Publications by authors named "David M Shackleford"

71 Publications

Systematic evaluation of structure-property relationships and pharmacokinetics in 6-(hetero)aryl-substituted matched pair analogs of amiloride and 5-(N,N-hexamethylene)amiloride.

Bioorg Med Chem 2021 May 23;37:116116. Epub 2021 Mar 23.

Illawarra Health and Medical Research Institute, Wollongong, NSW 2522, Australia; School of Chemistry and Molecular Bioscience, University of Wollongong, NSW 2522, Australia; Molecular Horizons, University of Wollongong, NSW 2522, Australia; CONCERT-Translational Cancer Research Centre, NSW 2750, Australia.

The K-sparing diuretic amiloride elicits anticancer activities in multiple animal models. During our recent medicinal chemistry campaign aiming to identify amiloride analogs with improved properties for potential use in cancer, we discovered novel 6-(hetero)aryl-substituted amiloride and 5-(N,N-hexamethylene)amiloride (HMA) analogs with up to 100-fold higher potencies than the parent compounds against urokinase plasminogen activator (uPA), one of amiloride's putative anticancer targets, and no diuretic or antikaliuretic effects. Here, we report the systematic evaluation of structure-property relationships (lipophilicity, aqueous solubility and in vitro metabolic stability in human and mouse liver microsomes) in twelve matched pair analogs selected from our 6-substituted amiloride and HMA libraries. Mouse plasma stability, plasma protein binding, Caco-2 cell permeability, cardiac ion channel activity and pharmacokinetics in mice (PO and IV) and rats (IV) are described alongside amiloride and HMA comparators for a subset of the four most promising matched-pair analogs. The findings combined with earlier uPA activity/selectivity and other data ultimately drove selection of two analogs (AA1-39 and AA1-41) that showed efficacy in separate mouse cancer metastasis studies.
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http://dx.doi.org/10.1016/j.bmc.2021.116116DOI Listing
May 2021

Discovery of Potent and Fast-Acting Antimalarial Bis-1,2,4-triazines.

J Med Chem 2021 Apr 24;64(7):4150-4162. Epub 2021 Mar 24.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville Campus), 381 Royal Parade, Parkville, VIC 3052, Australia.

Novel 3,3'-disubstituted-5,5'-bi(1,2,4-triazine) compounds with potent activity against parasites were recently discovered. To improve the pharmacokinetic properties of the triazine derivatives, a new structure-activity relationship (SAR) investigation was initiated with a focus on enhancing the metabolic stability of lead compounds. These efforts led to the identification of second-generation highly potent antimalarial bis-triazines, exemplified by triazine , which exhibited significantly improved metabolic stability (8 and 42 μL/min/mg protein in human and mouse liver microsomes). The disubstituted triazine dimer was also observed to suppress parasitemia in the Peters 4-day test with a mean ED value of 1.85 mg/kg/day and exhibited a fast-killing profile, revealing a new class of orally available antimalarial compounds of considerable interest.
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http://dx.doi.org/10.1021/acs.jmedchem.1c00044DOI Listing
April 2021

Carvedilol blocks neural regulation of breast cancer progression in vivo and is associated with reduced breast cancer mortality in patients.

Eur J Cancer 2021 Apr 24;147:106-116. Epub 2021 Feb 24.

Drug Discovery Biology Theme, Monash Institute of Pharmaceutical Science, Monash University, Parkville, VIC, 3052, Australia; Peter MacCallum Cancer Centre, Division of Cancer Surgery, Melbourne, VIC, 3000, Australia. Electronic address:

Purpose: The sympathetic nervous system drives breast cancer progression through β-adrenergic receptor signalling. This discovery has led to the consideration of cardiac β-blocker drugs as novel strategies for anticancer therapies. Carvedilol is a β-blocker used in the management of cardiovascular disorders, anxiety, migraine and chemotherapy-induced cardiotoxicity. However, little is known about how carvedilol affects cancer-related outcomes.

Methods: To address this, we investigated the effects of carvedilol on breast cancer cell lines, in mouse models of breast cancer and in a large cohort of patients with breast cancer (n = 4014).

Results: Treatment with carvedilol blocked the effects of sympathetic nervous system activation, reducing primary tumour growth and metastasis in a mouse model of breast cancer and preventing invasion by breast cancer cell lines. A retrospective analysis found that women using carvedilol at breast cancer diagnosis (n = 136) had reduced breast cancer-specific mortality compared with women who did not (n = 3878) (5-year cumulative incidence of breast cancer deaths: 3.1% versus 5.7%; p = 0.024 and 0.076 from univariate and multivariable analyses, respectively) after a median follow-up of 5.5 years.

Conclusions: These findings provide a rationale to further explore the use of the β-blocker carvedilol as a novel strategy to slow cancer progression.
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http://dx.doi.org/10.1016/j.ejca.2021.01.029DOI Listing
April 2021

Selecting an anti-malarial clinical candidate from two potent dihydroisoquinolones.

Malar J 2021 Feb 19;20(1):107. Epub 2021 Feb 19.

Department of Pharmaceutical Sciences, University of Kentucky College of Pharmacy, Lexington, KY, 40536, USA.

Background: The ongoing global malaria eradication campaign requires development of potent, safe, and cost-effective drugs lacking cross-resistance with existing chemotherapies. One critical step in drug development is selecting a suitable clinical candidate from late leads. The process used to select the clinical candidate SJ733 from two potent dihydroisoquinolone (DHIQ) late leads, SJ733 and SJ311, based on their physicochemical, pharmacokinetic (PK), and toxicity profiles is described.

Methods: The compounds were tested to define their physicochemical properties including kinetic and thermodynamic solubility, partition coefficient, permeability, ionization constant, and binding to plasma proteins. Metabolic stability was assessed in both microsomes and hepatocytes derived from mice, rats, dogs, and humans. Cytochrome P450 inhibition was assessed using recombinant human cytochrome enzymes. The pharmacokinetic profiles of single intravenous or oral doses were investigated in mice, rats, and dogs.

Results: Although both compounds displayed similar physicochemical properties, SJ733 was more permeable but metabolically less stable than SJ311 in vitro. Single dose PK studies of SJ733 in mice, rats, and dogs demonstrated appreciable oral bioavailability (60-100%), whereas SJ311 had lower oral bioavailability (mice 23%, rats 40%) and higher renal clearance (10-30 fold higher than SJ733 in rats and dogs), suggesting less favorable exposure in humans. SJ311 also displayed a narrower range of dose-proportional exposure, with plasma exposure flattening at doses above 200 mg/kg.

Conclusion: SJ733 was chosen as the candidate based on a more favorable dose proportionality of exposure and stronger expectation of the ability to justify a strong therapeutic index to regulators.
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http://dx.doi.org/10.1186/s12936-021-03617-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7893776PMC
February 2021

The Anti-Inflammatory Drug Aspirin Does Not Protect Against Chemotherapy-Induced Memory Impairment by Paclitaxel in Mice.

Front Oncol 2020 14;10:564965. Epub 2020 Dec 14.

Drug Discovery Biology Theme, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia.

Inflammation has been proposed to play a causal role in chemobrain which-if true-would represent an opportunity to repurpose existing anti-inflammatory drugs for the prevention and treatment of chemobrain. Here, we show that the chemoagent paclitaxel induces memory impairment and anhedonia in mice within 24 h of treatment cessation, but inflammation is not present until 2 weeks after treatment. We find no evidence of brain inflammation as measured by cytokine analysis at any time point. Furthermore, treating with aspirin to block inflammation did not affect paclitaxel-induced memory impairment. These findings suggest that inflammation may not be responsible for memory impairment induced by paclitaxel. These results contrast with recent findings of a causal role for inflammation in cancer-induced memory deficits in mice that were prevented by treatment with oral aspirin, suggesting that cognitive impairment in cancer patients undergoing treatment may arise from multiple convergent mechanisms.
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http://dx.doi.org/10.3389/fonc.2020.564965DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7768078PMC
December 2020

Development of Novel 4-Arylpyridin-2-one and 6-Arylpyrimidin-4-one Positive Allosteric Modulators of the M Muscarinic Acetylcholine Receptor.

ChemMedChem 2021 Jan 25;16(1):216-233. Epub 2020 Sep 25.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, 3052, Victoria, Australia.

This study investigated the structure-activity relationships of 4-phenylpyridin-2-one and 6-phenylpyrimidin-4-one M muscarinic acetylcholine receptor (M mAChRs) positive allosteric modulators (PAMs). The presented series focuses on modifications to the core and top motif of the reported leads, MIPS1650 (1) and MIPS1780 (2). Profiling of our novel analogues showed that these modifications result in more nuanced effects on the allosteric properties compared to our previous compounds with alterations to the biaryl pendant. Further pharmacological characterisation of the selected compounds in radioligand binding, IP accumulation and β-arrestin 2 recruitment assays demonstrated that, despite primarily acting as affinity modulators, the PAMs displayed different pharmacological properties across the two cellular assays. The novel PAM 7 f is a potential lead candidate for further development of peripherally restricted M PAMs, due to its lower blood-brain-barrier (BBB) permeability and improved exposure in the periphery compared to lead 2.
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http://dx.doi.org/10.1002/cmdc.202000540DOI Listing
January 2021

In the Loop: Extrastriatal Regulation of Spiny Projection Neurons by GPR52.

ACS Chem Neurosci 2020 07 7;11(14):2066-2076. Epub 2020 Jul 7.

Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, Victoria 3010, Australia.

GPR52 is a Gα-coupled orphan receptor identified as a putative target for the treatment of schizophrenia. The unique expression and signaling profile of GPR52 in key areas of dopamine and glutamate dysregulation suggests its activation may resolve both cortical and striatal dysfunction in the disorder. GPR52 mRNA is enriched in the striatum, almost exclusively on dopamine D-expressing medium spiny neurons (MSNs), and to a lesser extent in the cortex, predominantly on D-expressing pyramidal neurons. Synthetic, small molecule GPR52 agonists are effective in preclinical models of psychosis; however, the relative contribution of cortical and striatal GPR52 is unknown. Here we show that the GPR52 agonist, 3-BTBZ, inhibits phencyclidine-induced hyperlocomotor activity to a greater degree than amphetamine-induced motor effects, suggesting a mechanism beyond functional antagonism of striatal dopamine D receptor signaling. Using DARPP-32 phosphorylation and electrophysiological recordings in either striatopallidal or striatonigral MSNs, we were surprised to find no significant effect of 3-BTBZ in striatopallidal MSNs, but GPR52-mediated effects in striatonigral MSNs, where its mRNA is absent. 3-BTBZ increases phosphorylation of T75 on DARPP-32 in striatonigral MSNs, an effect that was dependent on cortical inputs. A similar role for GPR52 in regulating extrastriatal glutamatergic drive onto striatonigral MSNs was also evident in recordings of spontaneous excitatory postsynaptic currents and was shown to be dependent on the metabotropic glutamate (mGlu) receptor subtype 1. Our results demonstrate that GPR52-mediated regulation of striatal function depends heavily on extrastriatal inputs, which may further support its utility as a novel target for the treatment of schizophrenia.
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http://dx.doi.org/10.1021/acschemneuro.0c00197DOI Listing
July 2020

Lead Optimization of a Pyrrole-Based Dihydroorotate Dehydrogenase Inhibitor Series for the Treatment of Malaria.

J Med Chem 2020 05 16;63(9):4929-4956. Epub 2020 Apr 16.

Departments of Biochemistry, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd, Dallas, Texas 75390-9135, United States.

Malaria puts at risk nearly half the world's population and causes high mortality in sub-Saharan Africa, while drug resistance threatens current therapies. The pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase (DHODH) is a validated target for malaria treatment based on our finding that triazolopyrimidine DSM265 () showed efficacy in clinical studies. Herein, we describe optimization of a pyrrole-based series identified using a target-based DHODH screen. Compounds with nanomolar potency versus DHODH and parasites were identified with good pharmacological properties. X-ray studies showed that the pyrroles bind an alternative enzyme conformation from leading to improved species selectivity versus mammalian enzymes and equivalent activity on and DHODH. The best lead DSM502 () showed efficacy at similar levels of blood exposure to , although metabolic stability was reduced. Overall, the pyrrole-based DHODH inhibitors provide an attractive alternative scaffold for the development of new antimalarial compounds.
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http://dx.doi.org/10.1021/acs.jmedchem.0c00311DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7394244PMC
May 2020

Structure-Activity Relationship of Antischistosomal Ozonide Carboxylic Acids.

J Med Chem 2020 04 19;63(7):3723-3736. Epub 2020 Mar 19.

College of Pharmacy, University of Nebraska Medical Center, Nebraska Medical Center, Omaha, Nebraska 986125, United States.

Semisynthetic artemisinins and other bioactive peroxides are best known for their powerful antimalarial activities, and they also show substantial activity against schistosomes-another hemoglobin-degrading pathogen. Building on this discovery, we now describe the initial structure-activity relationship (SAR) of antischistosomal ozonide carboxylic acids OZ418 () and OZ165 (). Irrespective of lipophilicity, these ozonide weak acids have relatively low aqueous solubilities and high protein binding values. Ozonides with -substituted carboxymethoxy and -benzylglycine substituents had high antischistosomal efficacies. It was possible to increase solubility, decrease protein binding, and maintain the high antischistosomal activity in mice infected with juvenile and adult by incorporating a weak base functional group in these compounds. In some cases, adding polar functional groups and heteroatoms to the spiroadamantane substructure increased the solubility and metabolic stability, but in all cases decreased the antischistosomal activity.
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http://dx.doi.org/10.1021/acs.jmedchem.0c00069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7182039PMC
April 2020

Discovery of Acylsulfonohydrazide-Derived Inhibitors of the Lysine Acetyltransferase, KAT6A, as Potent Senescence-Inducing Anti-Cancer Agents.

J Med Chem 2020 05 19;63(9):4655-4684. Epub 2020 Mar 19.

School of Pharmaceutical Sciences, Nanjing Tech University, No. 30 South Puzhu Road, Nanjing 211816, People's Republic of China.

A high-throughput screen designed to discover new inhibitors of histone acetyltransferase KAT6A uncovered CTX-0124143 (), a unique aryl acylsulfonohydrazide with an IC of 1.0 μM. Using this acylsulfonohydrazide as a template, we herein disclose the results of our extensive structure-activity relationship investigations, which resulted in the discovery of advanced compounds such as and . These two compounds represent significant improvements on our recently reported prototypical lead WM-8014 () as they are not only equivalently potent as inhibitors of KAT6A but are less lipophilic and significantly more stable to microsomal degradation. Furthermore, during this process, we discovered a distinct structural subclass that contains key 2-fluorobenzenesulfonyl and phenylpyridine motifs, culminating in the discovery of WM-1119 (). This compound is a highly potent KAT6A inhibitor (IC = 6.3 nM; = 0.002 μM), competes with Ac-CoA by binding to the Ac-CoA binding site, and has an oral bioavailability of 56% in rats.
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http://dx.doi.org/10.1021/acs.jmedchem.9b02071DOI Listing
May 2020

An in vitro toolbox to accelerate anti-malarial drug discovery and development.

Malar J 2020 Jan 2;19(1). Epub 2020 Jan 2.

Medicines for Malaria Venture, PO Box 1826, 20 Route de Pré-Bois, CH-1215, Geneva 15, Switzerland.

Background: Modelling and simulation are being increasingly utilized to support the discovery and development of new anti-malarial drugs. These approaches require reliable in vitro data for physicochemical properties, permeability, binding, intrinsic clearance and cytochrome P450 inhibition. This work was conducted to generate an in vitro data toolbox using standardized methods for a set of 45 anti-malarial drugs and to assess changes in physicochemical properties in relation to changing target product and candidate profiles.

Methods: Ionization constants were determined by potentiometric titration and partition coefficients were measured using a shake-flask method. Solubility was assessed in biorelevant media and permeability coefficients and efflux ratios were determined using Caco-2 cell monolayers. Binding to plasma and media proteins was measured using either ultracentrifugation or rapid equilibrium dialysis. Metabolic stability and cytochrome P450 inhibition were assessed using human liver microsomes. Sample analysis was conducted by LC-MS/MS.

Results: Both solubility and fraction unbound decreased, and permeability and unbound intrinsic clearance increased, with increasing Log D. In general, development compounds were somewhat more lipophilic than legacy drugs. For many compounds, permeability and protein binding were challenging to assess and both required the use of experimental conditions that minimized the impact of non-specific binding. Intrinsic clearance in human liver microsomes was varied across the data set and several compounds exhibited no measurable substrate loss under the conditions used. Inhibition of cytochrome P450 enzymes was minimal for most compounds.

Conclusions: This is the first data set to describe in vitro properties for 45 legacy and development anti-malarial drugs. The studies identified several practical methodological issues common to many of the more lipophilic compounds and highlighted areas which require more work to customize experimental conditions for compounds being designed to meet the new target product profiles. The dataset will be a valuable tool for malaria researchers aiming to develop PBPK models for the prediction of human PK properties and/or drug-drug interactions. Furthermore, generation of this comprehensive data set within a single laboratory allows direct comparison of properties across a large dataset and evaluation of changing property trends that have occurred over time with changing target product and candidate profiles.
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http://dx.doi.org/10.1186/s12936-019-3075-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6941357PMC
January 2020

Preoperative β-Blockade with Propranolol Reduces Biomarkers of Metastasis in Breast Cancer: A Phase II Randomized Trial.

Clin Cancer Res 2020 04 21;26(8):1803-1811. Epub 2019 Nov 21.

Division of Cancer Surgery, Peter MacCallum Cancer Centre, Victorian Comprehensive Cancer Centre, Parkville, Melbourne, Australia.

Purpose: The majority of deaths from breast cancer occur following the development of metastatic disease, a process inhibited by β-blockers in preclinical studies. This phase II randomized controlled trial evaluated the effect of preoperative β-blockade with propranolol on biomarkers of metastatic potential and the immune cell profile within the primary tumor of patients with breast cancer.

Patients And Methods: In this triple-blind placebo-controlled clinical trial, 60 patients were randomly assigned to receive an escalating dose of oral propranolol ( = 30; 80-160 mg daily) or placebo ( = 30) for 7 days prior to surgery. The primary endpoint investigated the effect of propranolol on prometastatic and proinflammatory gene expression within the primary tumor.

Results: Propranolol downregulated primary tumor expression of mesenchymal genes ( = 0.002) without affecting epithelial gene expression ( = 0.21). Bioinformatic analyses implicated downregulation of Snail/Slug ( = 0.03), NF-κB/Rel ( < 0.01), and AP-1 ( < 0.01) transcription factors in structuring the observed transcriptome alterations, and identified changes in intratumoral neutrophil, natural killer cell, and dendritic cell recruitment (all < 0.01). Patients with clinical evidence of drug response (lowered heart rate and blood pressure) demonstrated elevated tumor infiltration of CD68 macrophages and CD8 T cells.

Conclusions: One week of β-blockade with propranolol reduced intratumoral mesenchymal polarization and promoted immune cell infiltration in early-stage surgically-resectable breast cancer. These results show that β-blockade reduces biomarkers associated with metastatic potential, and support the need for larger phase III clinical trials powered to detect the impact of β-blockade on cancer recurrence and survival..
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http://dx.doi.org/10.1158/1078-0432.CCR-19-2641DOI Listing
April 2020

SAR of a new antischistosomal urea carboxylic acid.

Bioorg Med Chem Lett 2018 12 25;28(23-24):3648-3651. Epub 2018 Oct 25.

College of Pharmacy, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, NE, United States. Electronic address:

Urea carboxylic acids, products of aryl hydantoin hydrolysis, were recently identified as a new antischistosomal chemotype. We now describe a baseline structure-activity relationship (SAR) for this compound series. With one exception, analogs of lead urea carboxylic acid 2 were quite polar with Log D values ranging from -1.9 to 1.8, had high aqueous solubilities in the range of 25-100 µg/mL, and were metabolically stable. None of the compounds had measurable in vitro antischistosomal activity or cytotoxicity, but four of these had moderate worm burden reduction (WBR) values of 42-70% when they were administered as single 100 mg/kg oral doses to S. mansoni-infected mice. These data indicate that with the exception of the gem-dimethyl substructure and the distal nitrogen atom of the urea functional group, the rest of the structure of 2 is required for in vivo antischistosomal activity.
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http://dx.doi.org/10.1016/j.bmcl.2018.10.039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6301076PMC
December 2018

Enhancing the Oral Absorption of Kinase Inhibitors Using Lipophilic Salts and Lipid-Based Formulations.

Mol Pharm 2018 12 13;15(12):5678-5696. Epub 2018 Nov 13.

ARC Centre of Excellence in Convergent Bio-Nano Science and Technology , Monash University , 381 Royal Parade , Parkville , Victoria 3052 , Australia.

The absolute bioavailability of many small molecule kinase inhibitors (smKIs) is low. The reasons for low bioavailability are multifaceted and include constraints due to first pass metabolism and poor absorption. For smKIs where absorption limits oral bioavailability, low aqueous solubility and high lipophilicity, often in combination with high-dose requirements have been implicated in low and variable absorption, food-effects, and absorption-related drug-drug interactions. The current study has evaluated whether preparation of smKIs as lipophilic salts/ionic liquids in combination with coadministration with lipid-based formulations is able to enhance absorption for examples of this compound class. Lipophilic (docusate) salt forms of erlotinib, gefitinib, ceritinib, and cabozantinib (as example smKIs demonstrating low aqueous solubility and high lipophilicity) were prepared and isolated as workable powder solids. In each case, the lipophilic salt exhibited high and significantly enhanced solubility in lipidic excipients (>100 mg/g) when compared to the free base or commercial salt form. Isolation as the lipophilic salt facilitated smKI loading in model lipid-based formulations at high concentration, increased in vitro solubilization at gastric and intestinal pH and in some cases increased oral absorption (∼2-fold for cabozantinib formulations in rats). Application of a lipophilic salt approach can therefore facilitate the use of lipid-based formulations for examples of the smKI compound class where low solubility limits absorption and is a risk factor for increased variability due to food-effects.
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http://dx.doi.org/10.1021/acs.molpharmaceut.8b00858DOI Listing
December 2018

Drug-receptor kinetics and sigma-1 receptor affinity differentiate clinically evaluated histamine H receptor antagonists.

Neuropharmacology 2019 01 22;144:244-255. Epub 2018 Oct 22.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia. Electronic address:

The histamine H receptor is a G protein-coupled receptor (GPCR) drug target that is highly expressed in the CNS, where it acts as both an auto- and hetero-receptor to regulate neurotransmission. As such, it has been considered as a relevant target in disorders as varied as Alzheimer's disease, schizophrenia, neuropathic pain and attention deficit hyperactivity disorder. A range of competitive antagonists/inverse agonists have progressed into clinical development, with pitolisant approved for the treatment of narcolepsy. Given the breadth of compounds developed and potential therapeutic indications, we assessed the comparative pharmacology of six investigational histamine H agents, including pitolisant, using native tissue and recombinant cells. Whilst all of the compounds tested displayed robust histamine H receptor inverse agonism and did not differentiate between the main H receptor splice variants, they displayed a wide range of affinities and kinetic properties, and included rapidly dissociating (pitolisant, S 38093-2, ABT-239) and slowly dissociating (GSK189254, JNJ-5207852, PF-3654746) agents. S 38093-2 had the lowest histamine H receptor affinity (pK values 5.7-6.2), seemingly at odds with previously reported, potent in vivo activity in models of cognition. We show here that at pro-cognitive and anti-hyperalgesic/anti-allodynic doses, S 38093-2 preferentially occupies the mouse sigma-1 receptor in vivo, only engaging the histamine H receptor at doses associated with wakefulness promotion and neurotransmitter (histamine, ACh) release. Furthermore, pitolisant, ABT-239 and PF-3654746 also displayed appreciable sigma-1 receptor affinity, suggesting that this property differentiates clinically evaluated histamine H receptor antagonists and may play a role in their efficacy.
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http://dx.doi.org/10.1016/j.neuropharm.2018.10.028DOI Listing
January 2019

Using Human Plasma as an Assay Medium in Caco-2 Studies Improves Mass Balance for Lipophilic Compounds.

Pharm Res 2018 Sep 17;35(11):210. Epub 2018 Sep 17.

Centre for Drug Candidate Optimisation, Monash Institute of Pharmaceutical Sciences, 381 Royal Parade, Parkville, VIC, 3052, Australia.

Purpose: To examine the utility of human plasma as an assay medium in Caco-2 permeability studies to overcome poor mass balance and inadequate sink conditions frequently encountered with lipophilic compounds.

Methods: Caco-2 permeability was assessed for reference compounds with known transport mechanisms using either pH 7.4 buffer or human plasma as the assay medium in both the apical and basolateral chambers. When using plasma, P values were corrected for the unbound fraction in the donor chamber. The utility of the approach was assessed by measuring the permeability of selected antimalarial compounds using the two assay media.

Results: Caco-2 cell monolayer integrity and P-gp transporter function were unaffected by the presence of human plasma in the donor and acceptor chambers. For many of the reference compounds having good mass balance with buffer as the medium, higher P values were observed with plasma, likely due to improved acceptor sink conditions. The lipophilic antimalarial compounds exhibited low mass balance with buffer, however the use of plasma markedly improved mass balance allowing the determination of more reliable P values.

Conclusions: The results support the utility of human plasma as an alternate Caco-2 assay medium to improve mass balance and permeability measurements for lipophilic compounds.
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http://dx.doi.org/10.1007/s11095-018-2493-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6156755PMC
September 2018

Isoxazolopyrimidine-Based Inhibitors of Dihydroorotate Dehydrogenase with Antimalarial Activity.

ACS Omega 2018 Aug 15;3(8):9227-9240. Epub 2018 Aug 15.

Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd, Dallas, Texas 75390-9038, United States.

Malaria kills nearly 0.5 million people yearly and impacts the lives of those living in over 90 countries where it is endemic. The current treatment programs are threatened by increasing drug resistance. Dihydroorotate dehydrogenase (DHODH) is now clinically validated as a target for antimalarial drug discovery as a triazolopyrimidine class inhibitor () is currently undergoing clinical development. We discovered a related isoxazolopyrimidine series in a phenotypic screen, later determining that it targeted DHODH. To determine if the isoxazolopyrimidines could yield a drug candidate, we initiated hit-to-lead medicinal chemistry. Several potent analogues were identified, including a compound that showed in vivo antimalarial activity. The isoxazolopyrimidines were more rapidly metabolized than their triazolopyrimidine counterparts, and the pharmacokinetic data were not consistent with the goal of a single-dose treatment for malaria.
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http://dx.doi.org/10.1021/acsomega.8b01573DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6120730PMC
August 2018

Synthesis and profiling of benzylmorpholine 1,2,4,5-tetraoxane analogue N205: Towards tetraoxane scaffolds with potential for single dose cure of malaria.

Bioorg Med Chem 2018 07 17;26(11):2996-3005. Epub 2018 May 17.

Research Centre for Drugs and Diagnostics, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, United Kingdom.

A series of aryl carboxamide and benzylamino dispiro 1,2,4,5-tetraoxane analogues have been designed and synthesized in a short synthetic sequence from readily available starting materials. From this series of endoperoxides, molecules with in vitro IC50s versus Plasmodium falciparum (3D7) as low as 0.84 nM were identified. Based on an assessment of blood stability and in vitro microsomal stability, N205 (10a) was selected for rodent pharmacokinetic and in vivo antimalarial efficacy studies in the mouse Plasmodium berghei and Plasmodium falciparum Pf3D70087/N9 severe combined immunodeficiency (SCID) mouse models. The results indicate that the 4-benzylamino derivatives have excellent profiles with a representative of this series, N205, an excellent starting point for further lead optimization studies.
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http://dx.doi.org/10.1016/j.bmc.2018.05.006DOI Listing
July 2018

Muscarinic M receptors modulate ethanol seeking in rats.

Neuropsychopharmacology 2018 06 5;43(7):1510-1517. Epub 2018 Feb 5.

The Florey Institute of Neuroscience and Mental Health, Parkville, VIC, 3052, Australia.

Despite the cost to both individual and society, alcohol use disorders (AUDs) remain a major health risk within society, and both relapse and heavy drinking are still poorly controlled with current medications. Here we demonstrate for the first time that a centrally active and selective negative allosteric modulator for the rat M muscarinic acetylcholine receptor (mAChR), ML375, decreases ethanol self-administration and attenuates cue-induced reinstatement of ethanol seeking in ethanol-preferring (iP) rats. Importantly, ML375 did not affect sucrose self-administration or general locomotor activity indicative of a selective effect on ethanol seeking. Based on the expression profile of M mAChRs in the brain and the distinct roles different aspects of the dorsal striatum have on long-term and short-term ethanol use, we studied whether intra-striatal microinjection of ML375 modulated ethanol intake in rats. We show in iP rats with an extensive history of ethanol intake that intra-dorsolateral (DL), but not intra-dorsomedial, striatal injections of ML375 reduced ethanol self-administration to a similar extent as the nicotinic acetylcholine receptor ligand varenicline, which has preclinical and clinical efficacy in reducing the reinforcing effects of ethanol. These data implicate the DL striatum as a locus for the effects of cholinergic-acting drugs on ethanol seeking in rats with a history of long-term ethanol use. Accordingly, we demonstrate in rats that selectively targeting the M mAChR can modulate both voluntary ethanol intake and cue-induced ethanol seeking and thereby provide direct evidence that the M mAChR is a potential novel target for pharmacotherapies aimed at treating AUDs.
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http://dx.doi.org/10.1038/s41386-017-0007-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5983544PMC
June 2018

Progress in antischistosomal N,N'-diaryl urea SAR.

Bioorg Med Chem Lett 2018 02 29;28(3):244-248. Epub 2017 Dec 29.

College of Pharmacy, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, NE, United States. Electronic address:

N,N'-Diaryl ureas have recently emerged as a new antischistosomal chemotype. We now describe physicochemical profiling, in vitro ADME, plasma exposure, and ex vivo and in vivo activities against Schistosoma mansoni for twenty new N,N'-diaryl ureas designed primarily to increase aqueous solubility, but also to maximize structural diversity. Replacement of one of the 4-fluoro-3-trifluoromethylphenyl substructures of lead N,N'-diaryl urea 1 with azaheterocycles and benzoic acids, benzamides, or benzonitriles decreased lipophilicity, and in most cases, increased aqueous solubility. There was no clear relationship between lipophilicity and metabolic stability, although all compounds with 3-trifluoromethyl-4-pyridyl substructures were metabolically stable. N,N'-diaryl ureas containing 4-fluoro-3-trifluoromethylphenyl, 3-trifluoromethyl-4-pyridyl, 2,2-difluorobenzodioxole, or 4-benzonitrile substructures had high activity against ex vivo S. mansoni and relatively low cytotoxicity. N,N-diaryl ureas with 3-trifluoromethyl-4-pyridyl and 2,2-difluorobenzodioxole substructures had the highest exposures whereas those with 4-fluoro-3-trifluoromethylphenyl substructures had the best in vivo antischistosomal activities. There was no direct correlation between compound exposure and in vivo activity.
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http://dx.doi.org/10.1016/j.bmcl.2017.12.064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6026081PMC
February 2018

The novel compound PBT434 prevents iron mediated neurodegeneration and alpha-synuclein toxicity in multiple models of Parkinson's disease.

Acta Neuropathol Commun 2017 06 28;5(1):53. Epub 2017 Jun 28.

The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Melbourne, VIC, 3010, Australia.

Elevated iron in the SNpc may play a key role in Parkinson's disease (PD) neurodegeneration since drug candidates with high iron affinity rescue PD animal models, and one candidate, deferirpone, has shown efficacy recently in a phase two clinical trial. However, strong iron chelators may perturb essential iron metabolism, and it is not yet known whether the damage associated with iron is mediated by a tightly bound (eg ferritin) or lower-affinity, labile, iron pool. Here we report the preclinical characterization of PBT434, a novel quinazolinone compound bearing a moderate affinity metal-binding motif, which is in development for Parkinsonian conditions. In vitro, PBT434 was far less potent than deferiprone or deferoxamine at lowering cellular iron levels, yet was found to inhibit iron-mediated redox activity and iron-mediated aggregation of α-synuclein, a protein that aggregates in the neuropathology. In vivo, PBT434 did not deplete tissue iron stores in normal rodents, yet prevented loss of substantia nigra pars compacta neurons (SNpc), lowered nigral α-synuclein accumulation, and rescued motor performance in mice exposed to the Parkinsonian toxins 6-OHDA and MPTP, and in a transgenic animal model (hA53T α-synuclein) of PD. These improvements were associated with reduced markers of oxidative damage, and increased levels of ferroportin (an iron exporter) and DJ-1. We conclude that compounds designed to target a pool of pathological iron that is not held in high-affinity complexes in the tissue can maintain the survival of SNpc neurons and could be disease-modifying in PD.
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http://dx.doi.org/10.1186/s40478-017-0456-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5490188PMC
June 2017

A tetraoxane-based antimalarial drug candidate that overcomes PfK13-C580Y dependent artemisinin resistance.

Nat Commun 2017 05 24;8:15159. Epub 2017 May 24.

Research Centre for Drugs and Diagnostics, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK.

K13 gene mutations are a primary marker of artemisinin resistance in Plasmodium falciparum malaria that threatens the long-term clinical utility of artemisinin-based combination therapies, the cornerstone of modern day malaria treatment. Here we describe a multinational drug discovery programme that has delivered a synthetic tetraoxane-based molecule, E209, which meets key requirements of the Medicines for Malaria Venture drug candidate profiles. E209 has potent nanomolar inhibitory activity against multiple strains of P. falciparum and P. vivax in vitro, is efficacious against P. falciparum in in vivo rodent models, produces parasite reduction ratios equivalent to dihydroartemisinin and has pharmacokinetic and pharmacodynamic characteristics compatible with a single-dose cure. In vitro studies with transgenic parasites expressing variant forms of K13 show no cross-resistance with the C580Y mutation, the primary variant observed in Southeast Asia. E209 is a superior next generation endoperoxide with combined pharmacokinetic and pharmacodynamic features that overcome the liabilities of artemisinin derivatives.
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http://dx.doi.org/10.1038/ncomms15159DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5458052PMC
May 2017

Antimalarial efficacy of MMV390048, an inhibitor of phosphatidylinositol 4-kinase.

Sci Transl Med 2017 04;9(387)

Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa.

As part of the global effort toward malaria eradication, phenotypic whole-cell screening revealed the 2-aminopyridine class of small molecules as a good starting point to develop new antimalarial drugs. Stemming from this series, we found that the derivative, MMV390048, lacked cross-resistance with current drugs used to treat malaria. This compound was efficacious against all life cycle stages, apart from late hypnozoites in the liver. Efficacy was shown in the humanized mouse model, and modest reductions in mouse-to-mouse transmission were achieved in the mouse model. Experiments in monkeys revealed the ability of MMV390048 to be used for full chemoprotection. Although MMV390048 was not able to eliminate liver hypnozoites, it delayed relapse in a monkey model. Both genomic and chemoproteomic studies identified a kinase of the parasite, phosphatidylinositol 4-kinase, as the molecular target of MMV390048. The ability of MMV390048 to block all life cycle stages of the malaria parasite suggests that this compound should be further developed and may contribute to malaria control and eradication as part of a single-dose combination treatment.
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http://dx.doi.org/10.1126/scitranslmed.aad9735DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5731459PMC
April 2017

Structure-Activity Relationship of the Antimalarial Ozonide Artefenomel (OZ439).

J Med Chem 2017 04 18;60(7):2654-2668. Epub 2017 Jan 18.

College of Pharmacy, University of Nebraska Medical Center , 986125 Nebraska Medical Center, Omaha, Nebraska 68198, United States.

Building on insights gained from the discovery of the antimalarial ozonide arterolane (OZ277), we now describe the structure-activity relationship (SAR) of the antimalarial ozonide artefenomel (OZ439). Primary and secondary amino ozonides had higher metabolic stabilities than tertiary amino ozonides, consistent with their higher pK and lower log D values. For primary amino ozonides, addition of polar functional groups decreased in vivo antimalarial efficacy. For secondary amino ozonides, additional functional groups had variable effects on metabolic stability and efficacy, but the most effective members of this series also had the highest log D values. For tertiary amino ozonides, addition of polar functional groups with H-bond donors increased metabolic stability but decreased in vivo antimalarial efficacy. Primary and tertiary amino ozonides with cycloalkyl and heterocycle substructures were superior to their acyclic counterparts. The high curative efficacy of these ozonides was most often associated with high and prolonged plasma exposure, but exposure on its own did not explain the presence or absence of either curative efficacy or in vivo toxicity.
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http://dx.doi.org/10.1021/acs.jmedchem.6b01586DOI Listing
April 2017

Revisiting the SAR of the Antischistosomal Aryl Hydantoin (Ro 13-3978).

J Med Chem 2016 12 28;59(23):10705-10718. Epub 2016 Nov 28.

College of Pharmacy, University of Nebraska Medical Center , 986125 Nebraska Medical Center, Omaha, Nebraska 68198, United States.

The aryl hydantoin 1 (Ro 13-3978) was identified in the early 1980s as a promising antischistosomal lead compound. However, this series of aryl hydantoins produced antiandrogenic side effects in the host, a not unexpected outcome given their close structural similarity to the antiandrogenic drug nilutamide. Building on the known SAR of this compound series, we now describe a number of analogs of 1 designed to maximize structural diversity guided by incorporation of substructures and functional groups known to diminish ligand-androgen receptor interactions. These analogs had calculated polar surface area (PSA), measured LogD, aqueous kinetic solubility, and estimated plasma protein binding values in ranges predictive of good ADME profiles. The principal SAR insight was that the hydantoin core of 1 is required for high antischistosomal activity. We identified several compounds with high antischistosomal efficacy that were less antiandrogenic than 1. These data provide direction for the ongoing optimization of antischistosomal hydantoins.
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http://dx.doi.org/10.1021/acs.jmedchem.6b01410DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5150661PMC
December 2016

Nitroheterocyclic drugs cure experimental Trypanosoma cruzi infections more effectively in the chronic stage than in the acute stage.

Sci Rep 2016 10 17;6:35351. Epub 2016 Oct 17.

Department of Pathogen Molecular Biology, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK.

The insect-transmitted protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease, and infects 5-8 million people in Latin America. Chagas disease is characterised by an acute phase, which is partially resolved by the immune system, but then develops as a chronic life-long infection. There is a consensus that the front-line drugs benznidazole and nifurtimox are more effective against the acute stage in both clinical and experimental settings. However, confirmative studies have been restricted by difficulties in demonstrating sterile parasitological cure. Here, we describe a systematic study of nitroheterocyclic drug efficacy using highly sensitive bioluminescence imaging of murine infections. Unexpectedly, we find both drugs are more effective at curing chronic infections, judged by treatment duration and therapeutic dose. This was not associated with factors that differentially influence plasma drug concentrations in the two disease stages. We also observed that fexinidazole and fexinidazole sulfone are more effective than benznidazole and nifurtimox as curative treatments, particularly for acute stage infections, most likely as a result of the higher and more prolonged exposure of the sulfone derivative. If these findings are translatable to human patients, they will have important implications for treatment strategies.
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http://dx.doi.org/10.1038/srep35351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5066210PMC
October 2016

A Triazolopyrimidine-Based Dihydroorotate Dehydrogenase Inhibitor with Improved Drug-like Properties for Treatment and Prevention of Malaria.

ACS Infect Dis 2016 12 4;2(12):945-957. Epub 2016 Oct 4.

Centre for Drug Candidate Optimisation, Monash Institute of Pharmaceutical Sciences, Monash University , Parkville, VIC 3052, Australia.

The emergence of drug-resistant malaria parasites continues to hamper efforts to control this lethal disease. Dihydroorotate dehydrogenase has recently been validated as a new target for the treatment of malaria, and a selective inhibitor (DSM265) of the Plasmodium enzyme is currently in clinical development. With the goal of identifying a backup compound to DSM265, we explored replacement of the SF-aniline moiety of DSM265 with a series of CF-pyridinyls while maintaining the core triazolopyrimidine scaffold. This effort led to the identification of DSM421, which has improved solubility, lower intrinsic clearance, and increased plasma exposure after oral dosing compared to DSM265, while maintaining a long predicted human half-life. Its improved physical and chemical properties will allow it to be formulated more readily than DSM265. DSM421 showed excellent efficacy in the SCID mouse model of P. falciparum malaria that supports the prediction of a low human dose (<200 mg). Importantly DSM421 showed equal activity against both P. falciparum and P. vivax field isolates, while DSM265 was more active on P. falciparum. DSM421 has the potential to be developed as a single-dose cure or once-weekly chemopreventative for both P. falciparum and P. vivax malaria, leading to its advancement as a preclinical development candidate.
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http://dx.doi.org/10.1021/acsinfecdis.6b00144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5148661PMC
December 2016

Quantifying Uncertainty in the Ratio of Two Measured Variables: A Recap and Example.

J Pharm Sci 2016 11 14;105(11):3462-3463. Epub 2016 Sep 14.

Centre for Medicine Use and Safety, Faculty of Pharmacy and Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia.

Estimating uncertainty in the ratio of 2 measured variables can be achieved via 2 seemingly different approaches: by determining the variance of the first-order Taylor approximation to the ratio, or by the so-called "Propagation of Error" approach. This Lesson Learned shows that the 2 approaches are mathematically equivalent, and provides an example of the approach.
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http://dx.doi.org/10.1016/j.xphs.2016.07.019DOI Listing
November 2016

Positive Allosteric Modulation of the Muscarinic M1 Receptor Improves Efficacy of Antipsychotics in Mouse Glutamatergic Deficit Models of Behavior.

J Pharmacol Exp Ther 2016 Nov 14;359(2):354-365. Epub 2016 Sep 14.

Drug Discovery Biology (K.H.C.C., D.T.M, C.J.L, P.M.S, J.R.L, A.C.), Centre for Drug Candidate Optimization (D.M.S., R.T.P.), and Medicinal Chemistry (S.N.M, P.J.S.), Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Australia; and Melbourne Neuropsychiatry Centre, Department of Psychiatry and Centre for Neural Engineering, University of Melbourne, Melbourne, Australia (C.P.)

Current antipsychotics are effective in treating the positive symptoms associated with schizophrenia, but they remain suboptimal in targeting cognitive dysfunction. Recent studies have suggested that positive allosteric modulation of the M muscarinic acetylcholine receptor (mAChR) may provide a novel means of improving cognition. However, very little is known about the potential of combination therapies in extending coverage across schizophrenic symptom domains. This study investigated the effect of the M mAChR positive allosteric modulator BQCA [1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid], alone or in combination with haloperidol (a first-generation antipsychotic), clozapine (a second-generation atypical antipsychotic), or aripiprazole (a third-generation atypical antipsychotic), in reversing deficits in sensorimotor gating and spatial memory induced by the N-methyl-d-aspartate receptor antagonist, MK-801 [(5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine]. Sensorimotor gating and spatial memory induction are two models that represent aspects of schizophrenia modeled in rodents. In prepulse inhibition (an operational measure of sensorimotor gating), BQCA alone had minimal effects but exhibited different levels of efficacy in reversing MK-801-induced prepulse inhibition disruptions when combined with a subeffective dose of each of the three (currently prescribed) antipsychotics. Furthermore, the combined effect of BQCA and clozapine was absent in M mice. Interestingly, although BQCA alone had no effect in reversing MK-801-induced memory impairments in a Y-maze spatial test, we observed a reversal upon the combination of BQCA with atypical antipsychotics, but not with haloperidol. These findings provide proof of concept that a judicious combination of existing antipsychotics with a selective M mAChR positive allosteric modulator can extend antipsychotic efficacy in glutamatergic deficit models of behavior.
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http://dx.doi.org/10.1124/jpet.116.235788DOI Listing
November 2016

Enantiomers of nifurtimox do not exhibit stereoselective anti-Trypanosoma cruzi activity, toxicity, or pharmacokinetic properties.

Antimicrob Agents Chemother 2015 6;59(6):3645-7. Epub 2015 Apr 6.

Drugs for Neglected Diseases initiative (DNDi), Geneva, Switzerland

With the aim of improving the available drugs for the treatment of Chagas disease, individual enantiomers of nifurtimox were characterized. The results indicate that the enantiomers are equivalent in their in vitro activity against a panel of Trypanosoma cruzi strains; in vivo efficacy in a murine model of Chagas disease; in vitro toxicity and absorption, distribution, metabolism, and excretion characteristics; and in vivo pharmacokinetic properties. There is unlikely to be any therapeutic benefit of an individual nifurtimox enantiomer over the racemic mixture.
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http://dx.doi.org/10.1128/AAC.05139-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4432195PMC
February 2016