Ljubica Medić-Mijačević

Ribavirin reduces clinical signs and pathological changes of experimental autoimmune encephalomyelitis in Dark Agouti rats.

The effect of ribavirin on development of experimental autoimmune encephalomyelitis (EAE) was investigated. The disease was induced in genetically susceptible Dark Agouti rats with syngeneic spinal cord homogenate in complete Freunds adjuvant (SCH‐CFA). Depending on the amount of mycobacteria in CFA, the animals developed either moderate or severe EAE. Ribavirin (1‐β‐D‐ribofuranosyl‐1,2,4‐triazole‐3‐carboxamide) was applied i.p. at a daily dosage of 30 mg/kg in two treatment protocols: from the start of immunization (preventive treatment) or from the onset of the first EAE signs after the induction (therapeutic treatment). Signs of EAE began between 7 and 9 days after induction and peaked at days 11–13. In moderate EAE (mean maximal severity score 3.33 ± 0.21), the recovery was completed by days 23–26, whereas, in severe EAE (mean maximal severity score 4.5 ± 0.23), obvious recovery was not detected. Preventive ribavirin treatment significantly decreased clinical signs after both moderate (score 1.75 ± 0.25, P < 0.05) and severe (score 3.62 ± 0.31, P < 0.015) immunization. Also, disease manifestations were reduced by therapeutic treatment of ribavirin (mean maximal severity score 2.5 ± 0.2 vs. 3.33 ± 0.21 in controls, P < 0.005) but less so in comparison with preventive treatment. Analysis of the effects of ribavirin on histopathologic changes in the spinal cord tissue revealed a reduction of mononuclear cell infiltrates, composed of T cells and macrophages/microglia, and the absence of demyelination, which were pronounced in control EAE animals. Beneficial effects of preventive and therapeutic treatment with ribavirin on development of EAE suggest this nucleoside analogue as a useful candidate for therapy in multiple sclerosis.

A novel rearrangement of steroidal α-hydroxy oximes

Abstract By the action of acidic titanium trichloride upon 16-oximino-17α-benzyl-17β-hydroxy derivatives in the androstane and estrane series the 16-oxo-17β-benzyl-17α-hydroxy derivatives 6 and 7 with inversed configuration at C 17 were obtained. A mechanism for this novel rearrangement is proposed.

Sulfinosine-induced cell growth inhibition and apoptosis in human lung carcinomas in vitro

In spite of tremendous effort for improvedtherapy, lung cancer remains the leadingcause of cancer-related deaths worldwide.In the present study, we used the novelpurine ribunocleoside sulfinosine andevaluated its antiproliferative andapoptotic outcome on the non-small celllung carcinoma cell line (NSCLC) and thesmall cell lung carcinoma cell line (SCLC).Using a BrdU incorporation-test sulfinosineinhibited cell growth in a dosedependent-manner. ID50 values were4.65 ± 0.17 μM in the case of NSCLCcells, and 3.59 ± 0.81 μM in thecase of SCLC cells. MTT testing revealedthat IC50 values were 6.24 ±0.77 μM for NSCLC and 5.68 ±0.58 μM for SCLC. Inhibitoryconcentrations (IC50 and ID50)for sulfinosine were nonsignificantly lowerin SCLC cells compared to NSCLC cells,indicating similar susceptibility of thecells. Flow-cytometric analysis, TUNELstaining, DNA laddering and cell deathELISA test were used to investigateapoptotic cell death. Our resultsdemonstrated that high concentrations ofsulfinosine can cause typical DNAladdering, a hallmark for apoptosis.Evidence of free nucleosomes and enzymaticlabeling of fragmented DNA confirmedapoptosis involvement in sulfinosinecytotoxicity. In addition, flow-cytometricanalysis showed that 25 μM sulfinosinearrested cell cycle progression atthe G2M phase and induction ofapoptosis in both cell lines. From theseresults, we concluded that sulfinosine mayact as an anticancer agent and furtherstudies may prove its efficacy in lungcancer cells. Thus the biological effectsof sulfinosine may be due to modulation ofcell growth, cell death, and cell cycleregulatory molecules.

A nucleoside analogue, 7-thia-8-oxoguanosine stimulates proliferation of thymocytes in vitro

7-thia-8-oxoguanosine (immunosine) is a nucleoside analogue with immunoenhancing activity. In this work, its effects on proliferation of thymocytes in vitro were studied. It was found that immunosine stimulated proliferation of thymocytes both of mice and rats. The stimulatory effect depended on antigen presenting cells (APC), since thymocytes depleted of accessory cells did not proliferate to immunosine. In addition, pretreatment of APC with immunosine for 24 h significantly increased proliferation of thymocytes. Immunosine stimulated interleukin 2 (IL-2) production and the expression of activation markers (CD25 and CD71). The upregulation of CD25 (alpha subunit of IL-2R) was detected both on thymocytes and thymic dendritic cells. Proliferation of thymocytes in the presence of immunosine was predominantly mediated by IL-2 since blocking IL-2Ralpha by specific monoclonal antibodies inhibited cell proliferation by 65-85%.

A novel fragmentation-cyclization reaction of steroidal α-hydroxy oximes

Abstract By a novel “one pot” fragmentation-cyclization reaction 17β-hydroxy-17α-substituted-16-oximino derivatives in the androstane and estrane series were converted to a new type of D-homo derivative.

Immunosine (7-thia-8-oxoguanosine) acts as a cofactor for proliferation of T cells

Abstract— Immunosine (7‐thia‐8‐oxoguanosine) is a novel guanosine analogue showing immunostimulatory activity both in vivo and in vitro. This compound acts on different components of the immune system including B cells, natural killer (NK) cells and antigen‐presenting cells (APC). However, its influence on functions of T cells is poorly understood. In this work we studied the effect of immunosine on proliferation of total rat splenocytes and purified T cells triggered by different mitogens and the mechanisms involved. The results demonstrate that immunosine significantly stimulates proliferation of T cells. The effect was dose‐dependent and also depended on concentrations of specific stimulators. Maximal stimulation was seen using 250 μM immunosine. The stimulatory effect of immunosine on lymphocyte proliferation triggered by Concanavalin A (Con A) correlated with increased interleukin 2 (IL‐2) production and upregulation of the IL‐2 receptor α (IL‐2Rα) expression. The dependency of T‐cell proliferation on IL‐2/IL‐2R was confirmed using neutralizing anti‐IL‐2Rα monoclonal antibodies (mAbs). Higher concentrations of immunosine in the presence of optimal concentrations of Con A (5 μg/mL) inhibited proliferation of T cells. A similar stimulatory effect of immunosine on proliferation of purified T cells and IL‐2 production was observed using an anti‐T‐cell receptor (TCR) mAb and a combination of anti‐TCR mAb and IL‐2. However, the guanosine analogue did not significantly modulate proliferation of T cells triggered by IL‐2 alone. When the combination of phorbol myristate acetate (PMA) and ionomycin was used for T‐cell stimulation different results were obtained. Under lower cell stimulation immunosine significantly potentiated T‐cell proliferation, expression of IL‐2Rα and IL‐2 production. In the presence of suboptimal stimulation the compound stimulated T‐cell proliferation and IL‐2Rα expression, whereas under maximal stimulation an enhancing effect on IL‐2 production was seen. Since direct stimulatory effect of immunosine on T‐cell growth in culture was rather weak it can be postulated that the compound acts as a cofactor for T‐lymphocyte proliferation.

17α-Benzyl-3β,17β-dihydroxy-5-androstene

The asymmetric unit of the title compound, 17α-benzyl-5-androst-5-ene-3β,17β-diol, C 26 H 36 O 2 , contains two molecules, which differ slightly in the conformation of the D ring. Of the four OH groups in the two symmetry-independent molecules, A and B, three participate in O-H...O hydrogen bonding. Crystal packing involves molecules of A forming helix-like chains along the b axis, with hydrophobic loops occupied by one part of the B molecules; their other parts are involved in hydrogen-bonded bridges to the neighbouring chains

D-Secoestrone derivatives. VII. 3-Methoxy-17-keto-17-benzyl-16,17-secoestra-1,3,5(10)-trien-16-nitrile and (17S) 3-methoxy-17-hydroxy-17-benzyl-16,17-secoestra-1,3,5(10)-trien-16-nitrile

The two title compounds 3-methoxy-17-keto-17-benzyl-16,17-secoestra-1,3,5(10)-trien-16-nitrile, C26H29NO2, (I) and (17S)-3-methoxy-17-hydroxy-17-benzyl-16,17-secoestra-1,3,5(10)-trien-16-nitrile, C26H31NO2, (II), have similar conformations in the solid state. The models from molecular mechanics calculations show an improvement in the similarity of the calculated (modeled) structures. However, they also revealed that the global energy minimum structure of (I) is a structure with a rotated 17-benzyl moiety. Thus, the difference in the bio logical activity of the two compounds ((I) a moderate anti-oestrogen, (II) biologically inactive) must be primarily caused by the difference in the orientation of the 17-benzyl moiety in the free molecules. In (II), the molecules are linked by weak O—H…N hydrogen bonds, forming spirals along the b direction.

Structure-activity relationships in 16- and 17-substituted 5-androstenes : 3β-Acetoxy-17β-picolyl-5-androsten-16-one

The title compound, 3β-acetoxy-17β-picolyl-5-androsten-16-one, C 27 H 35 NO 3 , was synthesized from a mixture of Z and E isomers of 3β-acetoxy-17-picolinylidene-5-androsten-16-one. The significant difference in these three compounds in the crystalline state lies in the conformation of the five-membered D ring, as well as in the orientation of the bulky substituent at C17. After molecular-mechanics calculations on the individual molecules, the conformational differences of the D ring disappeared, but the differences in the orientation of the substituent at C17 increased. The remarkable differences in biological activity of these compounds could therefore be due to the different orientations of the bulky C17 substituent.

17α-benzyl-17β-hydroxy-16-hydroxyimino-3-methoxyestra-1,3,5(10)-triene

The asymmetric unit of the title compound, 17α-benzyl-16-hydroxyimino-3-methoxyestra-1,3,5(10)-trien -l7β-ol, C 26 H 31 NO 3 , contains two molecules which differ in the orientations of the methoxy groups at C(3). The 17-hydroxy and 16-hydroxyimino moieties are involved in intramolecular N...O and intermolecular N...O and O...O hydrogen bonds. The hydrogen-bond network accounts for the differences in bond and torsion angles of the α-hydroxyimino moieties of the symmetry-independent molecules. This has been confirmed by molecular-mechanics calculations on the individual molecules which indicate that they have the same geometry in their energy minimum states.