Twelve Years of Drug Prioritization to Help Accelerate Disease Modification Trials in Parkinson's Disease: The International Linked Clinical Trials initiative.

In 2011, the UK medical research charity Cure Parkinson's set up the international Linked Clinical Trials (iLCT) committee to help expedite the clinical testing of potentially disease modifying therapies for Parkinson's disease (PD). The first committee meeting was held at the Van Andel Institute in Grand Rapids, Michigan in 2012. This group of PD experts has subsequently met annually to assess and prioritize agents that may slow the progression of this neurodegenerative condition, using a systematic approach based on preclinical, epidemiological and, where possible, clinical data. Over the last 12 years, 171 unique agents have been evaluated by the iLCT committee, and there have been 21 completed clinical studies and 20 ongoing trials associated with the initiative. In this review, we briefly outline the iLCT process as well as the clinical development and outcomes of some of the top prioritized agents. We also discuss a few of the lessons that have been learnt, and we conclude with a perspective on what the next decade may bring, including the introduction of multi-arm, multi-stage clinical trial platforms and the possibility of combination therapies for PD.

Neuroprotective role for RORA in Parkinson's disease revealed by analysis of post-mortem brain and a dopaminergic cell line.

Parkinson's disease (PD) is almost twice as prevalent in men, which has largely been attributed to neuroprotective effect of oestradiol in women. RORA (retinoic acid receptor-related orphan receptor alpha) regulates the transcription of central aromatase, the enzyme responsible for local oestradiol synthesis, simultaneously, RORA expression is regulated by sex hormones. Moreover, RORA protects neurones against oxidative stress, a key mechanism contributing to the loss of dopaminergic neurones in PD. Therefore, we hypothesized that there would be sex differences in RORA expression in the substantia nigra pars compacta (SNpc), which could contribute to sex differences observed in PD prevalence and pathogenesis. In a case control study, qPCR and western blot analyses were used to quantify gene and protein expression in the SNpc of post-mortem brains (n = 14 late-stage PD and 11 age and sex matched controls). The neuroprotective properties of a RORA agonist were then investigated directly using a cell culture toxin-based model of PD coupled with measures of viability, mitochondrial function and apoptosis. RORA was expressed at significantly higher levels in the SNpc from control females' brains compared to males. In PD, we found a significant increase in SNpc RORA expression in male PD compared to female PD. Treatment with a RORA agonist showed a significant neuroprotection in our cell culture model of PD and revealed significant effects on intracellular factors involved in neuronal survival and demise. This study is the first to demonstrate a sex specific pattern of RORA protein and gene expression in the SNpc of controls post-mortem human brains, and to show that this is differentially altered in male and female PD subjects, thus supporting a role for RORA in sex-specific aspects of PD. Furthermore, our in vitro PD model indicates mechanisms whereby a RORA agonist exerts its neuroprotective effect, thereby highlighting the translational potential for RORA ligands in PD.

Iron, Neuroinflammation and Neurodegeneration.

Disturbance of the brain homeostasis, either directly via the formation of abnormal proteins or cerebral hypo-perfusion, or indirectly via peripheral inflammation, will activate microglia to synthesise a variety of pro-inflammatory agents which may lead to inflammation and cell death. The pro-inflammatory cytokines will induce changes in the iron proteins responsible for maintaining iron homeostasis, such that increased amounts of iron will be deposited in cells in the brain. The generation of reactive oxygen and nitrogen species, which is directly involved in the inflammatory process, can significantly affect iron metabolism via their interaction with iron-regulatory proteins (IRPs). This underlies the importance of ensuring that iron is maintained in a form that can be kept under control; hence, the elegant mechanisms which have become increasingly well understood for regulating iron homeostasis. Therapeutic approaches to minimise the toxicity of iron include N-acetyl cysteine, non-steroidal anti-inflammatory compounds and iron chelation.

Correction: Data Sharing Goals for Nonprofit Funders of Clinical Trials.

[This corrects the article DOI: 10.2196/23011.].

Is Chelation Therapy a Potential Treatment for Parkinson's Disease?

Iron loading in some brain regions occurs in Parkinson's Disease (PD), and it has been considered that its removal by iron chelators could be an appropriate therapeutic approach. Since neuroinflammation with microgliosis is also a common feature of PD, it is possible that iron is sequestered within cells as a result of the "anaemia of chronic disease" and remains unavailable to the chelator. In this review, the extent of neuroinflammation in PD is discussed together with the role played by glia cells, specifically microglia and astrocytes, in controlling iron metabolism during inflammation, together with the results of MRI studies. The current use of chelators in clinical medicine is presented together with a discussion of two clinical trials of PD patients where an iron chelator was administered and showed encouraging results. It is proposed that the use of anti-inflammatory drugs combined with an iron chelator might be a better approach to increase chelator efficacy.

Data Sharing Goals for Nonprofit Funders of Clinical Trials.

Sharing clinical trial data can provide value to research participants and communities by accelerating the development of new knowledge and therapies as investigators merge data sets to conduct new analyses, reproduce published findings to raise standards for original research, and learn from the work of others to generate new research questions. Nonprofit funders, including disease advocacy and patient-focused organizations, play a pivotal role in the promotion and implementation of data sharing policies. Funders are uniquely positioned to promote and support a culture of data sharing by serving as trusted liaisons between potential research participants and investigators who wish to access these participants' networks for clinical trial recruitment. In short, nonprofit funders can drive policies and influence research culture. The purpose of this paper is to detail a set of aspirational goals and forward thinking, collaborative data sharing solutions for nonprofit funders to fold into existing funding policies. The goals of this paper convey the complexity of the opportunities and challenges facing nonprofit funders and the appropriate prioritization of data sharing within their organizations and may serve as a starting point for a data sharing toolkit for nonprofit funders of clinical trials to provide the clarity of mission and mechanisms to enforce the data sharing practices their communities already expect are happening.

Iron and inflammation: in vivo and post-mortem studies in Parkinson's disease.

In these present studies, in vivo and and post-mortem studies have investigated the association between iron and inflammation. Early-stage Parkinson's disease (PD) patients, of less than 5 years disease duration, showed associations of plasmatic ferritin concentrations with both proinflammatory cytokine interleukin-6 and hepcidin, a regulator of iron metabolism as well as clinical measures. In addition ratios of plasmatic ferritin and iron accumulation in deep grey matter nuclei assessed with relaxometry T2* inversely correlated with disease severity and duration of PD. On the hand, post-mortem material of the substantia nigra compacta (SNc) divided according to Braak and Braak scores, III-IV and V-VI staging, exhibited comparable microgliosis, with a variety of phenotypes present. There was an association between the intensity of microgliosis and iron accumulation as assayed by Perl's staining in the SNc sections. In conclusion, markers of inflammation and iron metabolism in both systemic and brain systems are closely linked in PD, thus offering a potential biomarker for progression of the disease.

GDNF and Parkinson's Disease: Where Next? A Summary from a Recent Workshop.

The concept of repairing the brain with growth factors has been pursued for many years in a variety of neurodegenerative diseases including primarily Parkinson's disease (PD) using glial cell line-derived neurotrophic factor (GDNF). This neurotrophic factor was discovered in 1993 and shown to have selective effects on promoting survival and regeneration of certain populations of neurons including the dopaminergic nigrostriatal pathway. These observations led to a series of clinical trials in PD patients including using infusions or gene delivery of GDNF or the related growth factor, neurturin (NRTN). Initial studies, some of which were open label, suggested that this approach could be of value in PD when the agent was injected into the putamen rather than the cerebral ventricles. In subsequent double-blind, placebo-controlled trials, the most recent reporting in 2019, treatment with GDNF did not achieve its primary end point. As a result, there has been uncertainty as to whether GDNF (and by extrapolation, related GDNF family neurotrophic factors) has merit in the future treatment of PD. To critically appraise the existing work and its future, a special workshop was held to discuss and debate this issue. This paper is a summary of that meeting with recommendations on whether there is a future for this therapeutic approach and also what any future PD trial involving GDNF and other GDNF family neurotrophic factors should consider in its design.

Novel 1-hydroxypyridin-2-one metal chelators prevent and rescue ubiquitin proteasomal-related neuronal injury in an in vitro model of Parkinson's disease.

Ubiquitin proteasome system (UPS) impairment, excessive cellular oxidative stress, and iron dyshomeostasis are key to substantia nigra dopaminergic neuronal degeneration in Parkinson's disease (PD); however, a link between these features remains unconfirmed. Using the proteasome inhibitor lactacystin we confirm that nigral injury via UPS impairment disrupts iron homeostasis, in turn increasing oxidative stress and promoting protein aggregation. We demonstrate the neuroprotective potential of two novel 1-hydroxy-2(1H)-pyridinone (1,2-HOPO) iron chelators, compounds C6 and C9, against lactacystin-induced cell death. We demonstrate that this cellular preservation relates to the compounds' iron chelating capabilities and subsequent reduced capacity of iron to form reactive oxygen species (ROS), where we also show that the ligands act as antioxidant agents. Our results also demonstrate the ability of C6 and C9 to reduce intracellular lactacystin-induced α-synuclein burden. Stability constant measurements confirmed a high affinity of C6 and C9 for Fe3+ and display a 3:1 HOPO:Fe3+ complex formation at physiological pH. Reducing iron reactivity could prevent the demise of nigral dopaminergic neurons. We provide evidence that the lactacystin model presents with several neuropathological hallmarks of PD related to iron dyshomeostasis and that the novel chelating compounds C6 and C9 can protect against lactacystin-related neurotoxicity.

DREADD Activation of Pedunculopontine Cholinergic Neurons Reverses Motor Deficits and Restores Striatal Dopamine Signaling in Parkinsonian Rats.

The brainstem-based pedunculopontine nucleus (PPN) traditionally associates with motor function, but undergoes extensive degeneration during Parkinson's disease (PD), which correlates with axial motor deficits. PPN-deep brain stimulation (DBS) can alleviate certain symptoms, but its mechanism(s) of action remains unknown. We previously characterized rats hemi-intranigrally injected with the proteasomal inhibitor lactacystin, as an accurate preclinical model of PD. Here we used a combination of chemogenetics with positron emission tomography imaging for in vivo interrogation of discrete neural networks in this rat model of PD. Stimulation of excitatory designer receptors exclusively activated by designer drugs expressed within PPN cholinergic neurons activated residual nigrostriatal dopaminergic neurons to produce profound motor recovery, which correlated with striatal dopamine efflux as well as restored dopamine receptor 1- and dopamine receptor 2-based medium spiny neuron activity, as was ascertained with c-Fos-based immunohistochemistry and stereological cell counts. By revealing that the improved axial-related motor functions seen in PD patients receiving PPN-DBS may be due to stimulation of remaining PPN cholinergic neurons interacting with dopaminergic ones in both the substantia nigra pars compacta and the striatum, our data strongly favor the PPN cholinergic-midbrain dopaminergic connectome as mechanism for PPN-DBS's therapeutic effects. These findings have implications for refining PPN-DBS as a promising treatment modality available to PD patients.

Precompetitive Consensus Building to Facilitate the Use of Digital Health Technologies to Support Parkinson Disease Drug Development through Regulatory Science.

Innovative tools are urgently needed to accelerate the evaluation and subsequent approval of novel treatments that may slow, halt, or reverse the relentless progression of Parkinson disease (PD). Therapies that intervene early in the disease continuum are a priority for the many candidates in the drug development pipeline. There is a paucity of sensitive and objective, yet clinically interpretable, measures that can capture meaningful aspects of the disease. This poses a major challenge for the development of new therapies and is compounded by the considerable heterogeneity in clinical manifestations across patients and the fluctuating nature of many signs and symptoms of PD. Digital health technologies (DHT), such as smartphone applications, wearable sensors, and digital diaries, have the potential to address many of these gaps by enabling the objective, remote, and frequent measurement of PD signs and symptoms in natural living environments. The current climate of the COVID-19 pandemic creates a heightened sense of urgency for effective implementation of such strategies. In order for these technologies to be adopted in drug development studies, a regulatory-aligned consensus on best practices in implementing appropriate technologies, including the collection, processing, and interpretation of digital sensor data, is required. A growing number of collaborative initiatives are being launched to identify effective ways to advance the use of DHT in PD clinical trials. The Critical Path for Parkinson's Consortium of the Critical Path Institute is highlighted as a case example where stakeholders collectively engaged regulatory agencies on the effective use of DHT in PD clinical trials. Global regulatory agencies, including the US Food and Drug Administration and the European Medicines Agency, are encouraging the efficiencies of data-driven engagements through multistakeholder consortia. To this end, we review how the advancement of DHT can be most effectively achieved by aligning knowledge, expertise, and data sharing in ways that maximize efficiencies.

Quantification of blood-brain barrier transport and neuronal toxicity of unlabelled multiwalled carbon nanotubes as a function of surface charge.

Nanoparticles capable of penetrating the blood-brain barrier (BBB) will greatly advance the delivery of therapies against brain disorders. Carbon nanotubes hold great potential as delivery vehicles due to their high aspect-ratio and cell-penetrating ability. Studies have shown multiwalled carbon nanotubes (MWCNT) cross the BBB, however they have largely relied on labelling methods to track and quantify transport, or on individual electron microscopy images to qualitatively assess transcytosis. Therefore, new direct and quantitative methods, using well-defined and unlabelled MWCNT, are needed to compare BBB translocation of different MWCNT types. Using highly controlled anionic (-), cationic (+) and non-ionic (0) functionalized MWCNT (fMWCNT), we correlate UV-visible spectroscopy with quantitative transmission electron microscopy, quantified from c. 270 endothelial cells, to examine cellular uptake, BBB transport and neurotoxicity of unlabelled fMWCNT. Our results demonstrate that: (i) a large fraction of cationic and non-ionic, but not anionic fMWCNT become trapped at the luminal brain endothelial cell membrane; (ii) despite high cell association, fMWCNT uptake by brain endothelial cells is low (<1.5% ID) and does not correlate with BBB translocation, (iii) anionic fMWCNT have highest transport levels across an in vitro model of the human BBB compared to non-ionic or cationic nanotubes; and (iv) fMWCNT are not toxic to hippocampal neurons at relevant abluminal concentrations; however, fMWCNT charge has an effect on carbon nanotube neurotoxicity at higher fMWCNT concentrations. This quantitative combination of microscopy and spectroscopy, with cellular assays, provides a crucial strategy to predict brain penetration efficiency and neurotoxicity of unlabelled MWCNT and other nanoparticle technologies relevant to human health.

Label-Free Time-of-Flight Secondary Ion Mass Spectrometry Imaging of Sulfur-Producing Enzymes inside Microglia Cells following Exposure to Silver Nanowires.

There are no methods sensitive enough to detect enzymes within cells, without the use of analyte labeling. Here we show that it is possible to detect protein ion signals of three different H2S-synthesizing enzymes inside microglia after pretreatment with silver nanowires (AgNW) using time-of-flight secondary ion mass spectrometry (TOF-SIMS). Protein fragment ions, including the fragment of amino acid (C4H8N+ = 70 amu), fragments of the sulfur-producing cystathionine-containing enzymes, and the Ag+ ion signal could be detected without the use of any labels; the cells were mapped using the C4H8N+ amino acid fragment. Scanning electron microscopy imaging and energy-dispersive X-ray chemical analysis showed that the AgNWs were inside the same cells imaged by TOF-SIMS and transformed chemically into crystalline Ag2S within cells in which the sulfur-producing proteins were detected. The presence of these sulfur-producing cystathionine-containing enzymes within the cells was confirmed by Western blots and confocal microscopy images of fluorescently labeled antibodies against the sulfur-producing enzymes. Label-free TOF-SIMS is very promising for the label-free identification of H2S-contributing enzymes and their cellular localization in biological systems. The technique could in the future be used to identify which of these enzymes are most contributory.

The Qualification of an Enrichment Biomarker for Clinical Trials Targeting Early Stages of Parkinson's Disease.

As therapeutic trials target early stages of Parkinson's disease (PD), appropriate patient selection based purely on clinical criteria poses significant challenges. Members of the Critical Path for Parkinson's Consortium formally submitted documentation to the European Medicines Agency (EMA) supporting the use of Dopamine Transporter (DAT) neuroimaging in early PD. Regulatory documents included a comprehensive literature review, a proposed analysis plan of both observational and clinical trial data, and an assessment of biomarker reproducibility and reliability. The research plan included longitudinal analysis of the Parkinson Research Examination of CEP-1347 Trial (PRECEPT) and the Parkinson's Progression Markers Initiative (PPMI) study to estimate the degree of enrichment achieved and impact on future trials in subjects with early motor PD. The presence of reduced striatal DAT binding based on visual reads of single photon emission tomography (SPECT) scans in early motor PD subjects was an independent predictor of faster decline in UPDRS Parts II and III as compared to subjects with scans without evidence of dopaminergic deficit (SWEDD) over 24 months. The EMA issued in 2018 a full Qualification Opinion for the use of DAT as an enrichment biomarker in PD trials targeting subjects with early motor symptoms. Exclusion of SWEDD subjects in future clinical trials targeting early motor PD subjects aims to enrich clinical trial populations with idiopathic PD patients, improve statistical power, and exclude subjects who are unlikely to progress clinically from being exposed to novel test therapeutics.

Molecular Neuroimaging of the Dopamine Transporter as a Patient Enrichment Biomarker for Clinical Trials for Early Parkinson's Disease.

The Critical Path for Parkinson's (CPP) Imaging Biomarker and Modeling and Simulation working groups aimed to achieve qualification opinion by the European Medicines Agency (EMA) Committee for Medical Products for Human Use (CHMP) for the use of baseline dopamine transporter neuroimaging for patient selection in early Parkinson's disease clinical trials. This paper describes the regulatory science strategy to achieve this goal. CPP is an international consortium of three Parkinson's charities and nine pharmaceutical partners, coordinated by the Critical Path Institute.

The histone deacetylase inhibitor nicotinamide exacerbates neurodegeneration in the lactacystin rat model of Parkinson's disease.

Histone hypoacetylation is associated with dopaminergic neurodegeneration in Parkinson's disease (PD), because of an imbalance in the activities of the enzymes responsible for histone (de)acetylation. Correction of this imbalance, with histone deacetylase (HDAC) inhibiting agents, could be neuroprotective. We therefore hypothesize that nicotinamide, being a selective inhibitor of HDAC class III as well as having modulatory effects on mitochondrial energy metabolism, would be neuroprotective in the lactacystin rat model of PD, which recapitulates the formation of neurotoxic accumulation of altered proteins within the substantia nigra to cause progressive dopaminergic cell death. Rats received nicotinamide for 28 days, starting 7 days after unilateral injection of the irreversible proteasome inhibitor, lactacystin, into the substantia nigra. Longitudinal motor behavioural testing and structural magnetic resonance imaging were used to track changes in this model of PD, and assessment of nigrostriatal integrity, histone acetylation and brain gene expression changes post-mortem used to quantify nicotinamide-induced neuroprotection. Counterintuitively, nicotinamide dose-dependently exacerbated neurodegeneration of dopaminergic neurons, behavioural deficits and structural brain changes in the lactacystin-lesioned rat. Nicotinamide treatment induced histone hyperacetylation and over-expression of numerous neurotrophic and anti-apoptotic factors in the brain, yet failed to result in neuroprotection, rather exacerbated dopaminergic pathology. These findings highlight the importance of inhibitor specificity within HDAC isoforms for therapeutic efficacy in PD, demonstrating the contrasting effects of HDAC class III inhibition upon cell survival in this animal model of the disease. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

L-DOPA functionalized, multi-branched gold nanoparticles as brain-targeted nano-vehicles.

The blood-brain barrier (BBB) is a protective endothelial barrier lining the brain microvasculature which prevents brain delivery of therapies against brain diseases. Hence, there is an urgent need to develop vehicles which efficiently penetrate the BBB to deliver therapies into the brain. The drug L-DOPA efficiently and specifically crosses the BBB via the large neutral amino acid transporter (LAT)-1 protein to enter the brain. Thus, we synthesized L-DOPA-functionalized multi-branched nanoflower-like gold nanoparticles (L-DOPA-AuNFs) using a seed-mediated method involving catechols as a direct reducing-cum-capping agent, and examined their ability to cross the BBB to act as brain-penetrating nanovehicles. We show that L-DOPA-AuNFs efficiently penetrate the BBB compared to similarly sized and shaped AuNFs functionalized with a non-targeting ligand. Furthermore, we show that L-DOPA-AuNFs are efficiently internalized by brain macrophages without inducing inflammation. These results demonstrate the application of L-DOPA-AuNFs as a non-inflammatory BBB-penetrating nanovehicle to efficiently deliver therapies into the brain.

The S100A4 Protein Signals through the ErbB4 Receptor to Promote Neuronal Survival.

Understanding the mechanisms of neurodegeneration is crucial for development of therapies to treat neurological disorders. S100 proteins are extensively expressed in the injured brain but S100's role and signalling in neural cells remain elusive. We recently demonstrated that the S100A4 protein protects neurons in brain injury and designed S100A4-derived peptides mimicking its beneficial effects. Here we show that neuroprotection by S100A4 involves the growth factor family receptor ErbB4 and its ligand Neuregulin 1 (NRG), key regulators of neuronal plasticity and implicated in multiple brain pathologies. The neuroprotective effect of S100A4 depends on ErbB4 expression and the ErbB4 signalling partners ErbB2/Akt, and is reduced by functional blockade of NRG/ErbB4 in cell models of neurodegeneration. We also detect binding of S100A4 with ErbB1 (EGFR) and ErbB3. S100A4-derived peptides interact with, and signal through ErbB, are neuroprotective in primary and immortalized dopaminergic neurons, and do not affect cell proliferation/motility - features which make them promising as potential neuroprotectants. Our data suggest that the S100-ErbB axis may be an important mechanism regulating neuronal survival and plasticity.

Pathological histone acetylation in Parkinson's disease: Neuroprotection and inhibition of microglial activation through SIRT 2 inhibition.

Parkinson's disease (PD) is associated with degeneration of nigrostriatal neurons due to intracytoplasmic inclusions composed predominantly of a synaptic protein called α-synuclein. Accumulations of α-synuclein are thought to 'mask' acetylation sites on histone proteins, inhibiting the action of histone acetyltransferase (HAT) enzymes in their equilibrium with histone deacetylases (HDACs), thus deregulating the dynamic control of gene transcription. It is therefore hypothesised that the misbalance in the actions of HATs/HDACs in neurodegeneration can be rectified with the use of HDAC inhibitors, limiting the deregulation of transcription and aiding neuronal homeostasis and neuroprotection in disorders such as PD. Here we quantify histone acetylation in the Substantia Nigra pars compacta (SNpc) in the brains of control, early and late stage PD cases to determine if histone acetylation is a function of disease progression. PD development is associated with Braak-dependent increases in histone acetylation. Concurrently, we show that as expected disease progression is associated with reduced markers of dopaminergic neurons and increased markers of activated microglia. We go on to demonstrate that in vitro, degenerating dopaminergic neurons exhibit histone hypoacetylation whereas activated microglia exhibit histone hyperacetylation. This suggests that the disease-dependent increase in histone acetylation observed in human PD cases is likely a combination of the contributions of both degenerating dopaminergic neurons and infiltrating activated microglia. The HDAC SIRT 2 has become increasingly implicated as a novel target for mediation of neuroprotection in PD: the neuronal and microglial specific effects of its inhibition however remain unclear. We demonstrate that SIRT 2 expression in the SNpc of PD brains remains relatively unchanged from controls and that SIRT 2 inhibition, via AGK2 treatment of neuronal and microglial cultures, results in neuroprotection of dopaminergic neurons and reduced activation of microglial cells. Taken together, here we demonstrate that histone acetylation is disease-dependently altered in PD, likely due the effects of dopaminergic neurodegeneration and microglial infiltration; yet SIRT 2 remains relatively unaltered with disease. Given the stable nature of SIRT 2 expression with disease and the effects of SIRT 2 inhibitor treatment on degenerating dopaminergic neurons and activated microglia detected in vitro, SIRT 2 inhibitors warrant further investigation as potential therapeutics for the treatment of the PD.