Browsing by Author "Spasojevic, Ivan"

Now showing 1 - 20 of 22

Open Access
A high-throughput in vitro drug screen in a genetically engineered mouse model of diffuse intrinsic pontine glioma identifies BMS-754807 as a promising therapeutic agent.
(PLoS One, 2015) Halvorson, Kyle G; Barton, Kelly L; Schroeder, Kristin; Misuraca, Katherine L; Hoeman, Christine; Chung, Alex; Crabtree, Donna M; Cordero, Francisco J; Singh, Raj; Spasojevic, Ivan; Berlow, Noah; Pal, Ranadip; Becher, Oren J
Diffuse intrinsic pontine gliomas (DIPGs) represent a particularly lethal type of pediatric brain cancer with no effective therapeutic options. Our laboratory has previously reported the development of genetically engineered DIPG mouse models using the RCAS/tv-a system, including a model driven by PDGF-B, H3.3K27M, and p53 loss. These models can serve as a platform in which to test novel therapeutics prior to the initiation of human clinical trials. In this study, an in vitro high-throughput drug screen as part of the DIPG preclinical consortium using cell-lines derived from our DIPG models identified BMS-754807 as a drug of interest in DIPG. BMS-754807 is a potent and reversible small molecule multi-kinase inhibitor with many targets including IGF-1R, IR, MET, TRKA, TRKB, AURKA, AURKB. In vitro evaluation showed significant cytotoxic effects with an IC50 of 0.13 μM, significant inhibition of proliferation at a concentration of 1.5 μM, as well as inhibition of AKT activation. Interestingly, IGF-1R signaling was absent in serum-free cultures from the PDGF-B; H3.3K27M; p53 deficient model suggesting that the antitumor activity of BMS-754807 in this model is independent of IGF-1R. In vivo, systemic administration of BMS-754807 to DIPG-bearing mice did not prolong survival. Pharmacokinetic analysis demonstrated that tumor tissue drug concentrations of BMS-754807 were well below the identified IC50, suggesting that inadequate drug delivery may limit in vivo efficacy. In summary, an unbiased in vitro drug screen identified BMS-754807 as a potential therapeutic agent in DIPG, but BMS-754807 treatment in vivo by systemic delivery did not significantly prolong survival of DIPG-bearing mice.
Open Access
A new SOD mimic, Mn(III) ortho N-butoxyethylpyridylporphyrin, combines superb potency and lipophilicity with low toxicity.
(Free radical biology & medicine, 2012-05) Rajic, Zrinka; Tovmasyan, Artak; Spasojevic, Ivan; Sheng, Huaxin; Lu, Miaomiao; Li, Alice M; Gralla, Edith B; Warner, David S; Benov, Ludmil; Batinic-Haberle, Ines
The Mn porphyrins of k(cat)(O(2)(.-)) as high as that of a superoxide dismutase enzyme and of optimized lipophilicity have already been synthesized. Their exceptional in vivo potency is at least in part due to their ability to mimic the site and location of mitochondrial superoxide dismutase, MnSOD. MnTnHex-2-PyP(5+) is the most studied among lipophilic Mn porphyrins. It is of remarkable efficacy in animal models of oxidative stress injuries and particularly in central nervous system diseases. However, when used at high single and multiple doses it becomes toxic. The toxicity of MnTnHex-2-PyP(5+) has been in part attributed to its micellar properties, i.e., the presence of polar cationic nitrogens and hydrophobic alkyl chains. The replacement of a CH(2) group by an oxygen atom in each of the four alkyl chains was meant to disrupt the porphyrin micellar character. When such modification occurs at the end of long alkyl chains, the oxygens become heavily solvated, which leads to a significant drop in the lipophilicity of porphyrin. However, when the oxygen atoms are buried deeper within the long heptyl chains, their excessive solvation is precluded and the lipophilicity preserved. The presence of oxygens and the high lipophilicity bestow the exceptional chemical and physical properties to Mn(III) meso-tetrakis(N-n-butoxyethylpyridinium-2-yl)porphyrin, MnTnBuOE-2-PyP(5+). The high SOD-like activity is preserved and even enhanced: log k(cat)(O(2)(.-))=7.83 vs 7.48 and 7.65 for MnTnHex-2-PyP(5+) and MnTnHep-2-PyP(5+), respectively. MnTnBuOE-2-PyP(5+) was tested in an O(2)(.-) -specific in vivo assay, aerobic growth of SOD-deficient yeast, Saccharomyces cerevisiae, where it was fully protective in the range of 5-30 μM. MnTnHep-2-PyP(5+) was already toxic at 5 μM, and MnTnHex-2-PyP(5+) became toxic at 30 μM. In a mouse toxicity study, MnTnBuOE-2-PyP(5+) was several-fold less toxic than either MnTnHex-2-PyP(5+) or MnTnHep-2-PyP(5+).
Open Access
A Testosterone Metabolite 19-Hydroxyandrostenedione Induces Neuroendocrine Trans-Differentiation of Prostate Cancer Cells via an Ectopic Olfactory Receptor.
(Frontiers in oncology, 2018-01) Abaffy, Tatjana; Bain, James R; Muehlbauer, Michael J; Spasojevic, Ivan; Lodha, Shweta; Bruguera, Elisa; O'Neal, Sara K; Kim, So Young; Matsunami, Hiroaki
Olfactory receptor OR51E2, also known as a Prostate Specific G-Protein Receptor, is highly expressed in prostate cancer but its function is not well understood. Through in silico and in vitro analyses, we identified 24 agonists and 1 antagonist for this receptor. We detected that agonist 19-hydroxyandrostenedione, a product of the aromatase reaction, is endogenously produced upon receptor activation. We characterized the effects of receptor activation on metabolism using a prostate cancer cell line and demonstrated decreased intracellular anabolic signals and cell viability, induction of cell cycle arrest, and increased expression of neuronal markers. Furthermore, upregulation of neuron-specific enolase by agonist treatment was abolished in OR51E2-KO cells. The results of our study suggest that OR51E2 activation results in neuroendocrine trans-differentiation. These findings reveal a new role for OR51E2 and establish this G-protein coupled receptor as a novel therapeutic target in the treatment of prostate cancer.
Open Access
Challenges encountered during development of Mn porphyrin-based, potent redox-active drug and superoxide dismutase mimic, MnTnBuOE-2-PyP5+, and its alkoxyalkyl analogues
(JOURNAL OF INORGANIC BIOCHEMISTRY, 2017-04-01) Rajic, Zrinka; Tovmasyan, Artak; de Santana, Otávio L; Peixoto, Isabelle N; Spasojevic, Ivan; do Monte, Silmar A; Ventura, Elizete; Rebouças, Júlio S; Batinic-Haberle, Ines
Open Access
Comprehensive pharmacokinetic studies and oral bioavailability of two Mn porphyrin-based SOD mimics, MnTE-2-PyP5+ and MnTnHex-2-PyP5+.
(Free radical biology & medicine, 2013-05) Weitner, Tin; Kos, Ivan; Sheng, Huaxin; Tovmasyan, Artak; Reboucas, Julio S; Fan, Ping; Warner, David S; Vujaskovic, Zeljko; Batinic-Haberle, Ines; Spasojevic, Ivan
The cationic, ortho Mn(III) N-alkylpyridylporphyrins (alkyl=ethyl, E, and n-hexyl, nHex) MnTE-2-PyP(5+) (AEOL10113, FBC-007) and MnTnHex-2-PyP(5+) have proven efficacious in numerous in vivo animal models of diseases having oxidative stress in common. The remarkable therapeutic efficacy observed is due to their: (1) ability to catalytically remove O2(•-) and ONOO(-) and other reactive species; (2) ability to modulate redox-based signaling pathways; (3) accumulation within critical cellular compartments, i.e., mitochondria; and (4) ability to cross the blood-brain barrier. The similar redox activities of both compounds are related to the similar electronic and electrostatic environments around the metal active sites, whereas their different bioavailabilities are presumably influenced by the differences in lipophilicity, bulkiness, and shape. Both porphyrins are water soluble, but MnTnHex-2-PyP(5+) is approximately 4 orders of magnitude more lipophilic than MnTE-2-PyP(5+), which should positively affect its ability to pass through biological membranes, making it more efficacious in vivo at lower doses. To gain insight into the in vivo tissue distribution of Mn porphyrins and its impact upon their therapeutic efficacy and mechanistic aspects of action, as well as to provide data that would ensure proper dosing regimens, we conducted comprehensive pharmacokinetic (PK) studies for 24h after single-dose drug administration. The porphyrins were administered intravenously (iv), intraperitoneally (ip), and via oral gavage at the following doses: 10mg/kg MnTE-2-PyP(5+) and 0.5 or 2mg/kg MnTnHex-2-PyP(5+). Drug levels in plasma and various organs (liver, kidney, spleen, heart, lung, brain) were determined and PK parameters calculated (Cmax, C24h, tmax, and AUC). Regardless of high water solubility and pentacationic charge of these Mn porphyrins, they are orally available. The oral availability (based on plasma AUCoral/AUCiv) is 23% for MnTE-2-PyP(5+) and 21% for MnTnHex-2-PyP(5+). Despite the fivefold lower dose administered, the AUC values for liver, heart, and spleen are higher for MnTnHex-2-PyP(5+) than for MnTE-2-PyP(5+) (and comparable for other organs), clearly demonstrating the better tissue penetration and tissue retention of the more lipophilic MnTnHex-2-PyP(5+).
Open Access
Differential coordination demands in Fe versus Mn water-soluble cationic metalloporphyrins translate into remarkably different aqueous redox chemistry and biology.
(Inorganic chemistry, 2013-05-06) Tovmasyan, Artak; Weitner, Tin; Sheng, Huaxin; Lu, MiaoMiao; Rajic, Zrinka; Warner, David S; Spasojevic, Ivan; Reboucas, Julio S; Benov, Ludmil; Batinic-Haberle, Ines
The different biological behavior of cationic Fe and Mn pyridylporphyrins in Escherichia coli and mouse studies prompted us to revisit and compare their chemistry. For that purpose, the series of ortho and meta isomers of Fe(III) meso-tetrakis-N-alkylpyridylporphyrins, alkyl being methyl to n-octyl, were synthesized and characterized by elemental analysis, UV/vis spectroscopy, mass spectrometry, lipophilicity, protonation equilibria of axial waters, metal-centered reduction potential, E(1/2) for M(III)P/M(II)P redox couple (M = Fe, Mn, P = porphyrin), kcat for the catalysis of O2(•-) dismutation, stability toward peroxide-driven porphyrin oxidative degradation (produced in the catalysis of ascorbate oxidation by MP), ability to affect growth of SOD-deficient E. coli, and toxicity to mice. Electron-deficiency of the metal site is modulated by the porphyrin ligand, which renders Fe(III) porphyrins ≥5 orders of magnitude more acidic than the analogous Mn(III) porphyrins, as revealed by the pKa1 of axially coordinated waters. The 5 log units difference in the acidity between the Mn and Fe sites in porphyrin translates into the predominance of tetracationic (OH)(H2O)FeP complexes relative to pentacationic (H2O)2MnP species at pH ∼7.8. This is additionally evidenced in large differences in the E(1/2) values of M(III)P/M(II)P redox couples. The presence of hydroxo ligand labilizes trans-axial water which results in higher reactivity of Fe relative to Mn center. The differences in the catalysis of O2(•-) dismutation (log kcat) between Fe and Mn porphyrins is modest, 2.5-5-fold, due to predominantly outer-sphere, with partial inner-sphere character of two reaction steps. However, the rate constant for the inner-sphere H2O2-based porphyrin oxidative degradation is 18-fold larger for (OH)(H2O)FeP than for (H2O)2MnP. The in vivo consequences of the differences between the Fe and Mn porphyrins were best demonstrated in SOD-deficient E. coli growth. On the basis of fairly similar log kcat(O2(•-)) values, a very similar effect on the growth of SOD-deficient E. coli was anticipated by both metalloporphyrins. Yet, while (H2O)2MnTE-2-PyP(5+) was fully efficacious at ≥20 μM, the Fe analogue (OH)(H2O)FeTE-2-PyP(4+) supported SOD-deficient E. coli growth at as much as 200-fold lower doses in the range of 0.1-1 μM. Moreover the pattern of SOD-deficient E. coli growth was different with Mn and Fe porphyrins. Such results suggested a different mode of action of these metalloporphyrins. Further exploration demonstrated that (1) 0.1 μM (OH)(H2O)FeTE-2-PyP(4+) provided similar growth stimulation as the 0.1 μM Fe salt, while the 20 μM Mn salt provides no protection to E. coli; and (2) 1 μM Fe porphyrin is fully degraded by 12 h in E. coli cytosol and growth medium, while Mn porphyrin is not. Stimulation of the aerobic growth of SOD-deficient E. coli by the Fe porphyrin is therefore due to iron acquisition. Our data suggest that in vivo, redox-driven degradation of Fe porphyrins resulting in Fe release plays a major role in their biological action. Possibly, iron reconstitutes enzymes bearing [4Fe-4S] clusters as active sites. Under the same experimental conditions, (OH)(H2O)FePs do not cause mouse arterial hypotension, whereas (H2O)2MnPs do, which greatly limits the application of Mn porphyrins in vivo.
Open Access
Disrupting the vicious cycle created by NOX activation in sickle erythrocytes exposed to hypoxia/reoxygenation prevents adhesion and vasoocclusion.
(Redox biology, 2019-07) MacKinney, Anson; Woska, Emily; Spasojevic, Ivan; Batinic-Haberle, Ines; Zennadi, Rahima
In sickle cell disease (SCD), recurrent painful vasoocclusive crisis are likely caused by repeated episodes of hypoxia and reoxygenation. The sickle erythrocyte (SSRBC) adhesion plays an active role in vasoocclusion. However, the effect of prolonged reoxygenation after hypoxic stress on the molecular mechanisms in SSRBCs involved in onset of episodic vasoocclusion remain unclear. Exposure of human SSRBCs to hypoxia followed by 2 h reoxygenation, increased reactive oxygen species (ROS) production. Using specific pharmacological inhibitors, we show that excess ROS production in both reticulocytes and mature SSRBCs is regulated by NADPH oxidases (NOXs), the mitogen-activated protein kinase (ERK1/2), and G-protein coupled-receptor kinase 2 (GRK2). Consequently, SSRBC ROS create an intracellular positive feedback loop with ERK1/2 and GRK2 to mediate SSRBC adhesion to endothelium in vitro, and vasoocclusion in a mouse model of vasoocclusion in vivo. Importantly, reducing ROS levels in SSRBCs with redox-active manganese (Mn) porphyrins, commonly known as mimics of superoxide dismutase (SOD), disrupted the cycle created by ROS by affecting NOX and GRK2 activities and ERK1/2 phosphorylation, thus abrogating RBC-endothelial interactions. Inhibition adhesion assays show that LW (ICAM-4, CD242) blood group glycoprotein and CD44 are the RBC adhesion molecules mediating endothelial binding. Conversely, hypoxia/reoxygenation of normal RBCs failed to activate this feedback loop, and adhesion. These findings provide novel insights into the pathophysiological significance of the deleterious cycle created by NOX-dependent ROS, GRK2 and ERK1/2 within SSRBCs activated by hypoxia/reoxygenation, and involved in SSRBC adhesion and vasoocclusion. Thus, this loop in SSRBCs, which can be disrupted by Mn porphyrins, likely drives the profound SCD vasculopathy, and may point to new therapeutic targets to prevent chronic vasoocclusive events.
Open Access
Disruption of wild-type IDH1 suppresses D-2-hydroxyglutarate production in IDH1-mutated gliomas.
(Cancer research, 2013-01) Jin, Genglin; Reitman, Zachary J; Duncan, Christopher G; Spasojevic, Ivan; Gooden, David M; Rasheed, B Ahmed; Yang, Rui; Lopez, Giselle Y; He, Yiping; McLendon, Roger E; Bigner, Darell D; Yan, Hai
Point mutations at Arg132 of the cytoplasmic NADP(+)-dependent isocitrate dehydrogenase 1 (IDH1) occur frequently in gliomas and result in a gain of function to produce the "oncometabolite" D-2-hydroxyglutarate (D-2HG). The mutated IDH1 allele is usually associated with a wild-type IDH1 allele (heterozygous) in cancer. Here, we identify 2 gliomas that underwent loss of the wild-type IDH1 allele but retained the mutant IDH1 allele following tumor progression from World Health Organization (WHO) grade III anaplastic astrocytomas to WHO grade IV glioblastomas. Intratumoral D-2HG was 14-fold lower in the glioblastomas lacking wild-type IDH1 than in glioblastomas with heterozygous IDH1 mutations. To characterize the contribution of wild-type IDH1 to cancer cell D-2HG production, we established an IDH1-mutated astrocytoma (IMA) cell line from a WHO grade III anaplastic astrocytoma. Disruption of the wild-type IDH1 allele in IMA cells by gene targeting resulted in an 87-fold decrease in cellular D-2HG levels, showing that both wild-type and mutant IDH1 alleles are required for D-2HG production in glioma cells. Expression of wild-type IDH1 was also critical for mutant IDH1-associated D-2HG production in the colorectal cancer cell line HCT116. These insights may aid in the development of therapeutic strategies to target IDH1-mutated cancers.
Open Access
Enhanced Drug Delivery to the Skin Using Liposomes.
(Plastic and reconstructive surgery. Global open, 2018-07-09) Blueschke, Gert; Boico, Alina; Negussie, Ayele H; Yarmolenko, Pavel; Wood, Bradford J; Spasojevic, Ivan; Fan, Ping; Erdmann, Detlev; Schroeder, Thies; Sauerbier, Michael; Klitzman, Bruce
Enhancing drug delivery to the skin has importance in many therapeutic strategies. In particular, the outcome in vascularized composite allotransplantation mainly depends on systemic immunosuppression to prevent and treat episodes of transplant rejection. However, the side effects of systemic immunosuppression may introduce substantial risk to the patient and are weighed against the expected benefits. Successful enhancement of delivery of immunosuppressive agents to the most immunogenic tissues would allow for a reduction in systemic doses, thereby minimizing side effects. Nanoparticle-assisted transport by low temperature-sensitive liposomes (LTSLs) has shown some benefit in anticancer therapy. Our goal was to test whether delivery of a marker agent to the skin could be selectively enhanced.In an in vivo model, LTSLs containing doxorubicin (dox) as a marker were administered intravenously to rats that were exposed locally to mild hyperthermia. Skin samples of the hyperthermia treated hind limb were compared with skin of the contralateral normothermia hind limb. Tissue content of dox was quantified both via high-performance liquid chromatography and via histology in skin and liver.The concentration of dox in hyperthermia-treated skin was significantly elevated over both normothermic skin and liver. (P < 0.02).We show here that delivery of therapeutics to the skin can be targeted and enhanced using LTSLs. Targeting drug delivery with this method may reduce the systemic toxicity seen in a systemic free-drug administration. Development of more hydrophilic immunosuppressants in the future would increase the applicability of this system in the treatment of rejection reactions in vascularized composite allotransplantation. The treatment of other skin condition might be another potential application.
Open Access
Fe Porphyrin-Based SOD Mimic and Redox-Active Compound, (OH)FeTnHex-2-PyP4+, in a Rodent Ischemic Stroke (MCAO) Model: Efficacy and Pharmacokinetics as Compared to Its Mn Analogue, (H2O)MnTnHex-2-PyP5+.
(Antioxidants (Basel, Switzerland), 2020-06) Li, Litao; Tovmasyan, Artak; Sheng, Huaxin; Xu, Bin; Sampaio, Romulo S; Reboucas, Julio S; Warner, David S; Batinic-Haberle, Ines; Spasojevic, Ivan
Mn(III) meso-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin, (H2O)MnTnHex-2-PyP5+ (MnHex) carrying long hexyl chains, is a lipophilic mimic of superoxide dismutase (SOD) and a redox-active drug candidate. MnHex crosses the blood-brain barrier, and improved neurologic outcome and decreased infarct size and inflammation in a rat middle cerebral artery occlusion (MCAO) ischemic stroke model. Yet, the dose and the therapeutic efficacy of Mn porphyrin were limited by an adverse effect of arterial hypotension. An equally lipophilic Fe analog, (OH)FeTnHex-2-PyP4+ (FeHex), is as redox-active and potent SOD mimic in vitro. With different coordination geometry of the metal site, FeHex has one hydroxo (OH) ligand (instead of water) bound to the Fe center in the axial position. It has ~2 orders of magnitude higher efficacy than MnHex in an SOD-deficient E. coli model of oxidative stress. In vivo, it does not cause arterial hypotension and is less toxic to mice. We thus evaluated FeHex versus MnHex in a rodent MCAO model. We first performed short- and long-term pharmacokinetics (PK) of both porphyrins in the plasma, brain, and liver of rats and mice. Given that damage to the brain during stroke occurs very rapidly, fast delivery of a sufficient dose of drug is important. Therefore, we aimed to demonstrate if, and how fast after reperfusion, Fe porphyrin reaches the brain relative to the Mn analog. A markedly different plasma half-life was found with FeHex (~23 h) than with MnHex (~1.4 h), which resulted in a more than 2-fold higher plasma exposure (AUC) in a 7-day twice-daily treatment of rats. The increased plasma half-life is explained by the much lower liver retention of FeHex than typically found in Mn analogs. In the brain, a 3-day mouse PK study showed similar levels of MnHex and FeHex. The same result was obtained in a 7-day rat PK study, despite the higher plasma exposure of FeHex. Importantly, in a short-term PK study with treatment starting 2 h post MCAO, both Fe- and Mn- analogs distributed at a higher level to the injured brain hemisphere, with a more pronounced effect observed with FeHex. While a 3-day mouse MCAO study suggested the efficacy of Fe porphyrin, in a 7-day rat MCAO study, Mn-, but not Fe porphyrin, was efficacious. The observed lack of FeHex efficacy was discussed in terms of significant differences in the chemistry of Fe vs the Mn center of metalloporphyrin; relative to MnHex, FeHex has the propensity for axial coordination, which in vivo would preclude the reactivity of the Fe center towards small reactive species.
Open Access
Growth hormone mitigates against lethal irradiation and enhances hematologic and immune recovery in mice and nonhuman primates.
(PLoS One, 2010-06-16) Chen, Benny J; Deoliveira, Divino; Spasojevic, Ivan; Sempowski, Gregory D; Jiang, Chen; Owzar, Kouros; Wang, Xiaojuan; Gesty-Palmer, Diane; Cline, J Mark; Bourland, J Daniel; Dugan, Greg; Meadows, Sarah K; Daher, Pamela; Muramoto, Garrett; Chute, John P; Chao, Nelson J
Medications that can mitigate against radiation injury are limited. In this study, we investigated the ability of recombinant human growth hormone (rhGH) to mitigate against radiation injury in mice and nonhuman primates. BALB/c mice were irradiated with 7.5 Gy and treated post-irradiation with rhGH intravenously at a once daily dose of 20 microg/dose for 35 days. rhGH protected 17 out of 28 mice (60.7%) from lethal irradiation while only 3 out of 28 mice (10.7%) survived in the saline control group. A shorter course of 5 days of rhGH post-irradiation produced similar results. Compared with the saline control group, treatment with rhGH on irradiated BALB/c mice significantly accelerated overall hematopoietic recovery. Specifically, the recovery of total white cells, CD4 and CD8 T cell subsets, B cells, NK cells and especially platelets post radiation exposure were significantly accelerated in the rhGH-treated mice. Moreover, treatment with rhGH increased the frequency of hematopoietic stem/progenitor cells as measured by flow cytometry and colony forming unit assays in bone marrow harvested at day 14 after irradiation, suggesting the effects of rhGH are at the hematopoietic stem/progenitor level. rhGH mediated the hematopoietic effects primarily through their niches. Similar data with rhGH were also observed following 2 Gy sublethal irradiation of nonhuman primates. Our data demonstrate that rhGH promotes hematopoietic engraftment and immune recovery post the exposure of ionizing radiation and mitigates against the mortality from lethal irradiation even when administered after exposure.
Open Access
H₂O₂-Driven Anticancer Activity of Mn Porphyrins and the Underlying Molecular Pathways.
(Oxidative medicine and cellular longevity, 2021-01) Batinic-Haberle, Ines; Tovmasyan, Artak; Huang, Zhiqing; Duan, Weina; Du, Li; Siamakpour-Reihani, Sharareh; Cao, Zhipeng; Sheng, Huaxin; Spasojevic, Ivan; Alvarez Secord, Angeles
Mn(III) ortho-N-alkyl- and N-alkoxyalkyl porphyrins (MnPs) were initially developed as superoxide dismutase (SOD) mimics. These compounds were later shown to react with numerous reactive species (such as ONOO^-, H₂O₂, H₂S, CO₃ ^•-, ascorbate, and GSH). Moreover, the ability of MnPs to oxidatively modify activities of numerous proteins has emerged as their major mechanism of action both in normal and in cancer cells. Among those proteins are transcription factors (NF-κB and Nrf2), mitogen-activated protein kinases, MAPKs, antiapoptotic bcl-2, and endogenous antioxidative defenses. The lead Mn porphyrins, namely, MnTE-2-PyP⁵⁺ (BMX-010, AEOL10113), MnTnBuOE-2-PyP⁵⁺ (BMX-001), and MnTnHex-2-PyP⁵⁺, were tested in numerous injuries of normal tissue and cellular and animal cancer models. The wealth of the data led to the progression of MnTnBuOE-2-PyP⁵⁺ into four Phase II clinical trials on glioma, head and neck cancer, anal cancer, and multiple brain metastases, while MnTE-2-PyP⁵⁺ is in Phase II clinical trial on atopic dermatitis and itch.
Open Access
Hyperthermia Improves Solubility of Intravesical Chemotherapeutic Agents
(Bladder Cancer, 2020-12-14) Grimberg, Dominic; Shah, Ankeet; Tan, Wei Phin; Etienne, Wiguins; Spasojevic, Ivan; Inman, Brant
BACKGROUND: Nearly 70% of all new cases of bladder cancer are non-muscle invasive disease, the treatment for which includes transurethral resection followed by intravesical therapy. Unfortunately, recurrence rates approach 50% in part due to poor intravesical drug delivery. Hyperthermia is frequently used as an adjunct to intravesical chemotherapy to improve drug delivery and response to treatment. OBJECTIVE: To assess the solubility profile of intravesical chemotherapies under varying conditions of pH and temperature. METHODS: Using microplate laser nephelometry we measured the solubility of three intravesical chemotherapy agents (mitomycin C, gemcitabine, and cisplatin) at varying physical conditions. Drugs were assessed at room temperature (23°C), body temperature (37°C), and 43°C, the temperature used for hyperthermic intravesical treatments. To account for variations in urine pH, solubility was also investigated at pH 4.00, 6.00, and 8.00. RESULTS: Heat incrementally increased the solubility of all three drugs studied. Conversely, pH largely did not impact solubility aside for gemcitabine which showed slightly reduced solubility at pH 8.00 versus 6.00 or 4.00. Mitomycin C at the commonly used 2.0 mg/mL was insoluble at room temperature, but soluble at both 37 and 43°C. CONCLUSIONS: Hyperthermia as an adjunct to intravesical treatment would improve drug solubility, and likely drug delivery as some current regimens are insoluble without heat. Improvements in solubility also allow for testing of alternative administration regimens to improve drug delivery or tolerability. Further studies are needed to confirm that improvements in solubility result in increased drug delivery.
Open Access
Increasing O-GlcNAcylation is neuroprotective in young and aged brains after ischemic stroke.
(Experimental neurology, 2021-05) Wang, Zhuoran; Li, Xuan; Spasojevic, Ivan; Lu, Liping; Shen, Yuntian; Qu, Xingguang; Hoffmann, Ulrike; Warner, David S; Paschen, Wulf; Sheng, Huaxin; Yang, Wei
Spliced X-box binding protein-1 (XBP1s) together with the hexosamine biosynthetic pathway (HBP) and O-GlcNAcylation forms the XBP1s/HBP/O-GlcNAc axis. Our previous studies have provided evidence that activation of this axis is neuroprotective after ischemic stroke and critically, ischemia-induced O-GlcNAcylation is impaired in the aged brain. However, the XBP1s' neuroprotective role and its link to O-GlcNAcylation in stroke, as well as the therapeutic potential of targeting this axis in stroke, have not been well established. Moreover, the mechanisms underlying this age-related impairment of O-GlcNAcylation induction after brain ischemia remain completely unknown. In this study, using transient ischemic stroke models, we first demonstrated that neuron-specific overexpression of Xbp1s improved outcome, and pharmacologically boosting O-GlcNAcylation with thiamet-G reversed worse outcome observed in neuron-specific Xbp1 knockout mice. We further showed that thiamet-G treatment improved long-term functional recovery in both young and aged animals after transient ischemic stroke. Mechanistically, using an analytic approach developed here, we discovered that availability of UDP-GlcNAc was compromised in the aged brain, which may constitute a novel mechanism responsible for the impaired O-GlcNAcylation activation in the aged brain after ischemia. Finally, based on this new mechanistic finding, we evaluated and confirmed the therapeutic effects of glucosamine treatment in young and aged animals using both transient and permanent stroke models. Our data together support that increasing O-GlcNAcylation is a promising strategy in stroke therapy.
Open Access
Methoxy-derivatization of alkyl chains increases the in vivo efficacy of cationic Mn porphyrins. Synthesis, characterization, SOD-like activity, and SOD-deficient E. coli study of meta Mn(III) N-methoxyalkylpyridylporphyrins.
(Dalton transactions (Cambridge, England : 2003), 2011-04) Tovmasyan, Artak G; Rajic, Zrinka; Spasojevic, Ivan; Reboucas, Julio S; Chen, Xin; Salvemini, Daniela; Sheng, Huaxin; Warner, David S; Benov, Ludmil; Batinic-Haberle, Ines
Cationic Mn(III) N-alkylpyridylporphyrins (MnPs) are potent SOD mimics and peroxynitrite scavengers and diminish oxidative stress in a variety of animal models of central nervous system (CNS) injuries, cancer, radiation, diabetes, etc. Recently, properties other than antioxidant potency, such as lipophilicity, size, shape, and bulkiness, which influence the bioavailability and the toxicity of MnPs, have been addressed as they affect their in vivo efficacy and therapeutic utility. Porphyrin bearing longer alkyl substituents at pyridyl ring, MnTnHex-2-PyP(5+), is more lipophilic, thus more efficacious in vivo, particularly in CNS injuries, than the shorter alkyl-chained analog, MnTE-2-PyP(5+). Its enhanced lipophilicity allows it to accumulate in mitochondria (relative to cytosol) and to cross the blood-brain barrier to a much higher extent than MnTE-2-PyP(5+). Mn(III) N-alkylpyridylporphyrins of longer alkyl chains, however, bear micellar character, and when used at higher levels, become toxic. Recently we showed that meta isomers are ∼10-fold more lipophilic than ortho species, which enhances their cellular accumulation, and thus reportedly compensates for their somewhat inferior SOD-like activity. Herein, we modified the alkyl chains of the lipophilic meta compound, MnTnHex-3-PyP(5+) via introduction of a methoxy group, to diminish its toxicity (and/or enhance its efficacy), while maintaining high SOD-like activity and lipophilicity. We compared the lipophilic Mn(III) meso-tetrakis(N-(6'-methoxyhexyl)pyridinium-3-yl)porphyrin, MnTMOHex-3-PyP(5+), to a hydrophilic Mn(III) meso-tetrakis(N-(2'-methoxyethyl)pyridinium-3-yl)porphyrin, MnTMOE-3-PyP(5+). The compounds were characterized by uv-vis spectroscopy, mass spectrometry, elemental analysis, electrochemistry, and ability to dismute O(2)˙(-). Also, the lipophilicity was characterized by thin-layer chromatographic retention factor, R(f). The SOD-like activities and metal-centered reduction potentials for the Mn(III)P/Mn(II)P redox couple were similar-to-identical to those of N-alkylpyridyl analogs: log k(cat) = 6.78, and E(1/2) = +68 mV vs. NHE (MnTMOHex-3-PyP(5+)), and log k(cat) = 6.72, and E(1/2) = +64 mV vs. NHE (MnTMOE-3-PyP(5+)). The compounds were tested in a superoxide-specific in vivo model: aerobic growth of SOD-deficient E. coli, JI132. Both MnTMOHex-3-PyP(5+) and MnTMOE-3-PyP(5+) were more efficacious than their alkyl analogs. MnTMOE-3-PyP(5+) is further significantly more efficacious than the most explored compound in vivo, MnTE-2-PyP(5+). Such a beneficial effect of MnTMOE-3-PyP(5+) on diminished toxicity, improved efficacy and transport across the cell wall may originate from the favorable interplay of the size, length of pyridyl substituents, rotational flexibility (the ortho isomer, MnTE-2-PyP(5+), is more rigid, while MnTMOE-3-PyP(5+) is a more flexible meta isomer), bulkiness and presence of oxygen.
Open Access
Mutant IDH1 is required for IDH1 mutated tumor cell growth.
(Oncotarget, 2012-08) Jin, Genglin; Pirozzi, Christopher J; Chen, Lee H; Lopez, Giselle Y; Duncan, Christopher G; Feng, Jie; Spasojevic, Ivan; Bigner, Darell D; He, Yiping; Yan, Hai
Frequent somatic hotspot mutations in isocitrate dehydrogenase 1 (IDH1) have been identified in gliomas, acute myeloid leukemias, chondrosarcomas, and other cancers, providing a likely avenue for targeted cancer therapy. However, whether mutant IDH1 protein is required for maintaining IDH1 mutated tumor cell growth remains unknown. Here, using a genetically engineered inducible system, we report that selective suppression of endogenous mutant IDH1 expression in HT1080, a fibrosarcoma cell line with a native IDH1(R132C) heterozygous mutation, significantly inhibits cell proliferation and decreases clonogenic potential. Our findings offer insights into changes that may contribute to the inhibition of cell proliferation and offer a strong preclinical rationale for utilizing mutant IDH1 as a valid therapeutic target.
Open Access
Neuroprotective efficacy from a lipophilic redox-modulating Mn(III) N-Hexylpyridylporphyrin, MnTnHex-2-PyP: rodent models of ischemic stroke and subarachnoid hemorrhage.
(The Journal of pharmacology and experimental therapeutics, 2011-09) Sheng, Huaxin; Spasojevic, Ivan; Tse, Hubert M; Jung, Jin Yong; Hong, Jun; Zhang, Zhiquan; Piganelli, Jon D; Batinic-Haberle, Ines; Warner, David S
Intracerebroventricular treatment with redox-regulating Mn(III) N-hexylpyridylporphyrin (MnPorphyrin) is remarkably efficacious in experimental central nervous system (CNS) injury. Clinical development has been arrested because of poor blood-brain barrier penetration. Mn(III) meso-tetrakis (N-hexylpyridinium-2-yl) porphyrin (MnTnHex-2-PyP) was synthesized to include four six-carbon (hexyl) side chains on the core MnPorphyrin structure. This has been shown to increase in vitro lipophilicity 13,500-fold relative to the hydrophilic ethyl analog Mn(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnTE-2-PyP). In normal mice, we found brain MnTnHex-2-PyP accumulation to be ∼9-fold greater than MnTE-2-PyP 24 h after a single intraperitoneal dose. We then evaluated MnTnHex-2-PyP efficacy in outcome-oriented models of focal cerebral ischemia and subarachnoid hemorrhage. For focal ischemia, rats underwent 90-min middle cerebral artery occlusion. Parenteral MnTnHex-2-PyP treatment began 5 min or 6 h after reperfusion onset and continued for 7 days. Neurologic function was improved with both early (P = 0.002) and delayed (P = 0.002) treatment onset. Total infarct size was decreased with both early (P = 0.03) and delayed (P = 0.01) treatment. MnTnHex-2-PyP attenuated nuclear factor κB nuclear DNA binding activity and suppressed tumor necrosis factor-α and interleukin-6 expression. For subarachnoid hemorrhage, mice underwent perforation of the anterior cerebral artery and were treated with intraperitoneal MnTnHex-2-PyP or vehicle for 3 days. Neurologic function was improved (P = 0.02), and vasoconstriction of the anterior cerebral (P = 0.0005), middle cerebral (P = 0.003), and internal carotid (P = 0.015) arteries was decreased by MnTnHex-2-PyP. Side-chain elongation preserved MnPorphyrin redox activity, but improved CNS bioavailability sufficient to cause improved outcome from acute CNS injury, despite delay in parenteral treatment onset of up to 6 h. This advance now allows consideration of MnPorphyrins for treatment of cerebrovascular disease.
Open Access
Novel Manganese-Porphyrin Superoxide Dismutase-Mimetic Widens the Therapeutic Margin in a Preclinical Head and Neck Cancer Model.
(International journal of radiation oncology, biology, physics, 2015-11) Ashcraft, Kathleen A; Boss, Mary-Keara; Tovmasyan, Artak; Roy Choudhury, Kingshuk; Fontanella, Andrew N; Young, Kenneth H; Palmer, Gregory M; Birer, Samuel R; Landon, Chelsea D; Park, Won; Das, Shiva K; Weitner, Tin; Sheng, Huaxin; Warner, David S; Brizel, David M; Spasojevic, Ivan; Batinic-Haberle, Ines; Dewhirst, Mark W
Purpose
To test the effects of a novel Mn porphyrin oxidative stress modifier, Mn(III) meso-tetrakis(N-n-butoxyethylpyridinium-2-yl)porphyrin (MnBuOE), for its radioprotective and radiosensitizing properties in normal tissue versus tumor, respectively.
Methods and materials
Murine oral mucosa and salivary glands were treated with a range of radiation doses with or without MnBuOE to establish the dose-effect curves for mucositis and xerostomia. Radiation injury was quantified by intravital near-infrared imaging of cathepsin activity, assessment of salivation, and histologic analysis. To evaluate effects of MnBuOE on the tumor radiation response, we administered the drug as an adjuvant to fractionated radiation of FaDu xenografts. Again, a range of radiation therapy (RT) doses was administered to establish the radiation dose-effect curve. The 50% tumor control dose values with or without MnBuOE and dose-modifying factor were determined.
Results
MnBuOE protected normal tissue by reducing RT-mediated mucositis, xerostomia, and fibrosis. The dose-modifying factor for protection against xerostomia was 0.77. In contrast, MnBuOE increased tumor local control rates compared with controls. The dose-modifying factor, based on the ratio of 50% tumor control dose values, was 1.3. Immunohistochemistry showed that MnBuOE-treated tumors exhibited a significant influx of M1 tumor-associated macrophages, which provides mechanistic insight into its radiosensitizing effects in tumors.
Conclusions
MnBuOE widens the therapeutic margin by decreasing the dose of radiation required to control tumor, while increasing normal tissue resistance to RT-mediated injury. This is the first study to quantitatively demonstrate the magnitude of a single drug's ability to radioprotect normal tissue while radiosensitizing tumor.
Open Access
Pharmacokinetics, Brain Hippocampus and Cortex, and Mitochondrial Accumulation of a New Generation of Lipophilic Redox-Active Therapeutic, Mn(III) Meso Tetrakis(N-n-butoxyethylpyridinium-2-yl)porphyrin, MnTnBuOE-2-PyP5+, in Comparison with its Ethyl and N-hexyl Analogs, MnTE-2-PyP5+ and MnTnHex-2-PyP5+
(Free Radical Biology and Medicine, 2013-11) Spasojevic, Ivan; Weitner, Tin; Tovmasyan, Artak; Sheng, Huaxin; Miriyala, Sumitra; Leu, David; Rajic, Zrinka; Warner, David S; Clair, Daret St; Huang, Ting-Ting; Batinic-Haberle, Ines
Open Access
Preclinical Testing of a Novel Niclosamide Stearate Prodrug Therapeutic (NSPT) Shows Efficacy Against Osteosarcoma.
(Molecular cancer therapeutics, 2020-07) Reddy, Gireesh B; Kerr, David L; Spasojevic, Ivan; Tovmasyan, Artak; Hsu, David S; Brigman, Brian E; Somarelli, Jason A; Needham, David; Eward, William C
Therapeutic advances for osteosarcoma have stagnated over the past several decades, leading to an unmet clinical need for patients. The purpose of this study was to develop a novel therapy for osteosarcoma by reformulating and validating niclosamide, an established anthelminthic agent, as a niclosamide stearate prodrug therapeutic (NSPT). We sought to improve the low and inefficient clinical bioavailability of oral dosing, especially for the relatively hydrophobic classes of anticancer drugs. Nanoparticles were fabricated by rapid solvent shifting and verified using dynamic light scattering and UV-vis spectrophotometry. NSPT efficacy was then studied in vitro for cell viability, cell proliferation, and intracellular signaling by Western blot analysis; ex vivo pulmonary metastatic assay model; and in vivo pharmacokinetic and lung mouse metastatic model of osteosarcoma. NSPT formulation stabilizes niclosamide stearate against hydrolysis and delays enzymolysis; increases circulation in vivo with t _1/2 approximately 5 hours; reduces cell viability and cell proliferation in human and canine osteosarcoma cells in vitro at 0.2-2 μmol/L IC₅₀; inhibits recognized growth pathways and induces apoptosis at 20 μmol/L; eliminates metastatic lesions in the ex vivo lung metastatic model; and when injected intravenously at 50 mg/kg weekly, it prevents metastatic spread in the lungs in a mouse model of osteosarcoma over 30 days. In conclusion, niclosamide was optimized for preclinical drug delivery as a unique prodrug nanoparticle injected intravenously at 50 mg/kg (1.9 mmol/L). This increased bioavailability of niclosamide in the blood stream prevented metastatic disease in the mouse. This chemotherapeutic strategy is now ready for canine trials, and if successful, will be targeted for human trials in patients with osteosarcoma.

Browsing by Author "Spasojevic, Ivan"

Results Per Page

Sort Options

Purpose

Methods and materials

Results

Conclusions