Intravenous 2-Hydroxypropyl-Β-Cyclodextrin (Trappsol® Cyclo™) Demonstrates Biological Activity and Impacts Cholesterol Metabolism in the Central Nervous System and Peripheral Tissues in Adult Subjects with Niemann-Pick Disease Type C1: Results of a Phase 1 Trial

2022, Social Science Research Network

UCSF UC San Francisco Previously Published Works Title Intravenous 2-hydroxypropyl-β-cyclodextrin (Trappsol® Cyclo™) demonstrates biological activity and impacts cholesterol metabolism in the central nervous system and peripheral tissues in adult subjects with Niemann-Pick Disease Type C1: Results of a p... Permalink https://escholarship.org/uc/item/10f7x3t7 Journal Molecular Genetics and Metabolism, 137(4) ISSN 1096-7192 Authors Hastings, Caroline Liu, Benny Hurst, Bryan et al. Publication Date 2022-12-01 DOI 10.1016/j.ymgme.2022.10.004 Peer reviewed eScholarship.org Powered by the California Digital Library University of California Molecular Genetics and Metabolism 137 (2022) 309–319 Contents lists available at ScienceDirect Molecular Genetics and Metabolism journal homepage: www.elsevier.com/locate/ymgme Intravenous 2-hydroxypropyl-β-cyclodextrin (Trappsol® Cyclo™) demonstrates biological activity and impacts cholesterol metabolism in the central nervous system and peripheral tissues in adult subjects with Niemann-Pick Disease Type C1: Results of a phase 1 trial Caroline Hastings a,b,⁎, Benny Liu c,d, Bryan Hurst e, Gerald F. Cox f, Sharon Hrynkow f a Department of Pediatric Hematology Oncology, UCSF Benioff Children's Hospital Oakland, 747 52nd Street, Oakland, CA 94609-1809, USA Department of Pediatrics, University of California San Francisco, San Francisco, CA, USA c GI & Liver Clinics, Highland Hospital, Alameda Health System, Highland Hospital, Oakland, CA, USA d Division of Gastroenterology & Hepatology, Highland Hospital, Alameda Health Systems, Highland Care Pavilion 5th ﬂoor, 1411 East 31st Street, Oakland, CA 94602, USA e Boyd Consultants, Electra House, Electra Avenue, Crewe CW1 6GL, UK f Cyclo Therapeutics, Inc., 6714 NW 16th St., Ste B, Gainesville, FL 32653, USA b a r t i c l e i n f o Article history: Received 8 June 2022 Received in revised form 5 October 2022 Accepted 12 October 2022 Available online 17 October 2022 Keywords: Cyclodextrin Niemann-Pick Disease Type C Lysosome Pharmacodynamics Pharmacokinetics a b s t r a c t Background: Niemann-Pick Disease Type C1 (NPC1) is a disorder of intracellular cholesterol and lipid trafﬁcking that leads to the accumulation of cholesterol and lipids in the late endosomal/lysosomal compartment, resulting in systemic manifestations (including hepatosplenomegaly and lung inﬁltration) and neurodegeneration. Preclinical studies have demonstrated that systemically administered 2-hydroxypropyl-β-cyclodextrin (HPβCD; Trappsol® Cyclo™) restores cholesterol metabolism and homeostasis in peripheral organs and tissues and in the central nervous system (CNS). Here, we assessed the safety, pharmacokinetics, and pharmacodynamics of HPβCD in peripheral tissues and the CNS in adult subjects with NPC1. Methods: A Phase 1, randomized, double-blind, parallel group study enrolled 13 subjects with NPC1 who received either 1500 mg/kg or 2500 mg/kg HPβCD intravenously every 2 weeks for a total of 7 doses (14 weeks). Subjects were 18 years or older, with a conﬁrmed diagnosis of NPC1 and evidence of systemic involvement on clinical assessment. Pharmacokinetic evaluations in plasma and cerebrospinal ﬂuid (CSF) were performed at the ﬁrst and seventh infusions. Pharmacodynamic assessments included biomarkers of systemic cholesterol synthesis (serum lathosterol) and degradation (serum 4β-hydroxycholesterol), secondary sphingomyelin storage (plasma lysosphingomyelin-509, now more accurately referred to as N-palmitoyl-O-phosphocholineserine [PPCS]), and CNS-speciﬁc biomarkers of neurodegeneration (CSF total Tau) and cholesterol metabolism (serum 24(S)hydroxycholesterol [24(S)-HC]). Safety monitoring included assessments of liver and kidney function, infusion related adverse events, and hearing evaluations. Results: Ten subjects completed the study, with 6 at the 1500 mg/kg dose and 4 at the 2500 mg/kg dose. One subject withdrew following the ﬁrst infusion after experiencing hypersensitivity pneumonitis, and 2 subjects withdrew after meeting a stopping rule related to hearing loss. Overall, HPβCD had an acceptable safety proﬁle. The observed pharmacokinetic proﬁle of HPβCD was similar following the ﬁrst and seventh infusions, with a plasma half-life of 2 h, a maximum concentration reached at 6 to 8 h, and no evidence of accumulation. Serum biomarkers of cholesterol metabolism showed reduced synthesis and increased degradation. Compared to Baseline, ﬁlipin staining of liver tissue showed signiﬁcant reductions of trapped unesteriﬁed cholesterol at both dose levels at Week 14. Plasma PPCS levels were also reduced. HPβCD was detected at low concentrations in the CSF (maximum, 33 μM) at both dose levels and persisted longer in CSF than in plasma. Total Tau levels in CSF decreased in most subjects. Serum levels of 24(S)-HC, a cholesterol metabolite from the CNS that is exported Abbreviations: 17D-NPC-CSS, 17-Domain Niemann-Pick disease Type C-Clinical Severity Scale; 24(S)-HC, 24(S)-hydroxycholesterol; HPβCD, 2-hydroxypropyl-β-cyclodextrin; AEs, adverse events; ALT, alanine aminotransferase; AUC0-∞, area under the concentration-time curve estimated from time zero to inﬁnity; AST, aspartate aminotransferase; ABR, auditory brainstem response; CNS, central nervous system; CSF, cerebrospinal ﬂuid; CRP, C-reactive protein; CK-MB, creatinine kinase MB isoenzyme; t1/2, elimination half-life; GFAP, glial ﬁbrillary acid protein; ITT, Intent-To-Treat; INR, international normalized ratio; IND, investigational new drug; LC-MS/MS, liquid chromatography tandem mass spectrometry; Cmax, maximum observed plasma concentration; NPC1, Niemann-Pick Disease Type C1; PP, Per protocol Population; PBMCs, peripheral blood mononuclear cells; SAEs, serious adverse events; SD, standard deviation; Tmax, time to maximum concentration; TEAEs, treatment-emergent adverse events; UCSF, University of California San Francisco; URI;, upper respiratory infection; Vss., volume of distribution at steady state. ⁎ Corresponding author at: Department of Pediatric Hematology Oncology, UCSF Benioff Children's Hospital Oakland, 747 52nd Street, Oakland, CA 94609-1809, USA. E-mail address: caroline.hastings@ucsf.edu (C. Hastings). https://doi.org/10.1016/j.ymgme.2022.10.004 1096-7192/© 2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). C. Hastings, B. Liu, B. Hurst et al. Molecular Genetics and Metabolism 137 (2022) 309–319 across the blood-brain barrier and into the circulation, decreased after both the ﬁrst and seventh doses. Hence, pharmacodynamic assessments in both peripheral and CNS-related tissue show target engagement. While not the aim of the study, subjects reported favorable impacts on their quality of life. Conclusions: The plasma pharmacokinetics and pharmacodynamics of HPβCD administered at two intravenous dose levels to subjects with NPC1 were comparable to those observed in preclinical models. HPβCD cleared cholesterol from the liver and improved peripheral biomarkers of cholesterol homeostasis. At low CSF concentrations, HPβCD appeared to be pharmacologically active in the CNS based on the increased efﬂux of 24(S)-HC and reduction in CSF total Tau, a biomarker of CNS neurodegeneration. These data support the initiation of longerterm clinical trials to evaluate the safety and efﬁcacy of intravenous HPβCD in subjects with NPC1. (ClinicalTrials.gov numbers: present trial, NCT02939547; open-label extension of the present trial, NCT 03893071; global pivotal trial, NCT04860960). © 2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/). studies, HPβCD released trapped cholesterol from the late endosomal/ lysosomal compartment in a dose-dependent manner, ameliorated hepatosplenomegaly and neurological symptoms, and prolonged survival [23–28]. Based on these pre-clinical studies, HPβCD was used in humans for the ﬁrst time as an investigational new drug (IND) for named subject use in 2009. Treatment regimens were diverse in terms of dose level, frequency, and route of administration. In a case report study, 12 subjects ranging in age from 1 to 27 years with mild to severe disease were treated with intravenous HPβCD for over 7 years [29]. The treatment was well tolerated, and no serious AEs were attributed to the drug. In general, subjects experienced slowing of disease progression and reported improved quality of life. These encouraging named subject data supported the initiation of a formal Phase 1 clinical trial of HPβCD administered intravenously in NPC1 subjects to evaluate its pharmacokinetics and pharmacodynamics as assessed by cholesterol metabolism and homeostasis in blood, liver, and the central nervous system (CNS) and additionally by biomarkers of sphingomyelin storage and neurodegeneration. 1. Introduction Niemann-Pick Disease Type C (NPC) is a rare, pan-ethnic, autosomal recessive lysosomal storage disorder whose birth prevalence is estimated to be 1 in 90,000 to 100,000 worldwide [1–4]. The disease is caused by loss of function mutations in the NPC1 (90% to 95% of subjects) or NPC2 (4% of subjects) genes, resulting in impaired intracellular trafﬁcking of cholesterol and lipids and leading to the toxic accumulation of unesteriﬁed cholesterol, glycosphingolipids, and GM2 and GM3 gangliosides [4–9]. Although the underlying pathological mechanisms are not fully understood, the accumulation of cholesterol and lipids in lysosomes and late endosomes is likely a crucial event in disease pathogenesis [4,10]. The symptoms associated with NPC vary in severity and with age of onset and include visceral manifestations (such as hepatomegaly, splenomegaly, and lung dysfunction), neurological manifestations (including hearing loss, cerebellar ataxia, dystonia, dysmetria, dysarthria, dysphagia, and vertical supranuclear gaze palsy), as well psychiatric symptoms and progressive cognitive impairment [1,5,6,11]. The disease is highly heterogeneous and may present from the perinatal period to the seventh decade of life, although most subjects develop symptoms in childhood and die between 10 and 25 years of age [1,4–6,12,13]. The diagnosis of NPC is often delayed in part due to the wide spectrum of clinical phenotypes, which are likely related to underlying genotypes (515 estimated pathogenic mutations) and epigenetic modiﬁers [5,6,14–16]. Currently, there is no treatment that ameliorates both the systemic and neurological manifestations of the disease [12]. Various therapeutic strategies have been attempted in the pre-clinical and clinical setting [6]. In the United States (US), the current standard of care is symptom-based management and palliative approaches including antiepileptic, anticholinergic, antidepressant, and antipsychotic drugs [1,12]. Miglustat (Zavesca®), a glucosylceramide synthase inhibitor, is approved for the treatment of NPC in the European Union but not in the US, where it is commonly used off-label [12,17]. Miglustat reduces the accumulation of glycosphingolipids (glucosylceramide, lactosylceramide, and GM2 and GM3 gangliosides) in the brain, which slows the progression of the neurological symptoms in some subjects and increases median survival by approximately 10 years [12,18]. However, this drug does not impact the systemic manifestations of the disease, and the majority of subjects experience adverse events (AEs), most commonly diarrhea and tremor, that may preclude long-term use [19,20]. Therefore, there is a critical need for novel treatments for NPC that can impact disease progression, quality of life, and survival. The cyclic oligosaccharide, 2-hydroxypropyl-β-cyclodextrin (HPβCD [Trappsol® Cyclo™]), has a hydrophilic exterior and a hydrophobic core, which allows it to form complexes with hydrophobic compounds and has led to its widespread use as a delivery vehicle to improve the solubility, stability, and bioavailability of various medicinal products [21,22]. Additionally, HPβCD is routinely used in cell culture systems to modulate cellular cholesterol content. In pre-clinical NPC1 mouse 2. Materials and methods 2.1. Study design This Phase 1, randomized, double-blind, parallel-group study enrolled adult subjects (18 years and older) with a conﬁrmed diagnosis of NPC1 at the University of California San Francisco (UCSF) Benioff Children's Hospital Oakland in Oakland, California, US (ClinicalTrials. gov number, NCT02939547). The objective of the study was to assess the safety, tolerability, pharmacokinetics and pharmacodynamics of two different doses of intravenous HPβCD (1500 mg/kg body weight and 2500 mg/kg body weight; Cyclo Therapeutics, Gainesville, FL), speciﬁcally through serum, plasma and cerebrospinal ﬂuid (CSF) biomarkers and liver assessments after 7 infusions (14 weeks). Eligible subjects had to meet the following inclusion and exclusion criteria. Subjects had to have an NPC1 diagnosis conﬁrmed by one of the following: two NPC1 mutations; one NPC1 mutation and positive ﬁlipin staining (current or prior); vertical supranuclear gaze palsy plus either one or more NPC1 mutations or positive ﬁlipin staining. The subject's clinical severity score based on the 17-Domain Niemann-Pick disease Type C-Clinical Severity Scale (17D-NPC-CSS) could not exceed 30 (severe impairment), with no more than 4 individual major domains scoring ≥3 (maximum score is 61, including 5 for each of the 9 major domains and 2 for each of the 8 minor domains) [30]. The major domains of the 17D-NPC-CSS are eye movement, ambulation, speech, swallow, ﬁne motor skills, cognition, hearing (sensorineural), memory, and seizures. The minor domains are gelastic cataplexy, narcolepsy, behavior, psychiatric, hyperreﬂexia, incontinence, auditory brainstem response (ABR), and respiratory (history of pneumonia). The total score reﬂects the sub-totals of the major and minor domains [30]. Additionally, all subjects had at least one systemic manifestation of NPC1, 310 C. Hastings, B. Liu, B. Hurst et al. Molecular Genetics and Metabolism 137 (2022) 309–319 and on Day 15 post doses 1 and 7. Unesteriﬁed cholesterol was measured by ﬂuorescence microscopy of ﬁlipin-stained sections of liver tissue obtained at Baseline and 2 weeks post dose 7. Liver biopsies were performed using standard ultrasound-guided needle or transjugular catheter procedures, and the tissue was ﬂash-frozen in liquid nitrogen. Tissue sections (8 μm) were stained with either hematoxylin and eosin or ﬁlipin III (ﬁlipin; Sigma, St. Louis, MO, USA) or phosphate buffered saline (Oxoid, Thermo Fisher Scientiﬁc, Inc., Waltham, MA, USA). All samples were digitally scanned using a Zeiss Axio Scan whole ﬂuorescence slide scanner and Zeiss ZEN Lite software (Carl Zeiss Microscopy, Jena, Germany). Study-speciﬁc algorithms were developed to deﬁne weak, moderate, strong, and maximum intensity ﬁlipin staining using either the Halo® image analysis platform (Indica Labs, Albuquerque, NM, USA) or a qualitative assessment of mild, moderate, or marked change in ﬁlipin staining compared to Baseline was performed by a qualiﬁed technical reader at Histologix Ltd. (Nottingham, UK). Exploratory assessments included hepatic elasticity, a measure of liver stiffness related to tissue composition [32] measured by ultrasonography at Baseline and Week 14, as well as ﬁlipin staining in skin tissue at Baseline and Week 14. Skin tissue obtained using a standard punch biopsy procedure was ﬂash-frozen and processed using the same procedure as for liver tissue. such as clinically detectable hepatomegaly and/or either aspartate aminotransferase (AST) or alanine aminotransferase (ALT) levels above the normal range, clinically detectable splenomegaly, or impaired respiratory function due to NPC1. Female subjects of childbearing age had to have a negative pregnancy test prior to treatment and were required to use contraception during the study. Subjects with NPC2 mutations were excluded, as were subjects with grade 3 renal impairment or evidence of acute liver disease. Subjects using miglustat were permitted to enter the study provided that their dose was stable for at least 3 months prior to entry. Subjects were scheduled to receive 7 intravenous infusions of HPβCD (Trappsol® Cyclo™, a proprietary formulation of HPβCD, Cyclo Therapeutics, Inc., at 1500 mg/kg [low-dose] or 2500 mg/kg [high-dose]; 25% diluted in 1000 mL) over 8–9 h every 2 weeks for a 12-week period, followed by further evaluation for 14 days after the ﬁnal infusion (Week 14). Drug was administered using peripheral venous catheters. Subjects weighing >100 kg were infused a maximum volume of 1250 mL, and infusion times were lengthened to approximately 10–11 h. The study was conducted at a single site in the US but was open to subject enrollment on an international basis. A Safety Review Committee met periodically to review AEs, study progress, and laboratory results. Informed consent was obtained from all subjects or their legal guardians prior to the initiation of treatment or the performance of assessments in accordance with the local Institutional Review Board and principles of ethical research according to the Declaration of Helsinki [31]. 2.4. Clinical outcome measures Clinical outcome measures were assessed at Baseline and Week 14 using the 17D-NPC-CSS total and individual domain scores. Liver and spleen sizes were assessed by ultrasound using the longest twodimensional axis measurement and reviewed by a central reader. 2.2. Pharmacokinetics Plasma samples were collected to determine drug concentration and evaluate the time to maximum concentration (Tmax), maximum observed plasma concentration (Cmax), volume of distribution, and elimination half-life (t1/2) after the ﬁrst and seventh (last) doses of HPβCD. Samples were collected at time 0 just prior to the infusion; at 2, 4, and 6 h after the start of the infusion; and at 0.5, 1, 2, 4, 8, and 12 h after the end of the infusion (approximately 20–21 h after the start of the infusion). Lumbar spinal catheters were placed at the time of the ﬁrst infusion to obtain sequential timed CSF samples for pharmacokinetic analysis. CSF samples after the ﬁrst dose were collected at the start of the infusion (0 h, Baseline), 4 h after the start of the infusion, at the end of the infusion (approximately 8–9 h from the start), and 4 h after completion of the infusion (approximately 12–13 h from the start). Lumbar puncture was performed at the end of the seventh infusion (approximately 8–9 h from the start) to obtain the ﬁnal CSF sample. All samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/ MS; Medpace Bioanalytical Laboratories [MBL], Cincinnati, OH). 2.5. Safety outcome measures Subjects underwent routine local laboratory assessments, including complete blood counts, comprehensive metabolic panels, liver and renal function panels, creatinine kinase MB isoenzyme (CK-MB), C-reactive protein (CRP), prothrombin, international normalized ratio (INR), and urinalyses performed at Baseline and periodically throughout the study. Automated total cell counts (white and red blood cells) were performed on CSF samples obtained following doses 1 and 7, as per protocol. A 24-h urine collection was obtained at Baseline and Week 14 to measure urinary hydroxyproline as a biomarker of bone turnover (MBL). Adverse events and concomitant medications were recorded at each visit. As hearing loss is associated with NPC natural disease progression, it is one of the 17 domains measured in the 17D-NPC-CSS, which is scored 0–5 based on the pure tone average across all measured frequencies (ranges tested, 0.5 to 8 kHz). Subjects underwent audiologic testing using behavioral assessment or the auditory brainstem response (ABR), if needed, at Baseline and at Weeks 2, 4, 6, 8, and 14 and as required for AE assessments. It should be noted that grade shifts in hearing may not result in a signiﬁcant change in the pure tone average that changes the hearing score in the 17D-NPC-SS. 2.3. Pharmacodynamics Plasma samples were collected at Baseline and at Weeks 2, 4, 8, 12, and 14 to assess inﬂammation (cathepsin S, lysozyme) by immunoassay (MBL) and lipid metabolism (plasma lysosphingomyelin-509, now more accurately referred to as N-palmitoyl-O-phosphocholineserine [PPCS]) by LC-MS/MS (Centogene, Rostock, Germany). CSF biomarkers of neurodegeneration and inﬂammation, including total Tau, interleukin-8, tumor necrosis factor-α, and glial ﬁbrillary acid protein (GFAP), were measured by immunoassay (MBL) at Baseline and following the seventh dose of HPβCD. Serum cholesterol precursors (lanosterol, lathosterol, and desmosterol) and cholesterol metabolites/ bile acid precursors (4β-, 24(S), 25-, and 27-hydroxycholesterol) were measured at Baseline and on Days 2, 3, 5, 8, 11, and 15 post doses 1 and 7 by gas chromatography (GC)-MS. The relative acidic compartment volume of peripheral blood mononuclear cells (PBMCs) was measured using the LysoTracker assay (Oxford University) assay at Baseline 2.6. Statistical analysis As this was an exploratory Phase 1 study with a blinded, randomized, parallel-group design, no formal statistical analysis was conducted. Instead, descriptive statistics are presented here. The sample size was determined on a practical basis with a plan to recruit 12 subjects randomized 1:1 to receive one of the two dose levels (1500 mg/kg or 2500 mg/kg; six subjects per dose level). The study population was described by demographics, baseline characteristics, medical history, and concomitant medications to assess the comparability of treatment groups using descriptive statistics and data listings. Efﬁcacy was evaluated on all randomized subjects (Intent-To-Treat [ITT] population). The Per Protocol population (PP) included all subjects 311 C. Hastings, B. Liu, B. Hurst et al. Molecular Genetics and Metabolism 137 (2022) 309–319 treatment with miglustat at the time of enrollment: 1 subject in the low-dose group for 4.6 years, and 2 subjects in the high-dose group for 6.9 years. who completed the study and did not have major protocol deviations. Summary statistics included N, mean, median, standard deviation (SD), minimum and maximum for continuous data, and count and percentage for categorical data. The pharmacokinetic analyses included subjects with sufﬁcient data to enable estimation of key parameters (e.g., Tmax, Cmax, t1/2), with subjects grouped according to the treatment dose received. Individual and mean plasma and CSF HPβCD concentration versus time data were tabulated and plotted by dose level. The plasma and CSF pharmacokinetics of HPβCD were summarized by estimating Tmax, Cmax, and volume of distribution. It was not possible to measure the half-life in CSF at the last time point measured, as it was at the maximum or close to maximum concentration. For pharmacodynamic assessments, descriptive statistics and change from baseline measurements were used. 3.2. HPβCD safety proﬁle Both dose levels of HPβCD showed an acceptable safety and tolerability proﬁle with no clinically signiﬁcant events, changes, or trends noted across safety labs, physical examinations, vital signs, or electrocardiograms considered related to study treatment. Urinary hydroxyproline levels were unchanged from Baseline to the end of the study (data not shown), suggesting no effect on bone turnover for either dose group. Of the 13 subjects enrolled, 10 (77%) completed the study (6 in the low-dose group and 4 in the high-dose group). Three (23%) subjects discontinued prematurely: 1 after the ﬁrst dose due to hypersensitivity pneumonitis, and 2 after multiple doses due to transient changes in hearing. A total of 44 AEs were reported in the study, with 13 in the 1500 mg/kg group and 31 in the 2500 mg/kg group (Table 2). Of these, 40 were treatment-emergent adverse events (TEAEs), with 13 in the 1500 mg/kg group and 27 in the 2500 mg/kg group. The most common TEAE was hearing loss/reduction (deafness) occurring in 6 (46%) subjects: 1 subject (16.7%) in the 1500 mg/kg group and 5 subjects (71.4%) in the 2500 mg/kg group. The next most common TEAEs were vomiting, headache, and hematuria, which occurred in two subjects each (one in each dose group for vomiting and hematuria, both in the 1500 mg/kg group for headache). A total of 8 serious adverse events (SAEs) in 4 subjects were reported in the study, all in the 2500 mg/kg group. Two subjects in the 2500 mg/kg group experienced a Grade 3 hearing loss (change from Baseline of 25 dB averaged over 3 contiguous frequencies) that was temporally related to the study drug infusion; the loss resolved within 2 weeks post-infusion in both subjects. After two further doses in both subjects, Grade 3 hearing loss was observed again, both of which improved signiﬁcantly within 2 weeks. Both subjects were withdrawn from the study. The transient hearing loss in both subjects was at predominantly higher frequencies, and neither subject perceived a change in hearing. One of these subjects wore hearing aids prior to study enrollment. One subject in the 2500 mg/kg group experienced hypersensitivity pneumonitis and a concurrent Grade 3 hearing loss with the initial dose. This subject was removed from the study, and 3 months later, the pulmonary symptoms resolved completely, and the hearing was 3. Results 3.1. Subject demographics and baseline disease characteristics Subject accrual began in October 2017 and was completed in February 2020. The baseline demographics and disease characteristics of the 13 enrolled study subjects are summarized in Table 1. Subjects had a mean age of 35.7 years, and most were male (61.5%) and white (84.6%). The high-dose group was on average 20 years younger than the low-dose group (26 vs 46 years). Overall disease features were similar in both dose groups as determined by the total 17D-NPC-CSS: both dose groups were in the mild to moderate range in disease severity. Eleven (85%) of the 13 subjects had some degree of hearing loss at Baseline. Three (23%) subjects had a history of familial hearing loss that was not associated with NPC (two were siblings whose mother had otosclerosis, wore a hearing aid, and had a family history of hearing loss at young age due to otosclerosis; and one subject had a family history of hearing loss of unknown etiology). Two (15%) subjects wore hearing aids (both in the low-dose group, 1500 mg/kg, one with moderate and one with severe hearing loss), and an additional two (15%) subjects tested in the range for hearing aids (moderate hearing loss) at Baseline (one each in the low- and high-dose groups) and were due to receive hearing aids. Additionally, slight to mild hearing loss was noted in 3 (23%) subjects in the low-dose group and 4 (31%) subjects in the high-dose group. Two (15%) subjects had normal hearing at study entry, both in the low-dose group. Three (23%) subjects were receiving Table 1 Demographics and baseline disease characteristics. Age (years) Sex Race Ethnicity Hepatosplenomegaly Hepatomegaly-only Splenomegaly-only 17D-NPC-CSS Mean (range) Male n (%) Female n (%) White n (%) Asian n (%) American Indian/Alaska Native n (%) Black/African n (%) Native Hawaiian/Paciﬁc Islander n (%) Other Multiple Hispanic or Latino n (%) Not Hispanic or Latino n (%) Mean (SD) HPβCD 1500 mg/kg (N = 6) HPβCD 2500 mg/kg (N = 7) Total (N = 13) 46.7 (20–69) 4 (66.7) 2 (33.3) 6 (100) 0 0 0 0 0 0 0 6 (100) 5 0 1 15.2 (4.49) 26.3 (18–40) 4 (57.1) 3 (42.9) 5 (71.4) 2 (28.6) 0 0 0 0 0 1 (14.3) 6 (85.7) 5 1 1 17.7 (7.36) 35.7 (18–69) 8 (61.5) 5 (38.5) 11 (84.6) 2 (15.4) 0 0 0 0 0 1 (7.7) 12 (92.3) 10 1 2 16.5 (6.10) 17D-NPC-CSS, 17-Domain Niemann-Pick disease Type C-Clinical Severity Scale; HPβCD, 2-hydroxypropyl-β-cyclodextrin (Trappsol® Cyclo™); SD, standard deviation. 312 C. Hastings, B. Liu, B. Hurst et al. Molecular Genetics and Metabolism 137 (2022) 309–319 Table 2 Summary of adverse events by treatment. Parameter Total Number of AEs Total Number of TEAEs Common (n ≥ 2) TEAEs by preferred term Deafness (hearing loss/reduction) Vomiting Headache Hematuria Total Number of Serious AEs Number of Related Serious AEs Total Number of Serious TEAEs Number of Subjects with at Least One TEAE Number of Subjects with at Least One Related TEAE Number of Subjects with at Least One Severe (Grade ≥ 3) TEAE Number of Subjects with at Least One TEAE Leading to Treatment Discontinuation Number of Subjects with at Least One TEAE Leading to Death Number of Subjects with at Least One Serious TEAE HPβCD 1500 mg/kg (N = 6) n (%) HPβCD 2500 mg/kg (N = 7) n (%) Total (N = 13) n (%) 13 13 31 27 44 40 1 (16.7) 1 (16.7) 2 (33.3) 1 (16.7) 0 0 0 4 (66.7) 0 0 0 0 0 5 (71.4) 1 (14.3) 0 1 (14.3) 8 6 6 7 (100) 5 (71.4) 4 (57.1) 3 (42.9) 0 3 (42.9) 6 (46.2) 2 (15.4) 2 (15.4) 2 (15.4) 8 6 6 11 (84.6) 5 (38.5) 4 (30.8) 3 (23.1) 0 3 (23.1) AE, adverse event; HPβCD, 2-hydroxypropyl-β-cyclodextrin (Trappsol® Cyclo™); TEAE, treatment emergent adverse event. back to baseline other than mild loss at very high frequencies. Another subject in the 2500 mg/kg group had hearing loss at the end of the study and an upper respiratory infection (URI) and congestion at that time; the audiology assessment was consistent with conductive rather than sensorineural hearing loss. This hearing loss also improved back to Baseline 4 weeks later. All AEs of hearing loss were detected by protocol-mandated audiometry and were not perceived by the subjects themselves or their families. To summarize the results on hearing based on the 17D-NPC-CSS, 6/10 (60%) subjects who completed the study had hearing that was unchanged at Week 14 compared to Baseline (4 in the low dose group and 1 in the high-dose group; note that one subject in the low-dose group had Baseline scores of 4 in one ear and 3 in the other, while at the end of the study had a score of 4 in both ears); 3 (30%) subjects had worsened hearing scores compared to Baseline (all in the high-dose group, including the subject with the URI at time of assessment that reverted to baseline post-URI; note that none of these subjects required a change in their hearing aids during the trial period); and 1 (10%) subject had improved hearing scores compared to Baseline (low-dose group). 3.3. HPβCD pharmacokinetics in plasma and cerebrospinal ﬂuid HPβCD showed similar plasma pharmacokinetic proﬁles after the ﬁrst and seventh infusions, with a mean t1/2 of 2 h and mean Tmax of 6 and 9 h after the start of the infusion, respectively, followed by a rapid decrease in concentration after the end of the infusion (Fig. 1A). Based on the Cmax (1500 mg/kg: 1.96 to 2.07 mg/mL; 2500 mg/kg: 2.66 to 2.45 mg/mL) and the area under the concentration-time curve estimated from time zero to inﬁnity (AUC0-∞; 1500 mg/kg: 18.1 to 19.5 mg·h/mL; 2500 mg/kg: 22.5 to 22.4 mg·h/mL), systemic exposure was predicted to increase on average 1.2- to 1.5-fold for a doubling in dose. However, the increase in systemic exposure was less than dose-proportional across the dose range studied. The geometric mean terminal t1/2 was 1.93 to 2.38 h, the geometric mean clearance (76.9–112 mL/h/kg) was similar to the glomerular ﬁltration rate (125 mL/h/kg), and the geometric mean volume of distribution at steady state (Vss) ranged from 236 to 268 mL/kg. The coefﬁcient of variation for clearance and Vss was between 10.0% and 27.7%. Albumin was not detected in the CSF pharmacokinetic samples, indicating no contamination from peripheral blood. HPβCD was ﬁrst Fig. 1. HPβCD had a 2-h half-life in plasma and persisted longer in cerebrospinal ﬂuid (CSF). Mean HPβCD plasma concentration (ng/mL) curves over time (hours) after the ﬁrst (squares) and seventh (triangles) infusions with low (1500 mg/kg; clear symbols) and high (2500 mg/kg; ﬁlled symbols) doses (A). Ratio of HPβCD concentration in the CSF and plasma over time (hours) after infusion with low (1500 mg/kg; squares) and high (2500 mg/kg; triangles) doses (B). 313 C. Hastings, B. Liu, B. Hurst et al. Molecular Genetics and Metabolism 137 (2022) 309–319 Additionally, serum levels of lathosterol, a marker of whole-body cholesterol synthesis, decreased within days of the ﬁrst HPβCD infusion, reaching a low of 54.1% (n = 6) of the baseline value of 1.687 mg/L at Week 1, Day 3 (Fig. 3A). The mean serum lathosterol level decreased with repeated infusions, but to a lesser extent. After the last infusion, the mean serum lathosterol levels were at 81.3% (n = 10) of the baseline levels, and then ﬂuctuated around or above the baseline value. In contrast, serum levels of 4β-hydroxycholesterol, a marker of cholesterol catabolism, increased after the ﬁrst infusion to a high of 175.5% (101.08 μg/L) of the baseline value of 61.57 μg/L at Week 1, Day 5 (Fig. 3B). This pattern was also observed to a lesser extent after the last infusion, with an increase in the mean metabolite level from 82.4% of the baseline value at Week 12, Day 2, to 105.1% at Week 12, Day 8. There was no evidence of any dose-relationship in these changes. Subjects also had a considerable and prolonged reduction in plasma PPCS (lysosphingomyelin-509) levels, the acyl-phosphorylcholine metabolite of sphingomyelin, which accumulates secondarily in NPC disease as a result of excess cholesterol storage in lysosomes. The lowest value, at Week 12, was 46.8% of the baseline value of 5.9 ng/mL (Fig. 3C). detected in the CSF 4 h following the start of the infusion (ﬁrst timepoint measured) for both dose groups. The highest CSF concentrations measured were 48.3 μg/mL (mean at 12 h, 33 μM) for the 1500 mg/kg group and 37.8 μg/mL (mean at 12 h, 25 μM) for the 2500 mg/kg group. CSF samples were not collected after this time point, precluding the ability to calculate a t1/2 for the drug in CSF. The HPβCD concentration ratio between CSF and plasma increased from 1.7% at 8 h post start of infusion to between 11% and 16% at 12 h post start of infusion (Fig. 1B), suggesting that HPβCD persisted in the CSF for several hours after the IV infusion had ended. 3.4. HPβCD effect on peripheral tissue cholesterol The pharmacodynamics of HPβCD was assessed by ﬁlipin staining for cholesterol in liver tissue sections and by serum measures of cholesterol homeostasis. All 10 subjects who completed 7 doses of study drug and had paired liver biopsies at Baseline and the end of study treatment showed reductions in ﬁlipin staining (Fig. 2A). Subjects had different degrees of reduction in ﬁlipin staining (Fig. 2B). All subjects who received the high-dose showed marked reduction, while in the low-dose group the reduction in ﬁlipin staining was more varied, from minimal to marked (Fig. 2C). Filipin staining in skin tissue was inconclusive with respect to change between Baseline and the end of study treatment. LysoTracker staining of PBMCs did not show a clear trend (data not shown). 3.5. HPβCD effect on the CNS Serum levels of 24(S)-hydroxycholesterol (24(S)-HC), a cholesterol metabolite produced in the CNS that is transported across the blood- Fig. 2. Release of trapped liver cholesterol following treatment with HPβCD. The percentage of ﬁlipin III (ﬁlipin)-stained positive tissue area in liver tissue samples from 8 NPC1 subjects at Baseline and 2 weeks after the seventh HPβCD infusion. Two samples represented in panel C were not available at the time of the quantitative assessment and were not able to be evaluated subsequently (A). Representative images of ﬁlipin staining of liver tissue at Baseline and 14 weeks post-treatment with low (1500 mg/kg) and high (2500 mg/kg) doses of HPβCD (B). Level of reduction (minimal, mild, moderate, and marked) in ﬁlipin staining for each subject (each bar represents a subject) according to the dose of HPβCD received (C). 314 C. Hastings, B. Liu, B. Hurst et al. Molecular Genetics and Metabolism 137 (2022) 309–319 Fig. 3. Trapped cholesterol is released from lysosomes and metabolized following treatment with HPβCD. Mean serum levels (as a % of the baseline mean value) of lathosterol (A), 4β-hydroxycholesterol (B), and PPCS (lysosphingomyelin-509) (C). Data combined across dose levels. D, day; W, week; EOS, end-of-study. doses of HPβCD (Fig. 4B). Nine of 11 (82%) subjects for whom data were available showed an increase in CSF total Tau at the end of the ﬁrst infusion (Table 3). The mean serum 24(S)-hydroxycholesterol (ng/L) level increased in subjects from both dose groups after the ﬁrst (Week 1 Day 2, W1D2) and seventh (Week 12 Day 3, W12D3) infusions with HPβCD and then returned to baseline levels. The peak following the seventh infusion was reduced compared to that of the ﬁrst infusion (A). Total Tau levels measured in the cerebrospinal ﬂuid collected from subjects following 7 doses compared to baseline pretreatment (B). brain barrier into the circulation, increased following the ﬁrst infusion with HPβCD, with a peak in the mean serum concentration at 128.02% of the baseline value of 60 ng/mL (n = 13) on Week 1, Day 3 (Fig. 4A). After the ﬁnal dosing, the next time point of observation, a similar pattern of increase was observed albeit with a smaller peak. Although a dose-response relationship could not be established, there was a tendency towards a post-treatment reduction in total Tau levels in the CSF in 6/10 (60%) subjects with the mean level increasing after the ﬁrst infusion and decreasing after the last infusion in both dose groups (Table 3). Of the 10 subjects, 7 had a signiﬁcant reduction, 1 had a mild reduction, and 2 had an increase in total Tau levels after 7 315 C. Hastings, B. Liu, B. Hurst et al. Molecular Genetics and Metabolism 137 (2022) 309–319 Fig. 4. HPβCD impacts cholesterol metabolism and a biomarker of neurodegeneration, total Tau, in the central nervous system. 3.6. Efﬁcacy outcomes 4. Discussion There was little to no change in the sizes of the livers and spleens of subjects during the course of the study. In the low-dose group, the mean liver size increased from 16.88 cm to 17.55 cm, and the mean spleen size decreased from 16.42 cm to 16.33 cm. In the high-dose group, the mean liver size decreased from 17.34 cm to 17.18 cm, and the mean spleen size decreased from 14.94 cm to 13.50 cm. These changes did not reach statistical signiﬁcance. Hepatic elasticity was unchanged in both dose groups. The mean (SD) total 17D-NPC-CSS score decreased from 15.2 (4.49) to 14.8 (4.83) in the low-dose group (n = 6) and increased from 17.7 (7.36; n = 7) to 19.8 (6.34; n = 6) in the high-dose group (one subject with a very low score exited the study following dose 1, and no further scoring was done). Collectively, the baseline scores for all subjects averaged 16.5 (6.10; range 8 to 26) for 13 subjects and 17.3 (5.97; range 10 to 26) for 12 subjects at Week 14 or end of study. Three subjects showed improvements in the swallowing domain, 2 in the low-dose group (1 subject with a change of −2 and 1 subject with a change of −1) and 1 in the high-dose group (change of −1). During regular infusion visits, these subjects reported improvements within 24 h of dosing and lasting for up to a week before waning and appearing at the next dosing. These subjects, who reported acute improvements in swallow, also tended to have improvements in speech, though not to levels meeting criteria for a scoring change as measured by the 17D-NPC-CSS. Additionally, individual subjects and/or their caregivers reported improvement in gait as well as increased energy levels, ability to focus, and likelihood of social engagement, observations not able to be assessed on the 17D-NPC-CSS. The results from this Phase 1 study support the mechanism of action of systemically (intravenous) administered HPβCD in mobilizing intracellular cholesterol stores in subjects with NPC1 as demonstrated previously by preclinical studies [26,27]. Speciﬁcally, HPβCD showed positive pharmacodynamic effects in plasma (normalization of cholesterol homeostasis), liver (reduction in stored cholesterol), and CSF (reduction in total Tau), and the drug was well tolerated. As in a previous report [21], there was no signiﬁcant change in plasma pharmacokinetic parameters or drug accumulation with repeated dosing, and the mean plasma clearance rate was consistent with elimination via renal ﬁltration. Dosing of HPβCD by body weight (mg/kg) is supported by a volume of distribution that was slightly larger than the extracellular ﬂuid compartment, which is proportionate to body weight (20%). HPβCD was detected in CSF at low concentrations (maximum, 33 μM) that persisted longer than in plasma. These results support the rationale for the use of this dosage regimen in future studies. The dose and frequency of HPβCD administration for this study was determined based on pre-clinical data of npc1−/− mice treated subcutaneously with 4000 mg/kg 2HPβCD. At this dose 2HPβCD was cleared from the mice in 24 h [33], and the effective dose 50 (ED50) in mice was approximately 230 mg/kg [34]. It was also demonstrated that the duration of action of 2HPβCD was approximately 7 days [35]. Based on these data and the observation that cholesterol accumulation in humans is 13 times slower when compared to mice [36], as well as in vitro data on potential cellular toxicity at high concentrations of 2HPβCD [37], initial dosing in humans was calculated to achieve a serum concentration between 0.1 mM and 1.0 mM [29]. Pharmacokinetic data obtained in compassionate use patients conﬁrm that a dose of 2500 mg/kg could achieve this serum concentration range, which had been determined in pre-clinical studies to be non-toxic to neurons and peripheral tissues and not to deplete cholesterol stores that would subsequently drive cholesterol synthesis [38]. In order to maximize clinical beneﬁt and safety while considering the tolerability of undergoing 2HPβCD biweekly infusions of 1500 mg/kg and 2500 mg/kg were chosen. Treatment with HPβCD released accumulated cholesterol from cells in peripheral organs and restored cholesterol homeostasis. Until recently, ﬁlipin staining in cultured ﬁbroblasts or a liver biopsy was the gold standard test for diagnosing NPC [1]. With widespread availability of genetic testing, ﬁlipin staining is less in use today as a diagnostic tool, but it still holds value as a way to visualize accumulated cholesterol within lysosomes. The reduction in ﬁlipin staining of the liver biopsy samples provides direct evidence of the release of trapped cholesterol Table 3 % Change from Baseline in CSF total Tau levels. Low-dose (1500 mg/kg) High-dose (2500 mg/kg) Subject number End of Infusion 1 End of Infusion 7 Subject number End of Infusion 1 A B F G H K +44% +43% +23% Mean +26% −11% J C D E L M N Mean +11% +10% +4% +12% −25% −36% −42% −36% +77% −4% +10% −7.0% No change +240% +44% End of Infusion 7 +76% −72% −86% −19% −25% 316 C. Hastings, B. Liu, B. Hurst et al. Molecular Genetics and Metabolism 137 (2022) 309–319 subjects with neurodegenerative disorders, including NPC, where its level correlates with neuronal degeneration and loss [12]. We observed an increase in the mean Tau levels after the ﬁrst infusion, indicating the release of a Tau bolus from neurons. Nine of the eleven subjects for whom data were available showed an increase in some level in Tau at the end of the ﬁrst infusion. This was followed by a reduction in the mean Tau levels after the last (seventh) infusion. Eight of ten subjects for whom data were available showed a reduction of some level of Tau at the end of the seventh infusion (the low-dose group averaged a reduction of 29%; and the high-dose group of 59%). The downward trend in total Tau levels observed suggests that HPβCD may be reducing the rate of apoptosis and degeneration of cells in the CNS. Subject heterogeneity is likely a key factor in the Tau ﬁndings, as some subjects had baseline levels of total Tau in CSF that were in the range of healthy adults (<200 pg/mL in CSF), while others had elevated levels. The observations showing an overall downward trend in CSF total Tau levels following the seventh infusion cannot be explained by concomitant use of miglustat, which has been shown in a pilot study to reduce levels of total Tau in NPC subjects [45], as only three subjects in the present study used miglustat. Furthermore, these 3 subjects had been taking miglustat for more than 4 years prior to the start of the study; therefore, it would be expected that any beneﬁt from miglustat would have been realized prior to enrollment in the present study. This is consistent with results from the pilot study mentioned above where 2 subjects who were already being treated with miglustat at Baseline remained stable with respect to total Tau levels in CSF during the course of the study [45]. In the present study, in addition to the 3 subjects on stable doses of miglustat, 4 subjects not using miglustat showed levels of Tau reduction, compared to baseline, of between 19% and 42%. Finally, the rapid change in Tau levels after the ﬁrst infusion of HPβCD in most subjects further supports its action on neurons in the CNS, apart from a presumed steady state effect of miglustat in the 3 subjects using that drug. The variability of results suggests that a longer observational period may be useful. In a Phase 1/2 study of HPβCD in NPC1 subjects that used the same administration schedule, reductions in CSF total Tau also were observed in the CSF after 24 and 48 weeks (manuscript in preparation; data presented at WORLD Symposium 2021) [46]. Preclinical studies suggest that little HPβCD penetrates the CNS through the blood-brain-barrier [47]. Presence of a drug in the CSF may also be a measure of drug transport across the choroid plexus, which forms the blood–CSF barrier and is leakier than the blood brain barrier [48]. Regardless of the mechanism of entry of HPβCD into the CSF after systemic administration, a previous study found that direct (intrathecal) administration of HPβCD to the brain did not result in additional improvements over what would have been expected from systemic administration alone, which showed signs of a CNS effect [29]. Therefore, it is possible that systemic administration results in sufﬁcient amounts of HPβCD being present in the CSF to exert a beneﬁcial effect. In this study, subjects in the high-dose group were more likely to experience transient hearing loss, providing further evidence of a CNS effect, even at low CSF drug concentrations. A high sensitivity of the inner ear hair cells to HPβCD, especially at high CSF drug concentration [49,50], may explain why all doses of intrathecally (IT) administered HPβCD (up to 1200 mg) in a Phase 1/2 study were associated with hearing loss as well as clinically signiﬁcant post-administration fatigue and unsteadiness at doses >600 mg [51], supporting the systemic route of administration as being safer. A subsequent report on the long-term treatment of 3 NPC1 patients with IT HPβCD, through an investigational new drug application for expanded access, also reported permanent hearing loss while maintaining disease stability [52]. As this trial had a short duration and a small sample size, the full clinical effects of treatment could not be evaluated. The overall stability in the 17D-NPC-CSS for most subjects at the end of the study could therefore be related to beneﬁts from the drug or to the short duration of the study. Nevertheless, 3 subjects showed an improvement in the swallowing domain of the 17D-NPC-CSS and reported acute changes in from the liver cells of subjects with NPC1. The observed difference between ﬁlipin baseline levels in the two dose groups was not considered signiﬁcant due to the genetic and clinical heterogeneity of the disease. The release of free cholesterol was accompanied by a reduction in the plasma levels of the cholesterol precursor, lathosterol, and an increase in the plasma levels of the cholesterol metabolite, 4β-hydroxycholesterol. That is, cholesterol synthesis was reduced, and cholesterol catabolism was increased, as expected with the restoration of intracellular cholesterol trafﬁcking out of the late endosomal/lysosomal compartment and into other regions of the cells. The return to baseline levels with repeated infusions was expected as cholesterol turnover returned to normal and may be indicative of a decline in stored cholesterol or a slow build-up between infusions. In addition to this effect on stored cholesterol, there was also a consistent reduction in the plasma levels of PPCS (lysosphingomyelin-509), a fatty-acyl derivative of sphingolipid that secondarily accumulates in the plasma of NPC subjects and serves as a prognostic biomarker for disease severity [39]. The normalization of sterol levels in cells may lead to a reduction in cellular damage and functional recovery over time. A key aspect that impacts the quality of life of subjects with NPC1 is the neurodegeneration caused by the disease. Therefore, any effective drug must have a therapeutic effect in the brain in addition to treating the visceral pathology [6]. Accordingly, the impact of HPβCD in the CNS was assessed in this study. While the volume of distribution was limited, detection of HPβCD in CSF suggests that systemically administered HPβCD has access the CNS. Although it is not clear how HPβCD reaches the CNS, it was detected at low levels in CSF (<0.1 mM) that are in the range of brain tissue concentrations associated with CNS efﬁcacy in animal models of NPC1 [40], including preventing degeneration of Purkinje cells in adult animals [28]. A recent in vitro study using ﬁbroblasts from NPC subjects showed that a 300 μM level of HPβCD affected the expression of over 400 genes [41], further supporting the statement that low levels of the drug in the CNS may underpin the clinical beneﬁts observed in the present study and in compassionate use IV programs described elsewhere [29]. Further, CSF turns over completely on the order of 4 to 5 times daily, suggesting that molecules found in the CSF could persist for greater periods than in plasma [42]. The present ﬁnding that HPβCD is detected in the CSF at 4 h after the end of the IV infusion is consistent with its longer half-life in the CSF than in plasma. Measurements of red blood cells and albumin levels in the CSF samples were made to determine if blood contamination from the lumbar puncture could explain any change in biomarkers or drug levels in the CSF. There was no evidence of blood contamination. Therefore, the HPβCD measured in the CSF was not introduced by the procedure. Here, we show direct evidence that the drug is present in the CSF following intravenous administration. Whereas the plasma HPβCD levels declined rapidly after the end of the infusion, CSF levels were maintained longer, indicating that the drug persists in the CNS longer than the plasma, which was consistent with the Vss values measured. At low concentrations (<1 mM), such as those found in the CSF, HPβCD acts as a cholesterol shuttle, transporting cholesterol between membranes [25,43]. Therefore, the presence of HPβCD in the CSF, which gives the drug direct exposure to the brain surface even at low concentrations, indicates its potential to release unesteriﬁed cholesterol from neuronal cells, which may restore their function or prevent further loss of function. The baseline 24(S)-HC serum levels measured in this study (~60 ng/mL) are similar to those reported in the plasma (64 +/− 14 ng/mL) [44]. The rise in this brain-derived cholesterol metabolite (24(S)-HC) in serum correlated temporally with the IV infusion, peaking roughly at 3 days post dose for both doses studied before returning to baseline. Our interpretation is that HPβCD is active in the brain, and that it contributes to the reduction of stored cholesterol in the CNS. Additionally, there was a post-treatment effect in total Tau levels in the CSF in most subjects. Tau is a microtubule-associated protein found primarily in axons of CNS neurons, and it is elevated in the CSF of some 317 C. Hastings, B. Liu, B. Hurst et al. Molecular Genetics and Metabolism 137 (2022) 309–319 Metabolic Consultant, Salford Royal Foundation Trust, UK, contributed signiﬁcantly as a Safety Review Committee member. We are indebted as well to Dr. Bryan Murray, Boyd Consultants, UK, our Medical Monitor for the study; to Medpace Laboratories (Cincinnati, OH) for laboratory support and to HistologiX (UK) for histology work. We acknowledge with gratitude the excellent support from Summit Global Health in preparation of this manuscript. This work would not have been possible without the active participation and commitment of time by the subjects and families who participated. We are grateful indeed for the teamwork in moving this study forward. speech and swallow after each dose. Many of the subjects reported improvements in quality-of-life areas such cognition, socialization abilities (increased focus and social interactions), and motor coordination (improved stance and gait). Considering the short duration of the trial, the clinical efﬁcacy ﬁndings should be interpreted with caution. None of the subjects in this trial experienced clinically signiﬁcant effects, trends, or clinical safety laboratory ﬁndings. Given that another Phase 1/2 trial using HPβCD administered intrathecally led to permanent hearing losses at mid-to-high-frequencies in all participants [51], the present trial included close monitoring for hearing changes. A transient effect on hearing was observed in some subjects, particularly those in the high-dose group (2500 mg/kg). It is important to note that hearing loss is part of the natural history of NPC [4], and that 11 of the 13 subjects who enrolled in this study had hearing loss of some degree at Baseline. Given that 6/10 (60%) subjects who completed the trial had no change in hearing, one subject's hearing improved, and 3 subjects had high-frequency hearing loss that was imperceptible and showed evidence of recovery post dosing, we conclude that HPβCD administered intravenously at the doses studied does not pose a signiﬁcant risk to subject hearing. Hearing should continue to be monitored in future clinical studies. The results from this study suggest that intravenously administered HPβCD has the potential to treat both the systemic and neurological manifestations of NPC. Slowing down disease progression through cholesterol mobilization is an important consideration for subjects with established disease, who can expect to experience neurodegeneration without treatment. A natural history study of 18 subjects has shown on average a 1.4-point increase per year in the 17D-NPC-CSS, and a retrospective review of 19 subjects showed a 1.9 point increase per year [30]. Considering the progressive nature of NPC and the short duration of this study (14 weeks), the results presented here are very encouraging as the observed efﬁcacy signals are likely to be only a proportion of the full treatment effect. The visceral effects of IV HPβCD may prove to be especially beneﬁcial to NPC patients approaching liver failure. Indeed, a recent open label trial in NPC infants with liver disease administered low dose HPβCD (250 mg/kg, 500 mg/kg, and 1000 mg/kg) to 3 children (and an additional patient under an emergency investigational new drug study, who expired from the underlying progressive disease) showed encouraging biomarker changes and demonstrated safety in this small cohort [53]. US residents who completed the Phase 1 study and are presented here were eligible to enroll in an extension study, and all eight eligible subjects enrolled (ClinicalTrial.gov number, NCT03893071). Foreign-based participants were offered continuous access to HβCD through compassionate/named subject programs, and all 4 eligible subjects are either currently enrolled or in the process of enrolling. A Phase 1/2, 48-week study (ClinicalTrials.gov number, NCT02912793) has also been completed and the results will be presented in a future publication. Additionally, a multicenter, doubleblind, randomized, placebo-controlled Phase 3 clinical trial to evaluate the long-term safety, tolerability, and efﬁcacy of HPβCD is currently enrolling (ClinicalTrials.gov number, NCT04860960). References [1] T. Geberhiwot, A. Moro, A. Dardis, U. Ramaswami, S. Sirrs, M.P. Marfa, M.T. Vanier, M. Walterfang, S. Bolton, C. Dawson, B. Heron, M. Stampfer, J. Imrie, C. Hendriksz, P. Gissen, E. Crushell, M.J. Coll, Y. Nadjar, H. Klunemann, E. Mengel, M. Hrebicek, S.A. Jones, D. Ory, B. Bembi, M. Patterson, R. International Niemann-Pick Disease, Consensus clinical management guidelines for Niemann-Pick disease type C, Orphanet J Rare Dis. 13 (2018) 50, https://doi.org/10.1186/s13023-018-0785-7. [2] C.A. Wassif, J.L. Cross, J. Iben, L. Sanchez-Pulido, A. Cougnoux, F.M. Platt, D.S. Ory, C.P. Ponting, J.E. Bailey-Wilson, L.G. Biesecker, F.D. Porter, High incidence of unrecognized visceral/neurological late-onset Niemann-Pick disease, type C1, predicted by analysis of massively parallel sequencing data sets, Genet Med. 18 (2016) 41–48, https://doi.org/10.1038/gim.2015.25. [3] M.C. Patterson, P. Clayton, P. Gissen, M. Anheim, P. Bauer, O. Bonnot, A. Dardis, C. Dionisi-Vici, H.H. Klunemann, P. Latour, C.M. Lourenco, D.S. Ory, A. Parker, M. Pocovi, M. Strupp, M.T. Vanier, M. Walterfang, T. Marquardt, Recommendations for the detection and diagnosis of Niemann-Pick disease type C: an update, Neurol. Clin. Pract. 7 (2017) 499–511, https://doi.org/10.1212/CPJ.0000000000000399. [4] M.T. Vanier, Niemann-Pick disease type C, Orphanet J Rare Dis. 5 (2010) 16, https:// doi.org/10.1186/1750-1172-5-16. [5] N.-C.G.W. Group, J.E. Wraith, M.R. Baumgartner, B. Bembi, A. Covanis, T. Levade, E. Mengel, M. Pineda, F. Sedel, M. Topcu, M.T. Vanier, H. Widner, F.A. Wijburg, M.C. Patterson, Recommendations on the diagnosis and management of Niemann-Pick disease type C, Mol. Genet. Metab. 98 (2009) 152–165, https://doi.org/10.1016/j. ymgme.2009.06.008. [6] N. Hammond, A.B. Munkacsi, S.L. Sturley, The complexity of a monogenic neurodegenerative disease: more than two decades of therapeutic driven research into Niemann-Pick type C disease, Biochim. Biophys. Acta Mol. Cell Biol. Lipids 2019 (1864) 1109–1123, https://doi.org/10.1016/j.bbalip.2019.04.002. [7] J. Newton, S. Milstien, S. Spiegel, Niemann-Pick type C disease: the atypical sphingolipidosis, Adv. Biol. Regul. 70 (2018) 82–88, https://doi.org/10.1016/j.jbior. 2018.08.001. [8] W.D. Park, J.F. O’Brien, P.A. Lundquist, D.L. Kraft, C.W. Vockley, P.S. Karnes, M.C. Patterson, K. Snow, Identiﬁcation of 58 novel mutations in Niemann-Pick disease type C: correlation with biochemical phenotype and importance of PTC1-like domains in NPC1, Hum. Mutat. 22 (2003) 313–325, https://doi.org/10.1002/humu. 10255. [9] S. Wheeler, D.J. Sillence, Niemann-Pick type C disease: cellular pathology and pharmacotherapy, J. Neurochem. 153 (2020) 674–692, https://doi.org/10.1111/jnc. 14895. [10] S.U. Walkley, M.T. Vanier, Secondary lipid accumulation in lysosomal disease, Biochim. Biophys. Acta 1793 (2009) 726–736, https://doi.org/10.1016/j.bbamcr. 2008.11.014. [11] L.A. Abel, M. Walterfang, M. Fietz, E.A. Bowman, D. Velakoulis, Saccades in adult Niemann-Pick disease type C reﬂect frontal, brainstem, and biochemical deﬁcits, Neurology. 72 (2009) 1083–1086, https://doi.org/10.1212/01.wnl.0000345040. 01917.9d. [12] I. Cariati, L. Masuelli, R. Bei, V. Tancredi, C. Frank, G. D’Arcangelo, Neurodegeneration in Niemann-Pick Type C disease: an updated review on pharmacological and nonpharmacological approaches to counteract brain and cognitive impairment, Int. J. Mol. Sci. 22 (2021) https://doi.org/10.3390/ijms22126600. [13] G. Trendelenburg, M.T. Vanier, S. Maza, G. Millat, G. Bohner, D.L. Munz, R. Zschenderlein, Niemann-Pick type C disease in a 68-year-old patient, J. Neurol. Neurosurg. Psychiatry 77 (2006) 997–998, https://doi.org/10.1136/jnnp.2005. 086785. [14] L. Lopez de Frutos, J.J. Cebolla, L. Aldamiz-Echevarria, A. de la Vega, S. Stanescu, C. Lahoz, P. Irun, P. Giraldo, New variants in Spanish Niemann-Pick type c disease patients, Mol. Biol. Rep. 47 (2020) 2085–2095, https://doi.org/10.1007/s11033-02005308-7. [15] B.K. Burton, A.G. Ellis, B. Orr, S. Chatlani, K. Yoon, J.R. Shoaff, D. Gallo, Estimating the prevalence of Niemann-Pick disease type C (NPC) in the United States, Mol. Genet. Metab. (2021) https://doi.org/10.1016/j.ymgme.2021.06.011. [16] J. Imrie, L. Heptinstall, S. Knight, K. Strong, Observational cohort study of the natural history of Niemann-Pick disease type C in the UK: a 5-year update from the UK clinical database, BMC Neurol. 15 (2015) 257, https://doi.org/10.1186/s12883-0150511-1. [17] M.C. Patterson, D. Vecchio, H. Prady, L. Abel, J.E. Wraith, Miglustat for treatment of Niemann-Pick C disease: a randomised controlled study, Lancet Neurol. 6 (2007) 765–772, https://doi.org/10.1016/S1474-4422(07)70194-1. [18] M.C. Patterson, W.S. Garver, R. Giugliani, J. Imrie, H. Jahnova, F.J. Meaney, Y. Nadjar, M.T. Vanier, P. Moneuse, O. Morand, D. Rosenberg, B. Schwierin, B. Heron, Long-term Funding This study was funded by Cyclo Therapeutics, Inc. Data availability The data that has been used is conﬁdential. Acknowledgements The authors are grateful to Dr. Pinar Karakas-Rothey, UCSF Benioff Children's Hospital Oakland, for her kind assistance as the central reader for ultrasound images. Professor Alan Boyd, UK, provided excellent leadership for the Safety Review Committee and Dr. Reena Sharma, Senior 318 C. Hastings, B. Liu, B. Hurst et al. [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] Molecular Genetics and Metabolism 137 (2022) 309–319 survival outcomes of patients with Niemann-Pick disease type C receiving miglustat treatment: a large retrospective observational study, J. Inherit. Metab. Dis. 43 (2020) 1060–1069, https://doi.org/10.1002/jimd.12245. N. Belmatoug, A. Burlina, P. Giraldo, C.J. Hendriksz, D.J. Kuter, E. Mengel, G.M. Pastores, Gastrointestinal disturbances and their management in miglustattreated patients, J. Inherit. Metab. Dis. 34 (2011) 991–1001, https://doi.org/10. 1007/s10545-011-9368-7. P. Karimzadeh, S.H. Tonekaboni, M.R. Ashraﬁ, Y. Shafeghati, A. Rezayi, S. Salehpour, M. Ghofrani, M.M. Taghdiri, A. Rahmanifar, T. Zaman, O. Aryani, B.N. Shoar, F. Shiva, A. Tavasoli, M. Houshmand, Effects of miglustat on stabilization of neurological disorder in niemann-pick disease type C: Iranian pediatric case series, J. Child Neurol. 28 (2013) 1599–1606, https://doi.org/10.1177/0883073812464526. M.E. Brewster, T. Loftsson, Cyclodextrins as pharmaceutical solubilizers, Adv. Drug Deliv. Rev. 59 (2007) 645–666, https://doi.org/10.1016/j.addr.2007.05.012. T. Loftsson, M.E. Brewster, Cyclodextrins as functional excipients: methods to enhance complexation efﬁciency, J. Pharm. Sci. 101 (2012) 3019–3032, https://doi. org/10.1002/jps.23077. F. Camargo, R.P. Erickson, W.S. Garver, G.S. Hossain, P.N. Carbone, R.A. Heidenreich, J. Blanchard, Cyclodextrins in the treatment of a mouse model of Niemann-Pick C disease, Life Sci. 70 (2001) 131–142, https://doi.org/10.1016/s0024-3205(01)01384-4. C.D. Davidson, N.F. Ali, M.C. Micsenyi, G. Stephney, S. Renault, K. Dobrenis, D.S. Ory, M.T. Vanier, S.U. Walkley, Chronic cyclodextrin treatment of murine Niemann-Pick C disease ameliorates neuronal cholesterol and glycosphingolipid storage and disease progression, PLoS One 4 (2009), e6951, https://doi.org/10.1371/journal.pone. 0006951. B. Liu, Therapeutic potential of cyclodextrins in the treatment of Niemann-Pick type C disease, Clin. Lipidol. 7 (2012) 289–301, https://doi.org/10.2217/clp.12.31. B. Liu, H. Li, J.J. Repa, S.D. Turley, J.M. Dietschy, Genetic variations and treatments that affect the lifespan of the NPC1 mouse, J. Lipid Res. 49 (2008) 663–669, https://doi.org/10.1194/jlr.M700525-JLR200. B. Liu, S.D. Turley, D.K. Burns, A.M. Miller, J.J. Repa, J.M. Dietschy, Reversal of defective lysosomal transport in NPC disease ameliorates liver dysfunction and neurodegeneration in the npc1−/− mouse, Proc. Natl. Acad. Sci. U. S. A. 106 (2009) 2377–2382, https://doi.org/10.1073/pnas.0810895106. C.M. Ramirez, B. Liu, A.M. Taylor, J.J. Repa, D.K. Burns, A.G. Weinberg, S.D. Turley, J.M. Dietschy, Weekly cyclodextrin administration normalizes cholesterol metabolism in nearly every organ of the Niemann-Pick type C1 mouse and markedly prolongs life, Pediatr. Res. 68 (2010) 309–315, https://doi.org/10.1203/00006450-20101100100604. C. Hastings, C. Vieira, B. Liu, C. Bascon, C. Gao, R.Y. Wang, A. Casey, S. Hrynkow, Expanded access with intravenous hydroxypropyl-beta-cyclodextrin to treat children and young adults with Niemann-Pick disease type C1: a case report analysis, Orphanet J Rare Dis. 14 (2019) 228, https://doi.org/10.1186/s13023-019-1207-1. N.M. Yanjanin, J.I. Velez, A. Gropman, K. King, S.E. Bianconi, S.K. Conley, C.C. Brewer, B. Solomon, W.J. Pavan, M. Arcos-Burgos, M.C. Patterson, F.D. Porter, Linear clinical progression, independent of age of onset, in Niemann-Pick disease, type C, Am. J. Med. Genet. B Neuropsychiatr. Genet. 153B (2010) 132–140, https://doi.org/10. 1002/ajmg.b.30969. A. World Medical, World medical association declaration of Helsinki: ethical principles for medical research involving human subjects, JAMA. 310 (2013) 2191–2194, https://doi.org/10.1001/jama.2013.281053. G. Ferraioli, P. Parekh, A.B. Levitov, C. Filice, Shear wave elastography for evaluation of liver ﬁbrosis, J. Ultrasound Med. 33 (2014) 197–203, https://doi.org/10.7863/ ultra.33.2.197. B. Liu, C.M. Ramirez, A.M. Miller, J.J. Repa, S.D. Turley, J.M. Dietschy, Cyclodextrin overcomes the transport defect in nearly every organ of NPC1 mice leading to excretion of sequestered cholesterol as bile acid, J. Lipid Res. 51 (2010) 933–944, https:// doi.org/10.1194/jlr.M000257. C.M. Ramirez, B. Liu, A. Aqul, A.M. Taylor, J.J. Repa, S.D. Turley, J.M. Dietschy, Quantitative role of LAL, NPC2, and NPC1 in lysosomal cholesterol processing deﬁned by genetic and pharmacological manipulations, J. Lipid Res. 52 (2011) 688–698, https://doi.org/10.1194/jlr.M013789. A. Aqul, B. Liu, C.M. Ramirez, A.A. Pieper, S.J. Estill, D.K. Burns, B. Liu, J.J. Repa, S.D. Turley, J.M. Dietschy, Unesteriﬁed cholesterol accumulation in late endosomes/lysosomes causes neurodegeneration and is prevented by driving cholesterol export from this compartment, J. Neurosci. 31 (2011) 9404–9413, https://doi.org/10. 1523/JNEUROSCI.1317-11.2011. J.M. Dietschy, S.D. Turley, Control of cholesterol turnover in the mouse, J. Biol. Chem. 277 (2002) 3801–3804, https://doi.org/10.1074/jbc.R100057200. K.B. Peake, J.E. Vance, Normalization of cholesterol homeostasis by 2hydroxypropyl-beta-cyclodextrin in neurons and glia from Niemann-Pick C1 [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] 319 (NPC1)-deﬁcient mice, J. Biol. Chem. 287 (2012) 9290–9298, https://doi.org/10. 1074/jbc.M111.326405. C. Hastings, D. Killilea, J.W. Suh, R. Browne, Clinical experience and biomarker assessment with intravenous and intrathecal infusions of hydroxypropyl-βcyclodextrin in identical twin patients with Niemann-Pick Type C disease, Ara Parseghian Scientiﬁc Conference on Niemann-Pick Type C Research, 2011 , Notre Dame, Indiana. M.S. Breilyn, W. Zhang, C. Yu, M.P. Wasserstein, Plasma lyso-sphingomyelin levels are positively associated with clinical severity in acid sphingomyelinase deﬁciency, Mol. Genet. Metab. Rep. 28 (2021), 100780, https://doi.org/10.1016/j.ymgmr.2021. 100780. C.H. Vite, J.H. Bagel, G.P. Swain, M. Prociuk, T.U. Sikora, V.M. Stein, P. O’Donnell, T. Ruane, S. Ward, A. Crooks, S. Li, E. Mauldin, S. Stellar, M. De Meulder, M.L. Kao, D.S. Ory, C. Davidson, M.T. Vanier, S.U. Walkley, Intracisternal cyclodextrin prevents cerebellar dysfunction and Purkinje cell death in feline Niemann-Pick type C1 disease, Sci. Transl. Med. 7 (2015) 276ra26, https://doi.org/10.1126/scitranslmed. 3010101. J.L. Rodriguez-Gil, L.L. Baxter, D.E. Watkins-Chow, N.L. Johnson, C.D. Davidson, S.R. Carlson, A.A. Incao, N.C.S. Program, K.L. Wallom, N.Y. Farhat, F.M. Platt, R.K. Dale, F.D. Porter, W.J. Pavan, Transcriptome of HPbetaCD-treated Niemann-Pick disease type C1 cells highlights GPNMB as a biomarker for therapeutics, Hum. Mol. Genet. 30 (2021) 2456–2468, https://doi.org/10.1093/hmg/ddab194. S.T. Proulx, Cerebrospinal ﬂuid outﬂow: a review of the historical and contemporary evidence for arachnoid villi, perineural routes, and dural lymphatics, Cell. Mol. Life Sci. 78 (2021) 2429–2457, https://doi.org/10.1007/s00018-020-03706-5. A.E. Christian, H.S. Byun, N. Zhong, M. Wanunu, T. Marti, A. Furer, F. Diederich, R. Bittman, G.H. Rothblat, Comparison of the capacity of beta-cyclodextrin derivatives and cyclophanes to shuttle cholesterol between cells and serum lipoproteins, J. Lipid Res. 40 (1999) 1475–1482. I. Burkard, K.M. Rentsch, A. von Eckardstein, Determination of 24S- and 27hydroxycholesterol in plasma by high-performance liquid chromatography-mass spectrometry, J. Lipid Res. 45 (2004) 776–781, https://doi.org/10.1194/jlr. D300036-JLR200. N. Mattsson, H. Zetterberg, S. Bianconi, N.M. Yanjanin, R. Fu, J.E. Mansson, F.D. Porter, K. Blennow, Miglustat treatment may reduce cerebrospinal ﬂuid levels of the axonal degeneration marker tau in niemann-pick type C, JIMD Rep. 3 (2012) 45–52, https://doi.org/10.1007/8904_2011_47. J. Raiman, M. Paucar-Arce, O. Staretz-Chacham, R. Spiegel, A. Boyd, C. Hastings, S. Hrynkow, Update on Safety and Efﬁcacy Results for Phase I/II trial of Trappsol® Cyclo™ Hydroxypropyl Betacyclodextrin Administered Intravenously in Subjects with Niemann-Pick type C1 Disease. Poster #209. in WORLDSymposium, 2021. C.C. Pontikis, C.D. Davidson, S.U. Walkley, F.M. Platt, D.J. Begley, Cyclodextrin alleviates neuronal storage of cholesterol in Niemann-Pick C disease without evidence of detectable blood-brain barrier permeability, J. Inherit. Metab. Dis. 36 (2013) 491–498, https://doi.org/10.1007/s10545-012-9583-x. W.M. Pardridge, CSF, blood-brain barrier, and brain drug delivery, Expert Opin. Drug Deliv. 13 (2016) 963–975, https://doi.org/10.1517/17425247.2016.1171315. M.A. Crumling, L. Liu, P.V. Thomas, J. Benson, A. Kanicki, L. Kabara, K. Halsey, D. Dolan, R.K. Duncan, Hearing loss and hair cell death in mice given the cholesterolchelating agent hydroxypropyl-beta-cyclodextrin, PLoS One 7 (2012), e53280, https://doi.org/10.1371/journal.pone.0053280. X. Liu, D. Ding, G.D. Chen, L. Li, H. Jiang, R. Salvi, 2-hydroxypropyl-beta-cyclodextrin ototoxicity in adult rats: rapid onset and massive destruction of both inner and outer hair cells above a critical dose, Neurotox. Res. 38 (2020) 808–823, https:// doi.org/10.1007/s12640-020-00252-7. D.S. Ory, E.A. Ottinger, N.Y. Farhat, K.A. King, X. Jiang, L. Weissfeld, E. Berry-Kravis, C.D. Davidson, S. Bianconi, L.A. Keener, R. Rao, A. Soldatos, R. Sidhu, K.A. Walters, X. Xu, A. Thurm, B. Solomon, W.J. Pavan, B.N. Machielse, M. Kao, S.A. Silber, J.C. McKew, C.C. Brewer, C.H. Vite, S.U. Walkley, C.P. Austin, F.D. Porter, Intrathecal 2hydroxypropyl-beta-cyclodextrin decreases neurological disease progression in Niemann-Pick disease, type C1: a non-randomised, open-label, phase 1-2 trial, Lancet. 390 (2017) 1758–1768, https://doi.org/10.1016/S0140-6736(17)31465-4. E. Berry-Kravis, J. Chin, A. Hoffmann, A. Winston, R. Stoner, L. LaGorio, K. Friedmann, M. Hernandez, D.S. Ory, F.D. Porter, J.A. O’Keefe, Long-term treatment of NiemannPick type C1 disease with intrathecal 2-hydroxypropyl-beta-cyclodextrin, Pediatr. Neurol. 80 (2018) 24–34, https://doi.org/10.1016/j.pediatrneurol.2017.12.014. M. Reynolds, L.A. Linneman, S. Luna, B.B. Warner, Y.P. Turmelle, S.S. Kulkarni, X. Jiang, G. Khanna, M. Shinawi, F.D. Porter, D.S. Ory, F.S. Cole, P.I. Dickson, A phase 1/2 open label nonrandomized clinical trial of intravenous 2-hydroxypropyl-betacyclodextrin for acute liver disease in infants with Niemann-Pick C1, Mol. Genet. Metab. Rep. 28 (2021), 100772, https://doi.org/10.1016/j.ymgmr.2021.100772.