Antiviral screen identifies EV71 inhibitors and reveals camptothecin-target, DNA topoisomerase 1 as a novel EV71 host factor.

Enterovirus 71 (EV71) is one of the causative agents of hand, foot and mouth disease (HFMD) associated with severe neurological disease. EV71's pathogenesis remains poorly understood and the lack of approved antiviral has led to its emergence as a clinically important neurotropic virus. The goals of this study were to: (i) identify novel anti-EV71 compounds that may serve as lead molecules for therapeutics; and (ii) investigate their targets in downstream studies. We screened a 502-compound library of highly purified natural products for anti-EV71 activities in a cell-based immunofluorescence assay that were then confirmed in viral plaque reduction assays. Along with known antivirals, novel inhibitors of EV71 were also identified. We selected camptothecin for downstream studies and found that it is a limited spectrum enterovirus inhibitor that inhibits coxsackievirus A16 but not ECHOvirus 7. Camptothecin, a DNA topoisomerase 1 (TOP1) inhibitor, inhibits both viral RNA replication and translation based on luciferase replicon studies. Depletion of TOP1 using siRNA was then able to rescue EV71 infection from camptothecin inhibition. Interestingly, EV71 viral RNA replication and translation were also in TOP1 depleted cells. We found that nuclear TOP1 was relocalized to cytoplasmic replication vesicles during EV71 infection and localized with viral 3CD using confocal microscopy and proximity-ligation assays. Our findings reveal camptothecin to be a limited spectrum antiviral against enteroviruses that functions in a TOP1-dependent but cytotoxicity-independent manner. TOP1 is in turn needed for maximal EV71 viral RNA replication and viral protein synthesis.

TU-100 exerts a protective effect against bacterial translocation by maintaining the tight junction.

PURPOSE: We previously reported that TU-100 suppresses irinotecan hydrochloride (CPT-11)-induced inflammatory cytokines and apoptosis. However, the mechanism underlying this effect has not been fully elucidated. The aim of this study was to further clarify the mechanism of CPT-11-induced bacterial translocation (BT) and the effect of TU-100 on BT. METHODS: Cell cytotoxicity was assessed in vitro by a WST-8 assay. For the in vivo experiments, rats were randomly divided into 3 groups: the control group, the CPT-11 group (250 mg/kg i.p. for 2 days), and the CPT-11 and TU-100 co-treated group (1000 mg/kg, p.o. for 5 days). All of the rats were sacrificed on day 6 and their tissues were collected. RESULTS: CPT-11 and TU-100 co-treatment improved CPT-11 the related cytotoxicity in vitro. All CPT-11-treated rats developed different grades of diarrhea and BT was observed in 80% of the rats. CPT-11 caused a significant increase in the expression of TLR4, IL-6, TNF-α, IL-1ß and caspase-3 mRNAs in the large intestine. The expression of tight junction (TJ) marker mRNAs (occludin, claudin-1 and 4, and ZO-1) was significantly decreased in comparison to the control group. TU-100 co-treatment significantly reversed diarrhea, BT, and the expression of TLR2, IL-6, TNF-α, IL-1ß and caspase-3, and improved the expression of occludin, claudin-4 and ZO-1. CONCLUSIONS: TU-100 can suppress the adverse effects associated with CPT-11 and improve the function of the TJ. It is possible that this occurs through the TLR pathway.

O emprego de sistemas de liberação como estratégia para melhorar as propriedades terapêuticas de fármacos de origem natural: o exemplo da camptotecina e seus derivados / Drug delivery systems as a way to improve the therapeutic properties of natural drugs: the example of camptothecin and its derivatives

As plantas têm sido utilizadas para a obtenção de um grande número de substâncias biologicamente ativas. Entretanto, muitos compostos naturais quando empregados sem qualquer modificação química não resultaram em medicamentos eficazes, por não apresentarem as características desejáveis para administração. Neste sentido, a melhoria das propriedades terapêuticas de compostos isolados de plantas por meio da sua incorporação em sistemas de liberação de fármacos consiste em uma importante estratégia na obtenção de novos medicamentos, na qual ainda existe muito a ser explorado. Tais sistemas são caracterizados por apresentar a capacidade de prolongar e controlar a liberação de substâncias ativas, proteger as moléculas frente à degradação no meio biológico, veicular fármacos hidrofóbicos e reduzir os efeitos colaterais indesejáveis. A camptotecina, um alcalóide proveniente do arbusto Camptotheca acuminata (Descaisne, Nyssaceae), é um fármaco que apresenta elevada atividade antitumoral, cujo mecanismo envolve a inibição da topoisomerase I, uma enzima altamente expressa nos tumores. Entretanto, a utilização deste fármaco na terapêutica foi limitada, durante anos, em virtude de suas características de baixa solubilidade aquosa, elevada instabilidade em meio fisiológico e elevada toxicidade. Neste artigo é realizada uma revisão sobre o potencial terapêutico da camptotecinae seus análogos no tratamento do câncer, dando ênfase aos estudos conduzidos com o intuito de contornar as limitações da administração destes fármacos e que resultaram na melhoria das propriedades terapêuticas. Estratégias como a microencapsulação, nanoencapsulação e solubilização em micelas poliméricas, entre outras, são discutidas e os principais resultados de atividade antitumoral in vitro e in vivo são apresentados.

St. John's wort attenuates irinotecan-induced diarrhea via down-regulation of intestinal pro-inflammatory cytokines and inhibition of intestinal epithelial apoptosis.

Diarrhea is a common dose-limiting toxicity associated with cancer chemotherapy, in particular for drugs such as irinotecan (CPT-11), 5-fluouracil, oxaliplatin, capecitabine and raltitrexed. St. John's wort (Hypericum perforatum, SJW) has anti-inflammatory activity, and our preliminary study in the rat and a pilot study in cancer patients found that treatment of SJW alleviated irinotecan-induced diarrhea. In the present study, we investigated whether SJW modulated various pro-inflammatory cytokines including interleukins (IL-1beta, IL-2, IL-6), interferon (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) and intestinal epithelium apoptosis in rats. The rats were treated with irinotecan at 60 mg/kg for 4 days in combination with oral SJW or SJW-free control vehicle at 400 mg/kg for 8 days. Diarrhea, tissue damage, body weight loss, various cytokines including IL-1beta, IL-2, IL-6, IFN-gamma and TNF-alpha and intestinal epithelial apoptosis were monitored over 11 days. Our studies demonstrated that combined SJW markedly reduced CPT-11-induced diarrhea and intestinal lesions. The production of pro-inflammatory cytokines such as IL-1beta, IFN-gamma and TNF-alpha was significantly up-regulated in intestine. In the mean time, combined SJW significantly suppressed the intestinal epithelial apoptosis induced by CPT-11 over days 5-11. In particular, combination of SJW significantly inhibited the expression of TNF-alpha mRNA in the intestine over days 5-11. In conclusion, inhibition of pro-inflammatory cytokines and intestinal epithelium apoptosis partly explained the protective effect of SJW against the intestinal toxicities induced by irinotecan. Further studies are warranted to explore the potential for STW as an agent in combination with chemotherapeutic drugs to lower their dose-limiting toxicities.

St. John's Wort modulates the toxicities and pharmacokinetics of CPT-11 (irinotecan) in rats.

CPT-11 is a DNA topoisomerase I inhibitor for the therapy of colorectal cancer, whereas St. John's Wort (Hypericum perforatum, SJW) is a widely used herbal anti-depressant. This study aimed to investigate the effects of co-administered SJW on the toxicities and pharmacokinetics of CPT-11 and the underlying mechanisms. The body weight loss, gastrointestinal and hematological toxicities induced by CPT-11, and the pharmacokinetic parameters of CPT-11 were evaluated in rats pretreated with SJW or vehicle. Rats treated with CPT-11 alone experienced rapid decrease in body weight, whereas co-administration of SJW with CPT-11 resulted in lesser body weight loss. The gastrointestinal and hematological toxicities following CPT-11 injection were both alleviated in the presence of SJW. The rat pharmacokinetics of both CPT-11 and its metabolite SN-38 were significantly altered in presence of SJW. In conclusion, co-administered SJW significantly ameliorated the toxicities induced by CPT-11. The protective effect of SJW may be partially due to pharmacokinetic interaction between CPT-11 and SJW.

Effects of St. John's wort on irinotecan metabolism.

St. John's wort (SJW), a widely used herbal product, has been implicated in drug interactions resulting from the induced expression of the cytochrome P450 CYP3A4 isoform. In this study, we determined the effect of SJW on the metabolism of irinotecan, a pro-drug of SN-38 and a known substrate for CYP3A4. Five cancer patients were treated with irinotecan (350 mg/m(2), intravenously) in the presence and absence of SJW (900 mg daily, orally for 18 days) in an unblinded, randomized crossover study design. The plasma levels of the active metabolite SN-38 decreased by 42% (95% confidence interval [CI] = 14% to 70%) following SJW cotreatment with 1.0 micro M x h (95% CI = 0.34 micro M x h to 1.7 micro M x h) versus 1.7 micro M x h (95% CI = 0.83 micro M x h to 2.6 micro M x h) (P =.033, two-sided paired Student's t test). Consequently, the degree of myelosuppression was substantially worse in the absence of SJW. These findings indicate that patients on irinotecan treatment should refrain from taking SJW because plasma levels of SN-38 were dramatically reduced, which may have a deleterious impact on treatment outcome.

Protective effects of kampo medicines and baicalin against intestinal toxicity of a new anticancer camptothecin derivative, irinotecan hydrochloride (CPT-11), in rats.

In clinical use, irinotecan hydrochloride (CPT-11; 7-ethyl-10-[4-(piperidino)-1-piperidino]carbonyloxycamptothecin), a novel antitumor agent, causes a relatively high incidence of severe forms of diarrhea. We investigated whether baicalin, an inhibitor of beta-glucuronidase, which deconjugates the glucuronide of the active metabolite of CPT-11, SN-38 (7-ethyl-10-hydorxycamptothecin), and Japanese herbal medicines (Kampo medicines) which contain baicalin can ameliorate CPT-11-induced intestinal toxicity in rats. CPT-11 (60 mg/kg i.v. once daily for 4 consecutive days) induced intestinal toxicity characterized by diarrhea, loss of body weight, anorexia and disruption of intestinal epithelium. Treatment with baicalin (25 mg/kg p.o. twice daily) or Kampo medicines (TJ-14 and TJ-114; 1 g/kg p.o. twice daily) from the day before to 4 or 10 days after the start of CPT-11 administration resulted in significantly decreased weight loss, improved anorexia and delayed onset of diarrheal symptoms. Histological examination revealed that Kampo medicine-treated animals had less damage to the intestinal epithelium and that damage was repaired more rapidly than in control rats. These results suggest that the prophylactic use of Kampo medicines (TJ-14 and TJ-114) may be of value against CPT-11-induced intestinal toxicity.