Phenylarsonous Acid Research Articles

Adenine nucleotide translocase: Current knowledge in post-translational modifications, regulations and pathological implications for human diseases.

Adenine nucleotide translocases (ANTs) are central to mitochondrial integrity and bioenergetic metabolism. This review aims to integrate the progresses and knowledge on ANTs over the last few years, contributing to a potential implication of ANTs for various diseases. Structures, functions, modifications, regulators and pathological implications of ANTs for human diseases are intensively demonstrated here. ANTs have four isoforms (ANT1-4), responsible for exchanging ATP/ADP, possibly composing of pro-apoptotic mPTP as a major component, and mediating FA-dependent uncoupling of proton efflux. ANT can be modified by methylation, nitrosylation and nitroalkylation, acetylation, glutathionylation, phosphorylation, carbonylation and hydroxynonenal-induced modifications. Compounds, including bongkrekic acid, atractyloside calcium, carbon monoxide, minocycline, 4-(N-(S-penicillaminylacetyl)amino) phenylarsonous acid, cardiolipin, free long-chain fatty acids, agaric acid, long chain acyl-coenzyme A esters, all have an ability to regulate ANT activities. ANT impairment leads to bioenergetic failure and mitochondrial dysfunction, contributing to pathogenesis of diseases, such as diabetes (deficiency), heart disease (deficiency), Parkinson's disease (reduction), Sengers Syndrome (decrease), cancer (isoform shifting), Alzheimer's Disease (coaggregation with Tau), Progressive External Opthalmoplegia (mutation), and Fascioscapulohumeral muscular dystrophy (overexpression). This review improves the understanding of the mechanism of ANT in pathogenesis of human diseases, and opens a window for novel therapeutic strategies targeted on ANT in diseases.

Preclinical Assessment of [68Ga]Ga-Cell Death Indicator (CDI): A Novel hsp90 Ligand for Positron Emission Tomography of Cell Death.

4-(N-(S-glutathionylacetyl)amino) phenylarsonous acid (GSAO) when conjugated with a bifunctional chelator 2,2'-(7-(1-carboxy-4-((2,5-dioxopyrrolidin-1-yl)oxy)-4- oxobutyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (NODAGA) (hereafter referred to as Cell Death Indicator [CDI]), enters dead and dying cells and binds to 90kDa heat shock proteins (hsp90). This study assesses stability, biodistribution, imaging, and radiation dosimetry of [68Ga]- Ga-CDI for positron emission tomography (PET). Preparation of [68Ga]Ga-CDI was performed as previously described. Product stability and stability in plasma were assessed using high-performance liquid chromatography. Biodistribution and imaging were conducted in ten healthy male Lewis rats at 1 and 2 h following intravenous [68Ga]Ga-CDI injection. Human radiation dosimetry was estimated by extrapolation for a standard reference man and calculated with OLINDA/EXM 1.1. Radiochemical purity of [68Ga]Ga-CDI averaged 93.8% in the product and 86.7% in plasma at 4 h post-synthesis. The highest concentration of [68Ga]Ga-CDI is observed in the kidneys; [68Ga]Ga-CDI is excreted in the urine, and mean retained activity was 32.4% and 21.4% at 1 and 2 h post-injection. Lower concentrations of [68Ga]Ga-CDI were present in the small bowel and liver. PET CT was concordant and additionally demonstrated focal growth plate uptake. The effective dose for [68Ga]Ga-CDI is 2.16E-02 mSv/MBq, and the urinary bladder wall received the highest dose (1.65E-02 mSv/Mbq). [68Ga] Ga-CDI is stable and has favourable biodistribution, imaging, and radiation dosimetry for imaging of dead and dying cells. Human studies are underway.

The ArsH Protein Product of the Paracoccus denitrificans ars Operon Has an Activity of Organoarsenic Reductase and Is Regulated by a Redox-Responsive Repressor.

Paracoccus denitrificans ArsH is encoded by two identical genes located in two distinct putative arsenic resistance (ars) operons. Escherichia coli-produced recombinant N-His6-ArsH was characterized both structurally and kinetically. The X-ray structure of ArsH revealed a flavodoxin-like domain and motifs for the binding of flavin mononucleotide (FMN) and reduced nicotinamide adenine dinucleotide phosphate (NADPH). The protein catalyzed FMN reduction by NADPH via ternary complex mechanism. At a fixed saturating FMN concentration, it acted as an NADPH-dependent organoarsenic reductase displaying ping-pong kinetics. A 1:1 enzymatic reaction of phenylarsonic acid with the reduced form of FMN (FMNH2) and formation of phenylarsonous acid were observed. Growth experiments with P. denitrificans and E. coli revealed increased toxicity of phenylarsonic acid to cells expressing arsH, which may be related to in vivo conversion of pentavalent As to more toxic trivalent form. ArsH expression was upregulated not only by arsenite, but also by redox-active agents paraquat, tert-butyl hydroperoxide and diamide. A crucial role is played by the homodimeric transcriptional repressor ArsR, which was shown in in vitro experiments to monomerize and release from the DNA-target site. Collectively, our results establish ArsH as responsible for enhancement of organo-As(V) toxicity and demonstrate redox control of ars operon.

Toxicokinetics of the tumour cell mitochondrial toxin, PENAO, in rodents.

PENAO (4-(N-(S-penicillaminylacetyl)amino)phenylarsonous acid) is a second-generation peptide arsenical that inactivates mitochondria in proliferating tumour cells by covalently reacting with mitochondrial inner-membrane adenine nucleotide transferase. The toxicokinetics of PENAO has been investigated in Sprague-Dawley rats to inform route of administration and dosing for human clinical trials. PENAO was well tolerated at 3.3mg/kg daily intravenous injections but associated with significant toxicity at 10mg/kg, primarily in the males. The major target organ for toxic effects was the kidney, with changes observed in tubular dilation, presence of casts, basophilic tubules, lymphoid aggregates and interstitial fibrosis. Kidney function was impaired in males with dose-dependent increase in serum creatinine concentration. The severity of the microscopic lesions was reduced in the females, but not the males, at the completion of the four-week recovery period. The elimination phase half-life of PENAO varied between 0.4 and 1.7h and volume of distribution ranged from 0.25 to 0.88L/kg for the different dose groups and treatment days, suggesting that PENAO distributes in the extracellular fluids at the doses tested. The area under the curve and clearance values indicate that male rats had reduced elimination of PENAO compared to females, which may account for the increased toxicity in males. PENAO is significantly better tolerated in rodents than its predecessor, GSAO. As GSAO was generally well tolerated with few side effects in a phase I trial in patients with solid tumours, these findings bode well for the tolerability of intravenous dosing of PENAO in patients.

Biodistribution and imaging of an hsp90 ligand labelled with 111In and 67Ga for imaging of cell death

Background4-(N-(S-glutathionylacetyl)amino) phenylarsonous acid (GSAO) when conjugated at the γ-glutamyl residue with fluorophores and radio-isotopes is able to image dead and dying cells in vitro and in vivo by binding to intracellular 90-kDa heat shock proteins (hsp90) when cell membrane integrity is compromised. The ability to image cell death has potential clinical impact especially for early treatment response assessment in oncology. This work aims to assess the biodistribution and tumour uptake of diethylene triamine pentaacetic acid GSAO labelled with 111In ([111In]In-DTPA-GSAO) and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid GSAO labelled with 67Ga ([67Ga]Ga-DOTA-GSAO) in a murine subcutaneous tumour xenograft model and estimate dosimetry of [67Ga]Ga-DOTA-GSAO.ResultsThere was good tumour uptake of both [111In]In-DTPA-GSAO and [67Ga]Ga-DOTA-GSAO (2.44 ± 0.26% injected activity per gramme of tissue (%IA/g) and 2.75 ± 0.34 %IA/g, respectively) in Balb c nu/nu mice bearing subcutaneous tumour xenografts of a human metastatic prostate cancer cell line (PC3M-luc-c6). Peak tumour uptake occurred at 2.7 h post injection. [111In]In-DTPA-GSAO and [67Ga]Ga-DOTA-GSAO demonstrated increased uptake in the liver (4.40 ± 0.86 %IA/g and 1.72 ± 0.27 %IA/g, respectively), kidneys (16.54 ± 3.86 %IA/g and 8.16 ± 1.33 %IA/g) and spleen (6.44 ± 1.24 %IA/g and 1.85 ± 0.44 %IA/g); however, uptake in these organs was significantly lower with [67Ga]Ga-DOTA-GSAO (p = 0.006, p = 0.017 and p = 0.003, respectively). Uptake of [67Ga]Ga-DOTA-GSAO into tumour was higher than all organs except the kidneys. There was negligible uptake in the other organs. Excretion of [67Ga]Ga-DOTA-GSAO was more rapid than [111In]In-DTPA-GSAO. Estimated effective dose of [67Ga]Ga-DOTA-GSAO for an adult male human was 1.54 × 10− 2 mSv/MBq.Conclusions[67Ga]Ga-DOTA-GSAO demonstrates higher specific uptake in dead and dying cells within tumours and lower uptake in normal organs than [111In]In-DTPA-GSAO. [67Ga]Ga-DOTA-GSAO may be potentially useful for imaging cell death in vivo. Dosimetry estimates for [67Ga]Ga-DOTA-GSAO are acceptable for future human studies. This work also prepares for development of 68Ga GSAO radiopharmaceuticals.

Preparation of a Dithiol-Reactive Probe for PET Imaging of Cell Death.

Conjugates of 4-(N-(S-glutathionylacetyl)amino)phenylarsonous acid (GSAO) with optical or radionuclide probes are able to image cell death in vivo. GSAO conjugates are retained in the cytosol of dying and dead cells via the formation of covalent bonds between the As(III) ion and the thiol groups of proximal cysteine residues. Here we describe the method for preparing a NODAGA-GSAO conjugate and its radiolabeling with gallium-68 (68Ga-NODAGA-GSAO) for positron-emission tomography (PET) imaging of cell death.

Flow Cytometry Assessment of Procoagulant Platelets Using a Dithiol-Reactive Probe.

Flow cytometry assessment of platelets using the combination of GSAO [4-(N-(S-glutathionylacetyl)amino)phenylarsonous acid], a dithiol-reactive probe, and P-selectin, a platelet activation marker, is a novel and powerful assay in the identification and quantification of the procoagulant subpopulation of platelets that has the capacity to support thrombin generation. In this chapter, we provide the flow cytometry protocols aimed at the study of procoagulant platelets under resting and agonist-stimulated conditions in whole blood and washed platelets of both human and murine (mouse) samples.

Length of the Stabilizing Zwitterionic Poly(2-methacryloyloxyethyl phosphorycholine) Block Influences the Activity of the Conjugated Arsenic Drug in Drug-Directed Polymerization-Induced Self-Assembly Particles.

We report on the synthesis of poly(2-methacryloyloxyethyl phosphorycholine-co-PENAO)-block-poly(methyl methacrylate) core-shell nanoparticles which carry different chain lengths of zwitterionic 2-methacryloyloxyethyl phosphorycholine (MPC) on a nanoparticle surface. The particles, 30-40 nm in size, were readily obtained by polymerization-induced self-assembly (PISA) of the corresponding arsenic-based MPC polymers as the stabilizer block and methyl methacrylate (MMA) as the core-forming block. Zwitterionic nanoparticles are ideal candidates for protein-repellent materials. Herein, we show how the decrease of zwitterionic chain lengths tunes the reactivity and cytotoxicity of the organoarsenical anticancer drug PENAO (4-(N-(S-penicillaminylacetyl)amino) phenylarsonous acid). More cytotoxic (5-fold) nanoparticles were obtained when the MPC chain lengths were condensed from 37 to 13 repeating units. To gain a better understanding of the behavior of the drug-directed PISA particles, small-angle neutron scattering (SANS) experiments were conducted, evidencing that having PENAO located in the hydrophilic building block indeed influences the physiochemical micelle structure in terms of core radius (rcore), SLD, shell thickness, and aggregation number.

Direct Polymerization of the Arsenic Drug PENAO to Obtain Nanoparticles with High Thiol-Reactivity and Anti-Cancer Efficiency.

PENAO (4-(N-(S-penicillaminylacetyl)amino) phenylarsonous acid), which is a mitochondria inhibitor that reacts with adenine nucleotide translocator (ANT), is currently being trialed in patients with solid tumors. To increase the stability of the drug, the formation of nanoparticles has been proposed. Herein, the direct synthesis of polymeric micelles based on the anticancer drug PENAO is presented. PENAO is readily available for amidation reaction to form PENAO MA (4-(N-(S-penicillaminylacetyl) amino) phenylarsonous acid methacrylamide) which undergoes RAFT (reversible addition-fragmentation chain transfer) polymerization with poly(ethylene glycol methyl ether methacrylate) as comonomer and poly(methyl methacrylate) (pMMA) as chain transfer agent, resulting in p(MMA)-b-p(PEG-co-PENAO) block copolymers with 3-15 wt % of PENAO MA. The different block copolymers self-assembled into micelle structures, varying in size and stability (Dh = 84-234 nm, cmc = 0.5-82 μg mol-1) depending on the hydrophilic to hydrophobic ratio of the polymer blocks and the amount of drug in the corona of the particle. The more stable micelle structures were investigated toward 143B human osteosarcoma cells, showing an enhanced cytotoxicity and cellular uptake compared to the free drug PENAO (IC50 (PENAO) = 2.7 ± 0.3 μM; IC50 (micelle M4) = 0.8 ± 0.02 μM). Furthermore, PENAOs arsonous acid residue remains active when incorporated into a polymer matrix and conjugates to small mono and closely spaced dithiols and is able to actively target the mitochondria, which is PENAO's main target to introduce growth inhibition in cancer cells. As a result, no cleavable linker between drug and polymer was necessary for the delivery of PENAO to osteosarcoma cells. These findings provide a rationale for in vivo studies of micelle M4 versus PENAO in an osteosarcoma animal model.

Methylated Phenylarsenical Metabolites Discovered in Chicken Liver

AbstractWe report the discovery of three toxicologically relevant methylated phenylarsenical metabolites in the liver of chickens fed 3‐nitro‐4‐hydroxyphenylarsonic acid (ROX), a feed additive in poultry production that is still in use in several countries. Methyl‐3‐nitro‐4‐hydroxyphenylarsonic acid (methyl‐ROX), methyl‐3‐amino‐4‐hydroxyphenylarsonic acid (methyl‐3‐AHPAA), and methyl‐3‐acetamido‐4‐hydroxyphenylarsonic acid (or methyl‐N‐acetyl‐ROX, methyl‐N‐AHPAA) were identified in such chicken livers, and the concentration of methyl‐ROX was as high as 90 μg kg−1, even after a five‐day clearance period. The formation of these newly discovered methylated metabolites from reactions involving trivalent phenylarsonous acid substrates, S‐adenosylmethionine, and the arsenic (+3 oxidation state) methyltransferase enzyme As3MT suggests that these compounds are formed by addition of a methyl group to a trivalent phenylarsenical substrate in an enzymatic process. The IC50 values of the trivalent phenylarsenical compounds were 300–30 000 times lower than those of the pentavalent phenylarsenicals.

Methylated Phenylarsenical Metabolites Discovered in Chicken Liver.

We report the discovery of three toxicologically relevant methylated phenylarsenical metabolites in the liver of chickens fed 3‐nitro‐4‐hydroxyphenylarsonic acid (ROX), a feed additive in poultry production that is still in use in several countries. Methyl‐3‐nitro‐4‐hydroxyphenylarsonic acid (methyl‐ROX), methyl‐3‐amino‐4‐hydroxyphenylarsonic acid (methyl‐3‐AHPAA), and methyl‐3‐acetamido‐4‐hydroxyphenylarsonic acid (or methyl‐N‐acetyl‐ROX, methyl‐N‐AHPAA) were identified in such chicken livers, and the concentration of methyl‐ROX was as high as 90 μg kg−1, even after a five‐day clearance period. The formation of these newly discovered methylated metabolites from reactions involving trivalent phenylarsonous acid substrates, S‐adenosylmethionine, and the arsenic (+3 oxidation state) methyltransferase enzyme As3MT suggests that these compounds are formed by addition of a methyl group to a trivalent phenylarsenical substrate in an enzymatic process. The IC50 values of the trivalent phenylarsenical compounds were 300–30 000 times lower than those of the pentavalent phenylarsenicals.

First-in-man trial of 4-(N-(S-penicillaminylacetyl)amino) phenylarsonous acid (PENAO) as a continuous intravenous infusion (CIVI), in patients (pt) with advanced solid tumours.

e14025Background: PENAO is an organic arsenical with anti-mitochondrial activity mediated by covalent inhibition of adenine nucleotide translocase (ANT). In vitro studies identified potential activ...

Necrotic platelets provide a procoagulant surface during thrombosis

AbstractA subpopulation of platelets fulfills a procoagulant role in hemostasis and thrombosis by enabling the thrombin burst required for fibrin formation and clot stability at the site of vascular injury. Excess procoagulant activity is linked with pathological thrombosis. The identity of the procoagulant platelet has been elusive. The cell death marker 4-[N-(S-glutathionylacetyl)amino]phenylarsonous acid (GSAO) rapidly enters a subpopulation of agonist-stimulated platelets via an organic anion-transporting polypeptide and is retained in the cytosol through covalent reaction with protein dithiols. Labeling with GSAO, together with exposure of P-selectin, distinguishes necrotic from apoptotic platelets and correlates with procoagulant potential. GSAO+ platelets form in occluding murine thrombi after ferric chloride injury and are attenuated with megakaryocyte-directed deletion of the cyclophilin D gene. These platelets form a procoagulant surface, supporting fibrin formation, and reduction in GSAO+ platelets is associated with reduction in platelet thrombus size and fibrin formation. Analysis of platelets from human subjects receiving aspirin therapy indicates that these procoagulant platelets form despite aspirin therapy, but are attenuated by inhibition of the necrosis pathway. These findings indicate that the major subpopulation of platelets involved in fibrin formation are formed via regulated necrosis involving cyclophilin D, and that they may be targeted independent of platelet activation.

ATPS-09A MITOCHONDRIAL METABOLISM INHIBITOR RADIO-SENSITIZES GLIOBLASTOMA TUMOURS IN MICE

PENAO (4-(N-(S-penicillaminylacetyl)amino)phenylarsonous acid) is a mitochondrial metabolism inhibitor that is currently in Phase I clinical testing in patients with solid tumors refractory to standard therapy. PENAO is a cysteine mimetic trivalent arsenical that enters cells via an organic ion transporter and accumulates in the mitochondrial matrix where the arsenical moiety cross-links Cys160 and Cys257 on the matrix face of adenine nucleotide translocase (ANT), which inactivates the transporter. ANT inactivation leads to partial uncoupling of oxidative phosphorylation, increase in superoxide production, proliferation arrest and ultimately apoptosis of the cell. PENAO only reacts with ANT when cells are proliferating as Cys160 and Cys257 appear to be disulfide bonded in growth quiescent cells, and so unreactive towards PENAO. PENAO inhibited the proliferation of a panel of 13 glioblastoma cells, including cells from both primary and recurrent tumors, with IC50 values ranging from 0.3-4.5 µM. The IC50 values for proliferation arrest of normal lung fibroblasts and astrocytes are 9 µM and 7 µM, respectively, indicating selectivity for glioblastoma cells. Administration of PENAO (3 mg/kg/day) to mice bearing subcutaneous glioblastoma xenografts resulted in significant inhibition of tumor growth (7 partial and 3 complete responses). Ki67 and TUNEL staining of tumor sections showed that PENAO induced tumor cell growth arrest and death, respectively. Pharmacokinetic analysis demonstrated that PENAO readily crossed the blood-brain-barrier and accumulated specifically in tumor tissue. There were also no signs or symptoms of treatment toxicity. As PENAO induces superoxide production in glioblastoma cells, we hypothesized that PENAO in combination with radiation would synergise. Combining 2 Gy/day radiation (20 Gy total) with intraperitoneal administration of 20 mg/kg/day PENAO, resulted in significant improvement in median survival in an orthotopic glioblastoma murine model. These in-vitro and in-vivo results show that PENAO is a potential new therapeutic for glioblastoma, alone or in combination with radiation therapy

Dual-targeting of aberrant glucose metabolism in glioblastoma.

BackgroundGlioblastoma (GBM) is the most common and malignant primary brain tumor. In contrast to some other tumor types, aberrant glucose metabolism is an important component of GBM growth and chemoresistance. Recent studies of human orthotopic GBM in mice and in situ demonstrated GBM cells rely on both glycolysis and mitochondrial oxidation for glucose catabolism. These observations suggest that the homeostasis of energy metabolism of GBM cells might be further disturbed by dual-inhibition of glucose metabolism. The present study aimed to evaluate the efficacy and the mechanisms of dual-targeting therapy in GBM cells.MethodsRepresentative GBM cells (immortalized GBM cell lines and patient-derived GBM cells) and non-cancerous cells were treated with 4-(N-(S-penicillaminylacetyl)amino) phenylarsonous acid (PENAO), an in-house designed novel arsenic-based mitochondrial toxin, in combination with dichloroacetate (DCA), a pyruvate dehydrogenase kinase inhibitor. The efficacy of this combinatorial therapy was evaluated by MTS assay, clonogenic surviving assay and apoptotic assays. The underlying mechanisms of this dual-targeting treatment were unraveled by using mitochondrial membrane potential measurements, cytosol/mitochondrial ROS detection, western blotting, extracellular flux assay and mass spectrometry.ResultsAs monotherapies, both PENAO and DCA induced proliferation arrest in a panel of GBM cell lines and primary isolates. PENAO inhibited oxygen consumption, induced oxidative stress and depolarized mitochondrial membrane potential, which in turn activated mitochondria-mediated apoptosis. By combining DCA with PENAO, the two drugs worked synergistically to inhibit cell proliferation (but had no significant effect on non-cancerous cells), impair the clonogenicity, and induce mitochondria-mediated apoptosis. An oxidative stress of mitochondrial origin takes a prominent place in the mechanism by which the combination of PENAO and DCA induces cell death. Additionally, PENAO-induced oxidative damage was enhanced by DCA through glycolytic inhibition which in turn diminished acid production induced by PENAO. Moreover, DCA treatment also led to an alteration in the multidrug resistance (MDR) phenotype of GBM cells, thereby leading to an increased cytosolic accumulation of PENAO.ConclusionsThe findings of this study shed a new light with respect to the dual-targeting of glucose metabolism in GBM cells and the innovative combination of PENAO and DCA shows promise in expanding GBM therapies.

Targeting of two aspects of metabolism in breast cancer treatment

Deregulated metabolism is gaining recognition as a hallmark of cancer cells, and is being explored for therapeutic potential. The Warburg effect is a metabolic phenotype that occurs in 90% of tumors, where glycolysis is favored despite the presence of oxygen. Dichloroacetate (DCA) is a pyruvate dehydrogenase kinase (PDK) inhibitor that can reverse the Warburg effect. PENAO (4-(N-(S-penicillaminylacetyl)amino) phenylarsonous acid) is a novel anti-mitochondrial agent that targets the adenine nucleotide transporter in mitochondria and is currently in clinical trials for solid tumors. We have investigated the targeting of two aspects of metabolism, using DCA to promote mitochondrial activity combined with PENAO to inhibit mitochondrial activity, in breast and other carcinoma cell lines. PENAO was effective at low uM concentrations in luminal (T-47D) and triple negative (MDA-MB-231) breast cancer cells, in normoxia and hypoxia. The cytotoxicity of PENAO was enhanced by DCA by a mechanism involving increased reactive oxygen species in both T-47D and MDA-MB-231 cells, however further investigations found it did not always involve PDK2 inhibition or reduction of the mitochondrial membrane potential, which are the accepted mechanisms for DCA induction of apoptosis. Nevertheless, DCA sensitized all cancer cell lines tested toward apoptosis of PENAO. DCA and PENAO are both currently in clinical trials and targeting cancer metabolism with these drugs may offer options for difficult to treat cancers.

DD-04 * PENAO: A POTENT MITOCHONDRIAL TARGETED INHIBITOR FOR GLIOBLASTOMA

PENAO (4-(N-(S-penicillaminylacetyl)amino)phenylarsonous acid) is a promising new treatment for glioblastoma patients and is currently in Phase-I clinical trial testing. PENAO selectively targets cancer-specific mitochondrial metabolism by binding to the inner-mitochondrial membrane protein adenine nucleotide translocase. This results in mitochondrial membrane pore transition and reactive oxygen species (ROS)-related cell death. PENAO, as a monotherapy, has shown potent efficacy however we have recently found significant synergism with PENAO and the blocking of glutathione. In a panel of 13 glioblastoma cell lines, including both commercial and primary patient-derived lines (from both primary and recurrent tumours), PENAO demonstrated marked anti-proliferative activity with IC50 values ranging from 0.3-4.5 µM. This was up to 50 and 23-fold more specific than for normal MRC5 lung fibroblasts and astrocytes, respectively. Moreover, PENAO was 440-fold more potent than temozolomide, the frontline chemotherapeutic used clinically for glioblastoma. PENAO efficacy was recapitulated in-vivo in glioblastoma mice models. Administration of PENAO (3mg/kg/day) in mice bearing subcutaneous glioblastoma xenografts resulted in a significant inhibition of tumor size (7 partial and 3 complete responses), as well as Ki67 and TUNEL staining showing that PENAO significantly induced tumor growth arrest and death, respectively. Pharmacokinetic analysis demonstrated that PENAO readily crossed the blood-brain-barrier and accumulated specifically in tumor tissue. There were also no signs or symptoms of treatment toxicity. As PENAO induces cytotoxic ROS in glioblastoma cells, we hypothesized that inhibitors of the glutathione system would synergise with PENAO. Combining the glutathione inhibitor, sulfasalazine with PENAO, resulted in significant synergy (up to 93% increase in cell death) in all lines tested. Additionally, concomitant treatment with inhibitors of ABCC1/2 transporters and glutathione synthesis increased the efficacy of PENAO by up to 472-fold. Together these in-vitro and in-vivo models of glioblastoma show that PENAO is a highly promising new therapeutic for glioblastoma, alone or in combination with glutathione inhibition.

Noninvasive molecular imaging of cell death in myocardial infarction using 111In-GSAO.

Acute insult to the myocardium is associated with substantial loss of cardiomyocytes during the process of myocardial infarction. In this setting, apoptosis (programmed cell death) and necrosis may operate on a continuum. Because the latter is characterized by the loss of sarcolemmal integrity, we propose that an appropriately labeled tracer directed at a ubiquitously present intracellular moiety would allow non-invasive definition of cardiomyocyte necrosis. A trivalent arsenic peptide, GSAO (4-(N-(S-glutathionylacetyl)amino)phenylarsonous acid), is capable of binding to intracellular dithiol molecules such as HSP90 and filamin-A. Since GSAO is membrane impermeable and dithiol molecules abundantly present intracellularly, we propose that myocardial localization would represent sarcolemmal disruption or necrotic cell death. In rabbit and mouse models of myocardial infarction and post-infarct heart failure, we employed In-111-labelled GSAO for noninvasive radionuclide molecular imaging. 111In-GSAO uptake was observed within the regions of apoptosis seeking agent- 99mTc-Annexin A5 uptake, suggesting the colocalization of apoptotic and necrotic cell death processes.

Elimination of the antimicrobial action of the organoarsenical cancer therapeutic, 4-(N-(S-glutathionylacetyl)amino) phenylarsonous acid, before finished product sterility testing

Arsenical compounds have been used therapeutically for over 2000 years finding particular relevance as antimicrobials. After being replaced by more selective and consequently less toxic antibiotics in the last century, arsenicals have recently made a resurgence as anticancer drugs (specifically arsenic trioxide and its derivatives). Arsenical parenteral formulations require post-manufacture sterility testing; however, their intrinsic antimicrobial activity must be neutralised before testing to eliminate the possibility of false (no-growth) test results. A range of thiol-containing compounds was screened to establish a suitable deactivation agent for the novel organoarsenical compound, 4-(N-(S-glutathionylacetyl)amino) phenylarsonous acid (GSAO). Dimercatopropanol (DMP) was found to successful deactivate GSAO and was validated according to pharmacopoeial sterility test guidelines (specifically the method suitability test/sterility validation test). DMP is an effective way of deactivating GSAO before sterility testing and can be used for pharmacopoeial sterility tests. Our results affirm previous research highlighting the sensitivity of Staphylococcus aureus to arsenical compounds A method of deactivating the arsenical drug GSAO before the post-manufacture sterility test was established and validated. DMP is a commonly used chelator/deactivation agent so this work may have implications for other inorganic therapeutic agents.

Optical Imaging of Treatment-Related Tumor Cell Death Using a Heat Shock Protein-90 Alkylator

The ability to assess in near-real time the tumor cell killing efficacy of chemotherapy regimens would improve patient treatment and survival. An ineffective regimen could be abandoned early in favor of a more effective treatment. We sought to noninvasively image treatment-related tumor cell death in mice using an optically labeled synthetic heat shock protein-90 (Hsp90) alkylator, 4-(N-(S-glutathionylacetyl)amino)phenylarsonous acid (GSAO). The Hsp90 chaperone is an important element in oncogene addiction and tumor cell survival, and its expression is enhanced by chemotherapy. These factors were predicted to favor the detection of tumor cell death using GSAO. GSAO specifically labeled apoptotic and necrotic tumor cells in culture and cells of comparable morphology in subcutaneous human pancreatic carcinoma tumors in mice. A near-infrared fluorescent conjugate of GSAO was used to noninvasively image cyclophosphamide-induced tumor cell death in murine orthotopic human mammary tumors. The GSAO conjugate did not accumulate in healthy organs or tissues in the mouse, and unbound compound was excreted rapidly via the kidneys. There was a significant increase in the GSAO fluorescence signal in the treated tumors measured either in vivo or ex vivo, and the fluorescence signal colocalized with apoptotic cells in sectioned tumors. The favorable biodistribution of optically labeled GSAO, the nature of its tumor cell target, and its capacity to noninvasively detect tumor cell death should facilitate the application of this compound in studies of the efficacy of existing and new chemotherapeutics.