Genetic Targets and Applications of Iron Chelators for Neurodegeneration with Brain Iron Accumulation

Neurodegeneration with brain iron accumulation (NBIA) is a group of neurodegenerative diseases that are typically caused by a monogenetic mutation, leading to development of disordered movement symptoms such as dystonia, hyperreflexia, etc. Brain iron accumulation can be diagnosed through MRI imaging and is hypothesized to be the cause of oxidative stress, leading to the degeneration of brain tissue. There are four main types of NBIA: pantothenate kinase-associated neurodegeneration (PKAN), PLA2G6-associated neurodegeneration (PLAN), mitochondrial membrane protein-associated neurodegeneration (MKAN), and beta-propeller protein-associated neurodegeneration (BPAN). There are no causative therapies for these diseases, but iron chelators have been shown to have potential toward treating NBIA. Three chelators are investigated in this Review: deferoxamine (DFO), desferasirox (DFS), and deferiprone (DFP). DFO has been investigated to treat neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD); however, dose-related toxicity in these studies, as well as in PKAN studies, have shown that the drug still requires more development before it can be applied toward NBIA cases. Iron chelation therapies other than the ones currently in clinical use have not yet reached clinical studies, but they may possess characteristics that would allow them to access the brain in ways that current chelators cannot. Intranasal formulations are an attractive dosage form to study for chelation therapy, as this method of delivery can bypass the blood-brain barrier and access the CNS. Gene therapy differs from iron chelation therapy as it is a causal treatment of the disease, whereas iron chelators only target the disease progression of NBIA. Because the pathophysiology of NBIA diseases is still unclear, future courses of action should be focused on causative treatment; however, iron chelation therapy is the current best course of action.


■ INTRODUCTION
Neurodegeneration with brain iron accumulation (NBIA) is an overall term to describe a group of rare neurodegenerative diseases that are inherited through a variety of genes that result in the presentation of iron overload in the brain.NBIA is characterized by extrapyramidal movement disorders and abnormal iron accumulation in the deep basal ganglia nuclei. 1 The cumulative term for NBIA diseases comes from the genes that are responsible for the iron overload in the brain, but the exact mechanism for this manner of neurodegeneration is not completely understood. 2Nomenclature for NBIA diseases has generally been regulated with the "(mutant protein)-associated neurodegeneration" convention. 1 NBIA can be classified as an orphan disease, affecting 1−3/1,000,000 in a population, with autosomal dominant, autosomal recessive, or X-linked dominant inheritance. 3Treatments for NBIA that have been developed have been symptomatic in nature; there are no causal therapies targeting iron accumulation.Iron chelation therapy is a potential symptomatic therapy.Iron chelating agents have been proven to induce iron excretion and a negative iron balance in patients with thalassemia major and hemochromatosis. 4These therapies have potential to be used in cases of NBIA and have been used with some benefits to mitigate brain iron accumulation.In this paper, we will review the mechanisms of NBIA and the role of iron in neurological function, the main genetic targets of NBIA and the use of iron chelators to treat this disease, and whether iron chelation is a viable therapy option to treat these diseases.We will also discuss some other potential new therapies that can be applied as well.
■ MECHANISMS OF NBIA Metals such as iron, zinc, and copper are vital to the function of multiple biological processes in the brain including nerve transmission, the synthesis and metabolism of neurotransmitters, and oxygen transport. 5Iron is present in dopaminergic neurons, where it is vital in dopamine synthesis and electron transport chain production. 4MECHANISM OF IRON TOXICITY Most iron in the body is related to the heme moiety of hemoglobin and myoglobin transport proteins. 6Iron plays a vital role in oxygen transport through the blood, in electron transport within cells, and as a cofactor for various enzymes in the body. 6However, an overabundance of this metal can lead to extensive damage of cells and organs.Oxidative stress is a deviation from the steady state redox balance of the body's metabolic system, or redox homeostasis. 7Transition metals such as iron can induce oxidative stress by reacting with hydrogen peroxide to produce hydroxyl radicals in a process known as the Fenton reaction, seen below. 8 The Fenton reaction plays a role in a larger mechanism, the Haber-Weiss reaction, in which iron plays the role of catalyst; as the reaction is thermodynamically unfavorable in biological systems, the presence of iron is what allows for the reaction to proceed. 8Below are the steps of the Haber Weiss reaction.
Net equation: The reaction indicates a direct relationship between the redoxactive iron and the hydroxyl radical, which is incredibly reactive and therefore very dangerous. 9The hydroxyl ion is an example of a reactive oxygen species (ROS) that is produced by iron, which can damage lipids and proteins. 10Iron accumulation leading to an increase in the labile iron pool (LIP) is therefore known to be associated with tissue damage due to oxidative stress. 11The LIP is characterized as a deposit of excess chelatable and redox-active iron in both its ferric and ferrous forms and could potentially participate in redox cycling, which refers to the process of iron being reduced and oxidized to form the hydroxyl radical. 12,13Excess iron that enters the LIP can react with the byproducts of cellular respiration, such as hydrogen peroxide, to produce ROS that are responsible for cellular damage. 14Some examples of the ways in which ROS can damage a cell are by inducing apoptosis, autophagia, necroptosis, and ferroptosis. 15nother detrimental consequence of the production of ROS is lipid peroxidation.The reaction takes place in three steps: initiation, propagation, and termination.The radical ROS initiates the reaction by reacting with a polyunsaturated fatty acid (PUFA) to create a lipid radical, which then reacts with molecular oxygen to form a reactive lipid peroxide radical LOO • and terminates following a hydrogen transfer from a neighboring lipid to create LOOH, a lipid peroxide. 16Lipid peroxidation alters physical properties of lipid bilayers, thus affecting membrane assembly, structure, and dynamics, and lipid peroxide radicals can further propagate by contributing to the production of variety of ROS. 17 Due to its high lipid content, the brain is most susceptible to peroxidative damage, which directly correlates to regional iron concentration. 18

Homeostasis of Iron in the Body
Regulation of iron in the body is important because there is no efficient physiological pathway to excrete iron from the body. 19erritin, an iron-storage protein found in cells throughout the body, is a key factor in iron homeostasis due to its capability to store excess iron and release it as needed. 20Dietary iron enters the body and is transported in two forms: heme iron and nonheme iron.Heme iron is present in hemoproteins such as myoglobin (stored in muscle cells) or hemoglobin (stored in red blood cells), whereas nonheme iron is directly transported in the blood by transferrin. 21The absorption of dietary iron can only occur in the form of heme iron or when the iron atom is in the ferrous (Fe 2+ ) state.More specifically, nonheme iron must be transformed from its insoluble ferric (Fe 3+ ) form into ferrous iron by specialized enzymes on the brush border of enterocytes before it can be transported across membranes by an iron export protein known as ferroportin 1 (FPN1). 22An important transport protein present in the intestinal epithelium is the divalent metal transporter 1 (DMT1), which transports ferrous iron across the apical membrane of the brush border, whereas FPN1 is located on the basolateral membrane of the cell. 23It is these epithelial enterocytes and their transmembrane proteins transporters that line the villi in the gastroduodenal junction that are responsible for controlling heme and nonheme iron absorption in the body, where it then passes through the gut lumen into the plasma. 19ven after it has been absorbed, iron transport must be strictly managed by a multitude of molecules to maintain homeostasis.Iron regulation in the body is controlled by hepcidin, a peptide produced in the liver, which binds to FPN1 to be internalized and degraded, inhibiting the excessive release of iron into the blood circulation. 24Transferrin, a liver protein able to bind two ferric ions, can safely circulate regulated iron to most cells in the body, including neurons, without causing any ROS production. 24This process is facilitated by hephaestin, a membrane-bound ferroxidase (iron oxidizing enzyme), which oxidizes Fe 2+ back to Fe 3+ prior to transport by transferrin. 25Transferrin binds to the transferrin receptors TfR1 and TfR2, which have slightly different cellular uptake pathways that result in varied phenotypic presentations in cells: TfR1 downregulation results in low iron levels in the tissues, while TfR2 downregulation can result in hemochromatosis. 26−29

Iron Regulation in the Brain
The brain is different from other organs in the body because of the vascular barrier that it lies behind, which regulates the physical material exchange between blood and fluids and the brain tissue; because of this, iron cannot be directly absorbed into the brain from systemic circulation. 30Iron uptake into the brain is regulated by the blood-brain barrier (BBB), made up of cerebrovascular endothelial cells, that controls the passage of large molecules across the membrane in a multistep transcellular process. 30Systemic circulation is separated by the BBB from the central nervous system (CNS), which does have the branching capillaries of systemic circulation that allow for passage of drugs and nutrients. 31Studies on oligodendrocytes, glial cells of the CNS that produce myelin, found that iron is integral for their function. 32Traces of ferritin, transferrin and iron were found in these cells, and a direct correlation between iron acquisition and myelin production was discovered. 33he choroid plexus is a network of capillaries in the brain that has many functions, with one of them being the production of cerebrospinal fluid (CSF) and separating the brain from the bloodstream. 34The capillaries of the choroid plexus extend into the four ventricles of the brain and regulate the drugs and nutrients that pass through. 31A wide range of transporters are expressed in the choroid plexus, such as Menke's and Wilson's metal transporters and DMT1, that could play a role in iron regulation in the brain along with the BBB. 35fter iron attached to holo-transferrin, or bound-transferrin, enters the brain, whether is it through the choroid plexus or the BBB, it travels into the ventricles via interstitial fluid or cerebrospinal fluid (CSF) and binds to surface cellular transferrin receptor 1 (TFRC); the holo-transferrin is then endocytosed into endothelial cells in the CNS. 25 Astrocyte end-feet that sheath the endothelial cells play an important role in the maintenance of the BBB and the regulation of iron transport across the membrane by regulating the hydrogen ion concentration, which is directly correlated with the release of iron in the endothelial cells. 36The clearance of iron from the CNS is debated, but one potential pathway is through CSF drainage; iron efflux may be related to the iron concentration gradient in the CSF and interstitial fluid. 37

■ GENETIC TARGETS Pantothenate Kinase-Associated Neurodegeneration (PKAN)
There are many various types of NBIA that can be differentiated by the mutated genes that cause the disorder as well as magnetic resonance imaging (MRI).The most prevalent form of NBIA, pantothenate kinase-associated neurodegeneration (PKAN), also known as NBIA1, makes up ∼35−50% of the NBIA patient population. 38PKAN is caused by the gene PANK2 located on chromosome 20p12.3and presents in two variations: typical and atypical PKAN. 39ANK2 is an autosomal recessive gene that causes an error in the phosphorylation of Vitamin B 5 , also known as pantothenate, which is a micronutrient vital in the production of coenzyme A (CoA) in the mitochondria. 40Pantothenate kinase is the enzyme that catalyzed the reaction of pantothenate into CoA, allowing it to directly affect cellular energy metabolism. 41The downstream effect that the PANK2 mutation has on the biosynthesis of CoA, affecting the ratelimiting step of the pathway, is associated with decreased acetylation of histones and tubulin, which is implicated in the impairment in neuronal functional that can lead to neurodegeneration. 42The impaired neurons have decreased respiratory ability in the mitochondria, increasing the levels of ROS in PKAN patients compared to baseline levels, which directly links oxidative stress to the PANK2 deficiency mutation. 43Typical PKAN is early onset with a rapid progression of symptoms such as dystonia, dysarthria, spasticity, hyperreflexia, and extensor toe signs; atypical PKAN presents with a later onset and slower progression of the typical symptoms, with speech difficulty and psychiatric symptoms also developing. 44PKAN is differentiated from other forms of NBIA by its specific "eye of the tiger sign" on radiographic imagery, consisting of a hypointensity of the global pallidus, hypointensity of the substantia nigra, and dentate nucleus. 45he globus pallidus is part of the basal ganglia, masses of neuronal cell bodies located within the cerebrum.Also known as the subcortical nuclei, the basal ganglia control voluntary functions.The globus pallidus is the medial portion of the lentiform nucleus, the lower mass of the most prominent basal ganglia, known as the corpus striatum.The substantia nigra is another nucleus in the brain and is the primary source of input to the basal ganglia via its dopaminergic neural projections. 46he nigrostriatal pathway is heavily implicated in the pathology of the motor deficits that are present in NBIA diseases. 46The dentate nucleus is the largest deep cerebellar nucleus and is involved in the efferent modulation of motor neurons; disruption of dentate nucleus function is associated with cerebellar ataxia. 47

PLA2G6-Associated Neurodegeneration (PLAN)
PLA2G6-associated neurodegeneration (PLAN) is aptly named for the autosomal recessive PLA2G6 mutation that causes the disorder.The most common type of this disorder is infantile neuroaxonal dystrophy (INAD) presents with severe psychomotor symptoms such as rapidly progressing hypotonia, hyperreflexia, and tetra paresis and cerebellar atrophy with brain iron accumulation. 48The PLA2G6 gene is vital in cellular membrane homeostasis as it encodes for the iPLA 2 -Via protein, a calcium-independent phospholipase, which has an underlying connection to the axonal pathology of PLAN, indicating that neuron axons are damaged when the mutation is expressed. 49europathology studies indicate that the PLA2G6 mutation results in axonal spheroids in the cerebral cortex, striatum, cerebellum, brainstem and spinal cord. 50Axonal spheroids are bead-like swellings along axons and are a frequent characteristic of axonal degeneration. 51Lewy bodies (LBs) are commonly found in many neurodegenerative diseases and are made up of aggregated forms of the protein alpha-synuclein (α-Syn), which form beta-sheet-rich amyloid fibrils that contribute to the pathology of the diseases. 52LBs are reportedly found in cases of PLAN and were confirmed through genetic testing, indicating that the PLA2G6 mutation potentially induces the aggregation of α-Syn. 52PLAN shared similar pathological characteristics with Parkinson's disease (PD) and Alzheimer's disease (AD), which could be caused by the Lewy bodies present in the neurological diseases. 48Both PKAN and PLAN involve a mutation associated with the mitochondria and lipid metabolism, which in turn could alter the regulation of iron transport and utilization in the brain. 48hough the exact pathology of the forms of NBIA are not yet fully understood, it can be inferred that this pathway plays an instrumental role.

Mitochondrial Membrane Protein-Associated Neurodegeneration (MPAN)
The next most prevalent form of NBIA is mitochondrial membrane protein-associated neurodegeneration (MPAN), accounting for ∼6−10% of all NBIA cases. 38Caused by the autosomal recessive gene C19orf12, MPAN usually presents in childhood or adolescence with symptoms such as dystoniaparkinsonism, optic atrophy, and axonal motor neuropathy. 532-weighted MRI displays hypo-intensities indicative of iron accumulation in the globus pallidus interna and externa and iso-intensities in the medial medullary lamina. 53Compared to PKAN, MPAN has a later onset and a more gradual psychomotor regression; the characteristic "eye-of-the-tiger" sign on MRI scans is not present, whereas a prominent T2 hypointensity in the substantia nigra is present. 54Lewy bodies and neurites are widespread in the globus pallidus, corpus striatum, midbrain substantia nigra, neocortex, and hippocampus and are present in a substantially higher volume than in cases of sporadic Lewy body disease. 55The C19orf12 gene is located on chromosome 19q12 and encodes for the C19orf12 protein. 56The function of this protein is not clear, but the expression of the gene in fat cells and its coregulation with other genes involved in fatty acid metabolism implicate its role in lipid metabolism. 57

Beta-Propeller Protein-Associated Neurodegeneration (BPAN)
The last of the four most common types of NBIA is betapropeller protein-associated neurodegeneration (BPAN).The gene responsible for this disorder is WDR45, which lies on the X chromosome, making BPAN the only NBIA disorder caused by an X-linked dominant gene. 58BPAN is the most recently discovered form of NBIA, with the de novo WDR45 mutation leading to iron accumulation in the substantia nigra and globus pallidus, along with axonal spheroids, gliosis, severe neuronal loss, and a significant decrease in Purkinje cells. 59The WDR45 gene encodes a protein belonging to the LIS1 family, all important for neuronal survival, and can be characterized as a beta-propeller scaffold, referring to the tertiary structure of the protein, that allows for protein−protein interactions that are critical in the process of autophagy. 60utophagy is the degradation of cellular components that is activated because of low nutrient availability to dispose of organelles, misfolded/aggregated proteins, or infectious agents. 61,62As mentioned before, protein aggregation resulting in cellular protein degradation is linked to the mechanism of NBIA.While much is not yet understood about the pathogenesis of BPAN, a mutation causing a defect in the pathways of autophagy could certainly be implicated in playing an instrumental role in the development of iron accumulation due to the iron homeostasis pathways mentioned previously.
Axial T-2 imaging indicates hypointensity in the globus pallidus consistent with iron accumulation, cortical atrophy, and even more obvious hypointensity in the substantia nigra. 63xial T-1 imaging show a combination of hypointensity in the mesencephalic peduncles with a linear hyperintensity surrounding it, often referred to as a "halo sign" typical of BPAN and may be unique to this disorder. 63able 1 summarizes each subtype of NBIA with the monogenetic mutation, inheritance pattern, prevalence, and therapeutics available or in development.

■ IRON CHELATION THERAPY
After identifying the most common types of NBIA and the mechanisms behind those specific diseases, the possible avenues of treatment can now be explored.Chelators are substances that can form complexes with metal ions.Iron chelation therapy is a promising treatment for the disease, but it is still symptomatic and not causal, or addressing the root of the illness.Iron chelators have a high affinity to bind to iron but must also be able to cross or bypass the blood-brain barrier to access the excess iron without causing significant iron depletion. 66The three iron chelators currently available for clinical use are deferoxamine (DFO), desferasirox (DFS), and deferiprone (DFP).
DFO is the oldest and the most widely used iron chelator currently on the market.Available as a subcutaneous infusion because of its short half-life in humans, DFO has had a consistently beneficial effect on the long-term survival of patients with thalassemia. 67It is a hexadentate chelator and binds to iron in a 1:1 molar ratio to be excreted through the urine or bile. 68The pharmacokinetic and biodistribution properties of the drug were improved using thioketal-crosslinked polymeric nanogels postfunctionalized with DFO moieties (rNG-DFO), yielding good elimination and low cytotoxicity profiles while also reducing iron-mediated oxidative stress. 69 65 results posted 65 While is it considered to be a safe and effective treatment for transfusional iron overload, low patient compliance and poor pharmacokinetics of the free drug administered parentally hinders its use and effectiveness in patients.Furthermore, DFO has a low lipophilicity and a large molecular weight, barring it from being able to cross the BBB to treat NBIA. 70Oral dosage forms of iron chelators are of interest for their increased patient compliance but DFO has been reported to have poor oral bioavailability.Thus, oral formulations that are reported are prophylactic in nature and focus on chelating dietary iron in the gut to reduce overall dietary iron uptake in patients with nontransfusional hemochromatosis. 71The parenteral route of delivering various types of nanoformulations of DFO to reduce body iron stores in transfusional hemochromatosis conditions remains the most popular, yet few of these studies have focused on designing IV nanoformulations that can efficiently cross the BBB.
Overall, the intranasal (IN) route seems to be the most favorable for bypassing the BBB and treating a multitude of neurodegenerative diseases with DFO.For example, IN-DFO was shown to target the CNS to reduce the infarct volume of middle cerebral artery occlusion and significantly improve spatial memory in mouse models with AD. 72,73 Chronic IN-DFO was used in an α-syn rAAV vector-based model of PD and showed an improvement in motor performance and a reduction in the number and size of α-syn aggregates. 74DFO can be rapidly delivered to the brain through this route along the olfactory and trigeminal nerves with negligible systemic adverse side effects. 75Intranasal formulations have been preferred in many cases over parenteral and oral formulations, but there are concerns regarding limitations of IN efficiency due to the protective barriers of the nasal mucosa, which result in the necessity for both frequent and high doses of IN formulations, leading to irritation of the nasal mucosa. 76FP was the first oral chelator that proceeded to enter extensive human trials. 67It was shown to have short-term safe and effective iron chelation abilities. 77It is a bidentate chelator, binding in a 2:1 chelator/iron ratio that is also excreted through the urine along with the free drug. 78DFS is tridentate oral iron chelator used to treat thalassemia and binds to iron in a 3:1 molar ratio. 79Clinical studies comparing the therapeutic efficiency and the tolerability of DFS to DFO report that DFS was noninferior to DFO and produced higher satisfaction and compliance. 79DFP is a hydroxypyridinone (HPO)-based chelator, a class of compounds that have a high selectivity for iron and a scaffold that is capable of a variety of biological actions. 80HPOs can be made more lipophilic by N-alkylation so that they are capable of crossing the BBB, which makes them promising candidates for treating NBIA. 81The downside to DFP is that it is rapidly metabolized in the liver, and there are dose-related toxicity concerns due to side effects such as agranulocytosis and mild neutropenia. 70DFS faces similar concerns, but its toxicity is much more severe, as there are reported fatalities associated with long-term use of the drug. 70ut of these three chelating agents, the applications of DFP and DFS in NBIA have been studied due to their oral formulation. 82DFP has been used in multiple clinical trials to treat PKAN.It was first used to treat NBIA in 2008 in a patient who had severe gait impairment and choreic dyskinesias incidence, both of which was improved significantly by treatment with DFP. 83A phase II pilot trial treated PKAN patients with DFP for 6 months and observed a significant reduction in iron levels in the globus pallidus, revealed by MRI scans. 84Another trial observed the long-term use of DFP to reduce brain iron overload and improve neurological manifestations; the 4-year follow up confirmed the safety of the drug and the efficacy of DFP as a therapeutic option in 83% of adult patients at early stages of the disease. 85One specific patient case study reported that a daily treatment of DFP combined with baclofen in a patient with classic PKAN decreased dystonia and increased quality of life. 86The TIRCON2012 V1 trial was an 18-month long, randomized, double-blind, placebo-controlled study conducted in hospitals in Germany, Italy, England, and the United States. 87Patients with PKAN were dosed with oral DFP twice a day, and results suggested slowed disease progression, though not significantly enough to be conclusive. 87An extension of the study was conducted, TIRCON-EXT (Table 2).This trial allowed for integral information to be collected on determining end points and understanding the natural history of the disease that has been used to design future clinical trials to study DFP to treat NBIA.No evidence was found of this trial progressing to the next phase of clinical trial, which is a cause for concern and leads to questions regarding how safe and effective the deferiprone treatment was.
Due to it making up the largest percentage of NBIA patients, PKAN studies are most prevalent in the literature.However, there have been studies done to assess different therapeutic options for other forms of NBIA.For example, docosahexaenoic acid (DHA) is integral in the function of the calciumindependent phospholipase A 2 β (iPLA 2 β) that is implicated in the pathology of PLAN. 91iPLA 2 β selectively hydrolyzes DHA, but when the protein is defective, as it is in cases of PLAN, the metabolism and signaling pathways of DHA in the brain are significantly reduced, increasing its vulnerability to neuroinflammation. 91Another study reported decreases in not only DHA metabolism in iPLA 2 β-deficient mice but also altered expression and concentrations of other brain phospholipases and fatty acids, which highlights how important iPLA2β is in brain lipid metabolism and how any disturbances can cause neuropathological abnormalities. 92DHA has been proven to have an antioxidant effect in the brain, reducing the production of ROS and oxidative stress. 93DHA has been combined with metal chelation therapy (iron and copper) to treat colorectal cancer cells, specifically targeting ROS that induces cancer cell toxicity and triggers degradation. 94A combined therapy of DHA with an EGCG-derivative showed an improvement in the antioxidant capacities of EGCG, which can be applied to developing treatments for neurodegenerative diseases. 95

■ NOVEL IRON CHELATION THERAPIES
Along with the current small molecule iron chelating agents that are being studied for their applications toward NBIA, there is a multitude of promising multifunctional agents that can also be applied to these diseases (Table 3).Epigallocatechin-3-gallate (EGCG), a polyphenol derived from tea and also known as a catechin, possesses ROS-scavenging capabilities as well as an affinity for binding and chelating metals such as copper and iron. 96Green tea polyphenols such as EGCG may be involved in the regulation of antioxidant protective enzymes as well; many preclinical animal studies have shown EGCG to have increased levels of antioxidant enzymes and to inhibit protein aggregation. 97R-Apomorphine (R-APO) is a dopamine D 1 -D 2 receptor agonist that has been successfully used to treat PD and has been proven to have neuroprotective properties. 98Apomorphine's neuroprotective properties can be attributed to its antioxidant and free radical scavenging characteristics. 99Apomorphine also has metal chelating abilities for iron and copper ions. 100Benzylisoquinolines other than apomorphine have been shown to reduce Fe 3+ and inhibit the hydroxyl radical production in the Fenton reaction, which can be applied to treatment the pathology of many neurodegenerative diseases caused by iron overload. 101urcumin (CURC), capsaicin (CAP), and S-allylcysteine (SAC), which are active agents in spices such as turmeric, chili, and garlic, can bind to ferrous ions (Fe 2+ ) to reduce lipid peroxidation by decreasing the amount of available iron to the Fenton reaction. 102Two newly developed iron chelators, VK-28 and M30, were studied against lactacystin-induced nigrostriatal degeneration and were found to be able to chelate iron successfully at the same potency as DFO, but unlike DFO, these chelators can cross the blood-brain barrier, which opens them up to a variety of possible clinical uses. 103VK-28 and M30 are derivatives of 8-hydroxyquinolines, a family of compounds that can form complexes with divalent metal cations such as iron. 104−107 These studies have made clioquinol an attractive compound for neurodegeneration treatments, but clioquinol has also been proven to cause cytotoxicity in astrocyte-derived KT-5 cells by depleting ATP levels and increasing ROS in the cells. 108Though clioquinol may not be a viable therapy for NBIA, related compounds such as VK-28 and M30 have the same chelation properties without the cytotoxicity of their relative, so their application toward these diseases has potential.

■ POTENTIALS OF GENE THERAPY
While iron chelators show much promise toward the treatment of NBIA, the drugs only target the iron accumulation in the brain and are not able to address the mutation that causes the genetic disorder.Gene therapy is a precision treatment for these monogenic disorders because it would correct the genetic mutation at the root of the neurodegeneration. 120This would be a causal treatment, where the current treatments are symptomatic.New technological developments in antisense oligonucleotide (ASO) therapy, adeno-associated virus (AAV) vector gene delivery, and CRISPR-Cas9 genome editing could be used as a clinical treatment for NBIA. 120SO therapy utilizes oligonucleotides, approximately 10−30 nucleotides long, that are capable of binding to cellular RNA and affecting multiple RNA processes, such as splicing, transcription, and more; while they are currently unable to cross the BBB, ASOs can be injected into CSF to experience neuronal uptake into the brain.121 These characteristics of ASOs give the molecules a high target specificity and allow them to be applied as treatment for undruggable diseases.122 Amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases are being tested with ASO treatments and have determined that ASOs are a strong candidate for monogenetic disorder therapies.123 Adeno-associated virus (AAV) vectors are another possible gene therapy that has shown promise in treating diseases like NBIA.Recombinant AAVs (rAAVs) have all viral encoding removed, except for protein coding sequences, and are instead encoded with "therapeutic gene expression cassettes," which are then released into the nucleus to be expressed in the host cells.124 AAV vectors have a low cytotoxicity and immunogenicity in vivo, can cross the BBB, no longer available Ferriprox (deferiprone) 100 mg/mL oral solution and produce lifelong transgene expression, making them viable treatment candidates for NBIA.124,125 AAV vectors have been studied in PD clinical trials with promising results.109 Lastly, clustered regularly interspaced short palindromic repeat (CRISPR)-Cas-associated nucleases are an avenue of gene therapy that have the potential to introduce heritable genome changes; the Cas9 protein, specifically, has been proven to be reliable and robust in function, making it a suitably programmable tool to generate DNA.126 AAVs have been used as a delivery system for CRISPR genome editing, along with Cas9 ribonucleoprotein, due to their versatility and adaptability to many different types of host cells.126 CRISPR-Cas9 genome editing has been used to treat AD, PD, ALS and other neurodegenerative diseases and were shown to be successful in either reducing or reversing the mutation expression of disease pathologies.127 Challenges that arise with this form of therapy lie in target delivery, and current animal models do not illustrate the exact motor or cognitive features of the human disease.120 However, with future studies in larger animals with more similar phenotypes for NBIA, there is potential in gene therapy to be used in the future to causally treat NBIA.
■ DISCUSSION/CONCLUSION Though several novel small molecule iron chelation therapies have not yet reached clinical trials to be tested in humans with neurodegenerative diseases, their applications toward NBIA provide many possible treatment options in the future.The genes that cause NBIA do not have many similarities in location or function, but the result of the mutations are associated with membrane or mitochondrial protein malfunction, leading to the accumulation of iron and harmful ROS in the brain.Enough is known about the mechanisms of NBIA to affirm that iron accumulation plays a pivotal role through the production of ROS, which leads to oxidative stress and lipid peroxidation in the brain, thus causing tissue damage and neurodegeneration.Lipid metabolism and the integrity of the phospholipid membrane are equally as important in metabolic pathways in neural cells, as well as other neuropathological features mentioned in this paper, such as Lewy bodies and altered mitochondrial function. 128These characteristics seem to be unifying theories throughout the different types of NBIA.Understanding how they all link together in the pathophysiology of neurodegenerative diseases caused by iron accumu-lation is the key to treating the cause of the disease, not only its progression.
DFP has been used in the treatment of PKAN with promising results in clinical trials.Its use in other forms of NBIA is a possibility for future studies, as well as other iron chelators once they have reached clinical studies.However, these trials have not progressed in the last five years, which raises questions whether the adverse side effects in patients are worth the chelating ability of the drugs.Because NBIA diseases are orphan diseases, there is not much motivation in funding larger clinical trials to further assess chelating treatments.There is no standard course of treatment for NBIA, and the only drugs that are approved for use are to alleviate the neuromuscular symptoms of iron overload.For example, baclofen is used to treat muscle spasms and cannot target the iron accumulation directly.However, new discoveries and developments in iron chelation therapies used to treat illnesses such as AD and PD may be applied toward NBIA, since iron overload is characteristic in many neurodegenerative diseases and its presence is positively correlated with increased oxidative damage.For example, the applications of DFO to AD and PD was reported in preclinical studies to decrease oxidative stress and improve behavior. 129IN-DFO has been studied in AD and PD models with encouraging results; therefore, intranasal formulations of DFP and DFS could also be an alternate route of delivery to the CNS while diminishing the chances for adverse systemic side effects.DFP has been studied to treat diseases similar to NBIA, most recently in a phase II trial to treat PD.Results showed that treatment with deferiprone decreased nigrostriatal iron content, but DFP was also associated with worsened symptoms of parkinsonism when compared to the placebo and patients presented with serious adverse events such as agranulocytosis and neutropenia. 130he largest challenge that iron chelators present when being used as treatment for NBIA is dose-related toxicities in the brain.Both DFP and DFS have been reported to have side effects that range from mild to fatal.A pharmacokinetic and safety profile of DFP in patients with sickle cell disease indicated that the drug was well tolerated with no major safety concerns. 131Studies of DFP in brain iron overload have also proven that the drug is well tolerated in the body but also produce some severe adverse effects, mentioned previously. 132he dose of DFP used in the phase II PD trial was lower than those used to treat systemic iron overload (30 mg/kg vs 100  102,112,113 Antioxidant, anti-inflammatory, antitumor, and antibacterial effects; upregulator of antioxidant proteins Hypoxia-induced myocardial infarction injury and toxic chemical induced liver injury Capsaicin (CAP) 102,114 Analgesic, antioxidant, and anti-inflammatory effects; tumor growth inhibition; lipid oxidation inhibitor; free radical scavenger Arthritis, diabetic neuropathy, gastric lesions, and cardiac excitability S-allylcysteine (SAC) 9,115−117 ROS scavenger; enzymatic and nonenzymatic antioxidant activator; prooxidant enzyme and lipid peroxidation inhibitor Hepatocellular carcinoma and brain ischemia VK-28 103,118 Brain permeable; peroxidation inhibitor; iron chelator Lactacystin-induced nigrostriatal degeneration and Parkinson's disease M30 92,119 Iron chelator; free radical scavenger; brain permeable; dopamine, serotonin, and noradrenaline activator Lactacystin-induced nigrostriatal degeneration and Parkinson's disease mg/kg for transfusion-dependent thalassemia), but similar adverse effects were seen. 130Further research should be conducted to gain a deeper understanding of the toxicity of iron chelation therapy in the brain, as the results are unclear with comparison to systemic toxicity.Iron chelators that have been tested in clinical trials have been successful in reducing iron content in the brain, but the adverse events that occur along with treatment are significant enough to question whether the rewards of chelation therapy outweigh side effects.At present, the optimal iron chelator for oral formulation is DFP, but DFO is the most versatile chelator overall due to its 1:1 binding ratio and structure which allows for easier chemical manipulation and modifications as needed for delivery via other routes such as intravenous, IN, or oral to improve bioavailability (IV) or uptake (oral, IN).Oral formulations have the most patient compliance and thus are an attractive route of administration for therapies, yet IN formulations can enter the brain without having to address the issue of crossing the BBB.It is our opinion that DFO shows the most promise in treating NBIA and that future research should be directed toward IN and nanoformulations of the drug.
Iron chelators still require significant development and testing before they are a practical treatment to be given to patients with NBIA.Intranasal dosage forms and nanoformulations show potential regarding chelation therapy, but the dosing limitations compared to parenteral and oral formulations are an obstacle that must be overcome for them to go into widespread use.The most current research on NBIA focuses on the use of deferiprone to treat the disorders, but gene therapy provides another pathway of study that would target the direct cause of NBIA.These therapies could allow for correction of the genetic defect before the onset of physical symptoms, negating the need for iron chelators (if diagnosis came after the accumulation of brain iron, then iron chelation therapies would have relevance to prevent further neuronal death).Gene therapies have been shown to have beneficial effects on neurodegeneration and studies have shown great promise for NBIA, such as the use of AAV vectors and CRISPR-Cas9 genome editing in cases of AD, PD, and other neurodegenerative diseases.
Iron chelation therapy may provide a treatment to NBIA, whereas gene therapy may provide a cure.Gene therapies are becoming more accessible to the public, as seen with the recent approval by the FDA of Casgevy and Lyfgenia for use in patients 12 and older with sickle cell disease. 133Though this is a major accomplishment, gene therapy is incredibly expensive and is far from being developed for use against an orphan disease such as NBIA.Though there are still many issues to be discussed and questions to be answered, iron chelation is the most realistic treatment that can currently be applied to NBIA, but in the future we hope to see gene therapy used as well.The pathophysiology of NBIA has not yet been completely discovered, but as more research is conducted on the mechanisms of iron accumulation and its associations with neurodegeneration, developing causal treatments for all types of NBIA becomes more possible.

Table 1 .
Currently Known Subtypes of NBIA Categorized by Gene, Percentage of Total NBIA Cases, and Current Therapies and Their Stages of Development

Table 2 .
Clinical Trials Studying DFP as the Intervention for PKAN mL oral solution will be administered twice daily (b.i.d.) for 18 months.An initial dose of 5 mg/kg b.i.d. will be administered for 6 weeks.The dose will then be escalated to 10 mg/kg b.i.d. and finally to 15 mg/kg b.i.d.

Table 3 .
Novel Iron Chelation Therapies with Potential Applications toward NBIA