Long-term efficacy of intrathecal cyclodextrin in patients with Niemann-Pick disease type C

Background and objectives: Niemann – Pick type C (NPC) is a rare lysosomal storage disease characterized by hepatosplenomegaly and progressive neurological deterioration due to abnormal intracellular cholesterol transport. Cyclic oligosaccharide 2-hydroxypropyl-β -cyclodextrin (HPBCD) is an effective treatment for NPC; however, few reports have shown its long-term efficacy and safety. To demonstrate long-term efficacy and safety of intrathecal HPBCD (IT-HPBCD) treatment for NPC, we herein reports five patients with NPC treated using IT-HPBCD for 4 – 11 years. Cases and results: Patients ’ ages at the onset ranged from 1.5 to 20 years. Notably, all patients showed rapid disease progression despite treatment with miglustat before IT-HPBCD treatment. Similarly, some patients showed transient improvement; however, all patients ’ conditions stabilized after long-term IT-HPBCD therapy. Mild-to-moderate hearing loss was observed in three patients. Furthermore, long-term treatment with IT-HPBCD may suppress neurological deterioration in patients with NPC; however, patients still experience some disease progression. Conclusions: Long-term treatment with IT-HPBCD may suppress neurological deterioration in patients with NPC; however, the treatment outcome is dependent on the neurological status at the time of diagnosis, and disease progression is not completely inhibited. Awareness of the disease and newborn screening is needed for earlier disease detection. In addition, further optimization of the treatment protocol and additional treatments are needed to improve patient outcomes.


Introduction
Niemann-Pick type C (NPC) is a rare lysosomal storage disease characterized by hepatosplenomegaly and progressive neurological deterioration due to abnormal intracellular cholesterol transport caused by pathogenic variants of the NPC1 or NPC2 gene variants [1].Disrupted intracellular cholesterol transport causes the accumulation of unesterified cholesterol in lysosomes and cholesterol utilization disorders, leading to lysosomal dysfunction and abnormal autophagy.
The clinical manifestations and progression of NPC vary depending on the age at the onset of neurological symptoms, and types of NPC include early infantile, late infantile, juvenile, and adult.Disease progression is typically rapid and severe in patients with early-onset NPC [1].Miglustat, which inhibits glucosylceramide synthesis, is the only drug approved to treat NPC in Japan as it effectively slows the progression of neurological symptoms.However, its effect is limited, especially in patients with early-onset NPC [2].
In 2009, the effectiveness of 2-hydroxypropyl-β-cyclodextrin (HPBCD), a cyclic oligosaccharide, was reported in an NPC mouse model [3,4].HPBCD forms inclusion compounds with cholesterol in its inner pores and acts as an alternative to NPC1 and NPC2.In April 2009, HPBCD was administered to twins with NPC under the US Food and Drug Administration Individual Investigation of New Drug Exemptions [5].Five months later, our department began treating patients with intravenous NPC [6].The systemic, intravenous administration of HPBCD is partially effective for hepatosplenomegaly and neurological symptoms, which may be due to challenges crossing the blood-brain barrier.Furthermore, lung toxicity has been reported in patients undergoing long-term treatment with intravenous HPBCD [7].
Then, the intracisternal administration of HPBCD was tested in mice [8] and cats [9] with NPC, revealing favorable efficacy; however, ototoxicity was reported [10,11].Clinical trials regarding the intrathecal (IT) or intracerebroventricular (ICV) administration of HPBCD in patients with NPC have demonstrated beneficial effects on patients' neurological symptoms; however, hearing loss has frequently been reported [7,12,13].Overall, HPBCD appears to be the most promising treatment for NPC; however, few studies have reported its long-term efficacy and safety [14].This report presents five patients with NPC who underwent long-term (4-11 years) IT-or ICV-HPBCD.

Ethics
The treatment protocol used in the present study was approved by the Ethics Committees of Saga University (no.2009-05-04), Saga University Clinical Research Review Board (C20190201), Nara Medical University Ethics Committee (13-Ken037 and 16-Ken009), and Jichi Medical University Ethics Committee (S17-052).Informed consent for the conduction and publication of this study was obtained from the patients' guardians.The patients' details are provided in Table 1.

Case 1
A 17-year-old female patient with a history of fetal hepatosplenomegaly was diagnosed with NPC based on pathological NPC1 variants (c.581_592delinsG, Y1088C) at 2 months of age.At 3 years of age, she began to exhibit rapid neurological deterioration, including progressive ataxia, cataplexy, dysarthria, dysphagia, and convulsions.Intravenous HPBCD was initiated when the patient was 4 years old, and miglustat was initiated when the patient was 5 years old.However, the patient became bedridden, lost the ability to speak, and required tube feeding through gastrostomy.IT-HPBCD therapy (10 mg/kg of 20% HPBCD diluted with 6 mL saline administered through a lumbar puncture every other week) was initiated when the patient was 6 years old.No adverse effects were observed within 2 months of initiating HPBCD therapy; therefore, an Ommaya reservoir was implanted to administer 20 mg/kg HPBCD weekly.The dose was then increased to 22.5 mg/kg (450 mg) weekly.Fifteen months later, the dose was changed to 200 mg twice weekly based on the HPBCD concentration of the cerebrospinal fluid (CSF).Twenty-one months later, the dose was increased to 300 mg twice a week; however, it was reduced shortly after due to an increased total tau (T-tau) in the CSF.The intravenous administration of HPBCD was discontinued 12 months after initiating IT-HPBCD; however, miglustat was continued, and anticonvulsants were adjusted as clinically indicated.

Case 2
An 11-year-old male patient had a history of cholestatic jaundice and hepatosplenomegaly during the neonatal period and interstitial pneumonia at 2 months of age.The patient was diagnosed with NPC based on pathological variants of NPC1 (p.C243R, p.E332fsX) at 8 months.Miglustat was initiated when the patient was 1 year old.At that time, he could stand while balancing with an object.He could walk with assistance (cruise) at 2 years old but never walked alone.By 3 years of age, the patient could no longer stand.Therefore, IT-HPBCD was initiated (5 mg/kg dose 20% HPBCD diluted with saline to a final volume of 3 mL) and administered through a lumbar puncture monthly.The dose was subsequently increased to 500 mg (20 mg/kg) monthly.

Case 3
A 44-year-old male patient dropped out of school at 20 years of age due to delusions of persecution and psychomotor excitation.Subsequently, he experienced seizures and apneic spells with a gradual intellectual deterioration.At 25 years of age, the patient was referred to an outside hospital for a diagnostic workup.Mild liver dysfunction, splenomegaly, ataxia, supranuclear gaze palsy, and athetoid movements were observed, as well as worsened intellectual deterioration and dysphagia.The patient underwent percutaneous endoscopic gastrostomy when he was 34 years old.He was referred to our department at age 35 years after his brother's diagnosis (Case 4).The patient's diagnosis was confirmed through the presence of compound heterozygosity for a novel splice variant, IVS6-3C>G (c.882-3C>G), and a known pathogenic variant of NPC1, p.G992R (c.2974G>C).The patient presented with ocular fixation, severe dysarthria, dysphagia, poor expectoration of sputum, and few spontaneous movements.He could stand and move in a wheelchair with assistance but could not walk alone due to truncal ataxia.His horizontal eye movement and speech slightly improved after 3 months of miglustat treatment; however, his neurological symptoms continued to deteriorate.IT-HPBCD was initiated at 100 mg 20% HPBCD diluted to 6 mL with saline each month administered through a lumbar puncture.The dose was subsequently increased to 400 mg monthly.

Table 1
Patients treated with intrathecal or intracerebroventricular HPBCD.

Case 4
A 35-year-old male patient successfully completed high school and proceeded to technical school.However, he dropped out of technical school at the age of 20 years due to poor academic performance after a mild intellectual impairment diagnosis.He is the brother to Case 3. At the age of 23 years, the patient developed seizures.Ataxia and intellectual deterioration were observed when the patient was 25 years old, and he was referred to our department one year later with a gait disturbance and a tendency to fall.Neurologically, the patient presented with intellectual deterioration, supranuclear downward-gaze palsy, dysarthria, dysphagia with choking, dystonia of the upper extremities, limb and truncal ataxia, and splenomegaly.He could not use chopsticks due to clumsiness and presented with a slow and unsteady gait due to ataxia and dystonia.His dysphagia and ataxia transiently improved after 3 months of miglustat; however, his neurological symptoms continued to deteriorate after that.IT-HPBCD was initiated at 28 years of age at a dose of 100 mg 20% HPBCD diluted to 6 mL with saline administered monthly through a lumbar puncture.The dose was subsequently increased to 400 mg monthly.

Case 5
An 11-year-old female with a history of a slightly unsteady gait at 3 years of age had psychomotor retardation when she was 5 years old.At that time, the patient had a full-scale IQ of 73 with the Wechsler Intelligence Scale for Children, Fourth Edition.One year later, the patient developed dystonia, ataxia, and vertical gaze palsy.Genetic examination revealed complex heterozygous pathogenic variants in the NPC1 gene (c.3618del., c.3634G>A; p.Lys1206Asnfs*36, p.Val1212Met).A diagnosis of NPC was confirmed using positive filipin staining of the skin fibroblasts.Miglustat was initiated at the time of diagnosis; however, the patient's neurological symptoms gradually deteriorated, and she experienced an epileptic seizure at 7 years of age.IT-HPBCD treatment was initiated when the patient was 7 years and 6 months old.At that time, the patient could walk and eat soft foods with assistance; however, her speech was slow and unclear.Therefore, IT-HPBCD was initiated at a dose of 400 mg 20% HPBCD diluted to 6 mL with saline administered through a lumbar puncture every other week.The dose was subsequently increased to 900 mg every other week.

Modified disability scale for NPC
The neurological status of each patient was assessed using a previously-published functional disability scale originally developed by Pineda et al. [15].This scale evaluates the patient's ambulation, manipulation, language, swallowing, seizures, and ocular movements using a three-, four-, or five-point scale.The original scoring system was modified for this study by assigning scores from 1 (best) to 5 (worst) with equal weighting for each parameter.Ambulation was rated across five severity categories from normal (score = 1) to wheelchair-bound (score = 5).The language was rated across five categories from normal (score = 1) to absence of communication (score = 5).Manipulation and swallowing were both rated across four severity categories: normal (score = 1) to severe dystonia/dysmetria (score = 4) for manipulation and normal (score = 1) through nasogastric tube or gastric button feeding (score = 4) for swallowing.Seizures and ocular movements were rated across three severity categories as occasional seizures (score = 1) to seizures resistant to antiepileptic drugs (score = 3) for seizures and slow ocular pursuit (score = 1) to complete ophthalmoplegia (score = 3) for ocular movements.The patients' disabilities were evaluated during diagnosis, treatment initiation, and final clinical contact.

CSF levels of T-tau
The CSF levels of T-tau were measured using an Enzyme linked immunosorbent assay Kit (R&D Systems, Minneapolis, MN, USA) following the manufacturer's protocol.The measured values were used to determine the axonal damage.

Changes in clinical status
Notably, all patients had rapid disease progression despite treatment with miglustat before administering IT-HPBCD (Fig. 1).Transient improvements were observed in Cases 2, 3, and 4, and all patients' symptoms had stabilized at the final follow-up.

Adverse events
Mild-to-moderate hearing loss was observed in Cases 1, 2, and 5.At the last follow-up, the mean hearing threshold was 37 dB in Case 1, 40 dB in Case 2, and 52 dB in Case 5.
An intractable perianal abscess developed in Case 1 at the age of 13 years, accompanied by multiple skin granulomatous lesions in the axilla and buttocks.The patient was diagnosed with Crohn's disease at the age of 16 years.At 17 years of age, she developed bacterial meningitis due to Roseomonas mucosa infection.The patient recovered completely; however, the Ommyer reservoir and the intrathecal baclofen device were removed.The IT-HPBCD therapy was then discontinued.

Magnetic resonance imaging
Diffuse brain atrophy was observed in all patients who underwent MRI after long-term IT-HPBCD treatment (Fig. 2).MRI was not conducted more than one year after initiating therapy in Case 3.

T-tau in the CSF
The CSF levels of T-tau were not correlated with the severity of the neurological status, as the patient with the most severe symptoms (Case 1) had low T-tau levels in the CSF (Fig. 3).However, this may be due to extensive brain damage in this patient, as low levels of creatine kinase are observed in patients with progressed muscular dystrophy.Patients with early-onset NPC had higher levels of T-tau in the CSF than those with adult-onset NPC.
High doses of HBPCD were correlated with high T-tau levels in the CSF during the first four years of treatment in Case 1 (Fig. 3A).The T-tau Cases 3 and 4 had gradual increases in the T-tau levels in the CSF (Fig. 3B).
High T-tau levels in the CSF were observed in Cases 2 and 4 (Fig. 3C).The T-tau levels in the CSF peaked 1-2 years after treatment in Case 5, which correlated with the development of frequent seizures in this patient (Fig. 2D).

Discussion
Several studies have reported the short-term clinical efficacy of IT-HPBCD for patients with NPC [7,12,13].However, only one study on the long-term outcomes of IT-HPBCD treatment has been reported [14].The previous study included three patients with NPC treated with IT-HPBCD for approximately 3 years.These patients exhibited stable or slightly improved NPC scores, consistent with the results of the current study, suggesting that the long-term administration of IT-HPBCD stabilizes disease progression.However, the disease progression was not completely inhibited in any patient in the current study.
There are several possible explanations for the inability of IT-HPBCD to completely inhibit disease progression.The optimizations of the dosage and schedule remain insufficient.The median effective dose of ICV-HPBCD is approximately 0.5 mg/kg in mice with NPC, and the effect of HPBCD with 35 mg/kg ICV-HPBCD lasted one week [16].The CSF HPBCD concentration 15 min and 1h after the IT injection of 200-300 mg (10-13 mg/kg) ranged from 205 μg/mL (0.15 mM) to 2,122 μg/mL (1.53 mM) [7].The optimum HPBCD concentration in the CSF was 0.1 mM in an in vitro study using primary cultures of neurons and glial cells from mouse models of NPC, and it was determined that 10 mM HPBCD is toxic for cells [17].In Case 1, the dose and frequency of ICV-HPBCD were increased to 300 mg twice weekly; however, the dose was subsequently reduced due to increased T-tau levels in the CSF.Therefore, increased doses and schedules may result in HPBCD overdose or toxicity.T-tau levels in CSF may increase for various reasons other than drug toxicity, such as neuronal injury by disease or frequent seizures.Immunological status may also affect neuronal injury in patients with NPC [18,19].Therefore, the data should be carefully interpreted for each patient's clinical circumstances.
The optimal IT dose of HPBCD is estimated as 900-1200 mg, based on the increased plasma concentration of 24(S)-hydroxy cholesterol, mainly derived from neurons following improved cholesterol homeostasis, as reported in a clinical trial [20].Another study reported that biweekly dosing may be more efficacious than monthly dosing [12].This long-term study's results indicated no obvious difference between the monthly and biweekly dosing.However, the clinical efficacy of HPBCD may depend on the patient's neurological status at initiation.Therefore, if NPC had been detected earlier and treated with HPBCD at an earlier stage, the disease progression may have been better controlled.
The shuttle and sink theory has been proposed to explain the potential mechanisms contributing to the efficacy and safety of cyclodextrin [21,22].HPBCD forms a complex with cholesterol at a 1:1 M ratio and a 2:1 M ratio complex at high concentrations.As the 2:1 complex has a higher affinity for cholesterol than the 1:1 complex, the cholesterol is trapped from the cell membrane surface, leading to cell membrane instability and cytotoxicity.HPBCD can form 1:1 and 2:1 complexes with unesterified cholesterol to normalize intracellular cholesterol trafficking, serving as a shuttle.Cellular unesterified cholesterol efflux is associated with cytotoxicity, acting as a sink in NPC model cells.Therefore, it is essential to maintain the optimal concentration of HPBCD to maximize its efficacy and safety.
Long-term IT-HPBCD treatment is associated with hearing loss in the current study.Hearing loss was reported in previous clinical trials after more than 18 months or 2.5-3 years of HPBCD treatment.Mild-tomoderate hearing loss was observed in three patients in the current study; however, the degree of hearing loss did not worsen with longterm treatment.Therefore, careful and regular follow-up of the patient's hearing function is needed during IT-HPBCD treatment.
In this study, Case 1 was complicated by an intractable perianal abscess and multiple granulomatous skin lesions.This patient was diagnosed with Crohn's disease at the age of 16 years.Crohn's disease is common in patients with NPC [23].Schwerd et al. reported 14 patients with NPC with inflammatory bowel disease with a Crohn's disease phenotype and granuloma formation.The previous study reported that NPC1 defects impair the elimination of bacteria in macrophages due to autophagy dysfunction.Furthermore, HPBCD did not enhance antibacterial autophagy.A recent study revealed that NPC1 interacts with STING, a key molecules for the innate immune system, and recruits it to the lysosomes for degeneration.Loss of NPC1 protein causes priming and boosting of STING signaling, resulting in neuroinflammation and degeneration [19].Therefore, HPBCD may not completely restore all NPC1 functions, which may be why the disease progression was not completely inhibited.
This study included only five patients, and the treatment protocol differed for each patient.Notably, all patients had been using miglustat for at least one year before IT-HPBCD treatment; however, using miglustat did not impact the efficacy of IT-HPBCD as all patients had rapid deterioration of their neurological symptoms with miglustat treatment before IT-HPBCD treatment.A previous study reported the annual increases in disability score as 4.97 [95% confidence interval (CI): 3.48-6.46]in patients with infantile onset treated with miglustat for less than one year, 0.78 [95% CI: 0.50-1.06] in patients with juvenile onset treated for less than one year, 0.37 [95% CI: 0.27-0.48] in patients with adult onset treated for less than one year and as 2.06 [95% CI: 1.07-2.43] in patients with infantile onset treated with miglustat for more than one year, 0.34 [95% CI: 0.16-0.51] in patients with juvenile onset treated for more than one year, and 0.37 [95% CI: 0.14-0.61] in patients with adult onset treated for more than one year [24].In this study, the annual increase disability scores during IT-HPBCD treatment were 0.09 in Case 1, 0.125 in Case 2, 0 in Cases 3 and 4, and 0.6 in Case 5.The score of Case 1 was limited by the ceiling effect.This study did not have a control group; however, comparing the natural disease course and miglustat treatment, these data clearly demonstrate the clinical benefits of IT-HPBCD treatment.These results support needs of longterm IT-HPBCD treatment studies in larger patient populations.

Conclusion
Long-term treatment with IT-HPBCD may suppress neurological deterioration in patients with NPC; however, the treatment outcome was dependent on the neurological status at the time of diagnosis, and the disease progression is not completely inhibited.Physicians must have an awareness of the disease, and newborn screening is needed for earlier disease detection.In addition, further optimization of the treatment protocol and additional treatments are needed to improve patient outcomes.