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.

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.

[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.

[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.

Enhanced resistance to experimental systemic candidiasis in tilorone-treated mice.

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.

In vivo activation of NK cells induces inhibition of lung colonization of H-2 positive and H-2 negative fibrosarcoma tumor clones.

The role of different tilorone analogs in the abrogation of the metastatic spread of H-2 positive and H-2 negative tumor clones was studied. Pre-treatment of BALB/c mice with RMI 10,874DA compound completely abolished lung colonization of an H-2 negative (GR9.B9) MCA-induced fibrosarcoma clone in an experimental metastasis assay. This effect was also evident when clones were treated with other tilorone analogs (R11,567DA or R11,513DA). Other H-2 positive and H-2 negative chemically induced fibrosarcoma clones were also tested. The effect was not due to direct toxicity of the tilorone analog on tumor cells, but instead was dependent on NK cells; this was suggested by the finding that treatment of mice with anti-asialo GM1 abrogated the effect of the tilorone analog (RMI 10,874DA compound). Interestingly, the inhibition of lung colonization after intravenous injection was again observed regardless of the H-2 phenotype of the tumor clones, and H-2+ and H-2- clones were similarly inhibited. In vitro assays of NK sensitivity of tumor clones showed that lysis varied depending on the H-2 phenotype of tumor clones, indicating an absence of correlation between in vivo and in vitro results.

[Radiosensitizing effect of interferon synthesis inducers]. / Radiosensibiliziruiushchee deistvie induktorov sinteza interferona.

The preinjection of inductors of leukocytic interferon synthesis of rapid (poly I.poly C, dextransulfate) and slow (tyloron) types to mice bearing inoculated solid sarcoma 37 considerably increases the efficiency of X-irradiation of tumors: the coefficient of tumor growth inhibition (kappa *) exceeds 1.0, and the number of animals with the completely regressed tumors increases. The effectiveness of the procedure depends on the time of the injection of the preparations and modes of irradiation.

Keratopathy in rats after treatment with tilorone.

According to clinical reports, the antitumor drug tilorone induces corneal opacities in patients. The present communication shows that keratopathy can be experimentally reproduced in rats and describes the cellular alterations underlying the corneal opacities. Tilorone was applied either orally (60-90 mg/kg) for several weeks or topically (2%) for a few days. Biomicroscopic examination performed after treatment for 6 weeks or longer revealed fine punctate opacities throughout the corneal stroma. Ultrastructurally, the keratocytes were swollen due to large, optically empty vacuoles in the cytoplasm. Similar, albeit smaller, vacuoles were also numerous in the endothelium and less frequent in the epithelium. Histochemical experiments showed that the cellular alterations represented lysosomal storage of polyanionic substances, most probably sulfated glycosaminoglycans, thus mimicking the cytological picture of mucopolysaccharidosis. Upon discontinuation of drug treatment, the alterations tended not to recede. This keratopathy in rats is part of a generalized mucopolysaccharidosis-like disorder induced by tilorone.

Prophylaxis and therapy of an experimental bladder cancer with biological response modifiers.

We have previously reported that the intraperitoneal injection of viable bacillus Calmette-Guerin (BCG) reduces the incidence of tumor takes and the rate of tumor growth and also increases tumor regression rate and survival in C3H/HeN mice challenged with the syngeneic MBT-2 bladder cancer. We have now investigated the immunoprophylactic effect of BCG and other biological response modifiers. Groups of 12 to 15 female C3H/HeN mice were challenged with 5 X 10(5) MBT-2 viable cells and treated with BCG, poly I:C, tilorone, levamisole or a combination of these agents. Appropriate controls were included in each experiment. In this study we confirmed previous findings that BCG alone is effective in prophylaxis and can also decrease the rate of tumor growth in those animals not protected against tumor takes. Both i.p. levamisole and oral tilorone lacked activity against the MBT-2 tumor. A single i.p. injection of poly I:C was also ineffective although its repeated administration reduced the rate of tumor growth and induced a significant number of tumor regressions. Combined therapy of BCG with either levamisole or tilorone offered no therapeutic advantage over BCG alone. Heat-inactivated BCG also failed to induce anti-tumor activity in C3H/HeN mice challenged with MBT-2 cells. These results indicate that live BCG remains the most active immunoprophylactic and immunotherapeutic agent against the MBT-2 murine bladder cancer. Furthermore, the anti-tumor effect of viable BCG is not potentiated further by the addition of other immune modulating agents.

[Effect of tilorone on the immunological indices of guinea pigs with synestrol-induced glandular hyperplasia of the endometrium]. / Vliianie tilorona na nekotorye immunologicheskie pokazateli u morskikh svinok s zhelezistoi giperplaziei éndometriia, indutsirovannoi sinéstrolom.

The immune system state was studied before and after administration of tilorone (an immunologic adjuvant) to guinea pigs with synestrol-induced glandular hyperplasia of endometrium. The total amount of T- and B-lymphocytes. T-lymphocytes with receptors to the Fc-fragment of IgM and IgG, the content of immune complexes were determined. Immune and nonimmune adherence of neutrophils was investigated as well. It was established that the total amount of T-lymphocytes decreased and the quantity of T-lymphocytes and immune complexes increased with the development of glandular hyperplasia of the endometrium. The tilorone administration normalized the structure of endometrium and T-lymphocyte immune controlling system.

Potentiation of antitumor and antimetastatic activities of alpha-difluoromethylornithine by interferon inducers.

The objective of the present investigation was to study the potentiation of antitumor and antimetastatic activities of DL-alpha-difluoromethylornithine (DFMO) by inducers of interferon, namely, tilorone and polyriboinosinic:polyribocytidilic acid complex [poly(l) X poly(C)]. The results of this study indicate that these interferon inducers enhance the antitumor activity of DFMO against B16 melanoma and Lewis lung carcinoma in mice. In B16 melanoma, DFMO, tilorone, or poly(l) X poly(C), when administered alone, showed 85, 39, and 39% of inhibition of tumor growth, respectively. However, a combination of DFMO and tilorone or poly(l) X poly(C) resulted in 98 and 95% inhibition of growth, with about 20% of animals showing no detectable tumors. This potentiation appears to be related to the ability of the compounds to induce interferon, since an analogue of tilorone, MDL 10,842, neither induced interferon nor potentiated the antitumor activity of DFMO. The data also indicate that this combination is particularly effective when the tumor burden is relatively low. When tilorone was given 7 days after tumor inoculation, it did not show any potentiation of antitumor activity of DFMO. The studies with Lewis lung carcinoma also showed that the interferon inducers potentiated both the antitumor and antimetastatic activities of DFMO. DFMO or tilorone administered alone showed 28 and 46% inhibition of tumor growth and 80 and 58% inhibition of metastases, respectively. Poly(l) X poly(C) by itself did not have any effect on the tumor growth and metastases. However, a combination of DFMO and tilorone brought about 78% inhibition of tumor growth and 99.5% inhibition of metastases, with 87% of the animals free of any metastases. A combination of DFMO and poly(l) X poly(C) also showed a potentiation of both antitumor activity (58% inhibition) and antimetastatic activity (94% inhibition), with 62% of the animals free of any detectable metastases. The mechanism underlying this tumor suppression by combination of DFMO and interferon inducers is not yet known. Enhancement of host immune response or interferon-mediated cytotoxicity could account for the observed marked suppression of tumor growth. Previous studies using interferon and the data reported here with interferon inducers, along with the relatively nontoxic nature of DFMO, suggest a potential use for the inhibitors of polyamine biosynthesis in combination with interferon or interferon inducers in cancer chemotherapy and other proliferative states.

Pre-Sézary syndrome.

Eighteen patients with erythroderma, recurrent cycles of circulating Sézary cells of less than 1,000 cells/mm3, and a chronic course were followed for a mean time of nearly 5 years and were diagnosed as having pre-Sézary syndrome. Only one patient died, and none developed lymphoproliferative disease. All ten patients who underwent patch testing showed positive results. The elevation of IgE was striking when this group was compared with a group with Sézary syndrome. Most patients achieved partial or complete remission on low-dose chlorambucil and prednisone therapy. Some patients had lymphocytic or lymphomatoid bands on skin biopsy specimens and were like previously reported patients with pre-Sézary syndrome whose condition progressed to Sézary syndrome. A nontoxic chemotherapy or an anti-T cell treatment program can control this chronic erythroderma state.

Tilorone hydrochloride in the treatment of T cell lymphoproliferative cutaneous disease.

Tilorone hydrochloride was used to treat eleven patients with T cell cutaneous disease ranging from pre-Sézary syndrome to tumor-stage mycosis fungoides. Cutaneous histologic study, Sézary counts, delayed skin tests, patch tests, and quantitative T cell counts were monitored. The study revealed that the response to tilorone depends in part on the type and stage of disease, as well as the characteristics and responsiveness of the individual lymphocyte population. Patients with pre-Sézary syndrome are most likely to benefit from tilorone. The effect of tilorone on the T cell population is manifested by changes in responses to patch and skin tests, as well as histologic improvement. Intact immune responses and elevated levels of IgE may be important prognostic clinical parameters in these patients. Tilorone is ineffective in patients with mycosis fungoides and advanced Sézary syndrome. Keratopathy can be a limiting but reversible complication of therapy. The drug may provide an effective therapeutic step in the treatment of early T cell cutaneous disease or an adjunctive therapy to leukapheresis and chemotherapy.

Antitumor activity of tilorone hydrochloride on a transplantable rat adenocarcinoma.

Tilorone HCL, administered in drinking water, retarded and slowed significantly the growth of the transplantable R3230 AC rat adenocarcinoma in Fischer 344 rats. This effect is dose-related; from the average three dosage levels studied, daily intake of 30 mg/kg rat/day was found to be optimal. However, Stadie slices of tumors from Tilorone-treated animals when transplanted, took, and showed a growth curve similar to tumors from untreated rats. Exposure to Tilorone does not select tumor cell clones of lesser malignancy or different histopathology.