2.3. Diagnostic Tools
The distinct heterogeneity of this disease makes it difficult to diagnose. There are several options to diagnose NPC: skin and liver biopsy for filipin staining of cultured fibroblasts; electron microscopic analysis of vacuolation or hepatocytes containing “myelin figures” (
Figure 2)
[27][28][29]; molecular genetic analysis with direct sequencing of
NPC1 and
NPC2 gene mutations
[28]; bone marrow aspiration for the detection of foamy histiocytes
[30][31]; and use of cholesterol esterification assays and oxysterol assay-based screening to measure the increase of cholestane-3β,5α,6β-triol (cholesterol oxidation product, “triol”)
[32][33][34][35]. A possible non-chemical biomarker and treatment control may consist in olfactory testing, since olfactory deficits may mirror the progress of the disease
[36]. However, as yet there are no human data available
[27].
Figure 2. Phenotypes of NPC1 in the endocrine and nervous system. (A) Ovary of an Npc1−/− mouse. The oocyte (O) contains enlarged endoplasmic reticulum (ER) with myelin-like deposits (arrowhead), as does the surrounding follicular epithelial cell (asterisk). Large accumulations are seen in a macrophage (arrows). (B) Neuroendocrine cell in the anterior pituitary. Arrows point at myelin-like inclusions between secretory vesicles. (C) Some alpha motor neurons in the anterior horn of the spinal cord are filled with light material replacing the darker perinuclear Nissl substance of the endoplasmic reticulum. (D) Similar damage is seen in dorsal root ganglion cells (arrows). (F) Corpus callosum: A longitudinal nerve fiber is enlarged and congested by autophagosome content (asterisks) that interrupts the continuity of neurofilaments and neurotubuli. The myelin sheath (arrows) has also thinned and disintegrated. R, node of Ranvier.
2.4. Therapies
So far, there is no causal therapy of NPC1, though the iminosugar miglustat (Zavesca
®) is the only approved drug in Europe used for supporting and symptomatic therapy in NPC1
[37]. Miglustat is a small molecule that inhibits glycosylceramid synthase, one of the key components of the glycosphingolipid biosynthesis, therefore reducing intracellular lipid storage
[38]. Long-term therapy with miglustat has been shown to increase lifespan and stabilize neurological functions. Additionally, miglustat has been ascribed activity against oxidative stress
[39]. However, limitations consist in mainly gastrointestinal side effects such as diarrhea, weight decrease, and flatulence, but also tremor
[40]. A further promising drug, 2-hydroxypropyl-β-cyclodextrin (HPβCD)—a cyclic oligosaccharide—is used as an enabling excipient in pharmaceutical formulations, as well as a cholesterol modifier in vivo. Therapy results in delayed onset of neurological symptoms with increased lifespan
[37][38][41]. Matsuo et al.
[42] reported in a clinical trial that HPβCD was effective in NPC1 patients, suggesting that HPβCD is a promising drug candidate in NPC1 disease. HPβCD overcomes the transport defect leading to excretion of accumulated cholesterol as bile acid, as shown in
Npc1−/− mice
[43]. It has been suggested that cholesterol efflux is mediated by the ATP binding cassette subfamily G member 1 (ABCG1), which promotes biliary excretion of sterols, ameliorating liver function
[43][44]. Unfortunately, HPβCD administration also has side effects, particularly on the survival of outer hair cells, leading to hearing loss. This major side effect occurs in a dose- and duration-dependent manner
[45][46]. What is more, in an open-label, dose-escalation phase 1–2a study, promising effects of HPβCD were recorded
[47]; however, preliminary results of a current multinational phase 2b/3 clinical study involving about 50 patients treated with 200 mg/kg intrathecally applied HPβCD every 2 weeks indicate doubts that HPβCD achieves benefits when compared to a placebo
[48][49][50].
Another promising therapy, so far applied only in animal models, consists of a combination of miglustat, the neurosteroid allopregnanolone, and HPβCD
[13][51][52][53], resulting in further prevention of cerebellar Purkinje cell loss, improved motor function, reduced intracellular lipid storage, and prolonged life span in
Npc1−/− mice.
Another therapeutic approach showed that the activity of the liver X receptor β (LXR β) can regulate the cholesterol flux from the brain, which leads to a reduction of neuroinflammation and slows therefore the neurodegeneration process. However, these positive effects result only in a modest lifespan prolongation
[54][55]. Nevertheless, LXR β activation by treatment with an LXR agonist (T1317) can be useful in combination, e. g., with HPβCD.
In the absence of a causal treatment, there is still, a need to identify novel treatment strategies. Currently, histone deacetylase inhibitors (HDACi) are a focus of interest, due to the findings that they can reduce cholesterol accumulation in LE/LY
[55][56][57][58]. These enzymes mediate posttranslational deacetylation of many types of proteins, e.g., histones, transcription factors, and chaperones
[59]. In spite of its interaction with many different proteins and signaling pathways, it has been shown that HDACi increases expression of the low-activity mutant NPC1 protein
[56][57], at least in vitro.
A further treatment option is FTY720 (fingolimod), a sphingosin analog. This drug is already approved for human use to treat multiple sclerosis
[60]. FTY720 can enter the cell nucleus, where it is phosphorylated by sphingosine kinase 2 (SphK2). This active form is an inhibitor of class I histone deacetylases. The advantage of this drug over available HDACi is to regulate the expression of only a limited number of genes, which are restricted to cholesterol and sphingolipid metabolism, compared with the large number (thousands of genes
[61]), which are activated by HDACi
[60].
Another treatment approach is the application of arimoclomol, a coinducer of heat shock protein 70 (HSP 70) that improves the binding of several sphingolipid-degrading enzymes to their essential cofactor bis(monoacyl)glycerophosphate in vitro
[62][63]. Beneficial effects for NPC patients have also been observed with drugs such as ursodeoxycholic acid
[64][65] and acetyl-DL-leucine
[66].
Moreover, an increased level of functional NPC1 can be achieved using gene therapy
[67]. In some studies, it has been shown that the adeno-associated virus (AAV) 9 vector may successfully transfer the
NPC1 gene into the CNS of
Npc1−/− mice
[67][68][69]. Systemic delivery of a functional
NPC1 gene into
Npc1−/− mice significantly extends the lifespan, ameliorates neurodegeneration, and improves behavioral abnormalities
[67][68][69].