Tilorone increases glucose uptake in vivo and in skeletal muscle cells by enhancing Akt2/AS160 signaling and glucose transporter levels.

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.

Repurposing Pyramax®, quinacrine and tilorone as treatments for Ebola virus disease.

We have recently identified three molecules (tilorone, quinacrine and pyronaridine tetraphosphate) which all demonstrated efficacy in the mouse model of infection with mouse-adapted Ebola virus (EBOV) model of disease and had similar in vitro inhibition of an Ebola pseudovirus (VSV-EBOV-GP), suggesting they interfere with viral entry. Using a machine learning model to predict lysosomotropism these compounds were evaluated for their ability to possess a lysosomotropic mechanism in vitro. We now demonstrate in vitro that pyronaridine tetraphosphate is an inhibitor of Lysotracker accumulation in lysosomes (IC50 = 0.56 µM). Further, we evaluated antiviral synergy between pyronaridine and artesunate (Pyramax®), which are used in combination to treat malaria. Artesunate was not found to have lysosomotropic activity in vitro and the combination effect on EBOV inhibition was shown to be additive. Pyramax® may represent a unique example of the repurposing of a combination product for another disease.

The effects of coadministration of tilorone dihydrochloride and culture supernatants from Lactobacillus reuteri on the mouse hepatoma cell line.

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 IC50 (TCIC50) 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.

Pulmonary administration of a dry powder formulation of the antifibrotic drug tilorone reduces silica-induced lung fibrosis in mice.

The aim of this work was to study the antifibrotic effect of pulmonary administration of tilorone to lung fibrosis. L-leucine coated tilorone particles were prepared and their aerosolization properties were analyzed using two dry powder inhalers (Easyhaler and Twister). In addition, the biological activity and cell monolayer permeation was tested. The antifibrotic effect of tilorone delivered by oropharyngeal aspiration was studied in vivo using a silica-induced model of pulmonary fibrosis in mice in a preventive setting. When delivered from the Easyhaler in an inhalation simulator, the emitted dose and fine particle fraction were independent from the pressure applied and showed dose repeatability. However, with Twister the aerosolization was pressure-dependent indicating poor compatibility between the device and the formulation. The formulation showed more consistent permeation through a differentiated Calu-3 cell monolayer compared to pristine tilorone. Tilorone decreased the histological fibrosis score in vivo in systemic and local administration, but only systemic administration decreased the mRNA expression of type I collagen. The difference was hypothesized to result from 40-fold higher drug concentration in tissue samples in the systemic administration group. These results show that tilorone can be formulated as inhalable dry powder and has potential as an oral and inhalable antifibrotic drug.

Alternative pre-approved and novel therapies for the treatment of anthrax.

BACKGROUND: Bacillus anthracis, the causative agent of anthrax, is a spore forming and toxin producing rod-shaped bacterium that is classified as a category A bioterror agent. This pathogenic microbe can be transmitted to both animals and humans. Clinical presentation depends on the route of entry (direct contact, ingestion, injection or aerosolization) with symptoms ranging from isolated skin infections to more severe manifestations such as cardiac or pulmonary shock, meningitis, and death. To date, anthrax is treatable if antibiotics are administered promptly and continued for 60 days. However, if treatment is delayed or administered improperly, the patient's chances of survival are decreased drastically. In addition, antibiotics are ineffective against the harmful anthrax toxins and spores. Therefore, alternative therapeutics are essential. In this review article, we explore and discuss advances that have been made in anthrax therapy with a primary focus on alternative pre-approved and novel antibiotics as well as anti-toxin therapies. METHODS: A literature search was conducted using the University of Manitoba search engine. Using this search engine allowed access to a greater variety of journals/articles that would have otherwise been restricted for general use. In order to be considered for discussion for this review, all articles must have been published later than 2009. RESULTS: The alternative pre-approved antibiotics demonstrated high efficacy against B. anthracis both in vitro and in vivo. In addition, the safety profile and clinical pharmacology of these drugs were already known. Compounds that targeted underexploited bacterial processes (DNA replication, RNA synthesis, and cell division) were also very effective in combatting B. anthracis. In addition, these novel compounds prevented bacterial resistance. Targeting B. anthracis virulence, more specifically the anthrax toxins, increased the length of which treatment could be administered. CONCLUSIONS: Several novel and pre-existing antibiotics, as well as toxin inhibitors, have shown increasing promise. A combination treatment that targets both bacterial growth and toxin production would be ideal and probably necessary for effectively combatting this armed bacterium.

[Detection of the interferon deficiency in inflammatory gynecological diseases and its correction with interferon inducers].

Interferons deficiency has a negative influence on the development of infection and inflammation in general. The use in the complex of anti-inflammatory therapy of interferon inducers (Meglumine acridоnacetate, Tilorone), combining antiviral, immunomodulatory, interferon correction effects with etiopathogenic action leads to the correction of the interferon system defects and eliminate etiological infectious agents, that is confirmed by laboratory data and clinical efficacy.

[Experimental and clinicolaboratory evaluation of complex therapy efficacy in arboviral infections].

Search for drugs efficient in prophylaxis and treatment of dangerous infections (especially arboviral ones) is rather actual, since no specific therapy is available. Many-year investigations of interferon inductors showed that they had immunomodulating, antiviral and antiinflammatory effects and were low toxic. The present study demonstrated that the protective effect was the following: Venezuelan equine encephalitis (VEE)--cycloferon > amixin = ridostin, Rift Valley fever (RVF)--cycloferon > amixin > ridostin, predator pox (PP)--cycloferon > amixin = ridostin, that was obvious that cycloferon was the most active agent in the treatment of VEE, RVF and PP, thus making it possible to acknowledge its priority in prophylaxis and therapy of dangerous viral infections (DVI). Ribavirin in combination with cycloferon solution or cycloferon tablets provided shorter periods of the fever, minimized the intoxication syndrome, promoted earlier resolution of hemorrhagic eruption and lowered the frequency of complications, which was in favour of the disease prognosis.

[Perspectives of prevention and treatment of herpes symplex with regular recurrence].

AIM: To assess efficacy of two-stage treatment of severe genital herpes (GH) with regular recurrences: combined use of antivirus medicines with interferon preparations and its inductors followed by antirecurrence vaccine therapy. MATERIAL AND METHODS: Two-stage treatment was given to 100 patients suffering from GH with severe regular recurrences. One-stage treatment included famvir in combination with interferon (viferon) or interferon inductors (amixin, cycloferon) and antioxidants. Stage two treatment consisted of vaccine therapy of patients who failed prophylactic standard vaccination by allergometric technique. RESULTS: The first stage treatment prolonged recurrence-free period 2-3-fold in more than 85% patients, improved quality of life. Stage-two treatment resulted in long-term clinicoimmunological remission which is necessary for conduction of anti-recurrence vaccine treatment. CONCLUSION: Changes in therapeutic-preventive policy in patients with recurrent GH with regular recurrence (2 stage of treatment) prolong recurrence-free intervals, improve quality of life and social adaptation of patients.

[Lavomax in outpatient treatment of urogenital chlamidiasis in males].

Sixty four males (age 21-45 years) with urogenital chlamidiasis were divided into two groups. 34 patients of the study group received interferon inductor and lavomax. 30 patients of the control group--interferon inductor and cycloferon. Treatment efficacy in the study group was higher.

[Antiviral effectiveness of the combined use of amixine and virasole in experimental hemorrhagic fever with renal syndrome in sucking albino mice].

The antiviral effectiveness of the combined and single use of superlow-dose amixine and virasole on the course of experimental hemorrhagic fever with renal syndrome was studied in sucking albino mice parenterally infected with their virus Hantaan. The co-administration of virasole and amixine was shown to protect 52% of the infected animals from death, which is superior to the effect of their monotherapy. The combined use of the drugs substantially prolongs the survival of albino mice after their infection and the level of brain viral reproduction suppression ( delta = 3.21 g) in the experimental group as compared to the controls and to the mice given only one of the drugs.

[Antiviral activity of an interferon inducer amixin in experimental West Nile Fever]. / Protivovirusnaia aktivnost' induktora interferona amiksina pri éksperimental'noi forme likhoradki Zapadnogo Nila.

Experimental research was undertaken to investigate the use of amixin in prevention, emergency prevention schemes and treatment of mice infected with West Nile fever (WNF) agent, strain Eg-101; the results are indicative of the drug efficiency both in its peroral and subcutaneous administrations. Amixin was shown to be most effective in the former case when administered, 10 mg/kg, in 96 hours before mice were infected as well as during the entire incubation period: lethality protection--46%. In the latter case, the drug was effective, when 3 administration schemes were in use, 10 mg/kg. The maximum degree of protection efficiency was registered with amixin administration according to the emergency prevention scheme: lethality protection--33%. The drug suppresses effectively the WNF virus reproduction in cerebral tissues.

[Intestinal microflora in rats under the conditions of a chronic toxic lesion of the liver]. / Mikroflora kishechnika krys v usloviiakh khronicheskogo toksicheskogo porazheniia pecheni.

Intestinal microflora in healthy rats and its changes under the conditions of experimental chronic toxic hepatitis were studied. The study revealed that in intact animals the microflora of the small intestine was represented by bacteria of the genera Escherichia, Enterobacter, Moraxella, Alcaligenes, Staphylococcus, Streptococcus. Bacteria of the genera Escherichia, Enterobacter, Moraxella, Alcaligenes, Staphylococcus, Corynebacterium and Clostridium were isolated from the large intestine. No bacteria were found in the systemic blood, the contents of the portal vein, as well as in the liver parenchyma and the mesenterial lymph nodes. As the result of dysbiosis induced by the introduction of kanamycin and in chronic hepatitis caused by carbon tetrachloride the sharp decrease in the species composition of microbial communities (up to 2-3 species) in the small intestine and was observed along with penetration of bacteria into the blood stream, the mesenterial lymph nodes and the liver parenchyma. The tendency towards the restoration of the quantitative and qualitative microflora composition was noted following administration into experimental animals of bactisubtil and amixin--an inductor of interferonogenesis.

[Interferon status of patients with non-specific ulcerous colitis and its correction with interferon inducers]. / Interferonovyi status bol'nykh nespetsificheskim iazvennym kolitom i ego korrektsiia induktorami interferona.

AIM: To examine validity of using interferon inductors (amixin and cycloferon) in combined treatment of patients with non-specific ulcerous colitis (NUC). MATERIAL AND METHODS: 113 NUC patients received basic antiinflammatory therapy (glucocorticoids and preparations of 5-aminosalycylic acid). Patients of groups 1 and 2 received interferon inductors (amixin and cycloferon in tables), respectively. Patients of groups 1a and 2a received placebo. All the patients were examined clinically with evaluation of interferon status and cellular immunity before and after the treatment. Colon mucosa biopsies obtained endoscopically were studied histologically. RESULTS: Alpha- and gamma-interferon production by leukocytes was substantially suppressed in all the examinees against normal levels of serum and spontaneous interferon. The interferon inductors stimulated relief of clinical symptoms and eliminated endoscopic signs of the disease. Amixin and cycloferon normalized interferon status in 36.3 and 33.3% of the treated patients, respectively. No response was registered in 9.1 and 8.4%, respectively. Improvement was seen in 54.6 and 58.3%, respectively. CONCLUSION: Cycloferon and amixin, interferon inductors, are effective in the treatment of NUC.

[Use of amixin in the therapy and prevention of some viral infections]. / Opyt primeneniia amiksina pri lechenii i profilaktike nekotorykh virusnykh infektsionnykh zabolevanii.

The trial of oral interferon inductor amixin for effectiveness in chronic viral hepatitis (CVH) and hemorrhagic fever with renal syndrome (HFRS) has shown that amixin in CVH improved general condition of the patients, removed yellowness of the skin and sclera, normalized activity of aminotransferases and blood bilirubin level. Virus replication was stopped in 25 and 1.6% in CVHB and CVGC, respectively. Amixin in HFRS was effective if received early. Preventive amixin therapy in population groups with high HFRS risk prevents development of HFRS and acute respiratory viral infection.

[Use of amiksin in complex therapy of cerebral gliomas]. / Zastosuvannia amiksynu v kompleksnomu likuvanni hliomy holovnoho mozku.

Indices were studied for cell-bound immunity during administration of a multimodality therapy treatments with making use of amiksin in patients with glial tumours of the brain IV degree anaplasia. Processes of lymph formation and differentiation of lymphocytes into natural killer cells have been found out to return to normal. No total stimulating action was recorded of the drug that would lead to stimulation of progressive growth of gliomas.

[Efficacy of amixine in experimental hantavirus infection]. / Effektivnost' amiksin pri éksperimental'noi khantavirusnoi infektsii.

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.

[Evaluation of amyxin effect in prophylaxis of acute respiratory viral infections]. / Otsenka profilakticheskogo éffekta amiksina v otnoshenii ostrykh respiratornykh virusnykh infektsii.

The results of the evaluation of the oral inductor of endogenic interferon (amyxin), manufactured in Russia are presented. The use of amyxin was found to produce a drop in morbidity in acute respiratory virus infections (ARVI) among medical workers 3.4 times, i.e. the preparation exhibited a pronounced prophylactic effect with respect to ARVI. The use of the preparation was accompanied by a decrease in the number of manifest forms of ARVI. Persons given the preparation often had ARVI in a mild or asymptomatic form.

[Evaluation of amixine reactivity and efficacy for prophylaxis of acute respiratory tract infections]. / Otsenka reactogennykh svoistv amikcina i ego c'ffektivnosti pri profilaktike ostrykh infektsii respiratornogo trakta.

Amixine reactivity and tolerability were evaluated in controlled trial at the risk group of medical personal at the period of flu and respiratory viral infection season. Drugs safety was estimated according to anamnesis, direct observation and hemogram. High efficacy of the drug for the infections prophylaxis and treatment was demonstrated. The drug was well tolerated and had no side effects. Amixine unreactivity was proved.

[Chlamydial laryngitis: clinical picture and amixine treatment]. / Klinika i lechenie khlamidioznogo laringita s primeneniem amiksina.

Chlamydia trachomatis DNA was detected by PCR in 12 patients with continuously recurrent catarrhal pharyngolaryngitis. These included 9 males and 3 females whose age ranged 16 to 72 years. All the patients were found to have degrees II-III alpha- and g-INF deficiencies. The interferon inducer amixine in combination with sumamed was used in the treatment of patients with chlamydial laryngitis. After treatment, there was a significant improvement in two thirds of patients. The pain syndrome was relieved, hoarseness diminished, and the laryngoscopic and microlaryngostroboscopic picture and interferon status became normal. Control studies showed no Ch. trachomatis in this group of patients. Despite positive changes in the course of the disease, the control study again detected Ch. trachomatis in 4 patients, which required repeated treatment courses.

[The treatment of laryngeal papillomatosis with interferon inducers]. / Lechenie papillomatoza gortani induktorami interferona.

Seventy three patients aged 16 to 88 years who had laryngeal papillomatosis (LP) were followed up. Microsurgical endolaryngeal removal of laryngeal papillomas was made in all the patients. The interferon inducers amixine and cycloferon as antirecurrent drugs were used in 45 patients by the regime the authors developed by taking into account the interferon status and cellular immunity of patients. The criteria for the efficiency of treatment were their improved interferon status and longer remission. The efficiency of treatment with amixine and cycloferon was 72 and 80%, respectively. Thus, the use of a sparing microsurgical intervention in combination with interferon inducers may be regarded as the method of choice in the LP treatment.