Iron accumulation in Patients with Parkinson’s Disease PHM142 Fall 2015 Coordinator: Dr. Jeffrey Henderson Instructor: Dr. David Hampson Iron accumulation in Patients with Parkinson’s Disease Mohammed Ali Chehade, Xin Yi Huang, Brianna Kispal, Jae Eun Shim PHM 142 University of Toronto

What is Parkinson’s Disease? Parkinson’s disease is a progressive disorder of the nervous system that involves the neurodegeneration of dopaminergic neurons of the substantia nigra. Since it is a progressive disorder, symptoms continue and worsen over time. It is the second most common neurodegenerative disease, behind Alzheimer’s, which affects approximately 1% of the population. PD is a progressive disorder of the nervous system that involves the neurodegeneration of dopaminergic neurons of the substantia nigra. Symptoms continue and worsen over time. This is the second most common form of a neurodegenerative disease behind alzheimers that affects approximately 1% of the population.

Symptoms Associated with Parkinson’s Disease Symptoms at Different Stages of Parkinson’s Disease Stage 1: Early Stage Stage 2: Moderate Stage 3: Advanced Tremors, joint pain, weakness, and fatigue Slow movement, stiff muscles, poor coordination, problems with posture and balance (speech and writing problems) Muscle stiffness and severe tremors, decrease in self-care function and wheelchair usage PD is a progressive disease, therefore, symptoms start out mild and become more serious as the disease progresses through the following stages: Early: tremors is the first symptom that will appear, as well as some weakness in joints and muscles Moderate: the most common symptom of PD is bradykinesia which is slow movement. the muscles become stiff at this point in the disease and people will start to notice changes in their speech and writing abilities. Advanced: tremors become more severe and frequent and self-care function decreases to the point where the person may be confined to a wheelchair.

Causes of PD The cause or causes of Parkinson’s disease is not known, however, there are many current theories. Genetics: PD can be hereditary, however, only a small percent of people with PD report having an immediate family member with the disease. It is possible that there are abnormal genes that are hereditary that may like early-onset PD in families. Oxidative Stress: When oxidative stress is put on the body, reactive oxygen species form, where the production of these molecules can damage macromolecules, which lead to cellular degeneration.Although the body has developed mechanisms to counteract oxidative stress, the brain is more susceptible to this damage, therefore, neuronal cells are more easily destroyed. Synaptic Function: Degeneration of neuronal cells, leads to a depletion of the neurotransmitter, dopamine, which can lead to the symptoms mentioned earlier. Environmental: Exposure to well water, manganese, and pesticides have been linked to PD. Lewy Bodies: abnormal aggregates of proteins that develop inside nerve cells, which can lead to PD Age: biological function decreases with age, which may be a link to PD

Increased oxidative stress in dopaminergic neurons MAO, FAD DOPAMINE DOPAC HVA Dopamine (DA) metabolism by monoamine oxidase (MAO) generates H2O2 as a byproduct: DA ---(MAO, FAD)---> DOPAC, HVA, H2O2 H2O2 generates hydroxyl radicals through the Fenton reaction: H2O2 + Fe2+ → OH* + OH- + Fe3+ H2O2 + Fe2+ OH* + OH- + Fe3+

Iron accumulation in Substantia Nigra(SN) PD results from the death of dopaminergic neurons in the substantia nigra. SN has constitutively high levels of iron and dopamine, and iron levels increase with age. In patients with PD, there is pathological iron accumulation. Potential causes of iron accumulation are due to iron homeostasis imbalance demonstrated in PD rat models: DMT1 (an iron import channel) up-regulated and ferroportin 1 (an iron export channel) down-regulated this is due to abnormal IRP-IRE interactions ferroportin 1 mRNA has IRE on 5’ end; DMT1 mRNA has IRE on 3’ end increased ROS levels activate IRPs, which then bind IREs IRP-IRE complex on ferroportin 1 mRNA increases translation while IRP-IRE complex on DMT1 mRNA decreases translation increased iron import and decreased iron export lead to increased intracellular iron

Iron accumulation in Substantia Nigra(SN) Potential causes of iron accumulation are due to iron homeostasis imbalance demonstrated in PD rat models. DMT1 (an iron import channel) is up-regulated and ferroportin 1 (an iron export channel) is down-regulated: ferroportin 1 mRNA has IRE on 5’ end; DMT1 mRNA has IRE on 3’ end increased ROS levels activate IRPs, which then bind IREs IRP-IRE complex bound on ferroportin 1 mRNA increases translation while IRP-IRE complex bound on DMT1 mRNA decreases translation increased iron import and decreased iron export lead to increased intracellular iron Another potential pathway is ceruloplasmin (CP) dysfunction, which facilitates iron export by oxidizing ferrous (Fe2+) to ferric (Fe3+) iron. CP dysfunction has been observed in the SN of PD patients, and this may be due to inactivation of CP by ROS. Studies have shown that CP-knockout mice develop PD within 6 months. How do high levels of iron confer neurotoxicity?

Iron as a cause of PD Increased intracellular iron saturates neuromelanin, an iron chelator in substantia nigra dopaminergic neurons. Overwhelming these iron chelators eventually lead to increased free iron intracellular iron and increased oxidative stress, and eventual cell death. This releases neuromelanin extracellularly, and can activate microglia (macrophages in the CNS) and cause neuroinflammation. inflammatory cytokines also affect iron homeostasis in astrocytes and microglia: proinflammatory TNF-alpha increases iron uptake and retention in astrocytes and microglia anti-inflammatory TGF-beta1 promotes iron efflux in astrocytes and iron retention in microglia Iron homeostasis imbalance as a primary cause of PD is controversial. There are studies showing conflicting results. Some observed iron storage in SN at various stages of PD, while other studies did not observe nigral iron accumulation.

Iron as a cause of PD Therefore, Iron chelation may be used as a means of PD treatment Despite the debate,((over iron deposition and parkinson’s disease whether the relationship was causal,)) Here is a well-established study in 2003 supporting the causal relationship. They made transgenic modification so more ferritin is secreted. By doing so, MPTP induced neuronal decrease was no longer significant. This shows that free iron does result in brain damage leading PD and therefore iron chelation may become a means of PD treatment. (rat tyrosine hydroxylase promoter (pTH), which drives expression of human Heavy ferritin gene. They added this to mice embryo as human ferritin binds to iron much more tightly than mouse isoform. Tg, ferritin transgenic, Wt. wild type. Effects of Acute MPTP Administration on Dopaminergic SN Neuronal Cell Number in pTH-Ferritin Transgenics versus Wild-Type. Kaur, D et al. Neuron 37(6), 899-909 (March 2003)

Possible iron chelator for PD- Deferiprone Iron chelators: Inhibition of Fe2+ reduction, protection against damages from the reduction Possible problem? Systemic chelation vs Cellular specific chelation Deferiprone(DFP): Phase II clinical research Benefits: additional antioxidant properties for comprehensive mode of action. effective cellular specific chelation. Hoffbrand, A., Taher, A., Cappellini, M. Blood 120(18), 3657-3669 (Novermber 2012) Mounsey, R. and Teismann, P. Int J Cell Biol 2012, (March 2012) As you can see on the left, there are many possible iron chelators for parkinson’s disease, with various targets. Most drugs work to decrease the ferrous iron oxidation, as well as various outcomes from that oxidation. Deferiprone is one of the chelators, currently undergoing phase II of clinical research and funded by the European Union. The drug leads to reduction of free iron, then binds to ferric iron. This allows to decrease possible damages. Not only that, this also has additional antioxidant properties to protect from ROS. To be an iron chelator targeting PD, it must not disturb systemic homeostasis of irons and many studies support that deferiprone does this. Deferiprone crosses the blood brain barrier effectively, which is another benefit crucial for our purpose. *Deferiprone is an iron chelator that binds to ferric ions (iron III) and forms a 3:1 (deferiprone:iron) stable complex and is then eliminated in the urine. Deferiprone is more selective for iron in which other metals such as zinc, copper, and aluminum have a lower affinity for deferiprone. *cellular specific: their ability to donate chelated iron to unsaturated transferrin (64, 65), thus avoiding the body iron losses generated by other chelators in clinical use.

Targeting Chelatable Iron as a Therapeutic Modality in Parkinson's Disease Partial recovery of dopaminergic cells within the SN through oral chelator Deferiprone(DFP) Here, we are looking at a study that used DFP for parkinson symptom induced mice. Here, you can see that the mice with the symptoms sginificantly recovers to the original states. Nigral dopaminergic cells as well as dopamine level are partially recovered. Also, as you can see in the graph, this is related to reduced DNA oxidation. This study supports the clinical effect of iron chelator in parkinson induced mice. 8-OHdG:(i.e., 8-oxo-deoxyguanosine formation) MPTP:1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) is a neurotoxin precursor to MPP+, which causes permanent symptoms of Parkinson's ??????disease by destroying dopaminergic neurons in the substantia nigra of the brain. Mouse: Saline- and MPTP-treated mice (5-month-old male C57Bl/6J mice, weighting 28–30 g, from Janvier Le Genest St Isle, France) (MPTP 4×20 mg/kg i.p. over 24 h) received 100 or 150 mg/kg i.p. DFO or p.o. DFP (ApoPharma) twice a day for 10 days (starting 3 days before saline or MPTP intoxication). Dopaminergic cells: tyrosine hydroxylase positive cells. (TH is L-DOPA(Dopamine precursor) producing enzyme.) drug's mode of action: Direct reduction in cellular labile iron pools and the ensuing detoxification and/or indirectly via induction of cell protective measures, and (ii) via a reduction in enzymatic dopamine catabolism and/or through nonenzymatic oxidation of the naturally produced dopamine or supplemented dopamine substitutes. (We base the latter assumption on the fact that DFP decreased the 3-O-methyl-DOPA/dopamine ratio in a dose-related manner in control and MPTP-intoxicated animals, indicating the possible inhibition of COMT activity via metal chelation and/or by interaction with the enzyme, due to the similarity between DFP hydoxypyridine structure and the enzyme's native substrates (68).) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4060813/ Reduced oxidation products of DNA Partial recovery of dopamine through DFP treatment Devos, D et al. Antioxid Redox Signal 21(2), 195–210 (July 2014)

Human Iron Chelation Study - Deferiprone Grolez et al. (2015) 40 PD patients recruited for an 18 month clinical trial given a 30mg/kg daily dose of DFP substantia nigra (SN) iron levels and disease progression were assessed Results: DFP resulted in lower SN iron levels and reduced disease progression additionally, ceruloplasmin (CP) activity was increased *D544E CP polymorphism responded better to chelation Recently, researchers have started moving on from animal models to using Deferiprone (DFP) in patient studies -an article was published this year where DFP was used in an iron chelation protocol on patients with Parkinson's Disease Protocol: -In this study, 40 PD patients were recruited in a 6-month delayed-start paradigm for a trial lasting 18 months -between months 0 and 6 patients were either given liquid DFP orally at a dose of 15mg/kg or a placebo -from months 6 to 18 all patients were given 30mg/kg of DFP Assessment: -all patients were assessed for disease progression, and the levels of iron in the SN were measured -SN iron levels were measured using magnetic resonance imaging -disease progression was gauged using the "Unified Parkinson's Disease Rating Scale" (UPDRS), and this was used as a measure of motor handicap -In the UPDRS, examiner rates items such as speech, facial expression, tremors, etc through a scored survey to assess PD symptoms -higher score means more severe symptoms and therefore more advanced disease progression Results: -the results of the study showed that iron chelation through DFP administration resulted in both a reduced accumulation of iron in the SN, and a reduction in UPDRS motor score (which, as mentioned, indicates reduced PD symptoms) -the researchers also found that ceruloplasmin activity was increased as a result of DFP administration -interestingly, the researchers noted that patients with a certain CP polymorphism (D544E polymorphism) had lower CP activity and ended up retaining more cellular iron, and this resulted in them responding better to chelation therapy...so while CP deficiency is associated with higher accumulation of iron in the SN, it also allowed for greater improvement through chelation

Implication to pharmacists Discovery of pharmacological treatment rather than symptom management! This is an exciting news for the pharmacy field. Although iron chelating treatment may not recover back to normal state, they can be a drug that cures, rather than manages the symptoms of the parkinson’s disease. Deferiprone Excessive levels of iron have been identified in the substantia nigra of PD patients correlating with disease severity.20 Deferiprone is a licensed treatment for iron chelation, known to cross the blood brain barrier.21 In a further delayed start design trial, deferiprone reduced levels of iron in the SN seen using T2* MRI, associated with a two point improvement in the UPDRS motor subscore.22 This effect size is clinically important although these data cannot yet be interpreted as neuroprotection given that it remains possible that there is some interaction between iron chelation and dopaminergic treatment. A further pilot trial is ongoing. (Clinical trials.gov NCT01539837). http://www.acnr.co.uk/2014/09/regenerative-drugs-for-parkinsons-disease/ Prioritised in 2012, a pan-European study is going ahead thanks to funding by the European Commission’s Horizon 2020. The trial will start recruiting in 2016. The Cure Parkinson’s Trust is fully supporting this trial in terms of recruitment and providing information. http://www.cureparkinsons.org.uk/prioritising-drugs Iron chelators (ie DeferipronePD)

Summary *Parkinson’s disease is a progressive disorder of the nervous system that involves the neurodegeneration of dopaminergic neurons of the substantia nigra. *Common causes of Parkinson’s disease include: genetics, oxidative stress, disturbances to synaptic function, environmental factors, presence of Lewy bodies, and age. *Dopamine metabolism creates oxidative stress as H2O2 is produced as a byproduct. *Iron accumulation in Parkinson’s disease is a result of iron homeostasis imbalance where DMT1 is over-expressed and ferroportin 1 is under-expressed. *High levels of iron saturates neuromelanin and increases oxidative stress. Neural cells are killed and neuromelanin is released in the extracellular environment. Free neuromelanin causes microglia activation and neuroinflammation, which can further disturb iron homeostasis. *Iron chelation can be a treatment for Parkinson’s Disease (currently under investigation) *Deferiprone(DFP): Iron chelator that reduces free iron and binds to the ferric iron. It crosses blood brain barrier and maintains iron homeostasis in the body which makes it viable drug for parkinson’s disease. *Preliminary patient studies with DFP have shown promising reductions in iron levels of the substantia nigra and improved motor function in Parkinson’s disease patients

References Ayton, S et al. "Iron Accumulation Confers Neurotoxicity to a Vulnerable Population of Nigral Neurons: Implications for Parkinson's Disease." Molecular Neurodegeneration 9 (2014): 27. Bharath, Srinivas, et al. "Glutathione, Iron and Parkinson's Disease." Biochemical Pharmacology 64 (2002): 1037-048. Calne, Donald B. "The Treatment of Parkinson's Disease." Drug Therapy 329.14 (1993): 1021-027. Devos, D et al. “Targeting chelatable iron as a therapeutic modality in Parkinson's disease.” Antioxidant & Redox Signaling 21.2 (2014): 195–210. Hoffbrand, A., Taher, A., and Cappellini, M. “How I treat transfusional iron overload.” Blood 120.18 (2012): 3657-3669. Jiang, H et al. "Up-Regulation of Divalent Metal Transporter 1 in 6-Hydroxydopamine Intoxication is IRE/IRP Dependent." Cell research 20.3 (2010): 345-56. Kaur, D et al. “Genetic or Pharmacological Iron Chelation Prevents MPTP-Induced Neurotoxicity In Vivo: A Novel Therapy for Parkinson’s Disease.” Neuron 37(6) (2013):899-909 . Meiser, J et al. "Complexity of Dopamine Metabolism." Cell Communication and Signaling 11.1 (2013): 34. Mounsey, R. and Teismann, P. “Chelators in the treatment of iron accumulation in Parkinson’s disease.” International Journal of Cell Biology 2012 (2012). Song, N et al. "Ferroportin 1 but Not Hephaestin Contributes to Iron Accumulation in a Cell Model of Parkinson's Disease." Free Radical Biology and Medicine 48.2 (2010): 332-41. "What Is Parkinson's Disease?" Parkinson's Disease Foundation. Web. 31 Oct. 2015. Grolez, G et al. "Ceruloplasmin activity and iron chelation treatment of patients with Parkinson’s disease." BMC Neurology 15:74 (2015).