Iron Chelator Deferiprone Research Articles

Puerarin Induces Ferroptosis in Colorectal Cancer Cells via Triggering NCOA4 Upregulation

Puerarin shows promise as an anti-cancer compound, but its mechanism of action remains unclear. Here we explored whether and how it promotes ferroptosis in a colorectal cancer cell line. The level of ferroptosis and expression of autophagy proteins were compared between puerarin-treated HT-29 cells expressing normal or reduced levels of the autophagy protein ATG5 or the ferritinophagy protein nuclear receptor coactivator 4 (NCOA4). Puerarin increased lipid peroxidation and inhibited cell proliferation in a dose-dependent manner, indicating the induction of ferroptosis. These effects were partially reversed by ferrostatin-1, a scavenger of reactive oxygen species; by the iron chelator deferiprone; by repression of autophagy through administration of 3-methyladenine or knockdown of autophagy-related gene 5 (ATG5); or by repression of ferritinophagy through NCOA4 knockdown. Puerarin may induce the proliferative inhibition of colorectal cancer cells by triggering ferroptosis through a mechanism requiring NCOA4 ferritinophagy.

Distinct regulation of iron homeostasis in rat heart and liver in response to systemic iron deficiency

Chronic heart failure (HF) is characterized by severe cardiac mitochondrial dysfunction. Cellular energetic function is importantly regulated by iron since mitochondrial enzymes involved in oxidative phosphorylation, antioxidative defense and oxygen transport require iron-containing cofactors (heme, Fe-S clusters). In HF, systemic iron deficiency (ID) shows high prevalence (30–50%) and is associated with worse exercise capacity and increased mortality. Human and animal studies showed discordant results concerning iron content in failing heart, even reporting mitochondrial iron overload in some instance. This leads to a controversy of using iron chelation or iron supplementation as metabolic therapy for HF. Our aim was to investigate the regulation of key actors of cellular iron metabolism in the left (LV) and the right (RV) ventricles of two rat models of systemic ID. A comparative study was also performed in the liver, the main iron storage organ known to sense systemic ID. We developed two rat models of systemic ID without anaemia and cardiac remodelling, using low iron diet for 60 days, combined either with two distinct blood withdrawals at day 0 and 30 or with iron chelator deferiprone from day 1 to 60. Control rats were fed on a normal iron diet. Rats were sacrificed at day 60, serum and tissue iron contents were determined using a photometric colorimetric method. mRNA and protein levels of transferrin receptor 1 (TfR1), L-ferritin (L-Ft) and ferroportin (Fpn), involved in iron uptake, storage and export respectively were measured in biopsies of RV, LV and liver. Liver hepcidin, regulating systemic iron homeostasis, was analysed at mRNA level as well as cardiac hepcidin required for autonomous cellular iron homeostasis. Our main results showed that LV and RV maintained their iron content in a physiological range while the liver drastically decreased its iron pool in response to ID. In this condition, the RV exhibited a post-transcriptional regulation of both TfR1, L-Ft and Fpn similarly to the one found in the liver. In contrast, the LV showed a post-translational regulation of TfR1, a potential post-transcriptional regulation of L-Ft and no regulation for Fpn in ID. Our study showed that both ventricles initiate an adaptative response to maintain their physiological iron pool under systemic ID. These results should be confirmed in a HF model with systemic ID to better establish the benefits of iron chelation or supplementation in HF patients.

RP-HPLC Method Development for Simultaneous Estimation of Oral Iron Chelator Deferiprone and its related Impurity

Purpose: This study aims to develop and validate a RP-HPLC method for simultaneous estimation of deferiprone and its synthesized impurity. Method: Structure of impurity was confirmed by spectral analysis while their purity was confirmed by HPLC analysis. The chromatographic separation was achieved on Agilent Zorbax Bonus-RP (250 x 4.6mm, 5µ) using mobile phase of 20 ACN-80 0.1% TFA. Detection was carried out at 280 nm using PDA detector with flow rate of 1 ml/min and temperature of 30℃. Result: The method was validated as per ICH guidelines. The percent recovery was found to be within limit and %RSD for deferiprone and the impurity was found to be 0.09 and 0.48. The standard curve was linear over the concentration range of 60-140µg/ml and shows correlation coefficient (r2) of 1. The LOD and LOQ values for deferiprone were found to be 0.21µg/ml and 0.62µg/ml respectively. The method was found to be robust even by change in flow rate of ±0.2ml/min. Conclusion: Validation studies demonstrated that the proposed HPLC method is specific, reliable, rapid and reproducible. The developed method can be successfully employed for routine analysis for deferiprone in pharmaceutical dosage form.

Palmitic acid-induced ferroptosis via CD36 activates ER stress to break calcium-iron balance in colon cancer cells.

Ferroptosis, featuring an iron-dependent peroxidation of lipids, is a novel form of programmed cell death that may hold great potential in cancer therapy. Our study found that palmitic acid (PA) inhibited colon cancer cell viability in vitro and in vivo, in conjunction with an accumulation of reactive oxygen species (ROS) and lipid peroxidation. The ferroptosis inhibitor Ferrostatin-1 but not Z-VAD-FMK (a pan-caspase inhibitor), Necrostatin-1 (a potent necroptosis inhibitor), or CQ (a potent inhibitor of autophagy), rescued the cell death phenotype induced by PA. Subsequently, we verified that PA induces ferroptotic cell death through excess iron as cell death was inhibited by iron chelator deferiprone (DFP), while it was exacerbated by a supplement of ferric ammonium citrate (FAC). Mechanistically, PA affects intracellular iron content by inducing endoplasmic reticulum (ER) stress leading to ER calcium release and regulating transferrin transport through increasing cytosolic calcium levels. Furthermore, we observed that cells with high expression of CD36 were more vulnerable to PA-induced ferroptosis. Altogether, our findings reveal that PA engages in anticancer properties by activating ER stress/ER calcium release/transferrin-dependent ferroptosis, and PA might serve as a compound to activate ferroptosis in colon cancer cells with high CD36 expression.

Oxidative Stress and Renal Function in Pediatric Patients with Beta Thalassemia Major (β-TM) Receiving Deferiprone and Deferasirox: A Cross-Sectional, Single Center Study

Oxidative stress in β-TM (beta-thalassemia major) patients is associated with increased of malondialdehyde (MDA) level and also decreased of superoxide dismutase (SOD) level. Deferiprone and deferasirox, which are used for the treatment of iron overload, exhibit antioxidant potential. However, various clinical studies have shown an increase in creatinine levels in pediatric patients receiving oral iron chelator. There was limited study assessed oxidative stress and impact of β-TM on the renal function, especially in children that growing up with β-TM and receiving iron chelator. Therefore, the aim of the study is to investigate renal function and oxidative stress between β-TM patients at Ulin Hospital Banjarmasin who received deferasirox and deferiprone. Ninety β-TM patients (aged 2-≤18 years) with regular iron chelators (deferiprone or deferasirox) use at Ulin Hospital between October-December 2020, were included in this cross-sectional study. Laboratory examinations included complete peripheral blood count, serum ferritin, urea, creatinine, MDA and SOD. Statistical analysis was used to compare all parameters between two groups.There was no significant difference in the levels of MDA (p= 0.663), SOD (p= 0.102), urea (p= 0.597), creatinine (p= 0.067) and glomerular filtration rate (p= 0.792) between the two groups. In this study, 9 patients had decreased GFR, of which 3 patients (33.3%) were taking DFX. Thus, 13.6% of DFX users (3 of 22 patients) and 8.8% of DFP users (6 of 68 patients) had decreased renal function. In general, the mean glomerular filtration rate (GFR) of the patients in this study ranged from 126.74± 32.71ml/kg/min/1.73m2. For conclusion, deferiprone and deferasirox had no significant difference in terms of protection against oxidative stress. However, the decline in renal function occurred slightly higher in deferasirox users. Early recognition will be an important key to prevent renal complication

Iron bioavailability regulates Pseudomonas aeruginosa interspecies interactions through type VI secretion expression

The cystic fibrosis (CF) respiratory tract harbors pathogenic bacteria that cause life-threatening chronic infections. Of these, Pseudomonas aeruginosa becomes increasingly dominant with age and is associated with worsening lung function and declining microbial diversity. We aimed to understand why P. aeruginosa dominates over other pathogens to cause worsening disease. Here, we show that P. aeruginosa responds to dynamic changes in iron concentration, often associated with viral infection and pulmonary exacerbations, to become more competitive via expression of the TseT toxic effector. However, this behavior can be therapeutically targeted using the iron chelator deferiprone to block TseT expression and competition. Overall, we find that iron concentration and TseT expression significantly correlate with microbial diversity in the respiratory tract of people with CF. These findings improve our understanding of how P. aeruginosa becomes increasingly dominant with age in people with CF and provide a therapeutically targetable pathway to help prevent this shift.

In vitro effect of the iron chelator deferiprone on the antimicrobial susceptibility and biofilms of Burkholderia pseudomallei

This study evaluated the effect of the iron chelator deferiprone (DFP) on antimicrobial susceptibility and biofilm formation and maintenance by Burkholderia pseudomallei. Planktonic susceptibility to DFP alone and in combination with antibiotics was evaluated by broth microdilution and biofilm metabolic activity was determined with resazurin. DFP minimum inhibitory concentration (MIC) range was 4–64 µg/mL and in combination reduced the MIC for amoxicillin/clavulanate and meropenem. DFP reduced the biomass of biofilms by 21 and 12% at MIC and MIC/2, respectively. As for mature biofilms, DFP reduced the biomass by 47%, 59%, 52% and 30% at 512, 256, 128 and 64 µg/mL, respectively, but did not affect B. pseudomallei biofilm viability nor increased biofilm susceptibility to amoxicillin/clavulanate, meropenem and doxycycline. DFP inhibits planktonic growth and potentiates the effect of β-lactams against B. pseudomallei in the planktonic state and reduces biofilm formation and the biomass of B. pseudomallei biofilms.

Lipocalin-2 promotes acute lung inflammation and oxidative stress by enhancing macrophage iron accumulation.

Lipocalin-2 (LCN2) is an acute-phase protein that regulates inflammatory responses to bacteria or lipopolysaccharide (LPS). Although the bacteriostatic role of LCN2 is well studied, the function of LCN2 in acute lung damage remains unclear. Here, LCN2 knockout (KO) mice were used to investigate the role of LCN2 in LPS-treated mice with or without recombinant LCN2 (rLCN2). In addition, we employed patients with pneumonia. RAW264.7 cells were given LCN2 inhibition or rLCN2 with or without iron chelator deferiprone. LCN2 KO mice had a higher survival rate than wild-type (WT) mice after LPS treatment. In addition to elevated LCN2 levels in serum and bronchoalveolar lavage fluid (BALF), LPS treatment also increased LCN2 protein in alveolar macrophage lysates of BALF. LCN2 deletion attenuated neutrophil and macrophage infiltration in the lungs of LPS-treated mice as well as serum and BALF interleukin-6 (IL-6). Circulating proinflammatory cytokines and LCN2-positive macrophages were prominently increased in the BALF of pneumonia patients. In addition to increase of iron-stained macrophages in pneumonia patients, increased iron-stained macrophages and oxidative stress in LPS-treated mice were inhibited by LCN2 deletion. In contrast, rLCN2 pretreatment aggravated lung inflammation and oxidative stress in LPS-treated WT mice and then resulted in higher mortality. In RAW264.7 cells, exogenous LCN2 treatment also increased inflammation and oxidative stress, whereas LCN2 knockdown markedly diminished these effects. Furthermore, deferiprone inhibited inflammation, oxidative stress, and phagocytosis in RAW264.7 cells with high LCN2 levels, as well as LPS-induced acute lung injury in WT and LCN2 KO mice. Thus, these findings suggest that LCN2 plays a key role in inflammation and oxidative stress following acute lung injury and that LCN2 is a potential therapeutic target for pneumonia or acute lung injury.

Trial of Deferiprone in Parkinson’s Disease

BackgroundIron content is increased in the substantia nigra of persons with Parkinson’s disease and may contribute to the pathophysiology of the disorder. Early research suggests that the iron chelator deferiprone can reduce nigrostriatal iron content in persons with Parkinson’s disease, but its effects on disease progression are unclear.MethodsWe conducted a multicenter, phase 2, randomized, double-blind trial involving participants with newly diagnosed Parkinson’s disease who had never received levodopa. Participants were assigned (in a 1:1 ratio) to receive oral deferiprone at a dose of 15 mg per kilogram of body weight twice daily or matched placebo for 36 weeks. Dopaminergic therapy was withheld unless deemed necessary for symptom control. The primary outcome was the change in the total score on the Movement Disorder Society–sponsored revision of the Unified Parkinson’s Disease Rating Scale (MDS-UPDRS; range, 0 to 260, with higher scores indicating more severe impairment) at 36 weeks. Secondary and exploratory clinical outcomes at up to 40 weeks included measures of motor and nonmotor disability. Brain iron content measured with the use of magnetic resonance imaging was also an exploratory outcome.ResultsA total of 372 participants were enrolled; 186 were assigned to receive deferiprone and 186 to receive placebo. Progression of symptoms led to the initiation of dopaminergic therapy in 22.0% of the participants in the deferiprone group and 2.7% of those in the placebo group. The mean MDS-UPDRS total score at baseline was 34.3 in the deferiprone group and 33.2 in the placebo group and increased (worsened) by 15.6 points and 6.3 points, respectively (difference, 9.3 points; 95% confidence interval, 6.3 to 12.2; P<0.001). Nigrostriatal iron content decreased more in the deferiprone group than in the placebo group. The main serious adverse events with deferiprone were agranulocytosis in 2 participants and neutropenia in 3 participants.ConclusionsIn participants with early Parkinson’s disease who had never received levodopa and in whom treatment with dopaminergic medications was not planned, deferiprone was associated with worse scores in measures of parkinsonism than those with placebo over a period of 36 weeks. (Funded by the European Union Horizon 2020 program; FAIRPARK-II ClinicalTrials.gov number, NCT02655315.)

Ferroptosis inhibitor liproxstatin-1 alleviates metabolic dysfunction-associated fatty liver disease in mice: potential involvement of PANoptosis.

Ferroptosis is a new form of regulated cell death characterized by excessive iron accumulation and uncontrollable lipid peroxidation. The role of ferroptosis in metabolic dysfunction-associated fatty liver disease (MAFLD) is not fully elucidated. In this study we compared the therapeutic effects of ferroptosis inhibitor liproxstatin-1 (LPT1) and iron chelator deferiprone (DFP) in MAFLD mouse models. This model was established in mice by feeding a high-fat diet with 30% fructose in water (HFHF) for 16 weeks. The mice then received LPT1 (10 mg·kg-1·d-1, ip) or DFP (100 mg·kg-1·d-1, ig) for another 2 weeks. We showed that both LPT1 and DFP treatment blocked the ferroptosis markers ACSL4 and ALOX15 in MAFLD mice. Furthermore, LPT1 treatment significantly reduced the liver levels of triglycerides and cholesterol, lipid peroxidation markers 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA), and ameliorated the expression of lipid synthesis/oxidation genes (Pparα, Scd1, Fasn, Hmgcr and Cpt1a), insulin resistance, mitochondrial ROS content and liver fibrosis. Importantly, LPT1 treatment potently inhibited hepatic apoptosis (Bax/Bcl-xL ratio and TUNEL+ cell number), pyroptosis (cleavages of Caspase-1 and GSDMD) and necroptosis (phosphorylation of MLKL). Moreover, LPT1 treatment markedly inhibited cleavages of PANoptosis-related caspase-8 and caspase-6 in MAFLD mouse liver. In an in vitro MAFLD model, treatment with LPT1 (100 nM) prevented cultured hepatocyte against cell death induced by pro-PANoptosis molecules (TNF-α, LPS and nigericin) upon lipid stress. On the contrary, DFP treatment only mildly attenuated hepatic inflammation but failed to alleviate lipid deposition, insulin resistance, apoptosis, pyroptosis and necroptosis in MAFLD mice. We conclude that ferroptosis inhibitor LPT1 protects against steatosis and steatohepatitis in MAFLD mice, which may involve regulation of PANoptosis, a coordinated cell death pathway that involves apoptosis, pyroptosis and necroptosis. These results suggest a potential link between ferroptosis and PANoptosis.

Combined administration of membrane-permeable and impermeable iron-chelating drugs attenuates ischemia/reperfusion-induced hepatic injury.

The underlying pathophysiological mechanisms of hepatic ischemia-reperfusion (I/R) injury have not been completely elucidated. However, it is well known that oxidative stress, caused by a burst of reactive oxygen species (ROS) production during the reperfusion phase, plays a crucial role. A growing body of evidence indicates that the intracellular availability of free iron represents a requirement for ROS-induced adverse effects, as iron catalyzes the generation of highly reactive free radicals. The aim of this study was to examine whether a combination of iron chelators with varying lipophilicity could offer enhanced protection against I/R by diminishing the conversion of weak oxidants, like H2O2, to extremely reactive ones such as hydroxyl radicals (HO.). HepG2 cells (hepatocellular carcinoma cell line) were exposed to oxidative stress conditions after pre-treatment with the iron chelators desferrioxamine (DFO) and deferiprone (DFP) alone or in combination. Labile iron pool was estimated using the calcein-acetoxymethyl ester (calcein-AM) method and DNA damage with the comet assay. We subsequently used a rabbit model (male New Zealand white rabbits) of hepatic I/R-induced injury to investigate, by measuring biochemical (ALT, ALT, ALP, γGT) and histological parameters, whether this may be true for in vivo conditions. The combination of a membrane-permeable iron chelator (DFP) with a strong membrane-impermeable one (DFO) raises the level of protection in both hepatic cell lines exposed to oxidative stress conditions and hepatic I/R rabbit model. Our results show that combinations of iron chelators with selected lipophilicity and iron-binding properties may represent a valuable strategy to protect against tissue damage during reperfusion after a period of ischemia.

Novel Antidepressant-Like Properties of the Iron Chelator Deferiprone in a Mouse Model of Depression

Depressed individuals who carry the short allele for the serotonin-transporter-linked promotor region of the gene are more vulnerable to stress and have reduced response to first-line antidepressants such as selective serotonin reuptake inhibitors. Since depression severity has been reported to correlate with brain iron levels, the present study aimed to characterise the potential antidepressant properties of the iron chelator deferiprone. Using the serotonin transporter knock-out (5-HTT KO) mouse model, we assessed the behavioural effects of acute deferiprone on the Porsolt swim test (PST) and novelty-suppressed feeding test (NSFT). Brain and blood iron levels were also measured following acute deferiprone. To determine the relevant brain regions activated by deferiprone, we then measured c-Fos expression and applied network-based analyses. We found that deferiprone reduced immobility time in the PST in 5-HTT KO mice and reduced latency to feed in the NSFT in both genotypes, suggesting potential antidepressant-like effects. There was no effect on brain or blood iron levels following deferiprone treatment, potentially indicating an acute iron-independent mechanism. Deferiprone reversed the increase in c-Fos expression induced by swim stress in 5-HTT KO mice in the lateral amygdala. Functional network analyses suggest that hub regions of activity in mice treated with deferiprone include the caudate putamen and prefrontal cortex. The PST-induced increase in network modularity in wild-type mice was not observed in 5-HTT KO mice. Altogether, our data show that the antidepressant-like effects of deferiprone could be acting via an iron-independent mechanism and that these therapeutic effects are underpinned by changes in neuronal activity in the lateral amygdala.

Abstract LB135: Deferiprone alters macrophage function and metabolism

Abstract We studied the effects of the intracellular, clinically approved, iron chelator deferiprone (DFP) on macrophage characteristics, function, and metabolism. This research expands on our previous studies investigating the ability of DFP to alter metabolism of breast and prostate cancer tumors and enhance treatment response. We used the murine macrophage cell line RAW 264.7, unpolarized (M0) &amp; polarized (to M1 by LPS, to M2 by IL-4), in vitro to evaluate the effects of DFP on macrophage (i) doubling times (cell count assay), (ii) polarization (flow cytometry), (iii) functional response by measuring cytokine (IL-12, IL-10, TNFα), secretion (Luminex multiplex cytokine detection system, Millipore), phagocytosis (IgG-coated latex beads), and reactive oxygen species production (fluorometric detection), (iv) glycolytic and oxidative metabolism (Agilent xFe96 analyzer), and (v) glycolytic metabolism (measuring incorporation of 1-13C-glucose by 1H &amp; 13C magnetic resonance spectroscopy (MRS) of cell extracts and supernatants). The EC50s of 48 h DFP exposure are 79±5 µM, 93±3 µM, and 114±5 µM for M0, M1, and M2-polarized RAW 264.7 cells, respectively. The effect of DFP is cytostatic and increases cell doubling times. Exposure to 100 µM DFP for 48 h increases the number of M1-polarized cells in M0 and M1 cultures, but not in M2-polarized cells. While M2-polarized cells secreted more anti-inflammatory IL-10 and less pro-inflammatory TNFα than M0 cells, their cytokine secretion was unaffected by DFP. M1-polarized cells stimulated the secretion of TNFα and IL-10. The IL-10 secretion may be a feedback mechanism to curtail the pro-inflammatory effects of LPS. DFP decreased IL-10 secretion by ~33% in M1-polarized cells, enhancing their M1 activation state. IL-12 secretion was minimal and inconsistent. The ~21% phagocytosis in M0 cells remained unchanged by M2 polarization and increased to ~37% in M1-polarized cells. DFP exposure increased phagocytosis efficiency in M0, M1, and M2-polarized cells but highest in M1 cells. While no detectable H2O2 was produced, RAW 264.7 cells make detectable nitric oxide (NO), with M1-polarized cells yielding the most NO. NO yield was unchanged by DFP. Like the results above, we found the greatest effect of DFP to be on mitochondrial metabolism in M1 macrophages, specifically, significant reductions of maximal, basal, and ATP-linked respiration. The increased suppression of oxidative metabolism in M1-polarized macrophages by DFP conforms with the observed reduction of anti-inflammatory cytokines. The DFP increased phagocytosis, specifically dominant in M1 macrophages, suggests an increased reliance on glycolysis. To support the above observations, we currently study the incorporation of 1-13C-Glucose into cellular metabolites of M0, M1, and M2-polarized cells without &amp; with DFP. Our data support a pro-inflammatory effect of DFP on macrophages, which may enhance their anti-tumor effect in vivo through altering tumor and macrophage metabolism. Citation Format: Ellen Ackerstaff, Sadna Budhu, Dov P. Winkleman, Soe S. Min, Jenny N. Ijoma, Inna S. Serganova, Ronald G. Blasberg, Taha Merghoub, Jason A. Koutcher. Deferiprone alters macrophage function and metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB135.

Binding of Citrate-Fe3+ to Plastic Culture Dishes, an Artefact Useful as a Simple Technique to Screen for New Iron Chelators.

NaCT mediates citrate uptake in the liver cell line HepG2. When these cells were exposed to iron (Fe3+), citrate uptake/binding as monitored by the association of [14C]-citrate with cells increased. However, there was no change in NaCT expression and function, indicating that NaCT was not responsible for this Fe3+-induced citrate uptake/binding. Interestingly however, the process exhibited substrate selectivity and saturability as if the process was mediated by a transporter. Notwithstanding these features, subsequent studies demonstrated that the iron-induced citrate uptake/binding did not involve citrate entry into cells; instead, the increase was due to the formation of citrate-Fe3+ chelate that adsorbed to the cell surface. Surprisingly, the same phenomenon was observed in culture wells without HepG2 cells, indicating the adsorption of the citrate-Fe3+ chelate to the plastic surface of culture wells. We used this interesting phenomenon as a simple screening technique for new iron chelators with the logic that if another iron chelator is present in the assay system, it would compete with citrate for binding to Fe3+ and prevent the formation and adsorption of citrate-Fe3+ to the culture well. This technique was validated with the known iron chelators deferiprone and deferoxamine, and with the bacterial siderophore 2,3-dihydroxybenzoic acid and the catechol carbidopa.

The neurosteroid pregnenolone is synthesized by a mitochondrial P450 enzyme other than CYP11A1 in human glial cells

Neurosteroids, modulators of neuronal and glial cell functions, are synthesized in the nervous system from cholesterol. In peripheral steroidogenic tissues, cholesterol is converted to the major steroid precursor pregnenolone by the CYP11A1 enzyme. Although pregnenolone is one of the most abundant neurosteroids in the brain, expression of CYP11A1 is difficult to detect. We found that human glial cells produced pregnenolone, detectable by mass spectrometry and ELISA, despite the absence of observable immunoreactive CYP11A1 protein. Unlike testicular and adrenal cortical cells, pregnenolone production in glial cells was not inhibited by CYP11A1 inhibitors DL-aminoglutethimide and ketoconazole. Furthermore, addition of hydroxycholesterols increased pregnenolone synthesis, suggesting desmolase activity that was not blocked by DL-aminoglutethimide or ketoconazole. We explored three different possibilities for an alternative pathway for glial cell pregnenolone synthesis: (1) regulation by reactive oxygen species, (2) metabolism via a different CYP11A1 isoform, and (3) metabolism via another CYP450 enzyme. First, we found oxidants and antioxidants had no significant effects on pregnenolone synthesis, suggesting it is not regulated by reactive oxygen species. Second, overexpression of CYP11A1 isoform b did not alter synthesis, indicating use of another CYP11A1 isoform is unlikely. Finally, we show nitric oxide and iron chelators deferoxamine and deferiprone significantly inhibited pregnenolone production, indicating involvement of another CYP450 enzyme. Ultimately, knockdown of endoplasmic reticulum cofactor NADPH-cytochrome P450 reductase had no effect, while knockdown of mitochondrial CYP450 cofactor ferredoxin reductase inhibited pregnenolone production. These data suggest that pregnenolone is synthesized by a mitochondrial cytochrome P450 enzyme other than CYP11A1 in human glial cells.

Iron overload and impaired iron handling contribute to the dystrophic pathology in models of Duchenne muscular dystrophy.

BackgroundOxidative stress is implicated in the pathophysiology of Duchenne muscular dystrophy (DMD, caused by mutations in the dystrophin gene), which is the most common and severe of the muscular dystrophies. To our knowledge, the distribution of iron, an important modulator of oxidative stress, has not been assessed in DMD. We tested the hypotheses that iron accumulation occurs in mouse models of DMD and that modulation of iron through the diet or chelation could modify disease severity.MethodsWe assessed iron distribution and total elemental iron using LA‐ICP‐MS on skeletal muscle cross‐sections of 8‐week‐old Bl10 control mice and dystrophic mdx mice (with moderate dystrophy) and dystrophin/utrophin‐null mice (dko, with severe dystrophy). In addition, mdx mice (4 weeks) were treated with either an iron chelator (deferiprone 150 mg/kg/day) or iron‐enriched feed (containing 1% added iron as carbonyl iron). Immunoblotting was used to determine the abundance of iron‐ and mitochondria‐related proteins. (Immuno)histochemical and mRNA assessments of fibrosis and inflammation were also performed.ResultsWe observed a significant increase in total elemental iron in hindlimb muscles of dko mice (+50%, P < 0.05) and in the diaphragm of mdx mice (+80%, P < 0.05), with both tissues exhibiting severe pathology. Iron dyshomeostasis was further evidenced by an increase in the storage protein ferritin (dko: +39%, P < 0.05) and ferroportin compared with Bl10 control mice (mdx: +152% and dko: +175%, P < 0.05). Despite having features of iron overload, dystrophic muscles had lower protein expression of ALAS‐1, the rate‐limiting enzyme for haem synthesis (dko −44%, P < 0.05), and the haem‐containing protein myoglobin (dko −54%, P < 0.05). Deferiprone treatment tended to decrease muscle iron levels in mdx mice (−30%, P < 0.1), which was associated with lower oxidative stress and fibrosis, but suppressed haem‐containing proteins and mitochondrial content. Increasing iron via dietary intervention elevated total muscle iron (+25%, P < 0.05) but did not aggravate the pathology.ConclusionsMuscles from dystrophic mice have increased iron levels and dysregulated iron‐related proteins that are associated with dystrophic pathology. Muscle iron levels were manipulated by iron chelation and iron enriched feed. Iron chelation reduced fibrosis and reactive oxygen species (ROS) but also suppressed haem‐containing proteins and mitochondrial activity. Conversely, iron supplementation increased ferritin and haem‐containing proteins but did not alter ROS, fibrosis, or mitochondrial activity. Further studies are required to investigate the contribution of impaired ferritin breakdown in the dysregulation of iron homeostasis in DMD.

Interleukin-6 triggers toxic neuronal iron sequestration in response to pathological α-synuclein.

SUMMARYα-synuclein (α-syn) aggregation and accumulation drive neurodegeneration in Parkinson’s disease (PD). The substantia nigra of patients with PD contains excess iron, yet the underlying mechanism accounting for this iron accumulation is unclear. Here, we show that misfolded α-syn activates microglia, which release interleukin 6 (IL-6). IL-6, via its trans-signaling pathway, induces changes in the neuronal iron transcriptome that promote ferrous iron uptake and decrease cellular iron export via a pathway we term the cellular iron sequestration response, or CISR. The brains of patients with PD exhibit molecular signatures of the IL-6-mediated CISR. Genetic deletion of IL-6, or treatment with the iron chelator deferiprone, reduces pathological α-syn toxicity in a mouse model of sporadic PD. These data suggest that IL-6-induced CISR leads to toxic neuronal iron accumulation, contributing to synuclein-induced neurodegeneration.

Triumvirate to treat mucormycosis: Interplay of pH, metal ions and antifungal drugs

Mucormycosis is a rare fungal infection mainly affecting immunocompromised patients. Recently, an alarming rise in cases of mucormycosis was observed in patients undergoing COVID-19 treatment. Commonly, this fungal infection is caused by Rhizopus spp., Mucor spp., and Lichtheimia spp., although the genera of other Mucorales such as Saksenaea, Rhizomucor, Cunninghamella, and Apophysomyces may also be involved. A number of factors such as high concentration of zinc, iron and hypoxia induced acidosis promote the growth of fungi, thereby increasing the risk of mucormycosis multiple folds. Iron chelators (deferasirox and deferiprone) and zinc chelators (clioquinol, phenanthroline and N,N,N′,N′-tetrakis-(2-pyridylmethyl)ethane-1,2-diamine) have been reported to inhibit the growth of fungi in a number of in vitro and animal studies. Correction of metabolic acidosis by administration of bicarbonate or glycine reduces the susceptibility of patients to the invasion by fungal species. However, these factors have largely been ignored while deciding the treatment strategy for mucormycosis and first line treatment for the management of mucormycosis continues to be the expensive lipid based formulation of amphotericin B. Treatment with isavuconazole which is proposed as the second choice is reported to be more cost effective. Therefore, it is proposed that a combination of antifungal agent (isavuconazole) with zinc and iron chelators (deferasirox and clioquinol respectively) along with alkalizer (glycine buffer) could be an effective and multi-target approach for the treatment of mucormycosis while being cost effective also.

Measuring Autophagic Cargo Flux with Keima-Based Probes.

Autophagy and autophagy-associated genes are implicated in a growing list of cellular, physiological, and pathophysiological processes and conditions. Therefore, it is ever more important to be able to reliably monitor and quantify autophagic activity. Whereas autophagic markers, such as LC3 can provide general indications about autophagy, specific and accurate detection of autophagic activity requires assessment of autophagic cargo flux. Here, we provide protocols on how to monitor bulk and selective autophagy by the use of inducible expression of exogenous probes based on the fluorescent coral protein Keima. To exemplify and demonstrate the power of this system, we provide data obtained by analyses of cytosolic and mitochondrially targeted Keima probes in human retinal epithelial cells treated with the mTOR-inhibitor Torin1 or with the iron chelator deferiprone (DFP). Our data indicate that Torin1 induces autophagic flux of cytosol and mitochondria to a similar degree, that is, compatible with induction of bulk autophagy, whereas DFP induces a highly selective form of mitophagy that efficiently excludes cytosol.

S123: SAFETY AND EFFICACY OF EARLY-START DEFERIPRONE IN INFANTS AND YOUNG CHILDREN WITH BETA-THALASSEMIA (START STUDY)

Background: Patients with beta-thalassemia may need regular red blood cell transfusions in infancy.1 In the absence of iron chelation therapy, frequent transfusions cause iron to accumulate, which can lead to morbidity, organ damage, and death. However, in very young children, current practice is to delay chelation therapy until receipt of 10–20 transfusions, or until serum ferritin (SF) has reached 1000 μg/L, due to concerns over excessive iron depletion with deferoxamine.1,2 Unfortunately, this delay may increase the risk of iron accumulation in endocrine glands where toxicities could later manifest.3 Aims: START (NCT03591575) evaluated the safety and efficacy of the oral iron chelator deferiprone (DFP) in children with transfusion-dependent beta-thalassemia who did not yet meet the criteria for starting chelation therapy per standard practice. Methods: Infants and children receiving regular blood transfusions (2 minimum) and whose SF level was between 200–600 μg/L were randomly assigned 1:1 to DFP oral solution or placebo until SF exceeded 1000 μg/L at 2 consecutive visits or 12 months of therapy. DFP was initiated at 25 mg/kg/d and increased to 50 mg/kg/d after 2 weeks; based on iron load, DFP was increased to 75 mg/kg/d. Efficacy was assessed by monthly SF measurements to monitor iron load. The primary endpoint was the percentage of patients in each group with SF <1000 μg/L. All patients provided informed consent or assent. Results: The study enrolled 64 patients; 32 per group. The mean (SD) age was 3.03 (2.42) years in the DFP group and 2.63 (1.70) years in the placebo group; participants were 62.5% and 65.6% male in the DFP and placebo groups, respectively. There were no group statistical differences in the baseline demographics. The primary reason for withdrawal was SF levels that exceeded 1000 μg/L at 2 consecutive visits, which occurred in 25% of patients receiving DFP vs 63% of patients receiving placebo (P=0.0051). After completing 12 months of treatment, 65.6% of patients receiving DFP had a SF level <1000 mg/L vs 37.5% receiving placebo (P=0.0446). The percentage of patients who reached the 1000 mg/L SF threshold increased more rapidly in the placebo group vs the DFP group, and the difference in rates between the 2 groups was significant (P=0.0407). A summary of adverse events (AEs) is shown in Table 1. There were no significant group differences in the number of overall AEs (P=1.0000), serious AEs (P=0.4258), or the number of AEs that were considered to be at least possibly related to the study treatment (P=1.0000). Two patients receiving DFP withdrew from the study due to AEs: 1 patient experienced agranulocytosis and 1 patient experienced neutropenia of moderate severity and both patients recovered. Conclusions: Initiation of DFP chelation therapy at a lower threshold of SF values than currently recommended was safe and efficacious in preventing iron overload in most transfusion-dependent children. After 12 months of treatment, the number of patients with a SF level <1000 μg/L was significantly higher in the DFP group vs the placebo group. Moreover, there were significantly more patients who withdrew due to elevated SF levels in the placebo group (62.5%) vs the DFP group (25%). The safety and tolerability profile of DFP administered for up to 12 months in infants and young children was comparable to the profile established in older age groups and there were no instances of iron depletion.