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Tilorone dihydrochloride (tilorone) is an orally active interferon inducer with anticancer effects. The present study aimed to evaluate the anticancer effects of tilorone in breast cancer. MTT assay was done to measure the proliferation of MCF-7 and MDA-MB-231 breast cancer cells after treatment with tilorone. Mammary carcinogenesis was induced by subcutaneous injection (35 mg/kg, 0.5 mL) of dimethylbenz[a]anthracene (DMBA) in mammary pads of Sprague Dawley (SD) rats. Tumors were allowed to grow for 16 weeks till their sizes reached to 550-700 mm 3 , and then treated with 10 and 20 mg/kg of tilorone and standard drug doxorubicin (4 mg/kg) twice a week for 3 weeks. Normal and disease-control animals received normal saline. Tumor volumes and body weights were measured. Tumors were isolated to measure the levels of interferon-β (IFN-β), vascular endothelial growth factor-A (VEGF-A), P53 and inflammatory markers by enzyme-linked immunosorbent assay (ELISA). Serum biochemistry, lipid peroxidation (LPO) and antioxidant enzymes were measured by standard methods. Histopathology and immunohistochemistry (IHC) of P53 was done in tumor sections. Tilorone reduced the proliferation of MCF-7 and MDA-MB-231 cells with IC 50 concentrations at 34.08 µM and 14.27 µM, respectively. Tilorone treatment showed reduced tumor volume, and increased survival with no significant changes in the body weights. Tilorone treatment also decreased levels of inflammatory markers and VEGF-A and increased IFN-β and P53 levels. Further, treatment with tilorone also decreased LPO and increased antioxidants levels. Histopathology of tumor sections showed normalizing morphology of treated animals. IHC of tumor sections showed increased levels of P53. In conclusion, tilorone has potential anticancer effects against breast cancer., (© 2024 Wiley Periodicals LLC.)

Skeletal muscle plays a major role in whole-body glucose metabolism. Insulin resistance in skeletal muscle is characterized by decreased insulin-stimulated glucose uptake resulting from impaired intracellular trafficking and decreased glucose transporter 4 (GLUT4) expression. In this study, we illustrated that tilorone, a low-molecular-weight antiviral agent, improves glucose uptake in vitro and in vivo. Tilorone increased bone morphogenetic protein (BMP) signaling in C2C12 myoblasts, the transcription of multiple BMPs (BMP2, BMP4, BMP7, and BMP14), Smad4 expression, and the phosphorylation of BMP-mediated Smad1/5/8. The activation of Akt2/AS160 (TBC1D4) signaling, the critical regulator of GLUT4 translocation, was also increased, as well as the levels of GLUT4 and GLUT1, leading to enhanced uptake of the radioactively labeled glucose analog 18 F-fluoro-2-deoxyglucose ( 18 FDG). However, this excess glucose content did not result in increased ATP formation by mitochondrial respiration; both basal and ATP-linked respiration were diminished, thereby contributing to the induction of AMPK. In differentiated myotubes, AS160 phosphorylation and 18 FDG uptake also increased. Moreover, tilorone administration further increased insulin-stimulated phosphorylation of Akt2 and glucose uptake of myotubes indicating an insulin-sensitizing effect. Importantly, during in vivo experiments, the systemic administration of tilorone resulted in increased 18 FDG uptake of skeletal muscle, liver, and adipose tissue in C57BL/6 mice. Our results provide new perspectives for the treatment of type 2 diabetes, which has a limited number of treatments that regulate protein expression or translocation., (© 2023 The Authors. Journal of Cellular Physiology published by Wiley Periodicals LLC.)

In the wake of the COVID-19 pandemic, drug repurposing has been highlighted for rapid introduction of therapeutics. Proposed drugs with activity against SARS-CoV-2 include compounds with positive charges at physiologic pH, making them potential targets for the organic cation secretory transporters of kidney and liver, i.e., the basolateral organic cation transporters, OCT1 and OCT2; and the apical multidrug and toxin extruders, MATE1 and MATE2-K. We selected several compounds proposed to have in vitro activity against SARS-CoV-2 (chloroquine, hydroxychloroquine, quinacrine, tilorone, pyronaridine, cetylpyridinium, and miramistin) to test their interaction with OCT and MATE transporters. We used Bayesian machine learning models to generate predictions for each molecule with each transporter and also experimentally determined IC 50 values for each compound against labeled substrate transport into CHO cells that stably expressed OCT2, MATE1, or MATE2-K using three structurally distinct substrates (atenolol, metformin and 1-methyl-4-phenylpyridinium) to assess the impact of substrate structure on inhibitory efficacy. For the OCTs substrate identity influenced IC 50 values, although the effect was larger and more systematic for OCT2. In contrast, inhibition of MATE1-mediated transport was largely insensitive to substrate identity. Unlike MATE1, inhibition of MATE2-K was influenced, albeit modestly, by substrate identity. Maximum unbound plasma concentration/IC 50 ratios were used to identify potential clinical DDI recommendations; all the compounds interacted with the OCT/MATE secretory pathway, most with sufficient avidity to represent potential DDI issues for secretion of cationic drugs. This should be considered when proposing cationic agents as repurposed antivirals. SIGNIFICANCE STATEMENT: Drugs proposed as potential COVID-19 therapeutics based on in vitro activity data against SARS-CoV-2 include compounds with positive charges at physiological pH, making them potential interactors with the OCT/MATE renal secretory pathway. We tested seven such molecules as inhibitors of OCT1/2 and MATE1/2-K. All the compounds blocked transport activity regardless of substrate used to monitor activity. Suggesting that plasma concentrations achieved by normal clinical application of the test agents could be expected to influence the pharmacokinetics of selected cationic drugs., (Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics.)

We studied the effect of tilorone on the dynamics of IFNα, IFNγ, and IL-1β levels in the lung tissue and blood serum in relation to viral load in the lungs of BALB/c mice with pneumonia caused by influenza virus A/Aichi/2/68 (H3N2). Tilorone was administered per os in doses of 40, 150, and 540 μg per mouse 6, 30, and 78 h postinfection, which simulated the drug regimen used in the clinic for the treatment of influenza and acute respiratory viral infections in Russia and post-Soviet countries. Tilorone reduced viral load with the maximum amplitude (2-3 lg) after 1-2 administrations. The results of studying the dynamics of the cytokine levels in the infected animals in general support the previous hypothesis that, in repeated dosing, tilorone enhances the IFN response (compensates for its deficiency) at the early stages of acute respiratory viral infections and suppresses (damps) excessive production of IFN and proinflammatory cytokines at the later stages., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Acetylcholinesterase (AChE) is an important drug target in neurological disorders like Alzheimer's disease, Lewy body dementia, and Parkinson's disease dementia as well as for other conditions like myasthenia gravis and anticholinergic poisoning. In this study, we have used a combination of high-throughput screening, machine learning, and docking to identify new inhibitors of this enzyme. Bayesian machine learning models were generated with literature data from ChEMBL for eel and human AChE inhibitors as well as butyrylcholinesterase inhibitors (BuChE) and compared with other machine learning methods. High-throughput screens for the eel AChE inhibitor model identified several molecules including tilorone, an antiviral drug that is well-established outside of the United States, as a newly identified nanomolar AChE inhibitor. We have described how tilorone inhibits both eel and human AChE with IC 50 's of 14.4 nM and 64.4 nM, respectively, but does not inhibit the closely related BuChE IC 50 > 50 μM. We have docked tilorone into the human AChE crystal structure and shown that this selectivity is likely due to the reliance on a specific interaction with a hydrophobic residue in the peripheral anionic site of AChE that is absent in BuChE. We also conducted a pharmacological safety profile (SafetyScreen44) and kinase selectivity screen (SelectScreen) that showed tilorone (1 μM) only inhibited AChE out of 44 toxicology target proteins evaluated and did not appreciably inhibit any of the 485 kinases tested. This study suggests there may be a potential role for repurposing tilorone or its derivatives in conditions that benefit from AChE inhibition.

Tilorone is a 50-year-old synthetic small-molecule compound with antiviral activity that is proposed to induce interferon after oral administration. This drug is used as a broad-spectrum antiviral in several countries of the Russian Federation. We have recently described activity in vitro and in vivo against the Ebola virus. After a broad screening of additional viruses, we now describe in vitro activity against Chikungunya virus (CHIK) and Middle Eastern respiratory syndrome coronavirus (MERS-CoV)., (Copyright © 2020 American Society for Microbiology.)

For the last 50 years we have known of a broad-spectrum agent tilorone dihydrochloride (Tilorone). This is a small-molecule orally bioavailable drug that was originally discovered in the USA and is currently used clinically as an antiviral in Russia and the Ukraine. Over the years there have been numerous clinical and non-clinical reports of its broad spectrum of antiviral activity. More recently we have identified additional promising antiviral activities against Middle East Respiratory Syndrome, Chikungunya, Ebola and Marburg which highlights that this old drug may have other uses against new viruses. This may in turn inform the types of drugs that we need for virus outbreaks such as for the new coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Tilorone has been long neglected by the west in many respects but it deserves further reassessment in light of current and future needs for broad-spectrum antivirals.

Quinacrine hydrochloride is a small-molecule, orally bioavailable drug that has been used clinically as an antimalarial and for many other applications. A machine learning model trained on Ebola virus (EBOV) screening data identified quinacrine as a potent (nanomolar) in vitro inhibitor. In the current study, quinacrine 25 mg/kg was shown to protect 70% of mice (statistically significant) from a lethal challenge with mouse-adapted EBOV with once-daily intraperitoneal dosing for 8 days., (Copyright © 2019 American Society for Microbiology.)

Tilorone hydrochloride, a low-molecular-weight synthetic compound, induces interferon production and has been reported to have both antiviral and antitumor activities. Here, we have demonstrated the ability of tilorone to activate NK cells and specific subpopulations of cytotoxic CD4+ and CD8+ T lymphocytes that recognize immune-evasive tumor cells and kill them via the FasL-Fas interaction. We have also performed a comparative analysis of characteristics between lymphocytes activated in the fraction of human peripheral blood mononuclear cells (PBMCs) upon treatment with different stimulants of the immune response: tilorone, innate immunity protein Tag7, and cytokine IL-2, a regulator of adaptive immunity. The results show that all the three stimulants, regardless of their nature, activate lymphocytes that are identical with respect to the spectrum of target cells, phenotype, and mechanism of cytotoxic action However, these stimulants induce different mechanisms of lymphocyte activation at early stages of the immune response. © 2018 IUBMB Life, 71(3):376-384, 2019., (© 2018 International Union of Biochemistry and Molecular Biology.)

Context: Tilorone dihydrochloride is a therapeutic agent with a different mechanism in cancer. The species of Lactobacillus have an important role in cytotoxic effect., Aims: Because of unknown effects of tilorone and culture supernatants from Lactobacillus reuteri on hepatoma, the aim of this study is to evaluate apoptotic, cytotoxic, and therapeutic effects of tilorone on mouse hepatoma cell line with and without culture supernatants from L. reuteri., Materials and Methods: To do so, after cell line culture, cells were divided into different groups such as negative control, treatment with four doses of tilorone, positive control of supernatant (single dose), and combination therapy groups of different doses of tilorone with supernatant (constant doses), for 48 h. All groups were studied with pathologic tests, biochemical study, tetrazolium dye (3-(4, 5- dimethylthiazol -2-yl)-2, 5-diphenyltetrazolium bromide [MTT]) assay, and absolute real-time-polymerase chain reaction (RT-PCR) were done to assess Bax and Bcl-2 genes expression, as molecular studies., Results: MTT assay results revealed that the tilorone tissue culture IC 50 (TCIC 50 ) on the Hepa1-6 cell line was 50 μg/ml. RT-PCR analysis showed that tilorone dihydrochloride induced upregulation and downregulation in expression of Bax and Bcl-2, respectively. Simultaneous, antioxidant effect has also seen in a way that prevented necrosis, in biochemical analysis. These results were dose dependent and statistically significant compared to the control group., Conclusions: Based on these results, it appeared that this agent could be a good candidate for further evaluation as effective chemotherapy acting through the induction of apoptosis in hepatoma. The cell death caused through bacterial supernatant was rather necrosis than apoptosis., Competing Interests: None

Tilorone dihydrochloride (tilorone) is a small-molecule, orally bioavailable drug that is used clinically as an antiviral outside the United States. A machine-learning model trained on anti-Ebola virus (EBOV) screening data previously identified tilorone as a potent in vitro EBOV inhibitor, making it a candidate for the treatment of Ebola virus disease (EVD). In the present study, a series of in vitro ADMET (absorption, distribution, metabolism, excretion, toxicity) assays demonstrated the drug has excellent solubility, high Caco-2 permeability, was not a P-glycoprotein substrate, and had no inhibitory activity against five human CYP450 enzymes (3A4, 2D6, 2C19, 2C9, and 1A2). Tilorone was shown to have 52% human plasma protein binding with excellent plasma stability and a mouse liver microsome half-life of 48 min. Dose range-finding studies in mice demonstrated a maximum tolerated single dose of 100 mg/kg of body weight. A pharmacokinetics study in mice at 2- and 10-mg/kg dose levels showed that the drug is rapidly absorbed, has dose-dependent increases in maximum concentration of unbound drug in plasma and areas under the concentration-time curve, and has a half-life of approximately 18 h in both males and females, although the exposure was ∼2.5-fold higher in male mice. Tilorone doses of 25 and 50 mg/kg proved efficacious in protecting 90% of mice from a lethal challenge with mouse-adapted with once-daily intraperitoneal (i.p.) dosing for 8 days. A subsequent study showed that 30 mg/kg/day of tilorone given i.p. starting 2 or 24 h postchallenge and continuing through day 7 postinfection was fully protective, indicating promising activity for the treatment of EVD., (Copyright © 2018 American Society for Microbiology.)

Mitochondrial nicotinic acetylcholine receptors (nAChRs) control permeability transition pore formation and cytochrome c release in the presence of apoptogenic factors. This study demonstrates that pharmacological agents amixin and agmatine affect mitochondrial nAChR functioning: they slightly suppress cytochrome c release from mouse brain and liver mitochondria stimulated with apoptogenic dose of Са2+ and prevent the effect of α7 nAChR agonist PNU282987. We conclude that mitochondria may be one of therapeutic targets of amixin and agmatine.

Tilorone dihydrochloride (1) has great potential for inducing interferon against pathogenic infection. In this paper, we describe a convenient preparation method for 2,7-dihydroxyfluoren-9-one (2), which is a usual pharmaceutical intermediate for preparing tilorone dihydrochloride (1). In the novel method, methyl esterification of 4,4'-dihydroxy-[1,1'-biphenyl]-2-carboxylic acid (4) was carried out under milder conditions with higher yield and played an important role in the preparation of compound 2. The structures of the relative intermediates and target compound were characterized by melting point, IR, MS, and ¹H-NMR. Furthermore, the synthesized tilorone dihydrochloride exhibited an obvious effect on induction of interferon-α (IFN-α) in mice within 12 h, and the peak level was observed until 24 h. This fruitful work has resulted in tilorone dihydrochloride becoming available in large-scale and wide application in clinics, which has a good pharmaceutical development prospects.

Cyclin-dependent kinase 5 (CDK5) is a potential target for prostate cancer treatment, the enzyme being essential for prostate tumor growth and formation of metastases. In the present study, we identified agents that target prostate cancer cells based on CDK5 expression. CDK5 activity was suppressed by transfection of PC3 prostate cancer cells with a dominant-negative construct (PC3 CDK5dn). PC3 CDK5dn and PC3 control cells were screened for compounds that selectively target cells based on CDK5 expression, utilizing the Johns Hopkins Drug Library. MTS proliferation, clonogenic and 3D growth assays were performed to validate the selected hits. Screening of 3,360 compounds identified rutilantin, ethacridine lactate and cetalkonium chloride as compounds that selectively target PC3 control cells and a tilorone analog as a selective inhibitor of PC3 CDK5dn cells. A PubMed literature study indicated that tilorone may have clinical use in patients. Validation experiments confirmed that tilorone treatment resulted in decreased PC3 cell growth and invasion; PC3 cells with inactive CDK5 were inhibited more effectively. Future studies are needed to unravel the mechanism of action of tilorone in CDK5 deficient prostate cancer cells and to test combination therapies with tilorone and a CDK5 inhibitor for its potential use in clinical practice.

A series of 2,7-diamidofluorenones were designed, synthesized, and screened by SRB assay. Some synthesized compounds exhibited antitumor activities in submicromolar range. Ten compounds (3a, 3b, 3c, 3g, 3j, 3l, 4a, 4h, 4i, and 4j) were also selected by NCI screening system and 3c (GI50=1.66 μM) appeared to be the most active agent of this series. Furthermore, 3c attenuated topoisomerase I-mediated DNA relaxation at low micromolar concentrations. These results indicated that fluorenones have potential to be further developed into anticancer drugs., (Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.)

Primase DnaG is an essential bacterial enzyme that synthesizes short ribonucleotide primers required for chromosomal DNA replication. Inhibitors of DnaG can serve as leads for development of new antibacterials and biochemical probes. We recently developed a nonradioactive in vitro primase-pyrophosphatase assay to identify and analyze DnaG inhibitors. Application of this assay to DnaG from Bacillus anthracis (Ba DnaG), a dangerous pathogen, yielded several inhibitors, which include agents with DNA intercalating properties (doxorubicin and tilorone) as well as those that do not intercalate into DNA (suramin). A polyanionic agent and inhibitor of eukaryotic primases, suramin, identified by this assay as a low-micromolar Ba DnaG inhibitor, was recently shown to be also a low-micromolar inhibitor of Mycobacterium tuberculosis DnaG (Mtb DnaG). In contrast, another low-micromolar Ba DnaG inhibitor, tilorone, is much more potent against Ba DnaG than against Mtb DnaG, despite homology between these enzymes, suggesting that DnaG can be targeted selectively.

Tilorone is an interferon inducer with anticancer activity. Twenty-two novel tilorone analogs were synthesized by improvements of fluorenone skeleton, side chains and amino groups to screen new anticancer agents. In vitro evaluation showed that ten new compounds had better anticancer activities than tilorone. Among them, 2c (IC50 < 7 μM against cancer cell lines and IC50 > 35 μM against non-cancer cell lines) and 5d (IC50 < 10 μM against cancer cell lines and IC50 > 53 μM against non-cancer cell lines) exhibited the best anticancer activities and selectivities. Pharmacophore modeling of highly active compounds was carried out by Molecular Operating Environment (MOE) to generate a visualized model for compound design in future study., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with a poor prognosis and very few therapeutic options. On the molecular level, patients with IPF have increased amounts of the bone morphogenetic protein (BMP) inhibitor gremlin in their lungs, which results in decreased BMP signaling, and an increase in transforming growth factor-β signaling. Based on these findings, we hypothesized that restoration of the impaired BMP signaling would offer a novel strategy for the prevention of fibrosis progression or for the treatment of pulmonary fibrosis. We used reporter cell lines and high-throughput screening of a chemical compound library as an approach to finding molecules that increase BMP signaling in lung epithelial cells, without increasing transforming growth factor-β signaling. The most promising candidate drug was analyzed further by studying its effects on BMP target gene expression, Smad protein phosphorylation, and a mouse model of silica-induced pulmonary fibrosis. The most promising drug candidate, tilorone, induced BMP signaling in the reporter cells and increased the expression of BMP-7 and a BMP target gene, Id3, in lung epithelial A549 cells. In a mouse model of pulmonary fibrosis, tilorone decreased lung hydroxyproline content and the expression of collagen genes Col1A1 and Col3A1. Mice treated with tilorone showed markedly decreased histological changes, compared with untreated mice. These findings indicate that tilorone has biologically significant antifibrotic properties.

The early changes of electrophoretic mobility (EPM) of murine T lymphocytes induced by structural analogues of amixine-dihydrochloryde 4,4'-bis-[2(diethylamino)ethoxy]diphenyl (compound 1) and dihydrochloryde 2-methoxycarbonil-4,4'-bis-[2(diethylamino)ethoxy]diphenyl (compound 2) were studied by electrophoresis technique. During the interval 0-2 hours all compounds increased the absolute values of EPM in comparison with control. These changes were of the same kind--distinctions were quantitative. Amixine and compound 1 during the interval 2-4 hours additionally increased the EPM. The compound 2, on the contrary, decreased the EPM. It was shown that the opposite effects of the aforementioned compounds were caused by the fact that amixine and compound 1 induce, and compound 2 does not induce IFN production in T lymphocytes in vitro. The results of our experiments are important for understanding of the mechanisms of immunomodulating effect of amixine and its structural analogues.

Two 6H-indoloquinoxaline derivatives were studied in different doses and schemes of application for their INFgamma-inducing potential and ability to effect functional activity of phagocytic cells. Tested compounds were shown to possess comparable or higher activity than reference drug Amixin in analogous doses. One indoloquinoxaline significantly elevated metabolic activity of macrophages and increased their potential for phagocytosis. Application of multiple treatments and higher doses allowed us to reveal differences between studied derivatives that were not obvious in previous in vivo experiment. Capacity of 6H-indoloquinoxalines to induce vast IFN amounts on in vivo level was demonstrated for the first time.

The well-known interferon-inducer tilorone was found to possess potent affinity for the agonist site of the α7 neuronal nicotinic receptor (K(i)=56 nM). SAR investigations determined that both basic sidechains are essential for potent activity, however active monosubstituted derivatives can also be prepared if the flexible sidechains are replaced with conformationally rigidified cyclic amines. Analogs in which the fluorenone core is replaced with either dibenzothiophene-5,5-dioxide or xanthenone also retain potent activity., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

In experiments on two model systems L929/VSV and RF/ HSV-1 in vitro we have studied the antiviral activity of new structural analogues of tilorone--4,4'-bis[2-(diethyl-amino)ethoxy]diphenyl dihydrochloride and 2-methoxy-carbonyl-4-4'-bis[2-(diethylamino)ethoxy]diphenyl dihydrochloride. In experiments the tested substances were administered in preventive and therapeutic schemes. The preventive scheme confirms the existence of a correlation between IFN production under the influence of tested substances and their antiviral activity. It is shown that diphenyls are able to inhibit the development of viral cytopathic effect induced by DNA- and RNA-containing viruses. Diphenyl derivatives are less toxic than tilorone and could be considered as promising substances for further research to develop new antiviral drugs.

The influence of tilorone dihydrochloride and its analogues--diphenyl derivatives on the changes of transmembrane potential of mitochondrial membranes of the isolated rat hepatocytes has been estimated. Authors have shown a significant increase in mitochondrial potential thirty minutes after the introduction of the test compounds to the cells using the fluorescent probe JC-1. These results indicate the rapid activation with tilorone and its analog--dihydrochloryde 4,4'-bis-[2-(diethylamino)ethoxy]diphenyl--of the RLR signaling pathway. The final stage of this pathway is the cell production of IFN type I. The authors concluded that there is an increasing of the organelles resistance to the extra/intracellular damaging agents under the influence of the test compounds.

The amixine-induced early changes in the electrophoretic mobility (EPM) of murine splenic T lymphocytes were studied in vitro by the microelectrophoresis technique. It has been found that T lymphocytes treated with amixine have a greater EPM within the first hours of amixine addition than control cells. This change in EPM depends on the concentration of amixine in the medium and the duration ofamixine exposure. It was concluded that the amixine-treated cells have a greater net negative surface charge density than control cells. This effect may play an important role in the cell-cell interaction during the immune response.

A major challenge for neurological therapeutics is the development of small molecule drugs that can activate a panoply of downstream pathways without toxicity. Over the past decade our group has shown that a family of enzymes that regulate posttranscriptional and transcriptional adaptive responses to hypoxia are viable targets for neuronal protection and repair. The family is a group of iron, oxygen, and 2-oxoglutarate-dependent dioxygenases, known as the HIF prolyl 4-hydroxylases (HIF PHDs). We have previously shown that pluripotent protection offered by iron chelators is mediated, in part, via the ability of these agents to inhibit the HIF PHDs. Our group and others have implicated the transcriptional activator HIF-1 in some of the salutary effects of iron chelation-induced PHD inhibition. While some iron chelators are currently employed in humans for conditions such as hemochromatosis, the diverse utilization of iron in physiological processes in the brain makes the development of HIF activators that do not bind iron a high priority. Here we report the development of a high throughput screen to develop novel HIF activators and/or PHD inhibitors for therapeutic use in the central nervous system (CNS). We show that tilorone, a low-molecular weight, antiviral, immunomodulatory agent is the most effective activator of the HIF pathway in a neuronal line. We also show that tilorone enhances HIF protein levels and increases the expression of downstream target genes independent of iron chelation and HIF PHD inhibition in vitro. We further demonstrate that tilorone can activate an HIF-regulated reporter gene in the CNS. These studies confirm that tilorone can penetrate the blood-brain barrier to activate HIF in the CNS. As expected from these findings, we show that tilorone provides effective prophylaxis against permanent ischemic stroke and traumatic spinal cord injury in male rodents. Altogether these findings identify tilorone as a novel and potent modulator of HIF-mediated gene expression in neurons with neuroprotective properties.

Prion diseases are a group of fatal neurodegenerative diseases affecting humans and animals. The only identified component of the infectious prion is PrP(Sc), an aberrantly folded isoform of PrP(C). Glycosaminoglycans, which constitute the main receptor for prions on cells, play a complex role in the pathogenesis of prion diseases. For example, while agents inducing aberrant lysosomal accumulation of GAGs such as Tilorone and Quinacrine significantly reduced PrP(Sc) content in scrapie-infected cells, administration of Quinacrine to prion-infected subjects did not improve their clinical status. In this study, we investigated the association of PrP(Sc )with cells cultured with Tilorone. We found that while the initial incorporation of PrP(Sc) was similar in the treated and untreated cells, clearance of PrP(Sc) from the Tilorone-treated cells was significantly impaired. Interestingly, prolonged administration of Tilorone to mice prior to prion infection resulted in a significant delay in disease onset, concomitantly with in vivo accumulation of lysosomal GAGs. We hypothesize that GAGs may complex with newly incorporated PrP(Sc) in lysosomes and further stabilize the prion protein conformation. Over-stabilized PrP(Sc) molecules have been shown to comprise reduced converting activity.

Background and Purpose: The alpha7 nicotinic acetylcholine receptor (nAChR) has attracted considerable interest as a target for cognitive enhancement in schizophrenia and Alzheimer's Disease. However, most recently described alpha7 agonists are derived from the quinuclidine structural class. Alternatively, the present study identifies tilorone as a novel alpha7-selective agonist and characterizes analogues developed from this lead., Experimental Approach: Activity and selectivity were determined from rat brain alpha7 and alpha4beta2 nAChR binding, recombinant nAChR activation, and native alpha7 nAChR mediated stimulation of ERK1/2 phosphorylation in PC12 cells., Key Results: Tilorone bound alpha7 nAChR (IC(50) 110 nM) with high selectivity relative to alpha4beta2 (IC(50) 70 000 nM), activated human alpha7 nAChR with an EC(50) value of 2.5 microM and maximal response of 67% relative to acetylcholine, and showed little agonist effect at human alpha3beta4 or alpha4beta2 nAChRs. However, the rat alpha7 nAChR maximal response was only 34%. Lead optimization led to 2-(5-methyl-hexahydro-pyrrolo[3,4-c]pyrrol-2-yl)-xanthen-9-one (A-844606) with improved binding (alpha7 IC(50) 11 nM, alpha4beta2 IC(50)>30 000 nM) and activity at both human and rat alpha7 nAChR (EC(50)s 1.4 and 2.2 microM and apparent efficacies 61 and 63%, respectively). These compounds also activated native alpha7 nAChR, stimulating ERK1/2 phosphorylation in PC12 cells., Conclusions and Implications: Tilorone, known as an interferon inducer, is a selective alpha7 nAChR agonist, suggesting utility of the fluorenone pharmacophore for the development of alpha7 nAChR selective agonists. Whether alpha7 stimulation mediates interferon induction, or whether interferon induction may influence the potential anti-inflammatory properties of alpha7 nAChR agonists remains to be elucidated.

Properties of interferon productivity of several inducers of interferon, analogs of amixin, have been studied in vivo. It has been shown, that under the influence of injected agents the content of endogenic interferon in the blood serum of laboratory animals increased and their non-fractionated cells of the peripheral blood (splenocites and macrophages) synthesized alpha- and gamma-interferon.

The authors studied the antiviral effect of an interferonogenic yeast RNA-tilorone molecular complex (MC) compared to the Virolex, videly used antiherpetic drug, and standard interferon (IFN) alpha/beta inducer poly(I)poly(C) in Vero cells culture infected with herpes simplex type I virus (HSV-1). The tilorone contained by MC has been shown to be twice less toxic and twice more active against HSV than its free molecules. The value of chemotherapeutic index (CI) of Virolex in experiments with Vero cells reaches 2500, CI poly(I)poly(C) and MC being 324; the last value meets the requirements for promising drugs.

Macrophages play a key role in the regulation of cytochrome P450 activity induced by immunostimulants in mammals. We investigated the effects of immunostimulants (LPS, dextran sulfate and tilorone) on biotransformation and macrophage activities in carp. The major effect of LPS was its capacity to inhibit 3-MC-induced cytochrome P450 activities in the liver and head kidney. Basal phase I activities were reduced by tilorone and dextran sulfate in immune organs. Tilorone and dextran sulfate differently modulated total cytochrome P450 contents and P4501A activities suggesting differential sensitivity for P450 classes. In immune organs, tilorone and dextran sulfate inhibited basal EROD activity. Tilorone inhibited 3-MC-induced EROD activity whereas dextran sulfate enhanced this activity. LPS and dextran sulfate increased ROS production by macrophages and all the immunostimulants induced macrophage activating factor (MAF) production. This study demonstrates for the first time in fish the capacity of CYP-regulated immunostimulants to activate macrophages and provides initial insight into the capacity of macrophages to regulate CYP activity induced by immunostimulants in fish.

Pharmacokinetics of amixin was studied after repeated administration (5 days) to animals. Perorally administered amixin is characterized by high bioavailability and is present in the circulation in high concentrations for a long time. The main pharmacokinetic parameters were estimated by the method of linear regression because of slow elimination of amixin from organs and tissues. Our results indicate that repeated treatment with amixin holds much promise for the prevention and therapy of chronic diseases (particularly hepatitides).

The action of amixin and cycloferon on the expression of genes in the systems of interferon (IF) and cell apoptosis (CA) was studied by semi-quantitative RT-PCR in human blood microsamples before and after the administration of the drugs. Individual changes were determined in the transcription activity of genes of IF (alpha, beta, gamma), enzymes 2',5' oligoadenylatesynthetase (OAS), RNSase L, dsRNA-dependent proteinkinase (dsPK) and of CA effectors (FasAg, bcl-2, gamma-actin) registered dynamically in 24 h and 48 h. The activity parameters of IF genes were compared with the results of biological titration of IF activity in blood samples in vivo and in vitro. A pronounced ability of cycloferon to stimulate selectively the activity of genes of human IF, type I (beta IF--by 100 times and alpha IF--by 10 times), without affecting essentially the activity of other genes in blood cells, was detected. Amixin was found to inhibit the titration of genes with high activity levels. (alpha-, beta-IF, RNAases L, bcl-2 and gamma-actin). The antiviral and IF-induced properties of the drug are explained to a great extent by the apoptotic effect (activation of genes Fas, gamma-IF, OAS and affected transcription of gene bcl-2). A positive correlation was observed between the processes of activation of IF-genes transcription and the production of the total circulating IF. Antagonistic relations between type I and II IFs in human blood cells were shown.

In vitro experiments the authors have studied a property of yeast RNA--tilorone hydrochloride complex covalently linked to spheron to induce the synthesis of interferons type I (alpha- and beta-interferons) in the culture of peripheral mononuclear human cells. Such a complex is shown to possess a marked interferonogenic activity. The data obtained appear to be a proof of the interferon induction to be realised by a mechanism needing at the first stage the contact between the inducer and the cell surface without its penetration into the cell.

The participation of NK cells in the activation of splenic macrophages or in resistance to systemic candidiasis is still a matter of debate. We had previously reported that there is a correlation between natural killer cell activation and resistance to systemic candidiasis. In those experiments we had used tilorone to boost NK cell activity in mice. Here we show a mechanism elicited by tilorone in splenic macrophages which could explain their effect on mouse survival during acute disseminated Candida albicans infection. The results demonstrate that tilorone treatment elicits, by a direct effect, the production of proinflammatory cytokines (interleukin-6 [IL-6], tumor necrosis factor alpha [TNF-alpha], and IL-12) by splenic macrophages. In addition, it increases the capacity of splenic macrophages to phagocytize C. albicans through activation of NK cells. We also demonstrate that the presence of NK cells is essential for maintaining a basal level of phagocytic activity, which characterizes splenic macrophages of naïve control mice. The results demonstrate that it is possible to identify two phenotypically and functionally peculiar cell populations among splenic macrophages: (i). cells of the "stimulator/secretor phenotype," which show high levels of major histocompatibility complex (MHC) class II surface expression, are poorly phagocytic, and synthesize the proinflammatory cytokines IL-6, TNF-alpha, and IL-12, and (ii). cells of the "phagocytic phenotype," which express low levels of MHC class II molecules, are highly phagocytic, and do not secrete proinflammatory cytokines.

The experimental studies conducted on 2-week suckling mice infected with Hantaan virus, Strain 76-118) treated with oral and subcutaneous amoxine showed its prophylactic, therapeutical-and-prophylactic, and therapeutical efficiencies. Oral amoxine exhibited the highest efficiency when used in a dose of 10 mg/kg-1 96 hours before infection and throughout the incubation period. The protective efficiency was 61%. Subcutaneously, the agent was effective when three schemes for injection in a dose of 1 mg/kg-1. Its maximum effect was observed when amoxine was given by the therapeutical-and-prophylactic scheme. The death protection rate was 65%. The agent is effective in suppressing the reproduction of Hantaan virus in the brain tissue.

The polyethiology of the respiratory diseases and the lack of effective vaccines to prevent them (except for influenza) underline the need for clinical trials and the practical application of immunomodulatory preparations that could become part of the group of anti-viral drugs with a wide range of activity. It has been demonstrated that amixin and poludan, the drugs made in our country, cause an increase of the serum IFN level, enhance the ability of leukocytes and lymphocytes to produce IFN-alpha and IFN-gamma, and lead to the activation of NK and peripheral blood phagocytes. In other words, they have an immunostimulating activity. The number of CD3(+), CD4(+), CD8(+), CD19(+) cells, the level of the Ig A, IgG, IgM and several other parameters of the immune system were not affected by these drugs. It has been demonstrated that amixin and poludan show good drug tolerance, without side effects on the hemogram in individuals who were taking these drugs just for disease prevention. The studied immunomodulators have a significant prophylactic activity in the cases of the polyethiologic group of acute respiratory viral infection during the seasonal peak of the disease, with a coefficient of efficiency of 3.6 (amixin) and 2.1 (poludan), and corresponding protection indices of 72.1% and 52.7%. Having the same chance of getting infected, individuals protected with these drugs often have the disease in a milder or asymptomatic form.

Antiviral and interferon inducing activity of the amixine and its some derivatives, as well as their influence on the proteolytic enzymes activity, monooxygenase activity of the microsomal fraction, level of the lipids peroxidation were studied. Lack of correlation between antiviral and interferon inducing activity in the investigated series of compounds was found. Vice versa, the good correlation between interferon inducing activity and the elastase-like activities inhibition ability of the compounds was observed. It allows to state the assumption, that only one ability of compounds to induce of an interferon doesn't suffice for obtaining of high titres of interferon, and while their rather high antiproteolitic activity is necessary. It's shown, that except for one compound the influence of amixine and its derivatives on the red-ox enzymes activity well correlates with their ability to the interferon-inducing. All presented above allows to attribute amixine and its derivatives to polymodal antiviral agents.

The influence of amyxin, the Russian oral analog of tilorone, on the human interferon and immune status has been evaluated. As revealed by this investigation the administration of amyxin has produced a rise in the content of serum interferon, an increase in the capacity of leukocytes and lymphocytes for synthesizing alpha- and gamma-interferon, the activation of NK and phagocytizing cells of peripheral blood. No essential influence of amyxin on the amount of B and T lymphocytes, their subpopulations and the levels of the main classes of immunoglobulins has been established.

The virus-inhibitory activity of a molecular complex (MC) of tilorone and yeast RNA was studied in vitro on three virus-cell systems: vesicular stomatitis virus (VSV) - murine fibroblast L929 cells, Venezuelan equine encephalittis virus (VEEV) - swine embryo kidney (SEK) cells and encephalomyocarditis virus (EMCV) - established piglet testicular (EPT) cells. In all these systems the MC exerted an antiviral effect similar to that of polynucleotide interferon (IFN) inducers such as poly(I)-poly(C), larifan and ridostin. The antiviral effect of the MC was similar when the compound was applied before or after virus adsorption to cells. The MC may be regarded as a perspective antiviral agent of common use.

Candida albicans is an increasingly important opportunistic fungal pathogen in immunocompromised patients. Natural killer (NK) cells constitute an important immune effector mechanism and are involved in the response to different pathological disorders. We wished to determine if this immune mechanism is involved in the specific response to C. albicans. Tilorone hydrochloride and related compounds have been described to display antiviral and antitumoral activity, as well as to enhance NK cell activity. In this study, we show the antimicrobial activity of different tilorone analogues and the enhanced resistance of tilorone-treated mice in experimental systemic candidiasis. We also present data suggesting that there is a correlation between NK cell activation and the resistance to experimental systemic candidiasis. Thus, it seems that the immunosurveillance of metastatic spread and the infection by C. albicans share some immune effector mechanisms, in particular activation of NK cells.

The present studies were undertaken to determine the effect of tilorone dihydrochloride (a synthetic interferon inducer) on the in vivo acetylation of 2-aminofluorene and DNA-2-aminofluorene adducts. For in vivo examination, pretreatment with tilorone dihydrochloride (50 mg/kg) 48 hr prior to the administration of 2-aminofluorene (50 mg/kg) resulted in a 35% and 29% increase in the fecal and urinary recovery of N-acetyl-2-aminofluorene and a 52% increase in the metabolic clearance of 2-aminofluorene to N-acetyl-2-aminofluorene. Following the exposure of rats to the 2-aminofluorene with or without pretreatment with tilorone dihydrochloride, DNA-2-aminofluorene adducts were determined in liver, bladder, lung and also in circulating leukocytes. The DNA-2-aminofluorene adducts in liver were increased by pretreatment with tilorone dihydrochloride. This is the first demonstration of tilorone-induced increasing in N-acetylation of carcinogens and DNA-carcinogen adducts in vivo.

Dicationic amphiphilic drugs such as the immunomodulator tilorone [2,7-bis-[2-(diethylamino)ethoxy]fluoren-9-one] are accumulated in lysosomes and disturb the degradation of sulphated glycosaminoglycans (GAGs) thus leading to generalized lysosomal GAG storage (mainly dermatan sulphate) in vivo and in cultured cells. In the present study, the time course of the tilorone-induced GAG storage was determined in cultured bovine corneal fibroblasts by a radiochemical approach and by morphological examination. In contrast to the rapid lysosomal accumulation of the drug as reported previously, it took approximately 42 h to reach 50% of the GAG storage obtained after 96 h. This is thought to be due to the fact that the temporal development of storage of undigested GAGs depends on the natural delivery of GAGs towards the lysosomal apparatus.

The spectrophotometric study of complex formation between phage MS2 single-stranded RNA and tilorone has been carried out. It was found that with components concentration forming "strong linkage" the absorption spectra of RNA-tilorone complex in UV-region experience hyperchromic and bathochromic shifts; besides, a new absorption maximum appears at 280 nm. The changes of spectral properties have been also detected in the long-waved region of the complex spectra. The conclusion has been made that in the course of single-stranded RNA with tilorone interactions a complex formation takes place due to interacalation between spontaneously appeared two-stranded regions in the structure of a single-stranded RNA and tilorone; which component stabilizes these structures and assures there by the previously described interferonogenic activity of a single-stranded RNA--tilorone complexes.

Anti-HIV activity of the molecular complex forming during interaction between yeast RNA and tilorone was studied in vitro on the models of acute and chronic infection. Addition of this agent to the cells infected with HIV-1/IIIB and HIV-1/BRU decreased the virus reproduction controlled by assessing the viability of cells, syncytium production, and accumulation of p24 antigen in culture medium. The authors hypothesize that the detected anti-HIV effect is due to its capacity to produce type I interferon and direct antiviral action.

The present study determines the amounts and patterns of glycosaminoglycans stored in cultured corneal fibroblasts after treatment with tilorone and three related compounds. The compounds have immunomodulatory properties and have been shown to impair the lysosomal degradation of glycosaminoglycans as a side effect. This side effect has been described as drug-induced mucopolysaccharidosis because the induced lysosomal storage of glycosaminoglycans leads to cellular lesions resembling those in patients with inherited mucopolysaccharidosis. In the present study, the dose-dependency of glycosaminoglycan storage was analyzed after treatment (96 hr) of bovine corneal fibroblasts. The investigated drug concentrations ranged from low concentrations inducing cytological lesions typical of drug-induced mucopolysaccharidosis to high concentrations at the borderline of cytotoxicity. The intracellular amounts of dermatan sulfate, heparan suflate, and chondroitin sulfate were quantified by densitometric scanning of Alcian Blue-stained bands after electrophoresis. All investigated compounds induced a predominant dermatan sulfate storage (3-4-fold accumulation) at low drug concentrations. With rising drug concentrations, a shift of the pattern of stored glycosaminoglycans was observed, characterized by the additional accumulation of heparan sulfate (up to 5-fold of control levels). In cultured human fibroblasts, tilorone also caused a marked dermatan sulfate storage, reaching maximum values at 5 microM and marked heparan sulfate storage at 20 microM. The present data provide evidence: (a) that selective dermatan sulfate accumulation is a characteristic feature of drug-induced glycosaminoglycan storage in cultured bovine and human fibroblasts, if these cells are treated with low concentrations (< or = 5 microM), that are likely to reflect the situation in vivo; and (b) that additional heparan sulfate storage is induced in vitro only by treatment with high concentrations that induce nonspecific cellular lesions.

We studied the effect of a tilorone analogue (RMI 10,874DA) and anti-asialo GM(1) serum on the survival of BALB/c and C57B1/6 mice after i.v. injections of different syngeneic murine tumour cells. Tumour lines used were different clones from chemically (GR9 wild type, GR9.B9, B7.1.B4, B7.1.B5, B7.2.38), and ultraviolet light (GRUV3)-induced sarcomas; B16 melanoma and LSTRA and YC8 lymphomas. Pretreatment of mice with tilorone inhibited metastatic colonization and increased survival significantly in all cases. In some tumour systems, the effect was attenuated when high numbers of cells were injected. Abrogation of NK cells with anti-asialo GM(1) serum significantly decreased (in all tumours and at different cell doses) survival in comparison with untreated mice injected with tumours, regardless of cell dose used. These results clearly suggest that NK cell activation in vivo by the tilorone analogue we tested prolongs survival and inhibits metastasis formation in mice, even when pretreatment consists of a single dose of the analogue.

Molecular complexes forming as a result of interaction between yeast RNA preparations with 2,7-bis[-(diethylaminoethoxy)-fluorene]-9-on dihydrochloride (tilorone) administered parenterally to mice cause the appearance of interferon in high titers compatible to those induced by standard inductors of polyribonucleotide origin, poly(I)-poly(C) and larifan. Some physiologic conditions of interferonogenesis have been studied. The above molecular complexes in the dose range used experimentally were completely nontoxic. Hence, these complexes are promising agents for interferon induction.

The murine bone marrow cellular chromosomal aberration technique was used to reveal anticlastogenic effects of the natural interferon inductors ridostin and larifan, as well as the synthetic ones polyguacil and amyxin. The action of the interferon inductors was timed to the maximum production of interferon in the body. Anticarcinogenic, antitumor, and antimetastatic properties of the above interferon inductors were found by using classical experimental models of carcinogenesis. Some mechanisms of antimutagenic and coupled anticarcinogenic action of interferon inductors were considered. The natural interferon inductor larifan was found to have an antipromoter activity. The treatment regimen by using interferon inductors in combination with the immunomodulator thymosin, a thymic hormone, was under discussion.

Tilorone (2,7-bis[2-(diethylamino)ethoxy]-fluoren-9-one) and several other bis-basic compounds are known to induce lysosomal glycosaminoglycan (GAG) storage. The responsible pathomechanism has not been elucidated yet. The assumption of an unspecific disturbance of lysosomal proenzyme targeting due to elevation of endosomal pH is opposed by the hypothesis of formation of a complex between tilorone and GAGs within the lysosomes, which renders GAGs indigestible to glycosidases. In cultures of bovine corneal fibroblasts the amounts of intracellular GAGs [dermatan sulphate (DS), heparan sulphate (HS) and chondroitin sulphate (CS)] were quantified. The fibroblasts were exposed to tilorone (5 microM), which was found to be readily taken up by the cells and to be accumulated within acidic compartments to finally achieve millimolar concentrations. Under these conditions the GAG storage is predominantly due to the accumulation of DS; however, the DS secretion into the culture medium was not affected. The HS accumulation was much less pronounced, accounting only for 3% of total GAG storage. Ammonium chloride (10 mM), which is known to diminish lysosomal enzyme activity by interfering with the mannose 6-phosphate receptor-mediated transport, prevents both HS and DS breakdown. By means of NMR spectroscopy it was shown that tilorone itself tends to display a concentration-dependent aggregation which was enhanced in the presence of GAGs. The diethylamino groups of tilorone interact physicochemically with DS, and to a smaller extent with HS, but not with chondroitin 4-sulphate. Thus, the strength of the interaction between tilorone and the different GAGs in vitro correlates with the potency of tilorone to inhibit the breakdown of the individual GAGs in cultured bovine fibroblasts. The results support the hypothesis of a specific interaction between tilorone and particular GAGs, rendering these resistant to enzymic degradation.