| 5α, 8α-cyclicobioxygen-24-bimethyl-6-vinyl-313-cholesterol(SC) is a lead compound withindependence intellectual property rights, its microsphere preparation could be a veryprospective new anti-HBV drug. These researches are to reveal SC activities of anti-HBV,immunity opsonization in mouse, the effects of anti-HBV and toxicity for its microspherepreparation.1 Anti-HBV activities and toxicity to cell of SC1.1 Anti-HBV activitiescell lines: L2.2.15; method: rt-PCR, Southern Blot and Elisa to test HBV quantitation,HBsAg and HBeAg.SC has no dose-effect relationship for inhibiting HBV replication at dose 0.1-12.5μg/ml, ithas stable and higher inhibit ratio especially at dose 0.1 and 12.5μg/ml. At 4d, inhibit ratio ofdoses are relative lower and straggling, maybe because of short action time; At 8d, it shows thehighest inhibit ratio(＞85％) during the whole experiment time; At 11d, inhibit ratio decreased,maybe cell exponent proliferation cycle comes; At 14d, no significant change for inhibit ratio, it isan evidence the effect of SC acting L2.2.15 cell line dose not disappear immediately. SouthernBlot test shows SC can inhibit rcDNA replication, this may be a cue in vivo to inhibit cccDNA.Action of SC inhibiting secretion Dane partical of L2.2.15 cell is complicated. According totime, the trend of action is inhibiting→prornoting→reversion, this result equal to Southern Blottest. The result of Southern Blot shows SC influence replication of HBV scDNA. At 4d, theinhibition ratio is upper than 70％; at 7d, the influence changed to promote(highest 200％); at10d(drug withdraw), almost no influence.SC also inhibits HBsAg(40％) and HBeAg(90％) secretion in L2.2.15 cell line, and inhibitoryactions show direct dose-effect relationship. From 4d to 14d, different doses, trend of inhibitionratio is increase→decrease, 8d＞11d＞4d≈14d; SC=12.5μg/ml, inhibition ratio 14d＞4d.1.2 toxicitycell lines:L2.2.15, HepG2 and CCC-HEL-1; method: MTS to test inhibition ratio of cellproliferation.SC influences proliferation of these three cell lines, it has different level for each cell line.Above all, low dose promoting and high dose inhibiting. Basicly, inhibiting level is also directdose-effect relationship. It is a crisis concentration SC=8μg/ml for each cell line, if lower,inhibiting level is not obvious(＜30％), by chance promoting. There is an evidence of safetymedication dose for SC. If SC＞16μg/ml, inhibition ratio of proliferation will be upper than 50％for these three cell lines, thus it would be toxicity to human embryo liver cell line. Specially, whiledifferent SC concentration for 48 hours, it has 40～80％inhibition ratio to L2.2.15 cell line, on thecontrary, to the other two cell lines, it have not. It indicates SC maybe possess selective activity ofcell toxicity.To sum up, SC has potential anti-HBV activities in vitro. To L2.2.15 cell line, safetyconcentration, these activities will be increase earlier, after peak, decrease, then stable relatively,no rebound. The inhibition ratio for HBsAg and HBeAg increase according to dose raising andtime lasting.2 SC immunifaetion in mouseSC 0.2mg/kg i.p, qd, 7 times, concanavalin A induced proliferation of lymphocyteincreasing(p＜0.05), with thymic hyperplasia(p＜0.05). SC 0.4 or 0.8mg/kg i.p, qd, 7 times,concanavalin A induced proliferation of lymphocyte decreaseing(p＞0.05), thymichyperplasia(p＞0.05). Index number of spleen is increasing follow as concentration of SC,while SC=0.8mg/kg, there is significant difference(p＜0.02) with control group. SC 0.2, 0.4 or 0.8mg/kg i.p, qd, 7 times, phagocytosis of peritoneal macrophage increasesobviously(p＜0.01), while group 0.8mg/kg, phagocytosis promotion ratio decreased relatively.Natural killer(cell) cytoactive is promoting equally(group 0.2mg/kg, p＜0.05, group 0.8mg/kg,p＜0.01). SC i.p, no manifest influence to carbon clearance in mouse(p＞0.05).Avove all, SC i.p group 0.2 or 0.4mg/kg maybe up-regulate immunological function inmouse, on the contrary group 0.8mg/kg suppresses.3 Functions of SC-MS inhibits DHBV-DNA in duck hepatitis B virus model in vivoObjective: to test the influence of pharmacodynamic for three administration styles. Injectionform: sc, ip and iv. Dosage: 25, 50 and 100mg/kg, b.i.d×10(sc, ip); 2.5, 5.0 and 10.0mg/kg, 5d adun vic(iv), positive control: 3TC, po, 50mg/kg, b.i.d×10. Sampling: before administration(T0), ad.5dOT5, ad. 10d(T10) and after drug withdraw 3d(P3), get plasma samples, dot blot, detect andmake statistics.3.1 ad. scSC 100mg/kg, b.i.d×10, very significant or significant difference(p＜0.01 or p＜0.05) to theinbit ratio of DHBV-DNA. SC 50mg/kg, b.i.d×10, unvarying inhibition. SC 25mg/kg, b.i.d×10,no apparent inhibition.3.2 ad. ipSC 100mg/kg, b.i.d×10, very significant or significant difference(P3, p＜0.01; T5, T10,p＜0.05) to the inbit ratio of DHBV-DNA. SC 50mg/kg, b.i.d×10, very significant or significantdifference(P3, p＜0.01; T10, p＜0.05). SC 25mg/kg, b.i.d×10, no apparent inhibition.3.3 ad. ivSC 10mg/kg, 5d ad un vie, significant difference(P3, T10, p＜0.05) to the inbit ratio ofDHBV-DNA. SC 5mg/kg, 5d ad un vic, same to 10mg/kg group. SC 2.5mg/kg, 5d ad un vic, noapparent inhibition.To sum up, statistical pharmacodynamics show iv. SC-MS is the optimizationadministration, because of lower dosage and higher curative effect. These effects might becaused by iv. passive-liver-target. Especially, no matter each administration style, there is norebound like 3TC. This result might come from two actions, the one is long acting preparationfor SC-MS, the other one maybe different anti-DHBV mechanism from 3TC.4 SC-MS therapeutical effect to carbon tetrachloride induced acute hepatic injury in ratsModel: carbon tetrachloride induced acute hepatic injury. Dosage: high, middle, low dosagegroups, each SC-MS(equivalent SC) 0.084, 0.042 and 0.021mg/kg, ip, s.i.d×7, positive control:inj. Solution of diammonium glycyrrhizinate(13.5mg/kg), ip, s.i.d×7.Results: SC-MS high group in the same inj. Solution of diammonium glycyrrhizinate couldsignificantly step down GPT and GOT in model rats(p＜0.05). Moreover, each SC-MS high andmiddle group could significantly depress hepatomegaly induced by CC14 in rats((p＜0.05).5 Toxicity of SC-MS in vivo.SC-MS iv, once, equal volume, different concentrations, in mouse and rats. Observe animalsreactions and deathasis, for 1～2 weeks. Results: LD50(mouse)=580.93mg/kg, confidenceinterval(95％) 483.07～704.44mg/kg; LD50(rats)=578.59mg/kg, confidence interval(95％)489.79～694.26mg/kg.long-term toxicity test in rats is agoing. Dosage: SC-MS iv. high, middle, low dosage groups,each SC-MS 120, 60 and 20mg/kg, once a week. By now(13 weeks), SC-MS iv, no influence toanimals weight and ingestion for each group.Hematology and biochemical indicator: ♂, low dosage group (contrast control group), Hb and PLT increasing(p＜0.01); MOincreasing(p＜0.05), GR decreasing(p＜0.05); BUN decreasing(p＜0.05).♀, each group(contrast control group), Hb and PLT decreasing(p＜0.01); high group, WBCincreasing(p＜0.01); MO decreasing(p＜0.05), GR increasing(p＜0.05); high or middle group, ALBdecreaing(p＜0.05); each group, ALT decreasing(only low group, p＜0.05).Organ index number:♂, each group(contrast control group), spleen multiplication(p＜0.05), lung multiplication(p＜0.01), liver multiplication(no significant).♀, each group(contrast control group), spleen multiplication(p＜0.01), lung multiplication(p＜0.01 or p＜0.05), liver multiplication(p＜0.05, high or middle group).Pathology half quantitative observation:SC-MS, iv, 13 weeks, in rats. Livers, spleens and lungs of rats become diversity extentpathology changes, spleens most severity, next livers and lungs.Above all, SC-MS, iv, 13 weeks, in rats. Toxicity is visualization. Main targeting organ isspleen, and lung, and liver. Especially, sex differences show up.With time goes on, come to the end of the toxicity experiment, more certain and preciseresult would be discovered. |
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