Toxicity and antiviral activity of LY253963 against respiratory syncytial and parainfluenza type 3 viruses in tissue culture and in cotton rats

LY253963, the sodium salt of 1,3,4-thiadiazol-2-ylcyanamide, was evaluated in tissue culture and in cotton rats for toxicity and antiviral efficacy against respiratory syncytial (RSV) and parainfluenza type 3 (PIV3) viruses. The selective index (ratio of the median toxic dose: median efficacious dose) of LY253963 in HEp2 tissue culture cells was greater than 100 against both RSV and PIV3. When given intraperitoneally to cotton rats, the minimum protective dose of LY253963 against both of these viruses was between 1 and 3 mg/kg/day. In contrast, doses of LY253963 as high as 30 mg/kg/day, administered orally after experimental inoculation of virus, did not significantly reduce pulmonary virus titers in treated animals compared to control animals given placebo. No toxic effects were noted in cotton rats, even in those given 20 mg/kg/day for eight consecutive days.     

Broad-spectrum antiviral activity of the carbocyclic analog of 3-deazaadenosine

The carbocyclic analog of 3-deazaadenosine (C-c3 Ado) was found to inhibit in vitro the replication of several DNA and RNA viruses, including vaccinia, reo, measles, parainfluenza and vesicular stomatitis, at a concentration of 0.2-1 microgram/ml, while not being toxic for the host (primary rabbit kidney, HeLa, Vero) cells at a concentration of 400 micrograms/Ml. In its activity against vesicular stomatitis virus, parainfluenza virus, measles and reo virus, C-c3 Ado proved about 100 times more potent than other established broad-spectrum antiviraL agents such as ribavirin (virazole) and (S)-DHPA ((S)-9-(2,3-dihydroxypropyl)adenine). In vivo, C-c3 Ado protected newborn mice against a lethal infection of vesicular stomatitis virus when administered as a single dose of 20, 100 or 500 micrograms per mouse 1 h after virus infection.     

Antiviral activity of NMSO3 against respiratory syncytial virus infection in vitro and in vivo

NMSO3, a sulfated sialyl lipid was evaluated for its efficacy against respiratory syncytial virus (RSV) and other myxovirus infections in cell culture. The median effective concentration (50% effective concentration, EC(50)) of NMSO3 against replication of the Long strain of RSV in HEp-2 cells was 0.2 and 0.32 microM by optical ELISA and the plaque reduction method, respectively. On the other hand, the corresponding values for ribavirin were 10.5 and 11.2 microM, respectively. NMSO3 showed potent activity against other laboratory strains as well as fresh clinical isolates of RSV, and the average EC(50) was similar to that for Long strain. NMSO3 exhibited minimal cytotoxicity against HEp-2, MDCK, HMV-2 and Vero cells for which the median cytotoxic concentration (CC(50)) was more than 685 microM. The selectivity index [SI=(CC(50) for HEp-2/EC(50))] of NMSO3 for RSV exceeded 2978 and that of ribavirin was 6. The EC(50) of NMSO3 against influenza virus (FluV) A (H3N2) was 23.8 by the MTT method using HMV-2 cells, and 17.8 microM by the TCID(50) method using MDCK cells. NMSO3 did not inhibit replication of influenza B virus, parainfluenza virus type 2 and canine distemper virus at 103 microM. NMSO3 inhibited RSV infection of HEp-2 cells when it was added between 0 and 1.5 h after virus infection. By a temperature shift experiment during the period of contact between the virus and cells, NMSO3 inhibited both the binding of RSV to the cells and its penetration into the cells. Prophylactic and therapeutic efficacy of NMSO3 against RSV infection in cotton rats was examined. Intraperitoneal administration of 100 mg/kg per day of NMSO3 to cotton rats from 1 day before or 1 h after to 3 days after the RSV infection, once a day every day, decreased the RSV titer in lungs to 10(-1.26) to 10(-1.63) compared to the control rats which were infected with RSV and left untreated.     

Comparison of the inhibition of human metapneumovirus and respiratory syncytial virus by NMSO3 in tissue culture assays

Human metapneumovirus (hMPV) is a recently elucidated respiratory virus pathogen for which there are no agents currently licensed to prevent or treat infections caused by it. However, NMSO3 has been reported to inhibit replication of human respiratory syncytial virus (hRSV), a virus that is closely related to hMPV, both in vitro in tissue culture cells and in vivo in cotton rats. For this reason, experiments were performed to compare the antiviral activity of NMSO3 against both hRSV and hMPV in tissue culture-based assays. Heparin and ribavirin, two other compounds known to inhibit hRSV, and two other paramyxoviruses, human parainfluenza virus type 3 (PIV3) and measles virus (MV), were included in these tests for comparison. All three compounds significantly inhibited the replication of subtype A and B strains of hRSV and serotypes 1 and 2 hMPV. However, unlike ribavirin, NMSO3 and heparin inhibited only hMPV and hRSV and not PIV3 or MV. Also unlike ribavirin, the activity of the two sulfated molecules was most effective if these materials were present during virus attachment and penetration of host cells. Interestingly, NMSO3, but not heparin, was able to limit secondary infection and spread of both viruses.     

Dynamics of the antiviral activity of N-methanocarbathymidine against herpes simplex virus type 1 in cell culture

N-Methanocarbathymidine [(N)-MCT], a thymidine analogue, exhibits potent activity in cell culture against herpes simplex virus1 (HSV-1). (N)-MCT showed higher antiviral activity than ganciclovir (GCV). Continuous treatment of Vero cells with (N)-MCT immediately or 10 h post-infection (p.i.) fully prevented the development of viral infection. However, when infected cells were treated with (N)-MCT at 12 h p.i., there was only a partial inhibition (ca. 50%). Additionally, continuous treatment of infected cells with (N)-MCT for about 48 h was sufficient to achieve full prevention of viral infection without further treatment. These findings suggest the complete loss of herpes simplex thymidine kinase (HSV-tk) activity occurs after 48 h of treatment with (N)-MCT. This study helps to understand the mechanism and dynamics of antiHSV activity of (N)-MCT, which is necessary for its future development as an antiviral drug.     

Antiviral efficacy of VP14637 against respiratory syncytial virus in vitro and in cotton rats following delivery by small droplet aerosol

VP14637, the lead compound in a series of substituted bis-tetrazole-benzhydrylphenols developed by ViroPharma Incorporated, was evaluated for antiviral efficacy against respiratory syncytial virus (RSV) in vitro in cell culture and in vivo in cotton rats. A selective index of >3000 (> or =2000 times greater than that observed for ribavirin) was determined in the in vitro studies for this compound against both RSV A and B subtypes. In cotton rats, animals given as little as 126 microg drug/kg by small droplet aerosol in divided doses starting 1 day after experimental virus infection with either a RSV A or B subtype consistently had significantly lower mean pulmonary RSV titers and reduced histopathological findings than mock-treated animals or cotton rats given placebo (vehicle-treated animals). No cotton rat treated with aerosols of VP14637 during these studies manifested any evident untoward responses. Thus, VP14637 exhibited good selective antiviral efficacy both in vitro and in vivo.     

展枝马尾藻硫酸多糖对呼吸道合胞病毒的抗病毒活性及其机理研究

目的研究从褐藻展枝马尾藻Sargassumpatens中分离到的一种硫酸多糖SP2的抗病毒活性及抗病毒作用机理。方法通过细胞致病效应实验、MTT测定和空斑形成抑制实验对该多糖的抗病毒活性、细胞毒性和可能的抗病毒机理进行测定。结果通过空斑形成抑制实验测定结果显示,该多糖对呼吸道合胞病毒有好的抗病毒活性,其EC50值为1.1但没有抗流感病毒A和B、副流感病毒的活性。多糖的CC50高达3000μg.mL-1。其对呼吸道合胞病毒的抗病毒选择性指数被估计大于2727。用SP2预处理宿主细胞,不显示任何对病毒感染的保护作用。SP2对呼吸道合胞病毒有一定的杀病毒活性,其EC50值超过100μg.mL-1。时间加入实验证明,只有在37℃呼吸道合胞病毒吸附时期加入SP2才能完全抑制病毒的复制。结论SP2可能是一种有潜力的抗呼吸道合胞病毒感染的多糖。其对呼吸道合胞病毒的抗病毒作用机制与抑制病毒的吸附有关。     

Medical burden of respiratory syncytial virus and parainfluenza virus type 3 infection among US children. Implications for design of vaccine trials

Respiratory syncytial virus (RSV) and parainfluenza virus type 3 (PIV3) are two leading causes of lower respiratory illness (LRI) in infants. Many efforts have been directed to develop vaccines against these two viruses. Licensure of new vaccines includes three phases of clinical trials to evaluate safety, immunogenicity, and efficacy. To design an efficacy trial, age-specific incidence rates of suitable clinical endpoints need to be available. In this review, historical data are summarized to estimate the age-specific rates of acute respiratory illness (ARI), LRI, and hospitalization caused by RSV and PIV3 among US children <5 years of age. Nation-wide data are available for hospitalization but not ARI and LRI. Age-specific rates of RSV and PIV3-related ARI or LRI can vary 9-fold or 5-fold, respectively, in different studies conducted in different populations using different clinical and laboratory definitions. The annual medical burden for RSV and PIV3 in children <5 was estimated respectively to be about 4.19 and 3.24 million cases of medically-attended ARI, 2.1 and 1.08 million cases of LRI, and 113 and 29 thousand cases of hospitalization, respectively. The impact of three important variables including age at vaccination, clinical endpoints, and laboratory diagnosis in designing efficacy trials is discussed. A RSV vaccine which is safe and effective in the first six months of life is optimal to reduce the severe disease burden of LRI and hospitalization, however, interference of maternal antibody may reduce vaccine efficacy in this age group. LRI occurs more frequently than hospitalization and may be the most feasible clinical endpoint for designing efficacy trials. Since age-specific rates of RSV and PIV3-related LRI can vary significantly in different populations, collecting age-specific LRI rates in phase 1 and 2 trials to further understand this variability appears warranted.     

Antiviral activity of diarylheptanoid stereoisomers against respiratory syncytial virus in vitro and in vivo

We previously showed that (5S)-5-hydroxy-7-(4-hydroxyphenyl)-1-phenylhept-3-one (AO-0011) and (5S)-5-methoxy-1,7-diphenylhept-3-one (AO-0016) isolated from Alpinia officinarum exhibited stronger anti-influenza virus activity and anti-respiratory syncytial virus (RSV) activity, respectively, than the other isolated diarylheptanoids. In this study, we synthesized an enantiomer (AO-0503) and racemate (AO-0504) of AO-0011 and an enantiomer (AO-0514) of AO-0016. The anti-RSV activities of the three stereoisomers (AO-0503, AO-0504, and AO-0514) and AO-0011 were examined in vitro and in vivo to evaluate the stereoisomeric effect on anti-RSV activity. In a plaque reduction assay using human epidermoid carcinoma cells, all four diarylheptanoids significantly exhibited anti-RSV activity, and AO-0514 and AO-0016 exhibited stronger anti-RSV activity than AO-0503, AO-0504, and AO-0011. In a murine RSV infection model, all four diarylheptanoids with anti-RSV activity in vitro were also significantly effective in reducing virus titers in the lungs of RSV-infected mice. In the histopathological analysis of RSV-infected lungs, the oral administration of even AO-0514, which showed the lowest reduction of virus titers in the lungs, was significantly effective in reducing the infiltration of lymphocytes and in reducing the interferon-γ level, which is a marker of severity of pneumonia due to RSV infection, in bronchoalveolar lavage fluids prepared from RSV-infected mice. Although the stereoisomeric effects of diarylheptanoids on anti-RSV activity varied moderately, all four diarylheptanoids examined were suggested to ameliorate pneumonia and have a potential anti-RSV activity in vivo. They are possibly mother compounds for the development of an anti-RSV drug in the future.     

Potent antiviral activity of brequinar against the emerging Cantagalo virus in cell culture

In the present work, the antiviral activity of brequinar (BQR) against the replication of Cantagalo virus was evaluated. BQR is a potent inhibitor of cellular dihydroorotate dehydrogenase, an enzyme of the de novo pyrimidine biosynthetic pathway. Infection in the presence of 0.5 μM BQR reduced virus progeny production by >90%, revealing an EC₅₀ (drug concentration required to inhibit 50% of virus replication) of 0.017 μM. Replication of other orthopoxviruses was also inhibited by BQR at similar levels. In the presence of the drug, virus early proteins accumulated to control levels, whereas late gene expression was severely impaired. This result was confirmed by indirect immunofluorescence assays and analysis of time-regulated expression of a reporter gene under the control of a virus promoter. Both assays revealed nearly 90% inhibition of late gene expression. BQR also blocked virus DNA replication, which accounted for the subsequent inhibition of virus late gene expression. The ablation of virus DNA replication, late gene expression and infectious progeny production was restored to control levels when infected cells were co-treated with uridine (URD) and BQR. These data demonstrated that BQR targeted virus DNA synthesis by depleting the cellular pyrimidine pool, which was bypassed by the salvage pathway when URD was added to the cell cultures.     

In vitro evaluation of the antiviral activity of SP-303, an euphorbiaceae shrub extract,against a panel of respiratory viruses

The antiviral dn cytotoxic activity of SP-303, a naturally occurring polyphenolic polymer (average M.W. = 2,100 daltons) isolated from a Euphorbiaceae shrub, was evaluated in tissue culture. The compound exhibited selective antiviral activity (ratio IC₅₀/EC₅₀ ≥10) against parainfluenza virus type 1, respiratory syncytial virus, influenza A viruses, and influenza B viruses; marginal selective antiviral activity (IC₅₀/EC₅₀ ratio >2 to <10) against parinfluenza virus type 3; and no selective antiviral activity (IC₅₀/EC₅₀ ratio ≤1) against measles virus or adenovirus type 5. Hemagglutination and other studies suggested that SP-303 may at least partially inactivate virus by direct interaction with virus or host cell lipid membranes. © Wiley-Liss, Inc.     

Evaluation of the safety, tolerability and pharmacokinetics of ALN-RSV01, a novel RNAi antiviral therapeutic directed against respiratory syncytial virus (RSV)

Small interfering RNAs (siRNAs) work through RNA interference (RNAi), the natural RNA inhibitory pathway, to down-regulate protein production by inhibiting targeted mRNA in a sequence-specific manner. ALN-RSV01 is an siRNA directed against the mRNA encoding the N-protein of respiratory syncytial virus (RSV) that exhibits specific in vitro and in vivo anti-RSV activity. The results of two safety and tolerability studies with ALN-RSV01 involving 101 healthy adults (65 active, 36 placebo, single- and multiple dose, observer-blind, randomized dose-escalation) are described. Intranasal administration of ALN-RSV01 was well tolerated over a dose range up through 150mg as a single dose and for five daily doses. Adverse events were similar in frequency and severity to placebo (normal saline) and were transient, mild to moderate, with no dose-dependent trend. The frequency or severity of adverse events did not increase with increasing ALN-RSV01 exposure. All subjects completed all treatments and assessments with no early withdrawals or serious adverse events. Physical examinations, vital signs, ECGs and laboratory tests were normal. Systemic bioavailability of ALN-RSV01 was minimal. ALN-RSV01 appears safe and well tolerated when delivered intranasally and is a promising therapeutic candidate for further clinical development.     

Neuroprotective activity of chemokines against N-methyl-D-aspartate or beta-amyloid-induced toxicity in culture

We have examined the effect of various chemokines on neuronal toxicity in culture. In mixed cortical cultures, challenged with a brief pulse of N-methyl-D-aspartate (NMDA, 60 microM, 10 min), chemokines were either present for 2 h preceding the pulse or they were co-applied with NMDA and then kept in the medium for the following 20-24 h. Interleukin-8 (IL-8), regulated on activation of normal T cells expressed and secreted (RANTES) and macrophage/monocyte chemoattractant protein-1 (MCP-1), were neuroprotective under both conditions, whereas stromal cell-derived factor 1alpha (SDF-1alpha) was protective only when applied during and after the NMDA pulse. Mixed or pure neuronal cultures were also exposed for 48 h to a toxic fragment of the beta-amyloid peptide (beta-amyloid peptide-(25-35), 12.5 or 25 microM) in the absence or presence of chemokines. Among a number of chemokines, only RANTES was neuroprotective against beta-amyloid peptide-(25-35)-induced neurotoxicity in both cultures. We conclude that activation of chemokine receptors differentially affects neuronal degeneration induced by excitotoxins or beta-amyloid peptide in cortical cultures.     

Evaluation of the antiviral activity of kaempferol and its glycosides against human cytomegalovirus

The antiviral activity of seven flavonoids, belonging to the kaempferol series, was evaluated against human cytomegalovirus (HCMV) by a rapid method of detection of the immediate-early (IE) antigen, induced by the virus in infected cells. Flavonoids bearing acyl substituents were found to be the most active compounds.     

Evaluation of active specific immunization against paraquat toxicity in rats

Four groups of Wistar rats were immunized against a BSA-paraquat derivative antigen, when lethally poisoned with paraquat dichloride via intraperitoneal route. No significant correlation in survival rate was observed between immunized (5/45) and control (0/26) rats, but a significant correlation (p less than 0.05) in the mean survival time was noted in immunized rats as compared to controls (9.1 +/- 16.8 days and 2.0 +/- 0.8 days versus 2.1 +/- 2.4 days and 1.0 +/- 0.1 days respectively).     

New in vitro method for evaluating antiviral activity of acyclic nucleoside phosphonates against plant viruses

A new method was developed for testing antiviral compounds against plant viruses based on rapidly growing brassicas in vitro on liquid medium. This method enables exchange of media containing tested chemicals in various concentrations and simultaneous evaluation of their phytotoxicity and antiviral activity. While using ribavirin as a standard for comparison, phytotoxicity and ability of the acyclic nucleotide analogues (R)-PMPA, PMEA, PMEDAP, and (S)-HPMPC to eliminate ssRNA Turnip yellow mosaic virus (TYMV) were evaluated by this method. Double antibody sandwich ELISA and real-time PCR were used for relative quantification of viral protein and nucleic acid in plants. Ribavirin had the most powerful antiviral effect against TYMV. On the other hand, (R)-PMPA and PMEA had no antiviral effect and almost no phytotoxicity compared to the control. (S)-HPMPC and PMEDAP showed moderate antiviral effect, accompanied by higher phytotoxicity. The tested compounds can be screened within 6-9 weeks in contrast to the 6 months for traditionally used explants on solid medium. The method enables large-scale screening of potential antivirals for in vitro elimination of viruses from vegetatively propagated crops and ornamentals.     

Analysis of the in vitro antiviral activity of certain ribonucleosides against parainfluenza virus using a novel computer aided receptor modeling procedure

The in vitro antiviral activity of 28 nucleosides against the parainfluenza virus type 3 has been analyzed by using a novel computer aided receptor modeling procedure. The method involves an extensive modification of our earlier work (Ghose, A. K.; Crippen, G. M. J. Med. Chem. 1985, 28, 333). It presents a more straightforward algorithm for the steps that suffered from subjectivity in the earlier method. The method first determines the possible low-energy conformations of the nucleosides, and assigns a priority value for each conformation of each molecule. It then performs the following steps repeatedly, until it finds an acceptable solution. Starting from the conformation of highest priority, the various energetically allowed conformations of the other molecules are superimposed on it. On the basis of the physicochemical property matching (or overlapping), the best superposition is determined. The superimposed molecules are dissected into a minimum number of parts and the local physicochemical properties at different regions are correlated with their binding data (antiviral activity). A modified version of distance geometry has been used for geometric comparison of the structure of the molecules. On the basis of the virus rating (VR) of 28 ribonucleosides, this procedure hypothesized the minimum-energy conformation of 6-(methylthio)-9-beta-D-ribofuranosylpurine as a reference conformation and used three physicochemical properties, namely hydrophobicity, molar refractivity, and formal charge density for property matching. The binding-site cavity was divided into seven regions or pockets to differentiate the nature of interaction quantitatively. The model suggests that the 2- and 3-positions of the purine ring and the corresponding atoms of the other rings get some steric repulsion, and nucleosides having a single five-membered heterocyclic ring will better fit this virus. The methylthio group gets a strong attraction from dispersive interaction. Both hydrophilic and dispersive groups are attractive here. Although our calculation supports the previously suggested active conformation of ribavirin, it shows that it is not the global minimum-energy conformation. The difference lies in the orientation of the amide group. The calculated viral rating from this model showed a correlation coefficient of 0.971 with the observed values, and the explained variance and the standard deviation of the fit were 0.880 and 0.125, respectively.     

Hepatoprotective activity of chitosan against isoniazid and rifampicin-induced toxicity in experimental rats

Tuberculosis is a dangerous disease and its death toll is increasing year by year. Intake of isoniazid and rifampicin, the most common antitubercular drugs, lead to fatal hepatotoxic condition. We have studied the protective effect of chitosan supplementation against the hepatotoxicity induced by antitubercular drugs with respect to the changes in the levels of protein, albumin-globulin ratio, urea and bilirubin in the serum and diagnostic marker enzymes (alanine amino transferase, aspartate amino transferase, acid phosphatase and alkaline phosphatase), protein, glycoprotein conjugates (hexose, hexosamine and sialic acid), lipid peroxidation and reduced glutathione in the liver tissue of normal and experimental groups of rats. Co-administration of chitosan was found to significantly prevent the antitubercular drugs-induced alterations in the levels of diagnostic marker enzymes, bilirubin and albumin/globulin ratio in experimental groups of rats. Isoniazid and rifampicin-induced lipid peroxidation was also found to be prevented by the administration of chitosan. Further, chitosan administration increased the levels of urea and protein (in serum and liver) in experimental groups compared to hepatotoxicity-induced group of rats. Levels of glycoconjugates were also maintained to near normal level by chitosan co-administration. From the results obtained, it can be concluded that chitosan is beneficial against antitubercualr drugs-induced hepatoxicity.     

Hepatoprotective activity of Tephrosia purpurea against arsenic induced toxicity in rats

Aim:

The present study was conducted to evaluate the hepatoprotective activity of Tephrosia purpurea (TP) against sodium arsenite (NaAsO2) induced sub-acute toxicity in rats.

Materials and Methods:

Twenty four wistar albino rats of either sex were randomly divided into three groups. Group II and III were orally administered with sodium arsenite (10 mg/kg) daily in drinking water for 28 days. Additionally Group III was orally treated with hydro-alcoholic extract of Tephrosia purpurea (TP) @ 500 mg/kg daily for the same time period, whereas only deionized water was given to Group I (control). Serum biomarker levels, oxidative stress parameters and arsenic concentration were assessed in liver. Histopathology was also conducted.

Results:

It has been seen that TPE (500 mg/kg) significantly (P < 0.01) reduced serum ALT, AST, ALP activity and increased total protein and reduced necrosis and inflammation in liver of group III compared to group II. A significantly (P < 0.01) higher LPO and lower GSH levels without change in SOD activity in liver was also observed in group II compared to group III, though there was no significant difference in arsenic accumulation between them. The plant extract also protects the animals of group III from significant (P < 0.01) reduction in body weight.

Conclusion:

Our study shows that supplementation of Tephrosia purpurea extract (500 mg/kg) could ameliorate the hepatotoxic action of arsenic.KEY WORDS: Arsenic, hepatotoxicity, rats, sub-acute toxicity, Tephrosia purpurea