Targeting chelatable iron as a therapeutic modality in Parkinson's disease

Abstract

Aims: The pathophysiological role of iron in Parkinson's disease (PD) was assessed by a chelation strategy aimed at reducing oxidative damage associated with regional iron deposition without affecting circulating metals. Translational cell and animal models provided concept proofs and a delayed-start (DS) treatment paradigm, the basis for preliminary clinical assessments.

Results: For translational studies, we assessed the effect of oxidative insults in mice systemically prechelated with deferiprone (DFP) by following motor functions, striatal dopamine (HPLC and MRI-PET), and brain iron deposition (relaxation-R2*-MRI) aided by spectroscopic measurements of neuronal labile iron (with fluorescence-sensitive iron sensors) and oxidative damage by markers of protein, lipid, and DNA modification. DFP significantly reduced labile iron and biological damage in oxidation-stressed cells and animals, improving motor functions while raising striatal dopamine. For a pilot, double-blind, placebo-controlled randomized clinical trial, early-stage Parkinson's patients on stabilized dopamine regimens enrolled in a 12-month single-center study with DFP (30 mg/kg/day). Based on a 6-month DS paradigm, early-start patients (n=19) compared to DS patients (n=18) (37/40 completed) responded significantly earlier and sustainably to treatment in both substantia nigra iron deposits (R2* MRI) and Unified Parkinson's Disease Rating Scale motor indicators of disease progression (p<0.03 and p<0.04, respectively). Apart from three rapidly resolved neutropenia cases, safety was maintained throughout the trial.

Innovation: A moderate iron chelation regimen that avoids changes in systemic iron levels may constitute a novel therapeutic modality for PD.

Conclusions: The therapeutic features of a chelation modality established in translational models and in pilot clinical trials warrant comprehensive evaluation of symptomatic and/or disease-modifying potential of chelation in PD.

Trial registration: ClinicalTrials.gov NCT00943748.

Figures

FIG. 1.

Effects of deferiprone (DFP) in the 1-methyl-4-phenyl-1,2,3,6-tetrapyridine (MPTP) mouse model of Parkinson's disease (PD). Mice treated (or not) with MPTP (n=10 per group) were given equivalent doses of i.p. deferoxamine (DFO) or p.o. DFP: either 200 mg (100 mg twice a day) or 300 mg (150 mg twice a day). (B–F)+Denotes a significant difference versus controls and *denotes a significant difference versus MPTP-treated mice. Mean and SEM are presented. (A) Immunohistochemistry of tyrosine hydroxylase (TH)-stained sections of the right substantia nigra (SN), illustrating the level of neuroprotection afforded by iron chelators. (B) TH+ cell counts of both sides of the SN. +MPTP: p<0.0001; +DFO 200: p=0.002; *DFO 200: p=0.009; *DFP 200–300: p<0.0001. (C ) Total iron levels in the SN, as measured by atomic absorption spectrometry. +MPTP: p=0.035; +DFP 300: p<0.0001; *p<0.0001. (D) R2* multiple-echo spin echo value (7-Tesla MRI) in the SN (using the same mice as in experiments c and d, before sacrifice for spectrometry measurements) (R2*=1/T2*(ms−1)×103) +p=0.001; *p=0.001. (E) Correlation between R2* values measured by MRI and total iron measured by spectrometry in the SN (values were pooled from the different conditions; Spearman test: r=0.6; p=0.0001). (F, G) Motor handicap scores (measured in a 10-min actimetry test). (F) Number of rearing +p=0.004; *DFP 300: p=0.01; *DFP 200: p=0.8; (G) maximum speed: +p=0.03; *DFP 300: p=0.01; *DFP 200: p=0.02. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 2.

Effects of DFP on the oxidative stress response in the MPTP mouse model of PD. (A, B, C) Mean and SEM amounts of oxidative stress markers in the substantia nigra (SN). N=10 per group for all experiments. +Denotes a significant difference versus controls and *denotes a significant difference versus the MPTP condition: (A) For the ratio between reduced glutathione (GSH) and oxidized glutathione (GSSG) (μM/g of protein): +p=0.026; *DFP 300: p=0.002; DFP 200: p=0.04. (B) For malonaldehyde: +p=0.003; DFP 200, DFP 300: *p=0.01. (C) For 8-oxo-deoxyguanosine (8-oxodG): +p=0.02; DFP 200, DFP 300: *p=0.03. DNA: deoxyribonucleic acid. (D)Mitochondrial labile iron pool. Mitochondria isolated from rat brain were loaded with calcein-AM as described in Materials and Methods section and treated with either DFP or DFO at the indicated concentrations for 20 min at 37°C. Fluorescence of calcein (given in arbitrary instrument units) was measured with a spectrofluorimeter (set at zero level with a sample of unlabeled mitochondria) (mean values of n=3 experiments). The size of the arrow denotes the increment in fluorescence intensity attained after addition of either 100 or 300 μM of the permeant chelator DFP over that attained with an equivalent concentration of the impermeant chelator DFO (that reveals iron bound to extramitochondrial calcein) (*p=0.005 and 0.001, respectively). The size of the arrows is a measure of the intramitochondrial labile (=DFP-chelatable) iron pool (in calcein fluorescence units—a.u.—that are 1:1 equivalent 1:1 to labile iron).

FIG. 3.

Effects of DFP on the dopamine system in the MPTP mouse model of PD. (A) Mean [18F]-DOPA distribution in an axial view of co-registered brain MRI-PET images at the striatal level. [18F]-DOPA levels were significantly lower after acute MPTP intoxication than in controls (p=0.029) and were significantly higher after acute MPTP intoxication with deferiprone treatment at 300 mg/day than after acute MPTP intoxication alone (p=0.03). (B) Mean (SEM) striatal levels of dopamine (DA) and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). Compared with the control condition: +DA-MPTP: p=0.008; +DOPAC-MPTP: p=0.01; +HVA-MPTP: p=0.04; (DA-DFP 300: p=0.2); +HVA-DFP 300: p=0.012; Compared with the MPTP condition: *DA-MPTP-DFP 300: p=0.0011; *DA- MPTP-DFP 200: p=0.0019. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 4.

Effects of DFP on oxidative-stressed cells.+Denotes a significant difference relative to control and *denotes a significant difference relative to the intoxication. (A–C) Human dopaminergic neurons. Results are given as the mean and SEM from three independent experiments: Data for the application of DFP or DFO (100 μM or 300 μM) to control cells are not shown, as there were no statistically significant changes. (A) Forty-eight hours of MPP intoxication (5 μM): cell viability:+p<0.0001; *DFP at 100 μM: p=0.003; DFP 300 μM: p=0.002; DFO 100 μM: p=0.01. ATP production:+0.004; *DFP 100 μM: p=0.003; DFP 300 μM: p=0.002. (B) 3 h of menadione (MEN) intoxication (200 μM): cell viability: +p<0.0001; *DFP 100 μM: p=0.002; *DFP 300 μM: p=0.002; DFO: no protection. ATP production:+p<0.0001; *DFP 100 μM: p=0.03; DFP 300 μM: p=0.002. (C) 18 h of N-ethylmaleimide (NEM) intoxication (5 μM): cell viability: +p=0.029; *DFP 100 μM: p=0.026; DFP 300 μM: p=0.02; DFO 100 and 300 μM: p=0.04. For all conditions: the addition of 75 μM of iron reversed the effect of chelators. (D–F) Effect of DFP on lymphocytes. Lymphocytes, obtained from 5 early-stage PD patients (mean time since diagnosis: 2.9±0.6 years; mean age: 61.4±3.6 years) by cytapheresis were prechelated with either DFO or DFP for 1 h (supplemented or not with 100 μM ferric ammonium citrate) and treated with MEN (200 and 300 μM) for 3 h and assessed by fluorescence (D)cell viability:+p=0.01; *DFP 100 μM: p=0.04; DFP 300 μM: p=0.02. (E)ROS production: +p=0.002; *DFP 100 μM: p=0.01; DFP 300 μM: p=0.01. (F) Labile cell iron pool in human lymphocytes treated with MEN (as in D). ΔF is the mean fluorescence change after addition of excess DFP to control or pretreated lymphocytes (6, 54). Fe: +p=0.03; DFP: +p=0.01; MEN: +p=0.004; *p=0.02.

FIG. 5.

Effect of DFP treatment on the R2* MRI parameter in the substantia nigra (SN) of PD patients. The clinical study was carried out as described in Supplementary Figure S1, with patients who started treatment at month 0 denoted as “ES” (early start) or at month 6 denoted as “DS” (delayed start). All patients continued DFP treatment until month 18 and were then randomized in a drug cessation paradigm between months 18 and 24; the “EC” (early cessation) group stopped DFP altogether and the “DC” (delayed cessation) group continued DFP until month 24. (A) Depicts an image from a parametric T2*-weighted gradient-echo 3-Tesla MRI. The demarcated area of the SN was used as the region of interest for calculating the relaxation time R2* (R2*=1/T2*), as described in the Materials and Methods section. (B) The R2* values calculated for the SN and the putamen (Pu) (quoted as the mean±SEM) for each group of patients represent the change in R2* (=ΔR2*) from baseline following either 6 or 12 months of DFP treatment: SN: *p=0.0001; SN: #p=0.03; Pu: #p=0.01; covariance analysis adjusted for baseline. (C) The change in R2* (=ΔR2*) from month 18 to month 24 treatment in the EC and DC groups: p=0.039; the covariance analysis was adjusted for baseline.

FIG. 6.

Laboratory parameters. Changes after 6 months of treatment with DFP. Means±SEM are given. (A) CSF assays of dopamine (DA) metabolites in DA-treated patients (n=20: 10 ES patients and 10 DS patients). The levels of the DA metabolites HVA and 3,4-dihydroxyphenylacetic acid (DOPAC) are given relative to those of DA and are baseline adjusted. The HVA/DA ratio declined after 6 months of DFP (covariance analysis: F(1,14)=8.8; p=0.014). Inset: Ferritin levels (relative to the baseline value; * p=0.0001; see also Table 1) (B) Serum assays of the L-dopa metabolite 3-O-methyldopa in L-dopa-treated patients (n=17: 9 ES patients and 8 DS patients). Levels of 3-O-methyldopa are given relative to L-dopa; they declined significantly (*) after 6 months of treatment (baseline-adjusted covariance analysis: F(1,14)=5.5; p=0.03) in the ES group relative to the DS group but stabilized after 12 months (p=0.7). (C) Serum assays of DA auto-oxidation in DA- and L-dopa-treated patients (n=17: nine ES patients and eight DS patients). The DA auto-oxidation product 5-cysteinyldopamine (5-cyst-Dopa) was not detected in the CSF or plasma of early-stage PD patients treated with DA agonists. In PD patients treated with L-dopa, the 5-cyst-Dopa/DA ratio decreased significantly following 6 months of DFP treatment in the ES group (n=9) relative to the DS group (n=8) (baseline-adjusted covariance analysis: F(1,10)=5.7; p=0.029) and in the DS group after 6 months of treatment (i.e., between months 6 and 12). (D)Ex vivo viability of lymphocytes challenged with hydrogen peroxide. Lymphocytes isolated at month 6 from 10 ES patients and 10 DS patients were challenged with hydrogen peroxide and assessed for viability. The proportion of viable cells is quoted as a percentage of the number of control (nonchallenged) cells. Challenged lymphocytes from ES patients (after 6 months of DFP treatment) had a greater proportion of viable cells (*p=0.035) than those from DS patients (no DFP during the first 6 months). (E) Hematological parameters. Hematocrit (hct) and haemoglobin (Hb) level. According to baseline-adjusted covariance analysis, there were no significant changes over the course of treatment. (F) Changes (relative to baseline values) in serum ferritin. *Indicates a significant difference between groups at 6 months (p=0.018; see Table 1), but there were no significant differences at 12 and 18 months.

FIG. 7.

Motor handicap (the UPDRS motor score) over the course of DFP treatment in a delayed-start paradigm (Supplementary Fig. S1). Details of the dopaminergic drug treatments are provided in Supplementary Table S1 and did not change during the course of the study. Left part: Mean±SEM change after 6 to 12 months of treatment (relative to baseline): early-start (ES) group: n=20; delayed-start (DS) group: n=19; *significant difference at 6 months: p=0.002; at 12 months: p=0.04, according to a baseline-adjusted covariance analysis. Right part: Mean±SEM change between 18 and 24 months: early cessation (EC) group: n=18; delayed cessation (DC) group: n=16; *significant difference p=0.003, according to a covariance analysis adjusted for the month 18 visit.