Treatment Of Niemann Pick C Using Miglustat Biology Essay

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Niemann-Pick diseases are an autosomal recessive form, which results in a build up of toxic materials by either enzyme deficiency or gene mutation. So far, there are limited treatments for it regardless of the extensive research conducted on how to control this disease, due to its unclear pathophysiology. This review will give a brief outline on the disease, its pathology and the different treatment options available, focussing ultimately on the drug, miglustat.

Type C affects the brain and many organs in the body and results in a progression of neurological dysfunction. Patients that are found to have neurological dysfunction at an early age have a more "aggressive form" of the disease but the progression of the disease varies for each patient. 1 in 120,000 births have been diagnosed with this disease, especially in Western Europe, Australia, Netherlands and Nova Scotia (Vanier, 2010). To diagnose type C, skin biopsy is performed. Fibroblasts are stained using filipin and viewed under ultraviolet light to observe the cholesterol storage in the cells. Recently, breakthrough research has shown elevated cholesterol oxidation products are found in the blood in Niemann-Pick C infected mice. From this it can be suggested that a simple blood test can be done to detect NPC disease earlier in children (Porter et al., 2010). If this test gets approved, it will no doubt prove to be an invaluable tool in the diagnosis of NPC.

Type D found to be an "allelic variant" of type C and has a similar phenotype to type C. It is most prevalent in French Arcadian population in Nova Scotia, which is where the name is derived from. Like type C, it is a result of gene mutation and results in cholesterol and sphingomyelin accumulation (Greer et al., 1998).

Type E is the rarest form of all Niemann-Pick diseases and the least has been investigated about it but it mainly affects adults (Kaneshiro, 2009).

NPC disease is responsible for 4% of lysosomal storage disorders. It can manifest from a very early age, with a median age of 9.3 years (Meikle, 1999) and can last till the patient is in their seventh decade of life (Vanier, 2010), with the main signs and symptoms occurring during adolescence. Typically, a patient with NPC will have progressive neurological dysfunction in addition to hepatosplenomegaly (enlargement of the liver and spleen).

Figure : An overview of the whole process involved in cholesterol trafficking. The malfunctioning of the receptors, NPC 1 and NPC 2, interrupts the cholesterol trafficking to TGN and hence no lipid raft is formed. Abbreviations used: LDL = Low density lipoprotein, PM = Plasma membrane, EE = early endosome, NPC 1 = Niemann-Pick C receptor type 1, NPC 2 = Niemann-Pick C receptor type 2, LE/LYS = late endosome/lysosomes, TGN = trans Golgi network, ER = endoplasmic reticulum, SREBP = sterol regulatory element-binding protein, SCAP = sterol cleavage-activating protein, ACAT1 = acyl-cholesterol acyltransferase isoenzyme type 1, CEH = cholesteryl ester hydrolase [Source: (Urano et al., 2008)]Niemann-Pick C disease occurs due to mutation of two genes, NPC 1 and NPC 2, which results in intracellular accumulation cholesterol in lysosomes and leads to impaired membranes as illustrated in Figure 1. 95% of patients experience NPC 1 gene mutation and 5% experiencing the other (Imrie et al., 2006).

1.2 NPC 1 and NPC 2 receptor function:

NPC 1 receptor is part of the membrane in vesicles, late endosomes and also cycles the trans-Golgi network. In mice and humans examined, NPC 1 has been found mainly in the liver controlled by sterol metabolism. Its function is to detect cholesterol levels. However, gene mutation on chromosome 18q11-q12, means that it loses its ability to detect if levels of cholesterol increase, resulting in the endoplasmic reticulum not being able to sense it and so continues to overproduce cholesterol (Frolov, 2003). It has been suggested that a sterol-binding domain is required to bind to cholesterol however there are limitations to the finding because it is yet to discover whether the specificity of sterol binding and the nature of sterol binding to the receptor is direct or indirect (Karten et al., 2009).

NPC 2 receptor is a soluble lysosomal glycoprotein which contains a hydrophobic pocket available for binding to cholesterol with high affinity. It can also bind to fatty acids but with a lower affinity. Gene mutation, mapped on chromosome 14q24.3, in HE1 protein led to NPC 2 disease by inhibiting the binding and possible transporting mechanism (Naureckiene, 2000). NPC 2 receptors are bound to mannose-6-phosphate receptors (MPRs). The function of MPRs is to transport mannose-6-phosphate from trans-Golgi network to late endosome-lysosome complex for hydrolysis of materials inside the cells. The receptors are recycled back to trans-Golgi network to ferry more mannose-6-phosphate (Grosshans et al., 2006).

The mode of action in which these two receptors can act synergistically is yet to be confirmed but there are two models suggested. One is that NPC 1 binds to cholesterol in the late endosome-lysosome complex membrane. Then the soluble NPC 2 receptors obtain cholesterol from NPC 1 and transfer it to the outer membrane for the cholesterol to be redistributed by an unknown mechanism. The second model is that NPC 2 extracts cholesterol from the inside of the late endosome-lysosome complex and passes it to NPC 1 to be transferred out of the cell (Karten et al., 2009). The second model is more feasible as NPC 2 receptors are more mobile and have a higher affinity to bind to cholesterol. NPC 1 receptors are integrated in the membrane so it would need cholesterol transported to them in some way before binding to them.


2.1 Main organelles and cholesterol trafficking:

The main organelles involved in this disease are late endosome-lysosome complex, Golgi network and endoplasmic reticulum. Gene mutations lead to an improper trafficking of unesterified cholesterol from late endosome-lysosome complex. Unesterified cholesterol is found to be the cause of peripheral manifestations, for example hepatomegaly. Glycolipids are storage compartments in the brain and cause central manifestations, for example neurological dysfunction.

Cholesterol is an essential part of cell membranes hence it is necessary to ensure that it can be made available to the body as and when it is required. In normal cells, cholesterol enters a cell bound to low density lipoprotein (LDL) which binds to LDL receptor on the cell membrane. To obtain the free cholesterol from LDL, the LDL receptors need to be metabolised and recycled as illustrated in Figure 2 (Kantharaj, 2006).

Figure : Model of how LDL receptors enter a cell via endocytosis. The coated LDL receptor-cholesterol vesicle fuses into the early endosomes where cholesterol is cleaved and passed through to lysosomes. The LDL receptors are then transported back to plasma membrane to await more cholesterol. Abbreviations used: LDL- low-density lipoprotein. [Source: (Kantharaj, 2006)]

Usually, cholesterol is hydrolysed in early endosomes in acidic conditions. This cholesterol is then transferred as the early endosomes mature into late endosomes and lysosomes. From this, cholesterol is then transported to different destinations including plasma membrane, endoplasmic reticulum and Golgi network. In the endoplasmic reticulum, the LDL-derived cholesterol is re-esterified by acyl-CoA: cholesterol acyltransferase (ACAT) enzymes. There are two main types of these: ACAT-1 and ACAT-2. Majority of the cells in the body will have ACAT-1, whereas the ACAT-2 enzymes are found mainly in the intestinal enterocytes in the gut (Urano et al., 2008). However in NPC disease this cholesterol cannot reach endoplasmic reticulum and Golgi body. Hence sterol synthesis remains unregulated resulting in cholesterol accumulation inside the cell (Xie et al., 1999). Figure 2 illustrates how cholesterol is trafficked to other organelles in most cells, including liver and spleen. Figure 3 shows how glycosphingolipids (GSLs) can be transported in the brain.

Figure : Model of how GSLs are internalised into early endosomes which then convert to late endosomes. Late endosomes communicate with the Golgi complex via rab9 route and release cholesterol along with other chemicals. Once all the contents are trafficked to the Golgi, the cell converts itself into a lysosome via rab7 route. Abbreviations used: GSLs = glycosphingolipids;

SLSD = sphingolipid storage disease [Source: (Marks & Pagano, 2002)]

Studies have shown how to distinguish between an early and a late endosome. Early endosomes are formed once vesicles containing GSLs lose their clathrin coat and fuse together. Early endosomes have a pH of 6.0 to allow receptors to be released and recycled. They also have rab5 sorting signal attached to guanine diphosphate (GDP) which is responsible for the fusion on vesicles. For an early endosome to convert to a late endosome it must lower its pH to around pH 5.0 and its receptors change from rab5 to rab7 but it is still associated with GDP. Rab7-GDP and communication to the Golgi apparatus is essential for cholesterol release. Late endosomes are particularly distinguished by the presence of lysobisphosphatidic acid (LBPA): a lipid molecule. Late endosomes then turn into lysosomes once all the material has been released to the Golgi. Lysosomes are filled with lysosomal associated membrane proteins (LAMP) and acid hydrolases. Lysosomes are vital for survival as they help to regulate trafficking of critical nutrients, for example cholesterol. They also regulate cellular stores of different molecules by enzyme degradation. Hence when lysosomes are defected, they can cause fatal diseases such as Gaucher's disease, Tay-Sach's, Sandhoff's disease and Niemann-Pick diseases (Meikle, 1999).

2.2: Function of rab proteins in NPC:

As mentioned above, rab proteins play a very important role in all aspects of membrane trafficking. They are small GTPase molecules and different rab proteins are found in different organelles. The table below has a brief introduction of some of the rab proteins involved in the body and their main localisation. For the purposes of this article, only rab7 and rab9 will be focused on as they play a role in cholesterol transport.

Table 1: Intracellular compartmentalisation of some rab proteins [Source: (Open University, n.d.)]

Type of rab

Cellular Compartment


ER and Golgi network


cis Golgi network


medial and trans Golgi network


clathrin-coated vesicles

Rab4 and Rab5C

early endosomes


late endosomes


late endosomes


basolateral secretory vesicles


synaptic and secretory vesicles

Rab7 is especially important. It localises itself in acidic organelles such as late endosomes and lysosomes. It is also involved in maturation of late endosomes from early endosomes (McCray et al., 2009) as illustrated in Figure 2. Membrane cholesterol may have a part in regulating rab7 function as there is an association made between cholesterol and endosomes motility. Hence increased cholesterol levels alter membrane bilayer and increases membrane-bound rab7. It does this by inhibiting the cycle between active GTP-bound and inactive GDP-bound forms of GTPases, rab7. In NPC, cholesterol accumulation has been thought to inhibit guanine diphosphate factor's (GDF) ability to cleave rab7 from guanine diphosphate disassociation inhibitor, hence reducing rab7 expression on the membrane (Cataldo & Nixon, 2007).!via/oucontent/course/484/s377book3chapter12_f025hi.jpg

Figure : Model of how Rab protein is cleaved from membrane in a normal functioning cell. Rab-GDP is present in the cytosol, bound to a GDI. GDF displaces the inhibitor and GEF activates Rab, exposing the prenyl group, which attaches to the membrane. Rab-GTP then binds to Rab effector proteins, which recruit a specific set of proteins to the cluster. Abbreviations used:

GDP: Guanine diphosphate; GDI: guanine diphosphate dissocation inhibitor; GTP: guanine triphosphate [Source: (Open University, n.d.)]

Its function is highly sensitive to cholesterol fluctuation and hence accumulation of cholesterol affects them too. For instance, cholesterol stabilises rab9 on the membrane (Ganley, 2006). One study showed that overexpression of rab proteins actually could be used to stop the progression of the disease. In NPC infected cells, they injected wild type constructs of rab7 and rab9 and this resulted in an improved Golgi targeting network and also had a significant decrease in intracellular accumulation of cholesterol. Perhaps this could be used in the future to investigate further therapeutic approaches for treating this condition (Choudhury et al., 2002).

2.3: Neurological pathology of NPC:

In the brain, it has been found that due to the gene mutations of NPC 1 and NPC 2, the neurons produced are faulty. There is formation of meganeurites, displaced dendrites, and axonal spheroids. As the disease progresses, the neurological dysfunction increases. This is found to be due to patterned loss of Purkinje cells. These cells are found in the cerebellum, which plays a prominent role in movement of the body. Purkinje cells are the only source of carrying signals out of the cerebellum hence any loss of function in this area can lead to serious complications. It is yet to be confirmed whether it is due to cell dysfunction or cell death however there are several theories related to degradation of Purkinje cells, with the majority regarding the accumulation of cholesterol and other lipids that can alter membrane rigidity and cause the cells to malfunction.

Another theory is that NPC 1 function loss results in the release of pro-inflammatory cytokines that cause cell degradation. Even though this theory is unclear, anti-inflammatory agents have been used in treatment of NPC. Microglia cells have been found in abundance in Purkinje cells in NPC infected mice, this is indicative of an inflammatory response in neurological cells (Ko et al., 2005).

It has been argued if neurofibrillary tangles are associated with NPC or not. One study found no incidence of neurofibrillary tangles growing in NPC (Zhang et al., 2008) but a few years later, it was found that they are present in NPC. They play a role in deregulating cholesterol metabolism and these tangles start to grow (Walkley & Suzuki, 2004).

There is also defragmentation of the Golgi apparatus which leads to major problems in the rest of the body. One of the initial signs of NPC is reduced lysosomal calcium levels and this could be a therapeutic target in treating this disease (Lloyd-Evans et al., 2008). Lysosomal sphingosine storage leads to depletion in calcium levels inside the organelle. This leads to cholesterol, sphingomyelin and glycosphingolipids storage.

The neurodegenerative aspect of this disorder seems to share some similarities with other neurological disorders, for example Alzheimer's Disease (AD). One main aspect of this is the build up of amyloid plaques. It has been already been proven that amyloid plaques building up is a cause of AD but now research into NPC has shown a similar property, but this is also associated with abnormal binding to gangliosides namely GM2 and GM3. GM2 and GM3 levels are significantly increased in brains of NPC patients however the levels are not elevated in foetal brains. The main amyloid substances indicated to aggregate are C99, Aβ40, and Aβ42, with Aβ42 being of most concern as it is more toxic and aggregates easily (Jin et al., 2004).

As illustrated in Figure 5, there is accumulation of cholesterol which can be seen under microscope using filipin staining. Different parts of mice brain have been stained and they have different levels of cholesterol accumulation.

Figure : Detection of neuronal cholesterol accumulation by filipin staining. Coronal brain sections (5 μm) taken from sibling WT (NPC1+/+) and NPC (NPC1−/−) mice perfuse-fixed with 4% paraformaldehyde were stained with filipin (blue) and neurotrace (green) and examined under confocal microscopy. Filipin stains intracellular cholesterol-rich domain(s), indicated by arrows; neurotrace stains neuronal perikarya, indicated by asterisks (*). Images shown are high-magnification images obtained from WT and NPC brains at postnatal days 9 and 22. Abbreviations used: CBL, cerebellum; CTX, cortex; TH, thalamus; HC, hippocampus CA3 region; DG, dentate gyrus. WT: wild type [Source: (Reid, 2003)]

Treatment of NPC:

There is no cure for NPC however, treatment aims to delay disease progression and improve quality of life. There are limited treatment regimes available at the moment for NPC.

3.1 Curcumin and beta-cyclodextrin

One of the therapeutic targets could be aimed to elevate calcium levels, curcumin was used to do this and it worked successfully and increased the life expectancy of NPC-infected mice by 35% and delayed progression by 3 weeks (Lloyd-Evans et al., 2008). However there needs to be research done in clinical trials to prove its efficacy in human models.

Another treatment which has been found beneficial is using β-cyclodextrins (βCD). They help to delay disease progression in mice model. Their mode of action is still unknown but there are several theories to support how βCD depletes cholesterol storage inside the cell membrane. βCD are hydrophobic molecules which rules out its action inside the brain as it does not cross the blood brain barrier. It has been argued that exposing cells to βCD alters the relative distribution of cholesterol between intracellular compartments and plasma membrane. Cholesterol is a component of membrane rafts that "compartmentalise cellular processes". βCD has been shown to extract cholesterol from these rafts (Zidovetzki & Levitan, 2007).

In NPC-infected mice, a study showed that short term administration of cyclodextrin reduced intracellular storage of cholesterol and glycosphingolipids. There was little or no accumulation of cholesterol in cerebral cortex and diminished accumulation of gangliosides which supports its use in NPC (Davidson et al., 2009). Discovering this treatment may help some patients however the lack of neurological action renders it unsuitable in young children.

3.2 Problems to overcome - formulation issue:

One of the biggest issues for finding a treatment for NPC is that it is difficult to develop drugs with particle sizes that are hydrophobic and small enough to cross the robust blood brain barrier. There has been research done on non specific therapy to try and overcome the issue. These include bone marrow transplant and haematopoietic stem cell transplantation. Unfortunately, these methods are more commonly found as part of treatment for Types A and B of this disease with a smaller portion for Type D.

3.2.1 Receptor access

NPC 1 and NPC 2 are structurally different and perhaps this is why the treatment has to be tailored, depending on which mutation is responsible for the dysfunction of lysosomes. NPC 1 protein is imbedded in the membrane, therefore harder to treat than NPC 2. There have been case studies to observe the efficacy of bone marrow transplants in animal models. It was shown to only partially reduce cholesterol accumulation barring any effects on neurological progression. Similar effect was found in a patient where they performed a liver transplant but again, this proved to be insufficient to stop the neurological advancement (Patterson et al., 2001).

NPC 2 receptor is a soluble protein and it can be envisaged to treat this using stem cell transplantation. One study performed in NPC 2 infected neonates found that haematopoietic stem cell transplantation was successful however there is still a risk as the long term effects have not been investigated (Verot et al., 2007).

3.2.2 Alternative Treatments

Another approach to treatment is manipulating rab proteins. As mentioned above, over-expression of rab proteins can help to delay disease progression. It has been found that rab7 and rab9 overexpression can reduce cholesterol accumulation and can be a therapy for NPC however the research is inconclusive of how effective this actually is in NPC (Choudhury et al., 2002).

Other types of treatment have been proposed to act on the symptoms of NPC rather than actually depleting cholesterol or glycosphingolipids. These include cholesterol lowering agents; anti-inflammatory agents; anti-epileptic drugs and anti-cholinergic drugs. They have proved to be of some use in adjunctive therapy in lysosomal storage disorders. (Patterson et al., 2001; Schiffmann, 2010).

3.3 Miglustat

Substrate reduction therapy is another way of reducing accumulation of metabolic subunits known to be found in storage disorders. Miglustat (also known as N-butyldeoxynojirimycin [NB-DNJ]) acts by inhibiting glycosylceramide synthase, which is the first enzyme step of the formation of glycosphingolipids, and in turn reducing accumulation of glycosphingolipids (see Figure 6). This delayed clinical onset of symptoms and increased lifespan in NPC infected mice by 30% (Zervas et al., 2001).

Figure : Biosynthesis of glycosphingolipids (GSLs) in humans.

The first step in the GSL biosynthetic pathway: the transfer of glucose [in the form of uridine diphosphate glucose (UPD-glucose)] to ceramide (N-acyl sphingosine). This step is catalysed by the enzyme ceramide-specific glucosyltransferase, which is inhibited by the imino sugars N-butyldeoxynojirimycin (NB-DNJ) also known as miglustat. [Source: (Platt & Butters, 2004)]

In 1999, it was found that miglustat helped to reduce the abnormal storage of glycosphingolipids, GM2, in a Tay-Sach's infected mouse (Liu et al., 1999). Since there is accumulation of GM2 in NPC too, then theoretically, it can be used as a therapeutic agent for NPC. Miglustat may also modulate intracellular calcium levels that end to increase in NPC disease. It also reduces the potentially neurotoxic accumulation of gangliosides GM2 and GM3, lactosylceramide and glucosylceramide (Wraith & Imrie, 2009). A recent study followed the patients' progress over 12 months and showed that miglustat could stabilise neurological progression in adults and juvenile adults (Wraith et al., 2010).

Currently, miglustat has been approved by the FDA and is now the choice of drug to give in this case. There is a strict protocol to be followed that has been discussed and agreed upon by a panel of experts in the field as shown in Figure 7. The dosing has to be individualised based on body surface area and the safety and tolerability of each patient to be monitored. Generally, the drug is well tolerated by all ages with the most common side effects being mild to moderate diarrhoea, flatulence and weight loss. It is however contraindicated in pregnant women as it can harm the foetus and in male patients as there is contradicting evidence of how miglustat affects male fertility. It is also found to be of little use in infants with accelerating neurological dysfunction and dementia. (Wraith & Imrie, 2009)

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Figure : Protocol for starting patient on miglustat as agreed upon by experts. aBiochemical and/or molecular-genetic diagnosis, with or without systemic or other clinical signs and symptoms; bPatients asymptomatic or with isolated splenomegaly, and with one or more older siblings in whom the time of neurological symptom onset and rate of progression are known. [Source: (Wraith et al., 2009)]


Miglustat is a small iminosugar compound that can cross the blood-brain barrier so it is thought to be the best choice of drug in the market. It has multiple indications including Gaucher's disease and it is currently under review for its efficacy in cystic fibrosis. It has only recently been approved as treatment for NPC surpassing cyclodextrin injections and other adjunctive therapy. More research has to be conducted to fill in the gaps regarding fertility in males and perhaps finding alternative treatment for pregnant women. I will be looking at the efficacy of miglustat to support or reject previous trials and research.