Publications by authors named "Nuri Gueven"

66 Publications

Bioactivity Profiles of Cytoprotective Short-Chain Quinones.

Molecules 2021 Mar 4;26(5). Epub 2021 Mar 4.

School of Pharmacy and Pharmacology, University of Tasmania, Hobart, TAS 7005, Australia.

Short-chain quinones (SCQs) have been investigated as potential therapeutic candidates against mitochondrial dysfunction, which was largely thought to be associated with the reversible redox characteristics of their active quinone core. We recently reported a library of SCQs, some of which showed potent cytoprotective activity against the mitochondrial complex I inhibitor rotenone in the human hepatocarcinoma cell line HepG2. To better characterize the cytoprotection of SCQs at a molecular level, a bioactivity profile for 103 SCQs with different compound chemistries was generated that included metabolism related markers, redox activity, expression of cytoprotective proteins and oxidative damage. Of all the tested endpoints, a positive correlation with cytoprotection by SCQs in the presence of rotenone was only observed for the NAD(P)H:quinone oxidoreductase 1 (NQO1)-dependent reduction of SCQs, which also correlated with an acute rescue of ATP levels. The results of this study suggest an unexpected mode of action for SCQs that appears to involve a modification of NQO1-dependent signaling rather than a protective effect by the reduced quinone itself. This finding presents a new selection strategy to identify and develop the most promising compounds towards their clinical use.
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http://dx.doi.org/10.3390/molecules26051382DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7961879PMC
March 2021

Standard of care versus new-wave corticosteroids in the treatment of Duchenne muscular dystrophy: Can we do better?

Orphanet J Rare Dis 2021 03 4;16(1):117. Epub 2021 Mar 4.

Institute for Health and Sport (IHeS), Victoria University, Melbourne, VIC, Australia.

Background: Pharmacological corticosteroid therapy is the standard of care in Duchenne Muscular Dystrophy (DMD) that aims to control symptoms and slow disease progression through potent anti-inflammatory action. However, a major concern is the significant adverse effects associated with long term-use. MAIN: This review discusses the pros and cons of standard of care treatment for DMD and compares it to novel data generated with the new-wave dissociative corticosteroid, vamorolone. The current status of experimental anti-inflammatory pharmaceuticals is also reviewed, with insights regarding alternative drugs that could provide therapeutic advantage.

Conclusions: Although novel dissociative steroids may be superior substitutes to corticosteroids, other potential therapeutics should be explored. Repurposing or developing novel pharmacological therapies capable of addressing the many pathogenic features of DMD in addition to anti-inflammation could elicit greater therapeutic advantages.
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http://dx.doi.org/10.1186/s13023-021-01758-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7934375PMC
March 2021

Novel Short-Chain Quinones to Treat Vision Loss in a Rat Model of Diabetic Retinopathy.

Int J Mol Sci 2021 Jan 20;22(3). Epub 2021 Jan 20.

School of Pharmacy and Pharmacology, University of Tasmania, Hobart, TAS 7005, Australia.

Diabetic retinopathy (DR), one of the leading causes of blindness, is mainly diagnosed based on the vascular pathology of the disease. Current treatment options largely focus on this aspect with mostly insufficient therapeutic long-term efficacy. Mounting evidence implicates mitochondrial dysfunction and oxidative stress in the central etiology of DR. Consequently, drug candidates that aim at normalizing mitochondrial function could be an attractive therapeutic approach. This study compared the mitoprotective compounds, idebenone and elamipretide, side-by-side against two novel short-chain quinones (SCQs) in a rat model of DR. The model effectively mimicked type 2 diabetes over 21 weeks. During this period, visual acuity was monitored by measuring optokinetic response (OKR). Vision loss occurred 5-8 weeks after the onset of hyperglycemia. After 10 weeks of hyperglycemia, visual function was reduced by 65%. From this point, the right eyes of the animals were topically treated once daily with the test compounds. The left, untreated eye served as an internal control. Only three weeks of topical treatment significantly restored vision from 35% to 58-80%, while visual acuity of the non-treated eyes continued to deteriorate. Interestingly, the two novel SCQs restored visual acuity better than idebenone or elamipretide. This was also reflected by protection of retinal pathology against oxidative damage, retinal ganglion cell loss, reactive gliosis, vascular leakage, and retinal thinning. Overall, mitoprotective and, in particular, SCQ-based compounds have the potential to be developed into effective and fast-acting drug candidates against DR.
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http://dx.doi.org/10.3390/ijms22031016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7864174PMC
January 2021

Idebenone: When an antioxidant is not an antioxidant.

Redox Biol 2021 01 25;38:101812. Epub 2020 Nov 25.

Victoria University, Institute for Health and Sport, Melbourne, Victoria, Australia.

Idebenone is a well described drug that was initially developed against dementia. The current literature widely portrays this molecule as a potent antioxidant and CoQ analogue. While numerous papers seem to support this view, a closer look indicates that the pharmacokinetics of idebenone do not support these claims. A major discrepancy between achievable tissue levels, especially in target tissues such as the brain, and doses required to show the proposed effects, significantly questions our current understanding. This review explains how this has happened and highlights the discrepancies in the current literature. More importantly, based on some recent discoveries, a new framework is presented that can explain the mode of action of this molecule and can align formerly contradictory results. Finally, this new appreciation of the molecular activities of idebenone provides a rational approach to test idebenone in novel indications that might have not been considered previously for this drug.
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http://dx.doi.org/10.1016/j.redox.2020.101812DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7708875PMC
January 2021

Targeting Nrf2 for the treatment of Duchenne Muscular Dystrophy.

Redox Biol 2021 01 18;38:101803. Epub 2020 Nov 18.

College of Health and Biomedicine, Victoria University, Melbourne, Victoria, Australia; Institute for Health and Sport, Victoria University, Melbourne, Victoria, Australia; Australian Institute for Musculoskeletal Science, Victoria University, St Albans, Victoria, Australia. Electronic address:

Imbalances in redox homeostasis can result in oxidative stress, which is implicated in various pathological conditions including the fatal neuromuscular disease Duchenne Muscular Dystrophy (DMD). DMD is a complicated disease, with many druggable targets at the cellular and molecular level including calcium-mediated muscle degeneration; mitochondrial dysfunction; oxidative stress; inflammation; insufficient muscle regeneration and dysregulated protein and organelle maintenance. Previous investigative therapeutics tended to isolate and focus on just one of these targets and, consequently, therapeutic activity has been limited. Nuclear erythroid 2-related factor 2 (Nrf2) is a transcription factor that upregulates many cytoprotective gene products in response to oxidants and other toxic stressors. Unlike other strategies, targeted Nrf2 activation has the potential to simultaneously modulate separate pathological features of DMD to amplify therapeutic benefits. Here, we review the literature providing theoretical context for targeting Nrf2 as a disease modifying treatment against DMD.
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http://dx.doi.org/10.1016/j.redox.2020.101803DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7695875PMC
January 2021

Development of a High-throughput Agar Colony Formation Assay to Identify Drug Candidates against Medulloblastoma.

Pharmaceuticals (Basel) 2020 Nov 5;13(11). Epub 2020 Nov 5.

School of Pharmacy and Pharmacology, College of Health and Medicine, University of Tasmania, Hobart TAS 7005, Tasmania, Australia.

Medulloblastoma (MB) is the most common malignant childhood brain cancer. High-risk MB tumours have a high incidence of metastasis and result in poor patient survival. Drug screens, commonly used to identify potential novel therapeutic agents against MB, focus on 2D cell proliferation and viability assays given that these assays are easily adaptable to high-throughput regimes. However, 2D models fail to address invasive characteristics that are crucial to MB metastasis and are thus not representative of tumour growth in vivo. In this study, we developed a 3D 384-well agar colony formation assay using MB cells of molecular subgroup 3 that is associated with the highest level of metastasis. Two fluorescence substrates, resazurin and glycyl-phenylalanyl-aminofluorocoumarin (GF-AFC) that measure cell viability via distinct mechanisms were used to assess the growth of MB cells in the agar matrix. The assay was optimised for seeding density, growth period, substrate incubation time and homogeneity of the fluorescent signals within individual wells. Our data demonstrate the feasibility to multiplex the two fluorescent substrates without detectable signal interference. This assay was validated by assessing the concentration-dependent effect of two commonly used chemotherapeutic agents clinically used for MB treatment, vincristine and lomustine. Subsequently, a panel of plasma membrane calcium channel modulators was screened for their effect on the 3D growth of D341 MB cells, which identified modulators of T-type voltage gated and ORAI calcium channels as selective growth modulators. Overall, this 3D assay provides a reproducible, time and cost-effective assay for high-throughput screening to identify potential drugs against MB.
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http://dx.doi.org/10.3390/ph13110368DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7694510PMC
November 2020

Dimethyl Fumarate and Its Esters: A Drug with Broad Clinical Utility?

Pharmaceuticals (Basel) 2020 Oct 13;13(10). Epub 2020 Oct 13.

Institute for Health and Sport, Victoria University, Melbourne, VIC 8001, Australia.

Fumaric acid esters (FAEs) are small molecules with anti-oxidative, anti-inflammatory and immune-modulating effects. Dimethyl fumarate (DMF) is the best characterised FAE and is approved and registered for the treatment of psoriasis and Relapsing-Remitting Multiple Sclerosis (RRMS). Psoriasis and RRMS share an immune-mediated aetiology, driven by severe inflammation and oxidative stress. DMF, as well as monomethyl fumarate and diroximel fumarate, are commonly prescribed first-line agents with favourable safety and efficacy profiles. The potential benefits of FAEs against other diseases that appear pathogenically different but share the pathologies of oxidative stress and inflammation are currently investigated.
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http://dx.doi.org/10.3390/ph13100306DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7602023PMC
October 2020

Idebenone Protects against Spontaneous Chronic Murine Colitis by Alleviating Endoplasmic Reticulum Stress and Inflammatory Response.

Biomedicines 2020 Sep 28;8(10). Epub 2020 Sep 28.

Gut Health Laboratory, School of Health Sciences, College of Health and Medicine, University of Tasmania, Launceston 7250, Tasmania, Australia.

Endoplasmic reticulum (ER) stress in intestinal secretory goblet cells has been linked to the development of ulcerative colitis (UC). Emerging evidence suggests that the short chain quinone drug idebenone displays anti-inflammatory activity in addition to its potent antioxidant and mitochondrial electron donor properties. This study evaluated the impact of idebenone in mice, that are characterized by spontaneous chronic intestinal inflammation and ER stress caused by a missense mutation in the mucin gene. Idebenone (200 mg/kg) was orally administered daily to 5-6 weeks old mice over a period of 21 days. Idebenone treatment substantially improved body weight gain, disease activity index (DAI), colon length and histopathology score. Immunohistochemistry revealed increased expression of MUC2 protein in goblet cells, consistent with increased mRNA levels. Furthermore, idebenone significantly reduced the expression of the ER stress markers C/EBP homologous protein (), activating transcription factor 6 () and X-box binding protein-1 () at both mRNA and protein levels. Idebenone also effectively reduced pro-inflammatory cytokine levels in colonic explants. Taken together, these results indicate that idebenone could represent a potential therapeutic approach against human UC by its strong anti-inflammatory activity and its ability to reduce markers of ER stress.
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http://dx.doi.org/10.3390/biomedicines8100384DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7601570PMC
September 2020

Comparative In Vitro Toxicology of Novel Cytoprotective Short-Chain Naphthoquinones.

Pharmaceuticals (Basel) 2020 Aug 7;13(8). Epub 2020 Aug 7.

School of Pharmacy and Pharmacology, University of Tasmania, Hobart, TAS 7005, Australia.

Short-chain quinones (SCQs) have been identified as potential drug candidates against mitochondrial dysfunction, which largely depends on the reversible redox characteristics of the active quinone core. We recently identified 11 naphthoquinone derivatives, - from a library of SCQs that demonstrated enhanced cytoprotection and improved metabolic stability compared to the clinically used benzoquinone idebenone. Since the toxicity properties of our promising SCQs were unknown, this study developed multiplex methods and generated detailed toxicity profiles from 11 endpoint measurements using the human hepatocarcinoma cell line HepG2. Overall, the toxicity profiles were largely comparable across different assays, with simple standard assays showing increased sensitivity compared to commercial toxicity assays. Within the 11 naphthoquinones tested, the -phenylalanine derivative consistently demonstrated the lowest toxicity across all assays. The results of this study not only provide useful information about the toxicity features of SCQs but will also enable the progression of the most promising drug candidates towards their clinical use.
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http://dx.doi.org/10.3390/ph13080184DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7463972PMC
August 2020

Mixed alkoxy/hydroxy 1,8-naphthalimides: expanded fluorescence colour palette and in vitro bioactivity.

Chem Commun (Camb) 2020 Jun 20;56(50):6866-6869. Epub 2020 May 20.

School of Life and Environmental Sciences, Deakin University, Waurn Ponds, 3216, Australia.

An efficient and functional group tolerant route to access hydroxy 1,8-naphthalimides has been used to synthesise a range of mono- and disubstituted hydroxy-1,8-naphthalimides with fluorescence emissions covering the visible spectrum. The dialkoxy substituted compounds prepared possess high quantum yields (up to 0.95) and long fluorescent lifetimes (up to 14 ns). The method has been used to generate scriptaid analogues that successfully inhibit HDAC6 in vitro with tubulin acetylation assays confirming that these compounds are more effective than tubastatin.
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http://dx.doi.org/10.1039/d0cc01251cDOI Listing
June 2020

Micro RNA Expression after Ingestion of Fucoidan; A Clinical Study.

Mar Drugs 2020 Feb 28;18(3). Epub 2020 Feb 28.

Marinova Pty Ltd., Cambridge, TAS 7170, Australia.

Fucoidans are a class of fucose-rich sulfated polysaccharides derived from brown macroalgae that exert a range of biological activities in vitro and in vivo. To generate an unbiased assessment of pathways and processes affected by fucoidan, a placebo-controlled double-blind pilot study was performed in healthy volunteers. Blood samples were taken immediately before and 24 h after ingestion of a single dose of 1 g of fucoidan (UPF) or placebo. Levels of isolated miRNAs were analyzed using Taqman Open Array Human MicroRNA panels. Out of 754 miRNAs screened, UPF affected a total of 53 miRNAs. Pathway analysis using the TALOS data analysis tool predicted 29 different pathways and processes that were largely grouped into cell surface receptor signaling, cancer-related pathways, the majority of which were previously associated with fucoidans. However, this analysis also identified nine pathways and processes that have not been associated with fucoidans before. Overall, this study illustrates that even a single dose of fucoidans has the potential to affect the expression of genes related to fundamental cellular processes. Moreover, it confirms previous data that fucoidans influence immunity, cancer cells, inflammation, and neurological function.
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http://dx.doi.org/10.3390/md18030143DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7143719PMC
February 2020

Metabolic Stability of New Mito-Protective Short-Chain Naphthoquinones.

Pharmaceuticals (Basel) 2020 Feb 12;13(2). Epub 2020 Feb 12.

Australian Centre for Research on Separation Sciences (ACROSS), School of Natural Sciences-Chemistry, College of Science and Engineering, University of Tasmania, Hobart TAS 7000, Australia.

Short-chain quinones (SCQs) have been identified as potential drug candidates against mitochondrial dysfunction, which is largely dependent on their reversible redox characteristics of the active quinone core. We recently synthesized a SCQ library of > 148 naphthoquinone derivatives and identified 16 compounds with enhanced cytoprotection compared to the clinically used benzoquinone idebenone. One of the major drawbacks of idebenone is its high metabolic conversion in the liver, which significantly restricts is therapeutic activity. Therefore, this study assessed the metabolic stability of the 16 identified naphthoquinone derivatives - using hepatocarcinoma cells in combination with an optimized reverse-phase liquid chromatography (RP-LC) method. Most of the derivatives showed significantly better stability than idebenone over 6 h ( < 0.001). By extending the side-chain of SCQs, increased stability for some compounds was observed. Metabolic conversion from the derivative to and reduced idebenone metabolism in the presence of were also observed. These results highlight the therapeutic potential of naphthoquinone-based SCQs and provide essential insights for future drug design, prodrug therapy, and polytherapy, respectively.
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http://dx.doi.org/10.3390/ph13020029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7169385PMC
February 2020

Adenylosuccinic acid therapy ameliorates murine Duchenne Muscular Dystrophy.

Sci Rep 2020 01 24;10(1):1125. Epub 2020 Jan 24.

Institute for Health and Sport, Victoria University, Melbourne, Victoria, 8001, Australia.

Arising from the ablation of the cytoskeletal protein dystrophin, Duchenne Muscular Dystrophy (DMD) is a debilitating and fatal skeletal muscle wasting disease underpinned by metabolic insufficiency. The inability to facilitate adequate energy production may impede calcium (Ca) buffering within, and the regenerative capacity of, dystrophic muscle. Therefore, increasing the metabogenic potential could represent an effective treatment avenue. The aim of our study was to determine the efficacy of adenylosuccinic acid (ASA), a purine nucleotide cycle metabolite, to stimulate metabolism and buffer skeletal muscle damage in the mdx mouse model of DMD. Dystrophin-positive control (C57BL/10) and dystrophin-deficient mdx mice were treated with ASA (3000 µg.mL) in drinking water. Following the 8-week treatment period, metabolism, mitochondrial density, viability and superoxide (O) production, as well as skeletal muscle histopathology, were assessed. ASA treatment significantly improved the histopathological features of murine DMD by reducing damage area, the number of centronucleated fibres, lipid accumulation, connective tissue infiltration and Ca content of mdx tibialis anterior. These effects were independent of upregulated utrophin expression in the tibialis anterior. ASA treatment also increased mitochondrial viability in mdx flexor digitorum brevis fibres and concomitantly reduced O production, an effect that was also observed in cultured immortalised human DMD myoblasts. Our data indicates that ASA has a protective effect on mdx skeletal muscles.
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http://dx.doi.org/10.1038/s41598-020-57610-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6981178PMC
January 2020

Idebenone Protects against Acute Murine Colitis via Antioxidant and Anti-Inflammatory Mechanisms.

Int J Mol Sci 2020 Jan 12;21(2). Epub 2020 Jan 12.

Department of Laboratory Medicine, School of Health Sciences, College of Health and Medicine, University of Tasmania, Launceston, Tasmania 7250, Australia.

Oxidative stress is a key player of the inflammatory cascade responsible for the initiation of ulcerative colitis (UC). Although the short chain quinone idebenone is considered a potent antioxidant and a mitochondrial electron donor, emerging evidence suggests that idebenone also displays anti-inflammatory activity. This study evaluated the impact of idebenone in the widely used dextran sodium sulphate (DSS)-induced mouse model of acute colitis. Acute colitis was induced in C57BL/6J mice via continuous exposure to 2.5% DSS over 7 days. Idebenone was co-administered orally at a dose of 200 mg/kg body weight. Idebenone significantly prevented body weight loss and improved the disease activity index (DAI), colon length, and histopathological score. Consistent with its reported antioxidant function, idebenone significantly reduced the colonic levels of malondialdehyde (MDA) and nitric oxide (NO), and increased the expression of the redox factor NAD(P)H (nicotinamide adenine dinucleotide phosphate) dehydrogenase quinone-1 (NQO-1) in DSS-exposed mice. Immunohistochemistry revealed a significantly increased expression of tight junction proteins, which protect and maintain paracellular intestinal permeability. In support of an anti-inflammatory activity, idebenone significantly attenuated the elevated levels of pro-inflammatory cytokines in colon tissue. These results suggest that idebenone could represent a promising therapeutic strategy to interfere with disease pathology in UC by simultaneously inducing antioxidative and anti-inflammatory pathways.
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http://dx.doi.org/10.3390/ijms21020484DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7013829PMC
January 2020

Effect of Combination l-Citrulline and Metformin Treatment on Motor Function in Patients With Duchenne Muscular Dystrophy: A Randomized Clinical Trial.

JAMA Netw Open 2019 10 2;2(10):e1914171. Epub 2019 Oct 2.

Division of Pediatric Neurology, University Children's Hospital Basel, Basel, Switzerland.

Importance: Nitric oxide precursors, such as the amino acid l-arginine and the biguanide antidiabetic drug metformin, have been associated with metabolism and muscle function in patients with Duchenne muscular dystrophy (DMD). The treatment of DMD remains an unmet medical need.

Objective: To evaluate the benefits and harms of a combination of l-citrulline and metformin treatment among patients with DMD.

Design, Setting, And Participants: A single-center randomized double-blind placebo-controlled parallel-group clinical trial was conducted between December 12, 2013, and March 30, 2016, at the University Children's Hospital Basel in Switzerland. A total of 47 ambulant male patients aged 6.5 to 10 years with genetically confirmed DMD were recruited locally and from the patient registries of Switzerland, Germany, Austria, and France. Data were analyzed from April 6, 2016, to September 5, 2019.

Interventions: Patients in the treatment group received 2500 mg of l-citrulline and 250 mg of metformin (combination therapy) 3 times a day for 26 weeks compared with patients in the control group, who received placebo.

Main Outcomes And Measures: The primary end point was the change in transfer and standing posture, as assessed by the first dimension of the Motor Function Measure, version 32, from baseline to week 26. Secondary end points included assessments of timed function, quantitative muscle force, biomarkers for muscle necrosis, and adverse events. The 2 prespecified subgroups comprised patients who were able to walk 350 m or more in 6 minutes (stable subgroup) and patients who were not able to walk 350 m in 6 minutes (unstable subgroup) at baseline.

Results: Among 49 ambulant male children with DMD who were screened for eligibility, 47 patients with a mean (SD) age of 8.2 (1.1) years were randomized to a treatment group receiving combination therapy (n = 23) or a control group receiving placebo (n = 24), and 45 patients completed the study. No significant differences between groups were found in the results of timed function and muscle force tests for overall, proximal and axial, and distal motor function. Among patients receiving combination therapy, the Motor Function Measure first dimension subscore decrease was 5.5% greater than that of patients receiving placebo (95% CI, -1.0% to 12.1%; P = .09). The administration of combination therapy had significantly favorable effects on the first dimension subscore decrease among the 29 patients in the stable subgroup (6.7%; 95% CI, 0.9%-12.6%; P = .03) but not among the 15 patients in the unstable subgroup (3.9%; 95% CI, -13.2% to 20.9%; P = .63). Overall, the treatment was well tolerated with only mild adverse effects.

Conclusions And Relevance: Treatment with combination therapy was not associated with an overall reduction in motor function decline among ambulant patients with DMD; however, a reduction in motor function decline was observed among the stable subgroup of patients treated with combination therapy. The statistically nonsignificant difference of distal motor function in favor of combination therapy and the reduced degeneration of muscle tissue appear to support the treatment concept, but the study may have lacked sufficient statistical power. Further research exploring this treatment option with a greater number of patients is warranted.

Trial Registration: ClinicalTrials.gov identifier: NCT01995032.
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http://dx.doi.org/10.1001/jamanetworkopen.2019.14171DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6824222PMC
October 2019

Identification of Key Pro-Survival Proteins in Isolated Colonic Goblet Cells of Winnie, a Murine Model of Spontaneous Colitis.

Inflamm Bowel Dis 2020 01;26(1):80-92

School of Health Sciences, University of Tasmania, Launceston, Tasmania, Australia.

Background: Accumulating evidence suggests that the goblet cell-derived mucin-2 (Muc2) is a major component of the immune system and that perturbations in Muc2 lead to an ulcerative colitis-like phenotype. The animal model Winnie carries a missense mutation in Muc2 that causes Muc2 misfolding, accumulation in goblet cells, and ER stress. Excessive ER stress is a hallmark of many diseases, including ulcerative colitis, cancer, diabetes and Parkinson's disease. However, rather than committing to cell death, which is the typical outcome of unresolved ER stress, Winnie goblet cells are characterized by hyperproliferation, suggesting additional regulation of this cellular stress response.

Methods: To elucidate the molecular mechanisms underlying ulcerative colitis in the Winnie model, we isolated goblet cells from Winnie and wild-type mice and used label-free quantitative proteomics and bioinformatics to understand the functional consequences of Muc2 misfolding and accumulation.

Results: A large number of changes were identified that highlight a dramatic reprogramming of energy production, including enhanced utilization of butyrate, a key energy source of colonic cells. A major finding was the marked upregulation of the coiled-coil-helix-coiled-coil-helix domain proteins Chchd2, Chchd3, and Chchd6. In particular, we identified and confirmed the upregulation and nuclear translocation of Chchd2, a protein known to inhibit oxidative stress induced apoptosis.

Conclusions: This study is the first to apply proteome-level analysis to the preclinical Winnie model of ulcerative colitis. Identification of proteins and pathways affected in isolated Winnie goblet cells provides evidence for novel adaptive mechanisms underlying cell survival under conditions of chronic ER stress.
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http://dx.doi.org/10.1093/ibd/izz179DOI Listing
January 2020

Amide linked redox-active naphthoquinones for the treatment of mitochondrial dysfunction.

Medchemcomm 2019 Mar 16;10(3):399-412. Epub 2019 Jan 16.

School of Natural Sciences - Chemistry , University of Tasmania , Hobart , TAS 7001 , Australia . Email: ; ; Tel: +61 3 6226 2182.

Naphthoquinones have been investigated as potential therapeutic molecules for neurodegenerative disorders, which is largely based on their anti-oxidative potential. However, a theoretical framework for the pleiotropic protective effects of naphthoquinone derivatives is largely missing. We synthesized a library of novel short chain 2,3-disubstituted naphthoquinone derivatives and measured their redox characteristics to identify a potential connection with their biological activity. Using two cell lines with different reducing potential, the compounds were tested for their inherent toxicity, acute rescue of ATP levels and cytoprotective activity. For the first time, a structure-activity-relationship for naphthoquinones has been established. Our results clearly demonstrate that it is the group on the alkyl side chain and not solely the redox characteristics of the naphthoquinone unit or lipophilicity that determines the extent of cytoprotection by individual compounds. From this, we developed a number of amide containing naphthoquinones with superior activity in ATP rescue and cell viability models compared to the clinically used benzoquinone idebenone.
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http://dx.doi.org/10.1039/c8md00582fDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6430093PMC
March 2019

Pathway Analysis of Fucoidan Activity Using a Yeast Gene Deletion Library Screen.

Mar Drugs 2019 Jan 14;17(1). Epub 2019 Jan 14.

School of Medicine, University of Tasmania; Hobart TAS 7001, Australia.

Fucoidan, the sulfated fucose-rich polysaccharide derived from brown macroalgae, was reported to display some anti-cancer effects in in vitro and in vivo models that included apoptosis and cell cycle arrest. The proposed mechanisms of action involve enhanced immune surveillance and direct pro-apoptotic effects via the activation of cell signaling pathways that remain largely uncharacterized. This study aimed to identify cellular pathways influenced by fucoidan using an unbiased genetic approach to generate additional insights into the anti-cancer effects of fucoidan. Drug⁻gene interactions of fucoidan were assessed by a systematic screen of the entire set of 4,733 halpoid gene deletion strains. Some of the findings were confirmed using cell cycle analysis and DNA damage detection in non-immortalized human dermal fibroblasts and colon cancer cells. The yeast deletion library screen and subsequent pathway and interactome analysis identified global effects of fucoidan on a wide range of eukaryotic cellular processes, including RNA metabolism, protein synthesis, sorting, targeting and transport, carbohydrate metabolism, mitochondrial maintenance, cell cycle regulation, and DNA damage repair-related pathways. Fucoidan also reduced clonogenic survival, induced DNA damage and G1-arrest in colon cancer cells, while these effects were not observed in non-immortalized human fibroblasts. Our results demonstrate global effects of fucoidan in diverse cellular processes in eukaryotic cells and further our understanding about the inhibitory effect of fucoidan on the growth of human cancer cells.
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http://dx.doi.org/10.3390/md17010054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6356313PMC
January 2019

Towards complete identification of allergens in Jack Jumper (Myrmecia pilosula) ant venom and their clinical relevance: An immunoproteomic approach.

Clin Exp Allergy 2018 09 3;48(9):1222-1234. Epub 2018 Aug 3.

Jack Jumper Allergy Program, Royal Hobart Hospital, Hobart, TAS, Australia.

Background: The venomous stings of Jack Jumper ant (JJA; species of the Myrmecia pilosula taxonomic group) are a significant public health issue in parts of south-eastern and south-western Australia, causing anaphylaxis in approximately 3% of the population. Three allergenic peptides, Myr p 1, Myr p 2 and Myr p 3, and one histamine-releasing peptide, pilosulin 5, have been fully described, but there are at least 5 additional high molecular weight IgE-binding components that have not been identified.

Objective: To identify IgE-binding components in JJA venom (JJAV) and to relate the IgE recognition of these components to relevant clinical parameters.

Methods: Identification of IgE-binding components and determination of their sensitizing prevalence was performed using SDS-PAGE immunoblot assay and sera from 90 patients with confirmed allergy to JJAV. Tandem mass spectrometry was used for identification of novel JJAV components fractionated by size exclusion chromatography (SEC) and SDS-PAGE.

Results: Using SDS-PAGE immunoblot, 10 IgE-binding bands were identified in JJAV, two of which were recognized by 81% and 47% of the population studied. Mass spectrometry identified 17 novel JJAV proteins, including 2 glycoproteins, and confirmed the presence of 4 known Myr p and pilosulin peptides in JJAV. Most of the newly identified IgE-binding proteins were enzymes, including phospholipase A , hyaluronidase, arginine kinase and dipeptidyl peptidase IV. Correlations were found between recognition of certain IgE-binding bands with JJAV-specific IgE titre by ImmunoCAP, intradermal test threshold and treatment-related issues.

Conclusions And Clinical Relevance: This study has for the first time revealed the identity of various proteins with IgE-binding capacity in the venom of JJA and demonstrated their clinical relevance in the diagnosis and treatment of JJAV allergy.
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http://dx.doi.org/10.1111/cea.13224DOI Listing
September 2018

Data on prolonged morphine-induced antinociception and behavioral inhibition in older rats.

Data Brief 2018 Aug 9;19:183-188. Epub 2018 May 9.

Division of Pharmacy, School of Medicine, University of Tasmania, Australia.

This article contains supportive data related to a research article entitled "Age-dependent antinociception and behavioral inhibition by morphine" (Paul et al., 2018) [1]. Antinociceptive latencies of 8 and 24-week old rats were obtained from tail-flick and hot plate assays after morphine treatment. Motor behavioral effects were measured at different time-points using automated infrared tracking in an open-field arena. Residual morphine content in post-mortem tissues were measured 240 min post-treatment. Concurrent measurements of antinociception, motor behavior and residual morphine content in post-mortem tissues of 8-week and 24-week old morphine-treated rats provide an integrated assessment of age-related differences.
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http://dx.doi.org/10.1016/j.dib.2018.05.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5992970PMC
August 2018

Age-dependent antinociception and behavioral inhibition by morphine.

Pharmacol Biochem Behav 2018 05 13;168:8-16. Epub 2018 Mar 13.

Division of Pharmacy, School of Medicine, University of Tasmania, Australia.

In current clinical practice, morphine is dosed in older patients based on patient-weight, with different calculations for adjustment. However, at present, neither clinical experience nor the literature offers a clear evidence base for the relationship between antinociception, behavioral effects and morphine administration in older patients. In this study, we compared the nociceptive response of 8 and 24 week old rats after subcutaneous administration of morphine per body weight and analyzed their behavior using an advanced multi-conditioning system. Residual morphine in all major tissues was determined. We observed prolonged morphine-induced antinociception in older rats compared to younger rats. Moreover, morphine significantly stimulated locomotor and rearing behavior 180 min after injection, which was significantly higher in the 8 week compared to 24 week old rats. Tissue analysis from animals extracted 240 min post-injection revealed a significantly higher concentration of residual morphine in the brains of older versus younger animals when standardized on tissue weight. However, this effect was not observed when residual morphine was standardized on protein content. Collectively, our data suggest that in older rats morphine exhibits higher antinociception and increased behavioral inhibition compared to younger animals. This effect is likely due to a significantly higher accumulation of morphine in the brain of older animals.
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http://dx.doi.org/10.1016/j.pbb.2018.03.003DOI Listing
May 2018

Time-Resolved Pharmacological Studies using Automated, On-line Monitoring of Five Parallel Suspension Cultures.

Sci Rep 2017 09 4;7(1):10337. Epub 2017 Sep 4.

School of Medicine and ACROSS, Faculty of Health Sciences, University of Tasmania, Private Bag 26, Hobart, Tasmania, 7001, Australia.

Early stage pharmacological studies rely on in vitro methodologies for screening and testing compounds. Conventional assays based on endpoint measurements provide limited information because the lack in temporal resolution may not determine the pharmacological effect at its maximum. We developed an on-line, automated system for near real-time monitoring of extracellular content from five parallel suspension cultures, combining cell density measurements with a high-resolution separations every 12 minutes for 4 days. Selector and switching valves provide the fluidic control required to sample from one culture during the analysis of the previous sample from another culture, a time-saving measure that is fundamental to the throughput of the presented system. The system was applied to study the metabolic effects of the drugs rotenone, β-lapachone and clioquinol using lactate as metabolic indicator. For each drug, 96 assays were executed on the extracellular matrix at three concentrations with two controls in parallel, consuming only 5.78 mL of media from each culture over four days, less than 60 μL per analysis. The automated system provides high sample throughput, good temporal resolution and low sample consumption combined with a rugged analytical method with adequate sensitivity, providing a promising new platform for pharmacological and biotechnological studies.
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http://dx.doi.org/10.1038/s41598-017-10472-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5583285PMC
September 2017

Morphine dosing strategy plays a key role in the generation and duration of the produced antinociceptive tolerance.

Neuropharmacology 2017 Jul 25;121:158-166. Epub 2017 Apr 25.

Division of Pharmacy, School of Medicine, University of Tasmania, Australia; Medical School, University of Cyprus, Nicosia, Cyprus.

Antinociceptive tolerance after repetitive administration of morphine severely limits its clinical use. Despite increased mechanistic understanding of morphine tolerance, little is known about the influence of dosing regimens in its development. We hypothesized that the starting dose of morphine, dosing frequency and dose increments, influence antinociception and the manifestation of antinociceptive tolerance in rats. Male rats were randomly divided into four groups with different intermittent starting-doses of daily morphine (b.i.d.) followed by different increments of single-dose morphine upon development of antinociceptive tolerance, for 2-3 weeks: 2.5 (b.i.d.)→5 → 10→15 mg/kg/day, 5 (b.i.d.)→10 mg/kg/day, 5 (b.i.d.)→15 mg/kg/day, 10 (b.i.d.)→20 mg/kg/day. Antinociception was assessed daily pre-treatment and at several time-points over 2 h post-administration, using tail-flick and hot-plate assays. Tolerance was defined as significant antinociceptive desensitization and was presented as significant reduction of the maximum and total antinociceptive efficacy upon morphine administration. Rats commenced on 2.5 mg/kg/day (b.i.d.) morphine developed tolerance faster than those started on 5 or 10 mg/kg/day (b.i.d.). Comparatively, higher starting and maintenance doses of morphine produced prolonged antinociception and delayed tolerance. Whereas, lower starting and maintenance doses of morphine produced less total antinociception during the course of treatment and did not delay the onset of tolerance, but require smaller dose-increments to reach antinociception after development of antinociceptive tolerance. These results suggest that morphine starting dose, dosing frequency, increments and timing determine the manifestation of antinociceptive tolerance and extent of antinociception. In addition, our results also highlight the need for generally standardized and validated assay protocols and procedures to compare different studies, as a prerequisite to translate pre-clinical results into the clinic.
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http://dx.doi.org/10.1016/j.neuropharm.2017.04.034DOI Listing
July 2017

Alzheimer's Disease and NQO1: Is there a Link?

Curr Alzheimer Res 2018 ;15(1):56-66

Division of Pharmacy, School of Medicine, Faculty of Health, University of Tasmania, Hobart, TAS 7001. Australia.

Background: Alzheimer's disease (AD) is a neurodegenerative disease characterised by a progressive decline in cognitive function and represents a major healthcare challenge worldwide. Increasing evidence indicates that mitochondrial dysfunction mediated oxidative stress plays a significant role in the pathophysiological process of AD. Therefore, the physiological activation of antioxidant enzymes that respond to increased oxidative stress is thought to prevent neuropathology. One of those endogenous defences is NADPH quinone oxidoreductase 1 (NQO1). NQO1 is a cytosolic homodimeric flavoprotein that catalyses the two-electron reduction of quinones and related molecules aimed at increasing their solubility and excretion. In line with its role as a phase II stress response protein, altered NQO1 expression is associated with several pathological conditions and disorders including AD.

Conclusion: This review summarizes the association between NQO1 and AD pathology. Understanding this association will provide further insight into the pathogenesis of the disease. More importantly, recent interest in drugs that affect NQO1 expression or its activity provides hope that this approach could lead to novel therapeutic options for the treatment of AD.
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http://dx.doi.org/10.2174/1567205014666170203095802DOI Listing
March 2019

Treatment with L-citrulline and metformin in Duchenne muscular dystrophy: study protocol for a single-centre, randomised, placebo-controlled trial.

Trials 2016 08 3;17(1):389. Epub 2016 Aug 3.

Division of Neuropaediatrics, University of Basel Children's Hospital, Basel, Switzerland.

Background: Duchenne muscular dystrophy (DMD) is an X-linked recessive disease that affects 1 in 3500-6000 male births. Despite broad research aiming to improve muscle function as well as heart and brain function, sufficient therapeutic efficacy has not yet been achieved and current therapeutic management is still supportive. In a recent pilot trial, oral treatment with L-arginine and metformin showed consistent changes of muscular metabolism both in vitro and in vivo by raising NO levels and expression of mitochondrial proteins in the skeletal muscle tissue of patients with DMD. This randomised, double-blind, placebo-controlled trial aims to demonstrate the superiority of L-citrulline and metformin therapy over placebo in DMD patients with regard to the Motor Function Measure (MFM) D1 subscore (primary endpoint) as well as additional clinical and subclinical tests.

Methods/design: A total of 40-50 ambulant patients with DMD will be recruited at the outpatient department of the University of Basel Children's Hospital (Switzerland), as well as from the DMD patient registries of Switzerland, Germany and Austria. Patients will be randomly allocated to one of the two arms of the study and will receive either a combination of L-citrulline and metformin or placebo for 26 weeks. Co-medication with glucocorticoids is allowed. The primary endpoint is the change of the MFM D1 subscore from baseline to week 26 under L-citrulline and metformin therapy. Secondary endpoints will include the motor function measure (MFM) and its items and subscores, the 6-minute walking test, timed function tests and quantitative muscle testing. Furthermore, quantitative muscle MRI assessment to evaluate the muscle fat fraction as well as safety and biomarker laboratory analyses from blood will be included. For comparison, muscle metabolism and mitochondrial function will be analysed in 10-20 healthy age-matched male children.

Discussion: The aim of this study is to test if a 6-month treatment of a combination of L-citrulline and metformin is more effective than placebo in preventing loss of motor function and muscle degeneration in DMD. The MFM D1 subscore is used as a clinical outcome measure and a quantitative muscle MRI assessment as the surrogate outcome measure of fatty muscle degeneration.

Trial Registration: ClinicalTrials.gov: NCT01995032 . Registered on 20 November 2013.
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http://dx.doi.org/10.1186/s13063-016-1503-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4973063PMC
August 2016

Targeting mitochondrial function to treat optic neuropathy.

Mitochondrion 2017 09 28;36:7-14. Epub 2016 Jul 28.

Pharmacy, School of Medicine, University of Tasmania, Hobart, TAS, Australia.

Many reports have illustrated a tight connection between vision and mitochondrial function. Not only are most mitochondrial diseases associated with some form of vision impairment, many ophthalmological disorders such as glaucoma, age-related macular degeneration and diabetic retinopathy also show signs of mitochondrial dysfunction. Despite a vast amount of evidence, vision loss is still only treated symptomatically, which is only partially a consequence of resistance to acknowledge that mitochondria could be the common denominator and hence a promising therapeutic target. More importantly, clinical support of this concept is only emerging. Moreover, only a few drug candidates and treatment strategies are in development or approved that selectively aim to restore mitochondrial function. This review rationalizes the currently developed therapeutic approaches that target mitochondrial function by discussing their proposed mode(s) of action and provides an overview on their development status with regards to optic neuropathies.
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http://dx.doi.org/10.1016/j.mito.2016.07.013DOI Listing
September 2017

Capillary electrophoresis for automated on-line monitoring of suspension cultures: Correlating cell density, nutrients and metabolites in near real-time.

Anal Chim Acta 2016 05 26;920:94-101. Epub 2016 Mar 26.

School of Medicine, Faculty of Health Sciences, University of Tasmania, Australia; School of Medicine and ACROSS, Faculty of Health Sciences, University of Tasmania, Australia. Electronic address:

Increasingly stringent demands on the production of biopharmaceuticals demand monitoring of process parameters that impact on their quality. We developed an automated platform for on-line, near real-time monitoring of suspension cultures by integrating microfluidic components for cell counting and filtration with a high-resolution separation technique. This enabled the correlation of the growth of a human lymphocyte cell line with changes in the essential metabolic markers, glucose, glutamine, leucine/isoleucine and lactate, determined by Sequential Injection-Capillary Electrophoresis (SI-CE). Using 8.1 mL of media (41 μL per run), the metabolic status and cell density were recorded every 30 min over 4 days. The presented platform is flexible, simple and automated and allows for fast, robust and sensitive analysis with low sample consumption and high sample throughput. It is compatible with up- and out-scaling, and as such provides a promising new solution to meet the future demands in process monitoring in the biopharmaceutical industry.
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http://dx.doi.org/10.1016/j.aca.2016.03.034DOI Listing
May 2016

Improved Muscle Function in Duchenne Muscular Dystrophy through L-Arginine and Metformin: An Investigator-Initiated, Open-Label, Single-Center, Proof-Of-Concept-Study.

PLoS One 2016 22;11(1):e0147634. Epub 2016 Jan 22.

Division of Neuropaediatrics, University of Basel Children's Hospital, Basel, Switzerland.

Unlabelled: Altered neuronal nitric oxide synthase function in Duchenne muscular dystrophy leads to impaired mitochondrial function which is thought to be one cause of muscle damage in this disease. The study tested if increased intramuscular nitric oxide concentration can improve mitochondrial energy metabolism in Duchenne muscular dystrophy using a novel therapeutic approach through the combination of L-arginine with metformin. Five ambulatory, genetically confirmed Duchenne muscular dystrophy patients aged between 7–10 years were treated with L-arginine (3 x 2.5 g/d) and metformin (2 x 250 mg/d) for 16 weeks. Treatment effects were assessed using mitochondrial protein expression analysis in muscular biopsies, indirect calorimetry, Dual-Energy X-Ray Absorptiometry, quantitative thigh muscle MRI, and clinical scores of muscle performance. There were no serious side effects and no patient dropped out. Muscle biopsy results showed pre-treatment a significantly reduced mitochondrial protein expression and increased oxidative stress in Duchenne muscular dystrophy patients compared to controls. Post-treatment a significant elevation of proteins of the mitochondrial electron transport chain was observed as well as a reduction in oxidative stress. Treatment also decreased resting energy expenditure rates and energy substrate use shifted from carbohydrates to fatty acids. These changes were associated with improved clinical scores. In conclusion pharmacological stimulation of the nitric oxide pathway leads to improved mitochondria function and clinically a slowing of disease progression in Duchenne muscular dystrophy. This study shall lead to further development of this novel therapeutic approach into a real alternative for Duchenne muscular dystrophy patients.

Trial Registration: ClinicalTrials.gov NCT02516085.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0147634PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4723144PMC
July 2016

Targeting mitochondrial function to protect against vision loss.

Expert Opin Ther Targets 2016 Jun 13;20(6):721-36. Epub 2016 Jan 13.

a Division of Pharmacy, School of Medicine, Faculty of Health , University of Tasmania , Hobart , Australia.

Introduction: Mitochondria, essential to multicellular life, convert food into ATP to satisfy cellular energy demands. Since different tissues have different energy requirements, mitochondrial density is high in tissues with high metabolic needs, such as the visual system, which is therefore highly susceptible to limited energy supply as a result of mitochondrial dysfunction.

Areas Covered: Vision impairment is a common feature of most mitochondrial diseases. At the same time, there is mounting evidence that mitochondrial impairment contributes to the pathogenesis of major eye diseases such as glaucoma and might also be involved in the reported vision impairment in neurodegenerative disorders such as Alzheimer's disease.

Expert Opinion: Rather than relying on symptomatic treatment, acknowledging the mitochondrial origin of visual disorders in mitochondrial, neurodegenerative and ocular diseases could lead to novel therapeutics that aim to modulate mitochondrial function in order to protect against vision loss. This approach has already shown some promising clinical results in inherited retinal disorders, which supports the idea that targeting mitochondria could also be a treatment option for other optic neuropathies.
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http://dx.doi.org/10.1517/14728222.2015.1134489DOI Listing
June 2016

Heparins in ulcerative colitis: proposed mechanisms of action and potential reasons for inconsistent clinical outcomes.

Expert Rev Clin Pharmacol 2015 25;8(6):795-811. Epub 2015 Aug 25.

a 1 Division of Pharmacy, School of Medicine, Faculty of Health, University of Tasmania , Hobart, Tasmania, Australia.

Current drug therapies for ulcerative colitis (UC) are not completely effective in managing moderate-to-severe UC and approximately 20% of patients with severe UC require surgical interventions. Heparins, polydisperse mixtures of non-anticoagulant and anticoagulant oligosaccharides, are widely used as anticoagulants. However, heparins are also reported to have anti-inflammatory properties. Unfractionated heparin was initially used in patients with UC for the treatment of rectal microthrombi. Surprisingly, it was found to be effective in reducing UC-associated symptoms. Since then, several pre-clinical and clinical studies have reported promising outcomes of heparins in UC. In contrast, some controlled clinical trials demonstrated no or only limited benefits, thus the potential of heparins for the treatment of UC remains uncertain. This review discusses potential mechanisms of action of heparins, as well as proposed reasons for their contradictory clinical effectiveness in the treatment of UC.
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http://dx.doi.org/10.1586/17512433.2015.1082425DOI Listing
June 2016