Acid
F
T
D
o
C
C
C
v
T
T
(inactive vorinostat analog, IVA)
Figure 1. Inactive vorinostat control. (A) Structures of suberoylanilide hydroxamic acid (vorinostat) and
I1061T/I1061T
suberoylanilide acid (inactive vorinostat, IVA). (B) Dose response of NPC1
fibroblasts to vorinostat
and IVA. After 72h of treatment, cells were PFA fixed and stained with filipin to detect cholesterol. The total
instensity of lysosomal storage organelle (LSO) filipin per cell area was determined using the LSO
comparatment ratio (total intensity above a high threshold/number of pixels above low threshold). Data are
shown as mean±SEM (n=3). (C) Assessment of acetyaltion status of histone H3 (H3) in mice by western
blotting. Wild type C57BL/6J were given a single dose of the specified compound and harvested 1h post
injection. Data are presented as mean ± SEM of the ratio of acetylated H3 to total H3, after normalization to an
Hsp90 loading control. All groups are n=4 and statistical analysis was done versus vehicle control using an
ANOVA with Dunnett’s test to determine significance. *, p<0.05; #, p<0.01.
3.2. Pharmacokinetic analysis of TCF formulation components
In order to evaluate the potential interaction between the individual components of TCF (Table 1), we
performed PK studies for vorinostat, HPCD and the PEG400 vehicle. Mice were administered a single
intraperitoneal dose of the specified treatment, and plasma, liver, and brain samples were harvested at 0.25, 1,
2, 4, and 7 hours after injection. Vorinostat, HPCD and PEG400 concentrations were determined by LC-
MS/MS. The Cmax for vorinostat in plasma was not significantly different between vorinostat in vehicle and in
TCF, though the area under the curve (AUC ) for vorinostat in TCF was increased 31%. In the liver tissue,
last
TCF increased vorinostat Cmax and AUClast by 18% and 27%, respectively (Table 2). Similarly, in the brain
tissue, TCF increased vorinostat Cmax and AUClast by 55% and 50%, respectively. These findings suggest that
in the presence of the PEG400 and HPCD there is reduced clearance of the drug from plasma, resulting in
increased tissue penetration. By contrast, PEG400 altered plasma HPCD PK by lowering the Cmax and
increasing time to peak concentration (Tmax). This was accompanied by a significant increase in AUClast for
HPCD in all formulations containing PEG400 (CD/vehicle, TCF active and TCF/IVA), as compared to water
vehicle (Table 3). Compared with CD/water, the liver tissue exposure (AUC ) for the HPCD-containing
last
formulations was increased 36%, 37% and 25% in the CD/vehicle, TCF active and TCF/IVA groups,
respectively, and the brain tissue exposure (AUC ) for the HPCD-containing formulations was increased
last

ACCEPTED MANUSCRIPT
12
106%, 129% and 95% in the CD/vehicle, TCF active and TCF/IVA groups. There was no clear relationship
between the PEG400 PK with respect to the presence or absence of HPCD or the HDACi, although addition
of either component increased Cmax and AUClast as compared to vehicle alone (Supplemental Table 1). Taken
together, these findings demonstrate that inclusion of the PEG400 vehicle in the formulations increased tissue
exposures for vorinostat and HPCD by increasing plasma concentrations of the drugs.
5
4
% DMSO,
5% PEG
2000 mg/kg 4000 mg/kg
50 mg/kg
vorinostat
30 mg/kg
CI-994
Treatment
H O
2
HPβCD
HPβCD
vehicle
+
+
CD/vehicle
+
+
CD/H O
+
+
2
CD/H O (3x/wk)
+
2
vorinostat
IVA
+
+
+
+
+
active
inactive
active
TCF active
TCF/IVA
TCF/CI994
+
+
+
inactive
+
vehicle
+
+
+
CD/vehicle
TCF active
+
+
active
I1061T/I1061T
-/-
Table 1. Composition of formulations. NPC1
mice and Npc1 were allocated to one of the
following treatment groups and were treated weekly (with the exception of the CD 3x/wk group) over 6-weeks.
The compositions of all formulations are listed below, and the same compositions were used for PK studies.
Vorinostat Pharmacokinetic Parameters
Treatment
TCF
active
vorinostat
Matrix Parameters
Unit
ng/mL
hr
Cmax
Tmax
10495
0.25
10683
0.25
Plasma
Liver
AUClast
AUCinf
t1/2
nghr/mL
nghr/mL
hr
7535
7604
0.68
9893
9897
0.326
Cmax
Tmax
ng/mL
hr
209
0.25
513
247
0.25
650
AUClast
nghr/mL

ACCEPTED MANUSCRIPT
13
Cmax
Tmax
ng/mL
hr
1163
0.25
1238
1800
0.25
1857
Brain
AUClast
nghr/mL
Table 2. Pharmacokinetics for vorinostat in plasma, liver, and brain tissues. C57BL/6J wild type mice
were administered a single dose of vorinostat or TCF active and harvested 0.25, 1, 2, 4, or 7 hours post-
injection. Vorinostat drug levels were measured via LC-MS/MS. PK values were calculated from the average of
n=4 mice per time point, and are shown for plasma, liver, and brain samples.
Cyclodextrin Pharmacokinetic Parameters
Treatment
TCF
CD/vehicle CD/H O
TCF/IVA
2
active
Matrix
Parameters
Cmax
Unit
μg/mL
hr
3880
1
5375
0.25
4556
4558
0.62
4718
1
3477
1
Tmax
Plasma
AUClast
AUCinf
t1/2
μghr/mL
μghr/mL
hr
6730
6731
0.53
9883
9887
0.54
6100
6101
0.51
Cmax
Tmax
μg/mL
hr
182
1
153
0.25
562
166
1
144
1
Liver
Brain
AUClast
μghr/mL
767
770
702
Cmax
Tmax
μg/mL
hr
156
0.25
258
145
0.25
126
120
1
188
0.25
246
AUClast
μghr/mL
289
Table 3. Pharmacokinetics for HPCD in plasma, liver, and brain tissues. C57BL/6J wild type mice were
administered a single dose of vorinostat or TCF active and harvested 0.25, 1, 2, 4, or 7 hours post-injection.
HPCD drug levels were measured using LC-MS/MS. PK values were calculated from the average of n=4
mice per time point, and are shown for plasma, liver, and brain samples.
•3.3. Evaluation of efficacy of TCF formulation on lipid storage in liver tissue
I1061T/I1061T
I1061T/I1061T
To evaluate the efficacy of TCF, we conducted a short-term study in Npc1
mice. Npc1
mice
harbor a point mutation in the NPC1 protein that models the most prevalent human NPC1 mutation, causing
rapid ER-associated degradation of the misfolded protein [8, 22]. In cell-based models, HDACi have been
1061T
shown to stabilize the NPC1I
protein by facilitating its exit from the ER and targeting of the mutant, but
I1061T/I1061T
correctly folded protein, to the lysosome, where it is functional [8, 21]. Therefore, the Npc1
mouse
model is an ideal model to test proteostatic regulators, such as HDACi. Starting at four weeks of age, mice

ACCEPTED MANUSCRIPT
14
were entered into a six-week treatment protocol, in which they were randomly allocated to one of nine
treatment groups: PEG400/DMSO vehicle, HPCD in water (CD/H O), HPCD in vehicle (CD/Vehicle),
2
HPCD in water delivered 3x per week (CD/H O [3x/wk]), vorinostat, IVA, TCF active, TCF/IVA, and TCF/CI-
2
994 (Table 1). CI-994 is a pan-HDAC inhibitor that has improved CNS penetration as compared to vorinostat.
We reasoned that if TCF provides benefit by improving the ability of the HDACi to cross the blood brain barrier,
a greater benefit might be seen with CI-994 than with vorinostat. Mice were treated weekly and euthanized at
10 weeks of age. Lipidomic profiling was performed to assess the effect of the formulations on lipid storage
(
Figure 2). We surveyed 42 sphingolipid and sterol species previously shown to be elevated in NPC1 mouse
models and that respond to HPCD treatment [34]. Significant reductions in lipid storage were observed in
sterols (3,5,6-cholestentriol and cholesterol), mono- and dihexosylceramides (MCer and DCer), and
gangliosides (GA2, GM1 and GM2), and were limited almost exclusively to the treatment groups receiving
HPCD-containing formulations. By contrast, none of the treatment groups administered formulations
containing vorinostat, CI994, or IVA alone showed evidence of lipid reduction, with the exception of a modest
reduction in only a single lipid species (MCer 24:0) in the vorinostat group. We further assessed the effect of
the different formulations on liver cholesterol by performing histochemical staining of liver sections from mice in
the six-week trial using filipin, a fluorescent polyene antibiotic. Consistent with the lipidomic results, significant
reduction in hepatocyte filipin staining (i.e., cholesterol storage) were found only in mice treated with HPCD-
containing formulations (Figure 3). These assessments focused on filipin staining of hepatocytes rather than
Kupffer cells because HPCD has previously been shown to reduce hepatocyte accumulation of cholesterol
while enhancing storage in Kupffer cells [37]. Vorinostat in vehicle alone did not alter cholesterol levels. Taken
together, these findings strongly support that the reduction in lipid storage is attributable to the actions of
HPCD rather than HDAC inhibition.

ACCEPTED MANUSCRIPT
15
6
5
4
3
2
1
0
5
†
†
†
†
†
†
†
†
†
†
†
†
#
#
#
#
#
*
4
4
3
3
2
1
2
1
0
0
le
O
le
)
t
A
e
4
A
l
e
O
le
)
t
IVA
e
4
IVA
le
O
le
k
)
a
t
t
A
e
v
4
A
k
a
v
I99 /IV
k
a
v
9
I99 F/IV
hic
2
hic
w
t
I
V
i
i
c
H 2
hic
w
t
i
9
hic
2
hic
w
I
V
i
t
H
(3x
s
t
/
s
t
I
/
/
H
(3x
s
/
o
c
F
C
h
(3x
o
c
F
o
c
a
e
D
/ve
rin
a
F/C
e
D
/ve
rin
a
F/C
e
D
/ve
rin
F/C
v
C
v
C
C
v
C
F
C
T
F
T
F
T
D
C
O
o
C
C
D
O
o
C
C
D
O
o
C
C
T
C
H
2
v
T
T
C
H
2
v
T
T
C
H
2
v
T
/
/
/
D
D
D
C
C
3
5
4
4
3
2
1
0
†
†
†
#
†
†
†
†
†
†
†
†
†
†
†
†
†
†
†
*
2
3
2
1
1
0
0
le
O
le
)
t
A
e
4
A
l
e
O
l
e
)
t
A
e
4
A
l
e
O
l
e
k
)
a
t
IVA
e
4
IVA
/
k
a
v
I99 /IV
k
a
v
I99 /IV
t
s
v
I99
hic
2
hic
w
t
I
V
c
i
h
i
c
H 2
h
i
c
w
t
I
V
c
i
i
c
H 2
i
c
w
t
i
H
(3x
s
t
/
(3x
s
t
h
/
h
(3x
c
/
o
F
C
o
F
C
o
F
e
D
/ve
rin
a
F/C
e
D
/ve
rin
a
F/C
e
D
/ve
rin
a
F/C
C
v
C
v
C
v
C
F
F
T
F
T
T
D
O
o
C
C
D
C
O
o
C
C
D
O
2
o
C
C
T
v
T
C
H
2
v
T
T
C
H
2
v
T
T
C
/
H
/
/
D
D
D
C
C
4
3
2
1
0
†
†
†
†
†
†
le
O
le
)
t
A
e
4
A
k
a
i
v
t
I99 /IV
hic
2
hic
w
t
I
V
c
H
(3x
s
/
o
F
e
D
/ve
rin
a
F/C
v
C
C
F
T
D
C
O
o
C
C
T
C
H
2
v
T
/
D
I1061T/I1061T
Figure 2. Lipidomics in liver tissue of Npc1
mice treated with TCF regimens. Beginning at four
I1061T/I1061T
weeks of age Npc1
mice were treated weekly with one of the following regimens: vehicle (n=8),
HPCD in water (n=8), HPCD in vehicle (n=8), vorinostat (n=7), inactive vorinostat (n=8), TCF active (n=8),
TCF inactive (n=7), 4000mg/kg HPCD in water delivered 3x/week (n=7), and TCF with CI-994 (n=9). After six
weeks of treatment, animals were euthanized, liver tissue harvested, and lipid and sterol species quantified in
liver homogenate using LC-MS/MS. Data for representative species are shown as mean ± SEM. Statistics
were performed using ANOVA and significance determined versus vehicle using Dunnett’s test. *, p<0.05; #,
p<0.01; †, p<0.001.

ACCEPTED MANUSCRIPT
16
WT
vehicle
CD/H O
CD/vehicle
CD/H O (3x/wk)
2
2
5
4
3
2
1
0
#
†
†
#
#
*
vorinostat
IVA
TCF Active
TCF/CI994
TCF/IVA
)
e
icle
)
t
e
4
x
l
O
k
a
IVA
v
9
IVA
T
i
c
H 2
w
s
t
t
i
9
o
veh
/
veh (3x ino
r
/
c
C
I
/
F
N
D
a
/
C
T
(
C
/
F
F
T
D
O
2
o
C
C
T
v
T
W
C
H
/
D
C
I1061T/I1061T
I1061T/I1061T
Figure 3. Filipin staining in liver tissue of Npc1
mice treated with TCF regimens. Npc1
mice were treated for six weeks with the various regimens as described in Figure 2 and liver tissue was
analyzed for effect on cholesterol accumulation using filipin staining. Liver sections were analyzed in a blinded
fashion on a scale of 0 – 5 (0 = no accumulation, 5 = extensive storage). Data are shown as mean ± SEM.
Statistics were performed using ANOVA and significance determined using Dunnett’s test. *, p<0.05; #,
p<0.01; †, p<0.001. versus vehicle. Representative images (20x) are shown for all treatments.
3.4. Evaluation of efficacy of TCF formulation on lipid storage in brain tissue
39 sphingolipid and sterol species profiled in the liver were also measured in brain tissue (Figure 4). Significant
reduction in 3,5,6-cholestentriol, an established NPC1 disease marker, was observed in the HPCD
x/week, TCF active and TCF/CI994 groups. Reductions in GA2 ganglioside 18:0 and MCer 24:0 were also
3
evident in the HPCD 3x/week and TCF/CI994 groups. Overall, the effect of the different formulations on bulk
lipid storage was less dramatic than in liver tissue both in the number of species affected and the magnitude of
the responses. Selective examination of lipid storage in the cerebral cortex by immunohistochemical staining
proved more sensitive, demonstrating decreased filipin and GM2 ganglioside staining in the HPCD/vehicle,
HPCD 3x/week, and TCF active groups (Figure 5). No reductions were observed with the non- HPCD
containing formulations.

ACCEPTED MANUSCRIPT
17
3
2
1
0
5
3
2
1
0
†
#
†
†
#
4
3
2
1
0
l
e
O
le
k
)
a
a
a
t
t
t
IVA
v
e
9
4
IVA
le
O
le
k
)
a
t
A
v
e
I99 F/IV
4
A
l
e
O
l
e
k
)
a
t
IVA
i
v
t
e
I99
4
IVA
/
t
t
t
i
hic
2
hic
t
V
i
c
H 2
c
t
i
c
H 2
hic
w
s
s
s
t
I
9
/
H
w
s
I
t
i
i
w
s
h
/
(3x
o
c
F
/
(3x
o
c
h
/
h
(3x
o
c
F
e
D
/ve
rin
a
F/C
e
D
/ve
rin
a
F/C
e
D
/ve
rin
a
F/C
v
C
C
v
C
C
T
v
C
F
C
F
T
F
T
D
O
o
C
C
D
C
O
o
C
C
D
O
o
C
C
T
C
H
2
v
T
T
T
T
C
H
2
v
T
T
C
/
H
2
v
T
/
/
D
D
D
C
C
5
3
4
†
†
†
4
3
3
2
2
1
0
2
1
1
0
0
le
O
le
)
A
e
4
A
l
e
O
l
e
)
t
A
e
4
A
l
e
O
/
l
e
)
t
A
ive
t
4
A
/IV
k
v
I99 /IV
k
a
v
I99 /IV
k
a
9
I
hic
2
hic
w
I
V
c
i
h
i
c
H 2
h
i
c
w
t
I
V
c
i
i
c
H 2
i
c
x
w
t
I
V
H
(3x
t
/
(3x
s
t
s
9
/
o
F
C
o
F
C
h
/
h
o
c
e
D
/ve
rin
a
F/C
e
D
/ve
rin
a
F/C
e
D
e
3
rin
a
F/C
F
v
C
v
C
v
C
v
(
C
F
T
F
T
F
T
D
O
o
C
C
D
O
o
C
C
D
C
O
o
C
C
T
C
H
2
v
T
C
H
2
v
T
T
C
/
H
2
v
T
/
/
D
D
D
C
C
5
4
#
3
2
1
0
le
O
le
)
A
e
4
A
k
v
I99 /IV
hic
2
hic
w
I
V
c
a
i
t
/
H
(3x
o
F
C
e
D
/ve
rin
F/C
v
C
F
T
D
O
o
C
C
T
C
H
2
v
/
D
C
I1061T/I1061T
I1061T/I1061T
Figure 4. Lipidomics in brain tissue in Npc1
mice treated with TCF regimens. Npc1
mice were treated for six weeks with the various regimens as described in Figure 2, and lipid and sterol
species were measured in brain homogenate using LC-MS/MS. Data for representative species are shown as
mean ± SEM. Statistical analyses were performed by ANOVA and significance determined using Dunnett’s
test. #, p<0.01; †, p<0.001 with respect to vehicle.

ACCEPTED MANUSCRIPT
18
WT
vehicle
CD/H O
CD/vehicle
CD/H O (3x/wk)
2
2
A
5
4
3
2
1
0
†
†
†
vorinostat
IVA
TCF Active
TCF/CI994
TCF/IVA
)
icle
icle
)
t
e
4
9
x
O
k
sta IVA ctiv
IVA
/
T
H 2
w
I
9
/
veh (3x ino
/
F
o
veh
C
N
D
a
/
F
C
(
C
/
r
F
T
T
W
D
O
2
o
C
C
T
C
v
T
/
H
D
C
WT
vehicle
CD/H O
CD/vehicle
CD/H O (3x/wk)
2
2
B
5
4
3
2
1
0
†
†
#
vorinostat
IVA
TCF Active
TCF/CI994
TCF/IVA
)
icle
icle
)
t
e
4
9
x
O
k
sta IVA ctiv
a
IVA
/
T
H 2
w
I
9
/
veh (3x ino
/
F
o
veh
C
N
D
/
F
C
(
C
/
r
F
T
T
D
O
2
o
C
C
T
v
T
W
C
H
/
D
C
I1061T/I1061T
Figure 5. Filipin and GM2 ganglioside staining in cerebral cortex of Npc1
mice treated with
I1061T/I1061T
TCF regimens. Npc1
mice were treated for six weeks with the various regimens as described in
Figure 2, and neuropathology was assessed. (A) Filipin labeling of unesterified cholesterol and (B)
immunoflourescent staining for GM2 ganglioside were performed on sections of the upper neocortex (layers 2-
3). Representative images (20x) are shown for all. Samples were analyzed in a blinded fashion on a scale of 0
–
5 with (0 = no accumulation, 5 = extensive storage). All data are shown as mean ± SEM. Statistics were
performed using ANOVA and significance determined using Dunnett’s test. #, p<0.01; †, p<0.001 versus
vehicle.
3.5. Effect of TCF formulations on histone acetylation
HDACi act by maintaining the acetylation levels of histones and non-histone proteins, such as molecular
chaperones, by inhibiting their deacetylation by HDAC enzymes. This action could affect the proteostatic

ACCEPTED MANUSCRIPT
19
environment of NPC1 protein [17, 38]. To examine the pharmacodynamic effect of the HDACi in the
formulations, we determined the acetylation status of histone H3 in liver and brain tissues of the mice at the
end of the six-week treatment trial (Figure 6). Samples were harvested one hour after the final dose, a time at
which we previously had shown provides the maximal acetylation response. In liver, treatment with vorinostat,
TCF active and TCF/CI994 resulted in a significant increase in the ratio of acetylated histone H3 relative to
total histone H3 (Figure 6A), in agreement with the acute acetylation studies (Figure 1). In brain tissue, only
vorinostat among the HDACi-containing formulations showed an increase in the ratio. The response for TCF
active and TCF/CI994 was attenuated at 6 weeks, possibly due to less favorable PK in the setting of chronic
administration. Unexpectedly, HPCD 3x/week also showed an elevated ratio (Figure 6B). Since HPCD did
not increase H3 acetylation acutely (Figure 1), it is possible that the increased acetylation relates to the
consequences of chronic administration of the drug and/or to changes in disease progression.
A
B
0
0
0
0
0
0
.5
.4
.3
.2
.1
.0
1.5
#
†
†
†
†
1
0
.0
.5
0.0
le
O
le
)
t
A
e
4
A
l
e
O
l
e
k
)
a
t
A
e
4
A
k
a
v
I99 /IV
t
i
v
t
c
I99 /IV
hic
2
hic
w
t
I
V
c
i
h
i
c
H 2
h
i
c
w
I
V
/
H
(3x
o
s
t
F
C
/
(3x
o
s
F
C
e
D
/ve
rin
a
F/C
e
D
/ve
rin
a
F/C
v
C
v
C
F
F
T
T
D
C
O
o
C
C
D
C
O
o
C
C
T
C
H
2
v
T
T
C
/
H
2
v
T
/
D
D
I1061T/I1061T
Figure 6. Histone H3 acetylation in brain tissue in Npc1
mice treated with TCF regimens.
I1061T/I1061T
Npc1
mice were treated for six weeks with the various regimens as described in Figure 2, and
acetylation of histone H3 was quantified via western blotting. In (A) liver and (B) brain, acetylated H3 and total
H3 were normalized to the Hsp90 loading control, then taken as a ratio. Data are presented as mean ± SEM
and statistical analysis was performed via ANOVA with Dunnett’s test versus vehicle to determine significance.
#, p<0.01; †, p<0.001.
3.6. Efficacy of TCF is independent of NPC1 protein expression
HDACi are hypothesized to reduce cholesterol storage in cells harboring NPC1 missense mutations by
correcting NPC1 mutant protein misfolding, thereby facilitating the movement of the correctly folded but mutant
I1061T/I1061T
protein to post-ER compartments. Although readily demonstrated in Npc1
fibroblasts [21, 22],
1061T
whether this occurs in vivo is controversial [24, 26]. Here we performed western blot analysis of the NPC1I
protein expression in liver and brain tissues from the mice treated with the TCF formulations (Figure 7). In none
I1061T
of the groups did we find evidence for stabilization of the mutant NPC1
protein in liver tissue; on the
contrary, we observed reduction of NPC1 protein in CD/vehicle, CD/H O and vorinostat groups. In brain tissue,
2
NPC1 protein was increased only in the CD/vehicle group, but not in mice treated with any of the HDACi-

ACCEPTED MANUSCRIPT
20
containing formulations. Thus, there is no correlation between the efficacy of the formulations with respect to
I1061T
reduction in lipid storage and stabilization of the mutant NPC1
protein.
A
B
1
1
0
0
.5
.0
.5
.0
6
4
2
0
†
†
#
*
*
*
le
O
le
)
t
A
e
4
A
l
e
O
l
e
k
)
tat
IVA
e
i
v
t
4
IVA
/
k
a
v
I99 /IV
I99
hic
2
hic
w
t
I
V
c
T
i
i
c
H 2
i
c
w
s
H
(3x
s
t
h
/
h
(3x
c
/
o
F
C
o
F
e
D
/ve
rin
a
F/C
e
D
/ve
rin
a
F/C
C
T
v
C
v
C
F
F
T
D
O
o
C
C
D
O
o
C
T
C
C
H
2
v
T
C
/
H
2
v
T
/
D
D
C
C
I1061T/I1061T
Figure 7. NPC1 Expression in liver and brain tissue in Npc1
mice treated with TCF regimens.
I1061T/I1061T
Npc1
Npc1
mice were treated for six weeks with the various regimens as described in Figure 2, and
I1061T/I1061T
mice (see Figure 2) by western blot analysis. (A) immunoprecipitations were performed in liver
homogenates and samples were internally normalized to vehicle control after quantification. (B) SDS-PAGE
and western blotting of membrane fractions isolated from brain. Samples were normalized to p63 loading
control. Data are presented as mean ± SEM and statistical analysis was performed via ANOVA and
significance determined using Dunnett’s test. *, p<0.05; #, p<0.01; †, p<0.001 versus vehicle.
To further test the dependence NPC1 protein expression for the attenuation of lipid storage, we examined the
-/-
effect of the formulations in Npc1 mice, which are null for NPC1 protein [29]. Four-week-old mice were
randomly allocated to a six-week treatment protocol with PEG400/DMSO vehicle, CD/vehicle, or TCF active.
The mice were treated weekly and euthanized at 10 weeks of age. Lipidomic profiling in liver (Figures 8) and
brain tissue (Figure 9) was performed to assess the effect of the formulations on lipid storage. Both CD/vehicle
and TCF active were equally effective in lowering lipid storage when compared to vehicle alone.
Immunohistochemical staining of tissue sections showed reduced cholesterol staining in liver (Figure 10A) and
cerebral cortex (Figure 10B) for both groups, as well as reduced GM2 ganglioside staining in cortex for the
TCF active group (Figure 10C). Taken together with the unchanged NPC1 protein expression, these findings
indicate that reduction in lipid storage resulting from the TCF formulations is independent of NPC1 proteostatic
regulation.

ACCEPTED MANUSCRIPT
21
5
4
3
2
1
0
5
4
3
2
1
0
†
†
†
†
†
†
4
3
2
1
0
cle
cle
i
ve
cle
cle
i
t
c
ve
le
le
l
le
ve
hic
hic
cti
i
i
t
i
i
h
h
c
h
h
e
e
a
e
e
/
v
a
e
/ve
a
v
v
v
v
/
F
F
F
D
C
D
C
D
C
C
T
C
T
C
T
5
4
4
5
4
†
†
†
†
†
†
3
2
3
3
2
1
2
1
1
0
0
0
le
ve
le
le
e
cle
cle
ive
hic
hic
cti
a
hic
hic
tiv
i
i
t
c
a
c
h
h
e
/ve
e
e
a
e
v
e
v
v
v
v
F
C
/
F
/
D
F
D
D
C
C
C
T
C
T
C
T
5
4
4
3
2
5
4
†
†
†
†
†
†
3
3
2
1
2
1
1
0
0
0
cle
cle
ve
i
e
le
e
v
le
le
ve
c
hic
i
hic
hic
cti
i
i
t
c
i
t
h
h
h
c
e
v
e
/
v
a
e
/ve
a
e
/ve
a
v
v
F
F
F
D
C
D
C
C
D
C
C
T
T
C
T
-/-
-/-
Figure 8. Lipidomics in liver tissue in Npc1 mice treated with TCF regimens. Npc1 mice were treated
for six weeks with PEG/DMSO vehicle (n=9), 2000mg/kg HPCD in water (n=9), TCF with vorinostat (n=9), or
untreated (n=8). Lipid and sterol species were measured in liver homogenate using LC-MS/MS. Data for
representative species are reported as mean ± SEM. Statistical analyses were performed by ANOVA and
significance determined using Dunnett’s test. †, p<0.001 versus vehicle.

ACCEPTED MANUSCRIPT
22
3
2
1
0
3
2
1
0
5
†
†
†
†
*
4
3
2
1
0
le
le
ve
l
e
le
e
v
le
le
ve
hic
hic
cti
i
c
hic
i
t
hic
hic
hic
hic
cti
h
c
e
/ve
a
e
/ve
a
e
/ve
a
v
v
F
v
F
F
D
C
D
C
D
C
C
T
C
T
C
T
4
3
2
4
3
5
4
†
†
3
2
1
0
2
1
1
0
0
le
le
ve
le
le
ve
le
le
e
hic
hic
cti
a
hic
hic
/ve
cti
hic
tiv
c
e
/ve
e
v
a
e
e
/
a
v
v
v
F
C
F
F
D
D
C
C
D
C
C
C
T
T
T
4
4
3
2
1
0
5
4
†
†
†
†
†
†
3
2
3
2
1
1
0
0
e
le
e
le
le
ve
l
e
le
e
v
l
v
c
i
t
i
c
hic
t
i
hic
hic
cti
i
h
c
h
c
a
e
/ve
a
e
/ve
a
e
/ve
v
v
v
F
F
F
D
C
D
C
D
C
C
T
C
T
C
T
-/-
-/-
Figure 9. Lipidomics in brain tissue in Npc1 mice treated with TCF regimens. Npc1 mice were treated
for six weeks with the various regimens as described in Figure 8. Lipid and sterol species were measured in
brain homogenate using LC-MS/MS. Data for representative species are reported as mean ± SEM.Statistical
analyses were performed by ANOVA and significance determined using Dunnett’s test. *, p<0.05; †, p<0.001
versus vehicle.

ACCEPTED MANUSCRIPT
23
A
untreated
vehicle
CD/vehicle
TCF active
5
4
3
2
1
0
†
†
d
cle
cle
ive
e
t
i
i
t
c
a
h
h
e
e
e
/
v
a
r
t
v
n
F
u
D
C
T
C
B
untreated
vehicle
CD/vehicle
TCF active
5
4
3
2
1
0
†
†
d
cle
cle
ive
e
t
i
i
t
a
h
h
e
v
c
a
e
e
r
t
v
n
/
F
u
D
C
C
T
untreated
vehicle
CD/vehicle
TCF active
C
5
4
3
2
1
0
*
d
cle
cle
ive
e
t
i
i
t
a
h
h
e
v
c
a
e
e
r
t
v
n
/
F
u
D
C
C
T
-/-
Figure 10. Filipin and GM2 ganglioside staining in liver and cerebral cortex of Npc1 mice treated with
-/-
TCF regimens. Npc1 mice were treated for six weeks with the various regimens as described in Figure 8.
Unesterified cholesterol in (A) hepatocytes and (B) upper neocortex sections were detected by filipin staining
and analyzed in a blinded fashion on a scale of 0 – 5 with (0 = no accumulation, 5 = extensive storage). (C)
Immunofluorescence staining for GM2 ganglioside was performed on the upper neocortex and analyzed in the
same manner. Data are shown as mean ± SEM. Statistics performed using ANOVA and Dunnett’s test to
determine significance versus vehicle control. *, p<0.05; †, p<0.001. Representative images (20x) are shown.

ACCEPTED MANUSCRIPT
24
I1061T/I1061T
3
.7. Effect of TCF on neurological function and survival of the Npc1
mice
I1061T/I1061T
To determine whether the results from short-term treatment trials correlated with survival in the Npc1
mouse model, mice were randomly allocated to one of nine treatment groups (Table 1). Untreated wild type
I1061T/I1061T
mice and Npc1
mice served as controls. Starting at four weeks, mice in the treatment groups were
treated weekly (with the exception of the 3x/week HPCD) with one of the nine regimens. Mice were
terminated when they showed clinical signs of end-stage disease [10]. Weekly weights plotted versus time
show a clear separation of groups receiving HPCD-containing formulations (red lines) versus groups
receiving formulations without HPCD (blue lines) (Figure 11). The median survival for all groups receiving
HPCD-containing formulations, including TCF/IVA, were significantly increased compared to either untreated
mutant mice or mice treated with the PEG400/DMSO vehicle (Figure 12). As expected, treatment with HPCD
3x/week resulted in the longest median survival. Neither vorinostat nor IVA extended lifespan.
A
B
3
2
2
1
0
5
0
5
WT (No Tx)
mut (No Tx)
vehicle
TCF Active
CD/H2O
3
0
CD/vehicle
IVA
CD/H O (3x/wk)
2
25
vorinostat
TCF/IVA
TCF/CI994
20
15
10
5
0
)
)
e
t
e
icle
)
4
9
x
x
l
IVA
a
v
O
k
IVA
T
T
i
c
s
t
t
i
2
w
I
9
H
veh (3x
/
/
o
o
veh
rino
c
/
F
/
C
(
N
(N
a
D
C
F
C
/
T
F
T
t
o
C
u
v
C
D
O
T
W
m
T
C
H 2
/
Approximate Age (days)
D
C
I1061T/I1061T
Figure 11. Growth curves for Npc1
mice in TCF treatment survival study. (A) Weights of wild
I1061T/I1061T
type (WT) and Npc1
mice treated with the indicated regimens beginning at 4 weeks were recorded
weekly (n=4 for HPCD administered 3x/week, all other regimens n=8). For survival study, mice were
euthanized when they showed clinical signs of end-stage disease. (B) Body weight distribution of mice in
survival study at p.98. Data are displayed as mean ± SEM, and are color-coded (WT, black; non-HPCD
containing formulas, blue; and HPCD-containing formulas, red).

ACCEPTED MANUSCRIPT
25
A
B
1
80
1
00
#
†
†
#
#
*
WT (No Tx)
mut (No Tx)
vehicle
160
140
120
CD/vehicle
CD/H2O
7
5
2
5
0
5
0
vorinostat
IVA
1
00
TCF active
TCF/IVA
8
0
TCF/CI994
CD/H O (3x/wk)
2
60
4
2
0
0
0
)
e
icle
)
t
e
4
x
l
O
k
a
IVA
v
9
I
9
IVA
T
i
c
H 2
w
s
t
t
i
o
h
/
h
x
/
o
c
/
C
F
F
/
(N
v
e
D
/ve
(
3
rin
a
C
C
F
T
t
o
C
T
u
D
O
v
C
T
0
5
0
5
0
5
0
5
0
5
0
5
0
5
C
2
0
0
1
1
2
2
3
3
4
4
5
5
6
6
m
/H
1
1
1
1
1
1
1
1
1
1
1
1
1
1
D
C
age (days)
C
Mean
Median
Treatment Group
Lifespan
Lifespan
(days)
364.0
120.0
114.5
124.5
129.0
148.5
114.0
115.5
127.0
123.5
126.0
(days)
WT
Mutant
vehicle
342.6
116.8
115.6
125.2
129.5
150.4
113.8
118.0
127.2
124.5
127.0
CD/H
CD/vehicle
CD/H O (3x/wk)
2
O
2
vorinostat
IVA
TCF active
TCF/CI994
TCF/IVA
I1061T/I1061T
Figure 12. Survival for Npc1
mice in TCF treatment survival study. (A) Kaplan-Meier survival
I1061T/I1061
curves were generated for Npc1
mice treated weekly with the indicated regimens beginning at 4
weeks. (B) Average lifespan for each group are shown mean ± SEM. Statistical significance was determined
I1061T
by unpaired t-test with Welch’s correction in comparison to NPC1
mice (mut (No Tx)). *, p<0.0332; #,
p<0.0021; †, p<0.0002. (C) Mean and median lifespan for each treatment group.
We further examined the effect of the TCF formulations on the neurological disease progression in the mice
using automated gait analysis, which has been previously validated in NPC1 mouse models [39]. Gait analysis
was performed weekly during the survival study. Results are shown for standing on the diagonal, right front-
right hind leg coupling, and forepaw stride length at 4 weeks (pre-treatment), 8 weeks and 12 weeks of age
I1061T/I1061T
(
Figure 13 and Supplemental Movie 1). At 12 weeks, the untreated and vehicle-treated Npc1
mice
have profound deficits in standing on the diagonal, as compared to wild type mice. There is, however,
significant preservation of function in all of the groups receiving formulations containing cyclodextrin and
PEG400/DMSO vehicle (CD/H2O treatment group shows a trend (p=0.0767) compared to vehicle). Similar
results were obtained for analysis of forepaw stride length, and to a lesser extent with the right front-right hind
leg coupling. Together, these finding demonstrate that the survival benefit and preservation of neurological
function in the treated mice derive exclusively from HPCD exposure and not from the inclusion of HDACi in
the TCF. Moreover, the concordance between results from the six week studies and the survival studies

ACCEPTED MANUSCRIPT
2
6
I1061T/I1061T
provide validation for use of the short-term treatment paradigm to evaluate drug therapies in the Npc1
mouse model.
A. Standing on Diagonal
4
weeks
8 weeks
12 weeks
1
00
100
80
60
40
20
0
100
80
60
40
20
0
†
#
†
†
†
†
*
8
6
4
2
0
0
0
0
0
)
)
e
icle
)
tat
T
e
4
)
)
icle
icle
)
t
e
4
)
)
icle
icle
)
t
e
4
x
x
l
O
k
IVActiv
9
IVA
x
x
O
k
a
IVA
v
9
IVA
x
x
O
k
a
IVA
v
9
IVA
T
T
i
c
2
w
s
9
T
(
T
2
w
s
t
t
i
9
T
T
(
T
2
w
s
t
t
i
9
T
/
H
veh (3x ino
/
I
F
/
o
o
veh
/
H
veh (3x ino
/
c
I
F
/
o
o
veh
/
H
veh (3x ino
/
c
I
F
/
o
o
(N veh
C
C
C
N
D
C
a
/
C
N
N
D
a
/
C
N
N
D
a
/
C
(
C
/
r
F
F
(
C
/
r
F
F
(
C
/
r
F
F
T
W
u
m
t
D
O
o
C
C
T
T
t
D
O
o
C
C
T
t
D
O
2
o
C
C
T
u
v
T
u
v
2
v
T
W
C
2
T
W
C
T
/
H
m
/H
m
/H
D
D
D
C
C
C
B. RF RH Coupling
4
weeks
8 weeks
12 weeks
#
1
00
100
80
60
40
20
0
100
80
60
40
20
0
†
*
*
8
6
4
2
0
0
0
0
0
)
)
e
icle
)
tat
T
e
4
)
)
icle
icle
)
tat
T
e
4
)
)
icle
icle
)
tat
T
e
4
9
x
x
l
O
k
IVActiv
9
IVA
x
x
O
k
IVActiv
9
IVA
x
x
O
k
IVActiv
IVA
T
T
i
c
2
w
s
9
T
T
2
w
s
9
T
T
2
w
s
9
H
/
o
(N veh
/
veh (3x ino
I
/
(N (N veh
/
H
veh (3x ino
/
I
/
(N (N veh
/
H
veh (3x ino
/
I
/
F
o
C
F
o
o
C
F
o
o
C
N
D
a
/
C
D
a
/
C
D
a
/
F
C
(
C
/
r
F
F
C
/
r
F
F
C
/
r
F
t
D
o
C
T
T
t
D
o
C
T
T
W
t
D
o
C
T
T
T
u
O
C
u
O
C
u
m
O
C
W
m
C
H
2
v
T
W
m
C
H 2
v
T
C
H 2
/
v
/
/
D
D
D
C
C
C
C. Fore Paw Stride Length
4
weeks
8 weeks
12 weeks
1
0
8
6
4
2
0
10
10
#
#
†
†
#
†
#
#
8
6
4
2
0
8
6
4
2
0
)
)
icle
icle
)
tat
T
e
4
)
)
icle
e
)
tat
T
e
4
)
)
icle
icle
)
tat
T
e
4
x
x
O
k
IVActiv
9
IVA
x
x
O
icl
k
IVActiv
9
IVA
x
x
O
k
IVActiv
9
IVA
T
T
H 2
w
s
9
T
T
2
x/w
s
9
T
T
H 2
w
s
9
/
h
x
/
o
I
F
/
o
(N veh
/H
veh (3
I
F/
h
/
h
/
I
/
o
o
h
C
o
ino
C
o
o
x
o
C
F
(
N
(N
v
e
C
D
/ve
(
3
rin
F
a
F
/
C
(
N
C
D
/
r
F
a
F
/
C
(N (N
v
e
C
D
/ve
(
3
rin
F
a
F
/
C
T
ut
T
ut
D
O
o
C
C
T
u
t
D
O
o
C
C
T
T
D
O
2
o
C
C
T
T
W
m
C
H
2
v
T
W
m
C
H 2
v
T
W
m
C
/
H
v
/
/
D
D
D
C
C
C
I1061T/I1061T
Figure 13. Gait analysis for Npc1
mice in TCF treatment survival study. Gait analysis data was
I1061T/I1061
performed weekly to monitor cerebellar function in Npc1
mice treated with various regimens described
in Figure 2. (A) Standing on Diagonal, (B) right forepaw to right hindpaw coupling, and (C) forepaw stride
length are shown at three representative time points representative of different stages of disease progression
I1061T/I1061
in the Npc1
mice: 4 weeks (pre-symptomatic), 8 weeks (onset of symptoms), 12 weeks (late stage
disease). Data are displayed as mean ± SEM. Statistical analysis was performed using unpaired t-tests with
Welch’s correction. *, p<0.0332; *#, p<0.0021; †, p<0.0002; versus untreated mutant.

ACCEPTED MANUSCRIPT
27
3.8. Lack of efficacy of HDACi in bone marrow-derived macrophages
I1061T/I1061T
One possible explanation for the lack of efficacy of vorinostat in Npc1
mice while there is a robust
effect in tissue culture cells is that most cell populations in vivo are slowly dividing as compared to rapidly
dividing cells in tissue culture. The effect of vorinostat was tested on slowly dividing terminally differentiated
bone marrow-derived macrophages. BMMs were treated with vorinostat at eight different doses ranging from 4
nM to 10 µM for 48h or 72h, and the level of cholesterol accumulation in LSOs was measured using a filipin
imaging assay that measures cholesterol in LSOs, which accumulate near the center of NPC1 mutant cells
(
Figure 14) [20, 21]. Vorinostat treatment was toxic to BMMs at concentrations above 1 µM. Each data point in
the plot shown in the figure, is normalized to corresponding DMSO treated cells. Hence, the value of one
indicates no correction. Although, 330 nM treatment showed slight decrease in cholesterol accumulation based
I1061T
on filipin intensity, it was not as robust as the response observed in NPC1
fibroblasts. Thus, vorinostat is
not effective at reducing the cholesterol accumulation in slowly dividing, differentiated macrophages.
A
B 1.5
1
1
0
0
.5
.0
.5
.0
vorinostat - 48h
vorinostat - 72h
vorinostat - 48h
vorinostat - 72h
1.0
0.5
0
.0
4
2
7
0
0
0
0
4
2
7
.11
.33
.00
0
1
3
1
3
0
.01
.03
0
0
0
0
1
3
.
0
.
0
0
0
1
0
.
0
.
0
.
0
.
0
.
1
0
0
concentration (mM)
concentration (mM)
Figure 14. Vorinostat does not reduce cholesterol storage in ex vivo bone marrow-derived
I1061T/I1061T
macrophages. (A) Differentiated BMMs and (B) Npc1
human fibroblasts were plated in 384 well
plates and incubated overnight. Cells were then treated with various doses (4-6 wells per dose) and incubated
for an additional 48 or 72 hours. For the final 24 hours the medium was supplemented with 20 µg/ml acetylated
LDL to increase cholesterol loading. Finally, the cells were washed with PBS, fixed with 2% PFA and stained
with filipin and Draq5. Four fluorescence images per well were acquired. Images were analyzed to measure
filipin intensity in lysosome like organelles (LSO) per cell. The LSO ratios in vorinostat treated cells were
normalized to corresponding DMSO treated cells. Hence, LSO values less than one indicate reduction of
stored cholesterol. Each data point is an average from 2-5 independent experiments and total of 48-80 images.
Although the 330 nM treatment at 48h showed a slight decrease in cholesterol accumulation in BMMs, it did
not achieve significance when corrected for multiple pair-wise comparisons. Error bars ± SEM.
4
. DISCUSSION
In cell-based assays HDACi have shown promise for correction of the lipid storage caused by NPC1 deficiency
19-21], but there are conflicting reports as to whether HDACi are able to mitigate the NPC1 phenotype in vivo
[

ACCEPTED MANUSCRIPT
2
8
I1061T/I1061T
[
24, 26, 27]. In the present study we examined the efficacy of HDACi in the context of TCF in Npc1
mice. Although we found that TCF containing vorinostat was effective in delaying neurodegeneration in the
NPC1 mice, TCF containing the inactive form of vorinostat was equally efficacious, clearly demonstrating that
the benefit of TCF was independent of HDACi activity. Instead, we show that efficacy of TCF was attributable
to enhanced PK of the HPCD component in the formulation. While we acknowledge that differences between
I1061T/I1061T
nmf164
the Npc1
and Npc1
models may account for differences in pharmacokinetic findings, the TCF
nih
was similarly efficacious in the NPC1-null BALB/c Npc1 mice, indicating that the benefit of TCF was
independent of NPC1 gene and protein expression or strain differences. Furthermore, HDACi were ineffective
I1061T/I1061T
in alleviating cholesterol storage in primary bone marrow derived macrophages from Npc1
mice,
suggesting that this therapeutic paradigm may not be applicable to slowly dividing cells. Taken together, these
findings provide an explanation for the lack of benefit of HDACi in vivo in the absence of the HPCD carrier
[
24, 26, 27].
As the neuroprotective benefits of HPCD in NPC1 disease models are well established [10-12], its inclusion in
TCF presents a major confounder for interpretation of the formulation’s efficacy [26]. The goal of our study was
not a precise replication of the experiments by Alam and colleagues, but to test which component or interaction
of the TCF was responsible for the mitigation of the NPC phenotype. In order to deconvolute the effects of
HPCD from the other TCF components, we rigorously examined the impact of TCF on the PK each of the
components. We excluded day 7 and day 15 HPCD pretreatment of the TCF group, a potential confounder,
and included a key control, HPCD in PEG/DMSO vehicle. We found that the benefits of TCF with respect to
I1061T/I1061T
reduction of lipid storage in brain tissue and prolonged survival in the Npc1
mice could be predicted
solely on the basis of the PK properties of HPCD in the different formulations. Inclusion of the PEG400/DMSO
co-solvents reduced renal clearance and markedly enhanced the plasma and tissue AUClast for HPCD, as
compared to water vehicle. Not only was HPCD in PEG/DMSO vehicle more efficacious than HPCD in
water, but it was also equally efficacious to TCF containing HDACi. Thus, we conclude that the active
component in TCF is the HPCD and that the PEG/DMSO vehicle simply enhanced its pharmacokinetics.
Although our vorinostat PK analysis was consistent with the previous report by Alam and colleagues – TCF
increased the vorinostat Cmax and AUClast in brain tissue by 55% and 50%, respectively – the increased survival
of TCF-treated NPC1 mice, as noted above, was independent of the pharmacokinetics and activity of the
HDACi. It is unclear how HPCD increased brain exposure of vorinostat, but could relate to cyclodextrin’s
known ability to inhibit P-glycoprotein (P-gp)-mediated efflux of drugs across the blood-brain barrier [40]. By
including an IVA control in our study, we excluded a role for HDAC inhibition since substitution of inactive for
the active form of vorinostat in the TCF had no impact on its efficacy. Furthermore, we found no evidence of
increased HDACi activity in brain tissue in the context of chronic delivery (Alam et al. reported increased
HDACi activity in brain tissue but only following acute TCF administration). Together, these findings argue

ACCEPTED MANUSCRIPT
29
strongly that the efficacy of TCF is neither dependent on the presence of vorinostat nor requires its HDACi
activity.
HDACi are proposed to correct the NPC1 phenotype through proteostatic regulation of the mutant NPC1
protein [8, 19-21], possibly via transcriptional upregulation of the NPC1 gene [26] and/or of endogenous
molecular chaperones [17]. Indeed, in cell-based models, vorinostat has been shown to prolong the half-life of
I1061T
the mutant NPC1
protein, which is normally rapidly degraded [21]. In the present study, however, none of
I1061T
the formulations examined led to an increase in NPC1
protein expression in either liver or brain tissue.
nmf164
This is consistent with an earlier report by Munkacsi and colleagues using the NPC1
mice [24], though
differs from Alam and colleagues who showed increased NPC1 mutant protein in the same mousemodel [26].
nih
To directly address the requirement for NPC1 mutant protein expression for TCF efficacy, we treated NPC1
mice that are null for NPC1 protein with the formulations. We found that both TCF active vorinostat and
HPCD in PEG/DMSO vehicle attenuated lipid storage in liver and brain tissue, and that these formulations
were equally efficacious. Thus, we conclude that the benefit conferred by TCF in the NPC1 mouse models is
independent of NPC1 protein expression.
Previous studies have shown that HDAC inhibition alleviates cholesterol storage in a variety of NPC1-deficient
I1061T/I1061T
cell lines, including patient fibroblasts and murine embryonic fibroblasts from the Npc1
mice [19-22].
Nevertheless, translation of the findings from cultured cells to in vivo NPC1 mouse models has been elusive
[
24, 26]. In the present study we show that treatment with HDACi failed to reduce cholesterol storage ex vivo in
I1061T/I1061T
bone marrow-derived macrophages harvested from Npc1
mice. While the explanation for these
divergent findings is uncertain, the absence of response to HDAC inhibition may reflect the fact that bone
marrow-derived macrophages are slowly dividing or post-mitotic whereas cultured fibroblasts are actively
dividing. It seems plausible that HDACi might be more effective in actively dividing cells with more open
chromatin than in non-dividing cells with more condensed chromatin [41]. This is consistent with the clinical
use of HDACi for treatment of cancers, and would potentially explain the failure of vorinostat to mitigate lipid
storage in liver tissue, even when tissue levels are at the EC for the drug [24].
50
The finding that PEG400 enhanced HPCD exposure in brain tissue begs the question as to whether the
addition of the co-solvent to HPCD formulations might offer intravenous delivery as an alternative to the
current more invasive intrathecal approach [13]. Our PK studies showed that HPCD in the PEG400/DMSO
achieved roughly twice the brain exposure of HPCD in water. Therefore, even in the setting of a peripheral
dose of 4000 mg/kg HPCD – a dose that is toxic in the NPC1 cat model and cannot be safely delivered in
humans [11] – the maximum drug concentration achieved would be ~15 M [16]. This is well below the 1000
M drug concentration achieved in CSF following direct CNS administration of HPCD [31], suggesting that