**2.2. Animal studies**

VTS-270 (Kleptose® HPB, Roquette Pharma, France) and Trappsol® Cyclo™ (CTD Holdings, Inc., Alachua, FL) are HP-β-CD products under investigation as novel treatments for NPD-C. Differences between these two products have been studied based on ion distribution and abundance profiles using mass spectrometry methodology as a means to assess key molecular distinctions between products. Trappsol® Cyclo™ was found to have a higher degree of substitution compared with VTS-270, with a greater number of hydroxypropyl groups and increased levels of dimeric ions. Additional differences in ion mobility profiles were found, there is a much greater level of non-specific chemical "noise" associated with Trappsol®

These two products are not chemically equivalent and therefore may not be biochemically equivalent or lead to comparable formulations from a clinical development perspective. The data suggest that biological and potential therapeutic equivalence should not be assumed. Further studies are needed to examine potential differences in biological and therapeutic

The efflux of cholesterol from cells in culture to cyclodextrin acceptors has been reported to be substantially more rapid than efflux induced by other known acceptors of cholesterol. A comparison of the time course of cellular [3H]cholesterol efflux mediated by HDL3 or by various concentrations of cyclodextrins showed the release of 50–90% of L cell [3H]cholesterol after 8 hours of incubation with HP-β-CD and methyl-β-cyclodextrin (M-β-CD) at 10 mM. The order of efficiency in accepting cholesterol was found to be M-β-CD > HP-β-CD > beta-cyclodextrin. The kinetics of the cholesterol efflux time course studies suggested that incubation of L cells with cyclodextrin resulted in the rapid equilibration of labeled cholesterol between

Several studies have shown that cholesterol released from late endosomes/lysosomes of NPCproteins deficient cells by HP-β-CD reaches the cytosolic compartment and is accessible to the endoplasmic reticulum (ER). In cultured cerebellar neurons, astrocytes, and microglia from NPC1-deficient mice, the sequestered cholesterol was mobilized to the ER by low concentra-

In murine models of NPC1, cell culture studies have shown that M-β-CD is more potent in exchanging cholesterol than HP-β-CD. Efficacy comparison of M-β-CD and HP-β-CD in reducing cholesterol accumulation in late endosome/lysosome in human fibroblasts NPC1/ NPC2 deficient after treatment with 300 μM M-β-CD and HP-β-CD for 1 day have shown to reduce the cholesterol accumulation detected by filipin labeling. After 2 and 3 days after treatment, cholesterol accumulation started to increase, but there was still a significant reduction. In concordance with animal studies, M-β-CD produces effects equivalent to those of HP-β-CD

Cyclo™.

effects of Trappsol® Cyclo™ and VTS-270 [11].

**2. Preclinical studies**

98 Cyclodextrin - A Versatile Ingredient

**2.1.** *In vitro* **experience**

cells and medium. [12].

tions (0.1–1.0 mM) of HP-β-CD [13].

The unexpected discovery of the utility of cyclodextrin in NPD-C was observed in a study in Npc1−/− mice treated with a combination therapy of two drugs, one of them (allopregnanolone) formulated in a cyclodextrin complex [15]. A later study showed the same cholesterol intraneuronal storage reduction and longevity increase with the combination of allopregnanolone and cyclodextrin than with the control arm, treated only with the cyclodextrin [16]. These results led researchers to perform studies to address the role of cyclodextrin as a possible treatment. The Npc1−/− mice receiving subcutaneous (SC) or intraperitoneal cyclodextrin every other day for 2 weeks revealed a slight decrease of intraneuronal accumulation of either cholesterol or gangliosides. Both routes showed similar outcome, but SC administration seemed to be a slightly more efficacious [16]. In another study, Npc1−/− mice treated with a single SC injection at 7 days of age of HP-β-CD (4000 mg/kg body weight) prolonged the average life (108 days).

Treatment with cyclodextrin improved hepatic dysfunction and decreased neurodegeneration, increasing the number of Purkinje cells surviving at 49 days of age nearly threefold respect to untreated mice [16]. In previous studies, no significant toxicity was observed following the administration of HP-β-CD except for increased macrophage infiltration of the lungs found at post mortem examination [16, 17]. Other studies in Npc1−/− mice showed that 1500 mg/kg HP-BC-D administered weekly caused a decrease in hepatic unesterified cholesterol concentrations without substantial effect on neurological signs The slight effects of the HP-β-CD on neurological symptoms at low doses may be partially due to their apparent nonpermeation of the blood-brain barrier (BBB) [18].

The efficacy of HP-β-CD was also tested in a feline model of NPD-C. Cats affected with NPD-C were first administered the drug at 3 weeks of age, prior to the onset of clinical signs of disease, and continued to receive the drug weekly. Cats were placed into one of five groups: received no HP-β-CD; received a weekly dose of 1000 mg/kg; 4000 mg/kg; 8000 mg/ kg body weight HP-β-CD subcutaneously; or 4000 mg/kg brain weight (120 mg for a 30 g brain weight) HP-β-CD intrathecally every 2 weeks. The preliminary data suggested a similar requirement for doses equal to or greater than 4000 mg/kg to positively affect neurological disease. Nevertheless, doses of 4000 mg/kg body weight resulted in an increase in hearing threshold only after repeated dosing and doses of 8000 mg/kg body weight resulted in significant increases in hearing threshold in both normal cats and cats with NPD-C following the administration of a single dose [19].

Studies in mouse models have shown that systemic administration of HP-β-CD, starting in early neonatal life, diminishes unesterified cholesterol accumulation in most organs, slows disease progression, and extends lifespan. Studies in adult Npc1−/− mice who received four weekly subcutaneous 4000 mg/kg body weight HP-β-CD at 49 days of age showed reduced whole-liver cholesterol content at 77 days. Comparable improvements were seen in other organs, such as spleen, and lifespan was extended [20]. On the whole, preclinical studies in animals showed that young animals respond more favorably, whereas the older ones may benefit less.

The orphan designation of HP-β-CD was obtained in May 2010 by FDA [28] and 2 months later by the European Medicines Agency (EMA) [29]. The orphan drug brand Trappsol® Cyclo™ was approved specifically for treating NPD-C. New findings suggested that the passage of HP-β-CD across the BBB was limited [21, 24, 25, 30], and physicians who treated the first patients reported slight benefit with IV route [26]. The FDA approved the request for IT delivery of HP-β-CD in September 2010 [31]. Another orphan drug of HP-β-CD was designated by FDA in February 2013, Kleptose™ (brand VTS-270). Both drugs were being evaluated in clinical trials, moreover compassionate use outside clinical trials has been reported

Use of 2-Hydroxypropyl-Beta-Cyclodextrin for Niemann-Pick Type C Disease

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The IV administration was the first route tested for HP-β-CD in humans. When different reports in animals demonstrated that HP-β-CD administered intravenously cross in very little proportion the BBB [21, 24, 25, 30], this route was changed or combined with IT/ICV administration of the same drug. Theoretically, IT and ICV routes deliver directly the drug in the central nervous system (CNS), the area mainly affected in NPD-C, especially in the neurological symptoms. There is still controversy over whether the IV route contributes in NPD-C therapy. Preclinical reports showed effectiveness in the peripheral manifestations of the disease in organs [20, 25], including the liver, the spleen, and, to a lesser extent, the lungs. However, the

influence of HP-β-CD in CNS after IV infusion remains in humans unknown.

**4. Experience in humans: efficacy of the intravenous administration** 

The first two patients who received IV HP-β-CD (Trappsol® Cyclo™) in USA were treated since April 2009 with 2500 mg/kg weekly over 8 hours, and later modified to administrations every 2 weeks for convenience. The twins were diagnosed at 3 years old with initial symptoms of hepatosplenomegaly, ataxia and seizure activity, and previously were treated with miglustat, without significant benefits. After 18 months of therapy, any objective improvement was reported, and the disease continued to progress as evidenced by positron emission tomography (PET) imaging and neurological assessments [26]. Both patients were described a transient appearance of slight scattered nodules in lungs during bronchoscopy, which were identified as xanthomas, a deposition of yellowish cholesterol-rich material typical of NPD-C,

Five different approaches have been employed in HP-β-CD therapy:

• IV at the beginning, and later changed to IT or ICV.

resolved without treatment changes in IV therapy [32].

worldwide [32].

• IV and IT simultaneously.

**of cyclodextrin**

• IV only. • IT only. • ICV only.

Brain uptake of 2-hydroxypropyl-[14 C]-propyl-β-cyclodextrin was determined in Npc+/+ and Npc1−/− mice using two methods: *in situ* brain perfusion and multi-time-point regression analysis flowing intraperitoneal administration. None of the data collected indicated that HP-β-CD enters the brain [21]. Other experiments examining cyclodextrins with regard to permeability using an *in vitro* model of the BBB have indicated that a small percentage of cyclodextrin may be transported across the barrier [22].

Intrathecal (IT) HP-β-CD (120 mg in 0.6 ml saline) every 2 weeks therapy of feline NPD-C delayed the clinical manifestations of neurological disease, but had no effect on hepatic or pulmonary disease. IT-treated cats showed amelioration of neuronal swelling and axonal spheroid formation in many but not all brain regions, and preservation of Purkinje cell numbers [23]. Research in mouse models of NPD-C also has shown that direct administration of HP-β-CD into the IT or intracerebroventricular (ICV) space at low concentrations has a similar or superior effect on delaying the onset of neurological symptoms as that observed following high systemic doses [24].

Recent researches in NPD-C cats showed that direct administration of HP-β-CD into the cisterna magna prevented the onset of cerebellar dysfunction for greater than a year and reduced in Purkinje cell loss and near normal concentrations of cholesterol and sphingolipids. Cats receiving 1000 mg/kg SC HP-β-CD had a similar occurrence of neurological dysfunction and survival than untreated cats. Nevertheless, cats that received 4000 mg/kg SC HP-β-CD showed modest amelioration of neurological disease and survived age than any untreated cats. Pulmonary toxicity limited the continued dosing of cats in the 8000 mg/kg group. Dose-dependent elevations in mean hearing threshold in cats receiving SC HP-β-CD were observed. In cats receiving intracisternal HP-β-CD, a significant elevation in the auditory threshold was also observed. [25].
